Feasibility of using the National Marine Mammal Tissue Bank for retrospective exploratory studies of perfluorinated alkyl acids.

Perfluorinated alkyl acids (PFAAs) have been used for 50+ years in materials such as stain-resistant treatments for paper and clothing, lubricants, and foam fire extinguishers. PFAAs are characterized by a fully fluorinated alkyl chain with a terminal acid group. Their long half-lives and ubiquitous environmental distribution create considerable concern for wildlife and human exposure. There is interest in examining temporal trends of PFAAs using the National Marine Mammal Tissue Bank (NMMTB), but NMMTB tissues are frozen and cryohomogenized in polytetrafluoroethylene (PTFE)-based materials. Because PTFE supplies may leach PFAAs into samples, this study mimicked collection, processing and storage steps of NMMTB samples and measured PFAA leaching to determine the feasibility of using this sample archive for PFAA temporal trends. We also explored concentrations in Atlantic white-sided dolphin (Lagenorhynchus acutus, WSDs) and rough-toothed dolphin (Steno bredanensis, RTDs) blubber (n=3 and 0) and liver (n=48 and 12, respectively). The materials used in NMMTB protocols may add up to 0.968ng/g perfluorooctanoic acid (PFOA), 0.090ng/g perfluorononanoic acid (PNFA), and 0.221ng/g perfluorooctane sulfonate (PFOS) to each archived sample. Leaching of PFNA and PFOS from supplies compared to dolphin levels was negligible, but PFOA contributions were substantially higher than levels found in most dolphin liver samples. Therefore, monitoring PFOA temporal trends from the NMMTB would require careful consideration. RTDs had significantly higher levels of PFOS and PFNA than WSDs. Both species have similar life history, trophic status, and foraging behaviors in deep pelagic waters, so differences could be from latitudinal variation in contamination. RTDs stranded in Florida; WSDs stranded farther north mostly in Massachusetts. Juveniles had significantly higher levels of PFOS and PFNA than adults in both species, suggesting growth dilution as they approach maturity. PFOS significantly decreased after 2001 in both species as expected based on changes in production.

Per- and polyfluoroalkyl substances (PFAS) in American Red Cross adult blood donors, 2000-2015.

In 2015, thirteen per- and polyfluoroalkyl substances (PFAS), including perfluorohexanesulfonate (PFHxS), perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), and perfluorodecanoate (PFDA) were analyzed in human plasma that were collected from a total of 616 American Red Cross male and female blood donors (ages 20-69) at 6 regional blood collection centers. Plasma samples were analyzed using a validated solvent precipitation-isotope dilution direction-liquid chromatography tandem mass spectrometry method. The data were analyzed in conjunction with prior cross-sectional investigations [2000-2001 (n =645), 2006 (n =600), and 2010 (n =600)] to determine PFAS trends. Age- and sex-adjusted geometric mean serum (2000-2001) and plasma (2006, 2010, 2015) concentrations (ng/mL) were, respectively: PFHxS (2.3, 1.5, 1.3, 0.9); PFOS (35.1, 14.5, 8.4, 4.3); PFOA (4.7, 3.4, 2.4, 1.1); PFNA (0.6, 1.0, 0.8, 0.4); and PFDA (0.2, 0.3, 0.3, 0.1). The percentage decline in these geometric mean concentrations from 2000-2001 to 2015 were: PFHxS (61%); PFOS (88%); PFOA (77%); PFNA (33%); and PFDA (50%). The results indicate a continued decline of PFHxS, PFOS, and PFOA concentrations in American Red Cross blood donors. For the remaining PFAS measured in 2015, including the shorter chain perfluoroalkyls perfluorobutanesulfonate (PFBS) and perfluorohexanoate (PFHxA), the majority of samples were below the lower limit of quantitation.

Polyfluorinated substances in abiotic standard reference materials.

The National Institute of Standards and Technology (NIST) has a wide range of Standard Reference Materials (SRMs) which have values assigned for legacy organic pollutants and toxic elements. Existing SRMs serve as homogenous materials that can be used for method development, method validation, and measurement for contaminants that are now of concern. NIST and multiple groups have been measuring the mass fraction of a group of emerging contaminants, polyfluorinated substances (PFASs), in a variety of SRMs. Here we report levels determined in an interlaboratory comparison of up to 23 PFASs determined in five SRMs: sediment (SRMs 1941b and 1944), house dust (SRM 2585), soil (SRM 2586), and sludge (SRM 2781). Measurements presented show an array of PFASs, with perfluorooctane sulfonate being the most frequently detected. SRMs 1941b, 1944, and 2586 had relatively low concentrations of most PFASs measured while 23 PFASs were at detectable levels in SRM 2585 and most of the PFASs measured were at detectable levels in SRM 2781. The measurements made in this study were used to add values to the Certificates of Analysis for SRMs 2585 and 2781.

Temporal trends of perfluoroalkyl concentrations in American Red Cross adult blood donors, 2000-2010.

Eleven perfluorinated alkyl acids (PFAAs) were analyzed in plasma from a total of 600 American Red Cross adult blood donors from six locations in 2010. The samples were extracted by protein precipitation and quantified by using liquid chromatography tandem mass spectrometry (HPLC/MS/MS). The anions of the three perfluorosulfonic acids measured were perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS). The anions of the eight perfluorocarboxylic acids were perfluoropentanoate (PFPeA), perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnA), and perfluorododecanoate (PFDoA). Findings were compared to results from different donor samples analyzed at the same locations collected in 2000-2001 (N = 645 serum samples) and 2006 (N = 600 plasma samples). Most measurements in 2010 were less than the lower limit of quantitation for PFBS, PFPeA, PFHxA, and PFDoA. For the remaining analytes, the geometric mean concentrations (ng/mL) in 2000-2001, 2006, and 2010 were, respectively, PFHxS: (2.25, 1.52, 1.34); PFOS (34.9, 14.5, 8.3); PFHpA (0.13, 0.09, 0.05); PFOA (4.70, 3.44, 2.44); PFNA (0.57, 0.97, 0.83); PFDA (0.16, 0.34, 0.27), and PFUnA (0.10, 0.18, 0.14). The percentage decline (parentheses) in geometric mean concentrations from 2000-2001 to 2010 were PFHxS (40%), PFOS (76%), and PFOA (48%). The decline in PFOS suggested a population halving time of 4.3 years. This estimate is comparable to the geometric mean serum elimination half-life of 4.8 years reported in individuals. This similarity supports the conclusion that the dominant PFOS-related exposures to humans in the United States were greatly mitigated during the phase-out period.

Determination of perfluorinated alkyl acid concentrations in biological standard reference materials.

Standard reference materials (SRMs) are homogeneous, well-characterized materials used to validate measurements and improve the quality of analytical data. The National Institute of Standards and Technology (NIST) has a wide range of SRMs that have mass fraction values assigned for legacy pollutants. These SRMs can also serve as test materials for method development, method validation, and measurement for contaminants of emerging concern. Because inter-laboratory comparison studies have revealed substantial variability of measurements of perfluoroalkyl acids (PFAAs), future analytical measurements will benefit from determination of consensus values for PFAAs in SRMs to provide a means to demonstrate method-specific performance. To that end, NIST, in collaboration with other groups, has been measuring concentrations of PFAAs in a variety of SRMs. Here we report levels of PFAAs and perfluorooctane sulfonamide (PFOSA) determined in four biological SRMs: fish tissue (SRM 1946 Lake Superior Fish Tissue, SRM 1947 Lake Michigan Fish Tissue), bovine liver (SRM 1577c), and mussel tissue (SRM 2974a). We also report concentrations for three in-house quality-control materials: beluga whale liver, pygmy sperm whale liver, and white-sided dolphin liver. Measurements in SRMs show an array of PFAAs, with perfluorooctane sulfonate (PFOS) being the most frequently detected. Reference and information values are reported for PFAAs measured in these biological SRMs.

Analysis of a homologous series of perfluorocarboxylates from American Red Cross adult blood donors, 2000-2001 and 2006.

The purpose of this study was to determine the concentration trends of a nine-target-analyte homologous series of perfluorocarboxylates from six American Red Cross adult blood donor centers. A total of 645 serum and 600 plasma samples were obtained in 2000-2001 and 2006, respectively, with samples stratified for each 10-year (20-69) age- and sex-group per each location. Samples were extracted by protein precipitation and quantified by using tandem mass spectrometry. The nine perfluorocarboxylates were perfluorobutanoate (PFBA, C(3)F(7)CO(2)(-)), perfluoropentanoate (PFPeA, C(4)F(9)CO(2)(-)), perfluorohexanoate (PFHxA, C(5)F(11)CO(2)(-)), perfluoroheptanoate (PFHpA, C(6)F(13)CO(2)(-)), perfluorooctanoate (PFOA, C(7)F(15)CO(2)(-)), perfluorononanoate (PFNA, C(8)F(17)CO(2)(-)), perfluorodecanoate (PFDA, C(9)F(19)CO(2)(-)), perfluoroundecanoate (PFUnA,C(10)F(21)CO(2)(-)), and perfluorododecanoate (PFDoA, C(11)F(23)CO(2)(-)). The majority of measurements were less than the lower limit of quantitation for PFPeA, PFHxA, and PFDoA. For the remaining targeted analytes, the geometric mean serum and plasma concentrations (ng/mL) for 2000-2001 and 2006 were, respectively, as follows: PFBA 2.61 vs 0.33, PFHpA 0.13 vs 0.09, PFOA 4.70 vs 3.44, PFNA 0.57 vs 0.97, PFDA 0.16 vs 0.34, and PFUnA 0.10 vs 0.18. Estimates of the 95th percent tolerance limits (ng/mL) were as follows: PFBA 5.3 vs 1.4, PFHpA 0.4 vs 0.4, PFOA 12.3 vs 7.7, PFNA 1.4 vs 2.2, PFDA 0.4 vs 0.8, and PFUnA 0.3 vs 0.5. Important observations were the decline in PFBA and increase in PFNA, PFDA, and PFUnA concentrations between 2000-2001 and 2006. The longer chain length perfluorocarboxylates were also highly correlated with each other.

Determination of perfluorinated compounds in fish fillet homogenates: method validation and application to fillet homogenates from the Mississippi River.

We report herein a simple protein precipitation extraction-liquid chromatography tandem mass spectrometry (LC/MS/MS) method, validation, and application for the analysis of perfluorinated carboxylic acids (C7-C12), perfluorinated sulfonic acids (C4, C6, and C8), and perfluorooctane sulfonamide (FOSA) in fish fillet tissue. The method combines a rapid homogenization and protein precipitation tissue extraction procedure using stable-isotope internal standard (IS) calibration. Method validation in bluegill (Lepomis macrochirus) fillet tissue evaluated the following: (1) method accuracy and precision in both extracted matrix-matched calibration and solvent (unextracted) calibration, (2) quantitation of mixed branched and linear isomers of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) with linear isomer calibration, (3) quantitation of low level (ppb) perfluorinated compounds (PFCs) in the presence of high level (ppm) PFOS, and (4) specificity from matrix interferences. Both calibration techniques produced method accuracy of at least 100±13% with a precision (%RSD) ≤18% for all target analytes. Method accuracy and precision results for fillet samples from nine different fish species taken from the Mississippi River in 2008 and 2009 are also presented.

Determination of perfluorinated alkyl acid concentrations in human serum and milk standard reference materials.

Standard Reference Materials (SRMs) are certified reference materials produced by the National Institute of Standards and Technology that are homogeneous materials well characterized with values for specified properties, such as environmental contaminant concentrations. They can be used to validate measurement methods and are critical in improving data quality. Disagreements in perfluorinated alkyl acid (PFAA) concentrations measured in environmental matrices during past interlaboratory comparisons emphasized the need for SRMs with values assigned for PFAAs. We performed a new interlaboratory comparison among six laboratories and provided, for the first time, value assignment of PFAAs in SRMs. Concentrations for perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and other PFAAs in two human serum and two human milk SRMs are reported. PFAA concentration measurements agreed for serum SRM 1957 using different analytical methods in six laboratories and for milk SRM 1954 in three laboratories. The interlaboratory relative standard deviation for PFOS in SRM 1957 was 7%, which is an improvement over past interlaboratory studies. Matrix interferences are discussed, as well as temporal trends and the percentage of branched vs. linear isomers. The concentrations in these SRMs are similar to the present-day average concentrations measured in human serum and milk, resulting in representative and useful control materials for PFAA human monitoring studies.

Comparison of extraction and quantification methods of perfluorinated compounds in human plasma, serum, and whole blood.

Perfluorinated compounds are ubiquitous in the environment and have been reported to occur in human blood. Accurate risk assessments require accurate measurements of exposures, but identification and quantification of PFCs in biological matrices can be affected by both ion suppression and enhancement in liquid chromatography-tandem mass spectrometry techniques (LC/MS-MS). A study was conducted to quantify potential biases in LC/MS-MS quantification methods. Using isotopically labeled perfluorooctanoic acid ([(13)C(2)]-PFOA), perfluorononanoic acid ([(13)C(2)]-PFNA), and ammonium perfluorooctanesulfonate ([(18)O(2)]-PFOS) spiked tissues, ion-pairing extraction, solid-phase extraction, and protein precipitation sample preparation techniques were compared. Analytical accuracy was assessed using both solvent calibration and matrix-matched calibration for quantification. Data accuracy and precision of 100+/-15% was demonstrated in both human sera and plasma for all three sample preparation techniques when matrix-matched calibration was used in quantification. In contrast, quantification of ion-pairing extraction data using solvent calibration in combination with a surrogate internal standard resulted in significant analytical biases for all target analytes. The accuracy of results, based on solvent calibration was highly variable and dependent on the serum and plasma matrices, the specific target analyte [(13)C(2)]-PFOA, [(13)C(2)]-PFNA, or [(18)O(2)]-PFOS, the target analyte concentration, the LC/MS-MS instrumentation used in data generation, and the specific surrogate internal standard used in quantification. These results suggest that concentrations of PFCs reported for human blood using surrogate internal standards in combination with external solvent calibration can be inaccurate unless biases are accounted for in data quantification.

Decline in perfluorooctanesulfonate and other polyfluoroalkyl chemicals in American Red Cross adult blood donors, 2000-2006.

In 2000, 3M Company, the primary global manufacturer, announced a phase-out of perfluorooctanesulfonyl fluoride (POSF, C8F17SO2F)-based materials after perfluorooctanesulfonate (PFOS, C8F17SO3-) was reported in human populations and wildlife. The purpose of this study was to determine whether PFOS and other polyfluoroalkyl concentrations in plasma samples, collected in 2006 from six American Red Cross adult blood donor centers, have declined compared to nonpaired serum samples from the same locations in 2000-2001. For each location, 100 samples were obtained evenly distributed by age (20-69 years) and sex. Analytes measured, using tandem mass spectrometry, were PFOS, perfluorooctanoate (PFOA), perfluorohexanesulfonate (PFHxS), perfluorobutanesulfonate (PFBS), N-methyl perfluorooctanesulfonamidoacetate (Me-PFOSA-AcOH), and N-ethyl perfluorooctanesulfonamidoacetate (Et-PFOSA-AcOH). The geometric mean plasma concentrations were for PFOS 14.5 ng/mL (95% CI 13.9-15.2), PFOA 3.4 ng/ mL (95% CI 3.3-3.6), and PFHxS 1.5 ng/mL (95% CI 1.4-1.6). The majority of PFBS, Me-PFOSA-AcOH, and Et-PFOSA-AcOH concentrations were less than the lower limit of quantitation. Age- and sex-adjusted geometric means were lower in 2006 (approximately 60% for PFOS, 25% for PFOA, and 30% for PFHxS) than those in 2000-2001. The declines for PFOS and PFHxS are consistent with their serum elimination half-lives and the time since the phase-out of POSF-based materials. The shorter serum elimination half-life for PFOA and its smaller percentage decline than PFOS suggests PFOA concentrations measured in the general population are unlikely to be solely attributed to POSF-based materials. Direct and indirect exposure sources of PFOA could include historic and ongoing electrochemical cell fluorination (ECF) of PFOA, telomer production of PFOA, fluorotelomer-based precursors, and other fluoropoly-mer production.

An interlaboratory study of perfluorinated alkyl compound levels in human plasma.

We conducted an interlaboratory study which differed from the typical study of this type because of its emphasis on comparing intralaboratory variability in results. We sent specimens to six laboratories experienced in the analysis of perfluorinated alkyl compounds in blood matrices and that use stringent procedures to control and assure accuracy and precision. Each received an identical set of 60 plasma specimens that were analyzed in six completely independent batches. Split specimens were included so that within- and between-batch coefficients of variation could be calculated. All laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorohexanesulfonate (PFHxS) measured in the specimens in general showed a high level of agreement, although in some cases the agreement was only moderate. The average within- and between-batch coefficient of variation for PFOS was 9.1% and 9.3%; for PFOA was 14.5% and 14.5%; and for PFHxS was 14.5% and 17.0%. The recent availability of labeled internal standards, among other advances, has facilitated improvement in the accuracy and precision of the assays. Considering the degree of between-subject variation in levels among people in background-exposed populations, the results indicate that biomarker-based epidemiologic studies of associations with health could have reasonable precision.

Preliminary evidence of a decline in perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) concentrations in American Red Cross blood donors.

The purpose of this pilot study was to determine whether perfluorooctanesulfonate (PFOS,C(8)F(17)SO(3)(-)) and perfluorooctanoate (PFOA,C(7)F(15)CO(2)(-)) concentrations in American Red Cross blood donors from Minneapolis-St. Paul, Minnesota have declined after the 2000-2002 phase-out of perfluorooctanesulfonyl-fluoride (POSF, C(8)F(17)SO(2)F)-based materials by the primary global manufacturer, 3M Company. Forty donor plasma samples, categorized by age and sex, were collected in 2005, and PFOS and PFOA concentrations were compared to 100 (non-paired) donor serum samples collected in 2000 from the same general population that were analyzed at the time using ion-pair extraction methods with tetrahydroperfluorooctanesulfonate as an internal standard. Eleven of the 100 samples originally collected were reanalyzed with present study methods that involved (13)C- labeled PFOA spiked into the donor samples, original samples, control human plasma, and the calibration curve prior to extraction, and was used as a surrogate to monitor extraction efficiency. Quantification was performed by high performance liquid chromatography tandem mass spectrometry methods. Among the 100 serum samples analyzed for PFOS, the geometric mean was 33.1 ng ml(-1) (95% CI 29.8-36.7) in 2000 compared to 15.1 ng ml(-1) (95% CI 13.3-17.1) in 2005 (p<0.0001) for the 40 donor plasma samples. The geometric mean concentration for PFOA was 4.5 ng ml(-1) (95% CI 4.1-5.0) in 2000 compared to 2.2 ng ml(-1) (95% CI 1.9-2.6) in 2005 (p<0.0001). The decrease was consistent across donors' age and sex. To confirm these preliminary findings, additional sub-sets of year 2000 samples will be analyzed, and a much larger biomonitoring study of other locations is planned.

Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry.

A selective and sensitive method for analysis of perfluorooctanoic acid (PFOA) in human serum and plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and thoroughly validated to satisfy strict FDA guidelines for bioanalytical methods. A simple, automated sample preparation procedure, involving extraction of the target analyte with acetonitrile on protein precipitation media in a 96-well plate format was developed, allowing efficient handling of large numbers of samples. The proposed method uses the calibration standards prepared in a surrogate matrix (rabbit serum or plasma) and (13)C-labeled PFOA as the internal standard to account for matrix effects, instrument drift, and extraction efficiency. Human serum and plasma could not be used for matrix matching of calibration standards as endogenous levels of PFOA observed in the control human serum and plasma significantly exceeded the targeted lower limit of quantitation (LLOQ) of the method. Precision and accuracy of the method were demonstrated by analysis of rabbit serum and plasma control samples fortified at 0.5, 5, and 40 ng/mL PFOA and human serum and plasma fortified at 1.0, 5.0, 40 ng/mL PFOA. The LLOQ of 0.5 ng/mL PFOA was experimentally demonstrated for rabbit and human serum and plasma. Within-day precision and accuracy, short-term stability, freeze-thaw stability, equivalence of response between PFOA and APFO (the ammonium salt of PFOA), and dilution of concentrated samples were also investigated. The results of the validation experiments comply with the precision and accuracy limits defined by the FDA guidance document: "Guidance for Industry, Bioanalytical Method Validation", May 2001.

Analytical techniques and method validation for the measurement of selected semivolatile and nonvolatile organofluorochemicals in air.

The widespread use of semi- and nonvolatile organofluorochemicals in industrial facilities, concern about their persistence, and relatively recent advancements in liquid chromatography/mass spectrometry (LC/MS) technology have led to the development of new analytical methods to assess potential worker exposure to airborne organofluorochemicals. Techniques were evaluated for the determination of 19 organofluorochemicals and for total fluorine in ambient air samples. Due to the potential biphasic nature of most of these fluorochemicals when airborne, Occupational Safety and Health Administration (OSHA) versatile sampler (OVS) tubes were used to simultaneously trap fluorochemical particulates and vapors from workplace air. Analytical methods were developed for OVS air samples to quantitatively analyze for total fluorine using oxygen bomb combustion/ion selective electrode and for 17 organofluorochemicals using LC/MS and gas chromatography/mass spectrometry (GC/MS). The experimental design for this validation was based on the National Institute of Occupational Safety and Health (NIOSH) Guidelines for Air Sampling and Analytical Method Development and Evaluation, with some revisions of the experimental design. The study design incorporated experiments to determine analytical recovery and stability, sampler capacity, the effect of some environmental parameters on recoveries, storage stability, limits of detection, precision, and accuracy. Fluorochemical mixtures were spiked onto each OVS tube over a range of 0.06-6 microg for each of 12 compounds analyzed by LC/MS and 0.3-30 microg for 5 compounds analyzed by GC/MS. These ranges allowed reliable quantitation at 0.001-0.1 mg/m3 in general for LC/MS analytes and 0.005-0.5 mg/m3 for GC/MS analytes when 60 L of air are sampled. The organofluorochemical exposure guideline (EG) is currently 0.1 mg/m3 for many analytes, with one exception being ammonium perfluorooctanoate (EG is 0.01 mg/m3). Total fluorine results may be used to determine if the individual compounds quantified provide a suitable mass balance of total airborne organofluorochemicals based on known fluorine content. Improvements in precision and/or recovery as well as some additional testing would be needed to meet all NIOSH validation criteria. This study provided valuable information about the accuracy of this method for organofluorochemical exposure assessment.