identifying-per-polyfluorinated-chemical-species-with-combined

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

U.S. Environmental Protection Agency

Ten different perfluoroalkyl acids (PFAAs), including perfluooctane sulfonate (PFOS), were measured in 30 common carp (Cyprinus carpio) fillets collected from three sites on the Upper Mississippi River in Minnesota in an effort to evaluate the potential impact of PFAA emissions in this area. Samples upstream of the city of St. Cloud (reference site) had median PFOS concentrations of 8.1 ng/g wet weight (ng/g wet wt), but median levels increased significantly downstream in the Minneapolis-St. Paul urban area, with concentrations from the Pig's Eye Lake site at 26 ng/g wet wt (p = 0.0015) and the Spring Lake site at 40 ng/g wet wt (p = 0.0004). This latter PFOS concentration is within the advisory range for limiting fish consumption to one meal a week according to the Minnesota Department of Health. Other PFAAs were also found to increase significantly between the reference site and the Minneapolis-St. Paul area, but maximal concentrations remained below 2.0 ng/g wet wt. This study demonstrates the bioaccumulation of PFAAs in a ubiquitous fish species in a major urban area known to have historical inputs of various PFAA compounds. The full extent of this contamination and the potential for accumulation in other species remain to be evaluated.

Perfluorinated compounds in common carp (Cyprinus carpio) fillets from the Upper Mississippi River.

The perfluoroalkyl acids (PFAAs), including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), have come under increasing scrutiny due to their persistence, global distribution, and toxicity. Given that their human exposure routes remain poorly characterized, the potential role of house dust needs to be more completely evaluated. In this study, new methods for the analysis of 10 PFAAs and three fluorinated telomer alcohols (FTOHs) were developed for dust samples collected from homes (n = 102) and day care centers (n = 10) in Ohio and North Carolina in 2000-2001. FTOHs were measured by GC/ MS and PFAAs were analyzed by LC-MS/MS. PFOS and PFOA were the most prominent compounds detected, occurring in over 95% of the samples at median concentrations of 201 and 142 ng/g of dust, respectively. Maximal concentrations of PFOS were 12 100 ng/g (95th percentile, 2240 ng/g), PFOA 1960 ng/g (95th percentile, 1200 ng/g), and perfluorohexanesulfonate (PFHS) 35 700 ng/g (95th percentile, 2300 ng/g). The 8:2 FTOH, which is volatile and can degrade to PFOA, had a maximum concentration of 1660 ng/g dust (95th percentile, 669 ng/g). These results indicate that perfluorinated compounds are present in house dust at levels that may represent an important pathway for human exposure.

Perfluorinated compounds in house dust from Ohio and North Carolina, USA.

The number of studies involving the analysis of perfluorooctanoic acid (PFOA) has increased recently because PFOA is routinely detected in human blood samples from around the world. Recent studies with mice have shown that dosing pregnant dams with PFOA during gestation gives rise to a dose-dependent mortality in the litters, a reduction in neonatal body weight for the surviving pups, and subsequent deficits in mammary gland development when compared to control animals. The actual body burdens of PFOA in dams and pups associated with these endpoints have not been determined, in part due to a lack of robust analytical methods for these matrices. The goal of the current study was to develop reliable methods with acceptable performance characteristics for the analysis of PFOA in several matrices relevant to pregnant mouse studies. Dam and pup serum, amniotic fluid, urine, milk, mammary tissue, and whole mouse pups were isolated for method development and analysis. The resulting method provided excellent accuracy (92.1-111%) and reproducibility (relative standard deviation 4.3-21%) making them very useful for future studies. These methods were then applied to dosed animal fluids and tissues in order to conduct a thorough evaluation of the pharmacokinetics in utero. Resulting tissue specific measurements of PFOA in serum, amniotic fluid, urine, milk, mammary tissue, and whole pup homogenate will be used to more completely describe the dose-response relationships for the most sensitive health outcomes and inform pharmacokinetic models that are being developed and evaluated.

Analysis of PFOA in dosed CD1 mice. Part 1. Methods development for the analysis of tissues and fluids from pregnant and lactating mice and their pups.

Guest comment: perfluoroalkyl acid focus issue.

In order to compare between in vivo toxicity studies, dosimetry is needed to translate study-specific dose regimens into dose metrics such as tissue concentration. These tissue concentrations may then be compared with in vitro bioactivity assays to perhaps identify mechanisms relevant to the lowest observed effect level (LOEL) dose group and the onset of the observed in vivo toxicity. Here, we examine the perfluorinated compounds (PFCs) perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS). We analyzed 9 in vivo toxicity studies for PFOA and 13 in vivo toxicity studies for PFOS. Both PFCs caused multiple effects in various test species, strains, and genders. We used a Bayesian pharmacokinetic (PK) modeling framework to incorporate data from 6 PFOA PK studies and 2 PFOS PK studies (conducted in 3 species) to predict dose metrics for the in vivo LOELs and no observed effect levels (NOELs). We estimated PK parameters for 11 combinations of chemical, species, strain, and gender. Despite divergent study designs and species-specific PK, for a given effect, we found that the predicted dose metrics corresponding to the LOELs (and NOELs where available) occur at similar concentrations. In vitro assay results for PFOA and PFOS from EPA's ToxCast project were then examined. We found that most in vitro bioactivity occurs at concentrations lower than the predicted concentrations for the in vivo LOELs and higher than the predicted concentrations for the in vivo NOELs (where available), for a variety of nonimmunological effects. These results indicate that given sufficient PK data, the in vivo LOELs dose regimens, but not necessarily the effects, could have been predicted from in vitro studies for these 2 PFCs. 

Dosimetric anchoring of in vivo and in vitro studies for perfluorooctanoate and perfluorooctanesulfonate.

Creatinine (CR) is an endogenously produced chemical that is routinely assayed in urine specimens to assess kidney function and sample dilution. The industry-standard method for CR determination, known as the kinetic Jaffé (KJ) method, relies on an exponential rate of a colorimetric change, and can therefore require automated processing equipment for moderate- to high-throughput analysis (hundreds to thousands of samples per day). This study evaluates an alternative colorimetric method, the "plateau Jaffé" (PJ) method, which utilizes the chemistry of the KJ method, a commercially available kit, and a multipoint calibration curve. This method is amenable to moderate-throughput sample analysis and does not require automated processing equipment. Thirty-two spot urine samples from healthy adult volunteers were analyzed for creatinine concentration (CRc) using the KJ and PJ methods. Samples were also analyzed using a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) method, which acted as an analytical control. Replicate measurements of spot samples (natural log-transformed values) were used to estimate method precision, and linear regression models were used to evaluate method accuracy (LC-TOF/MS measurements were considered the analytical benchmark). Measurement precision was comparable across all three methods, with coefficent of variation estimates ranging from 3 to 6%. Regression models generally showed good agreement across methods with R(2) estimates ranging from .996 to .998, slope estimates ranging from .944 to .986, and y-intercept estimates ranging from 0.111 to 0.303. Minor bias (between 2 and 16%) was observed across methods at the tails of the measurement distributions. The provided regression equations can be used to adjust for this bias and to improve CR measurement comparisons across studies employing different methods. Considering these results, the PJ method is a suitable alternative to the industry standard KJ method for urinary CRc determination. It can be implemented for moderate-throughput sample analysis using modest and commonly available lab instrumentation and manual sample preparation techniques. 

Evaluating an alternative method for rapid urinary creatinine determination.

Fipronil is a phenylpyrazole insecticide that is widely used in residential and agricultural settings to control ants, roaches, termites, and other pests. Fipronil and its transformation products have been found in a variety of environmental matrices, but the source[s] which makes the greatest contribution to fipronil in surface water has yet to be determined. A sampling effort designed to prioritize known fipronil inputs (golf courses, residential areas, biosolids application sites and wastewater facilities) was conducted in North Carolina to learn more about the origins of fipronil in surface water. High resolution mass spectrometry (HRMS) analysis indicated that fipronil and its known derivatives were routinely present in all samples, but concentrations were substantially elevated near wastewater treatment plant outfalls (range 10-500ng/L combined), suggesting that they predominate as environmental sources. Corresponding recycled wastewater samples, which were treated with NaOCl for disinfection, showed disappearance of fipronil and all known degradates. HRMS and nuclear magnetic resonance (NMR) analysis techniques were used to determine that all fipronil-related compounds are oxidized to a previously unidentified fipronil sulfone chloramine species in recycled wastewater. The implications of the presence of a new fipronil-related compound in recycled wastewater need to be considered. 

Comparison of fipronil sources in North Carolina surface water and identification of a novel fipronil transformation product in recycled wastewater.

2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate was designed to replace perfluorooctanoic acid (PFOA), which has been mostly phased out of U.S. production due to environmental persistence, detectable human and wildlife serum concentrations, and reports of systemic toxicity. In rodent models, PFOA exposure suppresses T cell-dependent antibody responses (TDAR) and vaccine responses in exposed humans. To determine replacement compound effects on TDAR and related parameters, male and female C57BL/6 mice were gavaged with 0, 1, 10, or 100 mg/kg/day for 28 days. Mice immunized with antigen on day 24 were evaluated for TDAR and splenic lymphocyte subpopulations five days later. Serum and urine were collected for test compound concentrations and liver peroxisome proliferation was measured. Relative liver weight at 10 and 100 mg/kg and peroxisome proliferation at 100 mg/kg were increased in both sexes. TDAR was suppressed in females at 100 mg/kg. T lymphocyte numbers were increased in males at 100 mg/kg; B lymphocyte numbers were unchanged in both sexes. Females had less serum accumulation and higher clearance than males, and males had higher urine concentrations than females at all times and doses. While this PFOA-replacement compound appears less potent at suppressing TDAR relative to PFOA, it produces detectable changes in parameters affected by PFOA; further studies are necessary to determine its full immunomodulatory profile and potential synergism with other per- and polyfluoroalkyl substances of environmental concern.

Evaluation of the immunomodulatory effects of 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate in C57BL/6 mice.

Legacy perfluorinated compounds exhibit significant environmental persistence and bioaccumulation potential, which has spawned an ongoing effort to introduce replacement compounds with reduced toxicological risk profiles. Many of these emerging chemical species lack validated quantitative methods, and, frequently, appropriate analytical standards for accurate monitoring and identification. To fill this knowledge gap, a general method for the quantitative determination of perfluoroether carboxylic acids (PFECAs) by LC-MS/MS was single-lab validated on spike-recovery samples in surface, drinking, and wastewater for a variety of perfluorinated ether standards. Relative error measurements for spike-recovery samples in each matrix ranged from 0.36% to 25.9%, with an average error of 10% overall. Coefficient of variation (CV) for each compound ranged from 10 to 28% with an average of 17%. The quantitative methodology was applied during repeated weekly monitoring of the Cape Fear River during remediation of PFECA hexafluoropropyloxide dimer-acid (HFPO-DA), known by the brand name "GenX." Semi-quantitative concentration estimates for emerging PFECA compounds lacking analytical standards was also carried out using surrogate calibration curves and mass labeled HFPO-DA as an internal standard. Estimates of the emerging compounds were possible using matched standards, but application of the estimation methodology to compounds with known concentration revealed that such estimates may possess up to an order of magnitude, or more, in uncertainty due to the difficulty of matching with an appropriate standard. Nevertheless, the estimation biases are primarily systematic (extraction efficiency and instrument response) rather than stochastic, enabling the collection of time-course data; both HFPO-DA and the emerging compounds were reduced in surface water and drinking water concentration of several orders of magnitude after removal of the source waste stream.

Validation of quantitative measurements and semi-quantitative estimates of emerging perfluoroethercarboxylic acids (PFECAs) and hexfluoroprolyene oxide acids (HFPOAs).

Mark Strynar

National Exposure Research Laboratory

U.S. Environmental Protection Agency

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow