Cross-Platform Comparison of Amino Acid Metabolic Profiling in Three Model Organisms Used in Environmental Metabolomics.

Environmental metabolomics is a promising approach to study pollutant impacts to target organisms in both terrestrial and aquatic environments. To this end, both nuclear magnetic resonance (NMR)- and mass spectrometry (MS)-based methods are used to profile amino acids in different environmental metabolomic studies. However, these two methods have not been compared directly which is an important consideration for broader comparisons in the environmental metabolomics field. We compared the quantification of 18 amino acids in the tissue extracts of Daphnia magna, a common model organism used in both ecotoxicology and ecology, using both 1H NMR spectroscopy and liquid chromatography with tandem MS (LC-MS/MS). 1H NMR quantification of amino acids agreed with the LC-MS/MS quantification for 17 of 18 amino acids measured. We also tested both quantitative methods in a D. magna sub-lethal exposure study to copper and lithium. Again, both NMR and LC-MS/MS measurements showed agreement. We extended our analyses with extracts from the earthworm Eisenia fetida and the plant model Nicotiana tabacum. The concentrations of amino acids by both 1H NMR and LC-MS/MS, agreed and demonstrated the robustness of both techniques for quantitative metabolomics. These findings demonstrate the compatibility of these two analytical platforms for amino acid profiling in environmentally relevant model organisms and emphasizes that data from either method is robust for comparisons across studies to further build the knowledge base related to pollutant exposure impacts and toxic responses of diverse environmental organisms.

In Vivo Transformation of a Novel Polyfluoroether Surfactant.

Per- and polyfluoroalkyl substances are a class of fluorochemicals that can degrade into perfluoroalkyl acids, which are well known to be persistent in the environment. It is thus important that novel fluorinated surfactants be designed to degrade into small, nonbioaccumulative products. We report the biotransformation and elimination kinetics of one such novel polyfluorinated surfactant, di(polyfluoroether thioether(S)-oate) sulfonate (diFESOS), and its metabolites. Biotransformation was investigated in vitro using S9 liver fractions and in vivo in Sprague-Dawley rats. Rats dosed by oral gavage with diFESOS were found to have relatively fast elimination kinetics, with half-lives on the order of hours, compared with legacy fluorinated surfactants such as the disubstituted polyfluoroalkyl phosphates that have half-lives on the order of days. To interrogate degradation of the polyfluorinated chain, rats were then dosed with a polyfluoroether thioether alcohol (a suspected product of carboxylate cleavage of diFESOS) either orally or intravenously, and the novel metabolite 2H-3:2 polyfluoroether sulfonic acid (2H-3:2 PFESA) was identified. Perfluoropropionic acid was detected in rat urine and is likely a terminal product. The blood of orally dosed rats contained higher levels of metabolites than the blood of intravenously dosed rats, suggesting the importance of metabolism in the gut and liver. Elimination kinetics of all the novel metabolites were faster than their fully fluorinated counterparts. Environ Toxicol Chem 2021;40:3328-3336. © 2021 SETAC.

(1)H NMR-based metabolomics investigation of Daphnia magna responses to sub-lethal exposure to arsenic, copper and lithium.

Metal and metalloid contamination constitutes a major concern in aquatic ecosystems. Thus it is important to find rapid and reliable indicators of metal stress to aquatic organisms. In this study, we tested the use of (1)H nuclear magnetic resonance (NMR) - based metabolomics to examine the response of Daphnia magna neonates after a 48h exposure to sub-lethal concentrations of arsenic (49µgL(-1)), copper (12.4µgL(-1)) or lithium (1150µgL(-1)). Metabolomic responses for all conditions were compared to a control using principal component analysis (PCA) and metabolites that contributed to the variation between the exposures and the control condition were identified and quantified. The PCA showed that copper and lithium exposures result in statistically significant metabolite variations from the control. Contributing to this variation was a number of amino acids such as: phenylalanine, leucine, lysine, glutamine, glycine, alanine, methionine and glutamine as well as the nucleobase uracil and osmolyte glycerophosphocholine. The similarities in metabolome changes suggest that lithium has an analogous mode of toxicity to that of copper, and may be impairing energy production and ionoregulation. The PCA also showed that arsenic exposure resulted in a metabolic shift in comparison to the control population but this change was not statistically significant. However, significant changes in specific metabolites such as alanine and lysine were observed, suggesting that energy metabolism is indeed disrupted. This research demonstrates that (1)H NMR-based metabolomics is a viable platform for discerning metabolomic changes and mode of toxicity of D. magna in response to metal stressors in the environment.

Is indirect exposure a significant contributor to the burden of perfluorinated acids observed in humans?

In comparison to other persistent organic pollutants, human fluorochemical contamination is relatively complicated. This complication arises at least in part from a disparity between the chemicals used commercially and those measured in the environment and humans. Commercial fluorochemical products are dominated by fluorinated polymers used in textile or carpet applications, or fluorosurfactants used in applications ranging from personal care products, leveling and wetting agents, to greaseproofing food-contact materials. Investigations into environmental and human fluorochemical contamination have focused on perfluorinated acids (PFAs), either the perfluorinated carboxylates (PFCAs) or sulfonates (PFSAs). In this review we will present an overview of data related to human fluorochemical exposure including a discussion of fluorochemical production, concentrations in exposure media, biotransformation processes producing PFAs, and trends in human sera. These data will be presented in the context of how they can inform sources of human PFA contamination, specifically whether the contamination results from direct PFA exposure or indirect exposure via the biotransformation of commercial fluorochemicals or their residuals. Concentrations of both perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) began to decrease in human sera around the year 2000, a change that mirrored the 2000-2002 phase-out of perfluorooctane sulfonyl fluoride (POSF) production. These temporal trends suggest exposure to current-use POSF-based materials was a significant source of PFOA and PFOS exposure prior to 2000. Relatively slow PFOA elimination and increasing concentrations of the C9 and C10 PFCAs in human sera suggest continued PFCA exposure, without similar exposure to PFOS, which is consistent with indirect exposure via the biotransformation of fluorotelomer-based materials. Conversely, human exposure models have suggested direct exposure to PFAs present in food items is the major source of human contamination. The data set presented here cannot unequivocally delineate between direct and indirect human exposure, however temporal trends in human sera and exposure media are consistent with indirect exposure representing a significant portion of observed human PFA contamination.

Exploring indirect sources of human exposure to perfluoroalkyl carboxylates (PFCAs): evaluating uptake, elimination, and biotransformation of polyfluoroalkyl phosphate esters (PAPs) in the rat.

BACKGROUND: Perfluorinated carboxylic acids (PFCAs) are ubiquitous in human sera worldwide. Biotransformation of the polyfluoroalkyl phosphate esters (PAPs) is a possible source of PFCA exposure, because PAPs are used in food-contact paper packaging and have been observed in human sera. OBJECTIVES: We determined pharmacokinetic parameters for the PAP monoesters (monoPAPs) and PAP diesters (diPAPs), as well as biotransformation yields to the PFCAs, using a rat model. METHODS: The animals were dosed intravenously or by oral gavage with a mixture of 4:2, 6:2, 8:2, and 10:2 monoPAP or diPAP chain lengths. Concentrations of the PAPs and PFCAs, as well as metabolic intermediates and phase II metabolites, were monitored over time in blood, urine, and feces. RESULTS: The diPAPs were bioavailable, with bioavailability decreasing as the chain length increased from 4 to 10 perfluorinated carbons. The monoPAPs were not absorbed from the gut; however, we found evidence to suggest phosphate-ester cleavage within the gut contents. We observed biotransformation to the PFCAs for both monoPAP and diPAP congeners. CONCLUSIONS: Using experimentally derived biotransformation yields, perfluorooctanoic acid (PFOA) sera concentrations were predicted from the biotransformation of 8:2 diPAP at concentrations observed in human serum. Because of the long human serum half-life of PFOA, biotransformation of diPAP even with low-level exposure could over time result in significant exposure to PFOA. Although humans are exposed directly to PFCAs in food and dust, the pharmacokinetic parameters determined here suggest that PAP exposure should be considered a significant indirect source of human PFCA contamination.

Uptake and elimination of perfluorinated phosphonic acids in the rat.

The mono- and di-substituted perfluorinated phosphonic acids (mono-PFPAs and di-PFPAs) are high production volume fluorinated surfactants. Mono-PFPAs have been observed in Canadian surface waters and wastewater treatment plant (WWTP) effluent. The first observation of the di-PFPAs in the environment is reported here, with the observation of the C6/C6 and C6/C8 di-PFPAs in the National Institute for Standards and Technology (NIST) WWTP sludge standard reference material (SRM) 2781. To characterize the risk associated with human exposure to the mono-PFPAs and di-PFPAs, we determined uptake and elimination parameters in the rat. After oral gavage all of the mono-PFPA and di-PFPA congeners were observed in the blood of the dosed animals. Half-lives after intraperitoneal dosing ranged from 0.96 +/- 0.11 to 2.8 +/- 0.5 days for the mono-PFPAs and 1.8 +/- 0.1 to 9.3 +/- 1.5 days for the di-PFPAs. These half-lives are of similar magnitude to those for perfluorooctane sulfonate (PFOS, 8 to 30 days in male rats) and perfluorooctanoate (PFOA, 6 days in male rats and 1 hour in female rats). Both PFOS and PFOA have human half-lives on the order of years, and so the mono-PFPAs and di-PFPAs may also have significant lifetimes in the human body. The analysis of paired whole blood and plasma samples demonstrated that the mono-PFPAs may bind to blood cells underestimating their concentration in plasma and sera samples. The biological fate of the mono-PFPAs and di-PFPAs determined here suggest there is the potential for human exposure and that if exposure does occur, they may be long-lived in the body.

Determining the molecular interactions of perfluorinated carboxylic acids with human sera and isolated human serum albumin using nuclear magnetic resonance spectroscopy.

Perfluorooctanoate (PFOA) is ubiquitous in North American human sera and has a serum half-life of 3.5 years in humans. The molecular interactions that lead to the bioaccumulation of these hydrophobic and lipophobic molecules in human blood are not well understood. Perfluorohexanoic acid (PFHxA) and PFOA were used as model perfluorinated carboxylic acids (PFCAs) to characterize the major site of PFCA interaction in human sera. Using novel heteronuclear saturation transfer difference nuclear magnetic resonance spectroscopy experiments, human serum albumin (HSA) was identified as the major site of interaction for both PFHxA and PFOA in human sera. Heteronuclear single quantum coherence nuclear magnetic resonance experiments were then performed to interrogate site-specific interactions of PFHxA and PFOA with isolated HSA. Perfluorohexanoic acid was found to bind specifically to Sudlow's drug-binding site II, whereas PFOA interacted preferentially with Sudlow's drug-binding site I at the lower concentration, with additional interactions developing at the higher concentration. These experiments highlight the utility of nuclear magnetic resonance spectrometry as a tool to observe the in situ interactions of chemical contaminants with biological systems. Both PFCAs displaced the endogenous HSA ligand oleic acid at concentrations lower than observed for the drugs ibuprofen and phenylbutazone, which are established HSA ligands. Interactions between PFCAs and HSA may affect the pharmacokinetics and distribution of fatty acids and certain drugs in the human body and warrant further investigation.

Observation of a commercial fluorinated material, the polyfluoroalkyl phosphoric acid diesters, in human sera, wastewater treatment plant sludge, and paper fibers.

Sources of human exposure to perfluorinated carboxylic acids (PFCAs) are not well-characterized. Polyfluoroalkyl phosphoric acids (PAPs) are fluorinated surfactants used in human food contact paper products. PAPs can migrate into food and food simulants, and their bioavailability and biotransformation into PFCAs has been demonstrated using a rat model. To characterize human exposure to PAP materials, we analyzed pooled human sera samples collected in 2004 and 2005 (n = 10) and 2008 (n = 10) from the midwestern United States for the 4:2 through 10:2 PAP diesters (diPAPs). The 2004 and 2005 sera samples contained 4.5 microg/L total diPAPs, with the 6:2 diPAP dominating the congener profile at 1.9 +/- 0.4 microg/L DiPAP concentrations observed in the 2004 and 2005 human sera samples were similar to those of the C8 to C11 PFCAs (0.13 +/- 0.01 to 4.2 +/- 0.3 microg/L) monitored in the same samples. 6:2 diPAP was also consistently observed in the 2008 human sera samples at a mean concentration of 0.63 +/- 0.13 microg/L As diPAPs have been shown to degrade to PFCAs in vivo, our observation of diPAPs in human sera may be a direct connection between the legacy of human PFCA contamination and PAPs commercial applications. Wastewater treatment plant (WWTP) sludge and paper fibers were analyzed for diPAPs as a proxy for human use and potential exposure to diPAPs. DiPAPs were observed in WWTP sludge at concentrations ranging from 47 +/- 22 to 200 +/- 130 ng/g, a range similar to perfluorooctane sulfonic acid (PFOS) (100 +/- 70 ng/g) and greater than the C8 to C11 PFCAs (1.6 +/- 0.6 to 0.17 +/- 0.10 ng/g) observed in the same samples. DiPAPs were observed in paper fiber extracts at concentrations ranging from 34 +/- 30 to 2200 +/- 400 ng/g. The high diPAP concentrations in WWTP sludge suggest PAP materials may be prevalent in our daily lives.

Perfluorinated phosphonic acids in Canadian surface waters and wastewater treatment plant effluent: discovery of a new class of perfluorinated acids.

The environmental prevalence of a new class of perfluorinated acids, the perfluorinated phosphonic acids (PFPAs), was determined in Canadian surface waters and wastewater treatment plant (WWTP) effluent. For quality control and comparison, the C8- to C11-perfluorinated carboxylic acids and perfluorooctane sulfonic acid were included in the analysis. Water samples were extracted using weak anion-exchange solid-phase extraction cartridges. Perfluorinated phosphonic acids were observed in 80% of surface water samples and in six of the seven WWTP effluent samples. The C8-PFPA was observed at concentrations ranging from 88 +/- 33 to 3400 +/- 900 pg/L in surface waters and from 760 +/- 270 to 2500 +/- 320 pg/L in WWTP effluent. To our knowledge, this is the first observation of PFPAs in the environment. Given their structural similarities with perfluorinated carboxylic and sulfonic acids, PFPAs are expected to be persistent. The observation of PFPAs in the majority of samples analyzed here suggests they are prevalent environmental contaminants and should be considered in future environmental monitoring campaigns to better understand the total burden of fluorinated materials in the environment.

Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): exploring routes of human contamination.

Perfluorinated acids are detected in human blood world-wide, with increased levels observed in industrialized areas. The origin of this contamination is not well understood. A possible route of exposure, which has received little attention experimentally, is indirect exposure to perfluorinated acids through ingestion of chemicals applied to food contact paper packaging. The current investigation quantified the load of perfluorinated acids to Sprague-Dawley rats upon exposure to polyfluoroalkyl phosphate surfactants (PAPS), nonpolymeric fluorinated surfactants approved for application to food contact paper products. The animals were administered a single dose at 200 mg/kg by oral gavage of 8:2 fluorotelomer alcohol (8:2 FTOH) mono-phosphate (8:2 monoPAPS), or the corresponding di-phosphate (8:2 diPAPS), with blood taken over 15 days post-dosing to monitor uptake, biotransformation, and elimination. Upon completion of the time-course study the animals were redosed using an identical dosing procedure, with sacrifice and necropsy 24 h after the second dosing. Increased levels of perfluorooctanoic acid (PFOA), along with both 8:2 PAPS congeners, were observed in the blood of the dosed animals. In the 8:2 monoPAPS-dosed animals, 8:2 monoPAPS and PFOA blood concentrations peaked at 7900 +/- 1200 ng/g and 34 +/- 4 ng/g respectively. In the 8:2 diPAPS-dosed animals, 8:2 diPAPS peaked in concentration at 32 +/- 6 ng/g, and 8:2 monoPAPS and PFOA peaked at 900 +/- 200 ng/g and 3.8 +/- 0.3 ng/g, respectively. Several established polyfluorinated metabolites previously identified in 8:2 FTOH metabolism studies were also observed in the dosed animals. Consistent with other fluorinated contaminants, the tissue distributions showed increased levels of both PFOA and the 8:2 PAPS congeners in the liver relative to the other tissues measured. Previous investigations have found that PAPS can migrate into food from paper packaging. Here we link ingestion of PAPS with in vivo production of perfluorinated acids.

Atmospheric chemistry of N-methyl perfluorobutane sulfonamidoethanol, C4F9SO2N(CH3)CH2CH2OH: kinetics and mechanism of reaction with OH.

Relative rate methods were used to measure the gas-phase reaction of N-methyl perfluorobutane sulfonamidoethanol (NMeFBSE) with OH radicals, giving k(OH + NMeFBSE) = (5.8 +/- 0.8) x 10(-12) cm3 molecule(-1) s(-1) in 750 Torr of air diluent at 296 K. The atmospheric lifetime of NMeFBSE is determined by reaction with OH radicals and is approximately 2 days. Degradation products were identified by in situ FTIR spectroscopy and offline GC-MS and LC-MS/MS analysis. The primary carbonyl product C4F9SO2N(CH3)CH2CHO, N-methyl perfluorobutane sulfonamide (C4F9SO2NH(CH3)), perfluorobutanoic acid (C3F7C(O)OH), perfluoropropanoic acid (C2F5C(O)OH), trifluoroacetic acid (CF3C(O)OH), carbonyl fluoride (COF2), and perfluorobutane sulfonic acid (C4F9SO3H) were identified as products. A mechanism involving the addition of OH to the sulfone double bond was proposed to explain the production of perfluorobutane sulfonic acid and perfluorinated carboxylic acids in yields of 1 and 10%, respectively. The gas-phase N-dealkylation product, N-methyl perfluorobutane sulfonamide (NMeFBSA), has an atmospheric lifetime (>20 days) which is much longer than that of the parent compound, NMeFBSE. Accordingly,the production of NMeFBSA exposes a mechanism by which NMeFBSE may contribute to the burden of perfluorinated contamination in remote locations despite its relatively short atmospheric lifetime. Using the atmospheric fate of NMeFBSE as a guide, it appears that anthropogenic production of N-methyl perfluorooctane sulfonamidoethanol (NMeFOSE) contributes to the ubiquity of perfluoroalkyl sulfonate and carboxylate compounds in the environment.