MAPPING POTENTIAL HUMAN EXPOSURE TO PER- AND POLYFLUOROALKYL SUBSTANCES (PFAS) FROM DOMESTIC AND IMPORTED FOOD PACKAGING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1024269
• Grant No.: 2020-67017-33075
• Cumulative Award Amt.: $479,712.00
• Proposal No.: 2020-03449
• Project Start Date: Jan 1, 2021
• Project End Date: Dec 31, 2024
• Grant Year: 2021
• Program Code: [A1332]- Food Safety and Defense

Recipient Organization
UNIV OF PITTSBURGH
(N/A)
PITTSBURGH,PA 15260

Performing Department
Civil & Environmental Engr

Non Technical Summary
Consumption of contaminated food is a major route of human exposure to toxic chemicals, in the United States and globally. One way that chemicals unintentionally enter into foods is through migration from food packaging. A class of chemicals that has received growing attention due to health concerns and that is highly relevant for packaged foods is the class of per- and polyfluorinated alkyl substances (PFAS). These chemicals are widely used because of their non-stick properties and their ability to repel oil. They are present in both paper-based and plastic-based food packaging, and people who consume a high proportion of packaged foods, including fast food and take-out foods, have higher concentrations of these chemicals in their blood. Some PFAS, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are now known to be bioaccumulative and toxic. These have been voluntarily withdrawn from much of the US market, and are restricted in the European Union, but continue to be used and produced in other countries. Unfortunately, there is still a lack of information about which other PFAS are present in most packaged foods, though a number of PFAS-containing food packaging products are approved by the FDA. The class of PFAS includes more than 5,000 compounds across a wide array of industries, and very little is known about the toxicity of most of them.In this project, we will perform the first systematic survey of PFAS present in both domestic and imported packaged foods. We will build a database of the types and origins of packaged foods that are available in both national supermarket chains and local international food stores, and come up with a representative selection of samples for PFAS analysis. The food packaging samples will be tested for their potential to impart PFAS to foods through extraction (a more extreme case) and through migration assays that simulate actual conditions when in contact with foods (a more typical case). Using state-of-the-art methods, we will analyze the mixtures that result from extraction and migration studies to identify which PFAS are present and at what quantity. By employing recently developed non-target analysis technology, we will be able to resolve the chemical structures of PFAS present even if they are previously unknown and/or have never been analyzed before. This non-targeted analysis will be followed up by more traditional high-performance targeted analysis to measure the concentration of PFAS that have analytical standards available. Finally, we will establish whether the types of PFAS present in these food packaging samples at the concentrations detect pose a risk to consumers by evaluating their potential to accumulate in the human body and to cause toxic effects. This will be done using a combination of computer simulations and experiments with zebrafish. The simulations will be used to understand whether the PFAS will bind to key proteins and receptors in the body, leading for example to accumulation in the liver or to changes in blood chemistry. The experiments on zebrafish, an animal model often used to anticipate effects in humans, will determine at what dose the PFAS cause toxicity, and in particular if there are measurable developmental effects that could be of concern to vulnerable populations such as children.Based on the outcomes of this project, better policy around the use of PFAS in food packaging can be developed, regulators will be made aware of the types of food imports more likely to contain toxic PFAS, consumers will have guidance on what specific types of packaging to avoid in order to reduce their exposure to toxic chemicals, and scientists will have better information about how specific PFAS structures and or mixtures of PFAS lead to increased toxicity, informing them in how to better design less hazardous chemicals.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
711	5010	2000	67%
711	5010	1150	33%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
5010 - Food;

Field Of Science
2000 - Chemistry; 1150 - Toxicology;

Keywords

pfas

food packaging

migration

exposure

non-target analysis

toxicology

mass spectrometry

Goals / Objectives
The major goalof our project is to conduct the first systematic study of per- and polyfluorinated alkyl substances (PFAS) in imported and domestic packaged foods. We will specifically evaluate which PFAS are present in current food packaging, whether those PFAS vary according to the country of origin of the packaged foods and what risks these PFAS may pose to US consumers through migration into food and subsequent human exposure. PFAS are widely used to provide non-stick and oil repellency characteristics to food packaging, but several PFAS have now been identified as bioaccumulative and/or toxic. Unfortunately, there are a wide range of PFAS potentially used in food packaging and very little is known about their structural identity, propensity to migrate into foods, or potential to exert toxic impacts.We will pursue these goals through three complementary objectives:1. In the first objective we will survey local supermarkets and international food stores in order to determine the types and geographic origins of packaged foods available and representatively sample them (circa 100 packaged food samples total), with a focus on foods targeted to children and seniors as potentially vulnerable populations.2. In the second objective, we will conduct high-resolution non-target analysis of packaging subjected to extraction and migration experiments to identify major types of PFAS present in these samples, This non-target analysis will be followed by targeted analysis in order to quantify the major PFAS present.3. In the third objective, we will evaluate the potential toxicity of the identified PFAS and their mixtures using molecular modeling for initial screening and prioritizationfollowed by zebrafish embryo toxicity assays to validate and further expand upon the modeling predictions.Successful completion of our objectives will provide a highly useful data set to support risk assessment for PFAS in food packaging and identify potential exposure hot spots in our food system.

Project Methods
For the packaged food surveys, project personnel will personally visit a variety of stores and document the types and origins of packaged foods available across a variety of categories and food types. The data gathered will be organized into a database containing information about packaging type, intended "audience" (e.g. food for children, general, food for seniors), and origin (market where purchased, domestic vs. imported and, for imported, country of origin). These data will be analyzed using statistical methods such as aStudent's t test to evaluate differences across markets and regions. Based on these results, we will select samples for collection, with a target of at least 100 samples.Extraction of the collected food packaging materials will be conducted following established US FDA protocols for PFAS in food packaging materials. The extracts will be analyzed using liquid chromatography (LC) high resolution accurate mass (HRAM) mass spectrometry (MS) with QExactive Plus orbital ion trap MS (ThermoFisher Scientific), providing resolution up to 140,000 (FWHM). This non-targeted approach will allow us to identify previously unknown PFAS and resolve their structures. Those sampleswith the highest PFAS content (about 25% of the samples) will be selected for migration studies. Migration assays will be conducted in accordance with the US FDA Guidance for Industry migration protocols for food contact substances. Recommended food simulants vary by food type: aqueous, acidic, alcoholic, fatty orsolid. For example, common food simulants for fatty food are miglyol oil, iso-octanol or 95% ethanol:5% water and for aqueous food - 10% ethanol. All migration studies will be conducted in triplicate and will include a blank or control using an identical treatment per US FDA recommendations.Identification of PFAS compounds will be based on full-scan mass spectra (MS) followed by dd-MS2 analysis with mass error <5 ppm. Full scan data will be analyzed using Compound Discoverer 3.2 and Free Style (ThermoFisher Scientific). The MS data will be analyzed for common features, i.e., ions characteristic for various PFAS classes. Identification will be confirmed with predicted composition, m/z cloud, ChemSpider and Mass list searches, and available high-resolution accurate mass databases based on full MS and dd-MS2 with mass error <5 ppm.Extracts from the migration and extraction essays will further be analyized by high-performance liquid chromatography coupledmass spectrometryfortargeted quantification of concentrations of a wide range individual PFAS compounds:forty-two representative ionic and neutral PFAS, including short- and long-chained molecules will be analyzed. PFAS from several structural groups will be included in the analysis: 12 perfluoroalkyl carboxylates, 10 perfluoroalkyl sulfonates, 8 perflourooctane sulfonamides and sulfonamidoethanols, 3 fluorotelomer sulfonates, 4 fluorotelomer alcohols, 5 fluorotelomer acrolytes and methacrylates. In addition, methods for the measurement of the major PFAS identified as a result of the non-targeted analysis will be developed and these PFAS will also be analyzed in all samples. This data analysis will provide information on the levels of PFAS compounds in each food packing sample analyzed. Based on these data, most abundant PFAS in these samples and food / packaging types with highest PFAS levels will be identified.The PFAS identified and quantified above will be evaluated for toxicity and bioaccumulation potential.We will first use molecular docking and dynamics to predict the relative interaction affinities between these PFAS and two key proteins, L-FABP and serum albumin. We will also predict relative strength of interaction between the PFAS and three PPAR isoforms, PPAR- α, - δ, and - ??, and between the PFAS and human transthyretin (TTR), a thyroid hormone transporter that transports thyroxine (T4) and retinol-binding protein. These will serve as potential markers for lipid disruption and endocrine-disrupting effects, respectively. These simulations are meant to serve as an in silico screen for bioaccumulation potential and disruption of lipid metabolism. The results of the simulations will therefore be compared with affinities of "positive control" compounds known to bioaccumulate and/or to serve as agonists or antagonists of the PPARs and TTR. Following our previously published approaches (Cheng & Ng 2018, Ng & Hungerbuehler 2015) we will evaluate uncertainty and calculate the standard errors around the predictions, and test for statistical differences between the test compounds and known bioaccumulative and/or toxic control compounds. The control compounds may be previously studied PFAS (e.g. PFOA, PFOS) or drug compounds with high-quality toxicological data available.Finally,we will conduct a complementrary screenof theidentified PFAS and the PFAS mixtures derived from migration and extraction for toxicity using the OECDzebrafish embryo toxicity assay (Test No. 236).Data collected will include mortality (% of tested embryos), dose-response curves, and qualitative and quantitative descriptions of developmental malformations. Statistical analysis will identify significant differences between treatment groups. The OECD guidelines for this standard test recommends 20 embryos per treatment group, and dose-response curves constructed using at least 5 test concentrations, the highest of which should result in 100% lethality. In our case, the highest concentration for extracts and migration test mixtures will be the "as-received" concentration, and the other four concentrations will be achieved using serial dilutions. A range-finding pilot experiment will be run to determine the appropriate level of dilution. Assays will use 20 embryos per concentration except for the range-finding study which will use 10 embryos per concentration. Statistical tests such as the Student's t-test will be used to determine whether significant differences exist between various treatments (single PFAS, extracts, migration mixtures) and controls (fish system water as negative controls, solvent controls, and positive controls will be included; 3,4-dichloroaniline is often used as a positive control for determining LC50).

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:The target audiences for this project are other researchers (including analytical chemists, exposure scientists, risk assessors, and food packaging scientists) as well as regulators interested in the presence of and potential human exposure to per- and polyfluoroalkyl substances (PFAS) in food packaging. To facilitate communication to these audiences, we published the findings from this research in the peer-reviewed journal Chemosphere. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Following their collaboration on the non-targed method development, Dr. Stroski, Sapozhnikova and Ng collaborated on and submitted a research proposal (not funded) which provided Dr. Stroski as an early career scientists a valuable training opportunity on proposal writing. How have the results been disseminated to communities of interest?The publication on non-targeted analysis was published in Chemosphere. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the final year of the project, we further developed the non-targeted workflow and published the associated manuscript in Chemosphere. We also carried out the zebrafish embryo toxicity assay for PFAS.From the non-target work, a total of 45 PFAS were tentatively identified, and five compounds were confirmed with analytical standards: 6:2 fluorotelomer phosphate diester (6:2 diPAP) and 6:2 fluorotelomer unsaturated carboxylic acid (6:2 FTUCA, one of its intermediate breakdown products), perfluoropentadecanoic acid (PFPeDA), perfluorohexadecanoic acid (PFHxDA) and perfluorooctadecanoic acid (PFOcDA), which are long-chain perfluoroalkyl acids and were not expected to be found in current use packaging due to a general phase-out of long-chain perfluoroalkyl acids. Longer perfluorocarboxylic acids including C17 and C19 to C24 were also found present within a foil sample. These results demonstrated that both emerging and legacy PFAS are prevalent in food packaging and highlighted the strength of pairing targeted and non-targeted analytical approaches in evaluating food packaging for PFAS. From the zebrafish work, morphological assays indicated that failed swim bladder inflation was the most commonly observed effects, and that severity of malformation as well as lethality generally increased with chain length. However, some short-chain PFAS showed specific malformations (e.g. bent tail, short trunk) that were not observed for long-chain PFAS, suggesting some unique toxicological effects for short-chain PFAS.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Kevin M. Stroski, Yelena Sapozhnikova, Raegyn B. Taylor, Andrew Harron, Non-targeted analysis of per- and polyfluorinated substances in consumer food packaging, Chemosphere, Volume 360, 2024, 142436, ISSN 0045-6535, https://doi.org/10.1016/j.chemosphere.2024.142436.

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:During the duration of this project, the target audiences were other researchers (including analytical chemists, exposure scientists, risk assessors, and food packaging scientists) as well as regulators interested in the presence of and potential human exposure to per- and polyfluoroalkyl substances (PFAS) in food packaging. To facilitate communication to these audiences, we published the findings from this research in the following relevant peer-reviewed journals: Chemosphere, Analytica Chimica Acta, and the Journal of Chromatography Open. We also presented the findings at research conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Ms Megha Bedi, who received her PhDin PI Ng's research group, spent part of her summer term in 2022 at Dr. Sapozhnikova's laboratory in Wyndmoor, PA. She learned PFAS targeted and non-targeted analysis with the developed protocols for this project, and coordinated with Postdoctoral Fellow Raegyn Taylor in the Sapozhnikova lab to determine which PFAS and PFAS mixtures identified in the extraction and migration assays for toxicity testing with zebrafish. Dr. Salamova's laboratory provided PFAS mixtures for toxicity testing in the Ng laboratory. Kevn Stroski, postdoctoral scholar in the Sapozhnikova lab further developed the non-targeted workflow to identify additional long-chain and emerging PFAS in the food packagign materials. Finally, the collaboration opened new collaborative opportunities for the trainees. For example, Dr. Stroski and Dr. Sapozhnikova used an incidental observation from the collected food packaging to develop a method for identifying unintentional PFAS contamination in plastic food storage bags that was recently published. Dr. Sapozhnikova worked with Dr. Megha Bedi on a related project measuring PFAS and POPs in seafood (funded through an internal Pitt grant) which resulted two additional publications. How have the results been disseminated to communities of interest?Results have been disseminated through peer-reviewed publications as well as presentations at national meetings and poster presentations by the co-PIs and trainees. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To address the major objectives of this project, we collected a total of 88food packaging samples from 13retail outlets across the city of Pittsburgh. These represented 24different national origins, andincluded shelf stable, refrigerated and frozen items across categories such as ready meals, bakery items, deli meats, snacks and dairy. We developed and optimized extraction, migration, and PFAS analysis procedures for these various collected food packagingsamples. For targeted analysis, PFAS mixtures covereda total of 34 native compounds and 20 mass-labeled standards. Food packaging extraction was optimized for solvent, extraction time, and technique (sonication, shaking, both) using spiked cardboard paper. A targeted LC-HRMS method for the 34 PFAS was developed and evaluated. Concentrations of total targeted PFAS in extracts ranged from 0.01 to 24.6 nmol F/g, with highest concentration in salami plastic/paper film from the US, while total precursors, determined via the total oxidizable precursor (TOP) assay were as high as 73 nmol F/g in a paper popcorn bag from the US. In addition, we applied the total oxidizable precursor (TOP) assay to quantify precursors capable of oxidizing to terminal carboxylic acids (PFCAs). Preliminary experiments were conducted to assess accuracy of the method using three precursor standards (NEtFOSAA, 6:2FTS, and 5:3FTCA) and showed >30 mM of potassium persulfate was needed to provide nondetectable levels of precursors. Recoveries were between 73 and 78% for NEtFOSAA and 22 and 27% for 6:2 FTS, which is likely due to missing fluorine content from short-chain products not quantified by our targeted method. Precursor content reported in this study in likely an underestimation of true precursor levels and is considered semi-quantitative. Based on the PFAS measured in extracts, frequency of detection, and TOP assay results, 25 food packaging samples with highest frequency of detection and highest measured PFAS levels were selected for migration tests. For room temperature applications (shelf stable foods), US FDA migration protocol recommends a test temperature of 40C (104F). For refrigerated and frozen food applications, the recommended test temperature is 20C (68F). Depending on the food type as defined by the US FDA, food simulants were selected as 10% ethanol/water for aqueous and acidic foods and 95% ethanol/water for fatty foods. Migration tests were conducted in triplicate with blanks for 2 food simulants at both temperatures. Aliquots of migration samples were collected and analyzed at 2, 24, 96 and 240 hours. Internal standards wereadded to replicate 2 of each sample to monitor HRMS instrument performance and guide in identification and semiquantitative analysis. Based on targeted analysis, PFHxS, PFHpA and PFHxA were detected, confirmed with fragment ions, and measured in migration extracts at 0.05-0.71 μg/kg levels. We then created and tested non-targeted workflows using available PFAS analytical standards. Retention times (tR) were aligned with ChromAlign feature) based on a reference file containing PFAS analytical standards. Extracted ion chromatogram (XIC) traces were created for fragments generated from specific molecular structures. Environmental and Food Safety (EFS) HRAM database (1634 compounds), extractables and leachables HRAM database (1741 compounds), PFAS EPA master list (10901 compounds), PFAS suspect list (4951 compounds) and PFAS master list with predicted tR (10762 compounds) were used as mass list databases. Analysis of food packaging migration extracts using this MS1 workflow resulted in 255 hits. Hits were searched against available information on CAS#s, reports, publications, and available analytical standards to confirm identity.A total of 45 PFAS were tentatively identified based on thisworkflow.Five tentatively identified compounds were confirmed with analytical standards: 6:2 fluorotelomer phosphate diester(6:2 diPAP) and 6:2 fluorotelomer unsaturated carboxylic acid(6:2 FTUCA, one of its intermediate breakdown products), perfluoropentadecanoic acid (PFPeDA), perfluorohexadecanoic acid (PFHxDA) and perfluorooctadecanoic acid (PFOcDA), which are long-chain perfluoroalkyl acids and were not expected to be found in current use packaging due to a general phase-out of long-chain perfluoroalkyl acids. Longer perfluorocarboxylic acids including C17and C19to C24were also found present within a foil sample. Concentrations of 6:2 FTUCA ranged from 0.78 to 127ngg−1in methanolic extracts and up to 6ngg−1in food simulant after 240h migration test. These results demonstrated that bothemerging and legacy PFAS are prevalent in food packaging and highlighted the strength of pairing targeted and non-targeted analytical approaches in evaluating food packaging for PFAS. Finally, the zebrafish embryo toxicity assaywas employed to evalute the toxicity of some of themost commonly detected PFAS in the food packaing extracts. Morphological assays indicated that failed swim bladder inflation was the most commonly observed effects, and that severity of malformation as well as lethality generally increased with chain length. However, some short-chain PFAS showed specific malformations (e.g. bent tail, short trunk) that were not observed for long-chain PFAS, suggesting some unique toxicological effects for short-chain PFAS.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Sapozhnikova, Y.?, Taylor, R., Bedi, M., and Ng, C. Assessing per- and polyfluoroalkyl substances in globally sourced food packaging. Chemosphere 337(139381). DOI:10.1016/j.chemosphere.2023.139381.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Kevin M. Stroski, Yelena Sapozhnikova, Raegyn B. Taylor, Andrew Harron, Non-targeted analysis of per- and polyfluorinated substances in consumer food packaging, Chemosphere, Volume 360, 2024, 142436, ISSN 0045-6535, https://doi.org/10.1016/j.chemosphere.2024.142436.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The target audience for this project includes exposure scientists and public health researchers, analytical chemists, food safety professionals, and risk assessors. We reached these target audiences through peer-reviewed publications and presentations at conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Megha Bedi successfully defended her PhD dissertation in April 2023. Dr. Kevin Stroski joined Dr. Sapozhnikova's lab as a postdoctoral scholar to work on the non-targeted analysis of PFAS in food packaging. How have the results been disseminated to communities of interest?Results were disseminated through a peer-reviewed publication in Chemosphere as well as through presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will wrap up the project with zebrafish embryo assay experiments to test the toxicity of the PFAS found in food packaging.

Impacts
What was accomplished under these goals? In project year 3, we published our findings on PFAS in food packaging extracts and from migration studies, which indicated that short-chain PFAS asd well as 6:2 diPAP were prevalent. Overall, we found PFAS present in >80% of tested food packaging samples. As reported in our publication in Chemosphere: " 6:2 diPAP [was found] most frequently and at the highest levels (224 ng/g). Other frequently detected substances (15-17% of samples) were PFHxS, PFHpA and PFDA. Shorter chain perfluorinated carboxylic acids PFHpA (C7), PFPeA (C5) and PFHxS (C6) were present at levels up to 51.3, 24.1 and 18.2 ng/g, respectively. Average ΣPFAS levels were 28.3 ng/g and 381.9 ng/g before and after oxidation with the TOP assay. The 25 samples with highest frequency of detection and amounts of measured PFAS were selected for migration experiments with food simulants to better understand potential dietary exposure. PFHxS, PFHpA, PFHxA and 6:2 diPAP were measured in the food simulants of five samples at concentrations ranging from 0.04 to 12.2 ng/g and at increasing concentrations over the 10-day migration period. To estimate potential exposure to PFAS that had migrated from food packaging samples, weekly intake was calculated and ranged from 0.0006 ng/kg body weight/week for PFHxA exposure in tomato packaging to 1.1200 ng/kg body weight/week for PFHxS exposure in cake paper. These values were below the established EFSA maximum tolerable weekly intake (TWI) of 4.4 ng/kg body weight/week for the sum of PFOA, PFNA, PFHxS and PFOS." In addition, we further developed the non-target analysis approach to identifying further legacy and emerging PFAS in food packaging of various types.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Sapozhnikova, Y.?, Taylor, R., Bedi, M., and Ng, C. Assessing per- and polyfluoroalkyl substances in globally sourced food packaging. Chemosphere 337(139381). DOI:10.1016/j.chemosphere.2023.139381.

Progress 01/01/22 to 12/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Ms Megha Bedi, a PhD candidate in PI Ng's research group,spent part of her summer term in 2022 at Dr. Sapozhnikova's laboratory in Wyndmoor, PA. She learned PFAS targeted and non-targeted analysis with the developed protocols for this project, and coordinated with Postdoctoral Fellow Raegyn Taylor in the Sapozhnikova lab to determine which PFAS and PFAS mixtures identified in the extraction and migration assays should be considered for toxicity testing during project year 3. This has been a valuable training opportunity for Ms. Bedi's future career goals. How have the results been disseminated to communities of interest?The project team is currently working on a publication describing the results of the extraction and migration assays, to be submitted to a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the focus will be on testing the toxicity of the PFAS identified in the food packaging, with a particular focus on creating mixtures that reflect the composition and concentrations found in the migration studies. This should result in a highly relevant exposure scenario. The toxicity assays will be conducted using the zebrafish embryo assay, which traditionally has a focus on tracking morphological changes, but we will add a component of gene expression analysis which should serve as a more sensitive indicator of low-dose toxicity and may give insight into toxic pathways potentially activated by the tested compounds and mixtures.

Impacts
What was accomplished under these goals? In year two we developed and optimized extraction, migration, and PFAS analysis procedures for collected food packaging samples. PFAS mixtures covering a total of 34 native compounds and 20 mass-labeled standards were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Sample Extraction Food packaging extraction was optimized for solvent (methanol, 1% acetic acid in methanol, 1% formic acid in methanol, and 95% ethanol in water), extraction time (30 min, 1 hour, and 2 hours), and technique (sonication, shaking, both) using spiked cardboard paper. A targeted LC-HRMS method for 34 PFAS was developed and evaluated. Concentrations of total targeted PFAS in extracts ranged from 0.01 to 24.6 nmol F/g, with highest concentration in salami plastic/paper film from the US, while total precursors, determined via the total oxidizable precursor (TOP) assay were as high as 73 nmol F/g in a paper popcorn bag from the US. Total Oxidizable Precursor Analysis The TOP assay was performed using previously reported methods to quantify precursors capable of oxidizing to terminal carboxylic acids (PFCAs). Preliminary experiments were conducted to assess accuracy of the method using three precursor standards (NEtFOSAA, 6:2FTS, and 5:3FTCA) and showed >30 mM of potassium persulfate was needed to provide non-detectable levels of precursors. Recoveries were between 73 and 78% for NEtFOSAA and 22 and 27% for 6:2 FTS, which is likely due to missing fluorine content from short-chain products not quantified by our targeted method. Precursor content reported in this study in likely an underestimation of true precursor levels and is considered semi-quantitative. Task 2.2. Migration assays Based on the PFAS measured in extracts, frequency of detection, and TOP assay results, 25 food packaging samples with highest frequency of detection and highest measured PFAS levels were selected for migration tests. For room temperature applications (shelf stable foods), US FDA migration protocol recommends a test temperature of 40?C (104?F). For refrigerated and frozen food applications, the recommended test temperature is 20?C (68?F). Depending on the food type as defined by the US FDA, food simulants were selected as 10% ethanol/water for aqueous and acidic foods and 95% ethanol/water for fatty foods. Migration tests were conducted in triplicate with blanks for 2 food simulants at both temperatures. Aliquots of migration samples were collected and analyzed at 2, 24, 96 and 240 hours. Internal standards were added to replicate 2 of each sample to monitor HRMS instrument performance and guide in identification and semi-quantitative analysis. Based on targeted analysis, PFHxS, PFHpA and PFHxA were detected, confirmed with fragment ions, and measured in migration extracts at 0.05-0.71 µg/kg levels. Liquid chromatography-mass spectrometry (LC-MS) analysis A Waters Acquity LC System equipped with the Waters PFAS solutions kit (Milford, MA, USA) and a 1.7 μm, 2.1 x 100 mm ACQUITY BEH C18 column and 1.7 μm, 2.1 x 5 mm Acquity BEH C18 guard column (Waters Corp., Milford, MA, USA) maintained at 50°C was used for chromatographic separation. The Acquity system was coupled to both HRMS and MS/MS triple quadrupole systems by a contact closure connection. Full-scan high-resolution mass spectrometry (HRMS) allowed for simultaneous target and non-target analysis on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap™ system (Thermo Fisher Scientific, Bremen, Germany). Mass calibration was performed before every analytical batch. After preliminary non-target screening with Compound Discoverer 3.3, an inclusion list was generated for suspected fluorinated chemicals. MS2 data was collected in ddMS2 mode on pooled extract samples. Data Processing Quantitation was completed in Tracefinder™ (Version 4.1, Thermo Fisher Scientific). Peak areas were generated using the summation peak integration function and quantified by 1/X weighted internal standard calibration curves. Suspect screening of non-target data was completed in Compound Discoverer 3.3 using a generic workflow that generates a peak list of chemical features (annotated by m/z and retention time) at levels >5× that of the method blank, with a peak rating >5, signal to noise ratio >5, and chemical formula prediction that contains at least CHF. Noisy baseline peaks were removed by visual inspection before an inclusion list was generated from the remaining features. ddMS2 data was added to the workflow as "identification only" file types and used to screen against the EPA PFAS Master list and mzCloud for potential matches. Fluoromatch was also used to aid in identification of PFAS not included in the targeted method; to the best of our knowledge, this is the first application of Fluoromatch for PFAS determination in food packaging. Development and evaluation of non-targeted workflows for PFAS analysis We created and tested non-targeted workflows using available PFAS analytical standards. Retention times (tR) were aligned with ChromAlign feature) based on a reference file containing PFAS analytical standards. Extracted ion chromatogram (XIC) traces were created for fragments generated from specific molecular structures. Environmental and Food Safety (EFS) HRAM database (1634 compounds), extractables and leachables HRAM database (1741 compounds), PFAS EPA master list (10901 compounds), PFAS suspect list (4951 compounds) and PFAS master list with predicted tR (10762 compounds) were used as mass list databases. Analysis of food packaging migration extracts using this MS1 workflow resulted in 255 hits. Hits were searched against available information on CAS#s, reports, publications, and available analytical standards to confirm identity. In the MS/MS (MS2) workflow, the same parameters as for MS1 workflow were used for selecting spectra and aligning tR. Mass traces for XIC MS2 with mass tolerance of 50 ppm were created, as well as pattern trace for isotopic ratios of PFOS (C8HF17O3S) and PFOA (C8HF15O2) with 5 ppm mass tolerance and 30% intensity tolerance. Predicted composition and mass defects were determined similar to MS1 workflow.ChemSpider was searched with: EPA Toxcast; FDA UNII - NLM; Food and Agriculture Organization of the United Nations; FooDB; Toxin, Toxin-Target Database databases; MzCloud was searched for the following compound classes: Excipients/Additives/Colorants; Extractables/Leachables; Illegal Additives; Perfluorinated Hydrocarbons; Small Molecule Chemicals; Textile Chemicals/Auxiliary/Dyes; and the following mass list databases were used: EFS HRAM Compound Database, Extractables and Leachables HRAM Compound Database, PFAS EPA Master List, PFAS Suspect List, PFAS Master list with predicted tR. A total of 45 PFAS were tentatively identified based on this MS2 workflow. Our efforts in confirming identities of tentatively identified PFAS resulted in one confirmation based on tR and mass MS1 and MS2 spectra, confirmed with reference analytical standard as 6:2 fluorotelomer phosphate diester (6:2 diPAP). It was semi-quantified in extracted food packaging and migration study samples. In extracted food packaging samples diPAP was frequently detected, with 74-100% detection among different food packaging types with concentrations ranging from 0.10-44.1 ng/g. In migration samples, diPAP was found in food packaging for bakery and meat in concentrations 0.2-12.2 µg/kg food with levels increasing over the 10-day migration test. Additionally, Cumulative Estimated Daily Exposure (CEDI) was calculated by multiplying the estimated daily intake (EDI) by the consumption factor (CF) for the packaging material, which are provided by the US FDA for determining exposure estimates. To our knowledge, there is no published information on this PFAS compound reported migrating from food packaging. Future efforts are focused on quantifying diPAP in migration samples by a targeted approach.

Publications

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:During this reporting period, we had one outreach activity that involved high school students (primarily juniors) in a research class. PI Ng mentored one high school student (September 2021-ongoing) on an independent research project that also involves surveying her research class at Allderdice High School in Pittsburgh. The project will culminate in a presentation by the studentat the Carnegie Science Center science fair. Changes/Problems:Our project experienced substantial COVID-related disruptions due to the shutdown and subsequent occupancy restrictions at our laboratories, especiallyat the EERC lab where material extractions and migration experiments will be conducted. The USDA EERC laboratory is still operaating under 100% telework. In July 2021, researchers were allowed to return to the laboratory at 25% capacity, and that restriction continues today. This prevents the execution of 10-day migration experiments at this time since laboratories are off limits on weekends. This will hopefully change in 2022. If not, the university laboratories (Pittsburgh and/or Emory) will aid in executing these planned experiments. Finally, supply chain disruptions affecting the availability of supplies, parts, chemicals and consumables have slowed down a number of experiments, and these also persist. What opportunities for training and professional development has the project provided?While COVID-related disruptions decreased our access to laboratory space and instrument time, particularly at EERC, it prompted the PI at the Univeristy of Pittsburghto develop the capability to conduct some of the targeted analysis in house in order to maintain progress and provide opportunity for graduate student Megha Bedi to learn the PFAS analysis method. This also provided the opportunity to mentor the high school student on a food packaging related project, an unplanned outreach opportunity for the project as a whole that is having a positive impact on the ongoing relationship between the Swanson School of Engineering at the University of Pittsburgh and Allderdice High School. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, packaging material extractions will begin at EERC under the direction of co-I Sapozhnikova, which will then allow for extracts to be sent to co-I Salamova for targeted PFAS analysis. Moreover, PI Ng will use the extracts to begin the zebrafish toxicity experiments proposed under Objective 3. In addition, longer-term (10-day) migration simulations will begin to evaluate the potential for PFAS in food packaging to transfer tofood.

Impacts
What was accomplished under these goals? The impact of the proposed work will be to provide new information on the presence of PFAS in food packaging and, importantly, whether that PFAS has the potential to migrate into food and thus pose a threat to human health. We first seek to understandwhich packaged foods are consumed in the US and whether these vary by shopping location (including communities served and price point) and food origin (to highlight potential risks associated with imported foods or particular diets). To work towards this impact under Objective 1,we conducted a survey of packaged foods across 12 different retail stores in the City of Pittsburgh, including grocery chains, independent international food markets, drug stores that also sell food, and wholesale-type stores to describe the types and origins of packaged foods present. Results of the survey indicate that the majority of packaging was coated paper, carboard, and plastic;all categories that may contain PFAS as a surface protectant. These covered six food categories: bakery, dairy, deli meants, produce, and packaged meals.The foods represented 25 different countries of origin across North America, Europe, Asia, and South America. After discussion among the project partners, we selected a set of 90 samples that were purchased and sentto the ERRC for analysis. Sample collection considered a mix of stores, prices, origins (50% US, 50% imported), and packaging materials. Based on this selection of packaged foods, we next seek to determine whether PFAS are present in this packaging and, if so, whether it can migrate to foods. To prepare for the analyses required to answer these questions (planned to begin in the next reporting period), weevaluated theextraction efficiency of PFAS from packaging materials using a variety of relevant solvents (methanol, 1% acetic acid in methanol, 1% formic acid in methanol, and 95:5 (v/v) ethanol:water). We simulated packagingby spiking paper cardboard 34 different PFAS representing a wide variety of chemical structures including carboxylates, sulfonates, telomer sulfonates, sulfonamides and several "emerging PFAS."Samples were dried forfive days and then analyzed via liquid chromatography-high resolution mass spectrometry. Overall, the extraction solvent had no significant effect on recovery from spiked paper.The C4, C6, and C8 perfluorinated sulfonamides had the lowest recoveries ranging between 66 and 80%. This could be due to the volatility of sulfonamides making them more likely to escape to air before analysis. Studies investigating indoor exposure to PFAS typically find higher levels of sulfonamides in air samples than in dust, so additional experiments will investigatethe recovery of sulfonamides without the aging process (5 days in air). However, these results suggest sulfonamide levels in food packaging may be lower than other compound classes due to volatility. Additionally, sulfonamides are precursors to other fluorinated alkyl substances (e.g., FOSA degrades into more stable PFOS and PFOA in the environment). We will evaluate whether this migh be occurring within the packaging itself. Due to solvent type showing no significant effect on extraction efficiency, unadjusted methanol was used to optimize extraction time with sonication (5 or 30 min), shaking (5 or 30 min), and a combination of the two (5 min sonication followed by 5 min shaking). The same approach for testing extraction solvents was utilized, but these samples were only left to dry for 10 min. For most compounds, recoveries fell within an acceptable range (80-120%). Overall, five minutes of sonication provided the lowest recoveries (86 ± 9%) while 5 minutes of shaking gave excellent recoveries for all analytes (105 ± 11%). However, increasing shaking time from 5 to 30 minutes increased the recovery of the fluoroether HFPO-DA (commonly known as a "GenX" compound)from 72 to 97%. Because this list of targeted analytes only represents a subset of the possible fluorinated compounds present in food packaging, a longer 30-minute extraction time will be used to enhance extraction of unknown compounds, as well as HFPO-DA. Additionally, as expected, skipping the aging process led to increased recovery of the more volatile sulfonamides. Extraction of collected packaging materials (n=90) is underway using the optimized protocol to screen and identify extractable organic fluorinated compounds.

Publications