Project for degree of Master of Science in Chemistry Independent project work
2019/2020
Total oxidizable precursor assay: Applications and limitations. A study on
the occurrence of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in
Chinese Wastewater Treatment Plants
Author: Pontus Larsson Supervisor: Leo Yeung Examiner: Anna Kärrman Date: 2020-06-01
Summary
Total oxidizable precursor (TOP) assay is an in-lab method which oxidatively converts precursor compounds of poly- and perfluoroalkyl substances (PFASs) into measurable perfluorinated alkyl acids (PFAAs). The method is a very strong tool to semi-quantify PFASs that would otherwise be missed in conventional targeted analysis using liquid chromatography - tandem mass spectrometry (LC-MS/MS). However, there are still challenges for it to be a fully quantitative tool. This report aims to increase the knowledge in using measures to improve the quality assurance of the assay, as well as investigate potential limitations of applying TOP assay outside the scope of what it was originally developed for. Furthermore, the objective was also to apply an evaluated TOP assay method on sample extracts from influent and effluent water from wastewater treatment plants (WWTPs) in China to investigate the occurrence of both legacy and novel PFASs.
To achieve improved quality assurance of the method, the use of isotopically labelled standards in the TOP assay were investigated. 13C8-FOSA was used as a model compound to measure the oxidation efficiency in the assay; the results showed that the molar yield of 13C8-FOSA to 13 C8-PFOA was 99% ± 2% (n=12) for the WWTP samples. This indicated it to be a good tool to monitor the oxidation, but more research is needed to understand the fate and reaction rate of other precursors. Oxidation of sample extracts were performed without evaporating solvent before oxidation to minimize loss of volatile precursors. This procedure was shown to be possible but require additional oxidation agent and base and may lead to different product pattern after oxidation. Further, performing TOP assay on sample extracts will introduce bias in which types of precursors are extracted and may lead to an underestimation of total PFAAs precursor present, depending on the extraction method or solvent type used. Additionally, the stability of two novel compound groups in the assay were investigated: 1) The Pre 2002 and Post 2002 formulas of Scotchgard™ fluorinated side-chain polymers were both degraded in the assay. For the Pre 2002 formula the primary degradation product was PFOA and for the Post 2002 formula, PFBA was the degradation product. Additionally, both the Pre 2002 and Post 2002 formulas were shown to undergo hydrolysis in high pH conditions, producing perfluoroalkyl sulfonic acids (PFSAs). 2) Per- and polyfluorinated ether acids (PFEAs) were also investigated in the TOP assay, which indicated that perfluorinated ether acids are stable in high pH oxidative conditions but may degrade in low pH oxidative conditions, while polyfluorinated ether acids may be degraded under oxidative conditions in both high and low pH.
The analysis of influent and effluent water from Chinese WWTPs was done by screening for 14 legacy PFASs, 16 novel PFASs, and performing TOP assay on sample extracts as well as analysis of extractable organic fluorine for mass balance analysis. Perfluorobutanoic acids (PFBA) had the highest detection rate among the legacy PFASs, and hexafluoropropylene oxide
dimer acid (HFPO-DA) was the most prevalent novel PFASs detected. TOP assay on the
anionic fraction lead to varying degree increase in PFAS concentration (0-15x) while performing the oxidation on the neutral/cationic fraction did not lead to any observable increase in PFASs. Even with TOP assay, the proportion of extractable organic fluorine still unidentified was 11% to 98%.
Table of content
Summary ... 2
Acknowledgements ... 4
1. Introduction ... 5
1.1. Research questions and aim ... 5
2. Background and theory ... 6
2.1. Per and polyfluoroalkyl substances (PFASs) ... 6
2.2. Synthesis, uses and application ... 6
2.3. Sources and base of concerns ... 6
2.4. Regulation ... 7
2.5. Limitation of current monitoring strategy ... 7
2.6. Mass balance of fluorine analysis ... 8
2.7. TOP assay – Principle, applications and limitations ... 8
2.8. Oxidative conversion ... 9
2.9. Chemicals of interest in current investigation ... 10
2.9.1. Scotchgard™ products ... 10
2.9.2. Per- and polyfluorinated ether acids (PFEAs) ... 10
3. Method ... 11 3.1. Chemicals ... 11 3.2. Samples ... 12 3.3. Extraction ... 12 3.4. TOP assay ... 13 3.5. Oxidation of PFEAs ... 14
3.6. TOP assay on WWTP sample extracts ... 14
3.7. Instrumental analysis ... 14
3.8. Quality control ... 15
4. Results and discussion ... 16
4.1. Improving the quality assurance of TOP assay ... 16
4.2. Discussion on the limitations of TOP assay on sample extract ... 18
4.3. Oxidation of fluorotelomers ... 19
4.4. Oxidation of fluorinated alkyl sulfonamides under different conditions ... 19
4.5. Stability of novel compounds in the TOP assay ... 22
4.5.1 Fate of Scotchgard™ fabric stain repellants under oxidative and high pH conditions. ... 22
4.5.2 Fate of fluoroalkyl ether acids under oxidative conditions ... 25
4.6. Application of TOP assay on wastewater samples ... 28
5. Conclusion and recommendations ... 33
References ... 34
Acknowledgements
Dr. Leo Yeung, in addition to other staff and students, of Örebro University are greatly thanked for help and support throughout this project. Dr. Anna Kärrman is thanked for meaningful discussions. Dr. Bei Wang, Felicia Fredriksson, Rudolf Aro, Mio Skagerkvist and Mohammad Sadia are thanked for contributions and discussions. Professor Jia-Yin Dai, students and staff, of the Institute of Zoology, Chinese academy of Sciences, Beijing, are gratefully thanked for contributions and kind hospitality for making this project succeed. Dr. Yitao Pan, Dr. Nan Sheng, Jinghua Wang, Jinzhi Yao, Ruina Cui and others of the group are thanked for insightful discussions and contributions to the project. Mobility grant provided by The Swedish Foundation for International Cooperation in Research and Higher Education (STINT) for the Joint China Sweden Mobility Programme 2018 (Dnr: CH2018-7805) is acknowledged.
1.
Introduction
Per- and polyfluoroalkyl substances (PFASs) are a group of anthropogenic chemicals whose history can be traced back to the accidental discovery and synthesis of Teflon in 1938 (Trier et al., 2017). The thermally and chemically stable fluorinated alkyl chain together with its lipophilic and hydrophilic properties have led to them being used extensively in both industrial and consumer applications (Kissa, 2001, Buck et al., 2011).
Due to the widespread use, bioaccumulative potential and the toxicological effects of PFASs, they have become increasingly regulated. However, regulation have so far only targeted a few selected PFASs (USEPA, 2017, Livsmedelsverket, 2020). Furthermore, it is estimated that thousands of PFASs exist on the market today (KEMI, 2015), but only a small fraction of these are currently being monitored and analyzed using standardized targeted analytical approaches (USEPA 2016; ISO 25101; UNEP, 2015). This is mainly because the structures are either not known or that analytical reference standard for the compounds are not available. Furthermore, a large proportion of these unknown PFASs are believed to be precursor compounds that may degrade to persistent perfluoroalkyl acids (PFAAs) in the environment (Amin et al., 2019). As a way to tackle this problem, Houtz and Sedlak (2012) developed a protocol for rapid, in-lab oxidation of PFAA precursors, converting them into measurable perfluoroalkyl carboxylic acids (PFCAs) of which analytical standards are readily available for their quantitation. They called this method the “Total Oxidizable Precursor (TOP) assay” and was originally applied to urban runoff water samples. Since then, the method has been applied numerous times on different matrices and samples.
However, there are still challenges in using the assay outside the scope it was intended for, i.e. direct oxidation on water samples. By first performing a sample extraction (e.g., water or soil extraction) before TOP assay, errors may be introduced due to bias in extraction, loss of precursors during sample preparation (e.g., evaporation of precursors during solvent evaporation). Further, improving the quality control during oxidation is desirable such as determining the oxidation efficiency and whether the oxidation of precursors is complete. There is also a need to broaden the understanding of which compounds are stable in the assay and which compounds that may degrade and contribute to PFAAs after the oxidation. Last, in an Australian interlaboratory study (Ventia, 2019) it was suggested that the very high alkaline environment in the TOP may lead to some precursors undergoing hydrolysis instead of oxidation, leading to the formation of different PFAA patterns; this warrants further investigation.
1.1. Research questions and aim
• How can the quality assurance of the oxidation efficiency be monitored or improved?
• What are the implications of performing TOP assay on sample extracts? • Will the formation of products change in different oxidative conditions (i.e.
increased pH or high organic load)?
• Can side-chain fluorinated polymers and novel fluoroalkyl ether acids be degraded in the TOP assay?
Overall, the main objective of the study was to broaden the knowledge in implementing measures to improve the quality assurance of the TOP assay as well as identifying limitations of working with TOP assay on sample extract. Specific task was also to apply TOP assay on
wastewater samples from wastewater treatment plants in China and to investigate the distribution of novel and legacy PFASs therein.
2.
Background and theory
2.1. Per and polyfluoroalkyl substances (PFASs)
PFASs refers to a group of fluorinated compounds that contain one or more carbon of which all hydrogen atoms have been replaced with fluorine atoms, generating the fluoroalkyl group CnF2n+1- (Buck et al., 2011).
Figure 1. The chemical structure of an eight carbon, fully fluorinated sulfonic acid - perfluorooctane sulfonic acid (PFOS).
Two common groups of PFASs are perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonic acids (PFSAs); these belong to the group of perfluorinated alkyl acids (PFAAs) (Trier et al., 2017). Polyfluorinated PFASs refers to compounds such as 6:2 fluorotelomer sulfonic acid, which are not fully fluorinated but have one or more fluorine atoms replaced with hydrogen or another atom. (Ahrens, 2011).
2.2. Synthesis, uses and application
There are two main synthesis pathways used in the manufacturing of PFASs; electrochemical fluorination (ECF) and telomerization. Historically, ECF have been used the most (80-90% in 2000) for the production of PFCAs (Prevendouros et al., 2006) and revolves around immersing a chemical such as a carboxylic acid in hydrogen fluoride (HF), which exchanges all hydrogen atoms to fluoride. ECF does not produce a pure product; branched isomers of both even and odd number of carbons are created during the procedure which have the same chain length as the starting material (Trier et al., 2017). The PFASs produced using ECF can be expected to be a mixture of primarily linear structure and branched isomers up to 30 wt% (Prevendouros et al., 2006). Telomerization manufacturing instead leads to a mixture of linear, even number PFASs. One telomerization process involves reacting telogens (CF3CF3I) to produce telomer iodine in a mixture of different chain length. The telomer iodine is reacted with ethylene to produce perfluoroethylene iodide, which in turn can be further reacted to produce fluorotelomer alcohols used as intermediates for surfactant manufacturing.
ECF and telomerization based PFASs have both overlapping uses and applications. Both have been used to produce PFOA, which has been used as a processing aid in the manufacturing of polytetrafluoroethylene (PTFE). Other uses include the use as surfactants in the manufacturing of hard chrome plated products, aqueous film forming form (AFFF) for firefighting, and side-chain fluorinated polymers used in surface treatment of paper and fabric (Wang et al., 2014)
2.3. Sources and base of concerns
The sources of PFCAs and PFSAs in the environment can be traced from two different categories: direct emission or indirect emission. With direct emission, it describes emission of PFAA directly from either a manufacturing facility, PFCA/PFSA impurities in e.g., consumer
or industrial products and use of AFFF. Indirect sources include degradation of precursors, such as POSF-based AFFF, fluorotelomer-based (e.g., from the use of AFFF) (Wang et al., 2014) and precursors in food contact material (FCM) (Trier et al., 2017). Wang at al. (2014) estimated that between 2610 to 21400 metric tons of PFCAs (C4-C14) have been released into the environment between the years 1951 to 2015.
Prior to 2002, the manufacturing of PFASs related products was located mostly in North America, Europe and Japan, but since then the manufacturing locations have shifted to Asia, primarily China. This has led to the industrial emission sources of PFASs shifting to these locations (Wang et al., 2014).
After the extensive use and release of PFASs from production facilities’ wastewater, there has been a widespread emission into the environment. Identification and detection of these compounds in biotic and abiotic samples have later been reported. In 1980, Ubel et al., reported that blood of workers from fluorochemical manufacturing plants contained organic fluorine, and that a large proportion of this was confirmed to be perfluorooctanoic acid (PFOA). Since then, numerous reports of the widespread detection of PFASs have been published; perfluorooctane sulfonic acid (PFOS) in the environment (Key et al., 1997), in wildlife (Geisy and Kannan, 2001), and humans (Kärrman et al., 2006) where numerous PFASs were detected in human milk and blood. Multiple studies have later shown the toxicity of PFASs in animals (Lau et al., 2007; Tsuda, 2016; Sheng et al., 2018).
2.4. Regulation
Due to the increasing awareness regarding their toxicity, persistence, bioaccumulation potential and widespread occurrence, PFASs have been increasingly regulated. The Stockholm Convention on persistent organic pollutants regulates PFOS and its parent molecule perfluorooctane sulfonyl fluoride (POSF), as well as PFOA and related compounds. Currently, perfluorohexane sulfonic acids (PFHxS) are under review to be added to the list (UNEP 2017, UNEP 2019). PFASs are also regulated under the European chemical regulation REACH and in July 2020, an EU legislation will come into effect that prohibits the import and use of PFOA and related compounds in products exceeding 25 µg/kg of the chemical (Trier et al., 2017). For drinking water, the United States Environmental Protection Agency (EPA) have set a health advisory level of 70 ng/L for PFOA and PFOS, combined or individual (USEPA, 2017). In Sweden, the National Food Agency (Livsmedelsverket) have an action limit of a sum of ten PFAAs (C4-C10 PFCAs, C4, C6 and C8 PFSAs) and one polyfluorinated precursor (6:2 fluorotelomer sulfonic acid, 6:2 FTSA) to 90 ng/L (Livsmedelsverket, 2020). Further, there is a proposal in 2020 to the water directive (5813/20) from the European Union to set two new guidelines for regulatory limits for drinking water in the member states: (1) The sum of 20 PFASs (C4 -C14 PFCAs and C4-C14 PFSAs) is limited to 100 ng/L. (2) “PFASs - Total”, which is intended to include all fluoroalkyl substances with three or more carbons and fluorinated ether acids that contains two or more carbons. The limit for the PFASs total is set to 500 ng/L.
2.5. Limitation of current monitoring strategy
As of now, standardized analytical methods using liquid chromatography – tandem mass spectrometry (LC-MS/MS) to measure PFASs such as ISO 25101, UNEPs standard operational procedure (SOP) (UNEP, 2015) and U.S. EPA Method 537.1/333, only target a very small fraction of all PFASs that exists. Among these, the two EPA methods combined includes the most PFASs, targeting 29 compounds. However, in a study by the Swedish Chemical Agency (KEMI, 2014) they were able to identify more than 2000 PFASs on the market and their
estimate of the total number of PFASs on the market was around 4000. This means that a large proportion of PFASs in existence are currently not being monitored or measured in routine analysis and complete environmental assessments may therefore be challenging. Techniques other than target analysis to cover a wider range of PFASs have been developed. Some techniques rely on measuring the organic fluorine using particle-induced gamma ray emission (PIGE) spectroscopy (Ritter et al., 2017) or combustion ion chromatography (CIC) (Yeung et al., 2008), which have introduced concept such as total fluorine (TF), adsorbable organic fluorine (AOF) and extractable organic fluorine or EOF (Koch et al., 2019) which is further elaborated on in section 2.6.
2.6. Mass balance of fluorine analysis
To get an estimation of the amount of unknown PFASs that potentially exists in a sample, a comparison between organic fluorine and sum of PFASs expressed as mass of fluorine equivalence, can be made. This is referred to as fluorine mass balance analysis. The PFASs fluorine mass can be calculated according to the following equation.
𝐶! =
𝑛!𝑀𝑊!
𝑀𝑊"!#$∗ 𝐶"!#$
The total fluorine (TF) is sum of organic fluorine (OF) + inorganic fluorine (IF). The organic fluorine can further be divided into extractable organic fluorine (EOF) and non-extractable organic fluorine (NEOF). Furthermore, the EOF can be divided into identified EOF fluorine and unidentified EOF (Koch et al., 2019). The value of identified EOF is derived from the sum of PFASs expressed as fluorine, as described above. This concept is explained visually in figure 2.
Figure 2. Mass balance of fluorine analysis. Illustrated with help from Koch et al., 2019. Total fluorine (TF) = extractable organic fluorine (EOF) + inorganic fluorine (IF) + non-extractable organic fluorine (NEOF).
2.7. TOP assay – Principle, applications and limitations
Another technique, developed to indirectly measure unknown precursors that contribute to the indirect emission of PFAAs, is a method called Total oxidizable precursor (TOP) assay. This concept was first conceived by Houtz and Sedlak in 2012 and have since been applied by many more (Houtz et al., 2013; Houtz et al., 2016; Robel et al., 2017; Martin et al 2019; Zhang et al 2019; Janda et al., 2019). The aim of the technique was to indirectly measure and estimate the precursor concentration, by oxidative conversion of unknown and known precursors of PFCAs and PFSAs into measurable, known PFCAs (Figure 3).
Figure 3. Example of using TOP assay to convert perfluorooctane sulfonamide (precursor) into a stable end product, PFOA.
The distinct feature of the assay is that it converts all precursors to PFCAs as stable, terminal products, as opposed to in the environment where some precursors may be transformed into PFSAs as terminal products. The principle behind the assay is a hydroxyl radical oxidation initiated by thermolysis of potassium persulfate under basic conditions (60 mM potassium persulfate, 150 mM sodium hydroxide, Houtz and Sedlak, 2012).
Samples are amended with the reagents and let react for 6h in an 85ºC water bath to undergo oxidative conversion. Initially, Houtz and Sedlak (2012) performed direct oxidation on the water sample, but other studies (Houtz et al., 2013; Robel et al., 2017; Janda et al., 2019) have later performed the oxidation on sample extracts where direct oxidation was not possible or practically unfavorable. Previously, studies using sample extract on TOP assay have evaporated the solvent prior to oxidation to remove the solvent that might otherwise interfere with the oxidation.
Quality assurance (QA) of the TOP assay have previously been done in various ways. Houtz and Sedlak, 2012, for example, performed the oxidation in triplicates in addition to having a heated control with no potassium persulfate or sodium hydroxide added and other various spiked experiments. A recent interlaboratory study from Australia (Ventia, 2019) put a forward quality assurance recommendation that concentrations of precursors before and after oxidation should be monitored and suggested that a limit of 5% of initial starting concentration of precursor after oxidation should be acceptable for a successful oxidation. They also discussed QA measures such as spiking native and isotopically labelled standards in the samples to monitor the oxidation. Further, Koch et al., 2019 suggested duplicate oxidation where one sample is diluted 10x. If both samples show similar concentrations post oxidation, it is assumed to be successful.
2.8. Oxidative conversion
As described in the previous section, PFCAs and PFSAs are resistant to degradation by hydroxyl radicals which bases the premise of the TOP assay (Houtz and Sedlak, 2012). However, PFCAs have been shown to degrade in persulfate activated systems where the primary oxidizing species are sulfate radicals, while PFSAs where shown to be stable under the same conditions (Bruton and Sedlak, 2017). The reaction mechanism of hydroxyl radical oxidation is primarily driven by hydrogen abstraction (Olmez-Hanci and Arslan-Alaton, 2013), while sulfate radical oxidation of PFCAs is thought to be initiated via electron transfer from the carboxylic head to the sulfate radical species, followed by HF elimination, producing shorter chain PFCAs until mineralization (Yin et al., 2016). Maintaining a high pH in the system is therefore crucial to shift the primary oxidation species to hydroxyl radical and limit or remove the possibility of degradation of the formed PFCAs during the TOP assay. The thermolysis of persulfate in basic condition to produce hydroxyl radicals are visualized in the equation below.
2.9. Chemicals of interest in current investigation
2.9.1. Scotchgard™ products
A recent study (Yeung et al. 2017) found that a large proportion of the organic fluorine found in sludge and leachate from WWTP are still of unknown origin. One hypothesis (Fredriksson et al., 2020) has been that part of the unknown fluorine could be explained by side-chain fluorinated co-polymers that can be found in consumer products, food packaging and water and oil repellant products (Trier et al., 2017), and may degrade to PFAAs. However, there is currently limited information on the efficiency of TOP assay on side-chain fluorinated co-polymers such as the 3M Company Scotchgard™ fabric stain repellent products.
In early 2000, 3M voluntary phased out of PFOS that led to the change in formulation of the Scotchgard™ products (Chu and Letcher, 2014); these two formulations are in herein referred to as “Pre 2002” and “Post 2002”. According to a report from Trier et al., (2017), that assembled a list with input from Trier et al., 2011 and Benskin et al., 2013, the Pre 2002 formula included monomers of various types fluoroacrylates such as
N-methyl-/N-ethyl-perfluorooctanesulfonamido ethyl methacrylate and
N-methyl-/N-ethyl-perfluorooctanesulfonamido ethyl acrylate. In 2014, Chu and Letcher provided mass spectral data that showed that the major component of the Pre 2002 formula contained the N-ethyl-perfluorooctancesulfonyl chemical group. Similarly structured but C4 chemistry based, N-ethyl-perfluorobutanesulfonyl was found in the Post 2002 formula (Chu and Letcher, 2014). In the same paper, they showed that the in vitro metabolites from the two products were perfluorooctane sulfonamide (FOSA) and perfluorobutane sulfonamide (FBSA), respectively. Chu and Letcher later in 2017 designated the chemical structure of the major component in both Pre and Post 2002 as fluorinated side chain co-polymers (Chu and Letcher 2017). However, the chemical structure of the polymer backbone is a 3M Company trade secret and is currently not published information. As other studies (Houtz and Sedlak, 2012) have shown that PFOS precursors such as N-ethyl perfluorooctane sulfonamido acetic acid (EtFOSAA) and N-methyl-perfluorooctane sulfonamido acetate (MeFOSAA) are transformed in a 1:1 molar ratio to PFOA in the TOP assay, it can be hypothesized that the Scotchgard™ products may be oxidizable in the TOP assay and that expected products may be PFOA for the Pre 2002 formula and PFBA for the Post 2002 formula.
2.9.2. Per- and polyfluorinated ether acids (PFEAs)
PFEAs are similarly structured as PFCAs and PFSA but instead have one or more ether linkages in the fluorinated chain. Because of the ether linkage, they were initially thought to be a more degradable alternative to legacy PFASs but have later been shown to be very persistent (Strynar et al., 2015). See figure 4 below for an example of a PFEA.
Figure 4. Chemical structure of hexafluoropropylene oxide dimer acid (HFPO-DA),
sometimes called GenX, referring to Chemours’ tradename of the ammonium salt of HFPO-DA.
Pan et al., 2018, recently revealed the widespread occurrence of PFEAs with the worldwide detection in Asia, Europe and the United states. Further, recent studies (Sheng et al., 2017, Guo et al., 2019, Wang et al., 2019) have shown the toxicological relevance of these emerging compounds. Furthermore, it has previously been suggested that some PFEAs, such as 1,1,2,2-tetrafluoro-2-(perfluorohexyloxo) ethane sulfonate (tradename F-53) and the 6:2 chlorinated polyfluoroalkyl ether sulfonate (commonly referred to as 6:2 Cl-PFESA or by its tradename F-53b) may degrade to PFSAs (KEMI, 2015). Later studies that have shown that the 6:2-Cl PFESA are resistant to both aerobic biodegradation and is stable in unbuffered 50ºC persulfate oxidation (Chen et al., 2018), while other studies have indicated that 6:2 Cl-PFESA are degraded in higher temperature persulfate oxidation systems (Du et al., 2016). A recent study was published by Zhang et al., 2019 where they investigated the fate of PFEAs in the TOP assay. Their results indicated that perfluorinated ether acids such as 6:2 Cl-PFESA are stable in the TOP assay, but some polyfluorinated ether acids such as 4,8-dioxa-3H-perfluorononanic acid (ADONA) may degrade. In the case of ADONA, the degradation product was perfluoro-3-methoxypropanoic acid (PFMOPrA), another PFEA. They achieved a molar transformation of 98% ± 20%, indicating that PFMOPrA was the terminal product of ADONA.
3. Method
3.1. Chemicals
The majority of the laboratory work was conducted at MTM research centre at Örebro University. The following chemicals were used: MilliQ water (18.2 MΩ·cm) was produced in lab using a Millipore system. Methanol (HPLC grade, LC/MS grade), 37 % hydrochloric acid and 25% ammonium hydroxide was bought from Fisher Scientific (Loughborough, UK). Acetic acid was bought from Merck (Darmstadt, Germany). Potassium persulfate, sodium carbonate, sodium bicarbonate and ammonium acetate were bought from Sigma-Aldrich (St Luis, MO, USA). Sodium hydroxide was from KEBO. Analytical standards of perfluroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates, fluorotelomer sulfonates (FTSAs) and fluoroalkyl sulfonamides (FASAs) and 13C labelled standards where bought from Wellington Labs (Guelph, Canada). The targeted PFCAs were: butanoic acid (PFBA), perfluoro-n-pentanoic acid (PFPeA), perfluoro-n-hexanoic acid (PFHxA), perfluoro-n-heptanoic acid (PFHpA), PFOA, perfluoro-n-nonanoic acid (PFNA) and perfluoro-n-decanoic acid (PFDA). The targeted PFSAs were: perfluoro-n-butane sulfonate (PFBS), perfluoro-n-hexane sulfonate (PFHxS), PFOS, and perfluoro-n-decane sulfonate (PFDS). The targeted FTSA was 6:2 FTSA and the target FASAs were perfluorobutane sulfonamide and perfluorooctane sulfonamide. Additional work was also performed at the Key laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences. Description of chemicals and laboratory materials can be found in Pan et al., 2017 and Pan et al., 2018. The target novel
compounds were hexafluoropropylene oxide dimer acid (HFPO-DA), hexafluoropropylene
oxide trimer acid, C7 (HFPO-TrA), hexafluoropropylene oxide trimer acid, C8 (HFPO-TrA),
hexafluoropropylene oxide trimer acid, C9 (HFPO-TrA), hexafluoropropylene oxide tetramer acid (HFPO TeA), perfluoro-2-methoxyacetic acid (PFMOAA), perfluoro-3,5-dioxahexanoic acid (PFO2HxA), perfluoro-3,5,7-trioxaoctanoic acid (PFO3OA), perfluoro-3,5,7,9-tetraoxadecanoic acid (PFO4DA), perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoA), 4,8-dioxa-3H-perfluorononanic acid (ADONA), 4:2 chlorinated polyfluorinated ether sulfonate (4:2 Cl-PFESA), 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), 6:2 polyfluorinated ether sulfonate (6:2 H-PFESA) 8:2 chlorinated polyfluorinated ether sulfonate (8:2 Cl-PFESA) and perfluoro (2-ethoxyethane) sulphonic acid (PFEESA). Structural information of these novel compounds can in appendix 2B.
3.2. Samples
Influent and effluent samples had previously been collected from three wastewater treatment plants (WWTPs) and were stored in freezer until sample extraction. The samples from Quzhou, Zhejiang were collected in December of 2015, and the recipient water was from industrial origin. Samples from the two WWTPs in Wuhan, Hubei were also collected in December 2015. The type of recipient water (industrial/domestic) from these sites are unknown.
Figure 5. Map of the location of the three WWTPs. Map sourced from Wikipedia.org. 3.3. Extraction
The samples preparation started with thawing frozen samples in an 8ºC fridge. The samples were then sonicated for 10 minutes to promote desorption of PFASs from the container wall and particles. Prior to extraction the samples were filtered using a 1000 mL Duran filtration apparatus (Millwill, NJ, US), with 0.45 µm Agilent (Santa Clara, CA, US) RC filters; these filters were investigated and showed satisfactory background levels of PFASs. To monitor background levels throughout the filtering and extraction blank samples containing LC water was used. After filtering the samples were stored in new, pre-cleaned HDPE bottles and the pH were adjusted to 4-5 using concentrated sulfuric acid. For the first batch of samples, around 200
mL of samples was used and for the second batch around 50 mL of sample was used, with the exact weight of the sample noted. Two sample later needed to be re-extracted because of suspected overloading of the cartridge, due to very high concentration of PFASs in these samples. For these re-extracts, around 10 mL of sample was used. For each sample, extraction was carried out in duplicate; one replicate containing internal standard for targeted analysis of novel PFASs (fraction A) and one replicate with no internal standard added (fraction B). This fraction was used later for TOP assay analysis of legacy PFASs and mass balance analysis. Before extraction, 10 µL of a 100 ppb internal standard mix was added to all samples of ‘fraction A’. For the second batch of samples, 20 µL of the internal standard mix was instead added as higher concentrations of PFASs was suspected. The samples were then subjected to solid phase extraction using Oasis WAX (Waters, Milford, MA, US) cartridges. The procedure followed ISO 25101 with some modifications. The cartridges were first conditioned with 5 mL 0,1% NH4OH in methanol, followed by 5 mL methanol and 5 mL LC-water. The samples were then loaded and passed through the cartridge with a speed of approximately 1 drop/second. After loading the samples, the sample bottles were cleaned with methanol and the rinsate added to the cartridges. The cartridges were then washed with 20 mL of 0,01% NH4OH in LC-water to remove any inorganic fluorine, followed by 30 mL of LC-water and 4 mL of 20 mM sodium acetate buffer at pH 4. The samples were then eluted in 15 mL polypropylene tubes using 4 mL of methanol for neutral and cationic PFASs (fraction 1) and 4 mL of 0,1% NH4OH in methanol for anionic PFASs (fraction 2). Both fractions were then evaporated down under a gentle stream of nitrogen to around 200 µL. As all novel PFASs analyzed in this study were anionic, only fraction 2 were further processed.
Fraction 2A was evaporated to dryness and later resuspended in a 200 µL mixture of 50:50 methanol:water. After shaking the samples for 2 minutes in at 500 RPM, the polypropylene tubes were centrifuges at 2000 RPM for 5 minutes to settle the extract. The extracts were then transferred to LC-vial before 10 µL of recovery standard mix was added prior to instrumental analysis. Fraction 2B was stored for later analysis.
3.4. TOP assay
Oxidation of sample matrices and individual compounds was tested in different settings throughout this project. The starting method used can be found described in Houtz and Sedlak, 2012. As explained in this report in section 2.7, their published method involves hydroxyl radical oxidation initiated by thermolysis of potassium persulfate under basic conditions (150 mM NaOH, 60 mM K2S2O8, 85ºC for 6h). In current work, the dose was kept the same as Houtz and Sedlak (2012), but in some cases the dose was proven insufficient and a double dose of both oxidant and base were amended. Different containers were also used throughout testing: 15 mL polypropylene tubes, 50 mL polypropylene tubes and 50 mL HDPE bottles.
In all experiments the samples were heated to 85ºC using a temperature-controlled water bath. The reaction time varied between 6-20h in different tests.
Extraction after oxidation was accomplished the same way as described in section 3.3. Precipitated salt was noted to be present in the ionic fraction after elution. For this reason, the additional washing step of 20 mL of 0,01% NH4OH in LC-water, followed by 30 mL of MilliQ water, was done even though analysis of organic fluorine was not done post oxidation. The reason for this was to attempt to remove anions to mitigate the problem of salt in the extract that could otherwise induce ion suppression in later mass spectrometry analysis. After elution, the samples were evaporated using a Labconco RapidVap system. The extracts were evaporated
in 55ºC in 400 mBar of pressure, until 100 µL of extract remained. After this, the extract was vortexed for a few seconds at 900 RPM, and subsequently centrifuged at 8000 RPM for 3 minutes to settle the extract and to separate the salt precipitation remaining in the extract. After the supernatant was transferred to a LC-vial the process was repeated with an additional 100 µL of methanol. The final volume was adjusted to 200 µL before recovery standard was added to the sample. An aqueous phase containing 2 mM ammonium acetate in MilliQ water was then added to achieve a water to methanol ratio of 40:60.
3.5. Oxidation of PFEAs
For the experiments with PFEAs, four different oxidative environments were tested with two different concentrations of PFEAs:
1) 5 mM K2S2O8, 150 mM NaOH, pH ≥13, [PFEA] 670 ng/L 2) 60 mM K2S2O8, 150 mM NaOH, pH ≥13 [PFEA] 130 ng/L 3) 100 mM K2S2O8, 250 mM NaOH, pH ≥13 [PFEA] 130 ng/L 4) 100 mM K2S2O8, 20 mM H2SO4, pH ≤2 [PFEA] 130 ng/L
Treatment 1) had a higher concentration than the rest of the treatment to simulate a low oxidant to PFEA ratio. To minimize errors in measuring the concentrations before and after oxidation the following methodology was used: four polypropylene tubes were prepared for each oxidative treatment according to above; two of these were spiked with a mix of 11 PFEAs before the reaction. The remaining two test tubes were spiked with the same mix after the reaction. This was done to rule out that any change in concentration post oxidation would be due to differences in matrix (different salt concentrations) leading to different recoveries in the subsequent extraction or potential ionization differences in the instrumental analysis. After oxidation and post spike, the samples spiked with 10 µL of a 100 ppb internal standard mix and were pH adjusted to around 5. The extraction was then carried out as described above in section 3.3. Extraction to determine PFAAs in the samples after oxidation.
3.6. TOP assay on WWTP sample extracts
The sample extract was diluted with MilliQ water to an appropriate amount prior to oxidation. The oxidation was performed in 50 mL polypropylene tubes. Before oxidation, the tubes were amended with 750 µL of a 10 M NaOH solution and filled up to 50 mL with a solution containing 60,9 mM K2S2O8 in MilliQ water. During each batch of samples, two blanks and two QA samples were included, to both of which oxidant and base were added. 50 µL of sample extract were added to the samples, before adding 5 µL of a solution containing 1 µg/mL 13 C8-FOSA to the WWTP samples and to the QA samples. The samples were then loaded into a preheated bath (85ºC) to initiate thermolysis of persulfate. After 2h, the samples were quickly cooled down to around 40ºC, before adding a second dose of oxidant and base (0.81g K2S2O8, 750 µL 10 M NaOH). The samples were then reheated and further reacted for an additional 4h. After reaction the samples were cooled down to room temperature and pH adjusted to 4 using concentrated hydrochloric acid.
3.7. Instrumental analysis
The analysis of PFCAs, PFSAs, FASAs and FTSAs was done using liquid chromatography – tandem mass spectrometry (LC-MS/MS). The chromatography system used was an Acquity ultra-performance liquid chromatography (UPLC) system using an Acquity bridged ethylene hybrid (BEH) C18 column (100x2.1 mm) with a 1.7 mm pore size. The mass spectrometer used was a XEVO TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, US), operated in electrospray negative mode. Mobile phase A consisted of 2 mM ammonium acetate in MilliQ
water and mobile phase B was made up of 2 mM ammonium acetate in a methanol:water mixture of 70:30. The quantification was done using isotopic dilution.
The instrumental analysis of the PFEAs was done using a different system that had shown to give higher sensitivity of HFPO homologues (Pan et al., 2018). An AB Sciex 5500 mass spectrometer (Framingham, MA, US.) was used and for separation, an Acquity (BEH) C18 column (75x2.1 mm) with a 1.7 mm pore size was utilized.
The organic fluorine was measured using a combustion ion chromatography (CIC) system. The ion chromatography consisted of a Compact IC flex with an adsorber module and ion exchange column (Metrohm, Herisau, Switzerland), which was connected to a combustion module (Analytik Jena AG, Jena, Germany). The eluent used was carbonate buffer containing 64 mM sodium carbonate and 20 mM sodium bicarbonate. The samples were either injected as pure extract or diluted prior to injection to fit within calibrated range. The quantification was done using external calibration using a 5-point calibration curve. The standards consisted of PFOS in concentration equal to a fluorine mass balance calculated concentration of 50 ng F/L to 1000 ng F/L. Between each injection, a combustion blank was run. The signal of the combustion blank was subtracted from the signal of the real sample.
3.8. Quality control
The limit of detection (LOD) for the target analysis was determined by calculating the mean plus three times the standard deviation of signals in the procedural blank. If no contamination were found in the blanks, the lowest point in a 7-point calibration curve was used.
Throughout experiments, consumables and apparatus were cleaned by sonicated in detergent and deionized water and washed with ethanol and methanol. Glassware used was burned in 450ºC for 12 hours before use to remove any residual contaminants.
In the initial sample extraction, two procedural blanks in each batch was included; one with mass labelled standards added and one without mass labelled standards added. For TOP assay experiments, two procedural blanks were included in each bath, both with mass labeled standards added. Results from these are discussed in section 4.6. Further, two quality control samples were also included that was spiked with mass labelled precursors. Mass labelled precursors were also spiked into real samples to monitor the oxidation efficiency.
In addition to extraction blanks, injection blanks were also used consisting of 100% methanol and were used to evaluate carry over between injections. Before each instrumental analysis a quality assurance sample containing selected analytes were also analyzed to evaluate instrument performance.
In each batch, one standard was prepared containing native and mass labelled standards; this was used for quantification using isotopic dilution method. If no mass labelled standard were available, compounds with similar structure and retention time was used (see appendix 2A for information regarding extraction standard used).
The recovery was evaluated on by-sample basis by calculating the recovery of the internal standard, however, this was only done on analytes were a mass labelled standard was available. Acceptable recovery was set between 50-150%. The overall recovery performance of the extraction method was evaluated by triplicate spiked recovery test of native standards in a Chinese river water sample. The results from this can be found in appendix A1.
Quality assurance and quality control regarding oxidative performance and repeatability in the TOP assay will be discussed in section 4.1.
4.
Results and discussion
4.1. Improving the quality assurance of TOP assay
As described in chapter 2.7., Ventia, 2019 suggested that assessment of the completion of oxidation could be based on a limit of 5% of the initial concentrations of precursors after oxidation. This may be challenging as there may be occurrences were no known precursors can be detected prior to oxidation, or that some measured precursors are intermediates and may not be a reliable way of determining the oxidation efficiency (I.e. 6:2 FTAB -> 6:2 FTSA -> C7 and shorter-PFCAs). In these cases, this approach would be challenging. As also discussed in Ventia, 2019, spiking native standards in the samples to monitor the oxidation could be a functional way to monitor the oxidation; however, this likely requires additional time and cost of analysis due to increased number of samples. This is also the case with the suggestion put forward by Koch et al., 2019 that suggests duplicate oxidation where one sample is diluted 10x. In this work, a 13C mass-labelled oxidation standard was used to monitor the oxidation efficiency in the TOP assay. A number of mass-labelled standards may serve as oxidation standards (e.g., fluoroalkyl sulfonamides (FASAs) or FTSAs). However, using a 13C mass-labelled fluorotelomer-based compound as an oxidation standard would be theoretically possible, but because it produces a variety of PFCAs upon oxidation it is practically challenging as it may interfere with 13C mass-labelled extraction and injection standards (Ventia, 2019) used for correcting the recovery loss and ionization effect (Figure 6). The 13C mass-labelled FOSA offers more practical advantages, as it will produce a same length PFCA that is 13C mass-labelled (13C8-PFOA) after oxidation that will not interfere with the corresponding extraction (13C4-PFOA) and injection standard (13C2-PFOA) used in current investigation.
Figure 6. Possible oxidation products of 13C8-FOSA and 13C7-6:2 FTSA subjected to TOP
assay. The mass labelled FASA (13C8-FOSA) may produce a same-length mass labelled PFCA
(confirmed). Mass labelled FTSA (13C7-6:2 FTSA) instead may produce a suit of mass labelled
PFCAs (Speculated based on oxidation of native 6:2 FTSA).
In this work, during each batch of oxidation, 5 ng of 13C8-FOSA was added to the samples as well as to two control samples. The control samples did not contain any matrix or additional methanol in order to monitor the oxidation efficiency under optimal conditions. For the control samples (n=4), the molar yield of 13C8-FOSA to 13C8-PFOA was 98% (SD ± 4%) and for the WWTP sample extracts (n=20) the molar yield was 99% (SD ± 2%). This indicates that the repeatability of the oxidation is satisfactory. Because 13C8-PFOS was used as an injection standard, it could not be used to monitor possible conversion of 13C8-FOSA to 13C8-PFOS, but as discussed in later sections, the conversion to 13C8-PFOS is estimated to be maximum a few percent. For the applications used here, this is treated as negligible.
It is important to consider the amount of oxidation standard added. Optimally, the sum of precursors present in a sample should be lower than the concentration of the 13C-labelled oxidation standard added. In this work, this was not always the case and may lead to an underestimation of precursor if all were not fully converted during the oxidation (Koch et al., 2019). However, it should be noted that this may not always practically possible as half the radicals will be consumed by the 13C-labelled oxidation standard and may lead to an unreasonable large amount of oxidant needed.
Further, even if complete oxidation of the oxidation standard 13C8-FOSA is observed and the concentration of the oxidation standard is estimated to be higher than the precursor, it does not mean that all potential PFAS precursor have been fully converted. Other unknown compound groups with different chemical moieties than -SO2NH2 may require additional oxidation agent or increased reaction time. For this reason, it is also important to further conduct studies on the rate of degradation of known precursors in the assay to determine if there is any significant
different in the rate of oxidation due to different chemical structures. By selecting the precursor with the lowest degradation rate as the optimal oxidation standard, it can be assumed that other precursors have also been oxidized in the assay.
4.2. Discussion on the limitations of TOP assay on sample extract
As described in section 2.7, the common way of to perform TOP assay on sample extract is to first remove the extraction solvent by evaporation prior to oxidative treatment. However, this practice may lead to loss of some volatile precursor compounds such as fluorotelomer alcohols, fluoroalkyl sulfonamides or other volatile or semi-volatile precursors.
In current study, during preliminary experiments it was noted that around 50% of FBSA was lost during evaporation to dryness of methanolic extract under a gentle stream of nitrogen. It can be theorized that different materials and/or shape of the container may lead to varying degree of evaporation depending on surface area and how well the precursor can adsorb to the material in the container upon evaporation. This can potentially have significant implications if samples contain a large proportion of volatile or semi-volatile precursors and should be further considered. Different ways of tackling this problem was discussed or tested in this study. These included:
• Adding an inert material to the extract before evaporation that would act as a trap for the precursors but not interfere with the subsequent oxidation;
• Perform sample elution after solid phase extraction with a solvent that could be used during the oxidation without evaporating the solvent and would therefore need to not interfere with the oxidation;
• Dilute with water and evaporate (repeatably) to remove as much methanol as possible; or
• Oxidize in the presence of methanol and increase the dose of oxidant and base to offset the hydroxyl radicals scavenged by methanol (tested in this study).
The strategy that was ultimately decided on was to oxidize in the presence of methanol and increase the dose of both oxidant and base. However, this strategy has its drawbacks; increasing the dose of oxidant may not lead to an equal increase in overall oxidative efficiency in the system, due to radical on radical interaction. Further, methanol is likely to degrade in multiple step to CO2 as an end product, leading to radicals being scavenged in multiple steps. The likely formation of CO2 from degradation of methanol was observed during oxidation testing when gas bubbles started to come out of solution as the pH was readjusted to around neutral after oxidation. To mitigate the challenges of methanol radical scavenging and radical on radical interactions, additional persulfate and base was used but were amended in separate doses to theoretically achieve a lower maximum concentration of radicals in the system.
Furthermore, in this study the TOP assay was performed on sample extracts from the elutes of SPE of water sample, which makes it possible to determine charge on the precursor; meaning if the precursor is anionic or neutral/cationic by oxidizing the two SPE eluent fractions separately. Oasis WAX used herein, is a mixed mode SPE cartridge, i.e. using both anion-exchange and reversed phase mechanism to retain compounds (Waters, 2005). However, it is clear that not all PFASs are retained on this cartridge - specifically large molecules may not be. This will inevitably lead to bias in which precursors that will later be oxidized in the assay. This is true for water samples extracted using SPE or similar method but is also true for solid samples such as soil or sludge as the extraction recovery and efficiency of different precursors will depend on the extraction solvent used (this is discussed further in section 4.5.1.). Hence, in spite
of the method still referred to as the total oxidizable precursor assay, a more correct terminology would be total extractable oxidizable precursor assay when oxidizing sample extracts.
4.3. Oxidation of fluorotelomers
To ensure that current TOP assay can be compared with other studies (Houtz and Sedlak, 2012, Martin et al., 2018, Zhang et al., 2019), the oxidation product of a model compound (6:2 FTSA) used in other studies, was included. The reaction parameters chosen was the same as Houtz and Sedlak (2012): 60 mM K2S2O8, 150 mM NaOH, 6h reaction time. Figure 7 and table 1 show the results from three replicates of 6:2 FTSA oxidation.
Figure 7. The figure displays the molar yield of 6:2 FTSA oxidation in the TOP assay. The results are based on triplicates and the error bars display the standard deviation across the triplicates.
The major degradation products were PFBA (28% ± 1% in molar yield), PFPeA (35% ± 1%), PFHxA (29% ± 1%); whereas the molar yield of PFHpA was comparably low (3% ± 1%). These results are overall in good agreement to others (Houtz and Sedlak, 2012, Martin et al., 2018, Zhang et al., 2019) regarding the composition profile after oxidation, but exhibited a slight bias towards higher yields in current study compared to the others; see table 1. Houtz and Sedlak (2012) reported molar yields of 22% ± 5% for PFBA, 27% ± 2% for PFPeA, 22% ± 2% for PFHxA and 2% ±1% for PFHpA.
Table 1. Molar yields after TOP assay on 6:2 FTSA.
Product after oxidation Current study, n=3 Houtz and Sedlak, 2012, n=8
PFBA 28% ± 1% 22% ±5 %
PFPeA 35% ± 1% 27% ± 2%
PFHxA 29% ±1 % 22% ± 2%
PFHpA 3% ± 1% 2% ± 2%
4.4. Oxidation of fluorinated alkyl sulfonamides under different conditions
Two fluorinated alkyl sulfonamides (FOSA and FBSA) were also chosen as model compounds for oxidation tests. The results from the oxidation tests are shown in Figure 8 and Table 2.
Figure 8. Oxidation of C8 (FOSA) and C4 (FBSA) fluoroalkyl sulfonamides under different conditions (see table 2). The average molar yield for all treatments are displayed in the staples. The error bars denote the standard deviation measured between replicates and different treatments. Starting precursor were not detected post oxidation in any sample. n=8.
In this work, the average molar yield FOSA to PFOA was 105,7% ± 36,6% (SD), across all experiments (n=8). The fate of FOSA in TOP assay has also been investigated earlier by others (Houtz and Sedlak 2012, Martin et al., 2019, Zhang et al., 2019, Janda et al., 2019) where they reported molar conversion recoveries of 87% ± 20% (Zhang 2019) to 103%, RSD 16% (Janda et al., 2019).
The relatively high variability across replicates in this study could be explained by challenges in reliably measuring the initial starting concentration. This could be due to FOSA potential to adsorb to the reaction container. For example, when the starting concentration of FOSA was determined per Houtz and Sedlak (2012) method: heating before adding the reactants at 60ºC for 30 min, and subsample was taken for instrumental analysis. Using this method in this work, the concentration may have been underestimated because FOSA may have adsorbed onto the wall of the container which may have been the reason for molar conversion of 140-160% after oxidation.
In contrast to what have been reported by others, PFOS was detected after oxidation (molar yield of 1,3% to 3,9%, see figure 8 and table 2) indicating that some FOSA was either oxidized to PFOS or that some of the FOSA had been hydrolyzed to PFOS before oxidation could occur. For example, the samples that produced the lowest molar yield was oxidized free from methanol and matrix, using the standard dose of oxidant and base (60 mM K2S2O8, 150 mM NaOH, Houtz and Sedlak, 2012) (molar yield: 1.4% ± 0.3%) and the samples oxidized in 2x standard dose but with the presence of matrix and methanol (molar yield: 1.3% ± 0.2%). The highest yield of PFOS was observed in the presence of methanolic reference matrix with the standard dose, giving a molar yield of about 4%. In all replicates of this treatment, the pH dropped to 8 after oxidation, suggesting that the dose of NaOH was not enough to maintain a pH >12 during the reaction.
FBSA achieved almost molar transformation to PFBA (92,4% ± 6,7% SD). This result is in agreement to what Janda et al (2019) showed, where they reported PFBA as the oxidation product of FBSA and achieved the slightly lower molar yield of 65% (RSD 7%). However, in
this work, FBSA was shown to also transform to PFBS during the oxidation process. During different oxidative conditions (see table 2) the molar yield of PFBS was between 0,6 % to 4,5 %. The lowest molar yield of PFBS was in samples oxidized free from methanol and matrix using the standard dose of oxidant and base, n=3 (Houtz and Sedlak, 2012: 60 mM K2S2O8, 150 mM NaOH). The highest yield of PFBS was observed when the oxidation was taken placed in the presence of methanolic reference matrix (n=2) using the same standard dose of oxidant. The pH was measured as 8 after oxidation, suggesting that the dose of NaOH was not enough to maintain a pH >12 during the reaction. Addition of a second standard dose of both oxidant and base after 2h of oxidation (in the presence of methanolic reference matrix), the PFBS yield was 1,4% (n=2). This dose showed to be sufficient to maintain a pH ≥13 during the reaction. One explanation for the formation of PFSAs in the TOP assay could be that in samples with large amount of radical scavengers (i.e. samples of high organic content or/in methanol) the hydroxyl radicals first reacts with methanol, leaving the FASAs to undergo hydrolysis in the high pH environment, which in turn converts the precursor to equivalent chain length PFSA (Figure 9).
Figure 9. A suggested explanation to the observed formation of PFBS/PFOS after TOP assay on FBSA/FOSA in the presence of methanol. Note that this displays a minor degradation pathway; the majority of FBSA/FOSA were oxidized to PFBA/PFOA even in presence of methanol (see table 2).
Table 2. Description of the different treatments. The concentrations of precursor in the pretreatment (1) was 625 ng/L and in (2) and (2) 250 ng/L for each precursor. Note that pretreatments (2) and (3) were not evaporated to dryness but oxidized in the presence of methanol. Three ways of measuring the starting concentration was tested; A) measuring the concentration directly in the sample by subsampling after heating up to 60°C for 30 min and B) duplicate bottles that was spiked in parallel but heated with 50:50 MeOH:Water (Houtz and Sedlak, 2012), B) showed most reliable results.
Sample description and
treatment PFOA/ FOSA PFOS/ FOSA PFBA/ FBSA PFBS/ FBSA
(1) Standard solution containing either FOSA or FBSA without matrix spiked in reaction vessel and evaporated to dryness.
149,2% ± 10,9% 1,4% ± 0,3% 85,0% ± 2,3% 0,5% ± 0,07%
Standard dose1 of oxidant and
base. n=3
(2) 50 µL methanolic extract from reference sample2 spiked with
standard solution containing the two FASAs.
Standard dose of oxidant and base. n=3 80,4% ± 2,3% 3,9% ± 0,4% 96,5% ± 2,0% 4,7% ± 0,4%
(3) 50 µL methanolic extract from reference sample spiked with standard solution containing the two FASAs.
Initial standard dose of oxidant and base. Second standard dose amended after 2h2. n=2 78,2% ± 2,7% 1,3% ± 0,2% 97,4% ± 5,7% 1,4% ± 0,3%
1Standard dose of oxidant and base denotes the concentration used by Houtz and Sedlak, 2012 (60
mM K2O8S2, 150 mM NaOH).
2Reference matrix was analyzed in duplicates separately in the TOP assay; the amount of PFASs in
the reference matrix was subtracted from the spiked samples.
3Second dose amended first after cooling the sample down to around 40°C to minimize loss of
precursors trapped in the gaseous phase of the reaction vessel.
In a report by Ventia Ltd. in conjunction with others (Ventia, 2019) it was suggested that at very high alkaline environment, hydrolysis of some precursors to PFSA may occur. In this work, for FBSA -> PFBS, the molar yield was higher for the samples in higher concentration of NaOH (0.5% for 150 mM versus 1.4% for 300 mM) but for FOSA ->PFOS it was not (1.4% for 150 mM and 1.3% for 300 mM). However, the samples oxidized in presence of radical scavengers (methanolic matrix) and standard dose of oxidant and base showed the highest molar yield of both FBSA -> PFBS (4.7%) and FOSA -> PFOS (3.9%). This result indicates that the presence scavengers (in high pH) may be a more important factor than only very high pH.
4.5. Stability of novel compounds in the TOP assay
4.5.1 Fate of Scotchgard™ fabric stain repellants under oxidative and high pH conditions.
To evaluate the results from the TOP assay it is important to determine whether a precursor is fully oxidized in the assay. This can be done by measuring the precursor after oxidation to verify complete oxidation. This was not done here as the side-chain fluorinated co-polymers in the Scotchgard™ Pre and Post 2002 formula are not retained by the WAX sorbent material used for extraction and could therefore not be extracted. Instead, the degree of oxidation under the conditions in the TOP assay was evaluated by varying the degree of oxidation in three different treatments: 1) standard dose of oxidant and base with 6h reaction time (60 mM K2S2O8, 150 mM NaOH), 2) 2x standard dose, 6h reaction time and 3) 2x standard dose with 20h reaction time. To minimize potential sorption of the co-polymers to the reaction container during the reaction, the bottles were sonicated at 0h, 2h and 5h. Furthermore, preliminary experiments indicated that the test chemicals may undergo hydrolysis in high pH which could therefore impact the products produced in the TOP assay (i.e., producing PFSA instead of PFCA as terminal products). To test this hypothesis, duplicate samples were therefore included that was subjected to 150 mM NaOH, but no oxidant added. To evaluate any conversions or transformations of the products due to heating, heated control samples were used. The summarized results can be found below in figure 10. Detailed results can be found in table 3.
Figure 10. Overview of the results from hydrolysis and oxidation experiments on Scotchgard™ Pre and Post 2002 formulas. Detailed results can be found in table 3. Spike amount of product tech mix was 1 µg (Concentration in reaction 67 µg/L). The bars display the average recovered mass after oxidation. The hydrolysis was conducted in duplicate. The oxidation was done with three different oxidative treatments with duplicate samples in each treatment (total number of oxidation replicates=6).
Table 3. Recovered mass from experiments on Scotchgard™ Pre 2002 and Post 2002 technical mix formulations. All treatments were heated in an 85ºC temperature-controlled water bath.
Samples and reaction time (1) Heated control n=1 20h reaction time (2) Hydrolysis n=2 20h reaction time (3) Oxidation n=2 6h reaction time (4) Oxidation n=2 6h reaction time (5) Oxidation n=2 20h reaction time
Treatment MilliQ 150 mM NaOH 1x std dose (60 mM K2S2O8, 150 mM NaOH)
START: 1x std dose (60 mM K2S2O8, 150 mM NaOH) After 2h: One additional std dose START: 1x std dose (60 mM K2S2O8, 150 mM NaOH) After 2h: One additional std dose 1000 ng Scotchgard™ Pre 2002
PFBA <LOQ <LOQ <LOQ 49 pg ± 14 pg <LOQ PFPeA <LOQ <LOQ 49 pg ± 1 pg 52 pg ± 2 pg 51 pg ± 7 pg PFHxA <LOQ 29 pg ± 9 pg 155 pg ± 11 pg 137 pg ± 3 pg 166 pg ± 2 pg PFHpA <LOQ <LOQ 95 pg ± 4 pg 93 pg ± 5 pg 111 pg ± 24 pg PFOA <LOQ 24 pg ± 3 pg 1010 pg ± 108 pg 786 pg ± 12 pg 880 pg ± 1 pg C9-C10 PFCA <LOQ <LOQ <LOQ <LOQ <LOQ
C4 -C7 PFSA <LOQ <LOQ <LOQ <LOQ <LOQ
PFOS <LOQ 17 pg ± 4 pg 17 pg ± 7 pg 34 pg ± 16 pg 33 pg ± 6 pg C9-C10 PFSA <LOQ <LOQ <LOQ <LOQ <LOQ
FOSA <LOQ <LOQ <LOQ <LOQ <LOQ
FBSA <LOQ <LOQ <LOQ <LOQ <LOQ
1000 ng Scotchgard™ Post 2002
PFBA <LOQ <LOQ 22 363 pg ± 111 pg 20 701 pg ± 1308 pg 22 066 pg ± 323 pg C5-C10 PFCA <LOQ <LOQ <LOQ <LOQ <LOQ
PFBS <LOQ 1631 pg ± 2 pg 49 pg ± 7 pg 57 pg ± 7 pg 57 pg ± 8 pg C5-C10 PFSA <LOQ <LOQ <LOQ <LOQ <LOQ
FOSA <LOQ <LOQ <LOQ <LOQ <LOQ
In the control samples, no PFASs were found above the limit of quantification (LOQ). Pre 2002 that underwent hydrolysis gave rise to very low levels of PFOA, PFHxA and PFOS. Overall, no observable difference in mass recovered after oxidation was observed between the different oxidative treatment groups (treatment 3-5, see table 3). For Pre 2002, the most prominent compound detected after oxidation was PFOA, while C4-C7 PFCAs and PFOS was found in lower amounts. Further, no PFASs with chain-length over C8 was found in any of the treatment. For the Post 2002, hydrolysis of the sample led to a significant level of PFBS after oxidation, but all other PFASs analyzed were below LOQ. Oxidative treatment of the Post 2002, gave rise to high levels of PFBA and minor levels of PFBS. Similar to the Pre 2002 samples, no observable difference after oxidation in PFASs mass were noted between the different treatments (3-5, table 3). For both the Pre 2002 and Post 2002, this indicates that the side-chain fluorinated polymers were able to be fully oxidized in the conditions tested here, as no increase in mass after oxidation was noted with increased dose of oxidant and base or increased time of oxidation.
It is possible that the Pre 2002 formula has branched isomers in its fluorinated side chains, as peaks with the same m/z eluted just before linear PFOA which may indicate that branched isomers of PFOA are present in the sample after oxidation. However, branched isomers of PFOA were not quantified in the investigation, and the values displayed in table 3 and figure 10 only includes linear PFOA. This observation is theoretically possible as it is reasonable to assume that the starting material of the Scotchgard™ Pre 2002 is of POSF/ECF source based on the current understanding of the chemistry of the fluorinated side-chains (Trier et al., 2017, Chu and Letcher, 2014, Fredriksson, 2020) and when ECF is known to produce branched isomers (Wang et al, 2014).
Interestingly, C4-C7 PFCAs was also detected after oxidation of Pre 2002. Two explanations for this are suggested: 1) the product itself contains a mix of fluorinated side-chains of different chain length, or 2) that the oxidation itself produces shorter chains than C8 due to “unzipping” of the alkyl chain during the reaction. This phenomenon has been suggested to occur during oxidation of fluorinated sulfonamides by Martin et al. (2019) where they noted that oxidation of MeFOSA, EtFOSA and FOSA produced up to 2 mol% of PFHpA. The second explanation is believed to be more probable as previous studies have not detected any shorter chain products than C8 in the Post 2002 formulation (Chu and Letcher, 2014).
As previously discussed, the chemical structure and thereby molar mass of the major component in the two Scotchgard™ Pre and Post 2002 formulations are proprietary, therefore no molar comparison before and after TOP assay could be done. Instead, a comparison before and after reaction can be done on a fluorine mass balance approach. In another study (Fredriksson et al., 2020), the fluorine mass fraction for the Pre 2002 technical mixture was measured as 0,6% ± 0,1% of the total mass, and for the Post 2002 formula the fluorine mass fraction was 2% ± 0,1%.
By comparing the fluorine mass fraction from Fredriksson et al., (2020) to this study, the fluorine mass balance before and after TOP assay can be evaluated. The fluorine after oxidation was measured by calculating the ΣPFAS fluorine mass after reaction (for details on how this calculation is done, see section 2.6). The mass of fluorine in Pre 2002 that was accounted for after oxidation by the ΣPFAS, was 1% ± 0,1% for hydrolysis and 14% ± 0,3% for oxidation. The
Post 2002 accounted for after reaction was higher for both the hydrolysis reaction and the oxidation and was 5% ± 0,2% for the hydrolysis and 68% ± 4% for the oxidized sample. Table 4. Summary from mass balance analysis of Scotchgard™ Pre 2002 and Post 2002 in hydrolysis and oxidative treatment. The results show the amount of fluorine that is explain by products detected post hydrolysis and oxidative treatment, compared to the total fluorine in the products. Assembled with CIC data comparison from Fredriksson, 2020.
% of total fluorine explained by PFASs produced after
Hydrolysis
% of total fluorine explained by PFASs produced after
Oxidation
Scotchgard™ Pre 2002 1% ± 0,1% 14% ± 0,3%
Scotchgard™ Post 2002 5% ± 0,2% 68% ± 4%
The results suggest that the Scotchgard™ Pre 2002 tech mix still contains a significant amount of fluorine still unaccounted for after hydrolysis and oxidative treatment of the product (only 1% accounted for from the hydrolysis and 14% for oxidative treatment), while in the Post 2002 tech mix, the proportion of fluorine that can be accounted for after hydrolysis and oxidation is higher (5% for hydrolysis and 68% for oxidation). This result may suggest that Post 2002 may be easier to degrade than the Pre 2002 formulation. The summarized results from the mass balance analysis can be found in table 4.
Furthermore, the results show that it is possible to capture side-chain fluorinated polymers that may degrade to PFSA/PFCA in the environment, in the TOP assay. Previous works by Chu and Letcher 2020 and Fredriksson 2020 have shown significant concentration of both the Scotchgard™ Pre 2002 and Post 2002 in biosolids and sludge samples in Canada and Sweden. In some cases, the levels were higher than PFCAs and PFSAs. This shows that side-chain fluorinated co-polymers may be a large contributor of PFCAs in oxidation experiments of sludge samples. However, it should be mentioned that current target PFAS analysis on sludge (Yeung et al., 2017) and soil (Janda et al., 2019) commonly uses methanol or basic methanol as the extraction solvent which may not optimized for extraction of side-chain fluorinated co-polymers. Chu and Letcher (2017) and Fredriksson et al., (2020) have instead reported using a 50:50 mixture of acetone and hexane. This dissimilarity of extraction solvents may lead to an underestimation of the level of potential PFAA precursor present, if the sample contains a significant proportion of side-chain fluorinated polymers. This should be considered in future studies of precursor oxidation from solid samples (i.e. soil, biosolids or sludge).
4.5.2 Fate of fluoroalkyl ether acids under oxidative conditions
In this study, a range from 5 mM persulfate and 670 ng/L PFEA concentration (simulating a small PFEA to persulfate ratio) to 100 mM persulfate and 130 ng/L PFEA concentration (simulating a large PFEA to persulfate ratio) was used. In addition, acidic persulfate oxidation was also tested to evaluate whether the PFEAs was stable in persulfate radical oxidation. The pH was not maintained >12 in 100 mM persulfate using 150 mM sodium hydroxide, therefore the concentration of sodium hydroxide was increased to 250 mM. The summaries result can be found in table 5.
Table 5. Summary of the percent recovered PFEA concentration after oxidation for the different concentrations. The ratio is calculated as: [PFEA] (after oxidation)/[PFEA]before oxidation. 100% means no change in concentration after oxidation. 250 mM NaOH was used in (3) as 150 mM NaOH was shown to be inadequate to maintain a pH>12 using 100 mM K2S2O8.
