REPORT NUMBER: FD 05/23. Date: 28 Feb 2006 PFOS and PFOA in Total Diet Study samples

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1 PERFLUOROCTANSULPHONIC ACID (PFOS), PERLUOROOCTANOIC ACID (PFOA) AND RELATED COMPOUNDS IN FOOD, DEVELOPMENT AND VALIDATION OF A METHOD AND ANALYSIS OF TOTAL DIET STUDY SAMPLES REPORT NUMBER: FD 5/23 Author: D B Clarke Date: 28 Feb 26 Project Title: PFOS and PFOA in Total Diet Study samples Sponsor: Dr Martin Gem Food Standards Agency Room 77C Aviation House 125 Kingsway London WC2B 6NH Sponsor's Project Number: C257 CSL Contract Number: M6GZ CSL File Reference: FLN 8282 Principal Workers: D Clarke, V Bailey Project Manager: M Rose Distribution: 1-5. Dr M Gem (FSA) (+ electronic copy) 6. Prof J Gilbert 7. Dr J Dennis 8. Dr M Rose 9. Dr D Clarke 1. S White 11. V Bailey 12. CSL Library Central Science Laboratory Sand Hutton York YO41 1LZ Telephone: Fax:

2 SUMMARY 1. An analytical method was developed and validated for the detection of PFOS and related fluorochemicals at low levels in food and beverages. The method was further validated by participation in the collaborative trial - 1 st worldwide interlaboratory study on perfluorinated compounds in human and environmental matrices. 2. This method was then applied to the analysis of the 24 Total Diet Study samples. The individual food samples had been composited into single pools for each of the 2 designated food groups. 3. PFOS type chemicals were provisionally identified at low levels in TDS group 12 potatoes, TDS group 13 other vegetables and TDS group 14 canned vegetables. 4. Higher concentrations of PFOS chemicals can be observed in other individual samples, such as fish, that are not composited. 2

3 1. List of Contents perfluoroctansulphonic acid (PFOS), perluorooctanoic acid (PFOA) and related compounds in food, development and validation of a method and analysis of total diet study samples...1 Introduction...5 Initial instrument setup...6 Chromatography...7 Detection...8 Methods...8 Samples...8 Chemicals...9 Plasma : TBAS ion pairing-mtbe...9 Serum : ammonium acetate buffer...9 Milk : formic acid...9 Fish liver : TBAS sodium carbonate...1 Taniyasu sodium hydroxide digestion....1 Contaation control...11 Analytical quality control...13 Results and discussion...14 Method validation...14 Collaborative trial participation...15 PFOS in TDS samples...16 Conclusions...17 References...18 Tables...2 Table 1. Names, masses, abbreviations, purity and suppliers of standards..2 Table 2. Summary of LC-MS/MS detection parameters Table 3. Comparison of recovery of low molecular weight analytes through anion exchange and C8 SPE phases...22 Table 4. Individual TDS food groups Table 5. Concentrations of PFOS and related observed fluorochemicals in TDS food groups μg/kg (ppb) Table 6. Concentrations of PFOS and related observed fluorochemicals in other environmental samples μg/kg (ppb)

4 Figures...26 Figure 1. Chromatography of PFOS analytes in a 1 μg/l aqueous solution standard...26 Figure 2. Detector linearity and variations in suppression with matrix...27 Figure 3. Comparison of linearity of recovery through WAX-SPE cartridges over 1-5 μg/l range Figure 4. Chromatograms of the C 4 F 9 SO 3 channel in TDS Group-12, potatoes Figure 5. Chromatograms of the C 6 F 13 SO 3 and C 8 F 17 SO 3 channels in TDS Group-12, potatoes...3 Figure 6. Chromatograms of the C 4 F 9 SO 3 - channel in TDS Group-13, other vegetables...31 Figure 7. Chromatograms of the C8F17SO3- channel (PFOS) in TDS Group-14 canned vegetables...32 Figure 8. Chromatograms of the C 4 F 9 SO 3 - channel (C4 sulphonic acid) in TDS Group-8 oils and fats

5 INTRODUCTION 1.1 PFOS is now the generally accepted term referring to the individual chemical PerfluoroOctaneSulphonic acid and any closely related derivatives that contain the PFOS moiety (C 8 F 17 SO 2 ), including those that may degrade to release it in the environment (Brooke 24). This term is often used interchangeably with other similar acronyms: PerFluoroOrganic Substances (PFOS), PerFluoro Compounds (PFC s), or PerFluoroAlkylated Substances (PFAS). The chemicals under consideration are best described as a homologous series of fullyfluorinated carboxylic acids, which can also be present as the sulphonic acids or as sulphonamide derivatives. Various larger compounds from which these contaants are derived also need consideration. The actual list of chemicals that require monitoring as contaants, or as source pollutants is therefore in the order of 2 individual analytes. Measuring every possible PFC is unrealistic at this point. Many of the source chemicals are unstable and degrade to identical contaants, which are then stable. The list of priority analytes was restricted to the simple carboxylic acids, the sulphonic acids and the amides. As in this work we are also severely restricted by the availability of standards this lead to an analyte list of 17 individual PFOS chemicals. 1.2 PFCs are, or have been used extensively in the industrial polymers (Teflon, SilverStone ), waterproofing agents (Gore-Tex ), stain repellents (ScotchGard, Stainmaster ), fire-fighting foams (AFFF) and in greaseproof food packaging materials (Zonyl ). 1.3 The monomers and low molecular weight products are liquid surfactants, while the higher molecular weight oligomers, polymers and esters are used for stationary surface treatments. 1.4 An analytical method was developed to screen UK TDS food samples for the presence of PFOS and related perfluoroalkyl contaants, with a target LOD of 1-5 μg/l of each analyte. 1.5 The UK Total Diet Study (TDS) is a cost effective and reliable means to provide information on dietary exposure of the general population and different sub-groups to nutrients and contaants. Analysis of yearly composites of each of 2 food groups ensures a diverse base of individual 5

6 samples that adequately represents average national dietary intake.experimental Initial instrument setup 2.1 Before embarking on the extraction of food samples it was necessary to develop a robust and sensitive separation and detection method. Because of the ubiquitous nature of PFOS in the environment, specifically the likely presence of PFOS in many items of common laboratory equipment, the final method needed to have a imal number of manual manipulation steps and be based on an instrument giving good sensitivity on extracts with little cleanup. LC-MS/MS is now the application of choice for such demanding analyses. 2.2 A number of chromatographic systems are reported in the literature, a number of these were investigated. While all could be reproduced in our laboratory, it became apparent that there were problems in extension of literature methods to include analytes with both smaller and larger alkyl groups. Most literature methods restrict analyses to a few key analytes. The standard C18 phases retain the larger e.g. C18 acids and these are not adequately eluted with 1 organic mobile phase. Where they do elute at 1 organic, peak broadening limits the usefulness of these chromatographic peaks for quantitation. Swapping to specific C8 phases allowed these larger acids to be eluted earlier in an aqueous to organic gradient with no degradation of peak shape. The SunFire C8 phase behaved more like a C18 phase and was discarded, the Thermo C8 and the new Waters BEH phase have similar properties and were both used. The BEH column had impressive between sample retention reproducibility and was therefore the favoured analysis column. 2.3 The next problem to address was early elution of the smaller acids. Literature methods focussed on the eight-carbon analytes can use isocratic elution, but a gradient elution pumping method is required to retain the C5 acid while simultaneously allowing elution of the strongly absorbed C18 acid. We selected a 1 acetonitrile aqueous starting condition, changing linearly to 1 acetonitrile. The retention times of the smallest acids C3 and C4 were variable with matrix and were excluded from the method. Retention times of the remaining analytes gave excellent reproducibility under all matrix conditions. 6

7 2.4 As expected, we encountered background interference problems within the instrument. The largest contributions to the PFOS background appeared to be introduced through the pumping system. After either long flushing periods at high flow rates, or after extended periods of use for chromatography, these analytes were gradually removed from the system. Dedicated solvent reservoirs are required for this application. Similarly carryover between injections was found to require a x-4 needle wash setting to remove analyte between injections. It was found that running the needle wash option with the water: methanol (1:1) mixture used as the pump seal wash solvent did not adequately clean the needle between injections and that putting in a further dedicated solvent line delivering 1 acetonitrile around the outside of the injection needle resolved the carryover issues. 2.5 It is critical to pump clean solvents, especially the aqueous phase, to prevent an on-line concentration effect. This results in analytes concentrating on the front end of the analytical column during equilibration, which are released when the organic content is increased during the elution method cycle. 2.6 A robustness test while experiencing system contaation showed that analyte ionisation was not adversely affected by simplifying the buffer composition. Ammonium acetate buffer at 1-2 mm adjusted to ph 4. is generally used in literature methods and we started from this point. In order to eliate a possible contaation source we purchased preprepared LC-MS grade ammonium acetate at.1 in water as the aqueous buffer. This was used directly from the supplied 2.5l Winchester bottle without decanting, as was the acetonitrile. 2.7 Using these four solvent delivery channels for cleaning and elution gave a stable instrumental platform with nondetectable background levels of all PFOS analytes across all mass channels, which when in place could be refilled and maintained indefinitely (until dissembled for other projects). Chromatography 2.8 A chromatographic method was readily developed which retains and separates all of the perfluorinated carboxylic acids from C 5 -C 18 and the other related analytes. This method gives partial separation of the i- branched and n-straight chain isomers of e.g. PFOS and PFOSA. The chosen HPLC column was Waters Bridged Ethylene Hybrid (BEH) C8 (2.1 x 15 mm) held at 3 o C. The injection volume was generally 25 ul. A 7

8 gradient programme was used starting from 1 acetonitrile with 9 aqueous ammonium acetate buffer (.1), and changing to 1 acetonitrile at 15, with a 5 hold at 1 acetonitrile. The programme returns to initial conditions at 21 for a 25 cycle time. The injector was programmed to provide an exterior wash of acetonitrile around the needle on an extended solvent wash (x-4). Detection 2.9 The detector was a Micromass Ultima triple quadrupole mass spectrometer equipped with an electrospray source operating in negative ion mode. Data were acquired by tandem mass spectrometry using a multiple reaction monitoring (MRM) method that monitored parent to daughter ion transitions for each analyte. The desolvation temperature was 3 o C and the source block temperature was 1 o C. The desolvation gas flow was 7 L h -1 and the capillary voltage was 2.8 Kv. 2.1 The names, abbreviations, formulae, masses, suppliers and stated purities of the PFOS chemicals are summarised in Table 1. Detection parameters for each individual analyte were detered and are summarised in Table 2. A representative chromatogram of an aqueous solvent standard at 1 ppb showing the individual analytes and their separation is show as Figure 1. METHODS Samples 3.1 Retail food samples are collected every fortnight from a different location in the UK, prepared for consumption and then combined into 2 food groups. In the current survey these were pooled further to give yearly composites for each food group. The primary objective of this project was to analyse Total Diet Study samples. A set of pre-homogenised and frozen samples representing the 24 diet set were provided through the FSA. These were subsampled and extracted without further treatment such as freeze-drying or homogenisation. 8

9 3.2 All other food samples and reference materials referred to in this report were obtained locally by the authors and were used principally for method validation. Chemicals 3.3 A summary of the chemicals used as analytical standards, their common names, abbreviations, molecular weights, suppliers and stated purities are given as Table Any other solvents and chemicals are reagent grade or specific LC-MS grade. Literature extraction methods 3.4 At the time this project was initiated, literature searching suggested the four following methods represented the state-of-the art for PFOS extraction. Plasma : TBAS ion pairing-mtbe 3.5 Plasma (1 ml) mixed with tetrabutylammonium hydrogen sulphate (.5 M, 1 ml) and sodium carbonate buffer (.25 M, ph1, 2 ml). This was extracted into MBTE (5 ml), shaken for 2 and centrifuged. Repeat 3 times. Methanol (.5 ml), was added and the sample evaporated and filtered (Guruge 24). Serum : ammonium acetate buffer 3.6 Serum (.1 ml) and ammonium acetate buffer (4 μl, 5 mm in water) were vortex mixed (3 sec) in a polypropylene tube. The internal standard THFOS (5 μl, 2 ppb) was added and the sample was vortex mixed again. MTBE (3 ml) was added with further vortex mixing. The mixture was then placed on an agitater/roller for 1. The mixture was centrifuged and the MBTE layer removed, evaporated to dryness and reconstituted in Mobile Phase, 3:7 water/meoh each with 2 mm ammonium acetate. The extract was vortex mixed and centrifuge at 3 rpm for 2 before analysis (Olsen 23). Milk : formic acid 3.7 Formic acid (.1 M, 3 ml), milk (1 ml) and IS were combined, vortex mixed and sonicated for 2. The mixture was cleaned up by Oasis HLB SPE (6 mg, 3 ml). Cartridges were conditioned with MeOH/Formic acid.1 and the entire aqueous extract was loaded. Columns were washed with.1 formic, 1:1 formic acid/methanol, followed by 1 Ammonium hydroxide. 9

10 Columns were vent dryied, and the analytes were then eluted with basic acetonitrile (NH 4 OH 1, 1 ml) (Kuklenyik 24). Fish liver : TBAS sodium carbonate 3.8 Fish liver (.5 g) Na 2 CO 3 (.5 M, 3 ml), water (1 ml), and ion pairing reagent TBAS (.5 M, ph 1, 1 ml), were combined with IS (1 μl). Samples were extracted into MTBE (5 ml), with shaking (1 ) then centrifuged. Repeat twice, then dry extracts, take up the residue in water/meoh 1:1 and filter (nylon.2 μm) before analysis (Martin 24). 3.9 These methods or adaptations of them where investigated with limited success. At this stage it was difficult to produce a batch with consistently low background levels of PFOS in all samples. Spot contaation was the most serious problem where a number of extracts in a given batch would have high levels of contaation. Taniyasu sodium hydroxide digestion. 3.1 A new extraction and cleanup strategy was presented by Taniyasu at the Fluorous meeting in Toronto, Canada (August 18-2, 25). This relied on a gentle sodium hydroxide digestion to release PFOS and solubilise it, before an anion exchange treatment of the basic aqueous extract. This method appeared to have considerable advantage over the previous strategies. As the total extract can be loaded straight onto the anion exchange SPE and the analytes elute in a small volume of methanolic extract, this method has fewer manual manipulations and more importantly does not require the evaporation of large volumes of organic solvent prior to the SPE step. We believed this drying step was where the majority of our contaation was occurring Our interpretation of the Taniyasu method is as follows: A portion of food (5 g) is placed in a polypropylene tube with potassium hydroxide (4 ml,.1 M) and the tubes were rotated on a mechanical roller overnight (16 h). Oasis WAX SPE cartridges (15 mg/6 cc/3 μm) were conditioned at atmospheric pressure with; ammonia MeOH (.1, 4 ml), methanol (4 ml), aqueous formic acid (.1 M, 4 ml), and lastly, aqueous KOH (.1 M, 4 ml). The digests are centrifuged (4 rpm, 5 ) and the entire volume of supernatant were loaded onto the conditioned cartridges with increasing vacuum as required to maintain flow. Cartridges were washed with ammonium acetate (25 mmol, 4 ph 4.5 [acetic acid]). All solvent was 1

11 expelled from the cartridge under vacuum before the analytes were eluted with ammonia in MeOH (.1, 3 ml) This differed from the original method in that there was no need to elute the neutral compounds (amides e.g. PFOSA) with methanol as a separate fraction before eluting the acidic compounds with basic methanol. We found this step worked as described, but created a separate fraction without an internal standard. We preferred to keep all the analytes in one fraction In order to achieve low limits of detection it was necessary to conduct a complex analysis schedule. From this point glassware in contact with extracts for extended periods was silyanised to imise surface binding losses. The eluent from the anion exchange SPE was analysed directly (3 μl, 1 extract) for the low weight carboxylic acids. The smallest acids would not survive concentration, due to volatility. The remaining extracts (2.7 ml) were allowed to reduce to 2 μl by positioning of vials under the airflow through the front of a closed fume hood. This concentrated extract was then injected three times, once to quantify acids (2 acquisition windows), once for sulphonic acids and once for amides (PFOSA), quantifying against tetra hydro-pfos (TH-PFOS) in each instance. Due to the differences in the number of mass transitions being measured simultaneously, this gave comparable sensitivity in all classes of analytes and overcomes the order or magnitude difference in ionisation between e.g. PFOSA and PFOA. The actual ions measured are summarised in Table 2 and also show in Figure The identical method was used on all TDS food groups without deviation. Contaation control 3.15 We encountered a number of contaation issues in the early phase of the project. This was predictable, as PFOS can in theory be present in fluorinated plastics, cleaning products and dust. Contaation readily transfers by deposition and surface-to-surface contact As we had multiple point source contaations occurring simultaneously, a system was devised to establish each source, while still operating a contaated detection system. When the LC-MS injector is programmed to deliver zero sample and the pumps and detector acquire a chromatogram, this is a system blank and establishes the level of contaation due to the instrument itself, primarily from the solvent delivery channels. Contaation can be present in solvent reservoirs as the fully solubilised 11

12 form, or can be due to historic contaation leaching slowing out of the degasser membrane and from other surfaces. When an injection of 25 μl of sample is injected, any increase in PFOS peaks above that seen in the system blank is contributed to the contents of the sample vial. A reagent blank is a water sample taken through the cleanup systems and introduced as a QC of the combined extraction/spe steps. Lastly, chemical blanks are where an individual solvent or reagent is suspected of contributing peaks in a system or reagent blank. A more concentrated stock is prepared in order to seek a concurrent increase in the contaant peak size. Even when the system blank is running with serious contaation it is possible to track the source to a specific point e.g. a batch of contaated water and then eliate the contaation from e.g. the chromatographic solvent. All of these contaation problems needed to be addressed simultaneously (qualitatively) to deliver a consistently clean system blank. Clear system and reagent blanks were necessary, before any trace level quantitation was possible Contaation of reagents was frequent at first, especially in those reagents prepared as aqueous solutions, this was controlled by preparing bulk quantities as concentrated stocks and reusing the same bottles/flasks for preparing the next batch of the identical working strength reagent without cycling glassware through standard laboratory cleaning procedures. By analysing each new preparation of reagent by LC-MS for contaation this systematic approach allowed a clean set of reagents was prepared and used with confidence This project was moved to a separate laboratory with dedicated space. The preparation and storage of stock solutions, dilution of these to working solutions of mixed standards and the overspiking and low-level food extraction processes were conducted on three different laboratory benches. All glassware was scrupulously cleaned, large items and volumetric flasks by repeatedly washing with methanol, all smaller items including: beakers, vials, Pasteur pipettes were cleaned by muffling, i.e. wrapping in aluium foil and baking off all organic matter by heating to 5 o C for 16 hr. Clean glassware was kept stoppered or wrapped in foil until required. All contact with samples was with glass Pasteur pipettes in place of spatulas e.g. for weighing out chemicals and food. All vial tops, plastic disposable pipette tips, single use polypropylene tubes (5 ml) were kept sealed at all times when not in use. Chemical bottles and Winchesters of solvent were reserved 12

13 for this project only and were only sampled by direct decanting or intermediate transfer from muffled glassware. Analytical quality control 3.19 Stock standard solutions were prepared a 1mg/ml in acetonitrile and stored at ambient temperature. Dilutions of these were prepared weekly as diluted solutions were unstable due to binding losses. 3.2 A working solution of mixed standards prepared at.5 μg/ml in water was used for food spiking. Foods (5 g) were overspiked at 1 and 5 μg/kg (ppb) by addition of 1 & 5 μg/ml. Tetrahydro-PFOS (TH-PFOS) was used as an internal standard by addition of 5 5 μg/ml giving a 5 μg/kg addition level. This analyte has a relatively poor ionisation and was used at a higher concentration Each sample was analysed in duplicate through the entire extraction method to ensure that advantageous point contaation was not mistaken for the presence of native analyte. Each sample was also overspiked at two concentrations (1 & 5 ppb) bracketing the expected worst case reporting limits. In order to prove the absence of a given analyte, the internal standard must be present in all extracts, the blank extract must be blank and the overspiked extracts must show a peak for the analyte at the required retention time. As the different foods behave differently with respect to extraction into potassium hydroxide (e.g. eggs are completely soluble), recovery of analytes through the SPE stages and matrix induced suppression of ionisation, a single LOD for all foods or all analytes was not possible, even through the same extraction method was being used. An inspection of each chromatogram was undertaken to estimate an LOD for each individual analyte, the LOD s differ considerably for analytes within a single food and between food groups. Method validation 3.22 Linearity of instrument response was excellent for all analytes. Response was compared between solvent based standards in both water and methanol, the water based standards after passage through SPE and a matrix matched set, prepared by passing extract of a soya/wheat flour (1:9 mix) through SPE (anion exchange) and the addition of the analyte to the eluent. Absolute response and the ratio of response of e.g. the C8 acid and TH-PFOS varied between the different calibration sets as reflected by the different y-axis 13

14 ranges, but all analytes in all calibration sets responded in a near linear manner, allowing line fitting Figure Before analysing TDS samples the various extraction and cleanup sets were assessed for robustness. The weak anion exchange SPE step was assessed against the equivalent C8 based SPE product. The data were assessed by ratioing to the TH-PFOS internal standard. The quantitation therefore relies on the internal standard behaving in an identical manner to the other classes of analytes. Where sorption and elution differ between sulphonic acids and carboxylic acids recoveries will differ widely from 1. A graphical representation of analytical recovery is given as Figure 3. Note that the Y- axis is in an arbitrary unit related to the instrument response factors for each analyte. Plotting these lines as percentages recovered would have resulted in overlapping the majority of the curves. These data are also tabulated as percentage recoveries for the smaller analytes, which were known to exhibit poor recovery on C8 and C18 SPE as Table 3. RESULTS AND DISCUSSION Method validation 4.1 The simplest extraction cleanup and concentration strategy, that based on potassium hydroxide digestion and anion exchange SPE gave the best recovery and more reproducible quantitation. This was particularly important for the lower molecular weight analytes, those with smaller alkyl chains such as pentanoic acid and butanesulphonic acid, are not adequately retained by a reverse phase C8 based binding interaction. The anion exchange based mechanism retains all acids groups irrespective of the alkyl chain length (Table 3). 4.2 As PFOS has a high solubility in water; 519 mg/l and a moderate solubility of 12 mg/l in seawater, a wholly aqueous based extraction was investigated, without use of an organic solvent. PFOS is believed to exist in the environment in the ionised form, as salts, which are readily water-soluble. This solubility is further enhanced by use of a basic digestion system ensuring complete conversion to the potassium salts at the extraction interface. Food is overspiked with analyte in water directly onto the surface 14

15 of the food prior to extraction. The analytes are quantitatively extracted into the potassium hydroxide extractant. 4.3 We had encountered a significant problem with the ion pairing MTBE organic extract approach co-extracting large volumes of fat along with PFOS, which subsequently blocked C8 based SPE cartridges. Adopting the anion exchange step and loading aqueous extractant directly onto the cartridge also overcame the fat extraction problem. The only remaining issue was blockage of columns with particulates, even after centrifugation there was often difficulty in passing the entire extract through a single SPE cartridge. Those based in the 3 cc barrels have an inadequate surface area, whereas the 6 cc volume at the larger 3 μm pore size allowed all sample types to be cleaned up. Polymer base SPE phases can be conditioned at atmospheric pressure and vacuum applied as the cartridges begin to exhibit reduced flow through partial blockage. These partial blockages are subsequently cleared from the cartridge bed during the ammonium acetate wash step, leaving a clean cartridge for elution. Collaborative trial participation 4.4 As part of our in-house validation we participated in: The 1 st Worldwide interlaboratory study on perfluorinated compounds in environmental and human samples. This was co-ordinated as a workpackage of the PERFORCE EU project by the Netherlands Institute for Fisheries Research (ASG-RIVO). Our data were returned as participant number 28. Our measurements are considered acceptable and were close to assigned values for the priority analytes. Our data were comparable to those submitted by other laboratories including those indicating >3 years previous experience in this area of analysis (van Leeuwen S., Karrman A., Zammit A., van Bavel B., van der Veen I. 1 st Worldwide interlaboratory study on perfluorinated compounds in human and environmental matrices. RIVO Netherland Institute for Fisheries Research, Report nr. CO7/5 October 25.). 4.5 While participation is collaborative trial provides a reassurance of accuracy, there is as yet no formal estimate of measurement uncertainty for this analytical measurement. As deterations were measns of duplicate extarctions with overspiking at the expected concentrations, we consider values at 1-1 ppb have an associated error of 2, or ± 2 ppb. 15

16 PFOS in TDS samples 4.6 A number of samples would appear to contain what are best described as trace levels of PFOS at concentration around the limit of detection. It was not possible to provide confirmation of these observations as by definition the primary ion transition used for the initial screening is the most intense of the possible ion transitions and attempts to confirm the identity of peak by use of other secondary transitions resulted in signal intensity well below the LOD for all but standard solutions. 4.7 Some foods, TDS groups 12, 13, 14 may contain PFOS analytes (Figures 4 8). As potatoes (TDS 12 Figures 4 & 5) would appear to contain three different classes of analytes; carboxylic acids, sulphonic acids and an amide, this should be viewed as a strong probability of being present, while those such as TDS groups 8 & 16 with only a single acid highlighted should be viewed with extreme caution. 4.8 The most significant observation from this screening of TDS foods was the peak cluster around the n-perfluorobutanesulphonic acid peak. This is illustrated in Figures 4 & 6. In TDS group 12 (potatoes) there are three satellite peaks at different retention times to the analytical standard, in TDS group 13 (other vegetables), two of these peaks are also present. As the ratio of these three peaks and the n-alky standard is different between these two samples and these peaks are not present in other foods, these must be considered real peaks that are unlikely to be due to procedures in the laboratories preparing or analysing the foods. These peaks are therefore real and relate to the food sample. 4.9 The next consideration is the nature of these peaks, while they contribute the same mass fragments as the standards, are they structural isomers or are they unrelated isobaric interferences eluting at broadly similar retention times? If they are unrelated to PFOS then only the n-alkyl contributions of 1 and 3.5 ppb should be considered in place of the 25 and 5 total values. Insufficient work has been conducted on technical mixtures to be sure of peak assignments. It is known that the branched isomers elute before the straight chain isomers (Twahig 25), so it is possible that the three peaks observed are due to the three other possible alky isomers: t-butyl-so 3 H, 1-methylpropyl-SO 3 H, and 2-methyl-propyl-SO 3 H, but no data exists to support or refute this theoretical possibility. 16

17 4.1 As a validation exercise we exaed a small number of individual fish samples, these were individual freshwater fresh from different UK locations. All contained PFOS (5-15 μg/kg) and other related analytes. Where we dissected out the internal organs from a number of carp and analysed a single liver composite an even higher concentration of PFOS was observed. This was estimated as 25 μg/kg. These findings are comparable to other studies on fish and in the absence of certified reference materials serve both to validate the extraction method on naturally incurred residues and to support the finding of low levels of PFOS across the TDS sample set. CONCLUSIONS 5.1 An analytical method for measuring PFOS in food has been established. This has indicated that vegetable products may contain PFOS at low levels. 5.2 While PFOS was not detected at high concentrations in any TDS sample, it can be present at much higher levels in environmental samples and in individual foods such as fresh water fish. 17

18 REFERENCES Austin M.E., Kasturi S.B., Barber M., Kannan K., MohanKumar P.S., MohanKumer S.M.J. Neuroendocrine effects of Perfluorooctane sulfonate in rats. Environ. Health Perspec., 23, 111(12), Brooke D., Footitt T.A., Nwaogu T.A. Environmental risk evaluation report: Perfluoroctanesulphonate (PFOS) Draft report prepared for the UK Environment Agency. By Building Research Establishment, Watford, and Risk and Policy Analysts, Loddon, Norfolk Environmental Working Group Assessment of EPA Draft Human Health Risk Assessment for the Teflon Chemical PFOA. Fluoros -Symposium website. Download of presentations and posters. August 18-2, Guruge K.S., Taniyasu S., Yamashita N., Wijeratna S., Mohotti K.M., Seneviratne H.R. Perfluorinated organic compounds in human blood serum and seal plasma : a study of urban and rural tea worker populations in Sri Lanka. J. Environ. Monit., 25, 74(4), Kuklenyik Z., Reich J.A., Tully J.S., Needham L.L., Calafat A.M. Automated solid-phase extraction and measurement of perfluorinated organic acids and amides in human serum and milk. Environ. Sci. & Tech., 24, 38(13), Olsen G.W., Church T.R., Larson E.B., van Belle G., Lundberg J.K., Hansen K.J., Burris J.M., Mandel J.H., Zobel L.R. Serum concentrations of perfluorooctanesulfonate and other fluorochemicals in an elderly population from Seattle, Washington. Chemosphere, 24, 54, Olsen G.W., Church T.R., Miller J.P., Burris J.M., Hansen K.J., Lundberg J.K., Armitage J.B., Herron R.M., Medhdizadehkashi Z., Nobiletti J.B., O'Neill E.M., Mandel J.H., Zobel L.R. Perfluorooctanesulfonate and other fluorochemicals in the serum of American Red Cross adult blood donors. Environ. Health Perspect., 23, 111(16),

19 Martin J.W., Smithwick M.M., Braune B.M., Hoekstra P.F., Muir D.C.G., Mabury S.A. Identification of long-chain perfluorinated acids in biota from the Canadian Arctic. Environ. Sci. & Tech., 24, 38 (2), Risk and Policy Analysts Limited. Proposal for regulation on PFOS-related substances. Partial regulatory impact assessment. 9 Sept 24, Prepared for DEFRA, by Risk and Policy Analyts Limited, Loddon, Norfolk UK. Taniyasu S., Kannan K., Ka So M., Gulkowska A., Sinclair E., Okazawa T., Yamashita N. Analysis of fluorotelomer alcohols, fluorotelomer acids, and short- and long-chain perfluorinated acids in water and biota. J Chrom. A., 25, in press. Twahig M., Ellor N., Worrall K., Jenkins T., Kearney G. Separation of branched PFOS isomers by UPLC with MS/MS detection van Leeuwen S., Karrman A., Zammit A., van Bavel B., van der Veen I. 1 st Worldwide interlaboratory study on perfluorinated compounds in human and environmental matrices. RIVO Netherland Institute for Fisheries Research, Report nr. CO7/5 October 25. Environmental Working Group Assessment of EPA Draft Human Health Risk Assessment for the Teflon Chemical PFOA. 19

20 TABLES Table 1. Names, masses, abbreviations, purity and suppliers of standards. Class Abbrev Formulae Name Isotopic MW CAS number Supplier Purity Amides PFOSA C8F17SO2NH2 Perfluorooctanesulphonamide ABCR 97 Sulphonates PFBSH C4F9SO3H Perfluorobutane sulphonic acid sigma 97 PFHxSH C6F13SO3H Perfluorohexane sulphonic acid PFHxSK C6F13SO3K Perfluorohexane sulphonate potassium salt fluka 98 PFOSH C8HF17SO3H Perfluorooctane sulphonic acid PFOSK C8F17S3K Perfluorooctanesulphonate potassium salt fluka 98 Acids PFPeA C4F9COOH Perfluoropentanoic acid sigma 97 PFHxA C5F11COOH Perfluorohexanoic acid fluka 97 PFHpA C6HF13COOH Perfluoroheptanoic acid sigma 99 PFOA C7HF15COOH Perfluorooctanoic acid sigma 96 PFNA C8HF17COOH Perfluorononanoic acid sigma 97 PFDeA C9HF19COOH Perfluorodecanoic acid sigma 98 PFUnA C1F21COOH Perfluoroundecanoic acid sigma 95 PFDoA C11F23COOH Perfluorododecanoic acid sigma 95 PFTdA C13F27COOH Perfluorotetradecanoic acid sigma 97 PFHdA C15F31COOH Perfluorohexadecanoic acid lancaster 95 PFOdA C17F35COOH Perfluorooctadecanoic acid ABCR 95 TH-acids TH-HxA C3F7C2H4COOH Tetrahydro-perfluorohexanoic acid ABCR Internal std TH-PFOS C8H5F13SO3 Tetrahydro-perfluorooctanesulphonic acid ABCR 2

21 Table 2. Summary of LC-MS/MS detection parameters. Primary Ion Secondary Ion Rt [M-H] - Prod Cone Prod Cone Tetrahydro C6 Acid TH-PFHxA n-c5 Acid PFPeA n-c6 Acid PFHxA C4 Sulphonate PFBS n-c7 Acid PFHpA Tetrahydro PFOS THPFOS n-c8 Acid PFOA C6 Sulphonate PFHxS n- C9 Acid PFNA n-c1 Acid PFDeA n-c8 Sulphonate n-pfos n-c11 Acid PFUnA n-c12 Acid PFDoA n-c8 Amide PFOSA n-c14 Acid PFTdA n-c16 Acid PFHdA n-c18 Acid PFOdA

22 Table 3. Comparison of recovery of low molecular weight analytes through anion exchange and C8 SPE phases. Recovery through KOH-WAX SPE Recovery through C8-SPE Analyte/concentration μg/l C5 Acid C6 Acid C7 Acid C8 Acid C4 Sulphonic acid C6 Sulphonic acid C8 Sulphonic acid C8 Amide

23 Table 4. Individual TDS food groups. Food group Food items sampled 1 Bread 2 Miscellaneous cereals 3 Carcass meat 4 Offal 5 Meat products 6 Poultry 7 Fish 8 Oils and fats 9 Eggs 1 Sugars and preserves 11 Green vegetables 12 Potatoes 13 Other vegetables 14 Canned vegetables 15 Fresh fruit 16 Fruit products 17 Beverages 18 Milk 19 Dairy products 2 Nuts 23

24 Table 5. Concentrations of PFOS and related observed fluorochemicals in TDS food groups μg/kg (ppb). 1 Bread 2 Cereals 3 Meat 4 Offal 5 Meat prod 6 Poultry 7 Fish 8 Oils & fats 9 Eggs 1 Sugars 11 Green veg 12 Potatoes 13 Other veg 14 Canned veg 15 Fresh fruit 16 Fruit 17 Beverages 18 Milk 19 Dairy prod 2 Nuts C8 amide PFOSA <2 <2 <2 <5 <2 <2 <5 <5 <5 <5 <5 5 <1 <2 <2 <1 <.1 <.2 <2 <1 C4 sulphonate <2 <5 <5 <5 <2 <5 <1 <.5 <.5 <1 <1 1* 1* <1 <1 <1 <.5 <.5 <2 <2 C6 sulphonate <5 <1 <1 <1 <2 <5 <2 <1 <1 <1 <2 1 <2 <2 <2 <2 <1 <1 <2 <2 C8 sulphonate PFOS <2 <1 <1 <2 <1 <1 <5 < <3 1 <3 2 <2 <1 <.5 <.5 <5 <2 TH-C6 acid <5 <5 <5 <5 <5 <5 <1 <1 <1 <1 <1 <1 <1 <1 <2 <2 <1 <1 <5 <2 C5 acid <1 <1 <1 <1 <1 <1 <5 <5 <5 <5 <5 <5 <2 <5 <5 <5 <5 <5 <2 <5 C6 acid <5 <5 <5 <1 <5 <5 <3 1 <1 <3 <1 <3 <1 <1 <.5 <.5 <1 <2 <5 <5 C7 acid <5 <5 <5 <5 <5 <3 <5 <5 <3 <1 <5 <3 <5 <1 <1 <1 <1 <1 <1 <2 C8 acid PFOA <5 <5 <2 <2 <2 <2 <3 <1 <1 <1 <1 1 <1 <5 <5 <5 <.5 <.5 <5 <5 C9 acid <5 <5 <5 <5 <5 <5 <.5 <.5 <.5 ~ <.5 <.5 5 <5 <5 <1 <5 <.5 <.5 <1 <5 C1 acid <5 <5 <5 <5 <5 <5 <2 <.5 <5 # <.5 <5 1 <5 <5 <5 <5 <.1 <.1 <5 <5 C11 acid <5 <5 <5 <5 <5 <5 <2 <.5 <2 # <.5 <5 1 <5 <1 <5 <5 <.2 <.4 <5 <5 C12 acid <1 <5 <2 <1 <1 <3 <2 <1 <5 # <1 <1 5 <1 <1 <5 <5 <. <.1 <5 <2 C14 acid <2 <2 <5 <2 <2 <1 <5 <5 <5 # <5 <1 1 <2 <2 nd <1 <.1 <.4 <2 <2 C16 acid <5 <5 <1 <2 <2 <1 <5 <5 <5 <5 <5 <5 nd <5 nd <5 <.1 <1 nd nd C18 acid nd nd <5 <5 nd <5 nd nd nd nd nd nd nd nd nd nd <1 <1 nd nd *Peak sets comprised of only 1ppb n-c 4- SO 3 H as used for calibration. Other peaks were observed at 24 and 47 ppb (gps 12 and 13), these may be isobaric interferences, or the other possible alkyl isomers e.g. t-butyl-so 3 H, 1-Me-C 3 -SO 3 H, and 2-Me-C 3 -SO 3 H. One replicate of eggs (TDS 9) had peaks at about the LOD, this group probably contains trace levels of the C 9-14 acids. #One replicate of fruit products (TDS 16)had a peaks at about the LOD, this group probably contains trace levels of the C 6 acids, oils and fats (TDS 8) may also contained a trace of the C 6 acid. 24

25 Table 6. Concentrations of PFOS and related observed fluorochemicals in other environmental samples μg/kg (ppb). CSL Fish liver Fish Site-3 Fish Site-4 Fish Site-5 Fish Site-7 C6 amide PFHxSA 1 <5 <5 <5 1 C8 amide PFOSA 25 <5 nd <5 3 C4 sulphonate <1 <5 <1 <1 <1 C6 sulphonate <1 <5 <5 <5 <1 C8 sulphonate PFOS TH- C6 acid <2 <1 <1 <1 <1 C5 acid nd <1 nd nd nd C6 acid nd nd nd nd <1 C7 acid <2 <1 <1 <1 <1 C8 acid PFOA <2 <1 <1 <1 <1 C9 acid <2 <1 <1 <1 2 C1 acid 5 2 <1 <1 5 C11 acid 1 2 <1 <1 3 C12 acid 1 5 <5 <5 4 C14 acid <2 <1 <1 <1 <1 C16 acid nd <1 <1 <1 <1 C18 acid nd nd nd nd nd 25

26 FIGURES Figure 1. Chromatography of PFOS analytes in a 1 μg/l aqueous solution standard > e > e > e > e > e > e > e > e > e > e > e > e > e > e > e > e > e4 Time Time Time

27 Figure 2. Detector linearity and variations in suppression with matrix A) Analytes prepared in water B) Analytes prepared in methanol Compound name: C8 Acid 413.1>369.1 Coefficient of Deteration: R^2 = Calibration curve: e-5 * x^ * x Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: Null, Axis trans: None Compound name: C8 Acid 413.1>369.1 Coefficient of Deteration: R^2 = Calibration curve: * x^ * x Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: Null, Axis trans: None In water In methanol Re sp 4. on se 3. Re sp 1. on se ng/ml ng/ml C) Analytes passed through SPE D)Flour matrix passed through SPE and added to analytes Compound name: C8 Acid 413.1>369.1 Coefficient of Deteration: R^2 = Calibration curve: * x^ * x Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: Null, Axis trans: None Compound name: C8 Acid 413.1>369.1 Coefficient of Deteration: R^2 = Calibration curve: * x^ * x Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area ) Curve type: 2nd Order, Origin: Exclude, Weighting: Null, Axis trans: None Re 6. spo nse Aqueous standards with SPE 3. Standards with SPE Matrix eluent 2.5 Re 2. sp on se ng/ml ng/ml The absolute response and the ratio of response of e.g. the C8 acid and TH-PFOS varied between the different calibration sets as reflected by the different y-axis ranges, all analytes in all calibration sets responded in a near linear manner, allowing line fitting. 27

28 Figure 3. Comparison of linearity of recovery through WAX-SPE cartridges over 1-5 μg/l range 12 C5 acid C4 sulp acid 1 C6 acid C7 acid Arbitary scale of resposone units C6 sulp acid C8 acid C9 acid C8 sulp acid C1 acid C11 acid C8 Amide C12 acid C14 acid Concentratation of analytes ug/l C16 acid C18 acid 28

29 Figure 4. Chromatograms of the C 4 F 9 SO 3 - channel in TDS Group-12, potatoes A) Without spiking, with TH-PFOS as internal standard 2 Other possible isomers of C 4 F 9 SO 3 H O S O OH O S O OH * = 12.3 ppb 1.49 O S O OH * = 2.2 ppb * = 9.3 ppb C4 Sulp 299.1> = 1ppb 299.1> e >8.1 IS-THPFOS 427.1>8.1; 11.65; ; e B) With 1 ppb n-c4 Sulphonic acid, and internal standard >8.1 C4 Sulp 299.1> e Standard of n-c 4 F 9 SO 3 O S OH 17 O 427.1>8.1 IS-THPFOS 427.1>8.1 ; 11.65; ; e Without comparison to further standards, it is not possible to exclude the possibility that the peaks at 9.7, 1.5, 1.6 in the 299>8 mass channel may be due to the other 4-carbon alkyl isomers of butane sulphonic acid. This sample may therefore contain ca. 1 ppb n-pfbsh and 24 ppb other isomers. 29

30 Figure 5. Chromatograms of the C 6 F 13 SO 3 - and C 8 F 17 SO 3 - channels in TDS Group-12, potatoes A) unspiked B) With 1ppb C6 sulphonic acid C6 Sulp 399.1> * F2:MRM of 3 channels,es > e+3 C6 Sulp 399.1> * F2:MRM of 3 channels,es > e IS-THPFOS 427.1> > e+4 IS-THPFOS 427.1> > e C) unspiked D) With 1ppb C8 sulphonic acid (PFOS) C8 Sulp 499.1> F2:MRM of 3 channels,es > e+3 C8 Sulp 499.1> F2:MRM of 3 channels,es > e IS-THPFOS 427.1> > e+4 IS-THPFOS 427.1> > e Chromatograms suggest the presence of ca. 1 ppb of C6 sulphonate and ca. 1 ppb of PFOS In this potato sample (TDS 12) 3

31 Figure 6. Chromatograms of the C 4 F 9 SO 3 - channel in TDS Group-13, other vegetables A) Without spiking, with TH-PFOS as internal standard * = 4.1 ppm 299.1> e * = 6.7 ppm C4 Sulp 299.1> = 3.5 ppm IS-THPFOS 427.1>8.1;11.62;352.24; > e B) With 1 ppb PFOS, and internal standard * 299.1> e * C4 Sulp 299.1> * >8.1 IS-THPFOS 427.1>8.1;11.62; ; e Chromatograms suggest ca. 3.5 ppb n-c4 sulphonate and 47 ppb other possible isomers in other vegetable TDS

32 Figure 7. Chromatograms of the C8F17SO3- channel (PFOS) in TDS Group-14 canned vegetables A) Without spiking, with TH-PFOS as internal C8 Sulp 499.1> F2:MRM of 3 channels,es > e standard 9 IS-THPFOS 427.1> > e B) With 1 ppb PFOS, and internal standard C8 Sulp 499.1> F2:MRM of 3 channels,es > e IS-THPFOS 427.1> > e Chromatograms suggest ca. 2ppb of n-pfos in this sample of canned vegetables. 32

33 Figure 8. Chromatograms of the C 4 F 9 SO 3 - channel (C4 sulphonic acid) in TDS Group-8 oils and fats A) Without spiking, with 5ppb TH-PFOS as internal standard 299.1> e+3 Unknown 299.1> C4 Sulp > e+4 IS-THPFOS 427.1> B) With 1 ppb C4 Sulphonic acid, and internal standard 299.1> e+4 C4 Sulp 299.1> Unknown isomer or co-elutant > e+4 IS-THPFOS 427.1> Chromatograms suggest that a peak at ca..5 ppb is not n-c4 sulphonic acid as the peak under consideration elutes.2 before the authentic standard when overspiked. 33