Project in chemistry, 15 HP Spring 2015
Analysis of per- and polyfluorinated
substances (PFASs) in eggs and hen
food from Örebro County, Sweden
Theolinda Hanell Supervisor: Ulrika Eriksson
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Index
Sammanfattning ... 2
Abstract ... 3
1. Introduction ... 4
1.1 Chemical properties, synthesis and applications of perfluorinated compound s ... 4
1.2 Exposure and toxic effects of perfluorinated compounds ... 5
1.3 Aim ... 7
2. Method ... 7
2.1 Samples ... 7
2.2 Chemicals ... 8
2.3 Sample extraction ... 8
2.4 Quality assurance and quality control ... 9
2.5 Sample analysis ... 10
3. Results and discussion ... 12
3.1 Quality control and assurance ... 12
3.2 PFAS ... 14 3.3 diPAPs ... 23 4. Conclusions ... 24 5. Acknowledgements ... 25 6. References ... 25 Appendix ... 29
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Sammanfattning
Perfluorerade alkylsubstanser (PFAS) har använts för olika ändamål i årtionden och har visat sig vara giftiga. Huvudkällan för mänsklig exponering verkar vara genom maten där ägg, fisk, kött och mjölkprodukter är de största källorna. Målet för den här studien var att analysera både ekologiska och konventionella ägg och hönsfoder för detektion av PFAS och dess föregångare diPAPs. Prover samlades från sju olika äggproduktioner och PFAS extraherades med solid phase extraction (SPE). Ultra performance liquid chromatography och tandem mass spectrometry (UPLC-MS/MS) användes för analyserna.
Resultaten visade att äggen innehöll fler och andra PFAS homologer än hönsfodret, vilket indikerar att det fanns fler källor för intag än genom fodret. PFOS och PFUnDA kunde detekteras mer frekvent i ekologiska ägg- och hönsfoderprover än i konventionella ägg- och hönsfoderprover vilket kan bero på innehållet av fiskmjöl i ekologiskt hönsfoder. Dock var utbytet för internstandarderna under 20 % i många av äggproverna. 6:2 FTSA och PFBS detekterades i äggproverna vilket de inte verkar ha gjort i tidigare studier. Produktionen av PFBS har ökat på senare år då det ersätter PFOS. diPAPs kunde inte hittas i något av foderproverna medan flera hittades i äggproverna.
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Abstract
Perfluorinated alkyl substances (PFASs) have been used for various purposes for decades and have shown to have toxic properties. The main source of human exposure seems to be through the dietary where eggs, fish, meat and dairy products are the main sources. The aim of this study was to analyze both organic and conventional eggs and hen food for detection of PFASs and their precursors diPAPs. Samples from seven different egg productions where collected and the PFASs were extracted by solid phase extraction (SPE). Ultra performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) was used for analysis. The results showed that the eggs contained more and other homologues of PFASs than the hen food, indicating that there are more sources of ingestion of PFASs than the hen food. PFOS and PFUnDA could be more frequently detected in organic hen food and egg samples than in conventional hen food and egg samples, which could be due to the content of fish meal in organic hen food. However, the recovery of the internal standards was below 20% for several of the egg samples. 6:2 FTSA and PFBS were detected in the egg samples which they do not seem to have been in earlier studies. The production of PFBS has increased in later years, as it replaces PFOS. None of the diPAPs could be detected in the hen food sample whilst several were found in the egg samples.
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1. Introduction
Per- and polyfluorinated alkyl substances (PFAS) includes thousands of chemicals. PFAS are first divided into two categories; polymers and nonpolymers. In general, polymers have different chemical, physical and biological properties than nonpolymers of a light molecular weight. Nonpolymers are further divided into perfluoroalkyl and polyfluoroalkyl substances. In perflourinated compounds, all of the hydrogens in the hydro-carbon backbone have been substituted with fluorine atoms whereas in polyfluorinated compounds, some of the
hydrogens have not been fluorinated (Buck et al., 2011). There are also different groups of perfluorinated carboxylic acids (PFCAs), perfluorinated sulfonic acids (PFSAs), high molecular weight flouropolymers, low-molecular weight perfluoroalkanamides and fluorotelomer alcohols (Ståhl et al., 2011). The most studied and well known compounds belong to the two first groups and are perfluorooctane sulfanate (PFOS) and perfluorooctanoic acid (PFOA) (Lindstrom et al., 2011).
1.1 Chemical properties, synthesis and applications of perfluorinated compounds
Perfluoroalkylated and polyfluoroalkylated substances have been used for both industrial and consumer applications as components and precursors for surfactants and surface since the 1950’s (Borg et al., 2012). Perfluoroalkyl compounds have a very high thermal and chemical stability, making them persistent and some of them bioaccumulate in the environment as they move through food cycles (Lindstrom et al., 2011). If the perfluorinated compound has a long carbon chain, consisting of 8 or more carbons with fluorinated carbons, it is generally more bioaccumulative than those with 7 or less (Conder et al., 2008). An exception is PFOA, which has seven fluorinated carbons and still seems to be able to accumulate in human serum
(Emmet et al., 2006).
The high stability of perfluorinated compounds is due to the very strong carbon-fluorine bond and effective shielding of carbon by fluorine atoms. Because of the small atomic radius of a covalent bonded fluorine (72 Å), they can shield a perfluorinated carbon atom without any steric stress. The unique chemistry of perfluorinated compounds is not fully understood, however the stability of the strong C-F bond makes them stable to acids, alkali, reduction, and oxidation, even at high temperatures. Fluorine atoms have a high ionization potential and low
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polarizability which lead to a very low surface tension and contributes to their hydrophobic and lipophobic properties (Kissa 2001).
The chemical and thermal stability of PFASs in addition to its hydrophobic and lipophobic nature give the compounds several applications. They have been used as coatings on
packaging to overcome wetting and dewetting problems, as oil and solvent repellents on paper and paperboard, and as water repellents in textiles. PFASs have also been used in fire-fighting foams, insecticides, in the electronic industry and for many more applications (Kissa 2001).
Two different procedures, telomerization and electrochemical fluorination, are used to produce PFASs. The telomerization process produces primarily or only linear PFASs, whereas the electrochemical fluorination produces a mixture of linear and branched isomers (Buck et al., 2011). The main global producer of PFASs, 3M, used electrochemical
fluorination for their production. When they phased out the production of perfluorooctyl-based chemicals (including PFOA) in 2002, the production of PFOA by telomerization increased and is now the main manufacturing process (Prevedouros et al., 2006).
1.2 Exposure and toxic effects of perfluorinated compounds
PFASs are present in the environment worldwide and have been detected in water, plants, different kinds of foodstuffs, in several animals, and in human breast milk and blood (Ståhl et al., 2011). Potential sources for human exposure are through food and drinking water, and also through inhalation of household dust (D’Hollander et al., 2010). After analyzing food from Sweden and Norway, the main sources of exposure of PFASs through the dietary seems to be fish, meat, diary products and eggs (Haug et al., 2010; Vestergren et al., 2012). In one studie from Sweden, the dietary intake of PFOS and PFOA was a factor 6 to 10 higher than the exposure through ingestion of household dust and drinking water (Vestergren et al., 2012). Another potential source of exposure to the chemicals is precursor compounds which can be metabolized and form PFOS and PFOA. The precurors include for example fluorotelomer alcohols (FTOHs), perfluoroalkyl sulfonamides (PFOSAs) and perfluoroalkyl sulfonamide alcohols (PFOSEs) (Xu et al., 2006; Nabb et al., 2007). However, precursors in food and food-packaging seems to have a minor contribution to the daily intake of PFOS and PFOA (Vestergren et al., 2008). Fairly recent studies have shown that polyfluorinated alkyl phosphate esters (PAPs) are precursors to PFCA (D’Eon et al., 2007). They belong to the group of polyfluorinated alkyl surfactants and can have various chain lengths. PAPs exists as
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phosphate mono-, di-, and triesters. They have been detected in indoor dust and are used in grease proof food packaging where they are able to migrate from the packaging into the food. The toxicology of PAPs is still not well studied but recent studies indicates that they can interfere with sex hormone synthesis in vitro. Disturbance of the sex hormone synthesis can affect male reproductive health and cause disturbance (Taxwig et al., 2014).
The main global producer of PFASs (3M) ceased their production on products based on perfluorooctanyl chemistry between 2000-2002 (3M, 2000) and since then, the levels of PFOA and PFOS found in eggs and PFOS found in fish have decreased over time (Glynn et al., 2012) At the same time other types of PFASs have remained stable or even increased. The diversity and high production of the compounds make it difficult to measure global trends. Even though some producers in USA, Japan and Europe ceased their productions, some developing countries, for instance China and India have started to produce perfluorinated compounds. This is making it difficult to determine temporal trend times and sources. The production of PFAS is likely to continue due to the high applicability of the compounds (Ståhl et al., 2011).
According to earlier studies, the daily uptake of PFOS ranges between 2 and 200 ng/kg BW/day and the uptake of PFOA ranges between 3-14 ng/kg BW/day. (Ståhl et al., 2011) The compounds have a long half-life in human, studies have showed that it is approximately 5.4 years for PFOS and 3.8 years for PFOA (Olsen G.W et al. 2007). There is no known
metabolism of the compounds in the body (Ståhl et al. 2011) and their long half-life gives concerns about potential health effects. Animal studies shows that PFASs can be taken up through both ingestion and inhalation. When being taken up by the body, they accumulate primary in the liver and if the exposure is high, they also accumulate in the blood and other organs. It has been shown that different PFASs have different half-lifes in different animals and in some species, the gender of the animal affects how long the half-life is. Animal tests have only showed small direct toxic effects but a longer exposure can give several health effects (Ståhl et al., 2011). Hepatotoxic effects with enlarged liver (Lindstrom et al., 2011) and changes in the lipid metabolism are common in laboratory animals. Also effects like weight loss, tumors and cancer have been observed after a long exposure (Ståhl et al., 2011). When a pregnant mouse has been exposed for high concentrations of either PFOS or PFOA it has resulted in high mortality and reduced growth for the pups (Lau C. et al. 2006).
7 1.3 Aim
The aims of this study are to analyze PFCA/PFSA and diPAPs in both commercial and organic eggs to detect and compare the concentrations and homologue distributions of the different compounds. Hen food from each farm where the eggs were collected will be analyzed for detection of PFASs to see if the hen food is a source of PFASs in the eggs. The concentrations and homologue distributions of PFASs in organic and commercial eggs will also be compared to detect any differences that can be traced to the way of production.
2. Method
2.1 SamplesFive different farms around Örebro County were visited in the beginning of April for collection of the samples. All of the farms had such a large production that they supplied supermarkets with eggs. At least three eggs and a few deciliters of hen food were collected at each farm. Two of the farms had an organic egg production, two had a commercial and the last one produced both commercial, organic and eggs with a golden yolk. In total eggs and food from seven different farms or productions were collected. The hens from the commercial productions were only indoors, whilst the hens from one organic production had been released outdoors the week before sampling.
According to earlier analysis, different eggs from the same farm have had high variations of PFOS levels (D’Hollander et al., 2011) and therefore three egg yolks from each farm and each department were pooled before the extraction. The egg whites and the yolks were separated by hand before mixing the yolks together. A few ml of hen food were crushed to a powder using a pestle. Both the egg-and hen food samples were weighed up to 0.25 g in
polypropylene tubes (15ml) and the exact weight was noted.
Table 1. Sampling location and production type for the hen food and egg samples.
Sampling location
Production type Possibility to be outdoors
Farm 1 Organic Yes
Farm 2 Organic Not yet Farm 3 Conventional No Farm 4 Conventional No Farm 5A Organic Not yet Farm 5B Conventional No Farm 5C Conventional, golden yolk No
8 2.2 Chemicals
Methanol (HPLC and LC-MS grade) were purchased from Fisher Scientific UK, Leics UK. Hydrochloric acid for the extraction was in solution 1 mol/l and obtained from Scarlau, Sentmenat Spain. Sodium thiosulfate pentahydrate, ammonia solution (25%) and acetic acid (100%) were all purchased from Merck, Darmstadt Germany. Ammonium acetate ≥99% for HPLC, used for the SPE was obtained from Fluka, Stenheim Netherlands. Superclean™ ENVI-Carb™ SPE bulk packing was purchased from Sigma-Aldrich, St. Louis USA. 1-Methylpiperidine ≥98% used for analysis of diPAPs was purchased from Safc, St. Louis USA. All 13C-PFAS and 13C-diPAPs in the internal standard, all 13C-PFAS in the recovery standard and all PFAS and diPAPs in the native standard were obtained from Wellington Laboratories, Guelph ON, Canada. The chemical purity was > 98% for all compounds in the standards and the isotopic purity was ≥ 99% for the internal standard and the recovery standard. A list of all PFAS and diPAPs in the different standards can be seen in table 2.
2.3 Sample extraction
The method used for extraction was based on previous methods (Taniyasu et al., 2005; Powley et al., 2005; Domingo et al., 2012). Both the egg samples and the hen food samples were extracted using the same method.
The samples were divided into two batches, one with the egg samples and one with the hen food samples. Before extraction, all equipment were cleaned with methanol. 10 µl of an internal standard containing 13C-PFCA/PFSA (0.2 ng/µl) and 20 µl of an internal standard containing 13C- 6:2,8:2 diPAP (0.2 ng/µl) and was added to 0.25 g sample. Hamilton syringes were used for the standards.
NaOH (300 µl, 0.2 M) was added to each sample and they were left to soak for 30 minutes. After that, 4 ml of methanol was added and the samples were then were vortexed, followed by 15 min in an ultrasonication bath and thereafter 15 minutes in a shaker. The samples were neutralized with 60 µl of 1 M HCl and put in a centrifuge for 30 min at 8000 g. The extract was transferred to clean test tubes and 4 ml of methanol was added to the samples. The vortexing, ultrasonication, shaking and centrifugation were repeated in the same way as before and the extracts were then transferred to the new test tubes. The samples were evaporated by using nitrogen gas down to a volume of 1 ml before adding 4 ml of Milli-Q H2O.
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Solid phase extraction (SPE) was performed as clean up. Before the SPE procedure, the samples were cloggy and were therefore centrifuged for 15 min. Oasis® WAX (Weak Anion
eXchange) 6cc Cartridge 150mg 30µm cartridges were used for the SPE. They were conditioned with 4 ml MeOH followed by 4 ml H2O before loading the samples onto the
cartridges. A vacuum pump was used to speed up the flow rate to about one drop per second. When the samples had run through, they were washed with 4 ml NaAc 0.025 M, pH 4, and then with 4 ml of a 20% MeOH solution. Vacuum was used to dry the cartridges before eluting the samples with 4 ml MeOH followed by 4 ml 2% NH4OH. The samples were eluted
into test tubes containing 40-50 mg ENVI-Carb and 100 µl acetic acid. The tubes were vortexed before evaporating the samples to a final volume of 1 ml. The samples were filtered by using Acrodisc® Syringe Filter 13 mm, 0.2 µm GHP into LC-vials containing 5 µl of a recovery standard (13C-PFCA/PFSA RS, 0.2 ng/µl). They were evaporated to a final volume of 200 µl and the samples were splitted into two fractions for analysis of PFCAs/PFSAs and diPAPs.
Before analysis of diPAPs, 100 µl NH4Ac 2mM, 1-MP 5mM in MeOH and 50 µl NH4Ac
2mM, 1-MP 5mM in H2O were added to each sample and to the standards (40 µl of MeOH
was added to the standards as well).
Before analysis of PFCA/PFSA, 150 µl NH4Ac (aq, 2 mM) was added to each sample. 300 µl
NH4Ac (aq, 2 mM) and 175 µl MeOH was added to the standard.
Samples that were cloggy either went through an extra filtration step or were centrifuged and transferred to a new vial.
2.4 Quality assurance and quality control
Native standards were used for calibration and internal standards were used for quantification and qualification. A recovery standard for PFCA/PFSA was used but none was available for diPAPs. A blank sample was prepared together with both the egg samples and the hen food samples and treated the same way as the other samples. A duplicate of the sample from farm one was made in mistake for both the hen food- and egg samples. It should have been spiked with native standard. The differences in concentrations for the samples from farm one and the duplicate is presented in the appendix, table 2.
10 2.5 Sample analysis
Analysis were performed on a Waters Acquity UPLC system coupled to a XEVO triple quadrupole MS/MS. The column used for the UPLC-MS/MS analysis was an Acquity UPLC
© BEH C18 with particle size 1.7µm, width and length 2.1 x 100 mm. The temperature of the
column was 50°C. The MS settings when analyzing PFASs were set on negative electrospray, source temperature 150°C, desolvation temperature 400°C, capillary voltage 0.7 kV, cone gas flow 150 L/Hr and desolvation gas flow 800 L/Hr. For analysis of diPAPs, the desolvation temperature was 350°C, the capillary voltage was 3 kV and the other settings were the same as for PFASs. The injection volume of the samples were 10 µl.
For the analysis of PFCAs/PFSAs, mobile phase A consisted of 70% H2O, 30% MeOH and 2
mM NH4Ac. Mobile phase B consisted of 100% MeOH and 2 mM NH4Ac. When analyzing
diPAPs, both mobile phases contained 5 mM 1-methylpiperidine as well. For the analysis of PFCAs/PFSAs, 100% of mobile phase A was used and for the analysis of diPAPs 53% of mobile phase A and 47% of mobile phase B were used. The flow rate was 0.30 mL/min.
MassLynx was used to control the instrument and to achieve the results.
Table 2. All PFASs and diPAPs analyzed and the MS settings for each compound.
Name Precursor/ product quantification Cone voltage (V) Collision energy (V) Precursor/ product qualification Cone voltage (V) Collision energy (V) PFBA RS 13C 3-PFBA IS 13C 4-PFBA 212.97 > 169.00 215.97 > 172.00 216.97 > 172.00 20.0 11.0 PFPeA 262.97 > 219.00 20.0 8.0 PFBuS 298.90 > 98.90 20.0 26.0 298.90 > 79.96 20.0 26.0 PFHxA IS 13C 2-PFHxA 312.97 > 269.00 314.97 > 270.00 20.0 9.0 312.97 > 118.95 20.0 26.0 PFHpA 362.97 > 319.00 20.0 10.0 362.97 > 168.97 20.0 16.0 PFHxS RS 13C 3-PFHxS IS 13C 4-PFHxS 398.90 > 98.90 401.90 > 98.90 402.90 > 102.90 20.0 30.0 398.90 > 79.96 398.90 > 119.01 20.0 20.0 34.0 28.0 PFOA IS 13C 4-PFOA RS 13C 8-PFOA 412.97 > 369.00 416.97 > 372.00 420.97 > 376.00 20.0 10.0 412.97 > 118.93 412.97 > 168.97 20.0 20.0 30.0 18.0 PFNA 462.99 > 419.00 20.0 12.0 462.99 > 219.00 20.0 18.0
11 IS 13C 5-PFNA RS 13C 9-PFNA 467.99 > 423.00 471.99 > 427.00 19.0 12.0 PFOS IS 13C 4-PFOS RS 13C 8-PFOS 498.97 > 98.96 502.97 > 98.96 506.97 > 98.96 20.0 38.0 498.97 > 79.96 498.97 > 169.03 498.97 > 419.00 20.0 44.0 34.0 35.0 PFDA IS 13C 2-PFDA RS 13C 6-PFDA 512.97 > 469.00 514.97 > 470.00 518.97 > 474.00 20.0 11.0 512.97 > 219.00 20.0 18.0 PFUnDA IS 13C 2-PFUnDA RS 13C 7-PFUnDA 562.97 > 519.00 564.97 > 520.00 569.97 > 525.00 20.0 12.0 562.97 > 268.99 20.0 18.0 PFDS 598.97 > 98.90 20.0 42.0 598.97 > 79.96 20.0 58.0 PFDoDA IS 13C 2-PDoDA 612.97 > 569.00 614.97 > 570.00 34.0 14.0 612.97 > 168.96 40.0 22.0 PFTrDA 662.90 > 619.00 20.0 14.0 662.90 > 168.96 20.0 26.0 PFTDA IS 13C 2-PFTDA 712.90 > 669.00 714.90 > 670.00 20.0 14.0 712.90 > 168.97 20.0 28.0 PFHxDA IS 13C 2-PFHxDA 812.90 > 769.00 814.90 > 770.00 30.0 15.0 812.90 > 168.96 30.0 32.0 PFOcDA 912.90 > 168.96 912.90 > 869.00 36.0 15.0 912.90 > 168.96 36.0 36.0 6:2 FTSA IS 13C 2-6:2FTSA 427.00 > 407.00 429.00 > 409.00 20.0 20.0 427.00 > 81.00 20.0 28.0 4:2/6:2 diPAP 688.90 > 97.00 64.0 28.0 688.90 > 242.91 688.90 > 342.91 688.90 > 442.91 688.90 > 542.91 64.0 18.0 6:2 diPAP IS 13C 2-6:2 diPAP IS 13C 2-6:2 diPAP 788.90 > 97.00 788.90 > 442.91 792.90 > 97.00 792.90 > 444.91 64.0 64.0 64.0 64.0 28.0 18.0 28.0 18.0 788.90 > 242.91 788.90 > 342.91 788.90 > 542.91 788.90 > 642.91 64.0 18.0 8:2 diPAP IS 13C 2-8:2 diPAP IS 13C 2-8:2 diPAP 988.78 > 96.94 988.78 > 542.81 992.78 > 96.94 992.78 > 544.81 68.0 68.0 68.0 68.0 34.0 26.0 34.0 26.0 988.78 > 342.81 988.78 > 442.81 988.78 > 642.81 988.78 > 742.81 68.0 26.0 6:2/8:2 diPAP 888.78 > 96.94 66.0 34.0 888.78 > 342.81 888.78 > 442.81 888.78 > 542.81 66.0 26.0
12 888.78 > 642.81 8:2/10:2 diPAP 1088.78 > 96.94 68.0 34.0 1088.78 > 442.81 1088.78 > 542.81 1088.78 > 642.81 1088.78 > 742.81 68.0 26.0 10:2 diPAP 1188.78 > 96.94 68.0 34.0 1188.78 > 442.81 1188.78 > 542.81 1188.78 > 642.81 1188.78 > 742.81 1188.78 > 842.81 68.0 26.0
3. Results and discussion
3.1 Quality control and assuranceMost of the internal standards in the hen food samples when analyzing PFAS had an approved recovery, over 20 % (seen in table 3). PFUnDA and PFDoDA could not be quantified, they are long chained compounds and therefore often receive lower recovery values. The egg samples had lower recoveries than the hen food samples. The recoveries in table 4, showed that only 6:2 FTS, PFBA, PFOA and PFHxA were possible to quantify in all the egg samples. The samples with recovery 5-20% and an area at least 3 times larger than the blank area were included in the results. Those levels are marked in the text and should be interpreted tentatively. The problem with low recovery may be due to matrix effects. Egg yolks have a high lipid percentage and without successful clean up steps, lipids and other matrix
constituents can lead to enhancement or suppression of the electrospray ionization. This results in inaccuracies (van Leeuwen et al., 2007). Egg samples often have lower recovery values for PFASs in the internal standard than other types of food samples which can be seen in earlier studies (Haug et al., 2012; Gebbink et al., 2015). The extraction method used in this study was not optimized for egg samples and it was beyond the scope of this study to do a method development. The egg samples were diluted 1:5 and run again in the UPLC-MS/MS to see if the low recovery values were due to suppression of the signal due to matrix effects. The recoveries did not increase after the dilution. Matrix effects might have been possible to overcome with a higher dilution factor or the extraction method may not have been
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The recovery for diPAPs in both egg- and hen food samples were very low for both 6:2 diPAP and 8:2 diPAP (< 20%)(table 5 and 6), making most of the diPAPs impossible to quantify. The samples were diluted 1:5 with an 80% methanol solution and run again in the UPLC-MS/MS. The recoveries did not increase and therefore, two of the samples were spiked with native diPAPs and analyzed again. The high signal of the diPAPs in the spiked samples indicated that the low recovery was not due to matrix effects. The extraction method may not have been successful.
Table 3. Recovery in % for the internal standards in each hen food sample.
Compound Blank Farm
1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
Org. Org. Org. Conv. Conv. Conv. Golden yolk 13C-8:2FTSA 69.5 39.2 51.3 47.8 60.7 46.0 74.8 72.9 13C-6:2FTSA 89.6 149.5 135.7 144.1 107.3 119.5 133.1 136.3 13C-PFBA 53.7 92.0 84.3 81.0 83.8 78.9 68.5 58.9 13C-PFHxA 85.0 90.1 80.5 77.6 100.7 90.7 100.9 98.2 13C-PFHxS 88.0 85.6 84.1 82.0 83.6 81.8 81.8 80.8 13C-PFOS 84.8 38.3 49.5 44.5 68.9 46.3 54.8 70.0 13C-PFOA 87.6 83.5 81.0 77.2 83.3 76.6 74.5 78.7 13C-PFNA 86.4 63.5 65.9 59.4 77.6 59.7 61.5 73.1 13C-PFDA 85.3 31.0 39.1 35.5 58.8 30.8 41.3 56.6 13C-PFUnDA 79.5 10.3 15.6 17.5 30.1 11.0 27.0 31.0 13C-PFDoDA 45.2 2.6 5.7 10.5 9.9 3.5 18.6 21.3
Table 4. Recovery in % for the internal standards in each egg sample.
Compound Blank Farm
1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
Org. Org. Org. Conv. Conv. Conv. Golden yolk 13C-8:2FTSA 38.2 7.4 26.8 10.0 11.4 4.3 16.8 49.3 13C-6:2FTSA 58.5 62.3 94.2 81.3 81.1 76.9 74.6 109.0 13C-PFBA 69.3 82.4 78.4 81.2 74.9 76.1 70.3 71.8 13C-PFHxA 73.9 69.4 71.3 72.4 66.9 73.4 72.9 72.3 13C-PFHxS 70.4 15.6 32.2 22.9 21.5 24.9 20.6 28.1 13C-PFOS 69.4 6.7 24.4 7.5 7.4 6.3 8.8 19.4 13C-PFOA 67.5 21.2 78.2 27.8 25.3 29.9 24.4 31.9 13C-PFNA 68.9 9.8 26.4 12.7 11.6 12.1 12.3 21.4
14 13C-PFDA 67.0 6.4 24.4 7.6 7.3 5.8 9.0 20.2
13C-PFUnDA 60.5 5.6 24.4 6.3 6.2 4.4 7.7 20.6 13C-PFDoDA 9.7 5.4 18.9 6.1 6.0 3.1 7.9 23.4
Table 5. Recovery including matrix effects in % for the internal standards when analyzing diPAPs in hen food samples.
Blank Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
Org. Org. Org. Conv. Conv. Conv. Golden yolk
13C-6:2 diPAP 34.8 0.4 1.2 7.4 1.6 1.2 9.0 12.4 13C-8:2 diPAP 2.2 0.0 0.0 0.4 0.0 0.2 1.8 3.6
Table 6. Recovery including matrix effects in % for the internal standards when analyzing diPAPs in egg samples.
Blank Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
Org. Org. Org. Conv. Conv. Conv. Golden yolk
13C-6:2 diPAP 35.0 6.2 10.9 6.6 6.4 4.8 8.8 20.6 13C-8:2 diPAP 1.8 3.8 5.8 3.8 4.4 2.6 5.8 12.4
3.2 PFCA/PFSA
Table 7 and table 8 shows the concentrations of PFCA/PFSA detected in the samples. The compounds in both tables were present in concentrations over the limit of detection in at least one of the samples.
Table 7. Concentrations of PFCA/PFSA (ng/g) in the hen food samples.
LOD (ng/g)
Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
PFOS 0.04 1.47 0.12 0.19 < 0.04 0.09 < 0.04 < 0.04 PFUnDA 0.12 0.55 (nq) 0.24 (nq) 0.21 (nq) < 0.12 0.29 (nq) 0.13 < 0.12 PFDoDA 0.10 0.58 (nq) 0.28 (nq) 0.21 (nq) 0.20 (nq) 0.48 (nq) < 0.10 0.18
PFTrDA 0.12 1.50 (nq) 0.39 (nq) 0.29a 0.26b 0.79b < 0.12 < 0.12
PFTDA 0.16 nq nq nq nq 0.65b < 0.16 < 0.16
a Recovery < 20 % and > 10%, area > 3 * blank area
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Table 8. Concentrations of PFCA/PFSA (ng/g) in the egg samples.
LOD (ng/g)
Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
PFBS 0.35 0.60 < 0.35 0.46 0.45 < 0.35 0.52 0.40 PFOA 0.93 1.44 < 0.93 < 0.93 < 0.93 < 0.93 < 0.93 < 0.93 PFNA 0.17 2.38a nq nq nq nq nq nq PFOS 0.04 3.63b 0.76 1.44 b nq nq nq nq PFDA 0.43 2.02b 0.45 1.37 (nq) 1.25 (nq) 1.69 (nq) 1.05 (nq) 0.47 PFUnDA 0.15 0.86 (nq) 0.38 0.64 (nq) nq nq nq < 0.15 6:2 FTSA 1.00 1.58 1.26 1.01 1.30 1.10 1.02 1.16
a Recovery < 20 % and > 10%, area > 3 * blank area
b Recovery < 10 % and > 5%, area > 3 * blank area
Figure 7. The average concentrations of PFCA/PFSA ± SD for both the hen food samples and egg samples.
Figure 7 shows that in average, there were higher concentrations of PFCA/PFSA present in the egg samples than in the hen food samples. The average concentrations in the hen food samples had a higher distribution. However, only one compound in each hen food sample was quantified, except for the sample from farm 3 were no PFCA/PFSA was found.
n = 6 n = 16 -0,4 -0,2 0 0,2 0,4 0,6 0,8 1 1,2
Hen food samples Egg samples
n
g/g
16
Figure 8. Average concentrations of PFCA/PFSA ± standard deviation in organic and conventional eggs.
The concentration of the organic hen food samples shows a high variation between samples. The other three sample types shows a lower variation. The egg samples contained higher concentrations of PFCA/PFSA than the hen food samples and the organic hen food samples also contain higher concentrations than the conventional hen food samples. PFOS was found in four of the hen food samples, of which three were organic. The concentrations of PFOS were higher in all of the organic hen food samples than in the conventional hen food sample. Levels of PFDoDA and PFUnDA were detected in two of the organic hen food samples. This may be due to the content of fish meal (see appendix) in organic hen food. Several studies of PFASs in fish have shown high levels of PFOS and presence of PFDoDA and PFUnDA. (Vestergren et al., 2012; Haug et al., 2010; Gebbink et al., 2015).
n= 3 n = 3 n = 8 n = 8 -0,4 -0,2 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
Organic hen food Conventional hen food Organic eggs Conventional eggs
n
g/g
17
Figure 1. All PFCA/PFSA present in each hen food sample. *Recoveries below 20%.
The samples from farm 1, 2 and 5A are organic and the others are conventional. The sample from farm 5C gives a golden yolk. The levels of analyzed PFASs in the hen food samples were in most cases below LOD. PFOS was quantified in four of the samples and in the samples from farm 5B and 5C PFUnDA respectively PFDoDA could be quantified.
* * * * * * * * * * * * * * * 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
n
g/g
PFCA/PFSA in hen food samples
18
Figure 2. All PFCA/PFSA present in each egg sample. *Recoveries below 20%.
6:2-FTSA was detected in all of the egg samples and PFBS was present in all samples except in the samples from farm 2 and 4. PFOA could only be detected in the sample from farm 1, possibly due to high concentrations in the blank, giving a high LOD (0.93 ng/g). PFDA could be quantified in the sample from farm 2 and 5C due to higher recovery for those samples.
When comparing the compounds present in the hen food samples (picture 1) to the
compounds present in the egg samples (picture 2), only PFOS and PFUnDA were present in both the hen food- and the egg samples. They were not possible to quantify in the egg samples but it indicates that they were present and the source of ingestion could be the hen food. The source of ingestion of the other PFASs in the egg samples seems to be from another source then the hen food. Most PFASs in the hen food samples had an approved recovery and the concentrations were below the LOD which further indicates that the ingestion of PFASs is from more sources than only the hen food. It could probably be from the drinking water or the environment inside the farm. 6:2 FTSA which is a fluorotelomer sulfonate was found in all egg samples but not in the hen food samples. Fluorotelomer sulfonates have been used for decades in firefighting foams, used at fire training facilities, for example at military bases, and to put out fires. 6:2 FTSA have been detected in ground water, soil and biota (Oakes et al., 2010; Schultz et al., 2004). * * * * * * * * * * 0 0,5 1 1,5 2 2,5 3 3,5 4
Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
n
g/g
PFCA/PFSA in egg samples
19
Table 9. Concentrations of PFCA/PFSA in eggs (ng/g) from earlier studies. Article Vestergren et al., 2012 Gebbink et al., 2015 Haug et al., 2010 Noorlander et al., 2011 This study Country and year of sampling Sweden, 2010 Sweden, 2005 Sweden, 1999 Sweden, 1999 Norway, 2008-2009 Neatherlands, 2009 Sweden, 2015 PFBS nr nr nr nr nq <LOD 0.48 PFOA 0.039 0.007 0.031 0.036 nq <LOD 1.44 PFOS 0.039 0.013 1.280 1.078 0.039 nq 0.76
PFHxA nq nq nq <LOD 0.013 <LOD <LOD
PFDA nq nq 0.015 0.006 nq 0.011 0.46
PFHxS 0.003 <LOD 0.039 0.034 0.004 <LOD <LOD PFUnDA <LOD nq 0.038 0.041 0.010 <LOD 0.38
PFNA <LOD nq 0.022 0.024 <LOD 0.006 <LOD PFDoDA <LOD <LOD 0.010 0.010 <LOD <LOD <LOD PFTrDA <LOD <LOD 0.014 0.016 nr <LOD <LOD PFTeDA <LOD <LOD <LOD 0.003 nr <LOD <LOD
6:2 FTSA nr nr nr nr nr nr 1.20
nr = not reported nq = not quantified
When looking at six earlier analysis of PFASs in eggs (table 9), the concentrations are over all lower than the concentrations detected in this study. In the earlier studies, the whole eggs were analyzed whilst in this study, only the egg yolks were analyzed. One third of an egg consists of egg yolk and if the PFASs accumulate in the egg yolk, it can be a reason why the concentrations are higher in this study.
PFOS is the most frequent detected compound and has been detected in all of the studies whilst PFOA has been detected in most of the studies. Gebbink and Vestergren used the same samples for their analysis in 1999. The samples from 1999 have much higher concentrations of PFOS than the later samples, showing that the concentrations are decreasing. This could be due to the 3 M phase out of PFOS among other PFASs, however the levels of PFASs in fish have not decreased over time (Vestergren et.al., 2012). There were more PFASs present in total in the two studies from 1999. In this study, PFOS could be quantified in the hen food samples and there are no known studies of levels of PFAS in hen food samples for
comparison. PFOS was quantified in the egg sample from farm 2 in concentrations of 0.76 ng/g. That is higher levels than in the other recent studies but still lower than in the studies from 1999. PFOA could be detected in one organic egg sample and the concentration was
20
higher (1.44 ng/g) than the concentrations in the earlier studies. PFDA was found in two commercial egg samples at 0.47 and 0.45 ng/g which also is higher than the concentrations found in the earlier studies. Both 6:2 FTSA and PFBS were detected in several egg samples in this study. PFBS was not detected in the earlier studies. The levels of PFBS in this study may be connected to the increased production of the compound as replacement for PFOS. 3 M started their production of PFBS in 2003 (Renner, 2006). 6:2 FTSA has not been analyzed previously in egg samples.
Figure 3. Concentrations of PFOS in egg and hen food samples.*Recoveries below 20%.
PFOS could be quantified in four of the hen food samples and in one of the egg samples. However, PFOS was present in both eggs and hen food in the samples from farm 1, farm 2 and farm 5A.
* * 0 0,5 1 1,5 2 2,5 3 3,5 4
Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
n
g/g
PFOS
21
Figure 4. The concentrations of PFOS in the hen food and egg samples from each farm were plotted against each other to see the correlation.
Figure 4 shows the correlation between the concentrations of PFOS in hen food and PFOS in egg samples. Even though PFOS could not be quantified in all of the egg samples, it is present in the samples from farm 1, farm 2 and farm 5A. The r-value close to one shows that the concentrations in the samples are correlated. This indicates that hen food is a source of PFOS in eggs. Ingestion of soil is also a possible contamination source of PFOS in eggs
(D’Hollander et al., 2010). The hens must then be outdoors which only the hens from farm 1
were. The eggs from farm 1 seems to have had the highest levels of PFOS (table 3), however the levels of PFOS in the food was also high and the recovery was below 20 % for the egg sample so the concentration could not be established.
R² = 0,9097 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 Egg s amp le s
Hen food samples
22
Figure 5. Concentrations of PFUnDA in egg and hen food samples.*Recoveries below 20%.
As seen in figure 5, PFUnDA could only be quantified in one hen food sample (farm 5B) and in one egg sample (farm 2).
Figure 6. The concentrations of PFUnDA in the hen food and egg samples from each farm were plotted against
each other to see the correlation.
The r-value of 0.59 indicates that the concentrations of PFUnDA in the egg samples and in the hen food samples have a positive association.
* * * * * * 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
Farm 1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
n
g/g
PFUnDA
Hen food samples Egg samples
R² = 0,5876 (0,10) 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 1,00 0 0,1 0,2 0,3 0,4 0,5 0,6 Egg s amp le s
Hen food samples
23 3.3 diPAPs
Table 10. Concentrations of diPAPs (ng/g) in hen food samples.
LOD Farm 1 Farm 2 Farm 3 Farm 4 Farm 5A Farm 5B Farm 5C
6:2 diPAP 0.23 nq nq nq nq nq <0.23 <0.23 8:2 diPAP 0.36 nq nq nq nq nq nq nq 6:2/8:2 diPAP 1.60 nq nq nq nq nq < 1.60 < 1.60 10:2 diPAP 0.02 nq nq nq nq nq nq nq
None of the diPAPs could be detected in the hen food samples due to low recoveries of the internal standard (table 5) and due to high blank levels for some of the homologues.
Table 11. Concentrations of diPAPs (ng/g) in egg samples.
LOD Farm 1 Farm 2 Farm 3 Farm 4 Farm 5A Farm 5B Farm 5C 6:2 diPAP 0.33 1.59b nq 4.58b nq 1.85b nq 0.39 8:2 diPAP 0.27 1.46 (nq) 0.28a 6.24 (nq) 1.66 (nq) 2.08 (nq) 0.35b 0.65a 6:2/10:2 diPAP 0.48 2.90 (nq) nq 3.38 (nq) 9.89 (nq) 2.65 (nq) 0.58b 0.65a 6:2/8:2 diPAP 3.66 nq nq 12.82 (nq) nq 4.61 (nq) nq < 3.66 10:2 diPAP 0.08 nq nq 0.90 (nq) nq 0.29 (nq) nq 0.19 (nq)
a Recovery < 20 % and > 10%, area > 3 * blank area
b Recovery < 10 % and > 5%, area > 3 * blank area
2,89833 3,3796 9,886853 2,646351 0,581065 0,545061
The recoveries for the egg samples were better than for the hen food samples. 6:2 diPAP in the sample from farm 5C was the only compound with recovery >20%.
24
Figure 10. diPAPs in egg samples. *Recoveries below 20%.
6:2 diPAP could be quantified in the egg sample from farm 5C in concentrations of 0.39 ng/g. In 1999, Gebbink detected lower concentrations of 6:2 diPAP in Swedish eggs. They were measured to be 0.016 ng/g. In that study, 6:2 diPAP and 6:2/8:2 diPAP were the only diPAPs detected in egg samples (Gebbink et al., 2015). However the sample from farm 3 has all diPAPs present and it looks like the concentrations are very high but it could not be confirmed due to the low recoveries. Method development is needed for analysis of diPAPs in these matrices.
4. Conclusions
Further method development is needed to evaluate the performance of the method but that is beyond the scope of this project work. Samples should have been spiked before and after extraction to see if the low recoveries were due to matrix effects. Another possible factor for low recoveries was the extraction method.
A comparison of the concentrations in the egg samples and hen food samples showed that there were more and different PFASs present in the egg samples, indicating that the compounds origins from more sources than the hen food. Compared to earlier studies of PFASs in eggs, the concentrations in this study were higher, however the compounds detected in most samples (PFBS and PFHxA) had not been detected in the earlier studies. The levels of
* * * * * * * * * * * * * * * 0 2 4 6 8 10 12 14
Farm1 Farm 2 Farm 5A Farm 3 Farm 4 Farm 5B Farm 5C
n
g/g
diPAP in egg samples
25
PFDA and PFOA were also higher in this study compared to earlier studies. The presence of PFOS, PFUnDA and PFDoDA in the organic hen food samples can possibly be explained by the content of fish meal in organic hen food. The levels of PFOS in the egg samples seemed to be correlated to the levels in the hen food samples. The organic eggs also contained higher levels of PFAS then the conventional eggs but that does not seem to be only due to the hen food, because the PFAS profile of the eggs differed from the profile of the hen food.
diPAPs could not be detected in the hen food samples due to very low recoveries and levels below LOD. Several compounds were detected in the egg samples but the recovery values were low, showing that method development is needed for these matrices to receive accurate results.
5. Acknowledgements
I would like to thank my supervisor Ulrika Eriksson for all great help, advice and
guidance. I would also like to thank the farmers who helped and supplied me with all the samples.
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Appendix
Table 1. Standards for calibration
Native
standards (0.2 ng/µL)
Standard isoPAP Standard diPAP Standard PFCA/PFSA 10:2 diPAP 40 µL 6:2, 8:2 diPAP 20 µL 6:2/8:2 diPAP 20 µL PFCA/PFSA CS1 10 µL Internal standards (0.2 ng/µL) 13C- 6:2,8:2 diPAP 20 µL 20 µL 13C-PFCA/PFSA 10 µL
Table 2. Differences in concentrations in % between the hen food sample from farm 1 and a replicate.
Table 3. Differences in concentrations in % between the egg sample from farm 1 and a replicate.
PFOS 12% PFUnDA 16% PFDoDA 23% PFTrDA 68% PFTDA 36% PFBuS 38% PFOA 14% PFNA 2% PFOS 8% PFDA 44% PFUnDA 15% 6:2 FTSA 46%
30
Table 4. Perfluorinated carboxylic acids (PFCAs) analyzed
Abbreviation Name Structure
PFBA Perfluorobutanoic acid PFPeA Perfluoropentanoic acid PFHxA Perfluorohexanoic acid PFHpA Perfluoroheptanoic acid PFOA Perfluorooctanoic acid PFNA Perfluorononanoic acid PFDA Perfluorodecanoic acid PFUnDA Perfluoroundecanoic acid
31 PFDoDA Perfluorododecanoic acid PFTrDA Perfluorotridecanoic acid PFTDA Perfluorotetradecano ic acid PFHxDA Perfluorohexadecano ic acid PFOcDA Perfluorooctanoicdec anoic acid
Table 5. Perfluorinated sulfonic acids (PFSAs) analyzed
Abbreviation Name Structure
PFBuS Perfluorobutane
sulfonic acid
PFHxS Perfluorohexane
32
PFOS Perfluorooctane
sulfonic acid
PFDS Perfluorodecane
sulfonic acid
Table 6. Fluorotelomer sulfonates (FTSs) analyzed
Abbreviation Name Structure
6:2 FTSA 6:2 Fluorotelomer
sulfonic acid
8:2 FTSA 8:2 Fluorotelomer
sulfonic acid
Table 7. Fluorotelomer sulfonate diesters (diPAPs) analyzed
Abbreviati on Name Structure 4:2/6:2 diPAP 4:2/6:2 Fluorotelomer phosphate diester 6:2 diPAP 6:2 Fluorotelomer phosphate diester 8:2 diPAP 8:2 Fluorotelomer phosphate diester
33 6:2/8:2 diPAP 6:2/8:2 Fluorotelomer phosphate diester 8:2/10:2 diPAP 8:2/10:2 Fluorotelomer phosphate diester 10:2 diPAP 10:2 Fluorotelomer phosphate diester
Ingredients in the hen food from the different farms
Farm 1: Organic wheat, organic oat, organic peas, lime, fish meal (7 %), organic barley,
organic linseed cake and concentrate.
Farm 2: Complete feed consisting of; organic wheat, organic oat, organic barley, lime, fish
meal (8.3 %), corn gluten meal, organic sunflower expeller, potato protein, monocalcium phosphate, sodium bicarbonate.
Farm 3: Wheat, oat, lime and concentrate; soy flour, colza flour, barley, colza seed, oat,
wheat and vegetable fatty acids.
Farm 4: Complete feed consisting of ; wheat, soy flour, oat, calcium carbonate, colza seed,
vegetable fatty acids, heat-treated colza seed expeller, premix additives, monocalcium phosphate, sodium chloride, sodium bicarbonate.
Farm 5A: Wheat, triticale, oat, peas, lime and concentrate consisting of; fish meal, wheat
(organic), corn gluten, soy expeller (organic), calcium carbonate, lucerne (organic), monocalcium phosphate and sodium chloride.
34 Farm 5B: Wheat, oat, colza seed, lime and concentrate consisting of; flour of peeled soybean,
wheat, colza seed, monocalcium phosphate, sodium chloride, sodium bicarbonate.
Farm 5C: Wheat, oat, colza seed, lime and concentrate consisting of; flour of colza seed,
peas, colza seed, wheat, flour of peeled soybean, malt sprouts, monocalcium phosphate, sodium chloride, calcium- and magnesium carbonate, dried algae, Haematococcus pluvialis, wheat middlings.
