Document gVDMR2GKX6jo5JXvbEVVzY8Q

Australian Government Departmen( of Industry, Innovation and Science Nal Measurement Institute Proficiency Test Report AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 6 US00001934 US00001935 ACKNOWLEDGMENTS This study was conducted by the National Measurement Institute (NMI). Support funding was provided by the Australian Government Department of Industry, Innovation and Science. I would like to thank the management and staff of the participating laboratories for supporting the study. It is only through widespread participation that we can provide an effective service to laboratories. The assistance of the following NMI staff members in the planning, conduct and reporting of the study is acknowledged. Raluca Iavetz Geoff Morschel Gavin Stevenson Jesuina De Araujo I would also like to thank Professor Jochen Mueller and Jennifer Braeunig from National Research Centre for Environmental Toxicology (ENTOX) - University of Queensland for performing homogeneity analysis on fish samples. Paul Armishaw Manager, Chemical Reference Values PO Box 138, North Ryde NSW 1670 Phone: 61-2-9449 0149 Fax: 61-2-9449 0123 paul.armishaw@measurement.gov.au N A T A Accredited for compliance with ISO/IEC 17043 TECHNICAL C O U W W iTCN CE 1 US00001936 This page is intentionally blank US00001937 TABLE OF CONTENTS SUMMARY 1 INTRODUCTION 1.1 NMI Proficiency Testing Program 1.2 Study Background 1.3 Study Aims 1.4 Study Conduct 2 STUDY INFORMATION 2.1 Study Timetable 2.2 Participation 2.3 Test Material Preparation 2.4 Test Material Homogeneity and Stability Testing 2.5 Laboratory Code 2.6 Sample Storage, Dispatch and Receipt 2.7 Instructions to Participants 2.8 Interim Report 3 PARTICIPANT LABORATORY INFORMATION 3.1 Test Methods Reported by Participants 3.2 Basis of Participants' Measurement Uncertainty Estimates 3.3 Participants' Comments 4 PRESENTATIONOF RESULTS AND STATISTICAL ANALYSIS 4.1 Results Summary 4.2 Assigned Value 4.3 Between-Laboratory Coefficient of Variation 4.4 Target Standard Deviation 4.5 z-Score 4.6 En-Score 4.7 Traceability and Measurement Uncertainty 4.8 Robust Average 5 TABLES AND FIGURES 6 DISCUSSION OF RESULTS 6.1 Assigned Value 6.2 Measurement Uncertainty Reported by Participants 6.3 z-Score 6.4 En-Score 6.5 Participants' Methods 6.6 Effects of Sample Matrix 7 REFERENCES APPENDIX 1 - PARTICIPATING LABORATORIES APPENDIX 2 - SAMPLE PREPARATION, HOMOGENEITY TESTING AND STABILITY CHECK A2.1 Sample Preparation A2.2 Sample Analysis and Homogeneity Testing 1 2 2 2 2 2 3 3 3 3 3 3 4 4 4 5 5 14 16 18 18 18 18 18 19 19 19 19 20 58 58 58 59 63 63 65 66 67 68 68 68 iii US00001938 A2.3 Stability Testing for Fish Samples S1 and S2 APPENDIX 3 - ROBUST AVERAGE AND ASSOCIATED UNCERTAINTY APPENDIX 4 - ACRONYMS AND ABBREVIATIONS 79 82 84 IV US00001939 SUMMARY AQA 16-06 was conducted in June 2016. Twenty-four laboratories registered to participate and submitted results. Six test samples were prepared in the NMI North Ryde laboratory and consisted of: Two fish samples, one from a contaminated site (SI) and one formulated to contain 64.9 pg/kg total PFOS and 69.9 pg/kg PFOA (S2). Two soil samples, one from a contaminated site (S3) and one formulated to contain 25.7 pg/kg total PFOS and 14.8 pg/kg PFOA (S4). Two water samples, one from a contaminated site (S5) and one formulated to contain 7 pg/L total PFOS and 12 pg/L PFOA (S6). The samples were sufficiently homogeneous and stable for evaluation of participants' performance. Of a possible 321 numeric results a total of 242 (75%) were submitted. The assigned values were the robust average of participants results. The associated uncertainties were estimated from the robust standard deviation of the participants' results. Traceability: The consensus of participants' results is not traceable to any external reference, so although expressed in SI units, metrological traceability has not been established. The outcomes of the study were assessed against the aims as follows: to compare the performances o fparticipant laboratories and to assess their accuracy in the measurement o f total and linear PFOS and PFOA infish, water and soil matrices; Laboratory performance was assessed using both z-scores and En-scores. Of 232 z-scores, 213 (92%) were satisfactory with |z| < 2. Of 232 En-scores, 174 (75%) were satisfactory with |E,,| < 1. Laboratories 1, 5, 8, 9, 10, 15, 17, 22 and 24 returned satisfactory z and En-scores for all the matrices that they analysed. Laboratory 23 performance was unsatisfactory for both z and E,,-scores. evaluate the laboratories 'methods; Participants used a variety of methods for extraction. No correlation between results and method was evident. The analytical detection method of choice was LCMS/MS. develop the practical application o f traceability and measurement uncertainty and provide participants with information that will be useful in assessing their uncertainty estimates. Two hundred and thirty of two hundred and forty-two numeric results (95%) were reported with an associated estimate of expanded measurement uncertainty. The magnitude of these expanded uncertainties was within the range 0% to 72% of the reported value. Twenty-one were less than 10% relative, which the study coordinator believes is unrealistically small for a routine PFOS/PFOA measurement. Laboratory 16 and 18 did not report expanded measurement uncertainties for all the analytes. AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 1 US00001940 1 INTRODUCTION 1.1 NMI Proficiency Testing Program The National Measurement Institute (NMI) is responsible for Australia's national measurement infrastructure, providing a range of services including a chemical proficiency testing program. Proficiency testing (PT) is: `evaluation of participant performance against pre-established criteria by means of interlaboratory comparison.'1 NMI PT studies target chemical testing in areas of high public significance such as trade, environment, law enforcement and food safety. NMI offers studies in: pesticide residues in fruit and vegetables, soil and water; petroleum hydrocarbons in soil and water; metals in soil, water, food and pharmaceuticals; controlled drug assay; folic acid in flour; and allergens in food. 1.2 Study Background Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are synthetic fluorinated chemicals used as a surface-active agent and in a variety of products such as firefighting foams, coating additives and cleaning products. They are persistent in the environment and resistant to environmental degradation processes. PFOS and PFOA were in 2010 added to the list of chemicals regulated under the international Stockholm Convention for Persistent Organic Pollutants, to which Australia is a signatory. 1.3 Study Aims The aims of the study were to: compare the performances of participant laboratories and assess their accuracy in the measurement of total and linear PFOS and PFOA in fish, soil and water matrices; evaluate the laboratories' test methods; and develop the practical application of traceability and measurement uncertainty and provide participants with information that will be useful in assessing their uncertainty estimates. 1.4 Study Conduct The conduct of NMI proficiency tests is described in the NMI Chemical Proficiency Testing Study Protocol.2 The statistical methods used are described in the NMI Chemical Proficiency Statistical Manual.3 These documents have been prepared with reference to ISO 170431and The International Harmonized Protocol for Proficiency Testing of (Chemical) Analytical Laboratories.4 The study falls within the scope of NMI's accreditation as a proficiency testing provider. Testing for homogeneity of fish samples was subcontracted to National Research Centre for Environmental Toxicology (ENTOX) from University of Queensland. 2 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001941 2 STUDY INFORMATION 2.1 Study Timetable The timetable of the study was: Invitation issued Samples dispatched Results due Interim report issued 12 May 2016 20 June 2016 12 August 2016 24 August 2016 2.2 Participation One hundred and twenty six Australian and international laboratories were invited to participate. Twenty-four laboratories participated (see Appendix 1) and submitted results by the due date. 2.3 Test Material Preparation Six test samples were prepared in April-May 2016. Care was taken to avoid any Teflon contamination during sample preparation. Two fish samples SI and S2 each 5 g, one with incurred contaminants and one spiked with a commercial standard of PFOS and PFOA isomers. Two soil samples S3 and S4 each 10 g, one from a contaminated site and one spiked with a commercial standard of PFOS and PFOA isomers. Two water samples S5 and S6 each 2x50 mL, one from a contaminated site and one spiked with a commercial standard of PFOS and PFOA isomers. Analytical standards used for spiking Samples S2, S4 and S6 were purchased from Sigma Aldrich. On the analytical report provided with the standards, PFOA (product 33824, batch SZBD308XV) has a stated purity of 99.4% and total PFOS (product 33829, batch SZBE107XV) of 98%. No value was provided for linear PFOS. 2.4 Test Material Homogeneity and Stability Testing The preparation of the samples and their testing for homogeneity is described in Appendix 2. Sample preparation has been demonstrated to yield sufficiently homogeneous samples for all three matrices. Stability study for soil and water was conducted in AQA 15-035 and found no significant changes in analytes' concentration. No stability study for these matrices was conducted in this PT study. Participants' results gave no reason to question the stability of soil and water test samples. A stability study of the fish samples was carried out to cover the storage condition during transport, at room temperature, for one month starting 17/06/2016 (before sample dispatch). The stability results for total and linear PFOS and PFOA are given in Appendix 2. Fish samples were demonstrated to be sufficiently stable for use in this PT study. 2.5 Laboratory Code All laboratories that agreed to participate were assigned a confidential code number.3* AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 3 US00001942 2.6 Sample Storage, Dispatch and Receipt Prior to dispatch soil and water samples were refrigerated at 4 C while fish samples were kept in the freezer at -80 C. Participants were sent 5 g fish in Greiner tubes for each of Sample SI and S2, 10 g soil in Greiner tubes for each of Sample S3 and Sample S4 and two 50 mL water in HDPE bottles for each of Sample S5 and Sample S6. The samples were packed in a foam box with a cooler brick and sent by courier on 20 June 2016. The following items were packaged with the samples: a covering letter which included a description of the test samples and instructions for participants; and a faxback form for participants to confirm the receipt and condition of the samples. An Excel spreadsheet for the electronic reporting of results was e-mailed to participants. 2.7 Instructions to Participants Participants were instructed as follows: Quantitatively analyse the samples using your normal test method. Report results in units of pg/kg on as received basis for fish and soil samples Report results in units of ug/L for water samples For each analyte in each sample report a single result expressed as if reporting to a client (ie correct for recovery or not, according to your standard procedure). This figure will be used in all statistical analysis in the study report. For each analyte in each sample report the associated expanded measurement uncertainty (eg 0.50 0.02 pg/kg). Report any analyte not tested as NT. No limit of reporting has been set for this study. Report results as you would to a client, applying the limit of reporting of the method used for analysis. Report the basis of your uncertainty estimates (eg uncertainty budget, repeatability precision, long term result variability). Return the completed results sheet by e-mail proficiencv@measurement.gov.auJ. Please return completed result sheet by 12 August 2016. Late results cannot be included in the study report. 2.8 Interim Report An interim report was emailed to participants on 24 August 2016. 4 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001943 3 PARTICIPANT LABORATORY INFORMATION 3.1 Test Methods Reported by Participants Table 1 Test methods - Samples SI and S2 fish Lab Sample Code weight (g) Sample pretreatment Extraction technique Extraction solvent(s): Clean-up Equipment Column type 11 21 3 1 Frozen 41 0.3 7 0.23 9 1 200 mM NaOH Sonication Acetonitrile / ammonium hydroxide Solvent QuEChERS Acetonitrile Acetonitrile Sonication Methanol Sonication Methanol Sonication Acetonitrile Weak anion Low resolution LC-MSMS C18 SPE Filtration 0.2 um cellulose Active Carbon LC-MSMS LC-MSMS (Qtrap) C18 C18 LC-MSMS C18 BEH High resolution LC-MSMS Low resolution LC-MSMS Nucleodur Sphinx C18 Gemini NX 10 0.5 LLE Acetonitrile SPE-WAX LC-MSMS C18 4.3 11 4.7 Mechanical Methanolic potassium agitation hydroxide SPE LC-MSMS C18 Enzyme 12 0.05 treatment, hexane extraction Ion Pairing MTBE Low resolution LC-MSMS ODS AQ Column Specifications: 5cm x 2.0mm x 1.6pm 5cm x 2.1mm x 1.8pm 4.6cm x 100mm x 2.6pm 5cm x 2.1mm x 1.7pm 3 pm 100/2 5cm x 2.0mm x 3.0pm 15cm x 2.0mm x 3.0pm 10cm x 2.1mm x 3.5pm 15cm x 2.0mm x 3pm Extra column for blank separation Yes No Yes No No No Yes No Yes AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 5 US00001944 Lab Sample Code weight (g) Sample pretreatment Extraction technique Extraction solvent(s): Clean-up Equipment Column type 14 1 Buffered MTBE LC-MSMS C18 15 0.5 Alkalinization SPE 50 mM NaOH MeOH 16 0.5 MTBE/TBAS ion pairing 18 1 Solid/Liquid Acetonitrile 0.087/0.435 Enzyme 24* 0.051/0.255 treatment, 0.076/0.38 hexane 0.087/0.435 extraction Ion pairing, LLE *Laboratory 24 sample weight represents sample/water (g/mL). MTBE SPE C18, Carbon LC-MSMS Low resolution LC-MSMS Low resolution LC-MSMS HSS PFP C18 C18 LC-MSMS ODS AQ Column Specifications: 10cm x 3.0mm x 2.6pm 15cm x 2.1mm x 1.8pm 5cm x 2.0mm x 1.6pm 10cm x 2.1mm x 1.8pm Extra column for blank separation No Yes No No 15cm x 2.0mm x 3pm Yes 6 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001945 Table 2 Test methods - Samples SI and S2 fish (cont'd) Lab code Internal std* 1 13C4PFOS; 13C4PFOA 2 13C4PFOS; 13C4PFOA 3 13C4PFOS; 13C4PFOA 4 13c 4p f o a 7 13C4PFOS; 13C4PFOA 9 13C4PFOS; 13C4PFOA 10 13C4PFOS; 13C4PFOA Recovery std** 13C8PFOS; 13C8PFOA PFOS, PFOA PFNA 13C8PFOA 13C8PFOS; 13C8PFOA 180 2PF0S; 13C8PFOA Recovery correction Yes No Yes No Yes Yes Yes 11 13C4PFOS; 13C2PFOA 13C2PFOUEA; 13C4PFOA Yes If yes, which method? 13C Int. std 13C Int. std Isotope dilution Isotope dilution 13C Int. std 13C Int. std Isotope dilution Standard Method used No 04-068 No No No No No No 12 13C4PFOS; 13C4PFOA No No Blank corrected No No No No Yes No Yes No No 14 13C4PFOS; 13C4PFOA No No 15 13C4PFOS; 13C4PFOA 16 13C8PFOS; 13C4PFOA 18 13C4PFOS; 13C4PFOA 13c 4p f o s 13C Int. std Yes Isotope dilution No Yes 13C Int. std No No No 24 13C4PFOS; 13C4PFOA No No Note: * Internal standard refers to a labelled compound which is spiked directly into samples prior to extraction. ** Recovery standard refers to a labelled compound which is added to the final sample extract prior to instrumental analysis.5 No Yes No No No AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 7 US00001946 Lab Code Sample weight (g) Sample pretreatment Extraction technique 1 0.5 Sonication 21 3 1 Cool Solvent 41 Sonication 51 Sonication 7 0.02 Sonication 0.1/1 8 0.1 Sonication 91 Sonication 11 21.2 23.3 Acidification Mechanical agitation 5 13 5 Table 3 Test methods - Samples S3 and S4 soil Extraction solvent(s): Clean-up Equipment Column type Methanol / ammonium hydroxide Acetonitrile Acetonitrile/methanol Methanol Methanol Methanol Methanol Methanol / ammonium hydroxide Methanolic potassium hydroxide Methanol/water Carbon SPE Low resolution LC-MSMS C18 SPE Filtration 0.2 um cellulose DSPE (active carbon) LC-MSMS LC-MSMS (Qtrap) LC-MSMS High resolution LTQ-Orbitrap High resolution LC-MSMS C18 C18 C18 BEH C18 Nucleodur Sphinx SPE LC-MSMS C8 Active Carbon Low resolution C18 Gemini LC-MSMS NX SPE LC-MSMS C18 LC-MSMS C18 Zorbax extended Column Specifications: 5cm x 2.0mm x 1.6pm 5cm x 2.1mm x 1.8pm 4.6cm x 100mm x 2.6pm 5cm x 2.1mm x 1.7pm 15cm x 2.1mm x 3pm 3pm 100/2 3cm x 100mm x 3.5pm 5cm x 2.0mm x 3.0pm 10cm x 2.1mm x 3.5pm 5cm x 2.1mm x 1.8pm Extra column for blank separation Yes No Yes No No No No No No 8 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001947 Lab Code Sample weight (g) Sample pretreatment Extraction technique Extraction solvent(s): Clean-up Equipment Column type 14 5 1% NH4OH in methanol LC-MSMS C18 16 2 MTBE/TBAS ion pairing Low resolution LC-MSMS C18 17 2 Methanol/acetonitrile Low resolution LC-MSMS C18 18 1 Digestion with base Solid/Liquid Methanol Carbon Low resolution LC-MSMS C18 19 1 Tumble Methanol in 0. l%acetic acid and methanol in 0.1% ammonium hydroxide Filter LC-MSMS C18 Column Specifications: 10cm x 3.0mm x 2.6pm Extra column for blank separation No 5cm x 2.0mm x 1.6pm No 10cm x 2.1mm x 1.7pm Yes 10cm x 2.1mm x 1.8pm No 5cm x 2.0mm x 1.6pm Yes 20 1 10.22 0.67 N NaOH 21 10.15 mixed with sample, pH adjusted to < 2 Leach 5.02 22 5.04 Shaker/sonic ation Methanol Methanol Methanol/KOH LC-MSMS C18 Filtration Low resolution LC-MSMS C18 SPE LC-MSMS C18 7.5cm x 3.0mm x 2.7pm 15cm x 2.0mm x 15pm 15cm x 3.0mm x 1.7pm No No AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 9 US00001948 Table 4 Test methods - Samples S3 and S4 soil (cont'd) Lab code Internal std 1 13C4PFOS; 13C4PFOA Recovery std 13C8PFOS; 13C8PFOA Recovery correction Yes If yes, which method? 13C Int. std Method used No Blank corrected No 2 13C4PFOS; 13C4PFOA PFOS, PFOA PFNA No 04-068 No 3 13C4PFOS; 13C4PFOA Yes 13C Int. std No No 4 13c 4p f o a No No No 5 13C4PFOS; 13C4PFOA Yes 13C Int Std No No 7 13C4PFOS; 13C4PFOA 13C8PFOA Yes Isotope Dilution No Yes 8 13C8PFO; 13C8PFOA [13C7]-PFUdA; [13C3]-PFHxS No No No 9 13C4PFOS; 13C4PFOA 13C8PFOS; 13C8PFOA Yes Isotope Dilution No No 11 13C4PFOS; 13C2PFOA 13C2PFOUEA; 13C4PFOA Yes 13 13c 8p f o s 13C8PFOA, 13C2PFHxA, 13C2- 6:2FTS, d5-N-EtFOSAA 13C4PFOS, 13C2PFOA, d3-NMeFOSAA Yes 14 13C4PFOS; 13C4PFOA No 13C Int. std, Isotope dilution Isotope Dilution No USEPA 537 USEPA 537 No No No 16 13C8PFOS; 13C4PFOA 13c 4p f o s No No No 17 13C4PFOS; 13C4PFOA No DIN38414 No 18 13C4PFOS; 13C4PFOA Yes 13C Int. std No No 19 13c 4p f o s 13C8PFOS; 13C4PFOA Yes 13C Int. std ASTMD 7969-14 No 20 PFOS; PFOA No EPA No 21 13C4PFOS; 13C4PFOA 13C8PFOS; 13C8PFOA Yes Isotope Dilution No No 22 13C4PFOS; 13C4PFOA Yes Isotope Dilution No No Note: * Internal standard refers to a labelled compound which is spiked directly into samples prior to extraction.** Recovery standard refers to a labelled compound which is added to the final sample extract prior to instrumental analysis.5 10 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001949 Lab Code Sample weight (mL) Sample pretreatment Extraction technique 1 (50 mL 1 SPE) 1 Direct injection SPE (for PFOA) Direct injection 2 10 SPE 3 100 Filter through glass microfibre 5 10 DLLME 61 30 7 5 9 50/1 66.8 11 65.9 Direct injection SPE SPE/Direct injection SPE Table 5 Test methods - Samples S5 and S6 water Extraction solvent(s): Clean-up Equipment Column type Dilution Methanol Methanol / NH4OH Dilution Methanol Methanol Octanol Methanol Methanol RC filter SPE RC filter Low resolution LC-MSMS SPE LC-MSMS SPE LC-MSMS (Qtrap) C18 C18 C18 High resolution LTQ-Orbitrap C18 Low resolution LC-MSMS C18 BEH High resolution Nucleodur LC-MSMS Sphinx SPE Low resolution C18 Gemini LC-MSMS NX SPE LC-MSMS C18 Column Specifications: Extra column for blank separation 5cm x 2.0mm x 1.6pm Yes 5cm x 2.1mm x 1.8pm 4.6cm x 100mm x 2.6pm 15cm x 2.1mm x 3pm 5cm x 2.1mm x 1.7pm 3pm 100/2 5cm x 2.0mm x 3.0pm 10cm x 2.1mm x 3.5pm No Yes No No No No No AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 11 US00001950 Lab Code Sample weight (mL) Sample pretreatment Extraction technique 13 50 0.5 g Trizma SPE 14 0.5 16 10 Direct injection SPE 18 10 SPE 19 0.01 Direct injection Extraction solvent(s): Methanol/water Clean-up Equipment Column type SPE LC-MSMS C18 Zorbax extended LC-MSMS Low resolution LC-MSMS Low resolution LC-MSMS C18 C18 C18 Column Specifications: 5cm x 2.1mm x 1.8pm Extra column for blank separation 10cm x 3.0mm x 2.6pm 5cm x 2.0mm x 1.6pm 10cm x 2.1mm x 1.8pm No No No MeOH to stabilise Filter LC-MSMS C18 5cm x 2.0mm x 1.6pm Yes 20 50 131.8 21 133.5 66.3 22 66.5 0.05 23 0.05 SPE Methanol SPE LC-MSMS C18 SPE 10% Methanol/NH4OH Low resolution LC-MSMS C18 SPE Direct injection Methanol/NH4OH SPE LC-MSMS High resolution Orbitrap C18 C18 7.5cm x 3.0mm x 2.7pm 15cm x 2.0mm x 15pm 15cm x 3.0mm x 1.7pm 10cm x 2.1mm x 1.7pm No No No 12 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001951 Table 6 Test methods - Samples S5 and S6 water (cont'd) Lab code Internal std* Recovery std** Recovery correction If yes, which method? Method used 1 13C4PFOS; 13C4PFOA 13C8PFOS; 13C8PFOA Yes 13C Int. std No 2 13C4PFOS; 13C4PFOA PFOS, PFOA PFNA No 04-068 3 13C4PFOS; 13C4PFOA Yes 13C Int. std EPA 537 5 13C4PFOS; 13C4PFOA Yes 13C Int Std No 6 PFOS and PFOA spikes No No 7 13C4PFOS; 13C4PFOA 13C8PFOA Yes Isotope Dilution ISO 9 13C4PFOS; 13C4PFOA 13C8PFOS; 13C8PFOA Yes Isotope Dilution No 11 13C4PFOS; 13C2PFOA 13C2PFOUEA; 13C4PFOA Yes 13C Int. std Isotope dilution No 13 14 13C4PFOS; 13C4PFOA No USEPA 537 16 13C8PFOS; 13C4PFOA 13c 4p f o s No No 18 13C4PFOS; 13C4PFOA Yes 13C Int. std No 19 13c 4p f o s 13C8PFOS; 13C4PFOA Yes 13C Int. std USEPA 537 20 PFOS; PFOA No EPA 21 13C4PFOS; 13C4PFOA 13C8PFOS; 13C8PFOA Yes Isotope Dilution No 22 13C4PFOS; 13C4PFOA Yes Isotope Dilution No 23 13C4PFOS; 13C4PFOA Yes 13C Int. std ISO 17025 Note: * Internal standard refers to a labelled compound which is spiked directly into samples prior to extraction. ** Recovery standard refers to a labelled compound which is added to the final sample extract prior to instrumental analysis.5 Blank corrected No No No No No Yes No No No No No No No No No No AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 13 US00001952 3.2 Basis of Participants' Measurement Uncertainty Estimates Table 7 Basis of Participants' Uncertainty Estimate Lab Approach to Estimating MU Code Information Sources for MU Estimation Precision Method Bias Guide Document for Estimating MU Top Down - precision and 1 estimates of the method and laboratory bias Control samples Duplicate analysis Recoveries of sample spike Nata Technical Note 33 Standard Purity 2 Method validation data Control samples Duplicate analysis Instrument calibration Instrument calibration Recoveries of sample spike NMI Uncertainty Course Standard Purity Bottom Up (ISO/GUM, fish 3 bone/ cause and effect diagram) Control samples Duplicate analysis CRM Eurachem/CITAC Guide Top Down - precision and 4 estimates of the method and laboratory bias Duplicate analysis Recoveries of sample spike ISO/GUM Top Down - precision and 5 estimates of the method and laboratory bias Control samples Recoveries of sample spike Eurachem/CITAC Guide Top Down - precision and 6 estimates of the method and laboratory bias Duplicate analysis Recoveries of sample spike Nata Technical Note 33 Standard Purity 7 Standard deviation of replicate analyses multiplied by 2 or 3 Control samples ISO/GUM Top Down - precision and 8 estimates of the method and laboratory bias Duplicate analysis Recoveries of sample spike Eurachem/CITAC Guide Standard Purity Control samples 9 Standard deviation of replicate analyses multiplied by 2 or 3 Duplicate analysis Instrument calibration Eurachem/CITAC Guide Recoveries of sample spike Instrument calibration 10 Standard deviation of replicate analyses multiplied by 2 or 3 Duplicate analysis In-house SOP Top Down - precision and 11 estimates of the method and laboratory bias Control samples Duplicate analysis Instrument calibration CRM ISO/GUM Recoveries of sample spike 12 Standard deviation of replicate analyses multiplied by 2 or 3 Duplicate analysis Recoveries of sample spike 13 QC DATA - Control Chart Control samples Duplicate analysis Instrument calibration CRM Instrument calibration Recoveries of sample spike Within-laboratory data on bias and precision has been calculated as per the procedures outlined in ASTM E2554-13 Control samples 14 Duplicate analysis Instrument calibration Recoveries of sample spike Instrument calibration 14 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001953 Lab Approach to Estimating MU Code Bottom Up (ISO/GUM, fish 15 bone/ cause and effect diagram) Top Down - precision and 16 estimates of the method and laboratory bias Information Sources for MU Estimation Precision Method Bias Guide Document for Estimating MU Duplicate analysis Instrument calibration Instrument calibration Standard Purity ISO/GUM Control samples Duplicate analysis Instrument calibration Laboratory bias from PT studies Recoveries of sample spike Nata Technical Note 33 Top Down - precision and 17 estimates of the method and laboratory bias DIN38414 18 Standard deviation of replicate analyses multiplied by 2 or 3 Control samples Top Down - precision and 19 estimates of the method and laboratory bias Control samples Duplicate analysis Recoveries of sample spike Nata Technical Note 33 Top Down - precision and 20 estimates of the method and laboratory bias Control samples Instrument calibration Recoveries of sample spike ISO/GUM Standard Purity Top Down - precision and 21 estimates of the method and laboratory bias Control samples Recoveries of sample spike Laboratory QA Manual Top Down - precision and 22 estimates of the method and laboratory bias Control samples Standard practice for Recoveries of sample spike laboratories utilizing US EPA's SW-846 document. 23 Professional judgment Instrument calibration Recoveries of sample spike 24 Standard deviation of replicate analyses multiplied by 2 or 3 Instrument calibration Recoveries of sample spike AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 15 US00001954 3.3 Participants' Comments The study co-ordinator welcomes comments or suggestions from participants about this study or possible future studies. Participants' comments are reproduced in Table 6. Table 8 Participants' Comments Lab Sample Code Participants' Comments Study manager response 1 All Total PFOS is quantified using linear stds. Both total and linear PFOS were quantified using linear PFOS calibration standards. PFOA and PFOS results 2 All are calculated using PFOA-C4 and PFOS-C4 Internal standards respectively. For Sample S3 PFOS results are out side the range of the validated method. The method was modified for sample weights. 3 S3, S5 A dilution was performed on PFOS, reported recovery and S6 was from the non-diluted data. Total PFOS and linear PFOS were quantified with the linear PFOS standard. Average results of 6 replicates 5 All for Samples S3 and S4 with RSD<13% and <11% and average results of 3 replicates for Samples S5 and S6 with RSD<6% and <2%. 9 All Calculation of Total PFOS based on linear PFOS standard 10 SI and S2 Linear PFOS contains branched isomer #2 (Potassium 1-trifhioromethylperfhiorohepatanesulfonate) 11 All Samples were received above our method specifications at ambient temperature. Stability studies for soil and water were performed in AQA 15-03. Analytes stability in fish samples was checked in this study. Samples were confirmed stable at room temperature for at least one month. The concentrations of PFOS and PFOA in the The PT study was designed to cover a 12 S2 unknown fish sample were too high compared to the range of concentrations based on our real samples. participants' capabilities. 13 All Should include the PFHxS as per recent enHealth advice. PFHxS will be included in the next PT study. Measured against non-matrix matched standards. 14 All Internal standard used for instrument correction only for samples S3 to S6. 15 SI Total PFOA is not detected. Reported Total PFOA result is linear PFOA isomer only. Branched PFOA isomers not tested. Reported Total PFOS (sum of di, mono and linear isomers) used All linear PFOS standard for calculation. Total PFOS using br-PFOS standard for calculation is SI 16 ug/Kg, S2 35 ug/Kg, S3 200 ug/Kg, S4 18 ug/Kg, S5 1.9 ug/L and S6 3.6 ug/L. 20 S3, S4, S5 PFOA data calculated on the anion-basis and not and S6 calculated on the salt basis. 16 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001955 Lab Code Sample Participants' Comments Date analyzed above was date of last sample analysed. Total PFOA results are based on Linear PFOA standard. For Samples S3 and S4 two aliquots of the sample were received and were composited prior to taking sample aliquot for processing. Uncertainty is sd from calculated LCS measured amount. (Spike amount 20 ug/kg; n=53) Laboratory evaluates IS recovery but does not report 21 All percent recovery for IS. For Sample S3, the IS recoveries were outside of acceptance limits; sample showed signs of matrix interference For Samples S5 and S6 standard uncertainty is sd from calculated LCS measured amount. (Spike amount 0.2 ug/L; n=63) Laboratory evaluates IS recovery but does not report percent recovery for IS For Sample S6 results reported from the analysis of a diluted extract (5x) due to high concentration of the target analyte in the analysis of the undiluted extract. As a result of dilution, IS did not meet criteria. 22 S3, S4, S5 and S6 The laboratory reports only total PFOS for client data. The Total PFOS value is above the upper calibration limit for the instrument for S3, S5 and S6. Study manager response AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 17 US00001956 4 PRESENTATION OF RESULTS AND STATISTICAL ANALYSIS 4.1 Results Summary Participant results are listed in Tables 6 to 12 with the summary statistics robust average, mean, median, maximum, minimum, robust standard deviation (SDrob) and robust coefficient of variation (CVrob)- Bar charts of results and performance scores are presented in Figures 2 Laboratory Figure 1 Guide to Presentation of Results 4.2 Assigned Value The assigned value is defined1as: `value attributed to a particular property of a proficiency test item.' In this study property is the mass fraction of analyte. Assigned values were the robust average of participants' results; the expanded uncertainties were estimated from the associated robust standard deviations. 4.3 Between-Laboratory Coefficient of Variation The between laboratory coefficient of variation is a measure of the between laboratory variation that in the judgement of the study coordinator would be expected from participants given the analyte concentration. It is important to note this is a performance measure set by the study coordinator; it is not the coefficient of variation of participant results. 4.4 Target Standard Deviation The target standard deviation (a) is the product of the assigned value (X) and the betweenlaboratory coefficient of variation (CV). This value is used for calculation of participant z-score. a = X * CV Equation 1 18 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001957 4.5 z-Score For each participant result a z-score is calculated according to Equation 2 below: z=fc l u Equation 2 where: z X X a is z-score is participant result is the study assigned value is the target standard deviation from equation 1 A z-score with absolute value (|z|): |z| < 2 is satisfactory; 2 < |z| < 3 is questionable; |z| > 3 is unsatisfactory. 4.6 En-Score The En-score is complementary to the z-score in assessment of laboratory performance. En-score includes measurement uncertainty and is calculated according to Equation 3 below: (X-X) Equation 3 where: En is En-score X is a participant's result X is the assigned value U is the expanded uncertainty of the participant's result Ux is the expanded uncertainty of the assigned value An En-score with absolute value (|E,,|): |En| < 1 is satisfactory; |En| > 1 is unsatisfactory. 4.7 Traceability and Measurement Uncertainty Laboratories accredited to ISO/IEC Standard 17025:20156must establish and demonstrate the traceability and measurement uncertainty associated with their test results. Guidelines for quantifying uncertainty in analytical measurement are described in the Eurachem /CITAC Guide.7 4.8 Robust Average The robust averages and associated expanded measurement uncertainties were calculated using the procedure described in `ISO 13528:2015(E), Statistical methods for use in proficiency testing by interlaboratory comparisons'.819 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 19 US00001958 5 TABLES AND FIGURES Sample Details Sample No. Matrix. Analyte. Units S1 Fish Linear PFOS ug/kg Table 9 Participant Results Lab Code Result 1 NT 2 19 3 18.6 4 12 5 NT 6 NT 7 18.7 8 NT 9 19 10 18.841 11 18.7 12 19.86 13 NR 14 NR 15 17.642 16 NT 17 NT 18 18 19 NT 20 NT 21 NT 22 NT 23 NT 24 18.49 Uncertainty NT 4.0 2.9 2.514 NT NT 5.2 NT 2.3 1.839 3.5 1.66 NR NR 1.844 NT NT NR NT NT NT NT NT 0.14 Recovery NT NR 84 77.9 NT NT 42 NT 90 96.8 69.5 86.05 NR NR 92.67 NT NT 101 NT NT NT NT NT 77 Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 18.62 Not spiked 18.6 18.62 18.70 18.08 11 19.86 12 0.50 2.7% 0.38 2.2 0.38 0.30 z-Score 0.10 -0.01 -1.78 0.02 0.10 0.06 0.02 0.33 -0.26 -0.17 -0.03 En-Score 0.09 -0.01 -2.60 0.02 0.16 0.12 0.02 0.73 -0.52 -1.63 -0.32 20 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001959 Results: S1 - Linear PFOS Results (ug/kg) z-Scors z-Scores: S1 - Linear PFOS En-Scores: S1 - Linear PFOS 4 3 2 1 0 -1 -2 -3 -4 15 18 24 3 11 7 10 Laboratory Figure 2 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 12 21 US00001960 En-Score Sample Details Sample No. Matrix. Analyte. Units S1 Fish PFOA ug/kg Table 10 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result <0.2 <1 <0.10 <5 NT NT <0.148 NT < 0.05 0.714 <1.2 0.06 NR <2 <0.010 <1 NT <1 NT NT NT NT NT 0.022 Uncertainty NR NR NR 1.048 NT NT 0.03 NT NR 0.259 1.2 0.00 NR NR NR NR NT NR NT NT NT NT NT 0.00015 Recovery 80 NR 136 77.9 NT NT 50.4 NT NR 103.0 78.7 65.22 NR 9 93.38 91 NT 87 NT NT NT NT NT 152 Statistics* Assigned Value Spike N Not Set Not Spiked 3 *Not enough results to calculate statistics on this analyte. 22 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001961 Results (ug/kg) Kernel Density Results; S1 - PFOA Figure 3 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 23 US00001962 Sample Details Sample No. Matrix. Analyte. Units S1 Fish Total PFOS ug/kg Table 11 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 23 21 20.9 15 NT NT 21 NT 22 20.453 22.5 22.35 NR 12 19.463 12 NT 20 NT NT NT NT NT 20.86 Uncertainty 7 4.0 3.8 5.493 NT NT 5.2 NT 2.5 2.308 4.3 1.81 NR 2.4 7.422 NR NT NR NT NT NT NT NT 0.12 Recovery 67 NR 84 77.9 NT NT 42 NT 90 96.8 69.5 86.05 NR 30 92.67 100 NT NR NT NT NT NT NT 77 Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 20.6 Not Spiked 20.4 20.6 20.9 19.4 14 23 12 1.90 9.2% 1.3 2.4 1.3 1.1 z-Score 0.58 0.10 0.07 -1.36 0.10 0.34 -0.04 0.46 0.42 -2.09 -0.28 -2.09 -0.15 0.06 En-Score 0.34 0.10 0.07 -0.99 0.07 0.50 -0.06 0.42 0.79 -3.15 -0.15 -6.62 -0.46 0.20 24 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001963 Results; S1 - Total PFOS Results (ug/kg) z-Scors z-Scores: S1 - Total PFOS Target Standard Deviation 4.1 ug/kg (Equivalent te 20% CV) -1 -2 1 -3 -4 14 16 15 18 10 24 3 Laboratory 2 En-Scores: S1 - Total PFOS 12 11 Figure 4 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 25 US00001964 En-Score Sample Details Sample No. Matrix. Analyte. Units S2 Fish Linear PFOS ug/kg Table 12 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result NT 40 34.2 23 NT NT 36 NT 38 39.768 40.1 45.40 NR NR 41.056 NT NT 29 NT NT NT NT NT 39.75 Uncertainty NT 8 5.3 8.423 NT NT 14 NT 4.5 9.240 7.6 1.18 NR NR 4.852 NT NT NR NT NT NT NT NT 0.017 Recovery NT NR 71 79.8 NT NT 53.6 NT 95 93.9 88.5 75.83 NR NR 89.33 NT NT 105 NT NT NT NT NT 78 z-Score 0.18 -0.57 -2.02 -0.34 -0.08 0.15 0.19 0.88 0.32 -1.24 0.15 Statistics Assigned Value Spike* Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 38.6 NA 41.8 38.6 39.8 36.9 11 45.4 23 2.8 7.3% 2.1 5.4 2.1 1.7 *Sample was spiked with a technical mixture, linear PFOS value has not been certified. En-Score 0.17 -0.77 -1.80 -0.18 -0.12 0.12 0.19 2.82 0.46 -4.57 0.55 26 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001965 Results (ug/kg) Kernel Density Results; S2 - Linear PFOS z-Scores: S2 - Linear PFOS z-Score E,,-Score 4 -r 321 0 -1 -3 - -4 -l--------------- -------------------------------------------------------------------------------------------------------------------------------------- 4 18 3 7 9 24 10 2 11 15 12 Laboratory Figure 5 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 27 US00001966 Sample Details Sample No. Matrix. Analyte. Units S2 Fish PFOA ug/kg Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 55 57 29.5 33 NT NT 46.2 NT 52 61.190 58.8 52.42 NR 37 58.003 53 NT 46 NT NT NT NT NT 50.19 Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 50.5 69.9 51.8 50.5 52.2 49.2 14 61.19 29.5 8.6 17% Table 13 Uncertainty 10 12 5.3 6.914 NT NT 9.2 NT 6.5 14.672 10.0 0.79 NR 7.4 21.852 NR NT NR NT NT NT NT NT 0.074 Recovery 80 NR 90 79.8 NT NT 57.7 NT 92 88.6 76.3 84.06 NR 145 94.26 99 NT 75 NT NT NT NT NT 113 5.7 3.5 6.7 5.7 5.1 z-Score 0.45 0.64 -2.08 -1.73 -0.43 0.15 1.06 0.82 0.19 -1.34 0.74 0.25 -0.45 -0.03 En-Score 0.39 0.49 -2.70 -1.95 -0.40 0.17 0.68 0.72 0.33 -1.45 0.33 0.44 -0.79 -0.05 28 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001967 Kernel Density Results; S2 - PFOA Results (ug/kg) z-Scors z-Scores: S2 - PFOA Target Standard Deviation 10.1 ug/kg (Equivalent to 20% CV) ----------- ^.............* 0.0 , - 0,6 0.4 ,.... ::..... ,.... - .....r.... : : .....f.............I 2- ~3 b 3 4 14 18 7 24 9 12 16 1 2 15 11 10 Laboratory En-Scores: S2 - PFOA Figure 6 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 29 US00001968 En-Score Sample Details Sample No. Matrix. Analyte. Units S2 Fish Total PFOS ug/kg Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 70 61 46.8 34 NT NT 55.5 NT 56 54.273 58.7 67.75 NR 32 56.320 42 NT 43 NT NT NT NT NT 61.09 Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 53.7 64.9 57.7 53.7 55.8 52.8 14 70 32 11.3 21% Table 14 Uncertainty 22 12 8.3 12.451 NT NT 14 NT 6.6 10.174 11.1 1.76 NR 6.4 16.064 NR NT NR NT NT NT NT NT 1.55 Recovery 70 NR 71 79.8 NT NT 53.6 NT 95 93.9 88.5 75.83 NR 19 89.33 98 NT NR NT NT NT NT NT 78 7.6 3.2 7.4 7.6 6.1 z-Score 1.52 0.68 -0.64 -1.83 0.17 0.21 0.05 0.47 1.31 -2.02 0.24 -1.09 -1.00 0.69 En-Score 0.70 0.51 -0.61 -1.35 0.11 0.23 0.05 0.37 1.80 -2.18 0.15 -1.54 -1.41 0.95 30 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001969 Kernel Density Results; S2 - Total PFOS Results (ug/kg) z-Score z-Scores: S2 - Total PFOS Target Standard Deviation 10.7 ug/kg (Equivalent to 20% CV) 14 16 18 10 7 9 15 11 Laboratory En-Scores; S2 - Total PFOS 4 -,----------------------------------------------------------------------------------------------------------------3- 24 12 -1 ----------------------------------------------------------------------------------------------- -2 -............ '`i -- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -3 - - 4 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- 14 4 16 18 3 10 7 9 15 11 2 24 12 1 Laboratory Figure 7 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 31 US00001970 En-Score Sample Details Sample No. Matrix. Analyte. Units S3 Soil Linear PFOS ug/kg Table 15 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result NT 195 119 150 217 NT 194 NT 183 NT 178 NT 212.6 NR NT NT NT 170 140 146 268 NT NT NT Uncertainty NT 40 18.0 28.110 28 NT 140 NT 21 NT 28 NT 42.5 NR NT NT NT 44 35 52.6 2.6 NT NT NT Recovery NT NR 50 83.5 74.7 NT 76.1 NT 85 NT 60.1 NT NR NR NT NT NT 90 85 NR NR NT NT NT Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 179 Not Spiked 194 179 180 181 12 268 119 40 22% 29 39 29 29 z-Score 0.45 -1.68 -0.81 1.06 0.42 0.11 -0.03 0.94 -0.25 -1.09 -0.92 2.49 En-Score 0.32 -1.76 -0.72 0.94 0.10 0.11 -0.02 0.65 -0.17 -0.86 -0.55 3.06 32 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001971 Kernel Density Results: S3 - Linear PFOS Results (ug/kg) z-Score z-Scores: S3 - Linear PFOS 4 Target Standard Deviation 38 ug/kg (Equivalent to 20% CV) 3 2 1 0 -1 - -2 -3 -4 19 20 18 11 9 Laboratory 13 21 En-Scores: S3 - Linear PFOS 4 -,-------------------------------------------------------------------------------------------------------------------------------------------------------------- 3- r 2- 1- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ -1 -2 - ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------0 3 7J ' -3 - - 4 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------3 19 20 4 18 11 9 7 2 13 5 21 Laboratory Figure 8 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 33 US00001972 E,,-Score Sample Details Sample No. Matrix. Analyte. Units S3 Soil PFOA ug/kg Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 5.4 6.0 5.58 <5 6.11 NT 5.9 4.9 5.7 NT 5.08 NT 5.83 6.9 NT 7.45 6.08 6.0 4.0 4.97 5.97 7.00 NT NT Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 5.83 Not Spiked 5.9 5.83 5.90 5.82 17 7.45 4 0.56 9.6% Table 16 Uncertainty 1.0 1.6 0.99 1.051 0.69 NT 2.9 1.0 0.5 NT 0.66 NT 1.2 1.4 NT 2.563 1.22 1.4 0.9 1.8 0.91 3.15 NT NT Recovery 83 NR 93 83.5 99.3 NT 64.8 87.8 87 NT 79.9 NT 97 120 NT 102 75 100 85 NR NR 98 NT NT 0.34 1.2 0.34 0.24 z-Score -0.37 0.15 -0.21 0.24 0.06 -0.80 -0.11 -0.64 0.00 0.92 1.39 0.21 0.15 -1.57 -0.74 0.12 1.00 En-Score -0.41 0.10 -0.24 0.36 0.02 -0.88 -0.22 -1.01 0.00 0.74 0.63 0.20 0.12 -1.90 -0.47 0.14 0.37 34 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001973 Kernel Density Results; S3 - PFOA Results (ug/kg) z-Score z-Scores: S3 - PFOA Target Standard Deviation 1.17 ug/kg (Equivalent to 20% CV) , , - 1 0 < 0 < 3.2 3 ; \.......... ...................... 1 - ' 5 3 - 3 - " ' 1 ......................... 19 8 20 11 1 3 13 7 21 18 2 1? 5 14 22 18 Laboratory En-Scores; S3 - PFOA 4 3 2 1 0 -1 -2 -3 -4 19 8 20 11 1 3 9 13 7 21 18 2 17 5 14 22 18 Laboratory Figure 9 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 35 US00001974 En-Score Sample Details Sample No. Matrix. Analyte. Units S3 Soil Total PFOS ug/kg Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 250 330 176 210 272 NT 282 267 275 NT 269 NT 336.6 260 NT 164 267.74 260 170 214.4 NT 290 NT NT Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 262 Not Spiked 270 262 267 253 17 336.6 164 29 11% Table 17 Uncertainty 80 99 34.4 39.354 35 NT 140 53 31 NT 43 NT 67.3 52 NT 27.2 66.94 NR 45 77.2 NT 154 NT NT Recovery 100 NR 50 83.5 74.7 NT 76.1 88.1 85 NT 60.1 NT 94 107 NT 97 86 NA 85 NR NT 51 NT NT 18 54 18 13 z-Score -0.23 1.30 -1.64 -0.99 0.19 0.38 0.10 0.25 0.13 1.42 -0.04 -1.87 0.11 -0.04 -1.76 -0.91 0.53 En-Score -0.15 0.68 -2.22 -1.20 0.25 0.14 0.09 0.36 0.15 1.07 -0.04 -3.00 0.08 -0.11 -1.90 -0.60 0.18 36 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001975 Results (ug/kg) Kernel Density Results; S3 - Total PFOS z-Scores: S3 - Total PFOS z-Score E,,-Score 4 3 2 1 0 -1 -2 -3 -4 18 19 3 4 20 1 14 18 8 17 11 5 7 22 2 13 Laboratory Figure 10 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 37 US00001976 Sample Details Sample No. Matrix. Analyte. Units S4 Soil Linear PFOS ug/kg Table 18 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result NT 20 14.1 11 18.75 NT 15.5 NT 17 NT 19.1 NT 19.56 NR NT NT NT 15 12 11.1 23.7 NT NT NT Uncertainty NT 4.0 2.2 3.804 5.24 NT 11 NT 2 NT 3.0 NT 3.9 NR NT NT NT 3.9 3 4 2.6 NT NT NT Recovery NT NR 94 70.1 103 NT 57.5 NT 87 NT 77.7 NT NR NR NT NT NT 96 86 NR NR NT NT NT z-Score 1.13 -0.67 -1.63 0.75 -0.25 0.21 0.86 1.00 -0.40 -1.32 -1.60 2.27 Statistics Assigned Value Spike* Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 16.3 NA 22.7 16.3 16.2 16.4 12 23.7 11 4.4 27% 3.1 4.5 3.1 2.9 *Sample was spiked with a technical mixture, linear PFOS value has not been certified. En-Score 0.73 -0.58 -1.08 0.40 -0.07 0.19 0.65 0.65 -0.26 -1.00 -1.03 1.83 38 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001977 Results (ug/kg) Results: S4 - Linear PFOS Assigned Value = 16.3 3.1 ug/kg 30 4 20 15 3 15 ? 9 . 11 13 - 1 10 12 18 18 17 22 23 24 8 Laboratory z-Scores: S4 - Linear PFOS Md R 4 HV z-Score En-Score -3 - "4 *..................................................................................................................................................................... ................ 4 20 19 3 18 7 9 5 11 13 2 21 Laboratory Figure 11 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 39 US00001978 Sample Details Sample No. Matrix. Analyte. Units S4 Soil PFOA ug/kg Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 11 13 9.96 5.8 11.98 NT 12.1 10.2 13 NT 12.6 NT 12.92 12 NT 19.8 13.62 13 8.6 9.43 12.3 14.2 NT NT Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 12.0 14.80 15.2 12.0 12.20 11.97 18 19.8 5.8 1.7 14% Table 19 Uncertainty 2 3.0 1.78 1.219 1.35 NT 6 2.0 1.1 NT 1.6 NT 2.6 2.4 NT 5.93 2.72 3.1 2 3.4 0.91 6.39 NT NT Recovery 79 NR 111 70.1 93.3 NT 69.1 86.3 82 NT 97.4 NT 101 120 NT 95 60 103 86 NR NR 91 NT NT 1.0 0.70 3.0 1.0 0.74 z-Score -0.42 0.41 -0.85 -2.59 -0.01 0.04 -0.75 0.41 0.25 0.38 0.00 3.24 0.67 0.41 -1.42 -1.07 0.12 0.91 En-Score -0.45 0.31 -1.00 -3.94 -0.02 0.01 -0.81 0.67 0.31 0.33 0.00 1.30 0.56 0.30 -1.52 -0.73 0.21 0.34 40 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001979 Kernel Density Results: S4-PFOA Results (ug/kg) z-Score z-Scores: S4 - PFOA Target Standard Deviation 2.4 ug kg {Equivalent to 20% CV) -j r*149 -3 -4 4 18 20 3 8 1 14 7 21 11 13 18 Laboratory 2 8 17 22 16 En-Scores: S4 - PFOA 4 3 2 1 0 -1 -2 -3 -4 19 20 3 8 1 5 14 7 21 11 13 18 2 9 17 22 18 Laboratory Figure 12 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 41 US00001980 E,,-Score Sample Details Sample No. Matrix. Analyte. Units S4 Soil Total PFOS ug/kg Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 20 32 18.9 17 22.27 NT 22.6 19.2 25 NT 26.0 NT 30.42 24 NT 21.8 22.61 22 15 16.4 NT 23.5 NT NT Statistics Assigned Value Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 22.0 25.7 26.5 22.0 22.3 22.3 17 32 15 4.2 19% Table 20 Uncertainty 8 6.0 3.7 5.879 7.79 NT 11 3.8 2.8 NT 4.2 NT 6.1 4.8 NT 4.09 5.65 NR 4 5.9 NT 12.5 NT NT Recovery 97 NR 94 70.1 103 NT 57.5 89.1 87 NT 77.7 NT 103 107 NT 104 93 NR 86 NR NT 43 NT NT 2.6 1.3 5.3 2.6 2.1 z-Score -0.45 2.27 -0.70 -1.14 0.06 0.14 -0.64 0.68 0.91 1.91 0.45 -0.05 0.14 0.00 -1.59 -1.27 0.34 En-Score -0.24 1.53 -0.69 -0.78 0.03 0.05 -0.61 0.79 0.81 1.27 0.37 -0.04 0.10 0.00 -1.47 -0.87 0.12 42 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001981 Results (ug/kg) Kernel Density Results; S4 - Total PFOS z-Scores: S4 - Total PFOS z-Score En-Score 4 3 -3 - .4 -.............................................................................................................................................................................................................. 19 20 4 3 8 1 18 18 5 ? 1? 22 14 9 11 13 2 Laboratory Figure 13 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 43 US00001982 Sample Details Sample No. Matrix. Analyte. Units S5 Water Linear PFOS ug/L Table 21 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result NT 2.1 1.09 NT 1.90 1.9 1.88 NT 1.6 NT 2.13 NT 1.79 NR NT NT NT 1.4 1.3 0.94 3.14 NT 3.374 NT Uncertainty NT 0.6 0.44 NT 0.25 0.4 0.49 NT 0.23 NT 0.40 NT 0.4 NR NT NT NT 0.21 0.3 0.56 0.016 NT 0.944 NT Recovery NT NR 49 NT 106 NR 52.3 NT 93 NT 70.8 NT NR NR NT NT NT 99 87 NR NR NT NR NT Statistics Assigned Value* Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 1.65 Not Spiked 2.00 1.81 1.88 1.89 13 3.374 0.94 0.49 27% 0.32 0.40 0.34 0.25 *Robust average excluding laboratories 21 and 23. z-Score 1.36 -1.70 0.76 0.76 0.70 -0.15 1.45 0.42 -0.76 -1.06 -2.15 4.52 5.22 En-Score 0.66 -1.03 0.62 0.49 0.39 -0.13 0.94 0.27 -0.65 -0.80 -1.10 4.65 1.73 44 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001983 Kernel Density Results: S5 - Linear PFOS Results (ug/L) z-Score z-Scores: S5 - Linear PFOS 4 Target Standard Deviation 0.33 ug/L (Equivalent to 20% CV) 3 2 1~ 0 " 3l 5 3 1i 0 -1 -2 -3 .... > - .... 03 .............................................................................................................. -4 20 3 19 18 9 13 7 5 6 2 11 21 23 Laboratory En-Scores: SS - Linear PFOS Laboratory Figure 14 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 45 US00001984 E,,-Score Sample Details Sample No. Matrix. Analyte. Units S5 Water PFOA ug/L Table 22 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result <0.01 <0.02 0.002 NT 0.087 5.8 0.00214 NT 0.0016 NT <0.01 NT < 0.01 <0.1 NT <0.001 NT <0.001 <0.01 <0.05 <0.0379 0.00791 0.153 NT Uncertainty NR NR 0.0008 NT 0.010 1.2 0.00043 NT 0.0001 NT 0.01 NT 0.01 NR NT 0.0002 NT NR NR 0.018 0.017 0.0030 0.042 NT Recovery 101 NR 141 NT 94 NR 77.8 NT 94 NT 81.2 NT 100 100 NT 91 NT 99 87 NR NR 43 NR NT Statistics Assigned Value Spike Robust Average Median Mean N Max. Min. Not Set Not Spiked 0.011 0.0079 0.86 7 5.8 0.0016 0.011 0.0087 46 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001985 Results (ufl/L) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -I-------- ,----- 3 Results; S5 - PFOA 1 11 19 2 Laboratory 21 20 14 10 12 15 17 24 8 Figure 15 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 47 US00001986 Sample Details Sample No. Matrix. Analyte. Units S5 Water Total PFOS ug/L Table 23 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 3.4 4.2 1.94 NT 2.54 2.7 3.84 NT 2.8 NT 3.64 NT 3.84 3.2 NT 3.290 NT 2.6 1.8 1.88 NT 3.23 5.42 NT Uncertainty 1.1 1.2 0.25 NT 0.33 0.5 0.49 NT 0.46 NT 0.69 NT 0.8 0.64 NT 0.5659 NT NR 0.4 .68 NT 1.87 1.518 NT Recovery 72 NR 49 NT 106 NR 52.3 NT 93 NT 70.8 NT 103 95 NT 84 NT NR 87 NR NT 85 NR NT Statistics Assigned Value* Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 3.00 Not Spiked 3.24 3.09 3.22 3.14 16 5.42 1.8 0.92 30% *Robust average excluding laboratory 23. 0.55 0.65 0.57 0.49 z-Score 0.67 2.00 -1.77 -0.77 -0.50 1.40 -0.33 1.07 1.40 0.33 0.48 -0.67 -2.00 -1.87 0.38 4.03 En-Score 0.33 0.91 -1.75 -0.72 -0.40 1.14 -0.28 0.73 0.87 0.24 0.37 -0.73 -1.76 -1.28 0.12 1.50 48 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001987 Kemel Density Results: S5 - Total PFOS Results (ug/L) z-Score z-Scores: S5 - Total PFOS Target Standard Deviation 0.60 ug/L (Equivalent to 20% CV) 19 20 18 6 9 14 22 16 1 11 13 Laboratory En-Scores: SS - Total PFOS 4 -,----------------------------------------------------------------------------------------------------------------32- 23 -2 - . 5 .. 3 -3 - - 4 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 19 20 3 5 18 6 9 14 22 16 1 11 13 7 2 23 Laboratory Figure 16 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 49 US00001988 En-Score Sample Details Sample No. Matrix. Analyte. Units S6 Water Linear PFOS ug/L Table 24 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result NT 5.1 3.27 NT 4.70 5.1 5.03 NT 4.3 NT 5.80 NT 4.87 NR NT NT NT 3.2 3.1 2.74 6.53 NT 8.478 NT Uncertainty NT 1.5 0.75 NT 0.62 1.0 0.8 NT 0.6 NT 1.10 NT 1.0 NR NT NT NT 0.48 0.7 1 0.016 NT 2.373 NT Recovery NT NR 41 NT 89 NR 79.9 NT 93 NT 72.4 NT NR NR NT NT NT 107 91 NR NR NT NR NT z-Score 0.67 -1.37 0.22 0.67 0.59 -0.22 1.44 0.41 -1.44 -1.56 -1.96 2.26 4.42 Statistics Assigned Value* Spike** Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 4.5 Not Spiked 4.97 4.7 4.87 4.79 13 8.478 2.74 1.5 32% 1.0 0.99 1.0 0.83 *Robust average excluding laboratory 23. **Sample was spiked with a technical mixture, linear PFOS value has not been certified. En-Score 0.33 -0.98 0.17 0.42 0.41 -0.17 0.87 0.26 -1.17 -1.15 -1.24 2.03 1.54 50 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001989 Kernel Density Results; S8 - Linear PFOS Results (ug/L) z-Score z-Scores: S6 - Linear PFOS Target Standard Deviation 0.9 ug/L (Equivalent to 20% CV) ,- -02 n 0.7 0.7 | ...........................|-.......................... ; 5 20 19 18 3 5 13 ? 2 Laboratory En-Scores; S6 - Linear PFOS 4 -,-------------------------------------------------------------------------------------------------------------------32 -*20 8 11 21 23 -1 - ........... ............ ......................... .................................................... ............................................................................................................................................................................................................................ -2 - ' ` -3 - 4 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 20 19 18 3 9 5 13 7 2 6 11 21 23 Laboratory Figure 17 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 51 US00001990 En-Score Sample Details Sample No. Matrix. Analyte. Units S6 Water PFOA ug/L Table 25 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 12 12 9.78 NT 10.02 19 10.6 NT 9.7 NT 11.7 NT 11.36 10.4 NT 13.54 NT 12 9.1 10.1 11.8 9.01 18.598 NT Uncertainty 3 2.4 2.47 NT 1.12 3.8 2.1 NT 0.87 NT 2.0 NT 2.3 2.1 NT 2.302 NT 3.4 2 3.6 0.017 3.51 5.207 NT Recovery 131 NR 47 NT 93 NR 97.4 NT 94 NT 103 NT 112 100 NT 94 NT 95 91 NR NR 62 NR NT Statistics Assigned Value* Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 10.83 12.00 11.5 11.2 11.36 11.81 17 19 9.01 1.79 16% 0.89 0.60 2.3 1.1 0.96 *Robust average excluding laboratories 6 and 23. z-Score 0.54 0.54 -0.48 -0.37 3.77 -0.11 -0.52 0.40 0.24 -0.20 1.25 0.54 -0.80 -0.34 0.45 -0.84 3.59 En-Score 0.37 0.46 -0.40 -0.57 2.09 -0.10 -0.91 0.40 0.22 -0.19 1.10 0.33 -0.79 -0.20 1.09 -0.50 1.48 52 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001991 Results (ug/L) Kernel Density Results: S6 - PFOA z-Scores: S6 - PFOA Target Standard Deviation 2.17 ug/L (Equivalent to 20% CV) i "' --------- [---------1--------- C = ^ ^ = ....................... ^ 3, ss = : ,--------- 1--------- 1--------- 1 I I t.......... 1.......... 1...........1.......... 1.......... 1....................... .................. 22 19 9 3 5 20 14 7 13 11 21 1 18 2 16 23 6 Laboratory En-Scores: S6 - PFOA z-Scors En-Score Figure 18 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 53 US00001992 Sample Details Sample No. Matrix. Analyte. Units S6 Water Total PFOS ug/L Table 26 Participant Results Lab Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Result 8.3 8.4 4.56 NT 5.60 6.5 8.22 NT 6.3 NT 7.85 NT 7.85 6.5 NT 6.979 NT 4.9 3.9 4.12 NT 6.81 11.387 NT Uncertainty 2.6 2.5 1.06 NT 0.74 1.3 0.8 NT 1.0 NT 1.49 NT 1.6 1.3 NT 1.200 NT NR 0.8 1.5 NT 3.95 3.188 NT Recovery 48 NR 41 NT 89 NR 79.9 NT 93 NT 72.4 NT 118 95 NT 90 NT NR 91 NR NT 95 NR NT Statistics Assigned Value* Spike Homogeneity Value Robust Average Median Mean N Max. Min. Robust SD Robust CV 6.5 7.00 7.5 6.6 6.7 6.8 16 11.387 3.9 1.90 29% *Robust average excluding laboratory23. 1.1 0.40 1.5 1.2 1.1 z-Score 1.38 1.46 -1.49 -0.69 0.00 1.32 -0.15 1.04 1.04 0.00 0.37 -1.23 -2.00 -1.83 0.24 3.76 En-Score 0.64 0.70 -1.27 -0.68 0.00 1.26 -0.13 0.73 0.70 0.00 0.29 -1.45 -1.91 -1.28 0.08 1.45 54 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001993 Kernel Density Results; S8 - Total PFOS Results (ug/L) z-Score z-Scores: S6 - Total PFOS Target Standard Deviation 1.3 ug/L (Equivalent to 20% CV) i-------- r 19 20 18 14 6 22 16 Laboratory 11 En-Scores; S6 - Total PFOS 4 -,----------------------------------------------------------------------------------------------------------------3- 13 23 1 - ---------------------------------------------------------------------------------------- - ------ - -- -----------m ^ -1 - ......... ........................................ ......................^ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -2 --- ^ ' ' ,'5 -3 - - 4 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------19 20 3 18 5 9 14 6 22 16 11 13 7 1 2 23 Laboratory Figure 19 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 55 US00001994 En-Score z-Score 10 8 e 4 2 0 u i:-2 -4 -e i' -8 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 21 2223 24 Figure 20 z-Score Dispersal by Laboratory e e o A!! Z-Score OOOOOOOOOOOOOOOO mm m m Figure 21 z-Score Dispersal by Analyte 56 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001995 En-Score 10 Figure 22 En-Score Dispersal by Laboratory AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 57 US00001996 6 DISCUSSION OF RESULTS 6.1 Assigned Value The robust average of participants' results was used as the assigned values for all samples. The robust averages and associated expanded uncertainties, were calculated using the procedure described in `ISO 13528:2015(E), Statistical methods for use in proficiency testing by interlaboratory comparisons'.8 The calculation of the expanded uncertainty for the robust average of PFOA in Sample S6 is presented in Appendix 3. Results less than 50% and greater than 150% of the robust average were removed before calculation of the assigned value.3,4 Assigned values for spiked Samples S2, S4 and S6 were within the range 72 - 92% of the spiked concentration for that analyte (Table 27). No assigned values were calculated for PFOA in Samples SI and S5, the levels in the incurred samples were very low and only a few laboratories were able to report numeric results. Traceability: The consensus of participants' results is not traceable to any external reference, so although expressed in SI units, metrological traceability has not been established. Table 27 Comparison of Assigned Value and Spiked Concentration. Sample Matrix Analyte S2 Fish PFOA S2 Fish Total PFOS S4 Soil PFOA S4 Soil Total PFOS S6 Water PFOA S6 Water Total PFOS Units Pg/kg Pg/kg Pg/kg Pg/kg pg/L pg/L Spiked Concentration Assigned Value 69.9 50.5 64.9 53.7 14.8 12.0 25.7 22.0 12.0 10.83 7.0 6.5 Assigned/ Spike (%) 72 83 81 86 90 93 6.2 Measurement Uncertainty Reported by Participants Participants were asked to report an estimate of the expanded uncertainty associated with their results and the basis of this uncertainty estimate (Table 4). It is a requirement of the ISO Standard 17025 that laboratories have procedures to estimate the uncertainty of chemical measurements and to report this uncertainty in specific circumstances, including: `when the client's instruction so requires.' Two hundred and thirty of two hundred and forty-two results (95%) were reported with an associated estimate of expanded measurement uncertainty. Laboratories 16 and 18 reported expanded measurement uncertainties only for some analytes. The magnitude of the reported expanded uncertainties was within the range 0% to 72% of the reported value. Twenty-one were less than 10% relative, which the study coordinator believes is unrealistically small for a routine PFOS/PFOA measurement. Results returning a satisfactory z-score but an unsatisfactory En-score may have underestimated the uncertainty. Some participants attached an estimate of the expanded measurement uncertainty to a result reported as less than their limit of reporting. 58 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001997 In some cases the results were reported with an inappropriate number of significant figures. The recommended format is to write uncertainty to no more than two significant figures and then to write the result with the corresponding number of decimal places (for example instead of 210 39.354 pg/kg better report 210 39 pg/kg)7. 6.3 z-Score A target standard deviation equivalent to 20% coefficient of variation (CV) was used to calculate z-scores. The between laboratory coefficient of variation predicted by the modified Horwitz equation9 and the between laboratories CV are presented for comparison in Table 28. Table 28 Target standard deviation, between laboratories CV and modified Horwitz values Sample Analyte Assigned value Unit SI Linear PFOS 18.62 Pg/kg SI Total PFOS 20.6 Pg/kg S2 Linear PFOS 38.6 Pg/kg S2 PFOA 50.5 Pg/kg S2 Total PFOS 53.7 Pg/kg S3 Linear PFOS 179 Pg/kg S3 PFOA 5.83 Pg/kg S3 Total PFOS 262 Pg/kg S4 Linear PFOS 16.3 Pg/kg S4 PFOA 12.0 Pg/kg S4 Total PFOS 22.0 Pg/kg S5 Linear PFOS 1.65 pg/L Target SD (as CV, %) 20 20 20 20 20 20 20 20 20 20 20 20 Modified Horwitz CV (%) 22 22 22 22 22 22 22 20 22 22 22 22 Between laboratories' CV (%) 2.7 9.2 7.3 17 21 22 9.6 11 27 14 19 27 S5 Total PFOS 3.00 pg/L 20 22 30 S6 Linear PFOS 4.5 pg/L 20 22 32 S6 PFOA 10.83 pg/L 20 22 16 S6 Total PFOS 6.5 pg/L 20 22 29 The dispersal of participants' z-scores is graphically presented in Figure 20 and by analyte in Figure 21. Of the two hundred and thirty-two results for which z-scores were calculated, 213 (92%) returned a satisfactory z-score of |z| <2. Nine laboratories analysed all three matrices. Laboratories 1, 7, 9, 11 and 18 returned satisfactory z-scores for all analytes tested for which z-scores were calculated. Seven laboratories analysed two matrices. Laboratories 5, 13, 22 and 19 returned satisfactory zscores for all analytes tested for which z-scores were calculated. Eight laboratories analysed only one matrix. Laboratories 8, 10, 12, 15, 17 and 24 returned satisfactory z-scores for all analytes tested for which z-scores were calculated. Laboratory 23 enrolled for water matrix only. This laboratory returned |z| > 3 for all test samples demonstrating an unsatisfactory performance. AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 59 US00001998 Scatter plots of z-scores for all analytes are presented in Figures 23-29. Scores are predominantly plotted in quadrants I and III, indicating laboratory bias is the major contributor to the variability of results. S1 and S2 - Total PFOS S1 Figure 23 Fish z-score scatter plot Total PFOS S1 and S2 - Linear PFOS S1 Figure 24 Fish z-score scatter plot Linear PFOS 60 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00001999 S3 and S4 - Total PFOS S3 Figure 25 Soil z-score scatter plot Total PFOS S3 and S4 - Linear PFOS S3 Figure 26 Soil z-score scatter plot Linear PFOS AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 61 US00002000 S3 and S4 - PFOA S3 Figure 27 Soil z-score scatter plot PFOA S5 and S6 - Total PFOS S5 Laboratory 23 is off-scale. Figure 28 Water z-score scatter plot Total PFOS 62 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002001 S6 and S5 - Linear PFOS S5 Laboratories 21 and 23 are off-scale. Figure 29 Water z-score scatter plot Linear PFOS 6.4 En-Score Where a laboratory did not report an uncertainty estimate an uncertainty of zero (0) was used to calculate the En-score. Of two hundred and thirty-two En-scores, 174 (75%) were satisfactory with |En| < 1. Laboratories 1 and 9 that analysed three matrices returned satisfactory En-score for all analytes tested for which En-scores were calculated. Laboratory 5 that analysed two matrices and laboratories 8, 10, 15, 17, 22 and 24 that analysed one matrix returned satisfactory E,,-score for all analytes for which En-scores were calculated. Laboratory 23 analysed one matrix, water, and returned |En| > 1 for all test samples. This indicates that the reported results did not agree with the assigned value and that the uncertainties associated with the results have been understated. The dispersal of participants' E,,-scores is presented in Figure 22. 6.5 Participants' Methods Participants were requested to analyse the samples using their normal test method and to report a single result as they would normally report to a client. Results reported in this way reflect the true variability of results reported to clients. The method descriptions provided by participants are presented in Tables 1 to 6. The study coordinator thanks all laboratories that completed the method questionnaire. A summary is presented below. Fish samples Pre-treatment: freezing (1), enzyme treatment (2) and alkalinization (2). Extraction technique: sonication (4), liquid-liquid (1), solid-liquid (1), ion pairing (3) and mechanical agitation (1). AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 63 US00002002 Clean-up: weak anion (1), SPE (4), filtration (1) active carbon (1) and C18, Carbon (1). Extraction solvent: acetonitrile (6), methanol (4) and MTBE (4). Calibration standard for PFOS analysis: mixed (8) and linear (6). Soil samples Pre-treatment: cooling (1), acidification (1), digestion with base (1) and pH adjustment (1). Extraction technique: sonication (7), tumble (1), mechanical agitation (1) and leaching (1). Clean-up: DSPE (Carbon) (4), SPE (1) and filtration (3). Extraction solvent: methanol (14), acetonitrile (1), acetonitrile/methanol (2) and MTBE (1). Calibration standard for PFOS analysis: mixed (9) and linear (9). Water samples Pre-treatment: Trizma buffer (1). Extraction technique: direct injection (6), filtration (1), SPE (11) and dispersive liquid-liquid microextraction (1). Extraction solvents: methanol (7), ammonia/methanol (3) and octanol (1). Calibration standard for PFOS analysis: mixed (7) and linear (10). For all matrices the analytical detection method of choice was LC-MSMS. Laboratories used different extraction and sample preparation methods. Due to the limited amount of data and the variety of analytical methods used no significant trend with extraction and sample preparation was identified. Participants used either mixed or linear standards to quantify total and linear PFOS. A plot of z-scores for total and linear PFOS vs mixed or linear calibration standards is presented in Figure 30. 10 1 8 - Total PFOS Linear PFOS 6- 42- <U 0 o (O-29 -4 - ik || __________ 1___________________ -6 - -8 - -10 J 1| S' f &" Q. n c/) (/) to !+ r+ D CD S' s 3 D 13 13 CL CL CL CL 01 01 QJ 01 --5 " ? --5 Q_ Q_ Q- Q_ ^Laboratory 23 participated only in water samples and performance was unsatisfactory for all z-scores. Figure 30 Linear vs mixed calibration standards for total and linear PFOS measurements 64 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002003 6.6 Effects of Sample Matrix Samples SI and S2 were fish, Samples S3 and S4 were soil and Samples S5 and S6 were water. Water samples received the least number of percent satisfactory z-scores. Sample SI Fish S2 Fish S3 Soil S4 Soil S5 Water S6 Water Table 29 Satisfactory z-scores for each matrix Incurred Spiked Incurred Spiked Incurred Spiked Expected number of z-scores 34 51 54 54 34 51 Actual number of z-scores (% of expected no of z-scores) 25 (74%) 39 (76%) 46 (85%) 47 (87%) 29 (85%) 46 (90%) Satisfactory 23 37 45 43 25 41 % satisfactory 92 95 98 91 86 89 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 65 US00002004 7 REFERENCES [1] ISO/IEC 17043:2010, Conformity assessment - General requirements for proficiency testing, ISO, Geneva. [2] NMI Chemical Proficiency Testing Study Protocol. http://www.measurement.gov.au --products and services --chemical proficiency testing -- details of our program --Study Protocol [3] NMI Chemical Proficiency Testing Statistical Manual. http://www.measurement.gov.au --products and services --chemical proficiency testing -- details of our program --Statistical Manual [4] Thompson, M. Ellison S. L. R and Wood, R., The international harmonized protocol for proficiency testing of (chemical) analytical laboratories, Pure Appl. Chem. 78, 145-196, 2006. [5] National Measurement Institute AQA 15-03 PFOS/PFOA in Water and Soil, http://www.measurement.gov.au/Publications/ProficiencvStudvReports/Pages/default.aspx [6] ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories, ISO, Geneva. [7] Eurachem/CITAC Guide Quantifying Uncertainty in Analytical Measurement, Third edition, (2012), http://www.eurachem,org/images/stories/Guides/pdf/OUAM2012-P1,pdf (Accessed July 2016) [8] ISO 13528:2015(E), Statistical methods for use in proficiency testing by interlaboratory comparisons, ISO, Geneva. [9] Thompson, M., Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing, Analyst, 125, 385-386, 2000. [10] Thompson, M. and Fearn, T., A new test for sufficient homogeneity, Analyst, 126, 1414-1417, 2001. 6 6 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002005 APPENDIX 1 - PARTICIPATING LABORATORIES ACS Laboratories (Australia), VIC ALS Czech Republic, s.r.o., Czech Republic AsureQuality Limited, NEW ZEALAND AXYS Analytical Services Ltd, CANADA CHEMCENTRE WA Eulji University, KOREA Envirolab Services NSW Eurofins GFA Lab Service GERMANY Government Laboratory HONG KONG Office of Environment and Heritage, Department of Premier and Cabinet Environmental Protection Science, NSW SGS Leeder Consulting VIC TestAmerica Sacramento California, USA Advanced Analytical Australia, NSW ALS Laboratory Group Ontario, CANADA Australian Laboratory Services, NSW Korea University, SOUTH KOREA National Centre Food Safety Risk Assessment, CHINA Eco-Research, ITALY Eurofins mgt, QLD Health Canada, Ottawa, CANADA NRCET ENTOX, Queensland University, QLD SGS Belgium Division IAC, BELGIUM TestAmerica Denver, USA University of A Coruna (UDC), SPAIN AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 67 US00002006 APPENDIX 2 - SAMPLE PREPARATION, HOMOGENEITY TESTING AND STABILITY CHECK A2.1 Sample Preparation Analytical standards used for spiking Samples S2, S4 and S6 were purchased from Sigma Aldrich. On the analytical report provided with the standards, PFOA (product 33824, batch S ZBD308XV) has a stated purity of 99.4% and PFOS (product 33829, batch SZBE107XV) of 98%. No value was supplied for linear PFOS. Sample S1: Dusky flathead (Platycephalus fuscus) from a contaminated site were composited. Sample S2: Yellow bream (Acanthopagrus australis) and sand whiting (Sillago ciliate) from a control site were composited. A portion of the composited fish was sub-sampled and placed in the -80C freezer for use as Sample S2 unspiked. Approximately 955.6 g of fish composite was placed on a tray and sprayed with a mist of the spiking solution using a commercial pump. This was repeated until all spiking solution was used. The fish was thoroughly mixed. Both Samples SI and S2 were placed on stainless steel trays as patties of no more than six centimeters square and frozen overnight in -80C freezer. The patties of fish were embrittled by immersion in liquid nitrogen and then passed through a Retsch SM2000 mill that was cooled with liquid nitrogen and dry ice. After grinding, samples were placed in a refrigerator in a covered Pyrex dish. The next day after all the carbon dioxide sublimed, 5 to 8 g portions of fish were packed into labelled 50 mL Greiner tubes. Sample S3: Soil obtained from a contaminated site and uncontaminated dried soil were sieved through 212 pm sieve. 903 g of contaminated soil and 310 g of uncontaminated soil were mixed for 90 min using a V-mixer. The mixture was divided into 20 g, packed into labelled 50 mL Greiner tubes. Sample S4: 1181 g of dried uncontaminated soil sieved through 355 pm sieve was placed in a stainless steel pot. A slurry was produced by adding acetone. The slurry was spiked with solutions of PFOA and PFOS prepared in methanol. The slurry was stirred using an IKA stirrer, left to evaporate overnight and purged with nitrogen for further drying. The soil was then mixed, dispensed into 20 g portions and packed into labelled 50 mL Greiner tubes.. Sample S5: Contaminated water was filtered through an Advantec Toyo GB 140 glass fibre filter. 305 g of filtered contaminated water was diluted with Milli-Q water to a final volume of 4578 g. Water was placed in a stainless steel pot, mixed with an IKA stirrer and dispensed into labelled 65 mL HDPE containers. Sample S6: 7167 g of autoclaved tap water was spiked with standard solutions of PFOA and PFOS prepared in methanol. The spiked water was stirred using an IKA stirrer and dispensed into labelled 65 mL HDPE containers. Fish samples were stored in -80C freezer and the soil and the water samples in a cool room at 4C prior to dispatch to participants. A2.2 Sample Analysis and Homogeneity Testing Homogeneity testing was based on that described in the International Protocol.4 Seven bottles were selected at random from each set of samples and analytes measured. The measurements were made under repeatability conditions in random order. A2.2.1 Fish Fish samples were prepared at NMI North Ryde (see A2.1) and analysed by National Research Centre for Environmental Toxicology (Entox) from University of Queensland. 6 8 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002007 Fish Samples SI and S2 were analysed in duplicate by weighing 1 g of sample and spiking with 20 pi (0.2 ppm) 13C-labelled PFAS solution mixture. The samples were digested with 200 mM NaOH/MeOH and subsequently extracted twice by ultra-sonication in acetonitrile and acidified with 4M HCl/MeOH. The supernatants were combined and reduced to 2 ml, then liquid-liquid extracted three times with n-hexane. The extract was reduced to 250 pi and reconstituted to 1 ml using methanol, then loaded onto a 100 mg BondElut carbon cartridge (Agilent Technologies) and eluted with methanol. The final extract was reduced to 400 pi and reconstituted with 5 mM ammonium acetate in water to 1 ml, then spiked with 20 pi (0.2 ppm) 13C8-PFOS and 13C8-PFOA solution mixture. Instrument analysis was performed using high performance liquid chromatography (HPLC, Shimadzu Corp., Kyoto Japan) coupled to a tandem mass spectrometer (QTrap 5500 AB-Sciex, Concord, Ontario, Ca) operating in negative electrospray ionisation mode and using scheduled multiple reaction monitoring (SMRM). For separation a volume of 5 pi was injected onto a Gemini NX C l8 column (50 x 2 mm, 3 pm, 110 A, Phenomenex, Lane Cove, Australia) held at a constant temperature of 50 C. PFASs were separated by gradient elution on the HPLC using mobile phase 10 % (A) and 90 % (B) methanol, respectively, with 5mM ammonium acetate. Identification and confirmation of peaks was done using retention times and comparing the ratios of MRM transition intensity between the samples and the calibration standards in the same batch of analysis. Quantification was conducted using 13C-labelled PFASs. Calibration standards were made up in 1000 pi (400 pL methanol/600 pL using 5 ml ammonium acetate in water). The concentration range of the eight prepared calibration standards was 0.1- 100 ppb (0.1; 0.4; 1; 4; 10; 20; 40; 100). Calibration standards were injected three times in each batch of samples. Quality control standards, including duplicate samples, native spikes and deionized water procedural blanks and instrument blanks were added to each batch and treated in the same way as real samples. All values reported are corrected for recovery of the surrogate standards. Results of the fish homogeneity testing are presented in tables 30-34. For each sample, the mean of the 14 measurements were used as the homogeneity value. All samples were found to be sufficiently homogeneous for use in this PT study. Table 30 Sample SI, Linear PFOS homogeneity testing results Bottle Fill Number 1 16 17 30 32 39 43 Mean CV Linear PFOS pg/kg Replicate 1 Replicate 2 18.0 16.5 18.7 17.5 18.6 18.1 20.1 19.0 19.3 18.2 19.4 18.7 19.8 18.9 18.6 5.0% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.29 0.20 0.363 Critical 0.73 0.5 3.41 Result Pass Pass Pass AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 69 US00002008 Table 31 Sample SI, Total PFOS homogeneity testing results Bottle Fill Number 1 16 17 30 32 39 43 Mean CV Total PFOS pg/kg Replicate 1 Replicate 2 19.7 18.0 20.4 19.2 20.3 19.9 20.8 21.8 19.8 21.0 21.2 20.5 21.6 20.7 20.4 4.9% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.35 0.19 0.441 Critical 0.73 0.5 3.97 Result Pass Pass Pass Table 32 Sample S2, Linear PFOS homogeneity testing results Bottle Fill Number 3 Linear PFOS pg/kg Replicate 1 Replicate 2 42.7 39.0 23 44.4 40.0 39 43.0 46.9 42 43.0 40.1 44 44.2 37.1 45 40.5 39.5 48 44.1 41.3 Mean 41.8 CV 6.3% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.43 0.34 0.000 Critical 0.73 0.5 25.09 Result Pass Pass Pass AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002009 Table 33 Sample S2, Total PFOS homogeneity testing results Bottle Fill Number 3 Total/Linear PFOS gg/L Replicate 1 Replicate 2 55.5 59.6 23 60.5 54.8 39 60.1 63.6 42 57.6 55.4 44 50.5 61.0 45 54.2 56.2 48 62.2 57.3 Mean 57.7 CV 6.2% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.54 0.33 0.000 Critical 0.73 0.5 46.14 Result Pass Pass Pass Table 34 Sample S2, PFOA homogeneity testing results Bottle Fill Number 3 PFOA pg/kg Replicate 1 Replicate 2 48.5 49.8 23 50.1 49.4 39 51.1 55.2 42 55.2 49.6 44 50.7 54.7 45 48.7 50.9 48 53.7 57.2 Mean 51.8 CV 5.5% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.38 0.24 2.276 Critical 0.73 0.5 28.79 Result Pass Pass Pass AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 71 US00002010 Soil and water samples were prepared (see A2.1) and analysed at NMI North Ryde. A brief description of analysis method is presented below. A2.2.2 Soil and Water Soil samples S3 and S4 were prepared in duplicate by accurately weighing 1 g of sample and spiking with 60 pL of labelled surrogate standard in methanol. The samples were extracted by overnight tumbling in alkaline methanol (0.01 N potassium hydroxide), then centrifuged and a portion was filtered and evaporated. The filtered extract was reconstituted to 600 pL in mobile phase and spiked with 10 pL labelled recovery standard in methanol. Water samples S5 and S6 were prepared by accurately weighing the entire content of the sample bottles (~50 mL) then spiking with 30 pL of labelled surrogate standard in methanol. The samples were pre-treated with IN acetic acid then extracted by solid phase extraction (Waters Oasis HLB, 6 mL, 500 mg, 60 pm particle size) under vacuum and eluted using methanol. After evaporation under nitrogen, the extract was reconstituted to 600 pL in mobile phase and spiked with 10 pL labelled recovery standard in methanol. All chemicals are analytical reagents or LCMS grade solvents. Instrument analysis was performed using an Agilent 1100 High Performance Liquid Chromatography (HPLC) coupled with an ABSciex 4000 Qtrap Mass spectrometer, operating in multiple reaction monitoring mode. 5 pL of extract was injected onto a Waters XBridge BEH column (1 mm x 150 mm x 3.5 pm, 130 A)with a mobile phase gradient consisting of water:methanol (2 mM ammonium acetate). Two mass transitions were monitored for each target analyte and labelled surrogate, and abundance ratios checked. The instrument mass accuracy is calibrated annually during preventative maintenance, and the six point calibration curve established for each analytical batch. A solvent batch blank is extracted and analysed with each batch, and sample results must be at least three times the level of any analyte detected in the batch blank to be reported. Quantification is based on the use of the labelled surrogates using relative retention factors from the multipoint calibration, and is corrected for surrogate recoveries. The analysis is based on USEPA 537 and used calibration, surrogate and recovery standards supplied by Wellington Laboratories, Canada. Chromatographic separation of the main four PFOS isomers was performed by optimising column and mobile phase conditions. Total PFOS was determined by integrating all peaks that eluted from the LC column within the retention time window established during analysis of a technical PFOS mixture standard (Wellington Laboratories). Linear PFOS was determined by integrating the peak that co eluted with the linear labelled surrogate PFOS. Both total and linear PFOS were quantitated using linear PFOS calibration standards. Results of the soil homogeneity testing are presented in tables 35-40. For soil samples, the mean of the 14 measurements were used as the NMI homogeneity value. All soil samples were found to be sufficiently homogeneous for use in this PT study. 72 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002011 Table 35 Sample S3, Linear PFOS homogeneity testing results Bottle Fill Number 1 Linear PFOS |ig/kg Replicate 1 Replicate 2 186 190 16 214 193 17 201 176 30 195 187 32 192 186 39 218 182 43 196 194 Mean 194 CV 5.9% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.52 0.34 0.000 Critical 0.73 0.5 537.13 Result Pass Pass Pass Table 36 Sample S3, Total PFOS homogeneity testing results Bottle Fill Number 1 16 17 30 32 39 43 Mean CV Total PFOS pg/kg Replicate 1 Replicate 2 258 261 297 268 279 252 272 269 266 257 298 256 275 270 270 5.2% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.51 0.29 0.000 Critical 0.73 0.5 904.12 Result Pass Pass Pass AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 73 US00002012 Table 37 Sample S3, PFOA homogeneity testing results Bottle Fill Number 1 PFOA pg/kg Replicate 1 Replicate 2 5.8 5.3 16 6.3 6.0 17 5.9 5.8 30 6.2 6.1 32 5.6 5.6 39 6.2 5.8 43 6.2 6.6 Mean 5.9 CV 5.6% Thompson and Fearn Homogeneity Tests Test Cochran s 2sam Value 0.29 0.19 0.063 Critical 0.73 0.5 0.34 Result Pass Pass Pass Table 38 Sample S4, Linear PFOS homogeneity testing results Bottle Fill Number 3 15 21* 27 29 35 38 Mean CV Linear PFOS pg/kg Replicate 1 Replicate 2 21.6 21.8 21.6 22.8 27.4 20.8 23.8 23.8 23.2 22.1 23.9 22.1 23.4 22.1 22.7 4.0% Thompson and Fearn Homogeneity Tests Test Value Cochran 0.43 Critical 0.78 Result Pass 0.18 0.5 Pass s 2sam 0.193 5.18 Pass *Results on bottle fill 21 were not included in the test for homogeneity being identified as an analytical outliers (test Sa/o) due to the difference between replicates.10 74 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002013 Table 39 Sample S4, Total PFOS homogeneity testing results Bottle Fill Number 3 15 21* 27 29 35 38 Mean CV Total PFOS pg/kg Replicate 1 Replicate 2 25.3 25.6 25.9 26.5 31.8 24.8 27.3 27.7 27.3 25.8 27.4 25.8 27.3 25.7 26.5 3.3% Thompson and Fearn Homogeneity Tests Test Value Cochran 0.32 Critical 0.78 Result Pass 0.15 0.5 Pass s 2sam 0.107 6.71 Pass *Results on bottle fill 21 were not included in the test for homogeneity being identified as an analytical outliers (test Sa/o) due to the difference between replicates.10 Table 40 Sample S4, PFOA homogeneity testing results Bottle Fill Number 3 15 21* 27 29 35 38 Mean CV PFOA pg/kg Replicate 1 Replicate 2 15.2 15.4 13.7 14.7 17.2 12.8 15.0 13.8 16.3 15.6 16.5 16.6 15.7 14.3 15.2 6.4% Thompson and Fearn Homogeneity Tests Test Value Cochran 0.40 Critical 0.78 Result Pass 0.21 0.5 Pass s 2sam 0.608 2.81 Pass *Results on bottle fill 21 were not included in the test for homogeneity being identified as an analytical outliers (test Sa/o) due to the difference between replicates.10 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 75 US00002014 Results of the water homogeneity testing are presented in tables 41-45. Since the entire water sample was used in each analysis, it was not possible to apply analysis of variance (ANOVA) to determine if samples were sufficiently homogeneous. When it is not possible to conduct replicate measurements, the standard deviation of the results (sd) will be compared with the target standard deviation of the PT (a) calculated as described in section 4.4. The proficiency test samples may be considered sufficiently homogeneous i f : sd < 0.3 a.8 For water samples, the mean of the 10 measurements were used as the NMI homogeneity value. All soil samples were found to be sufficiently homogeneous for use in this PT study. Table 41 Sample S5, Linear PFOS homogeneity testing results Bottle Fill Number 2 3 5 15 27 30 31 58 63 67 Mean sd Target a Linear PFOS Hg/L 1.99 2.05 1.93 2.19 2.01 2.07 2.06 2.05 1.87 1.81 2.00 0.10 0.33 Test sd/cr < 0.3 Value 0.30 Critical 0.3 Result Pass Table 42 Sample S5, Total PFOS homogeneity testing results Bottle Fill Number 2 3 5 15 27 30 31 58 63 67 Mean sd Target a Total PFOS Hg/L 1.99 2.05 1.93 2.19 2.01 2.07 2.06 2.05 1.87 1.81 3.24 0.14 0.60 Test sd/cr < 0.3 Value 0.23 Critical 0.3 Result Pass 76 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002015 Table 43 Sample S6, Linear PFOS homogeneity testing results Bottle Fill Number 6 17 21 31 38 55 62 64 77 96 Mean sd Target a Linear PFOS Hg/L 5.15 4.95 5.24 5.02 4.95 4.70 4.96 4.95 5.05 4.78 4.97 0.16 0.90 Test sd/cr < 0.3 Value 0.18 Critical 0.3 Result Pass Table 44 Sample S6, Total PFOS homogeneity testing results Bottle Fill Number 6 17 21 31 38 55 62 64 77 96 Mean sd Target a Total PFOS Hg/L 7.75 7.61 7.67 7.50 7.70 7.22 7.44 7.62 7.59 7.27 7.54 0.18 1.3 Test sd/cr < 0.3 Value 0.13 Critical 0.3 Result Pass AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 77 US00002016 Table 45 Sample S6, PFOA homogeneity testing results Bottle Fill Number 6 17 21 31 38 55 62 64 77 96 Mean sd Target a PFOA Hg/L 11.6 11.3 12.0 11.4 11.5 11.0 11.5 11.6 11.9 11.3 11.5 0.29 2.17 Test sd/cr < 0.3 Value 0.13 Critical 0.3 Result Pass AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002017 A2.3 Stability Testing for Fish Samples S1 and S2 For stability testing fish Samples SI and S2 were analysed at NMI North Ryde. Samples were prepared in duplicate by accurately weighing 1 g of the sample then spiking with 60 pL of labelled surrogate standard in methanol. The samples were extracted by overnight tumbling in alkaline methanol (0.01 N potassium hydroxide), then centrifuged and a portion was diluted in milliQ water and extracted by solid phase extraction (Waters Oasis HLB, 6 mL, 500 mg, 60 pm particle size) under vacuum and eluted using methanol. After evaporation under nitrogen, the extract was reconstituted to 600 pL in mobile phase and spiked with 10 pL labelled recovery standard in methanol. All chemicals are analytical reagents or LCMS grade solvents. Instrument analysis was performed as described in section A2.2.2. A stability study of the fish samples was carried out. A factor considered to affect the stability of fish samples was storage condition during transportation. Storage condition during transportation was considered at room temperature and assessed by comparing the average of measurements performed on two jars of each samples starting 17/06/2016 (sample dispatch) for one month to 18/07 2016. Results were in good agreement with each other, with the spiked level and the assigned value (Figures 31 and 32). os 10 < M Q_ m 10 < w Figure 31 Total and linear PFOS stability results and assigned value (AV) in incurred fish Sample SI (room temperature) AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 79 US00002018 fi ll. 30 hi KOJ *s2*r < 3 uQ_. < <<. < M Figure 32 Stability results, spike level and assigned value (AV) in fish Sample S2 (room temperature) 80 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002019 To confirm the stability during transportation and over time, results returned by participants were combined with the reported participant date of analysis and subjected to a trend analysis. No trend was evident providing further evidence of samples stability. An example for Total PFOS and PFOA in Sample S2 are presented in Figure 33. S2-Total PFOS 80 70 "3 ^O3) 60 (ou0_ 50 a. 40 re ,2 30 70 56 61 55' # 54.273 46.8 43 34 56.32 67.75 61.09 58.7 42 32 20 10 0 3----- 9/06/2016 19/06/2016 29/06/2016 9/07/2016 19/07/2016 29/07/2016 8/08/2016 18/08/2016 Date of analysis 70 -i 60 - 50 O) o3>40 <O 30 20 S2-PFOA 55 52 61.19 57 46.2 46 33 29.5 58.003 53 58.8 52.42 50.19 37 10 - 0 -I------------------------------------ i------------------------------------ i------------------------------------ i------------------------------------ i------------------------------------ i------------------------------------ i------------------------------------ 1 9/06/2016 19/06/2016 29/06/2016 9/07/2016 19/07/2016 29/07/2016 8/08/2016 18/08/2016 Date of analysis Figure 33 Participant results and date of sample analysis trend for Total PFOS and PFOA in fish Sample S2 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 81 US00002020 APPENDIX 3 - ROBUST AVERAGE AND ASSOCIATED UNCERTAINTY The robust average was calculated using the procedure described in TSO13258:2015(E), Statistical methods for use in proficiency testing by interlaboratory comparisons - Annex C'7the uncertainty was estimated as: Urob av 1.25 *Srob av / *\JP Equation 4 where: urobav Srobav p robust average standard uncertainty robust average standard deviation number of results The expanded uncertainty (Urobav) is the standard uncertainty multiplied by a coverage factor of 2 at approximately 95% confidence level. The robust average of all seventeen results was calculated (Table 46). Table 46 Robust average for all PFOA results in Sample S6 Lab. Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Robust average PFOA (gg/kg) 12 12 9.78 NT 10.02 19 10.6 NT 9.7 NT 11.7 NT 11.36 10.4 NT 13.54 NT 12 9.1 10.1 11.8 9.01 18.598 NT 11.23 82 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002021 Result from laboratories 6 and 23 was removed as this is outside of a range 50% of the robust average. The robust average and associated uncertainty of the remaining sixteen results were then used as the assigned value for this analyte (Table 47). Table 47 Uncertainty estimate for PFOA in Sample S6 after removing outlier No. results (p) 15 Robust Average ^Crob av 10.83 pg/L 1.37 pg/L Mrobav 0.443 pg/L k2 Urobav 0.89 pg/L The assigned value for PFOA in Sample S6 is 10.83 0.89 pg/L. AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER 83 US00002022 APPENDIX 4 - ACRONYMS AND ABBREVIATIONS c v Coefficient of Variation EPA Environment Protection Authority ISO International Standards Organisation LC Liquid Chromatography Max Maximum value in a set of results Md Median Min Minimum value in a set of results MS Mass Spectrometry NMI National Measurement Institute (of Australia) NR Not Reported NT Not Tested PFOA Perfluorooctanoic acid PFOS Perfluorooctane sulfonate PT Proficiency Test Robust CV Robust Coefficient of Variation Robust SD Robust Standard Deviation S Spiked or formulated concentration of a PT sample SPE Solid Phase Extraction Target SD Target Standard Deviation a Target standard deviation s 84 AQA 16-06 PFOS/PFOA IN FISH, SOIL AND WATER US00002023