Document Yrj2J09oLjLnG6j5panQVzMMk

3M Study Title Inherent A erobic A q uatic B io deg radability of Fluoroaliphatic Polymeric Ester o ? [ 1 Data Requirement 40 CFR 792 Authors Study Completion Date Date of signing Performing Laboratory 3M Environmental Technology and Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Project Identification E02-0913 Total Number of Pages 142 This page has been reserved for specific country requirements/ Page 2 of 142 E02-0913 ^ ^Biodgradation Study GLP Compliance Statement Study Titje: Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester of Study Identification Number: EQ2-0913 This study was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 CFR 792, with the exceptions listed below: Exceptions to GLP compliance: 40 CFR 792.130(e): The authenticated hardcopy printouts are considered the original raw data. ' . . 40 CFR 792.105: The purity fo r f J have not been determined. 40 CFR 792.105 (b): The stability of the reference substances was not determined prior to study initiation. Date i Page 3 o f 142 Quality A ssurance Statement Study Title: Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester of fJ Study Identification Number: E02-0913 This study was audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU), as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates 10/18/02 10/24/02 11/25/02 1/13/03-1/28/03 Phase Protocol In-Phase (dosing) In-Phase (SPE) Data/Report Date Reported to Management Study Director 10/18/02 - i 10/18/02 10/28/02 10/28/02 11/25/02 11/25/02 1/28/03 1/28/03 [, QAU Representative 1 J l-IO -o S Date Page 4 of 142 Ta b le o f C o ntents GLP Compliance Statem ent 3..................................................................................... ....................................................... ......................... Quality Assurance Statement 4........................................................................................................................................... ......................... Table of C ontents .................................... 5................................................................................................ ......................... List of T ables.................................................................................................................................................................................................... .................. . . . 6 Study Information............................................................................... 7......................... Summary............................................................................................ ...........9 Introduction.......... ............................................................................... .........10 Test Substance............................ .................................. .................... .........12 Reference Substances...................................................................... ......... .13 . Control Substances............................................................................ ........ 14 Test S ystem 14.................................................................................................................. <................................................. .................................. .................... Method S u m m a rie s 16.................................................................................................................................................................................................. Preparatory M ethods 16........................................................................................................................................................ .................... Analytical M ethod /. 16.................................................................................................................. ............................................... .................... Analytical R e s u lts 17.................................................................................................................................................................................... .................. Data Sum m ary 20.................................................................................................................................................................................................................... Statistical Methods and Calculations 22.............................................................................................................. .................. Statement o f Conclusion 23............................................................................................................................................................ .................. List of A tta ch m e n ts 23................................................................................................................................................................................. .................. Signature P a g e 24.............................................................................................................................................................................................. .................. Attachment A: Extraction and Analytical M ethods 25................................................................. .................. Attachment B: Data T a b le s 46................................................................................................................................................. .................. Attachment C: Sample Chromatograms 61.......................................................................... .......................... .................. Attachment D: Test Substance Information....................................... ........104 Attachment E: Protocol, Protocol Amendments and Deviations....... ........108 . Page 5 of 142 List of Tables Table 1: Medium A Exposed to Uninhibited Sludge) Percent Degradation Results1......................................................... ...... .............. 9 Table 2. Test Substances......................................................... 12 Table 3. Reference Substances................................................13 Table 4. Control Substances........................... ........................14 Table 5: Test System Preparation........................................... 15 Table 6: CCV Summary from 11/19/02 analysis...................... 18 .. Table 7: Out of specification precision results.......................... 19 Table 8: Out of Specification Matrix Spikes............................. . 19 Table 9: Medium A/ Results1.........' ...... ^Exposed to Uninhibited Sludge) Percent Degradation 20 Table 10: Medium B Results............................................... .....20 Table 11: Medium C Results....................................................21 1 Table 12: Accuracy of Analytical Results.................................. 21 / Page 6 of 142 Study Information Sponsor r1 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Study Director r7 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Principal Investigator ri 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Study Location Testing Facility 3M Environmental Laboratory 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Study Personnel 1 Study Dates Study Initiation: 10/24/02 Experimental Initiation: 10/24/02 Experimental Completion: 3/5/03 Study Completion: Date of signing Page 7 of 142 Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory according to 40 CFR Part 792. The test substance and analytical reference standard reserve samples are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. / Page 8 of 142 Summary This study was undertaken to determine the aerobic, aquatic biodegradation potential of the test substance (a complex mixture of fluoroaliphatic polymeric esters) when exposed to municipal wastewater treatment sludge. This was accomplished by utilizing aspects from the following guidelines: USEPA Zahn-Wellens/EMPA Test (OPPTS 835.3200) and USEPA Modified SCAS (OPPTS 835.3210). The test substance was suspended into three mediums: Medium A (mineral salts medium and sludge), Medium B (mineral salts medium, sludge, and antimicrobial . agent) and Medium C (mineral salts medium and antimicrobial agent). After incubation for varying intervals, study samples of polymer were prepared using solid phase extraction (SPE) and analyzed using High Performance Liquid Chromatography / Mass Spectrometry (HPLC/MS). The target analytes are the predicted degradation products , perfluorobutane sulfonate (PFBS), and The target analytes are based on the biodegradation study of , as reported in 3M Environmental Laboratory study # by the Microbial Activity Present in Municipal Wastewater Treatment Sludge" Samples were semi quantitatively analyzed for using two different techniques. Initially, was quantified using the response factor of ... Subsequently, a standard of was synthesized and characterized. The sample extracts of Medium A were reanalyzed and quantified versus a calibration curve of Due to the polymeric nature of , the predicted degradation products were measured rather than direct measurement of the loss of the starting material. The analytical results demonstrate that under the conditions of the study, the test substance is biodegraded (OPPTS 835.3210 "Modified SCAS Test" defines a greater than 70% loss of starting material as ultimate biodegradability). The major metabolites identified from the test cultures were ; : (representing Jegradation of the initial concentration of on day 0) and M: (representing 114% degradation of the initial concentration o f' , on day 4). Observed minor metabolites include ' . and PFBS. Complete mass balance between parent material and measured degradation products appears to have been achieved on day 4. See Table 1. Table 1: Medium A Analyte PFBS3 I <Exposed to Uninhibited Sludge) Percent Degradation Results1 DayO Day4 Day14 Day28 Vo 1.0% 0.40% <0.15% 0.84% 114% 58% 63% <3.5% 1.2% 0.47% 0.26% <0.92% 1.4% ! 1.9% 2.4% <1.0% ! <0.20% 0.12% 0.37% <0.33% 0.28% 0.97% 2.3% percentages reported in Table 1 were determined by dividing the average amount of analyte found in each test vessel by the theoretical amount if .otally biotransformed into that specific analyte.' as calculated using1 standard, hrhe analytical accuracy for all analytes is 20% or better. Page 9 of 142 Introduction ' The primary objective of this investioation is to identify the inherent aerobic aquatic biodegradation potential of the fluoropolymer. _ , when exposed to the microbial populations present in wastewater treatment sludge. This was accomplished by utilizing aspects from the following guidelines: USEPA Zahn-Wellens/EMPA Test (OPPTS 835.3200) and USEPA Modified SCAS (OPPTS 835.3210). . The test substance, is a complex mixture of fiuoroaliphatic polymeric esters rather than discreet monomeric material. The present investigation was conducted using the noted EPA methods as guidelines and incorporating portions of these guidelines to accommodate the specific testing requirements, such as individual samples per sampling event and specific target analysis instead of COD, DOC o rC 0 2. The present study is designed to utilize municipal wastewater treatment sludge as the inoculum. The focus is to determine the ability of viable microbial populations to degrade or transform into predicted fluorochemical breakdown products based on the biological degradation study o f1 . as reported in 3M Environmental Laboratory study # ' Biodegradation of by the Microbial Activity Present in Municipal Wastewater Treatment Sludge." The proposed biodegradative pathway is illustrated in Figure 1. Page 10 of 142 Figure 1: The Proposed ) Biocegrdtiv Pathway R F F FF O F--------------------------------- S-----0. I l I II F F FF 0 PFBS Page 11 of 142 Test S ubstance Table 2. Test Substances TestSubstance IUPAC Name Chemical Formula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCR Number . Physical Description . Purity Solubility** - . 3M Specialty Chemicals 07/27/06 Room Temperature 1 ' Yellow Viscous Oil 97.5% . in ASTM type I water ` Based on NMR data, " From 3M Environmental Laboratory study' Page 12 of 142 Reference S ubstances Table 3. Reference Substances Reference Substance ' IUPAC Name ' Chemical Formula Identifier Potassium perfluorobutanesulfonate c 4f 9s o 3k 29420-49-3* Source Aldrich Chemical 3M Specialty Chemicals ` Expiration Date Storage Conditions . ^ Chemical Lot Number TCR Number Physical Description Purity Reference Substance 5/1/2010 Frozen Clear liquid 99% 12/4/2006 Frozen White powder 96.7% IUPAC Name Chemical Formula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Description Purity Reference Substance 3M Specialty Chemicals 01/30/07 Frozen White crystals 97.25% 3M Specialty Chemicals Not Provided Frozen White powder . Not determined . 3M Specialty Chemicals 01/30/07 Ambient White crystals 95.55% IUPAC Name Chemical Formula Identifier Source Pace Analytical Services 3M Specialty Chemicals 3M Specialty Chemicals Expiration Date 2/4/2013 12/ 1/2010 11/29/2005 Storage Conditions Frozen Frozen Frozen Chemical Lot Number TCR Number Physical Description White Powder Light yellow powder Off White powder Purity 50.1% Not Determined 98.63% *CAS Number, **3M Identifier Code. The location of the documentation o f the methodfsVof synthesis of the test, control, and reference items are the same as the source of the compound. concentrations. Page 13 of 142 In order to quantify the predicted degradation product _ was initially used. *was chosen to quantify , because it is the to _ . The molecular formula for is Since both compounds are similar with exception of carbon chain lengths, it was assumed that they have similar response factors. Subsequent to analysis, was synthesized and the 'response factors of similar calibration standards were compared under the same conditions to provide a better estimate of the concentrations in the original analysis. It should be noted that the purity for used for this study is not thoroughly established and therefore the results for should be treated as semi-quantitative only. ' calibration curve used was compared to a characterized standard, providing a correction factor of %. The semi-quantitative results reported here are corrected using this purity information. - Control Substances Table 4. Control Substances ControlSubstances SodiumLauryi Sulfate IUPAC Name Use Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Description Purity Surrogate Standard 3M Specialty Chemicals 8/31/2006 Frozen White powder 86.9% Sodium Lauryi Sulfate Internal Standard for LC/MS analysis 3M Specialty Chemicals 10/18/2006 Frozen Toxicity and Reference Control Mallinckrodt 2/26/2007 Room Temperature 7718 V16603 White powder 98.6% TN-A-6021 White powder 99% ' T est System Mixed liquor suspended solids (MLSS) were obtained from the aeration units at the Metro Wastewater Treatment Plant, St. Paul, MN. The suspended sludge was allowed to settle for 24 hours at room temperature. The approximate percentage of sludge per volume of container was 19%. Mediums A, B, and C were then prepared. The mineral salts medium used was based on the USEPA Zahn-Wellens/EMPA Test (OPPTS 835.3200). After the sludge settled for 24 hours, Medium A was prepared by adding 200 mL of the settled sludge to 4.0 L of mineral salts medium. The total suspended solids for this medium was determined to be 0.747 g/L which is within the specification of 0.2 to 1.0 g dry matter/L as given by OPPTS 835.3200. Medium A was then used to create Medium B. Medium B consisted of a portion of Medium A and 130 pg/mL chloramphenicol. Medium C consisted of mineral salts medium and 126 pg/mL chloramphenicol. Page 14 of 142 Individual culture vessels were prepared by dispensing 25 mL of the appropriate medium into 125 mL glass Erienmeyer flasks. Then each flask was directly spiked with the test or control substance (or nothing in. the case of the blanks) as appropriate. Sample flasks were spiked as indicated in table 4. Day 0 samples were frozen and the rest of the samples were placed in incubators at 24 3C. All samples except for the day 28 samples maintained this temperature range. For the first 8 days of the study the temperature of the day 28 samples ranged from 20.8 to 28.0C. After day 8 these samples were maintained at the specified 24 3C .. Since the temperature was well within an acceptable range for supporting viable bacteria, the samples associated with day 28 were not rejected. Exposure to slightly elevated temperatures is not expected to adversely affect the quality of the data. , Table 5: Test System Preparation . .. .; SampleDescription of Test/Control Replicates Substanceadded* Reference Substances added** Medium Analysistobe Added conducted / `- .' . Blank Controls 2 None No A Reference Substances ' Blank Controls Inhibited 2 None No B Reference Substances Abiotic Controls 2 None No C Reference Substances Test Substance 3 I Test No Reference A Substances Test Substance Matrix spike 2` Test i Yes A Reference Substances Test Substance Inhibited 3 Test No B Reference Substances Test Substance Inhibited Matrix spike 2 Test / Yes B Reference Substances Abiotic Test Substance 3 , Test No c Reference Substances Abiotic Test Substance Matrix spike 2 Test Reference Yes c Substances , Toxicity Control 3 Test and SLS No A SLS Control Substance (SLS) 2 SLS No A SLS 2 No C 2 No B *The test substance was at a concentration of 36 ma/L. eauivalent to 20mg Carbon / L per FPA recommended guideline. SLS was spiked at a concentration of 40 mg/L and was spiked at a concentration of mg/L. "` Reference substances were added as post extraction matrix spikes at a nominal concentration of 500 ng/mL. Page 15 of 142 Method S ummaries Preparatory Methods Samples were prepared using ETS-8-39.0 "Solid Phase Extraction of Soils, Sediments and Sludges." In summary, samples were allowed to thaw after removal from the freezer. The sample was then vortex mixed for approximately 15 seconds and 0.25 mL of glacial acetic acid was added to the sample (to reach a nominal concentration of 1% acetic acid). A plug of glass wool was placed in the solid phase extraction (SPE) cartridge to prevent suspended solids from plugging the cartridge. The SPE cartridges were conditioned with at least two 5 m l washes of methanol and at least two 5 mL washes with 1% acetic acid, taking care not to run the column to dryness. The selected sample was then decanted into the SPE cartridge and collected as eluent one. The column was allowed to run to dryness. Ten mL of tetrahydrofuran (THF) was then added to the Erlenmeyer flask that originally contained the sample, swirled, and decanted into the SPE cartridge and collected as eluent two. The column was allowed to run to dryness. Eluent 2 was analyzed via LC/MS. Analytical Method Samples were analyzed via ETS-8-155.1 "Analysis of Waste Stream, Water Extracts or Other Systems Using HPLC-Electrospray/Mass Spectrometry." After the samples'were prepared they were diluted (if appropriate) and aliquoted into autovials for analysis via LC/MS. The following parameters were used. Analytical Equipment Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C18 2x50mm-, 5pm particle size Column temperature: 30 C Stop Time: 9.0 minutes Flow rate: 300 pL/min Injection volume: 2 or 5 pL ! Mobile phase components: Solvent A: 2.0 mM ammonium acetate in ASTM Type I water Solvent B: HPLC Grade Methanol Solvent Gradient: Time 0.00 0.50 3.00 5.50 6.00 9.00 %B 15% 15% 100% 100% 15% 15% Page 16 of 142 Mass S pectrom eter: Hewlett-Packard Sries 1100 API/Mass Spectrometer Detector Software: Agilent ChemStationTM A.08.03 Capillary Voltage: 4000 V Gain = 1.0 EMV Mode: Electrospray Negative Gas Temperature: 350 C Drying Gas: 8.0 L/m in. . Nebulizer Pressure: 30 psig Analysis Type: Single Ion Monitoring (SIM) ' . Compound A nalytical R esults For a detailed listing of analytical results, refer to Attachment B: Data Tables. The analytical results consist of data taken from 10 analytical sequences. / R egressions. Quadratic curve fits were applied to calibration standards and sample data to improve quantitation over the concentration range appropriate to the data. All calibration curves had least-square fits of 0.990 or greater. C alibration Standards. Standards ranging in concentration from approximately 2.5 to 750 ng analyte per mL tetrahydrofuran were used for the calibration curves. Calibration curves were originally prepared in mineral salts medium and extracted using SPE cartridges. High/low calibration standards that were not within the 80%-120% (70%-120% for the lower limit of quantitation) criteria were deactivated with the exception o f Jon 11/19/02 and/ Jon 12/05/02. For^ ith e 25.23 ng/mL standard (the lowest standard in the curve) had a recovery of 60%. This standard was kept in order to maintain a five point calibration curve. Since the 25.23 ng/ml_f Standard had an area count of 10866 and the highest sample area count for this analysis was 3475, the 25.23 ng/mL standard was accepted as the lower limit of quantification for this analysis. For|' J th e 10 ng/mL standard was kept in order to keep the 25 ng/mL standard within the 70%-120% criteria for the LLOQ. The LLOQ for this analysis was raised to 25 ng/mL. There should be no adverse affect on the data. Page 17 of 142  C ontinuing Calibration V erification (CCV). At least one calibration check was analyzed at least every ten samples to monitor instrumental drift. All continuing calibration verification checks were within 70%-120% with the exception of the analysis on 11/19/02. For all analytes the 10 ng/mL and 50 ng/mL standards were preceded by a matrix spike, which had at least a nominal concentration of 500 ng/mL. For and PFBS, ,, due to this high concentration, analyte carryover occurred which caused the 10 ng/mL CCV to be >120% and for just (2 out of 3) and ^1 out of 3) the 50 ng/mL CCV was >120%. However, the next CCV run immediately after the >120% CCVs was within the limits as specified by the protocol. The 10 ng/mL CCV for ,was below the lower limit of quantitation (25.23 ng/mL) for this analysis. Since all of the samples were either below the lower limit of quantitation, diluted and reanalyzed, or had acceptable precision (<20%), the results were accepted. There should be no adverse affect on the data. z7 :*3 Table 6: CCV Summary from 11/19/02 analysis ContinuingCalibrationVerificationStandards(ng/mL) 10 50 250 10 50 250 1Q 50 126% 117% 94% 124% 116% 92% 138% 116% 193% 107% 107% 169% 106% 104% 166% 105% 258% 125% 91% 223% 123% 91% 213% 120% 182% 116% 105% 266% .124% 107% 238% i 118% 107% 101% NR* 119% ; 97% NR* 104% 250 91% 103% 89% 104% 97% *NR: Not reported for this analyte. The CCV concentration is below the LOQ for this data set. Lower Limit of Quantitation (LLOQ). The LLOQ was equal to the lowest standard in the calibration curve, with a level of accuracy within 70%-120% with the exception of on 11/19/02 as noted above. The level of analyte in the LLOQ was also greater than two times the response of analyte in the blank samples. System Suitability. Five system-suitability standards were analyzed before the initial calibration curve. The criterion of <5% relative standard deviation (RSD) for the mean repeatability was achieved except fo r r (8.12%) on 11/19/02 and (5.2%) on 3/5/03. For . the samples were accepted since all of the samples were either less than the LLOQ or the samples had to be diluted and reanalyzed because they were above the upper limit of quantitation. For the samples were accepted since all of the accepted samples had acceptable precision and the CCVs for the analysis passed. The criterion of <2% RSD for the retention time repeatability was achieved for each analyte. Page 18 of 142  Duplicate Frequency / Acceptable Precision. Precision was determined for each set of duplicate or triplicate samples. Out of 160 sets of samples, 152 of the sample sets had . precisions of < 20%. Eight out of the 160 sample sets had precisions >20% and are described in Table 9. The >20% precision results for PFBS, and appear to follow the trends of the other time points and are not suspected to have an adverse affect on the data. For the sample precision was 132%. It is suspected that there was some sample contamination in the two samples used for the precision determination since three out of the five samples for this time point were below the lower limit of quantitation. Refer to attachment B for individual precision results. Table 7: Out of specification precision results Analyte -- - * Medium .A A B B B B C C; Day 14 14 14 14 28 28 28 28 RelativeStandardDeviation 24.6% 37.8% 38.3% 26.7% . 22.2% . 34.5% 25.7% . 132% Matrix Spikes. Samples were SDiked post-extraction at a nominal concentration of 500 ng/mL for all reference compounds except , which was not available at the time. Percent recovery was determined for duplicate pairs of matrix spikes. Out of 60 pairs of matrix spikes, 47 were within 70%-120%. Thirteen of the 60 pairs of matrix spikes were not within 70%-120% are indicated in table 8. Table 8: Out of Specification Matrix Spikes Analyte Medium Day Percent Recovery PFBS A 14 132% A0 154% IB C .0 0 ' 148% 141% _--_; :-_-_-- 1 A B 14 ' 14 150% . 138% C 14 149% A 28 136% B 28 129% C 28 134% A0 168% C 14 126% C 28 187% The high spike recoveries for may have occurred due to high concentrations of the analyte in the samples or signal enhancement due to matrix effect. The one high spike recovery for PFBS may be an outlier (using the Grubbs outlier test at the 99% confidence limit). For , it is unknown why the matrix spike recoveries were so high (ranging from 111 %-154% with an average of 135%). Since the Laboratory Control Spikes had very good recoveries (ranging from 94.1% - 105%) it is not expected that these high matrix spike recoveries will have an adverse affect on the data. Refer to attachment B for individual matrix spike results. No matrix spikes were run since no reference material was available at the time the samples were extracted. Page 19 of 142  Laboratory Control Spikes. All laboratory control spikes were within 70%-12G% recovery. Refer to Attachment B for individual laboratory control spike results. No laboratory control spikes were run since no reference material was available at the time the samples were extracted. Solvent Blanks. All solvent blanks were less than 'A the lower limit of quantitation. Matrix Blanks. All matrix blanks were less than 1/2 the lower limit of quantitation. Data S ummary The following tables are summaries of the percent of theoretical degradation of each analyte in each medium. The percent theoretical degradation is based on the amount of fluorine available in ' The percentages reported-in Tables 9,10, and 11 are from the average amount of analyte found in the test vessel divided by the theoretical amount if totally biotransformed into that specific analyte. For a summary of individual results, refer to attachment B. The lower limit of quantitation (LLOQ) for each time point varied depending on the dilution required to measure the major analyte for that time point. . Table 9: Medium A ( Exposed to Uninhibited Sludge) Percent Degradation Results1 Analyte..... . DayO Day4 Day14 Day28 1.0% 0.40% <0.15% 0.84% 114% 58%, 63% <3.5% 1.2% 0.47% 0.26% <0.92% 1.4% 1.9% 2.4% <1.0% <0.20% 0.12% i 0.37% <0.33% 0.28% 0.97% 2.3% T T h e percent degradation is based on the theoretical amount of fluorine available in the initial dose of The percentages reported in Table 1 were determined by dividing the average amount nf analyte found in each tesf vessel by the theoretical amount if totally biotransformed into that specific analyte. as calculated using standard. hTie analytical accuracy for all analytes is 20% or better. Table 10: Medium B Results ^Analyte DayO Day4 n ' ._ 5.7% <4.0% N/A2 <3.5% 1.3% <0.92% 0.60% I <1.0% <0.20% <0.33% 0.045% as calculated usin g ' 2N/A: Not analyzed hTie analytical accuracy for all analytes is 20% or better. Day 14 1.3% 35% 0.64% 1.3% <0.081% 0.092% Day28 0.63% 32% 0.35% 1.3% <0.081% 0.10% Page 20 of 142 Table 11: Medium C Results Analyte DayO 1.4% <4.0% <3.5% ( : ' <0.92% <1.0% PFBS3 <0.33% Day4 3.8% N/A2 0.67% 0.069% <0.20% <0.041%. Tl/A: Not analyzed 'The analytical accuracy for all analytes is 20% or better. Day14 6.1% <4.0% <3.5% <0.92% <1.0% <0.33% Day28 14% 3.5% 0.27% <0.018% <0.081% <0.074% The aerobic, aquatic biodegradation of the test substance was observed beginning in the Day 0 sample set. In medium A, by day 0, the test substance degraded to form 1 Joof the theoretical value). After Day 0, decreases in concentration as other reference substances start to appear. Medium B shows the same patterns as medium A, however at a slower rate. It appears that the antimicrobial agent only partially inhibited the activity of the microbial population, allowing biodegradation to occur in Medium B at a reduced rate. Medium C showed a different degradation route, which is consistent with the hydrolytic degradation route as shown in a previous study. The control substance sodium lauryl sulfate (SLS) demonstrated the required degradation in the toxicity control and control substance samples of 70 percent degradation after 14 days. At day 14, SLS had degraded to <5.8% (>94.2%) in the presence o f; _ and <8.2% , yin its' ' absence, showing that the sludge had good activity and was not inhibited by the test substance. The control substance showed some disappearance during the coarse of the study. However, it is believed that this can be attributed to retaining on the glass walls of the Erienmeyer flasks and adsorption onto the sludge as seen from a previous study, " Soil Adsdrption/Desorption of The results of duplicate laboratory control spikes in each of the three media were used to define the accuracy of results. Each laboratory control sample consisted of reference substance spiked in control media with subsequent extraction using SPE. . Table 12: Accuracy of Analytical Results Analyte Accuracy PFBS ~ j-! : ' 6% 16% 6% 2% . iiii 6% . 4%1 1Because the analytical standard was not available when the lab control spikes were prepared, the analytical accuracy was derived from results obtained from lab control spikes from study , "Inherent Aerobic Aquatic Biodegradability Page 21 of 142 Table 11: Medium C Results Analyte DayO 1.4% <4.0% <3.5% ' I ' <0.92% <1.0% PFBS3 <0.33% Day4 3.8% 2 | 0.67% 0.069% <0.20% <0.041% i ^N/A: Not analyzed JThe analytical accuracy for all analytes is 20% or better. Day14 6.1% <4.0% <3.5% <0.92% <1.0% <0.33% Day28 14% . 3.5% 0.27% <0.018% <0.081% <0.074% The aerobic, aquatic biodegradation of the test substance _ was observed beginning in the Day 0 sample set. In medium A, by day 0, the test substance degraded to form ' i) of the theoretical value). After Day 0, decreases in concentration as other reference substances start to appear. Medium B shows the same patterns as medium A, however at a slower rate. It appears that the antimicrobial agent only partially inhibited the activity of the microbial population, allowing biodegradation to occur in Medium B at a reduced rate. Medium C showed a different degradation route, which is consistent with the hydrolytic degradation route as shown in a previous study. The control substance sodium lauryl sulfate (SLS) demonstrated the required degradation in the toxicity control and control substance samples of 70 percent degradation after 14 days. At day 14, SLS had degraded to <5.8% (>94.2%) in the presence o f; _ _ and <8.2% (>91.8%) in its' absence, showing that the sludge had good activity and was not inhibited by the test substance. The control substance showed some disappearance during the coarse-of the study. However, it is believed that this can be attributed to retaining on the glass walls of the Erfenmeyer flasks and adsorption onto the sludge as seen from a previous study, " Soil Adsdrption/Desorption of The results of duplicate laboratory control spikes in each of the three media were used to define the accuracy of results. Each laboratory control sample consisted of reference substance spiked in control media with subsequent extraction using SPE. . Table 12: Accuracy of Analytical Results . \ Analyte Accuracy PFBS 6% . - ,j ' 16% 6% i 2% i 6% j . 4%1 ' Because the analytical standard was not available when the lab control spikes were prepared, the analytical accuracy was derived from results obtained from lab control spikes from study , "Inherent Aerobic Aquatic Biodegradability Page 21 of 142 Statistical Methods and Calculations The standard curve is subjected to a quadratic regression calculation. Additional statistical methods were limited to calculating means, standard deviations and relative standard deviations. Theoretical Percent Degradation The theoretical percent degradation was calculated based on the total amount of fluorine available from the test substance that could biotransform completely to a specific analyte. This calculation was as follows: Total available fluorine was determined by: Total A vailable Fluorine (jug) = C x V x F w here: C = Concentration o f m test vessel (pig/mL) V = Volume o f Sample (mL) F = Percent Fluorine Com position o f Next, the percentage of fluorine was determined from the molecular weight from a reference substance: N um ber o f fluorine atoms x 18.998-- P ercent Fluorine = _________ _______________ mol m olecular weight o f compound mol The total amount of the reference compound that could be formed from the available amount of fluorine was determined by: _ . .. pig Total A v ailable F luorine (jug) 1 T heoretical A m ount -- = --------;---------- :---------------- x ----------------------------------------- mL Percent Fluorine Extraction volum e (ml) ng A m ount o f A nalyte in Sam ple ---- P ercent D egradation = _________ ;___________________ mL T heoretical A m ount ng mL For example usingf ] Theorectical Amount So, for Day zero, medium A the percent degradation would be: L 7P ercent Degradation 7ng Jm L % inui Page 22 of 142 Statement of Conclusion The analytical results demonstrate that under the conditions of the study, the test substance/ Jis biodegraded (OPPTS 835.3210 "Modified SCAS Test" defines a greater than 70% loss of starting .material as ultimate biodegradability). The major metabolites identified fromihe test cultures were / ^representing 26% degradation of the initial concentration of[ _ /on day 0) and 1 [(representing 114%rdegradation of the initial concentration o f [ bn day 4). Observed minor metabolites include^ and PFBSJComplete mass balance between parent material and measured degradation products appears to nave been achieved on day 4. List of Attachments Attachment A: Extraction and Analytical Methods Attachment B: Data Tables Attachment C: Sample Chromatograms Attachment D: Test Substance Information Attachment E: Protocol, Protocol Amendments and Deviations Page 23 of 142 S ignature Page We certify that this report is a true and complete representation of the data for this study: DJJ/s,/o? Sate 7 03 ho Date Q 3 //'c/ q ^ Date / Page 24 o f 142 Attachment A: Extraction and A nalytical Methods Page 25 of 142 3M Environmental Laboratory Method Solid Phase Extraction of Soils, Sediments and Sludges Method Number: ETS-8-39.0 Adoption Date: Upon Signing Effective Date: Upon Signing Approved By: Date ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 1 o f 10 1 Scope and Application This is a performance-based method that describes the extraction of target analytes from soil, sediment, sludge, or solutions thereof, using solid phase extraction (SPE) and either extracted matrix or unextracted matrix (solvent) calibrations. This method may also be extended to other matrices provided that the data quality objectives are met. 2 Method Summary An amount of soil, sediment or sludge, wet or dry, is prepared in an aqueous l % solution of acetic acid. The sample is capped, mixed, and put on the centriiuge to clarify the supernatant, if needed. The supernatant is passed through a pre conditioned Ci8 SPE column, at which time the analytes are adsorbed onto the stationary phase. Finally, the analytes of interest are eluted from the SPE cartridge and analyzed using LC/MS or LC/MS/MS. 3 Definitions 3.1 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica or polymer sorbents at the other end. It is designed to retain the compounds of interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult matrices and introduced into appropriate solvents for analysis. 3.2 Reagent grade water Water with no detectable target analyte. 4 Warnings and Cautions / 4.1 Health and Safety W arnings Always wear appropriate gloves, eyewear, and clothing when working with solvents, samples and/or equipment. 4.2 Cautions Take care not to allow the SPE column to run to dryness after the methanol and water pre-conditioning steps. After the SPE column is conditioned, add sample and then allow the column to run to dryness. 5 Interferences Contaminants in solvents, reagents, glassware and other sample processing or other analysis hardware may cause interference. The routine analysis of laboratory method blanks must be used to demonstrate that there is no interference under the conditions of the analysis. 6 Instrumentation, Supplies, and Materials 6.1 Instrum entation Vortex mixer Vacuum Pump ' ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 2 of 10 Page 27 of 142 SPE Extraction Manifold Centrifuge . Shaker Balance (+/- 0.1000 g) Solvent trap - . 6.2 Supplies and M aterials Disposable pipettes, plastic or glass Volumetric flasks, glass, type A Vials, various sizes and materials, as appropriate Centrifuge tubes, various sizes and materials as appropriate Labels . Syringes, graduated Bottle-Top Dispenser SPE extraction cartridge, 1 g, Sep-Pak 6 cc tri-functional C!8 (Waters), or equivalent Other SPE extraction cartridges, characteristics to be determined by analyst Crimp cap glass autovials and caps Crimpers 7 Reagents and Standards Reagent grade water Acetonitrile, HPLC grade or equivalent Methanol, HPLC grade or equivalent Tetrahydrofuran, HPLC grade or equivalent Acetic Acid, glacial, ACS grade or equivalent / . 8 Sample Handling Samples should be stored according to instructions from the study director, manufacturer, chain of custody form, or as determined by the analyst. Storage conditions will be documented. Allow samples to-equilibrate to room temperature prior to extraction. Typically fresh matrix standards are prepared with each study. Extracted standards and samples are stored in capped autovials until analysis. ,1 If analysis will be delayed, extracted standards and samples may be refrigerated at approximately 4C for up to 6 months, or may be stored at room temperature. If the samples are not analyzed immediately, post-extraction control samples may be evaluated to demonstrate extract stability. ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 3 o f 10 Page 28 of 142 9 Method Perform ance Data Quality Objectives Application o f the method allows for usage of either an extracted matrix calibration or an unextracted matrix calibration (external solvent calibration). The following method quality control performance criteria will be met in the application o f the method: . 9.1 Extracted Matrix Calibration An extracted calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, and analyzed before each analytical set. It will consist of a minimum of nine (9) levels and may include a blank. The equation of the calibration curve will be determined by regression analysis using the peak areas of the analyte. The accuracy of each level will be verified. Any level outside 25% deviation from nominal must be deactivated and the regression recalculated, except the LLOQ which must be within 30% of nominal. All levels must show a response greater than twice that of the blank. 9.2 Unextracted Solvent Calibration (external calibration) A calibration curve of a minimum of nine (9) levels from unextracted solvent standards will be prepared, and may include a blank. - .The standard curve equation will be determined by regression analysis using the peak areas of the analyte. The accuracy at each level will be verified. Any level outside 25% deviation from nominal must be deactivated and the regression recalculated, except the LLOQ which must be within 30% of nominal. All levels must show a response greater than twice that of the blank. 9.3 Limits of Quantitation (LOQ) The lower level of quantitation (LLOQ) for each target analyte is determined by the lowest'calibration level that shows a recovery o f 30% for the target analyte and a response greater than twice that of the blank. Should the LLOQ level calibration point be deactivated in a particular set the practical limit of quantitation for this set will be raised to the next acceptable level. / 9.4 Continuing Calibration Verification (CCV) For both extracted and unextracted calibration curves, the continued accuracy of the calibration will be verified. This will be accomplished by the re-injection o f one of the original curve points, preferably one in the mid-range of the curve. A maximum of ten (10) samples maybe injected before the injection of a CCV, and then more samples maybe analyzed. A CCV is then analyzed again at the end o f the sample set. The CCV's must show a recovery within 25% to be considered acceptable. Samples will then be bracketed by the calibration curve and acceptable CCV's. 9.5 Blanks 9.5.1 Solvent Blank An aliquot o f methanol, or other appropriate solvent, is used as a solvent blank. Solvent blanks are not extracted. 9.5.2 Method Blank A 1.0 mL aliquot of water, or other appropriate amount, is used as a method blank. Method blanks are extracted and analyzed with each set following this procedure. 9.5.3 Matrix Blank An aliquot of matrix (wet or dry, as appropriate) is extracted and analyzed to determine the endogenous level of target analyte(s) in the matrix, if any. The frequency and exact makeup of the matrix blanks will depend on the scope o f the study and the availability of additional matrix. Specific requirements, if any, will be addressed in the study protocol and/or the raw data. ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 4 of 10 Page 29 of 142 il t $ if 9.6 Sample Replicate Samples replicates are prepared according to each study protocol or will be documented in the raw data. 9.7 Surrogate standard If surrogate standard is a component of the study, all samples are spiked with surrogate standard prior to extraction, with the exception of blank samples. . 9.8 Internal standard If internal standard is a component of the study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range of the calibration curve. , 9.9 Pre-Extraction Matrix Spike Pre-extraction matrix spikes consist of matrix spiked with a known amount of target analyte and extracted in the same manner as the study samples. The amount of target analyte present in the matrix prior to the addition o f the spike must . be known. This may be accomplished by the concurrent extraction of an additional aliquot o f the same matrix. Analysis of these samples provides a measure of extraction efficiency. Recoveries of target analytes should be within 30% of the theoretical concentration. If the samples fail to fall in this range the data for the study samples should be rejected. If the samples are to be accepted sufficientjustification for their use should be provided in the report. The number and frequency of pre-extraction matrix spikes will be addressed either by the protocol or the project lead. 9.10 Post-Extraction Control Sample Post-extraction control samples may be prepared for each set of extracted samples (if applicable to the study) and analyzed to determine extraction efficiency. The samples are prepared by adding additional target analyte to the final extract, and after any dilution. Post-extraction control sample duplicates may be prepared periodically to measure the precision associated with the analysis. These samples should be analyzed in the same run as the unspiked sample. Post-extraction control sample concentrations should fall in the mid-range o f the initial calibration curve or should be prepared at 1.5-5 times the endogenous concentration o f the.analyte. Spike concentrations should fall in the low-range of the initial calibration curve if extremely low levels are expected. 9.11 Sam ple Dilution Any sample extract with an area greater than that of the highest acceptable calibration standard will need to be diluted into the range of the calibration curve. All dilutions of the extract will be documented in the raw data. 10 Procedures - 10.1 Soil Sam ple Preparation and Extraction Weigh approximately 1g of sample, or other appropriate amount, into a tared sample vessel. Record this weight on the ' appropriate data form. Return the unused portion of the sample to storage. Spike target analyte(s) into the soil as appropriate. Add 25mL of 1% acetic acid in reagent water, or other appropriate amount, to the sample vial. Record the volume of solution added to the sample on the appropriate data form. Cap and mix the contents of the vessel thoroughly. Centrifuge the vessel until the solution becomes clear, if appropriate. Solutions containing less than 5% soil may not require the centrifugation step. Attach the reservoir to the SPE cartridge and attach this reserveir/cartridge unit to a vacuum manifold. Condition the SPE cartridge by washing twice with at least 5mL of methanol, or other appropriate solvent, followed by at least two 5mL aliquots of aqueous 1% acetic acid, taking care not to allow the column to run to dryness after each wash. ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 5 o f 10 Page 30 of 142 NOTE: Depending on the nature of the target compounds, the original sample filtrate may need to be collected and analyzed. If this is the case, place collection vessels under each cartridge at this point, prior to the addition of the sample. - After conditioning is complete, decant the aqueous portion of the sample into the reservoir/cartridge unit and allow all of the liquid to pass through the column to dryness. Take care not to pour the wet soil into the cartridge, it will hamper the progress o f liquids through the column. Run the vacuum on high for at least 2 minutes to adequately dry each SPE cartridge. Add 5mL, or other appropriate amount, of methanol, or other appropriate solvent, to the original sample vessel (where the soil remains), recording the volume on the appropriate data sheet. Mix contents thoroughly and centrifuge until the contents become clear. Place collection vessels under each cartridge and decant the solvent into the cartridge as above, again allowing the column to go to dryness. Repeat this solvent elution step with fresh collection vessels if needed. [f an internal standard is desired, transfer a known amount of eluate into an appropriate sample vial, then add the appropriate amount o f internal standard. Transfer remaining samples into the appropriate vials for the type of analysis being carried out, if needed. 10.2 Fluid Sample (Suspended Sludge) Preparation Fluid samples should be. at approximately room temperature for preparation. ` Vortex mix the fluid sample for approximately 15 seconds. Determine the volume of the original sample to extract. Record this volume on the appropriate data form. Return the unused portion o f the sample to storage. If additional dilution of the sample is desired at this stage, add water and record the volume on the data form. Spike target analyte(s) into the aqueous solution as appropriate. Add glacial acetic acid to the sample vial to reach a nominal concentration of 1%. Record the volume of acid added to the sample on the appropriate data form. Attach the reservoir to the SPE cartridge and attach this reservoir/cartridge unit to a vacuum manifold. Condition the SPE cartridge by washing twice with at least 5mL of methanol, or appropriate solvent, followed by at least two 5mL aliquots of aqueous 1% acetic acid, taking care not to allow the column to run to dryness after each wash. NOTE: Depending on the nature of the target compounds, the original sqmple filtrate may need to be collected and analyzed. If this is the case, place collection vessels under each cartridge at this point, prior to the addition of the sample. . After conditioning is complete, decant the sample into the reservoir/cartridge unit and allow all of the liquid to pass through the column to dryness. Samples containing a high percentage of sludge may necessitate the addition o f a plug of glass wool on top o f the SPE packing material to prevent the plugging o f the column. Run the vacuum on high for at least 2 minutes to adequately dry each SPE cartridge. Add 5mL, or other appropriate amount, of methanol, or other appropriate solvent, to the original sample vessel, recording the volume on the appropriate data sheet. Mix contents thoroughly and centrifuge until the contents become clear. Place collection vessels under each cartridge and decant the solvent into the cartridge as above, again allowing the column to go to dryness. Repeat this solvent elution step with fresh collection vessels if needed. If an internal standard is desired, transfer a known amount of eluate into an appropriate sample vial, then add the appropriate amount of internal standard. Transfer remaining samples into the appropriate vials for the type o f analysis being carried out, if needed. ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 6 of 10 11 Data Analysis and Calculations 11.1 C alculations If other calculations are used than those listed, they will be documented in the raw data. Calculate the total sample Dilution Factor: Dilution Factor (DF) = {(OW + DWyOW} x (EV/SV) x any additional dilution of final eiuate Calculate theoretical concentrations of analyte in final eiuate: Concentration = (Concentration of Analytical Standard x Volume of standard added) / EV Convert observed result to original sample result: Original sample result = Observed result x DF Calculate spike percent recoveries using the following equation: % Recovery Observed Result - Matrix Blank x 100 Theoretical Concentration OW = Original sample weight DW = Diluent weight, assume density of water = 1 / EV = Eiuate volume (volume of final extract) SV = Sample volume removed for extraction DF = Dilution factorCalculate relative standard deviation using the following equation: Relative Standard Deviation Standard Deviation x 100 Mean Calculate percent deviation using the following equation: % Deviation = Theoretical Cone. - Measured Cone, x 100 Theoretical Cone. 12 Pollution Prevention and Waste M anagem ent Sample waste is disposed of in low BTU containers. Flammable solvent waste is disposed of in high BTU containers. Glass pipette waste is disposed of in broken glass containers located in the laboratory. ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 7 o f 10 Page 32 of 142 13 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw data. , Summarize data using suitable software and store in the study folder. 14 Attachments Attachment A: Samples of Extraction Worksheets 15 References None 16 Affected Documents None 17 Revisions Revision Number Revision Description / Revision Date ETS-8-39.0 Solid Phase Extraction o f Soils, Sediments and Sludges Page 8 o f IO Sample ID i Sample Description Sample Weight, g Spike Added, uL Volume of 1% Acetic Acid Added, mL Cap and Mix Vessel Centrifuge? Y/N Precondition Extraction Column Pass Sample through cartridge. Collect Aqueous portion? Y/N Volume of Methanol added to Original Soli Sample, mL Cap and Mix Vessel Centrifuge? Y/N Pass sample through cartridge, collect eluate Repeat Solvent Extraction (Columns ID13)? Y/N Additional Dilution, if Needed [Addition of Internal [standard, If needed, uL Page 34 o f 142 Attachment A: Samples o f Extraction Worksheets S xtii.E xtractio n .W ork-sheet, M eth n ri JETjS^R-xxx J). S t u d y N u m b e r ................ ........... ..... .... _ _____ _____________ ______ ______ ______ ______ ____________T y p e o f C o l u m n U s e d 1 Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx C o l 3: Balance ID T /D /l _____________________ Col 4: Standard ID Standard Concentration:. Amount Standard Added:. T / D / l _____________________ C o l 5: Standard ID : Vo lu m e A dd ed: _________ T / D / l _____________________ Col 6: T/D/l 2 3 4 5 67 8 9 10 11 12 13 14 15 16 Co l 7: Centrifuqe ID: Sp ee d: ______________________________ _rcf/rpm D u r a t i o n : ________________________________ T/D/l Col 8: T/D/l Col 9: T/D/l Col 10: M e O H ID: Volume add ed : ____ j_________________________ T / D / l ______________________________________ Col 11: T/D/l Co l 12: Centrifuqe ID: Speed: rcf/rpm Duration:___________________________ ______ T/D/l Col 13: T/D/l Col 14: T/D/l Sa mp le s stored In refrigerator/freezer ID ______________________________ a t _________________________degrees C until analysis. T/D/l. ETS-8-39.0 Page 9 of 10 Solid Phase Extraction o f Soils, Sediments and Sludges Sample ID Sample Description Sample Volume, mL Additional Water Added, mL, if desired Spike Added, uL . Volume of Glacial Acetic Acid Added, mL Cap and Mix Vessel Precondition Extraction Column Pass Sample through cartridge. Collect Aqueous portion? Y/N Elute sample with mL of MeOH Eiute sample a second time? No, Yes-wlth mL of MeOH Additional Dilution, If Needed Addition of Internal standard, If needed, uL Comments Page 35 of 142 Attachment A: Samples o f Extraction Worksheets Fluid_S-am pie ExtraatiO-D-Work.sh-eel,.Method E T.S -8j3iL (l S t u d y N u m b e r ............ _........ ................................. '_____ ______ ______ T y p e of C o l u m n U s e d 1 Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Fxx-xxxx-xxxxxx Exx-xxxx-xxxxxx Fxx-xxxx-xxxxxx Exx-xxxx-xxxxxx Fxx-xxxx-xxxxxx Exx-xxxx-xxxxxx F.xx-xxxx-xxxxxx Fxx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Fxx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Col 3: T/D/l Col 4: Wat er ID Amount Water Added:. T/ D/ l _________________ Col 5: Standard ID: Volume Added:______ T/ D / l _________________ Co l 6: Standard ID: Volume Add ed; ______ T/ D/ l _________________ 2 3 4 5 6 78 9 10 Col 7: T/D/l Col 8: T/D/l Col 9: T/D/l Col 10: M eO H ID: Volume added:___ T /D /l _____________ C o M 1 : M eO H ID: Volume added:___ T/ D/ l ____ L_____ _ S a m ple E-Xlxa.ct S t o r a g e Samples stored in refrigerator/freezer ID _____________________________ a t ________________________ degrees C until analysis. 11 12 13 Col 12:Diluter ID: Volume of sample added:__ Volume of Diluent Added: Diluent ID_________________ Final Dilution Factor: T/ D / l _________ ____________ Col 13:lnternalStandard ID Internal standard Cone.____ Amount Added:____________ Volume of sample: IS Concentration__________ T/D/ l ______________________ T/D/l. ETS-8-39.0 Page 10 o f 10 Solid Phase Extraction o f Soils, Sediments and Sludges 14 3M Environmental Laboratory M ethod Analysis of Waste Stream, Water Extracts or Other Systems Using , HPLC-Electrospray/Mass Spectrometry M ethod Number: ETS-8-155.1 Adoption Date: I j ^l 00 Revision Date: ^ 3 / ^ Author: Approved By: / Date J Date / Page 36 of 142 1.0 Scope and A pplication 1.1 Scope: This method describes the analysis of waste stream, water samples or other systems using HPLC-electrospray/mass spectrometry. 1.1.1 Specific analytes, ions, matrices, solvents, solutions, quality controls, internal standard(s) and other parameters will be defined in the protocol or the sample . preparation worksheet(s). 1.2 Applicable Compounds: Electrospray ionizable compounds. 1.3 Matrices: 1.3.1 Waste Streams and other systems may consist of aqueous and/or organic solvent systems or as designated in the protocol or sample preparation worksheet(s). 1.3.2 Water Samples include tap water, ground water, wastewater and other aqueous solutions. The matrix will be defined by the protocol or sample preparation . worksheet(s). 2.0 Su m m a r y o f M e t h o d 2.1 This method describes the analysis of electrospray ionizable compounds, using HPLCelectrospray mass spectrometry (HPLC-ES/MS). The analysis is performed by the mass selection of a single ion characteristic of a particular compound. 3.0 D e f i n i t io n s 3.1 Atmospheric Pressure Ionization (API): Cdmmercially available HPLC-ES/MS single quadrupole systems allow for various methods of ionization by utilizing a variety of sources, probes, and interfaces. These include but are not limited to*. Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization in these processes occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): A method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrom eter (MS): Commercially manufactured MS systems are equipped with single quadrupole mass selective detectors. Ions are selected on the basis of mass to charge ratio (m/z) and subsequently detected. 3.4 Micromass MassLynx / HP ChemStation Software: System software designed for the specific operation of an HPLC-ES/MS. Please refer to the manual for the specific instrument software. 3M Environmental Laboratory ETS-8-155.1 Analysis Using HPLC-Eiectrospray/Mass Spectrometry Page 2 o f 10 4.0 W arnings and Cautions 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the electrospray probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective clothing, gloves, and eyewear. 4.2 Cautions: 4.2.1 Operate solvent pumps below a backpressure of 400 bar (5800 psi). If the backpressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences 5.1 To minimize interferences when analyzing samples, Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E q u ip m e n t ____________ . _____________________________________________________ 6.1 Equipment listed below may be modified in, order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Platform LCZ Mass Spectrometer (or equivalent) equipped with an electrospray ionization source. ' 6.1.2 HP 1100 low pulse solvent pump, solvent degasser, column compartment, and autosampler of equivalent HPLC system. 7.0 Supplies and M aterials 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (or house air system.). 7.1.2 HPLC analytical column, such as a Betasil C l8 column (50x2mm, 5 pm particle size) or equivalent. . 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 Reagents and Standards____________________________________ ;_____________ 8.1 Reagents: 8.1.1 Methanol, HPLC grade or equivalent. 3M Environmental Laboratory ETS-8-155.1 Analysis Using HPLC-EIectrospray/Mass Spectrometry Page 3 o f 10 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or another vendor. 8.1.3 Ammonium acetate, reagent grade or equivalent. 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 L volumetric flask, add the appropriate volume of Milli-Q water, mix until all solids are dissolved. Store at room temperature. 8.1.4 Other solvents and solutions may be used as stated in the protocol or the sample preparation worksheet. 8.2 Calibration Standards: 8.2.1 Typically two method blanks (Milli-Q water), two matrix blanks, and a minimum of 5 solvent standards are analyzed with each group of samples. 9.0 Sa m p l e H a n d l in g 9.1 Standards and prepared samples may be stored in capped autovials, capped 15 mL centrifuge tubes or other suitable containers until analysis. 9.2 If analysis will be delayed, standards and prepared samples may be refrigerated at approximately 4 C until analyses can be performed (refrigerator temperatures may have a detrimental affect on the solubility of saturated solutions). 10.0 Q u a l it y C o n t r o l ____________________________________________________________________ 10.1 Blanks: 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each sample set to determine contamination or carryover. 10.1.1.1 When the study matrices consist of highly purified solvents such as Type 1 water or HPLC grade organic solvents, the method and matrix blanks may be represented by a single solvent blank. 10.1.2 Solvent blanks should be analyzed prior to each calibration curve. Method blanks and matrix blanks should be analyzed after the initial calibration curve but prior to the study samples. If carryover is a problem, consecutive solvent blanks may be necessary. 10.2 M atrix Spikes: 10.2.1 Matrix spikes may be prepared for each set of extracted samples (if applicable to the study) and analyzed to determine exfraction efficiency. 3M Environmental Laboratory ETS-8-155.1 Analysis Using HPLC-Elcctrospray/Mass Spectrometry Page4of1Q Page 39 of 142 10.2.2 Matrix spike duplicates may be prepared periodically to measure the precision , associated with the analysis. 10.2.3 Analyze the matrix spike and matrix spike duplicate (if prepared) in the same run as the original sample. 10.2.4 Matrix spike and matrix spike duplicate concentrations should fall in the mid range of the initial calibration curve or should be prepared at 1.5-5 times the endogenous concentration of the analyte. Spike concentrations should fall in the low-range of the initial calibration curve if extremely low levels are expected. 10.3 Continuing Calibration Verifications: 10.3.1 Continuing calibration verifications (CCV) are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per sample set. . 10.4 Internal Standard/Surrogate Standard: 10.4.1 An internal standard (IS) may be used to quantify the target analytes by establishing a relationship between the ratio of analyte response to IS response and a known concentration of the analyte of interest. The IS should be spiked at , an amount that will fall within the mid-range of the calibration curve. The IS should be added after the extraction process and before analysis. 10.4.2 A surrogate standard may be used for quality control. The surrogate is used to quantitatively evaluate the entire analytical procedure including sample preparation and analysis. The surrogate should be spiked at the beginning of the sample preparation and should fall within the low to mid-range of the calibration curve. 11.0 C a l i b r a t i o n a n d St a n d a r d i z a t io n 11.1 Analyze the standard curves prior to and following each set of samples. The average of two standard curves may be plotted by linear regression (y = mx + b) weighted 1/x, or quadratic fit (y = ax2+ bx + c) using MassLynx or other suitable software. The calibration curves should not be forced through zero. 11.1.1 The closing calibration curve may be excluded if the CCV's meet acceptable criteria. If only the first curve is used, the calibration and standardization parameters are the same. 11.2 If the initial calibration curve does not meet acceptance criteria perform routine maintenance or prepare a new standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range. Example: when 3M Environmental Laboratory ETS-8-1S5.1 A nalysis Using HPLC-Electrospray/Mass Spectrometry Page 5 o f 10 Page 40 of 142 attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 11.4 High and/or low points may be excluded from the calibration curves to provide a better fit over the linear range appropriate to the data or because they did not meet the pre determined,acceptance criteria. Low-level curve points should also be excluded if their area counts are not at least twice that of the method blanks. Justification for exclusion of calibration curve points will be noted in the raw data. 11.4.1 A minimum of 5 points will be used to construct the calibration curve. 12.0 P r o c e d u r e s * 12.1 Acquisition set-up - please reference the SOP that pertains to the specific instrum ent. Actual parameters will be recorded on the instrument printouts. 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using the first letter of the name of the instrument (T for Tucker), the year (00 for 2000), the month (04 for April), and the day (T001012 for October 12, 2000 on Tucker). If more than one list is made on the same day, use increasing letters of the alphabet starting with A at the end of the list. 12.1.1.2 Assign a method (MS) file. . 12.1.1.3 Assign an HPLC program (Inlet file). 12.1.1.4 Type in sample descriptions and vial position numbers. 12.1.2 To create a method, click on method in the Acquisition control panel then mass spectrometer headings and select SIR. Set ionization mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected in addition to the SIRs. Save acquisition method. See the Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of solvent standards. 12.1.4 Samples are analyzed with a continuing calibration verification (CCV) injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor for possible analyte carryover. 12.2 Using the Autosampler/Column Heater 12.2.1 Place sample vials into the sample tray according to the sample list prepared in Section 12.1.1. 3M Environmental Laboratory ETS-8-155.I Analysis Using HPLC-Electrospray/Mass Spectrometry Page 6 o f 10 Page 41 of 142 12.2.2 Attach the proper analytical column in the column heater compartment. If using the switching valve, ensure that the tubing is run to the appropriate ports. 12.3 Using the Inlet Editor 12.3.1 Set-up the HP 1100 using the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.3.1.1 Sample size = 10 pL injection 12.3.1.2 Flow rate = 300 pL/min. 12.3.13 Cycle time = 10.0 minutes , 12.3.1.4 Mobile phase components: Solvent A: 2.0 mM Ammonium Acetate in Water Solvent B: Methanol (MeOH) Solvent Gradient: Time (min.) %B 0.00 5.00 % 1.00 5.00 % 4.50 95.0% 8.00 95.0 % 8.50 5.00 % 10.0 Stop 12.4 Instrument Set-up ) 12.4.1 Refer to the Platform LCZ User's Guide, the MassLynx NT User's Guide, the ETS-9-36, "Operation and Maintenance of the Micromass Platform LCZ Electrospray/Mass Spectrometer", or the SOP that pertains to the specific instrument. 12.4.2 Check the solvent level in reservoirs and refill if necessary. 12.4.3 Check the tip of the stainless steel capillary at the end of the probe with an eyepiece. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.4.4 Turn on the nebulizing gas. 12.4.5 Open the tune page. Click on `Operate' to initiate the desolvation heaters. 12.4.6 Open the Inlet Editor. 12.4.6.1 Set HPLC pump to "On". 12.4.6.2 Set the solvent flow to the desired flow rate. 12.4.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be . expelled with no nebulizing gas leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 3M Environmental Laboratory ETS-8-155.1 Analysis Using HPLC-Eiectrospray/Mass Spectrometry Page 7 o f 10 Page 42 of 142 12.4.6.4 Allow to equilibrate for at least 10 minutes.' 12.4.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response. Actual parameters will be recorded on the instrument printouts. 12.4.7.1 Drying gas 250-425 liters/hour 12.4.7.2 ESI nebulizing gas 10-15 liters/hour 12.4.7.3 HPLC constant flow mode, flow rate 10 - 500 pL/min 12.4.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.4.7.5 Source Block temperature 150. . 12.4.7.6 Desolvation temperature 250. 12.4.8 Print the tune page with its parameters, the Inlet page, sample list, mass spectrometer information, and all other applicable information and store it in the study binder with copies taped into the instrument run logbook. 12.4.8.1 All copies must be initialed and dated. 12.4.9 Click on start button on the MassLynx toolbar. Ensure beginning and ending sample numbers encompass all samples to be analyzed. 13.0 13.1 D a t a A n a l y s is a n d C a l c u l a t io n s . . Calculations (including, but not limited to): : 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.3 Calculate actual concentration of analyte in matrix (pg/mL): On-Column Concentration (pg/mL) x Dilution Factors = Calculated Concentration 14.0 M e t h o d P e r f o r m a n c e 14.1 The Limit of Quantitation (LOQ) is method, analyte, and matrix specific. For many analytes, the LOQ concentration is selected as the lowest acceptable non-zero standard in the calibration curve. ` 3M Environmental Laboratory ETS-8-155.1 Analysts Using HPLC-Electrospray/Mass Spectrometry Page 8 o f 10 14.2 Solvent and method blank area counts must be < % that of the lowest standard used in the calibration curve. 14.3 The coefficient of determination (r2) value for the calibration curve must be greater than or equal to 0.980. . 14.4 Continuing Calibration Verification (CCV) percent recoveries must be 30% of the standard concentration. . 14.5 Internal Standard recoveries should be within 50% of the spiked concentration. 14.6 If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the raw data. 14.7 If data is to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. . 16.2 Print the tune page, sample list, acquisition method and all other applicable information to include in the appropriate study folder. Copy these pages and tape into the instrument run logbook. 16.3 Plot the calibration curve then print these graphs and store in the study folder. . 16.4 Print data integration summary, integration method, and chromatograms and file in the study binder. 16.5 Summarize data using suitable software and store in the study binder. 16.6 Back-up electronic data to appropriate medium. Record the file names and location of backed-up electronic data in the study binder. 16.7 Documentation of analyte(s) and all reference substances will include trace numbers, lot #'s, purity, expiration, and storage conditions. 17.0 Attachments_________________________________________________________ __ 17.1 None 3M Environmental Laboratory ETS-8-155.1 Analysis Using HPLC-Eiectrospray/Mass Spectrometry ' Page 9 o f 10 Page 44 of 142 18.0 References 18.1 Platform LCZ User's Guide, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9LZ; United Kingdom. . 18.2 MassLynx NT User's Guide, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9LZ; United Kingdom. 18.3 MassLynx NT Guide To Data Acquisition, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9LZ; United Kingdom. 18.4 ETS-9-34.0, "Operation and Maintenance of Hewlett Packard HPLC 110G System Equipped with a Mass Spectrometer Detector and/or a Photo Diode Array Detector". 18.5 ETS-9-3 6.0, "Operation and Maintenance of the Micromass Platform LCZ Electrospray/Mass Spectrometer". 19.0 19.1 Affected Documents None 20.0 R e v is io n s Revision Number Reason For Revision Revision Date 1 Title Change 09 May 01 Section 1: Enlarged the scope of the method by including additional matrices. Also added comments about specific parameters will be defined in the protocol or prep sheets. Sections 1 and 2: Removed references to specific compounds. Section 3: Removed paragraph on Conventional vs. Z-Spray probe interface. Section 3: Changed to allow different systems and software. Section 8: Added paragraph allowing for the use of alternative solvents and solutions. Section 9: Broadened the types o f storage containers and conditions. Section 10: Added comments to allow for flexibility in quality controls for different types o f studies and applicability. ' Section 11: Added a paragraph allowing for the excluding o f the second curve. Section 11: Added a paragraph (11.4) allowing for the dropping of curve points and minimum number of curve points needed. Section 12: Added comments to reference the specific SOP's for set-up. Section 12: Corrected numbering. Section 16: Added paragraph on records o f analytes and substances. Section 18: Added reference to the method for the Hewlett-Packard LC/MS. 3M Environmental Laboratory ETS-8-I55.1 Analysis Using HPLC-Electrospray/Mass Spectrometry Page 10 o f 10 Attachment B: Data Tables Page 46 of 142 Medium A PFBS Sample E02-09I3-0I3 E02-09I3-014 EO2-09I3-0I5 E02-913-043 E02-09I3-044 E02-0913-045 E02-09!3 -103 E02-09I3-104 E02-0913-105 E02-09 f3 -163 E02-09I3-I64 E2-0913-l65 Result Average Timepoint (ng/mL) (ng/mL) Day 0 <50.06 Day 0 <50.06 <50.06 Day 0 . <50.06 Day 4 45.09 Day 4 38.44 42.17 Day 4 42.98 Day 14 152.4 Day !4 I49.3 I48.2 Day I4 143.0 Day 28 389.4 Day 28 ! 325.6 345.0 Day 28 320.0 Average (ng/mL) anion* <44.28 37.30 S3 1.1 305.2 Standard Deviation (ng/mL) N/A 3.398 4,790 38.58 * Average was multiplied fay a factor o f 0.8X46 to correct to anion ** Based on the total amount o f fluorine in PFBS, r N/A *Not applicable Relative Standard Deviation N/A Percent Degradation** <0.33% ' 8.06% 0.28% 3.23% 0.97% II.2% 2.3% Sample E02-0913-013 E02-0913-OI4 E02-09I3-015 E02-0913-043 E02-0913-044 E02-0913-045 E02-09I3-103 E02-09I3-I04 E02-09I3-105 E02-0913-163 E02-09I3-164 E02-0913-165 Result Average Timepoint (ng/mL) (ng/mL) Day 0 <490.8 Day 0 <490.8 <490.8 Day 0 <490.8 Day 4 167.5 Day 4 168.3 165.4 Day 4 160.3 Day 14 75.95 Day 14 61.90 66.66 Day 14 62.13 I Day 28 29.20 Day 28 41.11 36.90 Day 28 40.38 * Based on the total amount o f fluorine in Standard Deviation (ng/mL) N/A 4.406 8.046 I 6.675 /i Relative Standard Deviation . N/A 2.66% 12.1% 18.1% Percent Degradation* <3.5% 1.2% 0.47% 0.26% N/A = Not applicable Sample E02-0913-013 E02-0913-014 E02-0913-015 Result Average Timepoint (ng/mL) (ng/mL) Day 0- <126.1 Day 0 <126.1 <126.1 Day 0 <126.1 Standard Deviation (ng/mL) N/A ' E02-0913-043 E02-0913-044 E02-0913-045 E02.-0913-103 E02-0913-104 E02-0913-S05 E02-0913-163 E02-0913-164 E02-09I3-165 Day 4 j <25.23. Day 4 <25.23 Day 4 <25.23 Day 14 16.57 Day 14 4:89 Day 14 15.02 Day 28 46.24 Day 28 44.27 Day 28 48.20 <25.23 15.49 46.24 1 N/A 0.9347 1.965' * Based on the total amount of fluorine in ' N/A - Not applicable' Relative Standard Deviation N/A Percent Degradation* <1.0% N/A <0.20% 6.03% 0.12% 4.25% 0.37% Page 47 of 142 Mtfdiuin A Sample E02-09I3-0I3. E02-093-014 E02-09I3-015 E02-0913-043 E02-0913-044 E02-0913-045 E02-0913-I03 E02-0913-104 Result Timcpoint (ng/mL) . Day 0 <125.2 DayO <125.2 Day 0 <125.2 Day 4 ' 205.2 Day 4 185.5 Day 4 191.4 Day 14 ' 257.8. Day 14 ' 274.6 Average (ng/mL) <125.2 194.0 261.2 E02-0913-105 E02-0913-163 E02-0913-l 64 Dav 14 Day 28 Dav 28 251.1 365.4 311.6 320.8 E02-0913-165 Day 28 285.5 * Bas*d on the total amount o f fluorine in N/A - Not applicable Standard Deviation (ng/ml.) N/A Relative Standard Percent Deviation Degradation'" . N/A <0.92% 10.11 ' 5.21% 1.4% 12.10 4.63% l.9 aS 40.72 12.7% 2.4% Sample Timcpoint E02-0913-013 Day 0 EQ2-Q913-014 DayO E02-0913-015 Day 0 E02-0913-043 . Day 4 E02-09I3-044 Day 4 E02-0913-045 I Day 4 02-0913-103 Day 14 E02-0913-104 Day 14 E02-0913-105 Day 14 E02-0913-163 Day 28 E02-0913- 164 Day 28 E02-09I3-165 Day 28 Result (ng/mL) 4004 3961 4422 181.1 149.6 153.5 82.02 50.50 62.15 <25.09 <25.09 <25.09 Average (ng/mL) 4129 161.4 64.89 <25.09 * Based on the total amount o f fluorine in in/ a = .noc aoolicaDle Standard Deviation (ng/mL) 254.3 Relative Standard Percent Deviation Degradation* 6.16% 26% 17.17 10.6% 1.0% 15.94 1 24.6% 0.40% N/^ N/A <0.15% Sample Timepoint Result (ng/mL) Times Correction Factor (ng/mL) ** E02-0913-013 02-0913-014 Day 0 Day 0 <500.0 <500.0 <665.0 <665.0 E02-0913-015 Day 0 <500.0 <665.0 E02-0913-016 Day 0 <500.0 <665.0 E02-0913-017 Day 0 <500.0 <665.0 E02-0913 -103 Day 14 6247 8309 E02-0913-104 E02-0913-105 Day 14 1 12680 Day 14 6508 16865 8656 E02-0913-106 Day 14 5670 7542 E02-0913-17 Day 14 E02-0913-163 Day 28 6883 4717 9155 6274 E02-0913-164 E02-0913-165 Day 28 Day 28 4874 4051 6483 5388 E02-0913-166 Day 28 E02-0913-167 Day 28 4744 5001 6309 6652 * Based on the total amount of fluorine ir Average (ng/mL) <665.0 10105 6221 Standard Deviation (ng/mL) Relative Standard Deviation Percent Degradation* N/A N/A <4.0% 3824 37.8% ! 60% 489.4 7.87% : 37% ** A correction factor was calculated to be and ' N/A ~ Not applicable Page 48 of 142 Medium Sample E02-0913 - 0 13 . E02-09!3-014 E02-0913-015 E02-0913-043 E02-0913-044 EQ2-09[3-045 E02-0913-103 E02-09I3-104 E02-0913 -163 E02-09I3-64 E02-0913-165 Result Average Timepoint (ng'inl.) (ng'ml.) DayO I57.8 DayO Day 0 I4I.5 126.3 141.9 Day 4 Day 4 19494 19133 19243 Day 4 I9101 I Day 14 Day 14 9572 9761 9666 Day 28 ' Day 28 10336 Il79l 10599 Day 28 | 9670 * Based on the total amount o f fluorine in ' Standard Deviation (ng/mL) 15.78 218.2 133.4 1084 Relative Standard Percent Deviation Degradation-" 11.1% 0.84% 1.13% 114% 1.38% . 58% 10.2% 63% N/A = Not applicable / Page 49 of 142 Medium B PFBS Result Sample Timepoint (ng/ml.) E02-0913-0! 8 Day 0 <50.0'6 E02-0913-0! 9 DayO <50.06 E02-0913-020 Day 0 <50.06 E02-09! 3-048 Day 4 8.188 E02-09 13-049 Day 4 5.804 E02-0913-050 Day 4 6.823 E02-0913-I08 Day 14 * E02-0913-109 Day 14 10.26 E02-09 13-110 Day 14 I7.87 E02-09 13-168 Day 28 4.27 E02-0913-169 Day 28 16.19 E02-0913-170 Day 28 15.02 Average (ng'mL) <50.06 6.938 14.07 15.16 Average (ng/mL) anion** <44.28 Standard Deviation (ng/mL) N/A 6.138 1.196 12.44 5.381 13.41 i 0.968 * SPE dogged, unable to extract ** Average was multiplied by a factor o f 0,8846 to correct to anion M * Based on the total amount of fluorine ir PFBS, r N/A * Not applicable Relative Percent Standard Degradation Deviation *** N/A <0.33% 17.2% 0.045% 38.3% 0.092% 6.38% 0.10% Result Sample Timepoint (ng/mL) E02-0913-01.8 DayO <490.8 E02-09 13-019 Day 0 <490.8 Average (ng/mL) <490.8 Standard Deviation (ng/mL) N/A E02-0913-020 Day 0 <490.8 E02-0913-048 i Day 4 E02-0913-049 Day 4 192.5 187.9 180.6 16.73 E02-0913-050 E02-0913-108 E02-0913-109 Day 4 Day 14 Day 14 161.5 * 94.69 90.46 5.982 E02-0913-110 Day 14 86.23 E02-0913-168 E02-0913-169 E02-09 3-170 Day 28 Day 28 Day 28 44.25 43.29 63.04 50.19 11.14 !V * SPE dogged, unable to extract ** Raved on the total am ount o f fluorine in Relative Percent Standard Degradation Deviation ** N/A : <3.5% 9.26% 1.3% 6.61% 0.64% 22.2% 0.35% N/A = Not applicable Result Sample Timepoint (ng/mL) E02-0913-018 Day 0 <126.1 E02-0913-019 Day 0 <126.1 E02-0913-020 Day 0 <126.1 E02-09 13-048 Day 4 <25.23 E02-0913-049 Day 4 <25.23 E02-0913-050 Day 4 <25.23 E02-0913-108 Day 14 * E02-09 13-109 Day 14 <10.09 E02-0913-110 Day 14 <10.09 E02-09 13-168 Day 28 <10.09 E02-09 13-169 I Day 28 <10.09- .E02-09 13-170 Day 28 <10.09 Average (ng/mL) <126.1 <25.23 <10.09 <10.09 * SPE clogged, unable to extract m* Bused on the total amount of fluorine ip PFBA, N/A =*Not applicable Standard Deviation (ng/mL) N/A N/A N/A N/A Relative Percent Standard Degradation Deviation ** N/A <1.0% N/A <0.20% N/A <0.081 N/A <0.08! Page 50 of 142 Medium B Sample E02-9I3-0I8 E02-0913-I9 E02-09I3-02Q E02-0913-048 E02-0913-049 E02-09! 3-050 E02-0913-1S E02-0913-!09 E02-0913-1 10 E02-0913-168 E02-0913-169 E02-0913-170 Result Timepoint (ng/mL) DayO <125.2 DayO <125.2 Day 0 <125.2 Day 4 79.17 Day 4 76.13 Day 4 Day 14 88.06 Day 14 ! 162.9 Day 14 187.9 Day 28 163.3 Day 28 156.4 Day 28 209.9 Average (ng/ml.) <125.2 81.12 175.4 176.5 " SPE clogged, unable to extract Based nn rh~ amount a f fluorine in N/A * Not applicable Standard Relative Percent Deviation Standard Degradation (ng/mL) Deviation N/A N/A <0.92% 6.199 7.64% 0.60% 17.68 10.1% 1.3% 29.15 16.5% 1,3% Sample E02-0913-018 E02-0913-019 E02-0913-020 E02-0913-048 E02-0913-049 E02-0913-050 E02-0913-I08 E02-0913-109 E02-0913-110 E02-0913-168 E02-0913-169 E02-0913-I70 Result Average Timepoint (ng/mL) (ng/mL) DayO Day 0 3815 3978 3990 Day 0 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 4178 851.2 857.9 . 1044 *' 173.7 254.38 917.7 214.0 Day 28 Day 28 116.2 61.21 101.1 Day 28 126.0 * SPE dogged, unable to extract ** Based on the total amount of fluorine in Standard Deviation (ng/mL) Relative Percent Standard Degradation Deviation ** 181.8 4.56% 25% 109.4 11.9% 5.7% 57.05 26.7% 1.3% 34.92 / 34.5% 0.63% ' Sample E02-0913-018 Timepoint Day 0 Result (ng/mL) <500.0 Times Correction Factor (ng/mL) *** <665.0 E02-0913-019 E02-0913-020 Day 0 DayO <500.0 <500.0 <665.0 <665.0 E02-0913-021 E02-0913-022 E02-0913-108 E02-0913-109 E02-0913-110 E02-0913-1 11 E02-0913-112 E02-0913-168 Day 0 . Day 0 Day 14 Day 14 Day 14 Day 14 Day 14 Day 28 <500.0 <500.0 * 5090 4201 * 4052 3796 <665.0 <665.0 * 6770 5587 * 5389 5049 E02-0913-169 Day 28 3914 5206 E02-0913-170 E02-0913-171 E02-0913-172 Day 28 Day 28 Day 28 4054 4399 4198 5392 5850' 5584 SPE clogged, unable to extract ** Based on the total amount of fluorine in Average (ng/mL) <665.0 5915 5416 Standard Relative Percent. Deviation Standard Degradation (ng/mL) Deviation ** N/A , N/A <4.0% 746 12:6% 35% 314.8 5.81% , 32% ** A correction factor was calculated to be i . for the differences in response factor, and N/A = Not applicable \ L _____ ; Page 51 of 142 'Tv . ; .h . PF BS Vdium C E02-0913-023 E02-0913-024 E02-0913-025 E02-09I3-053 E02-09I3-054 E02-0913-055 E02-09!3 - 113 E02-0913-114 E02-0913-I 5 E2-09I3-173 E02-09 3-174 E02-0913-175 Day 0 Day 0 Day 0 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 <50.06 <50.06 <50.06 <6.258 <6.258 <6.258 <50.06 <50.06 <50.06 <10.01 <10.01 <10.01 <50.06 <6.258 <50.06 <10.01 * Average was multiplied by a factor ** Based on the total amount of fluorine in P F B S ,' N/A = Not applicable <44.28 <5.536 <44.28 <8.855 N/A N/A N/A N/A N/A ' <0.33% N/A <0.041% N/A <0.33% N/A <0.074% Result Sample Timcpoint (ng/mL) E02-0913-023 Day 0 <490.8 E02-0913-0241 DayO <490.8 E02-0913-025 Day 0 <490.8 E02-0913-053 Day 4 1 96.23 E02-0913-054 Day 4 85.91 E02-0913-055 Day 4 100.7' E02-0913-113 Day 14 <490.8 E02-0913-114 Day 14 <490.8 E02-0913-115 Day 14 <490.8 E02-0913-173 Day 28 47.23 E02-0913-174 ! Day 28 ! 33.10 E02-0913-175 Day 28 35.85 Average (ng/mL) <490.8 Standard Deviation (ng/mL) N/A 94.28 7.585 <490.8 N/A 38.73 1 7.491 ! * Based on the total amount of fluorine ir N/A = Not applicable Relative Standard Percent Deviation Degradation* N/A <3.5% 8.05% 0.67% N/A <3.5% 19.3% 0.27% Result Average Sample Timcpoint (ng/mL) (ng/mL) E02-0913-023 Day 0 E02-0913-024 Day 0 <126.1 <126.1 <126.1 E02-0913-025 DayO <126.1 E02-0913-053 E02-0913-054 E02-0913-055 E02-0913-113 E02-0913-114 E02-0913-! 15 E02-0913-173 E02-0913-174 E02-0913-175 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 <25.23 ' <25.23 <25.23 <126.1 <126.1 <126.1 <10.09 <10.09 <10.09 <25.23 <126.1 <10.09 * Based on the total amuunt of fluorine in Standard Deviation (ng/mL) N/A N/A N/A N/A N/A ~ Not applicable Relative Standard Percent Deviation Degradation* N/A <1.0% N/A <0.20% N/A <1.0% N/A <0.081% Page 52 of 142 (Viediuni C Sample E02-913-023 E02-09 3-024 Timcpoint Day 0 Day 0 Result (ng/mL) <125.2 <125.2 Average (ng/mL) <125.2 E02-0913-025 Day 0 <125.2 E02-09I3-053 E02-09I3-054 E02-0913-055 E2-09I3-!13 E02-0913-114 Day 4 Day 4 ' Day 4 Day 14 Day 14 9.331 <2.504 <2.504 <125.2' <125.2 9.331 <125.2 E02-0913-I 5 E02-0913- 173 E02-09I3-174 . E02-0913-175 Day 14 Day 28 Day 28 Day 28 <125.2 <2.504 <2.504 <2.504 <2.504 * B ut! on the total amount o f fluorine in N/A * Not applicable Standard Deviation (np/mL) N/A Relative Standard Percent Deviation Degradation* - N/A <0.92% N/A N/A 0.069% N/A N/A , <0.92% N/A , N/A <0.018% Sample E02-0913-023 E02-0913-024 E02-0913-025 E02-0913-053 E02-0913-054 E02-0913-055 E02-0913-113 E02-0913-114 E02-0913-115 E02-0913-173 Timcpoint Day 0 Day 0 Day 0 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 Day 28 Result (ng/mL) 228.0 1 223.5 216.1 605.3 624.7 602.7 996.7 978.3 982.3 1997 Average (ng/mL) 222.5 610.9 985.8 E02-0913-174 Day 28 3016 2327 E02-0913-175 Day 28 i 1966 * Based on the total amount of fluorine in Standard Deviation (ng/mL) 6.009 Relative Standard Percent Deviation Degradation* 2.70% 1.4% 12.02 1.97% 3.8% 9.692 0.983% 6.1% 597.5 25.7% /1 14% 1 Result Sample Timcpoint (ng/mL) E02-0913-023 Day 0 i <500.0 Times Correction Factor (ng/mL)** <665.0 E02-0913-024 Day 0 <500.0 <665.0 E02-0913-025 E02-0913-026 E02-0913-027 E02-0913-113 Day 0 Day 0 Day 0 Day 14 <500.0 <500.0 <500.0 <500.0 <665.0 <665.0 <665.0 <665.0 E02-0913-114 E02-913-!15 E02-0913-116 E02-0913-117 E02-0913-173 Day 14 Day 14 Day 14 Day 14 Day 28 <500.0 <665.0 <500.0 ! <665.0 <500.0 <665.0 <500.0 <665.0 858.2 1141.4 E02-0913-174 E02-0913-175 E02-0913-176 E02-0913-177 Day 28 Day 28 Day 28 Day 28 30.21 <10.00 <10.00 <10.00 40.2 <19.88 <19.88 <19.88 * U n c ^ l rwr* rki> fitfid n m n in l n f f lu o rin e in Average (ng/mL) Standard Deviation (ng/mL) <665.0 ! N/A <665.0 N/A 590.8 778.7 "* A correction factor was calculated to be or the differences in response factor, N/A - Not applicable Relative Standard Percent Deviation Degradation* N/A <4.0% N/A <4.0% 132% 3.5% Page 53 of 142 M atrix Spikes PFBS Sample . E2-913-016 E02-0913 - 0 17 E02-09 13-021 E02-09! 3-022 E02-0913-026 E02-0913-027 E02-0913-046 E2-0913-047 E02-0913-05! E02-0913-052 E02-0913-056 E02-0913-057 E02-0913-106 E02-0913-107 E02-0913-111 E02-913-112 E02-0913-116 E02-0913-117 E02-0913-166 E02-0913-167 E02-09 3-171 E02-0913-172 E02-0913-176 E02-0913 -177 Timepoint Medium . Day 0 A Day 0 A Day 0 B. Day 0 B Day 0 C Day 0 C Day 4 A Day 4 A Day 4 B Day 4 B Day 4 C Day 4 C Day 14 A Day 14 A Day 14 B Day 14 B ! Day 14 C Day 14 C Day 28 A Day 28 A Day 28 B Day 28 B Day 28 C Day 28 C. * S P E clogged, unable to extract N/A --N ot applicable Result Ing/inL) 490.75 491.55 429.05 434.15 430.18 428.55 712.25 698.01 504.95 490.38 506.81 509.42 596.05 634.31 ! * 456.75 439.70 429.04 877.21 755.87 460.10 460.13 416.77 397.95 _ Average (ng/mL) 491.2 431.6 429.4 705.1 497.7 508.1 615.2 456.8 434.4 816.5 460.1 407.4 Standard Deviation (ng/mL) 0.5698 Relative Standard Average Sample Deviation Result (ng/mL) 0.12% <50.06 True Value (ng/mL) 500.6 3.609 0.84% <50.06 500.6 1.156 0.27% <50.06 500.6 10.07 1.4% 42.17 500.6 10.30 2.1% 6.938 500.6 1.846 0.36% <6.258 500.6 27.05 4.4% ' 148.2 500.6 N/A N/A . 14.07 500.6 7.535 1.7% <50.06 500.6 85.80 10.5% 345.0 500.6 0.02121 0.0046% 15.16 500.6 13.31 3.3% <10.01 - 500.6 AVERAGE Percent Recovery 98.1% 86.2% 85.8% 132% 98.0% 102% 93.3% 88.4% 86.8% 94.2% 88.9% 81.4% 94.6% Sample E02-0913-016 E02-0913-017 E02-0913-021 E02-0913-022 E02-0913-026 E02-0913-027 E02-0913-046 E02-0913-047 E02-0913-051 E02-0913-052 E02-0913-056 E02-0913-057 E02-0913-106 E02-0913-107 E02-0913-111 E02-0913-112 E02-Q913-116 E02-0913-1 17 E02-0913-166 E02-0913-167 E02-09 13-171 E02-0913-172 E02-0913-176 E02-0913-1 77 Result Timepoint Medium (ng/mL) Day 0 A 551.63 Day 0 A 548.74 Day 0 B 533.57 Day. 0 B 531.15 Day 0 1 C 556,73 Day 0 C 526.33 Day 4 A 760.03 Day 4 A 637.31 Day 4 B 651.36 Day 4 B Day 4 1 c Day 4 c 688.90 ' 598.84 598.31 Day 14 A 504.58 Day 14 Day 14 A 556.54 B* Day 14 B ! 504.07 Day 14 C 547.88 Day 14 C 530.41 : Day 28 A 567.33 Day 28 A 579.25 Day 28 B 570.29 Day 28 B 581.15 Day 28 C 495.79 Day 28 C 460.98 * SPE dogged, unable to extract . N/A = Not applicable Average (ng/mL) 550.2 Standard Deviation (ng/mL) 2.045 Relative Standard Average Sample Deviation Result (ng/mL) 0.37% <490.8 True Value (ng/mL) 490.8 Percent Recovery 112% 532.4 1.705 0.32% <490.8 490.8 108% 541.5 ' 21.50 4.0% <490.8 490.8 110% 698.7 86.77 12% 165.4 490.8 109% 670.1 26.55 4.0% 180.6 490.8 99.7% 598.6 0.375 0.06% 94.28 490.8 103% 530.6 36.74 6.9% 66.66 490.8 94.5% 504.1 N/A N/A 90.46 490.8 / 84.3% 539.1 12.36 2.3% <490.8 490.8 110% 573.3 8.43 1.5% 36.90 490.8 109.3% 575.7 7.679 1.3% 50.19 490.8 107.1% 478.4 24.61 5.1% 38.73 490.8 AVERAGE 89.6% 103.1% Page 54 of 142 Matrix Spikes Sample E02-0913-016 E2-09I3-017 E02-0913-021 E02-913-022 E02-0913-026 E02-0913-027 E02-0913-046 E02-9I3-047 E02-0913-051 . E02-0913-052 E02-0913-056 E02-0913-057 E02-913 -106 E02-09 L3-107 E02-0913-111 E02-0913-12 E02-0913-116 E02-0913-117 E02-0913-166 E02-09I3-167 E02-0913-171 E02-0913-172 E02-0913-176 E02-0913-177 Timcpoint Day O' Day 0 Day 0 Day 0 Day 0 Day 0 Day 4 Day 4 Day 4 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 Day 28 Day 28 Day 28 Medium A A B B C C A A B B C C A A B B C C A A B B C C * SPE clogged, unable to extract N/A = Not applicable Result (ng/mL) 768.4 780.7 740.4 750,1 699.7 718.5 625.9 558.4 597.6 520.0 608.5 564.1 750.3 791.7 * 696.6 739.7 763.0 716.4 748.5 691.2 607.0 663.0 693.0 Average (ng/inL) 774.5 745.3 709.1 592.1 558.8 586.3 771.0 696.6 751.4 732.4 649.1 678.0 ` Standard Deviation (ng/mL) 8.675 Relative Standard Average Sample Deviation Result (ng/mL) 1.12% <126.1 True Value (ng/mL) 504.5 6.830 0.916%. <126.1 504.5 3.29 ; 1.87% <126.1 . 504.5 47.74 8.06% <25.23 504.5 54.85 9.82% <25.23 504.5 31.39 5.35% <25.23 504.5 29.33 3.80% 15.49 504.5 N/A N/A <10.09 504.5 16.50 2.20% <126.1 504.5 22.65 3.09% 46.24 504.5 59.56 9.18% <10.09 504.5 21.23 3.13% <10.09 504.5 AVERAGE Percent Recovery 154% 148% 141% 117% 111% 116% 150% 138% 149% 136% 129% 134% 135% Sample E02-0913-016 E02-0913-017 E02-0913-021 E02-0913-022 E02-0913-026 E02-0913-027 E02-0913-046 E02-0913-047 E02-0913-051 E02-0913-052 E02-0913-056 E02-0913-057 E02-0913-106 H02-0913-107 E02-0913-11! E02-0913-112 E02-0913-116 E02-0913-117 E02-0913-166 E02-0913-167 E02-0913-171 E02-0913-172 E02-0913-17 E02-0913-177 Result Timepoint Medium (ng/mL) Day 0 A 486.6 Day 0 A 488.1 Day 0 B 471.4 Day 0 B 473.2 Day 0 Day 0 C c 468.1 472.1 Day 4 A 711.5 Day 4 A 655.7 Day 4 Day 4 Day 4 B B C 547.7 568.0 503.2 Day 4 C 498.7 Day 14 A 717.8 ' Day 14 Day 14 A B 752.7 * Day 14 : B 603.7 Day 14 C 477.9 Day 14 C 471.0 Day 28 A 757.6 Day 28 Day 28 A B 794.7 626.6 Day 28 B . 620.0 Day 28 C , 437.2 Day 28 C 425.1 * SPE clogged, unable to extract N/A = N o t applicable Average (ng/mL) 487.4 Standard Deviation (ng/mL) 1.047 Relative Standard Average Sample Deviation Result (ng/mL) 0.215% . <125.2 True Value (ng/mL) 500.7 472.3 1.259 0.266% <125.2 500.7 470.1 2.843 0.605% <125.2 500.7 . 683.6 39.50 5.78% 194.0 500.7 557.8 14.36 2.57% 81.12 500.7 500.9 - 3.175 0.634% 9.331 500.7 735.2 24.64 3.35% 261.2 500.7 603.7 . N/A N/A 175.4 500.7 474.4 4.929 1.04% <125.2 500.7 776.1 26.18 3.37% 320.8 500.7 623.3 4.660 0.748% 176.5 500.7 431.2 8.535 1.98% <2.504 500.7 AVERAGE Percent Recovery 97.3% 94.3% 93.9% 97.8% 95.2% 98.2% . 94.7% 85.5% 94.8% 90.9% 89.2% 86.1% 93.2% Page 55 of 142 M atrix Spikes Result Sample Timepoint Medium (ng/mL) E02-0913-016 Day 0 A 4997 E02-0913-017 Day 0 A 4942 E02-0913-021 Day 0 B 4604 E02-0913-022 Day 0 -B 4523 E02-0913-026 Day 0 C 737.3 E02-0913-027 DayO C - 741.5 E02-0913-046 Day 4 A 759.4 E02-0913-047 Day 4 A 720.2 E02-09L3-051 Day 4 B 1453 E02-0913-0S2 Day 4 B 1445 E02-0913-056 Day 4 C 1218 E02-0913-057 Day 4 C 1200 E02-0913-I6 Day 14 A 591.4 E02-0913-107 E02-0913-111 Day 14 Day 14 A B 646.4 E02-0913-112 Day 14 B 620.0 E02-0913-116 E02-0913-117 Day 14 C Day 14 c - 1530 1703 E02-0913-166 Day 28 A 554.7 E02-09I3-167 Day 28 A 534.6 E02-0913-171 Day 28 B 617.4 EQ2-0913-172 Day 28 B 601.6 E02-0913-176 Day 28 C 3384 E02-0913-177 Day 28 C 3143 * SPE dogged, unable to extract N/A = Not applicable Average (ng/mL) 4970 Standard Deviation (ng/mL) 39.25 4563 . 57.23 739.4 2.991 739.8 27.68 1449 5.862 1209 12.59 618.9 38.83 620.0 N/A 1616 122.6 544.7 14.21 609.5 11.17 3264 170.4 _ Relative Standard Deviation 0.790% Average Sample True Value Result (ng/mL) (ng/mL) 4129 501.8 Percent Recovery 168% 1.25% 3990 501.8 114% 0.405% 222.5 501.8 103% 3.74% 161.4 501.8 115% 0.405% 917.7 501.8 106% 1.04% 610.9 501.8 119% 6.27% 64.89 501.8 ' 110% N/A 214.0 501.8 80.9% 7.59% . 985.8 501.8 126% 2.61% <25.09 501.8 109% 1.83% 101.1 501.8 101% 5.22% 2327 501.8 AVERAGE 187% 120% / Page 56 of 142 PFBS Laboratory Control Spikes Sam ple E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913-007 E02-0913-008 M edium A A B B C C R esult (ng/m L) 1307 1330 1319 1258 1105 1267 A ve ra g e (ng/m L) 1319 / S ta n d a rd D evia tion (ng/m L) 15.98 R elative S ta n d a rd D evia tion 1.21% T ru e V a lu e Percent (ng/m L) Recovery 1261 105% 1288 43.08 3.34% 1261 102% 1186 114.4 9.64% 1261 94.1% Page 57 of 142 Laboratory Control Spikes Sam ple E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913-007 E02-0913-008 M ediu m A A B B C C R esult (ng/m L) 1198 1261 1256 1210 1234 1243 A v e ra g e (ng/m L) 1230 1233 1239 S ta n d a rd D evia tion (ng/m L) 44.80 32.43 6.674 R elative S ta n d a rd D evia tion 3.64% T rue V alue (ng/m L) 1252 P e rce n t R e co ve ry 98.2% 2.63% 1252 98.5% 0.539% 1252 98.9% - - ............. Sam ple E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913-007 E02-0913-008 M edium A A B B C C R esu lt (ng/m L) 1257 1315 1331 1287 1331 1336 A ve ra g e 1286 1309 1333 S ta n d a rd D evia tion (ng/m L) 41.04 30.58 3.451 R elative S ta n d a rd D e via tio n 3.19% T rue V alue (ng/m L) 1254 P e rce n t R e co ve ry 103% 2.34% ' 1254 104% 0.259% 1254 106% / Page 58 of 142 Sodium Lauryl Sulfate Toxicity Control Sam ple R esult T im epoint (n g /m L ) , E02-0913-028 Day 0 61167 E02-0913-029 Day 0 52407 E02-0913-030 Day 0 58327 E02-0913-058 Day 4 <3330 E02-0913-059 Day 4 3388 E02-0913-06-0 ' Day 4 <3330 E02-0913-118 Day 14 <3330 E02-0913-119 Day 14 <3330 E02-0913-12 Day 14 <3330 E02-0913-178 Day 28 <111 E02-0913-179 Day 28 <111 E02-0913-180 Day 28 <111 A ve ra g e (ng/m L) 57300 3388 <3330 <111 S ta n d a rd D eviation (ng/m L) R elative S ta n d a rd D e via tio n Percent from Day 0 .4470 7.80% 100% _1 N/A N/A 5.9% N/A N/A ' <5.8% N/A N/A - <0.19% Control Substance -- Sam ple E02-0913-031 E02-0913-032 E02-0913-061 E0.2-0913-062 E02-0913-121 E02-0913-122 E02-0913-181 E02-0913-182 T im epo int ! Day 0 DayO Day 4 Day 4 Day 14 Day 14 Day 28 Day 28 R esult (ng/m L) 43921 36825 <3330 <3330 <3330 <3330 <111 <111 A v e ra g e (ng/m L) 40373 <3330 <3330 <111 Standard R elative D evia tion Standard (n g /m L ) D evia tion i 5018 12.4% Percent from DayO 100% N/A N/A <8.2% N/A N/A - <8.2% N/A . N/A <0.27% Page 59 of 142 A biotic R esult Sam ple T im e p o in t (ng/m L) E02-0913-033 DayO ; 266488 E02-0913-034 Day 0 265277 E02-0913-063 Day 4 266328 E02-0913-064 Day 4 249312 E02-0913-123 Day 14 25547-1 E02-0913-124 Day 14 266400 E02-0913-183 Day 28 179799 E02-0913-184 Day 28 207682 S ta n d a rd R elative Average D evia tion : Standard (ng/m L) (ng/m L) 1 D evia tion '265882 855.9 0.322% 257820 12032 4.67% 260936 7728 2.96% 193741 19716 10.2% Percent from DayO 100% . 97% 98% 73% Inhibited R esult A verage Sam ple T im e p o in t (ng/m L) (n g /m L ) E02-0913-035 DayO E02-0913-036 Day 0 198883 227774 213328 E02-0913-065 Day 4 02-0913-066 Day 4 142445 175943 159194 E02-0913-125 Day 14 E02-0913-126 Day 14 152035 154768 153401 E02-0913-185 Day 28 E02-0913-186 Day 28 101296 * 101296 * SPE clogged, unable to extract S ta n d a rd D evia tion (ng/m L) 20429 R elative S ta n d a rd D e via tio n 9.58% 23687 ' 14.9% 1933 1.26% N/A N/A Percent from DayO 100% 75% 72% 47% Attachm ent C: S am ple C hrom atogram s /> Page 61 of 142 ^g C i^'V b2-o^lJ i i I l A t t a c h m e n t D : T e s t S u b s t a n c e In f o r m a t io n Page 104 of 142 Ew en g RESEARCH fck Precise Research. Proven Results. CERTIFICATE OF ANALYSIS Exygen Research COA Reference #: 3M Product: Lot/Batch Number: 1 Test Control Reference #: Purity: 97.5% Test Name Purity1 Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate Organic Acids J (IQ Elemental Analysis": 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Result 97.5% Conforms Positive 1. 0.005 wt./wt.% 2. 0.002 wt./wt.% 3. 0.050 wt./wt.% 4. <0.001 wt./wt.% 5. <0.001 wt./wt.% 6. <0.001 wt/wt.% 7. <0.001 wt./wt. % 2.4 wt./wt.% None Detected None Quantified <0.01 w t/w t.% None Detected <0.013 wt./wt.% <0.004 wt/wt.% <0.035 wt/wt.% <0.008 wt/wt.% <0.005 wt/wt.% <0.006 wt/wt.% 0.089 wt/wt.% <0.1 wt/wt.% <0.1 wt./wt. % <0.1 wt/wt.% <0.2 wt/wt.% COA023-026 X 3058 Research Drive Staite College, PA 16801, USA VT: 8 o te F: 814.272.1019 exygen.com  Proven Results. ! CERTIFICATE OF ANALYSIS Exygen Research COA Reference # Date of Last Analysis: 07/27/00 Expiration Date: 07/27/01 Storage Conditions: Room Temperature Re-assessment Date: 07/27/06 Purity = 100% - (sum of metal impurities, 0.057% +NMR impurities, 2.4%+Inorganic Sulfate, 0.089) \ Total impurity from all tests = 2.55% Purity = 100% - 2.55% = 97.5% 2NMR: 3. .. : j 4Theoretical value calculations based on the empirical formula, obtained from the NMR analysis. This Work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160) Prepared By: Scientist, Exygen Research Reviewed By: Laboratory Manager, Exygen Research COA023-026 ? /x 02=^ Date y / j/ s X ' Date V o30s58 Research Drive Staite College, PA 16801, USA \ T: 8ofe$i n f ir F: 814.272.1019 exygen.com . Page 106 of 142 3M Environmental Laboratory Note to File The expiration dates for may be extended 5 years (07/27/06) as stability was demonstrated by GC and Total Fluorine analyses with report number i Date 06/24/02 O L I ^ i , Form ETS-4-15.0 Exact Copyof Original [As Initial oi* I h Id2 D a ta Page 107 of 142 A t t a c h m e n t E : P r o t o c o l , P r o t o c o l A m e n d m e n t s a n d D e v ia t io n s Page 108 of 142 3M w r a xxi5r PROTOCOL Inherent Aerobic Aquatic Biodegradability of Fluoroaiiphatic Polymeric Ester Data Requirement 40 CFR 792 // Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project identification ET&SS E02-0913 P ag e 1 of 15 Page 109 of 142 Protocol E02-0913 E02-0913 Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Este Test Substance Sponsor Study Director , Principal Investigator (PI) Study Location(s) Testing Facility 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 3M Environmental Laboratory 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Proposed Study Timetable Experimental Start Date October 24, 2002 Experimental Termination Date December 31, 2002 Page 2 of 15 Page 110 of 142 Protocol E02-0913 1. In t r o d u c t io n Microbial populations in nature make up about one-half of the biomass on earth and are associated with the major biochemical cycles of elements and nutrients, besides the decomposition of organic matter. Changes in microbial populations or effects on functions of the microbial communities could result in interference with their natural degradative functions that are essential to self-purification processes in the aquatic and terrestrial environments. In order to assess the environmental fate of a compound, it is pertinent that its chemical properties, biological behavior and transport processes (sorption properties) be addressed. 2. P u rpo se The primary objective of this investigation is to identify the inherent aerobic aquatic biodegradation potential of the fluoropolymer as mediated by the microbial activity of populations obtained from wastewater treatment sludge. This will be accomplished by utilizing aspects from the following guidelines: USEPA Zahn-Wellens/EMPA Test (OPPTS 835.3200) and USEPA Modified SCAS (OPPTS 835.3210). The test substance, , is a complex mixture of fluoroaliphatic polymeric esters rather than a discreet monomeric material. The present investigation will obtain information on the potential for biological degradation and/or biotransformation of the polymeric material as well as yielding information on the intermediates, end products and insight into the potential for partitioning. The present investigation will be conducted using the noted EPA methods as guidelines and incorporating portions of these guidelines to accommodate the specific testing requirements, such as individual samples per sampling event and specific target analysis instead of COD, DOC or CO2. (Refer to Section 8 for specifics on the modifications.) The present study is designed to utilize municipal wastewater treatment sludge as the inoculum. The focus is to determine the ability of viable microbial populations to degrade or transform into fluorochemical breakdown products based on the biological degradation study of" _ _ _ "' - -- -- - .j j ie precjjc{eci breakdown products o f described in section 7. .hat are going to be monitored are the reference substances as 3. R e g u l a t o r y C o m p l ia n c e This study will be conducted in accordance the United States Environmental Protection Agency Good Laboratory Practice Regulations for Non-clinica! Laboratory Studies, 40 CFR 792. 4. Q u a l it y A s s u r a n c e The 3M Environmental Laboratory Quality Assurance Unit will audit the study conduct, raw . data, and final report to determine compliance with Good Laboratory Practice Regulations, this protocol, and 3M Environmental Laboratory Standard Operating Procedures. Page 3 of 15 Page 111 of 142 Protocol E02-0913 5. Te s t S u b s t a n c e Table 1 Test Substances T est Substance IUPAC Name Chemical Formula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Description Purity 'Based on NMR data 3M Specialty Chemicals 07/27/06 Room Temperature Yellow Viscous Oil 97.5% The test substance. is a complex mixture of fluoroaliphatic polymeric esters. is a viscous liquid with a distinctive odor. It has a boiling point of at standard temperature and pressure it has a vapor pressure of mm Hg, a specific gravity o f 1.1, a measured pH range of , and viscosity o f ' 1. Lot# was obtained from 3M Specialty Chemicals, Chemical characterization data was performed by: 3M Specialty Chemicals (boiling point, viscosity, specific gravity, pH, vapor pressure, flash point, results of GC analysis, results of gel-permeation chromatography, and results of NMR analysis) and Centre Analytical (total amount of fluorine containing moieties present). The total amount of organic fluorine in 1 vas determined to by "Total Fluoride Analysis". A report of these findings ' is on file in the 3M Environmental Laboratory. 5.1 Sample Retention A retention sample will be taken and stored in the dark at ambient temperature. The sample will be archived for 10 years from the study completion date, or as long as a reliable analysis can be performed. 5.2 Disposition Should the retention sample be discarded, the final disposition will be documented in the 3M facility archive records. Page 4 of 15 Page 112 of 142 Protocol E02-0913 5.3 Safety Precautions When handling samples or solvent's wear protective gloves, eyewear and clothing. The operator must be familiar with instrumentation and their associated hazards, such as, but limited to, high, temperature, effluent venting, solvent use, and vacuum systems. All material safety data sheets or chemical hazard information should be reviewed as , appropriate. 6. C o n t r o l S u b s t a n c e s Table 2. Control Substances Control Substances L F orm ula .. : : : m P Sodium Lauryl Sulfate C uH aO SO jN a IUPAC Name S od iu m Lauryl Sulfate U se Source Expiration Date Storage C onditions Chem ical Lot Num ber TCR Number Physical Description Purity Surrogate Standard 3M Specialty Chem icals 8/31/2006 Frozen W hite powder 86.9% internal Standard for LC/M S analysis 3M Specialty Chem icals 10/18/2006 Frozen W hite pow der 98.6% T oxicity and R eference Control M aliinckrodt 2/26/2007 R oom Temperature 7718 V I6603 , TN -A-6021 W hite powder 99% / Page 5 of 15 Page 113 of 142 7. R e f e r e n c e S u b s t a n c e s Table 3. Reference Substances ReferenceSubstance Chem ical Formala Identifier Source Expiration Date Storage Conditions Chem ical Lot Number TCR Number Aldrich Chemical 5/1/2010 Frozen 3M Specialty Chem icals 12/4/2006 Frozen Protocol E02-0913 3M Specialty Chem icals N ot Provided Frozen IU PAC N ame Chem ical Formula Identifier Source 3M Specialty Chem icals 3M Specialty C hem icals Expiration Date N ot provided N o t Provided Storage Conditions Frozen Am bient Chemical Lot Number TCR Number Physical Description W hite crystals W hite crystals Purity 97.25 95.55 *C AS N um ber, **3M Identifier C ode. The location o f the docum entation o f the m ethod(s) o f synth esis o f the test, control, and reference items are the sam e as the source o f the compound. Page 6 of 15 Page 114 of 142 Protocol EO2-0913 8. E x p e r i m e n t a l D e s i g n 8.1 Preparation of the Test System The following table describes which substances will be added to which medium. There are no contaminants expected in the materials used for preparing and dosing the test system. Information on the preparation of the medium and the concentrations required for the test, control, and reference substances are also described below. Table 4: Test System Preparation__ ______ Sam ple Description #of R ep licates Test/C'ontrol Substance added Blank C ontrols 2 None R eference Substances added No M edium A dded1 A Analysis to be conducted R eference Substances Blank C ontrols Inhibited 2 None N o B Reference Substances Abiotic C ontrols 2 None No C Reference Substances Test Substance 3 Test No A Reference Substances Test Substance M atrix spike 2 Test Y es A Reference Substances Test Substance Inhibited Test Substance Inhibited M atrix spike A biotic Test Substance A biotic Test Substance Matrix spike T oxicity Control 3 2 3 2 3 Test Test Test Test y Test and SLS No Y es No Y es No B Reference Substances B Reference Substances c Reference Substances C Reference Substances . A SLS Control Substance (S L S ) 2 2 SLS No A SLS No c 2 See definitions o f mediums under section 8.1.2 No B Page 7 of 15 Page 115 of 142 Protocol 02-0913 8 .1 .1 S lu d g e Inoculum . Arrangements will be made to have Pace Analytical Services, Field Laboratory, Minneapolis, MN personnel obtain fresh mixed liquor suspended solids (MLSS) . from the aeration units at the Metro Wastewater Treatment Plant, St. Paul, MN. This sludge has been used in previous studies at 3M and Pace. Approximately six to eight liters of MLSS will be collected in either NalgeneTM polypropylene bottles or a two-gallon CubitainerTM or equivalent. The suspended sludge in the containers will be allowed to settle for at least 24 hours. The approximate percentage of settled sludge per volume of container will be noted. Previous studies had values that were approximately 20% (e.g. 200 mL o f sludge in a 1 liter container). This value is for comparison purposes only and is not meant as a criterion for passing the collected sludge. 8 .1 .2 M in era l S alts M edium The mineral salts medium employed will be based on the USEPA ZahnWellens/EMPA Test (OPPTS 835.3200). M edium A. Prepare this solution by adding approximately 100 mL of settled sludge to 2 liters of mineral salts medium. This solution should be swirled regularly during dispensing in order to keep the mixture homogenous. The mixed liquor suspended solids (MLSS) should be determined for this medium. The final result should be between 0.2 g/L and 1.0 g/L. If the MLSS is outside this range, justification should be given in the final report. M edium B. A portion of Medium A will be treated with 100 pg/mL of chloramphenicol as a microbial grov/th inhibitor. This solution should be swirled regularly during dispensing in order to keep the mixture homogenous. M edium C. Mineral salt medium, prepared without the sludge inoculum, will be treated with 100 pg/mL of chloramphenicol as a microbial growth inhibitor. This will be labeled as Medium C and used to prepare the 25 mL abiotic culture vessels. (To assess the potential for abiotic mechanisms, e.g. hydrolysis.) 8 .1 .3 C u ltu re Vessel Setup The individual culture vessels will be prepared by dispensing 25 mL of the appropriate medium into 125 mL glass Erlenmeyer flasks containing labels with the appropriate information. The vessels will be covered with a loose cap in order to reduce evaporation. The route of administration will be directly spiking the medium (this is the most direct route using solutions) in the culture vessel as detailed below. Page 8 of 15 Page 116 of 142 Protocol E02-0913 8 .1 .4 T est S u bstan ce S tock S olution The concentration of the test substance should be approximately 36 mg/L in each appropriate culture vessel (per table 4) as per the suggestion of the EPA guidelines .of 20 mg Carbon/L. The concentration was calculated by the theoretical value calculations based on the empirical formula, obtained from the NMR analysis (see report ). The initial stock solution should be prepared in acetone but any subsequent dilutions should be.made in mineral salts medium without inoculum. 8 .1 .5 C on trol S u bstan ces ' The concentration of the control substance, sodium lauryl sulfate, should be approximately 40 mg/L in each appropriate culture vessel (per table 4) as per the suggestion of the EPA guidelines of 20 mg Carbon/L. The biodegradation of this compound must reach at least 70 percent within 14 days. will also be added as a control substance to mediums B and C (as per table 4). The concentration of added in the 25 mL culture vessel should be approximately 112 mg/L. There should be minimal degradation of throughout the course of the study. will be added as the last step of the preparation, process prior to analysis by LC/MS. It should be added at an approximate concentration of 250 ng/mL. All initial stock solutions should be prepared in acetone but any subsequent dilutions should be made in mineral salts medium without inoculum. 8 .1 .6 R eferen ce S u bstan ces y Reference substances will be added to laboratory control spikes and post extraction matrix spikes to determine recovery. These substances will be added at a nominal concentration of 500 ng/mL. All initial stock solutions should be prepared in acetone but any subsequent dilutions should be made in mineral salts medium without inoculum for the laboratory control spikes and in tetrahydrofuran for the post extraction matrix spikes. 8 .1 .7 S a m p le C ollection The experiment will be set up for determination of biodegradation and/or biotransformation over a six week period, with triplicate samples set up, except for the blanks, post spikes and control substances, which will be duplicated only. Samples will be collected on days 0, 4, 7, 14, 21, 28 and 42 for each medium type. Analysis will be conducted initially on selected early time points. Based on these . results, it will be determined if it is necessary to analyze the other time points. Table 4 shows the sample preparation scheme for the investigation. On day zero, samples will be prepared and immediately extracted or placed in a freezer which is maintained at -19 7C. All other test vessels will be placed in a temperature controlled orbital shaker incubator which is maintained at 24 3C under dark conditions for up to 42 days. Page 9 of 15 Page 117 of 142 P rotei, L -v c -v v i o During sampling events, the samples will be removed from the incubator and will be either extracted immediately or frozen. 8.2 Sample Extraction Method ETS-8-39 will be used as the sample extraction method. In summary, an amount of sludge is prepared in an aqueous 1% solution of acetic acid. The sample is capped, mixed, and put on the centrifuge to clarify the supernatant, if needed. The supernatant is passed through a pre-conditioned Cis SPE column, at which time the analytes are adsorbed onto the stationary phase. Finally, the analytes of interest are eluted from the SPE cartridge and analyzed by appropriate methodology. 8.3 Analytical Methods Samples are to be analyzed via HPLC/Electrospray MS as per ETS-8-155. The following table describes the SIM ions to use. . Table 5. SIM tons________________ _____________ SIM IONS_________ Compound [ SIM Ion ( r r J z ) 9. M e t h o d s f o r C o n t r o l o f B ia s Control of bias is accomplished using analytical spikes (as an indicator of sample recovery and accuracy) and matrix blanks (for evaluation of possible contamination of the matrix). Solvent blanks will also be analyzed (possible sample contamination during the dilution process). Triplicate sam ple results are an indicator o f precision and the internal standard will be used to measure instrument variability. 10. D a ta Q u a l it y O b j e c t iv e s 10.1 Samples Sample precision should be < 20% relative standard deviation. Page 10 of 15 Page 118 of 142 Protocol E02-0913 10.2 Calibration Standards. Samples will be bracketed by a calibration curve and passing CCVs. Calibration standards used to generate an external calibration curve should be prepared in medium A and extracted in the same manner as the samples. The number of calibration standards and the concentration levels should be sufficient to encompass the expected concentrations of the study samples. The coefficient of determination (r2) of the standard curve must be equal to or greater than 0.990. If the calibration curve residuals are greater than 20% deviation (LOQ 70%-120%) from the theoretical value, quadratic curve fitting and/or dropping low/high curve points may be required if data review shows this to be a consistent and more accurate representation of the instrument response. Deviations will be documented in the raw data with technical justification. The Study Director will be consulted for direction and for final acceptance or rejection of the data. 10.3 Continuing Calibration Verification (CCV). Analyze a mid-range calibration standard after a maximum of every ten samples. Acceptable CCV values are 70 -120% of true value. 10.4 Solvent blank. Solvent blanks are mn before and after every calibration curve, and after every CCV. Solvents blanks may be mn before, and after matrix and control blanks if contamination is noted. Acceptable values for the blanks are values below 50% of the limit of quantitation (LOQ). If analytecarryover is a problem, use back-to-back solvent blanks and use the last solvent blank to evaluate carryover, 10.5 Laboratory Control Spikes / Two laboratory control spikes (LC'S) will be prepared for each medium (medium A, B, and C) for the Day 0 samples. The LCS will be spiked at a level that is expected in the samples. The analyst shall accept percent spike recoveries between 70% and 120% of theoretical value. Spike recoveries outside of this range should be noted and used with other criteria to evaluate the condition of the analytical mn or necessity for repeat analysis. Consult with the Study Director for direction and final acceptance or rejection of the analytical ran. 10.6 Matrix spikes. The analyst shall accept percent spike recoveries between 70% and 120% of theoretical value. Spike recoveries outside of this range should be noted and used with other criteria to evaluate the condition of the analytical run or necessity for repeat analysis. Consult with the Study Director for direction and final acceptance or rejection of the analytical run. . Page 11 of 15 Page 119 of 142 Protocol E02-0913 10.7 Limit of Quantitation (LOQ). The lowest concentration that can be reliably measured within specified limits of accuracy during routine laboratory operating conditions. The LOQ is defined as the lowest non-zero standard (70%-l20%) in the calibration curve that is greater than or ' equal to 2 times the level of the matrix blank. Sample LOQ are highly matrix-dependent. 10.8 internal Standard. Internal standard will be added to sample as the last step of the preparatory procedure. The same amount of internal standard will also be added to the standards. This will solely be used to monitor instrument performance and not used for quantitation. 10.9 Demonstration of Specificity. The analytical technique of HPLC/MS provides for chromatographic separation of targeted materials and detection/quantitation of selected ions which are characteristic of the targeted compounds. 10.10 System Suitability Five injections of a mid-level standard will be run prior to each calibration curve. The area counts should have a relative standard deviation <5% and the retention times should be <2%. 11. S tatistical A n a ly s is Standard deviations will be calculated using either Microsoft Excel (Version 8.0e or newer) or Microsoft Access. , The built in function contains the following quation, which is based on the individual entities (n) being less than 30*. i nl x 2 -C Z x)2 V n ( n - 1) Sample precision will be reported as % relative standard deviation (%RSD) for three or more replicates. Means will be calculated by adding the individual entities and dividing the resultant sum by the number of individual entities. Page 12 of 15 Page 120 of 142 Protocol E02-0913 12. R e p o r t Areport of the results of the study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: Name and address of the facility performing the study, Dates upon which the study was-initiated and completed. A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards. Objectives and procedures as stated in the approved protocol, including any amendments to the original protocol. The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the . Sponsor. Stability and the solubility of the test substances under the conditions of administration, if provided by the Sponsor. A description of the methods used to conduct the test(s). A description of the test system. A description of any circumstances that may have affected the quality or the integrity of the data. . The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study./ A description of the transformatio ns, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions . drawn from the analyses. Statistical methods used to evaluate the data, if applicable. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study, if applicable. The location where raw data and the final report are to be stored. A statement prepared by the Quality assurance unit listing the dates that study inspections and audits were made and the dates of any findings reported to the Study Director and Management. If it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. Page 13 of 15 Page 121 of 142 Protocol E02-0913 13. L o c a t io n o f R a w D a t a , R e c o r d s , a n d F in a l R e p o r t Original data or copies thereof, will be available at 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including those items listed below will be retailed in the archives of 3M Environmental Laboratory for a period of 10 years following signing of the final report. ; The following raw data and records will be retained in the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures. Approved protocol and amendments Study correspondence . Shipping records . Raw data Approved final report (original signed copy) Electronic copies of data The following supporting records will be retained separately from the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: Training records Calibration records Instrument maintenance logs / Standard Operating Procedures, Equipment Procedures, and Methods Appropriate specimens 14. D a ta / S a m p l e R eten tio n Extracts will be kept for 6 months, or as long as the preparation affords evaluation. Other raw data will be kept for 10 years following the effective date of the applicable final test rule. 15. P r o t o c o l A m e n d m e n t s a n d d e v ia t io n s Planned changes to the protocol will be in. the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. Any other changes (unplanned) will be in the form of written deviations, signed by the Study Director and filed with the raw data. All changes to the protocol and the reason for the changes will be indicated in the final report. Page 14 of 15 Page 122 of 142 16. Sig n atu r e s Protocol EO2-0913 Date Date / Page 15 of 15 Page 123 of 142 P rotocol EQ2-0913 A m endm ent #1 Study Title Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester PROTOCOL AMENDMENT NO. #1 Amendment Date; December 13, 2002 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification E+&SS E02-0913 Page 1 of 5 Page 124 of 142 Protocol E02-0913 A m endm ent #1 This am endm ent modifies the following portion(s) of the protocol: P R O T O C O L :r e a d s Section 8.1.2 "The mixed liquor suspended solids (MLSS) should be determined for this medium. The final result should be between 0.2 g/L and 1.0 g/L." A M E N D TO r e a d : The mixed liquor suspended solids (MLSS) should be determined for this medium. The final result should be between 0.2 g/L and 1.0 g/L. The procedure for this determination is given in attachment A. REASON: To add the procedure for the mixed liquor suspended solids determination. P R O T O C O L r e a d s : Section 10.2 "Calibration standards used to generate an external calibration curve should be prepared in medium A and extracted in the same manner as the samples". A M E N D TO r e a d : Calibration standards used to generate an external calibration curve should be prepared in mineral salts medium and extracted in the same manner as the samples REASON: Standard curves should be prepared in mineral salts medium, not in medium A. P R O T O C O L r e a d s : Section 7. Reference Substances are , PFBS, A m en d to r e a d : R eference Substance IUPAC Name Chemical Formula !b ........... - Identifier Source Expiration Date S toraqe Conditions 3M Specialty Chemicals 12/1/2010 Frozen Chemical Lot Number TCR Number Physical Description Liqht yellow powder Purity . Not Determined will be used to quantify ions that are related to the possible degradation product R e a s o n : To add ' s a reference substance. Page 2 of 5 Page 125 of 142 Protocol E02-0913 A m endm ent #1 P R O T O C O L r e a d s : Section 10.1 "Sample precision should be <20% relative standard deviation." . A m e n d t o R E A D : Sample precision should be <20% relative standard deviation. Sample precision outside of this range should be noted and used with other criteria to evaluate the condition of the analytical run or necessity for repeat analysis. Consult with the Study Director for direction and final acceptance or rejection of the analytical run. REASON: To clarify original intent o f how precisions >20% should be evaluated. P r o t o c o l r e a d s : Section 8.3 Table 5: SIM ion for A m e n d t o r e a d : Section 8.3 Table 5: SIM ion REASON: After method development, the ion had a better signal than the / Page 3 of 5 Page 126 of 142 P ro to co l E 0 2 -0 9 13 A m endm ent #1 Attachment A Determination of Mixed Liquor Suspended Solids (M LSS)1 Equipment Needed: 1. Glass Fiber Filter Disks 2. Aluminum Weighing Tins 3. Filtering apparatus with pump 4. Desiccator 5. Drying Oven . 6. Analytical Balance The determination of Mixed Liquor Suspended Solids will be determined as follows: 1. Place a glass fiber filter disk in an aluminum weighing tin. 2. Weigh the tin and filter on an analytical balance to the nearest 0.1 mg. 3. Remove the filter from the aluminum weighing tin and place the filter paper on the filtering apparatus and wet wiih Milli-Q water. Turn on the pump and ensure that the filter is properly seated in the apparatus. 4. Take a 5 mL aliquot from medium A. This aliquot should be taken approximately halfway down the flask and halfway between the wall of the flask and vortex of the solution. Dispense the solution onto the filter. 5. Rinse the filter three times with Milli-Q water and allow the filter to sit for at least three minutes while the pump is still on after the last rinse. 6. Turn off the pump and remove the filter. Place the filter on the original aluminum weighing tin. 7. Place the filter and tin in a drying oven (temperature >90C) for approximately two hours. Remove the filter and tin and place in a desiccator for at least an hour. 8. Weigh the glass fiber filter and aluminum weighing tin to nearest 0.1 mg. 9. Calculate MLSS as , Final Weight (mg) - Initial weight (mg) MLSS (g/L) = Volume used (mL) This is based on a modified procedure from APHA, AWWA, WEF "Standard Methods for the Examination of Water and Wastewater", Section 2540D "Total Suspended Solids at 103-105C" 19th Edition. Page 4 of 5 Page 127 o f 142 Amendment Approval Protocol E02-0913 Amendm ent #1 _____________ o ///y /o 3 sntative Date fy Date / Page 5 of 5 Page 128 of 142 Protocol tu z - u a u Am endm ent #2 Study Title Inherent Aerobic Aquatic Biodegradability of Fiuoroaiiphatic Polymeric Ester PROTOCOL AMENDMENT NO. #2 Amendment Date: February 19, 2003 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS E02-0913 Page 1 of 3 Page 129 of 142 P rotocol E02-0913 Amendm ent #2 This amendment modifies the following portion(s) of the protocol: PROTOCOL r e a d s : Section 7. Reference Substances are T PFBS, A mend to read: R eference Substance IUPAC Name Chemical Formula Identifier Source Expiration Date Storaae Conditions Chemical Lot Number TCR Number Physical Description Purity Not Available Pace 2/4/2013 Frozen White Powder ' 50.1% REASON: To add as a reference substance. / Page 2 of 3 Page 130 of 142 Amendment Approval Protocol E02-0913 Am endm ent #2 live Date Date t) Page 3 of 3 Page 131 of 142 P rotocol E02-0913 Am endm ent #3 Study Title inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester PROTOCOL AMENDMENT NO. #3 Amendment Date: February 27, 2003 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS E02-0913 Page 1 of 3 Page 132 of 142 P rotocol E02-0913 Am endm ent #3 This amendment modifies the foliowing portion(s) of the protocol: PROTOCOL r e a d s : Section 7. Reference anc A m end to r e a d : q PFR.9 IUPAC Name Chemical Formula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Description Purity 3M Specialty Chemicals 11/29/2005 Frozen . Off white powder 98.63% REASON: To add the additional1 eference substance used for this study. '/ Page 2 of 3 Page 133 of 142 Amendment Approval Protocol E02-0913 Amendment #3 itativ Date ^ p / p y / o ;^ Date / Page 3 of 3 Page 134 of 142 Protocol E02-0913 Am endm ent #4 Study Title inherent Aerobic Aquatic Biodegradability of Fiuoroaliphatic Polymeric Ester PROTOCOL AMENDMENT NO. #4 Amendment Date: March 6, 2003 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS E02-0913 Page 1 of 3 Page 135 o f 142 P rotocol E02-0913 Am endm ent #4 This amendment modifies the following portion(s) of the protocol: PROTOCOL r e a d s : Section 10.2 "Calibration standards used to generate an external calibration curve should be prepared in mineral salts medium and extracted in the same manner as the samples." AMEND TO r e a d : "Calibration standards used to generate an external calibration curve should be prepared in mineral salts medium and extracted in the same manner as the samples. Selected timepoints will be analyzed using *unextracted standards." REASON: Due to the late edition of these compounds to the study, unextracted standards are to be used to quantitate these compounds. / Page 2 of 3 Page 136 of 142 Amendment Approval t'roto co E02-0913 Am endm ent #4 presentative Date Date ) Page 3 of 3 Page 137 of 142 Nimrn 3M Confidential Record of Deviation Study / Project No. E02-0913 Deviation type (Check one) Document number E02-0913 Protocol 1. Identification . "SOP" ..... Method Equipment Procedure X Protocol Other: ; Date(s) of occurrence Sequence D021119 (Dudejr 11/19/02) 11. Description Required prqcedure/process: ~ 1. Section 10.3: "Acceptable CCV values are 70-120% of true value." 2. Section 10.2: "I f the calibration curve residuals are greater than 20% deviation (LOQ 70%-120%) from the theoretical value, quadratic curve fitting and/or dropping low/high curve points may be required if data review..." Actual procedure/process: 1. For all analytes the 10 pg/pL CCV was not within the specified criteria and out o f 3) the 50 pg/pL standard was outside the specified criteria. 2. The lowest standard for was not within the specified criteria (60%). (2 out o f 3) and Hi. Actions Taken - _________________________ ( s u c h a s a m e n d m e n t i s s u e d , S O P r e v i s i o n , e t c . ) __________ 1. For all analytes a 10 pg/pL CCV was run after a high level spike (500 pg/pL). For PFBS ' ______ the level o f the matrix spike caused instrument carryover into the next injection, which resulted in a high CCV. Since the other CCVs passed (50 and 250 pg/pL) and saniples repeated within 20% RSD, it is suspected that carryover was not a problem with these study samples. For the 10 pg/pL CCV was outside the calibration range used so it was discarded. Fot most of the associated samples (with exception o f the blank controls) were well above the 50 pg/pL CCV (closer to the passing 250 pg/pL CCV). However, the day four medium C samples ranged from 86 pg/pL to 101 pg/pL for , Since these samples had repeated <20 % RSD they were accepted. If analyte carryover was a problem, the samples would not have repeated within specification and would of shown decreasing concentrations. For all analytes, the study control blanks were all below the LOQ. Given all this, it is not suspected that the out of specification CCVs will have an adverse affect on the data. . 2. The 25.23 pg/pL standard was kept in order to maintain a 5 point calibration curve. Also, all of the study samples (with exception of the matrix spikes) were all well below the 25.23 pg/pL standard. Given this, it was deemed more accurate to state that the samples were below 25.23 pg/pL instead of 50.45 pg/pL. Recorded by : Date ;__________ _________ oi/a&/Q3 ' ; Date . __________________________ / Za 2 h i ' Deviation No. \7 (assigned by Study Director or Project Lead at the end of study or project) Page 138 of 142 3M Confidential Record of Deviation Study / Project No. E02-0913 Deviation type (Check one) Document number E02-0913 Protocol 1. Identification s o p ........... Method Equipment Procedure X Protocol Other: Date(s) of occurrence ! Sequence D021119 (Dudejr 11/19/02) II. Description Required procedure/process: Section 10.16: "The area counts should have a relative standard deviation o f <5% ..." Actual procedure/process: ,.12%) was outside "the specification. ill. Actions Taken _______________________________ ( s u c h a s a m e n d m e n t i s s u e d , S O P r e v i s i o n , e t c . ) ___________________________ _ Since all of the samples were either diluted and reinjected at a later date (required reanalysis) or less than the LLOQ it was decided to accept the results since the repeatability really doesn't matter below the LLOQ and any o f the samples that were detected were reanalyzed (required reanalysis due to being above the upper limit of quantitation). Recorded by ; Date Date . ___________________ - : / A 2./Q ? Deviation No. ...X / ..^ . (1 by Study Director or Project Lead at the end of study or project) Page 139 o f 142 3M Confidential Record of Deviation Study / Project No. E02-0913 Deviation type (C heck one) Document number E02-0913 Protocol /. Identification SOP X Protocol Method Equipment Procedure Other: Date(s) of occurrence . Sequence 1021231 (Itchy 12/31/02) II. Description Required procedure/process: Section 10.3 "Acceptable CCV values are 70-120% o f true value." Actual procedure/process: The 25.00 pg/pL CCV for was not within the 70-120% criteria (123%, 127%, and 128%). For this study III. Actions Taken (s u c h a s a m e n d m e n t is s u e d , S O P re v is io n , e tc .) is used as a surrogate to approximate another compound. In this analysis, the 25.00 pg/pL CCV did' not pass the criteria as specified by the protocol. of the samples that have values to be reported were either below the lower limit of quantitation or closer to th 250 pg/pL CCV which passed (CCV range 111%- U 7% , sample range 189.81 - 3 4 4 .7 pg/pL). Since the LLOQ for this analysis was 25.00pg/pL and the CCV was high, the high recovery would have shown if any samples were at or near 25.00 pg/pL. Given this and that all of the rest o f the sample hits were closer to the 250 pg/pL CCV, the results were accepted. No affect on the "data is expected. ?Acnrded bv Date Date O! / / S/O? Deviation No. js Director or Project Lead at the end of study or project) Page 140 of 142 &WT?jj-n 3M Confidential Record of Deviation __________________________ I. Identification Study / Project No. E02-0913 Deviation type SOP X Method Equipment Procedure (Check one) Protocol Other: Document number Date(s) o f occurrence ETS-8-39.0 11/18/02, 11/19/02, 11/22/02, 11/25/02, 11/26/02, and 12/19/02 _________ _________________ II. Description __________________ Required procedure/process: Section 10.2 o f the method states "Mix conte nt thoroughly and centrifuge until contents become d e a r". Actual procedure/process: Samples were not centrifuged. Instead, a glass wool plug was added to the SPE apparatus and was used to catch any particulates that may be suspended in the solution used to elute the compounds o f interest. HI. Actions Taken ______________ _______________ ( s u c h a s a m e n d m e n t i s s u e d , S O P r e v i s i o n , e t c . ) No impact on the study Recorded bv Date ector or Project Lead at the end of study or project) Page 141 of 142 3M Confidential Record of Deviation Study / Project No. 02-0913 ........... Deviation type (Check one) Document number E02-0913 Protocol l. Identification SOP Method Equipment Procedure X Protocol Other: __ _ __ _ _ ; Date(s) of occurrence ________________________: Sequence D030305 (Dudejr 3/5/03) __________//. Description________________ Required procedure/process: __ _ __________ _______ _ Section 10.10: "The area counts should have a relative standard deviation o f <5% ..." Actual procedure/process:___ _ 5.2%)_w>$ outside the specification. ill. Actions Taken ______________________________ ( s u c h a s a m e n d m e n t i s s u e d , S O F r e v i s i o n , e t c . ) _____________________________ After evaluating the data, since the calibration curves passed, the CCVs passed and the accepted sample replicates Showed repeatability within the specified criteria (<20%)^ the data was accepted. Recorded by ; Date ;____________ 1 3 /7 /tf3 : ! Date / / ________ ; /iation No.____ 5________ :ctor or Project Lead at the end of study or project) | 1 Page 142 of 142