Document 6B32R2QyNMRDoY53odL1j2X19

Study Title INHERENT AEROBIC AQUATIC BIODEGRADABILITY OF FLUOROALIPHATIC POLYMERIC ESTER OFf ]" - Data Requirement 40 CFR 792 [ Authors Study Completion Date Date of signi ng Performing Laboratory 3M Environmental Technology and Safety SeNices I 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN,,55106 Project Identification E02-0913 Total Number of Pages 142 Page 1 of 142 This page has been reserved for specific country requirements. 1 Page 2 of 142 E02-0913{ jSioc/egrudl.lliot! SlllCZV GLP COMPLIANCE STATEMENT [ Study Title: ~J Inherent Aerobic Aquatic Biodegradability of .Fluoroaliphatic Polymeric Ester of Study Identification Number: E02-0913 ,. Thisstudy 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 forE jhave 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 of 142 QUALITY ASSURANCE STATEMENT rStudy ]Title: Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester of 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 10/18/02 10/28/02 10/28/02 11/25/02 1/28/03 I 11/25/02 1/28/03 ( QAU Repres~tive 7 J ~ -lO-o~ Date Page 4 of 142 TABLE OF CONTENTS GLP Compliance Statement ; Quality Assurance Statement.. Table of Contents List of Tables Study Information Summary Introduction : ~ Test Substance Reference Substances Control Substances Test System Method Summaries Preparatory Methods Analytical Method / Analytical Results ; Data Summary Statistical Methods and Calculations Statement of Conclusion List of Attachments Signature Page 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 3 . 4 5 6 7 9 10 ; 12 13 14 14 16 16 16 17 20 22 23 23 24 25 46 61 104 ..108 Page 5 of 142 LIST OF TABLES Table 1: Me?ium A( Results ' . ]Exposed to Uninhibited Sludge) Percent Degradation 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: Me1dium A~I...;"'xipt:osed to Uninhibited Sludge) Percent Degradation Results : 20 Table 10: Medium B Results 20 Table 11: Medium C Results 21 Table 12: Accuracy of Analytical Results 21 i I Page 6 of 142 STUDY INFORMATION rSponsor 'i ,j 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Study Director ( 7 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 . St. Paul, MN 55106 Principal Investigator I' ] 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 l L 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 ftuoroaliphatic 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 tarqet analytes are the predicted deqradation products perftuorobutane 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 of 1 on day 4). Observed minor metabolites include I 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 'I Exposed to Uninhibited Sludge) Percent Degradation Results1 Analyte Day 0 Day 4 Day 14 Day 28 Yo 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% -, PFBS3 <0.33% 0.28% 0.97% .. , The percent degradation is based on the theoretical amount of fiuonne available In the Initial dose of 2.3% The 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 using r standard. Jrhe analytical accuracy for all analytes is 20% or better. Page 9 of 142 INTRODUCTION The primary objective of this if1vpstio8tion is to identify the inherent aerobic aquatic biodegradation potential of the f1uoropolymer _ when exposed to the microbial populations present in wastewater treatment sludge. This was accomplished by utilizing aspects from the following guidelines: USEPA Zahn-Wellens/EM'PA Test ~OPPTS 835.3200) and USEPA Modified SeAS (OPPTS 835.3210). The test substance, is a complex mixture of f1uoroaliphatic 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 or DOz. 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 f1uorochemical breakdown products based on the biological degradation study of ' _ . as reoorted in 3M Environmental Laboratory study # r 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 ) BioClegradative Pathway R F+H+~-O F F F F 0 II F F F F 0 PFBS ! - ......... Page 11 of 142 TEST SUBSTANCE Table 2. Test Substances Test Substance 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 Yellow Viscous Oil in ASTM type I water *Based on NMR data, **From 3M Environmental Laboratory study' Page 12 of 142 REFERENCE SUBSTANCES Table 3. Reference Substances Reference Substance - IUPAC Name Chemical Formula Identifier PFBS Potassium perfluorobutanesulfonate C4FgS03K 29420-49-3 Source Expiration Date Storage Conditions Chemical Lot Number TCRNumber Physical Description Purity Reference Substance Aldrich Chemical 5/1/2010 Frozen 3M Specialty Chemicals 12/4/2006 Frozen Clear liquid 99% White powder 96.7% IUPAC Name Chemical Formula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Description Purity 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% IUPACName Chemical Formula Identifier Source Expiration Date Pace Analytical Services 2/4/2013 3M Specialty Chemicals 12/1/2010 3M Specialty Chemicals 11/29/2005 Storage Conditions Frozen Frozen Frozen Chemical Lot Number TCR Number Physical Description Purity White Powder 50.1% Light yellow powder Not Determined Off White powder 98.63% *CAS Number, **3M Identifier Code. The location of the documentation of the method(s)ofsynthesis 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 Control Substances Formula 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 Lauryl Sulfate Internal Standard for LC/MS analysis 3M Specialty Chemicals 10/18/2006 Frozen White powder 98.6% Sodium Lauryl Sulfate Toxicity and Reference Control Mallinckrodt 2/26/2007 Room Temperature 7718 V16603 TN-A-6021 White powder 99% TEST 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 gIL 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 IJg/mL chloramphenicol. Medium C consisted of mineral salts medium and 126 IJg/mL chloramphenicol. Page 14 of 142 Individual culture vessels were prepared by dispensing 25 mL of the appropriate medium into 125 mL glass Erlenmeyer 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 3e. 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 28.0o e. After day 8 these samples were maintained at the specified 24 3e. 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 Sample Description fiof Replicates ... Test/Control Substance added* .- - Reference . Substances added* Medium Added ~ .. Analysis to be conducted Blank Controls 2 None Reference No A Substances Blank Controls Inhibited 2 None Reference No B Substances Abiotic Controls 2 None Reference No C Substances Test Substance I3 Test Reference Nc A Substances Test Substance Matrix spike 2 Test Reference Yes A Substances Test Substance Inhibited 3 Test Reference No B Substances Test Substance Inhibited 2 Matrix spike Reference Test / Yes B Substances Abiotic Test Substance 3 Test ! Reference No C Substances Abiotic Test Substance 2 Matrix spike 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 moiL. eaUivalent to 20mg Carbon I L per EPA 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 SUMMARIES 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 approximate-ly 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 mL 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 Analytical column: Keystone BetasilTM C18 2x50mm; 5~m particle size Column temperature: 30 C Stop Time: 9.0 minutes Flow rate: 300 ~Umin Injection volume: 2 or 5 ~L Mobile phase components: Solvent A: 2.0 mM ammonium acetate in ASTM Type I water Solvent B: HPLC Grade Methanol Solvent Gradient: system Time 0.00 0.50 3.00 5.50 6.00 9.00 %8 15% 15% 100% 100% 15% '15% Page 16 of 142 Mass Spectrometer: Hewlett-PackardID Series Software: Agilent ChemStation r.. A.OS.03 = Capillary Voltage: 4000 V Gain 1.0 EMV Mode: Electrospray Negative Gas Temperature: 350C Drying Gas: 8.0 L Imino Nebulizer Pressure: 30 psig Analysis Type: Single Ion Monitoring (SIM) 1100 API/Mass Spectrometer Detector Compound SLS PFBS Ion (m/z) 265 299 Fragmentor (volts) SO 120 SO 100 90 100 120 70 '140 80 ANALYTICAL RESULTS For a detailed listing of analytical results, refer to Attachment B: Data Tables. The analytical results consist of data taken from 10 analytical sequences. I Regressions. 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 orgreater. Calibration 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% ('(0%-120% for the lower ILlJlit of Quantitation) criteria were deactivated with the exception off 10n 11/19/02 and! Jon 12/05/02. Fori Jthe 25.23 ng/mL standard (the lowest standard in the curve) had a res:overy 0(60%. this standard "Yas kept in order to maintain a five point calibration curve. Since the 25.23 ng/mL[. ltandard had an area count of 10866 and the highest sample area count for this analYsis was 3475, the 25.~3 Fort J ng/mLtandard was accepted as the lower limit of quantification for this analysis. the 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  Continuing Calibration Verification (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 Ii 9/02. 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, I, 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 (S20%), the results were accepted. There should be no adverse affect on the data. Table 6: CCV Summary from 11/19102 analysis Anal e Continuing Calibration Verification Standards (nglmL) 10 50 250 '10 50 250 10 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% 118% NR* 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 a,ccuracy 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-suitabilit/standards were analyzed before the initial calibration curve. The criterion of <5% relative standard deviation (RSD) for the mean repeatability was achieved except for r (8.12%) on 11 Ii 9/02 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 th~ 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 I Acceptable Precision. Precision was determined for each set of duplicate or triplicate samples. Out of 160sets of samples, 152 of the sample sets had precisions of:5 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. Fat 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 ~." - - f-- ~ '- - - - Medium Day A 14 A 14 B 14 B 14 B 28 I B 28 C 28 C 28 Relative Standard Deviation 24.6% 37.8% 38.3% 26.7% 22.2% 34.5% 25.7% 132% Matrix Spikes. Samples were soiked 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 PFBS Medium A Day i4 Percent Recovery 132% - A 0 - i B 0 C 0 f-- A 14 f----. B 14 f-- C 14 ~ A 28 f-- B 28 !- 154% 148% 141% 150% 138% 149% 136% 129% C 28 -- AC 104 - _ C 28 ....;~-~ -.-.-..-..0-.. ,,-_._.- 134% 168% 126% 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%-120% 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 ;h the lower limit of quantitation. Matrix Blanks. All matrix blanks were less than % the lower limit of quantitation. DATA SUMMARY 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 fhe 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 I Exposed to Uninhibited Sludge) Percent Degradation Results1 An I Day 0 i , , 0.84% " <3.5% .. <0.92% Day 4 1.0% 114% 1.2% 1.4% Day 14 0.40% 58%, 0.47% 1.9% Day 28 <0.15% 63% 0.26% 2.4% <1.0% <0.20% 0.12% 0.37% <0.33% 0.28% 0.97% ... . The percent degradation IS based on the theoretical amount offluonne avarlable In the Initial dose of 2.3% The percentages reported in Table 1 were determined by dividing ttie average amount nf analyte found in each te~t vessel by the theoretical amount if :otally biotransformed into that specific analyte. as calculated using standard. :>-rhe analytical accuracy for all analytes is 20% or better. Table 10: Medium 8 Results Analyte Day 0 Day 4 <4.0% 5.7% N/A2 <3.5% 1.3% <0.92% 0.60% <1.0% <0.20% '--;as calculated uSing <0.33% 0.045% 2N/A: Not analyzed 111e analytical accuracy for aU analytes is 200/0 or better. Day 14 1.3% 35% 0.64% 1.3% <0.081% 0.092% Day 28 0.63% 32% 0.35% 1.3% <0.081% 0.10% Page 20 of 142 Table 11: Medium C Results Analyte Day 0 ~ - rr- . . : ; . ._ - PFBS3 1.4% <4.0% <3.5% <0.92% <1.0% <0.33% Day 4 3.8% N/A2 0.67% 0.069% <0.20% <0.041% "N/A: Not analyzed ~e analytical accuracy for all analytes is 20% or better. Day 14 6.1% <4.0% <3.5% <0.92% <1.0% <0.33% Day 28 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 j _ 6 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 d~gradation after 14 days. At day 14, SLS had degraded to <5.8% (>94.2%) in the presence of' and <8.2% , j in its' - absence, showing that the sludge had good activity and was notInhibited 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 Erlenmeyer flasks and adsorption onto the sludge as seen from a previous study, .. Soil AdsOrption/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 i .! 6% 16% 6% [ 2% i I 6% I 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 Arlalyte . f- - I-- . ' . . . . _. . . . . . . . PFBS3 '- Day 0 1.4% <4.0% <3.5% <0.92% <1.0% <0.33% Day 4 3.8% N/A2 0.67% 0.069% <0.20% <0.041% "N/A: Not analyzed 111e analytical accuracy for all analytes is 20% or better. Day 14 6.1% <4.0% <3.5% <0.92% <1.0% <0.33% Day 28 14% 3.5% 0.27% <0.018% <0.0810/; <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 fonn ' bof the theoretical value). After Day 0, decreases in concentration as other reference substances start to appear. Medium 8 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 8 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 d~gradation after 14 days. At day 14, SLS had degraded to <5.8% (>94.2%) in the presence of; ._ and <8.2% (>91.8%) in its' absence, showing that the sludge had good activity and was notlnhibited 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 Erlenmeyer flasks and adsorption onto the sludge as seen from a previous study, " Soil AdsrptioniDesorption of The results of duplicate laboratory 90ntrol 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% 16% 6% 2% 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 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 Clvaifable from the test substance that could biotransform completely to a specific analyte. This calculation was as follows: Total available fluorine was determined by: Total Available Fluorine (pg) = C x V x F where: C = Concentration of ill test vessel (pglmL) V = Volume of Sample (mL) F = Percent Fluorine Composition of Next, the percentage of fluorine was determined from the molecular weight from a reference substance: Percent Fluorine Number of fluorine atoms x 18.998~ mol molecular weight of compound ~ mol The total amount of the reference compound that could be formed from the available amount of fluorine was determined by: . IA J1g Theoretlca mount-= Total Available Fluorine ( . . tug) x - - - .1- - - - - mL .' Percent Fluonne ExtractIOn volume (ml) Amount of Analyte in Sample ~ Percent Degradation = ' mL Theoretical Amount ~ mL For example usingr ] BlUl Theorectical Amount So, for Day zero, medium A the percent degradation would be: L ];~ - - ng Percent Degradation =L_--=Jm=L- Page 22 of 142 STATEMENT OF CONCLUSION The analytical results demonstrate that under the conditions of the study, the test substancer ]is biodegraded (OPPTS 835.3210 "Modified SeAS Test" defines a greater than 70% loss of starting material as ul~mate biodegradability). The major metabolites identified fron;Lthe testcultures w.ere lrepresenting 26% degradation of the initial concentrati9n of j Jon day 0) and ](representing 114%...degradation of the initial concentration Of[ -bn day 4). Observed minor metabolites includel and PFBS}:omplete mass balance between parent material and measured degradation products appears to Rave been achieved on day 4. LIST OF ATTACHMENTS Attachment A: Extraction and Analytical Methods Attachment 8: Data Tables Attachment C: Sample Chromatograms Attachment D: Test Substance Information Attachment E: Protocol, Protocol Amendments and Deviations Page 23 of 142 SIGNATURE PAGE We certify that this report is a true and complete representation of the data for this study: J B'ate / lJ3 JtJ 03 Date J Date Page 24 of 142 ATTACHMENT A: EXTRACTION AND ANALYTICAL METHODS Page 25 of 142 3M ~nvironmentalLa~ory Method Solid ~hase Extraction of Soils, Sediments and Sludges Method Number: ETS8..39.0 Adoption Date: Upon Signing Effective Date: Upon Signing Approved By: ETS-8-39.0 Solid Phasl~ Extraction of Soils, Sediments and Sludges Page 1 of 10 Page. 26 of 142 1 Scope and Application This is a perfonnance-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 I% solution of acetic acid. The sample is capped, mixed, and put on the centrifuge to clarift the sUpernatant, if needed. The supernatant is passed through a preconditioned 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 LCMS or LCIMSMS. 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 Ii 4.1 Health and Safety Warnings 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 oflaboratory 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 Instrumentation Vortex mixer Vacuum Pump ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 2 of 10 Page 27 of 142 SPE Extraction Mariifold Centrifuge Shaker Balance (+/- 0.1000 g) Solvent trap 6.2 Supplies and Materials 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, I g, Sep-Pak 6 cc tri-fimctional CI8 (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 fonn, 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. 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 of Soils, Sediments and Sludges Page 3 of 10 Page 28 of 142 9 Method Performance Data Quality Objectives Application of 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 ofthe method: 9.1 Extracted Matrix Calibration An extracted calibration curve will be prepared from extracted matrix standards, in the same ma:trix as the samples, and analyzed before each analytical set. It will consist ofa minirnwn ofiline (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 \\!ithin 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 minimwn of nine (9) levels from unextracted solvent standards will be prepared, and may include a blank. The standard curve equation \\!ill be determined by regression analysis using the peak areas ofthe 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 ofquantitation (LLOQ) for each target analyte is determined by the lowest'calibration level that shows a recovery of 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 \\!ill 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 \\!ill be accomplished by the re-injection ofone of the original curve points, preferably one in the mid-range of the curve. A maximwn of ten (10) samples may be injected before the injection of a CCY, and then more samples may be analyzed. A CCY is then analyzed again at the end of the sample set. The CCY's must show a recovery \\!ithin 25% to be considered acceptable. Samples will then be bracketed by the calibration curve and acceptable CCY's. 9.5 Blanks 9.5.1 Solvent Blank An aliquot of 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, ifany. The frequency and exact makeup ofthe matrix blanks will depend on the scope of the study and the availability of additional matrix. Specific requirements, ifany, will be addressed in the study protocol and/or the raw data. ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 4 of 10 Page 29 of 142 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 ofmatrix 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 of the spike must be known. This may be accomplished by the concurrent extraction ofan additional aliquot of the same matrix. Analysis of these samples provides a measure ofextraction 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 sufficient justification 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 ofextracted samples (if applicable to the study) and analyzed to determine extraction efficiency. The samples are prepared by adding additional targetanalyte to the final extract, and after any dilution. Post-extractio'n 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 ofthe initial calibration curve or'should be prepared at 1.5-5 times the endogenous conc~ntration ofthei:malyte. Spike concentrations should fall in the low-range of the initial calibration curve if extremely low levels are expected. 9.11 Sample 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 ofthe extract will be documented in the raw data. 10 Procedures - 10.1 Soil Sample Preparation and Extraction Weigh approximately Ig 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 fonn. 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 reservoir/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 I% acetic acid, taking care not to allow the column to run to dryness after each wash. ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 5 of 10 Page 30 of 142 NOTE: Depending on the nature of the target compoLmds, the original sample [titrate may need to be collected and analyzed. If this is the case, place collection vessels under each cartridge at this pOlnt, 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 ofliqulds 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. 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 of analysis being carried out, if needed. 10.2 Fluid Sample (Suspended Sludge) Preparation Fluid samples should beat 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 fonn. Return the unused portion of 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 dre 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 ofa plug ofglass wool on top of the SPE packing material to prevent the plugging of 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 thecontents become clear. Place collection vessels under each cartridge and decant the solvent into the cartridge as ~bove, again allowing the column to go to dryness. Repeat this solvent elution step with fresh collection vessels ifneeded. 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 of analysis being carried out, if needed. ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 6 of 10 Page 31 of 142 11 Data Analysis and Calculations 11.1 Calculations 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 eluate Calculate theoretical concentrations of analyte in final eluate: Concentration = (Concentration of Analytical Standard x Volume of standard added) lEV 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 = Eluate volume (volume offinal 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 Management Sample waste is disposed of in low BTU containers. Flammable solvent waste is disposed of in high BTU containers. Glass pipette waste is disposed ofin broken glass containers located in the laboratory. ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 7 of 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. . Swnmarize data using suitable software and store in the study folder. 14 Attachments Attaclunent A: Samples ofExtractiori Worksheets 15 References None 16 Affected Documents None 17 Revisions Revision Number Revision Description Revision Date ETS-8-39.0 Solid Phase Extraction of Soils, Sediments and Sludges Page 8 of 10 Page 33 of 142 C'J ~ ~ ~ o ~ 0) Q) Attachment A: Samples ofExtractio~ Worksheets tJ1 CO Q Study Num ber Soil Extraction Worksheet Method FIs-a-xxx 0 ~<: o ~ 9 o<l> ~ ~ <l> <l> Ci. Ci. (/) (/) Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xx xx -x xxxx x 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 -xxx xx x Exx-xxxx-xxxxxx Exx -x xx x-xx xxx x Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Col 3: Balance 10 Col 4: siandard lD S la nda rd C once n tra lion : Amount Standard Added: Col 5: Standard 10: Volume' Added: o 1'"j :~cE 3: > EC> oell ..c: ..J :::l q: ..J ~E ell 0) "0; 't:; o .,... "l:l >< <l> 'tl ~ l'ell 'tl -o "el:lll 0) <l> Ci. q: ~ ell 'tl E q: '0 ~ :J "l:l Ci. "0 '0 0- ell '" '" > o o (J) (/) 4: 7 Col 7: CentrifuQe 10: Speed~ Dura tion : TIDII _ _ Col 8: TIOII Col 9: TIOII _ Co110: MeOH 10: Vo Iu mea d d ed: ~<: w~ <o: E E 'tl <: o :::l ~ "0 0... 0 ..c: :z g' ;;:: e tf 8-~ = .2 Ql 0 t i5. 0 E ai (/l '" (/) OJ "l:l :0J U) 0t: <U '" t :::l 0I.I..I '0" q0:" Tvpe of Column Used _ '0 ~ '" '0 > o (J) 'iii <: - III ..c: IcI:I ~ Q) 'OJ ~ 'I: ..J >< E. ~ Eell .... Ql '0 Ci. "l:l <: .g '" .2 E 0- >o ''0" '" (J) onl 10 11 ell ..0c:>.'2" :o::l ..Q..l ... 0 ;;:::z -S ~ Ql "0 ~ Ci. 0 E OJ ei :::l 'U"l "Ol:ll t en "i:: Ul t:: ~ '" '" o 0... 0 12 13 rcUrpm '- _ _ I Col 11:TIDII Col 12: CenlrifuQe 10: Speed: o uration: T/DII Co113: TIDII Co114: T/OII >: ~Ul <: E :::l C0 Q>l 0 ~ "0 <: (/) ,2 :z ni U <~l> >f<<l'?- cr= UJ 14 :t: c: .2 o:2 iii g 'tl :e .g:z 'tl Cll 'tl Cll <t 15 ..J :::l ai -0- ~ ~ .c.:. "'l":l 15 ~ .;2 ~ <: 'ti "l:l c: "~l:l t':" 16 reflrpm _ Col 6: TIOII Sample Extract Storage Samples slored in refrigeratorllreezer ________________________ at degrees C until analysis. ETS-8-39.0 Page 9 of 10 Solid Phase Extraction of Soils, Sediments and Sludges Attachment A: Samples of Extraction Worksheets Fluid Sample Extraction Worksheet, Method FTS8.39 0 Study Number Tvpe of Column Used -0 <ll ~ c: :x: -0 :x: -I 2: -0 [ ~ -c t: -I -0 CI> () ,0 a; .t: OJ >- ~ oc: 0 III :I n; 'Ii ~ ~ ;e"Ot ::l ~ j g ~ ; o E c( <ll > -I ~ -I <ll :I ~E III 0 .t: oS! 0 .t: ::i: '0 ::i: o : ~ '0 c: .. ~ e ~ g ~- -g.= ~~~ o = :0 -0 CI> 5-0 >< c: ~ .Q - U0 Q. Q. CI> -I "Q.E .-E cII:I '0 '" ] g ::; g g E :1 -g ~ ~ ~ III :e-o E 0.. < o0.. "<Qll. g<cl:l ~.II.I ~-0 -<oEll --<00ll c cU .. <ll OJ :I E E en -0 0 E- "'iii 'E- E~ :c "-:' ~ ~ ~ -0-1 ~ :I -0 "0.. "0 '0 Q. () :I f! 0 .. '1: <ll .. 1:: :0 CI> "5 .5 ~ ,.::! E -0 .. -0 '0 c: E o J ~ ~ en en en c( E en >< 11. U tIS 11. lQ () 0D" iiJ III ;:l CI> 0 Z U 3 10 11 12 13 14 Exx-xxxx-xxxxxx Exx-x xxx-x xxxx x Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx IF xx-xxxx-xxxxxx F xx-xxxx-xxxxxx 'F xX.Xxxx-xxxxxx iF Xx-x Xxx-x xxX"" lEx x-x xxx -xx xxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx IF xx -x xxx-x xxxxx IFxx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx Exx-xxxx-xxxxxx IFXX-XXXX-xxxxxX iF xx-x xxx-x xx xxx iF xx-x xxx-xxxxxx IF xx-xx XX-XXX""" Exx-xxxx-xxxxxx Col 3: TIDII Col 4: Water 10 Amount Water Added: T/OII Col 5: Standard 10: Volum e Added: T/OII _ _ _ _ Col 7: T/OII CoI8:T/01I Co19: T/OII Coli0: MeOH 10: Vblum e added: T/OII Col11: MeOH 10: Volum e added : _ _ _ Col 12:0iluter 10: Volume of sample added: Volume of Oiluent Added: OiluentIO _ Final'Oilution Factor: T/OII ~ Co 113: Internal Standard 10 Internal standard C onc. Amount Added: Volume of sample: IS Con ce otra tioo _ _ _ _ _ Sample Extract Storage Samples stored in refrigerator/freezer ________________________ at degrees C until analysis. III ETS-8-39.0 Page 10 of 10 Solid Phase Extraction of Soils, Sediments and Sludges C'\I "I\t ~ o4..- l.!J 0') Q) t:n cti Q. i 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF WASTE STREAM, WATER EXTRACTS OR OTHER SYSTEMS USING HPLC-ELECTROSPRAy/MASS SPECTROM:.ETRY , Method Number: ETS-8-15S.1 Adoption Date: iJ J~ 100 Revision Date: <;;/3 0 10 ( Author: Approved By: Date Page 36 of 142 1.0 SCOPE AND ApPLICATION 1.1 Scope: This method describes the analysis of waste stream, water samples or other systems using HPLC-electiospray/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 SUMMARY OF METHOD 2.1 This method describes the analysis of electrospray ionizable compounds, using HPLCelectrospray mass spectrometry (HPLC-ESI1vfS). The analysis is performed by the mass selection of a single ion characteristic of a particular compound. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): Cbmmercially available HPLC-ESIMS 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 (ES!), Atmospheric Pressure chemical Ionization (APel), 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 tjny charged droplets. These droplets are produced by the application of a 'strong electrical field. 3.3 Mass Spectrometer (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-ESIMS. Please refer to the manual for the specific instrument software. 3M Environmental Laboratory ETS-8-155.1 Analysis Using HPLC-Electrospray/Mass Spectrometry Page2of10 Page 37 of 142 4.0 WARNINGS 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 HPIIOO will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To mlliimize 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 EQUIPMENT 6.1 Equipment listed below may be modified in, order to optimize the system. Docwnent 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 HPIIOO low pulse solvent pump, solvent degasser, column compartment, and autosampler or equivalent HPLC system. 7.0 SUPPLIES AND MA.TERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (or house air system.). 7.1.2 7.1.3 HPLC analytical column, such as a Betasil CI8 column (50x2mm, 5 Jlffi particle size) or equivalent. Capped autovials orcapped 15 mL centrifuge tubes. 8.0 REAGENTS AND STANDARDS 8.1 Reagents: 8.1.1 Methanol, HPLC grade or equivalent. 3M Environmental Laboratory ETS-g-155.1 Analysis Using HPLCElectrospraylMass Spectrometry Page 3 of10 Page 38 of 142 8.1.2 Milli-QTM water (ASTM type 1), all water used in this method should be Milli-QTM water or equivalent, and may be provided bya 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 mMammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 L volumetric flask, add the appropriate volume ofMilli-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: 8.1.1 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 SAMPLE HANDLING 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 40 C until analyses can be performed (refrigerator temperatures may have a detrimental affect on the solubility of saturated solutions). . I 10.0 QUALITY CONTROL 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 Matrix Spikes: 10.2.1 Matrix spikes may be prepared for each set of extracted samples (if applicable to the study) and analyzed to determine extraction efficiency. 3M Environmental Lab<Jratory ETS-g-155.1 Analysis Using HPLC-ElectrospraylMass Spectrometry Page 4 of10 Page 39 of 142 10.2.2 Matrix spike duplicates may be prepared periodically to measure the predsion 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 midrange 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. TheIS should be added after the extraction process and before analysis. 10.4.2 A surrogate standard may be used fODquality 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 CALIBRATION AND STANDARDIZATION 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 = fiX + b) weighted l/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 paramet~rs 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 ET58155.1 Analysis Using HPLC-ElectrospraylMass Spectrometry Page 5 of10 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 ofthe 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 predetenninedacceptance 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 12.1 PROCEDURES Acquisition set-up - please reference the SOP that pertains to the specific instrument. 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). Ifmore 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. i 12.1.1.3 Assign an HPLC program (Irilet 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-&-155.l Analysis Using HPLC-ElectrospraylMass Spectrometry Page 6 of10 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 HPII00 using the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditi'ons in the ins~entlogbook: 12.3.1.1 12.3.1.2 Sample size = 10 J-LL injection Flow rate = 300 ~/min. 12.3.1.3 Cycle time =' 10.0 minutes 12.3.1.4 Mobile phase components: Solvent A: 2.0 roM Ammonium Acetate in Water Solvent B: Methanol (MeOH) Solvent Gradient: Time (min.) %B 0.00 5.00 % 1.00 5.00 % 4.50 95.00/0 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 Platfonn LCZ ElectrospraylMass 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 fme 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-Elc:ctrospraylMass Spectrometry Page 7 of10 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 j.lL/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 DATA ANALYSIS AND CALCULATIONS I 13.1 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 (uglmL): On-Column Concentration (llg/mL) x Dilution Factors = Calculated Concentration 14.0 METHOD PERFORMANCE 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 Analysis Using HPLC-EleetrosprayfMass Spectrometry Page8of10 Page 43 of 142 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 (r) 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 16.1 16.2 16.3 .16.4 16.5 16.6 16.7 RECORDS Each page generated for a study must ,p.ave 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. 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. Plot the calibration curve then print these graphs and store in the study folder. Print data integration summary, integration method, and chromatograms and file in the study binder. Summarize data using suitable software and store in the study binder. Back-up electronic data to appropriate medium. Record the file names and location of backed-up electronic data in the study binder. Documentation of analyte(s) and all reference substances will include trace numbers, lot #'s, puri.tY, expiration, and storage conditions. 17.0 AITACHMENTS 17.1 None 3M Environmental Laboratory ETS8155.1 Analysis Using HPLC-ElectrospraylMass Spectrometry Page 90f10 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 9L2; United Kingdom. 18.3 MassLynx NT Guide To Data Acquisition, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9L2; United Kingdom. 18.4 ETS-9-34.0, "Operation and Maintenance of Hewlett Packard HPLC 1100 System Equipped with a Mass Spectrometer Detector and/or a Photo Diode Array Detector". 18.5 ETS-9-36.0, "Operation and Maintenance of the Micromass Platform LCZ ElectrospraylMass Spectrometer". 19.0 AFFECTED DOCUMENTS 19.1 None 20.0 REVISIONS Revision Number Reason For Revision Revision Date Title Change 09 May 01 Section I: Enlarged the scope of the method by including additional matrices. Also added comments about specific parameters will be defmed 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 ofstorage containers and conditions. . Section 10: Added comments to allow for flexibility in quality controls for different types of studies and applicability. Section 11: Added a paragraph allowing for the excluding of the second curve. Section II: 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 of analytes and substances. Section 18: Added reference to the method for the Hewlett-Packard LCIMS. 3M Environmental Laboratory ETS-8-155.l Analysis Using HPLC-ElectrospraylMass Spectrometry Page lO of 10 Page 45 of 142 ATTACHMENTS: DATA TABLES i I Page 46 of 142 Medium A PFBS Sample E02 -09 13 -0 l3 E02-0913-0 1-1 E02-0913-015 E02-0913-043 E02-0913-0-l4 E02-0913-045 E02-0913-103 E02-0913-104 E02-0913-105 E02-09i3-163 E02-0913-164 E02~0913:"165 Timepoint Day () Day 0 Day 0 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 Result (ng/mt) <50.06 <50.06 <50.06 45.09 38.44 42.98 152.4 149.3 143.0 389.4 325.6 320.0 Average (nglanL) <50.06 42.17 148.2 345.0 Average (ng/mL) anion* <-+-1.28 37.30 131.1 305.2 Standard Deviation (nglmL) N/A 3.398 4.790 38.58 Averngewas multiplied by a factor of O.8X46 to correct to anion Based on the total amount of fluorine in PFBS,r N/A.. ~ Not applicable Sample E02-0913-0 13 E02-0913-014 E02-0913-015 E02-0913-043 E02 -0913 -044 E02-0913-045 E02-0913-103 E02-0913-104 E02-0913-105 E02-0913- I63 E02-0913-164 E02-0913-165 Timepoint Day 0 Day 0 Result (ng/mt) <490.8 <490.8 Average (nglroL) <490.8 Day 0 <490.8 Day 4 Day 4 Day 4 167.5 168.3 160.3 165.4 Day 14 Day 14 75.95 61.90 66.66 Day 14 62.13 Day 28 Day 28 29.20 41.11 36.90 Day 28 40.38 Based on the total amount of fluonne in Standard Deviation (ng'roL) N/A 4.406 8.046 6.675 I Relative Standard Deviation . N/A 2.66% 12.1% 18.1% NIA ~ Not applicable Relativc Standard Deviation N/A Pcn:cnt Dc!!radation** <0.33% 8.06% 0.28% 3.23% 0.97% 11.2% 2.3% Percent Degradation* <3.5% 1.2% 0.47% 0.26% Sample E02-0913-0 13 E02-0913-014 E02-09lJ-0 15 E02-0913-043 E02-0913-044 E02-09lJ-045 E02-0913-103 E02-0913-104 E02-0913-105 E02-0913-163 E02-0913-164 E02-0913-165 Timepoint DayO DayO Result (ng/roL) <126.1 <126.1 Average (ng/roL) <126.1 Day 0 Day 4 Day 4 <126.1 <25.23. <25.23 <25.23 Day4 <25.23 Day 14 Day 14 16.57 14:89 15.49 Day 14 15.02 Day 28 Day 28 46.24 44.27 40.24 Day 28 48.20 Based on the total amount of fluorine in Standard Deviation (ng/roL) N/A N/A 0.9347 1.965 . N/A ~ Not applicable Relative Standard Deviation N1A Percent Degradation* <1.0% N/A <0.20% 6.03% 0.12% 4.25% 0.37% Page 47 of 142 MtldiUlll A Sample E02-09 13-011 E02-091J-014 E02-091J-0 15 E02-0913-043 E02-0913-044 E02-0913-045 E02-091J-103 E02-0913-104 E02-0913-105 E02-0913-163 E02-0913-164 E02-09l3-165 Timcpoint Day 0 Result tng/ml) <125.2 Average lng/mt) DayO <125.2 <125.2 Day 0 <125.2 Day 4 205.2 Day 4 185.5 194.0 Day 4 191.4 Day 14 257.8 Day 14 274.6 261.2 Day 14 251.1 Day 28 365.4 Day 28 311.6 320.8 Day 28 285.5 B:l.S<:d on the to!Jl1 amount of fluorine ;n N/A ~ Not applicable Standard Deviation lng/ml.) N/A Relative Standard Deviation N/A Percent Degradation* <0.92% 10.11 5.21% 1.4% 12.10 4.63% 1.9'ti 40.72 12.7% 2.4% Sample E02-0913-013 E02-0913-014 E02-09IJ-0 15 E02-0913-043 E02-0913-044 E02-09IJ-045 E02-09IJ-IOJ E02-091J-I04 E02-09IJ-105 E02-09IJ-16J E02-0913-164 E02-09IJ-165 Result Timepoint (ng/ml) Average (ng/ml) Day 0 4004 Day 0 3961 4129 Day 0 4422 Day 4 181.1 Day 4 149.6 161.4 Day 4 153.5 Day 14 82.02 Day 14 50.50 64.89 Day 14 62.15 Day 28 <25.09 Day 28 <25.09 <25.09 Day 28 <25.09 Based on thc to!Jl1 amount of fluorine in Standard Deviation lng/ml) 254.3 Relative Standard Deviation 6.16% Percent Degradation* 26% 17.17 10.6% 1.0% 15.94 24.6% 0.40% N/f' N/A <0.15% N/f\. : ;\lot aoOl1cable Sample E02-0913-0IJ E02-0913-014 E02-09IJ-0 15 E02-0913-0 16 E02-0913-0 17 E02-0913-103 E02-0913-104 E02-0913- 105 E02-091J-I06 E02-0913-107 E02-0913-16J E02-09IJ-164 E02-09IJ-165 E02-09IJ-166 E02-0913-167 Timepoint Result (ng/ml) Times Correction Factor (ng/mL)** Day 0 <500.0 <665.0 Day 0 <500.0 <665..0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 14 6247 8309 Day 14 12680 16865 Day 14 6508 8656 Day 14 5670 7542 Day 14 6883 9155 Day 28 4717 6274 Day 28 4874 648J Dav 28 4051 5388 Dav 28 4744 6309 Day 28 5001 6652 Based on the total amount of fluonne Ir Average (ng/ml) <665.0 10105 6221 Standard Deviation (ng/ml) N/A 3824 489.4 Relative Standard Deviation Percent Degradation* N/A <4.0% 37.8% 60% 7.87% J7% A cUITcction lactor was calculated 10 b" and' N/A ~ :'lot applicable Page 48 of 142 Medium A Sample E02-0913-013 E02-0913-0 14 E02-0913-0 15 E02-0913-043 E02-0913-044 E02-09l3-045 E02-0913-103 E02-0913-104 E02-0913-163 E02-09l3-164 E02-09l3-165 Timcpoint Result (11l.!'mt.l Avemge (l1g'mt.) Day 0 157.8 Day 0 141.5 141.9 Day 0 126,3 Day '4 19494 Day 4 19133 19243 Day4 19101 Day 14 9572 Day 14 9761 9666 Day 28 . 10336 Day 28 11791 10599 Day 28 9670 Based on the total amoWlt of fluorine m NtA = Not applicable Standard Deviatioll (nl.!'mL) 15.78 218.2 l33.4 1084 RcI:ttivc Standard Dcvi:atioll Percent Dcgmdatiol1* 11.1% 0.84% 1.13% 1.38% 10.2% 114% 58% 63% Page 49 of 142 Medium B PFBS Sample E02-091 3-0 13 E02-091 3-019 E02"0913-020 E02-0913-Q.l3 E02-0913-049 E02-0913-050 E02-0913-108 E02-0913-109 E02-0913-110 E02-09 13-168 E02-0913-169 E02-0913-170 Timepnint Day 0 Day 0 Day 0 Day 4 Day 4 Day 4 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 Result (n!!lml.) <50.0'6 <50.06 <50.06 3.133 5.804 6.823 10.26 17.87 14.27 16.19 15.02 Aventgc (n~'ml) <50.06 6.938 14.07 15.16 Averdgc (ng/lUl) &lnion*'" <44.23 6.138 12.44 13.41 Stundard Deviation (ng/lUL) N/A 1.196 5.38l 0.968 SPE clugged, unable to e~tr,jct Avenge w"" multiplied by a factor of 0.8846 to correct til anion B""ed on the total .mount of fluonne i, PFBS. N/A =Not applicable Relutivc Stund:ml Dcvi;ttiun Itercent i)cgr:td;ttion **~ N/A <0.33% 17.2% 0.045% 38.3% 0.092% 6.38% 0.10% Sample E02-0913-0 [8 E02-09 13-019 E02-09l3-020 E02-0913-048 E02-0913-049 E02-0913-050 E02-09 13-108 E02-0913-109 E02-0913-110 E02-0913-168 E02-09 13-169 E02-0913-170 Timepoint Day 0 Day 0 Result (ng/ml) <490.8 <490.8 Averdge (ng/mL) <490.8 Day 0 <490.8 Day 4 192,5 Day 4 187.9 180.6 Day 4 Day 14 Day 14 161.5 94.69 90.46 Day 14 86.23 Day 28 Day 28 44.25 43.29 50.19 Day 28 63.04 SPE clogged, unable to extnlct Rll.,ed on the totat amount of fluorine in Standard Devi&ltion (ng/mL) N/A 16.73 5.982 11.14 i Relative Standard Deviation N/A Percent Degmdation ** <3.5% 9.26% 1.3% 6.61% 0.64% 22,2% 0.35% N/A = Not applicable Sample E02-091 3-0 18 E02-0913-0 19 E02-0913-020 E02-09 [3-048 E02-0913-049 E02-0913-050 E02-0913-108 E02-0913-109 E02-09 13-1 10 E02-0913-168 E02-0913-169 E02-0913-170 Timepoint Day 0 Day 0 Day 0 Day 4 Day4 Day 4 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 Result (ng/tnl) <126,1 <126.1 <126.1 <25.23 <25.23 <25.23 <10.09 <10.09 <10.09 <10.09. <10.09 Average (ng/mL) <126.1 <25.23 <10.09 <10.09 SPE clogged. unable to extract Bused. un the tueal amount of tluurine ip PFBA. N/A = Not applicable St.tndard Deviation (ng/tnt) N/A NlA NlA N/A Relative Standard Deviation N/A Percent Degradation ** <1.0% NlA <0.20% N/A <0.081 N/A <0.081 Page 50 of 142 Medium B S;,mplc: E02-09 I3-0 l::l E02-09 13-0 19 E02-0913-020 E02-091 J-048 E02-091 J-049 E02-0913-050 E02-09\ J-\ 08 E02-09lJ-I09 E02-091 J-\ 10 E02-091 J-\68 E02-091 J-169 E02-0913-170 Timcpoint Day 0 Day 0 Result (ng/roL) <125.2 <125.2 Avemge (ng!ml.l <125.2 Day 0 <125.2 Day 4 Day 4 79.17 76.13 81.12 Day 4 Day 14 88...06 Day 14 162.9 175..+ Day 14 187.9 Day 28 Day 28 16J.3 156.4 176.5 Day 28 209.9 SPE clogged, unable to extmct Based on rh... f",f,,:1 amnunt of t1uorinc tv: N/A Not applicable Stilndilrd Deviiltion (nwmL) N/A Reliltive St;Indurd Ileviatiun I)ercent D.:g.....dution +* N/A <0.92% 6.199 7.64% 0.60% 17.68 10.1% l.J% 29.15 16.5% 1,.3% Sample E02-09lJ -0 18 E020913-0 I9 E02-0913 020 E020913048 E02-09 1J -049 E02-0913-050 E020913-108 E02-0913109 E02-0913-110 E02-0913-168 E02-0913-169 E0209\3-170 Timepoint Day 0 Dayb Result (nglmL) 3815 3978 Average (nglmL) 3990 Day 0 4178 Day 4 Day4 851.2 857.9 917.7 Day 4 Day 14 Day 14 10..44 173.7 214,0 Day 14 254.38 Day 28 Day 28 116.2 61.21 10 1.I Day 28 126.0 SPE clogged, unable to extract Based on the total amount of fluorine in Standard Deviation (nglmL) 181.8 Relative Standard Deviution Percent Dcgr"dation ** 4.56% 25% 109.4 11.9% 5.7% 57.05 26.7% 1.3% 34.92 I 34.5% 0.63% Sample E02-0913-0 18 E02-09lJ-0 19 E02-09 13-020 E02091 J-021 E02-0913-022 E02-09 13-1 08 E02-09 13-109 E02-0913-110 E02-0913-111 E02-0913-112 E02~0913168 E02-0913-169 E02-091 J-170 E02-091J-17\ E02-09\3-172 Timepoint Day 0 Result (nglmL) <500.0 Times Correction Factor (nglmL) *** <665.0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 . Day 0 Day 14 .<500.0 <66..5.0 Day 14 5090 6770 Day 14 Day 14 42.0. 1 55..87 Day 14 4052 5389 Day 28 3796 5049 Day 28 3914 5206 Day 28 4054 5392 Day 28 4J99 5850 Day 28 4198 5584 SPE clogged, unable to extract .C1 Basr.:J un the !Otul amount of tluurine in Average (nglmL) <665.0 5915 5416 A t:oITcctiun factur was cakulatcd to bt: f ,fur the Jiftcn.:m.:cs in respunse factor, 'lnu NIA = Not applicable Standard Deviation (nglmL) Relative Standard Deviation Percent Degradation ** N/A N/A <4.0% 746 12.6% 35% 314.8 5.81% 32% Page 51 of 142 lVtcdiunl C PFB~ Sample Timepllint E02-09 [3 -023 Day 0 E02-09 [3-024 DayO Result (ng/ml.) <50.06 <50.06 Average (n1!fml.) <50.06 E02-0913-025 Day 0 <50.06 E02-0913-053 E02-0913-054 Day 4 Day 4 <6.258 <6.258 <6.258 E02-0913-055 Day 4 <6.258 E02-09 IJ-113 Day 14 <50.06 E02-09 13- [ [4 Day [4 <50.06 <50.06 E02-0913-[ 15 Day 14 <50.06 E02-09IJ-173 E02-09 13-1 74 Day 28 Day 28 <[0.01 <10.01 <10.01 E02-0913- [75 Day 28 <10.01 Averngc was multIplied by a foctor B"""d on the total amount of fluorine ir PFBS, ' N/A = Not applicable Average (ug/ml) anion'" <44.28 <5.536 <44.28 <8.855 Standard Deviation (ngfml.) N/A Rel'ltive Standurd Ileviiltion I'crccnt I>egmdation "''it N/A <0.33% N/A N/A <0.041% N/A N/A <0.33% N/A N/A <0.074% Sample Timepoint E02-0913-023 DayO E02-09 [3-024 Day 0 Result (ng/mL) <490.8 <490.8 Average (ng/mL) <490.8 E02-0913-025 Day 0 <490.8 E02-09 I3-053 E02-0913-054 Day4 Day4 96.23 85.91 94.28 E02-0913-055 Day 4 100.7 E02-09 I3-1 [3 E02-0913-114 Day 14 Day [4 <490.8 <490.8 <490.8 E02-0913-115 Day 14 <490.8 E02-0913- [73 Day 28 47.23 E02-0913-174 Day 28 33.10 38.73 E02-0913-175 Day 28 35.85 Based on the total amoont of fluorine ir N/A = Not applicable Standard Deviation fng/mL) N/A 7.585 N/A '7.491 f Relative Standard Deviation N/A Percent Degradation* <3.5% 8.05% 0.67% NlA <3.5% 19.3% 0.27% -- Sample Result Average Timcpoint (ng/mL) Cng/mL) E02-0913-023 DayO <126.1 E02-09 [3-024 DayO <126.1 <126.1 E02-09 [3-025 Day 0 <126.[ E02-0913-053 Day4 <25.23 E02-0913-054 Day 4 <25.23 <25.23 E02-0913-055 Day4 <25.23 E02-09[3-113 Day 14 <126.1 E02-09 IJ-114 Day 14 <126.1 <126.[ E02-0913-115 Day 14 <126.[ E02-0913-173 Day 28 <10.09 E02-0913-174 Day 28 <10.09 <10.09 E02-0913-175 Day 28 <[0.09 Bascd on thc total amwn! of fluonnc in NIA - Not applicable Standard Deviation (ng/mt) N/A N/A N/A N/A Relative Standard Deviation N/A Percent Degradation'" <1,0% N/A <0.20% N/A <1.0% N/A <0.081% Page 52 of 142 Mcdiuni C Sample E02-0913-023 E02-09 I3-024 E02-0913-025 E02-091J-053 E02-091J-054 E02-091J-055 E02-0913-113 E02-0913-1 14 E02-0913-[ 15 E02-0913-173 E02-0913-174 E02-0913-175 Result Timcpuint (ng!ml) Avernge (ng/mLl Day 0 <125.2 Day 0 <125.2 <[25.2 Day 0 <125.2 Day 4 9.33 [ Day 4 <2.504 9.331 Day 4 <2.504 Day 14 <125.2 Day 14 <125.2 <1252 Day [4 <125.2 Day 28 <2.504 Day 28 <2.504 <2.504 Day 28 <2.504 Sas..-U on the total amUlnt of tluOI1ne In ; NIA = Not applicable Standllrd Deviation (ng!mL) N/A N/A N/A N/A Relative Stundard Dcviution N/A Pen:ent Degradation* <0.92% N/A 0.069% N/A <0.92% 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 E02-0913-174 E02-0913-175 Result Timepoint (ng/ml) Average (ng/ml) Day 0 228.0 Day 0 223.5 222.5 Day 0 216.1 Day 4 605.3 Day 4 624.7 610.9 Day 4 602.7 Day 14 996.7 Day 14 978.3 985.8 Day 14 982.3 Day 28 1997 Day 28 3016 2327 Day 28 1966 Based on the total amwnt of fluOI1ne In Standard Deviation (ng/ml) 6.009 12.02 9.692 597.5 I Relative Standard Deviation 2.70% Percent Degradation* 1.4% 1.97% 3.8% 0.983% 6.1% 25.7% 14% Sample E02-0913-023 E02-0913-024 E02-0913-025 E02-0913-026 E02-0913-027 E02-0913-113 E02-0913-114 E02-0913-1 15 E02-0913-116 E02-0913-117 E02-0913-173 E02-0913-174 E02-0913-175 E02-0913-176 E02-0913-177 Times Correction ~ Result Factor Timepoint (ng/ml) (ng/mL>** Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 0 <500.0 <665.0 Day 14 <500.0 <665.0 Day 14 <500.0 <665.0 Day 14 <500.0 <665.0 Day 14 <500.0 <665.0 Day 14 <500.0 <665.0 Day 28 858.2 1141.4 Day 28 30.21 40.2 Day 28 <[0.00 <19.88 Day 28 <10.00 <19.88 Day 28 <10.00 <19.88 .. u:~ ..... t l"U'\ rh.~ tnf~d ~'Hnf .. tnt of fluonnc In .~. A cOlTcction factor was calculated to be Jr the llitTcrcnccs in response factor, N/A = Not applicable Average (ng/ml) <665.0 <665.0 590.8 Standard Deviation (ng/mL) Relative Standard Deviation Percent Degradation* N/A NJA <4.0% N/A N/A <4.0% 778.7 132% 3.5% Page 53 of 142 PFBS Sample E02-0913-0 16 E02-091 3-0 17 E02-0,) 13-021 E02-09 [3-022 E02-0913-026 E02-0,) [3-027 E02-0913-046 E02-09 tJ-04 7 E02-0913-05 [ E02-0,) 13-052 E02-0913-056 E02-0913-057 E02-0913-[ 06 E02-0913-107 E02-0913- [ [ I E02-0913-[ 12 E02-0913-116 E02-0913-[ [7 E02-0913-166 E02-0913- [67 E02-0913-17[ E02-0913-172 E02-0913-176 E02-0913- [77 Timepoint M,,:dium 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 [4 B Day 14 B Day 14 C Day [4 C Day 28 A Day 28 A Day 28 B Day 28 B Day 28 C Day 28 C SPE clogged. unable to extract NtA =Not applicable Result tng/mLl 490.75 491.55 429.05 434.15 430.[8 428.55 712.25 698.01 504:95 490.38 506.81 509.42 596.05 634.3 I '" 456.75 439.70 429.04 877.21 755.87 460.10 460.13 416.77 397.95 Matfix Spikes AveUlge Cng/mLl 491.2 Standard Deviation cng/mLl 0.569~ Relative Stundard Dcvi~ltion 0.12% Average Sample Result tng/mLl <50.06 True Value CnglmL) 500.6 431.6 3.60') 0.84% <50.06 500.6 429.4 1.156 0.27'% <50.06 500.6 705.1 [0.07 [.4% 42.17 500.6 497.7 10.30 2. I'Yo 6.938 500.6 508.1 1.846 0.36% <6.258 500.6 615.2 27.05 4.4% 148.2 500.6 456.8 N/A N/A 14.07 500.6 434.4 7.535 1.7% <50.06 500.6 816.5 85.80 10.5% 345.0 500.6 460.1 0.02[2[ 0.0046% 15. [6 500.6 407.4 13.3 [ 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% 8[.4% 94.6Y0o Sam Ie E02-0913-0 16 E02-09 [3-017 E02-0913-021 E02-0913-022 E02-0913-026 E02-0913-027 E02-0913-046 E02-09 [3-047 E02-0913-05 [ E02-09 [J-052 E02-09 [3-056 E02-09 [3-057 E02-0913- [ 06 E02-09 [3- 107 E02-09 [3-11 \ E02-09[3-[ 12 E02-09 13-1 16 E02-09 [3-1 17 E02-09 13- 166 E02-0913-167 E02-0,) 13-171 E02-0,) 13-172 E02-0913- [76 E02-09IJ-I77 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 c Day 2g c Day 2~ SPE dogged. unable to exlr.lct NtA =Not applicable Result (nglmL) 551.63 548.74 533.57 531.15 556.73 526.33 760.03 637.31 65[.36 688.90 598.84 598.31 504.58 556.54 504.07 547.88 530.41 567.33 579.25 570.29 581.15 495.79 460.98 Average (ng!mL) 550.2 532.4 541.5 698.7 670.1 598.6 530.6 504.\ 539.1 573.3 575.7 478.4 Standard Deviation (nwmL) 2.045 Relative Standurd AveUlge Sample Deviation Result (ng!mL) 0.37% <490.8 True Value (ng/mL) 490.8 1.705 0.32% <490.8 490.8 21.50 4.0% <490.8 490.8 86.77 12% 165.4 490.8 26.55 4.0% 180.6 490.8 0.375 0.06% 94.28 490.8 36.74 6.9% 66.66 490.8 N/A N/A 90.46 490.8 12.36 2.3% <490.8 490.8 8.43 1.5% 36.90 490.8 7.679 1.3% 50.1') 490.8 24.61 5.1% 38.73 490.8 AVERAGE Percent Recovery 112% 108% 110% 109% 99.7% 103% 94.5% 84.3% 110% 109.3% 107.1% 89.6% 103.1% Page 54 of 142 Matrix Spikes Sample E02-09 13-0 16 E02-0913-0 [7 E02-09 13-021 E02-0913-022 E02-0913-026 E02-0913-027 E02-09 [3 -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- [ 12 E02-0913-116 E02-0913-1 [7 E02-0913-[ 66 E02-0913-167 E02-0913-171 E02-0913-172 E02-0913-176 E02-0913- [77 Timcpoint 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 Day28 A Day 28 B Day 28 B Day 28 C Day 28 C SPE clogged, unable to extract N/A; Not applicable Result (og/mL) 768.4 780.7 740.4 750. [ 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 Avcmge (og/roL) 774.5 745.3 709.1 592.1 558.8 586.3 771.0 696.6 751.4 732.4 649.1 678.0 Sttmdard Deviation (oglmL) 8.675 6.830 13.29 47.74 54.85 31.39 29.33 N/A 16.50 22.65 59.56 21.23 Relative Standard Deviation 1.12% Avemge Sample Result (og/mL) <126.1 'frue V~,lue (og/ant) 504.5 0.916%. <126.1 504.5 1.81% <126.1 594.5 8.06% <25.23 504.5 9.82% <25.23 504.5 5.35% <25.23 504.5 3.80% 15.49 504.5 N/A <10.09 504.5 2.20% <126.1 504.5 3.09% 46.24 504.5 9.18% <10.09 504.5 3.13% <10.09 504.5 AVERAGE Percent Recovery 154% 148% 141% 117% 111% 116% 150% 138% 149% 136% 129% 134% 135 o/c0 Sample E02-0913-016 E02-09 [3-017 E02-0913 -021 E02-0913-022 E02-0913-026 E02-0913-027 E02-0913-046 E02-0913-047 E02-09 13-05 1 E02-0913-052 E02-0913-056 E02-0913-057 E02-0913-106 E02-0913- 107 E02-0913-111 E02-0913- I 12 E02-0913-116 E02-0913-117 E02-09 13-166 E02-0913-167 E02-0913-171 E02-0913-172 E02-0913-17b E02-09 13-177 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 [4 C Day 28 A Day 28 A Day 28 B Day 28 B Day 28 C Day 28 C SPE dogged, unable to extmct N/A ;Notapplicable Result (og/mL) 486.6 488.1 471.4 473.2 468.1 472.1 711.5 655.7 547.7 568.0 503.2 498.7 717.8 752.7 * 603.7 477.9 471.0 757.6 794.7 626.6 620.0 437.2 425.1 Average (og/rot) 487.4 472.3 470.1 683.6 \ 557.8 500.9 735.2 603.7 474.4 776.1 623.3 43[ .2 Standard Deviation (og/mL) 1.047 1.259 2.843 39.50 14.36 3.175 24.64 N/A 4.929 26.18 4.660 8.535 Relative Standard Deviation 0.215% Average Sample Result (og/mL) <125.2 True Value (og/mt> 500.7 0.266% <125.2 500.7 0.605% <125.2 500.7 5.78% 194.0 500.7 2.57% 81.12 500.7 0.634% 9.331 500.7 3.35% 26[.2 500.7 N/A 175.4 500.7 1.04% <125.2 500.7 3.37% 320.8 500.7 0.748% 176.5 500.7 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 Matrix Spikes Sam Ie E02-09lJ-Ol6 E02-091 J-O 17 E02-09IJ-021 E02-0913-022 E02-0913-026 E02-0913-027 E02-0913-046 E02-09l3-047 E02-09l3-051 E02-0913-052 E02-0913-056 E02-0913-057 E02-0913-106 E02-0913-107 E02-0913-111 E02-0913-112 E02-0913-1l6 E02-0913117 E02-09l3-166 E02-09l3-167 E02-0913-17l E02-0913-172 E02-09l3-176 E02-0913-177 Timc oint l\kdillln DayO A DayO 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 Day4 C Day 4 C Day 14 A Day 14 A D'ay 14 B Day l4 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 PE clogged, unp.ble to e~tract NlA =Not applicable Itcsult (nu/mLl 4997 4942 4604 4523 737.3 741.5 759.4 720.2 1453 1445 1218 1200 591.4 646.4 620.0 1530 1703 554.7 534.6 617.4 601.6 3384 3143 Avemgc (nw'mL) 4970 4563 739.4 739.8 1449 1209 618.9 620.0 1616 544.7 609.5 3264 Stmulard Dcvi~lIi()n (ng/Illl.) 39.25 57.23 2.991 27.68 5.862 12.59 38.83 N/A 122.6 14.21 11.17 170.4 Relative St~,"dard Deviation 0.790% Average SalllJlle Result (ng/IUI.) 4129 Tme V~t1ue (ng/I11M 501.8 1.25% 3990 50l.8 0.405% 222.5 50l.8 3.74% 16l.4 50l.8 0.405% 917.7 50l.8 1.04% 610.9 501.8 6.27% 64.89 501.8 N/A 214.0 501.8 7.59% 985.8 501.8 2.61% <25.09 501.8 1.83% 10l.1 501.8 5.22% 2327 501.8 AVERAGE Percent Recovcry 168% 114% 103% 115% 106% 119% 110% 80.9% 126% 109% 101% 187% 120% Page 56 of 142 PFBS Sample Medium E02-0913-003 A E02-0913-004 A E02-0913-005 . B E02-0913-006 B E02-0913-007 C E02-0913-008 C Laboratory Control Spikes Result (ng/ml) 1292 1353 1278 1248 1285 1294 Average (ng/ml) 1323 1263 1290 Standard Deviation (ng/ml) 43.54 21.12 6.147 Relative Standard Deviation 3.29% True Value (ng/ml) 1251 Percent Recovery 106% 1.67% 1251 101% 0.477% 1251 103% Sample E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913-007 E02-0913-008 Medium A A B B C C Result (ng/ml) 1025 1048 1050 1014 1046 1046 Average Cng/mL) 1037 Standard Deviation . (ng/ml) 16.24 1032 25.52 1046 0.142 Relative Standard Deviation 1.57% True Value (ng/mL) 1227 Percent Recovery 84.5% 2.47% 1227 84.1% 0.0136% 1227 85.3% Sample E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913 ..007 E02-0913-008 Medium A A B B C C Result (ng/ml) 1307 1330 1319 1258 1105 1267 Average (ng/ml) 1319 1288 1186 Standard Deviation (ng/ml) 15.98 43.08 114.4 Relative Standard Deviation 1.21% True Value (ng/mL) 1261 Percent Recovery 105% 3.34% 1261 102% 9.64% 1261 94.1% Page 57 of 142 Laboratory Control Spikes Sample E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913-007 E02-0913-008 Medium A A B B C C Result (ng/mL) 1198 1261 1256 1210 1234 1243 Average (nglmL) 1230 1233 1239 Standard Deviation (ng/mL) 44.80 32.43 6.674 Relative Standard Deviation 3.64% Tme Valuc (ng/mL) 1252 Pcrcent Recovery 98.2% 2.63% 1252 98.5% Q.539% 1252 98.9% Sample E02-0913-003 E02-0913-004 E02-0913-005 E02-0913-006 E02-0913-007 E02-0913-008 Medium A A B B C C Result (ng/mL) 1257 1315 1331 1287 1331 1336 Average (nglmL) 1286 1309 1333 Standard Deviation (ng/mL) 41.04 30.58 3.451 Relative Standard Deviation 3.19% True Value (nglmL) 1254 Percent Recovery 103% 2.34% 1254 104% 0.259% 1254 106% Page 58 of 142 T .. c Sample 02-09 13-028 02-0913-029 02-0913-030 02-0913-058 02-0913-059 02-0913-060 02-0913-118 02-0913-119 02-0913-120 02-0913-178 02-0913-179 E02-0913-180 Sodium Lauryl Sulfate Timepoint Day 0 Day 0 Day 0 Day4 Day 4 Day4 Day 14 Day 14 Day 14 Day 28 Day 28 Day 28 Result (ng/mL) 61167 52407 58327 <3330 3388 <3330 <3330 <3330 <3330 <Ill <Ill <Ill Average (ng/mL) 57300 3388 <3330 <Ill St,mdard Deviation (ng/mL) -4470 N/A N/A N/A Relative Standard Deviation 7.80% N/A N/A N/A Percent from Day 0 100% 5.9% <5.8% <0.19% Control Substance' .. Sample 02-0913-031 02-0913-032 02-0913-061 02-0913-062 E02-0913-121 E02-0913-122 E02-0913-181 E02-0913-182 Timepoint Day 0 Day 0 Day 4 Day 4 Day 14 Day 14 Day 28 Day 28 Result (ng/mL) 43921 36825 <3330 <3330 <3330 <3330 <Ill <Ill Average (ng/mL) 40373 I <3330 <3330 <Ill Standard Deviation (ng/mL) 5018 N/A N/A N/A Relative Standard Deviation . 12.4% N/A N/A N/A Percent from Day 0 100% <8.2% <8.2% <0.27% Page 59 of 142 Abiotic .. Sample E02-0913-033 E02-0913-034 E02-0913-063 E02-0913-064 E02-0913-123 E02-0913-124 E02-0913-183 E02-0913-184 Timepoint DayO Day 0 Day4 Day4 Day 14 Day 14 Day 28 Day 28 Result (ng/mL) 266488 265277 266328 249312 25547"1 266400 179799 207682 Avcmge Cng/lUL) 265882 257820 260936 193741 Standard Relative Deviation " Standard Cng/lULl Deviation 855.9 0.322% 12032 4.67% 7728 2.96% 19716 10.2% Percent from Day 0 100% 97% 98% 73% Inhibited Sample E02-0913-035 E02-0913-036 E02-0913-065 :202-0913-066 E02-0913-125 E02-0913-126 E02-0913-185 E02-0913-186 Result Timepoint (ng/mL) Day 0 198883 DayO 227774 Day4 142445 Day4 175943 Day 14 152035 Day 14 154768 Day 28 101296 Day 28 * * SPE clogged, unable to Avemge Cng/lULl 213328 159194 153401 101296 'extract Standard Deviation (ng/mL) 20429 23687 1933 N/A Relative Standard Deviation 9.58% 14.9% 1.26% N/A Percent from Day 0 100% 75% 72% 47% .Page 60 of 142 ATTACHMENT C: SAMPLE CHROMATOGRAMS I I Page 61 of 142 f- 0 6 - l C)'!S5 ~&-i+ t 62.- Dq 15 revqe g ~~-~ J I "3 ATTACHMENT D: TEST SUBSTANCE INFORMATION Page 104 of 142 E~~~nMCH Proven Results. CERTIFI(~ATE OF ANALYSIS Exygen Research eOA Reference #: 3M Product: LotIBatch Number: 1 Test Control Reference #: Purity: 97.51<> Test Name Purity Appearance Identification NMR Metals (ICPfMS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LCIMS) Total % Impurity (GCIMS) 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 (IC) , Specifications Report value Yellow viscous oil 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.lwt.% 6. <0.001 wt./wt.% 7.. <0.001 wt./wt.% 2.4 wt./wt.% None Detected None Quantified <0.01 wt./wt.% None Detected 1. <0.013 wt./wt.% 2. <0.004 wt./wt.% 3. <0.035 wt.lwt.% 4. <0.008 wt.lwt.% 5. <0.005 wt./wt.% 6. <0.006 wt./wt% 7. 0.089 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt./wt.% 4. <0.2 wt.lwt.% COA023-026 ~05B Research Drive . State College, PA 16801 / USA T: ao8.~.1:ff92 F: 814.272.1019 exygen.com Page 105 of 142 E~g~~I"CH 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 2NMR: 3. 4Theoretical value calculations based on the empirical formula, )btmned from the NJ...fR analysis. This Work was conducted under EPA Good Laboratory Practice Standards (40 CPR 160) Prepared By: Scientist, E~yg~n Research Reviewed By: Laboratory Manager, Exygen Research COA023-026 =c,h.,lQk Date ~058 Research Drive , State College, PA 16801, USA T: so8.~ 3:N92 F: 814.272.1019 exygen.com Page 106 of 142 3IVIENVIRONMENTAL 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 -_.__ _-._ __ - ..; ..- -_._-_ -- .-._.. -- - _..-. ....- .-.-.---.-.-- ---..-.-....- ..- - - - -.--...--- .. "-'-'-'- '''''--- ---- .-.------.--.-..-..- - - - ..,... - - _ --..:1 Recorded By: I Date 06/24/02 Form ETS-4-l5.0 Exact Copy of OrIginal LAs o~b.yIDl initial Data Page 107 of 142 ATTACHMENT E: PROTOCOL, PROTOCOL AMENDMENTS AND DEVIATIONS Page 108 of 142 PROTOCOL Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester Data Requirement 40 CFR 792 '; I Performing Laboratory 3M Environmenta.l Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS E02-0913 Page 1 of 15 Page 109 of 142 Protocol E02-0913 E02-0913 Inherent Aerobic Aquatic Biodegrad;lbility of Fluoroaliphatic Polymeric Estf" 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. Pall}, 1YfN 55106 3M Enviromnental Laboratory 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 81. Paul, MN 55106 Proposed Study Timetable Experimental Start Date Experimental Termination Date October 24, 2002 December 31, 2002 Page 2 of 15 Page 110of142 Protocol E02-0913 1. INTRODUCTION Microbial populations in nature make up about one-half of the biomass on earth and are associated with the major biochemical cydes of elements and nutrients, besides the . decomposition of organic matter. Change::; in microbial populations or effects' on fiinctions ofthe microbial communities could result in inh~rference with their natural degradative functions that are essential to self-purification processes in the aquatic and terrestrial environments. IIi order to assess the environmental fate of a componnd, it is pertinent that its chemical properties, biological behavior and transport processes (sorption properties) be addressed. 2. PURPOSE The primary objective of this investigation is to identify the inherent aerobic aquatic biodegradation potential of the fluoropol:>mer 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-Wel1ens/EMPA Test (OPPTS 835.3200) and USEPA Modified SeAS (OPPTS 835.3210), The test substance, , is a complex mixture offluoroaliphatic 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 conduct(~d 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 C02. (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 o~ transform into fluorochemical breakdown products based on the biologieal degradation study of' - -- . ~ - j: The predicted breakdown products of' 'nat are going to be monitored are the reference substances as described in section 7. 3. REGULA TORY COMPLIANCE This study will be conducted in accordance the United States Environmental Protection Agency Good Laboratory Practice Regulations for Non.;.clinical Laboratory Studies, 40 CFR 792. 4. QUAL/TY ASSURANCE 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. TEST SUBSTANCE Table 1 Test Substances -; Test Substance IUPACName Chemical Fonnula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCRNurnber 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 :nm Hg, a specific gravity of 1.1, a measured pH range of ' and viscosity of' ,. Lot # was obtained from 3M Specialty Chemicals, Chemical characterization data was performl;d by: 3M Specialty Chemicals (boiling point, viscosity, specific gravity, pH, vapor pressure, flash point, results of GC analysis, results of gel-penneation chromatography, and results ofNMR analysis) and Centre Analytical (total amount of fluorine containing moieties present). The total amount of organic fluorine in 1 Nas 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 \-Vhen handling samples or solvems wear protective gloves, eyewear and clothing. The operator must be familiar with ins~rumentation 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 infonnation should be reviewed as appropriate. 6. CONTROL SUBSTANCES Table 2. Control Substa-n-ce-s--'""'1 Control Substances Formula SodiulD Lauryl Sulfate IUPAC Name Sodium Lauryl Sulfate Use Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Descript'ion Purity Surrogate Standard 3M Specialty Chemicals 8131/2006 Frozen White powder 86.9% Internal Standard for LC/MS analysis 3M Specialty Chemicals 10118/2006 Frozen White powder 98.6% Toxicity and Reference Control MaIlinckrodt 2/26/2007 Room Temperature 7718 V16603 TN-A-602J White powder 99% Page 5 of 15 Page 113 of 142 Protocol E02-0913 7. REFERENCE SUBSTANCES Table 3. Reference Sub ....- stances - - IUPACName Chemical Formula Identifier Source Expiration Date Storage Conditions Chemical Lot Number TCR Number Physical Description Purity Aldrich Chemical 5/1/2010 Frozen 3M Specialty Chemicals 12/4/2006 Frozen 3M Specialty Chemicals Not Provided Frozen White powder Not determined IUPACName Chemical Formula Identifier Source 3M Specialty Chemicals 3M Specialty Chemicals Expiration Date Not provided Not Provided Storage Conditions Frozen Ambient Chemical Lot Number TCRNumber Physical Description White crystals White crystals Purity 97.25 95.55 *CAS Number, **3M Identifier Code. The location ofl:he documentation of the methodes) of synthesis of the test, control, and reference items are the same as the source of the compe,und. Page 6 of 15 Page 114 of 142 Protocol EO2-0913 8. EXPERIMENTAL DESIGN 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. T bl 4 T t S t P ti SalOple Description #of Ileplicates Test/Control Substance aclded Ileference SubstllDces addeel l\leclium ,\eldecl t ..\n:alysis to be (ondueted Blank Controls 2 None No A Reference Substances Blank Controls 2 Inhibited None No B Reference Substances Abiotic Controls 2 None No C Reference Substances Test Substance 3 Test No A Reference Substances Test Substance Matrix 2 Test spike Yes A Reference Substances Test Substance 3 Test No Inhibited Test Substance 2 Test Yes Inhibited Matrix spike Abiotic Test Substance 3 Test f No ,I Abiotic Test Substance 2 Test Yes Matrix spike Toxicity Control 3 Test and SLS No B Reference Substances B Reference Substances C Reference Substances C Reference Substances A SLS Control Substance 2 SLS (SLS) 2 I- 2 I See defim.tl.Ons of medIUms under sectIon 8.1.2 No A SLS No C No B - Page 7 of 15 Page 115 of 142 Protocol E02-0913 8.1.1 Sludge Inoculum. Arrangements will be made to have Pace Analytical Services, Field Laboratory, Minneapolis, MN personn(~l obtain fresh mixed liquor suspended solids (MLSS) from the aeration units at the l'vfetro Wastewater Treatment Plant, St. Paul, lvfN. This sludge has been used in previous studies at 3M and Pace. Approximately six to eight liters ofMLSS 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 pl~rcentage of settled sludge per volume of container will be noted. Previous studies had values that were approximately 20% (e.g. 200 mL of 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 lrfineral Salts fr'fedium The mineral salts medium employed will be based on the USEPA ZahnWellenslEMPA Test (OPPTS 835.3200). Medium 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 (W[LSS) should be detennined for this medium. The final result should be between 0.2 giL and 1.0 giL. If the MLSS is outside this range, justification should be given in the final report. Medium B. A portion ofl\1edium A will be treated with 100 )lglmL of chloramphenicol as a microbial groWth inhibitor. This solution should be swirled regularly during dispensing in order to keep the mixture homogenous. Medium C. Mineral salt medium, prepared without the sludge inoculum, will be treated with 100 )lglmL 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 . Culture 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 infonnation. The vessels will be covered with a loose cap in order to reduce evaporation. Th(~ 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 Test Substance Stock Solution The concentration of the test substance should be approximately 36 mglL in each appropriate culture vessel (per table 4) as per the suggestion of the EPA guidelines of 20 mg CarboniL. The concentration was calculated by the theoretical value ca1culatic1ns 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 Control Substances - The concentration of the control substance, sodium lauryI"sulfate, should be approximately 40 mglL in each appropriate culture vessel (per table 4) as per the suggestion of the EPA guidelines of20 mg CarboniL. The biodegradation of this compound must reach at least 70 percent within 14 days. will also be added as a control substance to mediums Band 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 la'st step of the preparation process prior to analysis by LCIMS. It should be added at an approximate concentration of 250 ngimL. 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 Reference Substalzces Reference substances will be added ito laboratory control spikes and post extraction matrix spikes to determine recovery. These substances will be added at a nominal concentration of 500 ngimL. 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 Sample Collection The experiment will be set up for determination of biodegradation and/or biotransformation over a SlX 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 deterrnin,::d 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 PratGvuI c.;;.vL.-U::71J 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 enl 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 HPLClElectrospray MS as per ETS-8-155. The following table describes the STh1 ions to use. c== Table 5. SIM .;.;.lo_ns _ ~J-nd-"";IS;;.I;.M;.;.I;.O..:.N.=.S.;.S.;.;:IM;...-lo-n-(mJ-z)---1 9. METHODS FOR CONTROL OF BIAS 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 sample results are an indicator of precision and the internal standard will be used to measure instrument variability. 10. DA TA QUALITY 'OBJECTIVES 10. 1 Samples Sarpple 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 lowlhigh 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 run before and after every calibration curve, and after every CCV. Solvents blanks may be run 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 analyte carryover is a problem, use back-to-back solvent blanks and use the last solvent blank to evaluate carryover. 10.5 Laboratory Control Spikes I Two laboratory control spikes (LCS) 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 ofthe analytical run or necessity for repeat analysis. Consult with the Study Director for direction and final acceptance or rejection of the analytical run. 10.6 Matrix spikes. The analyst shall accept percent spike recoveries between 70% and 120% of theoretical value. Spike recoveries outside ofthis 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 directitJn 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%-120%) 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 detectioniquantitation 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. STA TIS TICAL ANAL YS/S Standard deviations will be calculated using either Microsoft Excel (Version 8.0e or newer) or Microsoft AccessCXl The built in function contains the following equation, which is based on the individual entities (n) being less than 30: Jnu' _(Lx)' n(n-l) Sample precision will be reported as % relative standard deviation (%RSD) for three or more replicates. Means will be calculated by adding the indi vidual entities and dividing the resultant sum by the number of individual entities. Page 12 of 15 Page 120 of 142 Protocol E02-0913 12. REPORT A report of the results of the study will be prepared by 3!vl Environmental Laboratory. The report will include, but not be limited to, the foHowing, when applicable: Name and address ofthe 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 testes). 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 transformations, 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 evaluaJ:e the data, if applicable. The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. The locatio;n 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 date:s 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. LOCATION OF RAw DATA, RECORDS, AND FINAL REPORT Original data or copies thereof, will be available at 3M Environmental Laboratory to facilitate audits of the study during its progress and. before acceptance of 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 f Standard Operating Procec.ures, Equipment Procedures, and tyfethods Appropriate specimens 14. DATA / SAMPLE RETENTION 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. PROTOCOL AMENDMENTS AND DEVIA TlONS 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. SIGNA TURES Protocol E02-0913 Date I Date Page 15 of 15 Page 123 of 142 Protocol E02-0913 Amendment #1 Study Title Inherent Aerobic Aquatic Biodegradability of FluoroaJiphatic Polymeric Ester PROTOCOL AMENDMENT NO. #1 Anlendment Date: December 13, 2002 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paull MN 55106 Laboratory Project Identification ET&SS E02-0913 Page 1 of 5 Page 124 of 142 Protocol E02-0913 Amendment #1 This amendment modifies the following portion(s) of the protocol: PROTOCOL READS: Section 8.1.2 "The mixed liquor suspended solids (MLSS) should be determined for this medium. The final result shou'ld be between 0.2 giL and 1.0 giL." AMEND TO READ: The mixed liquor suspended solids (MLSS) should be determined for this medium. The final result should be between 0.2 giL and 1.0 gIL. The procedure for this determination is given in attachment A. REASON: To add the procedure for the mixed liquor suspended solids determination. PROTOCOL READS: Section 10.2 "Calibration standards used to generate an external calibration curve should be prepare,d in medium A and extracted in the same manner as the samples". in AMEND TO READ: Calibration standards used to generate an external calibration curve should be prepared in mineral salts medium and extracted the same manner as the samples REASON: Standard curves should be prepared in mineral salts medium, not in medium A. PROTOCOL READS: Section 7. Refen:nce Substances are }PFBS, .- AMEND TO READ: --- - .. - IUPAC Name Chemical Formula I - Identifier Source 3M Specialty Chemicals Expiration Date Storaoe Conditions Chemical Lot Number 12/1/2010 --" Frozen TCR Number Phvsical Description Liqht yellow powder Purity Not Determined wiLl be used to quanttfy lOns that are related to the possible degradation product REASON: To add' s a reference substance. Page 2 of 5 Page 125 of 142 Protocol E02-0913 Amendment #1 PROTOCOL READS: Section 10.1 '''Sample precision should be ::::20% relative standard deviation." AMEND TO READ: Sample precision should be S20% 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 rej ection of the analytical run. REASON: To clarify original intent of how precisions >20% should be evaluated. PROTOCOL READS: Section 8.3 Table 5: SIM ion for AMEND TO READ: Section 8.3 Table:;: S1M ion REASON: After method development, the ion had a better signal than the Page 3 of 5 Page 126 of 142 Protocol E02-0913 Amendment #1 Attachment A Determination of Mixed Liquor Suspended Solids (MLSS)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 detennined 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 with Milli-Q water. Tum 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. Tum 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 MLSS (gIL) =: Final "'eight (fig) -- Initial weight (mg) Volume used (mL) 1 This is based on a modified procedure fTorr.. APHA, AWWA, WEF "Standard Methods for the Examination of Water and Wastewater", Section 25400 "Total Suspended Solids at 103-105C" 19th Edition. Page 4 of 5 Page 127 of 142 Amendment Approval Protocol E02-0913 Amendment #1 ~ntative Date I I Date Page 5 of 5 Page 128 of 142 Protocol cUZ-U~:J1j Amendment #2 Study Title Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester PROTOCOL AMENDMENT NO. #2 AmendmelJt 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 Protocol E02-0913 Amendment #2 This amendment modifies the following portion(s) of the protocol: PROTOCOL READS: Section 7. Reference Substances are ~ , PFBS, AMEND TO READ: .- - ~~PA~ Na'~:I1~ Chemical Formula Identifier Source Exoiration Date Storaae Conditions Chemical Lot Number TCR Number Physical Descriotion 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 Amendment #2 Uve Date J I Page 3 of 3 Page 131 of 142 Protocol E02-0913 Amendment #3 Study Title Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester ' PROTOCOL AMENDMENT NO. #3 Amendmen,t Date: February 27J 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 Protocol E02-0913 Amendment #3 This amendment modifies the following portion(s) of the protocol: PROTOCOL READS: Section 7. Reference C'"1-.."'''",..,,,,0<, aT'" ane AMEND TO READ: PFR~ Reference Substance IUPAC Name Chemical Formula Identifier Source 3M Specialty Chemicals Expiration Date 11/29/2005 Storage Conditions Frozen Chemical Lot Number TCR Number Physical Description Purity Off white powder 98.63% REASON: To add the additional' reference substance used for this study. Page 2 of 3 Page 133 of 142 Amendment Approval Protocol E02-0913 Amendment #3 ltative Date Date Page 3 of 3 Page 134 of 142 Protocol E02-0913 Amendment #4 Study Title Inherent Aerobic Aquatic Biodegradability of Fluoroaliphatic Polymeric Ester PROTOCOLAMENDMENTNO.~ Amendmen1 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 of 142 Protocol E02-0913 Amendment #4 This amendment modifies the following portion(s} of the protocol: PROTOCOL READS: 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 READ: "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 i 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 136of142 Amendment Approval ~rotoco/ E02-0913 Amendment #4 'p resentative Date Date J / Page 3 of 3 Page 137 of 142 3M Confidential Record of Deviation Study 1Project No. E02-0913 Deviation type (Check one) Document'number E02-0913 ProtocoI I. Identification SOP" X Protocol :Nfethod EquIpment ProcedUre" Other: -I5"ate(s) of"occurrence ,. -_._._.~-. - _.-. Sequence D021119 (Dudejr 11/19/02) II. Description Required procedure/process: 1. 'SectIon 10.3: "Acceptable CC\i'"vaiues an:-7(fTio% oftroeYilue." 2. Section 10.2: i'ifthe calibration curve residuals are greater than 20% deviation (LOQ 70%-120%)'from the theoretical value, quadratic curve fitting and/':Jr dropping lowlbighcurve points may be required if data review... " Actual procedure/process: '1.' For all analvtes' the'l 0 pg/f.LL ccV was not"wlthin the"-speafie'ci cnterl"iancl .. out of 3) the '50 pglf.LL standard was outside the' speCified criterla:- - 2~The lowest standard for was not Wlthin the specified criteria (60%). (2 out of3fand 11/. Actions Taken (such as amendment issued, SOP revision, etc.) 1. For all analytes a 10 pglf.LL CCY was run after a high level spike (500 pglf.LL). For PFBS ' the level offue rnatrTxsj:,lke-caus'e-d-iiistrument'ciiTYoverliito-the next mJecilori~wh1Ch'resultedin'a high CCV. Since the other CCYs passed (50 and 250 pg/llL) and samples repeated within 20% RSD,. it is suspected that carrYover was-not a probfen1"wlth these stUdysamples. For: .he 10 pgljl:CCCY was outside the""callbratloli" range used so it was discarded. Fo< most of the associated samples (with exception of the blank controls) were well above the 5(i"pg;f.LL tcY (closer to the p'assini 250 pg;~L ttV). were' However, the day foui me'dlum CsampIe~i'ranged from-86"pg7~Cto IOfpgif.LCfo-r,Sllcefuese samples had repeated' <20-0i0'RSD they' accepted." i"fllilalYtecarryover wasapro'blem:-i"fie samples'w'ouId neii- . have repeated within specifkation and would of shown- decreasmg'concentratTons~--For'al1analytes,ilie stUdy . haY-e control blanks were all below the LOQ. Givert anaclv'erse-affec"t""on tlie-data~---------------' all th_i._s_,._i_t...i.s_.n._ot-_su.s_p.e.c.t-e...d _th-_at...th__e._o-u-t.-_o.f_s.-p-e"-c-ification CCYs will ..... '2. The 2Sj3 pg/f.LL standardwask:epi morder tomamtam a 5pomt calibratIon 'curve:AIso, all of"ihe study samples-(with exception oftheniatrlx spikes:, were iiIiwelTbelowtfie 25.23 pglf.LL standard: Glventhls, ii"was Recorded f'-y'" -- ._ _.- . __._ . "Date' deemed more accurate to state that the sampll~s were below 25.23 pglllL instead of 50.45 pglf.LL. ~ _.~.~._._._._.~. ""~ _,",u_. ,.,., : Deviation No. Dat; J //~2b? \ I (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) . '''Documeiiin-umber E02-0913 Protocol I. Identification 'sop X Protocol Method .. EquIpment Procedure .....~- Other: _.- - _. . Date-(s) Of. occ"lirreiice-----~,----- ~.,.. . : Sequence 0021119 (Oudejr 11/19/02) II. Description 'have a Required procedure/process: section [0.16: "The'-area countS 'shouia relative standarc(deviatlonof <5%.~:;' Actual procedure/process: ,: 12%)"was'outslde'the'speCiflca.tlOli".. III. Actions Taken (such as amendment issued, SOP revision, etc.) Since all of the samples were either diluted and reinjected at a later date (required reanalysis) or less than the 'ILOQlfw'as decided to accept the restiit:s's'mce'the repeatabmiY'really <foesli'Tmatterbeiow'theLLOQ and . any of the samples that were detected were reanalyzed (rquired reanalysis due to be"tiig 'above the upper limit of . quantitation). . _, . ,_.-_.,. __.-"._- ,., ,' , . ............... _... .... ,.- ..... , : Date ot/9..0/(/~ Deviation No. Date . J /1 z/o"( J.. / ,- ,ed by Study Director or Project Lead at the end of study or project) Page 139 of 142 3M Confidential Record of Deviation Study 1Project No. E02-0913 Deviation type (Check one) . Docum'e'[it'iiumber E02-0913 Protocol I. Identification '861:> X Protocol 'Method Equipment.Procedure Other: Date(s) ofoccurrenc-e - - . Sequence I021231 (Itchy 12/31/02) II. Description value:';' Required procedure/process: Section Ib.3"AcceptafJie ccv values"are16l20%"oftrUe .- Actual procedure/process: The25.00I;gj~L cCV for was not within-the 70-120% critena (1 23 <fl;, l27%,'-iii"a 128%). III. Actions Taken (such as amendment issued, SOP revision, etc.) For this study is used as a surrogate to approximate another compound. In this analysis, the 25.00 Pg/llL ccv dIef rIOt paSs-the' cnteria as specifled"b"yfue'protocofAfl'of the samples.that have'values"to 'berep'o'rt"e'd 'were either below the lower limit of quantitation or closer to th,6 250 pgtllL CCV which passed (CCV range 111 %- "'rf70/0, samplercingi1'S9.81 - 344.fi pgJ~LY." . Since the L[OQ"for'this anaiysis was 25~O(fpg/J.1Lan(fthe"CCV waShlgb;'thehigh recovery would have shown if .. zmysampleswere at or neai25:6opi7~LGl,;enthis and that' arT of the rest ofthesampI"ehltSweiedoserto . the 250 Pg/llL CCV, the results-were accepted. No affect on the-data is expected. .. : Date -I Date } . tJl /J ?/D? Deviation No. '3 I / )irector or Project Lead at the end of study or project) Page 140 of 142 3M Confidential Record of Deviation Study / Project No. E02-0913 I. Identification Deviation type (Check one) Document number ETS-S':39.0 SOP Protocol -.-- . ----.~-- .. --.-. " X Method Other: ......-..... "Oate(s) of occurrence III 18/02, 11119/02, 11/22/02, 11/25/02, 11126/02, and 12/19/02 II. Description Required procedure/process: Section fO.l' ofthe"mcthodstates "MiX'content' thoro'ughIy"anclce'ntrlfugeuntlrconten""ts"i;ecome dear';': _, '"M'M ~ __"'_'"''''P''''' .,.... , ~ _ _ , _ __ . _ _ .. , ._.. .._.,. Actual procedure/process: 's'arnpies were' not centrifuged. Instead, a glass wool plug'was 'adcle'clio'the'SPE apparatus and was'useei' to catch any particulates th'iJ:t' maybe' suspended in -the 'solution used to-elute the co'mpoUnds of interesf""'-" No impact on the study. ' . HI. Actions Taken (such as amendment issued, SOP revision, etc.) Recorded bv - - Date evia tion No. --:-_~i:----:--_-:- :ector or Project Lead at the end of study or project) Page 141 of 142 3M Confidential Record of Deviation Study I Project No. E02-0913 Deviation type .-. (Check one) 'D;-;~~;t n~b~~"'" E02-0913 Protocol J. Identification SOP Method X Protocol Other: Equipment Procedure II. . . _J3:.~3~i~~~ P.E9_<:~ure~p'ro~es~~ : __~ '_' '__'_''''''_'__''_''_''_'''''''__'''_''_'' . Section 10.10: "The area counts should have a relative standard deviation of <5% ... " . Ac_t':l_~Lp~oced~~p..Ioces~~_._._ ._ _ _ __ . ~ 5~~L'i'I~.~ ~pt.~!.~e. .th.~ ~E~~.~fi.~~_ti?_n..:._ . _ __ ""'1 JlI. Actions Taken such as amendment issued, SOP revision, etc.. . . . .__. .. - - --- . After evaluating the data, since the calibration curves passed, the CCVs passed and the accepted sample replicates .~~~~~.~ ~:~~':t.~~~~ij~ ~~ :~:~~~~i~~~~~t.~?~~t2b~~~ ~~e~~~~.-\~~~ ~~~R~i..~.:: ~ , ._.... Recorded by A : Date .. - .....i dation No. __-=5 ~- ;:.:tor or Project Lead at the end of study or project) Page 142 of 142