Document Rjj1ZboJMEMonjy9M3abGa27X

Report E01-1256 PFOS: A Reproduction Study with the Mallard Study Title Analytical Phase R eport for PFOS: A Repro ductio n Stu d y w ith the Mallard Data Requirement FIFRA Subdivision E, Section 71-4 OECD Guideline 206 Author Lisa A. Stevenson Analytical Phase Initiation Date February 19, 2002 Analytical Phase Completion Date At Signing Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Environmental Laboratory Study # E01-1256 Wildlife International Ltd: 454-109 Total Number of Pages 216 3M Environmental Laboratory Page 1 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard GLP C o m p l ia n c e S ta te m e n t Study Title: Analytical Phase Report for PFOS: A Reproduction Study with the Mallard Study Identification Number: E01-1256 This analytical phase was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards with the exceptions listed below: Exceptions to GLP compliance: Automated data collection systems are not validated. The hardcopy printouts are considered as the original raw data. The stability of the samples has not been determined. ' Not all data generated were recorded directly or promptly, or initiated and dated on date of entry. Date Fm/irrknmntol I ohr\ra*r\rNr 3M Environmental Laboratory Page 2 C0 1 5 .NS Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Q uality A ssurance Statement Study Title: Analytical Phase Report for PFOS: A Reproduction Study with the Mallard Study Identification Number: E01-1256 The analytical phase 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 02/22/02 04/03/02-04/08/02, 04/17/02 05/13/02-05/15/02 Phase In-phase Data Final Report Date Reported to Management S t u d y D ir e c to r 02/22/02 02/22/02 04/08/02, 05/21/02 04/08/02,05/21/02 05/16/02 05/16/02 L /C ^ Quality Assurance Representative 3M Environmental Laboratory 3M Environmental Laboratory Paae 3 of 216 Page 3 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table of C o n ten ts GLP Compliance Statement...................................................................................................................... 2 Quality Assurance Statement...................................................................................................................3 List of Tables...............................................................................................................................................5 Summary..................................................................................................................................................... 7 Purpose....................................................................................................................................................... 8 Reference Substances.............................................................................................................................. 8 Specimen Receipt and Storage..................................................................................... i........................ 9 Method Summary......................................................................................................................................9 Preparatory Method...................................................................................................................................9 Sample Receipt, Preparation and Analysis.............................................................................................. 10 Analytical Method....................................................................................................................................... 11 Analytical Results................................................................................................................................. .....12 Data Summary............................................................................................................................................14 Statement of Conclusion........................................................................................................................... 21 Statistical Methods.....................................................................................................................................23 Statement of Data Quality......................................................................................................................... 23 References................................................................................................................................................. 24 List of Attachments..................................................................................................................................... 24 Signature P a g e .......................................................................................................................................... 25 Attachment A: Extraction and Analytical Method, ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" ...................................................... 26 Attachment B: Data Summary Tables...................................................................................................... 46 Attachment C: Sample Chromatograms, Calibration Information, and Instrument Information....... 55 Attachment D: Sample Preparation Sheets.............................................................................................144 Attachment E: Study Protocol, Protocol Amendments, and Deviations............................................... 178 Attachment F: Example Calculations....................................................................................................... 216 3M Fnvirnnmpntal I ahnratnrv 3M Environmental Laboratory Paae 4 of 216 Ci r- Page 4 r- Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard List of Tables Table 1. Summary Table, C8F17S 0 3' Concentration in Sera and Liver................................................7 Table 2. Summary Table, C8F17S 0 3' Concentration In Sera and Liver with Outliers Removed...... 8 Table 3. Reference Substances............................................................................................................... 8 Table 4. SPE Extraction Summary..........................................................................................................9 Table 5. Sample Analysis..........................................................................................................................10 Table 6. Serum QC Summary Table........................................................................................................ 13 Table 7. Serum Matrix Spike Summary Table..............i........................................................................13 Table 8 . Liver QC Summary T able.........................................................................................................13 Table 9. Liver Matrix Spike Summary Table.......................................................................................... 13 Table 10. C8F17S03' Data Summary, Female Mallard Sera (Adult and Offspring)..............................14 Table 11. C8F17S03' Data Summary, Male Mallard Sera (Adult and Offspring).................................. 16 Table 12. C8F17S 0 3"Data Summary, Female Mallard Liver (Adult and Offspring).............................. 18 Table 13. C8F17S03' Data Summary, Male Mallard Liver (Adult and Offspring)..................................20 Table 14. Summary Table, C8F17S 0 3' Concentration in Sera and Liver...............................................22 Table 15. Summary Table, C8F17S 0 3' Concentration in Sera and Liver with Outliers Removed..... 23 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 216 Page 5 001589 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Study Information Study Director Sean P. Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 Sponsor 3M Environmental Technology and Safety Services 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55106 Susan A. Beach, Sponsor Representative Analytical Chemistry Laboratory 3M Environmental Technology and Safety Services (ET&SS) 3M Environmental Laboratory (3M Lab) Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Lisa Stevenson, Principal Analytical Investigator Study Personnel Sean P. Gallagher, Wildlife International LTD., Study Director Joann B. Beavers, Wildlife International LTD., Laboratory Management William K. Reagen, 3M Environmental Laboratory Management Susan A. Beach, Sponsor Representative Harold O. Johnson, III, Original Principal Analytical Investigator Lisa A. Stevenson, Principal Analytical Investigator Kent R. Lindstrom, Senior Research Chemist Ognjenka Krupljanin, Analytical Chemist Marlene Heying, Analytical Chemist Experimental Dates Sample Analysis Initiation: 19 February 2002 Sample Analysis Completion: 11 April 2002 Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory according to 40 CFR Part 792. The analytical reference standard reserve samples, as well as the samples pertaining to the analytical phase of this study, are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. PmflVnnmAntal i ahnratnrv 3M Environmental Laboratory Paae 6 of 216 Page 6 Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Summary The concentration of the C8F17S 0 3' anion of periluorooctane sulfonate, potassium salt (PFOS, C8F17S03"K+) was determined in the liver and sera of adult mallards and their offspring in both a control group and a 10ppm dosed group from Wildlife feeding study #454-109. The following tables summarize the C8F 17S 0 3` concentrations found in the liver and sera of study samples. Extracted-matrix PFOS standards are used to establish the HPLC-ES/MS/MS instrument calibration curve for C8F17S 0 3`. Concentrations of C8F17S 0 3' are reported after correction for the purity and the potassium contribution to the mass of the PFOS reference substance. Table 1 summarizes all study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q-Test. Table 1. Summary Table, C8F17S 0 3' Concentration in Sera and Liver S ampling Event Male Control Group N umber of S era Samples A verage C a F i/S O ] Conc. S era Range of Results !i 1 <100 ng/mL (14 of 18) 18 NA 107-1400 ng/mL range (4 of 18) N umber of L iver Samples A verage CeFuSCh Cono. L iver ------------------------- n 20 <50 ng/g Range of Results NA Female Control Group Male 10ppm Group < 100ng/mL (15of20) NA 20 166-2680 ng/mL range (5 of 20) 20 20 89700 ng/mL 11300-210000 ng/mL 20 <50ng/g NA 60900 ng/g 24200-125000 ng/g Female 10ppm Group 20 20300 ng/mL 6350-91400 ng/mL 20 10800 ng/g 2480-30700 ng/g Male Offspring Control Group Female Offspring Control Group 3 7 NA <100 ng/mL (2 of 3) 103 ng/mL (1 of 3) NA <100 ng/mL (7 of 7) i 3 <50 ng/g 7 <50 ng/g NA NA Male Offspring 10ppm Group 3 4410 ng/mL 3670 - 5450 ng/mL 3 3170 ng/g 22004640 ng/g Female Offspring 10ppm Group 7 4960 ng/mL 3420-6760 ng/mL 7 3620 ng/g 2240-5930 ng/g For individual sample values see Tables 10 to 13 and Attachment B. ok4 I 3M Environmental Laboratory 7 a#04C Page 7 m rv ; v Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 2. Summary Table, C8F17SO3' Concentration in Sera and Liver with Outliers Removed S ampling E vent N umber of Sera Samples A verage C sF u SO:,' C onc. S era Male Control Group 18* Range of Results * N umber of L iver Samples 20* A verage C uF u S O j C onc. Liver * Range of Results * Female Control Group 17 NA <100 ng/mL (15 of 17) 166-172 ng/mL range (2 of 17) Male 10ppm Group 18 87300 ng/mL 68700-108000 ng/mL 20* 20* * * * * Female 10ppm Group 19 16600 ng/mL 6350-42800 ng/mL 20* * * Male Offspring Control Group Female Offspring Control Group 3* 7* ! * * * 3* * * 7* Male Offspring 10ppm Group Female Offspring 10ppm Group ! 3* 7* * * 3* * * 7* * * * No outliers removed from this data set. Refer to Table 1 for results. For individual sample values see Tables 10 to 13 and Attachment B. P urpose The purpose of the analytical phase of this study is to quantify CaF^SOa' in the liver and sera of both control and dosed mallards and in their offspring. Reference Substances Table 3. Reference Substances Reference Substance Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Purity PFOS 3M Internal 08/31/2006 Frozen-20C+ /- 10C SD-018, Lot #217 White Crystalline Powder 86.9% PFDA Oakwood Products, Inc. 12/01/2010 Frozen -20"C +/- 10C SD-036 White Solid 98% PFDA is nonadecafluorodecanoic acid, or Perfluorodecanoic add, C i0F10O 2H and is used solely to monitor for gross instrument failure and not as an internal standard for quantitation. 3M Environmental Laboratory Page 8 ( W l r -; < v > Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S pecimen R eceipt and S torage Mallard serum and liver samples were obtained from the sacrifice of the test animals upon the conclusion of the in-life phase of the study (#454-109) conducted at Wildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. The specimens were received at 3M Environmental Laboratory, assigned a laboratory tracking (LIMS) number (E01-1256, E01-1326, or E01-1367), and were stored at -20C +/- 10C until prepared for analysis. M ethod Summary The determination of C8Fi7S 0 3' concentration in the liver and sera was performed according to 3M Environmental Laboratory method ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices". The sera samples were prepared by measuring a volume of serum and diluting with water in a 1/20 ratio. The liver samples were prepared by measuring a weight of liver and diluting with water to a 1/10 ratio prior to homogenizing. Five mL of acetonitrile was added to 1.0mL of the diluted sera or 1.0mL of the homogenate of liver and was shaken for 20 minutes. These samples were then centrifuged for 10 minutes; the supernatant was decanted into 40 mL of water and passed through a conditioned solid phase extraction (SPE) column. After drying the column, 2.0 mL of methanol was passed through the column to elute the analyte. The samples were analyzed for CgF17S 0 3' using HPLC-ES/MS/MS. Preparatory M ethod ETS-8-231.1 "Solid Phase Extraction and Analysis of Fluorochemical Components from Biological matrices." This method describes the extraction of target analytes from biological matrices, such as sera and liver, using solid phase extraction (SPE). Sera samples were diluted with Kandiyohi (commercially bottled) water 1/20, before extraction. Liver samples were diluted 1/10, approximately 1g of liver and 9ml of Kandiyohi water, and then homogenized. A 1.0mL aliquot of the diluted sera or homogenate of liver was extracted by adding 5.0mL of acetonitrile, shaking for 20 minutes, and centrifuging for 10 minutes. The supernatant was transferred to a clean tube, diluted with 40 ml of Kandiyohi water, and filtered through a conditioned C18 (SPE) column. Analytes were eluted off the column, and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). Table 4. SPE Extraction Summary S tep Sample Preparation 1 Measure volume of sera or weigh portion of liver 2 Dilute sera (1/20) and liver (1/10) with water 3 Mix sera/water or homogenize liver/water 4 Aliquot 1.0ml of diluted/homogenized sample and add 5.0ml ofACN. Spike samples j accordingly. Shake for 20 minutes, centrifuge for 10 minutes. 5 Dilute supernatant with 40 ml of water 6 Filtersample through conditioned SPE C18 column. 7 Elute target analytes fromthe column with 2.0mL methanol (MeOH). 3M Environmental Laboratorv 3M Environmental Laboratory Paae 9 of 216 0015 Report E01-1256 Report EQ1-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Sam ple R eceipt, P reparation and A nalysis All sera and liver samples were received frozen and remained frozen until thawed prior to preparing samples for analysis. After dilution or homogenization steps, unused samples and diluted sera or homogenized liver were frozen again if extractions were not immediately performed. On each day of extraction, a water blank, a sera or liver blank, and a matrix spike and matrix spike duplicate were prepared with each set of samples. Frozen diluted sera/homogenized liver samples were allowed to thaw prior to transferring a representative sample for extraction. Samples were prepared and analyzed according to ETS-8-231.1. The following table describes the sample collection and preparation regimen. For clarity, a reference to "Day 1" refers only to the group of samples associated with the first batch of samples extracted. Table 5. Sample Analysis Step Procedure D ate P erformed 1 Aliquoting and preparation of the sera sample homogenates (all groups and dose levels) 19 February 2002 2 Weighing of liver samples (control group and dosed group offspring) 21 February 2002 3 Weighing of liver samples (control group and dosed group adults) 4 Homogenizing of liver samples (control group and dosed group offspring) 22 February 2002 22 February 2002 5 Homogenizing of liver samples (control group and portion of the dosed group adults) 25 February 2002 6 Homogenizing of liver samples (remainder of dosed group adults) 26 February 2002 7 Preparation of method blanks and spikes, calibration curve, I 25 February 2002 qc samples, and sera samples, control group, adults (Day 1 sera samples) 8 Analysis of Day 1 sera samples 25 February 2002 9 Preparation of method blanks and spikes, and sera samples, control and 10 ppmadults (Day 2 sera samples) 10 Analysis of Day 2 sera samples 11 Preparation of method blanks and spikes, and sera samples, 10 ppm adults (Day 3 sera samples) 12 Analysis of Day 3 sera samples 13 Preparation of method blanks and spikes, and sera samples, control group, and 10 ppmoffspring (Day 4 sera samples) 14 Analysis of Day 3 and Day 4 sera samples 15 Analysis of diluted sera samples 16 Preparation of method blanks and spikes, calibration curve, qc samples, and liver samples, control group, adults (Day 1liver samples) 17 Analysis of Day 1 liver samples 26 February 2002 26 February 2002 27 February 2002 27 February 2002 28 February 2002 28 February 2002 01 March 2002 04 March 2002 04 March 2002 18 Re-analysis of Day 1 liver samples 19 Preparation of method blanks and spikes, and liver samples, 10ppm dosed group, adults (Day 2 liver samples) 05 March 2002 05 March 2002 3M Environmental Laboratory 3M Environmental Laboratory Page 10 of 216 Page 10 e> 0 * r .JL 3 Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Step 20 Procedure Analysis of Day 2 liver samples 21 Preparation of method blanks and spikes, and liver samples, control group and 10ppm dosed group, adults (Day 3 liver samples) 22 Analysis of Day 3 liver samples 23 Preparation of method Hanks and spikes, and liver samples, control group and 10ppm dosedgroup, offspring (Day 4 liver samples) 24 Analysisof Day 4 liver samples 25 Analysis of female sera dilutions 26 Analysis of QC liver and sera samples 27 Analysis of diluted sera samples D ate Performed 06 March 2002 06 March 2002 06 March 2002 07 March 2002 07 March 2002 08 March12002 05 April, 2002 11 April, 2002 A nalytical M ethod Samples were analyzed using HPLC-ES/MS/MS. C8F17S 0 3' concentrations were determined using matrix-extracted calibration standards ranging in concentration from 0.25-1000 ng C8F17S 0 37mL methanol. This result was then used to back calculate the ng C8F17S 0 37mL sera or ng C8F17S 0 37gram liver as appropriate. An internal standard, Nonadecafluorodecanoic acid (PFDA), was used to monitor instrument performance for gross malfunction, but was not used to quantitate results. HPLC-EC/MS/MS analytical conditions are summarized as follows: The HPLC used for the study was a Hewlett-Packard 1100. The system consists of a vacuum degasser, binary pump, autosampler and temperature controlled column compartment. The run time is 10 minutes per sample. HPLC conditions include the use of a Keystone Scientific, Betasil C18 HPLC analytical column (2.1mm x 50mm, 5pm particle size), a 40C column compartment temperature, a 10pL injection volume and a 0.300pL/min pump flow rate. The HPLC gradient is listed below: T ime, M inutes 0 1.0 5.5 7.5 8 10 I % 2 m M Ammonium A cetate: 90 90 5 5 90 90 % M ethanol 10 10 95 95 10 10 The mass spectrometer used for the study was a Micromass Ultima. The system consists of Z-Spray interface equipped with an electrospray probe operated in negative mode. The instrument was setup to operate in MS/MS mode, passing the C8F17S 0 3" molecular anion (m/z = 499) into the collision cell, where it is collided with inert gas to further fragment to the characteristic ion m/z = 99, F S 0 3\ Similarly the PFDA transition TM Fnvironmftntal I ahoratorv 3M Environmental Laboratory Paae 11 of 216 Page 11 : V ` . .jL .3 > Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard from m/z = 513 to m/z = 299 was monitored. The collision gas, 95% Argon / 5% Methane, was maintained at a pressure of approximately 3.0x1 O'3 mBar. A nalytical R esults Data quality objectives outlined in the 3M Environmental Laboratory method and protocol were met, except as noted and discussed below (see Attachment A, ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" and Attachment E, Study Protocol, Protocol Amendments, and Deviations). Regressions. Quadratic curve fit, weighted 1/X, was applied to calibration standards and sample data. Each calibration curve had a coefficient of determination of > 0.985. Calibration Standards. Instrument response was calibrated daily prior to sample analysis using matrix-extracted standards ranging in concentration in the final solvent extract from 0.20 to 1000 ng C8F17S 0 37mL methanol, corresponding to a concentration of 8.00 to 40160 ng/mL of sera or 4.00 to 20080 ng/g liver. The calibration curve analyzed consisted of fourteen matrix-extracted standards bracketing a wide concentration of analyte. The accuracy of each level was evaluated. Any level outside 75%-125% of nominal was deactivated with the exception of the lowest curve point, which needed to meet 70%-130% of nominal. In some cases the lowest concentration curve point, although outside the 30% nominal requirement, was included when constructing the calibration curve, but it was not used to determine the limit of quantitation. The limit of quantitation was the next point on the curve meeting the 30% nominal requirement. Due to the quadratic response of matrix extracted standards using LC/MS/MS instrumentation, the calculated concentration of higherlevel standards was often indeterminant, that is a single result could not be calculated using the quadratic equation. The accepted upper concentration range for study samples was determined by the highest calibration standard meeting the 75%-125% accuracy criteria when back calculated to the original concentration spiked into the extracted matrix standards. All analytical results that are reported are within the accepted calibration range of the instrument. System Suitability Samples. System suitability was demonstrated prior to the start and at the end of each analytical run. Prior to the calibration curve and after the last sample, three mid-level unextracted calibration standards were analyzed. The peak area precision and retention time precision was monitored at the beginning and end of the run separately. The peak area precision was equal to or less than 5.0% RSD and the precision of the retention time was equal to or less than 2.5% RSD, meeting the stated data quality objectives to show that the instrumentation was providing suitable results throughout each analytical sequence. Continuing Calibration Verification (CCV). A mid-level matrix calibration check was analyzed every ten samples or less to monitor instrumental drift, with a limit of 25% deviation of the target concentrations. Only data bracketed by passing CCV's were reported. Samples bracketed by one or more failing CCV's were reanalyzed. Limit of Quantitation (LOQ). The lower limit of quantitation (LLOQ) was defined in liver (ng/g) and in serum (ng/mL) as the lowest acceptable extracted calibration curve point that is within the 70-130% nominal criteria and with an analyte peak area at least 2 times the blank. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest acceptable curve point used to calibrate the response of the instrument. Blanks. Method blanks (purchased Kandiyohi drinking water, sera, and/or liver taken through the entire sample preparation and analysis process) provided a measure of background contamination. Quality Control (QC) and Matrix Spikes. One set of nine QC samples in each matrix was extracted and analyzed and consisted of three levels of analyte in triplicate. Additionally, two matrix spike samples at the lowest concentration were prepared with each extraction set. Results are as follows: 3M Environmental Laboratory 3M Environmental Laboratory Pane 1? of 91 fi Page 12 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 6. Serum QC Summary Table S erum Q C S am ples QC-25ppb QC-250ppb C o n c e n tra tio n in D iluted S erum 1004 ng/mL 10040 ng/mL Mean recovery of tr ip lic a te s 145%* 97.9% R elative S tandard D eviation 9.8% 2.9% QC-4.0ppm 160600 ng/mL 84.7% 6.7% ' These samples were spiked using a 1.0 ul aliquot of spiking solution. Matrix spikes prepared on the same day using 50 ul of a diluted stock meet all QC requirements. Spiking technique of injecting 1.0 ul is suspect Table 7. Serum Matrix Spike Summary Table S erum M atrix S pike Sam ples* C o n c e n tra tio n in D ilu te d S e ru m A verage R ecoveries Four Matrix Spikes 1004 ng/mL 114% Four Matrix Spikes Duplicates 1004 ng/mL 112% ' Some matrix spike samples were injected in a second analytical batch. Both results are included in the average recoveries. Table 8. Liver QC Summary Table Liver Q C S am ples C o n c e n tra tio n in D iluted Liver M ean recovery of trip lic a te s R elative S tandard D eviation QC-25ppb 497 ng/g 109%* 13% QC-250ppb 4967 ng/g 94.3% 8.3% QC-4.0ppm 79470 ng/g 103% 0.72% 'These samples were spiked using a 1.0 ul aliquot of spiking solution. Matrix spikes prepared on the same day using 50 ul of a diluted stock meet all QC requirements. Spiking technique of injecting 1.0 ul is suspect Table 9. Liver Matrix Spike Summary Table Liver M atrix S pike S am ples* C o n c e n tra tio n in D ilu te d L iv e r A verage R ecoveries Four Matrix Spikes 497 ng/g 111% Four Matrix Spikes Duplicates 497 ng/g 107% ' Some matrix spike samples were injected in a second analytical batch. Both results are included in the average recoveries. The protocol states that the acceptance range is 75-125% but makes allowances for a percentage of results to be outliers. The observed number of outliers for the "QC-25ppb" spikes exceeds that permitted in the protocol but the overall impact on data quality is minimal or none at all. All study samples that were not "Day 0", show analyte levels significantly above the LOQ. Occasionally, Day 0 samples or other types of blanks show positive detections at levels near the LOQ. 3M Fnvironmental Laboratory 3M Environmental Laboratory Paae 13 of 216 Page 13 GOIS97 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Absolute Recoveries. The absolute recovery of the QC samples is determined by comparing the results of the extracted quality control spikes quantitated versus an extracted matrix curve and a solvent curve. The sera spikes quantitate higher using a solvent curve than when using a matrix-extracted curve. The low spikes are 11.1% higher, the medium spikes 8.3% higher, and the high spikes 8.3% higher. The 9.5% average higher quantitation of the three levels of spikes using a solvent curve versus an extracted matrix curve demonstrates that it is appropriate to use matrix-extracted standards. The same trend is observed for liver samples. The extracted liver matrix curves are 12.2% higher, 7.8% higher, and 5.6% higher respectively for the low, medium and high spikes of the liver matrix, again demonstrating that it is appropriate to use matrix-extracted standards. Internal Standard: The internal standard (PFDA) was added to all samples and standards. PFDA was not used for quantitation, but was used to monitor for gross instrument failure. Instrument Response Calibration. HPLC-ES/MS/MS instrument response'was calibrated using the concentration of the C8F17S 0 3 anion of perfluorooctane sulfonate, potassium salt (PFOS) that was adjusted for purity and the potassium ion contribution to the mass of PFOS used to prepare stock solutions. Any future calculations requiring concentrations to be expressed as a percentage of dietary in-take must adjust the dosed amounts as well. Data S ummary Tables 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The table displays C8F17S 0 3' concentrations for individual injections. All data are corrected for purity of the PFOS reference substance used for the extracted-matrix calibration curve. Table 10. C8F17S0 3' Data Summary, Female Mallard Sera (Adult and Offspring) 3M Sample N umber SAM PLE D ESC RIPTIO N E01-1256-28663 E01-1256-28665 E01-1256-28667 E01-1256-28669 E01-1256-28670 E01-1256-28672 E01-1256-28674 E01-1256-28676 E01-1256-28678 E01-1256-28679 E01-1256-28681 E01-1256-28683 E01-1256-28685 E01-1256-28687 E01-1256-28689 E01-1256-28691 E01-1256-28693 E01-1256-28695 454-109-3218,0 PPM, Female, Adult 454-109-3220,0 PPM, Female, Adult 454-109-3222,0 PPM, Female, Adult 454-109-3224,0 PPM, Female, Adult 454-109-3226,0 PPM, Female, Adult 454-109-3228,0 PPM, Female, Adult 454-109-3230,0 PPM, Female, Adult 454-109-3232,0 PPM, Female, Adult 454-109-3234,0 PPM, Female, Adult 454-109-3236, 0 PPM, Female, Adult 454-109-3238,0 PPM, Female, Adult 454-109-3240,0 PPM, Female, Adult 454-109-3242,0 PPM, Female, Adult 454-109-3244,0 PPM, Female, Adult 454-109-3246,0 PPM, Female, Adult 454-109-3248,0 PPM, Female. Adult 454-109-3250,0 PPM, Female, Adult 454-109-3252,0 PPM, Female, Adult AMOUNT OF ADDITIONAL SERA USED (ML) DILUTION CONG. OF C8Fi 7SOj IN S a m p i f (NG/m L of Sera) 0.5 1 <100 0.5 1 0.5 1 0.5 1 0.5 1 <100 <100 <100 <100 0.5 1 2680 0.5 1 0.5 1 0.5 1 0-5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 <100 172 <100 <100 166 <100 <100 <100 <100 0.5 1 0.5 1 398* <100 0.5 1 | 757* 3M Environmental Laboratory 3M Environmental Laboratory Paae 14 of 216 Page 14 00155*8 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S a m p le N umber SAM PLE DESCRIPTIO N E01-1256-28697 E01-1256-28699 E01-1256-28701 E01-1256-28703 E01-1256-28705 E01-1256-28707 E01-1256-28709 E01-1256-28711 E01-1256-28713 E01-1256-28715 E01-1256-28717 E01-1256-28719 E01-1256-28721 E01-1256-28723 E01-1256-28725 E01-1256-28727 E01-1256-28729 E01-1256-28731 E01-1256-28733 E01-1256-28735 E01-1256-28737 E01-1256-28739 E01-1256-28740 EOT1256-28742 E01-1256-28743 E01-1256-28744 E01-1256-28745 E01-1256-28747 E01-1256-28748 E01-1256-28751 E01-1256-28752 E01-1256-28753 E01-1256-28754 E01-1256-28755 E01-1256-28758 EOT1256-28759 454-109-3254,0 PPM, Female, Adult 454-109-3256,0 PPM, Female, Adult 454-109-3258,10 PPM, Female, Adult 454-109-3260,10 PPM, Female, Adult 454-109-3262,10 PPM, Female, Adult 454-109-3264,10 PPM, Female, Adult 454-109-3266,10 PPM, Female, Adult 454-109-3268,10 PPM, Female, Adult , 454-109-3270,10 PPM, Female, Adult 454-109-3272,10 PPM, Female, Adult 454-109-3274,10 PPM, Female, Adult 454-109-3276,10 PPM, Female, Adult 454-109-3278,10 PPM, Female, Adult 454-109-3280,10 PPM, Female, Adult 454-109-3282,10 PPM, Female, Adult 454-109-3284,10 PPM, Female, Adult 454-109-3286,10 PPM, Female, Adult 454-109-3288,10 PPM, Female, Adult 454-109-3290,10 PPM, Female, Adult 454-109-3292,10 PPM, Female, Adult 454-109-3294,10 PPM, Female, Adult 454-109-3296,10 PPM, Female, Adult 454-109-9583,0 PPM, Female, Offspring 454-109-9589,0 PPM, Female, Offspring 454-109-9592,0 PPM, Female, Offspring 454-1 0 9 -9 5 9 8 , 0 PPM , Female, Offspring 454-109-9603,0 PPM, Female, Offspring 454-109-9610,0 PPM, Female, Offspring 454-109-9573,0 PPM, Female, Offspring 454-109-9629,10 PPM, Female, Offspring 454-109-9633,10 PPM, Female, Offspring 454-109-9634,10 PPM, Female, Offspring 454-109-9645,10 PPM, Female, Offspring 454-109-9648,10 PPM, Female, Offspring 454-109-9654,10 PPM, Female, Offspring 454-109-9659,10 PPM, Female, Offspring AMOUNT OF SFRA USED ADDITIONAL DILUTION (ML) CONG. OF cf17s o 3` in Sam ple (n g /m L of Sera) 0.5 1 0.5 1 <100 <100 0.5 1 13600 0.5 1 9420 0.5 1 0.5 1 0.5 1 0.5 1 17000 6350 7900 6730 0.5 10 91400* 0.5 1 0.5 10 7710 29400 0.5 10 0.5 1 0.2 10 40600 9840 32200 0.5 1 0.5 1 0.5 10 0.5 10 0.5 1 0.5 1 10800 6570 28900 42800 16000 10400 0.5 1 0.5 1 7660 10800 0.15 1 0.2 1 0.2 1 0.1 1 0.2 1 0.2 1 0.05 1 <100 <100 <100 <100 <100 <100 <100 0.1 1 5130 0.1 1 3420 0.5 1 6760 0.1 1j 3750 0.2 1 4900 0.1 1 5450 0.1 1 5350 These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers 3M Environmental Laboratory 3M Environmental Laboratory Pace 15 of 216 Page 15 ' I P '>5 rrr L Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 11. CeFi7S 0 3` Data Summary, Male Mallard Sera (Adult and Offspring) 3M Sample N umber SAM PLE D ESCRIPTIO N E01-1256-28662 E01-1256-28664 E01-1256-28666 E01-1256-28668 E01-1256-28671 E01-1256-28673 E01-1256-28675 E01-1256-28677 E01-1256-28680 E01-1256-28682 E01-1256-28684 E01-1256-28686 E01-1256-28688 E01-1256-28690 E01-1256-28692 E01-1256-28694 E01-1256-28696 E01-1256-28698 E01-1256-28700 E01-1256-28702 E01-1256-28704 E01-1256-28706 E01-1256-28708 E01-1256-28710 E01-1256-28712 E01-1256-28714 E01-1256-28716 E01-1256-28718 E01-1256-28720 E01-1256-28722 E01-1256-28724 E01-1256-28726 E01-1256-28728 E01-1256-28730 E01-1256-28732 E01-1256-28734 E01-1256-28736 E01-1256-28738 E01-1256-28741 454-109-3217,0 PPM, Male, Adult 454-109-3219,0 PPM, Male, Adult 454-109-3221,0 PPM, Male, Adult 454-109-3223,0 PPM, Male, Adult 454-109-3227,0 PPM, Male, Adult 454-109-3229,0 PPM, Male, Adult 454-109-3231,0 PPM, Male, Adult 454-109-3233,0 PPM, Male, Adult 454-109-3237,0 PPM, Male, Adult 454-109-3239,0 PPM, Male, Adult 454-109-3241,0 PPM, Male, Adult 454-109-3243,0 PPM, Male, Adult 454-109-3245,0 PPM, Male, Adult 454-109-3247,0 PPM, Male, Adult 454-109-3249,0 PPM, Male, Adult 454-109-3251,0 PPM, Male, Adult 454-109-3253,0 PPM, Male, Adult 454-109-3255,0 PPM, Male, Adult 454-109-3257,10 PPM, Male, Adult 454-109-3259,10 PPM, Male, Adult 454-109-3261,10 PPM, Male, Adult 454-109-3263,10 PPM, Male, Adult 454-109-3265,10 PPM, Male, Adult 4 5 4-109-3267, 10 PPM , Male, Adult 454-109-3269,10 PPM, Male, Adult 454-109-3271,10 PPM, Male, Adult 454-109-3273,10 PPM, Male, Adult 454-109-3275,10 PPM, Male, Adult 454-109-3277,10 PPM, Male, Adult 454-109-3279,10 PPM, Male, Adult 454-109-3281,10 PPM, Male, Adult 454-109-3283,10 PPM, Male, Adult 454-109-3285,10 PPM, Male, Adutt 454-109-3287,10 PPM, Male, Adult 454-109-3289,10 PPM, Male, Adult 454-109-3291,10 PPM, Male, Adult 454-109-3293,10 PPM, Male, Adult 454-109-3295,10 PPM, Male, Adult 454-109-9591,0 PPM, Male, Offspring AMOUNT OF SCRA USED (ML) a d d it io n a l d il u t io n Conc. or C fiFuSO j IN S am p le (n g /m L of Sera) 0.5 1 107 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.3 1 <100 0.5 1 0.5 1 <100 1400 0.5 1 <100 0.5 1 <100 0.5 1 162 0.5 1 1070 0.5 1 <100 0.1 1 <100 0.5 1 <100 0.5 10 78800 0.2 10 83300 0.5 10 68700 0.06 100 210000. 0.2 10 0.5 10 93800 75700 0.5 10 80600 0.5 10 11300. 0.5 10 106000 0.5 10 80500 0.5 10 80400 0.5 10 103000 0.5 10 74300 0.5 10 91800 0.5 10 I 108000 0.3 10 78700 0.3 10 97900 0.3 10 73500 0.5 10 96800 0.5 10 99700 0.1 1 <100 3M Environmental Laboratory 3M Environmental Laboratory Page 16 of 216 Page 16 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Sam ple N umber SAM PLE D ESCRIPTIO N E01-1256-28746 E01-1256-28749 E01-1256-28750 E01-1256-28756 E01-1256-28757 454-109-9606,0 PPM, Male, Offspring 454-109-9578,0 PPM, Male, Offspring 454-109-9625,10 PPM, Male, Offspring 454-109-9636,10 PPM, Male, Offspring 454-109-9642,10 PPM, Male, Offspring AMOUNT OF SERA USED (ML) ADDITIONAL Dl.UTION CONC. OF C g F u S O j IN Sam ple (ng /m L of Sera) 0.2 1 0.2 1 103 <100 0.2 1 0.1 1 0.2 1 5450 3670 4110 These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. 3M Environmental Laboratory 3M Environmental Laboratory Pane 17 of 216 0 Page 17 ` i f. -Am . Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 12. C8F17SO3' Data Summary, Female Mallard Liver (Adult and Offspring) 3M Sample N umofr SAM PLE D ESC RIPTIO N ! AMOUNT OF LIVER ADDITIONAL WEIGHED (G) DI.UTION C o n g , of C8F17SO3 IN Sample I E01-1326-29482 0 ppm a.i. 3218 F Liver, adult 1.0095 E01-1326-29583 0 ppm a.i. 3220 F Liver, adult 1.0944 E01-1326-29601 0 ppm a.i. 3222 F Liver, adult 1.0435 E01-1326-29619 0 ppm a.i. 3224 F Liver, adult 1.0425 E01-1326-29637 0 ppm a.i. 3226 F Liver, adult 1.0703 E01-1326-29655 0 ppm a.i. 3228 F Liver, adult 1.0526 E01-1326-29673 0 ppm a.i. 3230 F Liver, adult 1.0411 E01-1326-29691 0 ppm a.i. 3232 F Liver, adult 1.1704 E01-1326-29709 0 ppm a.i. 3234 F Liver, adult 1.1290 E01-1326-29727 0 ppm a.i. 3236 F Liver, adult 1.0800 E01-1326-29745 0 ppm a.i. 3238 F Liver, adult 1.1170 E01-1326-29763 0 ppm a.i. 3240 F Liver, adult 1.0086 E01-1326-29781 0 ppm a.i. 3242 F Liver, adult 1.0907 E01-1326-29799 0 ppm a.i. 3244 F Liver, adult 1.0291 E01-1326-29817 0 ppm a.i. 3246 F Liver, adult 1.1294 E01-1326-29835 0 ppm a.i. 3248 F Liver, adult 1.1075 E01-1326-29853 0 ppm a.i. 3250 F Liver, adult 1.1709 E01-1326-29871 0 ppm a.i. 3252 F Liver, adult 1.0761 E01-1326-29889 0 ppm a.i. 3254 F Liver, adult 1.1344 E01-1326-29907 0 ppm a.i. 3256 F Liver, adult 1.0669 (NG/G OF LlVLR) 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 1 <50 E01-1326-29925 E01-1326-29943 E01-1326-29961 E01-1326-29980 E01-1326-29998 E01-1326-30016 E01-1326-30034 E01-1326-30052 E01-1326-30070 E01-1326-30088 E01-1326-30106 E01-1326-30124 E01-1326-30142 E01-1326-30160 E01-1326-30178 E01-1326-30196 E01-1326-30214 E01-1326-30232 E01-1326-30250 10 ppm a.i. 3258 F Liver, adult 10 ppm a.i. 3260 F Liver, adult 10 ppm a.i. 3262 F Liver, adult 10 ppm a.i. 3264 F Liver, adult 10 ppm a.i. 3266 F Liver, adult 10 ppm a.i. 3268 F Liver, adult 10 ppm a.i. 3270 F Liver, adult 10 ppm a.i. 3272 F Liver, adult 10 ppm a.i. 3274 F Liver, adult 10 ppm a.i. 3276 F Liver, adult 10 ppm a.i. 3278 F Liver, adult 10 ppm a.i. 3280 F Liver, adult 10 ppm a.i. 3282 F Liver, adult 10 ppm a.i. 3284 F Liver, adult 10 ppm a.i. 3286 F Liver, adult 10 ppm a.i. 3288 F Liver, adult 10 ppm a.i. 3290 F Liver, adult 10 ppm a.i. 3292 F Liver, adult 10 ppm a.i. 3294 F Liver, adult 1.0034 1 7080 1.1331 1 6780 1.1294 10 13600 1.1829 1 6080 1.0487 1 5380 1.1214 1 5730 1.0751 10 13200 1.0658 1 5800 1.1226 10 13800 1.0953 10 30700 1.1956 1 5960 1.0600 1 2480 1.0275 1 4560 1.0915 1 6330 1.0657 10 28200 1.0330 10 24100 1.0352 10 19800 1.0658 1 4600 1.0627 1 4760 3M Environmental Laboratory 3M Environmental Laboratory Paae 18 of 216 Page 18 fVt Pf JiL . /*> *f*v* Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Sample N umber SAM PLE DESC RIPTIO N E01-1326-30268 E01-1367-31295 E01-1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31337 E01-1367-31343 E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31404 E01-1367-31410 10 ppm a.i. 3296 F Liver, adult 0 ppm a.i. 9583 F Liver, offspring 0 ppm a.i. 9589 F Liver, offspring 0 ppm a.i. 9592 F Liver, offspring 0 ppm a.i. 9598 F Liver, offspring 0 ppm a.i. 9603 F Liver, offspring 0 ppm a.i. 9610 F Liver, offspring 0 ppma.i. 9573 F Liver, offspring 10 ppm a.i. 9629 F Liver, offspring 10 ppm a.i. 9633 F Liver, offspring 10 ppm a.i. 9634 F Liver, offspring 10 ppm a.i. 9645 F Liver, offspring 10 ppm a.i. 9648 F Liver, offspring 10 ppm a.i. 9654 F Liver, offspring 10 ppma.i. 9659 F Liver, offspring AMOUNT OF LIVER WEIGHED (O) i ! 1.1238 ADDITIONAL DILUTION CONC. OF CbFij SCE IN Sami ae (NG/G OF LiVER) 1! 6330 1.1308 1.0005 1.0390 1.1204 1.1550 1.0217 1.0122 1 1 1 1 1 1 1 O to V <50 <50 <50 <50 <50 <50 1.1676 1.0510 1.1638 1.1510 1.1125 1.0770 1.0581 1 1| 1 1 1 1 1 2950 2240 4520 2580 3500 5930 3580 3M Fnvirrtnmental I ahnratorv 3M Environmental Laboratory Paae 19 of 216 0'i Page 19 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 13. C8Fi 7S03' Data Summary, Male Mallard Liver (Adult and Offspring) 3M Sample N umber SAM PLE D ESC RIPTIO N E01-1326-29473 E01-1326-29574 E01-1326-29592 E01-1326-29610 E01-1326-29628 E01-1326-29646 E01-1326-29664 E01-1326-29682 E01-1326-29700 E01-1326-29718 E01-1326-29736 E01-1326-29754 E01-1326-29772 E01-1326-29790 E01-1326-29808 E01-1326-29826 E01-1326-29844 E01-1326-29862 E01-1326-29880 E01-1326-29898 E01-1326-29916 E01-1326-29934 E01-1326-29952 E01-1326-29970 E01-1326-29989 E01-1326-30007 E01-1326-30025 E01-1326-30043 E01-1326-30061 E01-1326-30079 E01-1326-30097 E01-1326-30115 E01-1326-30133 E01-1326-30151 E01-1326-30169 E01-1326-30187 E01-1326-30205 E01-1326-30223 E01-1326-30241 E01-1326-30259 0 ppm a.I. 3217 M Liver, adult 0 ppm a.i. 3219 M Liver, adult 0 ppm a.i. 3221 M Liver, adult 0 ppm a.i. 3223 M Liver, adult 0 ppm a.i. 3225 M Liver, adult 0 ppm a.i. 3227 M Liver, adult 0 ppm a.i. 3229 M Liver, adult 0 ppm a.i. 3231 M Liver, adult 0 ppm a.i. 3233 M Liver, adult 0 ppm a.i. 3235 M Liver, adult 0 ppm a.i. 3237 M Liver, adult 0 ppm a.i. 3239 M Liver, adult 0 ppm a.i. 3241 M Liver, adult 0 ppm a.i. 3243 M Liver, adult 0 ppm a.i. 3245 M Liver, adult 0 ppm a.i. 3247 M Liver, adult 0 ppm a.i. 3249 M Liver, adult 0 ppm a.i. 3251 M Liver, adult 0 ppm a.i. 3253 M Liver, adult 0 ppm a.i. 3255 M Liver, adult 10 ppm a.i. 3257 M Liver, adult 10 ppm a.i. 3259 M Liver, adult 10 ppm a.i. 3261 M Liver, adult 10 ppm a.i. 3263 M Liver, adult 10 ppm a.i. 3265 M Liver, adult 10 ppm a.i. 3267 M Liver, adult 10 ppm a.i. 3269 M Liver, adult 10 ppm a.i. 3271 M Liver, adult 10 ppm a.i. 3273 M Liver, adult 10 ppm a.i. 3275 M Liver, adult 10 ppm a.i. 3277 M Liver, adult 10 ppm a.i. 3279 M Liver, adult 10 ppm a.i. 3281 M Liver, adult 10 ppm a.i. 3283 M Liver, adult 10 ppm a.i. 3285 M Liver, adult 10 ppm a.i. 3287 M Liver, adult 10 ppm a.i. 3289 M Liver, adult 10 ppm a.i. 3291 M Liver, adult 10 ppm a.i. 3293 M Liver, adult 10 ppm a.i. 3295 M Liver, adult AMOUNT O ' LIVER WEIGHED (G) ADDITIONAL DILUTION CONC. O! C T , S O j - J Sample (ng/g of Liver ) 1.1169 1.1847 1.1181 1.0788 1.1043 1.0486 1.0336 1.0165 1.0395 1.0373 1.1679 1.1493 1.0619 1.0263 1.1541 1.0813 1.0263 1.0088 1.0294 1.0926 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 1.0654 1.1316 1.1330 1.1404 1.1480 1.0620 1.1687 1.0313 1.1399 1.1143 1.0062 1.0380 1.0669 1.0955 1.1981 1.0500 1.0251 1.0441 1.0979 1.0815 10 10 10 100 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 72100 49200 70100 125000 61700 49900 57900 63000 85000 54900 53900 69000 49900 67100 55500 52600 59300 46500 24200 50400 3M Environmental Laboratory 3M Environmental Laboratory Paae 20 of 216 Page 20 6 0 1 -0 - Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Sample Number E01-1367-31301 E01-1367-31331 E01-1367-31349 E01-1367-31356 E01-1367-31392 E01-1367-31398 SAM PLE D ESC RIPTIO N AMoui-n or LivrRvvLiGHi::" Ai'-1"};!it' >'.ja UiLUilOfJ C o r.c Of C r , , S O : if: S.AMF LI:. (G) (NG/G OF L.FVF-R) 0 ppm a.I. 9591 M Liver, offspring 0 ppm a.i. 9606 M Liver, offspring 0 ppm a.i. 9578 M Liver, offspring 10 ppm a.i. 9625 M Liver, offspring 10 ppm a.i. 9636 M Liver, offspring 10 ppm a.i. 9642 M Liver, offspring 1.0411 1.1314 1.0413 1.0166 1.1367 1.1252 1 1 1 1 1 1 <50 <50 <50 4640 2200 2680 Statement of C onclusion Sera results from the male control group portion of this study showed no detectable levels of C8F17S 0 3' in 14 of 18 mallards (<100ng/mL) and quantifiable concentrations of C8F17S03' in 4 of 18 (range 107-1400ng/mL, Table 14). The sera results from the female control group portion of this study showed no detectable levels of C8F17S 0 3' in 15 of 17 birds (<100ng/mL) and quantifiable concentrations of C8F17S 0 3' in 2 of 17 (range 166-172ng/mL, Table 15). Analytical results for eighteen sera samples from the 10ppm dosed male group showed an average of 87300ng/mL C8Fi7S 0 3' (range 68700-108000ng/mL, Table 15). Analytical results for nineteen sera samples from the 10ppm dosed female group showed an average of 16600ng/mL C8F17S 0 3' (range 6350-42800ng/mL, Table 15). Sera results from the male offspring control group showed no detectable levels of CBF17S 0 3` in 2 of 3 birds (<100ng/mL) and a quantifiable concentration in 1 of 3 (103 ng/mL, Table 14). The sera results from the female offspring control group portion of this study showed no detectable levels of CbF17S 0 3' in 7 of 7 birds (<100ng/mL, Table 14). Analytical results for three sera samples from the 10ppm dosed male offspring group showed an average of 4410ng/mL (range 3670-5450ng/mL, Table 14). Analytical results for seven sera samples from the 10ppm dosed female offspring group showed an average of 4960ng/mL (range 3420-6760ng/mL, Table 14). Liver results from the male control group showed no detectable levels of C8F17S 0 3' in 20 of 20 birds (<50ng/g, Table 14). The liver results from the female control group also showed no detectable levels of C8F17S 0 3' in 20 of 20 birds (<50ng/g, Table 14). Analytical results for twenty liver sam ples from the 10ppm dosed male group showed an average of 60900n g/g C bF 17S 0 3' (range 24200-125000ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed female group showed an average of 10800ng/g C8F17S 0 3' (range 2480-30700ng/g, Table 14). Liver results from the male offspring control group portion of this study showed no detectable levels of C8F17S 0 3' in 3 of 3 birds (<50ng/g, Table 14). The liver results from the female offspring control group portion of this study showed no detectable levels of C8F17S 0 3' in 7 of 7 birds (<50ng/g, Table 14). Analytical results for three liver samples from the 10ppm dosed male offspring group showed an average of 3170ng/g (range 2200-4640ng/g, Table 14). Analytical results for seven liver samples from the 10ppm dosed female offspring group showed an average of 3620ng/g (range 2240-5930ng/g, Table 14). 3M Environmental Laboratory 3M Environmental Laboratory Page 21 of 216 Page 21 Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 14. Summary Table, CgFiySCV Concentration in Sera and Liver Sampling E vent Male Control Group N umber of S era S amples A verage CeFuSO ; C ono. S era Range of Results N umber of Liver Samples A verage c 8f ,, s o 3' C onc. Liver <100ng/mL (14 of 18) 18 NA 20 <50 ng/g 107-1400 ng/mL range (4 of 18) Range of Results NA Female Control Group 20 <100 ng/mL (15 of 20) NA 166-2680 ng/mL range (5 of 20) Male 10ppm Group Female 10ppm Group Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 20 20 3 7 3 7 89700 ng/mL 20300 ng/ml NA NA 4410 ng/mL 4960 ng/mL 11300-210000 ng/mL 6350-91400 ng/mL <100 ng/mL (2 of 3) 103 ng/ml_ (1 of 3) <100 ng/mL (7 of 7) 3670 - 5450 ng/mL 3420 - 6760 ng/mL For individual samples values see Tables 10 to 13 and Attachment B. 20 20 20 3 7 3 7 <50ng/g NA 60900 ng/g 24200-125000 ng/g 10800 ng/g 2480-30700 ng/g <50 ng/g <50 ng/g 3170 ng/g 3620 ng/g NA NA 22004640 ng/g 2240-5930 ng/g 3M Environmental Laboratory Page 22 IS O * Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 15. Summary Table, C8F17SO3' Concentration in Sera and Liver with Outliers Removed S ampling E vent Male Control Group N umber of Sera S amples A verage CsFuSOs* C onc. S era ------------------------1 18* Rango o f Results N um ber of Liver S amples 20* Female Control Group 17 I NA <100 ng/mL (15 of 17) 166-172 ng/mL range (2 of 17) 20* Male 10ppm Group 18 87300 ng/mL 68700-108000 ng/mL j 20* A verage CFrSOj' Conc. L iver * * * Range of Results * * * Female 10ppm Group 19 16600 ng/mL 6350-42800 ng/mL 20* Male Offspring Control Group Female Offspring Control Group 3* 7* * *' * 3* 7* * * Male Offspring 10ppm Group Female Offspring 10ppm Group -- 3* r ! * * * 3* * * * 7* * No outliers removed from this data set. Refer to Table 14 for results. For individual samples values see Tables 10 to 13 and Attachment B. Statistical M ethods Statistical methods included calculating means, standard deviations, % coefficient of variation, linear regressions, percent recoveries, and the Dixons Q-Test. The Dixons Q -Test, used for the rejection of outliers, has values ordered from lowest to highest. A Q-value is determined from the difference of a result and the next closest result that is divided by the difference of the highest and lowest value in the group of values. A critical Q-value is determined based on the number of samples in the group and the desired confidence interval. If the Q-value is greater than the critical Q-value then the value will be rejected. Statement of D ata Q uality The only measurement is the quality control and matrix spike results. Accuracy (the observed amount in the QC and MS samples, divided by the amount spiked, expressed as a percent) was found to range from 79.7% to 172%; while precision, expressed as the standard deviation of the recoveries, was found to range from 0.717% to 15.7%. 3M Environmental Laboratory n p -.I Page 23 Report EOl-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard References 1. Wildlife International, Ltd. Protocol No. 454-109 "PFOS: A Reproduction Study with the Mallard". 2. Wildlife International, Ltd. Protocol No. 454/120700/MR/SUB454 (3M Environmental Laboratory Project Number U2723) "PFOS: A Reproduction Study with the Mallard'. 3. Centre Analytical Laboratories, Inc. Study 023-044, "Validation of Analytical Methods "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC Electrospray/Mass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC Electrospray/Mass Spectrometry in Quail Serum and Liyer." 4. 3M Environmental Laboratory Standard Operating Procedure ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds form Biological Matrices". List of Attachm ents Attachment A: Extraction and Analytical Method Attachment B: Data Summary Tables Attachment C: Sample Chromatograms, Calibration Information, and Instrument Parameters Attachment D: Sample Preparation Sheets Attachment E: Study Protocol, Protocol Amendments, and Deviations Attachment F: Example Calculations 3M Environmental Laboratory 3M Environmental Laboratory Paae 24 of 216 Page 24 001808 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S ignature Page We certify that this report is a true and complete representation of the data for this study: Lisa A. Stevenson Principal Analytical Investigator o ih x k i Date , William K. Reagen Laboratory Management Date 3M Environmental Laboratory 3M Environmental Laboratory Page 25 of 216 Page 25 G9 1 6 0 9 Report EO1-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachm ent A: Extractio n and A nalytical M eth o d , E T S -8 -2 3 1 .1, "S olid P hase Extraction and A nalysis of Fluo ro chem ical C o m po unds from B iological M atrices" 3M Environmental Laboratory 3M Environmental Laboratory Paae 26 nf 216 Page 26 001610 Report EO1-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Method Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Method Num ber ETS-8-231.1 Adoption Date: Ifju j o i Revision Date: Effective Date: s j t t/t>z * ApprovedBy: Willliam K. Reagen Laboratory Manager ------ L Date ETS-E-23U Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page l o t 19 3M Environmental Laboratory 3M Environmental Laboratory Page 27 of 216 Page 27 G O lG ii Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 1 Scope and Application This method describes the extraction of target analytes from fish, rat liver, rat sera, mouse liver, and mouse sera using solid phase extraction (SPE), This method may also be extended to other biological matrices provided that the data quality objectives are met. 2 Method Summary An amount o f biological material, determined by the analyst, is prepared (fluids diluted and tissues homogenized) at a I/6 dilution, or other dilution as determined by the analyst using reagent grade water. An aliquot o f the dilution/homogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile (ACN) is added as an extraction solvent and also serves to precipitate the proteins. The sample is capped, mixed, and put on the centrifuge to clarity the supernatant. The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned Cjg SPE cartridge. Finally, the analytes o f interest are eluted from the SPE cartridge and analyzed by high perfonnance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). 3 Definitions______________________________________________________________ 3.1 Dilution A dilution expressed as 1:5 or l/6 is defined as: 1 mL of sample + 5 mLs o f diluent for a total o f 6 mLs combined, unless otherwise noted. 3.2 SPE cartridge A column containing an open solvent reservoirat one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds o f interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable conccntration(s) of the target analyte(s). 3.4 Quality control sample Sample used to monitor the extraction efficiency (as a matrix spike) and to verify the continued accuracy of the initial calibration curve (as a continuing calibration verification). 4 Warnings and Cautions___________________________________________________ 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewear, and clothing when working with solvents, samples and/or equipment. Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 volts. 4.2 Cautions Take care not to allow the SPE column to run to dryness after the methanol and water washes. After washing is complete, add sample then allow all o f the liquid to pass through the SPE column to dryness. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 2 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 28 of 216 Page 28 ty 0 l i ) 1 2 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar. the HPLC will initiate automatic shutdown. Do not nut solvent pumps to dryness. 5 Interferences____________________________________________________________ To minimize interferences. Teflon should not be used tor sample storage or any part of instrumentation that comes in contact with the sample or extract. 6 Instrumentation, Supplies, and Materials___________________________________ The following instrumentation, supplies, and materials are used while performing this method. Equivalent instrumentation, supplies, and materials may be used in place of those listed. 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 2 Ultra-Turrax T2S tissue homogenizer Vacuum Pump SPE Extraction Manifold Centrifuge. Mistral 1000 or IEC Shaker, Eberbach or VWR Balance (+/'- 0.1000 g) Micromass, Quatcro II or Ultima triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HPUOO or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppendorf or disposable pipettes, plastic or glass Dissecting scalpels Polypropylene bottles, capable o f holding 50 mL to 1 L (Nalgene) Volumetric flasks, glass, type A 40 mL glass vials (1CHEM) Plastic sampule vials, Wheaton, 6 mL (or other appropriate size) Centrifuge htbes. polypropylene. 15 mL and 50 mL Labels Graduated pipettes, glass Syringes, capable of measuring 5 pL to 1000 pL Bottle-Top Dispenser (capable o f dispensing 5mL o f solvent) SPE extraction cartridge, 1 g, Sep-Pak fi cc tri-functional Cn (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 3 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 29 of 216 Page 29 1G13 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Crimpers HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7 Reagents and Standards Reagent grade water, Milli-QTM, Nanopure II, or equivalent Acetonitrile. HPLC grade or equivalent Methanol, HPLC grade or equivalent Ammoni um acetate, reagent grade or equivalent Biological fluids or tissues, frozen from supplier , 7.1 Reagents preparation 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing reagent grade water, mix until all solids are dissolved, bring to volume using reagent grade water. Store at room temperature. Note: When preparing different volumes than those listed in reagents preparation, target analyte standard preparation, and surrogate standard preparation, adjust accordingly. 7.2 Target analyte standard preparation Prepare target analyte standard(s) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations. Weigh approximately 100 mg of target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location. Bring to volume with methanol for a stock standard of approximately 1000 ppm (jfg/mL). Dilute the stock solution with methanol for a working standard l solution of approximately 50 ppm. Example calculation: 1000 |ig/mL x 5 mL/lOOmL = 50 |ig/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution of approx. 5.0 ppm. Example calculation: 50 |!g/mL x 10 niLTOOmL = 5.0 Jig'rnL. Dilute working standard I with methanol to produce a working standard 3 solution o f approx. 0,50 ppm. Example calculation: 50 flg/mL x 1.0 mL/100 mL = 0.5 pgfrnL. 7.3 Surrogate standard preparation F'repare surrogate standard(s). The following is an example only and may or may not be appropriate for all surrogate standard preparations. Weigh approximately 90-110 mg o f surrogate standard into a 100-mL volumetric flask and record the actual weight. Bring to volume with methanol for a surrogate standard stock of approximately 900 - 1100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL of surrogate standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard of 90-1lOpprn. Record the actual volume transferred and standard concentratioas in the standards logbook or other appropriate location. 7.4 Internal standard preparation Prepare internal standard(s). The following is an example only and may or may not be appropriate for all interna] standard preparations. Weigh approximately 90-110 mg o f internal standard into a IOO-mL volumetric flask and record the actual weight. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 4 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 30 of 216 Page 30 001614 Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Bring to volume with methanol for an internal standard stock of approximately 900 -1100 ppm. Prpaie an internal standard working standard. Transfer approximately 1 mL of internal standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard o f 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 8 Sample Handling All samples are received frozen and must be kept frozen until the extraction is performed. .Allow samples to thaw to room temperature prior to extraction. Typically fresh matrix standards are prepared with each analysis. Extracted standards and samples ire stored in capped autovials until analysis. If analysis will be delayed, extracted standards and samples may be refrigerated at approximately 4C indefinitely or may be stored at room temperature until analysis can be performed. 9 Quality Control 9.1 Blanks 9.1.1 Solvent Blank An aliquot of methanol is used as a solvent blank. Solvent blanks arc not extracted. 9.1.2 Method Blank An aliquot o f l .0 mL o f water, or other appropriate amount, is used as a method blank. Four method blanks are extracted and analyzed with each set following this procedure (two are spiked with surrogate and two are not spiked). 9.1.3 Matrix Blank An aliquot o f l .0 mL or 1.0 g o f matrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one of three sources: 1) a study control matrix from a study control animal received with a sample set; 2) a commercially obtained sample of the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but o f a different species than the study animal, (eg. if rat is used to generate standard curves and CCVs for a mouse study). The matrix to use is dependent on the m atrix vised fo r die curve. 9.1.3.1 Study control matrix curve - if the study control matrix is used for the curve, prepare four (4) matrix blanks using the study control matrix (two spiked with surrogate and two not spiked). 9.1.3.2 Commercially obtained (same species) matrix c u rv e -if the commercially obtained matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix (two spiked with surrogate and two not spiked). 9.1.3.3 Surrogate matrix curve - if a surrogate matrix is used for the curve, prepare four (4) matrix blanks using tire same commercially available matrix and prepare four (4) matrix blanks using a commercially available matrix of the same species as the study animals (two spiked with surrogate and two not spiked). 9.1.3.4 If limited matrix is available, the number o f method and matrix blanks may be adjusted and will be noted in the study protocol or in the raw data. 9.2 Sample Replicate Samples replicates are prepared according to each study protocol or project outline. ETS-8-231.I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 31 of 216 Page 31 v G1 G 5 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 9.3 Surrogate standard [f surrogate standard is a component of the study, all samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range of the calibration curve, with the exception of blank samples as described above. Typically surrogate standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. 9.4 Internal standard If internal standard is a component of Ore study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range o f the calibration curve. Typically internal standard is spiked into the 2.0 mL of extract in the 15 mL centrifuge tube, before transferring to the autovial. 9.5 Lab Control Sample Lab control samples are not a component of this method. 9.6 Quality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efficiency and to verify the continued accuracy of the initial calibration curve. Typically 1.0 mL. or other appropriate amount, o f the same matrix used to prepare the initial calibration curve is used for each QC sample. Twelve (12) quality control samples (QC) will be prepared for each matrix during the course of a study. A minimum of 3 QC samples must be prepared (one at each level) on each day of sample extraction, (e.g. If the study is such that samples will be extracted on three different days then four QC samples must be prepared on each day o f extraction for a total of twelve.) QC samples will consist o f four samples at each o f three levels o f analyte. The levels listed below may be used and may represent sample concentrations diluted into the range o f the calibration curve: Low level: 3X to 5X the LLOQ, Mid-level: equivalent to a point near the middle of the calibration curve, High level: 80% of the ULOQ Two QC sample levels are analyzed after every tenth sample injection starting after the last calibration standard injection, with a minimum of three QC per analysis. Solvent blanks are not considered samples but may be included as such for determining when QC samples will be analyzed. QC samples extracted with a particular sample set must be analyzed in the same analytical run. Any QC samples extracted during the course of the study may be included in subsequent analyses. If samples from multiple extraction dates are analyzed in one analytical run, then QC samples from the same sample extraction dates must be included in that analysis. Each QC is expected to show an accuracy o f 75-125% of expected. A minimum of 2 0 of all QC samples must meet this criteria, and a minimum of 1/2 of the QC samples at each level must meet this criteria. If not, the set must either be re-analyzed or re-extracted. 9.7 Sample Dilution Any sample with an area greater than that o f the highest acceptable standard will need to be diluted into the range of the calibration curve. If samples are diluted into the range o f the curve during analyses and enough sample remains, a post-run dilution validation will be performed to verily sample values. To perfonn tire dilution validation, one sample will be separated into two representative samples (i.e. two 1.0 mL aliquots for fluid samples or two 1.0 gram amounts for tissue samples, or other amount as determined by the analyst ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 6 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 32 of 216 Page 32 e s ig i Report E01-1256 Report E01 -1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard and documented in a note to file) then diluted using two procedures. The first procedure consists of diluting the sample with additional matrix prior to extraction (fluid adding fluid), while the second procedure consists o f diluting die extract with solvent post-extraction (methanol extract adding additional methanol solvent.) If die relative percent difference is not within 15% for these two samples; additional testing will be required to determine which value is a correct representation o f the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, per study. II will consist of a minimum of nine (9) levels. Additional calibration curves may be extracted on separate sample extraction dates, as determined by the analyst and documented in a note to file. Transfer I.O mL, or other appropriate amount, of diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weigbt/volumes sheet or extraction worksheet, as appropriate. Four 1.0 mL aliquots, or other appropriate amount, o f control matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one standard curve. A total o f 9 standards, four matrix blanks, and four method blanks are prepared in addition to the QC samples and test samples. The number of standards and blanks may be adjusted as determined by the analyst and documented in a note to tile. Use Attachment C. or other appropriate form, as an aid in calculating the concentrations o f the working standards. Refer to section 12 to calculate the actual concentration of analyte in each calibration standard and QC sample. Typically the target analyte standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, it may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction Analyze the extracted matrix standard curve prior to each set of extracts. The curve equation will be determined by regression analysis using the peak areas of the target analyte(s) using MassLynx or other suitable software. Any level outside 75% - 125% of nominal must be deactivated, and regression re-calculated, except the LLOQ which must be within 30% o f nominal. All levels must show a response greater than twice that of the blank. A maximum of three (3) levels may be deactivated in any one set, or the set will be re-analyzed. ETS-8-231.1 Solid Phase Extraction and Analysis ofFluorochemical Compounds from Biological Matrices Page 7 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 33 of 216 Page 33 001617 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 'fu ill 1 AlTItOM.M VI 1 Sl`lKIM. A.MOl MS 1Oli SI AMI Vltll-< VMl SIMM S tlSISC l.ll Ml. Ol M VI ItIV Working standard (approximate concentration) pL Approximate final concentration o f analyte Approximate final concentration o f analyte in M atrix diluted 1:5 in Final 2.0 mL volume - - Blank Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 lO.Ong/gor ng/mL 0.750 ng/mL 0.500 ug/mL 8.0 25.0 ng/g or ng/mL 2.00 ng/mL 0.500 ug/mL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug/mL 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL 14.0 ng/mL 5.00 ug/mL 8.0 250 ng/g or ng/mL 20.0 ng/mL 5.00 ug/mL 16 500 ng/g or ng/mL 40.0 ng/niL 5.00 ug/mL 24 750 ng/g or ng/mL 60.0 ng/mL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/g or ng/mL 100 ng/mL 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/mL 50.0 ug/mL 6.0 1750 ng'g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures_____________________________________________________________ 11.1 Tissue Sample Preparation Obtain frozen tissue samples Cut approximately 1.0000 g of tissue (+ /-0.1000 g). or other appropriate amount, using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the tissue to thaw. Weigh Ihe tissue directly into a tared plastic sampule vial. Record the weight on the weight/volume sheet, extraction worksheet, or other appropriate location. Return unused tissue to the freezer after extraction amounts have been removed. Add 2.5 mL o f reagent water to sampule vial, or other volume as determined by the analyst and documented in a note to file. Homogenize the sample. Put the Ultra-Turrax grinder probe in the sample and grind for approximately 2 minutes, or until (he sam ple is hom ogeneous. Rinse tire probe into tlie tube containing the sample with 2.5 mL o f reagent grade water, or other volume as determined by the analyst and documented in a note to file, using a pipette. Take the grinder apart and clean it with methanol after each sample. Refer to ETS-9-52 for more information. If an amount other than 1.0000 g (not within +/- 0.1000 g) is removed for an initial weight, adjust the water volume accordingly to maintain a 1/6 dilution, (eg. if 0.5 gis removed for extraction, add a total o f 2.5 mL o f water.), or other ratio as determined by the analyst and documented in a note to file. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Conrpounds from Biological Matrices Page 8 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 34 of 216 Page 34 001618 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 11.2 Fluid Sample Preparation Obtain frozen fluid sample and allow it to thaw at room temperature or in lukewarm water. Label a 15 mL polypropylene centrifuge tube with the study number, sample ID, extraction date and analyst initials. See attached worksheet (Attachment A or similar worksheet) tor documenting the remaining steps. Vortex mix the fluid sample for approximately 15 seconds, then transfer 1.0 mL o f fluid, or other appropriate amount to a plastic sampule vial, or other appropriate container. Return unused samples to freezer after extraction amounts have been removed. Add 5.0 mL of reagent water to the 1.0 mL o f fluid for a 1/6 dilution, or other dilution as determined by the analyst and documented in a note to file. If a volume other than 1.0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilution as above. 11.3 Tissue and Fluid Sample Extraction After tissue or fluid samples have been prepared according to sections 11.1 and 11.2, vortex mix or shake by hand the diluted/homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mL polypropylene centrifuge tube. Return unused diluted/homogenized portions to the freezer after extraction amounts have been removed Record die volume removed on the extraction worksheet. (Attachment A or similar worksheet). Spike blanks, samples, and standards, ready for extraction with surrogate standard as described in this method. Spike each calibration standard matrix with the appropriate amount of standard as described in this method for the calibration curve standards and each QC sample. Vortex mix the standard curve samples and QC samples for approximately 5 seconds. To each sample and standard, add 5.0 mL of acetonitrile, cap, and vortex mix or shake by hand approximately 15 seconds. Place all samples on the shaker at an appropriate speed for 20 minutes to adequately mix (a setting of approximately 300 rpm on the models listed in section 6.1). Remove from the shaker and centrifuge at an appropriate speed for 10 minutes to adequately pellet the precipitate (a setting of approximately 2000 rpm on the models listed in section 6 .1). Add 40.0 mL of reagent grade water to a clean 50 mL centrifuge tube. Remove samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduce any of the matrix solids into the solutioa Cap and mix by inverting several times. In this step the order of addition may be changed (i.e. tlie sample m ay be put into the ccntritugc tube and then the w ater added). Attach the reservoir to the SPE cartridge and attach this reservoir/cartridge unit to a vacuum manifold, NOTE: When running the vacuum, set the vacuum chamber at approximately 15 kPA - to give an approximate elution flow o f 5-7 mL/min. Flows may vary through cartridges and the kPA may be raised for slow tubes and drying after most have been drawn down. Prepare the SPE cartridge by washing twice with approximately 5.0 mL of methanol, followed by approximately two 5.0 mL aliquots o f water, taking care not to allow the column to run to dryness after each wash. After washing is complete, pour the sample into the reservoir/cartridge unit and allow all of the liquid to pass through the culumn to dryness. Run the vacuum on high for approximately 5 minutes to adequately dry each SPE cartridge. Place a collection 15 mL polypropylene centrifuge tube under each cartridge and elute with 2.0 mL of methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. E T S - 8 - 2 3 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 9 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 35 of 216 Page 35 021619 Report E01-1256 Report E 0 1-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Label each glass autovial, as appropriate, with the study number, vial file archive number, animal number/gender/timepoint or L1MS number, matrix, final solvent, analyte components (if needed), extraction type, extraction date, and analyses) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.1 Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-month-day, and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or A020204a 1 = Initial of the instrument name (A = "Amelia1') YY = Test year (02) MM - Test month (02) DD = Tcst day (04) a = First sample list (run) o f the day (the next sample list will end with b'. the next 'c'. and so on.) Assign a filename using the instrument designator letter, the last 2 digits of the test year-month-day, and a 3-digit sequential file number that starts with 1 and increases by one for each filename. Example filename: IYYMMDDlW# or A020204001 I = Initial of instrument name YY = Test year MM = Test month DD - Test day ### = 3-digit sequential file number starting with 1 through 999 (001) Also, as part o f die samplelist, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume, and sample descriptions. To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate mass(es). Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are em ployed, additional product ion fragm entation inform ation m ay he collected. See M icrom ass M assLynx "Guide to Data Acquisition" for additional information on MRM. Typically the analytical batch run sequence begins with system suitability, solvent blanks, and a set o f extracted matrix standards. Sample extracts are analyzed with two QC samples injected after every tenth sample injection. Solvent blanks should t analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be included as such. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 10 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 36 of 216 Page 36 &21G20 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 11.4.2 HPLC set-up Set up sample hay according to the sample list prepared above. Set up the HPLC to the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook, or other appropriate location: Sample size = 10 pL injection Inject/sample = l Cycle time - 10.0 minutes Flow rate = 300 pL/min Mobile phase: Solvent A = 2 mM Ammonium Acetate. Solvent B - Methanol I Solvent gradient program: 0.00 'V Solvent B 10% 1.00 10% 5.50 95% 7.50 95% 8.00 10% 11.4.3 Instrument set-up Refer to ETS-9-24, "Operation and Maintenance o f the Micromass Quattro 11Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source." for details. Check the solvent level in HPLC reservoirs and refill if necessary. Check the stainless steel capillary at tire end of the probe. Use an eyepiece to check the tip. 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. Turn on the nitrogen. Open the tune page. Click on operate to initiate source block and desolvation heaters. Open the Inlet Editor. D ow nload the H PLC m ethod and initiate solvent flow to begin system equilibrium. Set the flow to 10- 500uUmin or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out of the tip of the probe. A fme mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip of the probe if no mist is observed Allow to equilibrate for approximately 10 minutes. Typical instrument parameters include: Drying gas 250-400 liters/hour ES nebulizing gas 10-15 liters/hour ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 11 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 37 of 216 Page 37 001621 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction S tud y with the Mallard HPLC constant flow mode, flow rate 10-500 pL'min Pressure <400 bar (this parameter is not set. it is a guide to ensure the HPLC is operating correctly.) Source block temperature approximately 150C Desolvation temperature approximately 250C These settings may change in order to optimize the response Print the tune page, sample list, and acquisition method from MassLynx and store it in the study binder with a copy taped into the instrument log. Click on start button in the Acquisition Control Panel (the location of the start button may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). 1 12 Data Analysis and Calculations___________________________________________ 12.1 Calculations If other calculations are used than those listed, they will be documented in the raw data. Calculate the matrix amount contained in the initial dilution using the following equation: ,, , . ,, ,, , , , . , , IW (g) (or IV (mL)) Matrix Amount (g/m Lorm L/m L) = ------------------------------------- -- (fW(g) (or IV(mL))+ DV (mL) Calculate actual concentrations of analyte in calibration standards using the following equation: Concentration (ng/g orng/mL) Spike Concentration (ug/m L)x Spiked Amount (mL) 1000 ng SV (mL) x Matrix Amount (g/mL or mL/mL) l ug IW = Initial weight (where 1.0 g = 1.0 mL ) IV = Initial volume DV = Diluent volume (reagent grade water) SV = Sam ple vo lu m e rem oved to r extraction (typically 1.0 mL) AR = Analytical result from MassLynx summary DF = Dilution factor FV = Final volume MA = Matrix amount v curve = MA o f tissuc/fluid standard curve, assumed to be I g or 1 mL/5 mL water 3 sample - MA of tissue/tluid sample {__ g or mL of sample/5 mL water) Calculate spike percent recoveries using the following equation: ,, Observed Result - Matrix Blank Result % Recovery --------------------------------------------------------- x tot) Spiking Level ETS-8-231.1 Solid Phase Extraction and Analysts o f Fluorochemical Compounds from Biological Matrices Page 12 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 38 of 216 Page 38 eaiS22 Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Calculate relative standard deviation using the following equation: ,,. . _ .. Standard Deviation Relative Standard Deviation = -------------------------- x 100 Mean Calculate percent deviation using the following equation: ,,, .. Expected Cone. - Calculated Cone. "/Deviation = -- ------------------------------------------ x 100 Expected Cone. Calculate actual concentration of analyte in fluid (pg/mL): AR (ngfmt.) * DF * cl curve ImL/mLl * FV (mL) in Curve 1.0 in; = (p g'g) d sample (inL/tnL) FV (mL) in Matrix 1000 ng Calculate actual concentration of analyte in tissue (ge/g): AR (ng-g) x DF * 0 curve la/mL) * FV tmL) in Curve * 1.0 mr d sample (g/mL) FV (mL) in Matrix 1000 ng = (pg/g) 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample of the run three (3) mid-level unextraeted calibration standards will be analyzed. As applicable, the peak area precision, retention time precision, resolution, and peak asymmetry will be monitored at the beginning and the end of the run separately. The peak area precision must be equal to or less than 5.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD. the resolution must be > 2.0, and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is the asymmetry factor. If any item of the system suitability fails, system maintenance must be completed prior to running a second set o f system suitability samples and the system suitability must pass before starting the calibration. If system suitability fails at the completion of a run. the sample set must be reanalyzed. 13.2 Quantitation The coefficient of determination value for the calibration curve, plotted by regression using the peak areas of the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% of the theoretical value with the exception of the LOQ point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify' a data range that may be affected by background levels o f the analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. If the curve cannot meet these criteria, the sample set must be reanalyzed or reextracted. 13.3 Accuracy Two thirds of all quality control samples and 1/2 of each quality control sample at each level are expected to show an accuracy o f 75-125%. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluoroehemical Compounds from Biological Matrices Page 13 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 39 of 216 Page 39 001623 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Surrogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be reanalyzed to confirm If the second analysis confirms the original, the deviation will be documented in the raw data. If the second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed of in noninfectious biohazard waste containers. Flammable solvent waste is disposed of in high BTU containers. Glass pipette waste is disposed of in broken glass containers located in the laboratory. 15 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw data. Each page generated for a study must contain the following information (if applicable): study/project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Other information may be added if applicable to the study. Print the tune page, sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into the instrument runlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data. Print data integration summary, integration method, and chromatograms from MassLynx. and store with the study raw data. Summarize data using suitable software (Excel 7.0 or LIMS) and store in the study folder. Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 16 Attachments_____________________________________________________________ Attachment A: Extraction Worksheet Attachment B: Sample WeighCVolume Worksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References_____________________________________________________ EiTS-9-24. "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52. "Operation and Maintenance o f a Tissue Grinder" 18 Affected Documents____________________________________________________ None ETS-8-23LI Solid Phase Extraction and Analysis o f Fluorochemical Compounds front Biological Matrices Page 14 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 40 of 216 Page 40 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 19 Revisions Revision Number l Revision Description Minor formatting changes. Added detailed information to all sections concerning the extraction procedure, analytical procedure, and calculations. Added attachments and references. Revision Dale 02/18/02 ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 15 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 41 of 216 Page 41 C<1162 Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Study Number: Prep Date: Analysts initials: Box#: Attachment A - Extraction Worksheet Method Revision: ETS-8-231.1 Matrix: Sample Timepoint: Sample Number Sample Number Volume of Amount and or diluted sample Amount end surrogate spike description removed spike mix used mix used Type of column used and lot Elution solvent and volume Comments 1 1 Blank matrix______ TN-A-___ ____,___ ; Amount weighed/aliquoted:__________g/mL 1. Homogenize sample 2. Aliquot 1 mL ol diluted matrix into 15 mL polypropylene tube 3. Spike samples accordingly 4. A d d ___ mL of ACN (TN-A-________ _ _ ) to each diluted sample and shake or vortex mix 5. Shake sample for 20 min @ ______rpm (Shaker____ __________ ) 6. Centrifuga sample for 10 min @ _______rpm (Centrifuge_______ ________) 7. Add 40 mL o f ___________ water to 50 mL polypropelene centrifuge tube. 8. D e c a n t extract into centrifuge tu b e s with w ater 9. Shake sample slightly to ensure proper mixing 10. Attach 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum manifold 11. Condition column with two washes of - 5 mL MeOH (TN-A-__________) - do not allow column to go to dryness 12. Wash column with two washes of -5 m L __________water - do not allow column to go to dryness 13. Filter sample through conditioned column, discarding filtrate 14. Allow column to go to dryness. After dripping stops, draw a high vacuum through column for at least 5 minutes. 15. Elute column with solvent (__________ TN-A-__________ ) into appropriate 15 mL centrifuge tube 16. Spike samples w ith ________uL of internai standard U _____________________ , cone.____________________ 17. Transfer sample into appropriately labeled autoviai and cap Note: In vacuum steps above set the vacuum chamber at approximately 15 kPA - this should give approximately 5-7 mL/min elution flow Flows may vary through cartridges - kPA may be raised for slow tubes and drying after most have been drawn down and shut off. ETS-8-231. 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 16 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 42 of 216 Page 42 :J i>1 6 2 6 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Prep Date(s): Analyst(s): Sample Matrix: Method/Revision: Sample ID Attachment B - Sample W eight/Volume Worksheet Study Number: Equipment Number: Final Solvent & TN Number: Initial Wt./Vol. e/mL/L W ater Volume added (mL) Volume Removed (mL) Comments Forni Completion Verified By:. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 43 of 216 Page 43 031627 Report E01-1256 Report E 0 1-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Prep date(s): A n aly te(s): Sample matrix: M ethod/revlsion: Attachment C: Calibration Standard Concentration Worksheet Standard number: Equipment number: Final solvent and T N : Blank Tissue or Fiuid/identifier: Analyte mix std approx. 0.500 ug/mL: Analyte mix std approx. 5.00 ug/mL: Analyte mix std approx. 50.0 ug/mL: Surrogate std approx. 100 ug/mL: Actual concentrations o f standards in the analyte mix A n a ly te Std cone ug/mL 0.500 0.500 0.500 O.SOO 0.500 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 All Am't spiked mL 0.0015 0.0030 0.0080 0.0160 0.0320 0.0056 0.0080 0.0160 0.0240 0.0320 0.0400 0.005 0.006 AU Final Volume: mL 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 All Initial Fluid Dilution mL/mL 0.1667 0.1667 0 1667 01667 0.1667 0 1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 All Initial Tissue Density g'rnL 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 Calculated concentrations o f standards in relation to the final 2.0 mL solvent and 2.0 m L Final Volume Fluid M atrix Tissue M atrix Analyte Final cone. ng/mL 0.375 0.750 2.00 4.00 8.00 14.0 20.0 40.0 60.0 80.0 100 125 150 Surrogate Std cone ng/mL 100 Surrogat Final cone ng/mL 0.500 A n aly te Final cone. ng/mL 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL J00 Surrogate Final cone ng/mL 6500 Analyte Final cone. ng/g 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 250 1500 1750 Surrogate Std cone ng/ntL 100 Surrogate Final cone n's 6500 ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 18 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 44 of 216 Page 44 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Study: Dilution Date/Analyst: Box Number: Sample Num ber or Description 1/ Attachment D: Dilutions Summary W orksheet Solvent/TN Number: Extraction Date/Analyst: Matrix/Timepoint: Dilutions V 1/ 1/ 1/ 1/ 1/ Comments Verified By: 1 Notes: l / t 0 dilution = ________ o f sam ple + _______ __ o f solvent Form C om pletion V erified B y :_______________________________ ETS-8-23I.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 19 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 45 of 216 Report EOl-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachm ent B: Data S ummary Tables 3M Environmental Laboratory 3M Environmental Laboratory Paae 4fi of 216 Report EO1-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD SERA DATA SUMMARY, BLANKS AND SPIKES 3M Sample Number E01-1256-36231 E01-1256-36259 E01-1256-36263 E01-1256-36263 E01-1256-36267 E01-1256-36231 E01-1256-36232 E01-1256-36260 E01-1256-36264 E01-1256-36264 E01-1256-36268 E01-1256-36232 Sampled Date 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 Sample Description Blank H 20-day 1 Blank H 20-day 2 Blank H 20-day 3 Blank H 20-day 3 Blank H20-day 4 Blank H 20-day 1 Inst. File Name (1020225037 d020226025 <1020228024 (1020301099 (20228107 (20308036 amount of sora used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 dilu tio n (diluted sera) 40 40 40 40 40 40 additional d ilu tio n 1 1 Inst Cone. (ng/mL) Cone, o f PFOS in Sample (ng/mL) 1.00 2.51 2.51 1.00 1.00 2.51 40.0 100.4 100.4 40.0 40.0 100.4 !oq for control sa m ple s 40.0 100.4 100.4 40.0 40.0 100.4 Blank mallard sera-day 1 <1020225038 0.05 40 1 1.00 Blank mallard sera-day 2 d020226026 0.05 40 2.51 Blank mallard sera-day 3 d020228025 0.05 40 1 2.51 Blank mallard sera-day 3 d020301100 0.05 40 1 1.00 Blank mallard sera-day 4 d020228108 0.05 40 1 1.00 Blank mallard sera-day 1 (20308037 0.05 40 2.51 40.0 100.4 100.4 40.0 40.0 100.4 40.0 100.4 100.4 40.0 40.0 100.4 3M Sample Number E01-1256-36233 E01-1256-36261 E01-1256-36265 E01-1256-36265 E01-1256-36269 E01-1256-36233 Sam pled Date 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 Sam ple D escription Inst. File Name Mallard Sara MS-day 1 Mallard Sera MS-day 2 Mallard Sera MS-day 3 Mallard Sera MS-day 3 Mallard Sera MS-day 4 Mallard Sera MS-day 1 d020225039 (20226027 (20228026 d020301101 (20228109 d020308038 amount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 difution (diluted sera) 40 40 40 40 40 40 additional d ilu tio n 1 1 1 1 1 Cone, o f PFOS in Inst Cone. (ng/mL) Sample (ng/mL) 27.79 29.50 28.85 27.76 28.01 29.03 1112 1180 1154 1110 1120 1161 E01-1256-36234 E01-1256-36262 E01-1256-36266 E 0 1-1256-36266 E01-1256-36270 E01-1256-36234 Concentration <LOQ 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 Mallard Sera MSD-day 1 Mallard Sera MSD-day 2 Mallard Sera MSD-day 3 Mallard Sera MSD-day 3 Mallard Sera MSD-day 4 Mallard Sera MSD-day 1 d020225040 (20226028 (20228027 d020301102 (20228110 (20308039 0.05 0.05 0.05 0.05 0.05 0.05 40 40 40 40 40 40 Cone of PFOS In Sample (ng/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2 .0 mL/0.05 mL = 40 amount of sera in diluted sample (mL) = amount o f sera used/total amount o f sera + water (1/20 dilution). = 1/20 = 0.05 mL 1 30.44 1 29.80 1 24.76 1 24.01 1 28.23 1 31.34 for troth ms and msd avg% rac std dev of %rec avg cone std dev of cone 1218 1192 990 960 1129 1254 112.7 8.487 1132 85.21 9 Flee 110.7 117.5 114.9 110.6 111.6 115.7 true val. 1004 avg %rec 113.5 avg 1140 std dev 29.36 121.3 118.7 98.65 95.66 112.5 124.9 true val. 1004 avg %rec 111.9 avg 1124 std dev 122.3 O <.? H* Cfi 03 3M Environmental Laboratory R lanlre A T e artH U C e Page 47 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD SERA versus unextracted curve 3!VI Sample Number Sample D escription Inst File Name e01-1256-36250 QC- 25ppb -1 (1004 ng/mL diluted sera) d o 20405036 e01-1256-36251 QC- 25ppb -2 (1004 ng/mL diluted sera) (J020405037 e01-1256-36252 QC- 25ppb -3 (1004 ng/mL diluted sera) d020405038 e01-1256-36253 QC- 250ppb -1 (10040 ng/mL diluted sera) d020405039 e 0 1-1256-36254 QC- 250ppb -2 (10040 ng/mL diluted sera) (J020405040 e 0 1-1256-36255 QC- 250ppb -3 (10040 ng/mL diluted sera) d020405041 e01-1256-36256 QC- 4.0 ppm -1 (160640 ng/mL diluted send020405042 e01-1256-36257 QC- 4 .0 ppm -2 (160640 ng/mL diluted sen do20405043 e 0 1-1256-36258 QC- 4.0 ppm -3 (160640 ng/mL diluted send020405044 A = Not within +/-25% criteria am ount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (d ilu te d sera) 40 40 40 40 40 40 40 40 40 Study number E01-1256 - MALLARD SERA versus extracted curve original Absolute Recoveries a d d itio n a l dilu tio n 1 1 1 1 1 1 10 10 10 Inst Cone. (ng/mL of M e th a no l) 48.29 39.92 45.30 288.00 294.03 293.10 523.44 432.18 415.07 Cone, ol PFOS in Sample (ng/m L of Sera) 1932 1597 1812 11520 11761 11724 209376 172872 166028 A A A A for all QC samples (absolute recovery) avg %rec 135.8 std d e vof% re c 33.11 QC Sampie Recoveries % Rec 192.4 159.0 180.5 114.7 117.1 116.8 130.3 107.6 103.4 avg 177.3 std d e v 16.90 %RSD 9.531 avg 116.2 s td d e v 1.293 % RSD 1.113 avg 113.8 s td d e v 14.51 %RSD 12.75 3M Sample Number Sample Description Inst. File Name am ount of sera used (mL) d ilu tio n (diluted sera) a d d itio n a l dilu tio n Inst Cone. (ng/mL of M ethanol) C one, o f PFOS in Sample (ng/m L of Sera) o Rec e01-1256-36250 e01-1256-36251 801-1256-36252 e01-1256-36253 e01-1256-36254 801-1256-36255 e01-1256-36256 e01-1256-36257 e01-1256-36258 QC- 25ppb -1 (1004 ng/mL diluted sera) (20225071 QC- 25ppb -2 (1004 ng/mL diluted sera) d020225072 QC- 25ppb -3 (1004 ng/mL diluted sera) (3020225073 QC- 2 5 0 ppb -1 (10040 ng/mL (fluted sera) d o2 0 2 2 5074 QC- 250ppb -2 (10040 ng/mL diluted sera) (3020225075 QC- 250ppb -3 (10040 nq/mL diluted sera) (1020225076 Q C - 4.0 ppm -1 (160640 ng/mL diluted sen <1020308045 QC- 4.0 ppm -2 (160640 ng/mL diluted sen (3020308046 QC- 4.0 ppm -3 (160640 ng/mL diluted send020308047 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 40 40 40 40 40 40 40 40 40 1 39.15 1566 1 32.41 1296 1 37.84 1514 1 246.03 9841 1 252.86 10114 1 238.51 9540 10 320.09 128036 10 364.90 145960 10 335.08 134032 A 156.0 avg 145.3 A 129.1 s td d ev 14.24 A 150.8 %RSD 9.800 98.02 evg 97.93 100.7 std d ev 2.860 95.02 %RSD 2.920 79.70 avg 84.67 90.86 std d ev 5.680 83.44 %RSD 6.708 A = Not within +/-25% criteria for all QC sam ples original analysis (recovery) avg %rec std dev o f % rec 109.3 28.68 versus extracted curve reran 3M Sample Number Sample D escription Inst. File Name am ount of sera used (mL) d ilu tio n (d ilu te d sera) a d d itio n a l d ilu tio n Inst Cone. (ng/mL of M e th a no l) Cone, of PFOS in Sample (ng/mL o f Sera) Rec e01-1256-36250 e01-1256-36251 e01-1256-36252 e01-1256-36253 e01-1256-36254 e01-1256-36255 QC- 25ppb -1 (1004 ng/mL diluted sera) (3020405036 QC- 25ppb -2 (1004 ng/mL diluted sera) (3020405037 Q C - 25ppb -3 (1004 ng/mL diluted sera) (1020405038 QC- 250ppb -1 (10040 ng/mL diluted sera) <3020405039 Q C - 250ppb -2 (10040 ng/mL diluted sera) (1020405040 QC- 250ppb -3 (10040 nq/mL diluted sera) <3020405041 0.05 0.05 0.05 0.05 0.05 0.05 40 40 40 40 40 40 i 43.07 1723 i 35.30 1412 1 40.31 1612 1 264.06 10562 1 269.60 10784 1 268.75 10750 A 171.6 avg 157.6 A 140.6 std dev 15.69 A 160.6 %RSD 9.957 105.2 avg 106.6 107.4 s td d e v 1.189 107.1 %RSD 1.115 eOI-1256-36256 Q C - 4.0 ppm -1 (160640 ng/mL diluted sen <1020405042 801-1256-36257 QC- 4.0 ppm -2 (160640 ng/mL diluted sen <1020405043 e01-1256-36258 QC- 4.0 ppm -3 (160640 ng/mL diluted serid020405044 0.05 0.05 0.05 40 40 40 10 479.65 191860 10 395.97 158388 10 380.39 152156 119.4 98.60 94.72 avg 104.3 s td dev 13.29 %RSD 12.75 A = Not within +/-25% criteria o o Cone of PFOS in Sample (ng/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilu6on(diluted sera)) H- for all QC samples rerun analysis (recovery) avg %rec 122.8 std dev o f %rec 28.08 dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 m170.05 mL = 40 W amount of sera in diluted sample (mL) = amount of sera used/total amount of sera + water (1/20 dilution). = 1/20 = 0.05 mL 3M Environmental Laboratory ftlAnlrc n r .o a n d M Q c Page 48 Report EOI-1256 Study number E01-1256 - MALLARD SERA PFOS: A Reproduction Study with the Mallard 3M Sam ple Number Sam ple Description E01 *1256-28663 454-109-3218, 0 PPM, Female, Adult 01-1256-28665 454-109-3220, 0 PPM, Female, Adult 01-1256-28667 454-109-3222,0 PPM, Female, Adult E01-1256-28669 454-109-3224,0 PPM, Female, Adult 01-1256-28670 454-109-3226,0 PPM, Female, Adult E01-1256-28672 454-109-3228,0 PPM, Female, Adult EOt-1256-28674 454-109-3230,0 PPM, Female, Adult E0M256-28676 454-109-3232, 0 PPM, Female. Adult E01 *1256-28678 454-109-3234. 0 PPM, Female, Adult E01-1256-28679 454-109-3236, 0 PPM, Female, Adult E01-1256-28681 454-109-3238. 0 PPM, Female, Adult E01-1256-28683 454-109-3240, 0 PPM, Female, Adult E01-1256-28685 454-109-3242, 0 PPM, Female, Adult E01-1256-28687 454-109-3244, OPPM, Female, Adult E01-1256-28689 454-109-3246, OPPM, Female, Adult E0M256-28691 454-109-3248, 0 PPM, Female, Adult E01-1256-28693 454-109-3250, 0 PPM, Female, Adull E01-1256-28695 454-109-3252, 0 PPM, Female, Adult E01-1256-28697 454-109-3254, OPPM, Female, Adult EOI-1256-28699 454-109-3256, 0 PPM, Female, Adult E01-1256-28701 454-109-3258, 10 PPM, Female. Adult E01-1256-28703 454-109-3260, 10 PPM, Female, Adult E01-1256-28705 454-109-3262, 10 PPM, Female, Adull E01-1256-28707 454-109-3264, 10 PPM, Female, Adult E01-1256-28709 454-109-3266, 10 PPM, Female, Adult E01 *1256-28711 454-109-3268,10 PPM, Female, Adult E01-1256-28713 454-109-3270,10 PPM, FemateTAdult E01-1256-28715 454-109-3272,10 PPM, Female, A d i* EOI-1256-28717 454-109-3274,10 PPM, Female, Adult E01-1256-28719 454-109-3276,10 PPM, Female, Adull E01-1256-28721 454-109-3278,10 PPM, Female, Adult EOI-1256-28723 454-109-3280.10 PPM, Female, A d i* E01-1256-28725 454-109-3282,10 PPM, Female, Adult E01-1256-28727 454-109-3284,10 PPM, Female, A d t* E01-1256-28729 454-109-3286,10 PPM, Female, A * * E01-1256-28731 454-109-3288,10 PPM, Female. Adult E01-1256-28733 454-109-3290,10 PPM, Female, A d i* E01-1256-28735 454-109-3292,10 PPM, Female, Adult E01-12S6-28737 454-109-3294,10 PPM, Female, A d i* E01-1256-28739 454-109-3296,10 PPM, Female, Adult E01-1256-28740 454-109-9583, 0 PPM, Female, Offspring E01-1256-28742 454-109-9589,0 PPM, Female, Offspring E01-1256*28743 454-109-9592,0 PPM, Female, Oflspring E01-1256-28744 454-109-9598,0 PPM, Female, Offspring E01-1256-28745 454-109-9603,0 PPM, Female, Offspring E01-1256-28747 454-109-9610,0 PPM, Female, Offspring E01 -1256-28748 454-109-9573,0 PPM, Female, Oflspring E01-1256-28751 454-109-9629,10 PPM, Female, Offspring E01-1256-28752 454-109-9633,10 PPM, Female. Offspring E01-1256-28753 454-109-9634,10 PPM, Female. Offspring E01-1256-28754 454-109-9645,10 PPM, Female, Offspring E01-1256-28755 454-109-9648,10 PPM, Female, Offspring E01-1256-28758 454-109-9654,10 PPM, Female, Offspring E01-1256-28759 454-109-9659,10 PPM, Female, Offspring Concentration <LOQ fnst. File Name d020225047 d020225049 d020225051 dO20225053 0020225054 d020Z25061 d020225063 d020225065 d020226050 d020226051 d020226052 d020226063 d020226054 0020226055 d020226056 020226057 d020226058 d020226064 d020226065 d020226066 <1020226067 d020226068 d020226069 d020226070 d020226071 <1020228031 (J020228032 020228033 d02040S045 d020228035 <1020228036 d020308048 d020228038 d020228039 <1020308049 d020228044 020228045 d020228046 d020228047 d02022804S CJ020228114 d020228115 d020228116 d020228117 d020228118 020228119 020228120 020228121 020228122 <*>20228123 d020228127 <1020228128 d020228129 <1020228130 am ount of sera used (m L) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.15 0.2 OS 0.1 OS OS 0.05 0.1 0.1 0.5 0.1 0.2 0.1 0,1 diSution (diluted sera) 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 a d d itio n a l d ilu tio n 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 10 10 1 10 1 1 10 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Inst Cone, (ng/m L of M ethanol) 1.90 1.00 2.21 1.00 1.53 66.94 1.00 4.30 2.51 2.51 4.16 2.51 2.51 2.51 2.51 9.96 2.51 18.92 2.51 2.S1 340.30 235.54 425.04 158.72 197.38 168.16 228.48 192.81 73.64 101.64 245.98 80.62 268.84 164.14 72.23 106.99 400.80 260.05 191.39 270.66 2.51 2.51 2.51 2.51 2.51 2.51 2.51 12828 85.41 168.96 93.74 122.59 136.20 133.73 C one of PFO S in loq for Sam ple (ng/m L of control Sera) sam ples 76.0 40.0 88.4 40.0 61.2 40.0 * 40.0 40.0 40.0 40.0 2678 40.0 40.0 re je cte d * 40.0 172 100.4 100.4 40.0 * 100.4 * 100.4 166 100.4 100.4 100.4 100.4 398 100.4 757 100.4 100.4 100.4 * 100.4 * 100.4 * 100.4 * 100.4 100.4 re je cte d * 100.4 100.4 re je cte d * 100,4 * 100.4 With outliers avg 271 std dev 589 %RSD 218 Without outlier avg 9 3 * std dev 37.0 % RSD 39* 13612 9422 17002 6349 7895 6726 91392 re je cte d 7712 29456 40656 9639 32248 10754 6566 28892 With outliers avg 1 2 std dev 20385 %RSD 100 42796 16032 10402 7656 10826 Without outliers avg 16571 std dev 11965 %RSD 72.2 100.4 100.4 100.4 100.4 100.4 100.4 100.4 100.4 100.4 100.4 100.4 e 100.4 100.4 e 100.4 avg 100* std dev 0 %RSD - 5131 3416 6758 3750 4904 5448 5349 avg 4965 std dev 1118 %RSD 22.5 Cone of PFOS In Sample (ngriiL) = (Inst conc(ng/mL)) x (additional dilution) x (dilution(diluted sera)) O dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/0.05 mL = 40 amount of sera in diluted sample (mL) = amount of sera used/total amount of sera + water (1/20 dilution). = 1/20 =0.05 mL w W ------- 3M Environmental Laboratory Page 49 Report EOI-1256 Study number E01-1256 - MALLARD SERA PFOS: A Reproduction Study with the Mallard 3M Sam ple Number Sam ple Description E01-1256-28662 454-109-3217, 0 PPM, Male, Adult E 0 M 256-28664 454-109-3219,0 PPM, Male, Adult E01-1256-28666 454-109-3221,0 PPM, Male. Adult E01-1256-28668 454-109-3223,0 PPM, Male, Adult E01-1256-28671 454-109-3227.0 PPM. Male. Adult E01-1256-28673 454-109-3229,0 PPM, Mate, AduR E01-1256-28675 454-109-3231,0 PPM, Male, Adult E01-1256-28677 454-109-3233, 0 PPM, Male, Adult E01-1256-28680 454-109-3237, 0PPM , Male, Adult E01-1256-28682 454-109-3239, 0 PPM, Male. Adult E01-1256-28684 454-109-3241, OPPM, Male, Adult E01-1256-28686 454-109-3243, OPPM, Male. Aduft E01-1256-28688 454-109-3245, OPPM, Male, Adult E01-1256-28690 454-109-3247, OPPM, Male, Adult E01-1256-28692 454-109-3249, OPPM, Male, Adult E01-1256-28694 454-109-3251, OPPM, Mala, Adult E01-1256-28696 454-109-3253, 0 PPM, Male, Adult E01-1256-28698 454-109-3255, OPPM, Male, Adult E01-1256-28700 454-109-3257, 10 PPM, Mate, Adult E01-1256-28702 454-109-3259, 10 PPM, Mate, Aduft E01-1256-28704 454-109-3261, 10 PPM, Male, Adult 01-1256-28706 454-109-3263, 10 PPM, Mate, Adult E01-1256-28708 454-109-3265. 10 PPM, Male, Adult E01-1256-28710 454-109-3267,10 PPM, Mate, Adult E01-1256-28712 454-109-3269,10 PPM, Male, Adult E01-1256-28714 454-109-3271,10 PPM, Male, Adult E01-1256-28716 454-109-3273,10 PPM. Mate. Adult EO1-1256-20718 454-109-3275,10 PPM, Male, Adult EOI-1256-28720 454-109-3277,10 PPM, Male, Adult E01-1256-28722 454-109-3279,10 PPM, Male, Adult E01-1256-28724 454-109-3281, 10 PPM, Mate, Adult E01-1256-28726 454-109-3283,10 PPM, Mate, Adult E01-1256-28728 454-109-3285,10 PPM, Male, Adult E01-1256-28730 454-109-3287,10 PPM, Male, Adull E01-1256-28732 454-109-3289, 10 PPM. Mate, Adult E01-1256-28734 454-109-3291,10 PPM, Mate, Adult E01-1256-28736 454-109-3293,10 PPM. Mate, Adult E01-1256-28738 454-109-3295,10 PPM, Mate, Aduft E01-1256-28741 454-109-9591,0 PPM, Male, Offspring E01-1256-28746 454-109-9606, 0 PPM, Male, Offspring E01-1256-28749 454-109-9578,0 PPM, Mate, Offspring E01-1256-28750 454-109-9625,10 PPM, Male, Offspring E01-1256-28756 454-109-9636,10 PPM, Mate, Offspring 601-1256-28757 454-109-9642,10 PPM, Male, Offspring Concentration <LOQ Inst. File Name <1020225046 <1020225048 d020225050 d020225052 d020225060 d020225062 d020225064 020226034 d020226035 <1020226036 <1020226037 (1020226038 <1020226039 20226040 d020226041 20226042 20226043 20226049 d020301106 <1020301107 d02030110 20411024 <1020301110 <20228049 <1020228050 <1020228051 20228052 d020228053 (20228057 20228058 <20228059 <1020228060 <20228061 <1020228062 20228063 <20228064 20228065 d020228066 20228131 (20228132 20228133 <1020228134 <1020228135 <1020228136 am ount of sera used (m L) dilution (diluted sera) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.3 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.1 0.5 0.5 0.2 0.5 0.06 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.3 0.3 0.3 0.5 0.5 0.1 0.2 0.2 0.2 0.1 02 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 a d d itio n a l d ilu tio n 1 f 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 10 10 100 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 1 1 1 1 Inst Cone. (ng/m L of M e th a n o l: 2.68 1.49 1.00 1.07 1.99 1.00 1.20 2.51 2.51 2.51 34.88 2.51 2.51 4.05 26.82 2.51 2.51 2.51 196.96 208.27 171.66 52.51 234.38 189.30 201.59 28.18 265.08 201.27 201.00 258.46 185.77 229.40 269.40 196.75 244.83 183.85 242.13 249.20 2.51 2.57 2.51 136.31 91.65 102.66 C o n e , of P F O S in S am p le (n g /m l of Sera) loq for control sam ples 107 59.6 40.0 42.8 79.6 40.0 48.0 100.4 100.4 100.4 1395 100.4 100.4 162 1073 100.4 100.4 100.4 78784 83308 68664 210040 93752 75720 80636 11272 106032 80508 80400 103384 74308 91760 107760 78700 97932 73540 96852 99680 100.4 103 100.4 5452 <3000 4106 40.0 40.0 * 40.0 40.0 40.0 40.0 40.0 100.4 100.4 100.4 100.4 100.4 100.4 100.4 100.4 100.4 * 100.4 * 100.4 avg 214 std dev 376 %RSD 176 re je cte d re je cte d With outliers avg 89652 std dev 35035 %RSD 39.1 Without outliers avg 87318 std dev 12337 %RSD 14.1 100.4 avg 101.2 100.4 * 100.4 std dev 1.39 %RSD 1,37 avg 4408 std dev 931 %RSD 21.1 Cone of PFOS in Sample (ng/mL) - (Inst conc(ng/mL)) x (additional dilution) x (dilution{dfluted sera)) dilution(diluted sera) = final volume(mLVamotnt of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample (mL) = amount of sera used/totai amount of sera + water (1/20 dilution). = 1/20 = 0.05 mL 3M Environmental Laboratory Page 50 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD LIVER DATA SUMMARY, BLANKS AND SPIKES 3M Sam ple Number Sam ple D e scription EOT*1326*36778 Blank H20-day 1 EOT-1326-36778 Blank H 20-day 1 EQ1-1326-36807 Blank H20-day 2 E01-1326-36812 Blank H20-day 3 E 0 M 326-36817 Blank H2Q-day 4 E01-1326-36780 Blank m allard fiver-day 1 E01-1326-36780 Blank m allard liver-day 1 E01-1326-368O9 Blank m allard liver-day 2 E 0 M 326-368U Blank m allard fiver-day 3 E 0 M 326-36819 Blank m allard fiver-day 4 In st. Fife Name d020304037 4020305036 d020306024 d020306104 020307024 am ount of liv p r w ebbed (g) or w ater alquoted (m l) 1.00 1.00 1.00 1.00 1.00 W ater Added fo r Hom genizm g (m L) 9.00 9.00 9.00 9.00 9.00 F 'rta! Volum e (m L) 2 00 2.00 2.00 2.00 2.00 Am ount of hver in d ilu te d sam ple (g) or w ater m L 0.100000 0.100000 0.100000 0.100000 0.100000 a d d itio n a l d ilu tio n 1 1 1 1 1 Inst C o re. (ng'm L Cone, o f PFOS in Sam p'e (ng/g of of M ethanol) L ive r) foq fo r c o n tro l sam ples 2.51 1.00 2.51 2.51 2.51 50.2 20.0 50.2 50.2 50.2 * 50.2 * 20.G * 50.2 * 50.2 * 50.2 0020304038 d020305037 0020306025 d020306105 d020307025 5.0597 5.0597 5-0597 5.0597 5.0597 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 0.101073 0.101073 0.101073 0.101073 0.101073 1 1 1 1 1 2.51 1.00 2.51 2.51 2.51 50.2 20.0 50.2 50.2 50.2 * 50.2 * 20.0 50.2 50.2 * 50.2 3M Sample Number _ _ .. Sample Description E01-1326-36781 M allard Liver MS-day 1 E01-1326-36781 M allard Liver MS-day 1 E01-1326*36810 M allard Liver MS-day 2 E01-1326-36815 M allard Liver MS-day 3 E01-1326-36820 MaHard Liver MS-day 4 Inst. File Name <1020304039 4020305038 <1020306026 <1020306106 <1020307026 am ount cf fiver weighed (g) 5.0597 5,0597 5.0597 5.0597 5.0597 Water Added for Homgenizing (mL) 45.0 45.0 45.0 45.0 45.0 Final Volume {m l} 2.00 2.00 2.00 2.00 2.00 Am ount of liver m diluted sample {q `> 0.101073 0.101073 0.101073 0.101073 0.101073 additional dilution 1 1 1 1 1 Inst Cone (ng/mL cf Methanol) 28.50 26.91 27.95 27.72 27.84 Cone, of PFOS in Sample (ng'g of Liver) 564 532 553 549 551 E 0 M 326-36782 M allard Liver MSD-day 1 E 0 M 326-36702 M allard Liver MSD-day 1 E01-1328-36811 M allard U ver MSD-day 2 E01-1328-36816 M allard Liver MSD-day 3 E01-1326-36821 M allard Liver MSD-day 4 * Concentration <LOQ <3020304040 <1020305039 <1020306027 d020306107 d020307027 5.0597 5.0597 5.0597 5.0597 5.0597 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 0.101073 0.101073 0.101073 0.101073 0.101073 1 1 1 1 1 26.86 26.18 26.20 27.50 27.68 531 518 518 544 548 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/m L)} x (additional dilution) x (fin a l volume (m L))/am ount of fiver in diluted sam ple (g) amount of liver fit diluted sam ple (g) = amount of fiver weighed (gy (water added for homogenizing (mL=g)} + amount cf liver weighed(g)) for both ms and msd avg %rac stddev of %rac avg cmic std dev of ernie 108.8 3.059 540.9 15.20 " c Rec 113.5 107.1 111.3 110.4 110.8 true val. 497 avg%rec 110.6 avg 549.8 stddev 11.33 106.9 104.2 104.3 109.5 110.2 true val. 497 avg%mc 107.0 avg 532.0 aid dev 13.91 S S 3C 3M Environmental Laboratory Page 51 Report EOI-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD LIVER DATA SUMMARY versus unextracted curve 3M Sample . Number ,, ,^ Sample Description K Inst. File Name amount of liver weighed (g) Water Added for H o m g e n i2in g (m L) E01-1326-36798 Q C -25ppb-1 (497 n ^ g homogenate) E01-1326-36799 QC- 25ppb -2 (497 ng/g homogenate) E01-1326-36800 QC* 25opb -3 (497 ng/g homogenate) 0020305045 <3020305046 0020305047 5.0597 5.0597 5 0597 45.0 45.0 45.0 E01-1326*36801 Q C *250ppb*1 (4967 ng/g homogenate) E01-1326-36802 QC- 250ppb -2 (4967 ng/g homogenate) E01-1326-36803 QC- 250ppb -3 (4967 ng/g homogenate) d020305048 d020305049 d020305050 5.0597 5.0597 5.0597 45.0 45.0 45.0 E01-1326-36804 QC- 4.0 ppm -1 (79467 ng/g homogenate) 0020405065 5.0597 45.0 E01-1326-36805 QC* 4.0 ppm -2 (79467 ng/g homogenate) E01-1326-36806 QC* 4.0 ppm -3 (79467 ng/g homogenate) d020405066 (3020405067 5.0597 5.0597 45.0 45.0 A = Not within W-25% criteria Final Volume (mL) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Absolute Recoveries Amount of liver in diluted sample (g) 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 additional dilution 1 1 1 1 1 1 10 10 10 Inst Cone, (n g /m l of Methanol) 28.87 29.22 35.74 242.46 249.01 278.56 440.20 443.16 436.91 Cone, o l PFOS in Sample (ng/g of Liver) 571 578 707 A 4798 4927 5512 87105 87691 86454 c-c Rec. 114.9 116.3 142.3 96.59 99.20 111.0 109.6 110.3 108.8 lo r ai) QC sam ples (absolute recovery) avg % rac s td dev o f % rec 112.1 13.07 s v g 124 5 s td d e v 15.41 %RSD 12.37 avg 102.3 s td dev 7.662 %RSD 7.493 avg 109.6 s td dev 0.7785 %RSD 0.7104 Study number E01-1256 - MALLARD LIVER DATA SUMMARY versus extracted curve 3M Sample Nurroer ,, ,^ Sample Description E01-1326-36798 QC- 25ppb-1 (497 ng/g homogenate) E01-1326-36799 QC- 25ppb -2 (497 ng/g homogenate) E01-1326-36800 QC- 25ppb -3 (497 ng/g homogenate) EQ1-1326-36801 QC- 250ppb -1 (4967 ng/g homogenate) E01-1326*36802 QC* 250ppb *2 (4967 ng/g homogenate) E01-1326-36803 QC- 250ppb -3 (4967 ng/g homogenate) E01* 1326*36804 QC* 4.0 ppm -1 (79467 ng/g homogenate) E01-1326-36805 QC- 4.0 ppm *2 (79467 ng/g homogenate) E01-1326-36806 QC- 4.0 ppm -3 (79467 ng/g homogenate) A s Not within +/-25% c rte ria Inst. File Name (J020305045 d020305046 (1020305047 d020305048 d020305049 (1020305050 <3020405065 d020405066 d020405067 am ount of liver weighed (g) 5.0597 5.0597 5.0597 5 0597 5.0597 5.0597 5.0597 5.0597 5.0597 Water Added for Homgenizing (mL) 45.0 45.0 45.0 45.0 45.0 45-0 45.0 45.0 45.0 Final Volume (rrU 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Cone of PFOS in Sample (ng/g) * (lo s t eonc(ng/m l)) x (additional dilution) x (final volum e (m l))/ am ount of live r in diluted sam ple (g) amount of liver in diluted sam ple (g) = am ount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed{g)) QC Sample Recoveries Am ount of liver in diluted sample ig i 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 additional dilution 1 1 1 1 1 1 10 10 10 fr s t Cone (nq.'mL Cerne of PFOS in of Methanol; Samnie (ng/g) 25.41 25.71 31.24 221.45 228.29 260.35 415.43 418.16 412.40 503 509 618 4382 4517 5152 82204 82744 81604 fo r a ll QC sam ples (recovery) avg % rc 102.3 s td dev o f % rc 10.14 c >Pec 101.2 102.4 124.4 88.22 90.95 103.7 103.4 104.1 102.7 avg 109.3 s td d e v 13.07 %RSD 11.96 avg 94.30 s td dsv 8.274 %RSD 8.774 avg 103.4 s td d e v 0.7175 %RSD 0.6937 3M Environmental Laboratory Page 52 Report E01-1256 Study number E01-1256 - MALLARD LIVER DATA SUMMARY 3M Sample Number Inst. File Sample Description Name E01*1326-29482 Oppm a.i. 3218 F Liver E01-1326-29583 E 0 M 326-296O1 E 0 1-1326-29619 0 ppm a .i. 3220 F Liver 0 ppm a.i. 3222 F Liver 0 ppm a .i. 3224 F Liver E01-1326-29637 0 ppm a.i. 3226 F Liver E01-1326-29655 0 ppm a .i. 3228 F Liver E01-1326-29673 0 ppm a .i. 3230 F Liver E01-1326-29691 0 ppm a.i. 3232 F Liver E01-1326-29709 0 ppm a .i. 3234 F Liver E01-1326-29727 0 ppm a.i. 3236 F Liver E01-1326-29745 0 ppm a.i. 3238 F Liver E01-1326-29763 0 ppm a .i. 3240 F Liver E01-1326-29781 0 ppm a.i. 3242 F Liver E01-1326-29799 0 ppm a.i. 3244 F Liver E 01-1326*29817 0 ppm a.i. 3246 F Liver E01-1326-29835 0 ppm a.i. 3248 F Liver E01-1326-29853 0 ppm a.i. 3250 F Liver E01-1326-2987 0 ppm a.i. 3252 F Liver EO t-1326-29889 0 ppm a .i. 3254 F Liver E01-1326-29907 0 ppm a .i. 3256 F Liver E01-1326-29925 10 ppm a.i. 3258 F Liver E01-1326-29943 10 ppm a.i. 3260 F Liver E01-1326-29961 10 ppm a.i. 3262 F Liver E01-1326-29980 10 ppm a.i. 3264 F Liver E01-1326-29998 10 ppm a.i. 3266 F Liver E01-1326-30016 EOM 326-30034 10 ppm a .i 3268 F Liver 10 ppm a i. 3270 F Liver E01-1326-30052 10 ppm a.i. 3272 F liv e r EO M 326-30070 10 ppm a.i. 3274 F Liver E01-1326-30088 10 ppm a.i. 3276 F Liver E01-1326-30106 EO I-1326-30124 10 ppm a.i. 3278 F Liver 10 ppm a.i. 3280 F liv e r E01-1326-30142 10 ppm a.i. 3282 F Liver E01-1326-30160 10 ppm a,i. 3264 F Liver E01-1326-30178 E01-1326-30196 10 ppm a.i. 3286 F Liver 10 ppm a.i. 3288 F Liver E01-1326-30214 10 ppm a.i. 3290 F Liver E01-1326-30232 E01-1326-30250 10 ppm a.i. 3292 F Liver 10 ppm a.i. 3294 F Liver E01-1326-30268 10 ppm a.i. 3296 F Liver E01-1367-31295 0 ppm a .i. 9583 F Liver E 0M 367-31307 0 ppm a .i. 9589 F Liver E01-1367-31313 E1-1367-31319 0 ppm a .i. 9592 F Liver 0 ppm a .i. 9598 F Liver E01-1367-31325 0 ppm a .i. 9603 F Liver E01-1367-31337 0 ppm a .i. 9610 F Liver E 0 M 367-31343 0 ppm a .i. 9573 F Liver E01-1367-31362 10 ppm a .i. 9629 F Liver E01-1367-31368 10 ppm a.. 9633 F Liver E01-1367-31374 10 ppm a.i. 9634 F Liver E01-1367-31380 10 ppm a.i. 9645 F Liver E01-1367-31386 10 ppm a i- F Liver E01-1367-31404 10 ppm a.i. 9654 F liv e r E01-1367-31410 10 ppm a.i. 9659 F Liver * Concentration <LOQ (#020305059 d020305060 (#020305061 <$020305062 d020305063 d020305064 d020305065 d020306031 (1020306032 d020306033 (1020306034 d020306035 (#020306036 d02C306037 (#020306038 (#020306039 d020306040 (#020306044 d020306045 d020306046 d020306066 d020306067 (#020307057 (#020306111 d020306112 d 020306113 (#020307059 (#020306115 d020307060 (1020307061 (1020306118 (#020307062 d020306120 d020306124 (#020307063 (1020307064 (#020307065 (#020306129 d020306129 d020306130 <1020307031 (#020307032 (#020307033 0020307034 <$020307035 (1020307036 (1020307037 (#020307044 (1020307045 <1020307046 (#020307047 d020307048 (#020307049 d020307050 amount of !ivcr weighed (gi Water Acded for Homgpnizing (m L) 1.0095 1.0944 1.0435 1.0425 1.0703 1.0526 1.0411 1.1704 9.00 9 .0 0 9 00 9 .0 0 9 .0 0 9.00 9.00 9 .0 0 1.1290 9.00 1.0800 1.1170 1.0086 1.0907 1.0291 1.1294 9.00 9.00 9.00 9 .0 0 9.00 9.00 1.1075 9.00 1.1709 1.0761 1.1344 1.0669 9 .0 0 9 .0 0 9 .0 0 9 .0 0 1.0034 9.00 1.1331 1.1294 9 .0 0 9.00 1.1829 1.0487 1.1214 9.00 9.00 9.00 1.0751 1.0658 9.00 9 .0 0 1.1226 9 .0 0 1.0953 1.1956 1.0600 1.0275 1.0915 1.0657 9 .0 0 9 .0 0 9.00 9 .0 0 9.00 9.00 1.0330 1.0352 9.00 9 .0 0 1.0658 1.0627 1.1238 9.00 9 .0 0 9.00 1.1308 1.0005 1.0390 1.1204 1.1550 1.0217 9 .0 0 9.00 9.00 9 .0 0 9 .0 0 9.00 1.0122 9 .0 0 1.1676 9.00 1.0510 1.1638 9 .0 0 9.00 1.1510 9.00 1.1125 9.00 1.0770 1.0581 9.00 9.00 Fina! Volume (mL) Am ount of liver in diluted sam ple (g) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.100854 0.108417 0.103898 0.103809 0.106283 0.104709 0.103684 0.115079 0.111462 0.107143 0.110408 0.100773 0.1 (090 0.102611 0.111497 0.109572 0.115123 0.106797 0.111936 0.105981 0.100306 0.111822 0.111497 0.116165 0.104362 0.110795 0.106709 0.105883 0.110900 0.108496 0.117266 0.105368 0.102468 0.108160 0.105874 0.102960 0.103157 0.105883 0.105608 0.111006 0.111820 0.100045 0.103496 0.110707 0.113737 0.101949 0.101097 0.114835 0.104567 0.114504 0.113388 0.110012 0 106877 0.105199 additional d d u 'io n 10 10 10 10 10 10 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Cone o l PFOS in Sample (ng/g) = (Inst conc(ng/m t)) x (additional dilution) x (final volum e (m L)y am ount ot Rver in diluted sam ple (g) am ount o f liver in diluted sam ple (g) = am ount o f liver weighed (g )/ (w ater added for homogenizing (mL=g)) + amount of Bver w eighed(g)) 3M Environmental Laboratory PFOS: A Reproduction Study with the Mallard Inst Cone (ng/mL Cone o f PFOS in Sample (ng<rg of of Methanol) Liver) 1.00 1.00 1.00 100 1.00 1.00 1.00 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 355.07 379.32 76.07 352.91 280.63 317.25 70.37 307.09 76.85 166.74 349.71 130.88 233.84 342.26 149.38 12422 101.98 243.62 251.58 351.37 2.51 2.51 2.51 2.51 2.51 2.51 2.51 169.63 117.36 258.53 146.41 192.68 316.88 188.42 20.0 20.0 20.0 20.0 20.0 20.0 20.0 50.2 502 502 502 502 50.2 502 502 502 502 502 502 502 7080 6784 13645 6076 5378 5727 13189 5801 13859 30737 5964 2484 4564 6329 28218 24130 19772 4602 4764 6331 502 502 502 502 502 502 502 2954 2245 4516 2582 3503 5930 3582 loq for control sa m p le s 20.0 20.0 20.0 20.0 20.0 20.0 20.0 502 502 502 50.2 502 50.2 502 502 502 50.2 502 502 502 avg 39.6 std dev 14.8 % RSD 37.3 50.2 502 50.2 502 50.2 502 502 avg 10772 etd dev 8463 % RSD 78.6 avg 50.2 std davo %RSD- v g 3616 std dev 1263 %RSD 34.9 Page 53 Report E01-1256 Study number E01-1256 - MALLARD LIVER DATA SUMMARY 3M Sample Number In st. File Sample Description Nim e E01-1326*29473 0 ppm a .i. 3217 M Liver E01-1326-29574 Oppm a .i. 3219 M Liver E0M 326-29592 0 ppm a.i. 3221 M Liver E01-1326-29610 0 ppm a.i. 3223 M Liver E01-1326-29628 0 ppm a .i. 3225 M Liver E01-1326-29646 0 ppm a .i. 3227 M Liver E01-1326-29664 0 ppm a .i. 3229 M Liver E01-1326-29682 0 ppm a.i. 3231 M liv e r 01-1326-29700 0 ppm a .i. 3233 M Liver E01-1326-29718 0 ppm a .i. 3235 M Liver E 0 M 326-29736 0 ppm a .i. 3237 M Liver 01-1326-29754 0 ppm a .i. 3239 M Liver E01-1326-29772 0 ppm a .i. 3241 M Liver E01-1326-29790 0 ppm a .i. 3243 M Liver E01-1326-29808 0 ppm a .i. 3245 M Liver E 0 M 326-29826 0 ppm a.i. 3247 M Liver E01-1326-29844 0 ppm a .i. 3249 M Liver E01-1326-29862 0 ppm a .i. 32S1 M Liver E01-1326-29880 0 ppm a .i. 3253 M Liver E01-1326-29898 0 ppm a.i. 3255 M Liver E01-1326-29916 10 ppm a.i. 3257 M Liver E 01-1326-29934 10 ppm a.i. 3259 M Liver E 01-1326-29952 10 ppm a .i. 3261 M Liver E 0M 326-29970 10 ppm a .i. 3263 M Liver E 0 M 326-29989 10 ppm a.i. 3265 M Uver E01-132630007 10 ppm a .i. 3267 M Liver E01-1326-30025 10 ppm a .i. 3269 M Liver E01-132630043 10 ppm a.i. 3271 M Uver E01-1326-30061 10 ppm a.i. 3273 M Uver E01-1326-30079 10 ppm a.i. 3275 M Liver E01-1326-30097 10 ppm a.i. 3277 M Liver E01-1326-30115 10 ppm a.i. 3279 M Liver E01-132630133 10 ppm a.i. 3281 M Uver E01-1326-30151 10 ppm a.i. 3283 M Uver E01-1326-30169 10 ppm a.i. 3285 M Liver E01-1326-30187 10 ppm a.i. 3287 M Liver EQ M 326-30205 10 ppm a .i. 3289 M Uver E 01-1326-30223 10 ppm a .i. 3291 M Liver E01-132630241 10 ppm a.i. 3293 M Liver E01-1326-30259 10 ppm a.i. 3295 M Liver E01-1367-31301 0 ppm a .i. 9591 M Liver E01-1367-31331 0 ppm a .i. 9606 M Uver E01-1367-31349 0 ppm a .i. 9578 M Liver E01-1367-31356 10 ppm a .i. 9625 M Liver E01-1367-31392 10 ppm a .i. 9636 M Uver E01-1367-31398 10 ppm a.i. 9642 M Uver *Concentration <LOQ d020305071 d020305072 0020305073 d020305074 d020305075 d020305076 d020305077 d020306047 d020306048 d020306049 d020306050 d020306051 d020306052 d020306053 d020306057 d020306058 0020306059 d020306060 d020306061 0020306062 d020306069 d020306070 d020306071 d020307058 0020306131 0020306132 0020306133 0020306137 d020306138 0020306139 0020306140 0020306141 0020306142 0020306143 0020306144 0020306145 0020306146 0020306150 0020306151 0020306152 0020307038 d020307039 0020307040 0020307051 0020307052 0020307053 am ount of liver weighed (g) Water Added for Homger:.' 'ng (m l) 1.1169 1.1847 9 .0 0 9.00 1.1181 1.0788 1.1043 9 .0 0 9.00 9 00 1.0486 1.0336 9 .0 0 9 .0 0 1.0165 1.0395 1.0373 9 .0 0 9 .0 0 9 .0 0 1.1679 9 .0 0 1.1493 1.0619 9 .0 0 9 .0 0 1.0263 1.1541 1.0813 9.00 9.00 9 .0 0 1.0263 1.0088 1.0294 9.00 9 .0 0 9 .0 0 1.0926 9 .0 0 1.0654 1.1316 9.00 9.00 1.1330 1.1404 9 .0 0 9 .0 0 1.1480 9 .0 0 1.0620 1.1687 1.0313 9.00 9 .0 0 9.00 1.1399 1.1143 9.00 9.00 1.0062 9 .0 0 1.0380 1.0669 1.0955 1.1981 9 .0 0 9.00 9.00 9.00 1.0500 1.0251 1.0441 1.0979 9 .0 0 9.00 9 .0 0 9.00 1.0815 1.0411 1.1314 9 .0 0 9.00 9.00 1.0413 1.0166 1.1367 1.1252 9 .0 0 9 .0 0 9.00 9.00 Final Volume (nL) Amount of liver in diluted sample (g 2.00 2.00 2.00 2.00 200 2.00 2.00 2.00 2.00 2.00 2 00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.110399 0.116322 0.110505 0.107037 0.109290 0.104353 0.103014 0.101483 0.103541 0.103345 0.114861 0.113239 0.105537 0.102361 0.113659 0.107258 0.102361 0.100791 0.102638 0.108258 0.105848 0.111690 0.111613 0.112461 0.113126 0.105546 0.114931 0.102808 0.112417 0.110171 0.100558 0.103407 0.105981 0.108514 0.117483 0.104478 0.102253 0.103952 0.108726 0.107276 0.103684 0.111673 0.103702 0.101492 0.112137 0.111129 additional dilution 1 10 10 10 100 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 1 1 1 1 Cone of PFOS in Sample (ng/g) = (Inst conc(ng'm l)) x (additional dilution) x (final volume (mL)V am ount of liver in dfluted sam ple (g) am ount of liver in diluted sam ple (g) = am ount oi liver weighed (9)/ (water added 1or homogenizing (m L=g)) + am ount of liver w eighed{g)) PFOS: A Reproduction Study with the Mallard Cone, of PFOS in Inst Cone. (ng'rr.L Samo'e (nq/q of of f/lethn-D ) Livor} 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.51 2.51 2.51 2,51 2.51 2.51 251 2.51 2.51 2.51 2.51 2.51 2.51 381.46 274.77 392.11 70.46 349.14 263.55 332.69 323.76 477.80 302.45 271.03 356.95 264.50 363.65 325.94 274.59 303.06 241.84 131.53 270.60 2.51 2.51 2.51 235.62 123.08 149.00 20.0 20.0 20.0 2 0 .0 20.0 20.0 20.0 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50-2 72077 49202 70137 125306 61726 49940 57894 62983 85005 54906 53905 69038 49915 67061 55487 52564 59276 46529 24195 50449 50.2 50.2 50.2 4643 2195 2682 loq for control sa m p le s 20.0 20.0 20.0 20.0 20.0 20.0 20.0 50.2 50.2 50.2 502 502 502 502 502 502 502 502 502 502 avg 39.6 aid dev 14.8 % RSD 37.3 avg COSSO stddav 19580 %ASD32 5 0 2 avg 502 50.2 stddevO 502 %RSD - avg 3173 std dev 1296 %RSO 40.8 001638 3M Environmental Laboratory Page 54 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachm ent C: Sam ple C hrom atogram s, Calibration Info rm atio n, and Instrum ent Information Fm/imnmAntal I ah/>rafnn/ 3M Environmental Laboratory P a n e *5 n f Page 55 91639 Report E01-1256 Report E01-1256 Methanol Solvent Blanks Solvent Blank 3M Environmental Lab d020226033 Sm (Mn, 1x2) : 100~i i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 26-Feb-2002, 22:16:16 1: M RM of 1 Channel ES- 6.77 980 TIC 1.10e4 Area %- O A r - r - r , n . .......................... | r i i I ............. ... ' 1 7- 1- 1 1 , 1 , - n - . | 1 . . 1 | 1 v r i n r , | '' 1 ' ' | ' 1 1 ' I 1 ' 1 ' I 1 ' 1 I ' 1 ' ' I 1 ' 1 1 I _l"r ' 1 I ' 1 1' r T i m e j _________ 1,00 2.00 3.00 4.00 5.00 6.00 ____7.00 8.00 9 .00 10.00 : 3M Fnvirnnmpntal I ohnratnrv 3M Environmental Laboratory Dono f OIC Page 56 i* i 1 G4.0 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Fnvirnnm A ntal I ahrtratnrv 3M Environmental Laboratory Pane <>7 o f 01 ft Page 57 001641 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard --' ' IT-p-T', . ] m - r H ' ' ' | 11' ''I 1 ......... .. ' ' I ' ' ' 1" , T ' T-.TT-P t_r | ' I I 1[ T , , , | - , . | 1.00 2.00 3 .00 4.00 5.00 6.00 7.00 ____8.00 I , ...................... 1 I 9.00 ___ 10.00 Pnw irrm m ontal I ahr>ratr\rw 3M Environmental Laboratory Do/io. r\( O-i K Page 58 1642 Report EOI-1256 Report E01-1256 Water Blank Samples PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Fm/irrnmmtal I a lw a to rv 3M Environmental Laboratory cn o-i c Page 59 031643 Report E01-1256 Report E01-1256 Blank H20-day 3 3M Environmental Lab d020228024 Sm (Mn, 1x2) 100- PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 28-Feb-2002,14:57:35 6.74 672 1: M R M of 1 Channel ESTIC 8.90e3 Area 3M F m /ironm ontal l ^hnrotrvrv 3M Environmental Laboratory Page 60 03IS44 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnv/irnnm pntal I a h n ra tn iv 3M Environmental Laboratory Dona ni Olft Page 61 Report E01-1256 Report E01-1256 Sera Blank Samples Blank H20-day 1 3M Environmental Lab d020225037 Sm (Mn, 1x2) 100-i i i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 2S-Feb-2002, 23:49:30 6.77 709 1:M RM of 1 Channel ES TIO 8.64e3 Area %-i 3M Fnvirnnmental I ahnratnrv 3M Environmental Laboratory D o h a CO 04C Page 62 0 0 1 o '--i Report E01-1256 Report E01-1256 Blank mallard sera-day 3 3M Environmental Lab d020228025 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 28-Feb-2002, 15:08:42 1: M RM of 1 Channel ES6.74 TIC 873 1.07e4 Area i %- l O-S-r'i r-p-n . ; i i W| i i i . , . i . i | , i i ^ t . ,T [ , , i ittt,, r - r-r i | " riy i r r r p , , | n-i . | i . i I r n r i | ................' 'T 1 " I ' 1 ' ' I Time ______ 1,00 2.00 3.00 4.00 5.00 6.00 7.00___ 8.00 9.00 10.00__ 3M Environmental Laboratory 3M Environmental Laboratory p' " n f Page 63 1M7 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard : ' 1 .............. 1.00 2.00 P ' 1 ' I ..........I ' ,_ r i I j-'Tf 11 I I I I-I'I I I---' I I I I I I I M I I I I I I I I I-I I !"'' ' T 1 1 r~r l 3.00 4 .0 0 _____5.00 6.00 7.00 ___ 8.00 9.00 11 Ml 10 .0 0 F n \jirr\n m A rrta l l 5Hnratr>r\< 3M Environmental Laboratory G>< O* Page 64 O S I 4 Report E01-1256 Report E01-1256 Liver Blank Samples PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvironmpnfal I ahnratnrv 3M Environmental Laboratory Ri rif OIK Page 65 031649 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvirnnm pnial I ah n ratn rv 3M Environmental Laboratory Qooo fiA of Page 66 091650 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Pana R7 M 0-1R Page 67 1 5 1 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Adult Male Sera Samples, Diluted 1:10 454-109-3285,10 PPM, Male, Adult,d10 3M Environmental Lab Id020228061 Sm (Mn, 1x2) i 100~l 6.74 293159 davey 070799 28-Feb-2002, 21:48:27 1: M R M o f 1 Channel ESTIC 3.16e6 Area i %-> ii 0-^ . ' i' i 1.00 ' rr' ",r ~Tl i l '-'~t '' 11rrl r''T" i''1 2.00 3.00 4.00 5.00 6.00 .......... . i'rr-i 1 1 i11 1 1 1 1 1 1 Tim e i 8.00 9.00 10.00 ! 3M Fnvirnnmental I ahnratnrv 3M Environmental Laboratory Pana Afl n f 91A Pr aa#ge 68 D1S52 Report E01-1256 Report E 0 1-1256 454-109-3279,10 PPM, Male, Adult,d10 3M Environmental Lab d020228058 Sm (Mn, 1x2) ! 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 283947 davey 070799 28-Feb-2002, 21:15:08 1: M RM of 1 Channel ESTIC 3.08e6 Area %- i 0- 3M Environmental Laboratory 3M Environmental Laboratory <' rri .......... ......... .... Time 8.00 9 .0 0 ____10,00___ Pane 69 of 216 Page 69 01653 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 1.00 2.00 _ 3.00 4,00 5.00 6.00 7.00 ____B.00 9.00 10.00 3M Environmental Lahnratorv 3M Environmental Laboratory Paae 70 of 21R Page 70 Oil I S 5 4 Report EOl-1256 Report E01-1256 1454-109-3281, 10 PPM, Male, Adult.dIO 3M Environmental Lab |d020228059 Sm (Mn, 1x2) I 100n PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 217116 davey 070799) 28-Feb-2002, 21:26:16) 1: M RM of 1 Channel ESTIC 2.40e6, Area I i i i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Fnvirnnm pntaf I ahnratruv 3M Environmental Laboratory P a Oft 71 o f 71 fi Page 71 001655 Report E01-1256 Report E01 -1256 454-109-3277,10 PPM, Male, Adult,d10 3M Environmental Lab d020228057 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 231932 davey 070799 28-Feb-2002, 21:04:03 1: M RM of 1 Channel ESTIC 2.54e6 Area i i i i Time i 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 72 of 216 Page 72 051G56 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratnrv 3M Environmental Laboratory Pano 7^ nf 01 Page 73 091657 Report E01-1256 Report E01-1256 454-109-3273,10 PPM, Male, Adult,d10 3M Environmental Lab d020228052 Sm (Mn, 1x2) 10CH i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 289554 davey 070799 28-Feb-2002, 20:08:29 1: M RM of 1 Channel ESTIC 3.16e6 Area % O | ' I ......................| ! 1 I I | 1 I I I | I I '" | I 1 I I | ' " I | I|I III| I II I| IIII| ...................... Tim e ! 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Paae 74 of 216 Page 74 001658 Report E01-1256 Report E01-1256 454-109-3271,10 PPM, Male, Adult,d10 3M Environmental Lab d020228051 Sm (Mn, 1x2) 100i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 38763 davey 070799 28-Feb-2002, 19:57:23 V. M RM of 1 Channel ESTIC 4.70e5 Area %- 1.00 2.00 n-r-prrr 3.00 4.00 'rrrrrTT 5.00 6.00 7.00 8.00 .-i-rTT-rn-n-i'T-i Tim e 9.00 10.00 3M Fnvimnmental I ahnratorv 3M Environmental Laboratory Pane 75 of 216 Page 75 001659 Report EO1-1256 Report E01-1256 454-109-3269,10 PPM, Male, Adult,d10 3M Environmental Lab d020228050 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 232496 davey 070799 28-Feb-2002,19:46:16 1: M RM of 1 Channel ESTIC 2.51 e6 Area i % O-1- -r--n Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnvironmftntal I ahoratnrv 3M Environmental Laboratory P a a e 7 6 of 216 Page 76 016S0 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Fnvirrnrrntal I ahnratnrv 3M Environmental Laboratory Dono 77 nf *)1i? Page 77 001661 Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Adult Female Sera Samples 454-109-3280,10 PPM, Female, Adult 3M Environmental Lab d020228037 Sm (Mn, 1x2) I 1001 6.74 487070' davey 070799 28-Feb-2002, 17:21:53 1: MRM of 1 Channel ESTIC 5.03e6 Area %- 0- n ,n -n -.-rr- i it-........... . Tim e . 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnvirnnm ontal I ahnratnrv 3M Environmental Laboratory D ono 7ft /\f Olf? Page 78 091662 Report E01-1256 Report E01-1256 454-109-3276,10 PPM, Female, Adult 3M Environmental Lab d020228035 Sm (Mn, 1x2) 100-1 I PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 1276061 davey 070799 28-Feb-2002, 16:59:39 1: MRM of 1 Channel ESTIC 1.17e6 Area 3M Environmental Laboratory 3M Environmental Laboratory Paoe79of 216 Page 79 091663 Report E01-1256 Report E01-1256 454-109-3258, 10 PPM, Female, Adult 3M Environmental Lab d020226067 Sm (Mn, 1x2) 10Ch PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 360788 davey 070799 27-Feb-2002, 04:34:15 1: MRM of 1 Channel ESTIC 3.86e6 Area %-j 1 i 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time 10.00 3M Environmental Laboratorv 3M Environmental Laboratory Pane 80 of 216 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard P m /im n m o n tal I ahnratrkrw 3M Environmental Laboratory Pane 1 nf 01R Page 81 0016S5 Report E01-1256 Report E01-1256 454-109-3272,10 PPM, Female, Adult 3M Environmental Lab d020228033 Sm (Mn, 1x2) 100-1 i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 224019 davey 070799 28-Feb-2002, 16:37:28 1: M R M of 1 Channel ESTIC 2.53e6 Area 1 T im e 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental 1ahoratorv 3M Environmental Laboratory Paae 82 of 216 Page 82 001686 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Pnwirnnrrtonfal I ahnrotnrw 3M Environmental Laboratory Donn Q1 of O-tC Page 83 001667 Report EOl-1256 Report E01-1256 1454-109-3278,10 PPM, Female, Adult [3M Environmental Lab d020228036 Sm (Mn, 1x2) ! 100-] PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 273170 davey 070799 28-Feb-2002,17:10:45 1: M R M o f 1 Channel ESTIC 3.01e6 Area %- 0 Time 1.00 ' 2.00 "T oo ' 4.00 " ' " 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 84 of 216 Page 84 c a ie s s Report EOl-1256 Report E01-1256 454-109-3264, 10 PPM, Female, Adult 3M Environmental Lab d020226070 Sm (Mn, 1x2) 100-, PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 196760 davey 070799 27-Feb-2002, 05:07:36 1: MRM of 1 Channel ES TIO 2.19e6 Area i %- z o oO 1.00 |,'T r r ' ,| | i ' [ i ' 111' i ' 1.......... ... ' i 11111 111111rr, Tr rr i ' 111' 11' 1 1 " M Time 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M F m /im nm ontal I ah n ratn rv 3M Environmental Laboratory Dono f l* nf 04 C Page 85 Report E01-1256 Report E01-1256 454-109-3262, 10 PPM, Female, Adult 3M Environmental Lab d020226069 Sm (Mn, 1x2) 100-. PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.76 415844 davey 070799 27-Feb-2002, 04:56:30 1: M R M o f 1 Channel ESTIC 4.42e6 Area i %- 3M Environmental Laboratory 3M Environmental Laboratory Panp flfi of 91 fi Page 86 Report E01-1256 Report E01-1256 454-109-3260, 10 PPM, Female, Adult 3M Environmental Lab d020226068 Sm (Mn, 1x2) 10CH PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.76 273820 davey 070799 27-Feb-2002, 04:45:23 1: MRM of 1 Channel ES TIO 3.02e6 Area %- O T i -i 1.00 ' ' zoo" 3.00 4.00 5.00 ri-p-T-i-T-;........... I i 1' 1I........... I Time i 8.00 9.00 10.00 l 3M Fnvironmental I ahnratorv SM Environmental Laboratory Pane R7 nf 216 Page 87 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Male Offspring Sera Samples 1454-109-9642, 10 PPM, Male, Offspring 3M Environmental Lab d020228136 Sm (Mn, 1x2) davey 070799 0 1 -M a r -2 0 0 2 ,11:41:03 1: M RM of 1 Channel ESTIC 1.74e6 Area %- 1.00 2.00 3.00 4.00 l *-T-|--!--Tr 5.00 6.00 7.00 8.00 T ! Time 9.00 10.00 Frt\/imnmorfol I ahnratrtrw 3M Environmental Laboratory Darwsflfi rtf 94 Page 88 031S72 Report E01-1256 Report E01-1256 454-109-9625, 10 PPM, Male, Offspring 3M Environmental Lab d020228134 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 183965 davey 070799 0 1 -M a r-2 0 0 2 ,11:18:49 1: MRM of 1 Channel ESTIC 2.17e6 Area I %i C K '". ' I .................................... . i i I " i i : i r n .............. . 1.00 2.00 3.00 4,00 5.00 3M Environmental Laboratorv 3M Environmental Laboratory Time 10.00 Page 89 of 216 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 90 of 216 Page 90 G21674 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Female Offspring Sera Samples 454-109-9654, 10 PPM, Female, Offspring 3M Environmental Lab d020228129 Sm (Mn, 1x2) 11U0U0 168735831 davey 070799 01-Mar-2002,10:23:19 1: M RM of 1 Channel ESTIC 2.20e6 Area % 1 .0 0 ....... 2.00 ' 3 .OO 4.00 5.00 6.00 7.00 8.00 i h i- T1 1 1 .i t, Time 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 91 of 216 Page 91 Report E01-1256 Report E01-1256 454-109-9659, 10 PPM, Female, Offspring 3M Environmental Lab d020228130 Sm (Mn, 1x2) 11U0U0n PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 186078568 davey 070799 01-M a r-2 0 0 2 ,10:34:21 1: MRM of 1 Channel ESTIC 2.11e6 Area %- O i . 1 1 1 1n i'i-iT pri i 1 1 1 , n Tim e i 1.00 2.00 3.00 4,00 5.00 6.00 7,00 8.00 9.00 10.00 | 3M Environmental Laboratory 3M Environmental Laboratory Pacte 92 of 216 Page 92 G0G7G Report EO1-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M FnvirnnmAntal I hnratrvv 3M Environmental Laboratory Pana OQ r*f 91A Page 93 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Page 94 of 216 Page 94 0 0 1 S7 8 Report E01-1256 Report E01-1256 454-109-9634, 10 PPM, Female, Offspring 3M Environmental Lab d020228123 Sm (Mn, 1x2) 100-. PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 221915' davey 070799 01-Mar-2002, 09:16:50 1: MRM of 1 Channel ES- TIC 2.59e6 Area %- i Time 1.00 2.00 3.00 4.00 5.00 6 .0 0 7.00 8 .0 0 9.00 10.00 SM Fm orrinm A ntal I ahnratrtn/ 3M Environmental Laboratory Dono OR n* 04 C Page 95 001679 Report EOI-1256 Report E01-1256 454-109-9629, 10 PPM, Female, Offspring 3M Environmental Lab d020228121 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 1742961 davey 070799 01-Mar-2002,08:54:44 1: MRM of 1 Channel ESTIC 2.1066 Area %- O-Li .......................... " i 1 M11n npi i'ij 'i .Til 11111Time 1.00 2.00 3.00 4.00 5.00 ' ' ' ' 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 96 of 216 Page 96 031680 Report E01-1256 Report E01-1256 454-109-9633, 10 PPM, Female, Offspring 3M Environmental Lab 02022B122 Sm (M n, 1x2) 100-i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 120377' davey 070799 01-Mar-2002, 09:05:46 1: MRM of 1 Channel EST1C 1.50e6 Area % i-rrr r T T IT' ii l |i iii|l PTn-rrrn n-rp-rr r p i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 97 of 216 Page 97 0016! Report EOI-1256 Report E01-1256 Dosed Group Mate Liver Samples 10 ppm a.i. 3285 M Liver, d10 3M Environmental Lab d020306144 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 308324 davey 070799 0 7 -M a r-2 0 0 2 ,12:04:28 1: MRM of 1 Channel ESnc 3.32e6 Area r' 11" * rntn-mp 11" " 11" T-rnTime 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 AM Fnvim nm Antal I ah oralrw 3M Environmental Laboratory D e n a fl 04C Page 98 031S82 Report E01-1256 Report E01-1256 10 ppm a.l. 3289 M Liver, d10 3M Environmental Lab d020306146 Sm {M n, 1x2) 10Ch PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 _ 293135 davey 070799 0 7 -M a r -2 0 0 2 ,12:26:46 1: MRM of 1 Channel ESTIC 3.21 e6 Area i " 'T 1 1 11 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fm/irnnmontal I a lw a tn rv 3M Environmental Laboratory P a n o QQ rtf 01ft Page 99 C; n i Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 3M Environmental Lahnratnrv 3M Environmental Laboratory Pana 100 nf ?1fi Page 100 001684 Report E01-1256 Report E01-1256 10 ppm a.I. 3283 M Liver, d10 3M Environmental Lab d020306143 Sm (M n, 1x2) 10(h PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 331373 davey 070799 0 7 -M a r-2 0 0 2 ,11:53:20 1: MRM of 1 Channel ESTIC 2.68e6 Area %- ' I " 11I " " I " " I " 11I ' 1" I " " 'P " 1P I ' ' ' ' I ' ' ' ' I ' ' 1' I ' 1.00 2.00 3.00 4.00 5.00 6.00 n n " i ' ' (' ' ' 1I' ' ' ' I ' ' ' ' I ' i Time 7.00 8.00 9.00 10.00 Pmilmff>mer>ta! I a h n r a tiw 3M Environmental Laboratory Pant 101 nf 91K Page 101 001685 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Pm/irr>nmntal I ahnraforv 3M Environmental Laboratory Pans -If nf SIR Page 102 00JLG86 Report EOI-1256 Report E01-1256 10 ppm a.i. 3279 M Liver, d10 3M Environmental Lab d020306141 Sm (M n, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 327374 davey 070799 0 7 -M a r-2 0 0 2 ,11:31:06 1:MRM of 1 Channel ES- TIC 3.53e6 Area 3M Enwimnmnntal Lahoratnrv 3M Environmental Laboratory n Time P an a in .3 n f 91R Page 103 lt.s ? Report EOl-1256 Report E01-1256 10 ppm a.l. 3277 M Liver, d10 3M Environmental Lab d020306140 Sm (M n, 1x2) 10(h PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 270271 davey 070799 0 7 -M a r -2 0 0 2 ,11:19:58 1:MRM of 1 Channel ESTIC 2.44e6 Area ''I " " I 1" M" 11I1 !' ' '' !' 1.00 2.00 3.00 111" M i 1' 1111 4.00 5.00 6.00 11i ' i ' i ' ' 111' ' 11111*i Time 7.00 8.00 9.00 10.00 3M Environmental Laboratorv 3M Environmental Laboratory Pane 10d nf 91 Page 104 0 if 1 6 8 8 Report E01-1256 Report E01-1256 10 ppm a.l. 3273 M Liver, d10 3M Environmental Lab d020306138 Sm (M n, 1x2) 100-i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 384816' davey 070799 0 7 -M a r-2 0 0 2 ,10:57:41 1: MRM of 1 Channel ES- TIC 3.1766 Area m g*i n r Ji m i i T i m I 100 2.00 3.00 4.00 5.00 6.00 7.00 8,00 9,00 10.00 ; 3M Fnvim nm Antal I ah oratrw 3M Environmental Laboratory Pan la S n f 91R Page 105 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M FmrfmnmAntat I ahnratnrv 3M Environmental Laboratory P an rn fin f 51R Page 106 Report E01-1256 Report E01-1256 10 ppm a.t. 3275 M Liver, d10 3M Environmental Lab d020306139 Sm (M n, 1x2) 100-i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 292720 davey 07079! 0 7 -M a r-2 0 0 2 ,11:08:4* 1:MRM of 1 Channel ESTIC 3.16e6 Area %- 0 1' | | , > >| , r ' Tl TTI-T'TI M| M I I ,1 I I I | I MI | I I I I | I I I I ! I I I I | I I 1.00 2.00 3.00 4.00 5.00 6.00 p ri"'"i 7.00 8.00 ............. .. Time 9.00 10.00 3M Environmental Lahoratarv 3M Environmental Laboratory P a n a 107 0 91fi Page 107 Report E 01-1256 Report E01-1256 Dosed Group Female Liver Samples PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Pm /im nm A nfal I aho rato n r 3M Environmental Laboratory Paruk 10Pnt 91ft Page 108 Report EOI-1256 Report E01-1256 10 ppm a.I. 3276 F Liver 3M Environmental Lab d020306117Sm (Mn, 1x2) 100-, PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 703650 davey 070799 07*Mar-2002, 07:04:24 1: MRM of 1 Channel ESTIC 7.16e6 Area ' i"'1 " 1 1 " 11 " " i " " i " *, " 1 1 'r~r~>TrTr` rTr r " i 1' "i ^ * 1111 ' i 1111! 11 " i 1" M ' i 1' ' ' i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M &ivim nm nnff I ahoratorv 3M Environmental Laboratory P an a MKI n f Page 109 Report E01-1256 Report E01-1256 10 ppm a.!. 3274 F Liver 3M Environmental Lab d020306116 Sm (Mn, 1x2) 10(h PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 469679 davey 070799 07-Mar-2002, 06:53:18 1:MRM of 1 Channel ESTIC 4.98e6 Area %- CHn-r i-r-i [-m -t-p I II | Il I i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M P n d m n m A n lal ( ahnrafruv 3M Environmental Laboratory PaoA 110 o f 91 ft Page 110 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Paae 111 of 216 Page 111 C 31595 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 3M PnvimnmAnlal I ahnralrw 3M Environmental Laboratory PariA Page 112 001S96 Report E01-1256 Report E01-1256 10 ppm a.i. 3266 F Liver 3M Environmental Lab d020306112 Sm (Mn, 1x2) 10(h PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 277317 davey 070799 07-Mar-2002,06:08:49 1: MRM of 1 Channel ESTIC 3.16e6 Area %} 0 11i!ii '' I' ' 1111111111111 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 113 of 216 Page 113 001897 Report E01-1256 Report E01-1256 10 ppm a.i. 3268 F Liver 3M Environmental Lab d020306113 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 302664' davey 070799 07-Mar-2002,06:19:56 1: MRM of 1 Channel ESTIC 3.37e6 Area %- T -1 i-i-n i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 MM F nvirnnm nntal l ahn rato rv 3M Environmental Laboratory Pace 114 of216 Page 114 001698 Report E01-1256 Report E01-1256 10 ppm a.I. 3264 F Liver 3M Environmental Lab d020306111 Sm (Mn, 1x2) 10CH PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 _ 324993 davey 070799 07-Mar-2002,05:57:39 1: MRM of 1 Channel ESTIC 3.67e6 Area %- 6.00r-p-rr 1.00 2.00 3.00 4.00 5.00 1 i .... . Time 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 115 of216 Page 115 C01699 Report EOl-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Paoe 116 of 216 Page 116 001700 Report E01-1256 Report E01-1256 10 ppm a.i. 3258 F Liver 3M Environmental Lab d020306066 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 315337' davey 070799 0 6 -M a r-2 0 0 2 ,21:37:37 1:MRM of 1 Channel ES TIO 3.60e6 Area % O-Vl-rr-p 1.00 ' " I ' " ' I 11" I 1111I 1r,1,T l ' 11 I 1 2.00 3.00 4.00 5.00 6.00 " i " " i " '1 r Time 7.00 8.00 9.00 10.00 3M Pnuim nm A nbl I ahrtrotsin/ 3M Environmental Laboratory D an a 4 <1*7es4 04 C Page 117 001701 Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Male Offspring Liver Samples 10 ppm a.i. 9636 M Liver 3M Environmental Lab d020307052 Sm (Mn, 1x2) 100-, 6.70 129040' davey 070799 0 8 -M a r-2 0 0 2 ,00:39:44 1: MRM of 1 Channel ES TIO 1.52e6 Area % ' T1" 1I ' 1' ' T ' rTTT i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Pmrnnmantal I ahnraltvi/ 3M Environmental Laboratory (tana 1in nt 01R Page 118 C Z: 1.7 0 2 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard *3M C n v iim n m a n b l I a h /v o ln n / 3M Environmental Laboratory P a n a 110 nf 91ft Page 119 001703 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Pm rironm anfol I ah/watrvru 3M Environmental Laboratory P a n e HOA O tft Page 120 CD1 7 0 4 Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Female Offspring Liver Samples 3M Environmental l ahoratnrv 3M Environmental Laboratory P an e 101 o f 01 Page 121 Report E01-1256 Report E01-1256 10 ppm a.i. 9034 F Liver 3M Environmental Lab d020307046 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 671 _ 240373 davey 070799 07-Mar-2002,23:33:03 1: MRM of 1 Channel ES- TIC 2.76e6 Area % 1I " " H' i-i-H T " n Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fm/irnnmontnl I ahnratnrv 3M Environmental Laboratory P aon 1V> f 91R Page 122 01705 Report E01-1256 Report E01-1256 10 ppm a.1.9629 F Liver 3M Environmental Lab d020307044 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 170716 davey 070799 07-Mar-2002,23:10:50 1:MRMof 1 Channel S- TIC 2.0366 Area %- i 1,000 ` l << ' | T I I I I ' I I ' I I TT' I IT I 2.00 3.00 I |T I I l-p - l n 4.00 5.00 "| l I. .I..I .|.l..I ....I r |-| rMnI 1I1|1'. 1I 11111 6.00 7.00 8.00 ................. Time 9.00 10.00 'IM Pntrim nm ankil I ohnratrw r 3M Environmental Laboratory Pona Page 123 001707 Report E01-1256 Report E01-1256 10 ppm a.i. 9645 F Liver 3M Environmental Lab d020307047 Sm (Mn, 1x2) 100h PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.70 150376 davey 070799 0 7 -M a r-2 0 0 2 ,23:44:10 1:MRMof 1 Channel ESTIC 1.77e6 Area % " 1I ' 1" I 1' " I ' 11111r i' 11 1" lT VT' 'T " " Pi p. rTime 1.00 2.00 3.00 4.00 5.00 ' 6.00''' 7.00 8.00 "' ' 9?00 10.00 3M Fnvirnnmpntjal I ah n rafcw 3M Environmental Laboratory Pann HdriOIR Page 124 031708 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Pnx/irrinmAntal I ahrvratrwv 3M Environmental Laboratory Pan 19Rnf01fi Page 125 31709 Report E01-1256 Report E01-1256 10 ppm a.1.9633 F Liver 3M Environmental Lab d020307045 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 123667"! davey 070799 07-Mar-2002,23:21:57 1: MRM of 1 Channel ES TIO 1.52e6 Area %- T Ti ....................... i ''' *i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fm/imnmontal I ahnrafnrv 3M Environmental Laboratory P ana 19 r>fH R Page 126 001710 Report E01-1256 Report E01-1256 10 ppm a.i. 9654 F Liver 3M Environmental Lab d020307049 Sm (Mn, 1x2) 100n PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.T2 279003 davey 070799 0 8 -M a r-2 0 0 2 ,00:06:20 1:MRM of 1 Channel ESTIC 3.11e6 Area % i I i n i |''"i i H i i i i | i i i i-| i i n | ' I ' ' ' 1I' ' ' ' I ' ' ' 'I ' i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environm ontal lab o rato ry 3M Environmental Laboratory Pan 197 nf 91 Page 127 001711 Report EOI-1256 Report E01-1256 Control Group Male Sera Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M PmrimnmAntal I ahnratorv 3M Environmental Laboratory Dona 400 a# 0 4 0 Page 128 'w17.1.2 Report EOI-1256 Report E01-1256 Control Group Female Sera Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard C n tn 'm n m a n ta l I a h rtra M n / 3M Environmental Laboratory D oaub 1 0 Q rvf 0 4 fi Page 129 001713 Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Male Offspring Sera Sample Qlljl C m r l m n m o n t Q l I o t w a t n n r 3M Environmental Laboratory P ans n n nf Page 130 051714 Report E 01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Female Offspring Sera Sample Fm rlrnnm antal I a lw a tn iv 3M Environmental Laboratory Pan 131 nf 91 fi Page 131 021715 Report EOI-1256 Report E01-1256 Control Group Male Liver Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M F nvim nm antal I nhnratrw v 3M Environmental Laboratory D o n a 4 3 0 r J 04 ft Page 132 00171 Report EOI-1256 Report E01-1256 Control Group Female Liver Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M P n v lm n n w ital I ah n ratn n / 3M Environmental Laboratory Pano 1 3 3 n f 71ft Page 133 001717 Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Male Offspring Liver Sample 3M Environmental Labnratorv 3M Environmental Laboratory Pan * 1fU rf 0 p a^ -- ' <x f j Report EOI-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Female Offspring Liver Sample Fnvim nm enlal I ahnratruv 3M Environmental Laboratory Paae 135 of 216 Page 135 001719 Report EOI-1256 Report E01-1256 Extracted Sera Calibration Curve Point PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard P n \itrrv tm n b t I aK n ratn ro 3M Environmental Laboratory Dam i'aC O-tft Page 136 051720 Report EOI-1256 Report E01-1256 Extracted Liver Calibration Curve Point PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 4M Fnvirrnm ontai I ah o ratn rv 3M Environmental Laboratory Paae 137 of 216 Page 137 L 7 2 L Report E01-1256 Report E01-1256 Unextracted Calibration Curve Point PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Pnuim nm A ntal I ahnratnrv 3M Environmental Laboratory P a n a 14A nf Page 138 091722 Report EOl-1256 Report E01-1256 Extracted Sera Calibration Curve PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvim nm nntal Lahoratrwv 3M Environmental Laboratory Pan 139 of 21fi Page 139 001723 Report EOI-1256 Report E01-1256 Extracted Liver Calibration Curve PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environm ental Lahoratorv 3M Environmental Laboratory Pane 140 of 21 fi Page 140 001724 Report E01-1256 Report E01-1256 Mass Spectrometer Tune Settings PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Tuning Method Report Method: C :\MASSLYNX\DAVEY 07 0799. PRONACQUDB \CENTRE1 Printed: Tue Mar 19 10:22:06 2002 Page 1 MS SOURCE ( ESP- ) Capillary Cone Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. Set Rdbk 2.56 20 0.5 0.2 0.8 150 250 -2.48 -20 148 249 Pressures Analyser Vacuum Gas Cell Rdbk 3.8e-5 2.9e-3 Analyser LM Res 1 HM Res 1 IEnergy 1 Entrance Collision Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Gas Flows Cone Gas Desolvation Set Rdbk; 13.0 13.Q 0.7 2 11 1 11.0 11.0 1.0 650 1 11 0 -646 Rdbk 150.5 701.5 3M Environmental Laboratory 3M Environmental Laboratory Paae 141 of 216 Page 141 <03172 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard HPLC Settings Method Report Method File: Last Modified: Printed: Page 1 C:\MASSLYNX\DAVEY070799.PROyVi Monday, March 11,2002 08:01:01 Tuesday, March 19,2002 08:37:50 HP1100 LC Pump Initial Conditions Solvents A% B% C% D% 90.0 10.0 0.0 0.0 Valve A set to channel Valve B set to channel Flow (mltaiin) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left('C) Oven Temperature Right('C) 0.300 10.0 0 400 40.0 40.0 HP1100 LC Pump GradientTimetable The gradient Timetable contains 5 entries which are: Time A% B% C% D% 0.00 90.0 10.0 0.0 0.0 1.00 90.0 10.0 0.0 0.0 5.50 5.0 95.0 0.0 0.0 7.50 5.0 95.0 0.0 0.0 8.00 90.0 10.0 0.0 0.0 Flow 0.300 0.300 0.300 0.300 0.300 HP1100 LC Pump External Event Turntable The Timetable contains 3 entries which are : Time Initial 0.00 0.10 Column Switch Ofl Ofl Ofl On Ofl Ofl Contactl Off On Ofl Contact2 Ofl Ofl Ofl Contac Off On Off HP1100Autoaamplar Initial Condtfona Draw Speed Eject Speed (plfmin) Draw Position (mm) Stop Time (mins) Injection Vdumefpl) Vial Number Thermostat On Thermostat Tempersture(*C) 200.0 200 0.00 10.00 10.0 94 20.0 QM Pnwimnmantal I ghnrolivu 3M Environmental Laboratory Dana H A O nfOIf Page 142 e:;i7; Report E01-1256 Report E01-1256 Mass Spectrometer Scanning Parameters PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Scanning Method Raport Method: C :\MASSLYNX\DAVKY07 0799.PRO\ACQTOB\10MIH-PFOS -PFDA Last Modified: Mon Jan 28 15:20:24 2002 Printed: Tua Mar 19 08:38:22 2002 Solvent Delay < nine ) : 0.00 Function : 1 MRM of 1 Mesa Pair ( ESP- ) Inter Channel Delay 1[ Secs ) : Span ( Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone ( V 1 499.00 99.00 0.30 43 60 Function : 2 MRM of 1 Maas Pair ( ESP- ) Inter Channel Delay ( Secs ) : Span ( Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone 1 513.00 219.00 0.30 20 20 Page 1 Pm/lmnmckntal I ah n ratn rv 3M Environmental Laboratory Pano 149 nf 91 Page 143 001727 Report E01-1256 Report E01 -1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard At ta c h m e n t D : S a m p l e P r ep a r a tio n S h e e ts 3M Pnvm nm A ntal I ah nratnrv 3M Environmental Laboratory Pane 144 of 216 Page 144 0,fft f rv q JL 9 f(* C w sample number E01-1326-29473 E01-1326-29482 E01-1326-29574 E01-1326-29583 E01-1326-29592 E01-1326-29601 E01-1326-29610 E01-1326-29619 E01-1326-29628 E01-1326-29637 E01-1326-29646 E01-1326-29655 E01-1326-29664 E01-1326-29673 E01-1326-29682 E01-1326-29691 E01-1326-29700 E01-1326-29709 E01-1326-29718 E01-1326-29727 E01-1326-29736 E01-1326-29745 E01-1326-29754 E01-1326-29763 E01-1326-29772 E01-1326-29781 E01-1326-29790 E01-1326-29799 E01-1326-29808 E01-1326-29817 E01-1326-29826 E01-1326-29835 E01-1326-29844 E01-1326-29853 E01-1326-29862 E01-1326-29871 E01-1326-29880 E01-1326-29889 E01-1326-29898 E01-1326-29907 E01 -1326-29916 E01-1326-29925 E01-1326-29934 E01-1326-29943 E01-1326-29952 E01-1326-29961 E01-1326-29970 E01-1326-29980 E01-1326-29989 description 0 ppm a.i. 3217 M Liver 0 ppm a.i. 3218 F Liver 0 ppm a.i. 3219 M Liver 0 ppm a.i. 3220 F Liver 0 ppm a.i. 3221 M Liver 0 ppm a.i. 3222 F Liver 0 ppm a.i. 3223 M Liver 0 ppm a.i. 3224 F Liver 0 ppm a.i. 3225 M Liver 0 ppm a.i. 3226 F Liver 0 ppm a.i. 3227 M Liver 0 ppm a.i. 3228 F Liver 0 ppm a.i. 3229 M Liver 0 ppm a.i. 3230 F Liver 0 ppm a.i. 3231 M Liver 0 ppm a.i. 3232 F Liver 0 ppm a.i. 3233 M Liver 0 ppm a.i. 3234 F Liver 0 ppm a.i. 3235 M Liver 0 ppm a.i. 3236 F Liver 0 ppm a.i. 3237 M Liver 0 ppm a.i. 3238 F Liver 0 ppm a.i. 3239 M Liver 0 ppm a.i. 3240 F Liver 0 ppm a.i. 3241 M Liver 0 ppm a.i. 3242 F Liver 0 ppm a.i. 3243 M Liver 0 ppm a.i. 3244 F Liver 0 ppm a.i. 3245 M Liver 0 ppm a.i. 3246 F Liver 0 ppm a.i. 3247 M Liver 0 ppm a.i. 3248 F Liver 0 ppm a.i. 3249 M Liver 0 ppm a.i. 3250 F Liver 0 ppm a.i. 3251 M Liver 0 ppm a.i. 3252 F Liver 0 ppm a.i. 3253 M Liver 0 ppm a.i. 3254 F Liver 0 ppm a.i. 3255 M Liver 0 ppm a.i. 3256 F Liver 10 ppm a.i. 3257 M Liver 10 ppm a.i. 3258 F Liver 10 ppm a.i. 3259 M Liver 10 ppm a.i. 3260 F Liver 10 ppm a.i. 3261 M Liver 10 ppm a.i. 3262 F Liver 10 ppm a.i. 3263 M Liver 10 ppm a.i. 3264 F Liver 10 ppm a.i. 3265 M Liver Amount of liver weighed (g) weighed by/date Homogenized w/ water to (ml) homogenized by/date I 2&*2 o t I. M l? 1. n s t V 7 1w La l./o < *2 > < L Q 2 3 ) 1 i'O ^ e L l.oi _ L l2 s ^ L g __ to ^ s * / _____________ t . 33V... /. O j o f ^ 1 1. / n o / im l.ccA i O G ,/ef- A ii * i,o m / i, io i s / I. c o m \.0 TG/o, . ) i.M U J I ,oG>G>7 q 1.06 W t U ' f l i. l, 1.10 e) (- W W * i . m f o j __ ___$z _ _______ _____ : i _____ 3M Environmental Laboratory f Page 145 031729 sample number E01-1326-29998 E01-1326-30007 E01-1326-30016 E01 -1326-30025 E01-1326-30034 E01-1326-30043 E01-1326-30052 E01-1326-30061 E01-1326-30070 E01-1326-30079 E01-1326-30088 E01-1326-30097 E01-1326-30106 E01-1326-30115 E01-1326-30124 E01-1326-30133 E01 -1326-30142 E01-1326-30151 E01-1326-30160 E01-1326-30169 E01-1326-30178 E01-1326-30187 E01-1326-30196 E01-1326-30205 E01-1326-30214 E01-1326-30223 E01-1326-30232 E01-1326-30241 E01-1326-30250 E01-1326-30259 E01 -1326-30268 E01-1367-31295 E01 -1367-31301 E01-1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31331 E01-1367-31337 E01-1367-31343 E01-1367-31349 E01-1367-31356 E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31392 E01-1367-31398 E01-1367-31404 E01-1367-31410 PFOS: A description 10 ppm a.i. 3266 F Liver 10 ppm a.i. 3267 M Liver 10 ppm a.i. 3268 F Liver 10 ppm a.i. 3269 M Liver 10 ppm a.i. 3270 F Liver 10 ppm a.i. 3271 M Liver 10 ppm a.i. 3272 F Liver 10 ppm a.i. 3273 M Liver 10 ppm a.i. 3274 F Liver 10 ppm a.i. 3275 M Liver 10 ppm a.i. 3276 F Liver 10 ppm a.i. 3277 M Liver 10 ppm a.i. 3278 F Liver 10 ppm a.i. 3279 M Liver 10 ppm a.i. 3280 F Liver 10 ppm a.i. 3281 M Liver 10 ppm a.i. 3282 F Liver 10 ppm a.i. 3283 M Liver 10 ppm a.i. 3284 F Liver 10 ppm a.i. 3285 M Liver 10 ppm a.i. 3286 F Liver 10 ppm a.i. 3287 M Liver 10 ppm a.i. 3288 F Liver 10 ppm a.i. 3289 M Liver 10 ppm a.i. 3290 F Liver 10 ppm a.i. 3291 M Liver 10 ppm a.i. 3292 F Liver 10 ppm a.i. 3293 M Liver 10 ppm a.i. 3294 F Liver 10 ppm a.i. 3295 M Liver 10 ppm a.i. 3296 F Liver 0 ppm a.i. 9583 F Liver 0 ppm a.i. 9591 M Liver O ppm a.i. 9589 F Liver 0 ppm a.i. 9592 F Liver 0 ppm a.i. 9598 F Liver 0 ppm a.i. 9603 F Liver 0 ppm a.i. 9606 M Liver Oppm a.i. 9610 F Liver 0 ppm a.i. 9573 F Liver 0 ppm a.i. 9578 M Liver 10 ppm a.i. 9625 M Liver 10 ppm a.i. 9629 F Liver 10 ppm a.i. 9633 F Liver 10 ppm a.i. 9634 F Liver 10 ppm a.i. 9645 F Liver 10 ppm a.i. 9648 F Liver 10 ppm a.i. 9636 M Liver 10 ppm a.i. 9642 M Liver 10 ppm a.i. 9654 F Liver 10 ppm a.i. 9659 F Liver Amount of liver weighed (g) weigher by/date Homogenized w/ water to (ml) homogenized by/date t . O G & O 7, tJ S l . i-I U o <. 0 ? S U t.O Z & A , OQS^l ak _T L____\ ____ _____L /a 2 *7 ^ 'C Z H t) t-i 2 2 0 * i - . g g . 6 3 T_ (.0 O G 2 /r t, 0Gex>4 /- O ? 5^<? 1- 0*115 4 -L im y 1. * 2 5 7 , 1 *-i730a _ l, O 'b lq 1 f < ______i __ L_____ L l. o 15*1 1 _____!U M 3 o ^ a 2z\-2. >k (. 4L ;i3=____ 1. 1SSOa, j i-cm f i -t .gcffrG&f af * 1.1o n -G ^ 1. o s io * \ 1. HUH t.ijg J k - l .5 3 ^ ' P ilg a - --A\ J 3M Environmental Laboratory Page 146 021730 Report E01-1256 PFOS: A S P E C o lu m n s E xtraction W o rk sh eet ard ox Prep Date: 3/4/2002 Analysts initials: OK/HOJ Urns Assigned sample number Sample Number or description Volume of sample filtered (ml) Type of column used and lot Method Revision: ETS-8-231_. Study Number Matrix: Liver / Z & Amount and spike Elution solvent m ix used and volume Comments E01-1326-36778 E01-1326-36780 E01-1326-36781 E01-1326-36782 E01-1326-36783 E01-1326-36784 E01-1326-36785 E01-1326-36786 E01-1326-36787 E01-1326-36788 E01 -1326-36789 E01 -1326-36790 E01-1326-36791 E01-1326-36792 E01-1326-36793 E01-1326-36794 E01-1326-36795 E01 -1326-36796 E01 -1326-36797 E01-1326-36798 E01-1326-36799 Blank H20-day 1 Blank mallard liver-day 1 Mallard Liver MS-day 1 Mallard Liver MSD-day 1 Curve point-0.2 ppb In liver extract Curve point-0.5 ppb in liver extract Curve point-1 ppb in liver extract Curve polnt-2.5 ppb in Over extract Curve polnt-5 ppb in iv e r extract Curve poinl-10 ppb in iver extract Curve point-25 ppb in liver extract Curve polnl-50 ppb In iver extract Curve point-75 ppb in liver extract Curve point-100 ppb In Iver extract Curve point-100 ppb-2 in liver extract Curve point-250 ppb in iver extract Curve polnt-500 ppb in liver extract Curve point-750 ppb In liver extract Curve point-1000 ppb in iver extract Q C - 2 5 p p b -1 Q C - 2 5 ppb -2 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W 2M 5B 2 Waters, 1g, 6mt, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, tot W2045B2 vvaibrs, ig, omi, lot W2045B2 l-ot- 2ml of MeOH NA TN-A-0009 2mtof MeOH NA TN-A-0008 2ml of MeOH 50UI of 02001-66 TN-A-0009 2mtof MeOH 50ul of 02001 -66 TN-A-0009 2mlof MeOH 0.4ul of 02001 -66 TN-A-0009 2mlof MeOH 1ul of 02001-66 TN-A-0009 2ml of MeOH 2ul of 02001 -66 TN-A-0000 2ml of MeOH Sul of 02001-66 TN-A-0009 2mlof MeOH lOul of 02001 -66 TN-A-0009 2ml of MeOH 20ul of 02001-66 TN-A-0009 2ml of MeOH 50ul of 02001-66 TN-A-0009 2ml of MeOH 100ul Of 02001 -66 TN-A-6009 2mlof MeOH 150ul of 02001 -66 TN-A-0009 2mlof MeOH 2O0ul of 02001-66 TN-A-0009 2m laf MeOH 4ul of 02001-53 TN-A-0009 2ml of MeOH 10ui Of 02001-53 TN-A-0009 2ml of MeOH 20ul of 02001 -53 TN-A-0009 2mfofMeOH 30ul of 02001 -53 TNAt-0009 2ml of MeOH 40ul of 02001-53 TN-A-0009 1ul of 02001-53 1ul of 02001-53 2ml of MeOH TN-A-0009 mi of MeOhi TN-A-6009 A , / il/ Blank Liver TN- A- ; Amount of liver: 5 .0 S *5)-? g Homogenize liver with Kandiyohi water; Amount of water added. 4 ^ - ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- 4 * 4 ^ 1to allquated sample . ^ (m * > a , $, Shake sample for 20 min @ 300 rpm (Shaker V u i R j a fK ) 0 4 ( 6 ^ ^ Centrifuge sample for 10 min @ 2000 rpm (Centrifuge oK. f-i Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH ( T N - A - _ 6 ^ o _ 2 _ ) w ash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent ( H e O H TN -A -Q i ) Into appropriate 15 ml centrifuge tube S u i.Spike samples with internal standard PFDA standard number C & s x s | - 4 g ________ , conc.b a l f y A M amount added. Transfer sample into apprlpriatelly marked autovial . 3M Environmental Laboratory Page 147 Report EOl-1256 SPE Columns Ex.rac.ion W o S e * ith the Mallard us ornaci Prep Date: 3/4/2002 /x if o Analysts initials: OK/HOJ fi Urns Assgned sample number EOI-1326-36800 E O I-1326-36801 E01 -1326-36802 E01-1326-36803 E01-1326-36804 E01-1326-36805 E01-1326-38806 E01-1326-29473 EOI-1326-29482 E01 -1326-29574 EOI-1326-29583 E01-1326-29592 E01-1326-29601 EOI-1326-29610 EOI-1326-29619 E01-1326-29628 E01-1326-29637 E01-1326-29646 E01-1326-29655 EOI -1326-29664 E01 -1326-29673 Sample Number or description QC- 25ppb -3 QC- 250ppb -1 QC- 250ppb -2 QC- 250ppb -3 QC- 4.0ppm -1 QC- 4.0ppm -2 QC- 4.0ppm -3 O ppm a.1.3217 M Liver 0 ppm a.i. 3218 F Liver 0 ppm a.i. 3219 M Liver 0 ppm a.i. 3220 F Liver 0 ppm a.i. 3221 M Liver 0 ppm a.i. 3222 F Liver 0 ppm a.i. 3223 M Liver 0 ppm a.i. 3224 F Liver 0 ppm a.i. 3225 M Liver 0 ppm a.i. 3226 F Uver 0 ppm a.i. 3227 M Liver 0 ppm a.i. 3228 F Liver 0 ppm a.i. 3 2 2 0 M Liver 0 ppm a.i. 3230 F Liver Volume of sample filtered (ml) Type of column used and lot 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W 204582 Waters, 1g, 6ml, lot W204SB2 Waters, 1g,6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot VK2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g,6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W 204582 W aters, 1g, 6m ). lot W 2045B2 w aters, ig , omi, lot 1AG045B2 <*>>oK <f4Z. Method Revision: EETTSS--88--2231 Study Number: E01- Matrix: Liver /Z 5 3 - 4 " t - Amount and spike Elution solvent mix used and volume Comments 2m lof MeOH 1ul of 02001-53 TNA-6000 2m lof MeOH 10ui of 02001-53 TNA-6000 2ml of MeOH 10ui of 02001-53 TNA-6009 2m lofM eO H 10ui of 02001-53 TN-A-6000 2ml of MeOH 160ul of 02001-53 TNM-aooe 2ml of MeOH 160ul of 02001-53 TN-A-eooe 2m lof MeOH 160ul of 02001-53 TN-A-6009 NA 2m lo(M O H TrM -e009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TNA-6009 NA 2ml of MeOH TN-A-6009 NA 2mlofM eOH TNA-6000 NA 2mi of MeOH TNA-6000 NA 2m lof MeOH TNA-6000 NA 2ml of MeOH TNA-6000 NA 2m lof MeOH TNA-6009 NA 2ml of MeOH TNA-6000 NA 2ml of MeOH TNA-6000 NA NA 2m lofM eO H TNA-6000 2m lofM eO H TNA-6000 h iA ______________ 0/ Blank Liver TN-A- ( o o X 'ii ; Amount of liver: 5 OSc^ ~ l a Homogenize liver with Kandiyohi water; Amount of water added 4*5 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN fTN-A- ) to aliquated samole Shake sample for 20 min @ 300 rpm fShakerVjte) O ^ tU S ^ M Centrifuge sample for 10 min @ 2000 rpm (Centrifuge Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH ( T N - A - _ ^ 2 S 3 _ ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Alow column to go completelly dry Elute column with indicated amount of solvent ( tfe O H TN-A- _) Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number Q < P o o t - & S~ cone. . amount added. Transfer sample Into appripriatelly marked autovlal 3M Environmental Laboratory Page 148 001732 Report E01-1256 SPE Standard Curvw-Solids PFOS: A Reproduction Study with the Mallard Prep Dita(i): Analyit(a): Sample Matrix: Mctbad/Revisian: T a r jr t Aaalyte(): 03/04/02,03/05/02,03/06/02,03/07/02 OK/HOI Mallard Uver ETS-l-231.1 PFOS Study Number. E01-1256 Equipmcol Number; NA Pinal Sebent J eTN Number: MeOH/TN-A-6009 Blank TtaffWUcadfier: M alted Ljver/TN-A-6023 PC Mix Std Apprax. 1.00 ppm: PC Mix Std Apprex, 50.2 ppm: S urr. Std Apprex. 100.7 ppm: Q2001-66 02001-53 02001-45 PFDA prosActual Concentrations of Standards si the FC Mix Standard Number Std Cone. ue/mL E01-1326-36783,0.2 ppb 1.004 E 0I-1326-36784, 0.5 ppb 1 004 E0M326-3678S, 1.0 ppb 1.004 B01-1326-36786,2.5 ppb 1.004 E01-1326-367S7. 5 0 ppb 1.004 E01-1326-3678B, 10 ppb 1.004 BQl-1326-36789, 25 ppb 1.004 E01-I326-36790, 50 ppb 1.004 E0M326-36791, 75 ppb 1.004 EOl-1326-36792,100 ppb 1.004 EOl-1326-36793,100ppb-2 50.20 E01-1326-36794, 250 ppb 50.20 E01-1326-36795, 500 ppb $0.20 E01-1326-36796, 750 ppb 50.20 E01-1326-36797, 1000 oob 50.20 QC-25B* 30.20 OC-JiOppb 50.20 OC-tppm 50.20 Mallard Uver-MS/MSD-Day 1 1.004 Mallard Uver-MS/MSD-Day 2 1.004 M alted Uvar-MS/MSD-Day 3 1.004 Mallard Uver-MS/MSD-Dw 4 1.004 PPOS Am't Spiked mL 0.0004 o.ooto 0.0020 0.0050 0.0100 0.0200 0.0500 0.1000 0.1500 0.2000 0.0040 0.0100 0.0200 0.0300 0.0400 0.0010 0.0100 0.1600 0.0500 0.0500 0.0500 0.0500 Calculated Concentrations of Standards in the sample matrix Standard Number PFOS StdConc. PFDA StdConc. a t/l nsta E01-1326-36783,0.2 ppb 3.97 1993 E0M 326-36784,0.5 ppb 9.93 1993 EOl-1326-36785,1.0 ppb 19.9 1993 E0I-I326-36786,2.5 ppb 49.7 1993 E01-I326-36787,5.0 ppb 99.3 1993 EOl-1326-36788,10 ppb 199 1993 E01-1326-36789,25 ppb 497 1993 E01-1326-36790, 50 ppb 993 1993 E0M326-36791, 75 ppb 1490 1993 EOl-1326-36792,100 ppb 1987 1993 EOl-1326-36793, 100ppb-2 1987 1993 E01-1326-36794,250 ppb 4967 1993 E01-1326-36795,500 ppb 9933 1993 E01-1326-36796, 750 ppb 14900 1993 E01-1326-36797. 1000 rob 19867 1993 QC-25 ppb QC-230 ppb 497 4967 1993 1993 OC-4Km Mallard Liver-MS/MSD-Day l 79467 497 1993 1993 Mallard Liver-MS/MSD-Day 2 497 1993 M alted Liver-MS/MSD-Day 3 Mallard Liver-MS/MSD-Dav 4 497 497 1993 1993 Calculated Concentrations o f Standards in the final sofamt Standard PFOS PFDA Number Std Cane. ns/mL Std Cane. nx/mL E01-1326-36783,0.2 ppb 0201 101 E0M 326-36784,0.5 ppb 0.502 101 EOl-1326-36785,1.0 ppb 1.00 101 E0M326-36786, ZS ppb 2.51 101 EOl-1326-36787, 5.0 ppb 5.02 101 E01-I326-36788,10 ppb 10.0 101 E01-1326-36789,25 ppb 25.1 101 E0I-I326-36790, 50 ppb 50.2 101 E01-1326-36791,75 ppb 75.3 101 EOl-1326-36792, 100 ppb 100 101 EOl-1326-36793.100ppb-2 100 101 E01-1326-36794,250 ppb 251 I0I E0I-1326-36795, 500 ppb 502 101 E01-1326-36796,750 ppb 753 101 E01-1326-36797. 1000 ppb 1004 101 QC-25 ppb 25.1 10) QC-250 ppb 251 101 OC-tppm 4016 101 Mallard Uver-MS/MSD-Day 1 25.1 101 MaUard Liver-MS/MSD-Dey 2 25.1 101 M alted Uver-MS/MSD-Day 3 25.1 101 Mallard Uvcr-MS/MSD-Dev 4 25.1 101 PPDA Std Cone. ufl/mL 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 3M Environmental Laboratory AB Amt Spiked mL 0.002 0002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0002 0.002 0002 0002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 Initial homogenate: 5.0J97ft/50.D597f+mL 0.1011 j/m L Blank liver Initia] bam n a u t a (f/aiL) far curvea: 0.1011 Pinal aalvtnt volume (mL) far curvea; 2.000 All Initial Homogenate g/mL 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0 1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.101] 0.1011 O.tOll 0.1011 0.1011 AU Final Vd. mL 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2000 2.000 2.000 2.000 2.000 2.000 2.000 1000 2.000 2.000 Verified by; _ \A l l\ I n i b . Page 149 001733 Report EOl-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/4/02 ok 799 900 5 Acetonitrile tn-a-4145 CH l 2 3 4 5 Volume, (mL) 1 to 10 M o io e Mass of solvent (g) Density1 3.9613 3.9484 fe/ml) 0.7857 0.7857 4.0278 0.7857 3.9249 0.7857 3.9858 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Faii: Volume of Solvent 5.0417 5.0253 5.1264 4.9954 5.0729 5.0524 101.05 0.04997 0.99 PASS PASS PASS P er u rm an ce Specificiitions Accuracy, <%) 98.0 - 102 % Corrective Actions: C.V., (%) 1.0% oK) `Density values taken from the Merck Index, 12a>Edition, Copyright 1996 d5', = specific gravity at y-**'*" c referred to water at xd*ff*f*c n-Hexane d204 =0.660 Acetone d25# =0.788 Methanol d" 4 =0.7866 Methylene Chloride dM4 =1.3255 Acetonitrile dM4 = 0.7857 3M Environmental Laboratory Page 150 031734 Report E01-1256 PFOS: A Re. S P E C o lu m n s E xtraction W ork sh eet with the Mallard IAS tffc llu f& y. U t 1 Prep Date: Analysts initials: o f c J Urns Assgned sample number E01-1326-36807 E01 -1326-36809 E01 -1326-36810 E01-1326-36611 E01-1326-29682 E01-1326-29691 E01-1326-29700 E01-1326-29709 E01-1326-29718 E01-1326-29727 E01-1326-29736 E01-1326-29745 E01-1326-29754 E01-1326-29763 E01-1326-29772 E01-1326-29781 E01-1326-29790 E01-1326-29799 E01-1326-29808 E01-1326-29817 E01-1326-29826 Volume of sample filtered Sample Number or description (ml) Type of column used and lot Blank H20-day 2 Blank mallard liver-day 2 Mallard Liver MS-day 2 Mallard Liver MSD-day 2 0 ppm a.I. 3231 M Liver 0 ppm a.i. 3232 F Liver 0 ppm a.i. 3233 M Liver 0 ppm a.i. 3234 F Liver 0 ppm a.i. 3235 M Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, ml, lot W204SB2 Waters, 1g, 6ml, lo tW 2 0 4 5 B 2 Waters, 1g,6ml, lot VW045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W204SB2 0 ppm a.i. 3236 F Liver 0 ppm a.i. 3237 M Liver 0 ppm a.i. 3238 F Liver 0 ppm a.i. 3239 M Liver 0 ppm a.i. 3240 F Liver 0 ppm a.i. 3241 M Liver 0 ppm a.i. 3242 F Liver 0 ppm a.i. 3243 M Liver 0 ppm a.i. 3244 F Uver 0 ppm a.i. 3245 M Liver O ppm a.i. 3 2 4 6 F Liver 0 ppm a.i. 3247 M Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W 204562 Waters, 1g, 6ml, lot W2045B2 lA/aters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lo t W 2045B2 Waters, ig, otm, lot W2045B2 Method Revision: ETS-8-231 Study Number: E01-J3 Matrix: Liver n f ` 7 Amount and spike Elution solven mix used and volume 2ml of MeOH NA TN-A-6009 2m! of MeOH NA TNnA-6009 2m lof MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 5Oui of 02001-66 TN-A-6009 NA 2m lof MeOH TN-A-6009 2ml of MeOH , NA TN-A-6009 2ml at MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2m lof MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2m lofM eO H NA TN-A-6009 2ml of MeOH NA TN-A-6009 2m lof MeOH NA TN-A-6000 NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 A/A lt Comments L Blank Liver TN-A-6023; Amount of liver: 5 . 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly WO O j 3o Add 5ml of ACN (TN-A-4145) to aliquated sample Shake sample for 20 min @ 300 rpm (Shaker VWR S/N 041694) Centrifuge sample for 10 min Q 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 mi polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (MeOH TN-A-6009) Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul Transfer sample Into appriprlatelly marked autovial 3M Environmental Laboratory Page 151 Report E01-1256 PFOS: A R epi^ ct a i M w !' S P E C o lu m n s E xtraction W o rk sh eet y foy, i *f x iIAth$eoMtfaiilUlaxrd Prep Date: Analysts initiais: Urns Assgned sample number tfA ^ Sample Number or description Volume of sample nitered (ml) Type of column used and lot E01-1326-29835 E01-1326-29844 E 0 1 -1 3 2 6 -2 9 8 5 3 E01-1326-29862 E01-1326-29871 E01 -1326-29880 E01 -1326-29889 E01-1326-29898 E01-1326-29907 E01-1326-29916 E01-1326-29925 E01-1326-29934 E01-1326-29943 E01 -1326-29952 E01-1326-29961 E01-1326-29970 0 ppm a.I. 3248 F Liver 0 ppm a.I. 3249 M Liver 0 ppm a.i. 3250 F Liver 0 pp m a.i. 3251 M Liver 0 ppm a.i. 3252 F Liver 0 pp m a.i. 3253 M Liver 0 ppm a.i. 3254 F Liver 0 ppm a.i. 3255 M Liver 0 ppm a.i. 3256 F Liver 10 ppm a.i. 3257 M Liver 10 ppm a.i. 3258 F Liver 1 0 ppm a.i. 3 2 5 9 M Liver 10 ppm a.i. 3260 F Liver 1 0 ppm a.i. 326 1 M Liver 1 0 ppm a.i. 3 2 6 2 F Liver 10 ppm a.i. 3263 M Liver 40+6 40+6 40+6 40+6 40+8 40+6 40*6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot VK2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot VW045B2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g. 6ml, lo tW 2 0 4 5 B 2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Method Revision: ETS-8-231 Study Number: E 01-l3e& ^ iC #*i Matrix: Liver t Z f Amount and spike Elution solven mix used and volume Comments NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2m lof MeOH rN-A-eooe 2ml of MeOH TN-A-8009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH 1 TN-A-6009 2m lof MeOH TN-A-6009 2ml of MeOH TN-A-6009 Zm lofM eO H TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2m lof MeOH TN-A-6009 m _____ 1/ _ _ ___________ __ -------- " tw 081r W Blank Liver TN-A-6023; Amount of liver 5 . 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver Into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample Shake sample for 20 min 3 0 0 rpm (Shaker VWR S/N 041694) .?'SO X - Centrifuge sample for 10 min 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract Into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding nitrate Allow column to go completelly dry Elute column with indicated amount of solvent (MeOH TN-A-6009) Into appropriate 15 ml centrifuge tube Snllr rnmnlan with in te rn a l s ta n d a rd PFDA standard number 02001-45. cone. 100.7 DDm. amount added 2ul 3M E nvironm ental L aboratory -- Page 152 031736 Report EOI-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/5/02 ok 799 900 5 Acetonitrile tn-a-4145 3 , <t -q OK l 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9429 (g/ml) 0.7857 3.9904 0.7857 3.959 0.7857 3.967 0.7857 3.9712 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0183 5.0788 5.0388 5.0490 5.0543 5.0479 100.96 0.02210 0.44 PASS Perl ormance Specificaliions Accuracy, (%> 98.0 - 102 % C.V., (%) 1.0% Corrective Actions: PASS PASS OK/ 'Density values taken from the Merck Index, 12th Edition, Copyright 1996 dyx = specific gravity at ydegr*" c referred to water at xto*r***c n-Hexane dJ04 =0.660 Acetone d150 =0.788 Methanol d254 =0.7866 Methylene Chloride d2#4 =1.3255 Acetonitrile d" 4 = 0.7857 3M Environmental Laboratory Page 153 031737 Report EOl-1256 PFOS: A Re S P E C o lu m n s E xtraction W o rk sh eet t pS - Prep Date: Analysts initials: Lims Assgned sample number E01-1326-36812 E01-1326-36814 EOI-1326-36815 E01-1326-36816 EOI-1326-29980 E01-1326-29969 E01-1326-29998 E01-1326-30007 E01 -1326-30016 EOI-1326-30025 E01 -1326-30034 E01-1326-30043 E01-1326-30052 E01-1326-30061 EOI-1326-30070 E01-1326-30079 E01-1326-30088 E01-1326-30097 E01 -1326-30106 E 0 1 -1 3 2 6 -3 0 1 1 5 E01-1326-30124 E01-1326-30133 Sample Number or description Volume of sample filtered (ml) Type of column used and lot Blank H20-day 3 Blank mallard liver-day 3 Mallard Liver MS-day 3 Mallard Liver MSD-day 3 10 ppm a.1.3264 F Liver 10 ppm a.I. 3265 M Liver 10 ppm a.i. 3266 F Liver 10 ppm a.i. 3267 M Liver 10 ppm a.i. 3268 F Liver 10 ppm a.i. 3269 M Liver 10 ppm a.i. 3270 F Liver 10 ppm a.i. 3271 M Liver 10 ppm a.i. 3272 F Liver 10 ppm a.i. 3273 M Liver 10 ppm a.i. 3274 F Liver 10 ppm a.i. 3275 M Liver 10 ppm a.i. 3276 F Liver 10 ppm a.i. 3277 M Liver 10 ppma.i. 3278 F Liver 10 ppm a.i. 3 2 7 9 M Liver 10 ppm a.i. 3280 F Liver 10 ppm a.i. 3281 M Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+7 40+6 Walers, lg , ml, lot W2045B2 Waters, 1g, 6ml, lotVU2046B2 Walers, lg , 6ml, lot W204SB2 W a te t*, 1g, 6ml, M W 2045B 2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, tat W2045B2 Waters, 1g, 6ml, tat W2045B2 Waters, 1g, 6ml, lot W204SB2 Waters, lg , 6ml, lot W2045B2 Waters, 1g, 6ml, tat W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, lg , 6ml, lot W2045B2 Waters, 1g, 6ml, tat W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Walers, 1g, 6ml, tot W2045B2 Waters, lg , 6ml, lot W2045B2 Waters, 1g, 6m l, lo t W 2046B2 Waters, 1g, 6ml, tat W2045B2 waters, lg, omi, lot W2045B2 Method Revision: ETS-8-231 Study Number E 0 1 - l 2 6 < g > ^ 3 * * 'i - _ Matrix: Liver Amount and spike Elution solven mix used and volume NA 2m iofM eO H TN-A-6009 2m lofM eO H NA TN-A-6009 2ml of MeOH 50ulo f 02001-66 TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 NA 2mlofM eOH TN-A-0009 NA 2m lof MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2m lof MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2m fof MeOH TN-A-6009 NA 2m lofM eO H TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2m lof MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2mlofM eOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2m! of MeOH TN-A-6009 NA NA 2mlofM #OH TN-A-6009 2ml of MeOH TN-A-6009 m , A Comments Blank Liver TN-A-6023; Amount of liver: 5 . 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver Into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to allquated sample Shake sample for 20 min 0 3 0 0 rpm (Shaker VWR S/N 041694) Centrifuge sample for 10 min 0 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (MeOH TN-A-6009) Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, cone. 100.7 ppm, amount added 2ul Transfer sample into appripriatelly marked autovlal 3M Environmental Laboratory Page 154 C51738 Report EOl-1256 SPE Columns Extraction W o r k .K e i* ^ < g t o ^ ^ M a l l a r d Prep Date: Analysts initials: Urns Assgned sample number E01-1326-30142 E01-1326-30151 E01-1326-30160 EOI-1326-30169 E01-1326-30178 E01-1326-30187 E01-1326-30196 E01-1326-30205 E01-1326-30214 E01-1326-30223 E01-1326-30232 E01-1326-30241 EOI-1326-30250 E01-1326-30259 E01-1326-30268 QK Sample Number or description Volume of sample filtered (ml) Type of column used and tot 10 ppm a.1.3282 F Liver 10 ppm a.i. 3283 M Liver 10 ppm a.I. 3284 F Liver 10 ppm a.i. 3285 M Liver 10 ppm a.i. 3286 F Liver 10 ppm a.i. 3287 M Liver 10 ppm a.i. 3288 F Liver 10 ppm a.i. 3289 M Uver 10 ppm a.i. 3290 F Liver 10 ppm a.i. 3291 M Liver 10 ppm a.i. 3292 F Liver 10 ppm a.i. 3293 M Liver 10 ppm a.i. 3294 F Liver 10p p m a .i. 3295 M Liver 10 ppm a.i. 3296 F Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Walers, 1g, 6ml, iot W2045B2 Wafers, 1g, 6ml, lof W2045B2 Walers, 1g, 6ml, Iot W2045B2 Walers, 1g, 6ml, lof W2045B2 Walers, lg , 6ml, Iot W2045B2 Waters, 1g, 6ml, lotW2045B2 Walers, 1g, 6ml, Iot W2045B2 Walers, 1g, 6ml, Iot W2045B2 Waters, lg , 6ml, Iot W2045B2 Walers, 1g, 6ml, toi W2045B2 Waters, 1g, 6ml, Iot W2045B2 Waters, 1g, 6ml, lol W2045B2 Waters, 1g, 6ml, lol W2045B2 Waters, lg , 6ml, kH W2045B2 Waters, 1g, 6ml, Iot W2045B2 Method Revision: ETS-8-231 Study Number E01 Matrix: Liver Amount and spike Elution solveri mix used and volume NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2ml of MaOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MOH TN-A-6009 2ml of MeOH rN-A-eooe 2m lof M*OH TN-A-OOO 2ml of MeOH TN-A-600S 2ml of MeOH TN-A-800B 2m) of M*OH TN-A-6009 2m lofW O H TN-A-6009 2ml of MoOH T N ^-6000 2m lofM *O H TN-A-6009 2ml of MOH TN-A-6009 Zm tofM oO H TN-A-6009 2ml of MoOH TN-A-600 2ml of MeOH TN-A-6009 j0 4 f Comments ' 7.6 oSlftW . Blank Liver TN-A-6023; Amount of liver: 5 . 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to allquated sample Shake sample for 20 min @ 300 rpm (Shaker VWR S/N 041694) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to SO ml polypropelene centrifuge tube. Decant extract Into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with Indicated amount of solvent (MeOH TN-A-6009) Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul 3M E nvironm ental L aboratory Page 155 C21739 Report E01-125 6 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/6/02 ok 799 900 5 Acetonitrile tn-a-4145 4 ,G -o % oK J 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9506 (g/ml) 0.7857 3.9857 0.7857 3.9874 0.7857 3.9772 0.7857 3.983 0.7857 AverageVolume: Volume of Solvent 5.0281 5.0728 5.0750 5.0620 5.0694 5.0614 %Accuracy: 101.23 Std. Dev.: %C.V.: Pass/Fail: 0.01927 0.38 PASS Per nrmance Specifieritions Accuracy, (%) 98.0 - 102 % c.v., (%) 1.0% Corrective Actions: PASS PASS DM 1Density values taken from the Merck Index, 12th Edition, Copyright 1996 d5, = specific gravity at yd*g"*'c referred to water at xde*"*` c n-Hexane dM4 =0.660 Acetone d250 =0.788 Methanol d" 4 = 0.7866 Methylene Chloride d2^ =1.3255 Acetonitrile d24 = 0.7857 3M Environmental Laboratory Page 156 001740 Report E01-1256 SPE Columns Extraction ^-M s^ard Prep Date: Analysts initials: ^ 0i Urns Assgned sample number Sample Number description E01-1326-36817 E01-1326-36819 E01-1326-36820 E01-1326-36821 E01-1367-31295 E01-1367-31301 E01 -1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31331 E01-1367-31337 E01-1367-31343 E01-1367-31349 E01-1367-31356 E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31392 E 0 1 -1 3 6 7 -3 1 3 3 8 E01-1367-31404 E01-1367-31410 Blank H20-day 4 Blank mallard liver-day 4 Mallard Liver MS-day 4 Mallard Liver MSD-day 4 0 ppm a.i. 9583 F Liver 0 ppm a.1.9591 M Liver 0 ppm a.i. 9589 F Liver 0 ppm a.i. 9592 F Liver 0 ppm a.i. 9598 F Liver 0 ppm a.i. 9603 F Liver 0 ppm a.i. 9606 M Liver 0 ppm a.i. 9610 F Liver 0 ppm a.i. 9573 F Liver 0 ppm a.i. 9578 M Liver 10 ppm a.i. 9625 M Liver 10 ppm a.i. 9629 F Liver 10 ppm a.i. 9633 F Liver 10 ppm a.i. 9634 F Liver 10 ppm a.i. 9645 F Liver 10 ppm a.i. 9648 F Liver 10 ppm a.i. 9636 M Liver 10 ppm a.i. 9 6 4 2 M Liver 10 ppm a.i. 9654 F Liver 10 ppm a.i. 9659 F Liver Volume of oi sample filtered Type ofcolumn (ml) used and lot 40+6 40+6 40+6 40+6 40+6 W a te n , ig , bmi, lot W2045B2 Watare, lg , 6ml, lo tW 2 0 4 5 B 2 Walers, lg , 6ml, lot W2045B2 Walers, lg , 6ml, lot W2045B2 w a te n , lg , 6ml, lot W2045B2 W a te n , 1g, 6ml, 40+6 40+6 40+6 lo tW 2 0 4 5 B 2 W ateis, lg , 6ml, lot W2045B2 w a te n , lg , 6ml, lot W2045B2 W a te n , lg , 6ml, 40+6 lo tW 2 0 4 5 B 2 W a te n , lg , 6ml, 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 lot W2045B2 W a te n , lg , 6ml, lot W2Q45B2 W a le n , lg , 6ml, lot W2045B2 W a te n , lg , 6ml, lot W2045B2 W a te n , lg , 6ml, lot W2045B2 W ate n , lg , 6ml, lot W2045B2 W aten , lg , 6ml, lot W2045B2 W a le n , lg , 6ml, lot W2045B2 W aten, lg , 6ml, lot W2045B2 W a te n , 1g, 6ml, lot W2045B2 W a le n , lg , 6ml, lot W2045B2 W a le n , lg , 6ml, lot W2045B2 W a le rs, lg , 6m l, lo t W 2045B2 W aten, lg , 6ml, lotVW045B2 40+6 W a te n , 1g, 6ml, lot W2045B2 Method Revision: ETS-8-231 Study Number: E O l s i a i f l ^ 7 ^ Matrix: Liver 7^ Amount and spike Elution solven mix used and volume 2ml of MeOH NA TN-A-eooe 2ml of MeOH NA TN-A-6009 2m fofM eO H SOul of 02001-66 TN-A-6009 2m lofM eO H 50ul of 02001-66 TN-A-eooe 2ml of MeOH NA TN-A-6009 2mi of MeOH NA TN-A-6009 2m) ot MeOH NA TN-A-6009 2ml of MeOH NA TNA-6009 2m lof MeOH NA TN^-SOOS 2m lofM eO H NA TNA-6009 2ml ot MeOH NA TNA-6009 2m! of MeOH NA TNA-6009 2ml of MeOH NA TNA-6009 2m! of MeOH NA TNA-6009 2ml of MeOH NA TNA-6009 2m! of MeOH NA TNA-6009 2ml of MeOH NA TNA-6009 2mi of MoOH NA TNA-6009 2m lofM eO H NA TNA-6009 2m lof MeOH NA TNA-6009 2m lofM eO H NA TNA-6009 2ml of MeOH NA TNA-eooe 2 mt of MeOH NA TNA-6009 A/A NA TNA-6009 \V Comments Blank Liver TN-A-6023; Amount of liver: 5 . 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample Shake sample for 20 min 3 0 0 rpm (Shaker VWR S/N 041694) Centrifuge sample for 10 min 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract Into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completely dry Elute column with Indicated amount of solvent (MeOH TN-A-6009) Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul Transfer sample Into apprlprtatelly marked autovlal 3M Environmental Laboratory Page 157 001741 Report EOl-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/7/02 ok 799 900 5 Acetonitrile tn-a-4145 oK y Mass of solvent (g) Density1 Volume of (8/ml) Solvent 1 3.9564 0.7857 5.0355 2 3.9674 0.7857 5.0495 3 3.982 0.7857 5.0681 4 3.9753 0.7857 5.0596 5 3.9713 0.7857 5.0545 AverageVolume: 5.0534 %Accuracy: 101.07 Std. Dev.: %C.V.: Pass/Fail: 0.01215 0.24 PASS Volume, (mL) 1 to 10 Per ormance Specificsirions Accuracy, (%) 98.0 - 102 % C.V., (%)_ 1.0% Corrective Actions: ta te 'Density values taken from the Merck Index, 12th Edition, Copyright 1996 d5, = specific gravity at y***TM"c referred to water at x***" c n-Hexane d2#4 =0.660 Acetone d150 =0.788 Methanol d" 4 =0.7866 Methylene Chloride d" 4 =1.3255 Acetonitrile d20 = 0.7857 3M Environmental Laboratory Page 158 031742 Report E01-1256 PFOS: A Reproduction Study with the Mallard A ttachm ent D: D ilutions Sum m ary W orksheet Study: E01-1326/1367 Dilution Date/Analyst: CD Box Number: o f ~ (?& G Solvent/TN Number: MeOH/54571 Extraction Date/Analyst: '9'32/ 3 '& '`cA.f O (o Matrix/Timepoint: jfA iU L A fC b n Sample Number Dilutions or Description 1/10 1/100 11 1/ 1/ 1/ 1/ Comments Verified By: E01-1326-29961 E01-1326-30034 E01-1326-30070 E01-1326-30088 X X X X MA /* b ^ <J-" n E01-1326-30178 X E01-1326-30196 X E01-1326-30214 X E01-1326-29916 X E01-1326-29934 X E01-1326-29952 X V E01-1326-29970 v/v X E01-1326-29989 X M E0I-1326-30007 X / / / IKS Pila ilo i E01-1326-30025 X E01-1326-30043 X E01-1326-30061 X E01-1326-30079 X i Notes: 1/10 d ilu tio n = IooijJ. o f sa m p le + o f solvent . ifioo d b 'iu fkyio i ^ jP ^ -bQuLMpi5 t ^ O jJ ) o] '3C* 'U jl \ _ J _______ .. ->K . .. (A )e>ye OO CP -jb cb^ c^ /eh Ctt*1 Form Completion Verified By: f c M 4 - 3M Environmental Laboratory Pay. U 1 IAS oftalLi Page 159 031743 Report EOI-1256 PFOS: A Reproduction Study with the Mallard A ttachm ent D: D ilutions Sum m ary W orksheet Study: M H { fe>H2><9//36 ? Dilution Date/Analyst: Box Number: o i ' ^ ^ o t*b<- Number: MeOH/5457 Extraction Date/Analyst: Matrix/Timepoint: CiXjefL, Notes: 1/10 dilu tio n \o o jJ) o f sam ple + o f solvent Form C om pletion V erified By: 3M Environmental Laboratory (iii^ PAt L d 2. i t ^ US w aita- Page 160 001744 PFOS: A Reproduction Study with the Mallard ^1 sample number 01-1256-28662 E01-1256-28663 E01-1256-28664 E01-1256-28665 E01-1256-28666 E01-1256-28667 E01-1256-28668 E01-1256-28669 E01-1256-28670 E01-1256-28671 E01-1256-28672 E01-1256-28673 E01-1256-28674 E01 -1256-28675 E01-1256-28676 E01-1256-28677 E01-1256-28678 E01-1256-28679 E01-1256-28680 E01-1256-28681 E01-1256-28682 E01-1256-28683 E01-1256-28684 E01-1256-28685 E01-1256-28686 E01-1256-28687 E01-1256-28688 E01-1256-28689 E01-1256-28690 E01-1256-28691 E01 -1256-28692 E01-1256-28693 E01 -1256-28694 E01-1256-28695 E01-1256-28696 E01-1256-28697 E01-1256-28698 E01-1256-28699 E01-1256-28700 E01-1256-28701 E01-1256-28702 E01-1256-28703 E01-1256-28704 E01-1256-28705 E01-1256-28706 E01-1256-28707 E01-1256-28708 E01-1256-28709 E01-1256-28710 description 454-109-3217, 0 PPWl Male, Adu| 454-109-321 0 PPM, Fehnalei! Adlt 454-109-321)9, 0 PPM, Male, Adu| 454-109-32201 0 PPM, rmale,;Adlt 454-109-322, o 'PPM Male, Aduli 454-109-3222] 0 PPM, :emale, Adlt 454-109-322, 0 PPM Male, Adult 454-109-3224] 0 PPM. ;emale, Adult 454-109-3226! 0 p N , :6male, Ad^lt 454-109-3227, 0 PPM Male, Adult 454-109-3228, 0 PPM, :male,Ad(ilt 454-109-3229, 0 PPM Male, Adulf 454-109-3230, 0 PPM, female, Adult 454-109-3231, 0 PPM. Male, Adult 454-109-3232J 0 PPM, Female.Adult 454-109-3233, 0 PPM, Male, Adult 454-109-3234* 0 PPM, fem ale Adult 454-109-3236 0 FPM, Female, Adllt 454-109-323^, 0 PPM, Male, Adu(t 454-109-3238, 0 PPM, Female, Adult 454-109-3238, 0 PPM Male, Aduli 454-109-3240; 0 P|>M, Female, Adult 454-109-3241, 0 PPM] Male, Adult 454-109-3242] 0 PpM, Female, Aditili 454-109-3248, 0 PPMi Male, Adu| 454-109-3244; 0 PPM, Female, Adllilt 454-109-3246, 0 PPM] Male, Aduft 454-109-3246; 0 PPM, Fmale,; Adtilt 454-109-324f, 0 PPM] Male, Adult 454-109-3248; 0 PPM, Fmale, Adylt 454-109-324 9, 0 PPM[Male, AduR 454-109-325Q 0 PfM , remale, Adtilt 454-109-325 I.O flP M Male, Aduft 454-109-3252 0 PPM, ;emale, Adult 454-109-325 1, 0 PPM ,Mal, Adu| 454-109-3254 0 PPM, remale, Adtilt 454-109-325 j, OWPM Male, Adutt 454-109-3250, OPPM, remale, Ad ilt 454-109-3257[ T o m .'Male, Adi t 454-109-3258J 10 w , Female, Aqult 454-109-3251 , 10 PPM, Male, Adult 454-109-3260; 10 PPM, Gmale, Acjult 454-109-3261 , lO PPfA Male, Adult 454-109-3262, 10 ppM/FemaM, Adult 454-109-326; , 10 PPM, Male, Adift 454-109-3264, 10 Pi^M.iFemale, Aqult 454-109-3268,10 PPM, Male, Adiflt 454-109-3266,110 P^M,`Female, Actilt 454-109-3267,10 PPM, Male, Adi|t Amount of sera aliquated Diluted with by/date water to (ml) aliauated(ml) diluted by/date - C j j g _____ /tjZ fU u 16 g -fa o f* /* /S .T O ______ _____ i___ a ft C -TO ____ I L____ L o fd , .. 1 ...... 1____ / 4* ! ta fa (0 /t . 6 _____ _____ M f -6 Q -J % ._____ C& / /o /+ so ` O 'f'd al _ tJ T < r.f oX _____ . / G P / a *'J1 -- 9 \ ____ L _ <9 /a a 0 -- A) <r r 0 -f a.s +x oX of aS of o .f 4?/ / <s f to . oX of r e>' l * ____ r s* /d /a X ____ _ / / /fi t 7. /<* /a 'i > / 1 1 \ 1 I & --0 P ~x / S > M l _______ ox /a .2 , _____ 1 _ 4 _ 6 - r _____ _____ L _______ o r ____ i ___ f d & 0 ia-vTtp Ta+ J iiS tcT ** 2 5Af Environmental Laboratory Page 161 0ill 745 f Report EO1-1256 & ) 2 -A PFOS: A Reproduction Study with the Mallard Jero^ ^ E01-1256-28724 E01-1256-28725 E01-1256-28726 E01-1256-28727 E01-1256-28728 E01-1256-28729 E01-1256-28730 E01-1256-28731 E01-1256-28732 E01-1256-28733 E01-1256-28734 E01-1256-28735 454-109-3280, 1Q1PPMJ Fematt, Adult 454-109-3281, 1Q PPM. Male, AjetUlt ~ 454-109-3282,10 PPM; Female, X<jult~ 454-109-3283,10 PPM, Male, Adult 454-109-3284, 10 PPM, Femat, A0ult 454-109-3285,10 PPM, Male, Addlt 454-109-3286, lO l^ M j Pfejnale;Kdult 454-109-3287, 10 PPM. Male, Adult 454-109-3288; 10PPM, Female, Aijiult 454-109-3289, lOPPM, Male. Addlt 454-109-3290; IQpPM, i, Adult 454-109-3291, 1( Male^dult 454-109-3292,10 PPM, Female, Adult .0 eT ,-r ,.jr c -r *-r JL XL Q ,1 Y<t So YQ JsJ*e - & 3M Environmental Laboratory Page 162 031746 Report EOl-1256 SPE Columns Extraction Worl t W * "1 Prep Date: 2/25/2002 Method Revision: ETS-8-231.0 Analysts initials: OK/HOJ Study Number: E01-1256 Urns Assgned sample number Sample Number or description Volume of sample filtered (ml) JlJ-oQOW Matrix: Sera Type of column Amount and spike Elution solver used and lot mix used and volume Comments e01-1256-36231 e01-1256-36232 e01-1256-36233 e01-1256-36234 e01-1256-36235 e 0 1 -1 2 5 6 -3 6 2 3 6 e O l-1256-36237 e01-1256-36236 e01-1256-36239 e O l-1256-36240 eO l-1256-36241 e01-1256-36242 eO l-1256-36243 e01-1256-36244 e 01-1256-36245 eO l-1256-36246 e01-1256-36247 e01-1256-36248 e01-1256-36249 e 0 1 -1 2 5 6 -3 6 2 5 0 e01-1256-36251 e O l-1256-36252 Blank H20-day 1 Blank mallard sera-day 1 Mallard Sera MS-day 1 Mallard Sera MSD-day 1 Curve point-02 ppb in sera extract Curve polnt-0.5 ppb In sera extract Curve paint-1 ppb In sera extract Curve point-2.5 ppb in sera extract Curve point-5 ppb in sera extract Curve point-10 ppb in sera extract Curve point-25 ppb in sera extract Curve point-50 ppb In sera extract Curve point-75 ppb in sera extract Curve point-100 ppb in sera extract Curve polnt-100 ppb-2 in sera extract Curve point-250 ppb In sera extract Curve point-500 ppb in sera extract Curve point-750 ppb in sera extract Curve point-1000 ppb in sera extract Q C 25pb-1 QC 25pb-2 QC 25pb-3 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Wajers, 1g, 6ml, iot% eess Waters, 1g, 6ml, lot 1290B2 Wafers, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1280B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, k>l 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 129062 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 lot 1290B2 . 2mt ofMeOH NA t n -a S w ^ x 2ml of MeOH NA TN-A-5457 2mJo/MeOH soul of 02001-66 TN-A-5457 2ml of MeOH 50ul o f 02001-66 TN-A-5457 2ml of MeOH 0.4ul of 02001-66 TN-A-6457 2ml of MeOH 1ul o f 02001-66 TN-A-6457 2ml of MeOH 2ul of 02001-66 TN-A-5457 2ml of MeOH 5ul of 02001-66 TN-A-5457 2m lof MeOH 10ul of 02001-66 TN-A-5457 2ml of MeOH 20ul of 02001-66 TN-A-5457 2ml of MeOH 50u! of 02001-66 TN-A-5457 2ml of MeOH 100ul of 02001-66 TN-A-5457 2mt of MeOH ISOul of 02001-66 TN-A-5457 2ml of MeOH 200ul of 02001-66 TN-A-5457 I | 2ml of MeOH 4ul of 02001-53 TN-A-5457 2mlof MeOH 10ul of 02001-53 TN-A-5457 2ml of MeOH 20ut of 02001-53 TN-A-5457 2ml of MeOH 30ul of 02001-53 TN-A-5457 2ml ofMeOH 40ul of 02001-53 TN-A-5457 2m) ofMeOH 1ul o f 02001-53 TN-A-5457 1ul of 02001-53 1ul of 02001-53 2mt of MeOH TN-A-5457 2ml of MeOH TN-A-5457 % / NA I I \/ Blank Sera TNA* Amount of sera allquated: 4 Dilute Sera with Kandiyohi water; Amount of water added w _ml ml Aliquot 1ml of sera Into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- r ) to allquated sample Shake sample for 20 min @ 300 rpm (Shaker ^) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge __) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with M eOH (TN-A- ^ 0 , a - ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent into appropriate 15 ml centrifuge tube Spike samples with Internal standard PFDA standard number Q 2 q o ( nsfer sample into appripriatelly marked autovial O S e A ftA - /n &**>*J. trenta JW/4 O , cone. )00-?r 3amount added. j A r CAC, Ei/? Ch 3M Environmental Laboratory Page 163 001747 Report EOl-1256 SPE Columns Extraction W orlksiieb^ Reproduction Study with the Mallard Prep Date: Analysts initials: OH / HO Z7 j-zr-oj. @ Mallard ^ &i / a i |2 v- / f W - 1 -> Method Revision: ETS-8-231.0 Study Number: E01-1256 Matrix: Sera volume of Urns Assgned sample number Sample Number or description sample filtered Type of column Amount and spike Elution solver (ml) used and lot mix used and volume Comments e01-1256-36253 eOl-1256-36254 e01-1256-36255 e01-1256-36256 e01-1256-36257 e01-1256-36258 E01-1256-28662 E01-1256-28663 E01 -1256-28664 E01-1256-28665 E01-1256-28666 E01-1256-28667 E01-1256-28668 E01 -1256-28669 E01-1256-28670 E01-1256-28671 E01 -1256-28672 E01-1256-28673 E01-1256-28674 EOI-1256-28675 EOI-1256-28676 QC-250ppb -1 QC-250ppb -2 QC-250ppb -3 QC-4ppm -1 QC-4ppm -2 QC-4ppm -3 454-109-3217,0 PPM, Mala, Adult 454-109-3218,0 PPM, Female, Adult 454-109-3219,0 PPM, Male. Adult 454-109-3220,0 PPM, Female, Adult 454-109-3221,0 PPM, Male, Adult 454-109-3222,0 PPM, Female, Adult 454-109-3223,0 PPM, Male. Adult 454-109-3224,0 PPM, Female, Adult 454-109-3226,0 PPM, Female, Adult 454-109-3227,0 PPM, Male, Adult 454-109-3228,0 PPM, Female, Adult 454-109-3229,0 PPM, Male, Adult 454-109-3230,0 PPM, Female, Adul 454-109-3231.0 PPM. Male, Adult 454-109-3232,0 PPM. Female, Adult 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot T998B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1a, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1a. 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 \ Waters, 1g, 6ml, I lot 1290B2 1 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g. 6ml, lot 1280B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g,6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g. 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 129062 W aters, 1g, 6ml, lot 1290B2 waters, 1g, orrn, lot 1290B2 ^ 2mi of MeOH 10ul of 02001-53 r 2ml of MeOH lOul of 02001-53 TN-A-5457 2mt of MeOH 10ul of 02001-53 TN-A-5457 2ml of MeOH 160ul of 02001-53 TN-A-5457 2ml of MeOH 160td of 02001-53 TN-A-5457 2ml of MeOH 160ul of 02001-53 TN-A-5457 NA 2ml of MeOH TN-A-5457 NA 2ml of MeOH TN-A-5457 NA 2 m lo fM e O H TN-A-5467 NA 2ml of MeOH TN-A-5457 NA 2ml of MeOH TN-A-5457 NA 2mlof MeOH TN-A-5457 NA 2ml of MeOH TN-A-5457 NA 2mf of MeOH TN-A-5457 NA 2m lo fM eO H TN-A-5457 NA 2mt of MeOH TN-A-5457 NA 2mi of MeOH TN-A-5457 NA 2ml of MeOH TN-A-5457 NA 2ml of MeOH TN-A-5457 NA NA 2mi of MeOH TN-A-5457 2ml of MeH TN-A-5457 - A / A ___________________ 1 _________ ___ i \/ . Blank Sera TN-A- Amount of sera allquated:. 4 _ml I--D1ilute Sera with Kandiyohi water; Amount of water added. I Aliquot 1ml of sera Into 15 ml polypropelene tube ml Spike samples accordingly Add 5ml of ACN ( T N - A - _ $ / ^ 2 _ J to allquated sample Shake sample for 20 min 3 0 0 rpm (Shaker^ Centrifuge sample for 10 min 2000 rpm (Centrifuge Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixlgg Condition column with MeOH (TN-A- ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry ri ^ Elute column with Indicated amount of solvent into appropriate 15 ml centrifuge tube Spike samples with Internal standard PFDA standard number Q & o o l ~ f j .T r a n s fe r sample into apprlpriatelly marked autovial , cone. 1 ^ P fS* / amoun* added 3M Environmental Laboratory Page 164 091748 Report E01-1256 SPB Standard C urvee-Flutii PFOS: A Reproduction Study with the Mallard Prop D ata(a): AnalyeMa): Sam ple M atrix: M etbed/Revfcloa: T a ry t A m dytet): O V U m , 02/26/02,02/27/02,02/2IA 2 OK/HOl M allard Sem ETS-S-231.1 p ro s Stndy N um ber: E01-1256 E qalpm antN om ben NA Final Solvent * TN N um ber: MeOH/TN-A-OJ457 Blank T taeoe/ldeattftcr: M alted Scre-E02-0241-36386 PTOS S id : 1.00 ppm: m s S id : 502 ppm: S n rrS id A ppro*. 100.7 ppm: 02001-66 02001*53 02001-45 PFDA Actual CooceotmloM o f Standard! ia the PC Mix p ro s ms Sid C u e . uiAnL A in't Spiked m l. 1.00 0.0004 1.004 0.0010 1.004 0.0020 1.004 0.0050 1.004 0.0100 1.004 0.0200 1.004 0.0500 1.004 0.1000 1.004 0.1300 1.004 0.2000 50.20 0.0040 50.20 0.0100 50.20 0.0200 50.20 0X000 50.20 0.0400 5010 0X1010 50.20 0.0100 50.20 0.1600 1.004 0.0500 1.004 0X000 1.004 0X000 1.004 0X000 PPDA Sid C u e. UC/Mtl. 100.7 100.7 W0.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 PFDA A in't Spiked mL 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0002 0.002 0.002 0.002 0.002 AD Initial Dilution mLAnL 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 Calculated CooceBtratkma o f Standante la the sample matrix S tandard p ro s PPDA Num ber S tdC ooc. S tdC oac. m /m L 1/mL 601-1256-36235,0.2 ppb 1.03 4028 E01-1256*36236, O J ppb 20.1 4028 E01-1256-36237.1.0 ppb 40.2 4028 E01-12S6-36238,2.5 ppb 100 4028 E0I-12S6-36239,5.0 ppb 201 4028 601*1256-36240, lO ppb 402 4021 B0I-1256-36241.25 ppb 1004 4028 E01-1256-36242,50 ppb 2008 4028 601-1256-36243,75 ppb 3012 4028 601-1256-36244,100 ppb 4016 4028 601-1256-36245.100-2 ppb 4016 4028 01-1256-36246,250 ppb 10040 4028 601-1256*36247,500 ppb 20060 4028 601-1256-36248,750 ppb 30120 4028 E01-1256-36249.1000 tn*> 40160 4038 QC-25 ppb QC-250 ppb OC-4 mm 1004 10040 160640 4028 402t 4028 M illud S e n MS/MSD-Day 1 1004 4028 M allard S en MS/MSD-Day 2 1004 4028 M alted S en MS/MSD-Day 3 1004 4028 M alted S en MS/MSD-Day 4 1004 4028 Calculated C ooccatm ioaa o f Standards la the filial aolveia S ta n d ard p ro s PFDA Num ber S tdC ooc. S tdC ooc. as/m L aa/tnL E01*)256*36233,0.2 ppb 0201 101 E0J-1256-36236.0-5 ppb 0JO2 101 E01-1256-36237,1.0 ppb 1.00 101 01-1236*36238,2S ppb 2.51 101 E01-1256-36239,5.0 ppb 5.02 101 E01-1256-36240, lO ppb 10.0 101 E01-1256-36241,25 ppb 25.1 101 601-1256-36242,50 ppb 502 101 E01-1256-36243,75 ppb 75.3 101 601-1256-36244,100 ppb 100 101 E01-1256-36245,100-2 ppb 100 101 E01-1256-36246,250 ppb 251 101 EOt-1236-36247,500 ppb 502 101 E0I-1256-36248,750ppb 753 101 E01-I256-36249.1000 pob 1004 101 O C -23ppb 25.1 101 QC-250 ppb 251 to i OC-4 DOR) 4016 101 M allard Sera MS/MSD-Day 1 25.1 101 M alted Sera MS/MSD-Day 2 25.1 101 M alted S en MS/MSD-Day 3 25.1 101 M alted S en MS/MSD-Day 4 25.1 101 A fl Final Vol. mL 2.000 2.000 2.000 2.000 2.000 2.000 2.000 , 2.000 2.000 2.000 2X100 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 B lank earn In itial d ilation (m IJm L) fa r a rm : Final aoIvani volum e (aU^> fo r ( v m ; 0.0500 2.000 (jAAeA. >r osja^-lox 3M Environmental Laboratory Report EOI-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 25 Febuary 2002 7 2 hoj/ok -S^ 799 V 5 Acetonitrile tn-a-4145 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9739 (8/ml) 0.7857 3.9683 0.7857 3.9559 0.7857 3.9767 0.7857 3.9718 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0578 5.0507 5.0349 5.0613 5.0551 5.0520 101.04 0.01031 0.20 PASS Per ormance Specificiitions Accuracy, (%) 98.0 - 102 % C.V., <%) 1.0% Corrective Actions: PASS PASS 'Density values taken from the Merck Index, 12th Edition, Copyright 1996 dTz = specific gravity at yd'*" **c referred to water at x****"" 0 n-Hexane dM4 =0.660 Acetone d^o =0.788 Methanol d" 4 =0.7866 Methylene Chloride dM4 =1.3255 Acetonitrile d24 = 0.7857 3M Environmental Laboratory Page 166 -1750 Report EOl-1256 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet (A^ Ha|lord Bf/ll/oi Prep Date: Analysts Initials'. Urns Assgned sample number 2/26/2002 OK/HOJ Sample Number or description Volume of sample filtered (ml) Method R evisn: ETS-8-231 Study Number: E01-1256 Matrix: Sera Type of column Amount and spike Elution solved used and lot mix used and volume Commenta E01-1256-36259 E01 -1256-36260 E01 -1256-36261 E01-1256-36262 E01-1256-28677 EOI-1256-28678 E01-1256-28679 E01-1256-28680 E01-1256-28681 E01-1256-28682 EOI-1256-28683 EOl-1256-28684 E01-1256-28685 E01-1256-28686 EOI-1256-28687 EOI-1256-28688 EOl-1256-28689 E01-1256-28690 EOl-1256-28691 E01-1256-28692 E01-1256-28693 E01-1256-28694 Blank H20-day 2 Blank mallard sera-day 2 Mallard Sera MS-day 2 Mallard Sera MSD-day 2 454-109-3233,0 PPM , Male, Adult 454-109-3234,0 PPM, Female, Adult 454-109-3236, 0 PPM, Female, Adult 454-109-3237,0 PPM , Male, Adult 454-109-3238,0 PPM, Female, Adult 454-109-3239,0 PPM, Male, Adult 454-109-3240, 0 PPM, Female, Adult 454-109-3241,0 PPM, Male, Adult 454-109-3242,0 PPM, Female, Adult 454-109-3243,0 PPM, Male, Adult 454-109-3244,0 PPM, Female, Adult 454-109-3245,0 PPM, Male, Adult 454-109-3246, 0 PPM, Female, Adult 454-109-3247,0 PPM, Male. Adult 454-109-3248,0 PPM, Female, Adult 454-109-3249,0 PPM, Male, AduR 454-109-3250,0 PPM, Female, Adult 494*110*0^01, 0 IT M , M aie, AduR 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lo tW 2 0 3 6 B 2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot WQ036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2Q36B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g,6ml, MW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lo tW 2 0 3 6 B 2 Waters, 1g,6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 w aters, ig , omi, lot W2036B2 LlfV 2 m lo f MeOH TN-A-6012 4 50ul of 02001-66 50ul of 02001-66 tin 2 m lo f MeOH TN-A-6012 2ml of MaOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-5457 2 m lo f MeOH TN-A-5457 2m lofM eOH TN-A-5457 2rri of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2rrd of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2 m lo f MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2mt of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A -5457 11 2ml of MeOH TN-A-5457 mUMeW TN-A-5457 MA 1f Blank Sera TN-A- Amount of sera allquated: 4 Dilute Sera with Kandiyohi water; Amount of water added 3 E ^Aliquot 1ml of sera into 15 ml polypropelene tube _ml ml Spike samples accordingly Add 5mI of ACN (TN-A- ) to aliquated sample Shake sample for 20 min 3 0 0 rpm (Shaker Centrifuge sample for 10 min 2000 rpm (Centrifuge 9 |M 9 6 ^ ^ 1 3 ) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- & < P l S j ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent into appropriate 15 ml centrifuge tube Spike samples with Internal standard PFDA standard number Transfer sample into appripriatetly marked autovial s- n . _ n 3M Environmental Laboratory ______ (/ ODX)0\-<V S" . cone. bo.?, amount added - Q J L Page 167 if 1 7 5 1 Report E01-1256 Prep Date: Analysts initials: OK/HOJ PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet _ ^. Ritt, a il u s ,> 2/26/2002 Method Revision: ETS-8-231 Study Nunriber: E01-1256 Matrix: Sera Blank Sera TN-A- r ) o $ j : Amount of sera aliquated: ml Dilute Sera with Kandiyohi water; Amount of water added & ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- I to aliquated sam ple. Shake sample for 20 min @ 300 rpm (ShakerU Q f i # N>l | G ^ 4 Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3 H Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH fTN-A- f e o P ~ ) ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completely dry Elute column with indicated amount of solvent into appropriate 15 ml centrifuge tube SDike samDleswith internal standard PFDA standard number 3M Environmental Laboratory ) . cone. | 0 0 . ' f p r * u . amount added 2d Page 168 021752 Report EOI-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 26 Febuary 2002 ok 799 900 5 Acetonitrile tn-a-4145 **S?| 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9525 (g/ml) 0.7857 3.9576 0.7857 3.9768 0.7857 3.9686 0.7857 3.9994 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0305 5.0370 5.0615 5.0510 5.0902 5.0541 101.08 0.02354 0.47 PASS Per 'ormance Specificsliions Accuracy, (%) 98.0 - 102 % C.V., (%) 1.0% Corrective Actions: o l PASS PASS 'Density values taken from the Merck Index, 12111Edition, Copyright 1996 d% = specific gravity at yd**'*" c referred to water at xd*gr" *c n-Hexane d204 =0.660 Acetone dM0 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 = 1.3255 Acetonitrile d204 = 0.7857 3M Environmental Laboratory Page 169 001753 Report EOl-1256 PFOS: A SPE Columns Extraction Worksheet Prep Date: Analysts initials: Urns Assgned sample number E01-1256-36263 E01-1256-36264 EOI-1256-36265 E01 -1256-36266 E01-1256-2B710 E01-1256-28711 E01-1256-28712 E01-1256-28713 E01-1256-28714 E01-1256-28715 E01-1256-28716 E01-1256-28717 E01-1256-28718 E01-1256-28719 E01-1256-28720 E01-1256-28721 E01-12S6-28722 E01-1256-28723 E01-1256-28724 EO I-1256-28725 E01 -125628726 E01-1256-28727 E01-1256-28728 Sample Number or description Blank H20-day 3 Blank mallard sera-day 3 Mallard Sera MS-day 3 Mallard Sera MSD-day 3 454-109-3267,10 PPM, Mala, Adult 454-109-3268,10 PPM, Female, Adult 454-109-3269,10 PPM, Male, Adul 454-109-3270,10 PPM, Female, Adult 454-109-3271,10 PPM, Male, AduK 454-109-3272,10 PPM, Female, Adul 454-109-3273,10 PPM, Male, Adult 454-109-3274,10 PPM, Female, Adul 454-109-3275,10 PPM, Male, Adul 454-109-3276,10 PPM, Female, AduR 454-109-3277,10 PPM, Male, AduR 454-109-3278,10 PPM, Female, AduR 454-109-3279,10 PPM, Male, AduR 454-109-3280,10 PPM. Female, AduR 454-109-3281,10 PPM. Male, Adul 454-108-3282,10 PPM, Famalt, Adult 454-1093283,10 PPM, Male, AduR 454-1093284,10 PPM, Female, Adul ^ M - I U S - j4 l O D , lU l 'M V I , MSUS, Adult Method Revision: ETS-8-231.0 Study Number: E01-1256 Matrix: Sera Volume of sample filtered (ml) Type of column Amount and spike Elution soIvan used and lot mix used and volume Comments 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1|j, 6ml, lot W2036B2 Waters, 1g. 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2038B2 Waters. 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2038B2 Waters, 1g, 6ml, lot W2038B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot VM2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lo t W2036B2 Waters, 1g,6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 waters, 1 3 , bmi, lot W2036B2 aja 2m lof MeOH TN-A-6012 2ml of MeOH b TN-A-6012 2m1ofMeOH 50ul of 02001-66 TN-A-6012 50ul of 02001-66 UA 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml o f MeOH TN-A-8012 2ml of MeOH TN-A-6012 2mf of MeOH TN-A-6012 2m lofM eOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2m lo f MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2 m lofM eO H TN-A-6012 2ml of MeOH TN-A-0012 2ml of MeOH TNA-6012 2ml o f MeOH TNA-6012 1 2ml o(M -O H u TN-A-8012 NA If Blank Sera TN-A-; Amount of sera allquated:. Dilute Sera with Kandiyohi water; Amount of water added. 3 6 _ml ml 'Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- ) to a liquated sample f, Shake sample for 20 min @ 300 rpm (Shaker W R W n *r $ Centrifuge sample for 10 min @ 2000 rpm (Centrifuge WWJf a QL ^ ? Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water 5? CLL shake sample slightly to ensure proper mixing Condition column with MeOH (T N -A -6 0 1X l 1 Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with Indicated amount of solvent into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number _ A U t . 3M Environmental Laboratory amount added Page 170 001754 Report EOl-1256 SPE Columns Extraction W o t f f iir t Prep Date: S i X b o O Analysts initials: Q Lima Assgned sample number Sample Number or description E01-1256-28729 E01-1256-28730 E01-1256-28731 E01-1256-28732 E01-1256-28733 E01-1256-28734 E01-1256-28735 E01-1256-28736 E01-1256-28737 E01-1256-28738 E01-1256-28739 454-109-3286,10 PPM, Female, Adult 4 54 -109-3287,10 PPM, Male, Adult 454-109-3288,10 PPM, Female, Adult 454-109-3289,10 PPM, Male, Adult 454-109-3290,10 PPM, Female, Adult 454-109-3291,10 PPM, Male, Adult 454-109-3292,10 PPM, Female, Adult 454-109-3293,10 PPM, Male, Adult 454-109-3294,10 PPM, Female, Adult 454-109-3295,10 PPM, Male, Adult 454-109-3296,10 PPM, Female, Adult Method Revision: ETS-8-231.0 Study Number: E01-1256 Matrix: Sera Volume of sample filtered (ml) Type of column Amount and spike Elution solven used and lot mix used and volume Comments 40*6 40*6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot VIQ036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g , 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lo tW 2 03 6 B 2 /U A 1 2m! of MeOH TN-A-0012 2ml of MeOH TN-A-6012 2m lafMeOH TN-A 4 0 1 2 2m lofM eOH TN-A-6012 2m lofM eOH TN-A-8012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-0012 UA 40+6 Waters, 1g, 6ml, lot W2036B2 2 m lo T M e O H TN-A-6012 40+6 40+6 40+6 Waters, 1g, 6ml, lot W 203682 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 2mi oT MeOH TN-A-0012 2m! of MeOH TN-A-6012 2ml of MeOH H i TN-A-6012 1 ----- ------------------- v - ' ' - = j Blank Sara TN-A- o @ ~ ' : Amount of sera aliquated: 4 ml Dilute Sera with Kandiyohi water; Amount of water added 2>? ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- A ) to aliquated sample Shake sample for 20 min @ 300 rpm (Shaker 1C ^ J f Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3 H | 3> Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract Into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- Q O ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completely dry Elute column with indicated amount of solvent Into appropriate 15 ml centrifuge tube Snike samoles with internal standard PFDA standard number T f ^ A 3M E nvironm ental L aboratory -------- w 7. amount added Page 171 031755 Report EOI-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 27 Febuary 2002 ok 799 900 5 Acetonitrile tn-a-4145 1 Mass of solvent (g) Density1 Volume of (g/ml) Solvent 1 3.9579 0.7857 5.0374 2 3.9883 0.7857 5.0761 3 3.9754 0.7857 5.0597 4 3.9804 0.7857 5.0661 5 3.9907 0.7857 5.0792 AverageVolume: 5.0637 %Accuracy: 101.27 Std. Dev.: %C.V.: Pass/Fail: 0.01662 0.33 PASS Volume, (mL) 1 to 10 Peribrmance Specificsliions Accuracy, (%) 98.0 -102 % C.V., (%) 1.0% Corrective Actions: PASS PASS DaK3 .9 c fi `Density values taken from the Merck Index, 12th Edition, Copyright 1996 dy, = specific gravity at yd**rt" c referred to water at n-Hexane d" 4 =0.660 Acetone d25# =0.788 Methanol d" 4 =0.7866 Methylene Chloride d204 =1.3255 Acetonitrile dM4 = 0.7857 3M Environmental Laboratory Page 172 051755 Report E01-1256 SPE Columns Extraction W orfiltiSe^ Repr<fdapfc06^Sftv^^w^tJjuBi/ft^yarci Prep Date: M Method Revision: ETS-8-231 Analysts initials: Q f ( J Study Number: E01-1256 ________________ _______________________________________________ Matrix: Sera Volume of Lims Assgned sample Sample Number sample filtered Type of column Amount and spike Elution solvent number or description (ml) used and lot mix used and volume E01-1256-36267 E01-1256-3S268 Blank H20-day 4 Blank mallard sera-day 4 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 WA i 2mlof MeOH TN-A-600B 2ml of MeOH TN-A-6009 VA E01-1256-36269 Mallard Sera MS-day 4 40+6 Waters, 1g, 6ml, lot W2036B2 2ml of MeOH 50ul of 02001-66 rN-A-6006 E01-1256-36270 E01-1256-28740 Mallard Sera MSD-day 4 454-109-9583,0 PPM, Female, Offspring 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 50ul of 02001-66 MA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-eO0fl E01-125S-28741 E01-1256-28742 454-109-9591,0 PPM, Male, Offspring 454-109-9589,0 PPM, Female, Offspring 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lo tW 2 0 3 6 B 2 | / 2ml of MeOH TN-A-6006 2ml of MeOH TN-A-6009 , E01-1256-28743 454-109-9592, 0 PPM, Female, Offspring 40+6 Waters, 1g, 6ml, lot W2036B2 2ml of MeOH TN-A-6009 E01 -1256-28744 454-109-9598,0 PPM, Female, Offspring 40+6 Waters, 1g,6ml, lot W2036B2 2ml of MeOH TN-A-6009 E01-1256-28745 E01-1256-28746 E01-1256-28747 E01-1256-28748 E01-1256-28749 454-109-9603,0 PPM, Female, Offspring 454-109-9606,0 PPM. Male, Offspring 454-109-9610, 0 PPM, Female, Offspring 454-109-9573,0 PPM, Female, Offspring 454-109-9578,0 PPM, Male, Offspring 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, IO IW 2 0 3 6 B 2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 a rt of MeOH TN-A-6009 1 2ml of MeOH TN-A-6009 1 a rt off MeOH TN-A-6009 \ 2ml of MeOH TN-A-6009 \ 2ml of MeOH TN-A-6009 E01-1256-28750 454-109-9625,10 PPM, Male, Offspring 40+6 Waters, 1g, 6ml, lotW2036B2 2rrtof MeOH TN-A-6009 E01-1256-28751 454-109-9629,10 PPM, Female, Offspring 40+6 Waters, 1g, 6ml, lot W2036B2 2ml of MeOH TN-A-6009 E01-1256-28752 454-109-9633,10 PPM, Female, Offspring 40+6 Waters, 1g, 6ml, lot W2036B2 2ml of MeOH TN-A-6009 E01 -1256-28753 454-109-9634,10 PPM, Female, Offspring 40+6 Waters, 1g, 6ml, lot W 2036B2 2mt of MeOH TN-A-6009 E01-1256-28754 E01-1256-28755 E01-1256-28756 454-109-9645, 10 PPM, Female, Offspring 454-109-9648,10 PPM, Female, Offspring 454-109-9636,10 PPM, Male. Offspring 40+6 40+6 40+6 Waters, 1g, 6ml, lot W 203682 Waters, 1g, 6ml, lot W2036B2 Watara. 1g, 6m l, lot W 2036B2 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MaOH TN-A-6009 E01-1256-28757 454-109-9642,10 PPM, Male, Offspring 40+6 Waters, 1g , 6ml, lot W2036B2 2ml of MeOH TN-A-6009 E01-1256-28758 E01-1256-28759 454-109-9654,10 PPM, Female, Offspring iu rrwi, Female, Offspring 40+6 40+6 Waters, 1g , 6ml, lot W2036B2 wafers: tj, uni, lot W2036B2 {i 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 J/ Comments i o?v, Blank Sera TN-A- 6022: Amount of sera aliquated: 4 ml Dilute Sera with Kandiyohi water; Amount of water added 36 ml 'Aliquot fm l of sera into f 5 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min 3 0 0 rpm (Shaker VWR S/N 041694) w(j Centrifuge sample for 10 min @ 2000 rpm (Centrlftiae 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 6009 ) Wash Column with Kandiyohi w ater Filter sample through conditioned column, discarding nitrate Allow column to go completelly dry Elute column with indicated amount of solvent into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number J 3FDA conc._100.7ppm, 3M Environmental Laboratory , amount added 2ul Page 173 Report EOl-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 28 Febuary 2002 ok 799 900 5 Acetonitrile tn-a-4145 OK 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9524 (g/ml) 0.7857 3.9923 0.7857 3.9673 0.7857 3.9812 0.7857 3.9989 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0304 5.0812 5.0494 5.0671 5.0896 5.0635 101.27 0.02398 0.47 PASS Peri`ormance Specificsliions Accuracy, (%) 98.0 - 102 % c.v., (%) 1.0% Corrective Actions: PASS PASS 'Density values taken from the Merck Index, 12th Edition, Copyright 1996 dr, = specific gravity at ydeg,e" c referred to water at xd**r*" c n-Hexane dM4 =0.660 Acetone d25 =0.788 Methanol d" 4 =0.7866 Methylene Chloride dw4 =1.3255 Acetonitrile d20 = 0.7857 3M Environmental Laboratory Page 174 021758 Report E01-1256 PFOS: A Reproduction Study with the Mallard A ttachm ent D: D ilutions Sum m ary W orksheet Study: E01-1256 Dilution Date/Analyst: C P Box Number: ,, q SolventTN Number: MeOH/5457 ^ Extraction Date/Analyst: Matrix/Timepoint: H M LM Kb ^- 3^ Sample Number D ilu tion s or Description 1/10 1/100 1/ 1/ 1/ 1/ 1/ Comments Verified By: E01-1256-28713 X E01-1256-28719 X 4* - E01-1256-28723 X 1 E01-1256-28729 X E01-1256-28731 X E01-1256-28700 X E01-1256-28702 X E01-1256-28704 X *f E01-1256-28706 -- X E01-1256-28708 X E01-1256-28710 X UA l A3 0 r tltfl. E01-1256-28712 X E01-1256-28714 X E01-1256-28716 X E01-1256-28718 X E01-1256-28720 X EOI-1256-28722 X Notes: 1/10 d ilu tio n = toOpv kS^jT vl * o f sam p le + c} G lA- o f so lv en t * >( - * * * * 1 ip t t t ____ y r <J) Tw'Lwri'c^K, tortir //A le oW 77/e. aTmIX*'-? > w lM C 7 )c y i/ 0 % TWe ^ ~ a \ oK, v t-fa b i < @ P y ^ p "3) / D f * a c ^ S'o , Form Com pletion V erified By: 3M Environm ental L aboratory Po^t- l e t 1 IAS_ j , Page 175 001759 Report EOI-1256 PFOS: A Reproduction Study with the Mallard A ttachm ent D: D ilutions Sum m ary W orksheet Study: 01-125 Dilution Date/Analyst: CP Box Number: f t ) l - Solvent/TN Number: MeOH/5457 Extraction Date/Analyst: Matrix/Timepoint: ______ Notes: 1/10 dilution = o f sam ple + o f solvent A ^ u ? o f'S O U M /^ e -t 9 ^ / x i Form C om pletion V erified By iM Environmental Laboratory Page 176 001760 Report E01-1256 PFOS: A Reproduction Study with the Mallard A ttachm ent D: D ilutions Sum m ary W orksheet Study: io(- -e&PioC ^ ',S -- Dilution Date/Analyst: 4- ll o3, [ o K j Box Number: \ 2 C ^ Solvent/TN Number: f a o M t U 3 Extraction Date/Analyst: Matrix/Timepoint: 7 A M -<5V S y- Notes: 1/10 d ilu tio n = aM 11looOlU -= o f sam ple + y v /4 -- o f solvent s j ____ - ) K <r ' / J5 v ^ j @ /W * * 3M Environmental Laboratory Form Com pletion V erified By: 5^ tied * O'. 4^ (^ ) page l of l US w/aijox /"j&Jt. ** ^ Page 177 031761 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachm ent E: Study Protocol, Protocol A m endm ents, and D e v ia tio n s Cmerr^nmAnhsI I 3M E nvironm ental L aboratory P a r 17 ri 91 Page 178 031762 Report E01-1256 PFOS: A Reproduction Study with the Mallard W ild life In t e r n a t io n a l , ltd PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/MR/SUB454 AMENDMENT NO.: 9 SPONSOR: 3M Corporation EFFECTIVE DATE: January 24, 2002 PROJECT NO : 454-109, E01-1256 AMENDMENT: This amendment includes an attachment (Attachment A) which provides the information necessary to complete the analytical phase o f this study. REASON: The protocol did not include information for completing the analyses required by the sponsor. AMENDMENT: Harold Johnson is the Principal Analytical Investigator REASON. The sponsor requested that a principal analytical investigate' be added to the protocol. AMENDMENT: The 3M Environmental Laboratory Project Identification Number to be included with the study is E01-1256. REASON. The sponsor requested that foe 3M Project Identification Number be included in foe protocol. 3M Environmental Laboratory Page 1 o f2 Page 179 e a i7 6 3 Rfrefypozritl/ SEDrlI-l1f2r5S62. FAX 410 22 0632 WILDLIFE INTERNATIONPAFLOS: A Reproduction Study w00it0h2the Mallard | JAN. 23.2002 5;Z5PM ENVIRONMENTAL LAB 2 3E 09 NO. 5293 P. 3 PROJECTNO.. 454-109, W ildlife In ter n a tio n a l, ltd_______________________ E o i-i2 3 g Lilxjnrtoty Management, .--------HuoU O. Johnson, Ptigagil Analytical lovsatigtfor 4 ^ ^ *^ Date I 3M Environmental Laboratory P *g e 2 f2 Page 180 001764 Report E01-1256 PFOS: A Reproduction Study with the Mallard W il d life In t e r n a t io n a l , l t d _________ PROJECT NO.: 454-109, E01-1256 Sean P. Gallagher, Study Director Joann B. Beavers, W ildlife International, LTD, Laboratory Management Harold O. Johnson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date Date Date Date 3M Environmental Laboratory Page 2 o f2 Page 181 Q317S5 Report E01-1256 PFOS: A Reproduction Study with the Mallard Protocol Amendment # 9 - Attachment A Wildlife International Ltd. #464-109 STUDY TITLE PFOS: A Reproduction Study with the Mallard SPONSOR 3MEnvironmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PERFORMING ANALYTICAL LABORATORY 3MEnvironmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 LABORATORY PROJECT IDENTIFICATION 3MEnvironmental Laboratory: E01-1256 Wildlife International Ltd.: 454-109 3M Environmental Laboratory Page 182 031766 Report EOI-1256 PFOS: A Reproduction Study with the Mallard Sponsor S tudy Id e n tifica tio n PFOS : A Reproduction Study with the Mallard 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 P rin cip a l A n a lytica l In ve stig a to r S tudy Lo cation s A n a l y t ic a l Te s t in g L a b o r a t o r y A n a l y t ic a l Te s t in g La b o r a t o r y Harold Johnson 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 651-778-6479 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. PO Box 33331 St. Paul, MN 55133-3331 PACE Tier 2 1700 Elm Street, Suite 200 Minneapolis, MN 55414 P roposed S tu d y Tim etable A n a l y t ic a l E x p e r im e n t a l S t a r t D a t e A n a l y t ic a l Ex p e r im e n t a l Te r m in a t io n Da te 01/24/02 03/24/02 3M EnvironmentalLaboratory 3M E nvironm ental L aboratory 2 Page 183 001767 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1. P u r p o s e /O b j e c t iv e s In the in-life phase ofthis study, mallards were regularly exposed to nominal doses of perfluorooctanesulfonate, potassium salt (PFOS) in their feed in order to evaluate the effects ofdietary exposure upon adults over a 21-week period. The purpose ofthis analytical phase ofthe study is to quantify PFOS in the liver and sera ofboth control and dosed mallards. The analytical information may be used to compare with toxicological/histopathological results from the in-life portion ofthis bird study. The in life portion ofthe study was completed at Wildlife International, Ltd. in study 454-109. 2 . R e g u l a t o r y C o m p l ia n c e This analytical phase study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. Q u a l it y A s s u r a n c e The 3M Environmental Laboratory Quality Assurance Unit will audit analytical phase study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3MEnvironmental Laboratory Standard Operating Procedures. The Quality Assurance Unit will report all findings to the Sponsor Representative and the Study Director. 4. Reference Substance 4.1 Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity and Stability--The purity ofLot #217 has been determined to be 86.9%. PFOS has been shown to be stable in methanol solutions for up to 6 months at ambient temperature in the validation report from study PS013, "Method Validation and Instrument Detection Limit Determination for LC/MS and GC/MS Analysis ofFC807 Compounds in Methanol." Refer to the Certificate ofAnalysis (stored in the Test, Control, and Reference database in the 3M Environmental Laboratory) for further purity and stability information. 4.3 Storage Conditions--The test article may be stored at ambient temperature or lower. 4.4 Reserve Samples--A reserve sample ofPFOS from Lot #217 has been frozen at 20C 5C or colder and is kept in the 3M Environmental Laboratory. 4.5 CAS Number--The CAS number is: 2795-39-3. 4.6 Molecular Weight--The molecular weight is 538 (CgFnSOjK). Note: The molecular weight ofthe anion (PFOS) is 499 (CgFnSOs"). 3M Environmental Laboratory 3M Environmental Laboratory 3 Page 184 Report E01-1256 PFOS: A Reproduction Study with the Mallard 5. C o n t r o l M a t r ic e s Types ofcontrol matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. 6. Te s t Sy s t e m The test system is the mallard (Anasplatyrhynchos). The test system was dosed (at Wildlife International Ltd. during the in-life phase ofthis study) with food prepared by mixing PFOS with feed in a large mixer in appropriate portions to achieve the nominal 10 ppm level. A food dose verification validation study was performed by Wildlife International under study number 454C-110 (3M #U2723), "Analytical Method Verification for the Determination ofPFOS in Avian Diet." Wildlife International verified the actual dosed food for this study and the results will be contained in the in-life Wildlife phase report. Mallard serum and liver samples were collected by Wildlife International, Ltd. and sent to 3MEnvironmental Laboratory for analysis. 6.1 J u s tific a tio n o f th e S election o f th e T est S ystem Mallard dude represent wild bird populations and they are an EPA recommended species because they do well in a laboratory environment. The use ofthese matrices for the study is to determine ifPFOS was deposited in the liver and serum ofmating adults that were fed PFOS. Furthermore, analysis ofthese matrices from hatchlings will determine ifPFOS was passed on to mating adults' offspring. 7. S o u r c e o f S u p p l y f o r t h e Te s t S y s t e m The birds were obtained by Wildlife International, Ltd. See their protocol (study 454-109) for information about bird origination location. 8 . S p e c im e n a n d Sa m p l e R e c e ip t a n d M a in t e n a n c e Sera and liver were collected by Wildlife International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 21-week endpoint sera and liver samples received by the 3MEnvironmental Laboratory were from the following birds: 40 control adult birds (20 mating pairs) that ingested food containing 0 ppm PFOS 40 experimental adult birds (20 mating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppmPFOS Samples will be maintained frozen except when removed for extraction and analysis as described in the method. The samples will be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M Environmental Laboratory A Page 185 Report E01-1256 PFOS: A Reproduction Study with the Mallard 9. SUBCONTRACTED ANALYSIS 9.1 All sera and liver extractions as detailed in this protocol amendment will be performed at Pace Tier 2,1700 Elm Street, Suite 200, Minneapolis, MN 55414. 9.2 All sera and liver analyses as detailed in this protocol amendment will be performed at 3MEnvironmental Laboratory, Building 2-3E-09,935 Bush Avenue, St. Paul, MN 55106. 10. E x p e r im e n t a l C o n d u c t The 21-week endpoint liver and sera samples received (see section 8) willbe extracted and analyzed for PFOS quantities using the methods listed below. 1 10.1 Preparatory Methods 10.1.1 ETS-8-6, Extraction ofFluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.1.2 ETS-8-4, Extraction ofFluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 Analytical Methods 10.2.1 ETS-8-7, Analysis ofFluorochemicals in Liver Extracts Using HPLC- Electrospray / Mass Spectrometry 10.2.2 ETS-8-5, Analysis ofPotassium Perfluorooctanesulfonate or Other Fluorochemicals in SerumExtracts Using HPLC-Electrospray/Mass Spectrometry 11. M e t h o d Va l id a t io n Method validation for PFOS quantitation in mallard sera and liver was performed at Centre Analytical Laboratories, Inc. in study 023-044. The method will be applied to sample analyses without additional validation. 1 2 . D a t a Q u a l it y O b j e c t iv e s D uring Sa m p l e A n a l y s is The following criteria will be met using the validated calibration levels and fortification levels determined in section 11: 12.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample ofthe run three (3) mid-level unextracted calibration standards will be analyzed. The peak area precision and retention time precision and peak asymmetry will be monitored at the beginning and the cod ofthe run separately. The peak area precision must be equal to or less than 2.0% RSD, the precision ofthe retention time must be equal to or less than 2.5% RSD and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is the asymmetry factor. If any item of the system suitability fails, system maintenance must be completed prior to running a 3M Environmental Laboratory 3M Environmental Laboratory 5 Page 186 051770 Report E01-1256 PFOS: A Reproduction Study with the Mallard 12.2 12.3 second set o f Systran suitability samples and the system suitability must pass before starting the calibration. C alibration It will consist ofa minimum ofnine (9) levels, including a blank with surrogate and a blank without surrogate, the lowest standard will be at approximately 50% ofthe LLOQ and the highest standard will be approximately 50% higher than the ULOQ. The standard curve equation will be determined by regression analysis using the peak areas ofthe analyte. The accuracy ofeach level will be verified. Any level outside 80% -120% accuracy must be deactivated, and regression re-calculated. The curve must have a correlation ofdetermination (r2) greater than or equal to 0.985. All levels must show a response greater than two times that ofthe blank. The total number of standards excluded my not exceed 20%. Ifthe preceding criterion are not met, the curve and sample set will be reanalyzed. L im its o f Q ua ntita tion (LOCO The lower limit ofquantitation (LLOQ) was determined to be 10 ng/g in liver and 10 ng/mL serum during the method validation as described above in section 11. The LLOQ may be determined to be higher than that found in the validation study during a screening to be done for endogenous levels ofPFOS in the blank matrices available at 3MEnvironmental Laboratory. If endogenous levels ofPFOS are found to be higher than the LLOQ or ifthe levels are found to be highly variable, the LLOQ will be adjusted to accommodate this. The screening procedures and LLOQ determination will be recorded in a note to file and kept with the raw study data as well as being reported in the final report. Recovery efficiency at the LLOQ will be determined during each run using an extracted quality control sample spiked at the LLOQ level. If the quality control sample at the LLOQ level fails during a run, the LLOQ for that specific run would be considered to be the next lowest passing quality control sample and any samples below the new LLOQ must be reanalyzed. The upper limit ofquantitation (ULOQ) was determined to be 250 ng/g in liver and 250 ng/mL in serum during the method validation as described above in section 11. An extracted quality control sample spiked at the ULOQ will be used to measure recovery efficiency at the ULOQ. If the quality control sample at the ULOQ fails during a run the ULOQ for that specific run would be considered to be the next highest passing quality control sample and any samples above the new ULOQ must be reanalyzed. Any sample with an area greater than that ofthe highest acceptable standard will need to be diluted into the range ofthe calibration curve. If samples are diluted into the range ofthe curve during analyses and enough sample remains, a post-run dilution validation will be performed to verify sample values. To perform the dilution validation, one sample will be separated into two representative samples 3M EnvironmentalLaboratory 6 3M Environmental Laboratory Page 187 031771 Report E01-1256 PFOS: A Reproduction Study with the Mallard (i.e. two 1mL aliquots for fluid samples or two 1gram amounts for tissue samples) then diluted using two procedures. The first procedure consists of diluting the sample with additional matrix prior to extraction (sera adding sera), while the second procedure consists ofdiluting the extract with solvent post extraction (methanol extract adding additional methanol solvent). If the values are not within 15% ofeach other additional testing will be required to determine which value is a correct representation ofthe sample concentration. 12.4 C o n tin u in g C a lib ra tio n V e rifica tio n s An unextracted midlevel standard (continuing calibration verification - CCV) will be analyzed after every 10 injections (starting aft* the last injection ofthe calibration curve). The accuracy ofthe CCV must be within 20%. Data may only be reported if bracketed by passing CCV's (or a CCV and the same level calibration curve point). Data bracketed by one or more failing CCV's must be reanalyzed. 12.5 Q u a lity C o n tro l Sam ples A minimum ofthree (3) quality control samples (QC) will be prepared with each set of samples. They will consist ofblank matrix spiked with three levels of analyte. The levels are: Low level: at the LLOQ (10 ng/mL in serum and 10 ng/g in liver), Mid-level: 5 times the LLOQ, High level: at the ULOQ (25 times the LLOQ) 12.6 QC P erform ance C riteria Each QC is expected to show an accuracy o f80-120% of expected. A minimum of2/3 of all QC must meet these criteria. If not, the entire sample set must either be re-analyzed or re-extracted (as decided by the Study Director). 12.7 Use o f Confirmatory M ethods Confirmatory methods are typically not needed with LC/MS/MS analysis. 12.8 D em onstration o f S p e c ific ity PFOS specificity will be substantiated by chromatographic retention time, by the characteristic primary ion (499), and the characteristic product ion (99) using LC/MS/MS. 13. S ubcon tracted A nalysis 13.1 All fluid and tissue extractions as detailed in tins protocol amendment will be performed at Pace Tier 2,1700 Elm Street, Suite 200, Minneapolis, MN 55414. 13.2 All fluid and tissue analyses as detailed in this protocol amendment will be performed at 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 3M Environmental Laboratory 7 3M Environmental Laboratory 034^188 Report EOl-1256 PFOS: A Reproduction Study with the Mallard 13.3 Pace Analytical Services, Inc. will follow all applicable 3M Environmental Laboratory Standard Operating Procedures and the following Pace Analytical Inc. Standard Operating Procedures. PSS-Admin-01 Quality System PSS-Admin-04 Laboratory Facilities PSS-Admin-06 Training Record Files Maintained by QAU PSS-ARC-02 Disposition ofArchive Materials PSS-ARC-04 Archiving Data, Specimen and Logbooks PSS-DC-05 Lab Notebooks, Logbooks, and Phone Logs PSS-DM-03 Statistical Evaluation ofData PSS-OPS-01 Use and Maintenance of Ultra-Turrax T25 Homogenizer PSS-OPS-02 Raw Data PSS-OPS-03 Hazardous Waste Disposal PSS-OPS-04 Use and Maintenance ofN-Evap Analytical Evaporator PSS-OPS-05 Use ofCompressed Gases in the Laboratory PSS-OPS-06 Use and Maintenance ofNANOpure II Water Purification System PSS-OPS-07 Cleaning Non-disposable Volumetric Pipettes PSS-OPS-08 Laboratory Calculations PSS-OPS-09 Deviations fromEstablished Procedures PSS-OPS-10 Analytical Definitions PSS-OPS-11 Use and Calibration ofa Bottle-Top Dispenser PSS-OPS-13 Use and Maintenance ofLab Refrigerators, Freezers, and Ovens PSS-OPS-14 Use and Calibration of Syringes with a Hamilton Chaney Adapter PSS-OPS-15 Glassware Cleaning PSS-OPS-16 Use and Maintenance ofthe Fisher Scientific Isotemp Freezer PSS-OPS-17 Use and Maintenance ofUltrasonic Water Baths (Sonicators) PSS-OPS-18 Use and Maintenance ofVortex Shakers PSS-OPS-24 Use and Maintenance of a Desiccator PSS-OPS-31 Use and Maintenance of Shakers PSS-OPS-32 Operation, Maintenance and Calibration o f Laboratory PSS-OPS-37 Operation, Maintenance, and Calibration ofpH Meters and pH Electrodes PSS-MC-01 Purchasing ofLaboratory Supplies PSS-MC-02 Receipt ofLaboratory Supplies PSS-MTR-01 Calibration of Certified Weight Set PSS-MTR-02 Calibration ofCertified Thennometers/Thermocouples PSS-MTR-05 Calibration ofEppendorfPipettes PSS-MTR-06 Verification of Calibration ofthe EppendorfPipettor PSS-MTR-07 New Equipment Qualification (IQ, PQ, OQ) PSS-MTR-08 Verification ofCalibration and Use ofAnalytical Balances PSS-MTR-09 Use and Maintenance ofFisher ScientificElectronic Temperature Monitoring Devices PSS-Safety-01 General Laboratory Safety PSS-Safety-02 Use and Maintenance ofBiological Safety Cabinets 3M Environmental Laboratory 8 3M Environmental Laboratory Page 189 021773 Report E01-1256 PFOS: A Reproduction Study with the Mallard PSS-Safety-04 Emergency Evacuation Procedures PSS-Safety-05 Use and Maintenance ofLaboratory Fume Hoods PSS-TS-01 Training Procedures 1 3 .4 An amendment to the protocol will be written if extractions and analyses are performed at laboratories other than the Pace Tier 2 or 3M Environmental Laboratory, respectively. 1 4 . S t a t is t ic a l A n a l y s is Statistical methods will be limited to the calculation ofmeans and standard deviations. Examples ofthe calculations used in the analyses will be included in the analytical phase report. 16. Report A final report will be prepared by the 3M Environmental Laboratory. The final report will follow all CEP requirements, and will include a description of all materials and methods used, including a narrative discussion with a tabular presentation ofall analyses results. 1 6 . L o c a t io n o f R a w D a t a , R e c o r d s , a n d F in a l R e p o r t When the final report is completed, all original paper data, including protocol, phase report, study correspondence, sample receipt and tracking, sample preparation, and analytical data, will be retained in the archives of3MEnvironmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained other at Pace Tier 2 or the 3M Environmental Laboratory, as applicable. 1 7 . S p e c im e n R e t e n t io n Samples ofthe test substance (any leftover control sera or liver and samples) and the analytical standards will be maintained in the laboratory for a period oftime as specified by regulation or as long as the quality ofthe preparation affords evaluation. However, samples will not be maintained more than 10years after the effective date ofthe final test rule (if applicable). 3MEnvironmental Laboratory Standard Operating Procedures also apply to sample retention times. 18. References 1 8.1 Wildlife International, Ltd. Protocol No. 454-109, "PFOS: AReproduction Study with the Mallard". 1 8 .2 Centre Analytical Laboratories, Inc. Study 023-044, "Validation ofAnalytical Methods "Extraction ofPotassium Perfluoroocatanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC Electrospry/Mass Spectrometry" and "Extraction ofPotassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC Electrospray/Mass Spectrometry" in Mallard Serum and Liver." 3M Environmental Laboratory 3M Environmental Laboratory 9 Page 190 021774 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1 8 .3 3M Study U2723, Wildlife Study 454C-110, "Analytical Method Verification for the Determination ofPFOS in Avian Diet." 1 9. P r o t o c o l A m e n d m e n t s a n d D e v ia t io n s Planned changes to the protocol will be in the form ofwritten amendments signed by the Study Director and the Sponsor Representative. Amendments will be considered as part ofthe protocol and will be attached to the final protocol. Any other changes will be in the form ofwritten deviations, signed by the Study Director and Sponsor Representative and filed with the raw data. All changes to the protocol will be indicated in the final report. 3M Environmental Laboratory 3M Environmental Laboratory 10 Page 191 i. 1 7 7 5 KgpfM/B31-ll35e FAX 410 22 0632 WILDLIFE INTERNatIORSOS: A Reproduction Study Mallard W ildlife International, ltd PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE. PFOS. A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/MR/SUB454 AMENDMENT NO.: 10 SPONSOR: 3M Corporation PROJECT NO.: 454-109, E01-J 256 EFFECTIVE DATE: January 30, 2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase o f this study. This attachment A replaces foe attachment A in amendment 9. REASON: The amendment 9 attachment A was updated to include a different extraction and analytical method, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director liM X Date -V -- Joann B. Bhvers, Wildlife International, LTD, Laboratory Manage Harold O. Johnson, Prltuipal Analytical investigator William K. Reagen, 3M Environmental Laboratory, ^_---- Labor fRobideau, Sponsor's Representative Date A^SiXia, (DC Date O.'/ / y / q Date - W M / Z r Date 3M Environmental Laboratory 1 Of 9 Page 192 021776 Report EOI-1256 W ildlife International, ltd PFOS: A Reproduction Study with the Mallard PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/M R/SUB454 AMENDMENT NO.: 10 SPONSOR: 3M Corporation PROJECT NO.: 454-109, E01-1256 EFFECTIVE DATE: January 30,2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase o f this study. This attachment A replaces the attachment A in amendment 9. REASON: The amendment 9 attachment A was updated to include a different extraction and analytical method, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director Date Joann B. Beavers, W ildlife International, LTD, Laboratory Management Harold O. Johnson, Principal Analytical Investigator Date Date William K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date 3M Environmental Laboratory 1 of 9 Page 193 .'i-/ . n Xi yf *? y 9 Report E01-1256 PFOS: A Reproduction Study with the Mallard Protocol Amendment # 10 - Attachment A Wildlife International Ltd. #454-109 STUDY TITLE PFOS: A Reproduction Study w ith the Mallard SPONSOR 3M Environm ental Laboratory Building 2-3E -09 PO B ox 33331 St. Paul, M N 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M Environm ental Laboratory B uilding 2-3E-09 PO B ox 33331 St. P au l, M N 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: E 01-1256 W ildlife International Ltd.: 454-109 3M Environmental Laboratory 3M Environmental Laboratory 2 of 9 Page 194 0177 Report EOI-1256 PFOS: A Reproduction Study with the Mallard Sponsor Study Identification PFOS : A Reproduction Study w ith th Mallard 3M Environm ental Laboratory Building 2-3E -09 PO Box 33331 St. Paul, M N 55133-3331 Principal Analytical Investigator Study Locations A n a l y t ic a l Te s t in g La b o r a t o r y Harold Johnson 3M Environm ental Laboratory B uilding 2-3E -09 PO Box 33331 St. Paul, M N 55133-3331 651-778-6479 3M Environm ental Laboratory B uilding 2-3E -09 935 Bush Ave. PO Box 33331 St. Paul, M N 55133-3331 P ro p o s e d S tu d y T im etab le A n a l y t ic a l Ex p e r im e n t a l S ta r t Da t e A n a l y t ic a l Ex p e r im e n t a l Te r m in a t io n Da t e 01/31/02 03/31/02 3M Environmental Laboratory 3M Environmental Laboratory 3 of 9 Page 195 031779 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1. P u r p o s e /O b j e c t iv e s In the in-life phase o f this study, mallards were regularly exposed to nom inal doses o f perfluorooctanesulfonate, potassium salt (PFO S) in their feed in order to evaluate the effects o f dietary exposure upon adults over a 21 -w eek period. The purpose o f this analytical phase o f the study is to quantify PFOS in the liver and sera o f both control and dosed mallards. The analytical inform ation m ay be used to compare with toxicological/histopathological results from the in -life portion o f this bird study. The in life portion o f the study w as com pleted at W ildlife International, Ltd. in study 454-109. 2. R e g u l a t o r y C o m p l ia n c e This analytical phase study w ill be conducted in accordance with the United States Environmental Protection A gency Good Laboratory Practice Standards, 40 CFR 792. 3. Q u a l it y A s s u r a n c e The 3M Environmental Laboratory Q uality Assurance U nit w ill audit analytical phase study conduct, data, and the final report to determine com pliance w ith G ood Laboratory Practice Standards and w ith 3M Environmental Laboratory Standard Operating Procedures. The Q uality Assurance U nit w ill report all findings to the Sponsor Representative and the Study Director. 4. Reference Substance 4 .1 P hysical D escription- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4 .2 P u rity a n d S tab ility--The purity o f Lot #217 has been determ ined to b e 86.9% . PFOS has been shown to be stable in methanol solutions for up to 6 months at ambient temperature in the validation report from study PS013, "M ethod V alidation and Instrument D etection Lim it Determ ination for LC/M S and GC/M S A nalysis o f FC807 Compounds in M ethanol." Refer to the Certificate o f A nalysis (stored in the Test, Control, and Reference database in the 3M Environmental Laboratory) for further purity and stability information. 4 .3 S to rag e C onditions-- The test article m ay be stored at am bient temperature or lower. 4 .4 R eserve Sam ples--A reserve sam ple o f PFOS from Lot #217 has been frozen at 20C 5C or colder and is kept in the 3M Environmental Laboratory. 4 .5 C A S N um ber--The CAS number is: 2795-39-3. 4 .6 M o le cu la r W eight--The m olecular w eight is 538 (C&F17 SO3 K). Note: The m olecular w eight o f the anion (PFOS) is 499 (C8 F17 SO3 ). 3M Environmental Laboratory 3M Environmental Laboratory 4 of 9 Page 196 031780 Report E01-1256 PFOS: A Reproduction Study with the Mallard 5. C o n t r o l M a t r ic e s Types o f control m atrices and their source, physical description, storage requirements, and traceability numbers w ill be recorded in the raw data and included in the final report. 6. Te s t S y s t e m The test system is the mallard (A n a s p la ty r h y n c h o s ). The test system w as dosed (at W ildlife International Ltd. during the in -life phase o f this study) w ith food prepared by m ixing PFOS w ith feed in a large m ixer in appropriate portions to achieve the nom inal 10 ppm level. A food dose verification validation study w as perform ed by W ildlife International under study number 454C -110 (3M #U 2723), "A nalytical M ethod V erification for the Determ ination o f PFOS in A vian D iet." W ildlife International verified the actual dosed food for this study and the results w ill be contained in the in -life W ildlife phase report. M allard serum and liver sam ples w ere collected by W ildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. 6.1 Justification o f the Selection o f the Test System Mallard duck represent w ild bird populations and they are an EPA recom m ended species because they do w ell in a laboratory environm ent. The use o f these m atrices for the study is to determine i f PFOS w as deposited in the liver and serum o f m ating adults that were fed PFOS. Furthermore, analysis o f these m atrices from hatchlings w ill determine if PFOS w as passed on to m ating adults' offspring. 7. S o u r c e o f S u p p l y f o r th e Te s t S y s te m The birds w ere obtained by W ildlife International, Ltd. S ee their protocol (study 454109) for inform ation about bird origination location. 8. Sp e c im e n a n d Sa m p l e R e c e ip t a n d M a in te n a n c e Sera and liver were collected b y W ildlife International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 21-w eek endpoint sera and liver sam ples received b y the 3M Environm ental Laboratory were from the follow ing birds: 40 control adult birds (20 m ating pairs) that ingested food containing 0 ppm PFOS 40 experim ental adult birds (20 m ating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppm PFOS Sam ples w ill be m aintained frozen except when rem oved for extraction and analysis as described in the method. The sam ples w ill be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M Environmental Laboratory 5 of 9 Page 197 031781 Report E01-1256 PFOS: A Reproduction Study with the Mallard 9. SUB-CONTRACTED ANALYSIS A ll sera and liver extractions and analyses as detailed in this protocol amendment w ill be performed at 3M Environmental Laboratory, B uilding 2 -3 E -0 9 ,935 Bush A venue, St. Paul, M N 55106.55414. 10. E x p e r im e n t a l C o n d u c t The 21-w eek endpoint liver and sera sam ples received (see section 8) w ill be extracted and analyzed for PFOS quantities using the m ethods listed below . 10.1 Preparatory and Analytical Method 10.1.1 E T S-8-231 Solid Phase Extraction and A nalysis o f Fluorochem ical Compounds from B iological M atrices 11. D a t a Q u a l it y O b j e c t iv e s D u r in g Sa m p l e A n a l y s is M ethod validation for PFOS quantitation in rat and m ouse sera and liver is in process in the 3M Environmental Laboratory study E 01-1277. Lim ited Mallard control matrix preclude a cross validation, how ever, the E 01-1277 study m ethods w ill be applied to sample analyses o f Mallard sera and liver sam ples using the follow ing performance based criteria: 11.1 System Suitability System suitability w ill be determined prior to the start and at the com pletion o f each analytical run. Prior to the calibration curve and after the last sam ple o f the run three (3) m id-level unextracted calibration standards w ill be analyzed. The peak area precision and retention tim e precision w ill be m onitored at the beginning and the end o f the run separately. The peak area precision m ust be equal to or less than 2.0% RSD, the precision o f the retention tim e m ust be equal to or less than 2.5% RSD. If any item o f the system suitability fails, system m aintenance m ust be com pleted prior to running a second set o f system suitability sam ples and the system suitability must pass before starting the calibration. 11.2 Absolute Recovery The absolute recovery o f the method w ill be evaluated one tim e in both liver and serum. For each matrix a minimum o f triplicate sam ples w ill be fortified at a minimum o f two levels o f PFOS that bracket the range o f analyte determ ined for the sam ples (i.e., at near the highest level determined in sam ples and at -150% o f LOQ for sam ples). The sam ples w ill be extracted through the m ethod, and analyzed by com parison with an external calibration o f non-extracted standards. The non-extracted standard calibration curves w ill be prepared in m ethanol, and w ill consist o f a minimum o f nine (9) levels, including a m ethanol blank. The best appropriate regression w ill be used to describe this curve (for best accuracy at all levels o f the standards). 3M Environmental Laboratory 3M Environmental Laboratory 6 of 9 Page 198 05178, Report E01-1256 PFOS: A Reproduction Study with the Mallard 11.3 11.4 The accuracy (% absolute recovery) and precision (%CV o f the recoveries) w ill be determined at each level using the external calibration curve for quantitation. There is no control lim it for accuracy; precision m ust be better than 20% at each level. Should a level (high or low ) not m eet this precision, the m ethod w ill only be considered valid for levels betw een the tw o valid levels. Additional testing m ay be conducted at other levels to extend the acceptable range o f the m ethod. Calibration A calibration curve should be prepared in each matrix by spiking the matrix with known concentrations o f the analyte and surrogate. A calibration curve should consist o f at least nine standard curve points covering the expected range, including the LLOQ. The sim plest m odel that adequately describes the concentration-response relationship should be used. The accuracy o f each level w ill be verified. Any level outside 75% -125% o f nom inal m ust be deactivated, and regression re-calculated, except the LLOQ w hich must be w ithin 30% o f nom inal. A ll levels m ust show a response greater than tw ice that o f the matrix blank. A m aximum o f four (4) levels m ay be deactivated in any one set, or the set w ill be re-analyzed. Limits o f Quantitation (LOQ) The low er lim it o f quantitation (LLOQ) w ill be defined in liver (ng/g) and in serum (ng/m L) as the low est acceptable extracted calibration curve point that is w ithin the 70% -130% nom inal criteria and w ith an analyte peak area at least 2 tim es the blank. The LLOQ determination w ill be recorded in a note to file and kept w ith the raw study data as w ell as being reported in the final report. 11.5 The upper lim it o f quantitation (ULOQ) w ill be defined in liver and in serum as the highest acceptable extracted curve point that is within the 75% - 125% nom inal criteria and includes applicable dilution factor corrections. Quality Control Samples A m inimum o f one set o f nine (9) quality control sam ples (QC) w ill be prepared one tim e in each matrix. They w ill consist o f three levels o f analyte, each in triplicate. The quality control sam ples should be prepared in each matrix by spiking the control matrix with known concentrations o f the analyte. The levels bracket the sam ple concentrations determined in the study: Low level: equivalent to an analyte concentration near or below the low est level o f analyte determined in sam ples M id-level: equivalent to an analyte concentration near the m iddle o f the sam ple range H igh level: equivalent to an analyte concentration near or above the highest level o f analyte determined in sam ples 11.6 Quality Control Sample Acceptable Recoveries 3M Environmental Laboratory 3M Environmental Laboratory 7 of 9 Page 199 05178 Report E01-1256 PFOS: A Reproduction Study with the Mallard Each QC is expected to show an accuracy o f 75-125% o f expected. A minimum o f 2/3 (6 out o f 9) o f all QC m ust m eet these criteria and a m inimum o f one h alf (2 out o f 3) o f the QC at each level m ust m eet these criteria. If not, the entire sam ple set must either be re-analyzed or re-extracted (as decided by the Principal A nalytical Investigator). 11.7 Matrix Spikes Two matrix spikes w ill be extracted w ith each sam ple preparation. The matrix spikes w ill consist o f one fortification level in the range o f the sam ples, extracted in duplicate. If the matrix spike and/or matrix spike duplicate fall outside o f 25% accuracy, the curve, CCV's, matrix spikes and sam ples w ill be reanalyzed. 11.8 Blanks One control matrix and one water m ethod blank w ill be extracted w ith each sam ple preparation. 11.9 Continuing Calibration Verification Two extracted standards (continuing calibration verification - CCV) w ill be analyzed after every 10 injections (starting after the last injection o f the calibration curve). The accuracy o f the CCV m ust be w ithin 25%. Data m ay only be reported i f bracketed by passing C C V's (or a CCV and the sam e level calibration curve point). Data bracketed by one or more failing CCV m ust b e reanalyzed. 11.10 Use o f Confirmatory Methods Confirmatory m ethods are typically not needed w ith LC/M S/M S analysis. 11.11 Demonstration of Specificity PFOS specificity w ill be substantiated by chromatographic retention tim e, by the characteristic primary ion (499), and the characteristic product ion (99) using LC/M S/M S. 12. S u b - C o n t r a c t e d A n a l y s is 12.1 A ll fluid and tissue extractions and analyses as detailed in this protocol amendment w ill be performed at the 3M Environmental Laboratory, Building 2- 3E -09, 935 Bush A venue, St. Paul, M N 55106. 12.2 An amendment to the protocol w ill be written if extractions and analyses are performed at laboratories other than the 3M Environmental Laboratory. 13. S ta tistic a l A n a ly s is Statistical m ethods w ill be lim ited to the calculation o f means and standard deviations. Exam ples o f the calculations used in the analyses w ill be included in the analytical phase report. 3M Environmental Laboratory 3M Environmental Laboratory 8 of 9 Page 200 031784 Report E01-1256 PFOS: A Reproduction Study with the Mallard 14. Report A final report w ill be prepared by the 3M Environmental Laboratory. The final report w ill follow all GLP requirements, and w ill include a description o f all m aterials and m ethods used, including a narrative discussion w ith a tabular presentation o f all analyses results. 15. L o c a tio n o f Ra w Da ta , R e c o r d s , a n d F in a l R e p o r t When the final report is com pleted, all original paper data, including protocol, phase report, study correspondence, sam ple receipt and tracking, sam ple preparation, and analytical data, w ill be retained in the archives o f 3M Environmental Laboratory. A ll corresponding training records, calibration records, instrument m aintenance logs, standard operating procedures, equipment procedures, and m ethods w ill be retained at the 3M Environmental Laboratory, as applicable. 16. S p e c im e n R ete n tio n Sam ples o f the test substance (any leftover control sera or liver and sam ples) and the analytical standards w ill be maintained in the laboratory for a period o f tim e as specified by regulation or as long as the quality o f the preparation affords evaluation. H owever, sam ples w ill not be maintained more than 10 years after the effective date o f the final test rule (if applicable). 3M Environmental Laboratory Standard Operating Procedures also apply to sam ple retention tim es. 17. R eferences 18.1 18.2 W ildlife International, Ltd. Protocol N o. 454-109, "PFOS: A Reproduction Study w ith the Mallard". 3M Study U 2723, W ildlife Study 454C -110, "Analytical M ethod Verification for the Determ ination o f PFOS in A vian D iet." 18. P r o to c o l A m en d m e n ts a n d D e v ia tio n s Planned changes to the protocol w ill be in the form o f written amendments signed by the Study D i r e c to r and th e S p o n s o r R e p r e s e n ta tiv e . A m e n d m e n ts w ill b e c o n s id e r e d a s p a r t o f the protocol and w ill be attached to the final protocol. A ny other changes w ill be in the form o f written deviations, signed by the Study Director and Sponsor Representative and filed w ith the raw data. A ll changes to the protocol w ill be indicated in the final report. 3M Environmental Laboratory 3M Environmental Laboratory 9 of 9 Page 201 Report EOI-1256 PFOS: A Reproduction Study with the Mallard W ildlife International, ltd PROJECTNO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 SPONSOR: 3M Corporation AMENDMENT NO.: 11I PROJECT NO.: 454-109, E01-1256 EFFECTIVE DATE: March 27,2002 AMENDMENT: Section 11.1, Sentence 4 Change: The peak area precision must be equal to or less than 2.0% RSD, the precision o f the retention time must be equal to or less than 2.5% RSD. To: The peak area precision must be equal to or less than 5.0% RSD, the precisimi o f the retention time must be equal to or less than 2.5% RSD. REASON: Information received from our instrument supplier indicates that a 2.0 % RSD for the area count is common to UV detector specifications. Mass spectrometers, which we used for this study, are more sensitive, in general and the fact feat analyte ionization can be suppressed by other species present in the sample matrix and mobile phase make area reproducibility more challenging to maintain. Sample and source cleanliness are the determining factors, but < 2.0% is still a bit aggressive for mass spec. The application chemists at Micromass, the instrument vendor, suggest a value o f <5.0% . AMENDMENT. Section 11.3, Sentence 5. Change: Any level outside 75% - 125% o f nominal value must be deactivated, and regression recalculated, except fee LLOQ which must be within 30% o f nominal. To: In some cases the lowest curve point, although outside fee 30% nominal requirement, may be included in fee linear range to influence the lower end o f the curve. Only one point, fee lowest level point, can be left in for this purpose. It will not be used to determine the limit o f quantitation. The limit o f quantitation w ill be the next point on fee curve meeting the 30% nominal requirement. REASON: The removal o f fee lowest calibration curve point in some cases causes the next lowest calibration point to deviate by greater than 30%. Removal o f this point in turn causes fee next calibration point to deviate by greater than 30%. This continued deletion o f calibration points in some cases could continue until there are very few points 3M Environmental Laboratory Page lo f3 Page 202 Report E01-1256 PFOS: A Reproduction Study with the Mallard remaining in the curve. The inclusion o f a curve point with a deviation greater than 30% is used to influence the lower end o f the curve and is not used for determination o f foe LOQ. AMENDMENT: Section 11.3, the last sentence. Change: A maximum o f four (4) levels may be deactivated in any one set, or the set w ill be reanalyzed. To: The calibration curve will contain a minimum o f six (6) calibration curve points. REASON: In most cases the calibration curves contain greater than nine (9) calibration points. The large number o f points in the curve is used to select a range representative to the samples. Limiting the removal o f only four (4) points in some cases could cause a calibration range not representative o f the samples. AMENDMENT: Section 11.3 Add: The coefficient o f determination for the calibration curve should be > 0.985. REASON: No coefficient o f determination is included in the protocol. It was inadvertently removed prior to printing o f the final copy. 3M Environmental Laboratory Page 2 o f3 Report EOI-1256 W ildlife International, ltd PFOS: A Reproduction Study with the Mallard PROJECTNO.: 454-109, E01-1256 Sean P. Gallagher, Study Director Joann B. Beavers, W ildlife International, LTD Laboratory Management _______________ Harold O. Johnson>nhcipal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, ^ ^ e p r e f r a t areseSntave Date Date Date i 3M Environmental Laboratory Page3of3 Page 204 017S8 t e w s - p w ? ; ^ FAX 410 822 0632 u n . TM INrowTIoSPS: A R'P rod"cto" Study with fte Mallard 04/01^02 18:29 BSD INF TECH 2-2E-01 * *84108220915 NO.90S P004 PROJECTNO.: 454-J09, -- n ---------- ------------------------------------------------------ E 0H 236 ,i Sopivi tlalbgber,. btoy D in U . . \ --------- ---------------JoawtpigL Baver, WOdKffcIntonation!, LTD lifctiiaitoiy Mantienici* Hkrod'. Johnson, Primaptl Analytical Investigator Due hK V ov One V ^ Date W llfi6 k lL lto w .3 M liig iiC T m (^L aibcngory. -% " 3M Environmental Laboratory P03of3 Page 205 Report EOI-1256 W ildlife International, ltd PFOS: A Reproduction Study with the Mallard PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENT NO.: 12 SPONSOR: 3M Corporation EFFECTIVE DATE: August 16,2002 PROJECT NO.: 454-109, E01-1256 AMENDMENT: Change: The 3M Environmental Laboratory Project Identification Number (LIMS) to be included with the study is E01-1256. To: The main 3M Environmental Laboratory Project Identification Number (LIMS) to be included with the study is E01-1256. Additional tracking numbers associated with this study are U2723, E01-1326, and E01-1367. REASON: Mallard samples were received in the 3M Environmental at several time points. Upon arrival, samples were assigned a LIMS tracking number. Not aware of the scope of this or any other particular study pertaining to these samples, they were checked in to different 3M LIMS projects. LIMS project numbers assigned to this study include the following: U2723, E01-1256, E01-1326, and E01-1367. Samples in each of these projects pertaining to this study were analyzed under one main study number E01-1256. AMENDMENT: Change: Harold Johnson is the Principal Analytical Investigator. To: Lisa A. Stevenson is the Principal Analytical Investigator. REASON: To change role o f the Principal Analytical Investigator. 3M Environmental Laboratory Page 1 of 2 Page 206 031790 Report EOl-1256 PFOS: A Reproduction Study with the Mallard W ildlife International, ltd JL r\- Sean P. Gallagher, Study Director -__ -- Joann B5. Beaves, 'W ildlife International, LTD, Laboratory Management C d A . S k t o X H _______________________ Lisa A. Stevenson, Principal Analytical Investigator W illiam K. Reagn,*3M Environmental Laboratory, Laboratory Management or's Representative PROJECT NO.: 454-109, EOl-1256 lW v Date ___O s / 3 /) lo ir _ Date f y i/ n Date 3M Environmental Laboratory Page 2 of 2 Page 207 017SJ Report EOI-1256 PFOS: A Reproduction Study with the Mallard W ildlife International, ltd PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENT NO.: 13 , SPONSOR: 3M Corporation EFFECTIVE DATE: September 27, 2002 PROJECT NO.: 454-109, E01-1256 AMENDMENT: Change: Rochelle R. Robideau is the Sponsor Representative. To: Susan A. Beach is the Sponsor Representative. REASON: To change role of the Sponsor Representative. ________ Sean P. Gallagher, Study Director ' qV W Date Jo an n B . B eavers, W ildlif e International, LTD , Laboratory Management j* A jIi m . Lisa A. Stevenson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management 'e tc A. . Susan A. Beach, Sponsor's Representative Date o ' j / a ?/,b X Date Date Q ~ j Date 3M Environmental Laboratory Page 1 of 1 Page 208 Report E 01-1256 3M C onfidential PFOS: A Reproduction Study with the Mallard Record of Deviation I. Id e n tific a tio n Study / Project No. EOl-1256... ..... ....................................... ................................................ Deviation type SO P Q M ethod Q Equipm ent Procedure (Check one) Q Protocol Q Other: Document number ETS-4-4.0 I Date(s) of occurrence ! 2/19/02,2/25/02,2/26/02,2/27/02,2/28/02,2/22/02, ! 3/4/02,3/5/02, 3/6/02,3/7/02__________ II.Description Required procedure/process: ETS-4-4.0 Internal chain o f custody The internal chain o f custody form to be used to track all movement of samples Within the ab. The form should be filed with study data or other relevant sample documents. Actual procedure/process : Internal chains of custodies were not used to track samples and sample extracts. Since receipt samples were Stored in freezer F I. After homogenizing liver and aliquating sera samples were stored in Freezer F8 Until extraction date. Samples remained frozen until extraction. Deviation to SOP written. III.ActionsTaken (such as amendment issued, S O P revision, etc.) Authorized by , Snul) OirtCTW ' 0 h D ate f^ o 2 - D ate p v* \ i DeviationNo._____ [_______ \ - J J k K * * * * * ' ' (assigned by Study Director or Project Lead at the end o f study or project) a\ v A q v U >U Lj Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 5 of 1 Page 209 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation 1. Identification Study / Project N o.E 01-1256 D eviation Type (C h eck one) V SOP M ethod Equipment Procedure Protocol Other: Docum ent Num ber:ETS-8-231.1 ! D ate(s) o f occurrence2/25/02, 2 /2 6 /0 2 ,2 /2 7 /0 2 , 2/28/02, i 3/1/02, 3/4/02, 3/5/02, 3/6/02, 3/7/02, 3/8/02, II. Description: R equired P rocedure/process: Section 12.1, Calculations: I f other calculations are used than those listed, they w ill be docum ented in the raw data. _A ctu al P rocedure/p rocess: .. ................................. ...... .................... ....... ....... ........ ..... ..... ............... ........ Standard E01-1342-36010, Prep sheet description "Curve Point - lOppb in liver", was prepped by spiking 20uL o f02001-66 (1.00ug/mL) into 1 mL of liver homogenate. This homogenate consists of 5g liveT plus 45mL water. Since lmL out of 50mL homogenate is used to prep each extracted standard, 1/50 of the Sg (0.1 g) of liver is "spiked". After this homogenate is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... (20ul) (l.OOugPFOS'/mL MeOH) (l.OmL/lOOOuL) (lOOOng/l.Oug) = 20ng PFOS' onto SPE column 20ng PFOS' / 2.0mL MeOH = lOng PFOSVmL MeOH. (Since this standard is recorded as lOppb, the correct units are lOngPFOSVmL of MeOH.! These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ngPFOS`/ l.OmL homogenate) (l .OmL homogenate/O.lg liver) = 200ngPFOS'/g liver This relationship shows the factor of 20 used to "correct" the units when calculating the final result in ng/g from the chromatographic result that is in ng/mL. Sample Calculation: "E01-1342-30348, Oppm a.i. 333 M liver" Chromatographic result is 10.76 ng/mL; reported value is 208.4 ng/g. Since the 1.0333g initial sample weight o f liver is homogenized with 9.0mL water, and lmL (10% of total liver weighed, since a liver density of 1.00 is assumed) is used for the entire SPE procedure, the calculation is as follows: (10.76ng PFOS-/ mL MeOH) (2mL MeOH / 0.1033 g liver)- 208.4 ng PFOS- / g liver Sample Calculation: Sample "E01-1342-36017, OC@10ppb-r': This QC sample is prepared by adding 4uL of 02001-53 (5.020ug PFOS' / mL MeOH) spiked into lmL liver homogenate. ((4ul) (5.020ug/mL) (ImL/l OOOuL) (1000ng/ug)) / (0.1 g liver) = 200.8ng PFOS" / g liver Using the earlier calculation, the analytical result of 13.59ng/mL calculates to 271.8ng/g liver. Since the spike level is 200.8ng PFOS"/ g liver, the recovery is (271.8/200.8X100)=135.4%. 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 3-______ (assigned by Study Director or Project Lead at the end o f study or project) Page 210 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation III. Actions Taken: (s u c h as a m e n d m e n t issued, S O P re v is io n , e tc .) issuing o f this SOP deviation and a note to file. Recorded By ___ _____ IV. Impact on Study /Project There w ill be no im pact on the study as this is for clarification purposes. D ate 4- A uthorized B y ( S t u d u D ir e c t o r / P r o je c t L e a d ) b ^ j i c X o A t _____ Dir&dor'. J U L -f. <\\v a \ o v LoUruU^ Hnagtrrtf*V -- 08 D ate 4 1 ( * l < 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 3______ (assigned by Study Director or Project Lead at the end o f study or project) Page 211 031795 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation I. Id e n tific a tio n Study / Project No. E01-1256 D eviation Type (C heck one) SOP Method Equipment Procedure V Protocol Other: Docum ent Number: E T S-8-231.1 D ate(s) o f occurrence: Throughout the study II.Description: Required Procedure/process: Section 11.3, Sentence 5 states: Any level outside 75% -125% o f nominal value m ust be deactivated, and regression recalculated...____________________________________________ Actual Procedure/process: M any o f the high calibration points in this study are kept in the curve despite being above the apex o f the curve. W hen a calibration point lies above the apex, it is considered indeterminant (labeled with an I) and no concentration is calculated for the standard. If no concentration is calculated, no percent difference is calculated for evaluating whether or not that standard is within the stated criteria. Rem oval o f these points in m ost cases causes the next calibration point to then be above the apex o f the curve. This continued deletion o f points could continue until there are no points remaining on the curve. Therefore, calibration points above the apex were included in the range o f the curve with no evaluation o f percent deviation criteria.________________________ III.ActionsTaken: (such a s a m en d m e n t issued, S O P revisio n , e tc .) This deviation was written, a note to file was written, and data points which were above the next highest calibration point were reanalyzed.__________________________________________ Recorded By . Lisa A. S t e v e n s o n ^ A uthorized B y (S tu d y D i r e c t o r / P r o je c t L e a d ) D ate 08/16/02 o s l n , ^ D ate Daw w . Hrto^eme\V'. \* 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. (assigned by Study Director or Project Lead at die end o f study or project) 3M Environmental Laboratory Page 212 0017 Report EO1-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation I.identification Study / Project No. EOI-1256 D eviation Type (C h eck one) V SOP M ethod Equipment Procedure Protocol Other: Docum ent Number: E T S-8-231.1 D ate(s) o f occurrence: Entire Study II.Description: Required Procedure/process: Section 12.1, Calculations-. If other calculations are used than those listed, they will be documented in the raw data. Actual Procedure/process; Standard E01-1256-36240, Prep sheet description "10 ppb sera curve point", was propped by spiking 4uL of 02001-52 (3.020ug/mL) into 2 mL of pooled sera. This diluted sera consists of 2mL sera plus 38mL water. Since lmL out of 40mL diluted sera is used to prepare each extracted standard, 1/40 of the 2mL of sera is "spiked". After 1.0 mL of diluted sera is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... (5.020ug PFOS/mL MeOH) * (4ul) * (l.OmL/lOOOuL) * (lOOOng/l.Oug) = 20ng PFOS' onto SPE column 20ng PFOS / 2.0mL MeOH = lOng PFOS /mL MeOH. (Since this standard is recorded as lOppb, the correct units are lOng PFOS /mL MeOH.) These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ng PFOS / 1.OmLdiluted sera) * (40.0mL diluted sera/ 2.0mL initial sera) =*400ng PFOS'/ mL Sera This relationship shows the factor of 40 (or 2.0mL MeOH/ 0.05mL Sera) used to "correct" the units when calculating the final result in ng PFOS/mL Sera from the chromatographic result that is in ng PFOS/mL MeOH. Sample Calculation: "E01-1256-28701,454-109-3258,10 ppm female, adult Chromatographic result is 340.3 ng/mL MeOH; reported value is 13612 ng/mL Sera. Since the 1.0 mL initial sample volume of sera is diluted with 3&0mL water, and lmL isused for the entire SPE procedure, the calculation is as follows: 1*1.0, or i/aodilwW ( OkS u/aoloi (340.3ng PFOS-/ l.OraL MeOH) * (2.0mL MeOH/ 0.05mL Sera) 13612ng PFOS / mL Sera Sample Calculation: Sample "EOl-1256-36253, QC250ppb-l": This QC sample is prepared by adding lOuL of 02001-53 (50.20ug PFOS' / mL MeOH) spiked into lmL diluted sera (or 0.05 mL of initial sera). ((50.20ug/mL) * (lOul) * (lmL/lOOOuL) * (1000ng/ug)) / (0.05 mL Sera) = 10040 ng PFOS' / mL Sera Using the earlier calculation, the analytical result of 264.06 ng/mL calculates to 10562.4 ng/mL Sera. Since the spike level is 10040ng PFOS7 mL Sera, the recovery is (10562.4/10040)(100)=105.2%.___________________________________________ III.ActionsTaken: (such as a m en d m e n t issued, S O P revisio n , e tc .) This deviation was written and a note to file was written. Recorded By Lisa A. Stevenson ^ D ate . 08/16/02 0 S/U ,/oa. A uthorized B y (S tu d y D i r e c t o r / P r o je c t L e a d ) Date S k A * O irt-cW 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory \< C 2~ Deviation No. (assigned by Study D irecto r or Project Lead a t die end o f study o r p roject) Page 213 031797 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation I. Id en tific atio n Study / Project No. E01-1256 D eviation Type (C heck one) SOP V M ethod Equipment Procedure Protocol Other: Docum ent Number: E T S-8-231.1 Date(s) o f occurrence: Throughout the study II. Description: Required Procedure/process: Section 13.3 o f the m ethod states: Two thirds o f all quality control sam ples and 1/2 o f each quality control sam ple at each level are expected to show an accuracy o f 75-125% ._________ Actual Procedure/process: A ll 25 ppb mallard sera quality control sample recoveries were >125% . III. Actions Taken: (such as a m en d m en t issued, S O P revisio n , etc .) T his deviation was written, sera data reported at the low range o f the curve may be biased high, and quality control data not within criteria are flagged in the raw data.________________________ Recorded By Lisa A . Stevenson Authorized B y (S tu d y D i r e c t o r / P r o je c t L e a d ) OiftcW LUW j 10 i n o x te - D ate 0 8 /2 6 /0 2 3 /2 (,lo 3 - D ate 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 5 ________ (assigned by Study D irecto r o r Project Lead at the end o f study o r project) Page 214 01798 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study/Project No. E01-1256 Recrd of Deviation /. Identification Deviation Type (Check one) 7 SOP 0 Method Equipment Procedure D Protocol Other: Document Number:ETS-8-231.1 j Date(s) of occurrence2/25/02, 2/26/02, 2/27/02,2/28/02, 3/1/02, __________________________________ 13/4/02,3/5/02, 3/6/02, 3/7/02,3/8/02,_______________________ II. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedure/process;______ ______________ __________ _ ___________________________ This deviation supersedes deviation #2 explaining the liver calculation: Standard EOl-1326-36788, Prep sheet description "Curve Point - lOppb in liver", was propped by spiking 20uL of 02001-66 (l.OOug/mL) into 1 mL of liver homogenate. This homogenate consists of 5.0597g liver plus 45mL water. Since lmL out of 50mL homogenate is used to prep each extracted standard, 1/50 of the 5.0597g (0.101073g = 5.0597 g / (45.0 mL water + 5.0597 g liver) is "spiked". After this homogenate is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... (20 uL) * (1.00 ug PFOS/mL MeOH) * (1.0 mL/1000 uL) * (1000 ng/1.0 ug) = 20.0 ng PFOS' onto SPE column 20 ng PFOSV2.0 mL MeOH = 10 ng PFOS'/mL MeOH. (Since this standard is recorded as lOppb, the correct units are 10 ng PFOS /mL of MeOH.) These are the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20 ng PFOS'/1.0 mL homogenate) * (1.0 mL homogenate / 0.101073 g liver) = 198 ng PFOS'/ g liver Sample Calcuhtipn; "E01-1326-29916, lOpprn a.i. 3257 M liver" IChromatographic result is 381.46 ng/mL; reported value is 72077 ng/g. Since the 1.0654 g initial sample weight of liver is homogenized with 9.0 mL water, and 1 mL is used for the entire SPE procedure, the calculation is as follows: 1.0654 g / (9.0 mL water + 1.0654 g liver) = 0.105848 g of liver in 1.0 mL homogenate removed for extraction. (381.46 ng PFOS / mL MeOH) * (2 mL MeOH / 0.105848 g liver) * 10 = 72077 ng PFOS / g liver Sample Calculation: Sample "E01-1326-36798, OC@25pnb-l": This QC sample is prepared by adding luL of 02001-53 (50.20ug PFOS' / mL MeOH) spiked into lmL liver homogenate. ((luL) * (5.020ug/mL) * (lmL/1000uL) * (1000ng/ug)) / (0.101073gliver) =497 ngPFOS' / gliver Using the earlier calculation, the analytical result of 38.87 ng/mL calculates to 571 ng/g liver. Since the spike level is 497 ng PFOS7 g liver, the recovery is (571/497) * (100) = 114,9%._____________________________________________________ III. Actions Taken: _________________________(such as amendment issued, SOP revision, etc.)______________________ IRsesucoinrgdeodfBthyis SOP deviation. _____ _________ _______________________________D_a_te_____________ Lisa A. Stevenson H k * A jW e/v/o v\ 12/02/02 Authorized By (Study Director /Project Lead) Date S\u.iM OireeW; SUf. GftHttjkif. /WM'.&ACrtrvtnV. Vvo),^\)\t&|*v fttA tU n. 3M Environmental Laboratory 0 Form ETS-4-8.0 Deviation No. L (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 215 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Atta c h m en t F : E x a m ple C a lc u l a tio n s Fo r m u l a U s e d f o r S e r a a n a l y s e s in St u d y E01 -1256 AR (ng/mL MeOH) * DF * Dilution (diluted sera) = ng/mL Sera Calculation Used for E01 1256-28701,454-109-3256,10 ppm Female adult (Initial volume = 0.5 mL) 340.3 ng/mL MeOH * 1 * 40 = 13600 ng/mL Fo r m u l a U s e d f o r L iv e r A n a l y s e s in S t u d y E01 -1256 AR (ng/mL MeOH) x D F k Final Volume (mL) = ng/g Liver amount of liver in diluted sample (g) Calculation Used for E01-1326-29925,10 ppm, a.i. 3258 F Liver (Initial weight = 1.0034 g) 355.07 ng/mL MeOH x 1 x 2.0 mL MeOH = 7080 ng/g Liver 0.100306 g Liver AR = Analytical result from MassLynx summary DF = Dilution factor Dilution(diluted sera) = Final volume(mL)/amount of sera in diluted sample(mL) Amount of sera in diluted sample(mL) = amount of sera used(mL)/total amount of sera + water(mL) Amount of liver in diluted sample(g) = amount of liver weighed(g)/ amount of liver weighed(g) + water added for homogenizing(mL=g) Examples: Amount of sera in diluted sample(mL) = amount of sera used (0.5 mLytotal amount of sera + water (10 mL) = 0.05 mL Dilution(diluted sera)(mL) = final volume (2.0 mL)/amount of sera in diluted sample (0.05 mL) = 40 Amount of liver In diluted sample(g) = amount of liverweighed (1.0034 g)/amount of liver weighed (1.0034 g) + water added for homogenizing (9.00 mL) = 1.0034 g /10.0034 g = 1.00306 g 3M Environmental Laboratory/ 3M E nvironm ental L aboratory j Pace 216 of216 Page 216 7% ^ 091800