Document qmMVDp9bYRyw7kqQVOE8X0k2K

Report E01-1256 PFOS: A Reproduction Study with the Mallard AR226-1743 Study Title ANALYTICAPLHASE REPORTFOR PFOS: A REPRODUCTIOSNTUDY WITH 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 COMPLIANCSETATEMENT 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. 0 Not all data generated were recorded directly or promptly, or initiated and dated on date of entry. a A. Stevenson, Principal Analytical Investigator I v Susa\n A. Beach, Sponsor Representative 01 J&.7 Date 3M Environmental Laboratory 3M Environmental Laboratory Page 2 of 216 Page 2 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard QUALITYASSURANCSETATEMENT 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. 02/22/02 04lO3/02-#I08l02, #/7I/02 05113/02-05/15/02 In-phase Data Final Report 02/22/02 02/22/02 04/08/02,05/21102 04/08/02,05/21/02 05116/02 05116/02 A I L&iWG@iJ Quality Assurdnce Representative Date 3M Environmental Laboratory 3M Environmental Laboratory Page 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 TABLEOF CONTENTS GLP Compliance Statement............................................................................................................... 2 Quality Assurance Statement ............................................................................................................ 3 List of Tables ...................................................................................................................................... 5 Summary............................................................................................................................................ 7 Purpose.............................................................................................................................................. 8 Reference Substances....................................................................................................................... 8 Specimen Receipt and Storage ......................................................................................................... 9 Method Summary................................................................................. ............................................. 9 Preparatory Method ..................................................................................................... Sample Receipt. Preparationand 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 Page .................................................................................................................................. 25 Attachment A: Extraction and Analytical Method, ETS-8-231.I",Solid Phase Extraction and Analysis of FluorochemicalCompounds from Biological Matrices"................................................... 26 Attachment B: Data Summary Tables................................................................................................ 46 Attachment C: Sample Chromatograms, Calibration Information. a n d Instrument Information ........ 55 Attachment D: Sample Preparation Sheets ................ ................................................................... 144 Attachment E: Study Protocol, Protocol Amendments, and Deviations ............................................ 178 Attachment F: Example Calculations ............................................................ ................................. 216 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 4 of 216 Page 4 ~ Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard LISTOF TABLES Table 1. Summary Table. C8Fl7SOi Concentration in Sera and Liver.............................................. 7 Table 2. Summary Table. C8FI7SOi Concentration in Sera and Liver with Outliers Removed........8 Table 3. Reference Substances......................................................................................................... 8 Table 4. SPE ExtractionSummary .................................................................................................... 9 Table 5. Sample Analysis................................................................................................................... 10 Table 6. Serum QC Summary Table................................... ........................................ 13 Table 7. Serum Matrix Spike Summary Table ................................................................................... 13 Table 8. Liver QC Summary Table .................................................................................................... 13 Table 9. Liver Matrix Spike Summary Table ...................................................................................... 13 Table 10. C8F17S03D- ata Summary. Female Mallard Sera (Adult and Offspring)............................ 14 Table 11. C8FI7SO3 Data Summary, Male Mallard Sera (Adult and Offspring) ................................ 16 Table 12. C8Fl7S0; Data Summary, Female Mallard Liver (Adult and Offspring)............................ 18 Table 13. C8F17SO; Data Summary. Male Mallard Liver (Adult and Offspring)................................ 20 Table 14. Summary Table. C8FI7SO3-Concentration in Sera and Liver................................. Table 15. Summary Table. C8Fl7S03 Concentrationin Sera and Liver with Outliers Removed ......23 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 216 Page 5 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 f'. 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 0 .Johnson, I l l , Original Principal Analytical lnvestigator Lisa A. Stevenson, Principal Analytical Investigator Kent F!. 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. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 216 Page 6 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard SUMMARY The concentration of the C8Fl7S0; anion of perfluorooctane sulfonate, potassium salt (PFOS, C8F17S0iK+w) as 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 M54-109. The following tables summarize the C8Fl7S0i concentrations found in the liver and sera of study samples. Extracted-matrix PFOS standards are used to establish the HPLC-ESIMSIMS instrument calibration curve for C8FI7SO3-. Concentrations of C8Fl7S0i 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 I.Summary Table, CsF17SOSConcentration in Sera and Liver Male Control I 1 1 Group l8 NA Female Control I 1 Group ~ 2o ~ NA 4 0 0 ng/mL (14 of 18) 107-1400ng/mLrange(4of 18) 4 0 0 ng/mL (15of20) 1662680ng/mLrange(5of20) Male 1Oppm Group 20 89700 ng/mL 11300-210000 ng/rnL 20 Female 1Oppm 20 20300 ng/mL 6350-91400 ng/mL 20 Group I 1 1 1 MaleOffspring 3 NA 4 0 0 ng/mL (2 of 3) 3 Control Group 103 ng/mL (1 of 3) FemaleOffspring Control Group <IO0 ng/mL (7of 7) NA M1OaplepmOffGsproriunpg 3 4410 ng/mL 3670 - 5450 ng/mL 3 Fe1mOpaplemOGffrsopuripng 7 4960 ng/mL 3420 - 6760 ng/rnL 7 For individual sample values see Tables 10 to 13 and Attachment B. NA NA 60900 ng/g 10800 ng/g 24200-125000 ng/g 2480-30700 ng/g <50 ng/g NA 3170 ng/g 2200-4640 ng/g 3620 ng/g 2240-5930 ng/g 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 216 Page 7 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 2. Summary Table, CIF~TSOBConcentration in Sera and Liver with Outliers Removed I I I Male ControlGroup 18' * I 1 I I I * I Fem;;ontrol NA <100ng/m~(15of17) * 166-172 ndmL range (2 of 171 20* I I I I I I I * Male 1OppmGroup 18 87300ng/mL 68700-108000ng/mL 20* * I I 1 1 I 19 16600ng/mL 635042800ng/mL I 1 I ' 1 FemGarolueIOpppm 20* MCoanletrOolffGsproriunpg 3* * 3* t Female Offspring * I I I I I I ControlGroup 7* I 1 I 1 3 ' 1 MaleOffspring 3* ~ * * 7` I* I I*1 1Oppm Group Female Offspring 7* * 1Oppm Group * 7* f * PURPOSE The purpose of the analytical phase of this study is to quantify C8Fl7S0i in the liver and sera of both control and dosed mallards and in their offspring. REFERENCSEUBSTANCES Table 3. Reference Substances I Source I 3M Internal I Oakwood Products, Inc. I I I I I ExDiration Date I I I I Storage Conditions 3M Laboratory I I I IdentificationNumber I I I I Phvsical Description 08/31/2006 Frozen -20C +I-10C SD-O18,Lot#217 White Crystalline Powder 12/01I2010 Frozen -20C +I-10C SD-036 White Solid I Purity I 86.9% I 98% I PFDA is nonadecafluorodecanoicacid, or Perfluorodecanoicacid, CloF,aOzH and is used solely to monitor for gross instrumentfailure and not as an internal standardfor quantitation. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 of 216 Page 8 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A ReproductionStudy with the Mallard SPECIMEN RECEIPATND STORAGE 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 (M54-109) 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 EO'I-1367), and were stored at -20C +/- 10C until prepared for analysis. METHODSUMMARY The determination of C8Fl7S0; 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 l.OmL of the diluted sera or 1.OmL 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 C8F1,SO< using HPLC-ESIMSIMS. PREPARATORMYETHOD ETS-8-231.I "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 l g of liver and 9ml of Kandiyohi water, and then homogenized. A l.OmL 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 mI 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-ESIMSIMS). Table 4. SPE Extraction Summary Measure volume of sera or weigh portion of liver Dilutesera (1120)and liver (1110)with water I 3 I Mix serdwater or homogenize livedwater I Aliquot 1.Om1of dilutedhomogenizedsample and add 5.0ml of ACN. Spike samples accordingly. Shakefor 20 minutes, centrifugefor 10 minutes. Dilute supernatant with 40 ml of water Filtersample through conditionedSPE C18 column. Elute target analytes from the column with 2.0mL methanol (MeOH). 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 216 Page 9 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard SAMPLE RECEIPTP,REPARATION AND ANALYSIS 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 perforimed. 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 samplles were allowed to thaw prior to transferring a representative sample for extraction. Samples were prepared and analyzed according to ETS-8-231 .I. 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 (all groups and dose levels) 2 Weighing of liver samples (control group and dosed group offspring) 3 Weighing of liver samples (controlgroup and dosed group 1 adults) 1 4 Homogenizing of liver samples (con$ol group and dosed group offspring) 21 February 2002 I 22 February2o02 1-22 February 2002 5 Homogenizingof liver samples (control group and portion of 25 February 2002 I the dosed arouD adults) I 6 1 Homogenizingof liver samples (remainder of dosed group adults) 7 Preparation of method blanks and spikes, calibration curve, 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 ppm adults (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 ppm offspring (Day 4 sera samples) 14 Analysis of Day 3 and Day 4 sera samples 15 -1 Analysis of diluted sera samples ~~ I 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 March2002 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 Envir'onmentalLaboratory 3M Environmental Laboratory Page 10 of 216 Page 10 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 20 Analysis of Day 2 liver samples 21 Preparationof method blanks and spikes, and liversamples, control group and 10ppm dosed group, adults (Day 3 liver samples) 22 Analysis of Day 3 liver samples 23 Preparationof method blanks and spikes, and liversamples, control group and 10ppm dosed group, offspring (Day 4 liver samples) 24 Analysis of Day 4 liver samples 25 Analysis of female sera dilutions 26 Analysis of QC liver and sera samples 27 Analysis of diluted sera samples 06 March 2002 06 March 2002 06 March 2002 07 March 2002 07 March 2002 08 March2002 05 April, 2002 11 April, 2002 ANALYTICAMLETHOD Samples were analyzed using HPLC-ESIMSIMS. C8F17S09concentrations were determined using matrix-extracted calibration standards ranging in concentration from 0.25-1000 ng C8FI7SOi/mL methanol. This result was then used to back calculate the ng C8Fl7S0i/mL sera or ng C8Fl7S0;lgram liver as appropriate. An internal standard, Nonadecafluorodecanoicacid (PFDA), was used to monitor instrument performance for gross malfunction, but was not used to quantitate results. HPLC-ECIMSIMS 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.lmm x 50mm, 5pm particle size), a 40C column compartment temperature, a 1OpL injection volume and a 0.300pLlmin pump flow rate. The HPLC gradient is listed below: I 77..55 I 5 95 I I 8 I 90 I 10 I I 10 90 10 0 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 C8FI7SO9 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, FSOi. Similarly the PFDA transition 3M Environmental Laboratory 3M Environmental Laboratory Page 11 of216 Page 11 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 . 0 ~01-3mBar. ANALYTICARLESULTS 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 Biolo-gical Matrices" and Attachment Study Protocol, Protocol Amendments, and Deviations). Regressions. Quadratic curve fit, weighted 1/X, was applied to calibration standards and saimple data. Each calibration curve had a coefficient of determination of 2 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 C8FI7SO3-/mLmethanol, corresponding to a concentration of 8.00 to 40160 ngi/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 nolminal. In some cases the lowest concentration curve point, although outside the 30% nalminal 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 LCIMSIMS 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 re1:ention 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 CCVs 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 Enviironmental Laboratory 3M Environmental Laboratory Page 12 of 216 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 QC-25ppb 1004 ng1mL 145%* 9.8% QC-250ppb 10040 ng1mL 97.9% 2.9% QC4.0ppm 160600 nglmL 84.7% I I I 6.7% "Thesesamples were spiked using a 1.OuIaliquot of spikingsolution. Matrix spikes prepared on the same day using 50 uIof a diluted st& meet all QC requirements. Spiking technique of injecting 1.0 ul is-suspect. Table 7. Serum Matrix Spike Summary Table I 7 I ! Four Matrixypikes I Four Matrix Spikes Duplicates 1004 ng1mL 1004 ng1mL 114% 112% *Some rnatrix spike samples were injected in a second analytical batch. Both results are induded in the average recoveries. Table 8. Liver QC Summary Table I I I I I QC-25ppb I I I I I QC-250ppb 497 nglg 4967nglg ~____ 109%* 94.3% 13% 8.3% Table 9. Liver Matrix Spike Summary Table 1 Four Matrix Spikes Four Matrix Spikes Duplicates 497 nglg 497 nglg 111% 107% 'Some matrix spike samples were injected in a second analytical batch. Both results are induded in the average recoveries. The protocol states that the acceptance range is 75125% but makes allowances for a percentage of results to be outliers. The observed number of outliers for the "QC-25ppb" spiikes 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 ablove the LOQ. Occasionally, Day 0 samples or other types of blanks show positive detections at levels near the LOQ. 3M Environmental Laboratory 3M Environmental Laboratory Page 13 of 216 Page 13 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. I % 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, arid 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. 0 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 C8FI7SO3 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. DATASUMMARY Tables; 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The table displays c~F17s03c-oncentrations for individual injections. All data are corrected for purity of the PFOS reference substance used for the extracted-matrix calibration curve. Table I O . CsFI7SOi Data Summary, Female Mallard Sera (Adult and Offspring) E01-1256-28672 lE01-12!56-28674 lE01-12!56-28676 lE01-1256-28681 E01-1256-28691 E01-1256-28695 454-109-3218,O PPM, Female, Adult 454-109-3220.0 PPM. Female, Adult 1454-109-3222,O PPM, Female, Adult (454-109-3224,OPPM, Female, Adult 454-109-3226,O PPM, Female, Adult 454-109-3228,O PPM, Female, Adult 1454-109-3230.0 PPM. Female. Adult 1454-109-3232.0 PPM. Female. Adult ~~ 454-109Z234,O PPM, Female, Adult 454-109-3236,O PPM, Female, Adult 1454-109-3238.0 PPM. Female, Adult 454-109-3240,O PPM, Female, Adult 454-109-3242,O PPM, Female, Adult 454-109-3244.0 PPM. Female. Adult 454-109-3246,O PPM, Female, Adult 454-109-3248,O PPM, Female, Adult 454-109-3250,O PPM, Female, Adult 454-109-3252,O PPM, Female, Adult 3M Environmental Laboratory 3M Environmental Laboratory 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 < I00 < I00 <0I0 400 <IO0 0.5 1 2680 I 0.5 1 1 I 400 1 0.5 1 1 1 172 0.5 1 400 0.5 I 0.5 I 1 1 1 < I00 166 0.5 1 400 0.5 1 400 0.5 1 < I00 0.5 1 400 0.5 1 0.5 1 398 400 0.5 1 757 Page 14 of 216 Page 14 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard E01-1; E01-1; E01-12 EOl-li EOl-li EOl-li EOl-12 E01-12 E01-12 E01-12 E01-12 E01-12 E01-12 E01-12 E01-12 E01-12 E01-12 E01-1256-28731 E01-1256-28735 E01-1256-28737 E01-1256-28739 6. 10 PPM. Female. Adult 454-109-3288,lO PPM, Female, Adult 454-109-3290, 10 PPM, Female, Adult 454-109-3292. 10 PPM. Female. Adult 454-109-3294,lO PPM, Female, Adult 454-109-3296, 10 PPM, Female, Adult 0.5 10 0.5 1 0.5 1 0.5 1 0.5 1 E01-1256-28740 454-109-9583,O PPM, Female, Offspring 0.15 1 E01-1256-28742 454-109-9589,O PPM, Female, Offspring 0.2 1 E01-1256-28743 454-109-9592.0 PPM. Female. OffsDrina 0.2 1 42800 16000 10400 7660 10800 < I 00 400 4 00 0 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 Page 15 of 216 Page 15 Report E01-1256 Report E01-1256 - PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 11. C ~ F I ~ SDOata~ Summary, Male Mallard Sera (Adult and Offspring) lE01-1256-28671 E01-1256-28677 E01-1256-28684 lE01-1256-28686 E01-12.56-28696 E01-12!56-28698 E01-12!56-28700 E01-12!56-28706 t-E01-12!56-28712 E01-12!56-28714 E01-12!56-28720 lE01-12!56-28722 E01-1256-28728 454-109-3221,O PPM, Male, Adult 454-109-3223,O PPM. Male. Adult 1454-109-3227.0 PPM. Male. Adult 454-109-3229,O PPM, Male, Adult 454-109-3231,O PPM, Male, Adult 454-109-3233.0 PPM. Male. Adult 454-109-3237,O PPM, Male, Adult 454-109-3239, 0 PPM, Male, Adult 454-109-3241.0 PPM. Male. Adult 1454-109-3243.0 PPM. Male. Adult 454-109-3245,O PPM, Male, Adult 454-109-3247.0 PPM. Male. Adult 454-109-3249,O PPM, Male, Adult 454-109-3251,O PPM, Male, Adult 454-109-3253.0 PPM. Male. Adult 454-109-3255,O 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 1454-109-3265,lO PPM, Male, Adult (454-109-3267.10 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 1454-109-3279. 10 PPM. Male. Adult 454-109-3281, 10 PPM, Male, Adult 454-109-3283, 10 PPM, Male, Adult 454-109-3285. 10 PPM. Male, Adult 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,O PPM, Male, Offspring 0.5 0.5 1 0.5 I 0.5 0.5 0.5 0.3 0.5 0.5 1 0.5 1 0.5 0.5 0.5 0.5 0.1 0.5 1 1 1 I 1 1 1 1 1 1 1 I 1 1 1 1 1 1 400 < I00 400 4 00 400 < I 00 400 < I 00 1400 400 400 162 1070 400 < I00 400 0.5 0.2 0.5 0.06 0.2 0.5 0.5 0.5 0.5 0.5 0.5 I 0.5 I 0.5 0.5 0.5 0.3 0.3 0.3 0.5 0.5 10 10 10 100 10 10 10 10 10 10 10 10 I 10 10 10 10 10 10 10 10 78800 83300 68700 21 0000. 93800 75700 80600 11300 106000 80500 80400 103000 74300 91 800 108000 78700 97900 73500 96800 99700 0.1 1 400 3M Environmental Laboratory Page 16 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard EOI-12!56-28746 1454-109-9606,O PPM, Male, Offspring E01-12:56-28757 454-109-9625,lO PPM, Mz,Offspring 454-109-9636. 10 PPM. Male. OffsDrina 1454-109-9642, 10 PPM, Male, Offspring 0.2 1 0.2 1 0.1 1 0.2 1 103 5450 3670 41 10 3M Environmental Laboratory 3M Environmental Laboratory Page 17 of 216 Page 17 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 12. C8Fl;rSOiData Summary, Female Mallard Liver (Adult and Offspring) 3M Environmental Laboratory 3M Environmental Laboratory Page 18 of 216 Page 18 Report E01-1256 Report E01-1256 - PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 10 pprn a.i. 3296 F Liver, adult E01-1367-31295 0 pprn a.i. 9583 F Liver, offspring 0 pprn a.i. 9589 F Liver, offspring E01-1367-31313 0 mrn a.i. 9592 F Liver. offsorina 0 pprn a.i. 9598 F Liver, offspring 0 porn a.i. 9603 F Liver, offsorina lE01-1367-31337 10 DDrn a i 9610 F Liver. offsorina I lE01-1367-31343 10 porn a.i. 9573 F Liver, offsDrina 1 10 pprn a.i. 9629 F Liver, offspring 10 pprn a i . 9633 F Liver, offspring E01-1367,-31374 10 Dorn a.i. 9634 F Liver. offsDrina IE01-1367-31380 110 mrn a.i. 9645 F Liver. offsDrina I 10 pprn a.i. 9648 F Liver, offspring 10 own a.i. 9654 F Liver, offswina E01-1367 31410 110 ppm a.i. 9659 F Liver, offspring 1.1238 1 1.1308 1 1.0005 1 1.0390 1 1.1204 1 1.1550 1 1.0217 1 1 I 1.0122 I 1 I 1.1676 1 1.0510 1 1.1638 1 1.1510 I 1 I 1.I125 1 1.0770 1 1.0581 1 6330 e50 60 40 60 -60 e50 <50 2950 2240 4520 2580 3500 5930 3580 3M Environmental Laboratory 3M Environmental Laboratory Page 19 of 216 Page 19 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 13. CSF~~SODa<ta Summary, Male Mallard Liver (Adult and OffsDrinaI EO1-1326-29473 0 ppm a.i. 3217 M Liver, adult 1.1169 1 0 ppm a.i. 3219 M Liver, adult 1.1847 1 E01-1326-:!9592 0 ppm a i . 3221 M Liver, adult 1.1181 1 0 ppm a.i. 3223 M Liver, adult 1.0788 1 0 pprn a.i. 3225 M Liver, adult 1.IO43 1 E01-1326-29646 0 ppm a.i. 3227 M Liver, adult 1.0486 1 0 ppm a.i. 3229 M Liver, adult 1.0336 1 e50 e50 60 e50 40 40 ~50 3M Environmental Laboratory 3M Environmental Laboratory Page 20 of 216 Page 20 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard STATEMENT OF CONCLUSION Sera results from the male control group portion of this study showed no detectable levels of C8Fl7S0iin 14 of 18 mallards (<lOOng/mL) and quantifiable concentrations of C8FI7SO9in 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 C8Fl7S09 in 15 of 17 birds (<lOOng/mL) and quantifiable concentrations of C8F17S03-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 C8F17S03-(range 68700-108000ng/mL, Table 15). Analytical results for nineteen sera samples from the 10ppm dosed female group showed an average of 16600ng/mLC8Fl7S09 (range 6350-42800ng/mL, Table 15). Sera results from the male offspring control group showed no detectable levels of C8Fl7SO9in 2 of 3 birds (<lOOng/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 C8Fl7S09in 7 of 7 birds (<lOOng/mL, Table 14). Analytical results for three sera samples from the 10ppm dosed male offspring group showed an average of 441Ong/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 C8F17S09in 20 of 20 birds (<50ng/g, Table 14). The liver results from the female control group also showed no detectable levels of C8Fl7S0iin 20 of 20 birds (<50ng/g, Table 14). Analytical results for twenty liver samples from the lOppm dosed male group showed an average of 60900ng/g C8FI7SO3- (range 24200-125000ng/g, Table 14). Analytical results for twenty liver samples from the 1Oppm dosed female group showed an average of 10800ng/g C8Fl7S09(range 2480-30700ng/g, Table 14). Liver results from the male offspring control group portion of this study showed no detectable levels of C8Fl7S09in 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 C8Fl7S0; 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 1Oppm 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 E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A ReproductionStudy with the Mallard Table 14. Summary Table, CsF17SO; Concentration in Sera and Liver I Male Control Group 1 l8 1 NA 1 <IO0 ng/mL(14of 18) 107-1400ng/mLrange(4of18) 20 I Female Control Group 1 1 NA I 4 0 0 ng/mL (15 of 20) 16~2680ng/mLrange(5of20) Male 1Oppm 20 89700 ng/mL 11300-210000 ng/mL 20 Group <50ng/g 1 NA 1 NA 60900 ng/g 24200-125000 ng/g Female 1Oppm Group Male Offspring I 1 Control Group I l FCemonatlreoOl Gffrsopuripng Male Offspring 10ppm Group Female Offspring 1Oppm Group 20 31 7I 3 7 20300 ng/mL NAI NAl 4410 ng/mL 4960 ng/mL 6350-91400 ng/mL <IO0 ng/mL (2 of 3) 103 ng/mL (1 of 3) <IO0 ng/mL (7 of 7) 3670 - 5450 ng/mL 3420 - 6760 ng/mL 20 10800 ng/g 2480-30700 ng/g I 7 l <50ng/g I NA I 3 3170 ng/g 2200-4640 ng/g 7 3620 ng/g 2240-5930 ng/g For individual samples values see Tables 10 to 13 and Attachment B. 3M Environmental Laboratory 3M Environmental Laboratory Page 22 of 216 Page 22 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 15. Summary Table, CSF+~SOC<oncentration in Sera and Liver with Outliers Removed Male Control Group I I I I I I I * I 18* * 20* Female Control <100ng/mL(15of 17) I 1 1 I I I I * Group l7 NA 16&172ng/mLrange(20f17) 20* I I I I 1 1 I * I Male10ppmGroup 18 87300 ng/mL 68700-108000 ng/rnL 20* Female lOppm 19 16600 ng/mL 635042800 ng/mL 20* Group MCoanletrOolffGsproriunpg 3* t * 3* Female Offspring 7* Control Group Male Offspring 1Oppm Group 3' Female Offspring 7* 1Oppm Group * I * 7* * * T 3 *I * I * I 7' STATISTICAML 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 DATAQUALITY 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 3M Environmental Laboratory Page 23 of 216 Page 23 ~ Report E01-1256 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 "E:xtraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC ElectrospraylMass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC Electrospray/Mass Spectrometry in Quail Serum and Liver." 4. 3M Environmental Laboratory Standard Operating Procedure ETS-8-231.1, "Solid Phase Extraction and Analysis of FluorochemicalCompounds form Biological Matrices". LISTOF ATTACHMENTS 0 Attachment A: Extraction and Analytical Method 0 Attachment B: Data Summary Tables 0 Attachment C: Sample Chromatograms, Calibration Information, and Instrument Parameters 0 Attachment D: Sample Preparation Sheets 0 Attachment E: Study Protocol, Protocol Amendments, and Deviations 0 Attachment F: Example Calculations 3M Environmental Laboratory 3M Environmental Laboratory Page 24 of 216 Page 24 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard SIGNATURPEAGE We certify that this report is a true and complete representationof the data for this study: Lisa A. Stevenson Principal Analytical Investigator Date William K. Reagen Laboratory Management Date 3M Environmental Laboratory 3M Environmental Laboratory Page 25 of 216 Page 25 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard ATTACHMENAT: EXTRACTIOANND ANALYTICAMLETHOD,ETS-8-231.I, "SOLID PHASE EXTRACTIOANND ANALYSISOF FLUOROCHEMICAL COMPOUNDS FROM BIOLOGICAMLATRICES" 3M Environmental Laboratory 3M Environmental Laboratory Page 26 of 216 Page 26 Report E01-1256 Report E01-1256 - PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Method Solid Phase Extractionand Analysis of Fluorochemical CompoundsfromBiological Matrices Method Number: ETS-8-231.7 Adoption Date: ////j/i)/ Revision Date: 2//t?/oz Effective Date: 2118/62 Approved By: ygjw- - Willliam K. Reagen Date Laboratory Manager ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 1 of 19 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 27 of 216 Page 27 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 froin 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 datzi (qualityobjectives ace met. - 2 IMethod Summary An amount ofbiological material. deteiinined by the analyst. is prepared (fluids diluted and tissues homogenized)at a 1/6 dilution, or other dilution as detemined by the analyst using re.agent grade water. An aliquot of the tlilutionihornogenateis spiked with the appropriate surrogate or analyte mixture. Acetonitrile (ACN) is added ;ts au extraction solvent and also serves to precipitate the proteins. The sample is capped. mixed. and put on the centrifuge to clarifi the supeiiutnnt. The supernatant is transfell-ed to a clean tube, diluted with water, and passed through a preconditioned C1gSPE cartridge. Finally,the analytesofinterest are eluted from the SPE cartridge and analyzed by high perfonnatlce liquid chroinatography-electrospiaytandem inass spectrometiy(HPLC-ES;nIlSiMS). - 3 Definitions 3.1 Dilution .A dilution expressed as 1 :5 or 116 is defined as: 1 niL of sample + 5 m L s of diluent for a total of 6 niLs combined, unless otherwise noted. 3.2 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds of inteitst under some solvent conditiolis and elute them under others. A separation is thus achieved; coinpowids can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectableconcentmtion(s)ofthe target analyte(s). 3.4 Quality control sample Sampleused to inonitorthe extraction efficiency(as a inahix spke) and to verify the continued accuracyof the initial c.%libratiocnw c (as a continuing calibrationverification). 4 Warnings and Cautions 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewear. aid clothiig when workingwith solvents.samples a~uUoerquipnient. Use caution with the voltage cables for the probe, When engaged, the pmbe employs a voltage ofapproximately 5000 volts. 4.2 Cautions Take care not to allow the SPE colunln to run to diyness after the methanol and water washes. After washing is complete.add sample then allow all of the liquid to pass through the SPE colonn 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 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Study with the Mallard Do not operate solvent pimps above capacityof400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar. the HPLC will initiate autonutic shutdowi. Do not nui solvent pumps to dryness 5 llnterferences 'To minimize interferences.Teflon should not be used for sample storage or any part of instrumentationthat comes in contact with the sample or extract. - 6 Instrumentation, Supplies, and Materials The following instninientation, supplies, and nuterials are used while perfom'ng this method. instrumentation,supplies,and materialsmay be used in place ofthose listed. Equivalent 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 2 IJltra-Turrax T25 tissue homogenizer Vacuuni Pump SPE Extraction Manifold ('mtrifiige. Mistral 1OOO or IEC Shaker, Eberbach or V b X Eialance (+i-0.1000 g) Edicronmss. Quattro I1or Ultima triple quadrupoleMass Spectrometerequipped with an electrosprayionizationsource HPI 100or Agilent low pulse solventpunping system solvent degasser.colunmcompartment,and autos'mpler 6.2 Supplies and Materials Eppendorf or disposable pipettes, plastic or glass Dissecting scalpels P'olypropyleie bottles, capable of holding 50 mLto I L (Nalgene) Volumetric flasks, glass. type A 40 mL glass vials (ICHEM) Plastic sampulr vials. Wheaton, 6 mL (or other appropriatesize) Centrifugetubes. polypropylene. I5 niL and 50 i L L.abels Ciraduated pipettes. glass Syinges, capable of measuring5 pL to 1000@ Eottle-TopDispenser(capableof dispensing5nL of solvent) SPE extraction cartridge. 1 g, Sep-Pak 6 cc tri-furictional Cui (Waters) 75 niL sample reservoir (or other appropriate size) Crinip cap glass autovials and caps ETS-8-231. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 3 of 19 3 M Environmental Laboratory 3M Environmental Laboratory Page 29 of 216 Page 29 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Crimpers HPLC analytical colunn. specificsto be determinedby the analystand documentedin the raw data. - 7 Reagents and Standards ]Reagentgrade water. Milli-QTh'.Nanopm 11, or equivalent .4cetonitrile. HPLC grade or equivalent ]Methanol,HPLC grade or equivalent d4mnioniumacetate. reagent grade or equivalent 13iologicalfluidsor tissues, fi-ozen from supplier 7.1 Reagents preparation 2!.0 mM animonium acetate solution: Wei& approximately 0.300 g ammoilium acetate. Porn into a 2000 nlL volumetric container contaiiiingreagent grade water. mix until all solids are dissolved, bring to volume using reagent grade water. Store at room teniperature. Note: When preparing difTerent volumes than those listed in reagents preparation, target analyte standard preparation, zmd 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 tollowing is an example only and may or may not be appropriate for all standard preparations. Weigh approximately IO0 rng of target analyte into a 100 n L 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 ppni QlgnL). Dilute the stock solution with methanol for a working standard 1 solution of approximately SO ppm. Example calculation: I000 pgmL x 5 mUlOOmL = 50 pg'nlL. Dilute working standard 1 with nlethanol to produce a working standard 2 solution of approx 5.0 ppni. Emnple calculation:50 M n i L x 10mLil OOmL = 5.0 pg/niL. Dilute working standard 1 with methanol to produce a working standard 3 solution of approx. 0.50 ppm. Example calculation:SO p g h L x 1 .0 mL/100mL = 0.5 pg/mL. 7.3 Surrogate standard preparation Prepare smogate standard(s). The following is an example only and may or may not be appropiiate for all surrogate standard prepai-ations. Weigh approximately90-1 I O mg of surrogate standard into a I(x)-mL volinnetricflask and record the actual weight. Ekingto volume with methanol for a surrogatestandard stock of approximately900 - 1100 ppm. Prepare a surrogate standard working standard. Transfer approximately I mL of surrogate standard stock to a IO-mL volumetric flask and bring to volume with methanol for a working standard of 90-1 l0ppm. Record the actual volume transferredand standard concentrationsin the standardsloghook or other appropriatelocation. 7.4 Internal standard preparation Prepare internal standard(s). The following is an exaniple only and may or may not be appropriate for all internal standard preparations. Weigh approximately90-1 10 mg of internal standard into a 100-mLvolumetric flask and record the actual weight. ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 4 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 30 of 216 Page 30 Report E01-1256 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 s?ock of approximately900 - 1100ppm. Prepme an internal standard working standard. T m y f i r approximately 1 mL of internal standard stock to a 10-inL volinnetric flask and bring to volume with methanol for a working standard of90-I I0ppm. Record the actual volume hansferredand standard concentrationsin the staxlards logbook or other appropriate location. - 8 Sample Handling ,411saniples ait received fiozen and must be kept frozenuntil the extiaction is performed. Allow samples to thaw to room temperatureprior to extraction. `Typically Gesh matrix standards we prepared with each analysis. Extracted standards and samples are stored in capped autovials imtil analysis. If analysis will be delayed. extracted standards and samples may be refrigerated at approximately 4C indefinitely or tnay be stored at room temperatureuntil analysiscan be perfomxd. - 9 Quality Control 9.1 13lanks 9.1.1 Solvent Blank An aliquot of niethrznol is used as a solvent blank. Solvent blanks are not extracted. 9.1.2 Method Blank An aliquot of 1.0 mL of 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 of 1.O id or 1.Og of matrix (dilutedor honiogenized)is used as a inatrix blank. Other amountsinay be used, as appropriate. Matrix blanks are prepared from one of three sources: 1) a study control matrix from a study control anirnal received %ith a sample set; 21 a conunercially obtained sanyle of the same species as the study miinals; or 3) a surrogatemarrix: also obtained commercially: but of a different speciesthan the study animal. (eg. if rat is used to generate standard curves and CCVs for a mouse study). The nutrix to use is dependent on the matrix used for the curve. 9.1.3.1 Study control matrix curve - if the studycontrol matrix is used for the curve, prepare four (4)mltrix blanks using the study control matrix (two spiked with surrogatt and two not spiked). 9.1.3.2 Commerciallyobtained (same species)matrix curve- if the commerciallyobtained matrix is used for the curve. prepare four (4) nutrix blanksusing the same commerciallyavailablematrix (two spiked with surrogateand two not spiked). 9.13 . 3 Surrogate nutrix curye- if a surrogateinatrix is used for the curve. prepare four (4) matrixblanks usingthe same conunerciallyavailableinatrix and prepare four (4)matrix blanks using a comnierciallyavailable n z h x of the same species as the study animals(two spiked with surrogateand two not spiked). 9.1.3.4 If limited matrix is available,the number of method and matrix blanks may be adjusted and will be noted in the study protocol or in the raw data. 9.2 Sample Replicate Samplesreplicates are prepared accordingto each study protocol or project outline ETS-8-231.1 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 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Study with the Mallard 9.3 Surrogate standard If 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 stiuidard is spiked into the I .O mL dilutedihomogenizedsample removed for extraction. However, :surrogate may be spiked directly into the matrix prior to diluting with water. into the dilutedihomogenized sanlple ]priorto removingthe 1.O mL sample, or into the 1.0mL dilutedhomogenizedsaniple removed for extraction. 9.4 llnternal standard If internal standard is a component of the study, all samples are spiked with llitemal standard after extraction to obtain a concentrationin the nud-rangeof the calibrationcurve. Typically intaml standard is spiked into the 2.0 mnL of extract in the 15 mL centrifuge tube. before wmsfening to the iiu tovial. 9.5 Lab Control Sample Lab control saniples are not a component of this method. 9.6 Quality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efticiency and to veri@ the contiiiued acciuacy of thc initial calibration curve. Typically 1.0 n i . or other appropriate amount,of the m i e imtrix used to pKepare 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 mninknuni 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 raniples will be extracted on three different days then four QC samplesmust be prepared on each day of extraction for i:i total of twelve.) QC samples will consist of four samples at each of three levels of analyte. The levels listed below may be used and may represent sample concentrationsdiluted into the range of the calibrationcurve: Low level: 3X to 5X the LLOQ, Mid-level: equivalent to a point near the middle of the Cali bmtion curve, High level: 80% of the ULOQ Two QC; sample levels are analyzed after every tenth sample in,jection starting after the last calibration standard injection, with a minimum ofthree QC per analysis. Solvent blanks are not considered samples but nlay be included as such for deternlining when QC samples will be analyzed. ()C samples extracted with a particular simple set must be analymd in the same analytical run. Any QC samples extrwted duringthe course ofthe studym y be included in subsequentanalyses. If sclniples from multiple extraction dates are analyzed in one analyticalrun, then QC" saniples koni the same smple extraction dates must be included in that analysis. Each QC is expected to show an accuracy of75425% of expected. A minimum of213 of all QC samples must meet tlhis criteria, and a minimum of li2 of the QC samples at each level must nieet h s ciiteiia. If not, the set must either be re-analyzed or re-extmcted. 9.7 Sample Dilution Any sample with an area greater than that of the highest acceptable standard will need to be diluted into the range of tine calibration curve. If samplesare diluted into the range ofthe curve during analyses and enough sample rzmains. a post-run dilution validation will be pcrfonned to veii@ sample values. To pertom the dilution validation. one sample will be separated into two representative samples (Le. tvio I .O mL aliquots for fluid samples or two 1.0gram amounts for tissue samples, or other amount as determined by the analyst ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds fiom Biological Matrices Page 6 of 19 3 M Environmental Laboratory 3M Environmental Laboratory Page 32 of 216 Page 32 Report E01-1256 Report E01-1256 - PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard .md documented in a note to file) then diluted using two procedures. The first procedure consists of diluting the .sanlple with additioiid matrix prior to extraction (fluid adding tluid). while the second procedure consists of diluting h e extract with solventpost-extraction(methimol extmct adding additionalmethanol solvent.) Lf die relative percent dityerence is not within 15% for these two samples; additional testing will be required to determinewhich value is a correct representationof the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration ( h e calibration curve will be prepared from extracted matrix standards. in the same matrix as the samples, per study. It will consist of a minimum of nine (9) levels. Additional calibrdtioncurves may be extracted on separate sample extractiondates. a?determinedby the analyst and documentedin a note to file. Trmfer I .O mLI or other appropriate mount, of' diluted control fluid or homogenized control tissue to a 15 inL cenhihge tube using a disposable plastic pipette. This will he repeated ubile preparing aliquots for the standard curve. Be sure to mix or shake the control nutrix container between aliquots to eiwre a Iioniogenous sample is renioved. Record each standard volume on the weightkoluiies sheet or extractionworksheet,as appropriate. Four 1.0 inL aliquots, or other appropriate amount,of control matrix sene as matrix blanks. The staiidard concentrations and spiking mounts listed in Table 1 nuy be used, when appropriate, to spike one standard c w e . A total of 9 standards. four matrix blanks. and four method blarks are prepared in additiorito the QC samples and test samples. The number of standards and blanks may be adjusted as detennined by the analyst and clocuniented in a note to file. IJse Attachment C. or other appropriate foim. as an aid in calculating the concentrations ofthe working standards. FLefer to section 12 to calculate the actual concentrationof analyte in each calibration standard aid QC sample. Typically the target d y t e standard is spiked into the 1.0 mL dilutedhomogenized sample removed for extraction. However, it may be syiked directly into the matrix prior to diluting with water, into the dilutecthoinogenized sample prior to removing the 1.O n L sample, or into the 1.O mL dilutedihoinogenizedsanlple removed for extraction Analyze the extmcted matrix standard curve prior to each set ofextracts. The curve equation will be determined by regressionanalysisusing the peak areas ofthe target analyte(s) using MassLynx or other suitable software. Pny level oulside 75% - 125/~ of nominal niust be deactivated,and regressionrecalculated. except the LLOQ which niust be within 30% of nominal. All levels must show a response greater than twice that of the blank. A nuximum of thee (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 of Fluorochemical Compounds from Biological Matrices 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 19 Page 33 of 216 Page 33 Report E01-1256 Report E01-1256 - PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Working standard (approximate concentration) Approximate final Approxinlate final lLL concentration of analyte concentration of analyte in Matrix diluted 1:s in Final 2.0 mL volume 11 Procedures II.I Tissue Sample Preparation Obtain Frozen tissue samples Cut approximately 1.0000g of tissue (+/-0.1ooO g), or other appropriateamount,using a dissecting scalpel. This part of die prccedure is best pertolined quickly,not allowingthe tissue to thaw. Weigh the tissue directly into a tared plastic sampule vial. Record the weight on the weightholume sheet, extractionworksheet, or other appropriatelocation. R.ehun unused tissue to the freezerafter extractionamountshave been removed. Add 2.5 mL of reagent water to sampule vial, or other volume as determinedby the atlalystand documented in a note to file. Homogenize the sample. Put the Ultra-Turrax grinder probe in the saniple and grind for approxinutely 2 nzinutes, or until the sample is homogeneous. Rjme the probe hito the tubecontainingthe samplewith2.5 mL ofreagent grade water, or othervolume as determined by the analyst and documentedin a note to file, using a pipette. Take the giider apart and clean it uith methanol after each sample. Refer to ETS-9-52 fix more int~imlation. If' an amount other than I .OOO0 g (not within +/- 0.1000 g) is removed for an initial weight. adjust the water volume accordinglyto mnaintain a lib dilution.(e.g. if 0.5 g is removed for extnction, add a total of 2.5 mL of water. ). or other mtio as determinedby the analyst and documentedin a note to file. ETS-8-23 1. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 8 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 34 of 216 Page 34 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 temperatureor in lukewarm water. Label a 15 niL polypropylene centiifige tube with the study number, saniple ID, extraction date arid analyst initials. See attached worksheet(AttachmentA or similarworksheet)for documentingthe remaining steps. Vortex mix the fluid sample for approximately 15 seconds. then transfer 1 .0 mL of fluid, or other appropiate amount to a plastic sampule vial, or other appropriate container. Retuin unused samples to freezer after extraction aniounts have been removed. Add 5.0 mL of reagent nater to the 1.0 mL of fluid for a 1% dilution, or other dilution as determined by the analyst and documented in n 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 ARer tissue or fluid samples have been prepared according to sections 1 I . 1 and 1 1.2,vortex mix or shake by hand the dilutedhoniogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to n clean 15 mL polypropylenecentrifugetube. IZeturnunused dilutedihomogenizedportions to the freezer after extractionamountshave been removed Record the volunie removed on the extraction worksheet. (AttachmentA or similar worksheet) Spike blanks, samples,and standards,iwdy for extractionwith surrogatestandard as described in this method. Spike each calibration standard matrix with the appropriate amount of standard as described in this method for the c:alibrationcurve standards arid each QC sample. Vortex ink the standard curve samplesand QC samples for approximately5 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 arid centriikge at an appropriate speed for 10 minutes to adequately pellet the precipitate (a setting ofapproximately 2000 rpm on the models listed in section 6.1). Add 40.0 mL of reagent gmde water to a clean SO mL centrifLige tube. Remove samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduceany of the matrix solids into the solution Cap and mix by inverting several times. In this step the order of addition may be changed (Le. the sample m a y be put into the centrifbgetube and then the water added). Athcli the reservoir to the SPE cartridge and attach this reservoidcartridge uiut to a vacuum niiuiifold. NOTE: When running the vacuum. set the vacuum chamber at approximately 15 kPA - to give an approximate elution flow of 5-7 mL/min. Flows 'nay vary tluough cartridges and the kPA may be raised for slow tubes and drying afier rmst have been drawn down. F'repare the SPE cartridge by washing &ice with approxinmtely5.0 mL of nietlmol, followed by approxinmtelytwo 5.0mL alicpots of water. taking care not to allow the column to run to dryness after each wash. After washing is complete, pour the sample into the reservoidcartridge unit and allow all of the liquid to pass through the column to dryness. Run the vacuum on high for approximatelyS minutes to adequatelyclry each SPE cartridge Place a collection 15mL polypropylenecentrifugetube under each cartridge and elute with 2.0 I& of methanol. Spike extractedblanks, samples,and standardswith internalstandardas described in this method. ETS-8-231 . 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 9 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 35 of 216 Page 35 Report E01-1256 Report E01-1256 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 numnberi~enderitiniepoinot r LlMS nuniber, matrix, final solvent, analyte coinponents (if needed), extraction type, extractiondate. and analyst(s) perfohng the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis I 11.4.1 Software set-up On the MassLym inain page, set up a sample list name. Save the list as instrumentdesignator letter, last 2 digits of test year-month-day.and a letterthat will increasethrough the alphabet with each iulditionallist for that day. Example Sample List: IWMMDDa or A020204a I = Initial of the instrumentname (A = "Amelia") W = Test year (02) MM = Test month (02) DD = Test day (04) a = First sample list (run) ofthe day (thenext 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-monthday, and a 3digit sequentialfile number that starts with 1 and increasesby one for each filename. Emiple filename: I Y Y M M D W or A020204001 I = Initial of instrumentname YY = Test ymr MM = Test month DD = Test day ####= 3-digitsequentialfile nuniber startingwith 1 through 999 (001) Also, as part of tlie samplelist,assign a method (MS) for acquiring,an inlet file. a bottle number, an ipjection volume, and sample descriptions. To create a method. click on Method Editor button in the MS status Pane and select SIR (Sinde Ion Recording)or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and nzass to 499 or other appropriate mss(es). Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentationinfixmlrrtion may be collected. See Microomass MnssLyIx "Guide t o Data Acquisition"for additional infomition on MRM. Typically the analyticalbatch runsequencebegins with system suitability,solventblanks, and a set of extracted matrix standards. Sample extracts me analyzed with two QC samples injected after every tenth sample injection. Solvent blanks should be analyzed periodically to nionitor possible aialyte carryover and are not considered sample extracts but my be included as suck ETS-8-23 1. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 10 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 36 of 226 Page 36 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 sarnpletray according to the ~ n p l leist prepared above. Set up the HPLC to the following conditions or at conditions the analyst considers appropriate for optind response. Record acaial conditions in the instrumentlogbook, or other appropriate location: Sample size = 10 pL injection Inject/sample= I Cycle time 5 10.0minutes Flow mte = 300 pLhnin Mobile phase: Solvent A = 2 nln4 Anunonium Acetate, Solvent B =Methanol Solvent gradient progani: 10% 95% 95% 10% I I.4.3 Instrument set-up Refer to ETS-9-24, "Operationand Maintenanceof the Micromass Q~attroI1 Triple QuadrupoleMass Spectrometer Fitted with an AtmosphericPressure Ionization Source." for details. Lleck the solvent level in HPLC reservoirs and refill ifnecessmy Check the stainless steel capillary at the end ofthe probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. Ifthe tip is found to be unsatisfactory?disassemblethe probe and replace the stainlesssteel capillary. Turn on the nitrogen. Open the tune page. Click on operate to initiate s o m e block and desolvationheatas. Open the Inlet Editor. Download the F1PI-C method and initiate solvent flow to begin system equilibrium. Set the flow to 10-500uLiniin or as appropriate Set HPLC pimp to "On" Observe droplets or mist coining out of the tip of the probe. A fuie mist should be expelled withno nitrogen leakingaround the tip ofthe probe. Readjust the tip of the probe if no nV5t is obsewed Allow to equilibrate for approximately I O minutes. Typical instrumentparametersiiiclude: Dryinggas 25WOO litersihour ES nebulizing IS 10-1 5 Iitem'hour ETS-8-231. I Solid Phase Extraction and Analysis of Fluorochemical Compounds fiom Biological Matrices Page 1I of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 37 of 216 Page 37 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard HPLC constant flow mode. flowrate W500 &/inin Pressure <400 bar (this paranieter is not set, it is a guide to ensure the HPLC is operating correctly.) Sourceblock temperatureapproximately 150C Desolvation temperatureapproximately250C These settingsmay change in order to optimize the response Print the tune page, sample list, and acquisition method Gom MassLynx and store it in the study hinder with a copy taped into the instrument log. Click on start button in the Acquisition Control Panel (the location of the start button nlay vary among M a ~ s L y ~ x versions, refer to appropriate MassLynx User's Guide). 12 Data Analysis and Calculations 12.1 Calculations If other calculationsare used than those listed.they will be documented in the raw data. Calculatethe nlatrix aniount contained in the initial dilutionusing the followingequation: Matrix Amount (g,'niL or mL!'mL) = IW (8)(or IV (mL)) (IF'(@ (or IV(niL))+ DV (mL) Calculateactual concentrationsof analyte in calibrationstandardsusing the followingequation: Concentration (ngig or ng/mL) = Spike Concentration (ug/mL) X Spiked Amount (mL) 1000ng X- SV (niL)xMatrix Amount (g/niL or mL/mL) 1 ug IW = Initial weight (where 1.0g = 1. O I L ) IV = Initial volume I)\' = Diluent volunie (reagent grade water) SV = Sample volwne removed for extraction(typically 1.OmL) AR = Analyticalresult from MassLynxsummary DF = Dilution factor EY = Final volume MA =Matrix amount 3 curve = MA of tissueifluidstandard curve. assumed to be 1 g or I niLi5 mL water 3 sample = MA of tissueifluid sample (-g or mL of sainplei5 mL water) Calculatespike percent recoveries using the followingequation: '%Recovery = Observed Result - Matrix Blank Result x 100 Spiking Level ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds fioni Biological Matrices Page 12 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 38 of 216 Page 38 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard CXculaterelative standarddeviationusing the followingequation: Relative Standard Deviation = Stindard Deviation x 100 Mean Calculatepercent deviationusing the followingequation: Expected Conc. - Calculated Conc. % Deviation = x 100 Expected Conc. Calculateactual concentrationof analyte in fluid (&nL): AR (n&/inL)x DF x a curve (mLiniL)x n'(mL) in Curve x a sample ( i d i d ) FV (mL) in Matrix 1000 ng = bg'g) Calculateactual concentrationof analyte in tissue (pg/g). AR (nS/g)x DF x a curve (e/mLI x FV InL) in Curve x a sample (giniL) FV (mL) III Matrix & 1000 ng = @g;g) 13 Method Performance 13.1 System Suitability System suitibility will be determined prior to the start and at the conipletion of each analytical run. Prior to the calibration curve and alter the last sample of the run three (3) mid-level unextracted calibration standards will be analyzed. As applicable, tlie peak area precision, retention time precision, resolution, and peak aymmetry will be monitored at the beginning and the end of the run separately. The peak area precision must be equal to or less than S.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(frontingor tailing) must be 0.5<AF<2.0.where AF is the asymmetryfactor. If any i t a of the system suitability fails, syszem maintenance must be completed prior to running a second set of systemsuitabilitysamples and the system suitabilitymust pass before startingthe calibration. If system suitabilityfails at the completion ofa rim. the sample set inust be reanalyzed. 13.2 Quantitation The coefficient of determination value for the calibration ciiwe, plotted by regression using the peak areas of the analyte(s).must be 0.990 or better. All active calibration curve points nitist be w+thin25% of the theoretical value with the exception of the LOQ point, which may deviate up to 30%. Calibntion standards with peak areas less than two times the curve nmtdx bl`ank will be deactivated to disqualifj. a data range that may be a t k t e d by background levels ofthe analyte. A valid calibration curve must codain at least 6 active points above and includingthe LOQ. Ifthe curve cannot meet these criteria,the saniple set must be reanalyzed or reextracted. 13.3 Accuracy Two thirds of all quality control samples <andli2 of each quality control mnple at each level are expectedto show an accuracy of75-125%. ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 13 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 39 of 216 Page 39 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Siurogates and internal standards must have a percent deviation < 50%. Debiations outside this range will be reanalyzed to confimL If the second analysis confimls the original. the deviation will be documented in the raw data. Ifthe second 'mdysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Saniple waste is disposed of in noninfectiousbiohaziid %vasecontainers. Flammable solvent waste is disposed of in high BTU containers. Glasspipette waste is disposed of in broken glass containerslocated in the laboratory. 15 Records Conplete the extractionworksheetattached to this method, or other applicableworksheet,and store with the studylaw data. Each page generated for a study must contain the following infomution (if applicable): study/project or instrument number, acquisition nxthod, integration method. saniple name, extraction date. dilution factor (if applicable). and analyst. Other informationmay be added ifapplicableto the study. Print the tune page. sample list. and acquisition method fiom MassLynx to include with the study raw data. copy these pages and tape into the instrumentrmlog. Plot the calibration curve by the appropriate regression. Print these graphsand store vdh the shidy raw data Print data integrationsumnlary, integrationmethod, and clxoinatogrrunsfrom MassLynx. aid store with the study raw data. Sunxmrizc data using suitable software (Excel 7.0 or LIMS) and store in the study folder. Hack up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 16 Attachments Attachment A: Extrxtion Worksheet Attachment B: Saniple WeightWolunie Worksheet AttachmentC, Calibration Standard ConcentrationWorksheet AttachnieotD. Dilutions S u m m q Worksheet 17 References ETS-9-24. "Opeiation and Maintenance of tlie Micronlass Quattro I1 Triple Quadiupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52. "Operationand Maintenanceofa Tissue Grinder" 18 Affected Documents None ETS-8-231.I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 14 of 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 I 9 Revisions Revision - Nu- mber 1 Revision Description Minor formatting changes. Added detailed ii~f'om~atiotno all sections concerning the extraction procedure, analytical procedure, and calculations. Added attaclinients and references. Revision - Date 02i18/02 ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 15 of 19 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 41 of 216 Page 41 Report E01-1256 Report E01-1256 Study Number: Prep Date: Analysts initials: Box#: PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachment A - Extraction Worksheet Method Revision:ETS-8-231,I Matrix: Sample Timepoint: Blank matrix TN-A- : Amount weighed/aliquoted: g/mL 1. Homogenize sample 2. Aliquot 1 mL of diluted matrixinto 15 mL polypropylene tube 3. Spike samples accordingly 4. Add mL of ACN (TN-A- ) to each diluted sample and shake or vortex mix 5. Shake sample for 20 min @ rpm (Shaker ) 6. Cenlrifuge sample for 10 min @ rpm (Centrifuge 7. Add 40 mL of ___________ water to 50 mL polypropelene centrifuge tube 8. Decant extract into centrifuge tubes with water 9. Shake sample slightly to ensure proper mixing - IO. 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 mL water - do not allow column to go to dryness 13. Filter sample through conditioned column, discarding filtrate 14. Allow column to go to dryness. Afler dripping stops, draw a high vacuum through column for at least 5 minutes. 15. Elute column with solvent C_-____-..-TN-A- ) into appropriate 15 mL centrifuge tube 16. Spike samples with uL of internal standard # , conc. 17. Transfer sample into appropriately labeled autovial and cap Note: In vacuum steps above set the vacuum chamber at approximately 15 kPA - this should give approximately 5-7 mLlmin elution flow Flows may vary through cartridges - kPA may be raised for slow tubes and drying afler most have been drawn down and shut off. ETS-8-231 I Solid Phase Extraction and Analysis ofFluorochemica1 Compounds from Biological Matrices Page I 6 of19 3M Environmental Laboratory 3M Environmental Laboratory Page 42 of 216 Page 42 Report E01-1256 Report E01-1256 ~____ ~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Prep l)ate(r): Analyst(s): Sample Matrix: MethodRevision: Sample ID Attachment B - Sample WeightNolume Worksheet Study Number: Equipment Number: Final Solvent & TN Number: Initial Wt.lVol. g/mL/L Water Volume added (mL) Volume Removed (mL) Comments b - -.--.--.---..I . ~ ~ " ~ I-" I ".-_I l l l i i _.I.I .............~ Fomi Completion Veritied By: ETS-8-23 1 . 1 Solid Phase Extraction and Analysis of Fluorochemical Cornpounds from Biological Matrices Page 17 of I9 3M Environmental Laboratory 3M Environmental Laboratory Page 43 of 216 Page 43 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Prep date@): Analyte(s): Sample matrix: Method/revision: Attachment C: Calibration Standard Concentration Worksheet Standard number: Equipment number: Final solvent and TN: Blank Tissue or Fluid/identifier: 5.00 0.0320 2.00 0.1667 0. I600 5.00 0.0400 2.00 0.1667 0.1600 50.0 0.005 2.00 0.1667 0.1600 50.0 0.006 2.00 0.1667 0. I600 (lalculated concentntions of standards in relation to the final 2.0 mL solvent and initial matrix Analyte Final conc. Surrogate Std conc ng/inL Analyte Final COIlC. Surrogate Std conc Analyte Final conc. Surrogate Std conc ng!nL ETS-8-23I . 1 Solid Phase Extraction and Analysis of Fluorocheniical Compounds from Biological Matrices Page 18 of19 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: Attachment D: Dilutions Summary Worksheet Solvent/TN Number: Extraction Date/Analyst: MatrixlTimepoint: Form Conipletion Verified By: ETS-X-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Conipouiids ti-om Biological Matrices Page 19 of19 3M Environmental Laboratory 3M Environmental Laboratory Page 45 of 216 Page 45 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Study with the Mallard ATTACHMENBT: DATASUMMARTYABLES 3M Environmental Laboratory 3M Environmental Laboratory Page 46 of 216 Page 46 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E014256 - MALLARD SERA DATA SUMMARY, BLANKS AND SPIKES E01-1256-36231 E01-1256-36259 EOl-I 256-36263 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 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 Blank H2O-day 1 do20225037 0.05 40 1 1 .oo Blank H20-day 2 do20226025 0.05 40 1 2.51 Blank H20-day 3 do20228024 0.05 40 1 2.51 Blank H2O-day 3 d020301099 0.05 40 1 Blank H20-day 4 do20228107 0.05 40 1 1 .OO 1.oo Blank H 2 0 d a y 1 do20308036 0.05 40 1 2.51 Blank mallard sera-day 1 do20225038 0.05 40 1 1.oo Blank mallard sera-day 2 do20226026 0.05 40 1 2.51 Blank mallard sera-day 3 do20228025 0.05 40 1 2.51 Blank mallard sera-day 3 d020301100 0.05 40 1 1 .oo Blank mallard sera-day 4 d020228108 0.05 40 1 1.oo Blank mallard sera-day 1 do20308037 0.05 40 1 2.51 40.0 !00.4 100.4 40.0 40.0 100.4 40.0 100.4 100.4 40.0 40.0 100.4 * 40.0 * 700.4 ' 100.4 40.0 * 40.0 * 100.4 ' 40.0 * 100.4 100.4 ' 40.0 ' 40.0 * 100.4 E01-1256-36233 E01-1256-36261 E01-1256-36265 E01-1256-36265 E01-1256-36269 E01-1256-36233 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 Mallard Sera MS-day 1 do20225039 0.05 40 Mallard Sera MS-day 2 do20226027 0.05 40 Mallard Sera MS-day 3 do20228026 0.05 40 Mallard Sera MS-day3 do20301 101 0.05 40 Mallard Sera MS-day4 d020228109 0.05 40 Mallard Sera MS-day 1 do20308038 0.05 40 E01-1256-36234 EO1-1256-36262 E01-1256-36266 E01-1256-36266 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 d020225040 0.05 40 Mallard Sera MSD-day 2 do20226028 0.05 40 Mallard Sera MSD-day 3 do20228027 0.05 40 Mallard Sera MSDday 3 do20301 102 0.05 40 Mallard Sera MSD-day4 do20228110 0.05 40 Mallard Sera MSD-day 1 d020308039 0.05 40 Conc of PFOS in Sample (ng/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilulion(di1uted sera)) dilution(di1utedsera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample (mL) =amount of sera usedltotal amount of sera t water (1/20 dilution). = 1/20 = 0.05 mL 1 27.79 1 29.50 1 28.85 1 27.76 1 28.01 1 29.03 1 30.44 1 29.80 1 24.76 1 24.01 1 28.23 1 31.34 for both ms and msd avg %rec avg conc std dev of conc 1112 1180 1154 1110 1120 1161 1218 1192 990 960 1129 1254 112.7 85.21 11 0.7 117.5 114.9 110.6 111.6 115.7 121.3 118.7 98.65 95.66 112.5 124.9 true Val. 1004 avg %rec 113.5 avg 1140 std dev 29.36 true Val. 1004 avg %rec 111.9 avg 1124 std dev 122.3 3M EnEvxicrelo2n00m0 ental Laboratory Blanks QCs and MSs mallard sera samples summary 030122.xls printed: 1/22/2003, 11:28 AMPage 47 Report E01-1256 Study number E01-1256 - MALLARD SERA versus unextractedcurve PFOS: A Reproduction Study with the Mallard Absolute Recoveries e01-1256-36254 QC- 250 e01-1256-36257 QC- 4.0 Study number E01-1256 - MALLARD SERA for all QC samples (absolute recovery) avg %rec std dev of %rec 135.8 33.11 e01-1256-36250 e01-1256-36251 e01-1256-36252 e01-1256-36253 e01-1256-36254 e01-1256-36255 e01-1256-36256 QC- 25ppb -1 (1004 ng/mL diluted sera) d020225071 QC- 25ppb -2 (1004 ng/mL diluted sera) do20225072 QC- 25ppb -3 (1004 ng/mL diluted sera) do20225073 QC- 25Oppb -1 (10040 ng/mL diluted sera) do20225074 QC- 250ppb -2 (10040 ng/mL diluted sera) do20225075 QC- 25oppb -3 (10040 ng/mL diluted sera) do20225076 QC- 4.0 ppm -1 (160640 ng/mL diluted seri d020308045 e01-1256-36257 QC- 4.0 ppm -2 (160640 ng/mL diluted send020308046 e01-1256-36258 QC- 4.0 ppm -3 (I60640 ng/mL diluted send020308047 A = Not within +/-25% criteria versus extracted curve rerun 0.05 40 0.05 40 0.05 40 0.05 40 0.05 40 0.05 40 0.05 40 0.05 40 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 avg 145.3 A 129.1 std dev 14.24 A 150.8 %RSD 9.800 98.02 100.7 avg 97.93 std dev 2.860 95.02 %RSD 2.920 79.70 90.86 avg 84.67 std dev 5.680 83.44 %RSD 6.708 for all QC samples original analysis (recovery) avg %rec 109.3 std dev of %rec 28.68 e01-1256-36250 e01-1256-36251 e01-1256-36252 e01-1256-36253 QC- 25ppb -1 (1004 ng/mL diluted sera) do20405036 QC- 25ppb -2 (1004 ng/mL diluted sera) d020405037 QC- 25ppb -3 (1004 ng/mL diluted sera) do20405038 QC- 250ppb -1 (10040 ng/mL diluted sera) do20405039 e01-1256-36254 QC- 250ppb -2 (10040 ng/mL diluted sera) d020405040 e01-1256-36255 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 seri do20405043 e01-1256-36258 QC- 4.0 ppm -3 (160640 ng/mL diluted serid020405044 A = Not within +/-25% criteria 0.05 40 1 0.05 40 1 0.05 40 1 0.05 40 1 0.05 40 1 0.05 40 10 0.05 40 10 0.05 40 10 Conc of PFOS in Sample (ng/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilution(di1uted sera)) dilution(di1utedsera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample (mL) = amount of sera usedltotal amount of sera +water (1/20 dilution). = 1/20 = 0.05 mL 35.30 40.31 264.06 269.60 268.75 479.65 395.97 380.39 3M EnvExicreol 2n0m00ental Laboratory Blanks QCs and MSs mallard sera samples summary 030122.xls 1412 1612 10562 10784 10750 191860 158388 152156 avg %rec A A 122.8 140.6 160.6 105.2 107.4 107.1 119.4 98.60 94.72 avg 157.6 std dev 15.69 %RSD 9.957 avg 106.6 stddev 1.189 %RSD 1.115 avg 104.3 std dev 13.29 %RSD 12.75 printed: 1/22/2003, 11:28 AMPage 48 Report E01-1256 Study number E01-1 256 - MALLARD SERA PFOS: A Reproduction Study with the Mallard E0l-1256-28663 454-109-3218. 0 PPM, Female, Adult do20225047 0.5 40 1 E01-1256-28665 454-109-3220,O PPM, Female, Adult do20225049 0.5 40 1 E01-1256-28667 454-109-3222, 0 PPM, Female, Adult d020225051 0.5 40 1 E01-1256-28669 454-109-3224, 0 PPM, Female, Adult do20225053 0.5 40 1 E01-1256-28670 454-109-3226, 0 PPM, Female, Adult do20225054 0.5 40 1 E01-1256-26672 454-iO9-3228,O PPM, Femaie. Adult 6020225061 0.5 40 1 E01-1256-28674 454-109-3230,0 PPM, Female, Adult do20225063 0.5 40 1 E01-1256-28676 454-109-3232, 0 PPM, Female, Adult do20225065 0.5 40 1 E01-1256-28678 454-109-3234, 0 PPM, Female, Adult d020226050 0.5 40 1 E01-1256-28679 454-109-3236, 0 PPM, Female, Adult do20226051 0.5 40 1 E01-1256-28681 454-109-3238, 0 PPM, Female, Adult do20226052 0.5 40 1 E01-1256-28683 454-109-3240, 0 PPM, Female, Adult do20226053 0.5 40 1 E01-1256-28685 454-109-3242, 0 PPM, Female, Adult do20226054 0.5 40 1 E01-1256-28687 454-109-3244, 0 PPM, Female, Adult do20226055 0.5 40 1 E01-1256-28689 454-109-3246, 0 PPM, Female, Adult d020226056 0.5 40 1 E01-1256-28691 454-109-3248, 0 PPM, Female, Adult do20226057 0.5 40 1 E01-1256-28693 454-109-3250, 0 PPM, Female, Adult do20226058 0.5 40 1 E01-1256-28695 454-109-3252, 0 PPM, Female, Adult do20226064 0.5 40 1 E01-1256-28697 454-109-3254. 0 PPM. Female. Adult do20226065 0.5 40 1 E01-1256-28699 454-109-3256; 0 PPM. Female; Adult do20226066 0.5 40 1 E01-1256-28701 454-109-3258. 10 PPM, Female, Adult do20226067 0.5 40 1 E01-1256-28703 454-109-3260, 10 PPM, Female, Adult do20226068 0.5 40 1 E01-1256-28705 454-109-3262, 10 PPM, Female, Adult do20226069 0.5 40 1 E01-1256-28707 454-109-3264, 10 PPM, Female, Adult do20226070 0.5 40 1 E01-1256-28709 454-109-3266, 10 PPM, Female, Adult do20226071 0.5 40 1 E01-1256-28711 454-109-3268, 10 PPM, Female, Adult do20228031 0.5 40 1 E01-1256-28713 454-109-3270, 10 PPM, Female, Adult E01-1256-28715 454-109-3272. 10 PPM, Female, Adult do20228032 0.5 40 10 do20228033 0.5 40 1 E01-1256-28717 454-109-3274, 10 PPM, Female, Adult do20405045 0.5 40 10 E01-1256-28719 454-109-3276,lO PPM, Female, Adult do20228035 0.5 40 10 E01-1256-28721 454-109-3278, 10 PPM, Female, Adult do20228036 0.5 40 1 EO1-1256-28723 454-109-3280. 10 PPM, Female, Adult d020308048 0.2 40 10 E01-1256-28725 454-109-3282, 10 PPM. Female, Adult do20228038 0.5 40 1 E01-1256-28727 454-109-3284,lO PPM, Female, Adult do20228039 0.5 40 1 E01-1256-28729 454-109-3286, 10 PPM, Female, Adult do20308049 0.5 40 10 E01-1256-28731 454-109-3288. 10 PPM, Female, Adult do20228044 0.5 40 10 E01-1256-28733 454-109-3290, 10 PPM, Female, Adult do20228045 0.5 40 1 E01-1256-28735 454-109-3292,lO PPM, Female, Adult do20228046 0.5 40 1 E01-1256-28737 454-109-3294, 10 PPM, Female, Adult do20228047 0.5 40 1 E01-1256-28739 454-109-3296. 10 PPM. Female, Adult do20228048 0.5 40 1 E01-1256-28740 454-109-9583,O PPM, Female, Offspring do20228114 0.15 40 1 E01-1256-28742 454-109-9589,O PPM, Female, Offspring d020228115 0.2 40 1 E01-1256-28743 454-109-9592, 0 PPM, Female, Offspring do20228116 0.2 40 1 E01-1256-28744 454-109-9598,O PPM, Female, Offspring do20228117 0.1 40 1 E01-1256-28745 454-109-9603,O PPM, Female, Offspring do2022811 8 0.2 40 1 E01-1256-28747 454-109-9610,O PPM, Female, Offspring do2022811 9 0.2 40 1 E01-1256-28748 454-109-9573,O PPM, Female, Offspring do20228120 0.05 40 1 E01-1256-28751 454-109-9629,lO PPM, Female, Offspring do20228121 0.1 40 1 E01-1256-28752 454-109-9633,lO PPM, Female, Offspring do20228122 0.1 40 1 E01-1256-28753 454-109-9634,lO PPM, Female, Offspring do20228123 0.5 40 1 E01-1256-28754 454-109-9645.10 PPM, Female, Offspring do20228127 0.1 40 1 E01-1256-28755 454-109-964610 PPM, Female, Offspring do20228128 0.2 40 1 E01-1256-28758 454-109-9654,lO PPM, Female, Offspring do20228129 0.1 40 1 E01-1256-28759 454-109-9659,lO PPM, Female, Offspring do20228130 0.1 40 1 * Concentration <LOO Conc of PFOS in Sample (ns/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilution(di1utedsera)) dilution(di1uted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample (mL) = amount of sera usedhotal amount of sera + water (1/20 dilution). = 1/20 = 0.05 mL 1.90 1 .oo 2.21 1 .oo 1.53 66.94 1 .oo 4.30 2.51 2.51 4.16 2.51 2.51 2.51 2.51 9.96 2.51 18.92 2.51 2.51 340.30 235.54 425.04 158.72 197.38 168.1 6 228.48 192.81 73.64 1016 4 245.98 80.62 268.84 164.1 4 72.23 106.99 400.80 260.05 191.39 270.66 2.51 2.51 2.51 2.51 2.51 2.51 2.51 128.28 85.41 168.96 93.74 122.59 136.20 133.73 3M EnvExicreol 2n00m0 ental Laboratory summaty by sex mallard sera samples summaty 030122.xls 76.0 40.0 88.4 40.0 61.2 2678 40.0 172 100.4 100.4 166 100.4 100.4 100.4 100.4 398 100.4 757 100.4 100.4 13612 9422 17002 6349 7895 6726 91392 7712 29456 40656 9839 32248 10754 6566 28892 42796 16032 10402 7656 10826 100.4 100.4 100.4 100.4 100.4 100.4 100.4 5131 341 6 6758 3750 4904 5448 5349 40.0 * 40.0 40.0 * 40.0 40.0 40.0 rejected * 40.0 40.0 * 100.4 * 100.4 100.4 * 100.4 * 100.4 * 100.4 * 100.4 100.4 rejected * 100.4 100.4 rejected Ir 100.4 * 100.4 With outliers avg 271 std dev 589 %RSD 218 Wthout outliers avg 93.4 std dev 37.0 %RSD 39.6 rejected With outliers avg 20312 std dev 20385 %RSD 100 Wthout outliers avg 16571 std dev 11965 %RSD 72.2 * 100.4 * 100.4 " 100.4 * 100.4 * 100.4 avg 100.4 100.4 * 100.4 std dev 0 %RSD - avg 4965 std dev 1118 %RSD 22.5 printed: 1/22/2003, 1128 AMPage 49 Report E01-1256 Sfuay number EOi-i256 - MALLARD SERA PFOS: A Reproduction Study with the Mallard E01-1256-28664 E0l-1256-28666 E01-1256-28668 E01-1256-28671 E01-1256-28673 454-109-3219,O PPM, Male, 454-109-3221,O PPM, Male, 454-109-3223, 0 PPM, Male, 454-109-3227.0 PPM. Male. 454-109-3229,0 PPM, Male, Adult Adult Adult Adult Adult do20225048 0.5 40 1 do20225050 0.5 40 1 do20225052 0.5 40 1 do20225060 0.5 40 1 do20225062 0.5 40 1 E01-1256-28675 E01-1256-28677 E01-1256-28680 E01-1256-28682 E01-1256-28684 454-109-3231,O 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 do20225064 0.5 40 1 do20226034 0.5 40 1 do20226035 0.3 40 1 do20226036 0.5 40 1 do20226037 0.5 40 1 E01-1256-28686 454-109-3243, 0 PPM. Male, Adult do20226038 0.5 40 1 E01-1256-28688 E01-1256-28690 E01-1256-28692 E01-1256-28694 E01-1256-28696 454-109-3245, 0 PPM, 454-109-3247, 0 PPM. 454-109-3249, 0 PPM. 454-109-3251, 0 PPM, 454-109-3253, 0 PPM, Male, Adult Male, Adult Male, Adult Male, Adult Male, Adult do20226039 0.5 40 1 do20226040 0.5 40 1 do20226041 0.5 40 1 do20226042 0.5 40 1 do20226043 0.1 40 1 E01-1256-28698 454-109-3255, 0 PPM, Male, Adult do20226049 0.5 40 1 EO1-1256-28700 454-109-3257, 10 PPM. Male, Adult d020301106 0.5 40 10 E01-125628702 454-109-3259, 10 PPM, Male, Adult do20301107 0.2 40 10 E01-1256-28704 454-109-3261, 10 PPM, Male, Adult d020301108 0.5 40 10 E01-1256-28706 454-109-3263, 10 PPM. Male, Adult do20411024 0.06 40 100 E01-1256-28708 454-109-3265, 10 PPM, Male, Adult do20301110 0.2 40 10 E01-1256-28710 454-109-3267, 10 PPM, Male, Adult do20228049 0.5 40 10 E01-1256-28712 454-109-3269, 10 PPM, Male, Adult do20228050 0.5 40 10 E01-1256-28714 454-109-3271,lO PPM, Male, Adult do20228051 0.5 40 10 E01-1256-28716 454-109-3273,lO PPM, Male, Adult E01-1256-28718 454-109-3275,lO PPM, Male, Adult E01-1256-28720 454-109-3277,lO PPM, Male, Adult do20228052 0.5 40 10 do20228053 0.5 40 10 do20228057 0.5 40 10 E01-1256-28722 454-109-3279, 10 PPM, Male, Adult do20228058 0.5 40 10 E01-1256-28724 454-109-3281. 10 PPM, Male, Adult do20228059 0.5 40 10 E01-1256-28726 454-109-3283, 10 PPM, Male, Adult do20228060 0.5 40 10 E01-1256-28728 454-109-3285,lO PPM. Male, Adult do20228061 0.5 40 10 E01-1256-28730 454-109-3287, IO PPM, Male, Adult E01-1256-28732 454-109-3289, 10 PPM, Male, Adult do20228062 0.3 40 10 do20228063 0.3 40 10 E01-1256-28734 454-109-3291, 10 PPM, Male, Adult do20228064 0.3 40 10 E01-1256-28736 454-109-3293, 10 PPM, Male, Adult do20228065 0.5 40 10 E01-1256-28738 454-109-3295,lO PPM, Male, Adult do20228066 0.5 40 10 E01-1256-28741 454-109-9591,O PPM, Male, Offspring do20228131 0.1 40 1 E01-1256-28746 454-109-9606,O PPM. Male, Offspring do20228132 0.2 40 1 E01-1256-28749 454-109-9578,O PPM. Male, Oftspring do20228133 0.2 40 1 E01-1256-28750 454-109-9625, 10 PPM, Male, Offspring do20228134 0.2 40 1 EO1-1256-28756 454-109-9636.10 PPM, Male, Offspring do20228135 0.1 40 1 E01-1256-28757 454-109-9642,lO PPM, Male, Offspring do20228136 0.2 40 1 * Concentration<LOQ Conc of PFOS in Sample ( n g h L ) = (Inst conc(ng/mL))x (additional dilution) x (dilution(di1utedsera)) dilution(di1utedsera) =final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample (mL) = amount of sera usednotal amount of sera + water (1/20 dilution). = 1/20 = 0.05 mL 1.49 1 .oo 1.07 1.99 1.oo 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 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 3666 4106 40.0 * 40.0 40.0 4c.c * 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 rejected rejected 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 std dev 1.39 * 100.4 %RSD 1.37 avg 4408 std dev 931 %RSD 21.1 3M EnEvxicreol2n00m0 ental Laboratory sumrnaly by sex mallard sera samples summary 030122.xls printed: 1/22/2003, 11:28 AMPage 50 Report E01-1256 Study number E01-1256 - MALLARD LIVER DATA SUMMARY, BLANKS AND SPIKES PFOS: A Reproduction Study with the Mallard El-1326-36778 Blank H2O-day 1 E01-1326-36778 Blank HPO-day 1 E01-1326-36807 Blank HPO-day 2 E01-1326-36812 Blank HPO-day3 E01-1326-36817 Blank H2O-day 4 E01-1326-36780 Blank mallard liver-day 1 E01-1326-36780 Blank mallard liver-day 1 E01-1326-36809 Blank mallard liver-day 2 E0l-1326-36814 Blank mallard liver-day3 E01-1326-36819 Blank mallard liverday 4 do20304037 d020305036 d020306024 d020306104 d020307024 do20304038 d020305037 d020306025 d020306105 d020307025 1.oo 1.oo 1.Lm 1.Lm 1.oo 5.0597 5.0597 5.0597 5.0597 5.0597 9.00 9-00 9.00 9.00 9.00 45.0 45.0 45.0 45.0 45.0 2.00 0.100000 1 2.51 2.00 0.!00000 1 1.oo 2.00 0.100000 1 2.51 2.00 0.1OMx)O 1 2.51 2.00 0.1 00000 1 2.51 2.00 0.101073 1 2.51 2.00 0.101073 1 1.oo 2.00 0.101073 1 2.51 2.00 0.101073 1 2.51 2.00 0.101073 1 2.51 50.2 20.0 50.2 50.2 50.2 50.2 20.0 50.2 50.2 50.2 * 50.2 * 20.0 * 50.2 * 50.2 * 50.2 * 50.2 * 20.0 * 50.2 * 50.2 * 50.2 E01-1326-36781 MallardLiver MS-day 1 E01-1326-36810 MallardLiver MS-day 2 E01-1326-36815 Mallard Liver MS-day 3 E0l-1326-36820 MallardLiver MS-day 4 d020305038 5.0597 45.0 d020306026 5.0597 45.0 d020306106 5.0597 45.0 d020307026 5.0597 45.0 2.00 2.00 2.00 2.00 E01-1326-36782 Mallard Liver MSD-day 1 E01-1326-36782 MallardLiver MSD-day 1 E01-1326-36811 MallardLiver MSD-day2 E01-1326-36816 MallardLiver MSD-day 3 E01-1326-36821 Mallard Liver MSD-day 4 * Concentration <LOQ d020304040 5.0597 45.0 d020305039 5.0597 45.0 d020306027 5.0597 45.0 d020306107 5.0597 45.0 d020307027 5.0597 45.0 2.00 2.00 2.00 2.00 2.00 Conc of PFOS in Sample (ng/g) = (Inst conc(nghL)) x (additionaldilution) x (final volume (mL))/amount of liver in diluted sample (9) amount of liver in dilutedsample (9) = amount of liver weighed (g)/ (water addedfor homogenizing(mL=g)) + amount d liver weighed(g)) 0.101073 0.101073 0.101073 0.101 073 0.101073 0.101073 0.1 01073 0.1 01073 0.101073 1 26.91 532 1 27.95 553 1 27.72 549 1 27.84 551 1 26.86 531 1 26.18 518 1 26.20 518 1 27.50 544 1 27.68 548 for both ms and msd avg%rec std dev of %rec std dev of conc 108.8 3.059 15.20 107.1 1 11.3 110.4 1 10.8 106.9 104.2 104.3 109.5 110.2 trueval. 497 avg %rec 110.6 avg 549.8 std dev 11.33 true val. 497 avg %rec 107.0 avg 532.0 std dev 13.91 3M EnEvxicreol2n00m0 ental Laboratory Blanks ICs and MSs mallard liver samples summary 030122.xls printed: 1/22/2003. 11:28 AMPage 51 Report E01-1256 Study number E01-1256 - MALLARD LIVER DATA SUMMARY versus unextracted curve E01-1326-36798 QC- 25ppb -1 (497 ngfg homogenate) 6020305045 5 0597 45 0 E01-1326-36799 ac- 25ppb -2 (497 n&j homogenate) d020305046 5.0597 45.0 E01-1326-36600 QC- 25Dpb -3 (497 ngfg homogenate) d020305047 5 0597 45.0 E01-1326-36801 QC- 250ppb -1 (4967 ng/g homogenate) do20305048 5.0597 45.0 E01-1326-36802 QC- 250ppb -2 (4967ndg homogenate) d020305049 5.0597 45.0 E0l-1326-36803 QC- 250ppb -3 (4967 ng/g homogenate) d020305050 5.0597 45.0 E01-1326-36804 QC- 4.0 ppm -1 (79467 ngfg homogenate) do20405065 5.0597 45.0 01-1326-36805 QC- 4.0 ppm -2 (79467 ng/g homogenate) d020405066 5.0597 45.0 E01-1326-36806 QC- 4.0 ppm -3 (79467 ng/g homogenate) d020405067 5.0597 45.0 A = Not within +/-25% criteria Study number E01-1256 - MALLARD LIVER DATA SUMMARY PFOS: A Reproduction Study with the Mallard Absolute Recoveries 200 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.m 0.101073 0.101073 0.101 073 0.101073 0.101073 0.101 073 0.101 073 0.101073 0.101 073 1 28 87 1 29.22 1 35.74 1 242.46 1 249.01 1 278.56 10 440.20 10 443.16 10 436.91 571 578 707 4798 4927 5512 87105 87691 86454 1149 116.3 A i42.3 96.59 99.20 111.0 109.6 110.3 108.6 for all QC samoles (absoluterecovelvl I avg%rec stddevof%rec 112.1 13.07 I av-a 1245 stddev 15.41 XRSD 12.37 avg 102.3 std dev 7.662 %RSD 7.493 avg 109.6 stddev 0.7785 %RSD 0.7104 QC Sample Recoveries E01-1326-36798 QC- 25ppb -1 (497 ng/g homogenate) d020305045 5 0597 45 0 E01-1326-36799 QC- 25ppb -2 (497 nglg homogenate) d020305046 5 0597 45 0 _ _ E01-1326-36800 QC- 25ppb -3 (497 ngfghomogenate) do20305047 5 0597 45 0 E01-1326-36801 QC- 250ppb -1 (4967 nda homo. genate) d020305048 5 0597 45 0 E0l-1326-36802 QC- 250ppb -2 (4967 ngfg homogenate) d020305049 5.0597 45 0 E01-1326-36803 QC- 250ppb -3 (4967 ng/g homogenate) d020305050 5.0597 45.0 E01-1326-36804 QC- 4.0 pprn -1 (79467 ng/g homogenate) do20405065 5.0597 45.0 E01-1326-36805 Qc- 4.0 ppm -2 (79467 nglg homogenate) d020405066 5.0597 45.0 E01-1326-36806 QC- 4.0 ppm -3 (79467 nglg homogenate) do20405067 5.0597 45.0 A = Not within +/-25% criteria 200 2 00 200 2 00 200 2.00 2.00 2.00 2.00 Conc of PFOS in Sample (ng/g) = (Inst conc(nglmL)) x (additionaldilution) x (final volume (mL))/amount of liver in diluted sample (9) amount of liver in dilutedsample (9) = amount of liver weighed (g)l (water addedfor homogenbing(mL=g)) + amount of liver weighed(g)) 0 101073 0 101073 0 101073 0 101073 0 101073 0.101073 0.101073 0.101073 0.101073 1 25 41 1 25 71 1 31 24 1 221 45 1 228 29 1 260.35 10 415.43 10 418.16 10 412.40 503 509 618 4382 4517 5152 82204 82744 81604 101 2 102 4 124 4 86 22 90 95 103.7 103.4 104.1 102.7 1 for all QC samples (recovery) avg %rec 102.3 S t d dev of %ret 10.14 avg 1093 std dev 1307 -%RSD 1196 eva 94 30 Std dev 8 274 %RSD 8.774 avg 103.4 Stddev 0.7175 %RSD 0.6937 3M EnEvxicerlo20n00mental Laboratory BlanksQCs and MSs mallard liver samples summary 030122.~1s printed: 1/22/2003, 11:26 AMPage 52 - Report E01-1256 Studv number E01-1256 MALLARD LIVER DATA SUMMARY PFOS: A Reproduction Study with the Mallard E01-1326-29583 0 ppm a.i. 3220 F Lver 6020305060 1.0944 9.00 2.00 0.108417 1 E01-1326-29601 0 ppm a.i. 3222 F Liver d020305061 1.0435 9.00 2.00 0.103898 1 E01-1326-29619 0 ppm a.i. 3224 F Liver d020305062 1.0425 9.00 2.00 0.103809 1 EO1-1326-29637 0 ppm a.i. 3226 F Liver 4020305063 1 0703 9.00 2.00 0.1%283 1 E0l-1326-29655 0 ppm a.i. 3228 F Liver 6020305064 1.0526 9.00 2.00 0,104709 1 E01-1326-29673 0 ppm a.i. 3230 F Liver d020305065 1.0411 9.00 2.00 0.103684 1 E01-1326-29691 0 ppm a.i. 3232 F Liver d020306031 1.1704 9.00 2.00 0,115079 1 E01-1326-29709 0 ppm a.i. 3234 F Liver do20306032 1.1290 9 .00 2.00 0.111462 1 E01-1326-29727 0 ppm a.i. 3236 F Liver d020306033 1.0800 9.00 2.00 0.107143 1 E01-1326-29745 0 ppm a.i. 3238 F Liver do20306034 1.1170 9.00 2.00 0.110408 1 E01-1326-29763 0 ppm a.i. 3240 F Liver d020306035 1.0086 9.00 2.00 0.100773 1 E01-1326-29781 0 ppm a.i. 3242 F Liver d020306036 1.0907 9.00 2.00 0.108090 1 EO1-1326-29799 0 ppm a.i. 3244 F Liver 6020306037 1.0291 9.00 2.00 0.10261 1 1 E01-1326-29817 0 ppm a.i. 3246 F Liver d020306038 1.1294 9.00 2.00 0.111497 1 E01-1326-29835 0 ppm a.i. 3248 F Liver d020306039 1.1075 9.00 2.00 0.109572 1 E01-1326-29853 0 ppm a.i. 3250 F Liver d020306040 1.1709 9.00 2.00 0.115123 1 E01-1326-29871 0 ppm a.i. 3252 F Liver d020306044 1.0761 9.00 2.00 0.106797 1 E01-1326-29889 0 ppm a i 3254 F Liver do20306045 1.1344 9.00 2.00 0.1 1 1936 1 E01-1326-29907 0 ppm a.i. 3256 F Liver do20306046 1.0669 9.00 2.00 0.1 05981 1 E01-1326-29925 10 ppm a.i. 3258 F Liver d020306066 1.0034 9.00 2.00 0.100306 1 EO1-1326-29943 10 ppm ad. 3260 F Liver do20306067 1.1331 9.00 2.00 0.1 11822 1 E01-1326-29961 10 ppm a.i. 3262 F Liver d020307057 1.1294 9.00 2.00 0.111497 10 EO1-1326-29980 10 ppm a.i. 3264 F Liver 6020306111 1.1829 9.00 2.00 0.1 16165 1 EO1-1326-29998 10 ppm a.i. 3266 F Liver 6020306112 1.0487 9.00 2.00 0,104362 1 E01-1326-30016 10 ppm a.i. 3268 F Liver do20306113 1.1214 9 .00 2 .oo 0.1 10795 1 E01-1326-30034 10 ppm a.i. 3270 F Liver d020307059 1.0751 9.00 2.00 0.106709 10 E01-1326-30052 10 ppm a.i. 3272 F Liver d020306115 1.0658 9.00 2.00 0.105883 1 EO1-1326-30070 10 ppm a.i. 3274 F Liver d020307060 1.1226 9.00 2.00 0.110900 10 EO1-1326-30088 10 ppm a.i. 3276 F Liver d020307061 1.0953 9.00 2.00 0.108496 10 EO1-1326-30106 10ppm a.i. 3278 F Liver d020306118 1.1956 9 .00 2.00 0.1 17266 1 E01-1326-30124 10 pprn a.i. 3280 F L i e r 6020307062 1 ,0600 9.00 2.00 0.105368 1 E01-1326-30142 10 ppm a.i. 3282 F Liver d020306120 1.0275 9.00 2.00 0.102468 1 E01-1326-30160 10 ppm a.i. 3284 F Liver do20306124 1.091 5 9.00 2.00 0.1 08160 1 E01-1326-30178 10 ppm a.i. 3286 F Liver d020307063 1.0657 9.00 2.00 0.1 05874 10 E01-1326-30196 10 ppm a i 3288 F Liver d020307064 1.0330 9.00 2.00 0.102960 10 E0l-1326-30214 10 ppm a.i. 3290 F Lver d020307065 1.0352 9.00 2.00 0.103157 10 E01-1326-30232 10 ppm a.i. 3292 F Liver d020306128 1.0658 9.00 2.00 0.105883 1 EOl-1326-30250 10 pvm a i 3294 F Liver d020306129 1.0627 9.00 2.00 0.105608 1 E01-1326-30268 10 ppm a.i. 3296 F Liver d020306130 1.1238 9.00 2.00 0.1 11006 1 E01-1367-31295 0 Dvm a.i. 9583 F Liver 6020307031 1.1308 9.00 2.00 0.111620 1 E01-1367-31307 0 ppm a.i. 9589 F Liver d020307032 1.0005 9.00 2.00 0.100045 1 E01-1367-31313 0 ppm a.i. 9592 F Liver d020307033 1.0390 9.00 2.00 0.1 03496 1 E01-1367-31319 0 ppm a.i. 9598 F Liver d020307034 1.1204 9.00 2.00 0.1 10707 1 E01-1367-31325 0 ppm a.i. 9603 F Liver d020307035 1.1550 9.00 2.00 0,113737 1 E01-1367-31337 0 ppm a.i. 9610 F Liver 4020307036 1.0217 9.00 2.00 0.101949 1 E01-1367-31343 0 ppm a.i. 9573 F Liver 6020307037 1.0122 9.00 2.00 0.1 01097 1 E01-1367-31362 10 ppm a.i. 9629 F Liver 6020307044 1.1676 9.00 2.00 0.1 14835 1 E01-1367-31368 10 ppm a.i. 9633 F Liver 6020307045 1.0510 9.00 2.00 0.104567 1 E01-1367-31374 10 ppm a.i. 9634 F Liver do20307046 1.1638 9.00 2.00 0.114504 1 E01-1367-31380 10 ppm ai. 9645 F Liver do20307047 1.1510 9.00 2.00 0.113388 1 E01-1367-31386 10 ppm a.i. 9648 F Liver 6020307048 1.1125 9.00 2.00 0.110012 1 E01-1367-31404 lOppm a.i. 9654 F Liver d020307049 1.0770 9.00 2.00 0.106877 1 E01-1367-31410 10 ppm a.i. 9659 F Liver d020307050 1.0581 9.00 2.00 0.105199 1 * Concentration <LOQ 1 .oo 1 .OO 1 .oo 1.oo 1.oo 1 .oo 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 76.85 166.74 349.71 130.88 233.84 342.26 149.38 124.22 101.98 243.62 251.58 351.37 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 Conc of PFOS in Sample (ng/g) = (Inst conc(ng/mL))x (additionaldilution) x (finalvolume (mL))/ amount of liver in diluted sample (9) amount of liver in diluted sample (9) =amount of liver weighed (g)/ (water added for homogenizing (mL=g))+amount of liver weighed(g)) samples by sex 3M EnEvxicreol 2nMmx) ental Laboratory mallard liver samples summary 03012 2 . ~ 1 ~ 20.0 20.0 20.0 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 7080 6784 13645 6076 5378 5727 13189 5801 13859 30737 5964 2484 4564 6329 28218 24130 19772 4602 4764 6331 50.2 50.2 50.2 50.2 50.2 50.2 50.2 2954 2245 4516 2582 3503 5930 3582 20.0 20.0 20.0 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 avg 39.6 std dev 14.8 %RSD 37.3 * 50.2 * 50.2 * 50.2 * 50.2 * 50.2 * 50.2 50.2 avg 10772 sid dev 8463 %RSD 78.6 avg 50.2 siddev 0 %RSD- avg 3616 std dev 1263 %RSD 34.9 printed: 1/22/2003. 1 1:28 AMPage 53 Report E01-1256 PFOS: A Reproduction Study with the Mallard E01-1326-29574 0 ppm a.i. 3219 M Liver d020305072 1.1847 9.00 2.00 0.1 16322 1 E01-1326-29592 0 ppm a.i. 3221 M Liver d020305073 1.1181 9.00 2.00 0.1 10505 1 E01-1326-29610 0 ppm a.i. 3223 M Liver d020305074 1.0788 9.00 2.00 0.107037 1 E01-1326-29628 0 ppm a.i. 3225 M Liver d020305075 1.1043 9 00 2.00 O.!C192SO ! E01-1326-29646 0 ppm a.i. 3227 M Liver d020305076 1.0486 9.00 2.00 0.1 04353 1 E01-1326-29664 0 ppm a.i. 3229 M Liver do20305077 1.0336 9.00 2.00 0.103014 1 E01-1326-29682 0 ppm a.i. 3231 M Liver do20306047 1.0165 9.00 2.00 0.1 01483 1 EOl-1326-29700 0 ppm a.i. 3233 M Lwer d020306048 1.0395 9.00 2.00 0.103541 1 E01-1326-29718 0 ppm a.i. 3235 M Liver d020306049 1.0373 9.00 2.00 0.103345 1 EO1-1326-29736 0 ppm a.i. 3237 M Liver d020306050 1.1679 9.00 2.00 0.114861 1 E01-1326-29754 0 ppm a.i. 3239 M Liver d020306051 1.1493 9.00 2.00 0,113239 1 E01-1326-29772 0 ppm a.i. 3241 M Liver do20306052 1.0619 9.00 2.00 0.105537 1 E01-1326-29790 0 ppm a.i. 3243 M Liver d020306053 1.0263 9.00 2.00 0.102361 1 E01-1326-29808 0 ppm a.i. 3245 M Liver d020306057 1.1541 9.00 2.00 0.113659 1 E01-1326-29826 0 ppm a.i. 3247 M Liver do20306058 1.0813 9.00 2.00 0.107258 1 E01-1326-29844 0 ppm a.i. 3249 M Liver d020306059 1.0263 9.00 2.00 0.1 02361 1 EO1-1326-29862 0 ppm a.i. 3251 M Liver d020306060 1.0088 9.00 2.00 0.100791 1 E01-1326-29880 0 ppm a.i. 3253 M Liver d020306061 1.0294 9.00 2.00 0.102638 1 E01-1326-29898 0 ppm a.i. 3255 M Liver do20306062 1.0926 9.00 2.00 0.108258 1 E01-1326-29916 10 pprn a.i. 3257 M Liver d020306069 1.0654 9.00 2.00 0.105848 10 E01-1326-29934 10 ppm a.i. 3259 M Liver d020306070 1.1316 9.00 2.00 0.111690 10 E01-1326-29952 10 ppm a.i. 3261 M Liver d020306071 1.1330 9.00 2.00 0.1 11813 10 E01-1328-29970 10 ppm a.i. 3263 M Liver d020307058 1.1404 9.00 2.00 0.112461 100 E01-1326-29989 10 ppm a i 3265 M Liver d020306131 1.1480 9.00 2.00 0.1 13126 10 EO1-1326-30007 10 ppm a.i. 3267 M Liver do20306132 1.m20 9 .oo 2O. O 0.105546 10 E01-1326-30025 10 pprn a.i. 3269 M Liver do20306133 1.1687 9.00 2.00 0.114931 10 E01-1326-30043 10 ppm a i 3271 M Liver d020306137 1.031 3 9.00 2.00 0.102808 10 E01-1326-30061 10 ppm a.i. 3273 M Liver do20306138 1.1399 9.00 2.00 0.1 12417 10 E01-1326-30079 10 ppm a.i. 3275 M Liver do20306139 1.1 143 9.00 2.00 0.1 10171 10 E01-1326-30097 10ppm a.i. 3277 M Liver d020306140 1. w 6 2 9.00 2 .oo 0.100558 10 E01-1326-30115 10 pprn a.i. 3279 M Liver d020306141 1.0380 9.00 2.00 0.103407 10 E01-1326-30133 10 ppm a.i. 3281 M Liver do20306142 1.0669 9.00 2.00 0.105981 10 E01-1326-30151 10 ppm a.i. 3283 M Liver do20306143 1.0955 9.00 2.00 0.1 08514 10 E01-1326-30169 10 ppm a.i. 3285 M Liver do20306144 1.1981 9.00 2.00 0.1 17483 10 E01-1326-30187 10 ppm a.i. 3287 M Liver d020306145 1.0500 9.00 2.00 0.1 04478 10 E01-1326-30205 10 ppm a.i. 3289 M Liver d020306146 1.0251 9.00 2.00 0.1 02253 10 E01-1326-30223 10 ppm a.i. 3291 M Liver d020306150 1.3441 9.00 2.00 0.103952 10 E01-1326-30241 10 ppm a.i. 3293 M Liver d020306151 1.0979 9.00 2.00 0.108726 10 E01-1326-30259 10ppm a.i. 3295 M Liver d020306152 1.0815 9.00 2.00 0.107276 10 E01-1367-31301 0 ppm a.i. 9591 M Liver d020307038 1.041 1 9.00 2.00 0.1 03684 1 E01-1367-31331 0 ppm a.i. 9606 M Liver d020307039 1.1314 9.00 2.00 0.111673 1 E01-1367-31349 0 ppm a.i. 9578 M Liver d020307040 1.0413 9.00 2.00 0.1 03702 1 EO1-1367-31356 10ppm a.i. 9625 M Liver d020307051 1.0166 9.00 2.00 0.101492 1 E01-1367-31392 10 ppm a.i. 9636 M Liver d020307052 1.1367 9.00 2.00 0.112137 1 EOl-1367-31398 10 ppm a.i. 9642 M Liver d020307053 1.1252 9.00 2.00 0.1 11129 1 * Concentration cLOQ Conc of PFOS in Sample (ng/g) = (Inst conc(nq/mL))x (additional dilution) x (final volume (mL))/amountof liver in diluted sample (9) amount of liver in diluted sample (9) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) +amount of liver weighed(g)) 1.oo 1.oo 1.oo 1 .00 1.oo 1.oo 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 381.46 274.77 392.1 1 70.46 349.14 263.55 332.69 323.76 477.80 302.45 271.03 356.95 284.50 363.85 325.94 274.59 303.06 241.84 131.53 270.60 2.51 2.51 2.51 235.62 123.08 149.00 20.0 20.0 20.0 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 4991 5 67061 55487 52564 59276 46529 24195 50449 50.2 50.2 50.2 4843 2195 2682 20.0 20.0 20.0 20.0 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 avg 39.6 50.2 std dev 14.8 * 50.2 %RSD 37.3 * 50.2 * 50.2 * 50.2 avg 60880 std dev 19580 XRSD 32.2 avg 50.2 stddev 0 %RSD - avg 3173 std dev 1296 %RSD 40.8 3M EnEvxicreloZonom0 ental Laboratory samples by sex mallard liver samples summary 030122.xls printed: 1/22/2Mu. 1128 AMPage 54 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard ATTACHMENCT: SAMPLECHROMATOGRAMCSA,LBI RATION INFORMATION, AND INSTRUMENT INFORMATION 3M Environmental Laboratory 3M Environmental Laboratory Page 55 of 216 Page 55 Report E01-1256 Report E01-1256 Methanol Solvent Blanks m e n t Blank 100- PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey070799 26-Feb-2002, 22:16:16 1: MRM of 1 Channel ES6.7 98 1.10e4 Area %- 3M Environmental Laboratory 3M Environmental Laboratory Page 56 of 216 Page 56 Report E01-1256 Report E01-1256 /Solvent Blank 3M Environmental Lab 1,d020228126 Sm (Mn, 1x2) 100 I I I I I I % PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey070799 01-Mar-2002, 09:50:06 1: MRM of 1 Channel ES- 6.7; TIC 84C 1.07e4 Area 0 3M Environmental Laboratory 3M Environmental Laboratory Page 57 of 216 Page 57 Report E01-1256 Report E01-1256 m e n t Blank 3M Environmental Lab d020;!28056 Srn (Mn. 1x2) 1oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 0707991 28-Feb-2002,20:52:57 1: MRM of 1 Channel ES- 6.75 TIC 105; 1.33e4 Area % 0- 3M Environmental Laboratory 3M Environmental Laboratory Page 58 of 216 Page 58 Report E01-1256 Report E01-1256 Water Blank Samples /Blank H20-day 4 3M Environmental Lab d020228107 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 0707991 Ol-Mar-2002,06:19:15 1: MRM of 1 Channel ES- 6.7 TIC 71 8.84e3 Area I 3M Environmental Laboratory 3M Environmental Laboratory Page 59 of 216 Page 59 Report E01-1256 Report E01-1256 Blank H20-dav 3 1 3 E~nvironm&l Lab id020228024 Sm (Mn, 1x2) 1 1oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.7~ 672 davey070799 28-Feb-2002, 14:57:35 1: MRM of 1 Channel ESTIC 8.90e3 Area I % 3M Environmental Laboratory 3M Environmental Laboratory Page 60 of 216 Page 60 Report E01-1256 Report E01-1256 3M Environmental Lab ~d020225037Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070791 25-Feb-2002, 23:49:31 1: MRM of 1 Channel ES- 6.77. TI ( 709 8.64e: Are; % 3M Environmental Laboratory 3M Environmental Laboratory Page 61 of 216 Page 61 Report E01-1256 Report E01-1256 Sera Blank Samples m k H20-day 1 13M Environmental Lab 1dO, C;?,l25037 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard - 6.7 70 25-Feb-2002, 23:49:30 1: MRM of 1 Channel ES- 8.64e3 Area %- "" ;'.bo OL' ' " I~~' ' ' ' ' 2.50' ' ' 3.b'O" '4.00" I ' ' 5.00 " l 6 . b ' O ' ' " 7.00 " ' ' 8.00 Time ' 9?00 " I ' 'IOioo 3M Environmental Laboratory 3M Environmental Laboratory Page 62 of 216 Page 62 Report E01-1256 Report E01-1256 mallard sera-day 3 3M Environmental Lab d020228025 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 0707s 28-Feb-2002, 15:08:4 1: MRM of 1 Channel ES 6.71 TI 87: 1.07e Are % 3M Environmental Laboratory 3M Environmental Laboratory Page 63 of 216 Page 63 Report E01-1256 Report E01-1256 b & k mallard sera-dav 1 3M Environmental Lad d020225038 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079! 26-Feb-2002, 00:00:34 1: MRM of 1 Channel ES- 6.7f TIC 860 l.OleL Arez 3M Environmental Laboratory 3M Environmental Laboratory k Page 64 of 216 Page 64 Report E01-1256 Report E01-1256 Liver Blank Samples mallard male liver-day 4 3M Environmental Lab Isd020007025Sm (Mn, 1x2) ~ lot I I I I % I I I PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07075 07-Mar-2002, 19:39:5 1: MRM of 1 Channel ES 6.7 TI 93: 1.06e Are 3M Environmental Laboratory 3M Environmental Laboratory Page 65 of 216 Page 65 Report E01-1256 Report E01-1256 /Blank mallard liver-day 2 3M Environmental Lab d020306025 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey070799 06-Mar-2002, 14:02:19 1: MRM of 1 Channel ES- 6.7. 901 TIC 1.22e4 Area % I ~ 07' "_ ' _ 1,O_O I , ,, , , 2.00 .- 3.00 4.00 I, 11118 5.00 6.00 8 1 1 8I , I I , 7.00 8.b0 9.00 I , lITime 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 66 of 216 Page 66 Report E01-1256 Report E01-1256 m k mallard male liver-day 3 3M Environmental Lab d020306105 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.7; 112! % - 1.00 2.00 3.00 - 4.00 5.00 6.00 7.00 8.00 9.00 1 0 . 0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 67 of 216 Page 67 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 : l O 1454-109-3285, 10 PPM, Male, Adult,dlO 3M Environmental Lab do20228061 Sm (Mn, 1x2) i loo 6.74 29315! davey07079 28-Feb-2002, 21:48:2 1: MRM of 1 Channel ES. TI ( 3.16e Are, ' 1.00 ' 2.60 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 '-800 - , , ..... ' 9.00 10.00 -J 3M Environmental Laboratory 3M Environmental Laboratory Page 68 of 216 Page 68 Report E01-1256 Report E01-1256 10 PPM, Male, AduIt,d10 3M Environmental Lab Id020228058 Sm (Mn, 1x2) 100 ~ PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Study with the Mallard davey 07079! 28-Feb-2002, 21:15:01 1: MRM of 1 Channel ES- 6 74 TIC 3.08ef Are; 3M Environmental Laboratory 3M Environmental Laboratory Page 69 of 216 Page 69 ~~ Report E01-1256 Report E01-1256 'm O 9 - 3 2 8 3 , 10 PPM, Male, Adult,dlO 3M Environmental Lab d020228060 Sm (Mn, 1x2) I 1oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 25839t davey 07079! 28-Feb-2002, 21:37:2' 1: MRM of 1 Channel ES- TIC 2.74eE Arei % __ 1.00 2.00 -_-3.00 4.00 2.00 6.00 7.00- -_8.00 9.00 3M Environmental Laboratory 3M Environmental Laboratory Page 70 of 216 Page 70 Report E01-1256 Report E01-1256 - 1454-109-3281, 10 PPM, Male, Adult,dlO 3M Environmental Lab do20228059 Sm (Mn, 1x2) 1 1oc I PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 21711t davey 07079! 28-Feb-2002, 21:26:1( 1: MRM of 1 Channel ESTIC 2.40eE Are2 % 3M Environmental Laboratory 3M Environmental Laboratory 7.00 8.00 9.00 Time 10.00 -1 Page 71 of 216 Page 71 Report E01-1256 Report E01-1256 10 PPM, Male, Adult,dlO 3M Environmental Lab do20228057 Sm (Mn, 1x2) ~ 100 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 231932 davey 07079! 28-Feb-2002,21:04:0: 1: MRM of IChannel ESTIC 2.54ef Are: % _&E_ 0 " ' I ", 1"- 1 Time r ' ' 1 'TTTTt 7 ' " ' I " ' ' I " ' I ' " ' 1 " " 1 ' " ' 1 ~ ~ 1.00 2.00. 3.00 4.00 -5.00 6.00 7.00 9.00 10.00 - 1 3M Environmental Laboratory 3M Environmental Laboratory Page 72 of 216 Page 72 Report E01-1256 Report E01-1256 10 PPM, Male, Adult,dlO 3M Environmental Lab . , bo20228053 Sm fMn. 1x2) I 1oc I , PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 23219. davey070799 28-Feb-2002, 20:19:37 1: MRM of 1 Channel ESTIC 2.55e6 Area % 1 0 0 ~~ 2.00 3.00 4.00 5.00 6.00 r,l,s,,v4, ,,, 7.00 8.00 - -900 , S TTime j 010, . 3M Environmental Laboratory 3M Environmental Laboratory Page 73 of 216 Page 73 Report E01-1256 Report E01-1256 454-109-3273, 10 PPM, Male, Adult,dlO 3M Environmental Lab Ido20228052 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 28955C 28-Feb-2002, 20:08:29 1: MRM of 1 Channel ES- 3.16e6 Area %- 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 74 of 216 Page 74 Report E01-1256 Report E01-1256 454-109-3271, 10 PPM, Male, Adult,dlO 3M Environmental Lab I do20228051 1oc Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 3876: davey07079 28-Feb-2002, 19:57:2: 1: MRM of 1 Channel ES- TI( 4.70e! Are; I I % _._ _ _ - 1.00 2.00 3.00 -_4.00 5.00 6.00 ----l " " I " " I " " " " ' l " -_7.00 8.00 ^A_ Time 9.00 10.00-- 3M Environmental Laboratory 3M Environmental Laboratory Page 75 of 216 Page 75 ~~ Report E01-1256 Report E01-1256 454-109-3269, 10 PPM, Male, Adult,dlO 3M Environmental Lab do20228050 Sm (Mn, 1x2) I 100 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079! 28-Feb-2002, 19:46:11 1: MRM of 1 Channel ES- 6.73 TI( 2.51e( Are; ' 1.00 2.00 3.00 4.00 5.00 6.00 7.100 8.00 900 ' _ _ , v ' ' l Time 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 76 of 216 Page 76 Report E01-1256 Report E01-1256 100- 1 1 % PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 22058! davey070799 28-Feb-2002, 19:35:08 1: MRM of 1 Channel ES- TIC 2.38e6 Area 3M Environmental Laboratory 3M Environmental Laboratory Page 77 of 216 Page 77 Report E01-1256 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 do20228037 Sm (Mn. 1x2) 110c I 6.74 487071 - davey070799 28-Feb-2002, 17:21:53 1: MRM of 1 Channel ESTIC 5.03e6 Area I % I I 0 1'"' ~'"''I''' __ .- _1.0_0 2.00 ' ' ' ' I ' ' " I ' ' ~ ' / ~ ' ~" ' 'I "I " l " " I " " I 3.00- 4.00 5.00 6.00 P Time 00 _ _ 8.00 9.00 10.0.0. 3M Environmental Laboratory 3M Environmental Laboratory Page 78 of 216 Page 78 Report E01-1256 Report E01-1256 454-109-3276, 10 PPM, Female, Adult 3M Environmental Lab PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 127606 davey 070791 28-Feb-2002, 16:59:31 1: MRM of 1 Channel ESTIC 1.17eE Are: % 1.00 2.00 3:OO- 4.00 5.00 6.00 .OO 8.00 9.00 10.0T0ime I 3M Environmental Laboratory 3M Environmental Laboratory Page 79 of 216 Page 79 Report E01-1256 Report E01-1256 454-109-3258, 10 PPM, Female, Adult . . 3M Environmental Lab do20226067 Sm fMn. 1x2) I ~ 100 ~~~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 36078t davey070799 27-Feb-2002, 04:34:15 1: MRM of 1 Channel ESTIC 3.86e6 Area I - 0 4 ' ' ' I ' ' ' ' , ' ' ' I ' ' ' ' I I I ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' I I ' ' ' ' I ' I ' ' ' ' 1 ' ' ' ' I. LOO 2.00 3.00 4.00 5.00 6 . 0 L 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 80 of 216 Page 80 Report E01-1256 Report E01-1256 10 PPM, Female, Adult 3M Environmental Lab id020226071 Sm (Mn, 1x2) 1 O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.76 23695: davey 0707991 ?4 0 1.00 2.00 _ _ 3.00- 4.00 ' 5.00 6.00 7.b0 ' 8.00- 9.00 ' ' I ' Time 10!OOp 3M Environmental Laboratory 3M Environmental Laboratory Page 81 of 216 Page 81 Report E01-1256 Report E01-1256 /454-109-3272, 10 PPM, Female, Adult 3M Environmental Lab do20228033 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A ReproductionStudy with the Mallard 6.74 22401 C davey 07079! 28-Feb-2002, 16:37:21 1: MRM of 1 Channel ESTIC 2.53et Are: - 1.00 2.00__ 3 O L 4.00 5.00 6.00 p-7.00 8.00 9.00 10.00 ' 3M Environmental Laboratory 3M Environmental Laboratory Page 82 of 216 Page 82 Report E01-1256 Report E01-1256 j454-109-3274, 10 PPM, Female, Adult 3M Environmental Lab do20228034 Sm (Mn, 1x2) 1oc ~~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 45127 - 28-Feb-2002, 16:48:3 3M Environmental Laboratory 3M Environmental Laboratory Page 83 of 216 Page 83 Report E01-1256 Report E01-1256 1454-109-3278, 10 PPM, Female, Adult 3M Environmental Lab 16020228036 Sm (Mn,1x2) 1 100 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 273170 ~ ~~ davey 07079 % 0 ' ~~~1.00 2.00 3.00 4 0 0 ' 5.00 6.00 7.00 8.00 9.00 10LOO p~ 3M Environmental Laboratory 3M Environmental Laboratory Page 84 of 216 Page 84 Report E01-1256 Report E01-1256 10 PPM, Female, Adult 3M Environmental Lab d020226070 Sm (Mn, 1x2) I100 ~ I ~ ~~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 19676( davey07079 27-Feb-2002, 05:07:3 1: MRM of 1 Channel ES. TI( 2.19el Are; i 1% - ' ' 0 8 8 , Time 1.00 ' 2.00 3.00 ' 4.00 5.00.- 6.00 ' 7.00 8.bO---Ip 9.bO 1200 ~ p~ 3M Environmental Laboratory 3M Environmental Laboratory Page 85 of 216 Page 85 Report E01-1256 Report E01-1256 j454-109-3262, I O PPM, Female, Adult I3M Environmental Lab 1d020226069 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079! 27-Feb-2002, 04:56:3( 1: MRM of 1 Channel ES- 6.76 TIC 4.42eE Are2 1.00 2.00 3.00 4.00 5 0 0 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 86 of 216 Page 86 Report E01-1256 Report E01-1256 10 PPM, Female, Adult 3M Environmental Lab do20226068 Sm (Mn. 1x2) 1 . I 1O t - PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.76 27382C davey 07079! 27-Feb-2002, 04:45:2: 1: MRM of 1 Channel ESTIC 3.02eE Are2 % 0 I - Time - 1.00 2.00 3 . 0 0 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 3M Environmental Laboratory 3M Environmental Laboratory Page 87 of 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 /454-109-9642, 10 PPM, Male, OffsDrinn - - 3M Environmental Lab do20228136 Sm (Mn, 1x2) ' lo( - 6.74 14252f davey 070799 01-Mar-2002, 11:41:03 1: MRM of 1 Channel ES- 1.74e6 Area ~ _ _ _ 1_.00 2.00 3.00- 4.00 5.00 6.00 Time 00 8 0 0 9.00- 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 88 of 216 Page 88 Report E01-1256 Report E01-1256 10 PPM, Male, Offspring 3M Environmental Lab do20228134 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 183965 davey 07079! 01-Mar-2002, 11:18:4! 1: MRM of 1 Channel ESTIC 2.17eE Are; 3M Environmental Laboratory 3M Environmental Laboratory Page 89 of 216 Page 89 Report E01-1256 Report E01-1256 10 PPM, Male, Offspring 3M Environmental Lab do20228135 Sm (Mn, 1x2) ~ 1001 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 128462 davey 07079! Ol-Mar-2002,11:29:5: 1: MRM of 1 Channel ESTIC 1.54ef Are: OL'' 1 I ' ' " I " " .oo 1 " " I " " I " " I " ' -' ~ 2 :~ O L. ~~ 3.00 4.00 ' I ' " ' I " 1 ' I ' ' " I ' -Time I 5.00 6.00 _ _ 7.00 8.00 9.00 10.00 -. 3M Environmental Laboratory 3M Environmental Laboratory Page 90 of 216 Page 90 ~~ 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 ~ 3 MEnvironmentalLab d020228129 Sm (Mn,1x2) 100 6.75 183831 davey 07079! 01-Mar-2002, 10:23:1! 1: M R M of 1 Channel ES- TIC 2.20eE Are: I % o+-' ~ ' I ' ' "I "' ' I ' ' ' ' I' ' -~1.00 2.00 - ' I " " I ' ' " I 1 ' " I ' I ' ' I ' ' I ' " ' I " " I ' " ' I ' - 3.00 4.00 5.00 6.00 I , 1 ) 1 I ' ' I ' ' ' " ' ' " I ' ' ' ' I ' ' ' ' I Time 7.00 -8.00 9.00 lO.OO-- 3M Environmental Laboratory 3M Environmental Laboratory Page 91 of 216 Page 91 Report E01-1256 Report E01-1256 1454-109-9659, 10 PPM, Female, Offs.Drin-g j3M Environmental Lab d020228130 Srn (Mn, 1x2) ~ 10C ~~ ~~~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 180868 davey070799 01-Mar-2002, 10:34:21 1: MRM of 1 Channel ESTIC 2.11e6 Area _ _ c - , 1.00 200.b_- 3.00 ' 4.00 -' 5.00 6.00 ~ 1 0 0 " " ' b . 0 0 ' ' ' " 9 . 0 0 " ~ ' ' 1 0 ~ ~ ~ 3M Environmental Laboratory 3M Environmental Laboratory Page 92 of 216 Page 92 Report E01-1256 Report E01-1256 1454-109-9648, 10 PPM, Female, Offspring 13M Environmental Lab d020228128 Sm (Mn, 1x2) ~ 101 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 16735: 1.00 ~~~~ 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.0&-' - , , ..... 7.00 ' 8.00 ' 9.00 10.00 _. 3M Environmental Laboratory 3M Environmental Laboratory Page 93 of 216 Page 93 Report E01-1256 Report E01-1256 154-109-9645, 10 PPM, Female, Offspring IM Environmental Lab 1020228127 Sm (Mn, 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 131158 davey 07079! Ol-Mar-2002,10:01:0! 1: MRM of 1 Channel ES- TIC 1.59eE Are2 o/c c ' 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 94 of 216 Page 94 Report E01-1256 Report E01-1256 154-109-9634, 10 PPM, Female, Offspring )M Environmental Lab j020228123 Sm (Mn. 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 22191 davey07079 OI-Mar-2002,09:16:5 1: MRM of 1 Channel ES TI( 2.59e Are % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 95 of 216 Page 95 Report E01-1256 Report E01-1256 54-109-9629, I O PPM, Female, Offspring ;M Environmental Lab 1020228121 Sm (Mn, 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A ReprodudionStudy with the Mallard 6.75 174296 davey 07079! OI-Mar-2002,08:54:4~ 1: MRM of 1 Channel ESTI( 2.10e( Are; % C ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 8.00 9.00 10.00 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 96 of 216 Page 96 Report E01-1256 Report E01-1256 64-109-9633, 10 PPM, Female, Offspring MA Environmental Lab 1020228122 Sm (Mn, 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 120377 davey 07079 OI-Mar-2002,09:05:4~ 1: M R M of 1Channel ES. TI( 1.50e( Are; 1 ' 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 Report E01-1256 Report E01-1256 Dosed Group Male Liver Samples IO ppm a.l. 3285 M Liver, d10 IM Environmental Lab j020306144 S m (Mn, 1x2) IO( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 308321 davey070799 07-Mar-2002,12:04:28 1: MRM of 1 Channel ES- TI( 3.32e Are ' 1.00 2.00 I 3.00 I 4.00 I 5.00 I 6.00 I 7.00 8.00 9.00 10.00 - 3M Environmental Laboratory 3M Environmental Laboratory Page 98 of 216 Page 98 Report E01-1256 Report EOI-1256 IO ppm a.i. 3289 M Liver, d10 IM Environmental Lab 1020306146Srn I.Mn.. 1x2,) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 29313! davey07079 07-Mar-2002,12:26:4 1: MRM of 1 Channel ES. TI( 3.21e Are I ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 ' 8.00 ' 9.00 ' 1 O : O O ~ ~ ~ - 3M Environmental Laboratory 3M Environmental Laboratory Page 99 of 216 Page 99 Report E01-1256 Report EOI-1256 I O ppm a.i. 3287 M Liver, d10 3M Environmental Lab j020306145 Sm IMn. 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 27290: davey07079 07-Mar-2002,12:15:3' 1: MRM of 1 Channel ESTI( 2.40e( Are; % 3M Environmental Laboratory 3M Environmental Laboratory Page I00 of 216 Page 100 Report E01-1256 Report E01-1256 IO ppm a.i. 3283 M Liver, dlO IM Environmental Lab 1020306143 Sm (.Mn..1x21, 1 O( ~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 33137 davey07079 07-Mar-2002, 11:53:2 I:MRM of 1 Channel ES. TI( z.8aei Are, % ' ' " I ' I 1.00 I 2.b0 1 3.00 I 4.00 I 5 . p 6.bO 3M Environmental Laboratory 3M Environmental Laboratory Page 101 of 216 Page 101 Report E01-1256 Report E01-1256 IO ppm a.i. 3281 M Liver, d10 IM Environmental Lab j020306142 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 26537~ davey 070791 07-Mar-2002,11:42:1: 1: MRM of 1 Channel ES- TIC 2.84et Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 102 of 216 Page 102 Report E01-1256 Report E01-1256 IO ppm a.i. 3279 M Liver, dlO IM Environmental Lab j020306141 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 32737d davey07079 07-Mar-2002,11:31:0 1: MRM of 1 Channel ES. TI( 3.53e Are % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 103 of 216 Page 103 Report E01-1256 Report E01-1256 I O ppm a.i. 3277 M Liver, d10 3M Environmental Lab j020306140 Sm (Mn, 1x2) 1oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 27027 davey 07079 07-Mar-2002, 11:19:5 1: MRM of 1 Channel ES TI1 2.44e Are % 0 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 104 of 216 Page 104 Report E01-1256 Report E01-1256 IO ppm a.i. 3273 M Liver, d10 . . IM EnvironmentalLab i020306138 Sm IMn. 1x2,) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 38481f davey 07079 07-Mar-2002,10:57:4 I:MRM of 1Channel ES. TI( 3.17e Are w 1 p t Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 ' 9.00 ' 1O:OO~~~- 3M Environmental Laboratory 3M Environmental Laboratory Page 105of 216 Page 105 Report E01-1256 Report EOl-1256 1 ppm a.i. 3271 M Liver, d10 1Environmental Lab 120306137 Srn (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 306919 davey 07079 07-Mar-2002,10:46:2 1: MRM of 1 Channel ES. TI( 3.27e Are 3M Environmental Laboratory 3M Environmental Laboratory Page 106 of 216 Page 106 Report E01-1256 Report E01-1256 IO ppm a.i. 3275 M Liver, dlO IM Environmental Lab io20306139 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 29272( davey07079 07-Mar-2002,11:08:4 1: MRM of 1 Channel ES. TI( 3.16e Are % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 107 of 216 Page 107 Report E01-1256 Report E01-1256 Dosed Group Female Liver Samples IO ppm a.i. 3278 F Liver IM Environmental Lab 1020306118 Srn (.Mn,-1x2) I 1O( __ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 32308: davey07079 07-Mar-2002,07:15:3 1: MRM of 1 Channel ES. TI( 3.62el Are, % 0 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 108 of 216 Page 108 Report E01-1256 Report EOl-1256 10 ppm a.i. 3276 F Liver 3M Environmental Lab Id020306117 Sm IMn. 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 7036% davey07079 07-Mar-2002,07:04:2 1: MRM of 1 Channel ES. TI ( 7.1681 Are 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 109 of 216 Page 109 Report E01-1256 Report E01-1256 IO ppm a.i. 3274 F Liver IM Environmental Lab j020306116 Sm (Mn, 1x2) loa PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 46967G davey 07079! 07-Mar-2002,06:53:11 1: MRM of 1 Channel ES- TIC 4.98et Are2 % ' 0 I I ' I " '1'.00I ' I ' ' 2.60 I I 3 h 0 ' ' I '4.60 ' 5.60 ' ' I I ' 6.bO I 7.00 , 8.00 ' 9.00 ' 1o:oo - 3M Environmental Laboratory 3M Environmental Laboratory Page 110 of 216 Page 110 Report E01-1256 Report E01-1256 0 ppm a.i. 3272 F Liver IM Environmental Lab 1020306115 Sm (Mn, 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A ReproductionStudy with the Mallard 6.72 295878 davey07079 07-Mar-2002, 06:42:1 1: MRM of 1 Channel ES. TI( 3.43er Are; o/c C ' 1.00 ' 2.00 3.00 ' 4.00 5.00 6.00 3M Environmental Laboratory 3M Environmental Laboratory Page 111 of216 Page 111 Report E01-1256 Report E01-1256 IO ppm a.i. 3270 F Liver 3M Environmental Lab 1020306114 Srn (Mn, 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Study with the Mallard 6.71 426931 davey07079 07-Mar-2002,06:31 :Os 1: MRM of 1 Channel ES. TI( 4.65el Are; 3M Environmental Laboratory 3M Environmental Laboratory i Page112of216 Page 112 Report E01-1256 Report E01-1256 ppm a.i. 3266 F Liver Environmental Lab !0306112 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.7. 3 277317 davey 070792 07-Mar-2002,06:08:4I 1: MRM of 1 Channel ES- TIC 3.16e8 Area I 1.00 ' 2.b0 ' 3.b0 ' 4.00 ' 5.b0 6.00 ' 7.00 I I J I I I I I I I I l , I I I I I I I 81Time 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 113 of 216 Page 113 Report E01-1256 Report E01-1256 0 ppm a.i. 3268 F Liver IM Environmental Lab 1020306113 Sm (.Mn..1x2,) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 302664 davey07079 07-Mar-2002,06:19:5~ 1: MRM of 1 Channel ES. TI( 3.37e( Are; % C ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 6.00 3M Environmental Laboratory 3M Environmental Laboratory Page 114of216 Page 114 Report E01-1256 Report EOI-1256 IO ppm a.1. 3264 F Liver IM Environmental Lab io20306111 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 324993 davey 07079! 07-Mar-2002, b5:57:3! 1: MRM of 1 Channel ES- TIC 3.67et Are: ' IIIqII,IIIIIIIIIIIII I 1.00 2.00 3.00 11111 4.00 II I 5.00 I r p Time 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 115 of 216 Page 115 Report E01-1256 Report E01-1256 IO ppm a.i. 3260 F Liver IM Environmental Lab 1020306067 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 328881 davey07079 06-Mar-2002,21:48:4 1: MRM of 1 Channel ES. TI( 3.68ei Are; % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 116 of 216 Page 116 Report E01-1256 Report E01-1256 10 ppm a.i. 3258 F Liver 5M Environmental Lab 1020306066Sm (Mn,1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 31533 davey 07079 06-Mar-2002,21:37:3 1: MRM of 1 Channel ES TI1 3.60e Are 94 a 3M Environmental Laboratory 3M Environmental Laboratory Page 117 of 216 Page 117 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Male Offspring Liver Samples ppm a.i. 9636 M Liver I Environmental Lab 20307052 Sm (.Mn..1x21, 6.70 129040 davey 07079! 08-Mar-2002, 00:39:44 1: MRM of 1 Channel ESTI( 1.52e( Are; I.UU L.UU J.UU 4.uu 3.uu 0.uu I.UU 0.uu Y.UU IU.UU 3M Environmental Laboratory 3M Environmental Laboratory Page 118 of 216 Page 118 Report E01-1256 Report E01-1256 IO ppm a.i. 9642 M Liver )M Environmental Lab 1020307053 Sm (Mn, 1x2) 1oa PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 152685 davey 070794 1: MRM of 1 Channel ES- I TIC 1.Met Are2 % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 119 of 216 Page 119 Report E01-1256 Report E01-1256 10 ppm a.i. 9625 M Liver 1M Environmental Lab j020307051 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard i 6.72 22367: davey07079 08-Mar-2002,00:28:3 1 1.00 2.00 3.00 4.00 5.00 6.00 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 120 of 216 Page 120 Report E01-1256 Report EOl-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Female Offspring Liver Samples IO ppm a.i. 9659 F Liver IM Environmental Lab j020307050 Sm (Mn. 1x2) 1 O( 6.72 186526 davey 07079 08-Mar-2002,00:17:3 1: MRM of 1 Channel ES TI( 2.24e Are 3M Environmental Laboratory 3M Environmental Laboratory ,, 1 1 1 , 1 1 I Time .OO 8.00 9.00 10.00 Page 121 of 216 Page 121 Report E01-1256 Report E01-1256 IO ppm a.i. 9634 F Liver Nul Environmental Lab j020307046 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard ~ 6.71 24037: davey07079 07-Mar-2002,23:33:0 1: MRM of 1 Channel ES- TIC 2.76e Are ' 1.00 ' 2.00 ' 3.00 4.00 5.00 ' 6.00 ' 00-7 1 Time 3M Environmental Laboratory 3M Environmental Laboratory Page 122 of 216 Page 122 Report E01-1256 Report E01-1256 IO ppm a.i. 9629 F Liver IM Environmental Lab j020307044 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 170716 davey 07079 07-Mar-2002,231:0 5 I:MRM of 1Channel ESTIC/ 2.03e6 % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 123 of 216 Page 123 Report E01-1256 Report E01-1256 IO ppm a.i. 9645 F Liver 1M Environmental Lab 1020307047 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.70 150376 davey 07079 07-Mar-2002,23:44:11 1: MRM of 1 Channel ES- TI ( 1.77e( Are; 94 C 3M Environmental Laboratory 3M Environmental Laboratory Page 124of 216 Page 124 Report E01-1256 Report E01-1256 ppm a.i. 9648 F Liver . . I EnvironmentalLab 20307048 Srn (Mn. 1x21 I PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 190031 davey070799l 07-Mar-2002,23:55:1 1: MRM of 1 Channel TI( 2.29e Are ' c , I ,,,, ,,,, lime 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 8.00 I 9.00 I 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 125 of 216 Page 125 Report E01-1256 Report E01-1256 IO ppm a.i. 9633 F Liver 5M Environmental Lab 1020307045 Sm (Mn, 1x2) 1 oc PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 123667 davey 07079 07-Mar-2002,23:21:5' 1: MRM of 1 Channel ES- TIC 1.52e( Are; % 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 126 of 216 Page 126 Report E01-1256 Report E01-1256 IO ppm a.i. 9654 F Liver 1M Environmental Lab 1020307049 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 27900: davey07079 08-Mar-2002,00:06:2 1: MRM of 1 Channel ESTIC( 3.1 l e 6 Are 9: 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7x0 I 8.b0 9.bO ' I I I Time lO!OO 3M Environmental Laboratory 3M Environmental Laboratory Page 127 of 216 Page 127 Report E01-1256 Report E01-1256 Control Group Male Sera Sample 154-109-3243, 0 PPM, Male, Adult IM Environmental Lab io20226038 Sm (.Mn.. 1x2,) IO( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079 26-Feb-2002,23:11:4 1: MRM of 1 Channel ES 6.7 TI( 92 l.16e Are % 3M Environmental Laboratory 3M Environmental Laboratory Page 128 of 216 Page 128 Report E01-1256 Report E01-1256 Control Group Female Sera Sample 154-109-3244, 0 PPM, Female, Adult IM Environmental Lab j020226055 Sm I.Mn..1x2), PFOS: A Reproduction Study with the Mallard PFOS: A ReprodudtionStudy with the Mallard davey07079 27-Feb-2002,02:20:5 6.76 630 3M Environmental Laboratory 3M Environmental Laboratory Page 129 of 216 Page 129 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Male Offspring Sera Sample 154-109-9578, 0 PPM, Male, Offspring LM EnvironmentalLab j020228133 Sm (Mn, 1x2) ioa 63.9705 davey 07079 01-Mar-2002,11:07:4 1: MRM of 1 Channel ES. TI( 6.11e Are % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 130 of 216 Page 130 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Female Offspring Sera Sample 154-109-9589, 0 PPM, Female, Offspring IM Environmental Lab 1020228115 Sm (Mn, 1x2) 100 65.6756 davey 07079! Ol-Mar-2002,07:48:0' 1: MRM of 1 Channel ES- TIC 7.88e: Are; % 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 131 of 216 Page 131 Report E01-1256 Report E01-1256 Control Group Male Liver Sample 1 ppm a.i. 3243 M Liver IM-Environmental Lab j020306053Srn (.Mn..1x2,) IO( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079 06-Mar-2002, 19:13:1 1: MRM of 1 Channel ES 6.7: TI( 83 1.07e Are % 1 3M Environmental Laboratory 3M Environmental Laboratory Page 132of 216 Page 132 Report E01-1256 Report E01-1256 Control Group Female Liver Sample 10 ppm a.i. 3244 F Liver 3M-Environmental Lab do20306037 Srn (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079 06-Mar-2002, 16:15:2 1: MRM of 1 Channel ES. TI ( 1.13e Are % I S I IIII II I I s I 1.00 I I I I II I III I I II I II I I II III III I,I I II , , 2.00 3.00 4.00 5.00 6.00 7.00 ,, , , , , , , I , , , , Time 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 133 of 216 Page 133 Report E01-1256 Report EOl-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Male Offspring Liver Sample 1 ppm a.i. 9578 M Liver )M Environmental Lab j020307040 Sm (Mn, 1x2) 1O( davey 07079 07-Mar-2002,22:26:2 1: MRM of 1 Channel ES, TI( 9.33e Are OA 0 3M Environmental Laboratory 3M Environmental Laboratory Page 134 of 216 Page 134 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reprodudion Study with the Mallard Control Group Female Offspring Liver Sample I ppm a.i. 9610 F Liver ;MEnvironmental Lab 1020307036Sm (Mn, 1x2) 100 64.6701, davey 07079! 07-Mar-2002,21:42:0f 1: MRM of 1 Channel ESTIC 5.47e? Ar0E % 3M Environmental Laboratory 3M Environmental Laboratory Page 135 of 216 Page 135 Report E01-1256 Report E01-1256 Extracted Sera Calibration Curve Point irve point-1 ppb in sera extract 1 Environmental Lab 20228010 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 07079 28-Feb-2002,12:22:3 1: MRM of 1 Channel ES- 6.74 TI( 293: 3.45ed Arei 3M Environmental Laboratory 3M Environmental Laboratory Page 136 of 216 Page 136 Report E01-1256 Report E01-1256 -~ PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Extracted Liver Calibration Curve Point irve point-1 ppb in liver extract 1 EnvironmentalLab 20307010 Sm IMn. 1x2) 6.71 2291 ~~ 07-Mar-2d00a2v,e1y6:05730:3719 1: MRM of 1 Channel ES- TI( 2.95ed Are; 3M Environmental Laboratory 3M Environmental Laboratory n Page 137 of 216 Page 137 Report E01-1256 Report E01-1256 Unextracted Calibration Curve Point 12003-15-1, 2.5 ppb unextractedcurve IM Environmental Lab j020305008 Sm (Mn, 1x2) 1O( PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 507 davey 07079! 05-Mar-2002,14:21:2' 1: MRM of 1 Channel ES- TI( 6.06er Are; % a 3M Environmental Laboratory 3M Environmental Laboratory Page 138 of 216 Page 138 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Extracted Sera Calibration Curve 2ompound 1 name: pfos (499>99) func 1 Method File: d020228a 2oefficient of Determination: 0.992963 2alibration curve: -0.922531 * xn2 + 1329.17 x + 2035.89 qesponse type: External Std, Area 2uwe type: 2nd Order, Origin: Exclude, Weighting: l/x, Axis trans: None 5.74e5 a X Response I 0 3M Environmental Laboratory 3M Environmental Laboratory Page 139 of 216 Page 139 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Extracted Liver Calibration Curve :ompound 1 name: pfos (499>99) func 1 Method File: d020307a :oefficient of Determination: 0.986337 :ahbration curve: -0.825149 * xA2+ 1136.85 x + 1614.53 lesponse type: External Std, Area :urve type: 2nd Order, Origin: Exclude,Weighting: l/x, Axis trans: None 4.95e5 X iesponse 0 3M Environmental Laboratory 3M Environmental Laboratory Page 140 of 216 Page 140 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\DAVEY070799.PRO\ACQlJDB\CENTREl Printed: Tue Mar 19 10:22:06 2002 Page 1 MS SOURCE ( ESP- ) Capi11ary Cone Hexapole 1 Aperture 1 Hexarole 2 Source Block Desolvation Temp. 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 nM 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 13.0 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 Page 141 of 216 Page 141 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:\MASSLYNxU1AVEYO70799.PRO\A( Monday, March 11,2002 08:Ol:Ol Tuesday, March 19,2002 08:37:50 HP1100 LC PumpinitialCondltions Solvents A% B% C% D% 90.0 10.0 0.0 0.0 Valve A set to channel Valve B set to channel Flow (mvmin) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left("C) Oven Temperature Right(%) 0.300 10.0 0 400 40.0 40.0 HPllOOLC Pump GradientTlmetable The gradient Timetable contains 5 entries which are : Time A% B% C% D% Flow 0.00 90.0 10.0 0.0 0.0 0.300 1.00 90.0 10.0 0.0 0.0 0.300 5.50 5.0 95.0 0.0 0.0 0.300 7.50 5.0 95.0 0.0 0.0 0.300 8.00 90.0 10.0 0.0 0.0 0.300 W P I l Q o 1Jc Pumpb m a lEwnt~~~e The Timetable contains 3 entriaa which am : Time Initial 0.00 0,IO Column Switch Ufl off Qfl On Ofl Dff Contact1 QR On Mf Contact2 OR OR Qfl C~ntac uff On OR HPIla0Aukasampler Initid Cbndiiom Oraw Speed Eject S p e d [VVmin) Oraw Position [rnm) Sdop Time (ninsj Injection Volurne(pl) Vial Number Therrnostsd On Thermostat TemperaturePC] 200.0 200 0.ao 10.00 10.0 94 20.0 3M Environmental Laboratory 3M Environmental Laboratory Page 142 of 216 Page 142 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 Report Method : C:\MASSLYNX\DAVEYO7O799.PRO\ACQUDB\10#IN-PFOS-PFDA Last Modified: Mon Jan 28 15:20:24 2002 Printed: Tue Mar 19 08:38:22 2002 Solvent Delay ( mine ) : 0.00 Function : 1 MRM of 1 Maas Pair ( ESP- ) Inter Channel Delay ( Span ( Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Secs : 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 MRld of 1 Mass Pair ( ESP- ) Inter Channel Delay ( Span ( Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Secs ) : Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone ( V ) 1 513.00 219.00 0.30 20 20 Page 1 3M Environmental Laboratory 3M Environmental Laboratory Page 143 of 216 Page 143 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Study with the Mallard ATTACHMENDT: SAMPLEPREPARATIOSNHEETS 3M Environmental Laboratory 3M Environmental Laboratory Page 144 of 216 Page 144 Report E01-1256 PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Page 145 Report &E071/--/1?2Jd56 hu 2 `7C;aL sample number description PFOS: A Reproduction Study with the Mallard Amount of weighed liver byldate weighed (9) i Homogenized homogenized wl water to byldate (ml) 3M Environmental Laboratory Page 146 Report E01-1256 Prep Date: 3/4/2002 Analysts initials: OWHOJ ILimsAssigned sampll number E01-132636778 EO1-132636780 EO1-1326-36781 E01-1326-36782 E01-1326-36783 E01-1326-36784 E01-1326-36785 E01-1326-36786 EO1-1326-36787 E01-1326-36788 E01-1326-36789 EO1-1326-36790 EO1-1326-36791 E01-1326-36792 E01-1326-36793 EO1-1326-36794 SPE Columns Extraction PFOS: A Worksheet ReproductMiodnkScltudLy.uwwithbAetheoMalaallllaalrd @ 3 pa%e lota im?.wt 9 Method Revision: ETS-8-2 Study Number: E01- I Volume of I i Matrix: i Liver i EO1-1326-36796 . 9w-r 1 1 (3w su @tea Homogenize liverwith Kandiyohi water; Amount of water added 4 5 ml Aliquot 1ml of liver into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TN-A- 4' lq '7 ) to aliquated sample Shake sample for 20 min @300 rpm ( S h a k e r v k l w 3 s f 6 9 4 Centrifuge sample forOI min Q 2000 rpm (Centrifuge%+FWKQ Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Conditioncolumn with MeOH (TN-A-G- 9 ) 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 ( wq T N)- A - e ? Spike sampleswith internal standard PFDA standard number 0d-l- 45 Transfer sample into appripriatelly marked autovial i1nto appropriate 5 ml centrifuge tube , conc. b.=fp$U,amount added a-d 3M Environmental Laboratory Page 147 Report E01-1256 Analysts initials: OWHOJ SPE Columns Extraction PFOS: A Worksheet mc;;;yj Reproduction Study with @ lh! the Mallard oalzrlol Study Number: E01 Homogenke liver with Kandiyohiwater; Amount of water added 4 5 ml Aliquot Iml of liver into 15 ml polypropelenetube Spike samples accordingly 5!d Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min Q300 rpm (ShakergtdR Centrifuge sample for IO min Q 2000 rpm ( ~ e n ~ f u g ?e @~ 6~ $g 3 Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. 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 /"fP Elute columnwith indicated amount of solvent ( TN-A- 6- 9 ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number odoo 1-4 5 , conc. , amount added Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 148 Report E01-1256 SPE Standard Curves--Solids PFOS: A Reproduction Study with the Mallard Pmp Date(s): Andyat(a): Smpk M a e : Method/Rcvisiou: n e e t AMlyte(s): 03/04/02,03/05/02,o3~o6/o2,03/07/02 OKhIOJ Mallard Liver ETS-8-231I. PFOS Study Number: EOI-1256 Equipment Number: NA i Final Solvent & TN Number: MeOH/l%A-6009 Blank TiasudIdenlibr: Mallard Liver/IN-A-6023 FC M i x Std Appmx. 1.00ppm: FC M i x Std Appmx. 50.2ppm: Sum.Std Appmx. 100.7ppm: 02001-66 02001-53 02001-45 PFDA A d Cmcenlralim ofStandards Standard Number EOI-1326-36783.0.2ppb EOI-1326-36784.0.5ppb EO1-1326-36785,1.0ppb EOl-1326-36786,2.p5pb EO1-1326-36787,5.p0pb EOI-1326-36788,IOppb EOl-1326-36789,25ppb E01-1326-36790,50ppb EO1-1326-36791.75ppb EOI-1326-36792.100ppb EOI-1326-36793,100 ppb-2 EO1-1326-36794,25p0pb E01-1326-367955,00ppb E01-1326-36796,750ppb EOI-1326-36797,I000 ppb QC-25 ppb QC-250 ppb QC-4 ppm Mallard Liver-MSMSD-Day 1 Mallard Liver-MSMD-Day 2 W a r d Liver-MSMD-Day 3 MnllardLiver-MSMD-Day 4 bFC Mix PPOS Std Conc. ug/mL 1.004 1.004 1.004 1.004 1.004 1.004 1,004 1.004 1.004 1.004 50.20 50.20 50.20 50.20 50.20 50.20 50.20 50.20 1.004 1.004 PPOS PFDA All Am't b i k d Std Gmc. Am? S ~ k d I I m L I UglmL I o.ooo4 I 100.7 I mL 0.002 0.0010 100.7 0.002 0.0020 100.7 0.002 0.0050 100.7 0.002 0.0100 100.7 0.002 0.0200 100.7 0.002 0.0500 100.7 0.002 0.I000 100.7 0.002 0.1500 100.7 0.002 0.2Ooo 100.7 0.002 0.0040 100.7 0.002 0.0100 100.7 0.002 0.0200 100.7 0.002 0.0300 100.7 0.002 0.0400 100.7 I 0.0010 I 100.7 I 0.002 0.002 0.0100 0.002 0.0500 0.0500 0.002 0.0500 100.7 0.002 0.0500 100.7 0.002 Cdculatedcmeentrationsof Stan& Standard Number EOI-1326-36783.0.2ppb EOI-1326-367840,.5 ppb EOl-I326-36785,1.p0pb EOl-1326-36786,2.p5pb EO1-1326-36787,5.p0pb EOI-1326-36788,IO ppb EO1-1326-36789,25ppb EO1-1326-36790,50ppb E01-1326-36791,75ppb EO1-1326-36792,l00ppb E01-1326-36793,100ppb-2 EOl-I326-36794,250ppb E01-1326-36795,5OOppb EOI-I326-36796,75p0pb EOI-1326-36797,l000mb QC-25 ppb QC-250 ppb QC-4 ppm Mallard Liver-M.3MSD-D.y 1 Mallard Liver-MSMD-Day 2 Mallard Liver-MS/MSD-Day3 Mallard Liver-MSMD-Day 4 in the sampleI PFOS std Conc. np/g 3.97 9.93 19.9 49.7 99.3 199 497 993 1490 1987 1987 4967 9933 I4900 19867 497 4967 79467 497 497 497 497 rix PFDA std c m c . nglg 1993 1 993 1993 1993 1993 1993 1993 1993 1993 1993 1993 1 993 1993 1993 1993 1993 1993 1993 1993 1993 1 993 1 993 y CalculatedConcen!mtiom of Standru intheWsol PPOS std Cmc. ng/mL EOI-1326-36783.0.2ppb 0.201 EOI-1326-367840,.5 ppb 0.502 EOI-1326-36785,1.0 ppb 1.00 EO1-1326-36786,2.p5pb 2.51 E01-1326-36787,5.Oppb 5.02 EOl-1326-36788,IO ppb 10.0 ~01-13~6-3678p9p,b~5 25.1 EOl-1326-36790,50ppb 50.2 EO1-1326-36791,75ppb 75.3 EO1-1326-36792,100ppb I00 EOI-132636793,100ppb-2 100 E01-1326-36794,25Oppb 251 EOI-1326-36795,500ppb 502 EOI-1326-36796.750ppb 753 EOI-1326-36797,IO00 ppb 1004 QC-25 ppb 25.1 QC-250 ppb 251 QC-4 ppm 4016 W a r d Liver-MSMD-Day 1 25.1 Msllard Liver-MSMSD-Day 2 25.1 Mallard Liver-MSMSD-Day3 25.1 Mallard Liver-MSMSD-Day 4 25.1 t PFDA std Conc. ndmL 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 Jnitial homogmpte:5.0597d50.0597ghnL= 0.1011dmL BImkliverinitid b o m o ~ t e ( g h n L ) f O r c u m l : 0.1011 Find = k a t volume (mL) for CUWUL: 2.OOO Au Jnitial Homogenate gld 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 0.1011 0.1011 0.1011 0.1011 0.1011 Au Final Vol 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 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 3M Environmental Laboratory Page 149 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (d): Solvent: ID #: V W R ,5mL 3/4/02 ok 799 900 5 Acetonitrile tn-a-4 145 1 06 G.4.ou 1 ~ 2 3 4 I - Mass Of Solvent (g) 3.9613 3.9484 4.0278 3.9 249 Density' (g/ml) 0.7857 0.7857 0.7857 0.7857 Volume of Solvent 5.0417 5.0253 5.1264 4.9.9.5. 4 AverageVolume: %Accuracy: Std. Dev.: %C.V.: 5.0524 101.05 0.04997 0.99 PASS PASS Volume, (Id) 1 to 10 Accuracy, (%) 98.0 - 102 % Corrective Actions: C.V., (%) & 1.0 % 'Density values taken from the Merck Index, 12'bEdition, Copyright 1996 d', =specific gravity at ydemC referred to water at xdem n-Hexane d204=0.660 Acetone dz0 =0.788 Methanol dB4 =0.7866 Methylene Chloride dZo4=1.3255 Acetonitrile d204= 0.7857 3M En3v/4ir/o02n,m8:en1t7aAl MLaboratory Page 150 Report E01-1256 Prep Date: Analysts initials: 0 Lims Assgned E01-132636807 E01-1326-36809 EO1-1326-36810 E01-1326-29682 E01-1326-29691 E01-1326-29700 IE01-1326-29709 E01-1326-29718 E01-1 326-29745 ]EO1 -1326-29754 lEOl-1326-29763 1E01-1326-29772 SPE Columns Extraction PFOS: A Worksheet @ rose Reproduction Study with f%'lWilodi[W z ](ks the Mallard 6)/2llb2 Method Revision: ETS-8-231 Study Number: EOI-&32@@?! Matrix: Liver m f 4 I 1 I I Amount and spike Elution soben mix used and volume 1 Comments I INA NA 2ml of MeOH TN-A-6009 .pL i I I I I I I I NA Zml of MeOH TNA-6009 I I I NA 2ml of MeOH TN-A-6009 2ml of MeOH NA hNA-6000 1 I 2ml of MeOH TN-A-6009 NA 2ml of MeOH N-A-8009 2ml of MeOH TNA-6009 1I II --i E01-1326-29808 IE01-1326-29826 Blank Liver TN-A-6023; Amount of liver: 5.05979 Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot Iml of liver into 15 ml polypropelenetube Spike samples accordingly o4b.5 -0a / Add 5ml of ACN (TNA-4145) to aliquated sample Shake sample for 20 min e 3 0 0 rpm (Shaker WVR SIN 041694) Centrifuge sample for I O min Q 2000 rpm (Centrifuge3M# 769613) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. 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 TNA-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 appripriatelly marked autovial 3M Environmental Laboratory Page 151 Report E01-1256 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet @ ~~~~~~ BSod&z Prep Date: 3.5'02 Analysts initials: Lims Assgned sample number I Sample Number or description EO1-1326-29835 0 ppm a.i. 3248 F Liver Volume of sample filtered Type of column used and lot -,SF%% Method Revision: ETS-8-231 Study Number: E01 Matrix: Liver Amount and spike Elution solven mix used and volume Comments E01-1326-29844 I E01-1326-29853 I EOI-1326-29862 EO1-1326-29871 E01-1326-29880 E01-1326-29889 E01-1326-29898 EO1-1326-299O7 E01-1326-29916 E01-1326-29925 E01-1326-29934 E01-1326-29943 E01-1326-29952 E01-1326-29961 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 I O 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 EO1-1326-29970 I O m m a.i.3263 M Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, lg, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, I g , 6ml, lot W204582 Waters, lg. 6ml, lot ME04582 Waters, 1g,6ml, lot W204582 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W204582 40+6 40+6 40+6 40+6 40+6 40+6 Waters. lg. 6ml. lotW045B2 Waters, Ig, 6ml. lot W204582 Waters, Ig, 6ml, lot W204582 Waters, lg, 6ml, lot W204582 Waters, lg, 6ml, lotW204582 Waters, Ig, 6ml, lotW2045B2 NA NA NA . .. . NA NA NA NA NA . .. . NA NA NA NA NA NA 2ml of MeOH TNA-BOO9 2ml of MeOH TNA-6009 2ml of MeOH TN-A-BDOg 2ml of MeOH TN-A-62ml of MeOH TNA-6009 2ml of MeOH TN-A4009 Zml of MeOH TNA-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-8009 2ml of MeOH TNASOOe 2ml of MeOH TNA-6W9 2ml of MeOH TNAgOOg 2ml of MeOH TN-A4009 II I - / . . I08 ldbl I I 1 I Blank Liver TN-A-6023; Amount of liver: 5.0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot Iml of liver into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TNA-4145) to aliquated sample Shake sample for 20 min a 3 0 0 rpm (Shaker WVR SIN 041694) yZ-02- Centrifuge sample for 10 min Q 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TNA-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow columnto go completelly dry Elute column with indicated amount of solvent (MeOH TNA-6009) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul T3raMnsfEernsavmirpolenimntoenaptparlipLriaateblolyrmaatrokreyd autoviai Page 152 Report E01-1256 PFOS: A Reproduction Study with the Mallard BottleTop Dispenser Daily Calibration Check Manufacturer and Model: VWR ,5mL I Date: 3/5/02 Initials: Ok Dispenser ID: 799 Balance ID: 900 Volume (mL): 5 Solvent: Acetonitrile ID #: tn-a-4 145 PASS Volume, (W 1 to 10 Accuracy, (%) 98.0 - 102 % C.V., (%) f 1.0 % `Density values taken from the Merck Index, 12L Edition, Copyright 1996 dyx=specific gravity at yde""" referred to water at xdemC n-Hexane dZo4=0.660 Acetone ds0 =0.788 Methanol dZS4=0.7866 Methylene Chloride d204=1.3255 Acetonitrile dm4= 0.7857 3M En3v/i5r/o0n2,m8e:nOtOalALMaboratory Page 153 Report E01-1256 06 Prep Date: .n 3'bo6 Analysts initials: SPE Columns Extraction PFOS: A Worksheet Reproduction Study withustheaMpl&allard Method Revision: ETS-8-2&3BP1 Study Number: E 0 1 - w E01-1326-30025 E01-1326-30034 E01-1326-30043 EO1-1326-30052 EO1-1326-30061 E01-1326-30070 EO1-132630079 E01-1326-30088 E01-1326-30097 EO1-1326-30106 E01-1326-30115 E01-1326-30124 EO1-1326-30133 10 ppm a.i. 3269 M Liver I O ppm a.i. 3270 F Liver 10 ppm a.i. 3271 M Liver I O 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 I O 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 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 lot W204582 Waters, I g , 6ml, lot ~ 2 0 4 5 8 2 Waters, I g , 6ml. lot ~ 2 0 4 5 8 2 Waters, Ig, 6ml, lot ~ 2 0 4 5 8 2 Waters, I g , 6ml, lot W204582 Waters, Ig. 6ml, lot W204582 Waters. I g , 6ml, lot W2045B2 Waters, I g , 6ml, lot wo45B2 Waters, Ig, 6ml, lot ~ 2 0 4 5 8 2 Waters, I g , 6ml, lot ~ 2 0 4 5 8 2 Waters, l g , 6ml, lot\N204582 Waters, Ig, 6ml, lot\N204582 , I I ami, lot Wo45B2 NA NA NA NA NA NA NA NA NA NA NA NA NA TNA-BWg 2ml of MsOH TN-ASOOQ 2ml of MeOH TN-A4009 2ml of MeOH TNA-Bog 2ml of MeOH TN-A-BOM) 2ml of MeOH TN-ASOOg 2ml of MeOH TNA-Bo9 2ml of MeOH TN-A4Wg 2ml of MeOH TN-A-6009 2ml of MeOH TNA-6009 2ml of MeOH TN-A-BWQ 2ml of MaOH TNAgOOg 2ml of MeOH TN-A-6009 3M Environmental Laboratory Page 154 Report E01-1256 SPE Columns Extraction PFOS: A Worksheet R@eprodpIbupUacttioLn L~Swtud]y with the Mallard L R ~08jailoi Prep Date: 3- 6.04 Analysts initials: f$ Lims Assgned sample Sample Number number or description I I E01-I32630142 I I EOI-I32630151 10 ppm a.i. 3282 F Lver 10 ppm a.i. 3283 M Liver EOI-1326-30160 10 ppm a.i. 3284 F Liver EO1-1326-30169 10 ppm a.i. 3285 M Liver E01-1326-30178 10 ppm a.i. 3286 F Liver EO1-1326-30187 I O ppm a.i. 3287 M Liver E01-1326-30196 10 ppm a.i. 3288 F Lver E01-1326-30205 I O ppm a.i. 3289 M Liver I E01-1326-30214 I I O ppm a.i. 3290 F Liver EO1-1326-30223 EO1-1326-30232 10 ppm a.i. 3291 M Liver ~~~ 10 ppm a.i. 3292 F Liver E01-1326-30241 10 ppm a.i. 3293 M Liver Volume of sample filtered (ml) Type of column used and lot &au Method Revision: ETS-8-2 Study Number: E01- Matrix: Liver i236 Amount and spike Elution solven mix used and volume Comments 40+6 40+6 Waters, I g , 6mL lot W2045B2 Waters, I g , 6ml, lot W2045B2 NA 2ml of MeOH TN-ASOOg tJlA 2ml of MeOH NA TN-A-Bo9 40+6 Waters, Ig, 6ml, lot W2045B2 2ml of MeOH NA TNA-6009 40+6 Waters, Ig, 6ml, lotW204582 NA 2ml of MeOH TN-A4009 40+6 Waters, I g , 6ml. lotW2045B2 NA 2ml of MeOH TKASOOg 40+6 Waters, Ig. 6ml, lot W204582 2ml of MeOH NA TN-A-6009 40+6 Waters, l g , 6ml, lot W2045B2 2ml of MeOH NA TN-A-6009 40+6 Waters, I g , 6ml, lot W2045B2 2ml of MeOH NA TNA-6009 40+6 Waters, I g , 6ml, lotW2045B2 2ml of MeOH NA TNA-6009 I I I 40+6 Waters. l a . 6ml, 1101 W M & 2 2ml of MeOH NA TNA-6009 IWaters. l a-.. 6ml. I 12ml of MeOH 40+6 lotW2045B2 NA TNA- 40+6 Waters, I g , 6ml, lot W2045B2 2ml of MeOH NA TN-A4009 E01-1326-30250 10 ppm a.i. 3294 F Lver NA EO1-1326-30259 E01-1326-30268 10 ppm a.i. 3295 M Liver 10 ppm a.i. 3296 F Lver 40+6 40+6 lot W204582 Waters, l g , 6ml. lot W045B2 NA TN-A-6009 2ml of MeOH NA TN-A4009 3.6-2 fi I I I I I I I Blank Liver TN-A-6023; Amount of liver: 5.0597g Homogenize liver with Kandiyohiwater; Amount of water added 45 mi Aliquot 1mi of liver into 15 ml polypropeienetube Spike samples accordingly Add 5ml of ACN (TNA-4145) to aliquated sample Shake sample for 20 min a300 rpm (Shaker WVR SIN 041694) Centrifugesample for I O min 0 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TNA-6009) Wash Column with KandiyohiWater 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 2ui T3raMnsEfernsvaimroplneminteonatpaplripLriatbeollyrmataorkreyd autovial Page 155 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottl-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 4.6-02 OKJ (g/ml) Solvent I I 1- 3.9506 0.7857 5.0281 I I I ~ ~~~~ 2 I 3.9857 I 0.7857 I 5.0728 ~ 3 3.9874 0.7857 5.0750 4 3.9772 0.7857 5.0620 I I 5 3.983 0.7857 5.0694 IAverageVolume: 5.0614 I %Accuracy:l 101.23 I PASS I Std. Dev.:l 0.01927 I %C.V.: 0.38 PASS Pass/Fail: PASS Volume, (mL) 1 to 10 Performance Specifications I Accuracy, I t 98.0 - 102 % C.V., t %) f 1.0 % I Corrective Actions: I 1Density values taken from the Merck Index, 12* Edition, Copyright 1996 dyx=specific gravity at ydemC referred to water at xden-Hexane d204=0.660 Acetone dzsO=0.788 Methanol =0.7866 Methylene Chloride d204=1.3255 Acetonitrile dm4= 0.7857 3M En3v/i6ro/0n2m,e6n:t3a3lALMaboratory Page 156 o< 4 Report E01-1256 Prep Date: Analysts initials: 5.7 k SPE Columns Extraction WoPrFkOshSe: Aet ?$?iy3 Rep@roduction Study wli.M th tohed 2Mda0l2lard Method Revision: ETS-8-231 Study Number: EOliW-!~@ebJ 3-7"c, Matrix: Liver 1 Blank Liver TN-A-6023; Amount of liver: 5.05978 Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TNA-4145) to aliquated sample Shake sample for 20 min a 3 0 0 rpm (Shaker WVR SIN 041694) Centrifuge sample for 10 min Q 2000 rpm (Centrifuge3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelenecentrifugetube. 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 TNA-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 appripriatelly marked autovial 3M Environmental Laboratory Page 157 Report E01-1256 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 +.?a Ok 6u 799 900 5 Acetonitrile tn-a-4 145 (g/ml) Solvent 1 3.9564 0.7857 5.0355 2 - 3.9674 0.7857 5.0495 3 3.982 0.7857 5.068 1 4 3.9753 0.7857 5.0596 I I 5 3.9713 0.7857 5.0545 IAverageVolume: 5.0534 I %Accuracy:l 101.07 I PASS I Std. Dev.:l 0.01215 I %C.V.: 0.24 PASS Pass/Fail: PASS Volume, (mL) 1 to 10 Performance Specifications I Accuracy, I (%) 98.0 - 102 % C.V., f 1.0 % I Corrective Actions: I `Density values taken from the Merck Index, 12* Edition, Copyright 19% d', =specific gravity at ydemC referred to water at xdn-Hexane dZo4=0.660 Acetone dz50=0.788 Methanol dz54=0.7866 Methylene Chloride dzo4=1.3255 Acetonitrile dzo4= 0.7857 3M En3v/ir7o/n0m2,e7n:4t0alALMaboratory Page 158 Report E01-1256 PFOS: A Reproduction Study with the Mallard E01-1326-30196 x E01-1326-30214 x E01-1326-29916 x / II ,/ E01-1326-29934 E01-1326-29952 E01-1326-29970 x x NJA x I r / lpts I 08h\oa, I E01-1326-29989 x NA / E01-1326-30007 x \ E01-1326-30025 x E01-1326-30043 x E01-1326-30061 x // E01-1326-30079 x u Lb Form Completion Verified By: wl 4 C t"), 3M Environmental Laboratory Page 159 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet f k ? Study: lWl-iH6 15-Ol-f3aC Dilution DateIAnalyst: Box Number: E 0,-I%&, tal-- - # rei.- o vent/TN Number: MeOW5457 Extraction Date/Analyst: Matriflimepoint: ~ ~ u f i~mj v e ~ Notes: 1/10 dilution = +of sample + 9 - Aof solvent Form Completion Verified By: +A 4 - L C - d L _ 3M Environmental Laboratory Page 160 Report E01-1256 PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Page 161 Report E01-1256 PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Page 162 Report E01-1256 Prep Date: Analysts initials: I Lims Assgned sample number OWHOJ SPE Columns Extraction PFOS: A Worksheet *- 3 R@eproPdbuc\.tliWondStudy with hJ2 th0e 8Mbalblarldpx 212512002 Method Revision: ETS-8-231.O Study Number: E01-1256 Matrix: Sera Sample Number or description e01 -1 256-36231 3lank H2Odav I e01-1256-36232 3lank mallard seraday 1 e01-125636233 Mallard Sera MSday I e01 -1 256-36234 e01-1256-36235 e01 -1 256-36236 e01 -1 256-36237 e01 -1 256-36238 e01-125636239 e01 -1 256-36240 e01 -1256-36241 e01 -1256-36242 e01 -1 256-36243 e01-1256-3624 e01 -1 256-36245 e01 -1 256-36246 e01 -1 256-36247 e01 -1 256-36248 e01 -1 256-36249 Mallard Sera MSDday I Curve point-O.2 ppb in sera extract Curve pointO.5 ppb in sera extract Curve point-I ppb in sera extrad Curve point-2.5 ppb in sera extrad Curve point5 ppb in sera extract C u m point-IO ppb in sera extract Curve point-25 ppb in sera extract Curve point50 ppb in sera extract Curve point-75 ppb in sera extract Curve point-I00 ppb in sera ewtract Curve point-I00 ppb-2 in sera extract Curve point-250 ppb in sera extract Curve point500 ppb in sera extract Curve point-750 ppb in sera extract Curve point-I000 ppb in sera extract e01-1256-36250 ZC 25pb-1 e01-1256-36251 2C 25pb-2 e01 -1 256-36252 2_ C_ 2_ _5r~-b--3 Dilute Sera with Kandiyohiwater; Amount of water added 36 ml Aliquot Iml of sera into 15 ml polypropelenetube Spike samples accordingly 4- Add 5ml of ACN (TN-A- lq ) to aliquated sample 69 Shake sample for 20 min e300 rpm (Shaker o't( I . m- - 3/c,, ~ J ?76h/YJ'7-*cJ&d&?#?&Q wc"Lc#+G% 3-4.cCV4-H Id G4k3.L- 1 &A [J dgn4 Z 9 w C & & A). P.hA,-, E A - - h, - h f Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper,mixing Conditioncolumn with MeOH (TN-A- 6 OIR ) 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 e samples with internal standard PFDA standard number 4< 15 ml centrifugetube &bo(- sfer sample into appripriatelly marked autovial 8.\ q d 3M EnOviIrCo,nmental Laboratory --+= ,cone. b 7 ,amountadded Page 163 Report E01-1256 /m Prep Date: &$!5-0q Analysts initials: ot< 3 h3 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet @ Malbd- MS d 3 h 2 W aovz J Method Revision: ETS-8-231.O Study Number: E01-1256 Matrix: Sera I Lims Assgned sample number I Sample Number or description QC-250ppb -2 e01 -1 256-36255 e01 -1 256-36256 QC-XODDb -3 ~ ~_______ QC4ppm -1 e01 -1256-36257 QC4ppm -2 e01 -1 256-36258 E01-1256-28662 E01-1256-28663 E01-1256-28664 E01-1256-28665 E01-1256-28666 E01-1256-28667 E01-1256-28668 EO1-1256-28669 E01-1256-28670 QC4ppm -3 454-109-3217,OPPM, Male, Adult 454-1093218,OPPM, Female, Adult 454-1093219,OPPM, Male, Adult 454-1093220,OPPM, Female, Adult 454-1093221,OPPM, Male, A. diiH 454-1093222,OPPM, Female, Adult 454-1093223,OPPM, Male, Adult 454-1093224,OPPM, Female. Adult 454-1093226,OPPM, Female, Adult E01-1256-28672 E01-1256-28673 E01 -1256-28674 EO1-1256-28675 EO1-1256-28676 . 454-1093228,OPPM, Female. Adult 454-1093229,OPPM, Male, Adult 454-1093230.0PPM, Female, Adult 454-109-3231.0PPM. Male. Adult 454-1093232,OPPM, Female, Adult Blank Sera TN-A- ; Amount of sera aliquated: ilute Sera with Kandiyohi water; Amount of water added 3 4 ml 6ml liquot Iml of sera into 15 ml polypropelenetube pike samples accordingly Add 5ml of ACN (TN-A- 414G ) to aliquated sample Shake sample for 20 min @300 rpm (ShakerM 04169b Centrifuge sample for IO min Q 2000 rpm ( ~ e n t r i f u g e 3 M L 7 6 ' F j Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water 2 5 ) shake sample slightly to ensure proper mixi Condition column with MeOH (TN-A- Go I 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 centrifugetube Spike samples with internal standard PFDA standard number &- 1- ' 5 ransfer sample into appripriatelly marked autovial CJL 3M Environmental Laboratory , conc. Page 164 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOSSa: I .00ppm: PFOS SM:503ppm: SorrStdApprm. 100.7ppm: FTOS std Conc. UghnL 1,004 1.004 I ,004 1 ,004 1 .ow 1.004 I ,004 1.004 1.w4 1.004 5020 50.20 50.20 50.20 50.20 50.20 50.20 50.20 1.004 1.004 1,004 Studvd Number m5m.Omm.m 7 m .m m OKMOJ Mnllard Scra ETS-8-231.1 PFOS M y Number: EOl-1256 EqnlpmrntNumber: NA FhulWvent & TN Numkr: McOWM-AM457 Blank TInowIdmUfkr: M.llud ScrpEo2-0241-36386 02001-66 02W1-53 02001-45 FTDA PFOS h ' tSDkd mi 0.0004 0.0010 0.0020 0.0050 O.Ol00 0.0200 O.MW 0.1000 0.1500 0.2000 O.Oo40 0.0100 0.0200 o.mm 0.04oO 0.00lO O.Ol00 0.1m 0.0500 0.0500 0.0500 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 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 All ~ Initial Dilution InLJmL ' 0.0soo 0.0500 0.0500 0.0500 0.0500 0.MM) 0.0500 0.0500 0.0500 0.0500 OM00 0.M00 0.0500 0.0500 0.0500 0.0500 0.om 0.0500 OM00 OM00 O.05M 0.0500 Au Pa i Vol. 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 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 Blank sera lnttipl dilutim(-L) for curvu: Finsl rolvent volume (nL)for curves: 0.0500 2.000 PFOS stdcoac. PFDA stdconc. EOl-I256-36238,2.5ppb EOl-l256-36239.5.0ppb EOI-125636240,IOppb 1-1256-36242.50ppb 3-325636243.75 ppb 1-1256-36244l,00ppb l-1256-36245,l0&2ppb 1-1256-36246,250ppb MI-1256-36247.500ppb cnlcuwconeenmtiom of SIaJhdSin tbe final eolvent EOI-1256-36239.5.0ppb EOl-1256-36240,lOppb EOI-1256-36241.25 ppb EOI-125636242.50ppb EOI-1256-36243.75ppb EOI-125636244,I00ppb ~1-1256-36245,100p-p2b EOI-1256-36246.250ppb EOI-1256-36247.500ppb . .. QC-4 ppm Mallard Sera MSIMSD-Day I MallardSeraMWSD-Day 2 Mallard Sera MSIMSD-Dav 3 4016 101 25.1 101 25.1 101 25.1 101 3M Environmental Laboratory Page 165 Report E01-1256 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: m #: V W R ,5mL 25 Febuary 2002 hojlok ZSJW ~799 z @ ' * ~ 0 2 5 Acetonitrile tn-a-4145 (gm Solvent 1 3.9739 0.7857 5.0578 2 3.9683 0.7857 5.0507 3 - 3.9559 0.7857 5.0349 4 3.9767 0.7857 5.0613 5 3.9718 0.7857 5.055 1 PASS PASS Volume, 1 to 10 Accuracy, (%) 98.0 - 102 % Corrective Actions: C.V., (%) f 1.0 % 'Density values taken from the Merck Index, 12* Edition, Copyright 19% d', =specific gravity at yde- referred to water at xden-Hexane d204=0.660 Acetone dZs0=0.788 Methanol =0.7866 Methylene Chloride d204=1.3255 Acetonitrile dzo4= 0.7857 3M En2v/2ir5o/0n2m, e9n:t4a1l ALMaboratory Page 166 Report E01-1256 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet Prep Date: Analysts initials: Lims Assgned sample number 2/26/2002 OWHOJ Study Number: E01-1256 Sample Number or description Matrix: Sera I I I Volume of I I I I sample filtered Type of column Amount and spike Elution solven used and lot I mix used and volume Comments 3lank H20-day 2 IEOl-1256-36260 3lank mallard sera-dav 2 Aallard Sera MS-day 2 E01-1256-28680 IEOl-1256-28681 E01-1256-28682 lEO1-1256-28685 lEO1-1256-28686 IEOl-1256-28687 lEO1-1256-28688 - E01 1256-28689 Aallard Sera MSD-day 2 454-1093233,O PPM. Male, Adult 454-1093234,O PPM, Female, Adult 454-1093236,O PPM, Female, Adult 454-1093237,O PPM. Male, Adult 4561093238,O PPM, Female, Adult 454-1093239.0 PPM, Male. Adult 454-1093240,O PPM, Female, Adult 454-1093241,O PPM, Male, Adult 454-1093242,O PPM, Female, Adult 454-1093243.0 PPM. Male, Adult 454-1093244,O PPM, Female, Adult 454-1093245,O PPM, Male, Adult 454-1093246,O PPM, Female, Adult 454-1093247,O PPM, Male, Adult 454-1093248,O PPM, Female, Adult 454-1093249,O PPM, Male, Adult 454-1093250,O PPM, Female, Adult 434-1W-JLDT ,u-r Adult 40+6 Waters, lg, 6ml, lot W036B2 40+6 Waters, I g , 6ml, lot WZ036B2 40+6 Waters, Ig. 6ml, lo1 W036B2 lid 40+6 - II 40+6 Waters, lg, 6ml, W036B2 [Waters, lg, 6ml, lot W203682 40+6 lot W036B2 2ml of MeOH TN-AS457 2ml of MeOH TNA-5457 2ml of MeOH TN-A-5457 I 2ml of MeOH TN-A-5457 II TN-A-5457 ZmlofMeOH-- TNh-5457 Blank Sera T N - A - W X ;Amount of sera aliquated: Lt Dilute Sera with Kandiyohiwater; Amount of water added 3 6 liquot 1ml of sera into 15 ml polypropelenetube mi ml Spike samples accordingly Add 5ml of ACN (TN-A- 41 4 s ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker Centrifuge sample for 10 min Q 2000 rpm Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Conditioncolumn with MeOH ( T N - A - 6 0 1 U ) 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 centrifugetube Spike samples with internal standard PFDA standard number ransfer sample into appripriatelly marked autovial 199% 3M0%Environmental Laboratory @J 02mt-95 tm 9.9- , conc. boqTrvWam,ount added Page 167 Report E01-1256 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet i Prep Date: Analysts initials: Lims Assgned sample 2/26/2002 OWHOJ Sample Number or description volume or sample filtered (mi) Study Number: E01-1256 Matrix: Sera Type of column Amount and spike Elution solve used and lot mix used and volume Comments IE01-1256-28697 454-1093252,O PPM, Female, Adult 454-1093253,O PPM,Male, Adult 454-109-3254,O PPM, Female, Adult 40+6 40+6 40+6 Waters, lg, 6m1, lot W2036B2 Waters, Ig, 6ml, lot W036B2 Waters, lg, 6ml, lot W2036B2 uw 2ml of MeOH TNA-5457 2ml of MeOH TN-A-5457 II 2ml of MeOH TN-A-5457 454-1093255,O PPM, Male, Adult 40+6 Waters, Ig, 6ml, lot W2036B2 2ml of MeOH TN-A-5457 454-1093256.0 PPM. Female. Adult 40+6 Waters, Ig, 6ml. lot W 0 3 6 B 2 2ml of MeOH TNA-5457 E01-1256-28702 E01-1256-28703 454-1093257.10 PPM. Male, Waters, Ig, 6ml, Adult 40+6 lot W2036B2 454-1093258,lO PPM, Female, Adult I 454-1093259,lO PPM, Male, I Adult 454-1093260,lO PPM, 40+6 40+6 Waters, Ig, 6ml, lot WO36B2 Waters, Ig, 6ml, lot W2036B2 Female, Adult 40+6 lot W2036B2 2ml of MeOH TNA-5457 I 2ml of MeOH TN-A-5457 I 2ml of MeOH TN-A-5457 TNA-5457 IEOl-I256-28704 454-1093261,lO PPM, Male, Adult 40+6 Waters, lg, 6ml, lot W2036B2 2ml of MeOH TN-A-5457 4541093262.10 PPM. Female, Adult 40+6 Waters, lg. 6ml. lot W2036B2 2ml of MeOH TN-A-5457 E01-1256-28708 EO1 -1256-28709 454-1093263,lO PPM, Male, Adult 454-1093264,lO PPM, Female, Adult 4541093265,lO PPM,Male, Adult 454-1093266.10 PPM. Female, Adult Waters, lg, 6ml. ,% 1 I 40+6 lot W2036B2 Waters, Ig, 6ml, Waters, lg, 6ml, lot W2036B2 Waters, Ig, 6ml, 1lo1t_W0320&326B2 I I I I I 2ml of MeOH TNA-5457 12ml of MeOH TN-A-5457 2ml of MsOH N-A-5457 2ml of MeOH NA-5457 -- I I Blank Sera TN-A- &&& ; Amount of sera aliquated: 4 ml Dilute Sera with Kandiyohi water; Amount of water added 3 8 ml liquot 1ml of sera into 15 rnl polypropelenetube pike samples accordingly 195 dd 5ml of ACN (TN-A- 4 ) to aliquated sample ~ 4 Shakesamplefor20min Q300rpm ( S h a k e r w d \ q + \ 6 q 36 Centrifuge sample forOI min Q 2000 rpm (Centrifuge3 96 )? Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Conditioncolumn with MeOH (TN-A- 60A) ) 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 centrifu e tube ke samples with internal standard PFDA standard number 3M +EnsavmirpoleninmtoeanptparlipLriaateblloyrmaatrokerdyautovial Page 168 Report E01-1256 PFOS: A Reproduction Study with the Mallard BottleTop Dispenser Daily Calibration Check I 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 Volume, 1 to 10 Accuracy, 98.0 - 102 % C.V., (%) f 1.0 % PASS PASS 'Density values taken from the Merck Index, 12* Edition, Copyright 1996 dyx=specific gravity at ydm referred to water at xdem n-Hexane dZo4=0.660 Acetone dso =0.788 Methanol dZ4 =0.7866 Methylene Chloride dzo4=1.3255 Acetonitrile dZo4= 0.7857 3M En2v/i2ro6/n0m2e,n8t:a2l0ALMaboratory Page 169 Report E01-1256 ow Prep Date: $a?*o? , Analysts initials: Lims Assgned sample number Sample Number or description SPE Columns V- n-.l-u.r..n-a n-.f I sample filtered (mi) 3 Extraction PFOS: A Worksheet Reproduction @ fio\itc(.d. sS~tudy with the Mallard (AS OS)U)OS ppoc. \6PZ Method Revision: ETS-8-231.O Study Number: E01-1256 Matrix: Sera I I Type of column Amount and spike Elution solve1 used and lot mix used and volume Comments E01-1256-36264 E01-1256-36265 101-1256-28710 01-1256-2871I EO1-1256-28712 k01-1256-28713 NJA NA Hank H2O-day 3 b Hank mallard sera-day 3 40+6 40+6 Waters, Ig, 6ml, lot W036B2 Waters, Ig,6ml, lot W036B2 Waters, Ig, 6ml, 21-14 of MeOH TN-A-BO12 2ml of MeOH TNA-BO12 i 2ml of MeOH Aallard Sera MS-day 3 40+6 lot W036B2 50111of 0200146 TN-A-6012 Aallard Sera MSD-day 3 40+6 Waters, Ig,6ml, lot W036B2 2ml of MeOH 5 0 ~o1f 0200166 TN-A-BO12 1 454-109-3267,lO PPM, Male Adult 40+6 Waters, Ig,6ml, lot W036B2 UP 2ml of MeOH TNA-8012 454-1093268,lO PPM, Female, Adult 4541093269,lO PPM, Male Adult 40+6 40+6 Waters, Ig, 6ml, lot W036B2 Waters, Ig,6ml, lot W036B2 I 2ml of MeOH TN-A-8012 2ml of MeOH TNA-BO12 454-1093270,lO PPM, Female, Adult 454-1093271,lO PPM. Male Adult 40+6 40+6 Waters, Ig, 6ml, lot W036B2 Waters, Ig, 6ml, lot W036B2 I 2ml of MeOH TNASO12 2ml of MeOH TN-A-8012 4541093272,lO PPM, Waters, Ig,6ml. 01-1256-28715 Female, Adult 40+6 lot W036B2 454-1093273,lO PPM, Male aters, Ig, 6m1, EO1-1256-28716 Adult 454-1093274.10 PPM, Waters, Ig,6ml, PI-1256-28718 Female, Adult 454-1093275,lO PPM, Male Adult 454-1093276,lO PPM, Waters, lg. 6ml, Waters, Ig, 6ml. 01-1256-28719 Female. Adult 40+6 lot W036B2 454-1093277.10 PPM,Male Waters, Ig,6ml, Adult 40+6 lot W036B2 454-1093278,lO PPM, Waters, Ig, 6ml, [EO1-1256-28721 Female, Adult 40+6 lot W036B2 I E01-1256-28722 E01-1256-28723 454-1093279,lO PPM, Male Adult 454-1093280,lO PPM, Female, Adult 40+6 40+6 Waters, Ig, 6ml, lot W036B2 Waters, Ig,6ml, lot W036B2 454-109-3281,lO PPM, Male Waters, lg, 6ml. Adult 40+6 lot W036B2 2ml of MeOH TNA-BO12 I NA-8012 N-A-BO12 1 NA-6012 2ml of MeOH TNA-BO12 1 2ml of MeOH TN-AS012 2ml of MeOH TN-A4012 2ml of MeOH TN-AS012 I 2ml of MeOH TN-A-BO12 I 2ml of MeOH TNA-BO12 EO1-1256-28725 1.01-1256-28726 k01-1256-28727 lE01-1256-28728 454-1093282,lO PPM, Female. Adult 454-109-3283,lO PPM, Male Adult 454-1093284.10 PPM. Female. Adult Adult 40+6 Waters, Ig,6ml. lot W2036B2 40+6 Waters, Ig,6ml, lot W2036B2 - 40+6 Waters, Ig, 6ml, lot W036B2 4 0 4 lot W036B2 I 2ml of MeOH TN-A-BO12 2ml of MeOH TNA-BO12 2ml of MeOH v TNA-8012 I TN-A-8012 Blank Sera T N - A - b g k J ;Amount of sera aliquated: 4 ml Dilute Sera with Kandiyohiwater; Amount of water added 36 ml Spike samples accordingly Add 5ml of ACN (TN-A- 4 / 4 5 ) to sample for 20 min Q300 rpm (Shaker of Kandiyohi water to 50 ml polypropelene Decant extract into centrifuge tubes with water 80 12 slightly to ensure proper mixing Condition column with MeOH (TN-A- \ ) Wash Column with Kandiyohi Water conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent into appropriate 15 ml centrifugetube emA Spike sampleswith internal standard PFDA standard number 4$ 3M EnvironmenaptaprlipLriaatbelolyrmaatorkreyd autovial dk#*+9*q , conc. b0.w, amount added Page 170 Report E01-1256 SPE Columns Extraction PFOS: A Worksheet Reprodmuac^^ti^o^ nhS?tudy witOh tdhe~MJa1llaOrd z rc) Prep Date: 2.2%R Analysts initials: 0 I Lims Assgned sample number E01-1256-28729 Sample Number or description 454-1093286,lO PPM. Female, Adult 454-1093287,lO PPM, Male, Adult E01-1256-28731 4541093288,lO PPM, Female, Adult EOI -I256-28732 454-1093289,lO PPM, Male, Adult E01-1256-28733 I E01-1256-28734 454-1093290,lO PPM, Female, Adult 454-1093291,lO PPM, Male, Adult EO1-1256-28735 454-1093292.10 PPM. Female, Adult EO1-1256-28736 E01-1256-28737 EO1-1256-28738 I 7 - EO1-1256-28739 4541093293,lO PPM, Male, Adult 454-1093294,lO PPM, Female, Adult 454-1093295, 10 PPM, Male, Adult 454-1093296,lO PPM, Female, Adult Method Revision: ETS-8-231.O Study Number: E01-1256 Matrix: Sera 4 Blank Sera TN-A- ; Amount of sera aliquated: mi 36 Dilute Sera with Kandiyohiwater; Amount of water added ml rAliquot 1ml of sera into 15 ml polypropelenetube Spike samples accordingly 94Ic99 4 Add 5ml of ACN (TN-A- ) to aliquated sa ple yd Shake sample for 20 min Q300 rpm (Shakerdw(L Centrifugesample forOI min Q 2000 rpm (Centrifuge 5 H 764@ 3 B Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. v Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Conditioncolumn with MeOH (TN-A- 6 0 iZ ) 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 7-A `or0 ~.n*dA T3raMnsfeErnsavmirpolenimntoeanptparlipLriaateblloyrmaatrokerdyautovial (8 -+IOKQ.q.02 ,conc. b*@ ,amount & added Page 171 Report E01-1256 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# V W R ,5mL 27 Febuary 2002 ok 799 900 5 Acetonitrile tn-a-4145 OK) 1 2 3 4 5 Volume, (mu 1 to 10 Mass Of solvent (g) Density' (gW Volume of Solvent 3.9579 3.9883 3.9754 3.9804 3.9907 0.7857 0.7857 0.7857 0.7857 0.7857 AverageVolume: 5.0374 5.0761 5.0597 5.066 1 I 5.0792 5.0637 %Accuracy: 1012 7 PASS Std. Dev.: 0.01662 I %.. C.-V-..:-Pass/Fail:l 0.33 PASS PASS Accuracy, 98.0 - 102 % C.V., (%) f 1.0 % 1Density values taken from the Merck Index, 12" Edition, Copyright 1996 dyx=specific gravity at ydem referred to water at xden-Hexane dZo4=0.660 Acetone dso =0.788 Methanol ds4 =0.7866 Methylene Chloride azo4=1.3255 Acetonitrile d2'4 = 0.7857 3M Env2i/2ro7/n0m2,en7t:a0l8LAaMboratory Page 172 Report E01-1256 "4 n Prep Date: Ja'oo( Analysts initials: 0KJ SPE Columns Extraction WorPkFsOhSe:eAt Reproduction Study with the Mallard Method Revision: ETS-81231 Study Number: E01-1256 Matrix: Sera Blank Sera TN-A- 6022;Amount of sera aliquated: 4 m l Dilute Sera with Kandiyohi water; Amount of water a d d e d x m l "Aliquot l m l of sera into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min Q300 rpm (Shaker WVR SIN 041694) Centrifuge sample for 10 min Q 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. 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 into appropriate 15 ml centrifugetube 1 Spike samples with internal standard PFDA standard number PFDA , conc,l00.7ppm~, T3raMnsfEernsavmirpolenimntoeanptparlipLriaateblloyrmaatrokreyd autovial @&l--4r OK $-9-07 amount added-2ul- Page 173 Report E01-1256 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 #: V W R ,5mL I 28 Febuary 2002 Ok OtG 790909 o&%fQcJ 5 Acetonitrile tn-a-4 145 Volume, (mu 1 to 10 Accuracy, (%I 98.0 - 102 % C.V., (%) & 1.0 % PASS PASS 'Density values taken from the Merck Index, 12* Edition, Copyright 1996 dyx=specific gravity at ydm referred to water at xdem n-Hexane d204=0.660 Acetone dz0 =0.788 Methanol d254=0.7866 Methylene Chloride dm4=1.3255 Acetonitrile d204= 0.7857 3M En2v/i2ro8/n0m2e,8n:ta06l LAMaboratory Page 174 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet a Study: E01-1256 Dilution Date/Analyst: Box Number: go I- Q ~6 Solventfl" Number: MeOW5457 MI/ iZ5 T3A Extraction Date/Analyst: 22sd d a ? ' a ; Matrflimepoint: biAK-&SB Form Completion Verified By: & 6 ( 3M Environmental Laboratory Page 175 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study: E01-1256 - Dilution Date/Analyst: Box Number: &/ &SG Attachment D: Dilutions Summary Worksheet w - & / a a w SolventrTN Number: MeOW5457 Extraction Date/Analyst: J& Matrflimepoint: ICIALLA- x=* Form Completion Verified By: &J b (-62- 3M Environmental Laboratory Page 176 -- - ReportE01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet @OK Study: 601-1256 ~*'s*oL Dilution DateIAnalyst: 4.II@. /Q I.0 SolvenVTN Number: *&wps Extraction DateIAnalyst: Box Number: Q- Matriflimepoint: q#Lf+m Notes: 3M Environmental Laboratory Page 177 Report E01-1256 Report EOi-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard ATTACHMENET: STUDY PROTOCOL, PROTOCOLAMENDMENTASN, D DEVIATIONS 3M Environmental Laboratory 3M Environmental Laboratory Page 178 of 216 Page 178 Report E01-1256 PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL. LTD PROJECTNO. 454-109, EO 1- 1256 AMENDMENT TO STUDY PROTOCOL STUDYTITLE: PFOS: A REPRODUCTIONSTUDYWITH THEMALLARD PROTOCOLNO.: 454/120700/MR/SUB454 AMENDMENTNO.: 9 SPONSOR 3M Corporation PROJECTNO.: 454-109, EO1-1256 EFFECTNE DATE: January 24,2002 AMENDm This amendment includes an attachment(AttachmentA) which providesthe i n f o d o n necessafy to complete the analytical phase of this study. REASON: The protocol did not include information fixcompletingthe analyses requiredby the sponsor. AMENDImNT: Harold Johnson is the Principal Analytical Investigator REASON: The sponsorrequested that a principalanalyticalinvestigwr be addedtothe pratocol. AMENDMENT: The 3M Environmental LaboratoryProject IdentificationNumber tobe includedwith the study is E01-1256. REASON: The sponsor requested that the 3M Project Identification Number be included in the pratocol. / 3M Environmental Laboratory Page lof2 Page 179 o! Report E01-1256 01/24/02 17:5.2 FAX 410 822 0632 WILDLIFE PFOS: A Reproduction Study with the Mallard INTERNATIONAL @I002 JAN. 23. 2002 5:25PY ENVIRONMENTAL LAB 2 3E NO, 6293 P. 3 .- .- WILDLIFE INTERNATIONAL, LTD PROJECTNO.: 454469, EO1-1256 3M Environmental Laboratory PageZef2 Page 180 Report E01-1256 WILDLIFE INTERNATIONAL. LTD PFOS: A Reproduction Study with the Mallard PROJECT NO.:454-109, EO1- 1256 Sean P.Gallagha, Study Director Date Joann B. Beavers,Wildlife Interoational, LTD, Date LabOratolyManagrn& Harold0.Johnson.,Principal M y t i c a lJnvestigabr Date William K. Reagen, 3M EnvironmentalLaboratory, Date LaboratoryManagement Rochelle R Robideau, Spoasor's Representative Date 3M Environmental Laboratory hge2Of2 Page 181 Report E01-1256 PFOS: A Reproduction Study with the Mallard Protocol Amendment # 9 = Attachment A Wildlife InternationalLtd. # 454-109 STUDY TITLE PFOS: A Reproduction Study with the Mallard SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 ,PERFORMINGANALYTICAL LABORATORY 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. P d ,MN 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: EO1-1256 Wildlife International Ltd.: 454-109 3M Environmental Laboratory Page 182 Report E01-1256 PFOS: A Reproduction Study with the Mallard Sponsor Study Identification PFOS: A Reproduction Study with the Mallard 3M EnvironmentalLaboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 551333331 Principal Analytical rrrvestigator Study Locations ANALWEALTESW LAsoRAroRY ANALYTICATLESTING LABORATORY Harold Johnson 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 651-770-6479 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 Pmposed Study Timetable ANALYTICAELX P E R I M ~ ASLTM DATE ANALYTICAELXPERIMENTATL m w m DATE 01/24/02 03/24/02 3M Enimnmntal Labomtory 2 3M Environmental Laboratory Page 183 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1. ~LRRPOSE/OBJECTNES In the in-We phase of this study, mallards were regularly exposedto nominal doses of perfluorooctandonate, potassium salt (PFOS) in their feed in order to evaluatethe effects of dietary exposure upon adults over a 21-week period. The purpose of this analyticalphase of the study is to quantifjlPFOS in the liver and sera of both control and dosed mallards. The analyticalinformationmay be used to compare with toxicological/histopathologicalresults fiom the in-We portion ofthisbird study. The inWe portion of the study was completed at WdMe International,Ltd. in study 454-109. 2. REGULATORCYO-NCE This analyticalphase study will be conducted in accordancewith the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. QUAUTYASSURANCE The 3M EnvironmentalLaboratory Quality AssuranceUnit will audit analyticalphase study conduct, data, and the Einalreport to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures.The QualityAssurance Unit wiil report all findingsto the Sponsor Representative and the Study Director. 4. REFERENCSE~ T A N C E 4.1 Phydcal &%scriptJon-Perfluorooctanesulfbnate,potassium salt &omLot 217 is a white crystalline material. 4.2 PuMy and StabilityLThepurity of Lot #217 has been determined to be 86.9%. PFOS has been shown to be stable in methanol solutionsfor up to 6 months at ambient temperature in the validationreport fkom study PS013, "Method Validation and Instrument Detection Limit Detembation for LC/MS and GCMS Analysis of FC807 Compounds in Methanol."Refer to the Certificateof Analysis (stored in the Test, Control, and Reference database in the 3M Environmental Laboratory) for mer purity and stability idormation. 4.3 Stonage CondMm-The test article may be stored at ambient temperature or lower. 4.4 Reserve samples--A reserve sample of PFOS &om Lot #217 has been & o m at - 20C k5"C or colder and is kept in the 3M Envkonmentd Laboratory. 4.5 CAS Number-The CAS number is: 2795-39-3. 4.6 Molecular Wdgh+The molecular weight is 538 (CsFd3aJK). Note: The molecular weight of the anion (PFOS) is 499 (CSF~~SO~-). 3M EnvironmentalLaboratory 3 3M Environmental Laboratory Page 184 Report E01-1256 PFOS: A Reproduction Study with the Mallard 5. C O ~ O L M A T W C E S Types of control matrices and their source, physical description, storage requirements, and traceabilitynumbers will be recorded in the raw data and included in the h a l report. 6. TESTSYSTEM The test system is the mallard ( A n m p h t y r ~ h )T.he test system was dosed (at Wildlife InternationalLtd. during the in-We phase of this 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 v d c a t i o n validation study was performed by Wildlife International under study number 454C-110 (3M#U2723), "Analytical Method Verification for the Determination of PFOS in Avian Diet." Wildlife International verified the actualdosed food for this study and the results willbe contained in the in-life Wildlife phase report. Mallard serum and liver samples were collected by Wildlife International, Ltd. and sent to 3M EnvironmentalLaboratoq for analysis. 6.1 Justification of the Selection of the Test System Mallard duck represent wild bird populations and they are an EPA recommended species because they do well in a laboratory environment. The use of these matrices for the study is to determine ifPFOS was deposited in the liver and serum of mating adults that were fed PFOS. Furthermore, analysis of these matrices &om hatchlings will determine ifPFOS was passed on to mating adults' offspring. 7. SOURCE OFSWPLYFOR THE TEsTSYSTEII The birds were obtained by WiIdlife International, Ltd. See their protocol (study 454-109) for information about bird origination location. 8. SPECMENANSDAMPLERECBPTANDM m - Sera and liver were collected by Wildlife International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 21-weekendpoint sera and liver samples received by the 3M E n v k o n m d Laboratory were &om the followingbirds: 0 40 control adult birds (20 mating pairs) that ingested food containing 0 ppm PFOS 0 40experimental adult birds (20 mating pairs) that ingested food containing 10ppm PFOS 0 20 fkst generationofipring birds that ingested food containing0 ppm PFOS Samples willbe mahtainedfiozen except when removed for extraction and analysis as descriied in the method. The sampleswillbe kept isolated fiom the test substance during storage. 3M Environmntal Labomtoty 4 3M Environmental Laboratory Page 185 Report E01-1256 PFOS: A Reproduction Study with the Mallard 9. SUB-CONTRACTED 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 3MEnvironmentalLaboratory, Building 2-3&09,935 Bush Avenue, St. Paul, MN 55106. 10. EXPERME~ACLONDUCT The 21-week endpoint liver and sera samples received (see Section 8) will be extracted and analyzed for PFOS quantities using the methods listed below. 10.1 Preparatory Methods 10.1.1 ETS-8-6, Extraction of Fiuorwhemical Compounds&omLiver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.1.2 ETS-8-4, Extraction of Fluorwhemical Compounds &om Serum for Analysis Using HPLC-ElectrosprayMas Spectrometry 10.2 Analytical Methods 10.2.1 ETS-8-7, AnalysisofFluorochemicalsin Liver ExtractsUsing WLCElectrospray / Mass Spectrometry 10.2.2 ETS-8-5, Analysis of PotassiUm Perfluorooctandonate or Other Fluorochemicalsin Serum Extracts Using HPLC-Electrosprayhkiss Spectrometry 11. METHODVAL~DAT~ON Method validation for PFOS qmtitation in mallard sera and liver was pedormed at Centre Analytical Laboratories, Inc. in study 023-044.The method will be applied to sample analyses without additional validation. 72. DATAQUAUnOWECnvEs DWW@SAMPLEAMLYSCS The foIlowing criteria will be met using the validated calibration levels and fortiilcation levels determined in section 11: 12.1 System Suitability System suitabilitywill be determined prior to the start and at the completion of each analyticalrun. Prior to the calibration curve and aRer the last sample of the run three (3) mid-level unextractd calibration standardswillbe analyzed. The peak area precision and retention time precision and peak asymmetry will be monitored at the beginning and the end ofthe run separately. The peak area precision must be equal to or less than2.0%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 0.54J32.0, where AF is the asymmetry factor. If any item of the system suitabilityfails, system maintenance must be completed prior to running a 3M EnvironmentalLaboratory 5 3M Environmental Laboratory Page 186 Report E01-1256 PFOS: A Reproduction Study with the Mallard 12.2 12.3 second set of system suitability samples and the system suitability must pass before starting the calibration. Calibration It will consist of a minimum of nine (9) levels, including a blank with surrogate and a blank without surrogate, the lowest standard will be at approximately50% of the LLOQ and the highest standard will be approximately50% higher than the ULOQ. The standard curve equation will be determined by regression analysis using the peak areas of the analyte. The accuracy of each level will be verified. Any level outside 80% - 120% accuracy must be deactivated, and regression re-calculated. The curve must have a correlation of determination (?) greater than or equal to 0.985. All levels must show a response greater than two times that of the blank. The total number of standards excluded my not exceed 20%. If the preceding criterion are not met, the curye and sample set will be reanalyzed. Limits of Quantitation (LOQ) The lower limit of quantitation(LI,OQ) was determined to be 10ng/g in liver and 10 ng/mL serum during the method Validation as descriied above in section 11. The LLOQ may be determined to be higher than that found in the validation study during a screeningto be done for endogenous levels of PFOS in the blank matrices available at 3M EnvironmentalLaboratory. If endogenous levels of PFOS are found to be higher than the LLOQ or ifthe levels are found to be highly variable, the LLOQ willbe adjusted to accommodatethis. 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 fkal report. Recovery efficiency at the LLOQ will be determined during each run using an extracted quality control sample spiked at the UOQ level. Ifthe quality control sample at the LLOQ level fails during a run,the LLOQ for that specificrun would be considered to be the next lowest passing quality control sample and any samples below the new UOQ must be reanalyzed. The upper limit of quantitation(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 UU)Q will be used to measure recovery efEciency at the ULOQ. Ifthe quality control sample at the ULOQ fails during a run the ULOQ for that specificrun 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 of the highest acceptable standard will need to be diluted into the range of the &%ration curve. If samples are diluted into the range of the curve during analyses and enough sample remains, a post-run dilution validation willbe performed to v* sample values. To perform the dilution validation, one sample will be separated into two representative samples 3M EnvironmentalLaboratory 6 3M Environmental Laboratory Page 187 Report E01-1256 PFOS: A Reproduction Study with the Mallard (i.e. two 1 mL aliquots for fluid samples or two 1 gram amounts for tissue samples) then diluted using two procedures. The fist procedure consists of diluting the sample with additional matrix prior to extraction (sera adding sera), while the second procedure consists of diluting the extract with solvent postextraction (methanol extract adding additional methanol solvent). If the values are not within 15% of each other additional testing will be required to determine which value is a correct representation of the sample concentration. 12.4 Continuing Calibration Verifications An unextracted midlevel standard (continuing caliiration verification - CCV) will be analyzed after every 10 injections (starting &er the last injection of the calibration curve). The accuracy of the CCV must be within 20%. Data may only be reported ifbracketed by passing CCV's (or a CCV and the same level calibration curve point). Data bracketed by one or more fw CCV's must be reanalyzed. 1 2 s Quality Contrvl Samples A minimum of three (3) quality control samples (QC) will be prepared with each set ofsamples. They will consist ofblank matrix spiked with three levels of analyte. The levels are: 0 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 (25times the UOQ) 12.6 QC Perlbnnance Criteria Each QC is expected to show an accuracy of 8O-120% of expected. A minimum of 2/3 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). f2.7 Use of Conflnnatory Methodb C o b t o r y methods are typically not needed with LCIMSIMS analysis. 12.8 Demonstration of Speciticity PFOS specificity willbe substantiatedby chromatographic retention time,by the characteristicprimary ion (499),and the characteristicproduct ion (99) using LC/MS/MS. 13. SU~-CONXRACIEDAWYSB 13.1 All fluid and tissue extractions as detailed in this protocol amendment will be performed at Pace Tier 2,1700Elm Street, Suite 200,Minneapolis, MN 55414. 13.2 All fluid and tissue analyses as detailed in this protocol amendment will be performed at 3M EnvironmentalLaboratory, Building 2-3E-09,935Bush Avenue, St. Paul, MN 55106. 3M Environmental Laboratory Page 188 Report E01-1256 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-Ad&-a4 LaboratoryFacilities PSS-Admin-06 TrainingRecord Files Maintained by QAU PSS-ARC-02 Disposition of Archive Materials PSS-ARC-Q4 Archiving Data, Specimen and Logbooks PSS-Dc-05 Lab Notebooks, Logbooks, and Phone Logs PSS-DM-03 Statistical Evaluation of Data Pss-oPs-ol Use and Maintenance of Ultra-Tui~aT~25 Homogenizer PSS-OPS-02 Raw Data PSS-OPS-03 Hazardous Waste Disposal PSS-OPS-a4 Use and Maintenance of N-Evap Analytical Evaporator PSS-OPS-05 Use of Compressed Gases in the Laboratory PSS-OPS-06 Use and Maintenance of NANOpure 11Water Purification System PSs-OPS-07 CleaningNon-disposable VolumetricPipettes PSS-OPS-08 Laboratory Calculations PSS-OPS-09 Deviations fiom Established Procedures PSS-OPS-10 Analytical Dehitions PSS-OPS-11 Use and Calibration of a Bottle-Top Dispenser PSS-OPS- 13 Use and Maintenance of Lab Refigerators, Freezers, and Ovens PSS-OPS- 14 Use and Calibrationof Syringeswith a Hamilton Chaney Adapter PSS-OPS-15 Glassware Cleaning PSS-OPS-16 Use and Maintenance of the Fisher ScientificIsotemp Freezer PSS-OPS- 17 Use and Maintenance of Ultrasonic Water Baths (Sonicators) PSS-OPS-18 Use and Maintenance of Vortex Shakers PSS-OPS-24 Use and Maintenance of a Desiccator PSS-OPS-3 1 Use and Maintenance of Shakers PSS-OPS-32 Operation,Maintenanceand CalibrationofLaboratory PSs-OPS-37 Operation, Maintenance, and Calibration of pH Meters and pH Electrodes PSS-MC-0 1 Purchasing of Laboratory Supplies PSS-MC-02 Receipt of Laboratory Supplies P S S - m - 0 1 Calibrationof Certified Weight Set P S S - m - 0 2 Calibration of C d e d ThermometerdThermocouples PSs-m-05 Calibration of Eppendorf Pipettes PSS-MTR-06 Verification of Calibrationof the Eppeadorf Pipettor PSs - m - 0 7 New Equipment QualiEication(IQ, PQ, OQ) PSS-MTR-08 Verification of Calibration and Use of Analytical Balances P S S - m - 0 9 Use and Maintenance of Fisher ScientificElectronic Temperature Monitoring Devices PSS-Safety-01 G e n d Laboratory Safety PSS-safety-02 Use and Maintenance of Biological Safety Cabinets 3M Envillonmental Labomtory 8 3M Environmental Laboratory Page 189 Report E01-1256 PFOS: A Reproduction Study with the Mallard PSS-Safety-04 Emergency Evacuation Procedures PSS-Safety-05 Use and Maintenance of Laboratory Fume Hoods PSS-TS-01 Training Procedures 13.4 An amendment to the protocol willbe written if extractionsand analyses are perf`ormed at laboratories other than the Pace Tier 2 or 3M Environmental Laboratory, respectively. 14. S T A ~ n c A L A N A l Y S S Statistical methods will be limited to the calculation of means and standard deviations. Examples of the calculationsused in the analyseswill be included in the analyticalphase report. 1s. REPORT A finalreport will be prepared by the 3M EnvironmentalLaboratory. The h lreportwill follow all GLP requirements, and will include a descriptionof all materials and methods used, including a narrative discussionwith a tabularpresentation of all analysesresults. 16. L O C A ~OFNRAW DATA,RECORDS, AND FINAL REPORT When the fhal report is completed, all originalpaper data, including protocol, phase report, study correspondence, sample receipt and tracking, sample preparation, and analytical data, will be retained in the archives of 3M EnvironmentalLaboratory.All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained either at Pace Tier 2 or the 3M EnvironmentalLaboratory, as applicable. 17. SPECWENRETENITON Samples of the test substance (any leftom control sera or liver and samples) and the analyticalstandardswill be maintainedin the laboratory for a period of time as specified by regulation or as long as the quality of the preparation affordsevaluation.However, sampleswill not be maintained more than 10years aferthe effective date of the final test rule (if applicable). 3M Environmental Laboratory Standard Operating Procedures also apply to sample retention times. 18. 18.1 18.2 Wildlife International, Ltd. ProtocolNo.454-109, "PFOS: A Reproduction Study with the Mallard". Centre Analytical Laboratories, Inc. Study 023-044, "Validation of Analytical Methods "Extraction of Potassium Pduoroocatanesulfonateor Other Fluorochemical Compounds From Serum for Analysis Using HPLC Electrospry/MassSpectrometry"and "Extraction of Potassium Pduorooctanewlfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC Electrospmy/Mass Spectrometry" in Mallard Serum and Liver." 3M EnvironmentalLaboretory 9 3M Environmental Laboratory Page 190 Report E01-1256 PFOS: A Reproduction Study with the Mallard 18.3 3M Study U2723,Wildlife Study 454C-110, ``AnalyticalMethod Verification for the Determination of PFOS in Avian Diet." 79. PROmCOL AMENDMENEASND D ~ l ~ n o " Planned changesto the protocol will be in the form of written amendments signed by the Study Director and the Sponsor Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. Any other changeswill be in the form of written deviations, signed by the Study Director and SponsorRepresentativeand filed withthe raw data. All changesto the protocol will be indicated in the finalreport. 3M Envimnmenfal Laboratory 10 3M Environmental Laboratory Page 191 - - R0e1p/o3r0t/E0021-11285:628 FAX- . -410 822 0632 WILDLIFE INTERNATIOPNFAOL S: A Reproduction Study wait0h0t2he Mallard WILDLIFE INTERNATIONAL, LTD PROJECTNO.:454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDYTITLE: PFOS: A REPRODUCTION STUDY W1W "EMALLARD PROTOCOL NO.:454/120700/MWSUB454 AMENDhENTNO.: 10 SPONSOR 3M Corporation PROJECTNO.:454-109, EOI-I 256 EFFECTIVE DATE: January 30,2002 LlMEmMENT: This amendment includes an attachment (AttachmentA) which provides updated infonnation neccssaryto completethe analytical phase afthis study. This attachmentA rcplacesthe attachmentA in amcndment 9. REASON: The amendmmt 9 attachment A was updated to include a different extraction and analyt~caml ethod, to update the data quality objectivcs,and to c h g c the liver and sera cxtraction location. * \ - Joann E.BAvers, Wildlife International,LTD, Harold 0.Johnson,P b d p a l Analytical Investigator William R.Rcagen, 3M Environmental Laboratory, Date Date 3 4 0 -L 8 C 13102 DatC A 8,/3/ 3- Datc 3M Environmental Laboratory 1 of9 Page 192 Report E01-1256 WILDLIFE INTERNATIONAL, LTD PFOS: A Reproduction Study with the Mallard PROJECT NO.: 454-109, EO 1-1256 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, EO1-1256 EFFECTIVE DATE: January 30,2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase of this study. This attachment A replaces the attachment A in amendment 9. REASON: The amendment 9 attachment A was updated to include a different extractionand analyticalmethod, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director Date Joann B. Beavers, Wildlife International,LTD, Date Laboratory Management Harold 0.Johnson, Principal Analytical Investigator Date William K. Reagen, 3M Environmental Laboratory, Date Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date 3M Environmental Laboratory Io f 9 Page 193 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 with the Mallard SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: E01-1256 Wildlife International Ltd.: 454-109 3M Environmental Laboratory 3M Environmental Laboratory 2of9 Page 194 Report E01-1256 PFOS: A Reproduction Study with the Mallard Sponsor Study Identification PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 Principal Analytical Investigator Study Locations ANALYTICAL TESTING LABORATORY Harold Johnson 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 65 1-778-6479 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. PO Box 33331 St. Paul, MN 55133-3331 Proposed Study Timetable ANALYTICAL EXPERIMENTAL START DATE ANALYTlCAL EXPERIMENTAL TERMINATION DATE 0 1/31/02 03/3 1/02 3M Environmental Laboratory 3M Environmental Laboratory 3of9 Page 195 Report E01-1256 PFOS: A Reproduction Study with the Mallard I. PURPOSE/~BJECTIVES In the in-life phase of this study, mallards were regularly exposed to nominal doses of perfluorooctanesulfonate,potassium salt (PFOS) in their feed in order to evaluate the effects of dietary exposure upon adults over a 2 1-week period. The purpose of this analytical phase of the study is to quanti@ PFOS in the liver and sera of both control and dosed mallards. The analytical information may be used to compare with toxicologicaVhistopathologica1results fiom the in-life portion of this bird study. The inlife portion of the studywas completed at Wildlife International, Ltd. in study454-109. 2. REGULATORCYOMPLIANCE This analytical phase study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. QUALITAYSSURANCE 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 3M Environmental Laboratory Standard Operating Procedures. The Quality Assurance Unit will report all findings to the Sponsor Representative and the StudyDirector. 4. REFERENCSEUBSTANCE 4.1 Physical Description-Perfluorooctanesulfonate,potassium salt from Lot 217 is a white crystalline material. 4.2 Purify and Stability-The purity of Lot #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 PSO13, "Method Validation and Instrument Detection Limit Determination for L C N S and G C N S Analysis of FC807 Compounds in Methanol." Refer to the Certificate of Analysis (stored in the Test, Control, and Reference database in the 3M Environmental Laboratory) for further purity and stability information. 4.3 Storage CondifioncThe test article may be stored at ambient temperature or lower. 4.4 Reserve Samples-A reserve sample of PFOS from Lot #2 17 has been fiozen at - 20C *5"C 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 (CsF17S03K). Note: The molecular weight of the anion (PFOS) is 499 ( C ~ F I ~ S O ~ - ) . 3M Environmental Laboratory 3M Environmental Laboratory 4of9 Page 196 Report E01-1256 PFOS: A Reproduction Study with the Mallard 5. CONTROMLATRICES Types of control matrices and their source, physical description, storage requirements, and traceabilitynumbers will be recorded in the raw data and included in the final report. 6. TESTSYSTEM The test system is the mallard (Anasplutyrhynchos).The test system was dosed (at Wildlife International Ltd. during the in-life phase of this 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 of PFOS 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 3M Environmental Laboratory for analysis. 6.1 Justification of the Selection of the Test System Mallard duck represent wild bird populations and they are an EPA recommended species because they do well in a laboratory environment. The use of these matrices for the study is to determine if PFOS was deposited in the liver and serum of mating adults that were fed PFOS. Furthermore, analysis of these matrices from hatchlings will determine if PFOS was passed on to mating adults' offspring. 7. SOURCE OF SUPPLY FOR THE TESTSYSTEM The birds were obtained by Wildlife International, Ltd. See their protocol (study 454109) for information about bird origination location. 8. SPECIMEN AND SAMPLE RECEIPATND MAINTENANCE 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 3M Environmental 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 ppm PFOS 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 5of9 Page 197 - Report E01-1256 PFOS: A Reproduction Study with the Mallard 9. SUB-CONTRACTED ANALYSIS All sera and liver extractions and analyses as detailed in this protocol amendment will be performed at 3M Environmental Laboratory, Building 2-3E-09,935 Bush Avenue, St. Paul, MN 55106.55414. 70. EXPERIMENTCAOL NDUCT The 21-week endpoint liver and sera samples received (see section 8) will be extracted and analyzed for PFOS quantities using the methods listed below. 70.7 Preparatory and Analytical Method 70.7.47 ETS-8-231 Solid Phase Extraction and Analysis of Fluorochemical Compounds fiom Biological Matrices 77. DATAQUALITOYBJECTIVESDURINGSAMPLEANALYSIS Method validation for PFOS quantitation in rat and mouse sera and liver is in process in the 3M Environmental Laboratory study E01-1277. Limited Mallard control matrix preclude a cross validation, however, the E01-1277 study methods will be applied to sample analyses of Mallard sera and liver samples using the followingperformance based criteria: 7 7.7 System Suitability System suitabilitywill 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 unextracted calibration standards will be analyzed. The peak area precision and retention time precision will be monitored at the beginning and the end of the run separately. The peak area precision must be equal to or less than 2.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD. If any item of the system suitability fails, system maintenance must be completed prior to running a second set of system suitability samples and the system suitability must pass before starting the calibration. 77.2 Absolute Recovery The absolute recovery of the method will be evaluated one time in both liver and serum. For each matrix a minimum of triplicate samples will be fortified at a minimum of two levels of PFOS that bracket the range of analyte determined for the samples @e., at near the highest level determined in samples and at -150% of LOQ for samples). The samples will be extracted through the method, and analyzed by comparison with an external calibration of non-extracted standards. The non-extracted standard calibration curves will be prepared in methanol, and will consist of a minimum of nine (9) levels, including a methanol blank. The best appropriate regression will be used to describe this curve (for best accuracy at all levels of the standards). 3M Environmental Laboratory 3M Environmental Laboratory 6of9 Page 198 Report E01-1256 PFOS: A Reproduction Study with the Mallard The accuracy (% absolute recovery) and precision (%CV of the recoveries) will be determined at each level using the external calibration curve for quantitation. There is no control limit for accuracy; precision must be better than 20% at each level. Should a level (high or low) not meet this precision, the method will only be considered valid for levels between the two valid levels. Additional testing may be conducted at other levels to extend the acceptable range of the method. 11.3 Calibration A calibration curve should be prepared in each matrix by spiking the matrix with known concentrations of the analyte and surrogate. A calibration curve should consist of at least nine standard curve points covering the expected range, including the LLOQ. The simplest model that adequately describes the concentration-responserelationship should be used. The accuracy of each level will be verified. Any level outside 75% - 125% of nominal must be deactivated, and regression re-calculated, except the LLOQ which must be within 30% of nominal. All levels must show a response greater than twice that of the matrix blank. A maximum of four (4) levels may be deactivated in any one set, or the set will be re-analyzed. 11.4 Limits of Quantitation (LOQ) The lower limit of quantitation (LLOQ) will be 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 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. The upper limit of quantitation (ULOQ) will be defined in liver and in serum as the highest acceptable extracted curve point that is within the 75% - 125% nominal criteria and includes applicable dilution factor corrections. 11.5 Quality Control Samples A minimum of one set of nine (9) quality control samples (QC)will be prepared one time in each matrix. They will consist of three levels of analyte, each in triplicate. The quality control samples should be prepared in each matrix by spiking the control matrix with known concentrations of the analyte. The levels bracket the sample concentrations determined in the study: 0 Low level: equivalent to an analyte concentration near or below the lowest level of analyte determined in samples 0 Mid-level: equivalent to an analyte concentration near the middle of the sample range 0 High level: equivalent to an analyte concentration near or above the highest level of analyte determined in samples 11.6 Quality Control Sample Acceptable Recoveries 3M Environmental Laboratory 3M Environmental Laboratory 7 of 9 Page 199 Report E01-1256 PFOS: A Reproduction Study with the Mallard Each QC is expected to show an accuracy of 75-125%of expected. A minimum of 213 (6 out of 9) of all QC must meet these criteria and a minimum of one half (2 out of 3) of the QC at each level must meet these criteria. If not, the entire sample set must either be re-analyzed or re-extracted (as decided by the Principal Analytical Investigator). 11.7 Matrix Spikes Two matrix spikes will be extracted with each sample preparation. The matrix spikes will consist of one fortification level in the range of the samples, extracted in duplicate. If the matrix spike and/or matrix spike duplicate fall outside of 25% accuracy, the curve, CCV's, matrix spikes and samples will be reanalyzed. 11.8 Blanks One control matrix and one water method blank will be extracted with each sample preparation. 11.9 Continuing Calibration Verification Two extracted standards (continuing calibration verification - CCV) will be analyzed after every 10 injections (starting after the last injection of the calibration curve). The accuracy of the CCV must be within 25%. 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 must be reanalyzed. I I.10 Use of ConfirmatoryMethods Confimatory methods are typically not needed with LC/MS/MS analysis. 1I .11 Demonstration of Specificity PFOS specificity will be substantiated by chromatographic retention time, by the characteristicprimary ion (499), and the characteristicproduct ion (99) using LC/MS/MS. 12. SUB-CONTRACTAENDALYSIS 12.1 All fluid and tissue extractions and analyses as detailed in this protocol amendment will be performed at the 3M Environmental Laboratory, Building 23E-09,935 Bush Avenue, St. Paul, MN 55106. 12.2 An amendment to the protocol will be written if extractions and analyses are performed at laboratories other than the 3M Environmental Laboratory. 13. STATISTICAALNALYSIS Statistical methods will be limited to the calculation of means and standard deviations. Examples of the calculations used in the analyses will be included in the analytical phase report. 3M Environmental Laboratory 3M Environmental Laboratory 8 of 9 Page 200 Report E01-1256 PFOS: A Reproduction Study with the Mallard 14. REPORT A final report will be prepared by the 3M Environmental Laboratory. The fmal report will follow all GLP requirements, and will include a description of all materials and methods used, including a narrative discussion with a tabular presentation of all analyses results. 15. LOCATIOONF RAw DATA,RECORDSA,ND FINALREPORT 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 of 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained at the 3M Environmental Laboratory, as applicable. 16. SPECIMEN RETENTION Samples of the test substance (any leftover control sera or liver and samples) and the analytical standards will be maintained in the laboratory for a period of time as specified by regulation or as long as the quality of the preparation affords evaluation. However, samples will not be maintained more than 10 years after the effective date of the final test rule (if applicable). 3M Environmental Laboratory Standard Operating Procedures also apply to sample retention times. 17. REFERENCES 18.1 Wildlife International, Ltd. Protocol No. 454-109, "PFOS: A Reproduction Study with the Mallard". 18.2 3M Study U2723, Wildlife Study 454C-110, "Analytical Method Verification for the Determination of PFOS in Avian Diet." 18. PROTOCOL AMENDMENTASND DEVIATIONS Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. Any other changes will be in the form of written 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 9 of 9 Page 201 Report E01-1256 PFOS: A Reproduction Study with the Mallard WILDLIFIENTERNATIONLATDL, - PROJECTNO.: 454-109, EO 1 1256 AMENDMENT TO STUDY PROTOCOL STUDYTlTLE PFOS:A REPRODUCTION STUDYWITH THEMALLARD PROTOCOLNO.: 454/1207WQR/SUB454 AMENDMENTNO.: 11 SPONSOR 3M Corporaton PROJECI"Od: 454-109, Eo1-1256 EFFECTIVE DATE. March 27,2002 AMENDMI3NT: Section 11.1, S-4 Change: Thepeak areaprecisionmust be equal to or lessthan2.0%RSD,the precision of the retentiontime must be equal to or less than 2.5% RSD. To:Thepeak areaprecisian mustbe qual to or less than 5.0% RSD,theprecision of the retentiontime must be equalto or lessthan 2.5% RSD. REASON: M o d o n received from our instrument supplier indicatesthat a 2.0 %RSD for the area count is common to W detector specifications. Mass spectrometers, which we used for this study, are more sensitive, in general and the &that analyte ionization can be suppressed by other species present in the sample tnatrix and mobile phase makearea reproducibility morechallengingto maintain.Sampleand soufce cleaalhessarethe determ*m*ng hctors, but 5 2.0% is stilla bit aggressivefor mass spec.The applicationchemists at Micromass, the instnunentvendor, suggest a value of 2 5.0%. AMENDMENT: W u n 11.3, Sentence5. - Change: Any level outside75% 125%of nominal value mustbe deactivated, and regremion recalculated,exceptthe LLOQ which mustbe within 30%of nominal. To:In somecasesthe lowest curve point, althoughoutsidethe 30%nominal requirement,may be included in the linear m g e to influencethe lower end ofthe curve.Oniyonepoint, the lowest level point, can be left in for this purpose. It will not be used to determinethe limit of quantitation.Thelimit of guantitatcm will be the next point on the cuve meetingthe 30% nominal requirement. REASON: The removalofthe lowestcalibrationcurye point in somecasesc~usesthe next lowest calibrationpointtodeviatebygreaterthan30%. Removalofthispointinturncauses the next calibrationpoint to deviateby greater than 30%. This c o h d deletion of calibrationpoints in somecases could continueuntil there are very fewpoints 3M Environmental Laboratory Page 1 af3 Page 202 Report E01-1256 PFOS: A Reproduction Study with the Mallard remainingin the curve. The inclusion of a curvepoint with a deviation greater than 30% is used to influencethe lower end of thecwve and is not used for determination of the LOQ. AMENDMENT: Section 11.3, the last sentence. Change:A maximumof four (4) levels may be deactivatedin any one set, or the setwill be Uyzed. To:Thecalibrationcwve will contain aminimumof six (6) calibrationcurve points. REASON:In most cases the calibrationcurvw containgreabr tban nine (9) calibrationpoints. The large number of points inthe curve is usedto select a range representative to the samples. Limiting the removal of only four (4) points in some cases could cause a calibrationrange not representative of the samples. AMENDMENT: Section 11.3 Add! The coefficientof determinationfor the calibrationcurve should be 5 0.985. REASON:No mfficient of determination is included inthe protocol. It was inadvertently removed prior to printing of the f'mal copy. 3M Environmental Laboratory Page 2 of 3 Page 203 Report E01-1256 WILDLIFE INTERNATIONAL. LTD Sean P. Gallagher, Study Director PFOS: A Reproduction Study with the Mallard - PROJJXTNO.: 454-109, EO1 1256 Date A William K.*en, 3M Environmental Laboratory, 3M Environmental Laboratory Page 204 Report E01-1256 04/02/02 17:16 FAX 410 822 0632 PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL @0I04 84/81/82 18:29 FISD INF TECH 2-2E-01 + ~8416822e915 M.989 P884 i ! I. ? I 1. I I . .!; . . 3M Environmental Laboratory Pag83d3 I I Page 205 Report E01-1256 WILDLIFE INTERNATIONAL, LTD PFOS: A Reproduction Study with the Mallard PROJECTNO.: 454-109, EO 1- 1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENTNO.: 12 SPONSOR: 3M Corporation PROJECT NO.: 454-109, EO1-1256 EFFECTIVE DATE: August 16,2002 AMENDMENT: Change: The 3M Environmental Laboratory Project Identification Number (LIMS)to be included with the study is EO1-1256. 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, EO1-1326, and E01-1367. REASON: Mallard samples were received in the 3M Environmentalat several time points. Upon arrival, samples were assigned a LIMS tracking number. Not aware of the scope of this or any other particular study pertainingto these samples, they were checked in to different 3M LIMS projects. LIMS project numbers assigned to this study include the following: U2723, E01-1256, EO1-1326, and EO1-1367. Samples in each of these projects pertaining to this study were analyzed under one main study number EO1-1256. AMENDMENT: Change: Harold Johnson is the Principal Analytical Investigator. To: Lisa A. Stevenson is the Principal Analytical Investigator. REASON: To change role of the Principal Analytical Investigator. 3M Environmental Laboratory Page 1 of 2 Page 206 Report E01-1256 PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL, LTD Sean P. Gallagher, Study Direcfor 1% 1 Joann B. Beavels, Wildlife International, LTD, Laboratory Management J h Lisa A. Stevenson,Principal Analytical Investigator Laboratory Management Laboratory, PROJECTNO.: 454-109, EO 1-1256 Date Date lan /a Date Date 3M Environmental Laboratory Page 2 of 2 Page 207 Report E01-1256 PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-109, EO1-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WlTH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENT NO.: 13 SPONSOR: 3M Corporation PROJECT NO.: 454-109, E01-1256 EFFECTIVE DATE: September27,2002 AMENDMENT: Change: Rochelle R. Robideau is the SponsorRepresentative. To: Susan A. Beach is the Sponsor Representative. REASON: To change role of the SponsorRepresentative. Sean P. Gallagher, Study Director International, LTD, Laboratory Management dk A,S h s o h Lisa A. Stevenson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management A Susah A. Beach, Sponsor's Representative Date lo\,I G Date 09/ 2 ? h Date Date Date ' 3M Environmental Laboratory Page 1 of 1 Page 208 Report E01-1256 3M Confidential PFOS: A Reproduction Study with the Mallard Record of Deviation w Study I Project No. I EQ1-1256 I Deviationtype (Check one) gsop 0 Protocol I Document number ETS-4-4.Q 1. Identifieation 0 Method 0 Other: 0 Eq-ui-pment Procedure Date(s) of occurrence 2119/Q2,2125/Q2,2/26/02,2/27102,2/281Q2,2/221Q2, 314/Q2,3/5/02,3161Q2,3/7/Q2 11. Dwcdption Required procedure/process: ETS-4-4.QInternalchainof custody The internalchain of custody form to be used to track all movement of samples Within the lab. The form should be filed with study data or other relevant sample documents. Actual procedure/process: IInternalchains of custodIi-es-were- n.ot used to tra-ck__sam- I - p- les a_n"l d_l sl a_mple-extracts. Sincer-eceipIt s-ampleswere Storedin fieezerF1. After homogenizing liver and aliquatingsera samples were stored in FreezerF8 IUntil extractiondate. Samplesremained fiozenuntil extraction. 1 Deviationto SOPwritten. Ill. Actions Taken (suchas amendmentissued, SOP revision,etc.) hj?y*dQ*u Recorded by Authorized by " . - Date WoZ- Date Dir&: &p- Deviation No. 1 (assigned by Study Director or Project Lead at the end of study or project) 4\ Ob 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 I Study1Project No.EO1-1256 1. Identification Deviation Type (Check one) .ISOP 0 Method 0 Equipment Procedure 0 Protocol 0 Other: I 11" lll_l Document Number:ETS-8-23l.l-- - -- __- I Date(s) of occurrence2/25/02,2/i6/02,2/27/02,2/28/02, 3/1/02; 3/4/02,3/5/02,3/6/02,3/7/02,3/8/02, 11. Description: IRequired Procedure/process: Section 12.1,Calculations: those listed, they will be documented in the raw data. If other calculations are used than Actual Procedure/process:_ I lll_ ---I__ _ _ 1111 Standard E01-134236010, Prep sheet description "Curve Point - lOppb in liver", was prepped by s p i g g 20uL of O?OOl-66 (1.OOug/mL.)into 1mL of liver homogenate. This homogenate consists of 5g liver plus 45mL water. Since 1mL out of 5OmL homogenate is used to prep each extracted standard, 1/50of the 5g (0.lg) of liver is "spiked". After this homogenate is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... (20ul) (1.OOugPFOS/mLMeOH) (1.OmL/lOOOuL) (lOOOng/l.Oug) = 20ng PFOS onto SPE column 20ng PFOS-/ 2.OmL MeOH = long PFOS-/mL MeOH. (Since this standard is recorded as lOppb, the correct units are 10ngPFOS`/mL of MeOH.) These are therefore the units being measured and reported on the chromatographicsystem. Similarly,the following statement is true. (2Ong PFOS`/ 1. O m L homogenate) (1. O m L homogenate / 0.1g liver) = 200ng PFOS-/ g liver This relationship shows the factor of 20 used to "correct" the units when calculating the fmal result in ng/g fiom the chromatographicresult that is in ng/mL. Sample Calculation: "E01-1342-30348, Oppm a.i. 333 M liver" Chromatographicresult is 10.76ng/&, reported value is 208.4 ng/g. Since the 1.0333g initial sample weight of liver is homogenizedwith 9.0mL water, and 1mL (10% of total liver weighed, since a liver density of 1.OO 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 Samule Calculation: Sample "E01-1342-36017,OC@,l0~ubDb-Tl"h:is QC sample is prepared by adding 4uL of 02001-53 (5.020ug PFOS-/ mL MeOH) spiked into 1mL. liver homogenate. ((4ul) (S.OZOug/mL) (lmU1OOOuL) (100Onglug)) / (0.lg liver) = 200.8ng PFOS-/ g liver Using the earlier calculation, the analytical result of 13.59nglmL calculates to 271.8nglg liver. Since the spike level is 200.8ng PFOS-/ g liver, the recovery is (271.8/200.8)(100)=135.4%. ...u " ^ " _ _ . . . I. 3M Environmental Laboratoiy Form ETS-4-8.0 3M Environmental Laboratory . . -, . . Deviation No. x (assigned by Study Director or Project Lead at the end of study or project) Page 210 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this SOP deviation and a note to file. IV. Impact on Study/ Project There will be no impact on the study as this is for clarification purposes. Date 4 `P3-02- Date 3M Environmental Lhboratoly Form ETS-4-80 3M Environmental Laboratory Deviation No. a (assigned by Study Director or Project Lead at the end of study or project) Page 211 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation 1. Identification ............................................................................................................................................................... Study / Project No. E01-1256 . ~ ............ ~ ....... Deviation Type (Check one) 0 SOP 0 Method 0 Equipment Procedure d~rotocol Other: I................................................................ " _ ...... - ................. " -. Document Number: ETS-8-231.1 Date(s) of occurrence: Throughout the study I/. Description: Required Procedurdprocess: ............ " " " __ ~ ~ ~ ~ Section 11.3, Sentence 5 states: Any level outside 75% - 125% of nominal value must be deactivated, and regression recalculated.. . remaining on the curve. Therefore, calibration points above the apex were included in the ran e of the curve with no evaluation of percent deviation criteria. ---..-......." ~ ............ ~ . ............. -- .............. " " ...... ~ -."_ ..... ....... ..... ........ ..._"............................................................ ~ .." ... "" ~ " " . " ............ Ill. Actions Taken: b (such as amendment issued. SOP revision. etc. This deviation was written, a note to" file was written, and data p...o.....i...n. ts" which were above "t_h_ e ~ ~ next highest calibration point were reanalyzed. ' RecordedBy Lisa A. Stevenson d' A jb,,, I m e d By (StudyDirector /Project Lead) II Date 3M EnvironmentalLaboratory F o ET~S-4-8.0 3M Environmental Laboratory Deviation No. 3 (assigned by Study Director or Project Lead at the end of study or project) Page 212 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation 1. Identification Study / Project No. EO1-1256 Deviation Type 4 SOP 0 Method 0 Equipment Procedure (Check one) _ 0 Protocol 0 Other: ".-.._---___I" _l-..--.-.__..-"_..- ...I ...- .I...----___ -_. Document Number: ETS-8-231.1 i Date(s) of occurrence: Entire Study 11. Description: ............................. _ Required Procedurdprocess: Section"12.1, Calculations: If other calculation" sa....r.e used than those listed, they-w-il"l...b. e documented in the raw.._d."-a...t".a...l_. ~ _". Actual Procedurdprocess: Standard EO1-1256-36240,Prep sheet description"10 ppb sera curve point". was p- rep_ p_ ed by spiking4uL of 02001-52 (502Oug/mL) into 2 mLof pooled sera. This diluted sera consistsof 2mL sera plus 38mL water. Since 1mLout of 40mL diluted sera is used to prepare each extracted standard, 1/40of 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... (5.02Oug PFOS/mL MeOH) * (4ul) * (1.OmU1ooouL) * (lOoOng/l.Oug) = 2Ong PFOS- onto SPE column mn 2Ong PFOS / 2.omL MeOH = lOng PFOS/mL MeOH. (Since thisstandard is recorded as lOppb, the correct units are PFOS%nLMeOu.) These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (2Ong PFOS/ 1 . W diluted sera) * (40.OmL diluted sera/ 2.0mL initial sera) = 4OOng PFOSV mL Sera This relationship shows the factor of 40 (or 2.0mL MeOW 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 PFOSlmL MeOH. SamDle Calculation; "E01-1256-28701,454-109-3258.10ppm female, adult Chromatographic result is 340.3 ng/mL MeOH; reported value is 13612ng/mL Sera. Since the 1.0 mLinitial sample volume 01 sera is diluted with 3MmL water, and 1mLis used for the entire SPE procedure, the calculation is as follows: 19.0, or d;\di~f@ i IAs r\jaolol (340.3ng PFOS-/ 1.OmLMeOH) * ( 2 . W MeOW 0.05mL Sera) = 13612ngPFOS / mL Sera SamDl-e C Sample"Eol-1256-36253, QC@25Oppb-l":This QC sample is prepared by adding lOuL of 02oO1-53 (50.2Oug PFOS / mL MeOH) spiked into 1mL diluted sera (or 0.05 mL of initial sera). ((50.2Oup;lmL) * (1Oul) * (lmU1oooUL) * (lOoOng/ug)) / (0.05 mL Sera) = lo040 ng PFOS / mLSera Using the earlier calculation,the analytical result of 264.06 n g / d calculates to 10562.4 n g / d Sera. Since the spike level is 1oo4ong PFOS7 mL Sera, the recovery is (10562.4/10040)*(100)=105.2%. Ill. Actions Taken: ...._.._I . . (s.u. ch as amendment issued, _I_ ........._.........__I_.._-_...__.___-._...__._..___.-.I_.I._.I SOP revision, etc.) This deviation was written and a note to file was written. . .. Recorded By Lisa A. Stevenson .._........ . l.-.-........l. ..... Authorized By (StudyDirector /Project Lead) Date 08/16/02 0811L/Q .....".ll-_._.." Lnbo&,,y -1; 3M EnvironmentalLaboratory Form ETS-4-8.0 3M Environmental Laboratory &+Q/~L Deviation No. v (assignedby Study Director or Project Lead at the end of study or project) Page 213 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation ............................... ___............................................................. -. Study / Project No. EO1-1256 1. Identification ......... ..................................................................................................................................................................................... Deviation Type (Check one) 0 SOP 4 Method 0 Equipment Procedure 0 Protocol 0 Other: IDocument Number: ETS-8-231.1 1 Date(s) of occurrence: Throughout the study .................................................................................................. ................................. ............ 11. Description: ................ Required Procedurdprocess: -- Section 13.3 of the method states: Two thirds of all quality control samples and 112 of each quality control sample at each level are expected to show an accuracy of 75-125%. Actual Procedurdprocess: All 25 ppb mallard sera quality control sample recoveries were >125%. Ill. Actions Taken: SOP (such as amendment issued, revision, etc.) ............................... ~ - ~ ~ -- ........... ~ - " I . . . . This deviation was written, sera data reported at the low range of the curve may be biased high, and quality control data not within criteria are flagged in the raw data. Recorded By L i s a ~S.tevenson ds/Q. JL- ................................................. Date 08/26/02 2 8 lab 1 Authorized . By .........................................................................................~ .......................................................... (StudyDirector /Project Lead) -. ~ 1............. ~ . Date ~ . " ~ ". 3M EnvironmentalLaboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 5 (assignedby Study Director or Project Lead at the end of study or project) Page 214 Report E01-1256 PFOS: A Reproduction Study with the Mallard Recdrd of Deviation I Study I Project No. EO1-1256 1. Identification 4SOP 0 Method 0 Equipment Procedure -- (Check one) 0Protocol 0 Other: Document Number:ETS-8-23 1.1 Date(s) of occurrence2/25/02,2/26/O2, W27/02,2/28/02,3/1/02, 3/4/O2,3/5/O2,3/6/02,3/7/02,3/8/O2, II. Description: Required Procedurdprocess: Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. I - Actual Procedurdprocess: .................................................................. ................ . ................. ................ . This deviation supersedes deviation #2 explaining the liver calculation: .................................................. ..... .. " ... Standard EO1-1326-36788, Prep sheet description "Curve Point lOppb in liver", was prepped by spiking 2OuL of 02001-66 (1.OOuglmL) into 1 mL of liver homogenate. This homogenate consists of 5.0597g liver plus 45mL water. Since 1mLout 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.OOug PFOS-/mLMeOH) * (1.OmUl 000uL) * (lo00 ng/l .O ug) = 20.0 ng PFOS` onto SPE column 20 ng PFOSY2.0 mLMeOH = 10 ng PFOS-/mLMeOH. (Since this standard is recorded as lOppb, the correct units are 10ng 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) = 198ng PFOS7 g liver Samule Calculation: "EO1-1326-29916, lOppm a.i. 3257 M liver" Chromatographic 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 PFOS4 mLMeOH) * (2 mL MeOH / 0.105848 g liver) * 10 = 72077 ng PFOS / g liver Samole Calculation: Sample "EO1-1326-36798, OC@25DDb-1":This QC sample is prepared by adding 1uLof 02001-53 (50.2Oug PFOS-/ mL M&H) spiked into 1mL liver homogenate. ((luL) * (5.02OuglmL) * (lmU1000 uL) * (1OOOnglug))/ (0.101073g liver) = 497 ng PFOS` / g liver Using the earlier calculation, the analytical result of 38.87 nglmL calculates to 571 ng/g liver. Since the spike level is 497 ng -PFOS/ g liv~e"r, the recovery is (571/497) * (100) = 114.9%. ~ Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this SOP deviation. Form ETS-4-8.0 3M Environmental Laboratory (assigned by Study Director or Project Lead at the end of study or project) Page 215 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A ReproductionStudy with the Mallard ATTACHMENFT: EXAMPLCEALCULATIONS FORMULAUSED FOR SERA ANALYSES IN STUDY EO1-1256 AR (ng/mL MeOH) x DF x Dilution (diluted sera) = ng/mL Sera Calculation Used for E01-1256-28701,454-109-3258,10 ppm Female adult (initial volume = 0.5 mL) 340.3 ng/mL MeOH x 1 x 40 = 13600 ng/mL FORMULAUSED FOR LIVERANALYSESINSTUDY E01-1256 AR (ng/mL MeOH) x DF x Final Volume (mL) = nglg Liver amount of liver in diluted sample (9) Calculation Used for E01-1326-29925,lO ppm, a.i. 3258 F Liver (initial weight = 1.0034 g) 355.07ng/mLMeOH x 1 x 2.0mLMeOH = 7080nglgLiver 0.100306 g Liver AR = Analytical result from MassLynx summary DF = Dilution factor Dilution(di1uted sera) = Final volume(mL)/amount of sera in diluted sample(mL) Amount of sera in diluted sample(mL)= amount of sera used(mL)ltotalamount 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 mL)/totalamount of sera + water (10 mL) = 0.05 mL Dilution(di1utedsera)(mL) = final volume (2.0mL)lamount of sera in diluted sample (0.05 mL) = 40 Amount of liver in diluted sample(g) = amount of liver weighed (1.0034 gyamount of liver weighed (1.0034 g) +water added for homogenizing (9.00 mL) = 1.0034 g / 10.0034 g = 1.00306 g 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 216 of 216 Page 216