Document k96EXza3kjoxLabXnKLw6gJbn

Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study Title ANALYTICAL PHASE REPORTFOR PFOS: A REPRODUCTIOSNTUDYWITH THENORTHERBNOBWHITE Data Requirement FIFRA Subdivision E, Section 71-4 OECD Guideline 206 Author Lisa A. Stevenson Analytical Phase Initiation Date January 31,2002 Analytical Phase Completion Date April 18, 2003 Performing Laboratory 3M Environmental Laboratoly Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Environmental Laboratory Study # E01-1245 Wildlife International Ltd: 454-108 Total Number of Pages 226 No part of this report should be reproduced, except in full, without written approval of the 3M Environmental Laboratory. 3M Environmental Laboratory Page 1 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite GLP COMPLIANCSETATEMENT Study Title: Analytical Phase Report for PFOS: A Reproduction Study with The Northern Bobwhite Study Identification Number: E01-1245 This analytical phase was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards with the exceptions listed below: Exceptionsto GLP compliance: 0 Automated data collection systems are not validated. The hardcopy printouts are considered as the original raw data. 0 The stability of the reference substances and samples has not been determined. 0 Not all data generated were recorded directly or promptly, or initiated and dated on date of entry. Lisa A. Stevenson, Principal Analytical Investigator Date 3M Environmental Laboratory 3M Environmental Laboratory Page 2 of 226 Page 2 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite QUALITYASSURANCSETATEMENT Study Title: Analytical Phase Report for PFOS: A Reproduction Study with The Northern Bobwhite Study Identification Number: E01-1245 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. 10/08/01-10/09/01 02/01 I02 o41o31o2-o41o~1o2,o4t7i /02 05/20/02-05/21to2 Protocol In-phase Data Final Report 10/09/01 10/09/01 02/04/02 02/04/02 04/08/02, 05116/02 04/08/02, 05116/02 05121I02 05/21 I02 Quality Assurance Representaf6e Date 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 226 Page 3 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite TABLEOF CONTENTS GLP Compliance Statement ............................................................................................................... 2 Quality Assurance Statement ............................................................................................................. 3 List of Tables ...................................................................................................................................... 5 Study Information ............................................................................................................................... 6 Summary ............................................................................................................................................ 7 Purpose .............................................................................................................................................. 8 Reference Substances ....................................................................................................................... 8 Specimen Receipt and Storage.......................................................................................................... 9 Method Summary ............................................................................................................................... 9 PreparatoryMethod............................................................................................................................ 9 Sample Collection and Analysis ......................................................................................................... 10 Analytical Method ............................................................................................................................... 11 Analytical Results............................................................................................................................... 12 Data Summary.................................................................................................................................... 14 Statement of Conclusion .................................................................................................................... 23 Statistical Methods ............................................................................................................................. 25 Statement of Data Quality .................................................................................................................. 25 References......................................................................................................................................... 26 List of Attachments............................................................................................................................. 26 Signature Page................................................................................................................................... 27 Attachment A: Extraction and Analytical Method, ETS-8-231.l,"Solid Phase Extraction and Analysis of FluorochemicalCompounds from Biological Matrices"................................................... 28 Attachment B: Data Summary Tables ................................................................................................ 48 Attachment C: Sample Chromatograms, Calibration Information, and Instrument Information.........62 Attachment D: Sample PreparationSheets ....................................................................................... 155 Attachment E: Study Protocol, ProtocolAmendments, and Deviations............................................. 187 Attachment F: Example Calculations ................................................................................................. 226 3M Environmental Laboratory 3M Environmental Laboratory Page 4 of 226 Page 4 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite LISTOF TABLES Table 1. Summary Table. CBFl7S0; Concentration in Sera and Liver............................................. 7 Table 2. Summary Table. C8Fl7S03 Concentration in Sera and Liver with Outliers Removed .......8 Table 3. Reference Substances ........................................................................................................ 8 Table 4. SPE Extraction Summary.................................................................................................... 9 Table 5. Sample Analysis .................................................................................................................. 10 Table 6. Serum QC Summary Table................................................................................................. 13 Table 7. Serum Matrix Spike Summary Table .................................................................................. 13 Table 8. Liver QC Summary Table.................................................................................................... 13 Table 9. Liver Matrix Spike Summary Table ..................................................................................... 13 Table 10. C8F17S0, Data Summary. Female Quail Sera (Adult and Offspring)............................... 15 Table 11. C8Fl7S09 Data Summary. Male Quail Sera (Adult and Offspring)................................... 17 Table 12. C8F17S03D- ata Summary. Female Quail Liver (Adult and Offspring) .............................. 20 Table 13. C8Fl7S0i Data Summary. Male Quail Liver (Adult and Offspring)................................... 22 Table 14. Summary Table. C8Fl7S0; Concentration in Sera and Liver........................................... 24 Table 15. Summary Table. C8Fl7S0; Concentration in Sera and Liver with Outliers Removed .....25 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 226 Page 5 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite STUDYINFORMATION Study Director Sponsor Sean P. Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 Analytical Chemistry Laboratory 3M Environmental Technology and Safety Services 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55106 Susan A. Beach, Sponsor Representative 3M EnvironmentalTechnology and Safety Services (ET&SS) 3M Environmental Laboratory (3M Lab) Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Lisa Stevenson, Principal Analytical Investigator Study Personnel Sean P. Gallagher, Wildlife International LTD., Study Director Joann B. Beavers, Wildlife International LTD., Laboratory Management William K. Reagen, 3M Environmental Laboratory Management Susan A. Beach, Sponsor Representative Harold 0.Johnson, Ill, Principal Analytical Investigator Lisa A. Stevenson, Principal Analytical Investigator Kent R. Lindstrom, Senior Research Chemist Ognjenka Krupljanin, Analytical Chemist Marlene Heying, Analytical Chemist Experimental Dates Sample Analysis Initiation: 31 January 2002 Sample Analysis Completion: 15 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 pertainingto the analytical phase of this study, are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 6 of 226 Page 6 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite SUMMARY The concentration of the C8Fl7S0i anion of perfluorooctane sulfonate, potassium salt (PFOS, C8FI7SOiK+) was determined in the liver and sera of adult bobwhite quail and their offspring in both a control group and a 10 ppm, dosed group from Wildlife feeding study #454-108. The following tables summarize the C8Fl7S03 concentrations found in the liver and sera of study samples. These results only relate to the samples tested. Matrix-extracted PFOS standards were used to establish the HPLC-MS/MS instrument calibration curve for C8Fl7S0i. Concentrations of C8F17SOiare reported after correction for the purity and the potassium contribution to the mass of the PFOS reference substance. Table 1 summarizes all study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q-Test. Table 1. Summary Table, CSF~~SOCoSncentration in Sera and Liver 1 1 1 1 1 1 FemaleControl 17 Group NA <400ng/mL(16of 17) 20 <50.2ng/g' <50.2 ng/g (20 of 20)' 21 10 ng/mL (1 of 17) 1 Male 1Oppm Group 20 I 141000ng/mL I 94400-226000ng/rnL I 2o I 88500 ng/g* 50100 - 147000ng/g* Female Offspring 4 NA <400 ng/mL (4 of 4) 4 <20.0 ng/g" <20.0 ng/g (4 of 4)* Control Group 1 1 1 M10aplepmmsGprroinugp 5 12600ng/mL ~ 8220- 19200ng/mL ~ 5 7350ng/g* ~ 4940 - 13700 ng/g" 1 Female 7640-18500ng/mL 5 ~ 7180ng/g* ~ 4590 - 13900 ng/g* 1 Group For individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +I-30%. Sera QC samples met the acceptance criteria Liver QC samples did not meet the '/2 of QC for at the method. each level must be within 75% - 125% criteria for the method. All liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% - 125%. 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 226 Page 7 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 2. Summary Table, CaF17S03' Concentration in Sera and Liver with Outliers Removed Male Control Group 19" FemGalreoCuoontrol 16 19 <400 ng/mL <400 ng/mL (16 of 16) 20' <50.2 ng/g** <50.2 ng/g (19 of 19)** Male 1Oppm Group 20* 20* Female 1Oppm Group 17 8700 ng/mL 4710 - 14700ng/mL 20' MCoanletrOolffGsproriunpg 6' 6' FeCmonatleroOlGffsropuripng 4' 4' 1 5 * 1 1 MaleOffspring 5* lOppmGroup Female Offspring 1Oppm Group * I 4 5760 ng/g'* 4940 - 6520 ng/g** I 4 1 5490ng/g** 1 4590-7030ng/g** No outliers from this data set. Refer to Table 1 for results. For individualsample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. ** Liver QC samples did not meet the Yz of QC at each level must be within 75% - 125% criteria for the method. All liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% - 125%. PURPOSE The purpose of this analytical phase of the study is to quantify C8FI7SO9 in the liver and sera of both control and dosed Northern Bobwhite Quail and their offspring. REFERENCESUBSTANCES Table 3. Reference Substances Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Puritv 3M Internal 08/3112006 Frozen -20C +/- 10C SD-018, Lot # 217 White Crystalline Powder 86.9% Oakwood Products, Inc. 12/01/2010 Frozen -20C +/- 10C SD-036 White Solid 98% PFDA is nonadecafluorodecanoic acid, or Perfluorodecanoic acid, C10FI9O2H and is used solely to monitor for gross instrument failure and not as an internal standard for quantitation. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 8 of 226 Page 8 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite SPECIMEN RECEIPTAND STORAGE Northern Bobwhite serum and liver samples were obtained from the sacrifice of the test animals upon the conclusion of the in-life phase of the study (#454-108) conducted at Wildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. The specimens were received at 3M Environmental Laboratory on 09/05/01, 09/25/01, and 09/27/01; assigned a laboratory tracking (LIMS) number (E01-1245, E01-1342, and E01-1365, respectively), and were stored at -20C +/- 10C until extraction and analysis. METHODSUMMARY The determination of C8FI7SO9 concentration in the liver and sera was performed according to 3M Environmental Laboratory method ETS-8-231.1, "Solid Phase Extraction and Analysis of FluorochemicalCompounds from Biological Matrices". The sera samples were prepared by measuring a volume of serum and diluting with water in a 1/20 ratio. The liver samples were prepared by measuring a weight of liver and diluting with water to a 1/10 ratio prior to homogenizing. Five mL of acetonitrile was added to 1.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. Upon drying the column 2.0mL of methanol was passed through the column to elute the analyte. The samples were analyzed for C8FI7SO3- using HPLC-ES/MS/MS. PREPARATOMREYTHOD 0 ETS-8-231.1 "Solid Phase Extraction and Analysis of Fluorochemical Components from Biological matrices." This method describes the extraction of target analytes from biological matrices, such as sera and liver, using solid phase extraction (SPE). Sera samples were diluted with Kandiyohi (commercially bottled) water 1/20, before extraction. Liver samples were diluted 1/10, approximately l g of liver and 9ml of Kandiyohi water, and then homogenized. A 1.0 mL aliquot of the diluted sera or homogenate of liver was extracted by adding 5mL of acetonitrile, shaking for 20 minutes, and centrifuging for 10 minutes. The supernatant was transferred to a clean tube, diluted with 40 ml of Kandiyohi water, and filtered through a conditioned C18 (SPE) column. Analytes were eluted off the column, and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). Table 4. SPE Extraction Summary I 1 I Measure out volume of sera or weigh portion of liver I Dilute sera (1/20) or liver (1/10) with water or homogenize livedwater 4 Aliquot 1.Om1of diluted/homogenizedsample and add 5.0ml of ACN. Spike samples accordingly. Shake for 20 minutes, centrifuge for 10 minutes. 5 Dilute supematant with 40 ml of water I 6 1 Filter sample through conditioned SPE C18 column. I I 7 I Elute target analytes from the column with 2.0 mL methanol (MeOH). I 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 226 Page 9 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite SAMPLE COLLECTION 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 performed. On each day of extraction, a water blank, a sera or liver blank, and a matrix spike and matrix spike duplicate were prepared along with each set of samples. Frozen diluted serdhomogenized liver samples were allowed to thaw prior to transferring a representative sample for extraction. Samples were prepared and analyzed according to ETS-8-231.1. The following table describes the sample collection and preparation regimen. For clarity, a reference to "Day 1" refers only to the group of samples associated with the first batch of samples extracted. Table 5. Sample Analysis I 2 Preparation of method blanks and spikes, calibration curve, qc samples, and sera samples, control group, adults (day 1 sera samples), see 1 Attachment D for samdes Dretmed Analysis of day 1 sera samples I Preparation of method blanks and spikes, and sera samples, control and 10 ppm adults, control offspring, and IOppm offspring (day 2 sera Analysis of day 2 sera samples 31 January 2002 31 January2002 01 February2002 01 February 2002 Analysis of control, 10 ppm and offspring,from day 1 and dav 2 sera samDles 7 Weighing out of liver samples 8 Preparation of method blanks and spikes, calibration curve, qc samples, and samples, control group (day 1 liver samples), see Attachment D for samples prepped 9 Analysis of day 1 liver samples 10 Preparationof method blanks and spikes, and liver 1 samples, control group (day 2 liver samples), see Attachment D for samples prepped 11 Analysis of day 2 liver samples 12 Preparationof method blanks and spikes, and liver samples, 1Oppm group (day 3 liver samples) ,see Attachment D for samples prepped 13 Preparation of method blanks and qc samples at 5 and 200 m b 14 Analysis of qc samples 15 Analysis of day 3 liver samples 07,08 February 2002 11 February 2002 11 February 2002 12 February 2002 12 February 2002 13 February 2002 13 February 2002 13 February 2002 14 February 2002 3M Environmental Laboratoly 3M Environmental Laboratory Page 10 of 226 Page 10 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite samples, loppm group, control offspring, 1Oppm offspring (day 4 liver samples), see Attachment D for samples prepped 17 Analysis of day 4 liver samples 18 Re-analysis of day 3 liver samples 19 Re-analysis of QC samples 20 Re-analysisof QC samples 18 Februaly2002 01 March 2002 05 April 2002 10April 2002 ANALYTICALMETHOD 0 Samples were analyzed using HPLC-ES/MS/MS. C8Fl7S0; concentrations were determined using matrix-extracted calibration standards ranging in concentration from 0.25-1000 ng C8FI7SO3*/mL methanol. This result was then used to back calculate the ng C8FI7SO3-/mL sera or ng C8F17SO;/gram 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-EC/MS/MS analytical conditions are summarized as follows: 0 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 0.300mUmin pump flow rate. The HPLC gradient is listed below: 0 90 10 1 .o 90 10 5.5 5 95 I 7.5 I 5 95 181 90 10 I 10 I 90 I 10 I 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 c8F17sOi 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, FSO;. Similarly the PFDA transition from m/z 513 to m/z 299 was monitored. The collision gas, 95% Argon / 5% Methane, was maintainedat a pressure of approximately 3 . 0 ~ 1 0m~Bar. 3M Environmental Laboratory 3M Environmental Laboratory Page 11 of 226 Page 11 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ANALYTICALRESULTS 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 FluorochemicalCompounds from Biological Matrices" and Attachment E, Study Protocol, Protocol Amendments, and Deviations). 0 Regressions. Quadratic curve fit, weighted l/x, was applied to calibration standards and sample data. Each calibration curve had a coefficient of determinationof 2 0.985. Calibration Standards. Instrument response was calibrated daily prior to sample analysis using matrix-extractedstandards ranging in concentration in the final solvent extract from 0.20 to 1000 ng C8Fl7S09/mL methanol, correspondingto a concentration of 201 to 40160 ng/mL of sera or 3.99 to 19953 ng/g liver. The calibration curve analyzed consisted of nine sera-extracted standards and fourteen liver-extracted standards bracketing a wide concentration of analyte. The accuracy of each level was evaluated. Any level outside 75%125% of nominal was deactivated with the exception of the lowest curve point, which needed to meet 70%-130% of nominal. In some cases the lowest concentration curve point, although outside the 30% nominal requirement, was included when constructing the calibration curve, but it was not used to determine the limit of quantitation. The limit of quantitation was then the next point on the curve meeting the 30% nominal requirement. Due to the quadratic response of matrix-extractedstandards using LC/MS/MS instrumentation, the calculated concentration of higher-levelstandards was often indeterminate, 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 matrixextracted standards. All analytical results that are reported are within the accepted calibration range of the instrument. System Suitability Samples. System suitability was demonstrated prior to the start and at the end of each analytical run. Prior to the calibration curve and after the last sample, three mid-level unextracted calibration standards were analyzed. The peak area precision and retention time precision was monitored at the beginning and end of the run separately. The peak area precision was equal to or less than 5.0% RSD, the precision of the retention time was equal to or less than 2.5% RSD, meeting the stated quality objectives to show that the instrumentationwas 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 d a t a bracketed by passing CCV's were reported. Samples bracketed by one or more failing CCV's were reanalyzed. Limit of Quantitation (LOQ). The lower limit of quantitation (LLOQ) was defined in liver (ng/g) and in serum (ng/mL) as the lowest acceptable extracted calibration curve point that is within the 70-130% nominal criteria and with an analyte peak area at least 2 times the blank. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest acceptable curve point used to calibrate the response of the instrument. Blanks. Method blanks (purchased Kandiyohi drinking water, sera and/or liver taken through the entire sample preparation and analysis process) provided a measure of background contamination. Quality Control (QC) and Matrix Spikes. One set of nine QC samples in each matrix was extracted and analyzed and consisted of three levels of analyte in triplicate. Additionally, two matrix spike samples at the lowest concentration were prepared with each extraction set. Results are as follows: 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 12 of 226 Page 12 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 1 Table 6. Serum QC Summary Table I MS-~DD~* 75.2% I 3.0% I I I I I I QC-25ppb* I I I I -0Ppb 1000ng/mL 8030nglmL 115% 90.5% 8.3% 2.5% I I I I I QC-40ppm I I I I I QC-80ppm* 1610000 ng1mL 3230000nglmL 76.2% 114% 2.8% 5.7% Sera samples labeled as MS and QC were prepared following the same procedure on a different day and at different concentrations. These MS samples were inadvertently not labeled as QC. `QC samples were reanalyzed to confirm original results. Data from reanalysis were not included in calculating these results. [Table 7. Serum Matrix Spike Summary Table 1 Three Matrix Spikes 1 10000 ng/mL I Three Matrix Spikes Duplicates 10000 ng1mL 93.9% 91.8% I Table 8. Liver QC Summary Table I QC-1Oppb* ~~ QC-75ppb* 200 nglg 1500 nglg 138% I 2.9% I 115% 2.9% QC-l50ppb* - 2990 nglg 104% 2.0% *QC samples were reanalyzed to confirm original results. Data from reanalysis were not included in calculating these results. =Liver Matrix Spike Summary Table Four Matrix Spikes Four Matrix Spikes Duplicates 499 ng/g I 499 nglg 110% 112% The protocol and method state that 213 of all QC and a minimum of YZ of QC at each level must be within 75% - 125% criteria. All three of the liver "QC-1Oppb" spikes exceeded the stated criteria, therefore the liver data reported in the low range of the curve (Day 0 samples) 3M Environmental Laboratoly 3M Environmental Laboratory Page 13 of 226 Page 13 Report E01-1245 Report E01-1 245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite may be biased high. Since all study samples that were not "Day 0" show analyte levels significantly above the low range of the curve, the overall impact (or bias) on data is minimal or none at all. 0 Absolute Recoveries. The absolute recovery of the QC samples is determined by comparing the results of the extracted quality control spikes quantitated versus an matrixextracted 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 37.8% higher, the medium spikes 31.7% higher, and the high spikes 28.6% higher. The 32.5% average higher quantitation of the three levels of spikes using a solvent curve versus a matrix-extracted curve demonstrates that it is appropriate to use matrix-extractedstandards. The same trend is observed for liver samples. The liver spikes quantitate at 4.8% lower, 6.4% higher, and 8.4% higher respectively for the low, medium and high spikes of the liver matrix with an average of 3.0% higher using a solvent curve versus an extracted liver matrix curve, again demonstratingthat it is appropriate to use matrix-extractedstandards. 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. 0 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 tables display C8FI7SO9 concentrations for individual injections. All data are corrected for purity of the PFOS reference substance used for the matrix-extracted calibration curve. All results relate only to the samples tested. 3M Environmental Laboratory 3M Environmental Laboratory Page 14 of 226 Page 14 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 10. CsF17SO; Data Summary, Female Quail Sera (Adult and Offspring) E01-1245-28444 454-108-330, Oppm female, adult, 01-31-02 E01-1245-28446 454-108-332, Oppm female, adult, 01-31-02 I 0.4 0.4 <400 <400 E01-1245-28449 454-108-336, Oppm female, adult, 01-31-02 E01-1245-28451454-108-338, Oppm female, adult, 01-31-02 E01-1245-28453 454-108-340, Oppm female, adult, 01-31-02 E01-1245-28456 454-108-344, Oppm female, adult, 01-31-02 E01-1245-28458 454-108-346, Oppm female, adult, 02-01-02 I E01-1245-28460 454-108-348, Oppm female, adult, 02-01-02 E01-1245-28462 454-108-350, Oppm female, adult, 02-01-02 E01-1245-28464 454-108-352, Oppm female, adult, 02-01-02 E01-1245-28466 454-108-354, Oppm female, adult, 02-01-02 E01-1245-28468 454-108-356, Oppm female, adult, 02-01-02 EOI-1245-28470 454-108-358, Oppm female, adult, 02-01-02 E01-1245-28472 454-108-360, Oppm female, adult, 02-01-02 0.3 1 <400 <400 0.3 1 <400 0.3 1 <400 <400 0.3 1 <400 0.2 1 <400 <400 0.2 1 <400 0.2 0.2 I I 1I I I O0..1l I I 0.3 0.3 1 1 1 1 I 1 1 <400 <400 <400 <400 <400 <400 <400 <400 1 <400 1 0.4 1 <400 <400 0.4 1 <400 1 c400 <400 I I I 0.44 1 <400 I 0.3 I 0.3 1 0.3 1 <400 <400 0.3 1 <400 0.3 , 0.3 ~ 0.3 1 1 I c400 <400 2050 2180 <400 I 2110. E01-1245-28474 454-108-362, Oppm female, adult, 02-01-02 0.2 1 <400 <400 0.2 1 <400 E01-1245-28477 454-108-366, Oppm female, adult, 02-01-02 0.2 1 <400 <400 0.2 1 <400 E01-1245-28479 454-108-368, Oppm female, adult, 02-01-02 0.3 0.3 1 1 1 <400 <400 <400 E01-1245-284811454-108-370, 1Oppm female, adult, 02-01-02 0.3 I 9170 EO1-1245-284831454-108-372.lOmm female. adult. 02-01-02 0.4 1 9140 E01-1245-28485 454-108-374, 1Oppm female, adult, 02-01-02 0.2 1 8400 E01-1245-28487 454-108-376, 1ODum female, adult, 02-01-02 0.3 1 9280 E01-1245-284891454-108-378.lOmm female. adult. 02-01-02 1 0.1 I 1 I 4980 I E0l-1245-284911454-108-380,10ppm female, adult, 02-01-02 0.1 1 5790 3M Environmental Laboratory Page 15 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite * E01-1245-28518 454-108-246, Oppm female, offspring, 02-01-02 0.1 1 <400 <400 0.1 1 <400 I* E01-1245-28521 454-108-259, Oppm female, offspring,02-01-02 ! 0.1 EO,-1245-285241454-1 08-273, Oppm female, offspring,02-01-02 0.08 0.08 c400 <400 <400 I <400 <400 I* I I I E01-1245-28525!454-108-2E',Oppmfemale, offspring,02-01-02 o0.2 1 <400 I I I * E01-1245-28528 454-108-517, 1Oppmfemale, offspring,02-01-02 18500 16600 17600 E01-1245-28530 454-108-525, 10ppmfemale, offspring,02-01-02 0.03 1 12800 12500 0.03 1 12100 E01-1245-28532 454-108-538, 1Oppmfemale, offspring,02-01-02 0.08 1 14600 13400 0.08 1 12200 E01-1245-28535 454-108-557, 10ppmfemale, offspring,02-01-02 0.06 1 0.06 1 8330 7460 7900 0.05 1 11500 E01-1245-28536 454-108-561, 10ppmfemale, offspring,02-01-02 10800 0.05 1 10100 * There are two results reported for these samples. Both values are included and averaged in the "Average of Dup Sample" column. These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 16 of 226 Page 16 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 11. CsF17SOBData Summary, Male Quail Sera (Adult and Offspring) I E01-1245-28443 r454-108-329, Oppm male, adult, 01-31-02 * E01-1245-28445 454-108-331, Oppm male, adult, 01-31-02 E01-1245-28447 454-108-333, Oppm male, adult, 01-31-02 E01-1245-28448 454-108-335, Oppm male, adult, 01-31-02 E01-1245-28450 454-108-337, Oppm male, adult, 01-31-02 E01-1245-28452 454-108-339, Oppm male, adult, 01-31-02 * E01-1245-28454 454-108-341, Oppm male, adult, 01-31-02 E01-1245-28455 454-108-343, Oppm male, adult, 01-31-02 E01-1245-28457 454-108-345, Oppm male, adult, 01-31-02 I E01-1245-28459 454-108-347, Oppm male, adult, 02-01-02 , E01-1245-28461 454-108-349, Oppm male, adult, 02-01-02 E01-1245-28465 454-108-353, Oppm male, adult, 02-01-02 * E01-1245-28467 454-108-355, Oppm male, adult, 02-01-02 * E01-1245-28469 454-108-357, Oppm male, adult, 02-01-02 E01-1245-28471 454-108-359, Oppm male, adult, 02-01-02 1, E01-1245-28473 454-108-361, Oppm male, adult, 02-01-02 I* E01-1245-28475 1454-108-363, Oppm male, adult, 02-01-02 * E01-1245-28476 454-108-365, Oppm male, adult, 02-01-02 E01-1245-28478 454-108-367, Oppm male, adult, 02-01-02 0.4 1 0.4 1 0.4 1 0.4 1 0.2 1 0.2 1 0.1 1 0.1 1 0.2 1 0.2 1 0.2 1 0.2 1 0.1 1 0.1 1 0.2 1 0.2 1 I 0.2 ' 0.02 1 0.02 1 0.3 1 0.3 1 0.2 1 0.2 1 0.07 1 0.07 1 0.2 1 0.2 1 i 0.2 I 0.1 0.4 I 1 0.4 1 0.1 1 0.1 1 <400 <400 <400 c400 <400 <400 <400 e400 c400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 c400 <400 c400 <400 c400 <400 <400 <400 ~400 <400 <400 <400 <400 c400 <400 <400 c400 <400 1400 c400 <400 c400 .e400 <400 <400 3M Environmental Laboratory 3M Environmental Laboratory Page 17 of 226 Page 17 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite I E01-1245-28480 1454-108-369, 1Oppm male, adult, d l 00,02-01-02 I o0.22 I I l10o 0 o 107000 117000 I 112000 0.2 E01-1245-28482 454-108-371, 1Oppm male, adult, d l 00,02-01-02 0.2 100 181000 180000 100 179000 0.1 E01-1245-28484 454-108-373, 1Oppm male, adult, d l 00,02-01-02 0.1 100 144000 150000 100 156000 0.3 * E01-1245-28486 454-108-375, 1Oppm male, adult, d100,02-01-02 0.3 100 138000 138000 100 138000 0.07 100 94400 E01-1245-28488 454-108-377, 1Oppm male, adult, d l 00,02-01-02 95300 0.07 100 96100 0.1 * E01-1245-28490 454-108-379, 1Oppm male, adult, d l 00,02-01-02 0.1 100 150000 158000 100 166000 0.1 * E01-1245-28492 454-108-381, lOppm male, adult, d100,02-01-02 0.1 100 141000 145000 100 149000 0.3 E01-1245-28494 454-108-383, lOppm male, adult, d100,02-01-02 0.3 100 147000 151000 100 156000 0.1 E0l-1245-28495 454-108-385, 10ppm male, adult, d100,02-01-02 0.1 100 119000 121000 100 123000 , E01-1245-28497 454-108-387, 10ppm male, adult, d l 00,02-01-02 0.3 0.3 100 174000 185000 100 196000 E01-1245-28499 454-108-389, 1Oppm male, adult, d100,02-01-02 0.1 0.1 1 00 129000 132000 100 136000 1. I 1 1 1 0.06 * E01-1245-28501 454-108-391, 1Oppm male, adult, d l 00,02-01-02 0.06 E0l-1245-28503 /454-108-393,10ppmmale, adult, d100,02-01-02 o0.'2 100 141000 144000 100 147000 160000 165000 162000 0.2 * E01-1245-28505 454-108-395, IOppm male, adult, d l00,02-01-02 0.2 100 127000 131000 100 136000 I I 1 :l I I 0.3 E01-1245-28507 454-108-397, IOppm male, adult, d100,02-01-02 0.3 E01-1245-28509 454-108-399, 1Oppm male, adult, d100,02-01-02 0.1 100 ' 100 99600 107000 138000 159000 103000 148000 0.1 E01-1245-28510 454-108-401, lOppm male, adult, d100,02-01-02 0.1 100 203000 215000 100 226000 0.2 E01-1245-28512 454-108-403, 1Oppm male, adult, d i 00,02-01-02 0.2 100 127000 132000 100 138000 1. I I ;:: i 1 0.1 E01-1245-28514 454-108-405, 1Oppm male, adult, d l 00,02-01-02 0.1 E01-1245-28516 1454-108-407, 10ppm male, adult, d100,02-01-02 0.1 100 108000 112000 100 1 17000 99200 112000 105000 I 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 18 of 226 Page 18 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite , E01-1245-28519 454-108-253, Oppm male, offspring, 02-01-02 I 0.02 I E01-1245-28520 454-108-256, Oppm male, offspring, 02-01-02 , E01-1245-28522 454-108-266, Oppm male, offspring, 02-01-02 0.07 0.07 , E01-1245-28523 454-108-270, Oppm male, offspring,02-01-02 0.2 0.2 , E01-1245-28526 454-108-283, Oppm male, offspring, 02-01-02 , E01-1245-28527 454-108-291, Oppm male, offspring, 02-01-02 0.05 0.05 E01-1245-28529 1454-108-522, 10ppm male, offspring, 02-01-02 1 0.07 I , E01-1245-28531 454-108-533, 1Oppm male, offspring, 02-01-02 , E01-1245-28533 454-108-545, 1Oppm male, offspring, 02-01-02 0.04 0.04 1 1 1 1 1 1 1 10 I I 1 1 <400 c400 <400 I <400 <400 c400 c400 <400 I 19300 I 9310 8220 15500 13600 , E01-1245-28534 454-108-552, 10ppm male, offspring, 02-01-02 0.07 1 13600 0.07 1 11500 EO1-1245-28537 454-108-568, lOppm male, offspring, 02-01-02 I 0.1 0.1 1 1 1 1 10400 * There are two results reported for these samples. Both values are included and averaged in the "Average of Dup Sample" column. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. c400 c400 <400 <400 19300 8760 14600 12500 3M Environmental Laboratory 3M Environmental Laboratory Page 19 of 226 Page 19 Report E01-1245 Report E01-1 245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 12. CsFl7SOBData Summary, Female Quail Liver (Adult and Offspring) I E01-1342-30321 10 DDm a.i. 330 F Liver E01-1342-30339 0 ppm a.i. 332 F Liver E01-1342-30357 0 ppm a.i. 334 F Liver E01-1342-30375 0 m m a.i. 336 F Liver E01-1342-30393 0 ppm a.i. 338 F Liver E01-1342-30411 0 ppm a.i. 340 F Liver E01-1342-30429 0 rmm a.i. 342 F Liver I E01-1342-30447 IODDm a.i. 344 F Liver E01-1342-30465 0 pprn a.i. 346 F Liver E01-1342-30483 0 ppm a.i. 348 F Liver E01-1342-30501 0 ppm a.i. 350 F Liver E01-1342-30519 0 ppm a.i. 352 F Liver E01-1342-30537 0 DDm a.i. 354 F Liver E01-1342-30555 0 ppm a.i. 356 F Liver E01-1342-30573 0 ppm a.i. 358 F Liver E01-1342-30591 0 pDm a.i. 360 F Liver E01-1342-30609 0 ppm a.i. 362 F Liver E01-1342-30627 0 ppm a.i. 364 F Liver E01-1342-30645 0 ppm a.i. 366 F Liver E01-1342-30663 0 ppm a.i. 368 F Liver E01-1342-30681 1E01-1342-30699 E01-1342-30717 E01-1342-30735 E01-1342-30753 10 DDm a.i. 370 F Liver 110m m a.i. 372 F Liver 10 ppm a.i. 374 F Liver 10 ppm a.i. 376 F Liver 10 o m a.i. 378 F Liver rn a.i. 380 F Liver 1.0469 1 1.0232 1 1.0188 1 1.0543 1 1.0182 1 1.0904 1 I 1.0087 I 1 I 1.0998 I 1.1036 1 1.0237 1 1.1509 1 1.0578 1 1.0312 1 1.1031 1 1.0586 1 1.0424 1 1.0364 1 1.0577 1 1.0920 1 1.0868 I 1.0748 I 1.0790 1.1036 1.0021 1 1 1 1 1 1 <50.2* c50.2' <50.2* <50.2* ~50.2' c50.2* <50.2* <50.2* ~50.2' <50.2' c50.2* ~50.2' ~50.2' <50.2* <50.2* <50.2* c50.2* c50.2* <50.2* c50.2* 5920* 4430* 543w 3980* 3750* IE01-1342-30982 E01-1342-31000 E01-1342-31018 E0l-1342-31036 I 10 DDm a.i. 402 F Liver 10 ppm a.i. 404 F Liver 10 ppm a.i. 406 F Liver 10 DDm a.i. 408 F Liver 3M Environmental Laboratory 3M Environmental Laboratory 1 1.0876 1 1 1.0173 ' 1 1.0047 1 1.0044 1 5900* 5420* 587W 4560* Page 20 of 226 Page 20 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. Accuracy: Sample results are accurate to 100% +/- 30%. * Liver QC samples did not meet the Yi of QC at each level must be within 75% - 125% criteria for the method. All liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% - 125%. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 21 of 226 Page 21 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS:A ReproductionStudy with the Northern Bobwhite Table 13. C a F 1 7 S O 3 Data Summary, Male Quail Liver (Adult and Offspring) E01-1342-30312 10 ppm a.i. 329 M Liver E01-1342-30330 10 DDm a.i. 331 M Liver E01-1342-30348 10 ppm a.i. 333 M Liver E01-1342-30366 0 ppm a.i. 335 M Liver E01-1342-30384 0 m m a.i. 337 M Liver E01-1342-30402 0 ppm a.i. 339 M Liver E01-1342-30420 0 ppm a.i. 341 M Liver E01-1342-30438 0 ppm a.i. 343 M Liver E01-1342-30456 10 mrn a.i. 345 M Liver E0l-1342-30474 10 tmm a.i. 347 M Liver E01-1342-30492 0 ppm a.i. 349 M Liver E0l-1342-30510 0 ppm a.i. 351 M Liver E01-1342-30528 0 ppm a.i. 353 M Liver E01-1342-30546 IO DDm a.i. 355 M Liver E01-1342-30564 0 ppm a.i. 357 M Liver E01-1342-30582 0 pprn a.i. 359 M Liver E01-1342-30600 IO mrn a.i. 361 M Liver E01-1342-30618 0 ppm a.i. 363 M Liver E01-1342-30636 0 ppm a.i. 365 M Liver E01-1342-30654 0 DDm a.i. 367 M Liver E01-1342-30672 I10 ppm a.i. 369 M Liver E01-1342-30708 I1o DDm a.i. 373 M Liver E01-1342-30726 10 ppm a.i. 375 M Liver E01-1342-30744 10 ppm a.i. 377 M Liver E01-1342-30762 10 pprn a.i. 379 M Liver E01-1342-30780 10 ppm a.i. 381 M Liver E01-1342-30798 10 ppm a.i. 383 M Liver E0l-1342-30829 110 m m a.i. 385 M Liver E01-1342-30847 10 ppm a.i. 387 M Liver E01-1342-30865 10 ppm a.i. 389 M Liver E01-1342-30883 10 ppm a.i. 391 M Liver E01-1342-30901 10 mrn a.i. 393 M Liver E01-1342-30919 10 ppm a.i. 395 M Liver E01-1342-30937 10 ppm a.i. 397 M Liver E01-1342-30955 10 ppm a.i. 399 M Liver E01-1342-30973 10 tmm a.i. 401 M Liver E01-1342-30991 10 ppm a.i. 403 M Liver E01-1342-31009 10 ppm a.i. 405 M Liver E01-1342-31027 10 Dprn a.i. 407 M Liver 3M Environmental Laboratory 3M Environmental Laboratory I I I 1.0530 I 1.0338 1.0062 1.0269 0.0886 1.0235 1.0090 I 1.1046 1 I 1.0586 I 1.0358 1.0583 1.0183 I 1.0899 1 1.0382 1.0941 1 1.0084 I 1.0818 1.0916 1.0327 I 1 I 1 1 1 1 1 1 1 I 1 I 1 1 1 1 I 1 1 1 I 1 1 1 1.0010 10 I 1.0276 I 10 I 1.1008 10 1.0236 10 1.0312 10 1.0501 10 1.0110 10 ' I 1.0678 I 1.0315 10 I 10 1.0310 10 1.0947 10 1.0834 10 1.0489 10 1.0309 I 10 1.0276 10 1.0361 100 1.0019 100 1.0336 100 1.0956 100 <50.2* I <50.2* I 209 e* I <50.2* <50.2* 40.2' <50.2* <50.2* <50.2* I <50.2* I <50.2* <50.2* <50.2* <50.2' I <50.2* <50.2* <50.2* I <50.2* <50.2* ~50.2' 78600* 86300* I 70000* 8030V 90700* 80000* 735w 834W I 79300* 70300* 70400' 86800' 65100* 50100* 63000* 147000* 146000* 138000* 132000* Page 22 of 226 Page 22 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite E01-1365-31194 0 ppm a.i. 253 M Liver, Offspring 0.3806 1 <20.0* E01-1365-31199 0 ppm a.i. 256 M Liver, Offspring 0.5947 1 <20.0* E01-1365-31209 0 pprn a.i. 266 M Liver, Offsprinu 0.2673 1 <20.0* lE01-1365-31214 10m m a.i. 270 M Liver. OffsDrina I 0.5900 I 1 I <20.0* I E01-1365-31229 0 ppm a.i. 283 M Liver, Offspring 0.6666 1 <20.0* E01-1365-31234 0 ppm a.i. 291 M Liver, Offsprinu 0.6072 1 <20.0* E01-1365-31245 10 ppm a.i. 522 M Liver, Offspring 0.7325 10 E01-1365-31255 10 ppm a.i. 533 M Liver, Offspring 0.6903 1 E01-1365-31265 10 ppm a.i. 545 M Liver, Offspring 0.4624 1 E01-1365-31270 10 ppm a.i. 552 M Liver, Offspring 0.5353 1 E0l-1365-31285 10 ppm a.i. 568 M Liver, Offsprinu 0.7581 1 13700 ** 4940* 5580' 5990* 6520* 0 These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. Attachment B contains data summary tables. Accuracy: Sample results are accurate to 100% +/- 30%. * Liver QC samples did not meet the Yi of QC at each level must be within 75% - 125% criteria for the method. All liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% - 125%. STATEMENT OF CONCLUSION Sera results from the male control group portion of this study showed no detectable levels of c8Fl7Soi in 19 of 19 quail (c400 ng/mL). The sera results from the female control group portion of this study showed no detectable levels of C8F17soi in 16 of 16 birds (c400 ng/mL) (Table 15). Analytical results for twenty sera samples from the lOppm dosed male group showed an average of 141000 ng/mL C8Fl7S0i (range 94400 - 226000 ng/mL, Table 14). Analytical results for seventeen sera samples from the lOppm dosed female group showed an average of 8670 ng/mL c8F17SO(irange 4710 - 14700 ng/mL, Table 15). Sera results from the male offspring control group showed no detectable levels of C ~ F I ~ SinO6~ of 6 birds (c400 ng/mL). The sera results from the female offspring control group portion of this study showed no detectable levels of c8F17soi in 4 of 4 birds (c400 ng/mL, Table 14). Analytical results for five sera samples from the 10ppm dosed male offspring group showed an average of 12600 ng/mL (range 8220 - 19200 ng/mL, Table 14). Analytical results for five sera samples from the 10ppm dosed female offspring group showed an average of 12400 ng/mL (range 7640 - 18500 ng/mL, Table 14). Liver results from the male control group showed no detectable levels of C8Fl7S0; in 19 of 19 birds ( 4 0 . 2 ng/g, Table 15) and quantifiable concentrations of C8Fl7S0i in 1 of 20 (209 ng/g, Table 14). The liver results from the female control group showed no detectable levels of C8F17S03i-n 20 of 20 birds (c50.2 ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed male group showed an average of 88500 ng/g C8Fl7S09 (range 50100 - 147000 ng/g, Table 14). Analytical results for twenty liver samples from the lOppm dosed female group showed an average of 4900 ng/g c8Fl7So3(-range 3290 - 7550 ng/g, Table 14). 3M Environmental Laboratory 3M Environmental Laboratory Page 23 of 226 Page 23 Report E01-1245 Repoti E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Liver results from the male offspring control group portion of this study showed no detectable levels of C8Fl7S0i in 6 of 6 birds (-50.2 ng/g, Table 14). The liver results from the female offspring control group portion of this study showed no detectable levels of CSFl7SOiin 4 of 4 birds (c20.0ng/g, Table 14). Analytical results for four liver samples from the 1Oppm dosed male offspring group showed an average of 5760 ng/g (range 4940 - 6520 ng/g, Table 15). Analytical results for four liver samples from the lOppm dosed female offspring group showed an average of 5490 ng/g (range 4590 - 7030 ng/g, Table 15). Table 14. Summary Table, CsF17SOB Concentration in Sera and Liver I I 1 1 I 1 1 F e m ~ ~ ? ~ 1t7r o l NA <400 ng/mL (16 of 17) 20 <50.2ng/g* <50.2 ng/g (20 of 20)' 2110 ng/mL (1 of 17) I 1 I 1 Male10ppm 20 141000ng/mL 94400-226000ng/mL 88500 ng/g* 50100 - 147000ns/g* I I Group 2o 1 I 1 ! I I FemaGlerloopuppm 18 9990ng/mL 4710 - 31900 ng/mL 4900 ng/g* 3290 - 7550 ns/g* 2o 1 1 1 Maleoffspring 1 I I ControlGroup NA <400 ng/mL (6 of 6) 6 <20.0ng/g' <20.0 ng/g (6 of 6)' Female <400 n@mL(4 of 4) <20.0 ng/g (4 of 4)' Control Group 1 1 1 1 1 1 M1OapplemmGsropupring 5 Female 1 I 1 1 Offspring10ppm 5 12600ng/mL 8220 - 19200 ng/mL 5 7350ng/g' 12400 ng/mL 7640 - 18500 ng/mL ~ 5 ~ 7180ng/g' 4940 - 13700 ns/g* 4590 - 13900 ng/g* Group For individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. Liver QC samples did not meet the Yz' of QC at each level must be within 75% - 125%criteria for the method. All liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% - 125%. 3M Environmental Laboratory 3M Environmental Laboratory Page 24 of 226 Page 24 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 15. Summary Table, C ~ F I ~ SCOon~centration in Sera and Liver with Outliers Removed I I I I MaleControlGroup I I I I 19' I I 1 I I I FemaleControl 16 I Group 19 * <400ng/mL <400ng/mL(16of 16) 20* <50.2 ndg" <50.2 ng/g (19 of 1 9 r Male 1Oppm Group I I I I I I I 1 20* 20* Female 1Oppm I I I I I I I Group 17 8700ng/mL 4710-14700ng/mL 20' Male Offspring I I . I 1OppmGroup Female Offspring I I I I 1OppmGroup 5* 1 I I I 4 5760ng/g" 4940-6520ng/g*' I I I I 4 5490ng/g** 4590-7030ng/g** * No outliers from this data set. Refer to Table 1 for results. For individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. ** Liver QC samples did not meet the !h of QC at each level must be within 75% - 125% criteria for the method. All liver QC samples (3) spiked at 10 ppb were 2125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% - 125%. STATISTICAMLETHODS Statistical methods were limited to calculating means, standard deviations, % coefficient of variation 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 which 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 measurement of accuracy available at this time is the quality control and matrix spike results. For sera data, the accuracy (the observed amount in the QC and MS samples, divided by the amount spiked, expressed as a percent) was found to range from 74.1% to 126%; while precision, expressed as the standard deviation of the recoveries, was found to range from 2.14% to 9.56%. For liver data, the accuracy was found to range from 72.6% to 141%; while precision was found to range from 2.1 1Yoto 5.51Yo. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 25 of 226 Page 25 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite The protocol and method state that 213 of all QC and a minimum of YZ of QC at each level must be within 75% - 125% criteria. All three of the liver "QC-1Oppb" spikes exceeded the stated criteria, therefore the liver data reported in the low range of the curve (Day 0 samples) may be biased high. Since all study samples that were not "Day 0" show analyte levels significantly above the low range of the curve, the overall impact (or bias) on data is minimal or none at all. REFERENCES 1. Wildlife International, Ltd. Protocol No. 454-108 "PFOS: A ReproductionStudy with the Northern Bobwhite". 2. Wildlife International, Ltd. Protocol No. 454/120700/QR/SUB454 (3M Environmental Laboratory Project Number U2723) "PFOS: A Reproduction Study with the Northern Bobwhite". 3. Centre Analytical Laboratories, Inc. Study 023-044, `Validation of Analytical Methods "Extraction of Potassium Perfluorooctanesulfonate or Other FluorochemicalCompounds From Serum for Analysis Using HPLC Electrospray/Mass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other FluorochemicalCompounds From Liver for Analysis Using HPLC Electrospray/Mass Spectrometry in Quail Serum and Liver." 4. 3M EnvironmentalLaboratory Standard Operating Procedure ETS-8-231.1, "Solid Phase Extractionand Analysis of FluorochemicalCompounds form Biological Matrices". LISTOF A~ACHMENTS 0 Attachment A: Extraction and Analytical Method Attachment B: Data Summary Tables 0 Attachment C: Sample Chromatograms, Calibration Information, and Instrument Parameters 0 Attachment D: Sample PreparationSheets 0 Attachment E: Study Protocol, Protocol Amendments, and Deviations Attachment F: Example Calculations 3M Environmental Laboratory 3M Environmental Laboratory Page 26 of 226 Page 26 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite SIGNATURPEAGE We certify that this report is a true and complete representation of 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 27 of 226 Page 27 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ATTACHMENTA: EXTRACTIOANND ANALYTICAML ETHOD,ETS-8-231. I , "SOLID PHASE EXTRACTIOANND ANALYSISOF FLUOROCHEMICAL COMPOUNDS FROM BIOLOGICAMLATRICES" 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 28 of 226 Page 28 ~~ Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Method Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Method Number: ETS-8-23f.f Adoption Date: / / / / 3 / $ 1 Revision Date: ~ / L ? / O L EffectiveDate: 3/18/62 Approved By: Laboratory Manager ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 1 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 29 of 226 Page 29 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 1 ScoDe and Atmlication This method describes the extraction of target malytes from fish. rat liver, rat sera. mouse liver, and mouse sera using solid phase extraction (SPE). This method may also be extended to other biological matrices provided that the data quality objectives are met. 2 Method Summary An amount of biological miterial. detemunedby the analyst. is prepared (fluidsdiluted and tissues lioinogenized)at a lih dilution, or other dilution as determined by the analyst using reagent grade water. An aliquot of the dilutioili'homogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile (ACN) is added 3s an extraction solvent and also serves to precipitate the proteins. The saniple is capped. mixcd. and put on the centrihge to clarif?rthe supernatant, The supematant is transferred to a clean hibe. diluted with water, and passed through a pre- conditioned CIKSPE cartridge. Finally, the analytesof interest are eluted from the SPE cartridge and analyzed by high performance liquid chromatographyelectrospraytandem mass spectrometry(HPLC-ES/MSiMS). 3 Definitions 3.1 Dilution A dilution expressed as 1.5 or 16! is deffiiedas: 1 niL of sample + 5 m L s of diluent for a total o f 6 n L s combined, unless otherwise noted. 3.2 SPE cartridge A colunin 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 interest under wnie solvent conditions and elute them wider others. A separation is thus achieved; compounds C N ~be removed from difitcult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable concentratiods) of the target analyte(s). 3.4 Quality control sample Sample used to monitor the exhxtion efficiency (as a nutrix spike)and to veri@ the continued accuracy of the initial calibration cwve (as a continuing calibration verification). 4 Warnings and Cautions 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewear, and clothing when working with solvents, samples andior equipment. Use caution with die voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 volts. 4.2 Cautions Take care not to allow the SPE column to run to dryness afler the methanol and water washes. Afler washing is complete. add sample then allowall ofthe liquid to pass through the SPE column to dryness. ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 2 of 19 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 30 of 226 Page 30 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite UO not operate solvent pumps above capacity of400 bar (5x00 psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. Jh not mn solvent punips to dryness. 5 Interferences To niininiize interferences,Teflon should not be used for snmple storage or any part of instrunentationthat comes in contact with the sanipleor extiact. 6 Instrumentation, Supplies, and Materials The following instrunlentation, supplies. and materials are used while performing this method. Equivalent instmrnentiltioii. supplies. and nuterials niay be used in place ofthose listed. 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 3 Ultia-Turrax T25 tissue homogenizer \iacuun Pump SPE Extraction Manifold Cenhihge. Mistral 1000or IEC Shaker, Eberbach or VWR Balance (+!- 0.1000 g) Micromass. QuattroI1 or Ultima h<pk quadnipoleMass Spectrometerequipped with an electrospmy ionizihon source HPl 100or Agilent low pulse solvent pumping system. solvent degisser. column compartment.and autosampler 6.2 Supplies and Materials Eppendorf or disposable pipettes. plastic or glass Dissecting scalpels Polypropylene bottles, capable ofholding 50 mL to I L (Nalgene) Volumetric flasks, glass. type A 40 i d glass vials (ICHEM) Plastic sampule vials, Wheaton, 6 i d (or other appropriate size) Centrifuge tubes, polypropylene. 15 mL and 50 mL Lnbels (kaduated pipettes; glass Syinges. capable of measuring 5 pL to 1000 pL Bottle-Top Dispenser (capable of dispensing 51nL of solvent) SPEextraction cartridge, 1 g, Sep-Pak 6 cc tri-fiuictional Clx(Waten) 75 mL samplereservoir (or otherappropriate size) C h i p cap glass autovials and caps ETS-8-23I. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 3 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 31 of 226 Page 31 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Crimpers HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7 Reagents and Standards Reagent grade water. Milli-QT",Nanopwe 11. or equivalent Acetonitrile. HPLC grade or equivalent Methanol, HPIX _-de or equivalent Ammonium acetate, reagent gmde or equivalent Biological fluids or tissues. frozen from supplier 7.1 Reagents preparation 2.0 n M annionium acetate solution: Weigh approximately 0,300 g ammonium acetate. Pour into a 2000 inL volumetric container containing reagent grade water. mix until all solids are dissolved, bring to volume wing reagent grade water. Store at room temperature. Note: When preparing different volurnes than those listed in reagents preparation. target analyte standard preparation, and surrogate stanhrd piqmation, adjmt accordingly. 7.2 Target analyte standard preparation Prepare target analyte standard(s) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and nmy or may not be appmpriate for all standard preparations. Weigh approximately 100 mg of target analyte into a 100 niL voluinetric flask and record the acturtl weight in the standard logbook or other appropriate location Bringto volume with methanol for a stockstandard ofapproximately 1000ppm (pghiiL). Dilute the stock solution with methanol for a working standard I solution of approxinlately 50 ppm. Example calculation: 1000 pg'mLx 5 nlLil00 i d = 50 p&nL. Dilute working standard 1 with methanol to produce a working standard 2 solution of approx 5.0 ppm. Example wlculation: 50 pg/mL x IO mL!lOOniL = 5.0 pg`mL. Dilute working ,standard 1 with methanol to produce a working standard 3 solution of approx. 0.50 ppm. Example calculation: 50 p g h L x 1.O niLil00 mnL =0.5 pginiL. 7.3 Surrogate standard preparation Prepare surrogate standard(s). The follo\ving is an example only and nlay or may not be appropriate for all surrogate standard preparations. Weigh approximately 90-I I O ing of surrozate standard into a lM)-mL volumetric flask arid record the actual weixht. Bringto volume with niethanol for a surrogate standard stock ofapproxirnately900 - 1100ppm. Prepare a surrogate standard working standard. Transfer approximately I mL of surrogate standard stock to a IO-nL volumetric flask and bring to volunie with methanol for a cvorking standard of 90-110ppni. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 7.4 Internal standard preparation Prepare internal standard(s). The following is an example only and may or [nay not be appropriate for all internal standard prepantions. Weigh approximately 90-1I O mg of internal standard into a 100-mL volumetric flask and record the actual weight. ETS-8-23 1. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 4 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 32 of 226 Page 32 Report E01-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Bringto volume with methanol for an internal standard stock of approxinutely 900 - I 100ppm. Prepare ui internal standard working standard. Transfer approxinlately 1 n L of internal standard stock to a 10-1nL volumetric flask and bring to volunie with methanol for a working standard of90-1 IOppm. Record the actual volume tnnsfmed and standard concentrations in the standards logbook or other appropriatelocation. 8 Sample Handling All saniples are received kozen and must be kept frozen until the extraction is performed. Allow samples to thaw to rooni temperature prior to extraction Typically fresh matrix staiulards are prepared )pith each analysis. Extracted stai~hrdsand samples are stored in capped autovials until miilysis. If arlalysis will be delayed, extracted standardsand sanlples nmy be refiigerated at approximately4C indefinitely or may be stored at rooin temperature until analysis can be perfonned. 9 Qualitv Control 9.1 Blanks 9.1.1 Solvent Blank An aliquot ofniethanol is used as a solvent blank. Solvent blanks are not extracted. 9.1.2 Method Blank An aliquot of I .0 mL of mter. or other appropriateamount. is used as a method blank. Four niethod blanks are extracted and analyzed with each set following this procedure (two are spiked with surrogate and hvo are not spiked). 9.1.3 Matrix Blank An aliquot of 1.0inL or 1.0g ofinatrix (diluted or homogenized)is used as a nlatrixblank. Other amountsn t ~ bye used. as appropriate. Matrix blanks are prepared from one of three sources: I ) a study control nmtiix from a study control aniinal received with a sample set; 21 a conunercially obtained simple of the same species as the study aniinals; or 3) a sunogate matrix, also obtained commercially, but o f a different species than the study animal. (eg. if rat is used to generate standard curves and C'CVs for a mouse study). The nlatrix to use is dependent on the nlatrix used for the curve. 9.1.3.1 Study control matrix curve - ifthe study control matrix is used for the curve. prepare four (4) nmtrix blanks using the study control matrix (two spiked with surrogate a i d two not spiked). 9.1.3.2 Coinnierciallyobtained (samespecies) nlatrix cuive - if the commercially obtained nutrix is used for the curve. prepare four (4)matrix blanks using the same coniinercially available matrix (t\vo spiked with surrogateand two not spiked). 9.13.3 Surrogate nmtrix curve- ifa surrogateimtrix is used for the curve. prepare four (4) matrix blanks wing the same cotnmetdly availablematrix and prepare four (4) matrix blanks using a conunerciallyavailablematrix of the Same species as the study animals (twospiked with surrogate and two not spiked). 9.13 . 4 If limited matrix is available.the number ofmethod and matrix blanks m y be adjuyted and will be noted in the study protocol or in the raw data. 9.2 Sample Replicate Samplesreplicatesare prepared accordingto each study protocol or pro.iect outline ETS-8-23 I. 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 of 10 3M Environmental Laboratory 3M Environmental Laboratory Page 33 of 226 Page 33 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite 9.3 Surrogate standard If suiTogate standard is a component of the study, all samples arc spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range of the calibration cuwe. with the exception of blank sample above. Typically surrogate standard is spiked into the 1.0 niL dilutedhomogenizedsample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water. into the dilutedhomogenized saniple prior to removing the 1.01 n L sample, or into the 1.0n L dilutect'homogeaizedlsample removed for extraction. 9.4 Internal standard If intermil s%indardis a component of the shidy, dl samplesare spiked with internal standard after extraction to obtain a concentration in the mid-range of the calibration curve. Typically internal standard is spiked into the 2.0 mL of extract in the 15 mL centrifuge tube, before transfemng to the autovial. 9.5 Lab Control Sample L,ah control samples are not a coinpoilent ofthis method. 9.6 Quality Control (QC) Sample Prepare quality control (QC) saniples to monitor extrnction eficiency and to veri@ the continued accuracy of the initial calibration curve. Typically 1.0 mL. or other appropriate amount, of the same matrix used to prepare the initial calibration curve is used for each QC sample. Twelve (12) quality control samples (QC) bill be prepared for each matrix during the course of a study. A niuiinium of 3 QC samples must be prepared (one at each level) on each day of sample extraction. (e.g. If the study is such that samples will be extracted on three direrent days then four QC samplesmust be prepared on each day of extraction for a total oftwelve.) QC samples will consist of four samples at each of three levels of analyte. The levels listed below may he used and may represent sample concentrations diluted into the range of the calibration curve: Low level: 3X to SX the LLOQ, Mid-level: equivalent to a point near the middle of the calibration curve. High level: 8G"hof the LXOQ Two QC sample levels are anal)zed after eveT tenth sample injection starting after the last calibration standard injection, with a minimum of three QC: per analysis. Solvent blatks are not considered samples but may be included as such for determining when QC samples will be analyzed. QC saniples extracted with a particular sample set must be analyzed in the same analytical run. Any QC samples extracted diuing the course ofthe study may be included in subsequentanalyses. If sarnples fiorn rnultiple extraction dates are analyzed in one analytical run. then QC: samples from the same saniple eutr'action dates niust be included in h t analysis. Each is expected to show an accumcy of 75-125Yiofexpected. A minimum of 213 of all QC:samples must meet this criteria, and a minimum of 1 2 of the QC samples at each level must meet this criteria. If not, the set must either be re-analyzed or re-extncted. 9.7 Sample Dilution Any sample with an area greater than that ofthe highest acceptable standard wIll need to be diluted into the range of the calibration curve. If srtniplesare diluted into the range of the curve during analyses and enough sample remains, a post-run dilution validation will be performed to veri@ saniple values. To peilbnn the dilution validation one sample will be separated into two representative samples (Le. two I .O inL aliquots for fluid sctniples or two 1.0 &ramamounts for tissue samples. or other amount x determined by the analyst ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 6 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 34 of 226 Page 34 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite and documented in a note to tile) then diluted using two procedurcs. The first procedure comists of diluting the sarnple with additional matrix prior to extraction (fluid adding fluid). while the second procedure consists of diluting the extract with solvent postcxtraction (methanol extract adding additional methanol solvent.) If the relative peicent direrence is not within 15% for these two samples: additional testing will he required to deteimine which value is a cortect representation of the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibtation cuve mi11 be prepared from extracted matrix standards. in the sane matrix asthe samples, per study. It will consist of a ininununi of nine (9) levels. Additional calibration curves may he extracted on separate sample extraction dates, as determined by the analystand documented in a note to file. Transfer 1.0 nL. or other appropriate mount. of diluted control fluid or homogenized control tissve to a 15 mL centrifuge hibe using a disposable plastic pipette. This will he repeated while preparing aliquots for the standard curve. Be suc to nliv or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weight/volumes sheet or extraction worksheet as appropriate, Four I .O ILaliquots,or other appropriateamount, of control nlatrixserve as imtrix blanks. The standard concentrations and spiking amounts listed in Table 1 nlay be used, hhen appropriate, to spike one standard cin-ve. A total o f 9 standards, four matrix blanks. nnd four nietliod blaiks are prepared in addition to the QC sanples and test samples. The nuniber of staticlards and blanks nlay be adjusted as determined by the analyst and documented in a note to file. Use Attachment C. or other appropriate fonn. as ai aid in calculating the concentrationsof the working standards. Refer to section 12 to calculatethe actual concentration of analyte in each calibration standard and QC; sample. Typically the target amlyte standard is spiked into the 1.0 mL dilute&holnogcnized sample removed for extraction. Homcver, it may be spiked directly into the matrix prior to diluting With water. into the diluted/homogenizedsample piior to removing the 1.0 mL sample,or into the 1.0 mL diluted!homogeni7rd sampleremoved for extraction. Analyze the extracted matrix standard curie prior to each set of extracts. The curve equation will be deterniined by regression analysis using the peak areas ofthe target analyte(s)using MassLynx or other suitablesoftware. Any level outside 75% - 125%of nominal must he deactivated,and regressionrecalculated. except the LLOQ which must be within 30% ofnominal. All levels mitit show a response @eaterthan twice that ofthe blank. A maximum of three (3)levels may be deactivatedin any one set, or the set \ d l be re-analyzed. ETS-8-23I. 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 7 of 19 Page 35 of 226 Page 35 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Working standard (approximlte concentration) 0.500 ugimL 0.500 ug/mL 0.500 ug/mL 0.500 u g h L 0.500 u&iL Approxinute final Approximate final ILL concentration ofanalyte concentration ofanalyte in Matrix diluted 1:s in Final 2.0 mL volume Blnnk Blank 1.5 5.00 n g g or ng/niL 0.375 ngimL 3.0 10.0 ngig or ngniL 0.750 ngimL I 8.0 25.0 npig or n,@niL 16 I 50.0ng/g or n @ n L I 32 I 00 ng/g or ng/mL 2.00 nginiL 4.00 ng'mL 8.00 &mL I I I 50.0 uginiL 1750ngig or ngirnL I Surrogate Std 100 uginiL 10 6500 ngig or ngimL 11 Procedures 150 ng'niL 500 n@mL 11.I Tissue Sample Preparation Obtain fiozen tissue samples Cut approximately 1.COO0 g of tissue (+i-0.1ooO g), or other appropriate amount. using a dissecting scalpel. This part ofthe procedure is best performed quickly,not allowing the tissue to thaw. Weigh the tissue directly into a tared plastic sunpule vial. Record the weight on the weight~\duinesheet. cxtraction worksheet. or other appropriate location. Return unused tissue to the freezer after extraction amounts have been renioved. Add 2.5 n L of reagent water to sampule vial. or other volume as detennined by the analyst and documented in a note to tile. Homogenize the sample. hit the Ultia-Tunax grinder probe in the sample and grind for approximately2 minutes, or until the sample is homogeneous. Rinse the probe intothe tube containingthe smiple with 2.5 nlL ofreagent grade water. or other volume as determined by the analyst and documented in a note tu file. using a pipette. Take the grinder apart and clean it with methanol after each snmple. Refer to ETS-9-52 for more infommtion. If an amount other tltm 1.0ooO g (not within +i-0.1000 g) is removed for an initial weight. adjust the water volume accordingly to maintain a li6 dilution. (e.g. if 0.5 g is removed for extraction.add a totd of2.5 mL of water.). or other ixtio as determined by the analyst and documented in a note to file. ETS-8-23 I. 1 Solid Phase Extraction and Analysis of Fluorocheniical Compounds from Biological Matrices Page 8 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 36 of 226 Page 36 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 11.2 Fluid Sample Preparation Obtain frozen fluid sample and allow it to thaw at room tenipeiatureor in lukewaiin watcr. Label a IS niL polypropylene centrifuge tube with the study number, saniple ID, extraction date and analyst initials. See attached worksheet (AttachmentA or similar worksheet) for documenting the remaining steps. Vortex mix the fluid sample for approxinutely 15 seconds. then transfer 1.0 mL of fluid, or other appropriateamount to a plastic snnipule vial, or other appropriatecontainer. Retuni unused samplesto freezer after exhaaction miounts have been removed. Add 5.0 niL of reagent water to the 1.0 mL of fluid for a I,% dilution, or other dilution as deterniined by the analyst and documentedin a note to file. Ifa volume other tlian 1.0 n L is removed for ai initial volume. adjust the mater volume accordinglyto nuintain the sanie dilution as above. 11.3 Tissue and Fluid Sample Extraction After tissue or fluid sampleshave been prepared accordingto sections 11.1 and 11.2,vortex mix or shake by hand the dilutedhoniogenized sample for approximately 15 seconds then transfer 1.0 mL, o r other appropriate volume. to ;i clean 15 mL polypropylene centrihge tube. Retuin unused dilutedi'hoinogenizedportionsto the fieezzr after extraction amountshave been removed. Record the volume removed on the extractionworksheet, (Attachment A or similar worksheet). Spikeblanks. samples,and standards. ready for extraction with surogate standard as described in this method Spike each calibration standard nutrix with the appropriate amount of standard as described in this method for thc calibrationcurve standards and each QC sample. Vortex mix the stindad curve samples and QC samples for approximately 5 seconds. To each sample aid standard. add 5.0 i d 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 ofapproxinxitely 300 rpm on the n i d e l s listed in section 6. I ). Remove from the shaker and centrihge at an appropriatespeed for 10 minutes to adequately pellet the precipitate (a setting of approximately2000 rpni on the models listed in section6 .I ). Add 40.0 i d , of reagent gnde water to a clean SO id centrifuge tube. Remove samples fiom the centrifuge and decant the supernatant into the water in the SO mL tube, taking care not to introduce any of the matrix solids into the solution. Cap and mix by inverting several times. In this step the order of addition may he changed (i.e. the s`mple nxiy he put into the centrihge tube and then the water added). Attach the reservoir to the SPE cartridge and attach this reservoirkartridgeunit to a ~acuummanifold. NOTE: When runningthe vacuum. set the vacuum chamber at approxinuitely 15kPA - t o give an approxinute elution flow of 5-7 mLimin. Flows may vary through cartridges and the kPA may be raised fix slow tubes and drying after most have been drawn doun. Prepare the SPE cartridge by washing twice with approximately 5.0 niL of methanol, followed by approxinutely two 5.0 niL aliquotsof wter. taking care not to allow the columnto run to dryness after each wash. After washing is complete, pour the sample into the resewoiricartridge unit and allow all of the liquid to pass through the column to dryness. Run the vacuum on hi& for approximately 5 minutes to adequately dr);each SPE cartridge. Place a collection 15mL polypropylene centrihge tube under each cartridge and elute with 2.0 mL ofnietlanol. Spike extracted blanks, samples,and standruds l i t h internal standard as described in this method. ETS-8-23 I. 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 9 of 19 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 37 of 226 Page 37 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Label each glass autovial. as appropriate. with the study number, vial file archive number. aninml iiumberlgenderitirnepoint or LIMS number. matrix, final solvent, analyte components (if needed). extraction type. extraction date. and analyst(s) performing the extraction. Transfereach eluant to a glassautovial and cap. 11.4 Extract Analysis 1I.4.1 Software set-up On the MassLqnx main page. set up a saniple list name. Save the list as instruinent designator letter, last 2 digits of test year-month-day.and a letter that will increasethrough the alphabet with each additional list for that day. Example Sample List: IWMMDDa or A0202Ma 1 =Initial ofthe instmnient nanie (A = "Amelia") YY = Test year (02) MM = Test month (02) DD = Test day (04) a = First sample list (run) ofthe day (the next saniple list will end with 'V.the next IC', and so on.) Assign a filenanie using the instrument designator letter, the last 2 digits of the test sequential file nuniher that starts with 1and increasesby one for each filename. -month-day. and a 3-digit Example filename: IYYMMDDi#Mlor A0202W001 I =initial of instrumentname YY = Test yea1 MM = Test month DD = Test day ##i: = 3digit sequential file number starting with 1 through 999 (001) Also. as part ofthe saniplelist, assign a method (MS) for acquiring. an inlet file. a bottle number, an injection volume, and sample descriptions. To create a method. click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording)or MRM [Multiple Reaction Monitoring). Set Ionization Mode as appropriate and imss to 4'99 or other appropriate mass(es). Also set the acquisition start and stop times. Swe acquisition method. If MSiMS instruments are employed. additional product ion fragmentationinformation may be collected. See Micronlass MassLynx "Guideto Data Acquisition" for additional infornution on MRM. Typically the analytical batch nin sequence begins mith system suitability. solvent blanks, and a set of extncted nmtrix stiindards. Sample extracts are analyzed with two QC samples injected after every tenth saniple injection. Solvent blanks should he analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be included as suck ETS-R-73I. 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 10 of 10 3M Environmental Laboratory 3M Environmental Laboratory Page 38 of 226 Page 38 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 11.4.2 HPLC set-up Set up sample tmy accordingto the sample list prepared above. Set up the HPLC to the following conditions or at conditions the analyst consideis appropriate for optimal response. Record actual conditions in the instrumentlogbook, or other appropriate location: Sample size = 10pL injection 1n.ject'saniple= 1 Cycle time = 10.0minutes Flow rate = 300 @min Mobile phase: Solvent A = 2 niM Animonium Acet1te. Solvent B = Methanol Solvent Fadient program: I .oo 10% I I I 11.' 3 Instrumen set-up Refer to ETS-9-24, "Operation and Maintenance of the Micromass Quattro I1 Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionkition Source." for details. Check tlie solvent level in HPLC reservoirs 'and refill if necessluy. Check the stainless steel capillaiy at the end ofthe probe. Use an eyepieceto check the tip. The tip should be flat with nojagzed edges. Ifthe tip is found to be unsatisfactoiy, disassembletlie probe and replace the stainless steel capillaiy. T u n on the nitrogen. Open the tune page. Click on operate to initiate sowce block and desolvation henters. Open the Inlet Editor. Download the HPLC method and initiate solvent flow to begin system equilibrium. Set the flow to 1C-SOOuLhnin or as appropiiate Set HPLC pump to "On" Observe droplets or inist corning out ofthe tip ofthe probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip ofthe probe if no mist is observed Allow to equilibrate for approximately IO minutes lypical instrument parametersinclude: Drying gas 250-100 litersihour ES nebulizing gas IC15 literdhour ETS-8-23I. 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 11 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 39 of 226 Page 39 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite HPLC constant flow mode. flow rate 10-500 pLimin Pressure 400bar (this paranieter is not set. it is a guide to ensurethe HPLC is operating correctly.) Source block temperature approximately 150C Desolvationtemperature approximately250C These settings inay change in order to optimize the response Print the tune page. sample list, and acquisition method from MassLynx and store it in the study binder with a copy taped into the inaumcnt log. Click on start button in the Acquisition Control Panel (the location of the start button may vary aniong MassL~mx versions, refer to appropriate MassLynx User's Guide). 12 Data Analvsis and Calculations 12.1 Calculations If other calculations are used than those listed. they will be docuinented in the raw dah. Calculate the matrix amount contained in the initial dilution using the following equation: IW (g) (or 1V (niL)) Matrix Amount (gimLor mLimL) = (IW(g) (or IV(mL))+ DV (mL) Calculate actual concentrations ofanalyte in calibration standardsusing the followingequation: Concentration (ngig or ng/mL) = Spike Concentration (ug/mL) x Spiked Amount (mL) X- 1000ng SV (mL)xMatnx Amount (gimLor mLimL) 1 ug IW = Initial wei@t (where 1.Og = 1.0 mnL ) IV = Initial volume DV = Diluent volume (reagentgade water) SV = Sample voiuine removed for extraction (typically i.0 mL) AR = Analyticalresult from MassLyx summary DF =Dilution tictor FV = Final volume MA =Matrix amount 3 curve = MA oftissueiflnidstandard curve. assiimed to be I g or 1 m i 5 mL water 3 sample = MA oftissue/fluidsample L g or mL of samplei5 mL water) Calculate spike percent recoveries using the following equation: % Recovery = Observed Result - Matrix Blank Result x 100 Spiking Level ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 12 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 40 of 226 Page 40 Report E01-1245 Report E01-1 245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Calculate relative standard deviation using the following eqilation: Standard Deviation Relative Standard Deviation = x I00 Mean Calculate percent deviation using the following cquation: Expected Conc. -Calculated Conc. %, Deviation = x 100 Expected Conc. Calculate actual concentration of analyte in fluid @g/mL): AR (ngiid) x DF x d curve (&nL) x FV (mL)in Curve x sample(mL/mL) FV (inL) in Matrix 1000ng = (pg'g) Calculate actual concentration ofanalyte in tissue (ldg): AR (ng'g) x DF x (3 curve IdmL) X FV hnL) in Curve X r) sample (g/nL) FV (nL) in Matnx ].ou9 1000 ng = (pgg) 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start and at the completion of each walytical run. Prior to the wlibrdtion curve and after the last sainple of the run t h e e (3) mid-level unextracted culibmtion standards will be analyzed. As applicable, the peak area precision. retention time precision, resolution, and peak asymmetry will be monitored at the beginning and the end ofthe nm separately. The peak area precision must be equal to or less than 5,0% RSD. the precision of the retention time inust be equal to or less than 2.5% RSD. the resolution must be >: 2.0, and the peak asynnxhy (fionting or tailing) mwt be 0.5<AF<2.0.ubere AF is the asynunetry factor. If any item of the system suitability fjils, system miiintenance must he completed prior to running a second set of system suitability samples and the system suitabilitymust pass before starting the calibntion. If system suitability fails at the completion ofa nm. the saniple set niust be reanalyzed. 13.2 Quantitation The coeffcient of detemination value for the calibration curve, plotted by regression using the peak areas of the mlyte(s). must be 0.990 or better. All active calihration curve points must be within 25% of the theoretical value with the exception of the LOO point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify a data range that may be affectedby background levels ofthe analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. Ifthe curve cannot meet these criteria, the sanipleset must be reanalyzed or reextracted. 13.3 Accuracy Two thirds of all qilality control samples and 1/2 of each quality control sample at each level are expected to show an accuracy of 75- 125"/0. ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 13 of 1'3 3M Environmental Laboratory 3M Environmental Laboratory Page 41 of 226 Page 41 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Surrogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be re'malyzed to confirm. If the second analysis confirms the original, the deviation wll be documented in the raw data. Ifthe second analysis is within 50%. then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed of in noninfectiousbiohazard waste containers. Flammable solvent waste is disposed of in high BTU containers. Glass pipette waste is disposed of in broken glass containers located in the laboratory 15 Records Complete the extraction worksheet attached to this method. or other applicable worksheet,and store with the studyraw data. Each page generated for a study must contain the following infomation (if applicable): studyiproject or instrument number, acquisition method, integration method. sample name, extraction date. dilution factor (if applicable). and analyst. Other information nlay be added ifapplicable to the study. Print the tune page. sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into the instrumentnmlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data. Print data integration summary, integration method, and chrom1toganxs from MassLynx and store with the study raw data. Sumnnl;trize data using suitable s o h e (Excel7.0 or LIMS) and store in the study folder. Back up electronic data to appropriate medium. Record in sTudy notebook the file m n e and location of haclcup electronic data. 16 Attachments Attachment A Extraction Worksheet Attachment B: Sample WeighUVolune Worksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Sunlnlary Worksheet 17 References ETS-9-24, "Optxition and Maintenance of the Micromass Quattrn I1 Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-Y-52, "Operation and Maintenance ofa Tissue Grinder" 18 Affected Documents None ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 14 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 42 of 226 Page 42 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite 19 Revisions Revision Number 1 Revision Descrintion Minor formatting changes. Added detailed iuforniation to all sections concerning the extraction procedure, analytical procedure. and calculations. Added c~tttachmentsand references. Revision Uatt: 0211 Ki02 ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 15 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 43 of 226 Page 43 Report E01-1245 Report E01-1245 Study Number: Prep Date: Analysts initials: Box#: PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Attachment A - Extraction Worksheet Method Revision:ETS-8-231.1 Matrix: Sample Timepoint: Blank matrix TN-A- : Amount weighed/aliquoted: g/mL 1. Homogenize sample 2. Aliquot 1 mL of diluted matrix into 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 Q rpm (Shaker ) 6. Centrifuge sample for 10 min @I rpm (Centrifuge ) 7. Add 40 mL of water to 50 mL polypropdene centrifuge tube. 8. Decant extract Into centrifuge tubes with water 9. Shake sample slightly lo ensure proper mixing 10. Attach 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum manifold 11. Condition wlumn 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 ( TN-A- ) into appropriate 15 mL centrifuge tube 16. Spike samples with uL of internal standard t# , conc. 17. Transfer sample into appropriately labeled autovlal 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 after most have been drawn down and shut off. ETS-8-23 1.1 Solid Phase Estraction and Analysis ofFluorochemica1 Compounds from Biolopical Matrices Page I6 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 44 of 226 Page 44 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Prep Date(s): AWdlySt(S): Sample Matrix: MethodlRevision: Sample ID "......" Attachment B - Sample WeightiVoluine Worksheet Study Number: Equipment Number: Final Solvent & TN Number: Initial wt.nroi. g/mL/L Water Volume added (mL) Volume Removed (mI,) ................ " ".... .".... ............. Comments " ........... " "......"_ ...... lil "."l_lll"." ...." .............................................................. "......"..... ............ "._ .......................................................................... .- ................... ~ ............"..................." ................. .......""................................... ... ......."....... ...." " .- ..................... ."........." ................................................................................................................................................................................................................................................................0 ........................ ...""....... .................................. " "... ^" ""... ........... ".......... ................................................................................................................................................................................................................................................................ ..... ".............. "" "-".-.........".......I " I ... lllll.ll...." "........." - ........................... " .......................................... " 1 - ............ " " ~ ............... " ................... .. " ................................................................... ........... "..."".l. ........ "............ ._........... ..........."... "............................................................................................... Fonn Coiiipietion Verified By: ETS-8-23 1.1 Solid Phase Extraction and Analysis of Fluoroclmnical Coinpounds from Biological Matrices Page 17 of 19 3M Environmental Laboratory 3M Environmental Laboratory P a g e 45 of 226 Page 45 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Prep date(s): Analyte(s): Sample matrix: MetLod/revision: Attachment C: Calibration Standard Concentration Worksheet Standard number: Equipment number: Final solvent and TN: Blank Tissue or Fluidhdentifier: Analyte Std conc u@mL All Ani't spiked mL All Final Volume: mL All Initial Fluid Dilution All Initial Tissue Deiisi ty g'mL (1.1600 0.1600 0.1600 0.1600 0.1600 0. 1600 0.1600 0. I600 0.1000 0.1600 0 .I600 0.l600 0.1600 Calculated concentrationsofstruidxds in relation to the final 2.0 mL solvent and initial inatrix 2.0 mL Final Volume Fluid Matrix Tissue Matrix Analyte Final Surrogate Std conc Analyte Final Surrogate Std conc Analyte Final Surrogate Std conc 125 1500 1500 150 1750 I750 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 18 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 46 of 226 Page 46 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Study: Dilution I)ate/Analyst: Box Number: Sample Number I Attachment D: Dilutions Sunlmary Worksheet Solvent/TN Number: Extraction DatelAnalyst: Matrifliniepoint: Dilutions Notes: 1/10 dilution = of sample + of solvent Form Completion Verified By: ETS-8-23 I . 1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 19 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 47 of 226 Page 47 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite ATTACHMENBT: DATASUMMARTYABLES 3M Environmental Laboratory 3M Environmental Laboratory Page 48 of 226 Page 48 Report E01-1245 Study number E01-1245 - QUAIL SERA DATA SUMMARY, BLANKS AND SPIKES PFOS: A Reproduction Study with the Nothern Bobwhite E01-1245-35766 E01-1245-35825 E01-1245-35825 1/31/2002 2/1/2002 2/1/2002 E01-1245-35767 E01-1245-35826 E01-1245.35826 1/31/2002 2/1/2002 2/1/2002 Water blank-day 1 Blank H2O-day2 Blank H20-day 2 quail sera blank-day 1 Blank quail sera-day 2 Blank quail sera-day 2 d020207031 0.05 40 1 10.0 400 400 d0202030 19 0.05 40 1 10.0 400 400 d020207035 0.05 40 1 10.0 400 400 d020207032 0.05 40 1 10.0 400 400 d020203020 0.05 40 1 10.0 400 400 d020207036 0.05 40 1 10.0 400 400 E01-1245-35768 E01-1245-35827 E01-1245-35827 1/31/2002 2/1/2002 2/1/2002 E01-1245-35769 E01-1245-35828 E01-1245-35828 1/31/2002 2/1/2002 2/1/2002 quail Sera MS-day 1 quail Sera MS-day 2 quail Sera MS-day 2 quail Sera MSD-Day1 quail Sera MSD-Day2 quail Sera MSD-Day2 d020207033 0.05 d020203021 0.05 d020207037 0.05 d020207034 0.05 d020203022 0.05 d020207038 0.05 Conc of PFOS in Sample (ng/ml) = (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 samule = amount of sera used. 40 1 225.29 400 40 1 254.33 400 40 1 227.43 400 9012 10173 9097 40 1 217.03 400 40 1 254.55 400 40 1 219.54 400 8681 10182 8782 for both ms and msd I 3 avg % rec 92.84 std dev of % rec 6.980 avg conc. 9321 std dev of conc. 680.2 89.76 101.3 90.61 86.47 101.4 87.47 true value 10040 avg %rec 93.9 avg cone 9427 sld dev of cone 647.4 true value 10040 avg %rec 91.8 avg cone 9215 std dev of cone 839.0 3M EnEvxicrelo20n00mental Laboratory Blanks QCs and MSs quail sera samples summary 030418.xls printed: 4/18/2003, 8% AMPage 49 Report E01-1245 Study number E01-1245 - QUAIL SERA Absolute Recoveries PFOS: A Reproduction Study with the Nothern Bobwhite E0l-1245-35780 01/31/2002 qc-25ppb-2.01-31-02 (1004 n&L diluted seraj d0202131M 0.05 40 EOl-1245-35781 01/31/2002 qc-25ppb-3,01-31-02 (1004 ng/mL diluted sera) d020213103 0.05 40 E0l-1245-36155 02/13/2002 ms-5ppb-l,02-13-02 (201 ng/mL diluted sera) d020213104 0.05 40 E01-1245-36156 02/13/2002 ms-5ppb-2,02-13-02 (201 ngmL diluted sera) d020213105 0.05 40 E01-1245-36157 02/13/2002 ms-5ppb-3, 02-13-02 (201 ng/mL diluted sera) d020213106 0.05 40 E01-1245-36158 02/13/2002 ms-200ppb-1.02-13-02 (8032 ng/mL diluted sera) d020213107 0.05 40 E01-1245-36159 02/13/2002 ms-200ppb-2,02-13-02 (8032 ng/mL diluted sera) d020213108 0.05 40 E01-1245-36160 02/13/2002 ms-200ppb-3,02-13-02(8032 ng/mL diluted sera) dO20213109 0.05 40 Study number E01-1245 - QUAIL SERA versus unextracted curve E01-1245-35779 01/31/2002 qc-25ppb-1, 01-31-02 (1004 ng/mL diluted sera) d020405082 0.05 40 E01-1245-35780 01/31/2002 qc-25ppb-2, 01-31-02 (1004 ng/mL diluted sera) d020405083 0.05 40 E01-1245.35781 01/31/2002 qc-25ppb-3, 01-31-02 (1004 ng/mL diluted sera) d020405084 0.05 40 E01-1245-36155 02/13/2002 ms-5ppb-I, 02-13-02 (201 ng/mL diluted sera) d020405085 0.05 40 E01-1245-36156 02/13/2002 ms-5ppb-2.02-13-02 (201 ngmL diluted sera) d020405086 0.05 40 E01-1245-36157 02/13/2002 ms-5ppb-3,02-13-02 (201 ng/mL diluted sera) d020405087 0.05 40 E01-1245-36158 02/13/2002 ms-200ppb-1,02-13-02 (8032 ng/mL diluted sera) d020405088 0.05 40 E01-1245-36159 02/13/2002 ms-200ppb-2,02-13-02 (8032 ng/mL diluted sera) do20405089 0.05 40 E01-1245-36160 02/13/2002 ms-200ppb-3,02-13-02 (8032 ng/mL diluted sera) d020405090 0.05 40 A = Not within +/-25% criteria Conc of PFOS in Sample (ng/ml) = (Ins1conc(ng/rnl)) x (additional diluiion) x (dilution(di1utedsera)) dilution(di1utedsera) =final volume(mL)/amount of sera in diluted sample(mL) = 2.0mU0.05mL= 40 amount of sera in diluted sample =amount of sera used. 1 39.12 201 1 45.18 201 1 5.54 201 1 5.51 201 1 5 23 201 1 248.13 201 1 254.19 201 I 253.36 201 1 36.06 201 1 41.69 201 1 44.96 201 1 6.17 201 1 5.88 201 1 6.17 201 1 248.12 201 1 260.27 201 1 254.58 201 .=I"i" ,.,.c 1 1 1565 A 155.9 std dev 14.46 1807 A 180.0 %RSD 8.853 222 110.2 avg 108.0 220 109.7 std dev 3.403 209 104.1 %RSD 3.151 9925 10168 A 10134 A 123.6 126.6 126.2 avg 125.4 Std dev 1.636 %RSD 1.304 1442 A 143.7 avg 163.0 1668 A 166.1 std dev 17.97 1799 A 179.2 %RSD 11.03 247 122.8 avg 120.9 235 117.0 std dev 3.332 247 122.8 %RSD 2.757 9925 10411 A 10183 A 123.6 129.6 126.8 avg 126.7 std dev 3.027 %RSD 2.390 3M EnEvxicrelo20n0m0 ental Laboratory Blanks QCs and MSs quail sera samples summary 030418.xls printed: 4/18/2003, 8:53 AMPage 50 Report E01-1245 Study number E01-1245 - QUAIL SERA versus extracted curve QC Sample Recoveries oriainal of all and rerun of 8ODDm t0l-1245-35785 01/31/2002 qc-80ppm-1.01-31-02 (3227040 ng/mL diluted sera) d020201035 0.05 40 E01-1245-35786 01/31/2002 qc-80ppm-2, 01-31-02 (3227040 ng/mL diluted sera) dO20201036 0.05 40 E01-1245-35787 01/31/2002 qc-80ppm-2.01-31-02 (3227040 ng/mL diluted sera) d020201037 0.05 40 E01-1245-35779 01/31/2002 qc-25ppb-1, 01-31-02 (1004 ng/mL diluted sera) d020207044 0.05 40 E01-1245-35780 01/31/2002 qc-25ppb-2,01-31-02 (1004 ng/mL diluted sera) d020207045 0.05 40 E01-1245-35781 01/31/2002 qc-25ppb-2, 01-31-02 (1004 ng/mL diluted sera) d020207046 0.05 40 E01-1245-35782 01/31/2002 qc-40ppm-1, 01-31-02 (1613520 ng/mL diluted sera) d020207047 0.05 40 E01-1245.35783 01/31/2002 qc-40ppm-2, 01-31-02 (1613520 ng/mL diluted sera) d020207048 0.05 40 E01-1245-35784 01/31/2002 qc-40ppm-2. 01-31-02 (1613520 ng/mL diluted sera) do20207049 0.05 40 E0l-1245-35785 01/31/2002 qc-80ppm-I. 01-31-02 (3227040 ng/mL diluted sera) d020207050 0.05 40 E01-1245-35786 01/31/2002 qc-80ppm-2.01-31-02 (3227040 ng/mL diluted sera) d020207051 0.05 40 E01-1245.35787 01/31/2002 qc-80ppm-2,01-31-02 (3227040 ng/mL diluted sera) d020207052 0.05 40 Study number E01-1245 - QUAIL SERA versus extracted curve rF)n,n E01-1245-35780 01/31/2002 qc-25ppb-2.01-31-02 (1004 ng/mL diluted sera) d020405083 0.05 40 E0l-1245-35781 01/31/2002 qc-25ppb-3. 01-31-02 (1004 ng/mL diluted sera) d020405084 0.05 40 E01-1245-36155 02/13/2002 ms-5ppb-1,02-13-02 (201 ng/mL diluted sera) d020405085 0.05 40 E01-124536156 02/13/2002 ms-5ppb-2,02-13-02 (201 ng/mL diluted sera) d020405086 0.05 40 EOl-1245-36157 02/13/2002 ms-5ppb-3, 02-13-02 (201 ng/mL diluted sera) d020405087 0.05 40 E0l-124536158 02/13/2002 ms-200ppb-1,02-13-02 (8032 ng/mL diluted sera) d020405088 0.05 40 E01-1245.36159 02/13/2002 ms-200ppb-2,02-13-02 (8032 ng/mL diluted sera) d020405089 0.05 40 Eol-1245-36160 02/13/2002 ms-200ppb-3,02-13-02 (8032 ng/mL diluted sera) d020405090 0.05 40 E01-1245-36155 02/13/2002 ms-5ppb-I. 02-13-02 (201 ng/mL diluted sera) d020410019 0.05 40 E01-1245-36156 02/13/2002 ms-5ppb-2.02-13-02 (201 ng/mL diluted sera) d020410020 0.05 40 E0l-1245-36157 02/13/2002 ms-5ppb-3, 02-13-02 (201 ng/mL diluted sera) d020410021 0.05 40 A = Not within +/-25% criteria Cone of PFOS in Sample (ng/ml) = (Ins1conc(ng/ml)) x (additional dilution) x (dilution(di1utedsera)) dilution(dilutedsera) =final volume(mL)/amountof sera in diluted sarnple(mL) = 2.0 mU0.05 mL= 40 amount of sera in diluted sample = amount of sera used. PFOS: A Reproduction Study with the Nothern Bobwhite 1MX) 95.65 400 1000 93.45 400 1000 85.75 400 1 26.99 400 1 27.89 400 1 31.52 400 100 299.0 400 100 316.2 400 100 306.4 400 1000 88.66 400 1000 85.99 400 1000 79.33 400 3826000 3738000 3430000 1080 1116 1261 A 1196120 A 1264880 1225440 3546400 3439600 31 73200 for all in original analysis (recovery avg%rec 101.5 I Std dev 19.89 I 118.6 115.8 106.3 107.5 111.1 125.6 74.13 78.39 75.95 109.9 106.6 98.33 avg 113.6 std dev 6.444 %RSD 5.674 avg 114.7 std dev 9.555 %RSD 8.327 avg 76.16 std dev 2.138 %RSD 2.808 avg 104.9 std dev 5.956 XRSD 5.676 1 28.49 201 1 30.78 201 1 3.85 201 1 3.65 201 1 3.84 201 1 177.0 201 1 186.2 201 1 181.9 201 1 5.19 100 1 4.64 100 1 4.62 100 1140 1231 154 146 154 7080 7447 7275 208 186 185 113.5 122.6 avg 111.3 std dev 12.51 %RSD 112 4 76.62 avg 75.22 A 72.64 std dev 2.243 76.42 %RSD 2.981 88.15 92.72 avg 90.48 std dev 2.285 90.58 103.3 %RSD 2.525 avg 95.85 92.34 std dev 6.437 91.94 %RSD 6.716 3M EnEvxicreol 2n00m0 ental Laboratory Blanks QCs and MSs quail sera samples summary 030418.xls printed: 4/18/2003. 8:53 AMPage 51 Report E01-1245 Study number E01-1245 - QUAIL SERA PFOS: A Reproduction Study with the Nothern Bobwhite t E0l-1245.28444 * E01-1245-28446 * E01-1245 28449 06/15/2001 06/15/2001 06/15/2001 t E01-1245.28451 06/15/2001 E0l-1245-28453 06/15/2001 t E01 1245-28456 06/15/2001 E01-1245-28458 06/15/2001 * E01-1245-28460 06/15/2001 t E01-1245-28462 06/15/2001 * E01-1245-28464 * E01-1245-28466 06/15/2001 06/15/2001 * E01-1245-28468 06/15/2001 * E01-1245-28470 06/15/2001 * E01-1245-28472 06/15/2001 * E01-1245-28474 06/15/2001 * E01-1245-28477 06/15/2001 * E01-1245-28479 06/15/2001 +Concentration <LOO d020131055 04 40 1 454-108-330.Oppm female, adult, 01-31-02 -~ d020207059 40 1 d020131057 0 05 40 1 454-108-332, Oppm female, adult 01-31-02 d020207061 o5 40 1 d020131060 03 40 1 454-108-336, Oppm female, adult, 01-31-02 d020207064 40 1 -__________ d020131062 03 40 1 454-108-338,Oppm female, adult, 01 31-02 do20207066 ,3 40 1 -__________ do201 31 069 0 2 40 1 454-108-340,Oppm female, adult, 01-31 02 d020207073 -~ ~ _ _ _ _ _ _ _ _ _ 40 1 do201 31 072 0 2 40 1 454 108-344,Oppm female, adult, 01-31 02 - ~o~0207076 40 1 d020201043 0 1 40 1 454-108-346 Oppm female, adult, 02-01-02 d020207087 40 1 ~ d020201045 0 3 40 1 454-108-348 Oppm female, adult, 02-01-02 d020207089 0 3 40 1 454 108.350, Oppm female, adult, 02-01 02 454-108-352, Oppm female. adult, 02-01-02 d020201047 0 4 40 1 ~ ~ d020207091 ~0 4 : ~4 4 4 0 0 0 : ~ :1 1 1 454-108 354, Oppm female, adult, 02-01-02 454-108-356. Oppm female, adult, 02-01-02 454-108-358. Oppm female, adult, 02-01-02 454-108-360.Oppm female, adult, 02-01-02 454-108-362,Oppm female, adult, 02-01-02 454-108-366, Oppm female, adult, 02-01-02 454-108-368. Oppm female, adult, 02-01-02 40 1 dd002200220071009540 0 3 40 1 d020201052 40 1 d020207096 0 3 40 1 ~ : ~ ~ : d020201059 40 1 d020207103 0 3 40 1 0.3 40 1 o,3 40 1 40 1 d020201063 O2 d020207107 0.2 40 1 40 1 dd002200220071101606 O2 0.2 40 1 40 1 d020201073 0.3 d020207117 0.3 40 1 10 0 - 10 0 100 -roo100 10 0 10 0 10 0 10 0 100 10 0 10 0 10 0 100 10 0 10 0 10 0 ~ ~10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 ~1 0 51.27 54.43 ~~~~~~~ 10.0 10.0 10.0 10.0 10.0 10.0 ~0 400 400 400 400 400 400 400 400 400 400 400 400 400 400 -~ 400 400 - 400 400 400 400 400 400 400 400 400 ~ 400 400 400 . ~~ 400 400 ~~ ~~~ ~~ ~~ 400 400 ~~ ~ 400 400 400 400--400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 ~400 2051 2177 400 400 400 400 400 400 + 400 + + 400 -+_+ + + 400 + + 400 - + + 400 + + 400 + + 400 + ~~ 400 __ + + 400 + 400 + -- 400 + ~~ + 400 + . ?. + 400 . __ -~~ rejected rejected 400 + 400 + + + 400 There are two results reported for these samples. Both values are included in the average 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 = amount of sera used/total amount of sera + water. For all samples >0.05 mL this value is 0.05 mL. For samples 4 0 5 mL this value is = t o the amount of sera used For example, sample E01-1245.28519- dilution(di1uted sera)= 2.0 mL final volumel0.02 mL amount of sera used = 100 With outliers avg 500.8 std dev 409.7 %RSD 81.80 Without outliers avg 400 - std dev 0 %RSD 3M EnEvxicrelo20n0m0 ental Laboratory summary by sex quail sera samples summaly 030418.xls printed: 4/18/2003, 853 AMPage 52 Report E01-1245 Study number E01-1245 - QUAIL SERA PFOS: A Reproduction Study with the Nothern Bobwhite E01-1245-28485 28487 28489 28491 E0l-1245-28493 E01-1245-28496 E01-1245-28498 E01-1245-28500 E01-1245-28502 E01-1245-28504 EO1-1245-28506 E01-1245-28508 E01-1245-28511 E01-1245-28513 E01-1245-28515 E01-1245-28517 * E01-1245-28518 06/J5/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 0611 512001 0611 5/2001 0611 5/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06115/2001 06115/2001 06/15/2001 07/23/2001 * E01-1245-28521 * E01-1245-28524 07/23/2001 07/23/2001 * E01-1245-28525 07/23/2001 * E01-1245-28528 07/23/2001 * E01-1245-28530 07/23/2001 * E01-124528532 07/23/2001 * E01-1245-28535 07/23/2001 ~~ ~ E01-1245.28536 07/232001 + Concentration <LOQ 454-108-374, 10ppm female.adult 02-01-02 454 108-376 lOppm female, adult 02-01-02 454 108-378. lOppm female adult, 02 01 02 454-108 380 lOppm female, adult, 02 01 02 454-1013 382. IOppm female, adult 02-01-02 454-108-386. IOppm female, ad@, 02-01-02 454-108-388. IOppm female, adult, 02-01-02 454-108 390, IOppm female, adult. 02-01-02 454-108-392. IOppm female, adult, 02-01-02 454-108-394. IOppm female, adult, 02-01-02 454-108-396,lOppm female, adult, 02-01-02 454-108-398, lOppm female, adult, 02-01-02 454-108-402,loppm female, adult, 02-01-02 454-108-404,lOppm female, adult, 02-01-02 454-108-406,lOppm female, adult, 02-01-02 454 108-408, lOppm female, adult, 02 01 02 454-108-246 Oppm female, offspring, 02 01 02 4020203028 -d020%3029 d020203030 d020203031 dgO416019 d020?F3033 d020203CJM d020203038 d020203039 d0202030$0 d020203041 d020203042 d020203043 d020203044 d020203045 d020203046 dd002200220071115332 454-108 259, Oppm female offspring. 02 01 02 454-108-273 Oppm female, offspring, 02 01 02 454-108-277, Oppm female, offspring, 02-01-02 454-108-517, lOppm female, offspring, 02-01-02 454-108-525, loppm female. offspring, 02-01-02 454-108-538,lOppm female, offspring, 02-01-02 454-108-557, loppm female. offspring, 02-01-02 454-108-561, lOppm female, offspring,02-01-02 ~~~~~~~~~ dd002200220017115398 dd002200220071116309 dd002200220071116487 dd0022002200711147'0 dd002200220071172 d020201154 do20207175 dd002200220017117565 0 2 03 0 1 OL-_ 0 3 04 03 p 02 03 03 0 09 01 04 01 02 0 2 0 1 0 08 O2 0.2 00.'0022 00..0033 00.'0088 00..006E 00.'0055 40 I 210 00 40 1 231 92 40 1 124 46 4E ~ 1 144 86 40 - 10 7981 ~ 40 1 367 26 400 -~ 8400~ 400 ~- 9277__ 400 -~ 4978 - - 400 5794 201 31 924 400 14690 __ ___ __ - _ _ _ rejected 40 _ - - 1 289 04 - - ~ 400 11562 ~ 40 1 157 64 400 6306 With outliers 40 1 --- 235 08 400 'Lo 1 284 92 400 9403 11397 -- avg 9990 std dev 6010 40-_ - -1 -- 11784 400 4714 ~ %RSD 60.2 40 - 1 261 76 400 10470 . . 40 - 1 167 91 400 6716 - Without outliers 40 ~~1 226 40 4 0- 1 224 49 40 1 196 03 40 1 10 0 40 1 10 0 40 1 10 0 40 1 100 40 1 10 0 40 1 10 0 40 1 10.0 40 1 10.0 400 9056 ~~ ~~ 400 _ - 8980 400 7841 400 400 + 400 400 + 400 400 400 + 400 400 400 + ~-_____ 400 400 + 400 400 -+ 400 -- 400 400 + 400 400 400 + avg 8699 std dev 2557 %RSD 29.4 avg 400 std dev 0 %RSD - 100 1 185.25 400 100-~~ - -~ 1 166.M 400 18525 16606 17566 66.67 66.67 1 192.74 400 12849 12452 1 180.81 400 1?054 ~ ~ ~~ ~~~~~p 40 0 4 - - 1 - 330645..8434- - 440000 L - 1124169138- ~1- 3405 - . ~ --- 40 1 208.24 400 40 . 186.59 400 8330 7897 7464 ~ - ~ ~ - ~~- - ~ ~ _ _ _ _ _ _ _ _ _ _ avg of dups 12423 40 1 287.26 400 40 1 252.41 400 11490 10096 10793 Std dev 3457 %RSD 27.8 * There are two results reported for these samples. Both values are included in the average Conc of PFOS in Sample (ng/ml) = (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 = amount of sera used/total amount of sera +water. For all samples >0.05 mL this value is 0.05 mL. For samples <0.05 mL this value is = to the amount of sera used. For example, sample E01-1245-28519- dilution(di1utedsera)= 2.0 mL final volume/0.02 m L amount of sera used = 100 3M EnEvxcierlo20n00mental Laboratory summary by sex quail sera samples summary 030418.~1s printed. 4/18/2003. 8:53 AMPage 53 Report E01-1245 Study number E01-1245 - QUAIL SERA PFOS: A Reproduction Study with the Nothern Bobwhite * E01-1245-28443 - * E01-1245-28445 * E01-1245-28447 0611 5/2001 06/15/2001 0611 5/2001 E01-1245-28448 0611WOO1 * 01-1245-28450 * E01-1245-28452 * E01-1245-28454 * E01-1245-28455 * E01-1245.28457 * E01-1245-28459 0611 5/2001 06/1 5/2001 06/15/2001 06/15/2001 06/1 5/2001 06/15/2001 * E01-1245-28461 * E01-1245.28465 * E01-1245-28467 * E01-1245-28469 06/15/2001 0611 5/2001 06/15/2001 06/1 5/2001 E01-1245-28471 06/15/2001 * E01-1245-28473 06/15/2001 * 01-1245-28475 - p * E0l-1245.28476 -~ * E01-1245-28478 06/15/2001 06/15/2001 06/15/2001 +Concentration <LOCI 454-108-329.Oppm male, adult. 01-31-02 p - ~ ~- 454-108-331, Oppm male, adult, 01-31-02 - 454-108-333.Oppm male, adult, 01-31-02 _______~ 454-108-335.Oppm male, adult, 01-31-02 454-108-337, Oppm male, adult. 01-31-02 454-108-339. Oppm male, adult, 01-31-02 454-108-341. Oppm male, adult, 01-31-02 ~p 454-108-343,Oppm male, adult, 01-31-02 454-108-345,Oppm male, adult, 01-31-02 454-108-347, Oppm male, adult, 02-01-02 454-108-349. Oppm male, adult. 02-01-02 454-108-353.Oppm male, adult. 02-01-02 454-108-355,Oppm male, adult, 02-01-02 454-108-357,Oppm male, adult, 02-01-02 454-108-359,Oppm male, adult, 02-01-02 454-108-361, Oppm male, adult, 02-01-02 454-108-363, Oppm male, adult, 02-01-02 454-108-365,Oppm male, adult. 02-01-02 454-108-367,Oppm male, adult. 02-01-02 d020131054 d020207058- - do20131056 d020207060 do20131 058 d020207062 do20131 059 d020207063 do20131061 d020207065 do20131063 d020207067 do20131070 d020207074 d020131071 d020207075 do20131073 d020207077 d020201044 d020207088 d020201046 d020207090 d020201049 d020207093 d020201051 d020207095 d020201058 d020207102 d020201060 d020207104 d020201062 d020207106 d020201064 d020207108 d020201006 d020207109 d020201067 d020207111 02 0.2 ~~ 0.4 0.4 0.4 0.4-0.2 0.2 0.1 0.1- 0.2 0.2 0.2 0.2 0.1 0.1 0.2 0.2 0.2 0.2 0.02 0.02 0.3 0.3 0.2 0.2 0.07 0.07 0.2 0.2 0.2 0.2 0.1 0.1 0.4 0.4 0.1 01 40 1 40 1 40 1 - 40 ~ 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 100 1 100 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 100 loopp 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 ~ 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 ~~~ p ~ ~ 400 400 400 400 4M) 400 - ~ 400 400 ~ 400 400 400 400 400 400 400 p~~~ ~ 400 400 50% 400 400 ~ 400 400 400 400 --400 ~An-n 400 400 400 400 400 p-pp 400 400 400 400 ~~ 400 400 400 + 400 ++ 400 +~~ + 400 + . - - ~ - ~ p + 400 + - - ~ - p p + 400 + p~ + 400 + p - + 400 _ _ -- - + ~p + 400 + p~ + 400 + + p +p + + + + + 400 _ - p _ 400 p ~ 400 400 + 400 -Lp ~ + + 400 + 400 *++ -- -- + + + + 400 400 _.. ^-^4uu - ~ - avg 400 std dev 0 %RSD - There are two results reported for these samples. Both values are included in the average Conc of PFOS in Sample (nglml) = (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 o i sera in diluted sample =amount of sera useUtotal amount of sera +water. For all samples ~ 0 . 0 5mL this value is 0.05 mL. For samples 4 0 5 mL this value is = t o the amount of sera used. For example, sample E01-1245.28519- dilution(diluted sera)= 2.0mL final volume/0.02 rnL amount of sera used = 100 3M Environmental Laboratory Excel 2000 summary by sex quail sera samples summary 030418.xls printed: 4/18/2003, 853 APM age 54 Report E01-1245 Study number E01-1245- QUAIL SERA PFOS: A Reproduction Study with the Nothern Bobwhite or t E01-1245-28480 06/15/2001 454-108-369. 10ppm male, adult, d100.02-01-02 do20201074 0.2 -________~ ~ d020g07118 40 100 26.73 400 40 100 ~29-20 400 * E0l-1245-28482 * E01-1245.28484 0611 5/2OOl 06/15/2001 454-108-371, lOppm male, adult, d100.02-01-02 - ~- 454-108-373, lOppm male, adult, d100.02-01-02 d020201076 d020ZOzg do20201 078 d020207m 02 0~ 2 01 OL 40 100 45 31 400 40- 100 445 400 40 100 35 90 400 40- - - 100 ~ 3913- 400 t E0l-1245-28486 06/132001 454-108-375. 1Oppm male, adult. d100,02-01-02 d020201080 03 d020207121 03 40 100 34 52 400 40 ~- - 100 ~ 34% 400 * E01-1245-28488 06/15/2001 454-108-377. lOppm male, adult, d100,02-01-02 -~ do20201082 d0202071Z 0 07 0 07 40 100 23 60 400 40 1 00 ~~ 24 03 400 * E01-1245-28490 06/15/2001 454-108-379. lOppm male, adult, d100,02-01-02 d020201089 01 40 100 37 59 400 d020207128 01 40 10% ~ 41 62 400 d020201091 01 E01-1245-28492 06/15/2001 454-108-381, lOppm male, adult, d100.02-01-02 do20207129 01 40 100 35 26 400 40 100 37 13 400 -- ~~~ ~ * E01-1245-28494 06/15/2001 454-108-383, lOppm male, adult, d100,02-01-02 d020201093 03 d020207130 03 40 100 36 65 400 40 1?!- 39 06 400 * E01-1245-28495 06/15/2001 454-108-385, lOppm male, adult, d100,02-01-02 d020201094 01 do20207131 01 40 100 29 64 400 40 1 0 0 30 68 400 * E01-1245-28497 06/15/2001 454-108-387, lOppm male, adult, d100,02-01-02 d0202O1096 03 d020207132 03 40 100 43 62 400 40 - 100 49 02- 400 * E01-1245-28499 06/15/2001 454-108-389. 10ppm male, adult, d100,02-01-02 do20201 103 01 40 100 32 26 400 do20207133 01 40 ~- 100 -3~3 91 400 E01-1245-28501 06/15/2001 454-108-391. lOppm male, adult, dl00,02-01-02 do20201 105 0 06 40 100 35 28 400 do20207134 0 06 40 ~ 100 36 72 400 E01-1245-28503 06/15/2001 454-108-393. 1Oppm male. adult, d100,02-01-02 do20201 107 02 dg20207135 02 40 100 40 06 400 40 100 41 16 400 * E01-1245-28505 06/15/2001 454-108-395, lOppm male, adult, d100,02-01-02 d020201109 02 do20207136 02 * E01-1245-28507 06/15/2001 454-108-397. 1Oppm male. adult. d100,02-01-02 dO20201111 03 d020207137 03 40 100 31 80 400 40 _100 _ ~~ ~33 93 400 40 1 00 24 90 400 40 1 0 0 - -__2_6__63__ 400 E01-1245.28509 06/15/2001 454-108-399. lOppm male, adult. d100,02-01-02 do20201118 0 1 do20207143 01 40 100 34 45 400 40 100 -39 62- 400 t E01-1245-28510 06/15/2001 454-108-401, 1Oppm male, adult, d100,02-01-02 do20201 119 01 do20207144 01 40 100 50 85 400 40 ~ 1% 56 60 400 I E01-1245-28512 06/15/2001 454-108-403, lOppm male, adult, d100,02-01-02 do20201 121 02 do20207145 02 * E01-124528514 06/15/2001 454-108-405. lOppm male, adult, d100,02-01-02 d020201123 01 do20207146 01 E01-1245-28516 06/15/2001 454-108-407. lOppm male, adult. d100.02-01-02 do20201125 01 do20207147 0.1 40 100 31 64 400 40 100 ~34 39 400 40 100 26 94 400 40 - - 100 29 23 400 40 100 24 79 400 40 100 27.90 400 106920 1 16800 ~~~--- lllUbU 181240 179440 180340 ~~ 143600 156520 - 150060 138080 137920 94400 96120 138000 ~- - 95260 150360 166480 158420 141040 148520 146600 156240 118560 122720 - ~ 144780 151420 ~___ 120640 174480 196080 185280 129040 135640 141120 146880 132340 ~- 144000 160240 164640 127200 135720 - _ 162440 - _ - 131460 99600 106520 ~- 103060 -~ 137800 158600 148200 203400 226400 214900 126560 137560 107760 116920 132060 ~-- 1 12340 - 99160 111600 4nllon 4 "_).J)o" There are two results reportedfor these samples. Both values are included in the average Conc of PFOS in Sample (nglml) = (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 = amount of sera used/total amount of sera + Water. For all samples ~0.05mL this value is 0.05 mL. For samples <0.05 mL this value is = to the amount of sera used. For example, sample 01-1245-28519. dilution(di1utedsera)= 2.0 mL final volume/0.02 rnL amount of sera used = 100 avg of dups 141112 std dev 30225 %RSD 21.4 3M EnEvxcierlo20n00mental Laboratory summary by sex quail sera samples summary 030418.xls printed: 4/18/2003, 8.53 AM Page 55 Report E01-1245 Study number E01-1245 - QUAIL SERA PFOS: A Reproduction Study with the Nothern Bobwhite * E01-1245-28519 * E01-1245-28520 * EOl-1245-28522 * E01-1245-28523 07/23/2001 07/23/2001 07/23/2001 07/23/2001 E01-1245-28526 * E01-1245-28527 E01-1245-28529 * E01-1245-28531 07/23/2001 07/23/2001 07/23/2001 07/23/2001 E0l-1245-28533 07/23/2001 * E0l-1245-28534 07/23/2001 t E01-1245-28537 07/23/2001 + Concentration <LOQ d020201133 0.02 100 454-108-253, Oppm ma.le_ , offspring, 02-01-02~~ _ _ do20207154 _-o.02pppp_p1100 d020201134 0.02 454-108-256.Oppm male, offspring, 02-01-02 do20207155 0.02 ~ p ~ ~ ~ _ _ _ _ _ ~ _ 454-108-266,Oppm male, offspring, 02-01-02 d020201136 d020207157 0.07 o,07 ~ _ 100 100 40 ~40 ~ ~ 454-108-270.Oppm male, offspring, 02-01-02 - 454-108-283, Oppm male, offspring, 02-01-02 454-108-291, Oppm male, offspring, 02-01-02 d020201137 d020207158 d020201140 ~~ d020207161 d020201141 d020207162 0.2 0.2 0.02 0.02 0.05 0.05 40 40 100 loo_-- 40 40 454-108-522. 10ppm male, offspring, 02-01-02 d020405091 0.07 ~ 40 454-108-533, IOppm male, offspring, 02-01-02 _~ ~~ ~~~ ~~ 454-108-545, IOppm male, offspring, 02-01-02 ~~ d020201150 0.07 d020207_171- ~ -0,07_ ~ d020201152 0.04 do202071E ~~_ ~0-.04~_~ 40 40 50 50 ~~ d020201153 0.07 40 454-108-552, IOppm male, offspring, 02-01-02 d020207174 0.07 40 ~_ ~ ~ ~ ~ ~ _ _ _ _ _ _ _ _ 40 454-108-568, loppm male. offspring, 02-01-02 dd0022002200711157'7 0.1 40 1 10.0 400 10.0~~~~ 400 o e 1 10.0 400 1 1 400 1 10.0 400 1 10.0 ~p~~~ ~ 400 1 10.0 400 10.0 400 1 10.0 400 1 ~ ~ _ _1_0_.0_ _ _ _ _ _ - 400 1 10.0 400 1 10.0 400 10 48.13 201 1 232.67 400 1 205.40 400 1 309.79 400 1 ~ 272.51 400 ~ 1 339.60 400 1 ~~~ 286.47 _ 400 1 301 .89 400 1 260.08 400 400 ~ 400 400 400 400 -~ 400 400 .. .. 400 400 ~~ .. 400 400 400 _____ 19252 9307 . 8216 15490 ~ 13626 _ 13584 11459 12076 10403 + 400 ++ _____________ + 400 + 400 + + --..5 . + +... + + ~p_______ __ 400 ~~~p~~ 400 ~..... ~ - ~ p 400 avg 400 std dev 0 %RSD - ~~ ~_ 19252 8761 .~~ ~-~ ~ _ _ _ _ _ _ _ _ _ 14558 ~ p ~ 12521 11239 avg 12601 std dev 3374 %RSD 26.8 There are two results reported for these samples. Both values are included in the average Conc of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(dilu1ed sera)) + dilution(dilu1edsera) =final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample = amount of sera usedtotal amount of sera water. For all samples >0.05 mL this value is 0.05 mL. For samples 4 0 5 mL this value is = to the amount of sera used For example, sample E01-1245-28519-dilution(dilu1edsera)= 2.0 mL final volurne/0.02 m L amount of sera used = 100 3M Environmental Laboratory Excel 2000 summary by sex quail sera samples summary 030418.xIs printed 4/18/2003, 8:53 AMPage 56 Report E01-1245 Study number E01-1245- QUAIL LIVER DATA SUMMARY, BLANKS AND SPIKES PFOS: A Reproduction Study with the Nothern Bobwhite 01-1342-36000 02/1W O O 2 Blank HPO-day 1 d020211037 1.oooo 9.00 E01-1342-36026 02/12/2002 Blank HPO-day 2 d020212025 1.oooo 9.00 E01-1342-36031 02/13/2002 Blank HPO-day 3 d020301024 1.oooo 9.00 E01-1342-36036 02/15/2002 Blank HPO-day 4 d020218025 1.oooo 9.00 E01-1342-36001 02/11/2002 Blank female liver-day 1 d020211038 5.0353 45.0 E01-1342-36027 02/12/2002 Blank female liver-day 2 d020212026 5.0353 45.0 E01-1342-36032 02/13/2002 Blank female liver-day 3 d020301025 5.0353 45.0 E01-1342-36037 02/15/2002 Blank female liver-day 4 d020218026 5.0353 45.0 E01-1342-36002 02/11/2002 Blank male liver-day 1 d020211039 1.0015 9.00 E01-1342-36028 02/12/2002 Blank male liver-day 2 4020212027 1.0015 9.00 E01-1342-36033 02/13/2002 Blank male liver-day 3 4020301026 1.0015 9.00 E01-1342-36038 02/15/2002 Blank male liver-day 4 d020218027 1.0015 9.00 2.00 0.100000 1 2.51 50.2 50.2 2.00 0.100000 1 2.51 50.2 50.2 2.00 0.100000 1 0.502 10.0 10.0 2.00 0.100000 1 1.oo 20.0 20.0 2.00 0.100635 1 2.51 50.2 50.2 2.00 0.100635 1 2.51 50.2 50.2 2.00 0.100635 1 0.502 10.0 10.0 2.00 0.1 00635 1 1 .oo 20.0 20.0 2.00 0.100135 1 2.51 50.2 50.2 2.00 0.100135 1 2.51 50.2 50.2 2.00 0.100135 1 0 502 10.0 10.0 2.00 0.1001 35 1 1.oo 20.0 20.0 EOl-1342-36029 02/12/2002 Female liver MS-day 2 d020212028 5.0353 45.0 2.00 E01-1342-36034 02/13/2002 Female liver MS-day 3 d020301027 5.0353 45.0 2 00 E01-1342.36039 02/15/2002 Female liver MS-day 4 d020218028 5.0353 45.0 2.00 E01-1342-36004 02/11/2002 Female liver MSD-day 1 d020211041 5.0353 45.0 2.00 E01-1342.36030 02/12/2002 Female liver MSD-day2 d020212029 5.0353 45.0 2.00 E01-1342-36035 02/13/2002 Female liver MSD-day 3 d020301028 5.0353 45.0 2.00 EOI-1342.36040 02/15/2002 Female liver MSD-day 4 d020218029 5.0353 45.0 2.00 * Concentration cLOQ Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL))x (additionaldilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (9) = amount of liver weighed (g)/ (water added for homogenizing (rnL=g)) + amount of liver weighed(g)) 0.100635 0.100635 0.1 00635 0.100635 0.100635 O.lOE35 0.100635 1 28.77 50.2 1 26.03 10.0 1 26.33 20.0 1 29.78 50.2 1 28.53 50.2 1 26.99 10.0 1 27.02 20.0 for both ms and msd std dev of % rec 5.514 avg conc. 552.6 true Val. 499 572 114.6 avg %rec 109.7 517 103.7 avg conc 547.2 523 104.9 std dev of conc 31.25 592 118.6 true Val. 499 567 113.6 avg%rec I1I.8 536 107.5 avg conc 558.1 537 107.6 Stddev Of conc 26.67 3M Environmental Laboratory Excel 2WO Blanks QCs a n d MSs quail liver samples summary 030418 XIS printed 4/18/2003. 8 52 AMPage 57 Report E01-1245 Study number E01-1245 - QUAIL LIVER versus unertracted curve nrininil E01-1342-36017 qc-10ppb-1 (200ngJg) do20211047 5.0353 45.0 E01-1342-36018 qc-IOppb-2 (POOng/g) d020211048 5.0353 45.0 E01-1342-36019 qc-l0ppb-3 (2OOnglg) d020211049 5.0353 45.0 EO1-1342-36020 qc-75ppb-1 (1496 ngg) d020211050 5.0353 45.0 E01-1342-36021 qc-75ppb-2 (1496 ng/g) d020211051 5.0353 45.0 E0l-1342-36022 qc-75ppb-3 (1496 ng/g) d020211052 5.0353 45.0 EOI-1342.36023 qc-150ppb-l (2993 ng/g) d020211053 5.0353 45.0 E01-1342.36024 qc-150ppb-2(2993 ng/g) do20211054 5.0353 45.0 E01-1342-36025 qc-150ppb-3(2993 nq'g) do20211055 5.0353 45.0 Study number E01-1245 - QUAIL LIVER PFOS: A Reproduction Study with the Nothern Bobwhite Absolute Recoveries 2.00 0.100635 1 13.59 50.2 270 A 135.0 avg 131.2 2.00 0.100635 1 12.74 50.2 253 A 126.6 std dev 4.274 2.00 0.100635 1 13.28 50.2 264 A 132.0 %RSD 3.258 2.00 0.100635 1 95.65 50.2 1905 A 127.3 avg 123.4 2.00 0.100635 1 90.27 50.2 1794 119.9 std dev 3.725 2.00 0.100635 1 92.58 50.2 1840 123.0 %RSD 3.018 2.00 0.100635 1 169.63 50.2 3371 112.6 avg 113.1 2.00 0.100635 1 167.02 50.2 3319 110.9 std dev 2.395 2.00 0.100635 1 174.15 50.2 3461 115.6 %RSD 2.119 lor all QC in original analysis (absolute recovely) E01-1342-36017 qc-l0ppb-1 (200ngJg) do20405136 5 0353 45 0 200 E01 1342 36018 qc-loppb-2 (2OOn.gl~ g) do20405137 5 0353 45 0 2 00 E01-1342-36019 qc-1Oppb-3 (2OOnglg) d020405138 5.0353 45.0 2.00 E01-1342-36020 qc-75ppb-l (1496 ng/g) do20405139 5.0353 45.0 2.00 E01-1342-36021 qc-75ppb-2(1496 n. g/. g) d020405140 5.0353 45.0 2.00 E01-1342-36022 qc-75ppb-3 (1496 ng/g) d020405141 5.0353 45.0 2.00 E01-1342-36023 qc-l5Oppb-1 (2993 ndg) d020405142 5.0353 45.0 2.00 E01-1342-36024 qc-150ppb-2(2993 ng/g) d020405143 5.0353 45.0 2.00 E01-1342-36025 qc-150ppb-3(2993 ng/g) do20405144 5.0353 45.0 2.00 A = Not within +/-25% criteria. Conc of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additionaldilution) x (linal volume (mL))/amount of liver in diluted sample (9) amount 01 liver in diluted sample (9) = amount 01 liver weighed (g)/ (water added lor homogenizing(mL=g)) + amount 01 liver weighed(g)) 0 100635 0 100635 0.100635 0.100635 0.100635 0.100635 0 100635 0.100635 0.100635 1 14 86 100 1 14 24 100 1 14.77 100 1 100.51 100 1 94.89 100 1 101.10 100 1 187.94 100 1 172.20 100 1 180.27 100 295 A 283 A 294 A 1998 A 1886 A 2009 A 3735 3422 3583 lor all QC in rerun analysis (absolute recovery) 147 7 141 5 146.8 133.5 126.1 134.3 124.8 114.3 119.7 avg 145 3 Std dev 3 329 %RSD 2.291 avg 131.3 std dev 4.554 %RSD 3.468 avg 119.6 std dev 5.226 %ASD 4.369 3M Environmental Laboratory Excei 2WO Blanks QCs and MSs quail liver samples summary 030418.~1s Page 58 printed: 4/18/2003. 8:52 AM Report E01-1245 Study number E01-1245 - QUAIL LIVER versus extracted curve onainal PFOS: A Reproduction Study with the Nothern Bobwhite QC Sample Recoveries E01-1342-36018 qc-l0ppb-2 (200ng/g) d020211048 5.0353 45.0 E01-1342-36019 qc-l0ppb-3 (ZOOnq'g) d020211049 5.0353 45.0 E01-1342-36020 qc-75ppb-1 (1496 ng/g) d020211050 5.0353 45.0 E01-1342-36021 qc-75ppb-2 (1496 nq'g) d020211051 5.0353 45.0 E01-1342-36022 qc-75ppb-3 (1496 ng/g) d020211052 5.0353 45.0 E01-1342-36023 qc-150ppb-1 (2993 nq'g) d020211053 5.0353 45.0 EOI-1342.36024 qc-150ppb-2 (2993 nq'g) d020211054 5.0353 45.0 E01-1342-36025 qc-150ppb-3 (2993 nq'g) d020211055 5.0353 45.0 Study number E01-1245 - QUAIL LIVER versus extracted curve rerun 2.00 0.100635 1 13.41 50.2 267 A 133.3 Std dev 3.971 2.00 0.100635 1 13.91 50.2 276 A 138.2 %RSD 2.887 2.00 0,100635 1 89.60 50.2 1781 119.0 avg 115.5 2.00 0.100635 1 84.54 50.2 1680 112.3 std dev 3.378 2.00 0.100635 1 86.63 50.2 1722 2.00 0.100635 1 155.46 50.2 3090 115.1 103.2 %RSD 2.925 avg 103.6 2.00 0.100635 1 153.16 50.2 3044 2.00 0.100635 1 159.43 50.2 3168 101.7 105.9 std dev 2.106 %RSD 2.033 for all QC in original analysis (recovery) 01-1342-36017 qc-lOppb-1 (POOng/g) do20405136 5.0353 45.0 2.00 E01-1342-36018 qc-IOppb-2 (PWng/g) d020405137 5.0353 45.0 2.00 E01-1342-36019 qc-IOppb-3 (200ng/g) d020405138 5.0353 45.0 2.00 E01-1342-36020 qc-75ppb-I (1496 ng/g) do20405139 5.0353 45.0 2.00 EOl-1342-36021 qc-75ppb-2 (1496 nq'g) do20405140 5.0353 45.0 2.00 E01-1342-36022 qc-75ppb-3 (1496 ng/g) d020405141 5.0353 45.0 2.00 E01-1342.36023 qc-150ppb-1 (2993 ng/g) d020405142 5.0353 45.0 2.00 E01-1342-36024 qc-150ppb-2 (2993 ngg) d020405143 5.0353 45.0 2.00 E01-1342-36025 qc-l50ppb-3 (2993 nq'g) d020405144 5.0353 45.0 2.00 A = Not within +/-25% critena. Conc of PFOS in Sample (ng/g) = (Inst conc(ng/mL))x (additionaldilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (9) = amount of liver weighed (g)/ (water added lor homogenizing (mL=g))+ amount of liver weighed(g)) 0.100635 0.100635 0.100635 0.100635 0.100635 0,100635 0.100635 0.100635 0.100635 1 13.43 100 1 12.91 100 1 13.35 100 1 84.61 100 1 79.97 100 1 85.09 100 1 155.84 100 1 143.14 100 1 149.66 100 267 A 257 A 265 A 1682 1589 1691 3097 2845 2974 for all QC in rerun analysis (recovery) 133.5 128.3 132.7 112.4 106.2 113.0 103.5 95.05 99.38 avg 131.5 std dev 2.782 %RSD 2.1 16 avg 110.6 sld dev 3.756 %RSD 3.398 avg 99.30 std dev 4.217 %RSD 4.247 3M EnEvxicrelo20n00mental Laboratory Blanks QCs a n d MSs quail liver samples summary 030418 . ~ 1 ~ printed: 4/18/2003. 8-52AMPage 59 Report E01-1245 Study number E01-1245 - QUAIL LIVER DATA SUMMARY PFOS: A Reproduction Study with the Nothern Bobwhite E01-1342-30339 E01-1342-30357 06/19/2001 0 ppm a.i. 332 F Liver 04/30/2001 0 ppm a.i. 334 F Liver d020211064 d020211066 E01-1342-30375 06/19/2001 0 ppm a.i. 336 F Liver d020211068 E01-1342-30393 06/19/2001 0 ppm a i 338 F Liver d020211070 E01-1342-30411 E01-1342-30429 06/19/2001 0 ppm a.i. 340 F Liver 05/31/2001 0 ppm a.i. 342 F Liver d020212034 d020212036 E01-1342-30447 06/19/2001 0 ppm a.i. 344 F Liver d020212038 E01-1342-30465 06/19/2001 0 ppm a.i. 346 F Liver d020212040 E01-1342-30483 06/19/2001 0 ppm a.i. 348 F Liver d020212042 E01-1342-30501 06/19/2001 0 ppm a.i. 350 F Liver d020212047 E01-1342-30519 06/19/2001 0 ppm a.i. 352 F Liver d020212049 E01-1342-30537 06/19/2001 0 ppm a.i. 354 F Liver d020212051 E01-1342-30555 06/19/2001 0 ppm a.i. 356 F Liver d020212053 E01-1342-30573 06/19/2001 0 ppm a.i. 358 F Liver d020212055 E01-1342-30591 06/19/2001 0 ppm a.i. 360 F Liver d020212059 EO1-1342-30609 06/19/2001 0 pprn a.i. 362 F Liver dO20212061 EO1-1342-30627 06/19/2001 0 ppm a.i. 364 F Liver d020212063 E01-1342-30645 06/19/2001 0 ppm a.i. 366 F Liver d020212065 E0l-1342-30663 06/19/2001 0 ppm a.i. 368 F Liver d020212067 E01-1342-30681 06/20/2001 10 ppm a.i. 370 F Liver d020301032 E01-1342-30699 06/20/2001 10 ppm a.i. 372 F Liver d020301033 E01-1342-30717 06/20/2001 10 ppm a.i. 374 F Liver d020301034 E01-1342.30735 06/20/2001 10 ppm a.i. 376 F Liver d020301035 E01-1342.30753 06/20/2001 10 ppm a.i. 378 F Liver d020301036 E01-1342-30771 06/20/2001 10 ppm a.i. 380 F Liver d020301037 E01-1342-30789 06/20/2001 10 ppm a.i. 382 F Liver d020301038 E01-1342-30820 04/11/2001 10 ppm a.i. 384 F Liver d020301039 E01-1342-30838 06/20/2001 10 ppm a.i. 386 F Liver d020301040 E01-1342-30856 06/20/2001 10 ppm a.i. 388 F Liver d020301041 E01-1342-30874 06/20/2001 10 ppm ai. 390 F Liver d020301045 EO1-1342-30892 06/20/2001 10 ppm a.i. 392 F Liver d020301046 E01-1342-30910 06/20/2001 10 ppm a.i. 394 F Liver d020301047 E01-1342-30928 06/20/2001 10 ppm a.i. 396 F Liver d020301048 E01-1342-30946 06/20/2001 10 ppm a.i. 398 F Liver d020301049 E01-1342.30964 06/20/2001 10 ppm a.i. 400 F Liver d020218033 E01-1342-30982 06/20/2001 10 ppm a.i. 402 F Liver d020218034 E01-1342-31000 06/20/2001 10 ppm a.i. 404 F Liver d020218035 EO1-1342-31018 06/20/2001 10 ppm a.i. 406 F Liver d020218036 E01-1342-31036 06/20/2001 10 ppm a.i.408 F Liver d020218037 E01-1365-31184 07/23/2001 0 ppm a.i. 246 F Liver, Offspring d020218038 E01-1365-31204 07/23/2001 0 ppm a.i. 259 F Liver, Offspring d020218041 E01-1365-31219 07/23/2001 0 ppm a.i. 273 F Liver, Offspring d020218047 E01-1365-31224 07/23/2001 0 ppm a.i. 277 F Liver, Offspring do20218048 E01-1365-31240 07/23/2001 10 ppm a.i. 517 F Liver, Offspring d020218051 E01-1365-31250 07/23/2001 10 ppm a.i. 525 F Liver, Offspring do20218053 E01-1365-31260 E01-1365-31275 07/23/2001 10 ppm a.i. 538 F Liver, Offspring do20405158 07/23/2001 10 ppm a.i. 557 F Liver, offspring d020218061 E01-1365-31280 07/23/2001 10 ppm ad. 561 F Liver, Offspring d020218062 * Concentration <LOO 1.0469 1.0232 1.01 88 1.0543 1.0182 1.0904 1.0087 1.0998 1.1036 1.0237 1.1509 1.0578 1.0312 1.1031 1.0586 1.0424 1.0364 1.0577 1,0920 1.0868 1.0748 1.0790 1.1036 1.0021 1.0825 1.0122 0.6864 1.1215 1.0878 1.0078 1.0060 1.0238 1.0370 1.0240 1.0181 1 .OB76 1.0173 1.0047 1.0044 0.7302 0.4434 0.5004 0.4326 0.6570 0.5720 0.5211 0.4654 0.9417 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 6.30 9.00 9 00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 6.30 3.60 4.50 3.60 5.40 5.40 4.50 4.50 8.10 2.00 0.104201 1 2.51 50.2 2.00 0.102083 1 2.51 50.2 2.00 0.1 01 689 1 2.51 50.2 2.00 0.1 04861 1 2.51 50.2 2.M) 0.1 01 635 1 2.51 50.2 2.00 0.108063 1 2.51 50.2 2.00 0.100782 1 2.51 50.2 2.00 0.108893 1 2.51 50.2 2.00 0.1 09228 1 2.51 50.2 2.00 0.102128 1 2.51 50.2 2.00 0.113379 1 2.51 50.2 2.00 0.1 051 72 1 2.51 50.2 2.00 0.102799 1 2.51 50.2 2.00 0.109184 1 2.51 50.2 2.00 0.1 05243 1 2.51 50.2 2.00 0.1 03800 1 2.51 50.2 2.00 0.1 03264 1 2.51 50.2 2.00 0.1 051 63 1 2.51 50.2 2.00 0.108205 1 2.51 50.2 2.00 0,107745 1 319.18 10.0 2.00 0.106682 1 236.52 10.0 2.00 0.1 07054 1 290.48 10.0 2.00 0.109226 1 217.55 10.0 2.00 0.100189 1 187.79 10.0 2.00 0,107364 1 214.22 10.0 2.00 0.1 01 097 1 166.41 10.0 2.00 0.0982460 1 206.04 10.0 2.00 0.110804 1 418.20 10.0 2.00 0.1 07833 1 187.91 10.0 2.00 0.1 00701 1 245.00 10.0 2.00 0.100540 1 298.53 10.0 2.00 0.102137 1 256.81 10.0 2.00 0.103318 1 253.83 10.0 2.00 0.102155 1 224.76 10.0 2.00 0.101626 1 255.94 20.0 2.00 0.1 07816 1 318.13 20.0 2.00 0.101554 1 275.17 20.0 2.00 0.100423 1 294.79 20.0 2.00 0.1 00396 2.00 0.103866 2.00 0.109660 2.00 0.100072 2.00 0.1 07276 1 228.77 20.0 1 1 .oo 20.0 1 1.oo 20.0 1 1.oo 20.0 1 1 .oo 20.0 2.00 0.108470 1 308.44 20.0 2.00 0.0957803 1 336.48 20.0 2.00 0.103782 10 72.30 100 2.00 0.0937286 1 219.12 20.0 2.00 0.1 04151 1 238.83 20.0 Conc of PFOS in Sample (ngig) = (Inst conc(ng/mL))x (additional dilution) x (final volume (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)) 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 50.2 50.2 50.2 50.2 50.2 50.2 5925 4434 5427 3983 3749 3991 3292 4194 7548 3485 4866 5939 5029 4914 4400 5037 5901 5419 5871 4557 20.0 20.0 20.0 20.0 5687 7026 13933 4676 4586 * * * * * * * * * * * * * * ' ' * avg 50.2 * std dev 0 + %RSD - * ' * * rejected avg 4898 std dev 1035 %RSD 21.1 avg 20.0 std dev 0 %RSD - Without outliers avg 5494 std dev 1137 %RSD 20.7 With outliers avg 7182 std dev 3900 %RSD 54.3 3M Environmental Laboratory Excel 2000 samples by sex quail liver samples summary 030418 . ~ 1 ~ Page 60 printed.4/18/2003. 8:52 AM - Report E01-1245 Studv number E01-1245 QUAIL LIVER DATA SUMMARY PFOS: A Reproduction Study with the Nothern Bobwhite EOl-l%2-30312 E01-1342-30330 E01-1342.30348 E01-1342-30366 E01-1342-30384 E01-1342-30402 E01-1342-30420 E01-1342-30438 E01-1342-30456 EOl-1342.30474 E01-1342-30492 06/19/2001 0 ppm a.i. 329 M Liver 06/19/2001 0 ppm a.i. 331 M Liver 06/19/2001 0 ppm a.i. 333 M Liver 06/19/2001 0 ppm a.i. 335 M Liver 06/19/2001 0 ppm a.i. 337 M Liver 06/19/2001 0 ppm a.i. 339 M Liver 06/19/2001 0 ppm a.i. 341 M Liver 06/19/2001 0 ppm a.i. 343 M Liver 06/19/2001 0 ppm a.i. 345 M Liver 06/19/2001 0 ppm a.i. 347 M Liver 06/19/2001 0 ppm a.i. 349 M Liver do20211061 d020211063 d020211065 d020211067 do2021 1069 d020212033 d020212035 d020212037 d020212039 d020212041 d020212046 E01-1342-30510 EOl-1342.30528 E01-1342.30546 EO1-1342-30564 E01-1342-30582 E01-1342-30600 E01-1342-30618 E01-1342-30636 06/19/2001 0 ppm a.i. 351 M Liver 06/19/2001 0 ppm a.i. 353 M Liver 06/19/2001 0 ppm a.i. 355 M Liver 06/19/2001 0 ppm a.i. 357 M Liver 06/19/2001 0 ppm a.i. 359 M Liver 06/19/2001 0 ppm a.i. 361 M Liver 06/19/2001 0 ppm a.i. 363 M Liver 06/19/2001 0 ppm a.i. 365 M Liver d020212048 d020212050 d020212052 d020212054 d020212058 d020212060 d020212062 d020212064 E01-1342-30654 06/1912001 0 ppm a.i. 367 M Liver d020212066 E01-1342-30672 EO1-1342-30690 E01-1342-30708 E01-1342.30726 E01-1342-30744 E01-1342-30762 E01-1342-30780 E01-1342-30798 E01-1342-30829 E01-1342-30847 E01-1342-30865 E01-1342-30883 E01-1342-30901 EOl-1342-30919 E01-1342.30937 EO1-1342-30955 E01-1342-30973 E01-1342-30991 E01-1342-31009 06/20/2001 10 DDm a.i.369 M Lwer 06/20/2001 10 ppm a.i. 371 M Liver 06/20/2001 10 ppm a.i. 373 M Liver 06/20/2001 10 ppm a.i. 375 M Liver 06/20/2001 10 ppm a.i. 377 M Liver 06/20/2001 10 ppm ad. 379 M Liver 06/20/2001 10 ppm a.i. 381 M Liver 06/20/2001 10 ppm a.i. 383 M Liver 06/20/2001 10 ppm a.i. 385 M Liver 06/20/2001 10 ppm a.i. 387 M Liver 06/20/2001 10 ppm a.i. 389 M Liver 06/20/2001 10 ppm a.i. 391 M Liver 06/20/2001 10 ppm a.i. 393 M Liver 06/20/2001 10 ppm a.i. 395 M Liver 06/20/2001 10 ppm a.i. 397 M Liver 06/20/2001 10 ppm a.i. 399 M Liver 06/20/2001 10 ppm a.i. 401 M Liver 06/20/2001 10 ppm a.i. 403 M Liver 06/20/2001 10 DDm a.i. 405 M Liver d020301050 do20405148 do20405149 d020301053 d020301054 d020405150 do20405151 d020301060 d020405152 d020301062 d020301063 do20301 064 do20405153 d020301066 do20301 067 d020218064 d020405159 d020405160 d020405161 E01 1342-31027 E0l-1365.31194 E01-1365-31199 06/20/2001 10 ppm a I 407 M Liver do20405162 07/23/2001 0 ppm a I 253 M Liver. Offspring do20218039 07/23/2001 0 ppm a.i. 256 M Liver, Offspring d020218040 E01-1365.31209 E0l-1365-31214 07/23/2001 0 ppm a.i. 266 M Liver, Offspring d020218042 07/23/2001 0 ppm a.i. 270 M Liver, Offspring d020218046 E01-1365-31229 07/23/2001 0 ppm a.i. 283 M Liver, Offspring d020218049 E0l-1365-31234 07/23/2001 0 ppm a.i. 291 M Liver, Offspring d020218050 E0l-1365-31245 E01-1365-31255 E01-1365.31265 07/23/2001 10 ppm a.i. 522 M Liver, Offspring do20405157 07/23/2001 10 ppm a.i. 533 M Liver, Offspring do20218054 07/23/2001 10 ppm a.i. 545 M Liver, Offspring d020218059 E01-1365-31270 E01-1365-31285 07/23/2001 10 ppm a.i. 552 M Liver, Offspring d020218060 07/23/2001 10 ppm a.i. 568 M Liver, Offspring d020218063 * Concentration <LOCI 1.0050 1.0530 1.0338 1.0062 1.0269 1.0886 1.0235 1.0090 1.1046 1.0586 1.0358 1.0583 1.0183 1.0899 1.0382 1.0941 1.0084 1.0818 1.0916 1.0327 1.0010 1.0856 1.0276 1.1008 1.0236 1.0312 1.0501 1.0110 1.0678 1.0315 1.0310 1.0947 1.0834 1.0489 1.0309 1.0276 1.0361 1.0019 1.0336 10956 0 3806 0.5947 0.2673 0.5900 0.6666 0.6072 0.7325 0.6903 0.4624 0.5353 0.7581 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9-00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9.00 9 00 3 60 5.40 2.70 5.40 6.30 5.40 6.30 6.30 4.50 4.50 7.20 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2 00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2 00 2 00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.100450 1 0,104745 1 0.103032 1 0.100558 1 0.1 02415 1 0,107904 1 0.102110 1 0.100809 1 0.109317 1 0.105243 1 0.10321 1 1 0.105217 1 0.1 01644 1 0.10801 9 1 0.103425 1 0.1 08390 1 0.100755 1 0.1 07302 1 0.108169 1 2.51 2.51 10.76 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 2.51 2.51 2.51 0.102933 1 2.51 0.100090 10 393.21 0.107639 10 422.59 0.102477 10 441.96 0.108981 10 381.61 0.102119 10 409.88 0,102799 10 466.19 0,104487 10 417.98 0.100989 10 371.06 0.1 06061 10 442.30 0.102826 10 407.70 0.102781 10 361.37 0,108443 10 381.87 0.1 07444 10 466.09 0.1 04380 10 339.50 0.1 02772 10 257.69 0.1 02477 10 322.88 0.1 03237 100 76.08 0.100171 100 72.89 0.103014 100 712 4 0 108523 100 0 0956137 1 0.0992043 I 0.0900819 1 0.0984975 1 0.0956851 1 0.101079 1 71 64 1 00 1.oo 1 .lo 1 .oo 1 .oo 1.oo 0.104159 10 71.33 0.098751 1 1 244.12 0.0931807 1 259.80 0.106309 1 318.62 0.0952614 1 310.49 Conc of PFOS in Sample (ngig) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (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)) 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 50.2 50.2 50.2 50.2 50.2 50.2 50.2 10.0 100 100 10.0 10.0 100 100 10.0 100 10.0 10.0 10.0 100 10.0 10.0 20.0 100 100 100 100 20 0 20.0 20.0 20.0 20.0 20.0 100 20.0 20.0 20.0 20.0 50.2 50.2 209 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 50.2 50.2 50.2 50.2 78571 78520 86255 70032 80275 90699 80006 73485 83405 79299 70318 70428 86760 65051 50148 63015 147389 145531 138311 132028 200 20.0 20.0 20.0 20.0 20.0 13696 4944 5576 5994 6519 * * rejected * ' * * * * * * * ' * * ' * With outliers avg 58.1 std dev 35.5 %RSD 61.0 Without outliers avg 50.2 Std dev 0 %RSD - * * * * * rejected avg 88416 Std dev 28517 %RSD 32.2 avg 20.0 std dev 0 %RSD - Without outliers avg 5758 std dev 666 %RSD 11.6 With outliers avg 7346 std dev 3591 %RSD 49.0 3M Environmental Laboratory Excel 2CQO samples by sex quailliver samples summary 03041&xis Page 61 printed: 4/18/2003. 8 52 AM Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite A~ACHMENCT: SAMPLECHROMATOGRACMASL,IBRATIOINNFORMATION, AND INSTRUMENT INFORMATION 3M Environmental Laboratory 3M Environmental Laboratory Page 62 of 226 Page 62 Report E01-1245 Report E01-1245 Methanol Solvent Blanks nethanol blank IM Environmental Lab j020212032 Srn (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite davey 07079! 12-Feb-2002,20:01:4( 1:MRM of 1 Channel ES- 6.7; TIC a47 9.3313: Are; % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 63 of 226 Page 63 Report E01-1245 Report EO1-1245 JeOH blank iM EnvironmentalLab 1020201057Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the NorthernBobwhite davev 07079: 02-Feb-2002,03:48:24 1: MRM of 1 Channel ES- 6.79 TIC 961 9.76e? Aree % J 1 0 3M Environmental Laboratory 3M Environmental Laboratory Page 64 of 226 Page 64 Report E01-1245 Report E01-1245 100- PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite davey 07079! 03-Feb-2002, 16:08:2r 6-7-6 958 1: MRM of 1 Channel ES- TIC 1.08e4 Are: % 1 3M Environmental Laboratory 3M Environmental Laboratory Page 65 of 226 Page 65 Report E01-1245 Report E01-1245 Water Blank Samples 01-1342-36026, Blank H2O-day 2 M Environmental Lab 020212025 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite davey 070793 12-Feb-2002,18:44:Ot 1: MRM of 1 Channel ES- 6.77 TIC 974 1.06eL Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 66 of 226 Page 66 Report E01-1245 Report E01-1245 Blank H2O-day 4 IM EnvironmentalLab 1020228107Sm IMn. 1x21 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite davey 07079I Ol-Mar-2002,06:19:1! 1: MRM of 1 Channel ES- 6.7E TIC 715 8.84e: Ares % 3M Environmental Laboratory 3M Environmental Laboratory Page 67 of 226 Page 67 Report E01-1245 Report E01-1245 E01-1342-36036,Blank HZO-day 4 IM EnvironmentalLab 1020218025 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northem Bobwhite davey 07079! 18-Feb-2002,12:00:3' 1: MRM of 1 Channel ES- 6.7 TIC 714 8.14eC Arez $6 3M Environmental Laboratory 3M Environmental Laboratory Page 68 of 226 Page 68 Report E01-1245 Report E01-1245 Sera Blank Samples pe, sera blank-1, day 2, 02-01-02 M EnvironmentalLab 020201032 Sm /Mn. 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite davey 07079! Ol-Feb-2002,23:11:0' 1: MRM of 1 Channel ES- 6.77 TIC 1535 I.83& Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 69 of 226 Page 69 Report E01-1245 Report E01-1245 ipe, sera blank-I, day 2, 02-01-02 IM EnvironmentalLab 1020201032Sm I.Mn.. 1x2,) 100 PFOS: A Reproduction Study with the Nothern Bobwhite uction Study with the Northern Bobwhite davey 0707% 01-Feb-2002,23:1I:O' 1: MRM of 1 Channel ES- 6.77 TIC 153: 1 .me4 Are2 % 3M Environmental Laboratory 3M Environmental Laboratory Page 70 of 226 Page 70 Report E01-1245 Report E01-1245 ;pe, sera blank-1, day 2,02-01-02 IM EnvironmentalLab 1020207036Sm (Mn,1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS A ReproductionStudy with the Northem Bobwhite davey 07079I 07-Feb-2002,14:36:1r D 0.0 % 6.79 TIC 117( 1.344 Ares % 3M Environmental Laboratory 3M Environmental Laboratory Page 71 of 226 Page 71 Report E01-1245 Report E01-1245 Female Liver Blank Samples 01-1342-36001 Blank female liver-day 1 M Environmental Lab 320211038 Sm (Mn, 1x2) PFOS: A Reproduction Study with the Nothern Bobwhite PFQS: A ReproductionStudy with the NorthernBobwhite davey 070799 ll-Feb-2002,23:13:48 I:MRM of 1Channel ES- 6.72672 TIC 9.27e3 Area 1 % 3M Environmental Laboratory 3M Environmental Laboratory Page 7 2 of 226 Page 72 Report E01-1245 Report E01-1245 FO1-1342-36032, Blank female liver-day 3 IM Environmental Lab 1020213026 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reprqduction,$tudywittl the Northern Bobwhite I! davey 07079! 13-Feb-2002,21:18:2: 1: MRM of 1 Channel ES- 6.72 TIC 428 5.84e: Are: % u A 0 3M Environmental Laboratory 3M Environmental Laboratory VU I 1 Page 73 of 226 Page 73 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite t01-1342-36037, Blank female liver-day 4 IM EnvironmentalLab 1020218026 Sm (Mn, 1x2) 100 davey 07079 18-Feb-2002,12:11:3! 6.73641 1: MRM of 1 Channel ESTIC 7.05eC Are: 56 I I 0 ' 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 74 of 226 Page 74 Report E01-1245 Report E01-1245 Male Liver Blank Samples :01-1342-36002 Blank male liver-day 1 LM EnvironmentalLab i020211039 Sm (Mn, 1x2) ioa PFOS: A Reproduction Study with the Nothern Bobwhite PFYS: A 41 Re Wqct lli~ n Iq Study with the Northern Bobwhite davey 07079! ll-Feb-2002,23:24:5: 1: MRM of 1 Channel ES- TIC 6.65e: Are: 6.7; % 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 75 of 226 Page 75 Report E01-1245 Report E01-1245 1-1342-36033, Blank male liver-day 3 I EnvironmentalLab 20213027 Srn (Mn, 1x2) 10 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS:A T y T Study with the Northern Bobwhite 11 davey 07079: 13-feb-2002,21:29:2! 1: MRM of 1 Channel ES- 6.71 TIC 293 3.99e: Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 76 of 226 Page 76 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduation qtydy Jth the Northern Bobwhite D1-1342-36038, Blank male liver-day 4 !A EnvironmentalLab 120218027 Sm (.Mn.-1x2) ~ 00 davey 07079s 18-Feb-2(102,12:22:3I 1: MRM of 1Channel ES- 6.73 TIC 450 5.116: Are; % 0 3M Environmental Laboratory 3M Environmental Laboratory I Page 77 of 226 Page 77 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 4ththe Northern Bobwhite I Dosed Group Adult Male Sera Samplps, Diluted 1:I110 I, 4-108-369, IOppm male, adult, d100,02-01-0~ I EnvironmentalLab 20207118 Sm IMn. 1x2) 6.72 4770: davey 07079' 08-Feb-2002,05:46:4 1: MRM of 1 Channel ES- D( 4.98e! Are; 3M Environmental Laboratory 3M Environmental Laboratory Time .OO 8.00 9.00 10.00 , Page 78 of 226 Page 78 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReprcduFtionStudy with the Northem Bobwhite I! C108-371, 10ppm male, adult, d100,02-01-02 Environmental Lab !0207119 Sm I.Mn..1x2) 6.72 6891! davey 07079! 08-Feb-2002, 05575: 1: MRM of 1 Channel ES- TIC 5.84ef Are: 3M Environmental Laboratory 3M Environmental Laboratory 1 -b Time '.OO 8.00 9.00 10.00 Page 79 of 226 Page 79 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 1 1 1 PFOS: A Reproduction Study with the Northern Bobwhite i ~ 11 154-108-373, lOppm male, adult, dl00,02-01-02 IM Environmental Lab 1020207120 Sm (Mn, 1x2) 100 6.72 61202 davey 07079! 08-Feb-2002,06:08:51 1: MRM of IChannel ES- TIC 6.59eI Are; % a 3M Environmental Laboratory 3M Environmental Laboratory Page 80 of 226 Page 80 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northem Bobwhite I-108.375, 1Oppm male, adult, dl00,0291-02 Environmental Lab !0207121 Sm (Mn. 1x2) 6.72 54909 davey 07079! 08-Feb-2002,06:20:Of 1: MRM of 1 Channel ESTIC 5.6561 Are; 1,,1,,,,1"'11,,,,1,,',"1,,,,1',,,1,,,,,,,,,l,,,,l'',,l,,,, 1.00 2.00 3.00 4.00 5.00 6.00 3M Environmental Laboratory 3M Environmental Laboratory Page 81 of 226 Page 81 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite :A n Study with the Northem Bobwhite 54-108-377, 10ppm male, adult, d100,02-01-02 M Environmental Lab 020207122 Sm (Mn, 1x2) 100 6.72 40589 davev 07079! 08-Feb-2002, b6:31:lr 1: MRM of 1 Channel ES- TIC 3.99eI Are: %. a m r n Time 1.00 2.00 3.00 4.00 5.00 6.00 '.OO 8.00 9.00 10.00 , 3M Environmental Laboratory 3M Environmental Laboratory Page 82 of 226 Page 82 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PWS A Reproduction Stucly with the Northern Bobwhite 154-108-379, 10ppm male, adult, dl00,02-01-02 1M Environmental Lab 1020207128 Sm (Mn, 1x2) 100 6.7264561 davey 070791 08-Feb-2002,08:45:41 1: MRM of 1 Channel ES- TIC 6.87ef Are: % 3M Environmental Laboratory 3M Environmental Laboratory 7 - 1 Time .OO 8.00 9.00 10.00 Page 83 of 226 Page 83 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite I tudy with the Northern Bobwhite 54-108-381, 10ppm male, adult, d100,02-01-02 M EnvironmentalLab 020207129 Sm (Mn, 1x2) 100 6.71 58506 davey 0707991 08-Feb-2002,08:57:00 1: MRM of 1 Channel ES- TIC 6.06e5 Area % =-= i e-? i ? Time 1.00 2.00 3.00 4.00 5.00 6.00 '.OO 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 84 of 226 Page 84 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproducti the Northem Bobwhite 64-108-383, 10ppm male, adult, d100,02-01-02 . . 1M EnvironmentalLab 1020207130 Sm IMn. 1x2) I 100 6.72 6110s davey 07079! 08-Feb-2002,09:08:0! 1: MRM of 1Channel ES- TIC 5.37e: Are; % 1 0 ,I, ,,, ,, Time ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' '.OO 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 85 of 226 Page 85 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite the Northern Bobwhite I 154-108-385, 10ppm male, adult, d100,02-01-02 IM Environmental Lab 1020207131Sm (Mn, 1x2) 100 6.72 davey 07079! 08-Feb-2002,09:19:1' 1: MRM of 1Channel ES- TIC 5.28eI Are2 % 0 Time ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' '.OO 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 86 of 226 Page 86 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study with the Northern Bobwhite 1-54-108-387,10ppm male, adult, dl00,02-01-02 IM EnvironmentalLab 1020207132 Sm (Mn, 1x2) 100 764.4762: davey 07079! 08-Feb-2002, 09:30: l! 1: MRM of IChannel ES- TIC 7.92eI Are: % 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 I , , I , , I , I r "~ '~I~ , 1 , ~ ~ , , 1 , 1 , , 1 , , , , 1 1 , , , 1 , , , , 1 1 , , , 1 , , , , , 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 87 of 226 Page 87 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northem Bobwhite Dosed Group Adult Female Sera Samples 454-108-370, 10ppm female, adult, 02-01-02 3M EnvironmentalLab 14020203026 Sm (Mn, 1x2) I100 6.75 318463 davey 07079! 03-Feb-2002,16: 19:31 1: MRM of 1 Channel ES- TIC 3.39eZ Are2 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 t 6.00 ' 7.b0 ' 8zO ' -0 Time 3M Environmental Laboratory 3M Environmental Laboratory Page 88 of 226 Page 88 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 154-108-372, 10ppm female, adult, 02-01-02 IM Environmental Lab 1020203027 Sm (Mn, 1x2) 100 6.75 3175OC davey 07079! 03-Feb-2002,16:30:4( 1: MRM of 1 Channel ES- TIC 3.38ef Are: % r 1~'~~1"''l''"l'~"l''*l111111111l"~~l~~~'l~~'~l~'~'I ' " I 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 89 of 226 Page 89 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS:A Reprodqqn Study With ths Northern Bobwhite 154-108-374, 10ppm female, adult, 02-01-02 IM EnvironmentalLab 1020203028 Sm (Mn, 1x2) 100 6.74 295902 davey 07079! 03-Feb-2002, 16:41:4! 1: MRM of 1 Channel ESTIC 3.15e Arez % 0 ' 1.00 ' 2.00 3.60 * 4.00 5.00 6.00 7.00"""8'.ii0'"1'ii.oo""'io1oToime ' 3M Environmental Laboratory 3M Environmental Laboratory Page 90 of 226 Page 90 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PIF/OSI:A R s p/ rI~ $ ~ c ;St itu~d~y with the Northern Bobwhite 154-108-376, 10ppm female, adult, 02-01-02 IM Environmental Lab 1020203029 Sm I.Mn. -1x21, 100 6.75 321 554 davey 07079! 03-Feb-2002, 16:52:41 1: MRM of 1 Channel ESTIC 3.54ef Are2 % 0 t Time , ' 1.00 2.00 3.00 4.00 I 5.00 6.00 7.00 8.00 ' ' ' i o o ' ' "'io100 3M Environmental Laboratory 3M Environmental Laboratory Page 91 of 226 Page 91 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite A Seprod@on Study with the Northern Bobwhite 154-108-378, 10ppm female, adult, 02-01-02 IM Environmental Lab 1020203030 Sm (Mn, 1x2) 100 6.74 187898 davey 07079! 03-Feb-2002,17:03:5~ 1: MRM of 1Channel ES- TIC 2.06ef Are; % a 3M Environmental Laboratory 3M Environmental Laboratory I T- .OO 8.00 Time 9.00 10.00 Page 92 of 226 Page 92 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 4 qeprqduction Study with the Northern Bobwhite ii W-108.380, 10ppm female, adult, 02-01-02 IM Environmental Lab 1020203031Sm I.Mn. -1x2), 1oa 6.75 214792 davey07079 2.34ei Are; w ' 0 I I I Time ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 8.b0 9.00 I lO!OO , 3M Environmental Laboratory 3M Environmental Laboratory Page 93 of 226 Page 93 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A iReproduCbion Qtudy with the Northern Bobwhite 454-108-382, IOppm female, adult, 02-01-02 3M EnvironmentalLab Id020203032 Sm (Mn, 1x2) I loa 6.74. 99 davev 07079! 03-Feb-2002, i7:26:01 1: MRM of 1 Channel ES- TIC 7.13ef Are; ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 8.00 ' 9.00 ' l0:OO 1 3M Environmental Laboratory 3M Environmental Laboratory Page 94 of 226 Page 94 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 54-108-386, IOppm female, adult, 02-01-02 IM Environmental Lab 1020203033 Sm (Mn, 1x2) 100 6.74 461 Boa davey 07079! 03-Feb-2002,17:37:1( 1: MRM of 1 Channel ES- TIC 4.88ef Ares % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 95 of 226 Page 95 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Refpbction Study with the NorthernBobwhite ~ /I 154-108-388, 10ppm female, adult, 02-01-02 IM Environmental Lab 1020203034Sm (Mn, 1x2) 100 6.74 384563 davey 07079! 03-Feb-2002,1?:48:1 i 1: MRM of 1 Channel ES- TIC 4.20eE Are; % 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 3M Environmental Laboratory 3M Environmental Laboratory Page 96 of 226 Page 96 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite tudy with the Northern Bobwhite j I 154-108-390, IOppm female, adult, 02-01-02 IM EnvironmentalLab 1020203038 Sm (Mn, 1x2) 100 6.75 231266 davev 07079! 03-Feb-2002, \ 8:32:3; 1: MRM of 1 Channel ES- TIC 2.58ef Are2 % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 97 of 226 Page 97 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite with the Northem Bobwhite Dosed Group Male Offspring Sera Samples 154-108-522, 10ppm male, offspring, 02-01-02 IM Environmental Lab 1020207169 Sm (Mn, 1x2) 100 6.73 471945 davey 07079! 08-Feb-2002,-16:20:5: I:MRM of 1 Channel ES- TIC 4.74et Are: % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 98 of 226 Page 98 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite `I I y,@ththe T No em Bobwhite 154-108-533, 10ppm male, offspring, 02-01-02 IM Environmental Lab 1020207171 Sm (Mn, 1x2) 100 6.73 284576 davey 07079! 08-Feb-2002,16:43:0; 1: MRM of 1 Channel ES- TIC 2.94e Are: % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 99 of 226 Page 99 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reprwction Study with the Northem Bobwhite d ! i 154-108-545, 10ppm male, offspring, 02-01-02 1M EnvironmentalLab 1020207173 Sm (Mn, 1x2) 100 6.73 352415 davey 07079! 08-Feb-2002, 17:05:1( 1: MRM of 1 Channel ESTIC 3.66e Ares 96 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' t 7.b0 ' 8.bO ' 9.00 ' I I I Time lOl00 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 100 of 226 Page 100 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite dy with the Northern Bobwhite 154-108-552, 10ppm male, offspring, 02-01-02 IM Environmental Lab 1020207174 Sm (Mn, 1x2) 100 6.73 375404 davey 07079! 08-Feb-2002,17:16:1~ 1: MRM of 1 Channel ESTIC 3.83et Are? % 0 I. ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 3M Environmental Laboratory 3M Environmental Laboratory Page 101 of 226 Page 101 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite y yith the Northern Bobwhite '~ I 154-108-568, 1Oppm male, offspring, 02-01-02 IM EnvironmentalLab 1020207177 Sm (Mn, 1x2) 100 6.73 34602! davey070799 08-Feb-2002. 17:49:29 I: MRM of iChannel ES- TIC 3.53e6 Area ! i 3M Environmental Laboratory 3M Environmental Laboratory Page 102 of 226 Page 102 Report E01-1245 Report E01-1245 , PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Dosed Group Female Offspring Sera Samples 154-108-517, 10ppm female, offspring, 02-01-02 1M Environmental Lab 1020207168 Sm (Mn, 1x2) 100 6.73 236874 davey 070791 08-Feb-2002, 16:09:4! 1: MRM of 1Channel ES- TIC 2.49ef Are: % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 103 of 226 Page 103 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite yqy with the Northem Bobwhite 154-108-525, IOppm female, offspring, 02-01-02 1M EnvironmentalLab 1020207170Sm I.Mn. -1x21. 100 6.74 244713 davey 0707% 08-Feb-2002,16:31:5( 1: MRM of 1Channel ES- TIC 2.62ef Are: % 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 ' 8.00 ' 9.00 ' 1O:OO I 3M Environmental Laboratory 3M Environmental Laboratory Page 104of 226 Page 104 Report E01-1245 Report EO1-1245 IM Environmental Lab 1020207172 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite (ly with the Northern Bobwhite 6.73 393873 08-Feb-2002, ?6:54:05 1: MRM of 1 Channel ES- TIC 3.96e6 Area % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 105 of 226 Page 105 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northem Bobwhite 154-108-557, 10ppm female, offspring, 02-01-02 IM EnvironmentalLab 1020207175 Sm (Mn, 1x2) 100 6.74 260577 davev 07079! 08-Feb-2002,i 7:27:I! 1: MRM of 1 Channel ES- TIC 2.68et Ares % 3M Environmental Laboratory 3M Environmental Laboratory Page 106 of 226 Page 106 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite e Northern Bobwhite 154-108-561,lOppmfemale, offspring, 02-01-02 IM Environmental Lab 1020207176 Sm (.Mn.-1x2r) 100 6.73 333841 davey 07079! 08-Feb-2002, 17:38:2! 1:MRM of 1 Channel ES- TIC 3.43e Are2 % 0 ' 1.00 ' 2.00 3.00 ' 4.00 ' 5.00 ' 6.00 ' 1 - 7.b0 ' 8.00 9.00 Time 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 107of 226 Page 107 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite RTm,qli PFoS:A I u ion Study with the Northem Bobwhite ~ Dosed Group Male Liver Samples 101-1342-30762,lO ppm a.i. 379 M Liver,dlO 1M Environmental Lab 1020301058 Sm (Mn. 1x2) 100 6.74 466858 davey 07079: 02-Mar-2002,01:32:5( 1: MRM of 1 Channel ES- TIC 4.76eE Ares % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 108 of 226 Page 108 Report E01-1245 R IM EnvironmentalLab 1020301065 Sm (Mn, 1x2) 100 PFOS: A Reproduction Study with the Nothern Bobwhite ite 6.74 482069 02-Mar-2002,02:50:4( I:MRM of 1Channel ES- TIC 4.90ef Are; % 3M Environmental Laboratory 3M Environmental Laboratory Page 109 of 226 Page 109 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite ction Study with the Northern Bobwhite I t01-1342-30883,lO ppm a.i. 391 M Liver,dlO IM EnvironmentalLab 1020301064 Sm (Mn, 1x2) 100 6.74 421019 davey 07079! 02-Mar-2002,02:39:41 1: MRM of 1 Channel ES- TIC 4.39e Are: Yo . 0 I I VI Time 1 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 ' 8.00 ' 9.00 ' l0:OO ~ 3M Environmental Laboratory 3M Environmental Laboratory Page 110 of 226 Page 110 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 ' i~~~ 201-1342-30829,lO ppm a.l.385 M Liver,dlO IM EnvironmentalLab 1020301061 Sm (Mn, 1x2) 100 6.74 484813 With the Northern Bobwhite davey 07079! 02-Mar-2002,02:06:1; I:MRM of 1Channel ES- TIC 4.91 e( Are: 96 a 3M Environmental Laboratory 3M Environmental Laboratory Page 111 of 226 Page 111 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 ' y PIiiF~O'S: A Reproduction t Study with the Northem Bobwhite ~ /'I :i t01-1342-30865,lO ppm a.i. 389 M Liver,dlO IM Environmental Lab 1020301063 Sm (Mn, 1x2) 100 6.74 405774 davey 07079! 02-Mar-2002,02:28:2; 1: MRM of 1 Channel ES- TIC 4.22ef Are; % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 112 of 226 Page 112 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite with the Northern Bobwhite i01-1342-30847, 10 ppm a.i. 387 M Liver,dlO IM Environmental Lab 1020301062 Sm (Mn, 1x2) 100 6.74 439049 davey 07079! 02-Mar-2002,02:17:2( I:MRM of 1 Channel ES- TIC 4.50& Are; % 0 L Time ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 113 of 226 Page 113 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite :Ol-1342-30798, 10 ppm a.i. 383 M Liver,dlO IM Environmental Lab 1020301060 Sm (Mn, 1x2) 100 6.74 41 3083 davey 070791 02-Mar-2002,01:55:0( 1: MRM of 1 Channel ESTIC 4.34eZ Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 114 of 226 Page 114 Report E01-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite iO1-1342-30780,lOmrn a.i. 381 M Liver.dl0 M Environmental Lab 020301059 Sm (Mn, 1x2) 100 6.74 470298 % 3M Environmental Laboratory 3M Environmental Laboratory Page 115 of 226 Page 115 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 201-1342-30937,lO ppm a.i. 397 M Liver,dlO IM EnvironmentalLab 1020301067Sm (Mn, 1x2) 6.74 315950 davev 07079! 02-Mar-2002,03:12:5! I:MRM of 1Channel ES- TIC 3.35ef Arez 1.00 2.00 3.00 4.00 5.00 6.00 3M Environmental Laboratory 3M Environmental Laboratory Page 116 of 226 Page 116 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 '~ t01-1342-30919, 10 ppm a.i. 395 M Liver,dlO IM EnvironmentalLab 1020301066 Srn (Mn, 1x2) 100 6.74 388593 V@I the Northern Bobwhite davey 0707% 02-Mar-2002,03:01:5: I : MRM of 1 Channel ESTIC 3.98et Are; % 0 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 117 of 226 Page 117 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northem Bobwhite Dosed Group Female Liver Samples f01-1342-30771,lO ppm a.i. 380 F Liver IM Environmental Lab i020301037 Sm (Mn. 1x2) I . I 1oa 6.73 271982 davey O?O% OI-Mar-2002,21:39:2( I:MRM of 1Channel ES- TIC 3.13ef Arei % 0 ' 1.00 ' 2.00 ' 3.00 4.00 ' 5.00 3M Environmental Laboratory 3M Environmental Laboratory Page I1a of 226 Page 118 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS A ReproductionStudy with the Northern Bobwhite f01-1342-30838, 10 ppm a.i. 386 F Liver IM EnvironmentalLab j020301040 Sm (Mn, 1x2) 100 6.73 446002 davey 07079! 01-Mar-2002,22:12:4! 1: MRM of 1 Channel ES- TIC 4.76ef Are: % 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 119 of 226 Page 119 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy wlth the Northem Bobwhite iO1-1342-308561,0 ppm a.i. 388 F Liver IM Environmental Lab 1020301041 Sm (Mn, 1x2) 100 6.72 243562 davey 07079! Ol-Mar-2002,22:23:5: 1: MRM of 1 Channel ES- TIC 2.84et Are: % 0 IIIII,III,,,,I,,,,I,,,,(,,,,I,,,,I,,,,I,,,,I,,,,I,,I,I,,I(I,,,,I 1.00 2.00 3.00 4.00 5.00 6.00 3M Environmental Laboratory 3M Environmental Laboratory Page 120 of 226 Page 120 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342.30820, 10 ppm a.i. 384 F Liver, d10 IM Environmental Lab 1020301039 Sm (Mn, 1x2) 100 6.74 263292 davey 07079! Ol-Mar-2002,22:01:4: 1: MRM of 1 Channel ES- TIC 2.83et Are; % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 121 of 226 Page 121 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite I y with the Northern Bobwhite !01-1342-30735, 10 ppm a.i. 376 F Liver IM EnvironmentalLab 1020301035 Sm (Mn, 1x2) 100 6.73 275483 davey 07079! OI-Mar-2002,21:I7:14 1: MRM of I Channel ES- TIC 3.19et Are; % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 122 of 226 Page 122 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite f01-1342-30717, 10 ppm a.i. 374 F Liver IM Environmental Lab !020301034 Sm (Mn, 1x2) ioa 6.74 346653 davey 07079! Ol-Mar-2002,21:06:0! 1: MRM of 1 Channel ES- TIC 3.78et Are: % a 3M Environmental Laboratory 3M Environmental Laboratory Page 123 of 226 Page 123 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite udy Nththe NorthernBobwhite f01-1342-30789,lO ppm a.i.382 F Liver IM EnvironmentalLab 1020301038 Sm (Mn, 1x2) 100 6.72 219312 davey 07079! Ol-Mar-2002,21:50:3; 1: MRM of 1 Channel ES- TIC 2.60ef Are: % 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 3M Environmental Laboratory 3M Environmental Laboratory Page 124 of 226 Page 124 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFQS:A ReproolwctidnStudy with the Northern Bobwhite E01-1342-30681,lOppm a.i. 370 F Liver IM EnvironmentalLab 1020301032 Sm (Mn, 1x2) 100 6.74 371782 davey 07079! Ol-Mar-2002,20:43:5; 1: MRM of 1 Channel ESTIC 4.09et Are: % 1 0 t I I I Time ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.b0 I 8.bO 9.00 I lO!OO 3M Environmental Laboratory 3M Environmental Laboratory Page 125 of 226 Page 125 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite EOI-1342-30699,lOppm a.i. 372 F Liver IM Environmental Lab 1020301033 Sm (Mn, 1x2) 100 6.72 295007 davey 07079! Ol-Mar-2002,20:55:0! 1: MRM of 1 Channel ES- TIC 3.36ef Arec % 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 3M Environmental Laboratory 3M Environmental Laboratory Page 126 of 226 Page 126 ~~ Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study with the Northern Bobwhite E01-1342-30753, 10 ppm a.1. 378 F Liver IM EnvironmentalLab !020301036 Sm (Mn, 1x2) 100 6.73 243424 davey 0707% Ol-Mar-2002,21:28:2( 1: MRM of IChannel ESTIC 2.86ef Are; % a , , , , , , , , , , , 1 1 1 1 1 1 , 1 1 I , , , , , , , Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 127 of 226 Page 127 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 'I Dosed Group Male Offspring Liver QahpJlbs IO1-1365-31245,lO ppm a.i. 522 M Liver, offspring IM Environmental Lab j020218052 Sm (.Mn..1x2) I 100 6.72 426343 with the Northern Bobwhite 1 I davey070799 18-Feb-2002, 16:59:46 1: MRM of 1Channel ES- TIC 4.43et Are; % 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 128 of 226 Page 128 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PpPS A ReproductionStudy with the Northem Bobwhite 01-1365-31270,lO ppm a.i. 552 M Liver, offspring M Environmental Lab 020218060 Sm (Mn, 1x2) IO0 6.70 302473 davey 07079! 18-Feb-2002, -18:28:3r 1: MRM of 1 Channel ES- TIC 3.23ef Are: % a """~l'.OO""'2.b"b"""'4.OO''"'5.bb:'''''6.OO"' 7.00 ' 8.00 ' 9.00 ' l0:OO - ! 3M Environmental Laboratoty 3M Environmental Laboratory Page 129 of 226 Page 129 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 udy With the Northern Bobwhite ~ t01-1365-31285,lO ppm a.i. 568 M Liver, offspring IM Environmental Lab 1020218063 Sm (.Mn, -1x2), 100 6.72 296813 davey 07079! 18-Feb-2002, -19:01:51 1: MRM of 1 Channel ES- TIC 3.19d Are: % 0 3M Environmental Laboratory 3M Environmental Laboratory L^ I ,7iTime .OO 8.00 9.00 10.00 Page 130 of 226 Page 130 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite E01-1342-30955, 10 ppm a.i. 399 M Liver, d10 IM Environmental Lab j020218064 Sm (Mn, 1x2) 100 6.73 305393 davey 0707M 18-Feb-2002,19:1 2 5 6 1: MRM of 1 Channel ES- TIC 3.11e6 Area % I 0 ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 ' 8.00 ' 9.00 ' 1O:OO'"- 1 3M Environmental Laboratory 3M Environmental Laboratory Page 131 of 226 Page 131 Report E01-1245 Report EO1-1245 PFOS: A Reproduction Study with the Nothern Bobwhite y with the Northern Bobwhite :01-1365-31255,lO ppm a.i. 533 M Liver, offspring IM Environmental Lab j020218054 Sm (Mn, 1x2) 100 6.72 246631 davey 0707% 18-Feb-2002,i7:21:5; 1: MRM of 1 Channel ESTIC 2.74et Are: % 0 3M Environmental Laboratory 3M Environmental Laboratory - I .OO 8.00 9.00 Time 10.00 Page 132 of 226 Page 132 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudywith the Northern Bobwhite t01-1365-31265,lO ppm a.i. 545 M Liver, offspring IM Environmental Lab 1020218059 Sm (Mn, 1x2) 100 6.73 2591 29 daveyO7079d 18-Feb-2002, -l8:17:2 1: MRM of IChannel ES- 2.83TeI{6 Area % a 3M Environmental Laboratory 3M Environmental Laboratory Page 133 of 226 Page 133 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Dosed Group Female Offspring Liver Samples ,01-1365-31260,lOppm ami5.38 F Liver, offspring M Environmental Lab 020218055 Sm (Mn, 1x2) 100 6 73446716 davey 07079! l&Feb-2002, 17:33:0! I: MRM of 1 Channel ESTIC 4.59ef Arez % a 3M Environmental Laboratory 3M Environmental Laboratory Page 134of 226 Page 134 Report E01-1245 Report EOl-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFO$: A R e m q d i p n Study with the Northern Bobwhite E01-1365-31250,10 ppm a.i. 525 F Liver, offspring )M EnvironmentalLab io20218053 Sm (Mn, 1x2) ioa 6.73 314521 davey 070799 18-Feb-2002, 17:10:50 1: MRM of 1 Channel ES- TIC 3.38e6 Area ! % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 135 of 226 Page 135 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A PeproCludonStudy with the Northem Bobwhite r01-1365-31275, I O ppm a.i. 557 F Liver, offspring LM Environmental Lab 1020218061 Srn (Mn, 1x2) 100 6.71 22587i davey 07079: 18-Feb-2002, 18:39:3! 1: MRM of 1Channel ES- TIC 2.28e Are: % n 1.00 2.00 3.00 4.00 5.00 6.00 .OO 8.00 9.00 Time 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 136 of 226 Page 136 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the NorthernBobwhite iO1-1365-31280, I O ppm a.i. 561 F Liver, offspring M Environmental Lab 020218062 Srn (Mn, 1x2) 1 oa 6.72 242318 davey 07079t 18-Feb-2002, 18:50:41 1: MRM of 1 Channel ESTIC 2.73e6 Area % 3M Environmental Laboratory 3M Environmental Laboratory Page 137 of 226 Page 137 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite tudy yith the Northern Bobwhite iO1-1365-31240,lO ppm a.i. 517 F Liver, offspring M EnvironmentalLab 020218051 Sm (Mn, 1x2) 100 6.73 davey07079 18-Feb-2002, 16:48:4 1: MRM of 1Channel ES- TIC 3.21e6 Area % ' ' ' C b,,88 88n8rTime ' 1.00 ' 2.00 ' 3.00 ' 4.00 ' 5.00 ' 6.00 ' 7.00 8.b0 9.00 10.00 ~ 3M Environmental Laboratory 3M Environmental Laboratory Page 138 of 226 Page 138 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Control Group Male Sera Sample 54-108-357, Oppm male, adult, 02-01-02 M Environmental Lab 020201058 Sm (.Mn.. 1x2,) IO0 davey 07079! 02-Feb-2002,03:59:2! 1: MRM of 1 Channel ES- 6.77 TIC 1695 2.12tM Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 139 of 226 Page 139 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite :A udy with the Northern Bobwhite Control Group Female Sera Sample 54-108-366.- O.p.pm female, adult, 02-01-02 M EnvironmentalLab 320201066 Sm (Mn. 1x2) oa davey 070799 02-Feb-2002,05:28: 12 1: MRM of 1 Channel ES- 6.76- 590 TIC 7.4383 Area % I , ,0 , I I , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , I I I I I I I I I I I I I I I I 8 I I I I I I t I I q I I I I I I I I I I I I I I I I I I Time 1.00 2.b0 I 3.00 I 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 140 of 226 Page 140 Report E01-1245 Repott E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Control Group Male Offspring Sera Sample 54-108-253, Oppm male, offspring, 02-01-02 M Environmental Lab 10.0 % IO0 67.1784. davey 07079! OS-Feb-2002,1354:l! 1: MRM of 1Channel ES- TIC 8.47eI Are; % 3M Environmental Laboratory 3M Environmental Laboratory J Page 141 of 226 Page 141 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite y ylth the Northern Bobwhite I Control Group Female Offspring Sera Sample 54-108-259, Oppm female, offspring, 02-01-02 M Environmental Lab 020207156 Sm (Mn, 1x2) 100 65.1793' davey 07079! 08-Feb-2002,13:56:3' 1: MRM of 1 Channel ES- TIC 6.10e: Are; 0/1 1 3M Environmental Laboratory 3M Environmental Laboratory t Page 142 of 226 Page 142 Report E01-1245 Report E01-1245 Control Group Male Liver Sample PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A RepWJ1IF.blonStudy with the Northern Bobwhite j020212058 Sm (Mn, 1x2) 1: MRM of 1 Channel ES 6.74 TI( 2246 2.55e Are 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 143 of 226 Page 143 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northem Bobwhite Control Group Female Liver Sample 1-1342-30627,O ppm a.i. 364 F Liver Environmental Lab !0212063 Sm (Mn,1x2) 0 davey 07079! 13-Feb-2002,01:58:0: 1: MRM of 1 Channel ESTIC 8.13e: Are: c 3M Environmental Laboratory 3M Environmental Laboratory Page 144 of 226 Page 144 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS A Reproduotian study with the Northern Bobwhite Control Group Male Offspring Liver Sample 01-1365-311940,ppm a.i. 253 M Liver, offspring M Environmental Lab 020215039 Sm (Mn, 1x2) IO0 69.1765 davey 07079! 15Feb-2002,21:12:3; 1: MRM of 1 Channel ES- TIC 9.66eI Are: % 3M Environmental Laboratory 3M Environmental Laboratory Page 145 of 226 Page 145 ~ ~~ Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 8 orthem Bobwhite PI Control Group Female Offspring Liver Sample E01-1365-31184, 0 ppm a.i. 246 F Liver, offspring 1M Environmental Lab 1020215038 Sm (Mn, 1x2) I 0 0 610.7341. davey 07079! 15-Feb-2002,21:01:2; 1: MRM of 1 Channel ESTIC 1.06s Arez % 3M Environmental Laboratory 3M Environmental Laboratory Page 146 of 226 Page 146 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite th the Northern Bobwhite I Extracted Sera Calibration Curve Point bpb extracted curve point, sera 01-31-02 I Environmental Lab 20201020 Sm (Mn, 1x2) 6.76 13162 davey 07079! OI-Feb-2002,20:57:21 1: MRM of 1 Channel ES- TIC 1.Met Are; c 3M Environmental Laboratory 3M Environmental Laboratory f"."----"------""--^, Time 00 8.00 9.00 10.00 Page 147 of 226 Page 147 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report EO1-1245 111 j 1 I rudy withthe NorthernBobwhite Extracted Liver Calibration Curve Point :01-1342-36007 Curve point-1 ppb in liver extract IM EnvironmentalLab . . 1020211022 Sm IMn. 1x2,) 100 6.72 1969 davey07079d ll-Feb-2002,20:16:33 1: MRM of 1 Channel ES- TIC 2.53e4 Area % 0 3M Environmental Laboratory 3M Environmental Laboratory Page 148 of 226 Page 148 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite tpdy with the Northern Bobwhite Unextracted Calibration Curve Point 12003-15-1 2.61 ppb unext IM EnvironmentalLab 1020201008 Sm (Mn. 1x2) 100 6.74 4858' % 0 3M Environmental Laboratory 3M Environmental Laboratory -I 1 Time I .OO 8.00 9.00 10.00 ~ Page 149 of 226 Page 149 Report E01-1245 Report 01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite P-6: A Reprodudon Study wlth the Northern Bobwhite Extracted Sera Calibration Curve Dmpound 1 name: pfos (499>99) func 1 Method File: d020201a oefficient of Determination: 0.988137 alibration curve: -0.862805 * XY+ 1603.72 * x + 8692.86 esponse type: External Std, Area urvetype: 2nd Order, Origin: Exclude,Weighting: l/x, Axis trans: None 8.01e5 X Sesponse 3M Environmental Laboratory 3M Environmental Laboratory Page 150 of 226 Page 150 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite with the Northem Bobwhite Extracted Liver Calibration Curve Impound1name: pfos (499~99f)unc 1 MethodFile: d020215a lefficient of Determination: 0.988785 [librationcurve: -0.689309 * xA2 + 1283.97 * x + 845.279 sponse type: External Std, Area iwetype: 2nd Order, Origin: Exclude,Weighting: Ilx, Axis trans: None 6.43eE X Lesponsc 3M Environmental Laboratory 3M Environmental Laboratory Page 151 of 226 Page 151 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Mass Spectrometer Tune Settings Tuning Method Report Method : C:\M?SSLYNX\DAVEY070799.PRO\ACQUDB\CENTREl Printed: Tue Mar 19 10:22:06 2002 Page 1 MS SOURCE ( ESP- 1 Capillary Cone Hexapole 1 Aperture 1 Hexapole 2 Source Block Desolvation Temp. Temp. Set Rdbk 2.56 20 0.5 0.2 0.8 150 250 -2.48 -20 148 24 9 Pressures Analyser Vacuum Gas Cell Rdbk 3.8e-5 2.9e-3 Analyser LM Res 1 HM Res 1 IEnergy 1 Entrance Collision Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Gas Flows Cone Gas Desolvation Set Rdbk 13.0 13.0 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 152 of 226 Page 152 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS:A ReproductionStudy with the Northern Bobwhite HPLC Settings Method Report Method File: Last Modified: Printed: Page 1 C:\MASSLYNX\DAVEY070799.PRO\A( Monday, March 11,2002 08:Ol:Ol Tuesday, March 19, 2002 08:37:50 HPllOO LC Pump Initial Conditions Solvents A% 90.0 B % 10.0 C% 0.0 D% 0.0 Valve A set to channel Valve B set to channel Flow (mllrnin) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left("C) Oven Temperature Right("C) 0.300 10.0 0 400 40.0 40.0 HPllOO LC Pump Gradient Timetable 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 t - l P L~C~Pu~mpEmtlrfEvent Timetsbae The Timetable contains 3 entries which are : Time Initial 0.00 0 10 Cctlunn Switch OR Off OR On Ofl Off Contact1 CHI On off Contact2 OR OR Qfl Gantac 6ff On off HPIl o a Arahammplerlnixjd CcwlclEtions Draw S p e d Eject S p e d (vlhin) Draw Position [mm) S h p Time (rnins) Injection Volurne(pI] Vial Number Thema&& On Thermostat Twnpenture(nC) 2m.o 2or3 0.00 1B.00 10.0 94 20.0 3M Environmental Laboratory 3M Environmental Laboratory Page 153 of 226 Page 153 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite Mass Spectrometer Scanning Parameters Scanning Method Report Method: C:\~SSLYNX\DAVEYO7O799.PRO\ACQUDB\1OMIN-PFOS-PFDA Last Modified: Mon Jan 28 15:20:24 2002 Printed : Tue Mar 19 08:38:22 2002 Solvent Delay ( mins ) : 0.00 Function : 1 MRM 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 ) 1 499.00 99.00 0.30 43 60 Function : 2 MRM of 1 Mass Pair ( ESP- ) Inter Channel Delay ( Span ( Daltons 1 : 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 2 1 9 . 0 0 0.30 20 20 3M Environmental Laboratory 3M Environmental Laboratory Page 154 of 226 Page 154 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A ReproductionStudy with the Northern Bobwhite ATTACHMENTD: SAMPLEPREPARATIOSNHEETS 3M Environmental Laboratory 3M Environmental Laboratory Page 155 of 226 Page 155 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction Worksheet Prep Date: Analysts initials: I I Lims Assgned sample number E01-1245-35773 E01-1245-35774 1/31/2002 \ 1 3-i-.03caz Method Revision: ETS-8-231 .O Study Number: EO1-135d@*j (.j./ 02 Matrix: Sera I I Sample Number or description Volume of sample filtered (mi) Type of column used and lot Amount and spike Elution solv mix used and volurr 40+6 Waters, l g , 6ml, lot129082 2ml of MeOH NA TN-A-5457 40+6 Waters, l g , 6ml, lot 129082 NA 2ml of MeOH TN-A-5457 40+6 40+6 Waters, Ig, 6ml, lot 129082 Waters, I g , 6ml, lot 129082 1' Oul of 02001-53 .J 1OUl Of 02001-53 2ml of MeOH TN-A-5457 2ml of MeOH MA5457 I 40+6 40+6 Waters, l g , 6ml, lot I29082 Waters, l g , 6ml, lot 129082 ,//2ul of 02001-52 4ul Of 02001-52 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 40+6 Waters, l g , 6ml, 2ml of MeOH lot 129082 1' Oul of 02001-52 TN-A-5457 150 ppb sera curve I 100 ppb sera curve 40+6 40+6 Waters, l g , 6ml. lot 129082 !Waters, I g , 6ml, llot 129082 I ,/ 20ul of 02001-52 I./ I 4ul of 02001-53 2ml of MeOH 1TN-A-5457 12ml of MeOH ITN-A-5457 E01-1245-35775 250 oob sera curve E01-1245-35776 500 ppb sera curve E01-1245-35777 750 ppb sera curve E01-1245-35778 1000 ppb sera curve E01-1245-35779 QC-25 ppb-1 E01-1245-35780 QC-25 ppb-2 E01-1245-35781 QC-25 ppb-3 E01-1245-35782 QC-40 ppm-1 E01-1245-35783 QC-40 oom-2 E01-1245-35784 QC-40 ppm-3 E01-1245-35785 QC-80 ppm-1 E01-1245-35786 QC-80 ppm-2 E01-1245-35787 QC-80 ppm-3 n.~ l :@lank Sera TN-A- 597 z ; Amount of sera aliquated: Dilute Sera with Kandiyohiwater; Amount of water added 40+6 40+6 40+6 40+6 40+6 ~~ 40+6 40+6 40+6 40+6 2 ? fi Waters, I g , 6ml, lot 129082 Waters, l g , 6ml, Waters, l g , 6ml, Waters, l g , 6ml, lot I29082 Waters, l g , 6ml, lot I29082 Waters, I g , 6ml, lot 129082 Waters, l g , 6ml, lot 129082 .Waters, lg, 6ml, lot129082 Waters, l g , 6m!, lot I29082 Waters, l g , 6ml, lot I29082 Waters, l g . 6ml, lot 129082 waiers,79. bmi, lot 129082 ml ml J 20ul of 02001-53 ,r " 30ul of 02001-53 40Ul of 02001-53 1OUI Of 02001-52 .-1Oul of 02001-52 /l Oul of 02001-52 J 361.11of 02001-15 36ul Of 02001-15 J 361.11of 02001-15 / 7 2 ~o1f 02001-15 / 72ul of 02001-15 72ul of 02001-1 5 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 Add 5ml of ACN (TN-A- ) to aliquat d Sam le0 sample for 20 min a300 rpm (Shaker&'z-c&?TqL )y"/ogg94- Centrifugesample forOI min Q 2000 rpm (Centrifug&JWc I 3 ) Wash Column with KandiyohiWater conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicatedamount of solvent ( HA3H TN-A- 5?5+ standard PFDA standard number &" I - 6 5 OJCa b , 7 a 7 1 c 4 , &J / . / f . d~ ifi+wJ ~ ~ c . . D # ~ Gzf- 6AEx ~ ~ ~ ) into appropriate 15 ml centrifugetube , conc. 100.3 W s@ otU2802- 3M Environmental Laboratory Page 156 Report E01-1245 Prep Date: Analysts initials: 1/31/2002 WMOJ ~ ~~ PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns ExtractionWorksheet @ QW;~ &Sw1aijV2 %P Method Revision: ETS-8-231 .O Study Number: E01-126' r/..J (.J6 4 Matrix: Sera @ Lims Assgned sample number E01-1245-28443 EO 1-1245-28444 ,vAAZ 454-108-329,OPPM, Male, Adult 454-108-330,0 PPM, Female, Adult 454-108-331,OPPM, Male, sample filtered I Type of column used and lot Waters, l g , 6ml, 40+6 lot 129082 Waters, l g , 6ml, 40+6 lot 129082 Waters, Ig. 6m1, E01-1245-28446 E01-1245-28447 E01-1245-28448 E01-1245-28449 E0l-1245-28450 L-lLYJ-L64J 1 E01L - I L 4 J -n L 6n4' r>-L E0l-1245-28453 E01-1245-28454 Eo1-l,A2~,vA~c 454-108-332,OPPM, Female, .Adult 454-108-333,OPPM, Male, Adult 454-108-335,OPPM, Male, Adult 454-108-336,OPPM, Female, Adult 454-108-337,OPPM, Male, . .A_rli-il-t .- ...".. . 1454-108-338.0PPM, Female,[ I AAiilt I I I 454-108-339.0PPM. Male.' I I I Adult 454-108-340,OPPM, Female, Adult 454-108-341,OPPM, Male, Adult 454-108-343,OPPM, Male, 40+6 40+6 40+6 4.0- +-6 d.n- +-6 40+6 40+6 Waters, l g , 6ml, lot 129082 Waters, I g , 6ml, lot 129082 Waters, lg. 6ml. lot 129082 iWaters. lg. 6ml, lot 129082 I [Waters, l g , 6ml, 129082 Waters, I g , 6ml, lot129082 Waters, lg. 6ml, lot 129082 Waters. I g , 6ml, EO 1 - 1245-28456 l,Aq ,vAq7 -IATJ-LO-t,, 454-108-344,OPPM, Female, A.d_ ul.t I I 454-108-345,OPPM, Male, I Adult I I 40+6 IWaters, I g , 6ml, llot129082 I Amount and spike Elution solvent mix used and volume Comments 2ml of MeOH NA TN-A-5457 *13okAr NA 2ml of MeOH TN-A-5457 4&@4~[ NA 2ml of MeOH TN-A-5457 4d6L 2ml of MeOH -0 I 9 E d NA TN-A-5457 12mlofMeOH 1 1 &a,+-.& NA TN-A-5457 2ml of MeOH NA TN-A-5457 %owi 2ml of MeOH NA TN-A-5457 .. 2ml of MeOH NA TN-A-5457 TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 Q lank Sera TN-A- 3994 ; Amount of sera aliquated: ml 1ilute Sera with Kandiyohiwater; Amount of water 0 Aliquot 1ml of sera into 15 ml polypropelenetube Spike samples accordingly added 3 8 ml 3 . 3 t(0' +e&p'8E;oZ Add 5ml of ACN (TN-A-4145 )to aliquated sample- iShake sample for 20 min e300rpm (Shakervlr3c! ORa( FL/4'&N/G9$ Centrifugesample for 10 min 2000 rpm (Centrifuge.NftW613 Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decantextract into centrifugetubes with water shake sample slightly to ensure proper mixing Condition columnwith MeOH (TN-A- 754 ) Wash Column with KandiyohiWater Filtersample through conditionedcolumn, discarding filtrate /%)u Allow column to go completelly dry Elute column with indicatedamount of solvent ( Spike samples with internalstandard PFDA standard number c -T3raMnsfeEr nsavmirploeninmtoeanpptraiplriaLteallbyomraarkteodrayutovial r p z e oWz&2 I Page 157 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study: E01-1245 Dilution DateIAnalyst: Box Number: /+t-=+W@L4s' I Attachment D: Dilutions Summary Worksheet ii/.ZL/oz Solvent/TN Number: Meow5457 w/ IS PFDA Extraction Date/Analyst: 1/31/02 Matrixmimepoint: &c;, I S Q ~ @LIS 1 1 / 2 L / ~ 2 I Notes: - .-. 3M Environmental Laboratory Page 158 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Standard CuNes--Fhids Prep Dale(s): Analyst@): Sample Matrix: MethidlRevision: Target Analylets): PFOS Sld: 5.02 ppm: PFOS Sld: 50.2ppm: PFOS S t d 2241 ppm: Surr.Sld Appnir. 100.7 ppm: oiniio2. ozioiio2, oz113ioz OWHOl Quail Sera ETS-8-231. I PFOS 02001-52 02001-53 02001-15 02001-45 PFDA -tual Concentrdtians of Standards in PFOS Std Conc. ug/mL 5.02 5.02 5 .M 5.02 50.2 50.2 50.2 50.2 50.2 5.02 2241 5 FC Mix Am't Spiked 0.0020 n.wo n.nio0 n.0200 0.0040 0.0100 0.0200 0.0300 0.0400 0.0100 0.0360 0.0720 0.0100 Std Conc. u mL 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 0.0800 I 100.7 0.002 0.002 0.002 0.002 0,002 Standard Number EOI-1245-35770.5.0 pph EOl-1245-35771, IO pph EOI-1245-35772.25 pph EOI-1245.35773.50 pph EOI-1245-35774, 1 0 0 pph EOI-1245-35775,250 pph EOI-1245.35776.500 pph EOI-1245.35777.750 m h QC-25 pph pCC-40 ppm Quail Sera MSIMSD-Day 1 Quail Sera MSMSD-Day 2 Quail Sera MS-5 pph IQuail Sera MS-200 pph PFOS Std Conc. nUmL 201 402 1004 2008 4016 10040 20080 30120 40160 PFDA Std Conc. ng/mL 4028 4028 4028 4028 4028 4028 4028 4028 I 613520 3227040 loo40 I II 10040 4028 I 201 4028 I I R032 4028 Standard Number EOl-1245-35770,5.0 pph EOl-1245-35771,10 pph EOl-1245-35772,25 pph EOl-1245-35773,50 pph EOl-1245-35774, 100 pph EOl-I245-35775,250 pph EOI-1245-35776,5Wpph EOl-1245-35777,750 pph EOI-1245-3577R, loo0 pph W-25 pph O.C-40w..m pC-SO ppm Quail Sera MSIMSD-Day I Quail Sera MS-5 pph Quail Sera MS-200 pph PFOS Std Conc. ng/mL 5.02 10.0 25.1 50.2 100 25 I 502 753 1004 25.1 I 40338 RM76 I 251 PFDA Std Conc. nUmL 101 101 101 in1 101 101 in1 101 in1 101 I in1 101 I 101 5.02 101 201 101 Study Number: E01-1245 Equipment Number: NA Find Siilvrnt & TN Number: MeOHfIT-A-05457 Blank Tissneildentifier: Quail Sera-TN-A-5992 All mumL 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.05~ 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 All Final Vol. mL 2.OOO 2.W 2.W 2.000 2.ooO 2.000 2.ooO 2.ooO 2.W 2.W 2.W 2.W 2.ooO 2.ooO 2.W 2.W Blank sera initial dilution (mWmL) for curves: Final siilvent viilume (mL) for C U N ~ S : 0.0500 2 W Verified hy: , / ! . - 7/uL 3M Environmental Laboratory SPE-013 102-QUA1LSERA-02001-52(PFOS).xls Page 159 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Prep Date: Analysts initials: I Lims Assgned sample number E01-1245-35825 E01-1245-35826 E01-1245-35827 E01-1245-35828 E01-1 245-28458 EO1- 1245-28459 E01-1245-28460 E01-1245-28461 E01 -1245-28462 E01-1245-28464 E01-I 245-28465 E01-1 245-2846 E01-1 245-28467 E01 -124528468 E01 -1245-28469 E01-1 245-28470 E01 - 1245-28471 I E01-1245-28472 E01-1245-28473 E01-1 245-28474 I E01-1245-28475 && 211J2002 OWHOJ ac fi , c -"7_ I Sample Number or description ( t y ' Blank H2O-day 2 eIJp1c.ow 3i* ' L Bwlanhk-lsLerae-da0,y6 2 I,~I+LYL AAaHarrl Sera M5-day 2 I W A I L OK l 3 / * Sera MSD-Day2 1454-108-346,O PPM. Female, Adult 454-108-347,O PPM. Male, Adult 454-108-348,O PPM, Female, Adult 454-108-349, 0 PPM, Male, .Adult 454-108-350,O PPM. Female, Adult 454-108-351,O PPM, Male, Adult 454-108-352,O PPM, Female, Adult 454-108-353,O PPM, Male, Adult 454-108-354,O PPM. Female, Adult 454-108-355,O PPM, Male, Adult 454-108-356,O PPM, Female, Adult ,454-108-357,O PPM, Male, Adult 454-108-358,O PPM, Female, Adult 454-108-359,O PPM, Male, Adult 454-108-360,O PPM, Female, Adult I VUlUlIlt! VI sample filtered Type of column (.ml.) used and lot Waters, lg, 6ml, 40+6 lot 129082 Waters, lg, 6ml, 40+6 lot 129082 40+6 Waters, lg, 6ml, lot I29082 40+6 Waters, lg, 6ml, lot 129082 Waters, lg. 6ml, 40+6 Waters, Ig, 6ml, lot 129082 Waters, lg, 6ml, Waters, Ig, 6ml, . Adult - - u m,Male, Adult M+6 Waters, l g , 6ml, a ers, g, m I lot 129082 I Amount and spike mix used NA NA 1OUl Of 02001-53 1OUI Of 02001-53 Method Revision: ETS-8-231.O Study Number: E01-1245 Matrix: Sera and volume 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 I ux3-I+)*a \%L p D 'hLAr t ' 4 , \ u.\ .s-iPc&.oPx* e d 2ml of MeOH TN-A-5457 2ml of MeOH 2ml of MeOH TN-A-5457 20ba I 1 2ml of MeOH 1TN-A-5457 .c) 5RYd 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 5 1"' '4""r r45y shake sample slightlyto ensure proper mixi Condition column with MeOH (TN-A- ) Wash Column with KandiyohiWater Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute columnwith indicatedamount of solvent Spike sampleswith internalstandard PFDA standard number h Transfer sample into appripriatelly marked autovial qj2e ~es.ad& 3M Environmental Laboratory P h - 4 L&ll h%-/ut-A i:, ck@5@* Y@#7 -%w6 444 JX;rz;NJ / A L 66,'- Z/.z&L - 3.8, ~ U W L i. / / G < "- / d $ - V L Page 160 Report E01-1245 Prep Date: Analysts initials: 2/1/2002 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction Worksheet 2.I.02 Method Revision: ETS-8-231 .O lank Sera TN-A liquot 1ml of sera into 15 ml polypropelenetube pike samples accordingly ql4c dd 5ml of ACN (TN-A- ) to aliquated sample &?,w hake sample for 20 min @300 rpm (Shaker&$ 6 9 Centrifugesample for 10 min @ 2000 rpm (CentrifugAg 7 y/N0'&6f4 6~ dd 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Condition column with MeOH (TN-A- Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicatedamount of solvent & m k TN-A-f?fd ) into appropriate 15 ml centrifugetube Spike samples with internalstandard PFDA standard number 0 7 3 ~ 7-/& , conc./&. h!!4 , amount added L L T3rMansEfernsvaimrpolenimntoeanptparliprLiaatebllyomraartkoerdyautovial &% ~ Page 161 Report E01-1245 Prep Date: Analysts initials: 211 12002 OW,!OJ SPE Columns PFOS: Extraction e $;$: A Reproduction Study with Worksheet q j the Nothern Bobwhite LRS 08/2110s 42 .*a OIL Method Revision: ETS-8-231 .O Study Number: E01-1245 Lims Assgned sample number E01-1245-28498 E01 -1245-28499 E01-1 245-28500 EOl-1245-28501 E01-1245-28502 E01-1245-28503 E01-1245-28504 E01-1245-28505 E01-1245-28506 E01-1245-28507 Unl-1 IA<-?P<nP Y"L-.I7_I-I"_I"Y E01-1245-28509 E01-1245-285IO E01-1245-28511 E01-1245-28512 Sample Number or description Volume of sample filtered (mu Type of column used and lot Amount and spike Elution solvent mix used and volume Comments 454-108-388, 10 PPM, Female, Adult 40+6 Waters, Ig, 6mI. lot129082 2ml of MeOH NA TN-A-5457 454-108-389, IO PPM, Male, Adult .--. 454-108-390, 10PPM, Female, Arliilt I I I I I 454-108-391, IO"r"'riw iIv`li'ie, 1 H*U'UI`J ''II 454-108-392, 10PPM, Female, I Adult 454-108-393. IO"r""rw M'a'e, xa'u I 454-108-394, IOPPM,Female, I Adult I d 1454-108-395. 10 PPM, Male, Adul 454-108-396, lOPPM, Female, Adult 40+6 M.-+6 40+6 40+6 40+6 40+6 40+6 Waters, I g , 6ml, lot 129082 I Waters, I g , 6ml, llot 129082 Waters, I g , 6ml. lot129082 Waters, I g , 6ml, lot 129082 Waters, I g , 6ml. lot129082 Waters, l g , 6ml. lot 129082 Waters, I g , 6ml. lot 129082 NA 2ml of MeOH TN-A-5457 p oc 2ml of MeOH 9 NA ITN-A-5457 1 LC%k?& . Gf - 2ml of MeOH NA TN-A-5457 &a .-c/ 2ml of MeOH NA TN-A-5457 I " I/ 2ml of MeOH NA TN-A-5457 NA 2ml of MeOH TN-A-5457 3~ a y c , NA 2nd of MeOH TN-A-5457 z----c. 2ml of MeOH 0 NA TN-A-5457 70kL 454-108-397, lOPPM,Male, Add 454-108-398, IO PPM, Female, Adult 40+6 40+6 Waters, Ig. 6ml, lot Waters, I g , 6ml, lot 129082 NA 2ml of MeOH TN-A-5457 7- 2ml of MeOH NA TN-A-5457 .--" I Waters, Ig, 6ml, 454-108-399, IO PPM, Male, Adult 40+6 lot 129082 NA 454108401, IOPPM,Male, t" IWaters, l g , 6ml, 40+6 /lot129082 NA 454.108402, IO PPM, Female, Adult NA 2ml of MeOH TN-A-5457 2ml of MeOH -A3457 2ml of MeOH TN-A-5457 /OV..L I 454-108403, IO PPM, Male, Add 40+6 IWaters. l g , 6ml, lot 129082 I NA 2ml of MeOH ITN-A-5457 I @w[- 454-108-404,lO PPM, lank Sera TN-A- j / s 4 2 ; Amount of sera aliquated: &/A- ml ilute Sera with Kandiyohiwater; Amount of water added A&- ml Aliquot 1ml of sera into 15 ml polypropelenetube Spike samples according1 Add 5ml of ACN (TN-A- ?45 ) to Shake sample for 20 min @300 rpm Centrifugesample forOI min 2000 rpm (centrifuge2 3~y~t~ 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decantextract into centrifugetubes with water TTff shake sample slightlyto ensure proper mixing Condition column with MeOH (TN-A- ) Wash Column with KandiyohiWater Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry f [a, ?A/ Elute column with indicatedamount of solvent T N - A - 5 7 1 4 into appropriate 15 ml centrifugetube Spike samples with internalstandard PFDA standard number %?W !- , conc. ?&4, amount added -/-0 Tr3aMnsfeEr snavmiprloeninmtoeapnptraiplriaLteallby omraarkteodrayutovial Page 162 Report E01-1245 Analysts initials: 3 SPE Columns ExtraPcFtiOoSn:WAoRrekpsrhoeduecttion@StudQyucw*;lith the Nothern Bobwhite US ~8jail~2 PG@ Y o f Y Method Revision: ETS-8-231.O Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. D', ecant extract into centrifugetubes with water s& shake sample slightlyto ensure proper mixing Condition column with MeOH (TN-A- ) Wash Column with KandiyohiWater Filter sample through conditioned column, discarding filtrate fqr' Allow columnto go completelly dry Elute column with indicatedamount of solvent ( &a# TN-A- .Spike samples with internalstandard PFDA standard number 02-1 T3raMnsfEernsavmirpoleninmtoeanptpariplrLiataelblyomraartkoedrayutovial -41 J ) into appropriate 1 ml qentrifugetube , conc. d o $// amount added ZwL Zvd7/* Page 163 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite BOTTLE-TODPISPENSEDRAILYCALIBRATION CHECK Manufacturer andModel: 5d Date: 2 I ' 0 2 Initials: 0 p(l Dispenser ID: 07 9 9 BalanceID: q C u Volume: 5nhE-Q Solvent: A C d m#:W-A- 4I4< I Mass of solvent (g) I Density' I Volume of Solvent I Std. Dev.: ?A0C.V. : Passmail: Volume, I Accuracy, (d) 1 to 10 (%I 98.0 - 102 % 00.3% C.V., f 1.0 % 1Density values taken from the Merck Index, 12'h Edition, Copyright 1996 dy, =specific gravity at ydegreesreferred to water at xdegrees n-Hexane dZo4=0.660 Acetone d2504 . 7 8 8 Methanol d254=0.7866 Methylene Chloride dZo4=1.3255 3M Environmental Laboratory Page 164 Report E01-1245 SPE Columns ExtracPtiFoOn WS:oArkRsheeperot duction Study with the Nothern Bobwhite I Lims Assigned sample number 1E01-1342-30312 1E01-1342-30323 lE0l-1342-30330 ~_____ EOl-1342-30339 E01-1342-30348 E01-1342-30357 EO 1- 1342-30366 E01-1342-30375 E01-1342-30384 EO 1 - 1342-30393 IEO1 - 1342-30402 IEO1-1342-30411 ~~~~ EO 1-1342-30420 EO 1-1342-30429 EO 1-1342-30438 EO 1-1342-30447 E0l-1342-30456 EO 1-1342-30465 lE0l-1342-30474 1801-1342-30501 Sample Number or description 0 ppm a.i. 329 M Liver 0 ppm a.i. 330 F Liver 0 ppm a.i. 331 M Liver 0 ppm a.i. 332 F Liver 0 ppm a.i. 333 M Liver 0 ppm a i . 334 F Liver 0 ppm a.i. 335 M Liver 0 ppm a.i. 336 F Liver 0 ppm a.i. 337 M Liver 0 pprn a.i. 338 F Liver 0 ppm a.i. 339 M Liver 0 ppm a.i. 340 F Liver 0 ppm a.i. 341 M Liver 0 ppm a.i. 342 F Liver 0 ppm a.i. 343 M Liver 0 p. .pm a.i. 344 F Liver 0 ppm a.i. 345 M Liver 0 ppm a.i. 346 F Liver 0 ppm a.i. 347 M Liver 0 ppm a.i. 348 F Liver 0 ppm ai. 349 M Liver 0 ppm a.i. 350 F Liver Blank Liver TN-A- ; Amount of liver: g Homogenizeliver with Kandiyohiwater; Amount of water added ml Aliquot 1ml of liver into 15 rnl polypropelenetube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min a300 rpm (Shaker ) Centrifuge sample for 10 min Q 2000 rpm (Centrifuge ) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifugetubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- Amount of liver * I<OJ E;- 3M Environmental Laboratory Page 165 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction Worksheet Blank Liver TN-A- ; Amount of liver: g Homogenizeliver with Kandiyohiwater; Amount of water added ml Aliquot 1ml of liver into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min Q300 rpm (Shaker ) Centrifuge sample for 10 min Q 2000 rpm (Centrifuge ) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decant extract into centrifugetubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 3M Environmental Laboratory Page 166 Report E01-1245 SPE Columns ExtracPtiFoOnSW:oArkRsheeperot duction Study with the Nothern Bobwhite Prep Date: Analysts initials: C:+L I ~CJIJ Method Revision: C31- 8 . 23t. / Study Number: e-&/-/F?r Matrix: L-1 number E01-1342-30708 E01-1342-30717 or description Volume of sample Type of column used Amount and Elutionsolvent Amount of liver filtered (ml) and lot spike mix used and volume I Liver 10 ppm a.i. 374 F I ;\mr EO 1-1342-30865 Liver Liver EO 1- 1342-30892 I Liver 10 ppm a.i.392 b Liver EO 1-1342-30901 Liver EO 1-1 342-30910 -. Blank Liver TN-A- 514 3 ;Amount oh&: Homogenize liver with Kandiyohiwater; Amount of water addegd Aliquot 1ml of liver into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min e300 rpm (Shaker ) Centrifuge sample for 10 min Q 2000 rpm (Centrifuge ) Add 40 ml of Kandiyohiwater to 50 ml polypropelenecentrifugetube. Decantextract into centrifugetubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- ) Wash Column with KandiyohiWater Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elutecolumnwith indicatedamount of solvent ( TN-A- Spike samples with internalstandard PFDA standard number Transfer sample into appripriatelly marked autovial ) into appropriate 15 ml centrifugetube , conc. , amount added I 3M Environmental Laboratory Page 167 Report E01-1245 Lims Assigned sample number EO 1-1342-30919 Sample Number I or descriDtion I 10 ppm. .a.i. 395 M E01-1342-30928 EO1-1342-30937 - E01-1342-309476E%~ 2 E01-1342-30955 EO 1-1342-30964 E01-1342-30973 E0l-1342-30982 EO 1-1342-30991 I Liver It 12%10 ppmk an.iL. 3,98wF~ 10 ppm a i . 399 M Liver 10 ppm a.i. 400 F Liver 10 ppm a.i. 401 M Liver 10 ppm a.i. 402 F Liver 10 ppm. .a.i. 403 M 10 ppm a.i. 404 F 10 ppm a.i. 405 M EO 1-1342-31018 10 ppm a.i. 406 F EO 1-1342-31 0 2 7 EO 1-1342-31036 Liver 10 ppm a.i. 408 F Liver PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns ExtractionWorksheet Matrix: C / r Amount of live weighed (9) Comments Blank Liver TN-A- ; Amount of liver: g Homogenizeliver with Kandiyohiwater; Amount of water added ml Aliquot 1rnl of liver into 15 ml polypropelenetube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample \ V 7 3M Environmental Laboratory riate 15 ml centrifugetube , conc. , amount added Page 168 Report E01-1245 Prep Date: I Lims Assigned Sample number I Sample Number or description E01-1365-31199 EO1-1365-3 1214 I 0 ppm a.i. 256 M Liver F M 8". 0 ppm a.i. 270 M ' - Liver %.i2. 73 F I Liver PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns ExtractionWorksheet E01-1365-31270 EO1-1365-3 1275 E01-1365-31280 E01-1365-31285 10 ppm a.i.9ZlW Liver 10 ppm a.i. bbl F Liver 10 ppm a.i.561 I- Liver 10ppm 8.1.568 IT Liver Blank Liver TN-A- ; Amount of liver: g Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver into 15 ml polypropelenetube Spike samples accordingly / Shake sample for 20 min e300 rpm (Shaker Condition column with Wash Column with Kandiyhi Water /vM.&e-y 0./1 4d.J M b F 4 h Y m f Lurt*ry A 4 c - d A%- a k l k A e / a 0 < 3 3 --z-$-P& - G -49 - 0 J-9 C"b3 3 - k AL 4.5hL -5.44 6.3-j -k*J*,L 6 .87 -3-2-sL &2&* 2-8 q+- @ Tf(i5 j5 7-eut. m;C3 wv f=& ALC L , bfeqc3 we f q H e 3 id C>waY. 3M Environmental Laboratory Page 169 Report E01-1245 SPE Columns ExtractPioFnOWSo: rAksRheepertoduction Study with the Nothern Bobwhite Prep Date: 211 112002 Analysts initials: 0K.HOJ $./pa OK - I Lims Assigned sample number Sample Number or description 1 Volume of sample filtered (ml) 40+6 + I E01-1342-36003 E01-1342-36004 Female liver MSday 1 Female liver MSDday 1 40+6 40+6 40+6 I 40+6 E01-1342-36005 Curve point-0.2 ppb in liver extract 40+6 E01-1342-36006 Curve point-OS ppb in liver extract 40+6 E0 1- 1342-36007 Curve point-I ppb in liver extract 40+6 E01-1342-36008 Curve point-2.5 ppb in liver extract 40+6 E0 1-1342-36009 Curve point-5 ppb in liver extract 40+6 E01-1342-36010 Curve point-IO ppb in liver extract 40+6 E01-1342-36011 Curve point-25 ppb in liver extract 40+6 E01-1342-36012 Curve point50 ppb in liver extract 40+6 E01-1342-36013 Curve point-75 ppb in liver extract 40+6 E01-1342-36014 Curve point-I00 ppb in liver extract Curve point-I00 ppb-2 in extract 40+6 I I I 40+6 40+6 I @AL@ " Method Revision: ETS-8-231.o Study Number:E01-+34W36S , y B r @&d ?e/f.dz I Type of column Amount and spike used and lot mix used Matrix: Elution solver and volume Liver Comments C18, l g , 6cc, lot W202581 NA 2ml MeOH TNA-5754 C18, l g , 6cc. lot W202581 C18, lg, 6cc, lot W202581 C18, l g , 6cc. lot W202581 C18. Ig.6cc. lot W2025B1 C18, l g , 6cc, lot W2025B1 NA NA I I 50ul of 02001-66 I I I 2rnl MeOH TN-A-5754 2ml MeOH TNA-5754 2ml MeOH TN-A-5754 2mlMeOH 2mlMeOH C18, lg. 6cc, lot W202581 C18, l g , 6cc, lot W202581 C18, lg. 6cc, lot W202581 5Ul Of 02001-66 TNA-5754 C18, l g , 6cc, lot W2025B1 1OUl of 02001-66 2ml MeOH TNA-5754 CIS, lg, 6cc, lot W202581 20Ul Of 02001-66 2rnl MeOH TN-A-5754 C18, l g , 6cc. lot W202581 5 0 ~o1f 02001-66 2ml MeOH TN-A-5754 C18, l a . 6cc. lot W2025B1 1OOUI Of 02001-66 2rnl MeOH TN-A5754 C18, lg, 6cc, lot W202581 C18. l g , 6cc, lot W202581 150~O1f 02001-66 200~1Of 02001-66 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 W202581 41.11Of 02001-53 2ml MeOH TN-A-5754 W202581 1 0 ~O1f 02001-53 2ml MeOH TNA5754 W202581 C18, I g , 6cc, lot W2025B1 ti1LI. l a . occ, IO1 W2025B1 I I 20ul of 02001-53 I 1 I I 30ul of 02001-53 I I I I 40ul of 02001-53 2ml MeOH TN-A-5754 2mlMeOH TN-A-5754 L LrniMevn HALewfgI3 @ TN-A-5754 OA q . 4 e 2 Blank Liver T N - A - 5 1 4 3 ; Amount o d C)35'3g Blank Liver T N - A - S / 4 & ; Amount ofk&- 1 Homogenize liver with Kandiyohi water; Amount of water added qg ml Amount of water added 7 Aliquot 1ml of liver into 15 ml polypropelene tube 1 4 5 Spike samples accordingly Add 5ml of ACN (TN-A- '! ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker deb 3d/q+eL Centrifuge sample forOI min @ 2000 rpm (Centrifuge ~ - 7 9d6 9 g j 3 ' B4'6'9G Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 57 54 ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate g ml Allow column to go completelly dry 4 Elute column with indicated amount of solvent ( TN-A- 5? 5 2 , d Spike samples with internal standard PFDA standard number oac-at- Transfer sample into appripriatelly marked autovial ) in& appropriate 15 ml centrifuge tube 4 5 7 , conc. (00-pTM, amount added I\ c & $ ~ p O x h Q p A k W @ ' ! $7 6'** fa?-o2op( d. U 3M Environmental Laboratory Page 170 Report E01-1245 a - l r ' PrepDate: OR Analysts initials: 06 0 1-1342-360 18 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction Worksheet Method Revision: t7 5-P-231- Blank Liver TN-A- 2 14% ; Amount of liver: 9.0353g Homogenize liver with Kandiyohi water; Amount of water added Blank Liver TN-A- 5 I42J ; Amount of liver: /,O&sg 4 5 ml Amount of water added 9 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly 4/45 Add 5rnl of ACN (TN-A- ) to aliquated sample duoe ' Shake sample for 20 rnin Q300 rpm (Shaker s&04' M Centrifuge sample for IO min Q 2000 rpm (Centrifuge W t C 9 Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 5j44 ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry %Ov w3q Elute column with indicated amount of solvent ( TN-A- ) into appropriate 15 ml centrifuge tube o ? @ /4 5 Spike samples with internal standard PFDA standard number - Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 171 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Standard Curver--Sohds Prep Date($: Analysl(s): Sample Matrix: MethodlRevision: Target Analyte(s): FC Mix Std Apprar. I 00 ppm: FC Mix Sld Approx. 5 02 ppm FC Mix Std Approx. 50 2 ppm: Surr. Std Approx. 100 7 ppm: 0211 1/02. 02/12/02, 02/13/02, 02/15/02 OWHOJ Qual Liver ETS-8-231 1 PFOS 02001-66 02001-52 02001-53 02001-45 PFDA Actual Concentrationsof Standards Standard Number E01 -1342-36005 E0l-1342-36006 EO I - 1342-36007 E01-1342-36008 E01-1342-36009 E01-1342-36010 E01-1342-36011 E01-1342-36012 EOI-I 342-36013 E0l-1342-36014 EO1-1 342-36062 E01-1 342-36015 E0l-1342-36016 EO 1-1342-36060 E01-1342-36061 QC-IOppb QC-75 ppb QC-I50 ppb Qual Liver-MSMSD-Day 1 Qual Liver-MSNSD-Day 2 Qual Liver-MSMSD-Day 3 Qual Liver-MSMSD-Day 4 le FC Mix PFOS Std Conc UdmL 1004 I 004 I 004 I 004 1004 1004 I 004 I004 1 004 1 004 50 20 50 20 50 20 50 20 50 20 5 020 5 020 5 020 1 004 1 004 1004 I004 PFOS Am't Spiked mL 0 0004 0 0010 0 0020 0 0050 00100 0 0200 0 0500 0 1000 0 IS00 0 2000 0 0040 00100 0 0200 0 0300 0 0400 0 0040 0 0300 0 0600 0 OS00 0 0500 0 0500 0 0500 Calculated Concentrationsof Standa Standard Number E01-1342-36005 E01-1342-36006 01-1342-36007 EOI-1342-36008 E0l-1342-36009 EOI-1342-36010 EOI-1342-3601 I E01-1342-36012 E01-1342-36013 E01-1342-36014 EO1- 1342.36062 E0l-1342-36015 E0l-1342-36016 01-1342-36060 E01- 1342.36061 QC-IO ppb QC-75 ppb QC-150 ppb Qual Liver-MSMSD-Day I Qual Liver-MSMSD-Day 2 Qual Liver-MSMSD-Day 3 Quail Liver-MSNSD-Day 4 in the sample I PFOS Std Conc nglg 3 99 9 98 20 0 49 9 99 8 200 499 998 I496 1995 1995 4988 9977 14965 I9953 zoo 1496 2993 499 499 499 499 IIX PFDA Std Conc ndr: 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 200 I 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 PFDA Std Conc "g/mL 100 7 100 7 100 7 100 7 100 7 100 7 100 7 1007 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 100 7 Study Number: 01-I245 Equipment Number: NA Final Solvent & TN Number: M e o w - A - 5 7 5 4 Blank Tissuelldentifier: Qual Liver(female)KN-A-5 I43 Qual Liver(male)m-A-5 I42 h t i a l female homogenate = 5 0353d50 0353g+mL = 0 1006 g/mL hiha1 male homogenate = I 0015glIO 0015g+mL = 0 1001glmL Blank liver initial homogenate (g/mL) for curies. MS, QC: Final solvent volume (mL) for cwves, MS, QC: 3Initial L:ogenate mL g/mL 0002 ' 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 IO06 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 IO06 0 002 0 I006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 0 002 0 1006 All 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 0 1006 2 000 Calculated Concentrationsof Standa Standard Number EO 1-1 342-36005 EO 1-1 342-36006 01-1342-36007 E01-1342-36008 E01-1342-36009 E01-1342-36010 EOl-1342-3601 I EOI-1342.360 I2 01- I 342-360I 3 EOI-1342-36014 E01-I 342-36062 EOI-1342-36015 EO1 -1342-3601 6 EO I - 1342-36060 EOl-1342-36061 QC-l0ppb QC-75 ppb Qual Liver-MSMSD-Day I Oual Liver-MSMSD-Dav 2 Qual Liver-MSiMSD-Day 3 Qual Liver-MSMSD-Day 4 m the final sol PFOS Std Conc og/mL 0 201 0 502 100 2 51 5 02 IO0 25 I 50 2 75 3 100 IO0 25 I 502 753 1004 10 0 75 3 151 25 I 25 I 25 1 25 1 PFDA Std Conc nglmL 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 3M Environmental Laboratory SPE-021102-QUAILLIVER-02001-66(PFOXSI)S 0 1006 0 1001 Page 172 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite BOTTLE-TOPDISPENSERDAILYCALIBRATION CHECK Manufacturer VCUL and Model: Date: 67-(/.a2 Initials:& Dispenser ID: c-`35 6~ Balance ID: 900 Volume: Solvent: /dLy In #: ,T A. Q / 4 f Mass of solvent (g) Density' I Volume of Solvent I I Averagevolume: I 1 %Accuracy: Std. Dev.: f`- 0333/aa. '30 ,6 b 3987- Volume, (d) 1 to 10 Accuracy, (%> 98.0 - 102 % Ma& b44 Corrective Actions: &J 2 -C t - T L C.V., ("A) k 1.0% `Density values taken from the Merck Index, 12thEdition, Copyright 1996 dY,=specific gravity at ydegreesreferred to water at xdegrees n-Hexane dzo4=0.660 Acetone dzs0=0.788 Methanol d254=0.7866 Methylene Chloride dZo4=1.3255 3M Environmental Laboratory Page 173 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Prep Date: Analysts initials: Lims Assigned sample number E01-1342-36026 EO 1- 1342-36027 EO 1-1342-36028 EO 1-1342-36029 EO 1- 1342-36030 2/12/2002 Qt( OWHOJ 2.12.02 Sample Number or description Blank HZO-day 2 Blank female liver-day 2 Blank male liver-day 2 Female liver MSday 2 Female liver MSDday 2 Volume of sample filtered (ml) 40t6 40+6 40+6 40t6 40t6 Method Revision: ETS-8-231. Study Number: E01- O r -~ * - A?? Matrix: Liver Type of column Amount and used and lot spike mix used Cnmrnnntc C18, l g , 6cc, lot W2025B1 CIS, l g , 6cc. lot W2025B1 C18, l g , ~ C C l,ot W2025B1 C18, l g , ~ C C l,ot CISW, 2l g0.265cBc1, lot I' NA NA NA NA I NA I NA NA NA 2ml MeOH TN-A5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH '"LA5754 I - . ,2ml MA.e?OH-,.-. I mi M e v n -.,*r-,r, I I zmi M e w TNA-5754 2ml MeOH bf T-N-.A.-.57-5.4. Blank Liver TN-A- 6 / 4 3 ; Amount of liver: 3 0 3 s 3 g Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1rnl of liver into 15 ml polypropelene tube 'a Blank Liver TN-A- ; Amount of liver: I . G O I 5- 9 45 ml Amount of water added 9 ml Spike samples accordingly 414 Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min Q300 rpm (Shaker V W f L 5@ 041694 Centrifuge sample for IO min Q 2000 rpm (Centrifugew@ 3 6w13 Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixin Condition column with MeOH (TN-A- 575-4 ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry r 7 5 q Elute column with indicated amount of solvent ( HeoH TN-A- oaoo - m, Spike samples with internal standard PFDA standard number ) into appropriate 15 ml centrifuge tube 4% , conc. [*' =f. amount added Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 174 Report E01-1245 Prep Date: Analysts initials: 2/12/2002 OWHOJ 3 us SPE Columns ExtractioPnFWOoSr:kAshReeetproduction Study with the Noot~he/rn~Boabwl h~ite~ .a 2.Q.& Method Revision: ETS-8-231 Study Number: EOl-t5Mft365 f 9 r & Matrix: Liver Lims Assigned sample number E01-1342-30537 EO 1- 1342-30546 Sample Number or description 0 ppm a.i. 354 F Liver 0 ppm a i . 355 M Liver Volume of sample filtered (ml) 40+6 40t6 Type of column used and lot C18, l g , 6cc, lot W202581 C18, lg, 6cc, lot Amount and . spike mix used I Elutionsolvent and volume E01-1342-30555 E01-1342-30564 E01-1342-30573 EO 1-1342-30582 1E01-1342-30591 1E01-1342-30600 lE01-1342-30609 0 ppm a.i. 356 F Liver 0 ppm a.i. 357 M Liver 0 ppm a.i. 358 F Liver 0 ppm a.i. 359 M Liver I 0 ppm a.i. 360 F Liver I I 0 ppm a.i. 361 M Liver I I 0 ppm a.i. 362 F Liver I 40t6 40+6 40t6 40+6 40t6 40+6 40+6 C18, l g , 6cc, lot v-----. C18, l g , 6cc, lot W202581 C18. l g , 6cc, lot C18W, 2l g02, 65c8c1, lot I' I c1!8n'nlng ?c69 .cac+' lot I c1!8nsnl gn'?6ccnciDlot I innlng 'l6c'cnci ' lot Comments Blank Liver TN-A- 5 'h3 ; Amount of liver: ,0353 g Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver into 15 ml polypropelene tube Blank Liver TN-A-4 '42 ; Amount of liver: ).-)< 45 ml Amount of water added 9 g ml Spike samples accordingly 5 Add 5ml of ACN (TN-A- 1114 ) to aliquated sample ' Shake sample for 20 min @300 rpm (Shaker VU-)@ " y d " 67 6 Centrifuge sample for 1o min Q 2000 rpm (Centrifuge N/+3 5/M 04 I 69$ I 3 Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- s7s 7 ) 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 ( TN-A-ST5 Spike samples with internal standard PFDA standard number 4 ) into appropriate 15 ml centrifuge tube ~ 4-5 , conc. %w%amount added Transfer sample into appripriatelly marked autovial n 3M Environmental Laboratory Page 175 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite BOTTLE-TOPDISPENSEDRAILYCALIBRATIOCNHECK Manufacturer y,Q e,5& a and Model: Date: 3 .I L' 2. Initials: WC Dispenser ID: 07 94 Balance ID: ?cL> Volume: 5 CJ- Solvent: A C h j - ID#: W - A 4/45 p,<,me +* I I Mass of solvent (g) I Density' I Volume of S o l v p t I I Average Volume: %Accuracv: I I Std. Dev.: Y0C.V.: I 5.03- 100. F 0.o a g 3 Performance SDecifications I Volume, I Accuracy, I C.V., I Corrective Actions: 5 'Density values taken from the Merck Index, 12'hEdition, Copyri h t 1996 dY,=specific gravity a t ydegreesreferred to water a t xdegrees n-Hexane dZo4=0.660 Acetone d250=0.788 Methanol d254=0.7866 Methylene Chloride dZo4=1.3255 3M Environmental Laboratory Page 176 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction Worksheet Prep Date: 2113/2002 Analysts initials: OWHOJ I Lims Assgned sample number E01-1245-36153 I Sample Number or description Blank-H20 E01-1245-36154 Blank sera E01-1245-36155 Sera MSdppb in sera extract-1 E01-1245-36156 Sera MSBppb in sera extract-2 E01-1245-36157 Sera MS-5ppb in sera extract-3 E01-1245-36158 Sera MS-200ppb in sera extract-I E01-1245-36159 Sera MS-200ppb in sera extract-2 E01-1245-36160 Sera MS-200ppb in sera extract-3 Volume of ;ample filterec (mu 40+6 40+6 40+6 40+6 ~~ 40+6 40+6 40+6 40+6 Type of column used and lot C18, l g , 6cc, lot W202581 C18, l g , 6cc. lot W202581 C18, lg. 6cc, lot W202581 C18, l g , 6cc, lot W202581 C18, l g , 6cc, lot W202581 C18. l g , 6cc, lot W202581 C18, l g , 6cc. lot W202581 C18, l g , 6cc, lot W202581 Amount and spike mix used Method Revision: ETS-8-231.O Study Number: E01-1245 Matrix: Sera Elution solvent and volume Comments NA NA 2UI Of 02001-52 2UI Of 02001-52 2UI Of 02001-52 8 0 ~O1f 02001-52 80ul Of 02001-52 8 0 ~O1f 02001-52 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 I U Blank Sera TN-A- 577 2 ; Amount of sera aliquated: 2 ml Dilute Sera with Kandiyohi water; Amount of water added r m I Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- 45) to aliquated sample Shake sample for 20 min @300 rpm (Shaker VW@ $d @41$94 Centrifuge sample forOI min 2000 rpm (Centrifuge 3~wY76q613 Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- ps7i34 ) 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 ( tw'p TN-A- sT5G Spike samples with internal standard PFDA standard number C@*l.- ) 5 into appropriate 15 ,cone. m ml centrifuge ,7p< . ~tube & ~ J J A Transfer sample into appripriatelly marked autovial pxhc)csi.o&A dow bs Qoc. 3M Environmental Laboratory Page 177 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Bottle-Top Dispenser Daily Calibration Check ~ ~ - ~r ~ ~ p y I Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID #: VWR 5ml dispenser q-fe o K G i h j f K - 3 $.OR8 211 312002 OK 0799 900 5 ACN TN-A-4145 f'f6 Q.1" aw Mass of solvent (9) Density' Volume of (gW Solvent 1 3.965 0.7857 5.046 2 3.9715 0.7857 5.055 I 3 I 3.9818 I 0.7857 I 5.068 I 4 3.9484 0.7857 5.025 5 3.9788 0.7857 5.064 Averagevolume: 5.052 I Std. Dev.:! 0.0169 I I I %C.V.: 0.33 PASS PassIFail: PASS Volume, W) 1 to 10 - Accuracy, ("A> 98.0 - 102 0'9 Corrective Actions: C.V., ("h) Zk 1.0% 1Density values taken from the Merck Index, 12`hEdition, Copyright 1996 dY,=specific gravity a t ydegrees C referred to water a t xdegrees n-Hexane dZo,=0.660 Acetone dZsO=0.788 Methanol dZs4=0.7866 Methylene Chloride dZ", =1.3255 3M Environmental Laboratory Page 178 Report E01-1245 Prep Date: Analysts initials: 2/13/2002 OWHOJ SPE Columns ExtractioPnFWOoSr:kAshReeetproduction Study with the Nothern Bobwhite Study Number: E01- Matrix: Liver 50ul of 02001-66 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TNA-5754 ~~~~~~ Blank Liver TN-A- 5143 ; Amount of liver: 5.03539 Blank Liver TN-A- 5142 ; Amount of liver: 1.0015q Homogenize liver with Kandiyohi water; Amount of water added-&I-% Aliquot 1ml of liver into 15 rnl polypropelene tube 4 4 PA& 0 &.,3.&"2 Amount of water added s m l Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker C'%LLqq Centrifuge sample for I O min Q 2000 rpm (Centrifuge j# 764613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 5 3 Wash Column with Kandiyohi Water ct ) Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry kkc s75q Elute column with indicated amount of solvent ( kl TN-A- c2Gc Spike samples with internal standard PFDA standard number Transfer sample into appripriatelly marked autovial -4<) into appropriate 15 ml centrifuge tube , conc. 10" q.pf.%L, , amount added 'Ld e q p A epod 3 t W C 3M Environmental Laboratory Page 179 Report E01-1245 SPE Columns ExtractioPnFWOoSr:kAshReeetproduction Study with the Nothern Bobwhite Prep Date: Analysts initials: 211 312002 OWHOJ Method Revision: ETS-8-231.O Study Number: E01-1342/1365 Matrix: Liver Lims Assigned sample I I I I I I I I number I I I IEO1- 1342-30820 ..-----. Nh EO 1- 1342-30829 EO 1-1342-30838 EO 1-1342-30847 EO 1- 1342-30856 I EO1- 1342-30865 Sample Number or description 10 ppm a.i. 384 F Liver 10 ppm a.i. 385 M Liver 10 ppm a.i. 386 F Liver 10 ppm a.i. 387 M Liver 1 0 ppm a.i. 388 F Liver 1 0 ppm a.i. 389 M Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18, l g , 6cc. lot W~T~RI C18, l g , 6cc, lot W202581 C18, l g , 6cc, lot W202581 C18, l g . 6cc, lot - W 2 0 2U = Y = 4I C I.A_,I. nJ , 6cc, lot W202581 C18, l g , 6cc, lot W202581 Amount and Elution solvent and spike mix used volume NA 2ml MeOH TN-A5754 NA 2ml MeOH TN-A-5754 ,Ik l n Wl 2ml MeOH TN-A5754 NA 2ml MeOH TN-A5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A5754 Comments \ \ 1 NA 2ml MeOH TN-A-5754 2mlMeOH Blank Liver TN-A- 5143 ; Amount of liver: 5.03539 Blank Liver TN-A- 5142 ; Amount of liver: 1.0015q Homogenize liver with Kandiyohi water; Amount of water added>'c:,y Amount of water added A m 1 Aliquot I m l of liver into 15 ml polypropelene tube 97\3 Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample wk Shake sample for 20 min @300 rpm (Shaker 5 [ d)04\ bq4 a Centrifuge sample for I O min 2000 rpm (Centrifuges'JP?bcjQiP Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 6 7 G? ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry 5?&9 Elute column with indicated amount of solvent ( He L'H TN-A02-1' Spike samples with internal standard PFDA standard number /) 4 5 Transfer sample into appripriatelly marked autovial EYbefiOL c4c3-v.Q 08 into appropriate 15 ml centrifuge tube , conc. / O i . . 7 P , amount added 1' ow.& op 3M Environmental Laboratory Page 180 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Bottle-Top Dispenser Daily Calibration Check ' Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID #: VWR 5ml dispenser 211 312002 OK 0799 900 5 am et6 Q.13 ACN TN-A-4145 Mass of solvent (8) Density' Volume of (dml) Solvent 1 3.965 0.7857 5.046 2 3.9715 0.7857 5.055 I 3 I 3.9818 0.7857 5.068 4 I 3.9484 I 0.7857 I 5.025 I 5 I 3.9788 I 0.7857 I 5.064 I 5.052 PASS Volume, (mLI 1 to 10 Pass/Fail:l PASS I I Performance Specifications Accuracy, (%I I 98.0 - 102% C.V., * (%I 1.0% PASS 1Density values taken from the Merck Index, 12'hEdition, Copyright 1996 dY,=specific gravity a t ydegreesC referred to water a t xdegreesC n-Hexane dZn,=0.660 Acetone dz50=0.788 Methanol d25,=0.7866 Methylene Chloride dzn4=1.3255 3M Environmental Laboratory Page 181 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Attachment D: Study: E01-1245 Dilution Date/Analyst: 6) Box Number: E01-1245 - Dilutions SummaryWorksheet Solvent/TN Number: MeOW5457 W/IS PFDA Extraction Date/Analyst: 27"#&qgvc. r d?.* Matriflimepoint: Quail sera Notes: 1/100 dilution= lOul of sample +990ul of solvent D A l l dilutionswere made on day of extractionor the day after analysis. Form Completion Verified By: & 9'k07 3M Environmental Laboratory Page 182 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3 Study: E01-1342 Dilution Date/Analyst: Box Number: E01-1342 Attachment D: Dilutions Summary Worksheet - i 2 6'OQ?J k '*" g&ent/TN Number: MeOH/5457 W/IS PFDA Extraction Date/Analyst: 2/13/02 Matrixflimepoint: Quail liver E01-1342-30991 x E01-1342-31009 x I E01-1342-31027 x Notes: 1/10 dilution = lOOul of sample + 900ul of solvent 1/100dilution= 1Oul of sample +990ul of solvent 0All dilutions were made on day of extraction or the day after analysis. Form Completion Verified By: - c \ -o@iP9 - 3M Environmental Laboratory Page 183 Report E01-1245 Prep Date: Analysts initials: 211 512002 OWHOJ SPE Columns ExtractioPnFWOoSr:kAshReeetproduction Study with1t4h5eoNsloztzh]eornr Bobwhite Method Revision: ETS-8-231.O Study Number: E 0 1 - #& m/ z 4 r Matrix: Liver Blank Liver TN-A- 5143 ; Amount of liver: 5.03539 Blank Liver TN-A- 5142 ; Amount of liver: 1.0015q Homogenize liver with Kandiyohi water; Amount of water a d d e d A m l - Amount of water added 3rd Aliquot lrnl of liver into 15 ml polypropelene tube 9f q5 Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min a 3 0 0 rpm (Shaker WVR S/N 041694) Centrifuge sample for 10 min 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 575 4 ) 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 ( TN-A- 5 7% Spike samples with internal standard PFDA standard number 1- ) 4 into 7 appropriate , conc. 15 w , ml centrifuge tube amount added %$ Transfer sample into appripriatelly marked autovial opow 03 3M Environmental Laboratory Page 184 Report E01-1245 SPE Columns ExtractioPnFWOoSr:kAshReeetproduction Study with the Nothern Bobwhite Prep Date: Analysts initials: I Lims Assigned sample number E01-1365-31214 E01-1365-31219 E01-1365-3 1224 E01-1365-3 1229 E01-1365-31234 1E01-1365-31240 E01-1365-3 1245 E01-1365-31250 E01-1365-31255 E01-1365-31260 E01-1365-31265 E01-1365-31270 IE01- 1365-31275 ~~~ E01-1365-31280 E01-1365-31285 211 512002 OWHOJ I Sample Number or description 0 p p m a.i. 270 M Liver 0 p p m a.i. 273 F Liver 0 p p m a.i. 277 F Liver 0 p p m a.i. 283 M Liver 0 p p m a.i. 291 M Liver I 10 p p m a.i. 517 F Liver 10 p p m a.i. 522 M Liver 10 p p m a.i. 525 F Liver 10 p p m a i . 533 M Liver 10 p p m a.i. 538 F Liver 10 p p m a.i. 545 M Liver 10 p p m a.i. 552 M Liver I 10 ppm a.i. 557 F Liver 10 p p m a.i. 561 F Liver 10 p p m a.i. 568 M Liver QK VO~UmeOf Sample I I filtered (ml) 40+6 40+6 40+6 40+6 40+6 I 40+6 I 40+6 40+6 40+6 40+6 40+6 I I 40+6 40+6 40+6 40+6 Type of column used and lot C18, l g , 6cc, lot W202581 C18. l g , 6cc, lo1 W202581 C18. l g , 6cc. lot W202581 C18, l g , 6cc. lot W202581 C18, l g , 6cc. lot \N?n?L;R1 l A Dlng 76KcR'cI ' lot C18, lg. 6cc, lot W202581 C18, 1g,6cc, lot W202581 C18, l g , 6cc, lot W202581 C18, l g , 6cc, lot W202581 C18, 19, 6cc, lot W202581 C18. l g , 6cc, lot W~?SRI C18. lg, ~ C Clot, \nnn?s~i C18, l g , 6cc, lot W202581 C18, l g , 6cc, lot W202581 Method Revision: Study Number: E01- Matrix: I I I I Amount and Elution solvent and spike mix used volume NA 2ml MeOH TN-A-5754 NA 2mlMeOH 1 TN-A-5754 NA 2ml M e O H TN-A-5754 NA 2ml M e O H TN-A5754 NA 2ml M e O H TN-A-5754 NA 2ml MeOH TNA-5754 NA 2ml MeOH TN-A-5754 NA 2ml M e O H TN-A-5754 NA 2ml M e O H TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml M e O H TN-A-5754 NA 2ml M e O H TNA-5754 NA 2ml MeOH TN-A-5754 NA 2ml M e O H TNA-5754 NA 2ml M e O H Thl A C 7 C A Comments AJA I c- I Blank Liver TN-A- 5143 ; Amount of liver: 5.03539 Blank Liver TN-A- 5142 ; Amount of liver: 1.0015q Homogenize liver with Kandiyohi water; Amount of water a d d e d A m l - Amount of water added s m l Aliquot I m l of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- 4 / 4 5 ) to aliquated sample Shake sample for 20 min a 3 0 0 rpm (Shaker WVR SIN 041694) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 575 4 ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate bfpaw 4 Allow column to go completelly dry Elute column with indicated amount of solvent ( TN-A- ' ' f 5 Spike samples with internal standard PFDA standard number ) into appropriate 15 ml centrifuge tube 4s- , conc. 100,- , amount added 1' Transfer sample into appripriatelly marked autovial gsoz 3M Environmental Laboratory Page 185 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite BottleTop Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: I D #: VWR 5ml 211 512002 OK 0799 900 5 ACN TN-Ad 145 1 1 2 3 4 5 I Mass of solvent (g) I 3.8904 I I 3.9526 3.9724 Density' (gW 0.7857 0.7857 0.7857 I Volumeof Solvent 4.9515 I I 5.0307 5.0559 3.961 0.7857 5.0414 3.964 0.7857 5.0452 AverageVolume: I %Accuracy:l 5.0249 100.50 I PASS PASS Volume, (mLI 1 to 10 I I Performance Specifications Accuracy, ("/.I 98.0 - 102 % I Corrective Actions: C.V., ("/.I f 1.0% 1Density values taken from the Merck Index, 12'hEdition, Copyright 1996 dY,=specific gravity a t ydegreesreferred to water a t xdegrees n-Hexane d204=0.660 Acetone dZ5,=0.788 Methanol d254=0.7866 3M Environmental Laboratory Page 186 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ATTACHMENTE: STUDY PROTOCOL, PROTOCOL AMENDMENTSA,ND DEVIATI oNS 3M Environmental Laboratory 3M Environmental Laboratory Page 187 of 226 Page 187 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite WILDLIFE INTERNATIONAL. LTD PROJECTNO.: 454-108, EO1-1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENTNO.: 9 SPONSOR 3M Corporation PROJECTNO.: 454-108, E01-1245 EFFECTNE DATE: January 24,2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides the information necessary to complete the analflcal phase of this study. REASON: The protocol did not include information for completing the analyses required by the sponsor. AMENDMENT: Harold Johnson is the Principal Analflcal Investigator REASON: The sponsor requested that a principal analytical investigatorbe added to the protocol. AMENDMENT: The 3M Environmental Laboratory Project IdentificationNumber to be included with the study is E01-1245. REASON: The sponsor requested that the 3M Project IdentificationNumber be included in the protocol. 3M Environmental Laboratory Page 1 of 2 Page 188 - . _Re0p1o/r2t9E/0012-1214.65:25 FAX 410 a22 0632 JAN. 29. 2002 1 1 : 34AM ENVIEONh!ENTAL WILDLIFEPFIONTSE:RANARTeIpOrNoAdLuction Study with the Nothern Bobwhite LAB 2 3E 09 w UUL 110,6313 P. 3 WILDLIFE INTERNATIONAL, LTD PROJECTNO.; 454-108, EOl-1245 3M Environmental Laboratory Page 189 Report E01-1245 WILDLIFE INTERNATIONAL, LTD PFOS: A Reproduction Study with the Nothern Bobwhite PROJECTNO.: 454-108, EO 1- 1245 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 EnvironmentalLaboratory, Date Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date 3M Environmental Laboratory Page 2 of 2 Page 190 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Protocol Amendment # 9 = Attachment A Wildlife International Ltd. ## 454-108 STUDY TITLE PFOS: A Reproduction Study with the Northern Bobwhite SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 3333 1 St. Paul, MN 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M EnvironmentalLaboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: E01-1245 Wildlife International Ltd. : 454- 108 3M Environmental Laboratory Page 191 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Sponsor Study Identification PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 Principal Analytical Investigator Study Locations ANALYTICAL TESTING LABORATORY ANALmCAL TESTINGLABORATORY 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 Proposed Study Timetable ANALYTICAL mPERIMEMAL START DATE ANALMEAL EXPERIMENTAL TERMINATION DATE 01/24/02 03/24/02 3M Environmental Laboratory 2 3M Environmental Laboratory Page 192 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 1. PURPOSEIOBJECTIVES In the in-life phase of this study, Northern Bobwhite quail 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 five-month period. The purpose of this analytical phase of the study is to quanti@PFOS in the liver and sera of both control and dosed Northern Bobwhite quail. The analytical information may be used to compare with toxicologicalhstopathologicalresults from the in-life portion of this bird study. The in-life portion of the study was completed at Wildlife International, Ltd. in study 454-108. 2. REGULATORCYOMPLMNCE 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 Study Director. 4. REFERENCSEUBSTANCE 4.I Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity a n d Stabi/*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 PS013, "Method Validation and Instrument Detection Limit Determination for LCMS and GCMS 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 Conditions-The test article may be stored at ambient temperature or lower. 4.4 Reserve Samples--A reserve sample of PFOS from Lot #217 has been frozen 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 Mo/ecu/ar Weight-The molecular weight is 538 ( C ~ F I ~ S O ~NKo)t.e: The molecular weight of the anion (PFOS) is 499 ( C ~ F U S O ~ ) . 3M Environmental Laboratory 3 3M Environmental Laboratory Page 193 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 5. CONTROMLATRICES Types of control matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. 6. TESTSYSTEM The test system is Northern Bobwhite quail. 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-lie Wildlife phase report. Northern Bobwhite 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 Northern Bobwhite quail 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 454-108) for information about bird origination location. 8. SPECIMEN AND SAMPLE RECEIPATND MAINTENANCE Sera and liver were collected by Wildlie International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 20-week endpoint sera and liver samples received by the 3M Environmental Laboratory were fiom 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 pprn 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 4 3M Environmental Laboratory Page 194 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 9. SUB-CONTRACTED ANALYSIS 9.7 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 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 70. EXPERIMENTACLONDUCT 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 Methods 70.7.7 ETS-8-6, Extraction of Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry 70.7.2 ETS-8-4, Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrosprayhdass Spectrometry 70.2 Analytical Methods 70.2.7 ETS-8-7, Analysis of Fluorochemicalsin Liver Extracts Using HPLCElectrospray / Mass Spectrometry 70.2.2 ETS-8-5, Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicalsin Serum Extracts Using HPLC-ElectrosprayMass Spectrometry 77. METHODVAUDA~ON Method validation for PFOS quantitation in quail sera and liver was performed at Centre Analytical Inc. in study 023-054. The method will be applied to sample analyses without additional validation. 72. DATAQUALITYOBJECTIVESDURING SAMPLEANALYSIS The following criteria will be met using the validated calibration levels and fortification levels determined in section 11: 72.7 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample of the run three (3) mid-level unextracted calibration standards will be analyzed. The peak area precision and retention time precision and peak asymmetry will be monitored at the beginning and the 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 and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0,where AF is the asymmetry factor. If any item of the 3M Environmental Laboratory 5 3M Environmental Laboratory Page 195 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 12.2 12.3 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. 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 approximately 50% of the LLOQ and the highest standard will be approximately 50% higher than the ULOQ. The standard curve equation will be determined by regression analysis using the peak areas 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 (8)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 curve and sample set will be reanalyzed. Limits of Quantitation (LOQ) The lower limit of quantitation (LLOQ) was determined to be 10 ng/g in liver and 10ng/mL serum during the method validation as described above in section 11. The LLOQ may be determined to be higher than that found in the validation study during a screening to be done for endogenous levels of PFOS in the blank matrices available at 3M Environmental Laboratory. If endogenous levels of PFOS are found to be higher than the LLOQ or if the levels are found to be highly variable, the LLOQ will be adjusted to accommodate this. The screening procedures and LLOQ determinationwill be recorded in a note to file and kept with the raw study data as well as being reported in the final report. Recovery efficiency at the LLOQ will be determined during each run using an extracted quality control sample spiked at the LLOQ level. Ifthe quality control sample at the LLOQ level fails during a run, the LLOQ for that specific run would be considered to be the next lowest passing quality control sample and any samples below the new LLOQ must be reanalyzed. The upper limit 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 ULOQ will be used to measure recovery efficiency at the ULOQ. If the quality control sample at the ULOQ fails during a run the ULOQ for that specific run would be considered to be the next highest passing quality control sample and any samples above the new ULOQ must be reanalyzed. Any sample with an area greater than that of the highest acceptable standard will need to be diluted into the range of the calibration curve. If samples are diluted into the range of the curve during analyses and enough sample remains, a post-run dilution validation will be performed to v e m sample values. To perform the 3M EnvironmentalLaboratory 6 3M Environmental Laboratory Page 196 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite dilution validation, one sample will be separated into two representative samples (i.e. two 1 mL aliquots for fluid samples or two 1 gram amounts for tissue samples) then diluted using two procedures. The first procedure consists of diluting the sample with additional matrix prior to extraction (sera adding sera), while the second procedure consists of diluting the extract with solvent postextraction (methanol extract adding additional methanol solvent). If the values are not within 15% of each other additionaltesting 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 calibrationverification - 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 20%. Data may only be reported if bracketed by passing CCV's (or a CCV and the same level calibration curve point). Data bracketed by one or more failing CCV's must be reanalyzed. 12.5 Quality Control Samples A minimum of three (3) quality control samples (QC) will be prepared with each set of samples. They will consist of blank 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 (25 times the LLOQ) 12.6 QC Performance Criteria Each QC is expected to show an accuracy of 80-120%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). 12.7 Use of Confirmatory Methods Confirmatory methods are typically not needed with L C M S M S analysis. 12.8 Demonstration of Specificity PFOS specificitywill be substantiated by chromatographic retention time, by the characteristic primary ion (499), and the characteristic product ion (99) using LCMSMS. 13. SUB-CONTRACTEDANALYSIS 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. 3M Environmental Laboratory 7 3M Environmental Laboratory Page 197 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 13.2 All fluid and tissue analyses as detailed in this protocol amendment will be performed at 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 13.3 Pace Analytical Services, Inc. will follow all applicable 3M Environmental Laboratory Standard Operating Procedures and the following Pace Analytical Inc. Standard Operating Procedures. PSS-Admin-01 Quality System PSS-Admin-04 Laboratory Facilities PSS-Admin-06 Training Record Files Maintained by QAU PSS-ARC-02 Disposition of Archive Materials PSS-ARC-04 Archiving Data, Specimen and Logbooks PSS-DC-05 Lab Notebooks, Logbooks, and Phone Logs PSS-DM-03 StatisticalEvaluation of Data PSS-OPS-0 1 Use and Maintenance of Ultra-Turrax T25 Homogenizer PSS-OPS-02 Raw Data PSS-OPS-03 Hazardous Waste Disposal PSS-OPS-04 Use and Maintenance 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 Cleaning Non-disposable Volumetric Pipettes PSS-OPS-08 Laboratory Calculations PSS-OPS-09 P sS-OPS- 10 Deviations from Established Procedures Analytical Definitions PSS-OPS-11 Use and Calibration of a Bottle-Top Dispenser PSS-OPS-13 Use and Maintenance of Lab Refrigerators, Freezers, and Ovens PSS-OPS-14 Use and Calibration of Syringes with a Hamilton Chaney Adapter PSS-OPS-15 Glassware Cleaning PSS-OPS-16 Use and Maintenance of the Fisher Scientific Isotemp 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, Maintenance and Calibration of Laboratory 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 PSS-MTR-01 Calibration of Certified Weight Set PSS-MTR-02 Calibration of Certified ThermorneterdThermocouples PSS-MTR-05 Calibration of Eppendorf Pipettes PSS-MTR-06 Verification of Calibration of the Eppendorf Pipettor PSS-MTR-07 New Equipment Qualification (IQ, PQ, OQ) PSS-MTR-08 Verification of Calibration and Use of Analytical Balances 3M Environmental Laboratory 8 3M Environmental Laboratory Page 198 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PSS-MTR-09 PSS-Safety-01 PSS-Safety-02 PS S-Safety-04 PSS-Safety-05 PSS-TS-01 Use and Maintenance of Fisher Scientific Electronic Temperature Monitoring Devices General Laboratory Safety Use and Maintenance of Biological Safety Cabinets Emergency Evacuation Procedures Use and Maintenance of Laboratory Fume Hoods Training Procedures 13.4 An amendment to the protocol will be written if extractions and analyses are performed at laboratories other than the Pace Tier 2 or 3M Environmental Laboratory, respectively. 14. STATlsnCAL ANALYSIS 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. 15. REPORT A final report will be prepared by the 3M Environmental Laboratory. The final 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. 16. LOCAnON OF RAWDATA, RECORDSA,ND FINAL REPORT 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 either at Pace Tier 2 or the 3M EnvironmentalLaboratory, as applicable. I 7. SPECIMEN RETENllON 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. 18. REFERENCES 18.I Wildlife International, Ltd. Protocol No. 454-108, "PFOS: A Reproduction Study with the Northern Bobwhite". 18.2 Centre Analytical Laboratories, Inc. Study 023-054, "Validation of Analytical Methods "Extraction of Potassium PeffluoroocatanesuKonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC ElectrospryhAass Spectrometry" and "Extraction of Potassium 3M Environmental Laboratory 9 3M Environmental Laboratory Page 199 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Pefluorooctanesulfonateor Other Fluorochemical Compounds From Liver for Analysis Using HPLC ElectrosprayMass Spectrometry in Quail Serum and Liver." 18.3 3M Study U2723, Wildlife Study 454C-110, "Analytical Method Verification for the Determination of PFOS in Avian Diet." 19. PROTOCOLAMENDMENTANSD D E W ~ O N S 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 finalprotocol. 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 10 3M Environmental Laboratory Page 200 -. R0e1p/o3r0t/-.E.0021-11284:- 52 8 FAX - 410 8 2 2 0 6 3 2 WILDLIFE PIFNOTESR:NAATRIOeNpAroLduction Study with the Noth@ e0rnI0 3Bobwhite WILDLIFE INTERNATIONAL, LTD - PROJECT NO.: 454-108, EO 1 1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH TIE NORTHERN BOBWHITE PROTOCOL NO.:454/12070O/QUSUB454 SPONSOR: 3M Corporation AMENDMENT NO.: 10 PROJECT NO,: 454-108,E01-1245 EFFECTIVE DATE: January 30,2002 AMENDMENT: This amendment includes ai attachmeilt (AttachmentA) which provides updated informationnecessary IO complctc the analytical phase of this study. This attaclment A replaccs the attachment A in amendmmt 9. REASON: c The amendment 9 attachment A was updated to include 3 different extraction and analytical method, to and to changethe liver and sera cxtraction location. Sean P. Gdlagher, Study birector J o 3. ~havers, Wildlife International, LTD, Laboratory Management c William K.Reage4 3M Environmental Laboratory, Date Date 1 30 O L 1 L 0 1 /3//cQ- Date CW!3,/dL Date Date 3M Environmental Laboratory 1 of9 Page 201 Report E01-1245 WILDLIFE INTERNATIONAL, LTD PFOS: A Reproduction Study with the Nothern Bobwhite PROJECT NO.: 454-108, E01 -1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454112070O/QR/SUB454 AMENDMENT NO.: 10 SPONSOR: 3M Corporation PROJECT NO.: 454-108, E01-1245 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 extraction and analytical method, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director Date Joann B. Beavers, Wildlife International, LTD, Date Laboratory Management Harold 0.Johnson, Principal Analytical Investigator Date William K. Reagen, 3M EnvironmentalLaboratory, Date Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date 3M Environmental Laboratory 1 of9 Page 202 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Protocol Amendment # I O - Attachment A Wildlife International Ltd. # 454-108 STUDY TITLE PFOS: A Reproduction Study with the Northern Bobwhite 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: EO1-1245 Wildlife International Ltd.: 454-108 3M Environmenfal Laboratory 3M Environmental Laboratory 2 of 9 Page 203 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Sponsor Study Identification PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55 133-3331 Principal Analytical lnvestigator Study Locations ANALYTICATLESTINGLABORATORY 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 ANALYTICAELXPERIMENTASLTARTDATE ANALYTlCAL EXPERIMENTATLERMINTAION DATE 01/31/02 0313 1/02 3M Environmental Laboratory 3M Environmental Laboratory 3 of 9 Page 204 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite I. PURPOSE~~BJECTIVES In the in-life phase of this study, Northern Bobwhite quail 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 five month period. The purpose of this analytical phase of the study is to quantify PFOS in the liver and sera of both control and dosed Northern Bobwhite quail. The analytical information may be used to compare with toxicologicalhistopathological results from the in-life portion of this bird study. The in-life portion of the study was completed at Wildlife International, Ltd. in study 454- 108. 2. REGULATORYCOMPLIANCE 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 Study Director. 4. REFERENCSEUBSTANCE 4.I Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity a nd 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 M S and GUMS 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 Conditions-The test article may be stored at ambient temperature or lower. 4.4 Reserve Samples-A reserve sample of PFOS from Lot #217 has been frozen at 20C 4~5Cor 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 ( C ~ F I ~ S O ~NKo)t.e: The molecular weight of the anion (PFOS) is 499 (C*F,$03-). 3M Environmental Laboratory 3M Environmental Laboratory 4of9 Page 205 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 5. CONTROML ATRICES Types of control matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. 6. TESTSYSTEM The test system is the Northern Bobwhite quail. 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. Northern Bobwhite 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 Northern Bobwhite quail 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 454108) for information about bird origination location. 8. SPECIMEN AND SAMPLE RECEIPTAND 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: 0 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 0 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 5 of 9 Page 206 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 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. 10. EXPERIMENTAL CONDUCT 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 and Analytical Method I O . I.I ETS-8-231 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices 11. DATAQUALITYOBJECTIVESDURINGSAMPLE ANALYSIS Method validation for PFOS quantitation in rat and mouse sera and liver is in process in the 3M Environmental Laboratory study EO1-1277. Limited Northern Bobwhite control matrix preclude a cross validation, however, the E01-1277 study methods will be applied to sample analyses of Northern Bobwhite sera and liver samples using the following performance based criteria: 11.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample of the run three (3) mid-level 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. 11.2 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 (i.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 207 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 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-response relationship 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. 71.5 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 208 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Each QC is expected to show an accuracy of 75-125% of expected. A minimum of 2/3 (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. 7 I .I O Use of Confirmatory Methods Confirmatory methods are typically not needed with LC/MS/MS analysis. 7 1.1I Demonsfrafion of Specificity PFOS specificity will be substantiated by chromatographic retention time, by the characteristic primary ion (499), and the characteristic product ion (99) using LC/MS/MS. 12. SUB-CONTRACTED ANALYSIS 72-I All fluid and tissue extractions and analyses as detailed in this protocol amendment will be performed at the 3M Environmental Laboratory, Building 2- 3E-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 209 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 14. REPORT A final report will be prepared by the 3M Environmental Laboratory. The final 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. LOCATIONOF RAWDATA,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. I Wildlife International, Ltd. Protocol No. 454-108, "PFOS: A Reproduction Study with the Northern Bobwhite". 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 210 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-108, EO 1-1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOLNO.: 454/120700/QR/SuB454 AMENDMENTNO.: 11 SPONSOR 3M Corporation PROJECT NO.: 454-108, E01-1245 EFFECTIVE DATE: March 27,2002 AMENDMENT: Section 11.1, Sentence4 Change: 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. To: The peak area precision must be equal to or less than 5.0% RSD, the precision of the retentiontime must be equal to or less than2.5% RSD. REASON: Information received h m our instrument supplierindicates that 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 fiict that analyte ionization can be suppressedby other speciespresent in the sample matrix and mobile phase make area reproducibility more challenging to maintain. Sample and source cleanliness are the determining factors, but 5 2.0% is still a bit aggressive for mass spec. The applicationchemists at Micromass, the instrument vendor, suggest a value of 5 5.0%. AMENDMENT: Section 11.3, Sentence 5. Change: Any level outside 75% - 125%of nominal value must be deactivated, and regression recalculated, except the LLOQ which must be within 30% of nominal. To: In some cases the lowest curve point, although outsidethe 30%nominal requirement, may be included in the linear range to influence the lower end of the curve. Only one point, the lowest level point, can be left in for this purpose. It will not be used to determine the limit of quantitation. The limit of quantitation will be the next point on the curve meeting the 30% nominal requirement. REASON: The removal of the lowest calibration curve point in some cases causes the next lowest calibration point to deviate by greater than 30%. Removal of this point in turn causes the next calibrationpoint to deviateby greaterthan 30%. This continued deletionof calibration points in some cases could continue until there are very few points 3M Environmental Laboratory Page 1 of 3 Page 211 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite remaining in the curve. The inclusion of a curve point with a deviation greater than 30% is used to influence the lower end of the curve and is not used for determination of the LOQ. AMENDMENT: Section 11.3, the last sentence. Change: A maximum of four (4) levels may be deactivated in any one set, or the set will be reanalyzed. To: The calibration curve will contain a minimum of six (6) calibration curve points. REASON: In most cases the calibrationcurves contain greater thannine (9) calibration points. The largenumber of points in the curve is used to select a range representativeto the samples. Limiting the removal of only four (4) points in some cases could cause a calibration range not representative of the samples. AMENDMENT: Section 11.3 Add: The coefficient of determination for the calibration curve should be 0.985. REASON: No coefficient of determination is included in the protocol. It was inadvertently removed prior to printing of the final copy. 3M Environmental Laboratory Page 2 of 3 Page 212 - Report E01-1245 04/02/02 17:17 FAX 410 822 0632 .. PFOS: A Reproduction Study with the Nothern Bobwhite WILDLIFE INTERNATIONAL @lo07 OA'01f02 18:25 RSD INF TECH 2-E-01 + *84188228915 ~0.385 Pam PRoJEcrNO.: 456108, .TD EO1-1245 .. ' :!i !. 1 , I ' !. 1 , :I .' I I . 3M Environmental Laboratory i Page 213 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite WILDLIFE INTERNATIONAL, LTD PROJECTNO.:454-108, EO 1-1245 Sean P. Gallagher, Study Director Joann B. Beavers, Wildlife International, LTD harold 0.Jo- rincipal Analyt~caIl nvestigator Date Date 4-Z-OZ Date Laboratoi 3M Environmental Laboratory Page 3 of3 Page 214 Report E01-1245 WILDLIFE INTERNATIONAL, LTD PFOS: A Reproduction Study with the Nothern Bobwhite PROJECT NO.: 454-108, EO 1- 1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/120700/QlUSUB454 AMENDMENT NO.: 12 SPONSOR: 3M Corporation PROJECT NO.: 454-108, E01-1245 EFFECTIVE DATE: August 16,2002 AMENDMENT: Change: The 3M Environmental Laboratory Project Identification Number (LIMS) to be included with the study is E01-1245. To: The main 3M Environmental Laboratory Project Identification Number (LIMS) to be included with the study is E01-1245. Additional tracking numbers associated with this study are U2723, E01-1342, and EO 1-1365. REASON: Bobwhite samples were received in the 3M Environmental at several time points. Upon arrival, samples were assigned a LIMS tracking number. Not aware of the scope of this or any other particular study pertaining to these samples, they were checked in to different 3M LIMS projects. LIMS project numbers assigned to this study include the following: U2723, E01-1245, E01-1342, and E01-1365. Samples in each of these projects pertaining to this study were analyzed under one main study number E01-1245. 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 215 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite WILDLIFE INTERNATIONAL, LTD - - Sean P. Gallagher,Study director r ut- Joann B. Bea;ers, Wildlife International,LTD, Laboratory Management JLJOh h a A. Stevenson, Principal AnalyticalInvestigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management PROJECT NO.: 454-108, E01-1245 9 a5/oa Date 4 1 4o x Date Date &?/JG/O J- Date 3M Environmental Laboratory Page 2 of 2 Page 216 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite WILDLIFE INTERNATIONAL. LTD PROJECT NO.: 454-108, E01- 1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENT NO.: 13 SPONSOR: 3M Corporation PROJECT NO.: 454-108, E01-1245 EFFECTIVE DATE: September 27,2002 AMENDMENT: Change: Rochelle R. Robideau is the Sponsor Representative. To: Susan A. Beach is the Sponsor Representative. REASON: To change role of the Sponsor Representative. L P. W\+ Sean P. Gallagher, Study Directod - - Joann B. BeaLrs, Wildlife International, LTD, Laboratory Management A , d a S h t n s o n %sa A. Stevenson, Principal Analytical Investigator Wilxam K. Reagen, 3M EnvironmentalLaboratory, Date Labgratory Management Su&n G .& A aJL A. Beach, Sponsor's Representative Date 3M Environmental Laboratory Page 1 of 1 Page 217 Report E01-1245 3M Confidential ~ ~~ PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation Study I Project No. EO 1-1245 Deviation type (Check one) XSOP 0Protocol Document number ETS-4-4.0 I. Identification 0Method 0 Other: 0Equipment Procedure Date(s) of occurrence 1131102,211102,2113102,217102,218102,211 1/02, 2112102,2113102,2115/02 /I. Description Required procedure/process: ETS-4-4.0 Internal chain of custody The internal chain 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: Internal chains of custodies were not used to track samples and sample extracts. Since receipt samples were Stored in freezer F1. After homogenizing liver and aliquating sera samples were stored in Freezer F8 Until extraction date. Samples remained frozen until extraction. Deviation to SOP written. Ill. Actions Taken (such as amendment issued, SOP revision, efc.) Recorded by Authorized by Date 4 ?-wl Deviation No. Dat4e. t c . t , 2 I or Project Lead at the end of study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 5 of 1 Page 218 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation 1. Identification Study 1 Project No.EO1-1245 Deviation Type (Check one) 0 SOP dProtocol 0 Method 0 Equipment Procedure 0 Other: Document Number :Protocol # : 454/12070O/QR/SuB454, amendment #: 10 Date(s) of occurrence: :1131/02, 2/1/02,2/2/02,2/7/02, 2111/02,2/12/02,2/13/02, 2/14/02,2/18/02,4/11/02, 411 6/02 11. Description: Required Procedure/process: Section 11.1, System Suitability, second sentence says that a mid-level unextracted calibration standard will be analyzed. Actual Procedure/process: On occasion through the dropping of calibration points, the system suitability samples were on the high end of the curve or outside the working range of the curve. Although these were unextracted curve points their area counts were at the high end of the calibration curve range, they do pass the system suitability criteria and these data packet will be considered acceptable. The data from 2/7/02 was just above the calibration range. These data will also be considered acceptable. Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this protocol deviation. 3M Environmental Laboratory Page 219 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation 1. /denfificafion Study 1 Project No.EO1-1245 Deviation Type (Check one) .iSOP 0 Protocol 0 Method 0 Equipment Procedure 0 Other: Document Number:ETS-8-23 1.1 Date(s) of occurrence:1131/02, 2/1/02,2/2/02,2/7/02, 2111/02,2/12/02,2/13/02,2/14/02, 2/18/02,4/11/02, 411 6/02 II. Description: Required Procedure/process: Section 12.1,Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedure/process: Standard E01-1342-36010, Prep sheet description "Curve Point - IOppb in liver", was prepped by spiking 20uL of 02001-66 (1.OOug/mL) into 1 mL of liver homogenate. This homogenate consists of 5g liver plus 45mL water. Since ImL out of 5 O m L homogenate is used to prep each extracted standard, 1/50 of the 5g (0.Ig) of liver is "spiked". After this homogenate is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... ( 2 0 4 (1 .OOugPFOS-/mLMeOH) (1.OmL/lOOOuL) (1OOOngil.Oug) = 20ng PFOS- onto SPE column 20ng PFOS-/ 2.0mL MeOH = 1Ong PFOS/mL MeOH. (Since this standard is recorded as lOppb, the correct units are IOngPFOS-/mLof MeOH.) These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ng PFOS-/ 1. O m L homogenate) (1 .OmL homogenate / 0.1g liver) = 200ng PF0S-l g liver This relationship shows the factor of 20 used to "correct" the units when calculating the final result in ng/g from the chromatographic result that is in ng/mL. Samde Calculation: "EO1-1342-30348, Oppm a i . 333 M liver" Chromatographic result is 10.76 ng/mL; reported value is 208.4 ng/g. Since the 1.0333g initial sample weight of liver is homogenized with 9.0mL water, and ImL (10% oftotal liver weighed, since a liver density of 1.00 is assumed) is used for the entire SPE procedure, the calculation is as follows: (10.76ng PFOS-/ mL MeOH) (2mL MeOH / 0.1033 g liver) = 208.4 ng PFOS- / g liver Sample Calculation: Sample "E01-1342-36017, QCo-lOmb-I": This QC sample is prepared by adding 4uL of 02001-53 (5.020ug PFOS-/ mL MeOH) spiked into I m L liver homogenate. ((4ul) (5.020ug/mL) (lmL/1000uL) (1000ng/ug)) / (0.lg liver) = 200.8ng PFOS- / g liver Using the earlier calculation, the analytical result of 13.59ng/mL calculates to 271.8ng/g liver. Since the spike level is 200.8ng PFOS-/ g liver, the recovery is (271.8/200.8)(100)=135.4%. 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 3 (assigned by Study Director or Project Lead at the end of study or project) Page 220 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation 111. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this SOP deviation and a note to file. Recorded By &c-Lc &IV. Impact on Study / Project There will be no impact on the study as this is for clarification purposes. Date 4<(ei.oz Date 4 19.c-2- / 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 3 (assigned by Study Director or Project Lead at the end of study or project) Page 221 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation II Study / Project No. E01-1245 .-.____ ^~"_"I""""""""""""."""_llll"""llll.""" """"""".""_l^ """"".""^_^^"^. 1. Identification " ^..^^ .... .. ... .. ... .... Deviation Type 0 SOP 0 Method 0 Equipment (Check one) 4Protocol 0 Other: .........._.."._ "."_ ____ " __ _ "^" ,,,,, Procedure Document Number: ETS-8-23 1.1 Date(s) of occurrence: Throughout the study Required Procedure/process: Section 11.3, Sentence 5 states: Any level outside 75% - 125% of nominal value must be deactivated. and rea-ession recalculated.. . Actu__a__ l Procedure/proce~ ss: - -- _--. - - __ - Many of the high calibration points in this study are kept in the curve despitebeing above the apex of the curve. When a calibration point lies above the apex, it is considered indeterminant (flagged with an I) and no concentration is calculated for the standard. If no concentration is calculated, no percent difference is calculated for evaluating whether or not that standard is within the stated criteria. Removal of these points in most cases causes the next calibration point to then be above the apex of the curve. This continued deletion of points could continue until there are no points remaining on the curve. Therefore, calibration points above the apex were included in the range of the curve with no evaluation of percent deviation criteria. Ill. Actions Taken: ---_- ~ - -(such as amendment_i__ssu_e-d,.LSOP" re___ vision, etc.) I ~ I I_ ~~~ 111-- 1 1 1 1 1 I This deviation was written, a note to file was written, and data points which were above the next highest calibration point were reanalyzed. Lisa A. Stevenson Date 08116/0023 I J L I X L 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. ? (assigned by Study Director or Project Lead at the end of study or project) Page 222 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation I. Identification 1 1 1 1 1 1 I - - - __ -- - ".-.Is"o-p Study / Project No.-EO1-1245 "" " """"" " ". __"_l."" ~.II _l".""_"."__",."__.I~..-""_l."..."" .. . Deviation Type Method 0 Equipment Procedure (Check one) 0 Protocol 0 Other: "".. "" """_ Document Number: ETS-8-231.1 Date(s) of occurrence: Entire Study II. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedurdprocess: Standard EOi---1345-35771,Prep sheet description "10 ppb sera curve point", was prepped by spiking 4uL of 02001-52 -(5.02OuglmL)into 2 mL of pooled sera. This diluted sera consists of 2mL sera plus 38mL water. Since 1mLout of 40mL diluted sera is used to prepare each extracted standard, 1/40 of the 2mL of sera is "spiked". After 1.0 mL of diluted sera is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE.. . (5.02Oug PFOS-/mLMeOH) * (4111) * (1.OmUlOOOuL) * (1000ngll.Oug) = 20ng PFOS- onto SPE column 20ng PFOS / 2.0mL MeOH = long PFOS/mL MeOH. (Since this standard is recorded as IOppb, the correct units are long PFOS-/mL MeOH.) These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ng PFOS / 1. O m L diluted sera) * (40.0mL diluted sera/ 2.0mL initial sera) = 400ng PFOS-/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 PFOS/mL MeOH. Samde Calculation: "E01-1245-28481,454-108-370,10ppm female, adult, 02-01-02 Chromatographic result is 229.2 ng/mL MeOH; reported value is 9170 ng/mL Sera. Since the 1.0 mL initial sample volume of sera is diluted with 38fhnL water, and 1mL is used for the entire SPE procedure, the calculation is as follows: i"i.0, or o. Vao ciiluhcn @LAX ~ ~ l a o / i u (229.2ng PFOS-/ 1.0mL MeOH) * (2.0mL MeOH/ 0.05mL Sera) = 9170ng PFOS- / mL Sera Samule Calculation: Sample "E01-1245-36155, QC@lOOppb-1": This QC sample is prepared by adding 2uL of 02001-52 (5.02ugPF0S-ImL MeOH) spiked into 1mLdiluted sera-(or 0.05 mL of initiai sera): ((5.02ug/mL) * (2ul) * (lmU1000uL) * (1000nglug)) / (0.05 mL Sera) = 200.8 ng PFOS- I mL Sera Using the earlier calculation, the analytical result of 6.17 nglmL calculates to 246.8ndmL Sera. Since the spike level is 2.-0.0...8.-%....PF-.OS-../.mL""""S""e" ra9-t.h.e recove.r.y is (246.^8_/.2^_0""0".._8.")"*I(_1_.0..0..).=..1..2.2......9%...1 .. , , "" ," _" " ^"^^ "",," . ...... ...... .. . Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) T__h__i_s__d-eviation was_written-and a_ n_ o- -te to- file was _w__r_itten. __ - - I - I Recorded By Lisa A. Stevenson loa, Date 08/16/02 0 $ / 1 ~ Form ETS-4-8.0 3M Environmental Laboratory (assignedby Study Director or Project Lead at the end of study or project) Page 223 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation I 1. Identification Study / Project No. E01-1245 Deviation Type (Check one) 0 SOP 0 Protocol 4 Method 0 Equipment Procedure 0 Other: Document Number: ETS-8-231.1 Date(s) of occurrence: Throughout the study 11. Description: Required Procedure/process: Section 13.3 of the method states: Two thirds of all quality control samples and 1/2 of eac quality control -s_a_m__ple at each level are expected to show a_n_ accuracy of 75-12_5_ %- . Actual Procedure/process: A1125 ppb quail liver quality control sam- ple recoveries were >125%. @as 03111103 cLih2h3 . Ill. Actions Taken: J ,. (such as amendment issued, SOP revision, etc.) This deviation was written, liver data reported at the low range of the curve may be biased and quality control data not _w_ ithin criteria are flagged in the raw data. I 1 Recorded By h I fl-fiA Lisa A. Stevenson JLlllO? 1 Authorized By (Study Director /Project Lead) Date 08/26/02 fd Date 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. %L (assigned by Study Director or Project Lead at the end of study or project) Page 224 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation 1. Identification Study / Project No.EO1-1245 Deviation Type (Check one) 4 SOP Protocol E Method 0 Equipment Procedure [ 1 Other: Document Number:ETS-8-23 1.1 Date(s) of occurrence: 1131/02, 2/1/02, 2/2/02, 2/7/02, 2111/02, 2/12/02. 2/13/02. 2/14/02. 2/18/02.4/11/02.4/16/02 II. Description: Required Procedure/process:Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedurehrocess: This deviation supersedes deviation #3 explaining the liver calculation: Standard E01-1342-36010, Prep sheet description "Curve Point - lOppb in liver", was prepped by spiking 20uL of 02001-66 (l.OOug/mL) into 1 mL of liver homogenate. This homogenate consists of 5.03538 liver plus 45.0mL water. Since ImL out of 50.0353mL homogenate is used to prep each extracted standard, 1/50 of the 5.03538 (0.100635g = 5.0353 g/(9.0 of water + 5.0353 g of liver) is "spiked". After this homogenate is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE.. . (20 uL) * (1.000ug PFOS-/mLMeOH) * (1.0 mL/lOOO uL) * (1000 ngll.0 ug) = 20.0 ng PFOS-onto SPE column 20.0 ng PFOS- / 2.0 mL MeOH = 10.0 ng PFOS`/mL MeOH. (Since this standard is recorded as IOppb, the correct units are 10.0ng PFOS-/mLof MeOH.) These are the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20.0ng PFOS-/ l.0mL homogenate) * (l.OmL homogenate / 0.100635 g liver) = 199ngPFOS-/ g liver Samule Calculation: "E01-1342-30348, Oppm a.1. 333 M liver" Chromatographic result is 10.76 ng/mL; reported value is 209 ng/g. Since the 1.03388 initial sample weight of liver is homogenized with 9.0mL water, and 1.0 mL is used for the entire SPE procedure, the calculation is as follows: 1.0338 g / (9.0 mL water + 1.0338 g liver) = 0.103032 g of liver in 1.0 mL of homogenate removed for extraction. (10.76 ng PFOS-/ mL MeOH) * (2.0 mL MeOH IO.103032 g liver) = 209 ng PFOS- / g liver Sample Calculation; Sample "E01-1342-36017, OCQ1ODpb-l": This QC sample IS prepared by adding 4 uL of 02001-52 (5.020ug PFOS- / mL MeOH) spiked into 1mL liver homogenate. ((4ul) * (5.020ug/mL)* (lmL/IOOOuL)* (1000ng/ug)) / (0.1006358 liver) = 200 ng PFOS-/ g liver Using the earlier calculation, the analytical result of 13.59ng/mLcalculates to 270 ng/g liver. Since the spike level IS 200ng PFOS-/ g liver, the recovery is (270/200) * (100)=135.0%. Ill. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this S O P deviation. A\ Recorded By d Date Lisa A. Stevenson &.-..h &A/-f?nI-,, . Authorized By (Study Director /Project Lead) Form ETS-4-8.0 3M Environmental Laboratory (assigned by Study Director or Project Lead at the end of study or project) Page 225 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ATTACHMENTF: EXAMPLCEALCULATIONS FORMULA USED FOR SERA ANALYSES IN STUDY E01-1245 AR (ng/mL MeOH) x DF x Dilution(di1utedsera) = ng/mL Sera Calculation Used for E01-1245-28481,454-108-370,lO ppm Female, adult (initial volume = 0.3 mL) 229.24 ng/mL MeOH x 1 x 40 = 9170 ng/mL Sera FORMULUASED FOR LIVERANALYSES IN STUDY E01-1245 AR (ng/mL MeOH) x DF x Final Volume (mL) = nglg Liver amount of liver in diluted sample (9) Calculation Used for E01-1342-30672,lO ppm, a.i. 369 M Liver (initial weight = 1.0010 g) 393.21 ng/mL MeOH x 10 x 2.0 mL MeOH = 78600 ng/g Liver 0.100090 g Liver AR = Analytical result from MassLynx summary DF = Dilution factor Dilution(di1utedsera) = Final volume(mL)/amount of sera in diluted sample(mL) A m o u n t of sera in diluted sample(mL) = amount of sera used(mL)/total amount of sera + water(mL) Amount of liver in diluted sample(g) = amount of liver weighed(g)/ amount of liver weighed(g) + water added for homogenizing(mL=g) Examples: Amount of sera in diluted sample(mL) = amount of sera used (0.3 mL)/total amount of sera + water (6 mL) = 0.05 mL Dilution(di1utedsera)(mL) = final volume (2.0mL)/amount of sera in diluted sample (0.05 mL) = 40 Amount of liver in diluted sample(g) = amount of liver weighed (1.0010 g)/amount of liver weighed (1.0010 g) + water added for homogenizing (9.00 mL) = 1.0010 g / 10.0010 g = 1.00090 g Note: For sera samples with less than 0.05 mL sera received, additionalwater was added in order to remove 1.O mL of diluted sera for extraction. For these samples, the dilution(di1uted sera) value was updated to reflect the correct dilution. For example, if a sample was receivedwith 0.02mL sera available, 0.98 mL of water was added. the amount of sera in diluted sample = 0.02mU1 .O mL = 0.02mL. The dilution(di1utedsera)(mL)= 2.0 mU0.02 mL = 50 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 226 of 226 Page 226