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Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study Title A nalytical Phase Report for PFOS: A Reproduction Study with T he Northern Bobwhite 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 Laboratory 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 G LP C o m plia n c e Statem ent 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: Exceptions to GLP compliance: Automated data collection systems are not validated. The hardcopy printouts are considered as the original raw data. The stability of the reference substances and samples has not been determined. Not all data generated were recorded directly or promptly, or initiated and dated on date of entry. Lisa A. Stevenson, Principal Analytical Investigator Susan A. Beach, Sponsor Representative 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 Q u a lity A ssu ranc e Statem ent 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. Inspection Dates 10/08/01-10/09/01 /0 2 0 1 / 0 2 04/03/02-04/08/02, 04/17/02 05/20/02-05/21/02 Phase Protocol In-phase Data Final Report Date Reported to M anag em ent S t u d y D ir ec to r 10/09/01 10/09/01 02/04/02 02/04/02 04/08/02, 05/16/02 04/08/02, 05/16/02 05/21/02 05/21/02 Quality Assurance Representative 01mo? 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 Table of C ontents 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 Preparatory Method........................................................................................................................... 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-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" .................................................. 28 Attachment B: Data Summary Tables............................................................................................... 48 Attachment C: Sample Chromatograms, Calibration Information, and Instrument Information........ 62 Attachment D: Sample Preparation Sheets...................................................................................... 155 Attachment E: Study Protocol, Protocol Amendments, 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 L ist o f Ta b le s Table 1. Summary Table, C8F17S 03' Concentrationin Sera and Liver..............................................7 Table 2. Summary Table, C8Fi7S 03' ConcentrationinSera 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. C8F17S 03' Data Summary, Female Quail Sera (Adult and Offspring)............................... 15 Table 11. C8Fi7S 03' Data Summary, Male Quail Sera (Adult and Offspring)................................... 17 Table 12. C8F17S 03` Data Summary, Female Quail Liver (Adult and Offspring).............................. 20 Table 13. C8F17S 03' Data Summary, Male Quail Liver (Adult and Offspring)................................... 22 Table 14. Summary Table, C8F17S03' Concentration in Sera and Liver........................................... 24 Table 15. Summary Table, C8F17SQ3' 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 Stu d y Inform ation 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 Environmental Technology and Safety Services (ET&SS) 3M Environmental Laboratory (3M Lab) Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Lisa Stevenson, Principal Analytical Investigator Study Personnel Sean P. Gallagher, Wildlife International LTD., Study Director Joann B. Beavers, Wildlife International LTD., Laboratory Management William K. Reagen, 3M Environmental Laboratory Management Susan A. Beach, Sponsor Representative Harold O. Johnson, III, 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 S am ple Analysis Com pletion: 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 pertaining to the analytical phase of this study, are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 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 S ummary The concentration of the C8F17S03' anion of perfluorooctane sulfonate, potassium salt (PFOS, C8F17S 03'K+) 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 C8F17S 03' 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 C8F17S 03\ Concentrations of C8F17S 03' are reported after correction for the purity and the potassium contribution to the mass of the PFOS reference substance. Table 1 summarizes all study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q-Test. Table 1. Summary Table, C8F17S03' Concentration in Sera and Liver Sampling Event N umber of Sera Samples A verage C8Fi 7S03 Conc. Sera Range o f Results Number of L iver Samples A verage C8Fi 7S 0 3 C onc. L iver Range o f Results Male Control Group 19 NA <400 ng/mL (19 of 19) 20 <50.2 ng/g* <50.2 ng/g (19 of 20)* 209 ng/g (1 of 20)* Female Control Group 17 <400 ng/mL (16 of 17) NA 20 <50.2 ng/g* <50.2 ng/g (20 of 20)* 2110 ng/mL (1 of 17) Male 10ppm Group 20 141000 ng/mL 94400 - 226000 ng/mL 20 88500 ng/g* 50100 -147000 ng/g* Female 10ppm Group 18 9990 ng/mL 4710 - 31900 ng/mL 20 4900 ng/g* 3290 - 7550 ng/g* Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 6 4 5 5 NA <400 ng/mL (6 of 6) i NA <400 ng/mL (4 of 4) 12600 ng/mL 8220-19200 ng/mL 12400 ng/mL 7640-18500 ng/mL 6 <20.0 ng/g* <20.0 ng/g (6 of 6)* 4 <20.0 ng/g* <20.0 ng/g (4 of 4)* 5 7350 ng/g* 4940 -13700 ng/g* 5 7180 ng/g* 4590 -13900 ng/g* 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 >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, C8Fi7S03' Concentration in Sera and Liver with Outliers Removed Sampling Event N umber of S era Samples A verage C8F17SO 3 Conc. Sera Male Control Group 19' * Female Control Group Male 10ppm Group 16 20' <400 ng/mL * Range of Results Number of L iver Samples A verage C 8F 17S O 3 Conc. L iver * 19 <50.2 ng/g** <400 ng/mL (16 of 16) 20* * * 20* * Range of Results <50.2 ng/g (19 of 19)** * * Female 10ppm Group 17 8700 ng/mL 4710-14700 ng/mL 20* * * Male Offspring Control Group Female Offspring Control Group 6* 4* * * * 6* * * 4* * * * Male Offspring 10ppm Group Female Offspring 10ppm Group 5* 5* * * * 4 5760 ng/g** 4940 - 6520 ng/g** * 4 5490 ng/g** 4590 - 7030 ng/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 % 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 C8Fi7S03' in the liver and sera of both control and dosed Northern Bobwhite Quail and their offspring. Reference Substances Table 3. Reference Substances Reference Substance Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Purity PFOS 3M Internal 08/31/2006 Frozen -20C +/- 10C SD-018, Lot #217 White Crystalline Powder 86.9% PFDA Oakwood Products, Inc. /1 2 0 1 / 2 0 1 0 Frozen -20C+/-10C SD-036 White Solid 98% PFDA is nonadecafluorodecanoic acid, or Perfluorodecanoic acid, C10F19O2H and is used solely to monitor for gross instrument failure and not as an internal standard for quantitation. 3M Environmental Laboratory 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 Reproduction Study with the Northern Bobwhite S pecim en R ec eipt an d S to rag e 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. M ethod Summary The determination of C8Fi7S 03' concentration in the liver and sera was performed according to 3M Environmental Laboratory method ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices". The sera samples were prepared by measuring a volume of serum and diluting with water in a 1/20 ratio. The liver samples were prepared by measuring a weight of liver and diluting with water to a 1/10 ratio prior to homogenizing. Five mL of acetonitrile was added to 1.0mL of the diluted sera or 1.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 C8F17S 03' using HPLC-ES/MS/MS. Preparatory M ethod ETS-8-231.1 " Solid Phase Extraction and Analysis of Fluorochemical Components from Biological matrices." This method describes the extraction of target analytes from biological matrices, such as sera and liver, using solid phase extraction (SPE). Sera samples were diluted with Kandiyohi (commercially bottled) water 1/20, before extraction. Liver samples were diluted 1/10, approximately 1g of liver and 9ml of Kandiyohi water, and then homogenized. A 1.0 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 Step 1 2 3 4 5 6 7 Sample Preparation Measure out volume of sera or weigh portion of liver Dilute sera (1/20) or liver (1/10) with water Mix sera/water or homogenize liver/water Aliquot 1.0ml of diluted/homogenized sample and add 5.0ml of ACN. Spike samples accordingly. Shake for 20 minutes, centrifuge for 10 minutes. Dilute supernatant with 40 ml of water Filter sample through conditioned SPE C18 column. Elute target analytes from the column with 2.0 mL methanol (MeOH). 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 S a m p le C o lle c tio n and A nalysis All sera and liver samples were received frozen and remained frozen until thawed prior to preparing samples for analysis. After dilution or homogenization steps, unused samples and diluted sera or homogenized liver were frozen again if extractions were not immediately performed. On each day of extraction, a water blank, a sera or liver blank, and a matrix spike and matrix spike duplicate were prepared along with each set of samples. Frozen diluted sera/homogenized liver samples were allowed to thaw prior to transferring a representative sample for extraction. Samples were prepared and analyzed according to ETS-8-231.1. The following table describes the sample collection and preparation regimen. For clarity, a reference to "Day 1" refers only to the group of samples associated with the first batch of samples extracted. Table 5. Sample Analysis S tep 1 2 Procedure Preparation of method blanks and spikes, calibration curve, qc samples, and sera samples, control group, adults (day 1 sera samples), see Attachment D for samples prepped Analysis of day 1 sera samples Date Performed 31 January 2002 31 January 2002 3 Preparation of method blanks and spikes, and sera samples, control and 1 0 ppm adults, control offspring, and 1 0 ppm offspring (day 2 sera samples), see Attachment D for samples prepped 4 Analysis of day 2 sera samples 5 Analysis of 10ppm female sera samples 6 Analysis of control, 10 ppm and offspring, from day 1 and day 2 sera samples 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 1 0 Preparation of method blanks and spikes, and liver samples, control group (day 2 liver samples), see Attachment D for samples prepped 1 1 Analysis of day 2 liver samples 1 2 Preparation of method blanks and spikes, and liver samples, 10ppm group (day 3 liver samples), see Attachment D for samples prepped 13 Preparation of method blanks and qc samples at 5 and 2 0 0 ppb 14 Analysis of qc samples 15 Analysis of day 3 liver samples 01 February 2002 01 February 2002 03 February 2002 07 February 2002 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 Laboratory 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 Step 16 17 18 19 20 Procedure Preparation of method blanks and spikes, and liver samples, 1 0 ppm group, control offspring, 1 0 ppm offspring (day 4 liver samples), see Attachment D for samples prepped Analysis of day 4 liver samples Re-analysis of day 3 liver samples Re-analysis of QC samples Re-analysis of QC samples Date Performed 15 February 2002 18 February 2002 01 March 2002 05 April 2002 10 April 2002 A n a ly tic a l M ethod Samples were analyzed using HPLC-ES/MS/MS. C8F17S 03' concentrations were determined using matrix-extracted calibration standards ranging in concentration from 0.25-1000 ng C8F17S 03VmL methanol. This result was then used to back calculate the ng C8F17S 03'/mL sera or ng C8F17S 037gram liver as appropriate. An internal standard, Nonadecafluorodecanoic acid (PFDA), was used to monitor instrument performance for gross malfunction, but was not used to quantitate results. HPLC-EC/MS/MS analytical conditions are summarized as follows: The HPLC used for the study was a Hewlett-Packard 1100. The system consists of a vacuum degasser, binary pump, autosampler and temperature controlled column compartment. The run time is 10 minutes per sample. HPLC conditions include the use of a Keystone Scientific, Betasil C18 HPLC analytical column (2.1mm x 50mm, 5pm particle size), a 40C column compartment temperature, a 10pL injection volume and a 0.300mL/min pump flow rate. The HPLC gradient is listed below: T ime, M inutes 0 1.0 5.5 7.5 8 10 % 2 m M A mmonium A cetate 90 90 5 5 90 90 % Methanol 10 10 95 95 10 10 The mass spectrometer used for the study was a Micromass Ultima. The system consists of Z-Spray interface equipped with an electrospray probe operated in negative mode. The instrument was setup to operate in MS/MS mode, passing the C8F17S 03' 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, FS03\ Similarly the PFDA transition from m/z 513 to m/z 299 was monitored. The collision gas, 95% Argon / 5% Methane, was maintained at a pressure of approximately 3.0x1 O'3 mBar. 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 A n a ly tic a l R esults Data quality objectives outlined in the 3M Environmental Laboratory method and protocol were met, except as noted and discussed below (see Attachment A, ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" and Attachment E, Study Protocol, Protocol Amendments, and Deviations). Regressions. Quadratic curve fit, weighted 1/x, was applied to calibration standards and sample data. Each calibration curve had a coefficient of determination of > 0.985. Calibration Standards. Instrument response was calibrated daily prior to sample analysis using matrix-extracted standards ranging in concentration in the final solvent extract from 0.20 to 1000 ng C8F17S 037mL methanol, corresponding to 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-extracted standards using LC/MS/MS instrumentation, the calculated concentration of higher-level standards 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 instrumentation was providing suitable results throughout each analytical sequence. Continuing Calibration Verification (CCV). A mid-level matrix calibration check was analyzed every ten samples or less to monitor instrumental drift, with a limit of 25% deviation of the target concentrations. Only data bracketed by passing C C V 's w e re reported. Samples bracketed by one or more failing CCV's were reanalyzed. Limit of Quantitation (LOQ). The lower limit of quantitation (LLOQ) was defined in liver (ng/g) and in serum (ng/mL) as the lowest acceptable extracted calibration curve point that is within the 70-130% nominal criteria and with an analyte peak area at least 2 times the blank. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest acceptable curve point used to calibrate the response of the instrument. Blanks. Method blanks (purchased Kandiyohi drinking water, sera and/or liver taken through the entire sample preparation and analysis process) provided a measure of background contamination. Quality Control (QC) and Matrix Spikes. One set of nine QC samples in each matrix was extracted and analyzed and consisted of three levels of analyte in triplicate. Additionally, two matrix spike samples at the lowest concentration were prepared with each extraction set. Results are as follows: 3M Environmental Laboratory 3M Environmental Laboratory 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 Table 6 . Serum QC Summary Table Serum QC Samples Concentration in Diluted Serum Mean recovery of triplicates Relative Standard Deviation MS-5ppb* 201 ng/mL 75.2% 3.0% QC-25ppb* 1000 ng/mL 115% 8.3% MS-200ppb 8030 ng/mL 90.5% 2.5% QC-40ppm 1610000 ng/mL 76.2% 2.8% QC-80ppm* 3230000 ng/mL 114% 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 Serum Matrix Spike Samples* Concentration in Diluted Serum Average Recoveries Three Matrix Spikes 10000 ng/mL 93.9% Three Matrix Spikes Duplicates 10000 ng/mL 91.8% 'One matrix spike sample was injected in a second analytical batch. Both results are included in the average recoveries. Table 8 . Liver QC Summary Table Liver QC Samples Concentration in Diluted Liver Mean recovery of triplicates Relative Standard Deviation QC-10ppb* 200 ng/g 138% 2.9% QC-75ppb* 1500 ng/g 115% 2.9% QC-150ppb* 2990 ng/g 104% 2.0% *QC samples were reanalyzed to confirm original results. Data from reanalysis were not included in calculating these results. Table 9. Liver Matrix Spike Summary Table Liver Matrix Spike Samples* Concentration in Diluted Liver Four Matrix Spikes 499 ng/g Four Matrix Spikes Duplicates 499 ng/g Average Recoveries 110% 112% The protocol and method state that 2/3 of all QC and a minimum of 1/2 of QC at each level must be within 75% - 125% criteria. All three of the liver "QC-10ppb" spikes exceeded the stated criteria, therefore the liver data reported in the low range of the curve (Day 0 samples) 3M Environmental Laboratory 3M Environmental Laboratory Page 13 of 226 Page 13 Report E01-1245 Report E01-1245 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. 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-extracted standards. 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 demonstrating that it is appropriate to use matrix-extracted standards. Internal Standard: The internal standard (PFDA) was added to all samples and standards. PFDA was not used for quantitation, but was used to monitor for gross instrument failure. Instrument Response Calibration. HPLC-ES/MS/MS instrument response was calibrated using the concentration of the C8F17S03 anion of perfluorooctane sulfonate, potassium salt (PFOS) that was adjusted for purity and the potassium ion contribution to the mass of PFOS used to prepare stock solutions. Any future calculations requiring concentrations to be expressed as a percentage of dietary in-take must adjust the dosed amounts as well. Data S ummary Tables 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The tables display C8F17S 03' 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. C8Fi7S03' Data Summary, Female Quail Sera (Adult and Offspring) 3M Sam ple N umber S a m p l e D e s c r ip t io n . E01-1245-28444 454-108-330, Oppm female, adult, 01-31-02 , E01-1245-28446 454-108-332, Oppm female, adult, 01-31-02 ,, E01 -1245-28449 454-108-336, Oppm female, adult, 01-31-02 ,, E01-1245-28451 454-108-338, 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 , 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 , E01-1245-28470 454-108-358, Oppm female, adult, 02-01-02 , E01-1245-28472 454-108-360, Oppm female, adult, 02-01-02 , E01-1245-28474 454-108-362, Oppm female, adult, 02-01-02 , E01-1245-28477 454-108-366, Oppm female, adult, 02-01-02 , E01-1245-28479 454-108-368, Oppm female, adult, 02-01-02 E01-1245-28481 454-108-370,10ppm female, adult, 02-01-02 E01-1245-28483 454-108-372,10ppm female, adult, 02-01-02 E01 -1245-28485 454-108-374,10ppm female, adult, 02-01-02 E01-1245-28487 454-108-376,10ppm female, adult, 02-01-02 E01-1245-28489 454-108-378,10ppm female, adult, 02-01-02 E01-1245-28491 454-108-380,10ppm female, adult, 02-01-02 AMOUNT OF SERA USED (ML) 0.4 0.4 0.05 0.05 0.3 0.3 0.3 0.3 0 .2 0 .2 0 .2 0 .2 0 .1 0 .1 0.3 0.3 0.4 0.4 0.4 0.4 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0 .2 0 .2 0 .2 0 .2 0.3 0.3 0.3 0.4 0 .2 0.3 0 .1 0 .1 A D D IT IO N A L DILUTION CONC. OF C 8 F1 7 SO 3 IN Sam ple (ng/mL of Sera) AVERAGE OF DUP. SAMPLES 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 2050 1 2180 2 1 1 0 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 9170 1 9140 1 8400 1 9280 1 4980 1 5790 3M Environmental Laboratory 3M Environmental Laboratory Page 15 of 226 Page 15 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Sam ple N umber S a m p l e D e s c r ip t io n AMOUNT OF SERA USED (M L) A D D IT IO N A L DILUTION CONC. OF C g F 1 7 S O 3 in S a m p le (n g /m L of S era) AVERAGE OF DUP. SAMPLES E01-1245-28493 454-108-382,10ppm female, adult, 02-01-02 0.3 1 0 31900 E01-1245-28496 454-108-386,10ppm female, adult, 02-01-02 0.4 1 E01-1245-28498 454-108-388,10ppm female, adult, 02-01-02 0.3 1 E01-1245-28500 454-108-390,10ppm female, adult, 02-01-02 0 .2 1 E01-1245-28502 454-108-392,10ppm female, adult, 02-01-02 0.3 1 E01-1245-28504 454-108-394,10ppm female, adult, 02-01-02 0.3 1 E01-1245-28506 454-108-396,10ppm female, adult, 02-01-02 0.09 1 E01-1245-28508 454-108-398,10ppm female, adult, 02-01-02 0 .1 1 E01-1245-28511 454-108-402,10ppm female, adult, 02-01-02 0.4 1 E01-1245-28513 454-108-404, 10ppm female, adult, 02-01-02 0 .1 1 E01-1245-28515 454-108-406,10ppm female, adult, 02-01-02 0 .2 1 E01-1245-28517 454-108-408,10ppm female, adult, 02-01-02 0 .2 1 14700 11600 6310 9400 11400 4710 10500 6720 9060 8980 7840 . E01-1245-28518 454-108-246, Oppm female, offspring, 02-01-02 , E01-1245-28521 454-108-259, Oppm female, offspring, 02-01-02 ,, E01-1245-28524 454-108-273, Oppm female, offspring, 02-01-02 , EOT1245-28525 454-108-277, Oppm female, offspring, 02-01-02 0 .1 0 .1 0 .1 0 .1 0.08 0.08 0 .2 0 .2 1 1 1 1 1 1 1 1 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 , E01-1245-28528 454-108-517,10ppm female, offspring, 02-01-02 , E01-1245-28530 454-108-525,10ppm female, offspring, 02-01-02 , E01-1245-28532 454-108-538,10ppm female, offspring, 02-01-02 . E01-1245-28535 454-108-557,10ppm female, offspring, 02-01-02 E01-1245-28536 454-108-561,10ppm female, offspring, 02-01-02 * 0 .0 2 0 .0 2 0.03 0.03 0.08 0.08 0.06 0.06 0.05 0.05 1 1 1 1 1 1 1 1 1 1 18500 16600 12800 12100 14600 12200 8330 7460 11500 10100 17600 12500 13400 7900 10800 * 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 Environmental Laboratory 3M Environmental Laboratory Page 16 of 226 Page 16 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 1 1 . CsF^SCV Data Summary, Male Quail Sera (Adult and Offspring) 3M S a m p l e N umber S a m p l e D e s c r ip t io n . E01-1245-28443 454-108-329, 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 , 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 , E01-1245-28473 454-108-361, Oppm male, adult, 02-01-02 ,, E01-1245-28475 454-108-363, 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 AMOUNT OF SERA USED (ML) 0 .2 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 .0 2 0 .0 2 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 0 .1 A D D IT IO N A L DILUTION Cono, of C 8 F1 7 SO 3 IN S a m p l e (n g / m L of Sera) AVERAGE OF DUP. SAMPLES 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 -- <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <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 3M Sample N um ber Sam ple Description AMOUNT OF SERA USED (ML) E01-1245-28480 454-108-369,10ppm male, adult, d100,02-01-02 E01-1245-28482 454-108-371,10ppm male, adult, d100,02-01-02 , E01 -1245-28484 454-108-373, 10ppm male, adult, d100,02-01-02 , E01-1245-28486 454-108-375, 10ppm male, adult, d100,02-01-02 , E01-1245-28488 454-108-377,10ppm male, adult, d100,02-01-02 * E01-1245-28490 454-108-379,10ppm male, adult, d100,02-01-02 E01-1245-28492 454-108-381,10ppm male, adult, d100,02-01-02 , E01 -1245-28494 454-108-383,10ppm male, adult, d100,02-01-02 , E01-1245-28495 454-108-385,10ppm male, adult, d100,02-01-02 ,, E01-1245-28497 454-108-387,10ppm male, adult, d100,02-01-02 , E01-1245-28499 454-108-389,10ppm male, adult, d100,02-01-02 . E01-1245-28501 454-108-391,10ppm male, adult, d100,02-01 -02 , E01-1245-28503 454-108-393,10ppm male, adult, d100,02-01-02 E01-1245-28505 454-108-395, 10ppm male, adult, d100,02-01 -02 , E01-1245-28507 454-108-397,10ppm male, adult, d100,02-01-02 , E01-1245-28509 454-108-399,10ppm male, adult, d100,02-01-02 . E01 -1245-28510 454-108-401,10ppm male, adult, d100,02-01-02 , E01-1245-28512 454-108-403, 10ppm male, adult, d100,02-01 -02 . E01 -1245-28514 454-108-405,10ppm male, adult, d100,02-01-02 E01-1245-28516 454-108-407,10ppm male, adult, d100,02-01-02 * 0 .2 0 .2 0 .2 0 .2 0 .1 0 .1 0.3 0.3 0.07 0.07 0 .1 0 .1 0 .1 0 .1 0.3 0.3 0 .1 0 .1 0.3 0.3 0 .1 0 .1 0.06 0.06 0 .2 0 .2 0 .2 0 .2 0.3 0.3 0 .1 0 .1 0 .1 0 .1 0 .2 0 .2 0 .1 0 .1 0 .1 0 .1 ADDITIONAL DILUTION CONC. OF C8 F1 7 S O 3 IN Sam ple (ng/m L of Sera) AVERAGE OF DUP. SAMPLES 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 107000 117000 181000 179000 144000 156000 138000 138000 94400 96100 150000 166000 141000 149000 147000 156000 119000 123000 174000 196000 129000 136000 141000 147000 160000 165000 127000 136000 99600 107000 138000 159000 203000 226000 127000 138000 108000 117000 99200 112000 112000 180000 150000 138000 95300 158000 145000 151000 121000 185000 132000 144000 162000 131000 103000 148000 215000 132000 112000 105000 3M Environmental Laboratory 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 3M Sample Number Sam ple Description E01-1245-28519 454-108-253, Oppm male, offspring, 02-01-02 , E01-1245-28520 454-108-256, Oppm male, offspring, 02-01-02 , E01-1245-28522 454-108-266, Oppm male, offspring, 02-01-02 ,, E01-1245-28523 454-108-270, Oppm male, offspring, 02-01-02 , E01-1245-28526 454-108-283, Oppm male, offspring, 02-01-02 E01-1245-28527 454-108-291, Oppm male, offspring, 02-01-02 AMOUNT OF SERA USED (ML) 0 .0 2 0 .0 2 0 .0 2 0 .0 2 0.07 0.07 0 .2 0 .2 0 .0 2 0 .0 2 0.05 0.05 A D D IT IO N A L DILUTION Cono, of C8 F1 7 S O 3 IN Sam ple (ng/mL of Sera) AVERAGE OF DUP. SAMPLES 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 E01-1245-28529 454-108-522, 10ppm male, offspring, 02-01-02 ,, E01-1245-28531 454-108-533,10ppm male, offspring, 02-01-02 , E01 -1245-28533 454-108-545,10ppm male, offspring, 02-01-02 , E01-1245-28534 454-108-552,10ppm male, offspring, 02-01-02 0.07 0.07 0.07 0.04 0.04 0.07 0.07 10 1 1 1 1 1 1 19300 9310 8220 15500 13600 13600 11500 19300 8760 14600 12500 E01-1245-28537 454-108-568,10ppm male, offspring, 02-01-02 * 0 .1 0 .1 1 12100 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. 11200 3M Environmental Laboratory 3M Environmental Laboratory Page 19 of 226 Page 19 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 12. C8Fi7S03'Data Summary, Female Quail Liver (Adult and Offspring) 3M Sam ple N umber S a m p l e D e s c r ip t io n E01-1342-30321 0 ppm 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 ppm 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 ppm a.i. 342 F Liver E01-1342-30447 0 ppm a.i. 344 F Liver E01-1342-30465 0 ppm 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 ppm 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 ppm 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 E01-1342-30699 E01-1342-30717 E01-1342-30735 E01-1342-30753 E 0 1-1342-30771 E01-1342-30789 E01-1342-30820 E01-1342-30838 E01-1342-30856 E01-1342-30874 E01-1342-30892 E01-1342-30910 E01-1342-30928 E01 -1342-30946 E01-1342-30964 E01-1342-30982 E01-1342-31000 E01-1342-31018 E01-1342-31036 10 ppm a.i. 370 F Liver 10 ppm a.i. 372 F Liver 10 ppm a.i. 374 F Liver 10 ppm a.i. 376 F Liver 10 ppm a.i. 378 F Liver 10 ppm a.i. 380 F Liver 10 ppm a.i. 382 F Liver 10 ppm a.i. 384 F Liver 10 ppm a.i. 386 F Liver 10 ppm a.i. 388 F Liver 10 ppm a.i. 390 F Liver 10 ppm a.i. 392 F Liver 10 ppm a.i. 394 F Liver 10 ppm a.i. 396 F Liver 10 ppm a.i. 398 F Liver 10 ppm a.i. 400 F Liver 10 ppm a.i. 402 F Liver 10 ppm a.i. 404 F Liver 10 ppm a.i. 406 F Liver 10 ppm a.i. 408 F Liver AM O UNT O F LIVER W EIGHED (G) A D D IT IO N A L DILUTION C o n c . O F C8F17S03' in Sam ple (n g /g o f L iv e r ) 1.0749 1 <50.2* 1.0469 1 <50.2* 1.0232 1 <50.2* 1.0188 1 <50.2* 1.0543 1 <50.2* 1.0182 1 <50.2* 1.0904 1 <50.2* 1.0087 1 <50.2* 1.0998 1 <50.2* 1.1036 1 <50.2* 1.0237 1 <50.2* 1.1509 1 <50.2* 1.0578 1 <50.2* 1.0312 1 <50.2* 1.1031 1 <50.2* 1.0586 1 <50.2* 1.0424 1 <50.2* 1.0364 1 <50.2* 1.0577 1 <50.2* 1.0920 1 <50.2* 1.0868 1.0748 1.0790 1.1036 1 .0 0 2 1 1.0825 1 .0 1 2 2 0.6864 1.1215 1.0878 1.0078 1.0060 1.0238 1.0370 1.0240 1.0181 1.0876 1.0173 1.0047 1.0044 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5920* 4430* 5430* 3980* 3750* 3990* 3290* 4190* 7550* 3490* 4870* 5940* 5030* 4910* 4400* 5040* 5900* 5420* 5870* 4560* 3M Environmental Laboratory 3M Environmental Laboratory 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 3M Sam ple N umber E01-1365-31184 E01 -1365-31204 E01-1365-31219 E01 -1365-31224 E01 -1365-31240 E01-1365-31250 E01-1365-31260 E01-1365-31275 E01-1365-31280 S a m p l e D e s c r ip t io n AM O UNT OF LIVER W EIGHED (G ) A D D IT IO N A L DILUTION C O N C . O F C8F17S03' IN Sam ple (n g /g o f L iv e r ) 0 ppm a.i. 246 F Liver, Offspring 0 ppm a.i. 259 F Liver, Offspring 0 ppm a.i. 273 F Liver, Offspring 0 ppm a.i. 277 F Liver, Offspring 0.7302 0.4434 0.5004 0.4326 1 1 1 1 <2 0 .0 * <2 0 .0 * <2 0 .0 * <2 0 .0 * 10 ppm a.i. 517 F Liver, Offspring 10 ppm a.i. 525 F Liver, Offspring 10 ppm a.i. 538 F Liver, Offspring 10 ppm a.i. 557 F Liver, Offspring 10 ppm a.i. 561 F Liver, Offspring 0.6570 0.5720 0.5211 0.4654 0.9417 1 1 10 1 1 5690* 7030* 13900.* 4680* 4590* 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 1/2 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 21 of 226 Page 21 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 13. C8F17SO3' Data Summary, Male Quail Liver (Adult and Offspring) 3M Sample Number Sam ple Description E01-1342-30312 0 ppm a.i. 329 M Liver E01-1342-30330 0 ppm a.i. 331 M Liver E01-1342-30348 0 ppm a.i. 333 M Liver E01-1342-30366 0 ppm a.i. 335 M Liver E01-1342-30384 0 ppm 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 0 ppm a.i. 345 M Liver E01-1342-30474 0 ppm a.i. 347 M Liver E01-1342-30492 0 ppm a.i. 349 M Liver E01-1342-30510 0 ppm a.i. 351 M Liver E01-1342-30528 0 ppm a.i. 353 M Liver E01-1342-30546 0 ppm a.i. 355 M Liver E01-1342-30564 0 ppm a.i. 357 M Liver E01-1342-30582 0 ppm a.i. 359 M Liver E01-1342-30600 0 ppm 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 ppm a.i. 367 M Liver E01-1342-30672 10 ppm a.i. 369 M Liver E01-1342-30690 10 ppm a.i. 371 M Liver E01 -1342-30708 10 ppm 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 ppm 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 E01-1342-30829 10 ppm 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 ppm 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 ppm 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 ppm a.i. 407 M Liver AMOUNT OF LIVER WEIGHED (G) ADDITIONAL DILUTION CONC. OF C 8F 17S 0 3 'IN Sam ple (ng/g o f Liver) 1.0050 1.0530 1 1 <50.2* <50.2* 1.0338 1.0062 1.0269 0.0886 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 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 209 * <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* 1 .0 0 1 0 1.0856 1.0276 1.1008 1.0236 1.0312 1.0501 1 .0 1 1 0 1.0678 1.0315 1.0310 1.0947 1.0834 1.0489 1.0309 1.0276 1.0361 1.0019 1.0336 1.0956 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 100 100 100 100 78600* 78500* 86300' 70000* 80300* 90700* 80000* 73500* 83400* 79300* 70300* 70400* 86800* 65100* 50100* 63000* 147000* 146000* 138000* 132000' 3M Environmental Laboratory 3M Environmental Laboratory Page 22 of 226 Page 22 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M S am ple N umber S a m p l e D e s c r ip t io n E01-1365-31194 E01-1365-31199 E01-1365-31209 E01-1365-31214 E01-1365-31229 E01-1365-31234 E01-1365-31245 E01-1365-31255 E01-1365-31265 E01-1365-31270 E01-1365-31285 0 ppm a.i. 253 M Liver, Offspring 0 ppm a.i. 256 M Liver, Offspring 0 ppm a.i. 266 M Liver, Offspring 0 ppm a.i. 270 M Liver, Offspring 0 ppm a.i. 283 M Liver, Offspring 0 ppm a.i. 291 M Liver, Offspring 10 ppm a.i. 522 M Liver, Offspring 10 ppm a.i. 533 M Liver, Offspring 10 ppm a.i. 545 M Liver, Offspring 10 ppm a.i. 552 M Liver, Offspring 10 ppm a.i. 568 M Liver, Offspring AMOUNT OF LIVER W EIGHED (G) A D D IT IO N A L DILUTION C O N C . O F C8F17S03' IN Sam ple (n g /g o f L iv e r ) 0.3806 0.5947 0.2673 0.5900 0.6666 0.6072 1 1 1 1 1 1 <20.0* <20.0* <20.0* <20.0* <20.0* <20.0* 0.7325 0.6903 0.4624 0.5353 0.7581 10 1 1 1 1 13700* 4940* 5580* 5990* 6520* 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 Vi 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%. Statem ent o f C o n c lu sio n Sera results from the male control group portion of this study showed no detectable levels of C8F17S 03' in 19 of 19 quail (<400 ng/mL). The sera results from the female control group portion of this study showed no detectable levels of C8F17S 03` in 16 of 16 birds (<400 ng/mL) (Table 15). Analytical results for twenty sera samples from the 10ppm dosed male group showed an average of 141000 ng/mL C8F17S 03` (range 94400 - 226000 ng/mL, Table 14). Analytical results for seventeen sera samples from the 10ppm dosed female group showed an average of 8670 ng/mL C8F17S 03' (range 4 7 1 0 - 14700 ng/mL, Table 15). Sera results from the male offspring control group showed no detectable levels of C8F17S 03' in 6 of 6 birds (<400 ng/mL). The sera results from the female offspring control group portion of this study showed no detectable levels of C8F17S 03' in 4 of 4 birds (<400 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 C8F17S 03' in 19 of 19 birds (<50.2 ng/g, Table 15) and quantifiable concentrations of C8F17S 03' in 1 of 20 (209 ng/g, Table 14). The liver results from the female control group showed no detectable levels of C8F17S 03' in 20 of 20 birds (<50.2 ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed male group showed an average of 88500 ng/g C8F17S03' (range 50100 147000 ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed female group showed an average of 4900 ng/g C8F17SQ3' (range 3290 - 7550 ng/g, Table 14). 3M Environmental Laboratory 3M Environmental Laboratory Page 23 of 226 Page 23 Report E01-1245 Report 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 C8F17S (V 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 C8F17S 03' in 4 of 4 birds (<20.0 ng/g, Table 14). Analytical results for four liver samples from the 10ppm 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 10ppm dosed female offspring group showed an average of 5490 ng/g (range 4590 - 7030 ng/g, Table 15). Table 14. Summary Table, CsFiySCV Concentration in Sera and Liver S a m plin g Event Male Control Group N umber of Sera Samples Average C8Fi 7S03 Conc. Sera 19 NA Range o f Results N umber of L iver Samples Average C8Fi 7S 0 3 Conc. L iver <400 ng/mL (19 of 19) 20 <50.2 ng/g* Female Control Group 17 <400 ng/mL (16 of 17) NA 20 <50.2 ng/g* 2110 ng/mL (1 of 17) Male 10ppm Group 20 141000 ng/mL 94400 - 226000 ng/mL 20 88500 ng/g* Female 10ppm Group 18 9990 ng/mL 4710-31900 ng/mL 20 4900 ng/g* Range o f Results <50.2 ng/g (19 of 20)* 209 ng/g (1 of 20)* <50.2 ng/g (20 of 20)* 50100-147000 ng/g* 3290 - 7550 ng/g* Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 6 4 5 5 NA <400 ng/mL (6 of 6) NA <400 ng/mL (4 of 4) 12600 ng/mL 8220-19200 ng/mL 12400 ng/mL 7640 -18500 ng/mL 6 <20.0 ng/g* <20.0 ng/g (6 of 6)* 4 <20.0 ng/g* <20.0 ng/g (4 of 4)* 5 7350 ng/g* 4940 -13700 ng/g* 5 7180 ng/g* 4590 -13900 ng/g* 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 'A 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, C8Fi7S03' Concentration in Sera and Liver with Outliers Removed Sampling Event Number of Sera Samples Male Control Group Female Control Group Male 10ppm Group 19* 16 20* A verage C 8F 17S O 3 Conc. Sera * <400 ng/mL * Female 10ppm Group Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 17 8700 ng/mL 6* * 4* * 5* * 5* * Range o f Results * N umber of L iver Samples A verage C 8F17SO 3 Conc. L iver 19 <50.2 ng/g** <400 ng/mL (16 of 16) 20* * 4710-14700 ng/mL 20* 20* * * 6* * * 4* * * 4 5760 ng/g** * 4 5490 ng/g** Range o f Results <50.2 ng/g (19 of 19)** * * * * 4940 - 6520 ng/g** 4590 - 7030 ng/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 V2 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% . S t a t is t ic a l M e th o d s 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. Statem ent of Data Q u ality 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 2.11% to 5.51%. 3M Environmental Laboratory 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 2/3 of all QC and a minimum of Vz of QC at each level must be within 75% -125% criteria. All three of the liver "QC-10ppb" 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 Reproduction Study 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 Fluorochemical Compounds From Serum for Analysis Using HPLC Electrospray/Mass Spectrometry" and "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC Electrospray/Mass Spectrometry in Quail Serum and Liver." 4. 3M Environmental Laboratory Standard Operating Procedure ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds form Biological Matrices". L ist o f Attachm ents Attachment A: Extraction and Analytical Method Attachment B: Data Summary Tables Attachment C: Sample Chromatograms, Calibration Information, and Instrument Parameters Attachment D: Sample Preparation Sheets Attachment E: Study Protocol, Protocol Amendments, and Deviations Attachment F: Example Calculations 3M Environmental Laboratory 3M Environmental Laboratory 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 S ig nature Page We certify that this report is a true and complete representation of the data for this study: i A _____ Lisa A. Stevenson Principal Analytical Investigator oh/ ^ / dj 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 Attach m en t A : Extr ac tio n and A n a ly tic a l M etho d, E T S -8-2 31 .1 , " S o lid P hase Ex tr ac tio n and A n alysis of F lu o r o c h em ic al C o m po unds from B io lo g ic a l M atr ic es" 3M Environmental Laboratory 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 Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory Method Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: H flijd l Revision Date: Effective Date: Jl/ i / z Approved By: Willliam K. Reagen Laboratory Manager Date ' ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 1 o f 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 Scope and Application This method describes the extraction o f target analytes from fish, rat liver, rat sera, mouse liver, and mouse sera using solid phase extraction (SPE). This method may also be extended to other biological matrices provided that the data quality objectives are met. 2 Method Summary An amount o f biological material, determined by the analyst, is prepared (fluids diluted and tissues homogenized) at a 1/6 dilution, or other dilution as determined by the analyst using reagent grade water. An aliquot o f the dilution/homogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile (ACN) is added as an extraction solvent and also serves to precipitate the proteins. The sample is capped, mixed, and put on the centrifuge to clarify the supernatant. The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned Cis SPE cartridge. Finally, the analytes o f interest are eluted from the SPE cartridge and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). 3 Definitions 3.1 Dilution A dilution expressed as 1:5 or 1/6 is defined as: 1 niL o f sample + 5 mLs o f diluent for a total o f 6 mLs combined, unless otherwise noted. 3.2 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds o f interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable concentrationfs) o f the target analyte(s). 3.4 Quality control sample Sample used to monitor the extraction efficiency (as a matrix spike) and to verify the continued accuracy o f the initial calibration curve (as a continuing calibration verification). 4 Warnings and Cautions 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewear, and clothing when working with solvents, samples and/or equipment. Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 volts. 4.2 Cautions Take care not to allow the SPE column to run to dryness after the methanol and water washes. After washing is complete, add sample then allow all o f the liquid to pass through the SPE column to dryness. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 2 o f 19 3M Environmental Laboratory 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 Reproduction Study with the Northern Bobwhite Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. Do not mn solvent pumps to dryness. 5 Interferences_________________________________________________________ To minimize interferences, Teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6 Instrumentation, Supplies, and Materials_________________________________ The following instrumentation, supplies, and materials are used while performing this method. Equivalent instrumentation, supplies, and materials may be used in place o f those listed. 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 2 Ultra-Turrax T25 tissue homogenizer Vacuum Pump SPE Extraction Manifold Centrifuge, Mistral 1000 or IEC' Shaker, Eberbach or VWR Balance (+/- 0.1000 g) Micromass, Quattro II or Ultima triple quadnipole Mass Specuometer equipped wife an electrospray ionization source HP 1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppendorf or disposable pipettes, plastic or glass Dissecting scalpels Polypropylene bottles, capable o f holding 50 mL to 1 L (Nalgene) Volumetric flasks, glass, type A 40 mL glass vials (1CHEM) Plastic sampule vials, Wheaton, 6 mL (or other appropriate size) Centrifuge tubes, polypropylene, 15 mL and 50 mL Labels Graduated pipettes, glass Swinges, capable of measuring 5 pL to 1000 |xL Bottle-Top Dispenser (capable o f dispensing 5mL o f solvent) SPE extraction cartridge, 1 g, Sep-Pak 6 cc tri-functional C |8 (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 3 o f 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 Reproduction Study 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-QTM, Nanopure II, or equivalent Acetonitrile, HPLC grade or equivalent Methanol, HPLC grade or equivalent Ammonium acetate, reagent grade or equivalent Biological fluids or tissues, frozen from supplier 7.1 Reagents preparation 2.0 rnM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing reagent grade water, mix until all solids are dissolved, bring to volume using reagent grade water. Store at room temperature. Note: When preparing different volumes than those listed in reagents preparation, target analyte standard preparation, and surrogate standard preparation, adjust accordingly. 7.2 Target analyte standard preparation Prepare target analyte standard(s) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations. Weigh approximately 100 mg o f target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location Bring to volume with methanol for a stock standard o f approximately 1000 ppm (jlg/mL). Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. Example calculation: 1000 |Tg/mL x 5 rnL/100 mL = 50 |ig/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution o f approx. 5.0 ppm. Example calculation: 50 (Ig/mL x 10 mL./100mL = 5.0 |ig/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution of approx. 0.50 ppm. Example calculation: 5 0 |ig /m L x l.OmL/'lOOmL = 0.5 |ig/mL. 7.3 Surrogate standard preparation Prepare surrogate standard(s). The following is an example only and may or may not be appropriate for all surrogate standard preparations. Weigh approximately 90-110 mg o f surrogate standard into a 100-mL volumetric flask and record the actual weight. Bring to volume with methanol for a surrogate standard stock o f approximately 900 -1100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL o f surrogate standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard o f 90-1 lOpprn. Record the actual volume transfened 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 may not be appropriate for all internal standard preparations. Weigh approximately 90-110 mg o f internal standard into a 100-mL volumetric flask and record the actual weight. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 4 o f 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 Bring to volume with methanol for an internal standard stock of approximately 9(X) - 1100 ppm. Prepare an internal standard working standard. Transfer approximately 1 mL o f internal standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard o f 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 8 Sample Handling All samples are received frozen and must be kept frozen until the extraction is perforated. Allow samples to thaw to room temperature prior to extraction. Typically fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials until analysis. If analysis will be delayed, exUacted standards and samples may be refrigerated at approximately 4C indefinitely or may be stored at room temperature until analysis can be perfonned. 9 Quality Control 9.1 Blanks 9.1.1 Solvent Blank An aliquot o f methanol is used as a solvent blank. Solvent blanks are not extracted. 9.1.2 Method Blank An aliquot o f l .0 mL o f water, or other appropriate amount, is used as a method blank. Four method blanks are extracted and analyzed with each set following this procedure (two are spiked with surrogate and two are not spiked). 9.1.3 Matrix Blank An aliquot o f 1.0 mL or 1.0 g o f matrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one o f three sources: 1) a study control matrix from a study control animal received with a sample set; 2) a commercially obtained sample of the same species as the study animals; or 3) a sumogate matrix, also obtained commercially, but o f a different species than the study animal, (eg. if rat is used to generate standard curves and CCVs for a mouse study). The matrix to use is dependent on the matrix used for the curve. 9.1.3.1 Study control matrix curve - if the study control matrix is used for the curve, prepare four (4) matrix blanks using the study control matrix (two spiked with surrogate and two not spiked). 9.1.3.2 Commercially obtained (same species) matrix curve - if the commercially obtained matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix (two spiked with surrogate and two not spiked). 9.1.3.3 Surrogate matrix curve - if a surrogate matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix and prepare four (4) matrix blanks using a commercially available matrix of the same species as the study animals (two spiked with surrogate and two not spiked). 9.1.3.4 If limited matrix is available, the number o f method and matrix blanks may be adjusted and will be noted in the study protocol or in the raw' data. 9.2 Sample Replicate Samples replicates are prepared according to each study protocol or project outline. ETS-8-23L1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 o f 19 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 Reproduction Study with the Northern Bobwhite 9.3 Surrogate standard If surrogate standar d is a component o f the study, all samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range o f the calibration curve, with the exception o f blank samples as described above. Typically surrogate standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted'homogenized sample removed for extraction. 9.4 Internal standard If internal standard is a component o f the study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range o f the calibration curve. Typically internal standard is spiked into the 2.0 mL o f extract in the 15 mL centrifuge tube, before transferring to the autovial. 9.5 Lab Control Sample Lab control samples are not a component o f this method. 9.6 Quality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efficiency and to verify the continued accuracy o f the initial calibration curve. Typically 1.0 mL. or other appropriate amount, o f the same matrix used to prepare the initial calibration curve is used for each QC sample. Twelve (12) quality control samples (QC) will be prepared for each matrix during the course o f a study. A minimum o f 3 QC samples must be prepared (one at each level) on each day o f sample extraction, (e.g. If the study is such that samples will be extracted on three different days then four QC samples must be prepared on each day o f extraction for a total oftwelve.) QC samples will consist o f four samples at each o f three levels of analyte. The levels listed below' may be used and may represent sample concentrations diluted into the range o f the calibration curve: Low' level: 3X to 5X the LLOQ, Mid-level: equivalent to a point near the middle o f the calibration curve, High level: 80% o f the ULOQ Two QC sample levels are analyzed after every tenth sample injection starting after the last calibration standard injection, with a minimum o f three QC per analysis. Solvent blanks are not considered samples but may be included as such for determining when QC samples will be analyzed. QC samples extracted with a particular sample set nmst be analyzed in the same analytical run. Any QC samples extracted during the course o f the study may be included in subsequent analyses. If samples from multiple extraction dates are analyzed in one analytical run, then QC samples from the same sample extraction dates must be included in that analysis. Each QC is expected to show an accuracy o f 75-125% of expected. A minimum o f 2/3 o f all QC samples must meet this criteria, and a minimum o f 1/2 of the QC samples at each level must meet this criteria. If not, the set must either be re-analyzed or re-extracted. 9.7 Sample Dilution Any sample with an area greater than that o f the highest acceptable standard will need to be diluted into the range of the calibration curve. If samples are diluted into the range o f the curve during analyses and enough sample remains, a post-run dilution validation will be performed to verify sample values. To pertonn the dilution validation, one sample will be separated into tw'o representative samples (i.e. tw'o 1.0 mL aliquots for fluid samples or tw'o 1.0 gram amounts for tissue samples, or other amount as determined by the analyst ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 6 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 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 fde) then diluted using two procedures. The first procedure consists o f diluting the sample with additional matrix prior to extraction (fluid adding fluid), while the second procedure consists o f diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent.) If the relative percent difference is not within 15% for these two samples; additional testing will be required to determine which value is a correct representation o f the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, per study. It will consist o f a minimum o f nine (9) levels. Additional calibration curves may be extracted on separate sample extraction dates, as determined by the analyst and documented in a note to file. Transfer 1.0 mL. or other appropriate amount, o f diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weight/volumes sheet or extraction worksheet, as appropriate. Four 1.0 mL aliquots, or other appropriate amount, o f control matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one standard curve. A total o f 9 standards, four matrix blanks, and four method blanks are prepared in addition to the QC samples and test samples. The number o f standards and blanks may be adjusted as determined by the analyst and documented in a note to file. Use Attachment C, or other appropriate fonn, as an aid in calculating the concentrations o f the working standards. Refer to section 12 to calculate the actual concentration o f analyte in each calibration standard and QC sample. Typically the target analyte standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, it may be spiked directly into tire matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. Analyze the extracted matrix standard curve prior to each set of extracts. The curve equation will be determined by regression analysis using the peak areas o f the target analyte(s) using MassLynx or other suitable software. Any level outside 75% - 125% o f nominal must be deactivated, and regression re-calculated, except the LLOQ which must be W'ithin 30% o f nominal. All levels must show a response greater than twice that o f the blank. A maximum o f three (3) levels may be deactivated in any one set, or the set will be re-analyzed. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 7 of 19 3M Environmental Laboratory 3M Environmental Laboratory 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 1 Mil 1 1 A l 'I' R O X I M A 1 K S l 'l k l N G A M O U N T S K ) R S T A N D A R D S A N D S l ' IKKS U s i n g 1 . 0 m L, o t M a t r i x W orking standard (approximate concentration) pL Approximate final concentration o f analyte in M a tr ix d ilu te d 1:5 Approximate final concentration o f analyte in Final 2.0 m L volum e - - Blank Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 10.0 ng/g or ng/mL 0.750 ng/mL 0.500 ug/mL 8.0 25.0 ng/g or ng/mL 2.00 ng/mL 0.500 ug/ntL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug/mL 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL 14.0 ng/mL 5.00 ug/mL 8.0 250 ng/g or ng/mL 20.0 ng/mL 5.00 ug/mL 16 500 ng'g or ng/mL 40.0 ng/mL 5.00 ug/mL 24 750 ng/g or ng/mL 60.0 ng/mL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/'g or ng/'mL 100 ng/mL 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/ntL 50.0 ug/mL 6.0 1750 ng/g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures__________________________________________________________ 11.1 Tissue Sample Preparation Obtain frozen tissue samples Cut approximately 1.0000 g o f tissue (+/- 0.1000 g), or other appropriate amount, using a dissecting scalpel. This part o f the procedure is best performed quickly, not allowing the tissue to thaw. Weigh the tissue directly into a tared plastic sampule vial. Record the weight on the weight'Volume sheet, extraction worksheet, or other appropriate location. Return unused tissue to tire freezer after extraction amounts have been removed. Add 2.5 mL o f reagent water to sampule vial, or other volume as detennined by the analyst and documented in a note to tile. Homogenize the sample. Put the Ultra-Tunax grinder probe in the sample and grind for approximately 2 minutes, or until the sample is homogeneous. Rinse the probe into the tube containing the sample with 2.5 mL o f reagent grade water, or other volume as determined by the analyst and documented in a note to file, using a pipette. Take the grinder apart and clean it with methanol after each sample. Reter to ETS-9-52 for more information. If an amount other than 1.0000 g (not within +/- 0.1000 g) is removed for an initial weight, adjust the water volume accordingly to maintain a 1/6 dilution, (e.g. if 0.5 g is removed for extraction, add a total of 2.5 mL o f water.), or other ratio as determined by the analyst and documented in a note to file. E T S - 8 - 2 3 1.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 8 o f 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 temperature or in lukewarm water. Label a 15 mL polypropylene centrifuge tube with the study number, sample ID, extraction date and analyst initials. See attached worksheet (Attachment A or similar worksheet) for documenting the remaining steps. Vortex mix the fluid sample for approximately 15 seconds, then transfer 1.0 mL o f fluid, or other appropriate amount to a plastic sampule vial, or other appropriate container. Return unused samples to freezer after extraction amounts have been removed. Add 5.0 mL o f reagent water to the 1.0 mL o f fluid for a 1/6 dilution, or other dilution as determined by the analyst and documented in a note to file. If a volume other than 1.0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilution as above. 11.3 Tissue and Fluid Sample Extraction After tissue or fluid samples have been prepared according to sections 11.1 and 11.2, vortex mix or shake by hand the diluted/homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mL polypropylene centriliige tube. Return unused diluted/homogenized portions to the freezer after extraction amounts have been removed. Record the volume removed on the extraction worksheet, (Attachment A or similar worksheet). Spike blanks, samples, and standards, ready for extraction with surrogate standard as described in this method. Spike each calibration standard matrix with the appropriate amount o f standard as described in this method for the calibration curve standards and each QC sample. Vortex mix the standard curve samples and QC samples for approximately 5 seconds. To each sample and standard, add 5.0 mL o f 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 o f approximately 300 rpm on the models listed in section 6.1). Remove from the shaker and centriliige at an appropriate speed for 10 minutes to adequately pellet the precipitate (a setting o f approximately 2000 rpm on the models listed in section 6.1). Add 40.0 mL o f reagent grade water to a clean 50 mL centrifuge tube. Remove samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduce any o f the matrix solids into the solution. Cap and mix by inverting several times. In this step the order o f addition may be changed (i.e. the sample may be put into the centrifuge tube and then the water added). Attach the reservoir to the SPE cartridge and attach this reservoir/cartridge unit to a vacuum manifold. NOTE: When running the vacuum, set the vacuum chamber at approximately 15 kPA - to give an approximate elution flow o f 5-7 mL/'min. Flows may vary through cartridges and the kPA may be raised for slow tubes and drying after most have been drawn down. Prepare the SPE cartridge by washing twice with approximately 5.0 mL o f methanol, followed by approximately two 5.0 mL aliquots of water, taking care not to allow the column to run to dryness a fter each wash. After washing is complete, pour the sample into the reservoir/cartridge unit and allow all o f the liq u id to pass through the column to dryness. Run the vacuum on high for approximately 5 minutes to adequately dry each SPE cartridge. Place a collection 15 mL polypropylene centrifuge tube under each cartridge and elute with 2.0 mL o f methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. E T S - 8 - 2 3 1.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 9 o f 19 3M Environmental Laboratory 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 Reproduction Study with the Northern Bobwhite Label each glass autovial. as appropriate, with the study number, vial file archive number, animal number/gender/timepoint or LIMS number, matrix, final solvent, analyte components (if needed), extraction type, extraction date, and analyst(s) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.1 Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-month-day. and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or A020204a 1 = Initial o f the instalment name (A = "Amelia") YY = Test year (02) MM = Test month (02) DD = Test day (04) a = First sample list (run) o f the day (the next sample list will end with 'b', the next 'c', and so on.) Assign a filename using the instrument designator letter, the last 2 digits o f the test year-month-day, and a 3-digit sequential file number that starts with 1 and increases by one for each filename. Example filename: IYYMMDD### or A020204001 I = Initial o f instrument name YY = Test year MM = Test month DD = Test day ### = 3-digit sequential file number starting with 1 through 999 (001) Also, as part o f the samplelist, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume, and sample descriptions. To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate mass(es). Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx "Guide to D ata A cquisition" for additional information on M R M . Typically the analytical batch ran sequence begins with system suitability, solvent blanks, and a set o f extracted matrix standards. Sample extracts are analyzed with two QC samples injected after every tenth sample injection. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be included as such ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 10 o f 19 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 tray according to the sample list prepared above. Set up the HPLC to the following conditions or at conditions the analyst considera appropriate for optimal response. Record actual conditions in the instrument logbook, or other appropriate location: Sample size = 10 jtL injection Inject/sample = 1 Cycle time = 10.0 minutes Flow rate = 300 uL/min Mobile phase: Solvent A = 2 raM Ammonium Acetate, Solvent B = Methanol Solvent gradient program: Time 'Mi Solvent B 0.00 10% 1.00 10% 5.50 95% 7.50 95% 8.00 10% 11.4.3 Instalment set-up Refer to ETS-9-24, "Operation and Maintenance o f the Micromass Quattro 11 Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for details. Check the solvent level in HPLC reservoirs and refill if necessary. Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. Turn on the nitrogen. Open the tune page. Click on operate to initiate source block and desolvation heaters. Open the Inlet Editor. Download the HPLC method and initiate solvent flow to begin system equilibrium. Set the flow to 10-500uL/min or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out o f the tip o f the probe. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o f the probe if no mist is observed Allow to equilibrate for approximately 10 minutes. Typical instrument parameters include: Drying gas 250-400 liters/hour ES nebulizing gas 10-15 liters/hour ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 11 o f 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 Reproduction Study with the Northern Bobwhite HPLC constant flow mode, flow rate 10-500 gL/min Pressure <400 bar (this parameter is not set, it is a guide to ensure the HPLC is operating correctly.) Source block temperature approximately 150C Desolvation temperature approximately 250C These settings may change in order to optimize the response Print the tune page, sample list, and acquisition method from MassLynx and store it in the study binder w'itb a copy taped into the instrument log. Click on start button in the Acquisition Control Panel (the location o f the start button may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). 12 Data Analysis and Calculations 12.1 Calculations If other calculations are used than those listed, they will be documented in the raw data. Calculate the matrix amount contained in the initial dilution using the following equation: Matrix Amount (g/m Lor mL/mL) IW (g) (or IV (mL)) (IW(g) (or IV(mL))+ DV (mL) Calculate actual concentrations o f analyte in calibration standards using the following equation: Concentration (ng/g or ng/mL) Spike Concentration (ug/mL) x Spiked Amount (mL) 1000 ng SV (m L)x Matrix Amount (g/mL or mL/mL) 1ug IW = Initial weight (where 1.0 g = 1.0 mL ) IV = Initial volume DV = Diluent volume (reagent grade water) SV = Sample volume removed for extraction (typically 1.0 mL) AR = Analytical result from MassLynx summary' DF = Dilution factor FV = Final volume MA = Matrix amount 3 curve = MA of tissue/fluid standard curve, assumed to be 1 g or 1 mU5 mL water 0 sample = MA o f tissue/fluid sample (___g or mL o f sample/5 mL water) Calculate spike percent recoveries using the following equation: Observed Result - Matrix Blank Result , % Recovery = ------------------------------------------------------- x 100 Spiking Level ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 12 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 40 of 226 Page 40 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Calculate relative standard deviation using the following equation: Relative Standard Deviation Standard Deviation x 100 Mean Calculate percent deviation using the following equation: % Deviation Expected Cone. - Calculated Cone. x 100 Expected Cone. Calculate actual concentration o f analyte in fluid 0tg''mL): AR (ng/mL) x DF x 9 curve fmL/niL'l x FV (mLl in Curve x l.Q ug = (pg'g) 3 sample (mL/mL) FV (mL) in Matrix lOOOng Calculate actual concentration o f analyte in tissue fylg/g): A R(ng'g) x DF x 3 curve fg/mL) x FV (mLl in Curve x l.Q ug = (flg/g) 3 sample (g/rnL) FV (mL) in Matrix lOOOng 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start and at the completion o f each analytical run. Prior to the calibration curve and after the last sample o f the run th e e (3) mid-level unextracted calibration standards will be analyzed. As applicable, the peak area precision, retention time precision, resolution, and peak asymmetry will be monitored at the beginning and the end o f the run separately. The peak area precision must be equal to or less than 5.0% RSD, the precision o f the retention time must be equal to or less than 2.5% RSD, the resolution must be > 2.0, and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is the asymmetry factor. If any item o f the system suitability fails, system maintenance must be completed prior to running a second set o f system suitability samples and the system suitability must pass before starting the calibration. If system suitability fails at tire completion o f a run, the sample set must be reanalyzed. 13.2 Quantitation The coefficient o f determination value for the calibration curve, plotted by regression using the peak areas o f the analyte(s). must be 0.990 or better. All active calibration curve points must be within 25% o f the theoretical value with the exception o f 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 affected by background levels o f the analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. If the curve cannot meet these criteria, the sample set must be reanalyzed or reextracted. 13.3 Accuracy Two thirds o f all quality control samples and 1/2 o f each quality control sample at each level are expected to show an accuracy o f 75-125%. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 13 o f 19 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 Reproduction Study with the Northern Bobwhite Sunogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be reanalyzed to confirm. If the second analysis confirms the original, the deviation will be documented in the raw data. If the second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed o f in noninfectious biohazard waste containers. Flammable solvent waste is disposed o f in high BTU containers. Glass pipette waste is disposed o f in broken glass containers located in the laboratory. 15 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw data. Each page generated for a study must contain the following information (if applicable): study/project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Other information may be added if applicable to the study. Print the tune page, sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into the instrument runlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data. Print data integration summary, integration method, and chromatograms from MassLynx, and store with the study rawdata. Summarize data using suitable software (Excel 7.0 or L1MS) and store in the study folder. Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 16 Attachments_________________________________________________________ Attachment A: Extraction Worksheet Attachment B: Sample Weight/Volume Worksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References___________________________________ ______________ __ _____ ETS-9-24, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52, "Operation and Maintenance o f a Tissue Grinder" 18 Affected Documents______________________________________________ ____ None ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 14 o f 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 Reproduction Study with the Northern Bobwhite 19 Revisions Revision Number Revision Description M inor formatting changes. Added detailed information to all sections concerning the extraction procedure, analytical procedure, and calculations. Added attachments and references. Revision Date 02/18/02 ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 15 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 43 of 226 Page 43 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Study Number: Prep Date: Analysts initials: Box#: Attachment A - Extraction W orksheet Method Revision:ETS-8-231.1 Matrix: Sample Timepoint: Sample Number S a m p le Number or description Volume of Amount and diluted sam ple Am ount and surrogate spike removed spike mix used mix used Type of column used and lot Elution solvent and volume C o m m ents Blank m a trix ______ T N -A -____________ ; Am ount w e ighed/aliqu ote d:___________ g/mL 1. Hom ogenize sam ple 2. A liquot 1 m l. o f diluted m atrix into 15 mL polypropylene tube 3. Spike sam ples accordingly 4. A d d ____ m L o f A C N (TN-A-____________ ) to each diluted sam ple and shake or vortex mix 5. S hake sam ple fo r 20 m in @ ______ rpm (S h a k e r________________ ) 6. C entrifuge sam ple for 10 m in @ ________rpm (C e n trifu g e __________________ ) 7. Add 40 m L o f ____________ w ater to 50 m L polypropelene centrifuge tube. 8. Decant extract into centrifuge tubes with water 9. Shake sam ple slightly to ensure proper mixing 10. A ttach 6 mL C18 S P E cartridges and 75 mL reservoirs to vacuum manifold 11. C ondition colum n w ith tw o washes o f - 5 mL MeOH (TN-A-___________ ) - do not allow colum n to go to dryness 12. W ash colum n w ith tw o washes o f - 5 m L ___________ w ater - do not allow colum n to go to dryness 13. Filter sam ple through conditioned colum n, discarding filtrate 14. A llow colum n to go to dryness. After dripping stops, draw a high vacuum through colum n fo r a t least 5 m inutes. 15. Elute colum n w ith solvent (___________ TN -A -___________ ) into appropriate 15 mL centrifuge tube 16. S pike sam ples w ith _________ uL o f internal standard # ________________________, cone._______________________ 17. T ransfer sam ple into appropriately labeled autovial and cap Note: In vacuum steps above set the vacuum cham ber at approxim ately 15 kPA - this should give approxim ately 5-7 m L/m in elution flow Flows m ay vary through cartridges - kPA may be raised for slow tubes and drying after m ost have been drawn down and shut off. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 16 o f'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): Analyst(s): Sample M a trix : Method/Revision: Sample ID Attachment B - Sample Weight/Volume Worksheet Study Number: Equipment Number: Final Solvent & TN Number: In itia l W t/Vol. g /m L /L W ater Volume added (mL) Volume Removed (m L) Comments Forni Completion Verified By:. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 45 of 226 Page 45 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): A n a ly te (s ): Sample m a trix : M ethod/revision: A ttachm ent C: Calibration Standard Concentration W orksheet Standard number: Equipm ent num ber: F inal solvent and T N : B la n k Tissue o r F lu id /id e n tifie r: A nalyte m ix std approx. 0.500 ug/mL: A n a lyte m ix std approx. 5.00 ug/mL: A n a lyte m ix std approx. 50.0 ug/mL: S urrogate std approx. 100 ug/mL: Actual concentrations o f standards in the analyte mix A n a ly te Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 A ll Am't spiked mL 0.0015 0.0030 0.0080 0.0160 0.0320 0.0056 0.0080 0.0160 0.0240 0.0320 0.0400 0.005 0.006 A ll Final Volume: mL 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 A ll Initial Fluid Dilution mL/niL 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 A ll Initial Tissue Density g/mL 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 Calculated concentrations o f standards in relation to tire final 2.0 mL solvent and initial matrix 2.0 m L F in a l V olum e F luid M a trix Tissue M a trix A n a ly te Final cone. ng/mL 0.375 0.750 2.00 4.00 8.00 14.0 20.0 40.0 60.0 80.0 100 125 150 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 0.500 A n a ly te Final cone. ng/mL 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 S u rro g a te Std cone ng/mL 100 Surrogate Final cone ng/mL 6500 A n a ly te Final cone. ng/g 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 S u rro g a te Std cone ng/mL 100 Surrogate Final cone ng'g 6500 ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 18 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 46 of 226 Page 46 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Study: D ilution Date/Analyst: Box Number: Sample N um ber o r Description 1/ Attachment D: Dilutions Summary Worksheet Solvent/TN Number: Extraction Date/Analyst: M atrix/Tim epoint: D ilutions 1/ 1/ 1/ 1/ 1/ 1/ Com m ents V erified By: Notes: 1/10 dilution = ________o f sample + _________ o f solvent Form Completion V erified By: ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 19 o f 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 Reproduction Study with the Northern Bobwhite Attachm ent B : Data S ummary Tables 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 3M Sample Number S a m p le d Date 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 Sample Description Water blank-day 1 Blank H20-day 2 Blank H20-day 2 quail sera blank-day 1 Blank quail sera-day 2 Blank quail sera-day 2 Inst. File Name d020207031 d020203019 d020207035 d020207032 d020203020 d020207036 amount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (hom oginate) 40 40 40 40 40 40 additional dilution 1 1 1 Inst Cone. (ng/mL of M eth a n o l) 10.0 10.0 10.0 1 10.0 1 10.0 1 10.0 loq for control sam ples 400 400 400 400 400 400 Cone, o f PFOS in Sample (ng/mL of Sera) 400 400 400 400 400 400 3M Sample Number E01-1245-35768 E01-1245-35827 E01-1245-35827 S a m p le d Date 1/31/2002 2/1/2002 2/1/2002 Sample Description quail Sera MS-day 1 quail Sera MS-day 2 quail Sera MS-day 2 Inst. File Name d020207033 d020203021 d020207037 E01-1245-35769 E01-1245-35828 E01-1245-35828 1/31/2002 2/1/2002 2/1/2002 quail Sera MSD-Day1 quail Sera MSD-Day2 quail Sera MSD-Day2 d020207034 d020203022 d020207038 Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/0.05 mL = 40 amount of sera in diluted sample = amount of sera used. amount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (hom oginate) 40 40 40 40 40 40 additional dilution 1 1 1 Inst Cone. (ng/mL of M e th anol) 225.29 254.33 227.43 loq for samples 400 400 400 1 217.03 1 254.55 1 219.54 400 400 400 for both ms and msd avg % ree 92.84 std dev of % ree 6.980 avg cone. 9321 std dev of cone. 680.2 Cone, o f PFOS in Sample (ng/mL of Sera) 9012 10173 9097 % Rec 89.76 101.3 90.61 8681 10182 8782 86.47 101.4 87.47 true value 10040 avg %rec 93.9 avg cone 9427 std dev of cone 647.4 true value 10040 avg %rec 91.8 avg cone 9215 std dev of cone 839.0 3M Environmental Laboratory Page 49 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study num ber E01-1245 - QUAIL SERA versus unextracted curve 3M Sample Number Sampled Date Sample Description E01-1245-35779 E01-1245-35780 E01-1245-35781 E01-1245-36155 E 0 M 2 4 5 -3 6 1 5 6 E01-1245-36157 E 0 M 24 5-3 6158 E01-1245-36159 E01-1245-36160 01/31/2002 qc-25ppb-1,01-31-02 (1004 ng/m Ldiluted sera) 01/31/2002 qc-25ppb-2t 01-31-02 (1004 ng/mL diluted sera) 01/31/2002 qc-25ppb-3, 01-31-02 (1004 ng/m Ldiluted sera) 02/13/2002 m s-5ppb-1,02-13-02 (201 ng/mL diluted sera) 02/13/2002 ms-5ppb-2, 02-13-02 (201 ng/mL diluted sera) 02/13/2002 ms-5ppb-3, 02-13-02 (201 ng/mL diluted sera) 02/13/2002 ms-200ppb-1,02-13-02 (8032 ng/m Ldiluted sera) 02/13/2002 ms-200ppb-2, 02-13-02 (8032 ng/mL diluted sera) 02/13/2002 ms-200ppb-3, 02-13-02 (8032 ng/mL diluted sera) original Inst. File Name d020213101 d 0 2 0 2 13102 d0202l3103 d020213104 d 0 2 0 2 13105 d020213106 d020213107 d020213108 d020213109 Absolute Recoveries amount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 dilu tion (diluted sera) 40 40 40 40 40 40 40 40 40 additional dilution 1 1 1 1 1 1 1 1 1 Inst Cone. (ng/mL of M e th a n o l) 38.69 39.12 45.18 5.54 5.51 5.23 248.13 254.19 253.36 loq fo r samples 201 201 201 201 201 201 201 201 201 Cone, of PFOS in Sample (ng/mL of 1548 1565 1807 222 220 209 9925 10168 10134 mu A A A A Study num ber E01-1245 - QUAIL SERA versus unextracted curve rerun 3M Sample Number Sampled Date Sample Description Inst. File Name E01-1245-35779 01/31/2002 qc-25ppb-1, 01-31-02 (1004 ng/m Ldiluted sera) E01-1245-35780 01/31/2002 qc-25ppb-2, 01-31-02 (1004 ng/m Ldiluted sera) E01-1245-35781 01/31/2002 qc-25ppb-3, 01-31-02 (1004 ng/mL diluted sera) E01-1245-36155 02/13/2002 m s-5ppb-1,02-13-02 (201 ng/mL diluted sera) E01-1245-36156 02/13/2002 ms-5ppb-2, 02-13-02 (201 ng/mL diluted sera) E01-1245-36157 02/13/2002 ms-5ppb-3, 02-13-02 (201 ng/mL diluted sera) E01-1245-36158 02/13/2002 ms-200ppb-1, 02-13-02 (8032 ng/mL diluted sera) E01-1245-36159 02/13/2002 ms-200ppb-2, 02-13-02 (8032 ng/mL diluted sera) E01-1245-36160 02/13/2002 ms-200ppb-3, 02-13-02 (8032 ng/mL diluted sera) A = Not within +/-25% criteria d020405082 d020405083 d020405084 d020405085 d020405086 d020405087 d020405088 d020405089 d020405090 Cone o f PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(dfluted sera)} dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/0.05 mL = 40 amount of sera in diluted sample = amount of sera used. amount of diluted sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 dilu tion (diluted sera) 40 40 40 40 40 40 40 40 40 for all in original analysis (absolute recovery) avg %rec 132.3 std dev 25.62 additional dilution 1 1 1 1 1 1 1 1 1 Inst Cone, (ng/m l of M ethanol) 36.06 41.69 44.98 6.17 5.88 6.17 248.12 260.27 254.58 loq fo r samples 201 201 201 201 201 201 201 201 201 Cone, o f PFOS in Sample (ng/mL of Sera) 1442 1668 1799 247 235 247 9925 10411 10183 for all in rerun analysis (absolute recovery) avg %rec 136.6 std dev 21.84 A A A A A % Rec 154.1 155.9 180.0 110.2 109.7 104.1 123.6 126.6 126.2 % Rec 143.7 166.1 179.2 122.8 117.0 122.8 123.6 129.6 126.8 avg 163.3 s td dev 14.46 %RSD 8.853 avg 108.0 std dev 3.403 %RSD 3.151 avg 125.4 s td dev 1.636 %RSD 1.304 avg 163.0 std dev 17.97 %RSD 11.03 avg 120.9 std dev 3.332 %RSD 2.757 avg 126.7 std dev 3.027 %RSD 2.390 3M Environmental Laboratory Page 50 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample H Number Sam pled Date Sample Description * E0M245-28444 06/15/2001 * E01-1245-28446 06/15/2001 * E01-1245-28449 06/15/2001 * E01-1245-28451 06/15/2001 # E01-1245-28453 06/15/2001 * E01-1245-28456 06/15/2001 * E01-1245-28458 06/15/2001 * E01-1245-28460 06/15/2001 * E01-1245-28462 06/15/2001 * E01-1245-28464 06/15/2001 * E01-1245-28466 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 h Concentration <LOQ 454-108-330, Oppm female, adult, 01-31-02 454-108-332, Oppm female, adult, 01-31-02 454-108-336, Oppm female, adult, 01-31-02 454-108-338, Oppm female, adult, 01 -31 -02 454-108-340, Oppm female, adult, 01-31-02 454-108-344, Oppm female, adult, 01-31-02 454-108-346, Oppm female, adult, 02-01-02 454-108-348, Oppm female, adult, 02-01-02 454-108-350, Oppm female, adult, 02-01-02 454-108-352, Oppm female, adult, 02-01-02 454-108-354, 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 Inst. File Name d020131055 d020207059 d020131057 d020207061 d020131060 d020207064 d020131O62 d020207066 d020131069 d020207073 d020131072 d020207076 d020201043 d020207087 d020201045 d020207089 d020201047 d020207091 d020201048 d020207092 d020201050 d020207094 d020201052 d020207096 d020201059 d020207103 d020201061 d020207105 d020201063 d020207107 d020201066 d020207110 d020201073 d020207117 amount of sera used (mL) 0.4 0.05 0.05 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.1 0.1 0.3 0.3 0.4 0.4 0.4 0.4 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.3 0.3 dilution (diluted additional sera) d ilu tio n 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 1 40 i 40 1 40 1 40 1 40 i 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 40 1 Inst Cone. (ng/mL of M ethanol) 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 10.0 10.0 10.0 10.0 10.0 10.0 51.27 54.43 10.0 10.0 10.0 10.0 10.0 10.0 loq for samples (ng/m L of Sera) 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 " Cone, of PFOS in Sample (ng/mL of Sera) average of dup. sam ples 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 " + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 2114 400 400 400 rejected rejected * There are two results reported for these samples. Both values are included in the average Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 ml_/0.05 mL = 40 amount of sera in diluted sample = 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(diluted sera)= 2.0 mL final volume/0.02 m L 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 Environmental Laboratory Page 52 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number E01-1245-28481 E01-1245-28483 E01-1245-28485 E 0 1 - 1245-2 8487 E01-1245-28489 E01-1245-28491 E01-1245-28493 E01-1245-28496 E01-1245-28498 E01-1245-28500 E01-1245-28502 E01-1245-28504 E01-1245-28506 E01-1245-28508 E01-1245-28511 E01-1245-28513 E01-1245-28515 E01-1245-28517 E01-1245-28518 Sam pled Date Sample Description 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 07/23/2001 454-108-370, 10ppm female, adult, 02-01-02 454-108-372. 10DPm female, adult, 02-01-02 454-108-374, 10ppm female, adult, 02-01-02 454-108-376, 10ppm female, adult, 02-01-02 454-108-378.10ppm female, adult, 02-01-02 454-108-380,10ppm female, adult, 02-01-02 454-108-382,10ppm female, adult, 02-01-02 454-108-386.10opm female, adult, 02-01-02 454-108-388.10ppm female, adult, 02-01-02 454-108-390.10ppm female, adult, 02-01-02 454-108-392, tOppm female, adult, 02-01-02 454-108-394. 10ppm female, adult, 02-01-02 454-108-396.10ppm female, adult. 02-01-02 454-108-398,10ppm female, adult, 02-01-02 454-108-402.10ppm female, adult, 02-01-02 454-108-404, 10ppm female, adult, 02-01-02 454-108-406,10ppm female, adult, 02-01-02 454-108-408,10ppm female, adult, 02-01-02 454-108-246, Oppm female, offspring, 02-01-02 E01-1245-28521 07/23/2001 454-108-259, Oppm female, offspring, 02-01-02 E01-1245-28524 07/23/2001 454-108-273, Oppm female, offspring, 02-01-02 E01-1245-28525 07/23/2001 454-108-277, Oppm female, offspring, 02-01-02 * E01-1245-28528 07/23/2001 454-108-517,10ppm female, offspring, 02-01-02 * E01-1245-28530 07/23/2001 454-108-525,10ppm female, offspring, 02-01-02 * E01-1245-28532 07/23/2001 454-108-538, 10ppm female, offspring, 02-01-02 * E01-1245-28535 07/23/2001 454-108-557, 10ppm female, offspring, 02-01-02 * E01-1245-28536 07/23/2001 hConcentration <LOQ 454-108-561, 10ppm female, offspring, 02-01-02 Inst. File Name d020203026 d020203027 d020203028 d020203029 d020203030 0020203031 d020416019 d020203033 d020203034 d020203038 d020203039 d020203040 d020203041 d020203042 d020203043 d020203044 d020203045 d020203046 d020201132 d020207153 d020201135 d020207156 d020201138 d020207159 d020201139 d020207160 d020201147 d020207168 d020201149 d020207170 d020201151 d020207172 d020201154 d020207175 d020201155 d020207176 amount of sera used (mL) 0.3 0.4 0.2 0.3 0.1 0.1 0.3 0.4 0.3 0.2 0.3 0.3 0.09 0.1 0.4 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.08 0.08 0.2 0.2 0.02 0.02 0.03 0.03 0.08 0.08 0.06 0.06 0.05 0.05 dilution (diluted additional sera) d ilu tio n 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 100 100 66.67 66.67 40 40 40 40 40 40 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Inst Cone. (ng/m L of M eth a n o l) 229.24 228.41 210.00 231.92 124.46 144.86 79.81 367.26 289.04 157.64 235.08 284.92 117.84 261.76 167.91 226.40 224.49 196.03 10.0 10.0 10.6 10.0 10.0 10.0 10.0 10.0 185.25 166.06 192.74 180.81 365.44 304.83 208.24 186.59 287.26 252.41 loq for samples (ng/m L of Sera) 400 400 400 400 400 400 201 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 Cone, o f PFOS in Sample (ng/mL of Sera) 9170 9136 8400 9277 4978 5794 31924 14690 11562 6306 9403 11397 4714 10470 6716 9056 8980 7841 400 400 400 400 400 400 400 400 18525 16606 12849 12054 14618 12193 8330 7464 11490 10096 + + + + + + + + average of dup. sam ples rejected 400 400 400 ..................... 4. 0. 0. 17566 12452 13405 7897 10793 W ith outliers avg 9990 std dev 6010 %RSD 60.2 W ithout outliers avg 8699 std dev 2557 %RSD 29.4 avg 400 std dev 0 %RSD - avg of dups 12423 std dev 3457 %RSD 27.8 * There are two results reported for these samples. Both values are Included in the average Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(diluted sera)) dllution(dlluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/0.05 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(diluted sera)= 2.0 mL final volume/0.02 m L amount of sera used = 100 3M Environmental Laboratory Page 53 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number Sam pled Date Sample Description E01-1245-28443 06/15/2001 E 0 1 - 1245-28445 06/15/2001 E01-1245-28447 06/15/2001 E01-1245-28448 06/15/2001 E 0 M 24 5-2 8450 06/15/2001 E01-1245-28452 06/15/2001 E O M 245-28454 06/15/2001 E01-1245-28455 06/15/2001 E01-1245-28457 06/15/2001 E 0 M 24 5-2 8459 06/15/2001 E01-1245-28461 06/15/2001 E01-1245-28465 06/15/2001 E O M 245-28467 06/15/2001 E 0 1 - 1245-28469 06/15/2001 E01-1245-28471 06/15/2001 E01-1245-28473 06/15/2001 E 0 M 24 5-2 8475 06/15/2001 E01-1245-28476 06/15/2001 E01-1245-28478 06/15/2001 hConcentration <LOQ 454-108-329, Oppm male, adult, 01-31-02 454-108-331, 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, 01-31-02 454-108-343, 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 Inst. File Name d020131054 d020207058 d020131056 d02020706Q d020131058 d020207062 d020131059 d020207063 d020131061 d020207065 d020131063 d020207067 d020131070 d020207074 d020131071 d020207075 d020131073 d020207077 d020201044 d020207088 d020201046 d020207090 d020201049 d020207093 d020201051 d020207095 d020201058 d020207102 d020201060 d020207104 d020201062 d020207106 d020201064 d020207108 d020201006 d020207109 d020201067 d020207111 amount of sera used (mL) 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 0.1 dilution (diluted additional sera) dilution 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 Inst Cone. (ng/mL of M ethanol) 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. 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. 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 loq for samples (ng/m L of Sera) 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 Cone, o f PFOS in Sample (ng/mL of Sera) average of dup. sam ples 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 + * There are two results reported for these samples. Both values are included In the average Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/O.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(diluted sera)= 2.0 mL final volume/0.02 m L amount of sera used = 100 avg 400 std dev 0 %RSD - 3M Environmental Laboratory Page 54 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number * E01-1245-28480 * E01-1245-28482 * E01-1245-28484 * E01-1245-28486 * E01-1245-28488 * E01-1245-28490 E01-1245-28492 * E 0 M 245-28494 * E01-1245-28495 * E01-1245-28497 * E01-1245-28499 E01-1245-28501 * E01-1245-28503 * E0M245-28505 * E01-1245-28507 E01-1245-28509 * E01-1245-28510 * E01-1245-28512 * E01-1245-28514 E01-1245-28516 Sampled Date Sample Description 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 06/15/2001 454-108-369,10ppm male, adult, d100,02-01-02 454-108-371,10ppm male, adult, d100,02-01-02 454-108-373, 10ppm male, adult, d100,02-01-02 454-108-375, 10ppm male, adult, d100,02-01-02 454-108-377,10ppm male, adult, d100,02-01-02 454-108-379,10ppm male, adult, d100,02-01-02 454-108-381,10ppm male, adult, d100,02-01-02 454-108-383,10ppm male, adult, d100,02-01-02 454-108-385, 10ppm male, adult, d100,02-01-02 454-108-387, 10ppm male, adult, d100,02-01-02 454-108-389, 10ppm male, adult, d100,02-01-02 454-108-391, 10ppm male, adult, d100,02-01-02 454-108-393, 10ppm male, adult, d100,02-01-02 454-108-395, 10ppm male, adult, d100,02-01-02 454-108-397, 10ppm male, adult, d100,02-01-02 454-108-399, 10ppm male, adult, d100,02-01-02 454-108-401,10ppm male, adult, d100,02-01-02 454-108-403, 10ppm male, adult, d100,02-01-02 454-108-405, 10ppm male, adult, d100,02-01-02 454-108-407, 10ppm male, adult, d100,02-01-02 Inst. File Name d020201074 d020207118 d020201076 d020207119 d020201078 d020207120 d020201080 d020207121 <3020201082 d020207122 d020201089 d020207128 d020201091 0020207129 d020201093 d020207130 020201094 d020207131 <3020201096 d020207132 d020201103 d020207133 d020201105 d020207134 d020201107 d020207135 d020201109 d020207136 d020201111 d020207137 d02Q2O1118 d020207143 d020201119 d020207144 d020201121 d020207145 d020201123 d020207146 <3020201125 d020207147 amount of sera used (mL) 0.2 0.2 0.2 0.2 0.1 0.1 0.3 0.3 0.07 0.07 0.1 0.1 0.1 0.1 0.3 0.3 0.1 0.1 0.3 0.3 0.1 0.1 0.06 0.06 0.2 0.2 0.2 0.2 0.3 0.3 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 dilution (diluted additional sera) dilution 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 ' ' 40 ..... ' " 40 40 40 100 100 10 100 too 100 100 100 100 100 100 100 100 100 100 ~ 100 100 100 100 100 100 100 100 100 100 100 100 ~ 100 100 100 " ioo 100 100 100 100 100 100 100 100 100 Inst Cone. (ng/mL of Methanol) 26.73 29.20 45.31 44.86 35.90 39.13 34.52 34.48 23.60 24.03 37.59 41.62 35.26 37.13 36.65 39.06 29.64 30.68 43.62 49.02 32.26 33.91 35.28 36.72 40.06 41.16 31.80 33.93 24.90 26.63 34.45 39.65 50.85 56.60 31.64 34.39 26.94 29.23 24.79 27.90 loq for samples (ng/mL of Sera) 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 Cone, of PFOS in Sample (ng/mL of Sera) 106920 116800 181240 179440 1 4 3 6 0 0 " ...... 156520 138080 137920 94400 96120 150360 166480 141040 148520 146600 156240 118560 122720 174480 196080 129040 135640 141120 146880 160240 164640 127200 135720 99600 106520 137800 158600 203400 226400 126560 137560 107760 116920 99160 111600 * There are two results reported for these samples. Both values are included in the average Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dllution(diluted sera)) dilutlon(diluted sera) = final volume(mL)/amount of sera In diluted sample(mL) = 2.0 mL/0.05 mL = 40 ' amount of sera in diluted sample = 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(diluted sera)= 2.0 mL final volume/0.02 m L amount of sera used = 100 average of dup. samples 111860 1O V O H U 150060 138000 1IOKoR'AtO&Ou 1IAHAH7Pl OCU1 1IO IA 9 f) 1I9nvfOi 4H nU 1fig a r i 1IRO Ot ORH/iUn 1 A /in nn 1O t n H U 131460 11un Ro un fotun 148200 <01IAHQitUfYU 132060 1 1 9RAn 105380 avg of dups 141112 std dev 30225 %RSD 21.4 3M Environmental Laboratory Page 55 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL SERA 1 3M Sample H Number Sam pled Date Sample Description * E01-1245-28519 07/23/2001 * E01-1245-28520 07/23/2001 * E01-1245-28522 07/23/2001 * E01-1245-28523 07/23/2001 E01-1245-28526 07/23/2001 * E01-1245-28527 E01-1245-28529 * E01-1245-28531 07/23/2001 07/23/2001 07/23/2001 * E01-1245-28533 07/23/2001 * E01-1245-28534 07/23/2001 * E0M245-28537 07/23/2001 h Concentration <LOQ 454-108-253, Oppm male, offspring, 02-01-02 454-108-256, Oppm male, offspring, 02-01-02 454-108-266, Oppm male, offspring, 02-01-02 454-108-270, Oppm male, offspring, 02-01-02 454-108-283, Oppm male, offspring, 02-01-02 454-108-291, Oppm male, offspring, 02-01-02 454-108-522,10ppm male, offspring, 02-01-02 454-108-533, 10ppm male, offspring, 02-01-02 454-108-545, 10ppm male, offspring, 02-01-02 454-108-552, 10ppm male, offspring, 02-01-02 454-108-568, 10ppm male, offspring, 02-01-02 Inst. File Name amount of sera used (mL) dilution (diluted sera) d020201133 d020207154 0.02 0.02 100 100 d020201134 0.02 100 d020207155 d020201136 d020207157 d020201137 d020207158 0.02 0.07 0.07 0.2 0.2 100 40 40 40 40 d020201140 0.02 100 d020207161 0.02 100 d020201141 0.05 40 d020207162 0.05 40 d020405091 0.07 40 d020201150 0.07 40 d020207171 0.07 40 d02201152 0.04 50 ... d020207173 ____ 0 .0 4 ____ d020201153 0.07 d020207174 0.07 d020201156 0.1 d020207177 0.1 50 40 40 40 40 additional d ilu tio n 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 .... 1 1 r 1 Inst Cone. (ng/mL of M eth a n o l) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 48.13 232.67 205.40 309.79 272.51 339.60 286.47 301.89 260.08 loq for samples (ng/m L of Sera) 400 400 400 400 400 400 400 400 400 400 400 400 201 400 400 400 400 400 400 400 400 Cone, o f PFOS in Sample (ng/mL of Sera) 400 400 + 400 400 + 400 400 + 400 400 + 400 400 + 400 400 + 19252 9307 8216 ' 15490 " ' 13626 13584 11459 12076 10403 average of dup. sa m p le s 400 400 400 400 400 400 19252 8761 14558 12521 11239 * There are two results reported for these samples. Both values are included in the average Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/0.05 m l = 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- dllution(diluted sera)= 2.0 mL final volume/0.02 m L amount of sera used = 100 avg 400 std dev 0 %RSD - avg 12601 std dev 3374 %RSD 26.8 3M Environmental Laboratory Page 56 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL LIVER DATA SUMMARY, BLANKS AND SPIKES 3M Sample Number E01-1342-36000 E01-1342-36026 E01-1342-36031 E01-1342-36036 Sampled Date Sample D escription 02/11/2002 02/12/2002 02/13/2002 02/15/2002 Blank H20-day 1 Blank H20-day 2 Blank H20-day 3 Blank H20-day 4 Inst. File Name amount o f liver weighed (g) or water aliquoted (mL) Water Added for H o m g e n izin g (mL) d020211037 d020212025 d020301024 d020218025 1.0000 1.0000 1.0000 1.0000 9.00 9.00 9.00 9.00 Final Volume (mL) 2.00 2.00 2.00 2.00 Am ount of liver in diluted sam ple (g) or water (mL) additional dilution 0.100000 0.100000 0.100000 0.100000 1 1 1 1 Inst Cone. (ng/mL of M e th a n o l) 2.51 2.51 0.502 1.00 E01-1342-36001 E01-1342-36027 E01-1342-36032 E01-1342-36037 02/11/2002 02/12/2002 02/13/2002 02/15/2002 Blank female liver-day 1 Blank female liver-day 2 Blank female liver-day 3 Blank female liver-day 4 d020211038 d020212026 d020301025 d020218026 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 0.100635 0.100635 0.100635 0.100635 1 2.51 1 2.51 1 0.502 1 1.00 E01-1342-36002 E01-1342-36028 E01-1342-36033 E01-1342-36038 02/11/2002 02/12/2002 02/13/2002 02/15/2002 Blank male liver-day 1 Blank male liver-day 2 Blank male liver-day 3 Blank male liver-day 4 d020211039 d020212027 d020301026 d020218027 1.0015 1.0015 1.0015 1.0015 9.00 9.00 9.00 9.00 2.00 0.100135 1 2.51 2.00 0.100135 1 2.51 2.00 0.100135 1 0.502 2.00 0.100135 1 1.00 loq for s a m p le s (ng/g) 50.2 50.2 10.0 20.0 Cone, of PFOS in Sample (ng/g of Liver) 50.2 50.2 10.0 20.0 50.2 50.2 10.0 20.0 50.2 50.2 10.0 20.0 50.2 50.2 10.0 20.0 50.2 50.2 10.0 20.0 3M Sample Number E01-1342-36003 E01-1342-36029 E01-1342-36034 E01-1342-36039 Sampled Date Sample Description 02/11/2002 02/12/2002 02/13/2002 02/15/2002 Female liver MS-day 1 Female liver MS-day 2 Female liver MS-day 3 Female liver MS-day 4 Inst. File Name am ount o f liver Water Added for H o m g e n izin g weighed (g) (mL) Final Volume (mL) Am ount of liver in additional diluted sam ple (g) dilution d020211040 d020212028 d020301027 d020218028 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 0.100635 0.100635 0.100635 0.100635 1 1 1 1 Inst Cone. (ng/mL of M e th a n o l) 29.01 28.77 26.03 26.33 loq for sa m p le s (ng/g) 50.2 50.2 10.0 20.0 Cone, of PFOS in Sample (ng/g of Liver) % Rec 577 115.5 572 114.6 517 103.7 523 104.9 E01-1342-36004 02/11/2002 E01-1342-36030 02/12/2002 E01-1342-36035 02/13/2002 E01-1342-36040 02/15/2002 * Concentration <LOQ Female liver MSD-day 1 Female liver MSD-day 2 Female liver MSD-day 3 Female liver MSD-day 4 d020211041 d020212029 d020301028 d020218029 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 0.100635 0.100635 0.100635 0.100635 1 29.78 50.2 1 28.53 50.2 1 26.99 10.0 1 27.02 20.0 592 118.6 567 113.6 536 107.5 537 107.6 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) for both ms and msd avg % ree 110.7 std dev of % ree 5.514 avg cone. 552.6 std dev of cone. 27.51 true vai. 499 avg %rec 109.7 avg cone 547.2 std dev of cone 31.25 true vai. 499 avg %rec 111.8 avg cone 558.1 std dev of cone 26.67 3M Environmental Laboratory Page 57 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL LIVER Absolute Recoveries versus unextracted curve___________________________ original________________________________________________________________________________________ 3M Sample Number Sample Description Water Added for am ount o f liver Inst. File Name weighed (g) Homgenizing (mL) Final Volume (mL) Am ount of liver in additional diluted sam ple (g) dilution Inst Cone. (ng/mL of M e th a n o l) E01-1342-36017 E01-1342-36018 E01-1342-36019 E01-1342-36020 E01-1342-36021 E01-1342-36022 E01-1342-36023 E01-1342-36024 E01-1342-36025 qc-10ppb-1 (200ng/g) qc-10ppb-2 (200ng/g) qc-10ppb-3 (200ng/g) qc-75ppb-1 (1496 ng/g) qc-75ppb-2 (1496 ng/g) qc-75ppb-3 (1496 ng/g) qc-150ppb-1 (2993 ng/g) qc-150ppb-2 (2993 ng/g) qc-150ppb-3 (2993 ng/g) d020211047 d020211048 d020211049 d020211050 d020211051 d020211052 d020211053 d020211054 d020211055 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 2.00 0.100635 1 13.59 2.00 0.100635 1 12.74 2.00 0.100635 1 13.28 2.00 0.100635 1 95.85 2.00 0.100635 1 90.27 2.00 0.100635 1 92.58 2.00 0.100635 1 169.63 2.00 0.100635 1 167.02 2.00 0.100635 1 174.15 loq for samples (ng/g) 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 Cone, of PFOS in Sample (ng/g of Liver) 270 253 264 1905 1794 1840 3371 3319 3461 for all QC in original analysis (absolute recovery) avg %rec 122.6 std dev 8.461 A A A A Study number E01-1245 - QUAIL LIVER versus unextracted curve rerun 3M Sample Number Sample Description Inst. File Name am ount o f liver weighed (g) Water Added for H o m g e n izin g (mL) Final Volume (mL) Am ount of liver in additional diluted sam ple (g) dilution Inst Cone. (ng/mL of M e th a n o l) loq for sa m p le s (ng/g) Cone, of PFOS in Sample (ng/g of Liver) E01-1342-36017 E01-1342-36018 E01-1342-36019 E01 -1342-36020 E01-1342-36021 E01-1342-36022 E01-1342-36023 E01-1342-36024 E01-1342-36025 A = Not within +/-25% criteria. qc-10ppb-1 (200ng/g) qc-10ppb-2 (200ng/g) qc-10ppb-3 (200ng/g) qc-75ppb-1 (1496 ng/g) qc-75ppb-2 (1496 ng/g) qc-75ppb-3 (1496 ng/g) qc-150ppb-1 (2993 ng/g) qc-150ppb-2 (2993 ng/g) qc-150ppb-3 (2993 ng/g) d020405136 d020405137 d020405138 d020405139 d020405140 d020405141 d020405142 d020405143 d020405144 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for 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 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 1 180.27 100 295 283 294 1998 1886 2009 3735 3422 3583 for all QC in rerun analysis (absolute recovery) avg % re c 132.1 std dev 11.79 A A A A A A % Rec. 135.0 126.6 132.0 127.3 119.9 123.0 112.6 110.9 115.6 avg 131.2 std dev 4.274 %RSD 3.258 avg 123.4 std dev 3.725 %RSD 3.018 avg 113.1 std dev 2.395 %RSD 2.119 % Rec. 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 %RSD 4.369 3M Environmental Laboratory Page 58 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study number E01-1245 - QUAIL LIVER versus extracted curve____________________________ original ____ QC Sample Recoveries 3M Sample Number E01 -1342-36017 E01-1342-36018 E01-1342-36019 E01-1342-36020 E01-1342-36021 E01-1342-36022 E01-1342-36023 E01-1342-36024 E01-1342-36025 Sample Description am ount o f liver W ater Added for Inst. File Name w eighed (g) H o m g e n izin g (mL) Final Volume (mL) Am ount of liver in diluted sam ple (g) additional dilution qc-10ppb-1 (200ng/g) qc-10ppb-2 (200ng/g) qc-10ppb-3 (200ng/g) qc-75ppb-1 (1496 ng/g) qc-75ppb-2 (1496 ng/g) qc-75ppb-3 (1496 ng/g) qc-150ppb-1 (2993 ng/g) qc-150ppb-2 (2993 ng/g) qc-150ppb-3 (2993 ng/g) d020211047 d020211048 d020211049 d020211050 d020211051 d020211052 d020211053 d020211054 d020211055 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 Inst Cone. (ng/mL of M e th a n o l) 14.20 13.41 13.91 89.60 84.54 86.63 155.46 153.16 159.43 loq for sa m p le s (ng/g) 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 Cone, of PFOS in Sample (ng/g of Liver) 282 267 276 1781 1680 1722 3090 3044 3168 for all QC in original analysis (recovery) avg % rec 118.9 std dev 15.17 A A A Study number E01-1245 - QUAIL LIVER versus extracted curve rerun 3M Sample Number E01-1342-36017 E01-1342-36018 E01-1342-36019 E01-1342-36020 E01-1342-36021 E01-1342-36022 E01-1342-36023 E01-1342-36024 E01-1342-36025 A = Not within +/-25% criteria. Sample Description Inst. File Name am ount o f liver w eighed (g) Water Added for Homgenizing (mL) Final Volume (mL) qc-10ppb-1 (200ng/g) qc-10ppb-2 (200ng/g) qc-10ppb-3 (200ng/g) qc-75ppb-1 (1496 ng/g) qc-75ppb-2 (1496 ng/g) qc-75ppb-3 (1496 ng/g) qc-150ppb-1 (2993 ng/g) qc-150ppb-2 (2993 ng/g) qc-150ppb-3 (2993 ng/g) d020405136 d020405137 d020405138 d020405139 d020405140 d020405141 d020405142 d020405143 d020405144 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) Am ount of liver in additional diluted sam ple (g) d ilu tion 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 1 1 1 1 1 1 1 1 1 Inst Cone. (ng/mL of M e th a n o l) 13.43 12.91 13.35 84.61 79.97 85.09 155.84 143.14 149.66 loq fo r s a m p le s (ng/g) 100 100 100 100 100 100 100 100 100 Cone, of PFOS in Sample (ng/g of Liver) 267 257 265 1682 1589 1691 3097 2845 2974 for all QC in rerun analysis (recovery) avg %rec 113.8 std dev 14.48 A A A % Rec. 141.1 133.3 138.2 119.0 112.3 115.1 103.2 101.7 105.9 avg 137.5 std dev 3.971 %RSD 2.887 avg 115.5 std dev 3.378 %RSD 2.925 avg 103.6 std dev 2.106 %RSD 2.033 % Rec. 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 2.116 avg 110.6 std dev 3.756 %RSD 3.398 avg 99.30 std dev 4.217 %RSD 4.247 3M Environmental Laboratory Page 59 Report E01-1245 Study number E01-1245 - QUAIL LIVER DATA SUMMARY PFOS: A Reproduction Study with the Nothern Bobwhite 3M Sample Number Sampled Date Sample Description Inst. File Name am ount of liver weighed (g) Water Added for H o m g e n izin g (mL) EO M 342-30321 06/19/2001 0 ppm a.i. 330 F Liver d020211062 E01-1342-30339 06/19/2001 0 ppm a.i. 332 F Liver d020211064 E01-1342-30357 04/30/2001 0 ppm a.i. 334 F Liver 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 06/19/2001 0 ppm a.i. 340 F Liver d020212034 E01-1342-30429 05/31/2001 0 ppm a.i. 342 F Liver 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 E01-1342-30609 06/19/2001 0 ppm a.i. 362 F Uver d020212061 E01 -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 E01-1342-30663 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 E01-1342-30856 06/20/2001 10 ppm a.i. 386 F Liver 06/20/2001 10 ppm a.i. 388 F Liver d020301040 d020301041 E01-1342-30874 06/20/2001 10 ppm a.i. 390 F Liver d020301045 E01-1342-30892 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 0020218035 E01-1342-31018 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 E01 -1365-31219 07/23/2001 0 ppm a.i. 259 F Liver, Offspring d020218041 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 d020218048 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 Uver, Offspring d020218053 E01-1365-31260 07/23/2001 10 ppm a.i. 538 F Uver, Offspring d020405158 E01-1365-31275 07/23/2001 10 ppm a.i. 557 F Uver, Offspring d020218061 E01 -1365-31280 07/23/2001 10 ppm a.i. 561 F Liver, Offspring d020218062 * Concentration <LOQ 1.0749 1.0469 1.0232 1.0188 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 1.0876 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 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 Final Volume (mL) Amount of liver in diluted sample (g) additional dilution 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 0.106691 0.104201 0.102083 0.101689 0.104861 0.101635 0.108063 0.100782 0.108893 0.109228 0.102128 0.113379 0.105172 0.102799 0.109184 0.105243 0.103800 0.103264 0.105163 0.108205 0.107745 0.106682 0.107054 0.109228 0.100189 0.107364 0.101097 0.0982480 0.110804 0.107833 0.100701 0.100540 0.102137 0.103318 0.102155 0.101626 0.107816 0.101554 0.100423 0.100396 0.103866 0.109660 0.100072 0.107276 0.108470 0.0957803 0.103782 0.0937286 0.104151 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 Inst Cone. (ng/mL of M e th a n o l) 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 2.51 2.51 2.51 2.51 319.18 236.52 290.48 217.55 187.79 214.22 166.41 206.04 418.20 187.91 245.00 298.53 256.81 253.83 224.76 255.94 318.13 275.17 294.79 228.77 1.00 1.00 1.00 1.00 308.44 336.48 72.30 219.12 238.83 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) loq for sa m p le s (ng/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 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 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 100 20.0 20.0 Cone, of PFOS in Sample (ng/g of Liver) 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 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 * * * * * * * * * ` * ` * * * * * * * ` * * * * rejected avg 50.2 std dev 0 %RSD - 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 Page 60 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL LIVER DATA SUMMARY_______________________________ 3M Sample Number Sampled Date Sample D escription Inst. File Name am ount of liver weighed (g) Water Added for H o m g e n izin g (mL) Final Volume (mL) Amount of liver in diluted sample (g) additional dilution Inst Cone. (ng/mL of M e th a n o l) E01-1342-30312 06/19/2001 0 ppm a.i. 329 M Liver d020211061 E01-1342-30330 06/19/2001 0 ppm a.i. 331 M Liver d020211063 E01-1342-30348 06/19/2001 0 ppm a.i. 333 M Liver d020211065 E01-1342-30366 06/19/2001 0 ppm a.i. 335 M Liver d020211067 E01-1342-30384 06/19/2001 0 ppm a.i. 337 M Liver d020211069 E01-1342-30402 06/19/2001 0 ppm a.i. 339 M Liver d020212033 E01-1342-30420 06/19/2001 0 ppm a.i. 341 M Liver d020212035 E01-1342-30438 06/19/2001 0 ppm a.i. 343 M Liver d020212037 E01-1342-30456 06/19/2001 0 ppm a.i. 345 M Liver d020212039 E01-1342-30474 06/19/2001 0 ppm a.i. 347 M Liver d020212041 E01-1342-30492 06/19/2001 0 ppm a.i. 349 M Liver d020212046 E01-1342-30510 06/19/2001 0 ppm a.i. 351 M Liver d020212048 E01-1342-30528 06/19/2001 0 ppm a.i. 353 M Liver d020212050 E01-1342-30546 06/19/2001 0 ppm a.i. 355 M Liver d020212052 E01 -1342-30564 06/19/2001 0 ppm a.i. 357 M Liver d020212054 E01-1342-30582 06/19/2001 0 ppm a.i. 359 M Liver d020212058 E01-1342-30600 06/19/2001 0 ppm a.i. 361 M Liver d020212060 E01-1342-30618 06/19/2001 0 ppm a.i. 363 M Liver d020212062 E01 -1342-30636 06/19/2001 0 ppm a.i. 365 M Liver d020212064 E01-1342-30654 06/19/2001 0 ppm a.i. 367 M Liver d020212066 E01-1342-30672 06/20/2001 10 ppm a.i. 369 M Liver d020301050 E01-1342-30690 06/20/2001 10 ppm a.i. 371 M Liver d020405148 E01-1342-30708 06/20/2001 10 ppm a.i. 373 M Liver d020405149 E01-1342-30726 06/20/2001 10 ppm a.i. 375 M Liver d020301053 E01-1342-30744 06/20/2001 10 ppm a.i. 377 M Liver d020301054 E01-1342-30762 06/20/2001 10 ppm a.i. 379 M Liver d020405150 E01-1342-30780 06/20/2001 10 ppm a.i. 381 M Liver d020405151 E01-1342-30798 06/20/2001 10 ppm a.i. 383 M Liver d020301060 E01-1342-30829 06/20/2001 10 ppm a.i. 385 M Liver d020405152 E01-1342-30847 06/20/2001 10 ppm a.i. 387 M Liver d020301062 E01-1342-30865 06/20/2001 10 ppm a.i. 389 M Liver d020301063 E01-1342-30883 06/20/2001 10 ppm a.i. 391 M Liver d020301064 E01-1342-30901 06/20/2001 10 ppm a.i. 393 M Liver d020405153 E01-1342-30919 06/20/2001 10 ppm a.i. 395 M Liver d020301066 E01-1342-30937 06/20/2001 10 ppm a.i. 397 M Liver d020301067 E01-1342-30955 06/20/2001 10 ppm a.i. 399 M Liver d020218064 E01-1342-30973 06/20/2001 10 ppm a.i. 401 M Liver d020405159 E01-1342-30991 06/20/2001 10 ppm a.i. 403 M Liver d020405160 E01-1342-31009 06/20/2001 10 ppm a.i. 405 M Liver d020405161 E01-1342-31027 06/20/2001 10 ppm a.i. 407 M Liver d020405162 E01-1365-31194 07/23/2001 0 ppm a.i. 253 M Liver, Offspring d020218039 E01-1365-31199 07/23/2001 0 ppm a.i. 256 M Liver, Offspring d020218040 E01-1365-31209 07/23/2001 0 ppm a.i. 266 M Liver, Offspring d020218042 E01-1365-31214 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 E0M 365-31234 07/23/2001 0 ppm a.i. 291 M Liver, Offspring d020218050 E01-1365-31245 07/23/2001 10 ppm a.i. 522 M Liver, Offspring d020405157 E01-1365-31255 07/23/2001 10 ppm a.i. 533 M Liver, Offspring d020218054 E01-1365-31265 07/23/2001 10 ppm a.i. 545 M Liver, Offspring d020218059 E01-1365-31270 07/23/2001 10 ppm a.i. 552 M Liver, Offspring d020218060 E01-1365-31285 07/23/2001 10 ppm a.i. 568 M Liver, Offspring d020218063 * Concentration <LOQ 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 1.0956 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 2.51 0.104745 1 2.51 0.103032 1 10.76 0.100558 1 2.51 0.102415 1 2.51 0.107904 1 2.51 0.102110 1 2.51 0.100809 1 2.51 0.109317 1 2.51 0.105243 1 2.51 0.103211 1 2.51 0.105217 1 2.51 0.101644 1 2.51 0.108019 1 2.51 0.103425 1 2.51 0.108390 1 2.51 0.100755 1 2.51 0.107302 1 2.51 0.108169 1 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 0.106061 10 442.30 0.102826 10 407.70 0.102781 10 361.37 0.108443 10 381.87 0.107444 10 466.09 0.104380 10 339.50 0.102772 10 257.69 0.102477 10 322.88 0.103237 100 76.08 0.100171 100 72.89 0.103014 100 71.24 0.108523 100 71.64 0.0956137 1 1.00 0.0992043 1 1.00 0.0900819 1 1.00 0.0984975 1 1.00 0.0956851 1 1.00 0.101079 1 1.00 0.104159 10 71.33 0.0987511 1 244.12 0.0931807 1 259.80 0.106309 1 318.62 0.0952614 1 310.49 loq for s a m p le s (ng/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 Cone, of PFOS in Sample (ng/g of Liver) 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 20.0 20.0 20.0 20.0 20.0 20.0 13696 4944 5576 5994 6519 rejected rejected With outliers avg 58.1 std dev 35.5 %RSD 61.0 Without outliers avg 50.2 std dev 0 %RSD - avg 88476 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 3597 %RSD 49.0 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) 3M Environmental Laboratory Page 61 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Attac h m en t C : S a m p le C hrom atogram s, Ca lib r a tio n In fo r m atio n , a n d In str u m en t Inform ation 3M Environmental Laboratory 3M Environmental Laboratory Page 62 of 226 Page 62 Report E01-1245 Report E01-1245 Methanol Solvent Blanks PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 63 of 226 Page 63 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFO: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 64 of 226 Page 64 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 65 of 226 Page 65 Report E01-1245 Report E01-1245 Water Blank Samples PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 66 of 226 Page 66 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 67 of 226 Page 67 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 68 of 226 Page 68 Report E01-1245 Report E01-1245 Sera Blank Samples PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 69 of 226 Page 69 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 70 of 226 Page 70 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 71 of 226 Page 71 Report E01-1245 Report E01-1245 Female Liver Blank Samples PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 72 of 226 Page 72 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 73 of 226 Page 73 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 74 of 226 Page 74 Report E01-1245 Report E01-1245 Male Liver Blank Samples PFOS: A Reproduction Study with the Nothern Bobwhite PFCjUS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 75 of 226 Page 75 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite i i Sil' ij iPFOS: A Reproduction Study with the Northern Bobwhite !!1 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 Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 77 of 226 Page 77 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 78 of 226 Page 78 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-371,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207119Sm (Mn, 1x2) 100-1 6.72 68915 davey 070799 08-Feb-2002, 05:57:53 1: MRM of 1 Channel ES- TIC 5.84e5 Area i 3M Environmental Laboratory 3M Environmental Laboratory Page 79 of 226 Page 79 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-373,10ppm male, adult, dlOO,02-01-02 3M Environmental Lab d020207120 Sm (Mn, 1x2) 100-1 6.72 _ 61202 davey 070799 08-Feb-2002, 06:08:59 1: MRM of 1 Channel ESTIC 6.59e5 Area %- i r p r r r j n 7 ^ m -|TT i 1.00 2.00 rrrrp -rr Ir' ' H 1 3.00 4.00 5.00 6.00 7.00 8.00 i Time 9.00 10.00 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 Northern Bobwhite 454-108-375,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207121 Sm (Mn, 1x2) 100-1 6.72 _ 54909 davey 070799 08-Feb-2002, 06:20:06 1: MRM of 1 Channel ESTIC 5.65e5 Area %- n -rp - i p i H 1 n r i p n 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 81 of 226 Page 81 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite jj ! F^OS: A Ffeprojdpction Study with the Northern Bobwhite 454-108-377,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207122 Sm (Mn, 1x2) 100-1 6.72 40589 davey 070799 08-Feb-2002, 06:31:14 1: MRM of 1 Channel ES TIC 3.99e5 Area I1r | II I 1.00 2.00 3.00 r i 111 if 'I m-p-n n - f p - r 4.00 5.00 6.00 7.00 8.00 ' i " r1n11 Time 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 PFOS: A Reproduction Study with the Northern Bobwhite 454-108-379,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207128 Sm (Mn, 1x2) 100-1 6.72 _ 64561 davey 070799 08-Feb-2002, 08:45:48 1: MRM of 1 Channel ES- TIC 6.87e5 Area %- I I I 'I | I1I n 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 83 of 226 Page 83 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 454*108-381,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207129 Sm (Mn, 1x2) 100- 6.71 58506 davey 070799 08-Feb-2002, 08:57:00 1: MRMof 1 Channel ESTIC 6.06e5 Area % T r i i p i Ti-p m - i - m r r m 1.00 2.00 3.00 r r i'p r r i 4.00 5.00 6.00 7.00 r i r i i i "i i TT r p m-| Time 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 Reproduction Study wify the Northern Bobwhite 454-108-383,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207130 Sm (Mn, 1x2) 1 0 0 -1 6.72 61109 davey 070799 08-Feb-2002, 09:08:05 1: MRM of 1 Channel ESTIC 5.37e5 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 85 of 226 Page 85 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-385,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207131 Sm (Mn, 1x2) 100-1 6.72 49730 davey 070799 08-Feb-2002, 09:19:11 1: MRM of 1 Channel ESTIC 5.28e5 Area %- 0 11 i i i -| r-1 ' i'T i n i'"| r r i'H 'i m | i t i i -| i t t i [' i T i r r r m i n h t t t t i -t t i it t i i - r r ; ........................................................................................... 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 86 of 226 Page 86 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 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 Reproduction Study with the Northern Bobwhite Dosed Group Adult Female Sera Samples 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 454-108-372,10ppm female, adult, 02-01-02 3M Environmental Lab d020203027 Sm (Mn, 1x2) 100-1 6.75 _ 317500 davey 070799 03-Feb-2002,16:30:40 1: MRM of 1 Channel ESTIC 3.38e6 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 89 of 226 Page 89 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 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 PFOS: A Reproduction Study with the Northern Bobwhite if I 454-108-376,10ppm female, adult, 02-01-02 3M Environmental Lab d020203029 Sm (Mn, 1x2) 100- 6.75 _ 321554 davey 070799 03-Feb-2002,16:52:48 1: MRM of 1 Channel ESTIC 3.54e6 Area 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 91 of 226 Page 91 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite A Reproduction Study with the Northern Bobwhite 454-108-378,10ppm female, adult, 02-01-02 3M Environmental Lab d020203030 Sm (Mn, 1x2) 100-1 6.74 _ 187898 davey 070799 03-Feb-2002,17:03:54 1: MRM of 1 Channel ESTIC 2.06e6 Area 0 -V t 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 92 of 226 Page 92 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite | PFOS: A Reproduction Study with the Northern Bobwhite 454-108-380,10ppiti female, adult, 02-01-02 3M Environmental Lab d020203031 Sm (Mn, 1x2) 100-1 6.75 _ 214792 davey 070799 03-Feb-2002,17:15:00 1: MRM of 1 Channel ESTIC 2.34e6 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 93 of 226 Page 93 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PF0S: A Reproduction Study with the Northern Bobwhite 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 3M Environmental Laboratory 3M Environmental Laboratory Page 95 ot 226 Page 95 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-388,10ppm female, adult, 02-01-02 3M Environmental Lab d020203034 Sm (Mn, 1x2) 100-1 6.74 _ 384563 davey 070799 03-Feb-2002,17:48:17 1: MRM of 1 Channel ESTIC 4.20e6 Area %- -m 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 96 of 226 Page 96 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite ireduction Study with the Northern Bobwhite 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 PFOS: A Reproduction Study with the Northern Bobwhite Dosed Group Male Offspring Sera Samples 454*108-522,10ppm male, offspring, 02-01-02 3M Environmental Lab d020207169 Sm (Mn, 1x2) 100-i 6.73 _ 471945 davey 070799 08-Feb-2002,16:20:52 1: MRM of 1 Channel ES TIO 4.74e6 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 98 of 226 Page 98 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite em Bobwhite 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 Reproduction Study with the Northern Bobwhite f 454-108-545,10ppm male, offspring, 02-01-02 3M Environmental Lab d020207173 Sm (Mn, 1x2) 100-1 6.73 352415' davey 070799 08-Feb-2002,17:05:10 1: MRM of 1 Channel ESTIC 3.66e6 Area %- 0 1111 1 1 1 " i 1" 11 l I I I I I 1.00 2.00 4.00 5.00 6.00 7.00 8.00 9.00 3M Environmental Laboratory 3M Environmental Laboratory Page 100 of 226 Page 100 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 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 PFOS: A Reproduction Study with the Northern Bobwhite 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 454-108-517,10ppm female, offspring, 02-01-02 3M Environmental Lab d020207168 Sm (Mn, 1x2) 1 0 (h 6.73 _ 236874 davey 070799 08-Feb-2002,16:09:45 1: MRM of 1 Channel ES- TIC 2.49e6 Area %- 1.00 2.00 3.00 4.00 5.00 6,00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 103 of 226 Page 103 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 104 of 226 Page 104 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite www with the Northern Bobyuhite 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 Northern Bobwhite 454-108-557,10ppm female, offspring, 02-01-02 3M Environmental Lab d020207175 Sm (Mn, 1x2) 100-1 6.74 260577' davey 070799 08-Feb-2002,17:27:19 1: MRM of 1 Channel ESTIC 2.68e6 Area %- 0 -r If-m -H r r r j m 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 106 of 226 Page 106 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Northern Bobwhite 454-108-561,10ppm female, offspring, 02-01-02 3M Environmental Lab d020207176 Sm (Mn, 1x2) 100-1 6.73 _ 333841 davey 070799 08-Feb-2002,17:38:25 1: MRM of 1 Channel ESTIC 3.43e6 Area n-rp-rr 1" 1r ',"'M| i 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 107 of 226 Page 107 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 108 of 226 Page 108 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 109 of 226 Page 109 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 110 of 226 Page 110 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 111 of 226 Page 111 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342-30865,10 ppm a.i. 389 M Liver,d10 3M Environmental Lab d020301063 Sm (Mn, 1x2) 100-1 6.74 405774 davey 070799 02-Mar-2002, 02:28:27 1: MRM of 1 Channel ESTIC 4.22e6 Area %- 3M Environmental Laboratory 3M Environmental Laboratory Page 112 of 226 Page 112 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 PFOS: A with the Northern Bobwhite E01>1342-30847,10 ppm a.i. 387 M Liver,d10 3M Environmental Lab d020301062 Sm (Mn, 1x2) 100-1 M il 6.74 _ 439049 davey 070799 02-Mar-2002, 02:17:20 1: MRM of 1 Channel ESTIC 4.50e6 Area %- 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 E01-1342-30798,10 ppm a.i. 383 M Liver,d10 3M Environmental Lab d020301060 Sm (Mn, 1x2) 100' 6.74 _ 413083 davey 070799 02-Mar-2002, 01:55:06 1: MRM of 1 Channel ESTIC 4.34e6 Area 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 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 E01-1342-30780,10 ppm a.i. 381 M Liver,d10 3M Environmental Lab d020301059 Sm (Mn, 1x2) 100 -i 6.74 _ 470298 davey 070799 02-Mar-2002, 01:43:59 1: MRM of 1 Channel ESTIC 4.79e6 Area %- 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 Il'llllfMf Study with the Northern Bobwhite E01-1342-30937,10 ppm a.i. 397 M Liver,d10 3M Environmental Lab d020301067 Sm (Mn, 1x2) 100-1 6.74 315950 davey 070799 02-Mar-2002, 03:12:59 1: MRM of 1 Channel ESTIC 3.35e6 Area -i-n 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 116 of 226 Page 116 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PfO S: A Fjl^ropluc^jon Study with the Northern Bobwhite E01-1342-30919,10 ppm a.i. 395 M Liver,d10 3M Environmental Lab d020301066 Sm (Mn, 1x2) 100- 6.74 388593 davey 070799 02-Mar-2002, 03:01:52 1: MRM of 1 Channel ES TIO 3.98e6 Area 1.00 ' ' " 2.00 rrm -p Time 5.00 6.00 7.00 8.00 " ' " 9.00 " ' ' 10.00 3M Environmental Laboratory 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 Reproduction Study with the Northern Bobwhite Dosed Group Female Liver Samples 3M Environmental Laboratory 3M Environmental Laboratory Page 118 of 226 Page 118 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342-30838,10 ppm a.i. 386 F Liver 3M Environmental Lab d020301040 Sm (Mn, 1x2) 100-1 6.73 446002 davey 070799 01-Mar-2002, 22:12:49 1: MRM of 1 Channel ESTIC 4.76e6 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 f T 'l I i ) I 7.00 8.00 [ Time 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 Reproduction Study with the Northern Bobwhite 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 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 PFOS: A Repfoduction Study with the Northern Bobwhite 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 E01-1342-30717,10 ppm a.i. 374 F Liver 3M Environmental Lab d020301034 Sm (Mn, 1x2) 1 0 0 -1 6.74 346653 davey 070799 01-Mar-2002, 21:06:09 1: MRMof 1 Channel ESTIC 3.78e6 Area %- 1.00 3M Environmental Laboratory 3M Environmental Laboratory -r-n Time 10.00 Page 123 of 226 Page 123 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFQSj: I Reproduction Study with the Northern Bobwhite E01-1342-30789,10 ppm a.i. 382 F Liver 3M Environmental Lab d020301038 Sm (Mn, 1x2) 100-1 6.72 219312 davey 070799 01-Mar-2002, 21:50:37 1: MRMof 1 Channel ESTIC 2.60e6 Area %I O V ttt 1.00 l "1' 1 I > M I I I I I I I I I I I I 2.00 3.00 4.00 5.00 6.00 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 PROS: A Reproduction Study with the Northern Bobwhite E01-1342-30681,10 ppm a.i. 370 F Liver 3M Environmental Lab d020301032 Sm (Mn, 1x2) 1 0 0 --i 6.74 _ 371782 davey 070799 OI-Mar-2002, 20:43:57 1: MRM of 1 Channel ESTIC 4.09e6 Area %- 0-Lt 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 125 of 226 Page 125 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342-30699,10 ppm a.i. 372 F Liver 3M Environmental Lab d020301033 Sm (Mn, 1x2) 100-1 6.72 295007 davey 070799 01-Mar-2002, 20:55:05 1: MRM of 1 Channel ESTIC 3.36e6 Area 3M Environmental Laboratory 3M Environmental Laboratory I Page 126 of 226 Page 126 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite the Northern Bobwhite E01-1342-30753,10 ppm a.i. 378 F Liver 3M Environmental Lab d020301036 Sm (Mn, 1x2) 100-1 6.73 243424 davey 070799 01-Mar-2002, 21:28:20 1: MRM of 1 Channel ESTIC 2.86e6 Area %- i-rrm TTi 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 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Dosed Group Male Offspring Liver Samples 3M Environmental Laboratory 3M Environmental Laboratory Page 128 of 226 Page 128 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1365-31270,10 ppm a.i. 552 M Liver, offspring 3M Environmental Lab d020218060 Sm (Mn, 1x2) 1 0 0 -1 6.70 302473 davey 070799 18-Feb-2002,18:28:34 1: MRM of 1 Channel ESTIC 3.23e6 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 129 of 226 Page 129 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 ipn Study with the Northern Bobwhite E01-1365-31285,10 ppm a.i. 568 M Liver, offspring 3M Environmental Lab -,d020218063 Sm (Mn, 1x2) 100 6.72 _ 296813 davey 070799 18-Feb-2002,19:01:50 1: MRM of 1 Channel ES TIC 3.19e6 Area I 11TT ' r 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 130 of 226 Page 130 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 131 of 226 Page 131 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1365-31255,10 ppm a.i. 533 M Liver, offspring 3M Environmental Lab d020218054 Sm (Mn, 1x2) 100-1 6.72 246631 davey 070799 18-Feb-2002,17:21:57 1: MRM of 1 Channel ES TIO 2.74e6 Area %- 3M Environmental Laboratory 3M Environmental Laboratory Page 132 of 226 Page 132 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PPOS: A Reproduction Study with the Northern Bobwhite E01-1365-31265,10 ppm a.i. 545 M Liver, offspring 3M Environmental Lab d020218059 Sm (Mn, 1x2) 100-1 6.73 259129 davey 070799 18-Feb-2002,18:17:25 1: MRM of 1 Channel ES TIO 2.83e6 Area %- 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 O ffspring Liver Samples E01-1365-31260,10 ppm a.i. 538 F Liver, offspring 3M Environmental Lab d020218055 Sm (Mn, 1x2) 100-1 6.73 446716 davey 070799 18-Feb-2002,17:33:05 1: MRM of 1 Channel ESTIC 4.59e6 Area %- . ..................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 134 of 226 Page 134 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1365-31250,10 ppm a.i. 525 F Liver, offspring 3M Environmental Lab d020218053 Sm (Mn, 1x2) 100-1 6.73 314522 davey 070799 18-Feb-2002,17:10:50 1: MRM of 1 Channel ESTIC 3.38e6 Area %- I i` I I'M i I | I I I i 1.00 11111111 2.00 3.00 4.00 5.00 6.00 7.00 8.00 T-n-i Time 9.00 10.00 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 FfFOS: A Reproduction Study with the Northern Bobwhite 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 Reproduction Study with the Northern Bobwhite 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 PFOS: A Study with the Northern Bobwhite 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 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 PFOS: A Study with the Northern Bobwhite Control Group Female Sera Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 140 of 226 Page 140 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite the Northern Bobwhite Control Group Male Offspring Sera Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 141 of 226 Page 141 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 If- I f Control Group Female Offspring Sera Sample ff the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 142 of 226 Page 142 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 Liver Sample 3M Environmental Laboratory 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 Northern Bobwhite Control Group Female Liver Sample 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 Reproduction Study with the Northern Bobwhite Control Group Male Offspring Liver Sample 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 the Northern Bobwhite Control Group Female Offspring Liver Sample 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 the Northern Bobwhite Extracted Sera Calibration Curve Point 3M Environmental Laboratory 3M Environmental Laboratory Page 147 of 226 Page 147 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Extracted Liver Calibration Curve Point 3M Environmental Laboratory 3M Environmental Laboratory Page 148 of 226 Page 148 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Report E01-1245 Unextracted Calibration Curve Point PFOS: A Reprodution ptudy with the Northern Bobwhite ll li' \: ; i i|;l! ; l ! 3M Environmental Laboratory 3M Environmental Laboratory Page 149 of 226 Page 149 Report E01-1245 Report E01-1245 Extracted Sera Calibration Curve PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 150 of 226 Page 150 Report E01-1245 Report E01-1245 Extracted Liver Calibration Curve PFOS: A Reproduction Study with the Nothern Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ilf 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 Reproduction Study with the Northern Bobwhite Mass Spectrometer Tune Settings Tuning Method Report Method: C:\MASSLYNX\DAVEY070799.PRO\ACQUDB\CENTRE1 Printed: Tue Mar 19 10:22:06 2002 Page 1 MS SOURCE ( ESP- ) Capillary Cone Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. Desolvation Temp. Set Rdbk 2.56 20 0.5 0.2 0.8 150 250 -2.48 -20 148 249 Pressures Analyser Vacuum Gas Cell Rdbk 3.8e- 5 2.9e- 3 Analyser LM Res 1 HM Res 1 IEnergy 1 Entrance Collision Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Gas Flows Cone Gas Desolvation Set Rdi 13.0 13.0 0.7 2 11 1 11.0 11.0 1.0 650 1 11 0 -6' 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 Reproduction Study with the Northern Bobwhite HPLC Settings Method Report Method File: Last Modified: Printed: Page 1 C:\MASSLYNX\DAVEY070799.PRO\A( Monday, March 11, 2002 08:01:01 Tuesday, March 19, 2002 08:37:50 HP1100 LC Pump Initial Conditions Solvents A% B% C% D% 90.0 10.0 0.0 0.0 Valve A set to channel Valve B set to channel Flow (ml/min) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left(C) Oven Temperature Right(C) 0.300 10.0 0 400 40.0 40.0 HP1100 LC Pump 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 HP110Q LC Pump Externa! Event Timetable The Timetable contains 3 entries which are : Time Initial 0.00 0.10 Column Switch Of! Off Of! On Ofl Off Contactl Off On Off Contact2 Ofl Ofl Ofl Corrtac Off On O ff HP110 Autosam pler Initiai Conditions Draw Speed Eject Speed (pKmin) Draw Position (mm) Stop Time (mi ns) Injection Vol ume(pl) Vial Number Thermostat On Thermostat TemperaturefC) 20.0 200 0.00 10.00 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 Reproduction Study with the Northern Bobwhite Mass Spectrometer Scanning Parameters Scanning Method Report Method: C :\MASSLYNX\DAVEY070799.PRO\ACQTJDB\ 10MIN-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 ( Secs ) : Span { Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone ( V ) 499.00 99.00 0.30 43 60 Function : 2 MRM of 1 Mass Pair ( ESP- ) Inter Channel Delay ( Secs ) : Span ( Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone ( V ) 1 513.00 219.00 0.30 20 20 Page 1 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 Reproduction Study with the Northern Bobwhite Attachment D: Sample Preparation Sheets 3M Environmental Laboratory 3M Environmental Laboratory Page 155 of 226 Page 155 Report E01-1245 SPE C olu m n s ExtracPFSfvAoRiRdf*10" W w Jtfk Prep Date: Analysts initials: 1/31/2002 OK/WOJ 3 to 3 0 (2 Method Revision: ETS-8-23E0 Study Number: E01 -1g 5 6 " j / 'i t Z Matrix: Sera *5 Llms Assgned sample number Sample Number or description Volume of sample filtered (ml) Type of column used and lot Amount and spike Elution solvent mix used and volume c TM n ts ^ (.^ . E01-1245-35766 E01-1245-35767 E01 -1245-35768 E01-1245-35769 E01-1245-35770 E01-1245-35771 E01-1245-35772 E01-1245-35773 E01-1245-35774 E01-1245-35775 E01-1245-35776 E01-1245-35777 E01-1245-35778 E01-1245-35779 E01-1245-35780 E01-1245-35781 E01-1245-35782 E01-1245-35783 E01-1245-35784 E01-1245-35785 E01-1245-35786 E01-1245-35787 Blank H20-day 1 BlankjnaHarrTsera-day 1 & ( /\l C K | 3 / t>7 Matiard Sera MS-day 1 Makerd Sera MSD 5 ppb sera curve 10 ppb sera curve 25 ppb sera curve 50 ppb sera curve 100 ppb sera curve 250 ppb sera curve 500 ppb sera curve 750 ppb sera curve 1000 ppb sera curve QC-25 ppb-1 QC-25 ppb-2 QC-25 ppb-3 QC-40 ppm-1 QC-40 ppm-2 QC-40 ppm-3 QC-80 ppm-1 QC-80 ppm-2 QC-80 ppm-3 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6mJ, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 ' Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters7 1 g,bm l, lot 1290B2 NA NA Oul of 02001 -53 / 10ul of 02001-53 ^ u l of 02001-52 \f 4ul of 02001-52 ^ lO u l of 02001-52 / 20ul of 02001 -52 y 4ul of 02001-53 10ul of 02001-53 s 20ul of 02001 -53 V 30ul of 02001 -53 V 40ul of 02001-53 y 10ul of 02001-52 10ul of 02001-52 y 10ul of 02001-52 y 36ul of 02001 -15 l' 36ul of 02001-15 y 36ul of 02001-15 y 72ul of 02001 -15 / 72ul of 02001-15 7 72ul of 02001-15 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 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-547 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 JV ______ aJ ^ Blank Sera TN-A- 5 c' t <l 'L - ; Amount of sera aliquated: _ml -J /U S c - fi Dilute Sera with Kandiyohi water; Amount of water added _ ml o b Aliquot 1ml of sera into 15 ml polypropelene tube Jjpike samples accordingly Add 5ml of AON (TN-A- ^ 5 ) to aliquated sample - - /b i_T /* * s 7 # jir C r" O tTc ^ / 'ro Shake sample for 20 min @300 rpm (Shaker Centrifuge sample for 10 min @ 2000 rpm (Centrifuged L) W3 ) ^ ^ ^^ 5 ^, Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. f r Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing ^ 0j - ^ A > ru / a* r2 'c - / * < - =- Condition column with MeOH (TN-A- b 7*^ 4 J ^ Wash Column with Kandiyohi Water a Filter sample through conditioned column, discarding filtrate ( ^ Allow column to go completelly dry , Elute column with Indicated amount of solvent ( H eO tf TN-A- 5 ? *5 V J into appropriate 15 ml centrifuge tube . a , ^ C/ Spike samples with internal standard PFDA standard number (Tffco I , cone. Kx?- ? p y t u ' q c k l ^ jJ / To Transfer sample into appripriatelly marked autovial ct o ^ /c ^ .A -if >&J f ^^ , --, 7 ? *-^ ;j oA ~ < D ^ ) O KS-ZXCSL 3M Environmental Laboratory Page 156 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction W orksheet ( > O w i i l S e r o X LAS p j /j i I d w arfx > Prep Date: Analysts initials: 1/31/2002 QK/HOJ c 'b I o 2 016- Lims Assgned sample number E01-1245-28443 E01-1245-28444 E01-1245-28445 E01-1245-28446 E01-1245-28447 E01-1245-28448 E01-1245-28449 E01-1245-28450 E01-1245-28451 E01-1245-28452 E01-1245-28453 E01-1245-28454 E01-1245-28455 E01-1245-28456 E01-1245-28457 Sample Number or description Volume of sample filtered (ml) Type of column used and lot 454-108-329, 0 PPM, Male, Adult 454-108-330, 0 PPM, Female, Adult 454-108-331,0 PPM, Male, Adult 454-108-332, 0 PPM, Female, Adult 454-108-333, 0 PPM, Male, Adult 454-108-335, 0 PPM, Male, Adult 454-108-336, 0 PPM, Female, Adult 454-108-337, 0 PPM, Male, Adult 454-108-338, 0 PPM, Female, Adult 454-108-339,0 PPM, Male, Adult 454-108-340, 0 PPM, Female, Adult 454-108-341, 0 PPM, Male, Adult 454-108-343, 0 PPM, Male, Adult 454-108-344, 0 PPM, Female, Adult 454-108-345, 0 PPM, Male, Adult 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Method Revision: ETS-8-231.0 Study Number: E01-12S6" r^J 7 ' * Z Matrix: Sera ^ Amount and spike Elution solvent mix used and volume Comments NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 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 2mt 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 r cJs T oo^c. 7 -c c > ^ ~ Z odu ,L- f a 3lank Sera TN-A- Amount of sera aliquated:. Dilute Sera with Kandiyohi water; Amount of water added _ Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly _ml ml g g 02- i^y rzA. / -dSc /i y-- Xc f - z e t/O c ^ u y . ycU / <5V 7 , Z 't A * . ?e> Add 5ml of ACN (T N -A -_ fti^ 2 _ _ _ ) to aliquated sample ' . Shake sample for 20 min @300 rpm (Shaker V V -/ j Centrifuge sample for 10 min @ 2000 rpm (Centrifuge Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water b f U - / fc *w C 2 -0 j T c c i-C_ /M L / `I m L J Y^L. V 5 shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- ' 7 *5 ^ Wash Column with Kandiyohi Water ) o Filter sample through conditioned column, discarding filtrate Allow column to go completely dry Elute column with indicated amount of solvent ( TN-A- ^ 7 * ^ / ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number o 2 .c c I ~~ *+5" .cone. lO c jJ b r M J 3M Environmental Laboratory Page 157 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Study: E01-1245 Dilution Date/Analyst: BoxNumber: Attachment D: Dilutions Summary Worksheet Solvent/TN Number: MeOH/5457 w/ IS PFDA Extraction Date/Analyst: 1/31/02 Matrix/Timepoint: Giu-Ci',1 Sem. tl/lvlol Sample Number or Description 1/1000 E01-1245-35785 E01-1245-35786 * E01-1245-35787 EM -1245-3577 7e ELU1245-35780 E01-1245-35781 j E01-1245-35782 E01-1245-35783 E01-1245-35784 1/100 1/ 1/ \ /-->s UK ITC* f . <-) >N sj < - Dilutions 1/ 1/ Comments Verified By: r )K U \f A T3 \C ? y y----------------- s' / /' if .r - W< o il fTX T-/ V _ 1/10 dilution = 'C o j J of sample +Q o ^ fjS l of solvent I jiva yv tc'f' a. - \0pSl -jCtLu ^ i|ieoo d jlu f '- J A ; I ,[ e j i e 4 ^ . V ** SZVOCk/C* f f j f v b o ^<9 h . 44 Form Completion Verified By: ^ $ 4 ,J o . ,, I , J V S ) . -a A . IA ^ ______________ O j 3M Environmental Laboratory Page 158 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Standard Curves--Fluids Prep Date(s): Analyst(s): Sample Matrix: Method/Revision: T arget Analyte(s): 01/31/02,02/01/02, 02/13/02 OK/HOJ Quail Sera ETS-8-231.1 PFOS Study N umber: E01-1245 Equipment Number: NA Final Solvent & TN N um ber: MeOH/TN-A-05457 Blank Tissue/Identifier: Quail Sera-TN-A-5992 PFOS Std: 5.02 ppm: PFOS Std: 50.2 ppm: PFOS Std: 2241 ppm: Surr.Std Approx. 100.7 ppm: 02001-52 02001-53 02001-15 02001-45 PFDA Actual Concentrations of Standards in the FC Mix PFOS PFOS Std Cone. Am't Spiked ug/mL mL 5.02 0.0020 5.02 0.0040 5.02 0.0100 5.02 0.0200 50.2 0.0040 50.2 0.0100 50.2 0.0200 50.2 0.0300 50.2 0.0400 5.02 0.0100 2241 0.0360 2241 0.0720 50.2 0.0100 50.2 0.0100 5.02 0.0020 5.02 0.0800 PFDA Std Cone. ug/mL 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 PFDA Am't Spiked mL 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0,002 All Initial Dilution mL/mL 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 All Final Voi. 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 Blank sera initial dilution (mL/mL) for curves: Final solvent volume (mL) for curves: 0.0500 2.000 sWV d d U c t fer c l c t r i i j j . ^ osji?l02. Calculated Concentrations of Standards in the sample matrix Standard PFOS PFDA Number Std Cone. Std Cone. ng/mL ng/mL E01-1245-35770, 5.0 ppb E01-1245-35771, lOppb E01-1245-35772, 25 ppb 201 402 1004 4028 4028 4028 E01-1245-35773, 50 ppb 2008 4028 E01-1245-35774, 100 ppb 4016 4028 EOl-1245-35775,250 ppb E01-1245-35776, 500 ppb 10040 20080 4028 4028 E01-1245-35777,750 ppb E01-1245-35778, 1000 ppb 30120 40160 4028 4028 QC-25 ppb 1004 4028 QC-40 ppm 1613520 4028 QC-80 ppm 3227040 4028 Quail Sera MS/MSD-Day 1 10040 4028 Ouail Sera MS/MSD-Dav 2 10040 4028 Quail Sera MS-5 ppb Quail Sera MS-200 ppb 201 8032 4028 4028 Calculated Concentrations of Standards in the final solvent Standard PFOS PFDA Number Std Cone. Std Cone. ng/mL ng/mL E01-1245-35770, 5.0 ppb EOl-1245-35771, lOppb 5.02 101 10.0 101 EOl-1245-35772, 25 ppb 25.1 101 EO1-1245-35773, 50 ppb E01-1245-35774, 100 ppb 50.2 101 100 101 EOl-1245-35775, 250 ppb E01-1245-35776, 500 ppb 251 101 502 101 EOl - 1245-35777, 750 ppb 753 101 E01-1245-35778, 1000 ppb 1004 101 QC-25 ppb 25.1 101 QC-40 ppm 40338 101 OC-80 ppm 80676 101 Quail Sera MS/MSD-Day 1 251 101 Ouail Sera MS/MSD-Dav 2 251 101 Quail Sera MS-5 ppb 5.02 101 Quail Sera MS-200 ppb 201 101 Verified by:: / W - Z f / l 7 t f i ' 2- 3M Environmental Laboratory Page 159 Report E01-1245 SPE Colum ns Extra.SF?vAjRePr0dftion(S^dywithS ^ Noher8 Bobwhite IW JE- t o M Prep Date: Analysts initials: Lims Assgned sample number 2/1/2002 OK/HOJ Q 'l'O o iA 2 - c - c 2_ Sample Number or description Volume of sample filtered (ml) Type of column used and lot Method Revision: ETS-8-231.0 Study Number: E01-1245 Matrix: Sera Amount and spike Elution solvent mix used and volume Comments E01-1245-35825 E01-1245-35826 Blank H20-day 2 (2 titffMC 1t-l-o-- Blank maHanf sera-day 2 40+6 40+6 E01-1245-35827 Mattard Sera MS-day 2 40+6 E01-1245-35828 E01-1245-28458 E01-1245-28459 k M lL C>f, f 3 l< & Mallard Sera MSD-Day2 454-108-346, 0 PPM, Female, Adult 454-108-347, 0 PPM, Male, Adult 40+6 40+6 40+6 E01-1245-28460 454-108-348,0 PPM, Female, Adult 40+6 E01-1245-28461 454-108-349, 0 PPM, Male, Adult 40+6 E01-1245-28462 454-108-350, 0 PPM, Female, Adult 40+6 (T ) E01-1245-28463 454-108-351,0 PPM, Male, Adult 40+6 E01-1245-28464 EOl-l245-28465 454-108-352, 0 PPM, Female, Adult 454-108-353, 0 PPM, Male, Adult 40+6 40+6 E01-1245-28466 454-108-354, 0 PPM, Female, Adult 40+6 E01-1245-28467 454-108-355, 0 PPM, Male, Adult 40+6 E01-1245-28468 454-108-356, 0 PPM, Female, Adult 40+6 EOI-1245-28469 454-108-357, 0 PPM, Male, Adult 40+6 EOl-1245-28470 454-108-358, 0 PPM, Female, Adult 40+6 E01-1245-28471 454-108-359, 0 PPM, Male, Adult 40+6 E01-1245-28472 454-108-360, 0 PPM, Female, Adult 40+6 E01-1245-28473 E01-1245-28474 E01-1245-28475 454-108-361,0 PPM, Male, Adult 454-108-362,OPPM,Female, Adult 404-1U6-303, u i-VM, iVlaie, Adult 40+6 40+6 40+6 (T ) tio ^ r? A ^ A H TCC FfoR T/fr3 O /^ M -1 o. Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g,6ml, lot 1290B2 Waters, ig , omi, lot 1290B2 NA NA 10ui of 02001-53 10ui of 02001-53 MA r 2mfofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 \r 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 / 2mlofMeOH TN-A-5457 2m!ofMeOH TN-A-5457 7*6 2mlofMeOH TN-A-5457 ~L<uX - 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mtofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 / I rC /S o Lk/L . 3 c& a s (- 2mIofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 3 e c <-L J^u/L-2mlofMeOH - TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 2mlofMeOH TN-A-5457 Jc c t-C o& sL 2 W.-r- A < \-c a a < n , / h , rQOp Spike samples accordingly - /o J ? ^ A c ^ r /< y ^ /) % > /// / V i.d 2 ) * ^ _Add 5ml of ACN (TNZ m . J to aliquated sample ' Shake sample for 20 min @300 rpm (Shaker***'#- -S'T'ft- 5 4 (C 4 & t-O o " /A 2 .. / >- I ` Centrifuge sample for 10 min @ 2000 rpm (Centrifuqe3fc-^~T6<7 ( 1 3 oi c Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. 6 - Decant extract into centrifuge tubes with water y shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- .Tt j T ) I Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (A t I f TN-A- J ~ L j4 ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number f) ? oc I - 4 5 _____ conc./e ? * . 1 //* 1 , amount added Transfer sample into appripriatelly marked autovial ^ a A- /tt e Q & ,t ~ / . ' /fc C < *L K / / P Avt <*Kt- ynae ( & $ & ) *S n u 3M Environmental Laboratory ^ m p t w n (b N i n f e e / Page 160 Report E01-1245 PFOS: A Ri SPE Columns Extraction W orksheet e NB{h?5n?jbwhite 2- H Prep Date: Analysts initials: Lims Assgned sample number E01-1245-28476 EO I-1245-28477 E O I-1245-28478 E01-1245-28479 E01-1245-28480 E01-1245-28481 E01-1245-28482 E01-1245-28483 E01-1245-28484 E 0 1 -1245-28485 E01-1245-28486 E01-1245-28487 E 01-1245-28488 E01-1245-28489 E01-1245-28490 E01-1245-28491 E01-1245-28492 E01-1245-28493 E01-1245-28494 E01 -1245-28495 E01-1245-28496 E01-1245-28497 2/ 1/2002 QK/HOJ e ( - c_ Sample Number or description Volume of sample filtered (ml) Type of column used and lot 454-108-365,0 P PM , M ale, A dult 454-108-366, 0 PPM , Fem ale, A dult 454-108-367, 0 P PM , M ale, A dult 454-108-368, 0 P PM , Fem ale, A dult 454-108-369,10 PPM , M ale, Adult 454-108-370, 10P PM , Fem ale, A dult 454-108-371, 10 P PM , M ale, A dult 454-108-372, 10 P PM , Fem ale, A dult 454-108-373, 10 PPM , M ale, A dult 454-108-374, 10 P PM , Fem ale, A dult 454-108-375,10 P P M , M ale, Adult 454-108-376,10 P PM , Fem ale, A dult 454-108-377,10 P PM , M ale, A dult 454-108-378,10 P PM , Fem ale, A dult 454-108-379,10 P PM , M ale, A dult 454-108-380, 10 PPM, Female, Adult 454-108-381,10 PPM, Male, Adult 454-108-382,10 PPM, Female, Adult 454-108-383,10 PPM, Male, Adult 454-108-385, 10 PPM, Male, Adult 454-108-386,10 PPM, Female, Adult 454-108-387,10 PPM, Male, , . Adult 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 w aters, ig, omi, lot 1290B2 Method Revision: ETS-8-231.0 Study Number: E01-1245 Matrix: Sera Amount and spike Elution solvent mix used and volume Comments 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 "Z. 2mlofMeOH NA TN-A-5457 / d c ? a *L ^ 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2m1ofMeOH NA TN-A-5457 /& r o C 2miofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 3 ^ ^ L 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 /J o e . 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 / c ro t^ . 2mlofMeOH NA TN-A-5457 / O U 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN -A -5457 /O C 2mlofMeOH NA TN-A-5457 / fe c -L 2mtofMeOH NA TN-A-5457 ~3 fBlank Sera TN-A- O n Amount of sera aliquated: a / a - ml Dilute Sera with Kandiyohi water; Amount of water added. ml Aliquot 1ml of sera into 15 ml polypropelene tube '^ > '4 -Spike samples accordingly r-p Add 5ml of ACN (TN-A-. J to aliquated sample jo & Xr tw - " Shake sample for 20 min @300 rpm (Shaker / ttf- Z ttto AL. ^ /V <?4 { C f * Centrifuge sample for 10 min @ 2000 rpm (Centrifuged # ^ - 7 </ ? Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. P) 0 K $ 2$o Z. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- ^ q 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- 4 ) into appropriate 15 ml centrifuge tube , Spike samples with internal standard PFDA standard number (T ? .a c r l ~ 4 f ______, cone/ c b j amount added 3M Environmental Laboratory 0 *, m ? , Page 161 Report E01-1245 SPE C olu m n s ExtraEE2S:yAoRePiOduettion(| tud ^ h thte N 0 ^ e rnfBO blhite Prep Date: Analysts initials: 2/ 1/2002 OK/HOJ Q- 3' O2 l/6 - Lims Assgned sample number E01-1245-28498 E O I-1245-28499 E O I-124 5-28500 EO I-1245-28501 E01-1245-28502 E01-1245-28503 E01-1245-28504 E01-1245-28505 E01-1245-28506 EO l-1245-28507 EO l-1245-28508 E01-1245-28509 E01-1245-28510 E01-1245-28511 E01-1245-28512 E01-1245-28513 E01-1245-28514 E01-1245-28515 E01-1245-28516 E01-1245-28517 E01-1245-28518 E01-1245-28519 Sample Number or description Volume of sample filtered (ml) Type of column used and lot 454-108-388,10 PPM , Fem ale, A dult 454-108-389,10 P PM , M ale, A dult 454-108-390, 10 PPM , Fem ale, A dult 454-108-391, 10 P PM , M ale, A dult 454-108-392,10 P PM , Fem ale, A dult 454-108-393.10 P P M M ale, A dult 454-108-394, 10 P PM , Fem ale, A dult 454-108-395, 10 P P M M ale, A dult 454-108-396, 10 P PM , Fem ale, A dult 454-108-397,10 P P M M ale, A dult 454-108-398, 10 P PM , Fem ale, A dult 454-108-399, 10 P P M M ale, A dult 454-108-401,10 P P M M ale, Adult 454-108-402,10 P PM , Fem ale, A dult 454-108-403, 10 P PM , M ale, Adult 454-108-404,10 PPM, Female, Adult 454-108-405,10 PPM, Male, Adult 454-108-406,10 PPM, Female, Adult 454-108-407,10 PPM, Male, Adult 454-108-408,10 PPM, Female, Adult 454-108-246, 0 PPM, Female, Offspring 4 M -iu d ^ S d , U'k k M, Male, Offspring 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, omi, lot 1290B2 Method Revision: ETS-8-231.0 Study Number: E01-1245 Matrix: Sera Amount and spike Elution solvent mix used and volume Comments 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 f i * ( /L - 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 ? ' * C 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 1 (X h A ^, 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 2mlofMeOH NA TN-A-5457 Z 2mlofMeOH NA TN-A-5457 / 2mlofMeOH NA TN-A-5457 / 2mlofMeOH NA TN-A-5457 Z 2mlofMeOH NA TN-A-5457 2mlofMeOH N A TN-A-5457 2mlofMeOH NA TN-A-5457 /& O L t 2mlofMeOH NA TN-A-5457 7 G \jC - ( / A , // x 1 [ Blank Sera TN-A- 7 ei ei ' 2 , : Amount of sera allquated: ml | Dilute Sera with Kandiyohi water; Amount of water added 1Aliquot 1ml of sera into 15 ml polypropelene tube ml o k \ db (Spike samples accordingly*. Add 5ml of ACN (TN-A- P 4 5 ) to aliquated sample b = j . <S? ' Shake sample for 20 min @300 rpm (Shaker^ ^ t - d flJ fc fc - s 0 * 4 / * 4 Centrifuge sample for 10 min @ 2000 rpm (Centrifuged * * ? 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- r a f f i 0- Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate f Allow column to go completely dry Elute column with indicated amount of solvent TN-A-f * S 4 , ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 9 f l o c U __ , cone. /C b o . amount added 7 J x L 3M Environmental Laboratory 7c Page 162 Report E01-1245 PFOS: Reproductioi SPE Columns Extraction W<DrKshee*t m the-Nothern. Bobwhite \ US ozialo} Hcf 4 J Prep Date: Analysts initials: Lims Assgned sample number E01-1245-28520 E01-1245-28521 E01-1245-28522 E 0 1 -1245-28523 E01-1245-28524 E01 -1245-28525 E01-1245-28526 E01-1245-28527 E01-1245-28528 E01-1245-28529 E01-1245-28530 E01-1245-28531 E01-1245-28532 E01-1245-28533 E01-1245-28534 E01-1245-28535 E01-1245-28536 E01-1245-28537 2/ 1/2002 5 - I - o 2 qr/HOJ 0\L 2 `1 Sample Number or description Volume of sample filtered Type of column (ml) used and lot 454-108-256, 0 P PM , M ale, O ffsp rin g 454-108-259, 0 PPM , Fem ale, O ffspring 454-108-266, 0 P PM , M ale, O ffsp rin g 454-108-270, 0 P PM , M ale, O ffsp rin g 454-108-273, 0 PPM , Fem ale, O ffsp rin g 454-108-277, 0 P PM , Fem ale, O ffsp rin g 454-108-283, 0 P PM , M ale, O ffsp rin g 454-108-291, 0 P PM , M ale, O ffsp rin g 454-108-517,10 P PM , Fem ale, O ffsp rin g 454-108-522, 10 P PM , M ale, O ffsp rin g 454-108-525, 10 P PM , Fem ale, O ffsp rin g 454-108-533,10 P PM , M ale, O ffspring 454-108-538,10P P M , Fem ale, O ffsp rin g 454-108-545,10 P PM , M ale, O ffsp rin g 454-108-552, 10 P PM , M ale, O ffspring 454-108-557,10 PPM, Female, Offspring 454-108-561,10 PPM, Female, Offspring 454-108-568,10 PPM, Male, Offspring 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Method Revision: ETS-8-231.0 Study Number: E01-1245 Matrix: Sera Amount and spike Elution solvent mix used and volume Comments 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 /d o o L 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2 C7 w L 2m I of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2 -c tx X. 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 J 2m! of MeOH NA TN-A-5457 Q iO 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 NA 2ml of MeOH TN-A-5457 4 (t^ c ^ CO 2ml of MeOH NA TN-A-5457 V *-. 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 _ _ _____ -- -------- IAS o s j u L i __ ___ __- -k-fa zBlank Sera TN Amount of sera aliquated:. cm Dilute Sera with Kandiyohi water; Amount of water added S '/ A r GI 'V71 Aliquot 1ml of sera into 15 ml polypropelene tube ml ml ( N , / r/? O c id b iip Q p J o g L . Spike samples accordingly Add 5ml of ACN (TN-A- 4 ( 4 S J to aliquated sample ~"Shake sample for 20 min @300 rpm (Shaker^ 'k ^ O t* A 6QJuup^-5 aH u o4ec/ far Centrifuge sample for 10 min @ 2000 rpm (Centrifuge?/^ ~HSiG<3 ,,.Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. p fo \ ffo -i . Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixina Condition column with MeOH (TN-A- ) g Wash Column with Kandiyohi Water '^ F ilte r sample through conditioned column, discarding filtrate Q Of o . Allow column to go completelly dry S' Elute column with indicated amount of solvent ( TN-A- 5 r 5 f ' ^ into appropriate 11^5 ml centrifuge tube Spike samples with internal standard PFDA standard number O T - O c l - 4 f /<f}0 \f/*U iamount added 3M Environmental Laboratory Page 163 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite B o t t l e - T o p D isp e n se r D a il y C a l ib r a t io n C h e c k Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume: Solvent: ID#: V/ 0 M -oO . o Kj ^co "5 A C lO Mass of solvent (g) 1 i. 4 2 3 4' 4 1 3 3 34 4 5 3 .4 4 1 3 Density1 (g/ml) Volume of Solvent c.f& G lo.qg 5 ^ G .O ioQ S O \Q p ' o-VZSi o .q ksq G.OXdL*? *5. o ~3lL \ to.'qk!57- G .o & Y Average Volume: 5 .OiGo %Accuracy: Jo. 2 % Std. Dev.: . o o S X o Y %C.V.: o . 1"/O Pass/Fail: Z Z 3 Z E Z Z I Volume, (mL) Ito 10 Perform ance Specifications Accuracy, C.V., (%) 98.0 - 102 % (%) 1 .0 % Corrective Actions: JU eue 1Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx=specific gravity at ydegreesc referred to water at xdegree5 c n-Hexane d24 =0.660 Acetone d2S0 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 =1.3255 3M Environmental Laboratory Page 164 Report E01-1245 Prep Date: <P- ? 'o Q -k Analysts initials: O K H e il SPE Columns ExtracflSOftcAReprOduction S^y/W^CtheCNotteern/BObWhite Method Revision: O f - 6 '^ 2 / -I Study Number: , Lims Assigned sample number E01-1342-30312 E01-1342-30321 E01-1342-30330 E01-1342-30339 E 0 1-1342-30348 E01-1342-30357 E01-1342-30366 E01-1342-30375 E01-1342-30384 E01-1342-30393 E01-1342-30402 E01-1342-30411 E01-1342-30420 E 0 1-1342-30429 E01-1342-30438 E01-1342-30447 E01-1342-30456 E01-1342-30465 E01-1342-30474 E01-1342-30483 EOI-1342-30492 E01-1342-30501 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 ppm 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 ppm 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 a.i. 349 M Liver 0 ppm a.i. 350 F Liver Volume of sample Type of column used Amount and filtered (ml) and lot spike mix used \ \ \ \\' -------------- v \ r\ Elution solvent Amount of liver and volume weighed (g) ----------1Ch a. / ' 63 l& * i . aS3Pa (. vC q ---------- --- tr (.a z c 4 < f \,0` i0 ( o ip X 1. 3 r a < ---- \p q o k a "" -------- --T----- i- ( `Oc--p i q \ \ \ .m i 0 \.\ -- (,, ozs 'J 1'Cjp> } q Comments NJA \ l Blank Liver TN-A-__________ ; Amount of liver:__________ g Homogenize liver with Kandiyohi water; Amount of water added. Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-__________ ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker__________ ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge, J 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- Wash Column with Kandiyohi Water Filter sample through conditiopetfcolumn, discarding filtrate Allow column to go corpptefelly dry Elute column witfrlndicated amount of solvent (__________ TN-A- Spike samples with internal standard PFDA standard number, Transfer sample into appripriatelly marked autovial ml ij - o J into appropriate 15 ml centrifuge tube , cone.____________ , amount added 3M Environmental Laboratory OOP) % h p c J j s h K A J p j o t^ 1 o W f c 2- Page 165 Report E01-1245 PFOS: A Reproduction Study WifeithSiNothem Bobwhite SPE Columns Extraction Worksheet (A) a o f s j D2/i2 (oi Prep Date: J- 7 - ^ Analysts initials: ^^ ' Method Revision: Study Number: Matrix: ??/ /2 V ~Z^cy Urns Assigned sample number E01-1342-30510 E01-1342-30519 E01-1342-30528 E01-1342-30537 E01-1342-30546 E01-1342-30555 E01-1342-30564 E01-1342-30573 E01-1342-30582 E01-1342-30591 E01-1342-30600 E01-1342-30609 E01-1342-30618 E01-1342-30627 EO1-1342-30636 E01-1342-30645 E01-1342-30654 E01-1342-30663 E01-1342-30672 E01-1342-30681 E01-1342-30690 E 0 1-1342-30699 Sample Number Volume of sample Type of column used Amount and or description filtered (ml) and lot spike mix used 0 ppm a.i. 351 M Liver 0 ppm a.i. 352 F Liver 0 ppm a.i. 353 M Liver 0 ppm a.i. 354 F Liver 0 ppm a.i. 355 M Liver 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 0 ppm a.i. 360 F Liver 0 ppm a.i. 361 M Liver 0 ppm a.i. 362 F Liver 0 ppm a.i. 363 M Liver 0 ppm a.i. 364 F Liver 0 ppm a.i. 365 M Liver U ppm a.i. 366 h Liver U ppm a.i. 3 6 / M Liver U ppm a.i. 366 h Liver 1U ppm a.i. 369 M Liver 1Uppm a.i. 3 /u F Liver 1U ppm a.i. 3/1 M Liver \ \ \ \ V i> . r">''--'N \ \-----------T~. V ---------------------\ 10 ppm a.i. 372 F Liver Elution solvent Amount of liver and volume weighed (g) /. 0 6 U 6 C < j< l (. /-O JP99J. 1, D l i l ^ L03?2% 1. I . cM 1, O ^ fQ tfa ---------------- & ls 6 / G j l o s y -7 1 ------------------ 7-- i,0 C tC ^ A 074A Comments AJA Blank Liver TN-A- ; Amount of liver: Homogenize liver with Kandiyohi water; Amount of water added _ Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-_________ ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker_________ ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge. J 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-_ Wash Column with Kandiyohi Water Filter sample through conditioned-cbiumn, discarding filtrate Allow column to go corripletSliy dry Elute column wjlMhdicated amount of solvent (_________ TN-A-_ Spike samples with internal standard PFDA standard number nsfer sample into appripriatelly marked autovial ml -o, 9s cA J into appropriate 15 ml centrifuge tube _______, cone.___________ , amount added.. 3M Environmental Laboratory Page 166 Report E01-1245 Prep Date: i? ^ o-2 Analysts initials: O 'K I o -i. SPE Columns ExtracfiSPS/tAkRfPpduction S t ^ Wit^thtNothern BobW^hiite Pay- Method Revision: d > j - g l i t - I Study Number: c~a(- Matrix: Lims Assigned sample number E01-1342-30708 E01-1342-30717 E01-1342-30726 E01-1342-30735 E01-1342-30744 E01-1342-30753 E01-1342-30762 E01-1342-30771 E01-1342-30780 E01-1342-30789 E01-1342-30798 E01-1342-30820 E01-1342-30829 E01-1342-30838 E01-1342-30847 E01-1342-30856 E01-1342-30865 E01-1342-30874 E01-1342-30883 E01-1342-30892 E 0 1-1342-30901 E01-1342-30910 Sample Number Volume of sample Type of column used Amount and or description filtered (ml) and lot spike mix used 10 ppm a.i. 373 M Liver 10 ppm a.i. 374 F Liver 10 ppm a.i. 375 M Liver 10 ppm a.i. 376 F Liver 10 ppm a.i. 377 M Liver 10 ppm a.i. 378 F Liver 10 ppm a.i. 379 M Liver 10 ppm a.i. 380 F Liver 10 ppm a.i. 381 M Liver 10 ppm a.i. 382 F Liver 10 ppm a.i. 383 M Liver 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 10 ppm a.i. 388 1- Liver 1U ppm a.i. 389 M Liver 10 ppm a.i. 390 h Liver 10 ppm a.i. 391 M Liver 10 ppm a.i. 392 F Liver 10 ppm a.i. 393 M Liver ----------- -- 10 ppm a.i. 394 F Liver Elution solvent Amount of liver and volume weighed (g) /. _ l--. ic^oL*, I- lo 3>Ga Ii*oo< ^ L --------- 1----v-- (. D SO I a ----------- d ~ i-OiQ-Q. ------------- J-- io m ^ ic n ti* / OZiCc* l /.C 9 f 7e /- QC&e ; ic a z & i Comments M r\ \i Blank Liver TN-A- J 1h ^ : Amount oftiver: q 7XJ-A- S l t f l Homogenize liver with Kandiyohi water; Amount of water added 7-S ml f d l t>. Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-_________ ) to aliquated sample C f yo Jl fo P ' E , kU . 'a (,0 i P C k L t-t ( O ixJ Shake sample for 20 min @300 rpm (Shaker_________ ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge, J Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water C xd 5 - O Q, n f< lJ / ^ shake sample slightly to ensure proper mixing 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 indicated amount of solvent (_________ TN-A- _) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number. _______, cone. _________ , amount added Transfer sample into appripriatelly marked autovial (j P O ^ 5 L<cqP q iJ [C p i c, f a v u & ^ p / ?c=>(y ckj b^ T -U e t o Rj A U At<^ <^ T h i 'S & T L X T (lAA J - e h ^ / wu ? / ^ l nij i.o e L + c i X < lU . 0$ 3M Environmental Laboratory Page 167 Report E01-1245 Prep Date: 9 T Analysts initials: Q , 2 - 7 c :iM ^ PFOS: A Reproduction Studywith, the Nothern Bobwhite SPE Columns Extraction Worksheet 3^ "U u a il L iw l i t , . P tty H el S Method Revision: `2.^? I* ( Study Number: t V - t Z 4 r , . Lims Assigned sample number E01-1342-30919 E01-1342-30928 E01-1342-30937 E01-1342-30947g^-:' ' V ---------------------------------- -C1a%i. E01-1342-30955 E01-1342-30964 E01-1342-30973 E01-1342-30982 E01-1342-30991 E01-1342-31000 E01-1342-31009 E01-1342-31018 E01-1342-31027 E01-1342-31036 Sample Number or description 10 ppm a.i. 395 M Liver 10 ppm a.i. 396 F Liver 10 ppm a.i. 397 M Liver 10 ppm a.i. 398 F B fa in O 'U e R 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 Liver 10 ppm a.i. 404 F Liver 10 ppm a.i. 405 M Liver 10 ppm a.i. 406 F Liver 10 ppm a.i. 407 M Liver 10 ppm a.i. 408 F Liver Volume of sample Type of column used filtered (ml) \\ and lot \\ Yr i \ Amount and spike mix used Elution solvent Amount of liver and volume weighed (g) I'OW Ly /O Z -fa a ----------- 7 7 ----1. 0 ^ 0 ^ i cJ L oi l. o3GI^ I- OS? 6 <CJ/---/.. OOt ^ c -1 1,0173 3 i. gxqJ ------------ ZT----l Yi fo Comments tJA Blank Liver TN-A- Amount of liver:. Homogenize liver with Kandiyohi water; Amount of water added _ Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-__________ ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker__________ ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge Add 40 ml of Kandiyohi water to 50 ml polypropelene-Centrifuge tube. Decant extract into centrifuge tubes with wati shake sample slightly to ensure proper milting Condition column with MeOH (TN>Ai__________) Wash Column with KandiyghrWater Filter sample through pefiditioned column, discarding filtrate Allow column togdcompletelly dry Elute colurprfwith indicated amount of solvent (__________ TN-A- Spike. amples with internal standard PFDA standard number Tr; er sample into appripriatelly marked autovial 0UUU kf ____ 'kP ' J into appropriate 15 ml centrifuge tube _______ , cone.____________ , amount added_ 3M Environmental Laboratory 1 Page 168 Report E01-1245 Prep Date: Analysts initials: fO M-f ; ~7 PFOS: A Reproduction Studyrwith theNqjthem Bobwhite SPE Columns Extraction Worksheet Method Revision: ( r ? S- g '2 .3 /. / Study Number: 'S/. 12 ^S~ Urns Assigned sam ple number E01-1365-31184 E01-1365-31194 E01-1365-31199 E01-1365-31204 E01-1365-31209 E01-1365-31214 E01-1365-31219 E01-1365-31224 E01-1365-31229 E01-1365-31234 E01-1365-31240 E01-1365-31245 E01-1365-31250 E01-1365-31255 E01-1365-31260 E01-1365-31265 E01-1365-31270 E01-1365-31275 E01-1365-31280 E01-1365-31285 Sample Number or description 0 ppm a.i. 246 F Liver 0 ppm a.i. 253 M Liver 0 ppm a.i. 256 M Liver 0 ppm a.i. 259 F Liver 0 ppm a.i. 266 M Liver 0 ppm a.i. 270 M Liver 0 ppm a.i. 273 F Liver 0 ppm a.i. 277 F Liver 0 ppm a.i. 283 M Liver 0 ppm a.i. 291 M Liver 10 ppm a.i. 517 F Liver 10 ppm a.i. 522 M Liver 10 ppm a.i. 525 F Liver 10 ppm a.i. 533 M Liver 10 ppm a.i. 538 F Liver 1U ppm a.i. b4b M Liver 10 ppm a.i. 662 M Liver 10 ppm a.i. b b / h Liver 10 ppm a.i. b b l h Liver 1U ppm a.i. 068 M Liver Volum e o f sam ple Type o f colum n used Am ount and filtered (ml) and lot spike mix used \ \ \ \ \ \ \ \ \ YW v o\ \ \ Elution solvent A m ount o f liver and volume weighed (g) 2 7 ^ o 2 ( j O . b fC & j G 4434^ o 0 , 5 C?CO ------------T-- 0. 0, 0 . ( p G & Q : e^_ 0- --------- Of 0. G i v i . 0, 9 -- 1------ 'd O f l'S S lq ----------- d ~ ~ Comm ents JOA Vf Blank Liver TN-A- ; Amount of liver: Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver into 15 mi polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-_________ ) to aliquated sj Shake sample for 20 min @300 rpm (Shaker Centrifuge sample for 10 min @ 2000 rpm (pntrifuge Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes wifn water shake sample slightly to ensure ppJper mixing Condition column with MeOH (Trl-A-_________ ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (_________ TN-A- Spike samples with internal standard PFDA standard number______ Transfer sample into appripriatelly marked autovial , K ' q S, okj /b c u u f a c> aud 3M Environmental Laboratory i *=> 'h u e y - c a- t i S u t'^ L e S I' o . / y k A Q e r A S % J A * + A 4 - / :i b < 5 -3 a ' 2 O -4<g - J ' fc ^ L o '^ 3 ,, A - A*- c -foji <s Zj O -^3 - f.4 - -2 a l - i l /*- ' ------- - * V & fri J 2 '^ < ( ^ ) T H / > <?s r ro *L T W fd L / \J T & ; '/0 7 > / / t a o T U ' V . 4-<7-c - I into appropriaiBTCml centrifuge tube _____ cone _______ , amount added W iW on < 5 v % O f J . l^ U J o q P L U ^ iu . Page 169 Report E01-1245 SPE Columns Extrac^Sftfc)A ReProduction W&k,fheuNo^erB 1 ! $ ^ Prep Date: 2 /1 1 /2 0 0 2 Analysts initials: OK.HOJ ^ If-O Q QjeL Lims Assigned sample number Sample Number or description Volume of sample filtered (ml) EO1-1342-36000 E01-1342-36001 E01-1342-36002 E01-1342-36003 E01-1342-36004 E01-1342-36005 E01-1342-36006 E01-1342-36007 E01-1342-36008 E01-1342-36009 E01-1342-36010 E01-1342-36011 E01-1342-36012 E01-1342-36013 E01-1342-36014 E01-1342-36062 E01-1342-36015 E01-1342-36016 E01-1342-36060 E01-1342-36061 Blank H20-day 1 Blank female liver-day 1 Blank male liver-day 1 Female liver MS-day 1 Female liver MSD-day 1 Curve point-0.2 ppb in liver extract Curve point-0.5 ppb in liver extract Curve point-1 ppb in liver extract Curve point-2.5 ppb in liver extract Curve point-5 ppb in liver extract Curve point-10 ppb in liver extract Curve point-25 ppb in liver extract Curve point-50 ppb in liver extract Curve point-75 ppb in liver extract Curve point-100 ppb in liver extract Curve point-100 ppb-2 in liver extract Curve point-250 ppb in liver extract Curve point-500 ppb in liver extract Curve point-750 ppb in liver extract Curve point-1000 ppb in liver extract 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18, 1g, 6ccplot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1gp6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6ccplot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1gp6cc, lot W2025B1 u ie , ig, occ, ioi W2025B1 Method Revision: ETS-8-231.0 Study Number: E01-1342/1305- /24*~ Matrix: Liver Amount and spike mix used NA NA NA 50ul of 02001-66 50ul of 02001 -66 0.4ul of 02001-66 1ul of 02001-66 2ul of 02001-66 5ul of 02001-66 10ui of 02001-66 20ul of 02001-66 50ul of 02001-66 10Oul of 02001-66 150ul of 02001-66 200ul of 02001-66 4ul of 02001-53 10ui of 02001-53 20ul of 02001-53 30ul of 02001-53 40ul of 02001-53 Elution solvent and volume 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 zmi MeUti TN-A-5754 Comments }0A / T -fr* Blank Liver TN-A - * 5 l k 2> ; Amount oKdver: 5 _ 0 _ 3 S ^ _ g Blank Liver TN-A- ^ 1 ; Amount oA d v e r t ) Homogenize liver with Kandiyohi water; Amount of water added ^ ml Amount of water added ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN fTN-A- ~H H S 1to aliquated sample . Shake sample for 20 min @300 rpm (Shaker d m R j ^ / aJ 't Centrifuge sample for 10 min @ 2000 rpm (Centrifuge i t }&(1 G il? 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-^ ^ ^ ^ ) 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 (. H e C H TN-A- 5 ? S q J into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number I - 4 * 3 , cone, t o o - ? amount added Transfer sample into appripriatelly marked autovial . !\ f ) s) m r\ <^@9. B/u4i4e exkQQT>'<9u Qet^ OK. a 3M Environmental Laboratory Page 170 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite SPE Columns Extraction Worksheet 2 .^ 2 j ^ PrepDate: Analysts initials: o K l t f O ^ Method Revision: f l "b Study Number: c o / - f S ^ f _________________________________________________________________________ Matrix: .v Lims Assigned Sample Number Volume of sample lype of column Amount and spike tlutio n solvent sample number or description filtered (ml) used and lot mix used and volume Comments E01-1342-36017 E01-1342-36018 E01-1342-36019 E01-1342-36020 E01-1342-36021 E01-1342-36022 E01-1342-36023 E01-1342-36024 E01-1342-36025 E01-1342-30312 E01-1342-30321 E01-1342-30330 E01-1342-30339 E01-1342-30348 E01-1342-30357 E01-1342-30366 E01-1342-30375 E01-1342-30384 E01-1342-30393 QC@10 ppb-1 QC@10 ppb-2 QC@10 ppb-3 QC@75 ppb-1 QC@75 ppb-2 QC@75 ppb-3 QC@150 ppb-1 QC@150 ppb-2 QC@150 ppb-3 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 ppm a.i. 338 F Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1gp6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 4ul of 02001-52 4ul of 02001-52 4ul of 02001-52 30ul of 02001-52 30ul of 02001 -52 30ul of 02001-52 60ul of 02001-52 60ul of 02001 -52 60ul of 02001 -52 NA NA NA NA NA NA NA NA NA NA 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 ya I I -- - ---- --- -- ---- \c o Blank Liver TN-A- I ; Amount of liver: a Blank Liver TN-A- *5 <^ f-3 j ; Amount of liver: /- g Homogenize liver with Kandiyohi water; Amount of water added _ ml Amount of water added ^ ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- 1to aliquated sample Shake sample for 20 min @300 rpm (Shaker y /d d R j Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3H ^ ~ K \G fL> 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- ) 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- ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number O S x Q t ' t Transfer sample Into appripriatelly marked autovial ________ ,, ccoonec..jk; >o - J & r m . aammoouunnt aadadaeda_ M^ ! . . n s \-anf)(o(A ^A(S)/\kH?rf >~( O/, 3M Environmental Laboratory - Page 171 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Standard Curves--Solids Prep Date(s): Analyst(s): Sample Matrix: Method/Revision: T arget Analyte(s): FC Mix Std Approx. 1.00 ppm: FC Mix Std Approx. 5 02 ppm: FC Mix Std Approx. 50.2 ppm: Surr. Std Approx. 100.7 ppm: 02/11/02, 02/12/02, 02/13/02, 02/15/02 OK/HOJ Quail Liver ETS-8-231.1 PFOS 02001-66 02001-52 02001-53 02001-45 PFDA Actual Concentrations of Standards in the FC Mix Standard PFOS Number Std Conc. ug/mL E01-1342-36005 1.004 H01 -1342-36006 1.004 E01-1342-36007 1.004 E01-1342-36008 1.004 E01-1342-36009 1.004 E 0 1-1342-36010 1.004 E01-1342-3 6011 1.004 E01 -1342-36012 1 004 HO1-1342-36013 1.004 E01 -1342-36014 1.004 E0I-1342-36062 50.20 E01 -1342-36015 50.20 E 01-1342-36016 50.20 E01-1342-36060 50.20 E01-1342-36061 50.20 QC-10 ppb 5.020 QC-75 ppb 5.020 QC-150 ppb 5.020 Quail Liver-MS/MSD-Day 1 1.004 Quail Liver-MS/MSD-Day 2 1.004 Quail Liver-MS/MSD-Day 3 1.004 Quail Liver-MS/MSD-Day 4 1.004 PFOS Am't Spiked mL 0.0004 0.0010 0.0020 0.0050 0.0100 0.0200 0.0500 0.1000 0.1500 0.2000 00040 0.0100 0.0200 0.0300 0.0400 0.0040 0.0300 0.0600 0.0500 0.0500 0.0500 0.0500 Calculated Concentrations of Standards in the sample matrix Standard PFOS PFDA Number Std Conc. Std Conc. E01-1342-36005 ng/g ng/g 3.99 2001 E 0 1-1342-36006 E01-1342-36007 E01-1342-36008 E0I-1342-36009 9.98 2001 20.0 2001 49.9 2001 99.8 2001 E01-1342-36010 E01-1342-36011 200 2001 499 2001 E01-1342-36012 998 2001 E01-1342-36013 E01 -1342-36014 1496 2001 1995 2001 E 01- ]342-36062 1995 2001 E 0 1 - 1342-36015 E01-1342-36016 E01-I342-36060 E01-1342-36061 QC-10 ppb 4988 9977 14965 19953 200 2001 2001 2001 2001 2001 QC-75 ppb QC-150 ppb 1496 2993 2001 2001 Quail Liver-MS/MSD-Day 1 Quail Liver-MS/MSD-Day 2 499 2001 499 2001 Quail Liver-MS/MSD-Day 3 Quail Liver-MS/MSD-Dav 4 499 2001 499 2001 Calculated Concentrations of Standards in the final solvent Standard PFOS Number Std Conc m>/mL E 01-1342-3 6005 0.201 E01-1342-36006 0.502 E01-1342-36007 1.00 E01-1342-3 6008 2.51 E01-1342-3 6009 5.02 E 0 1-1342-36010 10.0 E01-1342-3 6011 25.1 E01-1342-36012 50.2 E01-1342-3 601T 75.3 E 0 1-1342-36014 100 E01-1342-36062 100 E01-1342-36015 251 E01-1342-3 6016 502 E01-1342-36060 753 E01-1342-36061 1004 QC-10 ppb 10.0 QC-75 ppb 75.3 QC-150 ppb 151 Quail Liver-MS/MSD-Day 1 25.1 Quail Liver-MS/MSD-Day 2 25.1 Quail Liver-MS/MSD-Day 3 25.1 Quail Liver-MS/MSD-Day 4 25.1 PFDA Std Conc. n/mL 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 PFDA Std Conc ue/mL 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 Study Number: E01-1245 Equipment N um ber: NA Final Solvent & TN Num ber: MeOH/TN-A-5754 Blank Tissue/Identifier: Quail Liver(female)/TN-A-5143 Quail Liver(male)/TN-A-5142 Initial female homogenate = 5.0353g/50.0353g+mL = 0.1006 g/mL Initial male homogenate = 1.0015g/10.0015g+mL = 0.1001 g/mL Blank liver initial homogenate (g/mL) for curves, MS, QC: 0 .1006 Final solvent volume (mL) for curves, MS, QC: 2.000 All Am't Spiked mL 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 ' All Initial Homogenate g/mL 0.1006 0.1006 0.1006 0 1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 0.1006 All Final Voi. 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 0.1001 C q\ u \A " K. YM v a '.j Verified by: I M h x x Page 172 Report E01-1245 1 PFOS: A Reproduction Study with the Nothern Bobwhite B o t t l e - T o p D isp e n se r D a il y C a l ib r a t io n C h e c k Manufacturer \ J C \ / l L and Model: Date: 0 7 - i / c 2 Initials: Dispenser ID: Balance ID: Y>o Volume: Solvent: ID #: Mass of solvent (g) Density1 (g/ml) Volume of Solvent 1 A `i s z r A? e ? e> 5 2 3 3 T sr ,r 4 3 1 rrr f C 344 5 3 - r s u _______ ______ "S 7___________ 5 . 6 3 c l ' Average Volume: r .e n - r %Accuracy: /O .~ E Std. Dev.: %C.V.: 0 .(9 3 7 1 Pass/Fail: )/ F Volum e, (m L ) 1 to 10 Perform ance Specifications A ccuracy, C .V ., (%) 98.0 - 102 % (%) 1.0% Corrective Actions: 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx=specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 =1.3255 3M Environmental Laboratory Page 173 Report E01-1245 SPE Columns Extractit^ f^ ' ^ Njhf0rp Bobwhite Prep Date: Analysts initials: 2/12/2002 p* OK/HOJ Lims Assigned sample number E01-1342-36026 Sample Number or description Blank H20-day 2 E01-1342-36027 Blank female liver-day 2 E01-1342-36028 Blank male liver-day 2 E01-1342-36029 Female liver MS-day 2 E01-1342-36030 Female liver MSD-day 2 E01-1342-30402 0 ppm a.i. 339 M Liver E01-1342-30411 0 ppm a.i. 340 F Liver E01-1342-30420 0 ppm a.i. 341 M Liver E01-1342-30429 0 ppm a.i. 342 F Liver E01-1342-30438 0 ppm a.i. 343 M Liver E01-1342-30447 E01-1342-30456 E01-1342-30465 E01-1342-30474 E01-1342-30483 E01-1342-30492 E01-1342-30501 0 ppm 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 a.i. 349 M Liver 0 ppm a.i. 350 F Liver E01-1342-30510 0 ppm a.i. 351 M Liver E01-1342-30519 0 ppm a.i. 352 F Liver E01-1342-30528 0 ppm a.i. 353 M Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C I8 ,1 g , bcc, lot W2025B1 u le, ig, occ, lot W2025B1 U18, 'Ig, bcc, lot W2025B1 u io, ig, occ, io i W2025B1 u io, ig, occ, io i W2025B1 o ie , ig, occ, lot W2025B1 i/io , ig, occ, io i W2025B1 o io, ig, occ, lot W2025B1 c. io, ig, occ, lot W2025B1 i/ io , ig, occ, lor W2025B1 U'lts, ig, occ, lot W2025B1 o io, ig, occ, io i W2025B1 o'io, ig, occ, io i W2025B1 o io , ig, occ,ioi W2025B1 W2025B1 Method Revision: ETS-8-231.0/ 2 6~ Study Number: E01 1342/1365^3 Matrix: Liver Amount and Elution solvent and spike mix used volume Comments NA NA NA 50ul of 02001-66 50ul of 02001-66 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 AM \l Blank Liver T N- A- ; Amount of liver: *9 - 0 2 S g Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver into 15 ml polypropelene tube Blank Liver TN-A- ^ ; Amount of liver: 4 *5 ml Amount of water added ml Spike samples accordingly Add 5ml of ACN (TN-A- ^ ) to aliquated sample . Shake sample for 20 min @300 rpm (Shaker G t ' - 'r Centrifuge sample for 10 min @ 2000 rpm (Centrifuged # ^ 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-. S ? s 9 ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (_H e Q H tn-a - ' S 7 ^ .) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 1~ cone. 1 0 1*1 amount added A i J l Transfer sample into appripriatelly marked autovial ow L ( tf- C P f* 3M Environmental Laboratory oi Page 174 Report E01-1245 SPE Columns N O /fe^ Bobwhite Prep Date: 2/12/2002 ^ Analysts initials: OK/HOJ o . n . '^ L _____________________________ Matrix: Method Revision: ETS-8-231.(p Study Number: E01-1642/1365 f Liver Lims Assigned sample number E01-1342-30537 E01-1342-30546 E01-1342-30555 E01-1342-30564 E01-1342-30573 E01-1342-30582 E01-1342-30591 E01-1342-30600 E01-1342-30609 E01-1342-30618 E01-1342-30627 E01-1342-30636 E01-1342-30645 E01-1342-30654 E01-1342-30663 Sample Number or description 0 ppm a.i. 354 F Liver 0 ppm a.i. 355 M Liver 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 0 ppm a.i. 360 F Liver 0 ppm a.i. 361 M Liver 0 ppm a.i. 362 F Liver 0 ppm a.i. 363 M Liver 0 ppm a.i. 364 F Liver 0 ppm a.i. 365 M Liver 0 ppm a.i. 366 F Liver 0 ppm a.i. 367 M Liver 0 ppm a.i. 368 F Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C 18,1gp6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6co, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 Amount and Elution solvent and spike mix used volume NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 Comments vA _______-- --- --------- - ___ ----------I M ceinhi __ ' ------------------- ----------------- ---------------- i ____________ Blank Liver TN-A- S '* T b Amount of liver: ^ Q3S 3 g Blank Liver TN-A-2 ^ 2 ; Amount of liver: q Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver into 15 ml polypropelene tube ___ ml Amount of water added ml Spike sam ples accordingly Add 5ml of ACN (TN-A- 4 * ^ * ^ ) to aliquated sample Shake sample for 20 min @300 rpm fShaker l< 3^^ Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 7 Q> I Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- M) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completely dry _. Elute column with indicated amount of solvent (_ M f O t f t n -a - S 7 ^ ' H Spike samples with internal standard PFDA standard number q 3 q o >- ) into appropriate 15 ml centrifuge tube cone. 10 3 ^ - ^ ' A /s amount added_ 4 Transfer sample into appripriatelly marked autovial O f< < r< f^ 3M Environmental Laboratory s??5 fb a o fe j j Page 175 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite B o t t l e - T o p D isp e n se r D a il y C a l ib r a t io n C h e c k Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume: *> ^ J L Solvent: AC fO ID#: Mass of solvent (g) 1 2 7>, l 3 o, q Q - i q 4 4 T>~,cn - S'oq'-i 5 s. q q i> 4 Density1 (g/ml) Volume of Solvent MjOL VJ Average Volume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: *5 .0 '5o"T 5 .o 5 o 1 5-O<3 O `5 . o i , q 5". o 3 |Oo. . o J l? 3 o.s-(gJ!.G (fP>F Volum e, (m L ) 1 to 10 Perform ance Specifications A ccuracy, C .V ., (% ) 98.0 - 102% (% ) 1.0% `Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx=specific gravity at ydcsrccsc referred to water at xdcBrees n-Hexane d204 -0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 =1.3255 3M Environmental Laboratory Page 176 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite SPE Columns Extraction Worksheet Vt) &ua.l 1 IAS P a ^ c l c f ) ^ dsW dz Prep Date: 2/13/2002 Analysts initials: OK/HOJ Lims Assgned sample number Sample Number or description E01 -1245-36153 E01-1245-36154 E01-1245-36155 E01-1245-36156 E01-1245-36157 E01-1245-36158 E01-1245-36159 E01-1245-36160 Blank-H20 Blank sera Sera MS-5ppb In sera extract-1 Sera MS-5ppb in sera extract-2 Sera MS-5ppb in sera extract-3 Sera MS-200ppb in sera extract-1 Sera MS-200ppb in sera extract-2 Sera MS-200ppb in sera extract-3 Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 Method Revision: ETS-8-231.0 Study Number: E01-1245 Matrix: Sera Elution Amount and spike solvent and mix used volume Comments NA NA 2ul of 02001-52 2ul of 02001-52 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2m! of MeOH TN-A-5457 IA 2ul of 02001-52 80ul of 02001-52 80ul of 02001-52 80ul of 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 - -----------------7 02 h i -- -- Blank Sera TN-A- Q) : Amount of sera aliquated: .m l Dilute Sera with Kandiyohi water; Amount of water added ~ 3 ^ ~ ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker woft- o c<\<pc\ Li Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3h # i Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-_^ 7 * 5 4 J Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent ( TN-A- ) into appropriate 15 ml centrifuge tubeA Spike samples with internal standard PFDA standard number Transfer sample into appripriatelly marked autovial t iv h acfrou dotvc __h_________ , cone. ^-l0 ` 7 f f u , T 0t Ofi. 3M Environmental Laboratory r \ i/. 9 V Page 177 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Bottle-Top Dispenser Daily Calibration Check 11 " t v ?t r 'c p v of= c ? p * - i (Si > N f f \ ( ~ Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR 5ml dispenser 2/13/2002 OK 0799 900 5 ACN TN-A-4145 c j Mass of solvent (g) Density' (g/ml) Volume of Solvent 1 3.965 0.7857 5.046 2 3.9715 0.7857 5.055 3 3.9818 0.7857 5.068 4 3.9484 0.7857 5.025 5 3.9788 0.7857 5.064 AverageVolume: 5.052 %Accuracy: 101.03 Std. Dev.: %C.V.: Pass/Fail: 0.0169 0.33 PASS Volume, (mL) 1 to 1 0 fO l.k 'A ' Per brm ance Specifica tions Accuracy, (%) 98.0 - 102 % Corrective Actions: U o Kj c.v., (%) 1 .0 % j 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx=specific gravity at ydcgrcesCreferred to water at xde8'eesC n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d2(l4 =1.3255 3M Environmental Laboratory Page 178 Report E01-1245 SPE Columns ExtractiopF SkA R fP Bobwhite Prep Date: Analysts initials: 2/13/2002 OK/HOJ Lims Assigned sample number E01-1342-36031 E0M 342-36032 E01-1342-36033 E01-1342-36034 E01-1342-36035 E01-1342-30672 E01-1342-30681 E01-1342-30690 E01-1342-30699 E01-1342-30708 E01-1342-30717 E01-1342-30726 E01-1342-30735 E01-1342-30744 E01-1342-30753 E01-1342-30762 E01-1342-30771 E01-1342-30780 E01-1342-30789 E01-1342-30798 Sample Number or description Blank H20-day 3 Blank female liver-day 3 Blank male liver-day 3 Female liver MS-day 3 Female liver MS D-day 3 10 ppm a.i. 369 M Liver 10 ppm a.i. 370 F Liver 10 ppm a.i. 371 M Liver 10 ppm a.i. 372 F Liver 10 ppm a.i. 373 M Liver 10 ppm a.i. 374 F Liver 10 ppm a.i. 375 M Liver 10 ppm a.i. 376 F Liver 10 ppm a.i. 377 M Liver 10 ppm a.i. 378 F Liver 10 ppm a.i. 379 M Liver 10 ppm a.i. 380 F Liver 10 ppm a.i. 381 M Liver 10 ppm a.i. 382 F Liver 10 ppm a.i. 383 M Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 18,1gp6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1gp6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C 18,1gp6cc, lot W2025B1 C 18,1gp6cc, lot W2025B1 C 18,1gp6cc, lot W2025B1 Method Revision: ETS-8-231.0 Study Number: E01 -1342/1365 /2 (> Matrix: Liver Amount and Elution solvent and spike mix used volume Comments NA NA NA 50ul of 02001-66 50ul of 02001-66 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 k>A f ?G Blank Liver TN-A- 5143 : Amount of liver: 5.0353a Blank Liver TN-A-_ 5142 : Amount of liver: 1,0015g Homogenize liver with Kandiyohi water; Amount of water added 43 .'S' aqs+?j / t o A Aliquot 1ml of liver into 15 ml polypropelene tube O V*-- , Z Amount of water added 9ml Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker Centrifuge sample for 10 min @ 2000 rpm (Centrifuge j # 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- ^ ^ J Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (. _ TN-A-_^__^_^_ M ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number O ^ Q C t S . cone. I a . amount added_ Transfer sample into appripriatelly marked autovial e r e n o w T > e w i? $02^ 3M Environmental Laboratory CP<g) ofC W & U Page 179 Report E01-1245 SPE Columns ExtractiopF S kA R eProdUcti0n'StUdyW lthAth%NOthern Bbwhite Prep Date: Analysts initials: 2/13/2002 OK/HOJ Method Revision: ETS-8-231.0 Study Number: E01-1342/1365 Matrix: Liver Llms Assigned sample number E01-1342-30820 E01-1342-30829 E01-1342-30838 E01-1342-30847 E01-1342-30856 E01-1342-30865 E01-1342-30874 E01-1342-30883 E01-1342-30892 E01-1342-30901 E01-1342-30910 E01-1342-30919 E01-1342-30928 E01-1342-30937 E01-1342-30946 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 10 ppm a.i. 388 F Liver 10 ppm a.i. 389 M Liver 10 ppm a.i. 390 F Liver 10 ppm a.i. 391 M Liver 10 ppm a.i. 392 F Liver 10 ppm a.i. 393 M Liver 10 ppm a.i. 394 F Liver 10 ppm a.i. 395 M Liver 10 ppm a.i. 396 F Liver 10 ppm a.i. 397 M Liver 10 ppm a.i. 398 F Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18,1g, 6cct lot W2025B1 C 18,1gp6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 Amount and Elution solvent and spike mix used volume NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 n NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 2ml MeOH NA TN-A-5754 n _ Comments hiA i __________ i__________ ----- ------------------- Us os/aaloi _ J __________ i__________ Blank Liver TN-A- 5143 : Amount of liver: 5.0353a Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver Into 15 ml polypropelene tube Blank Liver TN-A- 5142 : Amount of liver: 1.0015g 4 + 4 Amount of water added 9ml g-i 3 0 ^ Spike sam ples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker vAHfeu &|rO )pAt Centrifuge sample for 10 min @ 2000 rpm (Centrifuge^ P 7 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- 1 ^ ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry _ Elute column with indicated amount of solvent ( TN-A- Spike samples with internal standard PFDA standard number c Q o o \ ' >Into appropriate 15 ml centrifuge tube ______ , cone. , amount added. Transfer sample into apprlpriatelly marked autovlal C fe u e ^ oJ. ^ ^ 0 It-z fZ - 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 2/13/2002 p fc K 0799 900 5 ACN TN-A-4145 o -)23 o 2 J 01 1 2 3 4 5 Volume, (mL) 1 to 1 0 Mass of solvent (g) 3.965 3.9715 3.9818 3.9484 3.9788 Density1 (g/ml) 0.7857 0.7857 0.7857 0.7857 0.7857 AverageVolume: Volume of Solvent 5.046 5.055 5.068 5.025 5.064 5.052 %Accuracy: 101.03 Std. Dev.: %C.V.: Pass/Fail: 0.0169 0.33 PASS P e r `o r m a n ce S p ecifica tio n s Accuracy, (%) 98.0 - 102% Corrective Actions: C.V., (%) + 1 .0 % * o Kj 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx=specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d254 =0.786 6 Methylene Chloride d204 =1.3255 3M Environmental Laboratory Page 181 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Attachment D: Dilutions Summary Worksheet Study: E01-1245 ^ Dilution Date/Analyst: { S Box Number: E01-1245 Solvent/TN Number: MeOH/5457 W/IS PFDA Extraction Date/Analyst: 2/t3/02p^ ^ c S - f - c Q .j Matrix/Timepoint: Quail sera Sample Number Dilutions or Description 1/10 1/100 1/ 1/ 1/ 1/ 1/ Com m ents E01-1245-28480 E01-1245-28482 VA X X --- E01-1245-28484 X E01-1245-28486 X E01-1245-28488 X E01-1245-28490 X E01-1245-28492 X E01-1245-28494 E01-1245-28495 X X LAS 0, h i o X E01-1245-28497 X E01-1245-28499 X E01-1245-28501 X E01-1245-28503 X E01-1245-28505 X E01-1245-28507 X E01-1245-28509 X E01-1245-28510 X / E01-1245-28512 X E01-1245-28514 X E01-1245-28516 Notes: X / 1/100 dilution= lOul of sample +990ul of solvent (? ) All dilutions were made on day o f extraction or the day after analysis. o K ) 9 Verified By: 1 _ J ________ -4 / Form Completion Verified By: 3M Environmental Laboratory Page 182 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Attachment D: Dilutions Summary Worksheet Study: E01-1342 >1 * Dilution Date/Analyst: Box Number: E01-1342 (& & >j ' SoWent/TN Number: MeOH/5457 W/IS PFDA Extraction Date/Analyst: 2/13/02 Matrix/Timepoint: Quail liver Notes: 1/10 dilution = lOOul of sample + 900ul of solvent 1/100 dilution= lOul of sample +990ul of solvent Q All dilutions were made on day of extraction or the day after analysis. f k \ AForm Completion Verified By: 2^ 3M Environmental Laboratory Page 183 Report E01-1245 Prep Date: Analysts initials: 2/15/2002 OK/HOJ SPE Columns Extracti oRFOS^kAReproductjonStUdyjWi'thiihecN/Othern Bobwhite C# 0 o L a Method Revision: ETS-8-231.0 Study Number: E01-1342/1 Matrix: Liver * /J' Lims Assigned sample number E01-1342-36036 E01-1342-36037 E01-1342-36038 E01-1342-36039 E01-1342-36040 E01-1342-30955 E01-1342-30964 E01-1342-30973 E01-1342-30982 E01-1342-30991 E01-1342-31000 E01-1342-31009 E01-1342-31018 E01-1342-31027 E 0 1 - 1 3 4 2 - 3 1036 E 0 1 -1 3 6 5 -3 1 184 E 0 1 -1 3 6 5 -3 1 194 E 0 1 -1 3 6 5 -3 1 199 E01-1365-31204 E01-1365-31209 Sample Number or description Blank H20-day 4 Blank female liver-day 4 Blank male liver-day 4 Female liver MS-day 4 Female liver MS D-day 4 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 Liver 10 ppm a.i. 404 F Liver 10 ppm a.i. 405 M Liver 10 ppm a.i. 406 F Liver 10 ppm a.i. 407 M Liver 10 ppm a.i. 408 F Liver 0 ppm a.i. 246 F Liver 0 ppm a.i. 253 M Liver 0 ppm a.i. 256 M Liver 0 ppm a.i. 259 F Liver 0 ppm a.i. 266 M Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18, 1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C 18,1g, 6ccplot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1gp6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18p1g, 6ccplot W2025B1 C18, 1g. 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18p1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 Amount and Elution solvent and spike mix used volume NA NA NA 50ul of 02001-66 50ul of 02001-66 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 2ml MeOH TN-A-5754 Comments m -- Blank Liver TN-A- 5143 : Amount of liver: 5.0353a Blank Liver TN-A- 5142 : Amount of liver: 1.0015g Homogenize liver with Kandiyohi water; Amount of water added__ 45 ml_ Amount of water added _9ml A liquot 1m l o f liver into 15 m l polypropelene tube Spike sam ples accordingly Add 5ml of ACN (TN-A- H N t ? ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker VWR S/N 041694 ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 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 ^ *5 ) 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 ( r j ^ E r f TN-A- ^ _ ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number & 9 o o l~ S . cone. KX> amount added_ Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 184 Report E01-1245 SPE Columns ExtractioBFOSkAReproductionStudywithjthecNOhe/n Bobwhite Prep Date: Analysts initials: 2/15/2002 OK/HOJ Q .//~ Method Revision: EETTSS-8--82-3213.01 .0 ^ Study Number: E01- Matrix: Lims Assigned sample number E01-1365-31214 E01-1365-31219 E01-1365-31224 E01-1365-31229 E01-1365-31234 E01-1365-31240 E01-1365-31245 E01-1365-31250 E01-1365-31255 E01-1365-31260 E01-1365-31265 E01-1365-31270 E 01-1365-31275 E01-1365-31280 E01-1365-31285 Sample Number or description 0 ppm a.i. 270 M Liver 0 ppm a.i. 273 F Liver 0 ppm a.i. 277 F Liver 0 ppm a.i. 283 M Liver 0 ppm a.i. 291 M Liver 10 ppm a.i. 517 F Liver 10 ppm a.i. 522 M Liver 10 ppm a.i. 525 F Liver 10 ppm a.i. 533 M Liver 10 ppm a.i. 538 F Liver 10 ppm a.i. 545 M Liver 10 ppm a.i. 552 M Liver 10 ppm a.i. 557 F Liver 10 ppm a.i. 561 F Liver 10 ppm a.i. 568 M Liver Volume of sample filtered (ml) 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Type of column used and lot C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g, 6cc, lot W2025B1 C18,1g, 6ccplot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 Amount and Elution solvent and spike mix used volume NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 NA 2ml MeOH TN-A-5754 Comments WA LAs D ??iaa.loi ----------- t Blank Liver TN-A- 5143 : Amount of liver: 5,0353a Blank Liver TN-A- 5142 : Amount of liver: 1.0015g Homogenize liver with Kandiyohi water; Amount of water added.. 45 ml Amount of water added 9ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly ^ Add 5ml of ACN (TN-A-. J to aliquated sample Shake sample for 20 min @300 rpm (Shaker VWR S/N 041694 ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 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- 91 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 ( n e O t V TN-A- ^ ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number O o ? o Q l ~ ^ S ~ . . amount added, Z A Transfer sample into appripriatelly marked autovial fy fa c fi& u cP& JQ d fO 3M Environmental Laboratory Page 185 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 2/15/2002 OK 0799 900 5 ACN TN-A-4145 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 (g/ml) 3.8904 0.7857 3.9526 0.7857 3.9724 0.7857 3.961 0.7857 3.964 0.7857 AverageVolume: Volume of Solvent 4.9515 5.0307 5.0559 5.0414 5.0452 5.0249 %Accuracy: 100.50 Std. Dev.: %C.V.: Pass/Fail: 0.04202 0.84 PASS PASS PASS Perl orm ance Specifica tions Accuracy, (%) 98.0 - 102 % Corrective Actions: c.v., (%) + 1.0% 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx=specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d204=0.660 Acetone d250=0.788 Methanol d254=0.7866 3MEnvironmental 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 Attachment E: Study Protocol, Protocol A mendments, and Deviations 3M Environmental Laboratory 3M Environmental Laboratory Page 187 of 226 Page 187 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite W il d l ife In t e r n a t io n a l , 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.: 9 SPONSOR: 3M Corporation PROJECT NO.: 454-108, E 01-1245 EFFECTIVE DATE: January 24, 2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides the information necessary to complete the analytical phase o f this study. REASON: The protocol did not include information for completing the analyses required by the sponsor. AMENDMENT: Harold Johnson is the Principal Analytical Investigator REASON: The sponsor requested that a principal analytical investigator be added to the protocol. AMENDMENT: The 3M Environmental Laboratory Project Identification Number to be included with the study is E01-1245. REASON: The sponsor requested that the 3M Project Identification Number be included in the protocol. 3M Environmental Laboratory Page 1 o f 2 Page 188 -RpOr$*E01-124^^FAX 410 822 0632 wILDLlFlPF9&:iAARipNALUCti0n Study with the Nothern Bobwhite 3M Environmental Laboratory Page 189 Report E01-1245 W ildlife In t e r n a t io n a l , ltd PFOS: A Reproduction Study with the Nothern Bobwhite PROJECT NO.: 454-108, E01-1245 Sean P. Gallagher, Study Director Joann B. Beavers, W ildlife International, LTD Laboratory Management Harold O. Johnson, Principal Analytical Investigator W illiam K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date Date Date Date 3M Environmental Laboratory Page 2 o f 2 Page 190 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Protocol Amendment # 9 - Attachment A W ildlife 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: 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 A nalytical Investigator Study Locations A n a l y t ic a l Te s t in g La b o r a t o r y A n a l y t ic a l Te s t in g La b o r a t o r y Harold Johnson 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 651-778-6479 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. PO Box 33331 St. Paul, MN 55133-3331 PACE Tier 2 1700 Elm Street, Suite 200 Minneapolis, MN 55414 Proposed Study Tim etable A n a l y t ic a l E x p e r im e n t a l S t a r t D a t e A n a l y t ic a l Ex p e r im e n t a l Te r m in a t io n Da te 01/24/02 03/24/02 3M Environmental Laboratory 3M Environmental Laboratory 2 Page 192 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 1. Purpose/Objectives 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 toxicological/histopathological 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. Regulatory Compliance This analytical phase study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. QualityAssurance 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. Reference Substance 4.1 Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity and Stability--The purity of Lot #217 has been determined to be 86.9%. PFOS has been shown to be stable in methanol solutions for up to 6 months at ambient temperature in the validation report from study PS013, "Method Validation and Instrument Detection Limit Determination for LC/MS and GC/MS 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 5C or colder and is kept in the 3M Environmental Laboratory. 4.5 CAS Number--The CAS number is: 2795-39-3. 4.6 Molecular Weight--The molecular weight is 538 (CgFnSO^K). Note: The molecular weight of the anion (PFOS) is 499 (CgFnSOs). 3M Environmental Laboratory 3M Environmental Laboratory 3 Page 193 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 5. Control Matrices 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. Test System 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-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 o f the Selection o f 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 Test System The birds were obtained by Wildlife International, Ltd. See their protocol (study 454-108) for information about bird origination location. 8. Specimen and Sample Receipt and Maintenance Sera and liver were collected by Wildlife 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 from the following birds: 40 control adult birds (20 mating pairs) that ingested food containing 0 ppm PFOS 40 experimental adult birds (20 mating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppm PFOS Samples will be maintained frozen except when removed for extraction and analysis as described in the method. The samples will be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M Environmental Laboratory 4 Page 194 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 9. SUB-CONTRACTED ANALYSIS 9.1 All sera and liver extractions as detailed in this protocol amendment will be performed at Pace Tier 2, 1700 Elm Street, Suite 200, Minneapolis, MN 55414. 9.2 All sera and liver analyses as detailed in this protocol amendment will be performed at 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 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 M ethods 10.1.1 ETS-8-6, Extraction of Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.1.2 ETS-8-4, Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 A nalytical M ethods 10.2.1 ETS-8-7, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray / Mass Spectrometry 10.2.2 ETS-8-5, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry 11. Method Validation 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. 12. Data Quality Objectives During Sample Analysis The following criteria will be met using the validated calibration levels and fortification levels determined in section 11: 12.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample 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. C a lib ra tio n 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 (r2) 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. Lim its o f Q uantitation (LOQ) The lower limit of quantitation (LLOQ) was determined to be 10 ng/g in liver and 10 ng/mL serum during the method validation as described above in section 11. The LLOQ may be determined to be higher than that found in the validation study during a screening to be done for endogenous levels 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 determination will be recorded in a note to file and kept with the raw study data as well as being reported in the final report. Recovery efficiency at the LLOQ will be determined during each run using an extracted quality control sample spiked at the LLOQ level. If the quality control sample at the LLOQ level fails during a run, the LLOQ for that specific run would be considered to be the next lowest passing quality control sample and any samples below the new LLOQ must be reanalyzed. The upper limit 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 verify sample values. To perform the 3M Environmental Laboratory 3M Environmental Laboratory 6 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 post extraction (methanol extract adding additional methanol solvent). If the values are not within 15% of each other additional testing will be required to determine which value is a correct representation of the sample concentration. 12.4 Continuing Calibration Verifications An unextracted midlevel standard (continuing 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 20%. Data may only be reported if bracketed by passing CCV's (or a CCV and the same level calibration curve point). Data bracketed by one or more failing CCV's must be reanalyzed. 12.5 Q uality C ontrol Sam ples 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: 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 Perform ance 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 o f Confirm atory M ethods Confirmatory methods are typically not needed with LC/MS/MS analysis. 12.8 Dem onstration o f 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. 13. Sub-Contracted Analysis 13.1 All fluid and tissue extractions as detailed in this protocol amendment will be performed at Pace Tier 2, 1700 Elm Street, Suite 200, Minneapolis, MN 55414. 3M Environmental Laboratory 3MEnvironmental Laboratory 7 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 Statistical Evaluation of Data PSS-OPS-01 Use and Maintenance of Ultra-Turrax T25 Homogenizer PSS-OPS-02 Raw Data PSS-OPS-03 Hazardous Waste Disposal PSS-OPS-04 Use and Maintenance of N-Evap Analytical Evaporator PSS-OPS-05 Use of Compressed Gases in the Laboratory PSS-OPS-06 Use and Maintenance of NANOpure II Water Purification System PSS-OPS-07 Cleaning Non-disposable Volumetric Pipettes PSS-OPS-08 Laboratory Calculations PSS-OPS-09 Deviations from Established Procedures PSS-OPS-10 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 U s e and M aintenance o f V ortex Shakers PSS-OPS-24 Use and Maintenance of a Desiccator PSS-OPS-31 Use and Maintenance of Shakers PSS-OPS-32 Operation, Maintenance and Calibration of Laboratory PSS-OPS-37 Operation, Maintenance, and Calibration of pH Meters and pH Electrodes PSS-MC-01 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 Thermometers/Thermocouples PSS-MTR-05 Calibration of EppendorfPipettes PSS-MTR-06 Verification of Calibration of the EppendorfPipettor PSS-MTR-07 New Equipment Qualification (IQ, PQ, OQ) PSS-MTR-08 Verification of Calibration and Use of Analytical Balances 3M Environmental Laboratory 3M Environmental Laboratory 8 Page 198 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PSS-MTR-09 PSS-Safety-01 PSS-Safety-02 PSS-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. Statistical 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. Location of Ra w Data, Records, and 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 Environmental Laboratory, as applicable. 17. Specimen Retention Samples of the test substance (any leftover control sera or fiver 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 18.1 18.2 Wildlife International, Ltd. Protocol No. 454-108, "PFOS: A Reproduction Study with the Northern Bobwhite". Centre Analytical Laboratories, Inc. Study 023-054, "Validation of Analytical Methods "Extraction of Potassium Perfluoroocatanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC Electrospry/Mass 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 Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using FIPLC Electrospray/Mass 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. Protocol Amendments and 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 10 Page 200 Reportai=J245-JiAA 4 1 0 , 822 0632 W ILD LIF E BFOStNAfReproduction Study with the Nothpsn obwhite W i l d l i f e In t e r n a t i o n a l , l t d PROJECT NO.: 454-108, E01-1245 % AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/J20700/QR/SUB454 AMENDMENT NO .. 10 SPONSOR: 3M Corporation PROJECT NO.: 454-108, E01-1245 EFFECTIVE DATE: January 30, 2002 AMENDMENT: Tliis amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase o f this study. This attachment A replaces the attachment A in amendment 9. REASON. The amendment 9 attachment A was updated to include a different extraction and analytical method, to uppdate the data qc uality objectives, and to change the liver and sera extraction location. T u . i . ILL^. 1 o oV Sean P. Gallagher, Studyv Direcltor Date -- --------------------------- --- Joann B. Beavers, Wildlife International, LTD, Laboratory Management Harold O. Johnson, Pnhei^ai Analytical Investigator Date Date C .. William K. Reagen, 3M Environmental Laboratory, Labrate g^SHe'R'^Robdeai o r s Representative Date Date f0 Z j 3M Environmental Laboratory 1 of 9 Page 201 Report E01-1245 W il d l if e In t e r n a t io n a l , 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.: 454/120700/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, Laboratory Management Harold O. Johnson, Principal Analytical Investigator Date Date William K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date 3M Environmental Laboratory 1 of 9 Page 202 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 3M Protocol Amendment # 10 - 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, M N 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, M N 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: E 0 1-1245 W ildlife International Ltd.: 454-108 3 M Environm ental 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 55133-3331 P rin c ip a l A n a ly tic a l In v e s tig a to r S tu d y L o c a tio n s A n a l y t ic a l Te s t in g L a b o r a t o r y Harold Johnson 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 651-778-6479 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. PO Box 33331 St. Paul, MN 55133-3331 P ro p o s e d S tu d y T im e ta b le A n a l y t ic a l Ex p e r im e n t a l S t a r t D a t e A n a l y t ic a l E x p e r im e n t a l Te r m in a t io n Da te 01/31/02 03/31/02 3M Environmental Laboratory 3M Environmental Laboratory 3 of 9 Page 204 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 1. Purpose/Objectives 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 toxicological/histopathological 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. Regulatory Compliance This analytical phase study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. Q u a l i t y A s s u r a n c e The 3M Environmental Laboratory Quality Assurance Unit will audit analytical phase study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The Quality Assurance Unit will report all findings to the Sponsor Representative and the Study Director. 4. Reference Substance 4.1 Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity and Stability--The purity 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 LC/MS and GC/MS 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 5C or colder and is kept in the 3M Environmental Laboratory. 4.5 CAS Number--The CAS number is: 2795-39-3. 4.6 Molecular Weight--The molecular weight is 538 (CgFnSOsK). Note: The molecular weight of the anion (PFOS) is 499 (CgF17S03'). 3M Environmental Laboratory 3M Environmental Laboratory 4 of 9 Page 205 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite 5. Control Matrices 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. Test System 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 J u s tific a tio n o f the S election o f the Test S ystem 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 Test System The birds were obtained by Wildlife International, Ltd. See their protocol (study 454 108) for information about bird origination location. 8. Specimen and Sample Receipt and Maintenance Sera and liver were collected by Wildlife International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 21-week endpoint sera and liver samples received by the 3M Environmental Laboratory were from the following birds: 40 control adult birds (20 mating pairs) that ingested food containing 0 ppm PFOS 40 experimental adult birds (20 mating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppm PFOS Samples will be maintained frozen except when removed for extraction and analysis as described in the method. The samples will be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M Environmental Laboratory 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 P re p ara tory an d A n a ly tic a l M ethod 10.1.1 ETS-8-231 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices 11. Data Quality Objectives During Sample Analysis Method validation for PFOS quantitation in rat and mouse sera and liver is in process in the 3M Environmental Laboratory study E01-1277. 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 S u ita b ility 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 A bsolu te R ecovery 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 6 of 9 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 C alibration 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 Lim its o f Q uantitation (LOQ) The lower limit of quantitation (LLOQ) will be defined in liver (ng/g) and in serum (ng/mL) as the lowest acceptable extracted calibration curve point that is within the 70% -130% nominal criteria and with an analyte peak area at least 2 times the blank. The LLOQ determination will be recorded in a note to file and kept with the raw study data as well as being reported in the final report. The upper limit of quantitation (ULOQ) will be defined in liver and in serum as the highest acceptable extracted curve point that is within the 75% - 125% nominal criteria and includes applicable dilution factor corrections. 11.5 Q uality C ontrol 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: Low level: equivalent to an analyte concentration near or below the lowest level of analyte determined in samples Mid-level: equivalent to an analyte concentration near the middle of the sample range High level: equivalent to an analyte concentration near or above the highest level of analyte determined in samples 11.6 Q uality C ontrol Sample Acceptable Recoveries 3M Environmental Laboratory 3MEnvironmental Laboratory 7 of 9 Page 208 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 M atrix 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 B la n k s One control matrix and one water method blank will be extracted with each sample preparation. 11.9 C ontinuing C alibration 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. 11.10 Use o f C onfirm atory Methods Confirmatory methods are typically not needed with LC/MS/MS analysis. 11.11 Dem onstration o f S pecificity 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 12.1 All fluid and tissue extractions and analyses as detailed in this protocol amendment will be performed at the 3M Environmental Laboratory, Building 23E-09, 935 Bush Avenue, St. Paul, MN 55106. 12.2 An amendment to the protocol will be written if extractions and analyses are performed at laboratories other than the 3M Environmental Laboratory. 13. Statistical 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. 3M Environmental Laboratory 3MEnvironmental 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. Location of Ra w Data, Records, and 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 at the 3M Environmental Laboratory, as applicable. 16. Specimen Retention Samples of the test substance (any leftover control sera or liver and samples) and the analytical standards will be maintained in the laboratory for a period of time as specified by regulation or as long as the quality of the preparation affords evaluation. However, samples will not be maintained more than 10 years after the effective date of the final test rule (if applicable). 3M Environmental Laboratory Standard Operating Procedures also apply to sample retention times. 17. References 18.1 Wildlife International, Ltd. Protocol No. 454-108, "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 Amendments and Devi ations 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 W ildlife In t e r n a t io n a l , ltd PROJECT NO.: 454-108, E01-1245 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDYWITHTHENORTHERN BOBWHITE PROTOCOLNO.: 454/120700/QR/SUB454 SPONSOR: 3M Corporation AMENDMENTNO.: 11 PROJECTNO.: 454-108, E01-1245 EFFECTIVE DATE: March 27, 2002 AMENDMENT: Section 11.1, Sentence 4 Change: The peak area precision must be equal to or less than 2.0%RSD, the precision ofthe 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 ofthe retention time must be equal to or less than 2.5%RSD. REASON: Information received fromour instrument supplier indicates that a 2.0 %RSD for the area count is common to UV detector specifications. Mass spectrometers, which we used for this study, are more sensitive, in general andthe feet that analyte ionization can be suppressed by other species present inthe sample matrix and mobile phase make area reproducibility more challenging to maintain. Sample and source cleanliness are the determining factors, but < 2.0%is still a bit aggressive for mass spec. The application chemists at Micromass, the instrument vendor, suggest a value of <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 outside the 30%nominal requirement, may be included in the linear rangeto influence the lower end ofthe 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 ofthe lowest calibration curve point in some cases causes the next lowest calibration point to deviate by greater than 30%. Removal of this point in turncauses the next calibration point to deviate by greater than 30%. This continued deletion of calibration points in some cases could continue until there are very few points 3M Environmental Laboratory Page 1 o f 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 ofthe 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 calibration curves contain greater than nine (9) calibration points. The large number of points inthe curve is used to select a range representative to the samples. Limiting the removal of only four (4) points in some cases could cause a calibration range not representative ofthe 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 ofthe final copy. 3M Environmental Laboratory Page 2 o f 3 Page 212 Reportm r m 17 fax 4 i o 822 0 0 3 2 wildlife 0 ^ /0 1 /0 2 1 8 :2S PSD INF TECH 2 -2 E -0 1 -> *84108220915 Study with the Nothern7Bobwhite NO.905 P007 W teD U F E INTERNATIONAL. LTD :/ iS qtf!. Gallagher, Study _ . '-- i JoannfalBB . B e a v x , WWi iild life International, LTD ;l^dhbiatory M anagem ent ; I > I Harold O. Johnson, Principal Analytical Investigator iWitHam K_ Rcageo, 3M Environmental Laboratory, PROJECT N O .: 454-108, E01-1245 ov Date Date Dato ft 3M Environmental Laboratory Page 3 of 3 Page 213 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite W ildlife In t e r n a t io n a l , ltd PROJECT NO.: 454-108, E01-1245 Sean P. Gallagher, Study Director Joann B. Beavers, Wildlife International, LTD Laboratory Management arold O. Jo] rincipal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management Date Date Date ____--o?) ~ ^ ^-- Date 3M Environmental Laboratory Page 3 o f 3 Page 214 Report E01-1245 W ildlife In t e r n a t io n a l , 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.: 454/120700/QR/SUB454 AMENDMENT NO.: 12 SPONSOR: 3M Corporation PROJECTNO.: 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 E01-1365. REASON: Bobwhite samples were received in the 3MEnvironmental 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 W ildlife In t e r n a t io n a l , ltd Sean P. Gallagher, Study director Joann B. Beavers, Wildlife International, LTD, Laboratory Management Yt \ L f i iS iln isa A. Stevenson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management obideau, Sponsor's Representative PROJECT NO.: 454-108, E01-1245 Date Date - O sjk*L Date Date 3M Environmental Laboratory Page 2 of 2 Page 216 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite W ildlife In t e r n a t io n a l , 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 SPONSOR: 3M Corporation EFFECTIVE DATE: September 27, 2002 AMENDMENT NO.: 13 PROJECT NO.: 454-108, E01-1245 AMENDMENT: Change: Rochelle R. Robideau is the Sponsor Representative. To: Susan A. Beach is the Sponsor Representative. REASON: To change role of the Sponsor Representative. Sean P. Gallagher, Study Directo: Joann B. BeSters, W ild life International, LTD, Laboratory Management Q)(\& A. Lisa A. Stevenson, Princ ipal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management ( I - fiifi s J U Suran A. Beach, Sponsor's Representative Date D ate o q /a i/o a Date 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 I. Id e n tifica tio n Study / Project No. E O 1-1245 ....................... D eviation type ^ S O P ......... Method Equipment Procedure (Check one) Protocol Other: D ocum ent num ber ETS-4-4.0 D ate(s) o f occurrence 1/31/02, 2/1/02, 2/13/02, 2/7/02, 2/8/02, 2/11/02, 2 /1 2 /0 2 , 2 /1 3 /0 2 , 2 /1 5 /0 2 ________________ II. D escrip tion Required procedure/process: ETS-4-4.0 Internal chain o f custody The internal chain o f custody form to be used to track all m ovem ent o f sam ples W ithin the lab. The form should be filed w ith study data or other relevant sam ple docum ents. Actual procedure/process: Internal chains o f custodies w ere not used to track sam ples and sam ple extracts. Since receipt sam ples w ere Stored in freezer F I . A fter hom ogenizing liver and aliquating sera sam ples w ere stored in Freezer F8 U ntil extraction date. Sam ples rem ained frozen until extraction. D eviation to SOP written. III. A c tio n s Taken (such as amendment issued, SOP revision, etc.) Recorded by Authorized by D i r t t 'W ks Date D eviation No. I (assigned by.MStudy Directoorr or Project Lead at the end o f study or project) A ttachm ent A 3M Environmental Laboratory E T S -4 -8 .2 D ocum entation o f D eviations Page 5 of 1 Page 218 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation I. Id e n tifica tio n Study / Project No.E01-1245 Deviation Type (Check one) SOP Method Equipment Procedure VProtocol Other: Document Number:Protocol #: 454/120700/QR/SUB454, amendment#: 10 Date(s) of occurrence: :1/31/02, 2/1/02, 2/2/02, 2/7/02, 2/11/02, 2/12/02, 2/13/02, 2/14/02, 2/18/02, 4/11/02, 4/16/02 II. D e scrip tio n : 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. ....... III. A c tio n s Taken: (such as amendment issued, SOP revision, etc.) Issuing of this protocol deviation. Date IV. Im p act on S tu d y / P ro je c t There will be no impact on the study as the injections involved were used for system suitability and were within the acceptable precision ranges even though above the extracted calibration curve. Authorized By/(Stufly D irector// Project Lead) Date 3M Environmental Laboratory Form ETS-4-8.0 / j. a. a. r-- 1_- i U L J ' 3M Environmental Laboratory .> Deviation No. a (assigned by Study Director or Project Lead at the end o f study or project) AW W Page 219 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation 1. Id e n tifica tio n Study / Project No.E01-1245 Deviation Type (Check one) V SOP Method Equipment Procedure Protocol Other: Document Number:ETS-8-231.1 Date(s) of occurrence: 1/31/02, 2/1/02, 2/2/02, 2/7/02, 2/11/02, 2/12/02, 2/13/02, 2/14/02, 2/18/02, 4/11/02, 4/16/02 II. D e scrip tio n : 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 EO1-1342-36010, Prep sheet description "Curve Point - 1Oppb in liver", was prepped by spiking 20uL of 02001 -66 (l.OOug/mL) into 1 mL of liver homogenate. This homogenate consists of 5g liver plus 45mL water. Since lmL out of 50mL homogenate is used to prep each extracted standard, 1/50 of the 5g (0. lg) of liver is "spiked". After this homogenate is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... (20ul) (LOOugPFOS/mL MeOH) (l.OmL/lOOOuL) (lOOOng/l.Oug) = 20ng PFOS' onto SPE column 20ng PFOS' / 2.0mL MeOH = lOng PFOS'/mL MeOH. (Since this standard is recorded as lOppb, the correct units are lOngPFOS'/mL of MeOH.1 These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ng PFOS' / 1.OmL homogenate) (1 .OmL homogenate / 0.lg liver) = 200ng PFOS"/ g liver This relationship shows the factor of 20 used to "correct" the units when calculating the final result in ng/g from the chromatographic result that is in ng/mL. Sample Calculation: "E01-1342-30348, Oppma.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.OmL water, and lmL (10% of total liver weighed, since a liver density of 1.00 is assumed) is used for the entire SPE procedure, the calculation is as follows: (10.76ng PFOS-/ mL MeOH) (2mL MeOH / 0.1033 g liver) = 208.4 ng PFOS- / g liver Sample Calculation: Sample "E01-1342-36017, QC@10ppb-l": This QC sample is prepared by adding 4uL of 02001-53 (5.020ug PFOS" / mL MeOH) spiked into lmL liver homogenate. ((4ul) (5.020ug/mL) (lmL/lOOOuL) (1000ng/ug)) / (O.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 UWcAt*^ 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 221 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation /. Id e n tifica tio n Study / Project No. EO1-1245 Deviation Type (Check one) SOP Method Equipment Procedure V Protocol Other: Document Number: ETS-8-231.1 Date(s) of occurrence: Throughout the study II. D e scrip tio n : Required Procedure/process: Section 11.3, Sentence 5 states: Any level outside 75% - 125% of nominal value must be deactivated, and regression recalculated... _____ Actual Procedure/process:_____ ______ _ _____ Many of the high calibration points in this study are kept in the curve despite being 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. III. A c tio n s Taken: _________ (such as amendment issued, SOP revision, etc . ) ______ _ This deviation was written, a note to file was written, and data points which were above the next highest calibration point were reanalyzed. Recorded By Lisa A. Stevenson Ar/- /t -I /t> Authorized By (Study Director / Project Lead) ! Date I 08/16/02 ! ........ Ctf/jfcjoTL : Date O.rexW :_________ jp v n m ^ p r t iiiv U W '. M l ) /0 /0 2 J 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 4 _______ (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 /. Id e n tifica tio n Study / Project No. EOI-1245 Deviation Type (Check one) VSOP Method Equipment Procedure Protocol Other: Document Number: ETS-8-231.1 i Date(s) of occurrence: Entire Study II. D e scrip tio n : 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 E01-1345-35771, Prep sheet description "10 ppb sera curve point", was prepped by spiking 4uL of 02001-52 (5.020ug/mL) into 2 mL of pooled sera. This diluted sera consists of 2mL sera plus 38mL water. Since lmL out of 40mL diluted sera is used to prepare each extracted standard, 1/40 of the 2mL of sera is "spiked". After 1.0 mL of diluted sera is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. THEREFORE... (5.020ug PFOSVmL MeOH) * (4ul) * (1.0mL/1000uL) * (1000ng/l .Oug) = 20ng PFOS onto SPE column 20ng PFOS / 2.0mL MeOH = lOng PFOS /mL MeOH. (Since this standard is recorded as lOppb, the correct units are 10ng PFOSVmL MeOH.) These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ng PFOS /1 .OmL diluted sera) * (40.0mL diluted sera/ 2.0mL initial sera) = 400ng PFOS'/ mL Sera This relationship shows the factor of 40 (or 2.0mL MeOH/ 0.05mL Sera) used to "correct" the units when calculating the final result in ng PFOS/mL Sera from the chromatographic result that is in ng PFOS/mL MeOH. Sample Calculation: "E01-1245-28481,454-108-370,10 ppm 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 2&0mL water, and lmL is used for the entire SPE procedure, the calculation is as follows: H.O, o r a i/je ciilwfiv*' (@LAS lljacjo i (229.2ng PFOS-/ l.OmL MeOH) * (2.0mL MeOH/ 0.05mL Sera) = 9170ng PFOS- / mL Sera Sample Calculation: Sample " E01-1245-36155, Q C @ 1 0 0 p p b -l" : This Q C sample is prepared by adding 2 uL o f 02001-52 (5.02ug PFOS' / m L MeOH) spiked into lmL diluted sera (or 0.05 mL of initial sera). ((5.02ug/mL) * (2ul) * (lmL/lOOOuL) * (1000ng/ug)) / (0.05 mL Sera) = 200.8 ng PFOS' / mL Sera Using the earlier calculation, the analytical result of 6.17 ng/mL calculates to 246.8ng/mL Sera. Since the spike level is 200.8ng PFOS7 mL Sera, the recovery is (246.8/200.8)*( 100)= 122.9%................................... ........... ............. ... ..... ........... III. A c tio n s Taken: (such as amendment issued, SOP revision, etc.) This deviation was written and a note to file was written. Recorded By Lisa A. Stevenson J\ , Date 08/16/02 ej/iu lo i Authorized By (Study Director /P roject Lead) Date S k U Oatc-U'. W / ^ //A J 0> 3M Environmental Laboratory fP rtrm O 3MEnvironmental Laboratory Deviation No. ____________ /nccicrnpH h v S tn H v D ir p r f n r n r Prnie.r*t Tp.nH a t thf* p.nH n f stn H v n r n r n ip r t^ Page 223 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite Record of Deviation I. Id e n tifica tio n Study / Project No. E01-1245 Deviation Type (Check one) SOP ^ Method Equipment Procedure Protocol Other: Document Number: ETS-8-231.1 Date(s) of occurrence: Throughout the study II. D escrip tion: Required Procedure/process: Section 13.3 of the method states: Two thirds of all quality control samples and 1/2 of each quality control sample at each leve 1are expected to show an accuracy of 75-125%. Actual Procedure/process:.......................................... ..... ............ A11J25 ppb quail liver quality control sample recoveries were >125%. io (gjlAS 03Jilio3 ...... III. A c tio n s Taken: (such as amendment issued, SOP revision, etc.) This deviation was written, liver data reported at the low range of the curve may be biased high, and quality control data not within criteria are flagged in the raw data. .......... ........... Recorded By . Lisa A. Stevenson Authorized By (Study Director / Project Lead) Piacivi-: A jQl-,-- & LuloraW^ Woi(\cxgtrt\tr>V' U CJQ> / 2 . /< T' X - I Date 1 ; 08/26/02 %kuloX 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 I. Identification Study / Project N o.E01-1245 D eviation Type (Check one) v/ S O P C M ethod Equipm ent Procedure Protocol O ther: D o c u m e n t N u m b e r:E T S -8 -2 3 1.1 D ate(s) o f occurrence: 1/31/02, 2/1/02, 2/2/02, 2/7/02, 2/11/02. 2 /1 2 /0 2 , 2 /1 3 /0 2 , 2 /1 4 /0 2 , 2 /1 8 /0 2 , 4 /1 1 /0 2 , 4 /1 6 /0 2 __________ II. Description: Required Procedure/process: Section 12.1, C alculations: If other calculations are used than those listed, they w ill be docum ented in the raw data. Actual Procedure/process:............................................................................................................ 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 (1.00ug/mL) into 1 mL of liver homogenate. This homogenate consists of 5.0353g liver plus 45.0mL water. Since lmL out of 50.0353mL homogenate is used to prep each extracted standard, 1/50 of the 5.0353g (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) * (1.000 ug PFOS/mL MeOH) *(1.0 mL/1000 uL) * (1000 ng/1.0 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 lOppb, the correct units are 10.0 ng PFOS'/mL of MeOH.) These are the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20.0ng PF0S7 l.OmL homogenate) * (l.OmL homogenate / 0.100635 g liver) = 199ng PFOS7 g liver Sample Calculation: "E01-1342-30348, Oppm a.i. 333 M liver" Chromatographic result is 10.76 ng/mL; reported value is 209 ng/g. Since the 1.0338g 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 / 0.103032 g liver) = 209 ng PFOS- / g liver Sample Calculation: Sample "E01-1342-36017, OC@ I0ppb-1": 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/1000uL) * (1000ng/ug)) / (0.100635g liver) = 200 ng PFOS' / g liver Using the earlier calculation, the analytical result of 13.59ng/mL calculates to 270 ng/g liver. Since the spike level is 200ng PFOS / g liver, the recovery is (270/200) * (100)=135.0%. .......................................................................................................... III. Actions Taken: (such as amendment issued, SO P revision, etc.) Issuing o f this SO P deviation Recorded By D ate 12/ 01/02 A uthorized By (Study D irecto r / P roject L ead) D ate F orm E T S-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 Attachment F: Example Calculations Fo r m u la Used for S er a A n a ly s e s in Stu d y E 0 1 -1 2 4 5 AR (ng/mL MeOH) x DF x Dilution(diluted sera) = ng/mL Sera Calculation Used for E01-1245-28481,454-108-370,10 ppm Female, adult (initial volume = 0.3 mL) 229.24 ng/mL MeOH x 1 x 40 = 9170 ng/mL Sera Fo r m u l a U s e d fo r L iver A n a l y s e s in St u d y E 01-1245 AR (ng/mL MeOH) x DF x Final Volume (mL) = ng/g Liver amount of liver in diluted sample (g) Calculation Used for E01-1342-30672,10 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(diluted sera) = Final volume(mL)/amount of sera in diluted sample(mL) A m o un t of sera in diluted sam ple(m L) = 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: Am ount of sera in diluted sample(mL) = amount of sera used (0.3 mL)/total amount of sera + water (6 mL) = 0.05 mL Dllutlon(diluted sera)(mL) = final volume (2.0 mL)/amount of sera in diluted sample (0.05 mL) = 40 Am ount 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 /10.0 010 g = 1.00090 g Note: For sera samples with less than 0.05 mL sera received, additional water was added in order to remove 1.0 mL of diluted sera for extraction. For these samples, the dilution(diluted sera) value was updated to reflect the correct dilution. For example, if a sample was received with 0.02mL sera available, 0.98 mL of water was added, the amount of sera in diluted sample = 0.02 mL/1.0 mL = 0.02 mL. The dilution(diluted sera)(mL) = 2.0 mL/0.02 mL = 50 3M Environmental Laboratory 3M Environmental Laboratory Page 226 of 226 Page 226