Document 1585wBNyNbRpn8nexeGYb1bLq

Report EO1-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study Title Analytical Phase R e po r t for PFO S : A R e pr o d u c tio n Stu d y w ith T he No r th e r n Bo b w h ite Data Requirement F IF R A Subdivision E, Section 7 1-4 O E C D Guideline 206 Author Lisa A. Stevenson Analytical Phase Initiation Date January 3 1 ,2 0 0 2 Analytical Phase Completion Date April 18, 2 00 3 Performing Laboratory 3M Environmental Laboratory Building 2-3E -09 935 Bush Avenue St. Paul, M N 55106 Project Identification 3M Environmental Laboratory Study # E 01-1245 W ildlife International Ltd: 4 5 4 -1 0 8 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite GLP C o m p lia n c e S tatem 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 EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Q uality A ssurance Statement 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. In s p e c t io n D a t e s 10/08/01-10/09/01 02/01/02 04/03/02-04/08/02, 04/17/02 05/20/02-05/21/02 P hase Protocol In-phase Data Final Report D ate R epo rted to M anagement S tu d y D ir e c t o 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 Representafive O V fth J , Date 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 226 Page 3 0 0 0 9 .MG Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothera Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table of Contents GLP Compliance Statement............................................................................................................................. 2 Quality Assurance Statement...........................................................................................................................3 List of T ab le s ....................................................................................................................................................... 5 Study Information...............................................................................................................................................6 Sum m ary.............................................................................................................................................................. 7 Purpose................................................................................................................................................................ 8 Reference Substances...................................................................................................... ................................8 Specimen Receipt and Storage....................................................................................................................... 9 Method Sum m ary............................................................................................................................................... 9 Preparatory Method........................................................................................................................................... 9 Sample Collection and Analysis......................................................................................................................10 Analytical M ethod.................................................................................................................................................11 Analytical Results.................................................................................................................................................12 Data Summary.....................................................................................................................................................14 Statement of Conclusion....................................................................................................................................23 Statistical M ethods.............................................................................................................................................. 25 Statement of Data Quality................................................................................................................................ 25 R eferences.......................................................................................................................................................... 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 Compoundsfrom Biological Matrices"..........................................................28 Attachment B: Data Summary Tables........................................................................................................... 48 Attachment C: Sample Chromatograms,Calibration Information, and Instrument Information...........62 Attachment D: Sample Preparation S h eets................................................................................................. 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 COO' Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite List of Tables Table 1. Summary Table, C8F i7S 0 3' Concentration in Sera and Liver.......................................... 7 Table 2. Summary Table, CBF i7S 0 3` Concentration in Sera and Liver with Outliers R em o ved ......8 Table 3. Reference Substances......................................................................................................................8 Table 4. SPE Extraction Summary................................................................................................................. 9 Table 5. Sample Analysis................................................................................................................................. 10 Table 6. Serum QC Summary T ab le .............................................................................................................. 13 Table 7. Serum Matrix Spike Summary T a b le ........... :............................................................................... 13 Table 8. Liver QC Summary T ab le................................................................................................................ 13 Table 9. Liver Matrix Spike Summary T a b le .................................................................................................13 Table 10. C 8F i7S 0 3` Data Summary, Female Quail Sera (Adult and Offspring).................................. 15 Table 11. C8F17S 0 3` Data Summary, Male Quail Sera (Adult and Offspring).......................................17 Table 12. C8F17S 0 3` Data Summary, Female Quail Liver (Adult and Offspring).................................20 Table 13. CBF17S 0 3` Data Summary, Male Quail Liver (Adult and Offspring).......................................22 Table 14. Summary Table, C8F i7S 0 3' Concentration in Sera and Liver................................................24 Table 15. Summary Table, CBF i7S 0 3' Concentration In Sera and Liver with Outliers R em o ved... 25 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 226 Page 5 C n Q 'V y 'i Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Stu d y Info rm atio n 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 Sam ple Analysis Com pletion: 15 April 2 0 0 2 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 Report E01-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite S ummary The concentration of the C8F i7S 0 3` anion of perfluorooctane sulfonate, potassium salt (PFOS, C8F17S 0 3'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 C8F i7S 0 3' 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 0 3`. Concentrations of C8F17S 0 3' are reported after correction for the purity and the potassium contribution to the mass of the PFOS reference substance. Table 1 summarizes all study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Qixon Q-Test. Table 1. Summary Table, CeF^SCV Concentration In Sera and Liver S a m p l in g E vent N umber of Sera S amples A verage C 8F i 7S O / C onc. S era Range of Results N umber of L iver S amples A verage C bF ,7S O / C o n c . L iver Range of Results Male Control Group 19 <50.2 ng/g (19 of 20)* NA <400 ng/mL (19 of 19) 20 <50.2 ng/g* 209 ng/g (1 of 20)* Female Control Group 17 <400 ng/mL (16 of 17) NA 20 <50.2 ng/g* < 50.2n g'g(20o f20)* 2110ng/rinL(1 of 17) Male lOppm 20 141000 ng/mL 94400-226000 ng/mL 20 88500 ng/g* 50100-147000 ng/g* Group 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) l NA <400 ng/mL (4 of 4) 12600 ng/mL 8220-19200 ng/mL 12400 ng/rnL 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* 4 590-13900 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 Vz of QC at each level must be within 75% -1 2 5 % criteria for the method. Ail 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 2. Summary Table, C8Fi7S0 3' Concentration in Sera and Liver with Outliers Removed S a m p l in g E v e n t N umber of S era S amples A verage C bF , 7S 0 3` C ono. S era Male Control Group Female Control Group 19' 16 * <400ng/mL Male 10ppm Group 20' * Range of Results * N umber o f L iver S amples r 19 A verage c 8f ,7s o 3` C o n c . L iver <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 Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 17 8700 ng/mL 6* I 4* * * 5* * 5* * 4710-14700 ng/mL * * 20* 6* 4* 4 4 * * 5760 ng/g** 5490 ng/g'* * * * 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 Vi of QC at each level must be within 75% -1 2 5 % 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 C8F17S 0 3` in the liver and sera of both control and dosed Northern Bobwhite Quail and their offspring. Reference S ubstances Table 3. Reference Substances Reference S ubstance Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Purity PFOS 3M Internal 08/31/2006 Frozen -20C +/- 10C SD-018, Lot # 217 White Crystalline Powder 86.9% PFDA Oakwood Products, Inc. 12/01/2010 Frozen-20C+/-10C SD-036 White Solid 98% PFDA is nonadecafluorodecanoic acid, or Perfluorodecanoic acid, C ioF,80 2H 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite S pecim en R eceipt and Storage Northern Bobwhite serum and liver samples were obtained from the sacrifice of the test animals upon the conclusion of the in-life phase of the study (#454-108) conducted at Wildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. The specimens were received at 3M Environmental Laboratory on 09/05/01, 09/25/01, and 09/27/01; assigned a laboratory tracking (LIMS) number (E01-1245, E01-1342, and E01-1365, respectively), and were stored at -20C +/- 10C until extraction and analysis. M ethod Summary The determination of C8F17S 0 3' concentration in the liver and sera was performed according to 3M Environmental Laboratory method ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices". The sera samples were prepared by measuring a volume of serum and diluting with water in a 1/20 ratio. The liver samples were prepared by measuring a weight of liver and diluting with water to a 1/10 ratio prior to homogenizing. Five mL of acetonitrile was added to 1.0mL of the diluted sera or 1.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 0 3' using HPLC-ES/MS/MS. Preparatory M ethod ETS-8-231.1 "Solid Phase Extraction and Analysis of Fluorochemical Components from Biological matrices." This method describes the extraction of target analytes from biological matrices, such as sera and liver, using solid phase extraction (SPE). Sera samples were diluted with Kandiyohi (commercially bottled) water 1/20, before extraction. Liver samples were diluted 1/10, approximately 1g of liver and 9ml of Kandiyohi water, and then homogenized. A 1.0 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 S ample Preparation M easure 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 2 0 minutes, centrifuge for 10 minutes. Dilute supernatant with 4 0 ml of water Filter sample through conditioned S P E C18 column. Elute target analytes from the column with 2 .0 m L methanol (M eO H). 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 226 Page 9 Report E 01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite S am ple C o llectio 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 apd 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 D ate P erformed 31 January 2002 31 January 2002 3 Preparation of method blanks and spikes, and sera samples, control and 10 ppm adults, control offspring, and 10ppm offspring (day 2 sera samples), see Attachment D for samples propped 4 Analysis of d ay 2 sera samples 5 Analysis of 10ppm fem ale 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 10 Preparation of method blanks and spikes, and liver samples, control group (day 2 liver samples), see Attachment D for samples prepped 11 Analysis of day 2 liver samples 12 Preparation of method blanks and spikes, and liver samples, 10ppm group (day 3 liver s am p les), 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 0 7 ,0 8 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 Cf c o s i v i Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite S tep 16 17 18 19 20 P rocedure Preparation of method blanks and spikes, and liver samples, 10ppm group, control offspring, 10ppm offspring (day 4 liver samples), see Attachment D for samples propped Analysis of day 4 liver samples Re-analysis of day 3 liver samples Re-analysis of Q C samples Re-analysis of Q C samples Date Performed 15 February 2002 18 February 2002 01 March 2002 05 April 2 002 10 April 2 002 A nalytical M ethod Samples were analyzed using HPLC-ES/MS/MS. C8F17S 0 3' concentrations were determined using matrix-extracted calibration standards ranging in concentration from 0.25-1000 ng C 8F17S 0 37mL methanol. This result was then used to back calculate the ng C BF i7 S 0 37mL sera or ng C8F17S 0 37gram liver as appropriate. An Internal standard, Nonadecafluorodecanoic acid (PFDA), was used to monitor instrument performance for gross malfunction, but was not used to quantitate results. HPLC-EC/MS/M S 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 im e , M in u tes 0 1.0 5 .5 7 .5 8 10 % 2 m M A m m o n iu m A cetate 90 90 5 5 90 90 % M ethanol 10 10 95 I 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/M S mode, passing the C8F17S 0 3` molecular anion (m/z 499) into the collision cell, where it is collided with inert gas to further fragment to the characteristic ion m/z 99, F S 0 3\ Similarly the PFDA transition 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 E 01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite A nalytical R esults Data quality objectives outlined in the 3M Environmental Laboratory method and protocol were met, except as noted and discussed below (see Attachment A, ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" and Attachment E, Study Protocol, Protocol Amendments, and Deviations). Regressions. Quadratic curve fit, weighted 1/x, was applied to calibration standards and sample data. Each calibration curve had a coefficient of determination of > 0.985. Calibration Standards. Instrument response was calibrated daily prior to sample analysis using matrix-extracted standards ranging in concentration in the final solvent extract from 0.20 to 1000 ng C8F17S 0 37mL methanol, corresponding to a concentration of 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% d evia tio n of th e ta rg e t concentrations. O n ly d a ta b rac ke ted by p assing C C V 's w e re repo rted. Samples bracketed by one or more falling 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 EO1-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 reanalysls 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% Q C -75ppb* 1 5 0 0 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 Diluled 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 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) 3M Environmental Laboratory 3M Environmental Laboratory Page 13 of 226 Page 13 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 C8F17S 0 3 anion of perfluorooctane sulfonate, potassium salt (PFOS) that was adjusted for purity and the potassium ion contribution to the mass of PFOS used to prepare stock solutions. Any future calculations requiring concentrations to be expressed as a percentage of dietary in-take must adjust the dosed amounts as well. D ata S ummary Tables 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The tables display C8F17S 0 3' 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 EO1-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 10. CBF17SO3' Data Summary, Female Quail Sera (Adult and Offspring) 3M Sam ple N um ber S a m p l e D e s c r ip t io n , E 0 1 -1245-28444 454-1 0 8 -3 3 0 , Oppm female, adult, 01-31-02 , E01 -12 4 5 -2 84 4 6 454-1 0 8 -3 3 2 , Oppm female, adult, 01-31-02 , E01-12 4 5 -2 84 4 9 4 5 4 -1 0 8 -3 3 6 , Oppm female, adult, 01-31-02 , E 01-1245-28451 454-1 0 8 -3 3 8 , Oppm female, adult, 01-31-02 , E 0 1 -1245-28453 4 5 4-108-340, Oppm female, adult, 01-31-02 , E 0 1 -1245-28456 4 5 4-108-344, Oppm female, adult, 01-31-02 , E 0 1 -1245-28458 4 5 4-108-346, Oppm female, adult, 02-01-02 ,, E 0 1 -1245-28460 4 5 4-108-348, Oppm female, adult, 02-01-02 , E 0 1 -12 4 5-28462 454 -1 0 8 -3 5 0 , Oppm female, adult, 02-01-02 . E 01-1245-28464 454 -1 0 8 -3 5 2 , Oppm female, adult, 02-01-02 ,, E 01-1245-28466 454-1 0 8 -3 5 4 , Oppm female, adult, 02-01-02 ,, E 01-12 4 5 -2 84 6 8 4 54-108-356, Oppm female, adult, 02-01-02 . E 0 1 -1245-28470 4 5 4 -1 0 8 -3 5 8 , Oppm female, adult, 02-01-02 . E 0 1 -1245-28472 454-1 0 8 -3 6 0 , Oppm female, adult, 02-01-02 , E 0 1 -12 4 5 -2 84 7 4 454 -1 0 8 -3 6 2 , Oppm female, adult, 02-01-02 . E 0 1 -1245-28477 4 5 4-108-366, Oppm female, adult, 02-01-02 . E 0 1 -1245-28479 4 5 4 -1 0 8 -3 6 8 , Oppm female, adult, 02-01-02 E 01-1245-28481 4 5 4 -1 0 8 -3 7 0 ,10ppm female, adult, 02-01-02 E 0 1 -12 4 5-28483 4 5 4 -1 0 8 -3 7 2 ,1 0ppm female, adult, 02-01-02 E 01-1245-28485 4 5 4 -1 0 8 -3 7 4 ,10ppm female, adult, 02-01-02 E 01-1 2 4 5 -2 84 8 7 4 5 4 -1 0 8 -3 7 6 ,10ppm female, adult, 02-01-02 E 0 1 -1 2 4 5 -2 84 8 9 4 5 4 -1 0 8 -3 7 8 ,1 0ppm female, adult, 02-01-02 E 01-1245-28491 4 5 4 -1 0 8 -3 8 0 ,1 0ppm female, adult, 02-01-02 A M O U N T OF SERA USED (M L) 0.4 0.4 0.05 0.05 0.3 1 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 D IL U T IO N C o n g , of C 8F , 7S 0 3' in S am ple (n g /m L of S era) AVERAGE O f DUFF 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 2110* 1 2180 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 C tW X X . Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 AMOUNT OF SERA USED (M l) ADDITIO NAL D IL U T IO N CONC. OF C 8F )7S O ;, ' in S a m p le (n g /m L of S era) AVERAGE OF DUP. SAMPLES E 0 1 -12 4 5-28493 4 5 4 -1 0 8 -3 8 2 ,10ppm fem ale, adult, 02-01-02 E 01-12 4 5-28496 4 5 4 -1 0 8 -3 8 6 ,10ppm female, adult, 02-01-02 E 01-12 4 5-28498 4 5 4 -1 0 8 -3 8 8 ,10ppm female, adult, 02-01-02 E01-12 4 5-28500 4 5 4 -1 0 8 -3 9 0 ,10ppm female, adult, 02-01-02 E 01-1245-28502 4 5 4 -1 0 8 -3 9 2 ,10ppm female, adult, 02-01-02 E 01-12 4 5-28504 4 5 4 -1 0 8 -3 9 4 ,10ppm female, adult, 02-01-02 E 01-12 4 5-28506 4 5 4 -1 0 8 -3 9 6 ,10ppm fem ale, adult, 02-01-02 E01-1245-28508 4 5 4 -1 0 8 -3 9 8 ,10ppm female, adult, 02-01-02 E 0 1 -1 24 5 -2 8 51 1 4 5 4 -1 0 8 -4 0 2 ,10ppm female, adult, 02-01-02 E 01-12 4 5 -2 85 1 3 4 5 4 -1 0 8 -4 0 4 ,10ppm fem ale, adult, 02-01-02 E 01-1245-28515 4 5 4 -1 0 8 -4 0 6 ,1 0ppm fem ale, adult, 02-01-02 E 01-1245-28517 4 5 4 -1 0 8 -4 0 8 ,10ppm female, adult, 02-01-02 , E01 -1245-28518 4 5 4-108-246, Oppm female, offspring, 02-01-02 ,, E01-1245-28521 454-1 0 8 -2 5 9 , Oppm female, offspring, 02-01-02 , E 0 1 -1245-28524 4 5 4-108-273, Oppm female, offspring, 02-01-02 , E 0 1 -1245-28525 4 5 4-108-277, Oppm female, offspring, 02-01-02 , E 01-1245-28528 4 5 4 -1 0 8 -5 1 7 ,10ppm female, offspring, 02-01-02 , E 0 1 -1245-28530 4 5 4 -1 0 8 -5 2 5 ,10ppm fem ale, offspring, 02-01-02 , E 01-12 4 5-28532 4 5 4 -1 0 8 -5 3 8 ,1 0ppm fem ale, offspring, 02-01-02 . E01 -1245-28535 4 5 4 -1 0 8 -5 5 7 ,1 0ppm fem ale, offspring, 02-01-02 E 01-1245-28536 4 5 4 -1 0 8 -5 6 1 ,1 0ppm female, offspring, 02-01-02 * 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 10 31900 1 14700 1 11600 1 6310 1 9400 1 11400 1 1 4710 1 10500 1 6720 1 9060 1 8980 1 7840 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 1 18500 17600 1 16600 1 12800 12500 1 12100 1 14600 13400 1 12200 1 8330 7900 1 7460 1 11500 10800 1 10100 * There are two results reported for these samples. Both values are included and averaged in the "Average of Dup Sample" column. These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. 3M Environmental Laboratory 3M Environmental Laboratory Page 16 of 226 f; Page 16 y Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 11. CsFiySCV Data Summary, Male Quail Sera (Adult and Offspring) 3M Sam ple N um ber ; 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 , E01-12 4 5-28447 454-108-333, Oppm male, adult, 01-31-02 , E 0 1 -1245-28448 454-108-335, Oppm male, adult, 01-31-02 , E 0 1 -1245-28450 454-108-337, Oppm male, adult, 01-31-02 . E 0 1 -1245-28452 454-108-339, Oppm male, adult, 01-31-02 , E 01-1245-28454 4 54-108-341, Oppm male, adult, 01-31-02 . E01-12 4 5-28455 454-108-343, Oppm male, adult, 01-31-02 , E01-1245-28457 454-108-345, Oppm male, adult, 01-31-02 , E 0 1 -1245-28459 4 5 4-108-347, Oppm male, adult, 02-01-02 , E 0 1-1245-28461 454-108-349, Oppm male, adult, 02-01-02 . E 01-1245-28465 4 54-108-353, Oppm male, adult, 02-01-02 . E 01-1245-28467 454-108-355, Oppm male, adult, 02-01-02 . E 0 1 -1245-28469 454-108-357, Oppm male, adult, 02-01-02 , E 01-1245-28471 454-108-359, Oppm male, adult, 02-01-02 , E 0 1 -1245-28473 4 54-108-361, Oppm male, adult, 02-01 -02 . E 0 1 -1245-28475 454-108-363, Oppm male, adult, 02-01-02 . E 0 1 -1245-28476 4 54-108-365, Oppm male, adult, 0 2 -01-02 E 0 1 -1245-28478 4 5 4-108-367, Oppm male, adult, 02-01-02 AMOUNT 0 : SERA USED (M L) 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 0.02 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 ADDITIO NAL D IL U T IO N CONC. Oi C 8F ,7S 0 3 IN S a m p l e (n g /m L of S era) 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 C0 1 0 0 0 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ! 3 M S a m p i e: N umber S a m p l e D e s c r ip t io n AMOUNT OF SERA USED (M L) E 0 1 -12 4 5-28480 4 5 4 -1 0 8 -3 6 9 ,10ppm male, adult, d100,02-01-02 . E 0 1 -1245-28482 4 5 4 -1 0 8 -3 7 1 ,10ppm male, adult, dlOO,02-01-02 ,, E 01-1245-28484 4 5 4 -1 0 8 -3 7 3 ,10ppm male, adult, d100,02-01-02 . E 01-1245-28486 4 5 4 -1 0 8 -3 7 5 ,10ppm male, adult, d100,02-01-02 . E 01-1245-28488 4 5 4 -1 0 8 -3 7 7 ,10ppm male, adult, d100,02-01-02 , E 01-1245-28490 4 5 4 -1 0 8 -3 7 9 ,10ppm male, adult, d100,02-01-02 , E 0 1 -1245-28492 4 5 4 -1 0 8 -3 8 1 ,10ppm male, adult, d100,02-01-02 , E01-1245-28494 4 5 4 -1 0 8 -3 8 3 ,10ppm male, adult, d100,02-01-02 ,, E 0 1 -1245-28495 4 5 4 -1 0 8 -3 8 5 ,10ppm male, adult, d100,02-01-02 , E 0 1 -1245-28497 4 5 4 -1 0 8 -3 8 7 ,10ppm male, adult, d100,02-01-02 , E 01-1245-28499 4 5 4 -1 0 8 -3 8 9 ,10ppm male, adult, d100,02-01-02 . E01-1245-28501 4 5 4 -1 0 8 -3 9 1 ,10ppm male, adult, d100,02-01-02 . E 01-1245-28503 4 5 4 -1 0 8 -3 9 3 ,10ppm male, adult, d100,02-01 -02 . E 0 1 -1245-28505 454-108-395, 10ppm male, adult, d 100,02-01-02 . E 01-1245-28507 4 5 4 -1 0 8 -3 9 7 ,10ppm male, adult, d100,02-01-02 , E 0 1 -12 4 5-28509 4 5 4 -1 0 8 -3 9 9 ,10ppm male, adult, d100,02-01-02 . E 01-1245-28510 4 5 4 -1 0 8 -4 0 1 ,10ppm male, adult, d100,02-01-02 , E 01-1245-28512 4 5 4 -1 0 8 -4 0 3 ,10ppm male, adult, d100,02-01-02 , E 0 1-1245-28514 454-108-405, 10ppm male, adult, d100,02-01-02 E 0 1 -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 AUDI i IONAI DILU I ION CONC. O " C 8F 17S 0 3"in S a m p l e (n g /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 COX'. Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M S am ple N umber S a m p _e D e s c r ip t io n , E 01-12 4 5 -2 85 1 9 454-1 0 8 -2 5 3 , Oppm male, offspring, 02-01-02 , E01-1 2 4 5 -2 85 2 0 454-1 0 8 -2 5 6 , Oppm male, offspring, 02-01-02 . E 01-1 24 5 -2 8 52 2 454-1 0 8 -2 6 6 , Oppm male, offspring, 02-01-02 , E 01-12 4 5 -2 85 2 3 4 54-108-270, Oppm male, offspring, 02-01-02 . E 01-12 4 5 -2 85 2 6 454-1 0 8 -2 8 3 , Oppm male, offspring, 02-01-02 , E01 -12 4 5-28527 454-1 0 8 -2 9 1 , Oppm male, offspring, 02-01-02 AMOUNT OF SERA USED (M L) 0.02 0.02 0.02 0.02 0.07 0.07 0.2 0.2 0.02 0.02 0.05 0.05 ADD TIONAI D ll UTTOIJ CONC. OF C 8F u S 0 3- in S a m p l e (n g /m L of S era) 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 E 01-1245-28529 4 5 4 -1 0 8 -5 2 2 ,10ppm male, offspring, 02-01-02 0.07 10 19300 , E 0 1 -1245-28531 4 5 4 -1 0 8 -5 3 3 ,10ppm male, offspring, 02-01-02 0.07 0.07 1 1 9310 8220 0.04 1 15500 , E 0 1-1245-28533 4 5 4 -1 0 8 -5 4 5 ,10ppm male, offspring, 02-01-02 0.04 1 13600 0.07 1 13600 , E 0 1 -12 4 5-28534 4 5 4 -1 0 8 -5 5 2 ,10ppm male, offspring, 02-01-02 0.07 1 11500 * E 01-1245-28537 4 5 4 -1 0 8 -5 6 8 ,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. 19300 8760 14600 12500 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 12. CaFnSCV Data Summary, Female Quail Liver (Adult and Offspring) 3M S am ple N umber S a m p l e D e s c r ip t io n E 01-1342-30321 0 ppm a.i. 330 F Liver E01-1 3 4 2 -3 0 3 3 9 0 ppm a.i. 332 F Liver E 0 1 -1 342-30357 0 ppm a.i. 334 F Liver E 01-13 4 2 -3 03 7 5 0 ppm a.i. 336 F Liver E 01-1 3 4 2 -3 03 9 3 0 ppm a.i. 338 F Liver E 0 1 -1342-30411 0 ppm a.i. 340 F Liver E 0 1 -13 4 2 -3 04 2 9 0 ppm a.i. 342 F Liver E01 -13 4 2 -3 04 4 7 0 ppm a.i. 3 4 4 F Liver E 01-1 3 4 2 -3 04 6 5 0 ppm a.i. 346 F Liver E 01-13 4 2 -3 04 8 3 0 ppm a.i. 348 F Liver E 0 1 -1342-30501 0 ppm a.i. 350 F Liver E 0 1 -13 4 2 -3 05 1 9 0 ppm a.i. 352 F Liver E 0 1 -13 4 2 -3 05 3 7 0 ppm a.i. 3 5 4 F Liver E 01-13 4 2 -3 05 5 5 0 ppm a.i. 3 56 F Liver E 01-13 4 2 -3 05 7 3 0 ppm a.i. 3 58 F Liver E 0 1 -1342-30591 0 ppm a.i. 3 6 0 F Liver E 0 1 -1342-30609 0 ppm a.i. 362 F Liver E 0 1 -13 4 2-30627 0 ppm a.i. 3 64 F Liver E 0 1 -1342-30645 0 ppm a.i. 3 66 F Liver E 01-13 4 2-30663 0 ppm a.i. 3 68 F Liver E01-1342-30681 E01-1342-30699 E01-1342-30717 E01-1342-30735 E01-1342-30753 E01-1342-30771 E01 -1342-30789 E01 -1342-30820 E01-1342-30838 E 01-1342-30856 E01-1342-30874 E01-1342-30B92 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. 3 70 F Liver 10 ppm a.i. 3 72 F Liver 10 ppm a.i. 3 74 F Liver 10 ppm a.i. 376 F Liver 10 ppm a.i. 3 78 F Liver 10 ppm a.i. 3 80 F Liver 10 ppm a.i. 382 F Liver 10 ppm a.i. 384 F Liver 10 ppm a.i. 3 86 F Liver 10 ppm a.i. 388 F Liver 10 ppm a.i. 390 F Liver 1 0 p p m a .i. 392 F Liver 10 ppm a.i. 394 F Liver 10 ppm a.i. 396 F Liver 10 ppm a.i. 3 98 F Liver 10 ppm a.i. 4 00 F Liver 10 ppm a.i. 4 02 F Liver 10 ppm a.i. 4 04 F Liver 10 ppm a.i. 4 06 F Liver 10 ppm a.i. 408 F Liver AMOUNT O F LIVER W EIG H ED (G) ADDITIONAL DILUTION C o n c . (V C ,F ,,S O S am ple (n g /g o f L iv e r ) 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 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <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* 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 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M S am 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 AMOUNT OF LIVER WEIGHED (G ) ADDITIONAL DILUTION C o n ic . o r C BF 17S 0 3` in S am ple (n g /6 o f L iv e r ) 0 ppm a.i. 2 4 6 F Liver, Offspring 0 ppm a.i. 2 5 9 F Liver, Offspring 0 ppm a.i. 2 73 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 * <20.0* <20.0* <20.0* 10 ppm a.i. 5 17 F Liver, Offspring 10 ppm a.i. 5 25 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 V2 of QC at each level must be within 75% -1 2 5 % 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% -1 2 5 % . 3M Environmental Laboratory 3M Environmental Laboratory Page 21 of 226 Report E01 -1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 13. C8F17SO3' Data Summary, Male Quail Liver (Adult and Offspring) 3M S am ple N umber S a m p l e D e s c r ip t io n E 0 1 -1 34 2 -3 0 31 2 0 ppm a.i. 329 M Liver E 0 1 -13 4 2 -3 03 3 0 0 ppm a.i. 331 M Liver E 0 1 -13 4 2-30348 0 ppm a.i. 333 M Liver E01 -13 4 2-30366 0 ppm a.i. 335 M Liver E 01-1 3 4 2 -3 03 8 4 0 ppm a.i. 337 M Liver E 0 1 -1342-30402 0 ppm a.i. 3 3 9 M Liver E01-1342-30420 0 ppm a.i. 341 M Liver E 01-13 4 2 -3 04 3 8 0 ppm a.i. 3 43 M Liver E 01-13 4 2 -3 04 5 6 0 ppm a.i. 3 45 M Liver E01-1342-30474 0 ppm a.i. 347 M Liver E01 -13 4 2-30492 0 ppm a.i. 3 4 9 M Liver E 01-1 3 4 2 -3 05 1 0 0 ppm a.i. 351 M Liver E01 -13 4 2-30528 0 ppm a.i. 3 53 M Liver E01-1342-30546 0 ppm a.i. 355 M Liver E 01-13 4 2 -3 05 6 4 0 ppm a.i. 357 M Liver E 0 1 -13 4 2-30582 0 ppm a.i. 3 5 9 M Liver E 0 1 -13 4 2 -3 06 0 0 0 ppm a.i. 361 M Liver E 0 1 -1 34 2 -3 0 61 8 0 ppm a.i. 3 6 3 M Liver E 01-13 4 2 -3 06 3 6 0 ppm a.i. 3 65 M Liver E 01-13 4 2 -3 06 5 4 0 ppm a.i. 3 67 M Liver E 0 1 -13 4 2-30672 10 ppm a.i. 3 69 M Liver E 01-13 4 2 -3 06 9 0 10 ppm a.i. 371 M Liver E 0 1 -13 4 2 -3 07 0 8 10 ppm a.i. 3 73 M Liver E 01-1 3 4 2 -3 07 2 6 10 ppm a.i. 3 75 M Liver E 0 1 -1 3 4 2 -3 07 4 4 10 ppm a.i. 377 M Liver E 01-13 4 2 -3 07 6 2 10 ppm a.i. 379 M Liver E 01-1 3 4 2 -3 07 8 0 10 ppm a.i. 381 M Liver E 01-1 3 4 2 -3 07 9 8 10 ppm a.i. 383 M Liver E 01-1 34 2 -3 0 82 9 10 ppm a.i. 385 M Liver E 01-13 4 2 -3 08 4 7 10 ppm a.i. 387 M Liver E 01-13 4 2 -3 08 6 5 10 ppm a.i. 3 89 M Liver E 01-1 3 4 2 -3 08 8 3 1 0 p p m a .i. 391 M Liver E 01-1342-30901 10 ppm a.i. 393 M Liver E 0 1 -13 4 2 -3 09 1 9 10 ppm a.i. 3 95 M Liver E 0 1 -13 4 2-30937 10 ppm a.i. 3 97 M Liver E 0 1 -1 34 2 -3 0 95 5 10 ppm a.i. 3 9 9 M Liver E 01-1 3 4 2 -3 09 7 3 10 ppm a.i. 401 M Liver E 0 1 -1342-30991 10 ppm a.i. 4 03 M Liver E 0 1 -1 3 4 2 -3 10 0 9 10 ppm a.i. 4 05 M Liver E 0 1 -1342-31027 10 ppm a.i. 407 M Liver A M O U N I OF LIVER WEIGHED (G ) ADDITIONAL DILU'I ION C o n c . o f C 8F 17S 0 3 in S am ple (n g /g o f L iv e r ) 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* 1.0010 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 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 C C I aj Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFQS: 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 AMOUNT OP LIVER WEIGHED (G) ADDI IONA! DILUTION C o n c . OF C8F17S 0 3' in S am ple (NG/G OF LIVER) E01-1365-31194 E01-1365-31199 E01-1365-31209 E01-1365-31214 E01-1365-31229 E01-1365-31234 0 ppm a.i. 2 5 3 M Liver, Offspring 0 ppm a.i. 2 56 M Liver, Offspring 0 ppm a.i. 2 6 6 M Liver, Offspring 0 ppm a.i. 2 70 M Liver, Offspring 0 ppm a.i. 2 8 3 M Liver, Offspring 0 ppm a.i. 291 M Liver, Offspring E01-1365-31245 E01-1365-31255 E01-1365-31265 E01-1365-31270 E01-1365-31285 10 ppm a.i. 522 M Liver, Offspring 10 ppm a.i. 533 M Liver, Offspring 10 ppm a.i. 545 M Liver, Offspring 110 ppm a.i. 552 M Liver, Offspring 10 ppm a.i. 568 M Liver, Offspring 0.3806 0.5947 0.2673 0.5900 0.6666 0.6072 0.7325 0.6903 0.4624 0.5353 0.7581 1 1 1 1 1 1 10 1 1 1 1 <20.0* <20.0* <20.0* <20.0* <20.0* <20.0* 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 Q C samples did not meet the Vi of QC at each level must be within 75% -1 2 5 % 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% -1 2 5 % . Statement of C onclusion Sera results from the male control group portion of this study showed no detectable levels of C8F17S 0 3' 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 0 3' 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 0 3` (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 0 3' (range 4 7 1 0 - 14700 ng/mL, Table 15). Sera results from the male offspring control group showed no detectable levels of C8F17S 0 3'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 0 3' in 4 of 4 birds (<400 ng/mL, Table 14). Analytical results for five sera samples from the 10ppm closed 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 0 3' in 19 of 19 birds (<50.2 ng/g, Table 15) and quantifiable concentrations of C8F17S 0 3' in 1 of 20 (209 ng/g, Table 14). The liver results from the female control group showed no detectable levels of C8F17S 0 3' 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 C8F17S 0 3' (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 EO1-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 CeF^SCV 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 0 3' 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, C8F17SO3' Concentration in Sera and Liver S ampling E vent N umber of S era S amples Average C isF u S O j" Conc. Sera Male Control Group 19 NA Range of Results N umber of Liver S amples Average C bF u S O / Co nc. Liver <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) MalelOppm Group 20 141000 ng/mL 94400 - 226000 ng/tnL 20 88500 ng/g* Female 10ppm Group 18 9990 ng/mL 4710 - 31900 ng/mL 20 4900 ng/g* Range of 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 I 6 4 Male Offspring tOppm Group Female Offspring 10ppm Group 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* 4 940-13700 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 Vi of QC at each level must be within 75% -1 2 5 % 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 15. Summary Table, C8Fi 7SC>3' Concentration in Sera and Liver with Outliers Removed S ampling E vent N umber of S era Samples A verage c ,,f 17s o ; Conc. S era Male Control Group 19* * Female Control Group Male 10ppm Group 16 20* <400ng/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 of Results N umber of Liver S amples A verage C eF ,, S O ; Co n c . L iver Range of Results * <400 ng/mL (16 of 16) 19 20* <50.2 ng/g** <50.2 ng/g (19 of 19)** i ... * 20* * * 4710-14700 ng/mL *j 20* 6* * 4* * * * * * * 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 Vi of QC at each level must be within 75% -1 2 5 % 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 t h 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 D ata Q uality 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 >- jf> IT*. L 38 Report EOl-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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% - 1 2 5 % 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 is t o f Atta c h m en ts 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 001009 Report EOl-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite S ig nature Page W e certify that this report is a true and complete representation of the data for this study: QftH A jj-fr/fruo*______ Lisa A. Stevenson Principal Analytical Investigator ___ Date William K. Reagen Laboratory Management ______ 0 / / / < / o 3 Date 3M Environmental Laboratory 3M Environmental Laboratory Page 27 of 226 Page 27 L '<>r ^IL( , -- * Report E 01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite At ta c h m e n t A: Extr a c tio n and A nalytical M e t h o d , E T S -8 -2 3 1 .1, "S o lid P hase Extractio n and A nalysis o f F luo ro chem ical C o m po unds from Biological M atrices" 3M Environmental Laboratory 3M Environmental Laboratory Page 28 of 226 n f: Page 28 .a . Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory Method Solid Phase Extraction and Analysis of Fluorochemicai Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: H jlijo l Revision Date: Effective Date: J ./l i f t z Approved By: Willliam K. Rcagen Laboratory Manager Date ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemicai Compounds from Biological Matrices Page 1of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 29 of 226 Page 29 c o irv i Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 1 Scope and Application This method describes the extraction o f target analytes from tish. rat liver, rat seta, 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 l/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, muted, 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 Ci SPE cartridge. Finally, the analytes of interest are eluted from the SPE cartridge and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPIX-ES/MS/MS). 3 Definitions 3.1 Dilution A dilution expressed as l :5 or l/6 is defined as: l mL of sample + 5 mLs of 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 al the other end. It is designed to retain the compounds of interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable concentration!s) of the target analyte(s). 3.4 Quality control sample Sample used to monitor the extraction efficiency (as a matrix spike) and to verify the continued accuracy o f the initial calibration ciuve (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 und/or equipment. Use caution with the voltage cables for the probe. When engaged, (lie probe employs a voltage of approximately 5000 volts. 4.2 Cautions Take care not to allow the SPE column to run to dryness after the methanol and water washes. After washing is complete, add sample then allow all of the liquid to pass through the SPE column to dryness. ETS-8-23U 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Do not operate solvent pumps above rapacity of 400 bar (5.XOO psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. Do not run 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 of those listed. 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 2 Ultra-Turrax T25 tissue homogenizer Vacuum Pump SPE Extraction Manifold Centriluge, Mistral lOOOorlEC' Shaker, Eberbach or VWR Balance (+/- 0.1000 g) Micromass, Quattro 11or Ultima triple quadrapole Mass Specuometer equipped with an electrospmy 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 of holding 50 mL to I L fNalgene) Volumetric flasks, glass, type A 40 mL glass vials (1CHEM) Plastic sampule vials, Wheaton, 6 m L (or other appropriate size) Centrifuge tubes, polypropylene, 15 mL and 50 mL Labels Graduated pipettes, glass Syringes, capable of measuring 5 pL to 1000 pL Bottle-Top Dispenser (capable of dispensing 5mL of solvent) SPE extraction cartridge, l g, Sep-Pak 6 cc tri-functional Ci, (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 3 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 31 of 226 Page 31 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 I 7.1 Reagents preparation 2.0 niM 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 of approximately 1000 ppm (fig/niL). Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. Example calculation: 1000 flg/mL x 5 mL/100 mL = 50 fig/rnL, 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/IOOmL = 5.0 |Xg/'mL. Dilute working standard 1 with methanol to produce a working standard 3 solution of approx. 0.50 ppm. Example calculation: 50 jig'ntL x 1.0 mL/100 mL = 0.5 flg/niL. 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 of surrogate standard into a 100-mL volumetric flask and record the actual weight. Bring to volume with methanol for a surrogate standard stock of approximately 900 -1100 ppm. Prepare a surrogate standard working standard. Transfer approximately I mL o f surrogate standard stock to a 10-rnL volumctric flask and bring to volume with methanol for a working standard of 90-1 lOppm. Record the actual volume transfened and standard concentrations in die 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 alt internal standard preparations. Weigh approximately 90-110 mg of internal standard into a 1Ofl-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 EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 transfen ed 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 perfonned. Allow samples to thaw to room temperature prior to extraction. Typically fresh matrix standards are prepared with each analysis. Extracted standards and sample^ are stored in capped autovials until analysis. If analysis will be delayed, extracted standards and samples may be refrigerated at approximately 4C indefinitely or may be stored at room temperature until analysis can be 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.O mL o f water, or other appropriate amount, is used as a method blank. Four method blanks are extracted and analyzed with each set following this procedure (two are spiked with surrogate and two are not spiked). 9.1.3 Matrix Blank An aliquot o f l.OmL or l.O g of matrix (diluted or homogenized) is used as a matnx blank. Odrer amounts may be used, as appropriate. Matrix blanks are prepared from one o f three sources: l ) a study contra! matrix from a study control animal received with a sample set: 2) a commercially obtained sample of the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but o f a different species than the study animal, teg. 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 die 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 of method and matrix blanks may be adjusted and will he noted in the study protocol or in the raw' data. 9.2 Sample Replicate Samples replicates arc prepared according to each study protocol or project outline ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 33 ot 226 Page 33 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 9.3 Surrogate standard If surrogate standar d is a component o f the study, ail samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range of the calibration curve, with the exception of blank samples as described above. Typically surrogate standard is spiked into the 1,0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL dilutedTiomogenizecI 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 of the calibration curve. Typically internal standard is spiked into the 2.0 mL of extract in the 15 mL centrifuge tube, before transferring to the autovial. 9.5 Lab Control Sample Lab control samples are not a component 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 of the initial calibration curve. Typically 1.0 mL. or other appropriate amount, o f tlie 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 of sample extraction, (c.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 of extraction for a total o f twelve.) QC samples will consist of four samples at each of three levels of analyte. The levels listed below may be used and may represent sample concentrations diluted into the range of 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 mast be analyzed in the same analytical run. Any QC samples extracted during the course of the study may be included in subsequent analyses. If samples from multiple extraction dates are analyzed in one analytical run, then QC samples from tire 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 of2/3 of all QC samples must meet this criteria, and a minimum of 1/2 of the QC samples at each level must meet this criteria. If not, the set must either he re-analyzed or re-extracted. 9.7 Sample Dilution Any sample with an area greater (Iran 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 of the curve during analyses and enough sample remains, a post-run dilution validation will be performed to verity sample values. To peifonrr the dilution validation, one sample will be separated into two representative samples (i.e. two 1.0 mL aliquots for fluid samples or two 1.0 gram amounts for tissue samples, or other amount as determined by the analyst E T S - 8 - 2 3 1.1 Solid Phase Extraction and Analysis o f Fluorocbemical Compounds from Biological Matrices Page 6 o f 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite and documented in a note to file) then diluted using two procedures. The first procedure coasists of diluting the sample with additional matnx prior to extraction (fluid adding fluid), while the second procedure coasists 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 vulue is a correct representation of the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibration curve will be prepared from extracted matrix standards, in die same matrix as the samples, per study. It will consist of a minimum of nine (9) levels. Additional calibration curves may be extracted on separate sample extraction dates, is 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. Tire standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one standard curve. A total of 9 standards, four matrix blanks, and four method blanks are prepared in addition to the QC samples and test samples. The number of standards and blanks n a y be adjusted as determined by the aialyst and documented in a note to file. Use Attachment C, or oilier appropriate form, as an aid in calculating the concentrations o f the working standards. Refer to section 12 to calculate the actual concentration of analyte in each calibration standard and QC sample. Typically the target analyte standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, it may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. Analyze tire extracted matrix standard curve prior to each set of extracts. The curve equation will be determined by regression analysis using the peak areas of the target analyte(s) using MassLynx or other suitable software. Any level outside 75% -125% o f nominal must be deactivated, and regression re-calculated, except tlie LLOQ which must be within 30% of 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 tire set will be re-analyzed. ETS-8-231.1 Solid Phase Extraction and Analysis of 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 1 Mil 1 1 /VlTROMM V11 Sl'IklV . A MIMA 1s IOH SI XMIAUIIS \\l> SI'IUI s I M m . I.it \ i l , o r M \ t u t\ Approximate final Approximate final Working standard (approximate concentration) fiL concentration of analyte in M atrix diluted 1:5 concentration of analyte in Final 2.0 mL volume - - Blank Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 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 .0 0 ng/mL 0.500 ug/mL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug-'ml. 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL )4.0ng/mL 1 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/tnL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/g or ng/mL 100 ng/mL 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/mL 50.0 ug/mL 6.0 1750 ng/g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures______________________________________________________________ 11.1 Tissue Sample Preparation Obtain frozen tissue samples Cut approximately l .0000 g o f tissue (+/- 0 .1000 g). or other appropriate amount, using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the tissue to thaw. Weigh 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 the freezer after extraction amounts have been removed. Add 2.5 mL of reagent water to sampule vial, or other volume as delennined by the analyst and documented in a note to file. Homogenize the sample. Put the Ultra-Turrax grinder probe in the sample and grind for approximately 2 minutes, or until the sample is liomogeneous. Rinse the probe into the tube containing the sample with 2.5 mL of reagent grade water, or other volume as determined by the analyst and documented in a note to file, using a pipette. Take the grinder apart and clean it with methanol after each sample. Refer to ETS-9-52 for more information. If an amount other than 1.0000 g (not within +/- 0.1 (XX) 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 o f 2.5 mL o f water.), or other ratio as determined by the analyst and documented in a note to file. ETS-8-2.31.1 Solid Phase Extraction and Analysis of Fluoroehemical Compounds from Biological Matrices Page 8 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 36 of 226 Page 36 0 1 0 ;./. 3 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 l .0 mL of fluid, or other appropriate amount to a plastic sampule vial, or other appropriate container. Return unused samples to freezer after extraction amounts have been removed. Add 5.0 mL of reagent water to the 1.0 inL of fluid for a l/6 dilution, or other dilution as determined by the analyst and documented in a note to file. If a volume other than 1.0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilution as above. 11.3 Tissue and Fluid Sample Extraction After tissue or fluid samples have been prepared according to sections 11.1 and 11.2, vortex mix or shake by hand the diluted/homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mL polypropylene centrifuge tube. Return unused diluted/homogenized portions to the freezer after extraction amounts have been removed. Record the volume removed on the extraction worksheet, (Attachment A or simi lar worksheet). Spike blanks, samples, and standards, ready for extraction with surrogate standard as described in this method. Spike each calibration standard matrix with tire appropriate amount of standard as described in this method for the calibration curve standards and each QC sample. Vortex mix the standard curve samples and QC samples for approximately 5 seconds. To each sample and standard, add 5.0 mL of acetonitrile, cap. and vortex mix or slake by land 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 centrifuge 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 sumples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduce any of the matrix solids into the solution. Cap and mix by inverting several times. In this step the order of addition may be changed (i.e. the sample may be put into the centriluge 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 of methanol, followed by approximately two 5.0 mL aliquots of water, taking care not to allow the column to run to dryness after each wash. After washing is complete, pour the sample into the reservoir/cartridge unit and allow all of the liquid 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. ETS-8-231.1 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 Nothem 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 analyses) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.1 Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-month-day. and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or A020204a 1= Initial of the instrument name (A = "Amelia") YY = Test year (02) MM = Test month (02) DD = Test day (04) a = First sample list (run) of 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-Uay, and a 3-digit sequential file number that starts with 1 and increases by one for each filename. Example filename: IYYMMDDfl## or A020204001 1= Initial of instrument name YY = Test year MM = Test month DD = Test day ### = 3-digit sequential file number starting with 1 through 999 (001) Also, as part of 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 Data Acquisition" for additional information on MRM. Typically the analytical batch run sequence begins with system suitability, solvent blanks, and a set of 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 of Fluorochemical Compounds from Biological Matrices Page 10 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 38 ol 226 Page 38 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 p L injection Inject/sample - l Cycle time = 10.0 minutes Flow rate = 300 pL/min Mobile phase: Solvent A = 2 mM Ammonium Accinte. Solvent B = Methanol 1 Solvent gradient program: Tun.' Si>!cut H 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 11Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for details. Check die solvent level in HPLC reservoirs and refill if necessary. Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the lip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace die 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- 500iiL/min or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out of the tip o f the probe. A fine mist should be expelled with no nitrogen leaking around the tip of 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 - r , , s Pae49 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite HPLC constant flow mode, flow rate 10-500 pL/min Pressure <400 bar (this parameter is not set, it is a guide to ensure the HPLC is operating correctly.) Source block temperature approximately 150C Desolvation temperature approximately 250C These settings may change in order to optimize the response Print the tune page, sample list, and acquisition method from MassLynx and store it in the study binder with a copy taped into the instrument log. Click on start button in the Acquisition Control Panel (the location of the start button may vary amopg 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 L or mL/mL) 1W (g) (or IV (mL)) (IW (g)(or IV(mL))+ DV (mL) Calculate actual concentrations of analyte in calibration standards using the following equation: Concentration (ng/g orng/mL) Spike Concentration (ug/mL) x Spiked Amount (mL) 1000 ng SV (m L)x Matrix Amount (g/mL or mL/mL) 1ug IW " Initial weight (where LO 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 bssue/fluid standard curve, assumed to be 1 g or 1 mU5 mL water 3 sample = MA oftissue/fluid sample (___ g or mL of 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 /'O ' 1 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Calculate relative standard deviation using the following equation: ,. , .. Standard Deviation Relative Standard Deviation = -------------------------- x 100 Mean Calculate percent deviation using the following equation: ,, .. Expected Cone. - Calculated Cone. , ,,,, % Deviation = -- :------------------------------------------ x 100 Expected Cone. Calculate actual concennation of analyte in fluid (jig/mL): AR (ng/inL) * DF * 0 curve (m Um D * FVtmLl in Curve x 1.0 ug = (pg/g) 0 sample (mL/mL) FV (mL) in Matrix 1000 ng Calculate actual concentration o f analyte in tissue (pg-'g): AR (ng'g) x DF x 0 curve (e/mLl x FV (m l) in Curve x 1,0 ue 0 sample (g/niL) FV (mL) in Matrix 1000 ng = (pg/g) 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start aixl at the completion o f each analytical run. Prior to the calibration curve and after the last sample o f the run three (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 of the run separately. The peak area precision must be equal to or less than 5.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD, the resolution must be > 2.0. and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0. where AF is the asymmetry factor. If any item of the system suitability tails, 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 of a am, the sample set must be reanalyzed. 13.2 Quantitation Tire coefficient o f determination value for the calibration curve, plotted by regression using the peak areas of the annlyte(s). must be 0.990 or better. All active calibration curve points must be within 25% of the theoretical value with the exception of the 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 l!2 of each quality control sample at each level are expected to show an accuracy o f 75-125%. ETS-8-231.1 Solid Phase Extraction and Analysis 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Surrogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be reanalyzed to confirm. If the second analysis confirms the original, the deviation will be documented in the raw data. If the second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed o f in noninfectious biohazard waste containers. Flammable solvent waste is disposed of in high BTU containers. Glass pipette waste is disposed of in broken glass containers located in the laboratory. 15 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw data Each page generated for a study must contain the following information (if applicable): study/project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Other information may be added if applicable to the study. Print the tune page, sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into die instrument runlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data. Print data integration summary, integration method, and chromatograms from MassLynx, and store with the study raw data Summarize data using suitable software (Excel 7.0 or 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 Attacliment B: Sample Weight/Volume Worksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References______________________________________________________________ ETS-9-24, "Operation and Maintenance of 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 of 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 19 Revisions Revision Number 1 Revision Description Minor 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 I E T S - 8 - 2 3 1.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 15 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 43 of 226 Page 43 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 Sample Number or description Volume of Amount and diluted sample Am ount and surrogate spike removed spike m ix used mix used Type of column used and lot Elution solvent and volume C o m m ents Blank m a trix ______ TN-A-___________ ; Am ount welghed/aliquoted:___________g/mL 1. Homogenize sample 2. Aliquot 1 mL o f diluted matrix into 15 mL polypropylene tube 3. Spike sam ples accordingly 4. A d d ____ mL o f ACN (TN-A-___________ ) to each diluted sample and shake or vortex mix 5. Shake sam ple fo r 20 min @ ______ rpm (S h a k e r________________) 6. Centrifuge sam ple for 10 m in @ _______ rpm (Centrifuge ______________ ) 7. Add 40 mL o f ____________ water to 50 mL polypropelene centrifuge tube. 8. Decant extract Into centrifuge tubes with water 9. Shake sam ple slightly to ensure proper mixing 10. Attach 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum manifold 11. C ondition column with two washes of - 5 mL MeOH (TN-A-__________ ) - do not allow column to go to dryness 12. W ash colum n with two w ashes o f - 5 m L __________ water - do not allow column to go to dryness 13. Filter sam ple through conditioned column, discarding filtrate 14. Allow colum n to go to dryness. After dripping stops, draw a high vacuum through colum n for at least 5 minutes. 15. Elute column with solvent (___________TN-A-___________ ) into appropriate 15 mL centrifuge tube 16. Spike sam ples w ith ________ uL o f internal standard # ______________________ , cone.______________________ 17. Transfer sam ple into appropriately labeled autovial and cap Note: In vacuum steps above set the vacuum cham ber at approximately 15 kPA - this should give approximately 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-8-23U Solid Phase Extraction and Analysis of Fluoiochemical Compounds from Biological Matrices Page 16 of \9 3M Environmental Laboratory 3M Environmental Laboratory Page 44 of 226 Page 44 Report EO1-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Prep Date(s): Analyst(s): Sample Matrix: Method/Revision: Sample ID Attachment B - Sample W eight/Volume Worksheet Study Number: Equipment Number: Final Solvent & TN Number: Initial Wt/VoL e/mL/L Water Volume added <mL) Volume Removed (ml,) Comments 1 Fonn Completion Verified By:. ETS-8-231.1 Solid Phase Extraction and Analysts of Fluorochemical Compounds from Biological Matrices Page 17 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 45 of 226 Page 45 Report E 01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Prep date(s): Analyte(s): Sample matrix: M ethod/revision: A ttachm ent C: C alibration Standard Concentration W orksheet Standard number: Equipment number: Final solvent and TN: Blank Tissue or Fluid/identiiler: Analyte mix std approx. 0.500 ug/mL: Analyte mix std approx. 5.00 ug/mL: Analyte mix std approx. 50.0 ug/mL: Surrogate std approx. 100 ug/mL: Actual concentrations o f standards in the analyte mix Analyte 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 All A nit spiked mL 0.0015 0.0030 0.0080 0.0100 0.0320 0.0056 0.0080 0.0100 0.0240 0.0320 0.0400 0.005 0 006 All Final Volume: mL 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 All Initial Fluid Dilution mL/mL 0.1607 0.1067 0.1067 0.1007 0.1067 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 All Initial Tissue Density &'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 the final 2.0 mL solvent and initial matrix 2.0 mL Final Volume Fluid Matrix Tissue Matrix A n aly 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 Analyte Final cone. ng/mL 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL 100 .Surrogate Final cone ng/mL 6500 Analyte Final cone. ne/E 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Sid cone ng/mL 100 Surrogate Fiual cone ng/g 6500 ETS-8-231.1 Solid Phase Extraction and Analysis o f FJuorochemical 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Study: Dilution Date/Analyst: Box Number: Sam ple Num ber or Description 1/ Attachment D: Dilutions Summary W orksheet Solvent/TN Number: Extraction Datc/Analyst: Matrix/Timepoint: Dilutions 11 1/ 1/ 1/ 1/ 1/ C om m ents Verified By: Notes: 1/10 dilution = ______ o f sample + ________ of solvent Form Completion Verified By: ETS-8-231.1 Solid Phase Extraction and Analysts o f Fluorochemtcal Compounds from Biological Matrices Page 19 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 47 of 226 Page 47 Report EOl-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Attachm ent B: D ata S ummary Tables 3M Environmental Laboratory 3M Environmental Laboratory Page 48 ot 226 Page 48 Report EO1-1245 Study number E01-1245 - QUAIL SERA DATA SUMMARY, BLANKS AND SPIKES PFOS: A Reproduction Study with the Nothem Bobwhite 3M Sample Number Sampled Date E01-1245-35766 E01-1245-35825 E01-1245-35825 1/31/2002 2/1/2002 2/1/2002 E01-1245-35767 E O M 245-35826 E01-1245-35826 1/31/2002 2/1/2002 2/1/2002 Sample Description Water blank-day 1 Blank H20-day 2 Blank H 20-day2 quail sera blank-day 1 Blank quail sera-day 2 Blank quail sera-dav 2 Inst. File Name d020207031 d020203019 0020207035 d020207032 4020203020 d020207036 amount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 dilution (h om ogin ate) 40 40 40 40 40 40 , additional dilution 1 1 1 Inst Cone, (,nq/,mL, of, 'a Methanol) 10.0 10.0 10.0 1 10.0 1 10.0 1 10.0 loq for control s a m p le s 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 E0M245-35768 E01-1245-35827 E01-1245-35827 Sampled Date 1/31/2002 2/1/2002 2/1/2002 Sample Description quail Sera MS-day 1 quail Sera M$-day 2 quail Sera MS-day 2 Inst. File Name 4020207033 4020203021 4020207037 E O I-1245-35769 E01-1245-35828 EOI-1245-35628 1/31/2002 2/1/2002 2/1/2002 quail Sera MSD-Dayl quail Sera MSD-Day2 quail Sera MSD-Day2 4020207034 4020203022 4020207038 Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilution(di!uted sera)) difution(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. amount ol sera used (m l) 0.05 0.05 0.05 0.05 0.05 0.05 dilution (homogmate) 40 40 40 40 40 40 additional dilution 1 1 1 Inst Cone. (ng/mL of Methanol) 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, of PFOS in Sample (ng-'mL of Sera) 9012 10173 9097 3- 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 o c,? 3M Environmental Laboratory Page 49 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA versus unextracted curve 3M Sample Number Sampled Sample _Description E01-1245-35779 E01-1245-35780 E01-1245-35781 E01* 1245-36155 E 0 M 245-36156 E01-1245-361S7 E01-1245-36158 E01-1245-36159 E0M245-3616O 01/31/2002 qc-25ppb-1,01 -31-02 (1004 ng/mL diluted sera) 01/31/2002 qc-25ppb-2,01-31-02 (1004 nj/m L diluted sera) 01/31/2002 qc-25pob-3.01-31-02 (1004 ng/mL diluted sera) 02713/2002 ms-5ppb-1,02-13-02 (201 ng/mL diluted sera) 02/13/2002 ms-5pptl-2, 02-13-02 (201 ng/mL diluted sera) 02/13/2002 ms-5ppb-3, 02-13-02 (201 nq/mL diluted sera) 02/13/2002 ms-200ppb-1,02-13-02 (8032 ng/mL diluted sera) 02/13/2002 ms-200ppb-2, 02-13-02 (8032 ng/m l diluted sera) 02/13/2002 ms-200ppb-3,02-13-02 (8032 nq/mL diluted sera) original Inst. File Name d020213101 d020213102 [20213103 d020213104 (J020213105 d020213106 d020213107 dQ20213108 J020213109 Absolute Recoveries amount of sera used (rnL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 dilution (diluted sera) 40 40 40 40 40 40 40 40 40 a d d it io n a l dilution 1 1 1 Inst Cone fng/mL of Methanol) 38.69 39.12 45.18 5.54 5.51 5.23 248.13 254.19 253.36 toq for samples 201 201 201 201 20 201 201 201 201 Cone of PFOS in Sample (n g /m l cf Sera) 1548 1565 1807 222 220 209 9925 10168 10134 Study number 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/mL diluted sera) E01-1246-35780 01/31/2002 qc-25ppb-2, 01-31-02 (1004 ng/mL diluted sera) E0M245-35781 01/31/2002 QC-25ppb-3, 01-31-02 (1004 n q /m l diluted sera) 01-1245*36155 02/13/2002 ms-5ppt>-1,02-13-02 (201 n g /m l diluted sera) E01-1245-36156 02/13/2002 ms-5ppb-2,02-13-02 (201 ng/mL diluted sera) E01-1245-3615? 02/13/2002 ms-5ppb-3, 02-13-02 (201 nq/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 n g /m l diluted sera) A * Not within +/-25% criteria d020405062 d020405083 d020405084 d0204050ft5 dO2O4O5086 0020405067 d020405086 d020405089 (5020405090 Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml)) x (additional dilution) x (dilulion(diiuted sera)) diluVion(diluted sera) = final volume(ml)/amount of sera in diluted sampie(mL) = 2.0 mUO.05 m l = 40 amount of sera in diluted sample = amount of sera used. amount of diluted sera used (m l) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 dilution (diluted sera) 40 40 40 40 40 40 40 40 40 for all in origina analysis (absolute recovery) avg %rec 132.3 std dev 25.62 additional dilution 1 1 1 1 1 1 1 1 1 Inst Cone, (n g /m l of M ethanol 36.06 41.69 44.98 6.17 5.88 617 248.12 260.27 254.58 !oq to r samples 201 201 201 201 201 201 201 201 201 Cone, of PFOS m Sample (n g /m l of Serai 1442 1668 1799 247 235 247 9925 10411 10183 for all in rerun analysis (absolute recovery) avg% rec 136.8 s td d e v 21.84 A A A A A A A A A A % Ree 154.1 155.9 180.0 110.2 109.7 104.1 123.6 126.6 126.2 avg 163.3 s td d e v 14.46 %RSD 8.853 avg 108.0 s td dev 3 403 %RSD 3.151 avg 125.4 s td d e v 1.636 %RSD 1.304 c0 Ree 143.7 166.1 179.2 122.8 117.0 122.8 123.6 129.6 126.8 avg 163.0 std dev 17.97 %RSD 11.03 avg 120.9 s ld 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 Nothem Bobwhite Study number E01-1245 - QUAIL SERA QC Sample Recoveries versus extracted curve original Of aB and rerun of 80ppm 3 M S a m p le Num ber S a m p le d D a te S a m p le D e s c rip tio n In s t. Fi e N am e E01-1245*35785 E01-1245-35786 E01-1245-35787 E01-1245-35779 E01-1245-35780 E0M245-35781 E01-1245-35782 E01-1245-35783 EOM 245-35784 EO M 245-35785 E01-1245-35786 E01-1245-35787 01/31/2002 qc-80ppm-1. 01-31-02 (3227040 ng/mL diluted sera) d020201035 01/31/2002 qc-80ppm-2,01-31-02 (3227040 ng/mL diluted sera) <1020201036 01/31/2002 qc-80ppm-2, 01-31-02 (3227040 ntj/m l diluted sera)d020201037 01/31/2002 qc-25ppb-1, 01-31-02 (1004 ng/mL diluted sera) d020207044 01/31/2002 qc-25ppb-2, 01-31-02 (1004 ng/mL diluted sera) d020207045 01/31/2002 qc-25ppb-2, 01-31-02 (1004 ng/mL diluted sera) d020207046 01/31/2002 qc-40ppm-1, 01-31-02 (1613520 ngfaiL diluted sera) d02Q207047 01/31/2002 qc-40ppm-2, 01-31-02 (1613520 ng/mL diluted sera) d020207048 01/31/2002 qc-40ppm-2,01-31-02 (1613520 ncyYnL diluted sera) <*020207049 01/31/2002 qc-80ppm-1, 01-31-02 (3227040 ng/mL diluted sera) d020207050 01/31/2002 qc-80ppm-2,01-31-02 (3227040 ng/mL diluted sera) d020207Q51 01/31/2002 qc-80ppm-2, 01-31-02 (3227040 nq/mL diluled sera) d020207052 am ount of sera u se d (m U 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (d ilu te d sera ) 40 40 40 40 40 40 40 40 40 40 40 40 Study number E01-1245 - QUAIL SERA versus extracted curve reran 3 M S a m p le Num ber S a m p le d D a te Sample Description Inst. File Name E01-1245-35779 01/31/2002 qc-25ppb-1,01-31-02 (1004 ng/mL diluted sera) EOM 245-35780 01/31/2002 qc-25ppb-2,01-31-02 (1004 ng /m l diluted sera) E01-1245*35781 01/31/2002 qc-25ppb-3, 01-31-02 (1004 ng/m l diluted sera) E0M245-36155 02/13/2002 m$-5ppb-1.02-13-02 (201 ng/mL diluted sera) E01-1245-36156 02/13/2002 ms-5ppb-2. 02-13-02 (201 ng/m l diluted sera) E0M245-36157 02/13/2002 ms-5ppb*3, 02-13-02 (201 ng'mL diluted sera) E0M245-36158 02/13/2002 ms-200ppb-l, 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) E 0 M 24 5-36155 02/13/2002 ms-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) A * Not within +7-25% criteria d020405082 d020405083 d020405084 <J020405085 d020405086 dO20405087 d020405068 d020405069 CJ020405090 020410019 d020410020 d020410021 Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/ml>) x (additional dilution} x (dilutton(diluted sera)) difution(diluted sera) = final volume(mL)/amount of sera in diluted sampte(mL) = 2.0 mL/0.05 m l * 40 amount of sera in diluted sample - amount of sera used. amount of sera used (m l) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (d ilu te d sera ) 40 40 40 40 40 40 40 40 40 40 40 40 a d d itio n a l d ilu tio n 1000 1000 1000 100 100 100 1000 1000 1000 In s t C on e. (n g /m L o f M e th a n o l) 95.65 93.45 85.75 26.99 27.89 31.52 299.0 316.2 306.4 88.66 85.99 79.33 lo q fo r s a m p le s 400 400 400 400 400 400 400 400 400 400 400 400 C o n e , o f P F O S in S a m p le (n q /m L o f S e ra i 3826000 3738000 3430000 1080 1116 1261 1196120 1264880 1225440 3546400 3439600 3173200 for an in original analysis (recovery) avg% rec 101J std dev 19.89 a d d itio n a l d ilu tio n 1 1 1 1 1 1 1 1 1 1 1 1 Inst Cone (ng/m l of M e th a n o l) 24.57 28.49 30.78 3.85 3.65 3.64 177.0 186.2 181.9 5.19 4.64 4.62 lo q fo r s a m p le s 201 201 201 201 201 201 201 201 201 100 100 100 for all in rerun analysis (recovery) avg %rec 95.57 std dev 14.18 ______ C o n e o f P F O S in S a m p le ( n g / m l of S e ra ) 983 1140 1231 154 146 154 7080 7447 7275 208 186 185 A A A R ec 118.6 115.0 106.3 107.5 111.1 125.6 74.13 78.39 75.95 109.9 106.6 98.33 avg 113.6 std dev 6.444 %RSD 5.674 avg 114.7 std dev 9.555 %RSD 8.327 avg 76.16 std dev 2.138 %RSD 2.808 avg 104.9 std dev 5.956 %RSD 5.676 Rec 97.89 113.5 122.6 76.62 72.64 76.42 68.15 92.72 90.58 103.3 92.34 91.94 avg 111.3 std dev 12.51 %RSD 11.24 avg 75.22 std dev 2.243 %RS0 2.981 avg 9 a 48 s td dev 2.285 %RSD 2.525 avg 95.85 std dev 6.437 %RS0 6.716 3M Environmental Laboratory Page 51 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number Sampled Date Sample Description * E01-1245-28444 06/15/2001 * E0M245-28446 06/15/2001 * E0M245-28449 06/15/2001 * E01-1245-28451 06/15/2001 E01-1245-28453 06/15/2001 . E01-1245-28456 06/15/2001 * 01*1245*28458 06/15/2001 * E0M245-28460 06/15/2001 * E01-1245-28462 06/15/2001 * E01-1245-28464 06/15/2001 * E0M245-28466 06/15/2001 * 01*1245*28468 06/15/2001 * E01-1245-28470 06/15/2001 * E0M245-2B472 06/15/2001 * E01-1245-28474 06/15/2001 * E01-1245-28477 06/15/2001 * E01-1245 28479 06/15/2001 + 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 20131055 (1020207059 20131057 20207061 20131060 20207064 20131062 20207066 20131069 20207073 20131072 (20207076 (20201043 20207087 20201045 20207089 20201047 20207091 20201048 20207092 (20201050 20207094 20201052 20207096 20201059 20207103 20201061 20207105 (20201063 20207107 20201066 20207110 20201073 <20207117 amount of sera used {m l} 0.4 0.4 0.05 0.05 0.3 0.3 03 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) dilution 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 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 40 1 Inst Cone. (ng/mL of Methanol} 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 10.0 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 (nq-'m l of Sera) 400 400 400 400 400 400 400 400 400 400 400 400 4 400 400 400 40 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 Cone of PFOS in Sample (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 2051 2177 400 400 400 400 400 400 4 4 4 + 4 4 4 + 4 + 4 4 4 4 + + + + + + + _4 4 + 4 4 4 4 4 4 4 4 # 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)) diiutk>n(diluted sera) = final volume(mL)/amount of sera in diluted sample(ml) a 2.0 ml70.05 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 dup 400 400 400 400 400 400 400 400 AAA 400 400 400 400 2114 00 00 400 rq tcte d rofBCtd With outliers avg 500.8 std dev 409.7 %RSD81.80 Without outliers avg 400 std dev 0 %RSD o a, V, : 3M Environmental Laboratory Page 52 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number Sampled Date Sample Description E01-1245-28481 E01-1245-28483 E0M245-284B5 E 0 M 245-28407 01-1245-28469 E01-1245-28491 01-1245-28493 E01-1245-28496 EO1-1245-20498 E01-1245-28500 E01-1245-28502 E01-1245-28504 E01-1245-28506 E01-1245-28508 E01-124S-28511 E01-1245-28513 E01-1245-28515 E01-1245-28517 01-1245-28518 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 06/15/20Q1 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. 10ppm female, adult, 02-01-02 454-108-374.10ppm female, adult, 02-01-02 454-108-376, lOppm female, adult, 02-01-02 454-108-378.10oom female, adult, 02-01-02 454-108-380,10oom female, adult, 02-01-02 454-108-382,10ppm lemale. adult, 02-01-02 454-108-386.10oom female, adult, 02-01-02 454-108-388. lOoom female, adult, 02-01-02 454-108-390.1 Doom female, adult, 02-01-02 454-108-392.10ppm female, adult, 02-01-02 454-108-394. 10oom female, adult, 02-01-02 454-108-396.toom female, adult. 02-01-02 454-108-398.10ppm female, adult, 02-01-02 454-108-402. lOoom 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*408.10oom 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 E0M245-28525 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 E0M245-28530 07/23/2001 454-108-525,10ppm female, offspring, 02-01-02 E 0 M 245-28532 07/23/2001 454-108-538,10ppm female, offspring, 02-01-02 E01-1245-28535 07/23/2001 454-108-5S7,10ppm female, offspring, 02-01 -02 E01-1245-28536 07/23/2001 + Concentration <LOQ 454-108-561, lOppm female, offspring, 02-01-02 Inst File Name 3020203026 3020203027 3020203028 3020203029 3020203030 3020203031 3020416019 3020203033 3020203034 3020203038 3020203039 d020203040 3020203041 3020203042 3020203043 3020203044 3020203045 3020203046 3020201132 3020207153 3020201135 3020207156 3020201138 3020207159 3020201139 3020207160 3020201147 3020207168 3020201149 3020207170 3020201151 32207172 3020201154 302(007175 3020201155 3020207176 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) 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 40 100 100____ 613.67 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 Inst Cone. (ng'm L of Methanol) 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 10.0 10.0 10-0 10.0 10. 10.0 185.25 166.06 192.74 180.81 365.44 304.83 208.24 186.59 287.26 252.41 lo q to r samples (nq/mL 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, of PFOS in Sample (ng/mL of Sera? average of dup s a m p le s 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 * 400 + + 400 + 400 + 400 + 17566 12452 13405 7897 10793 rejected W ith outliers avg 9990 std dev 6010 %RSD 60.2 Without outliers avg 8699 std dev 2557 %nSD 29.4 avg 400 std dev 0 %RSD * avg of dups 12423 std dev 3457 %RSD 27.8 * There are two resuits reported for these samples. Both values are included in the average Cone of PFOS in Sample (ng/m!) * (Inst conc(ng/mi)) x (additional dilution) x {dilutionfdiluted sera)) difution(di!uted sera) = final volume(mL)/amount of sera in diluted samp!e(mL) = 2.0 mL/0.05 mL = 40 amount of sera ft 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- dtiution(diluted sera)* 2.0 mL final volume/0.02 m L amount of sera used = 100 O 3M Environmental Laboratory P o rro Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number Sampled Date Sample Description * E0M245-28443 * E 0 M 245-28445 06/15/2001 06/15/2001 * E01-1245-28447 * E0M245-28448 06/15/2001 06/15/2001 * EO M 245-28450 06/15/2001 * 01-1245-28452 06/15/2001 * E0M245-28454 06/15/2001 * E01-1245-28455 06/15/2001 * E01-1245-28457 06/15/2001 * E01-1245-28459 06/15/2001 * E01-1245-28461 * E01-1245-28465 * E0M245-28467 06/15/2001 06/15/2001 06/15/2001 * E01-1245-28469 E01-1245-28471 06/15/2001 06/15/2001 * E01-1245-28473 06/15/2001 E01-1245-28475 06/15/2001 * E01-1245-28476 06/15/2001 * EO M 245-28478 06/15/2001 + Concentration <10Q 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 mate, 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 mate, 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 mate, adult. 02-01-02 454-108-365, Oppm mate, adult, 02-01-02 454-108-367. Oppm male, adult, 02-01-02 Inst. File Name 20131054 20207058 (20131056 (20207060 <3020131058 d020207062 <20131059 d020207063 d020131061 (20207065 d020131063 d020207067 <020131070 20207074 (20131071 (1020207075 20131073 20207077 20201044 20207088 20201046 20207090 20201049 20207093 20201051 (20207095 20201058 20207102 20201060 20207104 20201062 20207106 20201064 20207108 20201006 20207109 20201067 20207111 amount of sera used (m L) 0.2 0.2 0.4 0.4 0.4 0.4 0.2 0.2 o.i 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. (ncj/mL of Methanol) 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 10. 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 100 foq 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 Cone, of PFOS in Sample (n g /m l of Sera) average of dup. s a m p le s 400 + 400 400 + 400 f 400 400 f 400 + 400 400 + 400 400 + + 400 400 + 400 400 400 + 400 400 + 400 + 400 400 400 400 + + 400 400 + 400 400 + 400 + 400 400 + 400 + 400 400 400 + 400 400 + 400 + 400 400 + 400 + 400 400 + 400 + 400 400 + 400 + 400 400 + 400 + 400 400 + 400 + 400 400 + 400 400 400 + * There are two results reported for these samples. Both values are included in the average Cone of PFOS m Sample (ng/ml) = (Inst conc(ng/mI)) x (acfditional dilution) x (dilution(diluted sera)) dikition{diluted sera) * final volume(mL)/amount of sera in cSluted 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* dilution(di!uted sera)= 2.0 mL final volume/0 02 m l amount of sera used= 100 avg 400 std dev 0 %BSD - 3M Environmental Laboratory Page 54 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number E01-245-28480 E01-1245-28482 E01-1245-28484 01-1245-28486 E 0 M 245-28488 E01-1245-28490 E0M245-28492 E01-1245-28494 E01-1245-28495 E0M245-28497 E01-1245-28499 E01-1245-28501 E0M245-28503 E01-1245-28505 E01-1245-28507 EOf-1245-28509 E01-1245-28510 E01-1245-28512 E01-1245-28514 E01 -1245-28516 Sampled Date Sample De 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, lOppm male, adult. 0100,02-01-02 454-108-371,10ppm male, adult. 100.02-01-02 454-108-373.10ppm male, adult, d100,02*01-02 454-108-375, 10ppm male, adult, dlOO,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, lOppmmale. 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, lOppmmale, adult, d100,02-01-02 454-108-389, 10ppm mate, adult, d100,02-01-02 454-108-391, 10ppm male, adult, dlOO,02-01-02 454-108-393. 10ppm mate. adult, d100,02-01-02 454-108-395,10ppm mate, adult, dlOO,02-01-02 454-108-397, 10ppm male, aduR, dl00,02-01-02 454-108-399,10ppm male, adult, dlOO,02-01-02 454-108-401, lOppm male, adult, d100,02-01-02 454-108-403,10ppm mate. adult. dlOO,02-01-02 454-108-405. 10ppm male, aduR. dlOO,02-01-02 454-108-407, lOppm male, adult, d100,02-01-02 Inst. File Name 1020201074 1020207118 1020201076 1020207119 (1020201078 <1020207120 <1020201080 1020207121 0020201082 <1020207122 1020201089 <20207128 <20201091 <20207129 20201093 <20207130 <20201094 (20207131 0020201096 <20207132 <1020201103 020207133 1020201105 <20207134 <20201107 <1020207135 20201109 <20207136 (20201111 20207137 (20201118 <20207143 20201119 020207144 20201121 (20207145 020201123 <20207146 0020201125 (20207147 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 03 01 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 di'ution (diluted additional sera) dilution 40 100 40 100 40 100 40 100 40 Too 40 100 40 100 40 100 40 100 40 100 40 10" 40 100 40 100 40 100 40 Too 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 100 40 io o 40 100 40 100 40 100 40 100 40 100 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/m l of Serai 106920 116800 101240 179440 143600 156533_______ 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 average of dup s a m p le s 111860 180340 150060 138000 95260 158420 144780 151420 120640 185280 132340 144000 162440 131460 103060 148200 214900 132060 112340 105380 There ate two results reported for these samples. Both values are included in the average Cone of PFOS in Sample (ng/ml) = (Inst conc(ng/rrt)) x (additional dilution) x (di)ution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera tn diluted sample(mL) * 2.0 m l/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 avg o f dup* 141112 atddev 36225 %RSD 21.4 o .- S K 3M Environmental Laboratory Page 55 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL SERA 3M Sample Number Sampled Date Sample Description * EOt-1245-28519 * E01-1245-28520 * E01-1245-28522 * E01-1245-28523 E01-1245-28526 07/23/2001 07/23/2001 07/23/2001 07/23/2001 07/23/2001 * E01-1245-28527 E01-1245-28529 * E01-1245-2853t * E0M245-28533 * E01-1245-28534 07/23/2001 07/23/2001 07/23/2001 07/23/2001 07/23/2001 * E01-1245-28537 07/23/2001 f Concentration <LOO 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 mate, offspring. 02-01-02 454-108-545, lOppm male, offspring. 02-01-02 454-108-552,10ppm male, offspring, 02-01-02 454-108-568, lOppm male, offspring, 02-01-02 Inst. File Name <1020201133 (1020207154 d 0 2 0 2 0 l134 20207155 (1020201136 20207157 (1020201137 d020207158 <1020201140 (1020207161 20201141 (1020207162 <1020405091 20201150 20207J71 20201152 (1020207173 20201153 20207174 (1020201156 20207177 amount of sera used (mL) 0.02 0.02 0.02 0.02 0.07 0.07 0.2 0.2 0.02 0.02 0.05 0.05 0,07 0.07 0.07 " 0'4 0.04 _ _ 0.07 0.07 0.1 0.1 dilution (diluted sera) 100 100 100 100 40 40 40 40 100 100 40 40 40 40 40 50 50 40 40 40 40 additional dilution 1 1 1 1 1 1 1 1 1 1 i 1 10 1 1 1 1 1 ____ 1______ 1 1 Inst Cone. (ng/mL of Methanol) 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 !oq for samples (ng/mL o f Sera) 400 400 400 400 400 400 400 400 400 400 400 400 201 400 400 400 400 400 400 400 400 Cone, of PFOS in Sample (n g /m l 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. s a m p le s 400 400 400 400 400 400 19252 0761 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/m!)) x (additional dilution) x (dilution{diluted sera)) dilution(diluted sera)* final voiume(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- dtlufion(difuted 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 %RSO 26.8 wife 3M Environmental Laboratory Page 56 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL LIVER DATA SUMMARY, BLANKS AND SPIKES 3M Sample Number E01-1342-36000 EOM 342-36026 E01-1342-36031 E01-1342-36036 Sampled Date Sample Description 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 <1020211037 d02Q212025 0020301024 d020218025 amount o f liver weighed (g) or water a'rquofrd (mL) 1.0000 1.0000 1.0000 1.0000 Water Added for H o m g c n iz in g (mL} 9.00 9.00 9.00 9.00 Final Volume (mL) 2.00 2.00 2.00 2.00 Amount of liver in diluted sample (g) or water (ml 1 additional dilution 0.100000 0.100000 0.100000 0.100000 1 1 1 1 Inst Cone. (ng/mL of r.lefnano!) 2.51 251 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 fiver-day 3 Blank female liver-day 4 0020211038 (J020212026 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 mate fiver-day 1 Blank male liver-day 2 Blank male liver-day 3 Blank male liver-day 4 d020211039 <1020212027 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 1er samples irg/q) 50.2 50.2 10.0 20.0 Cone, of PpQS 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 50.2 50.2 10.0 10.0 20.0 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 amount o f liver weighed (g) Water Added for Homgenizing (mL) Final Volume (mL) 0020211040 d020212028 d020301027 0020218028 5.0353 5.0353 5.0353 5-0353 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 Amount of liver in additional diluted sample fg) dilution 0.100635 0.100635 0.100635 0.100635 1 1 1 1 tnst Cone. (ng/mL of Methanol) 29.01 28.77 26.03 26.33 loq for samples (ng/gi 50.2 50.2 10.0 20.0 Cone, of PFOS in Sample (ng/g of Livers 577 572 517 523 Ree 115.5 114.6 103.7 104.9 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 0020211041 (J020212029 120301028 320218029 5.0353 5.0353 5.0353 5.0353 45.0 45.0 45.0 45.0 2.00 0.100635 1 29.78 50.2 2.00 0.100635 1 28.53 50.2 2.00 0.100635 1 26.99 10.0 2.00 0.100635 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 tSIirtion) 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 devof % 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 eri cone 31.25 true vaf. 499 avg%rec 111.6 avg cone 558.1 std dev of cone 26.67 3M Environmental Laboratory Page 57 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL LIVER versus unextracted curve original Absolute Recoveries 3M S a m p le N u m b e r S a m p le D e s c rip tio n In s t. F ile N a m e a m o u n t o f liv o r w e ig h e d fq) W a te r A d d e d fo r H o m g c n iz m g (mL) F in a l V o lu m e (m L) A m o u n t of liv o r in d ilu te d s a m p le fq ) a d d itio n a l d ilu tio n In s t C one (n g /m L 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-l0ppb-1 (200ng/g) qc-10ppb-2 (200ng/g) qc-10ppb-3 (200ng/g) 0C-75DPb-1 (1496 nq/q) q c - 7 5 p p b -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) 0020211047 (J020211048 d020211049 d020211050 d02021l051 d020211052 (J020211053 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 95.85 2.00 0.100635 1 90.27 2.00 0.100635 92.58 2.00 0.100635 169.63 2.00 0.100635 167.X 2.00 0.100635 174.15 lo q to r s a m p le s in g /g ) 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 C o n e o f P F O S in S a m p le (n g /g o f L iv e r) 270 253 264 1905 1794 1840 3371 3319 3461 for ali OC 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 S a m p le N u m b e r S a m p le D e s c rip tio n In s t. F ite N a m e a m o u n t o f hve? w e ig h e d (q \ Water Added for H o m g e n iz in g (m L| F in a l V o lu m e (m L ) E01-1342-36017 E01-1342-36018 E01-1342-36019 E01-1342-36020 E01 -1342-36021 01 -1342-36022 E01-1342-36023 EOt-1342-36024 E01-1342-36025 A s Not within +/-25% criteria. qc-10ppt>1 (200ng/g) 0020405136 qc-10ppb-2 (200ng/g) d0204Q5137 qc-10ppb-3 (200ng/g) d020405138 qc-75ppb-1 (1496 ng/g) d020405139 qc-75opb-2 (1496 ng/g) d020405140 qc-75ppb-3 (1496 ng/g) dO20405141 qc-150ppb-1 (2993 ncg) d0204(S142 qC-150ppb-2 (2993 ng/g) d020405143 qc-150ppt>-3 (2993 ng/g) 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.X 2.00 2.00 2.00 Cone o! PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional tflution} 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)) A m o u n t of liv e r m d ilu te d s a m p le (g) a d d itio n a l d ilu tio n 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 C o n e (n g /m L of M e th a n o l) 14.86 14.24 14.77 1X.51 94.89 101.10 187.94 172.20 180.27 loq for s a m p le s ("9 -9 ) 1X 100 1X 1X 100 100 100 100 100 C o n e o f P F O S in S a m p le (n g /g o f L iv e r) 295 283 294 1996 1886 2009 3735 3422 3583 for ail QC in rerun analysis (absolute recovery) avg% rec 132.1 std dev 11.79 A A A A A A e R oc 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 v g 123.4 std dev 3.725 %RSD 3.018 avg 113.1 std dev 2.395 %RSD 2.119 ROC 147.7 141.5 146.8 133.5 126.1 134.3 124.8 114.3 119.7 avg 145.3 std dsv 3.329 %RSD 2.291 avg 131.3 std dev 4.554 %RSD 3 468 avg 1196 std dev 5 226 %RSQ 4.369 fV * 3M Environmental Laboratory Page 58 Report EO1-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study number E01-1245 - QUAIL LIVER versus extracted curve______ origina) QC Sample Recoveries 3M Sample Number Sample Description Inst Filo Name amount o f liver weighed (g) Water Added for Homgonizing (m l) Final Volume (mL) Amount of liver in additional diluted sample (g) dilution Inst Cone. (ng/mL of Methanol- 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-t (200ng/g) qc-10ppb-2 {200ng) qc-10ppb-3 (200ng/g) qc-75ppt>-1 (1496 ng/g) qc-75ppb-2 (1496 ng/g) qc*75ppb-3 (1496 ng/g) qc-150ppt>-1 (2993 ng/g) qc-1 S0ppb-2 (2993 ng/g) qc-150ppb-3 (2993 ng/g) (J020211047 CJ020211048 d020211049 d020211050 d020211051 d020211052 0020211053 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 45.0 450 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 1 14.20 1 13.41 1 13.91 1 89.60 1 84.54 1 86.63 1 155.46 1 153.16 1 159.43 loq tor samples i^ 'g i 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 Cene, of PFOS m Sample (ng/g ot Liver) 282 267 276 1781 1680 1722 3090 3044 3168 lor all QC in original analysis (recovery) evg%ree 118.9 std dev 15.17 A A A Study number E01-1245 - QUAIL LIVER versus extracted curve rerun 3M Sample Number Sample Description Inst. File Name amount o f liver weighed (gi Water Added for H o m g c n iz in g (m l) Final Volume (m l) Amount of liver in additional diluted sample (g) dilution Inst Cone. {ng/mL of Methanol) loq for samples (ng'g) Cone, of PFOS in Sample (ng'g of Ltver) E01-1342-36017 01*1342*36018 E 0 M 342-36019 E01-1342-36020 E01-1342-36G21 01-1342-36022 EOM 342*36023 E01-1342*36024 01-1342-36025 A = Not within +/-25% criteria. qc-10ppb-1 (200ng/g) qc-1Cppb-2 (200ng/g) qc-10ppb-3 (200ng/g) qc-75ppt>-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/q) 020405136 d020405137 d020405138 3020405139 d020405140 d020405141 d020405142 d020405143 (3020405144 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 weighedfg)) 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 0.100635 1 13.43 100 1 12.91 100 1 13.35 100 1 84.61 100 1 79.97 100 - 1 85.09 100 1 155.84 100 1 143.14 100 1 149.66 100 267 257 265 1682 1589 1691 3097 2845 2974 for all QC in rerun analysis (recovery) avg %rec 113.8 std dev 14.48 A A A Rcc. 141.1 133.3 138.2 119.0 112.3 115.1 103.2 101.7 105.9 avg 137.5 atd 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 c Rec 133.5 128.3 132.7 112.4 106.2 113.0 103.5 95.05 99.38 avg 131.5 std dsv 2.782 %RSD 2.116 avg 110.6 std dev 3.756 %RSD 3.398 avg 99.30 std dev 4.217 %RSO 4.247 O ,J $ k` 3M Environmental Laboratory Page 59 Report E01-1245 Study number E01-1245 - QUAIL LIVER DATA SUMMARY PFOS: A Reproduction Study with the Nothem Bobwhite 3M S a m p le Num ber S a m p le d D a te S a m p le D e s c rip tio n In s t F ile Nam e a m o u n t o f liv e r w e ig h e d (g ! W ate r A d d e d 1or H o m g e n iz in g (m l) E0M 342-X321 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 Oppm a.i. 334 F Uver d020211066 EOI-1342-30375 06/19/2001 0 ppm a.i. 336 F Uver *>20211068 E01-1342-30393 06/19/2001 0 ppm a.i. 338 F Uver d020211070 EOM 342-30411 06/19/2001 0 ppm a.i. 340 F Uver d020212034 E 0 M 342-30429 05/31/2001 0 ppm a.i. 342 F Liver d020212036 E 0 M 342-3O447 06/19/2001 0 ppm a.i. 344 F Liver d020212038 E01-1342-3O465 06/19/2001 0 ppm a.i. 346 F Uver 020212040 E01-1342-30483 06/19/2001 0 ppm a.i. 348 F Uver d020212042 E 0 M 342-30501 06/19/2001 0 ppm a.i. 350 F Liver d020212047 E 0 M 342-30519 06/19/2001 0 ppm a.i. 352 F Uver d020212049 E01-1342-30537 06/19/2001 0 ppm a.i. 354 F Uver CJO20212051 E01-1342-30555 06/19/2001 0 ppm a.i. 356 F Liver *>20212053 E01-1342-30573 06/19/2001 0 ppm a.i. 358 F Uver d020212055 E 0 M 342-3O591 E01-1342-30609 06/19/2001 0 ppm a.i. 360 F Uver 06/19/2001 Oppm a i. 362 F Uver (J020212059 *>20212061 E01-1342-30627 06/19/2001 0ppm a.i. 364 F Uver d020212063 E01-1342-30645 E 0 M 342-30663 06/19/2001 0 ppm a.i. 366 F Uver 06/19/2001 0 ppm a.i. 368 F Uver d020212065 d020212067 E01-1342-30681 06/20/2001 10 ppm a.i. 370 F Liver dO20301032 E01-1342-30699 01-1342-30717 06/20/2001 10 ppm a.i. 372 F Uver 06/20/2001 10 ppm a.i. 374 F Liver d020301033 d020301034 E01-1342-30735 06/20/2001 10 ppm a.i. 376 F Uver *120301035 E01-1342-30753 E01-1342-30771 06/20/2001 10 ppm a.i. 370 F Liver 06/20/2001 10 ppm a.i. 380 F Liver d020301036 {J020301037 E01-1342-30789 06/20/2001 10 ppm a.i. 382 F Uver d020301038 E01-1342-30820 04/11/2001 10 ppm a.i. 384 F Uver d020301039 E1-1342-30838 E01-1342-30856 E01-1342-30874 E01-1342-30892 E01-1342-30910 E01-1342-30928 E01-1342-30946 E01-1342-30964 E01-1342-30982 06/20/2001 10 ppm a.i. 386 F Liver 06/20/2001 10 ppm a.i. 388 F Uver 06/20/2001 10 ppm a.i. 390 F Liver 06/20/2001 10 ppm a.i. 392 F Uver 06/20/2001 10 ppm a.i. 394 F Uver 06/20/2001 10 ppm a.i. 396 F Uver 06/20/2001 10 ppm a.i. 398 F Liver 06/20/2001 10 ppm a.i. 400 F Uver 06/20/2001 10 ppm a.i. 402 F Liver d020301040 dO20301041 P020301045 d020301046 *>20301047 d020301048 d020301049 *>20218033 d020218034 E01-1342-31000 E0M342-31018 06/20/2001 10 ppm a.i. 404 F Liver 06/20/2001 10 ppm a.i. 406 F Uver d020218035 d020218036 E01-1342-31036 E01-1365-31184 E01-1365-31204 E01-1365-31219 E01-1365-31224 06/20/2001 10 ppm a.i. 408 F Uver 07/23/2001 0 ppm a.i. 246 F Uver, Offspring 07/23/2001 0 ppm a.i. 259 F Uver, Offspring 07/23/2001 0 ppm a.i. 273 F Liver, Offspring 07/23/2001 0 ppm a.i. 277 F liver. Offspring d020218037 (1020218038 (1020218041 <1020218047 (*>20218048 E0M365-31240 07/23/2001 10 ppm a.i. 517 F Uver, Offspring *>20218051 E01-1365-31250 E01-1365-31260 E01-1365-31275 07/23/2001 10 ppm a.i. 525 F Uver, Offspring d020218053 07/23/2001 10 ppm a.i. 538 F Uver, Offspring *>20405158 07/23/2001 10 ppm a.i. 557 F Uver, Offspring d02021806l E 0 M 365-31280 07/23/2001 10 ppm a.i. 561 F Uver, Offspring d020218062 ' Concentration <LOQ 1.0749 1.0469 1.0232 1.0188 1.0543 1.0182 1.0904 1.0087 1.0996 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 9.00 900 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 F in a l V o lu m e im L ) 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 .X 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 .X 2 .X 2 .X 2 .X 2 00 2 .X 2 .X 2 .X 2 .X 2.X 2.X A m o u n t o f liv e r in d ilu te d s a m p le fg ) a d d itio n a l d ilu tio n 0.106691 0.104201 0.102083 0.101689 0.104861 0.101635 0.108063 0.1X782 0.108893 0.109228 0.102128 0113379 0.105172 0.102799 0.1X184 0.105243 0.1038X 0.103264 0.105163 0.108205 0.107745 0.106682 0.107054 0.109228 0.1X189 0.107364 0.101097 0.0982480 0.11X04 0.107833 0.1X701 0.100540 0.102137 0.103318 0.102155 0.101626 0.107816 0.101554 0.100423 0.100396 0.103866 0.109660 0.10X72 0.107276 0.108470 0.0957803 0.103782 0.X37286 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 In s t C one (n g 'm L o f M c th a n o ij 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 251 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 319.18 236.52 2 X .4 8 217.55 187.79 214.22 166.41 2 X .0 4 410.20 187.91 245.X 298.53 256.81 253.83 224.76 255.94 318.13 275.17 294.79 228.77 1.X 1.X 1.00 1.00 308.44 336.48 72.X 219.12 238.83 Cone of PFOS in Sample (ng/g) = {Inst conc(ng/mL)) x (additional rflution} 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)) lo q fo r s a m p le s C o n e c f P F O S in S a m p le (n g 'g o f 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 502 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 1X 20.0 20.0 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 505 50.2 50.2 50.2 505 5925 4434 5427 3983 3749 3991 3292 4194 7548 3485 4866 S939 5029 4914 44X 5037 5X1 5419 5871 4557 20.0 20.0 20.0 20.0 5687 7026 1X33 4676 4586 avg 50.2 std dev 0 %RSD - re/eefed 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 o nVV Report E01-1245 Study number E01-1245 - QUAIL LIVER DATA SUMMARY 3M Sample Number Sampled Date Sample Description Inst. File Name Water Added amount of live' for weighed (g Homgenizing (m l) E 0 M 342-30312 06/19/2001 0 ppm a.i. 329 M Liver <20211061 E01-1342-30330 06/19/2001 0 ppm a.i. 331 M Liver d020211063 E01-1342-3034B 06/19/2001 0 ppm a.i. 333 M Uver 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 <20211069 E01-1342-30402 06/19/2001 0 ppm a.i. 339 M Liver <20212033 E01-1342-30420 06/19/2001 Oppm a.i. 341 M Liver 20212035 E01-1342-30438 06/19/2001 0 ppm a i. 343 M Uver d020212037 E01-1342-30456 06/19/2001 0 ppm a.i. 345 M Uver d020212039 E01-1342-30474 06/19/2001 0 ppm a.i. 347 M Uver d020212041 E01-1342-30492 06/19/2001 0 ppm a.i. 349 M Uver d020212046 E01-1342-30510 06/19/2001 Oppm a.i. 351 M Uver 20212048 E01-1342-30528 06/19/2001 Oppm a.i. 353 M Liver d020212050 E01-1342-30546 06/19/2001 0 ppm a.i. 355 M Uver d020212052 E01-1342-30564 06/19/2001 0 ppm a.i. 357 M Uver 20212054 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 Uver d020212060 E01-1342-30618 06/19/2001 Oppm a.i. 363 M Uver 20212062 E01-1342-30636 06/19/2001 0 ppm a.i. 365 M Uver 20212064 E01-1342-30654 06/19/2001 0 ppm a.i. 367 M Uver 20212066 E01-1342-30672 06/20/2001 10 ppm a.i. 369 M Uver 20301050 E01-1342-30690 06/20/2001 10 ppm a.i. 371 M Uver 20405148 E01-1342-30708 06/20/2001 10 ppm a.i. 373 M Uver 20405149 E01-1342-30726 06/20/2001 10 ppm a.i. 375 M Liver 20301053 E01-1342-30744 06/20/2001 10 ppm a.i. 377 M Uver 20301054 E01-1342-30762 06/20/2001 10 ppm a.i. 379 M Uver 20405150 E01-1342-30780 06/20/2001 10 ppm a.i. 381 M Uver 0020405151 E01-1342-30798 06/20/2001 10 ppm a.i. 383 M Uver 20301060 E01-1342-30829 06/20/2001 10 ppm a.i. 385 M Uver 20405152 E01-1342-30847 06/20/2001 10 ppm a.i. 307 M Uver 20301062 E01-1342-30865 06/20/2001 10 ppm a.i. 389 M Uver d020301063 E01-1342-30883 06/20/2001 10 ppm a.i. 391 M Uver 20301064 E01-1342-30901 06/20/2001 10 ppm a.i. 393 M Uver 20405153 E01-1342-30919 06/20/2001 10 ppm a.i. 395 M Uver 20301066 E01-1342-30937 06/20/2001 10 ppm a.i. 397 M Uver 20X1067 E01-1342-30955 06/20/2001 10 ppm a.i. 399 M Uver 20218064 E01-1342-30973 06/20/2001 10 ppm a.i. 401 M Uver 20405159 E01-1342-30991 06/20/2001 10 ppm a.i. 403 M Uver 20405160 E01-1342-31009 06/20/2001 10 ppm a.i. 405 M Uver 20405161 E01-1342-31027 06/20/2001 10 ppm a.i. 407 M Uver 20405162 E01-1365-31194 E01-1365-31199 07/23/2001 0 ppm a.i. 253 M Liver, Offspring 20218039 07/23/2001 0 ppm a.i. 256 M Uver, Offspring 20218040 E01-1365-31209 E01-1365-31214 07/23/2001 0 ppm a.i. 266 M Uver. Offspring 20218042 07/23/2001 0 ppm a.i. 270 M Uver, Offspring 20218046 E01-1365-31229 07/23/2001 0 ppm a.i. 283 M Uver, Offspring 20218049 E01-1365-31234 07/23/2001 0 ppm a.i. 291 M Uver, Offspring 20218050 E01-1365-31245 07/23/2001 10 ppm a.i. 522 M Uver, Offspring <20405157 E01-1365-31255 07/23/2001 10 ppm a.i. 533 M Liver. Offspring <20218054 E01-1365-31265 07/23/2001 10 ppm a.i. 545 M Uver, Offspring <20218059 E0M365-31270 07/23/2001 10 ppm a.i. 552 M Uver, Offspring <20218060 E01-1365-31285 07/23/2001 10 ppm a.i. 568 M Uver, Offspring <20218063 * Concentration <L0Q 1.0050 1.05X 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 .X 9.00 9.00 9.00 9.00 9.00 9 .X 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.X 6.X 4.50 4.50 7.20 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 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.Q0 2.00 2.00 2.00 2.00 2.X 2.00 2.00 2.00 2,00 2.00 2.00 2 00 2.00 2.00 2.00 2.00 2.00 Amount of liver a d d it io n a l in diluted sample (g) dilution 0.100450 0.104745 0.103032 0.100558 0.1X415 0.107904 0.102110 0.100809 0.109317 0.105243 0.103211 0.105217 0.101644 0.108019 0.103425 0.1083X 0.1X755 0.107302 0.108169 0.102933 0.100090 0.107639 0.1X477 0.108X1 0.1X119 0.102799 0.104487 0.100989 0.106061 0.1X826 0.102781 0.108443 0.107444 0.104380 0.102772 0.102477 0.10X37 0.1X171 0.10X14 0.108523 0.0X6137 0.0992043 0.0900819 0X84975 0.0956851 0.101079 0.104159 0.0987511 0.0931X7 0.1063X 0X52614 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 100 100 100 100 1 1 1 1 1 1 10 1 1 1 1 PFOS: A Reproduction Study with the Nothem Bobwhite Inst Cone ( n g m l ot Methanol) 2.51 2.51 10.76 2.51 2.51 2-51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 393.21 422.59 441.96 X I . 61 409.88 466.19 417.98 371.06 442.X 407.70 X 1 .3 7 381.87 466X 339 50 257.X 322.88 76.08 72.X 71.24 71.64 1.00 IX 1.00 1.00 1.00 1.00 71.33 244.12 259.80 318.X 31049 loqfor samples (ng/g) 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 X .2 50.2 X .2 X .2 X .2 X .2 502 X .2 X .2 X .2 X .2 X .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 IX 20.0 20.0 20.0 20.0 Cone, of PFOS in Sample (ng/g of Liver) X .2 50.2 209 50.2 X .2 50.2 50.2 50.2 X .2 50.2 50.2 50.2 X .2 X .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 867X 65051 X148 6X15 1473X 145531 138311 132028 20.0 20.0 20.0 20.0 20.0 20.0 1X96 4944 5575 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 dav 28517 %RSD 32J2 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 fiver weighed (g)/ (water added for homogenizing (mleg)) + amount of liver weighed(g)) 3M Environmental Laboratory Page 61 Report EOI-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Attachm ent C : S am ple C hrom atogram s, C alibration Info rm atio n, and In strum ent Information 3M Environmental Laboratory 3M Environmental Laboratory Page 62 of 226 Page 62 i ;v Report E01-1245 Report E01-1245 Methanol Solvent Blanks PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 63 of 226 Page 63 G-OdLO fj, Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 64 of 226 Page 64 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhlte 3M Environmental Laboratory 3M Environmental Laboratory Page 65 of 226 Page 65 Report E01-1245 Report E01-1245 W ater Blank Samples PFOS: A Reproduction Study with the Nothem 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 67 of 226 Page 67 . if i'jl A A Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 66 of 226 Page 68 Report E01-1245 Report E01-1245 Sera Blank Samples PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 69 of 226 c Page 69 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 71 of 226 Page 71 r - .-3 1 ' ` r X l ' V / i Report E01-1245 Report E01-1246 Female Liver Blank Samples PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite i .I 3M Environmental Laboratory 3M Environmental Laboratory Page 72 of 226 Page 72 rV - . C' ? 1-JL. *LAV* " 'f Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 73 of 226 Page 73 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 74 of 226 Page 74 Report EOI-1245 Report E01 -1245 Male Liver Blank Samples PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 75 of 226 Page 75 Report E01-1245 Report E0 1 -1 2 4 5 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 76 of 226 GOl Page 76 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 77 of 226 Page 77 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 78 of 226 Page 78 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-371,10ppm mate, adult, d100,02-01-02 3M Environmental Lab d 020207119 Sm (M n, 1x2) 100-1 6.72 _ 6891S davey 070799 08-Feb-2002, 05:57:53 1: M RM of 1 Channel ES T IC 5 .8 4 e 5 A rea 3M Environmental Laboratory 3M Environmental Laboratory Page 79 of 226 Page 79 r*JL Report E01-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 80 of 226 Page 80 mt Report E 01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 81 of 226 Page 81 0 /?> Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Ion Study with the Northern Bobwhite 454-108-377,10ppm male, adult, d100,02-01-02 3M Environmental Lab d 020207122 Sm (M n. 1x2) 100-1 6.72 40589 davey 070799 08-Feb-2002, 06:31:14 1: M RM o f 1 Channel EST IC 3 .9 9 e 5 A rea %- " IlM I 1.00 2.00 3 .0 0 t r t 111 m 4 .0 0 5.00 6.00 7 .0 0 8.00 " ' i" 9 .0 0 Tim e 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 83 of 226 Page 83 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite tudy with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 84 of 226 Page 84 Q .f''X .'O ; Report EOl-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproductor the Northern Bobwhite 454-108-383,10 ppm male, adult, d100,02-01-02 3M Environmental Lab d020207130 Sm (M n, 1x2) 100-1 6.72 . 61109 davey 070799 08-Feb-2002, 09:08:05 1: M RM o f 1 Channel E S T IC 5 .3 7e 5 A rea %- 0 n i ' ' ti i i 1.00 2.00 rri-irr rr rtTV 3 .0 0 4 .0 0 5.00 6.00 11 ' 7.00 8.00 9 .0 0 rr ri Tim e 1 0 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 85 of 226 Page 85 C: n i o 'll Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite R F Q S :A IReproduction Study with the Northern Bobwhite b i l h i !! i I i ! l . '! : 454-108-385,10ppm male, adult, d100,02-01-02 3M Environmental Lab d020207131 Sm (M n, 1x2) 1<Xh 6.72 49730' davey 070799 08-Feb-2002, 09:19:11 1: M RM of 1 Channel ES T IC 5 .2 8 e 5 A rea 3M Environmental Laboratory 3M Environmental Laboratory 7 .0 0 8.00 r r r r r r r r r rrnn Tim e 9.00 10.00 Page 86 ol 226 1> Page 86 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS;A the Northern Bobwhite 454-108-387,10ppm male, adult, d100,02-01-02 3M Environmental Lab d 020207132 Sm (M n, 1x2) 100n 6.72 74463' davey 070799 08-Feb-2002, 09:30:15 1; M RM of 1 Channel ES T IC 7 .9 2e 5 A rea %- 1.00 2.00 3 .0 0 rr-p 4 .0 0 n-p-r 5.00 6.00 i' r r r p r r 7.00 8.00 iT fiT i >i m >11 Tim e 9 .0 0 1 0 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 87 of 226 Page 87 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Dosed Group Adult Female Sera Samples 454*108-370,10ppm female, adult, 02-01-02 3M Environmental Lab d020203026 Sm (M n, 1x2) 100 -i 6.75 318463 davey 070799 0 3 -F e b -2 0 0 2 ,16:19:31 1: M RM o f 1 Channel ES T IC 3 .3 9e 6 A rea 1.00 2.00 3 .0 0 4 .0 0 5 .0 0 6.00 ttrrr 7 .0 0 " ..............I ' 8.00 9.00 Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 88 of 226 0^ Report EOI-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-372,10ppm female, adult, 02-01-02 3M Environmental Lab d020203027 Sm (M n, 1x2) 10(H 6.75 _ 317500 davey 070799 0 3 -F e b -2 0 0 2 ,16:30:40 1: M RM o f 1 C hannel E S T IC 3.3B e6 A rea %- 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 89 of 226 c * '; i Page 89 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A ReprojckKjtiqn Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 90 of 226 Page 90 ^ 1 aV Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 91 of 226 Page 91 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite r A Reproduction Study with the Northern Bobwhite i; ktl ' M - i)ll : : :: =! 3M Environmental Laboratory 3M Environmental Laboratory Page 92 of 226 Page 92 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 93 of 226 Page 93 G O iir - G Report EOI-1245 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A -Reproduction Study with the Northern Bobwhite I 1 3M Environmental Laboratory 3M Environmental Laboratory Page 94 of 226 Page 94 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 454-108-386,10ppm female, adult, 02-01-02 3M Environmental Lab d020203033 Sm (M n, 1x2) 100 -i 6.74 461800' davey 070799 0 3 -F e b -2 0 0 2 ,17:37:10 1: M RM o f 1 Channel E S - T IC 4 .8 8e 6 A rea %- i" 1 1.00 2.00 3 .0 0 4 .0 0 5 .0 0 6.00 7 .0 0 ' " l ' n 'r r rj Tr i 8 .0 0 9 .0 0 n Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 95 of 226 Page 95 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reprxfpctlon Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 96 ot 226 Page 96 G C X * lV Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 97 of 226 Page 97 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reprodurtion SjtMdy with the Northern Bobwhite Dosed Group Male Offspring Sera Samples 454-108-522,10ppm male, offspring, 02-01-02 3M Environmental Lab d020207169 Sm (M n, 1x2) 100-, 6.73 _ 471945 davey 070799 08-Feb-2002,16:20:52 1: MRM of 1 Channel ESTIC 4.74e6 Area %- 1.00 2.00 T-r-p 3 .0 0 4 .0 0 5.00 rr-p-i 6.00 7 .0 0 8.00 0 00 i Tim e 3M Environmental Laboratory 3M Environmental Laboratory Page 98 of 226 Page 98 Report EOI-1245 PFOS: A Reproduction Study with the Nothem 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite r 454-108-545,10ppm male, offspring, 02-01-02 3M Environmental Lab d 020207173 Sm (M n, 1x2) 100-1 6.73 _ 352415 davey 070799 08-Feb-2002,17:05:10 1:M R M of 1 Channel EST IC 3 .6 6e 6 A rea %- 3M Environmental Laboratory 3M Environmental Laboratory Page 100 of 226 Page 100 G O lll^ a Report E01-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A with the Northern Bobwhite i'ft 454-108-552,10ppm male, offspring, 02-01-02 3M Environmental Lab <1020207174 Sm (M n, 1x2) 100- 6.73 375404 davey 070799 08-Feb-2002,17:16:14 1: M RM erf 1 C hannel ES T IC 3 .8 3e 6 A rea %- 1.00 T -p -n 2.00 3 .0 0 4 .0 0 5.00 6.00 7 .0 0 8.00 ........... Tim e 9.nn m oo 3M Environmental Laboratory 3M Environmental Laboratory Page 101 of 226 Page 101 f ; r;' ,1- i Report EOI-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite with the Northern Bobwhite ! -i I 3M Environmental Laboratory 3M Environmental Laboratory Page 102 of 226 Page 102 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 (M n, 1x2) 100-1 6.73 236874 davey 070799 08-Feb-2002,16:09:45 1 :M R M o f 1 Channel ES T IC 2 .4 9e 6 A rea %- 1.00 i ' ' HTrT 2.00 3 .0 0 4 .0 0 I I I ! I I I I 1I I I I 5.00 6.00 7.00 T-|-TTT 'IT T T I 8.00 9 .0 0 i Tim e 1 0 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 103 of 226 Page 103 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite wtth the Northern Bobwhite 454-108-525, lOppm female, offspring, 02-01-02 3M Environmental Lab d 020207170 Sm (M n, 1x2) 100-1 davey 070799 08-Feb-2002,16:31:56 1: MRM of 1 Channel ES- %' 0 1.......1'! I l l ........ . . ..... I I I M l |T|"r i"|-r'i' i i ! ...... I _________ 1.00 2 .0 0 3 .0 0 4 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 104 of 226 Page 104 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite with the Northern Bobwhite 454*108-538,10ppm female, offspring, 02-01-02 3M Environmental Lab <1020207172 Sm (M n, 1x2) 100-1 6.73 393873' davey 070799 08-Feb-2002,16:54:05 1: M RM o f 1 C hannel E S T IO 3 .9 6 e6 A rea 1.00 " i ' ' ' i ' " i ' i ' 2.00 3 .0 0 " ' i ....... 4 .0 0 nm-p-r-i 11" 11 1' 5.00 6.00 7 .0 0 8.00 9 .0 0 T , Tim e in nn 3M Environmental Laboratory 3M Environmental Laboratory Page 105 of 226 Page 105 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 106 of 226 Page 106 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite the Northern Bobwhite 454-108-561,10ppm female, offspring, 02-01-02 3M Environmental Lab d 020207176 Sm (M n. 1x2) 100-1 6.73 333841 davey 070799 08-Feb-2002,17:38:25 1: M RM o f 1 Channel ES T IO 3 .4 3e 6 A rea %- 0 TI TI I " I I I PI I 1I " I " I " " 11111111 11111 " I 1.00 2 .0 0 3 .0 0 4 .0 0 3M Environmental Laboratory 3M Environmental Laboratory 9 .0 0 t t . Tim e 10.00 Page 107 of 226 Page 107 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A in Study vtfth the Northern Bobwhite Dosed Group Male Liver Samples E01-1342-30762,10 ppm a.i. 379 M Llver,d10 3M Environmental Lab d 020301058S m (M n, 1x2) 1 0 0 -, 6.74 466858' davey 070799 02-Mar-2002, 01:32:50 1:M R M of 1 Channel ES T IC 4 .7 6e 6 A rea % i 1 .0 0 2 .0 0 3 .0 0 4 .0 0 5 .0 0 6 ,0 0 7 .0 0 8 .0 0 9 .0 0 10.00 1 3M Environmental Laboratory 3M Environmental Laboratory Page 108 of 226 Page 108 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 109 of 226 Page 109 Report E 01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 110 of 226 Page 110 no i U r- ,J U Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 111 of 226 Page 111 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 112 of 226 Page 112 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Report E01 -1245 E01-1342-30847,10 ppm a.i. 387 M Liver,d10 3M Environmental Lab 0020301062 Sm (M n. 1x2) 100-1 PFOS: A Reproduction Stjudty with the Northern Bobwhite 0.74 439040- I davey 070799 02-Mar-2002,02:17:20 1: M RM o f 1 Channel ES T IC 4 .5 0e 6 A rea m tT T T n Tim e 1.00 2.00 3 .0 0 4 .0 0 5 .0 0 6.00 7 .0 0 8.00 9 .0 0 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 113 of 226 Page 113 Report EOI-1245 Report E01 >1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 114 of 226 Page 114 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 (M n, 1x2) 1 0 0 -: 6.74 470298 davey 070799 02-Mar-2002, 01:43:59 1 :M R M o f 1 Channel EST iC 4 .79e6 A rea %J 1.00 2.00 3 .0 0 4 .0 0 i- n - p - n 5 .0 0 6.00 7 .0 0 8.00 ' nr............... i Tim e 9 .0 0 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 115 of 226 Page 115 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 116 of 226 Page 116 Report EOl-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 117 o f 226 Page 117 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342-30838,10 ppm a.i. 386 F Liver 3M Environmental Lab d020301040 Sm (M n, 1x2) 100-1 6.73 446002 davey 070799 01-Mar-2002,22:12:49 1:M R M of 1 Channel ES T IO 4 .7 6e 6 A rea %- 'i" " i 1 1.00 ............. ' 1'"r r 2 .0 0 3 .0 0 I ! I I 1-i-p I M I I I I I I I rn I I 4 .0 0 5 .0 0 6 .0 0 7 .0 0 8.00 9 .0 0 i Tim e 1 0 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 119 of 226 Page 119 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 121 o f 226 Page 121 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Sti^dy with the Northern BobwhKe E01-1342-3073S, 10 ppm a.I. 376 F Liver 3M Environmental Lab d 020301035 Sm (M n, 1x2) 100-1 6.73 275483 davey 070799 01>Mar-2002,21:17:14 1:M R M o f 1 Channel ES T IO 3 .1 9e 6 A rea %- 1 .0 0 2 .0 0 3 .0 0 4 .0 0 5 .0 0 6 .0 0 7 .0 0 8 .0 0 9 .0 0 1 0 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 122 of 226 Page 122 *"v t * G O lli Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 123 of 226 Page 123 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS; f produrr the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 124 of 226 Page 124 c Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PROS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 125 of 226 Page 125 4 'V7 '' f , Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342-30099,10 ppm a.i. 372 F Liver 3M Environmental Lab d 020301033 Sm (M n, 1x2) 100-1 6.72 295007' davey 070799 01-Mar-2002,20:55:05 1: M RM of 1 Channel ES T IC 3 .36e6 A rea %- 1' i " 111' ' 11 1 1 1 1 1 1 1 1 1.00 2.00 rp -I 3 .0 0 4 .0 0 5 .0 0 rTT-T-i 6.00 7.00 i 8.00 ' r i r i-' i 9 .0 0 i Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 126 of 226 Page 126 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 127 of 226 Page 127 DO. * n Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Report E01-1245 Dosed Group Male Offspring Liver Sa I|Q & A foproduotion Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 128 of 226 Page 128 V. r ,,A. JL Report EOl-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Report E01 -1245 P f OS: A Reproduction Study with the Northern BobwWte !i: F # E01-1365-31270,10 ppm a.1.552 M Liver, offspring 3M Environmental Lab d 020218060 Sm (M n, 1x2) 100-1 6.70 302473" davey 070799 18-Feb-2002,18:28:34 1: M RM of 1 C hannel E S T IC 3 .2 3e 6 A rea 1.00 2.00 3 .0 0 4 .0 0 5.00 6.00 7 .0 0 " 11" 111" " i " ' 8 .0 0 9 .0 0 i Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 129 of 226 Page 129 O O il; Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 130 of 226 Page 130 / 'i f - J J Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite E01-1342-30955,10 ppm a.i. 399 M Liver, d10 3M Environmental Lab d 02 0 2 18064 Sm (M n, 1x2) 100-1 6.73 305393 davey 070799 18-Feb-2002,19:12:56 1: M RM o f 1 Channel E S T IC 3 .1 1e 6 A rea %- 1 .0 0 ____ 2.00 3 .0 0 4 .0 0 5 .0 0 6 .0 0 7 .0 0 8 .0 0 9 .0 0 1 0 .0 0 3M Environmental Laboratory 3M Environmental Laboratory Page 131 of 226 Page 131 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ; 11 E01-1365-31255,10 ppm a.i. 533 M Liver, offspring 3M Environmental Lab d 020218054 Sm (M n, 1x2) 100-1 8.72 246631' davey 070799 18-Feb-2002,17:21:57 1:M R M of 1 Channel EST IC 2 .7 4e 6 A rea %- ' " I " ' n 1.00 2.00 3 .0 0 4 .0 0 5 .0 0 6.00 7 .0 0 8.00 9 .0 0 -"-n Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 132 of 226 Page 132 C ib ili Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS; A Reproduction Study with the Northern Bobwhite . i. i . ; j ; . : i.-, m , ; i i. >: . 3M Environmental Laboratory 3M Environmental Laboratory Page 133 of 226 Page 133 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Dosed Group Female Offspring Liver Samples E01-1365-31260,10 ppm a.i. 538 F Liver, offspring 3M Environmental Lab d 020218055 Sm (M n, 1x2) 100-1 6.73 446716' davey 070799 18-Feb-2002,17:33:05 1: M RM o f 1 Channel ES T IC 4 .5 9e 6 A rea %- 6.00 8.001.00 2.00 3 .0 0 4 .0 0 5.00 7 .0 0 9.00 Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 134 of 226 Page 134 Report EOI-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study wHh the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 135 of 226 Page 135 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: 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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 137 of 226 Page 137 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 138 o1226 Page 138 m Report EOI-1245 Report EO I-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Control Group Male Sera Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 130 of 226 Page 139 C Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Report E01-1245 PFOS: A Raprqdi W\ i| j f f|j in Study with the Northern Bobwhite Control Group Female Sera Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 140 of 226 Page 140 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite ^jpii the Northern Bobwhite Control Group Male Offspring Sera Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 141 of 226 Page 141 0011, Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite i (tie Northern Bobwhite Control Group Female Offspring Sera Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 142 of 226 ^ r ^ S f 142,. Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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 EOI-1245 Report E01 -1245 PFOS: A Reproduction Study with the Nothem 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 EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite the ^orthem Bobwhite Control Group Female Offspring Liver Sample 3M Environmental Laboratory 3M Environmental Laboratory Page 146 of 226 i.' Page 146 io Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 147 of 226 Page 147 Report EOl-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite the Northern Bobwhite Extracted Liver Calibration Curve Point 3M Environmental Laboratory 3M Environmental Laboratory Page 148 of 226 Page 148 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 149 of 226 Page-449 mu Report EOI-1245 Report E01 -1245 Extracted Sera Calibration Curve PFOS: A Reproduction Study with the Nothem Bobwhite PFpS : A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 150 of 226 Report EOI-1245 Report E01-1245 Extracted Liver Calibration Curve PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory 3M Environmental Laboratory Page 151 of 226 Page 151 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Mass Spectrometer Tune Settings Tuning Method Report Method: C:\MASSLYNX\DAVEY070799.PRO\ACQUDB\CENTREl Printed: Tue Mar 19 10:22:06 2002 Page 1 MS SOURCE ( ESP- ) Capillary Cone Hexapole 1 Aperture 1 Hexapole 2 Source Block Temp. De so1vat ion 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 oas Flows Cone Gas Desolvation Set Rdbl 13.0 13.0 0.7 2 11 1 11.0 11.0 1.0 650 1 11 0 - 641 Rdbk. 150.5 701.5 3M Environmental Laboratory 3M Environmental Laboratory Page 152 of 226 Page 152 co Report EOl-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem 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% CD%% 90.0 10.0 00..00 Valve A set to channel Valve B set to channel Flow (ml/min) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Lefl(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% 0.00 90.0 10.0 0.0 0.0 1.00 90.0 10.0 0.0 0.0 5.50 5.0 95.0 0.0 0.0 7.50 5.0 95.0 0.0 0.0 8.00 90.0 10.0 0.0 0.0 Flow 0.300 0.300 0.300 0.300 0.300 H P1100 LC Pum p E xtern al E ven t Tim etab le T he Tim etable contains 3 entries which are : Tim e Initial 0.00 0.10 Colum n Switch O fl Off O ft On Ofl Ofl C o n tad i O ff On OH C o n tact2 O fl O fl Ofl Corrtac O ft On Off H P 1100 A u to sam p ler Initial C onditions Draw Speed Eject S peed (pVm in) D raw Position (m m ) Stop Tim e (m ins) Injection Volum e(pl) V ial Num ber Therm ostat On Therm ostat T e m p e ra tu re fC ) 2 0 0 .0 200 0 .0 0 1 0 .0 0 1 0 .0 94 2 0 .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 Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Mass Spectrometer Scanning Parameters Scanning Method Report Method: C:\MASSLYNX\DAVEY070799.PRO\ACQUDB\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 1 Coll Energy (eV) Cone ( V 1 499.00 99.00 0.30 43 60 Function : 2 MRM of 1 Mass Pair ( ESP- ) Inter Channel Delay Span ( Daltons ) : Start Time ( Mins ) End Time ! Mins ) : Repeats : Channel Parent 1 513.00 Secs ) : Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) 219.00 0.30 20 Cone ( V ) 20 Page 1 3M Environmental Laboratory 3M Environmental Laboratory Page 154 of 226 Page 154 GC 1.1.G Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with die Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite At t a c h m e n t D : S a m p l e P r ep a r a tio n S h e e ts 3M Environmental Laboratory 3M Environmental Laboratory Page 155 of 226 Page 155 Report E01-1245 SPE Columns E x tr a c ^ r M o e T '0" Prep Date: Analysts initials: Urns Assgned sample number 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 1/31/2002 OK/WOJ v 3 lo ^ (2 Sample Number or description Blank H20-day 1 BlankmaNarcTsera-day 1 eyiJAfc E 2 c k | 3 H > 2 MaHerd Sera MS-day 1 6 > U /\iC *>* t-2>t-o'2 Mattart) Sera MSD 5 ppb sera curve 10ppb 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 Volume of sample filtered Type of column (ml) used and lot 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 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, 6mi, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, tot 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 w iisrsn g , btvu,-- lot 1290B2 Amount and spike mix used NA NA ^1 Oul of 02001 -53 / 10ul of 02001-53 ^ u l of 02001-52 4ul Of 02001-52 ^lOul of 02001-52 / 20ul of 02001-52 4ul of 02001-53 y 10ul of 02001-53 y 20ul of 02001 -53 tr 30ul of 02001-53 y" 40ul of 02001-53 S 10ul of 02001-52 ^ 10ul of 02001 -52 V 10ul of 02001-52 s 36ulof02001-15 L- 36ul of 02001-15 / 36ul of 02001-15 s 72ul of 02001-15 7 72ui of 02001-15 7 72ul of 02001-15 Method Revision: ETS-8-231,.0 Study Number: E01 -1 g S ' / U Matrix: Sera +5 Elution solvent and volume 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 1 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2m I of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2 m fo f MeOH TN-A-547 2m l 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 ConYpnts >2 ( y~ < L Z ^ "Blank Sera TN-A- 7 'l-- ; Amount of sera aliquated: 2 . H EDilute Sera with Kandiyohi water; Amount of water added. ml ml P* 7&a / v t f c v i i / r t J c , A ir Aliquot 1ml of sera into 15 ml polypropelene tube Jspike samples accordingly ~^s>\ T<? - v a j s^T w cS c Add 5ml of ACN (TN-A- ) to aliquated sample A * * , X h i She W y f. g t Shake sample for 20 min @ 300 rpm ( S h a k e r 1 Centrifuge sample for 10 min @ 2000 rpm (Centrifugea i t ? C I 2> Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water D shake sample slightly to ensure proper mixing 3- Condition column with MeOH (TN-A- 's 7 ^ 4 J 1 2, O ja > r ~t *L t- h 0 ( c .< j~ r e 4 > o / Ac , J te * # Wash Column with Kandiyohi Water Q 6 Filter sample through conditioned column, discarding filtrate ( ^ Allow column to go completeiiy dry , \ Elute column with indicated amount of solvent ( H e O h TN-A- 5 7 'S V 1into appropriate 15 ml centrifuge tube . * . ja ^ Spike samples with internal standard PFDA standard number O J tco I - G -5 \O Q . 3 p f m \ c* * 3 * <^ J jV TO P * D < c Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 156 Report E01-1245 PFOS: A ReproductionStudy with the Nothem Bobwhite SPE Columns Extraction Worksheet rod 6W.l US ej/jilta 2* i f Prep Date: Analysts initials: 1/31/2002 QK/HOJ \ - 'b ) o '2 o \\Ll - Lims Assgned sample number E01-1245-28443 E01-1245-28444 E01-I245-28445 E01-1245-28446 E01-1245-28447 E01-1245-28448 E0M245-28449 E01-1245-28450 E01-1245-28451 E01-1245-28452 E01-1245-28453 E01-1245-28454 E01-1245-28455 E0M245-28456 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, tg, 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: E 0 1-12*6" Matrix: Sera Amount and spike Elution solvent mix used and volume Comments 2ml of MeOH NA TN-A-5457 It.O c iU , 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 2ml of MeOH NA TN-A-5457 'Z ^ L . 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 6 Q c *A ^ 2 m lo f MeOH NA TN-A-5457 T ook^ 2ml of MeOH NA TN-A-5457 '7 - c C - w v - - 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 ~ l d C 2 m lo f MeOH NA TN-A-5457 / d O t V 'L ' 2m I of MeOH NA TN-A-5457 2mJ of MeOH NA TN-A-5457 Q ^Blank Sera TN-A- ^ ^ j t : Amount of sera aliquated: 3 ml ^ Dilute Sera with Kandiyohi water; Amount of water added ml O Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN fTN-A- ^ ^ t 6? ) to aliquated sample Shake sample for 20 min @ 300 rpm (Shaker *U & o P ^ i 7jA>-/ ^4/(27 4 r- Centrifuge sample for 10 min @ 2000 rpm (Centrifuge -fcFtrSr'f, 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 f i r . t 7~A </4 4 -0 /* 4 r( ^ S ~ -+ **A ia : & u u * i4 . $ e +*-/ it v C 2-0 t e j T Oc> f c - L t/O c ^ u ^ f ; a-- t5Y /A t S' ^ v t. \y % 7 tj Condition column with MeOH (TN-A- ' ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate yQ dSb F 0 ' H o X Allow column to go completely dry Elute column with indicated amount of solvent ( TN-A- ) into appropriate 15 mli cc eentnrif^uge et tuubbee ^ ^ Spike samples with internal standard PFDA standard number o 2 g p I - ^ T cone. 10<? 3M Environmental Laboratory Page 157 Report E01-1245 PFOS: A Reproduction Study with the Nothera Bobwhite Study: E01-1245 Dilution Date/Analyst: Box Number: Attachment D: Dilutions Summary Worksheet u /j|,|oi Solvent/TN Number: MeOH/5457 w/ IS PFDA Extraction Date/Analyst: 1/31/02 Matrix/Timepoint: Qua.l S'ttu lllilcl Sam ple Num ber or Description 1/1000 E01-1245-35785 E 0 1-1245-35786 E 0 1-1245-35787 E frl-1 2 4 S -3 5 7 7 * -< A E ftl-1 2 4 5 -3 5 7 8 0 E01-1245-35781 J E 0 1-1245-35782 E 0 1-1245-35783 E01-1245-35784 1/100 1/ 1/ \y Q<. I}I\ :tAD Lli?- f"- ) ^, >O> X. - D ilu tion s 1/ 1/ Com m ents Verified By: ~ 7 ^/ r /S / l 1 ' /-^ / -- (J . r c- \t ` rf jr j b 'Z K o/ Notes: 1/10 dilution = ' C o of s a m p l e o f solvent -I jvO O yiiiL ih c k \. \0 pSl "jOLkXi. "V l|?C9C> 0*6'^U ^ C M : I ^ J T " t _ , i. \P&*>dew i pn ^0 0 W - o u I 'lS G - o Q ^ -* SDt ^^ okJ? vj ivfep'fet,.. nM J .$ ^ P e /u b Form Completion Verified By: LAj H / l U ^ ________________ 3M Environmental Laboratory Page 158 Report E 01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite SPE Standard Curves--Fluids Prep Date(s): Analyses): Sample Matrix: Method/Rcvision: Target Analyteisl: 01/31/02,02/01/02,02/13/02 OK/HOJ Quail Sera ETS-8-231.1 PFOS Study N um ber EOI-1243 Equipment Number: NA Final Solvent & TN Number: MeOH/TN-A-05457 Blank Tusue/ldcnUfler. Quail Sera-TN-A-5992 PFOS Std: 5.02 ppm: PFO SStd: 50.2 ppm: PFOS Sid: 2241 ppm: SnrrJStd Approx. 100.7 ppm: 02001-52 02001-53 02001-13 02001-45 PFDA Actual Quiccntrations (if Standards in the FC Mix PFOS PFOS Std G me. 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 0.0)00 5.02 0.0020 5.02 0.0800 PFDA Sid Cone. ufi/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 An 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. 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 (mlVmL) for curves: FkiBl solvent volume (mL) for curves: 0.0500 2.000 sVCixW ul&h m &c x d d t d - ittr c l o u ' ^ o s lttlo i Calculated Concentrations of Standards in the sample maina Standard Number E01 -1245-35770,5.0 ppb EOl-1245-35771, 10 ppb EOl-1245-35772,25 ppb EOl-1245-35773, 50 ppb EOM245-35774, 100 ppb EOM245-35775,250 ppb E01-1245-35776. 500 ppb E0I-1245-35777,750 ppb EOl-1245-35778. 1000 nob QC-25 ppb QC-40 ppm OC-80 nnm Quail Sera MS/MSD-Day 1 PFOS Std Cone. ne/mL 201 402 1004 2008 4016 10040 20080 30120 40160 1004 1613520 3227040 10040 PFDA Std Cone. ng/mL 4028 4028 4028 4028 4028 4028 4028 4028 4028 4028 4028 4028 4028 Quail Sera MS/MSD-Dav 2 Quail Sera MS-5 ppb Quail Sera MS-200 ppb 10040 201 8032 4028 4028 4028 Calculated Concentratings of Standards in the final solvent Standard PFOS PFDA Number Std Cone. Sid Cone. ng/mL ng/mL EEO0Il--11224455--33S5777701., 51.00ppppbb 5.02 101 10.0 101 EOl-1245-35772, 25 ppb 25.1 101 E0I-1245-35773.50 ppb EOI-1245-35774. >00 ppb 50.2 101 100 101 E0I -1245-35775, 250 ppb E0I-1245-35776, 500 ppb 251 101 502 101 E0I -1245-35777* 750 ppb 753 101 EOl-1245-35778. 1000 ppb 1004 >01 QC-25 ppb 25.1 101 QC-40 ppm OC-80 ppm 40338 80676 10) 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 Oiail Sera MS-200 ppb 201 101 Verifiedby: / V - X / l - ? /c7 2 -- 3M Environmental Laboratory Page 159 Report E01-1245 SPE Columns E x t r a l i v ^ 8 tlon d M hS te NoS f fl^ 'l'1[e ^ WJK- i o H Prep Date: Analysts initials: 2/1/2002 OK/HOJ Q` l o Method Revision: E TS -8-231.0 Study Number: E01-1245 Lims Assgned sample number 2 . Sample Number or description -*z . Volume of sample filtered (ml) Type of column used and lot Matrix: Sera Amount and spike Elution solvent mix used and volume Comments E01-1245-35825 E01-1245-35826 Blank H 20-day 2 ( 2 fc jy p it. OK l = "V ` Blank maHartr sera-day 2 40+6 40+6 E01 -1245-35827 0 * MaHsrti Sera MS-day 2 40+6 E01-1245-35828 E01-1245-28458 6Jl/A lL O ft. i 3 / ' ^ M aU m rti Sera MSD-Day2 454-108-346, 0 PPM, Female, Adult 40+6 40+6 E 0 M 24 5-2 8459 E01-1245-28460 4 54-108-347,0 PPM, Male, Adult 454-108-348,0 PPM, Female, Adult 40+6 40+6 E01-1245-28461 E O I-1245-28462 ( T ) EO I-1245-28463 EO l-1245-28464 E O I-1245-28465 EO I-1245-28466 E O I-1245-28467 454-108-349, 0 PPM, Male, Adult 454-108-350, 0 PPM, Female, Adult 454-108-351,0 PPM, Male. Adult 454-108-352,0 PPM, Female, Adull 454-108-353, 0 PPM, Male, Adult 454-108-354, 0 PPM, Female, Adult 454-108-355, 0 PPM, Male, Adult 40+6 40+6 40+6 40+6 40+6 40+6 40+6 EO I-1245-28468 454-108-356,0 PPM, Female, Adult 40+6 EO I-1245-28469 454-108-357, 0 PPM, Male, Adull 40+6 E O I-1245-28470 E01-1245-28471 454-108-358, 0 PPM, Female, Adult 454-108-359,0 PPM, Male, Adult 40+6 40+6 E01-1245-28472 454-108-360,0 PPM, Female, Adult 40+6 EO I-1245-28473 4 54-108-361,0 PPM, Male, Adult 40+6 EO I-1245-28474 454-108-362, 0 PPM, Fem ale, Adult 40+6 uo-sSo j, u HVOT," M3I6, E O I-1245-28475 Adult 40+6 (T ) Mo <^r?A SAN Pt e FoiR T^i S> w aters, 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 2mt o f MeOH N A TN-A-5457 NA 2ml of MeOH TN-A-5457 2ml of MeOH 10ui of 02001-53 TN-A-5457 2mt of MeOH 10ul of 02001-53 TN-A-5457 2ml of MeOH ____ MA_____ TN-A-5457 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, < lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2m! of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml o f MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2m I of MeOH TN-A-5457 Waters, 1g, 6ml, lot 129082 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2m lofM eOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 W aters, 1g, 6m l, lot 1290B2 uuatdrs, ig , w n rr tot 1290B2 2m! of MeOH TN-A-5457 2ml of MeOH TN.A-5457 2ml of MeOH TN-A-5457 \ \ in .C~ D rc^tV \ T tJ L * - T w a -T / / c d ttj- \ a o l~ 6 *^ T " 1 /S aocvL. Jiftru/LdbsL OM L? A,-1 q A o, /<o fv /t. T X z ^ COx* ~ e y Spike samples accordingly; " /} / r 2 ^ Add 5ml of ACN (TN-A- 4 ^ 7 3 ) to aliauafed sample''"' . 4 / fid ( s h a k e sample for 20 min @300 rpm (Shaker^ # 5 &" (U Centrifuge sample for 10 min @ 2000 rpm (Centrifuged 4 '~ ^ cf C ( 3 C Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. z, ^ / ^L- / A, i ( `4, - ' L ' * V `w t-- -1 6 & *>>C - t . Z n > C $j> t * L v ( i & 9 'c v * -* O ~Decant extract into centrifuge tubes with water y ~ shake sample slightly to ensure proper mixing 7 SO Condition column with MeOH (T N -A -0 5 ? 8? Wash Column with Kandiyohi Water ) S T i x t v - ^ `Si *~ L - Filter sample through conditioned column, discarding filtrate ^ Allow column to go completelly dry O Elute column with indicated amount of solvent ( A t ie t A TN-A- J t j Q ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number A ? cc I - 4 3 ______ , cone S3Ll .~ i- / / * i . amount added. A U . 2 2 L < > ~ * * r Transfer sample into appripriatelly marked autovial fy . vn2e cKQio2. (0 $ $ T ) 3M Environmental Laboratory fuPT V ' nO) ^\jrfr^ VOR / Page 160 * "** 'JL. Report E01-1245 PFOS SPE Columns Extraction Worksheet ion(\JV7 li' S f t f e * , " , Prep Date: Analysts initials: 2/ 1/2002 yH O J 5 7 -o o ) C M Urns Assgned sample number . Sample Number or description Volume of sample filtered (ml) Type of column used and lot E01-1245-28476 EOI-1245-28477 EOI-1245-28478 EOI-1245-28479 E01-1245-28480 E01-1245-28481 E01-1245-28482 E01-1245-28483 E01-1245-28484 EOI-1245-28485 EOM 245-28486 E01-1245-28487 E01-1245-28488 E01-1245-28489 EOI-1245-28490 E01-1245-28491 E01-1245-28492 E01-1245-28493 E01-1245-2B494 E01-1245-28495 E01-1245-28496 E01-1245-28497 454-108-365,0 PPM, Mile, Adult 454-108-366. 0 PPM, Female. Adult 454-108-367,0 PPM, Male. Adult 454-108-368. 0 PPM. Female. Adult 454-108-369,10 PPM, Male, Adull 454-108-370, 10 PPM, Female, Adult 454-108-371. 10PPM, Male. Adult 454-108-372.10PPM,Female. Adult 454-108-373, 10 PPM, Male, Adull 454-108-374.10 PPM. Female. Adult 454-108-375, 10 PPM, Male, Adult 454-108-376.10 PPM, Female. Adult 454-108-377,10 PPM, Male, Adult 454-108-378, 10PPM. Female. Adult 454-108-379,10 PPM, Male. Adult 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, 6 m l, tot 129062 Waters, 1g, 6ml, tot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, tot 1290B2 Waters, 1g, 6ml, tot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, tg , 6ml, lot 1290B2 Waters, 1g, 6m l, tot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, tot 1290B2 Waters, 1g, 6ml, tot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g,6ml, tot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, tot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 W a te rs , 1g, 6 m l, 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 2m 1of MeOH NA TN-A-5457 \o o o i^ . 2m! of MeOH NA TN-A-5457 `Z fffc v L -' 2ml of MeOH NA TN-A-5457 2mJ of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2ml of MeOH < NA TN-A-5457 2m! of MeOH NA TN-A-5457 7 o 6u A ~. c2ml of MeOH NA TN-A-5457 4 '< to 2m/ of MeOH NA TN-A-5457 jp s o L 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 ' oco, L 2m lo f MeOH NA TN-A-5457 J < -'< L 2ml of MeOH NA TN-A-5457 2 m /o f MeOH NA TN-A-5457 / 2ml of MeOH NA TN-A-5457 / O c . 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 Z ' 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 . 2ml of MeOH NA TN-A-5457 /O C L ^ i_ 2ml of MeOH NA TN-A-5457 / b + C 2ml of MeOH NA TN-A-5457 ~3 lank Sera TN-A- T T T f f i . : Amount of sera aliquated: >t / a - ml Dilute Sera with Kandiyohi water; Amount of water added. Aliquot 1ml of sera into 15 ml polypropelene tube ml C Spike samples accordingly Add 5ml of ACN (TN-A-. J to aliquated sample J"Shake sample for 20 min @300 rpm (Shaker C ? Z f t 4 { (9* Centrifuge sample for 10 min @ 2000 rpm (CentrifugeS t & /? % 0 K S 2 o &- _Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. shake sample slightly to ensure proper ~ Decant extract into centrifuge tubes with water *W Condition column with MeOH (TN-A' j Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding nitrate Allow column to go completelly dry Elute column with indicated amount of solvent ( / h o e # _TN-A- ^ nto appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number C V ? . a o l ~ 4 { ______ , cone./ ' 't> ^ ^ 1 amount added 3M Environmental Laboratory o* ? 232- T f-T 'V U x f ^ Page 161 Report E01-1245 SPE Columns Prep Date: Analysts initials: 2/ 1/2002 OK/HOJ i ' 0Ao l e' - Lims Assgned sample number EO1-1245-28498 EOI-1245-28499 E01-I245-28500 EOI-1245-28501 01-1245-26502 E01-1245-28503 E01-1245-28504 E01-1245-28505 E01-1245-28506 E0M245-28507 E01-1245-28508 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-] 08-388,10 PPM, Female, Adult 454-108-389,10 PPM, Male, Adull 454-108-390,10 PPM, Female, Adult 454-108-391,10PPM, Male. Adull 454-108-392,10 PPM. Female. Adult 454-108-393. 10 PPM, Male. Adult 454-108-394, 10 PPM, Female, Adult 454-108-395, 10 PPM, Male, Adull 454-108-396,10 PPM, Female, Adult 454-108-397,10 PPM. Male, Adull 454-108-398, 10 PPM, Female, Adult 454-108-399,10 PPM. Male, Adull 454-108-401, 10 PPM, Male, Adull 454-108-402,10 PPM, Female, Adult 454-108-403,10 PPM, Male, Adull 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 454-106-408,10 PPM, Female, Adult 454-108-246,0 PPM, Female, Oflspring 4D4-11KW50, U HHWl, Maie, 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 W aters, 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, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 W ald R , !g'. Bffll, lot 1290B2 Method Revision: E T S -8-231.0 Study Number: E01-1245 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 NA NA NA NA NA NA NA 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2m! of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH, TN-A-5457 2mI of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2mi of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2mI of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2m I of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2m I of MeOH TN-A-5457 2ml ofMeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 / " oL f a ( /i? C O ljE ' ~3c& A .-- **0 *^ 0 l / O d i ( ___ /< r o c L Z O i j c - { / Ate / / , 4 m I f Blank Sera TN-A- ` `t ' L . : Amount of sera aliquated: Dilute Sera with Kandiyohi water; Amount of water added zC/W ^ Aliquot 1ml of sera into 15 ml polypropelene tube ml ml QsM ^ ^ 3 ( j e n e oK O cA c sSpike samples accordingly* Add 5ml of ACN fTN-A- A 4 5 ) to aliquated sample . $ cn - P Shake sample for 20 min @ 300 rpm (Shaker/ 1 - ^ s I Centrifuge sample for 10 min @ 2000 rpm fCentrifuae2 * * 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- r y f ) 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 ( TN-A- i r j y 1 1nto appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number f ) 2 a e / ~ I j __ , cone. / o C s ^ j i f i k . amount a d d e d 7 c 3M Environmental Laboratory Page 162 O O i l 'iS Report E01-1245 PFOS: A Reproductioi SPE Columns Extraction Worksheet the-Nothem Bpbwhite X Yty, H ci H J o s h oj Prep Date: Analysts initials: 2/ 1/2002 o 2 0 \L Lims Assgned sample number E01-1245-28520 E0M245-28521 E01-1245-28522 EOI-1245*28523 E01-1245-28524 E01-1245-28525 E01-1245-28526 E01-124S-28527 E01-1245-28528 E01-1245-28529 EOI -i 245-28530 EOl-1245-28531 EOl-1245-28532 E01-1245-28533 EOI-1245-28534 E01-1245-28535 E01-1245-28536 E01-1245-28537 Sample Number or description 454-108-256,0 PPM, Male, Offspring 454-108-259,0 PPM, Female. Offspring 454-108-266. 0 PPM. Male, Offspring 454-108-270,0 PPM, Male, Offspring 454-108-273, 0PPM. Female, Offspring 454-108-277,0 PPM. Female, Offspring 454-108-283,0 PPM, Male, Offspring 454-108-291,0 PPM. Male, Offspring 454-108-517.10PPM, Female, Offspring 454-108-522,10 PPM, Male, Offspring 454-108-525,10 PPM. Female, Offspring 454-108-533,10 PPM, Male, Offspring 454-108-538,10PPM, Female, Offspring 454-108-545, 10 PPM, Male, Offspring 454-108-552, 10 PPM, Male, Offspring 454-108-557, 10 PPM, Female, Offspring 454-108-561, 10 PPM, Female, Offspring 454-108-568,10 PPM, Male, Offspring Volume of sample filtered (ml) Type of column used and lot 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 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, 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 / 4 0 i>L 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 <?C7 0 < ~ L s 2mI of MeOH NA TN-A-5457 2m of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 H o 2ml of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 2mf 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 2ml of MeOH NA TN-A-5457 d j ( T ~ ' (.-- 2ml of MeOH NA TN-A-5457 ^ 4 - r i r 1- 2ml of MeOH NA TN-A-5457 2mt of MeOH NA TN-A-5457 2ml of MeOH NA TN-A-5457 -------- -------------------- -- -------- -IAS OS I 2 L 1 fi)! Blank Sera TN-A- j Amount of sera aliquated: ml ^ / h ? -z c\i / Dilute Sera with Kandiyohi water; Amount of water added ml c p h iip O p J v flv c t - Aliquot 1ml of sera into 15 ml polypropelene tube g U . Spike samples accordingly Add 5ml of ACN (TN-A- J to aiiquated sample /''Shake sample for 20 min @ 300 rpm fShaker^ k # f i t f i l f K . J>!-a s O t * ^ vK - AW 6Qjuup$3-s f\ if ' f ' C ax)<K\jo4eQp far j Centrifuge sample for 10 min @ 2000 rpm (Centrifuge? **^ .y^Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. pfo c d i U e d h ' t . P iDecant extract Into centrifuge tubes with water P shake sample slightly to ensure proper mixina Condition column with MeOH ( T N - A - _ 3 z _ _ ) 6 Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate q Of ok Allow column to go completelly dry Elute column with indicated amount of solvent ( TN- A- s - r i T ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 0 7 . a u / - 4 f __ , cone. / d o )- M i t amount added C -y c ^ L 'T c 3M Environmental Laboratory 0 0 i ^ 3 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite B o t t l e - T o p D is p e n s e r D a il y C a l ib r a t io n C h e c k M anufacturer and Model: Date: <iM 'O Initials: & K j Dispenser ID: 07 ^ ^ Balance ID: ^ ^ Volume: S am^Solvent: AC tO ID #: TF>'A- 4 / ^ Mass of solvent (g) 1 2 3 -^4 (3 3 4 3 . q <a 5 3^414 Density1 (& W Volume of Solvent G.Oto c ISSI `Sr.Oi1 3 S. o ^ \ S .O Z & Y Average Volume: b .OlGo %Accuracy: Std. Dev.: lo o . 2>7o 7. ooSJoY Z Z Z 3 Z Z Z Z Z%C.V.: Pass/Fail: o. 1 Volume, (mL) 1to 10 P erform ance Specifications Accuracy, C.V., (%) 98.0 - 102% (%) 1.0% 'Density values taken from tbe M erck Index, 12th Edition, Copyright 1996 dv, =spccific gravity at y c referred to w ater at xdegr" ` n-Hexane d20=0.660 Acetone d250 =0.788 Methanol d2S4=0.7866 Methylene Chloride d204=1.3255 3M Environmental Laboratory Page 164 CO l i ''.7 Report EOI-1245 Prep Date: <P- ? ~c>3Analysts initials: OK SPE Columns ExtracBBSMotskStefHrbduction ite Method Revision: O f - S Study Number: <XSf - / ? 4 S ^ i* , Lims Assigned sample number E01-1342-30312 E01-1342-30321 E01-1342-30330 E01-1342-30339 EO1-1342-30348 E01-1342-30357 E01-1342-30366 E 0 1 -1342-30375 E01-1342-30384 E 0 1 -1342-30393 EO1-1342-30402 E 0 1 -1 3 4 2 -3 0 4 1 1 E01-1342-30420 E01-1342-30429 E01-1342-30438 E01-1342-30447 E01-1342-30456 EO 1-1342-30465 E01-1342-30474 E01-1342-30483 EO 1-1342-30492 E01-1342-30501 Sample Number Volume of sample Type of column used Amount and Elution solvent Amount of liver or description filtered (ml) and lot spike mix used and volume weighed (g) 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 \ \ \ '\ \ rA l 1 a, t< 1v (. i, / , $ Q, <7 10 ( (( h r , T ^e t l 4 2 *? ^ i.o q o t# \ \ \ \ i.m i. 0 i-C i> & (o q , 'J / . /v5 t. 1 'C js 3 } q Comments MA I \J Blank Liver TN-A- _____ ; Amount of liver:______ 9 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 cojpptifelly dry Elute column witMfidicated amount of solvent (__________ TN-A-. Spike sap es with Internal standard PFDA standard number _ Transfer sample into appripriatelly marked autovial ml 0*5-- J into appropriate 15 ml centrifuge tube , cone._____________, amount added ^OCHju ^ 3M Environmental Laboratory toP/ % h o o t / < ^ t.O fd 1 Page 165 0 0 1 :1 Report E01-1245 PFOS: A Reproduction Study Miiltha^dheiji Robwhite SPE Columns Extraction Worksheet (AC) a o t s j 0J'i2 i02- Prep Date: J - ? Analysts initials: (f/V I Method Revision: rr-i ^ ,' Study Number: Matrix: Cio^ * ~Z*o? Urns Assigned sample number E01-1342-305IO 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 E01-1342-30636 E01-1342-30645 . E01-1342-30654 E01-1342-30663 E01-1342-30672 E01-1342-30681 E01-1342-30690 E01-1342-30699 Sample Number Volume of sample Type of column used or description filtered (ml) and lot 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 0 ppm a.i. 366 h Liver u ppm a.i. 36f M Liver Oppm a.i. 366 f Liver 10 ppm a.i. 36SM Liver ID ppm a.i. 3/u iLiver 10 ppm a.i. 3/1 M Liver \\ \\ \ \ \ \ \ \ 'T F\ \ ...r r \ 10 ppm a.i. 372 F Liver Amount and spike mix used Elution solvent Amount of liver and volume weighed (g) i.e s & y i * i 1. i i y I.D 3 ? 2 % i. / t i l I-O ^ t/Ic x (.0 5 ^ <- O S t h 1, O Z G q J 1 0 5 ? / , .0 Z 2 * i.o c tc ^ / o ? 4 <P* Comments A J A ________ 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 allquated 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 conditionjd-cOlumn, discarding filtrate Allow column to go conjpleteliy dry Elute column wjttrtfidicated amount of solvent (__________ TN-A-_ Spike samples with internal standard PFDA standard number _ Pister sample into appripriatelly marked autovial ml & oS J into appropriate 15 ml centrifuge tube , cone._____________ , amount added <k Ito 3M Environmental Laboratory Page 166 Report EOI-1245 Prep Date: j ? . - C & a - - o Z . jAnalysts initials: o f ^ SPE Columns ExtracfigPft/o^kgKfSPduction S t i ^ ^ t h t . N o ^ g jg jw hhite Method Revision: < f h - g ?Jt- I Study Number: <- <?( Lims Assigned sample number Sample Number Volume of sample Type of column used Amount and Elution solvent Amount of liver or description filtered (ml) and lot spike mix used and volume weighed (g) Comments E01-1342-30708 E01-1342-30717 E01-1342-30726 E01-1342-30735 E01-1342-30744 E01-1342-30753 EO1-1342-30762 E01-1342-30771 E01-1342-30780 E01-1342-30789 E01-I342-30798 E01-1342-30820 E01-1342-30829 E01-1342-30838 E01-1342-30847 E01-1342-30856 E01-1342-3086J E01-1342-30874 E01-1342-30883 E01-1342-30892 E01-1342-30901 E01-1342-30910 10 ppm a.1.373 M Liver 10 ppm a.i. 374 F Liver 10 ppm a.i. 37S 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 fLiver 10 ppm a.t. 38a Liver 10 ppm a.i. 3b9 M Liver 1 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 i . o x ?<p j /. t c c >$ i./o lA i- o jlz p j L lV cP I cj L (. 17 f , 0 5 0 i 0| s )A ii o ?>t~l / o M c ^ | .O O i# ^ .c c o - \ l s if 7^ Blank Liver TN-A- 14 ^ ; Amount oftiver: 5 . 0 b o 7M -A - s i V i ;->i.oc? IH Homogenize liver with Kandiyohi water; Amount of water added 7 ^ ____ml /o E r e Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Cf . u i Add 5ml of ACN (TN-A-__________ ) to aliquated sample m e?g> ..p < o ^ l^ e Q ? Shake sample for 20 min @300 rpm (Shaker__________ ) M r*' T -u e to < o . X A U A f O l^ <" < ( W s U T V ^i tu -e o ^ - 1 l - \ 0 u t u c / ic :iU < / O ui ^ jn - c ^ s / r jj ' 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 A 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 completely 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 e e I f W v u o j p A j ? (p (y L e a p O t r i b?Y 5 f f o 'J . 3 VP ^ ^ 0 2 . 3M Environmental Laboratory Page 167 O ' O i l .Sf._A> Report E01-1245 ns Prep Date: / T Analysts initials: n .n fJ O r~ I P PFOS: A Reproduction Study.with.the Nothem Bobwhite ^ Q u a il liv e r 1 SPE Columns Extraction Worksheet (A t) H s i S J ^ OSm ^ K i Method Revision: t > T - i "2-i?I. ( Study Number: t y - t ~ 2 4 s ~ ^ i Matrix: ttsivi_ g r- \ -- Urns Assigned sample number E01-1342-30919 E01-1342-30928 E01-1342-30937 EO1 - 1 3 4 2 - 3 0 9 4 7 1% E01-1342-30955 E01-1342-30964 E01-1342-30973 E01-1342-30982 E01-1342-30991 E01-1342-31000 E01-1342-31009 EOM342-31018 E01-1342-31027 E01-1342-31036 Sample Number Volum e of sample Type of column used or description filtered (ml) and lot 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 Brain A * * 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 \ \ \ \ r h\ Amount and spike mix used Elution solvent Amount of liver and volume weighed (g) Comments _____ t _____ 1. 0 ? > o 9 ^ U \.o J2if O a ) i.p 3 G .ig i-cp -i? l-o o / .0 1 7 3 ^ icaC j i. ^ 4 ^ i i.o o q (jq 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 polypropelejjo-Centrlfuge tube Decant extract into centrifuge tubes with watt shake sample slightly to ensure properjniSing Condition column with MeOH (T { M \_ _________ ) Wash Column with Kandiyghi'Water Filter sample through pemitioned column, discarding filtrate Allow column togtfcompletelly dry Elute coluparr'with indicated amount of solvent (__________ TN-A- Spikesirnples with internal standard PFDA standard number___ Transfer sample into appripriatelly marked autovial f ____ i \ J into appropriate 15 ml centrifuge tube ________ , cone._____________, amount added_ ig n e noe mJ ; 3M Environmental Laboratory Page 168 0011^/ Report E01-1245 Prep Date: A A '^ P ^ Analysts initiais: mo a PFOS : A Reproduction Studvrwith the Nqthem Bobwhite SPE Columns Extraction Workksshheeeett Sdf P w jt Method Revision: e y y . Study Number: b / . Matrix: i 3 /. / . ' J "2 ^ r>~ Urns Assigned sample number EO1-1365-31184 E01-1365-31194 E01-1365-31199 E01-1365-31204 E01-1365-31209 E01-1365-31214 E01-1365-31219 EOI-1365-31224 E01-1365-31229 EO1-1365-31234 E01-1365-31240 EO1-1365-31245 E01-1365-31250 E01-1365-31255 EO1-1365-31260 E01-1365-31265 EOI-1365-31270 EOI-1365-31275 EOl-1365-31280 EOI -1365-31285 Sample Number Volume of sample Type of column used Amount and Elution solvent Amount of liver or description filtered (ml) and lot spike mix used and volume weighed (g) Comments 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. S4S M Liver 10 ppm a.i. 662 M Liver 10 ppm a.i. 557 F Liver 10 ppm a.i. &61 F Liver 10 ppm a.i. :>btt M Liver \ \ \ \\ \ \ \ \ Vvw .dS Y > \ \\ \ \ \ \ \ \ a n o i c * ______PA_______ c .b c t, > 5 < 3 o o < h / 0. O A -iJ L G y 0 .(p G G < y G e lA * Or S 0, O .if rj ' <3 -% n ^ d \{ 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 s Shake sample for 20 min @300 rpm (Shaker S ______ ) Centrifuge sample for 10 min @ 2000 rpm fp^ntrifuge Add 40 ml of Kandiyohi water to 50 ml ratypropelene centrifuge tube. Decant extract Into centrifuge tubes wrtn 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 completely dry Elute column with indicated amount of solvent (__________ TN-A-___ Spike samples with internal standard PFDA standard number______ Transfer sample into appripriatelly marked autovial O ./j A ^ e r A s Z M m M s ^ * 4 J * * t* . ?do^ f T H '<? i G ro ^ L A o V e a -* IX )^ ' ^ /M 77//t> y. 4 <7-c& . J into appropriaitff^5 ml centrifuge tube . cone ________ , amount added and ib -bt/ecfen 3M Environmental Laboratory s a t u ^ lo -s s m iij . Page 169 r 001152 Report E01-1245 Prep Date: 2/11/2002 Analysts initials: OK.HOJ SPE Columns E x tra c rf< iii^ o i^ ^ S lf0 ^uct*on Y C7 I ot 2 . D H-o3 ** QfcL Method Revision: ETS-8-231.0 Study Num ber E01- 134 2/1305- Matrix: Liver Urns Assigned sample number Sample Number or description Volume of sample filtered (ml) E 01-1342-36000 E 0 1 -1 3 4 2 -3 6 0 0 1 E01-1342-36002 E 0 1 -1342-36003 E 0 1 -1 3 4 2 -3 6 0 0 4 E 0 1-1342-36005 E 0 1-1342-36006 E01-1342-36007 E01-1342-36008 E01-1342-36009 E01-1342-36010 EO1-1342-36011 E01-1342-36012 E01-1342-36013 E01-1342-36014 E01-1342-36062 E 0 1-1342-36015 E 0 1-1342-36016 E01-1342-36060 EO 1-1342-36061 Blank H 20-day 1 Blank female liver-day 1 Blank male liver-day 1 Female liver MS-day 1 Female liver MSD-day 1 Curve point-O.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 polnt-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 id * 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 T ype of column used and lot C 18 ,1 g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 18 ,1 g , 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g . 6cc, lot W2025B1 C 18 ,1 g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2026B1 C 18 ,1 g , 6cc, lot W2025B1 C IS , 1g, 6cc, lot W2025B1 C 1 8 ,1g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C1B, 1g, 6cc. lot W2025B1 C 16,1g . 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 c i o , ig, ooc, io ! W2025B1 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 5Oui of 02001-66 100ul 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 2iYil M eUH TN-A-5754 Comments S)A VJ/ " Blank Liver TN-A- *5 )4 3 ; Amount Oliver: ^ o3*? "b> Homogenize liver with Kandiyohi water: Amount of water added. Blank Liver TN-A- ^ 1^ X j ; Amount oofAVfiverr:l 1/ Q-oit? g ml Amount of water added 1 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- i 4 5~ ) to aliquated sample Shake sample for 20 min @ 300 rpm fShaker v /m fij QH/VH-Pl*-- ^ Centrifuge sample for 10 min @ 2000 rpm (Centrifuge * * 4 9 6 9 C Y b 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 IT N -A -*3 ^ ^ ^ ) 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 f M f C H TN-A- U )into appropriate 1 5 ml centrifuge tube Spike samples with internal standard PFDA standard number t- 4 cone, t o o - amount added. Transfer sample innto appnripprriaaiteenlly marknedo auuitooviail if /} f l P K? C\ & A iiA e P x h o Q b ' & u o K . r $ ' G < * 0*< ? '" 3M Environmental Laboratory Page 170 G O I.;I / Report E01-1245 Prep Date: S i' O . Analysts initials: Or^ I H o ^ Lims Assigned sample number EO1-1342-36017 E 0 1 -1 3 4 2 -3 6 0 1 8 E01-1342-36019 E 01-1342-36020 E 0 1 -1342-36021 E01-1342-36022 E01-1342-36023 E 0 1 -1 3 4 2 -3 6 0 2 4 E01-1342-36025 E 0 1 -1 3 4 2-3 0 3 12 E01-1342-30321 E01-1342-30330 E 0 1 -1342-30339 E01-1342-30348 E01-1342-30357 E 0 1 -1 3 4 2 -3 0 3 6 6 E01-1342-30375 E 0 1 -1 3 4 2 -3 0 3 8 4 E01-1342-30393 --------- Sample Number or description Q C @ 10p pb-1 Q C @ 10 ppb-2 Q C @ 10 ppb-3 QC@ 75 ppb-1 Q C @ 75 ppb-2 Q C @ 75 ppb-3 Q C @ 150 ppb-1 Q C @ 150 ppb-2 Q C @ 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 ------ PFOS: A Reproduction Study with the Nothem Bobwhite SPE Columns Extraction Worksheet ($ - n- i f V jt , 3. o f 2 j 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 -- -- Type of column used and lot C 18,1g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C18, 1g, 6cc. lot W2025B1 C1, 1g, 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C 18,1g , 6cc. lot W2025B1 C 18,1g , 6cc. lot W225B1 C18, 1g, 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C 18 ,1 g . 6cc, lot W2025B1 C18, tg, 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C 1 8 ,1g, 6cc, lot W2025B1 C18, tg, 6cc, lot W2025B1 C 18,1g , 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 -- -- Method Revision: f l <b - Study Number: C*j~ Amount and spike mix used Matrix: -v yx^ R J ^ ? 4 i' tlution solvent and volume Comments 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 6Oui of 02001 -52 6Oui of 02001-52 NA NA NA NA NA NA NA NA NA NA ml 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 2 ml 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 _klA_______ | ---- ' --- -- ^ ft- Blank Liver TN-A- : Amount of liver: 9 . p ' b S h g Homogenize liver with Kandiyohi water; Amount of water added Blank Liver TN-A- *5 I Amount of liver: Uy> ml Amount of water added ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN fTN-A- 4 / 4 ^ ) to aliquated sample , Shake sample for 20 min @ 300 rpm (Shaker d i d R j IT 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 fTN-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 < p P O H TN-A- j into appropriate 15 ml centrifuge tube . Spike samples with internal standard PFDA standard number jjfa c d ~ _______ , cone. , amount added C X ^tJL_____ Transfer sample into appripriatelly marked autovial -i 3M Environmental Laboratory .f f . /] *,-k>rt)au velowvtrf VH OK. 'AFiv,. Page 171 001154 Report E01-1245 PFOS: A Reproduction Study with the Nothera Bobwhite SPE Standard Curves-Solids Prep Date(s): Analysl(s); Sample Matrix: M ethod/Kcvision : Target Analyte(s): FC Mix Std Approx. 1.00 ppm: FC Mix Std Approx. 5 02 ppm: FC Mix Std A pproi. $0.2 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. UR/mL EOM342-36O05 1 004 E 0t-1342*36006 1.004 E0I-I342-36O07 1.004 E01-1342-36008 1.004 E0M342-36009 1 004 E01 -1342-36010 1.004 E01 -1342-36011 1.004 E01 *1342-36012 1 004 E0I-1342-36013 1.004 E01-1342-36014 t 004 E0M342-36O62 50.20 E 0 1-1342-36015 50.20 E01- 1342-36016 50 20 EO1-1342-36060 50.20 EO1-1342-36061 50 20 QC-10 ppb 5 020 QC-75 ppb 5.020 QC-150 ppb 5 020 Quai! Uver-MS/MSD-Day 1 1 004 Quirl Uver-MS/MSD-Day 2 1.004 Quail Liver-MS/MSD-Day 3 1.004 Quail Liver-MS/MSD-Dav 4 1.004 PFOS Ain't Spiked mL 0 0004 0.0010 00020 00050 0.0100 0.0200 0 0500 0.1000 0.1500 0.2000 0 0040 0.0100 0.0200 0.0300 0.0400 0.0040 0.0300 0.0600 0.0500 0.0500 0.0500 00500 Calculated Concentrations of Standards in the sample matrix Standard PFOS PFDA Number Std Conc. Std Conc. E01-1342-36005 nete tlR/E 399 2001 E01-1342-36006 E01-1342-36007 E01-I342-36008 9 98 2001 200 2001 499 2001 EOI-I342-36O09 E 0 1-1342-36010 EO1-1342-36011 998 2001 200 2001 499 2001 E01-1342-36012 E01-1342-36013 EOl-l 342-36014 998 2001 1496 2001 1995 2001 E 0 I-1 342-36062 1995 2001 E01-1342-36015 E01-1342-36016 4988 9977 2001 2001 E01-1342-36060 E01-I342-36061 QC-lOppb 14965 19953 200 2001 2001 2001 QC-75 ppb 1496 2001 QC-150 ppb 2993 2001 Quail Liver-MS/MSD-Day 1 Quail Liver-MS/MSD-Day 2 Quail Liver-MS/MSD-Day 3 Quail Liver-MS/MSD-Dav 4 499 2001 499 2001 499 2001 499 2001 Calculated Concentrations of Standards in the final solvent Standard PFOS PFDA Number Std Conc Std Conc. ng/mL mt/mL E01*1342-36005 0.201 101 E01 -1342-36006 0.502 101 E01-1342-36007 1.00 101 EOI -1342-36008 2.51 10t E0M342-360O9 5 02 10! E01-1342-360l 0 10.0 101 EO1-1342-36011 25.1 101 EO1-134 2-36012 50.2 10! EO1-1342-36013 75.3 101 EO1-1342-36014 100 101 E01-1342-36062 100 101 E0I-I342-360I5 25t 101 EO1-1342-36016 502 101 E O l-l342-36060 753 101 EO1-1342-3 6061 1004 101 QC-lppb to o 101 QC-75 ppb 75.3 101 QC-150 ppb 15) 101 Quail Liver-MS/MSD-Day 1 25.1 101 Quail Liver-MS/MSD-Day 2 25 l 101 Quail Liver-MS/MSD-Dav 3 25.1 101 Quail Liver-MS/MSD-Dav 4 25.1 101 3M Environmental Laboratory PFDA Std Conc. uit/mL 100.7 100.7 100.7 100.7 J00.7 100.7 100.7 100.7 100.7 100 7 100.7 100.7 100.7 100 7 100.7 1007 100.7 100 7 100 7 100 7 100.7 100 7 Study Number: EOI-1245 Equipment Number: NA Final Solvent & TN Number: MeOH/TN-A-5754 Blank Tisiue/Idenrifter: Quail Liver(femaJe)/TN-A-5143 Quail Liver(maleyTN-A-5142 Initial female homogenate - 5 0353g/500153g+ml. = 0 1006 g/mL Initial male homogenate * I.0015g/l0.0015g+rnL * 0 1001 g/mL Blank liver initial homogenate (g/mL) for curves, MS, QC: 0.1006 Final solvent volume (raL) 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 0002 0002 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 2000 2.000 2.000 2000 2.000 2.000 2000 2.000 2.000 0.1006 0.1001 aeA. YM Verified bv: I M n Q b l& i Page 172 0011. Report EOI-1245 I PFOS: A Reproduction Study with the Nothem Bobwhite B o t t l e - T o p D is p e n s e r D a il y C a l ib r a t io n C h e c k M anufacturer and Model: Date: <??-// < Initials: Dispenser ID: Balance ID: tao Volume: Solvent: ____ it4s Mass of solvent (g) Density1 (g/ml) 1 2 *? 3 3 `T r ^ r 4 3 i rrr 5 3 AS TJ <0 ______ 1 r_______ Average Volume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 3 o5 5 4 . a3i2_ f.0344 3.6 3 r.oJT i/ 0/9. O, C.01-V2- Volume, (mL) 1 to 10 P erform ance Specifications Accuracy, c.v., (%) 98.0 - 102 % (%) 1.0% Corrective Actions: c -O ^ ^ * * ~ i-- < 'Density values taken from the M erck Index, 12th Edition, Copyright 1996 dyx=specific gravity at y<ltgre" c referred to water at xdeBrm n-Hexane d204 =0.660 Acetone dI50=0.788 Methanol d254=0.7866 Methylene Chloride d204 =1.3255 3M Environmental Laboratory Page 173 Report E01-1245 S PE Columns E x t r a c t i f M & k M ^ 0^ 0^ ^ ^ W fc1? Bobwhite Prep Date: Analysts initials: 2/12/2002 OK/HOJ -Q--o2> Lims Assigned sam ple number E01-1342-36026 E01-1342-36027 EOI-1342-36028 E01-1342-36029 E01-1342-36030 E01-1342-30402 E01-1342-30411 E01-1342-30420 E01-1342-30429 E01-1342-30438 E01-1342-30447 E01-1342-30456 E01-1342-30465 E01-1342-30474 E01-1342-30483 E01-1342-30492 EOI-1342-30501 E01-1342-30510 E01-1342-30519 E01-1342-30528 Sample Number or description Volume of sam ple filtered (ml) Blank H20-day 2 40+6 Blank female liver-day 2 40+6 Blank male liver-day 2 40+6 Female liver MS-day 2 40+6 Female liver MSD-day 2 40+6 0 ppma.i. 339 M Liver 0 ppm a.i. 340 F Liver 0 ppm a.i. 341 M Liver Oppm a.i. 342 F Liver 0 ppm a.i. 343 M Liver 40+6 40+6 40+6 40+6 40+6 0 ppm a.i. 344 F Liver 40+6 0 ppm a.i. 345 M Liver 40+6 0 ppm a.i. 346 F Liver 40+6 0 ppm a.i. 347 M Liver 40+6 0 ppma.i. 348 F Liver 40+6 0 ppm a.i. 349 M Liver 0 ppm a.i. 350 F Liver 0 ppm a.i. 351 M Liver 0 ppm a.i. 352 F Liver 0 ppm a.i. 353 M Liver 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 C1S. 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 u is , ig, occ, lot W2025B1 uns, ig, occ, tot W2025B1 uno, ig, occ, ii W2025B1 U1B,1fl|"6B8;"lot W2025B1 C18, IS, btc, lot W2025B1 uns, ig, occ, lot W2025B1 u ie, ig, occ, tot W2025B1 u ie, ig, occ, lot W2025B1 u ie , l","656," icx W2025B1 uiH. ig, ecc, lot W2025B1 C18, Ig, bit:, 16! W2025B1 uns, ig, occ, rot W2025B1 uns, ig, occ, tot W2025B1 U18, Ig. bCfcTioi W2025B1 W2025B1 Method Revision: E T S -8 -2 31 .0 ^,2 ^Study Number: E01 -104 2 /1 S6 6 ^ ^ Matrix: Liver 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 C om m ents iUA H( ? Blank Liver TN-A e i * * ? ) ; Amount of liver: <5 O S l b I Blank Liver TN-A' ^ : Amount of liver: Homogenize liver with Kandiyohi water; Amount of water added it * 5 ml Amount of water added ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly _ Add 5ml of ACN (TN-A- h I H ) to aliquated sample , Shake sample for 20 min @ 300 rpm (Shaker ^ 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 ( T N - A - _ ^ 2 _ 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 _, ' ) into appropriate 15 ml centrifuge tube 1~ 4 ^ , cone. 10 *1 amount added n c * jA-*- Transfer sample into appripriatelly marked autovial 9a Ja w ^ C9 ^ H o?- 3M E nvironm ental L aboratory oL Page 174 Report EOl-1245 SPE Columns Prep Date: Analysts initials: 2/ 12/2002 OK/HOJ OK s -a -^ ft? M i Bobwhite ^ 2- o i J Method Revision: ETS-8-231 Study Number: E O l- IS ^ s m e S 'W - r .r f d T / 2 - - Matrix: Liver Lims Assigned sam ple number E01-1342-30537 E01-1342-30546 E01-1342-30555 E01-1342-30564 E01-1342-30573 E01-1342-30582 E01-1342-30591 EOl -1342-30600 E0I-1342-30609 E01-1342-30618 E O l-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 MLiver 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 C18,1g, 6cc, lot W2025B1 C16,1g, 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 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W202SB1 C18,1g, 6cc, lot W2025B1 C18, 1g. 6cc, lot W2025B1 Amount and Elution solvent and spike mix used volume C om m ents NA 2ml MeOH TN-A-5754 _ J I A ___________ 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 ...__ --------- (AS O t ' l i & i ___------ ------ --- --------------------- L _____________ Blank Liver T N -A -^ ' ^ " --1 : Amount of liver: ^ 0 3 S g Homogenize liver with Kandiyohi water; Amount of water added Aliquot 1ml of liver into 15 ml polypropelene lube Blank Liver TN-A-*5 : Amount of liver: l' 0 0 ) ^ o ml Amount of water added ml Spike sam ples accordingly Add 5ml of ACN ( T N - A - _ j t _ ! i i 3 _ ) to aliquated sample Shake sample for 20 min 48)300 rpm (Shaker ^ rjfljr i* 'S /K ) 0 ^ \ Q cl E i Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 1 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 fTN-A- M) 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 T N -A --3 * v t into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number q 3 q o > cone. IC*0 ^ -^ r i/s amount added_ Transfer sample into appripriatelly marked autovial M. O ff f t 02' 3M E nvironm ental L aboratory bSLoK J Page 175 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite B o t t l e - T o p D is p e n s e r D a il y C a l ib r a t io n C h e c k M anufacturer and Model: Date: Initials: 0<_l Dispenser ID: Balance ID: Volume: Solvent: AC Kj ID#: TKj-A- Mass of solvent (g) 1 2>. * 1 ^ 1 ? 2 3 4 'O q''* 5 ------------------ <1--------- Density1 (g/ml) V.J Average Volume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent mJL S.O'io'J otno 1 5 c<S 3 o `5. SLLf 5 - o 3 'S o |O o. o. o. SGXC> (P>F Volume, (mL) 1 to 10 P erform ance Specifications Accuracy, C.V., (%) (%) 98.0 - 102% 1.0% Corrective Actions: - fa J h p 'V OKJ 'Density values taken from the Merck Index, 12th Edition, Copyright 1996 d \ =specific gravity at ydegr" ac referred to water at xdegr" s n-Hexane d2#4=0.660 Acetone di50=0.788 Methanol d2S,, =0.7866 Methylene Chloride dJ04=1.3255 3M E nvironm ental L aboratory Page 176 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite g ) Qua.) Sent ^ SPE Columns Extraction Worksheet o s jztjo x Prep Date: 2/13/2002 Analysts initials: OK/HOJ Urns 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 C18,1g, 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 2ul of 02001-52 80ul of 02001-52 80ul of 02001-52 80ul of 02001-52 2ml of MeOH TN-A-5457 2mI 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 J ' V * 03, 0 . 2 Jo 2 23.-LBlank Sera TN-A- 5 Amount of sera aliquated:. 5 Dilute Sera with Kandiyohi water; Amount of water added w .m l ml Aliquot 1m! of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN fTN-A- H & ' Z ) ) to aliquated sample Shake sample for 20 min @ 300 rpm (Shaker w jft- Centrifuge sample for 10 min @ 2000 rpm (Centrifuge Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-_ Wash Column with Kandiyohi Water J Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent ( T N-A- J into appropriatei 1lb5 mrml centrifuge tube Spike samples with internal standard PFDA standard number q 2 c g \ ~ M 'S \OQ, j Transfer sample into appripriloaticeliily mmaariikvecdu aauutiouvviaali K f ) J < 9 x .h Q (? b o M dow ^ OK. 3M E nvironm ental L aboratory \ An/) 'S jjJ t Page 177 c o lic o Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Bottle-Top Dispenser Daily Calibration Check 1^ M anufacturer and Model: Date: Initiais: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR 5ml dispenser 2/13/2002 OK 0799 900 5 ACN TN-A-4145 fi0 _ Pv OP e ? G 1 N/AC ^JT -o O f^ 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 10 P er brm an ce Specifica tions Accuracy, (%) 98.0 - 102% C.V., (%) 1.0% Corrective Actions: IOckX? T A ^ 'A CiI ,9 i~> v.-jj 'Density values taken from the Merck Index, 12lh Edition, Copyright 1996 d'', =specific gravity at ydt8" " c referred to water at xllcs,ce,c n-Hexane di04 =0.660 Acetone d250 =0.788 Methanol d15., =0.7866 Methylene Chloride d211., =1.3255 3M E nvironm ental L aboratory Page 178 c o t. Report E01-1245 SPE columns Bobwhite Prep Date: Analysts initials: 2/13/2002 OK/HOJ Lims Assigned sample number E01-1342-36031 E01-1342-36032 E01-1342-36033 EO1-1342-36034 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 E 0 1-1342-30744 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 MSD-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 C16,1g, 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 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6ccplot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W202SB1 C18,1g. 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W202SB1 Method Revision: ETS-8-231.0 Study Number: E01 -1342/1365 Matrix: Liver 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 VA 1' <? + Blank Liver TN-A- 5143 : Amount of liver: 5.0353a Blank Liver TN-A- 5142 : Amount of liver: 1.0015a Homogenize liver with Kandiyohi water; Amount of water added__4H_v* $ ' q+3 O i-n Aliquot 1mi of liver into 15 ml polypropelene tube 3. O --' c- 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 L lQ ^ -j ^jiySG*il>c' lT Centrifuge sample for 10 min @ 2000 rpm (Centrifuge P # 5| 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 mixjng Condition column with MeOH (TN-A- 5 7 ^ ^ ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry _ Elute column with Indicated amount of solvent ( N ^ L ' H TN-A- ^ ) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number G ^ O C l - ^ ^ 0 + , amount added Transfer sample into appripriatelly marked autovial 1 > 0 *O C O **' 3M E nvironm ental L aboratory Page 179 v* * **. Report E01-1245 SPE Columns E x t r a c t i o R ^ k ^ ^ r ^ ^ - * ! ^ ^ Bobwhite Prep Date: Analysts initials: 2/13/2002 OK/HOJ Method Revision: ETS-8-231.0 Study Number: E01-1342/1365 Matrix: Liver Lims A ssigned sam ple number E01-1342-30820 E01-1342-30829 E01-1342-30838 E01-1342-30847 E01-1342-30856 E01-1342-30865 E01-1342-30874 E 0 1-1342-30883 E01-1342-30892 E01-1342-30901 E01-1342-309I0 E01-1342-30919 E01-1342-30928 EO1-1342-30937 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 Lh/er 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, 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 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W202SB1 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 C om m ents AM . I I LAs 02]2J l o i < _^ Blank Liver TN-A- 5143 ; Amount of liver: 5.0353g Homogenize liver with Kandiyohi water; Amount of water added Blank Liver TN-A- 5142 : Amount of liver: 1.0015a (5 /v<^L Amount of water added 9ml Aliquot 1ml of liver into 15 ml polypropelene tube g -\3 0 '2 - Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min @ 300 rpm (Shaker vXvjCq ^ |rO f * t i Centrifuge sample for 10 min @ 2000 rpm ICentrifuge^ ?t /? d ' ^ Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH fTN-A- > 1 C,l~) 1 Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent ( H f* TN-A- Spike samples with internal standard PFDA standard number c Q c x > \ _ J into appropriate 15 ml centrifuge tube L* _______ , cone. I p A . amount added_ Transfer sample into appripriatelly marked autovial <)o K \ . O e rf 0 3M E nvironm ental L aboratory Page 180 C01.1/33 Report EOl-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Bottle-Top Dispenser Daily Calibration Check M anufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID #: VWR 5ml dispenser 2/13/2002 pfa 0K 0799 g* - & o U j 900 5 ACN TN-A-4145 1 2 3 4 5 Volume, (mL) 1 to 10 lO Q O P 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 Perl brm an ce Specifica tions Accuracy, (%) 98.0 - 102 % C.V., (%) 1.0% Corrective Actions: 1 0 /<j ^ PASS PASS 'Density values taken from the Merck Index, 12th Edition, Copyright 1996 dy,, =specific gravity at ydi*rM,c referred to water at xdegre" c n-Hexane d104=0.660 Acetone d2S0 =0.788 Methanol d" <=0.7866 Methylene Chloride d24 =1.3255 3M E nvironm ental L aboratory Page 181 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Attachment D: Dilutions Summary Worksheet Study: E01-1245 Dilution Date/Analyst: CI s Box Number: E01-1245 Solvent/TN Number: MeOH/5457 W/IS PFDA Extraction Date/Analyst: Matrix/Timepoint: Quail sera j Sample N um ber o r Description E01-1245-28480 E01-1245-28482 E01-1245-28484 E01-1245-28486 E01-1245-28488 E01-1245-28490 E01-1245-28492 E01-1245-28494 E01-1245-28495 EOI-1245-28497 E01-1245-28499 E01-1245-28501 E01-1245-28503 E01-1245-28505 E O I-1245-28507 E01-1245-28509 E01-1245-28510 E01-1245-28512 E01-1245-28514 E01-1245-28516 Notes: 1/10 1/100 mX X X X X X X X X X X X X X X X X X X X yL _ 1/ 1/ l / \j -- D ilu tio n s 1/ 1/ 1/ C om m ents L A S (7J 3llt>X Verified By: l 1 / I I I 1 / I 1 -- cV 1/100 dilution= lOul o f sample +990ul o f solvent "Q) 0^3 <fAll dilutions w ere m ade on day of extraction o r the day a fte r analysis. 9 Form Completion Verified By: \ o 3M E nvironm ental L aboratory Page 182 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Attachment D: Dilutions Summary Worksheet Study: E01-1342 c ? t - t z 4 r Dilution Date/Analyst: VI/ Box Number: E01-1342 ^ 1S^vent/TN Number: MeOH/5457 W/IS PFDA Extraction Date/Anaiyst: 2/13/02 Matrix/Timepoint: Quail liver Notes: 1/10 dilution = lOOul o f sample + 900ul o f solvent 1/100 dilution= 1Oul o f sample +990ul o f solvent G AH dilu tio n s w ere m ade on day o f extraction o r the day a fte r analysis. Form Completion Verified By: t M 4 3M E nvironm ental L aboratory Page 183 \j: - / jL ' f.-, 'W Report EOI-1245 SPE Columns E x tr a c tid R R ^ k & i^ r o d ^ o # Bobwhite Prep Date: Analysts initials: 2/15/2002 OK/HOJ Lims Assigned sam ple number E01-1342-36036 E01-1342-36037 E01-1342-36038 E01-1342-36039 E01-1342-36040 E01-1342-30955 E 0 1-1342-30964 E01-1342-30973 E01-1342-30982 E01-1342-30991 E01-1342-31000 E 0 1-1342-31009 E01-1342-31018 E01-1342-31027 E01-1342-31036 E01-1365-31184 E01-1365-31194 E01-1365-31199 E01-1365-31204 E01-1365-31209 Sample Number or description Volume of sam ple filtered (ml) Blank H20-day 4 40+6 Blank female liver-day 4 40+6 Blank male liver-day 4 40+6 Female liver MS-day 4 40+6 Female liver MSD-day 4 40+6 10 ppm a.i. 399 M Liver 40+6 10 ppm a.i. 400 F Liver 40+6 10 ppm a.i. 401 M Liver 40+6 10 ppm a.i. 402 F Liver 40+6 10 ppm a.i. 403 M Liver 40+6 10 ppm a.i. 404 F Liver 40+6 10 ppm a.i. 405 M Liver 40+6 10 ppm a.i. 406 F Liver 40+6 10 ppm a.i. 407 M Liver 40+6 10 ppm a.i. 408 F Liver 40+6 0 ppm a.i. 246 F Liver 40+6 0 ppm a.i. 253 M Liver 40+6 0 ppm a.i. 256 M Liver 40+6 0 ppm a .i. 259 F Liver 40+6 0 ppm a.i. 266 M Liver 40+6 Type of column used and lot C18,1gt 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, 6cq, 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, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18, 1g, 6cc, lot W2025B1 Method Revision: ETS-8-231.0 Study Number: E 0 1 - 4 ^ 4 2 /1 3 6 ^ > / g ^ ~ Matrix: Liver z ,/J ' Amount and Elution solvent and spike mix used volume C om m ents 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 WA________ \> Blank Liver TN-A- 5143 : Amount of liver: 5.0353o Blank Liver TN-A- 5142 : Amount of liver: 1.0015q Homogenize liver with Kandiyohi water; Amount of water a d d e d 45 ml Amount of water added 9ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike sam ples accordingly __ Add 5m! of ACN fTN-A- 4 ) to aliquated sam ple Shake sam ple for 20 min @ 300 rpm (Shaker VWR S/N 041694 ) Centrifuge sam ple for 10 min @ 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi w ater to 50 ml polypropelene centrifuge tube. D ecant extract into centrifuge tu b es with water shake sam ple slightly to ensure proper mixing Condition column with MeOH fTN-A- ) W ash Column with Kandiyohi W ater Filter sam ple through conditioned column, discarding filtrate Allow column to go completelly dry ._ Elute column with indicated am ount of solvent ( r e C f / TN-A- ^ ) into appropriate 15 ml centrifuge tube Spike sam p les with internal standard PFDA standard num ber p j ? P P l~ S~ cone. f ) Q amount added_ Transfer sam ple into appripriatelly marked autovial - 3M E nvironm ental L aboratory Page 184 Report EOI-1245 SPE Columns E xtractioR ^^k^i^pro^ticfti^tudj^w i^hj^e^t^ern Bobwhite Prep Date: Analysts initials: 2/15/2002 OK/HOJ Q -/`S o 2 , 0 1 O fL Method Revision: E T S -8-231.0 ^ Study Number: E 0 1 - m a 4 8 r ^ r Matrix: Lims Assigned sample number EO1-1365-31214 E 0 1-1365-31219 E01-1365-31224 EO1-1365-31229 E01-I365-3I234 E01-1365-31240 E01-1365-31245 E01-1365-31250 E01-1365-31255 E0I-1365-3126O E 0 I-1 365-31265 E01-1365-31270 E 0 1-1365-31275 E 0 1-1365-31280 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 MLiver 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 C16,1g. 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6cc, lot W2025B1 C18,1g, 6oc, 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 M<jOH 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-A5754 '> Comments AiA ias o sia a lo a Blank Liver TN-A- 5143 : Amount of liver: 5.0353q Blank Liver TN-A- 5142 : Amount of liver: 1 .0015a Homogenize liver with Kandiyohi water; Amount of water added 4 5 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- ^) 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 W TN-A- ^ pilfB camplp w ith internal stan d ard PFDA standard number O a ? 0 0 ( - ) Into appropriate 15 ml centrifuge tube . cone. IO & amount added_ Transfer sample into appripriatelly marked autovial t'k fc fi& A d U # O f0 c+ $2o7^ 3M E nvironm ental L aboratory Page 185 f' tI '.T.It., ,,' Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Bottle-Top Dispenser Daily Calibration Check M anufacturer 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) 3.8904 3.9526 3.9724 3.961 3.964 Density1 (g/ml) 0.7857 0.7857 0.7857 0.7857 0.7857 AverageVolume: "/Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 4.9515 5.0307 5.0559 5.0414 5.0452 5.0249 100.50 0.04202 0.84 PASS PASS PASS P e r b rm an ce Specifica tions Accuracy, (%) 98.0 - 102 % Corrective Actions: C.V., (%) 1 .0 % 'Density values taken from the Merck Index, 12,h Edition, Copyright 1996 dy, =specific gravity at ydc*rra c referred to water at xdeeree! c n-Hexane d104=0.660 Acetone d250 =0.788 Methanol diS4=0.7866 3M E nvironm ental L aboratory Page 186 GO:i.:;9 Report EOI-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Attachm ent E: Study P rotocol, P rotocol A m endm ents, and D eviations 3M Environmental Laboratory 3M E nvironm ental L aboratory Page 187 of 226 Page 187 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite W ildlife Internatio nal, 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 PROJECTNO : 454-108, E01-1245 EFFECTIVE DATE: January 24, 2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides the information necessary to complete the analytical phase of this study. REASON. The protocol did not include information for completing the analyses required by the sponsor. AMENDMENT: Harold Johnson is the Principal 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 E nvironm ental L aboratory Page 1of2 Page 188 -Rtt$39f-12il5_FAX 410 822 0632 WILDLI ^NALUCti0n Study with the Nothem Bobwhite 0002 3M E nvironm ental L aboratory Page 189 Report E01-1245 W ildlife In te r n a t io n a l, ltd PFOS: A Reproduction Study with the Nothem Bobwhite PROJECTNO.: 454-108, E01-1245 Sean P. Gallagher, Study Director Joann B. Beavers, Wildlife International, LTD Laboratory Management Harold O. Johnson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date Date Date Date 3M E nvironm ental L aboratory Page 2 of 2 Page 190 (-0 1 : '>r * Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Protocol Amendment # 9 - Attachment A Wildlife International Ltd. # 454-108 STUDY TITLE PFOS: A Reproduction Study with the Northern Bobwhite SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 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 E nvironm ental L aboratory Page 191 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Sponsor Study Identification PFOS : A Reproduction Study with the Northern Bobwhite 3M Environmental Laboratory Building 2-3E -09 PO Box 33331 St. Paul, MN 55133-3331 Principal Analytical Investigator Study Locations A n a l y t ic a l Te s t in g l a b o r a t o r y A n a l y t ic a l Te s t in g La b o r a t o r y Harold Johnson 3M Environmental Laboratory Building 2-3E -09 PO Box 33331 St. Paul, MN 55133-3331 651-778-6479 3M Environmental Laboratory Building 2-3E -09 935 Bush Ave. PO Box 33331 St. Paul, MN 55133-3331 PACE Tier 2 1700 Elm Street, Suite 200 Minneapolis, MN 55414 Proposed Study Timetable 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 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 3 M Environmental Laboratory 3M Environmental Laboratory 2 Page 192 C O ll'/S Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 1. P u r p o s e /O b j e c t iv e s In the in-life phase o f this study, N orthern B obw hite quail w ere regularly exposed to nominal doses o f perfluorooctanesulfonate, potassium salt (PFO S) in their feed in order to evaluate the effects o f dietary exposure upon adults over a five-m onth period. The purpose o f this analytical phase o f the study is to quantify PFO S in the liver and sera o f both control and dosed N orthern B obw hite quail. The analytical inform ation m ay be used to com pare with toxicological/histopathological results from the in-life portion o f this bird study. The in-life portion o f the study w as com pleted at W ildlife International, Ltd. in study 454-108. 2 . R e g u l a t o r y C o m p l ia n c e This analytical phase study will be conducted in accordance w ith the United States Environm ental Protection Agency G ood Laboratory Practice Standards, 40 C FR 792. 3 . Q u a l it y A s s u r a n c e The 3M Environm ental Laboratory Quality A ssurance Unit will audit analytical phase study conduct, data, and the final report to determ ine com pliance w ith G ood L aboratory Practice Standards and with 3M Environm ental Laboratory Standard O perating P rocedures. T he Q uality A ssurance U nit will rep o rt all findings to th e S ponsor Representative and the Study Director. 4. Reference S ubstance 4 . 1 P h y s ic a l D e s crip tio n - P erflu o ro o ctan esu lfo n a te, p o tassiu m salt from L o t 2 17 is a white crystalline material. 4 . 2 P u rity a n d S ta b ility -- T h e p u rity o f L o t # 2 17 h as been d eterm in ed to b e 8 6 .9 % . P F O S has been show n to be stable in m ethanol solutions for up to 6 m onths at am bient tem perature in the validation report from study PS013, "M ethod V alidation and Instrum ent D etection Limit D eterm ination for LC/M S and G C/M S Analysis o f FC807 C om pounds in M ethanol." R efer to the C ertificate o f Analysis (stored in th e Test, C ontrol, and R eference database in the 3M Environm ental Laboratory) for further purity and stability information. 4 .3 Storage Conditions--The test article may be stored at ambient tem perature o r lower. 4 .4 Reserve Sam ples-- A reserve sample o f PFO S from L o t #217 has been frozen at 20C 5C or colder and is kept in the 3M Environm ental Laboratory. 4 . 5 CAS Num ber-- The CA S num ber is: 2795-39-3. 4 .6 M o le c u la r W eight-- The m o lecu lar w eig h t is 538 (C gF|7S 0 3K ). N o te: T h e m o lecu lar w eight o f th e anion (P F O S ) is 4 9 9 (C g F n S O j ). 3 M Environm ental Laboratory 3M E nvironm ental L aboratory 3 Page 193 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 5. C o n t r o l M a t r ic e s Types o f control m atrices and their source, physical description, storage requirem ents, and traceability num bers will be recorded in the raw data and included in the final report. 6. T e s t S y s t e m The test system is N orthern B obw hite quail. T he test system w as dosed (at W ildlife International Ltd. during the in-life phase o f this study) w ith food prepared by mixing PFO S w ith feed in a large m ixer in appropriate portions to achieve th e nom inal 10 ppm level. A food dose verification validation study w as perform ed by W ildlife International under study num ber 454C -110 (3M #U 2723), "Analytical M ethod Verification fo r the D eterm ination o f PFO S in Avian D iet." W ildlife International verified the actual dosed food for this study and the results will be contained in the in-life W ildlife phase report. N orthern Bobw hite serum and liver samples w ere collected by W ildlife International, Ltd. and sent to 3M Environm ental L aboratory for analysis. 6.1 Justification o f the Selection o f the Test System N orthern B obw hite quail represent wild bird populations and they are an E P A recom m ended species because they do well in a laboratory environment. The use o f these m atrices for the study is to determ ine if PFO S w as deposited in the liver and serum o f mating adults that w ere fed PFOS. Furtherm ore, analysis o f these m atrices from hatchlings will determ ine if PFO S w as passed on to m ating adults' offspring. 7. S o u r c e o f S u p p ly fo r the Te s t S ystem The birds w ere obtained by W ildlife International, Ltd. See their protocol (study 454-108) for inform ation about bird origination location. 8 . S p e c im e n a n d S a m p l e R e c e ip t a n d M a in t e n a n c e Sera and liver w ere collected by W ildlife International, Ltd. and shipped to the 3M Environm ental Laboratory for analysis. The 20-w eek endpoint sera and liver sam ples received by the 3M Environm ental Laboratory w ere from the following birds: 40 control adult birds (20 m ating pairs) that ingested food containing 0 ppm PFOS 40 experim ental adult birds (20 m ating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppm PFO S Samples will be maintained frozen except w hen rem oved for extraction and analysis as described in the m ethod. The sam ples will be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M E nvironm ental L aboratory 4 Page 194 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 9. SUB-CONTRACTED ANAL YSIS 9.1 All sera an d liver ex tractio n s as detailed in this p ro to c o l am endm ent w ill b e perform ed at Pace Tier 2, 1700 Elm Street, Suite 200, M inneapolis, M N 55414. 9.2 All sera and liver analyses as detailed in this protocol am endm ent will be perform ed at 3M Environm ental Laboratory, B uilding 2-3E-09, 935 B ush A venue, St. Paul, M N 55106. 10. E x p e r im e n t a l C o n d u c t The 2 1-week endpoint liver and sera sam ples received (see section 8) will be extracted and analyzed for PFO S quantities using the m ethods listed below. 10.1 Preparatory Methods 10.1.1 E T S -8 -6 , E x tra c tio n o f F luorochem ical C o m p o u n d s from L iv er fo r A nalysis Using HPLC-Electrospray/M ass Spectrom etry 10.1.2 E T S -8 -4 , E x tra c tio n o f F luorochem ical C o m p o u n d s from S erum fo r A nalysis Using HPLC-Electrospray/M ass Spectrom etry 10.2 Analytical Methods 10.2.1 E T S -8 -7 , A nalysis o f F luorochem icals in L iv er E x tra c ts U sing H P L C - Electrospray / M ass Spectrom etry 10.2.2 E T S -8 -5 , A nalysis o f P o tassiu m P erflu o ro o ctan esu lfo n a te o r O th er Fluorochem icals in Serum E xtracts U sing H P L C -E lectrospray/M ass Spectrom etry 11. M e t h o d Va l id a t io n M ethod validation for PFO S quantitation in quail sera and liver w as perform ed at C entre Analytical Inc. in study 023-054. T he m ethod will be applied to sam ple analyses w ithout additional validation. 12. D a t a Q u a l it y O b j e c t iv e s D u r in g S a m p l e A n a l y s is The following criteria will be m et using the validated calibration levels and fortification levels determ ined in section 11: 12.1 System Suitability System suitability will be determ ined prior to the start and at the com pletion o f each analytical run. Prior to the calibration curve and after the last sample o f the run three (3) mid-level unextracted calibration standards will be analyzed. The peak area precision and retention tim e precision and peak asym m etry will be m onitored at the beginning and the end o f the run separately. The peak area precision m ust be equal to o r less than 2.0% RSD, the precision o f the retention tim e must be equal to or less than 2.5% R SD and the peak asym m etry (fronting or tailing) m ust be 0.5<A F<2.0, w here AF is the asym m etry factor. I f any item o f the 3 M E nvironm ental Laboratory 5 3M E nvironm ental L aboratory Page 195 0 0 i ' :;8 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 12.2 12.3 system suitability fails, system m aintenance m ust be com pleted prior to running a second set o f system suitability samples and the system suitability m ust pass before starting the calibration. Calibration It will consist o f a minimum o f nine (9) levels, including a blank w ith surrogate and a blank w ithout surrogate, the low est standard will be at approxim ately 50% o f the LLO Q and the highest standard will be approxim ately 50% higher than the U LO Q . The standard curve equation will be determ ined by regression analysis using the peak areas o f the analyte. The accuracy o f each level will be verified. A ny level outside 80% - 120% accuracy must be deactivated, and regression re-calculated. T he curve m ust have a co rrelatio n o f determ in atio n (r2) g re a te r th an o r eq u al to 0.985. All levels m ust show a response greater than tw o tim es that o f the blank. The total num ber o f standards excluded my not exceed 20% . If the preceding criterion are not m et, the curve and sam ple set will be reanalyzed. Limits of Quantitation (LOQ) The low er limit o f quantitation (LLO Q ) w as determ ined to be 10 ng/g in liver and 10 ng/m L serum during th e m ethod validation as described above in section 11. The LLO Q may be determ ined to be higher than that found in the validation study during a screening to be done fo r endogenous levels o f PFO S in th e blank m atrices available at 3M Environm ental Laboratory. I f endogenous levels o f PFO S are found to be higher than the LLO Q o r if the levels are found to be highly variable, the LL O Q will be adjusted to accom m odate this. T he screening procedures and LL O Q determ ination will be recorded in a note to file and kept w ith the raw study data as well as being reported in the final report. R ecovery efficiency at th e LL O Q will be determ ined during each run using an extracted quality control sam ple spiked at the LL O Q level. I f the quality control sam ple at the LLO Q level fails during a run, the LLO Q for that specific run w ould be considered to be the next low est passing quality control sample and any samples below the new LLO Q m ust be reanalyzed. The upper limit o f quantitation (U L O Q ) w as determ ined to be 250 ng/g in liver and 250 ng/m L in serum during the m ethod validation as described above in section 11. An extracted quality control sam ple spiked at th e U L O Q will be used to m easure recovery efficiency at the ULOQ. I f the quality control sam ple at the U L O Q fails during a run the U LO Q for that specific run w ould be considered to be the next highest passing quality control sample and any samples above the new U LO Q m ust be reanalyzed. Any sam ple w ith an area greater than that o f the highest acceptable standard will need to be diluted into the range o f the calibration curve. I f samples are diluted into the range o f the curve during analyses and enough sample remains, a post-run dilution validation will be perform ed to verify sam ple values. T o perform the 3 M E nvironm ental Laboratory 6 3M E nvironm ental L aboratory i pf ! ^ Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite dilution validation, one sample will be separated into tw o representative sam ples (i.e. tw o 1 mL aliquots for fluid sam ples o r tw o 1 gram am ounts for tissue samples) then diluted using tw o procedures. The first procedure consists o f diluting the sample w ith additional m atrix prior to extraction (sera adding sera), while the second procedure consists o f diluting the extract w ith solvent post extraction (m ethanol extract adding additional m ethanol solvent). If the values are not within 15% o f each other additional testing will be required to determ ine which value is a correct representation o f the sample concentration. 12.4 12.5 12.6 12.7 Continuing Calibration Verifications An unextracted midlevel standard (continuing calibration verification - C CV ) will be analyzed after every 10 injections (starting after the last injection o f the calibration curve). The accuracy o f the C C V m ust be within 20% . D ata m ay only b e re p o rte d if b ra ck eted by passing C C V 's (o r a C C V and th e sam e level calibration curve point). D ata bracketed by one o r m ore failing C C V 's m ust be reanalyzed. Quality Control Samples A minimum o f three (3) quality control samples (Q C ) will be prepared w ith each set o f samples. They will consist o f blank m atrix spiked w ith three levels o f analyte. The levels are: L ow level: at th e LLO Q (10 ng/m L in serum and 10 ng/g in liver), M id-level: 5 tim es the LLOQ, H igh level: at the U L O Q (25 tim es the LLO Q ) QC Performance Criteria Each QC is expected to show an accuracy o f 80-120% o f expected. A minimum o f 2/3 o f all QC m ust m eet these criteria. I f not, the entire sam ple set m ust either be re-analyzed or re-extracted (as decided by the Study Director). Use o f Confirmatory Methods C onfirm atory m ethods are typically not needed w ith L C /M S/M S analysis. 12.8 Demonstration o f Specificity PFO S specificity will be substantiated by chrom atographic retention tim e, by the characteristic primary ion (499), and the characteristic product ion (99) using LC/M S/M S. 13. S u b -C o n t r a c t e d A n a l y s is 13.1 All fluid and tissue extractions as detailed in this protocol am endm ent will be perform ed at Pace Tier 2, 1700 Elm Street, Suite 200, M inneapolis, M N 55414. 3 M E nvironm ental Laboratory 3M E nvironm ental L aboratory 7 Page 197 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 1 3 .2 1 3 .3 All fluid and tissue analyses as detailed in this protocol am endm ent will be perform ed at 3M Environm ental Laboratory, Building 2-3E-09, 935 B ush Avenue, St. Paul, M N 55106. P ace Analytical Services, Inc. will follow all applicable 3M Environm ental Laboratory Standard O perating Procedures and the following Pace Analytical Inc. Standard Operating Procedures. PSS-Admin-01 Quality System PSS-Adm in-04 Laboratory Facilities PSS-A dm in-06 Training R ecord Files M aintained by Q A U PS S-ARC-02 Disposition o f Archive M aterials PSS-A R C -04 Archiving D ata, Specimen and Logbooks P S S -D C -05 Lab Notebooks, Logbooks, and Phone Logs PSS-D M -03 Statistical Evaluation o f D ata PSS-OPS-01 Use and M aintenance o f U ltra-Turrax T25 Hom ogenizer PSS-OPS-02 Raw D ata PSS-OPS-03 H azardous W aste Disposal PSS-O PS-04 U se and M aintenance o f N -E vap Analytical E vaporator PSS-O PS-05 U se o f Com pressed G ases in the L aboratory PSS-O PS-06 U se and M aintenance o f N A N O pure II W ater Purification System PSS-O PS-07 Cleaning N on-disposable Volum etric Pipettes PSS-OPS-08 Laboratory Calculations PSS-O PS-09 Deviations from Established Procedures PSS-O PS-10 Analytical Definitions PSS-OPS-11 U se and Calibration o f a B ottle-Top Dispenser PSS-O PS-13 U se and M aintenance o f Lab Refrigerators, Freezers, and Ovens PSS-O PS-14 Use and Calibration o f Syringes with a Hamilton Chaney A dapter PSS-O PS-15 Glassware Cleaning PSS-O PS-16 U se and M aintenance o f the Fisher Scientific Isotem p Freezer PSS-O PS-17 Use and M aintenance o f Ultrasonic W ater Baths (Sonicators) PSS-OPS-18 U se and M aintenance o f V o rtex S hakers PSS-O PS-24 Use and M aintenance o f a D esiccator PSS-OPS-31 Use and M aintenance o f Shakers PSS-O PS-32 Operation, M aintenance and Calibration o f Laboratory PSS-O PS-37 Operation, M aintenance, and Calibration o f pH M eters and pH E lec tro d es PSS-M C-01 Purchasing o f Laboratory Supplies PSS-M C -02 R eceipt o f Laboratory Supplies PSS-M TR-01 Calibration o f Certified W eight Set PSS-M TR-02 Calibration o f Certified Therm om eters/Therm ocouples PSS-M TR-05 Calibration o f E ppendorf Pipettes PSS-M TR-06 Verification o f Calibration o f the E ppendorf Pipettor PSS-M TR -07 N ew Equipm ent Qualification (IQ, PQ, OQ) PSS-M TR -08 V erification o f Calibration and U se o f Analytical Balances 3 M Environm ental Laboratory 3M E nvironm ental L aboratory 8 Page 198 G O li Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PSS-M TR-09 PSS-Safety-01 PSS-Safety-02 PSS-Safety-04 PSS-Safety-05 PSS-TS-01 U se and M aintenance o f Fisher Scientific Electronic Tem perature M onitoring Devices General Laboratory Safety U se and M aintenance o f Biological Safety Cabinets Emergency Evacuation Procedures Use and M aintenance o f Laboratory Fume H oods Training Procedures 1 3 .4 An am endm ent to the protocol will be w ritten if extractions and analyses are perform ed at laboratories other than the Pace Tier 2 or 3M Environmental Laboratory, respectively. 14. S t a t is t ic a l A n a l y s is Statistical m ethods will be limited to the calculation o f m eans and standard deviations. Exam ples o f 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 Environm ental Laboratory. T he final report will follow all G LP requirem ents, and will include a description o f all m aterials and m ethods used, including a narrative discussion w ith a tabular presentation o f all analyses results. 16. L o c a t io n o f R a w D a t a , R e c o r d s , a n d F in a l R e p o r t W hen the final report is com pleted, 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 o f 3M Environm ental Laboratory. All corresponding training records, calibration records, instrum ent m aintenance logs, standard operating procedures, equipm ent procedures, and m ethods will be retained either at Pace T ier 2 or the 3M Environm ental Laboratory, as applicable. 17. S p e c im e n R e t e n t io n Samples o f the test substance (any leftover control sera or liver and samples) and the analytical standards will be m aintained in the laboratory for a period o f tim e as specified by regulation or as long as the quality o f the preparation affords evaluation. H ow ever, sam ples will not be m aintained m ore than 10 years after the effective date o f the final test rule (if applicable). 3M Environm ental Laboratory Standard O perating Procedures also apply to sample retention times. 18. R eferences 1 8.1 1 8 .2 W ildlife International, Ltd. P rotocol N o. 454-108, "PFO S: A R eproduction Study with the N orthern Bobwhite". C entre Analytical Laboratories, Inc. Study 023-054, "V alidation o f Analytical M ethods "E xtraction o f Potassium Perfluoroocatanesulfonate o r O ther Fluorochem ical Com pounds From Serum for Analysis U sing H PLC Electrospry/M ass Spectrom etry" and "Extraction o f Potassium 3 M Environm ental Laboratory 9 3M E nvironm ental L aboratory Page 199 0,011 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 1 8 .3 Perfluorooctanesulfonate or O ther Fluorochemical Com pounds From Liver for Analysis Using HPLC Electrospray/M ass Spectrom etry in Quail Serum and Liver." 3M Study U 2723, W ildlife Study 4 5 4 C -1 10, "Analytical M ethod V erification for the Determ ination o f PFO S in Avian D iet." 19. P r o t o c o l A m e n d m e n t s a n d D e v ia t io n s Planned changes to the protocol will be in the form o f w ritten am endm ents signed by the Study D irector and the Sponsor R epresentative. A m endm ents will be considered as part o f th e protocol and will be attached to the final protocol. Any other changes will be in the form o f w ritten deviations, signed by the Study D irector and Sponsor R epresentative and filed w ith th e raw data. All changes to the protocol will be indicated in the final report. 3 M Environm ental Laboratory 3M E nvironm ental L aboratory 10 Page 200 U 9 J i t 0632 WILDLIFE BF^AxRaj&rbduction Study with the Notjgpggjobwhite W il d l if e In t e r n a t io n a l , l t d PROJECT NO.: 454408, __ _________ E 014 2 4 5 AMENDMENT TO STUDY PROTOCOL STUDY TTLE: PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/120700/QRySUB454 AMENDMENT NO.. 10 SPONSOR. 3M Corporation PROJECT NO.: 4544 08, E014245 EFFECTIVE DATE: January 30, 2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase o f this study. This attachment A replaces the attachment A in amendment 9. REASON. The amendment 9 attachment A was updated to include a different extraction and analytical method, to update the data quality objectives, iand to change the liver and sera extraction location. Sean P. Gallagher, Study birector 1 3 o jp y Date Joann B. Beavers, Wildlife International, LTD, Laboratory Management Harroold O. Johnson, Pnftcipal Analytica l Investigator Date 2 *= - <S>< I Z ' U Date t* ' V William K. Reagen, 3M Environmental Laboratory, Labrn atopTMffl obideaui Sponsor's Representative Date a t / s j/ u L t . Date 3M E nvironm ental L aboratory 1 Of 9 Page 201 G il 1 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 Nothem 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 o f this study. This attachment A replaces the attachment A in amendment 9. REASON: The amendment 9 attachment A was updated to include a different extraction and analytical method, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director Date Joann B. Beavers, 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 E nvironm ental L aboratory 1 Of 9 Page 202 ( Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Protocol Amendment # 10 - Attachment A Wildlife International Ltd. #454-108 STUDY TITLE PFO S: A R eproduction Study w ith the N orthern B obw hite SPONSOR 3M Environm ental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, M N 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M Environm ental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, M N 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environm ental Laboratory: E01-1245 W ildlife International Ltd.: 454-108 3M Environmental Laboratory 3M E nvironm ental L aboratory 2 of 9 Page 203 cot Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Sponsor Study Identification PFOS: A Reproduction Study w ith the N orthern Bobwhite 3M Environm ental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, M N 55133-3331 Principal Analytical Investigator Study Locations Analytical Testing Laboratory Harold Johnson 3M Environm ental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, M N 55133-3331 651-778-6479 3M Environm ental Laboratory Building 2-3E-09 935 Bush Ave. PO Box 33331 St. P aul, M N 55133-3331 Proposed Study Timetable Analytical Experimental Start Date Analytical Experimental Termination Date 01/31/02 03/31/02 3M Environmental Laboratory 3M E nvironm ental L aboratory 3 of 9 Page 204 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 1. P u r p o s e /O b j e c t iv e s In the in-life phase o f this study, N orthern B obw hite quail w ere regularly exposed to nom inal doses o f perfluorooctanesulfonate, potassium salt (PFOS) in their feed in order to evaluate the effects o f dietary exposure upon adults over a five m onth period. The purpose o f this analytical phase o f the study is to quantify PFO S in the liver and sera o f both control and dosed N orthern B obw hite quail. The analytical inform ation m ay be used to com pare w ith toxicological/histopathological results from the in-life portion o f this bird study. T he in-life portion o f the study w as com pleted at W ildlife International, Ltd. in study 454-108. 2. R e g u l a t o r y C o m p l ia n c e This analytical phase study w ill be conducted in accordance w ith the U nited States Environm ental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3 . Quality A ssurance The 3M Environm ental Laboratory Q uality A ssurance U nit will audit analytical phase study conduct, data, and the final report to determ ine com pliance with Good Laboratory Practice Standards and w ith 3M Environm ental Laboratory Standard O perating Procedures. The Q uality A ssurance U nit w ill report all findings to the Sponsor Representative and the Study Director. 4. R eference S ubstance 4 .1 P h y s ic a l D e s c rip tio n - Perfluorooctanesulfonate, potassium salt from Lot 217 is a w hite crystalline m aterial. 4 .2 P u rity a n d S ta b ility --The purity o f Lot #217 has been determ ined to be 86.9% . PFOS has been show n to be stable in m ethanol solutions for up to 6 m onths at am bient tem perature in the validation report from study PS013, "M ethod V alidation and Instrum ent Detection Lim it D eterm ination for LC/M S and GC/M S Analysis o f FC807 C om pounds in M ethanol." Refer to the Certificate o f Analysis (stored in the Test, Control, and Reference database in the 3M Environm ental Laboratory) for further purity and stability information. 4 .3 S to ra g e C o n d itio n s -- The test article m ay be stored at am bient tem perature or lower. 4 .4 R es erve S am p les-- A reserve sam ple o f PFOS from Lot #217 has been frozen at 20C 5C or colder and is kept in the 3M Environm ental Laboratory. 4 .5 C A S N u m b e r-- The CAS num ber is: 2795-39-3. 4.6 M o le c u la r W e ig h t--T h e m o le c u la r w e ig h t is 538 (C g F n S 03K ). N ote: T h e m o le c u la r w eight o f the anion (PFO S) is 499 (C & F^SO j). 3M Environmental Laboratory 3M E nvironm ental L aboratory 4 of 9 Page 205 f Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 5. C o n t r o l M a t r ic e s Types o f control m atrices and their source, physical description, storage requirem ents, and traceability num bers will be recorded in the raw data and included in the final report. 6. T e s t S y s t e m The test system is the N orthern B obw hite quail. The test system w as dosed (at W ildlife International Ltd. during the in-life phase o f this study) w ith food prepared by m ixing PFO S w ith feed in a large m ixer in appropriate portions to achieve the nom inal 10 ppm level. A food dose verification validation study was perform ed by W ildlife International under study num ber 454C -110 (3M #U 2723), "Analytical M ethod V erification for the D eterm ination o f PFOS in A vian D iet." W ildlife International verified the actual dosed i food for this study and the results will be contained in the in-life W ildlife phase report. N orthern B obw hite serum and liver sam ples w ere collected by W ildlife International, Ltd. and sent to 3M Environm ental Laboratory for analysis. 6.1 Justification of the Selection o f the Test System N orthern Bobwhite quail represent w ild bird populations and they are an EPA recom m ended species because they do well in a laboratory environm ent. The use o f these m atrices for the study is to determ ine if PFOS was deposited in the liver and serum o f m ating adults that w ere fed PFOS. Furtherm ore, analysis o f these m atrices from hatchlings will determ ine if PFOS was passed on to m ating adults' offspring. 7. S o u r c e o f S u p p l y f o r t h e T e s t S y s t e m The birds w ere obtained by W ildlife International, Ltd. See their protocol (study 454108) for inform ation about bird origination location. 8. S p e c im e n a n d S a m p l e R e c e ip t a n d M a in t e n a n c e Sera and liver were collected by W ildlife International, Ltd. and shipped to the 3M Environm ental Laboratory for analysis. The 21-week endpoint sera and liver sam ples received by the 3M Environm ental Laboratory were from the follow ing birds: 40 control adult birds (20 m ating pairs) that ingested food containing 0 ppm PFOS 40 experim ental adult birds (20 m ating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppm PFOS Sam ples w ill be m aintained frozen except when rem oved for extraction and analysis as described in the method. The sam ples will be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M E nvironm ental L aboratory 5 of 9 Page 206 0 1 ..^ S Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 9. SUB-CONTRACTED ANAL YSIS All sera and liver extractions and analyses as detailed in this protocol am endm ent w ill be perform ed at 3M Environm ental L aboratory, B uilding 2-3E-09, 935 B ush A venue, St. Paul, M N 55106.55414. 10. E x p e r im e n t a l C o n d u c t The 21-week endpoint liver and sera sam ples received (see section 8) w ill be extracted and analyzed for PFO S quantities using the m ethods listed below. 10.1 Preparatory and Analytical Method 10.1.1 E T S -8-231 S olid P h ase E x trac tio n and A n aly sis o f F lu o ro ch em ical Com pounds from Biological M atrices 11. D a t a Q u a l it y O b j e c t iv e s D u r in g S a m p l e A n a l y s is M ethod validation for PFO S quantitation in rat and m ouse sera and liver is in process in the 3M Environm ental Laboratory study E01-1277. Lim ited N orthern Bobw hite control m atrix preclude a cross validation, however, the E 01-1277 study m ethods w ill be applied to sam ple analyses o f N orthern B obw hite sera and liver sam ples using the following perform ance based criteria: 11.1 System Suitability System suitability will be determ ined prior to the start and at the com pletion o f each analytical run. Prior to the calibration curve and after the last sam ple o f the run three (3) m id-level unextracted calibration standards will be analyzed. The peak area precision and retention tim e precision w ill be m onitored at the beginning and the end o f the run separately. The peak area precision m ust be equal to or less than 2.0% R SD , the precision o f the retention tim e m ust be equal to or less than 2.5% RSD. if any item o f the system suitability fails, system m aintenance m ust be com pleted prior to running a second set o f system suitability sam ples and the system suitability m ust pass before starting the calibration. 11.2 ' Absolute Recovery The absolute recovery of the m ethod will be evaluated one tim e in both liver and serum. For each m atrix a m inim um o f triplicate sam ples w ill be fortified at a m inim um o f two levels o f PFO S that bracket the range o f analyte determ ined for the sam ples (i.e., at near the highest level determ ined in sam ples and at ~ l 50% o f LOQ for samples). The sam ples will be extracted through the m ethod, and analyzed by com parison w ith an external calibration o f non-extracted standards. The non-extracted standard calibration curves will be prepared in m ethanol, and will consist o f a m inim um o f nine (9) levels, including a m ethanol blank. The best appropriate regression will be used to describe this curve (for best accuracy at all levels o f the standards). 3 M Environmental Laboratory 3M E nvironm ental L aboratory 6 of 9 Page 207 Report EO1-1245 PFOS: A Reproduction Study with the Nothem Bobwhite The accuracy (% absolute recovery) and precision (% CV o f the recoveries) will be determ ined at each level using the external calibration curve for quantitation. There is no control lim it for accuracy; precision m ust be better than 20% at each level. Should a level (high or low) not m eet this precision, the m ethod will only be considered valid for levels between the two valid levels. Additional testing m ay be conducted at other levels to extend the acceptable range o f the method. 11.3 Calibration A calibration curve should be prepared in each m atrix by spiking the m atrix with know n concentrations o f the analyte and surrogate. A calibration curve should consist o f at least nine standard curve points covering the expected range, including the LLOQ. T he sim plest m odel that adequately describes the concentration-response relationship should be used. The accuracy o f each level will be verified. A ny level outside 75% - 125% o f nom inal m ust be deactivated, and regression re-calculated, except the LLOQ w hich m ust be w ithin 30% o f nom inal. All levels m ust show a response greater than twice that o f the m atrix blank. A m axim um o f four (4) levels m ay be deactivated in any one set, or the set will be re-analyzed. 11.4 Limits of Quantitation (LOQ) The low er limit o f quantitation (LLOQ) will be defined in liver (ng/g) and in serum (ng/m L) as the low est acceptable extracted calibration curve point that is w ithin the 70% -130% nom inal criteria and w ith an analyte peak area at least 2 tim es the blank. The LLOQ determ ination w ill be recorded in a note to file and kept w ith the raw study data as well as being reported in the final report. The upper limit o f quantitation (ULOQ) will be defined in liver and in serum as the highest acceptable extracted curve point that is w ithin the 75% - 125% nom inal criteria and includes applicable dilution factor corrections. 1 1 .5 Q u a lity C o n tro l S a m p le s A m inim um o f one set o f nine (9) quality control sam ples (QC) will be prepared one tim e in each m atrix. They w ill consist o f three levels o f analyte, each in triplicate. The quality control sam ples should be prepared in each m atrix by spiking the control m atrix w ith know n concentrations o f the analyte. The levels bracket the sam ple concentrations determ ined in the study: Low level: equivalent to an analyte concentration near or below the low est level o f analyte determ ined in sam ples M id-level: equivalent to an analyte concentration near the m iddle o f the sam ple range High level: equivalent to an analyte concentration near or above the highest level o f analyte determ ined in sam ples 11.6 Quality Control Sample Acceptable Recoveries 3M Environmental Laboratory 3M Environmental Laboratory 7 of 9 Page 208 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Each QC is expected to show an accuracy o f 75-125% o f expected. A m inim um o f 2/3 (6 out o f 9) o f all QC m ust m eet these criteria and a m inim um o f one h alf (2 out o f 3) o f the QC at each level m ust m eet these criteria. If not, the entire sam ple set m ust either be re-analyzed or re-extracted (as decided by the Principal A nalytical Investigator). 11.7 Matrix Spikes Tw o m atrix spikes w ill be extracted with each sam ple preparation. The m atrix spikes w ill consist o f one fortification level in the range o f the sam ples, extracted in duplicate. If the m atrix spike and/or m atrix spike duplicate fall outside o f 25% accuracy, th e curve, C C V 's, m atrix spikes and sa m p les w ill b e rean aly zed . 11.8 Blanks One control m atrix and one w ater m ethod blank w ill be extracted w ith each sam ple preparation. 11.9 Continuing Calibration Verification Two extracted standards (continuing calibration verification - CCV ) w ill be analyzed after every 10 injections (starting after the last injection o f the calibration curve). The accuracy o f the CCV m ust be w ithin 25%. D ata m ay only be reported if bracketed by passing C C V 's (or a C C V and the sam e level calibration curve point). D ata bracketed by one or m ore failing CCV m ust be reanalyzed. 11.10 Use o f Confirmatory Methods C onfirm atory m ethods are typically not needed w ith LC/M S/M S analysis. 11.11 Demonstration o f Specificity PFOS specificity will be substantiated by chrom atographic retention tim e, by the characteristic prim ary ion (499), and the characteristic product ion (99) using LC/M S/M S. ' 1 2 . SUB-CONTRA CTED ANAL YSIS 1 2.1 All fluid and tissue extractions and analyses as detailed in this protocol am endm ent will be perform ed at the 3M Environm ental Laboratory, Building 23E-09, 935 B ush A venue, St. Paul, M N 55106. 12.2 An am endm ent to the protocol w ill be w ritten if extractions and analyses are perform ed at laboratories other than the 3M Environm ental Laboratory. 13. S t a t is t ic a l A n a l y s is Statistical m ethods w ill be lim ited to the calculation o f m eans and standard deviations. Exam ples o f the calculations used in the analyses will be included in the analytical phase report. 3M Environmental Laboratory 3M E nvironm ental L aboratory 8 of 9 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite 14. R eport A final report will be prepared by the 3M Environm ental Laboratory. The final report w ill follow all GLP requirem ents, and will include a description o f all m aterials and m ethods used, including a narrative discussion w ith a tabular presentation o f all analyses results. 15. L o c a t io n o f R a w D a ta , R e c o r d s , a n d F in a l R e p o r t W hen the final report is com pleted, all original paper data, including protocol, phase report, study correspondence, sam ple receipt and tracking, sam ple preparation, and analytical data, will be retained in the archives o f 3M Environm ental Laboratory. All corresponding training records, calibration records, instrum ent m aintenance logs, standard operating procedures, equipm ent procedures, and m ethods will be retained at the 3M Environm ental Laboratory, as applicable. 16. S p e c im e n R e t e n t io n Sam ples o f the test substance (any leftover control sera or liver and sam ples) and the analytical standards w ill be m aintained in the laboratory for a period o f tim e as specified by regulation or as long as the quality o f the preparation affords evaluation. How ever, sam ples will not be m aintained m ore than 10 years after the effective date o f the final test rule (if applicable). 3M Environm ental Laboratory Standard Operating Procedures also apply to sam ple retention times. 17. R eferences 1 8.1 1 8 .2 W ildlife International, Ltd. Protocol No. 454-108, "PFO S: A R eproduction Study with the N orthern Bobwhite" . 3M Study U 2723, W ildlife Study 454C -110, "Analytical M ethod V erification for the D eterm ination o f PFO S in A vian D iet." 18. P r o t o c o l A m e n d m e n t s a n d D e v ia t io n s Planned changes to the protocol will be in the form o f w ritten am endm ents signed by the Study D irector and the Sponsor Representative. A m endm ents w ill be considered as part o f the protocol and will be attached to the final protocol. A ny other changes w ill be in the form o f w ritten deviations, signed by the Study D irector and Sponsor R epresentative and filed with the raw data. All changes to the protocol will be indicated in the final report. 3M Environmental Laboratory 3M E nvironm ental L aboratory 9 of 9 Page 210 0 - 0 1 .1 3 3 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite W il d l if e 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 PROTOCOLNO.: 454/120700/QR/SUB454 SPONSOR: 3M Corporation AM EN D M EN TN O .: 11 PROJECT NO.: 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 o f the retention time must be equal to or less than 2.5% RSD. To: The peak area precision must be equal to or less than 5.0% RSD, the precision o f the retention time must be equal to or less than 2.5% RSD. REASON: Information received from our instrument supplier indicates that a 2.0 % RSD for the area count is common to UV detector specifications. Mass spectrometers, which we used for this study, are more sensitive, in general and the fact that analyte ionization can be suppressed by other species present in the sample matrix and mobile phase make area reproducibility more challenging to maintain. Sample and source cleanliness are the determining factors, but < 2.0% is still a bit aggressive for mass spec. The application chemists at Micromass, the instrument vendor, suggest a value of <5.0%. AMENDMENT: Section 11.3, Sentence 5. Change: Any level outside 75% - 125% o f nominal value must be deactivated, and regression recalculated, except the LLOQ which must be within 30% o f nominal. To: In some cases the lowest curve point, although outside the 30% nominal requirement, may be included in the linear range to influence the lower end o f the curve. Only one point, the lowest level point, can be left in for this purpose. It will not be used to determine the limit o f quantitation. The limit of quantitation will be the next point on the curve meeting the 30% nominal requirement. REASON: The removal o f the lowest calibration curve point in some cases causes the next lowest calibration point to deviate by greater than 30%. Removal o f this point in turn causes the next calibration point to deviate by greater than 30%. This continued deletion o f calibration points in some cases could continue until there are very few points 3M E nvironm ental L aboratory Page 1 of 3 Report EOI-1245 PFOS: A Reproduction Study with the Nothera Bobwhite remaining in the curve. The inclusion o f a curve point w ith a deviation greater than 30% is used to influence the lower end o f the curve and is not used for determination o f the LOQ. AMENDMENT: Section 11.3, the last sentence. Change: A maximum o f four (4) levels may be deactivated in any one set, or the set will be reanalyzed. To: The calibration curve will contain a minimum o f six (6) calibration curve points. REASON: In most cases the calibration curves contain greater than nine (9) calibration points. The large number o f points in the curve is used to select a range representative to the samples. Limiting the removal o f only four (4) points in some cases could cause a calibration range not representative of the samples. AMENDMENT: Section 11.3 Add: The coefficient o f determination for the calibration curve should be > 0.985. REASON : No coefficient o f determination is included in the protocol. It was inadvertently removed prior to printing o f the final copy. 3M E nvironm ental L aboratory Page 2 o f 3 Page 212 011^; R a p w t E Q k l ^ 17 FAX 410 822 0632 WILDLIFE P dUCti0" S,Udy Wth the aax0i/ a2 18:29 BSD INF TECH 2- 2E-01 * *84108220915 NO. 905 P007 VVfepuFE In t e r n a t io n a ^ ltd PROJECTNO.: 454-108, E01-1245 - <>w J o a g p B .~B e a V e r s ^ W ildlife International, LTD Laboratory Management HamU O. Johnson, Principal Analytical Investigator WlBtim K- Reagita, 1M E nm m ncatil Laboratory, Date Date cn. 3M E nvironm ental L aboratory Page 3 of 3 Page 213 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite W il d l if e 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 larold O. Jol 'rincipal Analytical Investigator William K. Reagh, 3M Environmental Laboratory, Laboratory Management Date Date 4 Z Date Date ^-- 3M E nvironm ental L aboratory Page 3 of 3 Page 214 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 Nothem 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 AM ENDM ENT NO.: 12 SPONSOR: 3M Corporation EFFECTIVE DATE: August 16,2002 PROJECT NO.: 454-108, E01-1245 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 E 0 1-1365. REASON: Bobwhite samples were received in the 3M Environmental at several time points. Upon arrival, samples were assigned a LIMS tracking number. Not aware of the scope of this or any other particular study pertaining to these samples, they were checked in to different 3M LIMS projects. LIMS project numbers assigned to this study include the following: U2723, E01-1245, E01-1342, and E01-1365. Samples in each of these projects pertaining to this study were analyzed under one main study number E01-1245. AMENDMENT: Change: Harold Johnson is the Principal Analytical Investigator. To: Lisa A. Stevenson is the Principal Analytical Investigator. REASON: To change role of the Principal Analytical Investigator. 3M E nvironm ental L aboratory Page 1 of 2 Page 215 ill Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite W il d l if e In t e r n a t io n a l , ltd jL .1 Ji J U u Sean P. Gallagher, Study Director Joann B. Beavers, W ildlife International, LTD, Laboratory Management A iS b __________ _______________ Lisa A. Stevenson, Principal Analytical Investigator _____--~ -------------------------------------------------W illiam K. Reagen, 3M Environmental Laboratory, Laboratory Management PROJECTNO.: 454-108, E01-1245 (/o i Date Date Date Date 3M E nvironm ental L aboratory Page 2 of 2 Page 216 0 -0 1 1 3 9 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-108, EOI-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 AM ENDM ENT 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 Director Joann B. B e y e rs , W ildlife International, LTD, Laboratory Management Q u * A. Lisa A. Stevenson, Princiipal Analytical Investigator W illiam K. Reagen, 3M Environmental Laboratory, Laboratory Management a -- C'X P u r i s t s , Suan A. Beach, Sponsor's Representative Date _____________ l & \ v \ c s T -- D ate o q /a i/o a Date Date j Vs Date 3M E nvironm ental L aboratory Page 1 of 1 Page 217 Report E01-1245 3M Confidential PFOS: A Reproduction Study with the Nothem Bobwhite Record of Deviation Study / Project No. EO1-1245 Deviation type SOP (Check one) 0 Protocol Document number ETS-4-4.0 I. identification D M ethod Q Equipm ent Procedure Other: Date(s) of occurrence 1/31/02, 2/1/02, 2/13/02, 2/7/02, 2/8/02, 2/11/02, _________ 2/12/02, 2/13/02,2/15/02___________________ li. Description____________________________ Required procedure/process: ETS-4-4.0 Internal chain of custody The internal chain o f custody form to be used to track all movement of samples Within the lab. The form should be filed with study data or other relevant sample documents. A ctual procedure/process: Internal chains of custodies were not used to track samples and sample extracts. Since receipt samples were Stored in freezer F I. After homogenizing liver and aliquating sera samples were stored in Freezer F8 Until extraction date. Samples remained frozen until extraction. Deviation to SOP written. III. Actions Taken (such as amendment issued, S O P revision, etc.) Recorded by A uthorized by Q r \, D ate f ? '0 2 _ L D ate 4 * r ^ Deviation No. I (assigned by.MStudy Director or Project Lead at the end of study or project) c\ \ > v\ o 'V Attachment A 3M E nvironm ental L aboratory ETS-4-8.2 Documentation of Deviations Page 5 o f 1 Page 218 O O i . `, ' O Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Record of Deviation I. Identification Study / Proj ect No.EO 1-1245 D eviation Type (Check one) SOP V Protocol M ethod Equipm ent Procedure Other: D ocum ent N um ber:Protocol #: Date(s) o f occurrence: :1/31/02, 2/1/02, 2/2/02, 2/7/02, 454/120700/Q R/SU B454, 2/11/02, 2/12/02, 2/13/02, 2/14/02, 2/18/02, 4/11/02, a m e n d m e n t# : 10____________________ 4/16/02______________________________________________ II. Description: R e q u ire d P ro c e d u re /p ro c e ss: Section 11.1, System Suitability, second sentence says that a m id-level unextracted calibration standard will be analyzed. A ctual P rocedure/process: On occasion through the dropping o f calibration points, the system suitability sam ples w ere on the high end o f the curve or outside the w orking range o f the curve. A lthough these were unextracted curve points their area counts were at the high end o f the calibration curve range, they do pass the system suitability criteria and these data packet w ill be considered acceptable. The data from 2/7/02 w as just above the calibration range. These data w ill also be considered acceptable. III. Actions Taken: (such as amendment issued, SO P revision, etc.) Issuing o f this protocol deviation. Recorded By D ate IV. Impact on Study / Project *7^ There will be no im pact on the study as the injections involved w ere used for system suitability and were w ithin the acceptable precision ranges even though above the extracted calibration curve. A uthorized B y ?(Stu(lv D ir e c to r // P roject Lead) D ate s Form E TS-4-8.0 /* O ..X . h lA ^ V 3M E nvironm ental L aboratory .. \ jX X W t^ Deviation No. . (assigned by Study Director or Project Lead at the end o f study or project) < \\W V Page 219 o n :*c X z - Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Record of Deviation I. Identification Study / Project No.E01-1245 D eviation Type (Check one) VSOP M ethod Equipm ent Procedure Protocol Other: D o c u m e n t N u m b e r:E T S -8 -2 3 1 .1 Date(s) o f 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 / 1 6 / 0 2 _______________________________________________ II. Description: 1 R e q u ire d P ro ce d u re/p ro c ess: Section 12.1, Calculations: If other calculations are used than those listed, they w ill be docum ented in the raw data. A ctual Procedure/process: Standard E 0 I-1 342-36010, Prep sheet description "Curve Point - lOppb in liver", was prepped by spiking 20uL o f 02001-66 (1 .OOug/mL) into 1 m L o f liver hom ogenate. This hom ogenate consists o f 5g liver plus 45m L water. Since lm L o u to f5 0 m L hom ogenate is used to prep each extracted standard, 1/50 o f the 5g (0. lg ) o f liver is "spiked" . A fter this hom ogenate is spiked and prepared using SPE , the final eluate is 2.0m L o f M eO H . T H E R E F O R E ... (2 0 u !)(1 .OOugPFOSVmL M eO H ) (l.OmL/lOOOuL) (lOOOng/l.Oug) = 20ng PFO S' onto SPE colum n 20ng PFOS' / 2.0mL M eOH = lOng PFO S'/m L MeOH. (Since this standard is recorded as 1Oppb, the correct units are lOnePFOSVmL o f M eO H .1 These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statem ent is true. (20ng P F O S ' / 1.OmL hom ogenate) (1 .OmL hom ogenate / 0 .1g Iiver) = 200ng P F O S '/ g liver This relationship shows the factor o f 20 used to "correct" the units when calculating the final result in ng/g from the chrom atographic result that is in ng/mL. Sample Calculation: "E 01-1342-30348, Oppm a.i. 333 M liver" C hrom atographic result is 10.76 ng/m L; reported value is 208.4 ng/g. Since the 1.0333g initial sam ple w eight o f liver is hom ogenized with 9.0m L w ater, and 1m L ( 10% o f total liver weighed, since a liver density o f 1.00 is assum ed) is used for the entire SPE procedure, the calculation is as follows: (10.76ng PFO S-/ mL M eOH) (2mL M eOH / 0.1033 g liver) - 208.4 ng PFOS- / g liver Sample Calculation: Sample "E 0 1 -1342-36017, OCioHOnDb-l": This QC sample is prepared by adding 4uL o f 02001-53 (5.020ug PFO S' / mL MeOH) spiked into lm L liver homogenate. I ((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 P F O S '/g liver, the recovery is (271.8/200.8>( 100)= 135.4%. 3M E nvironm ental L aboratory Form E TS-4-8.0 3M E nvironm ental L aboratory Deviation No. _____ 3_______ (assigned by Study Director or Project Lead at the end of study or project) Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Record of Deviation 3M E nvironm ental L aboratory Form E TS-4-8.0 3M E nvironm ental L aboratory Deviation No. ^_________ (assigned by Study Director or Project Lead at the end o f study or project) Page 221 c o ta o -i Report EOl-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Record of Deviation I. Identification S tudy / P ro ject N o. EO1-1245 D eviation Type (Check one) SOP M ethod Equipm ent Procedure VProtocol Other: D ocum ent Num ber: ETS-8-231.1 D ate(s) of occurrence: Throughout the study II. Description: Required Procedure/process: S ection 11.3, S entence 5 States: A ny level outside 75% -1 2 5 % o f nom inal valu m ust be deactivated, and regression recalculated... Actual Procedure/process: M any o f the high calibration points in this study are kept in the curve despite being above the apex o f the curve. W hen a calibration point lies above the apex, it is considered indeterm inant (flagged with an I) and no concentration is calculated for the standard. If no concentration is calculated, no percent difference is calculated for evaluating w hether o r not that standard is w ithin the stated criteria. R em oval of these points in m ost 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 rem aining on the curve. Therefore, calibration points above the apex were included in the range o f the curve w ith no evaluation o f percent deviation criteria. III. Actions 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 a / A cl. A uthorized B y (Study D irector / Project Lead) D i r t c _________________ RApre.stMc.Vwt *\v 4 o y T JZ J U D ate 08/16/02 etf/jfcJ c o . D ate 3M E nvironm ental L aboratory Form E TS-4-8.0 3M E nvironm ental L aboratory Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Page 222 R eport E 01-1245 PFOS: A R eproduction Study w ith the N othem Bobwhite Record of Deviation I. Identification Study / Project No. E01-1245 D eviation Type (Check one) TSO P M ethod Equipm ent Procedure Protocol Other: D o c u m e n t N um ber: E T S -8 -2 3 1.1 j D ate(s) o f o ccurrence: E n tire S tu d y II. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedure/process: Standard E 01-1345-35771, Prep sheet description " 10 ppb sera curve point", was prepped by spiking 4uL of 02001-52 (5.020ug/mL) into 2 mL o f pooled sera. This diluted sera consists o f 2mL sera plus 38mL water. Since lm L out of 40m L diluted sera is used to prepare each extracted standard, 1/40 o f the 2m L o f sera is "spiked". After 1.0 m L o f diluted sera is spiked and prepared using SPE, the final eluate is 2.0mL of MeOH. TH EREFO RE... (5.020ug P F O S/m L M eOH) * (4ul) * (l.OmL/lOOOuL) * (1000ng/l,0ug) = 20ng PFOS onto SPE column 20ng PFOS / 2.0mL M eOH = lOng PFOS /m L MeOH. (Since this standard is recorded as lOppb, the correct units are 10ne PFOSVmL M eO H .l These are therefore the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20ng PFO S / 1.OmL diluted sera) * (40.0m L diluted sera/ 2.0m L initial sera) = 400ng PFO S / m L Sera This relationship shows the factor of 40 (or 2.0mL M eOH/ 0.05m L Sera) used to "correct" the units when calculating the final result in ng PFOS/m L 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 Chrom atographic result is 229.2 ng/mL MeOH; reported value is 9170 ng/mL Sera. Since the 1.0 mL initial sample volum e of sera is diluted with 3&OmL water, and lm L is used for the entire SPE procedure, the calculation is as follows: n .o , or a ' / J o 4ilw4iw' (Rf)lA$ (229.2ng P F O S - /1.OmL M eO H ) * (2.0m L M eO H / 0.05m L Sera) = 9170ng PFO S- / m L Sera Sample Calculation: Sample "EOl-1245-36155, Q C @ 100ppb-l": This QC sample is prepared by adding 2uL o f 02001-52 (5.02ug PFOS" / mL M eOH) spiked into lm L 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 PFO S7 mL Sera, the recovery is (246.8/200.8)*(100)=122.9%. _ ___ III. Actions Taken: (such as amendment issued, SO P revision, etc.) This deviation was written and a note to file was written. Recorded By Lisa A. Stevenson J\ D ate 0 8 /1 6 /0 2 >*/((a I A uthorized B y (Study D irector /P ro je c t Lead) S k U D u b c -U '. W /^ D ate //A j/t ,' W lo o rak r^ 3M Environm ental Laboratory F n rn , T tT V .d .fl O 3M E nvironm ental L aboratory Deviation No. /n c c io iw l h v S m rlv D in rfn r n r P m ir c t Tm H a t Ihi nnri n f hiH v n r n m in rt^ Page 223 00120 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite Record of Deviation /. Identification Study / Project No. E01-1245 D eviation Type (Check one) SOP V M ethod Equipm ent Procedure Protocol Other: D ocum ent Num ber: ETS-8-231.1 Date(s) of occurrence: Throughout the study II. Description: Required Procedure/process:..................................................................................................... Section 13.3 o f the m ethod states: T w o thirds of all quality control sam ples and 1/2 o f each quality control sam ple at each level are expected to show an accuracy of 75-125% . Actual Procedure/process: .......................................... _ A11J25 p p b quail liv er q u a lity control sam ple recoveries w ere >125% . lo @ 1 A S 0 3 1il jo 3 c v / f x / c l .................................................................................. ................. ..................................................................................... .............. III. Actions Taken: (such as am endment issued, SO P revision, etc.) This deviation was written, liver data reported at the low range of the curve m ay be biased high, and quality control d ata not w ithin criteria are flagged in the raw data. Recorded By . Lisa A. Stevenson ^ ^ ; Date 08/26/02 , A uthorized By (Study D irector / Project Lead) D ate D.i^kr'. -A-Ajl-t--f, l oj \\ ftV ______ ;_______________ L aU u^ O Q /i { A 3M E nvironm ental L aboratory Form E TS-4-8.0 3M E nvironm ental L aboratory 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 Nothem Bobwhite Study / Project No.E01-1245 Record of Deviation I. Identification Deviation Type (Check one) V SOP Method Equipment Procedure Protocol H 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. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedure/process: This deviation supersedes deviation #3 explaining the liver calculation: Standard E01-1342-36010, Prep sheet description "Curve Point - lOppb in liver", was prepped by spiking 20uL of 02001-66 (l.OOug/mL) into 1 mL o f liver homogenate. This homogenate consists of 5.0353g liver plus 45.0m L water. Since lm L out of 50.0353m L hom ogenate 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 2.0m L of MeOH. T H E R E F O R E ... (20 uL) * (1.000 ug PFOS'/m L MeOH) * (1.0 mL/1000 uL) * (1000 ng/1.0 ug) = 20.0 ng PFOS' onto SPE column 20.0 ng PFO S' / 2.0 mL M eOH = 10.0 ng PFOS'/m L MeOH. (Since this standard is recorded as 1Oppb, the correct units are 10.0 ng PFO S'/m L o f M eO H .) These are the units being measured and reported on the chromatographic system. Similarly, the following statement is true. (20.0ng P F O S '/ l.OmL homogenate) * (l.OmL homogenate / 0.100635 g liver) = 199ng PFOS / g liver Sample Calculation: "E 01-1342-30348, Oppm a.i. 333 M liver" C hrom atographic result is 10.76 ng/m L; reported value is 209 ng/g. Since the 1,0338g initial sam ple weight o f liver is hom ogenized with 9.0m L w ater, and 1.0 mL is used for the entire SPE procedure, the calculation is as follows: 1.0338 g / (9.0 m L water + 1.0338 g liver) = 0.103032 g of liver in 1.0 mL o f 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: Sam ple "E 01-1342-36017, O C @ 10ppb-l' ': This QC sample is prepared by adding 4 uL o f 02001-52 (5.020ug PFO S' / m L MeOH) spiked into lm L liver homogenate. ((4ul) * (5.020ug/mL) * (lmL/lOOOuL) * (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) * ( l 00)= 135 091. III. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this SOP deviation. Recorded By . U S, A. Stevenson ^ ^ Authorized By (S tu d y D ire c to r /P r o j e c t L e a d ) . / . . D ate Ilio.U or Date PlrfctW, SeCtA GftlWukir____________ \ - Uloira+oi^. WntMjrfYYlWvl. W.thBiw foaw n 3M Environm ental Laboratory Form E TS-4-8.0 "- Deviation No. 1 (assigned by Study Director or Project Lead at the end of study or project) 3M E nvironm ental L aboratory Page 225 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Atta c h m e n t F: Ex a m ple C alculatio ns Fo r m u l a U s e d fo r S e r a A n a l y s e s in St u d y E 01-1245 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) i 229.24 ng/mL MeOH x 1 x 40 = 9170 ng/mL Sera Fo r m u la Use d fo r Liver An a ly se s in Stu 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 u n t of s e ra in diluted s a m p le (m L ) = amount of sera used(mL)/total amount of sera + water(mL) Am ount of liver in diluted sam ple(g) = am ount of liver w eig h ed (g )/ am ount of liver w eigh ed(g) + w a te r ad d e d for hom ogenizing(m L=g) Examples: Amount of sera in diluted sample(mL) = amount of sera used (0.3 mL)/total amount of sera + w ater (6 mL) = 0 .0 5 mL Dilution(diluted sera)(m L) = final volume (2.0 mL)/amount of sera in diluted sample (0.05 mL) = 40 Amount of liver in diluted sample(g) = amount of liver weighed (1.0010 g)/amount of liver weighed (1.0010 g) + w ater added for homogenizing (9.00 mL) = 1.0010 g / 10.0010 g = 1.00090 g Note: For sera samples with less than 0.05 mL sera received, 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 E nvironm ental L aboratory Page 226 of 226 Page 226 CCI