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Report E01-1256 PFOS: A Reproduction Study with the Mallard Study Title Analytical Phase Report for PFOS: A Reproduction Study with the Mallard Data Requirement FI FRA Subdivision E, Section 71-4 OECD Guideline 206 Author Lisa A. Stevenson Analytical Phase Initiation Date February 19, 2002 Analytical Phase Completion Date At Signing Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Environmental Laboratory S tudy# E01-1256 Wildlife International Ltd: 454-109 Total Number of Pages 216 3M Environmental Laboratory Page 1 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard GLP C o m p l ia n c e S t a t e m e n t Study Title: Analytical Phase Report for PFOS: A Reproduction Study with the Mallard Study Identification Number: E01-1256 This analytical phase was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards with the exceptions listed below: Exceptions to GLP compliance: Automated data collection systems are not validated. The hardcopy printouts are considered as the original raw data. The stability of the samples has not been determined. Not all data generated were recorded directly or promptly, or initiated and dated on date of entry. F m /im n m o n ta l I ahnratrvrv/ 3M Environmental Laboratory Porta OrtfOtft Page 2 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Q uality A ss u r a n c e S tatem ent Study Title: Analytical Phase Report for PFOS: A Reproduction Study with the Mallard Study Identification Number: E01-1256 The analytical phase was audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU), as indicated in the following table. The findings were reported to the study director and laboratory management. In s p e c t io n D a t e s 02/22/02 04/03/02-04/08/02, 04/17/02 05/13/02-05/15/02 PHASE In-phase Data Final Report Date Reported to Management S t u d y D ir ec to r 02/22/02 02/22/02 04/08/02, 05/21/02 04/08/02, 05/21/02 05/16/02 05/16/02 'A kJ L D m A , J Quality Assurance Representative il&zioz Date 3M Environmental Laboratory 3M Environmental Laboratory Paae 3 of 216 Page 3 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table of C o ntents GLP Compliance Statement.............................................................................................................. 2 Quality Assurance Statement........................................................................................................... 3 List of Tables..................................................................................................................................... 5 Summary........................................................................................................................................... 7 Purpose............................................................................................................................................. 8 Reference Substances...................................................................................................................... 8 Specimen Receipt and Storage........................................................................................................ 9 Method Summary................................................................................ ..............................................9 Preparatory Method.......................................................................................................................... 9 Sample Receipt, Preparation and Analysis....................................................................................... 10 Analytical Method................................................................................................................................11 Analytical Results................................................................................................................................12 Data Summary.................................................................................................................................. 14 Statement of Conclusion.................................................................................................................... 21 Statistical Methods............................................................................................................................. 23 Statement of Data Quality.................................................................................................................. 23 References........................................................................................................................................ 24 List of Attachments............................................................................................................................ 24 Signature Page................................................................................................................................. 25 Attachment A: Extraction and Analytical Method, ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" .................................................. 26 Attachment B: Data Summary Tables............................................................................................... 46 Attachment C: Sample Chromatograms, Calibration Information, and Instrument Information.......55 Attachment D: Sample Preparation Sheets...................................................................................... 144 Attachment E: Study Protocol, Protocol Amendments, and Deviations........................................... 178 Attachment F: Example Calculations................................................................................................ 216 3MFnvirnnmpntal lahnratnrv 3M Environmental Laboratory Paae 4 of 216 Page 4 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard L ist o f Ta b les Table 1. Summary Table, C8F17S 03' Concentration in Sera andLiver..............................................7 Table 2. Summary Table, C8F17S 03` Concentration In Sera andLiver with Outliers Removed...... 8 Table 3. Reference Substances......................................................................................................... 8 Table 4. SPE Extraction Summary.................................................................................................... 9 Table 5. Sample Analysis....................................................................................................................10 Table 6. Serum QC Summary Table...................................................................................................13 Table 7. Serum Matrix Spike Summary Table....................................................................................13 Table 8. Liver QC Summary Table.....................................................................................................13 Table 9. Liver Matrix Spike Summary Table.......................................................................................13 Table 10. C8F17S 03' Data Summary, Female Mallard Sera (Adult and Offspring)............................ 14 Table 11. C8F17S 03` Data Summary, Male Mallard Sera (Adult and Offspring)................................ 16 Table 12. C8F17S 03' Data Summary, Female Mallard Liver (Adult and Offspring)............................ 18 Table 13. C8F17S 03` Data Summary, Male Mallard Liver (Adult and Offspring)................................ 20 Table 14. Summary Table, C8F17S 03' Concentration in Sera and Liver............................................ 22 Table 15. Summary Table, C8F17S 03` Concentration in Sera and Liver with Outliers Removed.....23 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 216 Page 5 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S tu d y Info rm atio n Study Director Sean P. Gallagher Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 Sponsor 3M Environmental Technology and Safety Services 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55106 Susan A. Beach, Sponsor Representative Analytical Chemistry Laboratory 3M Environmental Technology and Safety Services (ET&SS) 3M Environmental Laboratory (3M Lab) Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Lisa Stevenson, Principal Analytical Investigator Study Personnel Sean P. Gallagher, Wildlife International LTD., Study Director Joann B. Beavers, Wildlife International LTD., Laboratory Management William K. Reagen, 3M Environmental Laboratory Management Susan A. Beach, Sponsor Representative Harold O. Johnson, III, Original Principal Analytical Investigator Lisa A. Stevenson, Principal Analytical Investigator Kent R. Lindstrom, Senior Research Chemist Ognjenka Krupljanin, Analytical Chemist Marlene Heying, Analytical Chemist Experimental Dates Sample Analysis Initiation: 19 February 2002 Sample Analysis Completion: 11 April 2002 Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory according to 40 CFR Part 792. The analytical reference standard reserve samples, as well as the samples pertaining to the analytical phase of this study, are archived at the 3M Environmental Laboratory according to 40 CFR Part 792. Fn\/irnnm ntal I ahnrsitnrv 3M Environmental Laboratory Paae 6 of 216 Page 6 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S ummary The concentration of the C8F17S 03' anion of perfluorooctane sulfonate, potassium salt (PFOS, C8F17S 03'K+) was determined in the liver and sera of adult mallards and their offspring in both a control group and a 10ppm dosed group from Wildlife feeding study #454-109. The following tables summarize the C8F17S 03' concentrations found in the liver and sera of study samples. Extracted-matrix PFOS standards are used to establish the HPLC-ES/MS/MS instrument calibration curve for C8F17S 03\ Concentrations of C8F17S 03' are reported after correction for the purity and the potassium contribution to the mass of the PFOS reference substance. Table 1 summarizes all study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q-Test. Table 1. Summary Table, C8Fi7S 0 3' Concentration in Sera and Liver S a m p l in g Event Male Control Group Number of S era Sam ples A verag e C 8F i 7S 0 3 Conc. S era Range o f Results N um ber o f L iv e r S am ples <100 ng/mL (14 of 18) 18 NA 20 107-1400 ng/mL range (4 of 18) A verage C 8F i 7S 0 3 C o n c . L iv e r <50 ng/g Range of R esults NA Female Control Group Male 10ppm Group Female 10ppm Group <100 ng/mL (15 of 20) 20 NA 20 166-2680 ng/mL range (5 of 20) 20 89700 ng/mL 11300-210000 ng/mL 20 20 20300 ng/mL 6350-91400 ng/mL 20 <50 ng/g NA 60900 ng/g 24200-125000 ng/g 10800 ng/g 2480-30700 ng/g Male Offspring <100 ng/mL (2 of 3) Control Group 3 NA 103 ng/mL (1 of 3) 3 <50 ng/g NA Female Offspring Control Group 7 NA <100 ng/mL (7 of 7) 7 <50 ng/g NA Male Offspring 10ppm Group 3 4410 ng/mL 3670 - 5450 ng/mL 3 3170 ng/g 22004640 ng/g Female Offspring 10ppm Group 7 4960 ng/mL 3420 - 6760 ng/mL 7 3620 ng/g 2240-5930 ng/g For individual sample values see Tables 10 to 13 and Attachment B. Oft A I ^ r-.-f, 3M Environmental Laboratory D0 /1/1 7 a# 04 Page 7 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 2. Summary Table, CsF17S03' 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 Sam ples Male Control Group 18* A verage C8F17SO3 C onc. S era Range of Results * N um ber of L iv e r Sam ples 20* A verage C8F17SO3 C o n c . L iv e r * Range of Results * Female Control Group 17 NA <100 ng/mL (15 of 17) 166-172 ng/mL range (2 of 17) 20* * * Male 10ppm Group 18 87300 ng/mL 68700-108000 ng/mL 20* * * Female 10ppm Group 19 16600 ng/mL 6350-42800 ng/mL 20* * * Male Offspring Control Group Female Offspring Control Group 3* 7* * * * 3* * * 7* * Male Offspring 10ppm Group 3* * * 3* * * Female Offspring 10ppm Group 7* * * 7* ' * * * No outliers removed from this data set. Refer to Table 1 for results. For individual sample values see Tables 10 to 13 and Attachment B. Purpose The purpose of the analytical phase of this study is to quantify C8F17S 03' in the liver and sera of both control and dosed mallards and in their offspring. Reference S ubstances Table 3. Reference Substances Reference Substance Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Purity PFOS 3M Internal 08/31/2006 Frozen -20C +/- 10C SD-018, Lot #217 White Crystalline Powder 86.9% PFDA Oakwood Products, Inc. 12/01/2010 Frozen -20C +/- 10C SD-036 White Solid 98% PFDA is nonadecafluorodecanoic acid, or Perfluorodecanoic acid, CioF190 2H and is used solely to monitor for gross instrument failure and not as an internal standard for quantitation. 3M Environmental Laboratory OHO Page 8 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S pecim en R eceipt a n d S to r ag e Mallard serum and liver samples were obtained from the sacrifice of the test animals upon the conclusion of the in-life phase of the study (#454-109) conducted at Wildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. The specimens were received at 3M Environmental Laboratory, assigned a laboratory tracking (LIMS) number (E01-1256, E01-1326, or EO'l-1367), and were stored at -20C +/- 10C until prepared for analysis. M ethod Summary The determination of C8F17S 03' concentration in the liver and sera was performed according to 3M Environmental Laboratory method ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices". The sera samples were prepared by measuring a volume of serum and diluting with water in a 1/20 ratio. The liver samples were prepared by measuring a weight of liver and diluting with water to a 1/10 ratio prior to homogenizing. Five mL of acetonitrile was added to 1.0mL of the diluted sera or 1.OmL of the homogenate of liver and was shaken for 20 minutes. These samples were then centrifuged for 10 minutes; the supernatant was decanted into 40 mL of water and passed through a conditioned solid phase extraction (SPE) column. After drying the column, 2.0 mL. of methanol was passed through the column to elute the analyte. The samples were analyzed for C8F17S 03' using HPLC-ES/MS/MS. Preparatory M ethod ETS-8-231.1 "Solid Phase Extraction and Analysis of Fluorochemical Components from Biological matrices." This method describes the extraction of target analytes from biological matrices, such as sera and liver, using solid phase extraction (SPE). Sera samples were diluted with Kandiyohi (commercially bottled) water 1/20, before extraction. Liver samples were diluted 1/10, approximately 1g of liver and 9ml of Kandiyohi water, and then homogenized. A 1.0mL aliquot of the diluted sera or homogenate of liver was extracted by adding 5.OmL of acetonitrile, shaking for 20 minutes, and centrifuging for 10 minutes. The supernatant was transferred to a clean tube, diluted with 40 ml of Kandiyohi water, and filtered through a conditioned C18 (SPE) column. Analytes were eluted off the column, and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). Table 4. SPE Extraction Summary S tep S a m p l e P r e p a r a t io n 1 Measure volume of sera or weigh portion of liver 2 Dilute sera (1/20) and liver (1/10) with water 3 Mix sera/water or homogenize liver/water 4 Aliquot 1.0ml of diluted/homogenized sample and add 5.0ml of ACN. Spike samples accordingly. Shake for 20 minutes, centrifuge for 10 minutes. 5 Dilute supernatant with 40 ml of water 6 Filter sample through conditioned SPE C18 column. 7 Elutetarget analytes from the column with 2.0mL methanol (MeOH). 3M Environmental Laboratorv 3M Environmental Laboratory Paae 9 of 216 Page 9 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S a m p le R eceipt, P reparation a n d A nalysis All sera and liver samples were received frozen and remained frozen until thawed prior to preparing samples for analysis. After dilution or homogenization steps, unused samples and diluted sera or homogenized liver were frozen again if extractions were not immediately performed. On each day of extraction, a water blank, a sera or liver blank, and a matrix spike and matrix spike duplicate were prepared with each set of samples. Frozen diluted sera/homogenized liver samples were allowed to thaw prior to transferring a representative sample for extraction. Samples were prepared and analyzed according to ETS-8-231.1. The following table describes the sample collection and preparation regimen. For clarity, a reference to "Day 1" refers only to the group of samples associated with the first batch of samples extracted. Table 5. Sample Analysis S tep P ro ced ure 1 Aliquoting and preparation of the sera sample homogenates (all groups and dose levels) 2 Weighing of liver samples (control group and dosed group offspring) 3 Weighing of liver samples (control group and dosed group adults) 4 Homogenizing of liver samples (control group and dosed group offspring) 5 Homogenizing of liver samples (control group and portion of the dosed group adults) 6 Homogenizing of liver samples (remainder of dosed group adults) 7 Preparation of method blanks and spikes, calibration curve, qc samples, and sera samples, control group, adults (Day 1 sera samples) 8 Analysis of Day 1sera samples Date P erformed 19 February 2002 21 February 2002 22 February 2002 22 February 2002 25 February 2002 26 February 2002 25 February 2002 25 February 2002 9 Preparation of method blanks and spikes, and sera samples, control a n d 10 pp m adults (D a y 2 s e ra s a m p les) 10 Analysis of Day 2 sera samples 11 Preparation of method blanks and spikes, and sera samples, 10 ppm adults (Day 3 sera samples) 12 Analysis of Day 3 sera samples 13 Preparation of method blanks and spikes, and sera samples, control group, and 10 ppm offspring (Day 4 sera samples) 14 Analysis of Day 3 and Day 4 sera samples 15 Analysis of diluted sera samples 16 Preparation of method blanks and spikes, calibration curve, qc samples, and liver samples, control group, adults (Day 1liver samples) 17 Analysis of Day 1liver samples 26 February 2002 26 February 2002 27 February 2002 27 February 2002 28 February 2002 28 February 2002 01 March 2002 04 March 2002 04 March 2002 18 Re-analysis of Day 1 liver samples 19 Preparation of method blanks and spikes, and liver samples, 10ppm dosed group, adults (Day 2 liver samples) 05 March 2002 05 March 2002 3M Environmental Laboratory 3M Environmental Laboratory Pape 10 of 216 Page 10 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S tep 20 Procedure Analysis of Day 2 liver samples 21 Preparation of method blanks and spikes, and liver samples, control group and 10ppm dosed group, adults (Day 3 liver samples) 22 Analysis of Day 3 liver samples 23 Preparation of method blanks and spikes, and liver samples, control group and 10ppm dosed group, offspring (Day 4 liver samples) 24 Analysis of Day 4 liver samples 25 Analysis of female sera dilutions 26 Analysis of QC liver and sera samples 27 Analysis of diluted sera samples Date P erformed 06 March 2002 06 March 2002 06 March 2002 07 March 2002 07 March 2002 08 March 2002 05 April, 2002 11 April, 2002 A nalytical M etho d Samples were analyzed using HPLC-ES/MS/MS. C8F17S 03' concentrations were determined using matrix-extracted calibration standards ranging in concentration from 0.25-1000 ng C8F17S 037mL methanol. This result was then used to back calculate the ng C8F17S 037mL sera or ng C8F17S 037gram liver as appropriate. An internal standard, Nonadecafluorodecanoic acid (PFDA), was used to monitor instrument performance for gross malfunction, but was not used to quantitate results. HPLC-EC/MS/MS analytical conditions are summarized as follows: The HPLC used for the study was a Hewlett-Packard 1100. The system consists of a vacuum degasser, binary pump, autosampler and temperature controlled column compartment. The run time is 10 minutes per sample. HPLC conditions include the use of a Keystone Scientific, Betasil C18 HPLC analytical column (2.1mm x 50mm, 5pm particle size), a 40C column compartment temperature, a 10pL injection volume and a 0.300pL/min pump flow rate. The HPLC gradient is listed below: T im e , M in u t e s % 2 m M A m m o n iu m A cetate % M ethanol 0 90 1.0 9 0 5 .5 5 7 .5 5 8 90 10 10 95 95 10 10 : i 90 10 I The mass spectrometer used for the study was a Micromass Ultima. The system consists of Z-Spray interface equipped with an electrospray probe operated in negative mode. The instrument was setup to operate in MS/MS mode, passing the C8F17S 03" molecular anion (m/z = 499) into the collision cell, where it is collided with inert gas to further fragment to the characteristic ion m/z = 99, FS03`. Similarly the PFDA transition 3M Environmental Laboratory 3M Environmental Laboratory Paae 11 of 216 Page 11 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard from m/z - 513 to m/z = 299 was monitored. The collision gas, 95% Argon / 5% Methane, was maintained at a pressure of approximately 3.0x1 O'3 mBar. A nalytical R esults Data quality objectives outlined in the 3M Environmental Laboratory method and protocol were met, except as noted and discussed below (see Attachment A, ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" and Attachment E, Study Protocol, Protocol Amendments, and Deviations). Regressions. Quadratic curve fit, weighted 1/X, was applied to calibration standards and sample data. Each calibration curve had a coefficient of determination of > 0.985. Calibration Standards. Instrument response was calibrated daily prior to sample analysis using matrix-extracted standards ranging in concentration in the final solvent extract from 0.20 to 1000 ng C8F17S 037mL methanol, corresponding to a concentration of 8.00 to 40160 ng/mL of sera or 4.00 to 20080 ng/g liver. The calibration curve analyzed consisted of fourteen matrix-extracted standards bracketing a wide concentration of analyte. The accuracy of each level was evaluated. Any level outside 75%-125% of nominal was deactivated with the exception of the lowest curve point, which needed to meet 70%-130% of nominal. In some cases the lowest concentration curve point, although outside the 30% nominal requirement, was included when constructing the calibration curve, but it was not used to determine the limit of quantitation. The limit of quantitation was the next point on the curve meeting the 30% nominal requirement. Due to the quadratic response of matrix extracted standards using LC/MS/MS instrumentation, the calculated concentration of higherlevel standards was often indeterminant, that is a single result could not be calculated using the quadratic equation. The accepted upper concentration range for study samples was determined by the highest calibration standard meeting the 75%-125% accuracy criteria when back calculated to the original concentration spiked into the extracted matrix standards. All analytical results that are reported are within the accepted calibration range of the instrument. System Suitability Samples. System suitability was demonstrated prior to the start and at the end of each analytical run. Prior to the calibration curve and after the last sample, three mid-level unextracted calibration standards were analyzed. The peak area precision and retention time precision was monitored at the beginning and end of the run separately. The peak area precision was equal to or less than 5.0% RSD and the precision of the retention time was equal to or less than 2.5% RSD, meeting the stated data quality objectives to show that the instrumentation was providing suitable results throughout each analytical sequence. Continuing Calibration Verification (CCV). A mid-level matrix calibration check was analyzed every ten samples or less to monitor instrumental drift, with a limit of 25% deviation of the target concentrations. Only data bracketed by passing CCV's were reported. Samples bracketed by one or more failing CCV's were reanalyzed. Limit of Quantitation (LOQ). The lower limit of quantitation (LLOQ) was defined in liver (ng/g) and in serum (ng/mL) as the lowest acceptable extracted calibration curve point that is within the 70-130% nominal criteria and with an analyte peak area at least 2 times the blank. The upper limit of quantitation (ULOQ) was defined as the concentration of the highest acceptable curve point used to calibrate the response of the instrument. Blanks. Method blanks (purchased Kandiyohi drinking water, sera, and/or liver taken through the entire sample preparation and analysis process) provided a measure of background contamination. Quality Control (QC) and Matrix Spikes. One set of nine QC samples in each matrix was extracted and analyzed and consisted of three levels of analyte in triplicate. Additionally, two matrix spike samples at the lowest concentration were prepared with each extraction set. Results are as follows: 3M Environmental Laboratory 3M Environmental Laboratory Pane 12 of 21R Page 12 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 6. Serum QC Summary Table Serum QC Samples Concentration in Diluted Serum Mean recovery of triplicates Relative Standard Deviation QC-25ppb 1004 ng/mL 145%* I 9.8% QC-250ppb 10040 ng/mL 97.9% 2.9% QC-4.0ppm 160600 ng/mL 84.7% 6.7% 'These samples were spiked using a 1.0 ul aliquot of spiking solution. Matrix spikes prepared on the same day using 50 ul of a diluted stock meet all QC requirements. Spiking technique of injecting 1.0 ul Is suspect. Table 7. Serum Matrix Spike Summary Table Serum Matrix Spike Samples* Concentration in Diluted Serum Average Recoveries Four Matrix Spikes 1004 ng/mL 114% Four Matrix Spikes Duplicates 1004 ng/mL 112% 'Some matrix spike samples were injected in a second analytical batch. Both results are included in the average recoveries. Table 8. Liver QC Summary Table Liver QC Samples Concentration in Diluted Liver Mean recovery of triplicates Relative Standard Deviation QC-25ppb 497 ng/g 109%* 13% QC-250ppb 4967 ng/g 94.3% 8.3% QC-4.0ppm 79470 ng/g 103% 0.72% *These samples were spiked using a 1.0 ul aliquot of spiking solution. Matrix spikes prepared on the same day using 50 ul of a diluted stock meet all QC requirements. Spiking technique of injecting 1.0 ul is suspect. Table 9. Liver Matrix Spike Summary Table Liver Matrix Spike Samples* Concentration in Diluted Liver Average Recoveries Four Matrix Spikes 497 ng/g 111% Four Matrix Spikes Duplicates 497 ng/g 107% 'Some matrix spike samples were injected in a second analytical batch. Both results are included in the average recoveries. The protocol states that the acceptance range is 75-125% but makes allowances for a percentage of results to be outliers. The observed number of outliers for the "QC-25ppb" spikes exceeds that permitted in the protocol but the overall impact on data quality is minimal or none at all. All study samples that were not "Day 0", show analyte levels significantly above the LOQ. Occasionally, Day 0 samples or other types of blanks show positive detections at levels near the LOQ. 3M Environmental Laboratory 3M Environmental Laboratory Paae 13 of 216 Page 13 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Absolute Recoveries. The absolute recovery of the QC samples is determined by comparing the results of the extracted quality control spikes quantitated versus an extracted matrix curve and a solvent curve. The sera spikes quantitate higher using a solvent curve than when using a matrix-extracted curve. The low spikes are 11.1% higher, the medium spikes 8.3% higher, and the high spikes 8.3% higher. The 9.5% average higher quantitation of the three levels of spikes using a solvent curve versus an extracted matrix curve demonstrates that it is appropriate to use matrix-extracted standards. The same trend is observed for liver samples. The extracted liver matrix curves are 12.2% higher, 7.8% higher, and 5.6% higher respectively for the low, medium and high spikes of the liver matrix, again demonstrating that it is appropriate to use matrix-extracted standards. Internal Standard: The internal standard (PFDA) was added to all samples and standards. PF:DA 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 03 anion of perfluorooctane sulfonate, potassium salt (PFOS) that was adjusted for purity and the potassium ion contribution to the mass of PFOS used to prepare stock solutions. Any future calculations requiring concentrations to be expressed as a percentage of dietary in-take must adjust the dosed amounts as well. Data S ummary Tables 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The table displays C8F17S 03' concentrations for individual injections. All data are corrected for purity of the PFOS reference substance used for the extracted-matrix calibration curve. Table 10. C8Fi7S 0 3' Data Summary, Female Mallard Sera (Adult and Offspring) 3M S a m p le Num ber SAMPLE DESCRIPTION E01-1256-28663 E01-1256-28665 E01-1256-28667 E01-1256-28669 E01-1256-28670 E01-1256-28672 E01-1256-28674 E01-1256-28676 E01-1256-28678 E01-1256-28679 E01-1256-28681 E01-1256-28683 E01-1256-28685 E01-1256-28687 E01-1256-28689 E01-1256-28691 E01-1256-28693 E01-1256-28695 454-109-3218,0 PPM, Female, Adult 454-109-3220,0 PPM, Female, Adult 454-109-3222, 0 PPM, Female, Adult 454-109-3224,0 PPM, Female, Adult 454-109-3226,0 PPM, Female, Adult 454-109-3228,0 PPM, Female, Adult 454-109-3230,0 PPM, Female, Adult 454-109-3232,0 PPM, Female, Adult 454-109-3234,0 PPM, Female, Adult 454-109-3236,0 PPM, Female, Adult 454-109-3238,0 PPM, Female, Adult 454-109-3240,0 PPM, Female, Adult 454-109-3242,0 PPM, Female, Adult 454-109-3244,0 PPM, Female, Adult 454-109-3246,0 PPM, Female, Adult 454-109-3248,0 PPM, Female, Adult 454-109-3250,0 PPM, Female, Adult 454-109-3252,0 PPM, Female, Adult AMOUNT OF SERA USED (ML) ADDITIONAL DILUTION CONO. OF C 8F i 7SC>3 in S a m p l e (n g /m L of S era) 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 <100 <100 <100 <100 <100 0.5 1 2680 0.5 1 <100 0.5 1 172 0.5 1 <100 0.5 1 <100 0.5 1 166 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.5 1 <100 0.5 1 0.5 1 398 <100 0.5 1 757 3M Environmental Laboratory 3M Environmental Laboratory Pace 14 of 216 Page 14 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S a m p le N um ber SAMPLE DESCRIPTION E01-1256-28697 E01-1256-28699 E01-1256-28701 E01-1256-28703 E01-1256-28705 E01-1256-28707 E01-1256-28709 E01-1256-28711 E01-1256-28713 E01-1256-28715 E01-1256-28717 E01-1256-28719 E01-1256-28721 E01-1256-28723 E01-1256-28725 E01-1256-28727 E01-1256-28729 E01-1256-28731 E01-1256-28733 E01-1256-28735 E01-1256-28737 E01-1256-28739 E01-1256-28740 E01-1256-28742 E01-1256-28743 E 01-1256-28744 E01-1256-28745 E01-1256-28747 E01-1256-28748 E01-1256-28751 E01-1256-28752 E01-1256-28753 E01-1256-28754 E01-1256-28755 E01-1256-28758 E01-1256-28759 454-109-3254,0 PPM, Female, Adult 454-109-3256,0 PPM, Female, Adult 454-109-3258,10 PPM, Female, Adult 454-109-3260,10 PPM, Female, Adult 454-109-3262,10 PPM, Female, Adult 454-109-3264,10 PPM, Female, Adult 454-109-3266,10 PPM, Female, Adult 454-109-3268,10 PPM, Female, Adult 454-109-3270,10 PPM, Female, Adult 454-109-3272,10 PPM, Female, Adult 454-109-3274,10 PPM, Female, Adult 454-109-3276,10 PPM, Female, Adult 454-109-3278,10 PPM, Female, Adult 454-109-3280,10 PPM, Female, Adult 454-109-3282,10 PPM, Female, Adult 454-109-3284,10 PPM, Female, Adult 454-109-3286,10 PPM, Female, Adult 454-109-3288,10 PPM, Female, Adult 454-109-3290,10 PPM, Female, Adult 454-109-3292,10 PPM, Female, Adult 454-109-3294,10 PPM, Female, Adult 454-109-3296,10 PPM, Female, Adult 454-109-9583,0 PPM, Female, Offspring 454-109-9589,0 PPM, Female, Offspring 454-109-9592,0 PPM, Female, Offspring 4 5 4 -1 0 9 -9 5 9 8 , 0 PPM , Fem ale, Offspring 454-109-9603,0 PPM, Female, Offspring 454-109-9610,0 PPM, Female, Offspring 454-109-9573,0 PPM, Female, Offspring 454-109-9629,10 PPM, Female, Offspring 454-109-9633,10 PPM, Female, Offspring 454-109-9634,10 PPM, Female, Offspring 454-109-9645,10 PPM, Female, Offspring 454-109-9648,10 PPM, Female, Offspring 454-109-9654,10 PPM, Female, Offspring 454-109-9659,10 PPM, Female, Offspring AMOUNT OF SERA USED (ML) ADDITIONAL DILUTION CONO. OF C8 F17SO 3 IN S a m p l e (n g / m L of S era) 0.5 1 0.5 1 <100 <100 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 13600 9420 17000 6350 7900 6730 0.5 10 91400 0.5 1 7710 0.5 10 29400 0.5 10 40600 0.5 1 9840 0.2 10 32200 0.5 1 10800 0.5 1 6570 0.5 10 28900 0.5 10 42800 0.5 1 16000 0.5 1 10400 0.5 1 7660 0.5 1 10800 0.15 1 0.2 1 0.2 1 0.1 1 0.2 1 0.2 1 0.05 1 <100 <100 <100 <100 <100 <100 <100 0.1 1 0.1 1 0.5 1 0.1 1 0.2 1 0.1 1 0.1 1 5130 3420 6760 3750 4900 5450 5350 These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers 3M Environmental Laboratory 3M Environmental Laboratory Pace 15 of 216 Page 15 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 11. C8F17SO3' Data Summary, Male Mallard Sera (Adult and Offspring) 3M S a m p le N um ber SAMPLE DESCRIPTION E01-1256-28662 E01-1256-28664 E01-1256-28666 E01-1256-28668 E01-1256-28671 E01-1256-28673 E01-1256-28675 E01-1256-28677 E01-1256-28680 E01-1256-28682 E01-1256-28684 E01-1256-28686 E01-1256-28688 E01-1256-28690 E01-1256-28692 E01-1256-28694 E01-1256-28696 E01-1256-28698 E01-1256-28700 E01-1256-28702 E01-1256-28704 E01-1256-28706 E01-1256-28708 E 01-1256-28710 E01-1256-28712 E01-1256-28714 E01-1256-28716 E01-1256-28718 E01-1256-28720 E01-1256-28722 E01-1256-28724 E01-1256-28726 E01-1256-28728 E01-1256-28730 E01-1256-28732 E01-1256-28734 E01-1256-28736 E01-1256-28738 E01-1256-28741 454-109-3217,0 PPM, Male, Adult 454-109-3219,0 PPM, Male, Adult 454-109-3221,0 PPM, Male, Adult 454-109-3223,0 PPM, Male, Adult 454-109-3227,0 PPM, Male, Adult 454-109-3229,0 PPM, Male, Adult 454-109-3231,0 PPM, Male, Adult 454-109-3233,0 PPM, Male, Adult 454-109-3237,0 PPM, Male, Adult 454-109-3239, 0 PPM, Male, Adult 454-109-3241,0 PPM, Male, Adult 454-109-3243,0 PPM, Male, Adult 454-109-3245,0 PPM, Male, Adult 454-109-3247,0 PPM, Male, Adult 454-109-3249,0 PPM, Male, Adult 454-109-3251,0 PPM, Male, Adult 454-109-3253,0 PPM, Male, Adult 454-109-3255,0 PPM, Male, Adult 454-109-3257,10 PPM, Male, Adult 454-109-3259,10 PPM, Male, Adult 454-109-3261,10 PPM, Male, Adult 454-109-3263,10 PPM, Male, Adult 454-109-3265,10 PPM, Male, Adult 454-109-3267, 10 PPM, Male, Adult 454-109-3269,10 PPM, Male, Adult 454-109-3271,10 PPM, Male, Adult 454-109-3273,10 PPM, Male, Adult 454-109-3275,10 PPM, Male, Adult 454-109-3277,10 PPM, Male, Adult 454-109-3279,10 PPM, Male, Adult 454-109-3281,10 PPM, Male, Adult 454-109-3283,10 PPM, Male, Adult 454-109-3285,10 PPM, Male, Adult 454-109-3287,10 PPM, Male, Adult 454-109-3289,10 PPM, Male, Adult 454-109-3291,10 PPM, Male, Adult 454-109-3293,10 PPM, Male, Adult 454-109-3295,10 PPM, Male, Adult 454-109-9591,0 PPM, Male, Offspring AMOUNT OF SERA USED (ML) ADDITIONAL DILUTION CONO. OF C8F17SO 3 IN S a m p le (n g /m L of S era) 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.3 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.1 1 0.5 1 107 <100 <100 <100 <100 <100 <100 <100 <100 <100 1400 <100 <100 162 1070 <100 <100 <100 0.5 10 78800 0.2 10 83300 0.5 10 68700 0.06 100 210000 0.2 10 93800 0.5 10 75700 0.5 10 80600 0.5 10 11300 0.5 10 106000 0.5 10 80500 0.5 10 80400 0.5 10 103000 0.5 10 74300 0.5 10 91800 0.5 10 108000 0.3 10 78700 0.3 10 97900 0.3 10 73500 0.5 10 96800 0.5 10 99700 0.1 1 <100 3M Environmental Laboratory 3M Environmental Laboratory Page 16 of 216 Page 16 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S a m p le N um ber SAMPLE DESCRIPTION E01-1256-28746 E01-1256-28749 E01-1256-28750 E01-1256-28756 E01-1256-28757 454-109-9606,0 PPM, Male, Offspring 454-109-9578,0 PPM, Male, Offspring 454-109-9625,10 PPM, Male, Offspring 454-109-9636,10 PPM, Male, Offspring 454-109-9642,10 PPM, Male, Offspring AMOUNT OF SERA USED (ML) ADDITIONAL DILUTION CONO. OF C8 F17SO 3 IN S a m p l e (n g /mL of S era) 0.2 1 0.2 1 103 <100 0.2 1 0.1 1 0.2 1 5450 3670 4110 These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. 3M Environmental Laboratory 3M Environmental Laboratory Pape 17 of 216 Page 17 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 12. C8Fi7S 0 3' Data Summary, Female Mallard Liver (Adult and Offspring) 3M S a m ple Num ber SAMPLE DESCRIPTION E01-1326-29482 E01-1326-29583 E01-1326-29601 E01-1326-29619 E01-1326-29637 E01-1326-29655 E01-1326-29673 E01-1326-29691 E01-1326-29709 E01-1326-29727 E01-1326-29745 E01-1326-29763 E01-1326-29781 E01-1326-29799 E01-1326-29817 E01-1326-29835 E01-1326-29853 E01-1326-29871 E01-1326-29889 E01-1326-29907 E01-1326-29925 E01-1326-29943 E01-1326-29961 E01-1326-29980 E01-1326-29998 E01-1326-30016 E01-1326-30034 E01-1326-30052 E01-1326-30070 E01-1326-30088 E01-1326-30106 E01-1326-30124 E01-1326-30142 E01-1326-30160 E01-1326-30178 E01-1326-30196 E01-1326-30214 E01-1326-30232 E01-1326-30250 0 ppm a.i. 3218 F Liver, adult 0 ppm a.i. 3220 F Liver, adult 0 ppm a.i. 3222 F Liver, adult 0 ppm a.i. 3224 F Liver, adult 0 ppm a.i. 3226 F Liver, adult 0 ppm a.i. 3228 F Liver, adult 0 ppm a.i. 3230 F Liver, adult 0 ppm a.i. 3232 F Liver, adult 0 ppm a.i. 3234 F Liver, adult 0 ppm a.i. 3236 F Liver, adult 0 ppm a.i. 3238 F Liver, adult 0 ppm a.i. 3240 F Liver, adult 0 ppm a.i. 3242 F Liver, adult 0 ppm a.i. 3244 F Liver, adult 0 ppm a.i. 3246 F Liver, adult 0 ppm a.i. 3248 F Liver, adult 0 ppm a.i. 3250 F Liver, adult 0 ppm a.i. 3252 F Liver, adult 0 ppm a.i. 3254 F Liver, adult 0 ppm a.i. 3256 F Liver, adult 10 ppm a.i. 3258 F Liver, adult 10 ppm a.i. 3260 F Liver, adult 10 ppm a.i. 3262 F Liver, adult 10 ppm a.i. 3264 F Liver, adult 10 ppm a.i. 3266 F Liver, adult 10 ppm a.i. 3268 F Liver, adult 10 ppm a.i. 3270 F Liver, adult 10 ppm a.i. 3272 F Liver, adult 10 ppm a.i. 3274 F Liver, adult 10 ppm a.i. 3276 F Liver, adult 10 ppm a.i. 3278 F Liver, adult 10 ppm a.i. 3280 F Liver, adult 10 ppm a.i. 3282 F Liver, adult 10 ppm a.i. 3284 F Liver, adult 10 ppm a.i. 3286 F Liver, adult 10 ppm a.i. 3288 F Liver, adult 10 ppm a.i. 3290 F Liver, adult 10 ppm a.i. 3292 F Liver, adult 10 ppm a.i. 3294 F Liver, adult AMOUNT OF LIVER W EIGHED (G) 1.0095 1.0944 1.0435 1.0425 1.0703 1.0526 1.0411 1.1704 1.1290 1.0800 1.1170 1.0086 1.0907 1.0291 1.1294 1.1075 1.1709 1.0761 1.1344 1.0669 1.0034 1.1331 1.1294 1.1829 1.0487 1.1214 1.0751 1.0658 1.1226 1.0953 1.1956 1.0600 1.0275 1.0915 1.0657 1.0330 1.0352 1.0658 1.0627 ADDITIONAL DILUTION 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 1 10 1 10 10 1 1 1 1 10 10 10 1 1 CONC. OF C8 F17SO 3 IN S am ple (n g /g o f L iv e r ) <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 7080 6780 13600 6080 5380 5730 13200 5800 13800 30700 5960 2480 4560 6330 28200 24100 19800 4600 4760 3M Environmental Laboratory 3M Environmental Laboratory Paae 18 of 216 Page 18 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S a m p l e Number SAMPLE DESCRIPTION E01-1326-30268 E01-1367-31295 E01-1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31337 E01-1367-31343 E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31404 E01-1367-31410 10 ppm a.i. 3296 F Liver, adult 0 ppm a.i. 9583 F Liver, offspring 0 ppm a.i. 9589 F Liver, offspring 0 ppm a.i. 9592 F Liver, offspring 0 ppm a.i. 9598 F Liver, offspring 0 ppm a.i. 9603 F Liver, offspring 0 ppm a.i. 9610 F Liver, offspring 0 ppm a.i. 9573 F Liver, offspring 10 ppm a.i. 9629 F Liver, offspring 10 ppm a.i. 9633 F Liver, offspring 10 ppm a.i. 9634 F Liver, offspring 10 ppm a.i. 9645 F Liver, offspring 10 ppm a.i. 9648 F Liver, offspring 10 ppm a.i. 9654 F Liver, offspring 10 ppm a.i. 9659 F Liver, offspring AMOUNT OF LIVER ADDITIONAL W EIGHED (G) DILUTION CONC. OF C8 F17SO 3 IN S am ple (n g /g o f L iv e r ) 1.1238 1 6330 1.1308 1.0005 1.0390 1.1204 1.1550 1.0217 1.0122 1 1 1 1 1 1 1 <50 <50 <50 <50 <50 <50 <50 1.1676 1 2950 1.0510 1 2240 1.1638 1 4520 1.1510 1 2580 1.1125 1 3500 1.0770 1 5930 1.0581 1 3580 3M Environmental I ahnratnrv 3M Environmental Laboratory Paae 19 of 216 Page 19 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 13. C8F17SO3' Data Summary, Male Mallard Liver (Adult and Offspring) 3M Sample Number SAMPLE DESCRIPTION AMOUNT OF LIVER W EIGHED (G) Cono. o fC8F17S03' in ADDITIONAL Sample DILUTION (ng/g of Liver) E01-1326-29473 E01-1326-29574 E01-1326-29592 E01-1326-29610 E01-1326-29628 0 ppm a.i. 3217 M Liver, adult 0 ppm a.i. 3219 M Liver, adult 0 ppm a.i. 3221 M Liver, adult 0 ppm a.i. 3223 M Liver, adult 0 ppm a.i. 3225 M Liver, adult 1.1169 1.1847 1.1181 1.0788 1.1043 1 1 1 1 1 <50 <50 <50 <50 <50 E01-1326-29646 E01-1326-29664 E01-1326-29682 E01-1326-29700 0 ppm a.i. 3227 M Liver, adult 0 ppm a.i. 3229 M Liver, adult 0 ppm a.i. 3231 M Liver, adult 0 ppm a.i. 3233 M Liver, adult 1.0486 1.0336 1.0165 1.0395 1 1 1 1 <50 <50 <50 <50 E01-1326-29718 0 ppm a.i. 3235 M Liver, adult E01-1326-29736 0 ppm a.i. 3237 M Liver, adult 1.0373 1.1679 1 1 <50 <50 E01-1326-29754 0 ppm a.i. 3239 M Liver, adult E01-1326-29772 0 ppm a.i. 3241 M Liver, adult 1.1493 1.0619 1 1 <50 <50 E01-1326-29790 0 ppm a.i. 3243 M Liver, adult 1.0263 1 <50 E01-1326-29808 0 ppm a.i. 3245 M Liver, adult 1.1541 1 <50 E01-1326-29826 0 ppm a.i. 3247 M Liver, adult 1.0813 1 <50 E01-1326-29844 0 ppm a.i. 3249 M Liver, adult E01-1326-29862 0 ppm a.i. 3251 M Liver, adult 1.0263 1.0088 1 1 <50 <50 E01-1326-29880 0 ppm a.i. 3253 M Liver, adult 1.0294 1 <50 E01-1326-29898 0 ppm a.i. 3255 M Liver, adult 1.0926 1 <50 E01-1326-29916 10 ppm a.i. 3257 M Liver, adult 1.0654 10 72100 E01-1326-29934 10 ppm a.i. 3259 M Liver, adult E01-1326-29952 10 ppm a.i. 3261 M Liver, adult 1.1316 1.1330 10 10 49200 70100 E01-1326-29970 10 ppm a.i. 3263 M Liver, adult 1.1404 100 125000 E01-1326-29989 10 ppm a.i. 3265 M Liver, adult E01-1326-30007 10 ppm a.i. 3267 M Liver, adult 1.1480 1.0620 10 10 61700 49900 E01-1326-30025 10 ppm a.i. 3269 M Liver, adult 1.1687 10 57900 E01-1326-30043 10 ppm a.i. 3271 M Liver, adult 1.0313 10 63000 E01-1326-30061 10 ppm a.i. 3273 M Liver, adult E01-1326-30079 10 ppm a.i. 3275 M Liver, adult 1.1399 1.1143 10 10 85000 54900 E01-1326-30097 E01-1326-30115 E01-1326-30133 10 ppm a.i. 3277 M Liver, adult 10 ppm a.i. 3279 M Liver, adult 10 ppm a.i. 3281 M Liver, adult 1.0062 1.0380 1.0669 10 10 10 53900 69000 49900 E01-1326-30151 E01-1326-30169 E01-1326-30187 10 ppm a.i. 3283 M Liver, adult 10 ppm a.i. 3285 M Liver, adult 10 ppm a.i. 3287 M Liver, adult . 1.0955 1.1981 1.0500 10 10 10 67100 55500 52600 E01-1326-30205 10 ppm a.i. 3289 M Liver, adult 1.0251 10 59300 E01-1326-30223 10 ppm a.i. 3291 M Liver, adult E01-1326-30241 10 ppm a.i. 3293 M Liver, adult 1.0441 1.0979 10 10 46500 24200 E01-1326-30259 10 ppm a.i. 3295 M Liver, adult 1.0815 10 50400 3M Environmental Laboratory 3M Environmental Laboratory Pane 20 of 216 Page 20 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S a m p le N um ber E01-1367-31301 E01-1367-31331 E01-1367-31349 E01-1367-31356 E01-1367-31392 E01-1367-31398 SAMPLE DESCRIPTION 0 ppm a.i. 9591 M Liver, offspring 0 ppm a.i. 9606 M Liver, offspring 0 ppm a.i. 9578 M Liver, offspring 10 ppm a.i. 9625 M Liver, offspring 10 ppm a.i. 9636 M Liver, offspring 10 ppm a.i. 9642 M Liver, offspring AMOUNT OF LIVER W EIGHED (G) 1.0411 1.1314 1.0413 1.0166 1.1367 1.1252 ADDITIONAL DILUTION CONC. O F C ^ y S O ^ IN S am ple (n g /g o f L iv e r ) 1 <50 1 <50 1 <50 1 4640 1 2200 1 2680 S tatem ent o f C o nclusio n Sera results from the male control group portion of this study showed no detectable levels of C8F17S 03' in 14 of 18 mallards (<100ng/mL) and quantifiable concentrations of C8F17S 03' in 4 of 18 (range 107-1400ng/ml_, Table 14). The sera results from the female control group portion of this study showed no detectable levels of C8F17S 03` in 15 of 17 birds (<100ng/mL) and quantifiable concentrations of C8F17S03' in 2 of 17 (range 166-172ng/mL, Table 15). Analytical results for eighteen sera samples from the 10ppm dosed male group showed an average of 87300ng/ml_ C8F17S 03' (range 68700-108000ng/ml_, Table 15). Analytical results for nineteen sera samples from the 10ppm dosed female group showed an average of 16600ng/mL C8F17S 03` (range 6350-42800ng/mL, Table 15). Sera results from the male offspring control group showed no detectable levels of C8F17S 03' in 2 of 3 birds (<100ng/mL) and a quantifiable concentration in 1 of 3 (103 ng/mL, Table 14). The sera results from the female offspring control group portion of this study showed no detectable levels of C8F17S 03' in 7 of 7 birds (<100ng/ml_, Table 14). Analytical results for three sera samples from the 10ppm dosed male offspring group showed an average of 4410ng/mL (range 3670-5450ng/mL, Table 14). Analytical results for seven sera samples from the 10ppm dosed female offspring group showed an average of 4960ng/mL (range 3420-6760ng/mL, Table 14). Liver results from the male control group showed no detectable levels of C8F17S 03` in 20 of 20 birds (<50ng/g, Table 14). The liver results from the female control group also showed no detectable levels of C8F17S 03' in 20 of 20 birds (<50ng/g, Table 14). Analytical results for twenty liver sam ples from th e 10ppm dosed m ale group show ed an a v erag e of 6 0 9 0 0 n g /g C 8F 17S 0 3` (range 24200-125000ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed female group showed an average of 10800ng/g C8F17S 03' (range 2480-30700ng/g, Table 14). Liver results from the male offspring control group portion of this study showed no detectable levels of C8Fi7S 03' in 3 of 3 birds (<50ng/g, Table 14). The liver results from the female offspring control group portion of this study showed no detectable levels of C8F17S 03' in 7 of 7 birds (<50ng/g, Table 14). Analytical results for three liver samples from the 10ppm dosed male offspring group showed an average of 3170ng/g (range 2200-4640ng/g, Table 14). Analytical results for seven liver samples from the 10ppm dosed female offspring group showed an average of 3620ng/g (range 2240-5930ng/g, Table 14). 3M Environmental Laboratory 3M Environmental Laboratory Page 21 of 216 Page 21 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 14. Summary Table, C8F17S 0 3' Concentration in Sera and Liver S a m p l in g Event Male Control Group Number of S era S am ples A verage C8F17SO 3 C onc. S era Range o f R esults N umber of L iv e r S am ples A verage C 8F17SO 3 C o n c . L iv e r <100 ng/mL (14 of 18) 18 NA 20 <50 ng/g 107-1400 ng/mL range (4 of 18) Range of R esults NA Female Control Group Male 10ppm Group Female 10ppm Group Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 20 20 20 3 7 3 7 <100 ng/mL (15 of 20) NA 166-2680 ng/mL range (5 of 20) 89700 ng/mL 11300-210000 ng/mL 20300 ng/mL NA NA 6350-91400 ng/mL <100 ng/mL (2 of 3) 103 ng/mL (1 of 3) <100 ng/mL (7 of 7) 4410 ng/mL 4960 ng/mL 3670 - 5450 ng/mL 3420 - 6760 ng/mL For individual samples values see Tables 10 to 13 and Attachment B. 20 20 20 3 7 3 7 <50 ng/g NA 60900 ng/g 24200-125000 ng/g 10800 ng/g 2480-30700 ng/g <50 ng/g <50 ng/g 3170 ng/g 3620 ng/g NA NA 2200-4640 ng/g 2240-5930 ng/g 3M Environmental Laboratory Page 22 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 15. Summary Table, C8Fi7S0 3' Concentration in Sera and Liver with Outliers Removed S a m p l in g E v e n t N um ber of S era S am ples A verage C8F17SO 3 C onc. S era Range of Results Number of L iv e r Sam ples A verage C8F17SO 3 Conc. L iv e r Range of Results Male Control Group Female Control Group 18* 17 <100 ng/mL (15 of 17) NA 166-172 ng/mL range (2 of 17) Male 10ppm Group 18 87300 ng/mL 68700-108000 ng/mL 20* 20* 20* * * * * * * Female 10ppm Group 19 16600 ng/mL 6350-42800 ng/mL 20* Male Offspring Control Group Female Offspring Control Group I 3* 7* * * * 3* * 7* * * Male Offspring 10ppm Group 3* * * 3* * * Female Offspring 10ppm Group 7* * * 7* * * * No outliers removed from this data set. Refer to Table 14 for results. For individual samples values see Tables 10 to 13 and Attachment B. S tatistical M etho ds Statistical methods included calculating means, standard deviations, % coefficient of variation, linear regressions, percent recoveries, and the Dixons Q -Test. T h e Dixons Q -Test, used for the rejection of outliers, has values ordered from lowest to highest. A Q-value is determined from the difference of a result and the next closest result that is divided by the difference of the highest and lowest value in the group of values. A critical Q-value is determined based on the number of samples in the group and the desired confidence interval. If the Q-value is greater than the critical Q-value then the value will be rejected. S tatem ent o f Data Q uality The only measurement is the quality control and matrix spike results. Accuracy (the observed amount in the QC and MS samples, divided by the amount spiked, expressed as a percent) was found to range from 79.7% to 172%; while precision, expressed as the standard deviation of the recoveries, was found to range from 0.717% to 15.7%. 3M Environmental Laboratory Page 23 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard References 1. Wildlife International, Ltd. Protocol No. 454-109 "PFOS: A Reproduction Study with the Mallard". 2. Wildlife International, Ltd. Protocol No. 454/120700/MR/SUB454 (3M Environmental Laboratory Project Number U2723) "PFOS: A Reproduction Study with the Mallard". 3. Centre Analytical Laboratories, Inc. Study 023-044, "Validation of Analytical Methods "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". List of Attachm ents Attachment A: Extraction and Analytical Method Attachment B: Data Summary Tables Attachment C: Sample Chromatograms, Calibration Information, and Instrument Parameters Attachment D: Sample Preparation Sheets Attachment E: Study Protocol, Protocol Amendments, and Deviations Attachment F: Example Calculations 3M Environmental Laboratory 3M Environmental Laboratory Pane 24 of 216 Page 24 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard S ig n a tu r e Pa g e We certify that this report is a true and complete representation of the data for this study: Lisa A. Stevenson Principal Analytical Investigator oi m l t , 3 Date William K. Reagen Laboratory Management Date 3M Environmental Laboratory 3M Environmental Laboratory Page 25 of 216 Page 25 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard At t a c h m e n t A : E x t r a c t io n a n d A n a l y t ic a l M e t h o d , ETS-8-231.1 , "S o lid P hase Extr a c tio n and A nalysis of F lu o r o c h em ic a l C o m po u n d s from B io lo g ical M atr ic es" 3M Environmental Laboratory 3M Environmental Laboratory Paae 26 of 216 Page 26 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environm ental Laboratory Method Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: II f f ijo l Revision Date: Effective Date: s j t t f 2- Approved By: Willliam K. Reagen Laboratory Manager Date ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 1 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 27 of 216 Page 27 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 1 Scope and Application This method describes the extraction o f target analytes from fish, rat liver, rat sera, mouse liver, and mouse sera using solid phase extraction (SPE). This method may also be extended to other biological matrices provided that the data quality objectives are met. 2 Method Summary An amount o f biological material, determined by the analyst, is prepared (fluids diluted and tissues homogenized) at a 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, mixed, and put on the centrifuge to clarify the supernatant. The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned C u SPE cartridge. Finally, the analytes o f interest are eluted from the SPE cartridge and analyzed by high perfonnance liquid chromatography-electrospray tandem mass spectrometiy (HPLC-ES/MS/MS). 3 Definitions 3.1 Dilution A dilution expressed as l :5 or 1/6 is defined as: 1 rnL o f sample + 5 mLs o f diluent for a total o f 6 rnLs combined, unless otherwise noted. 3.2 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds o f interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable concentration(s) o f the target analyte(s). 3.4 Quality control sample Sample used to monitor the extraction efficiency (as a matrix spike ) and to verify the continued accuracy o f the initial calibration curve (as a continuing calibration verification). 4 Warnings and Cautions_______________________________________________ 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewear, and clothing when working with solvents, samples and/or equipment. Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 volts. 4.2 Cautions Take care not to allow the SPE column to run to dryness after the methanol and water washes. After washing is complete, add sample then allow all o f the liquid to pass through the SPE column to dryness. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluoroehemical Compounds from Biological Matrices Page 2 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 28 of 216 Page 28 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. Do not am 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 Centrifuge. Mistral 1000 or IEC Shaker, Eberbach or VWR Balance (+/'- 0.1000 g) Micromass. Quattro II or Ultima triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppendorf or disposable pipettes, plastic or glass Dissecting scalpels Polypropylene bottles, capable o f holding 50 m L to 1 L (Nalgene) Volumetric flasks, glass, type A 40 mL glass vials (1CHEM) Plastic sampule vials, Wheaton, 6 mL (or other appropriate size) Centrifuge tubes, polypropylene. 15 mL and 50 mL Labels Graduated pipettes, glass Syringes, capable of measuring 5 pL to 1000 pL Bottle-Top Dispenser (capable o f dispensing 5nrL o f solvent) SPE extraction cartridge, 1 g, Sep-Pak 6 cc tri-functional Cis (Waters) 75 ntL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 3 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 29 of 216 Page 29 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Crimpers HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7 Reagents and Standards Reagent grade water, Milli-QTM, Nanopure II, or equivalent Acetonitrile. HPLC grade or equivalent Methanol, HPLC grade or equivalent Ammonium acetate, reagent grade or equivalent Biological fluids or tissues, frozen from supplier 7.1 Reagents preparation 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing reagent grade water, mix until all solids are dissolved, bring to volume using reagent trade water. Store at room temperature. Note: When preparing different volumes than those listed in reagents preparation, target analyte standard preparation, and surrogate standard preparation, adjust accordingly. 7.2 Target analyte standard preparation Prepare target analyte standard(s) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations. Weigh approximately 100 mg o f target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location. Bring to volume with methanol for a stock standard o f approximately 1000 ppm (flg/'mL). Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. Example calculation: 1000 |ig/niL x 5 mL/100mL = 50 |4g/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution o f approx 5.0 ppm. Example calculation: 50(ig/m L x lOmL/lOOmL = 5 .0 (Xg/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution o f approx. 0.50 ppm. Example calculation: 50 jJg'inL x 1.0 m L /100 mL = 0.5 |fg/mL. 7.3 S u rro g ate stan d ard preparation Prepare surrogate standard(s). The following is an example only and may or may not be appropriate for all surrogate standard preparations. Weigh approximately 90-110 mg o f surrogate standard into a 100-mL volumetric flask and record the actual weight. Bring to volume with methanol for a surrogate standard stock o f approximately 900 -1100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL o f surrogate standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard o f 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 7.4 Internal standard preparation Prepare internal standard(s). The following is an example only and may or may not be appropriate for all internal standard preparations. Weigh approximately 90-110 mg o f internal standard into a 100-mL volumetric flask and record the actual weight. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 4 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 30 of 216 Page 30 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Bring to volume with methanol for an internal standard stock o f approximately 900 -1100 ppm. Prepare an internal standard working standard. Transfer approximately 1 mL of internal standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard of 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 8 Sample Handling All samples are received frozen and must be kept frozen until the extraction is performed. Allow samples to thaw to room temperature prior to extraction. Typically fresh matrix standards are prepared with each analysis. Extracted standards and samples 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 performed. 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 procedute (two are spiked with surrogate and two are not spiked). 9.1.3 Matrix Blank An aliquot o f 1.0 mL or 1.0 g o f matrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one o f three sources: 1) a study control matrix from a study control animal received with a sample set; 2) a commercially obtained sample o f the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but o f a different species than the study animal, (eg. if rat is used to generate standard curves and CCVs for a mouse study). The matrix to use is dependent on the m atrix used for the curve. 9.1.3.1 Study control matrix curve - if the study control matrix is used for the curve, prepare four (4) matrix blanks using the study control matrix (two spiked with surrogate and two not spiked). 9.1.3.2 Commercially obtained (same species) matrix curve - if the commercially obtained matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix (two spiked with surrogate and two not spiked). 9.1.3.3 Surrogate matrix curve - if a surrogate matrix is used for tire 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, tire number o f method and matrix blanks may be adjusted and will be noted in tire study protocol or in the raw data. 9.2 Sample Replicate Samples replicates are prepared according to each study protocol or project outline. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 31 of 216 Page 31 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 9.3 Surrogate standard If surrogate standard is a component o f the study, all samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range o f the calibration curve, with the exception o f blank samples as described above. Typically surrogate standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. 9.4 Internal standard If internal standard is a component o f the study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range o f the calibration curve. Typically internal standard is spiked into the 2.0 mL o f extract in the 15 mL centrifuge tube, before transfening to the autovial. 9.5 Lab Control Sample Lab control samples are not a component of this method. 9.6 Quality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efficiency and to verify the continued accuracy o f the initial calibration curve. Typically 1.0 mL, or other appropriate amount, o f the same matrix used to prepare the initial calibration curve is used for each QC sample. Twelve (12) quality control samples (QC) will be prepared for each matrix during the course o f a study. A minimum o f 3 QC samples must be prepared (one at each level) on each day o f sample extraction, (e.g. If the study is such that samples will be extracted on three different days then four QC samples must be prepared on each day o f extraction for a total o f twelve.) QC samples will consist o f four samples at each o f three levels o f analyte. The levels listed below may be used and may represent sample concentrations diluted into the range o f the calibration curve: Low level: 3X to 5X the LLOQ, Mid-level: equivalent to a point near the middle of the calibration curve, High level: 80% o f the LJLOQ 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. Q C samples extracted with a particular sample set must be analyzed in the same analytical run. Any Q C samples extracted during the course of the study may be included in subsequent analyses. If samples from multiple extraction dates are analyzed in one analytical run, then QC samples from the same sample extraction dates must be included in that analysis. Each QC is expected to show an accuracy o f 75425% of expected. A minimum of2/3 o f all QC samples must meet this criteria, and a minimum o f 1/2 of the QC samples at each level must meet this criteria. If not, the set must either be re-analyzed or re-extracted. 9.7 Sample Dilution Any sample with an area greater than that o f the highest acceptable standard will need to be diluted into the range of the calibration curve. If samples are diluted into the range o f the curve during analyses and enough sample remains, a post-run dilution validation will be perforated to verify sample values. To perform 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 ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 6 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 32 of 216 Page 32 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard and documented in a note to tile) then diluted using twro procedures. The first procedure consists o f diluting the sample with additional matrix prior to extraction (fluid adding fluid), while the second procedure consists o f diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent.) If the relative percent difference is not within 15% for these two samples; additional testing will be required to determine which value is a correct representation o f the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, per study. It will consist o f a minimum o f nine (9) levels. Additional calibration curves may be extracted on separate sample extraction dates, as determined by the analyst and documented in a note to file. Transfer l.O mL, or other appropriate amount, of diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weight/volumes sheet or extraction worksheet, as appropriate. Four l .0 mL aliquots, or other appropriate amount, o f control matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one standard curve. A total o f 9 standards, four matrix blanks, and four method blanks are prepared in addition to the QC samples and test samples. The number of standards and blanks may be adjusted as detennined by the analyst and documented in a note to tile. Use Attachment C. or other appropriate form, as an aid in calculating the concentrations o f the working standards. Refer to section 12 to calculate the actual concentration o f analyte in each calibration standard and QC sample. Typically the target analyte standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, it may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. Analyze the extracted matrix standard curve prior to each set o f extracts. The curve equation will be determined by regression analysis using the peak areas o f the target analyte(s) using MassLynx or other suitable software. Any level outside 75% - 125% o f nominal must be deactivated, and regression re-calculated, except the LLOQ which must be within 30% o f nominal. All levels must show a response greater than twice that o f the blank. A maximum of three (3) levels may be deactivated in any one set, or the set will be re-analyzed. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 7 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 33 of 216 Page 33 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table1 A pproximate Spiking Am o i nts for Standards and Spikes Using 1.0 m L of Mai rix Working standard (approximate concentration) pL Approximate final concentration o f analyte Approximate final concentration o f analyte in M atrix diluted 1:5 in Final 2.0 mL volume - - Blank Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 10.0 ng/g or ng/mL 0.750 ng/mL 0.500 ug/mL 8.0 25.0 ng/g or ng/mL 2.00 ng/mL 0.500 ug/mL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug/mL 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL 14.0 ng/mL 5.00 ug/mL 8.0 250 ng/g or ng/mL 20.0 ng/mL 5.00 ug/mL 16 500 ng/g or ng/mL 40.0 ng/mL 5.00 ug/mL 24 750 ng/g or ng/mL 60.0 ng/mL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/g or ng/mL 100 n g m L 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/mL 50.0 ug/mL 6.0 1750 ng/g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures 11.1 Tissue Sample Preparation Obtain frozen tissue samples Cut approximately 1.0000 g o f tissue (+/- 0.1000 g), or other appropriate amount, using a dissecting scalpel. This part o f the procedure is best performed quickly, not allowing the tissue to thaw. Weigh the tissue directly into a fared plastic sampule vial. Record the weight on the weight/volume sheet, extraction worksheet, or other appropriate location. Return unused tissue to the freezer after extraction amounts have been removed. Add 2.5 mL o f reagent water to sampule vial, or other volume as determined by the analyst and documented in a note to file. Homogenize the sample. Put the Ultra-Turrax grinder probe in the sample and grind for approximately 2 minutes, or until the sam ple is hom ogeneous. Rinse the probe into the tube containing the sample with 2.5 mL o f reagent grade water, or other volume as determined by the analyst and documented in a note to file, using a pipette. Take the grinder apart and clean it with methanol after each sample. Refer to ETS-9-52 for more information. If an amount other than 1.0000 g (not within +/- 0.1000 g) is removed tor 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-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 8 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 34 of 216 Page 34 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 11.2 Fluid Sample Preparation Obtain frozen fluid sample and allow it to thaw at room temperature or in lukewarm water. Label a 15 mL polypropylene centrifuge tube with the study number, sample ID. extraction date and analyst initials. See attached worksheet (Attachment A or similar worksheet) for documenting the remaining steps. Vortex mix the fluid sample for approximately 15 seconds, then transfer 1.0 mL o f fluid, or other appropriate amount to a plastic sampule vial, or other appropriate container. Return unused samples to freezer after extraction amounts have been removed. Add 5.0 mL o f reagent water to the 1.0 mL o f fluid for a 1/6 dilution, or other dilution is 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 o r 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 similar worksheet). Spike blanks, samples, and standards, ready for extraction with surrogate standard as described in this method. Spike each calibration standard matrix with the appropriate amount o f standard as described in this method for the calibration curve standards and each QC sample. Vortex mix the standard curve samples and QC samples for approximately 5 seconds. To each sample and standard, add 5.0 mL o f acetonitrile, cap, and vortex mix or shake by hand approximately 15 seconds. Place all samples on the shaker at an appropriate speed for 20 minutes to adequately mix (a setting o f approximately 300 rpm on the models listed in section 6.1). Remove from the shaker and 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 samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, hiking care not to introduce any o f the matrix solids into the solutioa Cap and mix by inverting several times. In this step the order o f addition may be changed (i.e. tire sample m ay be put into the centrifuge tube and then the w ater added). Attach the reservoir to the SPE cartridge and attach this reservoir/cartridge unit to a vacuum manifold. NOTE: When running the vacuum, set the vacuum chamber at approximately 15 kPA - to give an approximate elution flow o f 5-7 mL/min. Flow's may vary through cartridges and the kPA may be raised for slow tubes and drying after most have been drawn down. Prepare the SPE cartridge by washing twice with approximately 5.0 mL of methanol, followed by approximately two 5.0 mL aliquots o f water, taking care not to allow the column to run to dryness after each wash. /liter 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 of methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluoroehemical Compounds from Biological Matrices Page 9 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 35 of 216 Page 35 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Label each glass autovial, as appropriate, with the study number, vial file archive number, animal number/gender/timepoint or L1MS number, matrix, final solvent, analyte components (if needed), extraction type, extraction date, and analyst(s ) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.1 Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-month-day, and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or A020204a I = Initial o f the instrument name (A = "Amelia") YY = Test year (02 ) M M = Test month (02) DD = Test day (04) a = First sample list (run) o f the day (the next sample list wall end with 'b', the next 'c', and so on.) Assign a filename using the instrument designator letter, the last 2 digits o f the test year-month-day, and a 3-digit sequential file number that stalls with 1 and increases by one for each filename. Example filename: IYYMMDD### or A020204001 I = Initial o f instrument name YY = Test year M M = Test month DD = Test day ### = 3-digit sequential file number starting with 1 through 999 (001) Also, as part o f the samplelist, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume, and sample descriptions. To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate mass(es). Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are em ployed, additional product ion fragm entation inform ation m ay be collected. See M icrom uss M assL ynx "G uide to Date Acquisition" for additional information on MRM. Typically the analytical batch run sequence begins with system suitability, solvent blanks, and a set o f extracted matrix standards. Sample extracts are analyzed with two QC samples injected after every tenth sample injection. Solvent blanks should 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 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 36 of 216 Page 36 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 11.4.2 HPLC set-up Set up sample tray according to the sample list prepared above. Set up the HPLC to the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook, or other appropriate location: Sample size = 10 pL injection Inject/sample = 1 Cycle time = 10.0 minutes Flow rate = 300 pL/min Mobile phase: Solvent A = 2 mM Ammonium Acetate, Solvent B = Methanol Solvent gradient program: Time % Solvent B 0.00 10% 1.00 10% 5.50 95% 7.50 95% 8.00 10% 11.4.3 Instrument set-up Refer to ETS-9-24, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fritted with an Atmospheric Pressure Ionization Source," for details. Check the solvent level in HPLC reservoirs and refill if necessary. Check the stainless steel capillary at the end o f tire probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. Turn on the nitrogen. Open the tune page. Click on operate to initiate source block and desolvation heaters. Open the Inlet Editor. D ow nload the H PL C m ethod and initiate solvent flow to begin system equilibrium . Set foe flow to 10-500uL/min or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out of foe tip of the probe. A fine mist should be expelled with no nitrogen leaking around foe tip o f the probe. Readjust foe tip o f foe 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 37 of 216 Page 37 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard HPLC constant flow mode, 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 o f the start button may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). 12 Data Analysis and Calculations________________________________________ 12.1 Calculations If other calculations are used than those listed, they will be documented in the raw data. Calculate the matrix amount contained in the initial dilution using the following equation: Matrix Amount (g/mL or mL/mL) 1W (g) (or IV (mL)) (IW(g) (or lV(m L))+ DV (mL) Calculate actual concentrations of analyte in calibration standards using the following equation: Concentration (ng/g or ng/mL) Spike Concentration (ug/mL) x Spiked Amount (mL) 1000 ng SV (mL) x Matrix Amount (g/mL or mL/mL) 1ug IW = Initial weight (where 1.0 g = 1.0 mL ) IV = Initial volume DV = Diluent volume (reagent grade water) SV' = S a m p le v o lu m e re m o v e d fo r e x tra c tio n (typically 1.0 m L ) AR = Analytical result from MassLynx summary DF = Dilution factor FV = Final volume MA = Matrix amount 0 curve = MA o f tissue/fluid standard curve, assumed to be 1 g or 1 mL/5 mL water o sample = MA o f tissue/fluid sample (___g or mL o f sample/5 mL water) Calculate spike percent recoveries using the following equation: Observed Result - Matrix Blank Result % Recovery ----------------------------------------------------------- - x 100 Spiking Level ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 12 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 38 of 216 Page 38 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Calculate relative standard deviation using the following equation: Standard Deviation Relative Standard Deviation = x 100 Mean Calculate percent deviation using the following equation: ,, .. % Deviation = Expected C one.- Calculated Cone. -- ----------------------------------------- x 100 Expected Cone. Calculate actual concentration o f analyte in fluid (jig/mL): AR (ng/mL) x DF x 0 curve fmL/mL) x FV (mL) inCurve x 1.0 ug = (pg/g) d sample (mlVmL) FV (mL) in Matrix 1000 ng Calculate actual concentration o f analyte in tissue (pg/g): AR (ng'g) x DF x 0 curve (g/mL) x FV 01110 in Curve x 1.0 ug d sample (g/mL) FV (mL) in Matrix 1000 ng = (pg/g) 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start and at the completion o f each analytical run. Prior to foe 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, foe precision o f the retention time must be equal to or less than 2.5% RSD, the resolution must be > 2.0, and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is foe asymmetry factor. If any item o f the system suitability fails, system maintenance must be completed prior to running a second set o f system suitability samples and the system suitability must pass before starting the calibration. If system suitability fails at foe completion o f a rim, the sample set must be reanalyzed. 13.2 Quantitation The coefficient o f determination value for the calibration curve, plotted by regression using foe peak areas o f the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% o f foe theoretical value with the exception of the LOQ point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify' a data range that may be affected by background levels o f the analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. If the curve cannot meet these criteria, the sample set must be reanalyzed or reextracted. 13.3 Accuracy Two thirds o f all quality control samples and 1/2 o f each quality control sample at each level are expected to show an accuracy o f75-125%. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 13 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 39 of 216 Page 39 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 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, tire deviation will be documented in the raw' data. If the second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed o f in noninfectious biohazard waste containers. Flammable solvent waste is disposed o f in high BTU containers. Glass pipette waste is disposed o f in broken glass containers located in the laboratory. 15 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw data. Each page generated for a study must contain the following information (if applicable): study/project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Other information may be added if applicable to the study. Print the tune page, sample list, and acquisition rnedrod from MassLynx to include with the study raw data. Copy these pages and tape into the instrument runlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data Print data integration summary, integration mediod, and chromatograms front MassLynx, and store with die study raw data. Summarize data using suitable software (Excel 7.0 or LIMS) and store in the study folder. Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 16 Attachments_________________________________________________________ Attachment A: Extraction Worksheet Attachment B: Sample Weight/Volume Worksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary' Worksheet 17 References__________________________________________________________ EiTS-9-24. "Operation and Maintenance o f die Micromass Quattro II Triple Quadtupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" E',TS-9-52. "Operation and Maintenance o f a Tissue Grinder" 18 Affected Documents__________________________________________ ________ None ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 14 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 40 of 216 Page 40 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 19 Revisions Revision Number ] Revision Description M inor formatting changes. Added detailed information to all sections concerning the extraction procedure, analytical procedure, and calculations. Added attachments and references. Revision Date 02/18/02 ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 15 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 41 of 216 Page 41 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Study Number: Prep Date: Analysts initials: Box#: Attachment A - Extraction Worksheet Method Revision:ETS-8-231.1 Matrix: Sample Timepoint: Sample Number Sample Number or description Volum e of Amount and diluted sample Am ount and surrogate spike removed spike mix used mix used Type of column used and lot Elution solvent and volume C o m m ents B lank m a trix ______ TN -A -______ ,, ___ ; A m oun t w e igh e d /a liq u o te d :____________ g/mL 1. Hom ogenize sam ple 2. A liquot 1 m L o f diluted m atrix into 15 mL polypropylene tube 3. S pike sam ple s accordingly 4. A d d ____ m L o f AC N (TN-A-____________ ) to each diluted sam ple and shake or vortex m ix 5. S h ake sam ple for 20 m in @ ______ rpm (S h a k e r________________ ) 6. C entrifuge sam ple fo r 10 min @ ________rpm (C e n trifu g e _________________ ) 7. A dd 40 m L o f ____________ w ater to 50 m L potypropelene centrifuge tube. 8. D e c a n t e x tra c t into ce n trifu g e tu b es w ith w a te r 9. S hake sam ple slightly to ensure proper mixing 10. A tta ch 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum m anifold 11. Condition colum n with tw o washes o f ~5 m L M eOH (TN-A-___________ ) - do not allow colum n to go to dryness 12. W ash colum n with tw o washes o f - 5 m L ___________ w ater - do not allow colum n to go to dryness 13. Filter sam ple through conditioned colum n, discarding filtrate 14. A llow colum n to go to dryness. A fte r dripping stops, draw a high vacuum through colum n fo r a t least 5 m inutes. 15. Elute colum n w ith solvent (___________ TN -A -___________ ) into appropriate 15 mL centrifuge tube 16. S pike sam ples w ith _________ uL o f internal standard # _______________________ , cone.___________ ___________ 17. T ransfer sam ple into appropriately labeled autovial and cap Note: In vacuum steps above set the vacuum cham ber at approxim ately 15 kPA - this should give approxim ately 5-7 m L/m in elution flow Flows m ay vary through cartridges - kPA m ay be raised fo r s lo w tubes and drying after m ost have been drawn dow n and shut off. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 16 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 42 of 216 Page 42 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Prep Date(s): Analyst(s): Sample M atrix: Method/Revision: Attachment B - Sample Weight/Volume Worksheet Study Number: Equipment Number: Final Solvent & TN Number: Sample ID Initial Wt./Vol. g/mL/L W ater Volume added (mL) Volume Removed (mL) Comments Forni Completion Verified By:. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 17 of 19 3M Environmental Laboratory 3M Environmental Laboratory Page 43 of 216 Page 43 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Prep date(s): Analyte(s): Sample matrix: Method/revision: Attachment C: Calibration Standard Concentration W orksheet Standard number: Equipment number: Final solvent and TN: Blank Tissue or Fluid/identifier: Analyte mix std approx. 0.500 ug/mL: Analyte mix std approx. 5.00 ug/mL: Analyte mix std approx. 50.0 ug/mL: Surrogate std approx. 100 ug/mL: Actual concentrations o f standards in the analyte mix 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 m 't spiked mL 0.0015 0.0030 0.0080 0.0160 0.0320 0.0056 0.0080 0.0160 0.0240 0.0320 0.0400 0.005 0.006 AU Final Volume: mL 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 All Initial Fluid Dilution mL/mL 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 Ail Initial Tissue Density g/niL 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 Analyte Final cone. ng/mL 0.375 0.750 2.00 4.00 8.00 14.0 20.0 40.0 60.0 80.0 100 125 150 Surrogate Std cone ng/mL 100 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. ng/g 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL too Surrogate Final cone n.a's 6500 ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 18 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 44 of 216 Page 44 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Study: Dilution Date/Analvst: Box Number: Sample Number or Description 1/ Attachment D: Dilutions Summary Worksheet Solvent/TN Number: Extraction Date/Analyst: Matrix/Timepoint: Dilutions 11 1/ 1/ 1/ 1/ 1/ Comments Verified By: Notes: 1/10 dilution = _______ of sample + _________ of solvent Form Completion Verified B y :_____________________________ ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds trom Biological Matrices Page 19 o f 19 3M Environmental Laboratory 3M Environmental Laboratory Page 45 of 216 Page 45 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachment B: Data S ummary Tables 3M Environmental Laboratory 3MEnvironmental Laboratory Pane 46 of 216 Page 46 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD SERA DATA SUMMARY, BLANKS AND SPIKES 3M Sam ple Number E01-1256-36231 E01-1256-36259 E01-1256-36263 E01-1256-36263 E01 -1256-36267 E01-1256-36231 E01-1256-36232 E01-1256-36260 E 0 1 -1256-36264 E01-1256-36264 E01-1256-36268 E01-1256-36232 Sam pled Date 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 Sam ple Description Blank H 20-day 1 Blank H 20-day 2 Blank H 20-day 3 Blank H 20-day 3 Blank H 20-day 4 Blank H 20-day 1 Inst. File Name d020225037 d020226025 d020228024 d020301099 d020228107 d020308036 amount of sera used (m L) 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (diluted sera) 40 40 40 40 40 40 a d d itio n a l d ilution 1 1 1 1 1 1 Inst C one. (ng/m L) C one, o f PFOS in Sam ple (ng/m L) loq for control sam ples 1.00 40.0 40.0 2.51 2.51 1.00 100.4 100.4 40.0 100.4 * 100.4 40.0 1.00 40.0 40.0 2.51 100.4 100.4 Blank mallard sera-day 1 d020225038 0.05 40 1 1.00 Blank mallard sera-day 2 d020226026 0.05 40 1 2.51 Blank mallard sera-day 3 d020228025 0.05 40 1 2.51 Blank mallard sera-day 3 d020301100 0.05 40 1 1.00 Blank mallard sera-day 4 d020228108 0.05 40 1 1.00 Blank mallard sera-day 1 d020308037 0.05 40 1 2.51 40.0 100.4 100.4 40.0 40.0 100.4 40.0 100.4 100.4 * 40.0 * 40.0 * 100.4 3M Sam ple N um ber E 0 1 -1256-36233 E01-1256-36261 E01-1256-36265 E01-1256-36265 E01-1256-36269 E01-1256-36233 S am pled Date 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 S am ple D escription In st. File Name Mallard Sera MS-day 1 Mallard Sera MS-day 2 Mallard Sera MS-day 3 Mallard Sera MS-day 3 Mallard Sera MS-day 4 Mallard Sera MS-day 1 d020225039 d020226027 d020228026 d 02 0 3 01 101 d020228109 d020308038 amount of sera used (m L) 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (diluted sera) 40 40 40 40 40 40 a d d itio n a l d ilu tio n 1 1 1 1 1 1 C one, o f PFOS in Inst Cone. (ng/m L) Sam ple (ng/m L) 27.79 29.50 28.85 27.76 28.01 29.03 1112 1180 1154 1110 1120 1161 E01-1256-36234 E01-1256-36262 E 0 1 -1256-36266 E01-1256-36266 E01-1256-36270 E01-1256-36234 * Concentration <LOQ 02/25/2002 02/26/2002 02/27/2002 02/27/2002 02/28/2002 02/25/2002 Mallard Sera MSD-day 1 Mallard Sera MSD-day 2 Mallard Sera MSD-day 3 Mallard Sera MSD-day 3 Mallard Sera MSD-day 4 Mallard Sera MSD-day 1 d020225040 d020226028 d020228027 d 02 0 3 01 102 d 020228110 d020308039 0.05 0.05 0.05 0.05 0.05 0.05 40 40 40 40 40 40 Cone of PFO S in Sam ple (ng/m L) = (Inst conc(ng/m L)) x (additional dilution) x (dilulion(diluted sera)) dilution(diluted sera) = final volum e(m L)/am ount of sera in diluted sam ple(m L) = 2 .0 m L/0.05 m L = 40 am ount of sera in diluted sam ple (mL) = am ount of sera used/total am ount o f sera + water (1/20 dilution). = 1/20 = 0.05 m L 1 30.44 1 29.80 1 24.76 1 24.01 1 28.23 1 31.34 for both ms and msd avg %rec std dev of %rec avg cone std dev of cone 1218 1192 990 960 1129 1254 112.7 8.487 1132 85.21 % Ree 110.7 117.5 114.9 110.6 111.6 115.7 true val. 1004 avg%rec 113.5 avg 1140 Std dev 29.36 121.3 118.7 98.65 95.66 112.5 124.9 true vai. 1004 avg %rec 111.9 avg 1124 std dev 122.3 3M Environmental Laboratory Rlanb-c C \ C 'c anH MQc Page 47 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD SERA versus unextracted curve ^N um ber* Sam ple D escription Inst. File Name e01 -1256-36250 Q C - 25ppb -1 (1004 ng/m L diluted sera) e 0 1-1256-36251 Q C -2 5 p p b -2 (1004 ng/mL diluted sera) e01-1256-36252 Q C -2 5 p p b -3 (1004 ng/mL diluted sera) d020405036 d020405037 d020405038 e01 -1256-36253 Q C - 250ppb -1 (10040 ng/m L diluted sera) d020405039 e01-1256-36254 QC- 250ppb -2 (10040 ng/mL diluted sera) d020405040 e 0 1-1256-36255 QC- 250ppb -3 (10040 ng/mL diluted sera) d020405041 e01 -1256-36256 Q C - 4 .0 ppm -1 (160640 ng/m L diluted sen d020405042 e01 -1256-36257 QC- 4.0 ppm -2 (160640 ng/m L diluted sen d020405043 e 0 1-1256-36258 QC- 4.0 ppm -3 (160640 ng/mL diluted sen d020405044 A = Not within +/-25% criteria am ount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (d ilu te d sera) 40 40 40 40 40 40 40 40 40 a dditional d ilu tio n 1 1 1 1 1 1 10 10 10 Study number E01-1256 - MALLARD SERA versus extracted curve original 3 N um ber'6 Sam ple D escription Inst. File Name e 0 1 -1256-36250 Q C - 2 5 p p b -1 (1004 ng/m L diluted sera) d020225071 e 0 1-1256-36251 Q C -2 5 p p b -2 (1004 ng/mL diluted sera) d020225072 e01-1256-36252 Q C -2 5 p p b -3 (1004 ng/mL diluted sera) d020225073 e01 -1256-36253 Q C - 250 p p b -1 (10040 ng/m L diluted sera) d020225074 e01-1256-36254 QC- 250ppb -2 (10040 ng/mL diluted sera) d020225075 e01 -1256-36255 Q C - 250ppb -3 (10040 ng/mL diluted sera) d020225076 e01 -1256-36256 Q C - 4 .0 ppm -1 (160640 ng/m L diluted sen d020308045 e01-1256-36257 QC- 4.0 ppm -2 (160640 ng/mL diluted sen d020308046 e01-1256-36258 QC- 4.0 ppm -3 (160640 ng/mL diluted send020308047 A = Not within +/-25% criteria am ount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n (d ilu te d sera) 40 40 40 40 40 40 40 40 40 a d d itio n a l d ilu tio n 1 1 1 1 1 1 10 10 10 versus extracted curve rerun 3M Sam ple Number Sam ple D escription Inst. File Name e01 -1256-36250 Q C -2 5 p p b -1 (1004 ng/m L diluted sera) d020405036 e01-1256-36251 Q C -2 5 p p b -2 (1004 ng/mL diluted sera) d020405037 e01-1256-36252 Q C -2 5 p p b -3 (1004 ng/mL diluted sera) d020405038 e01-1256-36253 Q C -2 50 p p b-1 (10040 ng/mL diluted sera) d020405039 e 0 1-1256-36254 Q C - 250ppb -2 (10040 ng/m L diluted sera) d020405040 e01-1256-36255 Q C- 250ppb -3 (10040 ng/mL diluted sera) d020405041 e01-1256-36256 Q C - 4.0 ppm -1 (160640 ng/m L diluted sen d020405042 e01-1256-36257 Q C- 4.0 ppm -2 (160640 ng/mL diluted sen d020405043 e01-1256-36258 QC- 4.0 ppm -3 (160640 ng/mL diluted sen d020405044 A = N ot within +1-25% criteria am ount of sera used (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 d ilu tio n ( d ilu te d sera) 40 40 40 40 40 40 40 40 40 a d d itio n a l d ilu tio n 1 1 1 1 1 1 10 10 10 Cone of PFO S in Sam ple (ng/m L) = (Inst conc(ng/m L)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volum e(m L)/am ount of sera in diluted sample(mL) = 2.0 mL/0.05 mL = 40 am ou n t o f sera in diluted sa m ple (mL) = am ount o f sera used/total am ount o f sera + water (1/20 dilution). = 1/20 = 0 .05 m L Absolute Recoveries Inst Cone. (ng/m L of M ethanol) 48.29 39.92 45.30 288.00 294.03 293.10 523.44 432.18 415.07 Cone, of PFOS in Sam ple (ng/m L of Sera) 1932 1597 1812 11520 11761 11724 209376 172872 166028 A A A A for all QC sam ples (absolute recovery) avg % rec 135.8 std dev o f % rec 33.11 % Rec 192.4 159.0 180.5 114.7 117.1 116.8 130.3 107.6 103.4 QC Sample Recoveries Inst Cone. (ng/m L of M ethanol) Cone, of PFOS in Sample (ng/m L of Sera) 39.15 32.41 37.84 246.03 252.86 238.51 320.09 364.90 335.08 1566 1296 1514 9841 10114 9540 128036 145960 134032 A A A % Rec 156.0 129.1 150.8 98.02 100.7 95.02 79.70 90.86 83.44 fo ra li QC sam ples original analysis (recovery) avg % rec std dev o f % rec 109.3 28.68 Inst Cone. (ng/m L of M ethanol) 43.07 35.30 40.31 264.06 269.60 268.75 479.65 395.97 380.39 Cone, of PFOS in Sam ple (ng/m L o f Sera) 1723 1412 1612 10562 10784 10750 191860 158388 152156 A A A % Rec 171.6 140.6 160.6 105.2 107.4 107.1 119.4 98.60 94.72 for all QC sam ples rerun analysis (recovery) avg % rec 122.8 std dev o f % rec 28.08 avg 177.3 s td d e v 16.90 %RSD 9.531 avg 116.2 s td d e v 1.293 % RSD 1.113 avg 113.8 s td d e v 14.51 %RSD 12.75 avg 145.3 s td d e v 14.24 %RSD 9.800 avg 97.93 s td d ev 2.860 %RSD 2.920 avg 84.67 s td d ev 5.680 %RSD 6.708 avg 157.6 s td dev 15.69 %RSD 9.957 avg 106.6 s td d e v 1.189 % RSD 1.115 avg 104.3 s td dev 13.29 %RSD 12.75 3M Environmental Laboratory R la n k c n ^ e an rl M .Q c Page 48 Report E01-1256 _ Study number EOI-1256 - M LL h D SERA PFOS: A Reproduction Study with the Mallard 3M Sample Sample Description E01-1256-28663 454-109-3218, 0 PPM, Female, Adult E01-1256-28665 454-109-3220,0 PPM, Female, Adult E01-1256-28667 454-109-3222, 0 PPM, Female, Adult E01-1256-28669 454-109-3224, 0 PPM, Female, Adult E01-1256-28670 454-109-3226, 0 PPM, Female, Adult E01-1256-28672 454-109-3228,0 PPM, Femaie, Adult E01-1256-28674 454-109-3230, 0 PPM, Female, Adult E01-1256-28676 454-109-3232, 0 PPM, Female, Adult E01-1256-28678 454-109-3234, 0 PPM, Female, Adult E01-1256-28679 454-109-3236, 0 PPM, Female, Adult E01-1256-28681 454-109-3238, 0 PPM, Female, Adult E01-1256-28683 454-109-3240, 0 PPM, Female, Adult E01-1256-28685 454-109-3242, 0 PPM, Female, Adult E01-1256-28687 454-109-3244, 0 PPM, Female, Adult E01-1256-28689 454-109-3246, 0 PPM, Female, Adult E01-1256-28691 454-109-3248, 0 PPM, Female, Adult E01-1256-28693 454-109-3250, 0 PPM, Female, Adult E01-1256-28695 454-109-3252, 0 PPM, Female, Adult E01-1256-28697 454-109-3254, 0 PPM, Female, Adult E01-1256-28699 454-109-3256, 0 PPM, Female, Adult E01-1256-28701 454-109-3258, 10 PPM, Female, Adult E01-1256-28703 454-109-3260, 10 PPM, Female, Adult E01-1256-28705 454-109-3262, 10 PPM, Female, Adult E01-1256-28707 454-109-3264, 10 PPM, Female, Adult E01-1256-28709 454-109-3266, 10 PPM, Female, Adult E01 -1256-28711 454-109-3268,10 PPM, Female, Adult E01-1256-28713 454-109-3270,10 PPM, Female, Adult E01-1256-28715 454-109-3272, 10 PPM, Female, Adult E01-1256-28717 454-109-3274, 10 PPM, Female, Aduit E01-1256-28719 454-109-3276,10 PPM, Female, Adult E01-1256-28721 454-109-3278, 10 PPM, Female, Adult E01-1256-28723 454-109-3280, 10 PPM, Female, Adult E01-1256-28725 454-109-3282,10 PPM, Female, Adult E01-1256-28727 454-109-3284,10 PPM, Female, Adult E01-1256-28729 454-109-3286,10 PPM, Female, Adult E01-1256-28731 454-109-3288,10 PPM, Female, Adult E01-1256-28733 454-109-3290,10 PPM, Female, Adult E01-1256-28735 454-109-3292,10 PPM, Female, Adult E01-1256-28737 454-109-3294,10 PPM, Female, Adult E01-1256-28739 454-109-3296,10 PPM, Female, Adult E01-1256-28740 454-109-9583, 0 PPM, Female, Offspring E01-1256-28742 454-109-9589,0 PPM, Female, Offspring E01-1256-28743 454-109-9592, 0 PPM, Female, Offspring E01-1256-28744 454-109-9598, 0 PPM, Female, Offspring E01-1256-28745 454-109-9603, 0 PPM, Female, Offspring E01-1256-28747 454-109-9610,0 PPM, Female, Offspring E01-1256-28748 454-109-9573, 0 PPM, Female, Offspring E01-1256-28751 454-109-9629,10 PPM, Female, Offspring E01-1256-28752 454-109-9633,10 PPM, Female, Offspring E01-1256-28753 454-109-9634,10 PPM, Female, Offspring E01-1256-28754 454-109-9645,10 PPM, Female, Offspring E01-1256-28755 454-109-9648,10 PPM, Female, Offspring E01-1256-28758 454-109-9654,10 PPM, Female, Offspring E01-1256-28759 454-109-9659,10 PPM, Female, Offspring * Concentration <LOQ Inst. File d020225047 d020225049 d020225051 d020225053 d020225054 d020225061 d020225063 d020225065 d020226050 d020226051 d020226052 d020226053 d020226054 d020226055 d020226056 d020226057 d020226058 d020226064 d020226065 d020226066 d020226067 d020226068 d020226069 d020226070 d020226071 d020228031 d020228032 d020228033 d020405045 d020228035 d020228036 d020308048 d020228038 d020228039 d020308049 d020228044 d020228045 d020228046 d020228047 d020228048 d020228114 d020228115 d020228116 d020228117 d020228118 d020228119 d020228120 d020228121 d020228122 d020228123 d020228127 d020228128 d020228129 d020228130 sera used (m L ) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.15 0.2 0.2 0.1 0.2 0.2 0.05 0.1 0.1 0.5 0.1 0.2 0.1 0.1 dilution (diluted 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 additional 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 10 10 1 10 1 1 10 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Cone of PFOS in Sample (ng/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilution(diluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mL/0.05 m L * 40 amount of sera in diluted sample (mL) = amount of sera used/total amount of sera + water (1/20 dilution). = 1/20 --0.05 mL (ng/mL of M e th a n o l) 1.90 1.00 2.21 1.00 1.53 66.94 1.00 4.30 2.51 2.51 4.16 2.51 2.51 2.51 2.51 9.96 2.51 18.92 2.51 2.51 340.30 235.54 425.04 158.72 197.38 168.16 228.48 192.81 73.64 101.64 245.98 80.62 268.84 164.14 72.23 106.99 400.80 260.05 191.39 270.66 2.51 2.51 2.51 2.51 2.51 2.51 2.51 128.28 85.41 168.96 93.74 122.59 136.20 133.73 Sample (ng/mL of Sera) control sa m ple s 76.0 40.0 88.4 40.0 61.2 2678 40.0 172 100.4 100.4 166 100.4 100.4 100.4 100.4 398 100.4 757 100.4 100.4 40.0 * 40.0 40.0 * 40.0 40.0 40.0 rejected * 40.0 40.0 * 100.4 * 100.4 100.4 * 100.4 * 100.4 * 100.4 * 100.4 100.4 rejected * 100.4 100.4 rejected * 100.4 * 100.4 With outliers avg 271 std dev 589 %RSD 218 Without outliers avg 93.4 std dev 37.0 %RSD 39.6 13612 9422 17002 6349 7895 6726 91392 7712 29456 40656 9839 32248 10754 6566 28892 42796 16032 10402 7656 10826 100.4 100.4 100.4 100.4 100.4 100.4 100.4 rejected With outliers avg 20312 std dev 20385 %RSD 100 Without outliers avg 16571 std dev 11965 %RSD 72.2 * 100.4 * 100.4 * 100.4 * 100.4 100.4 100.4 avg 100.4 std dev 0 * 100.4 %RSD - 5131 3416 6758 3750 4904 5448 5349 avg 4965 std dev 1118 %RSD 22.5 3M Environmental Laboratory Page 49 Report E01-1256 ______ Study number EOI-1256 - MALLARD SERA PFOS: A Reproduction Study with the Mallard 3M Sample Number Sample Description E01 -1256-28662 454-109-3217, 0 PPM, Male, Adult E01-1256-28664 454-109-3219,0 PPM, Male, Adult E01-1256-28666 454-109-3221,0 PPM, Male, Adult E01-1256-28668 454-109-3223, 0 PPM, Male, Adult E01-1256-28671 454-109-3227,0 PPM. Male. Adult E01-1256-28673 454-109-3229, 0 PPM, Male, Adult E01-1256-28675 454-109-3231,0 PPM, Male, Adult E01-1256-28677 454-109-3233, 0 PPM, Male, Adult E01-1256-28680 454-109-3237, 0 PPM, Male, Adult E01-1256-28682 454-109-3239, 0 PPM, Male, Adult E01-1256-28684 454-109-3241, 0 PPM, Male, Adult E01-1256-28686 454-109-3243, 0 PPM, Male, Adult E01-1256-28688 454-109-3245, 0 PPM, Male, Adult E01-1256-28690 454-109-3247, 0 PPM, Male, Adult E01-1256-28692 454-109-3249, 0 PPM, Male, Adult E01-1256-28694 454-109-3251, 0 PPM, Male, Adult E01-1256-28696 454-109-3253, 0 PPM, Male, Adult E01-1256-28698 454-109-3255, 0 PPM, Male, Adult E01-1256-28700 454-109-3257, 10 PPM, Male, Adult E01-1256-28702 454-109-3259, 10 PPM, Male, Adult E01-1256-28704 454-109-3261, 10 PPM, Male, Adult E01-1256-28706 454-109-3263, 10 PPM, Male, Adult E01-1256-28708 454-109-3265, 10 PPM, Male, Adult E01-1256-28710 454-109-3267, 10 PPM, Male, Adult E01-1256-28712 454-109-3269,10 PPM, Male, Adult E01-1256-28714 454-109-3271,10 PPM, Male, Adult E01-1256-28716 454-109-3273,10 PPM, Male, Adult E01-1256-28718 454-109-3275,10 PPM, Male, Adult E01-1256-28720 454-109-3277,10 PPM, Male, Adult E01-1256-28722 454-109-3279,10 PPM, Male, Adult E01-1256-28724 454-109-3281,10 PPM, Male, Adult E01-1256-28726 454-109-3283, 10 PPM, Male, Adult E01-1256-28728 454-109-3285,10 PPM, Male, Adult E01 -1256-28730 454-109-3287,10 PPM, Male, Adult E01-1256-28732 454-109-3289, 10 PPM, Male, Adult E01-1256-28734 454-109-3291,10 PPM, Male, Adult E01-1256-28736 454-109-3293,10 PPM, Male, Adult E01-1256-28738 454-109-3295,10 PPM, Male, Adult E01 -1256-28741 454-109-9591, 0 PPM, Male, Offspring E01-1256-28746 454-109-9606, 0 PPM, Male, Offspring E01-1256-28749 454-109-9578, 0 PPM, Male, Offspring E01-1256-28750 454-109-9625,10 PPM, Male, Offspring E01-1256-28756 454-109-9636,10 PPM, Male, Offspring E01-1256-28757 454-109-9642,10 PPM, Male, Offspring * Concentration <LOQ Inst. File Name d020225046 d020225048 d020225050 d020225052 d020225060 d020225062 d020225064 d020226034 d020226035 d020226036 d020226037 d020226038 d020226039 d020226040 d020226041 d020226042 d020226043 d020226049 d020301106 d020301107 d020301108 d020411024 d020301110 d020228049 d020228050 d020228051 d020228052 d020228053 d020228057 d020228058 d020228059 d020228060 d020228061 d020228062 d020228063 d020228064 d020228065 d020228066 d020228131 d020228132 d020228133 d020228134 d020228135 d020228136 amount of dilution (diluted sera used (mL) sera) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.3 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.1 0.5 0.5 0.2 0.5 0.06 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.3 0.3 0.3 0.5 0.5 0.1 0.2 0.2 0.2 0.1 0.2 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 additional d ilu tio n 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 10 10 100 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 1 1 1 1 Inst Cone. (ng/mL of M e th a no l) 2.68 1.49 1.00 1.07 1.99 1.00 1.20 2.51 2.51 2.51 34.88 2.51 2.51 4.05 26.82 2.51 2.51 2.51 196.96 208.27 171.66 52.51 234.38 189.30 201.59 28.18 265.08 201.27 201.00 258.46 185.77 229.40 269.40 196.75 244.83 183.85 242.13 249.20 2.51 2.57 2.51 136.31 91.65 102.66 Cone, of PFOS in Sample (ng/mL of Sera) loq for control sa m ple s 107 59.6 40.0 42.8 79.6 40.0 48.0 100.4 100.4 100.4 1395 100.4 100.4 162 1073 100.4 100.4 100.4 78784 83308 68664 210040 93752 75720 80636 11272 106032 80508 80400 103384 74308 91760 107760 78700 97932 73540 96852 99680 100.4 103 100.4 5452 3666 4106 40.0 40.0 * 40.0 40.0 40.0 * 40.0 40.0 * 100.4 * 100.4 * 100.4 100.4 * 100.4 * 100.4 100.4 100.4 * 100.4 * 100.4 * 100.4 avg 214 std dev 376 %RSD 176 rejected rejected With outliers avg 89652 std dev 35035 %RSD 39.1 Without outliers avg 87318 std dev 12337 %RSD 14.1 * 100.4 avg 101.2 100.4 * 100.4 std dev 1.39 %RSD 1.37 avg 4408 std dev 931 %RSD 21.1 Cone of PFOS in Sample (ng/mL) = (Inst conc(ng/mL)) x (additional dilution) x (dilutionfdiluted sera)) dilution(diluted sera) = final volume(mL)/amount of sera in diluted sample(mL) = 2.0 mU0.05 mL = 40 amount of sera in diluted sample (mL) = amount of sera used/total amount of sera + water (1/20 dilution). = 1/20 = 0.05 mL 3M Environmental Laboratory Page 50 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD LIVER DATA SUMMARY, BLANKS AND SPIKES 3M Sample Number Sample D escription E01-1326-36778 Blank H20-day 1 E 0M 326-36778 Blank H20-dav 1 E01-1326-36807 Blank H20-day 2 E01-1326-36812 Blank H20-day 3 E01-1326-36817 Blank H20-day 4 E01-1326-36780 Blank mallard liver-day 1 E01-1326-36780 Blank mallard liver-day 1 E01-1326-36809 Blank mallard liver-day 2 E01-1326-36814 Blank mallard liver-day 3 E01-1326-36819 Blank mallard liver-day 4 Inst. File Name d020304037 d020305036 d020306024 d020306104 d020307024 am ount of liver w eighed (g) or water aliquoted (mL) 1.00 1.00 1.00 1.00 1.00 Water Added for Hom genizing (mL) 9.00 9.00 9.00 9.00 9.00 Final Volum e (mL) 2.00 2.00 2.00 2.00 2.00 Am ount of liver in d ilu te d sam ple (g) or water (mL) 0.100000 0.100000 0.100000 0.100000 0.100000 a d d itio n a l d ilu tio n 1 1 Inst Cone. (ng/mL Cone, o f PFOS in Sample (ng/g of of Methanol) Liver) loq for control sam ple s 2.51 1.00 2.51 2.51 2.51 50.2 20.0 50.2 50.2 50.2 * 50.2 * 20.0 * 50.2 * 50.2 * 50.2 d020304038 d020305037 d020306025 d020306105 d020307025 5.0597 5.0597 5.0597 5-0597 5.0597 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 0.101073 0.101073 0.101073 0.101073 0.101073 1 1 1 1 1 2.51 1.00 2.51 2.51 2.51 50.2 20.0 50.2 50.2 50.2 50.2 20.0 50.2 50.2 50.2 | 3M Sample Number Sample Description E01-1326-36781 Mallard Liver MS-day 1 E01-1326-36781 Mallard Liver MS-day 1 E01-1326-36810 Mallard Liver MS-day 2 E01-1326-36815 Mallard Liver MS-day 3 E01-1326-36820 Mallard Liver MS-day 4 Inst. File Name d020304039 d020305038 d020306026 d020306106 d020307026 am ount of liver w eighed (g) 5.0597 5.0597 5.0597 5.0597 5.0597 Water Added fo r H o m g e n iz in g (mL) 45.0 45.0 45.0 45.0 45.0 Final Volum e (mL) 2.00 2.00 2.00 2.00 2.00 A m ount o f liver in diluted sam ple (g) 0.101073 0.101073 0.101073 0.101073 0.101073 a d d itio n a l d ilu tio n 1 1 1 1 1 Inst Cone. (ng/mL of M ethanol) 28.50 26.91 27.95 27.72 27.84 Cone, o f PFOS in Sample (ng/g of Liver) 564 532 553 549 551 E01-1326-36782 Mallard Liver MSD-day 1 E01-1326-36782 Mallard Liver MSD-day 1 E01-1326-36811 Mallard Liver MSD-day 2 E01-1326-36816 Mallard Liver MSD-day 3 E01-1326-36821 Mallard Liver MSD-day 4 * Concentration <LOQ d020304040 d020305039 d020306027 d020306107 d020307027 5.0597 5-0597 5.0597 5.0597 5.0597 45.0 45.0 45.0 45.0 45.0 2.00 2.00 2.00 2.00 2.00 0.101073 0.101073 0.101073 0.101073 0.101073 1 1 1 1 1 26.86 26.18 26.20 27.50 27.68 531 518 518 544 548 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) for both ms and msd avg %rec std dev of %rec avg cone std dev of cone 108.8 3.059 540.9 15.20 % Rec 113.5 107.1 111.3 110.4 110.8 true vai. 497 avg % rec 110.6 avg 549.8 std dev 11.33 106.9 104.2 104.3 109.5 110.2 true vai. 497 avg %rec 107.0 avg 532.0 std dev 13.91 3M Environmental Laboratory Page 51 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD LIVER DATA SUMMARY versus unextracted curve 3M Sample Number _ . _ . x. Sample D escription Inst. File Name am ount of liver w eighed (g) Water Added for H o m g e n izin g (mL) E01-1326-36798 Q C -25ppb -1 (497 ng/g homogenate) E01-1326-36799 QC- 25ppb -2 (497 ng/g homogenate) E01-1326-36800 QC- 25ppb -3 (497 ng/g homogenate) E01-1326-36801 Q C -250ppb-1 (4967 ng/g homogenate) E01-1326-36802 QC- 250ppb -2 (4967 ng/g homogenate) E01-1326-36803 QC- 250ppb -3 (4967 ng/g homogenate) d020305045 d020305046 d020305047 d020305048 d020305049 d020305050 5.0597 5.0597 5.0597 5.0597 5.0597 5.0597 45.0 45.0 45.0 45.0 45.0 45.0 E01-1326-36804 QC- 4.0 ppm -1 (79467 ng/g homogenate) E01-1326-36805 QC- 4.0 ppm -2 (79467 ng/g homogenate) E01-1326-36806 QC- 4.0 ppm -3 (79467 ng/g homogenate) d020405065 d020405066 d020405067 5.0597 5.0597 5.0597 45.0 45.0 45.0 A = Not within +/-25% criteria Final Volume (mL) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Absolute Recoveries A m ount of liver in diluted sam ple (g) 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 a d d itio n a l d ilu tio n 1 1 1 1 1 1 10 10 10 Cone, of PFOS in Inst Cone. (ng/mL Sample (ng/g of of Methanol) Liver) 28.87 571 29.22 35.74 578 707 A 242.46 249.01 4798 4927 278.56 5512 440.20 87105 443.16 436.91 87691 86454 % Rec. 114.9 116.3 142.3 96.59 99.20 111.0 109.6 110.3 108.8 tor atl QC samples (absolute recoveiy) avg %rec std dev of %rec 112.1 13.07 avg 124.5 s td dev 15.41 %RSD 12.37 avg 102.3 std dev 7.662 %RSD 7.493 avg 109.6 std dev 0.7785 %RSD 0.7104 Study number E01-1256 - MALLARD LIVER DATA SUMMARY versus extracted curve 3M Sample .. . Number _ ._ . Sample Description E01-1326-36798 Q C -25ppb -1 (497 ng/g homogenate) E01-1326-36799 QC- 25ppb -2 (497 ng/g homogenate) E01-1326-36800 QC- 25ppb -3 (497 ng/g homogenate) E01-1326-36801 Q C -250ppb-1 (4967 ng/g homogenate) E01-1326-36802 QC- 250ppb -2 (4967 ng/g homogenate) E01-1326-36803 QC- 250ppb -3 (4967 ng/g homogenate) E01-1326-36804 Q C -4 .0 p p m -1 (79467 ng/g homogenate) E01-1326-36805 QC- 4.0 ppm -2 (79467 ng/g homogenate) E01-1326-36806 QC- 4.0 ppm -3 (79467 ng/g homogenate) A = Not within +/-25% criteria Inst. File Name d020305045 d020305046 d020305047 d020305048 d020305049 d020305050 d020405065 d020405066 d020405067 am ount of liver weighed (g) 5.0597 5.0597 5.0597 5.0597 5.0597 5.0597 5.0597 5.0597 5.0597 Water Added for H o m g e n iz in g (mL) 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 45.0 Final Volume (mL) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Cone of PFOS in Sample (ng/g) = (Inst conc(ng/mL)) x (additional dilution) x (final volume (mL))/ amount of liver in diluted sample (g) amount of liver in diluted sample (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) QC Sample Recoveries A m ount of liver in diluted sam ple (g) 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 0.101073 a d d itio n a l d ilu tio n 1 1 1 1 1 1 10 10 10 Inst Cone. (ng/mL Cone, of PFOS in of Methanol) Sample (ng/g) 25.41 25.71 31.24 221.45 228.29 260.35 415.43 418.16 412.40 503 509 618 4382 4517 5152 82204 82744 81604 for all QC samples (recovery) avg%rec 102.3 stddevof% rec 10.14 % Rec. 101.2 102.4 124.4 88.22 90.95 103.7 103.4 104.1 102.7 avg 109.3 std dev 13.07 %RSD 11.96 avg 94.30 std dev 8.274 %RSD 8.774 avg 103.4 std dev 0.7175 %RSD 0.6937 3M Environmental Laboratory Page 52 Report E01-1256 Study number E01-1256 - MALLARD LIVER DATA SUMMARY 3M Sam ple Number Inst. File Sample Description Name E01-1326-29482 0 ppm a.i. 3218 F Liver E01-1326-29583 0 ppm a.i. 3220 F Liver E01-1326-29601 E01-1326-29619 0 ppm a.i. 3222 F Liver 0 ppm a.i. 3224 F Liver E01-1326-29637 0 Dpm a.i. 3226 F Liver E01-1326-29655 0 ppm a.i. 3228 F Liver E01-1326-29673 0 ppm a.i. 3230 F Liver E01-1326-29691 0 ppm a.i. 3232 F Liver E01-1326-29709 0 ppm a.i. 3234 F Liver E01-1326-29727 0 ppm a.i. 3236 F Liver E01-1326-29745 0 ppm a.i. 3238 F Liver E01-1326-29763 0 ppm a.i. 3240 F Liver E01-1326-29781 0 ppm a.i. 3242 F Liver E01-1326-29799 0 ppm a.i. 3244 F Liver E01-1326-29817 0 ppm a.i. 3246 F Liver E01-1326-29835 0 ppm a.i. 3248 F Liver E01-1326-29853 0 ppm a.i. 3250 F Liver E01-1326-29871 0 ppm a.i. 3252 F Liver E01-1326-29889 0 ppm a.i. 3254 F Liver E01-1326-29907 0 ppm a.i. 3256 F Liver E01-1326-29925 10 ppm a.i. 3258 F Liver E01-1326-29943 10 ppm a.i. 3260 F Liver E01-1326-29961 10 ppm a.i. 3262 F Liver E 01-1326-29980 10 ppm a.i. 3264 F Liver E01-1326-29998 10 ppm a.i. 3266 F Liver E01-1326-30016 10 ppm a.i. 3268 F Liver E01-1326-30034 10 ppm a.i. 3270 F Liver E01-1326-30052 10 ppm a.i. 3272 F Liver E01-1326-30070 10 ppm a.i. 3274 F Liver E01-1326-30088 10 ppm a.i. 3276 F Liver E01-1326-30106 10 ppm a.i. 3278 F Liver E01-1326-30124 10 ppm a.i. 3280 F Liver E01-1326-30142 10 ppm a.i. 3282 F Liver E01-1326-30160 10 ppm a.i. 3284 F Liver E01-1326-30178 10 ppm a.i. 3286 F Liver E01-1326-30196 10 ppm a.i. 3288 F Liver E01-1326-30214 10 ppm a.i. 3290 F Liver E01-1326-30232 10 ppm a.i. 3292 F Liver E01-1326-30250 10 ppm a.i. 3294 F Liver E01-1326-30268 10 ppm a.i. 3296 F Liver E01-1367-31295 0 ppm a.i. 9583 F Liver E01-1367-31307 0 ppm a.i. 9589 F Liver E01-1367-31313 0 ppm a.i. 9592 F Liver E01-1367-31319 0 ppm a.i. 9598 F Liver E01-1367-31325 0 ppm a.i. 9603 F Liver E01-1367-31337 0 ppm a.i. 9610 F Liver E01-1367-31343 0 ppm a.i. 9573 F Liver E01-1367-31362 10 ppm a.i. 9629 F Liver E01-1367-31368 10 ppm a.i. 9633 F Liver E01-1367-31374 10 ppm a.i. 9634 F Liver E01-1367-31380 E01-1367-31386 10 ppm a.i. 9645 F Liver 10 ppm a.i. 9648 F Liver E01-1367-31404 10 ppm a.i. 9654 F Liver E01-1367-31410 10 DDm a.i. 9659 F Liver * Concentration <LOQ d020305059 d020305060 d020305061 d020305062 d020305063 d020305064 d020305065 d020306031 0020306032 d020306033 d020306034 d020306035 d020306036 d020306037 d020306038 d020306039 d020306040 d020306044 d020306045 d020306046 d020306066 d020306067 d020307057 d 0 2 0 3 0 6 1 11 d020306112 d020306113 d020307059 d020306115 d020307060 d020307061 d020306118 d020307062 d020306120 d020306124 d020307063 d020307064 d020307065 d020306128 d020306129 d020306130 d020307031 d020307032 d020307033 d020307034 d020307035 d020307036 d020307037 d020307044 d020307045 d020307046 d020307047 d020307048 d020307049 d020307050 am ount of liver weighed (g) W ater Added for Hom geniiing (m L) 1.0095 1.0944 1.0435 1.0425 1,0703 1.0526 1.0411 1.1704 1.1290 1.0800 1.1170 1.0086 1.0907 1.0291 1.1294 1.1075 1.1709 1.0761 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 1.1344 1.0669 9.00 9.00 1.0034 1.1331 1.1294 1.1829 9.00 9.00 9.00 9.00 1.0487 1.1214 9.00 9.00 1.0751 1.0658 1.1226 1.0953 9.00 9.00 9.00 9.00 1.1956 1.0600 9.00 9.00 1.0275 1.0915 1.0657 9.00 9.00 9.00 1.0330 9.00 1.0352 9.00 1.0658 1.0627 9.00 9.00 1.1238 9.00 1.1308 1.0005 9.00 9.00 1.0390 1.1204 9.00 9.00 1.1550 1.0217 9.00 9.00 1.0122 9.00 1.1676 1.0510 1.1638 9.00 9.00 9.00 1.1510 9.00 1.1125 1.0770 9.00 9.00 1.0581 9.00 Final Volume (mL) Am ount of liver in d iluted sam ple (g) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.100854 0.108417 0.103898 0.103809 0.106283 0.104709 0.103684 0.115079 0.111462 0.107143 0.110408 0.100773 0.108090 0.102611 0.111497 0.109572 0.115123 0.106797 0.111936 0.105981 0.100306 0.111822 0.111497 0.116165 0.104362 0.110795 0.106709 0.105883 0.110900 0.108496 0.117266 0.105368 0.102468 0.108160 0.105874 0.102960 0.103157 0.105883 0.105608 0.111006 0.111620 0.100045 0.103496 0.110707 0.113737 0.101949 0.101097 0.114835 0.104567 0.114504 0.113388 0.110012 0.106877 0.105199 a d d itio n a l d ilu tio n 1 1 1 1 1 1 1 1 1 10 1 1 1 10 1 10 10 1 1 1 1 10 10 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 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 sam ple (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) 3MEnvironmental Laboratory PFOS: A Reproduction Study with the Mallard Cone, o f PFOS in Inst Cone. (ng/mL Sample (ng/g of of Methanol) Liver) loq for control sam ples 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 355.07 379.32 76.07 352.91 280.63 317.25 70.37 307.09 76.85 166.74 349.71 130.88 233.84 342.26 149.38 124.22 101.98 243.62 251.58 351.37 2.51 2.51 2.51 2.51 2.51 2.51 2.51 169.63 117.36 258.53 146.41 192.68 316.88 188.42 20.0 20.0 20.0 20.0 20.0 20.0 20.0 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 7080 6784 13645 6076 5378 5727 13189 5801 13859 30737 5964 2484 4564 6329 28218 24130 19772 4602 4764 6331 50.2 50.2 50.2 50.2 50.2 50.2 50.2 2954 2245 4516 2582 3503 5930 3582 20.0 20.0 20.0 20.0 20.0 20.0 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 * 50.2 " 50.2 * 50.2 * 50.2 " 50.2 avg 39.6 std dev 14.8 %RSD 37.3 avg 10772 std dev 8463 % RSD 78.6 avg 50.2 std dev 0 %RSD - avg 3616 std dev 1263 %RSD 34.9 Page 53 Report E01-1256 PFOS: A Reproduction Study with the Mallard Study number E01-1256 - MALLARD LIVER DATA SUMMARY________________________ 3M Sample Number Inst. File Sam ple Description Name E01-1326-29473 0 ppm a.i. 3217 M Liver E01-1326-29574 0 ppm a.i. 3219 M Liver E01-1326-29592 0 ppm a.i. 3221 M Liver E01-1326-29610 0 ppm a.i. 3223 M Liver E01-1326-29628 0 ppm a.i. 3225 M Liver E01-1326-29646 0 ppm a.i. 3227 M Liver E 0 M 3 2 6 -2 9 6 6 4 0 ppm a.i. 3229 M Liver E 0 M 32 6-2 9682 0 ppm a.i. 3231 M Liver E01-1326-29700 0 ppm a.i. 3233 M Liver E01-1326-29718 0 ppm a.i. 3235 M Liver E01-1326-29736 0 ppm a.i. 3237 M Liver E01-1326-29754 0 ppm a.i. 3239 M Liver E01-1326-29772 0 ppm a.i. 3241 M Liver E01-1326-29790 0 ppm a.i. 3243 M Liver E01-1326-29808 0 ppm a.i. 3245 M Liver E01-1326-29826 0 ppm a.i. 3247 M Liver E01-1326-29844 0 ppm a.i. 3249 M Liver E01-1326-29862 0 ppm a.i. 3251 M Liver E01-1326-29880 0 ppm a.i. 3253 M Liver E01-1326-29898 0 ppm a.i. 3255 M Liver E01-1326-29916 10 ppm a.i. 3257 M Liver E01-1326-29934 10 ppm a.i. 3259 M Liver E01-1326-29952 10 ppm a.i. 3261 M Liver E01-1326-29970 10 ppm a.i. 3263 M Liver E01-1326-29989 10 ppm a.i. 3265 M Liver E01-1326-30007 10 ppm a.i. 3267 M Liver E01-1326-30025 10 ppm a.i. 3269 M Liver E01-1326-30043 10 ppm a.i. 3271 M Liver E01-1326-30061 10 ppm a.i. 3273 M Liver E01-1326-30079 10 ppm a.i. 3275 M Liver E01-1326-30097 10 ppm a.i. 3277 M Liver E 0 M 3 2 6 -3 0 1 1 5 10 ppm a.i. 3279 M Liver E01-1326-30133 10 ppm a.i. 3281 M Liver E01-1326-30151 10 ppm a.i. 3283 M Liver E01-1326-30169 10 ppm a.i. 3285 M Liver E01-1326-30187 10 ppm a.i. 3287 M Liver E01-1326-30205 10 ppm a.i. 3289 M Liver E01-1326-30223 10 ppm a.i. 3291 M Liver E01-1326-30241 10 ppm a.i. 3293 M Liver E01-1326-30259 10 ppm a.i. 3295 M Liver E01-1367-31301 0 ppm a.i. 9591 M Liver E01-1367-31331 0 ppm a.i. 9606 M Liver E01-1367-31349 0 ppm a.i. 9578 M Liver E01-1367-31356 10 ppm a.i. 9625 M Liver E01-1367-31392 10 ppm a.i. 9636 M Liver E01-1367-31398 10 ppm a.i. 9642 M Liver * Concentration <LOQ d020305071 d020305072 d020305073 d020305074 0020305075 d020305076 d020305077 d020306047 d020306048 d020306049 d020306050 d020306051 d020306052 d020306053 d020306057 d020306058 d020306059 0020306060 d020306061 d020306062 d020306069 d020306070 d020306071 d020307058 d020306131 d020306132 d020306133 d020306137 d020306138 d020306139 d020306140 d020306141 d020306142 d020306143 d020306144 d020306145 d020306146 d020306150 d020306151 d020306152 d020307038 d020307039 d020307040 0020307051 d020307052 d020307053 am ount of liver w eighed (g) W ater Added fo r H o m g e n iz in g (mL) 1.1169 1.1847 1.1181 1.0788 1.1043 9.00 9.00 9.00 9.00 900 1.0486 9.00 1.0336 9.00 1.0165 1.0395 9.00 9.00 1.0373 1.1679 9.00 9.00 1.1493 1.0619 9.00 9.00 1.0263 1.1541 9.00 9.00 1.0813 1.0263 9.00 9.00 1.0088 1.0294 9.00 9.00 1.0926 9.00 1.0654 1.1316 9.00 9.00 1.1330 9.00 1.1404 9.00 1.1480 9.00 1.0620 1.1687 9.00 9.00 1.0313 1.1399 9.00 9.00 1.1143 1.0062 9.00 9.00 1.0380 1.0669 9.00 9.00 1.0955 1.1981 9.00 9.00 1.0500 1.0251 1.0441 1.0979 9.00 9.00 9.00 9.00 1.0815 1.0411 1.1314 9.00 9.00 9.00 1.0413 1.0166 1.1367 1.1252 9.00 9.00 9.00 9.00 Final Volum e (mL) Am ount of liver in diluted sam ple (g) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.110399 0.116322 0.110505 0.107037 0.109290 0.104353 0.103014 0.101483 0.103541 0.103345 0.114861 0.113239 0.105537 0.102361 0.113659 0.107258 0.102361 0.100791 0.102638 0.108258 0.105848 0.111690 0.111813 0.112461 0.113126 0.105546 0.114931 0.102808 0.112417 0.110171 0.100558 0.103407 0.105981 0.108514 0.117483 0.104478 0.102253 0.103952 0.108726 0.107276 0.103684 0.111673 0.103702 0.101492 0.112137 0.111129 a d d itio n a l d ilu tio n 1 1 10 10 10 100 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 1 1 1 1 Cone, o f PFOS in Inst Cone. (ng/mL Sample (ng/g of o f Methanol) Liver) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 381.46 274.77 392.11 70.46 349.14 263.55 332.69 323.76 477.80 302.45 271.03 356.95 264.50 363.85 325.94 274.59 303.06 241.84 131.53 270.60 2.51 2.51 2.51 235.62 123.08 149.00 20.0 20.0 20.0 20.0 20.0 20.0 20.0 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 72077 49202 70137 125306 61726 49940 57894 62983 85005 54906 53905 69038 49915 67061 55487 52564 59276 46529 24195 50449 50.2 50.2 50.2 4643 2195 2682 * * * loq for control sam ples 20.0 20.0 20.0 20.0 20.0 20.0 20.0 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 50.2 avg 39.6 std dev 14.8 %RSD 37.3 avg 60880 std dev 19580 %RSD 32.2 avg 50.2 std dev 0 %RSD avg 3173 std dev 1296 %RSD 40.8 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 sam ple (g) = amount of liver weighed (g)/ (water added for homogenizing (mL=g)) + amount of liver weighed(g)) 3M Environmental Laboratory Page 54 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Atta c h m en t C: S a m ple C h ro m ato g ram s, Calibratio n In fo r m a tio n , a nd In str u m en t Info rm ation 3IM P n \/im n m e n ta l I ahrratrw*\/ 3M Environmental Laboratory Panp 55 n f 915 Page 55 Report E01-1256 Report E01-1256 Methanol Solvent Blanks Solvent Blank 3M Environmental Lab d020226033 Sm (Mn, 1x2) 100n PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 26-Feb-2002, 22:16:16 1: MRM of 1 Channel ES- 6 .7 7 TIC 980 1.10e4 Area %- 3M Fnvirnnm pntal I ahnratnrv 3M Environmental Laboratory Dona rtf O IC Page 56 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M F n v lr n n m p n ta l I a h n r a tn r v 3M Environmental Laboratory P an e ft7 rtf 91ft Page 57 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M F n v /im n m o n ta l I a h n r a tn r v 3M Environmental Laboratory D o n a Aft r t f 01 A Page 58 Report E01-1256 Report E01-1256 Water Blank Samples Blank H20-day 4 3M Environmental Lab d020228107 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.76 7151 davey 070799 01-Mar-2002, 06:19:15 1: MRM o f 1 Channel ESTIC 8.84e3 Area %- T im e 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnv/lirnnmQntal I cthr\ratnr\/ 3M Environmental Laboratory en a o ic Page 59 Report E01-1256 Report E01-1256 Blank H20-day 3 3M Environmental Lab d020228024 Sm (Mn, 1x2) 100"! PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 28-Feb-2002, 14:57:35 1: MRM of 1 Channel ES 6.74 TIO 672 8.90e3 Area i %- 3M F m /irn n m o n ta l I a h n r a tn r v 3M Environmental Laboratory D o /ic fift /if o i c Page 60 Report E01-1256 Report E01-1256 Blank H20-day 1 3M Environmental Lab d020225037 Sm (Mn, 1x2) 10CH PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 25-Feb-2002, 23:49:30 1: MRM of 1 Channel ES 6.77 TIO 709 8.64e3 Area %- O-L' 11` " 1 .............. 1 1111h 1111111111m 11111111111" 1r ri 111" " i " 111111111111 ....... 11111* 11 1111 Time ... 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M F n \/irn n m p n ta l I a h n r a tn r v 3M Environmental Laboratory D o n o 4 r \ f 04 5 Page 61 Report E01-1256 Report E01-1256 Sera Blank Samples Blank H20-day 1 3M Environmental Lab d020225037 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 25-Feb-2002, 23:49:30 1: MRM o f 1 Channel ES6.77 TIC 709 8.64e3 Area %- ' 1 1 .0 0 .......2.00 T im e 3.00 " ' " 4.00 " ' " 5.00 " ' " 6.00 " ' " 7.00 " ' " 8 .0 0 ....... 9.00 " ' 10.00 3M Environmental I ahnratnrv 3M Environmental Laboratory D o c o fo ie Page 62 Report E01-1256 Report E01-1256 Blank mallard sera-day 3 3M Environmental Lab d020228025 Sm (Mn, 1x2) 100--i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 28-Feb-2002, 15:08:42 1: M R M o f 1 Channel ES6.74 TIC 873 1.07e4 Area %- i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory P ane R3 rtf 91R Page 63 Report E01-1256 Report E01-1256 Blank mallard sera-day 1 3M Environmental Lab d020225038 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 26-Feb-2002, 00:00:38 1: MRM of 1 Channel ES- 6-78 TIC 860 1.01e4 Area Time 1.00 1 2.00 ! 3.00 1 4.00 " ' " 5.00 ' 6 .0 0 ....... 7.00 ' 8.00 " ' " 9.00 " ' ' 10.00 3M F n v ir n n m p n ta l I a h n r a tn r v 3M Environmental Laboratory &AA fo-ift Page 64 Report E01-1256 Report E01-1256 Liver Blank Samples Blank mallard male liver-day 4 3M Environmental Lab d020307025 Sm (Mn, 1x2) 100-, PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 07-Mar-2002, 19:39:54 1: MRM of 1 Channel ES672 TIC 933 1,06e4 Area % 0 ' 11....... . 11111. 1111 >i : Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental I ahnratnrv 3M Environmental Laboratory Pone fit; r,f 0-1 Page 65 Report E01-1256 Report E01-1256 Blank mallard liver-day 2 3M Environmental Lab d020306025 Sm (Mn, 1x2) 100- PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard davey 070799 06-Mar-2002, 14:02:19 1: MRM of 1 Channel ES6.72 TIC 908 1.22e4 Area %- If V 111111 i 11111" 11111111111111111111 r r 111111111111111 " , i 111 11, 111111111 i ,, 11 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnvirnnmpntal I ahnratnrv 3M Environmental Laboratory Porte fi rtf o-ifi Page 66 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory P an e 7 nf 91 G Page 67 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Adult Male Sera Samples, Diluted 1:10 3M Environmental I ahoratnrv 3M Environmental Laboratory Pano fift nf 91 fi Page 68 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratorv 3M Environmental Laboratory Page 69 of 216 Page 69 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Lahnratorv 3M Environmental Laboratory Paae 70 of ?1fi Page 70 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M FnvirnnmAntal I ahnratnrv 3M Environmental Laboratory Paae 71 of 21fi Page 71 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Page 72 of 216 Page 72 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Pa no nf 91 Page 73 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Paae 74 of 216 Page 74 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvirnnmental Lahoratorv 3M Environmental Laboratory Paae 75 of 216 Page 75 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvironmantal I ahoratorv 3M Environmental Laboratory Paae 76 of 216 Page 76 Report E01-1256 Report E01-1256 454-109-3267, 10 PPM, Male, Adult,d10 3M Environmental Lab d020228049 Sm (Mn, 1x2) 100-, PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 220589 davey 070799 28-Feb-2002, 19:35:08 1: MRM o f 1 Channel ES- TIC 2.38e6 Area %- ! Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnvirnnm pnfpl I ahnratnrv 3M EnvironmentalLaboratory P ana 77 rtf 91 ft Page 77 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFO S: A R eproduction Study with the Mallard Dosed Group Adult Female Sera Samples 454-109-3280,10 PPM, Female, Adult 3M Environmental Lab d020228037 Sm (Mn, 1x2) 100-1 6.74 487070 davey 070799 28-Feb-2002, 17:21:53 1: MRM of 1 Channel ES TIO 5.03e6 Area 3M Fnvirnnm pntal I ahnratnrv 3M Environmental Laboratory P arto 7ft nf Page 78 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Paae 79 of 216 Page 79 Report E01-1256 Report E01-1256 454-109-3258, 10 PPM, Female, Adult 3M Environmental Lab d020226067 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 360788 davey 070799 27-Feb-2002, 04:34:15 1: MRM of 1 Channel ES TIO 3.86e6 Area %-j 3M Environmental Laboratory 3M Environmental Laboratory Paae 80 of 216 Page 80 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvirnnm ental I ahnratnrv 3M Environmental Laboratory P an e 1 rvf 91ft Page 81 Report E01-1256 Report E01-1256 454-109-3272,10 PPM, Female, Adult 3M Environmental Lab d020228033 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 224019 davey 070799 28-Feb-2002, 16:37:28 1: MRM of 1 Channel ES TIO 2.53e6 Area 1 3M Environmental Lahoratorv 3M Environmental Laboratory Paae 82 of 216 Page 82 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M P n \/irn n m on tal I ah n ratn rw 3M Environmental Laboratory D o n o QQ n f 0 4 C Page 83 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Page 84 of 216 Page 84 Report E01-1256 Report E01-1256 454-109-3264, 10 PPM, Female, Adult 3M Environmental Lab d020226070 Sm (Mn, 1x2) 100- PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 196760 davey 070799 27-Feb-2002, 05:07:36 1: MRM o f 1 Channel ESTIC 2.19e6 Area %- 1.00 2.00 3.00 4.00 5.00 6.00 7.00 3M Fnuirnnm pntal I ah n ratn rv 3M Environmental Laboratory Dana fi rtf 01 Page 85 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 11; rrl i 11 11 .................. 11 ' 1i ^11......... | i " i | | 1'| i | 11 11 | | 11i 11111 111| | | 11 | | ' i | " 11 | | | 11 " 1: i i ime 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 j 3M Environmental Laboratory 3M Environmental Laboratory P a n e ftfi nf 91R Page 86 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental I ahnratorv 3M Environmental Laboratory Paae 87 of ?1fi Page 87 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Male Offspring Sera Samples |454-109-9642, 10 PPM, Male, Offspring 3M Environmental Lab d020228136 Sm (Mn, 1x2) davey 070799 01-Mar-2002, 11:41:03 1: MRM of 1 Channel ES TIO 1.74e6 Area %- 0- F nv/irnnm ontal I ahnratnrv/ 3M Environmental Laboratory h r --............ Time 9.00 10.00 P a n o ft n f Q IR Page 88 Report E01-1256 Report E01-1256 454-109-9625, 10 PPM, Male, Offspring 3M Environmental Lab d020228134 Sm (Mn, 1x2) in n 1UU1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 183965 davey 070799 01-Mar-2002,11:18:49 1: MRM o f 1 Channel ESTIC 2.17e6 Area ! %- i 11it m ..........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 216 Page 89 Report E01-1256 Report E01-1256 454-109-9636, 10 PPM, Male, Offspring 3M Environmental Lab d020228135 Sm (Mn, 1x2) 100- PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 128462 davey 070799 01-Mar-2002,11:29:53 1: MRM o f 1 Channel ESTIC 1.54e6 Area %- " i ' " 1T' n rrr -'-H 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 90 of 216 Page 90 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Female Offspring Sera Samples 454-109-9654, 10 PPM, Female, Offspring 3M Environmental Lab d020228129 Sm (Mn, 1x2) in n 1UU1 6.75 183831 davey 070799 01-Mar-2002,10:23:19 1: MRM of 1 Channel ESTIC 2.20e6 Area % 3M Environmental Laboratory 3M Environmental Laboratory Page 91 of 216 Page 91 Report E01-1256 Report E01-1256 454-109-9659, 10 PPM, Female, Offspring 3M Environmental Lab d020228130 Sm (Mn, 1x2) 100t PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 180868 davey 070799 01-Mar-2002, 10:34:21 1: MRM of 1 Channel ES TIO 2.11e6 Area s %- 0^T " I ' " ' I " " i 1.00 rT~r ~r 2.00 3M Environmental Laboratorv 3M Environmental Laboratory ' a ...... i " ' ^ Time 9.00 10.00 Pane 92 of 216 Page 92 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvimnmfintal I ahnratnrv 3M Environmental Laboratory P an e O'* r \ f 0*1ft Page 93 Report E01-1256 Report E01-1256 454-109-9645, 10 PPM, Female, Offspring 3M Environmental Lab d020228127 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 6.74 _ 131156 davey 070799 01-Mar-2002,10:01:09 1:MRMof 1Channel ES TIO 1.59e6 Area m-p-n il m i y n 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 94 of 216 Page 94 Report E01-1256 Report E01-1256 454-109-9634, 10 PPM, Female, Offspring 3M Environmental Lab d020228123 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 221915 davey 070799 01-Mar-2002, 09:16:50 1: MRMof 1Channel ESTIC 2.59e6 Area % Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnx/m nmental I ahrtratrtrv 3M Environmental Laboratory D o n a OK n f 04 ft Page 95 Report E01-1256 Report E01-1256 454-109-9629, 10 PPM, Female, Offspring 3M Environmental Lab d020228121 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.75 174296 davey 070799 01-Mar-2002, 08:54:44 1: MRM of 1 Channel ESTIC 2.10e6 Area %- r f i i r ip r r Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 96 of 216 Page 96 Report E01-1256 Report E01-1256 454-109*9633, 10 PPM, Female, Offspring 3M Environmental Lab d020228122 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.74 120377 davey 070799 01-Mar-2002, 09:05:46 1: MRM of 1Channel ES TIO 1.50e6 A re a f rrrp -rr Timei' i ! '( i r i ' r - n 'T | 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Page 97 of 216 Page 97 Report E01-1256 Report E01-1256 Dosed Group Male Liver Samples 10 ppm a.l. 3285 M Liver, d10 3M Environmental Lab d020306144 Sm (Mn, 1x2) 100-) PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 308324 davey 070799 07-Mar-2002,12:04:28 1: MRMof 1 Channel ESTIC 3.32e6 Area %- " i 11" i 1 ' 1111111111111111111111Time 1.00 2.00 3.00 4.00 " ' " 5.00 6.00 7.00 8.00 9.00 10.00 I 3M Fnvimnmental I ahnratnrv 3M Environmental Laboratory Dono Q 04ft Page 98 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 3M FnvirnnmAntal I ahnratnrv 3M Environmental Laboratory Pane QQnf 91R Page 99 Report E01-1256 Report E01-1256 10 ppm a.i. 3287 M Liver, d10 3M Environmental Lab d020306145 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 6.72 _ 272903 davey 070799 07-Mar-2002,12:15:37 1: MRMof 1Channel ESTIC 2.40e6 Area ' i 111111 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 Pane 100 of 91fi Page 100 Report E01-1256 Report E01-1256 10 ppm a.i. 3283 M Liver, d10 3M Environmental Lab d020306143 Sm (M n, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 331373 davey 070799 07-Mar-2002,11:53:20 1:MRMof 1Channel ES- TIC 2.88e6 Area i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Fnvlrnnmontal I ahnratnrv 3M Environmental Laboratory Pana 101 nf 91fi Page 101 Report E01-1256 Report E01-1256 10 ppm a.i. 3281 M Liver, d10 3M Environmental Lab d020306142 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 6.72 265374 davey 070799 07-Mar-2002,11:42:12 1:MRMof 1Channel ES TIC 2.84e6 Area 1111111111 I Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnvirnnmontal I ahnratnrv 3M Environmental Laboratory Pano iOOnf 91fi Page 102 Report E01-1256 Report E01-1256 10 ppm a.i. 3279 M Liver, d10 3M Environmental Lab d020306141 Sm (Mn, 1x2) 100*i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 6.73 327374 davey 070799 07-Mar-2002,11:31:06 1: MRMof 1Channel ES- TIC 3.53e6 Area i i I i i i i I i i il I i i i i i i 1.00 2.00 3.00 4.00 5.00 -tt-h Time i.00 7.00 8.00 9.00 10.00 3M Environmental Lahoratorv 3M Environmental Laboratory Pane 103 nf 91R Page 103 Report E01-1256 Report E01-1256 10 ppm a.i. 3277 M Liver, d10 3M Environmental Lab d020306140 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 270271 davey 070799 07-Mar-2002,11:19:58 1: MRMof 1Channel ES TIO 2.44e6 Area %- 1.00 2.00 3.00 ' ' " 4.00..... 5.00...... 6.00 Time 7.00 8.00 9.00 10.00 3M Environmental Laboratorv 3M Environmental Laboratory Pane ir u nf 91 Page 104 Report E01-1256 Report E01-1256 10 ppm a.i. 3273 M Liver, d10 3M Environmental Lab d020306138 Sm (Mn, 1x2) 10Ch PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 6.73 384816 davey 070799 07-Mar-2002,10:57:41 1: MRMof 1Channel ESTIC 3.17e6 Area |*rTi " i 1 t h Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental I ahoratorv 3M Environmental Laboratory Pane 10R of 91R Page 105 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study w ith the M allard 3M FnvirnnmAntal I ahnratnrv 3M Environmental Laboratory Pane IflR n f 91R Page 106 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Pane 107 nf 91R Page 107 Report E01-1256 Report E01-1256 Dosed Group Female Liver Samples PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Pr\v/irnnmontal I ahnratnrv 3M Environmental Laboratory Pano 10ft nf 91ft Page 108 Report E01-1256 Report E01-1256 10 ppm a.i. 3276 F Liver 3M Environmental Lab d020306117 Sm (Mn, 1x2) 100-, PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Studywith the Mallard 6.72 703650 davey 070799 07-Mar-2002, 07:04:24 1: MRMof 1Channel ESTIC 7.16e6 Area 0 ' ' " i 1" 1111111" " i 1" 11" 111111111" 111" 11" " i 1" rr,"r'',Tr' iT T ^ t ^ i M11111 M1111111111111111 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 Pane 10Q nf 91fi Page 109 Report E01-1256 Report E01-1256 10 ppm a.i. 3274 F Liver 3M Environmental Lab d020306116Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 469679 davey 070799 07-Mar-2002, 06:53:18 1:MRM of 1Channel ES TIC 4.98e6 Area ""i"1 i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fm/imnmental I ahnratnrv 3M Environmental Laboratory Pan? 110 nf 91R Page 110 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study w ith the Mallard 3M Environmental Laboratory 3M Environmental Laboratory Paae 111 of 216 Page 111 Report E01-1256 Report E01-1256 10 ppm a.i. 3270 F Liver 3M Environmental Lab d020306114 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 426931 davey 070799 07-Mar-2002, 06:31:04 1: MRM of 1Channel ESTIC 4.65e6 Area 1I 111M1111I 1111I 1111I 1 Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Pnvirnnmpntal I ahnratnrv/ 3M Environmental Laboratory Pano 119 nf 91fi Page 112 Report E01-1256 Report E01-1256 10 ppm a.i. 3266 F Liver 3M Environmental Lab d020306112Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.73 277317 davey 070799 07-Mar-2002,06:08:49 1: MRMof 1Channel ESTIC 3.16e6 Area %- 3M Environmental Laboratory 3M Environmental Laboratory Page 113 of 216 Page 113 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratorv 3M Environmental Laboratory Paae 114 of 216 Page 114 Report E01-1256 Report E01-1256 10 ppm a.i. 3264 F Liver 3M Environmental Lab d020306111 Sm (Mn, 1x2) 100-t PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 _ 324993 davey 070799 07-Mar2002, 05:57:39 1: MRMof 1Channel ES TIC 3.67e6 Area T T [ Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Laboratory 3M Environmental Laboratory Pape 115 of 216 Page 115 Report E01-1256 Report E01-1256 10 ppm a.i. 3260 F Liver 3M Environmental Lab d020306067 Sm (M n, 1x2) 100-, PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 328881 davey 070799 06-Mar-2002,21:48:46 1: MRM o f 1 Channel ES TIC 3 .6 8 e 6 A re a i 1.00 I I I I I I I I II 2.00 3.00 4.00 5.00 6.00 TT 'f T T 7.00 8.00 T 9 .0 0 ! Tim e 10.00 3M Environmental Laboratory 3M Environmental Laboratory Pane 116 of 216 Page 116 Report E01-1256 Report E01-1256 10 ppm a.i. 3258 F Liver 3M Environmental Lab d020306066 Sm (Mn, 1x2) 10(h PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 315337 davey 070799 06-Mar-2002,21:37:37 1: MRMof 1Channel ESTIC 3.60e6 Area 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 naMon111 1I 1"m1nI Tnime 3MPnuirnnmonbl I tahnrtatnrv 3M EnvironmentalLaboratory Dona 417 ni Olft Page 117 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Male Offspring Liver Samples ftM Pnuirnnmanfal I ahnratnn/ 3M EnvironmentalLaboratory Pana -Mft rtf 9-1ft Page 118 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Pm/irnnmantal I ahnratnrv 3M Environmental Laboratory Pana 11Q nf 91ft Page 119 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the M allard i 3M Pnv/ironmontal I ahnratnrx/ 3M Environmental Laboratory P o n o 4 On r \ f O l ft Page 120 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Dosed Group Female Offspring Liver Samples 3M Environmental Laboratorv 3M Environmental Laboratory Pane 191 nf 91ft Page 121 Report E01-1256 Report E01-1256 10 ppm a.i. 9634 F Liver 3M Environmental Lab d020307046 Sm (Mn, 1x2) 10CH PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study w ith the Mallard 6.71 240373 davey 070799 07-Mar-2002,23:33:03 1: MRM of 1Channel ES- TIC 2.76e6 Area %- (Hr Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Environmental Lahnratnrv 3M Environmental Laboratory Pana 199 nf 91R Page 122 Report E01-1256 Report E01-1256 10 ppm a.i. 9629 F Liver 3M Environmental Lab d020307044 Sm (Mn, 1x2) 100-i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.71 170716 davey 070799 07-Mar-2002,23:10:50 1:MRMof 1Channel ESTIC 2.03e6 Area %- rq-n H T Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 *3M (!n\/irrtnmantal I ahnratnn/ 3M Environmental Laboratory Pano nf 01ft Page 123 Report E01-1256 Report E01-1256 10 ppm a.i. 9645 F Liver 3M Environmental Lab d020307047 Sm (Mn, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.70 150376 davey 070799 07-Mar-2002,23:44:10 1: MRMof 1 Channel ES- TIC 1.77e6 Area % i- r f n i Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 SM Fnvirnnmpntal I ahnratnrv 3M Environmental Laboratory PanA 194. o f 91 fi Page 124 Report E01-1256 Report E01-1256 10 ppm a.i. 9648 F Liver 3M Environmental Lab d020307048 Sm (M n, 1x2) 100-1 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 6.72 190031 davey 070799 07-Mar-2002,23:55:15 1: MRM o f 1 Channel ESTIC 2 .2 9 e 6 A re a i ' 111 i 11111" 1 TT-q Tim e 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnv/irnnmental I ahnratnrv 3M Environmental Laboratory Pane 19Rnf 91fi Page 125 Report E01-1256 Report E01-1256 10 ppm a.i. 9633 F Liver 3M Environmental Lab d020307045 Sm (Mn, 1x2) 100-i PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the M allard 6.71 _ 123667 davey 070799 07-Mar-2002, 23:21:57 1: MRM of 1Channel ESTIC 1.52e6 Area M' ' ''I ' 11111111 i [ Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 3M Fnvirnnm antal I ahnratnrv 3M Environmental Laboratory P a n a 19ft n f 91ft Page 126 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study w ith the M allard 3M Environmental Laboratnrv 3M Environmental Laboratory P a n 1 9 7 rtf 91ft Page 127 Report E01-1256 Report E01-1256 Control Group Male Sera Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the M allard FnvrnnmAntal I ahnratrtrv 3M Environmental Laboratory Dono 400 rsf04A Page 128 Report E01-1256 Report E01-1256 Control Group Female Sera Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard QM C n x fim n m n tal I oK nratnrx/ 3M Environmental Laboratory P an a 400 nf O i Page 129 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with th Mallard Control Group Male Offspring Sera Sample Cm /irnnm oniol I ahnrotnn/ 3M EnvironmentalLaboratory P a n o I S O rf 91 fi Page 130 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Female Offspring Sera Sample 3M Pm /irnnm ontal I ah nratnrv 3M Environmental Laboratory Pane 131 nf 91 fi Page 131 Report E01-1256 Report E01-1256 Control Group Male Liver Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvim nm ntal I ahnratrtrv 3M Environmental Laboratory P a n a 1 1 0 r\f01fi Page 132 Report E01-1256 Report E01-1256 Control Group Female Liver Sample PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fnvirnnm ental I ahnratnrv 3M Environmental Laboratory P a n e 133 nf 91A Page 133 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Male Offspring Liver Sample 3M Environmental Laboratorv 3M Environmental Laboratory Pana 134 nf 91R Page 134 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Control Group Female Offspring Liver Sample 3M Fnvirrnm pntal I a h n ratn rv 3M Environmental Laboratory Paae 135 of 216 Page 135 Report E01-1256 Report E01-1256 Extracted Sera Calibration Curve Point PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Pru/irrtnm ontal I ahnratrtrv 3M Environmental Laboratory D o n o 1 rxf 0-1 Page 136 Report E01-1256 Report E01-1256 Extracted Liver Calibration Curve Point PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Fm /irnnm antal I ah nratnrv 3M Environmental Laboratory Pane 137 of 216 Page 137 Report E01-1256 Report E01-1256 Unextracted Calibration Curve Point PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Fnvirnnm ontal I ahnratnrv 3M Environmental Laboratory P a n a I M n f 9-1K Page 138 Report E01-1256 Report E01-1256 Extracted Sera Calibration Curve PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Lahnratnrv 3M Environmental Laboratory Pane 139 of 21B Page 139 Report E01-1256 Report E01-1256 Extracted Liver Calibration Curve PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratorv 3M Environmental Laboratory Pane 140 of 216 Page 140 Report E01-1256 Report E 01-1256 Mass Spectrometer Tune Settings PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Tuning Method Report Method: C:\MASSLYNX\DAVEY070799.PRO\ACQUDB\CENTRE1 Printed: Tue Mar 19 10:22:06 2002 .................................................................................... 1 MS' " ......... x8 Page 1 1---- 1---- 1---- 1---- 1-- -- 1---- 1---- 1-5 56 57 53 5S 60 61 62 63 64 6 MS SOURCE ( ESP- ) Capillary Cone Hexapole 1 Aperture 1 Hexapole 2 Source Block Desolvation Temp. Temp. Set Rdbk 2.56 20 0.5 0.2 0.8 150 250 -2.48 -20 148 249 Pressures Analyser Vacuum Gas Cell Rdbk 3.8e-5 2.9e-3 Analyser LM Res 1 HM Res 1 IEnergy 1 Entrance Collision Exit LM Res 2 HM Res 2 IEnergy 2 Multiplier Gas Flows Cone Gas Desolvation Set Rdbk 13.0 13.0 0.7 2 11 1 11.0 11.0 1.0 650 1 11 0 -646 Rdbk 150.5 701.5 3M Environmental Laboratory 3MEnvironmental Laboratory Paae 141 of 216 Page 141 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard HPLC Settings Method Report Method File: Last Modified: Printed: Page 1 C:\MASSLYNX\DAVEY070799.PRO\A( Monday, March 11,2002 08:01:01 Tuesday, March 19,2002 08:37:50 HP1100 LC Pump Initial Conditions Solvents A% B% C% D% 90.0 10.0 0.0 0.0 Valve A set to channel Valve B set to channel Flow (ml/min) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left(C) Oven Temperature Right(C) 0.300 10.0 0 400 40.0 40.0 HP1100 LC Pump Gradient Timetable The gradient Timetable contains 5 entries which a re : 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 HP11O0 LG Pump External Event Timetable The Timetable contains 3 entries which are : Time Initial 0.00 0,10 Column Switch Ofl Off Ofl On Ofl Off Contact) Off On Off Contad2 Ofl Ofl Ofl Contac Off On Off HP1100 Autosampler Initial Conditions Draw Speed Eject Speed (pVmin) raw Position (mm) Stop Time (mins) Injection Volume(pl) Vial Number Thermostat On Thermostat Temperature(C) 2Q0.0 200 0.OQ 10.00 10.0 94 20.0 Pn\/irnnm A ntal I ah rtralnn/ 3M Environmental Laboratory D o n o iAO rtf 0 1 A Page 142 Report E01-1256 Report E01-1256 Mass Spectrometer Scanning Parameters PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Scanning Method Report Method: C :\MASSLYNX\DAVEY07 0799. PRO\ACQUDB \1OMIN-PFOS -PFDA Last Modified: Mon Jan 28 15:20:24 2002 Printed: Tue Mar 19 08:38:22 2002 Solvent Delay ( mins ) : 0.00 Function : 1 MRM of 1 Mass Pair ( ESP- ) Inter Channel Delay Span ( Daltons ) : Start Time ( Mins ) End Time ( Mins ) : Repeats : Channel Parent Secs ) : Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone ( V ) 1 499.00 99.00 0.30 43 60 Function : 2 MRM of 1 Mass Pair ( ESP- ) Inter Channel Delay ( Secs ) : Span ( Daltons ) : Start Time ( Mins ) : End Time ( Mins ) : Repeats : Channel Parent Daughter 0.03 0.00 0.00 10.00 1 Dwell (Secs) Coll Energy (eV) Cone 1 513.00 219.00 0.30 20 20 Page 1 3M Pnvirnnm ontal I ahnratnrv 3M Environmental Laboratory Pano 143 nf Page 143 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Atta c h m e n t D: S a m p le P reparation S heets 3M Fnvirnnm A ntal I ah n ratn rv 3M Environmental Laboratory Paoe 144 of 216 Page 144 sam ple num ber E01-1326-29473 E01-1326-29482 E01-1326-29574 E01-1326-29583 E01-1326-29592 E01-1326-29601 E01-1326-29610 E01-1326-29619 E01-1326-29628 E01-1326-29637 E01-1326-29646 E01-1326-29655 E01-1326-29664 E01-1326-29673 E01-1326-29682 E01 -1326-29691 E01-1326-29700 E01 -1326-29709 E01-1326-29718 E01-1326-29727 E01-1326-29736 E01-1326-29745 E01-1326-29754 E01-1326-29763 E01-1326-29772 E01-1326-29781 E01-1326-29790 E01-1326-29799 E01-1326-29808 E01-1326-29817 E01-1326-29826 E01-1326-29835 E01-1326-29844 E01-1326-29853 E01-1326-29862 E01-1326-29871 E01-1326-29880 E01-1326-29889 E01-1326-29898 E01-1326-29907 E01-1326-29916 E01 -1326-29925 E01-1326-29934 E01-1326-29943 E01-1326-29952 E01-1326-29961 E01-1326-29970 E01-1326-29980 E01-1326-29989 PFOS: allard description 0 ppm a. 3217 M Liver 0 ppm a 3218 F Liver 0 ppm a. 3219 M Liver 0 ppm a 3220 F Liver 0 ppm a. 3221 M Liver 0 ppm a 3222 F Liver 0 ppm a. 3223 M Liver 0 ppm a 3224 F Liver 0 ppm a. 3225 M Liver 0 ppm a 3226 F Liver 0 ppm a. 3227 M Liver 0 ppm a 3228 F Liver 0 ppm a. 3229 M Liver 0 ppm a 3230 F Liver 0 ppm a. 3231 M Liver 0 ppm a 3232 F Liver 0 ppm a. 3233 M Liver 0 ppm a 3234 F Liver 0 ppm a 3235 M Liver 0 ppm a 3236 F Liver 0 ppm a 3237 M Liver 0 ppm a 3238 F Liver 0 ppm a 3239 M Liver 0 ppm a 3240 F Liver 0 ppm a 3241 M Liver 0 ppm a 3242 F Liver 0 ppm a 3243 M Liver 0 ppm a 3244 F Liver Oppma 3245 M Liver 0 ppma 3246 F Liver 0 ppma 3247 M Liver 0 ppm a 3248 F Liver 0 ppm a 3249 M Liver 0 ppm a 3250 F Liver 0 ppma 3251 M Liver 0 ppma 3252 F Liver 0 ppm a 3253 M Liver 0 ppm a 3254 F Liver 0 ppm a 3255 M Liver 0 ppma 3256 F Liver 10 ppm a . 3257 M Liver 10 ppm i. 3258 F Liver 10 ppma . 3259 M Liver 10 ppm i. 3260 F Liver 10 ppm . 3261 M Liver 10 ppma i. 3262 F Liver 10 ppm . 3263 M Liver 10 ppm i. 3264 F Liver 10 ppm . 3265 M Liver Amount of w e ig h e d liver by/date weighed (g) 2#?-*2 ok /. 00^54 new? H o m o g en ized 1w w a te r to (m l) / _____ hom ogenized by/date 2 - 2 T.oZAb} \ I low * f-c'jsw l- Oy /. 1. OTo'bi I.O W q f 1 1 ,0 4 // / l.o(6G4 /- __ ICblSa. i, lozcfh T fm v I-//W im Icoi 1___ t.oion L OZ&iv, I.OW4 1 1&4i <5 i am * !,0MW 1,1OlsJ LOSLV* ___ 1___ 1. co$8* LOTGU ____ L _ 1.oQQ/ja 1.06 9V* l.0o34-<i U'lG 1. I& U l, I. /jW , i.\Z crt L___ ______\______ ______ \ _____ ______ ______ ______ _____ ______ _____ _____ ____ 3M Environmental Laboratory 't Page 145 sam ple num ber E01-1326-29998 E01-1326-30007 E01-1326-30016 E01-1326-30025 E01-1326-30034 E01-1326-30043 E01-1326-30052 E01-1326-30061 E01-1326-30070 E01-1326-30079 E01-1326-30088 E01-1326-30097 E01-1326-30106 E01-1326-30115 E01-1326-30124 E01-1326-30133 E01-1326-30142 E01-1326-30151 E01-1326-30160 E01-1326-30169 E01-1326-30178 E01-1326-30187 E01-1326-30196 E01-1326-30205 E01-1326-30214 E01 -1326-30223 E01-1326-30232 E01-1326-30241 E01-1326-30250 E01-1326-30259 E01-1326-30268 E01-1367-31295 E01-1367-31301 E01-1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31331 E01-1367-31337 E01-1367-31343 E01-1367-31349 E01-1367-31356 E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31392 E01-1367-31398 E01-1367-31404 E01-1367-31410 PFOS: A R description 10 ppm a 3266 F Liver 10 ppm a 3267 M Liver 10 ppm a 3268 F Liver 10 ppm a 3269 M Liver 10 ppm a 3270 F Liver 10 ppm a 3271 M Liver 10 ppm a 3272 F Liver 10 ppm a 3273 M Liver 10 ppm a 3274 F Liver 10 ppm a 3275 M Liver 10 ppm a 3276 F Liver 10 ppm a 3277 M Liver 10 ppm a 3278 F Liver 10 ppm a 3279 M Liver 10 ppm a 3280 F Liver 10 ppm a 3281 M Liver 10 ppm a 3282 F Liver 10 ppm a 3283 M Liver 10 ppm a 3284 F Liver 10 ppm a 3285 M Liver 10 ppm a 3286 F Liver 10 ppm a 3287 M Liver 10 ppm a 3288 F Liver 10 ppm a 3289 M Liver 10 ppm a 3290 F Liver 10 ppm a 3291 M Liver 10 ppm a 3292 F Liver 10 ppm a 3293 M Liver 10 ppm a 3294 F Liver 10 ppm a 3295 M Liver 10 ppm a 3296 F Liver 0 ppm a. 9583 F Liver 0 ppm a. 9591 M Liver 0ppm a. 9589 F Liver Oppma 9592 F Liver 0 ppm a 9598 F Liver 0 ppm a 9603 F Liver 0 ppm a. 9606 M Liver 0 ppm a 9610 F Liver 0 ppm a 9573 F Liver 0 ppm a. 9578 M Liver 10 ppm a 9625 M Liver 10 ppm a 9629 F Liver 10 ppm a 9633 F Liver 10 ppm 9634 F Liver 10 ppm 9645 F Liver 10 ppm 9648 F Liver 10 ppm a 9636 M Liver 10 ppm a 9642 M Liver 10 ppm . 9654 F Liver 10 ppm . 9659 F Liver Amount of w eig h e d liver by/date weighed (g) H o m o g en ized w 1 water to (m l) hom ogenized by/date l.O Q S .0 ^ m * ___ (iO tfa i. O J S f * t-o z& A i .O C S 'S i o k ___ A ? ______ \ \ Z - Z-< ' C i M o f u I/W & r / 7753? I.O O G ,D _ 1 / r O 'b S b * i , OG>ex> 4 (.0 ? LO^lS 4 t 1* 1< L O 'h T 2 $ i i .OG < l. OS 15} _____ I _____ L ____ h _ 22V2. JL2 \.tx?0 l.o V lo * 1.15S O i-c m ^ i.o u ta 1. 0 *iV b + i . 'Of Q(c><q. 1.1 3 I . O SlO i l. |IO ^ l.ia S lk I.U L W l.o? \ u ____ __ ___ 3M Environmental Laboratory Page 146 Report E01-1256 Prep Date: 3/4/2002 Analysts initials: OK/HOJ PFOS: A Reprj SPE Columns Extraction Worksheet t oX OK/ffO^ Method Revision: ETS-8-22 Urns Assigned sample number Sample Number or description Volume of sample filtered (ml) Type of column used and lot Amount and spike Elution solven mix used and volume Comments E01-1326-36778 E01-1326-36780 E01-1326-36781 E01-1326-36782 E01-1326-36783 E01-1326-36784 E01-1326-36785 E01-1326-36786 E01-1326-36787 E01-1326-36788 E01-1326-36789 E01-1326-36790 E01 -1326-36791 E01-1326-36792 E01-1326-36793 E01-1326-36794 E01-1326-36795 E01 -1326-36796 E01-1326-36797 E01-1326-36798 E01-1326-36799 Blank H20-day 1 Blank mallard liver-day 1 Mallard Liver MS-day 1 Mallard Liver MSD-day 1 Curve point-0.2 ppb in liver extract Curve point-0.5 ppb in liver extract Curve point-1 ppb in liver extract Curve point-2.5 ppb in liver extract Curve point-5 ppb in liver extract Curve point-10 ppb in liver extract Curve point-25 ppb in liver extract Curve point-50 ppb in liver extract Curve point-75 ppb in liver extract Curve point-100 ppb in liver extract Curve point-100 ppb-2 in liver extract Curve point-250 ppb in liver extract Curve point-500 ppb in liver extract Curve point-750 ppb in liver extract Curve point-1000 ppb in liver extract Q C- 25ppb -1 QC- 25ppb -2 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 waters, ig, omi, lot W2045B2 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 0.4ul o f 02001-66 TN-A-6009 2ml of MeOH 1ul o f 02001-66 TN-A-6009 2ml of MeOH 2ul o f 02001-66 TN-A-6009 2ml of MeOH 5ul o f 02001-66 TN-A-6009 2ml of MeOH 10ul of 02001-66 TN-A-6009 2ml of MeOH 20ul of 02001-66 TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 100ul o f 02001-66 TN-A-6009 2ml of MeOH 150ul of 02001-66 TN-A-6009 2ml of MeOH 200UI o f 02001-66 TN-A-6009 2ml of MeOH 4ul o f 02001-53 TN-A-6009 2ml of MeOH 10ui Of 02001-53 TN-A-6009 2ml of MeOH 20ul of 02001-53 TN-A-6009 2ml of MeOH 30ul of 02001-53 TN-A-6009 2ml of MeOH 40ul of 02001-53 TN-A-6009 1ul Of 02001-53 1ul o f 02001-53 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 \ / Blank Liver TN-A- ; Amount o f liver: f ^ i _ g Homogenize liver with Kandiyohi water; Amount o f water added. 4 s - oti mi cK 2 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 (ShakerV u jR jt ~3> r K ) < ^ 4 ( 6 ^ 4 Centrifuge sample for 10 min @ 2000 rpm (Centrifuge (S > KM +103 o K . t.x k o Z J fc Add 40 ml o f 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- < 0 0 ^ ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go com pletely dry Elute column with indicated amount o f solvent ( H f O H TN-A- ^ 0 0 ^ ) into appropriate 15 ml centrifuge tube * Spike samples with Internal standard PFDA standard number O e 3 a o |Transfer sample Into appripriatelly marked autovial _______ , cone. loo.^?ppAM . amount added & JLaX . ard x 4*2. 3M Environmental Laboratory Page 147 Report E01-1256 SPE Columns Extraction WorPkFsSeSetARePg d UCtn ^S; U^ WJthheeMlxlard Prep Date: 3/4/2002 Analysts initials: OK/HOJ 9 *3 O' Lims Assgned sample number E01-1326-36800 E01-1326-36801 E01-1326-36802 E01-1326-36803 E01-1326-36804 E01 -1326-36805 E01-1326-36806 E01-1326-29473 E01-1326-29482 E01-1326-29574 E01 -1326-29583 E01-1326-29592 E01-1326-29601 E01-1326-29610 E01 -1326-29619 E01 -1326-29628 E01-1326-29637 E01-1326-29646 E01-1326-29655 E 0 1 -1 3 2 6 -2 9 6 6 4 E01-1326-29673 Sample Number or description QC- 25ppb -3 QC- 250ppb -1 QC- 250ppb -2 QC- 250ppb -3 QC- 4.0ppm -1 QC- 4.0ppm -2 QC- 4.0ppm -3 0 ppm a.i. 3217 M Liver 0 ppm a.i. 3218 F Liver 0 ppm a.i. 3219 M Liver 0 ppm a.i. 3220 F Liver 0 ppm a.i. 3221 M Liver 0 ppm a.i. 3222 F Liver 0 ppm a.i. 3223 M Liver 0 ppm a.i. 3224 F Liver 0 ppm a.i. 3225 M Liver 0 ppm a.i. 3226 F Liver 0 ppm a.i. 3227 M Liver 0 ppm a.i. 3228 F Liver O p p m a .i. 3 2 2 9 M L ive r 0 ppm a.i. 3230 F Liver Volume of sample filtered (ml) Type of column used and lot 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 W aters, 1g, 6m l, lot W2045B2 WAfsrc, id, bun; lot W2045B2 Method Revision: ETTSS-8-22J1_ Study Number: E01- Matrix: Liver Amount and spike Elution solvent mix used and volume Comments 2ml of MeOH 1ul o f 02001-53 TN-A-6009 2ml of MeOH 10ui of 02001-53 TN-A-6009 2ml of MeOH 10ui of 02001-53 TN-A-6009 2ml of MeOH 10ui of 02001-53 TN-A-6009 2ml of MeOH 160ul of 02001-53 TN^A-6009 2ml of MeOH 160ul o f 02001-53 TN-A-6009 2ml of MeOH 160ul o f 02001-53 TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN+A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA NA 2 m l o f MeOH TN-A-6009 2ml of MeOH TN-A-6009 A /A ______ V.V Blank Liver TN-A- (BoXb ; Amount of liver: 5 -05^9 a Homogenize liver with Kandiyohi water; Amount o f water added. Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly ^ 45 mi fAP) HoiMOOt4/U&/UQ U&.2> <4><&n P u y o 'j^ Add 5ml of ACN (TN-A- 4 / ^ 5 ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker Centrifuge sample for 10 min @ 2000 rpm (Centrituoe3m* h ezgoS LoK 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 com pletely dry Elute column with indicated amount of solvent ( TN-A- S e c `L ) into appropriate 15 ml centrifuge tube s\ n Spike samples with internal standard PFDA standard number. D o lo o ! - & S . cone. . amount added Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 148 Report E01-1256 SPE Standard Curves-Solids PFOS: A Reproduction Study with the Mallard P rep Date(s): Analyses): Sample Matrix: M eth o d /R e v isio n : T arget Analyte(s): 03/04/02,03/05/02, 03/06/02,03/07/02 OK/HOJ Mallard Liver ETS-8-231.1 PFOS Study N um ber: E01-1256 Equipment Number: NA Final Solvent & TN N um ber: MeOH/TN-A-6009 B lank Tissue/Identifier: Mallard Liver/TN-A-6023 FC Mix Std A pprox. 1.00 ppm: FC Mix Std Approx. 50.2 ppm: S urr. Std A pprox. 100.7 ppm: 02001-66 02001-53 02001-45 PFDA Actual Concentrations o f Standards ir die FC Mix Standard PFOS Number Std Cone. u e/m L E01-1326-36783,0.2 ppb 1.004 E01-1326-36784,0.5 ppb 1.004 E01-1326-36785, 1.0 ppb 1.004 E01-1326-36786, 2.5 ppb 1.004 E01-1326-36787, 5.0 ppb E01-1326-36788, 10 ppb 1.004 1.004 E01-1326-36789,25 ppb 1.004 E01-1326-36790, 50 ppb 1.004 E01-1326-36791, 75 ppb 1.004 E01-1326-36792, 100 ppb 1.004 E01-1326-36793,100 ppb-2 50.20 E01-1326-36794, 250 ppb 50.20 E01-1326-36795, 500 ppb 50.20 E01-1326-36796,750 ppb 50.20 E01-1326-36797. 1000 oob 50.20 QC-25 ppb QC-250 ppb 50.20 50.20 OC-4ppm 50.20 Mallard Liver-MS/MSD-Day 1 1.004 Mallard Liver-MS/MSD-Day 2 1.004 Mallard Liver-MS/MSD-Day 3 1.004 Mallard Liver-MS/MSD-Dav 4 1.004 PFOS Am't Spiked mL 0.0004 0.0010 0.0020 0.0050 0.0100 0.0200 0.0500 0.1000 0.1500 0.2000 0.0040 0.0100 0.0200 0.0300 0.0400 0.0010 0.0100 0.1600 0.0500 0.0500 0.0500 0.0500 Calculated Concentrations o f Standards in die sample matrix Standard PFOS PFDA Number Std Cone. Std Caie. E01-1326-36783,0.2 ppb ng/g ng/g 3.97 1993 E01-1326-36784, 0.5 ppb 9.93 1993 E01-1326-36785,1.0 ppb 19.9 1993 E01-1326-36786,2.5 ppb 49.7 1993 E01-1326-36787, 5.0 ppb 99.3 1993 E01-1326-36788, 10 ppb 199 1993 E01-1326-36789,25 ppb 497 1993 E01-1326-36790, 50 ppb 993 1993 E01-1326-36791, 75 ppb 1490 1993 E01-1326-36792,100 ppb 1987 1993 E01-1326-36793, 100 ppb-2 1987 1993 E01-1326-36794,250 ppb 4967 1993 E01-1326-36795, 500 ppb 9933 1993 E01-1326-36796, 750 ppb 14900 1993 E01-1326-36797.1000 ppb QC-25 ppb QC-250 ppb 19867 497 4967 1993 1993 1993 OC-4 ppm 79467 1993 Mallard Liver-MS/MSD-Day 1 497 1993 Mallard Liver-MS/MSD-Day 2 497 1993 Mallard Liver-MS/MSD-Day 3 497 1993 Mallard Liver-MS/MSD-Dav 4 497 1993 Calculated Concentrations o f Standards in die final solvent Standard PFOS PFDA Number Std Cone. Std Caie. ne/mL ng/mL E01-1326-36783,0.2 ppb 0.201 101 E01-1326-36784, 0.5 ppb 0.502 101 E01-1326-36785,1.0 ppb 1.00 101 E01-1326-36786, 2.5 ppb 2.51 101 E01-1326-36787, 5.0 ppb 5.02 101 E01-1326-36788,10 ppb 10.0 101 E01-1326-36789,25 ppb 25.1 101 E01-1326-36790, 50 ppb 50.2 101 E01-1326-36791,75 ppb 75.3 101 E01-1326-36792, 100 ppb 100 101 E01-1326-36793, 100 ppb-2 100 101 E01-1326-36794, 250 ppb 251 101 E01-1326-36795, 500 ppb 502 101 E01-1326-36796, 750 ppb 753 101 E01-1326-36797.1000 DDb 1004 101 QC-25 ppb QC-250 ppb 25.1 101 251 101 OC-4 ppm 4016 101 Mallard Liver-MS/MSD-Day 1 25.1 101 Mallard Liver-MS/MSD-Day 2 25.1 101 Mallard Liver-MS/MSD-Day 3 25.1 101 Mallard Liver-MS/MSD-Dav 4 25.1 101 PFDA Std Cone. ue/mL 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 3M Environmental Laboratory All Am't Spiked mL 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0,002 Initial homogenate: 5.0597g/50.0597g-HnL = 0.1011 g/mL Blank liver initial homogenate (g/mL) fo r curves: 0.1011 Final solvent volume (m L) for curves: 2.000 AU Initial Homogenate g/mL 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 0.1011 AU Final Vol. mL 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 0 Verified by: \A y l i l n U a . Page 149 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/4/02 ok 799 900 5 Acetonitrile tn-a-4145 O l q M -o S j l 2 3 4 5 Volume, (mL) 1 to 10 UO io e Mass of solvent (g) Density1 3.9613 (g/ml) 0.7857 3.9484 0.7857 4.0278 0.7857 3.9249 0.7857 3.9858 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0417 5.0253 5.1264 4.9954 5.0729 5.0524 101.05 0.04997 0.99 PASS Per 'ormance Specificiitions Accuracy, w 98.0 - 102 % C.V., (%) 1.0 % Corrective Actions: PASS PASS oKi `Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx = specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d2#4 =0.660 Acetone d250 =0.788 Methanol d" 4 =0.7866 Methylene Chloride d2#4 =1.3255 Acetonitrile d204 = 0.7857 3M Environmental Laboratory Page 150 Report E01-1256 PFOS: A Re; SPE Columns Extraction Worksheet with the Mallard lAS >1(2i] m {Vy, let 1 Prep Date: ' j - S - o O i Analysts initials: o K s Lims Assgned sample number E01-1326-36807 E01 -1326-36809 E01-1326-36810 E01-1326-36811 E01-1326-29682 E01-1326-29691 E01-1326-29700 E01-1326-29709 E01-1326-29718 E01-1326-29727 E01-1326-29736 E01-1326-29745 E01 -1326-29754 E01-1326-29763 E01-1326-29772 E01-1326-29781 E01-1326-29790 E01-1326-29799 E01-1326-29808 E01-1326-29817 E01 -1326-29826 Sample Number or description Blank H20-day 2 Blank mallard liver-day 2 Mallard Liver MS-day 2 Mallard Liver MSD-day 2 0 ppm a.i. 3231 M Liver 0 ppm a.i. 3232 F Liver 0 ppm a.i. 3233 M Liver 0 ppm a.i. 3234 F Liver 0 ppm a.i. 3235 M Liver 0 ppm a.i. 3236 F Liver 0 ppm a.i. 3237 M Liver 0 ppm a.i. 3238 F Liver 0 ppm a.i. 3239 M Liver 0 ppm a.i. 3240 F Liver 0 ppm a.i. 3241 M Liver 0 ppm a.i. 3242 F Liver 0 ppm a.i. 3243 M Liver 0 ppm a.i. 3244 F Liver 0 ppm a.i. 3245 M Liver O ppm a.i. 3246 F Liver 0 ppm a.i. 3247 M Liver Volume of sample filtered (ml) Type o f column used and lot 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lo t W2045B2 waters, ig, omi, lot W2045B2 Method Revision: ETS--88--22:3 1 Study Number: E01 Matrix: Liver Amount and spike Elution solveri mix used and volume NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN^A-6009 NA 2ml of MeOH TN-A-6009 NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 NA 2ml of MeOH TN-A-6009 NA NA 2ml of MeOH TN-A-6009 2m! of MeOH TN-A-6009 A /A t j! Comments Blank Liver TN-A-6023; Amount of liver: 5 . 0597g Homogenize liver with Kandiyohi water; Amount o f water added 45 ml Aliquot 1ml o f liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample & ,, j -o 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-6009) Wash Column with Kandiyohi W ater Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount o f solvent (MeOH TN-A-6009) Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 151 Report E01-1256 PFOS: A Reproduction Study-Wth the Mallard SPE Columns Extraction Worksheet ^ ^1 l l t x j ^ osfiijbx Prep Date: ' 3 `5 C $ Analysts initials: rjA Lims Assgned sample number E01-1326-29835 E01 -1326-29844 E01-1326-29853 E01-1326-29862 E01-1326-29871 E01-1326-29880 E01-1326-29889 E01-1326-29898 E01-1326-29907 E01-1326-29916 E01-1326-29925 E01-1326-29934 E01-1326-29943 E01-1326-29952 E01-1326-29961 E01-1326-29970 Sample Number or description Volume of sample filtered (ml) Type o f column used and lot 0 ppm a.i. 3248 F Liver 0 ppm a.i. 3249 M Liver 0 ppm a.i. 3250 F Liver 0 ppm a.i. 3251 M Liver 0 ppm a.i. 3252 F Liver 0 ppm a.i. 3253 M Liver 0 ppm a.i. 3254 F Liver 0 ppm a.i. 3255 M Liver 0 ppm a.i. 3256 F Liver 10 ppm a.i. 3257 M Liver 10 ppm a.i. 3258 F Liver 10 ppm a.i. 3259 M Liver 10 ppm a.i. 3260 F Liver 10 ppm a.i. 3261 M Liver 10 ppm a.i. 3262 F Liver 10 ppm a.i. 3263 M Liver 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Method Revision: ETS-8-231 Study Number: E01-l3a ^iC ^I Matrix: Liver ' 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 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2mf of MeOH TN-A-6009 JJ_______________ Vi1 OS I'm Blank Liver TN-A-6023; Amount of liver: 5 . 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube j\t/ Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample ^-- Shake sample for 20 min @300 rpm (Shaker VWR S/N 041694) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3M# 769613) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go com pletely dry Elute column with indicated amount of solvent (MeOH TN-A-6009) into appropriate 15 ml centrifuge tube SnilfP sarrmlns with internal standard PFDA standard number 02001-45. conc.100.7 DDm. amount added 2ul 3MEnvironmental Laboratory ------ -- Page 152 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/5/02 ok 799 900 5 Acetonitrile tn-a-4145 OK 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9429 (g/ml) 0.7857 3.9904 0.7857 3.959 0.7857 3.967 0.7857 3.9712 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0183 5.0788 5.0388 5.0490 5.0543 5.0479 100.96 0.02210 0.44 PASS Perl'ormance Specificsitions Accuracy, (%) 98.0 - 102 % c.v., (%) 1.0 % Corrective Actions: PASS PASS K/ 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx = specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d2#4 =1.3255 Acetonitrile d204 = 0.7857 3M Environmental Laboratory Page 153 Report E01-1256 SPE Columns Extraction WorPkFshose:eat Repr^ u ctio n stu d y w iththe>MalLard t pS 3- Prep Date: 2>'rO^ Analysts initials: >*- Method Revision: ETS-8-2231 Study Number: E01-1326I Matrix: Liver tlf 3 4 7_ Lims Assgned sample number Sample Number or description Volume of sample filtered (ml) Type o f column used and lot Amount and spike Elution solven mix used and volume Comments E01-1326-36812 E01 -1326-36814 Blank H20-day 3 Blank mallard liver-day 3 40+6 40+6 Waters, 1g, 6ml, lot VU2045B2 Waters, 1g, 6ml, lot W204SB2 NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 m E01-1326-36815 E01-1326-36816 E01-1326-29980 Mallard Liver MS-day 3 Mallard Liver MSD-day 3 10 ppm a.i. 3264 F Liver 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 2ml of MeOH 50ul of 02001 -66 TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 NA 2ml of MeOH TN-A-6009 E01 -1326-29989 10 ppm a.i. 3265 M Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-29998 10 ppm a.i. 3266 F Liver 40+6 Waters, 1g, 6ml, lot W204SB2 NA 2ml of MeOH TN-A-6009 E01-1326-30007 10 ppm a.i. 3267 M Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30016 10 ppm a.i. 3268 F Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30025 10 ppm a.i. 3269 M Liver 40+6 Waters, 1g, 6ml, lotW2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30034 10 ppm a.i. 3270 F Liver 40+6 Waters, 1g, 6ml, lotW2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30043 10 ppm a.i. 3271 M Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30052 10 ppm a.i. 3272 F Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30061 10 ppm a.i. 3273 M Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30070 E01-1326-30079 10 ppm a.i. 3274 F Liver 10 ppm a.i. 3275 M Liver 40+6 40+6 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lotW204SB2 NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 E01-1326-30088 10 ppm a.i. 3276 F Liver 40+6 Waters, 1g, 6ml, lot W2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30097 10 ppm a.i. 3277 M Liver 40+6 Waters, 1g, 6ml, lotW2045B2 NA 2ml of MeOH TN-A-6009 E01-1326-30106 E O I-1326-30115 E01-1326-30124 E01-1326-30133 10 ppm a.i. 3278 F Liver 10 ppm a.i. 3279 M Liver 10 ppm a.i. 3280 F Liver 10 ppm a.i. 3281 M Liver 40+6 40+6 40+7 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W 2 0 4 5 B 2 Waters, 1g, 6ml, lot W2045B2 waters, ig, omi, lot W2045B2 NA NA NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 A Blank Liver TN-A-6023; Amount of liver: 5. 0597g Homogenize liver with Kandiyohi water; Amount of water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample Shake sample for 20 min @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-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount o f solvent (MeOH TN-A-6009) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 154 Report E01-1256 SPE Columns Extraction woriSFhO ItA R iproduc^ sidy,w ith fheM allard Prep Date: Analysts initials: Lims Assgned sample number E01-1326-30142 E01-1326-30151 E01-1326-30160 E01-1326-30169 E01-1326-30178 E01-1326-30187 E01-1326-30196 E01-1326-30205 E01 -1326-30214 E01-1326-30223 E01-1326-30232 E01 -1326-30241 E01-1326-30250 E01-1326-30259 E01-1326-30268 Volume of Sample Number sample filtered Type of column or description (ml) used and lot 10 ppm a.i. 3282 F Liver 10 ppm a.i. 3283 M Liver 10 ppm a.i. 3284 F Liver 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 10 ppm a.i. 3285 M Liver 40+6 Waters, 1g, 6ml, lot W2045B2 10 ppm a.i. 3286 F Liver 10 ppm a.i. 3287 M Liver 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot VU2045B2 10 ppm a.i. 3288 F Liver 10 ppm a.i. 3289 M Liver 10 ppm a.i. 3290 F Liver 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 10 ppm a.i. 3291 M Liver 10 ppm a.i. 3292 F Liver 10 ppm a.i. 3293 M Liver 10 ppm a.i. 3294 F Liver 10 ppm a.i. 3295 M Liver 10 ppm a.i. 3296 F Liver 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, 1g, 6ml, lotW2045B2 Waters, 1g, 6ml, lot W204SB2 Waters, 1g, 6ml, lot W2045B2 Method Revision: ETS-8-231 Study Number: EO1-i320'(ay**J7->**',2~ Matrix: Liver Amount and spike Elution solvent mix used and volume NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2mf of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 UA >t Comments ______________ > _____ IM Blank Liver TN-A-6023; Amount of liver: 5. 0597g Homogenize liver with Kandiyohi water; Amount o f water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample Shake sample for 20 min @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-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (MeOH TN-A-6009) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul 3MEnvironmental Laboratory Page 155 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/6/02 ok 799 900 5 Acetonitrile tn-a-4145 4 ,-C 'o Z OKJ 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 3.9506 (g/ml) 0.7857 3.9857 0.7857 3.9874 0.7857 3.9772 0.7857 3.983 0.7857 AverageVolume: Volume of Solvent 5.0281 5.0728 5.0750 5.0620 5.0694 5.0614 %Accuracy: 101.23 Std. Dev.: %C.V.: Pass/FaU: 0.01927 0.38 PASS Perl'ormance Specificsliions Accuracy, (%) 98.0 - 102 % C.V., (%) 1.0 % Corrective Actions: density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx = specific gravity at y degreesC referred to water at xdegreesc n-Hexane d204 =0.660 Acetone d2S0 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 =1.3255 Acetonitrile d204 = 0.7857 3M Environmental Laboratory Page 156 Report E01-1256 SPEColumns Extraction Prep Date: Analysts initials: 0i Urns Assgned sample number Sample Number description E01-1326-36817 E01-1326-36819 E01 -1326-36820 E01-1326-36821 E01-1367-31295 E01-1367-31301 E01-1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31331 E01-1367-31337 E01-1367-31343 E01-1367-31349 E01-1367-31356 E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31392 E01-1367-31398 E01-1367-31404 E01-1367-31410 Blank H20-day 4 Blank mallard liver-day 4 Mallard Liver MS-day 4 Mallard Liver MSD-day 4 0 ppm a.i. 9583 F Liver 0 ppm a.i. 9591 M Liver 0 ppm a.i. 9589 F Liver 0 ppm a.i. 9592 F Liver 0 ppm a.i. 9598 F Liver 0 ppm a.i. 9603 F Liver 0 ppm a.i. 9606 M Liver 0 ppm a.i. 9610 F Liver 0 ppm a.i. 9573 F Liver 0 ppm a.i. 9578 M Liver 10 ppm a.i. 9625 M Liver 10 ppm a.i. 9629 F Liver 10 ppm a.i. 9633 F Liver 10 ppm a.i. 9634 F Liver 10 ppm a.i. 9645 F Liver 10 ppm a.i. 9648 F Liver 10 ppm a.i. 9636 M Liver 10 ppm a.i. 9642 M Liver 10 ppm a.i. 9654 F Liver 10 ppm a.i. 9659 F Liver Volume of oi 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 40+6 waters, ig, bmi, lot VU2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lotW2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, 1g, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W2045B2 Waters, Ig, 6ml, lot W204SB2 Waters, Ig, 6ml, lo t W 2 0 4 5 B 2 Waters, ig, 6ml, lot W2045B2 40+6 Waters, 1g, 6ml, lot W2045B2 Method Revision: ETS-8-231 Study Number: Matrix: Liver / ^ Amount and spike Elution solvent mix used and volume NA 2ml of MeOH TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 50ul Of 02001-66 TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml of MeOH NA TN-A-6009 2ml o f MeOH NA T N -A -60 09 2ml of MeOH NA TN-A-6009 2ml or MeUH NA TN-A-6009 m \V Comments Blank Liver TN-A-6023; Amount of liver: 5. 0597g Homogenize liver with Kandiyohi water; Amount o f water added 45 ml Aliquot 1ml of liver into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-4145) to aliquated sample Shake sample for 20 min @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-6009) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent (MeOH TN-A-6009) into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number 02001-45, conc.100.7 ppm, amount added 2ul Transfer sample into appripriatelly marked autovial 3M Environmental Laboratory Page 157 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 3/7/02 ok 799 900 5 Acetonitrile tn-a-4145 o tL s 1 2 3 4 5 Volume, (mL) 1 to 10 Mass of solvent (g) Density1 (g/ml) 3.9564 0.7857 3.9674 0.7857 3.982 0.7857 3.9753 0.7857 3.9713 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0355 5.0495 5.0681 5.0596 5.0545 5.0534 101.07 0.01215 0.24 PASS Perlromance Specificiitions Accuracy, (%) 98.0 - 102 % C.V., (%) 1.0 % Corrective Actions: PASS PASS OH `Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx = specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d2#4 =0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d2#4 =1.3255 Acetonitrile d2#4 = 0.7857 3M Environmental Laboratory Page 158 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet Study: E01-1326/1367 Dilution Date/Analyst: CD Box Number: Solvent/TN Number: MeOH/5457. _ tt&X ^ Extraction Date/Analyst: .2>-4<?<2y3 ^ 0.2,( 3 -Q 'O Matrix/Timepoint: / / A / jLAF-T) C - IV C R J Sample Number Dilutions or Description 1/10 1/100 1/ 1/ 1/ 1/ 1/ Comments E01-1326-29961 X E01-1326-30034 X MA E01-1326-30070 X E01-1326-30088 X E01-1326-30178 X E01-1326-30196 E01-1326-30214 E01-1326-29916 E01-1326-29934 E01-1326-29952 E01-1326-29970 E01-1326-29989 E01-1326-30007 X X X X X w X X V X MA 1 Vas la it a E01-1326-30025 X E01-1326-30043 X E01-1326-30061 X E01-1326-30079 X Notes: 1/10 dilution = \oouJL *j Wo axufiovo V of sample + ^tooiS of solvent t oj jl eu-* ' iAje^e m * d < o u (p -JU o b c ^ / e e t u i Verified By: 643 n Form Completion Verified By: h M 4 . ^ 3M Environmental Laboratory (ftp Pay. U 1 IAS ay/aiUi Page 159 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet Study: E01-1256 Dilution Date/Analyst: Box Number: I q i ' f g lio ex- ent/TN Number: MeOH/5457 Extraction Date/Analyst: Matrix/Timepoint: U \je R ^ Notes: 1/10 dilution loo^J) of sample + of solvent Form Completion Verified By: 3M Environmental Laboratory f W , St t i i I, ^* US p a lw - Page 160 s ^ p o r t ^ l i 56 A3 PFOS: A Reproduction Study with the Mallard (f) Mallard LAS OSfat/tX fcy. 1of 1 / sample number E01-1256-28662 E01-1256-28663 E01-1256-28664 E01-1256-28665 E01-1256-28666 E01-1256-28667 E01-1256-28668 E01-1256-28669 E01-1256-28670 E01-1256-28671 E01-1256-28672 E01-1256-28673 E01-1256-28674 E01-1256-28675 E01-1256-28676 E01-1256-28677 E01-1256-28678 E01-1256-28679 E01-1256-28680 E01-1256-28681 E01 -1256-28682 E01-1256-28683 E01-1256-28684 E01-1256-28685 E01-1256-28686 E01-1256-28687 E01-1256-28688 E01-1256-28689 E01-1256-28690 E01-1256-28691 E01-1256-28692 E01-1256-28693 E01 -1256-28694 E01-1256-28695 E01-1256-28696 E01-1256-28697 E01-1256-28698 E01-1256-28699 E01-1256-28700 E01-1256-28701 E01-1256-28702 E01-1256-28703 E01-1256-28704 E01-1256-28705 E01-1256-28706 E01-1256-28707 E01-1256-28708 E01-1256-28709 E01-1256-28710 0 ^fL ^rtp description 454-109-32117, 0 PPML Male, Aduli 454-109-32181 0 PPM, Female, Adult 454-109-3210, 0 PPM, Male, Aduli 454-109-32201 0 PpM, Fmale,! Adtlt 454-109-322f1, 0 I I p MI Male, Adult 454-109-3222 0 PpM, Female, Adult 454-109-3220, 0 fPM Male, Aduli 454-109-3224* 0 PPM, Female, Adult 454-109-32261 0 PpM, iemale, Adult 454-109-3227, 0 PPM! Male, Aduli 454-109-3228! 0 PPM, Fanale, Adllt 454-109-322fe, 0 PPM, Male, Aduli 454-109-3230, 0 PPM, Fumale, Adult 454-109-3231, 0 PPM. Male, Adult 454-109-3232! 0 PPM, Fecale, Adult 454-109-3233, 0 PPM, Male, Adult 454-109-3234| 0 PPM, Female, Adult 454-109-3236, 0 PpM, Ridiale! AdUlt 454-109-3237, 0 pPlM Male, Adult 454-109-3238, 0 PPM, Female, Adult 454-109-3233, o f Pivi Male, Adult 454-109-3240, 0 PpM, Female, Adult 454-109-3241, 0 PPM, Male, dufc 454-109-3242, 0 PPM, Female, Adiilt 454-109-3248, 0 fPM i Male, Adult 454-109-3244,0 PPM, Female, Adult 454-109-3245, 0 PPM, Male, Adult 454-109-32465 0 PPM, Female, Adtlt 454-109-3247, 0 PPM, Male, Adult 454-109-3248 0 PPM, Female, Ad|ilt 454-109-324 9,0 PPM Male, Adult 454-109-3250 o tTMM. :tnalej Adult 454-109-329 I, 0 IfPM .Male, Aduli 454-109-3252 OfpM, remale, Adijilt 454-109-325 3,0 PPM Male, Adu| 454-109-3254 o p | m , remale, Ad|ilt 454-109-325 5, 0 PPM Male, Adu \ 454-109-3256 o r | m , remale, Adfit 454-109-3257', 10 fpiv , Male, Adi 454-109-3258] 10 PPM, Female, AcjSult 454-109-325 , 10 PPM, Male, Adult 454-109-3260] [10 PpM,1Female, Adult 454-109-3261 , 10! PPM, Male, Adilt 454-109-3262. 10 pilPMjFemald, Acjult 454-109-3263 , 10 PPM, Male, Adilt 454-109-3264] HO RPM, Remale, Adult 454-109-3268, 10 PPM, Male, j|Adi|t 454-109-3266J10 PpM, Female, Adult 454-109-3267,10 PPM, Male, Adult t+d S e?<s<i to T T 9 Amount of sera aliauated(ml) G.fO fX e G '* o&> 6.ft> gr-Td T Te rr>XO --GL,Sd - ____ <0 .5^ aliquated Diluted with by/date water to (ml) /XllfLi un* / i* l / /* f* t f* (O it AT fd /* / diluted by/date fr)Z-ru it) ` <px) ?`)1 tr.SL 5-f -> .r aY G -f o-ST >, / e ? -r oY e>Y a . r ______ T <1>X \ \ \ \ i | I I I I I I I I I X GX 6r _ l ______ ____ i L - ys**.. > / a /et a /O /a /*> /e /0 s<r /* //** / /* / Z /> /c? 'a 4 /e / / / e 'L /<> fi! -- -- __sa< 3M Environmental Laboratory Page 161 f RepDoo_rt_E__01_-_1_256 jTx^bf in ren n s t fr) PFOS: A Reproduction Study with the Mallard q) tAaUftc Sero^ ^ y~y ae% J sam ple num ber d e d ilp ti n E01-1256-28711 454-109-3268 E01-1256-28712 454-109-32^9^ E01-1256-28713 454-109-3270, 1 E01-1256-28714 454-109-3271, E01-1256-28715 454-109-3272, 1( E01-1256-28716 454-109-3273, E01-1256-28717 454-109-3274, IQjfflMj ftrlrajj Adult E01-1256-28718 454-109-3275, jP W ,i Mal;AUlt E01-1256-28719 454-109-3276; 10RPM^ Female, Adult E01-1256-28720 454-109-3277, I p f plfl, Mle,; Afliilt E01-1256-28721 454-109-3278^ 10 FPM, Female, Adult E01-1256-28722 454-109-3279, 10 Ppl^l,^Malj idilt E01-1256-28723 454-109-3280^ l M flJ Femai, Adult E01-1256-28724 454-109-3281,1Q PPM, Male, Adult E01-1256-28725 415544--110099--33228822^, 10 PPM, FPemr ale, Adult E01-1256-28726 454-109-3283,10 PPM, M.ale, Adult E01-1256-28727 454-109-3284; 10 PPM, F E01-1256-28728 454-109-3285,10 PPM, Male. Adtilt E01-1256-28729 454-109-3286, 10 PPM, Female, Adult E01-1256-28730 454-109-3287, t pFull,il^le, Adlillt E01-1256-28731 454-109-3288; 10 PPM, Female, Adult E01-1256-28732 454-109-3289, l j | p M , Male.iAdjt E01-1256-28733 454-109-3290,10 PPM, Female, Adult E01-1256-28734 454-109-3291, 1 # | N . Mle, M i t E01-1256-28735 ^454-109-3292,10 PPM,'Female, Aduit E01-1256-28736 454-109-3293, 10# M , Male, Adult E01-1256-28737 454-109-3294, id >PM,Spale, Adult E01-1256-28738 454-109-3295, 1Q PPM, Male, Adult E01-1256-28739 454-109-3296, 10f>pM,FilTial^ Afeilt E01-1256-28740 454-109-9583,0 PPM, Female, Offspring E01-1256-28741 454-109-9591,0 E01-1256-28742 454-109-9589, QP E01-1256-28743 454-109-9592, P E01-1256-28744 454-109-9598, 0 PF E01-1256-28745 454-109-9603, 0 PI E01-1256-28746 454-109-9606,: 0 E01-1256-28747 454-109-9610, 0 PF E01-1256-28748 454-109-9573, 0 PF E01-1256-28749 454-109-9578,0 E01-1256-28750 454-109-9625, 10 E01-1256-28751 454-109-9629, 1 E01-1256-28752 454-109-9633, 1 E01-1256-28753 454-109-9634, 1^3 H i E01-1256-28754 454-109-9645, 10Tl E01-1256-28755 454-109-9648,10 P E01-1256-28756 454-109-9636, 10 E01-1256-28757 454-109-9642, 10 E01-1256-28758 454-109-9654, 10 PPM, E01-1256-28759 454-109-9659, 10 PPM, Female, Amount of sera alkjuatedfm l^ c /.r &r o -'f' aliquated Diluted with by/date w ater to (ml) z>/ - /< J_ /o fa -r c -r a /O / 0 -f 0 .2^ cT .r o-r O-Y' 0 .3 fO JO fo /o /a ../o So : JS>JL O -f of J&S y`lO1 ' & .so cp -r &. 7, Q tf' 0*1 i A. /a /S fa 'd S ,o Z.., Z-O >>7 3 0-2 0*7-- g .f G. f J2jJL S .f o *Z er / t >0 4 Z0 /C Z -0 4.d 2 * Z diluted by/date 9 u <@ <3> ) <) e> 0 i 3M Environmental Laboratory Page 162 Report E01-1256 SPE Colum ns Extraction W o f f i A ^ Prep Date: Analysts initials: 2/25/2002 OK/HOJ Method Revision: ETS-8-231.0 Study Number: E01-1256 vow Matrix: Sera Volume of Lims Assgned sample number Sample Number or description sample filtered Type of column Amount and spike Elution solvein (ml) used and lot mix used and volume Comments e01-1256-36231 e01-1256-36232 e01-1256-36233 e01-1256-36234 e01-1256-36235 e01-1256-36236 e01-1256-36237 e01-1256-36238 e01-1256-36239 e01-1256-36240 e01-1256-36241 e01-1256-36242 e01-1256-36243 e01-1256-36244 e01-1256-36245 e01-1256-36246 Blank H20-day 1 Blank mallard sera-day 1 Mallard Sera MS-day 1 Mallard Sera MSD-day 1 Curve point-0.2 ppb in sera extract Curve point-0.5 ppb in sera extract Curve point-1 ppb in sera extract Curve point-2.5 ppb in sera extract Curve point-5 ppb in sera extract Curve point-10 ppb in sera extract Curve point-25 ppb in sera extract Curve point-50 ppb in sera extract Curve point-75 ppb in sera extract Curve point-100 ppb in sera extract Curve point-100 ppb-2 in sera extract Curve point-250 ppb in sera extract 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, ict^aeBe Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 Waters, 1g, 6ml, lot 1290B2 2ml of M eOH > NA 2ml of MeOH NA TN-A-5457 2ml of MeOH 50ul of 02001-66 TN-A-5457 2ml of MeOH 50ul of 02001-66 TN-A-5457 2ml of MeOH 0.4ul of 02001-66 TN-A-5457 2ml of MeOH 1ul of 02001-66 TN-A-5457 2ml of MeOH 2ul of 02001-66 TN-A-5457 2ml of MeOH 5ul of 02001-66 TN-A-5457 2ml of MeOH 10ul of 02001-66 TN-A-5457 2ml of MeOH 20ul of 02001-66 TN-A-5457 2ml of MeOH 50ul of 02001-66 TN-A-5457 2ml of MeOH 100ul of 02001-66 TN-A-5457 2ml of MeOH 150ul of 02001-66 TN-A-5457 2ml of MeOH 200ul of 02001-66 TN-A-5457 2ml of MeOH 4ul of 02001-53 TN-A-5457 2ml of MeOH 10ul of 02001-53 TN-A-5457 MA e01-1256-36247 e01-1256-36248 e01-1256-36249 e01-1256-36250 Curve point-500 ppb in sera extract Curve point-750 ppb in sera extract Curve point-1000 ppb in sera extract QC 25pb-1 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 2ml of MeOH 20ul of 02001-53 TN-A-5457 2ml of MeOH 30ul of 02001-53 TN-A-5457 2ml of MeOH 40ul of 02001-53 TN-A-5457 1ul o f 02001-53 2ml of MeOH TN-A-5457 e01-1256-36251 e01-1256-36252 QC 25pb-2 QC 25pb-3 40+6 40+6 Waters, 1g, 6ml, lot 1290B2 WAtbls, 10, bitH, lot 1290B2 . 1ul of 02001-53 1ul of 02001-53 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 \J I1 Blank Sera TNA" j Amount of sera aliquated:. _ml Dilute Sera with Kandiyohi water; Amount o f water added 3 6 ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN ( T N - A t o aliquated sample Shake sample for 20 min @300 rpm (Shaker ^ ^^ ) Centrifuge sample for 10 min @ 2000 rpm (Centrifuge ) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- C o IS . ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount o f solvent Into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number Q f f o o f - t S> nsfer sample into appripriatelly marked autovlal T***I^ S rt /W . 7* if. tS'eys y-b yi/c' , cone. loo- 7r amount added CAc. C xU r A * "* 0 /vLtf / jl ~ TE> 3M Environmental Laboratory Page 163 Report E01-1256 Prep Date: Analysts initials: Of< /HO ~3 _ , _ . ,, . . . PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet * . @ MalWd_ O i/aik f o ^ 1 oftl* J Method Revision: ETS-8-231.0 Study Number: E01-1256 Urns Assgned sample number e01-1256-36253 e01-1256-36254 e01-1256-36255 e01-1256-36256 e01-1256-36257 e01-1256-36258 E01-1256-28662 E01-1256-28663 E01-1256-28664 E01-1256-28665 E01-1256-28666 E01-1256-28667 E01-1256-28668 E01-1256-28669 E01-1256-28670 E01-1256-28671 E01-1256-28672 E01-1256-28673 E01-1256-28674 E01-1256-28675 E01-1256-28676 Sample Number or description QC-250ppb -1 QC-250ppb -2 QC-250ppb -3 QC-4ppm -1 QC-4ppm -2 QC-4ppm -3 454-109-3217, 0 PPM, Male, Adult 454-109-3218, 0 PPM, Female, Adult 454-109-3219, 0 PPM, Male, Adult 454-109-3220, 0 PPM, Female, Adult 454-109-3221,0 PPM, Male, Adult 454-109-3222, 0 PPM, Female, Adult 454-109-3223,0 PPM, Male, Adult 454-109-3224,0 PPM, Female, Adult 454-109-3226, 0 PPM, Female, Adult 454-109-3227,0 PPM, Male, Adult 454-109-3228, 0 PPM, Female, Adult 454-109-3229,0 PPM, Male, Adult 454-109-3230,0 PPM, Female, Adult 454-109-3231,0 PPM, Male, Adult 454-109-3232,0 PPM, Female, Adult Volume of sample filtered Type o f column Amount and spike Elution solvent (ml) used and lot mix used and volume 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, 2ml ofMeOH lot t2 9 6 B 2 lu ZdlGh2 10ul of 02001-53 T N n 4 W P | ^ Waters, 1g, 6ml, lot 1290B2 2ml of MeOH 10ul of 02001-53 TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH 10ul of 02001-53 TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH 160ul of 02001-53 TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH 160ul of 02001-53 TN-A-5457 Waters, 1g, 6ml, lot 1290B2 2ml of MeOH 160ul of 02001-53 TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 NA 2ml of MeOH TN-A-5457 Waters, 1g, 6ml, lot 1290B2 waters, ig, omi, lot 1290B2 {/ NA NA 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 < Comments I \ I I vL_____ Blank Sera TN-A- ( y c O -^ - Amount of sera aliquated: ^ _ml ^Dilute Sera with Kandiyohi water; Amount o f water added.~ w ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A-. _) to aliquated sample Shake sample for 20 min @300 rpm (Shaker^ 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 mixiqg Condition column with MeOH ( T N - A - _ j_ ~ _ ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount o f solvent into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number p & a p l - _____ , cone.. l o o . 7 f f n u . amount added. Transfer sample into appripriatelly marked autovial n1 /-m 3M Environmental Laboratory Page 164 Report E01-1256 SPE Standard Curves-FJuids PFOS: A Reproduction Study with the Mallard Prep Date(s): Analyses): Sample M atrix: M ethod/Revision: T arget Analyte(s): 02/25/02,02/26/02,02/27/02,02/28/02 OK/HOJ Mallard Sera ETS-8-231.1 PFOS Study N u m b er E1-1256 Equipm ent Num ber: NA fin a l Solvent & TN Num ber: MeOH/TN-A-05457 Blank Tissue/Identifler: Mallard Sera-E02-0241-36386 P F O S S td: 1.00 ppm: P F O S S td: 50.2 ppm: S u rrS td Approx. 100.7 ppm: 02001-66 02001-53 02001-45 PFDA Actual Concentrations of Standards in the PC Mix PFOS PFOS StdConc. Am't Spiked u a /m L mL 1.004 0.0004 1.004 0.0010 1.004 0.0020 1.004 0.0050 1.004 0.0100 1.004 0.0200 1.004 0.0500 1.004 0.1000 1.004 1.004 50.20 0.1500 0.2000 0.0040 50.20 0.0100 50.20 0.0200 50.20 0.0300 50.20 0.0400 50.20 0.0010 50.20 0.0100 50.20 0.1600 1.004 0.0500 1.004 0.0500 1.004 0.0500 1.004 0.0500 PFDA StdConc. u a/m L 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 100.7 Calculated Concentrations o f Standards in the sample matrix Standard PFOS PFDA Number StdConc. Std Cone. E01-1236-36235,0.2 ppb E01-1256-36236,0.5 ppb na/mL 8.03 20.1 n a/m L 4028 4028 E01-1256-36237,1.0 ppb 40.2 4028 E01-1256-36238,2.5 ppb 100 4028 E01-1256-36239,5.0 ppb 201 4028 E01-1256-36240,10 ppb E01-1256-36241,25 ppb 402 1004 4028 4028 E01-I256-36242,50 ppb 2008 4028 E01-1256-36243,75 ppb 3012 4028 E01-1256-36244,100 ppb 4016 4028 E01-1256-36245,100-2 ppb 4016 4028 E01-1256-36246,250 ppb 10040 4028 E01-1256-36247,500 ppb 20080 4028 E01-1256-36248,750 ppb 30120 4028 E01-1256-36249.1000 ppb QC-25 ppb QC-250 ppb QC-4 ppm 40160 1004 10040 160640 4028 4028 4028 4028 Mallard Sera MS/MSD-Day 1 Mallard Sera MS/MSD-Day 2 1004 1004 4028 4028 Mallard Sera MS/MSD-Day 3 1004 4028 Mallard Sera MS/MSD-Dav 4 1004 4028 Calculated Concentrations of Standards in the final solvent Standard PFOS PFDA Number StdConc. StdConc. n a /m L n a/m L E01-1256-36235,0.2 ppb 0.201 101 E01-1256-36236,0.5 ppb 0.502 101 E01-1256-36237,1.0 ppb 1.00 101 EOl-1256-36238,2.5 ppb 2.51 101 EOl-1256-36239,5.0 ppb 5.02 101 E01-1256-36240,10 ppb 10.0 101 E01-1256-36241,25 ppb 25.1 101 E01-1256-36242,50 ppb 50.2 101 E01-1256-36243,75 ppb 75.3 101 E01-1256-36244,100 ppb 100 101 EOl-1256-36245,100-2 ppb 100 101 E01-1256-36246,250 ppb 251 101 E01-1256-36247,500 ppb 502 101 E01-1256-36248,750 ppb 753 101 E01 -1256-36249.1000 ppb 1004 101 QC-25 ppb 25.1 101 QC-250 ppb 251 101 QC-4ppm 4016 101 Mallard Sera MS/MSD-Day 1 25.1 101 Mallard Sera MS/MSD-Day 2 25.1 101 Mallard Sera MS/MSD-Day 3 25.1 101 Mallard Sera MS/MSD-Dav 4 25.1 101 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 0.002 0.002 0.002 0.002 0.002 0.002 All Initial Dilution mL/mL 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 0.0500 AU Final Vol. mL 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 2.000 Blank sera initial dilution (mL/mL) for curves: Final solvent volume (mL) for curves: 0.0500 2.000 >r ^ o s la ilo i y -g g g fr-- 3M Environmental Laboratory Page 165 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 25 Febuary 2002 ? .7 hoj/ok d?V L 799 V -W'rtCO 5 Acetonitrile tn-a-4145 1 2 3 4 5 Volume, (mL) 1 to 10 A J z w iT Mass of solvent (g) Density1 3.9739 (g/ml) 0.7857 3.9683 0.7857 3.9559 0.7857 3.9767 0.7857 3.9718 0.7857 AverageVolume: Volume of Solvent 5.0578 5.0507 5.0349 5.0613 5.0551 5.0520 %Accuracy: 101.04 Std. Dev.: %C.V.: Pass/Fail: 0.01031 0.20 PASS PASS PASS Perlnrm ance Specificsliions Accuracy, (%) 98.0 - 102 % C.V., (%) 1.0 % Corrective Actions: -- 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx = specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d204 =0.660 Acetone d2S0 =0.788 Methanol d254 =0.7866 Methylene Chloride d2#4 =1.3255 Acetonitrile d2#4 = 0.7857 3M Environmental Laboratory Page 166 Report E01-1256 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction Worksheet ^ ~ Prep Date: Analysts initials: Urns Assgned sample number E01-1256-36259 E01-1256-36260 E01-1256-36261 E01-1256-36262 E01-1256-28677 E01-1256-28678 E01-1256-28679 E01-1256-28680 E01-1256-28681 E01-1256-28682 E01-1256-28683 E01-1256-28684 E0M256-28685 E01-1256-28686 E01-1256-28687 E01-1256-28688 E01-1256-28689 E01-1256-28690 E01-1256-28691 E01-1256-28692 E01-1256-28693 E01-1256-28694 2/26/2002 OK/HOJ Sample Number or description Blank H20-day 2 Blank mallard sera-day 2 Mallard Sera MS-day 2 Mallard Sera MSD-day 2 454-109-3233,0 PPM, Male, Adult 454-109-3234,0 PPM, Female, Adult 454-109-3236, 0 PPM, Female, Adult 454-109-3237,0 PPM, Male, Adult 454-109-3238,0 PPM, Female, Adult 454-109-3239, 0 PPM, Male. Adult 454-109-3240, 0 PPM, Female, Adult 454-109-3241,0 PPM, Male, Adult 454-109-3242,0 PPM, Female, Adult 454-109-3243,0 PPM, Male, Adult 454-109-3244,0 PPM, Female, Adult 454-109-3245,0 PPM, Male, Adult 454-109-3246, 0 PPM, Female, Adult 454-109-3247,0 PPM, Male, Adult 454-109-3248,0 PPM, Female, Adult 4 5 4 -1 0 9 -3 2 4 9 , 0 PPM, Male, A d u lt 454-109-3250, 0 PPM, Female, AduK 4b4-1Ub-j^o i, u r TM , rviaie, Adult Method Revision: ETS-8-231 Study Number: E01-1256 Matrix: Sera Volume of sample filtered (ml) Type o f column Amount and spike Elution solvent used and lot mix used and volume Comments 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot VIQ036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, MW2036B2 Waters, 1g, 6ml, lot W 2036 B 2 Waters, 1g, 6ml, lot W2036B2 waters, ig, omi, lot W2036B2 IA 2ml of MeOH TN-A-6012 2ml of MeOH l TN-A-6012 2ml of MeOH 50ul of 02001-66 TN-A-6012 50ul of 02001-66 MA 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2m! of MeOH TN-A-5457 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-54S7 2ml of MeOH TN-A-5457 2ml of MeOH I TN-A-5457 ma Lf o titR -v Z Blank Sera TN-A' Amount of sera aliquated:. 4 Dilute Sera with Kandiyohi water; Amount o f water added _ml ml r Aliquot 1ml of sera Into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker O U frP Centrifuge sample for 10 min @ 2000 rpm (Centrifuge l3 iM 13 ) Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper^mixing Condition column with MeOH (TN-A- 6 o i 3 j ) Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount of solvent into appropriate 15 ml centrifuge tube Spike samples with internal standard PFDA standard number T T f i A ____________, cone. Transfer sample into appripriatelly marked autovial sr 3M Environmental Laboratory ( / / C H c O \ - <V K9 amount added. Page 167 Report E01-1256 PFOS: A Reproduction Study with the Mallard SPE Columns Extraction W orksheet _ . ^ . .. (A^ M&UwcL Sera^ LA.S 0#/jl|b2. Prep Date: Analysts initials: Urns Assgned sample number E01-1256-28695 E01-1256-28696 E01-1256-28697 E01-1256-28698 E01-1256-28699 E01-1256-28700 E01-1256-28701 E01-1256-28702 E01-1256-28703 E01-1256-28704 E01 -1256-28705 E01 -1256-28706 E01-1256-28707 E01-1256-28708 E01-1256-28709 2/26/2002 OK/HOJ Sample Number or description 454-109-3252,0 PPM, Female, Adult 454-109-3253,0 PPM, Male, Adult 454-109-3254, 0 PPM, Female, Adult 454-109-3255, 0 PPM, Male, Adult 454-109-3256,0 PPM, Female, Adult 454-109-3257,10 PPM, Male, Adult 454-109-3258,10 PPM, Female, Adult 454-109-3259,10 PPM, Male, Adult 454-109-3260,10 PPM, Female, Adult 454-109-3261,10 PPM, Male, Adult 454-109-3262,10 PPM, Female, Adult 454-109-3263,10 PPM, Male, Adult 454-109-3264,10 PPM, Female, Adult 454-109-3265,10 PPM, Male, Adult 454-109-3266,10 PPM, Female, Adult Volume of sample filtered (ml) Type o f 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 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Method Revisii0on:^ ETS-8-231 Study Number: E01-1256 Matrix: Sera Amount and spike Elution solvent mix used and volume iUA. I \V 2ml of MeOH TN-A-5457 2ml of MeOH TN-A-5457 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 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 ;f Comments > ------- / *) __-- - ------------ Blank Sera TN-A- Amount of sera aliquated:. *1 Dilute Sera with Kandiyohi water; Amount o f water added 2> Q ml ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- ) to aliquated sam ple, Shake sample for 20 min @300 rpm (ShakerU t if iJ ^ N ilQ ^ l f f 3HCentrifuge 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 Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with Indicated amount o f solvent into appropriate 15 ml centrifuge tube SDike sarrmles with internal standard PFDA standard number 3M EnvironmentalLaboratory i-v * IO O .^tqpu . amount added 2 J Page 168 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 26 Febuary 2002 ok 799 900 5 Acetonitrile tn-a-4145 Mass of solvent (g) Density1 (g/ml) Volume of Solvent 1 3.9525 0.7857 5.0305 2 3.9576 0.7857 5.0370 3 3.9768 0.7857 5.0615 4 3.9686 0.7857 5.0510 5 3.9994 0.7857 5.0902 AverageVolume: 5.0541 %Accuracy: 101.08 PASS Std. Dev.: %C.V.: Pass/Fail: 0.02354 0.47 PASS PASS Volume, (mL) 1 to 10 Peri'ormance Specificiitions Accuracy, ( %) 98.0 - 102 % C.V., ( %) 1.0 % Corrective Actions: A jb (\te c*6 iQ & o g j 'Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx = specific gravity at ydegreesCreferred to water at xdegreesc n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 =1.3255 Acetonitrile d2#4 = 0.7857 3M Environmental Laboratory Page 169 Report E01-1256 PFOS: A SPE Columns Extraction Worksheet Prep Date: Analysts initials: Urns Assgned sample number E01-1256-36263 E01-1256-36264 E01-1256-36265 E01-1256-36266 E01-1256-28710 E01-1256-28711 E01-1256-28712 E01-1256-28713 E01-1256-28714 E01-1256-28715 E01-1256-28716 E01-1256-28717 E01-1256-28718 E01-1256-28719 E01-1256-28720 E01-1256-28721 E01-1256-28722 E01-1256-28723 E01-1256-28724 E01-12S6-2872S E01-1256-28726 E01-1256-28727 E01-1256-28728 Sample Number or description Blank H20-day 3 Blank mallard sera-day 3 Mallard Sera MS-day 3 Mallard Sera MSD-day 3 454-109-3267,10 PPM, Male, Adult 454-109-3268,10 PPM, Female, Adult 454-109-3269,10 PPM, Male, Adult 454-109-3270,10 PPM, Female, Adult 454-109-3271,10 PPM, Male, Adult 454-109-3272,10 PPM, Female, Adult 454-109-3273,10 PPM, Male, Adult 454-109-3274,10 PPM, Female, Adult 454-109-3275,10 PPM, Male, Adult 454-109-3276,10 PPM, Female, Adult 454-109-3277,10 PPM, Male, Adult 454-109-3278,10 PPM, Female, Adult 454-109-3279,10 PPM, Male, Adult 454-109-3280,10 PPM, Female, Adult 454-109-3281,10 PPM, Male, Adult 454-109-3282,10 PPM, F em ale, A d ult 454-109-3283,10 PPM, Male, Adult 454-109-3284,10 PPM, Female, Adult lua-j^oo, iu rriw, rvrare, Adult Method Revision: ETS-8-231.0 Study Number: E01-1256 Matrix: Sera Volume of sample filtered (ml) Type o f column Amount and spike Elution solvent used and lot mix used and volume Comments 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot VU2036B2 Waters, 1g,6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 w&er&, 13. bltil, lot W2036B2 aja 2ml of MeOH TN-A-6012 b 2ml of MeOH TN-A-6012 2ml of MeOH 50ul of 02001-66 TN-A-6012 2ml of MeOH 50ul of 02001-66 TN-A-6012 WA 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 '1u 2ml of MeOH TN-A-6012 2ml ol MeOH TN-A-6012 NA I I I [y Blank Sera TN-A-Cp 9 X j Amount of sera allquated: ^ _______ml Dilute Sera with Kandiyohi water; Amount o f water added 3 & ml "Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- tlk 'o ) to aliquated sample ., Shake sample for 20 min @300 rom (Shaker V W f a 0 4 1 SCentrifuge sample for 10 min @ 2000 rpm (Centrifuge WUJfa ? Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water cr >> shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- Q o I aJ ) > Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go com pletely dry Elute column with Indicated amount o f solvent into appropriate 15 ml centrifuge tube & Spike samples with internal standard PFDA standard number - r ____ e_________ i . _____ : :_! ,,II.. 3M Environmental Laboratory A _________ , cone. IQO 'IW A M , amount added_ ^ Page 170 Report E01-1256 SPE Columns Extraction Worksl P uy/1 o > ) Prep Date: m 9- . Analysts initials: Q Lims Assgned sample number E01-1256-28729 E01-1256-28730 E01-1256-28731 E01-1256-28732 E01-1256-28733 E01-1256-28734 E01-1256-28735 E01-1256-28736 E01-1256-28737 E01 -1256-28738 E01-1256-28739 Sample Number or description 454-109-3286,10 PPM, Female, Adult 454-109-3287,10 PPM, Male, Adult 454-109-3288,10 PPM, Female, Adult 454-109-3289,10 PPM, Male, Adult 454-109-3290,10 PPM, Female, Adult 454-109-3291,10 PPM, Male, Adult 454-109-3292,10 PPM, Female, Adult 454-109-3293,10 PPM, Male, Adult 454-109-3294,10 PPM, Female, Adult 454-109-3295,10 PPM, Male, Adult 454-109-3296, 10 PPM, Female, Adult 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 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Method Revision: ETS-8-231.0 Study Number: E01-1256 Matrix: Sera Amount and spike Elution solveh mix used and volume HA J_ _ _ 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 2ml of MeOH TN-A-6012 M V1 Comments Mallard ------ / -------------------^ s' "1 H Blank Sera TN-A- o Q - : Amount of sera aliquated: 4 ml Dilute Sera with Kandiyohi water; Amount o f water added 3 > ml Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN fTN-A- 4 14 ^ ) to aliquated sample Shake sample for 20 min @300 rpm ( S h a k e r 1 Q , ty Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 13 Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Qr Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing $ Condition column with MeOH (TN-A- Q O 'L L 1 Wash Column with Kandiyohi Welter Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount o f solvent into appropriate 15 ml centrifuge tube SDike samDles with internal standard PFDA standard number T f ^ A 3M Environmental Laboratory , cone. -TflP XA amount added - Page 171 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID#: VWR, 5mL 27 Febuary 2002 ok 799 O i6 900 S 8 - o 2 J 5 Acetonitrile tn-a-4145 Mass of solvent (g) Density1 Volume of (g/ml) Solvent 1 3.9579 0.7857 5.0374 2 3.9883 0.7857 5.0761 3 3.9754 0.7857 5.0597 4 3.9804 0.7857 5.0661 5 3.9907 0.7857 5.0792 AverageVolume: 5.0637 %Accuracy: 101.27 PASS Std. Dev.: %C.V.: Pass/Fail: 0.01662 0.33 PASS PASS Volume, (mL) 1 to 10 Peri'ormance Specificsitions Accuracy, (%) 98.0 - 102 % Corrective Actions: |0 o u e - A -S ik A C.V., (%) 1.0 % `Density values taken from the Merck Index, 12thEdition, Copyright 1996 dy%= specific gravity at ydegreesc referred to water at xdegreesc n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d2s4 =0.7866 Methylene Chloride d204 =1.3255 Acetonitrile d204 = 0.7857 3M Environmental Laboratory Page 172 Report E01-1256 SPE Columns Extraction Repr^pfcin^t^aywjtbwh/M^ilard P re p D ate: Sfk&, M ethod Revision: E T S -8 -2 3 1 Analysts initials: Study Number: E01-1256 ^ Matrix: Sera Lims Assgned sample number Sample Number or description Volume of sample filtered (ml) Type of column Amount and spike Elution solveri used and lot mix used and volume Comments E01-1256-36267 E01-1256-36268 E01-1256-36269 E01-1256-36270 E01-1256-28740 E01-1256-28741 E01-1256-28742 E01-1256-28743 E01-1256-28744 E01-1256-28745 E01-1256-28746 E01-1256-28747 E01-1256-28748 E01-1256-28749 E01-1256-28750 E01-1256-28751 E01-1256-28752 E01-1256-28753 E01-1256-28754 E01-1256-28755 E01-1256-28756 E01-1256-28757 E01-1256-28758 E01-1256-28759 Blank H20-day 4 Blank mallard sera-day 4 Mallard Sera MS-day 4 Mallard Sera MSD-day 4 454-109-9583,0 PPM, Female, Offspring 454-109-9591,0 PPM, Male, Offspring 454-109-9589, 0 PPM, Female, Offspring 454-109-9592, 0 PPM, Female, Offspring 454-109-9598,0 PPM, Female, Offspring 454-109-9603,0 PPM, Female, Offspring 454-109-9606,0 PPM, Male, Offspring 454-109-9610,0 PPM, Female, Offspring 454-109-9573,0 PPM, Female, Offspring 454-109-9578,0 PPM, Male, Offspring 454-109-9625,10 PPM, Male, Offspring 454-109-9629,10 PPM, Female, Offspring 454-109-9633,10 PPM, Female, Offspring 454-109-9634,10 PPM, Female, Offspring 454-109-9645,10 PPM, Female, Offspring 454-109-9648,10 PPM, Female, Offspring 454-109-9636, 10 PPM, Male, Offspring 454-109-9642,10 PPM, Male, Offspring 454-109-9654,10 PPM, Female, Offspring eofl-'iUy-boa, iu rrivt, Female, 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 40+6 40+6 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lotW2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, 1g, 6ml, lot W2036B2 Waters, i(j, oml, lotW2036B2 WA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 2ml of MeOH 50ul of 02001-66 TN-A-6009 WA 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH TN-A-6009 2ml of MeOH \ TN-A-6009 UA J/ -'R Q?^>tL Blank Sera TN-A- 6022: Amount of sera aliquated: 4 ml Dilute Sera with Kandiyohi water; Amount o f water added 36 ml 'Aliquot 1ml of sera into 15 ml polypropelene tube Spike samples accordingly Add 5ml of ACN (TN-A- 4145 ) to aliquated sample Shake sample for 20 min @300 rpm (Shaker VWR S/N 041694) QKj Centrifuge sample for 10 min @ 2000 rpm (Centrifuge 3M# 7696131 Add 40 ml of Kandiyohi water to 50 ml polypropelene centrifuge tube. Decant extract into centrifuge tubes with water shake sample slightly to ensure proper mixing Condition column with MeOH (TN-A- 6009 1 Wash Column with Kandiyohi Water Filter sample through conditioned column, discarding filtrate Allow column to go completelly dry Elute column with indicated amount o f solvent into appropriate 15 ml centrifuge tube Spike samples with Internal standard PFDA standard number^P F D A , conc._100.7ppm_ , amount added 3M Environmental Laboratory 2ul Page 173 Report E01-1256 PFOS: A Reproduction Study with the Mallard Bottle-Top Dispenser Daily Calibration Check i Manufacturer and Model: Date: Initials: Dispenser ID: Balance ID: Volume (mL): Solvent: ID th VWR, 5mL 28 Febuary2002 ok 799 900 5 Acetonitrile tn-a-4145 OK l 2 3 4 5 Volume, (mL) 1to 10 Mass of solvent (g) Density1 3.9524 (g/ml) 0.7857 3.9923 0.7857 3.9673 0.7857 3.9812 0.7857 3.9989 0.7857 AverageVolume: %Accuracy: Std. Dev.: %C.V.: Pass/Fail: Volume of Solvent 5.0304 5.0812 5.0494 5.0671 5.0896 5.0635 101.27 0.02398 0.47 PASS Perlrom ance Specificsitions Accuracy, (%) 98.0 - 102 % C.V., (%) 1.0 % Corrective Actions: Me J lffl- d lo K j `Density values taken from the Merck Index, 12thEdition, Copyright 1996 dyx =specific gravity at ydegreesc referred to water at xdegrcesc n-Hexane d204 =0.660 Acetone d250 =0.788 Methanol d254 =0.7866 Methylene Chloride d204 =1.3255 Acetonitrile d204 = 0.7857 3M EnvironmentalLaboratory Page 174 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet Study: E01-1256 Dilution Date/Analyst: CD Box Number: ^ 0 q Solvent/TN Number: MeOH/5457 Extraction Date/Analyst: LXS -c&J Matrix/Timepoint: MKLLWe ^ ,Q^-; Form Completion Verified By: 3MEnvironmental Laboratory P a ^ le t i LAS j , Page 175 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet Study: E01-1256 Dilution Date/Analyst: CP Box Number: f t ) I - / Q b Solvent/TN Number: MeOH/5457 Extraction Date/Analyst: Matrix/Timepoint: FormCompletion Verified By: &VJ 4 3M Environmental Laboratory Page 176 Report E01-1256 PFOS: A Reproduction Study with the Mallard Attachment D: Dilutions Summary Worksheet Study: o ( Dilution Date/Analyst: 4 -U -o A ( o K j Box Number: - Solvent/TN Number: f a o P o O f t ^ Extraction Date/Analyst: Matrix/Timepoint: 770*A-`SVS y~ Sample Number Dilutions or Description 1 / loo 1 / 1 / 1 / 1 / 1 / 1 / Comments fo l~ \7.^(o~ ^S '^o(o Verified By: I f i ] < rS'-o^__ ,0 1 ^ ---Notes: 1/10 dilution = aA of sample + of solvent ||l<5oC^txll'OU- fo jjS ) SOJLup^f + ^ 9 0 ^ i Q^f S C & b '& S J ( ^ ) K A * * /* * 3M Environmental Laboratory Form Completion Verified By: M S 4` r ^ r r '>n ______________ (^ Page, l of l IASoslujbx "j6j . , ,. Jt Page 177 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Attachment E: Study Protocol, Protocol A m endm ents, and D e v ia t io n s QM Pntiirnnm ntol I ohnrot/MV 3M EnvironmentalLaboratory P a n o 1 7 A n f 91 ft Page 178 Report E01-1256 PFOS: A Reproduction Study with the Mallard W ildlife International, ltd PROJECTNO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDYWITHTHE MALLARD PROTOCOLNO.: 454/120700/MR/SUB454 AMENDMENTNO.: 9 SPONSOR: 3M Corporation PROJECTNO.: 454-109, E01-1256 EFFECTIVE DATE: January 24, 2002 AMENDMENT: ' This amendment includes an attachment (Attachment A) which provides the information necessaryto complete the analytical phase ofthis study. REASON: The protocol did not include information for completing foe analyses requiredby foe sponsor. AMENDMENT: Harold Johnson is foe Principal Analytical Investigator REASON: The sponsor requestedthat a principal analytical investigator be addedto foe protocol. AMENDMENT: The 3M Environmental Laboratory Project IdentificationNumberto be included with foe study is E01-1256. REASON: The sponsor requestedthat foe 3M Project IdentificationNumberbe included infoe protocol. 3M Environmental Laboratory Page l o f 2 Page 179 le p o it/E 1 i2 3 6 FAX 410 822 0632 ,, . PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL F 002 I JAN. 23.2002 5:25PM ENVIRONMENTAL LAB 2 3E 09 NO. 5293 P. 3 W ildlife International, ltd M L . f- SesiaP. Gallagher, StudyDirector" Joann BL. oBceavear*s,. Wilkdflliiffe International, LTD, Laboratoiy Managern HaroldO. Johnson, PROJECTNO.: 454-109, E01-1256 11^1 Date t |t*4 |o 2 Date 4 - 4 **2- .. Date 3M Environmental Laboratory Page2 of 2 Page 180 Report E01-1256 W il d l if e In t e r n a t io n a l , l td PFOS: A Reproduction Study with the Mallard PROJECTNO.: 454-109, E01-1256 Sean P. Gallagher, Study Director Joann B. Beavers, Wildlife International, LTD, Laboratory Management HaroldO. Johnson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date Date Date Date 3M Environmental Laboratory Page 2 o f2 Page 181 Report E01-1256 PFOS: A Reproduction Study with the Mallard Protocol Amendment # 9 - Attachment A W ildlife International Ltd. # 454-109 STUDY TITLE PFOS: A Reproduction Study with the Mallard SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: E01-1256 Wildlife International Ltd.: 454-109 3M Environmental Laboratory Page 182 Report E01-1256 PFOS: A Reproduction Study with the Mallard Sponsor Study Identification PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 Principal Analytical Investigator Study Locations Analytical Testing Laboratory Analytical Testing Laboratory 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 Analytical Experimental Start Date Analytical Experimental Termination Date 01/24/02 03/24/02 3M EnvironmentalLaboratory 3M EnvironmentalLaboratory 2 Page 183 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1. Purpose/O bjecttves In the in-life phase o f this study, mallards were regularly exposed to nominal doses o f perfluorooctanesulfonate, potassium salt (PFOS) in their feed in order to evaluate the effects o f dietary exposure upon adults over a 21-week period. The purpose o f this analytical phase o f the study is to quantify PFOS in the liver and sera o f both control and dosed mallards. The analytical information may be used to compare with toxicological/histopathologicai results from the in-life portion o f this bird study. The in life portion o f the study was completed at Wildlife International, Ltd. in study 454-109. 2. Regulatory Compliance This analytical phase study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. Quality A ssurance The 3M Environmental Laboratory Quality Assurance Unit will audit analytical phase study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The Quality Assurance Unit will report all findings to the Sponsor Representative and the Study Director. 4. Reference Substance 4.1 Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity and Stability--The purity o f Lot #217 has been determined to be 86.9%. PFOS has been shown to be stable in methanol solutions for up to 6 months at ambient temperature in the validation report from study PS013, "Method Validation and Instrument Detection Limit Determination for LC/MS and GC/MS Analysis o f FC807 Compounds in Methanol." Refer to the Certificate o f Analysis (stored in the Test, Control, and Reference database in the 3M Environmental Laboratory) for further purity and stability information. 4.3 Storage Conditions--The test article may be stored at ambient temperature or lower. 4.4 Reserve Samples--A reserve sample o f PFOS from Lot #217 has been frozen at 20C 5C or colder and is kept in fee 3M Environmental Laboratory. 4.5 CAS Number--The CAS number is: 2795-39-3. 4.6 Molecular Weight--The molecular weight is 538 (C8F17SO3K). Note: The molecular weight o f fee anion (PFOS) is 499 (CgFnSOs ). 3M E nvironm ental Laboratory 3M Environmental Laboratory 3 Page 184 Report E01-1256 PFOS: A Reproduction Study with the Mallard 5. Control Matrices Types o f control matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. 6. Te s t S ystem The test system is the mallard (Anasplatyrhynchos). The test system was dosed (at Wildlife International Ltd. during the in-life phase o f this study) with food prepared by mixing PFOS with feed in a large mixer in appropriate portions to achieve the nominal 10 ppm level. A food dose verification validation study was performed by Wildlife International under study number 454C-110 (3M #U2723), "Analytical Method Verification for the Determination o f PFOS in Avian Diet." Wildlife International verified the actual dosed food for this study and the results will be contained in the in-life Wildlife phase report. Mallard serum and liver samples were collected by Wildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. 6.1 Justification o f the Selection o f the Test System Mallard duck represent wild bird populations and they are an EPA recommended species because they do well in a laboratory environment. The use o f these matrices for the study is to determine if PFOS was deposited in the liver and serum o f mating adults that were fed PFOS. Furthermore, analysis o f these matrices from hatchlings will determine if PFOS was passed on to mating adults' offspring. 7. Source o f Su pply for the Te s t S ystem The birds were obtained by Wildlife International, Ltd. See their protocol (study 454-109) for information about bird origination location. 8. Specim en an d Sam ple Rec eipt a nd Maintenance Sera and liver were collected by Wildlife International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 21-week endpoint sera and liver samples received by the 3M Environmental Laboratory were from the following birds: 40 control adult birds (20 mating pairs) that ingested food containing 0 ppm PFOS 40 experimental adult birds (20 mating pairs) that ingested food containing 10 ppm PFOS 2 0 first generation offspring birds that ingested food containing 0 ppm PFOS Samples will be maintained frozen except when removed for extraction and analysis as described in the method. The samples will be kept isolated from the test substance during storage. 3MEnvironmental Laboratory 3M Environmental Laboratory 4 Page 185 Report E01-1256 PFOS: A Reproduction Study with the Mallard 9. SUB-CONTRACTED ANALYSIS 9.1 All sera and liver extractions as detailed in this protocol amendment will be performed at Pace Tier 2 ,1 7 0 0 Elm Street, Suite 200, Minneapolis, MN 55414. 9.2 All sera and liver analyses as detailed in this protocol amendment will be performed at 3M Environmental Laboratory, Building 2-3E -09,935 Bush Avenue, St. Paul, MN 55106. 10. Experimental Conduct The 21-week endpoint liver and sera samples received (see section 8 ) will be extracted and analyzed for PFOS quantities using the methods listed below. 10.1 Preparatory Methods 10.1.1 ETS-8 -6 , Extraction o f Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.1.2 ETS-8-4, Extraction o f Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 Analytical Methods 10.2.1 ETS-8-7, Analysis o f Fluorochemicals in Liver Extracts Using HPLCElectrospray / Mass Spectrometry 10.2.2 ETS-8-5, Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry 11. Method Validation Method validation for PFOS quantitation in mallard sera and liver was performed at Centre Analytical Laboratories, Inc. in study 023-044. The method will be applied to sample analyses without additional validation. 12. Data Q uality Objectives D uring Sa m ple A nalysis The following criteria will be met using the validated calibration levels and fortification levels determined in section 1 1 : 12.1 System Suitability System suitability will be determined prior to the start and at the completion o f each analytical run. Prior to the calibration curve and after the last sample o f the run three (3) mid-level unextracted calibration standards will be analyzed. The peak area precision and retention time precision and peak asymmetry will be monitored at the beginning and the end o f the run separately. 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 and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is the asymmetry factor. If any item o f the system suitability fails, system maintenance must be completed prior to running a 3M E nvironm ental Laboratory 3M Environmental Laboratory 5 Page 186 Report E01-1256 PFOS: A Reproduction Study with the Mallard 12.2 12.3 second set o f system suitability samples and the system suitability must pass before starting the calibration. Calibration It will consist o f a minimum o f nine (9) levels, including a blank with surrogate and a blank without surrogate, the lowest standard will be at approximately 50% o f the LLOQ and the highest standard will be approximately 50% higher than the ULOQ. The standard curve equation will be determined by regression analysis using the peak areas o f the analyte. The accuracy o f each level will be verified. Any level outside 80% - 1 2 0 % accuracy must be deactivated, and regression re-calculated. The curve must have a correlation o f determination (r2) greater than or equal to 0.985. All levels must show a response greater than two times that o f the blank. The total number o f standards excluded my not exceed 20%. I f the preceding criterion are not met, the curve and sample set will be reanalyzed. Limits o f Quantitation (LOQ) The lower limit o f quantitation (LLOQ) was determined to be 10 ng/g in liver and 10 ng/mL serum during the method validation as described above in section 11. The LLOQ may be determined to be high- than that found in the validation study during a screening to be done for endogenous levels o f PFOS in the blank matrices available at 3M Environmental Laboratory. I f endogenous levels o f PFOS are found to be higher than the LLOQ or if the levels are found to be highly variable, the LLOQ will be adjusted to accommodate this. The screening procedures and LLOQ determination will be recorded in a note to file and kept with the raw study data as well as being reported in the final report. Recovery efficiency at the LLOQ will be determined during each run using an extracted quality control sample spiked at the LLOQ level. If the quality control sample at the LLOQ level fails during a run, the LLOQ for that specific run would be considered to be the next lowest passing quality control sample and any samples below the new LLOQ must be reanalyzed. The upper limit o f quantitation (ULOQ) was determined to be 250 ng/g in liver and 250 ng/mL in serum during the method validation as described above in section 11. An extracted quality control sample spiked at the ULOQ will be used to measure recovery efficiency at the ULOQ. If the quality control sample at the ULOQ fails during a run the ULOQ for that specific run would be considered to be the next highest passing quality control sample and any samples above the new ULOQ must be reanalyzed. Any sample with an area greater than that 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 performed to verify sample values. To perform the dilution validation, one sample will be separated into tw o representative samples 3MEnvironmentalLaboratory 6 3M Environmental Laboratory Page 187 Report E01-1256 PFOS: A Reproduction Study with the Mallard (i.e. tw o 1 mL aliquots for fluid samples or two 1 gram amounts for tissue samples) then diluted using tw o procedures. The first procedure consists o f diluting the sample with additional matrix prior to extraction (sera adding sera), while the second procedure consists o f diluting the extract with solvent post extraction (methanol extract adding additional methanol solvent). 12.4 12.5 If the values are not within 15% o f each other additional testing will be required to determine which value is a correct representation o f the sample concentration. Continuing Calibration Verifications An unextracted midlevel standard (continuing calibration verification - CCV) will be analyzed after every 1 0 injections (starting after the last injection o f the calibration curve). The accuracy o f the CCV must be within 20%. Data may only be reported if bracketed by passing CCV's (or a CCV and the same level calibration curve point). Data bracketed by one or more failing CCV's must be reanalyzed. Quality Control Samples A minimum o f three (3) quality control samples (QC) will be prepared with each set o f samples. They will consist o f blank matrix spiked with three levels o f analyte. The levels are: Low level: at the LLOQ (10 ng/mL in serum and 10 ng/g in liver), Mid-level: 5 times the LLOQ, High level: at the ULOQ (25 times the LLOQ) 12.6 QC 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 must meet these criteria. I f not, the entire sample set must either be re-analyzed or re-extracted (as decided by the Study Director). 12.7 Use o f Confirmatory Methods Confirmatory methods are typically not needed with LC/MS/MS analysis. 12.8 Demonstration o f Specificity PFOS specificity will be substantiated by chromatographic retention time, by the characteristic primary ion (499), and the characteristic product ion (99) using LC/MS/MS. 13. Sub-C ontracted A nalysis 13.1 All fluid and tissue extractions as detailed in this protocol amendment will be performed at Pace Tier 2 ,1 7 0 0 Elm Street, Suite 200, Minneapolis, MN 55414. 13.2 All fluid and tissue analyses as detailed in this protocol amendment will be performed at 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 3M E nvironm ental Laboratory 7 3M Environmental Laboratory Page 188 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1 3 .3 Pace Analytical Services, Inc. will follow all applicable 3M Environmental Laboratory Standard Operating Procedures and the following Pace Analytical Inc. Standard Operating Procedures. PSS-Admin-01 Quality System PSS-Admin-04 Laboratory Facilities PSS-Admin-06 Training Record Files Maintained by QAU PSS-ARC-02 Disposition o f Archive Materials PSS-ARC-04 Archiving Data, Specimen and Logbooks PSS-DC-05 Lab Notebooks, Logbooks, and Phone Logs PSS-DM-03 Statistical Evaluation o f Data PSS-OPS-Ol U se and Maintenance o f Ultra-Turrax T25 Homogenizer PSS-OPS-02 Raw Data PSS-OPS-03 Hazardous Waste Disposal P SS-O P S-04 P SS-O P S-05 P SS-O P S-06 U se and Maintenance o f N-Evap Analytical Evaporator U se o f Compressed Gases in the Laboratory U se and Maintenance o f NANOpure II Water Purification System PSS-OPS-07 Cleaning Non-disposable Volumetric Pipettes PSS-OPS-08 Laboratory Calculations PSS-OPS-09 Deviations from Established Procedures PSS-OPS-IO Analytical Definitions PSS-OPS-11 U se and Calibration o f a Bottle-Top Dispenser PSS-OPS-13 U se and Maintenance o f Lab Refrigerators, Freezers, and Ovens PSS-OPS-14 U se and Calibration o f Syringes with a Hamilton Chaney Adapter PSS-OPS-15 Glassware Cleaning PSS-OPS-16 U se and Maintenance o f the Fisher Scientific Isotemp Freezer PSS-OPS-17 U se and Maintenance o f Ultrasonic Water Baths (Sonicators) PSS-OPS-18 U se and Maintenance o f Vortex Shakers PSS-OPS-24 U se and Maintenance o f a Desiccator PSS-OPS-31 U se and Maintenance o f Shakers PSS-OPS-32 Operation, M aintenance and Calibration o f Laboratory PSS-OPS-37 Operation, Maintenance, and Calibration o f pH Meters and pH Electrodes PSS-MC-01 Purchasing o f Laboratory Supplies , PSS-MC-02 Receipt o f Laboratory Supplies PSS-MTR-01 Calibration o f Certified Weight Set PSS-MTR-02 Calibration o f Certified Thermometers/Thermocouples PSS-MTR-05 Calibration o f EppendorfPipettes PSS-MTR-06 Verification o f Calibration o f the EppendorfPipettor PSS-MTR-07 N ew Equipment Qualification (IQ, PQ, OQ) PSS-MTR-08 Verification o f Calibration and U se o f Analytical Balances PSS-MTR-09 U se and Maintenance o f Fisher Scientific Electronic Temperature Monitoring Devices PSS-Safety-01 General Laboratory Safety PSS-Safety-02 U se and Maintenance o f Biological Safety Cabinets 3M Environmental Laboratory 3M Environmental Laboratory 8 Page 189 Report E01-1256 PFOS: A Reproduction Study with the Mallard PSS-Safety-04 Emergency Evacuation Procedures PSS-Safety-05 U se and Maintenance o f Laboratory Fume Hoods PSS-TS-01 Training Procedures 13.4 An amendment to the protocol will be written if extractions and analyses are performed at laboratories other than the Pace Tier 2 or 3M Environmental Laboratory, respectively. 14. S tatistical A nalysis Statistical methods will be limited to the calculation o f means and standard deviations. Examples 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 Environmental Laboratory. The final report will follow all GLP requirements, and will include a description o f all materials and methods used, including a narrative discussion with a tabular presentation o f all analyses results. 15. L ocation of Ra w Data, Records, an d Final Repo rt When the final report is completed, all original paper data, including protocol, phase report, study correspondence, sample receipt and tracking, sample preparation, and analytical data, will be retained in the archives o f 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained either at Pace Tier 2 or the 3M Environmental Laboratory, as applicable. 17. Specimen Retention Samples o f the test substance (any leftover control sera or liver and samples) and the analytical standards will be maintained in the laboratory for a period o f time as specified by regulation or as long as the quality o f the preparation affords evaluation. However, samples will not be maintained more than 10 years after the effective date o f the final test rule (if applicable). 3M Environmental Laboratory Standard Operating Procedures also apply to sample retention times. 18. References 18.1 Wildlife International, Ltd. Protocol No. 454-109, "PFOS: A Reproduction Study 18.2 with the Mallard". Centre Analytical Laboratories, Inc. Study 023-044, "Validation o f Analytical Methods "Extraction o f Potassium Perfluoroocatanesulfonate or Other Fluorochemical Compounds From Serum for Analysis Using HPLC Electrospiy/Mass Spectrometry" and "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds From Liver for Analysis Using HPLC Electrospray/Mass Spectrometry" in Mallard Serum and Liver." 3M E nvironm ental Laboratory 3M Environmental Laboratory 9 Page 190 Report E01-1256 PFOS: A Reproduction Study with the Mallard 18.3 3M Study U2723, Wildlife Study 454C -110, "Analytical Method Verification for the Determination o f PFOS in Avian Diet." 19. Protocol A mendments an d Deviations Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations, signed by the Study Director and Sponsor Representative and filed with the raw data. All changes to the protocol will be indicated in the final report. 3M E nvironm ental Laboratory 3M Environmental Laboratory 10 Page 191 Report/B01-B3 5 6 8 FAX 410 8 2 2 0 6 3 2 W ILD LIFE INTERNAT I oRKDS: A Reproduction Study withthe Mallard W ildlife International, ltd PROJECTNO.: 454-109, E01-1256 am endm ent to study protocol STUDY TITLE: PFOS. A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/MR/SUB454 AMENDMENTNO.: 10 SPONSOR: 3M Corporation PROJECTNO.: 454-109, E01-1256 EFFECTIVE DATE: January 30, 2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase of this study. This attachment A replaces the attachment A in amendment 9. REASON: The amendment 9 attachment A was updated to include a different extraction and analytical method, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director Date _________ Joann B. Bhvers, Wildlife International, LTD, Laboratory Management Harold O. Johnson, Pratpal Analytical Investigator ____ ______________ Date & C / j i l o Z . ________ Date William K. Reagen, 3M Environmental Laboratory, --Laboral ____ 2 , 3 . Date ffCobideau, Sponsors Representative Date 3M Environmental Laboratory 1 of 9 Page 192 Report E01-1256 PFOS: A Reproduction Study with the Mallard W ildlife International, ltd PROJECT NO.: 454-109, .............. ..... E0 1 - 1 2 5 6 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/MR/SUB454 AMENDMENT NO.: 10 SPONSOR: 3M Corporation PROJECT NO.: 454-109, E01-1256 EFFECTIVE DATE: January 30,2002 AMENDMENT: This amendment includes an attachment (Attachment A) which provides updated information necessary to complete the analytical phase of this study. This attachment A replaces the attachment A in amendment 9. REASON: The amendment 9 attachment A was updated to include a different extraction and analytical method, to update the data quality objectives, and to change the liver and sera extraction location. Sean P. Gallagher, Study Director Date Joann B. Beavers, Wildlife International, LTD, Laboratory Management Harold O. Johnson, Principal Analytical Investigator Date Date William K. Reagen, 3M Environmental Laboratory, Laboratory Management Rochelle R. Robideau, Sponsor's Representative Date Date 3M Environmental Laboratory 1 Of 9 Page 193 Report E01-1256 PFOS: A Reproduction Study with the Mallard Protocol Amendment # 10 - Attachment A Wildlife International Ltd. #454-109 STUDY TITLE PFOS: A Reproduction Study with the Mallard SPONSOR 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 PERFORMING ANALYTICAL LABORATORY 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 LABORATORY PROJECT IDENTIFICATION 3M Environmental Laboratory: E01-1256 W ildlife International Ltd.: 454-109 3M Environmental Laboratory 3M Environmental Laboratory 2 of 9 Page 194 Report E01-1256 PFOS: A Reproduction Study with the Mallard Sponsor Study Identification PFOS: A Reproduction Study with the Mallard 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 Principal Analytical Investigator Study Locations Analytical Testing Laboratory Harold Johnson 3M Environmental Laboratory Building 2-3E-09 PO Box 33331 St. Paul, MN 55133-3331 651-778-6479 3M Environmental Laboratory Building 2-3E-09 935 Bush Ave. PO Box 33331 St. Paul, MN 55133-3331 Proposed Study Timetable Analytical Experimental Start Date Analytical Experimental Termination Date 01/31/02 03/31/02 3M Environmental Laboratory 3M Environmental Laboratory 3 of 9 Page 195 Report E01-1256 PFOS: A Reproduction Study with the Mallard 1. P urpose/O bjectives In the in-life phase o f this study, mallards were regularly exposed to nominal doses o f perfluorooctanesulfonate, potassium salt (PFOS) in their feed in order to evaluate the effects o f dietary exposure upon adults over a 21-week period. The purpose o f this analytical phase o f the study is to quantify PFOS in the liver and sera o f both control and dosed mallards. The analytical information may be used to compare with toxicological/histopathological results from the in-life portion o f this bird study. The in life portion o f the study was completed at W ildlife International, Ltd. in study 454-109. 2. Regulatory Compliance This analytical phase study w ill be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 3. Quality A ssurance The 3M Environmental Laboratory Quality Assurance Unit w ill audit analytical phase study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The Quality Assurance Unit w ill report all findings to the Sponsor Representative and the Study Director. 4. Reference Substance 4.1 Physical Description- Perfluorooctanesulfonate, potassium salt from Lot 217 is a white crystalline material. 4.2 Purity and Stability--The purity o f Lot #217 has been determined to be 86.9%. PFOS has been shown to be stable in methanol solutions for up to 6 months at ambient temperature in the validation report from study PS013, "Method Validation and Instrument Detection Limit Determination for LC/MS and GC/MS Analysis o f FC807 Compounds in Methanol." Refer to the Certificate o f Analysis (stored in the Test, Control, and Reference database in the 3M Environmental Laboratory) for further purity and stability information. 4.3 Storage Conditions--The test article may be stored at ambient temperature or lower. 4.4 Reserve Samples--A reserve sample o f PFOS from Lot #217 has been frozen at 20C 5C or colder and is kept in the 3M Environmental Laboratory. 4.5 CAS Number--The CAS number is: 2795-39-3. 4.6 Molecular Weight--The molecular weight is 538 (CgFnSOaK). Note: The molecular weight o f the anion (PFOS) is 499 (C8F17SO3 ). 3M Environmental Laboratory 3M Environmental Laboratory 4 of 9 Page 196 Report E01-1256 PFOS: A Reproduction Study with the Mallard 5. Control Matrices Types o f control matrices and their source, physical description, storage requirements, and traceability numbers w ill be recorded in the raw data and included in the final report. 6. Test S ystem The test system is the mallard (Anas platyrhynchos). The test system was dosed (at W ildlife International Ltd. during the in-life phase o f this study) with food prepared by mixing PFOS with feed in a large mixer in appropriate portions to achieve the nominal 10 ppm level. A food dose verification validation study was performed by W ildlife International under study number 454C -110 (3M #U 2723), "Analytical Method Verification for the Determination o f PFOS in Avian Diet." W ildlife International verified the actual dosed food for this study and the results w ill be contained in the in-life W ildlife phase report. Mallard serum and liver samples were collected by W ildlife International, Ltd. and sent to 3M Environmental Laboratory for analysis. 6.1 Justification of the Selection of the Test System Mallard duck represent wild bird populations and they are an EPA recommended species because they do w ell in a laboratory environment. The use o f these matrices for the study is to determine if PFOS was deposited in the liver and serum o f mating adults that were fed PFOS. Furthermore, analysis o f these matrices from hatchlings w ill determine if PFOS was passed on to mating adults' offspring. 7. Source o f Supply for the Test S ystem The birds were obtained by W ildlife International, Ltd. See their protocol (study 454 109) for information about bird origination location. 8. Specimen and Sam ple Receipt and Maintenance Sera and liver were collected by W ildlife International, Ltd. and shipped to the 3M Environmental Laboratory for analysis. The 21-week endpoint sera and liver samples received by the 3M Environmental Laboratory were from the following birds: 40 control adult birds (20 mating pairs) that ingested food containing 0 ppm PFOS 40 experimental adult birds (20 mating pairs) that ingested food containing 10 ppm PFOS 20 first generation offspring birds that ingested food containing 0 ppm PFOS Samples w ill be maintained frozen except when removed for extraction and analysis as described in the method. The samples w ill be kept isolated from the test substance during storage. 3M Environmental Laboratory 3M Environmental Laboratory 5 of 9 Page 197 Report E01-1256 PFOS: A Reproduction Study with the Mallard 9. SUB-CONTRACTED ANAL YSIS All sera and liver extractions and analyses as detailed in this protocol amendment w ill be performed at 3M Environmental Laboratory, Building 2-3E -09,935 Bush Avenue, St. Paul, MN 55106.55414. 10. Experimental Conduct The 21-week endpoint liver and sera samples received (see section 8) w ill be extracted and analyzed for PFOS quantities using the methods listed below. 10.1 Preparatory and Analytical Method 10.1.1 ETS-8-231 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices 11. Data Quality Objectives D uring Sample A nalysis Method validation for PFOS quantitation in rat and mouse sera and liver is in process in the 3M Environmental Laboratory study E01-1277. Limited Mallard control matrix preclude a cross validation, however, the E01-1277 study methods w ill be applied to sample analyses o f Mallard sera and liver samples using the following performance based criteria: 11.1 System Suitability System suitability w ill be determined prior to the start and at the completion o f each analytical run. Prior to the calibration curve and after the last sample o f the run three (3) mid-level unextracted calibration standards w ill be analyzed. The peak area precision and retention time precision w ill be monitored at the beginning and the end o f the run separately. 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. If any item o f the system suitability fails, system maintenance must be completed prior to running a second set o f system suitability samples and the system suitability must pass before starting the calibration. 11.2 Absolute Recovery The absolute recovery o f the method w ill be evaluated one time in both liver and serum. For each matrix a minimum o f triplicate samples w ill be fortified at a minimum o f two levels o f PFOS that bracket the range o f analyte determined for the samples (i.e., at near the highest level determined in samples and at -150% o f LOQ for samples). The samples w ill be extracted through the method, and analyzed by comparison with an external calibration o f non-extracted standards. The non-extracted standard calibration curves w ill be prepared in methanol, and w ill consist o f a minimum o f nine (9) levels, including a methanol blank. The best appropriate regression w ill be used to describe this curve (for best accuracy at all levels o f the standards). 3M Environmental Laboratory 3M Environmental Laboratory 6 of 9 Page 198 Report E01-1256 PFOS: A Reproduction Study with the Mallard The accuracy (% absolute recovery) and precision (%CV o f the recoveries) w ill be determined at each level using the external calibration curve for quantitation. There is no control limit for accuracy; precision must be better than 20% at each level. Should a level (high or low) not meet this precision, the method w ill only be considered valid for levels between the two valid levels. Additional testing may 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 matrix by spiking the matrix with known concentrations o f the analyte and surrogate. A calibration curve should consist o f at least nine standard curve points covering the expected range, including the LLOQ. The simplest model that adequately describes the concentration-response relationship should be used. The accuracy o f each level w ill be verified. Any level outside 75% -125% o f nominal must be deactivated, and regression re-calculated, except the LLOQ which must be within 30% o f nominal. A ll levels must show a response greater than twice that o f the matrix blank. A maximum o f four (4) levels may be deactivated in any one set, or the set w ill be re-analyzed. 11.4 Limits o f Quantitation (LOQ) The lower limit o f quantitation (LLOQ) w ill be defined in liver (ng/g) and in serum (ng/mL) as the lowest acceptable extracted calibration curve point that is within the 70% -130% nominal criteria and with an analyte peak area at least 2 times the blank. The LLOQ determination w ill be recorded in a note to file and kept with the raw study data as w ell as being reported in the final report. The upper limit o f quantitation (ULOQ) w ill be defined in liver and in serum as the highest acceptable extracted curve point that is within the 75% -125% nominal criteria and includes applicable dilution factor corrections. 11.5 Quality Control Samples A minimum o f one set o f nine (9) quality control samples (QC) w ill be prepared one time in each matrix. They w ill consist o f three levels o f analyte, each in triplicate. The quality control samples should be prepared in each matrix by spiking the control matrix with known concentrations o f the analyte. The levels bracket the sample concentrations determined in the study: Low level: equivalent to an analyte concentration near or below the lowest level o f analyte determined in samples M id-level: equivalent to an analyte concentration near the middle o f the sample range High level: equivalent to an analyte concentration near or above the highest level o f analyte determined in samples 11.6 Quality Control Sample Acceptable Recoveries 3M Environmental Laboratory 3M Environmental Laboratory 7 of 9 Page 199 Report E01-1256 PFOS: A Reproduction Study with the Mallard Each QC is expected to show an accuracy o f 75-125% o f expected. A minimum o f 2/3 (6 out o f 9) o f all QC must meet these criteria and a minimum o f one half (2 out o f 3) o f the QC at each level must meet these criteria. If not, the entire sample set must either be re-analyzed or re-extracted (as decided by the Principal Analytical Investigator). 11.7 Matrix Spikes Two matrix spikes w ill be extracted with each sample preparation. The matrix spikes w ill consist o f one fortification level in the range o f the samples, extracted in duplicate. If the matrix spike and/or matrix spike duplicate fall outside o f 25% accuracy, the curve, CCV's, matrix spikes and samples w ill be reanalyzed. 11.8 Blanks One control matrix and one water method blank w ill be extracted with each sample 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 must be within 25%. Data may only be reported if bracketed by passing CCV's (or a CCV and the same level calibration curve point). Data bracketed by one or more failing CCV must be reanalyzed. 11.10 Use of Confirmatory Methods Confirmatory methods are typically not needed with LC/MS/MS analysis. 11.11 Demonstration of Specificity PFOS specificity w ill be substantiated by chromatographic retention time, by the characteristic primary ion (499), and the characteristic product ion (99) using LC/MS/MS. 12. Sub-C ontracted A nalysis 12.1 A ll fluid and tissue extractions and analyses as detailed in this protocol amendment w ill be performed at the 3M Environmental Laboratory, Building 23E -09,935 Bush Avenue, St. Paul, MN 55106. 12.2 An amendment to the protocol w ill be written if extractions and analyses are performed at laboratories other than the 3M Environmental Laboratory. 13. Statistical A nalysis Statistical methods w ill be limited to the calculation o f means and standard deviations. Examples o f the calculations used in the analyses w ill be included in the analytical phase report. 3M Environmental Laboratory 3M Environmental Laboratory 8 of 9 Page 200 Report E01-1256 PFOS: A Reproduction Study with the Mallard 14. Report A final report w ill be prepared by the 3M Environmental Laboratory. The final report w ill follow all GLP requirements, and w ill include a description o f all materials and methods used, including a narrative discussion with a tabular presentation o f all analyses results. 15. L ocation of Ra w Data, Records, and Final Report When the final report is completed, all original paper data, including protocol, phase report, study correspondence, sample receipt and tracking, sample preparation, and analytical data, w ill be retained in the archives o f 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods w ill be retained at the 3M Environmental Laboratory, as applicable. 16. Specimen Retention Samples o f the test substance (any leftover control sera or liver and samples) and the analytical standards w ill be maintained in the laboratory for a period o f time as specified by regulation or as long as the quality o f the preparation affords evaluation. However, samples w ill not be maintained more than 10 years after the effective date o f the final test rule (if applicable). 3M Environmental Laboratory Standard Operating Procedures also apply to sample retention times. 17. References 18.1 18.2 W ildlife International, Ltd. Protocol No. 454-109, "PFOS: A Reproduction Study with the Mallard". 3M Study U2723, W ildlife Study 454C -110, "Analytical Method Verification for the Determination o f PFOS in Avian Diet." 18. Protocol Amendments and Deviations Planned changes to the protocol w ill be in the form o f written amendments signed by the Study Director and the Sponsor Representative. Amendments w ill be considered as part o f the protocol and w ill be attached to the final protocol. Any other changes w ill be in the form o f written deviations, signed by the Study Director and Sponsor Representative and filed with the raw data. A ll changes to the protocol w ill be indicated in the final report. 3M Environmental Laboratory 3M Environmental Laboratory 9 of 9 Page 201 Report E01-1256 PFOS: A Reproduction Study with the Mallard W il d l if e In t e r n a t io n a l , l t d PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOLNO.: 454/12070Q/QR/SUB454 AMENDMENT NO.: 11 SPONSOR: 3M Corporation PROJECTNO.: 454-109, E01-1256 EFFECTIVE DATE: March 27,2002 AMENDMENT: Section 11.1, Sentence 4 Change: The peak area precision must be equal to or less than 2.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD. To: The peak area precision must be equal to or less than 5.0% RSD, the precision of the 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 andthe fact that analyte ionization can be suppressed by other species present in foe sample matrix and mobile phase make area reproducibility more challenging to maintain. Sample and source cleanliness are foe determining factors, but < 2.0% is still a bit aggressive for mass spec. The application chemists at Micromass, the instrument vendor, suggest a value o f <5.0%. AMENDMENT: Section 11.3, Sentence 5. Change: Any level outside 75% -125% o f nominal value must be deactivated, and regression recalculated, except the LLOQ which must be within 30% of nominal. To: In some cases the lowest curve point, although outside the 30% nominal requirement, may be included in the linear 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 foe limit of quantitation. The limit of quantitation will be foe next point on foe curve meeting foe 30% nominal requirement. REASON: The removal o fthe lowest calibration curve point in some cases causes the next lowest calibration point to deviate by greater than 30%. Removal o fthis point in turn causes foe next calibration point to deviate by greater than 30%. This continued deletion of calibration points in some cases could continue until there are very few points 3M Environmental Laboratory Page 1 o f 3 Page 202 Report E01-1256 PFOS: A Reproduction Study with the Mallard remaining in the curve. The inclusion o f a curve point with a deviation greater than 30% is used to influence the lower end o f the curve and is not used for determination of the LOQ. AMENDMENT: Section 11.3, the last sentence. Change: A maximum of four (4) levels may be deactivated in any one set, or the set will be reanalyzed. To: The calibration curve will contain a minimum of six (6) calibration curve points. REASON: In most cases the calibration curves contain greater than nine (9) calibration points. The large number 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 of determination for the calibration curve should be > 0.985. REASON: No coefficient of determination is included in the protocol. It was inadvertently removed prior to printing of the final copy. 3M Environmental Laboratory Page 2 o f 3 Page 203 Report E01-1256 W il d l if e In t e r n a t io n a l , l t d PFOS: A Reproduction Study with the Mallard PROJECTNO.: 454-109, E01-1256 Sean P. Gallagher, Study Director Joann B. Beavers, Wildlife International, LTD Laboratory Management ^ .. .................... Harold 0 . Johnsoiif^rihcipal Analytical Investigator Date Date 3M Environmental Laboratory Page 3 o f 3 Page 204 PAI 41, <22 o n t m u r e intern*tioPFPS: A Reproductio" Study W M " MaJlald 94/Q1SQ2 18:29 BSD INF TECH 2-2E-01 -> 84108228915 NO.305 P004 W jg F E In ter n a tio n a l, ltd PROJECTNO.: 454-109, E0M2S6 S<^ap3CVbUagJghbeer, S lu DinciBr " u Joa^jBRL^even^ WdUfe International, LTD Harold Johnson, Principal Analytical Investigator WiU'ctmK. R&gea, 3M Environmental Laboratory. 4 |v | o v Date Date Date i i ! Iri I '! i 'i 3 M Environmental Laboratory \\ Page 3 of3 Page 205 Report E01-1256 W il d l if e In t e r n a t io n a l , l t d PFOS: A Reproduction Study with the Mallard PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 AMENDMENT NO.: 12 SPONSOR: 3M Corporation PROJECT NO.: 454-109, E01-1256 EFFECTIVE DATE: August 16,2002 AMENDMENT: Change: The 3M Environmental Laboratory Project Identification Number (LIMS) to be included with the study is E01-1256. To: The main 3M Environmental Laboratory Project Identification Number (LIMS) to be included with the study is E01-1256. Additional tracking numbers associated with this study are U2723, E01-1326, and E01-1367. REASON: Mallard samples were received in the 3M Environmental at several time points. Upon arrival, samples were assigned a LIMS tracking number. Not aware of the scope of this or any other particular study pertaining to these samples, they were checked in to different 3M LIMS projects. LIMS project numbers assigned to this study include the following: U2723, E01-1256, E01-1326, and E01-1367. Samples in each of these projects pertaining to this study were analyzed under one main study number E01-1256. AMENDMENT: Change: Harold Johnson is the Principal Analytical Investigator. To: Lisa A. Stevenson is the Principal Analytical Investigator. REASON: To change role of the Principal Analytical Investigator. 3M Environmental Laboratory Page 1 o f2 Page 206 Report E01-1256 PFOS: A Reproduction Study with the Mallard W il d l if e In t e r n a t io n a l , l t d Sean P. Giallagher, Study Director ------- Joann B. BBeeavers, Wildlife Iinternational, LTD, Laboratory Management Cdk- A - S lM A tO n Lisa A. Stevenson, Principal Analytical Investigator WiUiamK. Reagen, 3M Environmental Laboratory, Laboratory Management obideau, Sponsor's Representative PROJECT NO.: 454-109, E01-1256 Date vV ^ r\fr Date JJ I Date Date X /Z O /O Z . DatT 7 3M Environmental Laboratory Page 2 of 2 Page 207 Report E01-1256 PFOS: A Reproduction Study with the Mallard W ildlife In te r n a tio n a l, ltd PROJECT NO.: 454-109, E01-1256 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A REPRODUCTION STUDY WITH THE MALLARD PROTOCOL NO.: 454/120700/QR/SUB454 SPONSOR: 3M Corporation EFFECTIVE DATE: September 27, 2002 AMENDMENT NO.: 13 PROJECT NO.: 454-109, E01-1256 AMENDMENT: Change: Rochelle R. Robideau is the Sponsor Representative. To: Susan A. Beach is the Sponsor Representative. REASON: To change role of the Sponsor Representative. Joann :International, LTD, Laboratory Management Lisa A. Stevenson, Principal Analytical Investigator William K. Reagen, 3M Environmental Laboratory, Date Date Date Date 3M Environmental Laboratory Page 1 of 1 Page 208 Report E01-1256 3M Confidential PFOS: A Reproduction Study with the Mallard Record of Deviation I. Identification Study / Project No. E01-1256 Deviation type S O P ........... Method D Equipment Procedure (Check one) Q Protocol Other: Document number ETS-4-4.0 Date(s) of occurrence 2/19/02,2/25/02,2/26/02, 2/27/02,2/28/02,2/22/02, 3/4/02, 3/5/02, 3/6/02, 3/7/02___________________ II. Description Required procedure/process: ETS-4-4.0 Internal chain of custody The internal chain of custody form to be used to track all movement of samples Within the lab. The form should be filed with study data or other relevant sample documents. Actual procedure/process: Internal chains of custodies were not used to track samples and sample extracts. Since receipt samples were Stored in freezer FI. After homogenizing liver and aliquating sera samples were stored in Freezer F8 Until extraction date. Samples remained frozen until extraction. Deviation to SOP written. ill. Actions Taken (such as amendment issued, SOP revision, etc.) Recorded by . ^ e J ^ Q Authorized by Date f9 o Date . SVuAj* Dir&cfor- 0 h p * \i i Deviation No._____ [_______ (assigned by Study Director or Project Lead at the end of study or project) <\\vA ov lolorak^ KAfc^V. Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 5 of 1 Page 209 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation 1. Identification Study / Project No.E01-1256 Deviation Type (Check one) V so p Method Equipment Procedure Protocol Other: Document Number:ETS-8-231.1 ; Date(s) o f occurrence2/25/02,2/26/02,2/27/02,2/28/02, 3/1/02, 3/4/02, 3/5/02, 3/6/02, 3/7/02, 3/8/02, II. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations are used than those listed, they w ill be documented in the raw data. ................................ Actual Procedure/p rocess;.. ........... ................ ...................................... ............ .... ...................... ..... Standard E01-1342-36010, Prep sheet description "CurvePoint- lOppbin liver", was preppedby spiking 20uL of02001-66 (1.00ug/mL) into 1mLofliver homogenate. This homogenate consists of5g liver plus 45mLwater. Since lmL out of 50mL homogenate is used to prepeach extracted standard, 1/50 ofthe 5g (O.lg) of liver is "spiked". Afterthis homogenate is spiked andpreparedusing SPE, the final eluate is 2.0mL ofMeOH. THEREFORE... (20ul) (l.OOugPFOSVmLMeOH) (l.OmL/lOOOuL) (lOOOng/l.Oug) = 20ng PFOS' onto SPEcolumn 20ng PFOS' / 2.0mLMeOH= lOngPFOS'/mLMeOH. (Since this standardis recordedas lOppb, the correct units are lOnePFOS'/mLofMeOH.) These aretherefore the units being measuredandreportedon the chromatographic system. Similarly, the following statement is true. (20ngPFOS7 1.OmLhomogenate) (l.OmLhomogenate / O.lg liver) = 200ng PFOS7 g liver This relationship shows the factor of20 used to "correct"the units when calculating the final result in ng/g fromthe chromatographic result that is inng/mL. Sam ple Calculation: "E01-1342-30348, Oppma.i. 333 Mliver" Chromatographicresult is 10.76 ng/mL; reportedvalue is 208.4 ng/g. Since the 1.0333g initial sample weight of liver is homogenizedwith 9.OmLwater, and lmL (10%oftotal liver weighed, since aliver density of 1.00 is assumed) is used for the entire SPEprocedure, the calculation is as follows: (10.76ng PFOS-/mLMeOH) (2mLMeOH/ 0.1033 g liver) = 208.4 ng PFOS- / g liver Sample Calculation: Sample "E01-1342-36017, QC@10ppb-l":This QCsample is preparedby adding4uL of02001-53 (5.020ug PFOS' / mL MeOH) spiked into lmL liver homogenate. ((4ul) (5.020ug/mL) (lmL/lOOOuL) (1000ng/ug)) / (O.lg liver) = 200.8ng PFOS' / g liver Using the earlier calculation, the analytical result of 13.59ng/mLcalculates to 271.8ng/g liver. Since the spike level is 200.8ng PFOS"/ g liver, the recoveryis (271.8/200.8)(100)=135.4%. 3MEnvironmental Laboratory Form ETS-4-8.0 3M Environm ental L aboratory D eviatio n No. _____fb_______ (assigned by Study Director or Project Lead at the end of study or project) Page 210 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation Laloorukj^, HcmciglmAnV- 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. ______ (assigned by Study Director or Project Lead at the end of study or project) Page 211 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation I. Identification Study / Project No. EO1-1256 Deviation Type (Check one) SOP Method Equipment Procedure V Protocol Other: Document Number: ETS-8-231.1 Date(s) o f occurrence: Throughout the study II. Description: Required Procedure/process: Section 11.3, Sentence 5 states: Any level outside 75% -125% o f nominal value must be deactivated, and regression recalculated...___________________ Actual Procedure/process: Many o f the high calibration points in this study are kept in the curve despite being above the apex o f the curve. When a calibration point lies above the apex, it is considered indeterminant (labeled with an I) and no concentration is calculated for the standard. If no concentration is calculated, no percent difference is calculated for evaluating whether or not that standard is within the stated criteria. Removal o f these points in most cases causes the next calibration point to then be above the apex o f the curve. This continued deletion of points could continue until there are no points remaining on the curve. Therefore, calibration points above the apex were included in the range o f the curve with 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 ^ Authorized By (Study Director / Project Lead) rw X r. \ \^ V v Date 08/16/02 Or/iu/oo. Date 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Page 212 \ Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation I. Identification Study / Project No. EOI-1256 Deviation Type (Check one) . V SOP Protocol Method Equipment Procedure Other: Document Number: ETS-8-231.1 Date(s) o f occurrence: Entire Study II. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations areustritihanJhoselisted, they will be documentedin the rawdata. Actual Procedure/process; StandardE01-1256-36240, Prepsheet description "10ppb seracurvepoint", was preppedby spiking4uLof02001-52 (5.020ug/mL) into 2 mLof pooled sera. This diluted seraconsists of 2mLseraplus 38mLwater. Since lmL out of40mL diluted serais usedto prepareeach extractedstandard, 1/40ofthe 2mLof serais "spiked". After 1.0 mLofdiluted serais spiked andpreparedusing SPE, thefinal eluateis 2.0mLof MeOH. THEREFORE... (5.020ug PFOSVmLMeOH) *(4ul) *(l.OmL/lOOOuL) *(lOOOng/l.Oug) = 20ngPFOS"onto SPEcolumn 20ngPFOS/ 2.0mLMeOH= lOngPFOS/mLMeOH. (Since this standardis recordedas lOppb, thecorrectunits are 10ngPFOS'/mLMeQH.t These arethereforetheunits being measuredandreportedon thechromatographic system. Similarly, the following statement is true. (20ng PFOS/ l.OmLdiluted sera) *(40.0mLdiluted sera/ 2.0mLinitial sera) = 400ng PFOS/ mLSera This relationship shows the factor of40 (or2.0mLMeOH/ 0.05mLSera) used to "correct"theunits whencalculatingthe final result in ngPFOS/mLSerafromthe chromatographicresult thatis in ngPFOS/mLMeOH. Sample Calculation: "E01-1256-28701,454-109-3258,10 ppmfemale, adult Chromatographic result is 340.3 ng/mLMeOH; reportedvalueis 13612ng/mLSera. Since the 1.0 mLinitial sample volume of serais diluted with 38r0mLwater, and lmL is usedforthe entire SPEprocedure, thecalculation is as follows: 1*1.0, or i/ac. diluW IAS ujaoloa. (340.3ngPFOS-/ l.OmLMeOH) * (2.0mLMeOH/0.05mLSera) = 13612ngPFOS/ mLSera Sample Calculation: Sample"E01-1256-36253, QC@250ppb-l":This QCsampleis preparedby adding lOuLof 02001-53 (50.20ugPFOS"/ mL MeOH) spikedinto lmLdiluted sera(or0.05 mLofinitial sera). ((50.20ug/mL) * (lOul) * (lmL/lOOOuL) *(1000ng/ug)) / (0.05 mLSera) = 10040ngPFOS"/ mLSera Using theearliercalculation, the analytical result of 264.06 ng/mLcalculates to 10562.4 ng/mLSera. Since the spikelevel is 10040ngPFOS7mL Sera, therecoveryis (10562.4/10040)*(100)=105.2%.____________________________________ III. Actions Taken: (such as amendment issued, SOP revision, etc._) This deviation was written and a note to file was written. Recorded By Lisa A. Stevenson 0/\ Authorized By (Study Director / Project Lead) ^ Date . 08/16/02 Date SWAa OifLC-W UlooruW^ Heuvyijtmini 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory ///b L L Deviation No. (assigned by Study Director or Project Lead at the end of study or project) Page 213 Report E01-1256 PFOS: A Reproduction Study with the Mallard Record of Deviation I. Identification Study / Project No. E01-1256 Deviation Type (Check one) SOP y Method Equipment Procedure Protocol Other: Document Number: ETS-8-231.1 Date(s) o f occurrence: Throughout the study II. Description: Required Procedure/process: Section 13.3 o f the method states: Two thirds o f all quality control samples and 1/2 o f each quality control sample at each level are expected to show an accuracy o f 75-125%.________ Actual Procedure/process: All 25 ppb mallard sera quality control sample recoveries were >125%. Iff. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written, sera data reported at the low range of the curve may be biased high, and quality control data not within criteria are flagged in the raw data._______________________ Recorded By Lisa A. S t e v e n s o n ^ ^ j l b o w uthorized By (Study Director / Project Lead) 1,1 hi\\ x Date 08/26/02 a/jblo- Date 3M Environmental Laboratory Form ETS-4-8.0 3M Environmental Laboratory Deviation No. 5 ______ (assigned by Study Director or Project Lead at the end of study or project) Page 214 Report E01-1256 PFOS: A Reproduction Study with the Mallard Recdrd of Deviation I. Identification Study / Project No. E01-1256 Deviation Type (Check one) v SOP Method Equipment Procedure Protocol Other: Document Number:ETS-8-231.1 Date(s) of occurrence2/25/02, 2/26/02, 2/27/02, 2/28/02, 3/1/02, 3/4/02,3/5/02, 3/6/02, 3/7/02, 3/8/02,______________________ II. Description: Required Procedure/process: Section 12.1, Calculations: If other calculations are used than those listed, they will be documented in the raw data. Actual Procedure/process: This deviation supersedes deviation#2 explainingthe liver calculation: StandardE01-1326-36788, Prep sheet description "CurvePoint - lOppbin liver", was preppedby spiking 20uLof 02001-66 (l.OOug/mL) into 1mLofliver homogenate. This homogenate consists of 5.0597g liver plus 45mLwater. Since lmL out of 50mLhomogenate is used to prepeach extracted standard, 1/50 ofthe 5.0597g (0.101073g = 5.0597 g / (45.0 mLwater + 5.0597 g liver) is "spiked". Afterthis homogenate is spiked andpreparedusing SPE, the final eluateis 2.0mLofMeOH. THEREFORE... (20 uL) * (1.00 ug PFOS/mLMeOH) *(1.0 mL/1000uL) *(1000 ng/1.0 ug) = 20.0 ngPFOS' onto SPEcolumn 20 ngPFOS72.0 mLMeOH= 10 ngPFOS'/mLMeOH. (Since this standardis recorded as lOppb, the correctunits are 10ngPFOS'/mLofMeOH.) These aretheunits being measuredandreportedon the chromatographic system. Similarly, the following statement is true. (20 ng PFOS'/ 1.0 mLhomogenate) *(1.0 mLhomogenate/0.101073 g liver) = 198 ngPFOS'/ g liver Sample Calculation: "E01-1326-29916, lOppma.i. 3257 Mliver" Chromatographic result is 381.46 ng/mL; reportedvalue is 72077 ng/g. Since the 1.0654 g initial sample weight of liver is homogenized with 9.0 mLwater, and 1mLis used for the entire SPEprocedure, thecalculation is as follows: 1.0654 g/ (9.0 mLwater + 1.0654 g liver) = 0.105848 g of liver in 1.0 mLhomogenate removed for extraction. (381.46 ng PFOS/ mLMeOH) * (2 mLMeOH/ 0.105848 g liver) * 10 = 72077 ngPFOS / g liver Sample Calculation: Sample "E01-1326-36798, OC@25ppb-l":This QCsampleis preparedby adding luL of 02001-53 (50.20ug PFOS' / mL MeOH) spiked into lmL liver homogenate. ((luL) *(5.020ug/mL) * (lmL/1000 uL) *(1000ng/ug)) / (0.101073g liver) =497 ngPFOS' / g liver Using the earlier calculation, the analytical result of 38.87 ng/mLcalculates to 571 ng/g liver. Since the spike level is 497 ng PFOS7 g liver, the recovery is (571/497) *(100) = 114,9%._____________________________________________ III. Actions Taken: (such as amendment issued, SOP revision, etc.) Issuing of this SOP deviation. Recorded By Lisa A. Stevenson ^ ^ <*\ Authorized By (Study Director / Project Lead) SWAft Dircelo W GcAlftjW. 3M Environmental Laboratory Form ETS-4-8.0 Date 12/02/02 / Date Deviation No. JL. (assigned by Study Director or Project Lead at the end of study or project) 3M Environmental Laboratory Page 215 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Atta c h m en t F: Ex a m p le Calculatio ns Formula Used for Sera Analyses in Study E01 -1256 AR (ng/mL MeOH) x DF x Dilution (diluted sera) = ng/mL Sera Calculation Used fo r E01 -1256-28701,454-109-3258,10 ppm Female adult (initial volum e = 0.5 mL) 340.3 ng/mL MeOH x 1 * 40 = 13600 ng/mL Formula Used for Liver Analyses in Study E01-1256 AR (ng/mL MeOH) x DF x Final Volume (mL) = ng/g Liver amount of liver in diluted sample (g) Calculation Used for E01-1326-29925,10 ppm, a.i. 3258 F Liver (initial weight = 1.0034 g) 355.07 ng/mL MeOH x 1 x 2.0 mL MeOH = 7080 ng/g Liver 0.100306 g Liver AR = Analytical result from MassLynx summary DF = Dilution factor Dilution(diluted sera) = Final volume(mL)/amount of sera in diluted sample(mL) Amount of sera in diluted sample(mL) = amount of sera used(mL)/total amount of sera + water(mL) Amount of liver in diluted sample(g) = amount of liver weighed(g)/ amount of liver weighed(g) + water added for homogenizing(mL=g) Examples: Am ount o f sera in diluted sample(m L) = amount o f sera used (0.5 mL)/total amount o f sera + w ater (10 mL) = 0.05 mL Dilution(diluted sera)(m L) = final volum e (2.0 mL)/amount o f sera in diluted sam ple (0.05 mL) = 40 Amount o f liver in diluted sample(g) = amount o f liver weighed (1.0034 g)/amount o f liver weighed (1.0034 g) + water added for homogenizing (9.00 mL) = 1.0034 g /10.0034 g = 1.00306 g 3M Environmental Laboratorv 3M EnvironmentalLaboratory Pane 216 of 216 Page 216