Document 3Xx0EEzN7EyoBO5VoMYG2jgD

AR U 6 -0051 DETERMINATION OF THE WATER SOLUBILITY OF PFOS BY THE SHAKE FLASK METHOD -s WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454C-107 OECD Guideline for the Testing of Chemicals, 105 Water Solubility AUTHORS: Raymond L. VanHoven, Ph.D. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: February 10,1999 STUDY COMPLETION DATE: May 3,1999 AMENDED REPORT DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 35 AMENDED 000105 W il d l if e Internatio nal l t d . 2- - PROJECT NO.: 454C-107 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Determination of the Water Solubility of PFOS by the Shake Flask Method WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454C-107 3M LAB REQUEST NO. : U2723 STUDY COMPLETION: May 3,1999 AMENDED REPORT: April 26,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 792, 17 August 1989 and OECD Principles o f Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992, with die following exceptions: The test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance is being recharacterized in accordance with GLP. The stability of the test substance under storage conditions at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Wildlife International Ltd. SPONSOR APPPROVAL: /(-a& -oo DATE 000106 W il d l if e International l t d . -3 - PROJECT NO.: 454C-107 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 792, 17 August 1989 and OECD Principles of Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Sample Preparation Draft Report and Data Final Report Amended Report DATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 15,1999 February 15,1999 February 15,1999 February 17 and 18, 1999 February 18,1999 February 18,1999 March 16 and 17,1999 March 17,1999 March 23,1999 May 3,1999 May 3,1999 May 3,1999 April 26,2000 April 26,2000 April 26,2000 Quality Assurance Representative M -a DATE AMENDED 000107 W il d l if e Internatio nal l t d . -4- PROJECT NO.: 454C-107 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Determination of the Water Solubility of PFOS by the Shake Flask Method WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454C-107 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: MANAGEMENT: Willard B. Nixon, Ph.D. / Manager, Analytical Chemistry ^ 6-00 DATE DATE1 ' AMENDED OC0 1 0 5 W il d l if e In te r n a tio n a l l t d . -5 - PROJECT NO.: 454C-107 TABLE OF CONTENTS Title/Cover Page...................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement................................................................................................ 2 Quality Assurance Statement................................................................................................................................. 3 Report Approval......................................................................................................................................................4 Table o f Contents....................................................................................................................................................5 Summary.................................................................................................................................................................. 7 Introduction............................................................................................................................................................. 8 Purpose.................................................................................................................................................................... 8 Experimental Design...............................................................................................................................................8 Materials and Methods........................................................................................................................................... 8 Test Substance................................................................................................................................................9 Internal Standard............................................................................................................................................ 9 Reagents..........................................................................................................................................................9 Preliminary Test Procedure...........................................................................................................................9 Definitive Test Procedure......................................................................................................................... 9 Calibration Curve.........................................................................................................................................10 Reagent Blanks.............................................................................................................................................10 Example Calculations......................................................... 11 Results ................................................................ 11 Water Solubility.......................................................................................................................................... 11 Conclusions....... ................................................................................................................................................... 11 References............................................................................................................................................................. 12 TABLES Table 1. Typical LC/MS Operational Parameters........................................................................................... 13 Table 2. Reagent Blanks Analyzed Concurrently During Sample Analysis................................................... 14 Table 3. Solubility of PFOS in NANOpure Water....................................................................................... 15 000109 W il d l if e In ter n a tio n a l l t d . 6- - PROJECT NO.: 454C-107 TABLE OF CONTENTS - Continued - FIGURES Figure 1 - Analytical method flow chart for the analysis of PFOS in NANOpure W ater...........................16 Figure 2 - A typical calibration curve for PFOS................................................................................................17 Figure 3 - A representative ion chromatogram of a low-level standard.............................................................18 Figure 4 - A representative ion chromatogram of a high-level standard............................................................19 Figure 5 - A representative ion chromatogram of a reagent blank sample....................................................... 20 Figure 6 - A representative ion chromatogram of a Day 2 solubility sample................................................... 21 APPENDICES Appendix I -Protocol........................................................................................................................................... 22 Appendix II -Certificate of Analysis................................................................................................................... 33 Appendix III -Personnel Involved in the Study.................................................................................................... 34 Appendix IV -Report Amendment........................................................................................................................35 AMENDED O O O llO W il d l if e In te r n a tio n a l l t d . -7 - PROJECT NO.: 454C-107 SUMMARY STUDY TITLE: WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: SPONSOR: TESTING FACILITY: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: Determination of the Water Solubility of PFOS by the Shake Flask Method 454C-107 3M Corporation Wildlife International Ltd. Easton, Maryland 21601 Wildlife International Ltd. Easton, Maryland 21601 TEST SUBSTANCE: TEST DATES: PFOS Experimental Start - February 11,1999 Experimental Termination - February 20,1999 TEST SYSTEM: NANOpure Water SUMMARY: The overall mean solubility concentration o f PFOS in NANOpure water was 519 mg a.i./L (SD = 48.3; CV = 9.31%; N = 6). AMENDED 0C0111 W il d l if e Internatio nal l t d . 8- - PROJECT NO.: 454C-107 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. analytical chemistry facility in Easton, Maryland. A test was conducted to determine the water solubility of PFOS (perfluorooctane sulfonic acid, potassium salt). The experimental portion o f this study was conducted between February 11,1999 and February 20,1999. The original raw data generated by Wildlife International Ltd. and a copy of the final report are filed under Project Number 454C-107 in archives located on the Wildlife International Ltd. site. PURPOSE The purpose of this study was to determine the water solubility of PFOS at 20 0.5C by the shake flask method. Determination of water solubility by the shake flask method is applicable to test substances with water solubilities equal to or exceeding 0.01 gram/Liter (g/L). EXPERIMENTAL DESIGN The water solubility of PFOS was determined at a temperature o f 20 0.5C. A preliminary test was performed to estimate the water solubility. The test consisted of additions of increasingly large amounts of NANOpure water to a known weight o f test substance at room temperature. The definitive test consisted of equilibration of an excess amount of test substance with NANOpure water at 30C followed by equilibration at 20C and analyzing subsamples by high performance liquid chromatography with mass spectrometric detection (LCMS). MATERIALS AND METHODS This study was conducted according to the procedures outlined in the protocol, "Determination ofthe Water Solubility of PFOS by the Shake Flask Method." The protocol was based bn procedures outlined in the OECD Guideline for Testing of Chemicals, 105: Water Solubility (1). This study meets data requirements under this guideline and under U S. EPA Product Properties Test Guidelines, OPPTS, Series:830.7840, Water Solubility: 000112 W il d l if e In te r n a tio n a l l t d . -9 - PROJECT NO.: 454C-107 Column Elution Method; Shake FlaskMethod (2) for shake flask methodology and TSCA Title 40 ofthe Federal Code of Regulations, Part 796, Section 1840: Water Solubility (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International Ltd. identification number 4675 upon receipt. The test substance, a white powder, was identified as: FC-95, Lot Number: 217, Expiration Date: 2008 and a reported purity of 98.9%. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated March 9,2000 indicated a purity o f90.49%. The test substance was stored under ambient conditions. Internal Standard The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: IH, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions. Reagents All solvents used in the solubility analyses were of ACS reagent grade. Nanopure water (equivalent to ASTM Type II Designation D 1193-91) was used (4). Preliminary Test Procedure A preliminary test was conducted to estimate the water solubility of PFOS. The test was performed at room temperature by adding 10.0 mg of the test substance to a 50-mL volumetric flask. Increasing volumes of NANOpure water were added (10.0, 25.0 and 50.0 mL) to the flask. After each addition, the container was shaken vigorously for approximately 10 minutes and visually examined for undissolved test substance. After the addition of 50-mL of NANOpure water, no particulates were observed. Definitive Test Procedure Approximately 0.500 grams of the test substance was weighed into each of three labelled 16-ounce French square bottles. NANOpure water, 250 mL equilibrated at 30C for approximately 30 minutes, was added to AMENDED 000113 W il d l if e In t e r n a tio n a l l t d . - 10- PROJECT NO.: 454C-107 each bottle. The bottles were sealed with screwcaps and wrapped with parafilxn. The bottles were agitated in a shaker-water bath (Lindberg/Blue, Model No. SWB1122A) maintained at 30 1.0C. After one day, one bottle was removed and placed in a second constant temperature bath (Lindberg/Blue, Model No. SWB1122A, with agitation switched off) maintained at 20 0.5C for 24 hours with occasional shaking. Triplicate 10-mL subsamples were removed from the bottle, centrifuged at approximately 3000 rpm for approximately 15 minutes in a centrifuge regulated at 20C. Fifty microliters from each subsample were volumetrically removed and diluted to 500 mL with a solution of 50% methanol and 50% NANOpure water containing 0.05% v/v formic acid and 0.100 mg/L of the internal standard. The dilutions were placed into autosampler vials for analysis by reversephase high performance liquid chromatography (HPLC) using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer Turbo IonSpray ion source. The remaining two bottles were treated in a similar manner following initial equilibration periods o f 2 and 3 days at 30.0 1.0C. Instrumental parameters for the analysis of PFOS are summarized in Table 1 and a method flowchart is provided in Figure 1. Calibration Curve Calibration standards of PFOS, ranging in concentration from 9.15 to 45.7 pg a.i./L or 9.15 to 68.6 pg a.i./L, were analyzed with each sample set. Each calibration standard contained 0.100 mg/L of the internal standard. A linear regression equation was generated using the peak area responses (ratios o f PFOS to the internal standard) versus the respective concentrations o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o flow and high calibration standards are presented in Figures 3 and 4, respectively. Reagent Blanks Reagent blanks were analyzed to assess the presence of potential interferences. No chromatographic interferences were observed in the blanks (prepared from a solution of 50% methanol and 50% NANOpure water containing 0.05% v/v formic acid and 0.100 mg/L of the internal standard) at or above the limit of quantitation of 9.15 pg a.i./L (Table 2). A representative ion chromatogram of a reagent blank is presented in Figure 5. AMENDED 000114 W il d l if e In te r n a tio n a l l t d . - 11 - PROJECT NO.: 454C-107 Example Calculations Sample number 454C-107-2A, nominal concentration of 2000 mg/L in NANOpure water. Initial Volume: 0.0500 mL Final Volume: 500 mL Dilution Factor: 10,000 PFOS Peak Area: 687581 Internal Standard Peak Area: 603048 Peak Area Ratio: 1.140 Calibration curve equation. Slope: 0.020 Intercept: 0.068 Curve is weighted: (1/x) Calculated concentration: 539 mg a.i./L Note: manual calculations may differ. RESULTS Water Solubility Triplicate subsamples were removed from the appropriate bottles after one, two and three days of shaking in a water bath maintained at 30 1,0C and following one day o f a 20 0.5C equilibration period (Table 3). Analysis of aqueous subsamples after one day had a mean analytical result of 459 mg a.i./L (SD = 8.96, CV = 1.95%). For subsamples collected after two and three days, the mean concentrations were 537 mg a.i./L (SD = 27.6, C V = 5.14%) and 501 mg a.i./L (SD = 64.2, CV = 12.8%), respectively. A representative ion chromatogram of a Day 2 solubility sample is presented in Figure 6. CONCLUSIONS The Day 2 and Day 3 mean solubility concentrations were within 15% of each other and were averaged to obtain the overall mean solubility concentration. The overall mean solubility concentration of PFOS in NANOpure water was 519 mg a.i./L (SD = 48.3; CV = 9.31%; N = 6). AMENDED 000115 W il d l if e In t e r n a tio n a l l t d . - 12- PROJECT NO.: 454C-107 REFERENCES 1. Organisation for Economic Cooperation and Development. 1995. Guideline for Testing of Chemicals, 105: Water Solubility. 2. Product Properties Test Guidelines. 1996. OPPTS 830.7840. Water Solubility: Column Elution Method; Shake Flask Method. 3. TSCA Title 40 ofthe Federal Code of Regulations. 1994. Part 796, Section 1840: Water Solubility. 4. American Society for Testing and M aterials. 1991. Standard Specification for Reagent Water. D1193-91, ASTM Section II Water and Environmental Technology, Yol. 11.01:45-47. 000116 W il d l if e In te r n a tio n a l l t d . - 13 - PROJECT NO.: 454C-107 INSTRUMENT: Table 1 Typical LC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer Turbo IonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C]g column (100 mm x 2 mm, 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 10 minutes FLOWRATE: 0.220 mL/minute MOBILE PHASE: 72% Methanol : 28% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 50.0 pL PFOS RETENTION TIME: Approximately 7.07 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.85 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 000117 W il d l if e In te r n a tio n a l l t d . -14- PROJECT NO.: 454C-107 Table 2 Reagent Blanks Analyzed Concurrently During Sample Analysis Number (454C-107-) Blank t24 Blank tjg Sample Type Reagent Blank Reagent Blank Measured Concentration of PFOS (pg a.i./L)1 <LOQ <LOQ Blank Reagent Blank <LOQ 1 The limit of quantitation (LOQ) of 9.15 pg a.i./L was based upon the product of the lowest calibration standard analyzed (9.15 pg a.i./L) and the dilution factor of the respective blank sample (1.00). AMENDED 000118 W il d l if e In te r n a tio n a l l t d . -15 - PROJECT NO.: 454C-107 Sample Number (454C-107-) 1A IB 1C Table 3 Solubility of PFOS in NANOpure Water Day of Test Concentration of PFOS Fortified Measured (mg/L) (mg a.i./L) 1 2000 1 2000 1 2000 464 465 449 Mean SD CV Mean = 459 SD = 8.96 C V = 1.95% 2A 2 2B 2 2C 2 2000 2000 2000 539 Mean = 537 SD = 27.6 508 CV = 5.14% 563 3A 3 3B 3 2000 2000 562 Mean = 501 SD = 64.2 434 CV = 12.8% 3C 3 2000 507 Totals: Mean (Days 2 and 3)= SD = CV = 519 48.3 9.31% N= 6 Note: Results and corrections were generated using MacQuan version 8.3.3 software and manual calculations. Values have been rounded for reporting purposes. AMENDED 000119 W il d l if e In te r n a tio n a l Lt d . -16- PROJECT NO.: 454C-107 METHOD OUTLINE FOR THE PROCESSING OF PFOS IN NANOPURE WATER Prepare calibration standards in a solution of 50% methanol and 50% NANOpure water containing 0.05% v/v formic acid and 0.10 mg/L of the internal standard using volumetric flasks and gas-tight syringes. 1 Prepare solubility samples by weighing 0.500 grams into three 16-ounce French square bottles and adding 250 mL of Nanopure water to each. J After the appropriate equilibration periods, centrifuge triplicate 10-mL subsamples at approximately 3,000 rpm for approximately 15 minutes in a centrifuge regulated to 20C. Dilute subsamples to 500 mL with a solution of 50% methanol and 50% NANOpure water containing 0.05% v/v formic acid and 0.10 mg/L of the internal standard. I Transfer samples and standards to autosampler vials for analysis by LC/MS. Figure 1. Analytical method flow chart for the analysis o f PFOS in Nanopure water. 000120 W il d l if e Internatio nal l t d . - 17- PROJECT NO.: 454C-107 Figure 2. A typical calibration curve for PFOS. Slope = 0.020; Intercept = 0.068; R2= 0.990, Weighted (1/x). AMENDED 000121 W il d l if e In te r n a tio n a l l t d . - 18- PROJECT NO.: 454C-107 intensity: 8955 cps Figure 3. A representative ion chromatogram of a low-level (9.15 pg a.i./L) standard. AMENDED 000122 W il d l if e Internatio nal l t d . - 19- PROJECT NO.: 454C-107 Figure 4. A representative ion chromatogram o f a high-level (45.7 pg a.i./L) standard. AMENDED 000123 W il d l if e In te r n a tio n a l l t d . -20- PROJECT.NO.: 454C-107 Figure 5. A representative ion chromatogram of a reagent blank sample (454C-107-Blank-t4s). The arrow indicates the retention time of PFOS. 000124 W il d l if e In te r n a tio n a l l t d . -21 - PROJECT NO.: 454C-107 Figure 6. A representative ion chromatogram of a Day 2 solubility sample, 454C-107-2A (nominal concentration o f 2000 mg/L). 000125 W il d l if e In t e r n a t io n a l l t d . -22- APPENDIXI Protocol PROJECT NO.: 454C-107 000126 W il d l if e In te r n a tio n a l l t d . -23- PROJECT NO.: 454C-107 PROTOCOL DETERMINATION OF THE WATER SOLUBILITY OF PFOS BY THE SHAKE FLASK METHOD OECD Guidelinefor th Testing o f Chemicals, 105 W ater Solubity 3M Lab RequestNo. U2723 Submitted to 3M Corporation Environmental Laboratory P.O .B ox 33331 S t Paul, M innesota 55133 t Wildlife International ltd 8598Commerce Drive Easton, M aryland 21601 (410)822-8600 December 2,1998 PROTOCOLNO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000127 W il d l if e In te r n a tio n a l l t d . -24- PROJECT NO.: 454C-107 Wildlife International ltd. -2- DETERMINATION OF THE WATER SOLUBILITY OF PFOS BY THE SHAKE FLASK METHOD SPONSOR: 3M Corporation Environmental Laboratory P.O. Box 33331 S t Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms. Susan A Beach TESTING FACILITY: STUDY DIRECTOR: W ildlife International Ltd. 8598 Commerce Dave Easton, Maryland 21601 -Rondadacotar ffc^snA L V aai-llaipgn, Hi.TL -Senior Chem ist'S c/ten ) LABORATORY MANAGEMENT: W illardB . Nixon, PLD. Mining-n f flm n id iy FOR LABORATORY USE ONLY Proposed Dates: E xperim ental Start Date: P ro ject N o.: a J10/44 `t o t e - 1 0 0 T est Substance No.: Experim ental Termination Date: ...- 3 / j g / l l Int/Date: ______ Receipt Date: _ iftfAo/qg 1 I PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000128 W il d l if e Intern atio na l l t d . -25- PROJECT NO.: 454C-107 Wildlife International ltd. -3 - IN T R O D U C T IO N W ildlife International Ltd. w ill experimentally determine the water solubility o f PFOS (pcrihnxooctane sulfonic add, potassium salt). The study will be conducted at the W ildlife International Ltd. analytical chem istry facility in Gaston, Maryland. The study will be performed following foe guidance presented in foe OECD Guideline for Testing o f Chemicals, 105: Water Solubility (1) for shake flask methodology. This study is intended to meet data requirements under this guideline and iitvW EPA Product Properties T est Guidelines, OPPTS 830.7840, WaterSolubility: Column Elution M ethod; Shake F lask M ethod (2) and TSCA Title 40 o f foe Federal Code o f Regulations, P art 796, Section 1840: Water Solubility Q ). The method o f analysis for quantitation provided by foe Sponsor will be verified atW ildlife International Ltd. Raw data fo r all w ort performed at W ildlife International Ltd. and a copy o f foe final report w ill be filed ly project number in archives located on foe W ildlife International Ltd. site or at an alternative location to be specified in foe final report PURPOSE The purpose o fthis study is to determine the w ater solubility o f PFOS at 20 0.5 C by the shake flask m ethod. Determination o f w ater solubility by foe shake flask method is applicable to test substances w ith w ater solubilities equal to or exceeding 0.01 gram/Litcr (g/L). The methodology may not be applicable to surface active or volatile test substances. K.XPKRTMKNTAT. DESIGN The w ater solubility ofP F O S w l be determined at a temperature o f20 iO ^ C . A prelim inary test wi be performed to estimate the water sohM ily atroom temperature. The prelim inary test consists o f additions o f increasingly large amounts o f w ater to a known weight o f test substance until soiubilizalioaiseffectedorfoesohibility is estim ated to be less than 0.01 g /L F or substances w ith an estimatedwater solubility greater foan 0.01 g /L a definitive test w ill be performed. The definitive test coasists o f equilibration o f an excess amount o fte st substance with w ater a t an elevated tem perature, 30C , followed by equilibration at 20C. MATERIALSANDMcmOPS Test Substance Tbs test substance w ill be the highest purity available. The test substance should be a t least 99% pure by w eig h t The Sponsor w ill be asked to provide the following information concerning foe test substance, if available: PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000129 W il d l if e Intern atio na l Lt d . -26- PROJECT NO.: 454C-107 Wildlife International ltd. -4 - C hem icalN am e Empirical Formula and/or Molecular Weight Dissociation Constant in W ater Expiration Date Method o f Analysis Lot or Batch Number Chemical Purity. Vapor Pressure Storage Conditions The attached form IDENTIFICATION O F TEST SUBSTANCE BY SPONSOR (Appendix 1), along wife a Material Safity D ata Sheet, w iti be used to provide information necessary for the handling and testing o f the test substance. The Sponsor agrees to accept any unused test substance and/or test substance containers at the end o f the study. i : ! W ater that meets A S T M T ^pell standards (A ST M D 1193-91) w ill be used (4). Other solvents maybe neededfor the analytical method or preparation o f stock solutions and analytical standards. A ll solvents w ill be ACS reagent grade or better, and w ill be determined to be free o f contaminants that interfere w ith the quantitation o f the test substance. Preliminary Test Procedure A prelim inary test w ill be conducted to estimate the solubility o f the test substance at room tem perature. Approximately 10 mg o fth e test substance will be placed in a plastic tube. Increasing volumes o f w ater w ill be added stepwise as shown below. Total volume o fw ater added (mL) Approximate solubility (g/L) 0.1 0.5 1 2 10 >100 20 10 5 1 Aftereachadcfition ofw aterto yield the total volume indicated, the container w ill be capped and shaken vigorously for approximately 10 m inutes and visually examined for nadissolved test substance. I f the te st substance is not dissolved after addition o f 10 mL o f water, the contents o f the container w ill be transferred to a huger container and w ater added for a final volume o f 100 mL. Following u p p in g and shaking,tite containervoflbe visuatyebedorf for undissolved material, i.c.,undissotved m aterial would indicate a water solubility <0.1 g/L. Preliminary testing ofthe solubility at lower concentrations (<0.1 g/L) maybe performed by transferring the contents o f the 100-mL vessel to a 1-L vessel or reinitiating the test with a lesser amount o f test substance, e.g., 1 mg. Amounts and volumes noted above are only guides forconducto fthe prelim inary study; larger amounts o ftest substance with proportionally larger volumes o fw ater may be used. PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000130 W il d l if e Intern atio na l l t d . -27- PROJECT NO.: 454C-107 Wildlife International ltd. -5 - Definitive T est Procedure The quantity o ftest substance necessary to saturate the desired volume o f water is estimated from the prelim inary te st The volume o f w ater used w ill depend cm the analytical method and solubility range. G reater than (or equal to) five times the quantity o f test substance required to achieve water solubility w ill be transferred to a minimum o f three test vessels (plastic and/or other suitable container material). The desired volume o f w ater w ill be added to each vessel and the vessels sealed The dosed vesseb wifl be agitated (shakenor stirred) at 3 0 1.0 C in aw ater bath. A fter 1 day, one o f the vessels wfflbe removed and reequilibrated for 24 hours at 20 0 .5 C with occasional shaking or stirring. The contents o fth e vessel w ill then be centrifuged in a centrifuge regulated at 20C. The concentration o f the test substance in aminimum oftw o aliquots oftbe aqueous phase will be determined. The remaining tw o vessels are treated sim ilarly following initial equilibration periods o f 2 and 3 days at 30 1.0C. The test w ill be terminated if the concentrations o f die test substance in die aqueous phase o f two consecutive vessels agree within 15%. If greater variation is observed and/or at die request o f the Sponsor, the test may be reinitiated with longer equilibration periods. The analyticalmsthod for quantitation o fth c test substance w ill be based on procedures provided by the Sponsor. The method w l be appended to this protocol in Appendix II and referenced in the report The method may be verified as part o f the preliminary test(s) and may be modified to measure tbs observed test substance concentrations. Miyor modifications will be approved by the Sponsor and Study D irector, and documented in the raw data. C alculations The concentration o f die test substance in each vessel w ill be expressed in g/L, mg/L or pg/Lin water as appropriate. The average solubility for test vessels with solubilities within 15% o f each other w ill be averaged and reported. Sample Handling and Safety The Sponsor w ill identity any special handling or safety precautions to be used w ith the above referenced test substance. A ll normal precautions w ith respect to handling and storage w ill be taken. PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000131 Wil d l if e Intern atio na l l t d . -28- PROJECT NO.: 454C-107 W il d l if e In t e r n a t io n a l ltd. - 6- Sample and Test Substance Retention Upon com pletion o f testing, portions o f the test substance used as part o f this study w ill be disposed o f in accordance w ith federal, state and local regulations. Any unused portion o f the test substance w ill be returned to die Sponsor. RECORDS TO BE MAINTAINED R<wt<!tr>hr.immUamndftirdatageneratedbyWildlifeInternational Ltd,willinclude,butnotbe limitedto: 1. A copy o fth e signed protocol. 2. Identification nnd characterization o fth e test substance, ifprovided by the Sponsor 3. Dates o f initiation and completion o f the study. 4. Dates o f experimental start and termination. 5. Storage conditions o f the test substance. 6. Test substance use log. 7. "Concentration calculations and records o f solution preparation. 8. Instrum ent operating conditions and chromatograms, if applicable. 9. Statistical calculations. ' ' 1 0 . T est conditions. 11. A copy o fth e final report FINALRETORT A final report o f the results o f the study w ill be prepared by W ildlife International Ltd. The report w iU inchxk, b u t rwt be lim ited to the following, when applicable: 1. Name and address o fth e facility performing the study. 2. D ates upon which the study was initiated and completed. 3. A statem ent o f compliance signed by the Study Director addressing any exceptions to Good laboratory Practice Standards. 4. Prepare arxiprocedure, as stated in the approved protocol, including all amendments and deviations to the protocol. 5. T he test substance identification, including name or code number, purify, empirical formula, molecular formula, lot or batch number, method o f analysis, and any other inform ation provided by the Sponsor. 6. Description o f the test method or reference to foe method used along w ith any modifications made. PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000132 W il d l if e In te r n a tio n a l l t d . -29- PROJECT NO.: 454C-107 W il d l if e In t e r n a t io n a l ltd. -7 - 7. The individual concentrations o f each sample. 8. The means and standard deviations o f solubility determinations at each interval for each tem perature tested. 9. Description o f any problems experienced and how they were resolved. 10. A statem ent prepared by the Q uality Assurance U nit listing the dates that study inspections and audits were made and findings reported to die Study Director and Management CHANGING O F PROTO CO L Planned changes to the protocol w ill be in the fonn o f written amendments signed by the Study Director and the Sponsor. Amendments w ill be considered as part o fthe protocol and wffl be attached to the finalprotocol Any otherchanges wifibe m the form o fw ritten deviations filed w ith the raw data. A ll changes to the protocol w ill be indicated in the final report. GOOD LABORATORY PRACTICES This study w ill be conducted according to the Good Laboratory Practices described in OECD (ISBN 92-84-12367-9) and EPA (40 CFR P art 792). Each study conducted by W ildlife International Ltd. is routinety examined by the Wildlife International Ltd. Quality Assurance U nit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocoL A statem ent o f compliance with Good Laboratory Practices w ill be prepared for all portiocs o f the study conducted by W ildlife International Ltd. The Sponsor w ill be responsible for compliance w ith Good Laboratory Practices forprocedures performed by cither laboratories. Raw data for all work performed at W ildlife International Ltd, and a copy oftfac final reportwill be filed by project number in archives located on the W ildlife International Ltd, site or at an alternative location to be specified in the final report PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000133 W il d l if e Internatio nal l t d . -30- PROJECT NO.: 454C-107 Wildlife International ltd. - 8- REFERENCES 1 O rganisation fo r Econom ic C ooperation and D evelopm ent 1995. Guideline for Testing o f Chemicals, 105: Water Solubility. 2 P ro d u ct P roperties T est G uidelines. 1996. OPPTS 830.7840. Water Solubility: Column Elution M ethod; Shake Flask M ethod. 3 TSCA T itle 40 a t th e F ederal C ode o f R egulations. 1994. Part 796, Section 1840. Water Solubility. 4 Am erican Society fo r Testing and M aterials. 1991. Standard Specification for Reagent W ater. D 1193-91, ASTM Section II W ater and Environmental Technology,VoL 11.01:45-47. PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000134 W il d l if e In te r n a tio n a l l t d . -31 - PROJECT NO.: 454C-107 W il d l if e In t e r n a t io n a l ltd._________________________________________ -9 - APPENDEXI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor L Test Substance Identity (name to be used in the report): PFOS (Perfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A____________________________________ Internal Standard: 1.1-2-2ILHJLH Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch N um ber L ot217_______________________________________ T est Substance Purity (% Active Ingredient): 98,9______ Expiration Date: 2008_______________ n . Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately define the test substance and reference standard been determined prior to its use in this study in accordance w ith GLP Standards? Y es____ No X HL Test Substance Storage Conditions Please indicate the recommended storage conditions at W ildlife International Ltd- Ambient room tem perature ___________________________________________________________ Has the stability o fthe test substance under these storage conditions been determined in accordance w ith GLP Standards? Y es____ No X O ther pertinent stability information: ------------------------------------------------------------------------------------ IV. T est Concentrations: A djust test concentration to 100% a i. _ X _ based upon the purity (%) given above. Do not adjust test concentration to 100% _____a i. Test (he m aterial AS IS. V. Toxicity Information: M am m alian: R at LD50 251 mg/kg Mouse LD50 N/A A quatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Pw plrfoiiw E"11' 27m e/L __________ Rtwptow T rent; H weffl-- Daptmia maprtg- S0m g/L____________ EM&H&MfflSSSiLSilfflgffli. O ther Toxicity Information (mchiding findings o f chronic and subchronic tests): Please see MSDS----------------------------------------------------------------------------------------- PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000135 W il d l if e In te r n a tio n a l l t d . -32- PROJECT NO.: 454C-107 W il d l if e In t e r n a t io n a l ltd. -10- APPENDIXII ANALYTICAL METHOD Analytical Method for the Determination o f PFOS in W ater PFOS may be determined in w ater using high performance liquid chromatography with mass spectrometric detection (LCMS). Sample preparation for the fortification samples will involve dilution (using the same m atrix w ater) into the concentration range o f instrumental response. M atrix blanks w ill be directly injected. The following presents approxim ate chromatographic conditions for LCMS quantitation o f PFOS. Chromatographic conditions may be changed to provide conditions more optimized for retention and/or chromatographic separation o fPFOS. Instrument: Hewlett-Packard Model 1090A HPLC Column: Keystone Betacil C -1 8,100 mm x 2.0 mm, 3pan Mobile Phase: M ethanol: Ammonium Acetate Buffered W ater 65:35 (v:v), isocratic. Flow Rate: 250 L/minute Column Temperature: 40C Injection Volume: 100 L Ion Source: Turbo IonSpray Mass Spectrom eter Peridn-Elmer API100 RunTim e: 6 .0 minutes PROTOCOL NO.: 454/120298/830.7840/SUB454 3M LAB REQUEST NO. U2723 000136 W il d l if e In te r n a tio n a l l t d . -33 - APPENDIX II Certificate of Analysis PROJECT NO.: 454C-107 FC-95, Lot 217 March 9, 2000 R ichard M . Payfer This cam ple w as analyzed using LC/M S, `H-NM R, 1?F-NM R, and elem ental analyses techniques. The results o f these tests show tho.sam ple to contain the follow ing w eight percent com position: CjPsSO jT T C J^ g 0 i-jj+ GF SOjT T C ^itSO jT T C iF uS O jir S fh SOiTT C jFnSO jT f CsFjsSOjTC C ioFziSOjT T C i.F n S O iir CFs-COiTC SFsC tFiiSQ ilC 0.04% 0.83 % 13B % 1.30% 3.71 % 1.19% 90A9% 0.49% 0.13 % 0.04% 0.05% 0.35% Additionally, the isomer distribution o f the sample was determined using l,F-NMR techniques and found to contain fee following mole percent composition: C FstC F^-SO jK T (Normal chain, w here x 1*main}? 7) C K iO T d ^G F jK C F aJr s i ' & (Internal monomethylbranch, where r+ y is mainly 5. and x w O .y * 01 m ^ H C P ^ rB O tT C (Isopropyl branhh. where x is m ainly 5) C*Fa-CF(CF> S O ,' IT (A bba branch. where x is mainly 6) (t-butyl hrtrach. where x Is malnlv 41 C F K G F aM X C ftM C ftJr 8 0 s' K7 (Internal gem-dimethyl branch, w ho jrty is msdhly 4. ad x w 01 7041% 17.1% 103% 1.6% 03% 03% AMENDED 000137 W il d l if e International l t d . -34- PROJECT NO.: 454C-107 APPENDIX III Personnel Involved in the Study The following key Wildlife International Ltd. personnel were involved in the conduct or management of this study: 1. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 2. Raymond VanHoven, Ph.D., Scientist 3. Norman J. Glassbrook, M.S., Senior Chemist 4. Keri Leach, B.S., Technician 000138 W il d l if e In te r n a tio n a l l t d . -35- PROJECT NO.: 454C-107 APPENDIXIV Report Amendment 1. Original Report: Pages 1-4 and 6 Amendment: The pages were changed to include the amended report date, revised page numbers, and new signatures and dates due to the addition of the report amendment as Appendix VI. Reason: To reflect the issuing of an amended report. 2. Original Report: Page 9 Amendment: Information provided by the Sponsor reflecting the reanalysis o f the test substance, including the reanalysis date and the purity, was added to the Test Substance section. Reason: To reflect the current test substance information provided by the Sponsor. 3. Original Report: Entire report Amendment: All test substance concentrations were changed to reflect the purity ofthe test substance as determined by the Sponsor in a reanalysis of the test substance (FC-95, Lot 217). Test concentrations originally were based on the reported purity o f 98.9%. The certificate o f analysis dated March 9,2000 indicated a purity o f90.49%. Therefore, all data such as test substance concentrations, including nominal concentrations, measured concentrations, and statistical values, were recalculated and reported as mg a.i./L based on the 90.49% purity. Reason: To report the results of the test based on the test substance purity of 90.49% at the request of the Sponsor. Manager, Analytical Chemistry REVIEWED BY: f |. Quality Assurance "s DATE / DATE f / DATE AMENDED 000139