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BACK TO MAIN Study Title Characterization Study of PFOS, Primary Standard - Test Control Reference #TCR-00017-046 P h a s e : S o l u b il it y o f PFOS in W a t e r Data Requirement 40 CFR 160.105(b) Author Mark E. Ellefson Phase Completion Date Date of signing Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Environmental Laboratory Study# FACT-TCR002 (LIMS #E00-1716) Centre Analytical Laboratories Study # 023-021 Total Number of Pages 67 BACK TO MAIN GLP Compliance Statement Study Title: Characterization Study of PFOS, Primary Standard - Test Control Reference # TCR-00017-046, PHASE: Solubility of PFOS in Water. Study Identification Number: FACT-TCR002, Centre Analytical Laboratories Study # 023-021 This phase of the study was conducted in compliance with Environmental Protection Agency (EPA) Good Laboratory Practice (GLP) Standards 40 CFR 160 with the exceptions in the bulleted list below. Exceptions to GLP compliance: Some corrections were not made in the raw data per GLP requirements The electronic data systems in use have not been validated and there is not an electronic audit trail of corrections currently available (40 CFR 160.130 (e)). Authenticated hardcopies of chromatograms and associated documents will be considered as the original raw data. Not all data was dated on the day of entry and signed or initialed by the person entering the data. The SOPs (methods) used in the study were not approved by management prior to using them. William K. Reagen, Testing Facility Management Date FACT -TCR002 (LIMS #E00-1716), Page 2 of 67 BACK TO MAIN Quality Assurance Statement Study Title: Characterization Study of PFOS, Primary Standard - Test Control Reference # TCR-00017-046, PHASE: Solubility of PFOS in Water. Study Identification Number: FACT-TCR002, Centre Analytical Laboratories Study # 023-021 This phase of the study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Oates Phase 11/28/00,12/04,05/00 01/23/01,01/25/01, 01/26/01, 01/29/0101/31/01 Data Draft Phase Report Date Reported to Management Study Director 12/11/00 12/11/00 02/01/01 02/01/01 QAU Representative Date FACT -TCR002 (LIMS #E00-1716), Page 3 of 67 BACK TO MAIN Table of Contents GLP Compliance Statement.....................................................................................................................2 Quality Assurance Statement...................................................................................................................3 Study Information......................................................................................................................................6 S u m m a ry ....................................................................................................................................................7 P u rp o s e ...................................................................................................................................................... 7 Test Substance......................................................................................................................................... 7 Test System .............................................................................................................................................. 8 Method Summaries...................................................................................................................................8 Preparatory Methods................................................................................................................................ 8 Sample Collection and Analysis.............................................................................................................. 9 Analytical Method......................................................................................................................................10 Data Summary, Analyses, and Results................................................................................................... 11 Analytical Results......................................................................................................................................11 Data Sum mary.......................................................................................................................................... 12 Statistical Methods....................................................................................................................................14 Statement of Conclusion.......................................................................................................................... 14 References................................................................................................................................................ 14 List of Attachments....................................................................................................................................15 Signature Page.......................................................................................................................................... 16 FACT -TCR002 (LIMS #E00-1716), Page 4 of 67 BACK TO MAIN List of Tables Table 1. Summary Table of Solubility of PFOS TCR-00017-046 in Water.......................................... 7 Table 2. Characterization of the Test Substance and Analytical Reference Substances.................7 Table 3. Description of test systems used in this study....................................................................... 8 Table 4. Solubility Screen Test Sample Preparation........................................................................... 9 Table 5. Shake Flask Method Preparation for Study (FACT-TCR002)............................................... 9 Table 6. Sample Collection and Preparation for Study FACT-TCR002*............................................. 10 Table 7. Calibration Standard Reprocessing Summary........................................................................ 12 Table 8. PFOS TCR-00017-046 (PFOS) Data Summary.................................................................... 13 Table 9. Summary Table of Solubility of PFOS TCR-00017-046 in Water...........................................14 FACT -TCR002 (LIMS #E00-1716), Page 5 of 67 BACK TO MAIN Study Information Study Director John Flaherty Centre Analytical Laboratory 3048 Research Drive State College, PA 16801 Analytical Chemistry Laboratories Solubility Testing Certificate o f Analysis 3M Environmental Technology and Safety Services (ET&SS) 3M Environmental Laboratory Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Centre Analytical Laboratory 3048 Research Drive State College, PA 16801 John Flaherty, Study Director Dr. William K. Reagen, Laboratory Manager Mark E. Ellefson, Principal Analytical Investigator Kristin L. Terrell, Analytical Chemist Mark L. Anderson, Analytical Chemist Cindy M. Carlson, Analytical Chemist Sponsor Dr. William K. Reagen 3M Environmental Technology and Safety Services (ET&SS) 3M Environmental Laboratory 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Experimental Dates Sample Analysis Initiation: 13 July 2000 Sample Analysis Completion: 29 August 2000 Archives Reserve samples of the reference standards will be maintained by the 3M Environmental Laboratory. All study data for this phase and a copy of the phase report are archived by the 3M Environmental Laboratory. FACT -TCR002 (LIMS #E00-1716), Page 6 of 67 BACK TO MAIN Summary The approximate solubility of PFOS in water was determined by performing a solubility screen test, followed by a quantitative solubility determination via Shake Flask Method. Results are presented in table 1: Table 1. Summary Table of Solubility of PFOS TCR-00017-046 in Water. S a m p l in g E v e n t Day 1 Day 2 Day 3 Average Value PFOS S o l u b il it y in W a t e r (C o r r e c t e d f o r P u r it y ) I 621 pg/mL 717 pg/mL 702 pg/mL 680 pg/mL Standard Deviation 23 91 2 43 %CV 4% 13% 0.3% 6% The solubility of PFOS TCR-00017-046 in water was determined to be 680 pg/mL at 24-25 C. Purpose The purpose of this phase of the study was to determine the solubility of PFOS in water as part of the characterization of this test, control, and reference substance. Test Substance PFOS, Test Control Reference #TCR-00017-046 Table 2. Characterization of the Test Substance and Analytical Reference Substances T est S ubstance PFOS THPFOS Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Purity Re-crystallized by George Moore, 3M Specialty Materials, Bldg. 236-1B-10 01-01-2010 Frozen TCR-00017-046 White Powder 97.9% SynQuest Labs Not Available Frozen TCR-00017-047 Lot #Q75-30 White Powder * 'The purity of THPFOS (currently being determined by Centre Analytical Laboratory) will not affect any analytical findings. The same lot/source o f THPFOS was used throughout the entire study. FACT -TCR002 (LIMS #E00-1716), Page 7 of 67 BACK TO MAIN T est System Table 3. Description of test Systems used in this study Source Expiration Date Storage Conditions 3M Laboratory Identification Number Purity/Grade W ater Millipore System NA Ambient NA ASTM Type I Method Summaries The solubility determination of PFOS TCR-00017-046 in water was performed according to United States Environmental Protection Agency OPPTS 830.7840, "Water Solubility: Column Elution Method; Shake Flask Method" and OECD 105, "Water Solubility" Guidelines, using the 3M Environmental Laboratory methods ETS-8-170.1 "Solubility Screen Test: Approximate Solubility Determination of a Test Substance in Various Solvents;" and ETS-8-172.1 "Shake Flask Method; Solubility Determination of a Test Substance in Various Solvents" . All analyses were performed according to the 3M Environmental Laboratory method ETS-8-155.0 "Analysis of Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC- Electrospray/Mass Spectrometry". The Solubility Screen Test Method (ETS-8-170.1) was used to determine the solubility range of PFOS TCR-00017-046 in water. Incremental amounts of water were added to PFOS neat material and a qualitative determination of the solubility point was made. The screen test indicated a solubility of PFOS in water of greater than 5,000 pg/mL. The Shake Flask Method was used for the quantitative determination of PFOS solubility. Detailed descriptions of the Solubility Screen Test Method and Shake Flask methods used in this study are located in Attachment A. Preparatory Methods ETS-8-170.1 "Solubility Screen Test: Approximate Solubility Determination of a Test Substance in Various Solvents." This method is a pre-requisite to the shake flask and column elution methods and gives an estimate of the solubility point of the test substance in the solvent of choice. Approximately ten milligrams of PFOS was weighed into a glass vial, and varying amounts of water were added in a step-wise fashion. After each water addition, the vial was shaken, sonicated, and observed for particulates. The screen test estimated the solubility of PFOS in water to be greater than 5,000 pg/mL. This concentration estimation was used as a starting point for the shake flask method, and as a guide to determine the amount of dilution necessary to bring the final sample concentration into the analytical range of method ETS-8-155.0 (2.5 - 1002 ng PFOS/mL). FACT -TCR002 (LIMS #E00-1716), Page 8 of 67 BACK TO MAIN Table 4. Solubility Screen Test Sample Preparation S tep C e n t r if u g e T u b e s P r e p a r a t io n 1 10 mg test substance weighed into tared 4.0 mL glass screw-capped vial 2 0.1 mL of water added to glass screw-capped vial 3 Following the addition of water, vial was capped, shaken vigorously and vortexed, and sonicated. Mixing and sonication times recorded on the worksheet. 4 Sample checked visually for undissolved particles of the test substance. (If all of the test substance is dissolved, the solubility is determined to be greater than 10% (mass/volume) and no additional solubility testing is required. If a portion of the test substance remains undissolved, additional dilutions are performed using the appropriate solvent.) Observation made: undissolved particles present... continue to step 5. 5 Step 2 repeated with total volume of water of 0.5,1, and 2 mL, respectively. Following water addition, vial was shaken, sonicated, and observed for particulate. 6 Following the final water addition to bring the volume of water to 2 mL, it appeared as though the PFOS had dissolved. The concentration was approximately 5,000 pg PFOS/mL and used as a starting point for the shake flask method. ETS-8-172.1 "Shake Flask Method: Solubility Determination of a Test Substance in Various Solvents." Greater than 0.045 g PFOS was weighed into a tared 15 ml. plastic centrifuge tube. Water was added gravimetrically to approximately 10 g. Nine centrifuge tubes were prepared, along with three method blanks (tubes with 10 g 0.5 g water only). The centrifuge tubes were placed in a temperature-controlled orbital shaker set at approximately 30C. See the following table documenting the shake flask method preparation: Table 5. Shake Flask Method Preparation for Study (FACT-TCR002) S tep 1 2 3 4 C e n t r if u g e T u b e P r e p a r a t io n >0.045 g PFOS weighed into tared 15 mL centrifuge tube Water added to centrifuge tube such that total weight of PFOS and water is > 9.9-10.4 g. Method Blank Preparation- Approximately 10 mL water added to a tared centrifuge tube gravimetrically. Samples sealed with tape, vortex-mixed, and placed in a temperature-controlled orbital shaker set at approximately 30.0C, rotation speed set at approximately 225 rpm. R e p l ic a t e s 9 9 3 12 D a t e (s ) P erform ed I 07-31-00 07-31-00 07-31-00 07-31-00 Sample Collection and A nalysis On each of Days 1,2, and 3 (approximately 24, 48 and 72 hours after initial sample preparation) three centrifuge tubes (containing PFOS) and a method blank were removed for analysis. The centrifuge tubes with PFOS and method blanks were allowed to equilibrate at 24-25 C for 24 hours, centrifuged, and aliquots of supernatant were collected. The aliquots were diluted serially using two 100X dilutions (990 pL methanol:10 pL sample) to a final dilution factor of 10.000X. The final diluted samples were spiked with internal standard (11 pL of 22.2 pg FACT -TCR002 (LIMS #E00-1716), Page 9 of 67 BACK TO MAIN THPFOS/mL) and analyzed via HPLC/ES/MS according to analytical method ETS-8-155.0. Four injections of each final diluted sample were performed. The following table describes the sample collection and preparation regimen: Table 6. Sample Collection and Preparation for Study FACT-TCR002* S tep 1 2 3 4 5 P rocedure At 24,48, and 72 hours, 3 PFOS+ water, and 1 water (method blank) centrifuge tubes are removed from the incubator and equilibrated at 4-25 C for approximately 24 hours. R e p l ic a t e s 4 centri fuge tubes per day Day1 Day2 Day3 Aliquots of the supernatant (water containing dissolved PFOS or water only from the method blanks) were collected and dispensed into autovials. The aliquots were diluted 100X with methanol (using a dual syringe diluter). A second 100X dilution in methanol was performed to produce a final dilution factor of 10,000X. Internal Standard was added to the 1 0 .0 0 0 X sample dilutions (1 1 pL THPFOS solution at 2 2 . 2 pg /mL in methanol). Samples were analyzed via HPLC/ES/MS on HP 1100 LC/MSD 4 per centrifuge tube Day1 Day2 Day3 Day 1 Day 2 Day 3 Day 1 Day 2. Day 3 Day 1 Day 2 Day 3 D a t e (s ) P e r f o r m e d 8-1-00 8-2-00 8-3-00 8-2-00 8-3-00 8-4-00 6 -2 7 -0 0 8 -2 7 -0 0 8 -4 -0 0 8 -2 7 -0 0 8 -2 7 -0 0 (m eth o d blks only), an d 8 -2 9 -0 0 (sam p les only) 8 -7 -0 0 8 -2 7 -0 0 8-27-00, and 8-29-00 8 -2 5 -0 0 ` information pertains to sample preparation and dates of final reported data only. A nalytical Method ETS-8-155.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry" . Diluted samples (i.e. diluted supernatants) were analyzed using HPLC/ES/MS in the negative ion mode. PFOS levels were evaluated versus calibration standards ranging in concentration from 2.5-1002 ng PFOS/mL. Internal Standard quantification was used to normalize the data. Target ions were 499 m/z (PFOS anion), and 427 m/z (deprotonated THPFOS). Analytical Equipment Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C182x50mm, 5pm particle size Column temperature: Ambient Cycle Time: 10.5 minutes Flow rate: 300pL/min Injection volume: 5pL Mobile phase components: Solvent A: 2.0 mM ammonium acetate in water FACT -TCR002 (LIMS #E00-1716), Page 10 of 67 BACK TO MAIN Solvent B: Methanol Solvent Gradient: Time 0.00 1.00 4.50 7.50 8.00 10.50 %B 40% 40 % 95% 95% 40 % Stop Mass Spectrometer: Hewlett-Packard Series 1100 API/Mass Spectrometer Detector Software: HP ChemStationTM 6.0 Fragmentor Voltage: m/z 427=100 V; m/z 499= 140 V Capillary Voltage: 3500 V Gain = 2.0 EMV Mode: Electrospray Negative Gas Temperature: 350 C Drying Gas: 10.0 L /min. Nebulizer Pressure: 25 psig Analysis Type: Single Ion Monitoring (SIM) Data Summary, Analyses, and Results A nalytical Results Data quality objectives outlined in the 3M Environmental Laboratory method were met (see Appendix A). Regressions. Quadratic curve fit was applied to calibration standards and sample data to improve quantitation over the concentration range appropriate to the data. All calibration curves had least-square fits of 0.990 or greater (R2 values ranged from 0.9960 - 0.9998). Calibration Standards. Eleven standards ranging in concentration from approximately 2.5 to 1000 ng PFOS/mL methanol were used for the calibration curves. Calibration curves were run before and after every analytical sequence. Sample Replicates. Samples 073100-wPFOS-1.1.4, 073100-wPFOS-2.2.4, and 0731 OO-wPFOS-2.3.4 were excluded from the reported data using Dixon's Q-Test. Day 2, sample 1 was barely above the warning limit (RSD of 15.9%). Continuing Calibration Verification. For quantitative determinations, a mid-level matrix calibration check was analyzed every five samples to monitor instrumental drift, with a limit of 15% deviation of the target concentrations. Limit of Quantitation (LOQ). The LOQ is equal to the lowest standard in the calibration curve (either 2.5 or 5.0 ng/mL). FACT -TCR002 (LIMS #E00-1716), Page 11 of 67 BACK TO MAIN Table 7. Calibration Standard Reprocessing Summary D a t a S u b B a t c h , A n a l y s is D a t e C a l ib r a t io n S tandards Day 3 data: 08-25-00 5 - 500 ng/mL Day 1 data: 08-27-00 2.5 - 500 ng/mL Day 2 data: 08-29-00 Blanks. 5 - 500 ng/mL S t a n d a r d s n o t u s e d , r e a s o n f o r e x c l u s io n Std 1 (2.5 ng/mL), and Std 11 (1002 ng/mL) were excluded to better fit the data at the low end of the curve. Std 11 (1002 ng/mL) was excluded to better fit the data at the low end of the curve. Std 1 (2.5 ng/mL), and Std 11 (1002 ng/mL) were excluded to better fit the data at the low end of the curve. Method blanks (Milli-Q water taken through the entire sample preparation, dilution, and analysis process) provided a measure of laboratory contamination. Acceptable values for the blanks were less than 50% of the limit of quantitation (LOQ). Solvent blanks (methanol injections) provided a measure of instrument contamination. Acceptable solvent blanks must contain levels of target analyte less than 50% of the limit of quantitation (LOQ). The initial solvent blank in each of the sample run sequences for days 1 and 2 data had background levels of PFOS >50% of the LOQ. Day 1, run #19 exhibited a background level of PFOS>50% of the LOQ. Acceptable solvent blanks were analyzed before each calibration curve. Specificity (according to OPPTS 830.7840): The solubility determination as stated in the EPA Guidelines follows. This method should only be applied to: Pure substance. This study shows a purity of 97.9%. Substances that are stable in water. Hydrolytic studies have been conducted at the 3M Environmental Laboratory showing stability (Report # W1878). Data S ummary Table 8 summarizes individual sample data. Representative chromatograms are presented in Attachment C. The table displays PFOS concentrations for individual injections. Also included are the average concentrations for each Centrifuge Tube replicate, standard deviation, and % coefficient of variation. FACT -TCR002 (LIMS #E00-1716), Page 12 of 67 BACK TO MAIN Table 8. PFOS TCR-00017-046 (PFOS) Data Summary S a m p l e ID TCR-00017-046 PFOS ug /m L C o r r ec ted for P u r it y Day 1 Centrifuge Tube #1 Day 1 Centrifuge Tube #2 Day 1 Centrifuge Tube #3 Day 2 Centrifuge Tube#1 Day 2 Centrifuge Tube #2 Day 2 Centrifuge Tube #3 Day 3 Centrifuge Tube#1 Day 3 Centrifuge Tube #2 Day 3 Centrifuge Tube #3 Rep 1: 73100-wPFOS-1.l1 Rep 2: 73100-WPFOS-1.1.2 Rep 3: 73100-wPFOS-1.1.3 Rep 4: 73100-WPFOS-1.1.4 Rep 1: 73100-WPFOS-1.2.1 Rep 2: 73100-wPFOS-1.2.2 Rep 3: 73100-wPFOS-1.2.3 Rep 4: 731OO-wPFOS-1.2.4 Rep 1: 731OO-wPFOS-1.3.1 Rep 2: 73100-wPFOS-1.3.2 Rep 3: 731OO-wPFOS-1.3.3 Rep 4: 731OO-wPFOS-1.3.4 Rep 1: 73100-wPFOS-2.l1 Rep 2: 73100-wPFOS-2.1.2 Rep 3: 731OO-wPFOS-2.1.3 Rep 4: 731OO-wPFOS-2.1.4 Rep 1: 731OO-wPFOS-2.2.1 Rep 2: 73100-wPFOS-2.2.2 Rep 3: 731OO-wPFOS-2.2.3 Rep 4: 73100-wPFOS-2.2.4 Rep 1: 731OO-wPFOS-2.3.1 Rep 2: 73100-WPFOS-2.3.2 Rep 3: 73100-wPFOS-2.3.3 Rep 4: 73100-WPFOS-2.3.4 Rep 1: 73100-wPFOS-3.l1 Rep 2: 73100-WPFOS-3.1.2 Rep 3: 73100-wPFOS-3.1.3 Rep 4: 731OO-wPFOS-3.1.4 Rep 1: 73100-WPFOS-3.2.1 Rep 2: 73100-WPFOS-3.2.2 Rep 3: 73100-WPFOS-3.2.3 Rep 4: 731OO-wPFOS-3.2.4 Rep 1: 73100-WPFOS-3.3.1 Rep 2: 73100-wPFOS-3.3.2 Rep 3: 73100-WPFOS-3.3.3 Rep 4: 73100-WPFOS-3.34 594 579 613 2593* 659 653 599 556 667 659 595 680 646 718 617 911 580 572 656 1446* 851 932 696 3702* 648 723 723 717 705 702 712 702 683 685 718 711 . A v e r a g e u g /m L S td D ev %COEFFICIENT OF VARIATION 596* 14 2% *Rep 4 excluded via Q-Test 617 42 7% 650 33 5% 723 115 16% 603* 38 6% 'Rep 4 excluded via Q-Test 826* 98 12% 'Rep 4 excluded via Q-Test 703 32 5% 705 4 1% 699 15 2% FACT -TCR002 (LIMS #E00-1716), Page 13 of 67 BACK TO MAIN The average PFOS concentration and standard deviation for each day, as well as the final overall average for all non-excluded replicates and time points were as follows: Table 9. Summary Table of Solubility of PFOS TCR-00017-046 in Water. S a m p l in g E v e n t PFOS S o l u b il it y in W a t e r Standard (C o r r e c t e d f o r P u r it y ) Deviation Day 1 Day 2 Day 3 621 pg/mL 717 pg/mL 702 pg/mL 23 91 2 Average Value 680 pg/mL 43 %CV 4% 13% 0.3% 6% The solubility of PFOS TCR-00017-046 in water is 680 pg/mL at 24-25 C. Attachment B contains data summary tables. Statistical Methods Statistical methods were limited to calculating means and standard deviations. Data that did not meet acceptance criteria as described in the OPPTS and OECD guidelines was excluded using statistical justification provided by Dixon's Q-test. Refer to Attachment E for formulas and example calculations. . Statement of Conclusion Under the conditions of the present study, the solubility of PFOS TCR-00017-046 in ASTM Type I water is 680 pg/mL at 24-25 C. References 1. Fate, Transport and Transformation Test Guidelines Office of Prevention, Pesticides and Toxic Substances (OPPTS) 830.7840 Water Solubility: Column Elution Method; Shake Flask Method. EPA 712-C-96-041, August 1996. 2. OECD 105: Water Solubility. Adopted 27July, 1995. FACT -TCR002 (LIMS #E00-1716), Page 14 of 67 List of Attachments Attachment A: Extraction and Analytical Methods Attachment B: Data Summary Tables Attachment C: Sample Chromatograms Attachment D: Deviations from the Protocol Attachment E: Sample Calculations BACK TO MAIN FACT -TCR002 (LIMS #E00-1716), Page 15 of 67 BACK TO MAIN Signature Page We certify that this report is a true and complete representation of the data for this phase of the study: William K. Reagen Laboratory Manager Date FACT -TCR002 (LIMS #E00-1716), Page 16 of 67 BACK TO MAIN Attachment A: Extraction and A nalytical Methods FACT -TCR002 (LIMS #E00-1716), Page 17 of 67 3M Environmental Laboratory Solubility Screen Test: A pproximate Solubility Determination of a Test Substance in Various Solvents Method Number: ETS-8-I70.1 Adoption Date: 09/08/00 Effective Date: 03/14/01 Approved By: William K. Reagen, Laboratory Manager M ar'E llefson, Project Le 0 3 /; < v Date //v?t ' Dafe 1.0 Scope and Ap p l i c a t i o n ____________________________________________ 1.1 Purpose. According to methods set forth by the United States Environmental Protection Agency (US EPA) and the Organization for the Economic Cooperation and Development (OECD) a preliminary study, which will be outlined in this method, serves as a prerequisite to performing solubility testing via the Column Elution Method and Shake Flask Methods. The EPA and OECD solubility testing guidelines state that if the preliminary solubility determination test indicates a solubility of > 10'2g/L (10 ppm), the Shake Flask Method (ETS-8-172.0) is to be used. If preliminary testing indicates a solubility of < 10'2g/L (10 ppm) the Column Elution Method (ETS-8-171.0) will be used to determine the solubility in the solvent of interest. Additionally, due to the tendency of certain compounds to be highly soluble in particular solvents, no further solubility determination will be necessary for compounds having solubility greater than 10% (mass/volume). E x a c t O p y a s ' Test su^stance and degradation products for solubility testing. initial l> 0A PatR / o r $ / ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 18 of 67 Page 1 of 9 BACK TO MAIN 1.3 Acceptable Matrices. Aqueous (e.g. Milli-Q water, 0.01M CaCl2), Acetone, Methanol, or other solvent(s) of interest. 2.0 Summary of Method 2.1 Preliminary Solubility Determination in solvent. Weigh approximately 10 mg of test substance into a 2.5 mL-4 mL glass screw-capped vial. Add test solvent in varying increments to the vial. After each solvent addition, vortex mix for approximately 15 sec., and sonicate 2-5 minutes. Make observations of the solution and visually confirm whether or not particles are present; it may be necessary to allow the solution to sit for up to 5 minutes. Add test solvent in a stepwise fashion up to 2 mL. If the substance fully dissolves after the first solvent addition of 100 pL, the test substance shall be considered highly soluble, and no further testing is required. If there are still undissolved particles in the vial, weigh 10 mg of test substance into a 100 mL glass stoppered volumetric flask or graduated cylinder. Add test solvent until visual confirmation of the substance dissolution has been achieved. Record all observations on a standardized preparation sheet or logbook. Once 100 mL solvent has been reached, the approximate concentration is at 100 pg/mL. If the solubility limit has not yet been achieved, weigh approximately 10 mg of test substance and transfer to a 1 L graduated cylinder/volumetric flask (concentration is approximately 10 ppm). The container should be sonicated and allowed to sit overnight to allow for maximal dissolution. If the undissolved particles are still observed after sitting > 12 hours, the Column elution method will be utilized (ETS-8-171.0). If the test substance dissolves in 1 L or less of solvent, the shake flask method (ETS-8-172.0) will be used to determine the solubility. 3.0 Definitions_____________________________________________________________ 3.1 Test substance. A liquid or solid material for which the relative solubility in a specified solvent will be determined. 3.2 Test solvent. The matrix to which the test substance of interest is introduced. The test solvent may include but is not limited to aqueous matrices, including ASTM Type I Water and various salt matrices (e.g. 0.01M CaCl2(aq)); and organic solvent matrices, including methanol, and acetone. 4.0 Warnings and Cautions____________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Wear the proper lab attire for all parts of these procedures. Wear gloves and eye protection at all times. 4.1.2 Handle all solvents in a hood for all parts of the described sample preparation procedure. 4.1.3 For potential hazards of each chemical used, refer to material safety data sheets, packing materials, and 3M Environmental Laboratory's Chemical Hazard Review. 4.1.4 No mouth pipetting is allowed. ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 19 of 67 Page 2 of 9 BACK TO MAIN 4.2 Cautions: 4.2.1 Glassware in which standards are prepared should be triple rinsed with acetone and methanol to reduce the possibility of accidental contamination. 5.0 Interferences____________________________________________________ _________ 5.1 Impurities may significantly affect the solubility of the test substance. The purity of the test substance should be known and documented prior to starting the screening procedure. 6.0 Equipment__________________ ;_______________ ^_____________________ ________ 6.1 Analytical balance sensitive to 0.1 mg. 6.2 Vortex mixer. 6.3 Sonicating device. 7.0 Supplies and Materials____________________________________________________ 7.1 Disposable glass graduated pipettes, 1 mL to 100 mL. 7.2 Disposable glass Pasteur pipettes and rubber bulbs. 7.3 Glass beakers, various sizes. 7.4 2.5 mL-4 mL glass screw-top vial. 7.5 Glass volumetric flasks, 10 mL to 1000 mL. 7.6 10 pL-1000 pL PipettemanTM manual pipettor and plastic pipette tips, or equivalent. 8.0 Reagents and Standards_________ ^__________________________________________ 8.1 Methanol (MeOH), HPLC/SPEC/GC grade from EM Science, or equivalent. 8.2 Acetone, HPLC/SPEC/GC grade from EM Science or equivalent. 8.3 Water, ASTM Type I, or equivalent. 8.4 Calcium Chloride Dihydrate, Approximately 99% or better, from Sigma or equivalent. 8.5 0.01 M CaCl2, A 0.01 M CaCl2stock solution is prepared by weighing 1.5 g CaCl2 Dihydrate in a weigh boat and transferring to a 1 L volumetric flask and diluting to the mark with Milli-QTM water. 8.6 Test substance of known purity. 9.0 Sample Handling__________________________________________________________ 9.1 Record times of initial preparation and dilution on a sample preparation sheet or logbook. 9.2 Once the preliminary testing has been completed and the appropriate information is extracted, the screen test samples should be disposed into the proper waste stream. ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 20 of 67 Page 3 of 9 BACK TO MAIN 10.0 Quality Control 10.1 Not applicable. 11.0 Calibration and Standardization____________________________ _ 11.1 The compounds of interest must be of known characterization according to laboratory specifications. 11.2 All equipment used, such as the analytical balance, should be calibrated daily prior to use. 12.0 Procedures 12.1 Solubility screen. 12.1.1 Weigh 10 mg 1 mg of test substance into a 4 mL glass screw-top vial or equivalent. Record the weight (refer to attachment A for an example of a standardized prep sheet). 12.1.2 Add solvent (e.g. water, methanol, acetone) according to the table below to the test substance. 4 mL vial or equivalent Total volume solvent added (mL)... Approximate Solubility (pg/mL)... Solvent addition 1 0.1 Solvent addition 2 0.5 Solvent Solvent addition 3 addition 4 12 1OOOQ0____ ^O O gQ gjr ----- :--------- 10000 5000 12.1.3 Following addition of solvent, vortex mi^ipproximately 15 seconds, sonicate 2-5 minutes. Visually check for undissolved particles. If undissolved test substance remains, continue adding solvent according to the above chart. It may be necessary to allow the solution to settle for up to 5 minutes before making observations. Record observations on the standardized prep sheet. 12.1.4 If all particles dissolve, then estimate the approximate solubility and document the concentration. Because the concentration is >10 pg/mL, the shake flask method (ETS-8-172.0) will be used to determine the accurate solubility point. However, if the test substance dissolves after thefirst solvent addition of 100 pL the solubility is determined to be >10% (mass/volume). The test substance shall be regarded as "highly soluble," and no further testing is required. 12.1.5 If the test substance did not dissolve in the 2 mL of solvent, weigh 10 mg 1 mg of test substance into a 100 mL glass volumetric flask or equivalent. Record the weight (refer to attachment A for an example of a standardized prep sheet). 12.1.6 Add solvent according to the table below to the test substance to deliver the appropriate amount of solvent. As described in 12.1.3, vortex mix, sonicate, and allow the test solution to settle. Record all observations and procedures on the preparation sheet. ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 21 of 67 Page 4 of 9 BACK TO MAIN 100 mL volumetric Solvent addition 1 Solvent addition 2 Solvent addition 3 flask or equivalent Total volume solvent 10 50 100 added (mL)... Approximate Solubility (pg/mL)... 1000 200 100 12.1.7 If the solubility point is reached at or prior to the complete addition of 100 mL, the concentration estimation at which the substance is soluble should be documented and shall be used as a starting point for the shake flask method (ETS-8-172.0). 12.1.8 If the 100 mL solvent level has been reached without complete dissolution of test substance, weigh out 10 mg 1 mg test substance and transfer to a 1 L volumetric flask, graduated cylinder, or equivalent. Dilute to 1 L with test solvent. The approximate concentration of test substance is at 10 pg/mL. The flask should be allowed to sit 12-24 hours to allow for maximal dissolution. If the undissolved particles are still observed, the column elution method will be utilized (ETS-8171.0) . If no visible particulates are observed, the shake flask method (ETS-8172.0) shall be used to determine the solubility. 13.0 Data Analysis and Calculations__________________________________________ 13.1 The solubility determination in this method is qualitative/semi-quantitative. 13.2 The point to which the substance dissolves in solvent is confirmed visually. The solubility point is therefore a qualitative determination. 13.3 The concentration estimation is semi-quantitative in that the approximate concentration is calculated by the following equation: c = a/b Where: c= the semi-quantitative concentration pg/mL, a= amount of substance weighed out (pg), and b= the approximate amount of solvent added (mL). 14.0 Method Performance____________________________________________________ 14.1 Limitation of data. The accuracy to which the solubility is determined is subject to a large margin of error due to the way in which the solvent is added to the solute. Since large and varying increments of solvent are being added to the solute, this error margin must be considered when reporting the concentration estimation. 14.2 The data obtained through this study is an estimation only and should be treated as a qualitative estimation of the solubility of test substance in a given solvent. ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 22 of 67 Page 5 of 9 BACK TO MAIN 14.3 For an accurate measurement of the substance solubility concentration, the shake flask method, or column elution method will be used. 15.0 Pollution Prevention and Waste Management_____________________ _ 15.1 Dispose of sample waste by placing in high or low BTU containers as appropriate. Use broken glass containers to dispose of glass pipettes. 16.0 Records__________________________________________________________ ______ 16.1 Sign and date all observations and calculations. 17.0 Attachments_____________________________________________________________ 17.1 Attachment A-Example of a Standardized Sample Preparation Sheet: Solubility Screen Prep Sheet. 18.0 References______________________________________________________________ 18.1 Organization for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals. Water Solubility -OECD Guideline 105: pp. 1-7, Adopted 1995 18.2 United States Environmental Protection Agency. OPPTS 830.7860 Water Solubility (Generator Column Method). Prevention, Pesticides and Toxic Substances: Fate, Transport and Transformation Test Guidelines. EPA 712-C-96-042: pp. 1-17, 1996 18.3 United States Environmental Protection Agency. OPPTS 830.7840 Water Solubility: Column Elution Method; Shake Flask Method. Prevention, Pesticides and Toxic Substances: Fate, Transport and Transformation Test Guidelines. EPA 712-C-96-041: pp. 1-12, 1996 18.4 3M Environmental Laboratory Method ETS-8-171.0, "Column Elution Method: Solubility Determination of Test Substance in Various Solvents." 18.5 3M Environmental Laboratory Method ETS-8-172.0, "Shake Flask Method: Solubility Determination of Test Substance in Various Solvents." 19.0 A ffected Documents_______________________________________________________ 19.1 ETS-8-171.0, "Column Elution Method: Solubility Determination of Test Substance in Various Solvents." 19.2 ETS-8-172.0, "Shake Flask Method: Solubility Determination of Test Substance in Various Solvents." ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 23 of 67 Page 6 of 9 BACK TO MAIN 20.0 Revisions___________________________________________________ Revision Number Reason For Revision 1 It was desirable to make the method more universally applicable by removing references to specific test substances. Revision Date 03/13/01 ETS-8-170.1 Solubility Determination: Screen Test Method FACT -TCR002 (LIMS #E00-1716), Page 24 of 67 Page 7 of 9 Attachment A: Example Preparation Worksheet, page 1 o f2 BACK TO MAIN 3 M Fnvirnnm ental I ahnratory S O LU R ILITV SC.RFFN GLP Study Number: _______________________ Sample preparation worksheet Test Substance: lEUfc____ ------ Date: Source: Solvent: ID#: Step Source: 1 Weigh approximately 1 0 mg 1 mg of test substance into a 2.5 mL-4 mL glass screw-top vial. Weight o f test substance: __________ mg Balance ID:____________ Date/lnitials: 2nd replicate optional _mg Thermometer ID: 2 Add test solvent according to the table below. Following each addition o f solvent, shake vigorousiyA/ortex, and sonicate the mixture and visually check for undissolved particles. Analyst Analyst Analyst Analyst volume added (mL)/ approx cone, after addition (ug/mL) Sample Prep OBSERVATIONS/NOTES Date/Time- _ /_ Step 2-1 Total volume solvent added (mL) .1 Approximate Solubility (ug/mL) 100,000 Flask vortex-mixed? Yes/No Room TempFlask sonicated? Yes/No Time----------Flask allowed to settle? Yes/No Time__ _!C Is there solute still presenrYes-continue to step 2-2 NO- The solution is at 10%, and is considered "infinitely soluble * N3 further solubility testing is required. Date/Time- _/_ Step 2-2 Total volume solvent added (mL) Q.5 Approximate Solubility fua/mLI 20.000 Date/Time- _/_ Step 2-3 1Total volume solvent added (mL) Approximate Solubility (uq/mU .1.0,000 Date/Time_ Step 2-4 Total volume solvent added (mL) 2 Approximate Solubility fua/mLI 5,000 Flask vortex-mixed? Yes/No Room Temp. Flask sonicated? Yes/No Time----------Flask allowed to settle? Yes/No Time- is there solute still present? Yes/No Flask vortex-mixed? Yes/No Room 1 Flask sonicated? Yes/No Time-------Flask allowed to settle? Yes/No Time- Is there solute still present? Yes/No Flask vortex-mixed? Yes/No Room Temp Flask sonicated? Yes/No Time______ Flask allowed to settle? Yes/No Time__ _!C Is there solute still present? Yes/No Did the substance dissolve in 2 mL or less of solvent? Yes -Approximate concentration at which the test substance is soluble in solvent:____ The solubility of the test substance is to be determined via the shake flask method, or No Continue to page twofor further testing. ug/ml Notes/ additional comments: FACT -TCR002 (LIMS #E00-1716), Page 25 of 67 ETS-8-170.1 Solubility Determination: Screen Test Method Page 8 of 9 BACK TO MAIN Attachment A Cont.: Example Preparation Worksheet, page 2 of 2 3M Environmental Laboratory SOLUBILITY SCREEN GLP Study N u m b e r:___________________________ Sample preparation worksheet Test Substance:______________________ H:____________ Source:___________ Date:___________________ Solvent: ID#: Step (continued from page 1) Source:___________ ***Did the test substance dissolve in 2 mL? Yes/No If YES, the test is complete, and there is no need to continue w ith the screen test. If NO, continue to step 3 to determine the approximate solubility o f the substance. 3 Weigh approximately 10 mg 1 mg o f test substance into a 100 mL graduated stoppered cyiinderfvoiumetric flask. Weight of test substance: ma Balance ID: . 2 n d re p lica te o ptio n a l: ma Thermometer ID: . Date/lnitials: 4 Add test solvent according to the table below. Following each addition o f solvent, shake vigorously/vortex, and sonicate the mixture and visually check for undissolved particles. v o lu m e a d d e d (m L)/ a p p ro x co n e , a fte r S olubilitv data a d d itio n (u g /m L ) Sample Prep OBSERVATIONS/NOTES A na lyst D a te/T im e / S te p 4-1 T o ta l vo lu m e s o lv e n t add ed (m L )... 10 A p p ro x im a te S o lu b ility (u g /m L )... 1,000 Flask v o rte x-m ixe d ? Y e s /N o R o o m T e m D Flask s o n ic a te d ? Y e s /N o T im e Flask a llo w e d to s e ttle ? Y e s /N o T im e C m ip m in Is th e re s o lu te s till p re s e n t? Y e s /N o A na lyst D a te /T im e / S te p 4-2 T o ta l v o lu m e s o lv e n t a d d e d (m L ) ... SO A pp ro xim a te S o lu b ility (u g /m L )... 200 Flask v o rte x -m ix e d ? Y e s /N o R o o m T e m p Flask s o n ic a te d ? Y e s /N o T im e Flask a llo w e d to s e ttle ? Y e s /N o T im e C m in m in Is th e re s o lu te still p re s e n t? Y e s /N o A n a lyst D a te /T im e / S te p 4-3 T o ta l vo lum e so lven t added (m L )... 100 A p p ro x im a te S o lu b ility (u g /m L )... 100 Flask vo rte x-m ixe d ? Y e s/N o R o o m T e m D Flask s o n ic a te d ? Y e s /N o T im e Flask a llo w e d to s e ttle ? Y e s /N o T im e C m in m in Is th e r e s o lu te s till p r e s e n t? Y e s /N o 5 Weigh approximately 10 mg 0.5 mg of test substance into a 1 L graduated stoppered cylinder/votumetric flask/or equivalent. Weight of test substance:______ mo_________________Balance ID: .__________________ Date/lnitials: A na lyst D a te /T im e / S te p 5-1 1000 Flask vo rte x -m ix e d ? Y e s /N o R o o m T e m D Flask s o n ic a te d ? Y e s /N o T im e C Is th e re s o lu te s till p re s e n t? Y e s /N o m in A p p ro xim a te S o lu b ility (u g /m L )... 10 Conclusions: Did the substance dissolve In 1000 mL o r less o f solvent? Yes -Approximate concentration at which the test substance Is soluble in solvent: ______ ug/ml The solubility of the test substance is to be determined via the shake flask method. or No -The solubility of the test substance is to be determined via the column elution method. Notes/ additional comments: FACT -TCR002 (LIMS #E00-1716), Page 26 of 67 ETS-8-170.1 Solubility Determination: Screen Test Method Page 9 of 9 BACK TO MAIN 3M Environmental Laboratory Shake Flask M ethod: Solubility D etermination of a Test Substance in Various Solvents Method Number: ETS-8-172.1 Adoption Date: 09/08/00 Effective Date: 03/27/01 Authors: Kristin L. Terrell, Mark L. Anderson, and Mark E. Ellefson Approved By: 1.0 1.1 f A .4- Scope and Application_____________________________________________________ Purpose. According to the United States Environmental Protection Agency (U.S. EPA) and Organization for the Economic Cooperation and Development (OECD) guidelines, solubility determination of substances that have solubilities in a given test matrix (e.g. water/acetone/methanol) of greater than 100 pg/mL must be analyzed via the shake flask method (OECD Guideline 105, and OPPTS Guideline 830.7840). The prerequisite to this . method is a preliminary screen of the test compound for its approximate solubility level. Refer to ETS-8-170.0 for the preliminary solubility screen test procedures. c7 0 ^ 7 7 W * 'f'/ . ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 27 of 67 Page 1 of 11 BACK TO MAIN 1.2 Compatible Analytes. Test substance and degradation products for solubility testing. 1.3 Acceptable Matrices. Water, Acetone, and Methanol, or other solvents of interest. 2.0 Summary of Method__________ ;__________________________________ _ 2.1 Using the qualitative/semi-quantitative data obtained from the preliminary solubility screen test, weigh out more than five times the estimated soluble concentration of test substance into each of twelve screw-capped vessels (three time points with four test vessels each-sample, duplicate, triplicate, and matrix blank). Add the appropriate amount of test solvent gravimetrically. Cap the tubes, seal the caps with tape, and place horizontally on an incubator/shaker set to 30 C 2 C, shaking for approximately 24 hours. At approximately 24 hours, the first time point will be pulled and equilibrated at room temperature (about 20 C-26 C) for approximately 24 hours. The equilibrated samples are centrifuged and aliquoted into autovials. Samples are then diluted 1:10 or higher with a suitable solvent for analysis via LC/MS. At approximately 48 hours and 72 hours, the second and third sets, respectively, will be pulled, equilibrated, centrifuged, aliquoted and diluted with a suitable analytical solvent for LC/MS analysis. Depending on the analytical range, dilutions may be made using a diluter or syringe to make successive serial dilutions using a suitable analytical solvent to reach the desired concentration (e.g. 1:10 or higher sample:methanol/acetone serial dilutions), and an appropriate internal standard (e.g. 1H,1H,2H,2H tetrahydroperfluorooctane sulphonic acid (THPFOS)) will be added to the final dilution prior to analysis. Samples are to be analyzed via LC/MS against a standard curve containing the test substance and an appropriate internal standard. 3.0 Definitions___________________________________________________________________ 3.1 Method blank: An analyte-free matrix (e.g. methanol, water, or acetone) to which all reagents agree are added in the same volumes or proportions as used in the sample processing. The method blank is used to document contamination resulting from the entire sample treatment and analytical process. The method blank is carried through the complete sample preparation, treatment, and analytical procedure. 3.2 Solvent blank: A sample of analyte-free medium (e.g. methanol/water/acetone solution) that is not taken through the sample treatment process. This blank is used to evaluate instrument and reagent contamination. 3.3 Shake Flask Sample Triplicates: Three test vessels taken from and representative of the same sample source carried through all steps of the treatment, extraction, and analytical procedures in an identical manner. 3.4 Sample replicates: Replicate samples (two or more) taken from and representative of the same sample source (e.g. test vessel containing test analyte and solvent) and separately carried through analytical procedures in an identical manner. 3.5 Internal Standard (IS): A known amount of a compound similar in analytical behavior to the compound(s) of interest, added to all samples and standards, and carried through the entire measurement process. ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 28 of 67 Page 2 of 11 BACK TO MAIN 3.6 Calibration Standard: A dilution of various amounts of a stock, intermediate or purchased standard to achieve standard solutions in a concentration range of interest. 4.0 Warnings and Cautions________ ;_________________________________ _______ 4.1 Health and Safety Warnings: 4.1.1 Wear the proper lab attire for all parts of these procedures. Wear gloves and eye protection at all times. 4.1.2 Handle all solvents in a hood for all parts of the described sample preparation procedure. 4.1.3 For potential hazards of each chemical used, refer to material safety data sheets, packing materials, and 3M Environmental Laboratory's Chemical Hazard Review. 4.1.4 No mouth pipetting is allowed. 4.2 Cautions: 4.2.1 Glassware in which standards are prepared are to be triple rinsed with acetone and methanol to reduce the possibility of accidental contamination. 4.2.2 All test vessels (e.g. centrifuge tubes) are to be sufficiently rinsed with solvent prior to their usage. 5.0 Interferences__________________________________________________________ 5.1 Impurities may significantly affect the solubility of the test substance. The purity of the test substance should be known and documented prior to starting the preliminary solubility screening procedure. 5.2 Contaminants in solvents, reagents, glassware and other sample processing or analysis hardware may cause interference. The routine analysis of laboratory method blanks must be used to demonstrate that there is no interference under the conditions of the analysis. 6.0 Equipment__________________________________________________ ^__________ 6.1 Analytical balance sensitive to 0.1 mg. 6.2 Centrifuge capable of holding 15 mL centrifuge tubes or equivalent. 6.3 Incubator with heating/cooling capabilities. 6.4 Diluter, Hamilton Microlab 500 Series, or equivalent. 6.5 Vortex-mixer. 7.0 Supplies and M aterials _________________________________________ 7.1 Thermometer capable of reading at least 15 C-40 C. 7.2 5-250 mL polypropylene centrifuge tubes, or equivalent. 7.3 Disposable glass graduated pipettes, 1 mL to 10 mL. 7.4 Disposable glass pasteur pipettes and rubber bulbs. 7.5 Glass beakers, various sizes. 7.6 Crimp cap autovials-1.5 mL, caps, crimper, and decapper. 7.7 Hamilton Gastight syringes (precision 1% of the total volume), 5 pL to 1000 pL. 7.8 Pipette-man manual pipettor. ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 29 of 67 Page 3 of 11 BACK TO MAIN 8.0 Supplies and Materials 8.1 Methanol (MeOH), HPLC/SPEC/GC grade from EM Science, or equivalent. 8.2 Acetone, HPLC/SPEC/GC grade from EM Science or equivalent. 8.3 Water, ASTM Type 1. 8.4 Test substance of known purity. 8.5 Calcium Chloride Dihydrate, Approximately 99% or better, from Sigma. 8.6 0.01 M CaCh solution, Example: A 0.01 M CaCL stock solution is prepared by weighing 1.4 g CaCL in a weigh boat and transferring to a 1 L volumetric flask and diluting to the mark with Milli-QTM water. 9.0 Sample Handling 9.1 Record times of initial preparation, set-up, and sample aliquoting/analysis on a sample preparation sheet or logbook. 9.2 Once the samples have been diluted, they may be analyzed via LC/MS. Alternatively, the diluted samples may be kept in cold storage (e.g. approximately 1-5 C) in crimp-capped autovials until the time of analysis. 10.0 Quality Co n t r o l _____________________________________ 10.1 Shake Flask Sample Triplicates. Set up each test vessel in triplicate to provide a measure of the precision on sample preparation. 10.2 Sample Replicates. Prepare and analyze all samples (from each test vessel for each time point) in multiple replicates (2 or more) to provide a measure of the precision on analysis. 10.3 Quality Control Blank Samples. 10.3.1 Method blank. Set up a fourth test vessel without test substance to measure any contamination accrued throughout the sample preparation process. The method blank is carried through the same sample preparation procedures as the three test vessels with test substance, only no test substance is added throughout the entire procedure. 10.3.2 Solvent Blank. An aliquot of the dilution solvent (i.e. methanol) directly analyzed for possible contaminants. The solvent blank is used to document any possible contamination of the solvent(s) used during the sample preparation process, and to detect any instrumental contamination or background interferences. 11.0 Calibration and Standarization _______________;___________________ 11.1 The compounds of interest must be standardized according to laboratory specifications. 11.2 All equipment used, such as the analytical balance and automated diluter, should be calibrated prior to use (daily, weekly, etc.) as specified in its standard operating procedure. ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 30 of 67 Page 4 of 11 BACK TO MAIN 11.3 All samples analyzed will be run against a standard curve containing varying amounts of test substance, and a fixed amount of internal standard. 12.0 Calibration and Standarization______________________________ __________ 12.1 Preparation of Test Vessel. Record all data and observations on the example sample preparation worksheet (Attachment A) or equivalent. 12.1.1 Prepare a solution at least five times more concentrated than the concentration at which the substance was known to be dissolved at in the preliminary solubility screen test (ETS-8-170.0). 12.1.2 Weigh out test substance into a tared test vessel (e.g. 15 mL polypropylene centrifuge tube). 12.1.3 Add the test solvent to the test vessel gravimetrically until the desired volume has been obtained (note: solvents have differing densities, therefore the weight needed to achieve a specific volume will be dependent on the density.). Record all weights on a standardized preparation sheet or logbook. 12.1.4 Visually confirm that there are undissolved particles. If no particles are present, repeat 12.1.2 with more test substance or add more test substance and record the additional weight. 12.1.5 Prepare the solution described in 12.1.1-12.1.4 nine times and label (at minimum) the test vessels as Day 1, -Rep 1, -Rep 2, and -Rep 3; Day 2, -Rep 1, -Rep 2, and Rep 3; and Day 3, -Rep 1, -Rep 2, and -Rep 3. Also include the day of initial sample prep, the person(s) responsible for the sample, the test compound, nature of the study (e.g. water solubility), the solvent utilized, and the study number. 12.1.6 Prepare three test vessels as described in 12.1.3 without the test substance and label (at minimum) as Dayl,Rep4; Day2,Rep4; and Day3,Rep4 along with the information described under 12.1.5. These three test vessels are the "method blank" samples. 12.1.7 Seal the cap to the test vessel. Mix/shake test vessel to ensure contact between the test substance and the solvent. And wrap the cap with tape. 12.2 Equilibration of samples. 12.2.1 Place all of the test vessels on their side (horizontal) in an orbital incubator set to approximately 30 C 2 C and rotating at a considerable speed to ensure sufficient contact/mixing between the test substance and solvent. 12.2.2 At approximately 24 hours, four test vessels are removed: three containing test substance labeled "Day l,Repl", "Dayl,Rep2", "Dayl,Rep3", and "Dayl,Rep4". 12.2.3 The samples are shaken to ensure no test substance is stuck to the side of the tube. 12.2.4 The tubes are then placed in a stable temperature environment of approximately 20 26 C (record the actual temperature) for about a 24 hour period. 12.2.5 After about 24 hours of equilibration at 20 C-26 C, centrifuge the samples until the solution is visibly clear. If micelle formation is suspected, an additional high speed centrifugation step may be added (20,000 RCF for approximately 1 hour). The test vessels are now ready for dilution. See section 12.3 for further sample preparation. ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 31 of 67 Page 5 of 11 BACK TO MAIN 12.2.6 At approximately 48 hours of incubation time, the second set of test vessels may be pulled (labeled "Day2,Repl", "Day2,Rep2", "Day2,Rep3", and "Day2,Rep4") and equilibrated as described in 12.2.3-12.2.5. 12.2.7 At approximately 72 hours, the final set of samples is pulled (labeled "Day3,Repl", "Day3,Rep2", "Day3,Rep3", and "Day3,Rep4") and equilibrated as described in 12.2.3-12.2.5. 12.3 Sample preparation for analysis. 12.3.1 Observe the test solution. Due to the possibility of emulsions/or a concentrated layer of solute at the surface of the solution, it may be necessary to pipette off the top layer of solution (or an aliquot of sample may be centrifuged as stated in 12.2.5). The use of a pasteur pipette or equivalent is recommended. 12.3.2 Aliquot the test solution into each of four autovials (e.g. 1.5 mL polypropylene or glass crimp-cap vials). 12.3.2.1 Sample aliquots may be taken by submerging the pipette or syringe tip below the surface of the test solution. Note: when selecting the type o f pipette to be used, take into consideration the test substance's tendency to adsorb to certain materials (i.e. adsorption o f test substance to glass or plastic). 12.3.2.2 Prior to aliquoting sample to the autovial, the pipette tip must be equilibrated/saturated with the test solution by drawing and gently expelling the test solution in and out of the pipette tip, taking care not to disturb the solid particulate. 12.3.3 Label the four autovials and record the sample id. 12.3.4 Using a diluter, make a 1:10 or higher dilution of sample:extraction solvent (i.e. methanol, acetone) into a labeled autovial. 12.3.5 Using the solubility information obtained in the preliminary screen test, estimate the appropriate dilutions, if any, required to bring the concentration of sample into the appropriate analytical range (e.g. approximately 3 ng/mL to 3000 ng/mL test substance). Utilize the diluter to make serial dilutions of the sample using a suitable analytical solvent. 12.3.6 To the final analytical sample, add internal standard at a concentration suitable to the analytical method. 12.3.7 Samples will be analyzed via LC/MS against a standard curve of varied, known concentrations of test substance with a constant concentration of internal standard equal to the concentration in the samples. 13.0 Data Analysis and Calculations_________________________________________ 13.1 Samples will be analyzed via a calibration curve. The amount of test substance in the sample will be quantified against a standard curve. 13.2 Means will be calculated by adding the individual entities and dividing the resultant sum by the number of individual entities. 13.3 Standard deviations will be calculated using either Microsoft Excel or Microsoft Access to calculate standard deviation. The built in function contains the following equation which is based on the individual entities (n) being less than 30: ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 32 of 67 Page 6 of 11 BACK TO MAIN 13.4 Sample precision will be reported as %RSD (or %CV). Sample precision will be calculated using the following equation: A/B X 100 = Sample %RSD where: A= standard deviation of averaged samples B= average of samples Sample RPD will be calculated using the following equation: IA-B | /((A+B)/2)= Sample RPD where: A= concentration of first replicate B= concentration of second replicate 13.5 Exclusion of an outlier data point may be performed by utilizing Dixon's Q-Test. The questionable data point may err to the high or low end of the data set. Calculate the variable "Qobserved." If Qobserved > Qtabulated, then the data point may be rejected with 90% confidence (see table 1 below for Qtabulated values). Q observed= gap/range where: Gap= the difference between the questionable point and the nearest value. Range= total spread of the data. Table 1 Criteria for Rejection of Outlier Values: Number of observations Qtabulated, 90% Confidence Range 3 0.886 4 0.679 5 0.557 14.0 Data A nalysis and Calculations____________________________ ____________ _ 14.1 Precision of data. Sample data must have a percent relative standard deviation (%RSD) (or relative percent difference) of < 15%. Non-compliant data must be evaluated for obvious outliers. The Q-Test may be applied to exclude questionable data points. If an outlier value exists, sample average and precision is re-calculated and reported without the questionable data point. Document the non-compliant data on data summary sheets, and include results of the statistical analysis with the final results. 14.1.1 Sample replicate %RSD's should be <15%. If the average of the sample triplicate data is >15% RSD, evaluate the three values for an outlier value. To questionable data points, apply the Dixon's Q-Test. If the outlier value is rejected with 90% confidence, exclude the sample from further data calculations and calculate the average and RPD for the remaining two values. If no data points can be excluded and the data does not meet the criteria, then the data set may not be used in the final reported data. The remaining shake flasks for the timepoint shall be used to report the data. 14.1.2 The %RSD between shake flasks should agree within 15%. If this criteria is not met, apply the Q-test to outlier datum. If the questionable data point is discarded ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 33 of 67 Page 7 of 11 BACK TO MAIN via the Q-Test, then the non-compliant shake flask data is not to be used for the timepoint. Include justification for dropping the shake flask (e.g. q-test results). 14.1.3 And finally, the %RSD between the three days should be <15 % for the shake flask method. If this criteria is not met, it may be necessary to re-evaluate the data for possible outliers. Three agreeable time intervals are required to report the final solubility. If three consecutive timepoints do not agree within 15%, it may be necessary to repeat the entire test, or re-dilute/re-shoot samples to check for error. 14.2 Limit of Quantification. For this study, the LOQ will be equal to the lowest calibration standard used in the calibration curve containing more than twice the area counts of the highest Quality Control Blank. 14.3 Quality Control Blanks: Method and Solvent Blank samples. The level of analyte (test analyte OR internal standard analyte) shall be less than 50% of the area counts of the LOQ. If background levels of analyte exist in the initial method blanks, but subsequent blanks prior to the calibration standard curve are clean, the data may be accepted (provided that there are no other indicators that either the samples or the instrument contain significant background levels of analyte). 15.0 Pollution prevention and Waste Management 15.1 Dispose of sample waste by placing in high or low BTU containers as appropriate. Use broken glass containers to dispose of glass pipettes. 16.0 Records 16.1 Sign and date all observations and calculations. 16.2 Fill out all appropriate sample preparation worksheets. 17.0 Attachments 17.1 Attachment A-Example Sample Preparation Worksheet. 18.0 References 18.1 Organization for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals. Water Solubility -OECD Guideline 105: pp. 1-7, Adopted 1995 18.2 United States Environmental Protection Agency. OPPTS 830.7860 Water Solubility (Generator Column Method). Prevention, Pesticides and Toxic Substances: Fate, Transport and Transformation Test Guidelines. EPA 712-C-96-042: pp. 1-17, 1996 18.3 United States Environmental Protection Agency. OPPTS 830.7840 Water Solubility: Column Elution Method; Shake Flask Method. Prevention, Pesticides and Toxic ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 34 of 67 Page 8 of 11 BACK TO MAIN Substances: Fate, Transport and Transformation Test Guidelines. EPA 712-C-96-041: pp. 1-12,1996 18.4 ETS-8-170.0, Solubility Screen Test: Approximate Solubility Determination of a Test Substance in Various Solvents. 18.5 Harris, Daniel C. Statistics. Quantitative Chemical Analysis. 4th ed.; W.H. Freeman and Company: New York, 1982; p 70-71. 18.6 Natrella, Mary Gibbons. The Treatment of Outliers. Experimental Statistics, National Bureau of Standards Handbook 91; U.S. Government Printing Office: Washington, D.C., 1963; Pages 17-3, and T-27. 19.0 A ffected Documents ____________ ^ 19.1 ETS-8-170.0, "Solubility Screen Test: Approximate Solubility Determination of a Test Substance in Various Solvents." 20.0 Revisions Revision Number 1 Reason For Revision Section 1.2, references to specific test substances were removed. Section 3.6, removed "internal standard blank" from definition section. Section 12.2.5, an optional high-speed centrifugation step was added. Fixed the incorrect numbering of sections 13.5 through 14.3. Section 14.3, more clearly defined the acceptance criterion for method and solvent blanks. Revision Date 03-27-01 ETS-8-172.1 Solubility Test: Shake Flask Method FACT -TCR002 (LIMS #E00-1716), Page 35 of 67 Page 9 of 11 BACK TO MAIN A TTACHMENTA: Example Sample Preparation Sheet (page 1 o f 2). Temperature o f Study: __ !C 3M Environmental Laboratory Solubility Test: Shake Flask Method Sample preparation worksheet Study # A n a lv s tfs ): Test Substance: ID # : S ource: Date: Test Solvent: ID#: Test Vessel. 15ml centrifuge tube/O ther(spedfy): solvent density:___________ fl/m l S ource: iVas the solubility screen test performed prior to start? Y/N If so, what is theapproximate concentration? ug/ml Balance ID: Thermometer ID: Room Temo: SFM vials ID#: "samplelD" = Sample Description Total mass weight test substance (solute + solvent) added: added: 'internal Standard addition _______ uL/ mis soln IS added to dilution factor: samplelD-1.1.1 th ru -1.1.4 Day1, Smp1 1 samplelD-1.2.1 thru -1.2.4 Day1, Smp2 1 samplelD-1.3.1 thru -1.3.4 Day1, Smp3 1 samplelD-1.4.1 thru -1.4.4 Day1, Smp4 blank none added 1 samplelD-2.1.1 th ru -2.1.4 Day2, Smp1 1 samplelD-2.2.1 thru -2.2.4 Day2, Smp2 1 samplelD-2.3.1 thru -2.3.4 Day2, Smp3 1 samplelD-2.4.1 thru -2.4.4 Day2, Smp4 blank none added 1 samplelD-3.1.1 thru -3.1.4 Day3, Smp1 1 sample!D-3.2.1 thru -3.2.4 Day3, Smp2 1 samplelD-3.3.1 thru -3.3.4 Day3, Smp3 1 samplelD-3.4.1 thru -3.4.4 Day3, Smp4 blank none added 1 Date/Time: Initials: Test Vessels sealed w/ tape? Y/N Test Vessels shaken/vortex-mixed? Y/N Test vessels placed on orbital incubator? Y/N Incubator ID: SDeed: ' internal Standard: Int. Std. Cone.: mm Initial Temperature: ID#: -jjg /m l C Date/Time/Inltials: Observations/Comments: Day 1: Four "Day 1" samples are pulled and allowed to be placed in a temperature controlled environment for approx. 24 hours with no agitation. DaieiTlmeilnttials: Location of samples:____________________________________ Temperature of environment_______________!C Thermometer ID:_______ _ Visual observations: FACT -TCR002 (LIMS #E00-1716), Page 36 of 67 ETS-8-172.1 Solubility Test: Shake Flask Method Page 10 of 11 ATTACHMENTA CONTINUED (page 2 o f 2). BACK TO MAIN FACT -TCR002 (LIMS #E00-1716), Page 37 of 67 Day 3: Four "Day 3" samples are pulled and allowed to be placed in a temperature controlled environment for approx. 24 hours with no agitation. Location of samles: Temoerature of environment: C Thermometer ID: . Visual observations: Daie/rme/intais: . "Day 2" Test Vessels Centrifuged: Cent. ID: Visual observations: RPM or RCF readout: duration: min. Oatemme/initiais: .. . -Four aliquots from each of the four replicates are to be made into glass/plastic autovials (also, check autovials for correct labeling). Yes/No -immediately dilute the solutio 1: with methanol. Methanol TN-A . Additional dilutions: Dilution factor (test solution.methanolV. 1: X's how manv dilutions: =fmal dilution factor: 1: Diluter ID used: V -Store samples and extracts ir a cooler at until time of analysis. Amounts used: Cooler ID: uL methanol. Cooler Temp: C Date/Time/lnitials:------------------------------------------------ . ul samQles Date/Time/lnitials: .. Day 4: At approx. 24 hours of equilibration, the "Day 3" samples are to be aliquoted/extracted. Environment Temp.: Test Vessels Centrifuged: Cent. ID:___________________ RPM o r RCF readout:______ : duration:. Visual observations: ! Therm. ID:. min D ate/Tim e/lnitials: Four aliquots from each of the four samples are to be made into glass/plastic autovials (also, check autovials for correct labeling). Yes/No -Immediately dilute the solution 1:______with methanol. Methanol TN-A:___________________ ,. Additional dilutions: Dilution factor (test solution:methanol): 1:___________X's how many dilutions:____________ =final dilution factor 1:. Diluter ID used: Amounts used: _uL methanol, _ -Store samples and extracts in a cooler at until time of analysis. Cooler ID :. Cooler Temp:. D ate/Tim e/lnitials: u! samples D ate/T im e/lnitials: ETS-8-172.1 Solubility Test: Shake Flask Method Page 11 of 11 BACK TO MAIN 3M Environmental Laboratory M ethod Analysis o f P otassium Perfluorooctanesulfonate o r O th er F lu o roch em ica ls in W aste Stream o r W ater E xtracts Using H P L C -E lectrospray/M ass Spectrom etry Method Number: ETS-8-155.0 Author: Mark L. Anderson, Mark E. Ellefson Approved By: Adoption Date: l l j ( f o o Revision Date: Exact Copy of Original - J M ... Initial oe/ink ' Vate Word 97 ETS-8-155.0 Page 1 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 38 of 67 BACK TO MAIN 1.0 Scope and Application____________________________________________________ 1.1 Scope: This method describes the analysis of waste stream or water extracts using HPLCelectrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemicals or other electrospray ionizable compounds. 1.3 Matrices: Tap water, ground water, wastewater and other aqueous solutions. 2.0 Summary of Method___________________________________________________ __ 2.1 This method describes the analysis of fluorochemicals or other electrospray ionizable compounds extracted from water, using HPLC-electrospray mass spectrometry (HPLCES/MS). The analysis is performed by the mass selection of a single ion characteristic of a particular compound, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499 or perfluorooctanoate (PFOA), m/z = 413. 3.0 Definitions____________:___________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Platform LCZ single quadrupole system and other commercially available LC/MS systems allow for various methods of ionization by utilizing a variety of sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization in these processes occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): A method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometer (MS): The Platform LCZ and other commercially manufactured ES/MS systems are equipped with a single quadrupole mass selective detector. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. 3.4 Conventional vs. Z-spray probe interface: The Micromass Platform LCZ system utilizes a "Z-spray" conformation. The spray emitted from the probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.). Other commercially manufactured ES/MS systems may have similar features. 3.5 Mass Lynx Software: System software designed for the specific operation of Micromass LCZ Mass spectrometer. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (MassLynx NT User's Guide or Micromass Platform LCZ User's Guide). ETS-8-155.0 Page 2 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 39 of 67 BACK TO MAIN 4.0 Warnings and Cautions___________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective clothing, gloves, and eyewear. 4.2 Cautions: 4.2.1 Operate solvent pumps below a backpressure of 400 bar (5800 psi). If the backpressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences__________________________________________________________ 5.1 To minimize interferences when analyzing samples, Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Platform LCZ Mass Spectrometer equipped with an electrospray ionization source. HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler. 7.0 Supplies and Materials________ ._______________________ ,___________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (or house air system.). 7.1.2 HPLC analytical column, such as a Betasil C l8 column (50x2mm, 5 pm particle size) or equivalent. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. ETS-8-155.0 Page 3 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry ' FACT -TCR002 (LIMS #E00-1716), Page 40 of 67 BACK TO MAIN 8.0 Reagents and Standards________ ;__________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor. 8.1.3 Ammonium acetate, reagent grade or equivalent. 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 L volumetric flask, add the appropriate volume of Milli-Q water, mix until all solids are dissolved. Store at room temperature. 8.2 Calibration Standards 8.2.1 Typically two method blanks (Milli-Q water), two matrix blanks, and solvent standards are prepared during the sample extraction procedure. 9.0 Sample Handling_____________________________________________________ 9.1 Standards and sample extracts are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, standards and sample extracts may be refrigerated at approximately 4 C until analyses can be performed. 10.0 Quality Control________________________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each sample set to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared for each sample set and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared periodically to measure the precision associated with the analysis. 10.2.3 Analyze the matrix spike and matrix spike duplicate (if prepared) in the same run as the original sample. 10.2.4 Matrix spike and matrix spike duplicate concentrations should fall in the mid-range of the initial calibration curve or should be prepared at 1.5-5 times the endogenous ETS-8-155.0 Page 4 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 41 of 67 BACK TO MAIN concentration of the analyte. Spike concentrations should fall in the low-range of the initial calibration curve if extremely low levels are expected. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications (CCV) are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per sample set. 10.4 Internal Standard/Surrogate Standard 10.4.1 An internal standard (IS) may be used to quantify the target analytes by establishing a relationship between the ratio of analyte response to IS response and a known concentration of the analyte of interest. The IS should be spiked at an amount that will fall within the mid-range of the calibration curve. The IS should be added after the extraction process and before analysis. 10.4.2 A surrogate standard may be used for quality control. The surrogate is used to quantitatively evaluate the entire analytical procedure including sample preparation and analysis. The surrogate should be spiked to fall within the low to mid-range of the calibration curve. 11.0 Calibration and Standardization_________________________________ 11.1 Analyze the standard curves prior to and following each set of extracts. The average of two standard curves may be plotted by linear regression (y = mx + b) weighted 1/x, or quadratic fit (y = ax2+ bx + c) using MassLynx or other suitable software. The calibration curves should not be forced through zero. 11.2 If the calibration curve does not meet acceptance criteria perform routine maintenance or prepare a new standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures______________________________________________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using the first letter of the name of the instrument (T for Tucker), the year (00 for 2000), the month (04 for April), and the day (T001012 for October 12, 2000). If more than one list is made on the same day, use increasing letters of the alphabet starting with A at the end of the list. ETS-8-155.0 Page 5 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 42 of 67 BACK TO MAIN 12.1.1.2 Assign a method (MS) file. 12.1.1.3 Assign an HPLC program (Inlet file). 12.1.1.4 Type in sample descriptions and vial position numbers. 12.1.2 To create a method, click on method in the Acquisition control panel then mass spectrometer headings and select SIR. Set ionization mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected in addition to the SIRs. Save acquisition method. See the Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of solvent standards. 12.1.4 Samples are analyzed with a continuing calibration verification (CCV) injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor for possible analyte carryover. 12.2 Using the Autosampler/Column Heater 12.2.1 Place sample vials into the sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Attach the proper analytical column in the column heater compartment. If using the switching valve, make sure that the tubing is run to the appropriate ports. 12.3 Using the Inlet Editor 12.3.1 Set-up the HP1100 using the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.3.1.1 Sample size = 10 pL injection 12.3.1.2 Flow rate = 300 pL/min. 12.3.1.3 Cycle time = 10.0 minutes 12.3.1.4 Mobile phase components: Solvent A: 2.0 mM Ammonium Acetate Solvent B: Methanol (MeOH) Solvent Gradient: Time (min.') 0.00 1.00 4.50 8.00 8.50 10.0 %B 5.00 % 5.00% 95.0% 95.0% 5.00 % Stop ETS-8-155.0 Page 6 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 43 of 67 BACK TO MAIN 12.4 Instrument Set-up 12.4.1 Refer to the Platform LCZ User's Guide, the MassLynx NT User's Guide or ETS-936, "Operation and Maintenance of the Micromass Platform LCZ Electrospray/Mass Spectrometer". 12.4.2 Check the solvent level in reservoirs and refill if necessary. 12.4.3 Check the tip of the stainless steel capillary at the end of the probe with an eyepiece. 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. 12.4.4 Turn on the nebulizing gas. 12.4.5 Open the tune page. Click on `Operate' to initiate the desolvation heaters. 12.4.6 Open the Inlet Editor. 12.4.5.1 Set HPLC pump to "On". 12.4.5.2 Set the solvent flow to the desired flow rate. 12.4.5.3 Observe droplets coming out of the tip of the probe. A fine mist should be expelled with no nebulizing gas leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.4.5.4 Allow to equilibrate for at least 10 minutes. 12.4.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.4.6.1 Drying gas 250-425 liters/hour 12.4.6.2 ESI nebulizing gas 10-15 liters/hour 12.4.6.3 HPLC constant flow mode, flow rate 10 - 500 jiL/min 12.4.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.4.6.5 Source Block temperature 150. 12.4.6.6 Desolvation temperature 250. 12.4.7 Print the tune page with its parameters, the Inlet page, sample list, mass spec information, and all other applicable information and store it in the study binder with copies taped into the instrument run logbook. 12.4.7.1 All copies must be initialed and dated. 12.4.8 Click on start button on the MassLynx toolbar. Ensure start and end sample numbers include all samples to be analyzed. 13.0 Data Analysis and Calculations________________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: ETS-8-155.0 Page 7 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 44 of 67 BACK TO MAIN % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.3 Calculate actual concentration of analyte in matrix (jig/mL): On-Column Concentration (pg/mL) x Dilution Factors = Calculated Concentration 14.0 Method Performance____________;_______________________________________ 14.1 The Limit of Quantitation (LOQ) is method, analyte, and matrix specific. For many analytes, the LOQ concentration is selected as the lowest acceptable non-zero standard in the calibration curve. 14.2 Solvent and method blank values must be < Vi that of the lowest standard used in the calibration curve. 14.3 The coefficient of determination (r2) value for the calibration curve must be greater than or equal to 0.980. 14.4 Continuing Calibration Verification (CCV) percent recoveries must be 30% of the standard concentration. 14.5 Internal Standard recoveries should be within 50% of the spiked concentration. 14.6 If criteria listed in this method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the raw data. 14.7 If data is to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 P ollution P r e v e n t i o n a n d W a s t e M a n a g e m e n t ________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx and other applicable information to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve then print these graphs and store in the study folder. ETS-8-155.0 Page 8 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 45 of 67 BACK TO MAIN 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 16.5 Summarize data using suitable software and store in the study folder. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Attachments_____________________________________________________________ 17.1 None 18.0 References_______________________________________________________________ 18.1 Platform LCZ User's Guide, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9LZ; United Kingdom. 18.2 MassLynx NT User's Guide, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9LZ; United Kingdom. 18.3 MassLynx NT Guide To Data Acquisition, Micromass UK Limited, Tudor Road, Altrincham, WA14 5RZ; or Floats Road, Wythenshawe M23 9LZ; United Kingdom. 18.4 ETS-9-36.0, "Operation and Maintenance of the Micromass Platform LCZ Electrospray/Mass Sprectrometer". 19.0 Affected Documents_____________ .___________________________ 19.1 None 20.0 Revisions__________________________________________________________ Revision Number. Reason For Revision Revision Date ETS-8-155.0 Page 9 of 9 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Waste Stream or Water Extracts Using HPLC-Electrospray/Mass Spectrometry FACT -TCR002 (LIMS #E00-1716), Page 46 of 67 A ttachment B: Data S ummary Tables BACK TO MAIN FACT -TCR002 (LIMS #E00-1716), Page 47 of 67 Q p m 11-m o TCR-o/^ FACT -TCR002 (LIMS #E00-1716), Page 48 of 67 H082700.S Samples acquired on Hillary 08-27-00, Reprocessed by KLT on 08-2800. Sample ID Sample Pese Run# Standards Data/Performance 00027-43-11 00027-43-11 00027-43-00 00027-43-01 System Suitabiity Stds 00027-43-02 System SuRabilty Stds 00027-43-03 System Suitabiity Stds 00027-43-04 System Suttabtlty Stds 00027-43-05 System Suitabiity Stds 00027-43-08 System Suitabiity Stds 00027-43-07 System Suitabiity Stds 00027-43-08 System Suitabiity Stds 00027-43-09 System Suitabiity Stds 00027-43-10 System Suitabiity Stds 00027-43-11 System Suttabtlty Stds 00027-43-00 0 Std 00027-43-01 Std 1 (LOQ-2.5 ppb) 00027-43-02 Std 2 00027-43-03 Std 3 00027-43-04 Std 4 00027-43-05 Std 5 00027-43-06 Std 6 00027-43-07 Std 7 00027-43-08 Std 8 00027-43-09 Std 9 00027-43-10 Std 10 00027-43-11 Std 11 (Excluded to better fit data) 00027-43-06 CCV 00027-43-06 CCV 0QQ27-43-QQ 0 Std 00027-43-01 Std 1 (LOQ-2.5 ppb) 00027-43-02 Std 2 00027-43-03 Std 3 00027-43-04 Std 4 00027-43-05 Std 5 00027-43-06 Std 6 00027-43-07 Std 7 00027-43-08 Std 6 00027-43-09 Std 9 00027-43-10 Std 10 00027-43-11 Std 11 (Excluded to better fH data) 2 3 0 7 8 9 10 If 12 13 14 15 16 17 21 22 23 24 25 26 27 28 29 30 31 32 44 56 59 60 61 62 63 64 65 66 67 68 69 70 VU* Mtd Data Fila Vial# H62700B.M H82700B.M H62700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H62700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H8270QB.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H8270B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H62700B.M H8270B.M H82700B.M HILL0002.D HILL0003.D HILL0006.D HILL0007.D HLL0008.D HILL0009.D HILL0010.D H1LL0011.D HILL0012.D HILL0013.D HILL0014.D HIU0015.D HILL0016.D HILL0017.D HILL0021.D HILL0022.D H1LL0023.D HILL0024.D HILL0025.D HILL0026.D HILL0027.D H1LL0028.D HILL0029.D HILL0030.D H1LL0031.D HILL0032.D HILL0044.D HILL0056.D HIIL0059.D HILL0060.D HILL0061.D HILL0062.D HILL0063.D HJLL0064.D HILL0Q65.D HILL0068.D H1LL0067.D HILL0068.0 HILL0069.D HILL0070.0 12 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 7 7 1 2 3 4 5 6 7 8 9 10 11 12 Page 1 of 3 BACK TO MAIN Day 1/W ater/PFOS TCR-0QQ17-46 THPFOS PPb BI Area 250,0 250.0 250.0 250.0 250.0 250.0 250.0 250,0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 250.0 8.146 6.139 6 .1 4 1 6.143 6.140 8.147 6.138 6.139 6.142 6.146 6.140 8.143 6.146 6.143 6.137 8.139 8.144 6.147 6.148 8.142 6.142 6.153 6.148 6.143 6.145 6.140 6.139 6.144 6.136 6.148 6.140 6.137 6.147 6.146 6.141 6.139 6.142 6.139 6.140 8.138 2822194.3 2762785.5 2735708.0 2820521.3 2804231.5 2783125.8 2816558.0 2815918.5 2863963.3 2809055.3 2812877.5 2825108.3 2805414.5 2843693.3 2774394.0 2850030.0 2837082.3 2830421.8 2812000.3 2806759.3 2851692.8 2798432.0 2798688.8 2802453.3 2823974.3 2813255.0 2845795.3 2861852.0 2772065.0 2875237.0 2812862.5 2774225.3 2792990.3 2817281.0 2841104.8 2815518.5 2828082.3 2806456.5 2788683.3 2777985.3 PFOS HI Area PPb 6.543 6.541 6.542 6.543 6.541 6.544 8 .5 4 1 0.541 6.541 0.542 6.541 6 .5 4 4 6.541 6.542 6.544 0 .5 4 1 6 .5 4 3 6.541 6.541 8.543 6.541 6.541 0.540 6 .5 4 3 6.542 6.543 6 .5 4 2 6 .5 4 4 6.544 6 .5 4 3 8.542 6 .5 4 4 6 .5 4 4 0.541 6.541 6.541 541 6.543 6.544 0.541 35165920.0 34447728.0 88942.6 266916.0 339340.9 511958.0 1271388.0 2052451.9 2591958.0 3750088.3 5002989.0 11418789.0 19901130.0 33450226.0 63275.9 185661.4 299971.8 494519.7 1249220.0 1996589.8 2495294.5 3644372.5 4826660.0 11151017.0 19737748.0 33120708.0 2410067.3 2454911.8 56645.5 101235.6 282306.2 470515.5 1215011.4 1949906.5 2417009.5 3560460.0 4877907.5 10850738.0 19181934.0 32261558.0 0.0 0,0 1.2 4.8 0.3 10.0 25.5 41.9 52.4 78.5 106.0 258.9 516.4 0.0 0.7 3.1 5.4 9.4 25.1 40.9 50.6 70.5 102.7 254.3 506.0 0.0 48.9 49.6 0.5 2.6 5.1 9.1 24.8 39.7 49.2 74.2 98.2 246.1 495.1 0.0 124.3% 108.7% 94.1% 100.3% 102.0% 101.1% 101.8% 102.5% 101.0% 101.0% 97.7% 99.0% 103.4% 102.5% 91.1% 98.1% 99.1% 98.2% 98.7% 98.0% 98.3% 98.9% Data from Hilary-H 082700.s (-Hi' X>cD T C R -p S f'C -P BACK TO MAIN Day 1/Water/PFOS TCR-00017-48 S am ple Pata/Perform ance 731 OO-wPFOS 1.1.1 731 OO-wPFOS 1.1.2 731 OO-wPFOS 1.1.3 T C R -00017-48 T C R J tff PFOS Solubility In waterO Day 1 Samples MLA 8 -> q o TC R -00017-46 TCR-AOTPFOS SoWjWty In waterO Day 1 Sam ple* MLA B-Z-ao T C R -00017-48 TCR-0OTPFOS S olubiity In waterO Day 1 Sam ple* M LA 8^2*00 TCR-00017-48 TCR-ACCfPFOS SoMHSty in waterO Run # Mtd Data FUe Vial# DDb 35 H82700B.M HILL0035.D 21 250.0 36 H82700B.M HILL0Q38.D 22 250.0 37 H82700B.M HILL0037.D 23 250.0 __WA07nnR M -- 34-- , 250.0 731 OO-wPFOS 1.2.1 731 OO-wPFOSI .2.2 73100-W P FO S 1.2.3 731 OO-wPFOSI.2.4 TC R -00017-48 TCR-0&r1FOS Solubility In waterO Day 1 Samples MLA 8 -^ 0 6 TC R -00017-48 T C R 4 0 1 W 3 S Solubility in waterO Day 1 Samples MLA 8-24Sp TCR-00017-48 TCR-OW>PFOS S olubiity in waterO Day 1 Samples MLA 8 - 2 f 0 TCR-00017-48 TC R -O &fPFO S Solubility In waterO Day 1 Sample* MLA 8-2-00 40 H82700B.M HILL0040.D 25 41 H82700B.M H1LL0041.D 26 42 H82700B.M HILL0042.D 27 43 H82700B.M HILL0043.D 28 250.0 250.0 250.0 250.0 73100-WPFOS1.3.1 731 OO-wPFOSI .3.2 73100-W P FO S 1.3.3 731 OO-wPFOS 1.3.4 TCR-00017-48 T C R -S & P F O S S o M > l*y In waterD Day 1 Samples MLA 8 2 -0 0 TCR-00017-48 TC R -W T P F O S S olubiity in waterO Day 1 Sample* MLA 8-2-00 TCR-00017-48 T C R -9 9 T P F 0 S SoluWIty In waterO Day 1 Samples MLA 8-2-00 TCR-00017-48 TCR-O0TPFOS SoluWIty in waterO Day 1 Sample* MLA 8-2-00 47 H82700B.M HILL0047.D 29 48 H82700B.M HILL0048.D 30 49 H82700B.M HILL0049.D 31 50 H82700B.M HILL0050.D 32 250.0 250.0 250.0 250.0 731 OO -wPFOSI.4.1 731 OO -wPFOSI.4.2 731 OO -wPFOSI.4.3 731 OO-wPFOS 1.4.4 (j) TC R -00017-48 T C R -o W = F O S SolubIHy In waterD Day 1 Sample* MLA &2430 52 TCR-00017-48 TCR-tJOTPFOS Solubility in waterO Day 1 Samples M L^f-^-O O 53 TC R -00017-46 T C fW M PFOS S olubiity In w aterd Day 1 Sample* MLA8-.2-00 54 TC R -00017-48 T C R W * PFOS SoluWIty in waterO Day 1 Sample* MLA 8-2-00 .... _ ___________ ___ 5 5 _ rtod-TCKl-oo^. ~~ m 11-20 6 0 " H82700B.M HILL0052.D H82700B.M HILL0053.D H82700B.M HILL0054.D H 827 0 0 B M HILL0Q55.D 33 34 35 38 250.0 250.0 2500 250.0 A ve ra g e : Std Dev: %CV: FI 6.145 6.144 8.148 6.144 6.146 8.145 8.140 0.140 6.143 6.140 0.146 8.145 8.149 0.151 8.146 6.142 ppb Area________ _______ EI__ ___ baa___ Bute__ 2870339.3 6.544 2993830.5 80.7 Average PPM S td Dev ppm %CV 607.1 608.3 2893204.5 0.544 2943648.8 59.2 591.7 14.1 2862875.8 6.541 3078239.0 62.0 828.1 2.3% ---------------- 6 * 4 2 ------ 1 4 7 1 7 8 7 8 .0 --- 2 8 4 ^ ------ 2 6 4 8 8 --------- -?t2*paexelu d sd 2861874.0 2842725.5 2891975.3 2862384.8 2805592-0 2898872-3 2887931.3 2879242.8 8.542 6.541 0.543 0.543 3295552.3 3248570.0 3038282.0 2800103.5 6.540 0.543 8.541 0.540 3339678.3 3339718.3 3013332.0 3418030.3 67.3 66.7 61.2 56.8 68.1 67.3 60.7 69.4 072.8 867.5 011.7 588.0 681.2 073.1 607.3 094.4 830.0 43.0 6.8% 664.0 33.6 5.1% 2901136.3 2887013.5 2898727.8 2873989.3 6.544 6.542 6.542 8.542 41780.1 47069.1 34471.0 40063.7 2831206.4 38740.1 1.4% (S td * AND Sm pls Included) 0.2 0.3 0.0 0.2 Total Day 1 ppm 636.S Std Dev 40.6 %CV 8% FACT -TCR002 (LIMS #E00-1716), Page 49 of 67 Page 2 of 3 Data from t f la ry-H 082700.* TCR-ptfl ( M litf I'' Blank Oata/Perfonnapee Sample ID Sample Dese MeOH TNM484 Alquoted 25 Jul 00 MeOH TNA4464 A1quoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4484 Alquoted 25 Jul 00 MeOH 7NA4464 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jtd 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4404 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNM464 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4464 Aiquoted 25 Jul 00 MeOH TNA4404 Aiquoted 25 Jul 00 Run# 1 4 5 18 19 20 33 34 39 45 46 51 57 58 71 72 Mtd H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H62700B.M H82700B.M H82700B.M H82700B.M H82700B.M H82700B.M H827Q0B.M H82700B.M H62700B.M H82700B.M Date File HILL0001.D HILL0004.D HILL0005.D HILL0018.D H1LL0019.D HILL0020.D HILL0033.0 HILL0034.D HILL0039.D HILL0045.D HILL0046.D HILL0051.D HILL0057.D H1LL0058.D HILL0071.D HILL0072.D V ia)# 91 91 91 91 92 92 92 92 93 93 93 93 94 94 94 94 Page 3 of 3 BACK TO MAIN Day 1/Water/PFOS TCR-00017-46 p0p.0b 00..00 000...B000000I000 0000000000000.............0000000000000 0000000000000.............000000000000000000000000000000000000000 A0r.e0a 0.0 0.0 00..00 00000000........00000000 00..00 0.0 BI 6.543 6.542 6.542 0.541 0.542 0.543 6.542 0 .5 4 4 8 .5 4 3 6.542 0 .5 4 2 6.541 6 .5 4 0 0.543 6.543 0.542 Area 459292.2 60705.3 45505.9 82874.6 106202.0 05038.9 09268.1 53477.0 74133.6 54522.9 47028.0 48018.3 78030.4 40175.2 54503.8 40344.1 "35" LOQ Area p000p...000b > 5 0 * LOQ 0.0 00000.....00000 > 5 0 * LOQ 0000....0000 Q0..0Q 0.0 Data from HiBary-H082700.* &* (@Our ii-pqlJO TCR401 PFOS3olubty Day 2 Semole P f Acquired on HBarv 8-2KHI Sequence: H0629PQJ Standards Dat/Performanee Sample ID 00027-43-11 00027-43-11 00027-43-00 00027-43-01 00027-43-02 00027-43-03 00027-43-04 00027-43-05 00027-434)8 00027-43-07 00027-43-08 00027-43-09 00027-43-10 00027-43-11 00027-434 00027-43-01 00027-43-02 00027-43-03 00027-43-04 00027-43-05 00027-43-06 00027-43-07 00027-43-08 00027-43-09 00027-43-10 00027-43-11 00027-43-08 00027-43-06 00027-43-00 00027-434)1 00027-434)2 00027-434)3 00027-43-04 00027-434)5 00027-43-06 00027-43-07 00027-434)6 00027-434 00027-43-10 00027-43-11 Sampls Dssc System Suitabilty Samples System Suitabllty Samples System SuHatoflKy Samples System SuttabIMy Samples System Suitability Samples System SultabiMy Samples System Suitability Samples System Suttabilty Samples System Suitabllty Samples System Suitabllty Samples System SiritafaMy Samples System SuKebiHy Samples System Suitabllty Samples System Suitabllty Samples 0 Std Std 1 (Exobdad to better IN <Ma) Std 2 (LOO-5 ppb) Std 3 Std 4 Std 5 Std 6 Std 7 Std 6 Std 9 Std 10 Std 11 (Excluded to better lit data) CCV CCV 0 Std Std 1 (Excluded to bettor fit (Ma) Std 2 (LOO-5 ppb) Std 3 Std 4 Std 5 Std 6 Std 7 Std 6 Std 9 Std 10 Std 11 (Exdudod to better 6t data) Run# 2 3 6 7 6 9 10 11 12 13 14 15 16 17 21 22 23 24 25 26 27 26 29 31 32 44 66 66 60 61 62 63 64 65 66 87 68 66 70 Mid H082900A.M H062900A.M H08260QAJ4 H082900A.M H062900A.M H062900A.M H062900A.M H06290QA.M H082900A.M H062900A.M H062900A.M H062900A.M H062900Al.M H062900A.M HQ629Q0AM H062900A.M H062900A.M H062900A.M H062900A.M H082900A.M H082900A.M H062900AM H062900A.M H062900A.M H062900A.M H082900AM H082900A..M H082900A.M HQ62900A.M H062900A.M H062900A.M H082900A.M H0829004JUI H062900A.M H062900A.M H0829Q0A.M H082900A.M H062900A.M H082900A.M H062900A.M Data File HILL0002.D NLL0003.0 HILL0006.D HLL0007.0 HILL0008.D HLL0009.0 WLL0010.D HILL0011.0 HILL0012.0 HILL0013.D HLL0014.D HILL0Q15.O HILL0016.D HILL0017.0 HLL0021.D HILL0022.D HILL0023.D LL0O24.D HILL0025.D HILL0028.D HIU.0027.D HI110O26.D HJLL0029.D HILL0030.D HILL0031.D HLL0032.D HILL0044.D HILL0056.D HILL0059.D HILL0060.0 HILL0061.D MLL0062.D HILL0063.D HILL0064.0 HILL006S.D HIL0066.D HILL0067.D HIU.0066.D HILL0069.0 HILL0070.0 FACT -TCR002 (LIMS #E00-1716), Page 51 of 67 BACK TO MAIN Day 2/Water/PFOS TCR-00017-48 m m gK m m gK m ggK m ggm ggm m gii thpfps THPFOS fil 6.15 6.15 6.14 6.15 6.14 8.15 6.15 6.14 6.14 6.14 6.15 6.15 6.15 8.18 8.14 8.14 6.14 6.14 8.15 6.15 6.15 8.15 8.15 6.15 6.15 6.14 6.14 8.14 8.14 6.14 6.14 6.15 6.15 8.15 6.15 6.15 8.14 8.15 8.14 6.15 THPFOS Aras 1068757.1 1153380.6 1388342.3 1484845.6 1540848.0 1618214.0 1872720.0 1728031.1 1824887.0 1820424.6 1856518.4 1800033.6 1040071.6 1072672.0 2046207.4 2111404.0 2096061.6 2132470.0 2144005.3 2171590.8 2205455.0 2171843.3 2190710.0 22Q0Q04J 2175422.0 2224102.3 2341012.3 2375128.5 2358025.3 2355963.5 2369146.6 2354475.5 2380583.0 2378348.5 2422405.0 2380072.0 2373400.8 2401039.3 2356475.3 2300706.0 PFOS El 6.54 8 .5 4 6 .5 4 6.54 6.54 0.54 8.54 8.54 6 .5 4 6 .5 4 6.54 6 .5 4 0 .5 4 654 0 .5 4 6 .5 4 6.54 6 .5 4 6 .5 4 654 6.54 0 .5 4 6 .5 4 6.54 6.54 6.54 0.54 8.54 6.54 8.54 0.54 6 .5 4 6.54 6 .5 4 6.54 6 .5 4 6 .5 4 6.54 0 .5 4 6.54 PFOS PFOS Arsa ppb 25785074.0 0.0 6203012.0 0.0 09273.1 1.2 431029.4 14.2 316246.5 9.5 458310.6 13.6 1121098.5 35.0 1784189.6 55.1 2210081.3 65.5 3183340.6 06.0 4244007.0 127.8 0616081.0 300.6 16940810.0 634.1 28705198.0 0.0 60762.7 0.1 248047.4 X Excluded 270481.7 5.5 109.1% 451537.4 0.9 00.1% 1104108.5 20.4 105.4% 1771716.0 43.0 107.3% 2195559.5. 53.0 105.9% 3203310.3 60.2 106.8% 4271387.5 107.8 1078% 9714680.0 262.9 105.0% 16905772.0 533.6 106.5% 28831362.0 X Exciudad 2213222.8 50.2 100.2% 2157711.5 48.2 96.2% 55850.0 X 206739.0 X Exciudad 249832.4 4.2 84.0% 417615.9 8.1 80.6% 1075203.6 23.2 02.5% 1705371.0 37.5 03.7% 2167708.8 47.4 94.6% 3116886.6 70.fi 942% 4177286.0 06.7 96.5% 0566670.0 234.9 03.8% 16677574.0 460.0 93.8% 28617368.0 X Excluded Points E xdudsd to bs tts r fit data. 3M ETS Laboratory Acquisition RunH82OO on Hilary FACT -TCR002 (LIMS #E00-1716), Page 52 of 67 Paga 2 of 3 SM ETS Laboratory Acquisition Run H082900 on Hilary 5 I < s o o* <C 5 f =8 I r t S- HSqooooooooaoooaoo Ujo d o o d d o d o o d d o d d 3h 8 8 8 8 S 8 8 8 8 8 8 8 8 8 8 8 a"odood Sooooooooooooaooo |SS5SSSS83SS3S3SS aaaaaaooaoaaaaao s liliiiiiiiiiils i "i i i i i i i sssssssassss: .asajsssaacc 8888888888888888 ?i s1sssssIssssas?ssaa-s f5 m 5 3i iiis m 5i m ?i I s s s |s |s |5 |||||s s s assisasasJsasssi FACT -TCR002 (LIMS #E00-1716), Page 53 of 67 Pag* 3 of 3 3M ETS Laboratory Acquisition Run H02900 on Hilary cm d m m ii-x-co TCR-OM PFOS SdubiRty (toy b Standards Data/Parformance Sample ID 00027-43-00 00027-43-01 00027-43-02 00027-43-03 00027-43-04 00027-43-05 00027-43-0$ 00027-43-07 00027-43-05 00027-43-09 00027-43-10 00027-43-11 00027-43-06 00027-43-06 00027-43-00 00027-43-01 00027-43-02 00027-434)3 00027-43-04 00027-43-05 00027-43-06 00027-43-07 00027-43-08 00027-43*09 00027-43-10 00027-43-11 Sample Desc OStd Std 1 (Excluded to batter fit data) Std 2 (LOQ-5 ppb) Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Std 9 Std 10 Std 11 (Excluded to better fit data) CCV CCV OStd Std 1 (Excluded to better fit data) Std 2 (LOQ-5 ppb) Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Std 9 Std 10 Std 11 (Excluded to better fit data) Run# 3 4 5 6 7 8 9 10 11 12 13 14 26 38 41 42 43 44 45 46 47 48 49 50 51 52 Mtd H82500A4.M H82500A4.M H82500A4.M H62500A4.M H8250QA4.M H62500A4.M H82500A4.M H82500A4.M H62500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H62500A4.M H 825 0 0M .M H82500A4.M H62500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M Data FUe HILL0097.D HILL0098.D HILC0099.0 HILL0100.D HILL0101.D HILL0102.D HILL0103.D HILL0104 0 HILL0105.D HILL0106.D HILL0107.D HILL0108.D HILL0120.D HILL0132.0 HILL0135.D HILL0136.D HILL0137.D HKA.0138.D HILL0139.D HILL0140.D HILL0141.D HILL0142.D HILL0143.D HILL0144.D HILL0145.D HILL0146.D FACT -TCR002 (LIMS #E00-1716), Page 54 of 67 Page 1 of 3 Exact C opyofW glnaJ ^ M-- Initial BACK TO MAIN Day 3/Water/PFOS TCR-00017-46 V I aW 1 2 3 4 5 6 7 8 9 10 11 12 7 7 1 2 3 4 5 6 7 6 9 10 11 12 PPb 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 SI 6.15 6 .1 5 6.15 6.14 6.15 6.15 6.15 6.15 6.14 6.15 6.15 6.15 6.15 6 .1 5 6.15 6.15 6 .1 4 6.15 6.14 8.15 6.15 6.15 6.15 6.14 6.14 6.15 Area 2827919.5 2887205.8 2856399.5 2873539.5 2871680.0 3161595.5 2915268.8 2875385.0 2920716.5 2889778.3 2901318.3 2911203.5 2903577.8 2930110.3 2850464.3 2869825.5 2855512.3 2842810.8 3141249.8 2889577.3 3207130.3 2852557.5 3112386.0 2871272.3 2871562.3 3065178.3 SI 6 .5 4 6 .5 4 6.54 6.54 6 .5 4 6 .5 4 6.54 6 .5 4 6 .5 4 6 .5 4 6 .5 4 6 .5 4 6.54 6.54 6 .5 4 6 .5 4 6 .5 4 6 .5 4 8 .5 4 6 .5 4 6 .5 4 6.54 6 .5 4 6.54 6.54 6 .5 4 Area PPb 53350.4 1.5 166313.7 3.7 Excluded 298617.5 6.3 126.1% 507393.3 10.4 103.7% 1323633.9 26.6 106.3% 2040681.1 37.3 93.0% 2634008.8 52.3 104.4% 3827296.5 77.7 103.4% 5067724.5 102.2 102.0% 11650840.0 253.2 101.1% 20554404.0 34501388.0 501.6 0.0 100.2% Excluded 2595877.8 51.7 103.3% 2589949.8 51.2 102.1% 52082.5 1.5 158504.7 3.5 Excluded 297578.5 6.3 125.7% 506642.3 10.5 104.6% 1229335.4 22.6 90.3% 2049667.8 41.0 102.2% 2538722.5 45.8 91.4% 3766329.8 77.0 102.5% 4901655.0 92.4 92.2% 11477140.0 250.8 100.2% 20257486.0 35672188.0 499.0 0.0 99.6% Excluded Points Excluded lo better fit data. 3M ETE Laboratory Data fro m H ila ry H082500a Data from Hilary H082500a 3M E TS *.e brat<Ky o feli. < s o I- o* <C <3 s FACT -TCR002 (LIMS #E00-1716), Page 55 of 67 Page 2 of 3 Cibi (g) fa n-XrC TCR-ptf PFOS Solubiily Blank Data/Performance Sample ID MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 MeOH TNA4464 Sample Desc Alquoted 25 Juf 00 Alquoted 25 Jul 00 A lquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jil 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Alquoted 25 Jul 00 Run* 1 2 15 16 21 27 26 33 ' 39 40 53 54 55 Mid H82500A4.M H82500A4.M H82500A4.M H8250QA4.M HS2500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M H82500A4.M Data Fla HIU0095.D HILL0096.D HILL0109.D HILL0110.D HILL0115.D HILL0121.D HILL0122.D HILL0127.D HILL0133.D HILL0134.D HILL0147.D HILL0148.D HILL0149.D V ia l* 96 96 96 96 97 97 97 97 98 98 98 98 98 ppb 0 0 0 0 0 0 0 0 0 0 0 0 0 El 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 FACT -TCR002 (LIMS #EOO-1716), Page 56 of 67 Page 3 of 3 3M ETSefcewt#fy BACK TO MAIN Day 3/Water/PFOS TCR-00017-46 Area 6 1 t e a PPb 0.0 6.54 55538.7 0.4 0.6 6.54 48190.1 0.4 0.0 6.54 42932.7 0.4 0.0 8.54 41835.0 0.4 0.0 6.54 77874.9 0.4 0.0 6.54 54579.8 0.4 0.0 6.54 50519.9 0.4 0.0 6.54 50779.9 0.4 0.0 6.54 81693.0 0.4 0.0 6.54 61039.4 0.4 0.0 6.54 51639.5 0.4 0.0 6.54 45487.9 0.4 0.0 6.54 40381.1 0.4 LOQ-5ppb S td S3 Average LOO Area 149049.0 Data from Hillary H082500a Attachment C: Sample Chromatograms BACK TO MAIN FACT -TCR002 (LIMS #E00-1716), Page 57 of 67 LIS L U l i fM lil H U U 1 I I Data File C:\HPCHEM\1\DATA\H082900\HILL0012.D Sample Nan Injection Date 8/29/00 7:09:50 PM Seq. Line Sample Name 00027-43-06 Vial Acq. Operator KLT on "Hillary" Inj Inj Volume Acq. Method C :\HPCHEM\1\METHODS\PFOS825.M Last changed 8/29/00 4:33:39 PM by KLT Analysis Method C :\HPCHEM\1\METHODS\H082900A.M Last changed 8/30/00 11:31:37 AM . (modified after loading) Data Reprocessing on Workstation "Lefty" PFOS Quant using internal Std THPFOS KLT 8-30-00 : 12 :7 :1 : 5 pi BACK TO MAIN MSD1 427, EIC=426.7:427.7 (H082900\HILL0012.D) API-ES Negative 400000 350000-! 300000 250000 200000-i 150000 100000 -i 50000 Q-. MSD1 409, EIC=498.7:499.7(H082900\HILL0012.D) API-ES Negative 500000400000 7 300000 200000 -i 100000-i 0: x 6 CO P -L i A T 6 T 8 -r 8 10 10 Internal Standard Report Sorted By : Calib. Data Modified : Multiplier : Dilution : Sample ISTD Information: ISTD ISTD Amount Name # [ppb] 1 250.00000 THPFOS Signal Wednesday, August 30, 2000 11:17:18 AM 1.0000 1.0000 ' Exaa* C o p v f-r ^ ' ''gu ----------------------------. Ifeii Instrument 1 8/30/00 11:31:38 AM FACT -TCR002 (LIMS #E00-1716), Page 58 of 67 Page 1 of 2 Data File C:\HPCHEM\1\DATA\H082900\HILL0012.D Sample Na Injection Date 8/29/00 7:09:50 PM Seq. Line Sample Name 00027-43-06 Vial Acq. Operator KLT on "Hillary" Inj Inj Volume Acq. Method C:\HPCHEM\l\METHODS\PFOS825.M Last changed 8/29/00 4:33:39 PM by KLT Analysis Method C :\HPCHEM\1\METHODS\H082900A.M Last changed 8/30/00 11:31:37 AM (modified after loading) Data Reprocessing on Workstation "Lefty" PFOS Quant using internal Std THPFOS KLT 8-30-00 12 7 1 5 pi BACK TO MAIN Signal 1: MSD1 427, EIC=426.7:427.7 RetTime Type Area Amt/Area [min] ratio ^I 1 6.145 BB I 1.82489e6 ' 1.00000 Amount Grp Name [ppb] 250.00000 THPFOS Totals without ISTD(s) : 0.00000 Signal 2: MSD1 499, EIC=498.7 :499.7 RetTime Type [min] "1 6.540 VBA+ Area 2.21968e6 Amt/Area ratio 1 2.15268e-l Amount Grp Name [ppb] 65.45964 PFOS Totals without ISTD(s) : 65.45964 *** End of Report *** Instrument 1 8/30/00 11:31:38 AM **^ofO r na! jm S Z Date FACT -TCR002 (LIMS #E00-1716), Page 59 of 67 Page of 2 BACK TO MAIN Attachment D: Deviations from the Protocol FACT -TCR002 (LIMS #E00-1716), Page 60 of 67 NO.480 BACK TO MAIN Centre Analytical Laboratories. Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (614) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 Page PROTCK:O L DEVIATIO N Deviation Nuniber: 001 Date of Occurrence: Entire Study Centre Study Number. 023-021 SDonsor Studv Number: FACT-TCR002 001 .. DESCRIPTION OF DEVIATION Solubility studies of PFOS Primary Standard, Test Control Reference # TCR-00017-46, were not conducted in methanol or acetone due to limited quantities of test substance. ACTIONS TAKEN i.e,, amendment issued. SOP revision, etc... Protocol Deviation issued. Recorded By/Dateu. j / s' / ' f ............... IMPAC TONTHE STUDY Results in limited solubility data for th ; test substance. -Siudy Director Signature Sj^^& km agem ent Signature Centre QAU Re view j-L--s M ol i1 j/i/r Date a /g /o y Date " January 5,2001/3 Exact C o p y o f O riginal FACT -TCR002 (LIMS #E00-1716), Page 61 of 67 BACK TO MAIN Record of Deviation I. Identification Study / Project No. Deviation Type: (Check one) TCR-002 SOP Method Equipment Procedure JS1Protocol Other: Document Number: ETS-8-155.0 Date(s) of occurrence: 13 Jul to 29 ug 00 II. Description: Required Procedure/process: ETS-8-155.0 section 10.1.2 states, "Analyze a method blank and a matrix blank prior to each calibration curve. Actual Procedure/process: In this experiment, the method and matrix blanks are essentially the same. The method/matrix blanks were not ran before the calibration curves. The method blanks were ran after the first calibration curve. III. Actions Taken: (such as amendment issued, SOP revision, etc.) The ETS-8-155.0 method has been revised so that the method and matrix blanks are to be ran after the first calibration curve. Recorded By: Date: IV. Impact on Study / Project 0// 6 # / The impact of this deviation on the study is minimal. The placement of the blanks have no bearing on the results. See Corrective Action. 3MEnvironmental Laborofp Q Form ETS-4-8.0 C fln v f it O rininall Deviation No. _0__2_.__ W O py T w i l y (Qf^ihedby Study Director or Project Lead at the end of study or project) fr/tff/ol In itia l A . D a te r -TCR002 (LIMS #E00-1716), Page 62 of 67 BACK TO MAIN Record of Deviation I. Identification Study / Project No. Deviation Type: (Check one) TCR-002 SOP Method Equipment Procedure S-Protocol Other:. Document Number: ETS-8-155.0 Date(s) of occurrence: 13 Jul 00 to 29 Aug II. Description: Required Procedure/process: ETS-8-155.0 sectionlO.2.1 states, " Matrix spikes are prepared for each sample set and analyzed to determine the matrix effect on the recovery ^ c ie n c y ? ' Section 0.2.3 states, "analyze the matrix spike and the matrix spike duplicate ( if prepared) in the same run as the original sample." Actual Procedure/process: The matrix spikes were not prepared, therefore, they were not analyzed. See Corrective Action. -- ...... ... - ..--.... 2/J?/o/ Exact C opy o f Original! 7 III. Actions Taken: (such as amendment issued, SOP revision, etc.) The ETS-8-155.0 method has been revised so that matrix spikes do not need to be prepared when there is no need. See Corrective Action. Date: IV. Impact on Study / Project The impact of this deviation on the study is minimal. The lack of a matrix spike has no bearing on the final results. The matrix is well defined and characterized. The matrix spike is actually not possible in this study since the matrix is saturated with the analyte of interest. See Corrective Action. Authorized By: (Study Director / Project Lead) Date: Z>^/o IJot 3M Environmental Laboratory Form ETS-4-8.0 Deviation No. Q3 (assigned by Study Director or Project Lead at the end o f study or project) FACT -TCR002 (LIMS #E00-1716), Page 63 of 67 BACK TO MAIN Record of Deviation 1. Identification Study / Project No. Deviation Type: (Check one) TCR-002 SOP Method Equipment Procedure 0 Protocol Other: Document Number: ETS-8-155.0 Date(s) of occurrence: 13 Jul 66 to 29 Aug 66 II. Description: Required Procedure/process: In dealing with the standard curve, the curve must have a coefficient of determination (r2) value greater than or equal to 0.980 and individual curve points must have `Percent Deviations' equal to or less than +/- 30%. Actual Procedure/process: In some cases, high and/or low curve points were eliminated so that the ? value would meet the criteria. In other cases, high and/or low curve points were eliminated, as they were not within the +/-30% accuracy requirements. When this was done, the curve point(s) affected and the action(s) taken were annotated on the `Data Summary Sheet'. The following is a list of all curve points affected and the reasons why. Day 1, (H082700), points Hill0032 and HU10070. Day 2, (H082900), points Hill0022,0032, 0060 and 0070. Day 3, (H082500a), points Hill0098, 0108, 0136 and 0146. All of these points were excluded to provide a better fit over the range appropriate to the data. s u ON O III. Actions Taken: (such as amendment issued, SOP revision, etc.) The 3M Environmental Method ETS-8-155.0 was amended to take into account the elimination of curve points. Curve points may be omitted for the following reasons: (Comments are bulleted as they would appear on the summary sheets.) (1) "High/low curve points were excluded to provide a better fit over the range appropriate to the data." (2) "Low level poinf(s) were not 2x higher than the extraction blank; these points were excluded from the curve to disqualify a data range that may have been significantly affected by background levels of the analyte." (3) "High/low curve point(s) were excluded as they were not within the +/- 30% accuracy requirements of the method when the curves were evaluated over a range appropriate to the data." o a>* 03 <o0 & X u 3MEnvironmental Laboratory Form ETS-4-8.0 Deviation No. CA" (assigned by Study Director or Project Lead at the end o f study or project) FACT -TCR002 (LIMS #E00-1716), Page 64 of 67 BACK TO MAIN Recorded By: s Record of Deviation Date: IV. Impact on Study / Project 0 /fc j 0 / The elimination of curve points had a positive impact on this study. If curve points were not eliminated, than the curves would not be as accurate. Authorized By: (Study Director / Project Lead) Date: 0 * ^ 0 1/^1) oyfo/gj Date for In itia l 3MEnvironmental Laboratory Form ETS-4-8.0 Deviation No. CM- [Xtykk'2. (assigned by Study Director or Project Lead at the end of study or project) FACT -TCR002 (LIMS #EOO-1716), Page 65 of 67 Attachment E: Sample Calculations BACK TO MAIN FACT -TCR002 (LIMS #E00-1716), Page 66 of 67 BACK TO MAIN 3M Environmental Laboratory Report No. FACT-TCR002 Mean and standard deviation were calculated using functions provided in Microsoft Excel* software. Standard deviation population was used to measure the scatter about the mean o f a data set; thus, it can be used to estimate the precision of a method. Relative standard deviation presents a measure of the magnitude of the standard deviation and is calculated by dividing the standard deviation population by the mean. Means are calculated by adding individual entities and dividing the resultant sum by the number of individual entities. Standard deviation population was calculated using the following equation: -W here n is the number of observations, and x is sample concentration. A verage/Standard Deviation Populatfon/% RSD exam ple calculations: Sam ple ID Rep1 Rep 2 Rep 3 Rep 4 C oncentration pg /m L 647.9 723.4 723.0 717.3 C alculations Average (647.9+723.4+723.0+717.3)/4= 702.9 Std Dev Pop (see above equation) 31.8 %RSD (31.8/702 .9)* 4.5% Data that did not meet acceptance criteria as described in the OPPTS and OECD guidelines was excluded using statistical justification provided by Dixon's Q -tesl A data point may be excluded if "Qotved" <s greater than with 90% confidence. = gap/range where "gap" is the difference between the questionable data point and the closest value of the data set, and "range" is the difference between the highest and lowest value of the data s e t Q-tabulated (90% confidence) for 4 observations (or data points) is 0.679 and for 3 observations is 0.886. Q -Test Exam ple (Shaded numberrepresents the suspected outlier): Description Sam ple C one., Rep1 Sam ple C one., Rep2 Sam ple C one., Rep3 Sam ple C one., Rep4 Range (Rep4-Rep2): G ap (Rep4-Rep3): QohMMd (Range/G ap): No. observations: . Retain? no uo/m l 607.1 591.7 626.1 2 0 5 7 .1 4 2 0 2 2 .6 6 0.983 4 Q-Test example conclusion: Since excluded. __ ,_ 0.983, was greater than Qubuumi, 0.679, rep 4 may be FACT -TCR002 (UNIS #E00-1716), Page 67 of 67