3M Environmental Laboratory Report No. W1878 Study Title Hydrolysis Reactions of Pertluorooctane Sulfonate (PFOS)

Data Requirement: Based on OPPTS: 835.21 10 Author Thomas L. Hafield, Ph. D.

Study Completion Date March 27,2001 Performing Laboratory 3M Environmental Laboratory Building 2 3E 09,935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Laboratory Report No: W1878 Total Number of Pages 71 Page 1 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 This page has been reserved for specific country requirements.

Page 2 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Statement of Non Compliance StudyTitle: HydrolysisReactionsofPerfluorooctaneSulfonate( PFOS). StudyIdentificationNumber: W1878 This study does not comply with the requirements of the US EPA Good Laboratory Practices Standards at 40 CFR Part 792 (TSCA). However, many GLP standards were used in the development of the analytical method (Appendix A), and the quality assurance procedures followed in this study were based on the practices described in the GLP documentation.

Spo'nsor Representative Date Page 3 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Quality Assurance Statement StudyTitle: HydrolysisReactions of PemuorooctaneSulfonate( PFOS). StudyIdentificationNumber: W1878 The following table provides details of the audits performed by the 3M Environmental Laboratory Quality Assurance Unit (QAU).

Date Reported to Management Study Director Inspection Dates Phase I I 8/ 11/ 00, 8/ 16 17/ 00 I Dataand Draft Report I 8/ 18/ 00 I 8/ 18/ 00 I I 9/ 7,8, 11/ 00 I Dataand Draft Report I 9/ 11/ 00 I 9/ 11/ 00 1 3/ 13 14/ 0 1 I DraftReport I 3/ 14/ 01 1 3/ 14/ 01 1 Page 4 of 71 BACK TO MAIN 3M Environmental Laboratory Report No . W1878 Table of Contents Statement of Non Compliance ............................................................................................ 3 Quality Assurance Statement .............................................................................................. 4 List of Tables ........................................................................................................................ 6 List of Figures ....................................................................................................................... 6 Study Personnel and Contributors ....................................................................................... 6 Location of Archives ............................................................................................................. 7 Summary .............................................................................................................................. 8 Introduction .......................................................................................................................... 9 Summary of Kinetics Model ............................................................................................... 10 Materials and Methods ....................................................................................................... 11 Chemical Characterizations .......................................................................................... 11 Sample Preparation ...................................................................................................... 11 Sample Analysis ............................................................................................................ 12 Deviations ...................................................................................................................... 12 Results and Discussion ..................................................................................................... 14 Data Quality Objectives (DQO's) .................................................................................. 14 Anomalous Analytical Results ....................................................................................... 14 Statistical Methods and Calculations ............................................................................ 15 Data Summary and Discussion ..................................................................................... 15 Conclusions ........................................................................................................................ 19 References ......................................................................................................................... 20 Signatures .......................................................................................................................... 21 Appendix A: Analytical Method .......................................................................................... 22 Appendix B: Kinetics Model ............................................................................................... 38 Appendix C: Selected Analytical and Kinetics Results ..................................................... 48 Appendix D: Selected Chromatograms ............................................................................. 59 Page 5 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 List of Tables Table 1. Summary of Results Based on PFOS Concentrations ........................................ 8 Table 2. Summary of Results Based on the Mean and Precision of PFOS Measurements ...................................................................................................... 8 Table 3. Characterizations of Test and Reference Substances ...................................... 1 1 Table 4. Observed (50' C) Slopes of PFOS Concentrations in Aqueous Buffered Solution for Various pH Levels. .......................................................................... 15 Table 5. Calculated Slope of PFOS Concentrations in Aqueous Buffered Solutions Using Data Pooled Over All pH Levels ............................................................... 16 Table 6. Degradation Rate and Half Life of PFOS in Aqueous Buffered Solutions Based on the Concentration Mean and Standard Deviation ......................................... 18 List of Figures Figure 1. Structure of the Potassium Salt of PFOS ........................................................... 9 Figure 3. Pooled PFOS Data and Slope Regression. ...................................................... 17 Figure 2. Observed PFOS Degradation for Various pH levels. ....................................... 16 Study Personnel and Contributors Study Director Thomas L. Hatfield, Ph. D. 3M Environmental Laboratory Building 2 3E 09 935 Bush Avenue St. Paul, MN 55106 (651) 778 7863 Sponsor 3M Corporation Professional Services Contributing Personnel Kuruppu Dharmasiri, Ph. D. Kevin Macklin Mark T. McCann Anthony E. Scales Angela Schuler Joseph J. S. Tokos, Ph. D. (Pace Analytical Services, Inc., 1700 Elm St., Minneapolis, MN 55144)

Page 6 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Location of Archives The 3M Environmental Laboratory will retain the original data documents and digital copies of the original data related to this work for at least 10 years following the effective date of any related final ruling. Information may obtained through written inquiry addressed as follows:

3M Environmental Laboratory Building 2 3E 09 935 Bush Avenue St. Paul, MN 55106 Page 7 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Summary We report here the results of our study of the hydrolysis of perfluorooctane sulfonate (hereafter, PFOS). Our methods are described below and in Appendix A to this work; our results are based on the observed concentrations of PFOS in buffered aqueous solutions as a function of time. 3" s Environmental Laboratory staff developed the study procedures; they are based on EPAs OPPTS Guideline Document 835.21 I O ' but do not fulfill all the requirements of the guideline. The chosen analytical technique was high petformance liquid chromatography with mass spectrometry detection (HPLUMS). Table 1 summarizes the results of the study.

During this study, we prepared and examined samples at six different pH levels between 1.5 and 11 .O over a period of 49 days, and our results indicate no dependence of the degradation rate of PFOS on the sample pH level. We have excluded the "Day 0 samples because they were improperly stored before analysis; see the "Deviations" section below. Our observations of the PFOS concentrations for the 42 day incubation period (IDay 7" through "Day 49), pooled over the six observed pH levels, are presented in Table 1.

I Table I. Summary of ResultsBasedon PFOS Concentrations. I Calculated slope upper limit (20) Calculated slope Calculated slope lower limit (20) (day')

6.66 x IO ' 5.30 x 1 0 4 2.98 X 10" (day ') (day ')

These results indicate no degradation of PFOS; the derived positive slope does not provide a half life estimate.

The mean value and precision of PFOS concentrations do provide an estimate of the PFOS half life, presented in Table 2.

I Table 2. Summary of Results Based on the Mean and Precision of PFOS Measurements Half Life Page 8 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Introduction Three primary chemical routes of environmental degradation are hydrolysis, photolysis, and biodegradation. Studies of these routes provide information on the environmental persistence of both the "parent" compounds and their reaction products, and are ideally carried out over the range of chemical conditions pertinent to both environmental and metabolic processes. The hydrolysis of the potassium salt of PFOS (or, more generally, its degradation in the presence of H20) is addressed in this report. The structure of the PFOS salt is illustrated in Figure 1.

Fiaure I. Structure of the Potassium Salt of PFOS F F F F F F F F O ..

Page 9 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Summary of Kinetics Model A full mathematical description of the kinetics model employed in this study is presented in Appendix B. The data in similar studies typically allow two independent estimates of the hydrolytic half life, but only one estimate is available from our PFOS data.

A first estimate is usually based on the observed degradation of the compound in dilute, appropriately buffered aqueous solutions. Equation BIO describes the estimated half lifeintermsoftheestimatedtotalhydrolysisrate cp :

=  In( 2)

kP Eq. 1 We attempted to determine the quantity Gp from the experimental data as described in Appendix B. The data corresponding to "Day 7" were used to determine the relative concentration ratios (see the "Deviations" section below; see also Equations B8 and B9). The derived rates, both at various pH levels and when pooled over all pH levels, were poorly determined, and indicate only that PFOS does not degrade in aqueous solution.

A half life second estimate (see Equation B37) is available from the mean p and standard deviation 0 of the observed PFOS concentrations, assuming that they were essentially constant over the experimental portion of the study. This estimate is Eq. 2 where A t represents the sample incubation period.

All the samples used in this study were maintained a reaction temperature of 50" (& 3") C. The quoted results, valid for the reaction temperature 25" C, were calculated from our experimental results according to methods described in Appendix B (Eq. B38 and B39).

Page 10 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Materials and Methods Details of the characteristics of the test materials, sample preparation techniques, and analytical methods are presented in Appendix A (ETS 8 13.0, "Hydrolysis of Perfluorooctane sulfonate (PFOS) and Analysis by High Performance Liquid Chromatography with Mass Spectrometry Detection"). A summary of these items is provided below, as well as a description the known deviations from the procedures of Appendix A. 3M prepared and analyzed the samples included in this study between May 24 and July 12,1999.

Chemical Chamctemizations Table 3 describes the sources and properties of the materials used in this work. These materials were used to prepare both the samples and the quantitative standards used to quantify them. For this reason, and because Equation 1 involves only ratios of the parent and product concentrations, the resulting rate and half life estimates are largely independent of the material purity levels.

1 Table 3. Characterizations of Test and Reference Substances PFOS (Potassium Salt) THPFOS Source ICN Biomedicals 3M ICPIPCP Division Chemical Lot Number 1 Batch # 171 I Batch # 53406 I Physical Description I LightcoloredpowderBrownwaxysolid Molecular Weight (gm mole`) 538 428 Sample Preparation We prepared four 1 .O mL aqueous buffer samples (a sample, a duplicate, a triplicate, and a matrix spike at each of six pH levels (1.5,3, 5,7, 9 and 11) for analysis at eight time intervals (0, 7, 14,21,28, 35,42 and 49 days). Buffered solutions containing 501.5 ng/ mL of the potassium salt of PFOS and 368 ng/ mL of THPFOS (3, 3,4,4, 5, 5, 6,6, 7, 7, 8,8, 8 tridecafluorooctane sulfonic acid), the latter serving as a surrogate for the compound PFOS, formed the basis of all these samples. The chosen buffer solutions are described fully in Appendix A.

All the samples were prepared simultaneously, and all but the "Day 0 samples were placed in an orbital incubator/ shaker maintained at 50" (+ 3") C. After the appropriate incubation times, subsets of the sample vials were removed from the incubator; they were then spiked (as required) with the PFOS solution, diluted 1O: l with methanol containing the internal standard THPFOS, and refrigerated at 4" C. Similar treatment of the "Day 0 samples is prescribed by the analytical method (see Appendix A); they are Page 11 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 to be agitated for a minimum of three minutes, diluted and spiked as described immediately above, and then refrigerated rather than incubated. In this study, the "Day 0 samples were not refrigerated; see the "deviations" section below. Except during the relatively short periods of time required to prepare them, all the samples were shielded from light.

Eight calibration standards containing PFOS (301 to 802 ng/ ml) served as the quantitative basis of the study. All these standards were prepared at the appropriate pH levels using the buffer solutions described in Appendix A.

Sample Analysis The equipment we used for the HPLC/ MS analysis was a Hewlett Packard model 1 100 equipped with a Dionex lonPac@ NG 1 HPLC column (aqueous ammonium acetate/ methanol solvent gradient) and an ALS Model G1322A degassing module. An ALS Model G1315A column heater maintained the column temperature at 40° C, a quaternary pump supplied a column flow rate of 0.3 mumin, and an ALS Model G1313A auto sampler provided 5 pL sample injections. The detector was a Hewlett Packard MSD mass spectrometer, operated in negative mode electrospray ionization mode; anions of PFOS and THPFOS were detected at the mass to charge (m/ z) ratios 499, 427, respectively. We processed the resulting data using the computer program HP ChernStation for LC (Rev. A. 06.0). Further analytical details, including the gradient elution program, instrument and detector parameters, and performance specifications, are presented in Appendix A.

Deviations After incubation and prior to analysis, all samples were stored at temperatures ( 4" C or 20" C) rather than at the prescribed temperature of 4" C prescribed in the Method.

The pH meter calibration was performed with only two standards (at pH = 7.0 and 10.0) rather than at the three Method prescribed pH levels (4.0, 7.0, and 10.0).

Only three ions were monitored in the analysis, instead of the seven specified in the Method.

Two samples (PFOS 157 and PFOS 1 13) were quenched with 18 mL of MeOH rather than the prescribed 9 mL, and the concentration results were corrected to reflect this fact.

The buffer solutions prepared for use as "Day 0" samples were stored at room temperature for seven days prior to spiking with PFOS test analyk and processing. This led to evaporation of the solutions and artificially raised the "Day 0" concentrations. Two additional sets of samples were prepared and analyzed as origmlly intended for generation of the "Day 0" and "Day 7" data. The results of these additional analyses indicated no measurable PFOS degradation in the first seven days. On the basis of these additional results, we have excluded the "Day 0" data h m the data set used to estimate the PFOS degradation rates and half life; the "Day 7" data are used in the calculation of the concentration ratios of Equation B8. For these "Day 0" and "Day 7" re runs, the ammonium acetate solution was 5 mM rather than the 2 mM specified in the method, and Page 12 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 the sample injection was 10 pL rather than the specified 10 pL; the continuing calibration verification sample also exceeded the allowed 15% for these re runs samples.

Page 13 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Results and Discussion Data Quality Objedies (DQO's) We briefly describe the data quality objectives applied in this study below. Appendix A describes them in greater detail. With the exceptions of the anomalous results noted below, all the DQO's were met. Appendix C presents the results for each sample set, organized by pH level. Calibrations. The minimum acceptable coefficient of determination (P) for linear fits to calibration data is 0.990. The acceptance criterion for individual calibration points is that their values fall within k 25% of the linear fit value; data outside this range are excluded and the linear fit is recalculated. No more than two points may be rejected from a calibration data set. Data for the high or low calibration standards may be rejected, though this results in a smaller effective calibration range. The average results of calibrations performed before and after the analytical procedures are used to calculate the analyte concentrations. Continuing Calibration Verification (CCV). Selected calibration samples are examined at the beginning of, during, and at the end of each analytical procedure. The results may not deviate by more than k 15% of the known values. Matrix Spikes. The acceptable percent spike recovery range is 70% to 130%; recoveries outside this range % place the analysis out of control, and require intervention by the Team Leader or designee. Analyte specificity is demonstrated by acceptable analyte spike recoveries. Identically Prepared Samples. Triplicate sample results with relative standard deviations (RSDs) greater than 25% place the analysis out of control, and require intervention by the Team Leader or designee. Solvent Blanks. Concentration results for solvent blanks exceed neither 5% of the highest calibration standard nor 25% of the lowest calibration level. System Suitability. Suitability was demonstrated by either an abbreviated mass to charge (m/ z) check tune or performance of a full auto tune routine.

Anomalous Analytical Results With the following exceptions, our analytical results met or exceeded the data quality objectives of Appendix A.

0 Spike Recoveries. The triplicate samples PFOS 157 through 159 (pH = 7.0) failed to meet this DQO and were rejected.

Page 14 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Statistical Methods and Calculations Using functions provided in Microsoft@ Excel@ software, we calculated means, standard deviations, and first order rate constants (see Appendix B, Equation B8) for various subsets of the acquired data. Our linear regressions included the determination of constant terms, that is, the regression fits were not forced to pass through the origin.

As described in Appendix B (Equations B38 and B39), rates measured at 50° C were extrapolated to 25° C by dividing by a factor of 10; this approximation is valid for reactions, such as these, with Arrhenius heats of activation near 18 Kcal/ mole.*

Data Summary and Discussion The LOQ is defined as the concentration of the lowest (accepted) standard in the calibration set for which the known concentration exceeds 400% of the indicated solvent blank level (see Appendix A). During this study, the LOQ for PFOS was 308 ng/ mL. Results for the surrogate compound THPFOS were very consistent throughout the study. The percent relative standard deviations of the measured values, calculated for each pH level, ranged from 1.2 % to 3.4%.

Table 4 presents the results of the slope determinations (see Equation B8) at six pH levels and 50° C.

I ~~ ~~~__

Table 4. Observed (50" C) Slopes of PFOS Concentrations in Aqueous Buffered Solution for Various pH Levels.

I+ 3.0 5.0 7.0 9.0 11 Observed Slope (day")

0.00025 0.00032 0.0001 1 0.0001 5 0.00057 0.00030 Percent (20) Rate Uncertainty (day")

111 73 618 253 89 161 Figure 3 illustrates these values, which are all positive, and the fact that they are generally only poorly determined; their percent relative 20 (95% confidence) uncertainties range from 73% to 618%. The data do not indicate degradation of PFOS at any of the six pH levels.

In the absence of a clear trend relating the degradation rate to sample pH, it is appropriate to "pool" the data over pH level and determine the concentration slope using the entire data set. Figure 3 illustrates the results of this pooled analysis according to Equation 1, and Table 5 summarizes the results of the analysis. It also indicates no degradation of PFOS, and the positive slope does not provide a half life estimate.

Page 15 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Table 5. Calculated Slope of PFOS Concentrations in Aqueous Buffered Solutions Using Data Pooled Over All pH Levels.

Calculated slope Calculated slope Calculated slope lower limit (20) upper limit (20) (day ') 2.98 x 104 (day'') (day ') 6.66 x IO* 5.30 x Figure 2. Observed PFOS Degradation for Various pH levels.

0.04 ' 0.03

0.02

n 0.01

\

B E 0.00

0.01

0.02

0.03 ! I I I I _._ pH 1.5 pH 3. C , pH 5.0  ,pH 7. C . I

pH 9. C 1 1 pH 11 0 10 20 30 40 50 Time (days)

Page 16 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Figure 3. Pooled PFOS Data and Slope Regression.

0.05 0 Solid Line: Dashed Lines: y = 2.983E 04~  5.616E 03 20 limits( slopeandintercept) = 1.481E 01 0 10 20 30 40 50 60 time (days)

The mean and standard deviation of the PFOS data do provide a useful estimate of its half life. Details of the related calculations are presented in below (see Appendix B, Equations B36 and 837). The maximum degradation rate is given in Equation 3:

and the minimum half life is given in Equation 4 Eq. 3 Eq. 4 We note that in both Equations 3 and 4, the mean PFOS concentration (pp) and standard deviation ( op ) can be either molar or mass quantities. Table 6 presents the results of the calculation.

Page 17 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 I Table 6. Degradation Rate and Half Life of PFOS in Based on the Concentration Mean and Standard Deviation 1 Page 18 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Conclusions We have performed a study of the aqueous hydrolysis of perfluorooctane sulfonate (PFOS). Six different pH levels were included in the study, which were carried out at 50° C and extrapolated to 25° C. Our results based on direct observation of the PFOS concentration indicate no hydrolytic degradation at any individual pH, nor do the data pooled over all six pH levels. From the mean value and precision of PFOS concentrations, we estimate the hydrolytic half life of PFOS to be greater than 41 years.

Page 19 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 References ~~

1. "Fate, Transport and Transformation Test Guidelines: 835.21 I O : Hydrolysis as a Function of pH," U. S. EPA Office of Prevention, Pesticides and Toxic Substances, publication number 712 C 98 057, January 1998. 2. Experimental Physical Chemistry", F. Daniels, et al., McGraw Hill Book CO. (New York), p. 131, 1962.

Page 20 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Signatures Page 21 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Appendix A: Analytical Method Method ETS 8 13.0, "Hydrolysis of Perfluorooctane Sulfonate (PFOS) and Analysis by High Performance Liquid Chromatography with Mass Spectrometry Detection."

This Appendix presents the analytical method employed in this study.

Page 22 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 3M ENVIRONMENTAL LABORATORY METHOD HYDROLYSIS OF ~RFLUOROOCTANE SULFONATE (PFOS) AND ANALYSIS BY MASS SPECTROMETRY DETECTION HIGH PEFWORMANCE LIQUID cHR0MATOGRAPHYWIT. H MethodNumber: ETS 8 13.0 Adoption Date: a q los Effective Date:

Approved by:

Laboratory Page 23 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 1.0 SCOPE AND APPLICATION 1.1 This method defines the steps for analysis of perfluorooctane sulfonate (PFOS) hydrolysis products by high performance liquid chromatography (HPLC) with mass spectrometry (MS) detection and quantitation. It is based on EPA OPPTS: 835.21 10 (Reference 18.1). PFOS anion is detected and quantified by this method using the anion of 1 , 1,2,2 tetrahydro( trideca) fluorooctane sulfonic acid (THPFOS) as the internal standard. Representative chemical structures are shown in Attachment A.

tetrahydro( trideca) fluorooctane sulfonic acid (THPFOS). 1.2 Compatibleanalytes. Perfluorooctanesulfonate (PFOS) and 1, 1,2,2 1.3 Acceptable matrices for analysis. Aqueous solutions at various buffered pHs. 1.4 This method is defined as performance based. Target analyte or surrogate matrix spike recoveries (100 f 30%) are used for each sample matrix to evaluate method performance. (Refer to Section 10 for the quality control parameters to be analyzed by this method. Refer to Section 14 for the quality assurance evaluation criteria for this method,)

2.0 SUMMARY OF METHOD 2.1 Aliquots of PFOS stock solution are added to vials that contain b e e r s at pH 1.5,3.0, 5.0, 7.0,9.0, and 11.0. The vials are then placed in an orbital incubatorhhaker set at 50 4 3 "C. Sets of vials are removed at designated intervals and the date and time recorded. . The aqueous sample from the hydrolysis of PFOS is diluted 1 to 10 with methanol (MeOH) containing internal standard. PFOS, incubated in one of the several buffers is separated on a reverse phase Dionex IonPac@ NG 1 HPLC column using an ammonium acetateMeOH solvent gradient, with detection by electrospray ionization mass spectrometry in the negative mode.

3.0 DEFINITIONS 3.1 Method blank. An analyte free matrix to which all reagents are added in the same 3.2 3.3 3.4 volumes or proportions as used in the sampling processing. The method blank is carried through the complete sample preparation and analytical procedure. Because the reagent in a hydrolysis experiment is the aqueous buffer, which is the solvent, the classical definition of a method blank is limited in applicability. The method blank is used to document contamination resulting from the entire preparation and analytical process. Solvent blank. A sample of analyte free medium (for example, 10: 90 buffered water/ MeOH) that is not taken through the sample preparation process. This blank is used to evaluate instrument contamination. Sample triplicates. Three samples taken from and representative of the same sample source and separately carried through all steps of the extraction and analytical procedures in an identical manner. Matrix spike (MS). Prepared by adding a known mass of target analyte to specified amount of a sample matrix. This assumes that an independent estimate of target analyte concentration is available. Matrix spikes are used to determine the effect of the matrix on the method's recovery efficiency.

ETS 8 1 3 .O PFOS Hydrolysis and Analysis by HPLCiMS Page 2 of 15 Page 24 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 3.5 3.6 3.7 3.8 3.9 3.10 4.0 Internal standard (IS). A known amount of a compound or element similar in analytical behavior to the compound or eIement of interest, added to al1 samples and standards, and carried through the entire measurement process. IS responses provide a reference for evaluating and controlling the precision and bias of the applied analytical process. Calibration standard. A dilution of various amounts of a stock, intermediate or purchased standard to achieve standard solutions in a concentration range of interest. Hydrolytic half lives resulting fiom these analyses are calculated based on analytical ratios and not absolute numbers. Therefore, results do not depend on the purity of the standards used. Continuing calibration verification (CCV). Standards analyzed during an analytical run to verify the continued accuracy of the calibration curve. This solution may or may not be prepared fiom a different source or lot number than the calibration curve standards. Dilution. A step in the hydrolysis study procedure in which a solvent is added to the test analytehuffer solution to prepare it for instrumental analysis. This step occurs after the vials are removed from incubation and before the samples are analyzed. If the solvent used is miscible with the test analyte/ bufTer solution, the diluting solvent is merely added and mixed. If the diluting solvent is non miscible, a liquid liquid extraction is performed. IS( s) may be incorporated into the diluting solvent, if desired. Limit of quantitation (LOQ). The lowest concentration that can be reliably measured within specified limits of accuracy (see Sections 14.1 and 14.2) and precision (see Section 14.3) during routine laboratory operating conditions. The LOQ is generally 5 to 10 times the minimum concentration with a 99% confidence limit that the concentration is greater than zero. However, it may be nominally chosen within these guidelines to simplify data reporting. For many analytes, the LOQ is selected as the lowest non zero standard in the calibration curve that is greater than 4 times the level of the solvent blanks. Sample LOQ are highly matrix dependent. Residuals. The percentage difference between that actual known concentration in a standard, versus the result obtained fiom the back calculation using the calibration curve's linear regression formula. For the purposes of the study, the acceptance criteria for the residuals are k 25%.

Residual = known conc.  calculated conc. x 100 known conc.

WARNINGS AND CAUTIONS 4.1Healthandsafetywarnings 4.1.1 Wear the proper lab attire for all parts of this procedure. Wear gloves and proper eyewear at all times. 4.1.2 Handle all solvents in a hood for all parts of the described sample preparation procedure. Whenever possible and practical, dilute samples with solvent in a hood.

ETS 8 13 .O PFOS HydroIyis and Analysis by HPL# MS Page 3 of 15 Page 25 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 4.1.3 For potential hazards of each chemical used, refer to material safety data sheets, packing materials, and 3M Environmental Laboratories Chemical Hazard Review.

4.2 Cautions 4.2.1 Rinse all glassware in which standards are prepared with 1: l acetoneMeOH, then dry to reduce the possibility of contamination. 4.2.2 Ensure that the HPLC mobile phases are prepared fresh prior to beginning a run sequence, and that there is sufficient quantity to complete the run. Do not allow the pump to run dry.

the computer to save all run data. 4.2.3 Ensure that before starting the run sequence there is ample hard disk space on 4.2.4 Ensure that there is enough nitrogen in the supply tank to complete sequence rUnS.

5.0 INTERFERENCE 5.1 Contaminants in solvents, reagents, glassware, and other sample processing or analysis hardware may cause interference. Use the routine analysis of laboratory method blanks to demonstrate that there is no such interference.

interference at low detection levels. The routine analysis of solvent blanks must be used to demonstrate that there is no such interference. 5.2 , Contamination from columns, HPLC tubing, and detector components may cause 6.0 EQUIPMENT 6.1 Analyticalbalancesensitive to 0.1 mg 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.1 1 Shaker, incubator capable of maintaining temperature at 50 k 3 OC Hewlett Packard (HP) 1 100 HPLC System, or equivalent 6.3.1 Pump, quaternary, Model G13 1 1 A, or equivalent 6.3.2 Solvent degasser, Model G1322A or equivalent 6.3.3 Autosampler, ALSModelG1313A, variableinjectionvolumecapable 6.3.4 Columnheater, ModelG1316A Dionex IonPac@ NG 1 column, 35 mm x 4.0 mm, 10 pm packing , or equivalent Mass spectrometer. Hewlett Packard LC/ MSD, or equivalent, capable of operating in the seIected ion monitoring mode Clock, digital. Only one clock should be used, to insure unambiguous documentation of the correct performance of procedures. Centrifuge capable of maintaining 3000 rpm for 5 min pH meter. Corning Model 308 pWTemperature Meter with 3 in 1 gel filled combination electrode (pWreference/ temperature), or equivalent Refigerator, capable of maintaining 4 t 2 OC Data system. A PC computer capable of controlling the HPLC system as well as recording and processing signals from the detector Data analysis software: Hewiett Packard  ChemStation', Version A. 6.03 or more recent.

ETS 8 13.0 Page 4 of 15 PFOS Hydrolysis and Analysis by HPLUMS Page 26 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 7.0 SUPPLIES AND MATERIAIS 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 Vials, 40 mL VOA (I CHEM or equivalent) Crimp cap autovials, 1.5 mL Labels Graduated pipets, glass, disposable, l mL to 10 mL Pasteur pipets, glass, disposable Hamilton Gastight@ syringes (precision f 1 % of total volume), 1 0 pL to 1 000 pL Volumetric flasks, various sizes Beakers, glass, various sizes Automatic pipettor, capable of dispensing 10 5000 pL 8.0 REAGENTS AND STANDARDS 8.1 Methanol( MeOH): HPLC/ SPEC/ GCgradefromEMScience, or equivalent 8.2 8.3 8.4 8.5 8.6 8.7

18.0 Mi2 water. Water with lower resistance must not be used. Ammonium acetate (approximately 2 mM). This is chromatographic solvent A (see Section 12.3.1). Example: An acceptable buffer solution is made by weighing out 0.15 grams of ammonium acetate (reagent grade) into a weigh boat and then quantitatively transferring to a 1 L volumetric flask. Add 10 mL of MeOH as a preservative, dilute to the mark with 18.0 MSZ water and mix thoroughly. Calibration and Standard Stock Solutions All weights should be recorded to the nearest 0.0001 g 8.4.1 PFOS prepared in MeOH. (Example: A PFOS stock solution is prepared at a concentration of approximately 10,000 pg/ mL by weighing 0.1000 g of PFOS in a 10 mL volumetric flask and bringing to the mark with MeOH. This solution is diluted in MeOH to make additional standards as needed.) 8.4.2 THPFOS prepared in MeOH. (Example: A THPFOS stock solution is prepared at a concentration of approximately 10,000 pg/ mL by weighing 0.1000 g of THPFOS in a 10 mL. volumetric flask and bringing to the mark with MeOH. This solution is diluted in MeOH to make additional standards as needed.) Internal Standard (THPFOS)/ Diluting Solvent 8.5.1 THPFOSprepared in MeOH. ( Example: a THPFOS internal standard solution is prepared to a concentration of approximately 400 ng/ mL by diluting 100 pL of a 10,000 pg/ mL THPFOS stock standard (Section 8.4.2) into 2.5 L of MeOH. This solution will be used for the final MeOH dilution of samples prior to analysis. Buffers for calibration of pH Meter. Purchased pH calibration standards of pH 4.0, 7.0, and 10.0 (Mallinckrodt or equivalent). Buffer solutions for hydrolysis study. Prepare buffer solution of pH 5.0 using guidelines fiom Fate, Transport and Transformation Test Guidelines (Reference 1 8.1). Prepare buffer solutions of pH = 1.5,3.0,7.0,9.0, and 11.0 using guidelines f? om CRC Handbook of Chemistry and Physics (Reference 18.2). Prepare the buffer solutions in l L ETS 8 13.0 PFOS Hydrolysis and Analysis by HPLC/ MS Page 5 of 15 Page 27 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 quantities. Calibrate a portable pWtemperature meter using purchased pH calibration standards of pH 4.0,7.0, and 10.0, and measure the pH of all buffer solutions. Prepare buffer solutions of pH 1.5,3.0,5.0,7.0,9.0, and 11.0 at ambient room temperature. The concentrations are given below. Record final pH measurements of all buffers. Store buffers in sealed glass containers. 8.7.1 8.7.2 8.7.3 8.7.4 8.7.5 8.7.6 pH 1.5 a) 207 mL of 0.1 N HCl (reagent grade)

b) 125 mL of 0.2 M KC1 (reagent grade)

c) Adjust pH to 1.5 with additional 1 N HC1 d) Bring to a final volume of 1 L with 18.0 Ma water pH 3.0 a) 10.2 g potassium biphthalate (reagent grade) dissolved in approx. 600 mL b) pH adjusted with 1 N HCl to 3.0 c) Bring to a final volume of 1 L with 18.0 MQ water pH 5.0 a) Approximately 3.8777 g ammonium acetate (reagent grade) added to 250 b) Add 250 mL 0.052 M acetic acid (reagent grade)

c) Add 18.0 MQ water to about 900 mL d) Add to glacial acetic acid (about 0.5 mL) to adjust to pH 5.0 e) Bring to a final volume of 1 L with 18.0 Mi2 water pH 7.0 a) 500 mL 0.1 M KHzPO, buffer (reagent grade)

b) 291 mL 0.1 N NaOH (reagent grade)

c) Adjust pH to 7.0 with either 1 N HCl or 1 N NaOH d) Bring to a final volume of 1 L with 18.0 MQ water.

pH 9.0 a) 46 mL of 0.1 N HCl b) 125 mL of 0.1 M borax (NqBO,* H, O) (reagent grade)

c) Adjust pH to 9.0 with either 1 N HCI or 1 N NaOH d) Bring to a final volume of 1 L with 18.0 M a water.

p H 11.0 a) 500 mL 0.05 M NaIICO2 (reagent grade) b) 227 mL, 0.1 N NaOH 1 8.0 MQ water mL 18.0 MQ water ETS 8 13.0 Page 6 Of 15 Page 28 of 71 PFOS Hydrobsis and Analysis by HPLUMS BACK TO MAIN 3M Environmental Laboratory Report No. W1878 c) Add water to 950 mL d) Adjust pH to 1 1.0 with 1 N NaOH e) Bring to a final volume of 1 L with 18.0 MR water 8.8 Test AnalyteandSpikesolutions: 8.8.1 PFOS test analyte solution. [Example: An analyte solution of PFOS is prepared by adding 0.5 mL of 10,000 pg/ mL PFOS standard (Section 8.4.1) to st 1 0 mL volumetric flask and diluting to the mark with MeOH. A 10 pL aliquot of this solution is then added to 1 mL buffer (Section 12.1.5), resuIting in a final concentration of approximately 500 n g /d after MeOH dilution (Section 12.1.11)]. 8.8.2 Spiking solution. pxample: A spiking solution is preparedby adding 0.1 mL of an approximately 10,000 ppm PFOS standard (Section 8.4.1) to a 10 mL volumetric flask and diluting to the mark with MeOH. Final concentration after addition of the spike (Section 12.1.12) and MeOH dilution (Section 12.1.1 1) is approximately 100 n g /d of PFOS].

9.0 SAMPLE HANDLING 9.1 Record times of initial preparation and dilution on the fluorochemical degradation 9.2 Once the 9.0 mL of diluting solvent (Section 8.5.1) has been added to the hydrolysis (hydrolysis) analysis sample preparation sheet (Attachment B).

mixtures, the samples should be analyzed within 24 hours. Alternatively, the samples should be stored at 4 f 2 "C before dilution with MeOH and until analysis can be performed.

10.0 QUALITY CONTROL 10.1 Internal Standards. IS( s) are added in a constant concentration to all standards,

10.2 Sample triplicates. Prepare and analyze all samples in triplicate to provide a measure of samples, and matrix spikes.

the precision of analysis. The analyst shall accept RSD values (25%. RSD values of 25% or greater should be noted. Appropriate steps must be taken to correct the problem before analysis is allowed to proceed (e. g. sample re runs, additional blanks, etc.). Consult with the Team Leader or designee for direction and final acceptance or rejection of the analytical run.

study. Concentrations of the spike should be approximately equal to a mid range calibration standard. The matrix spike sample should be analyzed immediately following the sample triplicates to which it corresponds. The analyst shall accept percent spike recoveries of 100 rt 30%. Spike recoveries outside of this range should be noted. Appropriate steps must be taken to correct the problem before analysis is allowed to proceed (e. g. reruns, additional blanks or spikes, etc.). Before the analysis is allowed to proceed, consult with the Team Leader or designee for direction and final acceptance or rejection of the analytical run. 10.3 Matrix spikes. Prepare a post hydrolysis matrix spike for each of the pHs used in the ETS 8 13.0 PFOS Hydrolysis and Analysis by HPLUMS Page 7 of 15 Page 29 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 10.4 Solvent Blank. Solvent blanks should be run before and after every calibration curve, CCV, and after no more than 20 injections. Acceptable values for the blank are values below the limit of quantitation (LOQ) of the instrument (Section 3.9). If analyte carry .over is a problem, use back to back solvent blanks.

11.0 CALIBRATION AND STANDARDIZATION 11.1 Standard preparation. Prepare at least six calibration standards of PFOS in MeOH. Standards from approximately 300 n g /d to 800 n g /d PFOS are recommended. This solution should also contain appropriate concentrations of the internal standard. 11.2 Calibration standards. Analyze the set of calibration standards at the beginning of the m, after every 20 injections, and at end of the run. Use the data reduction software program for linear regression calculations to relate the analyte peak area ratio versus the amount ratio from the internal standard. ExternaI standard calibration may be used if data review shows a problem with the internal standard analysis. Consult with the Team Leader or designee for direction prior to performing the external calibration methodology.

12.0 PROCEDURES 12.1Sampleandspikepreparation 12.1.1 12.1.2 12.1.3 12.1.4 12.1s 12.1.6 12.1.7 12.1.8 12.1.9 Before spiking with any of the stock standards, transfer approximately 1 mL of the solution to an autovial and cap it. Use this smaller volume for spiking to minimize the effects of evaporation from stock solutions. Determine the number of time points that will be analyzed. Each time point will have at least four vials for each pH, multiplied by the number of pHs analyzed. One vial at each level will be labeled as "sample", "duplicate", triplicate", or "spike". Obtain the appropriate number of 40 mL VOA vials with caps and cardboard boxes. Prepare appropriate sample preparation worksheets and create labels and affix to the vials. The labels should include the sample number, pH, time point and initials of the analyst. Record the pH of each buffer solution, Remove the cap of the VOA vial and add 1 mL of the appropriate buffer solution to all of the pre labeled vials. Always replace the cap immediately after any addition to minimize evaporation. To all of the vials, add 10 pL of the PFOS test analyte solution (Section 8.8.1) with a 1 0 pL Gastight@ syringe. Record the time of addition for each vial. For "Time Zero" samples only, proceed to section 12.1.11. For all other samples, continue on to section 12.1.7. Make sure that the cap has been firmly tightened and place the samples back in the cardboard case. Place the case into a pre warmed incubatodshaker for the appropriate time. Record the time, temperature, and rate of shaking. The temperature is determined by the conditions of the experiment. Continue to manually monitor the incubator temperature daily during the entire incubation. Record the temperature on the sample preparation sheet (Attachment B). Remove the case from the incubator at the designated preset time.

ETS 8 13 .O PFOS Hy& olysis and Analysis by HPLC/ MS Page 8 of 15 Page 30 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 12.2 12.3 12.4 12.1.10 Remove the vials from the case and place in racks. Allow the vials to cool for approximately 15 minutes to room temperature. Alternatively, refrigerate the vials at 4 k 2 "C if solutions are to be quenched at a later date. 12.1.11 Using a 10 mL graduated pipet, add 9 mL MeOH containing approximately 400 ng/ mL THPFOS internal standard (Section 8.4.2) to each vial. 12.1.12 Using a 25 pL Gastight syringe, add 10 pL of PFOS spiking solution (Section 8.8.2) to the "spike" vials. Invert each vial several times to mix the contents. 12.1.13 Aliquot approximately 1 mL of each sample to the appropriately labeled autovial. Cap the vials and mark the bottom of the meniscus. 12.1.14 Place the vials in the HPLC autosampler. Instrument set up 12.2.1 Check that the appropriate HPLC column is in the instrument for each analysis. 12.2.2 Check that the correct eluent solutions are in bottles to be used and that enough 12.2.3 Place the samples in the autosampler tray and construct a sequence table with is available to complete the sequence run.

appropriate calibration standards, calibration check standards and solvent blanks.

information: sample or standard ID, method name, one injection per sample.

03 1499s). Save data to a subdirectory labeled with analysis date (e. g. 031499).

"STANDBY" on HP systems). 12.2.4 Verify that all samples and standards are positioned correctly. Enter sequence 12.2.5 Save sequence as analysis date (e. g. on March 14,1999 save sequence as 12.2.6 Set post sequence cornmand macro to shut down system (Example:

HPLC set up: 12.3.1 Analysis of PFOS Samples from pHs 1.5,3.0,5.0,7.0,9.0 and 11.0. Install the column: Dionex IonPac@ NG l, 4.0 x 35 mm, 10 pm or equivalent. Solvent A: 2mM Ammonium Acetate in 1% MeOH (Section 8.3)

Solvent B: MeOH Solvent Gradient:

Time, minutes 0.3 mIJmin 95 5 8.0 0.3 mL,/ min 95 5 4.0 0.3 mL/ min 40 60 1 .o 0.3 mLJmin 40 60 0.0 Flow Rate %B %A Post time: 5.0 minutes; Column Temperature: 40" C Recommended Mass Spectrometer set up; * 12.4.1 Analysis of PFOS at pHs 1.5,3.0,5.0,7.0,9.0 and 11.0,

ETS 8 13.0 PFOS Hydrolysis and Analysis by HPLCYMS Page 9 of 15 Page 31 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878  Drying Gas Temp 3500 V Capillary Voltage 300 "C `Example conditions are applicable to Hewlett Packard HPl 100 equipment only.

Time 1 SIM 1 Identity I Gain I Fragmentor I Dwell I . .. . 5.6 210 70 I .o PFOS aualitative ion 500 6.2 210 70 1 .o PFOS quantitation ion (M H) 499 6.2 210 70 1 .O PFHS 13C, (M H) qualitative ion 400 5.9 210 70 1 .O THPFOS (M H), quantitation ion 427 I I * "Quant" ion for THPFOS; **" Qua& ' ion for PFOS ..

1 2 3

12.5 12.6 13.0 pduorohexanesulfonaide perfluorohexanesulfonate perfluorooctanesulfonamide Autosampler set up*

Autosampler:

5.0 pL Injection volume: None Autosampler Program: ALS Model G13 13A *Example conditions are applicable to Hewlett Packard HP1100 equipment only.

Sample analysis 12.6.1 12.6.2 12.6.3 12.6.4 12.6.5 Enter the standard, sample and QC information into the sequence table. Analyze calibration standards, up to 20 injections, and the calibration standards again. If more than 20 injections are to be run, analyze a continuing calibration standard (CCV) after every 20 and run the calibration standards again at the end of the sequence. Run solvent blanks after the highest calibration standard, before and after the CCV,, and after the set of samples to check for any malyte carryover.

exceed five characters if the sequence contains more than 99 lines. Place the standards, samples, and QC (matrix spikes and sample blanks) into the autosampler tray according to the order they are listed in the sequence. Save the sequence table with a name corresponding to today` s date. (e. g. if today is December 1,1998, save the sequence as 120198.) Start the sequence. Identify the electronic acquisition files with an appropriate prefix. Do not DATA ANALYSIS AND CALCULATIONS 13.1 Peak Evaluation: Peaks must be symmetric in shape and identified by extracting compound specific ions. Peaks considered for calibration must have peak heights greater than 5 (five) times the baseline noise for that region of the chromatogram. Peak integration is from baseline to baseline through a peak using automatic or manual integration. Compounds with isomers present as a shoulder or as a discrete second peak ETS 8 13.0 PFOS Hydrolysis anddnalysis by HPLC'MS Page 10 of 15 Page 32 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 13.2 13.3 13.4 13.5 14.0 should be integrated with the parent compound unless otherwise noted. Quantitation data are calculated using THPFOS as the internal standard. However, external standard calibration may be acceptable. Consult with the Team Leader or designee for direction prior to performing the external calibration methodology. Calculation of k Calculate the PFOS concentrations in each of the pH matrices using the curves obtained from the calibrations and the internal standard. Assuming first order kinetics a rate constant (k) can be determined by plotting:

concentrations determined at some elapsed time t and at t = 0, respectively. The slope of the resulting line is k. The iL value for this plot should be > 0.800. For rZ values less than 0.800, consult the Team Leader or designee for direction. Calculate the PFOS concentrations in each of the pH matrices versus t using the curves obtained from the calibrations and the internal (or external) standard. Matrix spikes. Calculate the percent recovery for each of the matrix spikes. Using the observed matrix spike recoveries, calculate the average spike recovery. Calculate the matrix spike percent recoveries using the following equation:

YO Recovery = (observed spiked sample result  observed sample result) x actual amount spiked Sample triplicates. Calculate the relative standard deviation (RSD) for the triplicate samples:

RSD = StandardDeviationx 100% Mean Conc.

This parameter is also known as the coefficient of variation, a measure of the confidence of the mean.

METHOD PERFORMANCE 14.1 Coefficient of Determination (3. An acceptable coefficient of determination (13, for 14.2 linear curves is 0.990 or greater. The curves should be examined closely for linea& and intercept, particularly for accuracy of quantitation at the low and high ends of the curve. The acceptance criteria for curve fitting residuals (Section 3.10) is & 25%. Alternative methods of curve fitting (e. g. quadratic) may be necessary in some cases. An acceptable correlation coefficient (r) for qkdratic curves is 0.990 or greater. Record in the raw data the reasons for using quadratic equations. Matrix spikes. Performance based warning and control limits are recommended for measurements of percent spike recovery from matrix spike samples. The analyst shall accept percent spike recovery values of 100 & 30%. Spike recoveries outside of this range place the analysis out of control. Appropriate steps must be taken to correct the problem before analysis is allowed to proceed. Consult the Team Leader or designee for direction.

ETS 8 13.0 PFOS Hydrolysis and Analysis by HPLC/ MS Page 11 of 15 Page 33 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 14.3 14.4 14.5 14.6 15.0 Sample Triplicates. Performance based warning and control limits are recommended for measurements of relative percent difference of replicate samples. The analyst shall accept RSD values < 25 %. RSD values of 25% or greater place the analysis out of control. Appropriate steps must be taken to correct the problem before analysis is allowed to proceed. Before analysis is allowed to proceed, consult the Team Leader or designee. Continuing calibration verification. If the percent difference for the amount of measured analyte is greater than 15% from the true value, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration check standard will be used. Reanalyze remaining samples with a new calibration curve. Limit of Quantitation. The limit of quantitation is equal to the lowest standard in the calibration curve that is greater than 4 times the level of the solvent blanks. Solvent blanks. Solvent blanks should show no more than a 5% carry over from a high standard or calibration check standard. If so, two solvent blanks may be necessary to rule out instrumental contamination. If peaks with greater than 25% the peak area of a low standard value are observed in sequential solvent blanks, the run should be stopped. This indicates instrument contamination. The instrument shall be maintained by thoroughly cleaning the electrospray source, and replacing or cleaning columns, tubing, etc.

POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Dispose of sample waste by placing in high or low BTU containers as appropriate. Use 15.2 Collect HPLC solvent waste in the satellite accumulation can. Empty into the flammable 15.3 Use smaller bore columns when possible to minimize waste generation. broken glass containers to dispose of glass pipettes.

storage drum in the hazardous waste collection area on the 2nd floor.

16.0 16.1 16.2 163 16.4 16.5 16.6 16.7 16.8 REcoRlDs Print out hard copies of all graphics and data analysis summaries for archiving Sign and date all graphics and label with instrument ID. Fill out the hydrolysis sample preparation worksheet completely, making sure to include all initials and dates. Print chromatograms and internal standard reports for all analyses. Print calibration tables and curve information and store in the raw data file. Store hydrolysis sample preparation worksheets in the raw data file. Enter all standard preparation information in the standards preparation logbook. Make a photocopy of the logbook page and include the copy in the raw data file. Archive electronic data to appropriate media when necessary.

17.0 ATTACHMENTS 17.1 Attachment A. RepresentativeChemicalStructures 17.2 Attachment B. HydrolysisSampleLogsheet ETS 8 13.0 PFOS Hydroiysis and Analysis by HPLUMS Page 12 of 15 Page 34 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 18.0 BIBLIOGRAPHY 18.1 Fate, Transport and Transformation Test Guidelines Office of Prevention, Pesticides and Toxic Substances (OPPTS) 835.21 10 Hydrolysis as a Function of pH, EPA 712 C 98 057, January 1998.

Operational Definitions of pH," Robert C. Weast, Ph. D., 1988, p. D 87. 18.2 CRC Handbook of Chemistry and Physics, 1st Student Edition, `` Buffer Solutions 19.0 AFFECTED DOCUMENTS None.

20.0 REVISIONS Revision Number ETS 8 13 .O PFOS Hydrolysis and Analysis by HPLUMS Page 13 of 15 Page 35 of 71 BACK TO MAIN m c z 0 0 \\ ;o BACK TO MAIN ." .. BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Appendix B: Kinetics Model This Appendix includes a mathematical description of the kinetics model employed in the study. Appendix Page 38 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Kinetics Model BI. Reaction Components and Rates The arguments below are based on the following idealized set of reactions representing the hydrolysis of a parent compound P and its hydrolysis products A,, which number N. The actual hydrolysis reactions that occur under neutral, acidic, and basic conditions are subsumed in these equations, and are assumed to proceed with pseudo first order rates k,, (for the parent) and k,, (for the parent's hydrolysis products).

I ' + H, O w n, A,+ Y, (m= ltoN) krm A, + H20 w Y,, k h (m= 1 toN) (B2)

where the general symbols Y,, and Ym2 represent all the other hydrolysis products.

B2. Parent Compound Concentrations Equation B1 indicates that the pseudo first order differential change in the parent concentration P is given by which is equivalent to the separable differential equation dp=[ z P n, k,,] dt Equation B4 may be directly integrated .to obtain the general solution Ink]= x n, k,, t +C ( my, ]

With the initial condition P( t = 0) = Po, the specific solution to Equation B4 is P =Po exp [& n, k,, t]= P, e kpt using the additional definition of the total parent hydrolysis rate Page 39 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 N k, = n, k,, .

Equation B6 can be re written in a form that allows a least squares estimate of the total parent hydrolysis rate:

k, t = In (i)

Using the initial (t = 0) measured value of the parent concentration Po and later values P measured at later times t , one can calculate and plot the (linear) quantity [ In (P/ Po)] versus time and obtain a least squares estimate of the slope of the line.

The resulting slope is the least squares estimate c, of the total parent hydrolysis rate.

Equation B6 indicates that over a period of time TI': (the parent hydrolysis half life) the parent concentration P is reduced through hydrolysis by a factor of two, where A least squares estimate ? vt of the parent hydrolysis half life is therefore available from B3. ProductCompoundConcentrations The pseudo first order differential changes in the product concentrations 4, (using Equations B2 and B6) are dA, = ( n, kp, P  k,, A,) dt = ( nmkPmPO e kp  k,, A,) dt (B11)

and the (first order, non separable) differential equation governing the product concentrations is %+ k, A, = n, k,, Po e kpt . dt The "standard form" of Equation B12 is Page 40 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Ab + S (t) A, = Q( t)

where the "function" S (t) is actually a constant:

and Q( t) = n, k,, Po e kpt .

The general solution A, to Equation B12 is contained in where and There are two cases of Equation 818 to consider. In the circumstance that k, = k, , which occurs only when the hydrolysis rate of the mth product is identical to the total parent hydrolysis rate, the general solution to Equation B18 is (for k, = k,)

A, ekpt = n, k,, Po t + C and, using the initial condition A,( t = 0 ) = A, , the specific solution to Equation18 is (for k, = k, )

A, = (nmkPmPO t + A,) e kpt We note that when k, = k, = 0 (that is, when both the parent and potential product are hydrolytically stable), Equation B7 requires (also) that k,, = 0 , so Equation B20 becomes Page 41 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Am = A,

indicating, as required, that the productconcentration does not change with time.

The circumstance k, = kp is highly improbable, and is neglected in the remainder of this discussion. However, the reader should bear in mind that the expressions derived below do not hold when the parent hydrolysis rate k, and the product hydrolysis ratek, approach each other.

In the more probable case, for which k,, # k, (i. e. that the hydrolysis rate of the mth product is different from the total parent hydrolysis rate), the general solution to Equation B18 is and the specific solution to Equation B18 with the initial condition Am( t = 0) = Am, is nrnkPmPO e k, t  nmkPmPO e kp t kF'  kArn 1 kP  kAm Of greatest interest here is the case in which the product compounds are known to be hydrolytically stable, that is, when k, = 0 for all m. In this case, Equation B23 becomes (for hydrolytically stable products)

A, =AmO + nmkPmPO (1 e k p t ) . kP B4. Relationships Between the Parent and Compound Concentrations Equations B7 and B24 can be combined to obtain (for hydrolytically stable products)

Page 42 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 so that (for hydrolytically stable products)

or (for hydrolytically stable products)

If the changes in the product concentrations are all small compared to the original parent concentration, that is, if we may use the expression (valid for 1 5 X I 1 )

1 1 1 4 ln( l+ X)= X x2 + x3 x4+ ..... 3 2 L J and Equation 823 becomes (for hydrolytically stable products and Page 43 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 or (for hydrolytically stable products and Z A , Amo m k, t E m= l B5. Parent Half Life Estimates Based on Limits of Quantification of the Products In every experimental determination of k, , there is some set of values A r Q (the "limits of quantitation") below which the product concentrations A, cannot be reliably measured. If during an experiment carried out over the period of timeA t all the product concentrations A, remain below their limits of quantitation, then the maximum possible value of the rate k, is obtained by assuming (for all the products) that 1) A,, = 0 and 2) at time t = A t , the product concentrations have increased to the values A, = AZQ. With these assumptions, the experimental data indicate that the reaction rate k, is less than some maximum value (kp) as follows:

(for hydrolytically stable products at concentrations below the limits of quantitation)

Under the same circumstances and assumptions, the experimental data indicate that the parent half life TI': (see Equation B9) is greater than the value (TVi) . as follows: mm (for hydrolytically stable products at concentrations below the limits of quantitation)

TV2 1 = P h 42)  A t Po In( 2) (kP 1

The reader should note that Equations B32 and B33 are valid only when both 1) the products are hydrolytically stable and 2) the concentrations of all the potential products are measured. Otherwise, the quantity (k,),, in Equation B32 may not actually represent the maximum possible value of the rate constant k, , and the related result in Equation B33 for (TVi) . is also questionable. mm Page 44 of 79 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 B6. Parent Half Life Estimates Based on Limits of Quantification and Experimental Precision of Product Concentrations In certain experiments, some hydrolysis products are present at quantifiable but essentially constant concentrations over the time (A t ) of the experiment. In this case, it is the experimental precision of the measured product concentrations, rather than the limits of quantitation, which contribute to the estimate of the maximum value of the parent hydrolysis rate k, . If the set of concentrations measured for the mth product have the mean value p, and standard deviation CY,, the data do not exclude the possibility that the product concentration increased from the initial value G,,, p, to the value 0, + p, at time t = A t . Taking this possibility to be the actual case for the measured products, the maximum value of the quantity (A, A,, ) is 20,. This reasoning suggests that the following estimate of the maximum parent hydrolysis rate is appropriate:

(for hydrolytically stable products at either 1) constant measured concentrations with standard deviation Om or 2) concentrations below the limits of quantitation)

r 1 kp 5 k P ), = 1 I A r Q + c20, j.

'0 * Below LOQ Cons tan t Under these circumstances and assumptions, the experimental data indicate that the parent half life T1'i is greater than the value (T v i ) . as follows: nun (for hydrolytically stable products at either 1) constant measured concentrations with standard deviation Om or 2) concentrations below the limits of quantitation)

r 7 1 The reader should note that Equations B34 and B35 are valid only when both 1) the products are hydrolytically stable and 2) the concentrations of all the potential products are measured.

B6. Parent Half Life Estimates Based on the Experimental Precision of Parent Concentrations In certain experiments, the hydrolytic parent remains at an essentially constant concentration over the time (A t ) of the experiment. In this case, it is the experimental precision of the measured parent concentrations that determines the maximum value of the parent hydrolysis rate k, . If the set of concentrations measured for the parent have the mean value pLp and standard deviation op, the data do not exclude the possibility Page 45 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 where R = 1.99 x Kcal mole ' K ' is the ideal gas constant. Using the valueB2 AH, =I8 Kcallmole, the rate ratio kl/ k2 at the corresponding temperatures T, =298 K and T, =323 K is that the product concentration increased from the initial value pP  crP to the value pp + op at time t = A t . This reasoning suggests that the following estimate of the maximum parent hydrolysis rate is appropriate:

(for essentially constant parent concentrations with mean value pp and standard deviation Gp )

Under these circumstances and assumptions, the experimental data indicate that the parent half life T1'i is greater than the value (T v i ) . as follows: nun (for essentially constant parent concentrations with mean value pp and standard deviation Gp )

B8. Temperature Dependence of the Reaction Rate and Half Life In order to increase the speed of the reactions of interest, we conducted this experimental study using samples maintained at the temperature 50° C = 323 K. Of greater interest are the corresponding results for the environmentally important temperature 25° C = 298 K.

When the Arrhenius activation energy for a reaction is AH,, Equation 838 B1 provides the following relationship between the hydrolysis rates (kl and k2) for that reaction at two different absolute temperatures ( Tl and T2):

Page 46 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Equation B39 indicates that the hydrolysis reactions of interest proceed approximately ten times more slowly at 25° C than at the chosen experimental temperature of 50° C. Accordingly, the rate reactions reported here for the temperature 25° C are ten times lower than those measured at 50° C, and the hydrolysis half life estimates reported here for 25° C samples are ten times longer than those calculated from the 50° C experimental data.

References to Appendix B:

'' I. N Levine, "Physical Chemistry," McGraw Hill (New York), pp. 498 501 (1978).

'* F. Daniels, et ai., "Experimental Physical Chemistry", McGraw Hill (New York), p. 131 (1962).

Page 47 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Appendix C: Selected Analytical and Kinetics Results This Appendix includes selected sample data and their related kinetics results. For brevity, some solvent blank and continuing calibration verification results that did meet the data quality objectives have been excluded from the following tables. However, all data failing to meet the data quality objectives are included below.

Page 48 of 71 BACK TO MAIN r BACK TO MAIN z 0 ? E 2 a 1 BACK TO MAIN u A t * vl 0 BACK TO MAIN s E s s B m 5 8 g s N 6 r s E  s $0 s BACK TO MAIN ae r $5 0 z NNNNNNNNNNNNNNNN d d d d d d d d d d d d d d d d NNNNNNNP d d d d d d d u BACK TO MAIN z 0 BACK TO MAIN z 0 BACK TO MAIN co b 2 4 0 BACK TO MAIN 03 b 03 5 0 z BACK TO MAIN ,,

3M Environmental Laboratory Report No. W1878 Figure 3. Pooled PFOS Data and Slope Regression 0.05 n p"

+ 0.00 E4 0.05 0 0 /.o *

O F I S !

6  W 0 8 3 9. I I    0 e

8 0 0  V 0 __________._... ..... 0 0 Solid Line:

Dashed Lines: y = 2.983E 04~  5.616503 20 limits (slope and intercept) R2 = 1.481E 01 I I I 1 1 10 20 30 40 time (days) 50 60 SUMMARY OUTPUT Multiple R Regression Sfatisfics 0.384852267 R Square 0.148111267 Adjusted R Square 0,126267966 Standard Error 0.010265446 Observations 41 ANOVA . .. . df SS MS F Regression Signitiiance F 10.0007145380.0007145386.7806266280.0129706 Residual Total 39 0.0041097960.000105379 40 0.004824334 Coefficients Standard Enor Stat P value Intercept Lower95% 0.0056162880.003550759 1.5817147620.121791782 0.0127983690.001566793 0.0127983690.0015658 Upper 95% L m r 95.0% pper95.0%

X Valiable I 0.0002982790.0001145482.6039636380.0129706 6.65838Ea5 0.0005299736.65838E 050.00053 6.66E 055.30E 04 Page 58 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Appendix D: Selected Chromatograms A representative set of chromatograms from the present study is included in this Appendix.

Page 59 of 71 BACK TO MAIN Calib. Data Modified :

Calcuiate Based on Rel. Reference Window : Abs. Reference Window : Rel. Non ref. Window : Abs . Non ref . ' Window : MultTplFer .. Dilution Sample Amount Uncalibrated Peaks .' Partial Calibration : Correct All Ret. Tiines: *%.

9/ 24/ 1999 10: 08: 51 AM Y 5 ; 00.0 % I 0.000 min 1.0000' 1.0000 I 0.00000 I I not reported Yes, identified pe. aks are recalibrated No, only for identified peaks Cunie Type Origin Weight ' Linear Included * .E q ~d .

Recalibration Settings: Average Response Average allcalibrations Average Retention Time: FloatingAverage New 75%

Calibration Report Options : Printout of recalibrations within a sequence: Calibration Table after Recalibration Normal Report after Recalibration If the sequence is done with bracketing: Results of first cycle (ending previous bracket)

Signal 1: .MSD1 427, EIC= 426.7: 427.7 Signal 2,: MSDl 499, EIC= 498.7: 499.7 Signal 3: MSDl 399, EIC= 398.7: 399.7 RetTime Lvl Amount , , lPPbl I I 1 11 2'. 22 3 33 4 44 5 55 6 66 7 77 8 Area I 300.90000 300.90000 401.20000 401.20000 451.35000 451.35000 501.50000 501.50000 6.86155e4 541.62000 6.91006e4 541.62000 6.74844e4 601.80000 7.89434e4 601.80000 7.90989e4 .702. IOOOO 8.70628e4 702.10000 9.13195e4 802.40000 1.00442e5 _ 3.78048e4 3.80593e4 4.74013e4 5.14873@ 4 5.60681e4 5.86619e4 6.94725e4 Amt/ Area Re€ Grp Name 1 1 1 1 ~, 7.95930e 3 ., PFWS 7.90609e 3 8.46390e 3 7.79221e 3 a. 05003e 3 7.69409e 3 7.21869e 3 7.30884e 3 7.83813e 3 . 8.02586e 3 7.62318e 3 7.60820e 3 8.06430e 3 7.68839e 3 7.98866e 3 Page 60 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Method C:\ HPCHEM\ l\ METHODS\ O922 5O. M RetTime Lvl Amount Area Amt /Area [minl Sig [PPbl 1 1 1 ~ 88 802.40000 1.00128e5 8.01373e 3 5.931 1 1 367.92000 4.73070e5 7.77729e 4 2 367.92000 4.60726eS 7.98565e 4 3 367.92000 4.39572e5 8.36996e 4 ~ ~

4 367.92000. 4.90981e5 7.49357e 4 5 367.92000 4.67449e5 7.87080e 4 6 367.92000 4.57579e5 8.04058e 4 7 .367.92000 4.3.5372e5 8.45071e 4 8 367.92000 4.52504e5 8.13075e 4 11 367.92000 4.61022e5 7.98052e 4 22 367.92000 4.61213e5 7.97722e 4 44 367.92000 4.55268e5 8.08140e 4 55 367.92000 4.57342e5 8.04475e 4 6.6 367,92000 4.52098e5 ,8. '13806e 4 77 367.92000 4.56825eS 8.05384e 4 .88 367.92000'4.52021e5 8.. 13945e 4 21 300.90000 9.24706e5 3.25401e 4 2 401.20000'1.21379e6 3.30534e 4 22 401.20000 1.22485e6 3.27551e 4 3.. 451.35000 1.32239e6 3.41314e 4 33 451.35000 1.34445e6 3.35713e 4 4 501.50000 1.56794e6 3.19846e 4 44 501.50000 1.51715e6 3.30554e 4 5 541.62000 1.636.0le6 3.31062e 4 55 541.62000 1.63668e6 3.30926e 4 6 601.80000 1.76299e6, 3.41353e 4 66 601.80000 1.79159e6 3.35902e 4 7 702.10000 2.03577e6 3.44881e 4 77 102.10000 2.07740e6 3.37970e 4 8 802.40000.2.31841e6 3.46099e 4 8 8 802.40000 2.34731e6 3.41839e 4 . . . ..

33367.920004.53927e58.10526e 4 6.2222 1 300.900009.20859e53.26760e 4 PFOS. . I =  P= s== PIOIE= P= 10= I== I= 3rP= rPI91= P= P= P=~==~=== x~==================

Peak Sum Table '

'OI= P=~= Pe==== rpt= ll===~========~=~=~==~~~===~~~   0= 13= r Page 61 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Method C:\ HPCHEM\ 1\ METHODS\ O922_ 50. M THPFOS at exp. RT: 5.931 MSDl 427, EIC= 426.7: 427.7 Correlation: 0.99395 Residual Std. Dev.: 12673.93865 Formula: y = mx + b '

'.. m: 1244.66095 .

b: 3.54902e 10 x: Amount [ppbl y: Area PFOS at exp. RT: 6.222 MSDl 499, EIC= 498.7: 499.7 Correlation: 0.99860 Residual Std. Dev.: 31589.73759 Formula: y = mx + b m: 2878.88299 b: 51022.56267 x: Amount Cppbl y: Area.

Page 62 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Batch Run # 1 of 60 Data File D:\ DATA\ O92399\ PFOSOOOl. D Sample Name: MeOH Blank Sorted Signal Calib. Data Modified : 9/ 24/ 1999 10: 08: 51 AM Multiplier ,1.0000 . Dilution 1.0000 Signal 1: MSDl 427, EIC= 426 .` 7: 427.7 Totals : 0.00000 Totals : 0.00000 Page 63 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 I Batch Run # 1 of 60 Data File D:\ DATA\ O92399\ PFOSOOOl. D Sample Name: MeOH Blank Totals : 0.00000 1 Warnings or Errors :

warning : Calibrated compound( s1 not found'

Sorted By Signal Calib. Data Modified : ' 9/ 24/ 1999 10: 08: 51 AM . .  Multiplier 1.0000 Dilution *' i; oooo Signal 1: MSDl 427, EIC= 426 .7: 427.7 Totals : 0.00000 Name Totals : ' 0.00000 Signal 3: MSDI 399, EIC= 398,7: 399.7 Peak RetTime Type WidthArea Area Name #. [minl [minl %.  I;~ J J l l 1 5.642 0.0000 0.00000 0.0000 PFHS Totals : ' 0.00000 Page 64 of 71 BACK TO MAIN Batch Run # 1 qf 60 Data F i l e D:\ DATA\ O92399\ PFOSOOOl. D 1 Warnings or Errors : Sample Name: MeOH Blank +

Page 65 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Batch Run .# 6 of 60 I Data F i l e D:\ DATA\ O92399\ PFOSOOO6. D Sample Name: L4 Std, 501.50 Sorted By Signal Calib. DataModified : 9/ 24/ 1999 10: 08: 51 AM Multiplier 1.0000 Dilution 1.0000 Signal 1: MSDl 427, EIC= 426.7: 427.7 Page 66 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Batch Run # 6 of 60 Data File D:\ DATA\ O92399\ PFOSOOO6. D Sample Name: L4 Std, 501.50 Page 67 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Batch Run # 18 of 60 Data F i l e D:\ DATA\ 092399\ PPOSOOl€ i. D Sample Name: PFOS 035 Sorted By Calib. Data Modifi, ed : Multiplier .. . Dilution Signal I: MSDl 427, 61C0426 Page 68 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Batch Run # 18 of 60 Data File D:\ DATA\ O92399\ PFOSOO18. D Sample Name: PFOS 035 Page 69 of 71 BACK TO MAIN 3M Environmental Laboratory Report No. W1878 Batch Run # 19 of 60 '

Data File D:\ DATA\ O92399\ PFOSOOl9. D Sample Name: PFOS 036 '=== 13= IPI== 1==== t= 'DPP = s,=  === _ __ == r== n===== xs 'InjectionDate : 9/ 23/ 1999 2 :0 8 :5 8 PM Seq. Line : 19 Sample 1 : PFOS 036 Vial : 18 Acq. Operator: : MTM 'tnj : .I Acq. Method i : C:\ HPd\ l\ METHODS\ PFO5 NG2. M Last changed j : 9/ 23/ 1999 9: SO: OO AM by MTM Analysis Methbd : C:\ HPCHEM\ l\ METHOD5\ 0922_ 50. M Last changed : 9/ 24/ 1999 10: 12: 55 AM by MTM SIM Analysis 'jES) for THPFOS, PFOS, and PFHS using 4mmx35mm Dionex IonPac NG1 column, SlN 12879.

.MTM *,. '' InjVolume : 5 ,d (Results are from a previously saved Batch)

_C

Signal 2 : MSDl 499, EIC= 498.7: 499.7 RetTime Type Area Amt/ AreaAmount G r p ,Name [minl [PPbl I I l l l ~ '

6.231 BB 1.89862e63.38022e 4641.77689 PFOS Totals : 641.77689 Page 70 of 71 BACK TO MAIN Batch Run # 19 of 60 Data F i l e D:\ DATA\ o9,2399\ PFOSOOl9. D S i g n a l 3 : MSDl 399, EIC= 398.7: 399.7 RetTime Type Area Amt/ Area h i n l Totals. : 3M Environmental Laboratory Report No. W1878 Sample Name: PFOS 036 c 680.89256 Page 71 of 71 BACK TO MAIN