Document DDzRxGy3YKzmkQjbwm6y8ZJMM

RECYCLEO CD / AR226-2694 AR226-2694 DuPont EMSE Report No. 17-03 Study Title Adsorption/Desorption of Ammonium Perfluorooctanoate to Soil (OECD 106) Test Guideline(s) OECD 106 Guidelines for the Testing o f Chemicals; Adsorption-Desorption Using a Batch Equilibrium Method (January 21,2000) A u th o r Lynn Ann Dekleva Study Completion Date 17-April-2003 Test Facility E.I. du Pont de Nemours and Company Central Research & Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA Sponsor Association o f Plastics Manufacturers in Europe Avenue E. Van Nieuwenhuyse, 4 / Box 3 B-1160 Brussels Belgium EM SE Study /P roject Number E M SE R T 0111/14107 Report Number EMSER 17-03 T0111 /14107 Page 1of55 DuPont EMSE Report No. 17-03 Page Reserved for Specific Country Requirements T0111 /14107 Page 2 of 55 DuPont EMSE Report No. 17-03 Good Laboratory Practice Compliance Statement The study described in this report was conducted in compliance with the United States Environmental Protection Agency, (FIFRA), Title 40 Code o f Federal Regulations Part 160 (effective October 16, 1989), and TSCA Title 40 Code o f Federal Regulations Part 792 which are consistent with the OECD Principles on Good Laboratory Practice (ENV/MC/CHEM(98)17) (Paris, 1998). The test substance is a commercially available material. The certificate o f analysis (COA) was provided by the supplier and the accuracy o f the data was considered sufficient for the purposes of this study. Analysis to confirm concentration and uniformity of the stock solutions was not performed. This is believed not to affect the validity o f the study because the test substance was accurately measured and added to the test vessels, and the concentration o f test substance in the CaCL controls (test solutions, no soil) was determined throughout the study. Study Director William R. Berti, Ph.D. Senior Research Biologist DuPont Central Research & Development QjWf Date S p onsor/Subm itter Date T0111/14107 Page 3 o f 55 DuPont EMSE Report No. 17-03 Quality Assurance Statement Study Number EMSER 17-03 /14107 Study Title Adsorption/Desorption of Ammonium Perfluorooctanoate to Soil (OECD 106) The conduct of this study, or studies of the same type, was inspected periodically. Audit dates are given below: Study Phase Inspected Study Protocol Study Conduct Report Dates Findings Inspection/Audit Reported to Dates_____________ Study Director 15-Jul-2002 15-Jul-2002 02-Jan-2003 02-Jan-2003 5,7,10-Feb-2003 10-Feb-2003 Dates Findings Reported to Management Not Applicable 02-Jan-2Q03 17-Apr-2003 These inspections confirm that the methods, procedures, and observations are accurately and completely described in this report, and the reported results accurately and completely reflectthsr* raw data o f this study. jJlum bydiA Kimberly B. Brebnqf/ QA Auditor DuPont Co. ___ _ / 7- PrPfc-^-003 Date T0111/ 14107 Page 4 of 55 DuPont EMSE Report No. 17-03 Certification of Authenticity Adsorption/Desorption of Ammonium Perfluorooctanoate to Soil (OECD 106) We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record of the procedures and results. Report by: Htynad im . LymrAnn Dekleva Staff Engineer n-hp^L -Joo3 . Date Reviewed by: ________ William R. Berti, Ph.D. Senior ReseaschBiologist * f>Qfr-l-3oa3 Date - Approved by: iT. Gannon, Ph.D. Research Manager Date Study Initiation Date: 26-July-2002 Date Study Completed: 17-Apr-2003 Sponsor: Association of Plastics Manufacturers in Europe Avenue E. Van Nieuwenhuyse, 4 / Box 3 B-1160 Brussels Belgium T0111 /14107 Page 5 o f 55 DuPont EMSE Report No. 17-03 Table of Contents Title Page..............................................................................................................................................1 Page Reserved for Specific Country Requirements........................................................................... 2 Good Laboratory Practice Compliance Statement...................................................................... 3 Quality Assurance Statement.............................................................................................................. 4 Certification of Authenticity............................................................................................................... 5 Table of Contents.................................................................................................................................6 General Study Information................................................................................................................. 9 1.0 Summary................................................................................................................................... 11 2.0 Introduction............................................................................................................................... 13 2.1 Study Purpose..................................................................................................................... 13 2.2 Study Objectives................................................................................................................ 13 2.3 Test Guidelines...................................... 13 3.0 Materials and Methods............................................................................................................. 13 3.1 Test Substance.................................................................................................................... 13 3.2 Reagents and Solvents....................................................................................... 14 3.3 Application Information............................................... 14 3.4 Test System............................................................................................... 14 3.5 Test Soils and Sludge......................................................................................................... 15 3.6 Analytical M ethods............................................................................................................ 15 3.7 Experimental Design..........................................................................................................15 3.7.1 Phase 1 Preliminary Studies..................................................................... ........--15 Validation o f the Analytical Method.................................................................... 15 Adsorption to Test Vessel Containers.................................................................. 16 Determining the SoihSolution Ratio and Equilibration Tim e.............................16 Determination o f Test Substance Stability...........................................................17 3.7.2 Phase 2 Definitive Studies..................................................................................... 17 Adsorption Experiments....................................................................................... 17 Desorption Experiments....................................................................................... 18 3.8 Data Analysis..................................................................................................................... 18 3.8.1 Equilibration Time and Percent Adsorption......................................................... 18 3.8.2 Adsorption.............................................................................................................. 19 3.8.3 Desorption.................................................................................................... 19 3.8.4 Material Balance from Adsorption/Desorption Experiments.............................. 20 3.8.5 Adsorption Isotherms........................................................................................... -21 3.9 Statistical Methods and Control o f Bias............................................................................21 3.10 Deviations from Test Protocol........................................................................................ 22 T0111 / 14107 . Page 6 o f 55 DuPont EMSE Report No. 17-03 4.0 Results and Discussion...............................................................................................................22 4.1 Preliminary Studies............................................................................................................ 22 Validation o f the Analytical Method.............................................................................. 22 Adsorption to Test Vessel....................................................................................... -.......23 Soil Solution Ratio and Equilibration Tim e...................................................................23 Mass Balance Determinations........................................................................................ 24 4.2 Definitive Studies............................................................................................................... 24 Adsorption and Desorption Studies................................................................................ 24 5.0 Conclusions..................................................................................................................................26 6.0 Retention of Records......................................................................................... 26 7.0 Disposal o f Test Substance............ ................................................................................ 27 8.0 References....................................................................................................................................27 Tables Table 1 Chemical and Physical Properties o f Test M aterials............................................... 28 Table 2 Determination o f Test Substance in CaCl2 Solutions (No Soil)..............................29 Table 3 Determination o f Test Substance in CaCl2 Soil Extracts.........................................30 Table 4 Adsorption to Test Vessel..........................................................................................31 Table 5 Adsorption in Percent as a Function o f SoihSolution Ratio and Equilibration Time ........................................................................................... 32 Table 6 Mass Balance Calculations o f Drummer and Hidalgo Soils Performed at 1:1 Soil Solution Ratio after 24 Mixing........................................................................................ 33 Table 7 Definitive Adsorption Study; Percent Adsorption and Linear Adsorption Isotherm Parameters................................................................................................................. 34 Table 8 Definitive Adsorption Study; Freundlich Adsorption Isotherm Parameters...... .....35 Table 9 Desorption of Test Substance from Test Soils......................................................... 36 Figures Figure 1 Analytical Method Validation o f CaCl2 Controls (No Soil) and Extracts from Drummer and Cape Fear Soils....................................................................................... 37 Figure 2 Adsorption of Test Substance as a Function o f SoihSolution Ratio and Equilibration Time with Drummer Soil.................................................................................. 38 Figure 3 Adsorption of Test Substance as a Function o f SoihSolution Ratio and Equilibration Time with Hidalgo Soil.......................... 39 Figure 4 Average Kd vs Fraction o f Organic Carbon for Drummer, Hidalgo, Cape Fear and Keyport Soils.............................................................................................................40 Figure 5 Average Komvs Fraction o f Organic Carbon for Drummer, Hidalgo, Cape Fear and Keyport Soils................................ .'...........................................................................41 Figure 6 Average Kocvs Fraction o f Organic Carbon for Drummer, Hidalgo, Cape Fear and Keyport Soils.................................................................. .........................................42 Figure 7 Linear Adsorption Isotherm o f the Test Substance in Drummer Soil....................43 Figure 8 Linear Adsorption Isotherm of the Test Substance in Hidalgo Soil....................... 44 Figure 9 Linear Adsorption Isotherm of the Test Substance in Cape Fear So il...................45 T0111 /14107 Page 7 o f 55 DuPont EMSE Report No. 17-03 Figure 10 Linear Adsorption Isotherm o f the Test Substance in Keyport Soil..................... 46 Figure 11 Linear Adsorption Isotherm o f the Test Substance in Wilmington Sludge..........47 Figure 12 Freundlich Adsorption Isotherm of the Test Substance in Drummer S oil...........48 Figure 13 Freundlich Adsorption Isotherm of the Test Substance in Hidalgo Soil..............49 Figure 14 Freundlich Adsorption Isotherm of the Test Substance in Cape Fear Soil...........50 Figure 15 Freundlich Adsorption Isotherm o f the Test Substance in Keyport Soil..............51 Figure 16 Freundlich Adsorption Isotherm of the Test Substance in Wilmington S ludge........................................................................................................................................ 52 Figure 17 Total Desorption vs Fraction o f Organic Carbon for Drummer, Keyport and Cape Fear Soils.................................................................................................................. 53 Appendices Appendix 1 Ammonium Perfluorooetanoate Analytical Method............................. ............54 Appendix 2 Certificate o f Analysis o f Test Substance.......................................................... 55 T0111 /14107 Page 8 o f 55 DuPont EMSE Report No. 17-03 Adsorption/Desorption of Ammonium Perfluorooctanoate to Soil (OECD 106) General Study Information Study Purpose The purpose of this study was to test the adsorption behavior o f the test substance on four soil samples and one activated sludge sample to determine a sorption value that can be used to predict partitioning o f the test substance in the environment. Study Objectives ** Study the sorption behavior o f test substance in soils with varying soil characteristics. Provide data for determining the leaching and runoff potential o f the test substance in soil including determinations of the following parameters: adsorption coefficient, Kd adsorption coefficient as a function of organic matter, Kom adsorption coefficient as a function of organic carbon, Koc percent desorbed Freundlich adsorption isotherms in all test soils with greater than 10% adsorption Test System Justification The test system is outlined by the U.S. EPA and OECD and was requested by the sponsor. Study Personnel Management: John T. Gannon, Ph.D. Study Director: William R. Berti, Ph.D. Additional Study Personnel: Stanley F. Bachmura, Jr. Lynn Ann Dekleva Barbara S. Larsen, Ph.D. Testing Facility E.I. du Pont de Nemours and Company Central Research & Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA T0111/14107 Page 9 o f 55 DuPont EMSE Report No. 17-03 Analytical Facility E.I. du Pont de Nemours and Company Central Research & Development Corporate Center for Analytical Services Experimental Station Laboratory Building 228 Wilmington, DE 19808 USA Study Execution Dates Study Initiation Date: Experimental Start Date: Experimental Completion Date: Study Completion Date: 26-July-2002 22-Aug-2002 03-Feb-2003 17-Apr-2003 T0111 /14107 Page 10 of 55 DuPont EMSE Report No. 17-03 Adsorption/Desorption of Ammonium Perfluorooctanoate to Soil (OECD 106) 1.0 Summary The adsorption and desorption properties of ammonium perfluorooctanoate were investigated in four soil and one activated sludge samples. The adsorption coefficient (Kd), adsorption coefficient as a function o f organic matter (Kom), adsorption coefficient as a function o f organic carbon (Koc), and Freundlich isotherm parameters (Kf and 1/n) were determined. The adsorption of ammonium perfluorooctanoate was evaluated according to "OECD Guidelines for Testing of Chemicals 106" and was performed in two phases. Phase 1 consisted o f screening studies to determine the optimal soil to solution ratio, the equilibrium time for adsorption, potential for adsorption on the surfaces o f the test vessels, and the stability o f the test substance during the test. Phase 2 utilized a batch equilibrium soil slurry method to determine the linear and Freundlich adsorption isotherm parameters and evaluate desorption o f the test substance. The table below summarizes the results from this study. Drummer Hidalgo Wilmington Sludge Cape Fear Keyport < Adsorption (%) IQ (mL/g) Koc (mL/g) K,,m(mL/g) Kk(pgl l/n(mL),/ng"1) 1/n 79.3 - 88.9 4.25 - 8.86 73.8-111 42.8 - 89.2 5.64 0.994 27.8 - 43.8 0.41 - 0.83 53.0-108 30.8 - 62.6 0.59 1.00 69.5 - 87.3 12.6 - 36.8 20.5 - 59.6 11.9-34.6 3.90 1.36 54.1-73.7 1.19-2.84 95.9 - 229 55.6 - 133 3.23 0.885 64.5 - 80.8 1.82-4.26 48.9-115 28.4 - 66.5 1.64 1.11 Soils tested at 1:1 soil:solution ratio; Wilmington sludge tested at 1:5 so!ids:solution ratio 24 hour equilibration time ' w : witness* The average adsorption o f ammonium perfluorooctanoate during a 24-hour equilibration time at a 1:1 soilsolution ratio ranged from 40.8% for the Hidalgo to 81.8% for the Drummer soil. The test soils yielded Kd values o f 4.25 to 8.86 mL/g for the Drummer soil, 1.82 to 4.26 mL/g for the Keyport soil, 1.19 to 2.84 mL/g for the Cape Fear soil, and 0.41 to 0.83 mL/g for the Hidalgo soil. The Wilmington sludge sample had the highest average Kd value o f 22.5 mL/g, with Kd values that ranged from 12.6 to 36.8 mL/g. T0111 /14107 Page 11 o f 55 DuPont EMSE Report No. 17-03 The Koc values o f the soils ranged from 48.8 mL/g in the Keyport soil to 229 mL/g in the Cape Fear soil and the Komvalues ranged from 28.4 mL/g for the Keyport soil to 133 mL/g for the Cape Fear soil. The Wilmington sludge sample had an average KoCvalue o f 36.5 mL/g and average K<,mvalue o f 21.2 mL/g. There was a strong linear correlation (R2= 0.9465) between the fraction o f organic carbon and average K j values (Figure 4). The Freundlich adsorption coefficient (KF) and the constant (1/n) were determined from the linear form o f the Freundlich equation for all test materials. Freundlich adsorption coefficients ranged from 0.59 for the Hidalgo soil to 5.64 pg M/n (ml)1/ng'1for the Drummer soil. The KFoc values ranged from 44.2 to 261 pg !"1/n(ml)1/ng '1and the KFomvalues ranged from 25.6 to 151 pg M/n (ml)1/ng'1for the Keyport and Cape Fear soils, respectively. There was a strong inverse relationship (R2=0.8665) between the fraction o f organic carbon (foe) and the total % desorption o f the test compound in the Drummer, Keyport and Cape Fear soils. *. The Drummer soil which had the highest foe o f the soils used in this study (5.76%) had a 36.0% > average total desorption. Cape Fear with a foe o f 1.24% had an average desorption of 53.0%. wi The desorption results for the Hidalgo soil and the Wilmington Sludge sample were highly variable and ranged from 41.6 to 121% and 14.9 to 101%, respectively. T0111 / 14107 Page 12 o f 55 DuPont EMSE Report No. 17-03 2.0 Introduction 2.1 Study Purpose The purpose o f this study was to test the adsorption behavior o f the test substance on four soil samples and one activated sludge sample to determine a sorption value that can be used to predict partitioning o f the test substance in the environment. 2.2 Study Objectives The major objectives of this study were to Study the sorptive behaviour o f ammonium perfluorooctanoate in soils with varying soil characteristics Provide data for determining the leaching and runoff potential o f the test substance in soil including determinations o f the following parameters: adsorption coefficient, Kd adsorption coefficient as a function o f organic matter, Kom adsorption coefficient as a function o f organic carbon, KoC percent desorbed Freundlich adsorption isotherms in all test soils with greater than 10% adsorption 2.3 Test Guidelines The study design met the data requirements specified in the OECD Guideline for Testing o f Chemicals, "Adsorption-Desorption Using a Batch Equilibrium Method", Guideline 106, January 2000 and U.S. Environmental Protection Agency, Pesticide Assessment Guidelines (1982). 3.0 Materials and Methods 3.1 Test Substance Name: Synonyms: Active substance: CAS Name: Ammonium perfluorooctanoate Ammonium pentadecafluorooctanoate; Ammonium perfluorocaprylate; DS 101; Fluorad FC 143; Perflurorooctanoic acid ammonium salt; Unidyne DS 101;APFO Octanoic acid, pentadecafluoro-, ammonium salt ' Octanoic acid, pentadecafluoro-, ammonium salt T0111 /14107 Page 13 o f 55 DuPont EMSE Report No. 17-03 CAS Number: Supplier: Product Number: Lot Number: Molecular Formula: Formula Weight: Concentration o f a.s., nominal: Certificate of Analysis Date: Solubility: Appearance: Date Received: 3825-26-1 Fluka Chemical Company 1001 West St. Paul Milwaukee, WI 53233 USA 77262 421207/1 C8H4F15N02 431.1 g/mole >98.0% 24-Jan-01 1 g/10 ml White Powder 22-August-02 3.2 Reagents and Solvents All chemicals and solvents were of reagent grade or purer and were purchased from the following suppliers: Calcium chloride dihydrate (Mallinckrodt); Methanol used for extractions (Burdick & Jackson), Methanol HPLC grade (EM Science), and Ammonium Acetate ACS grade (EM Science). 3.3 Application Inform ation The test substance was administered from a 1000 mg/L stock solutionrin 0.01M CaCh prepared in a polypropylene volumetric flask (Nalgene 4000-0050). The stock solution was prepared the day of testing and stored at room temperature until use. 3.4 Test System The test system employed in this study consisted of individually capped 50-mL polypropylene conical tubes (Falcon BD 35 2070), uniquely identified, containing 0.01M CaCfe solution with and without test soils or sludge. The tubes were mixed using an end-over-end rotator (Glas-Col Rotator Model 099A RD4512) at 30-35 RPM at an ambient temperature of 19-25C. The temperature was monitored using a Dickson THDx Temperature recorder, which, had a resolution of 0.5C and accuracy o f 1C. Following incubation, the samples were centrifuged for 30 minutes at 6000 RPM (Sorvall RC-5 HS-4 Rotor) and supernatants were transferred to 50-mL polypropylene tubes ( Falcon BD 35 2070) using a disposable pipette (VWR 53283-706). Aliquots (5 mL) of the supernatants were filtered (Pall Gelman 4190) in 14-mL polypropylene tubes (Falcon 35 2059) using a 10-mL disposable syringe (BD 309695) and refrigerated or frozen until analysis. All experiments were conducted in duplicate. T0111 /14107 Page 14 of 55 DuPont EMSE Report No. 17-03 3.5 Test Soils and Sludge Four soil samples and one activated sludge sample were used in this study. The characterization of the soils and sludge was performed at MDS Harris Laboratories (Lincoln, Nebraska) and is presented in Table 1. The pH o f the soils ranged from 5.4 to 7.8 and organic carbon ranged from 0.77 to 5.76 %. Prior to use, the test soils were air-dried and sieved though a 2-mm mesh sieve. The activated sludge sample was collected from the dewatering facility o f the Wilmington Wastewater Treatment facility. Sludge samples were washed twice with distilled water, freeze-dried, pooled, and gamma irradiated. The moisture content of the test materials was determined on three aliquots (Ohaus AP 210-0) by heating at 100-105C (Yamato drying oven DX 300) for a minimum o f 12 hours. The moisture content was determined by the equation: % Moisture (Wet Weight - Dry Weight) xlOO Dry Weight 3.6 Analytical M ethods Ammonium perfluorooctanoate (APFO) was quantitated by liquid chromatography (LC) (Agilent 1090 Liquid Chromatograph) combined with tandem mass spectrometry (MS/MS) (Micromass Quatro). Negative ion electrospray was used to produce the molecular anion for the perfluorooctanoic acid, which was then subjected to collision-induced dissociation producing the decarboxylated anion. The MS/MS transition was specific to perfluorooctanoic acid and the area of the chromatogram was proportional to the concentration o f APFO. . The standard operating conditions for the method are provided in Appendix l .'tSamplesiwere diluted (if required) with 0.01M CaCl2and submitted for analysis in polypropylenewials (Agilent 5182-0567) sealed with a natural rubber crimp seal (Agilent 5182r 1210). Samples were refrigerated or frozen until analysis. - Samples were analysed by duplicate injections and quantified using a calibration curve generated the day of analysis. Sample concentrations were reported at or above the limit of quantitation (LOQ) for this study. 3.7 Experim ental Design 3.7.1 Phase 1 Preliminary Studies Validation of the Analytical Method The solubility o f the test substance was reported by the manufacturer to be 0.1 g/mL in H20 . The solubility of the compound was not determined in this study because the highest test concentration used in this study, 2500 pg/L, was significantly below the solubility limit for the compound reported by the manufacturer. ' T0111/14107 Page 15 o f 55 DuPont EMSE Report No. 17-03 The test substance was administered to the test systems by means o f diluting a 1000 mg/L stock solution with 0.01M CaCl2 solution (prepared using deionized water). Aliquots o f the test substance were added to polypropylene tubes containing 0.01M CaCl2 to achieve final test concentrations of 100, 500,1000 and 2500 pg/L. Blank tubes were included and contained only the 0.01M CaCl2 solution. Aliquots (5 mL) o f the samples were filtered (Gelman Pall 4190) into 14 mL polypropylene tubes (Falcon 35 2059) using a 10 mL disposable syringe (BD 309695) and refrigerated or frozen until analysis. All experiments were conducted in duplicate. Analytical samples were diluted with 0.01M CaCl2 solution prior to submission to achieve an analytical concentration in the range o f 10 to 150 pg/L. The analytical method was validated on two soils. Soil extracts from Drummer (5.76% organic carbon) and Cape Fear (1.24% organic carbon) soils were prepared by mixing 20 g o f soil with 20 mL 0.01M CaCl2 solution for 48 hours at 30-35 RPM at ambient temperature. The extracts were recovered after centrifugation (Sorvall RC-5B HS-4 rotor, 6000 RPM, 30 minutes). The test compound was added to the soil extracts, which were then filtered (Gelman Pall 4190) and submitted for analysis. Soil and CaCl2blanks and CaCl2 controls at the four test concentrations (100, 500, 1000 and 2500 pg/L) were included in the study. Replicate concentration measurements were done on two separate days. Adsorption to Test Vessel Containers ^ The potential for adsorption of the test substance to the test vessel was assessed by analyzing the test substance solutions containing |h.g.highest and lowest concentrations in the validation>nag tm: experiments (100 and 2500 pg/L). The test substance was added to 50 mL polypropylene'* centrifuge tubes containing 20 mL o f 0.01M CaCl2solution. The tubes were placed on a rotator for 24 hours at 30 to 35 RPM at ambient room temperature. The concentration o f the solution at 24 hours was compared to aliquots taken at set-up to determine the potential for adsorption o f the test substance to the test vessel. Determining the Soil:Solution Ratio and Equilibration Time The SoikSolution Ratio and Equilibration Time experiment was conducted with two soil types, Drummer and Hidalgo, and three soil-solution ratios (20 g soil:20 mL, 5 g soil:25 mL, and 1 g soil:25 mL). Soil samples were pre-equilibrated with the 0.01M CaCl2 solution using an end-over-end rotator at 30-35 RPM. Following pre-incubation (12 hr), the test substance was added to the test vessels to yield a final test concentration of 1000 pg/L. Control tubes (test substance in CaCl2), CaCl2 blanks and soil blanks were prepared and processed with the adsorption samples. T0111/14107 Page 16 o f 55 DuPont EMSE Report No. 17-03 The test vessels were placed on the end-over-end rotator at 30-35 RPM at ambient room temperature. Duplicate test vessels for each soihsolution ratio, including control and blank tubes were collected at 2 ,6 ,2 4 and 72 hours and were centrifuged. The supernatants were transferred to 50 mL polypropylene tubes, aliquots were filtered and submitted for determination o f the test compound. The percent adsorption was calculated at each time point on the basis o f the nominal initial concentration and the measured concentration at the sampling time. Soil tubes and supernatants were frozen for approximately one month until the extraction procedure was performed for determination o f test substance stability and mass balance calculations. Determination of Test Substance Stability A mass balance was performed after the soihsolution ratio experiment. The 1:1 soil solution ratio tubes from the 72-hour time point, were used for this experiment. Test and blank soil tubes were thawed at room temperature for approximately one hour before addition o f 20 mL o f methanol (Burdick & Jackson 232-1). The pellets were disrupted using a polystyrene weighing spoon (Bel Art H36940-000) and resuspended by vortexing. The tubes were mixed for a minimum o f 2 hours before centrifugation (6000 RPM, 30 minutes) and recovery o f the methanol. A second extraction was immediately performed. -nUK >; siiij Aliquots (5 mL) were filtered (Gelman Pall 4190) to remove any particulate material. Aliquots o f the filtered methanol extracts (1 mL o f the first and 3 mL o f the second) were transferred to 5 mL polypropylene test tubes and evaporated completely using a Pierce Reacti-Vap Evaporating Unit (Model 18780) with a Nitrogen purge (1 psi). The residue in the tubes was resuspended in one mL 0.01M CaCl2and submitted for analysis. "jhinitied 3.7.2 Phase 2 D efinitive Studies Adsorption Experiments The adsorption experiment was conducted on four soils and one sludge sample. The experiment was conducted, in duplicate, at a 1:1 soil: solution for the test soils and 1:5 soihsolution ratio for the sludge sample at 4 test concentrations (100, 500,1000 and 2500 pg/L). The samples were pre-equilibrated with 0.01M CaCl2solution overnight (12 hours) before addition o f the test substance. The tubes were mechanically agitated using an end-over-end rotator (30-35 RPM, ambient temperature) for 24 hours before the supernatants were recovered by centrifugation. Control tubes (test substance in CaCl2), CaCl2 blanks and soil blanks were prepared and processed with the adsorption samples. An aliquot (5 mL) o f the supernatants was filtered (Gelman Pall 4190), diluted (if required), and refrigerated or frozen until analysis. The percent adsorbed was determined by comparing the amount o f test substance in the aqueous phase at equilibrium to the amount of test substance added to the system (based on calculated concentration). T0111 / 14107 Page 17 o f 55 DuPont EMSE Report No. 17-03 Desorption Experiments Desorption experiments were conducted on all test soils immediately following the adsorption study. The soil pellets from the adsorption study were resuspended in 20 mL o f 0.01M CaCL solution (sludge tubes received 25 mL). The pellets were disrupted via a polystyrene weighing spoon (Bel Art H36940-000) and vortexed to ensure complete mixing. The samples were agitated using an end-over-end rotator for 24 hours at ambient temperature, centrifuged and supernatants recovered. A second desorption step was conducted immediately. Aliquots of the desorption supernatants were filtered and submitted for analysis. The percent test substance desorbed was determined by comparing the amount o f test substance in the aqueous phase following a given desorption interval to the amount o f test substance associated with the soil phases at the adsorption equilibrium. The total amount o f test substance desorbed was obtained by summing the percent desorption at each desorption interval. 3.8 Data Analysis 3.8.1 Equilibration Time and Percent Adsorption Equilibration time can be determined from plots o f the concentration of the test substance in the aqueous phase versus time or from plots o f percent adsorption (A) against time. The values of adsorption (A) were calculated according to the following equations. Svmbol A nisoii m0 Co Vo cw Vw m .. xlOO A ---- -- ------- m0 where mo = Co x Vo mSOii = m0- (Cw x Vw) Definition percent adsorption mass o f test substance adsorbed to soil at equilibrium mass o f test substance applied to test vessel initial concentration o f test solution volume o f test solution added to test vessel concentration o f test substance in aqueous phase at adsorption equilibrium volume o f aqueous phase in the adsorption test at equilibrium (test solution, V0, plus water present in the soil) Unit % Pg Pg pg/mL mL pg/mL mL T0111 / 14107 Page 18 of 55 DuPont EMSE Report No. 17-03 3.8.2 Adsorption The adsorption coefficient, Kd, for each test solution concentration were calculated for each test soil using the following equation. The Kd value will also be used to calculate Komand KoC values. Cs Kd = Cw _ Kd Kom om _ Kd Ko c : oc Svmbol Kd Cs Kom Koc oc om Definition adsorption coefficient concentration of the test substance in the soil phase at adsorption equilibrium adsorption coefficient based on organic matter adsorption coefficient based on organic carbon fraction of organic carbon (foe) = % organic carbon/100 fraction of organic matter =fraction of organic carbon *1.724 Unit mL/g Pg/g mL/g mL/g na na 3.8.3 Desorption The percent desorbed, D, was calculated according to the following equations. mw,Dl xlOO Dl = msoil mw,D2 100 D2= m soil Dt= Di +D2 where m WjDi = C Wjdi x VW;di - (Cwx Vret) mW;D2 = C W)D2 X V WjD2 (CWiDl x V ret,Dl) T0111 /14107 Page 19 of 55 DuPont EMSE Report No. 17-03 Symbol Di D2 Dj mSOii mWjDi Cw,di Vw,di Cw Vret mWjD2 CW,D2 VWjd2 VretjDi Definition__________ _________________________________ percent o f test substance desorbed after first desorption interval percent o f test substance desorbed after second desorption interval total percent of test substance desorbed after both desorption intervals mass o f test substance adsorbed to soil at equilibrium mass o f test substance in aqueous phase after first desorption interval concentration of test substance in aqueous phase after first desorption interval total volume of aqueous phase (added solution plus volume o f water retained in soil) after first desorption interval concentration o f test substance in aqueous phase at adsorption equilibrium volume o f water retained in soil at adsorption equilibrium mass o f test substance in aqueous phase after second desorption interval concentration of test substance in aqueous phase after second desorption interval total volume o f aqueous phase (added solution plus volume o f water retained in soil) after second desorption interval volume o f water retained after first desorption interval 3.8.4 M aterial Balancefro m Adsorption/Desorption Experim ents Units % % % pg pg pg/mL mL pg/mL mL pg pg/mL mL mL (Cw XV MB = + mw,Dl + mw,D2 xl00/i V co Svmbol MB Cw Vw Definition material balance concentration of test substance in aqueous phase at adsorption equilibrium total volume o f the aqueous phase (added solution plus volume of water present in the soil) Units % pg/mL mL T0111 /14107 Page 20 o f 55 DuPont EMSE Report No. 17-03 mwoi mw.D2 Vo Co mass o f test substance in aqueous phase following first desorption interval mass o f test substance in aqueous phase following second desorption interval volume o f solution added to test vessel concentration o f test solution added to test vessel ftg ftg mL pg/mL 3.8.5 Adsorption Isotherm s Linear and Freundlich adsorption isotherms were constructed for each soil in which adsorption exceeded >10%. Linear isotherms were constructed by plotting Cs (y-axis) and Cw(x-axis) for each soil tested. The Freundlich adsorption coefficient (KF) and the exponential constant (1/n), were determined from linear regression analysis of the Freundlich equation: Cs = KFx Cw1/n The linear form o f this equation is shown below. log(Cs) = log(Kp) + ( 1/n x log(Cw)) Plotting the linear form of the Freundlich equation with log (Cs) on the y-axis and log (Cw) on the x-axis will yield a line with a slope o f 1/n and a y-intercept o f log (KF). The Kf value was used to calculate KFomand KFoc values. KFc om = om KFBoe 3.9 Statistical M ethods and Control o f Bias Statistical methods including means, standard deviations, and regression lines were used as appropriate. Bias was effectively controlled through duplicate sampling, replicate analysis, sample spiking, and maintenance o f material balance. Microsoft Excel 2000 was used in all calculations and in the statistical t-Test. T0111 /14107 Page 21 o f 55 DuPont EMSE Report No. 17-03 3.10 Deviationsfro m Test Protocol The mass balance determinations from the preliminary study were not immediately performed after the completion o f the soiksolution ratio and equilibrium time study. The analysis o f the substance extracted from the solid phase (mass balance) studies was not completed until after the adsorption-desorption experiments were completed. This is not believed to have affected the definitive study and is discussed in more detail in the Results and Discussion section o f this report. One o f the soil types for the soihsolution ratio was recommended to have an organic carbon content less than 2.0% and pH o f less than 4.8. The measured pH from the original characterization of the Cape Fear soil (8-Dec-97) had a reported value of 4.9. The accuracy of the equipment utilized by Agvise laboratories for this analysis is unknown. The soil was re-characterized by Harris laboratories and had a reported pH o f 5.4. The pH deviation is not believed to have affected the results from soihsolution ratio or definitive studies. 4.0 Results and Discussion 4.1 Preliminary Studies Validation o f the Analytical M ethod Ammonium perfluorooctanoate (APFO) in CaCl2 solutions was quantified as the perfluorooctanoate anion using liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS). Liquid chromatography was used to separate the analyte o f interest using a linear gradient of Methanol (Appendix 1). The flow was diverted to waste for the first 3 minutes o f the run and then directed to the mass spectrometer. The MS/MS transition 423 > 369 was specific to perfluorooctanoic acid and the area of the chromatogram was proportional to the concentration of ammonium perfluorooctanoate. Calibration standards were prepared fresh each day by dilution o f a refrigerated stock solution of 1000 mg/L APFO. The instrument was calibrated using a 5-point calibration ranging from 10 to 150 pg/L. The calibration standards were run in duplicate followed by a low (20 pg/L) check standard, high (100 pg/L) check standard, and a blank injection. The standard injection volume for the method was 25 pL. The calibrations curves had correlation coefficients (R2) o f greater than 0.985 and the check standards were 10% the test concentration. The samples were analyzed from duplicate injections and the mean test concentration was reported. The limit o f quantitation (LOQ) (Reference 7) for this method was 10 pg/L with the method of detection limit (MDL) at 5 ppb. The coefficient of variation on 5 replicates was determined to be < 15%. The accuracy was determined to be 15% o f the mean value o f 5 replicates except at the LOQ where the accuracy was 20%. T0111 /14107 Page 22 of 55 DuPont EMSE Report No. 17-03 The analytical method was validated using filtered CaCb solutions and extracts prepared from Drummer and Cape Fear soils. The measured concentration for the CaCl2 tubes was < 20% of the expected value except for one of the 2500 pg/L Control tubes, which had a deviation of 25.4% from the nominal test concentration (Table 2). This deviation from the expected concentration was not observed in subsequent testing of CaCl2 control solutions, which had less than 20% difference from mean control tube measured values at all four test concentrations (Table 3). The CaCl2 blanks in this preliminary study resulted in measured concentrations of 15.5 and 15.6 pg/L for the test compound (Table 2) and were isolated to this experiment. CaCh in all subsequent testing resulted in measured concentrations o f less than the limit o f quantitation (LOQ) o f 10 pg/L. The soil extracts did not interfere with the detection of the test compound. The measured concentrations for the soil extracts were generally < 15% that o f the CaCl2 controls (no soil) prepared during the study and had similar linear correlations (Table 3, Figure 1). One of the Cape Fear soil extracts prepared at a test concentration o f 100 pg/L had a 33.1% difference from the mean value for the CaCl2 100 pg/L controls. Since this discrepancy was isolated to one tube and was detected in only one soil extract it was attributed to experimental error during the preparation o f the sample. Adsorption to Test Vessel The test compound did not adsorb to the test vessel during the 24-hour study. The difference between the measured concentrations at the initial and 24-hour time point were not significant (p=0.05) and averaged 1.36% for the 100 pg/L tubes and 16.3% for the^SOOqig/L tubes (Table 4). Soil Solution Ratio and Equilibration Time The soil solution ratio and equilibration time investigation was conducted at a test concentration of 1000 pg/L on two soil types, Drummer and Hidalgo, and at three soil solution ratios. The adsorption was calculated (Table 5) based on the nominal test concentration and was plotted against incubation time for the both soils (Figures 2 and 3). At 24 hours, the adsorption ranged from 29.3% for the 1:25 soiksolution ratio to 83.9% for the 1:1 soil solution ratio with the Drummer soil, and 20.1 % for the 1:25 soiksolution ratio to 42.2% for the 1:1 soil solution ratio with the Hidalgo soil. These values remained essentially unchanged or decreased slightly after 72 hours, and 24 hours was selected as the equilibration time for subsequent studies. To achieve adsorption values o f greater than 20% and preferably greater than 50% for all test soils, a 1:1 soiksolution ratio was selected for adsorption studies for the test soils. The low density o f the sludge sample required that the adsorption-desorption studies be performed at a 1:5 soiksolution ratio. However, this ratio met the desired adsorption of greater than 50% (Table 7). T0111 /14107 Page 23 of 55 DuPont EMSE Report No. 17-03 M ass Balance Determinations The mass balance for the Drummer and Hidalgo soil tubes was lower than the CaCl2 control tubes (Table 6) based on the nominal test concentration from the soihsolution ratio study. The Hidalgo soil had an average mass balance o f 84.4% and a range o f 79.0 to 89.8%. The mass balance was 68.6% determined from the one Drummer soil tube available from the soihsolution ratio equilibrium time experiment. The CaCl2 control tubes, which contained no soil, were included in the mass balance determinations. The supernatants recovered at the end o f the soihsolution ratio equilibrium time experiment accounted for an average o f 98.9% o f the added test compound. The methanol extraction in the control tubes (no soil, with test substance) resulted in an average mass balance of 110% with a range o f 100 to 119%. The less than 90% recovery o f the test substance from the soil pellets may be the result o f the difficulty in resuspending die soil pellets in the extraction solvent. This disruption step required the mechanical manipulation of the soil pellets which inadvertently led to some soil loss. The pellets were vigorously vortexed after disruption but because o f the soil matrix it was difficult to establish whether the slurry had a uniform particle dispersion or if there was some residual clumping. The Drummer soil pellet was extremely difficult to resuspend during the extraction phase and required more mechanical disruption than the Hidalgo soil pellets. This may account for the lower mass balance determinations between the two soils. The methanol extracts were not directly analyzed for the test compound but evaporated completely and resuspended in 0.01M CaCl2 solution before analysis. The evaporation step may, however, not account for the lower recovery o f the test compound in the soil extracts. The efficiency o f methanol'in * extracting the test substance from the soils was not determined in this study, but was*assumed to be a suitable extraction solvent for this compound. The effect of the time lag between the soihsolution ratio experiment and the extraction o f the soil pellets on the recovery o f the test compound is unknown. The soihsolution ratio experiment was concluded approximately one month before the extraction o f the soil pellets. This storage may have had some effect on the recovery o f the test compound from the soil pellets or on the nature o f the interaction between the test compound and the soil matrix. Although the recovery of the compound was < 90% in this experiment, the definitive adsorption desorption experiments are descriptive of the test compound's behavior in the presence o f soil matricies. The difficulties in pellet resuspension during extraction and subsequent evaporation o f the extraction solvent suggest that alternative experimental approaches may need to be developed and validated for subsequent mass balance studies. 4.2 D efinitive Studies . Adsorption and Desorption Studies ` The aqueous phase analysis o f the adsorption experiment agreed well with the 24-hour results from the preliminary soihsolution ratio and equilibrium time phase of the study for the Drummer and Hidalgo soils. After 24 hours, the Drummer soil had adsorbed an average of 83.6% and Hidalgo soil had adsorbed an average of 38.1%, based on nominal initial concentrations, when tested at 1000 pg/L (Table 7). T0111 /14107 Page 24 o f 55 DuPont EMSE Report No. 17-03 Linear isotherms were constructed by plotting Cw (concentration of the aqueous solution at equilibrium) vs Cs (mass adsorbed to soil/test soil dry weight) for the four test soils and the activated sludge sample (Figures 7-11). The linear adsorption isotherms had correlation coefficients, which ranged from 0.926 for the Hidalgo soil to 0.977 for the Drummer soil. The values for the adsorption coefficient, Kd, were calculated for all four test soils and the one activated sludge sample (Table 7). The average Kdvalues ranged from an average o f 0.61 mL/g for the Hidalgo soil to an average o f 5.63 mL/g for the Drummer soil. The test soils yielded Kd values of 4.25 to 8.86 mL/g for the Drummer soil, 1.82 to 4.26 mL/g for the Keyport soil, 1.19 to 2.84 mL/g for the Cape Fear soil, and 0.41 to 0.83 mL/g for the Hidalgo soil. The Wilmington sludge sample had the highest average Kd value o f 22.5 mL/g, with Kdvalues that ranged from 12.6 to 36.8 mL/g. There was a strong linear correlation (R2= 0.947) between the fraction of organic carbon and average Kd values (Figure 4). The adsorption coefficients were corrected for organic carbon and organic material content of the test materials to calculate the soil sorption coefficients Koc and Kom(Table 7). The fraction of organic carbon (foe) o f the test materials was determined by the Walkley-Black method and the fraction of organic matter was calculated based on the assumption that soil organic matter contains 58% carbon. The average KoCvalues ranged from 79.0 mL/g in the Hidalgo soil to 148 mL/g in the Cape Fear soil and the average Komvalues ranged from 45.8 mL/g for the Hidalgo soil to 56.7 mL/g for the Drummer soil. The Wilmington sludge sample had an average Koc value o f 36.5 mL/g and average Komvalue o f 21.2 mL/g. The Freundlich adsorption coefficient (KF) and the constant (1/n) were determined from the linear form of the Freundlich equation for all test materials. Adsorption isotherms were constructed for all test materials (Table 8, Figures 12-16) and resulted in Freundlich adsorption coefficients, which ranged from 0.59 for the Hidalgo soil to 5.64 pg 1-1/11(ml)1/ng-1 for the Drummer soil. The adsorption coefficients were corrected for organic carbon and organic material content for each test material (Table 8). The Kfocvalues ranged from 44.2 to 261 pg 1-1/11(ml)1/n g-1 and the KFomvalues ranged from 25.6 to 151 pg 1-1/n (ml)1/ng-1for the Keyport and Cape Fear soils, respectively. There was a strong inverse relationship (R2=0.8665) between the fraction o f organic carbon (foe) in the Drummer, Keyport and Cape Fear soils and the total desorption o f the test compound (Figure 17). The Drummer soil with a foe o f 5.76% had an average of 36.0% total desorption while Cape Fear with a foe o f 1.24% had an average desorption o f 61.7%. The Keyport soil with an intermediate foe o f 3.72% desorbed an average o f 43.3% o f the test compound, a result that is between the Drummer and Cape Fear soil. The amount o f test compound recovered in the second stage of desorption is generally comparable to that recovered in the first stage. There was no evidence o f "tailing o ff' which suggests that if further desorptions were performed more of the test compound would be recovered. T0111 /14107 Page 25 o f 55 DuPont EMSE Report No. 17-03 The desorption results for the Hidalgo soil and the Wilmington Sludge sample were highly variable and ranged from 41.6 to 141% and 14.9 to 101%, respectively. In addition, the amount of test substance recovered varied between desorption steps 1 and 2 with the Hidalgo and Wilmington Sludge samples while the test substance recovery was more uniform with the other test soils. The total desorption values were inversely related to the test concentration in the Wilmington Sludge samples and decreased from an average o f 98.9% at 100 pg/L to 14.9% at the 2500 pg/L test concentration. This behavior was isolated to the sludge materials and remains unexplained in the current study. The variability seen in both the desorption experiments and the mass balance experiments o f this study may be attributed to the difficulty in resuspending the soil pellets. The desorption and extraction phases required two sequential steps that required the mechanical disruption o f the pellet to ensure complete suspension. Although there were unquantifiable soils losses with this procedure the solid losses and variability were present in all test materials and did not affect the overall behaviour o f the test compound. 5.0 Conclusions The adsorption of the test compound ranged from 36.0 to 83.0% for a soihsolution ratio o f lg/mL for the five test materials. The average adsorption coefficient values, Ka, ranged from 0.61 mL/g for the Hidalgo soil to 5.63 mL/g for the Drummer soil. The activated sludge sample had the highest average Kd o f 22.5 mL/g. The Freundlich adsorption coefficients (KF) ranged from 0.59 pg M/n (ml)I/n g-1 for the Hidalgo soil to 5.64 pg M/n (ml),/n g"1for the Drummer soil. The two sequential desorption steps, at a soihsolution ratio o f 1 g/mL, recovered an average o f 36.0 to 90.6% o f the adsorbed test compound. 6.0 Retention of Records For the periods required by GLP guidelines and specific country requirements, study documents and materials will be stored in the archives o f DuPont CR&D Environmental & Microbiological Sciences and Engineering (EMSE) and/or Iron Mountain, Wilmington, Delaware USA, including but not limited to: study protocol; any protocol and/or report amendments or addenda or SOP deviations; all raw data; one original signed copy o f the final report; laboratory-specific or site-specific raw data such as personnel files, instrument, equipment, refrigerator, and/or freezer raw data. T0111/14107 Page 26 of 55 DuPont EMSE Report No. 17-03 Documents and materials are archived according to the principles of Good Laboratory Practice in the organization o f the testing facility. 7.0 Disposal of Test Substance After issuance of the final report, the remaining test substance will be stored at the DuPont EMSE laboratory until its expiration date and then destroyed by burning, unless other arrangements are made between the facility supplying the test substance and the Test Facility. 8.0 References 1. U.S. Environmental Protection Agency, Pesticide Assessment Guidelines, Subdivision N, EPA 540/9-82-021, pp. 64-71, 1982. 2. Organization for Economic and Cooperative Development (OECD) Guideline for the Testing of Chemicals 106, Adsorption/Desorption (January 21, 2000). 3. U.S. Environmental Protection Agency, Office of Prevention, Pesticides, and Toxic Substances (OPPTS), Fate, Transport and Transformation Test Guidelines, OPPTS 835.1110 Activated Sludge Sorption Isotherm, EPA 712-C-98-298, January 1998. 4. U.S. Environmental Protection Agency. 1989. Good Laboratory Practice Standards, 40 CFR, Part 160, Final Rule. EPA, Washington, D.C. 5. OECD Principles of Good Laboratory Practice published in ENV/MC/CHEM(98) 17, OECD, Paris, France. 6. DuPont Central Research & Development, Environmental and Microbiological Science and Engineering Standard Operating Procedure. Adsorption-Desorption Using a Batch Equilibrium Method: EMSE067-P (June 2002). 7. U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry Bioanalytical Method Validation, May 2001. T0111 /14107 Page 27 of 55 DuPont EMSE Report No. 17-03 Table 1 Chemical and Physical Properties of Test Materials Site Soil Location Description Depth Drummer Rochelle, minois Fallow Top 20 cm Hidalgo Wilmington Sludge Cape Fear Donna, Texas Wilmington, Delaware Cooper River, South Carolina Bedded Fallow Top 20 cm Grass, walking path 20 - 30 cm Keyport Newark, Delaware Fallow Top 20 cm Texture Classification t Particle Size Analysis 2mm-50pm < 2 pm (Sand) 50-2um (Silt) (Clay) (%) <%) (%) Silt Clay Loam 12.0 60.0 28.0 Sandy Clay Loam 52.0 20.0 28.0 Sandy Loam 68.0 16.0 16.0 Sandy Loam 68.0 16.0 16.0 Loam 48.0 36.0 16.0 Soil Drummer Hidalgo Wilmington Sludge Cape Fear Keyport Bulk Density (g/mL) 1.1 1.3 0.4 1.4 1.2 foe Walkley pH Black % Moisture (%) (%) 7.6 5.76 7.8 0.77 6.7 61.7 5.4 1.24 5.5 3.72 5.95 3.32 8.14 0.93 1.91 Cation Exchange Soil Capacity Hydrogen Potassium (meq/100g) (% ofCEC) (% of CEC) Drummer Hidalgo Wilmington Sludge Cape Fear Keyport 36.6 25.7 10.2 3.1 9.5 0 0.7 0 3.8 0 3.4 12.8 1.7 31.5 2.2 Magnesium (% of CEC) 12.8 10.2 22.7 11.7 10.5 Calcium Sodium (% of CEC) (% of CEC) 86.2 0.2 83.4 2.6 69.5 4.4 71.8 2.1 55.2 0.6 f USDA system (sand: 2mm-50pm, silt: 50-2pm, clay: <2pm) T0111/14107 Page 28 o f 55 DuPont EMSE Report No. 17-03 Table 2 Determination of Test Substance in CaCl2Solutions (No Soil) Sample Description CaC12 Blank CaC12 Blank 100-1 100-2 500-1 500-2 1000-1 1000-2 2500-1 2500-2 Nominal Concentration pg/L M easured Concentration f Pg/L Deviation from Expected Value { % 0 0 100 100 500 500 1.00E+03 1.00E+03 2.50E+03 2.50E+03 15.5 15.6 96.7 103 566 587 876 972 1.86E+03 2.03E+03 ND ND -3.30 2.90 13.1 17.4 -12.4 -2.79 -25.4 -18.7 f Limit of Quantification (LOQ) = 10 pg/L $ Deviation from Expected Value = (Measured-Nominal)/Nominal x 100 0 Not Determined T0111 /14107 Page 29 o f 55 DuPont EMSE Report No. 17-03 Table 3 Determination of Test Substance in CaCl2Soil Extracts Sample Description Measured Concentration pg/L Deviation from Deviation from Mean Control Tube Expected Value J Measured Value O %% CaC12 Blank CaC12 Blank Drummer Soil Blank 1 Drummer Soil Blank 2 Cape Fear Soil Blank 1 Cape Fear Soil Blank 2 CaCI2Controls (No Soil) 100 ppb 100 ppb 500 ppb 500 ppb 1000 ppb 1000 ppb 2500 ppb 2500 ppb Drummer Soil Extract 100 ppb 100 ppb 500 ppb 500 ppb 1000 ppb 1000 ppb 2500 ppb 2500 ppb Cape Fear Soil Extract 100 ppb 100 ppb 500 ppb 500 ppb 1000 ppb 1000 ppb 2500 ppb 2500 ppb <LOQf <LOQ <LOQ <LOQ <LOQ <LOQ 104 101 521 572 889 826 2.01E+03 2.51E+03 97.3 99.8 464 558 874 951 2.22E+03 2.19E+03 136 118 551 622 985 943 1.97E+03 2.34E+03 4.44 0.550 4.20 14.5 -11.1 -17.4 -19.4 0.516 -2.69 -0.200 -7.16 11.5 -12.6 -4.89 -11.3 -12.5 36.4 18.4 10.2 24.3 -1.46 -5.72 -21.1 -6.50 -5.06 -2.63 -15.1 1.99 1.91 10.9 -2.05 -3.41 33.1 15.5 0.805 13.7 14.9 9.90 -12.9 3.26 t LOQ= 10 pg/L t Deviation from Expected Value = (Measured-Nominal)/ Nominal x 100 0 Deviation from CaC12 Mean Control Tube Value = (Measured-Mean CaC12 Control)/Mean CaC12 Control x 100 T0111/14107 Page 30 o f 55 DuPont EMSE Report No. 17-03 Table 4 Adsorption to Test Vessel Sample Description CaC12 Blank-1Start CaC12 Blank-2 Start CaC12 Blank-1 24 hour CaC12 Blank-2 24 hour 100 ppb-1 Initial 100 ppb-2 Initial 100 ppb-1 24hr 100 ppb-2 24 hr 2500 ppb-1 Initial 2500 ppb-2 Initial 2500 ppb-1 24 hr 2500 ppb-2 24 hr Measured Concentration pg/L <LOQf < LOQ <LOQ <LOQ 93.2 98.1 90.8 98.3 1.64E+03 1.62E+03 1.19E+03 1.54E+03 Deviation from Initial Value $ % ND ND 5.07 -2.77 26.9 5.58 f LOQ Limit of Quantitation = 10 pg/L %Deviation from Initial Value = (Average Initial Measured Concentration-Measured Concentration at 24hr)/ Average Initial Measured Concentration* 100 ND= Not Determined T0111/14107 Page 31 of 55 DuPont EMSE Report No. 17-03 Table 5 Adsorption in percent as a Function of Soil:Solution Ratio and Equilibration Time Time, hours 2 2 6 6 24 24 72 72 Drummer 1:1 65.3 72.4 75.7 73.3 83.9 79.8 78.8 NDf Drummer 1:5 29.9 36.6 32.9 37.7 39.3 51.2 48.1 ND Drummer 1:25 24.8 27.7 4.11$ 25.4 33.0 29.3 23.4 33.3 Hidalgo 1:1 21.1 9.28 30.5 22.5 42.2 39.3 27.7 35.8 Hidalgo 1:5 7.84 6.38 16.6 16.5 24.4 24.2 29.7 23.9 Hidalgo 1:25 -0.01 -1.61 6.84 18.7 20.1 25.9 12.3 3.17 f ND = Not Determined; Sample was lost $ Result was an outlier and not reported in range o f adsorption values T0111 /14107 Page 32 o f 55 DuPont EMSE Report No. 17-03 Table 6 Mass Balance Calculations Of Drummer and Hidalgo Soils Performed at 1:1 Soil Solution Ratio after 24 Mixing Sample Description MEOH Blank MEOH Blank-2 Control Control Drummer Soil Blank 1 Drummer Soil Blank 2 Drummer 1000 ppb Test 1 Hidalgo Blank 1 Hidalgo Blank 2 Hidalgo 1000 ppb Test 1 Hidalgo 1000 ppb Test 2 Soil Dry Wt S 19.3 18.9 19.1 19.8 19.2 19.6 19.5 Measured Concentration of Aqueous Phase at Equilibrium (19-Dec-G2) pg/L 1.14E+03 972 <LOQ <LOQ 224 <LOQ <LOQ 763 678 Test Substance in Aqueous Phase at Equilibrium Mg 23.2 19.6 <LOQ <LOQ 2.92 <LOQ <LOQ 10.4 9.29 Measured Concentration of MEOH Extraction 1 {01-F e b -03) pg/L <LOQf <LOQ 12.7 11.4 <LOQ <LOQ 323 <LOQ <LOQ 262 218 Test Substance i MEOH Extraction Mg 0.245 0.223 6.69 5.45 4.52 Measured Concentration of MEOH Extraction 2 (01-Feb-03) pg/L <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 146 <LOQ <LOQ 72.3 70.0 Test Substance in MEOH Extraction 2 Mi <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2.83 <LOQ <LOQ 1.41 1.38 Trat Substance in Void Volume Mg <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1.11 <LOQ <LOQ 0.451 0.430 Total Test Compound Recovered Mg <LOQ <LOQ 23.5 19.8 <LOQ <LOQ 13.6 <LOQ <LOQ 17.7 15.6 Mass Balance % NCJ NC 119 100 NC NC 68.6 NC NC 89.8 79.0 t LOQ Limit o f Quantitation = 10 jig/L J NC = Not Calculated. Mass Balance could not be determined because samples <LOQ T0111/14107 Page 33 o f 55 DuPont EMSE Report No. 17-03 Table 7 Definitive Adsorption Study; Percent Adsorption and Linear Adsorption Isotherm Parameters Sample ID Test Soil % Adsorption t Cs Cw log Cs logCw Kd Koc Kom Soil 1 100 ppb Soil 1 100 ppb Soil 1 500 ppb Soil 1 500 ppb Soil 1 1000 ppb Soil 1 1000 ppb Soil 1 2500 ppb Soil 1 2500 ppb Soil 2 100 ppb Soil 2 100 ppb Soil 2 500 ppb Soil 2 500 ppb Soil 2 1000 ppb Soil 2 1000 ppb Soil 2 2500 ppb Soil 2 2500 ppb Soil 3 100 ppb Soil 3 100 ppb Soil 3 500 ppb Soil 3 500 ppb Soil 3 1000 ppb Soil 3 1000 ppb Soil 3 2500 ppb Soil 3 2500 ppb Soil 4 100 ppb Soil 4 100 ppb Soil 4 500 ppb Soil 4 500 ppb Soil 4 1000 ppb Soil 4 1000 ppb Soil 4 2500 ppb Soil 4 2500 ppb Soil 5 100 ppb Soil 5 100 ppb Soil 5 500 ppb Soil 5 500 ppb Soil 5 1000 ppb Soil 5 1000 ppb Soil 5 2500 ppb Soil 5 2500 ppb Fgfe pg/L Mg/g Fg/C mL/g mL/g mL/g Drummer 82.2 0.08 16.4 -1.07 1.21 5.17 89.7 52.0 Drummer 79.3 0.08 18.9 -1.09 1.28 4.25 73.8 42.8 Drummer 82.7 0.39 73.4 -0.41 1.87 5.35 92.9 53.9 Drummer 88.9 0.45 50.3 -0.35 1.70 8.86 154 89.2 Drummer 82.0 0.83 164 -0.08 2.22 5.04 87.6 50.8 Drummer 85.3 0.86 135 -0.07 2.13 6.39 111 64.3 Drummer 82.9 2.01 374 0.30 2.57 5.38 93.5 54.2 Drummer 80.6 1.95 425 0.29 2.63 4.60 79.8 46.3 Mean T 83.0 1 1 563 1' 972 1i 56.7 Hidalgo 33.4 0.03 60.4 -1.49 1.78 0.53 68.9 40.0 Hidalgo 29.5 0.03 64.3 -1.54 1.81 0.45 58.2 33.8 Hidalgo 41.2 0.20 267 -0.70 2.43 0.75 97.1 56.3 Hidalgo 43.8 0.21 253 -0.68 2.40 0.83 108 62.6 Hidalgo 39.7 0.38 545 -0.42 2.74 0.70 90.4 52.5 Hidalgo 36.6 0.35 574 -0.46 2.76 0.61 79.0 45.8 Hidalgo 27.8 0.65 1.60E+03 -0.18 3.21 0.41 53.0 30.8 Hidalgo 36.0 0.84 1.42E+03 -0.07 3.15 0.59 77.2 44.8 Mean | 36.0 1 1 061 1i 796 1 45.8 Wilmington Sludge 69.5 0.35 28.1 -0.45 1.45 12.6 20.5 11.9 Wilmington Sludge 70.8 0.37 26.8 -0.44 1.43 13.7 22.2 12.9 Wilmington Sludge 74.6 1.95 119 0.29 2.08 16.3 26.5 15.4 Wilmington Sludge 77.7 1.91 106 0.28 2.02 18.1 29.3 17.0 Wilmington Sludge 83.4 4.40 155 0.64 2.19 28.3 45.9 26.7 Wilmington Sludge 82.7 4.27 161 0.63 2.21 26.4 42.9 24.9 Wilmington Sludge 87.3 10.7 291 1.03 2.46 36.8 59.6 34.6 Wilmington Sludge 83.4 10.9 390 1.04 2.59 27.9 45.3 26.3 Mean | 78.7 1 11 22.53 1 36.5 1 21.2 Cape Fear 64.8 0.06 32.5 -1.22 1.51 1.87 151 87.5 Cape Fear 63.3 0.06 33.7 -1.23 1.53 1.73 140 80.9 Cape Fear 70.8 0.33 134 -0.49 2.13 2.43 196 114 Cape Fear 73.7 0.34 121 -0.46 2.08 2.84 229 133 Cape Fear 60.3 0.52 348 -0.28 2.54 1.51 21 70.4 Cape Fear 59.3 0.55 372 -0.26 2.57 1.48 119 69.1 Cape Fear 54.1 1.22 1.02E+03 0.09 3.01 1.19 95.9 55.6 Cape Fear 61.4 1.36 847 0.13 2.93 1.61 130 75.3 Mean | 63.5 1 1 1.83 1 147.8 1 852 Keyport 64.5 0.06 33.7 -1.21 1.53 1.82 48.9 28.4 Keyport 68.9 0.07 28.7 -1.19 1.46 227 61.1 35.4 Keyport 77.2 0.37 107 -0.43 2.03 3.48 93.4 54.2 Keyport 73.7 0.30 105 -0.52 2.02 2.90 77.9 45.2 Keyport 76.7 0.73 215 -0.14 2.33 3.39 91.0 52.8 Keyport 80.8 0.75 175 -0.13 2.24 4.26 115 66.5 Keyport 75.8 1.74 550 0.24 2.74 3.17 85.1 49.4 Keyport 70.6 1.64 655 0.21 2.82 2.50 67.2 39.0 Mt Aeadnsorptiondeter|mine6d3.1on1:11(soils)and1:5(sludge)soil:soludonratio11 242.8h5req1uili9b0ra2tion!1tim52e.6 1 1 1 1 1 T0111 /14107 Page 34 of 55 DuPont EMSE Report No. 17-03 Table 8 Definitive Adsorption Study; Freundlich Adsorption Isotherm Parameters TestSoil Slope Intercept (1/n) (logKK) Drummer 0.994 -2.25 Hidalgo 1.00 -3.23 CapeFear 0.885 -2.49 Keyport 1.11 Wilmington Sludge 1.36 -2.78 -2.41 R2 kf ^Foc (pg'-l,n(mL)1,ng1) (pg,1,"(mL)1"'g-1) 0.97 5.64 98.0 0.96 0.59 76.2 0.96 3.23 0.97 1.64 261 44.2 0.98 3.90 6.3 ^Fom 56.8 44.2 151 25.6 3.7 T0111 /14107 Page 35 of 55 DuPont EMSE Report No. 17-03 Table 9 Desorption of Test Substance From Test Soils Sample Description Drummer 100 ppb Drummer 100 ppb Drummer 500 ppb Drummer 500 ppb Drummer 1000 ppb Drummer 1000 ppb Drummer 2500 ppb Drummer 2500 ppb Hidalgo 100 ppb Hidalgo 100 ppb Hidalgo 500 ppb Hidalgo 500 ppb Hidalgo 1000 ppb Hidalgo 1000 ppb Hidalgo 2500 ppb Hidago 2500 ppb Wilmington Sludge 100 ppb Wilmington Sludge 100 ppb Wilmington Sludge 500 ppb Wilmington Sludge 500 ppb Wilmington Sludge 1000 ppb Wilmington Sludge 1000 ppb Wilmington Sludge 2500 ppb Wilmington Sludge 2500 ppb Cape Fear 100 ppb Cape Fear 100 ppb Cape Fear 500 ppb Cape Fear 500 ppb Cape Fear 1000 ppb Cape Fear 1000 ppb Cape Fear 2500 ppb Cape Fear 2500 ppb Keyport 100 ppb Keyport 100 ppb Keyport 500 ppb Keyport 500 ppb Keyport 1000 ppb Keyport 1000 ppb Keyport 2500 ppb Keyport 2500 ppb Measured Cpd Measured Cpd Concentration Recovered Desorbed Concentration Recovered Desorbed Total Cpd Desorp I Soin in Desorp 1 Stage 1 Desorp 2 Soin in Desorp 2 Stage 2 Recovered Desorption Total Itg/L 13.3 12.9 73.9 93.0 211.5 166.8 397.8 367.4 28.3 29.7 124.3 125.1 148.9 138.3 426.4 158.5 52.7 49.4 142.2 139.1 112.9 121.5 129.7 174.7 18.4 20.8 162.1 113.3 173.9 169.4 439.0 447.2 23.5 22.0 35.5 30.5 162.8 100.5 227.5 412.2 PS 0.26 0.24 1.39 1.77 3.97 3.17 7.68 6.96 0.53 0.57 2.33 2.31 2.79 2.62 7.99 3.02 1.27 1.19 3.45 3.25 2.70 2.90 4.03 4.25 0.36 0.40 3.04 2.16 3.26 3.23 8.19 8.25 0.44 0.41 0.66 0.58 3.10 1.90 4.15 7.76 % 16.0 15.4 18.9 20.7 25.2 19.2 20.0 18.7 85.5 102.2 60.6 57.0 37.7 38.5 62.4 18.4 78.1 72.3 38.8 35.0 13.6 14.9 8.0 8.6 29.8 34.1 46.5 31.7 31.0 29.7 33.8 30.5 37.1 31.8 9.1 9.8 21.8 12.8 11.8 24.3 W'L 9.53 11.6 53.8 48.0 113 75.8 182 190 7.99 7.86 45.1 43.3 89.6 102 137 141 11.4 12.6 53.2 29.3 70.7 45.9 66.8 96.8 10.2 10.4 70.6 64.6 161 145 221 197 7.66 10.2 61.7 68.0 152 143 221 201 PS 0.27 0.33 1.51 1.33 3.09 2.11 5.10 5.27 0.22 0.21 1.24 1.18 2.47 2.81 3.74 3.84 0.37 0.40 1.71 0.96 2.24 1.48 2.17 3.12 0.28 0,28 1.93 1.76 4.45 3.99 6.09 5.41 0.21 0.29 1.71 1.92 4.28 3.97 6.28 5.64 % 16.8 21.4 20.4 15.6 19.6 12.8 13.3 14.2 35.3 38.8 32.3 29.1 33.4 41.2 29.2 23.5 22.6 24.5 19.2 10.4 11.3 7.60 4.30 6.33 23.2 23.9 29.6 25.8 42.2 36.7 25.1 20.0 17.5 22.4 23.6 32.4 30.1 26.7 17.9 17.7 PS 0.53 0.56 2.90 3.09 7.06 5.27 12.8 12.2 0.75 0.78 3.57 3.49 5.26 5.43 11.7 6.86 1.64 1.60 5.16 4.21 4.94 4.39 6.20 7.37 0.64 0.68 4.97 3.92 7.71 7.22 14.3 13.7 0.65 0.69 2.37 2.50 7.38 5.87 10.4 13.4 % 32.9 36.7 39.3 36.3 44.7 32.0 33.3 32.9 121 141 92.9 86.0 71.2 79.7 91.6 41.9 101 96.8 58.0 45.4f 25.0 22.5 12.3$ 14.9 53.0 57.9 76.1 57.5 73.2 66.5 58.9 50.5 54.7 54.2 32.7 42.2 51.9 39.5 29.8f 41.9 t Soil tube spilled during processing; not included in average calculations %Adsorption supernatant added to soil tube during processing; not included T0111 /14107 Page 36 of 55 DuPont EMSE Report No. 17-03 Figure 1 Analytical Method Validation of CaCl2Controls (no soil) and Extracts from Drummer and Cape Fear Soils Target concentration, pg/L t y = 0.8928x + 23.403, R2 = 0.978 t y = 0.8749x + 27.432, R2 = 0.998 <Dy = 0.8463x + 73.34, R2 = 0.983 T0111 /14107 ' Page 37 o f 55 DuPont EMSE Report No. 17-03 Figure 2 Adsorption of Test Substance as a Function of Soil:Solution Ratio and Equilibration Time with Drummer Soil 100 80 ao H 60 a 40 T&uov3i 20 AA A A --------- A 1:1 SoihSolution Ratio A 1:5 Soil:Solution Ratio 1:25 SoikSolution Ratio A J----------- ]----- ----- ------------ 1----------- !-----------1----------- 1----------- 1-----------1 10 20 30 40 50 60 70 80 Equilibration Time, Hours oration T0111 / 14107 Page 38 o f 55 DuPont EMSE Report No. 17-03 Figure 3 Adsorption of Test Substance as a Function of Soil:Solution Ratio and Equilibration Time with Hidalgo Soil 100 +o* 80 H 60 coaut. 40 20 3 1 0+ 0 10 1:1 Soil:Solution Ratio A 1:5 SoilrSolution Ratio 1:25 Soil:Solution Ratio 20 30 40 50 60 Equilibration Time, Hours * A 70 80 T0111 / 14107 Page 39 of 55 DuPont EMSE Report No. 17-03 Figure 4 Average Kdvs Fraction of Organic Carbon for Drummer, Hidalgo, Cape Fear and Keyport Soils T0111 / 14107 Page 40 of 55 DuPont EMSE Report No. 17-03 Figure 5 Average KqMvs Fraction of Organic Carbon for Drummer, Hidalgo, Cape Fear and Keyport Soils T0111 / 14107 Page 41 of 55 DuPont EMSE Report No. 17-03 I Figure 6 Average KqCvs Fraction of Organic Carbon for Drummer, Hidalgo, Cape Fear and Keyport Soils ) Fraction of organic carbon, % T0111 /14107 Page 42 o f 55 DuPont EMSE Report No. 17-03 Figure 7 Linear Adsorption Isotherm of the Test Substance in Drummer Soil T0111 /14107 Page 43 o f 55 DuPont EMSE Report No. 17-03 Figure 8 Linear A dsorption Isotherm of the Test Substance in Hidalgo Soil T0111 /14107 Page 44 o f 55 DuPont EMSE Report No. 17-03 Figure 9 Linear Adsorption Isotherm of the Test Substance in Cape Fear Soil T0111 /14107 Page 45 o f 55 DuPont EMSE Report No. 17-03 Figure 10 Linear Adsorption Isotherm of the Test Substance in Keyport Soil T0111/14107 Page 46 o f 55 DuPont EMSE Report No. 17-03 Figure 11 Linear Adsorption Isotherm of the Test Substance in Wilmington Sludge T0111 /14107 Page 47 o f 55 DuPont EMSE Report No. 17-03 Figure 12 Freundlich Adsorption Isotherm of the Test Substance in Drummer Soil T0111 /14107 Page 48 of 55 DuPont EMSE Report No. 17-03 Figure 13 Freundlich Adsorption Isotherm of the Test Substance in Hidalgo Soil T0111/14107 . Page 49 o f 55 DuPont EMSE Report No. 17-03 Figure 14 Freundlich Adsorption Isotherm of the Test Substance in Cape Fear Soil T0111/14107 Page 50 o f 55 DuPont EMSE Report No. 17-03 Figure 15 Freundlich Adsorption Isotherm of the Test Substance in Keyport Soil T0111/14107 Page 51 o f 55 DuPont EMSE Report No. 17-03 Figure 16 Freundlich Adsorption Isotherm of the Test Substance in Wilmington Sludge T0111/14107 Page 52 o f 55 DuPont EMSE Report No. 17-03 Figure 17 Total Desorption vs Fraction of Organic Carbon for Drummer, Keyport and Cape Fear Soils Fraction of Organic Carbon, % T0111 /14107 Page 53 of 55 DuPont EMSE Report No. 17-03 Appendix 1 Ammonium Perfluorooctanoate Analytical Method Operating Conditions The LC column is equilibrated with 95% Solvent A (5% Methanol in 2mM Ammonium Acetate) and Solvent B (100% Methanol). The method utilizes a linear gradient with the following conditions: Time (min) 0 1 6 8 9 13 Solvent B % 5 5 100 100 .5 5 Flow (ml/min) 0.25 0.25 0.25 0.25 0.25 0.25 The flow is diverted to waste between 0-3 minutes and directed to the mass spectrometer between 3 to 12.5 minutes. The injection volume is 25 microliters. The mass spectrometer is optimized for the molecular anion at 413 m/z with the Capillary Voltage set at 3.0kV, Cone Voltage set at 10V, the source block temperature is set at 150C and desolvation temperature is set at 200C. The collision cell is maintained at a pressure of argon 1.0 E-3 and nitrogen flow is maintained at 100 psi. The mass spectrometer monitors the transition 423 > 369. T0111/14107 Page 54 of 55 DuPont EMSE Report No. 17-03 Appendix 2 Certificate of Analysis of Test Substance Sigjna-AMrkh Certificate ofAnalysis P*8 1o)r2 71 C e rtlfic a te o r n a ly s is la t e Lot N o) Find I TEST ProoiNam* ftro&iGttttiffitoer LO T {421207ft) R E S U L T S PenttKfeeaBtwrooatsnoie am ammonium satt mm PsvAPttf/n# tebhSmt'A / 1CFeAdSiraNiu*mfesr fumalaiValeht I1TRATIH(Mi)NCUH UH 3S25261 G^iNO 431.1 tffl.9% wwwjnitLOfi} S^mTY<lMIW W H IT E P O W D E R C O LO U RLESS C L E A R {O .S TE(F5> SOittMUY 0*81*00) *tsn m m m nsm m m ifRAf^O^CTRUIS4 GATEOFtC^tUilASt- 1 O jN 10 M L H 20 <0.1% <0.1 % C O R R ESPO N D S 24OAM01 F ltika g u a rttite a t the 'Sales-SfisaB satin' v a tu ** onljr, fffln-SpediSied M H tttay be kteKted M additional Inform alton. Tha suerent Salss-Specllcafens' stieat Is w a iia b te on raspest. F o rfu rtf f in q s , p iM a eontaet our T *n ie l Sam te. Ftaika w aran ls. ihat ite produds ttm im m to tt infofmaUon oonlamed in M s and other Ftaka publteattons. FurcSsaser must daiem iBi tto suitaWlity ef the product for iis partloaiar use. Sae reysrsa skte of b w ftsr additional tarm s and ccntfflens of SBe. The vafitos gieen on ihe Certificate of natysis* am the results dstemiinecS at the me of arralyais. Mwfcr Sr^S1bwk~'~~ W^lnfoew.si8ima-alWi*.com/cgi-bin^SX-S^W*|S<','COrAinf.RetmCOfA I/20Q003 T0111 /14107 Page 55 of 55