Document ZBqrmepBj0jwpJyx0QOxXKzDL

110> ACUTE TO X IC ITY TO TH E EARTHW ORM (Eisenia fetida) TE S T SUBSTANCE_______________________________________ __________ * id entity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC -95. (1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt, CAS # 2795-39-3) Rem arks: The test substance is a white powder. Sample w as taken from 3M lot number 217. Sam ple w as stored under ambient conditions prior to testing. Purity determined to be 86.9% by LC/M S, 1H-HM R, 19F-N M R and elemental analyses techniques. M ETHOD__________________________________________________________ M ethod: The study was conducted using a protocol based on procedures outlined in the Organization for Economic Cooperation and Development (O EC D ) Guideline No. 207. T est type: Static, artificial soil substrate GLP: Yes Y e a r C om pleted: 2002 Species: Eisenia fetida A n alytical m onitoring: Test substance concentrations in soil measured by LC/M S/M S at Days 0 and 14; in earthworm tissues after test term ination. S tatistical m ethods: LC5o values calculated by moving average method or binomial probability with non-linear interpolation using the computer software of C .E . Stephan. Treatm ent group means were compared to control means with Dunnett's test (a = 0.05) using SAS Version 8 to determine if significant differences from the control occurred. T es t organism source: Obtained from Worm Man's Worm Farm, R & K Trading Co., Monroe Township, NJ T es t organism age a t stu d y in itiatio n : Adult (with ciitellum) Test conditions T es t m edia: Artificial soil of the following composition: Quartz sand 31.5 kg Kaolin clay 9.0 kg Sphagnum peat 4.5 kg Calcium carbonate 1.125 kg Artificial soil prepared in bulk by blending 70% sand, 20% kaolin clay and 10% sphagnum peat. The pH of the bulk soil prior to hydration was adjusted to 6.0 using calcium carbonate. Moisture content adjusted to -3 3 % by weight. im jut 29 AH 9s25 CONTAIN NO CBI 000197 Lighting: Fluorescent lights, intensity measured as 494-775 lux. Photoperiod was 24-hours of continuous light. Stock and test solutions preparation: The test substance was first dry mixed with the test soil. Approximately 4 kg of each test concentration and the negative control was prepared separately. The soil, test substance and deionized w ater were mixed together for 20 minutes prior to separation into the appropriate exposure vessel. Test soils w ere prepared and held overnight at approximately 20C prior to adding worms to allow the soil to become less fluid. Exposure vessels: One liter Nalgene beakers containing approximately 750 g of prepared soil, except for the 625 mg/kg (m easured) concentration, where only 650 g would fit in the vessel. The beakers w ere covered with plastic wrap, which was perforated for air exchange. Number of replicates: four Number of test organisms per replicate: ten Number of concentrations: five plus a negative control Test media parameters: Initial and final % moisture (controls): 3 3 .9 ,3 2 .1 Initial and final % moisture (1042 mg/kg): 32.5, 34.5 Initial and final soil pH in water (controls): 6.9, 8.3 Initial and final soil pH in water (1042 mg/kg): 8.0, 8.3 Initial and final Temp., C(controls): 2 2 ,2 2 Initial and Final Temp., C (1042 mg/kg): 2 2 ,251 1 One outlier of 25C Element Basis: LC50 - mortality. NO EC - burrowing behavior, body weight and clinical signs of toxicity. A t test initiation, worms were placed on the surface of the soil in each test chamber and observed for burrowing behavior. On Day 7 and Day 14, the contents of each test cham ber were removed to determine the number of surviving worms and to evaluate sublethal effects. On Day 7, following observations, test soil was returned to the test chambers and the worms w ere placed on the soil surface in order to again observe burrowing behavior. Method of calculating means of measured concentrations: Arithmetic mean 00198 RESULTS Nominal concentrations: <LOQ, 7 8 .1 ,1 5 6 , 313, 6 2 5 ,1 2 5 0 mg/kg Measured concentrations (Day 0): <LOQ, 7 7 .0 ,1 4 1 , 2 8 9 ,4 8 8 ,1 0 4 2 mg/kg (as dry weight) Element values: 7-day LCso (95% C .I.): 3 9 8 (2 8 9 -4 8 8 ) mg/kg 7-day NOEC: 289 mg/kg 7-day LOEC: 488 mg/kg 14-day LC50 (95% C .I.): 373 (3 1 6 -4 4 0 ) mg/kg 14-day NOEC: 77 mg/kg 14-day LOEC: 141 mg/kg All elem ent values w ere calculated using the Day 0 measured concentrations in the test soils. Statistical Evaluation: The 7-day LCso value and 95% confidence interval were calculated by binomial probability. The 14-day elem ent values were determined using the moving average method. Analytical Methodology: Analyses of test soils and earthworms w ere performed at W ildlife International Ltd. using high performance liquid chromatography with triple quadrupole mass spectrometric detection (HPLC/M S/M S). W hen determining the concentration of the test substance in the samples, the sam e and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. On Day 14, following observations and body weight determination, all surviving worms from each treatm ent group were placed on moistened filter paper and allowed to purge their gut contents for approximately 24 hours. The worms were then euthanized by freezing and stored frozen prior to analysis. The LOQs (limits of quantitation) in soil and earthworms were 29.3 and 50.0 mg/kg, respectively, in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 87.3% in the soil and 86.6% in the earthworm tissue. Soil samples collected at test initiation had measured values from 78.1% to 98.6% of nominal. Measured values for samples taken on Day 14 ranged from 87.5 to 103% of nominal. 000199 Summary of analytical chemistry date for soil, Day 0: Nominal Test Concentration, mq/kq Measured Replicate Values, mg/kg (dry weight) Mean Concentration, mg/kg (dryw t) Percent of Nominal Negative Control All < LOQ (29.3) <LOQ (29.3) 78.1 7 4 .6 ,7 2 .7 ,1 0 4 , 7 4 .7 ,6 9 .4 , 66.5 77.0 98.6 156 144, 138 141 90.4 313 312, 266 289 92.3 625 5 2 6 ,4 4 9 488 78.1 1250 1152,906, 894, 93 0 ,1 4 0 5 , 966 1042 83.4 Summary of analytical chemistry date for soil, Day 14: Nominal Test Concentration, mg/kg Measured Replicate Values, mg/kg (dry weight) Mean Concentration, mg/kg (dry wt) Percent of Nominal Negative Control 78.1 156 313 625 1250 All < LOQ (29.3) 7 4 .9,8 6 .2 143, 142 302, 308 5 3 2 ,7 3 5 1 0 5 9 ,1 1 2 9 <LOQ (29.3) 80.6 143 305 634 1094 103 91.7 97.4 101 87.5 Summary of analytical chemistry data for earthworm(1), Day 14: Day 0 Mean Soil Concentration, mg/kg Measured Replicate Tissue Values, mg/kg (wet weight) Mean tissue Concentration, mg/kg (wet weight) Negative Control All < LOQ (50.0) <LOQ (50.0) 77.0 189, 201 195 141 2 0 9 ,1 9 6 203 289 240, 263 252 488 1042 1105 No surviving worms 1105(a) _ hours to purge gut contents. The entire mass of worms collected from each treatment group was composited, euthanized by freezing and stored frozen until analyzed. (2) Results may be unreliable as limited number of worms were available for analysis 000200 Biological observations after 96-hours: Earthworms in the negative control, and the 77.0 mg/kg treatm ents appeared healthy and normal throughout the test with no mortality or overt clinical signs of toxicity. On Day 7 there was 90% and 100% mortality in the 488 mg/kg group and the 1042 mg/kg group, respectively. On Day 14, the 1 4 1 ,2 8 9 , and 488 mg/kg groups exhibited 7.5, 2.5 and 95% mortality, respectively. Worms in the 488 and 1042 mg/kg groups exhibited an aversion to the soil during observations of burrowing behavior on Day 0. On Day 0 after one hour, some worms in the 488 mg/kg group were not burrowed, and in the 1042 mg/kg group many remained on the soil surface. After two hours, all but one worm in the 488 mg/kg group had burrowed but in the 1042 group most worms remained out of the soil. On Day 7, all surviving worms burrowed into the soil within 15 minjutes. On Day 7, worms found dead in the 1042 mg/kg group were on the side of the test chamber, not in the soil. Cumulative percent mortality: Day 0 Mean Soil Concentration, mg/kg Neg. Control 77.0 141 289 488 1042 Day 7 0 0 0 0 90 100 Control response: Satisfactory Day 14 0 0 7.5 2.5 95 100 Average Earthworm Body VVeights: Day 0 Mean Soil Concentration, mg/kg Neg. Control Day 0 weight, g 0 .2 9 ,0 .2 3 , 0.23, 0.26 Day 14 Weight, g 0 .2 1 ,0 .1 9 , 0.18, 0.20 Mean Total Weight Change, g -0.06 77.0 0.25, 0.25, 0.26, 0.25 0 .2 0 ,0 .1 9 ,0 .2 0 , 0.18 -0.06 141 0.26, 0 .2 7 ,0 .2 3 , 0.26 0 .2 1 ,0 .1 7 , 0.14, 0.20 -0 .0 7 289 0.27, 0 .2 5 ,0 .2 3 .0 .2 5 0 .1 7 ,0 .1 7 , 0.16, 0.18 -0.08 488 0.24, 0.25,0.23, 0.24 0 .0 8 * -0 .1 7 * 1042 0.25, 0.25, 0.24, 0.25 No surviving worms ` Statistically different (p < 0 .0 5 ) from the control group using D unnett's test. 000201 CO NCLUSIO NS The perfluorooctanesulfonate, potassium salt 14-day LCsofor the Earthworm, Eisenia fetida, was determined to be 373 mg/kg with a 95% confidence interval of 316 - 440 mg/kg. The 14-day no observed effect concentration was 77 mg/kg. Subm itter: 3M Corporation, Environmental Laboratory, P.O . Box 33331, St. Paul, Minnesota, 55133 DATA Q UALITY________________________ ___________________________ R eliability: Klimisch ranking 1. R E F E R E N C E S _____________________________________________________ This study was conducted at W ildlife International, Ltd. Easton, MD at the request of the 3M Company. O THER__________________________ Last changed: 6/17/02 000202 PFOS: AN ACUTE TOXICITY STUDY W ITH THE EARTHWORM IN AN ARTIFICIAL SOIL SUBSTRATE FINAL REPORT W ILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 Environmental Laboratory Project Number U2723 OECD Guideline 207 AUTHORS: Anne B. Sindermann John R. Porch Henry O. Krueger, Ph.D. Raymond L. Van Hoven, Ph.D. STUDY INITIATION DATE: October 17,2001 STUDY COMPLETION DATE: May 10,2002 SUBMITTED TO: 3M Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page l o f 52 000203 Wildlife International, Ltd. Project Number 454-111 2- - GOOD LABORATORY PRACTICE COM PLIANCE STATEM ENT SPONSOR: 3M TITLE: PFOS: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate W ILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 STUDY COMPLETION DATE: May 10,2002 T his study was conducted in compliance w ith Good Laboratory Practice Standards as published by the U.S. Environm ental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, ENV/MC/CHEM (98) 17, Paris, 1998; and Japan MAFF, 59 NohSan, Notification No. 3850, A gricultural Production Bureau, 10 August 1984. 000204 W ildlife International, Ltd. Project Number 454-111 -3 - QUALITY ASSURANCE STATEM ENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles o f Good Laboratory Practice, ENV/MC/CHEM (98) 17, Paris, 1998; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f all inspections and audits, and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation DATE CONDUCTED: November 2,2001 Test Substance Preparation November 19,2001 Observations - Day 7 Matrix Fortifications Body Weights - Day 14 Analytical Data, Draft Report Raw Data, Draft Report Final Report November 27,2001 November 27,2001 December 4,2001 January 11,2002 January 10 and 11,2002 May 6,2002 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: November 2,2001 November 5,2001 November 19,2001 November 20,2001 November 27,2001 November 28,2001 November 27,2001 November 28,2001 December 4,2001 December 5.2001 January 14,2002 January 11,2002 January 14, 2002 January 16, 2002 May 6,2002 May 10,2002 Jduties H. Coleman Quality Assurance Representative Date 000205 dlife International, Ltd. Project Number 454-111 SPONSOR: 3M -4 REPORT APPROVAL TITLE: PFOS: An Acute Toxicity Study with die Earthworm in an Artificial Soil Substrate W ILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 STUDY DIRECTOR- W ILDLIFE INTERNATIONAL. LTD. MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants 000206 W ildlife International, Ltd. Project Number 454-111 -5 - TABLE OF CONTENTS Title Page...................................................................................... 1 Good Laboratory Practice Compliance Statement.............................................. 2 Quality Assurance Statem ent.............................................................................................................. 3 Report Approval.................................................................................... 4 Table o f C ontents................................................................................................................... 5 Sum m ary..................................................................................................................... 7 Introduction....................................... 8 O bjective.......................................... 8 Experimental Design............................................................................................................................ 8 M aterials and Methods........................................................................................................................ 9 Test Substance................................................................... 9 Test Organism................................ 9 Artificial Soil and Test Soils Preparation............................................................................... 9 Analytical Sampling............................................................................................................... 10 Analytical T e stin g ............................... 11 Test Cham bers................................................................. 11 Physical Properties o f Test Soils............................................................................................11 Environmental Conditions............................................................. ........................................ 11 Observations........................................................................................................................... 12 Body W eights.......................................................................................................................... 12 Reference Toxicity T e st.........................................................................................................12 Statistical Analyses ............................................................................................................... 12 Results and D iscussion........................................................ 13 Environmental Conditions...................................................................................................... 13 Physical Properties o f Test Soils...................... 13 Observations........................................................................................................................... 13 Statistical A nalyses....................................... 14 Reference Toxicity T est.............................................................................................. 14 Sample Analysis............................................................................................... 15 Conclusions.......................... 15 000207 W ildlife International, Ltd. 6- TABLE OF CONTENTS - Continued - Project Number 454-111 References........................................................................................................................................... 16 Table 1. TABLES Moisture, pHw, and Temperature o f Test S oils..............................................17 Table 2. Cumulative M ortality and Observations o f Earthworms Exposed to PFOS in an Artificial Soil Substrate................................................................ 18 Table 3. Average Body W eights o f Earthworms Exposed to PFOS in an Artificial Soil Substrate.......................................................................... 19 Table 4. Day 14 Measured Values for PFOS in Earthworm Tissue .......................... 20 A ppendix 1. A ppendix 2. A ppendix 3, Appendix 4. A P P E N D IC E S Personnel Involved in the Study ......................................................................21 Changes to the Study Protocol ........................................................................ 22 Artificial Soil and Test Soils Preparation ......................................................23 Analytical C hem istry......................... 25 000268 W ildlife International, Ltd. Project Number 454-111 -7 SUMMARY SPONSOR: 3M Corporation TITLE : PFOS: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 TEST DATES: Study Initiation: October 17,2001 Experimental Start (OECD): November 2,2001 Experimental Start (EPA): November 20,2001 Experimental Termination: December 4,2001 Study Completion: M ay 10,2002 TEST ORGANISM: Earthworm (Eiseniafetida) AGE OF TEST ORGANISM : Adult (with clitellum) SOURCE OF TEST ORGANISM : Worm Man's Worm Farm R & K Trading Co. 116 Pergola Ave. Monroe Township, New Jersey 08831 MEAN MEASURED TEST CONCENTRATIONS: Negative Control 77.0 nag a .i / kg 141 mg a.i./ kg 289 mg a.i./ kg 488 mg a.i./ kg 1042 mg a.i./k g RESULTS: 7-Day LC50: 398 mg a.i./ kg 7-Day NOEC: 289 mg a.i./ kg 7-Day LOEC: 488 mg a.i./ kg 14-Day LC50: 373 mg a.i./ kg 14-Day NOEC: 77 mg a.i./k g 14-Day LOEC: 141m ga.i./kg 000209 W ildlife International, Ltd. Project Number 454-111 - 8- IN T R O D U C T IO N Wildlife International, Ltd. conducted this study for 3M at the W ildlife International, Ltd. toxicology facility in Easton, Maryland. The in-life portion o f this test was conducted from November 19, 2001 to December 4, 2001. Raw data generated a t W ildlife International, Ltd. and a copy o f the final report are filed under Project Number 454-111 in archives located on the W ildlife International, Ltd. site. Key personnel involved in the conduct or management o fthe study are listed in Appendix 1. O B JE C T IV E The objective o f this study was to determine the acute effects o f Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) on earthworms during a 14-day exposure period in an artificial soil substrate. EXPERIM ENTAL DESIGN Earthworms (Eisenia fetida) were exposed to a geometric series o f five concentrations o f PFOS in soil. A control group was maintained concurrently in soil without the addition o f PFOS. This route o f administration was selected because it was representative o f the natural exposure o f earthworms to chemicals. Four replicate test chambers were maintained in each treatm ent and control group, with 10 worms in each test chamber. Observations o f m ortality and clinical signs were conducted twice within the first four hours o f test initiation, and then on Day 7 and Day 14. The m ortality results were analyzed with the moving average method to determine an LC50 value. A t Wildlife International, Ltd., reference toxicity tests with a reference toxicant, chloroacetamide, are conducted periodically to assess fee sensitivity o f fee test species and test procedures. These studies are conducted under separate protocols, as independent studies. A summary o f fee results from fee most current reference toxicity test is presented in this report. 000210 W ildlife InternationalLtd. -9 - Project Number 454-111 M ATERIALS AND M ETHODS The study was conducted based upon the procedures outlined in the protocol, "PFOS: An Acute Toxicity Study with the Earthworm in an A rtificial Soil Substrate." The protocol was based upon procedures outlined in the Organization for Economic Cooperation and Development (OECD) Guideline No. 207, Guideline fo r Testing o f Chemicals, Earthworm, Acute Toxicity Tests (1). Changes to the protocol are listed in Appendix 2. T est Substance The test substance used was received from 3M on October 29, 1998 and assigned W ildlife International, Ltd. identification number 4675. The test substance was a white powder identified by the Sponsor as FC-95, lot 217. Information from the Sponsor indicated the purity o f the test substance to be 86.9% with an expiration date o f August 31,2006. The test substance was stored under ambient conditions. Test O rganism Adult earthworms (Eisenia ftida ) were obtained from Worm Man's W orm Farm , Monroe Township, New Jersey. Approximately 18.5 hours prior to the test, 280 worms were selected and placed in a container o f prepared artificial soil substrate adjusted to moisture content o f approximately 33% by weight, for the acclimation period. On the day o f test initiation, the worms were rinsed briefly with deionized water and indiscriminately distributed by pairs into groups o f 10 worms each. Each group o f worms was weighed tren placed on the soil surface in the appropriate test chamber. The worms were not fed during testing. A rtificial Soil and Test Soils P reparation The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay and 10% sphagnum peat (Appendix 3). The pH o f the bulk soil prior to hydration was adjusted to 6.0 using calcium carbonate. The bulk artificial soil was stored in a sealed container under ambient conditions until used to prepare the test sods. 000211 W ildlife International, Ltd. Project Number 454-111 -10- Test soils were prepared by stirring the test substance into a portion o f bulk soil before mixing it with the remainder o f the soil and deionized water (Appendix 3). Sufficient deionized w ater was added to the dry artificial soil to achieve a moisture content o f approximately 33% by weight. Test soil components were mixed for a total o f 20 minutes to ensure a homogeneous mix. Negative control soil was prepared in the same manner as test soil, but without the addition o f test substance. Seven hundred fifty grams o f prepared soil were added to each o f four test chambers for the control group, with the exception o f the 625 mg a.i./ kg concentration in which no more than 650 g would fit into the test chambers. Test concentrations were adjusted for the purity o f PFOS, which was reported to be 86.9% by the Sponsor. Therefore, test concentrations and the LC50 values are reported as milligrams o f test substance active ingredient per kilogram o f soil on a dry weight basis (mg a.i./kg). Test soils were prepared and held overnight at approximately 20 2C prior to adding worms to allow the soil to become less fluid. The target nominal concentrations were 78.1, 156, 313, 625, and 1250 mg a.iVkg dry soil. M ean measured concentrations at the time o f soil preparation were 77.0, 141, 289, 488, and 1042 mg a.i./kg soil. A nalytical Sam pling During the test, samples o f the experimental soils were collected to determine the homogeneity and stability o f the test substance in the artificial soil and to verify test concentrations. Samples were collected a t soil preparation to determine homogeneity, measure test concentrations, and establish Day 0 values for evaluating stability over the course of the exposure period. On Day 14, samples were collected from one replicate o f the control and two replicates o f each test concentration to assess stability o f the test substance under test conditions. A t test termination samples o f tissue from the earthworms in each treatm ent group were analyzed to determine the concentration o f PFOS in the worm tissue from each treatm ent group. All surviving worms from each treatment group were placed on moistened filter paper for approximately 24 hours to purge gut contents. The entire mass o f worms collected from each treatm ent group was composited, euthanized by freezing and stored frozen until analyzed. 000212 W ildlife International, Ltd. - 11- Project Number 454-111 A nalytical Testing Chemical analysis o f the soil and worm samples was performed using HPLC/MS/MS methodology (Appendix 4). Test Cham bers The test chambers were one-liter Nalgene beakers covered with plastic wrap which was perforated for air exchange. A11 test chambers were identified with the project number, test concentration and replicate. Physical Properties of T est Soils Soil temperature was measured in one replicate o f each treatm ent and control group a t test initiation and termination using a hand-held thermometer. M oisture content and pH measurements were made on soil samples collected from each batch o f soil prepared for treatm ent and control groups a t test initiation. An additional soil moisture content measurement was made after test initiation to verify the soil moisture. A t test termination, samples for moisture content and pH measurements were collected from one replicate o f each control and treatm ent group. Measurements o f pH were made on suspensions o f soil and deionized water using a waterproof hand-held pH meter, and are reported as pH* values. Soil moisture content was determined by measuring the initial weight o f the soil sample, then weighing the soil sample after drying for at least 24 hours at approximately 105C. The percent moisture was calculated using the following formula: % M oisture = [(wet weight - dry weight) wet weight] * 100 Environm ental Conditions During the test, the worms were maintained in an environmental chamber set to maintain a temperature o f approximately 20 2C. Air temperature was measured a t least once daily in the environmental chamber. The photoperiod during the test was 24 hours o f continuous light per day provided by overhead fluorescent bulbs. The target light intensify during the test was approximately 400 to 800 lux, and was verified on Day 7 o f the test. 000213 W ildlife International, Ltd. Project Number 454*111 -12- O bservations A t test initiation, the worms were placed on the surface o f the soil in each test chamber and were observed for burrowing behavior. On Day 7 and Day 14 o f the test, the contents o f each test chamber were removed to determine the number o f surviving worms. All surviving worms were observed for behavioral or pathological abnormalities. On Day 7, following observations, test soil was returned to the test chambers and the worms were placed on the soil surface in order to observe burrowing behavior. On Day 14, following observations and body weight determination, surviving worms were placed on m oist filter paper in dean test chambers and allowed to purge their gut contents for approximately 24 hours. The worms were then euthanized by freezing and held prior to analytical testing. Body W eights On Day 0, group weights for all replicates were collected prior to being placed in the test chambers. On Day 14, all surviving worms were removed from each replicate test chamber and weighed. Prior to weighing, worms were rinsed with deionized water and blotted dry on paper towels. Group body weights were measured from each test chamber, and average individual body weights were calculated. Reference Toxicity Test A reference toxicity test was conducted under a separate protocol to determine the LC50 value for earthworms exposed to the reference toxicant, chloroacetamide, in the soil (2). The test was conducted under conditions sim ilar to those used in this test, and with earthworms from the same source, to monitor the techniques used and sensitivity o f the test population. The worms were exposed to chloroacetamide in fire soil at nominal concentrations o f 7 .5 ,1 5 ,3 0 and 60 mg a.i./kg dry soil. S tatistical Analyses The LC50 value and 95% confidence interval were calculated using the computer program o f C.E. Stephan (3). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (4,5,6). In this study, the 7-day LC50 value was calculated using the binomial probability method and file 14-Day 000214 Wildlife InternationalLtd. Project Number 454-111 -13 - LC50 value was calculated using the moving average method. Mean body weights at test initiation and test termination as well as the mean change in body weight during the test were determined for each treatm ent and control group. Treatment group means were compared to control means with Dunnett's test (a=0.05) using SAS Version 8 (7) in order to determine if significant differences from the control occurred. RESULTS AND DISCUSSION Environm ental Conditions A ir tem perature in the environmental chamber was within the desired range o f approxim ately 20 2C, remaining at 22-23C throughout the test. The worms were maintained under continuous lighting a t an average intensity o f 627 83.9 lux, with a range over the surface o f the test chambers o f 494-775 lux. Physical P roperties o f T est Soils Measurements o f soil pHw, temperature and moisture content at test initiation and test termination are presented in Table 1. Soil pHwranged from 6.9 to 8.1 at test initiation and from 8.3 to 8.4 a t test termination. Soil temperature was 22C, within the desired range o f 20 2C a t test initiation and a t test termination. A ttest termination the 1042 mg a.i./kg Replicate A soil tem perature was 25C. It was suspected that the Replicate A chamber was placed too close to a light ballast and w as not indicative o f the soil temperature for that treatment group. R eplicate B was m easured and found to be 22C. Soil moisture content during the test ranged from 29.5 to 33.9% a t test initiation and from 31.4 to 34.5% at test termination, indicating that there was little change in soil moisture content during the test. O bservations The data from weekly observations o f the worms for mortality and other signs o f toxicity are presented in Table 2. There were no mortalities in the negative control group during tie 14-day te s t All control worms were normal in appearance and behavior throughout the test period. On Day 7 there was high m ortality in the 488 and 1042 mg a.i./kg treatm ent groups. On Day 14 there was some W ildlife International, Ltd. Project Number 454-111 -14- mortality in the 141 and 289 mg a.i./kg treatment groups and very high mortality in the 488 mg a.i./kg group and complete mortality in the 1042 mg a.i./kg group. Worms in the 488 and 1042 mg a.i./kg treatment groups exhibited an aversion to the soil during observations o f burrowing behavior on Day 0. On day 0 after one hour, some worms in the 488 mg a.i./kg group were not burrowed, and in the 1042 mg a.i./kg group many remained on the soil surface. After two hours, all but one worm in the 488 mg a.i./kg group had burrowed but in the 1042 mg a.i./kg group most worms remained out o f the soil. On Day 7, all surviving worms burrowed into the soil within 15 minutes. On Day 7, worms found dead in the 1042 mg a.i./kg group were on the side o f the test chamber not in the soil. Statistical Analyses A 7-Day LC50 estimation o f 398 mg a.i./kg with 95% confidence limits o f 289 and 488 mg a.i./kg soil was determined using the binomial probability method, since the data did not fit either the probit method or moving average method. A 14-Day LC50 was determined using the moving average method since the data did not fit the probit method. The moving average method was acceptable except for the indication that the confidence limits may be too close due to the lower mortality in the 289 mg a.i./kg soil than in the 141 mg a.i./kg soil treatm ent group. Therefore, the 14-day LC50 estimation for earthworms exposed to PFOS in an artificial soil substrate was determined to be 373 mg a.i./kg soil with 95% confidence limits o f 316 and 440 mg a.i./kg. After seven days o f exposure the no-observed-effect-concentration, NOEC, was 289 mg a.i./kg soil and the lowest-observed-effect-concentration, LOEC, was 488 mg a.i./kg soil. The 14-Day NOEC was 77 mg a.i./kg soil and the LOEC was 141 mg a.i./kg soil. Average individual body weights, and the change in body weight from test initiation to test termination, were calculated from the Day 0 and Day 14 replicate measurements (Table 3). A slight loss in body weight from test initiation to test termination was expected in both the control and treatment groups since the worms were not fed during the test. Mean body weight at test termination 000216 W ildlife International, Ltd. Project Number 454-111 -15- as well as the mean loss in body weight among worms in the treatment groups were statistically significant (p<0.05) at 488 mg a.i./kg dry soil compared to the control group. Table 4 contains the 14 day measured values for PFOS in earthworm tissue. Reference Toxicity Test The 14-day LC50 value for earthworms exposed to the reference substance, chloroacetamide, in an artificial soil substrate was approximately 19.4 mg a.i./kg dry soil with a 95% confidence interval o f 15 and 30 mg a.i./kg dry soil (2). These results are consistent with those observed in previous studies, and verify the adequacy and consistency of the methods used in this study (454-111). Sam ple Analysis Chemical analysis o f test soils and earthworm tissue was conducted at W ildlife International, Ltd. and is reported in Appendix 4. C O N C L U S IO N S The 7-day LC50 estimation for earthworms exposed to PFOS in an artificial soil substrate was determined to be 398 mg a.i./kg. The 7-day NOEC was determined to be 289 mg a.i./kg and the 7-day LOEC was determined to be 488 mg a.i./kg. The 14-day LC50 estimation for earthworms exposed to PFOS in an artificial soil substrate was determined to be 373 mg a.i./kg. The 14-day NOEC was determined to be 77 mg a.i./kg and the LOEC was 141 mg a.i./kg. 000217 :e International, L td, -16REFERENCES Project Number 454-111 1 OECD Guideline 207. 1984. Guideline fo r Testing o f Chemicals, Earthworm, Acute Toxicity Tests. Organization for Economic Cooperation and Development. 2 W ildlife International, L td. 2000. Chloroacetamide: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate. Project No. 100R-105. 3 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal Communication. 4 Finney, D.J. 1971. Statistical M ethods in Biological Assay. Second Edition, Griffin Press, London. 5 Thom pson, W .R. 1947. Bacteriological Reviews. Vol. H, 2:115-145. 6 Stephan, C .E. 1977. Methods for Calculating an LC50. Aquatic Toxicology and H azard Evaluations. American Society for Testing and M aterials. Pub. No. STP 634:65-84. 7 SAS Institute, Inc. 1999. SAS/STAT User's Guide, Version 8. Cary, NC, SAS Institute, Iric. 000218 W ildlife International, Ltd. Project Number 454-111 -17Table 1 Moisture, pHw, and Temperature o f Test Soils1 Mean Measured Concentration (mg a.i./kg) Negative Control Test Initiation M oisture Content2 (%) _ 33.9 Test Initiation pHw3 Test Initiation T em perature3 (C) Test Termination Moisture Content (%) Test T erm ination pHw Test T erm ination T em perature (C) 6.9 22 32.1 8.3 22 77.0 31.9 7.9 22 31.4 8.4 22 141 29.5 8.1 22 31.5 8.3 22 289 31.4 8.1 22 33.0 8.3 22 488 31.0 8.1 22 32.3 8.3 22 1042 32.5 8.0 22 34.5 'Measurements taken from one replicate test chamber at each concentration. Measurements taken from prepared soil two days alter preparation. Measurements taken from prepared soil on Day 0. Measurement of soil temperature in Replicate B was 22C. 8.3 254 000219 W ildlife International, Ltd. Project Number 454-111 -18- Table 2 Cumulative M ortality and Observations o f Earthworms Exposed to PFOS in an Artificial Soil Substrate M ean Day 7 M easured Concentration Number ( r o g a i /kg) Rep. Dead* Effects12 Negative Control A 0/10 B 0/10 C 0/10 D OflO 10 AN 10 AN 10 AN 10 AN Day 14 Number Dead Effects 0/10 10 AN 0/10 10 AN 0/10 10 AN 0/10 10 AN Replicate " Percent Mortality 0 0 0 0 Group Percent M ortality 0 77.0 A 0/10 B 0/10 10 AN 10 AN C 0/10 10 AN D 0/10 10 AN 0/10 10 AN 0/10 10 AN 0/10 10 AN 0/10 10 AN 0 0 0 0 141 A 0/10 B 0/10 10 AN 10 AN 2/10 8 AN 0/10 10 AN 20 0 C 0/10 9 AN, 1T+S 1/10 9 AN 10 D 0/10 10 AN 0/10 10 AN 0 0 7.5 289 A 0/10 B 0/10 10 AN 10 AN 0/10 10 AN 0/10 10 AN 0 0 2.5 C 0/10 10 AN 0/10 8 AN, 2 R 0 D 0/10 10 AN 1/10 9 AN 10 488 A 9/10 B 8/10 IA N 2 AN 10/10 . ._ 100 95 8/10 2 T + R 80 C 10/10 __ 10/10 -- 100 D 9/10 IA N 10/10 --- 100 1042 A 10/10 B 10/10 C 10/10 D 10/10 _ _ -- 10/10 10/10 10/10 10/10 _ -- -- -- 1Q0 100 100 100 100 1 Mortality date are presented as the cumulative number dead per number exposed. 2 Observed Effects: AN = normal in appearance and behavior, T=thin, S=soft, R=reduced reaction to mechanical stimuli 000220 W ildlife International, Ltd. Project Number 454-111 -19- T able3 Average Body Weights o f Earthworms Exposed to PFOS in an Artificial Soil Substrate Mean Measured Concentration (mg a.i./kg) Negative Control Replicate A B C D Mean Std. Dev. Average Earthworm Body Weights (g) Day 0 0.29 0.23 0.23 0.26 0.25 0.029 Day 14 0.21 0.19 0.18 0.20 0.20 0.013 Total Change -0.08 -0.04 -0.05 -0.06 -0.06 0.017 77.0 A 0.25 0.20 -0.05 B 0.25 0.19 -0.06 C 0.26 0.20 -0.06 D 0.25 0.18 -0.07 Mean Std. Dev. 0.25 0.005 0.19 0.010 -0.06 .0.008 141 A 0.26 0.21 -0.05 B 0.27 0.17 -0.10 C 0.23 0.14 -0.09 D 0.26 0.20 -0.06 Mean Std. Dev. 0.26 0.017 0.18 0.031 -0.07 .0.024 289 A 0.27 0.17 -0.10 B 0.25 0.17 -0.08 C 0.23 0.16 -0.07 D 0.25 0.18 0.07 Mean Std. Dev. 0.25 0.016 0.17 0.007 -0.08 0.014 488 A 0.24 B 0.25 0.08 -0.17 C 0.23 -- -- D 0.24 -- -- Mean Std. Dev. 0.24 0.008 0.08 1 -0.17 --* 1042 A 0.25 B 0.25 -- C 0.24 -- D 0.25 -- Mean Std. Dev. 0.25 0.005 --' -- _ -- -- -- -- 1Body weight Was statistically different from (p<0.05) the control groups using Dunnett's test. 2Change in body weight was statistically significant (p<0.05) at test concentration when compared to the control groups using Dunnett's test 000221 W ildlife International, Ltd. Project Number 454-111 - 20- Table 4 Day 14 Measured Values for PFOS in Earthworm Tissue Number (454-111-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) W -l 0.0 < LOQ2 - W -2A W -2B 78.1 78.1 189 195 201 W -3A W-3B 156 156 209 203 196 W -4A W-4B 313 313 240 252 263 W-5 625 1105 - 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. *The method limit ofquantitation (LOQ) in earthworm tissue was 50.0 mg ai./kg on a wet-weight basis and was calculated as the product ofthe lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor oflhe matrix blank sample (100,000 L/kg). 000222 W ildlife International, Ltd. Project Number 454-111 - 21- Appendix 1 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management o fthis study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. John R. Porch, Supervisor, Non-Target Plants and Insects 3. Anne B. Sindermann, Senior Biologist 4. Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry 000223 W ildlife International, Ltd. Project Number 454-111 -22Appendix 2 Changes to the Study Protocol This study was conducted according to the approved study protocol with the following changes: 1. Test soils were prepared, and analytical samples collected, one day prior to test initiation. 2. An additional soil moisture measurement was made. 3. Soil moisture for the 156 mg a.i./kg level was 29.5%, slightly below approximately 33%. 4. Test soil for the 625 mg a.i./kg level weighed 650 g rather than 750 g. 5. Earthworms used for foe study weighed less than between 300 and 600 mg. 6. Earthworms were conditioned for approximately 18.5 hours rather than 24. 7. The soil temperature in Replicate A o f the 1250 mg a.i./kg treatment group was 25C at test termination, receding foe 202C range specified in the protocol. 000224 W ildlife International, Ltd. Project Number 454-111 -23- Appendix 3 Artificial Soil and Test Soils Preparation A rtificial Soil Preparation H ie artificial soil was prepared in bulk by mixing the following constituents in a soil mixer for approximately 20 minutes: quartz sand 31.500 Isg kaolin clay 9.000kg sphagnum peat 4.500 kg calcium carbonate 1.125 kg T est Soil P reparation A total o f approximately 4 kg (wet weight) o f test soil with a calculated soil moisture content o f approximately 33% was prepared for the negative control and each of the treatm ent groups. Test substance concentrations were based on the dry weight portion o f the soil. The calculated dry weight o f 41 o f prepared soil, with a moisture content o f 33%, was 2.680 kg. To account for the 4% moisture content typically present in the bulk soil stored in ambient conditions, 1208 mL o f water were added to 2680 g. o f bulk soil minus the weight o f test substance. Amounts o f test substance, bulk soil and water were as follows: Concentration ime a.L/kg test soil) Negative control 78.1 156 313 625 1250 Nominal Weight* and Volumes to Prepare Test Soils Required Amount o f Actual Amount of Test Substance fg) Test Substance (g) ---- 0.241 0.2410 Bulk Soil (g) 2680.0 2679.8 0.482 0.4819 2679.5 0.965 0.9650 2679.0 1.927 1.9270 2678.1 3.854 3.8541 2676.2 W ater (mL) 1208 1208 1208 1208 1208 1208 The appropriate amount o f bulk soil was weighed into a tared Hobart mixer bowl. Approximately a handful o f dry soil was removed to a Nalgene beaker and the measured amount o f test substance added and mixed in with a metal spatula, after using dry soil to clean test substance off 000225 Wildlife InternationalLtd. Project Number 454-111 -24- o f the weigh boat. The soil and test substance mixture was added to the Hobart bowl. 1208 mL o f deionized water was added and the soil mixed for 5 minutes. After the 5-mmute mix, the sides o f the bowl were scraped and the soil was mixed for an additional 15 minutes. Prepared test soil, 750 g for each replicate, except for the 625 mg a.i./kg concentration, which received 650 g, was placed into test chambers labeled with concentration and replicate. Negative control soil was prepared in the same manner, without the addition of test substance, and 750 g was placed into each negative control test chamber. 000226 W ildlife International, Ltd. Project Number 454-111 -25Appendix 4 THE ANALYSIS OF PFOS IN ARTIFICIAL SOIL AND EARTHWORM TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECTNO.: 454-111 000227 W ildlife International, Ltd. Project Number 454-111 SPONSOR: 3M -26REPORT APPROVAL TITLE: PFOS: AN ACUTE TOXICITY STUDY W ITH THE EARTHWORM IN AN ARTIFICIAL SOIL SUBSTRATE W ILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-111 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 PRINCIPAL INVESTIGATOR: MANAGEMENT: W illard B. Nixon, Ph. Director, Analytical Chemistry 5-I -00~ DATE 000228 W ildlife International, Ltd. Project Number 454-111 -27- Introduction This study was conducted by Wildlife International, Ltd. for 3M at the Wildlife Internationa!, Ltd. analytical chemistry facility in Easton, Maryland. Samples were collected for the determination o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in artificial soil and earthworm tissue and were analyzed using high performance liquid chromatography with triple quadrupole mass spectrometric detection (HPLC/MS/MS). Samples were prepared and analyzed between November 27 and 28,2001 and December 13, 2001. Raw data generated by Wildlife International, Ltd. and a copy o f the final report are filed under Project Number 454-111 in archives located on the 'M idlife International, Ltd. site. A nalytical M ethodology A rtificial Soil Submitted artificial soil samples were analyzed for PFOS according to the validated procedures documented in WTL study number 454C-120. The entire submitted soil sample was blended for approximately two minutes prior to sub-sampling the requisite 10-g aliquot for the analytical determination. The artificial soil sub-samples were extracted with methanol, agitated for a minimum o f 30 minutes on a gyratory shaker table a t approximately 250 rpm and then vacuum filtered with qualitative filter paper. The filtrate was then transferred to a 200-mL volumetric flask and brought to volume with methanol. Approximately 20 m illiliters o f each sample was transferred to a separate vial and centrifuged for approximately 5 minutes a t 2000 rpm. Dilutions into the calibration range o f 1he HPLC/MS/MS methodology were performed with a solution o f 50% methanol (HPLC grade, 99.9+%) . and 50% NANOpure water. Samples were then analyzed by direct injection. Concentrations o f PFOS were determined by reverse-phase high performance liquid chromatography using a HewlettPackard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-EImer API 3000LC M ass Spectrometer equipped with a Peridn-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone Betasil C column (50 mm x 2.0 mm, 3pm particle size) fitted with a Keystone Javelin C]8 guard column (20 mm x 2.0 mm). A method flowchart is provided in Figure 1 and the instrument parameters are summarized in Table 1. 000229 Wildlife International, Ltd. Project Number 454-111 E arthw orm Tissue -28- Submitted earthworm tissue samples were analyzed for PFOS according to methodology developed a t W ildlife International, Ltd. Approximately one gram o f each earthworm tissue sample was weighed directly into tared scintillation vials to which 10.0 milliliters o f methanol extraction solvent were added. The samples were concurrently extracted and homogenized using a tissue shredder (Janice and Kunkel) followed by sonic dismembration. The sample extracts were centrifuged and diluted into the calibration range o f the HPLC/MS/MS methodology. Dilutions were performed w ith a solution o f 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection HPLC/MS/MS as described previously. A method flowchart is provided in Figure 2. P rim ary and Secondary Stock Solutions All prim ary and secondary stock preparations were adjusted for the purity o f the test substance (86.9% ). A 10.0 mg a.i./m L primary stock solution o f PFOS in methanol was prepared by weighing 1.1508 g o f 1he test substance and bringing to a final volume o f 100 m illiliters with methanol. Secondary stock solutions (1000,100,10.0,1.00, and 0.100 mg a.i./L) o f PFOS in methanol were prepared by serial volumetric dilution from the prim ary stock. C alibration Standards and C alibration Curves Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions o f the 1.00 mg a.i./L stock solutions o f PFOS in methanol. Calibration standards o f PFOS, ranging in concentration from 1.00 to 10.0 pg a.i./L, were analyzed with each artificial soil sample set and calibration standards o f PFOS, ranging in concentration from 0.500 to 5.00 pg a.i./L, were analyzed with the earthworm tissue sample se t The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration and study samples. No attem pt was made to quantify PFOS on the basis o f individual isomeric components. The calibration standard series was injected at the beginning and end o f each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using peak area responses versus the respective concentrations o fthe calibration standards. A typical calibration curve for PFOS in artificial soil and earthworm tissue is presented in Figure 3 and Figure 4, respectively. Representative ion 000230 W ildlife International, Ltd. Project Number 454-111 -29- chromatograms o f low and high calibration standards for the artificial soil are presented in Figures 5 and 6, respectively and representative ion chromatograms o f low and high calibration standards for PFOS in earthworm tissue are presented in Figure 7 and 8, respectively. The concentrations o f PFOS in the samples were determined by substituting peak area responses into the applicable linear regression equation. Concentrations o f PFOS in the samples were calculated on a dry weight (soil samples) or wet weight (earthworm tissue) basis using the at-instrumeat PFOS concentrations and the appropriate weight/volume dilution and/or moisture correction factors. M atrix Blanks and M atrix Fortifications A rtificial Soil Selected 20.0-g aliquots o f artificial soil were fortified with the appropriate stock solutions o f PFOS prepared in methanol using a gas-tight syringe. The fortified soils were then homogenized using a W aring blender for approximately 2 minutes. A 10-g aliquot o f each homogenized fortified soil sample was weighed into a tared weigh boat and transferred to an 8-oz. French-square bottle for extraction. The m atrix blanks were unfortified artificial soil. E arthw orm Tissue A 1.00-g aliquot o f negative control earthworm tissue was fortified with the 1000 mg a.i./L secondary stock solution o f PFO S prepared in m ethanol using a gas-tight syringe. A m atrix blanks was prepared with unfortified negative control earthworm tissue. Lim its o f Q uantitation A rtificial Soil The method limit o f quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a dry-weight basis and was calculated as the product o f the lowest calibration standard (0.00100 mg a.i./L), foe weight/volume dilution factor o f foe m atrix blank samples (20,000) divided by foe percent solids for foe test system soil (68.3%). 000231 W ildlife International, Ltd. Project Number 454-111 -30- Earthw orm Tissue The method lim it o f quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wet-weight basis and was calculated as the product o f the lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor o f the m atrix blank sample (100,000 L/kg). Exam ple Calculations Exam ple C alculations Sample number 454-111-5, nominal concentration o f 78.1 mg a.i./kg in artificial soil. First Initial Weight: 10.0 g First Final Volume: 200 xnL Second Initial Volume: 0.0500 mL Second Final Volume: 50.0 mL Dilution Factor: 20000 Mean Soil Moisture Content: 31.7% Percent Solids: 68.3% LCMS Dilution Factor: 20000/0.683 = 29283 PFOS Peak Area: 23914 Calibration curve equation: Slope: 8981195 Intercept: 1011.0532 ... . peak area - (v-interceot) PFOS (mg a.t./kg) measured a t instrument = ---------- --------------- PFOS (mg a.i./kg) in sample = PFOS measured at instrument (mg a.i./kg) x LCMS dilution factor 23914 - 1011.0532 8981195 x 29283 = 74.67 . ._ PFOS (mg a.i./K s) in sample __ Percent o f Nominal Concentration = p p o s ^ l/K g ) x 100 = 74.67x100 = 95.6% 78.1 Calculated with HPLC/MS/MS instrument software: MacQuan, version 1.6. 000232 Wildlife International, Ltd. Project Number 454-111 -31- RESULTS M atrix blank samples for each m atrix type were analyzed to determine possible interferences. No interferences were observed a t or above the corresponding LOQ {Tables 2 and 3). Representative ion chromatograms o f artificial soil and earthworm tissue m atrix blank samples are presented in Figures 9 and 10, respectively. M atrix fortification samples for artificial soil had recoveries ranging from 75.6% to 103.2% o f the nominal concentrations with a mean recovery o f 87.3% o f nominal (SD=11.2; CV=12.8). A representative ion chromatogram o f an artificial soil m atrix fortification is presented in Figure 11. The earthworm tissue m atrix fortification (Figure 12) had a recovery o f 86.6% o f the nominal concentration. A rtificial soil samples collected a t Day 0 from the 78.1 mg a.i./kg, 156 mg a.i./kg, 313 mg a.i./kg, 625 mg a.i./kg and 1250 mg a.i./kg treatm ent levels had mean measured PFOS concentrations o f 77.0 mg a.i./kg (SD=13.6; CV=17.7%, n=6), 141 mg a.i./kg (n-2), 289 mg a.i./kg (n=2), 488 mg a.i./kg (n=2), and 1042 mg a.i./kg (SD=201; CV=19.3%, n=6), respectively (Table 4). These values corresponded to mean percent recoveries o f 98.6%, 90.4%, 92.3%, 78.1% and 83.4% o f the nominal concentrations with an overall mean percent recovery o f 89.6% (SD=15.3; CV=17,1%, n -18). A representative ion chromatogram o f an artificial soil test sample is presented in Figure 13. Artificial soil samples collected a t Day 14 from the 78.1 mg a.i./kg, 156 mg a.i./kg, 313 mg a.iVkg, 625 mg a.i./kg and 1250 mg a.iA g treatm ent levels had mean measured PFOS concentrations o f 80.6 mg a.i./kg (n=2), 143 mg a.i./kg (n~2), 305 mg a.i./kg (n=2), 634 mg a.i./kg (n=2) and 1094 mg a x /k g (n=2), respectively (Table 5). These values corresponded to mean percent recoveries o f 103%, 91.7%, 97.4%, 101% and 87.5% o f the nominal concentrations with an overall mean percent recovery o f 96.3% (SD=10.7; CV=11.2%, n=10). Earthworm tissue analyzed on Day 14 had mean measured concentrations o f 195, 203 and 252 mg a.i./kg for nominal exposure levels o f 78.1, 156, and 313 mg a.i./kg, respectively (Table 6). The measured value (1105 mg a.i./kg) for the 625 mg a.i./kg exposure level was suspect due to the limited sample size (3 worms, approximately 0.2 g total weight) available from this treatm ent group. A representative ion chromatogram o f an earthworm tissue test sample is presented in Figure 14. OOO2 3 3 W ildlife International, Ltd. Project Number 454-111 -32REFERENCES 1. A m erican Society for Testing and M aterials. 1991. Standard Specification for Reagent W ater. D l 193-91, ASTM Section Ii W ater and Environmental Technology, Vol. 11.01:45-47. 000234 W ildlife International, Ltd. Project Number 454-111 IN STR U M EN T: -3 3 - T able 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 M ass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone Betasil Cig (50 mm x 2.0 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin C!S cartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 5.00 min FLO W RATE: M O B ILE PHASE: 250 fiL/min 30% NANOpure W ater w ith 0 .1% Form ic A cid: 70% M ethanol INJECTION VOLUME: 5.0 pL PFOS PEAK RETENTION TIM E: Approximately 4 minutes PFOS MONITORED MASS: 499.0 amu - 99.1 amu 000235 W ildlife International, Ltd. Project Number 454-111 -34Table 2 Artificial Soil M atrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number (454-111-) Sample Type M A B -1 M A B -2 Soil M atrix Blank Soil M atrix Blank PFOS Concentration ____ (mg a-i./kg)_______ Fortified Measured1 Percent Recovered1 0 . 0 < LOQ2 < LOQ2 _ -- M ean M easured (mg a.i./kg) _ M ean Percent Recovery -- M A S-1 M A S-4 Soil M atrix Fortification Soil M atrix Fortification 36.6 28.5 29.3 77.7 80.1 28.9 79.0 M A S-2 M A S -5 Soil M atrix Fortification Soil M atrix Fortification 586 565 533 96.4 90.9 549 93.7 M A S-3 M AS- 6 Soil M atrix Fortification Soil M atrix Fortification 1464 1503 1106 103 75.6 1305 89.1 O verall M ean = 87.3 Standard D eviation3 11.2 CV= 12.8 ____________ N = 6 __________ *Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a diy-weight basis and was calculated as the product ofthe lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor o f th e m atrix blan k sam ples (20,000> divided by th e percent solids for the te st system so il (68,3% ). 000236 Wildlife International, Ltd. Project Number 454-111 -35- Table 3 Earthworm Tissue M atrix Blank and Fortification Analyzed Concurrently During Sample Analysis Number (454-111-) Sample Type PFOS Concentration (m gal/kg) Fortified Measured1 Percent Recovered1 W M A B -1 W orm Tissue M atrix 0 .0 <LOQ3 B lan k - W M A S-1 W orm Tissue M atrix Fortification 100 8 6 .6 8 6 .6 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 1 The method limit of quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wetweight basis and was calculated as the product ofthe lowest calibration standard (0.000500 mg ai./L ) and the weight/volume dilution factor of the matrix blank sample (100,000 L/kg). 000237 W ildlife InternationalLtd. Project Number 454-111 -36- Table 4 Day 0 Recoveries for PFOS in Artificial Soil Sample Number (454-111-) Nominal Concentration PFOS Concentration (mg a l/k g ) M easured1 Percent Recovered1 M ean M easured (mg a.i./kg) M ean Percent Recovery 1 0 .0 < LOQ2 -- - -- 2 78.1 3 78.1 4 78.1 5 78.1 6 78.1 7 78.1 74.6 95.5 x =77.0 98.6 72.7 93.1 SD= 13.6 104 133 CV= 17.7% 74.7 95.6 69.4 88.9 66.5 85.1 8 156 9 156 144 92.1 141 90.4 138 88.4 1 0 313 1 1 313 312 99.6 289 92.3 266 85.0 1 2 625 526 84.1 488 78.1 13 625 - 4 4 9 71.9 14 1250 15 1250 16 1250 17 1250 18 1250 19 1250 1152 906 894 930 1405 966 92.1 72.4 71.5 74.4 112 77.3 x * 1042 SD= 201 CV= 19.3% 83.4 O verall M ean 89.6 Standard D eviation 15.3 CV= 17.1% N 18 ^Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 D ie method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a dry-weight basis and was calculated as the product o fthe lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor of the matrix blank samples (20.000) divided by the percent solids for the test system soil (68.3%). 000238 W ildlife International, Ltd. Project Number 454-111 -37- Table 5 Day 14 Recoveries for PFOS in Artificial Soil Number (454-111-) Sample Nominal Concentration PFOS Concentration (mg a.i./kg) M easured1 Percent Recovered* Mean M easured (mg a.i./kg) M ean Percent Recovery 2 0 0 .0 <LOQ2 -- _ - 2 1 78.1 2 2 78.1 74.9 95.9 80.6 103 8 6 . 2 110.4 23 156 24 156 143 91.4 143 91.7 142 91.1 25 313 26 313 302 96.5 305 97.4 308 98.3 27 625 28 625 532 85.1 634 101 735 118 29 1250 30 1250 1059 1129 84.7 1094 87.5 90.4 O verall M ean:= 96.3 Standard D eviation:- 10.7 CV == 1 1 .2 % N>= 10 1 Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.L/kg on a dry-weight basis and was calculated as the product ofthe lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor o f die matrix blank samples (20,000) divided by the percent solids for die test system soil (68.3%). 000239 ildlife International, Ltd. Project Number 454-111 -38- Table 6 Day 14 Measured Values for PFOS in Earthworm Tissue Number (454-111-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) M easured1 Mean M easured (m ga.i./kg) W -l 0 .0 <LOQz - W -2A W -2B 78.1 78.1 189 195 201 W -3A W -3B 156 156 209 203 196 W -4A W -4B 313 313 240 252 263 W-5 625 1105 - 1 Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The method limit of quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wet-weight basis and was calculated as the product ofthe lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor of the matrix blank sample (100,000 L/kg). 000240 W ildlife International, Ltd. -39- Project Number 454-111 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN ARTIFICIAL SOIL W eigh approxim ately 10.0 gram s o f each sam ple into a tared w eigh boat. T ransfer to 8 oz. French square bottles. 4 For each sample, m easure 100 mL o f m ethanol w ith a graduated cylinder and transfer into th e French square bottle. 4 Cap bottles and place on shaker table. Allow the sam ples to shake for a m inim um o f 30 m inutes a t approxim ately 250 ipm . 4 Vacuum filter w ith qualitative filter paper and rinse retained soil 3 tim es w ith m ethanol into filtrate. 4 T ransfer the filtrate into a 200-m L volum etric flask and bring to volum e w ith m ethanol. M ix w ell w ith several repeat inversions. 4' T ransfer approxim ately 20 mL o f each sam ple into a separate glass centrifuge tube or scintillation vial ami cap. Centrifuge sam ples for approxim ately 5 m inutes at 2000 rpm . 4 Prepare appropriate dilution(s) to bring final concentration into the calibration range o f th e LCMS methodology. For all dilutions, use 50% m ethanol: 50% NANOpure w ater dilution solvent, gas-tight syringes, and Class A volum etric glassware. 4 Am putate sam ples and subm it for LCMS analysis. F ig u re 1. A nalytical m ethod flow chart fo r the analysis o f PFOS in artificial soil. 000241 W ildlife International, Ltd. -40- Project Number 454-111 M ETHOD O U TLIN E FO R T H E ANALYSIS O F PFO S IN EARTHW ORM TISSUE W eigh approxim ately 1.00 gram s o f each sample into a tared scintillation. 4 Add 10.0 mL o f m ethanol to each sam ple w ith a glass Class A volum etric pipette. 4* Homogenize each test sample for approxim ately 1 m inute using a hand-held tissue shredder. Rinse the hom ogenizer w ith the appropriate solvents) in between samples. 4 Sonicate each sam ple for approxim ately 5 m inutes w ith a sonic dism em brator. 4 Cap the vials and shake well. Centrifuge the vials at 2000 rpm for approxim ately 5 m inutes. 4 Prepare appropriate dilutions o f study and Q C sam ples to w ithin the calibration range o f the PFOS LC/MS/MS m ethodology w ith 50% NANOpure :50% m ethanol. 4 Amputate sam ples and subm it for LCMS analysis. F ig u re 2. A nalytical method flow chart for th e analysis o f PFOS in earthw orm tissue. 000242 "Wildlife International, Ltd. -41- Area Project Number 454-111 Figure 3. A typical calibration curve for PFOS (artificial soil). Slope -8981195; Intercept =1011.0532; r =0.994. 000243 Wildlife InternationalLtd. -42- Area Project Number 454-111 Figure 4. A typical calibration curve for PFOS (earthw orm tissue). Slope =23387; Intercept =2337.9114; r =0.999. 000244 W ildlife International, Ltd. Project Number 454-111 -43- PF0S_1 STD 0.00100 m s .fA . 467SA-011O-16 4.98 In 1 period PFCS No Internai Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Ttues. 30.0 Quant Thres. 1.0 Min. Width 3 Mutt. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.04 Area 10695 Height 1146 Start Time End Time Integration Width Retention Time Integration Type 3.67 4.34 0.67 4.00 A -8B Thu. Nov 29, 2001 05:52 100i 90 80 70 60 SO 40 30 20 to o4 15 46 67 101 41 01 121 0.09 1.38 2.03 175 206 181 201 2.70 3.37 intensity: 10000 ops 239 241 4.04 203 281 Scan 4.71 Time Figure 5. A representative ion chromatogram o f a low-level (0.00100 mg a.i./L ) PFOS standard (artificial soil). 000245 W ildlife International, Ltd. -44- PFOS.S STD 0.0100 mg a .U l 4675A-0t 10-20 4.98 in 1 period pros No Internat Standard Use Area 1: 4.97 MRM, 298 scans 49S.0->99.1 Noise Thres. 30.0 Quant Thres. 1.0 Min. Width 3 M ult Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.04 Anta 9991S Height 10171 Start Timo EndTime Integration Whim Retention Time Integration Type 3.70 4.37 0.67 3.99 A -V B Thu. Nov 29, 2001 00:16 Project Number 454-111 intensity: 10000 eps Figure 6. A representative ion chromatogram o f a high-level (0.0100 mg a.i./L) PFOS standard (artificial soil). 000246 W ildlife International, Ltd. Project Number 454-111 -45- PFOS 1 S T O 0.500 u g a.i./t 467SA-011D-6 4.98 in 1 period p ro s No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Hires. 1.0 M ia Width 3 Mult. Width Base. W idh 6 40 HT Win. (secs) 20 Smooth Expected RT 1 4.05 Area 13949 Height 1470 Start Time 3.77 End Time Integration Width 4.41 0.64 Retention Time 4.05 Integration Type A -B B Fri, Dec 14, 2001 12:43 10(H 908070 60 SO 40 30 20 10 o4 intensity: 15000 cps 242 36 74 114 150 178 g l 41 81 121 161 201 241 0.69 1.36 2.03 2.70 3.37 4.04 283 281 Scan 4.71 Tim Figure 7. A representative ion chromatogram o f a low-level (0.000500 mg a.i./L) PFOS standard (earthworm tissue). 000247 W ildlife International, Ltd. -46- PFOS.5 STD 5.00 U9 a.UL 4875A-011D -t 0 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 4 9 9 .0 -> 9 9 .t Noise Thres. Quant Ihres. 30.0 1.0 Min. W idlfi 3 Mutt. Width 8 Base. Width 40 RT Win. (secs) 20 Smooth Expected RT 1 4.05 -Area 11B40S Height 12241 Start Time 3.70 End Time 4.29 Integration Width 0.59 Retention Tune 3.94 Intgration Type A -V B Fit. Dec 14. 2001 13:07 Project Number 454-111 intensity: 15000 cps Figure 8. A representative ion chromatogram of a high-level (0.00500 mg a.i./L) PFOS standard (earthworm tissue). 000248 W ildlife International, Ltd. -47- PFOS_7 454-111- MAB-1 ThU. NOV 29, 2001 06:28 4.98 In t period pros No Internal Standard Use Area 1: 4.97 MHM, 298 scans 499.0->99.1 Noiso Hires. 30.o Quant Hires. t.o Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth Expected RT 1 4.04 Area 0 Height 0 Start Time B id Time Integration Width Retention Time Integration Type 0.00 0.00 0.00 0.00 Project Number 454-111 intensity: 10000 eps Figure 9. A representative ion chromatogram of an artificial soil matrix blank sample (454-111-MAB-l). The arrow indicates the approximate retention time of PFOS. / 0 0 0 2 ''9 W ildlife International, Ltd, -48- PFOS_7 454-111- WMAB-1 Fri, Dec 14. 2001 13:20 4.98 in 1 period TOS No Internal Standard Use Area 1: 4.97 MHM. 298 scans 499.0->99.1 Noise Hires. 30.0 Quant Three. 1.0 Min. Width 3 Mutt. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.05 Ana o Height O Start Time 0.00 End Time 0.00 integration Width 0.00 Retention Time 0.00 Integration Type Project Number 454-111 intensity: 15000 eps Figure 10. A representative ion chromatogram of an earthworm tissue matrix blank sample (454-111 WMAB-1). The arrow indicates the retention time ofPFOS. 000250 W ildlife International, Ltd. -49- PFOSJB 454-111- MAS-2 Thu, Nov 29. 2001 06:40 4.98 In 1 period TOS No Internal Standard Use Ana 1: 4.97 MRM, 296 scans 499.0->99.1 Noise Thres. Quant H ires 30.0 0 .5 Min. Width MuH. Width 3 6 Base. Width 40 NT Win. (secs) 20 Smooth 1 Expected RT 4.04 Area 35671 Height 3641 Start Time 359 End Time 436 Integration Width 057 Retention Time 4.00 Integration Type A* - V0 Project Number 454-111 intensity: 10000 ops Figure 11. A representative ion chromatogram of an artificial soil matrix fortification sample (454-111MAS-2, nominal PFOS concentration of 586 mg a.i./kg, dilution factor = 146413x). 000251 W ildlife International, Ltd. Project Number 454-111 -50- PFCS_8 454-111- WMAS-1 Fri, Oec 14, 2001 13:26 4.S8 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->B9.1 Noise Thres. 30.0 Quant Thres. Mm. Width 1.0 3 M ult Width B u s . Width RT Win. (secs) Smooth Expected RT 6 40 20 1 4.0S Area 22590 Height 2367 Start Time 3.70 End Time Integration Width Retention Time Integration Type 4.34 0.64 3.94 A-VB 1001 Intensity: 15000 cps 90 80 70 60 50 40 30 20 235 10 04 19 81 79 41 81 116 141 176 204 121 161 201 0.69 1.36 2.03 2.70 3.37 278 241 281 Scan 4.04 4.71 Time Figure 12. A representative ion chromatogram of an earthworm tissue matrix fortification sample (454 111-WMAS-l, nominal PFOS concentration of 100 mg a.i./kg, dilution factor = lOOOOOx). 000252 W ildlife International, Ltd. -51- PF0S_13 454-111- Thu, Nov 29, 2001 07:04 4.98 In 1 period pros No Internal Standard Use Area 1: 4.97 MRM. 298 scans 489.0->99.1 Noise Thres. 30.0 Quant Ihres. 1.0 Min. Width Mutt Width Base. Width 3 6 40 RT Win. (secs) 20 Smooth 1 E je cte d RT 4.04 Area 23883 Height 2478 Statt Time End Time 3.69 4.36 Integration Width 0.67 Retention Time 3.99 Integration Type A - SB Project Number 454-111 intensity: 10000 ops Figure 13. A representative ion chromatogram of an artificial soil test sample (454-111-2, nominal PFOS concentration of78.1 mg a.i./kg, dilation factor = 29283x). 000253 W ildlife International, Ltd. -52- PF0S.1 454-111- W-2A Ftl, Dec 14, 2001 13:38 4.98 in 1 period PROS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Hues. 30.o Quant Hues. 1.0 Mk). Wktth 3 Mutt. W kitli 6 Basa. Widtti 40 RT Win. (secs) 20 Smooth 1 Expected FIT 4.05 Area 46847 Height 4953 Start Time End Time Integration Width Retention Time integration Type 3.70 4.37 0.87 3.94 A-BB Project Number 454-111 Intensity: 15000 cps Figure 14. A representative ion chrom atogram o f a n earthw orm tissue test sam ple (454-111-W -2A, dilution facto r= lOOOOOx). 000254