Document MJ2Q5yLnKXeXxBKEqvo1JvEGa

< BIO CO NCENTRATIO N - R EVISED AR2.3.G- IIO S TEST SUBSTANCE ____________________________________________ Identity: 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) Remarks: Sample from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/MS, 1H-HM R, 19F-NM R and elem ental analyses techniques. METHOD:_____________________________________ ____________________ Method/guideline followed: US EPA O PPTS 850.1730 and O ECD 305 Type: Flow-through exposure with flow-through depuration phase. GLP (Y/N): Yes Year: 2001, Report amended 2002 Species: Bluegill (Lepomis macrochirus) Supplier: Osage Catfisheries, Inc., Osage Beach, Missouri Length and weight at test termination: Mean length = 62 mm, range 56-66 mm Mean weight = 2.70 g, range 2.03 - 3.32 g Loading: 0.48 g fish/L/day (based on initial loading of 90 fish per tank, using mean fish weight at the end of the study and volume of w ater that passed through test chamber in 24-hours). Fish age: Approximately 7 months at test initiation Analytical monitoring: Concentration of PFOS in water and fish. Pretreatment: None Number of concentrations: Two plus a negative control Test concentrations (mean measured): Negative control, 0.086 and 0.87 mg/L Uptake period: 62-days (0.086 mg/L exposure) 35-days (0.87 mg/L exposure - this exposure ended after 35-days due to fish mortality) Depuration period: 56-days (0.086 mg/L exposure) None (0.087 mg/L exposure) Test conditions: Dilution water: M oderately-hard well water Dilution water chemistry: Specific conductance: 313 (310 - 315 umhos/cm) Hardness: 130 (128 - 132 mg/L) Alkalinity: 1 7 8 (1 7 6 -1 7 8 ) pH: 8.1 (8 .0 -8 .2 ) Measured during the 4-w eek period immediately preceding the test. cst CO c_ cr ~r c ro U3 3W IS up TO /M fs k! I ,\ f \ co C23 TO vO cat <*< v**> ro CONTAIN NO CBI 000283 Test conditions (cont.): S tock and te s t solution preparation: Two stock solutions were prepared at 10 and 100 mg a.i./L. Stock solutions stirred with an electric top-down mixer to aid in the solubilization of the test substance. After mixing, the stocks appeared clear and colorless. Stocks were prepared at approximately weekly intervals during the uptake phase. Stocks injected into the diluter mixing chambers at a rate of 3.5 mL/minute where they were mixed with dilution w ater at a rate of 350 mL/minute to achieve the desired test concentrations. All final test solutions appeared clear and colorless. D ilu ter flow rate: Approx. 6.3 volume additions per 24-hours Exposure vessels: 104 L stainless steel aquaria filled with approximately 80 L solution. N um ber o f replicates: None - one vessel per concentration N um ber o f fish per vessel: 90 D iet: Flake food, Ziegler Brothers, Inc., Gardners, PA W ater chem istry ranges during the study:_____________________ Neg. Control 0.086 mg/L 0.87 mg/L D issolved oxygen, mg/L: 6 .8 - 8 . 6 6 .8 -8 .6 6 .4 -8 .2 Temperature, C: 2 1 .8 -2 2 .0 2 1 .7 -2 2 .0 2 1 .7 -2 1 .9 - E * ____________________ 7 .9 - 8 .2 7 .9 -8 .2 7 .9 -8 .2 Photoperiod: 16 hours light and 8 hours dark with a 30 minute transition period. Light intensity: 278 lux at surface of the negative control vessel at test initiation C ollection o f tissu e sam ples: Fish were collected from test chambers by random selection at 12 time points during the 62-day uptake phase. They were euthanized, blotted dry, weighed and measured. Fish then rinsed with dilution water, blotted dry again and dissected into edible and nonedible tissue fractions. The fractions w ere individually weighed. The head, fins and viscera were considered to be nonedible tissue. The remaining tissue, including skin was considered to be edible tissue. S tatistical m ethods: W hole fish concentrations were calculated based on the sum of the edible and nonedible parts. Steady-state bioconcentration BCF values w ere originally calculated from the tissue concentrations at apparent steady-state using the BIOFAC model. Upon further investigation, this model was deemed inappropriate for use with this data set. The tissue concentrations had reached a point where there were not statistically significant differences between the last three sample days. However, a plot of the data clearly shows a trend of increasing concentrations in the tissues. In addition, the BIOFAC program is not accepted as appropriate for surfactants. An amended report was issued with BCFK values calculated as outlined in the draft O PPTS 850.1730 Guidance Document. The kinetic bioconcentration factor (BCFK), uptake rate (k i) and depuration rate (k2) w ere calculated for the edible, nonedible and whole fish exposed to 0.086 mg/L. These rate constants were then 000284 used to calculate a BCFK (BCFK = K1/K2) and half-lives for clearance for each tissue type. The results from this data reanalysis are presented below. R E S U L T S _________________________________________________ Nominal concentrations: Negative control, 0.1 and 1.0 mg/L Mean measured concentrations: < 0.05, 0.086 and 0.87 mg/L Kinetic Bioconcentration factors (BCFK): 0.086 mg/L exposure BCFK: Edible 1124 Nonedible 4013 Time to reach 50% clearance: 86 days 116 days Whole Fish 2796 112 days 0.87 mg/L exposure Although BCF values w ere calculated using the BIOFAC software, the results are not reported here. All of the fish had died or been sampled prior to achieving steady-state. As a result, the BCF values were underestimated and are not relevant. 000285 PFOS Concentrations in Tissues of Bluegill Exposed to 0.086 mg/L Values are from 4 individual fish at each sample period. Uptake Day Edible Tissue, Nonedible Tissue, mg/kg mg/kg 0 (4-hours) 0.167,0.155,0.144,0.182 0.415, 0.519, 0.417, 0.497 1 0.734, 0.726, 0.631, 0.806 1.68, 1.85,1.72, 2.07 3 1.73, 2.07, 2.03, 2.11 4.59, 5.50, 5.47, 5.97 7 3.73,4.25, 4.73, 6.25 10.2,10.6,11.9,15.2 14 11.4, 9.07,13.7,12.6 27.3, 23.2,35.3, 32.6 21 11.7,12.0, 12.9,10.6 33.3,22,7,24.6,24.4 28 18.3,13.7, 23.9, 23.1 49.4,40.7,65.3, 57.9 35 22.6, 27.7, 23.8, 20.6 67.1, 73.3,62.0, 59.1 42 27.6, 25.3, 21.2, 27.6 64.0, 68.1,54.4, 79.6 49 33.3,36.2, 39.0, 30.6 85.0,95.1,93.1,77.7 56 48.3,38.9,44.1,38.3 122,94.2,73.2,106 62 42.4, 66.2,42.2, 39.2 101,112,105, 96.4 Depuration Day 14 48.5,31.8, 31.6,42.0 124, 79.4,81.8,113 28 26.0, 33.3, 38.7, 55.8 85.7,95.1,85.7,94.8 42 24.1,31.2, 30.0,33.0 71.7, 80.6, 78.3, 82.1 56 21.1,37.6,32.9,31.2 57.7, 80.3,85.4, 84.4 Whole Fish Cone., mg/kg 0.293, 0.351, 0.286, 0.363 1.26,1.34,1.29, 1.53 3.21,4.04,4.18,4.38 7.33, 7.66, 8.73, 11.4 20.2,16.9,26.0, 24.6 23.3,18.4,19.8,18.5 35.3,29.2,45.4,44.1 46.3,53.8,46.6,40.9 50.1,49.4,40.9,56.3 62.8,69.6, 70.8, 57.4 90.6, 71.6, 63.3, 74.8 77.0,92.7, 79.6, 73.1 90.3,60.4, 61.6,85.3 58.2, 70.1,68.1,81.1 51.4, 61.4,61.0,62.2 41.6,66.5,65.8,62.1 PFOS Concentrations in Tissues of Bluegill Exposed to 0.87 mg/L Values are from 4 individual fish at each sample period. Uptake Edible Tissue, Nonedible Tissue, Day mg/kg mg/kg 0 (4-hours) 1.46,1.48, 1.19, 1.39 3.52, 4.37, 4.22,4.06 1 4.68,6.59,5.56,5.64 1 1.1,14.2,13.3, 12.1 3 17.3,15.8,19.0,20.8 39.3,42.0,43.8, 51.8 7 42.0,44.0, 57.7, 46.8 100,102,102,120 14 87.1,81.6, 90.7,73.3 177,207, 245, 214 21 79.4,117, 104,102 201,278,246,229 28^ 102,131,107,133 289,372,320,361 Whole Fish Cone., mg/kg 2.71,3.08, 2.84, 2.89 8.00,10.9,10.2, 9.47 3 0 .5 ,3 0 .7 ,3 4 .5 , 39.1 74.9, 77.0, 85.3, 89.8 141,157,180,158 146,210,185,172 205,267, 232,263 (1)Sampling offish sitopped after Uptake Day 28 due to mortality. Test organism mortality: Negative control: None during the uptake phase (62 days) or depuration phase (35 days) 0.086 mg/L exposure: One fish died after 49 days and one after 59 days of exposure in the uptake phase, none during the depuration phase (total of 2.2% mortality during the study). 00028G 0.87 m g/L exposure: Mortality first noted on Day 9 and continued through Day 35 of the uptake phase at which time all of the fish had either died or had been sampled A n alytical m ethodology: Analyses of test solutions and fish tissues were performed at W ildlife International, Ltd. W ater samples w ere diluted and analyzed by HPLC with single quadrupole mass spectrometric detection. Tissue samples w ere homogenized, extracted, diluted and analyzed by HPLC with triple quadrupole mass spectrometric detection. W hen determining the concentration of the test substance in the samples, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ w as 0.05 mg/L for w ater in this study. For tissue samples, the LOQ w as calculated on an individual basis for each sample since each entire submitted sample, of differing weight, was extracted without an adjustment to constant weight. Recovery was excellent in both w ater and fish tissues, ranging from 84.9 to 122% of fortification levels. Analytical results were not corrected for procedural recovery. CO NCLUSIO NS ________________________________________ ___ PFOS bioconcentrated in the tissues of bluegill sunfish during this study. The BCFK values calculated for the edible, nonedible and whole fish tissues from the 0.086 mg/L exposure w ere calculated to be 1 1 2 4 ,4 0 1 3 , and 2796, respectively. PFOS depurated slowly. The BIOFAC estimates for the time to reach 50% clearance for edible, nonedible and whole fish tissues from the 0.086 mg/L exposure were 8 6 ,1 1 6 and 112 days, respectively. DATA QUALITY___________ 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, M D at the request of the 3M Company, Lab Request number U2723. The report was amended and reissued June 6 ,2 0 0 2 . O THER____________________________________________________________ Last changed: 6/11/02 000287 PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW -THROUGH BIOCONCENTRATION TEST W ITH THE BLUEGILL (Lepomis macrochirus) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 ENVIRONMENTAL LABORATORY REQUEST NUMBER: U2723 U.S. Environm ental Protection Agency Series 850 - Ecological Effects T est Guidelines OPPTS Num ber 850.1730 and OECD Guideline 305 AUTHORS : K urt R. D rottar Raymond L. VanHoven, Ph.D. Henry O. Krueger, Ph.D.. STUDY INITIATION DATE: October 31,2000 STUDY COM PLETION DATE: June 21,2001 AMENDED STUDY COM PLETION DATE: June 6,2002 SUBM ITTED T O : 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, M innesota 55144St. Paul, M innesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, M aryland 21601 (410) 822-8600 Page 1 o f 134 AMENDED 000238 W ildlife International, L td Project Number 454A-134 - 2GOOD LABORATORY PRA CTICE COM PLIANCE STATEM ENT SPO N SO R : 3M Corporation T IT L E : Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test w ith the Bluegill (Lepomis machochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT N U M B ER : 454A-134 . STUDY COMPLETION: June 21,2001 AMENDED STUDY COMPLETION: June 6 ,2 0 0 2 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 7 9 2 ,1 7 August 1989. STUDY DIRECTOR: N on-Target Plants SPONSOR APPROVAL: d ( /U'id. X U D ate Jl D ate OR 000289 AMENDED W ildlife International, Ltd. Project Number 454A-134 -3 QUALITY ASSURANCE STATEMENT This stucfy was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 7 9 2 ,1 7 August 1989. The dates o f all inspections and audits and the dates that any findings were reported to the Stuffy Director and Laboratory M anagement were as follows: ACTIVITY: Test SubstancePreparation Matrix Fortification Water Chemistry DATE CONDUCTED: November 29,2000 December 8,2000 February 20,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: November 29,2001 November 30,2000 December 8,2000 December 11,2000 February 20,2001 February22,2001 Analytical Data and DraftRepoit May 14 - 1 7 and May 20 -24,2001 May 24,2001 June 2,2001 Biological Data and Draft Report Final Report Amended Final Report June 11-13,2001 June 20,2001 June S, 2002 June 13,2001 June 20,2001 June 5,2002 June 14,2001 June 20,2001 June 6,2002 000290 AMENDED W ildlife International, Ltd. -4 REPORT APPROVAL Project Number 454A-134 SPONSOR : 3M Corporation TITLE :Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUM BER : 454A-134 STUDY DIRECTOR: D irector o f Aquatic Toxicology and N on-Target Plants PRINCIPAL INVESTIGATOR: AMENDED 000291 W ildlife International, Ltd. Project Number 454A-134 -5 TABLE OF CONTENTS Titie/CoverPage.................................................... Good Laboratory Practice Compliance Statement. Q uality Assurance Statem ent Report A pproval..................... Table o f C ontents................... Summary ............................ In tro d u c tio n ............................ Objective ............................ Experimental D esign............. M aterials and M ethods Results and Discussion. C onclusions.................. References .................. .. 1 .2 .3 .4 .5 ,,8 .9 .9 .9 10 16 18 19 TABLES Table 1 M eans and Ranges o f W ater Quality Param eters.................................................................... 20 T able 2 Concentrations o f PFOS in W ater Samples During the Uptake Phase.................................... 21 T able 3 Concentrations o f PFOS in W ater Samples During the Depuration Phase..............................22 T able 4 PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues o f Bluegiil Exposed to 0.086 mg a.i./L ........................... .......................................... 23 T able S A pparent Steady-State BCF Values for Bluegiil Exposed to 0.086 mg a.i./L ........................ 26 T ab le 6 BCFK Model Estim ates for Bluegiil Exposed to 0.086 mg a.i./L .......................................... 27 AMENDED 000292 Wildlife International, Ltd, - 6- TABLE OF CONTENTS - Continued - Project Number 454A-134 T able 7 T able 8 TA BLES (C ont'd.) PFOS Concentrations in Edible, N onedible and W hole Fish Tissues o f Bluegill Exposed to 0.87 m g a.i./L .............................................................28 Day 28 BCF Values for Bluegill Exposed to 0.87 mg a.i./L ..................................... 30 F igure 1 F igure 2 F igure 3 F igure 4 FIG U RES Concentrations o f PFOS in Edible Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L .................................................................. ....................... .................31 Concentrations o f PFOS in Nonedible Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L ............................................................................................................32 Concentrations o f PFOS in W hole Fish Tissues o f Bluegill Exposes to 0.086 mg a.i./L ............................................................................................................33 Concentrations o f PFOS in Edible. Nonedible, and W hole Fish Tissues O f Bluegill Exposed to 0.87 m g a.i./L.......................................................................... 34 AMENDED 000293 W ildlife International, Ltd. Project Number 454A-134 -7 - TABLE OF CONTENTS - Continued - APPENDICES A ppendix 1 Specific Conductance, Hardness, A lkalinity and pH o f W ell W ater M easured1D uring the 4-W eek Period Immediately Preceding the T e st ............. 35 A ppendix 2 Analyses o f Pesticides, Organics and M etals in W ildlife International Ltd. W ell W ater................................................................... 36 A ppendix 3 The Analysis o f Perflurooctanesulfonate, Potassium Salt (PFOS) Concentrations in Freshwater and Bluegill Sunfish Tissue in Support o f W ildlife International L td Project No. 454A -134..................................................... 38 A ppendix 4 Tem perature and pH o f W ater in the T est C ham bers...................................................89 A ppendix 5 Dissolved Oxygen o f W ater in the T est C ham bers.......................................................91 A ppendix 6 Hardness, Alkalinity, Conductivity and TOC o f W ater in the N egative C ontrol........................................ .................................................................... 95 A ppendix 7 Cumulative M ortality and Treatm ent-Related E ffects..................................................96 A ppendix 8 A ppendix 9 Changes to Protcol............................................. 105 . -O' Protocol, Amendments and D eviations........... .............................................................106 A ppendix 10 Personnel Involved in the Study............................................ 130 A ppendix 11 Calculations O f The Kinetic Concentration Factor (BCFK), The Uptake Rate Constant (k\), The Depuration R ate C onstant (2), The Half-Life For Clearance, And The Tim e To Reach 90% O f Steady S tate 131 A ppendix 12 Report Amendment...................................................................................................... 133 AMENDED 000294 Wildlife International, Ltd. Project Number 454A-134 - 8- SUMMARY SPONSOR: 3M Corporation SPONSOR'S REPRESENTATIVE: M s. Susan A. Beach LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: W ildlife International L td Easton, MD 21601 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST (0.086 mg a.i./L): LENGTH OF TEST (0.87 m g al/L ): 454A -134 Perfluorooctanesulfonate, Potassium Salt (PFOS) Periluorooctanesulfonate, Potassium Salt (PFOS): A FlowThrough Bioconcentration Test w ith the Bluegill (Lepomis m acrochirus) Negative C ontrol, 0.10 and 1.0 mg a.i./L Negative Control, 0.086 and 0.87 m g a.i./L. Exposure to 0.87 mg a.i./L ceased after 35 days due to fish m ortality and tissue sam pling Experim ental S tart (OECD) - November 2 9,2000 Experim ental S tart (EPA) - December 5 ,2 0 0 0 Biological Term ination - A pril 2,2001 Experim ental Term ination - April 11,2001 118 Days (62-D ay Uptake, 56-D ay Depuration) 35 Days o f U ptake___________________________ TEST ORGANISM: Bluegill (Lepomis macrochirus) SOURCE OF TEST ORGANISMS: Osage C atfisheries, Inc. 1170 N ichols Road Osage Beach, M issouri 65065 AGE OF TEST ORGANISMS: Juveniles MEASUREMENTS OF 10 NEGATIVE CONTROL FISH COLLECTED AT TEST TERMINATION: WEIGHT (g): TOTAL LENGTH (mm): M ean= 2 .7 0 ; Range = 2.03 to 3.32 Mean = 62; Range = 56 to 66 RESULTS: (0.086 m g a.i./L) Kinetic Bioconcentration factor (BCFK): Edible 1124 N onedible 4013 W hole Fish 2796 RESULTS: (0.87 mg a.i./L) E d ib le N o n ed ib le W hole Fish DAY 28 BCF*: 136 386 278 Note: The BCFs were underestim ated due to fish m ortality prior to achieving steady-state. AMENDED 000295 W ildlife International, Ltd. Project Number 454A-134 -9 - INTRODUCTION A bluegill sunfish, Lepomis macrochirus, bioconcentration study was conducted for 3M Corporation at the W ildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f the test was conducted from December 5 ,2 0 0 0 to A pril 2,2001. Raw data generated by W ildlife International, Ltd. and a copy o f the final report are filed under Project Num ber 454A -134 in archives located cm the W ildlife International, Ltd. site. OBJECTIVE The objective o f this study was to determine the bioconcentration potential o f perfluorooctanesulfonate, potassium salt (PFOS) in the bluegill sunfish. EXPERIMENTAL DESIGN The bioconcentration test consisted o f a 62-day uptake phase followed by a 56-day depuration phase. During the uptake phase, die test organism s were exposed in one o f three groups: 1) A negative (dilution w ater) control; 2) A nominal concentration o f 0.10 mg active ingredient (a.i.)/L ; or 3) A nominal concentration o f 1.0 mg a.i./L. A t the start o f the depuration phase, stock flow to the treated groups was stopped and the bluegill were exposed to dilution w ater w ithout PFOS for the remainder o f the test. Each test chamber contained 90 bluegill a t test initiation, and one replicate was tested for each treatm ent and die negative control. W ater sam ples were collected on Day -4 (pre-test), Day -1 (pre-test) on uptake Days 0 (0 and - 4 hours), 1 ,3 ,7 ,1 4 ,2 1 ,2 8 ,3 5 ,4 2 ,4 9 ,5 6 and 62 and on depuration Days 14,28,42 and 56 during the test and analyzed for PFOS using liquid chromatography-mass spectrom etry (LC/MS). Tissue samples were also collected at selected water sam ple collection periods during the test and analyzed for PFOS by LC/M S. The results o f these analyses ware used to calculate the BCF values, uptake rates and depuration rates in edible tissue, nonedible tissue and whole fish. 000296 W ildlife International, Ltd. Project Number 454A-134 - 10- MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "Perfluorooctanesulfonate, Potassium Salt (PFOS) (A ppendix 9): A Flow-Through Bioconcentration Testwith the Bluegill (Lepomis macrochirus)". The protocol was based on procedures outlined in U .S. Environm ental Protection Agency Series 850 - Ecological Effects T est Guidelines OPPTS Num ber 850.1730 (1); ASTM Standard E l022-84 Standard Practice fo r Conducting Bioconcentration Tests with Pishes and Saltwater Bivalve M olluscs (2); and OECD Guideline for Testing o f Chemicals 305, Bioconcentration: Plow-Through Fish Test (3). Test and Reference Substances The test substance was received from 3M Corporation on O ctober29,1998 and was assigned W ildlife International, Ltd. identification num ber 4675. The test substance, a w hite powder,was identified as FC-95, Lot #217. Inform ation provided by the Sponsor indicated a purity o f 86.9% and an expiration date o f A ugust 31, 2001. The test substance was stored under am bient conditions. Preparation of Test Solutions The nom inal test concentrations were 0.10 and 1.0 mg a i./L . Two stock solutions were prepared at concentrations o f 10 and 100 m g a.i./L. The appropriate am ount o f test substance was weighed out and dissolved in dilution w ater for each stock. The stock solutions were stirred with an electric top-down m ixer to aid in the solubilization o f the test substance. A fter m ixing, the stock solutions appeared clear and colorless. Stock solutions were prepared a t approxim ately weekly intervals during the uptake phase o f the te s t The stock solutions were injected into the diluter m ixing chambers (at a rate o f 3.5 mL/minute) where they were mixed with dilution w ater (at a rate o f 350 m L/minute) to achieve the desired test concentrations. All test solutions appeared clear and colorless. Test Organism The bluegill, Lepomis macrochirus, was selected as the test species for this study. The bluegill is one o f the recommended freshwater fish species for use in bioconcentration tests (1 ,2 ,3 ). Bluegill used in the test were obtained from Osage Catfisheries, Inc., Osage Beach, M issouri. The fish were approxim ately 7 months old at test initiation. Identification o f the species was verified by the supplier. 000297 W ildlife International, Ltd. Project Number 454A-134 - 11- The bluegill were held in W ildlife International, Ltd. well w ater for 103 days prior to testing. The fish w ere acclimated to test conditions for approxim ately 49 hours p rior to test initiatioa During the holding and acclim ation periods the fish showed no signs o f disease o r stress. During the 14-day holding period preceding the test, w ater tem peratures ranged from 22.3 to 22.7C. The pH o f the w ater ranged from 7.8 to 8.2 and dissolved oxygen ranged from 7.6 to 8.2 mg/L. Instrum entation used for w ater measurements are described in the Environmental Conditions section o f this report. A t test initiation, the bluegill were collected from the acclim ation tank and indiscrim inately distributed 1 to 2 at a tim e into the test chambers until each cham ber contained 90 fish. During holding and during the test, the bluegill were fed flake food supplied by Zeigler B rothers, Inc., Gardners, Pennsylvania. The bluegill were fed at least once daily during holding and once daily during the test Feeding and sampling schedules were coordinated so that fish wore sampled at least four hours after feeding. A ll fish used in the test were from the same source and year class, and the standard length o fthe longest fish was no more than twice the length o fthe shortest. The length and weight o f fish in fee negative control were considered to be representative o f all fish used in the test. The m ean total length o f 10 negative control fish m easured at the end o f fee test was 62 mm w ife a range o f 56 to 66 mm. The average w et weight (blotted dry) was 2.70 gram s with a range o f 2.03 to 3.32 grams. Loading was defined as the total wet weight o ffish per liter o f test w ater that passed through the test chamber in 24 hours, and was determined to be 0.48 g fish/L/day. The loading rate was based on the average weight o f the fish at the end o f the test and the initial stocking density (90 fish per tank). ' T est A pparatus A continuous-flow diluter was used to deliver each concentration o f the test substance and a negative control. A peristaltic pum p (Cole-Parmer Instrum ent Company, Chicago, Illinois) was used to deliver fee test substance stock solutions into m ixing chambers assigned to each PFOS treatm ent. The stock solutions were mixed with dilution w ater in fee m ixing chambers prior to deliveiy to fee test chambers. The flow o f dilution water to fee test chambers was controlled by rotam eters. The delivery o f water from fee rotameters was checked prior to fee test and at approximately weekly intervals thereafter. Approxim ately 6.3 volume additions o f test water were delivered to fee test chambers every 24 hours. The general operation o f fee diluter was checked at least two times a day during fee test and once on fee last day o f fee test. 000298 W ildlife International, Ltd. Project Number 454A-134 - 12- Test cham bers were 104-L stainless steel aquaria filled w ith approxim ately 80 L o f test solution. The depth o f the test w ater in a representative cham ber w as approxim ately 19 cm. T est cham bers were indiscrim inately positioned in a tem perature-controlled w ater bath designed to m aintain a constant temperature. The w ater bath was enclosed in a plexiglass ventilation hood in order to m inim ize any potential for crosscontam ination. T est cham bers were siphoned daily and periodically cleaned during the test to rem ove excess feed and fecal m atter. Test cham bers were identified by the project num ber and test concentration. Dilution Water H ie w ater used for holding and testing was freshw ater obtained from a well approxim ately 45 m eters deep located on the W ildlife International, Ltd. site. The well w ater is characterized as m oderately-hard water. The specific conductance, hardness, alkalinity and pH m easurem ents o f the well w ater during the four-week period immediately preceding the test are presented in Appendix 1. The well w ater was passed through a sand filter to remove particles greater than approxim ately 25 pm , and pum ped into a 37,800-L storage tank and aerated w ith spray nozzles. Prior to use, the w ater again was filtered (0.45pm ) to remove m icroorganisms and particles. The results o f periodic analyses perform ed to measure the concentrations o f selected contam inants in well w ater used by W ildlife International, Ltd. are presented in Appendix 2. Environmental Conditions The target tem perature range for the test was 221C . Tem perature was recorded continuously in the negative control w ith a Fulscope ER/C Recorder (1900 J Series model no. A). Tem perature w as also measured in all test chambers a t the beginning and end o f the test and at weekly intervals during the test, w ith a liquid-inglass thermometer. Dissolved oxygen was m easured w ith a Yellow Springs InstrumentCompany, Inc. Model 5 IB dissolved oxygen meter. W ith the exception o f uptake Day 24, measurements were made daily in each test chamber. Dissolved oxygen measurem ents were not m ade on uptake Day 24 due to biologist oversight. M easurements o f pH were made in each test cham ber at the beginning and end o f the test and at weekly intervals during the test using a Fisher Accumet Model 915 pH meter. 000299 W ildlife International, Ltd. Project Number 454A-134 -13 - Hardness, alkalinity, conductivity and total organic carbon (TOC)ware measured in the negative control at the beginning and end o f the test, and at weekly intervals during the test. Hardness and alkalinity were measured by titration based on procedures in StandardM ethodsfo r the Examination ofW ater and Wastewater (4). Conductivity was m easured using a Yellow Springs Instrum ent Company, Inc. M odel 33 SalinityConductivity Temperature m eter. Total organic carbon was m easured using a Shimadzumodel TOC-5000 total organic carbon analyzer. Ambient room light was used to illum inate the test systems. Fluorescenttubes that emitted wavelengths sim ilar to natural sunlight (Colortone SO) were controlled by an automatic tim er toprovide a photoperiod o f 16 hours o f light and S hours o f darkness. A 30-m inute transition period o f low light intensity w as provided when lights w ent on and o ff to avoid sudden changes in light intensity. Light intensity at the surfaceo fthew ater (over the negative control) was 278 lux at test initiation. Light intensity was measured using a SPER Scientific Model 840006 light meter. O bservations All fish were observed once each day to evaluate the num ber o f m ortalities and the num ber o f individuals exhibiting signs o f abnormal behavior. P rocedures for E xposure o f F ish to PFOS The test chambers were conditioned by delivering PFOS to the diluter system for approximately 5 days before adding the fish. W ater samples were collected three times during die pre-test period to confirm that equilibrium concentrations o f test substance in the test chambers were achieved prior to adding the fish. A t the end o f the pre-test period, the uptake phase o f the test was initiated on December 5 ,2 0 0 0 by placing the fish in the test chambers. Bluegill were im partially removed from the holding tank in groups o f 1to 2. The groups o f bluegill were distributed among the test chambers until each test chamber contained 90 fish. The duration o f the uptake phase was 62 days for the low exposure concentration (0.1 mg a.i./L, nominal). The uptake phase for the high concentration (1.0 mg a.i./L, nom inal) was term inated after 35 days due to fish mortality. All subsequent references in this report to sam pling periods after Day 35 o f uptake refer to the 0.1 mg a.i./L (nominal) exposure concentration. A t the end o f the uptake phase, stock flow to the treatm ent groups was stopped and the bluegill were exposed to dilution water without PFOS for a period o f 56 days. 000300 Wildlife InternationalLtd. Project Number 454A-134 -14- C ollection an d A nalysis o f W a te r Sam ples W ater sam ples were collected on Days 0 (0 hours and 4 hours)., 1 ,3 ,7 ,1 4 ,2 1 ,2 8 ,3 5 ,4 2 ,4 9 ,5 6 and 62 o f the uptake phase. W ater sam ples were also collected on Days 1 4 ,2 8 ,4 2 and 56 o f the depuration phase. A t each w ater sam pling interval, tw o w ater sam ples were collected from die negative control and three samples were collected from each o f the tw o PFOS treatm ent groups. One negative control sample and tw o samples from each o f the PFOS treatm ent groups were analyzed for PFOS. The rem aining sam ples were held in reserve as backup sam ples. A ll w ater sam ples were collected from m id-depth o f each test cham ber using a glass pipette. The w ater sam ples were analyzed for PFOS by liquid chromatography-m ass spectrom etry (LCVMS). Procedures for analysis o f the w ater sam ples are provided in Appendix 3. W ater sam ples w ere analyzed as soon as possible after collection w ithout storage. C ollection an d A nalysis o fT issue Sam ples Tissue sam ples were collected on Days 0 (4 hours), 1 ,3 ,7 , 1 4 ,2 1 ,2 8 ,3 5 ,4 2 ,4 9 ,5 6 and 62 o f the uptake phase. Tissue sam ples w ere also collected on Days 1 4 ,2 8 ,4 2 and 56 o f the depuration phase. A t each tissue sam pling interval, a sufficient num ber o f fish were collected to provide two replicate sam ples o f negative control fish and four replicate sam ples o f each PFOS treatm ent group. Fish were im partially removed from the test chambers and euthanized by severing the spinal cord above the opercular region. The fish were blotted dry and m easured for total length and w et weight within approxim ately 15 minutes o f collection, when possible. Each fish was then rinsed w ith dilution w ater, blotted dry again and dissected into edible and nonedible tissue fractions. D issection was accomplished by m aking an incision from ju st posterior to the base o f the pectoralfin dorsally through the spinal cord. The head, fins and viscera were removed from the body and were considered to be nonedible tissue. The rem aining tissue (including skin) was considered the edible tissue. Tissue sam ples were transferred to tared scintillation vials and weighed. Procedures for extraction and analysis o f die tissue sam ples are provided in Appendix. All tissue sam ples were extracted immediately or stored at approxim ately 14C until extraction. ! 000301 W ildlife International, Ltd. Project Number 454A-134 -15- U ssu e L ipid C ontent Selected fish were collected to determ ine lipid content (Appendix 3). The determ ination o f perant lipids provides the potential to express BCF values in term s o f lipid content. Fish were sam pled on Day 0 o f uptake, on Day 62 o f uptake and on Day 56 o f depuration. A ll fish collected for lipid content were stored at approxim ately -14C until analysis. In this study, no attem pt was m ade to express bioconcentration in relation to lipid content. D ata A nalysis W hole fish concentrations were calculated based on the sum o f th e edible and nonedible parts. The steady-state bioconcentration factor (BCF) values were determ ined from the tissue concentrations at apparent steady-state divided by the average w ater concentration Tissue concentrations were consideredto be at apparent steady-state if three o r m ore consecutive sets o f tissue concentrations were not significantly diffrait (p > 0.05). Tissue concentrations were evaluated for norm ality and homogeneity o f variance using the Shapiro-W ilk's test and B artlett's test, respectively. If the data did not meet the assum ptions, the data was transformed in an attempt to correct the data. M ean tissue concentrations w ere then com pared using analysis o f variance and Dunnett's test. In the original report the tissue value's were considered to be at steady state and the com puter program BIOFAC was used to estim ate the kinetic concentration factor (BCFK), the uptake rate constant (k |), the depuration rate constant (k2), the estim ated time to reach 90% steady state, and the half-life for clearance. Although tissue concentrations from uptake days 4 9 ,5 6 and 62 were not significantly different (p >0.05), the concentrations o f PFOS in tissues appeared to still be increasing. Therefore, it can be argued that the fish did not reach an apparent steady state and that using the BIOFAC program may not have been the best way to estim ate these parameters. Therefore, the data were reanalyzed using the equations and graphical methods outlined in the draft OPPTS 850.1730 Guidance Document (Appendix 11). The recalculated rate constants were used to calculate a BCFK (BCFK = kl/k2). AMENDED 000302 W ildlife International, Ltd. Project Number 454A-134 -16- RESULTS AND DISCUSSION W ater C hem istry M eans and ranges o f tem perature, dissolved oxygen and pH o f the w ater in the test cham bers are presented in T able 1. The individual measurements are given in Appendicies 4 and 5. W atertemperatures in the test cham bers were w ithin the tem perature range o f 22 1C established for the testw ith one exception. O n Day 19 o f depuration, the continuous tem perature recorder m easured 20 C. The duration o f this tem perature deviation was approxim ately 2 hours. Dissolved oxygen concentrations rem ained >6.4 mg/L (74 percent o f saturation) throughout the test. M easurements o f pH ranged from 7.9 to 8.2. W eekly measurem ents o f hardness, alkalinity, conductivity and total organic carbon rem ained consistent throughout the test (Table 1 and A ppendix 6). O bservations o f M o rtality and C linical Signs O bservations o f m ortality and clinical signs are presented in Appendix 7. Bluegill in the negative control appeared norm al and healthy throughout the te s t Two bluegill died in the 0.1 mg a.i./L (nom inal) treatm ent group; all other fish appeared norm al and healthy. Bluegill in the 1.0 mg a.i./L (nom inal) treatm ent group started to die on Day 9 o f the uptake phase. By Day 35 o f the uptake phase, all fish in the 1.0 mg/L treatm ent group had either died o r been sam pled for tissue analyses. Any fish found dead in die aquaria were stored frozen. C oncentrations of PFO S in W ater Concentrations o f PFOS in the negative control were <LOQ (0.0500 mg a.i./L ) (Tables 2 and 3). M easured concentrations ofPFO S during the uptake phase in the 0.10 mg aiTL treatmentgroup ranged from 68 to 113% o f the nom inal test concentration (Appendix 3). W hen concentrations m easured during the uptake phase were averaged, the m ean m easured concentration was 0.086 m g a.i./L which represented 86% o f the nominal test concentration. M easured concentrations ofPFO S during the uptake phase in the 1.0 m g a.i./L treatm ent group ranged from 73 to 101% o f the nom inal test concentration. W hen concentrations m easured during the uptake phase were averaged, the mean m easured concentration was 0.87 mg a.i./L, which represented 87% o f the nominal test concentratioa Concentrations ofPFO S during the depuration phase were all <LOQ. 000303 W ildlife International, Ltd. Project Number 454A-134 -17- Concentrations of PFOS in Fish Tissues Concentrations o f PFOS in the negative control tissue sam ples contained no quantifiable PFOS concentrations (Appendix 3) The concentrations o f PFOS in tissues o ffish exposed to 0.086 mg a.i./L are presented in Table 4. Although tissue concentrations from uptake days 4 9 ,5 6 and 62 were not significantly different (p >0.05), the concentrations o f PFOS in tissues appeared to still be increasing. The mean m easured tissue concentrations from these last three uptake sam ples were 41 .6 ,9 6 .7 and 73.6 m g a.i./K g for edible, nonedible and whole fish, respectively. Steady-state BCF values calculated from the last three days o f uptake ranged from 484 in edible tissue to 1124 in nonedible tissue (Table 5), however, these values were believed to be unreliable since tissue concentrations were still increasing with time. It was believed th at better estim ates o f the bioconcentration factor could be obtained by determ ining the kinetic concentration factor (BCFK). In the original report computer program BIOFAC was used to estim ate the kinetic concentration factor (BCFK), the uptake rate constant (ki), the depuration rate constant (k2), the estim ated tim e to reach 90% steady state, and the half-life for clearance. However, after the report had been finalized it was determined that using the BIOFAC program m ay not have been the best way to estim ate these param eters. Therefore, the data were reanalyzed using the graphical methods outlined in the draft OPPTS 850.1730 Guidance Document (1). These new calculations are described in A ppendix 11. Plots o f the tissue concentrations versus tim e were constructed to evaluate the data. The raw data were plotted as weft as a curve connecting the mean values, and a curve through the predicted values from BIOFAC. This graphical approach was a sim ple way to evaluate how well the curves fit the data. The end o f the uptake phase (Day 62) was a key date to evaluate, since data from this day has a pronounced influence on the estim ates o f the uptake and depuration rate constants (&i and k2). The plotted results in Figures 1-3 show that the BIOFAC model predicted values for Day 62 are lower than the Day 62 means for all three tissue types (whole, edible, and non-edible fish tissue). Thus, the BIOFAC model slightly underestimated the uptake and depuration rate constants, and overestim ated die kinetic concentration factor, the estim ated tim e to reach 90% steady state, and the half-life for clearance in the original report. The recalculated BCFK values for edible, nonedible, and whole fish were 1124, 4013, and 2796, respectively (Table 6). AMENDED 000304 'Wildlife InternationalLtd. Project Number 454A-134 -18- Recalculated estim ates o f the tim e to reach 90% o f steady state in edible tissue, nonedible tissue and whole fish were 2 8 7 ,3 8 3 , and 371 days, respectively (Table 6). During the depuration phase o f the test, estim ates o fth e tim e to reach 50% clearance ofPFO S were 86,116, and 112 days for edible tissue, nonedible tissue and whole fish, respectively (Table 6). The concentrations o f PFOS in tissues o f fish exposed to 0.87 mg a.i./L are presented in Table 7. PFOS concentrations in edible, nonedible and whole fish tissues did not appear to reach steady-state by Day 28 (Figure 4). By Day 35, all fish in the 0.87 rag a.i./L treatm ent group had been sam pled o r were dead. Consequently, the only inform ation on bioconcentration that could be determ ined from the 0.87 mg a.i./Ltreatm ent group was the Day 28 BCF, Day 28 BCF values ranged from 136 in edible tissue to 386 in nonedible tissue (Table 8). Based on the length o f tim e required to reach 90% o f steady-state in the 0.086 m g/L treatm ent group, these values underestim ate the bioconccntration potential ofPFO S. C O N C L U S IO N S Perfluorooctanesulfonate, potassium salt (PFOS) bioconcentrated in the tissues o f bluegill sunfish (Lepomis macrochirus). Bluegill exposed to 0.87 mg a.i./L were either dead or had been sampled by Day 35 o f the uptake phase. Consequently, no reliable inform ation was gained from this treatm ent group. Apparent steady-state concentrations were not reached in the 0.086 m g a.i./L treatm ent group. PFOS concentrations appeared to be increasing up to Day 62, the last day o f exposure. BCFK values for edible, nonedible, and whole fish tissues were 1124,4013, and 2796, respectively. Estim ates for time to reach 50% clearance for _ edible, nonedible and whole fish tissues were 8 6 ,1 1 6 , and 112 days, respectively. AMENDED 000305 W ildlife International, Ltd, Project Number 454A-134 -19REFERENCES 1 U.S. E nvironm ental P ro tectio n A gency. 1996. Series 850 - Ecological Effects T est Guidelines (draft), OPPTS Number 850.1730: Fish BCF, 2 A STM S ta n d ard E 1Q 22-84.1988. Standard Practicefo r Conducting Bioconcentration Tests withFishes and Saltwater Bivalve Molluscs. American Society for Testing and M aterials. 3 O ECD G uideline fo r T esting o f C hem icals 305.1996. Bioconcentration: Flow-Through Fish Test. 4 A PHA, AW W A, W PCF. 1985. StandardM ethodsfo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. A m erican W ater W orks Association. W aterPollution Control Federation, New York. 5 B IO FA C . 1991. September 19,1991 v ersio a The Dow Chemical Company, M idland, M ichigan. 000306 W ildlife International, Ltd. Project Number 454A-134 - 20T able 1 M eans o f Ranges o f W ater Q uality Param eters Sponsor Test Sustance: Test Organism: Dilution Water Uptake Phase Nominal Concentration (mg a.iA ) 3M Corporation PFOS Bluegill,Lepomis machrochirus Well Water Temperature1 DO1 c o (mg/L) PH Conductivity (pmhos/cm) Alkalinity (mg/L as CaCQj) Negative Control 21.9 7.8 8.1 21.8-22.0 6.8-8.6 7.9-8.2 324 310-330 180 174-185 Hardness (mg/L as CaCOa) 127 104-138 TOC (mg C/L) <1 <1-<1 21.8 7.8 8.1 0.10 21.7-22.0 6 .8 - 8 6 7 .9 - 8 2 21.8 7.5 8.1 1.0 21.7-21.9 6 .4 -8 .2 7 .9 -8 .2 1Temperature measured continuously in the negative control ranged from 20.0 to 22.0C. 2At a temperature of 22C1the dissolved oxygen saturation concentration is 8,7 mg/L and 60% saturation is 5.2 mg/L. 000307 -21- Project Number 454A-134 Table 2 Concentrations o f PFOS in W ater Samples During the Uptake Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lspomis macrochirus Well Water Nominal Concentration (mg a.i./L) Negative Control 0 Hour <LOQl 4 Hours <LOQ 1 <LOQ 3 <LOQ 7 <LOQ Day of Uptake 14 , <LOQ 21 <LOQ 28 <LOQ 35 <LOQ 42 <LOQ 49 <LOQ 56 <LOQ Mean Measured Concentration 62 (mg a.i./L) <LOQ <LOQ Percent of Nominal ** 0.10 0.0717 0.0791 0.0702 0.0751 0.0826 0.0680 0.110 0.0822 0.0915 0.0983 0.103 0.0853 0.0887 0.086 0.0692 0.0741 0.0734 0.0717 0.0781 0.0709 0.113 0.0843 0,0914 0.110 0.103 0.0948 0.0914 1.0 0.797 0.891 0.867 0.813 0.845 0.900 0.988 0.913 0.838 0.802 0.930 0.820 0.734 0.818 0.875 1.01 0.925 0.871 __2 ** 0.87 86 87 1The Limit o f Quantitation (LOQ) was 0.0500 mg a.i./L, 2 Samples not collected due to 100% mortality. Note: Values presented for each sampling interval are two replicate samples collected from each test chamber. 000308 W ildlife International, Ltd. Project Number 454A -134 -22Table 3 Concentrations o f PFOS in W ater Sam ples During the Depuration Phase Sponsor: Test Substance: Test Organism: Dilution Water: Uptake Phase Mean Measured Concentration (mg a.i./L) 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Day o f Depuration (mg a.i./L) 14 28 42 Negative Control <LOQ' <LOQ <DQ <LOQ <LOQ 0.086 <LO0 <L0Q "t . . ..-- 1 The Limit o f Quantitation (LOQ) was 0.0500 mg a.i./L.` <LOQ <LO0 56 <LOQ <LOQ <LO0 000303 -23- Project Number 454A-134 Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E4/N4 E5/N5 E6/N6 E7/N7 Table 4 PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L 3M Corporation PFOS Bluegill, Lepomis macrochirus Well W ate- Uptake Day Edible Tissue Concentration (m* a.i./Kg) 0 (4 hours) 0.167 0 (4 hours) 0.155 0 (4 hours) 0.144 0 (4 hours) 0.182 Edible Tissue Weight (g> 2.0545 1.8960 2.5219 1.3561 Nonedible Tissue Concentration (mga,i./Kg) 0.415 0.519 0.417 0.497 Nonedible Tissue Weight (8) 2.1153 2.2103 2.7449 1.8377 E17/N17 E18/N18 E19/N19 E20/N20 1 1 1 1 0.734 1.6517 1.68 2.0826 0.726 1.9269 1.85 2.2925 0.631 1.2074 1.72 1.8419 0.806 1.0777 2.07 1.4272 E30/N30 E31/N31 E32/N32 E33/N33 3 3 3 3 1.73 1.7643 4.59 1.8836 2.07 1.3794 5.50 1.8505 2.03 1.2621 5.47 2.0994 2.11 1.1883 5.97 1.7014 E43/N43 E44/N44 E45/N45 E46/N46 7 7 7 7 3.73 2.2291 10.2 2.7943 4.25 1.6419 10.6 1.8984 4.73 1.2994 11.9 1.6429 6.25 1.2686 15.2 1.7110 E56/NS6 E57/N57 E58/N58 E59/N59 14 14 14 14 11.4 9.07 13.7 12.6 2.0423 2.1548 1.0709 1.1804 E69/N60 21 11.7 2.1161 E70/N70 E71/N71 E72/N72 21 21 21 12.0 1.2984 12.9 1.4092 10.6 1.6268 1Whole Fish Concentration = fechble wt. X edible conc.1 + fnonedible wt. X nonedible cone.) (edible wt. + nonedible wt.) 27.3 23.2 35.3 32.6 33.3 22.7 24.6 24.4 2.5002 2.6582 1.4046 * 1,7455 2.4712 1.9396 1.9910 2.1872 ; Whole Fish Concentration1 (mg a.i7Kg) 0.293 0.351 0.286 0.363 1.26 1.34 1.29 1.53 3.21 4.04 4.18 4.38 7.33 7.66 8.73 11.4 20.2 16.9 26.0 24.6 23.3 18.4 19.8 18.5 000310 Wildlife International, Ltd. -24- Project Number 454A-134 T able 4 (C ontinued) PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water ___ _____________________________________________________ ___________________ Edible Tissue Edible Tissue Nonedible Tissue Nonedible Tissue Uptake Sample ID______________Day Concentration (mg a.iTKg) Weight (g) Concentration (mga.i./Kg) Weight (g) E82/N82 28 18.3 1.4636 49.4 1.7564 E83/N83 28 13.7 1.4113 40.7 1.8903 E84/N84 28 23.9 1.2081 65.3 1.3062 E85/N85 28 23.1 0.8830 57.9 1.3512 E9S/N95 E96/N96 E97/N97 E98/N98 35 35 35 35 22.6 1.7888 67.1 2.0434 - 27.7 1.5897 73.3 2.1281 23.8 1.1136 62.0 1.6454 20.6 1.2226 59.1 1.3627 E103/N103 E104/N104 E105/N105 E106/N106 42 42 42 42 27.6 1.2037 64.0 1.9474 25.3 1.4942 68.1 1.9266 21.2 1.4367 54.4 2.0920 27.6 1.3033 79.6 1.6100 E lll/N lll E112/N112 E113/N13 E114/N114 49 49 49 49 33.3 1.7249 85.0 2.2873 36.2 1.2185 95.1 1.5993 39.0 1.5506 93.1 2.2184 30.6 1.4650 77.7 1.9337 El 19/N119 E120/N120 E121/N121 E122/N122 56 56 56 56 48.3 1.6058 122 2.1688 38.9 1.0946 94.2 1.5831 44.1 1.0521 73.2 2.0532 38.3 1.8592 106 2.1739 E127/N127 E128/N128 E129/N129 E130/N130 62 62 62 62 42.4 1.2209 66.2 1.3902 42.2 1.0832 39.2 1.6697 1Whole Fish Concentration = (edible wt. X edible conc.I+ (nonedible wt. X nonedible cone.1) (edible wt. + nonedible wt.) 101 112 105 96.4 1.7596 1.9170 1.5965 2.4378 Whole Fish Concentration1 (mga.i./Kg) 35.3 29.2 45.4 44.1 46.3 53.8 46.6 40.9 50.1 49.4 40.9 56.3 62.8 69.6 70.8 57.4 90.6 71.6 63.3 74.8 77.0 92.7 79.6 73.1 000311 -25- Project Number 454A-134 Table 4 (Continued) PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E135/N135 E136/N136 E137/N137 E138/N138 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Edible Tissue Uptake Concentration Day (mg a.i./Kg) 14 48.5 14 31.8 14 31.6 14 42.0 Edible Tissue Weight (g) 1.3634 1.4503 1.2626 0.9340 Nonedible Tissue Concentration (mg a.i./Kg) 124 79.4 81.8 113 E143/N143 E144/N144 E145/N145 E146/N146 28 28 28 28 26.0 1.7691 85.7 33.3 1.1358 95.1 38.7 0,8845 85.7 55.8 0.6909 94.8 E150/N150 E151/N151 E1S2/N152 E153/N153 42 42 42 42 24.1 31.2 30.0 33.0 1.7590 1.1220 0.8440 0.8672 71.7 80.6 78.3 82.1 E157/N1S7 E158/N158 E159/N159 E160/N160 56 56 56 56 21.1 1.5471 37.6 0.5892 32.9 0.6244 31.2 0.7223 1Whole Fish Qpncentrfttion " ^x ^ l f (nonedjble wt, %nQnedjble^pnJ (edible wt. + nonedible wt.) 57.7 80.3 85.4 84.4 Nonedible Tissue Weight (g) 1.6928 2.1909 1.8708 1.4560 2.0744 1.6772 1.4801 1.2803 2.3578 1.7635 1.5181 1.2687 1.9735 1.2353 1.0439 0.9975 Whole Fish Concentration1 (mg a.i./Kg) 90.3 60.4 61.6 85.3 58.2 70.1 68.1 81.1 51.4 61.4 61.0 62.2 41.6 66.5 65.8 62.1 000312 W ildlife International, Ltd, Project Number 454A-134 -26- Table 5 Apparent Steady-State BCF Values for Bluegill Exposed to 0.086 mg a.i7L Sponsor: T est Substance: Test Organism: Dilution W ater: Tissue Type Edible 3M Corporation PFOS Bluegill, Lepomis macrochirus W ell W ater M ean M easured Test C oncentration (mg &./L) 0.086 U ptake Days at A pparent Steady-State 4 9 ,5 6 and 62 M ean M easured A pparent Steady- State Tissue Concentration - (m g a.i./Kg) 41.6 A pparent Steady-State BCF 484 N onedible 0.086 4 9 ,5 6 and 62 96.7 1124 W hole Fish 0.086 4 9 ,5 6 and 62 73.6 856 000313 W ildlife International, Ltd. Project Number 454A -134 -27- Table 6 BCFK Estim ates for B luegill Exposed to 0.086 m g a.i./L Sponsor: 3M Corporation T est Substance: PFOS Test Organism: Bluegill, Lepomis m acrochins Dilution W ater: W ell W ater K in etic Bioconcentration U ptake R ate Factor C onstant Tissue Type (B C FK ) ft) Depuration Rate C onstant ft) E d ib le 1124 9.022 0.0080 Estim ate Time to Reach 90% o f Steady State (D ays) 287 Estim ated T im e to Reach 50% C learan ce (D ays) 86 N o n ed ib le W hole Fish 4013 2796 24.08 17.35 0.0060 0.0062 383 371 116 111 AMENDED 000314 Wildlife International,Ltd. Project Number 454A-134 -28- Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E 9/N 9 E 10/N 10 H U /N il E 12/N 12 T able 7 PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues o f Bluegill Exposed to 0.87 mg a.i./L 3M Corporation PFOS Bluegill, Lepomis macrochtrus W ell Water Edible Tissue Edible Tissue Nonedible Tissue Nonedible Tissue Concentration W eight Concentration W eight Uptake Day 0 (4 hours) 0 (4 hours) 0 (4 hours) 0 (4 hours) (mg a.i,/Kg) 1.46 1.48 1.19 1.39 (8) 0.9795 1.3226 1.6284 1.5205 (mg a.i./Kg) 3.52 4.37 4.22 4.06 (g) 1.5183 1.6397 1.9391 1.9489 E 22/N 22 E23/N23 E 24/N 24 E 25/N 25 1 1 1 1 4.68 1.7060 11.1 1.8323 6.59 1.3724 14.2 1.7515 5.56 1.1272 13.3 1.7081 5.64 1.0507 12.1 1.5273 E 35/N 35 E 36/N 36 E37/N37 E 38/N 38 3 3 3 3 17.3 0.9889 39.3 1.4781 15.8 1.2666 42.0 1.6727 19.0 0.8348 43.8 1.4005 20.8 1.1215 51.8 1.6070 E 48/N 48 E 49/N 49 E S 0/N 50 E51/N51 7 7 7 7 42.0 1.7575 44.0 1.5527 57.7 1.3391 46.8 0.9532 100 ` 2.3064 102 2.0505 102 2.2079 120 1.3562 E61/N61 E 62/N 62 E 63/N 63 P64/N 64 14 14 14 14 87.1 81.6 90.7 73,3 1.9813 1.2152 1.1290 1.0811 1Whole Fish Concentration = (edible wt, X ediblecpnc.) + (nonedible w L X nonedible.can cj (edible w t.+ nonedible wt.) 177 207 245 214 2.9193 1.8260 1.5376 1.6199 W hole Fish Concentration1 (mg a.i./Kg) 2.71 3.08 2.84 2.89 8 .0 0 10.9 10.2 9.47 30.5 30.7 34.5 39.1 7 4 .9 7 7 .0 85.3 89.8 141 157 180 158 000315 Wildlife International, Ltd. Project Number 454A-134 -29- T able 7 (C ontinued) PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues o f Bluegill Exposed to 0.87 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E74//N74 E75/N75 E76/N76 E77/N77 3M Corporation PFOS Bluegill, Lepomismacrochirus Well Water Edible Tissue Concentration Uptake Day___________(mg a.iJKg) 21 79.4 21 117 21 104 21 102 Edible Tissue Nonedible Tissue Weight Concentration (g)______________ (mg ai^Kg) 1.8300 . 201 1.4526 278 1.6824 246 1.4076 229 Nonedible Tissue Weight (g) 2.1803 1.9786 22130 1.7558 E87/N87 E88/N88 E89/N89 E90/N90 28 28 28 28 102 1.6679 131 1.0923 107 1.1675 133 1.1466 1Whole Fish rnnrentretw = (edible wt. X edible cone.) + (nonedible wt, X nonedible cone.) (edible wt. + nonedible w t) 289 372 320 361 2.0549 1.4045 1.6595 1.5186 Note: Sampling ended after Day 28. By Day 35 all the fish in this treatment group had died or were previously sampled for tissue analysis. Whole Fish Concentration1 (mg a.iVKg) 146 210 18S 172 205 267 232 263 000316 W ildlife International, Ltd. Project Number 454A-134 -30- T able 8 Day 28 BCF V alues for Bluegill Exposed to 0.87 mg a.i./L Sponsor: 3M Corporation T est Substance: PFOS T est Organism: Bluegill, Lepomis macrochirus D ilution W ater: W ell W ater M ean M easured T est Concentration Tissue Type (mg a.i./L) Edible 0.87 M ean Day 28 Tissue C o n cen tratio n (mg a/K g) 118 Non Edible 0.87 336 W hole Fish 0.87 242 Note: BCF Values presented here are biased low due to fish m ortality. Day 28 BCF 136 386 278 000317 Wildlife International, Ltd. -31- Project Number 454A-134 Figure 1. Concentrations o f PFOS in Edible Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L. 000318 AMENDED Wildlife International, Ltd. -32- Project Number 454A-134 Figure 2. Concentrations o f PFOS in Nonedible Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L. 000319 AMENDED \ Wildlife International, Ltd. -33- Project Numb1454A-134 Figure 3. Concentrations o f PFOS in W hole Fish Tissues o f Bluegill Exposed to 0.086 m g a.i./L. PFOS Concentration (mg/Kg 000320 AMENDED Wildlife International, Ltd. Project Number 454A-134 -34Figure 4. Concentrations o f PFOS in Edible, Noneible and W hole Fish Tissues o f Bluegill Exposed to 0.87 mg a.i./L. 000321 W ildlife International, Ltd. Project Number 454A-134 -35- A ppendix 1 Specific Conductance, Hardness, A lkalinity and pH o f W ell W ater M easured During the 4-W eek Period Im m ediately Preceding the Test Sponsor: T est Substance: T est Organism: D ilution W ater: 3M Corporation PFOS Bluegill, Lepomis macrochirus W ell W ater M ean Range Specific Conductance (pm hos/cm ) 313 (N = 4) 3 1 0 -3 1 5 H ardness (m g/L as C aC 03) 130 (N = 4) 128-132 A lkalinity (m g/L as CaCOj) 178 (N = 4) 1 7 6 -1 7 8 pH 8.1 (N = 4) 8 .0 -8 .2 000322 W ildlife International, Ltd. -36- A ppendix 2 _________A* nalyses o~ fPesticides, O--rpg--aniwcs ~and M e..t..a..ls. inW?i-l-d-l-i-f-e Inter-n-a--t-i-o-n--a--l,7Lt--d. W--e--ll W^ -a--t-err1 ............... Pesticides and Organics Component Aclonifen Alachlor Amebyn Atrazinc Azinphos-ethyl Azinphos-methyi Azoxystrobin Bifcnthrin Bioallcthrin Bitcrtano! Bromacil Bromophos Bromophos-cthyi Bromopropylate Bupirimate Carbary! Carbofuran Carboxin Cblorfenvinphos Chloridazon Chlorpropham Chloipyrifos Chlorpyrifos-methyl Chlorothalonil Coumaphos Cyanazine Cyfluthrin Cypermethrin Cyproconazole Deltamcthrin Demeton Demeton-O Desethyfatrazine Dcsisopropylatrazinc Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid Measured Concentration <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.2S pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <025 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L Component Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodcmorph Endosulfan-a Endosulfen-P Endosulfan-sulfale Epoxiconazole Eptam Esfenvalcrate Ethion Ethofumcsate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrotbion Fcnoxycarb Fenpiclonil Fenpropathrin Fenpropimorph Fenthion Fenvalerate Fluazifop-butyl Fluoroglycofcn-cthyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Measured Concentration <0.01 pg/L <025 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L 0 .0 2 pg/L 0 .0 5 pg/L 0 .0 2 pg/L 0 .0 1 pg/L 0 .0 2 pg/L 0 .0 2 pg/L 0 .0 1 pg/L 0 .0 5 pg/L 0 .0 2 pg/L 0 .0 5 pg/L 0 .0 2 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. Continued 000323 W ildlife International, Ltd. -37- A ppendix 2 (C ontinued) Analyses o f Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater1 PesticdesAnd Organics (Page 2) Component Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procyraidon Promcttyn Propachlor Preparine Propham Propiconazole Propoxur Propyzamide Prosulfocarb Pyrazophos Measured Concentration <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L" <0.02 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Component Mcthoxychlor Mctolachlor Metriburin Mevinphos Nitiothal-Isopropyl Pyrifersox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylarinc Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos TrifluraJin Vamidothion Vinclozolin Measured Concentration <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0,01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 13.2 mg/L 21 mg/L 35 mg/L <0.02 mg/L 6.2 mg/L <0.09 mg/L 0.72 pg/L <0.3 pg/L <0.6 pg/L <0.4 pg/L Metals Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.4 pg/L <1.0 pg/L <2.3 pg/L <0.7 pg/L <0.3 pg/L <0.3 pg/L <0.25 pg/L <0.025 pg/L <1 pg/L `Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. 000324 W ildlife International, Ltd. -38- Appendix 3 THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) CONCENTRATIONS IN FRESHW ATER AND BLUEGELL SUNFISH TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD, PROJECT NO.: 454A-134 000325 Wildlife International, Ltd. Project Number 454A-134 SPONSOR: -39REPORT APPROVAL 3M Corporation TITLE: Perfiuorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the Bluegill {Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 PRINCIPAL INVESTIGATOR: [L. VanHoven, Ph.D. Scientist WILDLIFE INTERNATIONAL. LTD. MANAGEMENT dialog D ate W illard B. Nixon, Ph,D / Director, Analytical Chemistry D ate' ' AMENDED 000326 W ildlife International, Ltd. -40- In tro d u c tio n F resh w ater sam ples and tissu e sam ples w ere collected from a flow -through aq u atic te s t to determ ine th e bioconcentration p otential o f perfluorooctanesulfonate, potassium salt (P F O S) in th e bluegill (Lepom is m acrochirus). T he study w as co n d u cted b y W ildlife In tern atio n al, L td . and identified as P ro ject N um ber 454A -134. T he analyses o f fresh w ater and tissu e sam ples w ere perform ed a t W ildlife In tern atio n al, L td . by h ig h perform ance liquid chrom atography (H P L C ) w ith m ass spectrom etric detection. W ater sam ples w ere diluted and analyzed by H PL C w ith single quadrupole m ass spectrom etric detection (L C /M S). T issue sam ples w ere hom ogenized, ex tracted , diluted, and analyzed by H PLC w ith trip le quadrupole m ass spectrom etric d etectio n (L C /M S/M S ). F reshw ater sam ples w ere collected and analyzed from D ecem ber 1 ,2 0 0 0 to A pril 2 ,2 0 0 1 . T issue sam ples w ere collected and analyzed from D ecem ber 5, 2000 to A pril 2, 2001. A dditional tissu e sam ples w ere collected and analyzed gravim etrically fo r lipid content. Sam ples fo r lipid co n ten t w ere collected o n D ecem ber 5 ,2 0 0 0 , F ebruary 5 ,2 0 0 1 and A pril 2 ,2 0 0 1 and analyzed betw een A pril 4 and A pril 1 1 ,2 0 0 1 . T est Substance and In tern al S tan d ard T he te st substance, PFO S, w as used to prepare calibration standards and m atrix fortification sam ples and w as identified as W ildlife International, L td. identification num ber 4675. T he internal standard w as received from 3M C orporation on July 2 ,1 9 9 8 and w as assigned W ildlife In tern atio n al L td. identification num ber 4 526 u p o n receipt. T he in tern al standard, a g ranular m aterial, w as identified as: 1H, 1H, 2H , 2 H P erflu o ro o ctan e S ulfonic A d d , C hem ical A b stract N um ber: 27619-97-2. T he internal standard, referred to hereafter as 4H PFO S, w as sto red under am bient conditions. A nalytical M ethod W ater and tissu e sam ples w ere analyzed fo r PFO S using high perform ance liquid ch ro m ato g rap h y (H P L C ) w ith m ass sp ectro m etric d etectio n . W ater sam ples w ere analyzed according to th e m ethod entitled "A nalytical M ethod V alidation fo r th e D eterm ination o f P erflu o ro o ctan e S ulfonic A cid, P otassium S alt (P F O S ) in F resh w ater, S altw ater and A lgal M edia" (W ildlife International, L td. P ro ject N o. 454C -109). T issue sam ples w ere analyzed according to th e m ethod en titled " A nalytical M eth o d V alidation fo r th e D eterm in atio n o f P erflu o ro o ctan e S u lfonic Acid, 000327 W ildlife International, Ltd. - 41 - Potassium Salt (PFOS) in Fish Tissues" (W ildlife International, Ltd. Project No. 454C -119). For water analyses, one m inor m odification from the validation was incorporated into the present study. This change was use o f 10 ppb in place o f 100 ppb 4HPFOS internal standard concentration in all calibration standards and study sam ple dilutions. The lower internal standard nominal concentration was closer to the range o f PFOS calibration used in the present work. The analytical methodology implemented for the determ ination o f lipid content in fish tissue is presented on page 42. F reshw ater Sam ples A m ethod flow chart for the analysis o f PFOS in freshw ater is presented in Figure 1. Sam ples were diluted in a 50% m ethanol (HPLC grade, 99.9+% ): 50% NANOpure w ater solution containing 10.0 pg 4H PFOS/L and 0.05% form ic acid (v/v) so th at they fell w ithin the calibration range o f the LCMS methodology. A liquots o f the dilutions were transferred to autosam pler vials and subm itted for analysis by direct injection. Concentrations o f PFOS in freshwater samples were determ ined by reverse-phase high perform ance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chrom atograph (HPLC) interfaced w ith a Perkin-Elm er API 3000LC m ass spectrom eter operated in selective ion m onitoring (SIM ) detection mode. The m ass spectrom eter was equipped w ith a Perkin-Elm er TurbolonSpray ion source. Chrom atographic separations were achieved using a K ey sto n e B etasil C m analytical column (50 mm x 2 mm I.D ., 3 pm particle size) fitted with a Keystone Javelin C Guard Cartridge (20 mm x 2 mm). The instrum ent param eters are summarized in Table 1. F reshw ater quality co n tro l (Q C ) sam ples (m atrix blanks and fortifications) w ere p rocessed in th e sam e m anner a s th e te s t sam ples. F resh w ater w as fortified w ith th e ap p ro p riate P FO S in m ethanol sto ck solution using a g as-tig h t syringe. M atrix blank sam ples w ere n o t fortified w ith th e te st s u b sta n c e . T issue Q C Sam ples B iuegill sunfish (Lepom is m acrochirus) tissu e s w ere obtained from a breeding stock m aintained a t W ildlife Intern atio n al, L td . A pproxim ately 2 0 0 individual fish (ag es 3 to 5 m onths) w ere rem oved from a breeding tan k and euthanized by severing th e spinal cord above the o p ercu lar region. H eads, fins and viscera w ere rem oved from th e body and w ere considered to b e nonedible tissue. T he balance o f th e tissu e w as considered to b e edible tissue. T he edible and nonedible tissues w ere separately 000328 W ildlife International, Ltd. -42- hom ogenized w ith a W aring stainless steel blender. A n appropriate num ber o f 1-gram aliquots o f edible and nonedible hom ogenate w ere w eighed in to separate, 20-m L glass scintillation vials. E ach vial w as uniquely identified and labeled w ith a facility lo g identification num ber. T he tissue Q C sam ples w ere stored frozen until they w ere used in th e preparation o f m atrix blank and m atrix fortification sam ples, o r sto rag e stability sam ples. T issue Sam ples A m ethod flow ch art fo r th e analysis o f PFO S in fish tissu es is presented in F igure 2. E dible and nonedible fish tissu e sam ples w ere collected from th e te st in 20-m L glass scintillation vials and sto red frozen, if necessary, until analysis. U pon analysis, tissu e sam ples w ere batched by sam pling interval and tissu e type. A t th at tim e, th ree o f th e appropriate tissue Q C sam ples also w ere rem oved from th e freezer. A ny frozen sam ples w ere allow ed to thaw . O ne tissu e Q C sam ple w as designated as th e m atrix blank sam ple. T he o th er tissu e Q C sam ples w ere designated as m atrix fo rtificatio n sam ples and w ere fortified w ith th e appropriate P FO S sto ck so lu tio n s) in m ethanol using g as-tig h t syringes. T est and Q C tissu e hom ogenates w ere extracted as follow s. T en m illiliters o f m ethanol w ere added to each vial. T he sam ples w ere hom ogenized w ith a tissue shredder fo r approxim ately one m inute. T he sam ples w ere th en sonicated w ith a sonic dism em brator fo r approxim ately five m inutes. T he sam ples w ere capped, shaken, and centrifuged a t approxim ately 2000 rp m fo r approxim ately 5 m inutes. A liquots o f th e ex tract w ere th en volum etrically diluted in to th e calibration range o f th e L C M S m ethodology w ith 50:50 m ethanol: N A N O pure w a te r dilution solvent. A liquots o f th e diluted ex tracts w ere tran sferred to autosam pler vials and subm itted fo r analysis. B ack-up tissu e sam ples w ere collected and sto red frozen. I f necessary, th e back-up tissue sam ples w ere rem oved from the freezer, allow ed to thaw and processed using th e sam e p rocedures described above. T issue Storage Stability Sam ples Stability sam ples w ere prepared at te s t initiation to establish te s t substance stability in fish tissues stored frozen during the study. T w o tissue Q C sam ples o f each fish tissu e type (edible and nonedible) w ere rem oved from the freezer and allow ed to thaw . The edible and nonedible fish tissues 000323 W ildlife International, L td -43- w ere each fortified a t 0.100 and 10.0 m g a.i./K g u sing th e appro p riate PFO S sto ck so lu tio n and a gastig h t syringe. T he stability sam ples w ere retu rn ed to th e freezer. Follow ing 119 days o f frozen sto rag e, th e stability sam ples w ere rem oved from th e freez er and analyzed. F resh fo rtificatio n and m atrix b lank sam ples also w ere p rep ared and analyzed a t th is tim e. T he sam ples (new and old) w ere p ro cessed using th e sam e p ro ced u res described fo r tissu e sam ple analyses. C oncentrations o f PFO S in fish tissu e w ore determ ined by reverse-phase high perform ance liquid chrom atography using a H ew lett-P ackard M odel 1100 H PL C interfaced w ith a P erkin-E lm er A P I 300OLC M ass S p ectro m eter o p erated in m ultiple io n reactio n m onitoring (M R M ) d etection m ode. T he m ass spectrom eter w as equipped w ith a Perkin-E lm er T urboIonS pray io n source. C hrom atographic sep aratio n s w e re achieved using a K eystone B etasil C l 8 analytical colum n (50 m m x 2 m m I.D ., 3 p m p artic le size) fitte d w ith a K ey sto n e Javelin Ci* G u ard C artrid g e (2 0 m m x 2 m m ). T he instrum ent param eters are sum m arized in T able 2. T issue Sam ples for L ipid C ontent A m ethod flo w -ch art fo r th e analysis o f lipid co n ten t in fish tissu e is p resen ted in F ig u re 3. E dible and nonedible fish tissu e sam ples to be analyzed fo r lipid co n ten t w ere co llected from th e te st at initiation, on D ay 62 o f th e u p tak e p h ase (D ay 0 depuration), and a t term ination (D ay 56 dep u ratio n ). O nly fish from th e N egative C o n tro l and 0.10 m g a.i./L nom inal te st level w ere sam pled fo r lipid content. Sam ples w ere collected in 20-m L g lass scintillation vials and sto red fro zen u n til analysis. Sam ple w eights w ere reco rd ed a t th e tim e o f collection. U pon analysis, tissu e sam ples desig n ated fo r lipid co n ten t determ ination w ere rem oved from th e freezer and allow ed to thaw . F o r each sam ple, 10 m L ofN A N O pure w ater w as added to th e fish tissu e in th e vial and th e sam ple w as hom ogenized for approxim ately one m inute using a tissu e shredder. E ach hom ogenate w as tran sferred to a 250-m L separatory funnel th a t contained 25 m L o f chloroform and 50 m L o f m ethanol. E ach sam ple vial w as rinsed w ith an additional 10 m L ofN A N O pure w ater and th e rinse w as poured into th e respective sep arato ry funnel. T he sep arato ry funnels w ere shaken w ith venting fo r approxim ately o n e m inute. F ifty m illiliters o f chloroform follow ed by 50 m L o f satu rated sodium chloride w ere added to each sep arato ry funnel. T he sep arato ry funnels w ere briefly sw irled w ith venting. T he phases w ere allow ed to sep arate. F o r each sam ple, th e chloroform layer w as drained th ro u g h a pow der funnel packed w ith 000330 W ildlife International, Ltd. -44- glass w ool and anhydrous sodium sulfate in to a 250-m L round- bottom flask. A n additional 50-m L aliquot o f chloroform w as added to each separatory funnel and th e extraction and draining procedures w ere rep eated . T h e ex tracts w ere ro ta ry evap o rated in a w aterb ath m aintained a t approxim ately 40 C to n ear dryness. E ach ex tract w as tran sferred to a pre-w eighed labeled scintillation vial. E ach250-m L ro u n d -b o tto m flask w as rinsed w ith a sm all volum e o f chloroform and th e rinse w as tran sferred to th e respective scintillation vial. T he rem aining solvent in each vial w as evaporated u n d er a g entle stream o fn itro g e n o rc le a n d ry a ir. The vials w ere rew eighed and th e w eights w ere recorded. L ipid content w as calculated fo r each sam ple as th e ratio o f lipid to fish tissu e w eights (m g/K g). C alibration Standards F o r w ater analyses, calibration standards o f P FO S prepared in a 50% m ethanol : 50% NAN Opure w a te r solution containing 10.0 p g 4H P FO S/L and 0.05% form ic acid (v/v), ranging in con cen tratio n from 0.500 to 5.00 p a.i./L , w ere analyzed w ith th e each sam ple set. P FO S w as fortified in to th e standards b y ap p ro p riate dilutions o f a 1.00 m g a.i./L sto ck so lu tio n o f P FO S in m ethanol. F ive calibration standards (different concentrations) w ere analyzed w ith each sam ple set. T he calibration standard series w as injected at th e beginning and end o f each run, and o n e standard w as in jected , a t a m inim um , a fte r every five sam ples. L in ear reg ressio n eq u atio n s w ere g en erated using peak area response ratio s (P F O S : internal standard) versus th e respective concentration ratios (PFO S: internal standard) o f th e calibration standards. A typical calibration curve from w ater analyses is presented in F igure 4. T he concentration o f PFO S in th e fresh w ater sam ples w as determ ined by su b stitu tin g th e p eak area response ratio s in to th e applicable lin ear regression equation. R epresentative ion chrom atogram s o f low and high calibration standards used fo r freshw ater analyses are presented in F igures 5 and 6, respectively. F o r tissu e analyses, calibration stan d ard s w ere p rep ared in 50:50 m ethanol: N A N O pure fw a te r by ap p ro p riate dilutions o f eith er a 1.00 m g a.i./L o r a 10.0 m g a.i./L sto ck so lu tio n o f P FO S in m ethanol. T w o separate PFO S calibration standard sets w ere em ployed in th e study. F o r D ays 0 th ro u g h 7 o f th e u p tak e phase, and fo r stability sam ple analyses, P FO S calibration standards ranging in concentration from 0.500 to 5.00 p g a.i./L , w ere analyzed w ith each sam ple set. F o r all subsequent sam pling intervals, a higher (5.00 to 50.0 PFO S p g a.i./L ) calibration range w as required to m inim ize dilution o f th e sam ple extracts. F o r each sam ple set, five calibration standards (different 000331 W ildlife International, Ltd. -45- co n cen tratio n s) w ere analyzed. T h e ap p ro p riate calib ratio n stan d ard series w as injected a t th e beginning and end o f each ru n , and o n e stan d ard w as in jected, a t a m inim um , afte r every five sam ples. L inear regression equations w ere generated using th e peak area responses versus th e respective con cen tratio n s o f th e calibration standards. A typical calibration curve from tissu e analyses is presented in F igure 7. C oncentration o f PFO S in fish tissu e sam ples w as determ ined by substituting th e peak area response into th e applicable linear reg ression equation. R epresentative io n chrom atogram s o f low and high calibration standards u sed fo r tissue analyses are p resented in F igures 8 and 9, respectively. F o r b o th w ater and tissu e analyses fo r P F O S , th e sam e and m o st prom inent p eak resp o n se fo r PFO S w as utilized to m o nitor PFO S in all calibration, quality con tro l, and stu d y sam ples. N o attem pt w as m ade to quantify PFO S o n th e basis o f individual isom eric com ponents. F ortification Stocks F reshw ater and fish tissu e hom ogenates w ere fortified w ith th e appropriate stock solution o f PFO S prep ared in m ethanol. E ach sto ck solution w as assigned a unique identification co d e th a t w as reco rd ed o n a sto ck p rep aratio n lo g sheet. L im its o f Q u a n tita tio n T he m ethod lim it o f q u antitation (L O Q ) fo r PFO S in fresh w ater sam ples w as 0.0500 m g a l/L , calculated as th e p ro d u ct o f th e low est PFO S calibration standard (0.0005 m g a.i./L ) and th e dilution fa c to r o f th e m atrix blank sam ple (100) analyzed concu rren tly w ith th e te s t sam ples. T he L O Q w as calculated o n an individual basis fo r each tissu e sam ple since each en tire subm itted sam ple ( o f differing w eig h t) w as ex tracted w ith o u t an adjustm ent to a co n stan t w eight. T he LO Q (m g a.i./K g) fo r a given tissu e analysis w as calculated as th e p ro d u ct o f th e low est PFO S calibration standard (0.0005 o r 0.005 m g a.i./L ) and th e overall dilution facto r (L /K g) o f th e tissu e sam ple. T o illu strate fo r a 1.207 gram sam ple analyzed w ith th e low -level calibration standard set, ex tra ctio n w ith 10 m L s o f m ethanol follow ed by a 1Ox v o lu m etric d ilu tio n (o v erall d ilu tio n fa c to r = 82.85 L /K g), gives an L O Q = 0 .0414 m g a.i./K g. 000332 W ildlife International, Ltd. -46- Freshw ater M atrix B lank and F ortification Sam ples A long w ith th e actual freshw ater sam ple analyses, 20 fresh w ater m atrix blank sam ples w ere analyzed a t periods throughout the study to determ ine possible interferences (T able 3). N o m atrix interferences w ere observed a t o r above th e lim it o f quantitation (0.0500 m g a.i./L ). A representative io n chrom atogram o f a freshw ater m atrix blank sam ple is presented in F igure 10. F reshw ater sam ples w ere fortified at tw o levels (0.0750 and 0.500 m g a.i./L ) o r th ree levels (0 .0 7 5 0 ,0 .5 0 0 and 2 .00 m g a.i./L ) a t each sam pling interval using appropriate stock solutions ofP F O S prep ared in m ethanol. F reshw ater m atrix fortifications w ere analyzed concurrently w ith each sam ple set and th e analytical resu lts w ere n o t co rrected fo r m ean procedural recovery. R ecoveries ranged from 84.9 to 120% o f nom inal concentration (T able 3). T he m ean and standard deviation o f fortification recoveries a t th e 0 .0 7 5 0 ,0 .5 0 0 and 2 .00 m g a.i./L fortification levels w ere 94.7% 5 .1 2 % (n = 2 0 ), 98.7% 6.60% (n = 2 0 ), an d 96.9% 2.9 5 % (n = 12), respectively. A rep resen tativ e ion chrom atogram o f a freshw ater m atrix fortification is presented in F igure 11. T issue M atrix B lank and F ortification Sam ples A long w ith th e actual tissu e sam ple analyses, 16 edible and 16 nonedible fish tissu e m atrix blank sam ples w ere analyzed to determ ine p o sa b le interferences (T ables 4 and 5, respectively). N o interferences w ere observed a t o r above th e applicable lim its o f quantitation during th e test. R epresentative ion chrom atogram s o f edible and nonedible fish tissue m atrix blank sam ples A re presented in Figures 12 and 13, respectively. E dible and nonedible fish tissu e hom ogenates w ere fortified w ith the appropriate stock so lu tio n ^ ) o f PFO S prepared in m ethanol. T issue m atrix fortifications w ere prepared and analyzed concurrently w ith each sam ple set and th e analytical resu lts w ere n o t corrected fo r m ean procedural recovery. The levels o f fortification w ere chosen to bracket th e expected concentration o f test sam ples. B ecause m easured concentrations increased w ith tim e for the duration o f th e u ptake phase, th e fortification levels w ere frequently revised. L ow -level fortification levels ranged from 0 .100 to 5.00 m g a.i./L and high-level fortification levels ranged from 10.0 to 500 m g a.i./L . E dible fish tissu e recoveries ranged from 94.8 to 111% o f nom inal concentration fo r th e low -level fortifications, and 000333 I Wildlife International, Ltd. -47- fro ra 9 2 .0 to 107% o f nom inal concentration fo r th e high-level fo rtificatio n s (T able 4 ). T he m ean and stan d ard deviation o f fo rtificatio n recoveries a t th e low - and high-level fortifications (n = 16) fo r th e edible fish tissu e w ere 102% 4 .6 1 % and 100% 4 .4 0 % , respectively. N onedible fish tissu e reco v eries ranged from 9 3 .9 to 116% and from 90.1 to 122% o f nom inal co n cen tratio n fo r lo w - an d high-level fortifications, respectively (T able 5). T he m ean and standard deviation o f fo rtificatio n recoveries at th e low - and high-level fortifications (n = 16) fo r th e nonedible fish tissu e w ere 106% 5.55% and 103% 8.49% , respectively. R ep resentative io n chrom atogram s o f edible and nonedible fish tissu e m atrix fortification sam ples a re p resented in F igures 14 and 15, respectively. Exam ple C alculations T he analytical resu lt and p ercent recovery fo r freshw ater sam ple 4 5 4 A -134-68, from th e 0 .10 m g a.i./L nom inal PFO S treatm ent group, w as calculated using th e follow ing equations: Peak A rea Ratio = Analyte Peak A rea/Intem al Standard Peak Area PFOS Area = 593240 Internal Standard A rea = 1295042 Peak area ratio = 0.4581 Internal Standard Concentration: 10.0 ng a.i./L Y -intercept = -0.0187 Slope = 5.2182 Initial volume (Vi) = 0.100 mL Final volume (Vf) = 10.0 mL D ilution factor (VfTVi) = 100 PFOS(/iga.i/L)at Instrument = * * -- * > * 1 ^ 5 ^ C oncent 0.4S 81-(-0.0187):c]i0 5.2182 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at Instrum ent x Dilution Factor = 0.0009137x100 = 0.09137 PFOS (mg a,i./L )in sam ple Percent o f Nom inal Concentration = xlO O PFO S (mg a.i./L ) nominal 0,09137 mg a .i./L _,100 0.10m ga.i./L = 91.4% 000334 Wildlife InternationalLtd. -48- T he analytical re su lt fo r edible fish tissu e sam ple 454A -134-E -43, from th e 0 .1 0 m g a.i./L nom inal P FO S treatm en t g ro u p , w as calculated using th e follow ing equations: Peak A rea = 11192 Y -in tercep t = 713.81 S lope = 6287.33 P rim ary D ilution: In itial W eight (W i) = 2.2291 g E x trac tio n V olum e (V e) = 10.0 m L S econdary D ilution: In itial V olum e (V i) * 0.0500 m L F inal V olum e (V f) = 2 5 .0 m L OveraU D ilu tio n F acto r (V e/W i x W V i) = 2 2 4 0 m L /g = 2 2 4 0 L /K g ,,,,,,,, . w ,, x (peak area - y - intercept) PFO S a t instrum ent (//g a.i./L )= ------------- -------------- -- (1 1 1 9 2 -7 1 3 .8 1 ) 6287.33 = 1667 p g a.i./L PFO S in sam ple (m g a.i./K g) = PFO S at instrum ent (p g a.i./L ) x overall dilution fa cto r x unit conversion facto r 1.667jug a.i. 2440L x _ h n g _ PFO Sin sam ple (mg a.i./K g) = L Kg 1000 /g = 3.73 m g a.i./K g R ESU LTS Freshw ater Sam ple A nalysis F reshw ater sam ples w ere collected and analyzed fo r PFO S concentrations o n D ays -4 and -1 (tw ice) during th e pre-uptake phase o f th e test and D ays 0 (0 and 4 hours), 1 , 3 , 7 , 1 4 , 2 1 , 2 8 , 3 5 , 4 2 , 4 9 ,5 6 , m id 6 2 during th e u p tak e p hase o f th e te s t. U p tak e w as suspended o n D ay 62 o f th e te s t and depuration began. D uring the depuration phase o f th e te st, freshw ater sam ples w ere collected and analyzed fo r PFO S concentrations o n D ays 1 4 , 2 8 , 4 2 , and 56. M easured concen tratio n s o f PFO S in th e p re -test d ilu ter verification (pre-u p tak e p hase) sam ples w ere <LO Q in th e negative controls. M easured concentrations in p re -te st sam ples ranged from 6 4 .6 to 95.4% o f th e nom inal concentration 000335 Wildlife InternationalLtd. -49- in th e 0 .1 0 m g a.i./L treatm en t g roup and from 71.8 to 111% o f th e nom inal co n centration in th e 1.0 m g a.i./L treatm en t g ro u p (T able 6). D uring th e u p tak e p h ase o f th e te st, m easured concentrations o f PFO S in th e negative co n tro l freshw ater sam ples w ere <LO Q . F reshw ater sam ples collected from th e 0 .1 0 and 1.0 m g a .i./L treatm en t g ro u p s ran g ed from 6 8 .0 to 113% and from 7 3 .4 to 101% o f th e nom inal concentration, respectively (T able 7). D uring th e dep u ratio n phase o f th e te s t, m easured co n cen tratio n s o f PFO S in all freshw ater sam ples w ere <LO Q . A representative ion chrom atogram o fa freshw ater sam ple is presented in F ig u re 16. T issu e S am p le A nalysis T issue sam ples w ere collected o n u p tak e D ays 0 (4 h ours), 1 , 3 , 7 , 1 4 , 2 1 , 2 8 , 3 5 , 4 2 , 4 9 , 5 6 , and 62 and depuration D ays 1 4 , 2 8 , 4 2 , and 56 o f th e test. M easured concentrations o f PFO S in all negative co n tro l edible and nonedible fish tissu e sam ples w ere <LO Q (T ables 8 and 9). T he results o f analyses o f edible and nonedible fish tissu e sam ples collected from th e 0.1 0 0 and 1.00 m g a.i./L treatm ent groups are presented in T ables 8 and 9, respectively. R epresentative ion chrom atogram s o f edible and nonedible tissu e sam ples from the sam e study fish are presented in F igures 17 and 18, re sp e c tiv e ly . S ta b ility S am ple A nalysis S tability sam ples w ere p repared a t te s t initiatio n (u p tak e D ay 0 ) and sto red frozen. T he results o f stability sam ple analyses are presented in T able 10. T hese d a ta in d icate th at th e te st su bstance w as stab le during relatively long term (1 1 9 days) fro zen sto rag e a t th e 0.100 and 10.0 m g a.i./K g concentration levels. D efinitive tissu e sam ple sto rag e did n o t exceed 7 days d uring th e conduct o f th e study. T issu e S am ple A nalysis fo r L ip id C o n ten t T issue sam ples w ere collected o n u p tak e D ays 0 (0 h o u r) and 62 and dep u ratio n D ay 56 to determ ine lipid co n ten t in fish tissues o n a w et-w eight basis. T he resu lts o f lipid analyses in edible and nonedible fish tissu es are p resen ted in T ables 11 and 12, respectively. 000336 W ildlife International, Ltd. -50Table 1 Typical HPLC/MS Operational Parameters for Analysis o f Aqueous Samples INSTRUMENT: Hewlett-Packard Model 1100 High Performance liq u id Chromatograph (HPLC) with a Perkin-Elmer A PI 3000 M ass Spectrometer equipped w ith a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion m onitoring mode (SIM). ANALYTICAL COLUMN: Keystone B etasil Clg column (50 mm x 2 mm I.D ., 2-p3m particle size) GUARD COLUMN: Keystone Javelin C column (20 m m x 2 mm I.D.) OVEN TEMPERATURE: 40 C STOP TIME: 5.00 minutes FLOW RATE. 250 pL/minute MOBILE PHASE: INJECTION VOLUME: 70.0% Methanol: 30.0% NANOpure W ater containing 0.1% Formic Acid 10 pL PFOS RETENTION TIME: Approximately 3.7 minutes INTERNAL STANDARD RETENTION TME: Approximately 2.5 minutes PFOS MONITORED MASS: 499am ux INTERNAL STANDARD MONITORED MASS: 427 amu 000337 W ildlife International, Ltd, -51- T able2 Typical HPLC/M S/M S Operational Param eters for Analysis o f Tissue Samples IN STR U M EN T: Hewlett-Packard M odel 1100 High Perform ance Liquid Chrom ato-graph (HPLC) w ith a Perkin-Elm er A PI 3000 M ass Spectrom eter quippedwith a Perkin-Elm er TurboIonSpray ion source. O perated in m ultiple ion reaction m onitoring (MRM ) mode. ANALYTICAL COLUMN: Keystone B etasil Ck colum n (50 mm x 2 mm I.D ., 2-p3m particle size) GUARD COLUMN: Keystone Javelin Cig colum n (20 mm x 2 mm I.D .) OVEN TEMPERATURE: 40 C STOP TIME: 5.00 minutes FLOW RATE: 250 pL/m inute MOBILE PHASE: INJECTIONVOLUME: 70.0% M ethanol: 30.0% NANOpure W ater containing 0.1% Form ic Acid 10.0 jtL PFOS RETENTION TIME: Approxim ately 4.1 minutes PFOS MONITORED MASS: 499.0 AMU PFOS MONITORED MASS: 499.0 amu -->99.1 amu 000338 W ildlife International, Ltd, -52- Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples SampleNutnbcr (434A-134-) PTtMAB-1 PT-MAS-1 PT-MAS-2 PT-MAS-3 -4 -4 -4 -4 Sampting Interval (Day) Pretest Concentration o fPerfluorooctanesulfonate, Potassium Salt (PFOS) (mg a,L/L) Fortified Measured1 0.00 0.0750 0.500 ' 2.00 <LOQ 0.0707 " 0.493 1.98 Percent Recovery1 -- 94.3 98.6 99,0 PT-MAB-2 PT-MAS-4 PT-MAS-5 PT-MAS-6 PT-MAB-2 PT-MAS-7 PT-MAS-8 PT-MAS-9 -1 -1 -1 -1 -1 -1 -1 -1 Pretest Pretest 0.00 0.750 0.500 2.00 0.00 0.0750 0.500 2.00 <LOQ 0.0779 0.464 1.91 <LOQ 0.0724 0.483 1.86 104 92.9 95.3 -- 96.5 96.5 93.2 MAB-1 MAS-1 MAS-2 MAS-3 0 Hour 0 - Uptake 0 0 0 0.00 0.0750 0.500 2.00 <LOQ 0.0712 0.488 2.00 95.0 97.5 99.9 MAB-2 M A S4 MAS-5 MAS-6 0 Hour4 -U ptake 0 0 0 0.00 0.0750 0.500 2.00 <LOQ 0.0728 0.490 1.97 . 97.1 98.0 98.6 MAB-3 MAS-7 MAS-8 MAS-9 MAB-4 MAS-10 MAS-11 MAS-12 1 1 1 1 3 3 3 3 Uptake Uptake 0.00 0.0750 0.500 2.00 0.00 0.0750 0.500 2.00 <LOQ 0.0745 0.498 1.95 <LOQ 0.0666 0.509 1.86 -- 99.3 99.7 97.6 -- 88.9 102 92.8 MAB-5 MAS-13 MAS-14 MAS-15 7 7 7 7 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0753 0.508 1.96 100 102 98.2 MAB-6 MAS-16 MAS-17 MAS-18 14 14 14 14 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0671 0.450 1.85 -- 89.5 90.0 92.7 5The limit o f quantitationQLOQ) was 0.0500 mg a.i./L, calculated as the product o f the lowest calibration standard (0.0005 m g a.i./L) and the dilution factor o f the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000339 W ildlife International, Ltd. -53- Tabie 3 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently w ith Freshw ater Samples Sample Number (454A-134-) MAB-7 MAS-19 MAS-20 MAS-21 21 21 21 21 Sampling Interval (Day) Uptake Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg ajJL ) Fortified Measured1 0.00 0.0750 0.500 2.00 <LOQ 0.0710 0.600 1.99 Percent Recovery2 94.6 120 99.6 MAB-8 MAS-22 MAS-23 MAS-24 28 28 28 28 Uptake 0.00 0.750 0.500 2.00 <LGQ 0.0659 0.517 1.91 - 87.6 103 95.4 MAB-9 MAS-25 MAS-26 MAS-27 35 35 35 35 Uptake 0.00 0.0750 0.500 2.00 <LQQ 0.0727 0.470 2.01 -- 96.9 94.0 101 MAB-10 MAS-28 MAS-29 42 42 42 Uptake 0.00 - 0.0750 0.500 <LOQ 0.0734 0.517 - 97.9 104 MAB-11 MAS-30 MAS-31 49 49 49 Uptake 0.00 0.0750 0.500 <LOQ 0.0712 0.463 -- 95.0 92.7 M A S-12 MAS-32 MAS-33 56 56 56 Uptake 0.00 0.0750 . 0.500 <LOQ 0.0701 0.477 --" 93.4 95.3 MAB-13 MAS-34 MAS-35 62 62 62 Uptake 0.00 0.0750 0.500 <LOQ 0.0745 0.481 993 96.1 MAB-14 MAS-36 MAS-37 14 14 14 Depuration 0.00 0.0750 0.500 <LOQ 0.0751 0.494 -- 100 98.7 MAB-15 MAS-38 MAS-39 28 28 28 Depuration 0.00 0.0750 0.500 <LOQ 0.0644 0.525 -- 85.8 105 1-The lim it o f quantitatir>n(I.OQ) w as Q.OSQQmg a.i./L, calculated as the product o f the lowest calibration standard (0.0005 m g a.i./L) and die dilution factor o f die matrix blank sample (100). 2ftesults were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000340 W ildlife International, Ltd. -54- Table 3 (Continued) M atrix Blanks and Fortifications A nalyzed Concurrently w ith Freshw ater Samples Sample Number Sampling Interval Potassium Salt(PFOS)(mg a l/L ) Percent (454A-134-) MAB-16 42 (Day) Depuration Fortified 0.00 M easured1 <LOQ Recovery2 - MAS-40 42 0.0750 0.0637 84.9 MAS-41 42 0.500 0.452 90.4 MAB-17 56 Depuration 0.00 <LOQ -- MAS-42 56 0.750 0.0712 94.9 MAS-43 56 0.500 0.484 96.9 lThe limit of quantitation(LOQ) was 0.0500 mg &i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.L/L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000341 W ildlife International, Ltd. -55- Tabic 4 Matrix Blanks and Fortifications Analyzed Concurrently with Edible Fish Tissue Samples Sample Number (454A -34-) E-M A B -1 E-M A S-1 E-M AS-2 E-M A B -2 E -M A S-3 E -M A S-4 E-M A B-3 E -M A S-5 E-M AS-6 E-M A B -4 E -M A S-7 E-M A S-8 E-M A B -5 E -M A S-9 E-M AS-10 E-M A B -6 E-M A S-11 E-M AS-12 E-M A B -7 E-M A S-13 E-M AS-14 E -M A B -8 E-M AS-15 E-M AS-16 E-M AB-10 E-M AS-19 E-M AS-20 0 0 0 1 1 1 3 3 3 7 7 7 14 14 14 21 21 21 28 28 28 35 35 35 42 42 42 Sampling Interval (D ay) Hour 4 -U p take Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS) (m g a.i./FCg) Fortified Measured1 0 .0 0 0 .1 0 0 10.0 < 0 .0 5 0 0 0 .0 9 7 0 9.65 Uptake 0 .0 0 0 .1 0 0 10.0 < 0 .0 5 0 0 0.0993 9.77 Uptake 0.00 0.500 50.0 < 0 .1 0 0 0 .4 9 4 48.6 Uptake 0 .0 0 1.00 100 * <0.200 0.948 95.0 Uptake 0.00 <1.00 5.00 5.31 250 268 Uptake Uptake - Uptake 0.00 5.00 250 0.00 5.00 250 0.00 5.00 100 < 1 .0 0 5.21 251 < 1 .0 0 5.27 249 <1.00 4.92 104 Uptake 0.00 <1.00 5.00 4.97 100 105 Percent Recovery2 9 7 .0 96.5 -- 99.3 97.7 .. 98.8 97.2 ... 9 4 .8 95.0 -- 106 107 -- 104 101 105 99.5 -- 98.3 104 -- 99.4 105 E-M AB-11 E -M A S-21 E-M AS-22 49 49 49 Uptake 0.00 <1.00 5.00 5.27 106 100 106 106 E-M AB-I2 E -M A S-23 E-M A S-24 56 56 56 Uptake 0.00 < 1 .0 0 '-- 5.00 5.31 106 200 208 104 `The limit o f quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product o f the lowest calibration standard (0.0005 m g a.i./L) and the dilution factor o f the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000342 Wildlife International, Ltd. -56- Table 4 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently w ith Edible Fish Tissue Samples Sample Number (454A -134-) E -M A B -13 E-M AS-25 E-M AS-26 62 62 62 Sampling Interval (Day) Uptake Concentration ofPerfiuoFooctanesulfonate, Potassium Salt (PFOS) (mg ad/K g) Fortified Measured1 0 .0 0 5.00 200 < 1 .0 0 5.54 192 Percent Recovery3 .. I ll 9 6 .0 E-M AB-14 E -M A S-27 E-M AS-28 14 14 14 Depuration 0 .0 0 5.00 200 < 1 .0 0 5.37 205 107 102 E-M AB-15 E-M AS-29 E-M AS-30 28 28 28 Depuration 0 .0 0 5.00 100 < 1 .0 0 5.15 104 103 104 E-M AB-16 E -M A S-31 E-M AS-32 42 42 42 Depuration 0 .0 0 5.00 100 <1.00 4.82 92.0 96.5 9 2 .0 E-M AB-17 E-M A S-33 56 56 Depuration 0 .0 0 5.00 <1.00 4.99 99.7 E-M AS-34 56 100 101 101 lLess than values correspond to limit o f quantitation (LOQ)! For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f the matrix Wank sample. A ll sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000343 W ildlife International, Ltd. -57- T a b le5 M atrix Blanks and Fortifications Analyzed Concurrently with Nonedible Fish Tissue Samples Sample Number (454A -134-) N-M AB-1 N-M AS-1 N-M AS-2 0 0 0 Sampling Interval (D ay) Hour 4 - Uptake Concentration ofPerfluorooctanesulfonate, Potassium Salt (PFOS) (m g a.i./Kg) Fortified Measured1 0 .0 0 0 .1 0 0 10.0 < 0 .0 5 0 0 0 .1 0 9 9.26 Percent Recovery2 -- 109 9 2 .6 N-M AB-2 N-M AS-3 N-M AS-4 N-M AB-3 N-M AS-5 N-M AS-6 N-M AB-4 N-M AS-7 N-M AS-8 N-M AB-5 N-M AS-9 N-M AS-10 N-M AB-6 N-M A S-11 N-M AS-12 N-M AB-7 N-M AS-13 N-M AS-14 N-M AB-8 N-M AS-15 N-M AS-16 1 1 1 3 3 3 7 7 7 14 14 14 21 21 21 28 28 28 35 35 35 Uptake Uptake Uptake Uptake Uptake Uptake - Uptake 0 .0 0 0 .1 0 0 10.0 0 .0 0 0 .5 0 0 5 0 .0 0 .0 0 1.00 100 0 .0 0 5 .0 0 250 0 .0 0 5 .0 0 500 0 .0 0 5 .0 0 500 0 .0 0 5 .0 0 100 < 0 .0 5 0 0 0 .1 0 6 9.36 < 0 .1 0 0 0 .4 7 0 45.8 < 0 .2 0 0 0 .9 7 5 90.1 < 1 .0 0 5.25 255 < 1 .0 0 4 .9 8 505 < 1 .0 0 5.25 5.16 < 1 .0 0 5.15 108 106 9 3 .6 ... 9 3 .9 91.6 97.5 90.1 .. 105 102 .. 99.7 101 -- 105 103 -- 103 108 N -M A B-IO N-M AS-19 N-M AS-20 42 42 42 Uptake 0 .0 0 < 1 .0 0 .. 5.00 5.26 105 100 104 104 N -M A B-11 N-M AS-21 N -M A S-21 49 49 49 Uptake 0.00 <1.00 5.00 5.40 108 100 108 108 N-M AB-12 N-M AS-23 56 56 Uptake 0 .0 0 < 1 .0 0 .. 5.00 5.41 108 N-M AS-24 56 200 245 122 'Less than values correspond to lim it o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f the matrix blank sample. A ll sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000344 Wildlife International, Ltd. -58- Table 5 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently with Nonedible Fish Tissue Samples Sample Number (454A -134-) N-M AB-13 N-M AS-25 N-M AS-26 62 62 62 Sampling Interval (D ay) Uptake Concentration ofPerfluorooctanesulfonate, Potassium Salt (PFOS) (m g a.i./Kg) Fortified Measured1 0.00 <1.00 5.00 5.68 200 212 Percent Recovery2 -- 114 106 N-M AB-14 N-M AS-27 N-M AS-28 14 14 14 Depuration 0 .0 0 5 .0 0 200 < 1 .0 0 5.29 207 106 . . 104 N-M AB-15 N-M AS-29 N-M AS-30 28 28 28 Depuration 0 .0 0 5 .0 0 100 < 1 .0 0 5.45 111 109 111 N-M AB-16 N-M AS-31 N-M AS-32 42 42 42 Depuration 0 .0 0 5.00 100 < 1.00 5.19 98.9 -- 104 98.9 N-M AB-17 N-M AS-33 56 56 Depuration 0 .0 0 5.00 < 1.00 5.80 .. 116 N-M AS-34 56 100 112 112 Less than values correspond to limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f the matrix blank sample. A ll sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000345 W ildlife International, Ltd. -59Table 6 M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Pre-Test D iluter V erification Samples Nominal Test Concentration (m ga.L ) 0 .0 0 (N egative Control) Sample Number (4S4A -134-) PT-1 PT-9 Phase Pre-Uptake Sampling Time (Day) -4 -1 Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m ga.i./L) <LOQ <LOQ PT-17 -1 <LOQ Percent of N om inal2 -- - -- 0.10 PT-3 PT-4 PT-11 PT-12 PT-19 PT-20 -4 0.0850 -4 0.0844 -1 0.0695 -1 0.0646 -1 0.0954 -1 0.0937 8 5 .0 8 4 .4 6 9 .5 6 4 .6 9 5 .4 9 3 .7 1.0 PT-6 -4 0.917 9 1 .7 PT-7 -4 0.949 9 4 .9 PT-14 -1 0.718 7 1 .8 PT-15 -1 0.738 7 3 .8 PT-22 PT-23 -1 1.11 -1 1.01 111 101 The limit o f quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product o f the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor o f the matrix blank sample (100). 2Results were generated using MacQuan, version 1,6 software. Manual calculations may differ slightly. 000346 W ildlife International, Ltd. -60- Table? M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Measured Concentration o f Nominal Test Concentration (m g a.i./L) Sample Number (45 4 A -1 3 4 -) Phase Sam pling Time (Day) Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./L) Percent of Nominal' 0 .0 (N egative 1 Uptake 0 ,0 hours 9 0 ,4 hours 17 1 25 3 33 7 41 14 49 21 57 28 65 35 73 42 78 49 83 56 88 62 <LOQ <LOQ <LOQ <LOQ <LOQ <X )Q <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ -- --- -- .-- .. -- -- -- " 93 Depuration 14 98 28 103 42 108 56 <LOQ <LOQ <LOQ <LOQ -- -- -- -- 0.10 3 Uptake 0 ,0 hours 4 0 ,0 hours 11 0 ,4 hours 12 - 0 ,4 hours 19 1 20 \ 27 3 28 3 35 7 36 7 43 14 44 14 51 21 52 21 59 28 60 28 67 35 68 35 75 42 76 42 80 49 81 49 85 56 86 56 90 62 91 62 0.0717 0.0692 0.0791 0.0741 0.0702 0 .0 7 3 4 0.0751 0.0717 0.0826 0.0781 0.0680 0.0709 0.110 0.113 0.0822 0.0843 0.0915 0.0914 0.0983 0.110 0.103 0.103 0.0853 0.0948 0.0887 0.0914 'T h elim it o f quantitation (LOQ) w as 0.0500 m g a.i./L, calculated as the product o f the lowest calibration standard (0.0005 m g a.i./L) and the dilution factor o f the m atrix blank sam ple (100). R e su lts w ere generated using M acQuan, version 1,6 software. M anual calculations may differ slightly. 71.7 6 9 .2 79.1 74.1 70.2 7 3 .4 75.1 7 1 .7 82.6 78.1 6 8 .0 7 0 .9 110 113 82.2 84.3 91.5 91.4 98.3 110 103 103 85.3 94.8 88.7 91.4 000347 Wildlife International, Ltd. -61- Table 7 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Measured Concentration o f Nominal Test Concentration (m g a.L/L) Sanqtle Number (454A -134-) Phase Sampling Time (Day) Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg Bd.IL) Percent of N om inal2 0 .1 0 95 Depuration 14 96 14 100 28 101 28 105 42 106 42 110 56 111 56 <LOQ <LOQ <LOQ <L0Q <LOQ <LOQ <LOQ <LOQ -- -- -- -- -- 1.0 6 Uptake 0 ,0 hours 0.797 7 0 ,0 hours 0.802 14 0 ,4 hours 0.891 15 0 ,4 hours 0.930 22 1 0.867 23 1 0.820 30 3 0.813 31 3 0.734 38 7 0.845 39 7 0.818 46 14 0.900 47 14 0.875 54 21 0.988 55 - 21 1.01 62 28 0.913 63 28 0.925 70 35s 0.838 ______ 11______ ____ 25!_____ 0.871 -- `THf! lim it ff gnantitatinn (T.QQ) w as 0 .0 5 0 0 m g a-i./L . calculated a s th e p rod uct oftfae low est calib ratio n 7 9 .7 8 0 .2 89.1 93.0 86.7 8 2 .0 81.3 7 3 .4 8 4 .5 81.8 90.0 8 7 .5 9 8 .8 101 91.3 92.5 8 3 .8 87.1______ standard (0.0005 m g a i./L ) and the dilution factor o f the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 3Sampling was suspended after Day 35 Uptake due to 100% mortality at the 1.0 mg a.i7L nominal test level. 000348 Wildlife International, Ltd. -6 2 - Table 8 M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Measured Concentration o f Nominal Test Concentration Sample Number Sampling Time Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./L) (454A -1340 Phase (Day) (m ga.i./L) 0 .0 E -l Uptake 0 ,4 hours < 0 .0 5 1 0 (N egative E-2 0 ,4 hours <3.0665 E -14 1 <0.0960 E-15 1 <0.0835 E-27 3 <0.114 E-28 3 <0.0910 E -40 7 <0.0965 E-41 7 <0.162 E-53 14 <0.468 E-54 14 <0.560 E -66 21 <0.730 E-67 21 <0.710 E-79 28 <0.740 E-80 28 <0.800 E-92 35 <0.970 E-93 35 <0.575 E-100 42 <0.399 E-101 42 <0.590 E -108 49 <0.575 E-109 49 <0.520 E -l 16 56 <0.775 E -l 17 56 <0.820 E -124 62 <0.660 E-l 25 62 <0.615 E -132 E-133 E-140 Depuration 14 14 28 <0.685 < 0 .7 1 0 <0.585 E -l 41 28 <0.590 E-147 42 <0.830 E-148 42 <0.825 E-154 56 <0.755 E-155 56 <1.07 0.10 E-4 Uptake 0,4 hours E-5 0 ,4 hours 0.167 0.155 E-6 0 ,4 hours 0.144 E-7 E-17 0 ,4 hours 1 0.182 0.734 E -l 8 E-19 1 0.726 1 0.631 E-20 1 0.806 E-30 E-31 E-32 E-33 3 1.73 3 2.07 3 2.03 3 ______________ 2JJ________________ `Less than values correspond to limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f the sam ple (L/Kg). 2Results w ere generated using M acQ uan, version 1.6 software. M anual calculations may differ slightly. 000349 W ildlife International, Ltd. -63- Table 8 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium S alt (PFOS) in Edible F ish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (m gai./L ) Sample Number (454A -134-) Phase Sampling T im e (Day) Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m g a.i./L) 0 E-43 Uptake 7 E -4 4 7 E -4 5 7 E-46 7 E -5 6 14 E-57 14 E -5 8 14 E-59 14 E-69 21 E -7 0 21 E-71 21 E-72 21 E -8 2 28 E -83 28 E-84 28 E-85 28 E -95 35 E -96 35 E -97 35 E-98 35 E -103 42 E -104 42 E -105 42 E -106 42 E -lll 49 E -112 49 E -113 49 E -114 49 E -119 56 E -120 56 E-121 56 E -122 56 E -127 62 E -128 62 E -129 62 E -130 62 3.73 4 .2 5 4.73 6 .2 5 11.4 9.07 13.7 12.6 11.7 12.0 12.9 10.6 18.3 13.7 2 3 .9 23.1 2 2 .6 27.7 23.8 20.6 27.6 25.3 2 1 .2 2 7 .6 33.3 36.2 3 9 .0 3 0 .6 4 8 .3 3 8 .9 44.1 38.3 42.4 6 6 .2 42.2 3 9 .2 E -135 Depuration 14 4 8 .5 E -136 14 31.8 E -137 14 31.6 E -138 14 42.0 E -143 28 26.0 E -144 28 33.3 E-14S 28 38.7 E -146 28 55.8 E -150 42 24.1 E-151 42 31.2 E -152 42 30.0 E -153 42 33.0 `Less than values correspond to lim it o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f die sample (L/Kg). 'Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000350 Wildlife International, Ltd. -6 4 - Table 8 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i ) 0 .1 0 Sample Number (4 5 4 A -1 3 4 -) E -157 E -158 E -159 E -160 Phase Depuration Sampling Time (Day) 56 56 56 56 Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m ga.i ) 21.1 3 7 .6 3 2 .9 31.2 1.0 E -9 Uptake 0 ,4 hours 1.46 E -10 0 ,4 hours 1.48 E -ll 0,4h ou rs 1.19 E -12 0 ,4 hours 1.39 E-22 1 4.68 E -23 1 6.59 E -24 1 5.56 E-25 1 5.64 E -35 3 17.3 E-36 3 15.8 E-37 3 19.0 E -38 E -48 3 20.8 7 42.0 E*49 7 44.0 E -5 0 7 57.7 E-51 7 E-61 - 14 4 6 .8 87.1 E -62 14 81.6 E-63 14 90.7 E -64 14 73.3 E-74 E -75 21 79.4 21 117 E-76 21 104 E-77 21 102 E-87 28 102 E-88 28 131 E -89 28 107 E-90 28 ______________ 133________________ 'Less than values correspond to limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the ^Results were generated sing MacQuan, version 1.6 software. Manual calculations may differ slightly. 000351 W ildlife International, Ltd. -65- Table 9 M easured Concentrations o f Perflnorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegjll Sunfish Bioconcentration T est Nominal Test Concentration (m g a.i7L) 0 .0 (N egative Sample Number (454A -134-) N -l N -2 N -14 N -1S N -27 N -28 N -40 N -41 N -53 N -54 N -66 N -67 N -79 N -80 N -92 N -93 N -100 N-101 N -108 N -109 N -l 16 N -l 17 N -l24 N -l25 Phase Uptake Sampling Time (Pay) 0 ,4 hours 0 ,4 hours 1 1 3 3 7 7 14 14 21 21 28 28 35 35 42 42 49 49 56 56 62 62 Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m ga.i./L) < 0 .0 4 5 8 < 0 .0 5 7 5 < 0 .0 8 2 0 < 0 .0 6 4 5 .. < 0 .0 8 2 0 < 0 .0 7 9 5 < 0 .0 8 0 5 < 0 .0 9 6 5 < 0 .4 3 4 <0.457 < 0 .5 6 0 < 0 .5 6 0 < 0 .5 8 0 < 0 .6 2 0 < 0 .5 9 0 < 0 .4 1 7 <0.363 < 0 .4 8 0 <0.555 <0.375 < 0 .5 4 0 < 0 .5 2 0 < 0 .5 0 5 < 0 .5 0 5 N -l 32 N -133 N -140 N -141 N -147 N -l 48 N -l 54 N -l 55 Depuration 14 14 28 28 42 42 56 56 < 0 .4 7 0 < .0 4 7 2 < 0 .4 4 9 < 0 .5 0 5 < 0 .5 9 0 < 0 .5 7 0 < 0 .6 3 0 < 0 .7 8 0 0 .1 0 N -4 Uptake 0 ,4 hours 0 .4 1 5 N -5 N -6 N -7 N -17 N -18 0 ,4 hours 0 ,4 hours 0 ,4 hours 1 1 0 .5 1 9 0.417 0.497 1.68 1.85 N -19 N -20 1 1.72 1 2.07 N -30 N-31 N -32 _____ N-33 3 4.59 3 5.50 3 5.47 3 5.97 Less than values correspond to lim it o f quantitation (LOQ). For each analysis, die LOQ was calculated as the product o f the low est calibration standard and the overall dilution factor o f the sam ple (L/Kg). 2R esults w ere generated using M acQ uan, v ersion 1.6 software. M anual calculations may differ slightly. 000352 W ildlife International, Ltd. - 66- Table 9 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegl Sunfish Bioconcentration Test Nominal Test Concentration (mg a i./L ) 0 .1 0 Sample Number (454A -134-) N -43 N -44 N -45 N -46 N -56 N -57 N -58 N -59 N -69 N -70 N -71 N -72 N -82 N -83 N -84 N -85 N -95 N -96 N -97 N -98 N -103 N -104 N -105 N -106 N -lll N -112 N -113 N -114 N -119 N -120 N -121 N -122 N -127 N -128 N -129 N -130 Phase Uptake Sam pling Time (Day) 7 7 7 7 14 14 14 14 21 21 21 21 28 28 28 28 35 35 35 35 42 42 42 42 49 49 49 49 56 56 56 56 62 62 62 62 Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m g a.i./L) 10.2 10.6 11.9 15.2 27.3 2 3 .2 35.3 3 2 .6 33.3 2 2 .7 2 4 .6 2 4 .4 4 9 .4 4 0 .7 65.3 5 7 .9 67.1 73.3 6 2 .0 59.1 6 4 .0 68.1 5 4 .4 7 9 .6 8 5 .0 95.1 93.1 77.7 122 94.2 7 3 .2 106 101 112 105 9 6 .4 N -135 N -136 Depuration 14 14 124 7 9 .4 N -137 N -138 N -143 N -144 N -145 N -146 N -150 14 81.8 14 113 28 85.7 28 95.1 28 85.7 28 94.8 42 71.7 N -151 42 80.6 N -152 42 78.3 N -153 42 ______ ;_______ m ________________ `Less than values correspond to lim it o f quantitation (LQQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f the sample (L/Kg). ^Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000353 W ildlife International,Ltd. -67Table 9 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (m gal/L ) 0 .1 0 Sample Number (454A -134-) N -157 N -158 N -159 N -1 6 0 Phase Depuration Sampling Time (Day) 56 56 56 56 Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m g a.i./L) 57.7 80.3 8 5 .4 84.4 1.0 N -9 Uptake 0,4 hours N -10 0,4 hours 3 .5 2 4.37 N -ll N -12 0,4 hours 0,4 hours 4.22 4 .0 6 N -22 N -23 N -24 N -25 1 11.1 1 14.2 1 13.3 1 12.1 N -35 N -36 3 39.3 3 42.0 N -37 3 N -38 3 N -48 7 N -49 7 N -50 7 N-51 - 7 N-61 14 N -62 14 4 3 .8 51.8 100 102 102 120 177 207 N -63 N -64 N -74 14 245 14 214 21 201 N -75 N -76 N -77 N -87 N -88 N -89 N -90 21 21 21 28 28 28 28________ 278 246 229 289 372 320 361 `Less than values correspond to lim it o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard and the overall dilution factor o f the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 000354' W ildlife International, Ltd. - 68- Table 10 M easured Concentrations o f Perfluorooctanesnlfonate, Potassium Salt (PFOS) in Tissue Storage Stability Samples from a Bluegill Sunfish Bioconcentration Test N om inal C oncentratim i _(mga.L/Kg) N eg ativ e Control 0.100 Sample Number (4 54A -134-) E-M AB-184 N -M A B -184 E-STM A S-l3 E-M A S-354 N -STM A S-13 N-M AS-354 Tissue Type Edible N o n ed ib le Edible Edible N o n ed ib ie N onedible M easured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./K g) <LOQ` <LOQl 0.109 0.111 0.114 0.112 Percent of N om inal2,5 -- -- 109 111 114 112 E -STM A S-23 Edible 10.0 E-M A S-364 E d ib le 8.43 11.3 84.3 113 N -STM A S-23 N o n ed ib le 9.30 93.0 N-M AS-364 N o n ed ib le 11.3 113 Less than values correspond to limit o f quantitation (LOQ). The LOQ was 0.0500 mg a.i./Kg, calculated as the product o f the lowest calibration standard (0.0005 mg a.i./:)) and the overall dilution factor o f the matrix blank sample (100 L/Kg). A ll sample weights = 1 OOgram. 2 Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 3 The stability sam ples were fortified on December 5 ,2 0 0 0 and stored in the freezer. The samples were removed from the freezer on April 3,2001 (after 119 days o f frozen storage) and analyzed. 4 T hese sam ples w ere p repared on A pril 3 ,2 0 0 1 and the resu lts w ere com pared to th e stability sam ple resu lts. s The criterion for storage was Percent o f Nominal between 8 0 and 120%. 000355 Wildlife International, Ltd. -69- Table 11 Lipid Content in Edible Fish Tissue Nom inal Test C o n cen tratio n (mg aiVL) 0.0 (Negative Control) Sam ple N um ber (454A -134-) ELI EL2 EL3 EL4 Phase U ptake Sam pling T im e (D ay) 0 ,0 hour 0 ,0 h o u r 0 ,0 hour 0 ,0 hour L ip id W eight (g) 0.0314 0.0453 0.0453 0.0463 Fish Tissue W eight (8) 1.7499 2.1445 1.8577 1.8335 EL6 U p tak e 62 0.0231 1.5122 EL7 62 0.0135 1.0670 EL8 62 0.0134 0.9726 EL9 62 0.0162 1.0650 EL 16 EL17 EL18 ELI9 D epuration 56 56 56 56 0.0254 0.0163 0.0158 0.0256 1.3549 1.0890 1.1234 1.3777 0.10 EL11 U p tak e 62 0.0162 1.0411 EL12 62 0.0327 1.3551 EL13 62 0.0329 1.4893 EL14 62 0.0188 0.9054 EL21 EL22 D epuration 56 56 0.0172 0.0190 1.2362 1.3483 EL23 EL24 56 0.0250 1.3303 56 0.0252 1.1040 'R atio calculated as flipid weight (g) 4- fish tissue w eight (g)l x 1000 mg/g x 1000 g/Kg. L ip id /F ish T issu e W eight1 (m g/K g) 17,900 21,100 24,400 25,300 15,300 12,700 13,800 15,200 18,700 15,000 14,100 18,600 15,600 24,100 22,100 20,800 13,900 14,100 18,800 22,800 000356 W ildlife International, Ltd. -70- Table 12 Lipid Content in N onedibk Fish Tissue Nominal Test Concentration (m g a.UL) 0 .0 (Negative Control) Sample Number (454A -134-) NL1 NL2 NL3 NL4 Phase Uptake Sampling Lipid Weight Time (Day) (B) 0 ,0 hour 0 ,0 hour 0 ,0 hour 0 ,0 hour 0.1363 0.2383 0.2258 0 .1 9 9 2 Fish Tissue W eight (B> 2.0649 2 .6 3 5 5 1.9568 2.0812 NL6 NL7 NL8 NL9 Uptake 62 0 .0 5 0 6 1.8821 62 . 0.0274 1.5901 62 0 .0 2 9 8 1.75% 62 0.0518 1.7302 NL16 NL17 NL18 NL19 Depuration 56 56 56 56 0 .0 6 1 6 0.0318 0.0360 0 .0 9 6 6 1.7263 1.6746 1.5847 1.8165 0.10 NL11 Uptake 62 0.0503 1.5484 NL12 62 0.1370 1.7642 NL13 62 0 .1 2 7 6 2.0113 NL14 62 0.1221 1.2898 NL21 Depuration 56 . 0.0343 NL22 56 0.0404 NL23 56 0.0710 NL24 56 0.0714 'Ratio calculated as [lipid weight (g)-^ fish tissue weight (g)l x 1000 m gfe x 1000 g/Kg. 1.7085 1.8394 1.7652 1.5682 Lipid/Fish Tissue W eight' (mg/Kg) 66,000 90,400 115,400 9 5 ,7 0 0 2 6 ,9 0 0 17,200 16,900 29,900 35,700 19,000 22,700 53,200 32,500 77,700 63,400 94,700 20,100 22,000 40,200 4S.500 000357 Wildlife International, Ltd. -71- METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFS) IN FRESHWATER Prepare matrix fortification samples in freshwater matrix by spiking the requisitevolarne ofPFOS stock solutions directly into freshwater. Perform fortifications with gas-tight syringes and Class A volumetric flasks. i Prepare appropriate dilutions o f study and QC samples (o within the calibration range o f the PFOS LCMS methodology: Partially fill Class A volumetric flasks with 50% methanol/50% water dilution solvent containing 0.0100 mgfL 411 PFOS intentai standard and 0.05% v/v formic add. Add appropriate volume o f sample and bring to volume with dilution solvent. Process matrix blank samples for a given matrix using the same dilution and aliquot volumes as for die lowest fortification level in that matrix. Mix w ell by several repeat inversions. i Ampulate samples and submit for LCMS analysis. F igure 1. M ethod flow chart for the analysis o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in freshw ater. CONTAIN NO CBI 000358 W ildlife International, Ltd. -72- m e t h o d OUTLINE FOR THE ANALYSIS OF PERFLUOROOCTANESULFONATE POTASSIUM SALT (PFOS) IN FISH TISSUES Quality control samples are prepared from aliquots (approximately 1 g) o f bulk control fish tissue homogenate Remove appropriate (edible or non-edible) aliquots from frozen storage and allow to thaw. Prepare fortification samples with the appropriate PFOS stock solution using gas-tight syringe(s). The matrix blank sample will be unfortified fish tissue homogenate. I Add 10.0 mL o f methanol to each samplewith a glass ClassA volumetric pipette. Homogenize each test sample for approximately 1 minute using a tissue shredder. Rinse the homogenizer with the appropriate solvent(s) in between samples. i Sonicate each sample for approximately 5 minutes with a sonic dismembrator i Cap die vials and shake well. Centrifuge the vials at approximately 2000 rpm for approximately S minutes. I Prepare appropriate dilutions o f study and QC samples to within the calibration range o f the PFOS LCMS methodology: Partially fill ClassA volumetric flasks with 50%: 50% metbanoI/NANOpare* water dilution solvent. Add appropriate volume of sample and bring to volume with dilution solvent Process matrix blank samples for a given matrix using the same dilution and aliquot volumes as for the lowest fortification level in that matrix Mix w ell by several repeat inversions. I T ransfer an aliquot o f each sam ple to an autosam pler vial and subm it fo r LC/M S/M S analysis. F igure 2. M ethod flow chart for the analysis o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in fish tissues. 0003S 9 Wildlife International, Ltd. -73 METHOD OUTLINE FOR THE ANALYSIS OF LIPIDS IN FISH TISSUES Remove vials to be analyzed from the freezer. Allow samples to thaw. 4* For each sample, add 10 mL r f NANOporc* water to fish tissue In vial and homogenize for approximately 1 minute usings hand-held tissue shredder. Rinse foe homogenizer with the appropriate solvent^ in between samples. Transfer each homogenate to a 250-mL separatory funnel that contains 25 niL o f chloroform and 50 mi. o f methanol. I Rinse each vial with an additional 10 mL o f NANOpure water and pour rinse into respective Shake one minute with venting. Add 50 mL o f chloroform followed try 50 mL o f saturated sodium chloride to each separatory funnel. Jr Briefly swirl each separatory funnel with venting. 1 Allow foe phases to separate. Foreach sample, drain foe chloroform layer through a powder funnel packed with glass wool and anhydrous sodium sulfate into a 250-mL round-bottom flask. Jr Add an additional 50-mL aliquot o f chloroform to each separatory funnel and repeat foe extraction and draining Rotary evaporate the extracts in a water bath maintained at approximately 40"C to neardryness. 1 T ransfer each extract to a prc-w eighed. labeled scintillation vial. Jr Rinse each 250-mL round-bottom flask with a small volume o f chloroform and transfer rinse to respective scintillation viai. Evaporate the remaining solvent in each vial under a gentle stream o f nitrogen or dean dry air. Reweieh each d al and record weight.________________________ F igure 3. Method flow (h art for the analysis o f lipids in fish tissues. 000360 W ildlife International, Ltd. -74- ' AreafFtatio) F ig u re 4 . A typical calibration curve for Perfiuorooctancsulfonatc, Potassium Salt (PFOS) in freshwater. Slope = 4.64; Intercept = -0.01; r = 0.99932. Curve is weighted (1/x). (m onitored m asses = 499 amu (PFOS) and 427 amu (4HPFOS internal standard)). 000361 W ildlife International, Ltd. -75- PF03L.1 STD O.SOO ug .LA. 4875A-01 ID -11 4 .9 6 rtp e rio d PTDS Memal Standard: 4HPFOS Use Ama Absoluto Betentlon Time 1: 4.97 Q1 Mi. 298 sean* 4 9 9 .0 NoiceTtves. Quant Thres. Mm. Wldlh MUR. WkMl Base-W k RT Win. (saca) 2 0 .0 1fi.O 3 6 40 so Smooth 1 ExpectedRT 3.80 Ama 2SI8S6 Haight 23873 Start Tima 3.61 E ndito fcdaQratkm Wtdlh 48B 0.67 Retontlon Tina integrado Type 383 A-BB Tu, Dec 6, 2000 10:48 intensity: 250000 cps 108 90 60 70 60 50 40 30 20 10 31 66 to s 228 167 199 A . .. 261 ` 41 ' *'1 J 11 161 21 2*1 261 Scan 089 186 202 2 69 386 4 8 3 4.70 Time PFOS.1 STD a.500 ug i.ITL 4673A-011D >11 4.S3tn 1 period 4HPFOS use a s Internal Standard 1: 4.87 Q1 Mi, 298 scant 427.0 N absT hrea. Quant Three. 2.0 1.0 Mn. Width Mutt. Width Base. Width 3 8 30 RT win. (sacs) Smooth Expected RT 20 1 2.53 A na 1134885 Height 141549 Start TOM 2.44 End tim e Integration Wtdh Retention Time Integration Type 2.95 050 2.54 A-BB Tub. Dec 5. 2000 10:48 intensity: 250000 cps F igure 5. A representative ion chromatogram o f a low-level (0.500 pg a.i./L ) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for freshwater analyses, (monitored m asses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). 000362 \ /Vildlife International, Ltd. -76- PF0SL9 STD SUM us JA 4675A-011D *15 4.98 In 1 period pres Internal Standard 4HPFOS Usa Area Absolut Retention Time 1: 4.97 Q1 Ml, ZSS scans 499.0 Mdse Three. 20.0 Quant Three. Min. Width Mutt Width 15.0 3 B Base. Width RT Win. (secs) Smooth Expected RT 40 30 1 3.80 Area 2505788 Height 227264 Start Time EM Time Integration Width 3JM 4J 0.87 Retention Time Integration Type 3JB3 A-BB Tue. Decs. 2000 11:10 100 90 60 70 SO 50 40 30 20 10 <r intensity: 250000 eps so 111 153 A 287 ' 4*1 ' 0*1 ' 121 0.68 1.36 2.02 161 248 21 3.36 241 ` 4.03 PFOSJ5 STD 5.00 ug a.ML 4675A-011D -15 4.96 in 1 period 4HPPOS use a s Internet Standard 1: 4.97 Q1 Ml. 298 scans 427.0 Hebe Three. 2.0 Quant Ihres. 1.0 Mia Width 3 MuL Width 6 Base. Width 30 R TW ia(secs) 20 Sm ooth 1 ExpectedRT 2.53 Area 1052782 Height 133897 Start Time 2.44 EM Time Integration Width 2.95 OSO Retention Time 2.S4 Integrstion Type A-BB Tue. D ec5, 2000 11:10 intensity: 250000 cps F igure 6. A representative ion chromatogram o f a high-level (5.00 pg a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for freshw ater analyses, (m onitored m asses - 499 am u (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). 000363 W ildlife International, Ltd, Amt -77- F igure 7. A typical calibration curve for Perfluoraoctanesulfonate, Potassium Salt (PFOS) in fish tissue. Slope = 4552.41; Intercept = 69.58; r = 0.99940. Curve is weighted (1/x). (m onitored m ass = 499 amu -> 9 9 .1 amu). 000364 W ildlife International, Ltd. -78- intensity; 2500 cps F ig u re 8. A representative ion chromatogram o f a low-level (0.500 pg a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for fish tissue analyses. From low-range (0.500 to 5.00 pg a.i./L ) calibration set. (monitored m ass = 499 amu --99.1 amu). 000365 W ildlife International, Ltd. -79- intensity: 30000 cps F ig u re 9 . A representative ion chromatogram o f a high-level (50.0 pg &.UL) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for fish tissue analyses. From high-range (5.00 to 50.0 pg a.i./L) calibration s e t (monitored mass - 499 amu --99.1 amu). 000366 W ildlife International, Ltd. -80- FFOSJP IIAB-1 4S4A-134- Tu, Dec S. 2000 11SB 4.88 in 1 period PFCS Internat Standard: 4HPPOS lisa Area Absoluta Ratenden Time 1; 4.97 Qt Ml. 298 scans 499.0 N ote Three. Quant H ires. Milt. Wkflh Mu*. Width Base. Width RTWin. (sacs) Smooth Expected RT 20.0 15.0 3 6 40 30 1 3.90 Area 0 Haight 0 Start Tima End Tima Integration Width 0430 0.00 0.00 Retenfion Tima Integration Typo QjOO intensity: 250000 cpa PFOSJT MAB-1 454A-134. 4.98 in t period 4HPFOS use as internal Standard Tuo. Dec S. 2000 11:22 t: 4.97 Q1 Mi. 298 scans 427.0 Noise Hues. 2.0 Quant Three. 1.0 3 ; Wkkh 30 RTlMn. (secs) 20 t JBT 2.63 1178468 180726 Start Time 2.44 End Time Integration Width 2.96 aso Ratenden Tin 2.S8 tntogration Typo A -B 8 intensity: 2S0000 cps F igure 10. A representative ion chromatogram o f a freshwater m atrix blank sam ple (454A - 1 3 4 - MAB - 1 , dilution = 1OOx). The arrow indicates the retention tim e o f Perfluorooctanesulfonate, Potassium Salt (PFOS). (m onitored m asses = 499 am u (PFOS - top) and 427 amu (4HPFOS internal standard - bottom . 000367 W ildlife International, Ltd. -81- PFOS_S UAS-2 454A -134- Tue, Dec S, 2000 1134 4.98 In 1 period PFCS Internal Standan4HPPOS Use Area Absolute Retention Time 1: 4.97 Q1 Ml, 298 scans 499.0 Noise Ttwes. Quant Tines. Min. Wktth Mult Width Basa. Wktth RTWin. (secs) Smooth Expected RT 20.0 15.0 3 < 40 30 1 3.90 Area 095049 Height 92S17 Start Time EndTime Integration Width Retention Timo integration Type 61 458 0.67 63 A -88 PFOS.a UAS-2 454A-134- 4.B8 in t period 4HPTOS use a s Internal Standard Tub, Dec 5. 2000 11.-34 1: 4.97 Q1 Ml. 238 scans 427.0 Noise Threi. Quant Tims. Mia Width 2.0 1.0 3 Mult. Width 6 Base. Wktth RT Win. (tecs) Smooth Expected RT 90 so 1 2.53 Area 1109335 Height 139303 Start Time EndTime Integration Wktth Retention Tima Megration type 43 93 0.50 256 A-BB Intensity: 250000 epe intensity: 2SOOOO cps F igure 11. A representative ion chromatogram o f a freshw ater m atrix fortification sam ple (454A-134M A S-2,0.500 m g a.i./L nominal concentration, dilution = 250x). (monitored m asses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom )). 003G8 Wildlife International, Ltd. -82- intensity: 2500 cps F igure 12. A representative ion chromatogram o f an edible fish tissue m atrix blank sam ple (454A-134E-MAB-1, overall dilution factor = 100 L/Kg). The arrow indicates the retention tim e o f Perfluorooctanesulfonate, Potassium Salt (PFOS). (monitored mass = 499 am u --99.1 amu). 000369 W ildlife International, L td -83- intensity: 2500 eps F igure 13. A representative ion chromatogram o f a nonedible fish tissue m atrix blank sam ple (454A134- N-M AB-1, overall dilution factor = 100 L/Kg). The arrow indicates die retention tim e o f Perfluorooctanesulfonate, Potassium Salt (PFOS). (m onitored m ass= 4 9 9 am u - 99.1 amu). 000370 W ildlife International, Ltd. -84- intensity: 3500 cps fig u re 14. A representative ion chromatogram o f an edible fish tissue m atrix fortification sample (454A -134-E-M A S-6,50.0 mg ai./K g nominal concentration, overall dilution factor = 20000 L/Kg). (monitored m ass = 499 amu --*99.1 amu). 000371 W ildlife International, Ltd. -85- intensity: 3000 cps F igure 15. A representative ion < (454A -134-N M A S-8,100 mg a.i./Kg nominal concentration, overall dilution facto r= 50000 L/Kg). (monitored m ass = 499 amu--99.1 amu). 000372 W ildlife International, Ltd. PF0S.1S 454A-134- Tue. Dec 5, 2000 1138 4.98 h 1 period HCS Internal Standard: 4HPFOS U*oArea Absolute Retention Time 1: 4.97 Q1 Ml. 298 scans 499.0 Noise Thres. 20.0 Quant Thres. ts.O Min. Width 3 MUR. WidUl 6 Basa. Width 40 RTIMn. (secs) 30 Smooth Expected RT 1 3.B0 Area 355173 Height 33158 Suit Time 3.63 EndTime 4.30 Integration Width 0.67 Retention TTme 3.83 Integration Type A - 8B - 86- RFOSJI3 454A-1 34* Tue. 060 5. 2000 11:58 4.98 ln 1 period 4WF0S um u Internal Standard i: 4.97 Q i Ml. 253 scans 427.0 Noise Three. 2.0 Quant Three. 1.0 Min. Width Mutt. Width 3 6 Base. Width RSmT oWoitnh. {sec*) Expected RT 30 20 2.53 Area 1094647 Height 138642 Start Time 2.44 End Time Integration Width 2.95 0.50 Retention Time integration Type 258 A-B8 intensity: 250000 cps Intensity: 250000 cps F igure 16. A representative ion chromatogram o f a freshwater sample (454A-134-3, dilution * lQOx) from die 0.10 mg a.i./L treatm ent group, (m onitored m asses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard -bottom )). 000373 Wildlife International, Ltd. -87- Intensity: 3500 cps F ig u re 17. A representative ion chromatogram o f an edible fish tissue sample (454A -134-E-30, overall dilution factor = 1420) from die .0.10 mg a.i./L treatm ent group), (m onitored m ass = 499 amu -->99.1 amu). 000374 W ildlife International, Ltd. - 88- 100- so SO TO SO SO 4030 2010o4~ 45 41 ' 0.69 -- I-- 81 1.36 " T 1-3"1n 121 2.03 intensity: 3000 cps 210 262 285 T 1'" 281 Scan 4.71 Tin F igure 18. A representative ion chromatogram o f a nonedible fish tissue sam pie (454A-134-N-30, overall dilution factor = 2 6 5 0 ) from the 0 .1 0 m g a.i./L treatm ent group), (m onitored m ass * 499 amu -99.1 amn). 000375 ProjectNumber 454A-134 -89- A ppendix 4 Temperature o f pH o f W ater in the Test Chambers Uptake Phase Sponsor: 3M Corporation Test Sustance: PFOS Test Organism: Bluegill, Lepomis machrochirus Dilution Water: W ell W ater ________________________________________ ________ Uptake Phase Day 0 Day 7 Day 14 Day 21 Mean Measured Concentration (mg a.i./L) Negative Control Temp1 CO 22.0 pH 8.2 Temp1 ro 21.9 pH 8.0 Temp1 CO 21.8 pH 8.0 Temp1 CO 21.8 pH 8.1 0.086 21.9 8.2 21.8 8.0 21.8 8.0 21.8 8.1 0.87 21.9 8.2 21.8 8.0 21.8 'Continuous measurements o f temperature ranged from 20,0 to 22.0C. 7.9 21.8 8.1 Dary28 Temp1 CO . 21.9 P 7.9 21.9 7.9 21.8 8.0 Uptake Phase Mean Measured Concentration (mg a.i./L) Negative Control Day 35 Temp1 CC) 21.8 pH 8.1 Day 42 Temp1 CO 21.8 pH 8.0 Day 49 Temp1 CC) 21.9 pH 8.0 0.086 21.7 8.1 21.8 7.9 21.8 0.87 21.7 8.2 __2 _2 > 1Continuous measurements of tem perature ranged from 20.0 to 22,0C. 2Measurements discontinued due to 100% mortality. 8.0 Day 56 Temp1 CO 21.9 pH 8.1 21.9 8.1 mm 000376 ProjectNumber 454A-134 -90- A ppendix 4 (C ontinued) _________________ Temperature o f pH o f W ater in the Test Chambers Depuration Phase Sponsor: Test Sustance: Test Organism: Dilution Water. 3M Corporation PFOS Bluegill, Lepomis machrochirus Well Water Uptake Phase DavO DayO Day 14 Day 21 Mean Measured Concentration Temp1 Temp1 Temp1 Temp1 (mg a i/L ) Negative Control (C) 21.9 pH 8.1 <C) pH 21.8 8.2 (C) pH 21.9 8.0 T c) 21.9 PH 8.1 0.086 21.8 8.1 21.8 8.2 21.9 'Continuous measurements o f temperature ranged from 20.0 to 22,0C. 8.1 21.8 8.1 Pay 28 Temp1 21.8 pH 8.1 21.7 8.1 Uptake Phase M ean M easured Concentration (mg a.i./L) Negative Control Day 35 Temp1 <C) 21.9 pH 8.1 Day 42 Temp1 (C) 21.9 pH 8.1 Day 49 Temp1 (C) 22.0 pH 8.1 0.086 21.8 8.1 21.9 8.1 22.0 1Continuous measurements of tem perature ranged from 20.0 to 22.0C. 8.1 Day 56 Tlrenm-LnprJ (c) 21.9 pH 8.1 21.9 8:1 000377 W ildlife International, Ltd. Project Number 454A-134 -91- A ppendixS Dissolved Oxygen (m g/L) o f W ater in the T est Cham bers1 Sponsor Test Sustance: Test Organism: D ilution Water: Day 3M Corporation FFOS Bluegill, Lepomis machrochirus W ell Water Negative Control Uptake Phase 0 .0 8 6 m g a.i./L 0.87 m g a.i./L 0 8.2 8.2 8.2 1 6.8 6.8 6.8 2 7.4 7.2 6.9 3 7.3 7.1 6.5 4 7.3 7.2 7.3 5 7.3 7.3 7.1 6 7.3 7.3 6.8 7 7.0 7.0 7.0 8 7.6 7 .6 7.4 9 7.6 7.4 7.4 10 7.8 7.6 7.2 11 7.7 7.6 7.5 12 7.5 7.3 7.0 13 7.8 7.6 7.1 14 7.3 7.1 6.4 15 7.5 7.2 6.7 16 7.5 7.3 7.4 17 7.9 7.8 8.0 18 8.0 8.0 8.0 19 7.4 7.5 7.5 20 7.8 7.8 7.7 21 7.8 7.8 7.8 22 7.8 7.6 7.7 23 7.8 7.8 7.9 24 NC2 NC NC 25 7.5 7.4 7.4 26 7.5 7.4 7.6 27 7.6 7.6 7.8 28 7.3 7.2 7.4 29 8.0 7.8 8.0 30 7.6 7.4 7.9 31 7.8 7.6 8.0 32 7.5 7.4 7.7 33 7.8 7.9 7.8 34 7.8 7.8 8.2 35 7.5 7 .4 8.1 1A dissolved oxygen concentration o f 52. mg/L represents 60% saturation in freshwater at 22C. 2 NC-Data inadvertently not collected. 000378 W ildlife Internationaly Ltd. -92- Project Number 454A-134 A ppendix 5 (C ontinued) Dissolved Oxygen (mg/L) o f W ater in the T est Chambers1 Sponsor 3M Corporation Test Sustancc: Test Organism: PFOS Bluegill, Lepomis machrocfurus Dilution Water: W ell Water Uptake Phase Day Negative Control 0.086 m e a.i7L 0.87m ga.i./L 36 7.8 7.6 37 7.7 7.8 -- 38 8.2 7.8 -- 39 7.8 7.6 -- 40 7.8 7.8 -- 41 7.8 7.7 -- 42 7.4 7.2 -- 43 7.7 7.4 -- 44 7.3 7.0 45 7.8 7.4 46 7.8 7.2 47 7.6 7.3 48 7.9 7.6 49 8.0 8.0 50 7.5 7.2 51 7.6 7.5 52 7.5 7.1 53 8.0 7.9 54 7.4 7.1 55 7.8 7.2 56 7.8 7.4 57 7.7 7.4 58 7.4 7.4 59 7.6 7.4 60 7.6 7.6 61 7.6 7.6 62 7.6 7.6 -- -- -- -- -- -- -- -- -- -- -- -- -- -- . -- -- -- -- 1A dissolved oxygen concentration o f 52. mg/L represents 60% saturation in freshwater at 22C. 2NC-Data inadvertently not collected. 000379 .W ildlife International, Ltd -93- Project Number 454A-134 Appendix 5 (Continued) Dissolved Oxygen (m g/L) ofW ater in the T est Cham bers1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochina Dilution W ater W ell Water Day Depuration Phase Negative Control 0,086 m g a l/L 1 7.5 2 7.6 3 8.3 4 8.0 5 8.2 6 8.0 7 8.4 8 8.3 9 7.9 10 8.0 11 8.0 12 7.9 13 8.0 14 7.9 15 7.6 16 8.1 17 8.0 18 7.9 19 7.8 20 8.2 21 8.0 22 8.1 23 8.4 24 8.4 25 8.1 26 7.5 27 7.9 28 7.5 29 8.2 30 8.0 31 8.3 32 8.4 33 8.0 34 8.3 35 8.5 7 .6 7 .7 8 .4 8 .0 8.2 8 .0 8 .4 8 .4 8 .0 8 .0 8 .0 8 .0 8 .0 7 .9 8 .0 8 .2 8.0 8 .0 7.8 8 .2 8.1 8.2 8 .4 8 .4 8 .2 7 .6 7 .9 7 .6 8.2 8.1 8.3 8.4 8.1 8.4 8.5 1A dissolved oxygen concentration o f 5.2 mg/L represents 6% saturation in freshwater at 22C. 000380 .W ildlife International, Ltd -94- Project Number 454A-134 Appendix 5 (Continued) Dissolved Oxygen (m g/L) o f W ater in the T est Chambers1 Sponsor. Test Substance: Test Organism: Dilution Water: 3M Corporation FFOS Bluegill, Lepomis macrochina W ell Water Day Depuration Phase Negative Control 0.086 m g a.i7L 36 8.4 37 8.2 8.5 8.1 38 8.0 39 8.0 40 8.1 41 7.7 42 7.8 43 8.2 44 7.8 8 .0 8 .0 8 .2 7.7 7 .8 8.3 7 .8 45 8.2 46 8.1 8.3 8.1 47 8.6 48 8.4 49 8.5 SO 8.1 SI 8.6 52 8.1 53 8.2 54 8.4 55 8.2 56 8.2 8 .6 8.4 8 .6 8.1 8 .6 8.1 8.2 8 .4 8.3 8.3 1A dissolved oxygen concentration o f 5.2 mg/L represents 6% saturation in freshwater at 22C 000381 ,W ildlife International Ltd. - 95 - Project Number 454A-134 A ppendix 6 Hardness, Alkalinity, Conductivity and TOC o f W ater in the Negative Control U ptake Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: W ell Water Parameter Day 0 7 14 21 28 35 42 49 Hardness 132 112 126 130 136 132 104 134 (mg/L as CaCO3) Alkalinity 183 174 179 183 181 183 183 184 (mg/L as CaCO3) Conductivity 310 325 330 330 325 325 310 320 (um hos/cm ) TOC <1 <1 <1 <1 <1 <1 <1 <1 ______ QngC/L)___ 56 130 181 330 <1 D epuration Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism Bluegill, Lepomis macrochirus Dilution W ater W ell Water Parameter Dav 1 7 14 21 28 35 42 49 56 Hardness 134 110 126 124 130 132 132 128 126 (mg/L as CaCO3) Alkalinity 179 178 178 180 183 180 175 179 178 (mg/L as CaCO3]! Conductivity 330 330 325 320 325 325 330 320 325 (pm hos/cm ) TOC (m gC/L) <1 <1 <1 <1 <1 <1 <1 <1 <1 000382 W ildlife Internationalj Ltd. -96- Project Number 454A-134 Appendix 7 Cumulative M ortality and Treatment-Related Effects1 Negative Control - Uptake Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: W ell Water Cumulative Number Number Day Observations Dead Remaining 0 AN 1 AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN 10 AN 11 AN 12 AN 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 90 85 80 80 75 75 75 75 70 70 70 70 70 70 70 65 65 65 65 65 65 65 60 60 60 60 60 60 60 55 55 55 55 55 55 55 1 O bserved Effects: AN = A ppears Normal Number Sampled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 000383 W ildlife International, Ltd. -97- Project Number 454A-134 Appendix 7 (Continued) Cum ulative M ortality and Treatm ent-Related E ffects1 N egative Control --U ptake Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: W ell Water Cumulative Number Number Day Observations Dead R em aining 36 , AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 A N 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN 50 AN 51 A N 52 AN 53 AN 54 AN 55 AN 56 AN 57 AN 58 AN 59 AN 60 AN 61 AN 62 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 50 50 50 50 50 50 45 45 45 45 45 45 45 40 40 40 40 40 40 40 35 35 35 35 35 35 1 Observed Effects: AN = Appears Normal Number Sam pled 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 10 0003S4 W ildlife International, Ltd. - 98 - Project Number 454A-134 A ppendix 7 (C ontinued) C um ulative M ortality and Treatm ent-R elated E ffects1 N egative Control - U ptake Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus W ell Water Cumulative Number Number Day Observations Dead IRemaining 1 AN 0 25 2 AN 0 25 3 AN 0 25 4 AN 0 25 5 AN 0 25 6 AN 0 25 7 AN 0 25 8 AN 0 25 9 AN 0 25 10 AN 0 25 11 AN 12 --* 0 0 25 25 13 AN 0 25 14 AN 0 25 15 AN 0 20 16 AN 0 20 17 AN 0 20 18 AN 0 20 19 AN 0 20 20 AN 0 20 21 AN 22 AN 0 0 20 20 23 AN 0 20 24 AN 0 20 25 AN 0 20 26 AN 0 20 27 AN 0 20 28 AN 0 20 29 AN 0 16 30 AN 0 16 31 AN 0 16 32 AN 0 16 33 AN 0 16 34 AN 0 16 35 AN 0 16 1Observed Effects: A N = Appears Normal Biological observations not recorded on this day. Number Sampled 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 * 000385 .W ildlife International, L td -99- ProjectNumber 454A-134 A ppendix 7 (C ontinued) Cum ulative M ortality and Treatm ent-Related Effects1 _______________________________ Negative C ontrol - U ptake Phase________ Sponsor 3MCorporation Test Substance: PFOS TestOrganism Bluegill,Lepomis macrochirus Dilution Water: Well Water Cumulative Number Number Day Observations Dead Remaining 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 16 16 16 16 16 16 12 12 12 12 12 12 12 12 12 12 12 12 12 12 1ObservedEffects: AN=Appears Normal Number Sampled 0 0 or 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 12 0003SG W ildlife InternationalJ Ltd. -100- ProjectNumber 454A-134 A ppendix 7 (C ontinued) Cumulative M ortality and Treatm ent-Related E ffects' 0.086 mg a.i./L - Uptake Phase Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill,Lepomis macrochirus Dilution Water: Well Water Cumulative Number Number Day Observations Dead Remaining 0 AN 0 1 AN 0 2 AN 0 3 AN 0 90 85 80 80 4 AN 0 5 AN 0 75 75 6 AN 7 AN 8 AN 9 AN 10 AN 11 AN 12 AN 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 75 75 70 70 70 70 70 70 70 65 65 65 65 65 65 65 60 60 60 60 60 60 60 55 55 55 55 55 55 55 1Observed Effects: AN =AppearsNormal Number Sampled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 000387 W ildlife International, Ltd. -101 Project Number 454A-134 A ppendix 7 (C ontinued) Cum ulative M entality and Treatm ent-Related E ffects1 __________0.086 m g a.iVL - Uptake Phase_________ Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bhiegl, Lepomis macrochirus Dilution Water: Well Water Cumulative Number Number Day Observations Dead Remaining 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN, IX 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN 57 AN 58 AN 59 AN, IX 60 AN 61 AN 62 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 50 50 50 50 50 50 50 45 45 45 45 45 45 45 39 39 39 39 39 39 39 34 34 34 33 33 33 1ObservedEffects: AN= AppearsNormal. X =Dead. Number Sampled 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 10 000388 W ildlife International, Ltd. -102- Project Number 454A-134 A ppendix 7 (C ontinued) Cumulative M ortality and Treatm ent-Related Effects1 0.086 mg a.i./L - Depuration Phase Sponsor: 3MCorporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus DilutionWater WellWater Cumulative Number Number Day Observations Dead Remaining 1 AN 2 23 2 AN 2 23 3 AN 2 23 4 AN 2 23 5 AN 2 23 6 AN 2 23 7 AN 2 23 8 AN 2 23 9 AN 2 23 10 AN 11 AN 2 2 23 23 12 2 23 13 AN 2 23 14 AN 2 23 15 AN 2 18 16 AN 2 18 17 AN 2 18 18 AN 2 18 19 AN 20 AN 21 AN 2 2 2 18 18 18 22 AN 2 18 23 AN 24 AN 2 2 18 18 25 AN 2 18 26 AN 2 18 27 AN 2 18 28 AN 2 18 29 AN 2 14 30 AN 2 31 AN 2 32 AN 2 14 14 14 33 AN 34 AN 35 AN 2 2 2 14 14 14 1ObservedEffects: AN= Appears Normal Biological observationsnot recorded on this day. Number Sampled 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0003S3 W ildlife International, Ltd. -103 - ProjectNumber 454A-134 Appendix 7 (Continued) Cumulative M ortality and Treatm ent-Related E ffects' 0.086 m g a .i /L - D epuration Phase_______ Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Cumulative Number Number Day Observations Dead Remaining 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 14 14 14 14 14 14 14 14 10 10 10 10 10 10 10 10 10 10 10 10 10 1ObservedEffects: AN= AppearsNormal Number Sampled 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 10 000390 W ildlife International, Ltd. -104- projectNumbcriMA-rn A ppendix 7 (C ontinued) Cumulative M ortality and Treatm ent-Related Effects1 0.087mg a.i./L - Uptake Phase Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water Well Water Cumulative Number Number Day Observations Dead Remaining 0 AN 0 1 AN 0 90 85 2 AN 0 3 AN 0 80 80 4 AN 0 75 5 AN 0 75 6 AN 0 75 7 AN 0 75 8 AN 0 70 9 AN, IX 10 AN 11 AN 1 1 1 70 69 69 12 AN, 1R 1 13 AN, IX 2 14 AN, 6R, 14X 16 15 AN, 8X 24 69 69 68 49 16 AN.6X 30 17 AN 30 18 AN 30 19 AN 30 20 AN 30 21 AN, 3R, 5X 35 22 AN, IR,4X 39 41 35 35 35 35 35 25 23 AN.IR.2X 41 24 AN, IX 42 21 19 25 AN.5R.1X 43 26 AN.6X 49 27 AN, 1R 49 28 AN.3X 52 18 17 11 11 29 AN, 1C, IX 53 30 1C, IX 54 31 1C 54 32 1C 54 3 2 1 1 33 1C 54 34 1C 54 35 IX 55 1 1 1 1Observed Effects: AN* AppearsNormal, C= Lethargic, R=LyingonBottom, X= Dead Number Sampled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 0 000391 ,W ildlife International Ltd. -105- ProjectNumber 454A-134 A ppendix 8 Changes to Protocol This study was conducted in accordance with the approved Protocol w ith the following changes: 1. Amendment: The proposed experim ental start and term ination dates were added to die protocol. 2. Amendment: The frequency o f light intensity m easurem ents was added to the protocol. 3. Amendment: The frequency o f TOC measurements w as added to the protocol. 4. Amendment: The methodology for TOC measurem ents was added to the protocol. 5. Amendment: Storage stability QC sam ples were added to the protocol. 6. Amendment: The protocol was clarified to indicate wheat fish would be sam pled for lipid analysis onDayO. 7. Amendment: The internal reference standard w as added to the protocol. 8. Deviation: Dissolved oxygen was not m easured on Day 24 o f the uptake phase. 9. Deviation: T est tem perature was out o f range for approxim ately 2 hours. 10. Deviation: The 0.87 m g a.iTL test concentration adversely affected the test organisms. 11. Deviation: Biological observations were not recorded on Day 12 o f the depuration phase. 12. Deviation: Four fish were collected from each treatm ent on Days 28 and 42 o f the depuration phase. 13. Deviation: The depuration phase was term inated on Day 56. 14 Deviation: Fish in the 0.87 mg a i./L treatm ent group were n ot collected for lipid analysis a t the end o f the uptake or depuration phases. 000392 W ildlife International, Ltd. - 106- ProjectNumber 454A-134 Appendix 9 Protocol, Amendments and Deviations 000393 W ildlife International, Ltd. -107- Project Number 454A-134 PROTOCOL PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL {JLepamismacrochtrus) U.S. Envinmmenlal ProtectionAgency Strict 850 - Ecological Effects TestGuidelines OPPTS Number 850.1730 im i OECD Guideliws 305 Environmental LaboratoryRequestNumber U2723 Submitted to 3M Coipoialkn Environmental Laboratory 935 BuA Avenue S t Paul. Minnesota 55144 Wildlife International, Ltd. 8598 Commerce thrive Easton. Maryland 21401 (410) 822-8601 October 18.2000 000394 W ildlife International, Ltd. -108- Project Number 454A-I34 W ildlife InternationalLtd. -2- PERTLUOROOCTANESULFONATE. POTASSIUMSALT (PFOS): A FLOW-THROUGH BIOCONCENTHATIONTESTWITH THEBLUEOILL(Lepomii macrochirus) SPONSOR: 3M Corporation Eaviraomental Laboratory P.O.Box 33331 S t Paal, MincesoU. 55133 SPONSOR'S REPRESENTATIVE: Itbit lilt. fA O U I: M. Satan A. React S59S CommerceDrive Easton, Maryland 21601 STUDY DIRECTOR: K uitR . Drottar LABORATORY MAMAQEMEMI: HenryO. Krueger, PitD. Director ofAquaticToxicology & Non-Target Plants _______________________FOR LABORATORY USE ONLY Proposed Dates: Experimental Experimental StartDate: _______________ ______ TerminationDate:___ ProjectNo.: 4S * 4 -A - 1 3 4 - Test Concentrations: NegativeControlJU_md 1.Qjam-UL-------------- TestSubstanceNo.: 4675 ReferenceSubstanceNo. (ifapplicable): PROTOCOL ATPMMftfc STUDY DIRECTOR * /4 * /* //< 3 0 DATO PROTOCOL NO : 454/10t 00/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 00039S .W ildlife International, Ltd L09- Project Number 454A-134 W ildlifc International, Ltd. -3- INTRODUCTJON Wildlife International, Ltd. wiQ conduct a hinconeeiaration test with the bloegill stmfish {Lepomlr macrocHrus) for the Spooler at the Wildlife latcmstionsl Ltd. aquatic toxicology facility in Easton, Maryland The study will be performed based on procedures in U S Eoviranmental Protection Agency Series 150 - Ecological Effects Test G adefina OFPTS Number 50.1730 (1); ASTM Standard E1022S4 Standard PracticeJar Conducting BioccncuU m tlon T a ts v tth Fisher and Saltw ater Bivalve M olluscs (2); tad OECD Guide!ino far Testing of Chemicals 305, Bioaccvmuiatlon: Flow-Through Fish Test(3). Raw dafa foraflvrork perforated at Wildlife Inleraaaortal, Ltd. and a copy of the final report will be Sled by project number in ardnves located oc the Wildlife fetanaiicnal, Ltd. site; or at a alternative locationto bospecified in the finalreport OBJECTIVE The objective of das study is to obtain laboratory data characterizing the biocoacentratw potential of Perfluorooctancnilfonatc (hereafter referred to as PFOS) in the bluegtll, Lspomis m aem hints. EXPERIMENTAL DESIGN Bluegill will be exposed to two test conctartrstians and a rtegativo coctroL Each group will consist o f one test chamber with up to apprrarimatcly 4*0 grams o f fish biomass in each chamber. The test will be divided into an uptake and a depuration phase. During the uptake phase, fish in the treatment groups will be exposed to subkthal concentrations ofPFOS, while fish ia the control group will be esposod to dHutkn water. The duration o f the uptake phase <3 burn so 38 days) and the depuration phase (6 hours to 60 days) may vaiy according to the time required to reach steady-cate. During both phases o f the study, test organisms and water samples am U ncled and analyzed for PFOS content. These values wBl be used to determine the uptake rate oneslant (hi), the depuratim sate constant (kj), the Inurtie bioconccntratioo factor (BCFK) and the steady-state biocooccntration factor (BCF) farwhole body tissues, erSbletissue (Uda on)and umedible tissue. MATERIALS ANDMETHODS T est Substance The test substance win be Perfluorobutanesulfonate, Potassium Salt, hereafter referred to as PFOS. Information oo ths characterization oftest, control or reference substances is required by Good PROTOCOL NO.: M4/101SOO/BLU-Bi(ySUB454 Environmental Laboratory Request Number U2723 000396 W ildlife International, Ltd. -110- Project Number 454A-134 WilcUife International, Ltd. -4- Laboratory Practice Standards (GLP). TheSponsors rapomuWe forprovidingWildlifeInternational, Ud. written verification that the test siibstanee has faeea characterized according to GLFs prior to initiation o f the study. If written verification ofGLP test mibttinco characterization U not provided to Wildlife International, Ltd., it willbe noted in the compliance statement ofthe final report The Sponsor is responsible for all formation related to the test substance, including the retention of a reserve sampler* b o k * or batch o f the test substanceused in this study. Tlic Sponsor also agrees to accept any unused test arfatance and/or test substance containers remaining t the end o fthe study. Preparation ofTest Concentrations The test substance will be administered to he test organisms ia water. This rente of administration was selected because ft represents the most likely route of exposure to aquatic otganisms. A primary stock solution of PFOS will be prepared by transferring tee PFOS to a volumetricflask and dilating to an appropriatevolumewith dilution water. Test Organism The test organism used in this study will be the bluegill, Lepom lt m aeroehina. B lu e tt are ww Qf hit fiflj fijf UK in bloccncentration tests (1,2,3). BhicgtQs will be obtained as juveniles fireni a commercial supplier, gradually acclimated to WildEfb International. Ltd. well water, and held for a period of at least 14 days prior to the test. If the fish are held at.water temperatures that differ from the test taaperahrra, they will be brought to test temperature at a rate not emending J'C per 72 baaa daring kokfing. If mortality exceeds 3% during She W-bour period munrriiatriy preceding the test, the entire batch o f fish will be rejected or held for an additional 14- day periodto ensurethat they are healthy. Fish will be bandied as little as possible, but when handling is necessary, it will be done carefkQy. gently, and quickly. To control bias, fish well be removed from bolding tanks with nets and impartially distributed to Shetest dorabera. No other forms o f bias are expected to affect the results ofthe study. PROTOCOL NO : 454/101SOO/BLU-BIO/SUB454 Environmental Laboratory Request NumberU2723 000397 W ildlife International, Ltd. -111- Project Number 454A-134 W ildUfc International, Ltd. -s- The minimum acceptable sine of individual lo t organisms will be determined by the quantity of tissue required for measurement o f (endues. Loading (the total wet weight o f fish per fiter oftest solution) will net exceed 1.0 gram o f fish per tier of solution that passes through a test chamber in 24 boors. BtaegSl will be fed flake food or mother commercial feed, onee daily. Exoess feed will be siphoned from the tanks approximately 30 minutes after feeding. Feeding and sampling schedules will be eootdhated so Hat fids will be sampled at least lour boors after feeding. Specifications for acceptable levels of contaminants in fish dels have not been established. However, them tie no known levels o f eosdamatanh seasonably expected to bo present in the diet that are considered to interferewith the purposeor coodnet ofthetest. Dilution Water Water used for the bolding and testing o f btuegiU will be obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The water will be passed through a sand filter and pumped into a 37,800-L storage tank where die water will be aerated with spray nozzles. Prior to use the water will be filtered to 045 im in order to remove fine particles. Wafer used for M d*"a and testing is characterized as moderately bard. Typical values for hardness, alkalinity, pH ind specific amdactaneeare approximately: Hardness,tng/L as CaCOs Alkalinity, mg/L as CaCOi 130 170 8.2 320 Hardness, alkalinity, pH and specific otmdncfeitce will be measured weekly to monitor the consistency o f the well water. Means and ranges of the measured parameters for the four-week period precedingthe test will be provided in the final report Analyses win be performed s t least once asmualiy to determine tbe cwmottratioas of selected organic and inorganic constituents o f die well waterand results ofthe mostreceat GLP analyseswill be aumnariaed m the final report Diluter System and Test Conditions A continuous-flow dihtter will be used to provide two concentrations of PFOS and a control. The flow ofdilution water to control and treatment test chambers will be regulated by roCareetenr. A PROTOCOL NO.: 454/101KWBLU-BKWSUB434 Environmental Laboratory Request Number U2723 000398 W ildlife International, Ltd. -112- ProjectNumber 454A-134 W ildlife International, Ltd. -6 - peristaltic pump will be used to inject the test substance w aiting stock solutioa into t mixing Flow rates will be e la te d m that each test dum ber receives at least six volume udditoes of water every 24 hours. Delivery of the ten substance working stock solution will be initiated at least 48 hours prior to the test Duplicate water samples four each test dum ber win bo collected at least tw i to verify the esttbliriiraem o f equilibrium eonceatiations o f PFOS in rise test chambers before introduction o f the test organisms (ia , measured conccaftartont should remain within 70 to 120% of nominal concentrations). The general operation o f fte dilator will be checked visually at least two times p day duringthe test Stainless steel aquaria filled Ah appradnuriefy 80 L o f test water will be used as test chambers. Test chambers will be placed in a water bath to maintain a test temperature of 22 a I*C. The test chambers and water bath will be enclosed in a plexiglass hood in order to miniirire potential cross-ccotamination Ambient light during testing v*Ul be provided by fluorescent tubes that emit wavelengths limilar to natural sunlight (e.g. C otonou^ 50 or equivalent). A photoperied of 15 hours of light and 8 bouts o f darkness will be controlled with an automatic timer. A 30-minute transition period of low ligfr intensity will be provided when fights go on or off to avoid sudden changes in light tensity. Testchambers willbe identified by the project number and test concentration. PROCEDURES The test win be divided into an uptake and a depuration phase. Organisms in the control group wilt be exposed to well water in tbc uptake and depuration phases. Organisms in the treatment groups will be exposed to the teat substance in the uptake phase and wdlsvaler in the deputation phase. Selection of Test Concentrations Tbs test concentrations selected will not stress, irritate, or otherwise adversely affect the organisms. The test concentrations will be selected in conjunction with the Sponsor and will be based on acute orchronictoxicity data. Tbc two exposure concentrations should differby a factoro f 10. PROTOCOL NO : 454/iQ180(VBLU-BIO/SUB454 Environmental Laboratory Request Number U2723 000399 W ildlife International, Ltd. -113- Project Number 454A-134 W ildlife International, Ltd. ChcraicabThyrical Measurements Temperature win be monitored and recorded continuously during rhe entire to t in one control replicate using a Fulscope ER/C Recorder (1900 I Seri Model A ), or equivalent. Recorder measurements will be verified prior to the test and at approximately weeldy intervals during the test using liquid-in-glass thermometer. Temperature also will be measured in every test dum ber at tbe beginning and end o f tbe test and at weekly intervals during fin test using a liquid-in-glass Dissolved oxygen will be measured daily in every test chamber during tbe test using a Yellow Spring instruments Model 51B dissolved oxygen meter, or equivalent, in tbe event that dissolved oxygen level] fidl below 60% saturation, appropriate actions will be taken after consultation with tbe Sponsor. Measurements of pH will bo made in every test chamber nt tbe beginning end end of the test and at weekly intervals during the test using a Fisher Accumet Model 913 pH meter, or equivalent Hardness, alkalinity, and conductivity will be measured in tbe control treatment at rite beginning and end o f the test and at weekly intervals duruig the test Hardness and alkalinity measurements will be rnutn by lim iim using procedures based on methods is Sketdard M ethods fo r rite Exam ination o f W ater and W astewater (4). Conductivitywill bo measured using a Yellow Springs Instrument Model 33 Salinity-Cooducth'ity-Tcmpaaturemeter, or equivalent Biologic*) Measurements Observations of behavior and mortality will be made daily. For a test to be considered valid, mortality and aboormal behavior should not be apparent m more than 10% eftbe fish in the treatment or centralgroups. Duration af Uptake Phase The statistically optimum durationo ftbe uptake phase (n) is near n1.6flQ ,lm t notmote dun 3.0*2, which is equivalent to 93 percent of steady state. The uptake phase will continue until the concentration of PFOS residues in body tissues reaches steady-state. The criterion for anainmest of slcudy-s&tc is that three consecutive PFOS residue concentrations in fish are net statistically different firmseach other. PROTOCOL NO.: 454/101I00/BUJ-BIO/SUB4S4 Environmental Laboratory Request Number U2723 000400 W ildlife International, Ltd. -114- Project Number 454A-134 W ildlife International, Ltd. Duratlon ofDepuration Phase I- 0.69/ka], bowever. the dputation phase 31 continue until 3 concentration o f PFOS tesidue in the organisn a ttira anyoaeoflte&BowiagthreecondrtiaM: 1. Rendues rcach les*thiB 10%o f stcady-siatc: or 2. ReaidacsfillbelowthoLOQior 3. Sixty (60) days ofdepunuioahave elapted. Simple Intervali fer W ater and K * Colectien Fuh and water wi be ixmpied do icss than fivc times dnring the uptake phase and four tinves durisg the dpuration phase of the study a*the same ralcrvalj In addition o u trai and trcated water 31 he sampicd no kss than twiee prier ta addition of fish ta the chamberj ta coafinn the eonoa Uratiens desirad. fa the ateence o f infonnalion on the bipaccnmulation potentiel o f the test substance and/or reliable physico-chemical data from which bioaceumulatkm potential may be estimated,tte recommended sampling schedule is a fellow. Phase Sample Interval Water1 Fish1 Action Pre-Uptake* -Ul X Start -"a X Intake 0 ~ 4 hows l day X XX XX Add fish 3 day XX 7 XX 14 X X 21 X X 28 X X Deparation 1 XX 3 XX 7 XX 10 X X 14* X X 'X J S ** "P****" *'water saapke w illU collectedat each interval listed for the analysis of A d ^ to sU ^ ^ re w ili be draw* as necesnry far Ite determination o f pH, hardness, 1 Sefiteia* fistiteli be collected to allow the preparation o ffa nr replicate samples from each treatment group and duplicate samples from the contici gm rp fw the analysts o f FFOS. Additiooalsamples will bedrawn i t necessary farthe analysiso f pid contest. Pre-Uptakewatersampling -n j ft confirmation oftreatment eveUprior to additionoffish. v aw atm n phase nmpfaig wiffcontinue unta the caocenUaticn o'PFOS residue is the organisms attains any oneo ftbecwmticinsactlined in the dnnrtiop o ffeoorat>cnphaseon case g. PROTOCOL NO.: 454/1O1I0Q/BLU-BKVSUB4S4 Environmental Laboratory RequestNumber U2723 000401 W ildlife International, Ltd. -115- Project Number 454A-134 W ildlife International, Ltd. .9 - The imraber tod frequency of sample intervals may be changed based on physico-chemical properties of the test substance from whichpeedietkw* of broaecunalatk* rate cm be derived andtor *1thedirectim of the Spootor. At etch tampSng interval, ton water samples win be collected fin s the control and three water tamplcs wifi be coUectad from the PFOS treatment groups. The ?FOS canocntratirm will be detenamed Sir a nuuhaam of one replicate o f a control and two replicates at each treatment level Sir each interval m which samples n s tnalyssd ( u specified by die Study Director}. The remaining replicates of samples draws fir PFOS analysis will be held in reserve end may be analyzed at die direction ofthe Study Director or Chemistry Principal Investigator. Theconcentrationof PFOS in the water at cadi sample interval will be presented as the average of the replicate measurements, as applicable. Additional samples will be drawn as necessary for foe determination o f pH, hardness, alkalinity, specific conductance, andtotal organic carbon (TOC). To assess das FFOS cantal in fids tissues, sufficient fish will be collected to sBew das preparation o f four replicate samples from each treatment group and duplicate samples fieni the oontnd group. One ritgticnal sample firm each group will be collected and held in reserve and may be analysed at the {Erection o f tile Study Director or Chemistry Principal Investigator. The emccnnatfam of PFOS ts Osh at each sample Interval wfflbe presented as the avenge o f dm replicate Fish sampled for determination of FFOS content will be impartially removed from foe test chambers, rinsed with dOadon water, blotted dry, and sacrificed. The wsgbt (wet weight, blotted dry) and total length ofeach fish will he determined within approximately IS mantes of collection, if possible. Each fish vriD be separated into edible and nanedible tissue and foe vet weight of eneh tissue action (i.e,, edible and nonedible) will be recorded. Fish will be stored frozen if not analyzed PROTOCOL NO t 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request NumberU2723 000402 W ildlife International, Ltd. -116- Project Number 454A-134 W ildlife International, Ltd. - 10- Additional samples will be drawnas necessary for the analysis oflipid content.. Analytical Method Wider and tissue samples will be analyzed for PFOS using liquid chromatography-mass spectrometry. Water samples will be analyzed according to the method entitled "Analytial Method Validation for the Determination o f Perfluorooctanc Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Saltwater and Algal Media'' (Wildlife international, Ltd Project No. 454C-109), Tissue samples will he amlyzed according to foe method entitled "Analytical Method Validation for the Determination of Perftuorooctsne Sulfonic Acid, Potassium Salt (PFOS) in Fish Tissues" (ProjectNo. 4S4C-119). Any raoddkatiarts to the above methodologies will be documented in the raw data and described in the find report Tisiu* Lipid Content At a minimum^ fish will be sampled from the control aquarium at Day 0 (upon transfer o f fish to aquaria), at an interval where the PFOS concentration in fish has reached steady stats (or following 28 days o f exposure), and at the end o f the depuration period. Fish will he sampled to generate four samples from the control and two treated aquaria a t each of the intervals noted. The lipid content of d m fiw a in linm wgHM liHi w in tv itrryttwjnnd hy rM nrnfnrm A ncfhanol extraction. Date Analysis Results o f tissue analyses wlli be presented on n wet weight basis. The steady-state bioconcentrarion factor for foe test substance (BCF), kinetic bioeopcentrarioc facte* (BCFK) uptake rate (kO, and depuration rate (kj) will be expressed for alible tissue, eonediblc tissue and whole fish W. RECORDS TO BE MAINTAINS Records to be maintained for data generated at Wildlife International, Ltd. will include but not be Smiledto: 1. A copy ofthe tigned protocol 2. Identification and characterization o f the test substance, ifprovidedby she Sponsor. 3. *>ritwriMi<wmrvtMmawmlinAffhwiMt 4. Test organismholding and aedimation records. PROTOCOL NO.. 4J4/10180WBLU-B1O/SUB454 Environmental Laboratory RequestNumber U2723 000403 W ildlife International, L td. -117- Project Number 454A-134 ildlife International, Ltd. -u- 5. Methods used 1a prepare stock cotutkms anddilutions ofdie testsubstance. 6. Daily observations. 7. Waterchemistry calculations (., hardnessand alkalinity). 8 . O ty nifffl H m w a irw iw i^ S. The methods used to analyze test substance concentrations and the results of analytical 10. Statistical calculation 11. A copy ofthe final report. FINAL REPORT A report o f the results o f the study will be prepared by Wildlife International, Ltd. The report Ytifiinclude, butnotbe ignited to,the Mowing: 1. Name and address ofthr.facility performing the study. 2. Dates on which the study was initiated and completed. It is the responsibility o fthe Sponsor to provide the final date that data ate recorded for chemistry, pathology and/ supporting evaluations that may be generatedat other laboratories. 3. A statement of compliance signed by the Study Director addressing any exceptions to Good Laboratory PracticeStandards. 4. Objectives end procedures stated in the approved protocol, including any changes in the original protocol. 5. Statistical methods employedtor analyzingthe data. i , The control nyf tefixtsM |jy ^ 1 iwtmbw a t cods number, strength, purity, and composition or other appropriate characteristics, if provided bydie Sponsor. 7. Stability and, when relevant to the conduct of the study, the solubility o f the test, control sad reference substances ruder fee conditions of sdwinistiaticn. if provided by fee Sponsor or fflfitnfflfpl ti7 UdL S. A description ofthe methods used. 9. A description o f fee test system need. Where applicable, fee final repast shall include the number of animals used, body weight range, source of supply, pedes, age, and procedure used tor identification. 10. A descriptiono fthe dosage, dosage regimen, routeofadministration, and duration. U. A descriptiono fall circumstances that strayhave affected the quality or integrity ofthe data. PROTOCOL NO.: 434/1QISOO/BLU-BIO/SUB434 Environmental Laboratory RequestNumber U2723 000404 .W ildlife International, L td -118- Project Number 454A-134 W ildlife International, Ltd. *12* 12. T terantooftivtStudyM rector, the sine* o fcflier scientists or professionals, snd the nameso f all supervisorypersonnel, in the study. 13. A description o f the trsnsfbnnatiooa, cslentations, or operations performed on the data, a stannary and analysis o f the data, and a statemento fthe conclusions drawn lie n the analysis. 14. Tbc signed and dated reports o f each of the individual scientists or other professionals involved in tbs atudy, if applicable. 15. The loeaticwhere afispeciinem, taw data, andthe final repeatarc to bo stored. 16. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the d a ta of any findings reported to the Study Directorand Management 17. If it is necessity to make correction* or addition! to a final report afler it la s been accepted, sucb changes shall be in tbc fotta o f amendmentby die Study Director. H e amendment should d tatly identify tbc part ofIbe oal Bport that is being added to or comctcd aod tits reasons ftf fee correction or addition. Amendments shall be rigeed sad dated by the Stud} Director. CHANGING OF PROTOCOL Planned changes to ibe protocol will be in tbc b a a of written amendments signed by Ibe Study D inaor and the Sponsor's Representative. Amendments will be constdercd as p u t of the protocol and wilS be attached to the final protocol. Any other changesnoil be in the fonn o f written deviations signed by the Study Director and filed with tbe taw data. All changes to the protocol will be indicated in flicfinal report. GOOD LABORATORY PRACTICES This atndy will be caoductcd in accordance with Good Laboratory Practice Standards for EPA (40 CFR Fait 160), OECD Principles o f Good Laboratory Practise (ENY/MC/CHEM (98) 17)-. ! Ibpae MAFF (39 NahSan. Notification No. 3830, Agricultural Ptodoctioo Bureau). Each study caoductcd fay WhBifb International, lid . it routinely examined by tbc Wildlife International, lid . Quality Assurance Unit for coupSancc with Good Laboratory Practices, Standard Operating Procedures and tbc spocificd protocol. A statement o f compliance with Good Laboratory Practices will be prepared far afl pcilkaa o fthe atudy conducted by Wildlife International, lid . The Sponsor will be topoiaftto& rcornpaaoe wifli Good Laborstoty Practices for procedures performed by other laboratories (e g , residue analyses or pathology). Saw data fer all work performed at Wildlife International, Ltd. and a copy ofthe final report will bo filed by project number in archives located on the Wildlife International, lid , site, or at an alternative location to be specified in the final! report. PROTOCOL NO.: 454M01800/BLU-B1O/SUB454 Environmental Laboratory RequestNumber U2723 000405 W ildlife International, Ltd. - 119- Project Number 454A-134 -13- RJEFERENCES 1. U.S. E ay inw aeotal Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (ftri& c .D ^ O P l> T S Number 850.1730: F ishB C F . 2. ASTM Standard E102Z-84. 1988. Standard P ra ctictf i t C onducting B ioconctniration T ests w ith F ishes a n d Saltw aterB ivalve M oihucs. American Society for Testing and M aterials. 3. OECD Guideline to r Testing of Chemicals 305. 1990. B ioconctniration: F lm t-T hrovgft F ish T est. 4. APHA, AWWA, W PCF. 1985. Standard M ethods f i r d ie E xam ination o f W ater and W astew ater. I&h Edition, American Public Health Association . American W ater Worics Association. W ater Pollution Control Federation.NewYosk. 5. G. E Blau and G. L. Agin. 1978. BIOFAC. The Dow Company. Midland, M l. PROTOCOL N O .: 454/101SOO/BLU-BIO/SUB454 Environmental Laboratory Request N um ber U2723 000406 W ildlife International, Ltd. -120- Project Number 454A-134 PROJECTNO.: 454A-134 W i l d l i f e In t e r n a t io n a l l t d .___________________________________ _________________ p * 1*2 AMENDMENTTO STUDYPROTOCOL STUDYTITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATION TESTWITH THEBLUEGILL(JUpamts m acrochin*) PROTOCOL NO.: 454/101SO0/BUJ-BIO/SUB434 AMENDMENT NO.: I SPONSOR: 3MCocponrion PROJECT NO.: 4S4A-134 EFFECTIVE DATE: Nowraber7,2000 ENVIRONMENTAL LABORATORYREQUEST NO.: U2733 AMENDMENT: Pigc2 Add: Expaimem*! Stsrl Date: 12/5/00 ExpcdmenUlTctminttion Date: 1/14/01 REASON: The sbovo infoinution was sotknownwheaths Study Directorsillied the protocol. AMENDMENT: Diluter System lad T ejt Conditlom,Page6 Add: Light intensity will bemeuuxed attestinitiation with a SPER Scientific Lid. light meter or equivalent REASON: To specify the freqsencj oflightintensity mrauremeot AMENDMENT: Chcmical/PhrucklM cuuraacaU. Pago7 Change: Hardness, BoUndy,!^odaivityv^ benHurM inthecontoltreatmemu thebeginningndend ofthe test andatvecUy inlenraliduringdie test Toe HjtnSoo. IValimty.crnrliicHyttyandtotal organicnarfeoa(TOC)w illbaracasraodm thecotfroitnattneiU at the beginning tad cod ofthe tostind *tweeklyinterval* during the tesL REASON: To specif the ttequeneyofTOC measurements. AMENDMENT: Chemicsi/PhysicalMeasurements, Page7 Add: TOC will be measuredmine a ShimudzuModel TOC-J9W TOC analjaer. REASON: Toaddlheroeftodology forTOC measurements. 9c 000407 W ildlife International, Ltd. 121- Project Number 454A-134 W il d l if e Intern a tio n a l lto. PROJECTNO.: 434A-134 Page 2 o f 2 AMENDMENT: A nalytical M ethod. Pago 10 Add: Tissue stability samples will be prepared at test initiation to establish testsubstance stability in fish tissues stored frozen during the study. REASON: To add storage stability QC samples. AMENDMENT: Tlssne L ipid C ontent, Page 10 Change: A t minimum, fish ill be sampled from the control aquaria at Day 0 . . . To: A t a minimum, fish w ill be sampled on Pay 0 . . . REASON: Fish sampled on Day 0 for lipid analysts will be collected priorto distribution to lb : test chambers. DATE 000408 W ildlife International, Ltd, -122- Project Number 454A-134 W lL D L IF E INTERNATIONAL. Ltd. PROJECTNO.: 454A-134 Peelofl AMENDMENTTO STUDY PROTOCOL STUDYTITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): A FLOW-THROUGH BIOCONCENTRATIONTESTWITHTHE BLUEGILL(Opomb m acrocklnu) PROTOCOL NO.: 454/10W0WBUWMCVSUB454 AMENDMENT NO.: 2 SPONSOR: JM Copontran PROJECT NO.; 454A-IJ4 EFFECTIVE DATE: November2>.2000 ENVIRONMENTAL LABORATORY REQUEST NO.: U2723 AMENDMENT: Pge2 Add: ReferenceSuhslenceNo.: 4324 REASON: To addthefctttmd referencestandard. ///S O /C O DATE SPONSOR'S REPRESENTATIVE $MU - V H 000409 W ildlife International, Ltd. -123- Project Number 454A-134 W il d l if e In ter n a tio n a llTM. PROJECTNO.: 454A-134 Page i c f I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): A FLOW-THROUGH BIOCONCENTRATION TESTWITH THE BLUEGILLOepomis macrocbirvs) PROTOCOL NO: 454/101800/BLU-BKVSUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PR O JEC T N O .: 454A -U 4 DATE OP DEVIATION: D tfa r2 9 .2000____________________ ___________________ ________ DEVIATION: Hie protocol stales Rot dissolved oxygen will be measured daily in every test chamber. Dissolved oxygenurn notmeasired on Day34 o fthe uptakephase o fthe test. REASON: Biologistoversight All dissolved oxygen measurementschaing thelea were60% o fsaturation. Consequently, it is the best judgement o f the Study Director that this deviation did not adversely aflect theresults o fthestudy. STUDY DIRECTOR LAB ORATORY MAi NAGEMENT DATE l 000410 W ildlife International, Ltd. -124- Project Number 454A-134 W il d l if e In tern a tio n a l lh>. PROJECTNG.: 4S4A-134 Page to il DEVIATIONTOSTUDYPROTOCOL STUDYTITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUCH BIOCONCENTRATIONTEST WITHTHEBLUEGILL(Lepomts macrochirm) PROTOCOL NO: 454/I018W/BLU-BIO/SUB454 DEVIATIONNO.: 2 SPONSOR: 3MCorporation PROJECTNOa 454A-134 DATE O F DEVIATION: Febnurv24.2001_____________________________________ ______________ DEVIATION: The protocol states that &s cut chambers m il be placed fa a water bath to maintain a test temperato of22* 1*C. OnDay 19of<fepuration,tlccfawwteniperaiarereccrderreahod 20*C. Tltef^m m vsaitrfn^fee#pp*wim M eJy2hduts. REASON; Unknown. Based on tlwslwrt durationo fthe temperaturedeviation,it is &ebestjudgement o fthe Study Direaor thatthis deviationdidnotadverselyaf&etthe remits o fthe study. _S7i23/<V DATE 000411 Wildlife International, Ltd. -125- Project Number 454A-134 W il d l if e In ter n a tio n a l lto. PROJECTNO.: 454A-134 Paget of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATION TEST WITHTHEBLUEOILL(JLtpamismacrochirus) PROTOCOLNO: 4S4/J(H*0<WBLU-BIO/SUB454 DEVIATION NO.: 3 SPONSOR: 3M Corporation PROJECT NO.; 454A-134 DATE OYDEVIATION; December 14.2000________________________________________________ DEVIATION: Uveprotocol sates flatthetestoooeeatnrttoia gdcctcdwiHnotstros,imtet^<rolhenMscaffict the organisms. The0.X7 mg *.i./L treatment group was actually chronicallytoxic to bluegill REASON: Infonnat#on theeluwdetoxicity ofPFOS to Muegjfflumnotknownvrtenthetest concentrations were selected. This deviation adverse!/ fleeted the results o f the study because adequate bioccnceatrationdata was not obtained fromthe0.87 mg a.UL treatmentgroup. to(IH01 DATE LABI E manaiGEM ENT 000412 W ildlife International, Ltd. -126- Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 434A -I34 Page 1 of l DEVIATIONTO STUDY PROTOCOL STUDYTITLE: PERPLUOROOCTANESUUONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATIONTESTWITH THEBLUEGILL (Lepom is m acrochtrus) PROTOCOL NO: 454/101S00/BLU-BIO/SUB454 DEVIATION NO.: 4 SPONSOR: 3MCorporation PROJECT NO.: 454A-134 PATE O F DEVIATION: February 17.2001_________________ _____________ _____________________ _ DEVIATION: The protocol staUa thatobservations ofbehavioroixJnvirtaHtywill be made daily. Biological observations were not recordedoa Day 12 ofdepuration. REASON: Biologist oversight Biological observations on Day 11 and 13 of depuration were the same. Consequently. itis the bestjudgemento fthe studydirector thatthis deviationdidnotadverselyaffect the results ofthe study. STUDY DIRECTOR ^. %/ LABORATORY MANAGEMENT DATE DATE 000413 Wildlife International, Ltd. -127- Project Number 454A-134 W il d u f e International ltd. PROJECT NO,: 454A-134 Page l o f i DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULEONATE.POTASSIUM SALT(PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEG1LL (L tpom it m aerochlrus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: S SPONSOR: 3M Corporation PRO JECTN O .: 4S4A-134 DATES OF DEVIATION: March S 4 1 9 .2 0 0 1 __________________________________________ DEVIATION: The protocol tea that w fffeiait fa ll willbe collected to Bowtecp rq n tio a o ffourreplicate samples t o n each treatment group and duplicate samples from tbe control group: One additional sample to n each group will be cotextsd andheld In reserve and maybe tnafyzed at tee <firetioa o f the Study DirectorC hem istry Principal Investigator. On Days 28 rad 42 o f depuration, tessrve samples were not collected. REASON: Reserve samples m xt not collected to conserve the number offhteremainmg in tee study. It is the bestjudgement o f the study director that this deviation did not adversely affect the results o f the study. STUDY DIRECTOR %JL LABORKATORY MANAGEMENT DATE H l /z j . DATE 000414 W ildlife International, Ltd. -128- Project Number 454A-134 W i l d u f e In t e r n a t io n a l ltd. PROJECTNO.: 454A-134 Page 1ofl DEVIATIONTO STUDYPROTOCOL STUDY T IT L E ; PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Jbepomis m a m xh iru s) PROTOCOL NO: 454/IQI8<XM3LU-BIO/SUB454 DEVIATION N O.: 6 SPONSOR: 3M Corporation PR O JEC T N O.: 454A-134 DATE OF DEVIATION: A nrg2-2001 _______________________________________________________ DEVIATION: The protocol states th at the depuration phase w ill continue unta the concentration o f PFOS residuein ttecqssm sro& toii anyone o fthe following tte o conditions: l ) Residues reachless d u n 10% ofstedy-sUtc,<x 2) Residues Callbelow the LOQ,o r 3) Sixty(60)dayso fdeputation hsveehpsed. The residuesdid n o tm eetl or2*boyeBt*Idepua-jeicnw*s terminated on Day 56 o f depuration. REASON: The Sponsor requested that sampling occur v7 w w 5 * 8 depuration, it th e best judgemento fthestudydirectorthat this deviation did not adversely affect theresults o fthe study. DATE 000415 Wildlife InternationalLtd. -129- Project Number 454A-134 W lL D U F E INHERNATIONAL. Lt d . PROJECT NO.: 4S4A-134 Page l o f i DEVIATION TO STUDY PROTOCOL STUDYTITLE: PERFUUOROOCTANESULFONATE, POTASSIUM SALT<PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEOILL (JLepomts m acrochina) PROTOCOL NO: 454/101OO/BLU-BIO/SUB454 DEVIATION NO.: 7 SPONSOR: 3M Ctxpontkm PR O JEC T N O .: 454A-134 DATES OF DEVIATION: February 5 and April 2,2001 ______________________________________ DEVIATION: Theprotocolstate* that EikwiU be u n c le s fix Upidanalysis listarfy-stateanr! attheendo fthe depuration phase. No fish were sampled fixaithe 0.S7 mg aJL/L treatm ent group. REASON: T la ^ wereno fih ic n u ii^ m d :0 .S 7 mg a.i7L treatmentpo op at the above tinea. Itisth obcst STUDY DIRECTOR 9 UL LABORA TORY MMAANNiAGEM ENT tb(/H /C > ! ___ 000416 Wildlife International, Ltd. ProjectNumber 454A-134 -130A ppendix 10 Personnel Involved in the Study The following key W ildlife International Ltd. personnel were involved in the conduct or management o f this study: 1. M ark Jaber, Director o f Research 2. Henry O. Krueger, Pb.D., Director, A quatic Toxicology and N on-Target Plants 3. W illard B. Nixon, Ph.D., Director, Analytical Chemistry 4. K urt R. D rottar, Senior Aquatic B iologist 5. Cary A. Sutherland, Laboratory Supervisor 6. Raymond L. Van Hoven, Ph,D ., Scientist 7. Susan T. Plantam a, Biologist AMENDED 000417 Wildlife International, Ltd. ProjectNumber 454A-134 -131- Appendix 11 Calculations O f The Kinetic Concentration Factor (BCFK), The Uptake R ate Constant (M), The D epuration Rate Constant (MX The Half-Life For Clearance, And The Tim e To Reach 90% O f Steady State. 1. The depuration rate constant ( k 2) was determ ined from data collected during the depuration phase and was defined as die slope o f the regression line where x equals days o f depuration and y equals the natural log o f the tissue concentration. Results o f all calculations were generated using Excel 2000 in full precision mode. M anual calculations may differ slightly. depuration rate constant ( k 2) values Edible Fish Tissue 0.0080 Non-Edible Fish Tissue 0.0060 W hole Fish Tissue 0.0062 2. The half-life for clearance in tissue during the depuration phase was determined by the following equation: tU2= ( l / k 2) i n2 Half-lives for Clearance (days) Edible Fish Tissue 86.4 Non-Edible Fish Tissue 115.5 W hole Fish Tissue 111.7 3. The uptake rate constant ( ,) was determined from the following equation: Cf k2 ' C ,,(l where C f is the concentration o f fish tissue from the uptake/depuration curve near the uptake midpoint (mean concentrations from day 35 were used in calculations); where C w is the mean measured concentration in water (0.086 mg a.i/L ); and where t = 35 days. AMENDED 000418 W ildlife International, Ltd. -132- ProjectNumber 454A-134 Edible Fish Tissue 9.022 A ppendix 11 (continued) uptake rate constant ( k t ) values Non-Edible Fish Tissue 24.08 W hole Fish Tissue 17.35 4. It is assum ed that the two com partment, two-param eter m odel applies. This m eans that the tim e to reach 90% steady state in the uptake phase is about equal to the tim e it takes to reach 90% o f depuration in the depuration phase. Therefore, k 2 , the depuration rate constant, was used in the following equation to determ ine the tim e to reach 90% steady state for uptake: 0 .9 = 1 - <?"** or t90= 2 3 l k 2 Edible Fish Tissue 287 Time to reach 90% o f steady-state (days) Non-Edible Fish Tissue 383 W hole Fish Tissue 371 5. BCFK. values ( f k 2) are provided below: BCFK values Edible Fish Tissue 1124 Non-Edible F ish Tissue 4013 W hole Fish Tissue 2796 AMENDED 000419 ,W ildlife International Ltd, -133- Project Number 454A-134 A ppendix 12 Report Amendment 1. Original Report: Pages 1-8 Amendment: The pages were changed to include the amended report date, revised page numbers, and new signatures and dates due to the addition o f the report amendment as Appendix 12. Reason: To reflect the issuing o f an amended report 2. Original Report: Page 2 and 4 Amendment: The Study Director was changed to Henry Kruegerand Management was changed to M ark Jaber. Reason: Reassignment o f responsibilities due to the departure o f the original Study Director. 3. Original Report: D ata Analysis Section, Page 15 Amendment: This section was m odified to clarify that the tests for norm ality and homogeneity m et the assumptions o f the tests. Additionally, the section was m odified to explain why the computer program BIOFAC was not used to estim ate the kinetic concentration factor (BCFK), the uptake rate constant (ki), the depuration rate constant (k2), the half-life for clearance, and the tim e to reach 90% o f steady state. However, after further review, the BIOFAC program may not be the best way to estim ate these param eters. Reason: A fter consultation with the Sponsor it was believed that the BIOFAC model underestim ates the uptake and depuration rate constants, and overestim ates the kinetic concentration factor, the estim ated time to reach 90% steady state, and die half-life for clearance. Therefore, the data have been reanalyzed using tire equations and graphical methods outlined in the draft OPPTS 850.1730 Guidance Document 4. Original Report: Pages 17-18 and 31-33 Amendment: The results and conclusion sections, as well as the Table 6 and Figures 1-3 have been changed to reflect the recalculated numbers. Reason: To report the results o f the test based on recalculated numbers. AMENDED 000420 W ildlife International, Ltd. -134 - Project Number 454A-134 A ppendix 12 -Continued* Report Amendment 5. O riginal Report: A ddition o f Appendix 11. Amendment: A ppendix 11 was added to detail how calculations in the amended report were perform ed. Reason: To provide adequate explanation o f how the new calculations were performed. AMENDMENT SIGNATURES: _JL. -J?A DATE AMENDED 000421