Document dD2XL6bnZNnoL4KvXxmmX0Ko9

PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A -I34 ENVIRONMENTAL LABORATORY REQUEST NUMBER: U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects T est Guidelines OPPTS Number 850.1730 and OECD Guideline 305 AUTHORS : Kurt R. Drottar Raymond L. VanHoven, Ph.D. Henry O. Krueger, Ph.D.. STUDY INITIATION DATE: October 31,2000 STUDY COMPLETION DATE: June 21,2001 AMENDED STUDY COMPLETION DATE: June 6,2002 SUBM ITTED TO : 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 W ildlife International, Ltd. Project Num ber 454A -134 - 2- GOOD LABORATORY PRA CTICE COM PLIANCE STATEM ENT SPONSOR : 3M Corporation TITLE : 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 79 2 ,1 7 A ugust 1989. STUDY DIRECTOR- D irector o f Aquatic Toxicology and N on-Target Plants SPONSOR APPROVAL: AMENDED Wildlife International, Ltd. Project Number 454A-134 -3 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792,17 August 1989. The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory M anagement w o e as follows: ACTIVITY: Teat SubstancePieparation DATE CONDUCTED: November 29,2000 DATEREPORTED TO: STUDY DIRECTOR: MANAGEMENT: November 29,2001 November 30,2000 Matrix Fortification Water Chemistry December 8,2000 February 20,2001 December 8,2000 February 20,2001 December 11,2000 February 22,2001 Analytical Data and DraftRepoit Biological Data and Draft Report Final Report May 14 -1 7 and May 20 - 24,2001 June 11-13,2001 June 20,2001 May 24,2001 June 13,2001 June 20,2001 June 2,2001 June 14,2001 June 20,2001 Amended Final Report June 5,2002 June 5, 2002 June <5,2002 M arsh T. Hynson Qualii ^Assurance Program Supervisor DATE AMENDED W ildlife International, Ltd. -4 REPORT APPROVAL Project Number 454A -134 SPONSOR : 3M Corporation TITLE :PerihiOTOoctanesulfonatc, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the BluegiU (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: A AMENDED W ildlife International, Ltd. Project Num ber 454A -134 -5 TABLE OF CONTENTS T itle/C o v erP ag e..................................................................................................................................................... 1 Good Laboratoiy Practice Compliance Statem ent............................................................................................ 2 Quality A ssurance Statem ent.............................................................................................................................. 3 R eport Approval................................................................................................................................................... 4 Table o f C ontents................................................................................................................................................. 5 Summary ........................................................................................................................................................... 8 In tro d u c tio n ........................................................................................................................................................... 9 Objective ...........................................................................................................................................................9 Experimental D esign............................................................................................................................................9 M aterials and M ethods.......................................................................................................................................10 Results and Discussion....................................................................................................................................... 16 C onclusions......................................................................................................................................................... 18 References ......................................................................................................................................................... 19 TABLES T ab le 1 M eans and Ranges o f W ater Q uality 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 ab le 4 PFOS Concentrations in Edible, Nonedible and W hole Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L ........... .......................................................... 23 T ab le 5 Apparent Steady-State BCF Values for Bluegill Exposed to 0.086 mg a.i./L ........................ 26 T ab le 6 BCFK Model Estimates for Bluegill Exposed to 0.086 mg a.i./L ........................................... 27 AMENDED , .W ildlife International Ltd -6 - TABLE OF CONTENTS - Continued - Project Num ber 4S4A -134 T able 7 T able 8 TABLES (Coat'd.) PFOS Concentrations in Edible, Noncdible and W hole Fish Tissues o f Bluegill Exposed to 0.87 mg 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 FIGURES 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 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 easured During 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 A nalysis o f Perflurooctanesulfbnate, 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 Temperature 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 Test Cham bers...................................................... 91 A ppendix 6 Hardness, Alkalinity, Conductivity and TOC o f W ater in the Negative C ontrol............................................................................................................ 95 A ppendix 7 Cumulative M ortality and Treatment-Related E ffects................................................. 96 A ppendix 8 A ppendix 9 Changes to Protcol............................................ 105 . -0 Protocol, Amendments and D eviations....................................................................... 106 A ppendix 10 Personnel Involved in the S tu d y ....................................................................................... 130 A ppendix 11 Calculations O f The Kinetic Concentration Factor (BCFK), The Uptake Rate Constant (Jki), The Depuration Rate Constant (k2), 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 .W ildlife International, Ltd Project Number 454A-134 - 8- SUMMARY SPONSOR: 3M Corporation SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: W ildlife International Ltd. 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 mg a/L): 454A -134 Perfluorooctanesulfonate, Potassium Salt (PFOS) Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow Through Bioconcentration Test w ith the Bluegill (Lepomis macrochirus) Negative Control, 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 dLi.lL ceased after 35 days due to fish m ortality and tissue sam pling Experimental S tart (OECD) - November 2 9,2000 Experim ental S tart (EPA) - December 5 ,2O0O Biological Term ination - A pril 2,2001 Experimental Term ination - A pril 11,2001 - 118 Days (62-Day Uptake, 56-Day Depuration) 35 Days o f Uptake TEST ORGANISM: Bluegill (Lepomis macrochirus) SOURCE OF TEST ORGANISMS: Osage C atfisheries, Inc. 1170 Nichols Roaa 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 = 2.70; Range = 2.03 to 3.32 Mean = 62; Range = 56 to 66 RESULTS: (0.086 mg a.i./L) Kinetic Bioconcentration factor (BCFK): E dible 1124 N onedible 4013 W hole Fish 2796 RESULTS: (0.87 mg a.i./L) Edible N onedible 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 W ildlife International, Ltd. Project Num ber 454A-134 -9 - INTRODUCTION A bluegiU sunfish, Leponas 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 April 2,2001. Raw data generated by W ildlife International, L td and a copy o f the final report are filed under Project Number 454A-134 in archives located on die 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, the test organisms were exposed in erne o f three groups: 1) A negative (dilution water) 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 te s t Each test chamber contained 90 bluegill at test initiation, and one replicate was tested for each treatm ent and the negative control W ater samples 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 sample collection periods during the test and analyzed for PFOS by LC/MS. The results o f these analyses were used to calculate the BCF values, uptake rates and depuration rates in edible tissue, nonedible tissue and whole fish. Wildlife InternationalLtd. Project Num ber 454A-134 - 10- MATERIALS AND METHODS The study w as 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 Test Guidelines OPPTS Num ber 850.1730 (1); A SJM Standard E1022-84 Standard Practice fo r Conducting Bioconcentration Tests -with Fishes and Saltwater Bivalve M olluscs (2); and OECD Guideline for Testing o f Chemicals 305, Bioconcentration: Flow-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 white powder, was identified as FC-95, Lot # 2 17. 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 ambient 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 mg 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 mixing, the stock solutions appeared clear and colorless. Stock solutions were prepared at 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 mLVminute) where they were mixed with dilution w ater (at a rate o f 350 mL/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. .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 o e acclimated to test conditions for approxim ately 49 hours prior to test initiation. During the bolding and acclimation periods the fish showed no signs o f disease or stress. During the 14-day holding period preceding the test, w ater tem peratures ranged from 22.3 to 22.TC . 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 forw ater measurements are described in the Environmental Conditions section o f this report. A t test initiation, the bluegill were collected from the acclimation tank and indiscrim inately distributed 1 to 2 at a tim e into the test chambers until each chamber contained 90 fish. During holding and during the test, the bluegill were fed flake food supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania. The bluegill were fed at least once daily during holding and once daily during die test Feeding and sam pling schedules were coordinated so that fish were 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 f the shortest. The length and weight o f fish in the negative control were considered to be representative o f all fish used in the test. The mean total length o f 10 negative control fish measured at the end o f the test was 62 mm with 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 f fish per liter o f test water 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 die test and the initial stocking density (90 fish per tank). ' Test A pparatus A continuous-flow diluter was used to deliver each concentration o f the test substance and a negative control. A peristaltic pump (Cole-Parmer Instrum ent Company, Chicago, Illinois) was used to deliver the test substance stock solutions into m ixing chambers assigned to each PFOS treatment. The stock solutions were m ixed with dilution water in the m ixing chambers prior to delivery to the test chambers. The flow o f dilution water to the test chambers was controlled by rotam eters. The delivery o f water from the rotameters was checked prior to the test and at approximately weekly intervals thereafter. Approximately 6.3 volume additions o f test water were delivered to the test chambers every 24 hours. The general operation o f the diluter was checked at least two tim es a day during the test and once on the last day o f the test. .W ildlife International, Ltd Project Num ber 454A -134 - 12- T est cham bers were 104-L stainless steel aquaria filled w ith approxim ately 80 L o f test so lu tio a The depth o f the test w ater in a representative d u m b er w as approxim ately 19 cm. T est cham bers were indiscrim inately positioned in a tem perature-controlled w ater bath designed to maintain a constant temperature. The w ater bath w as enclosed in a plexiglass ventilation hood in order to m inimize any potential for cross contamination. T est chambers were siphoned daily and periodically cleaned during the test to rem ove excess feed and fecal m atter. Test chambers were identified by the project number and test concentration. Dilation Water The 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 w ell w ater is characterized as m oderately-hard water. The specific conductance, hardness, alkalinity and pH measurem ents o f the well water during the four-week period immediately preceding the test are presented in A ppendix 1. The well w ater was passed through a sand filter to rem ove particles greater than approxim ately 25 pm, and pumped into a 37,800-L storage tank and aerated w ife spray nozzles. Prior to use, fee water again was filtered (0.45pm ) to remove microorganisms and particles. The results o f periodic analyses perform ed to measure fee concentrations o f selected contaminants in well w ater used by W ildlife International, Ltd. are presented in A ppendix 2. E nvironm ental C onditions The target tem perature range for fee test was 22 1C. Temperature was recorded continuously in the negative control w ife a Fulscope ER/C Recorder (1900 J Series model no. A). Temperature was also measured in all test chambers at fee beginning and end o f the test and at weekly intervals during the test, w ife a liquid-in glass thermometer. Dissolved oxygen was measured wife a Yellow Springs Instrument Company, Inc. Model 51B dissolved oxygen meter. W ith fee exception o f uptake Day 24, measurements were made daily in each test chamber. Dissolved oxygen measurements were not made on uptake Day 24 due to biologist oversight. M easurements o f pH were made in each test chamber at fee beginning and end o f fee test and at weekly intervals during fee test using a Fisher Accumet Model 915 pH meter. .W ildlife International, Ltd Project Number 454A -134 - 13- Hardness, alkalinity, conductivity and total organic carbon (TOC) w o e measured in the negative control at the beginning and end o f the test, and at weekly intervals during the te s t Hardness and alkalinity were m easured by titration based on procedures in StandardM ethodsfo r the Examination ofW ater end Wastewater (4). Conductivity was measured using a Yellow Springs Instrum ent Company, Inc. Model 33 SalinityConductivity Tem perature meter. Total organic carbon was m easured using a Shimadzumodel TOC-5000total organic carbon analyzer. Am bient room light was used to illuminate the test system s. Fluorescent tubes that emittedwavelengths sim ilar to natural sunlight (Colortone SO) were controlled by an automatic tim er to provide a photoperiod o f 16 hours o f light and 8 hours o f darkness. A 30-minute transition period o f low light intensity was provided when lights went on and o ffto avoid sudden changes in light intensity. Light intensity at the surface o fthe water (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 number o f m ortalities and the number o f individuals exhibiting signs o f abnormal behavior. P rocedures fo r E xposure o f Fish 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 the 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 ai./L , nominal). The uptake phase for the high concentration (1.0 mg a.iiL , nom inal) was term inated after 35 days due to fish mortality. All subsequent references in this report to sampling periods after Day 35 o f uptake refer to the 0.1 mg a.i./L (nominal) exposure concentration. At 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. .W ildlife International, Ltd Project Num ber 454A -134 -1 4 - 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 14,2 8 ,4 2 and 56 o f the depuration phase. A t each w ater sam pling interval, two w ater sam ples were collected from the negative control and three samples were collected from each o f the two PFOS treatm ent groups. One negative control sample and two samples from each oftheP F O S treatm ent groups were analyzed for PFOS. The remaining 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 (LC/MS). Procedures for analysis o f the w ater sam ples are provided in Appendix 3. W ater sam ples were analyzed as soon as possible after collection w ithout storage. ' C oflection an d A nalysis o f T 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 o o 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 number o f fish were collected to provide tw o 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 measured for total length and wet weight within approxim ately 15 minutes o f collection, when possible. Each fish was th a t raised w ith dilution w ater, blotted dry again and dissected into edible and oonedible tissue fractions. D issection was accomplished by m aking an incision from ju st posterior to the base o f the pectoral fin dorsally through the spinal cord. The head, fins and viscera were removed from the body andwereconsidered to be nonedible tissue. The remaining 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 the tissue samples are provided in Appendix. All tissue sam ples were extracted immediately or stored at approximately 14C until extraction. W ildlife International, Ltd. Project Num ber 454A -134 - 15- U ssue Lipid Content Selected fish were collected to determine lipid content (Appendix 3). The determ ination o f percent 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 fix: lipid content were stored at approxim ately -14C until analysis. In this study, no attem pt was made to express bioconcentration in relation to lipid content. D ata A nalysis W hole fish concentrations were calculated based on fire sum o f the edible and nonedible parts. The steady-state bioconcentration factor (BCF) values w o e determ ined from the tissue concentrations at apparent steady-state divided by the average water concentration. Tissue concentrations wereconsidered to be at apparent steady-state if three or more consecutive sets o f tissue concentrations were not significantly different (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 m eet the assum ptions, the data was transformed in an attempt to correct the data. M ean tissue concentrations were then compared using analysis o f variance and D unnett's te s t In the original report the tissue values 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 (kj), the depuration rate constant (k2), the estim ated tim e 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 = k l/k l). AMENDED W ildlife International, Ltd. Project Num ber 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 chambers are presented in Table 1. The individual measurements arc given in Appendicies 4 and 5. W atertemperatures in the test chambers were within the tem perature range o f 22 1C established for the test with one exception. On 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 m g/L (74 percent o f saturation) throughout the to st M easurements o f pH ranged from 7.9 to 8.2. W eekly m easurem ents o f hardness, alkalinity, conductivity and total organic carbon remained consistent throughout the test (Table 1 and Appendix 6). O bservations o f M o rtality an d C linical Signs O bservations o f m ortality and clinical signs are presented in Appendix 7. Bluegill in the negative control appeared normal and healthy throughout the test. Two bluegill died in the 0.1 mg a.i./L (nom inal) treatm ent group; all other fish appeared normal 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 or been sampled for tissue analyses. Any fish found dead in the aquaria were stored frozen C oncentrations o f 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 o f PFOS during the uptake phase inth e0 .1 0 m g a.i7 L treatmentgroup ranged from 68 to 113% o f the nominal test concentration (Appendix 3). W hen concentrations m easured during the uptake phase were averaged, the mean m easured concentration was 0.086 mg a i./L which represented 86% o f the nominal test concentration. M easured concentrations o f PFOS during the uptake phase in the 1.0 mg a.i./L treatm ent group ranged from 73 to 101% o f the nominal test concentration. W hen concentrations m easured during the uptake phase were averaged, the m ean m easured concentration was 0.87 mg a.i./L, which represented 87% o f the nominal test concentratioa Concentrations o f PFOS during the depuration phase were all <LOQ ,W ildlife International Ltd. Project Number 454A-134 -17- C oncentrations o f PFO S 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 f fish 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 samples 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 that 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 estimate the kinetic concentration factor (BCFK), the uptake rate constant (0 , the depuration rate constant (2), the estim ated time 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 may not have been the best way to estim ate these param eters. Therefore, the data were reanalyzed using die graphical methods outlined in the draft OPPTS 850.1730 Guidance Document (1). These new calculations are described in Appendix 11. Plots o f die tissue concentrations versus time were constructed to evaluate the data. The raw data were plotted as well 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 estimates o f the uptake and depuration rate constants (k\ 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 the kinetic concentration factor, the estim ated time 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 ,W ildlife International Ltd. Project Num ber 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 f the tim e to reach 50% clearance o f PFOS 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 mg a.i./L treatm ent group had been sampled or were dead. Consequently, the only inform ation on bioconcentration that could be determined 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 o f PFOS. CONCLUSIONS 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 mg 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 6,116, and 112 days, respectively. AMENDED .W ildlife International, Ltd Project Number 454A-134 -19REFERENCES 1 U.S. E nvironm ental P rotection A gency. 1996. Series 8S0 - Ecological Effects Test Guidelines (drafi), OPPTS Number 850.1730: Fish BCF. 2 ASTM S tan d ard E 1 0 2 2 -8 4 .1988. Standard Practicefo r Conducting Bioconcentration Tests withFishes and Saltwater Bivalve M olluscs. American Society for Testing and M aterials. 3 O ECD G uideline fo r T esting o f Chem 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. American W ater W orks Association. W aterPollution Control Federation, New York. 5 B IO FA G 1991. Septem ber 19,1991 version. The Dow Chemical Company, M idland, M ichigan. 'W ildlife International, Ltd Project Num ber 454A-134 - 20Table 1 M eans o f Ranges o f W ater Quality Parameters Sponsor; Test Sustance: Test Organism: Dilution Water: Uptake Phase Nominal Concentration (mg a.iJL) Negative Control 3M Corporation FFOS Bluegill,Lepomis machrochirus Well Water Temperature1 DO2 (C) (mg/L) PH 21.9 7.8 8.1 21.8-22.0 6 .8 -8 .6 7 .9 -8 2 Alkalinity Conductivity (mg/L as Ounhos/cm) CaCOi) 324 180 310-330 174-185 Hardness (mg/L as CaCOs) 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 *Temperature measured continuously in the negative control ranged from 20.0 to 22.0C. 2At a temperature of22C, the dissolved oxygen saturation concentration is 8.7 mg/L and 60% saturation is 5.2 mg/L. -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 Blucgill, Lepomis macrochirus Well W ater Nominal Concentration (mg a.i./L) Negative Control 0 Hour <LOQ' 4 Hours <LOQ 1 <LOQ 3 <LOQ 7 <LOQ Day of Uptake 14 21 28 <LOQ <LOQ <LOQ 35 <LOQ 42 <LOQ 49 <LOQ 56 <LOQ 62 <LOQ Mean M easured Concentration (mg a.i./L) <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 z - - -- 0.802 0.930 0.820 0.734 0.818 0.875 1.01 0.925 0.871 Jt 0.87 86 87 1The Limit of 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. W ildlife International, Ltd. Project Number 454A -134 - 22- Table 3 Concentrations o f PFOS in W ater Samples During the D epuration Phase Sponsor: Test Substance: Test Organism: Dilution Water: Uptake Phase Mean Measured Concentration (mg a.i./L) 3M Corporation PFOS Bluegill, Lepomis macrocMrus Well Water Day of Depuration (m g& iJL) 14 28 42 Negative Control <LOQ' <LOQ <LOQ <LOQ <LOQ i..T. he 0.086 L_.im.it o .f Q uantitation <LOO (LOQ) was 0,0500 mg <LOO a.i./L <LOQ <LO0 56 <LOQ <LOQ <LOQ -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 Water Uptake Day 0 (4 hours) 0 (4 hours) 0 (4 hours) 0 (4 hours) Edible Tissue Concentration .... (mg a.i./Kg) 0.167 0.155 0.144 0.182 Edible Tissue Weight (g) 2.0545 1.8960 2.5219 1.3561 Nonedible Tissue Concentration 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/N56 ' E57/N57 E58/N58 E59/N59 14 14 14 14 11.4 2.0423 27.3 2.5002 9.07 2.1548 23.2 2.6582 13.7 1.0709 35.3 1.4046 12.6 1.1804 32.6 1.7455 ` E69/N60 E70/N70 E71/N71 E72/N72 21 21 21 21 11.7 2.1161 12.0 1.2984 12.9 1.4092 10.6 1.6268 1Whole Fish Concentration = (edible wt. X edible cone.) + (nonedible wt. X nonedible cone.) 33.3 22.7 24.6 24.4 2.4712 1.9396 1.9910 2.1872 (edible wt. + nonediblc wt.) Whole Fish Concentration1 (ma a-iTKal 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 W ildlife International, Ltd. ' -24- Project Number 454A* 134 Table 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,Lepomiamacrochirua Dilution Water Well Water Sample ID E82/N82 E83/N83 E84/N84 E85/N85 Uptake Day 28 28 28 28 Edible Tissue Concentration (mg a.i/Kg) 18.3 13.7 23.9 23.1 Edible Tissue Weight (g) 1.4636 1.4113 1.2081 0.8830 Nonedible Tissue Concentration ... (mga.iyKg) 49.4 40.7 65.3 57.9 Nonedible Tissue Weight (e) 1.7564 1.8903 1.3062 1.3512 E95/N95 E96/N96 E97/N97 E98/N98 35 35 35 35 22.6 1.7888 67.1 2.0434 - 27.7 1.S897 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 Elll/Nlll E l 12/N112 E113/N113 El 14/N114 49 49 49 49 33.3 1.7249 85.0 2.2873 36.2 1.2185 95.1 1.S993 39.0 1.5506 93.1 2.2184 30.6 1.4650 77.7 1.9337 E119/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 = fedible w t X edible cone.) + (nonedible w t X nonedible conc.1 101 112 105 96.4 1.7596 1.9170 1.5965 2.4378 (edible w t + nonedible wt.) Whole Fish Concentration1 (mg a.i^Kg) 35.3 29.2 45.4 44.1 46.3 53.8 466 40.9 50.1 49.4 40.9 56.3 62.8 69.6 70.8 57.4 90.6 716 63 3 74.8 77.0 92.7 79.6 73.1 -25- Project Number 454A -I34 Table 4 (C ontinued) PFOS Concentrations in Edible, Noncdible and W hole Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E13S/N135 E136/N136 E137/N137 E138/N138 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Uptake Day Edible Tissue Concentration (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 E1SKN151 E152/N152 E153/N153 42 42 42 42 24.1 1.7590 ' 71.7 31.2 1.1220 80.6 30.0 0.8440 78.3 33.0 0.8672 82.1 E157/N157 E158/N158 E159/N1S9 E160/N160 56 56 56 56 21.1 1.5471 37.6 0.5892 32.9 0.6244 31.2 0.7223 1Whole Fish Concentration = (edible w t X edible cone.) + fnonedible wt X nonedible conc.l (edible w t.+ 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 85.3 582 70.1 68.1 81.1 51.4 61.4 61.0 62.2 41.6 665 65 8 62.1 W ildlife International, Ltd. Project Num ber 454A-134 -26- Table 5 A pparat Steady-State BCF Values for Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution W ater: 3M Corporation PFOS Bluegill, Lepomis macrochirus W ell W ater Tissue Type Edible N o n ed ib le W hole Fish M ean M easured Test C oncentration (mg a.i./L) 0.086 0.086 0.086 Uptake Days at A pparent Steady-State 4 9 ,5 6 and 62 4 9 ,5 6 and 62 49,5 6 and 62 Mean M easured Apparent Steady- State Tissue Concentration (m ga.i./K g) 41.6 A pparent Steady-State BCF 484 96.7 1124 73.6 856 W ildlife International, Ltd. Project Num ber 454A -134 -27- Table 6 BCFK Estim ates for Bluegill Exposed to 0.086 m g a.i./L Sponsor: 3M Corporation T est Substance Test Organism PFOS Bluegill, Lepomis macrochirus Dilution W ater W ell W ater K inetic Bioconcentration Uptake Rate Factor C o n stan t Tissue Type (B C FK ) <fc) Depuration Rate C onstant &) Edible 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 learance (D ays) 86 N onedible 4013 24.08 0.0060 383 116 W hole Fish 2796 17.35 0.0062 371 111 AMENDED ,W ildlife International Ltd. Project Number 454A -134 . -28- Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E9/N9 E10/N10 Ell/Nll E12/N12 T able 7 PFO S C oncentrations in Edible, N onedible and W hole F ish T issues o f B luegill Exposed to 0.87 m g a.i./L 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Uptake Day 0 (4 hours) 0 (4 hours) 0 (4 hours) 0 (4 hours) Edible Tissue Concentration (mg a.iVKg) 1.46 1.48 1.19 1.39 Edible Tissue W eight (g) 0.9795 1.3226 1.6284 1.5205 Nonedible Tissue Concentration (mg a.i./Kg) 3.52 4.37 4.22 4.06 Nonedible Tissue W eight (g) 1.5183 1.6397 1.9391 1.9489 E22/N22 E23/N23 E24/N24 E25/N25 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 E35/N35 E36/N36 E37/N37 E38/N38 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 E48/N48 E49/N49 ' E5Q/N50 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 E62/N62 E63/N63 ______E64/N64 14 14 14 _________14_________ 87.1 81.6 90.7 73,3 1.9813 1.2152 1.1290 1.0811 1Whole Fish Concentration = (fable Wt X fable <X>1K.),+(nonedible Wt. XnQMxfabk cfflM,) (edible wt. + nonedible wt.) 177 207 245 214 2.9193 1.8260 1.5376 .......1.6199 Whole Fish Concentration1 (mg a.i./Kg) 2.71 3.08 2.84 2.89 8.00 10.9 10.2 9.47 30.5 30.7 34.5 39.1 74.9 77.0 85.3 89.8 141 157 180 158 W ildlife International, Ltd. Project Number 454A -134 ' -29- T able 7 (C ontinued) PFOS Concentrations in Edible, Noncdible and W hole Fish Tissues o f Bluegill Exposed to 0.87 mg a.L/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 T s b u o Concentration Uptake Day (mg a.iJKg) 21 79.4 21 117 21 104 21 102 Edible Tissue Weight (B) 1.8300 . 1.4526 1.6824 1.4076 Nonedible Tissue Concentration (mg aiTKg) 201 278 246 229 E87/N87 E88/N88 28 28 102 1.6679 131 1.0923 E89/N89 28 107 1.1675 E90/N90 28 133 1.1466 1Whole Fish Concentration = (eflbl.vrtJS Edible cone.)+ (nonedible wt. XOTiedifrle con,) (edible wt. + nonedible wt.) 289 372 320 361 Nonedible Tissue Weight (8) 2.1803 1.9786 2.2130 1.7558 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.iJKg) 146 210 185 172 205 267 232 263 W ildlife International, Ltd. Project Number 454A-134 -30- Table 8 Day 28 BCF Values for Bluegill Exposed to 0.87 mg a.i7L Sponsor: 3M Corporation T est Substance: Test Organism: Dilution W ater: PFOS Bluegill, Lepomis macrochirus W ell W ater Mean M easured T est Concentration Tissue Type (m ga.i./L ) Edible 0.87 Mean Day 28 Tissue Concentration (m ga.i./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 mortality. Day 28 BCF 136 386 278 Wildlife International, Ltd. -31- PrqjtNmnbr454A.134 Figure 1. Concentrations o f PFOS in Edible Fish Tissues o f Bluegill Exposed to 0.086 mg a.i./L. AMENDED ,W ildlife 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. AMENDED Wildlife International, Ltd. - 33- Project Number 454A -134 Figure 3. Concentrations of PFOS in W hole Fish Tissues o f BluegiU Exposed to 0.086 m g a.i./L. PFOS Concentration (mg/Kg AMENDED Wildlife International, Ltd. Project Number 454A -134 -3 4 Figure 4. Concentrations o f PFOS in Edible, Noneible and W hole Fish Tissues o f Bluegill Exposed to 0.87 mg a.i./L. a Non-Edible x Whole Fish Edible 0 10 20 30 Day W ildlife International, Ltd. ProjectNumber454A*134 -3 5 - A ppendix 1 Specific Conductance, Hardness, A lkalinity and pH o f W ell W ater M easured During the 4-W eek Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution W ater: 3M Corporation PFOS Bluegill, Lepomis macrochirus W ell W ate- 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 lk alin ity (m g/L as CaCOj) 178 (N = 4) 1 7 6 -1 7 8 pH 8.1 ( N - 4) 8 .0 -8 .2 ,W ildlife International Ltd. -36- Appendix 2 Analyses o f Pesticides, Organics and Metals in Wildlife International, Ltd. W ell W ater1 Pesticides and Organics Component Aclonifen Alachlor Ametryn Atrazine Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifcnthrin Bioallcthrin Bitertanol Bromacil Bromophos Bromophos-cthyi Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin Chlorfenvinphos Chknidazon Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Chlorothalonil Coumaphos Cyanazine Cyfluthrin Cypermethrin Cyproconazole Deltamcthrin Dcmeton Demeton-O Desethytatrazine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorhenzamide Dichlorfenthion Dichlorlluanid 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 <025 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 ' <0.25 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 Dimcthoatc Dimethomorph Disulfbton DMST Dodemorph Endosulfan-oc Bndosulfan-p Endosulfan-sulfate Epoxiconazolc Eptam Esfenvalerate Ethion Ethofiimcaate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorph Fenthion Fenvalerate Fluazifop-butyl Fluoroglycofcn-cthyl Fluroxypyr-meptyl Flutolanil Fonophoa Furalaxyi 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 <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. Continued 'Wildlife International, Ltd. -37- Appendix 2 (Continued) Analyses o f Pesticides, Organics and Metals in Wildlife International, L td W ell Water1 PesticdeiAnd Organic* (Page 2) Component Malathion Metalaxyl Metamitron Metazachlor Mcthidathion Paclobutazte Parathion Parathion-methyl Penoonazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Promctryn Propachlor Propazine Propham Propiconazole Propaxur Propyzamide Ptosulfocarb Pyrazophoi 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 <O.0S pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L 0 .0 5 pg/L 0 .0 2 pg/L 0 .0 5 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 2 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 2 pg/L 0 .0 5 pg/L 0 .0 3 pg/L 0 .0 2 pg/L 0 .0 2 pg/L 0 .0 3 pg/L Component Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifcnox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazinc Tctrachlorvinphos Tetmhydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyi Tolylfluanid Triadimefon Triadimenol Triallate Triazophoa Trifluratin Vamidothion Vinclozolin Measured Concentration 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 2 pg/L 0 .0 1 pg/L 0 .0 5 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 2 pg/L 0 .0 1 pg/L 0 .0 2 pg/L 0 .0 5 pg/L 0 .0 5 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 1 pg/L 0 .0 5 pg/L 0 .0 1 pg/L 0 .0 5 pg/L 0 .0 4 pg/L 0 .0 1 pg/L 0 .0 4 pg/L 0 .0 5 pg/L 0 .0 5 pg/L 0 .0 2 pg/L 0 .0 2 pg/L 0 .0 2 pg/L 0 .0 1 pg/L 0.01 pg/L Magnesium Sodium Calcium Iran Potassium Aluminum Manganese Beryllium Chromium Cobalt 13.2 mg/L 21 mg/L 35 mg/L 0 .0 2 mg/L 6.2 mg/L 0 .0 9 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 .2 5 pg/L 0.025 pg/L <1 pg/L 'Analyses performed by TNONutrition and Food Institute on samples collected on November 15,2000. Wildlife InternationalLtd. -3 8 Appendix 3 THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) CONCENTRATIONS IN FRESHW ATER AND BLUEGILL SUNFISH TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-134 W ildlife International, Ltd. - 39- SPONSOR; REPORT APPROVAL 3M Corporation Project Number 454A -134 TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochims) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 PRINCIPAL INVESTIGATOR: WILDLIFE INTERNATIONAL. LTD. MANAGEMENT dialog Date W illard B. Nixon, Ph.D / Director, Analytical Chemistry AMENDED W ildlife International, Ltd. -4 0 - In tro d u c tio n F reshw ater sam ples and tissue 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 by W ildlife In tern atio n al, L td . an d 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 International, L td. by high p erform 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 d etection (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 etection (L C /M S/M S). F resh w 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 co llected 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, 2000, F ebruary 5, 2001 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 tandard T he te st substance, PFO S, w as used to p repare calibration standards and m atrix fortification sam ples and w as identified as W ildlife In ternational, 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 4526 u po n receipt. T he internal standard, a g ran u lar m aterial, w as identified as: 1H, 1H, 2H , 2H P erflu o ro o ctan e Sulfonic A d d , C hem ical A b stract N um ber: 27619-97-2. The internal standard, referred to hereafter as 4H PFO S, w as stored 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 In tern atio n al, L td. P ro ject N o. 454C -109). T issue sam ples w ere analyzed according to th e m ethod en title d "A nalytical M ethod V alidation fo r th e D eterm in atio n o f P erflu o ro o ctan e S u lfonic Acid, W ildlife International, Ltd. -4 1 - Potassium Salt (PFOS) in Fish Tissues" (W ildlife International, Ltd. Prefect 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 sample 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 freshwater is presented in Figure 1. Samples were diluted in a 50% methanol (HPLC grade, 99.9+%): 50% NANOpure* w ater solution containing 10.0 pg 4H PFOS/L and 0.05% formic acid (v/v) so that 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 determined by reverse-phase high performance 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-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a K ey sto n e B etasil Cig an aly tical column (50 mm x 2 mm I.D ., 3 pm particle size) fitted with a Keystone Javelin Guard Cartridge (20 nun x 2 mm). The instrum ent param eters are sum m arized in Table 1. F resh w ater quality control (Q C ) sam ples (m atrix blanks and fortifications) w ere processed in th e sam e m anner as th e te st sam ples. F reshw ater w as fortified w ith th e ap p ro p riate PFO 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 substance. T issue Q C Sam ples B luegill sunfish (Lepom is m acrochirus) tissu e s w ere obtained from a breeding stock m aintained a t W ildlife In tern atio n al, L td . A pproxim ately 2 00 individual fish (ag es 3 to 5 m onths) w ere rem oved from a breeding tank and euthanized by severing th e spinal co rd above th e o p ercular region. H eads, fins and viscera w ere rem oved from the body and w ere considered to be nonedible tissue. T he balance o f th e tissue w as considered to be edible tissue. T he edible and nonedible tissues w ere separately ,W ildlife International Ltd. -4 2 - 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 the 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 a rt fo r th e analysis o f PFO S in fish tissu es is presen ted in F ig u re 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, i f 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 QC sam ples also w ere rem oved from th e freezer. A ny frozen sam ples w ere allow ed to thaw . O ne tissue 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 PFO S stock 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 then 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 rpm fo r approxim ately S m inutes. A liquots o f th e ex tract w ere th en volum etrically diluted into th e calibration range o f th e L C M S m ethodology w ith 50:50 m ethanol: NAN Opure w a te r dilution solvent. A liquots o fth e diluted ex tra cts w ere tran sferred to autosam pler vials and subm itted fo r analysis. Back-up tissue samples were collected and stored frozen. If necessary, the back-up tissue samples were removed from the freezer, allowed to thaw and processed using the same procedures described above. T issue Storage Stability Sam ples S tability sam ples w ere prepared at te st initiation to establish te st substance stability in fish tissu es stored frozen during th e study. T w o tissu e 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 W ildlife International, Ltd. -4 3 - w ere each fortified a t 0 .100 and 1 0 .0 m g a.i./K g using th e appropriate PFO S stock solution and a gastig h t syringe. T he stability sam ples w ere retu rn ed to th e freezer. F ollow ing 119 days o f frozen sto rag e, th e stability sam ples w ere rem oved from th e freezer and analyzed. F resh fo rtificatio n and m atrix blank sam ples also w ere prepared and analyzed a t this tim e. T he sam ples (new and o ld ) w ere p ro cessed using th e sam e procedures described fo r tissu e sam ple analyses. C oncentrations o f PFO S in fish tissu e w ere determ ined by reverse-phase high perform ance liquid chrom atography using a H ew lett-P ackard M odel 1100 H PLC interfaced w ith a P erkin-E lm er A P I 3000L C 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 P erkin-E lm er T urboIonSpray ion source. C h ro m ato g rap h ic sep aratio n s w ere achieved u sin g a K ey sto n e B etasil C 18 analytical colum n (SO 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 * 2 m m ). T h e instrum ent param eters are sum m arized in T able 2. T issu e 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 content w ere collected from th e test at initiation, on D ay 62 o f th e uptake phase (D ay 0 depuration), and at term ination (D ay 56 depuration). O nly fish from th e N egative C ontrol and 0.10 m g a.i./L nom inal te st level w ere sam pled fo r lipid co n ten t. Sam ples were collected in 20-m L g lass scintillation vials and sto red frozen until analysis. S am ple w eights w ere recorded a t th e tim e o f collection. U pon analysis, tissu e sam ples d esignated 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 o fN A N O pure w a te r 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 tissue shredder. E ach hom ogenate w as transferred to a 250-m L sep arato ry funnel th at 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 o f N A N O pure w ater and th e rinse w as poured into th e respective sep a rato ry funnel. T he sep arato ry funnels w ere shaken w ith venting fo r approxim ately on 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 a rato ry funnel. T he sep arato ry funnels w ere briefly sw irled w ith venting. T he phases w ere allow ed to separate. F o r each sam ple, th e chloroform layer w as drained through a pow der funnel p acked w ith ,W ildlife International Ltd. -44- g lass w ool and anhydrous sodium sulfate into a 250-m L round- bottom flask. A n additional 50-m L aliq u o t o f chloroform w as added to each sep arato ry flannel and th e ex tractio n an d draining procedures w ere rep eated . T h e ex tracts w ere ro tary evaporated in a w aterb ath m aintained a t approxim ately 40 C to n ear dryness. E ach ex tra ct w as tran sferred to a pre-w eighed labeled scintillation vial. E ach250-m L rou 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 gentle stream o f nitrogen o r clean d ry air. 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 PFO 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 concentration 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 stock solution o f PFO 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 the beginning and end o f each run, and one standard w as in jected , a t a m inim um , a fte r every five sam ples. L inear reg ressio n equations w ere g en erated using peak area response ratio s (P F O S : internal standard) versus the 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. The concentration o f PFO S in th e freshw ater sam ples w as determ ined by su b stitu tin g th e p eak area response ra tio s into th e applicable lin ear regression equation. R epresentative io n chrom atogram s o f low and high calibration stan d ard s u sed 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 water by ap p ro p riate dilutions o f either a 1.00 m g a.i./L o r a 10.0 m g a.i./L sto ck solution o f PFO 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, PFOS calibration standards ranging in con cen tratio n from 0.500 to 5.00 pg 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 W ildlife International, Ltd. -45- co n cen tratio n s) w ere analyzed. T h e ap p ro p riate calibration standard series w as injected a t th e b eginning an d en d o f each ru n , and o n e stan d ard w as in jected , a t a m inim um , a fte r every fiv e sam ples. L inear regression equations w ere generated using th e peak area responses versus th e respective concentrations o f th e calibration standards. A typical calibration curve from tissu e analyses is presen ted in F ig u re 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 regression equation. R epresentative ion chrom atogram s o f low and high calibration standards used fo r tissu e analyses are p resented in F igures 8 and 9, respectively. F or b o th w ater and tissue analyses fo r P FO S, th e sam e and m ost prom inent p eak resp o n se fo r P FO S w as utilized to m onitor PFO S in all calibration, quality control, and study 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 tissue 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 on a sto ck p rep aratio n lo g sheet. Limits of Quantitation T he m ethod lim it o f q u an titatio n (L O Q ) for PFOS in freshwater sam ples w as 0.0500 m g a.i./L , calcu lated as th e p ro d u ct o f th e lo w est PFOS calib ratio n stan d ard (0.0005 m g a.i./L ) and th e d ilu tio n fa c to r o f th e m atrix blank sam ple (100) analyzed concurrently w ith th e te st sam ples. The L O Q w as calculated o n an individual basis fo r each tissu e sam ple since each entire sub m itted sam ple ( o f differing w eig h t) w as ex tra cted w ith o u t an adjustm ent to a co n stan t w eight. T he L O Q (m g a.i./K g ) fo r a given tissu e analysis w as calculated as th e p roduct o f the 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 tissue 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 ilution (overall d ilu tio n fa c to r = 82.85 L /K g), gives an LO Q = 0.0414 m g a.i./K g. W ildlife International, Ltd. -46- Freshw ater M atrix B lank and Fortification 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 th e 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 q uantitation (0.0500 m g a.i./L ). A rep resen tativ e 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 ) at each sam pling interval using appropriate stock solutions o fPFO S p rep ared in m ethanol. F reshw ater m atrix fortifications w ere analyzed concurrently w ith each sam ple set and th e analytical results w ere n o t corrected 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 fo rtificatio n reco v eries 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% 5.12% (n = 20), 98.7% 6.60% (n = 20), and 96.9% 2.95% (n = 12), respectively. A representative ion chrom atogram o f a freshw ater m atrix fortification is presented in F igure 11. T issue M atrix B lank and Fortification 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 possible 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 tissue hom ogenates w ere fortified w ith the appropriate stock so lu tio n s) 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 results w ere not corrected fo r m ean procedural recovery. T he 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 the low -level fortifications, and *W ildlife International, Ltd. -47- fro ra 92.0 to 107% o f nom inal concentration fo r th e high-level fortifications (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 fo rtifications (n - 16) fo r th e edible fish tissu e w o e 102% 4 .6 1 % and 100% 4 .4 0 % , respectively. N onedible fish tissue reco v eries ran g ed 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 - and high-level fortifications, respectively (T able S). T he m ean and standard d eviation o f fortification recoveries a t 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 epresentative ion chrom atogram s o f edible and nonedible fish tissu e m atrix fortification sam ples are presented in F igures 14 and 15, respectively. E xam ple C alculations T he analytical resu lt and p ercen t recovery for freshw ater sam ple 454A -134-68, from the 0 .1 0 m g a.i./L nom inal PFO S treatm en t group, w as calculated using th e follow ing equations: Peak A rea R atio = A nalyte Peak A rea/Intem al Standard Peak A rea PFOS A rea = 593240 Internal Standard A rea = 1295042 Peak area ra tio = 0.4581 Internal Standard C oncentration: 10.0 fig a.i./L Y -intercept = -0.0187 Slope = 5.2182 Initial volum e (V i) = 0.100 m L Final volum e (V f) = 10.0 mL D ilution facto r (VfTVi) = 100 - PFO S (p g a.i./L ) a t Instrum ent = ^>ea^ . 8163 m^ ceP$ x Internal S tandard C oncentration S lo p e _ 0 ,4 5 8 1 -(-0 .0 187) x j Q q 5 .2 1 8 2 PFO S (m g a.i./L ) in sam ple = PFO S (m g a.i./L ) at Instrum ent x D ilution Factor = 0 .0 0 0 9 1 3 7 x 1 0 0 = 0.09137 PFO S (m g a .i./L )in sam ple P ercent o f N om inal C oncentration = xlOO PFO S (m g a.i./L ) nom inal 0.09137 m g a.i./L -------------------------- x 100 0 .1 0 m g a.i./L = 91.4% .W ildlife International, Ltd -4 8 - T h e analytical re su lt fo r edible fish tissu e sam ple 4S 4A -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: P eak A rea = 1 1 1 9 2 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 O verall D ilu tio n F a c to r (V e/W i x VfTVi) = 2 2 4 0 m L /g = 2 2 4 0 L /K g PFOSai^arum. slope (1 1 1 9 2 -7 1 3 ,8 1 ) 6 2 8 7 .3 3 = 1667 p g a.i./L P FO S in sam ple (m g a.i./K g) = PFO S at instrum ent (p g a.i./L ) x overall dilution facto r x unit conversion facto r x 1 .6 6 7 / a.i. 2440L PF O S m sam ple (m g a .i./K g );---------- - ------- lm 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 P FO S concentrations o n D ays -4 and -1 (tw ice) during the 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 , 49, 56, and 62 during the uptake phase o f the test. U ptake w as suspended on D ay 62 o f th e te st 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 on D ays 1 4 ,2 8 ,4 2 , and 56. M easured concentrations o f PFO S in th e p re-test diluter verification (p re-u p tak e phase) sam ples w ere <LO Q in the negative controls. M easu red co n cen tratio n s in p re -te st sam ples ranged from 6 4.6 to 95.4% o f th e nom inal co ncentration Wildlife InternationalLtd. -4 9 in th e 0.10 m g a.i./L treatm ent group and from 71.8 to 111% o f th e nom inal concentration in th e 1.0 m g a.i./L treatm en t g roup (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 O .lO a n d 1.0 m g a.i./L treatm en t g ro u p s ranged from 6 8 .0 to 113% an d from 7 3 .4 to 1 0 1 % o fth e nom inal concentration, respectively (T able 7). D uring th e depuration phase o f th e te st, m easured concentrations 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 igure 16. T issu e S am ple A nalysis T issue sam ples w ere collected on uptake D ays 0 (4 hours), 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 P FO 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 0.100 and 1.00 m g a.i./L treatm ent g ro u p s 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 th e 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 p le A nalysis S tability sam ples w ere prepared a t te st initiation (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 ata indicate th a t th e te st substance w as stab le during relativ ely long term (119 days) fro zen sto rag e a t th e 0.1 0 0 and 10.0 m g a.i./K g co n cen tratio n levels. D efinitive tissu e sam ple sto rag e did n o t exceed 7 days during th e conduct o f th e s tu d y . 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 on u p tak e D ays 0 (0 h o u r) and 62 and depuration D ay 56 to determ ine lipid co n ten t in fish tissues o n a w et-w eig h t basis. T he resu lts o f lipid analyses in edible and n onedible fish tissu e s are p resen ted in T ables 11 an d 12, respectively. 'Wildlife International, Ltd. -50Table 1 Typical HPLC/MS Operational Parameters for Analysis o f Aqueous Samples INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000 M ass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion m onitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C u column (SO mm x 2 mm I.D ., 2-p3m particle size) GUARD COLUMN: Keystone Javelin C column (20 mm x 2 mm I.D.) OVEN TEMPERATURE: 40 C STOP TIME: S.00 minutes FLOWRATE. 2S0 pU m inute MOBILE PHASE: INJECTION VOLUME: 70.0% Methanol: 30.0% NANOpure W ater containing 0.1% Formic Acid 10 pL PFOS RETENTION TIM E: A pproxim ately 3.7 m inutes ' INTERNAL STANDARD RETENTION TME: Approximately 2.5 minutes PFOS MONITORED MASS: 499 amux INTERNAL STANDARD MONITORED MASS: 427 amu ,W ildlife International Ltd. -51- Table 2 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 Performance Liquid Chrom ato-graph (HPLC) w ith a Perkin-Elm er A PI 3000 M ass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in m ultiple ion reaction monitoring (M RM ) mode. ANALYTICAL COLUMN: Keystone Betasil C column (50 mm x 2 mm I.D ., 2-p3m particle size) GUARD COLUMN: Keystone Javelin C column (20 mm x 2 mm I.D.) OVEN TEMPERATURE: 40 C STOP TIME: 5.00 minutes FLOW RATE: 250 pL/minute MOBILE PHASE: INJECTION VOLUM E: 70.0% Methanol: 30.0% NANOpure W ater containing 0.1% Form ic Acid 10.0 pL PFOS RETENTION TIME: Approxim ately 4.1 minutes PFOS M ONITORED MASS: 499.0 AMU PFOS MONITORED MASS: 499.0 amu --*99.1 amu W ildlife International, Ltd. -52- Table 3 M atrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples Samplc Numbcr (454A-134-) PT-MAB-1 PT-MAS-1 PT-MAS-2 PT-MAS-3 -4 -4 4 4 Sampling Interval (Day) Pretest Concentration ofPerfluorooctanesulfonatc, Potassium Salt (PFGS)(mg a.iA ) Fortified Measured1 0.00 0.0750 0.500 ' 2.00 <LOQ 0.0707 ' 0.493 1.98 Percent Recovery* -- 94.3 98.6 99.0 PT-MAB-2 PT-MAS-4 PT-MAS-5 PT-MAS-6 -1 -1 -1 -1 Pretest 0.00 0.750 0.500 2.00 <LOQ 0.0779 0.464 1.91 104 92.9 95.3 PT-MAB-2 PT-MAS-7 PT-MAS-8 PT-MAS-9 -1 -1 -1 -1 Pretest 0.00 0.0750 0.500 2.00 <LOQ 0.0724 0.483 1.86 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 MAS-4 MAS-5 MAS-6 0 Hour4 -Uptake 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 1 1 1 1 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0745 0.498 1.95 -- 99.3 99.7 97.6 MAB-4 MAS-10 MAS-11 MAS-12 3 3 3 3 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0666 0.509 1.86 _ 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 w 89.5 90.0 92.7 *The limit o f quantitatian(LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor ofthe matrix blank sample (100). ^Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly W ildlife International, L td -53- Table 3 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently w ith Freshwater Samples Sample Number (454A-134-) MAB-7 MAS-19 MAS-20 MAS-21 21 21 21 21 Sampling Interval (Day) Uptake Concentration ofPeriluorooctanesulfonate, _____ Potassium Salt (PFOS)(mg aJTL) Fortified Measured' 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 <LOQ 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 <LOQ 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 MAB-12 MAS-32 MAS-33 56 56 56 Uptake 0.00 0.0750 . 0.500 <LOQ 0.0701 0.477 --" 93.4 953 MAB-13 MAS-34 MAS-35 62 62 62 Uptake 0.00 0.0750 0.500 <LOQ 0.0745 0.481 99.3 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 *The limit of quantitation(LOQ) was 0.0500 mg a.i /L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and die dilution factor o f the matrix blank sample (100). R esults were generated using MacQuan. version 1.6 software. Manual calculations may differ slightly. W ildlife International, Ltd. -54- Table 3 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently w ith Freshw ater Samples Sample Number (454A-134-) MAB-16 MAS-40 MAS-41 42 42 42 Sampling Interval (Day) Depuration Concentration ofPerfluorooctaneeulfonate, Potassium Salt(PFOS) (mg a.L/L) Fortified Measured' 0.00 0.0750 0.500 <LOQ 0.0637 0.452 Percent Recovery2 84.9 90.4 MAB-17 MAS-42 MAS-43 56 56 56 Depuration 0.00 0.750 0.500 <LOQ 0.0712 0.484 -- 94.9 96.9 lThe limit o f quantitationCLOQ) was 0.0500 mg a.iiL , 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. 1Wildlife International, Ltd. -55- T ablc4 M atrix Blanks and Fortifications Analyzed Concurrently with Edible Fish Tissue Samples Sample Number (454A -134-) E-MAB-1 E-MAS-1 E-MAS-2 0 0 0 Sampling Interval (Day) Hour 4 -U ptake Concentration o f PerfluorooctanesuHbnate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified M easured1 0.00 0.100 10.0 0 .0 5 0 0 0.0970 9.65 Percent Recovery2 -- 97.0 96.5 E-MAB-2 E-MAS-3 E-MAS-4 1 1 1 Uptake 0.00 0.100 10.0 0 .0 5 0 0 0.0993 9.77 99.3 97.7 E-MAB-3 E-MAS-5 E-MAS-6 3 3 3 Uptake 0.00 0.500 50.0 0 .1 0 0 0.494 48.6 -- 98.8 97.2 E-MAB-4 E-MAS-7 E-MAS-8 7 7 7 Uptake 0.00 0 .2 0 0 1.00 0.948 94.8 100 95.0 95.0 E-MAB-5 E-MAS-9 E-MAS-10 14 14 14 Uptake 0.00 <1.00 5.00 5.31 106 250 268 107 E-MAB-6 E-MAS-11 E-MAS-12 21 21 21 Uptake ' 0.00 5.00 250 <1.00 5.21 251 -- 104 101 E-MAB-7 E-MAS-13 E -M A S-14 E-MAB-8 E-MAS-15 E-MAS-16 28 28 28 35 35 35 Uptake Uptake 0.00 <1.00 -- 5.00 5.27 105 250 249 99.5 0.00 <1.00 -- 5.00 4.92 98.3 100 104 104 E-MAB-10 E-MAS-19 E-M A S-20 42 42 42 Uptake 0.00 <1.00 -- 5.00 4.97 99.4 100 105 105 E -M A B -11 E-MAS-21 E-MAS-22 49 49 49 Uptake 0.00 <1.00 -- 5.00 5.27 106 100 106 106 E-M AB-I2 56 Uptake 0.00 <1.00 -- E-MAS-23 56 5.00 5.31 106 E-MAS-24 56 200 208 104 'The limit o f quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product o fthe 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. W ildlife International, Ltd. -56- Table 4 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently w ith Edible Fish Tissue Samples Sample Number (454A-134-) E-MAB-13 E-MAS-25 E-MAS-26 62 62 62 Sampling Interval (Day) Uptake Concentration o fPcrfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.LKg) Fortified M easured1 0.00 <1.00 5.00 5.54 200 192 Percent Recovery2 -- Ill 96.0 E-MAB-14 E-MAS-27 E-MAS-28 14 14 14 Depuration 0.00 5.00 200 <1.00 5.37 205 107 102 E-MAB-15 E-MAS-29 E-MAS-30 28 28 28 Depuration 0.00 5.00 100 <1.00 5.15 104 103 104 E-MAB-16 E-MAS-31 E-MAS-32 42 42 42 Depuration 0.00 5.00 100 <1.00 4.82 92.0 96.5 92.0 E-MAB-17 56 Depuration 0.00 <1.00 E-MAS-33 56 5.00 4.99 99.7 E-MAS-34 56 100 101 101 Less than values correspond to limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor o f the matrix blank sample. All sample weights * 1.00 gram. R esults were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. Wildlife InternationalLtd. -57- T ab lc 5 M atrix Blanks and Fortifications Analyzed Concurrently w ith Nonedible Fish Tissue Samples Sample Number (454A -134-) N-MAB-1 N-MAS-1 N -M A S-2 0 0 0 Sampling Interval (Day) Hour 4 - Uptake Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified M easured1 0.00 0.100 10.0 <0.0500 0.109 9.26 Percent Recovery2 -- 109 92.6 N-MAB-2 N-MAS-3 N -M A S-4 1 1 1 Uptake 0.00 0.100 10.0 <0.0500 0.106 9.36 106 93.6 N-MAB-3 N -M A S-5 N -M A S-6 3 3 3 Uptake 0.00 0.500 50.0 0 .1 0 0 0.470 45.8 __ 93.9 91.6 N -M A B -4 N -M A S-7 N -M A S-8 7 7 7 Uptake 0.00 0 .2 0 0 1.00 0.975 97.5 100 90.1 90.1 N-MAB-5 N -M A S-9 N-MAS-10 14 14 14 Uptake 0.00 <1.00 -- 5.00 5.25 105 250 255 102 N -M A B -6 N -M A S-11 N-MAS-12 21 21 21 Uptake 0.00 <1.00 .. 5.00 4.98 99.7 500 505 101 N-MAB-7 N-MAS-13 N-MAS-14 28 28 28 Uptake 0.00 <1.00 -- 5.00 5.25 105 500 5.16 103 N -M A B -8 N-MAS-15 N -M A S-16 35 35 35 Uptake 0.00 <1.00 5.00 5.15 103 100 108 108 N -M A B -10 N -M A S-19 N -M A S-20 42 42 42 Uptake 0.00 <1.00 __ 5.00 5.26 105 100 104 104 N -M A B -11 N -M A S-21 N-M AS-21 49 49 49 Uptake 0.00 <1.00 .. 5.00 5.40 108 100 108 108 N-MAB-12 56 Uptake 0.00 <1.00 ,,,, N-MAS-23 56 5.00 5.41 108 N-MAS-24 56 200 245 122 Less than values correspond to limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor o f the matrix blank sample. All sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. W ildlife International, Ltd. -58- Table 5 (Continued) M atrix Blanks and Fortifications Analyzed Concurrently with Nonedible Fish Tissue Samples Sample Number (4S4A-134-) N-MAB-13 N-MAS-25 N -M A S-26 62 62 62 Sampling Interval (Day) Uptake Concentration o fPerQuorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified M easured1 0.00 <1.00 5.00 5.68 200 212 Percent Recovery2 -- 114 106 N-MAB-14 N-MAS-27 N -M A S-28 14 14 14 Depuration 0.00 5.00 200 <1.00 5.29 207 106 .. 104 N-MAB-15 N -M A S-29 N -M A S-30 28 28 28 Depuration 0.00 5.00 100 <1.00 5.45 111 .. 109 111 N -M A B -16 N -M A S-31 N-MAS-32 42 42 42 Depuration 0.00 5.00 100 <1.00 5.19 98.9 104 98.9 N-MAB-17 N-MAS-33 56 56 Depuration 0.00 5.00 <1.00 5.80 -- 116 N-MAS-34 56 100 112 112 `Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the matrix blank sample. All sample weights = 1.00 gram. R esults were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 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 (mga-iVL) 0.00 (Negative Control) Sample Number (454A-134-) PT-1 PT-9 Phase Pre-U ptake Sampling Time (Day) -4 -1 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.L/L) <L0Q <LOQ PT-17 -1 <LOQ Percent of Nominal' -- - -- 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 85.0 84.4 69.5 64.6 95.4 93.7 1.0 PT-6 -4 0.917 91.7 PT-7 -4 0.949 94.9 PT-14 -1 0.718 71.8 PT-15 PT-22 PT-23 -1 0.738 -1 1.11 -1 1.01 73.8 111 101 'The limit o f quantitation (LOQ) was 00500 mg a.i./L, calculated as the product o fthe lowest calibration standard (0.0005 mg a.i7L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacOuan, version 1,6 software. Manual calculations may differ slightly. W ildlife International, Ltd. - 60- T ablc7 M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Freshwater ____________ Samples from a Bluegill Snnfish Bioconcentration Test___________________ Measured Concentration of Nominal Test Concentration (mg a.i./L) Sample Number (454A-134-) Phase Sampling Time (Day) Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i7L) Percent of Nominal' 0.0 (Negative 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 <iO Q <LOQ <LOQ <LOQ <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 0.0717 4 0 ,0 hours 0.0692 11 0 ,4 hours 0.0791 12 0 ,4 hours 0.0741 19 1 0.0702 20 1 0.0734 27 3 0.0751 28 3 0.0717 35 7 0.0826 36 7 0.0781 43 14 0.0680 44 14 0.0709 51 21 0.110 52 21 0.113 59 28 0.0822 60 28 0.0843 67 35 0.0915 68 35 0.0914 75 42 0.0983 76 42 0.110 80 49 0.103 81 49 0.103 85 56 0.0853 86 56 0.0948 90 62 0.0887 _____ 91 62 0.0914 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). ^Results were gengated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 71.7 69.2 79.1 74.1 70.2 73.4 75.1 71.7 82.6 78.1 68.0 70.9 110 113 82.2 84.3 91.5 91.4 98.3 110 103 103 85.3 94.8 88.7 91.4 .W ildlife International, Ltd -61- Table 7 (Continued) M easured Concentrations o f Perfluorcxxtanesulfbnate, Potassium Salt (PFOS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Measured Concentration of Nominal Test Sample Perfluorooctanesulfonate, Percent Concentration Number Sampling Potassium Salt (PFOS)1 of (m gal/L ) (454A -134-) Phase Time (Day) (mg ai./L ) Nommai2 0.10 95 Depuration 14 <LOQ -- 96 14 <LOQ -- 100 28 <LOQ -- 101 28 <LOQ -- 105 42 <LOQ -- 106 42 <LOQ -- 110 56 <LOQ -- 111 56 <LOQ -- 1 .0 6 Uptake O.Ohours 0.797 7 0 ,0 hours 0.802 14 0 ,4 hours 0.891 15 0 ,4 hours 0.930 22 1 0.867 79.7 80.2 89.1 93.0 86.7 23 1 0.820 82.0 30 3 0.813 81.3 31 3 0.734 73.4 38 7 0.845 84.5 39 7 0.818 81.8 46 14 . 0.900 90.0 47 14 0.875 87.5 54 21 0.988 98.8 55 21 1.01 101 62 28 0.913 91.3 63 28 0.925 92.5 70 353 0.838 __________ Z 1 ___________ 353 0871 1The limit o f quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration 83.8 87-1 standard (0.0005 mg a.i./L) and die dilation factor o f the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. Sam pling was suspended after Day 35 Uptake due to 100% mortality at the 1.0 mg ai./L nominal test level. W ildlife International, Ltd. -62- T a b le 8 M easured Concentrations o f Perfluorooctancsulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegdl Sunfish Bioconcentration Test Measured Concentration of Nominal Test Sampling Perfluorooctancsulfonate, Concentration Sample Number Time Potassium Salt (PFOS)1 . (mg a.i./L) (454A-134-) Phase (Day) (mg a.i./L) 0.0 E -l Uptake 0 ,4 hours <0.0510 (Negative E-2 E-14 E-15 E-27 E-28 E-40 E-41 E-53 E-54 E-66 E-67 E-79 E-80 E-92 E-93 E-100 E-101 E-108 E-109 E -l 16 E -l 17 E-124 E-125 E-132 E-133 E-140 E-141 E-147 E-148 E-154 E-155 Depuration 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 14 14 28 28 42 42 56 56 0 .0 6 6 5 0.0 9 6 0 0 .0835 0 .1 1 4 0.0 9 1 0 0 .0965 0 .1 6 2 0 .4 6 8 0 .5 6 0 0 .7 3 0 0 .7 1 0 0 .7 4 0 0 .8 0 0 0 .9 7 0 0 .5 7 5 0 .3 9 9 0 .5 9 0 0 .5 7 5 0 .5 2 0 0 .7 7 5 0 .8 2 0 0 .6 6 0 0 .6 1 5 0 .6 8 5 0 .7 1 0 0 .5 8 5 <0.590 0 .8 3 0 0 .8 2 5 0 .7 5 5 <1.07 . 0.10 E-4 Uptake 0,4 hours 0.167 E-5 0 ,4 hours 0.155 E-6 0 ,4 hours 0.144 E-7 0 ,4 hours 0.182 E-17 1 0.734 E-18 1 0.726 E-19 1 0.631 E-20 1 0.806 E-30 3 1.73 E-31 3 2.07 E-32 3 2.03 E-33 3 ____________ 111___________ Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). R esults were generated using MacQuan, version 1,6 software. Manual calculations may differ slightly. ,W ildlife International Ltd. -63- 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./L) Sample Number (454A -134-) Phase S a n lin g Time (Day) Measured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.iVL) 0 E-43 Uptake 7 E-44 7 E-45 7 E-46 7 E-56 14 E-57 14 E-58 14 E-59 14 E-69 21 E-70 21 E-71 21 E-72 21 E-82 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 E-120 56 56 E-121 56 E-122 56 E-127 62 E-128 62 E-129 62 E-130 62 3.73 4.25 4.73 6.25 11.4 9.07 13.7 12.6 11.7 12.0 12.9 10.6 18.3 13.7 23.9 23.1 22.6 27.7 23.8 20.6 27.6 25.3 21.2 27.6 33.3 36.2 39.0 30.6 48.3 38.9 44.1 38.3 42.4 66.2 42.2 39.2 E-135 Depuration 14 48.5 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-145 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 limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product of 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. W ildlife International, Ltd. -64- Table 8 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a BlucgiU Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.10 Sample Number (454A-134-) E-157 E-158 E-159 E-160 Phase Depuration Sampling Time (Day) 56 56 56 56 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.iVL) 21.1 37.6 32.9 31.2 . 1.0 E-9 Uptake 0 ,4 hours E-10 0 ,4 hours E - ll 0 , 4 hours E-12 0 ,4 hours E-22 1 E-23 1 E-24 1 E-25 1 1.46 1.48 1.19 1.39 4.68 6.59 5.56 5.64 E-35 E-36 E-37 E-38 E-48 3 17.3 3 15.8 3 19.0 3 20.8 7 42.0 E-49 7 44.0 E-50 7 57.7 E-Sl 7 46.8 E-61 14 87.1 E-62 14 81.6 E-63 14 90.7 E-64 14 73.3 E-74 21 79.4 E-75 21 117 E-76 21 104 E-77 21 102 E-87 28 102 E-88 28 131 E-89 28 107 E-90 _____2? - 133 Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as die sroduct 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. W ildlife International, Ltd. -65- Table 9 M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.iVL) 0.0 (Negative Sample Number (454A -134-) N -l N-2 N-14 N-15 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 -l 25 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 of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (m ga.L /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 .4 5 7 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 .3 6 3 0 .4 8 0 0 .5 5 5 0 .3 7 5 0 .5 4 0 0 .5 2 0 0 .5 0 5 0 .5 0 5 N -l 32 N -l 33 N-140 N-141 N-147 N -l 48 N-154 N -l 55 Depuration 14 14 28 28 42 42 56 56 0 .4 7 0 <.0472 0 .4 4 9 0 .5 0 5 0 .5 9 0 0 .5 7 0 0 .6 3 0 0 .7 8 0 0.10 N -4 Uptake 0 ,4 hours 0.415 N-5 0 ,4 hours 0.519 N -6 0 ,4 hours 0.417 N-7 0 ,4 hours 0.497 N-17 1 1.68 N-18 1 1.85 N-19 1 1.72 N-20 1 2.07 N-30 3 4.59 N-31 3 5.50 N-32 3 5.47 ____ N-33_______ _____ 3________ ____________ 197_______________ 'Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor o f the sample (L/Kg). ^Results were generated using MacQuan, version l .6 software. Manual calculations may differ slightly. W ildlife International, Ltd. - 66- Table 9 (Continued) M easured Concentrations o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bhiegill Sunfish Bioconcentration Test Measured Concentration of Nominal Test Sampling Perfluorooctanesulfonate, Concentration Sample Number Time Potassium Salt (PFOS)1 (mfi aiVL) (454A -134-) Phase (Day) (mg a.i7L) 0.10 N-43 Uptake 7 N-44 7 N-45 7 N -46 7 N-56 N-57 N -58 14 14 14 N -59 N -69 14 21 N -70 21 N-71 21 N-72 21 N-82 N-83 N-84 N-85 28 28 28 28 N-95 35 N-96 35 N-97 N-98 35 35 N-103 N-104 N-105 N-106 N -lll 42 42 42 42 49 N-112 N-113 N -114 N-119 N-120 N-121 N-122 49 49 49 56 56 56 56 N-127 N-128 N-129 62 62 62 N-130 62 10.2 10.6 11.9 15.2 27.3 23.2 35.3 32.6 33.3 22.7 24.6 24.4 49.4 40.7 65.3 57.9 67.1 73.3 62.0 59.1 64.0 68.1 54.4 79.6 85.0 95.1 93.1 77.7 122 94.2 73.2 106 101 112 105 96.4 N-135 Depuration 14 124 N-136 14 79.4 N-137 14 81.8 N-138 14 113 N-143 28 85.7 N-144 28 95.1 N-145 28 85.7 N-146 28 94.8 N-150 42 71.7 N-151 42 80.6 N-152 42 78.3 _____ __________ 42 __________ 32J_____________ lLcss than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor ofthe sample (L/Kg). Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. W ildlife International, Ltd. -67- Table 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 gai./L ) 0.10 Sample Number (454A -134-) N-157 N-158 N-159 N-160 Phase Depuration Sampling Time (Day) 56 56 56 56 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./L) 57.7 80.3 85.4 84.4 1.0 N -9 Uptake 0,4 hours N-10 0,4 hours 3.52 4.37 N -ll N-12 N -22 0,4 hours 0,4 hours 1 4.22 4.06 11.1 N-23 N-24 N-25 1 14.2 1 13.3 1 12.1 N-35 N-36 N-37 N-38 N-48 N-49 N-50 N-51 N-61 N-62 N-63 3 39.3 3 42.0 3 43.8 3 51.8 7 100 7 102 7 102 7 120 14 177 14 207 14 245 N-64 N-74 14 214 21 201 N-75 21 278 N-76 21 246 N-77 21 229 N-87 28 289 N-88 28 372 N-89 28 320 ______ 2 2 _______ _____22_______ 361 'Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of 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 maty cliff slightly. W ildlife International, Ltd. 68- Table 10 M easured Concentrations o f Perfluorooctanesnlfonatc, Potassium Salt (PFOS) in Tissue Storage Stability Samples from a Bluegill Sunfish Bioconcentration Test N om inal Concentration (mg a.L/Kg) N eg ativ e C ontrol Sample Number (454A -134-) E-M A B -184 N -M A B -184 Tissue Type Edible N anedibie M easured Concentration o f Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./K g) <LOQ <LOQl P e r o it of N om inal2,5 -- -- 0.100 10.0 E-STM A S-13 E-M A S-354 N -STM A S-13 N -M A S-354 E -STM A S-23 E -M A S-364 Edible Edible Nonedible N anedibie Edible Edible 0.109 0.111 0.114 0.112 8.43 11.3 109 111 114 112 84.3 113 N -STM A S-23 N onedible 9.30 93.0 N -M A S-364 N an ed ib ie 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 fthe lowest calibration standard (0.0005 mg a.i./:)) and the overall dilution factor of the matrix blank sample (100 L/Kg). All sample weights - 1.00 gram. 2 Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 3 The stability samples were fortified on December 5,2000 and stored in the freezer. The samples were removed from the freezer on April 3,2001 (after 119 days o ffrozen storage) and analyzed 4 These samples were prepared on April 3,2001 and tbc results were compared to the stability sample results. 5The criterion for storage was Percent of Nominal between 80 and 120%. ,W ildlife International Ltd. -69- Tablell Lipid Content in Edible Fish Tissue Nom inal Test C o n cen tratio n (mg a.iiL ) 0.0 (Negative Control) S am ple Number (454A -134-) ELI EL2 EL3 EL4 Phase U p tak e S am pling T im e (D ay) 0 ,0 hour 0 ,0 hour 0, 0 hour 0 ,0 hour Lipid W eight <g) 0.0314 0.0453 0.0453 0.0463 Fish Tissue W eight (g) 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 EL16 EL17 EL18 EL19 D ep u ratio n 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 ep u ratio n 56 56 0.0172 0 .0 1 9 0 1.2362 1.3483 EL23 EL24 56 0.0250 1.3303 56 0.0252 1.1040 'R atio calculated as flipid weight (g) -r fish tissue weight (g)1 x 1000 m g/g x 1000 gflCg. 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 W ildlife International, Ltd. -70- Table 12 Lipid Content in Nonedible Fish Tissue Nominal Test Concentration (mga-iTL) 0.0 (Negative Control) Sample Number (454A -134-) NL1 NL2 NU NL4 Phase Uptake Sampling Time P a y ) 0 ,0 hour O.Ohour 0 ,0 hour O .O hour Lipid Weight (g) 0.1363 0.2383 0.2258 0.1992 Fish Tissue Weight (8) 2.0649 2.6355 1.9568 2.0812 NL6 Uptake 62 0.0506 1.8821 NL7 62 0.0274 1.5901 NL8 62 0.0298 1.7596 NL9 62 0.0518 1.7302 NL16 NL17 NL18 NL19 Depuration 56 56 56 56 0.0616 0.0318 0.0360 0.0966 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 0.1276 2.0113 NL14 62 0.1221 1.2898 NL21 Depuration 56 . 0.0343 NL22 56 0.0404 NL23 NL24 56 0.0710 56 0.0714 'R atio calculated as flipid weight Oft + fish tissue weight (g)] x 1000 mg/g x 1000 g/Kg. 1.7085 1.8394 1.7652 1.5682 Lipid/Fish Tissue W eight1 (mgflCg) 66,000 90,400 115.400 95,700 26,900 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 45,500 W ildlife International, Ltd. -71- METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN FRESHWATER Prepare matrix fortification samples in freshwater matrix by spiking the requisite volume o f PFOS stock solutions directly into freshwater. Perform fortifications with gas-tight syringes and Class A volumetric flasks. i Prepare appropriate dilutions o fstudy and QC samples to within the calibration range of the PFOS LCMS methodology; Partially fiU Class A volumetric flasks with 50% methanol/50% water dilution solvent containing 0.0100 mg(L 4H PFOS Internal standard and 0.05% v/v formic add. Add appropriate volume ofsample and bripg to volume with dilution solvent Process matrix blank samples for a given matrix using die same dilution and aliquot volumes as for the lowest fortification level in that matrix. Mix well by several repeat inversions. .i Ampulate samples and submit for LCMS analysis. F igure 1. M ethod flow chart for the analysis o f Perfluorooctancsulfonate, Potassium Salt (PFOS) in freshw ater. W ildlife International, Ltd. -72- METHOD OUTLINE FO R THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN FISH TISSUES Quality control samples are prepared to n aliquots (approximately 1 g) of 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 syringefs). The matrix blank sample will be unfortified fish tissue homogenate. I Add 10.0 mL of methanol to each sample with a glass ClassA volumetric pipette. Homogenize each test sample for approximately 1 minute using a tissue shredder. Rinse the homogenieer with the appropriate solvent(s) in between samples. i Sonicate each sample far approximately 5 minutes with a sonic dismembrator i Cap the vials and shake w ell Centrifuge dm vials at approximately 2000 rpm for approximately 5 minutes. i Prepare appropriate dilutions ofstudy and QC samples to within the calibration range of the PFOS LCMS methodology; Partially fill ClassA volumetric flasks with 50%: 50% methanoI/NANOpure* 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 well by several repeat inversions. I T r a n s f e r a n a liq u o t o f e a c h s a m p le to a n a u to s a m p lc r v ia l a n d s u b m it f o r L C /M S /M S a n a ly s is . F igure 2. M ethod flow chart for the analysis o f Perfluorooctanesulfonate, Potassium Salt (PFOS) in fish tissues. Wildlife InternationalLtd. -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 of NANOpnre* water to fish tissue in vial and homogenize for approximately 1 minute using a hand-held (Issue shredder. Rinse the homogenlzer with the appropriate solvent^ in between samples. Transfer each homogenate to a 250-mL separatory funnel that contains 25 mL o f chloroform and 50 mL of methanol. X Rinse each vial with an additional 10 mL o f NANOpore water and pourrinse into respective separatory funnel. Shake each separatory funnel for aj Briefly swirl each separatory funnel with venting. X Allow the phases to separate. For each sample, drain the chloroform layer through a powder funnel packed with glass wool and anhydrous sodium sulfate into a 250-mL round-bottom flask. Add an additional 50-mL aliquot of chloroform to each separatory funnel and repeat the extraction and drainingprocedures. Rotary evaporate the extracts in a water bath maintained at approximately 40"C to near dryness. X Transfer each extract to a prc-weighed, labeled srintiilaUoo vial. Rinse each 250-mL round-bottom flask with a small volume o f chloroform and transfer rinse to respective scintillation vial. Evaporate the remaining solvent in each vial under a gentle stream o fnitrogen or dean dry air. X _________________ Reweigh each vial and record weight______________________ F igure 3. M ethod flow chart for the analysis o f lipids in fish tissues. W ildlife International, Ltd. -74- AreafRatfo) F igure 4. A typical calibration curve for Pcxfluoxooctanesulfonate, Potassium Salt (PFO S) in freshwater. Slope = 4.64; Intercept = -0.01; r = 0.99932. Curve is weighted (1/x). (monitored m asses = 499 am u (PFOS) and 427 amu (4HPFOS internal standard)). ,'Wildlife International Ltd. -75- PT03L1 STD 0.5QQ ug (.LA. 4875A011D -11 ppjg4 .0 8 in 1 p e r io d to te m * S ta n d a rd : 4 H P F 0 8 Um Am Absolute Retention Time t : 4.07 Q1 Ml, 208 cans 4 9 9 .0 Noise Uvea. Quant Three Min. Wlddi M l*. WKMl Base. WidUl RTWkv (sac*) 2 0 .0 IS.O 3 6 40 30 S m o o th Expected n r i 3.60 Area 251666 Haight 23873 suit nm 3.61 E ndltna Intagration Width 4 J8 OS7 Ratantion Tkna Iraagration Typa 3.83 A BB Tua, Dec 5. 2000 10:48 P FO S.1 STD a.500 ug bJVL 4S75A-011D -11 4.98 in 1 period pros u s e * In te rn a l S ta n d a rd 1: 4.97 Q1 Ml, 298 scan 4 2 7 .0 Noise Dues. 2 .0 Quant Throe 1.0 MM. Width 3 Mutt Width 6 B ase Width 30 RT Win. (sacs) 20 Smootta 1 ExpadadRT 2.53 Area 1134885 Haight 141549 Start Tima 44 EndTima 98 Intagradon Width 050 Ratantion Time 54 lntsgraiien Type A- 88 Tim, Dec 8, 2000 10:48 intensity; 250000 cpa intensity: 250000 cpa 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, (m onitored m asses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). W ildlife International, Ltd. -76- PF0SL9 STD M O g g iJ J l 4S75A-011D -15 4 .9 6 in 1 p e r io d PFO S M a n a t S ta n d a rd 4 H P F O S U s a A ia a A b to iu t* R e te n tio n T im a 1 : 4 .9 7 Q 1 M l, 3 9 0 s c a n * 4 9 9 .0 N o fcaT h ras. Q u a n t T h ie a . M in . W id th UM w a n 3 0 .0 1 5 .0 3 a B a s e . W id th R T W in . ( s e c a ) 40 30 S m o o th B ead e d RT 1 8 .8 0 A IM 3505788 H a ig h t 337364 S ta rt T im a E n d T im a i n la g r a tio n width R a la n tlo n T im a In te g ra tio n T y p e 3JS 1 4 .2 8 057 353 A -BB Tue, Dec 5. 2000 11:10 PFO S.6 STO 5.00 g a JJL 467SA -011D -15 4 .9 8 in 1 p e r io d 4W TO S bwnutw a * S ta n d a rd 1: 4.97 Q1 Mt, 298 scant 437.0 Noita Three. 3.0 QuantUvaa. 1.0 Min. WW9l 3 M uL Width 6 Bat*. WM8) 30 RT Win. (sec*) s o S m o o th f Ejected RT 353 A na 1053783 H a ig h t 133897 Starl Tkn End T i m Intagralien W id th Ralantlon Um 3.44 3.95 0.80 SLS4 In ta g ra tio n T y p e A -BB Tu. Dae S. 3000 11:10 Intenalty: 350000 e p i intensity: 350000 cpt F igure 6. A representative ion chromatogram o f a high-level (S.00 pg a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for freshw ater analyses, (monitored m asses - 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom )). W ildlife International, Ltd. Atm -77- F igure 7. A typical calibration curve for Perfluorooctanesulfonate, 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 --99.1 amu). 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 s e t (monitored m ass = 499 amu -99.1 amu). W ildlife International, Ltd. -79- F ig u re 9. A representative ion chromatogram o f a high-level (50.0 ig a.i7L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for fish tissue analyses. From high-range (5.00 to 50.0 pg a.i/L ) calibratim i s e t (monitored m ass - 499 amu --99.1 amu). .W ildlife International, Ltd -80- PFO S_7 494A M 34* M A B -1 T im , D m 5 . 2 0 0 0 1 1 2 2 pros4 .9 # to 1 p t r io d In te rn a t S ta n d a rd : 4 H P T O S U ta A n a A b s o lu ts R a te n d e n n n e 1 : 4 .9 7 Q 1 M l. 2 M a c a n a 4 9 9 .0 N o k e T h ra * . Q uantH aas. 2 0 .0 1 S .0 M in . V M 9 l M u h . W id th B a s e . W kJP i 3 6 40 R T W In . (a o c s ) 30 S m o o th E x p e c te d R T 1 3 .9 0 A rea 0 H a lo h l 0 S ta r t T im a OJOO E n d T im a ln t* g > a U o n W id th 0 .0 0 O jO O R a te n lo n T h n a in te g ra tio n T y p o O jOO in te n s ity : 2 5 0 0 0 0 e p a P F O S .7 4 5 4 A -I3 4 - M A B -1 4 .9 8 in 1 p a rio d V R 9S u a a a s in M m a i S ta n d a r d T ua. D ec S . 2000 1 1 2 2 1 : 4 .9 7 Q 1 M L 2 9 8 s c a n s 4 2 7 .0 N o is e T h r u . Quant Three. Uh. VMUi MOL Width B a ta . VWdSi RTWIn. (so<a ) S m o o th EM adodRT 2.0 1 .0 3 30 20 2.53 A re a 1178488 H e ig h t 1507Z S 8 ta it U rn E n d T im a In te g r a tio n W id th R e u n io n T im e tnlagrstian T y p e 44 2 .9 6 aso 2 .5 8 A -BB In te n s ity : 2SQ O O O e p a F igure 10. A representative ion chromatogram o f a freshwater m atrix blank sample (454A - 1 3 4 - MAB - 1 , dilution = lOOx). The arrow indicates the retention tim e o f Perfluorooctanesulfonate, Potassium Salt (PFOS). (monitored m asses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). W ildlife International, Ltd\ -81- PFO S_S 4 5 4 A -1 3 4 - U A S -Z T iw , D e c 5 , 2 0 0 0 1 1 3 4 4 .9 8 In 1 p a rte d ms in te rn a l S ta n d o rt! 4 H P F O S U se A rea A b e o h ite R e te n tio n T im e 1 : 4 .9 7 Q l M ,2 9 S * c a n a 4 9 9 .0 N qIm T h re s , Q u a n t T h re e . M in . W id th M u lL W id th B a s e . W id th H T W in . ( s e c s ) S m o c ih E x p e c te d R T 2 0 .0 1 S .0 3 6 40 30 1 3 .8 0 A rea 995049 H e ig h t 92S 17 S ta r t T im e E nd T im I n te g r a tio n W id th R e te n tio n T im e In te g ra tio n T y p e 3 .8 1 428 087 3 .8 3 A -BB PFO S_9 4 5 4 A -1 3 4 - IIA S -2 4 .9 8 k i t p e rio d 4H PFQ S u s e a t In lo m il S ta n d a rd T u b , D o c 5 , 2 0 0 0 1 1 :3 4 1 : 4 .9 7 Q l M L 2 9 8 s c o n s 4N2o7is.e0 Hum . Q uant H w m . M h . W id th M u H W id th B a s e . W id th NTWin. (Mes) S m o o th E x p e c te d R T 2.0 1.0 a so aa t 253 A res 1109335 H e ig h t 139309 S ta rt lim e E n d T im s I n te g r a tio n W id th R e te n tio n T im e H e g ra tio n ty p e 2 .4 3 O9.S3O A-2B5 B8 in te n s ity : 2 5 0 0 0 0 o p t in te n s ity : 2 5 0 0 0 0 c p e F igure 11. A representative ion chromatogram o f a freshw ater m atrix fortification sam ple (454A-134M A S-2,0.500 mg a.i./L nominal concentration, dilution = 250x). (monitored m asses * 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). Wildlife InternationalLtd. -82- intensity: 2500 cps F igure 12. A representative iou chromatogram o f an edible fish tissue m atrix blank sam ple (454A-134E-M AB-1, overall dilution factor = 100 L/Kg). The arrow indicates the retention tim e o f Perfluorooctanesulfonate, Potassium Salt (PFOS). (monitored mass = 499 amu -- 99.1 amu). W ildlife International, Ltd. -83- Intanstty: 2500 cps 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). (monitored m ass = 499 amu --*99.1 amu). Wildlife 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 a.i7Kg nominal concentration, overall dilution factor = 20000 UKg). (monitored m ass = 499 amu -99.1 amu). Wildlife InternationalLtd. -85- intensity: 3000 cps F igure 15. A representative ion chromatogram o f a nonedible fish tissue m atrix fortification sample (454A -134-N M A S-8,100 mg a.i7Kg nominal concentration, overall dilution factor = 50000 L/Kg). (monitored m ass = 499 amu-->99.1 amu). W ildlife International, Ltd. PF09L 13 4 5 4 A -1 3 4 - 3 T im . M c S . 2 0 0 0 1 1 :5 8 4 ,8 8 In 1 p e r io d FF06 In te rn a l S ta n d a rd 4 H P P 0 S U se A n a A b s o lu te R te n tio n T im e 1 : 4 .9 7 O f M l. 2 9 8 s c a n s 4 9 9 .0 N o is e H u e s . 2 0 .0 O o n t T h re e . 1 5 .0 M m . W id th M u * . W id U i B a s e . W id th H T W in . ( s e e s ) S m o o th E x p e c te d H T 3 6 40 30 1 3 .8 0 A rea 355173 H e ig h t 33153 S ta rt T im e E n d T im e In te g r a tio n W id th 163 4 .3 0 0 .8 7 R e te n tio n T im e 183 In te g ra tio n T y p e Ai - 8 B PFO SJI3 454A-1 34* 3 DoeT u e . 5 . 2 0 0 0 1 1 :5 8 4 .W in 1 p e rio d 4H PF0S u m u In te rn a l S ta n d a rd 1: 4.97 Q i M l. 298 scene 4 2 7 .0 N a h e T h re e . 2.0 Q u a n t T h re e . 1.0 M in . W k fth U u H . W id th 3 8 30 RT Win. (sacs) Smootfi 2i 0 EapectadRT 2.53 Area 1094547 Height 138842 Start Tima 2.44 End Time 2.95 Integration Width 0.50 Retention Time 2.56 Integration Type A-B3 -86- in te n s ity : 2 5 0 0 0 0 c p s Intensity: 2SOOOO epe F igure 16. A representative ion chromatogram o f a freshwater sample (454A -134-3, dilution - lOOx) from the 0.10 m g a.i./L treatm ent group, (m onitored m asses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard -bottom )). ,W ildlife International, Ltd -87- Intensity: 3500 cp s F igure 17. A representative ion chromatogram o f an edible fish tissue sample (454A-134-E-30, overall dilution factor = 1420) from the 0.10 mg a.i./L treatm ent group), (m onitored m ass * 499 amu --*99.1 amu). ,W ildlife International Ltd. -88- 100 90 80 70 60 50 40 30 20 10 o- - 45 41 0.69 -- i-- -- 81 1.36 -- r1--3--1T 121 2.03 Intensity: 3000 cps 210 262 285 --r 281 Scan 4.71 Time F igure 18. A representative ion chromatogram o f a nonedible fish tissue sam ple (454A-134-N-30, overall dilution factor 2650) from the 0.10 m g a.i./L treatm ent group), (m onitored m ass 499 am u -->99 1 am u). -89- Project Number 454A-134 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 W ater: W ell W ater Uptake Phase Mean Measured Day 0 _____ D a y 7_____ Day 14 Day 21 Concentration (mg a.i./L) Negative Control Temp1 <C) 22.0 pH 8.2 Temp1 (C) 21.9 pH 8.0 Temp1 CO 21.8 pH 8.0 Tem p1 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 C ontinuous measurements o f temperature ranged from 20,0 to 22.0oC. 7.9 21.8 8.1 Day 28 Temp1 C O _ PH 21.9 7.9 21.9 7.9 21.8 8.0 Uptake Phase Mean Measured Concentration (mg a.i./L) Negative Control Day 35 Temp1 CO 21.8 pH 8.1 Day 42 T em p1 (C) 21.8 pH 8.0 Day 49 Temp1 CO 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 temperature ranged from 20.0 to 22.0C. 2M easurements discontinued due to 100% mortality. 8.0 Day 56 Temp1 CO 21.9 pH 8.1 21.9 8.1 mm -90- Project Number 454A-134 A ppendix 4 (C ontinued) Temperature o f pH o f W ater in the Test Chambers ________________________________________ __________ Depuration Phase Sponsor. 3M Corporation Test Sust&ncc: PFOS Test Organism: Bluegill, Lepomis machrochirus Dilution Water:____ Well Water_________________________________________________ __________ Uptake Phase Day 0 Day 0 Day 14 Day 21 Mean Measured Concentration (mg a/L ) Negative Control Temp1 CO 21.9 pH 8.1 Temp1 Temp1 CO p H ____ _________ pH 21.8 8.2 21.9 8.0 Temp1 C O ...... pH 21.9 8.1 0.086 21.8 8.1 21.8 8.2 21.9 'Continuous measurements o f tem perature ranged from 20,0 to 22.0C 8.1 21.8 8.1 Day 28 Temp1 ... C O 21.8 pH 8.1 21.7 8.1 Uptake Phase Mean M easured C oncentration (mg a.i./L) Negative Control Day 35 Temp1 CO 21.9 pH 8.1 Day 42 Temp1 CO 21.9 pH 8.1 Day 49 Day 56 T em p1 Temp' C O _ ..PH _ ____ C Q _____ e h 22.0 8.1 21.9 8.1 0.086 21.8 8.1 21.9 8.1 22.0 1Continuous measurements of temperature ranged from 20.0 to 22.0C 8.1 21.9 &1 W ildlife International, Ltd. Proj",N""b-454A-*34 -91- A ppendix 5 Dissolved Oxygen (m g/L) o f W ater in the Test Cham bers' Sponsor 3M Corporation Test Sustance: PFOS Test Organism: Bluegill, Lepomis machrochints Dilution Water: Well Water Uptake Phase Day Negative Control 0.086 mg 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 i n 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. Wildlife International, Ltd. -92- Project Number 454A-134 A ppendix 5 (C ontinued) Dissolved Oxygon (mg/L) o f W ater in die T est Chambers1 Sponsor Test Sustance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis machrochirus Well Water Uptake Phase Day Negative Control 0.086 m s a.L/L 0.87 mg a.iJL 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 of 52. mg/L represents 60% saturation in freshwater at 22C. 2 NC-Dala inadvertently not collected. - Wildlife InternationalLtd. -93- Project Num ber 454A -134 Appendix 5 (Continued) Dissolved Oxygen (mg/L) o f W ater in die T est Chambers1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution W ater Well Water Day Depuration Phase Negative Control 0.086 mg aJTL 1 7.5 7.6 2 7.6 7.7 3 8.3 4 8.0 5 8.2 8.4 8.0 8.2 6 8.0 8.0 7 8.4 8.4 8 8.3 8.4 9 7.9 8.0 10 8.0 8.0 . 11 8.0 12 7.9 13 8.0 8.0 8.0 8.0 14 7.9 15 7.6 7.9 8.0 16 8.1 17 8.0 8.2 8.0 18 7.9 19 7.8 8.0 7.8 20 8.2 8.2 21 8.0 8.1 22 8.1 23 8.4 24 8.4 25 8.1 26 7.5 8.2 8.4 8.4 8.2 7.6 27 7.9 28 7.5 29 8.2 7.9 7.6 8.2 30 8.0 8.1 31 8.3 32 8.4 33 8.0 34 8.3 35 8.5 8.3 8.4 8.1 8.4 8.5 1A dissolved oxygen concentration of 5.2 mg/L represents 6% saturation in freshwater at 22C. W ildlife International, Ltd. -94- Project Number 454A-134 Appendix 5 (Continued) Dissolved Oxygen (mg/L) o f W ater in the Test Cham bers' Sponsor. 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus M ution W ater WeQ W ater Day Depuration Phase Negative Control 0.086 mg a.i 36 8.4 8.5 37 8.2 8.1 38 8.0 8.0 39 8.0 8.0 40 8.1 8.2 41 7.7 7.7 42 7.8 7.8 43 8.2 8.3 44 7.8 7.8 45 8.2 8.3 46 8.1 47 8.6 8.1 8.6 48 8.4 8.4 49 . 8.5 8.6 50 8.1 51 8.6 8.1 8.6 52 8.1 8.1 53 8.2 8.2 54 8.4 8.4 55 8.2 8.3 56 8.2 8.3 1A dissolved oxygen concentration o f 5.2 mg/L represents 6% saturation in freshwater at 22C W ildlife International, Ltd. -95- Project Number 454A-134 Appendix 6 Hardness, Alkalinity, Conductivity and TOC ofW ater in die Negative Control Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochtrus Dilution Water: Well W ater Param eter Day 0. 7 14 21 28 35 42 49 56 Hardness 132 112 126 130 136 132 104 134 130 (mg/L as CaCO3) Alkalinity 183 174 179 183 181 183 183 184 181 (mg/L as CaCO3) Conductivity (pmhos/cm) 310 325 330 330 325 325 310 320 330 TOC <1 <1 <1 <1 <1 <1 <1 <1 <1 _____ (me c /l ) ________________________ D epuration Phase Sponsor: Test Substance: Test Organism: Dilution W ater 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Param eter Day 1 7 14 21 28 35 42 49 56 Hardness 134 110 126 124 130 132 132 128 126 (mg/L as CaCO3) A lkalinity 179 178 178 180 183 180 175 179 178 (mg/L as CaCO3) Conductivity (pm hos/cm ) 330 330 325 320 325 325 330 320 325 TOC (mR C/L) <1 <1 <1 <1 <1 <1 <1 <1 <1 W ildlife International, Ltd. -96- Project Number 454A-134 A ppendix 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: Well Water Cumulative Number Number Day Observations Dead Remaining 0 AN 0 90 1 AN 0 85 2 AN 0 80 3 AN 0 80 4 AN 0 75 5 AN 0 75 6 AN 0 75 7 AN 0 75 8 AN 0 70 9 AN 0 70 10 AN 0 70 11 AN 0 70 12 AN 0 70 13 AN 0 70 14 AN 0 70 15 AN 0 65 16 AN 0 65 17 AN 0 65 18 AN 0 65 19 AN 20 AN 21 AN 22 AN 0 0 0 0 65 65 65 60 23 AN 0 60 24 AN 0 60 25 AN 0 60 26 AN 0 60 27 AN 0 60 28 AN 0 60 29 AN 0 55 30 AN 0 55 31 AN 32 AN 0 0 55 55 33 AN 0 55 34 AN 0 55 35 AN 0 55 1Observed Effects: AN = Appears 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 W ildlife International, Ltd. -97- Project Num ber 454A-134 Appendix 7 (Continued) Cumulative M ortality and Treatm ent-Related Effects1 Negative Control --Uptake 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 0 0 50 50 38 AN 0 50 39 AN 0 50 40 AN 41 AN 0 0 50 50 42 AN 0 50 43 AN 0 45 44 AN 0 45 45 AN 0 45 46 AN 0 45 47 AN 0 45 48 AN 0 45 49 AN 0 45 50 AN 51 AN 0 0 40 40 52 AN 0 40 53 AN 0 40 54 AN 0 40 55 AN 0 40 56 AN 57 AN 38 A N 59 AN 0 0 0 0 40 35 35 35 60 AN 61 AN 0 0 35 35 62 AN 0 35 1 Observed Effects: AN = Appears Normal' 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 Wildlife InternationalLtd. -98- Project Num ber 454A -134 Appendix 7 (Continued) Cumulative M ortality and Treatm ent-Related Effects1 Negative Control - Uptake Phase Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Cumulative Number Number Day Observations Dead Remaining 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ' 26 27 28 29 30 31 32 33 34 35 AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN 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 25 25 25 25 25 25 25 25 25 25 25 25 25 25 20 20 20 20 20 20 20 20 20 20 20 20 20 20 16 16 16 16 16 16 16 Observed Effects: AN = 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 * W ildlife International, Ltd. -99- Project Number 454A-134 Appendix 7 (Continued) Cumulative M ortality and Treatm ent-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 36 AN 0 16 37 AN 0 16 38 AN 0 16 39 AN 0 16 40 AN 0 16 41 AN 0 16 42 AN 0 16 43 AN 0 12 44 AN 0 12 45 AN 0 12 46 AN 0 12 47 AN 0 12 48 AN 0 12 49 AN 0 12 50 AN 0 12 51 AN 0 12 52 AN 0 12 53 AN 0 12 54 AN 0 12 55 AN 56 AN 0 0 12 12 1 Observed Effects: A N = Appears Normal Number Sampled 0 0 Of 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 12 Wildlife International, Ltd. -100- Project Num ber 454A -134 Appendix 7 (Continued) Cumulative M ortality and Treatm ent-Related Effects1 0.086 mg a.i./L - U ptake Phase Spouses: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Cumulative Number Number Day Observations Dead Remaining 0 AN 0 90 1 AN 0 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 9 AN 0 10 AN 0 11 AN 0 70 70 70 70 12 AN 13 AN 14 AN 0 0 0 70 70 70 15 AN 0 65 16 AN 17 AN 18 AN 0 0 0 65 65 65 19 AN 20 AN 21 AN 22 A N 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 0 0 0 0 0 0 0 0 0 0 65 65 65 60 60 60 60 60 60 60 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN 0 0 0 0 0 0 0 55 55 55 55 55 55 55 1Observed Effects: AN Appears 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 W ildlife International, Ltd* -101 - project Number 454A-134 A ppendix 7 (C ontinued) Cumulative Mentality and Treatm ent-Related Effects1 0.086 m g a.iVL - U ptake Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: W ell Water Cumulative Number Number Day O bservations Dead Remaining 36 AN 0 50 37 AN 0 50 38 AN 0 50 39 AN 0 50 40 AN 0 50 41 AN 0 50 42 AN 0 50 43 AN 0 45 44 AN 0 45 45 AN 0 45 46 AN 0 45 47 AN 0 45 48 AN 0 45 49 AN, IX 1 45 50 AN 1 39 51 AN 1 39 52 AN 1 39 53 AN 1 39 54 AN 1 39 55 AN 1 39 56 AN 1 39 57 AN 1 34 58 AN 1 34 59 AN, IX 2 34 . 60 AN 2 33 61 AN 2 33 62 AN 2 33 1Observed Effects: AN = Appears Normal. 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 Wildlife International, Ltd. -102- Project Num ber 454A -134 A ppendix 7 (C ontinued) Cumulative M ortality and Treatment-Related Effects' _______ 0.086 mg a.i./L - Depuration Phase_______ Sponsor. 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochlrux Dilution Water. Well Water Cumulative Number Number Day Observations Dead Remaining 1 AN 2 23 2 AN 2 23 3 AN 2 23 4 AN 2 23 S AN 2 23 6 AN 2 23 7 AN 8 AN 9 AN 10 AN 2 2 2 2 23 23 23 23 11 AN 12 _ * 13 AN 14 AN 15 AN 16 AN 2 2 2 2 2 2 23 23 23 23 18 18 17 AN 18 AN 19 AN 20 AN 21 AN 2 2 2 2 2 18 18 18 18 18 22 AN 23 A N 24 AN 25 AN 26 AN 27 AN 28 AN 2 2 2 2 2 2 2 18 18 18 18 18 18 18 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN 2 2 2 2 2 2 2 14 14 14 14 14 14 14 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 4 0 0 0 0 0 0 0 W ildlife International, Ltd. -103- Project Num ber 454A -134 Appendiz 7 (Continued) Cumulative M ortality and Treatm ent-Related Effects1 0.086 m g a.i./L - D epuration Phase Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochina Dilution W ater Well Water Cumulative Number Number Day Observations Dead Remaining 36 AN 37 AN 38 AN 2 2 2 14 14 14 39 AN 2 14 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 2 2 2 2 2 2 2 14 14 14 14 10 10 10 47 AN 2 48 AN 2 49 AN 2 50 AN 2 51 AN 2 10 10 10 10 10 52 AN 53 AN 54 AN 55 AN 56 AN 2 2 2 2 2 10 10 10 10 10 Observed Effects: AN = Appears Normal Number Sampled 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 10 Wildlife International, Ltd. -104- project Number 454A-134 A ppendix 7 (C ontinued) Cum ulative M ortality and T reatm ent-R elated E ffects1 0.087m g a .i/L - U ptake Phase Sponsor 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution W ater 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 6 AN 0 75 75 75 7 AN 0 8 AN 0 75 70 9 AN, IX 10 AN 11 AN 1 1 1 70 69 69 12 AN, 1R 1 69 13 AN, IX 2 14 A N .6R .14X 16 IS AN, 8X 24 69 68 49 16 A N .6X 30 17 AN 30 18 AN 30 41 35 35 19 AN 30 20 AN 30 21 AN, 3R.5X 35 22 AN. IR, 4X 39 23 AN, 1R.2X 41 24 AN, IX 42 35 35 35 25 21 19 25 AN, 5R, IX 43 26 A N .6X 49 27 AN, 1R 49 28 AN.3X 52 29 AN, 1C, IX 53 30 1C, IX 54 31 1C 54 32 1C 54 33 1C 54 34 1C 54 35 IX 55 18 17 11 11 3 2 1 1 1 1 1 1Observed Effects: AN = Appears Normal, C ==Lethargic, R= Lying on Bottom, 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 W ildlife International, Ltd. -105- Project Num ber 4S4A-134 Appendix 8 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. Amendment: The proposed experim ental start and term ination dates were added to the protocol. 2. Amendment: The frequency o f light intensity m easurements 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 measurements was added to the protocol. 5. Amendment: Storage stability QC samples were added to the protocol. 6. Amendment: The protocol was clarified to indicate when fish would be sam pled for lipid analysis on Day 0. 7. Amendment: The internal reference standard was added to die protocol. 8. Deviation: Dissolved oxygen was not measured on Day 24 o f the uptake phase. 9. Deviation: Test tem perature was out o f range for approxim ately 2 hours. 10. Deviation: The 0.87 m g a.iVL 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: H ie depuration phase was term inated on Day 56. 14 Deviation: Fish in the 0.87 m g a.iVL treatm ent group were not collected for lipid analysis at the end o f the uptake or depuration phases. Wildlife International, Ltd. -106- Project Number 454A-134 Appendix 9 Protocol, Amendments and Deviations W ildlife International, Ltd. -107- Project Num ber 454A -134 PROTOCOL PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEOILL ('Jjepamis m acrochinu) U.S. Environmental Protection Agency Series 850 - Bcdqgical Effects TentGuidelines OPPTS Number 850.1730 and OECDGuideline 303 Environmental Laboratory Request Nnmber U27Z3 Submitted to BM Caqnm tko Environmental Laboratory 935 Burii Avenue S t Paul. Mntesela 55144 W ildlife International, Ltd. >598 Commerce Drive Easton, Maryland 21601 (410) >22-8601 October 18,2000 ,W ildlife International Ltd. -108- Project Num ber 454A-134 W ild life International, L td. -2- PHRTLUOROOCTANESULFONATE, POTASSIUM SALT [PFOS): A FLOW-THROUGH BIOCONCENTRATTONTEST WITH THE BLUEGILL {Ltpomit naerochinu) SPONSOR: SPONSORS REPRESENTATIVE 3MCorporation Eivircaneatal laboratory P.O. Bor33331 SLPaal,Minnesota 55133 Mi. SunnA. Beach TESTING FACILITY: STUDYDIRECTOR. W M ifclm enatiauI Ltd. 1591 CommerceDriva E uh, Maryland 21601 KurtR. Drottar LABORATORYMANAGEMENT: Hairy0. Krueger, Ph.D. DirectorofAquaticToxicology &Non-Target Plants TOR LABORATORY USE ONLY Proposed Dates: Experimental Experimental Start Date: _____________________ Termination D ate:___ ProjectNo.: 4 S <4-A - Test Concentrations: NegativeControl. 0.1 and I.O m gii/L -------------- TestSubstanceNo.: 4675 Reference SubstanceNo. (ifapplicable): PROTOCOL. APPROVAL STUDYDIRECTOR /> /*//o o DATE PROTOCOL NO.: 454/101JOO/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 .W ildlife InternationalLtd -109- Project Number 454A -134 W ildlife International, Ltd. -3- INTRODUCnON Wildlife liaeraaiiooil, Lei wiO cnadact a H oeoeeeam ioa test with the blorgiil sunfidi (Upomis macrochinu) fartheSponsorattheWildlifeInternationalLtd.aquatictoxicologyfacilityin Easton, Maryland. The study will be petfboaed based on procedures In U.S. Environmental Protection Agency Series ISO- Ecological Effects Test CmdeBnes OPPTSNumber 150.1730 (1); ASThtStandardP.1022-34StandardPracticefar ConductingBloconccntrtuion Tna withFichu and Saltwater Blvaht UoOutct (2); tad OECD Goidatine far Testing of Chemicals 305, Bioacaumdatkm: Flow-Thrawgkfish Tat(3). Raw data farlfl work perfanned alWOdUfaInterna tional, Ltd and a copyof fas final icpoit n il be filed by project number is archives located onfae WildlifeInternational, Ltd. tits , atanalternative locationto baspecifiedinthe finalreport OBJECTIVE The objective of da study is to obtain laboratory data dnrectorizing the bioconceutration potential of Perfluorooctanesulfanate (hereafter referred to as PFOS) in the bluegill, Ltpotnis awracMm. EXPERIMENTAL DESIGN Bluegill will be exposedto twolest oonceatrttioas anda itegativucontrol Eachgroupwill consistofonelest damher withuptoapprarimatdy4S0 gams offish biomass ineachchamber. Thetest willbedividedintoanuptakeanda depurationphase. Duringdieuptakephase, fishmfan treatmentgroupsstillbeexposedtosublethalceneobstioat ofCTOS, whilefishiathecontrolgroup wiBbeespenndto iMntionwater. The daritiuii ofthe uptakepbaao(3 bouts to 28 days) andthe dqmrafionphase(6 hootsto60 days)mayvaryaccodiagtothefiaierequiredtoteachSteady-state Duringboth phases ofthe study, test otganliuis andwatersimples areonBertwl andamdyaodfee PPOScontent Thesevalueswillbonasdtodemmdnadta uptakeramconsnmtOn), faedepsiition rate conatant Cfci), die kineticbiocoaecntretienfactor (BCFX) anddmsteady-stalebioenneentration factor(BCF)farwholebodytiatuca,nSMetieeue(eldaon)id nonedibletietua. MATERIALSANDMETHODS TcatSnbetance The test substance wigbe PaBuonobutaoeauUdnade, PotatgamSilt, hereafter referredtosi PFOS. Informationonthecharacterizationofteat,controlorreferencesubstance! it requiredbyGood PROTOCOL NO 454/10lftxyBUJ-BKVSUB434 Environmental Laboratory Request Number U7723 Wildlife InternationalLtd. - 110- Project Num ber 454A -134 W ild life International, Ltd. -4- LaboratrxyPracticeStandard)(CLP). TheSponsorsstesponsibUfoeprovidingWildlifeInternational, Ltd. writtenverificatioariot rimtest subaancohasbeta characterisedaccordingto OLFs prior to inilialtonofdiertndy. Iftiiitta verificationofOLFtestsubstancecharaeterisatioait notprovidedto WildhfeInlrrmtioosl,lid, itwillbenotedinthecompUsncestatementofthefinalreport The Spaoaor it fa aU SiMti related to the test adistance. including rim retsdioBoft rcserr*mmpliof(boktorbitchofthetortsubstanceusedinta t study. TheSponsor alio agreeloaccept anytamedtat snhwinceand/orteatubtftncc containersremainingtetheend ofdmstudy. PreparationofTestConcortroBow Tho test substance wffl he sdmiaistcrod to rite test organisms in water. Thil route of idinimitniioa was refected becaom it lepremats rimmost likely route of exposure to aquatic organisms. A primary dock solution of PFOS win be prepared by transferring the PFOS to a volumetricflaskand(HatingtoanappropriatewriomewidtdUutiflnwater. Teet Orgaaitas The teatorganism used at this study will be rim bluegjll, Ltpomlt nacrothina. Bltsegfl are one of rite wcaaanrjafcd fish species f a use la blooonocutrshcn tests (U 3X BtucgUs will bo obtained as juveniles frora a cemswrcial vft& a, gradually acclimate! to WadElfc internstioaal. Ltd. well water, aad held for s period of st least 14days priorto the test. If the fish sit held at.water tLiupuulmes that differ from die test tenpesatmB, they wiSbe brought to test temperature at a late not aaeeediug 3`C per 72 houn dartegtoM sg If mortality exceeds 3% during the 41-hour period iaanediatfdy pnxafing tbs te a , the entire batdi o f fids will be iqected or held for mn Afitinnal 14dsy periodtoseam that thayam healthy. Fish wiUbe handled as Btde at possible, but when handling ts accessary, it writ be dorm careftlly. gently,andquidcly. Tocantedbias, fishwiUberemovedfromholdingtankswithnetsand impartiallydistributedsofa testdatahem. Nootherformsofbiasaseexpectedto ai&ettheresults ofthestudy PROTOCOL NO.: 454/101KXVBLU-BIQ/SUB454 Envinxunadal Laboratory Rreprest Number 112723 Wildlife International, Ltd. - Ill - Project Num ber 454A-134 W ild life International, L td. -5 - The nunimumacceptable si oTindividual test organisms will be detennuwdbydiequantity oftissuerequiredfix measurementofresidaes. Loading(thetotalwetweightoffishperliteroftest solution)wil notexceed1.0gumoffishperliterofwhitenthatpassesthroughatestchamberin24 hours. Bhtsgill will be tedflake teedor another commercial teed, once daily. Excess teedwill be yipVnwj tie |ip|waaasj*>ly j Qminutey |%y pBCdlOgWd W will be eeeefiaatadaothatfishwillbesampledat least tear hems after Seeding. Specificationter acceptable levels of conUsmssats in fish diets have not been established. However, then are an known levels of contaminants seasonably expectedto be pressedindmdietthat are consideredto interftnwiththepwposeoreondaetofthetest. DilutionWater Waterusedter theholdingandtesta ofMuegillwfll be obtainedfroma well approximately 40 snatersdeepteemede e theWfldHft International, Ltd. ska. Thewater will bepassedthrougha rend filter and pureped into a 37,800-L storage tank where the water will be aerated withspray nozzles. Priorto usethewaterwill befiltered to 045 tm inordet toremovefineparticles. Water used fa and teste is characterised as moderately hand. Typical values fa hardness, alkalinity,pHsadspecificmndactanccareapproximately: Hardness,mg/L CsOOj Alkalinity, mg/Lss CaCCh ' ^iwa<iin|w y |^ | inih t t h 130 170 32t0.3 Hardneu, aUcalhty. pHand spneific conductance wl be meanred weddy to mnnitnr the ccnmsteacy cf the wdl wnter. Meus sut ranges e t the meumed paramete fbr the feurwretr periodprocdathetestwillbcprovidodmlhefinalreport. Analyseswlbcpctfcnncdatlcaxtonce maarallyto dcSsrminete concanratte at sdeetedpaie andinogsoic cransttwaits ofte wdl watersndrsulteofte mostrcentOLPadyseswillbesummariaeda thefinalreport DikaterSystem and Test Conditemi A ccntinuoui-flowdiluterwill beusedto previdetwonwmeetratinneof PFOS unda control. The flowofdilutionwatertocontrol andtreetnwnttedchamben willbe wgulitedby rotameters. A PROTOCOL NO : 454/l0l*0tVBUJ-BIO/SUB434 Environmental Laboratory Request Number U2723 W ildlife International, Ltd. - 112- Project Number 454A-134 W ild life International, L td. 6- peristaitic pump wiU be used to meet the test tnhttmrr working slock solution into a mixing Flowrates will beadjustedm thatcedi teat chamberreceive* at least sis volumeadiiiticw of vm a every 24 hour Deliveryofthetestsubstanceworkingnode solutionwill be initialedatleast 41 hem priorto theteat Duplicatewateraampics Soareachteatchamberwiflbecollectedat least w " * rS mynHhrimit wmmmciflni e t WPS inthetea dambas befbre introductionofthetestorganisms(ijn, measuredccnceatratloBt shouldremainwithin70to 120%of nominal concentration). Thegeneral operationof toe (flitter will be checkedvisoally at leasttwo timesperdayduringthetest. Stahdeis ateel aquaria filled with appmdmatefy SO L of test water will be used as test chamber! Test chambers will beplacedina waterbathtomaintain a test temperatureof22*1*C. Theteg chamberandwaterhathwillbeenclosedinaplexiglasshoodinordertominimisepotential cross-contamination. Ambient light daring tearing will be providedby fluorescent tubes that emit wavelengthssimilartonaturalsunlight (e.g. Colortooe*50orequivalent) Apbotoperiodof IS hours of light andI boon ofdarknesswill becontrolledwithan antomatietimer. A 30-mmutetransition periodof lowlight internitymil be providedwhenlights go onor offto avoidsoddenchanges in lightintensity. Testchamberswillbeidentifiedbytheprojectnumberandtestconcentration. PROCEDURES ThetestwiHbedividedintoanuptakeandadepurationphase. Organismsinthecontrolgroup will be eapoocd to wcB waterin theptitr>and deputationphases Organismsmthetreatmentgroups viu be mpnarit to th e test suhsUnc in <heuptake phase and w rit water in thruSfpnration phase. Selection ofTeat Concentrations The tost concentrations selected wiHnot stress, irritate, or otherwise adversely affect the organisms. ThetestwntratinMwiflbeselectedlaconjunctionwiththeSponsorandwillbebased onacuteorchronictoxicitydata. Thetwocxpooiinconcentrationsshoulddffierbyafhetorof10, PROTOCOL NO : 454/101SOO/BLU-B1CVSUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. -113- Project Number 454A-134 W ild life International, Ltd. Chcmkal/Ffcysica]Measurements Tcsgjcraturewill bemonbocedandrecordedmntlnuwitlytaringtheentireten fa onecontrol replicate using a Fulscope ER/C Recorder (1900 I Sene* Model A), or equivalent. Recorder measurements willbeverifiedpriortothetestandat approximatelyweeklyintervals duringthetest usiaga liquid-in-glassthermometer. Temperaturealsowiltbemeasuredineverytestdumberatthe beginning and end of the test and it weakly faterrab during Ob tad tidag a liqtrid-in-glass thermometer. Dissolvedoxygenwillbemeasureddailyineverytest chamberduringthetedusinga Yellow Spring Indnimiai Model 51B dissolvedoxygenmeter, or equivalent, hi the eventthat dissolved oxygenlevel61 Mow<0%saturation, appropriate actionwillbetakenafter consultationwiththe Sponsor. Mcasaicmcnte ofpHwil bomadeineverytest dtamherat thebeginning andendofthetest andat weekly intervalsdoting die ted usinga Fisher AecnmetModel 913 pHmeter, or equivalent Hardness, alkalinity, andconductivitywil bemeasuredinthecontrol treatmentatthebeginningand cadofthetestandat weeklyintervals duringthetest Hardnessandalkalinitymeasurementswffl be rarf by ik"iwi using on"dM inStandardMethodsfor (hr Examination o f Wattrand f'attrwaur (4). ConductivitywillbameasuredusingaYellowSpriags InstrumentModel 33 Salinity-Condactivity-Tempmtmc m eter.orequivalent. BiologicalMcasamncats Observationsofbehaviorandmortalitywillbemadsdaily. For a ted tobe consideredvalid, mortalityandabnormalbehaviorahouMnotbeapparentInmorethan 10%ofthefishiathetreatment of m h o ly o u p t DaraHonfUpta]Be?1ufte Ihc rtalisticallyoptimumduratila!oftheoptatephasc(u)isnearu* 1.6*2, bainetmorefinn 3.0*2, riddi la equivalent to 93 perceat ofsteadydate. Theaptakephase will coatimieuntil th oonoautratica ofPFOS reddueaInbodydasueatrochea uradystate, Tbe criterioibr datarne*of Urartystile iathatIhreeconsecutiveFFOSresiduecxxxxmrationsinfialiarerxxstitisticallydiflcrcnt trancadaother. PROTOCOL NO 34/101HXVBLU-BIO/SUB4S4 Enviraamcatal Laboratory Requcd Number U272J W ildlife International, Ltd. -114- Project Number 454A -134 W ild life International, Ltd. -i- DuraHonofDtprattanPhase Twotinicsa isusuallyawfficicnltimeforabout95 pacml removalofthebodyburden(t*<> O.MAcJ. however, thedeputationphaseill oootinueantil tbs concentrationofPFOS residueia Ihe otgamnwatiaimanyooeefttefoBowingthreeconditions: 1. Residues reach las (haa 10%ofsteady-stale or 2. Kendaca fallbelowthe LOQ;or 3. Stay(60}daysofdepantioahaveelapsed. ' Ssupls Intervals for WateraadTab Coledian Fishandwater willbe sampleddolessthat fived an daringtheuptake phaseandfair times duringtherirpmationpharcofthestudyHdiepaneintervalr IntddWnn.oorfrolandtreatedwater will he sampled no less dm twice prior to adfitkai of fish to the chambea to confirm the coimeemiuMdesired, h the abeam of ielbeinatiee oa the bioaccwimltlioa potential of the test suhstanec and/or reliable phyaico-chemical data from which bioaccamutation potential may be inilMteH Phase SampleInterval Water1 Fish1 Action Pre-Uptake* ftt X Start X Uptake 0X ~ 4hoars X X Addfish 1day X X 3day X X 7 XX 14 X X 21 X X 21 X X Transfix Deparaban 1 XX 3 XX 7 XX 10 X X 14* X X At teamfive repUcataacf wateraaaoleswil beeollectedat eachinterval liftedtordieaoalyaiaof _PalFkOalmS.ityA,sdpdeitciiofnicaelasaadmucptlatancc^ilalnbdetodtaolwgiaarsdneeccaersbeoanry(TfOarCt1he determination of pH, hardness, 13efficient fish wMbe eollectodto allowthe preparation of four replicate samples Aon each treatment group and *ySiwa sanples ftoa the contml group for the analysis of PFOS. ^Additionalsamplessrifibdrawnnsneeessanrforthetaalywofmidcontent *Pte-Uptakswatersampling(-ei.-nj forconfirmationoftwatinsntlsvcliss toadditionoffish. TaJtctpauntuataincnyopnbessofrtahmeepmlmndwitirilalioroocntltjanuedeiunntthiletdheeroaotioocnaoptfrdateipounroatfioPnFpOhSnareeosindupeaniaeth.eorganisms PROTOCOL NO.: 454/101I00/BLU-BKVSUB4S4 EovuanncaUl Laboratory RequestNumber U2723 W ildlife International, Ltd. -115- Project Number 454A -134 W ilcttife International, Ltd. -9- The tw ite tad frequocy of sampleservili may be thaagedbased a phyiierrchereieal propertiesoftheteataitataneftomwhichpredictionsofbiowrtwnnlaricii ratoctnbederivedtediar at thedirectionofde Spanar. At esdi anphi huenal, ten voter P fln HDbe cnllrrtni fccre da control wdthree waterMaples wi3beeeUectodEromthePFOS treatmentgroups. The PFOS concentrationwillbe fattenedIbraauahuuaiefonereplicateofa controlandtan replicatesateachtrerementtendfcr each interral inwhichtampla are analyzed(at specifiedby the StudyDirector}- The renaming rcpUcatcsofsamplesdrumfcr PFOS analysis waQbeheldinreserveandmaybe analyzedat die directionofdieStudyDirectororChemistryPriacipslInvestigator. TbsconcentrationofPFOSindie water at cadi sample interval will be presentedas die avenge of die replicate measurements, as applicable. Additional samples will be drawn as necessary lor the determination of pH, hardness, atohaity, specificconductance, andtotalorganiccarbon(TOC). FishSampling To assets dre PFOS contai in fish (stare, sufficient fish will be coltocted to allowdie preparation of fear replicate sampfas fromeach treatment groupandduplicate samples fromtbs oootiolgroup. OneadditionalsamplefromeachgroupwillbecollectedandhdUinreserveandmay bo aaalyzod at (be a-*TM of dmStudy Director or Chemistry Phacipul Investigator. Tbs wziotsialineofPFOS fate a t eachaasaptsIntervalwidbeprusereedastbsaverageoftbswpUcats Fidi sampled ibr dstenniaatiaa of PFOS nontcnt svili bo isnpattiaHy resnoved fiore tbe test charnbcn. tfcnodwithddurioowater, bkstoddry. and sacriflcod. Tbowcigbl (wet wetd*. Uottad dry)andtotallengdiofcadi fistiwdlbedetenninedwilhinapproedmatdy15annate! ofcoBeetioa.if pcusibla Hachfisbwill bo acpaiatcdiato odftloandacwidibls tissueandAc wet wdghtofcadi tisaneftaclian(i.o..edftlnandncnedible)wiSbeiecardcd. Pitbwill bestomi fiozen Haotanalyzaf xmaodtxy. PROTOCOL NO : 434/101(DOmLU-BIO/SUB4S4 Environmental Laboratory Request NumberU2723 W ildlife International, Ltd. - 116- Project Number 454A-134 W ild life International, L td. 10- AAfitional Naples will bedrawnas necesmy forfoeanalysisoflipidcontent.. AnalyticalMethod Water and time samples son be analyzed fix PFOS mint liquid chrcroatographyciass spcctnemetiy. Watersamples01 be analyzedaccordingto tbe methodentitled"Aaalytial Method Validation for tbe Dctenmaatkm of Perflnorooctane Sulfcmic Add, Potassium Salt (PFOS) in Freshwater, Saltwateraid Algal Media' (WildlifeInternational, LtdPtqjcctNo. 454C-I09). Tirsno luigilm will be analyzed according to the method entitled "Analytical Method Validatioa fix the DetenainatiooofPafinorooctaneSulfonicAeid, PotassiumSalt (PFOS) inH*hTissues"(ProjectNo. 4S4C-119). Anytaodificatiorato tig abovemethodolopcawillbe docamentodintherawdataand describedinthefinalreport. ThroeLipidContent At a wauaun. fishwill besampledSoot thecontrol aquarianat Day0 (upontransferofftdt toaquaria),ataaiatervalwherethePFOScoaccaiiztioainfishhas reachedsteadystate(orfollowing 21 daysofexpomie), andat (heendofthedepurationperiod. Fishwillhesampledtogeneratefar samplesfromthecontrolandtwotreatedaquariaateachoftbeintervalsacted. The Hpidcontentof dmriTnfcrrvy*'t>willhiUf>mimythyrMnrrdhrmAnrthannl (attartinn DataAnalysis Remits of tkme analyses wifi be presented on a wet weight basis. The steady-state bioconceutraticn fiutnr fer theteetaabstance (BCF), tactic bioconcentiation fata (BCFK) uptake ale fa), anddepunSionralefa) winbe expressedforcdfidetisane, noacdiblctissueandwholefish C*)- RECORDS TO BE MAINTAINED Recordstobe< !--t fa datageneratedit WfldKft International, Ltd. win includebutnet betailedto: 1. Acopyofthesqm!protocol 2. Identificationandcharacterizationofthetestsubstance, ifprovidedbytheSponsor. 3. Dale ofinitiation and terminationo f the test. 4. "fartargaiaunholdingandacclbwatroarecords. PROTOCOL NO.: 4542101800/BLU-&10/SUB454 Environmental Laboratory RequestNumber U2723 .W ildlife InternationalLtd -117- Project Number 454A -134 ildlife International, L td. -ii- 5. Methodsusedlapreparedocktohitkxutoddilutionofthetextsubstance. 6. Dailyobservations. 7. Watercbcaabycalculations(c^.,hardnessandalkalinity). I. OrganismweightandlengthmoBuemeaU. 9. The methods Died to analyat tot wbsranrr. mneentratioas and the results of analytical 10. II. Acopyoftbefinalreport. FINAL REPORT AreportofdieresultsofthettudywillbepreparedbyWildlifeInternational, Ltd. Thoreport willinclude,bataotbeSmiledto,thebfioning: 1. Nameandaddressofthefic9ityperfbnioglbetu<iy. 2. Data onwhichthestudywasinitiatedandcompleted KistheresponsibilityoftheSponsorto provide the final date that data am meoeded for chemistiy. pathology and/or supporting evaluationsthatmaybegeaentedatotherlaboratories. 3. A statement of ccmplianct signedbythe Study Director addressing any creep!ions to Good LaboratoryPracticeStandards. 4. Objectives and procedures stated ia the approved protocol, atrinHing any changes in the orgbuSprotocol 5. Statisticalmethodanployedbe analyzingthedata. 6. The teat, control andreference substanrre identified by name, chemical abstracts number or codenumber, strength, parity, andcompositionorotherappropriatecharacteristics, ifprovided bytheSponsor. 7. Stabilityand, whenrelevanttotheconduct ofthestudy, thesolubilityofthetest, controlaud reference substance under the "liii" of admmistntion, if provided by the Sponsor or I. A description o f the methodsusod. 9. A description of the test systemused. When applicable, the final report rial! iadude the umber ofanimalused,bodyweightrange,sourceofsupply,species,age, andprocedureused 10. Adescriptionofthedosage,dosageregimen,routeofadministrating, anddmation. II. Adescriptionofallcircumstancesthatmayhaveaffectedthequalityorintcjpityofthedata. PROTOCOL NO.: 454/10l*0<yBLU-B]O/SUB454 Environmental Laboratory RequestNumber U2723 W ildlife International, Ltd. - 118- Project Num ber 454A -134 W ild life International, L td. - 12- 12. Thenameoftte StudyDirector,thematesofothersmemtistsorpmfossicftalj, andthenamesof al mperviaorypenoaaal, involvedinthestudy. 13. A description of Ac transfonnatioas, calculations, or operation performed on lb* data, a stannaryandanalysisofthedata,and statcaKrt ofthecondusiotBdrawnfronttheanalysis. 14. Tbc signedaddatedreportofcadi oftheindividualscientists orotherprofessionals involved fattbastudy.ifapplieablo. 13. The location whereall pecinv.ni, raw data, andthefinal report are to be stored. 16. AstatementpreparedbytheQtalityAssuranceUtahlistingfoodatesthatstudyinspectionsand auditsweremadesadthedatesofanyfindingsreportedtotheStudyDirectorandManagement. 17. If it ia necessaryto make corrections or additions to a final report after k has beenaccepted. 4 A a i y Jw ll t m riu. Sum - f a iM tiM r t tiy tha Study Dieartar. The amendment should dearly identifythspartofthefinal repostthat isbeingaddedtoorcorrectedandthereasonsCor thecorrectionoraddition. Atnendracritt dullbesignedanddatedbytheStudyDirector. CHANGINGOF PROTOCOL Planned<*--g--totheimaad will be inthe (bratofwrittenamendmentsignedbytheStudy DirectorandtheSponsor's Representative. Amendments wfll be oomktaedas put of tbc pndoool *mAyin h* ^ rii--ftwi Anyotiicrchangeswallbe inthefontsofwrittendeviations signed by the Study Director and filed with the saw data. All charge to the protocol will be indicatedindiefinal report GOODLABORATORY PRACTICES This tendywill beconductedinacnnrriance withGoodLaboratoryPractice StandardsforEPA (40 CFR. Pan 1% OECDPlindpkst f OoodLaboratoryPractice(ENV/MC/CHEM(9t) 17)-,nmd lapan UAFF (39 NohSaa. Notiflration No. 3130. Agricultural Production Bucali). Badi study conrincfnd by WMife tntarnatinnal. Lid. is routinely remained by tbs Wildlife hdematinoai. Ltd. QuUty Assunnoe Unit to compliance with Oood Laboratory Practices, Standard Operating Procedura andfee specifiedprotocol. A statement ofcompliance wife GoodLaboratoryPractices will bepreparedtor all portionsofthestudyoondnrTwi byWildlife International, Ltd. TheSponsor willberesponribieto corepUaacewifeGoodLaboratoryPracticesforproceduresperformedbyother laboratories (ig , residue analytes or pathology). Rawdata to all work performed at WfldHfc International, Ltd. anda copyofthefinal reportwillbe filedbyproject numberinarchives locatedon theWildlifeInternational.Ltd,site,oratamalternativelocationtobe specifiedindiefinalreport. PROTOCOL NO.: 434/10IM0/BLU-61O/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. -119- Project Number 454A -134 -13RETERENCES 1- U-S. Environmcntal Protection Agency. 1996. Saies ISO Ecologie! Eficcts Test GuideEnes (Publie OPPTSNumber*50.1730: Flth BCF. 2. ASTM Standard E102Z-84. 19**. StandardPracttcafor ConducttngBioconctntration Tant wllh F iih a and SaltwatcrBivuivz Mouscs. AmericanSocietyforTestingsudMzleriali. 3. OECD Gukiellse fer Tcstinc of Chemicals 305. 1996. BfocM ctntratton: Flow-Throvg/t F ih T t. 4. APHA, AWWA, WPCF. 1913. Standard M tthodt fo r th t E m anation o f W altr and W cattwattr. 16ds BdMon, American Publie Healdi Association. American Water Works Association. Water Pollution Cantro! Fdration,New Yorit. 3. G. E. Bl*n andG. L. Agai. 1971. BIOFAC. TheDowCompany. Midlsad, MI. PROTOCOL NO.: 454/10 U00/BLU-B1O/SUB4S4 Environmental Laboratory Request Num ber U2723 W ildlife International, Ltd. -120- Project Num ber 454A-134 W il d l if e International lh>. PROJECTNO.: 454A-134 PT lof2 AMENDMENTTOSTUDYPROTOCOL STUDYTITLE; PERFUJOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATIONTESTWITHTHEBLUEGILL(Upontj maencklna) PROTOCOLNO.: 4S4/101SM/BLU-BIQ/SUB434 AMENDMENTNOj 1 SPONSOR; 3MCorporation PROJECTNO.: 434A-IJ4 EFFECTIVEDATE: November7,2000 ENVIRONMENTALLABORATORYREQUESTNO.: U2723 AMENDMENT: Page2 AAt ExperimentalStartDate: 12/5/00 ExperimentalTenniriitioaDate 1/16/01 REASON: TheaboveinformationwasnotknownwhentheStadyDirectoraignedtheprotocol. AMENDMENT: DilaterSystemandTestGenditioaa,Page6 Add: Lightintensitywigbemeasuredattedinitiationwitha SPERScientificLtd,lightmeterorcqtuvNent REASON: Tospecifythofroqecncyoflightintensitymcsurcrocnt AMENDMENT: O w tollhpliilMcnaurrmrnil.Pnge7 Change Hardnesa.alaiairfy, awlconductivitywillbemeasuredin theoontroltreatmentatthebegwingandend thetestandrtweddyinierrabduringthetest To: IVilinity.conductivityandtotal nraannartina (TOC)wilbamcsanrB<liathacCTaoltraannci<at Acbeginningandcadofthetaatandatweddpinterval*duringthetot REASON: ToparitytheacqueacyofTOCinnaunaMoU. AMENDMENT: OfwjcalffbyNtnlManunMent^Pnge7 Add: TOCwillbemeasuredusingaShimadiuModelTQC-5000TOCaaafyxcr. REASON: ToaddthcincdiodologyforTOCmeasurement QrtC ll-T-oo Wildlife International, Ltd. -121 - Project Number 454A -134 Wil d l if e International lto. PROJECT NO.: 434A-134 P ige2of2 AMENDMENT: Analytical M etbed. Page 10 Add: Tissuestability sampleswillbeprepared atlestinitiationto establishtestsubstancestability in fishtissues storedfrozenduringthe study. REASON: To add Stongestability QC samples. AMENDMENT: Tissuelip id Content, Page 10 Change: Ata minimum,fidswill be sampledfromthe control aquaria at Day0 ... To: Ata minimum,fish will besampledcn Day0 ... REASON: Fish sampledon Day 0 for lipid analyas will becollected priorto distribution to the test chandlers. DATE SPONSOR'S REPRESENTATIVE Wildlife International, Ltd. -122- Project Num ber 454A-134 Wil d l if e International ltd. PROJECT NO,: 454A-I34 Pagelofi AMENDMENTTO STUDY PROTOCOL STUDV TITLE; PERFLUOROOCTANESULfONATB.POTASSIUM SALT (FFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (U p oalt m ocrochim i) PROTOCOL NO.: 454/101X0/BLU-BKVSUB454 SPONSOR: 3M Corporation AMENDMENT NO.: 2 PROJECTWX: 454A-1J4 EFFECTIVE DATE: November 21.2000 ENVIRONMENTAL LABORATORY REQUEST NO.; U2723 AMENDMENT: P*e2 AAt ReferenceSubvienneNo.: 4526 REASON: To addthe internal reference sundard. SPONSOR'S REPRESENTATIVE HU U-W-e Wildlife International, Ltd. -123- Project Number 454A-134 W j l d u f e International ltd. PROJECTNO.: 454A-134 Page I of 1 DEVIATION TO STUDY PROTOCOL STUDYTITLE: PERFUJOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATIONTESTWITH THE 8LUEGILL(Upomis maavchina) PROTOCOL NO: 454/10180WBLU-BKySUB454 DEVIATION NO.: 1 SPONSOR: 3MCorporation PROJECTNO.: 454A-134 PATE OFDEVIATION: IMP_______________________________________ ___ DEVIATION: The protocol states that dissolved oxygen m il be measured daily in evoy test chamber. Dissolved oxygenwas not meativtd on Day 24 oTthe trptake phase o fthe test REASON: Biologistoversight. AS dissolved oxygen measurementsAxingthetost were ofsaturation. Consequently, it is the best judgement of the Shady Director that this deviation did not adversely affectthe results o fthe study. STUDY DIRECTOR LABI KY MANAGEMENT S ' A 3/C > f DATE W ildlife International, Ltd. -124- Project Num ber 454A-134 WlLDLIFE INTERNATIONALLto. PRQJECTNQ.: 454A-134 Page l o f i DEVIATION TO STUDY PROTOCOL STUDYTITLE; PERFLUOROOCTANESULFONATE, POTASSIUMSALT(PFOS): A FLOW-THROUGH BIOCONCENTRATKJNTEST WITH THE BLUEGHL (U pom ts nacrodtirus) PROTOCOL NO; 45V101800/BLU-BIO/SUB454 DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NOu 454A-134 DATE OP DEVIATION; FehmarvI4.2001__________________________________________ ______ DEVIATION: Tbs protocol states that tbs test dum bos will be placed in a water bath to maintain a test tcm pentareof221 1*C. On Day 19 rfdqw radon,thecentIwiouatexnpcratorereccrdcrreaped 20*C The u n p e rso n wm ant efiaugefcr approximately 2 hours. REASON: Unknown. Based on the short duration o fthe temperature deviation,it is tbe bestjudgement o fthe Study Director that this deviation did not adversely effectthe result* ofthe study. STUDY DIRECTOR LABORraAT' ORYm a Na g m en t DATE Wildlife International, Ltd. - 125- Project Number 454A-134 W il d u f e International lid. PROJECTNO.: 4S4A-134 Page 1o fl DEVIATIONTO STUDYPROTOCOL STUDYTITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATIONTEST WITHTHEBLUEG1LL(JLrpomis m acrochina} PROTOCOLNO: 4S4/101800/BLU-BIO/SUB454 DEVIATIONNO.: 3 SPONSOR: 3M Corporation PROJECT NO.: 4S4A-134 DATEOYDEVIATKIN- IVm iW U niflft___________________________________________ DEVIATION: Tlprotocol thattfaclestooocentrationirelatedwiMnotatrcM.krilate,orotfagwiaeaflto tbsorganisms. The0 X7mga.i7Ltreatmentgroupwasactuallychronicallytoxicin bkuegflL REASON: InfiwmalioaondiechroaJctoxicityofPFOSwbfcegjwasaocknovmwhenthetestcoaccctritions were elected. This deviation adversely affected (he tanks of the study because adequate bioccncentratiaodatawasnotobtainedfromthe0.87 mgjll/L.treatmentgroup. STUDY DIRECTOR &I/H/OI DATE Wildlife 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 lto PROJECT NO.: 434A-134 Page 1 of 1 DEVIATIONTO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (JLepomis macrochlnu) PROTOCOL NO: 454/1O1I00/BLU-BIO/SUB454 DEVIATION NO.: 4 SPONSOR: 3M Corporation PROJECT NO.: 454A-I34 PATE OF DEVIATION: Pebnm v 17.2001_____________________________________________________ DEVIATION: The protocol states fbat observations of behaviorand mortality will be made daily. Biological observations w e not recordedon Day 12 ofdeputation. REASON: Biologist oversight. Biological observations on Day 11 and 13 of depuration were the same. Consequently,itistho bestjudgementofthe study directorthatthis deviationdidnotadverselyafiect the results ofthe i tody . DATE Wildlife International, Ltd. -1 2 7 - Project Number 454A-134 W i l d u f e In t e r n a t i o n a l l t o . PROJECTNO: 434A-134 Page l o f i DEVIATIONTO STUDYPROTOCOL STUDYTITLE: PERFLUOROOCTANESULFONATE.POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATIONTESTWITHTHEBLUEG1LL(Ltpomis mac/ochina) PROTOCOLNO: 454/ 101MXYBLU4 KMSUB454 DEVIATIONNO- 3 SPONSOR: 3MCocpoc*tioo PROJECTNO-: 4S4A-I34 DATESOFDEVIATION: Marchi and 19.2001 DEVIATION: Theprotocolgatesflat rnfficicmfiahwillbecollectedtoallowtheprepwationoffourreplicate samples fran each treatment poop and dnpTtcato samples from e control troop. Oae ah&kmalample fromeachgraspwillbecollectedsodheldla serveandmayhe analyzedat the(fiscrat*oftheStudyDirectorcrChemistryPrincipalInvcatigatar. OnDays28 tad42 of depuntioo, reserve saaoplea werenotcaSectod. SEASON: Reservesampleswerenotcollecaedtoconservethe aumboroffishremainingin(hestudy. Itis the bestjudgaacat ofthe study dilator that this deviation did not adversely affect the resnltt of the W /Y / ( DATE b l l& t. DATE W ildlife International, Ltd. -128- Project Number 454A -134 W il d l if e International urn. PROJECTNO.: 454A-134 Page l o f i DEVIATIONTO STUDY PROTOCOL STUDYTITLE: PERFLUOROOCTANESULFONATE,POTASSIUMSALT(PFOS): AFLOW-THROUGH BIOCONCENTRATIONTESTWITH THE BLUEG1LL(fjcpomis macrocktrut) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 6 SPONSOR: 3M Corporation PROJECT NO,,- 4S4A-134 PATE OP DEVIATION: A rri2.200I ________________________________________________ DEVIATION: The protocol (tales that Ihc depuration phase will continue unti the concentmioa of PFOS residuein theorganism attainsanyoac ofthe followingthreecoaditionr 1}Residuereachless than 10Kofiteady-ittfe,or2)ResiduesfrllbclowtlieLOQ,or3) Sixty(60)dsyserfdepuration hsredapaed. T ic residuesdidnotmeet 1or2 tboyc anddepuration wasterminatedan Day S6 o f depuration. REASON: The Sponsor requested that sampling occur every two weeks during depuration. It is the best judgementofthe studydirector that this deviation did not adversely affect the results ofthe study. DATE Wildlife International, Ltd. -1 2 9 - Project Number 454A-134 Wil d u f e International Lin. PROJECT NO.: 454A-134 Page 1 o f I DEVIATION TO STUDY PROTOCOL STUDYTITLE: PERFLVOROOCTANESULFONATE, POTASSIUM SALT<PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUECHLL (Jjtpo*rt* macrxxhina) PROTOCOL NO: 454/101IOO/BLU-BIO/SUB454 DEVIATION NO.: 7 SPONSOR: 3M Corporation PR O JEC T N O.: 454A-134 DATES O F DEVIATION: FebraarrSm d AW3.200I_________________________________________ DEVIATION: Theprotocolstate* thatfish willbe simple* S x lipid anilyib at stedy-*tate anda ttheeadcfthe depuration phase. No fish a m sanlcd fin s the 0.17 mg aiTL treatment group. REASON: These icreix> fish ran*iaii^m t>K& S7n>gaiyL treatmentgroup at the above times. It b thebest judgement o fthe etaty director d o t M s deviation did not adversely affect the results o f the study. STUDY DIRECTOR U LLABORR9AATTOORRYYMMAANNzAGEMENT <>{W /Ol DATE Wildlife International, Ltd. -1 3 0 A ppendix 10 Project Number 454A-134 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, Ph.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 Biologist 5. Cary A. Sutherland, Laboratory Supervisor 6. Raymond L. Van Hoven, Ph.D., Scientist 7. Susan T. Plantains, Biologist AMENDED Wildlife International, Ltd. Project Number 454A-134 -131- A ppendix i l Calculations O f 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. 1. The depuration rate constant ( k 2) was determined from data collected during the depuration phase and was defined as the slope o f the regression line where x equals days o f depuration and y equals die natural log o f the tissue concentration. Results o f all calculations were generated using Excel 2000 in M l 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: *1/2 = ( l / k 2) l n 2 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 ( k , ) was determined from the following equation: t - C/* 2 ' C J l - e - * ' 1) where C f is the concentration o f fish tissue from the uptake/depuration curve near the uptake m idpoint (mean concentrations from day 35 were used in calculations); where C w is fee mean measured concentration in water (0.086 mg a.i./L); and where t = 35 days. AMENDED W ildlife International, Ltd. -132- Project Number 454A-134 Edible Fish Tissue 9.022 Appendixll (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 compartment, two-param eter model 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 , die depuration rate constant, was used in the following equation to determine the tim e to reach 90% steady state for uptake: 0 .9 = 1- e~kl' or /go = 2.3 t 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 S. BCFK values ( k ^ l k 2) are provided below: BCFK values Edible Fish Tissue 1124 Non-Edible Fish Tissue 4013 W hole Fish Tissue 2796 AMENDED 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 rep o rt 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 IS A m endm ent This section was modified to clarify that the tests for normality and homogeneity met the assumptions o f the tests. Additionally, the section was modified 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 estimate these parameters. Reason: A fter consultation with the Sponsor it was believed that the BIOFAC model underestimates the uptake and depuration rate constants, and overestim ates the kinetic concentration factor, the estim ated time to reach 90% steady state, andthe half-life for clearance. Therefore, the data have been reanalyzed using the equations and graphical methods outlined in the draft OPPTS 8S0.1730 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 \Yildlife International, Ltd. -134- Project Number 454A-134 A ppendix 12 -C ontinued- Report Amendment 5. Original Report: Addition o f Appendix 11. Amendment: Appendix 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: / ... DATE se. & L AMENDED