Document 7Mw5BvZdZ282Gd7g7wyqRvepj

\ 8) OECD 305-OPPTS 850.1730,Fish bioconcentration (bhiegil) PERFLUOROBDTANE SULFONATE, POTASSrUM SALT (PFBS): r* A M I T l - i r p x A FLOW-THROUGH BJOCONCENTRATION TEST WITH THE A N I T I Z E D BLUEGILL {Lepomis macrochirus) 1 FINAL REPORT DEC 0 9 2003 WILDLIFE INTERNATIONAL, LTD, PROJECT NUMBER: 454A-] 17 3M ENVIRONMENTAL LABORATORY REQUEST NUMBER: E00-1429 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test 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: June 29,2000 STUDY COMPLETION DATE: May 9,2001 Submitted to 3M Corporation Environmental Laboratory P.O.Box 33331 St. Paul, Minnesota 55133 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page I of 100 Wildlife Internationalr Ltd. Prnirrf Nnmher 4 UA.T 17 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Perfluorobutane Sulfonate, Potassium Salt (PFBS): A Flow-Through Bioconcentxation Tost with the Bluegili (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 45C A -li7 STUDY COM PLETION: M ay 9, 2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the US. Environmental Protection Agency in 40 CFR Part 792, 17 August 1989, with the following exceptions: The test substance was not characterized in compliance with Good Laboratory Practices prior to its use in the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original characterization purity. The stability of the test substance under conditions o f storage at the test site was not determined as required by Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R. Drottar Senior Biologist SPONSOR APPROVAL: DATE DATE Applicant/Submitter DATE Wildlife International, Ltd. -3- Project Number 4vtA -1 )7 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 Part 792, 17 August 1989. The dates of ail inspections and audits and the dates that any findings were reported to the Study Director and Laboratory- Management were as follows: A C T IV IT Y : DATE CONDUCTED: Test Substance Preparation July 13,2000 Matrix Fortiii cation July 24,2000 Tissue Sampling, Fish Lengths and Weight Measurements and Tissue Preparation August 22, 2000 Analytical D ata and Draft R ep o rt' September 29, October 2 - 6 and 9, 2000 DATE REPORTED TO: S T U D Y DIRECTOR: MANAGEMENT: July 13, 2000 July 17, 2000 July 24, 2000 July 25, 2000 August 22, 2000 October 9, 2000 August 23, 2000 October 16, 2000 Biological Data and Draft Report October 2 3 -2 6 , 2000 October 26, 2000 October 31, 2000 Final Report May 9, 200! May 9,2001 May 9, 2001 Marsh; [T. Hynson Qualit; Assurance Program Supervisor DATE Wildlife internationalr Ltd Pmiect Number 4S4A-117 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Perfluorobutane Sulfonate, Potassium Salt (PFBS): A Flow-Through Bioconcentration Test with the Bluegili (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-117 STUDY DIRECTOR: Senior Biologist MANAGEMENT: Director of Aquatic Toxicology and Non-Target Plants DATE W ildlifeInternational, Ltd.__________ p^ n^ ^ a-ht -5- TABLE OF CONTENTS Title/Cover Page...................................................................................................................................................... I Good Laboratory Practice Compliance Statement....................................... 2 Quality Assurance Statement................................................................................................................................ 3 Report Approval................ 4 Table of Contents....................................................................................................................................................5 Summary..,................................................ 8 Introduction.................................................. 9 Objective................................................................................................................................................................. 9 Experimental Design................ 9 Materials and Methods.................................................... 10 Results and Discussion........................ 15 Conclusions...... .......................................................................................................... 17 R e fe re n c e s ............................................................................................................................................ 18 TABLES T able 1 - Means and Ranges of Water Quality Parameters................................ .................. --.................... 19 Table 2 - Concentrations o f PFBS in Water Samples During the Uptake Phase..........................................20 T ab le 3 - Concentrations o f PFBS in Water Samples During the Depuration Phase.................................. 21 Table 4 PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues o f Bluegill Exposed to 0.53 mg a.i./L................................................................................. 22 T able 5 - Steady-State BCF Valnes for Bluegill Exposed to 0.53 mg a.i./L................................................25 Table 6 - BIOFAC Model Estimates for Bluegill Exposed to 0.53 mg a.i./L....:........................................... 26 ,Wildlife International Ltd 6- - Projerr Number 454 A- 1\~ f TABLE OF CONTENTS - Continued - TABLES (Cont'd.) Table 7 - PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues o f Bluegill Exposed to 5.2 mg a.i./L.................................................................................. 27 Table S - Steady-State BCF Values for Bluegill Exposed to 5.2 mg a.i./L.................................................. 30 Table 9 - BIOFAC Model Estimates for Bluegill Exposed to 5.2 mg a.i,/L................................................. 3 1 FIGURES Figure 1 Concentrations o f PFBS in Edible Fish Tissues o f Bluegill Exposed to 0.53 m ga.i./L...................... 32 Figure 2 Concentrations o f PFBS in Nonedible Fish Tissues o f Bluegill Exposed to 0.53 mg a.i./L............................................................................................. 33 Figure 3 Concentrations o f PFBS in Whole Fish Tissues of Bluegill Exposed to 0 .5 3 m g a.i./L ..................................................................................................................................34 Figure 4 Concentrations of PFBS in Edible Fish Tissues o f Bluegill Exposed to 5.2 mg a.i./L.................................................................................................. 35 Figure 5 Concentrations o f PFBS in Nonedible Fish Tissues of Bluegill Exposed to 5.2 mg a.i./L....................................................................................................................................36 Figure 6 Concentrations of PFBS in Whole Fish Tissues of Bluegill Exposed to 5.2 mg a.i./L................................ 37 Wildlife Internationalf Ltd. /- Project Number 4 vtA .l 17 TABLE OF CONTENTS - Continued - APPENDICES Appendix I Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Wcek Period Immediately Preceding the T est............................ 38 Appendix II Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater................................. ....................................... 39 Appendix III The Analysis of Perflurobutane Sulfonate. Potassium Salt (PFBS) Concentrations in Freshwater and Bluegill Sunfish Tissue in Support of Wildlife International, Ltd. Project No.: 454A-117........................ ................................. 41 Appendix IV Temperature and pH o f Water in the Test Chambers..........................................................89 Appendix V Dissolved Oxygen of Water in the Test Chambers.................................... 90 Appendix VI Hardness, Alkalinity, Conductivity and TOC of Water in the Negative Control............. 92 Appendix VII Cumulative Percent Mortality and Treatment-Related Effects........................................... 93 Appendix V III Changes to Protocol............................................ 99 Appendix IX Personnel Involved in the Study....................................................................:...................100 Wildlife Internationalr Ltd - 8- Project Number 4 ^ A-117 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA .AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Ms. Susan A. Beach Wildlife International, Ltd. Easton, Maryland 21601 1 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: . 4 5 4 A -1 17 Perfluorobutane Sulfonate, Potassium Salt (PFBS) Pcrfluorobutanc Sulfonate, Potassium Salt (PFBS): A Flow Through Bioconccntration Test with the Bluegill {Lepatnis macrochirus) Negative Control, 0.50 and 5.0 mg a.i./L Negative Control, 0.53 and 5.2 mg a.i./L Experimental Start (OECD) - July 13,2000 Experimental Start (EPA) - July 18,2000 Biological Termination --August 3 1, 2000 Experimental Termination - September 13, 2000 44 Days (28-Dav Uptake, 16-Day Depuration) j TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: MEASUREMENTS OF 10 NEGATIVE CONTROL FISH COLLECTED AT TEST TERMINATION: WEIGHT (g): TOTAL LENGTH (m m ); Bluegill (Lepomis macrochirus) Osage Catfisheries, Inc. 1170 Nichols Road Osage Beach, Missouri 65065 Juveniles Mean = 1.81; Ranee = 1.37 to 2.76 Mean = 55; Ranee = 50 to 6 1 ; i RESULTS: (0.53 mg a.i./L) STEADY-STATE BCF: I RESULTS: (5.2 mg a.i./L) 1 STEADY-STATE BCF: Edible 0.21 Edible 0.16 Nonedible 0.51 Nonedible 0.43 Whole Fish 0.38 Whole Fish 0.30 j Wildlife Internationals l td -9- rojer.t Nnmher 454A -I17 INTRODUCTION A bluegill sunfish, Lzpomis macrochims, bioconcentration study was conducted for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from July 18,2000 to August 31,2000. Raw data generated by Wildlife International, Ltd. and a copy of the final report arc filed under Project Number 45 4A-117 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to determine the bioconcentration potential ofperfluorobutane sulfonate, potassium salt (PFBS) in the bluegill sunfish. EXPERIMENTAL DESIGN The bioconcentration test consisted of a 28-day uptake phase followed by a 16-day depuration phase. During the uptake phase, the test organisms were exposed in one of three groups: 1) A negative (dilution water) control; 2) A nominal concentration o f 0.50 mg active ingredient (a.i.)/L; or 3) A nominal concentrationo f 5.0 mg a.i./L. Test concentrations were based on the potassium salt o f pcrfluorobutane sulfonate (PFBS). At the start of the depuratio n phase, slock flow to Cite treated groups w as sto p p ed and the bluegill were exposed to dilution water without PFBS for the rem ainder of the test Each test chamber contained 85 bluegill at test initiation, and one replicate was tested for each treatment and the negative control. Water samples were collected on Day -2 (pre-test), Day -1(pre-test) on uptake Days 0 (0 and 4 hours), 1,3,7,14,21 and 28 and on depuration Days 1 ,3 ,7 ,1 0 and 14 during the test and analyzed for PFBS using liquid chromatography-mass spectrometry (LC/MS). Tissue samples were also collected at selected water sample collection periods during the test and analyzed for PFBS 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. Tissue samples were also collected on Day 16 of depuration. These samples wore not analyzed due to the pattern o f depuration observed. ,Wildlife International Y.td - 10. Project Number 4 SdA.] 1 7 ' MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "Perfluorobutane Sulfonate, Potassium Salt (PFBS). A Flow-Through Bioconcentration Test with the Bluegili (Lepomis macrochints)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1730 ( 1); ASTM Standard 022-84 Standard Practice fo r Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs (2); and OECD Guideline for Testing of Chemicals 305, Bioconcentrarion: Flow-Through Fish Test (3). Test Substance The test substance was received from 3M Corporation on March 27, 2000 and was assigned Wildlife International, Ltd. identification number 5216, The test substance, a white powder, was identified as Potassium Perfluorobutane Sulfonate, lot H2, TCR-00017-71. Information provided by the Sponsor indicated a purity of 97.90% and an expiration date of March 2010. A subsequent rev ision of the certificate of analysis indicated a purity of 97.3% and an Expiration/Reassessment Date o f January 17, 2002. The test substance was stored under ambient conditions. Preparation o f Test Solutions The nominal test concentrations were 0.50 and 5.0 mg a.i./L. Two stock solutions were prepared at concentrations of 500 and 5000 mg a.i./L. The appropriate amount o f test substance was weighed out and dissolved in dilution water for each stock. The stock solutions were stirred with an electric top-down mixer to aid in the solubilization o f the test substance. After mixing, the stock solutions appeared dear and colorless, Stock solutions were prepared at approximately weekly intervals during the uptake phase of the test. The stock solutions were injecced into the diluter mixing chambers (at a rate o f 0.350 mL/minute) where they were mixed with dilution water (at a rate o f 350 mL/minute) to achieve the desired test concentrations. All test concentrations were adjusted for the original reported purity of the active ingredient (potassium perfluorobutane sulfonate) in the test substance (97.9%). Wildlife International, Ltd. - 11 Ero.ieccNiimhw4S4A.li7 Test Organism The bluegill, Lepomis macrockirus, was selected as the test species for this study. The bluegill is one of the recommended freshwater fish species for use in bioconcentraticn tests (1,2,3). Bluegill used in the test were obtained from Osage Catfisheries, Osage Beach, Missouri. Identification of the species was verified by the supplier. The bluegill were held in Wildlife International, Ltd. well water for 20 days prior to testing. The bluegill were fed at least once daily during holding. The fish were acctimated to test conditions for approximately 48 hours prior to test initiation. During the holding and acclimation periods the fish showed no signs of disease or stress. During the 14-day holding period preceding the test, water temperatures ranged from 21.7 to 22.2C: The pH of the water ranged from 8,0 to 8.3 and dissolved oxygen ranged from 8.0 to 8.6 mg/L. Instrumentation used for water measurements arc described in the Environmental Conditions section o f this report. At test initiation, the bluegill were collected from the acclimation tank and indiscriminately distributed 1 to 2 at a time into the test chambers until each chamber contained 85 fish, During the holding period preceding the test the bluegill were fed flake food supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania and brine shrimp nauplii suppled by Summit Artemia, Ogden, Utah. Bluegill were not fed during the acclimation period, however, they were fed flake food once daily during the test. Feeding and sampling schedules were coordinated so that fish were sampled at least four hours after feeding. All fish used in the test were from the same source and year class, and the standard length of the 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 of 10 negative control fish measured at (he end of the test was 55 mm with a range o f 50 to 61 mm. The average wet weight (blotted dry) was 1.81 grams with a range o f 1.37 to 2.76 grams. leading was defined as the total wet weight offish per liter o f test water that passed through the test chamber in 24 hours, and was determined to be 0.31 g fish/L/day Test Apparatus A continuous-flow dilutcr was used to deliver each concentration o f the test substance and a negative control. A peristaltic pump (Cole*Parraer Instrument Company, Chicago, Illinois) was used to deliver the test substance stock solutions control into mixing chambers assigned to each PFBS treatment. The stock solutions Wildlife InternationalTLtd. - 12- Project Number 454A-1!7 were mixed with dilution water in the mixing chambers prior to deliver)' to the test chambers. The flow of dilution water to the test chambers was controlled by rotameters. The delivery of water from the rotameters was checked prior to the test and at approximately weekly intervals thereafter. Approximately 6.3 volume additions of test water were delivered to the test chambers every 24 hours. The general operation of the dilutcr was checked at least two times a day during the test. Test chambers were 106-L stainless steel aquaria filled with approximately 80 L o f test solution. The depth o f the test water in a representative chamber was approximately 19 cm. Test chambers were indiscriminately positioned in a temperature-controlled water bath designed to maintain a constant temperature. The water bath was enclosed in a plexiglass ventilation hood in order to minimize any potential for cross contamination. Test chambers were siphoned daily and periodically cleaned during the test to remove excess feed, fecal matter, algae and bacterial growth. Test chambers were identified by the project number and test concentration. Dilution Water The water used for holding and testing was freshwater obtained from a well approximately 45 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as modcratcly-hard water. The specific conductance, hardness, alkalinity and pH measurements of the well water during the four-week period immediately preceding the test arc presented in Appendix I. The well water was passed through a sand filter to remove particles greater than approximately 25 fun, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water again was filtered (0.45 pm) to remove microorganisms and particles. The results o fperiodic analyses performed to measure the concentrations o f selected contaminants in well water used by Wildlife International, Ltd. are presented in Appendix II. Environmental Conditions The target temperature range for the test was 22 1C. Temperature was recorded continuously in the negative control with a Fulscope ER/C Recorder {1900 J Series mode! no. A). Temperature was also measured n ail test chambers at the beginning and end of the test and at weekly intervals during the test, with a liquid-in-gl ass thermometer. ,Wildlife International ltd -13- Projert Number 17 Dissolved oxygen was measured with a Yellow Springs Instrument Company, Inc. Model 5 IB dissolved oxygen meter. Measurements were made daily in each test chamber during the test, Measurements o f pH were mace in each test chamber at the beginning and end of the test and at weekly intervals during the test using a Fisher Accumet Model 915 pH meter. Hardness, alkalinity, conductivity and total organic carbon (TOC) were measured in the negative control at the beginning and end of the test, and at weekly intervals during the test Hardness and alkalinity were measured by titration based on procedures in Standard Methods fo r the Examination o f Water and Wastewater (4) Conductivity was measured using a Yellow Springs Instrument Company, Inc. Model 33 Salinity-Conductivity Temperature meter. Total organic carbon was measured using a Shmadzu model TOC-5000 total organic carton analyzer. Ambient room light was used to illuminate the test systems. Fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50) were controlled by an automatic timer to provide a photoperiod of 16 hours o f light and 8 hours of darkness. A 30-minute transition period o f low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Observations All fish w ere observed once each day to evaluate the num ber o f mortalities and the num ber o f individuals exhibiting signs o f abnormal behavior. Procedures for Exposure o f Fish to PFBS The test chambers were conditioned by delivering PFBS to the diluter system for approximately 5 days before adding the fish. Water samples were collected twice 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. At the end of the pre-test period, the uptake phase of the test was initiated on luly 18,2000 by placing the fish in the test chambers. Bluegill were impartially removed from the holding tank in groups of 1 to 2. The groups o f bluegill distributed among the test chambers until each test chamber contained 85 fish. The duration of .Wildlife International ltd -14- Project Number 4MA-1 17 the uptake phase was 28 days. At the end of the uptake phase, stock flow to the treatment groups was stopped and the bluegifl were exposed to dilution water without PFBS for a period of 16 days. Collection and Analysis o f Water Samples Water samples were collected on Days 0(0 hours and 4 hours), 1,3,7, 14,21 and 28 of the uptake phase. Water samples were also collected on Days 1,3,7, lOand 14 o f the depuration phase. At each water samphng interval, two water samples were collected from the negative control and three samples were collected from each ot the two PFBS treatment groups. One negative control sample and two samples from each o f the PFBS treatment groups were analyzed for PFBS. The remaining samples were held in reserve as backup samples. All water samples were collected from mid-depth of each test chamber using a glass pipette. The water samples were analyzed for PFBS by liquid chromatography-mass spectrometry (LC/MS). Procedures for analysis of the water samples are provided in Appendix III. Water samples were analyzed as soon as possible after collection without storage. Collection and Analysis of Tissue Samples Tissue samples were collected on Days 0 (4 hours), 1, 3, 7, 14, 2 1 and 28 of the uptake phase. Tissue samples were also collected on Days 1,3 ,7 ,10 and 14 of the depuration phase, At each tissue sampling interval, a sufficient number o f fish were collected to provide two replicate samples o f negative control fish and four replicate samples of each PFBS treatment group. Fish were impartially removed from the test chambers and euthanized by severing the spinal cord above the opercular region. The fish were bloctcd dry and m easured for total length and wet weight within approximately 15 minutes o f collection, when possible. Each fish was then rinsed with dilution water, blotted dry again and dissected into edible and nonedible tissue fractions. Dissection was accomplished by making an incision fromjust posterior to the base of the pectoral fin dorsally through the spinal cord. The head, fins and viscera were removed from the body and were considered to be nonedible tissue. The remaining tissue was considered the edible tissue. Tissue samples were transferred to tared scintillation vials and weighed. Procedures for extraction and analysis of the tissue samples are provided in Appendix 111. AH tissue samples were extracted immediately or stored at approximately -14C until extraction. .Wildlife Internationalt Ltd - 15 - Prnjftrt Number 4S4A-T |7 Tissue Lipid Content Selected fish were collected to determine lipid content (Appendix ill). The determination ofpercent lipids provides the potential to express BCF values in terms of lipid content. Fish were sampled on Day 0 of uptake, on Day 28 o f uptake and on Day 14 o f depuration. All fish collected for lipid content were stored at approximated -14C until analysis. D ata Analysis Whole fish concentrations were calculated based on the sum o f the edible and nonedible parts. The steady- state bioconccntration factor (BCF) values were determined from the tissue concentrations at apparent steadystate divided by the average water concentration. Tissue concentrations were considered to be at apparent steadystate if three or more consecutive sets o f tissue concentrations were not significantly different (p > 0.05). During this determination, tissue concentrations <LOQ were eliminated from the means. Tissue concentrations were evaluated for normality and homogeneity o f variance using the Shapiro-Wilk's test and Bartlett's test, respectively. If the data did not meet the assumptions, the data was transformed in an attempt to correct the data.. Mean tissue concentrations were then compared using analysis o f variance and the appropriate multiple comparison test (i.e., Dunnctt's test or the Bonferroni t-test). The kinetic bioconcentration factor (BCFK), uptake rate (K,) and depuration rate (K2) were calculated for edible, nonediblc and whole fish using BIOFAC computer software (5). BIOFAC is a nonlinear parameter estim ation routine which estim ates rate constants from a set of sequential time-concentration data. These rate constants are then used to calculate a BCFK (BCFK = Ki/Kj). Tissue concentrations less than the limit of quantitation (LOQ) were entered into the BIOFAC model as 0,001 mg a.i./Kg. This concentration was arbitrarily selected as a concentration close to zero (the BIOFAC model would not run with zeros). RESULTS AND DISCUSSION Water Chemistry Means and ranges of temperature, dissolved oxygen and pH o f the water in the test chambers are presented in Table 1. The individual measurements are given in Appendicies IV and V. Water temperatures in the test chambers were within the temperature range o f 22+ 1C established for the test, Dissolved oxygen concentrations remained 7.4 mg/L (85 percent of saturation) throughout the test. Measurements of pH ranged from 8.0 to 8.5. Wildlife Internationalr T.td - 16 mifxl Ntimher 45dA.M 7 Weekly measurements of hardness, alkalinity, conductivity and total organic carbon remained consistent throughout the test (Table 1 and Appendix VI). Observations of Mortality and Clinical Siena Observations of mortality and clinical signs are presented in Appendix VII. Bluegili in the negative control appeared normal and healthy throughout the test. One bluegili died in the 5.0 mg a.i./L (nominal) treatment group on Day 23 of the uptake phase o f the test. All other fish in the test appeared normal with no treatment-related signs o f toxicity'. Concentrations o f PFBS in Water Concentrations of PFBS in the negative control were <LOQ (0.125 mg a.i./L) (Tables 2 and 3) Measured concentrations ot PFBS duri ng the uptake phase in the 0.50 mg a.i./L treatment group ranged from 96 to 119% of the nominal test concentration. When concentrations measured during the uptake phase were averaged, the mean measured concentration was 0.53 mg a.i./L which represented 106% o f the nominal test concentration. Measured concentrations o f PFBS during the uptake phase in the 5.0 mg a.i./L treatment group ranged from 101 to 109%of the nominal test concentration. When concentrations measured during the uptake phase were averaged, the mean measured concentration was 5.2 rag a J /L which represented 104% of the nominal test concentration. Concentrations of PFBS during the depuration phase were all <LOQ. Concentrations o f PFBS in Fish Tissue* The majority o f the negative control tissue samples contained no quantifiable PFBS concentrations (Appendix III). However, three edible and four nonedible negative control tissue samples contained measurable PFBS. PFBS was not detected b the negative control water during the test. Consequently, the residues measured were most likely due to contamination during extraction or analysis, it should be noted that the study required the measurement of PFBS at low levels near the LOQ m a complex matrix. The concentrations o f PFBS in tissues of fish exposed to 0.53 mg a.i./L are presented in Table 4. PFBS concentrations in edible and nonedible tissues appeared to reach steady-state at Day 7 (Figures 1-3). Tissue concentrations from uptake days 7,14,21 and28 were not significantly different (p >0.05). The mean measured tissue concentrations during this period o f time were 0.113,0.272 and 0.203 mg a.i./Kg for edible, nonedible and whole fish, respectively. Steady-state BCF values ranged from 0.113 b edible tissue to 0.272 in nonedible tissue Wildlife international T.td. - 17- roierrNMrnhpx<md-| \ i (Table 5). BIOFAC estimates o f the time to reach 90% of steady state in edible tissue, nonediblc tissue and whole fish were 6.9,9.5 and 4.2 days, respectively (Table 6). During the depuration phase of the test, PFBS was eliminated rapidly with estimates of time to reach 50% clearance of 2.1, 2.9 and 1.3 days for edible tissue, nonedible tissue and whole fish, respectively. It should be noted that some of the highest tissue residues were measured on Day 1 o f depuration. In addition, the BCFK for edible tissue and whole fish appeared to be overestimated by the BIOFAC model as shown in Figures 1 and 3. Tne concentrations of PFBS in tissues o f fish exposed to 5.2 mg a.i./L are presented in Table 7. PFBS concentrations in edible, nonedibie and who le fish tissues appeared to reach steady-state at Day 3 (Figures 4 - 6) D um ctt's test showed that PFBS concentrations for uptake Days 3 - 28 were not .significantly different (p > 0.05). The mean measured tissue concentrations during this period o f time were 0.829,2.24 and 1.57 mg a i /K" for edible, nonedible and whole fish, respectively. Steady-state BCF values ranged from 0.16 in edible tissue to 0.43 in nonediblc tissue (Table 8). BIOFAC estimates of the time to reach 90% o f steady state in edible tissue, nonedible tissue and whole fish were 6.5,7.1 and 7.0 days, respectively (Table 9). During the depuration phase o f the test, PFBS was eliminated rapidly with estimates of time to reach 50% clearance of 1.9,2 .1and 2 .1davs for edible tissue, nonedible tissue and whole fish, respectively. CONCLUSIONS Perfluorobutane sulfonate (PFB S) residues rapidly achieved steady-slate in the tissues o f biuegiil sunfish (Lepomis macrochirus). Steady-state tissue concentrations were achieved after only 3 to 7 days o f exposure Although steady-state was achieved rapidly, PFBS did not bioconcentrate in biuegiil. Steady-state BCF values ranged from 0.16 to 0.51, indicating that tissue concentrations never reached exposure concentrations. During depuration, PFBS was eliminated rapidly. The BIOFAC estimates for time to reach 50% clearance ranged from 1.'3 to 2.9 days. Wildlife international. Ltd. - 18- Project Number 454A-I17 REFERENCES . 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft OPPTS Number 850.1730: Fish BCF. 1 J" 2 ASTM Standard E l 022-84. 1988. Standard Practice fo r Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs. American Society for Testing and Materials. .3 OECD Guideline for Testing of Chemicals 305. 1996. Bioconcentration: Flow-Through Fish Test. 4 APHA, AWWA, VVPCF. 1985. Standard Methodsfo r the Examination o f Water and Wastewater 16th Edition, American Public Health Association. American Water Works Association Water Pollution Control Federation, New York. ' 5 BIOFAC. 1991. September 19, 1991 version. The Dow Chemical Company, Midland, Michigan. ,Wildlife international Yfd 19- p rf-jpr:t Number dSdxty Table X Means and Ranges o f Water Quality Parameters Sponsor: Test Substance: Test Organism: Dilution Water: Uptake Phase Nominal Concentration (mg 0.L/L) 3M Corporation PFBS Bluegiil, Lepomti macrochims Well Water Temperature1 CC) 22.0 21.9-22.1 DO2 (mgrtO 8.2 7.4 - 8.7 PH 8.1 8.0-8.4 Conductivity Cumhos/cm) 324 300 - 330 Alkalinity (mg/L as CaCOj) 176 164 - 184 Hardness (mg/L as CaCOj) 119 104-128 TOC (mg C L ) <1 <! - <) 0.50 22.0 8.2 8.1 21.9-22.1 7.4-8.8 8.0-8.5 5.0 22.0 8 2 8.1 21.9-22.1 7 .4 -8 .8 8.0 -8 .5 j Temperature measured continuously in the negative control remained at approximately 22.0C. At a temperature of 22 C. toe dissolved oxygen saturation concentration is 8.7 mg/L and 60% saturation is 5.2 mg/L - 20Table 2 Project Number 454A-117 Concentrations o f PFBS in W ater Samples During die Uptake Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Bl uegill, Lepomis macrochirus Well Water ,, Nomlml Concentration (mg _____ 0 (0 Hour) 0 (4 Hours) 1 Negative Control <L O Q ' <LOQ <I0Q 0S0 0.515 0.5111 0.478 0.518 0.525 0.490 _" Day o f Study (mg a.L/L) 3 ______ 7 ________ 14_______ 21 <LOQ < I.O Q <LOQ <J.OQ 0 542 0 542 0.525 0 559 0.519 0.514 0.593 0.567 = -- ===== 28 <LOQ 0.515 0.516 Mean Measured Concentiatiou Percent of Nomina] <LOQ 0.S3 106 5 0 5.24 5.35 5.02 5.08 5.17 5.36 5.04 5.45 5 2 104 542 5.43 . 5.12 5.12 5.22 5 32 5.21 5.40 'The Limit o f Quantitation (l.OQ) was 0.125 mg ad 7b. O t0o> Wildlife Internationalf Ltd. -21 - Project Number 454A-117 Table 3 Concentrations o f PFBS in Water During the Depuration Phase Sponsor Test Substance: Test Organism: Dilution Water: Uptake Phase Mean Measured Concentration (mg a.i./L) 3M Corporation PFBS Bluegill, Lepomis m acrochim t Well Water 13 Negative Control ' <LOQ' <L0Q 0.53 <L0Q <LOQ <LOQ <LOQ 52 <LOQ <LOQ <LOQ <L0Q 1The Limit of Quantitation (LOQ) was 0.125 mg a.i./L. Day of Study (mg a.i./L) 7 <LOQ <LOQ <LOQ <LOQ <LOQ 10 <LOQ <LOQ <LOQ <LOQ <L0Q O 'e r OVj o_Vt 14 <LOQ <LOQ <LOQ -22- Table 4 PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues ofBluegill Exposed to 0.53 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution W ater JM Corporation PFBS Bluegill, Lfpom is macrochirus Well Water Sample ID E4/N4 E5/N5 E6/N6 E7/N7 Uptake Day 0 (4 hours) 0 (4 hours) 0 (4 hours) 0 (4 hours) E dita Tissue Concentration (inga.i./Kg) <fl053O3 <0.0585 <0.0393 <010590 Edible Tissue Weight - - (6) 0.9446 0.8512 1.2723 0.8483 E17/N17 1 00382 1.4848 E18/N18 1 00428 1.1682 E19/N19 1 00915 0.5462 E20/N20 1 <0.0650 0.7672 E3Q/N30 E31/N31 E32/N32 E33/N33 3 3 3 3 0.0874 00636 00911 0.0704 0.9636 2.0468 0.6166 0.9082 K43/N43 E44/N44 E45/N45 K46/N4G 7 7 7 7 0.116 0.136 0101 0.133 . 1.2395 0.7690 1.2201 1.0375 E56/N56 14 <0.0880 0.5688 E57/N57 14 <0.0715 0.6971 E58/N58 14 0.0869 1 2892 E59/N59 14 0.105 0 5636 1Whole Fish Conc-mlration = ( g d M e M J i edib!e.cancj J J a m e d ih le wt. X n o n ^ ib le cone.) (edible \vl + nonedible wt.) 2Less than values cone spend to the limit of quantitation (See Appendix ITT). Nonedible Tissue Concentration (mg a.i /Kg) <0.06.30 <0.0729 <0.0439 <0.0743 0.0786 0.0894 0.112 0.0730 0.180 0.138 0.173 0.141 0.265 0 293 0.263 0.307 0 235 0.181 0.236 0.179 Nonedible Tissue Weight (8) 1.2556 10863 1.8064 i 0570 1 6591 1 5313 0 7838 1 1590 1.2489 2 1180 0.8193 1 3600 10629 0 6806 0.9194 1 0075 0 8077 1.1184 1.6895 1.0733 Prfijecl Number 454A-117 Whole Fish Concentration1 (mg a.iVlCg) <LOQ <LOQ <LOQ vl.OQ 0.0595 <LOQ <L.OQ <LOQ 0.140 0.101 0.1 38 0.113 0.185 0.210 0 171 0 219 <LOQ <LOQ 0.171 0.154 p <oo0 - 23 - Table 4 (Continued) PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues o f Bluegill Exposed to 0.53 mg a.i./L Sponsor: Test Substance', l est Organism: Dilution Water: 3M Corporation PFBS Bluegill, Lepomir mocrochirus Well Water Sample ID E69/N69 E70/N70 E71/N71 E72/N72 Uptake Day 21 21 21 21 Edible Tissue Concentration (mg u VKr) 0.136 0.125 0.213 0.114 Edible Tissue Weight . ..(B) 1.4931 1.2111 0.9709 0.9750 E82/N82 E83/N83 E84/N84 E85/N85 28 28 28 28 0.0682 0.103 00619 0.0871 09166 1 3823 0.9593 0 8614 'Whole Fish Concenation = foliN c wi. X edible cone.) fnoncdible wt. X noncdiblc ernie.) (edible vM. + nonedible wt.) Nonediblc Tissue Concentration (mi a.iTKg) 0.341 0.314 0.579 0.323 0.185 0.262 0 153 0.243 Nonedihle Tissue Weight (B) 1.6291 1 5265 1 2477 1.1193 1.2116 1 7309 1 4567 1 1446 Project Number 454A -117 Whole Fish Concentration1 (mg a.i/Kg) 0.243 0.230 0.419 0.226 0.135 0 19] 0.117 0.176 p. 99 -24- Table 4 (Continued) PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues oT Rluegill Exposed to 0.53 mg a.i./L Sponsor; Test Substance; Test Organism: Dilution Water: 3M Coqioration PFBS Bluegill, Lepamis ntacrochirus Well Water Sample ID E95/N95 E96/N96 E97/N97 E98/N98 Depuration Day 1 1 1 1 Edible Tissue Concentration (m gii./K g) 1.19 3.31 0.209 3.98 Edible Tissue Weight <e) 0.9400 0.8289 0.8374 0.7705 E108/N108 K109/N109 HI 10/Nl 10 Elll/N TN 3 3 3 3 00758 0.0571 <0.0341J <0.0488 1.2572 1.2397 1.4670 l .0255 E121/N121 E122/N122 E123/N123 K124/N124 7 7 7 7 <0.0401 <0.0476 <0.0414 <00915 1.2473 1.0515 1.2077 0.5453 E134/N134 E135/NI35 E136/N136 E137/N137 10 10 10 10 <0.0459 0108 <0.0580 <0.0595 1.0910 1.0374 0.8632 0.8421 EM7/N147 E148/N148 E149/N149 E 1 5 0 /N 1 50 14 14 14 14 <0.0499 <00520 <0.0630 <0.0364 1.0021 0.9644 0.7932 1.3735 lWhole Fish Concentration = idibje tyt X edible cg n c J U . j ^ n e d i b l ^ J l i i oneJililecone.) (edible 1. + nonedible wt.)* *Less than values correspond to the limit o f quantitation (Set Appendix HI). Nonedible Tissue Concentration (rag a.i /Kg) 0.207 1.15 0.537 6.31 0.156 0.145 <0.0457 0.0934 0.0920 0 .0 5 1 5 <0.0562 <0.0982 <0 0634 <0.0620 <0.0645 <00755 0.0796 <0 0543 <0.080S <0 0490 Nonediblc Tissue Weight (g) 1 4752 1.3630 1 3587 I 1058 I.S573 1.5719 1 7321 1 3102 1.4536 I 5374 1.4110 0.8047 1.2482 1.2777 1.2273 1.0490 1.3124 1.4587 0.9847 1.6148 I'rojecl Number 454A-117 Whole Fish Concentration1 (mg a. ./Kg) 0 590 1.97 0.412 5.35 0.120 0.106 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ p. 101 Wildlife International, Ltd. Project Number 454A -117 . -25- Table 5 Steady-State BCF Values for Bluegill Exposed to 0.53 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Bluegill, Lepomis macrochirut Well Water Tissue Type Mean Measured Test Concentration (mg a.i./'L) Uptake Days at Steady-State Edible 0.53 7, 14, 21,28 Nonedible ' 0,53 7, 14,21,28 Whole Fish 0.53 7.14, 21,28 Mean Measured Steady-State Tissue Cuucenlration (mg ai/K g ) 0.113 0.272 0.203 Steady-State BCF 0.21 051 0 38 p. 102 Wildlife International, Ltd. -26- Project Number 454A-117 Table 6 BIOFAC Model Estimates for Blucgiil Exposed to 0.53 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: Tissue Type Edible 3M Corporation PFBS Bluegill, Lepomit mucrochirus Well Water Kinetic Bioconcentration Factor (DCFK) Uptake Rate Constant { ^ .L -K g '-D a y ') 0.73 0.24 Depuration Rate Constant (k2, Day'1) 0.33 Nonedible 0.86 0.21 0,24 Whole Fish 1.1 0.60 ' ' 0.54 Estimate Time to Reach 90% Steady State (Days) 6.9 9.6 4.2 Estimated Time to Reuch 50% Clearance (Days) 2.1 2.9 ; .3 -27 - Table 7 PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 5.2 mg a.i /L Sponsor: 3M Corporation 't est Suh.stanc.e-. PFBS lest Organism: Bluegill, Lepornis macrochirus Dilution Water: Well Water___________________________ ____________ ____________________________ Sample ID E9/N9 E10/N10 E11/N1I E12/N12 Uptake Day 0 (4 hours) 0 (4 Injurs) 0 (4 hours) (4 hours) Edible Tissue Concaitration (mg i.i./Kg) 0.127 0.0975 0.0811 0127 Edible Tissue Weigh! (E) 0.8480 0.8819 1.1497 0.6880 Nonedible Tissue Concentration (mg u.i /Kg) 0.233 0.173 0.178 0.247 E22/N22 E23/N23 E24/N24 E26/N26 1 1 1 1 0.240 0273 0.311 0234 0.8780 0.9052 0.5573 0.8420 0.726 0.730 0.702 0.598 E35/N35 E36/NT36 E37/N37 E38/N38 3 3 3 3 0.675 0487 0611 0.448 1.2035 0.8812 1.4409 1.4412 1.49 1.35 2.08 1 29 E48/N48 E49/N49 F.SO/N50 E51/N51 7 7 7 7 1.07 1.6347 3.10 1.19 1.6604 2 95 0.701 1.4695 2.04 1.50 0.9706 3.68 E61/N61 E62/N62 E63/N63 E64/NS4 14 14 14 14 0.438 1.12 115 (1.748 0.6935 0.9984 0.7593 2.1590 `Whole Fish Conce,,nation - (gdjlile wt, X edible Cone.) t j n onedible Wt. X WOcdible cone.) ______ _ (edible u t t- nonedible wt.)_______________ 0.S60 3.28 3.42 2.22 Noncdiblc Tissue Weight (E) 1.0907 1.2291 1.3879 0.9672 1.2179 1.1803 08573 1 3474 1.2976 1.1688 1.4651 1.4124 1 1526 1.2130 t .2203 0.8058 1.2983 1.2129 1 0165 2.3433 Project Number 15-1-117 Whole Fish Concentration1 (mga.iVKg) 0.187 0.141 0.134 0.197 0.525 0.532 0.548 0.458 1.10 0.979 1.35 0.865 1.91 1 93 1.31 2.49 0.778 2 31 2.45 1.51 p. 103 -28- Table 7 (Continued) PFBS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 5.2 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water, Sample ID K74/N74 U7S/N75 E76/N76 E77/N77 3M Corporation PFBS Rluegill, l.epomi.r m acm chinis Well W ater Uptake Day Edible Tissue Concentration (mga.i./Kg) 21 0.596 21 0.593 21 1.50 21 0.590 E87/N87 E88/N88 E89/N89 E 9 0 /N 9 0 28 28 28 28 0764 0.907 0.565 0,837 Edible Tissue Weight . ..... -(6)....... 1 0974 1 1861 1.1840 1.3253 1 1961 0.7393 0.9953 0.8194 Nonedible Tissue Concentration (mg a.i./Kg) 1.56 1.66 4.06 1.64 1.66 2 02 205 2.22 Nonedible Tissue Weigh! (8) 1 3836 1 6043 1.4418 1.6331 1.4016 l 1143 1.3411 1.2026 'W hole Fish Concentration _______________ ______ + (n pnedibie.wt. X n o n ed .b leoorc.) (edible \vt + nonedible wt.) ProjcctNumber454A-l 17 Whole Fish Concentration1 (mg a.i./Kg) 1 13 1 21 2 91 117 1.25 1.58 1.46 1.66 p. 104 - 29- Table 7 (Continued) Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E100/N100 E101/N101 E102/N102 E103/N103 PFBS Concentrations in Edible, Noncdible and Whole Fish Tissues of Bluegill Exposed to 5.2 mg a.i./L 1M Corporation PFBS Bluegill, Lepomis m acrochins Well Water Depuration Day 1 1 1 1 Edible Tissue Concent ration - (mg a i /Kg) 0718 0.522 1.05 0.467 Edible Tissue Weight (g) 1 1689 0.8329 0 8862 0.5526 Nonedible Tissue Concentration (mg a.i./Kg) 2.09 2.32 3.29 1.17 Nonedible Tissue Weight (fi) 1 4841 1 1449 1.1917 1.0147 E l 13/N113 E1I4/N114 E l 15/N115 E l 16/N116 3 3 3 3 0235 0197 0.601 0.413 1.0013 0.8749 1.5729 1.1804 0676 0.529 1.57 1.36 1 4174 1.2461 l.8050 l 6609 E126/N126 E127/N127 E128/N128 E129/N129 7 7 7 7 0.196 0.151 0.124 <0.087 93 1.2959 0.9239 0.9014 0.5686 0691 0.530 0.330 <0.0808 1.5717 1.0888 1 0053 0 9801 E139/N139 E140/N140 E141/N141 E142/N142 10 10 10 10 0.0791 <00740 0.0815 0.0930 1.0421 0.6779 0.6133 0 5384 0.181 <0.0800 <0.0887 <0.0903 1.1765 0.9876 0.8965 0 8802 . C1527N152 E153/N153 E I54/N I54 E155/N 155 14 14 14 14 0.0410 00454 0.0685 00705 1.2193 1.1015 0.7300 0 7080 `Whole Kish Concentration = (edible wt X edible cone.) f ,,(npnedible wt. X nonedible cone.) (edible tvt. + nonedible wt.) 3 I.ess than values correspond to the limit of quantitation (Se* Appendix IE). <0.0494 <0.0502 0 .0 7 7 3 <0.0847 1 6027 1.3159 1.0248 0.9371 Project Number 454A-117 Whole Fish Concentration1 (mg a.i./Kg) 1.49 1.61 2.34 0.922 0.493 0.392 1.12 0.967 0.467 0.356 0.233 <l.OQ 0.133 <LOQ <LOQ <LOQ <LOQ <I.OQ <LOQ <LOQ p. 105 p. 106 Wildlife International, Ltd. - 30 - Project Number 454A-1 i? Tabic 8 Steady-State BCF Values for Bluegill-Exposed to 5.2 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Bluegill, Lipomis macrochirus Well Water Tissue Type Edible Nonedible Whole Fish Mean Measured Test Concentration {mg a.i./L) . 5.2 Uptake Days at Steady-State 3,7, 14,21,28 5.2 3 ,7 , 14,21,28 5.2 3 ,7 ,1 4 ,2 1 ,2 8 Mean Measured Steady-State Tissue Concentration (mgn.i./Kg) 0.829 2.24 1.57 Steady-State BCF 0.16 0.43 0.30 p. 107 Wildlife International, Ltd. -31 - Project Number 4J4 -117 Table 9 BIOFAC Model Estimates for Biucgill Exposed to 5.2 mg a.i./L sponsor: Test Substance: Test Organism: Dilution Water: 3M uorporatiou PFBS Bluegill, Ltpom is macrocntrus Well Water Tissue Type Kinetic Bioconcentration Factor (BCFK) Uptake Rate Constant (kuL .K g-'.D a y 1) Depuration Rate Constant (Vcj.Day') Estimate Time to Reach 90% Steady State (Days) Edible o .is 0.066 0.36 6.5 Nonedible 0.50 0.16 0.32 7.1 Whole Fish 0.36 0.12 0.33 7.0 Estimated Time to Reach 50% Clearance (Days) 1.9 2.1 2.1 p. 108 Wildlife International, Ltd. -32- Projeci Number 454A-117 Figure 1. Concentrations o f PFBS in Edible Fish Tissues of Bluegill Exposed to 0.53 mg a.i./L. p. 109 Wildlife International, Ltd. -33 - Project Number 454A-1] 7 Figure 2, Concentrations of PFBS in Nonediblc F ist Tissues o f Blucgill Exposed to 0.5 3 mg a.i./L. ^ 5Wft--117 0.53 M.0 a . i . /L None d ' b Is T i s s u e p. 110 ,Wildlife International Ltd. -34- Project Number 454A -117 Figure 3. Concentrations o f PFBS in Whole Fish Tissues o f Bluegill Exposed to 0.53 mg a.i./L. " o 1 S U 0 - 1 1 7 0 . 5 3 m3 . i . / L Who I F l K p. 111 Wildlife International, Ltd. . . 35. Project Number 454A -] 17 Figure 4. Concentrations o f PFBS in Edible Fish Tissues of Bluegill Exposed to 5.2 mg a.i./L. UEUH-117 5.2 *g a . r. /L Edible- T i s s u e p. 112 .Wildlife International, Ltd -36- Preject Number 454A-117 Figure 5. Concentrations o f PFBS in Noncdiblc Fish Tissues o f Bluegill Exposed to 5.2 mg a.i./l. UpUp--11 7 S , ^ a . , y L Mon * d i b> I o T i * * u I./Kg p. 113 Wildlife International, Ltd.________ p^Numbc^A-n- - 37 Figure 6. Concentrations o f PFBS in Whole Fish Tissues of Bluegill Exposed to 5.2 mg a.i./L. 4540** 1 1 / 5 . 2 mg a . I . / L Who I o F f --1-------------- '-------------- -------------- v------------- v------------- 1--------- o ! <D--------- -________ i 0-0 6 - 12.0 18-0 2 4 .0 3 0 . 0 3G. 0 42.0 HB.0 Dog p. 114 Wildlife International, Ltd. -38 Project Number 454A-117 APPENDIX I Specific Conductance. Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Bluegill, Lepamis macrochirus Well Water Specific Conductance (mnhos/cm) Mean 320 (N = 4) Range 315-325 Hardness (mg/1- as CaCOj) 130 (N = 4) 128-132 Alkalinity (mg/L as CaCCb) 172 ( N - 4) 172 - 172 pH 8.1 ( N - 4 ) 8.0 - 8 . 2 p. 115 ,Wildlife International Ltd, -39- Project Number 454A-117 APPENDDCII Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Measured Concentration Component Measured Ccncer.tration Pesticides md Organics Aclonifcn Alnchlor Amctryn Atman Azinphos-ethyl Azinpho-methyl Azoxystradin Bifenclirin Biuallcthnn Bitertanol Bromacil Bromophos Bromopbos-elhyl Broompropy'aal Dupirimaat Carbary! Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyriphos. CMorpyriphos-nrethyi Chlorthalonil Coumnphos Cyanazin Cyfluthrin Cypermethrin Cyproconazole Dellamethrin Demeaon Demeton-o Desethylntrazin Desisopropylatrazin DcMnctryn Diaanon Dichiobcnil Dichloran Diuhlorbenzamide Dichlorfcnthion Dichiortluanid <0.03 pgL <0.01 pgL <0.01 pgL <0.01 pgL <0.04 ugL <0.08 pgL <0.25 pg/L <0.05 ug/L <0.05 pg/L 0 .0 5 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L 0 .0 2 pg/L <0.05 ug/L <0.05 ug/L 0 .0 3 pg/L <0.02 pg/L 0 ,0 2 pg/L <0,05 pgL 0 .0 2 pg/L <0.01 pg/L <0.01 pg/L <0 04 pg/L 0 .0 2 pg/L <0.05 pg/L <0 05 pg/L 0.25 pgL <0.05 pgL <0.02 pgL " 0.02 pgL 0.02 pgL <0.01 pgL <0.02 pg/L <0,01 pgL 0.01 pgL <0.01 pgL 0 .0 3 pg/L 0.02 pgL 0.01 pgL 0.03 usL Dimcthomorf Disulfaton DMST Dwlcmurf Hr.cosulfatwx Endojulfan- Endosuln-sulfaat Epoxicnnszole Eptam Esfenvaleraat Ethion Ethofumesaat Elhoprophos Etridiaiols Etrimphos Fenarimol Fenehlorphos Fenitrothion Fcnoxycarb Fenpiclonil Fenpropathrin Fenpropimnrf Ftmthion FenvaJcraet Fluaaifop-butyl Fluoroglycofcn-ciyl Fluroxypyr-meptyl Mutolanil Fonophos Furalaxyi Hcptcnophos Imazalil tprodinn Krcsoxim-mcKiyl Lcnacil Lindane Malathion Metalaxyl Mctamitron Metazachlor Methidathion <0.05 pgL <0.02 pgL <0.05 pgL 0.01 pgL <0.0! pgL <0.01 ugL <0.02 pgL <0.05 prL <0.02 pg/t. 0.02 pgL <0.05 ugL <0.02 ug/L <0.01 ig/L 0.02 pgL <0.05 pgL <0.05 pgL <0.01 pgL <0.03 ^ig/L <0.03 pgL <0.05 pgL 0.25 pgL <0.01 pgL <0.01 pgL <0.02 .ug/L, 0.02 pgL <0.02 pgL <0.05 pgL <0.02 pg/L <0.01 pgL <0.02 pgL <C,Q2 ug/L 0.01 PgL 0.05 pgL 0.02 pgL 0.05 pgL 0.02 pgL 0.02 pgL <0.05 pgL <0.05 pgL <0.02 pg/L <0.02 pgL 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October Id and 15,1999. p. 116 .Wildlife International, Ltd -40- Project Number 454A-1 ] 7 Appendix 11 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Pesticides And Organics (Page 2) Component Measured Concentration Component Measured Concentration Dichlorvos Dicofol Diethyltoluamide Difenoconazoie Dimethoate Padohutazule Parathicn Parothion-methyl Penconazole Pcndimethalir. Pcrmethrin-cis Permethrin-trans Phosalon Phownet Phosphamid on-cis Pirimicorb Pirimiphos-ethyl Pirimiphos-methyi Proehlotaz Fracymidon Promctryn Propachlor Propazin Propham ?ropicona20o| Propoxur Propyzatnide Prosulfocarb Pyrazoohos <0.01 pg/L <0.25 pg/L <0.02 pg/L 0 .0 3 pg/L 0 .0 2 pg/L 0 .0 5 pg/L 0 .0 1 pg/L <0.01 pg/L 0 .0 5 pgIL 0.03 pgL 0 .0 1 pg/L <0.01 pg/L 0 .0 5 fig/L 0 .0 2 pg/L 0 .0 5 pg/L 0 . 0 1 pg/L <0.01 pg/L <0.01 pg/L <0.02 Hgda <0.01 pg/L 0 .0 1 pg/L <0.01 pg/L <0.01 pg/L 0 .0 2 pg/L <0.05 pg/L -'0.03 Hg/L <0 02 pg/L 0 ,0 2 pg/L 0 .0 3 pg/L Metboxychlor Metoladilur Mctribu^in Mevinphos Nltrothal-Isopropyl Pyrifennx-1 Pyrifenox-2 Pyrimethanil Quizalofop-etliyl Simazin Sulfotep Tcbuconazolc Tcbufenpyrad Terbutryn Terbutyazin Tdrauhlorvinphos Tctrahydroftaalimidc Tetramethrin Thi&bervdazole Thiometon Tolclophos-methyl Tolytfluanid Triadimefon Triadimenol Triallflat Triazophos TrtHurulin Vamidothion Vinchlozolin <0.01 pg/L <0.01 ,ug/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0 01 pg/L <0.01 pg/L <0.02 pg/L O O l pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/1. <0.05 pg/L <0.01 pg/L <0.05 ug/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L 0.02 pgL <0.02 pg/L <0.01 pg/L <0.01 prL Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mgL 29 mg/L <0.01 5 mg']. 1.1 rng/L <0.02 mgL <0,1 pg/L <0.2 pg/L <0.5 pg/L _________ <0-2 ______ Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.1 pg/L <0.7 fig/L <0.25 pg/L <0.3 pg/L <0.2 pg/L <0.1 pg/L <0.5 pg/L <0.025 pg/L <0.5 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15,1999 Wildlife International, Ltd. .41 . Project Number 454A-U7 APPENDIX III THE ANALYSIS OF PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) CONCENTRATIONS IN FRESHWATER AND BLUEGILL SUNFISH TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-117 p. 118 Wildlife International, Ltd. 42- REPORT APPROVAL Project Number 454A-117 SPONSOR: 3M Corporation TITLE: Perfluorobutane Sulfonate, Potassium Salt (PFBS): A Flow-Through Bioconcentration Test with the Bluegiil (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-117 PRINCIPAL INVESTIGATOR- Raymond X Van Hoven, Ph D. Scientist WILDLIFE INTERNATIONAL LTD. MANAGEMENT: _____________ J jtL , Willard B, Nixon, Ph.Dr1 Manager, Analytical Chemistry Date Dati Wildlife International, Ltd. -43 Project Number 454A-1 ]7 Introduction Freshwater samples and tissue samples were collected from a flow-through aquatic test to determine the bioconcentration potential of perfluorobutane sulfonate, potassium salt (PFBS) in the bluegill (Lepomis macrochirus). The study was conducted by Wildlife International, Ltd. and identified as Project Number 454A-117. The analyses o f freshwater and tissue samples were performed at Wildlife International, Ltd. by high performance liquid chromatography (HPLC) with mass spectrometric detection. Water samples were diluted and analyzed by HPLC with single quadrupole mass spectrometric detection (LC/MS). Tissue samples were homogenized, extracted, diluted, and analyzed by HPLC with triple quadrupole mass spectrometric detection (LC/MS/MS) Freshwater samples were collected and analyzed from July 16, 2000 to August 29, 2000. Tissue samples were collected from July 18, 2000 to August 29, 2000 and analyzed from July 18, 2000 to September 5, 2000. Additional tissue samples were collected and analyzed gravimetrically for lipid content. Samples for lipid content were collected on July 18, 2000, August 15, 2000 and August 29, 2000 and analyzed between September 8 and 13, 2000. Test Substance The test substance, PFBS, was used to prepare calibration standards and matrix fortification samples and was identified as Wildlife International, Ltd. identification number 5216. A nalytical M ethod Water and tissue samples were analyzed for PFBS using high performance liquid chromatography (HPLC) with mass spectrometric detection. Water samples were analyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater" (Wildlife International, Ltd. Project No. 454C-115). Tissue samples were analyzed based on the method entitled "Analytical Method Validation for the Determination o f Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Fish Tissues" (Wildlife International, Ltd. Project No. 454C-116). For tissue analyses, two modifications from the validation study were incorporated into the present study. These changes were (1) use o f a sonication step to minimize binding effects, and (2) the use of triple quadrupole mass detection mode to reduce matrix interferences. The analytical methodology implemented for the determination of lipid content in fish tissue is presented below. p. 120 Wildlife International Ltd._____ -44- Project Number 454A-117 Freshwater Samples A method flow chart for the analysis of PFBS in freshwater is presented in Figure 1. Dilutions o f collected freshwater samples were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure* water. Aliquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in freshwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model U 00 High Performance Liquid Chromatograph (HPLC) interfaced with a Perkin-Elmer API 100LC mass spectrometer (single quadruple) operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (30 mm x 1.5 mm. 3-pm particle size) fitted with a Keystone Javelin C 1S Guard Cartridge (20mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flow chart is provided in Figure 1. Freshwater quality control (QC) samples (matrix blanks and fortifications) were processed in the same manner as the test samples. Freshwater was fortified with the appropriate PFBS stock solution using a gas-tight synngc (for matrix fortification samples). Matrix blank samples were not fortified with the test substance. Tissue QC Samples BiucgUl sunfish (Lepomis macrochirus) tissues were obtained from a breeding stock maintained at Wildlife International, Ltd. Approximately 80 individual fish were removed from a breeding tank and euthanized by severing the spinal cord above the opercular region. Dissection was accomplished by making an incision from just posterior to the base of the pectoral fin dorsaliv through the spinal column. Heads, fins and viscera were removed from the body and were considered to be nonedible tissue. The balance o f the tissue was considered to be edible tissue. The nonedible fillets were transferred to a 1-quart glass bottle and blended with an Uftra-Turrax homogenizer (Janke & Kunklc, Model T25) set at approximately 9500 rpm. This procedure was repeated for the edible tissue. An appropriate number o f L-gram aliquots of edible and nonedible homogenate were weighed into separate, 20-mL glass scintillation vials. Each vial was uniquely identified and labeled with a facility log identification number. The tissue QC samples were stored frozen until they were used in the preparation of matrix blank and matrix fortification samples or storage stability samples. Wildlife International Ltd. - 45 - Project Number 454A-117 Tissue Samples A method flow chart for the analysis of PF8S in fish tissues is presented in Figure 2. Edible and nonediblc fish tissue samples were collected from the test in 20-mL glass scintillation vials and stored frozen, if necessary, until analysis. Upon analysis, tissue samples were removed from the freezer, if necessary, and batched by sampling interval and tissue type. At that time, three of the appropriate tissue QC samples also were removed from die freezer. Test and QC tissue samples were allowed to thaw. One tissue QC sample was designated as the matrix blank sample. The other tissue QC samples were designated as matrix fortification samples and were fortified with the appropriate PFBS stock solution^) using gas-tight syringes. Test and QC tissue samples were extracted as follows. Ten milliliters o f methanol were added to each vial. The samples were homogenized with a tissue shredder for approximately one minute. The samples were then sonicated with a sonic dismembrator for approximately five minutes. The samples were capped and centrifuged at ~2000 rpm for ~5 minutes. Aliquots o f the extract were then volumetrically diluted into the calibration range o f the LC/MS/MS methodology with 50:50 methanol: NANOpure water dilution solvent. Aliquots o f the diluted extracts were transferred to autosampler vials and submitted for analysis. Back-up tissue samples were collected and stored frozen. If necessary, the back-up tissue sam ples w ere removed from the freezer, allowed to thaw and processed using the sam e procedures described above. Tissue Storage Stability Samples Stability samples were prepared at test initiation to establish test substance stability in fish tissues stored frozen during the study Two tissue QC samples o f each fish tissue type were removed from the freezer and allowed to thaw. The edible and nonedible fish tissues were each fortified at 0.100 and 10.0 mg a.i./Kg using the appropriate PFBS stock solution and a gas-tight syringe. The stability samples were returned to the freezer, Following 49 days o f frozen storage, the stability samples were removed from the freezer and analyzed Fresh fortification and matrix blank samples also were prepared and analyzed at this time. The samples (newly fortified and 49 days old) were processed using the same procedures described for tissue sample analyses. Wildlife International, Ltd. - 46 - Project Number 454A-U7 Concentrations o f PFBS in fish tissue were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 HPLC interfaced with a Perkin-Elmer API 3000LC Mass Spectrometer (triple quadrupole) operated in multiple reaction monitoring (MRM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (30 mm x 1.5 mm, 3-pm particle size) fitted with a Keystone Javelin C,5 Guard Cartridge (20mm x 2 mm). The instrument parameters are summarized in Table 2. Tissue Samples for Lipid Content A method flowchart for the analysis of lipid content in fish tissue is presented in Figure 3. Edible and nonedible fish tissue samples for lipid content were collected from the test in 20-mL glass glass scintillation vials and stored frozen until analysis. Sample weights were recorded at the time of collection. Upon analysis, tissue samples designated for lipid content determination were removed from the freezer and allowed to thaw. For each sample, 10 mL o f NANOpure water was added to the fish tissue in the vial and the sample was homogenized for approximately one minute using a hand-held tissue shredder. Each homogenate was transferred to a 250-mL separatory funnel that contained 25 mL o f chloroform and 50 mL o f methanol. Each sample vial was rinsed with an additional 10 mL of NANOpure water and the rinse was poured into the respective separatory funnel. The separatory funnels were shaken with venting for approximately one minute. Fifty milliliters of chloroform followed by 50 mL of saturated sodium chloride were added to each separatory funnel. The separatory funnels were briefly swirled with venting. The phases were allowed to separate. For each sample, the chloroform layer was drained through a powder funnel packed with Teflon wool and anhydrous sodium sulfate into a 250-mL round-bottom flask. An additional 50-mL aliquot of chloroform was added to each separatory funnel and the extraction and draining procedures were repeated. The extracts were rotary evaporated in a water bath maintained at approximately 40C to near dryness. Each extract was transferred to a pre-weighed labeled scintillation vial. Each 250-mL round-bottom flask was rinsed with a small volume o f chloroform and the rinse was transferred to the respective scintillation vial. The remaining solvent in each vial was evaporated under a gentle stream of nitrogen or clean dry air. The vials were reweighed and the weights were recorded. Lipid content was calculated for each sample as the lipid weight (mg) divided by the fish weight (Kg). Wildlife International, Ltd. -47- Project Number 454A-11 Calibration Stacks and Standards Calibration standards were prepared in 50:50 methanol: NANOpure w ater by appropriate dilutions o f a 1.00 mg a.i.7 L stock solution ofPFB S in methanol. Calibration standards o f PFBS, ranging ir. concentration from 0.500 to 5.00 pg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every' five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations o f the calibration standards. Typical calibration curves from water and tissue analyses are presented in Figures 4 and 5, respectively. Representative ion chromatograms o f low and high calibration standards used for water and tissue analyses are presented in Figures 6 through 9. The concentration o f PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. The concentration o f PFBS in the freshwater was determined by substituting the peak area responses into the linear regression equation as follows: PFBS in sample (mg a.i./L) = [(peak area - y-intercept)/slope]*dilution factor*unit conversion factor % Recovery = mg^-ure^-Pl g S c?qrcntration (m&a.i./L) x l00 ^ nominal PFBS concentration (mg a.i./L) The concentration of PFBS in the tissue and stability samples was determined by substituting the peak area responses into the linear regression equation as follows: PFBS in sample (mg a.i./Kg) = ((peak area - y-intercept)/slope] ' overall dilution factor Overall dilution factor for tissue samples (L/Kg) = primary dilution x secondary dilution, where: Primary dilution = [extraction volume (L)/sample weight (Kg)], and Secondary dilution = [final volume (mL)/initial volume (mL)] Fortification Stocks Freshwater and fish tissue homogenates were fortified with the appropriate stock solution of PFBS prepared in methanol. Each stock solution was assigned a unique identification code that was recorded on a stock preparation log sheet. p. 124 Wildlife International, Ltd._____ -48 - Project Number 454A-] 17 Limits of Quantitation The method limit o f quantitation (LOQ) for PFBS in freshwater samples was 0.125 mg a.i./L, calculated as the product o f the lowest PFBS calibration standard (0.0005 mg a.i./L) and the dilution factor o f the matrix blank sample (250) analyzed concurrently with die test samples. The LOQ was calculated on an individual basis for each edible tissue sample since the entire submitted samples (of differing weights) were extracted without an adjustment to a constant weight. Where appropriate, the LOQ (mg a.i./Kg) for a given edible tissue analysis was calculated as the product of the lowest PFBS calibration standard (0 0005 mg a.i./L) and the overall dilution factor (L/Kg) o f the edible tissue sample. To illustrate for a 1.000 gram sample, extraction with 10 mLs o f methanol followed by a lOx volumetric dilution (overall dilution factor =100 L/Kg), gives an LOQ = 0.0500 mg a.i./Kg. An apparent PFBS background (0.0395 0.0132 mg a.i./Kg) was measured in nonedible control tissue homogenates (see Figure 13 on page 83). The suspected origin of this background was residual laboratory cleaning detergent in the blender employed for bulk homogenization. Laboratory cleaning detergents had been suspected as a potential interfrent during method development trials. Contamination from PFBS itself was another possibility since this blender may have been used previously for homogenizations in studies conducted at significantly higher levels o f test substances containing PFB S. Therefore, a practical LO Q was defined for individual nonedible (where appropriate) tissue samples as the mean measured background plus three standard deviations o f the background measurements (0.0792 mg a.i./Kg for a one-gram sample), adjusted for the individual weights o f the tissue samples. Freshwater M atrix Blank and Fortification Samples Along with the actual freshwater sample analyses, 15 freshwater matrix blank samples were analyzed to determine possible interferences (Table 3). No matrix interferences were observed at or above the limit o f quantitation (0.125 mg a.i./L). A representative ion chromatogram o f a freshwater matrix blank sample is presented in Figure 10. Freshwater samples were fortified at 0.250, 2.00 and 7.50 mg a.i./L at each sampling interval using appropriate stock solutions of PFBS prepared in methanol. Freshwater matrix fortifications Wildlife International Ltd. -49-, Project Number 454A-I17 were analyzed concurrently with each sample set and the analytical results were not corrected for mean procedural recovery. Recoveries ranged from 89.7 to 115% o f nominal concentration (Table 3). The mean and standard deviation of fortification recoveries at the 0.250, 2 00 and 7.50 mg a.i./L fortification levels (n - 15) were 99.8% 7.02% , 97.1% 2.97%, and 99.4% = 2.33%, respectively. A representative ion chromatogram of a freshwater matrix tonification is presented in Figure 11. Tissue M atrix Blank and Fortification Samples Along with the actual tissue sample analyses, 12 edible and 12 nonedible fish tissue matrix blank samples were analyzed to determine possible interferences (Tables 4 and 5, respectively). No interferences were observed at or above the applicable limits of quantitation during the test. Representative ion chromatograms o f edible and nonedible fish tissue matrix blank samples are presented in Figures 12 and 13, respectively. Edible and nonedible fish tissue homogenates were fortified at 0.100 and 10.(1 mg a.iYKg using a stock solution o f PFBS prepared in methanol. Tissue matrix fortifications were prepared and analyzed concurrently with each sample set and the analytical results were not corrected for mean procedural recovery. Edible fish tissue recoveries ranged from 66.9 to 93.4% o f nominal concentration for the low-level fortification, and from 76.6 to 94.1% of nominal concentration for the high-level fortification (Table 4). The mean and standard deviation o f Fortification recoveries at the O.IOO an d 10.0 mg a i./K g fortification levels (n = 12) for the edible fish tissu e w ere 8 4 .4 % r 8. 10% and 83.2% t 5.00%, respectively. Nonedible fish tissue recoveries ranged from 80.4 to 132% and from 80.8 to 94 0% of nominal concentration for low- and high-level fortifications, respectively (Table 5). The mean and standard deviation o f fortification recoveries at the 0.100 and 10.0m ga.i./K g fortification levels (n = 12) for the nonedible fish tissue were 102% 15.0% and 88.0% 4.70%, respectively. Representative ion chromatograms of edible and nonedible fish tissue matrix fortification samples are presented in Figures 14 and 15, respectively. Example Calculations The analytical result and percent recovery for freshwater sample 454A-117-3, from the 0.500 mg a.i./L nominal PFBS treatment group, was calculated using the following equations: p. 126 Wildlife International, Ltd._____ -50- Project Number 454A-117 (peak area - y-intercept) PFBS in sample (mg a1 /L ) = slope x dilution factor x unit conversion factor Peak area = 152419 Y-intercept = 2116.42700 Slope = 72902.35156 - Initial volume (VO = 0.100 mL Final volume (Vf) = 25.0 mL Dilution factor (V/V,) = 250 (152419-2116.42700) .lm c PFBS in sample (mg a.i./L) = 72902.35156 x 250 x 1000 |Ag PFBS in sample (mg a.i./L) = 0.515 mg a.i./L measured PFBS concentration (mg a.i./L) ,, jqq % Recovery in sample = nominal PFBS concentration (mg a.i./L) % Recovery in sample - * 100 % Recovery in sample = 103 % The analytical result for edible fish tissue sample 454A-117-E-45, from the 0.500 mg ai./L nominal PFBS treatment group, was calculated using the following equation: PFBS in sample (mg a.i./Kg) = PFBS at instrument (pg a.i./L) x overall dilution factor x unit conversion factor Peak Area = 6639.89 Y-intcrcept = -946.98 Slope = 12296.93 Primary Dilution: Initial Weight (Ws) = 1.2201 g Extraction Volume (V4) = 10,0 mL Secondary Dilution: Initial Volume (V,) = 0.500 mL Finai Volume (Vf) = 10.0 mL Overall Dilution Factor (Ve/W, x V /V ,) - 163.92 mL/g = 164 L/Kg ____ . , . _. (peak area - v-intercept) PFBS at instrument (pg a.i./L) = -----------;------------ e~L Wildlife International, Ltd. -51 - Project Number 454-117 PFBS at instrument (pg a.i./L) = ( ~ ^ 6 98)) = 0 617 a ; / l PFBS in sample (mg a.i./Kg) = PFBS at instrument (pg a.i./L) x overall dilution factor x unit conversion factor . , , . ,,,, , 0.617 pga.i. 164 L PFBS m sample (mg a,./K g) = ------- x x Img PFBS in sample (mg a.i./Kg) = 0.101 mg a.i./Kg ' RESULTS Freshwater Sample Analysis Freshwater samples were collected and analyzed for PFBS concentrations on Days -2 and -1 during the pre-uptake phase o f the test and Days 0 (0 and 4 hours), 1, 3, 7, 14, 21, and 28 during the uptake phase of the test. Uptake was suspended on Day 28 o f the test and depuration began. During the depuration phase of the test, freshwater samples were collected and analyzed for PFBS concentrations on Days 1, 3, 7, 10 and 14. Measured concentrations of PFBS in the pre-test diluter verification (pre-uptake phase) samples were <LOQ in the control. Measured concentrations in pre test samples ranged from 100 to 103% of the nominal concentration in the 0.500 mg a.i./L treatment group and from 98.8 to 104% of the nominal concentration in the 5.00 mg a.i./L treatment group (Table 6). During the uptake phase of the test, measured concentrations o f PFBS in the control freshw ater sam ples were <LOQ. Freshwater samples collected from tho 0.500 and 5.00 mg a.i./L treatment groups ranged from 95.7 to 119% and from 101 to 109% o f the nominal concentration, respectively (Table 7). During the depuration phase of the test, measured concentrations of PFBS in all freshwater samples were <L0Q. A representative ion chromatogram of a freshwater sample is presented in Figure 16. Tissue Sample Analysis Tissue samples were collected on uptake Days 0 (4 hours), 1, 3, 7, 14, 21, and 28, and depuration Days 1, 3, 7, 10 and 14 o f the test. Measured concentrations of PFBS in all but three (454A-117-E40, 66 and 105) o f the 24 control edible fish tissue samples were <LOQ (Table 8), The results of analyses o f edible fish tissue samples collected from the 0.500 and 5.00 mg a.i./L treatment groups are presented in Table 8. Measured concentrations of PFBS in all but four (454A-117-N-40, 66, 67 and 105) o f the 24 control nonedible fish tissue samples were <LOQ (Table 9). Three o f these Wildlife International Ltd. -52 - Project Number 454A -J17 nonedible samples corresponded with the three edible samples that had measured PFBS concentrations above the LOQ (i.e. originated from the same fish). The results of analyses of nonedible fish tissue samples collected from the 0.500 and 5.00 mg a.i./L treatment groups arc presented in Table 9. Representative ion chromatograms of edible and nonedible tissue samples from the same study fish are presented in Figures 17 and 18, respectively. Stability Sample Analysis Stability samples were prepared at test initiation (uptake Day 0) and stored frozen. The results o f stability sample analyses arc presented in Table 10. These data indicate that the test substance was stable during relatively long term (49 days) frozen storage at the 0.100 and 10.0 mg a.i./Kg concentration levels, Definitive tissue sample storage did not exceed 5 days during the conduct of the study, Tissue Sample Analysis for Lipid Content Tissue samples were collected on uptake Days 0 (0 hour) and 28 and depuration Day 14 to determine lipid content in fish tissues on a wet-weight basis. The results o f lipid analyses in edible and nonedible fish tissues are presented in Tables 11 and 12, respectively. . Wildlife International Ltd. -53 - Project Number 454A-117 Table 1 Typical HPLC/MS Operational Parameters for Analysis o f Aqueous Samples INSTRUMENT: SOURCE: ANALYTICAL COLUMN: GUARD COLUMN OVEN TEMPERATURE: STOP TIME; FLOW RATE: MOBILE PHASE; INJECTION VOLUME: PFBS PEAK RETENTION TIME: PFBS M ONITORED MASS: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode Perkin-Elmer TurbolonSpray Keystone PRTSM RP (30 mm x 15 mm, 3-u.m particle size) Keystone Javelin 0 8 cartridge (20 x 2 mm) 40C 3.00 min 200 jjJVmin 25% NANOpure Water with 0.1% Ammonium Formate: 75% Methanol 5.0 p.L Approximately 1.7 minutes 299.amu p. 130 Wildlife International, Ltd.______________________ -54- SAN* . Z E D Project Number 454A-11? Table 2 Typical HPLC/MS/MS Operational Parameters for Analysis o f Tissue samples INSTRUMENT: . ION SOURCE: ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFBS PEAK RETENTION TIME: PFBS MONITORED MASS: Hewlett-Packard Mcde! 1100 High Performance Liquid Chromatograph with a Psrkin-Elmer API 100LC Mass Spectrometer operated in Multiple Reaction Ion Monitoring (MRM) Mode Perkin-Elmer TurboIonSpray Keystone PRISM RP (50 mm x 2 mm, 3-pm particle size) Keystone Javelin C18 cartridge (20 x 2 mm) 40C 5.00 min 200 pL/nnn 10% NANOpurc Water with 0.1% Ammonium Formate: 90% Methanol 5.0 pL Approximately 3,1 minutes 299.0 am i^ . p. 131 Wildlife International, Ltd._____ -55 - Project Number 454A -117 Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples Sarapie Nuraber (454A-1I7-) PT-MAB-I PT-MAS-1 PT-MAS-2 PT-MAS-3 PT-MAB-2 PT-MS-4 PT-MAS-5 FI-MAS -6 ' MAB-1 fciAS-l MAS-2 MAS-3 Concentration ofPejfluarobuUnc Sulfonate, Potauiua Sail (PPUS) (mg a.i/L) Fortified Measured^ 0.00 < LOQ 0.250 0.266 2.00 2.03 7.50 7.58 0.00 0.250 2.00 7.50 <LOQ 0.243 1.88 7.33 0.00 0.250 2.00 7,50 < LOQ 0.241 1.93 7.41 Percent Recovery7 106 101 101 _ 97.2 93.8 97.7 _ 96.5 96.3 98.8 MAB-2 MAS-4 MAR-5 MAS-6 0.00 0.250 2.00 7.50 < LOQ 0.245 2.01 7.56 97.9 101 101 MAB-3 MAS-7 MAS-8 MAS-9 MAB-4 MAS-10 MAS-lI MAS-12 0.00 0.250 2.00 . 7.50 0.00 0250 2.00 7.50 < LOQ 0.229 1.87 7.16 <LOQ 0.250 1 .99 7.63 91.5 93.3 95.5 _ 100 9 9 .4 102 MAB-5 MAS-13 MAS-14 MAS-15 0.00 0250 2.00 7.50 < LOQ 0.242 1.95 7.34 97.0 97.5 97 8 MAB- MAS-16 MAS-17 MAS-] 8 0.00 0.250 2.00 7.50 < LOQ ' 0.271 1.87 7.50 108 93 6 to o MAB-7 0.00 < LOQ _ io l/l MAS-19 MAS-20 MAS-21 2.00 7.50 0.232 1.92 7.74 92.8 95.9 103 1The limit of quantitation (LOQ) was 0.I25 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 rag a.i./L) and the dilution factor of the matrix blank sample (250). 7Results were generated. using MacQuan, version 1.6 software. Manual calculations may differ slightly. p. 132 Wildlife International, Ltd._____ -56- Project Number 454-117 Tabic 3 (continued) M atrix Blanks and Fortifications Analyzed Concurrently With Freshwater Samples Sample Number (454A-U7-) MAB-8 MAS-22 MAS-23 MAS-24 ConcentrationofPtrfluurvbulaneSulfonate, PotassiumSalt(PFBS) (ma a.i./L) Fortified Measured1 0.00 0.250 2.00 7.50 < LOQ 0.252 1.93 7.47 Percent Recovery2 ,, 11 96.4 99.6 MAB-9 MAS-25 MAS-26 MAS-27 ' 000 0.250 2.00 7.50 < LOQ 0287 1.96 7.74 ,, 115 98,1 103 MAB-10 MAS-2S MAS-29 MAS-30 0.00 0.250 2.00 7.50 <LOQ 0.270 1.90 7.20 _ 108 95.0 96.0 MAB-II MAS-31 MAS-32 MAS-33 ooo 0.250 2 00 7.50 < LOQ 0.236 1.89 7.34 __ 94.3 94.6 97.9 Ma D-12 MAS-34 MAS-35 MAS-36 0.00 0.250 2.00 7.50 < LOQ 0.224 2.071 7.32 89.7 103 97.7 MAB-13 MAS-37 MAS-38 MAS-39 0.00 0.250 2.00 750 < LOQ 0.254 1.96 7.53 102 97.9 100 lThe limit of quantitation (LOQ) was 0.125 mg a.i./L, calculated as the product of the lowest calibration standard (0 0005 mg a.i A ) and the dilution factor of tire matrix blank sample (250). 2Results were generated using MacQuart, version 1.6 software. Manual calculations may differ slightly ' Reporting average of duplicate injections of reel luted original fortification Original dilution not reported due to dilution error. Wildlife International, Ltd, -57- Project Number 454A- L17 Table 4 Matrix Blanks and Fortifications .Analyzed Concurrently With Edible Fish Tissue Samples Satnole Number (454A -117-) 2-MAB-l E-MAS-1 E-MAS-2 Concemralion of Perfluorobutane S u lfo n a te , Potarjium Salt(PFBS) (mg a.i./tCo) fortified 535 Measured1 <LOQ 0.100 0.0914 9.10 7.68 Percent Reco_very5 91.4 84.4 E-MAB-2 E-MAS- E-MAS-4 0.00 . 0.100 10.0 <LOQ 0.0880 8.61 88.0 86.1 E-MAB-3 E-MAS-5 E-MAS-6 0.00 0.100 10.0 <LOQ 0.0912 8.80 91.2 88.0 E-MAB-4 E-MAS-7 E-MAS-8 0.00 0.100 10.0 <LOQ 0.0785 8.04 78.5 RQ.4 E-MAB-5 E-MAS-9 E-MAS-10 0.00 0 100 10.0 <LOQ 0.0868 7.66 86.8 76.6 E-MAB-6 E-MAS-11 E-MAS-12 0.00 0.100 10,0 <LOQ 0.0867 7.91 86.7 79.1 E-MAB-7 E-MAS-13 E-MAS-14 E-MAB-S E-MAS-15 E-MAS-16 0.00 0.100 10.0 0.00 0.100 to.o <LOQ 0.0889 7,77 <LOQ 0.0857 8.06 88.9 77.7 85.7 80.6 E-MAB-9 E-MAS-17 E-MAS-IS E-MAB-10 E-MAS-19 E-MAS-20 E-MAB-1I E-MAS-21 E-MAS-22 0.00 0.100 10.0 000 0.100 10.0 0.00 0.100 10.0 <LOQ 0.0934 8.07 <LOQ 0.0838 8.35 <LOQ 0.0669 9.4! 93.4 80.7 . 83.8 835 . 66.9 94.1 E-MAB-12 E-MAS-23 H-MAS-24 0.00 0.100 0.0 <I.OQ 0.0718 866 71.8 86.6 1The LOQ was 0.0500 mg a.i/Kg, calculated as the product of the lowest calibration standard (0.0005 tug a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). All sample weights 1.00 gram. *Results were generated using MacQuan. version 1.6 software. Manual calculations may dilFer slightly.___________ p. 134 Wildlife International, Ltd._____ -58 - Project Number 454A-117 Table 5 Matrix Blanks and Fortifications Analyzed Concurrently With Noncdible Fish Tissue Samples SampleNumber (4J4A-U7-0 ConcentrationoFPerfluorobulaneSulionate. PotassiumSail(PFDS) (mga.iJtCs) ' fon Measured1 Percent Recovery3 N-MA3-1 N-MAS-1 N-MAS-2 0.0 0.100 10.0 <LOQ 0.101 9.13 101 91.3 N-MAR-2 N-MAS-3 N-MAS-4 0.00 0.100 10.0 <LOQ 0.114 9.16 114 91.6 N-MA3-3 N-MAS-3 N-MAS-6 o.oo 0.100 10.0 <LOQ 00911 .65 91.2 86.5 N-MAB-4 N-MAS-7 N-MAS-8 o.oo 0.100 10.0 <LOQ 0.0866 8.90 86.6 89.0 N-MAB-5 N-MAS-9 N-MAS-10 0.00 a.loo 10.0 <LOQ Q.092G S.23 . 92.6 82.3 N-MAB-6 N-MAS-I1 N-MAS-12 0.0(1 0.100 10.0 <LOQ 0.0804 8.16 80.4 816 N-MAB-7 N-MAS-13 N-MAS-14 o.oo 0.100 10.0 <LOQ 0.0988 8.56 . ' 98.8 85.6 N-MAB-8 N-MAS-M hl-MAS-16 (1.00 o1.io.Ua <LOQ 08,1.0381 8103.2 N-MAB-9 N-MAS-17 N-MAS-IS 0.00 <U3Q 0.100 0.121 10.0 9.17 - 121 9J.7 N-MAB-IO N-MAS-19 N-M.AS-20 0.00 0.100 10.0 <LOQ 0.111 9.40 ui 94.0 N-MAB-11 N-MAS-21 N-MAS-22 CI.0 01..100.00 <LOQ 0.105 9.39 w 105 93.9 N-MAB-12 N-MAS-23 N-M.4S-24 0.00 0.100 10.0 <LOQ 939 8.81 V 93.9 88.1 1Tbs LOQ 1 0.C792 mgs-i./Kg, calculatedastheproductofthemeanmeasuredbackgroundconcentrationplusthreestandarddeviations ofthemeasuredbackgroundconcentrations. Allsampleweights- 1-00gram. 1ResultsweregettersledusingMacQuan,version1.6software. Manualcalculationsmaydifferslightly. p. 135 Wildlife International, Ltd._____ -59- Project Number 454A-117 Table 6 Measured Concentrations o f Pcrfluorobutanc Sulfonate, Potassium Salt (PFBS) in Pre-Test Diluter Verification Samples Nominal Test Concentration (mgs.i;/L) 0.0 (NegaliveConlrul) Sample Number (454A-117-} PT-l PT-9 Ptusc Pre-Uptake Sampling Time COsy) -2 -1 MeasuredConcentrationor PcrfluorobutaneSulfonate, Polonium Salt(PFBS)1 (mga.i.'L) <LOO <LOQ Percent of Nominal3 - 0.50 rro PT-4 PT-l 1 pr-ir .2 0.515 m .2 O.iOt i0 -1 O.JOs 102 -l 0.500 100 5.0 PT-6 FT-7 PT-l4 PT-l5 -2 98.8 2 5.09 C2 A 5.07 10) -1 5.18 104 1The limit of quantitation(IOQ) wu 0.125 mgsu./L, calculatedastheproductofthelowest calibrationstandard(00005 mga-i./L) andthe dilutionfactorc/thematrixblanksample(250). 1Result*weregeneratedusingMacQuan,version1.6software. Manualcalculationsmaydifferslightly p. 136 Wildlife International, Ltd._____ . -60- Project Number 454A-117 Tabic 7 Measured Concentrations o f Pcrfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Nommai Test Concentration ima a.i./L) 0.0 (Negative Control) Sample Number (454A-117-) 1 9 17 25 33 41 49 57 65 73 81 89 97 Phase Uptake Depuration Sampling Time (Day) 0, 0 hours 0, 4 hours 1 3 7 14 21 28 I 3 7 10 14 Measured Concentration of Petti unrebutaue Sulfonate, Potassium Salt (PFBS)1 ( mg a.i./L) < LOQ <LOQ < LOQ < LOQ < LOQ <LOQ < LOQ < LOQ < LOQ < LOQ <LOQ <LOQ <LOQ ' Percent of Nominal - - -- - - 0.50 3 Uptake 0,0 hours 4 0 ,0 hours 11 0 ,4 hours 12 0 ,4 hours 19 1 20 l 27 3 28 3 35 7 36 7 43 14 44 14 51 21 52 21 59 28 60 28 0.515 0.518 0.519 0.525 0.478 0.490 0.542 0.542 0.525 O.S59 0.510 0.514 0.593 0.567 0.515 0.516 103 104 104 105 95.7 98.0 109 \ 0 `) 105 112 104 103 119 114 103 103 67 Depuration 68 75 76 83 84 91 92 59 100 1 1 3 3 7 7 IU 10 14 14 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ `The limit o f quantitation (LOQ) was 0.125 mg a.i./L, calculated as the product o f the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of die matrix blank sample (250). 3Results were generated using MacQuzn, version 1 6 software. Manual calculations may differ slightly. -- - - p. 137 Wildlife International, Ltd._____ -61 - Project Number 454A-117 Table 7 (continued) Measured Concentrations o f Perfluorobutane Sulfonate, Potassium Salt {PFBS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 5.0 Sample Number (454A-117-) 6 7 14 15 22 23 30 31 38 39 46 47 54 55 62 63 Phase Uptake ' Sampling Time (Day) 0, C hours 0,0 hours 0 ,4 hours 0,4 hours 1 1 3 3 7 7 14 14 21 21 28 28 Measured Concentration of Perfluorobutane Sulfcnute, Potassium Salt (PFBS)1 (mg a.i./L) 5.24 5.42 5.35 5.43 5.02 5.12 5.08 5.12 5 17 5.22 5.36 5.32 5.04 5.21 5.45 5.40 Percent of ^ Nominal" 105 108 107 109 101 102 102 102 103 104 107 106 101 104 109 108 70 Depuration .1 71 1 78 3 79 3 86 7 87 7 94 10 95 10 102 14 103 14 < LOO < LOQ < LOQ < LOQ < LOQ < LOO < LOQ < LOQ < LOQ < LOQ 'The limit of quantitation (LOQ) was 0.123 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 (250). 5Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly - - Wildlife International, Ltd, -62- Project Number 454A-117 Table 8 Measured Concentrations of Pcrfluorobutane Sulfonate, Potassium Salt (PFBS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nomimi Test Concentration (mgai) 0.0 (NegativeContro)) Sample Number (44MI7-) E-l E-2 F.-14 E-13 ' E-27 E-28 E-40 E-41 -53 E-54 E-66 E-67 E-79 -80 E-92 E-93 E-l05 E-106 E-l 18 E-l 19 E-131 E-132 E-l44 E-145 Phase Uptake Depuration Sampling Time (Day) 0,4 hours 0, 4hours 1 1 3 3 7 7 14 14 21 21 28 28 Y 1 3 5 7 7 10 10 14 14 MeasuredConcentrationof PerfluorobutaneSulfonate, PotassiumSalt (PFBS)1 (mgi.i./Kg) <0.0385 <00295 <0.0695 <0.0725 <0.0370 <0.0575 0.0548 <0.0515 <0.039* <0.0975 0.0788 <0.0352 <0.0334 <0*0238 <0.0710 <0.0649 a.uo <0.0598 <0.0425 <0.0488 <0.0428 <0*0625 <0.0530 <0.0953 0.50 E-4 Uptake 04huun E-5 0 ,4 hours <<00..00558350 E-6 . 0 ,4 huun <0.0393 E-7 0 ,4 hours <0.0590 E-l7 1 0.0382 E-18 l <0.0428 E-l9 1 <0.0915 E-20 1 <0.0650 E-30 3 0.0874 E-3J 3 0.0636 E-32 3 0.0911 E-33 3 0.0704 E-43 7 . 0.116 E-44 7 0.136 E-45 7 0.1CI E-4 7 3.133 ' Less than value! correspond la limit o f quantitation (LOQ) For each analysis, ihe LOQ w u calculated aa the product o f the lowest calibration standard (0.0003 mga.iTL) and the overall dilution factor of the sample (L'Kg). ! Result* were generated using MacQuan, version I f software. Manual calculations may differ slightly. p. 139 Wildlife International Ltd.____ -63 Project Number 454A-117 Table 8 (continued) Measured Concentrations o f Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Edible Fish Tissue Samples from a Bluegill Sunftsh Bioconcentration Test Nominal Test Concentration (mgai-.'L; 0.50 5.0 Sample Number (4MA-U7*) -56 -57 E-5* E-59 ' E-69 E-70 E-71 E-72 E-S2 E-83 E-84 E-35 E-95 -96 E-97 E-98 E-108 E-109 E-110 E-Ut E-121 E-122 -123 E-124 E-134 E-135 E~134 e-u7 E-I47 E-148 E-149 150 E-9 E-ta E-n E-n E-22 E-23 E-24 E-26 Phase Uptake Depuration Uptake SamplingTime (Day) 14 H 14 14 21 21 21 21 28 28 28 28 1 1 1 l 3 3 3 3 7 7 7 7 10 1C 10 1C 14 14 14 14 0, 4hours 0,4 hours 0,4 hours 0,4 houn t 1 1 l MeasuredConcentrationof PeriluurobuUncSulfonate. PotassiumSalt <PFR5)J (mgtWKg) 0.0880 <0.0715 0.0869 0.105 0.136 0.125 0.213 0.114 0.0682 0.103 0.0619 0.0871 1.19 3.31 0.209 398 0.0758 0.0571 <0.0341 <0.0488 <0.0401 <0.0476 <0.0414 <0.0915 <0.0459 0.108 C0058 <0.0595 <0.0499 <0.0520 <0.0630 <0.0364 0.127 0.0975 0-0811 0.127 0.240 0.273 0.311 0.234 1L eo than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ we* calculated a* the product o f the lowest calibration stuidaid (0.0005 mg e.i./L) and the overall dilution factor cf the sample (l>'Kg; 1 Results were generated using MacQuan, version 1.6 software Manual calculations may differ slightly. Wildlife International, Ltd, -64 - Project Number 454A-117 Table 8 (continued) Measured Concentrations of Pcrfluorobutanc Sulfonate, Potassium Salt (PFBS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg 5.0 Sample Number (454A -U 7-) E-35 E-36 E-37 E-38 E-48 E-49 E-O -51 E-61 E-62 E-63 E-64 E-74 E.73 E-76 E-77 E-87 E-88 E-89 E-90 E-100 E-101 E-102 E-103 E-I13 E-U 4 E -U E-116 F.-126 E-I27 E-128 E-129 -139 E-140 E*14J E-142 E-152 E-153 E-154 E-153 Phase Uptake Depuration Sampling Time (Day) 3 3 3 3 7 7 7 7 14 14 14 14 21 21 21 21 21 28 28 28 1 1 1 l 3 3 3 D 7 7 7 7 10 10 LO 10 14 14 14 14 Measured Concentration o f Perlluorobvtanc Sulfonate, Pcttustum Salt (?FBS)' {m ga.u /K g) 0.675 0.487 0.6 U 0.448 1.07 1.19 . 0.701 1.30 0.438 1.12 1.15 0.748 0.596 0.593 1.50 0.590 3.764 3.9Q7 0.656 0.8j 7 0.71* 0.622 1.05 0.467 0,233 0Il f1tl9l7l 0.413 0.196 0.151 0.124 <0.0879 0.0791 <0.0740 <0.0815 <0.0930 <0.0410 <0.0454 <0.0685 <0.0705 1La than values cuirespoixi 10 limit o f quantitation (LOQ). For each analysis, the LOQ was calculated as the product o f the lowest calibration standard (0.0005 mg a.i./L) and `Jte overall dilution factor of the sample (UKg). Results were generated using MacQuan, version 1.f>software. Manual calculations may ditt'er slightly. p. 141 __Wildlife International Ltd. -65 Project Number 454A-117 Table 9 Measured Concentrations of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Nonedible Fish Tissue Samples From a Bluegill Sunfish Bioconcentratiun Test dominai Ten Concentrator (rag a.i./L) 0.0 (Negative Control) Sample Number (454A-117-) N-J N-2 N-14 N-13 N-27 N-78 N^W N-41 N-33 N-54 N-66 N-67 N-79 N-80 Phase Uptake Sampling Time (Day) 0, 4 hours 0.4 hours 1 1 3 3 7 7 14 ' 14 21 2J 28 28 MeasuredConcentrationof Peifluurobuiane Sulfonate, Potassium Salt (P fB s y (mga.i,.'Kg) <0.0369 <0.94*1 <0.0713 <0,0824 <0.0307 <D.D7&2 0.164 <0.0887 <0.0443 <0.103 0.0320 0.0492 <0.0623 <0.0426 N-92 N-93 N-iOJ N-106 N-118 N-119 N -m N-132 N-144 N-143 Depuration 1 1 3 3 1 7 10 10 14 14 <0.0751 <0.0748 0.224 <0.0538 <0.0644 <0.0570 <0.0541 . <0.0732 <0.0990 <0.105 0.50 N-4 N-5 N-6 N-7 N-17 N*JH N-19 N-20 N-30 N-31 K-32 N-33 N-43 N-44 N-45 N^S Uptake 0,4 hours O, 4 haunt 0, 4 hours 0, 4boun i i i i 3 3 3 2 1 1 7 7 <0.0630 CO.7JO <0.0439 <0.0749 0.0786 .D894 0.112 0.0730 0.180 ans 0.173 0.141 0.265 0.293 0.263 0.307 1Lew than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the mean measured background (matrix blank) conccmralicns p'us'lhree standard deviations of the measured background (9.0792 mg a.i./Kg), adjusted for the individual weight of the tissue sample 1Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. ' p. 142 Wildlife International Ltd. -66 - Project Number 454A-117 Table 9 (continued) Measured Concentrations of PerJEIuorobutane Sulfonate, Potassium Salt (PFBS) in Nonediblc Fish Tissue Samples from a Blucgill Sun fish Bioconceutration Test Nominal Test Concentration (mga-L/L) a,jo Sample Number (434A-1I7-) N-5 N-J7 N-58 . N-59 N-69 N-70 N-71 N-73 N-82 N-83 N-84 N-85 Phase Uptake SamplingTime (Dy) 14 14 14 14 21 21 21 21 2;8 28 28 28 MeasuredConcentruirmof PeriluorobutAncSulfonate, Potassiumalt (PF3S)1 {mgi.i/Kg) 0.235 0 181 0,236 0.179 0.341 J14 0.379 0.323 0.185 0.262 0.133 0243 N-95 N-9 N-97 N-91 N-10S N-109 .V-110 N-1U N-12I N-122 N-123 N-124 K-134 KN-'113356 N-137 K-147 N-14S N-U9 N-liO Depuration 1 1 1 1 3 3 3 3 7 7 7 7 10 1to0 10 14 14 14 14 0.207 1.15 0-337 6.11 0,156 0.145 <0.0437 0.0934 0.0920 <0.0515 <0.0562 <0,0982 <0.0634 <<00..006*4230 <0,0735 0.0796 <0.0343 <0.0808 <0.0490 3.0 N-9 Uptake 0,4 hours N-ia 0,4hours N-n 0,4hours N-12 G,41lours N-22 i N-23 1 N-24 1 N-26 1 0233 0.173 0.178 0.247 0.726 0.730 0.702 0.598 1Lessthanvalues correspondto limit of quantitation (LOQ). for eachanalysis, the LOQwu calculated u themean measuredbackground (matrixblank) concentrationsplusthreestandarddeviationsofthemeasuredbackground(0.0792mga.i./XgXadjustedfortheindividual weight ofthetissuesample. 2ResultsweregeneratedusingMacQuan,version1.6software. Manualcalculationsmaydiftrslightly. p. 143 __Wildlife International, Ltd. -67- Projcct Number 454A-117 Table 9 (continued) Measured Concentrations o f Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test* Nominal Tesi Concentration (mg a.w'L) 50 Sample Number (454A-117.) N-35 N-36 N-37 . NO 8 N-48 N-49 N-50 N-I N-6t N-62 N-63 N-64 N-74 N-75 N-76 N-77 N-87 N-BS N-39 N-90 Phase Uptake Sampling Time (Day) 3 3 3 3 7 7 *7 7 14 14 14 14 21 21 21 21 78 28 28 28 Measured Concentralion of Perfluorobutane Sulfonate, Poussium Salt (PFBS)' (oi6L'K8) 1.49 1.35 2.08 1.29 3 10 2.95 2.04 368 0.960 3.28 3.42 2.22 - 1.56 1 .6 6 4.05 1 .6 4 1 .6 6 2.02 2,05 2.22 N-IC N-101 N-102 N-103 N-113 N-114 N -115 N-U6 N-12 N-127 N-128 N-129 N-139 N-140 N-141 N-142 N-152 NO 53 N-154 N-I55 Depuration l 1 l l 3 3 3 3 7 7 7 7 10 10 10 10 14 14 14 14 2.09 2.32 3.29 1.17 0.676 0.529 3.3 7 1.36 0,691 0.530 0.330 <0.0808 0.181 <0 0 800 <0.0887 <0.0903 <0 0494 <0.0602 <0.0773 <0.0847 . 1Less than values correspond la limit o f quantitation (LDQ). For each analysis, the I.UQ was calculated is the mean measured background (niaLiix blank) concentrations plus three standard deviations of the measured background (0.079? mg vi./Kg), adjusted for the individual weight of the tissue sample. *Result were generated using MacQuan, version 16 software. Manual calculation! may differ slightly. p. 144 Wildlife International Ltd. 68 - Project Number 454A -117 Tabic 10 Measured Concentrations o f Pcrfluorobutane Sulfonate, Potassium Salt (PFBS) in Tissue Storage Stability Samples from a Bluegill Sunfish Bioconcentration Test Numilia] Ccncoiiriiion (mga.i./Kg) NegativeControl 0.100 Sample Number (454A-117-) E-MAB-134 N-MAB-134 ' E-STMAS-11*4 E-MAS-234 N-STMAS.]' N-MAS-25* Tissue Type Edible Nontdiblc Edible Edible Nonedible Nonedible MeasuredConwentr&lioaof PcrfluorobutaneSulicnaie. PotassiumSal; (PFBS) (niga.i./Kg) <LCQ` <U3Q' ' 0.0732 0.0732 0.0923 0.0741 Percent oT Nominal* 73.2 73.2 92.3 74.1 10.0 E-STMAS-21 Edible 9.39 93.3 E-MAS-264 Edible 8.82 88.2 N-STMAS-2J N-MAS-264 Nonedible Nonedible 8.34 8.09 83.4 80.9 1Leu thanvaluescorrespondto limit ofquantitation(LOQ). The LQQforthe edibletissuematrix was0.0300 mga.i./Kg, calculatedasthe product ofthe lowest calibration standard(0.0003 mga.i/L) andthe uvcnUJ dilutionfactor of (he sample (100 UKg). The LOQfor the nortedihietissuematrixwas0.0792 mga.:./Kg. calculatedastheproduct ofthemeanmeasuredbackgroundconcentrationplusthreestandard deviationsofthemeasuredbackgroundconcentrations. Allsampleweights~ 1.00gram. ResultsweregeneratedusingM&cQuan,version1Asoftware. Manualcalculationsmaydifferslightly. *The stability samples were fortified on July 18, 2000 and stored in the freezer. The samples were removed fromthe freezer on September 5, 2000(after49daysoffrozenstorage) andanalyzed. . 4ThesesampleswerepreparedonSeptember3,2000andtlieresultswerecomparedtothestabilitysampleresulta. p. 145 ,Wildlife International Ltd. -69- Project Number 454A-117 Table 11 Lipid Content in Edible Fish Tissue NominalTest Concentration {mga.i./L) u.o (Negative Control) Sample Number (454A-117-) ELI ELI ELS EL4 EL7 ELS ELS ELIO ELZ1 EL22 EL23 EL24 ELU EL12 EL13 RLi4 EL26 EL27 EL28 EL29 Phase Uptake Uptake Depuration Upufce Depuration Sampling; Tune (D*y) 0, 0hour 0,0 hour 0.0 hour 0.0 hour 28 28 28 28 14 14 14 14 2 28 23 23 L4 14 14 14 Lipid Weight <> o.otui 0.0139 0.0097" 0.0039 0.0177 0.0184 0.0210 0.0139 0.0147 0.0108 o.uoy* 0.0168 C.0137 0.0158 C.0O93 C.0109 non? 0.0160 0.013J 0.0084 3.0 ELLS Uptake 2S 0.0091 ELI 7 23 0.0098 ELLS EL19 EL31 Depuration 23 28 14 QM 99 0.0086 0.0142 EL32 14 0.0161 EL33 KL34 14 o.oi n 14 0.0094 1Ratio calculated as [lipid weight (g) + fish tissue weight (g)j * 1000 mg^g * IDOO 6'K*. FishTissue Weigh! (s> o.y9i 1.0534 1.0629 0.5316 1.7044 1.5720 12386 1.1749 0.9663 0.6989 0.5332 1-1122 1.0888 1.3244 0.7595 0.8266 0.6912 0.7963 0.8681 0.6457 0.7488 0.6174 0.7166 0.6388 0.9453 0.9085 D.S717 0.rfS#l Ltpid'Fish Tissue Weight1 10200 13200 9130 11100 10400 11700 17000 11800 15200 35500 18300 15100 1261X3 11900 12200 13200 19100 20100 15600 13000 12200 15900 13800 13500 15000 17700 16800 14300 p. 146 .__Wildlife International, Ltd -70- Project Number 454A-117 Table 12 Lipid Content in Noncdible Fish Tissue NominalTeat Concentration (mga.i./L) UO (Negative Control) Sample Number (454A-117) NL1 NU NL3 NL4 .NX? NL8 NL9 NL1 NUl NL22 NL23 NL24 ?ha:e Uptalte UpUke Depuration Sampling Time (Day] 0,Chour 0,0 hour 00hour 0, 0hour 2S 28 28 28 14 14 14 14 Lipid Weight is) 0.0181 0.0335 0.0155 0.0134 0.0248 0.0271 0.0253 0.0195 0.0252 0.0167 00162 0.02X3 . 0.30 NLU Uptake NLL2 NU3 NL14 NL26 Depuration NL17 NL.2S NL29 2* 28 22 28 14 14 14 14 0.0315 0.0293 0.0188 0.0328 0.0201 0.0405 0.0284 0.0191 5.0 NL1 Uptake 28 0.0220 NU-7 28 0.0283 NUS 28 0,0240 NLI9 28 0.0186 NUl Depuration 14 0.0403 NU2 14 0.0394 MNLU343 14 0.0340 14 0.0127 1Ratiocalculatedas(Jipulweight(g)+ fishtissueweight(k)1a 1000mg/gx 1000g/Kg. FishTissue Weight is) 1.3008 1.3820 1.2944 0.7521 t.29U 1.2749 1.2049 1.2714 1.1730 0.8990 0.8678 0.9681 1.3192 1.6196 0.8519 1.1668 1.0539 1.0630 1.2032 1.0488 1.1230 0.9749 1.0825 0.9275 1-1694 1.0638 0.9449 0,9560 Lipid/Fish Tissue Weight' (mgi'Kg) 13900 242CO 120C0 17800 19200 21300 21000 15300 21500 18600 18700 29200 23900 18100 32100 28100 19100 38100 23600 18200 19600 29000 22200 20100 34500 37000 36(100 0300 p. 147 , ___Wildlife International Ltd. ~ 71 - Project Number 454A-117 METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) IN FRESHW ATER ' Prepare matrix fortification samples in freshwater matrix by spiking the requisite volume o f PFBS stock solutions directly into freshwater. Perform fortifications with gas-tight syringes and Class A volumetric flasks. Prepare appropriate dilutions o f study and QC samples to within the calibration range o f the PFBS LC/MS methodology: Partially fill Class A volumetric flasks with dilution solvent (50% methanol: 50% NANOpure water). Add appropriate volume o f sample and bring to volume with dilution solvnt. Process matrix blank samples using the same dilution and aliquot volumes as for the lowest fortification level. Mix well by several repeat inversions. i I Ampulate samples and submit for LC/MS analysis. Ii I Figure 1. Method flow chart for the analysis o f Perfluorobutane Sulfonate, Potassium Salt (PFBS) in freshwater. p. 148 ___Wildlife International, Ltd. -72- Project Number 454A-1 7 METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) IN FISH TISSUES Quality control samples are prepared from aliquots (approximately 1 g) o f bulk control fish tissue homogenate. Remove appropriate (edible or nonedible) aliquots from frozen storage and allow to thaw. Fortify the QC (MAS) samples with the appropriate PFBS stock solution using gas-tight syringe(s). The matrix blank (MAB) sample will not be fortified. '4 Add 10.0 mL of methanol to each sample with a glass Class A volumetric pipette. Homogenize each test sample for approximately 1 minute using a hand-held tissue shredder. Rinse the homogenizer with the appropriate solvent(s) in between samples. i Sonicate each sample for approximately 5 minutes with a sonic dismembrator i Cap the vials and shake well. Centrifuge the vials at approximately 2000 ipm for approximately 5 minutes, Prepare appropriate dilutions o f stu dy and QC samples to within ihc calibration range o f the PFBS LCMS methodology: Partially fill Class A volumetric flasks with 50:50 methanol;NANO*pure water dilution solvent. Add appropriate volume o f sample and bring to volume with dilution solvent. Process matrix blank samples for a given matrix using the same dilution and aliquot volumes as for the lowest fortification level in that matrix. Mix well by several repeat inversions. 4- Transfer an aliquot of each sample to an autosampier vial and submit for LC/MS/MS analysis. Figure 2. Method flow chart for the analysis o f Pcrfluorobutane Sulfonate, Potassium Salt (PFBS) in fish tissues. p. 149 .Wildlife International Ltd -73 Project Number 454-117 METHOD OUTLINEFORTHEANALYSISOFLIPIDS IN FISHTISSUES Removevialstobeanalyzedfromthefreezer. Allowample*tothaw. 4 F w each sample, add 10 mL of NlANOpure water to liih tissue in v iilin d hotnogcrizc far approximately I minute using a hand-held tissue shredder. Rinse the homogenises wrth the appropriate soiventfs) in between samples. 4 Transfereachhomogenatelos230-mLseparatoryfiittnel thatcontain] 23mLofchloroformand30mLofmethanol. 4 Rinseeachvial withaitadditional It) mLofNANOptire*waterandpourrinseintorespectiveseparatoryftmael 4 Shakeeachsepaiatoryfunnelforapproximatelyoneminutewithventing. .4 Add30mLofchloroformfollowedby30mLofsaturatedsodiumchloridetoeachseparatoryfunnel. 4 Brieflyswirl eachseparatoryfunnelwithventing. 4 Allowthephasestoseparate. Foreachsample,drainthechloroformlayerthroughapowderfunnelpockedwithTeflon"wooland anhydroussodiumsulfateintoa230-mLround-bottomflask. A d d Mil ltdd t t o n t i S0-VTl- a ^ g o t o f d h lu ro lo n r i I >> p w t O r y i u m l n d repeal M tir t/tC tio * A nd d i i n i n j f p r p M c lu m . I RoUxyevaporatetheextract*inawaterbathmaintainedatapproximately40*Ctoneardryness. ; Transfereachextracttoaprc-wcighcd,labeledscintillationvil. Rinseeach250-mLrcuud-bottomflaskwithasmall volumeofchloroformandtransferrinsetorespectivescintillationvial. Evaporatetheremainingsolvent ineachvial underagentlestreamofnitrogenorelcandryair. i Reweigheachvialandrecordweight Figure 3. Method flow chart for the analysis of lipids in fish tissues. __Wildlife International, Ltd. -74- p. 150 Project Number 454A-117 Figure 4. A typical calibration curve for Perfluorobutane Sulfonate, Potassium Salt (PFBS) in freshwater. Slope -- 72902,35156; Intercept = 2116.42700; r = 0.9998. Curve is weighted (1/x). __Wildlife International Ltd. ' -75 - p. 151 Project Number 454A-] 17 Figure 5. A typical calibration curve for Perfluorobulane Sulfonate, Potassium Salt (PFBS) in fish tissue. Slope = 15309.60; Intercept = -1042.05; r = 0.99793. Curve is weighted (!/x). ___Wildlife International, Ltd. -76- p. 152 Prqject Number 454A-117 intensity: 60000 cps Figure 6. A representative ion chromatogram of a low-level (0.500 pg a.i./L) Perfluorobutane Sulfonate, Potassium Salt (PFBS) standard for freshwater analyses. __Wildlife International, Ltd. a - p. 153 P ro ject N u m b er 45 4A - 117 intensity 60000 cps Figure 7. A representative ion chromatogram of a high-level (5.00 (i.g a,i./L) Perfluorobutane Sulfonate, Potassium Salt (PFBS) standard for freshwater analyses. , ___Wildlife International Ltd. -78- p. 154 Pfoject Number 454A-117 intensity: 6000 cps Figure 8. A representative ion chromatogram o f a low-level (0.500 jig a.iVL) Perftuorobutane Sulfonate, Potassium Salt (PFBS) standard for fish tissue analyses. , ___Wildlife International Ltd. -79- p. 155 Projsct Number 454A-117 intensity: 6 000 cps fig u re 9, A representative ion chromatogram of a high-level (3.00 pg a.i./L) Perfluorobutane Sulfonate, Potassium Salt (PFBS) standard for fish tissue analyses. , __Wildlife International Ltd. -80- p. 156 Projsct Number 454A-117 intensi ly: 60000 cps Figure 10. A representative ion chromatogram of a freshwater matrix blank sample (454A - 117 - M A B - 1, dilution = 250x). The arrow indicates the retention time of Perfluorobutane Sulfonate, Potassium Sait (PFBS). Wildlife International, Ltd. -81 - Project Number 454A-117 intensity: 60000 cps Figure 11. A representative ion chromatogram of a freshwater matrix fortification sample <454A -117-M A S-2, 2 .0 0 mg a.L./L nominal concentration, dilution -- IQOOx). p. 158 , __Wildlife International Ltd. -82 - Project Number 454A-117 1 DO90* 80 70 60 50 40 30 20 10* o-- imensiiy: eooo cps 1 I 714i87 111 141 18B I ' I - r'" I I " 41 81 121 161 201 0.69 1.36 2.03 2.70 3.37 254 2 4 1 ' 281 Scan 4.04 4.71 Time Figure 12. A representative ion chromatogram o f an edible fish tissue matrix blank sample (454A117-E-MAB-3, overall dilution factor = 100 L/Kg). The arrow indicates the retention time o f Perfluorobutane Sulfonate, Potassium Salt (PFBS). p. 159 __Wildlife International, Ltd. -83 - Project Number 434A-117 intensity: 6000 cps Figure 13. A representative ion chromatogram o f a nonedible fish tissue matrix blank sample (454A- 117-N-MAB-2, overall dilution factor = 100 L/Kg). The arrow indicates the retention time o f Perfluorobutane Sulfonate, Potassium Sait (PFBS). For a discussion o f the integrated peak in vicinity of the analyte retention time, see page 48. , __Wildlife International Ltd. -84 p. 160 Project Number 454A-117 intensity: 6000 cps Figure 14. A representative ion chromatogram o f an edible fish tissue matrix fortification sample {454A-117-E-MAS-5, 0.100 mg a.i,/Kg nominal concentration, overall dilution factor = 100 L/Kg). Note: fortification level = 2 x LOQ {0.0500 mg a.i./Kg). Wildlife International, Ltd. -85 - Project Number 454A-117 intensity: 6000 cps I______________________ I Figure 15. A representative ion chromatogram of a nonedible fish tissue matrix fortification sample (454A-117-N-MAS-3, 0 100 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg). Note interferent on shoulder o f analyte peak , .__Wildlife International Ltd -86- p. 162 Projec; "Number 454A-1 ] 7 Imansity: 60000 epa ------------------------------ ------------------------------------------------------ --- -------------------- --- -------------------1 Figure 16. A representative ion chromatogram o f a freshwater sample (454A-117-3, dilution = 250x) from the 0.50 m g a.i./L treatm ent group. p. 163 , __Wildlife International Ltd. -87 - Project Number 454A-117 intensity: 6000 cps Figure 17. A representative ion chromatogram of tin edible fish tissue sample (454A-117-E-33, overall dilution factor = ! 10) from the 0.50 mg a.i./L treatment group). __Wildlife International, Ltd. -88 - p. 164 Project Number 454A-117 intensity: 6000 cps Figure 18. A representative ion chromatogram of a nonedible fish tissue sample (454-U7-N-33, overall dilution factor = 73 .5) from the 0.50 mg a.i./JL treatment group) - 89- APPENDIX IV Temperature and pH of Water in the Test Chambers Uptake Phase_________ Sponsor: Test Substance: Test Organism: Dilution Water. 3M Corporation PFBS Bluegill, Lepomis macrochirus Well Water ____ Uptake Phase Mean Measured Concentration (mg o.i./L) Negative Control Day 0 Temp.1 22.1 th 8.2 Day 7 Temp.1 TO 22.0 pH 8.1 Day 14 Temp.1 TO) 22.0 pH 8.0 Day 21 Temp.1 TO PH 22.0 i 8.0 0.53 22.1 8 2 2 2 0 8.1 22.0 8.0 22.0 8.0 5.2 22.1 8.2 22.1 8.1 22.0 'Continuous measurements of temperature remained at approximately 22.0C. 8.0 Uptake Phase Mean Measured Concentration (mg a i./L) Negative Control Depuration Phase Day 7 Day 14 Temp.' rc) 22.0 PH 8.4 Temp.1 T O ... -P. 22.0 8.1 22.0 8.0 Day 16 Temp,1 TO 21.9 pH 8.0 0.53 21.9 85 22.0 8 1 21.9 8.1 52 21 9 8.5 22.0 8.2 21.9 1Continuous measurements of temperature remained at approximately 22.0C. 8.1 Project Number 454A -117 Day 28 Temp.' TO 220 pH 8.0 22.0 8.1 22.0 8 0 p. 165 p. 166 Wildlife International, Ltd. -90- Project Number 454A-11? Sponsor: Test Substance: Test Organism: Dilution Water: Day APPENDIX V Dissolved Oxygen (rog/L) o f Water in the Test Chambers1 3M Corporation PFBS Bluegill, Lepomis macrochirus Weil Water Negative Control Uptake Phase 0.53 rag a.i./L 5.2 rag a.i./L 0 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 8.7 7.7 7.8 8.3 8.2 8.2 8.1 8.6 8.4 8.5 , 8.4 8.4 8.6 8.6 7.6 7.9 7.4 8.1 8.2 8.1 7.9 7.9 8.L 8.1 8.0 8.2 8.1 8.7 8.0 . 8.7 7.7 7.8 8.3 8.1 8.2 8.0 8.6 8.4 8.5 8.3 8.4 8.5 8.6 7.7 7.9 7.4 8.0 8.) 8.1 7.9 8.0 7.9 8.0 8.2 8.3 8.1 8.6 8.1 8.7 7.6 7.8 8.3 8.0 8.2 8.0 8.6 8.5 8.5 8.3 8.4 8.5 8.5 7.7 7.8 7.4 8.0 8.1 8.1 7.9 7.8 7.9 7.8 8.2 8.2 8.2 8.8 8.4 lA dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22C. p. 167 Wildlife International, Ltd. -91 - Project Number 454A-117 Sponsor: Test Substance: Test Organism: Dilution Water: APPENDIX V (Continued) Dissolved Oxygen (m.q/L) of Water in the Test Chambers' 3M Corporation PFBS Bluegill, Lepomis macrochinas Well Water Depuration Phase Day Negative Control 0.53 mga.i./L 5.2 mg a.i./L 1 7.8 7.8 7.9 2 7.9 8.2 8.0 3 8.2 8.3 8.3 4 3.2 8.2 8.1 5 . 8.4 8.4 8.2 6 8.6 8.8 8.8 7 8.4 8.2 8.2 8 7.6 7.8 7.9 9 8.7 8.7 8.6 10 8.7 8.7 8.7 11 8.0 8.2 8.2 12 8.0 8.4 8.4 13 8.0 8.0 8.0 14 8.6 8.4 8.4 15 8.4 8.3 8.3 16 8.6 8.7 8.6 'A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22C p. 168 Wildlife International, Ltd. -92- Project Number 454A-117 APPEND1X VI Hardness, Alkalinity, Conductivity and TOC of Water in the Negative Control Uptake Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Bluegill, Lepomis macruchirus Well Water Parameter 0 Day ? 14 Hardness 128 128 104 (mg/L as C aC 03) Alkalinity (mg/L as CaCOj) 164 . 174 166 21 128 174 Conductivity Cumhos/cm) 330 330 330 TOC <1 <1 <1 <1 (mg CTO 1Conductivity inadvertently not recorded on Day 7 28 112 180 300 <1 Depuration Phase Spunsor: 3M Corporation Test Substance: PFBS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water_______ Parameter 7 Day 14 Hardness (mg/L as CaCOj) 116 116 Alkalinity (mg/L as CaCOj) 180 184 Conductivity 0-imhos/cm) 330 325 TOC (mg C/L) <1 <1 16 116 182 325 Not Measured p. 169 Wildlife International, Ltd. 93- Projcct Number 454A -1]7 APPENDIX VII Cumulative Percent Mortality and Treatment-Related Effects1 Negative Control - Uptake Phase Sponsor Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Bluegill, Lupomis macrochtrus Well Water Cumulative Number Day Observations Dead 0 AN I AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 . AN 9 AN 10 AN 11 AN 12 AN 13 AN 14 AN 15 AN 16 AN 17 AN IS AN 19 AN 20 AN 21 A N 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 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 'Observed Effects: AN " Appears Normal Number Remaining 85 80 75 75 70 70 70 70 65 65 65 65 65 65 65 60 60 60 60 60 60 60 55 55 55 55 55 55 55 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 D 0 0 10 p. 170 Wildlife International, Ltd. -94- Project N um ber 454A -117 APPENDIX VII (Continued) Cumulative Percent Mortality and Treatment-Related Effects' Negative Control - Depuration Phase Sponsor: Test Substance: Test Organism: Dilution Water; 3M Corporator) PFBS Hlueyill, I.epvniis macroc'nirui Well Water Cumulative Number Day Observations Dead 1 AH 2 AN 0 0 3 AN 0 4 AN . 0 5 AN 0 6 AN 0 7 AN 0 8 AN 0 9 AN 0 10 AN 0 U AN' 0 12 AN 0 13 AN 0 14 AN 0 15 AN 0 16 AN 0 iObserved Effects: AN *AppearsNormal. Number Remaining 45 . 40 40 35 35 35 35 30 30 30 25 25 25 25 15 15 Number Sampled 5 0 s 0 0 0 s 0 0 5 0 0 10 0 0 p. 171 Wildlife International, Ltd. -95 - Project Number 454A-117 APPENDIX VU (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 ; a.i./L - Uptake Phase Sponsor lest Substance: 'Jest Organism. Dilution Water: 3M Corporation PFBS Blacgill, Lepom is macfochiru.i Well Water Cumulative Number Div Observations Dead 0 1 2 3 3 5 6 7 8 9 10 1! 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 'Observed Effects: AN " Appears Normal Number Remaining 85 80 75 75 70 ' 70 70 70 65 65 65 65 65 65. 65 60 60 60 60 60 60 60 55 55 55 55 55 55 55 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 10 p. 172 Wildlife International, Ltd. ' -97- . Project Number 454A-117 APPENDIX V (Continued) Cumulative Percent Mortality and Treatment-Related Effects' 5.2 mg a.i./L - Uptake Phase Sponsor: Test Substance: I eat Organism: Dilution Water; 3M Corporation PFBS Bluegillf Lipom i* macrochirus Well Water Cumulative Number Day Observations Dead 0 AN 1 , AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN 10 AN n AN 12 AN . 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN 72 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 11 1 1 'Observed Effects: AN " Appears Normal Number Remaining 85 80 75 75 70 70 70 70 65 65 65 65 65 65 65 60 60 60 60 60 60 60 55 54 S4 54 54 54 54 Number Sampled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 O 0 0 0 0 0 10 p. 173 Wildlife International, Ltd. -98 - Project Number 454A-] 17 APPENDIX VII (Continued) Cumulative Percent Mortality and Treatment-Related Effects' 5,2 mg a.i./L - Depuration Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Biucgili, L ep o m is m acrochiruz Weil Water Day Observations 1 2 3` 4 5 6 7 8 9 10 11 12 12 14 IS 16 AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN `Observed Effects: AN " Appears Normal Cumulative Number Dead 1 1 I 1 l 1 1 l 1 1 1 1' I 1 1 1 Number Remaining 44 39 39 34 34 34 34 29 29 29 24 24 24 24 14 14 Number Sampled 5 0 5 0 0 0 0 c 5 0 0 0 10 0 0 p. 174 Wildlife International, Ltd. _______________________________Project Number 454A. I i 7 -99- APPENDIX vnr Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. Amendment: The proposed experimental start and termination dates were added to the protocol. 2. Amendment: The frequency o f TOC measurements was added to the protocol. 3. Amendment: The methodology for TOC measurements was added to the protocol. 4. Deviation: Conductivity of tile negative control water was not recorded on Day 7 o f the test, 5. Deviation: Day 0 tissue samples for lipid analysis were collected prior to distribution to the test chambers. 6. Deviation; A tissue storage stability test was performed 7. Deviation: Light intensity was measured at test initiation. 8. Deviation: Total organic carbon was not measured in tbe dilution water at test termination. 9. Deviation: Weekly fulscope verification was not documented four times during the test. p. 175 Wildlife International, Ltd.________________________________ Project Number 4S4A-117 -100- APPENDIXIX Personnel Involved in the Study The following key Wildlife Internationa], Ltd. personnel were involved in the conductor management of this study; 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Kurt R. Drottnr, Senior Aquatic Biologist 4. Cary A, Sutherland, Laboratory Supervisor 5. Raymond L. Van Hoven, Ph.D., Scientist 6. Susan T. Plantania, Biologist