Document omdMLLmq3D79r49KL7pOJ7yRr

18} OE CD 305-OPPTS 850 .1730,Fish bioconcentration (ioluegil l ) p . 76 p . 77 PERFLUOROBUTANE SULfiO1vATE, POTASSIUM SALT ( PFBS) ; CiaN~T~ZE~ A FLOW-THROUGH BJOCONCENTRATION TEST WITH THE ~7 1 BLUEGILL (Lepom;s macrochrrus) ` FINAL REPORT DE C O ~ ~QQ3 WILDLIFE INTERNATIONAL, LTD, PROJECT NUMBER : 454A- 11 7 3M ENVIRONMENTAL LABORATORY REQUEST NUrMBER : E00-1429 U .S . Environmental Protection Agency Seri es 850 - Ecological Effects Tcst Guidelines O PPTS N umber 8 5 0 .173 0 and OECD Guideline 305 AUTHORS ; Kurt R Drottar Raymond L . Van Hoven, Ph .D . Henry 0 . Krueger, Ph .D . STUDY INITIATION DATE : June 29, 2000 STUDY COMPLETION DATE : May 9, 2001 Submitted to 3~y1 Corpora tion Environmental Laboratory P .O. Box 3333 1 St . Paul, Minnesota 5513 3 Wildlife .tn tem ational, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 100 p . 78 ~Vrl~l~f~ In te"'atinngla Ltd -2 . GOOD LABORATORY PRACTICE COMPLIANCE STATE1ViENT SPONSOR: 3M Corporatio n TITLE : Ptrtiuorot;utane S ulfonate, Potassium Salt (PFBS) : A Flow-Through .Bioconcentration Test with the Blue ;ili (Lepomis macrochrrus ) WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER : 454A-117 STUDY COMPLETtON : May 9, 200 1 This study was corducted in compliance with Good Laboratory Practice Standards as published by the U .S . Environmental Protection Ageitcy in 40 CFR Part 792,17 August 1999, with the following exceptions : The test substance was not characte rized 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 pu rity. The stability of the test substance under conditions of storage at the test site was not determined as required by Good Laboratory Practice Standards . STUDY DIRECTOR : Kurt R. Drottar DATE Senior Biologis t SPONSOR 4PPROVAL DATE N v; ApplicandSubnvttzr DATE p . 79 Wrfdl7 P 171"l tPYf2L~'t~Q~ al,r Ltd. 3- PmjP ci N uaiher~ i4A-1 17 QUALITY ASSURANCE STATEMEN T 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, l7 August 1989 . The dates of all inspections and audits an d the dates that any findings wcrc reportcd to the Study Director and Laboratory Man ngeirter.t were as follows : VATS RE-PORT-ED'ro : AC'I'1 Vf T Y: DATE CONDUCTED: STUDY DIRECTOR : MANAGEMENT : Test Substance Preparation July 13, 2000 July 13, 2000 July 17, 20t)0 Matrix Fortification July 24, 2000 July 24, 2000 July 25, 2000 Tissue Sampling, Fish Lengths and Weight Measurements and Tissue Preparation Analytical Data and Draft Report " August 22, 2000 August 22, 2000 August 23, 2000 September 29, October 2- 6 October 9, 2000 October 16, 2000 and 9, 2000 Biological Data and Draft Report Oetubcr 23 - 26, 2000 October 26, 2000 October 31, 2000 Fuial Report May 9, 2001 May 3, 2001 May 9, 2001 ~ ~00 Ntarsh T. Hynson DAT E Qualit ssurance Program Supervisor I p . 80 Wildlife International , Ltd, -4- REPORT APPROVAL pmiect N,,mhec-4-54A 117 SPONSOR : 3M Corporation TITLE : Perfluorobutane Sulfonate, Potassium Salt (PFBS) : A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochirus ) WiLDLIF'E INTERNATIONAL, LTD . PROJECT NUMBER : 454A-117 STUDY DIRECTOR : Kurt R. Drottar Senior Biologist s~91 n DAT E MANAGEMENT : Henry 0 . eger, Ph.D . Dr'1T Director of Aquatic Toxicology and Non-Target Plants p. 81 Wildlife Lutemational, Ltd. -5 Pr e{t hL=Fp r A S A e_ IL 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 Materi als and Methods . . . . . . . . . . . . . . . . . .. . . . . .. . .. . . . . . . . . . . . . . . .. .. . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . 10 Results and Discussion., . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ._ . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . 15 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . 17 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 18 TABLE S Table 1 - Means and Ranges of Water Quali ty Parameters . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . .. . . . . ._ . .. . . . . . . . . . . . . . . . . . . . 19 Table 2- Concentrations of PFBS in Water Samples During the Uptake Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Table 3 - Concentrations of PFBS in Water Samples Du ri ng the Depuration Phase . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 21 Table 4- PFBS Concentrations in Edible, N onedible and Whole Fish Tissues of Bluegill Exposed to 0 .53 mg a .i./L . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Table 5- Steady-State BCF Values for Bluegill Exposed to 0 .53 mg a.i ./L . . . . . . . .. . . . . .. . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . 25 Table 6-$fOFAC Model Estimates for Blucgill Exposed to 0 .53 mg a .i .lL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 p . 82 ~ .~ Wildlife Iyaem atioytaLlP Ltd- -6- 1!rPjf'crN11mher454A-1 17 TABLE OF CONTENT S - Continued - TABLES (Cont'd .) Table 7 - PFBS Concentrations in Edible, Nonedible and Whole Fis h Tissues of Bluegill Exposcd to 5 .2 mg a .i.IL . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 27 Table 8- Steady-State BCF Values for Bluegil l Exposed to 5 .2 mg a.i.lL . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Table 9 - BIOFAC Model Estimates for Bluegill Exposed to 5 .2 mg a.i,fL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 31 FIGURES Figure 1 Concentrations of PFBS in Edible Fish Tissues of Bluegill Expose d to 0 .53 nzg a .i .ll.. . . .. . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . ., . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 32 Figure 2 Concen trations of PFBS in Noaedible Fish Tissues of Blucgill Expose d to 0 . 5 3 mg a.i . /L. . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 33 Figure 3 Concentrations of PFBS in Whole Fish Tissues of Bluegill Expose d to 0 .53 mg a.i .1L. . . .. .. . .. . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . .. . . . . . . . . . . . . . .. . 34 Figure 4 Concentrations of PFBS in Edible Fish Tissues of Bluegill Exposed to 5 .2 mg a .i ./L . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . -- . . . . . . . . . . . . 35 Figure 5 Concentrations of 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 Bluegilt Expose d to 52 mg a .i ./I,. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 p. 83 Wildlife MtematxnnaZ. Ltd. Project NrmhpL ;4A 1 7 TABLE OF CONTENTS - Continued - APPENDICES Appendix I Specific Conductance, Hardness, Alkalinity and pH of Well Wate r Measured During the 4-Wcck Period Immcdiately Preceding the Test .. . . . . . . . . . . . . . . . . . . . . . . . .: 38 Appendix II Analyses of Pesticides, Organics and Metal s in Wildlife International, Ltd . Wall Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Appendi x III The Analysis of Perflurobutane Sulfonate, Potassium Salt (PFBS) Concentrations in Fresh w ater and BluegilI Sunfish Tissue in Suppor t of Wildlife Intemational, Ltd . Project No. : 45 4A-1 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Appendix IV Temperature and pH of Water in the Test Chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Appendix V Dissolved Oxygen of Water in the Test Chambers . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Appendix V( Hardness, Alkalinitv, Conductivi ty and TOC of Water in the Negative Control . . . . . . . . .. . . 92 Appendix VII Cumulative Percent Mortality and Treatment-Rclatcd Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Appendix VIII Changes to Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99 Appendix IX Personnel Involved in the Study . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . .. . . 100 p . 84 Wildlife~T~te~~atio~al -8_ SUMMARY SPONSOR: 3M Corporation SPONSOR'S REPRESENTATIVE : Ms . Susan A . Beach LOCATION OF STUDY RAW DATA AND A COPY OF THE Wildlife International, Ltd . FINAL RE, OttT : Easton, Maryland 21601 WILDLIFE INT-n.;RNATIONAL , LTD . PROJECT NLP.vIBER : 454A-11 7 TEST SUBSTANCE : STUDY : NOMINAL TEST CONCENTRATIONS : MF'?AN MEASURED TEST CONCENTRATIONS : Perfluorobutane Sulfonate, Potassium Salt (PFBS ) Perfluorobutanc Sulfonate, Potassium Salt (PFBS) : A FlowThrough Bioconcentration Test with the $luegill (Lepnrnis macrochirus) Negative Control, 0 .50 and 5 .0 mg a.i .'L Negative Control, 0 .53 and 5 .2 mg a.i./L TEST DATES : Experimental Start (OECD ) - July 13, 2000 Experimental Start EPA) - July 18, 2000 Biological Terminatioa- August 31, 2000 Experimental Termination - September 13, 200 0 LENGTH OF TEST : 44 Days 28-Dav Uptake, 16-Da De uration TEST ORGANISM : Bluegill (Lepomis macrochirus) SOURCE OF TEST ORGANISMS : Osa g e Catfisheries, Inc . 1170 Nichols Road Osage Bcach, Missouri 65065 AGE OF TEST ORGANISMS : Juveniles MFASLIREMENTS OF 10 NEGATIVE CONTROL FISH COLLECTED AT TEST TERtifINATION : WEIGHT (g): Mean = 1 .81 ; Range ~ 1 .3? to 2 .76 TOTAI. LENGTH (mm) : Mean = 55 ; Range - 50 to 6 1 RESt7LTS : ( 0 .53 mg a.i ./L) Edible Nonedible STEADY-STAT: BCF : 0.21 0 .51 Whole Fish 0 .38 RESULTS : (5 .2 mg a .i .(L .) Edible Nonedible Whole Fish STEADY-STATE BCF: 0 .16 0.43 0 .30 p. 85 Wildlife Intemat anal. LU. - Pro' S ,7 9-INTRODUCTION A bluegill sunfish, Lepomis macroch ir,rs, bioconcentration study was conducted for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the testwas conducted from July 18, 2000 to August 31, 2000 . Raw data generated by Wildlife Internati onal, Ltd . and a copy of the final report are filed under Project Number 45 4A- 117 in archives located on the Wildlife Intcrnational, Ltd . site . OBJECTIVE The objective of this study was to determine the bioconcentration potential ofperfluorobutane sulfonate, potassium salt (PFBS) in the bluegill sunfish . EXPERIMENTAL DESIG N 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 we of three groups : 1) A negative ( dilution water) control ; 2) A nominal concentration of 0 .50 mg active ingredient ( a. i . )/I . ; or 3) A nommai concentration of 5 .0 mg a.i ./I.. . Test concentrations were based on the potassium salt of parfluorobutane sulfonate ( PFBS). At the start of the deputation phase, stock flow to the treated groups was stopped and the biuegill wcre exposed to dilution water without 1'FBS for the rcmainder of the test. Each test chamber contained 85 bluegill at test initiation, and one replicatc was tested for each treatment and the negative con tr ol . Water samples were collected on Day -2 ( pre-test), Day -I (pre -test) on uptake Days 0 ( 0 and 4 hours) ; 1, 3, 7, 14, 21 and 28 and on depuratiun Days 1, 3, 7, 10 and 14 during the test and analyzed for PFBS using liquid chromatography-mass spectrometry (LC/MS) . Tissue samples were also collected at selected water sun ple collection periods during the test and analyzed for PFBS by LC/MS . The results of 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 were not analyzed due to the pattern of deptrration observ ed . p . 86 Wildlife 1'h f.PrnatTon d , .t - I0 . Dr~irr+>tltl~:~r 454n i i 7 MAT E RIALS AND METHODS The s~udy was conducted according to the procedures outlined in the protocol, "Perfluorobutane Stdfonate, Potassium Salt (PFB S) . A Flow-Through 6ioconcentration Test with the BlueQill (Lepomis macrocnirus)" . The prorocol was based on procedures outlined in U .S . Environmental Protection Agency Series 850 - Ecolo gical Effects Test Guidelines OPPTS Number 850.1730 ( 1) ; ASn1 Standard E1022-84 Standard Prachce for C'onducring Bi oconcentration Tesrs with Fishes and Saltwater Bivalve ,.Wolluscs (2) ; and OECD Guideline for Testing of Chemicals 30 5 , B i oconcentrarivn : Flo w-Through Fish Test (3) . Test Substanc e The test substance was received from 3M Corporation on March 27, 2000 and was assigned Wildlife International, Ltd, identification number 52 E6, The test substance, a white powder, was identified as Potassium Pertluorobutane Sulfonate, lot 44 2, TCR-00017-71 . Information provided by the Sponsor indicated a puri ty of 97 .90% and an expiration date of March 2010 . A subsequent revision of the cert ificate of analysis indicated a puri ty of 97 .3% and an E:cpirationlReassessme.nt Date of January 17, 2002 . The test substance was stored under ambient conditions . PrepaX a tion of.Te t Solution s The nominal test concenrntrations were 0 .50 and 5,0 mg a .i ./L . Two stoc k solutions were prepared at concentrations of 5 00 and 5000 mg a.i .lL . The appropriate a tnount of =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 solubiiization of the test substance. After mi xing, the stock solutions appeared clear and colorless, Stock solutions were prepared at approximately weekly intervals during the uptake phase of the test . The stock solutions were injected i nto the diluter mixing chambers ( at a rate of 0 .3 5 0 mL/minute) wherc they were mixed with dilution water ( at a rate of 350 inL/minute) to achieve the desired test concentrauans . All test concentrations were adjusted for the original repo rted purity of the active ingredient (potassium perfl uorobutane sulfonate) in the test sub s tance (97 .9%) . p . 87 Wildlife jnt rnc~tinna r~ ~~td. -11- Frn * rr hbirnhcr i4,~ 117 Test Or g anism The bluegill, Lepomis macrochirus, was selected as the test species for this study . The bluegill is one of the recommended freshwater fish species for use in bioconcentratien tests (1, 2, 3) . Btuegill 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 inteneational, Ltd . well water for 20 days prior to testing . The bluegill were fed at least once daily during holding. The fish were acclimated 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 p re ceding 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 mpl. . Instrumentation used for water measurements are described in the Environmental Conditions section of th is repo rt . At test initiation, the bluegill were collected from the acclimation tank and indiscriminately dis tributed 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 A rtenua, Ogden, Utah . Bluegill were not fed during the acclimation pe riod, however, they were fed fl ake food once daily during the test. Feeding and sampling schedules were coordinated so that fish were sampled at least four hours after feeding. A ll fish used in the test were from the same source and year class, and the standard length of the longest fish was no more than twice the length of the shortest . The length and weight of tish in the negative control were considered to be representative of all fish used in the test. The mean total length of 10 negative control fish measured at the end of the test was 55 mm with a range of 50 to 61 mm . The average wet weight ( blo tted dry) was 1 .81 grams with a range or 1 .37 to 2 .76 grams . Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was determined to he 0 .31 g fishlL/day . Test Apparatus A continuous-fl ow diluter was used to deliver each concentration of the test substance and a negative control . A peristaltic pump (Cole- Partner lastrument Company, Chicago, Illinois) was used to deliver the test substance stock solutions control into mixing chambers assigned to each PFBS treatment. The stock solutions p . 88 Wildlife _ I",M. . 12- ern-j rrr Niimhe r were mixed with dilution water in the mixing chambers p rior to delivery to the test chambers . The Row of dilution water to the test chambers was controlled by rotameters. The delivery of water from the rotar.leters was checked prior to the test and at approximately weekly interv als thereafter . Approximately 6 .3 volume additions of test water were delivered to the test chambers evcry 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 aquari a filled with approximately 80 L of test solution . The depth of the test water in a representative chamber was approximately 19 cm . Test chambers were indiscriminately positioned in a temperature-con tr ollcd water bath designed to maintain a constant tcmperaturc, The water bath was enclosed in a plexiglass ventilation hood in order to m :nis.ize any potential for crosscontamination. 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 Wate r The water used for holding and tcsting was freshwater obtained from a well approximately 45 meters deep located on the Wildlife International, Ltd. site . The well water is characterized as moderatcly-hard water . The specific conductance, hardness, alkalinity and pH measurements of the well water during the four-week period immediately preceding the test are presented in Appendix 1 . The well water was passcd through a sand filter to remove particles greater than approximately 25 Fun, and pumped into a 37,80 0-L storage tank and aerated with spray nozzles . Pri or to use, the water again was filteret! (0 .45 pun) to remove microorganisms and particles . The results ofperiodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International, Ltd . are presented in Appendix H. Environmental Conditions The target temperature range for the test was 22 1C . Temperature was recorded continuouslv in the negative control with a pulscope ER/C Recorder (1900 J Series model no. A) . Temperature was also measured -in all test chambers at the beginning and end of the test and at weekly intervals during the test, with a liquid-in-gl ass thermometer . _ --..~. Ml<dlife In teYn atinn.xl, L td -13- p . 89 ----------------- Pr(uer t 1N i mhA QSQL4 1 i, Dissolved oxygen was measured with a Yellow Springs Instrument Company, Inc. Model 51B dissolved oxygen meter . Measurements were made daily in each test chamber during the test, Measurements of pH were made 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 alkalini ty were measured by titration based on procedures in Standard Methods foi r the Exa mination of Water and Wastewater (A) . Conductivi ty was measured using a Yellow Springs Instrument Company, Inc . Model 33 Salinity-Conductivity Temperature meter. Total organic carbon was measured using a Shimadzu model TOC-5000 total organic carbon analyzer. Ambient room light was used to illuminate the test systems. Fluorescent tubes that emitted wavelengths similar to natural sunlight ( Colortonem 50) were controlled by an automatic timer to provide a photoperiod of 16 hours oflight and 8 hours of darl.ztess . A 30-minute transition period of low light intensi ty was provided when lights went on and off to avoid sudden changes in light intensity . Obcervations All fi sh were observ ed once each day to evaluate Lhc number of mortalities and the nwnber of individuals exhibiting signs of abnormal behavior. Procedures for Exposw ; of Fish to PFB S The test chambers were conditioned by delivering PFBS to the diluter system for app roximately 5 days before adding the fish. Water samples were collected twice during the pre-test period to confirm that equilibrium concentrations of 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 July 18, 2000 by placing the fish in the test chambers . Bluegill were impartially removed from the ho lding tank in groups of I to 2 . The groups ofbluegi]1 distributed among the test chambers until each test chamber contained 85 fish. The duration of P . 90 ------------------- Wildlife In teYnatinna r d I, -14- Pr umhrr4~4A 11 ~ the uptake phase was 28 days . At the end of the uptake phase, stock flow to the treatment groups was stopped and the bluegill were exposed to dilution water without PFBS for a period of 16 days. Collection and Analysis Q Water Samples Water samples were collected on Days 0(0 hours and 4 hours), 1, 3, 7, 14, 21 and 28 of the uptakephase . Water samples were also collected on Days 1, 3, 7, 10 and 14 of the deputation phase . At each water sampling interval, two water samples were collected from the negative control and three samples were collected from each of the two PFBS treatment groups . One negative control sample and two samples from each of the PFBS treatment groups were analyzed for Pt':BS . 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 wster 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, 21 and 28 of the uptake phase . Tissue samples were also collected on Days 1, 3, 7, 10 and 14 of the depwation phase, At each tissuc sampling interval, a sufficient number of fish were collected to provide two replicate samples of 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 abovc the opercular region . The fish were blocted dry and measured for total length and wct weight within approximately 15 minutes of colloction, when possible . Each fish was then rinsed with dilution water, blotted dry again and dissected into edibic and nonedible tissue fractions . Dissection was accomplished by making an incision from just 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 . All tissue samples were extracted immediately or stored at approximately -14C until extraction . P . 91 W4 ldli& j n L[iI sLatZQILLL4 , Ltd. -15- p rn~lPftT\liimlu'rd5c?, 7] 7 Ti sue Lil2id Conten t Selected fish were collected to determine lipid content (Appendix III). The detertnination of percent lipids provides the potential to express BCF values in terms of 1 ipid content . Fish were sampled on Day 0 of uptake, on Day 28 of uptake and on Day 1 4 of depurztion . All fish collected for lipid content were stored at approximately -l4C until analysis . Data Analvsi s Whole fish concentrations were calculated based on the sum of the edible and nonedible parts . The steadystate bi.oconccntra.tion factor ( BCF) values were determined &om the tissue concentrations at apparent steadystate divided by the average water concen tr ation. Tissue concentrations were considered to be at apparent steady s:ate if :hree or more consecutive sets of tissue concentrations were not signific antly different (p > 0 .05). During this determination, tissue concentrations <LOQ were e li minated from the means . Tissue concentrations were evaluated for normality and homogeneity of variance using the Shapiro-Wilk's test and Bartlett's test, respcctively . 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 of variance and the approp ri ate multiple comparison test (i.e ., Dunnett's test or the Bonferroni t-test) . The kinetic bioconcentration factor (BCFK), uptake rate ( I{,) and depuration rate (Kz) were calculated for edible, nonedible and whole fish using BIOFAC computer software (5) . BIOFAC is a nonlinear parameter estimation routine which cstimates rate constants from a set of sequential timc-conecn tration data. These rate constants are thcn used to calculate a BCFK ( BCFK = KI/Ki). 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 B10 FAC model would not run with zeros) . RESULTS AND DISCUSSION Water Chemis W Means and ranges of temperature, dissolved oxygen and pH of the water in the test chambers are presented in Table 1 . The individual measurements are giv en in Appendicies IV and V . Water temperatures in the test chambers were within the temperature range of 22 i 1C established for the test, Dissolved oxygen concentrations remained L, 7 .4 mg/L ( 85 percent of saturation) t hroughout the test. Measurements of pH ranged from 8 .0 to 8 .5 . p . 92 Wildlife nternatinn ~~d _ _ rY1~tY~'t N~ n m PY 4 1 t Q 1 t 7 -16- Weekly measurements of hardness, alkalini ty , conductivity an d total organic carbon remained consistent throughout the test (Table I and Appendix VI) . Observations of Mortalitv and Clinical Si ms Observations of mo rtality and clinical signs are presented in Appendix VII . Bluegil: in the negative control appeared normal and healthy throughout the test. One bluegilf died in the 5 .0 mg a.i./L (nominal) treatment group on Day 23 of the uptake phase of the test . All other fish in the test appeared normal with no treatment-related signs of toxicity . Concentrations of PFBS in Water Concentrations of PFBS in the negative control were <LOQ (0 .12 5 mg a .i./1.,) (Tables 2 and 3) . Measured concentrations of PFBS duri ng the uptake phase in the 0 .50 mg a .i,/L treatment group ranged from 96 to 119/o of the nominal test concentration . When concen tr ations measured during the uptake phase were averaged, the mean measured concentration was 0 .53 mg a.i./L which represented 106% of the nominal test concentration. Measurr,d concentrations of 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 mg a.iJL which represented 1 0 4/,, of the nominal test conccntration, Concentrations of PFBS during the depuration phase were all <LOQ . Concentrations of PFBS in Fish Tissue s The majori ty of the negative con tr ol tissue samples conlained no qu anti fi able PFBS concen trations (Appcndix 1tI) . However, three edible and four nonedible negative control tissue samplcs contained measurable PFBS . PFBS was not detected in the negative control water du ri ng the test. Consequently, the nsidues mea.sured 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 in a complex ma trix. The concentrations of 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 and 28 tivere not significantly diffcrent (p >0 .05) . The mean measured tissue concentrations during this period of 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 inedible tissue to 0 .272 in noneclible tissuc p . 93 Wildlife Intematr'nnrx~l ~t~. . PrnjPr V"rnhvr Ql t A ~ t - 17 . (Table 5) . BIOFAC estimates of the time to reach 90% of steady state in edible tissue, nonedible 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 elim:nated rapidly with estimates of time to reach 50% clearance of 2 .1, 2 .9 and 1 .3 days for edible tissue, nonedible tissu-- and whole fish, respectively. It should be noted that some of the highest tissue residues were measured on Day 1 of depuration . In addition, the BCFK for edible tissue and whole fish appeared to be overestimated by the 810FAC model as shown in Figures 1 and 3 . The concentrations of PFBS in tissues of fish exposed to 5 .2 mg a.i./G are presented in Table 7 . PFBS concentrations in edible, nbnedible and whole fish tissues appeared to reach steady-state at Day 3 (Figures 4-6) . Dunnctt'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 of time were 0 . 829, 2 .24 and 1 .57 mg a .i./Kg for edible, nonedible and whole fish, respectively, Steady-state BCF values ranged from 0 .16 in edible tissue to 0 .43 in noncdiblc tissue (Table 8) . BfOFA(: estimates of the time to reach 90% of 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 of the test, PFBS was eliminated rapidly with estimates of time to reach 50% clearance of 1 .9, 2 .1 and 2 .1 days for edible tissue, nonedible tissue and whole fish, respectively . CONCLUSIONS Perfluorobutane sutfonate (PFBS) residues rapidly achieved steady-state in the tissues of bluegill sunfish (Lepomis macrnchirus). Steady-state tissue concentrations were achieved after only 3 to 7 days of expostvc . Although steady-state was achieved rapidly, PFBS did not bioconcentrate in bluegill . 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 5 0% clearance ranged from 1 :3 to 2 .9 days . p . 94 Wildlife Inte, 18 . REFERENCES 1 U .S . Environmental Protection Agency . 1996 . Series 85 0 -Ecological Effects Test Guidelines (draft), OPPTS Number 850 .1730 ; Fish 13CP 2 ASTM Standard E1022-84 . 1988 . Standard Practice for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs . American Society for Testing and Materials. 3 OECD Guideline for Testing of Chemicals 305. 1996 . Bioconceiuration : Flow-Through Fish Test. 4 APHA, AWWA, WPCF. 1995 . Standard Merhods for the ,6'xaminarion of Water and Wastewarer. 16th Edition, Ame rican Public Health Association . American Water Works Association_ Water Pollution Control Federation, New York. 5 BIUFAC. 1991 . September 19, 1991 version . The Dow Chemical Company, Midland, Michigan . p . 95 ---------------- Wildlife 1'nterna .inn~1,, r td .19- Ivip , Mirnher , <, ;, 7 Table I Meats and Ranges of Water Quality Parameters Sponsor: 3M Corporation Test Substance: YFD S Test Organism, Bluegill, Lrpomts macrochiru.r Dilution Water : Well Wate r Uptake Phase Nominal Concentration Temperature l D(7' Conducti%,ity Atkalinitv Hardness TO C (mg a .i./F.) ('C) (mg(L) PH (,:cmltos/cm) (rnpJL as C .:CO3) (~ p/i,ias (Cam COyg) C/L) Negative Control 22_0 8 .2 8 .1 324 176 119 <1 21 .9 - 22 . i 7-4-9 . -, 8.0-8 .4 300 - 330 164 - t84 109 - t 28 <! _<I 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 Temperature neasured continuously in the negativa control remained at approximately 22 . 1 At a ternperature of 22 C, be dssolvcd oxygen saturation concentration is 8 .7 mgf- and 60% saturation is 5 .2 mEJ1,. r~ ~ o ' ~-v z u M ~y C J 1c.1 :2 ,,-, v y O O ti v! r`C N ~n ' A !17 Q ~:3 ul bA I .~ L. ~ V Q ~ iA T frN1 N 4 V O C `~ ~ ^ N ~,, fr ,T . ' E" ~V Rf [~ VX WD~ y -4~ 0 1 N O O h w .~. + b I . 0 y N 7 v'1 00 N 'r+vi L 0w a g c v O ""~ ~ U r" h a N F ~ + ~ ! .. N vl M M7 c ^ 4 O V1 v1 I^ O ~ Co v N Qr (~ v c.~ ~ v_ O r, '~7r `"aOacoo c41 cis m u '^ IA Z; ~F? 9 A . U Z C E . .~~ p . 96 . . p. 97 Wildlife Internationa7, Ltd. .21- Project Number 454A-1 1-7 Table 3 Concentrations of PFBS in Water During the Depuration Phase Sponsor: 3M Corporation Test Substance : PFB S Test Organism : Biuegill, Lcoomis mucrochira.s Dilution Water : Well Water Uptake Phase Day of Study Mcan Measured (mg a.iJL1 Concentration (mg a .i./L} 1 3 7 to 14 Negative Control <T_OQI <LOQ <LOQ <LOi? <LOQ 0 .53 <:.OQ <LOQ <LOQ <LUQ <LO Q <LOQ <LOQ <LOQ <LOQ <LOQ 5 .2 <LOQ <LOQ <LOQ <LOQ The Limit of Quantitatioa (LOQ) was 0 .125 mg a.i./L . <LOQ <LOQ <I.nQ <LOQ <LOQ <[AQ ....._ .,~,... p . 98 C ^a c^o ?cs a a~` c~i_ " ~ G o~ v v v'~ o v av ca o~~ ri c o o ~j y o 0 U #= ~ornvc r,ooo c~o,,p a~v{n ~ ~rv,{-: u~r. . ~nn.oN ob x n r 0%0 .-mcac PO N v,0 r,0 ao n ,o ao o-- x . : ~G ~n ~~ r N x~1oav O~C C1 O pq r~p w u o o E ' O 4~ P[T b C O CJ l, b~ c~ u .`J . ..~ ~Q C'-R f"~ c,`~0 ~ oomh c y ay O O O C~ O p .~ Q r ..a _.. {GO.{n0 NN OC~CJ~ OOCD OQOO OOOC {n {o P noOMr 0000 p~, z ~O cJ ~ a u ~QNmn ooNNN NDaov, N oo .--N~O y ' 'L7 L'a _, 'yb v N ao Q aO ~G l~ en ~D {J oo P C O t~ . 00 l'~ ~ m ~ ~,T~ ~ C V'1 n ? OD ~ 7 {O ~O V .- O m \7 1 [r1 ~O P 70 ~O ~ v u v . G~ 00 N o0 C .- . {n n P O V+ T O {/'4 ~O N n' 00 .-0 N h N r--~00 tONpCt -O^~- CO- Q O p] "~ V O H ' . o u^ G C T ~7 x i C/2 w y~y'S ~L ~~O a cT N Nkm C3 C rb 1 _ O i0 M O m m ao n~p "' + .1 0 .i t! .a o G o p . oaoo o~o ....~~_ 0 p O o~ rnQ 0i .CCC7o ~o~'q~~~ ^1I 5~i . G~_ e V V Y 4'a~s OOd O 'C O V r,l _U i~' U v C~ C u' u~i m .ii'H rS ,h c E ~^ F~. .CC Y.~. .C . r .-. .r m {n ~n /n (~ r i~ n ~C Y ~I' V . . ~ .7~~( ? ~? c y :oooo 0 cn v aw 5 c ~ ' V U V ~. Q' C H f~ 00 O{ O ~--~ Cl rq n~ V1 ~Ci W~ 00 P G ? rv m r~7+mn 7vQ C yyV n 7nv~ yn U ~~3 c v{n~i+ n_ ao ac o-~ m ma{nC~ ~ a ~dn rn ., _? cA~' f~1W[7:W N r+M~nm ccvv {n{nu1~n 4i y~ y ~; W W W Lc : L] W W~.1 'si fs: W LJ W CU S: W v .- . u~ ~ ~ y { 1 ru-, ~ a 3 3 ~ N N VN ~,.~~ 00o0. o0 c7 0 0~ .~.. .~. .~r_.., P . 99 ^ G~ V 1~ P ~OsOv L'J7 v~O ~9 N'- N PV z fL R7 M e,7 ;; !ti N 00 \D IQ- .0 h'1 l`1 L/1 m - N N :~V ~ .COao CCOQ a I Z N 7 C .~+ + v ~'Ax . n ~_ s 4 . N b . W .1~ i ~, '~ rr1 ~+ ~O Q ~O N O, .~. U ~ 5 U p3 ~ F C 7 e V a a ~':oo a - O~. a a `n o -oo ~ w c I- c w C y D ~ {J :-' ~J ^ c!] ~ ~ mN ,.,~ p~h - ~ a+o ~ 0 ~0 m 0a~o a o~ + ~, W V `-' a u..~. h a C x m 00 co ~Q N N N N N N N N I G `a + ~. r .. ~p~~ .~ ~aq5 ~ m tri ~r=N oNOOooo c c U a N b c :7 W sl f'.I ONO ~ cvq 0~0 W Cfo~ W W ru H ~ t 5 ~..~ p . 100 u z N. r;, ~+~ ~ d ~ ~ c'"o 0o v v v v v v v v v v v v v v W V U ~ Fu c-_~u .~c~`^vt~ r ~mo n c.n ~ a ~ Nrov~~ + cno- n~rrovonr'r- nr-oC+aNti_cw, aa er a t~ ~o n o ~n n e, ._ n~. ~n Cc f~ v C M M r- ~n v7 (~ ~'1 C V'~ Q 00 N N N C? nI7 C~ ` O ^ ._ . ... --_^ _ . .r .- _,- .- .. . - -p C O f .~, LJr y y 7 .P ~~ N N ~r o ~n +n nn m o a-- ~ 9 8 v 3 e0 p d C~ .. O~ r"I vl C V~ N Y1 V1soCcc r~~DNK.C " ~D~~ f~ 'O ~ ~V~ l 00CC 1 N~ V1~^ a OQC ~JGOO a00 0 a; v v v v z ~ vC Tr ~ w=_ W t7 v~ Q C% v V1 00 !^~ O f - N C~ n O Vl m N. in [~ - wN {~ M h V1 -. P Q17 N C N vi M N N~ in [^ J t N e+1 l~ kn en %G N QO C O% M ~O 'rt O~'J U{, C~ ~0 00 ['- N N~ O N O N'~1 O Q~ bC O C+ ~ r'% ^ EE o $ r u ;G C7 y y X n c ~ ~~ . U Cl F~ ac r~ ^ G G c ~ 't7 yoo .o v~n a o In a o o a~ .~, Q~ ~ N~ C CO o r V1 a co Oa C~ N e~f ~i Ci l"i y ~ .M ~ a~ o~ O o .i O r^r O C , J P~ O 9 C o 6 cJ _.~ O O 4`-1 'F. V V Q V V V V J V V Y V V V/'"~ u y O ^ r W V o ^y . C ~ u ` U C X a I wi 4 ` C +1 S; ~ ^: C - ~ - - Zm - - nro rcC ~ OG ~~ ~ ~ w '~ ~ 'O C C m 9a O+ - N fn w C V1 %Q (- n CO O~ C C N e Vl ~.O i'- 00 Q_ O^^ N_ N_ N_ N M_ N` ~1 [+1 R_ V_ eT' t!? O CN (71 ol <K .~'r-~~ C~CC ~CCC CCCC C CCC CCCC ~ y v1 N. pp ~ c- - N n ef v-r, ND r r Co Cl O ~ p~~ G~ 01 C~ O~ - N N N N M r+1 r'f 1r1 C? Q Vl W J =0 WW slW k s WW WLtiL~ 11WWLV W_7is1W " > u ad Jr' a v~iH FA 't lu '~ p . 10 1 Wildlife International, Ltd. Project Number 454A- 11 7 -25- Table 5 Steady-State BCF Values for Bluegill Exposed to 0 .53 mg a.i ./I. Sponsor: 3M Corporation Test Substance: PF $S Test Organism Bluegill, Lepomis macrochirus D il u tion Water: Well Wate r Mean Measured Mean Measured Test Steady-State Tissue Tissue Type Concentration Uptake Days at Concentration (mg n .iJL) Steacy-Statc (mg a .i. /K g ) Steady-State BC? Edible 0 .53 7, :4,21,28 0 .I]3 0 . 2 1 Nonedible 0,53 7, 14,21,28 0 .272 05[ Mole Fish 0 .53 7, 14, 21, 28 0 .203 0.38 ~r. p . 102 Wildlife International, Ltd. -26- Project Number 454A-I 1 7 Table 6 BIOFAC Model Estimates for Bluegill Exposed to 0 .53 mg a . UL Sponsor: 3Iv[ Corporat.on Test Substance, PFBS Test Organism : Bluegill, I_epomi .s mucrochiru.r Dilution Water : Well Wate r Kinetic Estimate Time Estimated Time Bioconcentration Uptake Rate Depuration Rate to Reach 90 0/n to Reach 50% Factor Constant Coastant Steady State Clearance Tissue Type (BCFK) ( k , , LKg' ' Day"' ) (kZ, Day' ' ) m ays) (Days) Edible 0.73 0 .24 0.33 6 .9 2 .1 Nonedibie 0 .86 0 .21 0,24 9 .6 2 .9 Whole Fish 1 .1 0 .60 0.54 4 .2 1 .3 p . 103 ~w .....~~..~.~~ ~.~~.... r` v A _ a i- Vl N 00 00 91 v-i LO n em Ir I ?? cU~ ~^O ^~ D '~- QOO0 '1 O .'N C t a C~C ~ C_ , a . .,- .. .'.7 Nv ~ hl v :n VU ., ~ . . n c~ N c~ r1 rn v ~o oc - c tio O ., oo r, ,,~ ~r+ r, . a% n n N O t'~ t~ n p~ vt f4 e'V ~'1 O v1 CO N m 1~p'~ N 00 ~O r- 00 Y1 R L1 ~G ~C ~ v1 + N O <^ :Izo O N M 0} N GC M1 N-+ V: Q - N N Cl0 N N C M y LL u ~ O N m 'J% 00 A U1 f' 00 00 N N ~r1N .n ~N IN+t~~ Ya1 ~0N O~ O~O ~N C N C o4 'O 'JU L~ OO~JO 0000 N"' MNNM OMM N : o w o U c " ~t W-1 z 1 n rz en r ~ r- - 4 4 0 o N RS ~(j] N O ON n .'~a' ,~ a_3 tA Rw3 O N M O 'n N Gs N l+ V . v7 yVj 50 0o kn n N rn o .- a a1 o0 0~. OA C-0 7 00 C 70 n O Vl 7 O 90 V MNO 10 N O+ 1N- v. V1 .C1 ao m .^. .G oc Ot w! oo rv oo v v 10 Io 7 a~Q ~ n ;G ~C7-'~ 0000 .-.Cr~ .-. .. : .-.O OCO['7 Jt3 ~ ~ 5u S. ."' y ~ V p IT u ~ Vl W hO~~, - V v1 n .-- 00 .^ 70 C 00 00 C : LL p'~~, e4 O O O`~ Q O O O O O O O ~ v .1 .~i . ~ ir i-. - U y O y ~ .n 'G .`~ l <vv..~ v,a . o e a o 00 M o ~~ :i L y w { n M V3 r4 3 y T N N: Q~D h ~D n 00 00 f^l N PI N M e+1 r1 e+f V O~n ~N NM 4 'l~ ~O ~O tiL ~ .. ca V O. 5 O Ne n nN Q 00 G~ d N r ~ g~~ o ~ wc~ww N N N N wwww e+1 ~+1 ri .+~ C V~h V1 raww~t ~w~ :w w~,r7~'.ap w~qw~Q^[ .y, rlS n L A ~ y '^ o o'u~ + ~ C.~.~~oa._~~. .; - N Ih ar1 U tV cVo I % I O t to 0 Z N 04 CL ! F~ : W^ M, 07 ^-~4 M~ k^J Cd .-~i M ~ ~+; ^J ~ N A v Cai ~ ~ '~ m ull ~caa,z ~'+~ '3 v 0 N N N 03 N 6J ~OO+vi4~i 'r'~ .'': C 00 00 K4 C% t+1 O ~-! N U O r'1 er O~ 0,0 CG OO :a ~p o ~4 r I n r- vS npvi nviao cc o o^' o o v 6 6 . . TJ~~ A I W v V ~ v p p. N N c'`7 tV N N N N V n ax3l ' m~ ~ v ~ E a r~r-`' onoo~oxa .~~ '-' ZZ ?~ Z Z Z Z V y kn %Dn nccc , v~ C ~ /~ .c ':1un: n[l0W 0 0W 0 ouv J W ,~ ~-~~c p . 104 p . 105 . ~ = --~ y a ~^~~~ N O O U Q'~ , O O O ~ ~ 000 OC ~O V G V~ V V V V V O "~ p , ~~ G1 3"~ a) x, . a*t [~ e r q r~c O~ nr~ 1~ OQ .xu ^l vl .a V-1 N [~ o Onwo N a OO .r Av~ - . oa -r cr - - 7 o t~, r o0 0o r oo GN oc -- ry rn ' Q -- N OQ V ' ~ +n O~ C` ^ cT 00 OC ~ e+s O C . I z 7 r. .... .- - _., .- .-. . . ... .r ~ SJ q::3 u rnN r4 C4 m - 00 't \r Ch r, \0 0 CN r- 'r and 04o O O x oo~~ ooo~ og ~oov Q v~~ u z ., ci IN 0 "m ~ -; v p r ^-~ u O ~ Ep - 0N 0 N IL~ C`~ I N M O I n~ C1 C ~ C.p O N A h,n O 00 GO '~ 4 A 00 Q N~ v Omo ~ v~ S m v~1 ~ N~ 7+ Vi O~ ~ \D N~ n ti C Q C~ ^ O -~ ^ -- C O O -- O O O ^^- O O ~ Q W C. ~ Pl. C_ u t . ~ a ~/ .M Go r r ~p a _ ^ ~n 0 O ~- In In ' N O v1 tQ r~n L1 OM+ ~ h oo QN ~~~+ ~ u .; n ~n a c~ t0 a p o C O 4 O~ C' o o- o c o c 0 opQ o o d~ o V`a +,' W V~'! q .. 6 v rn en 1'n M n n Cr n o O O O ~ ~~~ a - ? V ~ O N C1 r"1 V v1 ~O ~4 l~ 00 G+ C, O-r7 N C'~ v km N ~~ ~ O C_ _ 0 _ Q_ r -_ - _ Nv N N ~ N M v ~l V v] v . y ~ U C = ~ N . y ae O th t1 v n~C ~O n 00 N N rt e vi ~ : t,; ..O Cy0 ~ ~ O O_ O_ Q_ ~_ -^ ~ N N N N M R t} - A ' A Vl vl ~ L H ~ O C ~/% Jl W U1 G] W i:: ~t7 W W ^. W~l L:S G:] W W G s7 W :3 p v ~ p . 10 6 Wrrdlrfe Intern ational, L td. -3U- Project Number 454A-11 7 Tablc 8 Steady-State BCF Values for Bluegill-Exposed to 5 .2 mg a .i ./1, Sponsor: 3M Corporation Test Substance : PFB S Test Organism : Bluegill, I,epomis rnoerochirus Dilution Water: Well Water Tissue Tv e Edible Mean Measured Mean Measured Steady-State Tissue Test Concentration Uptake Days Concentratio n (mg a . UL) at SteaCy-State (-n n.i .lKg) Steady-State $C F 5 .2 3,7,14, 2 1,28 0,829 0,16 Nonedible 5-2 3, 7,14,21,28 2 .24 () .43 Whole Fish 5 .2 3,7,14,21,28 1 .57 (),30 ------- p . 107 Wildlife International, L td. -31- Project Number 454A . 117 Table 9 BIOFAC Model Estimates for Biucgill Exposed to 5 .2 mg a .i ./L Sponsor 3M Corporatiou Test Saastance : PFBS Test (hgarusm : BLuegill, Lepon+ls macrochims Dilutior. Water : Well Water Knetio Estimate Time Estimated Tinie Bioconcentration Uptake Rate Depurataon Rate to Reach 90% to Reach 50% Factor Constant Constant Steady State Clearanc e Tissue Type (BCF:C) (hl, LKg'''Day'') (k2. Day-) (Days) (Days) Edible 0 .18 0 .066 0 .36 6 .5 1 .9 Nonedible 0 .50 0 .16 0 .32 7 .1 2.l 'Whole Fish 0.36 0 .12 0.33 7 .0 2A ~~.. . ~~ p . 108 WllG~l~fe International, t L d. -32- Project Number 454A-117 Figure 1 . Concentrations of PFBS in Edible Fish Tissues of Bluegiii Exposed to 0 .53 mg a.i.lL . 4 SUR-117 0_53 Ed161o Tloave o IS1 = 0 .243 ~ K,2 = 0 .33 4 03 K8 = 0 .7 3 c~ ._ tJ p ~ e o ti p O O a .0 8 .0 1 2 .0 18 .0 24 .0 30-0 3 6 .0 Dog u2_ 0 us-0 p . 109 Wildlife International, Ltd. -33 Project Number 454A- 11 7 Figure 2, Concentrations of PFSS in Nonediblc Fish Tissues of Bluegill Exposed to 0 . 5 3 ,ng a.i.1Z. 454 9-117 0 .53 n .~ ~ .i ./L hlonod'S1a Tiaeu B .8 ._ .~ o 0 g 0 ~ tS 1 = 0 . 207 tt2 = 0_240UU N8 d p_8 B rM , 0 .0 8r0 12 .0 1E3_0 24 .0 30 .0 3G .0 42 .C48 .0 Day p . 11 0 Wildlife International, Ltd. 34- Project Number 45 4A- 11 7 Figure 3 . Concentrations of PFBS in Whole Fish Tissues of Bluegill Exposed to 0 .53 mg a .i ./L. 11SUR-11 7 Q . 53 n~g e, i .1L Whole F i a K K1 - 0 .603 K2 = Q .54558 KB a 1 .1 1 x ~p on ... ~ d O O Q O 0 .J 6 .0 12 .0 16 .0 2 4 .0 3C .0 nay 36 .0 42 .0 40 .0 p . 11 1 Wird~fe International, Ltd. -35- Projecc Number 454 A-] 17 Figure 4 . Concentrations oPFBS in .Edible Fish Tissues of Bluegill Exposcd to 5 .2 mg A .i,/L. 45UR-117 $ .2 ns a_i ./L Edible- Tivsu o ~ A o ~_ ~ o o 8 ftl = 0 .066 K2 0 .35605 csflo,is 0 0 cn 0 .0 6 .0 12 .0 113 .0 24 .0 30 .0 36_0 Oay 42 .0 48 .0 p . 11 2 Wildlife International., Ltd. -36- PrcjQQt Number 454A-1 1 7 Figure 5 . Concentrations of PFI3S in P7onodialc Fish Tissues of Bluegill Exposed to 5 .2 mg a.iJL . U5UFI-137 5 .2 -ny a . ; . /L i`1.yn~diblo Ti . u G $ 0 o M8 p w (D = C3 O Q K1 = 0 .C63 ISZ = 0 .3 24 68 0 .50 Y o 00 ~ - ) fI O 0 .0 8 .0 12 . .0 16 .0 24 .0 30 .0 35 .0 42 .0 4B .0 Day p . 11 3 Wildlife International, Ltd. 37 Project Number 4 5 4A .11 7 Figure 6 . Concentrations of PFBS in Whole Fish Tissues of Bluegill Exposed to 5 .2 mg a .l ./L . o ~StlFa-l I! 5 .2 mg a . I .lL Who le Froth ~ " K1 o = Q .i17 K ? = 0 .32677 Ka 0 . 3 5 x .r 1 ~ v fl 1N -+~ C7 0_0 8 .0 1 2 .0 1 8_0 24 .0 30 .0 3 6 .0 42 .0 Doy 4 8 .0 p . 11 4 W1rdr fe International, Ltd. -38 Project Number 454A-1 17 APPENDIX I Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: 3M Corporatio n Test Substance : PFB S Test Organism : Bluegill, Lepomis macrochirus Dilution Water : Well Watcr Mean Range Specific Conductance 320 (N 4) 315 -325 (mhosicm ) Hardness 130 (N = 4) (mg/1, as CaC03) 128- 132 Alkalinity 172 (N 4) 172 - 172 (mg/L as CaCO3 ) pH 8 .1 (N=4) 8 .0-8 .2 p . 115 Wildlife International, L td. -39- Project Number 454A-11', APPENDIX 11 Analyses of Pesticides, Organics and Metals in Wildlife ]ntemational, Lt.d. Well Water t Component Mcasurcd Concentration Componcnt Measured Ccnccctration Pcaticides and Organics Aelonifcn <0 .03 gll.. Dimcthomorf <0 .05 glL AlflChlor <0 .01 u&'L Disuifoton <0 .02 ug'L Ainctrvn <0 .01 1. +g DMST <0 .05 g,L Atrnxin a] .01 pyL Dcxicmurf <D.01 Irg/L Azinphos-cthyl <0 .04 ugrL 1 :r.co4ulfan-ca c0,01 uFy7, Azinphos-mothyl <0.08 ugIL Endosulfart-R <001 ug/L Azoxystroain <].25 g/'L Endosulfan-sutfaat <0.02 LvZ, Bifenthrin <0.05 ugiL Epoxionnar,ole <0 .05 pg/L Biaatlctirrin -0.05 glL Eptam <0 .02 Ixell . Bitcrtanal <0 .05 gIL Eafcnveleraat 4 .02 g/t, Bromacil <30 .05 }ttyL Ethion <0 .05 uglL Bromophos <0 .02 e/L Ethofumesaat <0 .02 ug1L Bromophos-ethyl <0 .02 pg/I, Rthnprnphos <0 .01 p/L Broompropylaat < .02 upJL Etridiazoie <0 .02 ~tp/[, Dupirimaat <0.05 ug/i. Etrimphos <0.05 .4g/L Carbdtyl <0.05 ugJL cenarimol <0.05 g(L Carbofuran a0.03 uglL Fcnchlorphos <0,01 8/L Carboxin <0.02 }tg/L Fenitrothion <0.03 pg/L Chlorfenrinphos <0,02 up/I. Ecnoxycarb <0 .03 ug/L Chloridazon <0.05 ug/i. Fenpiclonil <0 .05 &(L Chlorpropham <0 .02 g/L Eenpropathrin <.0,25 }tgrL Chlorpyriphos . <0 .01 Ng/i. Fenpropimorf <0 .01 Ng'L Chlorpyriphos-methyl <0 .01 pg/L Fonthion <0 .01 pg,,L Chlorthalonil <0-04 pg/L Fenvaleraat <0 .02 ugrL Coumaphos <0 .02 p/L. Flua2ifop-butyl <0 U2 gn, Cyanazin <0 .05 vp/L Fluoroglyeofcn-cthyl <0.02 u:7, Cyfluthrin <0 .05 Iig/L Fluroxypyr-meptyl <0 .05 pg;L Cypcrmethrin 10 .25 ~ag/L. !7utolanil <0 .02 NgIL Cyproconazole <005 g/L Fonophos <0-{)I pg,q, Dchamethrin <0 .02 lrg/L Furalaxyi <0 .02 Ngli . Demon <0 .02 pfL Hcptcnophos <0.02 ujp'L Demeton-o <0 .02 pg/L Imazalil <10 .01 glL Desethylatrazin <0 .01 pg/L Tprodinn <0.05 &'L Desisopropylatrazin <0 .02 Ifg/f, Kresoxirn-mcthyl <0 .02 pg(L Dcxmctryn <0 .01 Np/L Lcnacil <A.05 glL Diazinon <0 .01 glL Lindane <0 .02 It g;L Dichlobcnil <0.01 gVI. Malathion <0 .02 p&'L Dicltloran <0 .03 ug/I . Mctalaxyl <0 .05 glL Dichlorbcnzamidc <0 .02 p8/1 . Mctamitron <0 .05 pg/L Dichlorfenthion @ .01 pg/L Metazachlor <0 .02 pg/L Dichlodtuanid <0 .03 nK/L Methidathion <0 .02 pg1L IAttalyses performed by TNO Nufition and Food Institute on ssmpies collected on October 14 and 15, 1999 . p . 11 6 Wildlife International, Ltd. - 40 Project Number 454A- 117 Apptrndix 11 (Continued) Analyses of Pesticides, Orgurtics and Metals in Wildlife Intcmational, Ltd. Well Water` Pesticides And Orgamcs (Page 2) Ccmpanent Measured Concen :ration Component MeHSUred Concentration Dichlorvos ~4 .01 gtL Methoxychlor <0 .01 pg/L Dicofol ~ .25 grL Mctolachlor <0,0] ug/L Dicthylt4luaa;ide <0 .02 pgtL Mctribu:ir. <0 .02 pg/L Difenoconaznlc N .03 }tgrL Mevinphos <0 .01 g/L Dimethoate <0_02 }igiL Nitrothal-Isopropyl <0 .05 g/I, PAntohutazolc <0 .05 gfL Pyrifeamt-S <0 .0] tigfi . Parathicn <0 .01 lcg/L Pyrifenox-2 <0 .01 pg/L parathion-methyl <0 .01 1tgiL PyrittuthaniI <0 .01 pg/L Fenconaxole < 0 .0 5 glL Cluizalofop-ethyl <0 .02 } g1L Pendimethalir, <0 .03 p grl. Simazin <0 U1 glL Permethrin-cis <0.01 }tg/L Sulfotep <0 .02 g/[, Pertnethrin-trans <0 .01 g/L Tcbuconazolc < 0 .05 grL Phosalon <0 .05 pg2 Tcbufenpyrad <0 .05 glL Phosr.tet <0.02 } :gJL Terbutryn <0 .01 E/L Phosphamidon-cis <0.05 pg/L Terbutylazin <0 .01 glL Pirimicarb <0.01 p g/L Tclrachlorrinphos <0 .01 g1, Pirimiphos-cthyl <0,01 }tg/L Tctrahydroftaalimidc <0 .05 ug/L Pirimiphos-methyl <0 .01 g/L Tetramethrin <0 .0 1 ;rgfL Prochloraz <0 .02 Vg/L Thiabandazok <4 .05 .ig/I. Procytnidon < 0 .01 pgfi. Thiometon <0 .04 }tg/L Prometryn <0 .01 pg/L Tolclophas-methyl <0 .0I g/L Propachlor <0 .03 g/[. Tolytfluanid <0 .04 QfL Propazin <0 .01 Pg/L Triadimefen <0 .05 pg/L ProphAm <U .02 }tg/L T7iadimenol --0 .05 }tg/L Propiconazool <0 .05 g/L Triallaat <0 .02 1rg/L Propoxur =9 .03 yg/L Triazophos --O,02 &/L Propyzamide <0 .02 }g!L Trillurulin <0 .021g1L Prosulfocarb <0,02 pg/L Vamidothion a0 .0i pp/L Pyrazophos c0.03 Pe_- Vinchlozolin <0 .01 lag'L Metals Magnesium 11 .0 m&'L Nickel <1 .1 pig/I. Sodium 18.0 mgfL Copper <0.7 It g/I. Calcium 29 mg/L Zinc <025 g/L Iron <0_015 mg.,] - Molybdenum <0.3 Itg/L Potassium 1 .1 mg,Z Silver <021rglL Aluminum <0 .02 mg/1, Cadmium <0.1 1tg/L Mangan= < 0,1 g/l. Arsenic <0 .5 ug/L Beryllium <0 .2 p/L Mercury <0,025 g,'- Chromium < 0 .5 gJf. Selenium <0 .51tgJL Cobalt <0 .2 glL 'Analyses performed by TNO Nutrition and Food Institute on samplra cnilected on October 14 and 15, 1999 . .~...~ . Wildli~e International, tL d. -41- p . 117 .~ Project N umber 434A-1 17 APPENDIX III THE ANALYSIS OFPERFLUOROBUTANE SULFONATE, POTASSIUM SALT {PFBS} CONCENTRATIONS IN FRESHWATER AND BLUEGILL SUNFISH TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD . PROJECT NO, ; 454A-I 17 p . 118 VV'rldrz fe International, L td. -42REPORT APPROVAL Project N umber 45,d A-117 SPONSOR : 3M Corporation TITLE : Pertluorobutane Sulfonate, Potassium Salt (PFBS) : A Flow-Through Bioconcentration Test with the Bluagiil ( Lepornis macrochirus) WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER : 454A-1 1 7 P RIN C IP A LINVESTIGATOR: Ra d Scientis t ,. . Van Hoven, Ph .D . WILDLIFE INTERNATIONAL, LTD . 'Ir1AN GEMENT: 5-q_o~ Date Willard B . Nixon, Ph .D Manager, Analytical Chemistry Dat p . 119 Wildlife International, Ltd. - 43 PrcjcctNumber 4 54A-117 Introduction Freshwater sampics and tissue samples were collected from a flow-through aquatic test to determine the bioconcentration potential of pertluorobutane sulfonate, potassium salt (PFBS) in the bluegill (Lepomis maerochirus) . The study was conducted by Wildlife (nternational, Ltd . and identified as Project Number 454A-117 . The analyses of freshwater and tissue samples were performcd 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 13, 2000 to September 5, 2000 . Additional tissue samples were collected and analyzed gravirnetrically for lipid coutent . 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 Substanc e The test substance, PFBS, was used to prepare calibration standards and matrix fortification samples and was identified as Wildlife International, Ltd . identification number 521 6 Analytical Method 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-1 .15) . Tissue samples were analyzed based on the method entitied "Analytical Method Validation for the Determination of 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 of 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 . 12 0 Wildli fe Interna tional, Ltd. -44- Project Number 45 4A-I1 7 Freshwater S amp l es A method flow chart for the analysis of PFBS in freshwater is presented in Figure 1 . Dilutions of collected freshw ater samples were performed with a solution of 5 0 .'a methanol (HPLC grade, 99 .9+%) and 50% NANdpureo water . Aliquots of the dilutions were transferred to autosampler vials and su bmitted 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 1100 High Performance Liquid Chromatograph ( HPLC) interfaced with a Perkin-Elmer API IOOLC mass spectrometer (single quadruple) operated in selective ion monito ring (SIM) detection mode . The mass spectrometer was equipped with a Perkin-Elmer TurbolonSpray ion source . Chromatographic separations were achieved using a Keystone PRISM RP column ( 3 0 mm x 1 .5 mm, 3-m particle size) fitted with a Keystone Javelin C,, Guard Cartridge ( 20mm x 2 mm) . The instrument parameters are summa ri zed in Table I and a method fl ow cha rt is provided in Figure 1 . Freshwater quality control ( QC) samples (matrix blanks and fort ifications) were processed in the same manner as the test samples . Freshwater was forti fi ed with the appropriate PFES stock solution usi n g a gas-tight syringe (for matrix fo rt ification samples) . Matrix blank samples were not fortifi ed with the test substance . Tissue QC Samples Biucgill sunfish (ir.epomis 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 dorsally through the spinal column . Heads, fins and viscera were removed from the body and were considered to be nonedible tissue . The balance of the tissue was considered to be edible tissue . The nonedible fillets were transferred to a I-quart glass bottle and blended with an Ultra--Currax homogenizer (Janke & Kunkl c , Model T25) set at approximately 9500 rpm. This p ro cedure was repeated for the edible tissue . An approp riate number of Igram aliquots of edible and nonedible homo genate 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 mat ri x blank and matrix fortification samples or storage stability samples . p . 12 1 Wi ldlife In ternational, Ltd. -45 - Project Number 4 54A-11 ? Tissue Samples A method flow chart for the analysis of PFBS in fish tissues is presented in Figure 2 . Edible and nonedible 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 tirne, duce of the appropriate tissue QC samples also were removed from the 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(s) using gas-tight syringes . Test and QC tissue samples were extracted as follows . Ten milliliters of 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 of the extract were then volumetrically diluted into the calibration range of the LC/MS/MS methodology with 50 :50 methanol : NANOpureo water dilution solvent . Aliquots of 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 samples were removed from the freezer, allowed to thaw and processed using the same 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 of each fish tissuc 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, FolIowing 49 days of 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 fort'sfied and 49 days old) were processed using the same procedures described for tissue sample analyses . p . 12 2 Wildlife International, L td. -46- Project Number 45 4A- 117 Concentrations of PFBS in fi sh tissue were dctcrmincd by reverse-phase high performan ce 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) dctcction mode . The mass spectrcmeter was equipped with a Perkin-Elmer TurbolonSpray ion source . Chromatographic separations were achieved using a Keystone PRISlyi RP column (30 nurc x 1 .5 nun, 3-rn particle size) fitted with a Keystone Javelin C,R Guard Cartridge (20mm x 2 rnrn), The instrument parameters are summarized in Table 2 . Tissue Samples for Lipid Conten t A method flowchart for the analysis of lipid content in fish tissue is presented in Figure ? . 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 of NANOpurem 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 trahsferred to a 250-mL separatory funnel that contained 25 mL of chloroform and 50 mL of methanol . Each sample vial was rinsed with an additional 10 tnL of NANOpurc 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 ~ivith a small volurne of 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) . p . 12 3 Wildlife International, L td. 47 Project Number 454A-1 17 Calibration Stocks and Standards Calibration standards were prepared in 50 :50 methanol : NAN Opurewater by appropriate dilutions of a 1 .00 mg a.iJ L stock solution of PF.BS in methanol. Calibration standards of PFBS, ranging in concentration from 0,500 to 5 .00 g a.i ./L, were an alyzed with each sample set . Five calibration standards (di fferent cencentrations) were an alyzed with the samples . The calibration standard seri es was injected at the beginning and end of each run, and one standard was injected, at a tnitumum, after every five samples . Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration st an dards . Typical calibration cu rves from water and tissue analyses are presented in Figures 4 and 5, respectively . Representative ion chromatograms of low an d high calibration standards used for water and tissue analyses are presented in Figures 6 through 9 . The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation . The concentration of PFBS in the freshwater was determined by substituting the peak area responses into the linear re gression equation as follows : PFBS in sample (mg a.i ./L) = [(peak area - y-intercept)/slopej*dilution factor*unit conversion factor % Recovery = measured PFBS concentration mg a .i ./L) U nominal PFBS concentration ( mg a .i ./L) " iU 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 .fKg) = [(peak area -- y-intercept)/slopej " overall dilution factor Overall dilution factor for tissue samples ( LlKg) = 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 an d fish tissue homogenates were fort i fied with the appropriate stock solution of PFBS p re pared 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 4_54A-l 17 Limits of Quantitatio n The method limit of quantitation ( LOQ) for PFBS in freshwater samples was 0.125 nig a .i ./L, calculated as the product of the lowest PFBS calibration standard (0 .0005 mg a .iJL) and the diluti on factor of the mat ri x blank sample ( 250) analyzed concurrently with the test samples . The LOQ was calculated on an individual basis for each edible tissue sample since the cntire submitted samples ( of differing weighcs) were extracted without an adjustment to a constant weight . Where approp riate, 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 (LJKg) of the edible tissue sample . To illustrate for a 1 .000 grarn sample, extraction with 10 mLs of methanol followed by a lOx volumetric dilution (overall dilution z5actor =100 L/Kg), gives an LOQ = 0 .0500 mg a .i ./Kg . An apparent PFBS background ( 0 .039 5 a-0 .0132 mg a .i,/Kg) was measured in nonedible control tissue homogenates (see Figure 13 on page 8 3 ) . The suspected origin of this background was residual laboratory cleaning detcrgent in the blender employed for bulk hamogcnization . Laboratory cle an ing detergents had been suspected as a potential interferent 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 of test substances containing PFBS . Therefore, a practical LOQ was dcSncd for individual nonedibie (wher e appropriate) tissu:, samples as the me an measured background plus three standard deviations of the background measurements (0 .0792 mg a .i ./Kg for a one-gram sample), adjusted for the individual weights of the tissue samples . Freshwater Matrix Blank and Fortific2tion Samples Along with the actual freshwater sample an alyses, 1 5 freshwater matrix blank samples were analyzed to determine possible interferences (Table 3) . No mat rix interferences were observed at or above the limit of quantitation (0 .125 mg a,i ./l..) . A representative ion chromatogram of a freshwater matri x blank sample is presented in Figure 10. Freshwater samples were fort ified at 0 .250, 2 .00 and 7 .50 mg a .i .lL at each sampling inte rv al using appropri ate stock solutions of PFBS prepared in meth anol . Freshwater matrix fortifications p . 125 MIAfe Interna tional, tL d. -49- Project Number 454A- 1 17 were analyzed concurrently with each sample set and the analytical results were not corrected for . mean procedural recovery . Recoveri es ranged from 89 .7 to 115% of 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% t 2 .97%, and 99 .4% ; 2 .33%, respectivelv . A representative ion chromatogram of a freshwater matrix fortification is presented in Figure 1 l . Tissue Matrix Blank and Fo rt ifi cation Samples Along with the actual tissue sample analyses, 12 edible and 12 nonediblc fi sh 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 tcst . Representative ion chromatograms of edible and nonedible fi sh tissue matrix blank samples are presented in Figures 12 and 13, respectively . Edible an d nonedibie fish tissue homogenates were fortificd at 0 . 1 00 and 10 .0 tng a .iJKg using a stock solution of 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 fr om 66 .9 to 93 .4% of nominal concentration for the low-level fortification, and from 76.6 to 94 .1% of nominal concentration for the high-level fo rtification (Table 4) . The mean and standard deviation of fo rti fi cation recoveries at the 0 .100 and 10 .0 mg a .iJKg fo rtification levels (n = 12) for the adih te fish tissue were 84 .4r,, y R .10% and 83 .2%+ 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 of forti ficati on recoveries at the 0 .100 and 10.0 mg a .i .IKg fortification levels (n = 12) for the nonedible fish tissue were 102% 15 .0% and 88 .0% 4 .70/a, respectively. Representative ion chromatograms of edible and nonedible fi sh tissue matrix fo rt ification samples are presented in Figures 14 and 15, respectively. Example Calculations The analytical result and percent recovery for freshwater sample 454A-1 17-3, from the 0,300 mg a_i .JL nominal PFBS treatment group, was calculated using the following equations : p . 12 6 Wild l i fe International, Ltd. -s o Project Number 454A-1 17 (peak area - y -intercept} PFBS in sample (nag a .i,/L) = slope x dilution fa=or x unit conversion facto r Peak area = 152419 Y-intercept - 2116 .42700 Slope = 72902 .35156 Initial volume (Vi) = 0 .100 mL Final volume (Vf) = 25 .0 mI. Dilution factor (V,( ;) = 250 (152419 - 21 16.42700) lmc PFBS in sample (mg a .i ./L) = 72902 .35156x 2510 0 x 0' 0 PFBS in sample (mg a.i.lL) = 0 .515 mg a .i ./L % k2ecovery in sample measured PFBS concentration (mg a.i ./L) = x 100 nominal PFBS concentration ( mg a .i .lL) % Recovery in sample = 0 .515 mg a .i .IL X 100 0 .500 mg a .i .!L % Recovery in samplc = 103 % The analytical result for edibie fish tissue sample 454A-117-E-45, from the 0 .500 mg a.i ./L nominal PFBS treatment group, was calculated using the following equation: PFBS in sample (mg a .iJKg) = PFBS at instrument ( g a .i ./L) x overall dilution factor x unit conversion factor Peak Arca = 6639 .89 Y-intcrccpt = - 946.98 Slope = 12296 .93 Prima ry Dilution : Initial Weight (W;) = 1,2201 g Extraction Volume (V.) = 10,0 mL Secondary Dilution : Initial Volume (Vi) = 0.500 mL Finai Volume (Vf) = 10 .0 rnL Overall Dilution Factor (V,,'W, x VfIV) = 163 .92 mL/g = 164 1.IKg (peak area - v-intercept) PFBS at instrument ( g a .i .l7.) = slopc p . 12 7 Wildri e International, Ltd. -51- Project Number 454A-1 1 7 (6639,89 - (-946 .98) ) PFBS at instrument ( pg a .i ./L) = 12296 .93 = 0 .617 g a.ilL PFBS in sample (mg a .iJKg) = PFBS at instrument (g a .i ./L) x overall dilution factor x unit conversion factor 0 .617 g a.i. 164 L I PFBS in samplc (mg a .i ./Kg) = L x Kg x 1000 sg PFBS in sample; ( mg a .iJKg) = 0 .101 mg a.i ./Kg RESULTS Freshwater Sample Analysi s Freshwater samples were collected and analyzed for PFBS concentrations on Days -2 and -1 during the pre-uptake phase of 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 of th e test and depuration began . Du ring the depuration phase of the test, freshwater samples were collected and an alyzed 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 pretest samples ranged from 100 to 103% of the nominal concentration in the 0 .500 nig 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) . Du ri ng the uptake phase of the test, measured concentrations of PFBS in the control freshwater samples were <LaQ . Freshwater samples collected from the 0.500 and 5 .00 mg a .i ./L treatment groups ranged from 95 .7 to 119% and from 101 to 109% of the nominal concentration, respectively (Table 7) . During the depuration phase of the test, measured concentrations of PFBS in all freshwater samples were <LOQ . 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 of the test . Measured concentrations of PFBS in all but three (454A- i 17-E40, 66 and 105) of the 24 control edible fish tissue samples were <LOQ (Table 8), The results of analyses of edible fi sh tissue samples collected from the 0 .500 and 5 .00 mg a.i ./L treatment groups are presented in Table S . Measured concentrations of PFBS in all but four ( 454A-117-N-40, 66, 67 and 105) of the 24 control nonedible fish tissue samples were <LOQ (Table 9)_ Three of these p . 12 8 Wrldllfe International,, Ltd. -52- Project Number 454A-! 17 nonedible samples corresponded with the three edible samples that had measured PFBS concentrations above the L OQ (i .e. originated from the same fish), The results of analyses of nonedible fi sh tissue samples collected fr om the 0 .500 and 5 .00 mg a .i .(L treatment groups are presented in Table 9. Representative ion chromatograms of edible and nonedible tissue samples fro m the same study fi sh are presented in Figures 17 and 18, respectively . Stability Sample Analysi s Stability samples were prepared at test initiation (uptake Day 0) and stored fro=n_ The results of stability sample analyses are 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 E0. 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 Conten t Tissue samples were collected on uptake Days 0 (0 hour) and 28 an d depu.ration Day 14 to determine lipid content in fish tissues on a wet-weight basis . The results of lipid analyses in edible and nonedible fish tissues are presented in Tables I 1 and 12, respectively . p . 12 9 Wildlife International, Ltd. -53- Project Number 454A-11 7 Table I Typical HPLCr`MS Operational Parameters for Analysis of Aqueous Samples INSTRUMENT : Hewle tt-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin -Elmer API 100LC Mass Spectrometer operated in Selective Ion Monitoring ( SIM) Mode SOURCE : ANALYTICAL COLUMN : Perkin-Elmer Turbolon5pray Keystone PRISM RP (30 mm x 1 .5 mm, 3-um particle size) GUARD COLUMN Keystone Javelin C 18 cartridge (20 x 2 mm) OVEN TEMPERATURE: 40C STOP TIME : 3 .00 min FLOW RATE: 200 L'min MOBILE PHASE : 25% NANOpure Water' with 0.1%Ammon ium Formate : 75% Methanol INJECTION VOLUME : 5.0 L PFBS PEAK RETENTION TIME : Approm mately 1 .7 minutes PFBS MONf1 ORED MASS : 299 .0 am~s Wildlife International, Ltd. -34- p . 130 ..~, SAs~ti ; . ZED P :'Oject Number 454A- 117 Table 2 Typical HPLC/MSJMS Operational Parameters for Analysis of Tissue samples INSTRUMENT : FIew1e!t-Packard Mcde : 1100 High Performance Liquid Chromatograph with a P ccicin-Elmer API 100LC Mass Spectrometer operated in Multiple Reaction Ion Monitoring (NIP-M) Mode ION SOURCE : Perlan-Elmer TurbolonSpray ANALYTICAL COLUMN : Keystone PRISM RP (50 mni x 2 mm, 3-.n particle size) GUARD COLUMN : Keystone Javelin CIS cartridge (20 x 2 mm ) OVEN TEMPERATURE: 400C STOP TIME : 5 .00 min FLOW RATE : 200 l./min MOBILE PHASE : 10% NANOpure Water with 0 .1% Ammonium Formate : 90% Methanol INJECTION VOLUME : 5 .0 L PFBS PEAK RETENTION TIME : Approumatety 3,1 minutes PFBS MONITORED MASS : 299 .0 am~ p . 13 1 Wildlife International, Ltd. -55 . Project Number 434A- 117 Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples Concentration of Aerfluorobulane Su Iforut:, P otauium Salt ( Pf HS ) Sarop ;e Number m a .iJL) Percent (454A-1 I7-) Fcr:f:ed Measured Recoveryz ?T-MAS-I 0.00 < LO .250 PT-MAS-2 2 .00 :).2 QP7'-1vIA50 56 106 2 .03 l 0I PT-MAS-3 7.50 7.58 101 PT-MAB-2 0 .00 < LOQ -- PTd4SASjt 0 .250 0 .243 97 .2 PT-MAS-5 2 .00 1 .88 93 .8 Y I'-11AAS-6 7 .50 7 .33 97 7 MAB-1 MAS-l NW-2 MAS-3 0 .00 < LOQ -0 .250 0 .241 96 .5 2 .00 1 .93 96 .3 7 .50 7 .41 98 .8 MAB-2 0.00 < LOQ MAS-4 0.250 0 .245 97 .9 MA5-5 2.00 2 .01 101 MAS-6 . ?.50 7 .56 101 MA8 .3 0 .00 < I .OQ MRS-7 0_250 0 .229 91 .5 MAS-8 2 .00 1 .87 93 .3 MAS-9 7 .50 7 .16 95 .5 MA$4 0 .00 < LOQ - MAS-10 0 .250 0 .250 100 IvL1S-1] 2 .00 1 .99 99 .4 IriAS-12 7 .50 7 .63 102 MAB-5 MAS-11 ; M5-14 NW-15 0 .00 < LQQ 0 .250 0 .242 97 .0 2 .00 1 .95 97 .5 7 .50 7.34 ()7 .8 MAB-6 MAS-16 MAS-17 MAS18 0.00 < LOQ 0.250 0.271 108 2.D0 1 .87 93 .6 7.50 7.50 100 MAB-7 0 .00 < LOQ -MAS-19 0.250 0.232 92 .8 MAS-20 2 .00 1 .92 95 .9 MAS-21 7 .50 7.74 103 he limit of quarctitat:on (LOQ) was 0 .125 mg a.iJl,, calculated as the product of the lowest calibration standard (0.0005 mg a .iJL) and the dilution factor of the matrix blank sample (250). Results were generatod using MacQwtn, version 1 .6 software. Manual calculat ions may differ slightly . p . 13 2 Wrldli fe Internation al, Ltd. -5b- Project Number 454A-1 1 7 Table 3 (continued) Matrix Blanks and Fortifications Analyzed Concurrently With Freshwater Samples Concentration ofPe ;tluurubutane Sulfonate, Potass ium Salt (?F3S) Sample Number (m2 a .i ./L) ?ercent (454A 117-) Fortified M easure d Recoveryl tvlA13-$ 0 .00 < LOQ -- MAS-22 0 .250 0 .252 101 MAS-23 2 .00 1 .93 96 .4 MAS-24 7 .50 7 .47 99.6 MAB-9 MAS-25 MAS-26 MAS-27 0.00 < LOQ -- 0.250 0 .287 115 2.00 1 .96 98,1 7.50 7 .74 103 IviAB-13 0 .00 < LOQ - MAS-28 0 .250 0 .270 108 MAS-29 2 .00 1 .90 95 .0 MAS-30 750 7.20 96.0 M?.13-11 MAS-31 MAS-32 MAS-33 0 .00 < LOQ - 0 .250 0.236 94 .3 2 .00 1 .99 94 .6 7.50 7 .34 97.9 ?rL1f3-12 0.00 < LUQ -MAS-34 0 .250 0.224 89 .7 MAS-35 2.00 2 .071 103 MAS-36 7.50 7 .32 97 .7 "-13 0.00 < LOQ - 1viA5-37 0 .250 0 .254 102 MAS-38 2 .00 1 .96 97 .9 MAS-39 7.50 7 .53 100 The limit of quantitation (LOQ) was 0 .125 mg a .i./L, calculated as the product of the lowest culibration standard (0 .0005 ing a.i ./L) and the dilution factor of die matr ix blank samplc (150) . 'Results were generated using MacQuan, version 1 .6 software. Manuel calculations may diffix slightl y 3Reporting average ofdupficat.e injections of reciiuted original fo rt ification . O:iginai dilution not reported due to dilution error. p . 133 Wildli e International, Ltd, - 5 7- Project Number 454A- 11 7 Table 4 Matrix Blanks and Forti fications Analyzed Concurrently With Edible Fish Tissue Samples Concentration of Pe~flucxobutane 3ulfonate, P otassium Salt (FTES) Sample Number m a .i./Ke) Peroent (4>4A-117-) fo;tiliecl Measured Recovery' i-IvIAB-i 0.00 .LpQ - E-4fAS-1 0. :00 0 .0914 91-4 E-MAS-2 9.10 7.68 84 .4 E-MAB-2 0 .30 <LOQ - W-Mr1S-3 0.100 0.0880 88 .0 I~ \24ti-4 10 .0 8 .61 86 .1 E-MAB-3 0 .00 <LOQ - E-MAS-S E-MAS-6 0 .100 0 .0912 91 .2 10.0 8 .80 88 .0 E-1vtAB4 0.00 <LOQ E-MAS-7 0.100 0.0785 78_5 E4 1AS-8 10 .0 8 .04 80 .4 E-&IAB-5 0.00 <LOQ - E-MAS-9 0.100 0.0868 86 .9 E-IvIAS-10 10 .0 7 .66 76 .6 E-MAB-6 0.00 <LOQ E-1r3AS-i 1 0 .100 0 .0867 86 .7 E-Iv1AS-12 10 .0 7 .91 79 .1 E-MAB-7 0 .00 <LOQ E-M.'4S-13 0.100 0.0889 8 9 .9 E-hLaS-14 I D.0 7 .77 77.7 E-MA i3-$ 0 .00 <LOQ - E-viAS-15 0.100 0 .0857 95 .7 B-:~IAS-16 1o .0 8_06 80.6 E-NW-9 0 .00 E-MAS-17 0 .100 E-ASAS-1 S 10 .0 <LOQ 0.0934 93 .4 8.07 80 .7 E-MAB-10 0 .110 <LOQ - E-MM-19 0 . 1 00 0.0838 83 .8 E-MAS-20 10 .0 8_35 83,5 E-1u.A$- l 1 0.00 <LQQ E-MAS-21 0 .101} 0 .0669 66 .9 E-Iv4AS-22 10 .0 9.41 94 . 1 E-MAB- 12 0 .00 <LOQ - E-IvIAS-23 0 .100 0.0718 71 .8 E-Y1AS-24 10 .0 8 .66 86 .6 The LOQ was 0 .0 500 mg &.i ./Kg, calculated as the prodtu :t of the lowest calibratio n standard (0 .0005 mg a .i .R.) and th e overall dilution factor of the maix blank samples ( 100 L/Kg). All sample weights = 3 .00 gram . 'Results were generated using MacQuan, version 1 .6 software . Manual calculations rncy differ slightly. p . 134 Wildlife International, Ltd . .5g- Project Number 454A-11 7 Table 5 Matrix Blanks and Fortifications Analyzed Concurrently With Nonedible Fish Tissue Samples Sample Number (454A-11^,) Coneantration oFParfluorobu l ane Sulibnate, Potassium Salt (PFDS) (mg a .iJKg) F'cutGird Mtaxrr& PcrcenL Racavery~ N-MAB-l 0 .00 <LOQ - N-MAS-1 (1,100 0.101 lO l N-MAS-2 10.0 9.13 91 .3 N-VAR2 N-MAS-3 N-MA.S-4 0.00 <LOQ 0.100 0.114 114 10 .0 9.16 91 .6 N-MAB-3 000 `-M AS-S 0.100 N-MA3-6 10 .0 <LOQ 00912 8.65 91.2 86 .5 N-MAB-4 0.00 h-MAS-7 0.100 N-MAS-8 10 .0 <LOQ - 0.0866 86 .6 8.90 89 .0 N-MAB-5 0 .00 <LOQ - i`7-M AS-9 0 .100 0 .0926 92.6 NMAS-10 10 .0 8.23 82 .3 N-MAB-6 N-MAS-11 N-MAS-12 0 .00 <lAQ - 0 .100 0 .0804 80.4 10 .0 8.16 81 .6 NMAB-7 0.00 SLOQ 54-4lAS-13 0 . 1 00 0,0988 N-MrLS-14 10 .0 8.56 98.3 35 .6 .4bsAH-8 0 .00 N-MAS- 15 0 .100 N-MA1-14 1010 <LO Q 0,132 8 .08 132 80.7f N-MAB-9 0.00 ' <I .OQ N-MAS-17 0 .100 0 .121 121 N-h1.AS-18 10 .0 9 . J7 91 .7 N-tv1AS-10 0.00 <LOQ N-MA$-19 0.100 0.111 N-MAS-20 10 .0 9 .40 1 1 1 94 .0 N-MAB-11 0 .00 <Ln Q N-&W-11 0.100 0.'05 1U5 N-MAS-22 10.0 9 .39 93.9 NNL4B-12 0 .00 <LOQ NM .4S-23 0 .100 0 093 9 ld-b4 .4S-24 10.0 8.8 1 93 .9 SK.1 The LOQ was O .C792 rug &.i.-'K& calcalatad as the product af (he mean rrsa.tiured background concentration plus tlsrre standard deviations 'VCc Liteo nron aauc nd e baen kgrolur rid atioa. All sample weights r 1 .00 Zra n Rcaults we re gcncratad using N(acQuaq veion 1 .6 software. Manual calculalioqs may differ slightly . ~~ . ~~... ..,~ p . 135 Wildrife International, tL d. -59- PrOject Number 454A-1 17 Table 6 Measured Concentrations of i'crtluorobutanc Sulfonate, Potassium Salt (PFBS) in Pre-Test Diluter Verificat :on Samples Nominal Txl Concentration (mga.i:~L) btcaFured Cuncer.tration of Ssmp(e Sampling Perfl uo robutane SuL'onace, Polaysiwn Parcenl Number Time Salt(PF9S), of (454A-i17-) Ptwac (Day') (mga.i . /i.) 1Jotnin .4' 0 .0 PT 1 Pre-Uptake -2 < LOQ -(Negative Contrul) PT9 -1 < lAQ -- 0 .50 Yr3 -2 PI--4 -2 PT-1. 1 -1 PT42 -1 0 .515 103 0 .506 l o t 0 .J08 102 0 .500 100 i .o FT-6 .2 FT-7 -2 PT-14 l PT-1S -l 4.94 98 .$ 5.09 tC2 5.07 101 5.18 104 ~ Ilia limit of quantitation (I .OQ) was 0.145 mg u.iJi., calculatod as the product of the lovm t calibration standard (0.0005 mg a_i.iL) and the dilulion fack;r olthe matrix blank "]a (250} Reeults wero generated uaing MacQuan, versinn 1 .6 w@ware . Manuel calcul>,tione may differslightly . p . 13 6 Wildlife International, Ltd. -60- Project Number 454A-1 17 Table 7 Measured Concentrations ofPerfluorobutane Sulfonatc, Potassium Salt (PFBS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Measured Concen tra :ion of Notninal'1'est sample 9ampiiuig Pclt]uorcbutane Suifonute, Percent Conceatration Number Time Potassium Salt (PFBS)' of -(me a_i ./L) (454A-117-) Phase (Day) (n-,g a .i .lL) Nominal 2 0_0 1 Uptake 0, 0 hours < LOQ -- (Negative 9 0, 4 hours <LOQ - Control) 17 1 < LOQ -- 25 3 < LOQ - 33 7 < LOQ -- 41 14 < LOQ - 49 57 2! < 28 LOQ < LOQ -- 65 Deptu'ation I < 1,OQ 73 3 < LOQ 81 7 <LOQ 89 10 <LOQ 97 14 < LOQ - -- - 0 .50 3 Uptake 0, 0 hours 0 .515 103 4 0, 0 hours 0 .518 104 11 0, 4 hours 0 .519 104 12 19 20 27 28 35 36 43 0, 4 hours 1 1 3 3 7 7 14 0 .525 105 0.478 95 .7 0.490 98 .0 0.542 109 0 .542 10') 0 .525 105 0.559 11 2 O .5[ 0 10 4 44 14 0.314 103 51 21 0 .593 1 19 52 21 0.567 114 59 28 0.515 103 60 28 0.5 i6 103 67 Depuration I < LOQ 68 1 < LO Q 75 3 < LOQ - 76 3 < LOQ - 83 7 < LOQ - 84 7 < LOQ -- 91 10 LOQ 92 10 < LOQ 99 14 < LOQ -- 100 14 < LOQ -- The limit of quantiratLoa (: ,OQ) was 0 .125 rng a.i./i., calculated as the product of the lowest calibration standard ( 0 .0005 mg a .iJL) and the dilution factor of the matrix blank sample (250) . Results wixe generated using Iv_4acQuan, version 1 .6 software . Manual calculations may differ slightly. p . 13 7 Wildtife International, L td. -61- Project Number 454A- 11 7 Table 7 (continued) Measured Concentrations of Perfluorobutane Suhlonate, Potassium Salt (PFBS) in Freshwater Samples from a B ;uegill Sunfish Bioconcentration Test Measured Concentration o f Nominal Test Sample Concentration Number sampling Time Perfluorooutane Sultor:ute, Percent Potassium Salt (PFBS)j of (mg a.i .lE .) (454A-117-) Phase (Day) (mg a .i49.) Nominal'- 5 .0 6 Uptake 0, C hours 5 .24 105 7 0, 0 hours 5 .42 108 14 0, 4 hours 5 .35 107 15 22 0,4 hours 5 .43 109 1 5.02 101 23 1 5 .12 102 30 3 5 .08 102 31 3 5 .12 ] 02 3$ 7 5 17 103 39 7 5 .22 104 46 14 5 .36 107 47 54 14 21 5 .32 106 5 .04 101 55 21 5 .21 104 62 28 5 .45 109 63 28 3 .40 108 70 Depuration . 1 < LOQ - 71 1 < LOQ 78 3 < LOQ 79 3 < LOQ 86 7 < LOQ 87 7 < LOO 94 10 < LOQ -- 95 10 < LOQ -- 102 14 < LQQ -- 103 14 < LOQ 'The limit of quantitatian (LOQ) was 0.123 mg a.i.l1., calculated as the product of the lowest calibration standard (0 .0005 mg a .i .lL) and the dilution factor of the matrix blank sample (250) . z Res ults were generated using MacQuan, version 1 .6 software. Manual calculations may differ slightly . ....r~ p . 138 Wildlife International, tL d. -62 Project Number 454A-117 Table 8 Measured Concentrations of Pcrfluorobutane Sulforiate, Potassium Salt (PFBS ) in Edible Fish Tisstte Samples from a Bluegill Sun6sh Bioconcentration Test Nominal Test Sample Concrntrat :an (mga .i.IL) Number (4 5 4A-117-j Phase sampling Ti i nc (Day) 4Saaurad Coacuntration of Pertluoro but:me Sulfonate. Potassium Salt (PFSS) ' (mga .i .rKY) 0.0 E-1 Uptake (Negati ve Control) F-2 0, 0, 4 hours <0 . 0355 4 hours <0 . 029 5 F.-14 1 <0_0695 E-15 1 <0.0725 E-27 3 <0 .0370 E-28 3 <0.0575 E-40 7 0.0548 E-41 7 <0.0515 E-53 14 <0,0398 E-54 14 <0.0975 E-66 21 0.0788 E-67 21 <0.033 2 E-79 2 8 <0 .0334 E-80 2 9 <U .0238 E-92 Depuration t 4 0.0710 E-93 <0.064 9 &10S 3 0 .110 E-106 3 <0.0598 E118 7 < 0.0423 E- ; 19 7 <0.0488 E-131 to <0.0428 E-132 t0 <0.0623 E-144 14 <0-0530 El43 l4 <0.095 3 0 .50 E-4 Up-k. O, 4 M- E-5 0, 4 haura <0 .05 9 5 E-6 . 0, 4 houa <0 .0393 E7 0. 4 hours <0.0390 E-17 I 0 .0382 E-18 l C0 .0478 fi-19 1 <0A915 E-20 1 <0 .0650 E-30 3 0 .0974 E-31 3 0 .0636 E-32 3 0.0911 E-33 E-43 E-44 3 7 7 0.0704 0 .116 0 .136 E-45 Fr46 7 7 0 .1C1 3 .1 :3 Less thaa values correspond to limit of quantitation (likQ ). For each analysis, the LOQ was calculated as the predict of the iowtst calibration stnndard (0 .0005 mg a.i JL) and the overall dilution Pac.tar of the srnple (!Az). Results were generated using MacQusw, vcrsion M . software . Manual cal= lationt may diffcr slightly . p . 13 9 Wildlz fe In ternational, Ltd. .63 . Project Number 454A-117 Table 8 (continued) Measured Concentratior-s of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Edible Fish Tissue Samples from a$luegill Sunfish Bioconcentration Test Measured Comcntrationof Nommal Teat S,rnplo Petflucxubutanc Sulfonzte, Pouysium Salt CoacenCition Number Sampling T`.me ( PFAS)' fmga. : ./L; (434p-1 :7-) Phase (Day) (mgLi .:Kg) 0 .50 E56 Uptake &17 14 E-58 E-59 E-u9 E-70 E-71 E-72 E-62 E-83 E-84 E-85 14 e-0 .0880 <0,0715 14 0 . 0 86 9 14 0.103 21 0.136 21 21 0.125 0.213 21 0.114 28 0.0682 28 0 .103 28 0 .0619 28 0_0871 E-93 Depuration 1 1 .19 E-96 1 3.31 E-97 1 0 .209 F.-98 1 ].98 E-108 3 0 .0758 E- 109 3 0 .0571 E-110 3 <0 .034I E-111 3 <0 .0488 E-12t 7 <0 .0401 E-122 7 <0 .0476 E-123 7 <0,04 14 E-124 7 <0.0915 E-134 10 <0 .0459 E-131 lC 0 .108 F-06 to e6.65 QO E-137 1r, <0 .0 59 5 E-147 14 <0.0499 E-14& 14 <0.0520 E-149 14 <0.0630 ElSO t4 <0.0364 5 .0 E-9 Uptake 0, 4 hours 0 .1 1 7 E10 0,4 hours 0 .0975 $.11 0, 4 hours 0-0811 F-13 0,4 hours 0.127 E-Z2 i E-13 1 0.240 0 .273 E-24 1 0 .311 E26 l 0 .234 L = than value6 Correspond to limit of quantitstion (LOQ) . For each analysis, the LOwQ wa: calculated as the product of the Iowan edlibrittiorl sturdard (0 .0005 mg a.i .IL) and the overall dilution factor cf thc sample (ljKg', Resu fts wera generaled using MacQuan, version 1 .6 sotlware. Maraual calculaliam may differ Slightly . p . 140 Wildlife Interna tional, Ltd. -64- Projcct Number 4 5 4A-1 17 Table 8 (continued) Measured Concentrations of Pclfluolobutanc Sulfonatc, Potassium Salt (PFBS) in Edible .Fish Tissue Samples from a Aluegill Sunfish BioconcentTation Test Meaaurad Cunczntra t ion of Numinal Teas Sample Sampling Perfluorobutane 3ulfunate, Pr.tassium Salt Concentration NLmber T ime (PF9S)' (mg a .i .iL) (454A-1 t7-) ?ham (Day) (mg airICg) 5 .0 E-35 Uptake 3 0.675 E-36 3 0.487 E37 3 0.6i 1 E-38 3 0 .448 E-48 7 1 .07 E-49 7 1 .19 E-50 7 . 0 .70E E-51 7 1 .30 E-6l 14 0 .439 E-62 14 1 .12 E-63 14 1 .15 E-64 14 0.74$ E-74 21 0 .596 E75 21 0 .593 E-76 21 1 .50 E-77 21 0 .590 E-87 28 3-7, 64 fi-9x 28 3 .907 E .89 28 0 .656 E-90 28 0 .837 E-100 Depuration 1 7.718 E-101 l 0.622 E-102 E-103 1 I 1 .05 0.467 E-I13 3 0,235 E-114 sits A E-t 16 E-126 E-127 3 0 .197 nrnt 3 0 .413 7 0 .196 7 0 .151 E-1Z8 7 0 .124 E-129 7 <0.0879 E-139 10 0.0791 E140 '.0 <0.0740 E141 1 0 <0 .0815 E-142 0 <0 .0930 g .I52 14 <0 .0410 E-153 14 <0 .0454 E-154 . 14 <0 .0685 F,155 14 <0 .0705 Lacs than values carrespornd to limit of quant3ation (LOQ) . For cach anulysis, the LOQ was caluulated as the pnxiu, oCthe lowest calibratio n standard ( 0 .0005 mg a .i.iL) and the overall dilution factor of the sampk (UKg). Rcsults were generated using N(aaQuan, version 1 .6 softwan. Manual calculations may differ Slightly. p . 14 1 Wildlife International, Ltd. -65- Project Numbcr 454A-11 7 Table 9 Measured Concentrations of Pcrfluorobutane Sulfonate, Potassium Salt (PFBS) in Nonecib3e Fish Tissue Samplcs from a Blueol Sunfish Biocoaceiltratiotl Test \ontiaai Test Conceotraron (rtte 3.1./I,) 0.0 (Negative Contra I) Measured Concentration of sample sampling Pert7uurotw 1. me Sulfonale, Potassium Salt Number That (PFBS Y (454A-117-) Phan ( Day) (atg a.i .lKg) NI Uptake D. 4 hours <0 .0569 N-2 0. 4 hours <0 :04fi 1 N-14 I <U .0713 N-1 S I <0.0824 P:-27 i <0.0307 N-78 3 <0. U782 N-4p 7 0 .164 N-41 7 a0 .0887 N-53 14 <0 .0443 N-54 14 <0 .101 N-66 21 0 .0530 N-67 21 0,0492 N-79 28 <0 .0625 N-80 28 <0 .0426 N-92 Depuration 1 <0 .0731 N-93 1 <0 .0748 N-I OS 3 0 .224 N-106 3 <0 .0638 N-118 7 <0 .0644 N-119 7 <0 .0570 N-131 10 <0 .0541 N-131 10 <0-0732 N-144 14 -~0 .0990 N-145 14 <0 .10 3 0 .30 N-4 Uptake 0, 4 ham <0.0630 N-S 0, 4 haurY 0_0727 N-6 0, 4 hours 13 1.0439 N7 0, 4 &w~ <0,0749 N-17 l 0.0786 N-18 1 0.0894 N-t9 1 0.112 N-20 ! 0.0730 N-30 3 9.180 N-31 3 0.139 N-32 3 0.1^3 N-33 3 0.141 tv-43 7 0.:65 N-44 7 0.293 N -4 3 7 0.263 N-46 7 0.307 La= than value, correspond to limst of qu>mtitation (LCQ) . For each anaivsis, the LOQ was caicusated as the mean meau i rcd background (matrix blank) conecnv atiens p:us litres standard deviations of the rnearwred background (0.D792 mg ai .1Kg), adjusted for the indi vidual weigh t nf the tissue samp la . Results were g=rated using MacQuan, version 1 .6 software. Manual calculatiottc may differ sli8ttiy . p . 14 2 M rdli fe International, Ltd. -66- Project Number 454A.1 17 Table 9 (continued) Measured Concentrations of PerfTuorobutane Sulfonatc, Potassium Salt (PFBS) in Nonedible Fish Tissue Samples from a Blucgjll Sunfish Bioconcetltration Test Me :ssurad Coneetsation of Nomina: Test Sample Perfluorobutane SuSonace, Pot,usium Salt Concentration Number sampling Time (PFr~S) L (mga_iJL) (4:4A-117-) Phase (Day) (mga.i ./Kg) 0, 50 N-56 l:ptak: 14 0 .235 N-]7 14 0 .181 N-58 14 0,236 N-39 14 0 .179 N-69 21 0 .341 N-70 21 0 .314 N-71 21 0 .579 N-72 21 0 .323 N-x2 29 0 .185 N-83 28 0.262 N-84 28 0 .153 N-85 28 0 .243 N-95 Depuration Tl-96 1 N-97 1 N-98 1 1 0.207 1 .13 0.337 6 .11 N-10g 3 0 .156 N-1D9 3 0 .145 V-110 3 <0,0457 N 11! N-121 3 7 0.0934 0.0920 N122 N-I23 N-124 7 7 <0.0515 7 <0 .0562 <0,682 N-134 10 <0 .0634 N-135 10 <0 .062 0 N436 to 10 .064 5 N-137 10 <0.0735 N,147 14 0.0796 N-148 14 0,D343 N449 14 <0.0808 N 4 50 14 <0 .049 0 3 .0 N-9 11pt.ike 0, 4 hours 0 .233 111-10 0,4 hours 0 .173 N-11 0, 4 hours 0 .178 N-12 0, 4 hours 0 .247 N-22 i 0 .726 N-23 0.730 N-24 1 0 .702 N-26 0 .599 [ass than values correspond to limit or quantitrtion (LOQ). For each analysis, the LCIQ was calculated as the mean measured background (matrix blank) concentrations plus three atanderd deviation: of the mesxusrad background (0 .0792 mg ai .:Xg), adjusted for the individual weight ofUte tissue sample. ' Results were generzted using kfacQuan, version 1 .6 software. Manual calculations may differ slightly . ~..~ ..~.. p . 143 Wildlife International, Ltd. -67- Project Number 4 54A-I17 Table 9 (continued) Measured Concentrations ofPer#luorobutane Sulfonate, Potassium Salt (PFBS) in Nonedible Fish Tissue Samples from a Bluegill Sunfi sh Bioconcentration Test lVleaenred C om enlral i on of' Nominal Tss1 Sample Sampling Perflucrobutanc SulTonate, Pousswm Salt Concentrition Number Time ( mga .i .iL) (454A-I17 .) Phase (Day) ( PF13S) ' (mga.iJKB) 50 N35 Uptaita 3 1 .49 N-36 3 1 .35 N-37 3 2-08 N-38 3 1 .29 N-48 7 3 10 N- 17 7 7.95 N-30 7 2.04 N-51 7 3.68 N-61 14 .0.960 N-62 14 3.28 N63 14 3.42 N-64 14 2.22 N-74 21 1 .36 N-7S 21 1 .66 N-76 21 4 .D6 N-77 21 : .6A N-B7 78 1 .66 N-88 28 2 .02 \-s9 28 2,05 N-90 28 2 .22 [Y-100 Depuratiat 1 2 .39 N-101 1 2 .32 N-102 1 3 .29 N-103 l 1 .17 N-113 . 3 0.676 N-114 3 0 .329 N-113 3 1- 5 7 N-116 3 1 .36 N126 7 0,691 N-127 7 0 .530 N-128 7 0 .370 N-129 7 <0.0808 N-139 10 0 .181 N-I40 10 c0 .0800 N- 141 10 <0.08 87 N-142 10 :0 .0903 N-152 14 <0,0494 N-153 14 <0 .060 7 N-154 14 <0 .0773 N- 1 55 14 <0 .0847 ' l.esa than values correspond to lima of quantitalion (LDQ,L For each >nalysia, the I .UQ was calculated a s the mean measured hmkgroun d (nclzix blank) coneantrationa plus tltroc otandssd 3eviation+ of !he r.eaaurad background (0.0792 mg a.i-/Kg), adjustad for the indiv idual w<ig,ht of the tissue sample. 2 Rzsults were generated using blac .Qumt, version 1 .6 software . Manual calculations may diffor slightly. .~_ p . 144 Wildli fe Interna tional, Ltd. -68- 1'roject Number 454A-117 Table L O Measured Concentrations ofPclflu .orobutane Sulfonate, Potassium Salt (PFBS) in Tissue Storage Stability Samples from a Bluegill Sunfish Bioconcentratitm Tes t Measured C onc ontrat ion o f Nominal Sample PerBuorobulana Sulicnate, Ponn.`ium Sa1: Pc:ee :u Cencer.uation Number Tissue (PFFSS) af (mga.i .IKg) (454A-t17) Type fmga .i .1K8) Norninal= Negrtive control EMA3-13' Edible <LCQ' DI-IvSAB13' Nonedible <!AQ' 0.100 E-STMAS-1' Edibie 0 .0732 73.2 E-btAS-25' Edible 0 .0732 73 .2 N-STMASly :Qonediblc 0 .0925 92 .5 N-MAS-25' Nonrdible 0 .0741 74 . 1 10 .0 E-STMAS-2' Edible 9.39 93 .~ oh1AS-26' Edible 8.82 88,2 V-STVSAS-2' i4-MAS-26' Nonedible 8 .34 Nonedible 8.09 83 .4 80 .9 Le v than values correspond to limit of quantitation (LOQ) . The LOQ for the edible ti ssue matrix was 0 .0500 mg DI/Kg. calculated es the product of the lowest calibrxtiua stand" (0.0005 mg s.i.lL) and the overall dilution factor of the sample (100 UKg). TI>C LOQ for the nonedihle tissue matrix was 0 .0792 mg a_i Ag calculated as the product of the mean measured background concentration plus three xhndard deviations of the measured background concentralions. All sample weights t 1 .00 gram. 'Results were generated using MaaQu.u4 version 1 .6 software. Manual calculations may differ slightly . 'Tile stability samples were fnrtified on luly 1 8, 2000 and stored in the freezer. September S, 7000 ( after 49 days of fra2en storage) and analyze & The samples were removed from the freezer on These samples were prepared on September 5, 2000 and the results were compared to the stability sample results. .., . ... ~ .. p . 145 YVildli fe International, Ltd. -69- Project Number 45 4A-11 7 Table 1 1 Lipid Content in Edible Fish Tissue NcminsiTcst 5zI:ple Concentration Number {uiga .i./L} (454A-117-) Samph:t.g Lipid Phase Tune Weight (Day) (g) Lapid'Ciah Fish Tissue Tissue 'Neighl weight , (g) (tnglKg) O,G (Negativs C(m itrol) ELI Uptake 0, 0 ho.u 0 .0101 0 .9891 10200 EL? 0, 0 hour 0.0139 1 .0534 13200 ELI 0. 0 hour 0.909T 1 .0629 MO10, EL4 0, 0 hour 0.0059 0.5316 11100 EL7 Uptake 28 0.0L77 1 .7044 10400 ELS 28 0.0184 1 .5720 11700 EL9 28 0.9210 1 .2 386 17000 ELLO 28 0:7139 1 .1749 11500 ELZ1 Acpuration 14 0.0147 0.9668 15200 EL22 14 0.0108 0.6989 15500 EL2 3 EL24 14 0?009 8 0.5352 18300 1 4 0.0168 1 . .11 2 2 15100 0_50 ELI 1 Uptake EL12 2R C .0137 1 .0888 1 2600 28 0.0158 1 .3244 11900 EL13 28 C.0093 0 .7595 12200 E1.1 4 28 C .0109 0 .8266 13200 EL76 Depur.ltioo 1 4 O .fll :i2 0 .69 1 2 19100 8L27 1 4 0 .0160 0 .7963 20100 EL28 14 0 .0 1 35 0 .8681 15600 EL29 1 4 0 .0084 0 .6457 13000 3.0 ELL6 Uptake 28 0 .009 1 0 .7488 12250 ELI7 28 0 .0098 0 .6174 15900 ELLB 28 0 .0099 0 .7166 13800 EL 19 28 0 .0086 0.6388 13 1 00 EL31 Dapuration 14 0 .0142 0.9458 1 5000 E1 32 14 0.0161 0.9085 17705 E133 14 0 .01 1 3 0.6717 1 6800 F,f_34 14 0 .0094 0. 6 581 14300 Rrtia calcu3alod as [iipid weight (g)+ fiaL tiarue wcight (g)j y 1000 meg K 1000 grKg. p . 146 ~.~~~...~ Wildlife International, Ltd. -70- Project Number 454A-! 1 7 Table 1 2 Lipid Content in Noncdible Fish Tissue tlutninal Teat Sample Sampling Lipid Concentration Number Time We;ght (mg a.iJL) (454A-117-) Ph me (Day,, (8) Li pid/Fish Fish Tissue 7 issue Weight Weipht' (g) (m WKY) 0 .0 (Negati ve Control) NLI Uptaka 0, C hour 0.0! 81 1 .3U08 139W NL2 0, 0 hour 0.0935 1 .3820 24200 NL3 0, 0 hour 0.0 :5S 1 .2944 120C0 NL4 0, 0 hour 0 .0134 0.7521 17800 NL7 Uptake 28 0 .0248 E .29 1 5 19200 iAfL8 28 0.0271 1 .2749 213C0 NL9 28 0.0353 1 .2049 21000 NI-10 28 0.0 :95 1 .2714 15300 NL2l Depurttioa 14 0 .0252 1 .1730 21 5 00 23L22 14 0 .0167 0.8990 18600 NL23 14 0 .0167 0.8678 J8700 NL24 14 0.0283 0.9681 29200 0 .30 NLLI Uptake 28 0 .0313 1 .3192 7 3900 NLL2 28 0.0293 1 .6196 18100 NLl3 28 0.0188 0.8519 22100 NL14 28 0.0328 1 .1668 28100 NL26 Deputation 14 0.0201 1 .0339 19100 NL27 14 0.0405 1 .0630 38100 NL28 1 4 0.0284 1 .203 1 23600 NL29 :4 0.01 9 1 1 .0488 18200 3 .0 4L16 Uptake 28 0. 0:20 1 .1230 19600 NL17 28 0,0283 0 .9749 29000 NUS 28 0,0240 1 .0825 22200 14L19 :8 0 .0186 0 .9275 20100 N13 1 Deputation 14 0 .0407 1 .1694 34500 N1 3 2 14 0 .0394 1 .0638 37000 NL33 14 14 1-34 14 0 .0340 0.9449 36000 6 .0i~7 0.9 5 60 133fri1 1 Ratio calculated as (lipid weieht ( g)= Ctsh tissue weightfig)] x 1000 rngJg x 1000 yJKg. 14Jildlife International, Ltd . -71- p . 14 7 ~ ..ww.~~....~.~ .~ Proj act Number 454A- 117 METHOD OUTLINE FOR THE ANALYSIS OF PFRFLUOROBUTAVE SULFOI'ATE, POTASSIUM SALT (PFBS) IN FRFSHWATFR Prepare matrix fortification samples in freshwater matrix by spiking the requisite volume of PFJ3S stock solutions directly into frcshwater. Perform fortifications with gas-tight svringes and Class A volumetri c #lasks. f Prepare appropriate dilutions of study and QC samples to within the calibration range of the pFBS i,C/11tS methodoloU: Partially fill Class A volumetric fl asks with dilution solvent (50% methano[ : 50% NANOpure water) . Add app ro p ri ate volume of sample and b ring to volume with dilution solvent . Process matrix blank samples using the same dilution and aliquot volumes as for the lowest fortification level . Mix well by sevcral repeat inversions . Arnpulate samples and submit for LC/MS analysis . Figure 1 . Method flow chart for the analysis of Perfluorobutan e Sulfonate, Potassium Salt (PFBS) in freshwater. W ildli fe International, tL d. -72- p . 148 ~~~.,..r~. ....s Project Number 454A-1 17 M>`:'1'HOD OUTLINE FOR THE ANALYSIS OF PERF'LUOROBUTANE SULFONATE. POTASSIUM SALT (PFBS) IN FISH TISSUES Quality control samples are prepared from aliquots (approximately I g) of bulk control rLsh 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 . y Add 10 .0 niL of methanol to each sample with a glass Class A volumetric pipette . Homogenize each test sample for approximately l minute using a hand-held tissue shredder . Rinse the homogenizer with the appropriate solvent(s) in between samples . ~ Sonicate each sample for approximately 5 minutes with a sonic dismernbrato r Cap the vials and shake well. Centrifuge the vials at approximately 2000 rpm for approximately 5 minutes , Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFBS LCMS methodology: Partially fill Class A volumetric flasks with 50:50 methanol :NANO'Opure water dilution solvent. Add appropriate volume of sample and bring to volume with dilution solvent . Process matrix blank samples for a given matrix using the same dilution and aliquot volumes as for the lowest fortification level in that matrix . Mix well by several repeat inversions . ~ Transfer an aliquot of each sample to an autosampler vial and submit for LCIMS/MS analysis . Figure 2 . Method flow chart for the analysis of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in fish tissues . p . 149 ti'Yl ld4fe International, Ltd. - 73 - Project Number 4 54A- 1 1? M ET1IOD OUTLINE FOR THE ANALYSIS OF LIPIDS IN FISH TISSUES Remove vials to be analyzed from the freezer . Allow samples to thaw . ~ I'r.r each sample, add 10 mL of VANUpure water to lish tissue in vial and homoger.ize for iporoximateJy )minute uing a hand-held lie=4 yhredder. Rinse the homogenizer wrth the rppropriate so.vent(s) in between snmplea . y Transfer each homogenate to a I30-ml, separato ry flmnel that cortfaitu 2] mL of chlorofomt atW 30 mL or methanol. Rinse each vial with ar, additional I0 nmL nf NANOpure water and pour rinse into respective separatary runnel . 1 Shake cock scpat atory funnel for appsoximately one minute with venting. y Add 5D mL af chlorofortn followed by 34 tnL of saturated sodium chloride to each cepantoty funnel. B rie fl y swirl each separatory ftulnci with venting. 1 Allow the phases to separatc . For each sample, drain the chloroform layer through a powder fume] packed with Teflon wool and anhydrous sodium sulfate inlua 25 0-rnL round-lwttoin flask. AJd .u~ Jditional SOH. .I . s} syxot of rhlwot'or. te . .elt eep.ratpry fu-1 . ..d .ep..t tSw -traction n ..d d<ain:nY p,...1- Rotary evaporate the extract.t in a water both maintained at approximately 401 C to now dryness. 1 Tramfer each extract to a pre-wei g tcd,labeled scintillation vial. Rinse each 230-tnL ruuu6 botYom flask with a small volume of chlurofomt and transfer rinse to respective scintillation vial . Evaporate the remaining solvent in each vial under a gentle stream of nitrogen or clean dry air. y Reweigh each viai and record waight- Figure 3 . Method flow chart for the analysis of lipids in fish tissues . p . 15 0 Wildlife International, Ltd. -74- ProjectNumber4 54A- 11 7 Ara a ~ 3600001 320000260000 ,240200000160000120U008000 0' 400000 .0 0.60 1 .20 1 .80 2 .40 3 .00 3 .60 4 .20 4 .80 Cooc_ ug a.U L Figure 4, A typical calibration curve for PerEluorobutane Sulfonate, Potassium Salt (PFBS) in freshwater . Slope = 72902 .35156 ; Intercept = 2116 .42700 ; r= 0 .9998 . Curve is weighted (1!x), p . 15 1 Wirdrife International, Ltd. -75 - Project Number 454A-1 17 Area 70000~ _~ If 60000J { 540001 4000 ~ 30000- 20000- 10000. 0 0 .0 0 .60 1 .20 1 .80 2 .40 3 .00 3 .60 4 .20 4 .ea Conc. ug a.iJ L F igure 5. A typical calibration curve for Pertluorobulane Sulfonate, Potassium Salt (PFBS) in fish tissue. Slope = 15309 .60 ; Intercept =-1042 .05 ; r = 0 .99793 . Curve is weighted ('!x) . Wildlife International, Ltd. -76- .M...~ ...~ p . 152 Project Number 4 5aa-117 intensity: 60000 op, 100 90- s 70 6 S 4 30- 2 t 102 fl 4 0 6 4_. 123 16 0 21 41 61 a1 1 6 1 121 141 161 0.35 0 .69 1 .02 1 .36 1 .69 2.03 2 .36 2 .70 scan Time Figure 6 . A representative ion chromatogram of a low-level (0 .500 g a .i .IL) Perfluorobutan e Sulfonatc . Potassium Salt (PFBS) standard for freshwater analyses . ~ Wa1dli e International, .~td. .77 . ... ~... p . 153 ProjcctNumher~~4A-117 fntensity~ 60000 cps 100, 102 9 8 70- . 5 40- 7~ff.i ;.. 30- 2 1 45 58 73 ` 15D]8 0 21 41 61 81 101 121 141 161 Scan 0 .35 0.89 1 .02 1 .36 1 .69 2.03 2.36 270 Time Figure 7 . A represcntative ion chromatogram of a high-tuvcl ( 5 .00 }lg a .i./L) Perfluorobutan e Sulfonate, Potassium Salt (PFBS) standard for freshwater analyses . p . 154 Wildlife International, Ltd. _78- Project Number 454A-11 7 intensity ; 6 000 cps 100- g0 70- 6 so 4 30- 20~~ Y I 0 15 51 72 94 126 ~ 21 8 41 81 12 1 161 201 241 281 Scan 0 .69 1 .36 2 .03 2.70 3 .37 4.04 4 .71 Time I - ------ ._---- .~.~_ _ ~ Figure 8 . A representative ion chromatogram of a low-level ( 0'.500 Ag a .iJL) Perftuorobutane Sulfonatc, Potassium Salt (PFBS) standard for fi sh tissue analyses . _ Wildlife International., Ltd. -79- .s.........~ p . 155 Project Number 454A-1 1 7 100 so- 706050403020, 0- intensity : 6000 cps 18 4 ii 25 6 4 i 81 121 161 201 241 2 81 Scan 0 .89 1 .36 2.03 2 .70 3 .37 4 .04 4 .71 Time f igure 9, A representative ion ch r omatogram of a high-level (5 .00 g a .i ./L) F'erfluorobutane Su(fonate, Potassium Salt (PFBS) standard for fish tissue analyses . p . 15 6 Wirdt~fe International, Ltd. -80- Pro}xtNumbcr454A-1]? intenclly: 60000 cps 10 90- S 70- 6 01 5 34 2 t 21 52 82 101 128 t55 172 Q 21 41 61 81 101 12t 14i 1 61 SCan 0.35 0.69 1 .02 1 .36 1 .69 2.03 2 .36 2.70 Tima Figure 10 . A representative ion ch romatogram of a freshwater matrix blank sampl e (454A - 1I7 - MA$ - 1, dilution = 250x) . The arrow indicates the retention time of Perfluorobutane Sulfonate, Potassium Salt (PFBS) . p . 15 7 Wildlife fe International, Ltd. -81- Project Number 454A-] 1 7 ~~ 1a 9 8 701 6 intensity- 50000 cps 40 102 20, 1 0 99-117 83 15 0 164 21 41 61 8 1 101 121 141 16j SMn 0.35 0.69 1 .02 1 .36 1 .69 2 .03 2 .36 2,70 Time tI Figure 11 . A representativc ion chromatogram of a freshwater matrix fortification sample (454A-117-M 45-2, 2 .00 mg a_i.IL nominal concentration, dilution = 1000a) . Wildlife International, Ltd. -82 . p . 15 8 r . ...r,~ Project ?Vum5er454 A -11 7 100- intensity : 600p cps go- so- 70- 60- so- 40- so- 20- 10- 261 01 ~ 7487 111 141 188 25 4 41 81 121 161 2 41 2 81 Scan 0 .69 1 .36 2 .03 2.70 3 .37 4 .04 4.71 Tim e 4 Figure 1 2 . A representative ion chromatogram of an edible fish tissue matrix blank sample (454A117-E-1V1AB-3, overall dilution factor = 100 IJKg) . The arrow indicates the retention time of Perfluorobutane Sulfonate, Potassium Salt (PFBS) . p . 15 9 Wildlife In ternational, Ltd. -83- Project Number 454A-1 1 7 Intensity : 6000 cps 10 0 go- 70- 60- 50- 403 1 20- 10- 186 a 46 78 23 6 41 8 1 121 161 201 241 281 Scan 0 .69 1 .36 2 .03 2 .70 3 .37 4.04 4 .71 Time Figure 13 . A representative ion chromatogram of a nonedible fish tissue matrix blank sample (4 5 4A117-t*I-'1fAB-2, overall dilution factor - 100 L/Kg) . The arrow indicates the retention time of Perftuorohutane Sulfonn.te, Potassium Salt (PFBS) . For a disaussion of the integrated peak in -vicinity of the analyte retention time, see page 48 . p . 16 0 Wildlife International, Ltd. -84- Proicet Number 454A-1 1 7 f 10 8 7060- 5 403020 10 intqnsity : 6000 cps 18 4 7186 114 138 231 27 1 41 81 121 1 6 1 201 24 1 281 Scan 0 .69 1 .36 2 .03 2 .70 3.37 4.04 4 .71 Time Figure 14 . A representative ion chromatogram of an edible fi sh tissue matrix fortification sample (454A-1 i7-F,-MAS-5, 0 .100 mg a .i ./Kg nominal concentration, overall dilution factor= I00 UKg) . Note : fortif 'scation level 2 x LOQ (0 .0500 mg a_i .lKg} . p . 16 1 Wirdlz fe Interna tion al, Ltd. -8 3 - Project Number 45 4A - 11 7 100go80706 5040- J 3 201 (3 intensity: 6000 cps 184 47 110 133 228 25 6 41 81 0,69 1 .36 121 181 201 241 281 Scan 2.03 2 .70 3.37 4 .04 4 .71 Time 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 interfcreat on shoulder of analyte peak p . 162 Wildlife In te7''-hLZtlOYIGdl, .L td. -8b- Project Numbcr 454A- 1 17 Intensity : 60000 cQs 10 7g 6 5 4 102 2 1 0 22 40 55 72 83 .` , 14B 16 4 21 41 61 81 1Q1 121 t41 161 SC~1 0 .35 0.89 1 .02 1_38 1 .69 2 .03 2 .36 2.70 lime Figure 16. A representative ion chromatogram of a freshwater sample (454A-[17-3, dilution= 250x} from the 0 .50 mg a .i .lL .. treatment group . p . 16 3 Wildlife International, Ltd. _87- Project Number 454A-1 1 7 intensity: 6000 cps to 90 80 7 6050403020- 18 4 y p 41 81 121 161 201 241 281 Scan 0 .69 1 .36 2 .03 2.70 3 .37 4 .04 4 .71 Time Figure 17 . A representative ion chromatogram of an edible fish tissue sample (454A-1 17-E-33, over-al l dilution factor = 110) from the 0 . 5 0 mg a .i ./L treatment group). p . 164 WZ ldl1fe International? d Lt . -88- Project Number 454A-1 17 intensity: 6000 cps 10 90- 80- 70- 60- HI____182 ~ 64 98 133 26 1 41 81 121 161 201 241 281 Scan 0 .69 1 .36 2 .03 2.70 3 .37 4 .04 4 .71 Time Figure 18 . A representative ion chromatogram of a nonedihle fish tissue sample (454A-[17-N-33 , overall dilution factor = 73 .3) from the 0 .50 mg a .i ./L traaunent group) . p . 165 i~7 Q ~ ~ N a. ~ x ad oo ~ ~a4 ~ o 0 N N N EL v N N N xc o 00 G x o ac o0 n ! o 0 O .~In . S vN Ny~GjUN c' Vf {r ,,. O O O 4 BCOU W+ I:L N C1 o0 00 ~o u s []~ G O !, T k O = 5 y r ~3 N N ~ ~ ~ O G O n r ..~ ~.. ~ . l U V v~ N l Id ` N Ei 7 IV n C4 70 ~ :~ _ ,d . a+ p' O O -' C-i .Q~LC a ~I ap n a* rp y ~ FL., cc Q ~' < v~ ,n u ac oc od V Q ~ U o ~ n 4 L T: A3 V N ( ~ 00 `C a~l+ N N y{.. ~ CO 06 7 N 1~- ~ .C Q fri Qy ` . .: U r~ ~ O G N! ~ ~" .~-r ^ . _ _ _ G r zC ~ ~ ~ ~ C o W ~ ~- ~ z y E U N N + V .... N N N N p u~~ U fI v ~ y f u y m ~ G ~ `y~ a ~b ~ U c-i vt O N + .: ~ Lc o r k c ac a ~~v~C~ ~~C~" ~ 2 u ~ Z :J p . 16 6 aiwrinrrs,nr..ur~~ Wildlife Intemational, Rd. -90- Project Number 454A . 1 17 APPENDIX V Dissolved Oaygc,n (mg/i .) of Water in the Test Chambers' Sponsor: 3M Corporatio S nTestSubacvPFB Test Organism : Bluegill, Lepomis macrochirus Dilution Water : Well Water Uptake Phase Day Negative Control 0 . 5 3 mg a .i ./L 5 .2 rng a .i.!L. 0 1 8 .7 7 .7 8.7 7 .7 8 .7 7 .6 2 3 4 5 6 7 8 9 10 11 7 .8 8 .3 8 .2 8 .2 8 .1 8 .6 8 .4 8 .5 8 .4 8 .4 7 .8 8 .3 8 .1 8 .2 8 .0 8 .6 8 .4 8 .5 8 .3 8 .4 '7 .8 8 .3 8 .0 8 .2 8 .0 8 .6 8.5 8 .5 3.3 8 .4 12 8 .6 8 .5 8 .5 13 8 .6 14 7 .6 15 . 7 .9 16 7 .4 17 9 .1 18 8,2 19 8 .1 20 7,9 21 7 .9 22 8 .1 . 23 8 .1 24 8 .0 25 8 .2 26 8 .1 27 28 $ .7 8 .0 8 .6 7 .7 7 .9 7 .4 8 .0 8 .1 8 .1 7 .9 8 .0 7 .9 8 .0 8 .2 8 .3 8 .1 3 .6 9 .1 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 'A dissolved oxyg en concentrazion of 5 .2 mgJl, represent s 60% saturation in freshwater at 22 0 C . p . 16 7 Wildlife International, L td. -91- Project Number 454A . i 17 APPENDIX V (Continued) Dissolved OZ.gen (rngl) of Water in the Test Chainbers' Sponsor : 3M Corporation Test Substancc : PFH S Test Organism : Bluegill, LeElorr.is macrochirus Dilution Water: Well Water Depuration Phase Day Negative Conrrol 0 .53 mg a . UL 5 .2 mg a-i,lL 1 2 3 4 7.8 7.9 8.2 3 .2 7_8 7 .9 8 .2 8_0 8 .3 8 .3 8 .2 8 .1 5 6 7 8 . 8.4 .8 .6 8 .4 7.6 8 .4 8 .2 8 .8 8 .8 8 .2 8 .2 7 .8 7,9 9 8 .7 10 8 .7 8,7 8 .7 8 .6 8 .7 11 8 .0 1 2 8 .0 8 .2 8 .4 8 .2 8 .4 13 8 .0 14 8 .6 8 .Q 8 .4 8 .0 8 .4 15 16 8 .4 8 .6 8.3 8 .7 8,3 8, 6 'A di sso l ved oxygen conce ntration of 5 .2 mg/L represcras 60% saturation in freshwater at 22C- p . 168 WxlG~l!fe International., d Lt . .92 . Projcct Numbe, 454A-117 APPENDIX VI Hardness, Alkalinity, Conductivity and TOC of Water in the Negativc Control _,__Uptake Phase sponsor : 3M Corporatio n Test Substaace : PFB S Test Or ;=ism : BtuegilI, Lepornis macrUChirus Dilutiot: Water: Well Water Parameter 0 7 Hardness 128 128 (mg/L as CaCO3) ~-----~~ Day 1 4 21 28 104 128 112 ALkulinity 164 174 166 174 .180 (mg/I, as CaCO3 ) Conductivity 330 --1 330 330 3 0 0 C.umhos/cm) 10(: (mg C'i. ) <1 <1 Conductivity inadvertently not recorded on Dav 7 <1 <1 <1 ma ru e~tisoe~ s Depuration Phas e . .. .. Spunsor : 3M Corporation Test Substance: PFB S Test Organism: Bluegill, Lepomis moc,nchirus Dilution Water : Well Water Paramctcr 7 Day 14 16 Hardness (mg/L as CaCO3) 116 116 116 Alkalir.ity (mgJL as CaCO3 ) 180 184 182 Conductivity 330 323 325 0.emhoslcm) TOC <I Not Measured (m8 CIL,) p . 16 9 Wildlife International, Ltd. -93- Project Nwnber 454A-1 1 7 APPENDIX VI I Cumulative Percent Mor:aIity and Treattnent-Related Effects' .Negative Control -,Uptake Pha se Sponsor : 3M Corporation Test Substance' PFES Test Organism : Bluegill, Leporrus rraacrotA+rus I)iiutinn Water: Well Water Cumulative Number Number Number Remaining Sampled Day Obscrv a4ons Dcad 0 I 2 3 4 5 AN 0 85 . AN 0 80 AN 0 75 AN 0 75 AN 0 70 AN 0 i0 5 5 0 5 0 0 6 7 AN AN 0 0 70 70 0 5 8 AN 0 65 0 9 10 II AN AN AN 0 0 0 65 65 65 0 0 0 12 13 14 15 16 AN 0 AN 0 AN 0 AN 0 AN 0 65 65 65 60 60 0 0 5 0 0 17 18 19 20 21 22 AN AN AN AN AN AN 0 0 0 0 0 0 60 60 60 60 60 55 0 0 0 0 5 0 23 24 25 AN AN AN 0 0 0 55 55 55 0 0 0 26 27 AN AN 0 0 55 55 0 0 28 AN 0 55 10 ' Ubservcd Effects : AN Appcars Normal p . 17 0 Wildlife InteYnational, Ltd. -94- Project Number 454A-117 APPENDIX VII (Continucd) Cumulativ e Pcrccnt Mortality and Treatment-Related Effects ' Negativo Control - Depuration Phasc Snonsor. 3M Corporation Test Substance ; PFBS T iM nrganisu: : 3tur8ill, f.epornis mecrochirtes Dilution Wa tcr: Well Water Cumulative Number Day Observations Dead Number Number Rcrnaini ng Sampled I AN 0 45 5 2 AN 0 40 0 3 AN 0 40 5 4 AN 0 35 0 5 6 7 AN AS AN 0 0 0 35 35 35 0 0 5 S AN 0 30 0 9 AN 10 AN 0 0 30 30 0 5 II AN 0 25 0 12 AN 0 25 0 13 AN 0 25 0 14 AN 0 25 10 15 AN 0 15 0 16 AN 0 l Clbservcd E1Teats: ACi - App earsNor.aa l . F---- ..~~ 15 - 0 p . 17 1 i ~r~wrw~ r Wildlife International, Ltd. -95- Project Number 454A-1 17 APPENDIX Vtt (Continued) Cumulative Percent Mortality and TreatinentRelat:xl Effects 0.53 mg a.i ./L--Uptake Phase Sponsor: 3NS Corporation Test Substanoc : PF13S 'f est Organism . 31 ungill, Lepnmis macroch[rus Dilution Water: well Water Cumuiativc Number Day -Observations Dead Number Number Remaining Sam p:ed 0 AN 0 85 5 I 2 3 4 S 6 7 S 9 AN 0 80 AN 0 75 AN 0 75 AN 0 ?0 AN 0 70 AN 0 70 AN 0 70 AN 0 65 AN 0 65 5 0 5 0 0 0 5 0 0 10 1? AN AN 0 0 65 65 0 0 l2 13 14 15 AN 0 65 AN 0 63 . AN 0 6 ; AN 0 60 0 0 5 0 16 17 AN AN 0 0 60 60 0 0 l8 AN 19 AN 20 AN 0 0 0 60 60 60 0 0 0 21 AN 0 60 5 22 AN 0 55 0 23 AN 0 55 0 24 AN 0 55 25 AN 0 55 26 AN 0 55 0 0 0 27 AN 0 55 0 28 AN 0 55 to 'Obscrscd E ffcnte : AN Appears Normal , ~_^~ p . 172 ~1~r irrw~wwr Wildlife International, L td. -97- Project Number 454A. 11 7 APPENDIX VIT (Continued) Cumulative Percent Mortality and Treattnent-Rclated Effects' 5 .2 mg a.i ./L-Uptake Phase Sponsor : 3M Corpo ration Test Substance : P1:B S Test Organism 131uegi1l, Lepomis macrxhirw Dilution Water. Well Water Cumulative Number Number Number Remaining Sampled Day Observafians Dcad 0 AN 0 85 5 ANT 0 80 5 2 3 4 AN AN AN 0 0 0 75 75 70 0 5 0 5 6 7 AN AN AN 0 0 0 70 70 70 0 0 5 8 9 AN AN 0 0 65 65 0 0 10 11 12 AN AN AN q 0 0 65 65 65 0 0 0 13 AN U 65 0 14 15 AN AN 0 0 65 60 5 0 16 17 18 AN AN AN 0 0 0 60 60 60 0 0 0 19 20 AN AN 0 0 60 60 0 0 21 22 23 AN 0 60 AN 0 55 AN 1 54 5 0 0 24 25 26 27 AN AN AN AN 1 54 154 1 54 1 54 0 0 0 0 28 AN 1 54 t0 'Observed EtIcols: AN- Appears Normal p . 17 3 Waldllfe International, Ltd. .98- Project Number 454A-] 17 APPENDIX VII (Continued) Cumulative Percent Morta[itv and Treatment-Related Effects' 5 .2 mg a.i./L - De uration Phas e Sponsor: 3M Co. poration Test Substance: PFB S Test Organism : $iuegill, Lepomir raacrochirres Dflution Water: Well Water Da Observations Cumulative Numbcr Dead lvumtw.r Number Remaining Sampled 1 AN 1 44 5 2 AN 1 39 0 3 AN I 39 5 4 5 6 AN AN AN 1 1 1 34 34 34 0 0 0 7 AN 1 34 5 8 AN l 29 Q 9 AN 1 29 0 10 AN I 29 5 I 1 AN 1 24 0 1 ., AN 1 24 0 13 AN 1 24 0 14 AN 24 10 15 16 AN 1 14 AN 1 14 0 0 'Observed Effects : AN = Appears Normal p . 174 Wildlife International, Ltd. -99- Project Number 454A . i7 APPENDIX VI[[ Changes to Protocol This study was conducted in aceordanco with the approved Protocol with the following changes : 1 . Amendment : The proposed experimental start and termination dates w ere added to the protocol . 2 . Amendment : The frequency of TOC measurements was added to the protocol. 3 . Amendment : The methodology for TOC measurements was added to the protocol. 4 . Deviation : Conductivity of the negative control water was not recorded on Day 7 of the test. 5 . Deviation- Day 0 tissue samples for lipid analysis were collected p ri or to distribution to the test chambers . 6 . Deviation : A tissue storage stability test was performed . 7 . Deviation : Light intensi ty was measured at test initiation . 8 . Deviation : Total organic carbon was not measured in the dilution water at test termination. 9. Deviation: Weekly fulscope verification was not documented four times during the test . p . 175 --- ~~ . _ Wildlife Intanatr'onat, Ltd. _100- Project Number 45 4A . 117 APPENDIX IX Personnel Involved in the Study The following key W ildlifc International, Ltd. personncl were involved in the conductor management of this study : 1 . Henry 0. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2 . Wi ll ard B . Nixon, Ph.D., Managcr, Analytical Chemistry 3 . Kurt R . Drattar, Senior Aquatic Biologist 4 . Cary A, Sutherland, Laboratory Superv isor 5 . Raymond L. Van Hoven, Ph .D ., Scientist 6, Susan T . Plantania, Biologist