16) OECD 203-OPPTS 850.1075, 96-Hour static acute toxicity test with the (bluegill), 454A-114
PERFLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS): A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE BLUEGILL (Lepomis macrochirus)
SANITIZED
FINAL REPORT
WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-114 3M LAB REQUEST NO. EOO- 1429
DEC 0 9 2003
U. S Environmental Protection Agency . Series 850 - Ecological Effects Test Guidelines
OPPTS Number 850.1075 and
OECD Guideline 203
AUTHORS:
Kurt R. Drottar Henry 0. Krueger, Ph.D.
STUDY INITIATION DATE: April 4,2000 STUDY COMPLETION DATE: March 20,200 1
Submitted to
3M Corporation Environmental LaboratoIy
Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 5 5 144
Wildlije Intemutional, Ltd.
8598 Commerce Drive Easton, Maryland 21601
(410) 822-8600
Page 1 .of 41
I
\
W I L D L I F EI N T E R N A T I O N A L
LTD.
PROJECT NO.: 454A-114
-2-
D E09m
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SANITIZED
SPONSOR: 3M Corporation
TITLE:
Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Bluegill (Lepomis macrochirus)
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-114
STUDY COMPLETION: March 20,2001
This study was conducted in compliance with Good Laboratory Practice Standardsas publishedby the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions:
The test substance was not characterizedin compliance with Good LaboratoryPracticesprior to its use in
the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original
characterization purity.
The stability of the test substance under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards.
STUDY DIRECTOR:
Kurt R Drottar Senior Biologist
SPON-SOR APPROVAL:
DATE
3/23 Id 1
DATE
WILDLIFE INTERNATIONALTLD.
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PROJECT NO.: 454A-114
QUALITY ASSURANCE STATEMENT
This study was examined for compliance with Good Laboratory Practice Standards as published by the
U.S.Environmental Protection Agency, 40 CFR Parts 160 and 792, 17August 1989;OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows:
ACTIWI'Y:
Test Substance Preparation
Matrix Fortification Observations Analytical Data and Draft Report
Biological Data and Draft Report
Final Report
DATE CONDUCTED: December 18,2000 December 18,2000 December 21,2000
January 15 and 16,2001
January 16 and 17,2001 March 16,2001
DATE REPORTED TO:
STUDY DIRECTOR:
MANAGEMENT:
December 18,2000
December 20,2000
December 18,2000
December 20,2000
December 21,2000
January 4,2001
January 16,2001
January 18,2001
January 17,2001 March 16,2001
January 18,2001 March 16,2001
Robert N. McGee Quality Assurance Representative
WILDLIFE INTERNATIONALTLD.
-4-
REPORT APPROVAL
PROJECT NO.: 454A-114
SPONSOR: 3M Corporation
TITLE:
Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Bluegill (Lepomis macrochirus)
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-114
STUDY DIRECTOR:
Kurt R Drottar Senior Biologist MANAGEMENT:
Director, Aquatic Toxicology and Non-Target Plants
3 / 4 0I
DATE
WILDLIFE INTERNATIONALTLD.
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PROJECT .NO. 454A-114
TABLE OF CONTENTS TitleKover Page................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement............................................................................................... 2 Quality Assurance Statement ............................................................................................................................... 3 Report Approval......................................................... :......................................................................................... 4
Table ofContents................................................................................................................................................. 5
summary............................... :............................................................................................................................... 7
Introduction .......................................................................................................................................................... 8 Objective .............................................................................................................................................................. 8 Experimental Design ............................................................................................................................................ 8 Materials and Methods ......................................................................................................................................... 9 Results and Discussion...................................................................................................................................... 12 Conclusions ....................................................................................................................................................... 13 References ......................................................................................................................................................... 14
TABLES
Table 1 .Summary ofhalflcal ChemistryData ........................................................................................... 15 Table 2 .Temperature. Dissolved Oxygen and pH of Water in the Test Chambers ....................................... 16 Table 3 .Cumulative Percent Mortality and Treatment-RelatedEffects......................................................... 17 Table 4 .LC50 Values...................................................................................................................................... 18
WILDLIFE INTERNATIONLTDA. L
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PROJECT NO.: 454A-114
TABLE OF CONTENTS
- Continued -
FIGURE Figure 1. Concentration-Response Curve (96-Hour Data) ............................................................................ 19
APPENDICES
Appendix 1 - Specific Conductance, Hardness, Alkalinity, and pH of Well Water
Measured During the 4-Week Period Immediately Preceding the Test .................................. 20
Appendix 2 - Analyses of Pesticides, Organics and Metals
in Wildlife International, Ltd. Well Water ............................................................................. 2 1
Appendix 3 - The Analysis of PFBS in Freshwater in Support of Wildlife
International, Ltd. Project No.: 454A- 114........................................................................... 23
Appendix 4 - Changes to Protocol ............................................................................................................... 40
Appendix 5 - Personnel Involved in the Study............................................................................................. 41
WILDLIFE INTERNATIONALTLD.
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PROJECT NO.: 454A-114
SPONSOR:
SPONSOR'S REPRESENTATIVE:
LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT:
SUMMARY
C 3 3M Corporation
Wildlife International Ltd. Easton, Maryland 2 1601
WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY:
MEAN MEASURED TEST
CONCENTRATIONS:
TEST DATES:
LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS:
454A-114
Perfluoro Butane Sulfonate, Potassium Salt (PFBS)
Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the B l u e d (Lepomismacrochims)
Negative Control, 629,131I, 2715,5252 and 9433 mg a.i./L
Experimental Start - December 18,2000 Biological Termination - December 22,2000
Experimental Termination - December 22,2000
96 Hours
Bluegill (Lepomis macrochims) Osage Catfisheries, Inc. Osage Beach, Missouri
AGE OF TEST ORGANISMS:
MEASUREMENTS OF 10
NEGATIVE CONTROL FISH:
WEIGHT (g): TOTAL LENGTH (m):
Juveniles
Mean= 1.0; Mean = 44;
Range = 0.39 to 1.6 Range = 33 to 53
' 96-HOUR LC50: 95% CONFlDENCE LIMITS:
NO MORTALITY CONCENTRATION:
NO-OBSERVED-EFFECTCONCENTRATION:
6452 mg a.i./L 5252 and 9433 mg a.i./L, 27 15 mg a.i./L
2715 mg a . i L
WILDLIFE INTERNATIONALTLD.
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SPONSOR:
SPONSOR'S REPRESENTATIVE:
LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT:
SUMMARY
[3M Corporation
3
Wildlife International Ltd. Easton, Maryland 2 1601
PROJECT NO.: 454A-114 *
WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY:
MEAN MEASURED TEST
CONCENTRATIONS:
TEST DATES:
LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS:
454A-114
Perfluoro Butane Sulfonate, Potassium Salt (PFBS)
Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour StaticAcute Toxicity Test with the Bluegdl(Lepomisrnacrochirus)
Negative Control, 629,13 11,2715,5252 and 9433 mg a.i./L
Experimental Start - December 18,2000
Biological Termination - December 22,2000 Experimental Termination - December 22,2000
96 Hours
Bluegill (Lepornis rnacrochirus) Osage Catfisheries, Inc. Osage Beach, Missouri
AGE OF TEST ORGANISMS: MEASUREMENTS OF 10
NEGATIVE CONTROL FISH:
WEIGHT (g): TOTAL LENGTH (m):
96-HOUR LC50: 95% CONFIDENCELIMITS:
NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION:
Juveniles
Mean = 1.0; Mean = 44;
Range = 0.39 to 1.6 Range = 33 to 53
6452 mg a.i./L 5252 and 9433 mg a.i./L 2715 mg a.i./L
2715 mg a.i./L
WILDLIFE INTERNATIONALIDL.
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PROJECT NO.: 454A-114
INTRODUCTION
This study was conducted by Wildlife InternationalLtd. for 3M Corporation at the WildlifeInternational Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from December 18,2000 to December 22,2000. Raw data generated by WildlifeInternational Ltd. and a copy of the finalreport are filed under Project Number 454A-114 in archives located on the Wildlife International, Ltd. site.
OBJECIWE
The objective of this study was to evaluate the acute toxicity of Perfluoro Butane Sulfonate, Potassium Salt (PFBS)to the bluegill,Lepomis macrochims,during a 96-hourexposure period under statictestconditions.
EXPERIMENTAL DESIGN
Bluegill were exposed to a geometric series of five test concentrations and a negative (cblution water) control. Two replicate test chambers were maintained in each treatment and control group, with 10 bluegill in each test chamber for a total of 20 bluegill per test concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an exploratory range finding toxicity test.
Nominal test concentrationsselected were 612,1224,2448,4895 and 9790 mg active ingredient(a.i.)/L. Mean
measured test concentrations were determined from samples of test water collected from each treatment and the control group at the beginning of the test, at approximately 48 hours, and at test termination.
Bluegill were indiscriminatelyassigned to exposurechambers at testinitiation. Observationsof mortality and other clinical signs of toxicity were made at approximately 3,24,48,72 and 96 hours after test initiation. Cumulativepercent mortality observed in the treatment groups was used to calculateLC50 values at 24,48,72 and 96 hours. The no mortality concentrationand the no-observed-effectancentration(NOEC)were determined by visual interpretation of the mortality and clinical observation data.
WILDLIFE INTERNATIONALLTD.
PROJECT NO.: 454A-114
-9MATERIALS AND METHODS
DEC 0 9 2003
SAN ITlZED
The study was conductedbased on the procedures outlined in the protocol, "PerfluoroButane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Bluegill (Lepomismacrochims)'~T. he protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecologcal Effects Test Guidelines, OPPTS Number 850.1075 (1): OECD Guideline for Testing of Chemicals 203: Fish, Acute Toxicity Test (2); and ASTM Standard E729-88aYStandard Guidefor ConductingAcute Toxiciw Tests with Fishes, Macroinvertebrates and Amphibians (3).
Test Substance
The test substance was received from 3M Corporation on June 28, 2000 and was assigned Wildlife
International Ltd. identification number 5292. The test substance was described as a white powder. It was
`
1
identified as Potassium Perfluoro butane sulfonate, AKAP jDevelopmental Product,AKA PFBS, fromlot L
2. Information provided by the Sponsor indicated a purity of 97.9%. A subsequent revision of the certificate of
analysis indicated a purity of 97.3% and an Expiratiofieassessment Date of January 17, 2002. The test
substance was stored at ambient room temperature.
Preuaration of Test Concentrations Nominal test concentrationswere 612,1224,2448,4895 and 9790 mg a.i./L. All materialswhichcame
into contact with the test substance during preparation of test concentrations were constructed of plastic or stainless steel. A 60-L primary stock solution was prepared in dilution water at a concentration of 9790 mg
a.i./L. The primary stock solution was mixed with an electric mixer for approximately 30 minutes to aid in the solubilization of the test substance. After mixing, the primary stock solution was proportionally diluted with dilution water to prepare the four additional test concentrations. The appropriate amount of primary stock was mixed with dilution water in the test chambers. All test solutions appeared clear and colorless. Test concentrations were corrected for the origmal purity of the active ingredient in the test substance (97.9%).
Test Organism The bluegdl, Lepomis macrochirus, was selected as the test species for this study. The bluegill is '
representative of an important group of aquatic vertebrates and was selected for use in the test based upon past
WILDLIFE INTERNATIONALTLD.
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PROJECT NO.: 454A-114
history of use in the laboratory. Bluegill used in the test were obtained from Osage Catfisheries, Inc., Osage Beach, Missouri.
Bluegill were held at approximately the same temperature as used during the test. The fish were held for approximately 173 days prior to testing. 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-dayholding period preceding the test, water temperaturesranged from 22.0 to 22.8"C. ThepH of the water ranged from 8.0 to 8.3 and dissolved oxygen ranged from 8.0 to 8.4 m a . Instrumentation and methods used for water measurements are described in the Environmental Conditions section of this report. At test initiation, the bluegill were collected from the acclimation tank and transferred to the test chambers.
During the holding period, bluegill were fed a commercially-prepared diet (Zeigler Brothers, Inc., Gardners, PA). The fish were not fed during the acclimation period (at least 48 hours prior to the test) or during the test.
All fish used in the test were from the same source and year class, and the total length of the longest fish was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the test was 44 mm with a range of 33 to 53 mm. The average wet weight (blotted dry) of 10negative controlfish at the end of the test was 1.Ogramswith a range of 0.39to 1.6grams. Loadingwas 0.70
g fish/Lof test water present in the test chambers at any given time.
Test Apparatus Test chambers were 25-L polyethylene aquaria containing 15 L of test solution. The depth of water in a
representative test chamber was approximately 16.1 cm. Test chambers were impartially positioned in an environmentalchamber set to maintain a temperatureof 22+2OC. The test chamberswere labeledwiththeproject number, test concentration and replicate.
Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately 40 meters
deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water.
WILDLIFE INTERNATIONLTDA.L - 11 -
PROJECT NO.: 454A-114
The specific conductance, hardness, akalinity, 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 passed through a sand filter to remove particles greater than approximately 25 ym, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water again was filtered (0.45 pm) to remove microorganisms and particles. The results of periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International Ltd. are presented in Appendix 2.
Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation and testing was
provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone@50). A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was approximately 220 lux at the surface of the water. Light intensity was measured using a SPER Scientific Ltd. light meter.
Temperature was measured in each test chamber at the beginning of the test and at approximately 24-hour intervals thereafter using a liquid-in-glass thermometer. Temperaturealsowas measuredcontinuouslyin
one negative control replicateusing a Fulscope EWC Recorder. The target test temperatureduringthe study was
22*2C. Dissolved oxygen and pH measurementswere made on water samples fiom all replicate test chambers of each treatment and control at test initiation and at approximately 24-hour intervals thereafter. Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation.
Measurementsof pH were made using a Fisher Accumet Model 915pH meter,and dissolvedoxygenwas measured using a Yellow Springs Instrument Model 5 1B dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperaturemeter. Hardness and alkalinitymeasurements were made by titrationbased on proceduresin StandardMethodsfor the Examination of Water and Wastewater (4).
WILDLIFE INTERNATIONALTLD. - 12-
PROJECT NO.: 454A-114
Observations Observations were made to determinethe number of mortalities. The number of individualsexhibiting
clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 3, 24,48,72 and 96 hours after test initiation.
Statistical Analyses The 24,48,72 and 96-hour LC50 values and the 95% confidenceintervalswerecalculatedwhenpossible
by probit analysis, the moving average method or binomial probability with non-linear interpolation (5,6, 7) using the computer soffwaie of C.E. Stephan (8). In this study, the probit method was used to evaluatemortality at 24 hours and binomial method was used to evaluate mortality at 48, 72 and 96 hours. The no mortality concentrationand NOEC were determined by visual interpretationof the mortality and clinicalobservationdata.
Analytical Chemistry Water samples were collected at mid-depth from each replicate test chamber of each treatment and
controlgroup at the beginningof the test, at 48 hours and at test terminationto measure concentrationsof thetest substance. The samples'were collected in glass vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3.
RESULTS AND DISCUSSION
Measurement of Test Concentrations Results of analyses to measure concentrations of PFBS in water samples collected during the test are
presented in Table 1and in the analyticalchemistryreport (Appendix 3). Nominal concentrationsselectedforuse in this study were 612, 1224,2448,4895 and 9790 mg a.i./L. Samplescollected at test initiation had measured values that ranged from 93 to 114% of nominal values. Measured values for samples taken at 48 hours ranged from 95 to 116% of nominal. Measured values for samples taken at 96 hours ranged from 99 to 124% of nominal. When measured concentrationsof the samples analyzed at test initiation,approximately48 hours and at test terminationwere averaged,the mean measured concentrationsfor this studywere 629,1311,2715,5252 and 19433 mg a.i./L. Mean measured concentrations were used in the calculation of LC50 values.
WILDLIFE INTERNATIOLNTDA. L
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PROJECT NO.: 454A-114
Observations and Measurements Measurements of temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were
withinthe 22k2OC range established for the test. Dissolved oxygen concentrationsremained 3 . 4m& (62%of saturation) throughout the test. Measurements of pH ranged from 8.0 to 8.4 during the test.
Daily observations of mortality and other clinical signs of toxicity observed during the test are shown in Table 3. Bluegill in the negative control, 629,13 11and 2715 mg a.i./L treatment groups appeared normal and healthy during the test. After 96-hours of exposure, mortality in the 5252 and 9433 mg a.i./L treatment groups was 15 and loo%, respectively. LCSO values and 95% confidence limits at 24, 48, 72 and 96 hours were calculated from the mortality data, and are shown in Table 4. A graph of the concentration-response curve is presented in Figure 1.
CONCLUSIONS
The 96-hour LC50 value for bluegill (Lepomis macrochirus) exposed to Perfluoro Butane Sulfonate, Potassium Salt (PFBS) was 6452 mg a.i./L with 95% confidence limits of 5252 and 9433 mg a.i./L. Thk 96hour no-mortality concentration and the NOEC were 27 15 mg a.i./L.
WILDLIFE INTERNATIONLTDA. L
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PROJECT NO.: 454A-114
REFERENCES
1 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines
(draft),OPPTS Number 850.1075: Fish Acute Toxicity Test,Freshwater andMarine.
2 Organisation for Economic Cooperation and Development. 1993. OECD Guidelines for Testing of
Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992.
3 ASTM Standard E729-88a. 1994. Standard Guidefor ConductingAcute ToxicityTestswithFishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials.
4 APHA, AWWA, WPCF. 1985. StandardMethodsfor the Examination of Waterand Wastewater. 16th Edition. American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York.
5 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication.
6 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. GriffinPress, London.
7 Thompson, W.R 1947. Bacteriological Reviews. Vol. 11, No. 2. Pp. 115-145.
8 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Publication Number STP 634, pp 65-84.
WILDLIFE INTERNATIONALTLD.
PROJECT NO.:454A-114
Table 1 Summary of Analytical Chemistry Data
Sponsor: Test Substance: Test Organism: Dilution Water:
3M Corporation PFBS Bluegill, Leponzis rnacrochims Well Water
Nominal Test
Sampling
Measured
Mean Measured
Percent
> Concentration
- mg a.i./L
Negative Control
Replicate A
Time 0
Concentration m ai5
<LOQ'
Concentration m a.i./L <LOQ
of Nominal
--
B
0
<LOQ
A
48
B
48
<LOQ <LOQ
A'
96
<LOQ
B
96
<LOQ
612
A
0
B
0
A
48
B
48
A
96
B
96
639
629
103
659
591
644
604
636
1224
A
0
B
0
A
48
B
48
A
96
B
96
1323 1325
1319 1308 1306
1284
1311
107
2448
A
0
B
0
A
48
B
48
A
96
B
96
2596
2789 2563
2837
2482 3022
2715
111
4895
A
0
B
0
A
48
B
48
A
96
B
96
5272 5256 5148 5357 51 19 5362
5252
107
9790
A
0
B
0
A
48
B
48
A
96
B
96
'The limit of quantitation (LOQ) was 50.0 mg a.i./L. 2Samplesnot collected due to 100% mortality.
9077 983 1 9270
95--22
--2
943 3
96
- 16-
Table 2 Temperature,Dissolved Oxygen and pH of Water in the Test Chambers
PROJECT NO.: 454A-114
Sponsor: Test Substance: Test Organism: Dilution Water:
3M Corporation PFBS Bluegill, Lepomis macrochins Well Water
Mean Measured
0 Hour'
Test Concentration
Temp2 DO3
(mg a.i./L)
Replicate ( 0 ~ ) ( m a ) H
Negative Control
A
22.4 8.0 8.3
B
23.6 8.1 8.3
24 Hours
Temp DO
("C) (mg/L) PH
22.2
7.1 8.3
23.6
7.0 8.3
48 Hours
Temp DO
("C) (mg/L) pH
22.1
6.3 8.2
23.5
6.0 8.2
72 Hours
Temp DO ("C) (mglL) PH 22.0 6.0 8.1 23.5 5.6 8.1
96 Hours
Temp DO
("C) (mg/L) pH
22.1
6.0
8.0
23.6
5.4 8.0
629
A
23.5
8.0 8.3
23.5
6.4 8.3
23.4
5.8 8.2
23.5
5.5 8.1
23.6 5.4 8.1
B
23.1
8.0 8.3
23.2
6.4 8.3
23.2
5.9 8.2
23.2 5.5 8.2
23.3
5.4 8.1
1311
A
23.6 8.1 8.3
23.9
6.4 8.3
23.9
6.1 8.3
23.8 5.6 8.2
23.9
5.4 8.2
B
23.6
8.0 8.3
23.9
6.6 8.3
23.8
6.4 8.3
23.7 6.1 8.2
23.9
5.4 8.2
2715
A
23.4 8.0 8.3
23.9
6.6 8.3
23.9
6.2 8.3
23.9
5.8 8.3
23.9
5.6 8.2
B
22.3
8.0 8.4
23.5
6.8 8.4
22.5
6.6 8.4
22.4 6.4 8.3
22.9
6.0 8.3
5252
A
22.9
8.2 8.3
23.8
6.5 8.3
23.8
6.1 8.3
23.7 . 5.8 8.3
23.8
5.4 8.3
B
22.7
8.1 8.3
23.9
7.0 8.3
23.3
6.6 8.3
23.4 6.2 8.3
23.5
6.0 8.3
' 9433
A
22.1
8.1 8.2
23.7
7.0 8.3
23.7
7.2 8.4
23.7
- 4
-
- --
B
22.1
8.0 8.2
23.7
7.0 8.3
23.7
6.2 8.4
23.6
- 4
-
- --
1 TTehme 0p-ehraotuurredimluetaiosunrwedatceornmtienausouurselmyednutrsinfogrthhaertdenset srsa,naglekdalfirnoimty aapnpdrsopxeicmifaitceclyo2n1d.u5cttoan2c2e.w5"eCre. 148 m-g 5 as CaC03, 190 m-g 5 as CaC03and 350 ymhodcm, respectively.
A dissolved oxygen concentration of 5.2 mglL represents 60% saturation at 22C in freshwater.
Measurements discontinued due to 100%mortality.
SpOtISOK
Test Substance: Test Organism: Dilution Water:
3M Corporation PFBS Bluegill, Lepomrs mocrochirus Well Water
Mean Measured Test Concentration
3 Hours
No.
No.
(mg ai.&)
Exposed Dead' E&&'
Negative Control
A
10
0
10 AN
B
10
0
10 AN
- 17-
Table 3 Cumulative Percent Mortality and Treatment-RelatedEffects
24 Hours
No. Dead
Effects
0
10 AN
0
10 AN
48 Hours
No. Dead
Effects
0
10 AN
0
10 AN
72 Hours.
No. Dead
Effects
0
10 AN
0
10 AN
Cumulative Percent Mortality 0
629
A
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
0
B
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
1311
A
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
0
B
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
2715
A
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
0
B
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
5252
A
10
0
10 AN
0
10 AN
0
10 AN
0
10 AN
15
B
10
0
10 AN
2
8AN
2
8AN
3
7AN
- 9433
A
10
0
9C, 1R
7
3"
10
-
10
100
B
10
0
5C, 5R
8
2AN
10
-
10
-
I Cumulative number of dead fish.
* ObservedEffects: AN = Appears Normal; C = Lethargic,R = Lying on Bottom
96 Hours
No.
Dead
Effects
0
10 AN
0
10 AN
Cumulative Percent
0
0
10 AN
0
0
10 AN
0
10 AN
0
0
IO AN
0
10 AN
0
0
10 AN
0
10 AN
15
3
7"
10 10
- -
100
~~~
~
~~
WILDLIFE INTERNATIONLTADL.
PROJECT NO.: 454A-114
Table 4 LC50 Values
Sponsor: Test Substance: Test Organism: > Dilution Water:
3M Corporation
PFBS
Bluegill, Lepomis macrochirus Well Water
Lower 95%
Confidence
LC50
. Limits
24 Hours
7710
6630
48 Hours
6606
5252
72 Hours
6452
5252
96 Hours
6452
5252
Upper 95% Confidence
Limits
9121
9433
9433
9433
Statistical Probit
Binomial Binomial Binomial
WILDLIFE INTERNATIONALLTD.
- 19-
Figure 1. Concentration-ResponseCwve (96-Hour Data)
PROJECT NO.: 454A-114
8 7 6
5
4 3
2
1 I00
1000 Mean Measured Concentration (mg a.i./L)
10000
WILDLIFE INTERNATIOLNTDA. L
- 20 -
PROJECT NO.: 454A-114
Appendix 1
Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test
Sponsor: Test Substance: Test Organism: Dilution Water:
3M Corporation PFBS Bluegill, Lepomis macrochirus Well Water
Specific Conductance , (ymhos/cm)
Mean 313 (N = 4)
Hardness ( m aas CaC03)
131 (N=4)
Alkalinity ( m a as CaC03)
178 (N= 4)
Range
310 - 315
128 - 132
176 - 178
PH
8.0 (N = 4)
8.0-8.1
WILDLIFE INTERNATIONALTLD. -21 -
PROJECT NO.:454A-114
Appendix 2
Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water'
Component
Measured Concentration
~
~~
Component
~
~~~
Measured Concentration
Pesticides and Organics
Aclonifen Alachlor
Amem Atrazine Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin C hlorfenvinphos C hloridazon Chlorpropham Chlorpyrifos C hlorpyrifos-methyl C hlorothalonil Coumaphos Cyanazine Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-0 Desethylatrazine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid
4.03 pgL 4.01 pgn
4.01 p g n 4.01 pgn
4 . 0 4 PgiL 4.08 pg/L ~ 0 . 2 5p g L 4.05 pg/L 4.05 pgL
4.05 pglL 4.05 pgL 4 . 0 2 pgiL
4.02 pgn
4 . 0 2 pg/L 4.05 p g 5 ~ 0 . 0 5pg/L ~0.03pg/L C0.02 p g L 4.02 pgfL 4.05 pglL
4.02 pgn
4 . 0 1 &L CO.01 pg/L 4 . 0 4 pg/L 4.02 pg/L 4.05 pgk 4.05 pg/L 4.25 pg/L 4 . 0 5 pg/L
4.02 pgn 4.02 pgn
4 . 0 2 pg/L 4 . 0 1 pg/L
4.02 pgn 4.01 pgn 4.01pgn 4.01 pgn
4.03 pPn, 4.02 p g n 4.01 ppn <0.03 pg/L
Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodemorph Endosulfan-a Endosulfan-j3 Endosulfan-sulfte Epoxiconazole Eptam Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb
Fenpiclonil
Fenpro pathrin Fenpropimorph Fenthion Fenvalerate Flumifop-butyl Fluoroglycofen-eth yl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Jprodion Kresoxim-methyl Lenacil Lindane
4 . 0 1 pg/L ~ 0 . 2 5pg/L C0.02 p g L 4 . 0 3 pg/L
4.02 pgn
4.05 pg/L C0.02 pg/L 4 . 0 5 pg/L 4.01 pgn 4 . 0 1 pg/L 4 . 0 1 p& 4.02 pgn 4 . 0 5 pg/L 4.02 pglL 4 . 0 2 pg/L <0.05 pglL 4 . 0 2 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L 4.05 p& 4.05 pg/L 4.01 pg/L ~ 0 . 0 3pg/L 4.03 p&
4 . 0 5 pg/L
4 . 2 5 pglL 4 . 0 1 pg/L 4.01 pg/L 4.02 pfl 4.02 p g 5 4.02 p g k 4.05 pg/L
4.02 p g n
4 . 0 1 pglL 4 . 0 2 pg/L C0.02 pg/L <0.01 pg/L ~0.05p g L 4.02 pglL 4.05 pg/L 4 . 0 2 pg/z
~~
'Analyses performed by TNO Nutrition and Food Institute on samplescollected on October 14 and 15,1999.
Continued
WILDLIFE INTERNATIONALTLD.
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PROJECT NO.: 454A-114
Appendix 2
Analyses of Pesticides, Organics and Metals in Wildlife International,Ltd.Well Water'
Page 2
~~~
Pesticides And Organics (Page 2)
Component
Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphosethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine
Propham
Propiconazole Propxur Proppmide Prosulfocarb Pyrazophos
Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt
Measured Concentration
Component
4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L -=0.02 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L ~ 0 . 0 5pg/L <0.03 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L ~ 0 . 0 5pg/L <0.02 pg/L ~ 0 . 0 5pg/L 4 . 0 1 pg/L C0.01 pg/L <0.01 pg/L 4 . 0 2 pg/L C0.01 pg/L 4 . 0 1 pg/L 4.01 pg5 4 . 0 1 pg/L
4 . 0 2 pglL
4 0 5 pglL 4 . 0 3 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4.03 pgL
Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon
Triadirnenol
Triallate Triazophos Trifluralin Vamidothion Vinclozolin
Metals
11.0mg/L 18.0 mg/L 29 mg/L 4 . 0 1 5 mg/L 1.1 mg/L 4 . 0 2 mg/L
4.1 C0.2 p g 5
4 . 5 vg/L 4.2
Nickel
Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium
Measured Concentration
<0.01 pg/L <0.01 pg/L 4 . 0 2 pig, 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L <0.01 pg/L 4 . 0 2 pglL 4 . 0 1 pg/L 4 . 0 2 pg/L <0.05 pg/L ~ 0 . 0 p5glL 4 . 0 1 pg/L <0.01 p g L 4 . 0 1 pg/L ~ 0 . 0 5p g 5 <0.01 pg/L ~ 0 . 0 p5g/L 4 . 0 4 pg/L <0.01 pg/L 4 . 0 4 pg/L 4 . 0 5 pg/L
4.05 pg/L
<0.02 pglL 4 . 0 2 pig, 4 . 0 2 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L
'Analyses performed by TNO Nutrition and Food Instituteon samples collected on October 14 and 15, 1999.
WILDLIFE INTERNATIONALTLD.
- 23 -
Appendix 3
PROJECT NO.: 454A-114
THE ANALYSIS OF PFBS IN FRESHWATER IN SUPPORT OF
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-114
WILDLIFE INTERNATIONALIDL.
- 24 -
PROJECT NO.: 454A-114
REPORT APPROVAL
SPONSOR 3M Corporation
TITLE:
PEIWLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS): A 96-HOUR STATIC ACUTE TOXICITY TEST with the BLUEGILL (Lepomis macrochirus)
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-114
3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429
PRINCIPAL INVESTIGATOR:
R a y m a flVan Hoven, Ph.D.
scientist
MANAGEMENT:
Willard B. Nixon, Ph.D. '
Director, Analytical Chemistry
63-30-01 DATE
DATE
WILDLIFE INTERNATIONALTLD.
PROJECT NO.: 454A-114
-25 -
DEC 0 9 2003
Introduction
Sk\,"l1'$'JZED
Freshwater samples were collected from a static acute aquatic toxicity study designed to determine the
effects of PFBS (Perfluoro Butane Sulfonate, Potassium Salt) to the bluegill (Lepomzs macrochims). This
study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-114. The analyses of these water samples were performed at Wildlife International, Ltd using hgh performance
liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis
on December 18, 20 and 22, 2000 and were analyzed on each sample receipt day.
Analytical Standard
The analytical standard was received from 3M Environmental Technology and Safety Services on
March 27, 2000, assigned Wildlife International, Ltd. Identification number 5216, and stored under
ambient conditions. The analytical standard, a white powder, was identified as: Potassium
[ Perfluorobutane Sulfonate
>Lot 2), expiration date: March 2010. The analytxal standard was
further identified with the 3M Environmental Laboratory test control and reference number TCR #
3 c The test substance had a reported purity of 97.90%. A subsequent revision of the certificate of
analysis indxated a purity of 97.3% and an Expiratiofleassessment Date of January 17, 2002. The
analytical standard was the same material and lot number as the test substance (Wildlife International, Ltd.
Identification number[ ] The analytical standard was used to prepare calibration and matrix
fortification samples.
Analytical Method Water samples were analyzed according to the method entitled "Analytical Method Validation for the
Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater" (Wildlife International, Ltd. Project No. 454C-115). Samples were diluted in a 50% methanol : 50% NANOpure' water solution so that they fell w i h the calibration range of the PFBS methodology. AIiquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in freshwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) interfaced with a P e r h Elmer API lOOLC mass spectrometer (single quadrupole) operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perlun-Elmer TurboIonSpray ion source.
Chromatographic separations were achieved using a Keystone PRISM RP column (30 mm x 1.5 mm, 3-pm
WILDLIFE INTERNATIONLTAD.L
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PROJECT NO.: 454A-114
particle size) fitted with a Keystone Javelin CIS Guard Cartridge (20 nun x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1.
Primarv and Secondary Stock Solutions, All primary and secondary stock preparations were adjusted for the purity of the analytical standard
(97.90%). A 10.0 mg a.i./mL primary stock solution of PFBS in methanol was prepared by weighing 1.024 g of the analytical standard and bringing to a final volume of 100 mL with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) of PFBS in methanol were prepared by serial volumetric dilution fiom the primary stock.
Calibration Standards and Calibration Curves Calibration standards were prepared in 5050 methanol: NANOpure@water by appropriate dilutions of
the 10.0 mg a.i./L stock solution of PFBS in methanol. The calibration standards of PFBS, ranging in concentration fiom 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end of each run,and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Figure 2. The
concentration of PFBS in the samples was determined by substituting the peak area responses into the
applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively.
Limit of Quantitation
The method limit of quantitation (LOQ)for these analyses was set at 50.0 mg a.i./L calculated as the
product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000).
Matrix Blank and Fortification Samdes Three matrix blank samples were analyzed to determine possible interference. No interferences were
observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a
matrix blank is presented in Figure 5.
WILDLIFE INTERNATIONLTADL.
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PROJECT NO.: 454A-114
Freshwater was directly fortified (ie. without use of carrier solvent) with PFBS at 100, 1500 and 12000 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 2). Sample concentrations were not corrected for the mean procedural recovery of 95.7%. A representative ion chromatogram of a matrix fortification is presented in Figure 6.
Example Calculations
Sample number 454A-114-4, nominal concentration of 612 mg a.i./L (625 m a ) in freshwater.
First Initial Volume: 0.100 mL
Calibration curve equation:
First Final Volume: 25 .O mL
Slope: 39918576
Second Initial Volume: 0.250 mL
Intercept: 107167.24219
Second Final Volume: 25.0 mL
Curve regression weighted l/x
Dilution Factor: 25000
PFBS Peak Area: 1159343
eak area - (y-intercept)
PFBS (mg a.i./L)measured at instrument =
slope
PFBS (mg a.i.L) in sample = PFBS measured at instrument (mg a.i./L) x llution fhctor
- 1159343 - 107167.24219 25000 39918576
= 659
PFBS m a.i./L in sam le
Percent of Nominal Concentration = pFBs((tg
no&l :
x 100
---665192 x 100= 108%
Calculated with HPLCMS instrument software: MacQuan, version 1.6.
WILDLIFE INTERNATIONLTAD.L
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PROJECT NO.: 454A-114
Sample Analvsis
RESULTS
Freshwater samples were collected from an acute toxicity study with the bluegill (Lepomis
macrochirus) at test initiation, December 18, 2000 (Day 0), on December 20, 2000 (Day 2), and at test
termination, December 22,2000 (Day 4). The measured concentrations of PFBS in the samples collected
at initiation of exposure of the test organisms (Day 0) ranged from 92.7 to 114% of the nominal
concentrations. Samples collected at Day 2 had a measured concentration range of 94.7 to 116% of
nominal values. Samples collected at test termination (Day 4) had a measured concentration range of
I
98.7%to 124% of nominal values (Table 3). A representative ion chromatogram of a test sample is shown
in Figure 7.
WILDLIFE INTERNATIONLATLD.
PROJECT NO.: 454A-114
Table 1
Typical HPLCMS Operational Parameters
INSTRUMENT:
Hewlett-Packard Model 1100 High Performance Liquid
Chromatograph with a Perkin-Elmer API lOOLC Mass Spectrometer
operated in Selective Ion Monitoring (SIM) Mode
ION SOURCE:
Perlun-Elmer TurboIonSpray
ANALYTICAL COLUMN:'
Keystone PRISM RP (30 mm x 1.5 mm, 3-pm particle size)
GUARD COLUMN:
Keystone Javelin C18 cartridge (20 mm x 2 mm)
OVEN TEMPERATURE:
40C
STOP TIME:
3.00 min
FLOW RATE:
200 pL/min
MOBILE PHASE:
25% NANOpure" Water with 0.1% Ammonium Formate: 75% Methanol
INJECTION VOLUME:
5.0 pL
PFBS PEAK RETENTION TIME: Approximately 2.3 minutes
PFBS MONITORED MASS:
299.0 m u
WILDLIFE INTERNATIONAL LTD. -30-
PROJECT NO.: 454A-114
Table 2 Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis
Sample Number (454A-114-)
MAB- 1 MAB-2 MAB-3
Sample Type
Matrix Blank Matrix Blank Matrix Blank
Concentrations of PFBS (mg a.i./L)
Fortified
0.00 0.00 0.00
Measured'
<LOQ* <LOQ <LOQ
Percent Recovered]
----
MAS-1
Matrix Fortification
100
104
104
MAS4
Matrix Fortification
100
82.0
82.0
MAS-7
Matrix Fortification
100
96.5
96.5
MAS-2
Matrix Fortification
1500
1480
98.6
MAS-5
Matrix Fortification
1500
1410
93.7
MAS-8
Matrix Fortification
1500
1450
96.5
MAS3
Matrix Fortification
12000
12600
105
MAS-6
Matrix Fortification
12000
11300
94.2
MAS-9
Matrix Fortification
12000
10900
90.8
Mean = 95.7 Standard Deviation = 6.92
CV = 7.23%
N=9
Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual
calculations may vary slightly.
2 The limit of quantitation (LOQ)was 50.0 mg a.i./L based upon the product of the lowest calibration
standard a d y z e d (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000).
WILDLIFE INTERNATIONLTAD.L
-31 -
PROJECT NO.: 454A-114
Table 3
Measured Concentrations of PFBS in Freshwater Samples from a Bluegill Static Acute Toxicity Test
Nominal Test Concentration
(mg a.i./L)
0.0
Sample Number (454A-114-)
1 2 13 14 25 26
Sampling Time (Day)
0 0 2 2 4 4
PFBS Measured Concentration' (ma a.i./L)
LOQ' < LOQ
LOQ < LOQ < LOQ
LOQ
Percent of
Nominal'
----
L-
--
--
612
3
0
4
0
15
2
16
2
27
4
28
4
639
104
659
108
59 1
96.5
644
105
604
98.7
636
104
1224
5
0
6
0
17
2
18
2
29
4
30
4
1323
108
1325
108
1319
108
1308
107
1306
107
1284
105
2448
7
0
2596
106
8
0
2789
114
19
2
2563
105
20
2
2837
116
31
4
2482
101
32
4
3022
124
Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software.
' Manual calculations may vary slightly. The limit of quantitation (LOQ) was 50.0 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000).
WILDLIFE INTERNATIONALTLD.
-32-
PROJECT NO.: 454A-114
Table 3 (Continued)
Measured Concentrations of PFBS in Freshwater Samples from a Bluegill Static Acute Toxicity Test
Nominal Test Concentration
(mg a.i./L)
4895
Sample Number
(454A-114-)
9 10 21 22 33 34
Sampling Time (Day)
0 0 2 2 4 4
PFBS Measured Concentration' (mg a.i./L)
5272 5256 5 148 5357 5119 5362
Percent
' of
Nominal
108 107 105 109 105 110
97903
11
0
9077
92.7
12
0
983 1
100
23
2
9270
94.7
24
2
--
4
--
4
9552
97.6
--
--
1 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software.
Manual calculations may vary slightly.
2 The limit of quantitation (LOQ) was 50.0 mg a.i./L based upon the product of the lowest calibration
standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000).
p -
was not sampled on Day 4 due to 100%mortality on Day 2.
WILDLIFE INTERNATIONALLTD.
- 33 -
PROJECT NO.: 454A-114
METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHWATER
Prepare each matrix fortification sample by weighing the requisite amount of PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with
freshwater. Rmse weighing paper and the sides of the flask with repeat freshwater rinses. Swirl the flask to dissolve the test substance and then bring to final volume with freshwater. Sonicate, as appropriate, and mix with several repeat inversions. The matrix blank is unfortified freshwater.
Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFBS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure" water dilution solvent. Add the appropriate volume of sample and bring to volume with dilution solvent. Perform secondary dilutions as necessary. Process matrix blank samples using the same dilution and
aliquot volume as for the lowest fortification level. Mix well by several repeat inversions.
J
Ampulate samples and submit for LCMS analysis.
Figure 1 . Analytical method flowchart for the analysis of PFBS in freshwater
WILDLIFE INTERNATIONALTLD.
Area
PROJECT NO.: 454A-114
Concentration pg a.i./L
Figure 2.
A typical calibrationcurve for PFBS. Slope = 39918576; Intercept = 107167.24219; r = 0.9983.
WILDLIFE INTERNATIONALTLD. -35 -
PROJECT NO.: 454A-114
intensity: 500000 cps
'101
8
5
19 36
69 84 106 I
o-llllllllllllllllll
31
61
91
121 151
Scan
0.52 1.02 1.52 2.03 2.53 Time
Figure 3. A representative ion chromatogram of a low-level (0.0100 mg a.i./L) PFBS standard.
WILDLIFE INTERNATIONALIDL. -36 -
PROJECT NO.: 454A-114
intensity: 500000 cps 100
70
50-1
137
'igure 4. A representative ion chromatogram of a high-level (0.0500 mg a.i./L) PFBS standard.
WILDLIFE INTERNATIONALTLD. - 37 -
PROJECT NO.:454A-114
intensity: 500000 cps
'460
j45004
1 o ~ 14l
i3 1 l 4l6 l 6l 8 l
31
61
0.52 1.02
1
8l 6 l 1i06 l l 1l 37l i 1l 67l l
91
121 151
scan
~
~~
1:52 2.03 2.53 Time
Figure 5. A representative ion chromatogram of a matrix blank sample (454A-114-MAB-1).
The arrow indicates the retention time of PFBS.
WILDLIFE INTERNATIONALTLD.
- 38 -
PROJECT NO.:454A-114
intensity: 500000 cps
4`3
137
31
61
91
121 151
scan
0.52
1.02
1.53
2.03
2.53
Time
Figure 6.
A representative ion chromatogram of a matrix fortification sample (454A-114-MAS-2, nominal PFBS concentration of 1500 mg a.i./L, dilution factor = 50000~).
WILDLIFE INTERNATIONALTLD.
- 39 -
PROJECT NO.: 454A-114
intensity: 500000 cps
60
137
20
10
0
31
61
91
121 151
scan
0.52
1.02 1.52
2.03
2.53
Time
Figure 7.
A representative ion chromatogram of a test sample (454A-114-7, nominal PFBS concentration of 2448 mg a.i./L, dilution factor = lOOOOOx).
WILDLIFE INTERNATIONLID.AL
- 40 -
PROJECT NO.: 454A-114
Appendix 4 Changes to Protocol
This study was conducted in accordance with the approved Protocol with the following changes:
1. The protocol was amended to add the proposed experimental start and termination dates and test concentrations.
~~
WILDLIFE INTERNATIONALLTD.
-41 -
PROJECT NO.: 454A-114
Appendix 5 Personnel Involved in the Study
The followingkey Wildlife InternationalLtd. personnelwere involved in the conduct or managementof this
study:
1. Henry 0. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Raymond L. VanHoven, Ph.D., Scientist 5 . Kurt R. Drottar, Senior Biologist 6. Molly McCoy, Biologist