Document Ev3m9E7d0BmBOYQGabBvk8XJx
PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH THE SHEEPSHEAD MINNOW (C'n'nodon vuriegutus) FINAL REPORT
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-146A ENVIRONMENTAL LABORATORY PROJECT NUMBER: U2723
U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines
OPPTS Number 850.1075
AUTHORS:
Susan J. Palmer Raymond L. VanHoven, Ph.D.
Henry 0.Krueger, PbD.
STUDY INITIATION DATE: May 16,2001 STUDY COMPLETIONDATE: January 7,2002
Submitted to
3M Corporation
Environmental Laboratory 935 Bush Avenue
St. Paul, Minnesota 55144
WildlifeInternational, Ltd.
8598 Commerce Drive Easton, Maryland 21601
(410) 822-8600
Page 1 of 46
WildlifeInternational, Ltd.
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Project Number 454A-146A
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation
TITLE:
Perfluorooctanesulfonate,Potassium Salt (PFOS): A 96-Hour Static-Renewal Acute Toxicity Test with the SheepsheadMinnow (Cyprinodonvariegatus)
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454A-146A
STUDY COMPLETION: January 7,2002
This studywas conducted in compliancewith Good Laboratory Practice Standards as published by 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, 10August 1984.
STUDY DIRECTOR:
C
F
susan J. Palmer
/
Senior Biologist
SPONSOR APPROVAL:
0 7*, q m /zDo2 Date /
WildlifeIntemational, Ltd.
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Project Number 454A-146A
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 160and 792,17 August 1989;OECD Principles of Good Laboratory Practice (ENV/MC/CHEM(98) 17); and Japan MAFF, 59 NohSan, NotificationNo. 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:
ACTIVITY:
DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR MANAGEMENT:
Initial Trial 454A-146: Test Substance Preparation
Matrix Fortifications
July 6,2001 July 10, 2001
July 6,2001 July 11,2001
July 11,2001 July 13,2001
Observations and Analytical Sampling July 12,2001
July 12,2001
July 16,2001
Definitive Test 454A-146A:
Test Substance Preparation
August 13,2001
Matrix Fortifications
August 13,2001
Observationsand Analytical Sampling August 17,2001
Analytical Data and Draft Report Biological Data and Draft Report
October 4 - 5,2001 October 3 - 5,2001
Final Report
January 7,2002
August 13,2001 August 13,2001 August 17,2001 October 5,2001 October 5,2001 January 7,2002
August 15,2001 August 15,200 1 August 23,2001 October 8,2001 October 9,2001 January 7,2002
Kimbetly A. Holder Quality Assurange Representative
/- 7-02
Date
WildlifeInternational, Ltd.
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Project Number 454A-146A
REPORT APPROVAL
SPONSOR: 3M Corporation
TITLE:
Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon vuriegutus)
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-146A
STUDY DIRECTOR:
susan J. Palmer [
Senior Biologist
WILDLIFE INTERNATIONAL. LTD. MANAGEMENT:
07a~~~,b
/ Date /
Director, Aquatic Toxicology and Non-Target Plants
WildlifeInternational. Ltd.
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Project Number 454A-146A
TABLE OF CONTENTS
TitleKover Page ..................................................................................................................................... 1 Good LaboratoryPractice Compliance Statement................................................................................... 2 Quality Assurance Statement .......................................... :....................................................................... 3 Report Approval...................................................................................................................................... 4 Table of Contents.................................................................................................................................... 5 summary ................................................................................................................................................. 7 Introduction............................................................................................................................................. 8
Objective................................................................................................................................................. 8 Experimental Design ............................................................................................................................... 8 Materials and Methods............................................................................................................................ 9
Test Substance............................................................................................................................ 9 Preparation of Test Concentrations ............................................................................................ 9 Test Organism ............................................................................................................................ 9 Test Apparatus ......................................................................................................................... 10 Dilution Water.......................................................................................................................... 10 Environmental Conditions........................................................................................................ 11 Observations............................................................................................................................. 12
StatisticalAnalyses................................................................................................................... 12
AnalyticalChemistry................................................................................................................ 12
Results and Discussion.......................................................................................................................... 12 Measurement of Test Concentrations ....................................................................................... 12 Observations and Measurements.............................................................................................. 13
Conclusions........................................................................................................................................... 13
References............................................................................................................................................. 14
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TABLE OF CONTENTS
- Continued -
TABLES AND FIGURES
Table 1. Summary of Analytical Chemistry Data ............................................................................... 15 Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers ............................ 17
Table 3. Cumulative Percent Mortality and Treatment-Related Effects.............................................. 19 Table 4. .LC50Values......................................................................................................................... 20
APPENDICES Appendix 1. Salinity and pH of Saltwater Measured During the 4-Week Period
Immediately Preceding the Test .....................................................................................21
Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd.
Saltwater......................................................................................................................... 22
Appendix 3. The Analysis of PFOS in Filtered Saltwater in Support of
Wildlife International, Ltd. Project No.: 454A-146A.................................................... 24
Appendix 4. Changes to Protocol ....................................................................................................... 45
Appendix 5. Personnel Involved in the Study..................................................................................... 46
WildlifeInternational. Ltd.
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Proiect Number 454A-146A
SUMMARY
I SPONSOR:
SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW
3M Corporation Ms. Rochelle Robideau
I DATA AND A COPY OF THE
FINAL REPORT:
Wildlife International, Ltd. Easton, Maryland 2 1601
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY:
MEAN MEASURED TEST CONCENTRATIONS: TEST DATES:
LENGTH OF TEST:
454A-146A
Perfluorooctanesulfonate,Potassium Salt (PFOS)
Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon vuriegutus)
Negative Control, Solvent Control and 15 mg a.i./L Experimental Start (OECD) - July 6,2001 Experimental Start (EPA) -August 13,2001 Biological Termination - August 17, 2001 Experimental Termination - August 17,2001
96 Hours
TEST ORGANISM:
SOURCE OF TEST ORGANISMS:
AGE OF TEST ORGANISMS:
MEASUREMENTS OF 10
NEGATIVE CONTROL FISH: WET WEIGHT: TOTAL LENGTH:
SheepsheadMinnow (Cyprinodon vuriegutus) Aquatic BioSystems, Inc. Fort Collins, Colorado 80524
Juveniles
Mean = 0.44 g Mean = 3.0 cm
Range = 0.21 to 0.66 g Range = 2.4 to 3.5 cm
96-HOUR LC50:
NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION:
>15 mg a.i./L 15 mg a.i./L
4 5 mg a.i./L
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Project Number 454A-146A
INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. An initial trial of the study was conducted under static conditions from July 10,2001 to July 14,2001. However, the analytical results were 165% of the nominal concentration during the test. Therefore, the test was repeated under staticrenewal test conditions. The in-life phase of the definitive test was conducted from August 13,2001 to August 17,2001. Raw data generated by WildlifeInternational, Ltd. and a copy of the finalreport are filed under Project Number 454A-146A in archives located on the Wildlife International, Ltd. site.
OBJECTIVE The objective of this study was to determine the acute effects of Perfluorooctanesulfonate, Potassium Salt (PFOS) to the sheepshead minnow, Cyprinodon vuriegutus, during a 96-hour exposure period under static-renewaltest conditions..
EXPERIMENTAL DESIGN Sheepshead minnows were exposed to a single limit concentration, a negative (dilution water) control and a solvent control (0.5 mL methanom) for 96 hours, with renewal of test solutions approximately every 24 hours. Three replicate test chambers were maintained in each treatment and control group, with 10 sheepshead minnows in each test chamber for a total of 30 fish per test concentration. Two abiotic replicate test chambers also were maintained at the limit concentration. The nominal test concentration was selected in consultation with the Sponsor, and was based upon the results
of exploratory range finding toxicity tests, and the solubility of the test substance in saltwater. The nominal test concentration selected was 20 mg active ingredient (a.i.)/L,. The mean measured test
concentration was determined from samples of test water collected from each treatment and the control group at the beginning of the test, from new and old test solution at approximately 24,48 and 72 hours of the test, and from old test solution at test termination.
Sheepshead minnows were indiscriminately assigned to exposure chambers at test initiation, and were transferred daily to freshly prepared test solutions. Observationsof mortalityand other clinicalsigns of toxicity were made at approximately 2.5, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment group was used to estimate LC50 values at 2.5,24,48,72 and
96 hours. The no mortality concentration and the no-observed-effect-concentration(NOEC) were
determined by visual interpretation of the mortality and clinical observation data.
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MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Static Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus)". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine (1); U.S. Environmental Protection Agency, Standard Evaluation Procedure, Acute Toxicity Test for Estuarine and Marine Organisms (2);and ASTM Standard E729-88a, Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3).
Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned
Wildlife International, Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 86.9% and an expiration date of August 3 1,2006. The test substance was stored at ambient room temperature.
Preparation of Test Concentrations
The nominal test concentration was 20 mg a.i./L, based on a test substance purity of 86.9%. On
each of Days 0, 1 , 2 and 3 of the test, a 250-mL primary stock solution was prepared in methanol at a concentration of 40 mg a.i./mL. The primary stock solution was sonicated for approximately 20 minutes and inverted to mix. Five replicates of the test solution (three biotic and two abiotic replicates) were
prepared at a concentration of 20 mg a.i./L by adding the appropriate volume of primary stock to dilution water in the test aquaria to achieve a final volume of 15 L. Each solution was stirred with a stainless steel whisk for approximately one minute. All test solutions appeared clear and colorless. Threesolvent control
replicates were prepared by adding the appropriate volume of methanol to dilution water in the aquaria. The solvent concentration in the treatment and solvent control groups was 0.5 m L L
Test Organism The sheepshead minnow, Cyprinodon variegatus,was selected as the test species for this study.
The sheepshead minnow is representative of an important group of aquatic vertebrates and was selected for
use in the test based upon past history of use in the laboratory. Sheepshead minnows used in the test were
obtained from Aquatic BioSystems, Inc., Fort Collins, Colorado. Identification of the species was verified by the supplier.
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The fish were held for approximatelyseven weeks prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 14-day holding period preceding the test, water temperatures ranged from 22.5 to 22.8"C. The pH of the water ranged from 7.9 to 8.1,salinitywas 20% (partsper thousand) and dissolved oxygen ranged from 7.0 to 7.2 mg/L (289% of saturation). Instrumentation and procedures used for water measurements are described in the Environmental Conditions section of this report. The fish were acclimated to test conditions for approximately5 1hours prior to test initiation. During the acclimationperiod, no mortalitiesoccurred and the fish showed no signs of disease or stress. At test initiation,the fish were collectedfrom the acclimation
tankand indiscriminatelydistributed two at a time to the test chambers until each contained 10 fish.
During the holding period, the fish were fed a commercially-prepareddiet (ZeiglerBrothers, Inc., Gardners, PA) ,as well asArtemia nauplii (Summit Artemia, Ogden, Utah). The fish were not fed during the acclimation period (at least two days prior to the test) or during the test.
All fish used in the test were from the same source and year class, and the 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 3.0 cm with a range of 2.4 to 3.5 cm. The averagewet weight (blotted dry) of 10 negative control fish at the end of the test was 0.44 g with a range of 0.21 to 0.66 g. Loading
was 0.30 g fish/Lof test water.
Test Apparatus Test chambers were 25-L polyethylene aquaria containing 15 L of test solution. The depth of
water in a representativetest chamber was approximately 17.1 cm. Test chambers were positioned in an environmental chamber set to maintain the desired temperature throughout the test. The test chambers were labeled with the project number, test concentration and replicate.
Dilution Water The water used for culturing and testing was natural seawater collected at Indian River Inlet,
Delaware that was filtered and diluted to a salinity of approximately 20% with well water. Salinity and pH measurements taken during the four-week period immediately preceding the test are presented in
Appendix 1.
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Project Number 454A-146A
The fi-eshly-collectedseawater was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank. The filtered saltwater then was diluted with freshwater from a well on the Wildlife International, Ltd. site and aerated with spray nozzles. The 20% water was filtered to 0.45 pm to remove microorganisms and fine particles prior to its use in the test. The results of periodic analyses performed to measure the concentrations of selected contaminants in the saltwater used by Wildlife International, Ltd. are presented in Appendix 2.
Environmental Conditions Lightingused to illuminatethe cultures and test chambers duringholding, acclimation and testing
was provided by fluorescenttubes 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 offto avoid sudden
changes in lighting. Light intensity at test initiation was approximately 139 lux at the surface of the water of one representative test chamber.
Temperaturewas measured in each test chamber at the beginning of the test, prior to and after test solution renewal, and at test termination using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope EWC Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. The target test temperature during the study was 22 f 2C. Dissolved oxygen and pH measurementswere made on water samplescollected fkom all replicate test chambers of each treatment and control at test initiation, prior to and after test solution
renewal, and at test termination. Salinitywas measured in the dilution water prior to test initiation and at
test termination.
Light intensity was measured using a SPER Scientific Ltd. Model 840006C light meter. Measurements of pH were made using a Fisher Accumet Model 9 15 pH meter, and dissolved oxygen was measured using a Yellow SpringsInstrument Model 5 1Bdissolved oxygen meter. Salinitywas measured using a Bio-Marine, Inc. Aquafauna refractometer.
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Project Number 454A-146A
Observations All organisms were observed periodically to determine the number of mortalities in each control
and treatment group. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 2.5,24,48,72 and 96 hours after test initiation.
Statistical Analyses The use of a single test concentration precluded the statistical calculation of LC50 values.
Therefore, since there were no mortalities during the study, the 2.5,24,48,72 and 96-hour LC50 values were estimated to be greater than the single concentration tested. The no mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data.
Analytical Chemistry Water samples were collected at mid-depth from each biotic test chamber of each treatment and
control group at the beginning of the test, prior to and after test solution renewal at approximately 24,48 and 72 hours of the test, and at test termination to measure concentrations of the test substance. Samples also were collected for analysis from the abiotic test chambers prepared at 20 mg a.i./L prior to and after test solution renewal at approximately 24, 48 and 72 hours, and at test termination. All samples were collected in plastic 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 PFOS in water samples collected during the test are presented in Table 1 and in the analytical chemistry report (Appendix 3). The single nominal concentration selected for use in this study was 20 mg a.i./L. Samplescollected from new test solutionsat test initiation and at approximately 24, 48 and 72 hours had measured concentrations that ranged from 74.9 to 92.9% of nominal concentrations. Samples collected from old test solutions at approximately 24, 48 and 72 hours and at test termination had measured concentrations that ranged from 55.6 to 89.9% of nominal concentrations. The abiotic 20 mg a.i./L sampleshad measured concentrationsthat ranged from 79.4 to 88.5% of nominal in the new test solutions, and from 45.0 to 54.4% of nominal in the old test solutions. The measured concentrations of PFOS in the abiotic samples from the old solutions were
WildlifeInternational, Ltd.
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Project Number 454A-146A
slightly lower than the concentrations of the 20 mg a.i./L treatment group samples with the fish present. The most plausible reason for this was increased deposition of test substance at the limit of solubility in the absence of the natural mixing action provided by the movement of the fish. When measured concentrations of the 20 mg a.i./L biotic samples analyzed at each sampling interval were averaged, the mean measured concentration for this study was 15 mga.i./L, representing 75% of the nominal concentration. The results of the study were based on the mean measured concentration.
Observationsand Measurements Measurementsof temperature, dissolved oxygen and pH are presented in Table 2. Temperatures
were within the 22 f 2OC range established for the test. Dissolved oxygen concentrationswere 27.0 mg/L (89% of saturation) in the new test solutions and ranged from 1.6 to 6.7 mgL (20 to 86% of saturation) in the old test solutions. Although dissolved oxygen concentrations dropped below 60% of saturation several times during the test, the biological results of the study indicate that this did not have an adverse effect on the results of the study. Measurements of pH ranged from 7.9 to 8.3 during the test. Salinity measurements of the dilution water at test initiation and termination were 20% (Table 2).
Daily observations of mortality and other signs of toxicity observed during the test are presented in Table 3. Sheepshead minnows in the negative control and solvent control groups appeared normal and healthy throughout the test period. No mortalities occurred in the 15 mg a.i./L treatment group during the test. However, upon transfer to new test solution at approximately48 and 72hours, some of the fish were observed swimmingerratically and turning a dark color. The fish appeared normal within approximately two hours, although one fish in the 15 mg a.i./L treatment group appeared discolored at test termination. LC50 values estimated from the mortality data at 2.5,24,48, 72 and 96 hours are shown in Table 4.
CONCLUSIONS The 96-hour LC50 value for the sheepshead minnow, Cyprinodon vuriegutus, exposed to Perfluorooctanesulfonate,Potassium Salt (PFOS) was >15 mg a.i./L, the single concentration tested. The
96-hour no mortality concentration was 15 mg a.i./L. Based on the transitory effects observed in the 15 mg a.i./L. treatment group after transfer to fresh test solutions, the NOEC was 4 5 mg a.i./L.
WildlifeInternational, Ltd.
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REFERENCES
Project Number 454A-146A
~-
1 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines
(drap),OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine.
2 U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity
Testfor Estuarine and Maine Organisms (Estuarine Fish 96-Hour Acute Toxicity Test). Hazard Evaluation Division. Office of Pesticide Programs. EPA-540/9-85-009. Washington, DC.
3 ASTM Standard E729-88a. 1994. Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials.
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Project Number 454A-146A
Table 1
Summary of Analytical Chemistry Data
Nominal Test Concentration
(mg a.i.iL)
~~
Negative Control
Replicate
A
B C A B C A B C A
B C A B C A B C A B C A B C
Sampling Time
(Hours)
0
0
0
24 (Old) 24 (Old) 24 (Old) 24 (New) 24 (New) 24 (New) 48 (Old) 48 (Old) 48 (Old) 48 (New) 48 (New) 48 (New) 72 (Old) 72 (Old) 72 (Old) 72 (New) 72 (New) 72 (New)
96 96 96
Measured Concentration
(mg a.i./L)'
<LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ
<LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ
Percent of
Nominal'
Mean Measured Concentration
(mg a.i./L)
Mean Measured Percent of Nominal
Solvent Control
A
0
<LOQ
B
0
<LOQ
C
0
<LOQ
A
24 (Old)
CLOQ
B
24 (Old)
<LOQ
C
24 (Old)
<LOQ
A
24 (New)
<LOQ
B
24 (New)
<LOQ
C
24 (New)
<LOQ
A
48 (Old)
<LOQ
B
48 (Old)
<LOQ
C
48 (Old)
<LOQ
A
48 (New)
<LOQ
B
48 (New)
<LOQ
C
48 (New)
<LOQ
A
72 (Old)
<LOQ
B
72 (Old)
<LOQ
C
72 (Old)
<LOQ
A
72 (New)
<LOQ
B
72 (New)
<LOQ
C
72 (New)
<LOQ
A
96
<LOQ
B
96
<LOO
P
' '
The limit of quantitation (LOQ)was 5.00 mg a.i./L. Results generated using MacQuan Version 1.6 software
in
full
precision
mode.
Manual calculationsmay
differ slightly.
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Project Number 454A-146A
Nominal Test Concentration
(mg a.i./L) 20
20 (Abiotic)
Replicate
A B C A B C A B C A B C A B C A B C A B C A B C
D E
D
E D E D E D E D E D
Table 1 (Continued)
Summary of Analytical Chemistry Data
Sampling Time (Hours)
0
0
0 24 (Old) 24 (Old) 24 (Old) 24 (New) 24 (New) 24 (New) 48 (Old) 48 (Old) 48 (Old) 48 (New) 48 (New) 48 (New) 72 (Old) 72 (Old) 72 (Old) 72 (New) 72 (New) 72 (New)
96 96 96
Measured Concentration (mg ai./L)'
16.4 15.7 16.0 15.2 13.3 15.2 16.2 15.7 15.0 13.4 11.3 12.7 17.4 18.2 18.6 15.2
18.0 16.2 17.0 16.6 17.4 13.7 11.1 13.4
Percent of
Nominal'
82.0 78.6
80.0 76.1 66.6 76.2 81.2 78.7 74.9 66.9 56.4
63.4 86.9
90.9 92.9 75.8
89.9 80.9 85.2 82.9 87.1 68.4 55.6
67.1
Mean Measured Concentration
(mg a.i./L)
15
24 (Old)
10.9
54.4
13
24 (Old)
9.22
46.1
24 (New)
16.1
80.3
24 (New)
16.6
83.0
48 (Old)
9.62
48.1
48 (Old)
9.69
48.5
48 (New)
16.6
83.2
48 (New)
17.5
87.3
72 (Old)
9.01
45.0
72 (Old)
9.25
46.3
72 (New)
17.7
88.5
72 (New)
15.9
79.4
96
9.84
49.2
Mean Measured Percent of Nominal 75
65
'
The limit of quantitation (LOQ) was 5.00 mg a.i./L. Results generated using MacQuan version 1.6 software
in
full
precision
mode.
Manual calculations may differ slightly.
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Project Number 454A-146A
Table 2
Temperature, Dissolved Oxygen and pH of Water in the Test Chambers
Mean Measured Concentration
(mg a.i./L)
Negative Control
Replicate
A B C
0 Hour'
Temp2 DO3
rc) ( m ~ ) p~
22.2
7.0 8.2
22.3
7.1 8.2
22.4
7.1 8.3
24 Hours (Old) Temp DO
~
22.4 6.2 8.1 22.4 6.0 8.1 22.6 5.9 8.2
24 Hours (New)
Temp DO ("C) (mg/L) PH
22.6 7.1 8.2 22.6 7.2 8.2 22.5 7.2 8.2
Solvent Control
A
B
C
22.5
7.1 8.3
22.5
7.1 8.3
22.6
7.1 8.3
22.8 5.8 8.1 22.8 5.9 8.2 22.9 5.9 8.2
22.6 7.3 8.3 22.6 7.3 8.3 22.7 7.3 8.3
15
A
22.7
7.1 8.3
23.0 5.6 8.1
22.6 7.3 8.3
B
22.8
7.1 8.3
23.1 5.6 8.1
22.7 7.3 8.3
C
22.8
7.1 8.3
23.1 5.4 8.1
22.7 7.3 8.3
D4 22.7
7.1 8.3
23.1 6.7 8.3
22.8 7.3 8.3
E4
22.8
7.1 8.3
23.1 5.2 8.2
22.7 7.3 8.3
The 0 and %-hour dilution water measurements for salinity were 20%.
* Temperature measured continuously during the test remained between 21.5 and 225C.
A dissolved oxygen concentration of 4.7 mgL represents 60% saturation at 22C in saltwater with a salinity of 20%. Abiotic replicates.
48 Hours (Old)
Temp DO
3("(
(mgn) PH
22.0
5.3 8.0
22.1
4.6 7.9
22.1
3.4 8.0
22.3
4.4 8.0
22.4
3.7 8.0
22.4
3.6 8.0
22.5
4.6 8.0
22.5
3.0 8.0
22.6
5.0 8.1
22.7
5.0 8.1
22.7
4.8 8.1
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Table 2 (Continued)
Temperature, Dissolved Oxygen and pH of Water in the Test Chambers
Mean Measured
- Concentration (mg a.i./L)
Negative Control
Replicate
A B C
48 Hours (New) Temp' DO3 ("c) ( m t m PH
22.1
7.2 8.2
22.1
7.3 8.2
22.2
7.3 8.2
72 Hours (Old) Temp DO ("C) ( m f m PH
22.2
5.5
8.0
22.3 3.9 7.9
22.3 3.7 8.0
72 Hours (New)
Temp DO ("C) ( m a ) PH
22.1 7.4 8.2 22.0 7.4 8.2 22.1 7.4 8.2
Solvent Control
A
B
C
22.3 , 7.2 8.2
22.4
7.2 8.2
22.4
7.2 8.2
22.3 4.0 8.0 22.4 3.0 7.9 22.4 4.0 8.0
22.2 7.4 8.2 22.2 7.5 8.2 22.3 7.6 8.2
15
A
22.4
7.2 8.2
22.5 3.6 7.9
22.2 7.6 8.2
B
22.5
7.2 8.2
22.5 3.6 8.0
22.2 7.5 8.2
C
22.5
7.2 8.2
22.6 3.3 7.9
22.3 7.4 8.2
D4 22.6
7.4 8.3
22.6 5.4 8.1
22.3 7.5 8.2
E4
22.5
7.4 8.3
22.6
5.5
8.1
22.3 7.5 8.2
' The 0 and 96-hour dilution water measurements for salinity were 20%.
Temperature measured continuously during the test remained between 2 1.5 and 22.5"C.
A dissolved oxygenconcentration of 4.7 m a represents 60% saturation at 22OC in saltwater with a salinity of 20%.
Abiotic replicates.
Project Number 454A-146A
96 Hours (Old) '
Temp DO ("C) (mg/L) PH 22.1 3.8 7.9 21.9 3.5 7.9 22.1 2.8 7.9 22.1 2.6 7.9 22.2 2.7 7.9 22.3 1.7 7.9 22.4 1.9 7.9 22.3 1.6 7.9 22.4 3.3 7.9 22.5 3.4 8.0 22.6 3.2 8.0
WildlifeInternational, Ltd.
Project Number 454A-146A
Table 3
Cumulative Percent Mortality and Treatment-Related Effects
Mean Measured Concentration
(mg a.i./L)
2.5 Hours
No.
No.
Rep. Exposed Dead2
Observed Effects'
24 Hours '
No. Observed Dead Effects
48 Hours I
No. Observed Dead Effects
72 Hours I
No, Observed Dead Effects
Negative Control A
10
B
10
C
10
10 AN 10 AN 10 AN
0
10 AN
0
10 AN
0
10 AN
10 AN 10 AN 10 AN
0 10 AN
0
10 AN
0
10 AN
Solvent Control A
10
B
10
C
10
10 AN 10 AN 10 AN
0
10 AN
0
10 AN
0
10 AN
10 AN 10 AN 10 AN
0 10 AN 0 10 AN 0 10 AN
15
A
10
B
10
C
10
10 AN 10 AN 10 AN
0
10 AN
0
10 AN
0
10 AN
2E,D; 8AN
0 3ED; 8AN
3E,D; 1R; 6AN 0 4E,D; 6AN
lE,D; 9AN
0 2E,D; 9AN
' All fish were transferred to fresh test solution at approximately24,48 and 72 hours.
Cumulative number of dead fish. 3 Observed Effects: AN = appear normal; E = erratic swimming; D = discoloration (dark); R = lying on bottom.
96 Hours
No. Observed Dead Effects
0
10 AN
0
10 AN
0
10 AN
0 10 AN 0 10 AN
0 10 AN
0 10 AN 0 10 AN 0 lD;9AN
Cumulative MPoerrctaelnitty
0
0
0
WildlifeInternational, Ltd.
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Project Number 454A-146A
Table 4 LC50 Values
Time
2.5 Hours
24 Hours 48 Hours 72 Hours 96 Hours
LC50 (mg a.i./L)
>15 >15 >15 >15 >15
95% Confidence Interval (mg a.i./L)
-_1
-- 1
--1
__ I
-- I
Statistical Method
NA~ NA~ NA~ NA~ NA~
I 95% confidence limits could not be calculated fiom the data. 2 NA = not applicable; LC50 value was estimated by visual interpretationof the mortality data.
Wild1ife Intemational, Ltd. - 21 -
Project Number 454A-146A
Appendix 1
Salinity and pH of Saltwater Measured During the 4-Week Period Immediately Preceding the Test
Parameter
Mean
~~~
~~
Range
Salinity (%)
PH
20 (n = 4) 8.2 (n = 4)
20 - 20 8.1 - 8.3
Wild1ife Intemational, Ltd.
Project Number 454A-146A
Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International,Ltd. Saltwater'
Component
Pesticides And Organics
Measured Concentration
Component
Measured Concentration
Aclonifea Alachlor
hew Atrazine Azinphosethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Chlorothalonil Coumphos
Cyanazine
cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-0 Desethylatmzine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid
~0.03pg/L CO.01 pg/L 10.01 pg/L co.01 pg5 c0.04 p g 5 <0.08 pg/L 10.25 p g 5 <0.05 pg/L co.05 p g 5 <0.05 p g 5 10.05 p g 5 <0.02 pg/L <0.02 p a <0.02 pg/L co.05 p g 5 10.05 pg/L <0.03 pg5 10.02 pg/L <0.02 pg/L <0.05 pg5 10.02 pg5 co.01 p g 5 <0.01 pg/L <0.04 pg/L
c0.02 pgiL
co.05 p g n
<0.05 pg/L . 10.25 pg5 CO.05 pgiL 10.02 p g 5 <0.02 p g 5 <0.02 pgiL 10.01 pgiL C0.02 pg/L 10.01 pg5 CO.01 pg/L co.01 pg5 <0.03p g 5 <0.02 pg5 CO.01 pg5 <0.03p g L
Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodemorph Endosulh-a Endosulfan$ Endosulfan-sulfate Epoxiconazole Eptam Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb
Fenpiclonil
Fenpropathrin Feapropimorph Fenthion Fenvalerate Fluazifop-butyl Fluoroglycofenethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane
<0.01 pg/L <0.25 p g 5 <0.02 pg/L <0.03 pg/L e0.02 pg/L <0.05 pg/L <0.02 pg/L <0.05 p g 5 co.01 p g 5 <0.01 p g 5 40.01 pg/L 40.02 pg/L CO.05 p g 5 40.02 pg/L 40.02 pg/L 40.05 p g 5 40.02 pg/L CO.01 pg/L 40.02 p g 5 ~ 0 . 0 5pg/L 40.05 pg/L <0.01 p g 5 10.03 p g 5 40.03 pg/L <0.05 pg/L <0.25 pg/L dO.01 pg5
co.01 p g n
10.02 p g 5 <0.02p g 5 <0.02 p g 5 10.05 pgiL c0.02 p g 5 10.01 pg5 <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L
c0.02p g 5 c0.05 p@
10.02 pg5
'Analyses performed by TNO Nutrition and Food Institute on samples collected on November IS, 2000.
Wild1ife Intemational, Ltd.
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Project Number 454A-146A
Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Saltwater'
Component
~
Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Pmethrincis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Propham
Propiconazole
Propoxur
Propyzamide Prosulfocarb Pyrazophos
Pesticides And Organics (Page 2)
Measured Concentration
Component
<0.02 p g 5 CO.05 p a <0.05 p g n C0.02 p g n C0.02p g n <0.05 p g 5
<0.01 jlgn
<0.01 pg/L <0.05 p g n <0.03 pg& <0.01 p g n
<0.01 p g n
<0.05 p g n <0.02 pg/L CO.05 p g 5 <0.01 pg/L XO.01 p g 5 <0.01 p g n C0.02 p g n <0.01 p g n <0.01 p g 5 <0.01 p g n
<0.01 p g n <0.02p g n
pa <0.05 p g 5
<0.03 <0.02 pg/L <0.02 pgL <0.03 pg/L
Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpymd Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydrofialimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Tnadimenol
Triallate
Triazophos
TriflWalin Vamidothion Vinclozolin
Measured Concentration
<0.01 p g n <0.01 p g n <0.02 p g 5 <0.01 pg/L <0.05 pg/L <0.01 p g n
<O.Ot p g n
CO.01 pa
<0.02 p g n
<0.01 pgn C0.02 p g 5
<0.05 p g n
<0.05 pg/L <0.01 pgn <0.01 pg/L
10.01 p g n <0.05 p g n
<0.01 p g n
<0.05 p g n
<0.04 p g 5 <0.01 p g 5 <0.04 pg& <0.05 p g n <0.05 pg5
(0.02 p g 5
<0.02 pa <0.02 p g n
<0.01 pg/L <0.01 p g n
Metals
Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium ChrOmiUm Cobalt
730 m g 5 5,800 m@
270 m f l ~ 0 . 0 m3 a 200 mg/L <O. 18 mgL 4.0 pg5 ~ 3 . p0g/L <6.0 pg/L <4.0 p g 5
Nickel Copper zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium
'Analyses Derformed by TNO Nutrition and Food Institute on samples collected on November IS, 2000.
WildlifeInternational, Ltd.
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Appendix 3
Project Number 454A-146A
THE ANALYSIS OF PFOS IN FILTERED SALTWATER
IN SUPPORT OF WILDLIFE INTERNATIONAL,LTD. PROJECT NO.: 454A-146A
WildlifeInternational, Ltd.
Project Number 454A-146A
REPORT APPROVAL
SPONSOR: 3M Corporation
TITLE:
PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus)
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-146A
PRINCIPAL INVESTIGATOR:
& d l L .Van Hoven, Ph.D.
MANAGEMENT:
Willard B. Nixon, Ph.b. Director, Analytical Chemistry
DATE DATE
Wildlife Xntemational, Ltd.
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Project Number 454A-146A
Introduction Filtered saltwater samples were collected from a 96-hour static-renewal acute toxicity test designed
to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the sheepshead minnow (Cyprinodon vuriegutus). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-146A. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLCMS). Samples were received for analysis on August 13, 14, 15, 16 and 17,2001 and were analyzed on each sample receipt day.
Test Substance and Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 4675.
The test substance, referred to hereafter as PFOS, was used to prepare calibration standards and matrix fortification samples.
The internal standard was received from 3M Corporation on July2,1998 and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: lH, lH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 2761997-2. The standard, referred to hereafter as 4H PFOS, was stored under ambient conditions.
Analytical Method The method used for the analysis of the filtered saltwater samples was developed at Wildlife
International, Ltd. and entitled "Analytical Method for the Determination of PFOS in Freshwater,
Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M
Corporation. Several modifications from the validated methodology were implemented for the present study. First, the concentration of the internal standard was changed from 100 pg/L to 10.0 pg/L to better match the calibration and test sample instrumental concentrations. Second, a guard cartridge was used in conjunction with a shorter (50 mm vs. 100 mm) analytical column for long-term protection of the analytical column and faster run times.
Wild1ife International, Ltd.
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Project Number 454A-146A
Samples were diluted in a 50% methanol : 50% NANOpure@water solution containing 10.0 pg 4H PFOS /L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology.
Concentrations of the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API lOOLC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C18 analytical column (50 mm x 2 mm I.D., 3-pm particle size) fitted with a Keystone Javelin C18 guard
cartridge (20 x 2 mm). The instrument parameters are summarized in Table 1. A method flowchart is
provided in Figure 1.
Calibration Curve and Limit of Quantitation Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure@water solution
containing 10.0 pg 4H PFOS (internal standard)/L,and 0.05% formic acid (v/v), ranging in concentration from 0.500 to 5.00 pg a.i./L, were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS :internal standard) of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration ofPFOS in the samples was determined by substitutingthe peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively.
The method limit of quantitation (LOQ) for these analyseswas set at 5.00 mg a.i./L calculated asthe product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (10,000).
WildlifeInternational, Ltd.
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Project Number 454A-146A
Matrix Blank and Fortification Samples Five matrix blank samples were analyzed to determinepossible interference. No interferenceswere
observed at or above the LOQ during samples analyses (Table 2). A representativeion chromatogram of a matrix blank is presented in Figure 5 .
Filtered saltwaterwas fortified at 18 and 22 mg a.i./L and analyzed concurrentlywith the samplesto
determine the mean procedural recovery (Table 3). Sampleconcentrationswere not correctedfor the mean procedural recovery of 99.2%. A representative ion chromatogramof a matrix fortificationis presented in Figure 6.
Example Calculations Sample number 454A-146A-9, nominal concentration of 20 mg a.i./L in filtered saltwater.
Peak Area Ratio = Analyte Peak A r e h t e r n a l Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration of Internal Standard Internal Standard Concentration: 0.0100 mg/L First Initial Volume: 0.100 mL First Final Volume: 10.0 mL Second Initial Volume: 0.100 mL Second Final Volume: 10.0mL Dilution Factor: 10,000
PFOS Peak Area: 252985
Internal Standard Peak Area: 469623 Peak Area Ratio: 0.5387
Calibration curve equation. Slope: 3.2154 Intercept: 0.0244 Curve is weighted (Ux)
PFOS (mg a.i./L) at instrument =Peak area Slo-pe
x Internal Standard Concentration
Wildlife International, Ltd.
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Project Number 454A-146A
-- 0.5387 - 0.0244 x 0.0100 3.2154
= 0.001599
PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor
= 0.001599 x 10,000
= 16.0
PFOS m Percent of Nominal Concentration = PFOS((:g
a.i./L
nomintl insam le x 100
---126o.0 x100
= 80.0% Quantitation software for recoveries: MacQuan, version 1.6.
RESULTS
Sample Analysis Filtered saltwater samples were collected from a 96-hour static-renewalacute toxicity test with the
sheepsheadminnow (Cjprinodonvun'egutus). New solutions were collected at test initiation, August 13,
2001 (Hour 0). New and old solutionswere collected on August 14 (Hour 24), August 15 (Hour 48), and August 16(Hour 72), 2001. Old solutionswere collected at test termination,August 17,2001 (Hour 96).
The measured concentrations of PFOS in the samples collected from the 20 mg a.i./L treatment group at initiation of exposure of the test organisms (Hour 0) had measured concentrations ranging from 15.7 to 16.4 mg a.i./L, corresponding to 78.6 to 82.0% of the nominal concentrations (Table 4). New samples collected at Hours 24, 48, and 72 had measured concentrations ranging from 15.0 to 16.2 mg a.i./L, 17.4 to 18.6mg a.i./L, and 16.6to 17.4 mg a.i./L, respectively. These values corresponded to percent of nominal ranging from 74.9 to 81.2%, 86.9 to 92.9%,and 82.9 to 87.1%, respectively. Old samples collected at Hours 24, 48, and 72 had measured concentrations ranging from 13.3 to 15.2 mg a.i./L, 11.3 to 13.4 mg a.i./L, and 15.2 to 18.0 mg a.i./L, respectively. These values corresponded to percent of nominal ranging from 66.6 to 76.2%, 56.4 to 66.9%, and 75.8 to 89.9%,respectively. The
WildlifeInternational, Ltd.
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Project Number 454A-146A
measured concentrations of PFOS in the samples collected at test termination (Hour 96) had measured concentrations ranging from 11.1 to 13.7 mg a.i./L, corresponding to 55.6 to 68.4% of the nominal concentrations.
The measured concentrations of PFOS from the abiotic 20 mg a.i./L treatment group were slightly lower (mean measured concentration = 13 mg a.i./L) than those from the 20 mg a.i./L treatment group (mean measured concentration= 15 mg a.i./L) with the fish present (Table 4). The most plausible reason for this was increased deposition of test substance at the limit of solubility in the absence of the natural mixing action provided by the movement of the fish. A representativeion chromatogram of a test sample is shown in Figure 7.
WildlifeInternational, Ltd.
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Project Number 454A-146A
INSTRUMENT:
Table 1
Typical HPLCNS Operational Parameters
Hewlett-Packard Model 1100High Performance Liquid Chromatograph
with a Perkin-Elmer API lOOLC Mass Spectrometer (HPLC/MS)
equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM).
ANALYTICAL COLUMN: GUARD COLUMN:
OVEN TEMPERATURE:
Keystone Betasil c18 column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin column (20 x 2 mm)
30C
STOP TIME:
5.00 minutes
FLOW RATE:
0.220 mL/minute
MOBILE PHASE:
72.0%Methanol :28.0%NANOpure@water containing 0.1% Formic Acid
INJECTION VOLUME:
25.0 pL
PFOS RETENTION TIME:
Approximately 4.4 minutes
INTERNAL STANDARD RETENTION TIME:
Approximately 3.0 minutes
PFOS MONITORED MASS: 499 amu
INTERNAL STANDARD MONITORED MASS:
427 amu
WildlifeInternational, Ltd.
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Project Number 454A-146A
Table 2
Matrix Blanks Analyzed Concurrently During Sample Analysis
Number (454A-l46A-)
MAB- 1
Sample
Type Matrix Blank
Measured Concentration of PFOS'
(mga.i./L) < LOQ
MAB-2 MAl3-3
Matrix Blank Matrix Blank
< LOQ
< LOQ
MAB-4
Matrix Blank
< LOQ
MAB-5
Matrix Blank
< LOQ
The limit of quantitation (LOQ) was 5.00 mg a.i./L based upon the product of the lowest calibration
standard analyzed (0.000500 mg a.i./L.) and the dilution factor of the matrix blank samples (10,000).
WildlifeInternational. Ltd.
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Project Number 454A-146A
Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis
Sample Number (454A-146A-)
MAS-1 MAS-3 MAS-5 MAS-7 MAS-9
CoInncceennttrations of PFOS (mg a.i./L)
Forrtified'
MMeeaassuurreedd''
18.0
16.2
18.0
17.9
18.0
16.6
18.0
18.4
18.0
18.1
Percent Recovered'
89.9 99.4 92.4 102 101
MAS-2
22.0
20.2
92.0
MAS-4
22.0
22.4
102
MAS-6
22.0
22.0
99.9
MAS-8
22.0
23.8
108
MAS- 10
22.0
23.0
105
Mean =99.2
cv Standard Deviation = 5.92 = 5.97%
' N =10 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly.
WildlifeInternational, Ltd.
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Project Number 454A-146A
Table 4
Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cypn'nodonvuriegutus)96-Hour Toxicity Test
Nominal Test Concentration
(mg ai./L)
0.0
(Negative Control)
Replicate
A B C A B C A B C A B C A B C A B C A B C A
B
C
Sampling Time
(Hours)
0 0 0 24 (Old) 24 (Old) 24 (Old) 24 (New) 24 (New) 24 (New) 48 (Old) 48 (Old) 48 (Old) 48 (New) 48 (New) 48 (New) 72 (Old) 72 (Old) 72 (Old) 72 (New) 72 (New) 72 (New) 96 96
96
Measured Concentration (mg ai./L)'
<LOQ <LOQ
<LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ
<LOQ
<LOQ
Percent of
Nominal'
---
Mean Measured Concentration
(mg a.i./L)
<LOQ
Mean Measured Percent of Nominal
--
The limit of quantitation(LOQ)was 5.00 mg a.iJL.
* Results were generated using MacQuan version 1.6softwarein full precision mode. Manual calculationsmay differ
slightlv.
WildlifeInternational, Ltd.
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Project Number 454A-146A
Table 4 (Continued)
Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cyprinodonvuriegutus)96-Hour Toxicity Test
Nominal Test Concentration
(mg ai&)
Replicate
Sampling Time
(Hours)
Measured Concentration (mg ai./L)'
Percent of
Nominal'
Mean Measured Concentration
(mg a.i./L)
Mean Measured Percent of
Nominal
0.0
A
0
<LOQ
B
0
<LOQ
(Solvent Control)
C
0
<LOQ
A
24 (Old)
<LOQ
B
24 (Old)
<LOQ
C
24 (Old)
<LOQ
A
24 (New)
<LOQ
B
24 (New)
<LOQ
C
24 (New)
<LOQ
A
48 (Old)
<LOQ
B
48 (Old)
<LOQ
C
48 (Old)
<LOQ
A
48 (New)
<LOQ
B
48 (New)
<LOQ
C
48 (New)
<LOQ
A
72 (Old)
<LOQ
B
72 (Old)
<LOQ
C
72 (Old)
<LOQ
A
72 (New)
<LOQ
B
72 (New)
<LOQ
C
72 (New)
<LOQ
A
96
<LOQ
B
96
<LOQ
C
96
<LOO-
' The Limit ofquantitation (LOQ) was 5.00 mg a.i./L. Results were generated using MacQuan version 1.6 software
in
full precision
mode.
Manualcalculationsm a y
differ
WildlifeInternational, Ltd.
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Project Number 454A-146A
Table 4 (Continued)
Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cypn'nodonvuriegutus)96-Hour Toxicity Test
Nominal Test Concentration
(mg a.i./L)
Replicate
Sampling Time (Hours)
Measured Concentration (mg ai./L)*
Percent of
Nominal'
Mean Measured Concentration
(mg a.i./L)
Mean Measured Percent of Nominal
20
A
0
16.4
82.0
15
75
B
0
15.7
78.6
C
0
16.0
80.0
A
24 (Old)
15.2
76.1
B
24 (Old)
13.3
66.6
C
24 (Old)
15.2
76.2
A
24 (New)
16.2
81.2
B
24 (New)
15.7
78.7
C
24 (New)
15.0
74.9
A
48 (Old)
13.4
66.9
B
48 (Old)
11.3
56.4
C
48 (Old)
12.7
63.4
A
48 (New)
17.4
86.9
B
48 (New)
18.2
90.9
C
48 (New)
18.6
92.9
A
72 (Old)
15.2
75.8
B
72 (Old)
18.0
89.9
C
72 (Old)
16.2
80.9
A
72 (New)
17.0
85.2
B
72 (New)
16.6
82.9
C
72 (New)
17.4
87.1
A
96
13.7
68.4
B
96
11.1
55.6
C
96
13.4
67.1
' The limit of quantitation(LOO)was 5.00 mg a.i./L.
Results were generated using MacQuan version 1.6 software in fullprecision mode. Manual calculationsmay differ
slightly
WildlifeInternational, Ltd.
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Project Number 454A-146A
Table 4 (Continued)
Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cyprinodon variegatus) 96-Hour Toxicity Test
Nominal Test Concentration
(mg ai&)
Replicate
Sampling Time (Hours)
Measured Concentration (mg ai.L)'
Percent of
Nominal2
Mean Measured Concentration
(mg a.i./L)
Mean Measured Percent of Nominal
20 (Abiotic)
D
24 (Old)
10.9
54.4
13
65
E
24 (Old)
9.22
46.1
D
24 (New)
16.1
80.3
E
24 (New)
16.6
83.0
D
48 (Old)
9.62
48.1
E
48 (Old)
9.69
48.5
D
48 (New)
16.6
83.2
E
48 (New)
17.5
87.3
D
72 (Old)
9.01
45.0
E
72 (Old)
9.25
46.3
D
72 (New)
17.7
88.5
E
72 (New)
15.9
79.4
D
96
9.84
49.2
E
96
9.22
46.1
I The limit of quantitation (LOQ)was 5.00 mg a.i.iL.
* Results were generated using h4acQuan version 1.6 software in full precision mode. Manual calculationsmay differ
WildlifeInternational, Ltd.
-38-
Project Number 454A-146A
METHOD OUTLINE FOR THE ANALYSIS OF PFOS
IN FILTERED SALTWATER
Prepare matrix fortification samples in filtered saltwater matrix by spiking the requisite volume of PFOS stock solutions directly into filtered saltwater. Perform fortifications with gas-tight syringes and class A volumetric flasks. The matrix blank is unfortified filtered saltwater.
Prepare appropriate dilutions of study and QC samples to within range of the PFOS HPLC/MS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure@water dilution solvent containing 10.0 pg 4H PFOSL and 0.05% (v/v) formic acid. Add appropriate volume of sample and bring the flask to volume with dilution solvent. Process the matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions.
Ampulate samples and submit for HPLCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in filtered saltwater.
,
WildlifeInternational, Ltd.
-39-
FFC6 499.0 internalStandard: 4HPFOS Weighted (llx)
Intercept = 0.0244 Slope = 3.2154 Correlation Coeff. = 0.9996
Area(Ratio)
Project Number 454A-146A
Conc. (Ratio) Figure 2. A typical calibration curve for PFOS.
Wildlqe International, Ltd.
-40-
Project Number 454A-146A
PFOS-1
STD 0.500 ug d J L
4675A-011D -21
4.98 in 1 period
R(3s
Internal Standard: 4HPFOS
Use Area
Absolute Retention Time
1: 4.98 Q1 MI. 297 scans
499.0
Noise Thres.
10.0
Quant Thres.
5.0
Min. Width
3
Muit. Width
6
Base. WKfth
80
RTWin. (secs) 2 0
Smooth
1
ExpectedUT
4.24
Area ' 74579
Height 7951
Start Time
4.02
End Time integration Width Retention Time Integration Type
4.60 0.57
-4.24
A VB
Mon, Aog 13, 2001 14:23
intensity: 80000 cps 10
9
8
7
6
5
4
3
2
1
252
41 0.69
81 121 1.36 2.04
161 2.71
201 3.38
241 4.06
281 Scan 4.73Time
PFOS-1
STD 0.500 ug r.lJL
4675A-011D -21
4.98 in 1 period
4HpFos
use as Internal Standard
1: 4.98 Q1 MI, 297 scans
427.0
NoiseThres.
2.0
Quant Thres.
1.O
Mh. Wath
3
Mult. Mdth
6
Base. w m
30
RT Win. (secs) 20
Smooth
1
CRT
3.00
Area 414322
Height 63988
Start Time
2.78
Endm
3.28
Integration Width
0.61
Retention Time Integration Type
2.93 A- 88
Mon, Aug 13, 2001 14:23
intensity: 80000 cps
i I
3
2
1 24 41 0.69
174
103 81 121 1.36 2.04
181 2.71
218 247 201 241 3.38 4.08
281 Scan 4.73 Time
Figure 3.
A representative ion chromatogram of a low-level (0.500 pg a.i./L) PFOS standard. (monitored masses = 499 amu (PFOS -top) and 427 amu (4HPFOS internal standard -
bottom)).
WildlifeInternational, Ltd.
-41 -
Project Number 454A-146A
PFOSJ
STD 5.00 ug alA
4675A-011D -25
5.00 in 1 period
Rc6
InternalStandard: 4HPFOS
U s e Area
Absolute Retention Time
1: 4.98 Q1 Mi, 297 scans
499.0
Noise Thres.
Quant Thres.
10.0 5.0
Min. Width
3
Mull. Width
6
Base. Wdth
80
RT Win. (secs) 2 0
Smooth
1
ExpectedRT
4.24
Area
680879
Height 68328
Start Time
4.03
End Time
4.75
Integration Width
0.72
Retention Time Integration Type
4.26
A - VB
Mon, Aug 13, 2001 14:48
'8 1
intensity: 80000 cps
n253
3
2
~ 10 1 8 %5 41 0.69
96 81 1.37
127 121 2.04
161 2.71
201 3.39
241 4.06
281 Smn 4.73 Time
PFOS-5
STD 5.00 ug riA
4675A-0110 -25
5.00 in 1 period 4tQFos use as Internal Standard
1: 4.98 Q1 MI, 297 scans
427.0
NoiseThres.
2.0
QuantThres.
1.0
Min. Width
3
Mutt. Width
6
Base. Wklth
30
RTWin. (secs) 2 0
Smooth
1
EXpactedRT
3.00
Area 420532
Height 56584
Start Time
2.78
EndT i
3.28
Integration Width
0.51
Retention Time integration Type
-2.93
A BB
Mon, Aug 13, 2001 14:48
intensity: 80000 cps
100-
90.
80. 70- 174
6&
50. 40.
30.
2e
. . . . . . 10.
04 1 1 4,7
101120
0
:
41 0.69
01 121 1.37 2.04
161 2.71
210 242 264
201 241 201 Scan
3.39 4.06 4.73 Time
Figure 4.
A representative ion chromatogramof a high-level (5.00 pg a.i./L)PFOS standard.
(monitored masses = 499 amu (PFOS -top) and 427 amu (4HPFOS internal standard -
bottom)).
Wildlgfe International, Ltd.
- 42 -
Project Number 454A-146A
PFOS-7
MAE-1
454A-146A-
Mon, Aug 13, 2001 16:Ot
4.98 in 1 period
rn
internal Standard: 4HPFOS
Use Area
Absolute Retention Time
1: 4.98 Q1 MI, 297 scans
499.0
Noise Thres.
10.0
Quanl Thres.
5.0
Mln. Width
3
Mull. Width . 6
Base. Wdh
80
RTWin.(seca) 2 0
Smooth
1
ExpectedRT
4.24
Area 4434
Height 496
Start Time
4.1 1
End Time
4.43
Integration Width
0.32
RetenUon Time Integration Type
-4.26
A BB
intensity: 80000 cps 10
:3
1
2
1
20
87
131 171
221 36%
' 4'1 ' 8'1 ' lil ' 181 261 ' 241 ' 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73 Time
PFOS-7
MAE1
454.4-146A-
Mon, Aug 13, 2001 15:Ol
4.98 in 1 period
4HpFos
use as Internal Standard
1: 4.98 Q1 MI, 297 scans
427.0
NoiseThres.
2.0
Quant Thres.
1.0
Mh. Width
3
Muk Width
6
Base. wl@l
30
RTWin.(sacs) 20
Smooth
1
EXp0CtedAT
3.00
Arep
474292
Helght 63384
start Time
2.78
EM n m
3.30
Integration Width
0.51
Retention lime Integration Type
-2.93
A 88
O01
8 7060.
60-
4030. 20. 1Ck
04 1 2I 2 8. 2 8 . .107 1 I .
intensity BOO00 cps
245 277
Figure 5.
A representative ion chromatogram of a matrix blank sample (454A-146A-MAB-1). Dilution factor = 10,000~T. he arrow indicates the retention time of PFOS. (monitored masses = 499 amu (PFOS -top) and 427 amu (4HPFOS internal standard bottom)).
WildlifeInternational, Ltd.
- 43 -
Project Number 454A-146A
PFOS-9
MAS-2
454A-146A-
M M . Aug 13, 2001 15:13
4.98 in 1 period
Ros
lntemal Standard: 4HPFOS
Use Area
Absolute Retention Time
1: 4.98 Q1 MI, 297 scans
499.0
Noise Thres.
10.0
Quant Thres.
5.0
Min. Width
3
Mult. Width
6
Base. Wdth
80
RTWin.(secs) 2 0
Smooth
1
Expected RT
4.24
Area 319919
Height 32768
Start Time
4.04
End Time
4.75
Integration Width Retention Time Integration Type
0.71
-4.26
A VB
100 90 80 70 60 50 40 30 20 10 0
41 0.69
Intensity: BO000 cps 253
PFOS-9
MAS-2
454A-146A-
Mm. Aug 13, 2001 15:13
4.98 in 1 period
4HpFos
use as Internal Standard
1: 4.98 Q1 MI. 297 Scans
427.0
NoiseThres.
2.0
QuantThres.
1.0
Mln. Width
3
Mult. Wdith
6
BW. width
30
RTWin. (secs) 2 0
SmoOth
1
EwpectedRT
3.00
Area 473996
Height 81883
Start Time
2.79
End Time
3.35
Intearation Width
0.66
ij10 6 4
3 2
intena : 8OOoo cps .I75
nn...
.e.
Figure 6.
A representative ion chromatogram of a matrix fortification sample (454A-146A-MAS-2). Nominal Concentration = 22 mg a.i./L, Dilution factor = 10,000~. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)).
WildlifeInternational, Ltd.
-44-
Project Number 454A-146A
PFOS-17
7
454A-146A-
Mon, Aug 13. 2001 16:04
4.98 in 1 period
pFI3s
lntemal Standard: 4HPFOS
Use Area
Absolute Retention Time
1: 4.98 Q1 MI, 297 scans
499.0
Noise mms.
10.0
Quant Thres.
6.0
Min. width
3
Mult. Width
6
Base. width
00
RTWin.(secs) 20
Smooth
1
Expected RT
4.24
Area 269764
Height 26978
Starl Time
4.02
~ nTdime
4.60
fntegration Width
0.66
Retention Time Integration Type
-4.26
A VB
10
44
3 2
1
intensity: 8OOOO cps
253
41 0.69
81 1.36
121 2.04
161 2.71
201 3.38
241 4.06
281 Scan
4.73Time
1: 4.90 01 MI, 297 scans
427.0
Noise Thres.
2.0
a u n t mms. 1.0
Min. W m
3
Mult. Width
6
Base. width
30
RTWln.(secs) 20
SmOOth
1
5tpectedRT
3.00
Area 471004
Height 82678
Start Time
2.79
EndTim Integration Wldth Retention Time
3.30 0.61
2.85
100SO. 80. 7060.
60
40-
30.
20. 10
2 0, 1 7rn
175
94 110 16
217 243 205
Figure 7.
A representative ion chromatogram of a test sample (454A-146A-7).
Nominal Concentration = 20 mg a.i./L., Dilution factor = 10,OOOx. (monitored masses = 499 amu (PFOS- top) and 427 amu (4HPFOS internal standard -
bottom)).
WildlifeInternational, Ltd.
-45-
Project Number 454A-146A
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, test concentrations, and test and reference substance identification.
2. The protocolwas amended to include the procedures and samplingscheme for conductinga limit test.
3. The protocol was amended to delete the analysis of feed since historical analyses of Wildlife International, Ltd. aquatic feed have shown that no contaminants are present at levels known to be capable of interfering with the study.
4. The protocol was amended to clarify the analytical methodology to be used during sample analyses.
5. The protocol was amended to change the proposed test dates and to include the procedures and sampling scheme to be used during a static-renewal test since the test was repeated under staticrenewal test conditions to maintain test concentrations during the exposure period.
6. The protocol was amended to include the use of methanol in the preparation of the test solutions, and to include a solvent control group.
7. Salinity of the dilution water was measured at test termination, as well as at test initiation. Since the
additional measurement confirmed that the salinity remained constant during the test, this change to the protocol had no impact upon the study
Wild1ife Intemational, Ltd. - 46 -
Project Number 454A-146A
Appendix 5 Personnel Involved in the Study
The followingkey WildlifeInternational,Ltd. personnelwere involved in the conduct or management of this study:
1. Henry 0. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Raymond L. Van Hoven, Ph.D., Scientist 5 . Susan J. Palmer, Senior Biologist 6. Michelle Stence, Biologist 7. Frank J. Lezotte, Chemist