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PFOS : A 96-HOUR STATIC-RENEWA L ACUTE TOXICITY TEST WITH Hyalella azteca
FINAL REPORT
WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER : 454A-253A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER : E07-0082
ASTM Standard E729-9 6
AUTHORS : Tui Minderhout, Ph .D . Jon A . MacGregor, B .S . Henry O . Krueger, Ph .D .
STUDY INITIATION DATE : February 15, 2007 STUDY COMPLETION DATE : March 29, 2007
SUBMITTED TO :
3M Corporation
^r -,
Environmental Laboratory
3M Center
Building 0260-05-N-17
Maplewood, MN 55144
Wildlife International, Ltd.
8598 Commerce Drive Easton, Ma ry land 21601 USA
1-410-822-8600
Page 1 of 4 3
CONTAINS NO C6
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Wildlife International, Ltd.
Project Number 454A-253 A
-2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR 3M Corporation TITLE PFOS : A 96-Hour Static-Renewal Acute Toxicity Test with Hyalella azteca WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER . 454A-253 A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER E07-0082
STUDY COMPLETION March 29, 200 7
This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989) with the following exception
Periodic analyses of well water for potential contaminants were performed using a certified laboratory and standard U .S . EPA analytical methods .
STUDY DIRECTOR :
ui Minderhout, Ph D . Senior Biologist
3 f 2.~ Ji 0b ~ Date
SPONSOR APPROVAL : sot Representative Dat e
31 0~;z
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Wildlzfe International, Ltd.
Project Number 454A-253 A
-3QUALITY ASSURANCE STATEMEN T
This study w as examined for compliance with Good Laboratory Practice Standards as published by th e U .S . Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989) . The dates of all inspections an d audits and the dates th at any findings were reported to th e Study Director and Laboratory Management were as follows :
DATE REPORTED TO :
ACTIVITY : DATE CONDUCTED : STUDY DIRECTOR : MANAGEMENT :
Protocol February 19, 2007 February 19, 2007
February 20, 2007
Initial Trial : 454A-253A Test Substance P reparation
February 16, 2007
February 16, 2007
February 21, 2007
Definitive Test : 454A-253A Mat rix Fortification March 9, 2007 March 9, 2007 March 15, 2007
Observations March 9, 2007 March 9, 2007 March 15, 2007
Analytical Data and Draft Report
March 20 and 21, 2007 March 21, 2007 March 22, 2007
Biological Data and Draft Repo rt
March 19 - 21, 2007 March 21, 2007 March 26, 2007
Final Report March 29, 2007 March 29, 2007 March 29, 2007
All inspections were study-b as ed unless otherwise noted .
C` ~d A . 7
J es H. Coleman Date Quality Assurance Representative
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Wildlife International, Ltd.
-4REPORT APPROVAL
Project Number 454A-253 A
SPONSOR: 3M Corporation TITLE : PFOS : A 96-Hour Static-Renewal Acute Toxicity Test with Hyalella azteca WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER: 454A-253 A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER : E07-0082
STUDY DIRECTOR:
I pt,l,l,l ~ I t1E~~~t~~~f
3 9 / 9_0C?
ui Minderhout, Ph .D . Date
Senior Biologist
PRINCIPAL INVESTIGATOR:
/On A . MacGregor, B . . /Scientist
Date
~~
WILDLIFE INTERNATIONAL . LTD . MANAGEMENT :
Hen rueger, Ph .D . Date Director of Aquatic Toxicology/Terrestrial Plants and Insects
Aim'( 4~
Willard B . Nixon, Ph .D . Director of Chemistry
Date
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WZldllfe International, Ltd.
-5TABLE OF CONTENT S
Project Number 454A-253A
Title Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Good Laboratory Practice Compliance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Quality Assurance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Repo rt Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Test Substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Test Organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Dilution Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Test Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Preparation of Test Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Analytical Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Analytical Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 1 Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3
Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Measurement of Test Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Observations an d Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 4
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6
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Project Number 454A-253A
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TABLES AND FIGURE S Table 1 . Measured Concentrations of PFOS in Freshwater Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 2 . Temperature, Dissolved Oxygen and pH of Water in the Test Chambers . . . . . . . . . . . . . . . . . . . . . . . . 18 Table 3 . Specific Conductance, Hardness and Alkalinity Measured in Dilution Water
at Test Initiation and in Composite of Negative Control at Test Termination . . . . . . . . . . . . . . . . . . 19 Table 4 . Cumulative Mo rtality and Clinical Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Table 5 . LC50 Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Figure 1 . Concentration-Response Curv e (96-Hour Mortality Data) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
APPENDICE S
Appendix 1 . Specific Conductance, Hardness, Alkalinity and pH of Well Wate r Measured During the 4-Week Period Immediately Preceding the Test . . . . . . . . . . . . . . . . . . 26
Appendix 2 . Analyses of Pesticides, Organics and Metals in Wildlife Inte rn ational, Ltd . Well Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Appendix 3 . The Analysis of PFOS in Freshwater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3 .1 Analytical Method Flowchart for the Processing of PFOS in Freshwater . . . . . . . . . . . . . . 30 3 .2 Typical HPLC/MS/MS Operational Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3 .3 Analytical Stocks Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3 .4 Example Calculations for a Representative Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3 .5 Quali ty Control Samples of PFOS in Freshwater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3 .6 Representative Calibration Curve for PFOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3 .7 Representative Chromatogram of a Low-level PFOS Calibration Standard . . . . . . . . . . . 37 3 .8 Representative Chromatogram of a High-level PFOS Calibration Standard . . . . . . . . . . 38 3 .9 Representative Chromatogram of a Matrix Blank Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3 .10 Representative Chromatogram of a Matrix Fort ification Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3 .11 Representative Chromatogram of a Test Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1
Appendix 4 . Changes to Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Appendix 5. Personnel Involved in the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
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W lldIlfe International, Ltd.
Project Number 454A-253A
-7SUMMARY
SPONSOR: 3M Corporatio n TITLE : PFOS : A 96-Hour Static-Renewal Acute Toxicity Test with Hyalella azteca WILDLIFE INTERNATIONAL, LTD . PROJECT NUMBER : 454A-253 A 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER : E07-0082
TEST DATES : LENGTH OF EXPOSURE :
Experimental Start : March 5, 2007 Biological Termination : March 9, 2007 Experimental Termination : March 9, 2007
96 Hours
TEST ORGANISMS : SOURCE OF TEST ORGANISMS :
AGE OF TEST ORGANISMS :
Amphipod (Hyalella azteca)
Environmental Consulting and Testing Superior, Wisconsin
12 days old at test initiation
TEST CONCENTRATIONS :
Nominal Mean Measured Negative Control < LO Q
13 mg a.i ./L 13 mg a .i./L 25 mg a.i ./L 25 mg a .i./L 50 mg a .i ./L 51 mg a .i ./L 100 mg a .i ./L 102 mg a.i ./L 200 mg a .i ./L 199 mg a.i ./L
RESULTS : Based on mean measured concentrations :
96-Hour LC50 : 95% Confidence Inte rv al : No-Mo rtality Concentration : No-Obse rv ed-Effect Concentration :
15 mg a .i ./L 4 .5 - 24 mg a .i ./L
<13 mg a .i./L <13 mg a .i ./L
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Project Number 454A-253A
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INTRODUCTIO N 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 was conducted from February 19 to 22, 2007 but was terminated due to unacceptable percent survival in the negative control possibly caused by stress of organisms during shipment . The in-life phase of the definitive toxicity test was conducted from March 5 to 9, 2007 . Raw data generated by Wildlife International, Ltd . and a copy of the final report are filed under Project Number 454A-253A 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) on the amphipod, Hyalella azteca, during a 96-hour exposure period under static-renewal test conditions .
EXPERIMENTAL DESIGN Amphipods were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 96 hours under static-renewal conditions . Ten replicate test chambers were maintained in each treatment groups while twenty replicates were maintained in the control group . A single organism was placed in each test chamber for a total of 10 amphipods per treatment concentration and twenty amphipods per negative control . Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of exploratory range finding toxicity data . Nominal test concentrations selected were 13, 25, 50, 100 and 200 mg PFOS active ingredient (a.i .)/L . Test solutions were renewed at approximately 48 hours . Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, prior to renewal at 48 hours, and at the end of the test .
Amphipods were impartially assigned to test chambers at test initiation . Observations of mortality and other signs of toxicity were made approximately 4 .5, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LC50 values at 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 observation data .
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MATERIALS AND METHOD S The study was conducted according to the procedures outlined in the protocol, "PFOS : A 96Hour Static-Renewal Acute Toxicity Test with Hyalella azteca" . The protocol was based on procedures outlined in the U .S . Environmental Protection Agency Report number 600/R-99/064 Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates (1) and ASTM Standard E729-96 Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians (2) .
Test Substance The test substance used to prepare the test solutions, analytical calibration standards and the
analytical matrix fortification samples for the study was received from 3M on October 29, 1998 . It was assigned Wildlife International, Ltd . identification number 4675A upon receipt and was stored under ambient conditions . The test substance, a white powder, was identified as : FC-95 ; Lot number 217 . The test substance contained 86 .9% active ingredient and had an expiration date of August 31, 2016 .
Test Organis m The amphipod, Hyalella azteca, was selected as the test species for this study . Amphipods
are representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use and ease of culturing in the laboratory . Amphipods used in the test were obtained from Environmental Consulting and Testing (ECT), Superior, Wisconsin . Amphipods were hatched on February 21, 2007 and were 12 days old at test initiation . Prior to the test, the organisms were held for 6 days in a container with pieces of gauze and overlying water from the same source and at approximately the same temperature as that used in the test . Amphipods were fed YCT (1 .8 g/L) and Tetramin flake food during the holding period and YCT on days 0 and 2 of the test .
During the 6 days immediately preceding the test, water temperatures in the holding container ranged from 21 .8 to 23 .4C, measured with a hand-held liquid-in-glass thermometer . The pH of the water ranged from 8 .1 to 8 .5, measured with a Fisher Scientific Accumet Model 915 pH meter . Dissolved oxygen ranged from 6 .4 to 8 .0 mg/L (>_75% of saturation), measured with a Yellow Springs Instruments Model 51 B dissolved oxygen meter .
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The organisms showed no signs of disease or stress during the holding period . At test initiation, amphipods were collected from the holding container and placed in one or more transfer containers, then indiscriminately transferred one at a time to each test chamber . All transfers were made below the water surface using wide-bore pipettes .
Dilution Wate r 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. The specific conductance, hardness, alkalinity and pH 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 m, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles . Prior to use, the water was filtered to 0 .45 m and passed through an ultraviolet (UV) sterilizer to remove fine particles and microorganisms . The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water are presented in Appendix 2 .
Test Apparatus Test chambers were 30-mL Nalgene plastic beakers filled with approximately 20 mL of
water. The depth of the test water in a representative chamber was 1 .9 cm . The cups had a piece of Nitex screen placed in each of the cup as substrate for the organisms . Test chambers were positioned in a temperature-controlled chamber to maintain a temperature of 23 f 1C . Test chambers were covered with plastic and were labeled with the project number, test concentration and replicate .
Preparation of Test Concentration s A stock solution was prepared at a nominal concentration of 200 mg a .i ./L, the highest
concentration tested, by mixing a calculated amount of PFOS into dilution water (Wildlife International, Ltd . UV sterilized well water) . The stock solution for day 0 was mixed by stirring overnight and was sonicated for approximately 5 to 10 minutes the following day . For the day 2 solution, the stock solution was mixed by stirring overnight and was sonicated for 5 minutes the next
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day . Both stock solutions appeared clear and colorless . Aliquots of the 200 mg a .i ./L stock solution were proportionally diluted with well water to prepare 300 mL of test solution at nominal concentrations of 13, 25, 50 and 100 mg a .i ./L . The solutions were mixed by inversion and all appeared clear and colorless . All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (86 .9%) . Test solutions were prepared for test initiation and renewal . All surviving amphipods were transferred from old to new solutions at approximately 48 hours . At test initiation and termination, all solutions appeared clear and colorless .
Analytical Samplin g At the beginning of the test and on day 2, samples were collected from the newly prepared
batches of test solution to determine concentrations of the test substance . Prior to renewal at approximately 48 hours and at test termination, samples of old test solutions were collected from each test chamber and pooled for analysis of test substance concentrations . All samples were collected at mid-depth, placed in plastic vials, and processed immediately for analysis .
Analytical Metho d The analytical method used for the analysis of PFOS in freshwater was developed at Wildlife
International, Ltd. The analytical method consisted of dilution of the samples 1 :1, v/v with acetonitrile, followed by secondary dilution using acetonitrile : HPLC-grade bottled water (50 :50, v/v), and analysis by direct injection high performance liquid chromatography with mass spectrometric (LC/MS/MS) detection .
Concentrations of PFOS in the samples were determined by LC/MS/MS using an Agilent Series 1100 Series High Performance Liquid Chromatograph interfaced with an Applied Biosystems / MDS Sciex API 3000 mass spectrometer (MS/MS) operated in negative ion multiple-reaction monitoring (MRM) detection mode . The mass spectrometer was equipped with a Turbolon Spray ion source . Chromatographic separations were achieved using an Agilent Zorbax RX-C8 column (150 mm x 2 .1 mm, 5m particle size) . A flow chart for the analysis of PFOS is provided in Appendix 3 .1 and typical instrumental parameters are summarized in Appendix 3 .2 .
Calibration standards of PFOS, ranging in concentration from 0 .0500 to 1 .00 g a .i ./mL, were prepared in acetonitrile : HPLC-grade bottled water solution (50 :50, v/v) using a stock solution of
4/l
p. 1 2
Wildlife International, Ltd.
Project Number 454A-253A
-12-
PFOS in methanol (Appendix 3 .3) . Quadratic (weighted 1/x) regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards using Analyst Version 1 .4 .1 software of the Applied Biosystems/MDS Sciex API 3000 mass spectrometer system . The concentration of PFOS in the samples was determined by substituting the peak area responses of the samples into the applicable regression equation . An example of the calculations for a representative sample is included in Appendix 3 .4 .
The method limit of quantitation (LOQ) for these analyses was set at 2 .00 mg a .i ./L, calculated as the product of the lowest calibration standard (0 .0500 g a.i ./mL) and the dilution factor of the matrix blank samples (40 .0) . Three matrix blank samples were analyzed to determine possible interferences . No interferences were observed at or above the LOQ during the sample analyses (Appendix 3 .5) .
Matrix fortification samples were prepared fresh on each sampling day and were analyzed concurrently with the samples . Samples of freshwater were fortified with a stock solution of the test substance in methanol at nominal PFOS concentrations of 5 .00, 50 .0 and 250 mg a .i ./L . The measured concentrations for the matrix fortification samples ranged from 98 .4 to 114% of nominal concentrations (Appendix 3 .5 )
A representative calibration curve is presented in Appendix 3 .6 . Representative chromatograms of low and high-level calibration standards are presented in Appendices 3 .7 and 3 .8, respectively. A representative chromatogram of a matrix blank sample is presented in Appendix 3 .9 and a representative chromatogram of a matrix fortification sample is presented in Appendix 3 .10 . A representative chromatogram of a test sample is presented in Appendix 3 .11 .
Environmental Conditions Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone 50) were
used for illumination of the cultures and test chambers . 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, measured using a SPER Scientific Model 840006C light meter, was 668 lux at the surface of the water of one representative test chamber .
6/
p. 1 3
Wildlife International, Ltd.
Project Number 454A-253A
- 13 -
The target test temperature during the study was 23 1C . Temperature was measured in two alternate replicates at the beginning of the test, prior to and after each renewal (old and new solutions) and at the end of the test (old solution) using a liquid-in-glass thermometer . Temperature was also monitored daily in a container of water adjacent to the test chambers in the environmental chambers using a continuous temperature recorder .
Dissolved oxygen and pH were measured in samples collected from batches of new test solutions or were measured in composite samples of old solutions. Old solutions from the replicates of each test group were combined and a composite sample collected for analysis . Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Model 525Aplus meter .
Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation and in a composite sample of negative control water at test termination . Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-ConductivityTemperature meter . Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (3) .
Observations Observations of mortality were made periodically in each treatment group . Lethality is
defined as the lack of visible movement in the amphipod . The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated . Observations were made approximately 4 .5, 24, 48, 72 and 96 hours after test initiation.
Statistical Analyse s The mortality data were analyzed using the computer program of C . E . Stephan (4) . The
program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation (5, 6 and 7) . In this study, the binomial probability was used to calculate the 24-hour LC50 value and the probit analysis was used to calculate the 48, 72 and 96-hour LC50 values . The no-mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data .
5~~.
p. 1 4
Wlldllfe International, Ltd.
Project Number 454A-253A
-14-
RESULTS AND DISCUSSIO N Measurement of Test Concentrations
Nominal concentrations selected for use in this study were 13, 25, 50, 100 and 200 mg a .i ./L . Results of analyses to measure concentrations of PFOS in the test solution samples collected during the test are presented in Table 1 . Samples collected at test initiation had measured concentrations that ranged from 100 to 105% of the nominal concentrations . Samples collected prior to renewal of the test solutions at 48 hours (old solutions), at renewal (new solutions) and at test termination had measured concentrations that ranged from 96 .4 to 103%, 95 to 100% and 100 to 102% of the nominal concentrations, respectively . When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 13, 25, 51, 102 and 199 mg a.i ./L, representing 100, 100, 102, 102 and 99 .5% of nominal concentrations, respectively . The results of the study were based on the mean measured concentrations .
Observations and Measurements Measurements of temperature, dissolved oxygen and pH of the water in each test chamber are
presented in Table 2 . Water temperatures were within the 23 1C range established for the test . Dissolved oxygen concentrations remained ?8 .1 mg/L (>_95% of saturation) throughout the test . Measurements of pH ranged from 8 .1 to 8 .6 . The measurements of hardness, alkalinity and specific conductance in the dilution water at test initiation were typical of Wildlife International, Ltd . well water (Table 3) .
Daily observations for mortality and signs of toxicity during the test are presented in Table 4 . Amphipods in the negative control group appeared normal throughout the test, with the exception of two lethargic amphipods that resulted in mortality at test termination . This was well within an acceptable level as indicated in the guideline . Percent mortality at test termination in the 13, 25 and 51 mg a .i ./L treatment groups was 40, 80, and 80%, respectively . There was 100% mortality in the 102 and 199 mg a .i/L PFOS treatment groups . The no-mortality concentration and the NOEC were both <13 mg a.i ./L . LC50 values at 24, 48, 72 and 96 hours were determined from the mortality data and are shown in Table 5 . A graph of the concentration-response curve is included in Figure 1 .
43
p. 1 5
Wildlife International, Ltd.
Project Number 454A-253A
-15-
CONCLUSIONS The amphipod, Hyalella azteca, was exposed for 96 hours under static-renewal conditions to five mean measured concentrations of PFOS ranging from 13 to 199 mg a .i ./L . The 96-hour LC50 value was 15 mg a.i ./L, with a 95% confidence inte rv al of 4 .5 to 24 mg a .i ./L . The slope of the concentration-response cu rv e was 2 .4 . The no-mo rtality concentration and the NOEC were both <13 mg a .i ./L .
p . 16
WZrdllfe International, Ltd.
-16REFERENCES
Project Number 454A-253 A
1 U .S . Environmental Protection Agency . 2000 . Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates . EPA 600/R-99/064 .
2 ASTM Standard E729-96 . 1996 . Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians . American Society for Testing and Materials .
3 APHA, AWWA, WPCF . 1985 . Standard Methods for the Examination of Water and Wastewater. 16th Edition, American Public Health Association . American Water Works Association. Water Pollution Control Federation, New York .
4 Stephan, C .E . 1978 . U .S . EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication .
5 Thompson, W .R. 1947 . Bacteriological Reviews . Vol . II, No . 2 . Pp . 115-145 .
6 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 .
7 Finney, D .J. 1971 . Statistical Methods in Biological Assay . Second edition . Griffin Press, London .
69
p. 1 7
Wildlife International, Ltd.
Project Number 454A-253A
-17Table 1 Measured Concentrations of PFOS in Freshwater Sample s
Measured Mean Mean
Nominal Test Sample Sampling Concentration Percent Measured Measured
Concentration Number Time PFOS of Concentration Percent o f
(mg a .i ./L) (454A-253A-) ( Hours) (mg a.i ./L)' Nominal
(mg a.i./L) Nominal
0 .00 1 0(new)
< LOQ2 -- < LOQ --
(Negative Control) 7 48(new) < LOQ --
13 48(old) < LOQ --
19 96(old) < LOQ --
13 2 0(new) 13 .4 103 13 100 8 48(new) 12 .3 95 . 0 14 48(old) 13 .5 103 20 96(old) 13 .0 100
25 3 0(new) 25 .1 100 25 100 9 48(new) 24 .3 97 . 0 15 48(old) 24 .9 99 .6 21 96(old) 25 .4 10 1
50 4 0(new) 52 .6 105 51 102 10 48(new) 49 .4 98 . 9 16 48(old) 50 .2 100 22 96(old) 50 .8 102
100
5 0(new) 102 102 11 48(new) 100 100 17 48(old) 103 103 23 96(old) 101 10 1
102
102
200
6 0(new) 204 12 48(new) --3
102 --3
18 48(old) 193 96 .4
24 96(old) 3 --3
199
99 .5
Results generated using Analyst version 1 .4.1 software . Manual calculations may vary . 2 The limit of quantitation (LOQ) was 2 .00 mg a.i ./L calculated as the product of the lowest calibration standard
( 0 .0500 g a .i ./mL) and the dilution factor of the mat rix blanks (40 .0) .
3 Study level not sampled due to 100% mo rtality . Value not used in statistical calculations .
p . 18
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WZ ldIZfe International, Ltd.
Project Number 454A-25 3
-19-
Table 3
Specific Conductance, Hardness and Alkalinity Measured in Dilution Water at Test Initiation and in Composite of Negative Control at Test Terminatio n
Paramet er Day 0 Day 4
Specific Conductance 295 320 (mhos/cm )
Hardness 132 136 (mg/L as CaCO3 )
Alkalinity 182 186 (mg/L as CaCO3)
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p. 24
Wildlife International, Ltd.
Project Number 454A-253A
-24Table 5 LC50 Values
Time
LC50
95% Confidence Interval Statistical
(mg a .i ./L) (mg a .i ./L) Metho d
24 Hours 161 >102 '
Binomial Probability
48 Hours 93 70 - 125 Probit Analysis
72 Hours 29 20 - 40 Probit Analysis
96 Hours 15 4 .5 - 24 Probit Analysi s
At a confidence level of 95% the binomial test shows that the LC50 is above 102 mg a .i ./L .
73
p. 25
Wildlife International, Ltd.
Project Number 454A-253A
-25Figure 1 Concentration-Response Curve (96-Hour Mo rt ality Data)
9-
8
E. 7 a
6 0
5
Slope = 2 .4
4-
3 10
100 1000 Concentration ( mg a.i ./L)
74
p . 26
Wildl fe International, Ltd.
Project Number 454A-253 A
-26-
Appendix 1
Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test
Parameter Mean Range
Specific Conductance 290 285 - 29 5 (mhos/cm) (N = 4 )
Hardness 136 132 - 140 (mg/L as CaCO3) (N = 4 )
Alkalinity 182 180 - 184 (mg/L as CaCO3) (N = 4 )
pH
8 .1 (N = 4 )
8 .1
~~
p. 27
Wildlife In ternational, Ltd.
Project Number 454A-253A
-27-
Append ix 2
Analyses of Pesticides, Org an ics an d Metals in Wildlife Inte rnational, Ltd . Well Water'
Pesticides and Org a nic s
Measured Concentration Measured Concentration Component (g/L) Component (g/L)
Aldrin < 0 .019 Heptachlor < 0 .0096 Alpha BHC < 0 .0096 Heptachlor Epoxide < 0 .0096 Alpha Chlordane < 0 .0096 Kepone < 0 .19 Beta BHC < 0 .038 Malathion < 1 .9 Bolstar < 1 .9 Merphos < 1 .9 Chlordane < 0 .48 Methoxychlor < 0 .096 Coumaphos < 2 .9 Methyl Parathion < 1 .9 Delta BHC < 0 .0096 Mevinphos < 1 .9 Demeton-O < 1 .9 Mirex < 0 .11 Demeton-S < 1 .9 Naled < 2 .9 Diazinon < 1 .9 o,p-DDD < 0 .019 Dichlorv os < 1 .9 o,p-DDE < 0 .019 Dieldrin < 0 .029 o,p-DDT < 0 .019 Disulfoton < 1 .9 p,p-DDD < 0 .019 Dursban (Chlorpyrifos) < 1 .9 p,p-DDE < 0 .019 Endosulfan I < 0 .0096 p,p-DDT < 0 .019 Endosulfan II < 0 .019 PCB-1016 < 0 .48 Endosulfan Sulfate < 0 .019 PCB-1221 < 0 .48 Endrin < 0 .019 PCB-1232 < 0 .48 Endrin Aldehyde < 0 .096 PCB-1242 < 0 .48 Endrin Ketone < 0 .019 PCB-1248 < 0 .48 EPN < 3 .8 PCB-1254 < 0 .48 Ethion < 1 .9 PCB-1260 < 0 .48 Ethoprop < 1 .9 Phorate < 1 .9 Ethyl Parathion < 1 .9 Ronne] < 1 .9 Famphur < 1 .9 Stirophos < 1 .9 Fensulfothion < 3 .8 Telodrin < 0 .0096 Fenthion < 1 .9 Tokuthion < 1 .9 Gamma BHC - Lindane < 0 .0096 Toxaphene < 0.96 Gamma Chlordane < 0 .096 T ri chloronate < 1 .9 Guthion (Azinphos-methyl) < 3 .8 T ri thion < 1 .9 HCB < 0.096
Analyses performed by Lancaster Laborato ri es on samples collected on December 15, 2005 .
76
p. 28
Wildlife International, Ltd.
Project Number 454A-253A
-28-
Appendix 2 (Continued )
Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd . Well Water'
Me ta ls Measured Concentration Measured Concentratio n Component (mg/L) Component (mg/L)
Aluminum < 0 .200 Magnesium 13 .3 Antimony < 0 .0200 Manganese < 0 .0050 Arsenic < 0 .0200 Mercury < 0 .00020 Barium < 0 .0050 Nickel < 0 .0100 Beryllium < 0 .0050 Nitrate Nitrogen < 0 .50 Bromide < 2 .5 Nitrite Nitrogen < 0 .50 Cadmium < 0 .0050 Potassium 7 .65 Calcium 33 .1 Selenium < 0 .0200 Chloride 2 .7 Silver < 0 .0050 Chromium < 0 .0150 Sodium 19 .1 Cobalt < 0 .0050 Sulfate < 5 .0 Copper < 0 .0100 Thallium < 0 .0200 Fluoride 0 .56 Vanadium < 0 .0050 Iron < 0 .200 Zinc < 0 .0200 Lead < 0 .020 0
Analyses p erforme d by Lancaster Laboratories on samples collected on December 15, 2005 .
77
p. 29
Wildlife International, Ltd.
Project Number 454A-253A
-29Appendix 3 The Analysis of PFOS in Freshwater
~g
p. 30
Wildlife International, Ltd.
Project Number 454A-253A
-30Appendix 3. 1 Analytical Method Flowchart for the Processing of PFOS in Freshwater
METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATE R
Prepare calibration standards in acetonitrile : HPLC-grade bottled water (50 :50,v/v) using volumetric flasks and gas-tight syringes, STORE REFRIGERATED .
Prepare matrix fortification samples in well water using volumetric flasks, volumetri c pipettes, 15-mL tubes and gas-tight syringes .
Dilute all samples initially 1 :1 with 100% acetonitrile using 15-mL culture tubes or equivalent, gas-tight syringes and/or class A volumetric pipettes . Mix well .
Volumetrically dilute solutions further, if necessary, with acetonitrile : HPLC-grade bottled water (50 :50,v/v) so that the final sample concentrations fall within the calibration standard range . Mix well. ~
Transfer aliquots of final sample dilutions and calibration standards to autosampler vials for analysis by LC/MS/MS .
79
p. 31
Wildlife International, Ltd.
Project Number 454A-253A
-31Appendix 3 .2 Typical HPLC/MS/MS Operational Parameters
INSTRUMENT : Agilent Series I 100 High Performance Liquid Chromatograph (HPLC) coupled with an Applied Biosystems/MDS Sciex API 3000 Mass Spectrometer (MS/MS) operated in the negative ion multiple-reaction monitoring (MRM) mode .
ION SOURCE : Turbolon Spray
ANALYTICAL COLUMN : Agilent Zorbax RX-C 8
(150 mm x 2 .1 mm, 5 m particle size)
STOP TIME :
5 .00 minute s
FLOW RATE :
0 .300 mL/minute
OVEN TEMPERATURE : 40C
MOBILE PHASE : 80% MeOH : 20% H20 containing 0 .1% formic acid INJECTION VOLUME : 10 .0 L
PFOS RETENTION TIME : Approximately 2 .8 minutes PFOS MONITORED MASS : 499 -* 99 amu
~~
p . 32
Wildlife International, Ltd.
Project Number 454A-253 A
-32Appendix 3 .3 Analytical Stocks and Standards Preparation
A stock solution of PFOS was prepared by weighing 1 .1507 g (corrected for purity) of the test substance on an analytical bal ance . The test substance was transferred to a 100-mL volumetric flask and brought to volume using methanol . This primary stock solution contained 10 .0 mg a .i ./mL of PFOS . Seconda ry stocks of PFOS in methanol (1 .00 and 0 .100 mg a .i ./n-iL) were prepared from the primary stock by volumetric dilution . The 10 .0 and 1 .00 mg a.i ./mL stock solutions were used to prepare concurrent matri x fo rt ification samples (QC) for this study . The 0 .100 mg a .i ./mL stock solution was used to prepare calibration standards . The calibration standards were prepared in acetonitrile : HPLC-grade bott led water (50 :50, v/v) . The following shows the dilution scheme for the set of calibration standards .
Stock Final Standard
Concentration Aliquot Volume Concentration
( mg a.i ./mL)
(ML)
mL
(.g a .i ./mL )
0 .100 0 .0500 100 0 .0500
0 .100 0 .150 100 0 .150
0 .100 0 .250 100 0 .250
0 .100 0 .500 100 0 .500
0 .100 1 .00 100 1 .00
p . 33
WZldllfe International, Ltd.
Project Number 454A-253 A
-33Appendix 3 .4
Example Calculations for a Representative Sampl e
The analytical result and percent recovery for sample number 454A-253A-2, an exposure
sample prepared at a nominal concentration of 13 mg a .i ./L, was calculated as follows using the
software algorithms of Analyst Version 1 .4 .1 of the Applied Biosystems/MDS Sciex API 3000 mass
spectrometer system . Regression was used to generate calibration equations for each analytical
sequence relating the measured peak areas of reference standard solution injections of PFOS with
their known concentrations . The curve was weighted 1/x with respect to concentration and expressed
as a quadratic function as follows :
y=ax2+bx+c
where :
y = instrumental peak area response of concentration x of PFOS in mg a.i ./L
a = quadratic coefficient
b = linear coefficient
c =constant coefficient (y_intercept )
Concentrations of PFOS in samples were determined by substituting peak area responses of the samples into the applicable rearranged regression equation as follows :
PFOS (mg a.i ./L) = Dilution Factor - Linear Coeffi cient +( Linear Coeffi ci en t)2 - [4 (Quadratic Coefficien t) (Y Interce pt - Peak Area) ~
2 (Quadratic Coefficien t)
where the Dilution Factor compensates for dilution of the water sample so that the peak response was bracketed by the standard calibration curve .
Data used for quantitation of PFOS in Sample Number 454A-253A-2 are summarized below :
Peak area = 146010 0 Constant Coefficient = -3901 .34 Linear Coefficient = 4406500 Quadratic Coefficient = -80517 .7 Dilution Factor (Vf,,a,/Vi,,;tial) : = 40 .0
~Z
p . 34
Wlldl fe International, Ltd.
Project Number 454A-253 A
-34Appendix 3 .4 (Continued) Example Calculations for a Representative Sampl e
- 4406500 + V(4406500)2 - [(4 (-80517 .7)) (-3901 .34 -1460100)] PFOS = 40 .0
2 (-80517 .7) PFOS = 40 .0 0 .33425 mg a .i ./L PFOS = 13 .4 mg a .i ./L The measured concentration was compared to the nominal concentrations as follows :
PFOS in sample (mg a.i ./L) Percent of nominal concentration = PFOS nominal concentration (mg a .i ./L) X 100
13 .4 mg/L 13 .0 mg/L X 100 = 103%
03
p. 35
Wildlife International, Ltd .
Project Number 454A-253A
-35Appendix 3 .5 Quality Control Samples of PFOS in Freshwater
Sample Sampling Number Time (454A-253A-) (Hours)
Concentration (mg a .i ./L )
Percent
Fortified
Measured 1,2 Recovery 1
MAB-1 0 0.0 MAB-2 48 0.0 MAB-3 96 0.0
< < <
LOQ LOQ LOQ
----
MAS-1 0 5 .00 5 .51 110 MAS-2 0 50 .0 51 .5 103 MAS-3 0 250 251 100
MAS-4 48 5 .00 5 .71 114 MAS-5 48 50 .0 52 .4 105 MAS-6 48 250 246 98 .4
MAS-7 96 5 .00 4 .98 99 .5 MAS-8 96 50 .0 49 .5 99 .0 MAS-9 96 250 246 98 . 5
X=103 S .D .=5 .62 C .V .=5 .46%
Results generated using Analyst version 1 .4 .1 software . Manual calculations may vary .
2 The limit of quantitation (LOQ) was 2 .00 mg a.i ./L calculated as the product of the lowest calibration standard (0 .0500 g a .i ./mL.) and the dilution factor of the matrix blanks (40 .0) .
W
p. 36
Wildlife International, Ltd.
Project Number 454A-253A
-36Appendix 3 . 6 Representative Calibration Curve for PFO S
T Wf507 Dard) (PFC6}"QerAic" Rag-("1 hr vagYfn9rv= A Q5er001X`z+4.41e 4 06x+3 ~ =Q ggRj
4.4e6 4.29 s 4.03s 38e6 3feB 34e6 3236 30a6 28e8 2696 24e6 2~W 20e6 1 .8e6 1 .6e6 1 .4e6 1 .236 1 .Q:6 80~a
4.M 206
QOi Q10 0.15 02) 025 0.3D 0.35 1140 0 45 OW 0 .55 Q OD 065 0 .70 0 75 080 Q85 090 Q95 1 .00 CbrcErtrzh a1 ig ailn t
Linear coefficient=4406500 ; constant coefficient=-3901 .34; quadratic coefficient= -80517 .7 ; r=0 .9999
8S
p. 37
Wlldl fe International, Ltd.
Project Number 454A-253A
-37Appendix 3 .7 Representative Chromatogram of a Low-level PFOS Calibration Standard
~,~, .., .. . .. .,w ~
Nominal concentration : 0 .0500 mg a .i ./L
]l
12 44
.
F~1D
p. 38
Wildlife International, Ltd.
Project Number 454A-253A
-38-
Appendix 3. 8 Representative Chromatogram of a High-level PFOS Calibration Standard
~.~.~~a .~ ~:
- -------- ----
I~~ > III z
, Nominal concentration : 1 .00 mg a.i ./L
p . 39
W ildllfe International, Ltd.
Project Number 454A-253 A
-39Appendix 3 . 9 Representative Chromatogram of a Matrix Blank Sample
~ ... ~.:
II
w,YAYa rto ra A o 05
,
. 1 q 19A3 1 ]a }JB }S]8 v.~
, T , 20 }1
:
4~ 4 . e
Sample number 454A-253A-MAB-1 . Dilution factor = 40 .OX . The arrow indicates the retention time of PFOS .
VV
p. 40
Wildlife International, Ltd.
Project Number 454A-253A
..~ ~
-40Appendix 3 .1 0 Representative Chromatogram of a Matrix Fortification Sample
~~..,~ .
- , ze
s. Sample number : 454A-253A-MAS-1, nominal concentration 5 .00 mg/L . Dilution factor = 40 .OX .
Lr' 7
p. 41
Wildlife International, Ltd.
Project Number 454A-253A
-41Appendix 3 .1 1 Representative Chromatogram of a Test Sample
.~-
~~.ti
.., ..
zt :
Sample number : 454A-253A-2, Day 0, nominal concentration 13 mg a .i ./L . Dilution factor = 40 .OX .
qO
p. 42
Wildlife International, Ltd.
Project Number 454A-253A
-42Appendix 4 Changes to Protoco l
This study was conducted in accordance with the approved Protocol with the following changes :
1 . The test chambers had a piece of Nitex screen placed in each of the beakers as substrate .
2 . The temperature was measured in two alternate replicates, rather than in each replicate, at the beginning of the test, prior to and after each renewal (old and new solutions) and at the end of the test (old solution) using a liquid-in-glass thermometer .
3 . The temperature was monitored daily in a container of water adjacent to the test chambers in the environmental chambers using a continuous temperature recorder, rather than in the negative control chamber .
4 . Dissolved oxygen and pH were measured in samples collected from batches of new test solutions or measured in composite samples of old solutions . Old solutions from the replicates of each test group were combined and a composite sample collected for analysis .
5 . A tray of clean water was placed in the environmental chamber to maintain the moisture content of the air in the environmental chamber and thus decrease the rate of evaporative loss of water from the test solutions in the test chambers .
6 . Test organisms were obtained from Environmental Consulting and Testing, Superior, WI .
7 . The Environmental Laboratory Project Number assigned by the Sponsor for this study was E07082 .
8 . Twenty replicate test chambers were maintained in the negative control group while ten replicate test chambers were maintained in each treatment group . A single organism was placed in each test chamber for a total of twenty organisms per negative control group, and ten organisms per each treatment group .
9 . Dilution water was passed through a UV sterilizer prior to use . This had no adverse impact on the study results .
10 . The test solution level in each chamber was not examined and brought up to the 20 mL mark daily with reverse osmosis water . This had no adverse impact on the study results .
~~
p. 43
Wildlife International, Ltd.
Project Number 454A-253A
-43Appendix 5 Personnel Involved in the Stud y
The following key Wildlife International, Ltd . personnel were involved in the conduct or management of this study :
1 . Henry O . Krueger, Ph .D ., Director of Aquatic Toxicology/Terrestrial Plants and Insects 2 . Willard B . Nixon, Ph .D ., Director of Chemistry 3 . Tui Minderhout, Ph .D ., Senior Biologist 4 . Amy S . Blankinship, Laboratory Supervisor, Aquatics 5 . Jon A . MacGregor, Scientist
7~