Document Jvd38eyEbmvzvr7xBpMQBJozB
P-1
finz-s'ny .
PFOA: A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH Chironomus tentans
AMENDED FINAL REPORT
WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-250 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0081
ASTM Standard E729-96
AUTHORS: Tui Minderhout, Ph.D. Jon A. MacGregor, B.S. Henry O. Krueger, Ph.D.
STUDY INITIATION DATE: February 7, 2007 STUDY COMPLETION DATE: March 2, 2007 AMENDED FINAL REPORT DATE: March 8, 2007
SUBMITTED TO: 3M Corporation
Environmental Laboratory 3M Center
Building 0260-05-N-17 Maplewood, MN 55144
Wildlife International, Ltd.
8598 Commerce Drive Easton, Maryland 21601 USA
1-410-822-8600
Page 1 of 45
CONTAINS NO CB
AMENDED
P-2
Wildlife International, Ltd.
Project Number 454A-250
2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR 3M Corporation TITLE PFOA A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentans WILDLIFE INTERNATIONAL, LTD PROJECT NUMBER 454A-250 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER E07-0081 STUDY COMPLETION March 2, 2007 AMENDED REPORT DATE March 8. 2007
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 exceptions
Periodic analyses of well water for potential contaminants were performed using a certified laboratory and standard U S EPA analytical methods
The test substance was characterized in compliance with Good Laboratory Practice Standards prior to the start of the test However, test solutions were prepared and used, and the test was earned out, after the test substance expiration date of October 31, 2006 while the Certificate of Analysis was being updated
STUDY DIRECTOR
'jmi fvyAkrf
Tui Minderhout, Ph D Senior Biologist
Date
m
AMENDED
P-3
Wildlife International, Ltd.
Project Number 454A-250
-3QUALITY ASSURANCE STATEMENT
This study was examined for compliance with Good Laboratory Practice Standards as published by the U S. Environmental Protection Agency (40 CFR Parts 160 and 792, 17 August 1989). 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: Protocol Test Substance Preparation Matrix Fortification Water Chemistry Analytical Data and Draft Report Biological Data and Draft Report Final Report Amended Final Report
DATE REPORTED TO. DATE CONDUCTED. STUDY DIRECTOR: MANAGEMENT.
February 12, 2007
February 12, 2007
February 15, 2007
February' 12, 2007
February 12, 2007
February 21, 2007
February 16, 2007
February 16, 2007
February 21, 2007
February 16, 2007
February 16, 2007
February 21, 2007
February 22 -2 3 , 2007 February 23. 2007
March 1, 2007
February' 22 - 23, 2007 February 23. 2007
February 27, 2007
March 2. 2007
March 2, 2007
March 2, 2007
March 8, 2007
March 8, 2007
March 8, 2007
All inspections were study-based unless otherwise noted
Jaofcs H Coleman Quality Assurance Representative
Date
AMENDED
P-4
Wildlife International, Ltd.
project Number 454A -250
-4REPORT APPROVAL
SPONSOR: 3M Corporation TITLE: PFOA: A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentons WILDLIFE INTERNATIONAL, LTD PROJECT NUMBER: 454A-250 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0081
STUDY DIRECTOR:
> ( k n ILui Minderhout, Ph D. Senior Biologist
1> l*lw 0 Date
j/g /< 7 7 Date
WILDLIFE INTERNATIONAL. LTD MANAGEMENT:
-1-1--Henry 0. Krueger. Ph D
Date
Director of Aquatic Toxicolog> /Terrestrial Plants and Insects
Willard B. Nixon. Pfi.D Director of Chemistry
Date
AMENDED
P-5
Wildlife International, Ltd.
Project Number 454A-250
-5TABLE OF CONTENTS
Title Page...................................................................................................................................................... 1
Good Laboratory Practice Compliance Statement.................................................................................... 2
Quality Assurance Statement..................................................................................................................... 3
Report Approval.......................................................................................................................................... 4
Table of Contents........................................................................................................................................ 5
Summary......................................................................................................................................................7
Introduction..................................................................................................................................................8
O b je c tiv e ...................................................................................................................................................... 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........................................................................................................................11 Environmental Conditions........................................................................................................... 12 Observations................................................................................................................................. 13 Statistical Analyses.......................................................................................................................13
Results and Discussion.............................................................................................................................. 14 Measurement of Test Concentrations..........................................................................................14 Observations and Measurements................................................................................................. 14
C onclusions................................................................................................................................................ 15
References.................................................................................................................................................. 16
P-6
Wildlife International, Ltd.
Project Number 454A-250
6- TABLE OF CONTENTS (Continued)
TABLES Table 1. Measured Concentrations of PFOA 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 Termination.........................................................................................19 Table 4. Cumulative Mortality and Clinical Observations................................................................ 20 Table 5. LC50 Values...........................................................................................................................23
APPENDICES
Appendix 1. Certificate of Analysis.................................................................................................24
Appendix 2. Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test..................27
Appendix 3. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water................................................................................................................. 28
Appendix 4. The Analysis of PFOA in Freshwater.........................................................................30 4.1 Analytical Method Flowchart for the Processing of PFOA in Freshwater..............31 4.2 Typical HPLC/MS/MS Operational Parameters....................................................... 32 4.3 Analytical Stocks and Standards Preparation............................................................ 33 4.4 Example Calculations for a Representative Sample................................................. 34 4.5 Quality Control Samples of PFOA in Freshwater..................................................... 35 4.6 Representative Calibration Curve for PFOA............................................................ 36 4.7 Representative Chromatogram of a Low-level PFOA Calibration Standard.......... 38 4.8 Representative Chromatogram of a High-level PFOA Calibration Standard......... 39 4.9 Representative Chromatogram of a Matrix Blank Sample...................................... 40 4.10 Representative Chromatogram of a Matrix Fortification Sample............................41 4.11 Representative Chromatogram of a Test Sample.......................................................42
Appendix 5. Changes to Protocol.....................................................................................................43
Appendix 6. Personnel Involved in the Study.................................................................................44
Appendix 7. Report Amendment..................................................................................................... 45
AMENDED
p.7
Wildlife International, Ltd.
Project Number 454A-250
-7SUMMARY
SPONSOR: 3M Corporation TITLE: PFOA: A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentons WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-250 3M ENVIRONMENTAL LABORATORY PROJECT NUMBER: E07-0081
TEST DATES: LENGTH OF EXPOSURE:
Experimental Start: Biological Termination: Experimental Termination:
96 Hours
February 12, 2007 February 16, 2007 February 16, 2007
TEST ORGANISMS:
Freshwater Midge (Chironomus tentans)
SOURCE OF TEST ORGANISMS: Environmental Consulting and Testing Superior, WI 54880
AGE OF TEST ORGANISMS:
2ndto 3rd instar larvae (approximately 10-day old) at test start
TEST CONCENTRATIONS:
Nominal Negative Control
63 mg a.i./L 125 mg a.i./L 250 mg a.i./L 500 mg a.i./L 1000 mg a.i./L
Mean Measured < LOQ
74 mg a.i./L 153 mg a.i./L 277 mg a.i./L 579 mg a.i./L 1090 mg a.i./L
RESULTS: Based on mean measured concentrations:
96-Hour LC50: No-Mortality Concentration: No-Observed-Effect Concentration:
>1090 mg a.i./L 277 mg a.i./L 277 mg a.i./L
m
p.8
Wildlife International, Ltd.
Project Number 454A-250
8- -
INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the definitive toxicity test was conducted from February 12 to 16, 2007. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-250 in archives located on the Wildlife International, Ltd. site.
OBJECTIVE The objective of this study was to determine the acute effects of perfluorooctanoate, ammonium salt (PFOA) on the freshwater midge, Chironomus tentans, during a 96-hour exposure period under static-renewal test conditions.
EXPERIMENTAL DESIGN Midge larvae 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 and control group, with a single organism in each test chamber for a total of 10 midges per concentration. 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 63, 125, 250, 500 and 1000 mg 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 and after renewal at 48 hours, and at the end of the test.
Midge larvae were impartially assigned to test chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 2.5, 25, 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/immobility concentration and the no observed-effect concentration (NOEC) were determined by visual interpretation of the mortality and observation data.
P-9
Wildlife International, Ltd.
Project Number 454A-250
-9-
MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOA: A 96-Hour Static-Renewal Acute Toxicity Test with Chironomus tentans". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Report number 600/R-99/064 Methods fo r Measuring the Toxicity and Bioaccumulation o f Sediment-Associated Contaminants with Freshwater Invertebrates (1) and ASTM Standard E729-96 Standard Guide fo r 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 Decmber 27, 2006. It was assigned Wildlife International, Ltd. identification number 7864 upon receipt and was stored under frozen conditions. The test substance, a solid, was identified as: FC-143; PFOA; Lot number 332; CAS number 3825-26-1. The test substance contained 95% active ingredient and had an expiration date of February 27, 2017 (Appendix 1).
Test Organism The midge, Chironomus tentans, was selected as the test species for this study. Midges 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. Midge larvae used in the test were second- to third-instar larvae (approximately 10 days old) at test initiation. Midge larvae were obtained from Environmental Consulting and Testing (ECT), Superior, Wisconsin and were 7 days old when received. During the 3-day period prior to the test, the organisms were held in an aquarium with sand and overlying water from the same source and at approximately the same temperature as that used in the test. Midges were fed a 56 g/L suspension of flake food during the holding period and a 4 g/L suspension of Tetramin flake food on days 0 and 2 of the test.
During the 3-day acclimation period immediately preceding the test, water temperatures in the holding aquarium ranged from 22.0 to 22.8C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.1 to 8.4, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 7.2 to 8.6 mg/L (>85% of saturation), measured with a Yellow Springs Instruments Model 5 IB dissolved oxygen meter.
AMENDED
Wildlife International, Ltd.
Project Number 454A-250
- 10-
The organisms showed no signs of disease or stress during the holding period. At test initiation, midge larvae were collected from the holding aquarium and indiscriminately transferred one at a time to each test chamber. All transfers were made below the water surface using wide-bore pipettes.
Dilution Water The water used for holding 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 2.
The well water was passed through a sand filter to remove particles greater than approximately 25 pm, 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 pm 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 3.
Test Apparatus Test chambers were 30-mL Nalgene plastic beakers filled with approximately 20 mL of
water. The cups had approximately one-eighth of a teaspoon of sand on the bottom of the cup to provide a monolayer of burrowing substrate for the midges. Test chambers were positioned in a temperature-controlled chamber to maintain a temperature of 23 1C. Test chambers were covered with plastic wrap and were labeled with the project number, test concentration and replicate.
Preparation of Test Concentrations A stock solution was prepared at a nominal concentration of 1000 mg a.i./L, the highest
concentration tested, by mixing a calculated amount of PFOA into dilution water. The stock solution was sonicated for five minutes and mixed by inversion, and appeared clear and colorless. Aliquots of the 1000 mg a.i./L stock solution were proportionally diluted with well water to prepare 300 mL of test solutions at nominal concentrations of 63, 125, 250 and 500 mg a.i./L. The solutions were mixed by inversion. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (95%). Test solutions were prepared on day 0 and day 2. All surviving
52
Wildlife International, Ltd.
Project Number 454A-250
- 11 -
midge larvae were transferred from old to new solutions at approximately 48 hours. At test initiation and termination, all solutions appeared clear and colorless in the test chambers.
Analytical Sampling 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 test solution were collected from each test chamber and pooled by treatment group for analysis of test substance concentrations. All samples were collected at mid-depth, placed in plastic vials, and processed immediately for analysis.
Analytical Method The analytical method used for the analysis of PFOA 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 PFOA in the samples were determined by LC/MS/MS using an Agilent 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 Turboion Spray ion source. Chromatographic separations were achieved using a Zorbax RX-C8column (150 mm x 2.1 mm, 5pm particle size). A flow chart for the analysis of PFOA is provided in Appendix 4.1 and typical instrumental parameters are summarized in Appendix 4.2.
Calibration standards of PFOA, ranging in concentration from 0.100 to 1.00 pg a.i./mL, were prepared in acetonitrile: HPLC-grade bottled water solution (50:50, v/v) using a stock solution of PFOA in acetonitile (Appendix 4.3). 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 PFOA in the samples was determined by substituting the peak area
Wildlife International, Ltd.
Project Number 454A-250
- 12 -
responses of the samples into the applicable regression equation. An example of the calculations for a representative sample is included in Appendix 4.4.
The method limit of quantitation (LOQ) for these analyses was set at 10.0 mg a.i./L, calculated as the product of the lowest calibration standard (0.100 pg a.i./mL) and the dilution factor of the matrix blank samples (100). Three matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix 4.5).
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 PFOA concentrations of 25.0, 250 and 1200 mg a.i./L. The measured concentrations for the matrix fortification samples ranged from 97.0 to 107% of nominal concentrations (Appendix 4.5)
A representative calibration curve is presented in Appendix 4.6. Representative chromatograms of low and high-level calibration standards are presented in Appendices 4.7 and 4.8, respectively. A representative chromatogram of a matrix blank sample is presented in Appendix 4.9 and a representative chromatogram of a matrix fortification sample is presented in Appendix 4.10. A representative chromatogram of a test sample is presented in Appendix 4.11.
Environmental Conditions Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone 50) were
used for illumination of the holding 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 776 lux at the surface of the water of one representative test chamber.
The target test temperature during the study was 23 1C. Temperature was measured in two alternate replicates of each treatment and control group at the beginning of the test, prior to and after the renewal (old and new solutions) and at the end of the test (old solution) using a liquid-in-glass
Wildlife International, Ltd.
Project Number 454A-250
- 13 -
thermometer. Temperature was also monitored daily in a container of water adjacent to the test chambers in the environmental chamber using a continuous temperature recorder.
Dissolved oxygen and pH were measured in samples collected from the batches of new test solution at 0 and 48 hours or were measured in composite samples of old solutions at 48 and 96 hours. 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 termination. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods fo r the Examination o f Water and Wastewater (3).
Observations Observations of mortality were made periodically in each treatment group. Lethality was
defined as the lack of visible movement in the midge. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 2.5, 25, 48, 72 and 96 hours after test initiation.
Statistical Analyses Less than 50% mortality in any of the PFOA treatment groups was observed during the test,
which precluded the statistical calculation of LC50 values. Therefore, the 24-, 48-, 72- and 96-hour LC50 values were estimated to be greater than the highest concentration tested. The nomortality/immobility concentration and the no-observed-effect concentration (NOEC) were determined by visual interpretation of the mortality, immobility and observation data.
Wildlife International, Ltd.
Project Number 454A-250
- 14-
RESULTS AND DISCUSSION Measurement of Test Concentrations
Nominal concentrations selected for use in this study were 63, 125, 250, 500 and 1000 mg a.i./L. Results of analyses to measure concentrations of PFOA 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 102 to 106% of the nominal concentrations. Samples collected prior to and after renewal of the test solutions at 48 hours had measured concentrations that ranged from 120 to 141% and 102 to 104%, respectively, of the nominal concentrations. Samples collected at test termination had measured concentrations that ranged from 107 to 160% of the nominal concentrations.
The concentration of the solutions collected after 48 and 96 hours were slightly higher than those of newly prepared solutions due in part to the evaporative loss of water from the tests solutions even though the test chambers were covered with plastic. The high surface area in comparison to the small volume of the solution and the position of the fan that circulated the air in the environmental chamber might have contributed to the evaporation rate. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 74, 153, 277, 579 and 1090 mg a.i./L, representing 118, 123, 111, 116 and 109% of nominal concentrations, respectively. The mean measured concentrations indicated no reduction in test concentrations in the test system during the study. 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 the test chambers are
presented in Table 2. Water temperatures were within the 23 1C range established for the test. Dissolved oxygen concentrations remained >7.6 mg/L (>89% of saturation) throughout the test. Measurements of pH ranged from 7.9 to 8.5. The measurements of hardness, alkalinity and specific conductance in the dilution water at test initiation and termination 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. A 10% mortality rate was observed in both the negative control group and in the 74 mg a.i./L
Wildlife International, Ltd.
Project Number 454A-250
- 15 -
treatment group after 72 and 96 hours of exposure, respectively. Since the mortality in the 74 mg a.i./L treatment group was comparable to the negative control, and was not dose-responsive, it was not considered to be treatment-related. No mortalities or signs of toxicity were observed among midges in the 153 and 277 mg a.i./L treatment groups during the test. Percent mortality at test termination in the 579 and 1090 mg a.i./L treatment groups was 30 and 40%, respectively. All surviving midges in the PFOA treatment groups appeared normal throughout the test. The no mortality concentration and the NOEC were both 277 mg a.i./L. LC50 values at 24, 48, 72 and 96 hours were estimated to be >1090 mg a.i./L, the highest concentration tested (Table 5).
CONCLUSIONS The midge, Chironomus tentans, was exposed for 96 hours under static-renewal conditions to five mean measured concentrations of PFOA ranging from 74 to 1090 mg a.i./L. The 96-hour LC50 value was >1090 mg a.i./L, the highest concentration tested. Based on the mortality seen in the 579 and 1090 mg a.i./L treatment groups, the 96-hour no-mortality concentration and the NOEC were both 277 mg a.i./L.
Wildlife International, Ltd.
Project Number 454A-250
- 16REFERENCES
1 U.S. Environmental Protection Agency. 2000. Methods fo r Measuring the Toxicity and Bioaccumulation o f Sediment-Associated Contaminants with Freshwater Invertebrates. EPA 600/R-99/064.
2 ASTM Standard E729-96. 1996. Standard Guide fo r 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 fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York.
Wildlife International, Ltd.
Project Number 454A-250
- 17 -
Table 1 Measured Concentrations of PFOA in Freshwater Samples
Nominal Test Concentration
(mg a.i./L)
Sample Number (454A-250-)
Sampling Time
(Hours)
Measured Concentration
PFOA (mg a.i./L )1
Percent of
N om inal1
Mean Measured Concentration (mg a.i./L)
Mean Measured Percent o f Nominal
0.00
1
0(new)
< LOQ2
< LOQ
(Negative Control)
7
48(new)
< LOQ
-
13
48(old)
< LOQ
-
19
96(old)
< LOQ
--
63
2
0(new)
65.4
104
74
8
4 8(new)
65.1
103
14
48(old)
75.5
120
20
96(old)
91.2
145
125
3
0(new)
130
104
153
9
48(new)
129
103
15
48(old)
154
123
21
96(old)
200
160
250
4
0(new)
259
104 277
10
4 8(new)
259
104
16
48(old)
302
121
22
96(old)
289
116
500
5
0(new)
532
106 579
11
48(new)
511
102
17
48(old)
707
141
23
96(old)
566
113
1000
6
0(new)
1020
102
1090
12
48(new)
1020
102
18
48(old)
1260
126
24
96(old)
1070
107
117 122 111 116 109
Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit o f quantitation (LOQ) was 10.0 mg a.i./L calculated as the product o f the lowest calibration standard
(0.100 pg a.i./mL) and the dilution factor o f the matrix blanks (100).
/6f
Wildlife International, Ltd,
Project Number 454A-250
- 18Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers
Mean Measured
Concentration (mg a.i./L)
Negative Control
q j jQur
Temp.1 DO2 (C) (mg/L)
22.5 8.7
22.3 -
pH
8.2
-
48 Hours (Prior to Renewal)
Temp.1 DO2 (C) (mg/L) pH
22.4 7.9 8.5
22.0 - -
48 Hours ______ (At Renewal)
Temp.1 DO2 c o (mg/L)
pH
22.4 8.7 8.3
22.2 - -
Temp.1 DO2 CO (mg/L)
22.4 8.0
22.6 -
pH
8.5
-
74
22.7 8.7 8.2
22.0 8.0 8.5
22.6 8.6 8.3
22.1 8.0 8.5
23.0 - -
22.0 - -
22.2 - -
22.1 - -
153
22.5 8.7 8.1
21.9 8.0 8.4
22.4 8.7 8.3
22.1 8.0 8.5
22.5 - -
21.8 - -
22.1 - -
22.5 - -
277
22.4 8.7 8.1
21.7 8.1 8.4
22.4 8.7 8.2
22.8 8.0 8.4
22.5 - -
22.4 - -
22.0 - -
22.7 - -
579
22.5 8.7 8.0
22.1 8.1 8.3
22.3 8.7 8.2
22.1 7.6 8.4
22.5 - -
22.8 - -
22.5 - -
22.8 - -
1090
22.3 8.7 7.9
23.0 - -
22.2 8.0 8.1
22.9 - -
22.7 8.7 8.0
22.3 - -
22.6 7.7 8.3
22.8 - -
1 Manual temperature measurements replicate: day 0 (A, B); 48-hr old (C, D); 48-hr new (E, F); 96-hr old (G, H). Temperature measured continuously during the test ranged from 22 to 24C.
2 A dissolved oxygen concentration o f 5.1 mg/L represents 60% saturation at 23.0C in freshwater.
Wildlife International, Ltd.
Project Number 454A-250
- 19-
Table 3 Specific Conductance, Hardness and Alkalinity Measured in Dilution Water
at Test Initiation and Termination
Parameter
Specific Conductance (pmhos/cm)
Hardness (mg/L as CaC03)
Alkalinity (mg/L as CaC03)
Day 0 290 132 180
Day 4 310 136 182
/ /
Wildlife International, Ltd
Project Number 454A-250
p. 20
/G Z
-20-
Table 4 Cumulative Mortality and Clinical Observations
Mean Measured Concentration
(mg a.i./L)
Negative Control
Replicate1
A B C D E F G H I J
2.5 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
74 A 0 B0 C0 D0 E0 F0 G0 H0 I0 J0
1 A single midge was exposed in each replicate. 2 Cumulative number of dead midges. 3 Observations: AN = appear normal, D = pale. 4 T = transferred to new test solution.
AN AN AN AN AN AN AN AN AN AN
25 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
48 Hours (T)4
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0D 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
72 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN
1 ... 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 0 AN 0 AN 0 AN 0D 0 AN 0 AN 0 AN 0 AN
96 Hours
No. Dead2 Obs.3
Cumulative Percent Mortality
0 AN 0 AN 0 AN
1 ... 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
10
0 AN 0 AN 0 AN 0 AN 0 AN
1 ... 0 AN 0 AN 0 AN 0 AN
10
Wildlife International, Ltd.
project Number 454A -250
p. 21
/ J
-21 -
Table 4 (Continued)
Cumulative Mortality and Clinical Observations
Mean Measured Concentration
(mg a.i./L)
153
Replicate1
A B C D E F G H I J
2.5 Flours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
277 A B C D E F G H I J
0 0 0 0 0 0 0 0 0 0
1 A single midge was exposed in each replicate. 2 Cumulative number of dead midges. 3 Observations: AN = appear normal. 4 T = transferred to new test solution.
AN AN AN AN AN AN AN AN AN AN
25 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
48 Hours (T)4
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
72 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
96 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
Cumulative Percent Mortality
0
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0
Wildlife International, Ltd.
Project Number 454A-250
fi?/
-22-
Mean Measured Concentration (mg a.i./L)
579
Replicate>
A B C D E F
2.5 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
H0 I0 J0
1090
A B C D E F G H I J
0 0 0 0 0 0 0 0 0 0
1 A single midge was exposed in each replicate. 2 Cumulative number of dead midges. 3 Observations: AN = appear normal, D = pale. 4 T = transferred to new test solution.
AN AN AN
AN AN AN AN AN AN AN AN AN AN
Table 4 (Continued) Cumulative Mortality and Clinical Observations
25 Hours
No. Dead2 Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
48 Hours (T)4
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
72 Hours
No. Dead2
Obs.3
0 AN 0 AN 0 AN 0 AN 0 AN 0 AN
0 AN 0 AN 1--
0 AN 0 AN 0 AN 0 AN 1-- 0 AN 0 AN 0 AN 0 AN 1--
0 AN 0 AN 1--
0D 0 AN 0 AN 0 AN 1 ... 0 AN 0 AN 0 AN 0 AN 1--
0 AN 0 AN 1--
1-- 0 AN 0D 0 AN 1 ... 0 AN 0 AN 0 AN 0 AN 1--
96 Hours
No. Dead2 Obs.3
0 AN 0 AN 0 AN 1-- 0 AN 0 AN
Cumulative Percent Mortality
30
0 AN 0 AN 1--
1-- 0 AN 1 ... 0 AN 1 ... 0 AN 0 AN 0 AN 0 AN 1 ...
40
p. 22
Wildlife International, Ltd.
Project Number 454A-250
-23 Table 5 LC50 Values
Time
LC50 (mg a.i./L)
95% Confidence Interval (mg a.i./L)
Statistical Method
24 Hours
> 1090
1 N A 12
48 Hours
> 1090
1 NA2
72 Hours
> 1090
1 NA2
96 Hours
> 1090
1 NA2
1 95% confidence limits could not be calculated with the mortality/immobility data obtained. 2 NA = not applicable; <50% mortality precluded statistical calculation of an LC50 value.
Wildlife International, Ltd.
-24Appendix 1 Certificate of Analysis
Project Number 454A-250
Wildlife International, Ltd.
-25 -
Project Number 454A-250
INTERIM CERTIFICATE OF ANALYSIS
Revision 3
Centre Analytical Laboratories COA Reference U: 023-034
3M Product: PFOA, Primary Standard
Test Control Reference #: TCR-99030-30
Purity'
_________ Purity: 95.0%
Test Name
Specifications
Result 95.0%
Appearance
White, crystalline solid
Conforms
Identification NMR
Positive
Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese
Total %Impurity (NMR) Total %Impurity (LC/MS) Total %Impurity (GC/MS)
Residual Solvents (TGA)
Purity by DSC Inorganic Anions (IC)
1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate Organic Acids2(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis3: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine
1. Theoretical Value = 22.3% 2. Theoretical Value = 0.935% 3. Theoretical Value = 3.25% 4. Theoretical Value = 0% 5. Theoretical Value = 66.1%
1. 0.001 wt./wt.% 2. <0.001 wt./wt.% 3. 0.001 wtVwt.% 4. <0.001 wt./wt.% 5. <0.001 wt./wt.% 6. <0.001 wt./wt.% 7. <0.001 wt./wt.%
0.36 wtAvt.% 4.68wt7wt.% None Quantified.% None Detected
99.8%
1. <0.015 wt7wt.% 2. <0.005 wt./wt.% 3. <0.040 wtVwt.% 4. <0.009 wt7wt.% 5. <0.006 wt./wt.% 6. <0.006 wt./wt.% 7. <0.040 wt./wt.%
1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt7wt.% 4. <0.25 wtVwt.%
1. 18.9 wt./wt.% 2. 1.27 wtVwt.% 3. 3.76 wtVwt.% 4. 4.38 wtVwt.% 5. 62.1 wtJv/t.%
Ammonium Analysis3 Ion Selective Electrode
Theoretical Value = 4.18%
2.94 wtAvt. %
COA023034 REVISION 3.doc
Page 1 o f 2
/ 7
AMENDED
Wildlife International, Ltd.
-26-
Project Number 454A-250
INTERIM CERTIFICATE OF ANALYSIS
Revision 3 Centre Analytical Laboratories COA Reference #: 023-034
3M Product: PFOA, Primary Standard Test Control Reference #: TCR-99030-30
Date of Last Analysis: 2/27/07
Expiration Date: 2/27/17 Storage Conditions: <-10C Re-assessment Date: 2/27/17 'Purity = 100% - (total metal impurities, 0.002% + Total NMR impurities, 0.36 + Total LC/MS impurities, 4.68 %)
Total impurity from all tests = 5.042% Purity = 100% - 5.042% = 95.0%
2 TFA HFBA NFPA PFPA
Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid
3Theoretical value calculations based on the empirical formula, CsFisCh* *NH4W (MW=431.1)
This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160).
LC/MS Purity Profile:
Peak# 1 2 3
Total
Retention Time (min) 12.144 13.533 14.238
Mass(s) 269.0
331,319 369 -
Identity C6
F,VC7 PFOA
Area 205859 903329 22630600 23739788
% Area 0.87
3.81 -
4.68
Prepared By:
Date
Reviewed By: Kevin Lloyd Exygen Research
Date
COA023034 REVISION 3.doc
Page 2 o f 2
/C$>
AMENDED
Wildlife International, Ltd.
Project Number 454A-250
-27-
Appendix 2
Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test
Parameter
Specific Conductance (pmhos/cm)
Hardness (mg/L as CaC03)
Alkalinity (mg/L as CaC03)
pH
Mean
293 (N = 4)
136 (N = 4)
183 (N = 4)
8.0 (N = 4)
1 00
Range 285 - 300 132-140 182-184
Wildlife International, Ltd
Project Number 454A-250
-28-
Appendix 3 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1
Component
Aldrin Alpha BHC Alpha Chlordane Beta BHC Bolstar Chlordane Coumaphos Delta BHC Demeton-0 Demeton-S Diazinon Dichlorvos Dieldrin Disulfoton Dursban (Chlorpyrifos) Endosulfan I Endosulfan II Endosulfan Sulfate Endrin Endrin Aldehyde Endrin Ketone EPN Ethion Ethoprop Ethyl Parathion Famphur Fensulfothion Fenthion Gamma BHC - Lindane Gamma Chlordane Guthion (Azinphos-methyl) HCB
Pesticides and Organics
Measured Concentration (Pg/L)
Component
<0.019 < 0.0096 < 0.0096 <0.038 < 1.9 <0.48 <2.9 < 0.0096 < 1.9 < 1.9 < 1.9 < 1.9 < 0.029 < 1.9 < 1.9 < 0.0096 <0.019 <0.019 <0.019 < 0.096 <0.019 <3.8 < 1.9 < 1.9 < 1.9 < 1.9 <3.8 < 1.9 < 0.0096 < 0.096 <3.8 < 0.096
Heptachlor Heptachlor Epoxide Kepone Malathion Merphos Methoxychlor Methyl Parathion Mevinphos Mirex Naled o,p-DDD o,p-DDE o,p-DDT p,p-DDD p,p-DDE p,p-DDT PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 Phorate Ronnel Stirophos Telodrin Tokuthion Toxaphene Trichloronate Trithion
Measured Concentration (Pg/L)
< 0.0096 < 0.0096 <0.19 < 1.9 < 1.9 < 0.096 < 1.9 < 1.9 <0.11 <2.9 <0.019 <0.019 <0.019 <0.019 <0.019 <0.019 <0.48 <0.48 <0.48 <0.48 <0.48 <0.48 <0.48 < 1.9 < 1.9 < 1.9 < 0.0096 < 1.9 <0.96 < 1.9 < 1.9
1Analyses performed by Lancaster Laboratories on samples collected on December 15, 2005.
m
Wildlife International, Ltd
Project Number 454A-250
-29-
Appendix 3 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1
Metals
Measured Concentration
Measured Concentration
Component__________________ (mg/L)_________________ Component__________________(mg/L)
Aluminum Antimony Arsenic Barium Beryllium Bromide Cadmium Calcium Chloride Chromium Cobalt Copper Fluoride Iron Lead
< 0.200 < 0.0200 < 0.0200 < 0.0050 < 0.0050 <2.5 < 0.0050 33.1
2.7 <0.0150 < 0.0050 <0.0100
0.56 < 0.200 < 0.0200
Magnesium Manganese Mercury Nickel Nitrate Nitrogen Nitrite Nitrogen Potassium Selenium Silver Sodium Sulfate Thallium Vanadium Zinc
13.3 < 0.0050 < 0.00020 <0.0100 <0.50 <0.50
7.65 < 0.0200 <0.0050 19.1 <5.0 < 0.0200 < 0.0050 < 0.0200
1Analyses performed by Lancaster Laboratories on samples collected on December 15, 2005.
17/
Wildlife International, Ltd.
Project Number 454A-250
-30Appendix 4 The Analysis of PFOA in Freshwater
/72
Wildlife International, Ltd.
Project Number 454A-250
-31 Appendix 4.1 Analytical Method Flowchart for the Processing of PFOA in Freshwater
METHOD OUTLINE FOR THE ANALYSIS OF PFOA IN FRESHWATER
Prepare calibration standards in acetonitrile : HPLC-grade bottled water (50:50,v/v) using volumetric flasks and gas-tight syringes, STORE REFRIGERATED. I
Prepare matrix fortification samples in well water using volumetric flasks, volumetric pipettes, 15-mL culture tubes and gas-tight syringes. i
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.
m
Wildlife International, Ltd.
Project Number 454A-250
-32 Appendix 4.2 Typical HPLC/MS/MS Operational Parameters
INSTRUMENT:
ION SOURCE: ANALYTICAL COLUMN: STOP TIME: FLOW RATE: OVEN TEMPERATURE: MOBILE PHASE: INJECTION VOLUME: PFOA RETENTION TIME: PFOA MONITORED MASS:
Agilent Series 1100 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.
Turboion Spray
Zorbax RX-Q (150 mm x 2.1 mm, 5 pm particle size)
5.00 minutes
0.300 mL/minute
40C
80% CH3OH : 20% H20 containing 0.1% formic acid
5.00 pL
Approximately 2.6 minutes
413 --369 amu
{ 7?
Wildlife International, Ltd.
Project Number 454A-250
-33 Appendix 4.3 Analytical Stocks and Standards Preparation
A calibration standard stock solution of PFOA was prepared by weighing 0.1053 g (corrected for purity) of the test substance on an analytical balance. The test substance was transferred to a 100-mL volumetric flask and brought to volume using acetonitile. This primary stock solution contained 1.00 mg a.i./mL of PFOA. A secondary stock of PFOA in acetonitile (0.100 mg a.i./mL) was prepared from the primary stock by volumetric dilution. The 0.100 mg a.i./mL secondary stock solution was used to prepare calibration standards. The calibration standards were prepared in acetonitrile: HPLC-grade bottled water (50:50, v/v). The following shows the dilution scheme for the set of calibration standards.
Stock Concentration ime a.i./mL)
0.100 0.100 0.100 0.100 0.100
Aliquot (mL) 0.100 0.250 0.500 0.750 1.00
Final Volume
(mL) 100 100 100 100 100
Standard Concentration ( u r a.i./mL)
0.100 0.250 0.500 0.750 1.00
A fortification standard stock solution of PFOA was prepared by weighing 1.0526 g (corrected for purity) of the test substance on an analytical balance. 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 PFOA and was used to prepare concurrent matrix fortification (QC) samples.
t75
Wildlife International, Ltd.
Project Number 454A-250
-34Appendix 4.4 Example Calculations for a Representative Sample
The analytical result and percent recovery for sample number 454A-250-2, an exposure sample prepared at a nominal concentration of 63 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 PFOA 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 PFOA in mg a.i./F
a = quadratic coefficient b = linear coefficient c =constant coefficient (y intercept)
Concentrations of PFOA in samples were determined by substituting peak area responses of the samples into the applicable rearranged regression equation as follows:
PFOA (mg a.i./L) = Dilution Factor
- Linear Coefficient + -^/(Linear Coefficien 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 PFOA in Sample Number 454A-250-2 are summarized below:
Peak area = 15456000 Constant Coefficient = 32311.9 Linear Coefficient = 26602900 Quadratic Coefficient = -4615070 Dilution Factor (Vfinai/Vinitiai): = 100
/7
W ildlife International, Ltd.
Project Number 454A-250
-35 Appendix 4.4 (Continued) Example Calculations for a Representative Sample
- 26602900 + V (-26602900)2 - [(4 (-4615070)) (32311.9-15456000)] PFOA= 100
' 2 (-4615070) PFOA= 100 0.654 mg a.i./L
PFOA = 65.4 mg a.i./F
The measured concentration was compared to the nominal concentrations as follows:
Percent
of
nominal
concentration
='
_____PFOA in PFOA nominal
sample (mg a.i./L)_____ concentration (mg a.i./L)
X
100
65.4 mg/L 63.0 mg/L X 100
= 104%
/77
Wildlife International, Ltd.
Project Number 454A-250
-36Appendix 4.5 Quality Control Samples of PFOA in Freshwater
Sample Number (454A-250-)
MAB-1 MAB-2 MAB-3
MAS-1 MAS-2 MAS-3
MAS-4 MAS-5 MAS-6
MAS-7 MAS-8 MAS-9
Sampling Time (Hours)
0 48 96
0 0 0
48 48 48
96 96 96
Concentration (mg a.i./L)
Fortified
0.0 0.0 0.0
25.0 250 1200
25.0 250 1200
25.0 250 1200
M easu red 1,2
< LOQ < LOQ < LOQ
26.9 258 1190
25.3 254 1200
25.9 256 1160
Percent Recovery 1
_ -- ~
107 103 99.1
101 102 99.7
104 102 97.0
Mean =102 S.D. = 2.93 C.V. = 2.87%
1 Results generated using Analyst version 1.4.1 software. Manual calculations may vary. 2 The limit of quantitation (LOQ) was 10.0 mg a.i./L calculated as the product of the lowest
calibration standard (0.100 pg a.i./mL) and the dilution factor of the matrix blanks (100).
/7
Wildlife International, Ltd,
Project Number 454A-250
-37-
Appendix 4.6
Representative Calibration Curve for PFOA
021207_D0.rdb (PFOA): "Quadratic" Regression ("1 / x" weighting): y = -4 .62e+006 x*2 + 2.66e+007 x + 3.23e+004 (..
2.3e7 2.2e72.1e72.0e7 1.9e71.8e71.7e7 1.6e7 1.5e7 1.4e7 1.3e7 1.2e7 1.1e7 1.0e79.0e6 8.0e6 7.0e6 6.0e6 5.0e6 4.0e63.0e6
0.1 0 2 0.3 0.4 0 5 0.6 0.7 0.8 0.9 1.0 Concentration, ug a.i.AnL
Linear coefficient=26602900; constant coefficient=32311.9; quadratic coefficient=-4615070; r=0.9999
Wildlife International, Ltd.
Project Number 454A-250
-38Appendix 4.7 Representative Chromatogram of a Low-level PFOA Calibration Standard
IsaiTpteMme"W L u u r s a n p s lu ` l LiXJug Po* Nan 'FTOA- Mass*) ` 41300690am/ Ctxrment 'FFOM* AnrWann *
Sample Index:
25
Sarple Type
Standard
Concent ram n
0.100 ug a . i . /mL
2/12/2003Calcuta ted Co* 0.099436 ug
Acq. Date:
2-Befr
3-04-20 PM
Modified: Analyst Classic
Bitching Factor *'**se Threstiold:
1 Th re sh o ld :
xp. Va 1. R atio: T Window: Expected RT-
5 Rei s tiv e AT-
21e6-
20e6-
17e6-|
1e&j
5I ee-|
14a6-|
JeJ
1Qe6| 90sS80e570e5-j 60frj 50 540530*5-' 205-| 105-
1276
Nominal concentration: 0.100 pg a.i./mL
/SO
Wildlife International, Ltd
Project Number 454A-250
-39Appendix 4.8 Representative Chromatogram of a High-level PFOA Calibration Standard
Nominal concentration: 1.00 pg a.i./mL
/ 3f
p. 40
Wildlife International, Ltd
Project Number 454A-250
-40Appendix 4.9 Representative Chromatogram of a Matrix Blank Sample
IsaiVie u n v k * _ uo/- s a n p s iu i* i Pu*NarTM TFOA- MastfM) "41300690amJ Crmwlt 454A250-" Anncticn "
Sample Index-
31
Sample Type-
Blanx
Acq. D ate: ' Acq. Time:
2/12/200 * 3:39 43 PM
28e6n 2 7e6-
26*6-
25*62.4*6-
23*6-
22*6-
2 1*6-
20*6
'9*6"
13*6-
12*6-
1 1*6-
1.0*6-
9.0*68 0*5-
70*6-
60*6 5.0*5-
4 0*6' 3 0*520*610*6-
0O--
Q2S 061j a ~ 05 ' '
1D
276 319_______ _______ ______ 464 !5 2D 2 5 30 3.5 40 45 ______________________ Tmamn__________________________________________________
Sample number 454A-250-MAB-1. Dilution factor = 100X. The arrow indicates the retention time of PFOA.
p. 41
Wildlife International, Ltd.
Project Number 454A-250
-41 Appendix 4.10 Representative Chromatogram of a Matrix Fortification Sample
'1
15e6i
H
3
'"i
15 20
Sample number: 454A-250-MAS-2, nominal concentration 250 mg a.i./L. Dilution factor = 100X.
p. 42
W ildlife International, L f J ,
Project Number 454A-250
-42 Appendix 4.11 Representative Chromatogram of a Test Sample
ISample r a r e v k a _u ic San pa
P a * Name"PFOA-
'13 0G69 0 a m i
CUmwit '454ASS0-* Anrctafcn "
Sample Index
3d
Sanple Type.
Sample number: 454A-250-3, Day 0, nominal concentration 125 mg a.i./L. Dilution factor = 200X.
p. 43
Wildlife International, Ltd.
Project Number 454A-250
-43 Appendix 5 Changes to Protocol
This study was conducted in accordance with the approved Protocol with the following changes:
1. The protocol was amended to add that the cups will have approximately one-eighth of a teaspoon of sand on the bottom of the cup to provide a monolayer of burrowing substrate for the midges.
2. The protocol was amended to state that the temperature will be 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 protocol was amended to add that the temperature will also be 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. The protocol was amended to state that dissolved oxygen and pH will be measured in samples collected from batches of new test solutions or measured in composite samples of old solutions.
5. The protocol was amended to state that the test concentrations will be 63, 125, 250, 500 and 1000 mg a.i./L rather than 16, 31, 63, 125 and 250 mg a.i./L as requested by the Sponsor to accommodate a no-effect study.
6. The protocol was amended to add the Environmental Laboratory Project Number of E07-0081 as assigned by the Sponsor after the protocol was executed.
7. The protocol was amended to state that test organisms will be obtained from a commercial supplier to complete the protocol as required by GLP.
8. Midge larvae used in the test were obtained from a commercial supplier and held until used in the test rather than hatched from egg masses. This had no adverse impact on the study results.
9. Plastic wrap rather than plastic or glass lids was used to cover the test chambers. This had no adverse impact on the study results.
10. The 24-hour observations were made after 25.25 hours rather than within 24 1 hour of test initiation. This had no adverse impact on the study results.
Wildlife International, Ltd.
Project Number 454A-250
-44Appendix 6 Personnel Involved in the Study
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, M.S., Laboratory Supervisor, Aquatics 5. Jon A. MacGregor, B.S., Scientist 6. Susan T. Thomas, B.S., Biologist
Wildlife International, Ltd.
Project Number 454A-250
1. Original Report: Amendment:
Reason:
-45-
Appendix 7
Report Amendment
Pages 1, 2, 3,4 and 6 The pages were changed to include the amended report date, the amended report audit date, revised page numbers, and/or new signatures and dates due to the addition of the report amendment as Appendix 7. To reflect the issuing of an amended report.
2. Original Report: Amendment:
Reason:
Page 9 The expiration date was changed in the Test Substance section of the report. A new Certificate of Analysis was supplied after the issuance of the original final report.
3. Original Report: Amendment: Reason:
Pages 25 and 26 The Certificate of Analysis was replaced. A new Certificate of Analysis was supplied after the issuance of the original final report.
AMENDMENT SIGNATURES:
iU . Study Director
3 / tfUMO } Date
Laboratory Management REVIEWED BY:
Date
C O tf m
AMENDED
