Document GKbBd72oaGY9vpqwQZwa87eGm
PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723
U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft)
OPPTS Number 850.2200 FIFRA Subdivision E, Section 7 1-2
OECD Guideline 205
AUTHORS:
Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven
STUDY INITIATION: April 21, 1999
STUDY COMPLETION: April 26,2000
AMENDED REPORT DATE: March 23,2004
SUBMITTED TO
3M Corporation Environmental Laboratory
935 Bush Avenue St. Paul, Minnesota 55 144
Wildlife International, Ltd.
8598 Commerce Drive
Easton, Maryland 21601
(410) 822-8600
Page 1 of 69
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PROJECT NO.: 454-103
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation
TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103
3M LAB REQUEST NO.: U2723
STUDY COMPLETION: April 26,2000
AMENDED REPORT DATE: March 23,2004
This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (OCDElGD(92) 32, Environment Monograph No. 45, Paris, 1992); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exception:
At the time the test was conducted the test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance was recharacterized in accordance with GLP standards following study completion.
The stability of the test substance and reference standard under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards.
Analyses of liver and sera samples conducted by 3M Corporation were not performed in accordance with GLP standards and results of these analyses are being reported separately.
STUDY DIRECTOR:
Sean P. Gallagher Senior Biologist
SPONSOR'S REPRESENTATIVE
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QUALITY ASSURANCE STATEMENT
This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (OCDE/GD (92) 32, Environment Monograph No. 45, Paris, 1992); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates that any findings were reported to the Study Director and Laboratory Management were as follows:
ACTIVITY
DATE CONDUCTED
DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT
Test Substance Prep. & Analytical Sampling April 22, 1999
April 22, 1999
April 23, 1999
Matrix Fortification
April 22, 999
April 22, 999
April 23, 1999
Feed Consumption & Analytical Sampling April 27, 999
April 27, 999
May4, 1999
Analytical Data and Draft Report
July 7, 8,9, 1999
July 9, 1999
July 16, 1999
Biology Data and Draft Report
August 26,27,30,31, 1999 August 3 1, 1999
September 13, 1999
Final Report
April 17-18,2000
April 18,2000
April 19, 2000
Amended Final Report
March 19,2004
March 19,2004
March 19,2004
X . aua-yl
Susan L. Coleman
?,&my
Senior Quality Assurance Representative
DATE 3-aa- 04
AMENDED
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PROJECT NO.: 454-103
AMENDED REPORT APPROVAL
SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723
STUDY DIRECTOR:
Sean P. Gallagher
Date
Senior Biologist, Avian Toxicology
CHEMISTRY PRINCIPAL INVESTIGATOR:
R a p W.%an Hoven, Ph.D Scientist, Analytical Chemistry
REPORT APPROVED BY:
Linda R. Mitchell Manager of Ecotox Operations
Willard B. Nixon, Ph.6. Director of Chemistry
&@/
Date
AMENDED
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PROJECT .NO. 454-103
TABLE OF CONTENTS TITLE PAGE ........................................................................................................................
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................... QUALITY ASSURANCE STATEMENT ........................................................................... REPORT APPROVAL ......................................................................................................... TABLE OF CONTENTS...................................................................................................... Tables and Appendices ....................................................................................................... SUMMARY .......................................................................................................................... INTRODUCTION ................................................................................................................ OBJECTIVE ......................................................................................................................... MATERIALS AND METHODS..........................................................................................
Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 8 Page 8
Test Substance .................................................................................................................... Treatment Groups ............................................................................................................... Duration of the Test ............................................................................................................ Test Birds ............................................................................................................................ Animal Diet ........................................................................................................................ Diet Preparation .................................................................................................................. Diet Sampling ..................................................................................................................... Analytical Method .............................................................................................................. Housing and Environmental Conditions ............................................................................. Observations ....................................................................................................................... Animal Body WeightdFeed Consumption ......................................................................... Gross Necropsy...................................................................................................................
Statistical Analyses .............................................................................................................
Page 8 Page 9 Page 9 Page 9 Page 10 Page 10 Page 10 Page 11 Page 13 Page 14 Page 14 Page 14 Page 14
RESULTS ............................................................................................................................. Page 15
Diet Analysis ...................................................................................................................... Page 15 Mortalities and Clinical Observations ................................................................................ Page 15 Body Weight and Feed Consumption................................................................................. Page 17 Gross Necropsy................................................................................................................... Page 17
CONCLUSION..................................................................................................................... Page 18
REFERENCES ..................................................................................................................... Page 19
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TABLE OF CONTENTS .continued .
TABLES AND APPENDICES
TABLE 1: TABLE 2: TABLE 3: TABLE 4: TABLE 5:
Cumulative Mortality from a Northern Bobwhite Acute Dietary Toxicity
Study with PFOS .......................................................................................... Page 20
Mean Body Weight (g) from a Northern Bobwhite Acute Dietary Toxicity
Study with PFOS .......................................................................................... Page 21
Mean Feed Consumption (ghirdday) from a Northern Bobwhite
Acute Dietary Toxicity Study with PFOS .................................................... Page 23
Group Gross Pathological Observations from a Northern Bobwhite
Acute Dietary Toxicity Study with PFOS .................................................... Page 25
Cumulative Mortality (Estimated Cumulative Dose. mg/kg) from a
Northern Bobwhite Acute Dietary Toxicity Study with PFOS .................... Page 26
APPENDIX I: Certificate of Analysis ........................................................................... Page 27
APPENDIX 11: Diet Formulation.................................................................................... Page 28
APPENDIX 111: Analytical Methods and Results ............................................................ Page 29
APPENDIX IV: Diet Preparation ..................................................................................... Page 43
APPENDIX V:
Cumulative Mortality by Pen from a Northern Bobwhite
Acute Dietary Toxicity Study with PFOS ............................................. Page 44
APPENDIX VI: Individual Body Weights (g) from a Northern Bobwhite
Acute Dietary Toxicity Study with PFOS ............................................. Page 45
APPENDIX VII: Feed Consumption (ghirdday) by Pen from a
Northern Bobwhite Acute Dietary Toxicity
Study with PFOS ...................................................................................
Page 58
APPENDIX VIII: Changes to Protocol ............................................................................... Page 61
APPENDIX IX: Personnel Involved in the Study............................................................ Page 62
APPENDIX X: Revised Study Summary........................................................................ Page 63
APPENDIX XI: Report Amendment................................................................................ Page 68
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SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 STUDY: PFOS: A Dietary LC50 Study with the Northern Bobwhite
RESULTS':
The dietary LC50 value for northern bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a.i. to 289 ppm a.i. The slope of the concentration-response curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 73.2 ppm a.i. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 ppm a.i. test concentration, the no-observed-effect concentration was 73.2 ppm a.i.
TEST DATES:
Hatch - April 12, 1999 Acclimation - April 12-22, 1999 Experimental Start -April 22, 1999 ExperimentalTermination- May 14, 1999
NOMINAL TEST CONCENTRATIONS: 0, 18.3,36.6, 73.2, 146,293,586 and 1171ppma.i.
TEST ANIMALS:
Northern Bobwhite (Colinusvirginianus)
AGE TEST ANIMALS: 10 days of age at test initiation
SOURCE TEST ANIMALS: Wildlife International Ltd. Production Flock 8598 Commerce Drive Easton, Maryland 21601
STUDY COMPLETION: April 26,2000
AMENDED REPORT DATE: March 23,2004
' See Appendix X for the revised nominal test concentrations and results based upon the reanalysis of the test
material following study completion.
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INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. avian toxicology facility in Easton, Maryland. The in-life portion of the test was conducted from April 22, 1999 to May 14, 1999. Raw data generated at Wildlife International Ltd. and a copy of the final report are filed under Project Number 454-103 in archives located on the Wildlife International Ltd. site.
OBJECTIVE The objective of this study was to evaluate the toxicity of a test substance to the Northern Bobwhite (Colinus virginianus) administered through the diet for five days. An LC50 value will be calculated, if possible.
MATERIALS AND METHODS The methods used in conducting this study are based upon procedures specified in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.2200 (1), Section 71-2 of the Environmental Protection Agency's Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms (2); OECD Guideline 205, Guideline for Testing of Chemicals, Avian Dietary Toxicity Test (3); and upon ASTM Standard E857-87, "Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species" (4).
Test Substance The test substance was received fiom 3M Corporation on October 29, 1998 and was assigned
Wildlife International Ltd. Identification Number 4675 upon receipt. The test substance was white powder identified as: FC-95; Lot No.:217. The reported punty of the test substance was 98.9%, with an expiration date of 2008. Following test termination, the test material was reanalyzed. The results of reanalysis indicate a test substance punty of 90.49%. All test concentrations have been adjusted to reflect the purity reported on the new Certificate of Analysis (Appendix I)'. The test substance was stored under ambient conditions.
The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular
I Following study completion, the test material was recharacterized in accordance with GLP standards and the results of reanalysis indicate a purity of 86.9%. While test concentrations have not been adjusted to reflect the newest purity, the revised study summary in Appendix X summarizes the effect of the change in purity.
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material, was identified as: IH, lH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 276 19-97-2. The standard was stored under ambient conditions.
Treatment Groups The test consisted of a geometric series of seven test concentrations and a control group. Thirty
northern bobwhite chicks were assigned to the control group and ten northern bobwhite chicks were assigned to each of the treatment groups. The birds were sorted by weight, then chosen indiscriminately from within each represented weight class for placement into control and treatment groups. The birds were housed in brooding pens containing five chicks each. Nominal dietary concentrations used in this study were 0, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 parts per million active ingredient (ppm ai.) of PFOS. The dietary concentrations were established based upon known toxicity data and information supplied by the Sponsor.
Each group was fed the appropriate test or control diet for five days. During the exposure period the control group received untreated feed. Following the five-day exposure period all groups were given untreated basal diet for three days. On Day 8, half of the surviving treatment and control birds were euthanized and liver tissue, blood, and bile samples were collected for analysis. The remaining birds were fed basal ration until Day 22. On Day 22, these birds were euthanized and also sampled for liver weight, blood, and bile.
Duration of the Test The primary phases of this test and their durations were:
1. Acclimation - 10 days. 2. Exposure - 5 days. 3. Post-exposure observation - 3 or 17 days
Test Birds All northern bobwhite (Colinus virginianus) were 10 days of age and appeared to be in good
health at initiation of the test. The birds were obtained from Wildlife International Ltd. Production Flock, Easton, MD and were hatched on April 12, 1999. Birds ranged in weight from 18 to 23 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex.
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All birds were from the same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day of hatch until initiation of the test.
Animal Diet Throughout acclimation and testing all test birds were fed a game bird ration formulated to
Wildlife International Ltd.'s specifications (Appendix 11). The chicks were given a vitamin supplement in their water from the day they were hatched until the initiation of the test. Water from the town of Easton public water supply, and feed were provided ad libitum during acclimation and testing. The birds received no form of antibiotic medication during acclimation or testing.
Diet Preparation The test substance was mixed directly into the ration. Mixing was done with a Hobart mixer
(Model Number AS200T). All dietary test concentrations were adjusted to 100% PFOS based upon the reported purity of the test substance. All dietary concentrations and the LC50 value are reported as ppm a.i. in the diet. Nominal dietary test concentrations used in this study were 18.3, 36.6, 73.2, 146,293, 586, 1171 ppm a.i. (Appendix IV).
Diet Sampling Samples of the test diets were collected to verify the test concentrations administered and to
confirm the stability and homogeneity of the test substance in the diets. Homogeneity of the test substance in the diet was evaluated by collecting six samples from the 18.3 ppm a i . a.i. test diet and six samples fiom the 1171 ppm a.i. test diet at preparation on Day 0. Homogeneity samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. The homogeneity samples also served as verification samples. One verification sample was collected from the control diet and two verification samples were collected from each remaining treatment group at preparation on Day 0. At the end of the exposure period (Day 5), one sample was collected from the control and two samples were collected from each treatment group to determine stability of the test substance in the diet under test conditions. The stability samples were collected from feed remaining in the feeders after being at ambient test pen conditions for five days. Samples were transferred immediately to Wildlife International Ltd. analytical chemistry.
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Analytical Method The method used for the analysis of the avian diet samples was based upon methodology
developed at Wildlife International Ltd. and entitled "Method Outline for the Determination of PFOS in Avian Feed".
Avian diet samples were extracted with methanol. Methanol was added to a requisite quantity of feed contained in a French-square glass bottle. Bottles were capped and shaken on a shaker table. Samples were vacuum filtered using qualitative filter paper. The retained feed was rinsed three times with methanol into the filtrate. The filtrate was transferred to a volumetric flask and brought to volume with methanol. As appropriate, samples were further diluted with methanol. Each sample then was diluted with a 50% methanol : 50% NANOpureB water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. A method flowchart is provided in Appendix Ill, Figure 1.
Concentrations of PFOS in the standards and samples were determined by reversed-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API lOOLC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C18 analytical column (100 mm x 2 mm I.D., 3 pm particle size). The instrument parameters are
summarized in Appendix 111, Table 1 .
Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpureB water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00229 to 0.0457 mg a.i./L were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) of the calibration standards. A typical calibration curve is presented in Appendix 111, Figure 2. The concentration of PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Appendix 111, Figures 3 and 4, respectively.
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The method limit of quantitation (LOQ) for these analyses was set at 1.15 ppm a.i. calculated as the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500).
Two matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during sample analyses (Appendix 111, Table 2). An interference in the feed appeared at approximately the same retention time as the peak of interest but it was well below the LOQ. A representative chromatogram of a matrix blank is presented in Appendix 111, Figure 5.
Avian diet was fortified at 4.57, 183 and 1830 ppm a.i. and analyzed concurrently with the samples to determine the mean procedural recovery (Appendix 111, Table 3). Sample concentrations were not corrected for the mean procedural recovery of 94.7%. A representative chromatogram of a matrix fortification is presented in Appendix 111, Figure 6.
An example calculation is presented for sample number 454-103-2, nominal concentration of 18.3 pprn a.i. in avian diet.
Initial Weight: 10.0 g Final Volume: 200 mL Dilution Factor: 100(intermediatedilution factor x final dilution factor) PFOS Peak Area: 113568 Internal Standard Peak Area: 4 13160 Peak Area Ratio: 0.2749
Calibration curve equation. Slope: 2.77397 Intercept: 0.01894 Curve is weighted (lix).
PFOS (mg a.i./L) at instrument
(Peak area ratio - (Y-intercept)) x I.S. Concentration
=
Slope
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- (0.2749 - 0.01894) x 0.100 mg/L 2.77397
= 0.00923 mg a.i./L
Note: I.S. = internal standard.
PFOS (ppm a i ) in sample
- - PFOS (mg a.i./L) at instrument x Final Volume (L) x Dilution Factor Initial Weight (Kg)
- 0.00923 x 0.200 x 100
-
0.01
= 18.5 ppm a.i.
Percent of"omina1 Concentration
PFOS (ppm a i ) in sample
=
x 100
PFOS (ppm a i ) nominal
--~ 18.5 x 100 = 101% 18.3
Housing and Environmental Conditions During acclimation and testing, all birds were housed indoors in batteries of thermostatically
controlled brooding pens manufactured by Beacon Steel Products Co. (Model No. B735Q). Each pen had floor space that measured approximately 72 X 90 cm. Ceiling height was approximately 23 cin.
External walls, ceilings and floors were constructed of galvanized steel wire and sheeting. Birds were sorted by weight, then chosen indiscriminately from within each represented weight class for assignment to pens. Each group of birds was identified by pen number and test concentration. Individual birds were identified by leg bands.
During the test the average temperature in the brooding compartment of the pens was 38C f 2C
(SD). Average ambient room temperature for this study was 27.3"C * 1.2"C (SD) with an average
relative humidity of 31% i 14% (SD). The photoperiod (maintained by a time clock) was sixteen hours of light per day during acclimation and throughout the test. The light source was fluorescent lights which closely approximate noon-day sunlight. The birds were exposed to an average of
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approximately 139 lux of illumination. Housing and husbandry practices were based on guidelines established by the National Research Council (5).
Observations During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical
injury were not used. Following test initiation and continuing until termination, all birds were typically observed at least twice daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior.
Animal Body WeightdFeed Consumption Individual body weights were measured at the initiation of the test, on Day 5 , Day 8, and on
Days 15 and Day 22 for all remaining birds. Average feed consumption values during the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) were determined by pen for each treatment group and the control group. Additionally, feed consumption was determined for Days 8-15 and 15-22 for the remaining treatment and control birds. Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over a given period of time. The accuracy of feed consumption values may have been affected by the unavoidable wastage of feed by the birds.
Gross Necropsv All test birds that died during the course of the test and all birds remaining at the termination were
subjected to a gross necropsy. Additionally, livers were weighed and liver tissue, blood, and bile were collected from birds euthanized on Day 8 and 22, and when possible from those that died during the course of the study.
Statistical Analvses Mortality data were analyzed using the computer program of C.E. Stephan (6). The program was
designed to calculate the LC50 value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (7,8,9). In this study, the LC50 value was determined using the probit method. The slope of the concentration-response curve and results of the goodness of fit test are reported. Body weight data were compared by Dunnett's test using TOXSTAT software (10,ll). No statistical analyses were applied to feed consumption data.
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RESULTS
Diet Analvsis Avian diet samples were collected from the 18.3 and 1171 pprn a.i. test concentrations and
analyzed to evaluate homogeneity of the test substance in the avian diet. The analysis of these samples also served as verification of test substance concentrations. Resulting mean measured concentrations, standard deviations and coefficients of variation (CV) for these test concentrations were 19.5 iz 2.13 pprn a i (CV = 10.9%) and 1196 k 70.2 ppm a.i. (CV = 5.87%), respectively (Appendix 111, Table 4). Control avian diet samples collected during the test showed no interferences above the LOQ. Samples collected during the test to verify the 36.6, 73.2, 146, 293 and 586 ppm a.i. test substance concentrations had mean measured concentrations of 40.2, 74.5, 174, 291 and 537 pprn ai., respectively. These values represented 110, 102, 119, 99.3 and 91.6% of the nominal concentrations, respectively (Appendix 111, Table 5). Analysis of avian diet samples collected from feeders after being held at ambient temperature for five days averaged 101, 122, 104, 101, 109, 114 and 102% of the Day 0 values for the 18.3, 36.6, 73.2, 146,293, 586 and 1171 ppm a i . test substance concentrations, respectively (Appendix 111, Table 6). A representative chromatogram of a test sample is shown in Appendix 111, Figure 7.
Mortalities and Clinical Observations One incidental mortality occurred in the control group during the course of the study (Table 1 and
Appendix V). On the morning of Day 5 , one bird was noted with a broken leg and was subsequently euthanized on Day 6. Additionally, two birds in the control group were intermittently noted with foot lesions associated with cage mate aggression. Otherwise, all control birds were normal in appearance and behavior throughout the test.
No treatment related mortalities or overt signs of toxicity were observed in the 18.3, 36.6, or 73.2 pprn a.i. treatment groups. One bird in the 18.3 pprn a.i. treatment group was noted as lame from Day 6 through Day 8, and blood of undetermined origin was noted on the underside of one bird from the 73.2 pprn a i . treatment group on Day 22. Otherwise, all birds in the 18.3, 36.6 and 73.2 ppm a.i. treatment groups were normal in appearance and behavior throughout the test period.
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There was 11% (1 of 9) mortality in the 146 pprn a.i. treatment group, 80% (8 of IO) mortality in the 293 ppm a.i. treatment group and 100% (10 of 10) mortality in the 586 and 1171 pprn a.i. treatment groups. In the 146 ppm a.i. treatment group, one bird was euthanized on Day 3 after sustaining a broken leg. This incidental mortality was not used in the calculation of the LC50 value. Additionally, there was one treatment-related mortality in the 146 ppm a i . treatment group, a bird found dead on the morning of Day 7. Clinical signs of toxicity were observed in this treatment group on Day 5 , when two birds displayed wing droop. All other birds at this test concentration were normal in appearance and behavior for the duration of the test.
In the 293 ppm a.i. treatment group there were eight treatment-related mortalities, occurring on Days 5 , 6 and 7. Signs of toxicity were first observed on the morning of Day 4 and continued to be exhibited through the morning of Day 8 for the single bird euthanized on Day 8, and through the afternoon of Day 8 for the single bird surviving until Day 22. Signs of toxicity included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, wing droop, loss of coordination, lower limb weakness and convulsions. The single remaining bird appeared to have recovered and was normal in appearance and behavior from the afternoon of Day 9 until test termination.
In the 586 ppm a.i. treatment group mortalities were first noted on Day 3 and continued to be observed through Day 7, at which point all birds had died. Overt signs of toxicity were first observed on the afternoon of Day 2 and continued through the morning of Day 7 , when the final birds were found dead. Signs of toxicity observed among birds in the 586 pprn a.i. treatment group included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, wing droop, loss of coordination, lower limb weakness, lower limb rigidity, prostrate posture, and convulsions.
In the 1171 pprn a.i. treatment group 100% mortality had occurred by the morning of Day 4. Signs of toxicity in the 1171 ppm a.i. treatment group were first observed on the afternoon of Day 2, with the first mortalities noted on the morning of Day 3. Signs of toxicity observed prior to death included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, loss of coordination, wing droop, and lower limb weakness and rigidity.
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Body Weight and Feed Consumption When compared to the control group, there were no apparent treatment related effects upon body
weight among birds in the 18.3, 36.6 or 73.2 ppm a i . treatment groups. However, there was a concentration responsive reduction in body weight gain or body weight loss in the 146, 293 and 586 pprn a.i. treatment groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). Differences from the control group were statistically significant at p < 0.05 for the 146 ppm a i level and at p<O.Ol for the 293 and 586 ppm a.i. levels. A statistically significant (p<O.Ol) reduction in body weight gain continued to be observed at the 146 ppm a.i. test concentration through Day 8 of the study. At the 293 ppm a i concentration a statistically significant (p<O.Ol) mean weight loss continued through Day 8 of the study and a marked reduction in weight gain was noted through Day 15 of the study. Due to total mortality, body weight effects could not be determined for the 1171 ppm a.i. level during the exposure period or for the 586 and 1171 ppm a i . treatment groups for postexposure period.
There were no apparent treatment related effects upon feed consumption at the 18.3, 36.6, 73.2 or 146 ppm a.i. test concentrations (Table 3 and Appendix VII). However, a reduction in feed consumption was noted at the 293, 586 and 1171 ppm a i treatment groups during the exposure period (Days 0-5). There were no treatment-related effects on feed consumption in any of the surviving treatment groups during the Day 6-8 post-exposure period. In the 293 ppm a i . treatment
group only one bird survived to Day 22. The reduction in feed consumption observed at the 293 pprn
a.i. test concentration during both the Day 8-1.5 and 15-22 post-exposure periods was the result of having only one bird in the pen, and was not considered to be treatment related.
Gross Necropsy During the course of the test, all birds that died were subjected to a gross necropsy. Necropsy
results for birds found dead were similar. Common observations included thin condition, loss of muscle mass, altered spleen color, autolysis of tissues and pale organs. Details of the necropsy findings are presented in Table 4.
Half of the surviving birds were subjected to gross necropsy on Day 8 and the remaining birds were necropsied on Day 22, following test termination. On Day 8, one bird in the 73.2 pprn a.i. treatment group was noted with a slightly pale liver. Due to the isolated nature of this finding, it was
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not considered to be related to treatment. The single bird euthanized from the 293 ppm a.i. treatment group was observed at necropsy to have with a lack of muscle mass and general thinness. Since these findings correlated with an impact upon body weight noted at this concentration, the findings were considered to be treatment related. Necropsy results were unremarkable for all other birds euthanized on Day 8. Similarly, Day 22 necropsy findings were unremarkable for all birds.
CONCLUSION
The dietary LC50 value for northern bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a i . to 289 ppm a i . The slope of the concentrationresponse curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 73.2 pprn a.i. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 pprn a.i. a.i test concentration, the no-observed-effect concentration was 73.2 ppm a i .
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REFERENCES
1. U.S. Environmental Protection Agency, 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.2200: Avian Dietary Toxicity Test.
2. U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildllfe and Aquatic Organisms, subsection 71-2, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C.
3. Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing of Chemicals. Guideline 205. Paris.
4. American Society for Testing and Materials. 1987. Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book of ASTM Standards, Vol. 11.04. Philadelphia, PA.
5. National Research Council. 1996. Guide for the Care and Use of Laboratovy Animals. Washington, D.C. National Academy Press. 125 pp.
6. Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication
7. Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 InAquatic Toxicology and
Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA.
8. Finney, D.J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London.
9. Thompson, W.R. 1947. Bacteriological Reviews, Vol2, No.2: 115-145.
10. Dunnet, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50:1096-1121.
11. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University of Wyoming.
WILDLIFE INTERNATIONAL, LTD
- 20 -
PROJECT NO. : 454-103
TABLE 1 Cumulative Mortality from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Day0
No. Dead Per No. Exposed Exposure Period
Day 1 Day2 Day3 Day4
Day5
No. Dead Per No. Exposed Post-Exposure Period
Day6 Day7 Days'
Control 0
0130 0130 0130 0130 0130 0130
1/30 1/30
1/30
Treatment 18.3 36.6 73.2 146 293 586 1171
Oil0 0110 0110 0110 Oil0 0110
0110 0110 0110 Oil0 0110 0110
0110 0110 0110 0110 0110 Oil0
0110 Oil0 Oil0 1/102 019
019
0110 0110 Oil0 Oil0 0110 2110
Oil0 0110 0110 1110 2/10 5/10
0110 0110 0110 3/10 10110 10110
0110 Oil0 Oil0 019 411 0 8110 10110
0110 011 0 0110 119 8110 10110 10110
0110 011 0 Oil0 119 8/10 10110 loll0
The LC50 value was calculated to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a.i. to 289 ppm a i .
' -No mortalities occurred in any of the control or treatment groups from Day 8 to Day 22. * ~ Bird euthanized on day 3 after sustaining a broken leg.
WILDLIFE INTERNATIONAL, LTD
-21 -
PROJECT NO. 454-103
TABLE 2 Page 1
Mean Body Weight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Exposure Period Change'
Day0 Day5 Day0-5
Post-Exposure Period
Change'
Day 8
Day 5-8
Total Change'
Control
0
Mean
20
30
10
38
8
18
SD
1
4
3
5
2
4
Treatment
18.3
Mean
21
31
11
40
9
20
SD
1
4
3
5
2
3
36.6
Mean
20
31
11
39
8
19
SD
2
3
2
3
1
2
73.2
Mean
20
30
9
37
7
16
SD
1
2
1
3
1
2
146
Mean
20
27*
7*
33*
6**
13**
SD
2
3
3
3
2
4
293
Mean
20
18**
-2 * *
18**
-2 * *
-1 **
SD
1
2
2
4
4
5
586
Mean
SD
20
16**
-4 * *
1
2
2
1171
Mean
20
SD
1
'Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). (-) = No data available due to mortality. *Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test).
WILDLIFE INTERNATIONAL, LTD
- 22 -
PROJECT NO.: 454-103
TABLE 2 Page 2
Mean Body Weight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Day8
Post-Exposure Period
Day 15
Change'
Day 8-15
Day 22
Change' Day 15-22
Total
Change'@-22)
Control 0
Mean SD
37
59
23
6
10
5
82
22
45
13
3
8
Treatment 18.3
Mean SD
40
68
24
6
8
3
87
23
47
7
3
3
36.6
Mean
SD
38
65
26
3
5
2
89
24
50
7
2
4
73.2
Mean
SD
35
60
24
3
4
2
79
20
44
4
2
2
146
Mean
SD
34*
58
24
3
3
1
79
21
45
2
2
1
293
Mean
2
SI)
21
35
14
55
20
34
586
Mean
SD
1171
Mean
SD
`Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). *n=1, could not be evaluated statistically with Dunnett's t-test. (-) = No data available due to mortality. *Statistically different from the control group at p < 0.05 )(Dunnett's t-test).
WILDLIFE INTERNATIONALLT,D
- 23 -
PROJECT NO.: 454-103
TABLE 3 Page 1
Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Control 0
Treatment 18.3 36.6 73.2 146 293 586 1171
Exposure Period Day 0-5
Mean
9
SD
2
9 8 10 9 5 6 4
Post-Exposure Period Day 6-8
10 2
11 12 13 10 9 19
(-) = N o data available due to mortality.
WILD LIFE I NTER NATIONAL , LTD
- 24 -
PROJECT NO.: 454-103
TABLE 3 Page 2
Mean Feed Consumption (ghirdday) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Control 0
Mean SD
Post-Exposure Period
Day 8-15
Day 15-22
9
13
2
1
Treatment 18.3 36.6 73.2 146 293 586 1171
10
12
14
15
13
15
11
14
8
9
(-) = No data available due to mortality.
WILD LIFE INTER NATIONAL , LTD
- 25 -
PROJECT NO.: 454-103
TABLE 4 Group Gross Pathological Observations from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
Birds that died during the course of the study
Finding
Control N= 1
146
N =2
Male, Female, and Undetermined
PPM A.I.
293
586
N =8
N =10
1171 N =10
Abdominal cavity, some autolysis
0
Abdominal cavity, autolysis throughout
0
Crop, empty
0
Emaciated
0
Fractured leg
1
G.I. tract empty
0
Gizzard contents bile stained
0
Heart, anterior portion mottled white color
0
Heart, pale
0
Intestinal contents tar-like
0
Keel, prominent
0
Kidneys, pale
0
Liver, pale and mottled
0
Loss of muscle mass
0
Muscular-skeletal, pale
0
Small in stature
0
Spleen, black
0
Spleen, dark
0
Spleen, grey
0
Spleen, grey-brown
0
Spleen, pale
0
Spleen, small
0
Spleen, small and pale
0
Thin
0
Not Remarkable
0
0
2
2
4
0
0
1
1
0
2
5
2
0
2
5
8
1
0
0
0
0
1
1
0
0
2
5
1
0
1
0
0
0
0
2
1
0
0
2
0
0
1
3
10
0
0
2
0
1
0
0
0
0
4
7
9
1
0
0
0
0
3
0
0
0
0
1
0
0
0
0
2
0
0
1
0
0
0
0
1
0
1
0
1
0
0
0
1
0
0
3
0
0
0
4
2
0
1
0
0
WILDLIFE INTERNATIONALL,TD
- 26 -
PROJECT NO. : 454- 103
Experimental Group
(ppm a.i.) Control
0 0 0 0 0 0 Treatment 18.3 18.3
Table 5
Cumulative Mortality (Estimated Cumulative Dose, mglkg') from a Northern Bobwhite
Acute Dietary Toxicity Study with PFOS
No. Dead Per No. Exposed (Cumulative Dose, mgkg) Exposure Period
No. Dead Per No. Exposed Post-Exposure Period
Pen Day0
Day 1
Day 2
Day 3
Day 4
Day 5
Day6 Day7 Day82
1 015
015
015
015
015
015
015
015
015
2 015
015
015
015
015
015
015
015
015
3 015
015.
015
015
015
Of5
015
015
015
4 015
015
Oh
015
015
015
015
015
015
5 015
015
015
015
015
015
1 I5
1 I5
115
6 015
015
015
015
015
015
015
015
015
1 015 2 015
015 (7) 015 (6)
015 (14) 015 (12)
015 (21) 015 (1 8)
015 (28) 015 (24)
015 (35) 015 (30)
015
015
015
015
015
015
36.6
1 015
015 (13)
015 (26)
015 (39)
015 (52)
015 (65)
015
015
015
36.6
2 015
015 (11)
015 (22)
015 (33)
015 (44)
015 ( 5 5 )
015
015
015
73.2
1 015
015 (33)
015 (66)
015 (99)
015 (132)
015 (165)
015
01 5
015
73.2
2 015
015 (32)
015 (64)
015 (96)
015 (128)
015 (160)
015
015
015
146
1 015
015 (46)
015 (92)
015 (138)
015 (184)
015 (230)
015
1 I5
115
146
2 015
015 (49)
015 (98)
115 (147)
115 (196)
115 (245)
1I5
115
115
293
1 015
015 (66)
015 (132)
015 (198)
015 (264)
115 (330)
115
415
415
293
2 015
015 (101)
015 (202)
015 (303)
015 (404)
115 (505)
315
415
415
586
1 015
015 (213)
015 (429)
115 (639)
215 (852)
215 (1065) 415
515
515
586
2 015
015 (178)
015 (356)
015 (534)
015 (7 12)
315 (890)
415
515
515
1171
1 015
015 (256)
015 ( 5 12)
315 (768)
515
515
515
515
515
1171
2 015
015 (256)
015 (512)
015 (768)
515
515
515
515
515
The LC50 value was calculated to be approximately 220 ppm a.i. with a 95% confidence interval of 164 to 289 ppm a.i..
*- Bird was euthanized due to a broken leg.
I - Estimated cumulative dose is based upon the average body weight and feed consumption over the 5-day exposure period, and serves as a rough
approxiinaton of the actual amount of test substance consumed.
*- No mortalities occurred in any of the treatment or control groups after Day 8.
WI LD LI FE INTERNATIoNAL , LTD
- 27 -
PROJECT NO.: 454- 103
APPENDIX I
Certificate Of Analysis
WILDLIFE INTERNATIONAL, LTD - 28 -
PROJECT NO.: 454-103
APPENDIX I1
DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION'
INGREDIENTS
Fine Corn Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos
Methionine Premix + Liquid
Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)* Salt Iodized Total
PERCENT ("A)
44.83 30.65 6.50 6.00 4.00 3.00 2.50
0.90 0.60 0.35 0.32 0.25 0.10 100.00
VITAMIN AND MINERAL PREMIX
AMOUNT ADDED PER TON
Vitamin D3 Vitamin A Riboflavin Niacin
Pantothenic Acid
Vitamin B12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (MenadioneDimethylpyrimidinolBisulfite) Manganese Zinc
Copper Iodine Iron Selenium
2,000,000 I.C.U. 7,000,000 I.U.
6 grams 40 grams
10 grams
8 mgs
600 mgs 64 mgs
1.2 grams 1.2 grams 20,000 I.U.
5.8 grams 102 grams
47 grams 6.8 grams 1.5 grams 51 grams
182 mgs
I The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum
of 5% crude fiber. 2Fermentation By-products (Source of Unidentified Growth Factors).
WILDLIFE INTERNATIONAL, LTD
- 29 -
PROJECT NO.: 454-103
APPENDIX I11 ANALYTICAL METHODS AND RESULTS
WILDLIFE INTERNATIONAL, LTD
- 30 -
PROJECT NO.: 454-103
INSTRUMENT:
APPENDIX I11
Table 1 Typical L C M S Operational Parameters
Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 1OOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM).
ANALYTICAL COLUMN:
Keystone Betasil CI8column (100 mm x 2 mm I.D., 3 pm particle size)
OVEN TEMPERATURE:
30C
STOP TIME:
10.0 minutes
FLOW RATE:
0.220 mL/minute
MOBILE PHASE:
72.0% Methanol : 28.0% NANOpureB Water containing 0.1?4n Formic Acid
INJECTION VOLUME:
25.0 pL
PFOS RETENTION TIME:
Approximately 7.0 minutes
INTERNAL STANDARD
RETENTION TIME:
Approximately 4.8minutes
PFOS MONITORED MASS:
INTERNAL STANDARD MONITORED MASS:
498.6 amu 426.7 amu
WILDLI FE INTER NATIoNAL, LTD
-31 -
PROJECT NO.: 454-103
APPENDIX I11 Table 2
Matrix Blanks Analyzed Concurrently During Sample Analysis
Number (454- 103-)
MAB-1
Sample
Type Matrix Blank
Measured Concentration of
PFOS'
-
(ppm a.i.)
< LOQ
MAB-2
Matrix Blank
< LOQ
' The limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard
analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500).
WILDLIFE INTERNATIONAL, LTD
- 32 -
PROJECT NO.: 454-103
APPENDIX 111
Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis
Sample Number (454-103-)
MAS- 1A MAS-4A
Concentrations of PFOS
(ppm a i )
Fortified
Measured
4.57
4.54
4.57
4.79
Percent Recovered
99.2 105
MAS-2
183
176
96.1
MAS-5
183
162
88.3
MAS-3 MAS-6
1830 1830
1576
86.1
1716
93.7
Mean =
94.7
Standard Deviation =
6.99
cv =
7.38
N=
6
Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes.
WILDLIFE INTERNATIONALL,TD
PROJECT NO.: 454-103
- 33 -
APPENDIX I11
Table 4
Nominal Concentration
(ppm a.i.)
18.3
Sample Number (S-454-103-)
2 3 4 5 6 7
Homoeeneitv of PFOS in Avian Diet
Location Sampled in
Mixing Vessel
PFOS Measured Concentration
(ppm a.i.)
Top Left
18.5
Top Right
23.4
Middle Left
18.3
Middle Right
17.3
Bottom Left
19.4
Bottom Right
19.9
Mean Measured Concentration (x)
Standard Deviation (SD) Coefficient of Variation (cv)'
x = 19.5 pprn a i
SD = 2.13 pprn a.i. CV = 10.9%
Mean Percent of
Nominal
107
1171
18
19
20
21
22
- 23
P
Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right
1239 1221 1118 1301 1163 1133
x = 1196 pprn a.i
SD = 70.2 ppm. a.i CV = 5.87%
Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations.
rounded for reporting purposes. 1Coefficient of variation was calculated using full precision of mean and standard deviation results.
102 Values have been
WILDLIFE INTERNATIONAL,LTD
PROJECT NO.: 454-103
- 34 APPENDIX 111
Table 5 Verification of PFOS Concentrations in Avian Diet
Nominal Concentration
(ppm a.i.)
0.0
18.3
Sample Number (S-454-103-)
1
__
Sampling Interval (Day)
0
__
PFOS Measured Concentration'
(ppm a.i.)
< LOQ
__
Percent of
Nominal
__
--
Mean Measured Concentration
(ppm a i )
-_
19.5'
Mean Percent of
Nomina 1
__
107'
36.6
8
0
45.7
125
40.2
110
9
0
34.6
94.5
73.2
10
0
11
0
77.8
106
74.5
102
71.2
97.3
146
12
0
13
0
176
120
174
119
172
117
293
14
0
15
0
274
93.8
29 1
99.3
307
105
586
16
0
17
0
550
93.9
537
91.6
523
89.4
1171
_-
_-
--
--
1196'
1022
Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes.
'The limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500). 'Result obtained from Table 4.
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
- 35 -
APPENDIX I11 Table 6
Ambient Stability of PFOS in Avian Diet During the Northern Bobwhite LCSO Study
Concentration (PPm a.1.)
0
Sample Number (S-454-103-)
1
Day 0'
Mean Measured Concentration
(PPm a.1.)
--
Mean Percent of
Nominal
__
Sample Number (S-454- 103-)
24
Dav 5
Measured concentration'
(PPm a.1.)
< LOQ
Mean Measured Concentration
(PPm a.1.)
_-
Mean Percent of
Day 0
--
18.3
2-7
19.5
107
25
19.2
19.6
101
26
19.9
36.6
8,9
40.2
110
27
44.4
49.1
122
28
53.8
73.2
10,ll
74.5
102
29
76.4
77.2
104
30
77.9
146
12, 13
1 74
119
31
177
176
101
32
174
293
14, 15
291
99.3
33
318
317
109
34
315
586
16, 17
537
91.6
35
560
613
114
36
665
1171
18-23
1196
102
37
1260
1224
102
38
1187
'Day 0 results obtained from Table 4 and Table 5. 'The limit of quantitation (LOQ) was 1.15 ppm a i . based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500).
WI LDLI FE INTERNAT IoNAL , LTD
- 36 -
PROJECT NO. 454- 103
APPENDIX 111
METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET
Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique.
1
Dry Mix Technique
For the high-level matrix fortification sample, weigh the requisite quantity of Wildlife International Ltd. (WIL) ration into a weigh boat.
1
Weigh the requisite quantity of test substance (PFOS) into a beaker.
1
Add % of the WIL ration and the test substance to a largcr bcakcr.
1
Rinse the bcaker that containcd the PFOS with small portions of thc remaining ration and transfer all portions to the larger beaker.
1
Mix the contents of the larger beaker well and transfcr the mixture to a Waring blcndcr.
1
Blend the mixture for -5 minutcs stopping at 1 minute intervals to scrape down the sides of thc blcndcr. During the third interval transfer the fortified feed to a beaker, mix well and return the mixture to the blcndcr to complete mixing in the
specified time.
1
Prcpare the next two matrix fortification levels by serial dilutions. Follow the same procedure described for the high-level matrix fortification except weigh the appropriate quantity of fortified matrix (high or
mid-level) rather than the test substance.
1
Weigh 10-g samples of the matrix blank, matrix fortification and test samples into wcigh boats and transfer to 16-oz. French-square glass bottles. Record the weights.
For each sample, measure 100 mLs of methanol with a graduated cylinder and transfer volume to the French-square bottle.
1
Cap bottles and place on shakcr tablc. Allow the samples to shake for a minimum of 30 minutes at 250 rpm.
I
Vacuum filter each sample with qualitative filter paper and rinse retained feed 3 times with mcthanol into the filtrate.
1
Transfer the filtrate to a 200-mL volumctric flask and bring the flask to volume with methanol.
1
Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. Use mcthanol for intermcdiatc dilutions, if required.
For all final dilutions usc 50% mcthanol : 50% NANOpureB water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v).
Ampulatc and submit samples for LC/MS analysis
Figure 1. Analytical method flowchart for the analysis of PFOS in avian diet.
WILDLIFE INTERNATIONALL,TD
- 37 -
APPENDIX 111
PROJECT NO.: 454-103
0.000
0.100
0.200
0.300
Concentration (Ratio)
8
1
0.400
0.500
Figure 2.
A typical calibration curve for PFOS. Slope = 2.77397; Intercept = 0.01894; r = 0.9981. Curve is weighted (l/x).
WILDLIFE INTERNATIONALLTD,
- 38 -
APPENDIX 111
PROJECT NO.: 454-103
intensity: 1995 cps 167 100 90 80 70 60 50 40 30
31
1.30
61
2.55
91
3.81
121
5.06
151
6.32
21 1
7.57 8.83
scan
Time
~~~
Figure 3.
A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard.
WILDLIFE INTERNATIONALL,TD
- 39 -
PROJECT NO. : 454- 103
intensity: 26954 cps
..
3.1
61
1.30
2.55
91
121
151
181
21 1
3.81
5.06
6.32
7.57
8.83
Time
Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard.
WILDLIFE INTERNATIONALL,TD
-40-
APPENDIX 111
PROJECT NO.: 454-103
10
9
4
8
7
6
5
4
3
2
1
-4 1 ;0
166
1
intensity: 230 cps
I
I
216
31
61
91
121
151
181
21 1
1.30
2.55
3.81
5.06
6.32
7.57
8.83
scan
Time
Figure 5.
A representative chromatogram of a matrix blank sample (454-103-MAB-1). The arrow indicates the retention time of PFOS.
WI LDLIFE INTERNATI oNA L, LTD
-41 APPENDIX I11
PROJECT NO.: 454-103
intensity: 6238 cps 66
"7
144
1.30 2.55 3.81 5.06 6.32 7.57 8.83
Time
Figure 6. A representative chromatogram of a matrix fortification sample (454-103-MAS-1A).
WILDLIFE INTERNATIONALLT,D
-42 APPENDIX I11
PROJECT NO.: 454-103
intensity: 7248 cps
-
Figure 7.
A representative chromatogram of a test sample (454-103-2).
WILDLIFE INTERNATIONALL,TD
- 43 -
APPENDIX IV DIET PREPARATION
PROJECT NO.: 454- 103
Weight and volume of constituents used to prepare test diets:
Nominal Concentrations
(ppm a.i.) 0 18.3
36.6 73.2 146 293 586 1171
SubTsetsatnce
I
Basal Ration
0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 1 1.6483
8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4
Diets were prepared as follows:
05000.0 g of basal ration was weighed into a tared Hobart mixing bowl.
*The test substance was weighed in a tared weigh boat.
*Approximately 100 g of basal ration was taken from the mixing bowl and placed in a Waring blender.
.The test substance was added to the blender and the weigh boat was rinsed with additional ration, with the rinse also being placed in the blender.
*The blender contents were blended for approximately 60 seconds and transferred to the mixing bowl. The blender was rinsed with additional ration, with the rinse also being placed in the
mixing bowl.
.The bowl was placed on a Hobart mixer and the contents were mixed for approximately six minutes. The remaining ration as added to the bowl and the contents were mixed for six more minutes.
.The diet was transferred to a labelled paper feed bag.
WILDLIFE INTERNATIONALL,TD
-44 -
PROJECT NO.: 454-103
Experimental Group
(ppm a.i.)
Control 0 0 0 0 0 0 Treatment 18.3 18.3 36.6 36.6 73.2 73.2 146 146 293 293 586 586 1171 1171
APPENDIX V Cumulative Mortality by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS
No. Dead Per No. Exposed
No. Dead Per No. Exposed
Pen
Exposure Period
Post-Exposure Period
Day0 Day 1 Day2 Day3 Day4 Day5
Day6 Day7 Day8l
1
015
015
015
015
015
015
2
015
015
015
015
015
015
3
015
015
015
015
015
015
4
015
015
015
015
015
015
5
015
015
015
015
015
015
6
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
1/5*
1I5
115
015
015
015
1
015
015
015
015
015
015
2
015
015
015
015
015
015
1
015
015
015
015
015
015
2
015
015
015
015
015
015
1
015
015
015
015
015
015
2
015
015
015
015
015
015
1
015
015
015
015
015
015
2
015
015
015
1/5*
115
1 I5
1
015
015
015
015
015
115
2
015
015
015
015
015
115
1
015
015
015
115
215
215
2
015
015
015
015
015
315
1
015
015
015
315
515
515
2
015
015
015
015
515
515
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
1 I5
115
1 I5
115
115
1 15
415
415
315
415
415
415
515
515
415
515
515
515
515
515
515
515
515
The LC50 value was calculated to be 220 ppm a.i. with a 95% confidence interval of 164 pprn a i to 289 ppm a.i.
* - Bird was euthanized due to a broken leg.
1 - No mortalities occurred in any of the control or treatment groups after Day 8.
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454- 103
Experimental Group
(ppm a.i.) 0
0
0
0
- 45 -
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 1
Bird
1 2 3 4 5 Mean SD
1 2 3 4 5 Mean SD
1 2 3 4
5
Mean SD
1 2 3 4 5 Mean SD
Day 0
19 20 21 22 23 21 2
19 20 20 22 22 21
1
19 20 20
22
22 21
1
19 19 19 21 22 20 1
Day 5
24 27 32 32 39 31 6
26 25 29 33 31 29 3
23 26 29
30
35 29 5
28 27 30 32 33 30 3
Change Day 0-5
5 7 11 10 16 10 4
7 5 9 11 9 8 2
4 6 9 8
13 8 3
9 8 11 11 11 10 1
Day 8
27 34 42 40 50 39 9
35 35 37 43 39 38 3
30 29 32
37
40 34
5
37 35 36 42 43 39 4
Change Day 5-8
3 7 10 8 11 8 3
9 10 8 10 8 9 1
7 3 3
7
5 5 2
9 8 6 10 10 9 2
Total Change
8 14 21 18 27 18 7
16 15 17 21 17 17 2
11 9 12
15
18 13 4
18 16 17 21 21 19 2
WILD LIFE INTER NATIONAL , LTD
PROJECT NO.: 454-103
Experimental Group
(ppm ai.)
0
-46-
APPENDIX VI
Individual Body Weights (8) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 2
Bird
1 2 3 4 5 Mean
Day 0
19 20 21 20 22 20
Day 5
28 27 32 30 34 30
Change Day 0-5
9 7 11 10 12 10
Day 8
35 36 44
44 40
Change Day 5-8
7 9 12
10 10
Total Change
16 16 23
22 19
0
1
18
30
12
39
9
21
2
21
35
14
46
11
25
3
19
29
10
37
8
18
4
21
32
11
41
9
20
5
22
32
10
42
10
20
Mean
20
32
11
41
9
21
SD
2
2
2
3
1
3
Group
Mean
20
30
10
38
8
18
Total
SD
1
4
3
5
2
4
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
-47-
APPENDIX VI
Individual Body Weights (8) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 3
Experimental Group
(ppm a.i.)
18.3
Bird
1 2 3 4 5 Mean SD
Day 0
19 19 21 22 22 21 2
Day 5
26 33 27 34 36 31 4
Change Day 0-5
7 14 6 12 14 11 4
Day 8
34 39 35 46 46 40 6
Change Day 5-8
8 6 8 12 10 9 2
Total Change
15 20 14 24 24 19
5
18.3
1
19
28
9
38
10
19
2
19
28
9
37
9
18
3
20
29
9
38
9
18
4
22
34
12
44
10
22
5
22
35
13
44
9
22
Mean
20
31
10
40
9
20
SD
2
3
2
3
1
2
Group
Mean
21
31
11
40
9
20
Total
SD
1
4
3
5
2
3
WILDLIFE INTERNATIONAL,LTD -48 -
PROJECT NO.: 454-103
APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite
Acute Dietary Toxicity Study with PFOS Page 4
Experimental Group
(ppm a.i.)
36.6
Bird
1 2 3 4 5 Mean
Day 0
18 19 19 21 21 20
Day 5
26 30 29 30 34 30
Change Day 0-5
8 11 10 9 13 10
Day 8
35 39 37 38 43 38
Change Day 5-8
9 9 8 8 9 9
Total Change
17 20 18 17 22 19
36.6
1
19
29
10
37
8
18
2
19
29
10
37
8
18
3
21
32
11
40
8
19
4
22
35
13
44
9
22
5
23
36
13
44
8
21
Mean
21
32
11
40
8
20
SD
2
3
2
4
0
2
Group
Mean
20
31
11
39
8
19
Total
SD
2
3
2
3
1
2
WILDLIFE INTERNATIONALL,TD
- 49 -
PROJECT NO. : 454- 103
APPENDIX VI
Individual Body Weights (8) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 5
Experimental Group
(ppm a.i.)
73.2
Bird
1 2 3 4 5 Mean SD
Day 0
18 19 20 21 21 20 1
Day 5
26 26 29 32 30 29
3
Change Day 0-5
8 7 9 11 9 9 1
Day 8
32 33 36 39 36 35 3
Change Day 5-8
6 7 7 7 6 7 1
Total Change
14 14 16 18 15 15 2
73.2
1
18
27
9
34
7
16
2
20
31
11
38
7
18
3
21
30
9
37
7
16
4
21
32
11
39
7
18
5
22
32
10
41
9
19
Mean
20
30
10
38
7
17
SD
2
2
1
3
1
1
Group
Mean
20
30
9
37
7
16
Total
SD
1
2
1
3
1
2
WILDLIFE INTERNATIONAL, LTD
- 50 -
PROJECT NO.: 454-103
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 6
Experimental Group
(ppm a.i.)
146
Bird
1 2 3 4 5 Mean SD
Day 0
19 19 20 22 23 21 2
Day 5
28 28 24 33 28 28
3
Change Day 0-5
9 9 4 11 5 8 3
Day 8
35 32
38 32 34
3
Change Day 5-8
7 4
5 4 5 1
Total Change
16 13
16 9 14 3
146
1
18
24
6
31
7
2
19
27
8
35
8
3
19
27
8
33
6
4
21
5
23
24
1
28
4
Mean
20
26
6
32
6
SD
2
2
3
3
2
Group
Mean
20
27*
7*
33*
6**
Total
SD
2
3
3
3
2
(-) = N o data available due to mortality. *Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different froin the control group at p < 0.01 (Dunnett's t-test).
13 16
14
5 12 5 13** 4
WILD LIFE INTER NATIONAL , LTD
-51 -
PROJECT NO.: 454-103
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 7
Experimental Group
(ppm a.i.)
293
Bird
1 2 3 4 5 Mean SD
Day 0
18 19 20 22 22 20 2
Day 5
20 17 18
Change Day 0-5
2 -2 -2
Day 8 21
20
-2
-1
21
2
Change Day 5-8
1
1
Total Change
3
3
293
1
19
19
0
15
-4
-4
2
19
3
20
20
0
4
20
17
-3
5
21
16
-5
Mean
20
18
-2
15
-4
-4
Group
SD
1
2
2
Mean
20
18**
-2 * *
18**
-2 * *
- 1**
Total
SD
1
2
2
4
4
5
(-) = No data available due to mortality. *Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test).
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
- 52 -
APPENDIX VI
Individual Body Weights (8) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 8
Experimental Group
(ppm a.i.)
586
Bird
1 2 3 4 5 Mean SD
Day 0
18 20 21 21 22 20 2
Day 5
Change Day 0-5
Day 8
13
-7
18
-3
17
-5
16
-5
3
2
Change Day 5-8
Total Change
586
1
19
16
-3
2
20
3
20
16
-4
4
19
5
21
Group
Mean
20
SD
1
Mean
20
16
-4
0
1
16**
-4 * *
Total
SD
1
2
2
(-) = No data available due to mortality. **Statistically different from the control group at p < 0.01 (Dunnett's t-test).
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
- 53 -
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 9
Experimenta1
Group
(ppm a.i.)
Bird
Day 0
1171
1
18
2
20
3
20
4
21
5
21
Mean
20
SD
1
Day 5
Change Day 0-5
Day 8
Change Day 5-8
Total Change
1171
1
18
2
20
3
20
4
22
5
21
Mean
20
SD
1
Group Total
Mean
20
SD
1
(-) = No data available due to mortality.
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
Experimental Group
(ppm a.i.) 0
0
- 54 -
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 10
Bird
1 2 3 4 5 Mean SD
1 2 3 4 5 Mean
Day 8
27 34 42 40 50 39 9
35 35 37 43 39 38
Day15
42 56 69 63 78 62 14
Change Day8-15
15 22 27 23 28 23
5
Day22
61 77 92 84 104 84 16
55
20
74
53
18
74
64
27
85
74
31
103
59
20
80
61
23
83
Change Day 15-22
19 21 23 21 26 22
3
19 21 21 29 21 22
Total Change
34 43 50 44 54 45
8
39 39 48 60 41 45
0
1
30
54
24
79
25
49
2
29
45
16
63
18
34
3
32
54
22
77
23
45
4
37
55
18
75
20
38
5
40
68
28
95
27
55
Mean
34
55
22
78
23
44
SD
5
8
5
11
4
8
Group
Mean
37
59
23
82
22
45
WI LDLI FE INTERNATIoNAL , LTD
PROJECT NO.: 454-103
- 55 -
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 11
Experimental Group
(ppm a.i.)
18.3
Bird
1 2 3 4 5 Mean SD
Day 8
34 39 35 46 46 40
6
Day 15
Change Day 8-15
Day 22
Change Day 15-22
53
19
78
25
65
26
85
20
60
25
86
26
73
27
95
22
71
25
92
21
64
24
87
23
8
3
7
3
Total Change
44 46 51 49 46 47
3 -
Experimental
Group
(ppm a.i.)
Bird Day 8
36.6
1
35
2
39
3
37
4
38
5
43
Mean
38
SD
3
Day 15
Change Day 8-15
Day 22
Change Day 15-22
60
25
81
21
65
26
91
26
65
28
91
26
61
23
83
22
72
29
97
25
65
26
89
24
5
2
7
2
Total Change
46 52 54 45 54 50 4
WILD LIFE INTERNATI oNAL , LTD
- 56 -
PROJECT NO.: 454-103
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 12
Experimental Group
(ppm a.i.)
73.2
Bird
1 2 3 4 5 Mean SD
Day 8
32 33 36 39 36 35 3
Day 15
Change Day 8- 15
Day 22
Change Day 15-22
54
22
76
22
58
25
77
19
62
26
79
17
65
26
87
22
59
23
78
19
60
24
79
20
4
2
4
2
Total Change
44 44 43 48 42 44
2
Experimental
Group
Change
Change
(ppm a.i.)
Bird
Day 8
Day 15 Day 8-15 Day 22 Day 15-22
146
1
35
58
23
81
23
2
32
55
23
77
22
3
4
38
63
25
81
18
5
32
57
25
78
21
Mean
34*
58
24
79
21
SD
3
3
1
2
2
(-) = No data available due to mortality. *Statistically different from the control group at p < 0.05 (Dunnett's t-test).
Total Change
46 45
43 46 45
1
WILDLIFE INTERNATIONAL,LTD
- 57 -
PROJECT NO.: 454-103
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 13
Experimental
Group
(ppm a.i.)
Bird
Day 0
293
1
21
2
3
4
5
Mean
21
SD
(-) = No data available due to mortality.
Day 5 35
Change Day 0-5
14
Day 8 55
Change Day 5-8
20
35
14
55
20
Total Change
34
34
WILD LIFE INTERNATIoNAL , LTD
PROJECT NO.: 454-103
- 58 -
APPENDIX VI1
Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 1
Experimental Group
(ppm a.i.)
Pen
Exposure Period Day 0-5
Post-Exposure Period Day 6-8
Control
1
9
12
2
7
12
3
8
7
4
12
12
5
9
9
6
10
9
Mean
9
10
SD
2
2
WILDLIFE INTERNATIONAL,LTD
PROJECT NO.: 454-103
- 59 -
APPENDIX VI1
Feed Consumption (ghirdday) by Pen from a Northern Bobwhite Acute
Dietary Toxicity Study with PFOS
Page 2
Experimental Group
Exposure Period
Post-Exposure Period
(ppm a.i.)
Pen
Day 0-5
Day 6-8
18.3
1
9
10
2
8
11
Mean
9
11
36.6
1
8
14
2
7
9
Mean
8
12
73.2
1
10
12
2
10
14
Mean
10
13
146
1
7
8
2
10
12
Mean
9
10
293
1
4
8
2
6
10
Mean
5
9
586
1
6
16
2
5
22
Mean
6
19
1171
1
4
2
4
Mean
4
- No data available due to adult mortality.
WILDLIFE INTERNATI oNAL , LTD
PROJECT NO.: 454-103
- 60 -
APPENDIX VI1
Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 3
Experimental Group
(ppm ai.)
Pen
Exposure Period Day 8-15
Post-Exposure Period Day 15-22
Control
10
14
10
12
7
12
Mean
9
13
SD
2
1
Experimental Group
(ppm a i )
Pen
18.3
1
36.6
1
73.2
1
146
1
293
1
Exposure Period Day 8-15 10 14 13 11 8
Post-Exposure Period Day 15-22 12 15 15 14 9
WILDLIFE INTERNATIONAL, LTD
-61 APPENDIX VI11 CHANGES TO PROTOCOL
PROJECT NO.: 454-103
1. The protocol was amended to indicate that bile will be collected from all study birds. The protocol was clarified to indicate the collection of liver from birds that died during the course of the study
2. Blood samples were collected on Day 8 and Day 22 in non-heparinized 5 ml borosilicate glass test tubes. The protocol indicated that heparinized vacutainers would be used.
3. The protocol was amended to change the test concentrations from 0,20,40, 80, 160,640 and 1280 ppm a i . , to 0, 18.3,36.6, 73.2, 146,293, 586 and 1171 pprn a.i. Test concentrations were changed to reflect the test substance purity given in the new certificate of analysis.
4. The temperatures from several brooder units on Day 4, 7, 8, and all brooder units on Day 22 were not recorded
5. The afternoon observations were inadvertently not recorded for 2 birds in the 640 ppm a.i. treatment group on April 26, 1999.
-
WILDLIFE INTERNATIONALLT,D
- 62 APPENDIX IX PERSONNEL INVOLVED IN THE STUDY
PROJECT NO.: 454-103
The following key Wildlife International Ltd. personnel were involved in the conduct or management of this study:
(1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) Sean P. Gallagher, Senior Biologist (4) Courtney Casey, M.S., Senior Biologist (5) Willard R. Nixon, Ph.D., Manager, Analytical Chemistry (6) Timothy Z. Kendall, Supervisor, Analytical Chemistry (7) Raymond L. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist
WILDLIFE INTERNATIONAL,LTD
- 63 APPENDIX X REVISED STUDY SUMMARY
PROJECT NO.: 454-103
This study was reported using a test substance purity of 90.49%. The test substance characterization was revised on 9/7/00, following study completion, and the punty was determined to be 86.9%. The Sponsor requested that the results of this study be revised to reflect the most recent purity analyses. The results previously presented were simply corrected by the ratio of the two purities or a factor of 0.9603. No attempt was made to recalculate analytical standard concentrations or measured concentrations from original raw data. As such, minor errors due to rounding may be present. Attached is the revised summary page from the final report and the most recent certificate of analysis.
AMENDED
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
-64REVISED STUDY SUMMARY BASED ON A PURITY OF 86.9%
SPONSOR'S REPPRESENTATIVE:
LOCATION OF STUDY, RAW DATA AND A COPY OF THE
Ms. Susan A. Beach
WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY:
NOMINAL TEST CONCENTRATIONS :
TEST DATES:
- LENGTH OF TEST:
TEST ORGANISM: SOURCE OF TEST ORGANISMS:
I1 AGE OF TEST ORGANISMS:
LC50:
95% CONFIDENCE LIMITS:
NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION:
454-1 03 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Dietary LC50 Study with the Northern Bobwhite 0, 17.6, 35.1, 70.3, 141, 281, 562 and 1125 ppma.i.
Experimental Start - April 22, 1999 Biological Termination - May 14, 1999 Experimental Termination - May 14, 1999 22 days Northern Bobwhite (Colinus virginianus) Wildlife International Ltd. Production Flock
I Easton, Maryland 21601
I 10 days at test initiation
212 pprn a.i. 158 and 278 ppm a.i.
70.3 ppm a.i.
70.3 ppm a.i.
AMENDED
WILDLIFE INTERNATIONALLTD,
- 65 -
PROJECT NO.: 454-103
Phone: (814) 231-8032
Inc. I Laboratories,
Ilege. PA 18801 www.cen1releb.m FQX'(814)P1-1253M(814)231-1580
LWTERIM CERTIFICATEOF ANALYSIS
Revision 3 Centre Analvticd Lnborntorles COA Reference # 023-018A
Twt N l w hrlty'
AppcIdentification
1. 'calcium' 2. Magnesium 3. sodium 4. Potassium' 5. Nickel
7. Mmganesc
. . W S )
Total % Impurity
I
POAA Residual Solvents (TGA)
Purity by DSC
inorganic Anions (IC) 1. chloride 2. Fluoride
' 3. Bmmide 4. NjtntC
5. Nitrite
6. Phorphate
7. Sulfate'
2. PFPA 3. HFBA
Elemental Analysis :
2. Hydrogen 3. Nitmgea 4. Sulfur
5 . Fluorine
COA023-018A
spednt~tiom Wbitc Crysbllme Powder
- Theoretical Valuc 17.8%
Thmtical Value = 0% ?hmretical Value = 0% 4. TheoreticalVdue = 5.95%
5. Theorrtical Value = W h
Ruult 86.9%
Cimfolms
Poaitivc
1. O.O05wt/wt.% 2. 0.001 W M . % 3. 1.439wtM.% 4. 6.849 wlhvt.% 5. co.oolWt/wt./D 6. 0.005 wtJwt.% 7. 6).001wt./Wc%
1.91 wt"iwt.% 8.41 wt/wt.%
None Detected
0.33 wtlwt.% None Detected Not Applicable'
1. co.015 Wt/W% 2. 0.59 wtJwt.99 3. 4.040wthvr% 4. *).009Wthvt% 5. d0.O06wt/wt% 6. -=0.007wLhuT4? 7. 8.76 Wt1wt.36
1. co.1 wt&%
2. <O.l wtlwt% 3. 0.10wt./Wt%
4. 0.28wt./Wt%
I . 12.48 wt.hwt.% 2. 0.244wt.h.%
3. 1.74wtJwt.% 4. 8.84wtJwt.% 5. 54.1 wtJwt%
P a s I of 3
AMENDED
WILDLIFE INTERNATIONALLT,D
- 66 -
PROJECT NO.: 454-103
Date of-
C u r t E Analytical Laboratorks, Inc.
3048 Research D h State College,PA 16601 www.centrehb.com
Phone: (814)231-8032
Fax: (814)231-1253or(E14)~1-1580
INTERIM CERTIFIC.4 TE OF ANALYSIS
Revirion 3
Centre Analytical LaboratorieJ COA Reference # 023418A
Analysis: 08/31/00
ExpirationDate: OW3 1/06
Storage Conditions: Frozen frloDC
Re-ass-cnt Date: 0813 1/06
'Purity = 1Wh- (sum of metat impurities, 1.45%+ L W impurities, 8.41%+hor@c
Fluoride, 0.59?h+NMRimpurities, 1.905%+0rganic acid impurities,0.38%+POAA.
0.33%)
- Total impurity from all tests = 13.07% purity 100% 13.07% = 86.9%
*Potassiumis expected in this salt form and is themfore not considered an impurity.
'Purity by DSC is generally not applicableto materialsof low purity. No mddthumwas observed for this sample.
'Sulfur in the sample appears to be convertedto SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determinationin the elemental analysis, lendingwnfidcncc to this intapretation. Based on the results,the SO,is not considered an impurity.
STPA KFEA NFTA PITA
Trifluoroacctic a c i d Heptaflmrobutyric acid Nonafluoropentwoic acid Pmtanuoropropanoic acid
%heoretical value calculationsbased on the cmpmcal formula, C~IF~~S@X+(MW=~~E)
This work was conducted under EPA Good LaboratoryPractice Stpndnrds (40 CFR 160).
COA023-018A
Page 2 Of 3
AMENDED
WILDLIFE INTERNATIONALL,TD
- 67 -
PROJECT NO.: 454-103
Cent= Analytical laboratories, Inc
E
r
n
Phone:(814) 231-8032
Fa:(814)231-1253 tX (814)231-1560
INTERM CERTIFICATEOF ANALYSIS
Rwfsion 3 Centre Analytical Laboratories COA Reference #: O23418A
LC/MS Purity Profile:
Imparity c4 c5 C6 c7
Total
Wwt % 1.22 1.33 4.72
1.14 8.41
Note: The C4 and C6 values were calculatedusingthe C4 and C6 standardcalibration curves, respactively. The C5 value was calculated using the average result from the C4
and C6 standard e w e s . Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves.
Scientist, Cenhe Analytical Laboratories
j6hnFlahnty
/
Date
Laboratory Manager,Centre Analytical Laboratories
COAO23-018A
Page 3 af 3
AMENDED
~
_
_
WILDLIFE INTERNATIONAL, LTD
PROJECT NO.: 454-103
- 68 APPENDIX XI
Report Amendment
1.
Original Report:
Title Page
Amended Report:
The amended report date was added. The total number of pages
was changed from 62 to 69.
Reason:
To indicate that the report was amended and note change in
pagination.
2.
Original Report:
Amended Report:
Reason:
Page 2 The amended report date was added and the exceptions to the compliance statement were revised. To show the amended report date, provide new signatures and dates for the amended report and to update the compliance statement.
3.
Original Report:
Amended Report:
Reason:
Page 3 The audit dates for the amended report were added. To show the amended report audit dates and to provide a new signature and date for the amended report.
4.
Original Report:
Page 4
Amended Report:
New signatures and dates were affixed and a title change was
made.
Reason:
To provide signatures and dates of approval for the amended
report and to update a manager's title.
5.
Original Report:
Amended Report:
Reason:
Page 6 Appendix X, Revised Study Summary, and Appendix XI, Report Amendment, were added to the Table of Contents. To revise the Table of Contents to include the revised summary and report amendment.
6.
Original Report:
Page 7
Amended Report:
A footnote and the amended report date were added to the
Summary page.
Reason:
To direct the reader to Appendix X, which contains the revised
study summary and to show the amended report date.
7.
Original Report:
Amended Report:
Reason:
Page 8 A footnote was added to the Test Substance text. To direct the reader to Appendix X, which contains the most recent Certificate of Analysis.
AMENDED
WILDLIFE INTERNATIONAL,LTD
- 69 APPENDIX XI Report Amendment (continued)
PROJECT NO.: 454-103
8.
Amended Report:
Reason:
9.
Amended Report:
Reason:
Pages 63-67, Revised Study Summary, were added to the report. To provide additional information.
Pages 68 and 69, Report Amendment, were added to the report. To provide details of changes made to the original report.
AMENDMENT SIGNATURES:
Study Director
Wildlife International, Ltd. Management
REVIEWED BY:
Quality Assurance
&cu?
Date Date
3-22 -04
Date
AMENDED
