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PFOS: A DIETARY LC50 STUDY WITH THE MALLARD
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 3M REQUEST NO. U2723
U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft)
OPPTS Number 850.2200
FIFRA Subdivision E, Section 71-2
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 55144
Wildlife International, Ltd.
8598 Commerce Drive Easton, Maryland 21601
(410) 822-8600
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GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation
TITLE: PFOS: A Dietary LC50 Study with the Mallard
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102
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, (OCDE/GD(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 o f 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:
__________________d a t e
Sean P. Gallagher
'
Senior Biologist, Avian Toxicology
________
SPONSOR'S REPRESENTATIVE
ms. ouscui /v Dcawu
DATE 3
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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 & 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
Matrix Fortification, Test Substance Prep., Analytical Sampling
April 22, 1999
Feed Consumption and Analytical Sampling April 27, 1999
Necropsy, Blood Collection,
Liver Weights and Bile
Collection
May 14, 1999
Analytical Data and Draft Report
July 7, 8, and 9, 1999
Biological Data and Draft Report
Aug. 30, 31, Sept. 1, 1999
Final Report
April 18, 2000
Amended Final Report
March 19, 2004
DATE REPORTED TO. STUDY DIRECTOR MANAGEMENT
April 22, 1999 April 27, 1999
April 23, 1999 May 4, 1999
May 14, 1999
May 21, 1999
July 9, 1999
July 16, 1999
September 1, 1999 April 18, 2000
September 3, 1999 April 19, 2000
March 19, 2004
March 19, 2004
____ 'S u L iA rtn ^ . G o l o n o O n _____________ DATE Susan L. Coleman
Senior Quality Assurance Representative
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AMENDED REPORT APPROVAL
SPONSOR: 3M Corporation
TITLE: PFOS: A Dietary LC50 Study with the Mallard
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102
3M LAB REQUEST NO.: U2723
STUDY DIRECTOR:
P.
Sean P. Gallagher
l
Senior Biologist, Avian Toxicology
QV\ Date
CHEMISTRY PRINCIPAL INVESTIGATOR:
Scientist, Analytical Chemistry
REPORT APPROVED BY:
Linda R. Mitchell Manager of Ecotox Operations
Willard B. Nixon, Ph.Dy Director of Chemistry
Z3 Date
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TABLE OF CONTENTS
TITLE PAGE.............................................................................................................................Page 1
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT................................ Page 2
QUALITY ASSURANCE STATEMENT..............................................................................Page 3
REPORT APPROVAL............................................................................................................. Page 4
TABLE OF CONTENTS.......................................................................................................... Page 5
Tables and Appendices........................................................................................................... Page 6 SUMMARY...............................................................................................................................Page 7 INTRODUCTION.................................................................................................................... Page 8 OBJECTIVE..............................................................................................................................Page 8
MATERIALS AND METHODS..............................................................................................Page 8
Test Substance.........................................................................................................................Page 8 Treatment Groups....................................................................................................................Page 9 Duration of the Test................................................................................................................ Page 9 Test Birds................................................................................................................................. Page 9 Animal D ie t..................................................................................................................................Page10 Diet Preparation.......................................................................................................................Page 10 Diet Sampling...............................................................................................................................Page10 Analytical Method........................................................................................................................Page10 Housing and Environmental Conditions..................................................................................... Page13 Observations............................................................................................................................Page 13 Animal Body Weights/Feed Consumption................................................................................. Page14 Gross Necropsy.............................................................................................................................Page14 Statistical Analyses.......................................................................................................................Page14
RESULTS.......................................................................................................................................Page14
Diet Analysis................................................................................................................................Page14 Mortalities and Clinical Observations................................................................................... Page 15 Body Weight and Feed Consumption......................................................................................... Page16 Gross Necropsy........................................................................................................................Page 17
CONCLUSION.............................................................................................................................. Page18
REFERENCES...............................................................................................................................Page19
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TABLE OF CONTENTS - continued -
TABLES AND APPENDICES
TABLE 1: Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS............................................................................................. Page 20
TABLE 2: Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS............................................................................................. Page 21
TABLE 3: Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS......................................................Page 23
TABLE 4: Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFO S................................................................ Page 25
TABLE 5: Cumulative Mortality (Estimated Cumulative Dose, mg/kg) from a Mallard Acute Dietary Toxicity Study with PFOS....................................... Page 26
APPENDIX I: Certificate of Analysis................................................................................ Page 27
APPENDIX II: Diet Formulation......................................................................................... Page 28
APPENDIX III: Analytical Methods and Results................................................................Page 29
APPENDIX IV: Diet Preparation.......................................................................................... Page 43 APPENDIX V : Cumulative Mortality by Pen from a Mallard
Acute Dietary Toxicity Study with PFOS.............................................. Page 44
APPENDIX VI: Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFO S.............................................. Page 45
APPENDIX VII: Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFO S.................................Page 60
APPENDIX VIII: Changes to Protocol.................................................................................... Page 64
APPENDIX IX: Personnel Involved in the Study................................................................ Page 65
APPENDIX X: Revised Study Summary.............................................................................Page 66
APPENDIX XI: Report Amendment..................................................................................... Page 71
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SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY: PFOS: A Dietary LC50 Study with the Mallard
RESULTS':
The dietary LC50 value for Mallards exposed to PFOS was determined to be 628 ppm a.i. with a 95% confidence interval o f 448 ppm a.i. to 958 ppm a.i. The slope o f the concentration-response curve was 3.67 and the chisquaTe value was 2.13. The no mortality concentration was 146 ppm a.i. Based upon reductions in body weight gain at the 73.2 ppm a.i. test concentration, the no-observed-effect concentration was 36.6 ppm a.i.
TEST DATES:
Hatch - April 13, 1999 Acclimation - April 14-22, 1999 Experimental Start-April 22, 1999 Experimental Termination-M ay 14,1999
NOMINAL TEST CONCENTRATIONS: 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586, and 1171 ppm a.i.
TEST ANIMALS:
Mallard (Anasplatyrhynchos)
AGE TEST ANIMALS: 10 days of age at test initiation
SOURCE TEST ANIMALS:
Whistling Wings, Inc. PO Box 1-A 113 Washington St. Hanover, IL 61041-3512
STUDY COMPLETION: April 26, 2000
AMENDED REPORT DATE: March 23, 20041
1See Appendix X for the revised nominal test concentrations and results based upon the reanalysis o f 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. 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 454-102 in archives located on the Wildlife International Ltd. site.
OBJECTIVE The objective o f this study was to evaluate the toxicity of a test substance to the mallard (Anas platyrhynchos) 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 o f 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 from 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 purity 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 purity of 90.49%. All test concentrations have been adjusted to reflect the purity reported on the new Certificate of Analysis (Appendix I)1. 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
1Following 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: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions
Treatment Groups The test consisted of a geometric series of eight test concentrations and a control group. Thirty
mallard ducklings were assigned to the control group and ten mallard ducklings were assigned to each of the treatment groups. 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 ducklings each. On Day 0, following experimental start, the Sponsor requested the addition of a 10 ppm test concentration. Birds from the same lot that remained after test initiation were used for the additional test concentration. Nominal dietary concentrations used in this study were 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 parts per million active ingredient (ppm a.i.). 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 treatment and control birds were euthanized and liver weight and tissue, blood, and bile samples collected for analysis. The remaining birds were fed basal ration until Day 22. On Day 22, these birds were sacrificed and also sampled for liver weight and tissue, blood, and bile.
Duration of the Test The primary phases of this test and their durations were:
1. Acclimation - 9 days. 2. Exposure - 5 days. 3. Post-exposure observation - 3 or 17 days
Test Birds All mallard (Anas platyrhynchos ) were 10 days of age and appeared to be in good health at
initiation of the test. The birds were obtained from Whistling Wings Inc., Hanover, IL. and were hatched on April 13, 1999. Birds ranged in weight from 96 to 204 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex. All birds were from the
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same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day after 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 II). 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 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 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 9.1 ppm a.i. a.i. and six samples from the 1171 ppm a.i. a.i. test diets 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. 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.
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 o f PFOS
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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% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so they fell within the calibration range of the PFOS methodology. A method flowchart is provided in Appendix III, 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 100LC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Ct8 analytical column (100 mm x 2 mm I.D., 3 pm particle size). The instrument parameters are summarized in Appendix III, Table 1.
Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure 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 III, 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 III, Figures 3 and 4, respectively.
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).
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Two matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during sample analyses (Appendix III, 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 III, 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 III, 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 III, Figure 6.
An example calculation is presented for sample number 454-102-2, nominal concentration of 18.3 ppm a.i. in avian diet.
Initial Weight: 10.0 g Final Volume: 200 mL Dilution Factor: 100 (intermediate dilution factor x final dilution factor) PFOS Peak Area: 113568 Internal Standard Peak Area: 413160 Peak Area Ratio: 0.2749
Calibration curve equation. Slope: 2.77397 Intercept: 0.01894 Curve is weighted (1/x).
PFOS (mg a.i./L) at instrument
(Peak area ratio - (Y-intercept)) x I.S. Concentration Slope
(0.2749 - 0.01894) x 0.100 mg/L 2/77397
= 0.00923 mg a.i./L
Note: I.S. = internal standard.
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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 --
= 18.5 ppm a.i.
Percent of Nominal 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 o f thermostatically
controlled brooding pens manufactured by Safeguard Products, Inc. Each pen had floor space that measured approximately 62 X 90 cm. Ceiling height was approximately 25.5 cm. 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 wing bands.
During the test the average temperature in the brooding compartment of the pens was 38C 2C (SD). Average ambient room temperature for this study was 25.2C 0.7C (SD) with an average relative humidity of 53% 18% (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 approximately 207 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
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observed at least once daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior.
Animal Body Weiehts/Feed Consumption Individual body weights were measured at the initiation o f the test, on Day 5, on Day 8 for all
birds, 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 half of the 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 Necropsy 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, liver weight and liver tissue, serum and blood solids, 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 Analyses 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 by 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 (10,11). No statistical analyses were applied to feed consumption data.
RESULTS
Diet Analysis Avian diet samples were collected from the 9.1, 18.3 and 1171 ppm 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
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concentrations, standard deviations and coefficients of variation (CV) for these test concentrations were 9.8 0.969 ppm a.i. (CV = 9.94%), 19.5 2,13 ppm a.i. (CV = 10.9%) and 1196 70.2 ppm a.i. (CV = 5.87%), respectively (Appendix III, 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 ppm a.i., respectively. These values represented 110, 102, 119, 99.3 and 91.6% of the nominal concentrations, respectively (Appendix III, Table 5). Analysis of avian diet samples collected from feeders after being held at ambient temperature for five days averaged 119, 97.4, 130, 104, 94.3, 101, 101 and 95.0% of the Day 0 values for the 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. test substance concentrations, respectively (Appendix III, Table). A representative chromatogram of a test sample is shown in Appendix III, Figure 7.
Mortalities and Clinical Observations No mortalities occurred in the control group, and all birds were normal in appearance and
behavior throughout the test. There were no treatment-related mortalities or overt signs of toxicity in the 9.1, 18.3, 36.6, 73.2, or 146 ppm a.i. treatment groups (Table 1 and Appendix V). However, there was 20% (2 of 10) mortality in the 293 ppm a.i. treatment group, 30% (3 of 10) mortality in the 586 ppm a.i. treatment group and 90% (9 of 10) in the 1171 ppm a.i. treatment group.
In the 293 ppm a.i. treatment group single mortalities were noted on the mornings of Day 7 and Day 8. Signs of toxicity were first observed on the morning of Day 4 and continued until termination for those birds sacrificed on Day 8 and until the afternoon of Day 8 for those birds sacrificed on Day 22. All birds in this treatment level displayed signs of toxicity. These included reduced reaction to stimuli (sound and motion), loss of coordination, ruffled appearance, lethargy, and lower limb weakness. All remaining birds appeared to have recovered and were normal in appearance and behavior from the morning of Day 9 until test termination.
At the 586 ppm a.i. treatment level there were three treatment-related mortalities, one each noted on the morning of Day 5, Day 6, and Day 7. Signs o f toxicity were first observed on the morning of Day 3 and continued until termination for those birds sacrificed on Day 8 and until the afternoon of Day 8 for those birds sacrificed on Day 22. All birds in this treatment level displayed signs of toxicity. These included reduced reaction to stimuli (sound and motion), ruffled appearance, lower
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limb weakness, and lethargy. All remaining birds appeared to have recovered and were normal in appearance and behavior from the morning of Day 9 until test termination.
In the 1171 ppm a.i. treatment group there were nine treatment-related mortalities. The first two mortalities were found on the morning of Day 4, followed by four mortalities on Day 5, two mortalities on Day 6 and the final mortality on Day 7. Overt signs of toxicity were first observed on the morning of Day 2 and the single surviving bird continued to display signs of toxicity through the afternoon of Day 8. Signs of toxicity observed among birds in the 1171 ppm a.i. treatment group included reduced reaction to stimuli (sound and motion), loss of coordination, prostrate posture, depression, convulsions, ruffled appearance, lower limb weakness, and lethargy. The one surviving bird appeared to have recovered and was normal in appearance and behavior from the morning of Day 9 until test termination.
Body Weight and Feed Consumption When compared to the control group, there were no apparent treatment related effects on body
weight among birds in the 9.1, 18.3, or 36.6 ppm a.i. treatment groups. There was a statistically significant (p<0.05) reduction in weight gain at the 9.1 ppm a.i. level for the Day 0-5 period that was reflected in significantly lower (p< 0.05) mean Day 5 body weight. When compared to the control group, reductions in weight gain were also statistically significant (p<0.05) for the Day 5-8 period, resulting in a significantly (p<0.05) lower Day 8 mean body weight. However, differences from the control group at the 9.1 ppm a.i. level appear to be due to a lower mean Day 0 body weight for the 9.1 ppm a.i. level and were not dose responsive. Therefore, these differences were not considered treatment related.
There was a marked, treatment-related, concentration-responsive effect on body weight in the 73.2, 146, 293, 586, and 1171 ppm a.i. treatment groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). These reductions were statistically significant at p<0.05 for the 73.2 ppm a.i. level and at p<0.01 at the 146, 293, 586 and 1171 ppm a.i. levels. During the Day 5-8 post-exposure period, weight gain continued to be reduced for surviving birds in the 293, 586 and 1171 ppm a.i. treatment groups. These reductions were statistically significant at p<0.01 for the 293 and 586 ppm a.i. levels. Differences at the 1171 ppm a.i. level were marked but could not be statistically compared. Body weight losses and reductions in weight gain at the 293, 586 and 1171 ppm a.i. levels were reflected in significantly (p<0.05 or p<0.01) lower mean body weights on Days 5, 8, 15, and 22
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of the test. At the 146 ppm a.i. level mean body weights on Days 8 and 22 were also significantly (p<0.05) lower than the control group. During the Day 8-15 and Day 15-22 post-exposure periods, body weight gain appeared comparable among all groups.
When compared to the control group, there was a marked reduction in feed consumption in the 293, 586, and 1171 ppm a.i. treatment groups during the exposure period (Days 0-5). A reduction in feed consumption continued to be observed through Day 15 in the 293, 586 and 1171 ppm a.i. treatment groups.
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, empty crops, and empty gastrointestinal tracts. Findings were considered to be treatment related. 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 on Day 22, following test termination. The necropsy results for birds euthanized on Day 8 and Day 22 from the 18.3, 146 and 1171 ppm a.i. treatment groups were unremarkable. One bird in the control group and one bird in the 36.6 ppm a.i. treatment group were noted with retained yolk sacs during necropsy of birds on Day 8. A single bird in the 9.1 ppm a.i. treatment group necropsied on Day 8 was noted as small, with a lack of muscle mass. In the 73.2 ppm a.i. treatment group one bird necropsied on Day 8 was small, with a lack of muscle mass, and had a 2 x 0.5 cm abscess containing caceous necrotic material on the right side of the abdominal wall. Due to the nature o f the lesions observed in birds, and the isolated incidence o f occurrence, the findings listed above in euthanized birds from the 9.1, 36.6, and 73.2 ppm a.i. treatment groups were not considered to be related to treatment. In addition, all remaining birds in the control, 9.1, 36.6, and 73.2 ppm a.i. treatment groups were non remarkable.
In the 293 ppm a.i. treatment group, one bird euthanized on Day 8 was noted with an incidental finding of a retained yolk sac. A second bird in the 293 ppm a.i. group, necropsied on Day 8, was noted as emaciated with a lack of muscle mass. This loss of muscle mass was consistent with reduced weight gain for the level and was considered treatment related. Another bird in the 293 ppm a.i. treatment group, euthanized on Day 22, was noted with 2 cm cysts containing yellow fluid on the
00706
W il d l if e In t e r n a t io n a l , ltd
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PROJECT NO.: 454-102
anterior portion of the left lobe of the liver. Due to the nature of the cysts and the isolated incidence of occurrence, this observation was not considered treatment related.
Three birds in the 586 ppm a.i. treatment level were euthanized on Day 8. At necropsy, they were noted to be thin and lacking muscle mass. One bird was noted with a slightly pale liver. These findings are considered to be treatment related. Four birds in the 586 ppm a.i. treatment level survived until Day 22. Necropsy results for these four birds were not remarkable.
CONCLUSION
The dietary LC50 value for Mallards exposed to PFOS was determined to be 628 ppm a.i. with a 95% confidence interval of 448 ppm a.i. to 958 ppm a.i.. The slope of the concentration-response curve was 3.67 and the chi-square value was 2.13. The no mortality concentration was 146 ppm a.i.. Based upon reductions in body weight gain at the 73.2 ppm a.i. test concentration, the no-observedeffect concentration was 36.6 ppm a.i..
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W il d l if e In ter n atio na l, ltd
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PROJECT NO.: 454-102
REFERENCES
1 U. S. Environmental Protection Agency, 1996. Series 850- Ecological Effects Test Guidlelines {draft), OPPTS Number 850.2200: Avian Dietary Toxicity Test.
2 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife 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 fo r the Care and Use o f Laboratory Animals. Washington, D.C. National Academy Press. 125 pp.
6 Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA.
7 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication.
8 Finney, D.J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London.
9 Thompson, W.R. 1947. Bacteriological Reviews, Vol 2, 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.
6-00708
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PROJECT NO.: 454-102
TABLE1 Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
No. Dead Per No. Exposed Exposure Period
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5
No. Dead Per No. Exposed Post-Exposure Period
Day 6 Day 7 Day 8 1
Control 0
0/30 0/30 0/30 0/30 0/30 0/30
0/30 0/30 0/30
Treatment 9.1 18.3 36.6 73.2 146 293 586 1171
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10 2/10 6/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 2/10 2/10 3/10 3/10 8/10 9/10 9/10
The LC50 value was calculated to be 628 ppm a.i. with a 95% confidence interval o f 448 ppm a.i. to 958
ppm a.i.
1- No mortalities occurred in any of the treatment levels from Day 8 to Day 22.
____
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Wild life International, ltd
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PROJECT NO.'. 454-102
TABLE 2 Page 1
Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Exposure Period Change
Post-Exposure Period Change1
Control 0
Mean SD
135 279 24 42
144 22
380 101 53 16
Treatment 9.1
Mean SD
119
226**
108**
14 36
23
317* 66
91 33
18.3 Mean SD
146 277 14 26
131 18
377 100 32 11
36.6 Mean SD
147 275 16 32
128 22
375 100 37 12
73.2 Mean SD
143 260 30 55
117* 33
343 82 90 42
146 Mean SD
143 242 19 37
100** 21
331 89 48 16
293 Mean SD
129 161** 32**
17 28
33
220** 55
57 44
586 Mean SD
144 137** -6**
23 27
26
175** 34
36 41
1171 Mean SD
147
112**
-37**
25 28
13
1852
-
Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI).
2n=l, could not be evaluated statistically using Dunnett's t-test. (-) = No data available due to mortality. *Statistically different from the control group at p < 0.05 (Dunnett's t-test).
3l2
-
245 34
198* 55
231 25
228 30
200* 73
188** 32
94** 54
39** 36
152
-
C9 0
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PROJECT NO.'. 454-102
TABLE 2 Page 2
Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Day 8
Post-Exposure Period
Change'
Day 15 Day 8-15
Day 22
Change'
Total
Day 15-22 Change1(8-22)
Control 0
Treatment 9.1
18.3
36.6
73.2
146
293
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
410 640 230 30 56 47
345* 33
384 27
377 33
364 82
332* 58
211** 49
575 32
625 42
620 44
579 97
564 56
467** 51
230 20
241 22
243 40
216 21
232 22
256 35
823 183 49 24
773 38
811 81
828 78
782 135
688* 86
701* 41
198 15
186 41
208 34
203 39
124* 90
234 35
413 42
427 34
427 62
451 71
418 56
356 106
490 54
586 Mean SD
174** 46
394** 82
221 37
613** 91
219 13
1171 Mean
1852
3732
1982
6342
2 5 12
SD
- --
--
'Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). 2n=l, could not be evaluated statistically using Dunnett's t-test. (-) = 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).
439 47
4492 -
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PROJECT NO.: 454-102
TABLE 3 Page 1
Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Control
0
Exposure Period Day 0-5
Mean SD
92 10
T reatm ent 9.1 18.3 36.6 73.2 146 293 586 1171
(-) = No data available due to mortality.
73 91 94 77 105 44 36 22
Post-Exposure Period Day 6-8
125 17
117 132 125 101 159 63 55 25
C0 0 7 1 2
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PROJECTNO.: 454-102
TABLE 3 Page 2
Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS
Experimental Group
(ppm a.i.)
Control
0
Mean SD
Post-Exposure Period
Day 8-15
Day 15
171 180 13 20
Treatment 9.1 18.3 36.6 73.2 146 293 586 1171
(-) = No data available due to mortality.
172 186 165 148 159 109 114 106
198 204 179 173 164 132 143 154
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PROJECT NO.: 454-102
TABLE 4
Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFOS
Birds that died during the course o f the study
Finding
Crop empty Emaciated G.I. Tract, primarily empty Gizzard contents bile stained Gizzard, empty Intestinal contents, black and tar-like Keel prominent Kidneys, pale Loss of muscle mass Spleen, grey Spleen, small and pale Spleen, pale Thin
Male, Female, and Undetermined _____________ PPM A.I.__________
293 586 1171 N=2 N=3 N=9
027 1 14 111 024 00 1 00 1 002 00 1 225 00 1 1 12 023 1 14
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cn
W il d l if e In t e r n a t io n a l , ltd
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PROJECT NO.: 454-102
APPENDIXI
Certificate O f Analysis
FC-95, Lot 217
March 9,2000
Richard M. Payfer
This sample w u analyzed using LC/MS, lH-NMR, I*F-NMR, and elementalanalyses techniques. The resalts Vi these tests &ow ttw.ssmple to contain tbs M ow ing weight p a te n t composition:
CjFsSOj'K*'
w sf cx& onr C sF iiS O air CsFhSOiTC S p ijSiTC C iF.rS O jT C C F ifS O iT C 7
:
"
C,oPs,SO jT^ _ C nM ^
:
ra rSFaO FisSO sX
0.04 34 0.83%
138 % 1.30% 3.71%
1.19% -scdS "" TP9H
0.13% 0.04% 05534 035%
Additionally, the isomer distribution o ftb s sample was determined using 'T-NM R tachniquea and found to contain the M ow ing mole percent composition;
CV^O/fr-SQ, X (Normal chain, where x is mainly 7)
C T K C F rlx ^ C F rH C F iy SOj-XT (Internal nnomethyl Winch,
where x fy Is mainly 3. and x * 0. y * d
(CF.JjCP-CCFjV S C h T ilsoOTonyl branch. where x is mainly 5)
ClFa 1-C F(C Fj)-SO j'K T (Aloha branch. w hen x la mainlv 6)
(CF)iC<CFi)i* 8 0 s' K1, (t-iwtyl branch, where x is maM v 4)
CTsKGFsMXCPiMCaPslr SOj 1C (Internal gem-dimethyl branch, where x+y
ism ninlv4.*ndx*0)
70.3%
17.1% 10.3% 1.6% 03%
0.234
G0 0 7 3 .6
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PROJECT NO.: 454-102
APPENDIX II
DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION1
INGREDIENTS
Fine Com 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)2 Salt Iodized Total
PERCENT (%)
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 Bn Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium
2.000. 0001.C.U. 7.000. 0001.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
'The guaranteed analysis is a minimum o f 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber.
fermentation By-Products (Source of Unidentified Growth Factors)
000717
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PROJECT NO.: 454-102
APPENDIX III ANALYTICAL METHODS AND RESULTS
000718
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PROJECT NO.: 454-102
INSTRUMENT:
Appendix III Table 1
Typical LC/MS Operational Parameters
Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM).
ANALYTICAL COLUMN:
Keystone Betasil Cis column (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% NANOpure Water containing 0.1% Formic Acid
INJECTION VOLUME:
25.0 pL
PFOS RETENTION TIME:
Approximately 7.0 minutes
INTERNAL STANDARD RETENTION TIME:
Approximately 4.8 minutes
PFOS MONITORED MASS:
INTERNAL STANDARD MONITORED MASS:
498.6 amu 426.7 amu
C CO7 1 .9
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PROJECT NO.: 454-102
Appendix III Table 2
Matrix Blanks Analyzed Concurrently During Sample Analysis
Number (454-102-)
MAB-1
Sample
Type Matrix Blank
Measured Concentration of " PFOS'
(ppm a.i.)
< LOQ
MAB-2
Matrix Blank
< LOQ
'T he limit o f quantitation (LOQ) was 1.15 ppm a.i. based upon the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500).
CC0720
W il d l if e Internatio nal, ltd
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PROJECT NO.: 454-102
Appendix III Table 3
Matrix Fortifications Analyzed Concurrently During Sample Analysis
Sample Number (454-102-)
M A S -1A MAS-4A
Concentrations o f 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 1716
86.1 93.7
Mean = Standard Deviation =
CV = N=
94.7 6.99 7.38
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.
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ati :b a k
( 0 ).
T e 3 y 5 ^ in i c ai ii e t A h h I a ( n a i is ir d :on e it it oi \ il e b t S 1 r t . 4 i y c 1 K u ir 1/ t a: i: ii i :a ci
H mm mm
O O O *\
*
W il d l if e In t e r n a t io n a l , ltd
-36-
PROJECT NO.: 454-102
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.
4
Dry Mix Technique
For the high-level matrix fortification sample, weigh the requisite quantity of Wildlife International Ltd. (W1L) ration into a weigh boat.
4
Weigh the requisite quantity of test substance (PFOS) into a beaker.
4
Add V4of the W1L ration and the test substance to a larger beaker.
4
Rinse the beaker that contained the PFOS with small portions of the remaining ration and transfer all portions to the larger beaker.
4
Mix the contents of the larger beaker well and transfer the mixture to a Waring blender.
4
Blend the mixture for -5 minutes stopping at 1 minute intervals to scrape down the sides of the blender. During the third interval transfer the fortified feed to a beaker, mix well and return the mixture to the blender to complete mixing in the specified
time.
4
Prepare 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.
4
Weigh 10-g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 16-oz. French-square glass bottles. Record the weights.
4
For each sample, measure 100 mLs of methanol with a graduated cylinder and transfer volume to the French-square bottle.
4
Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at 250 tpm.
4
Vacuum filter each sample with qualitative filter paper and rinse retained feed 3 times with methanol into the filtrate.
4
Transfer the filtrate to a 200-mL volumetric flask and bring the flask to volume with methanol.
4
Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. Use methanol for intermediate dilutions, if required.
For all final dilutions use 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v).
4
Ampulatc and submit samples for LC/MS analysis.
Figure 1.
Analytical method flowchart for the analysis of PFOS in avian diet.
c00725
W il d l if e In te r n a tio n a l , ltd
-37-
Appendix III
PROJECT NO.: 454-102
000726
C00727
W il d l if e In t e r n a t io n a l , ltd
-39-
Appendix III
PROJECT NO.: 454-102
Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard.
00728
W il d l if e In t e r n a t io n a l , ltd
-40-
Appendix III
PROJECT NO.: 454-102
Figure 5. A representative chromatogram o f a matrix blank sample (454-102-MAB-l). The arrow indicates the retention time of PFOS.
00729
W il d l if e In t e r n a t io n a l , ltd
-41 -
Appendix III
PROJECT NO.: 454-102
100908070 6050403020IQ-
24
31 1.30
7,1 f f l s ,
61 2.55
91 3.81
intensity: 6238 cps
Figure 6. A representative chromatogram of a matrix fortification sample (454-102-MAS-1A).
C0 0 7 3 0
W il d l if e In t e r n a t io n a l , ltd
-42-
Appendix III
PROJECT NO.: 454-102
10090807060504030-
20-
10o-V
intensity: 7248 cps 167
31 1.30
61 2.55
T--r-f 91
3.81
123 I
121
5.06
I ifnnijiHi i i t i i l
151 181 211 6.32 7.57 8.83
Scan Time
Figure 7. A representative chromatogram of a test sample (454-102-2).
W il d l if e In te r n a tio n a l , ltd
-43 -
PROJECT NO.: 454-102
APPENDIX IV DIET PREPARATION Weight and volume of constituents used to prepare test diets:
Nominal Concentrations
(ppm a.i.)
0 9.1 18.3 36.6 73.2 146 293 586 1171
Test Substance
(g)
0.0910 0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 11.6483
Basal Ration
(g) 9000.0 8999.9 8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4
Diets were prepared as follows:
5000.0 g o f 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.
C0 0 7 3 2
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- M'
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---- ------------------------------
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E er iei] 1
N Dd
ih Ih 17 u t Di i r To cil St ly
-- -- * -- _ 5^- =s_ s s r i . E )Oi 1
iti 'E 3
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Gr ip pr i.i.
en Di 0
)ay 1
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D '2
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*os y ay
101 rol
1 C 0/5 1 5 0, o/: /5
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lC
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/5
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i 0 0/5 1 5 0, o/: 15
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re m it .1 .1 8.: 8.: 6.f 6.i 3.: 3.: 46 46 93 93 86 86
i c 0/5 1 5 0, or. 15 2 c 0/5 I ) 0, or. 15 1 0 0/5 ( > 0, or. 15 l 0 0/5 I > 0, or. 15 1 0 0/5 i 5 0, or. 15 l 0 0/5 ( 5 0, or. 15 1 0 0/5 1 5 0/ or. 15 l 0 0/5 1 > 0/ or. 15 l 0 0/5 1 5 0/ or. 15 l 0 0/5 ( ) 0/ or. 15
1 0 0/5 1 5 0/ 0/i /5 l 0 0/5 ( > 0/ or. 15
0 3/5 c 0/ )/5 15 ) 0 3/5 c 0/. )/5 15
3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 1/5
17
0 3/5 c 0/. 75 15
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17
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i/5
he 3
/al w; cal lat to T: pp a.i /ith a W on len in va f4
a PI a.: 0 $ iPl i a.
**
- i smss aslsi st ur i i ny th rea ieri >rcon ol 22 aft D 8ss.s ss SS SS s s ss
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s ,, s __ 1F r i 3.
xf SU : F ri(
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i y
( ) )/: ( 5 )/: ( > )r. ( )/: ( j 3/5 C j 3/5
( > 3/5 ( j )/; ( i 3/5 ( 5 3/5 ( i 3/5 ( i 3/5 C > 3/5 ( > 3/5 C i 3/5 C i Mi C > Mi 1 ! ili 1 ; \i 2 ; Hi 5 ; Mi 4 i Mi
=_ ==
__ __ ggg T __
W il d l if e In t e r n a t io n a l , ltd
-45APPENDIX VI
PROJECT NO.: 454-102
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 1
Experimental Group
(ppm a.i.)
Bird
01 2 3 4 5
Mean SD
Day 0
132 136 154 158 169 150
15
Day 5
302 292 319 319 309 308
12
Change Day 0-5
170 156 165 161 140 158
12
Day 8
404 381 415 429 418 409
18
Change Day 5-8
102 89 96 110 109 101
9
Total Change (0-8)
272 245 261 271 249 260
12
0
1 136 282
146 399 117
2 142 284 142 394 110
3 152 299
147 409 110
4 152 312 160 430 118
5 177 345
168 484 139
Mean
152
304
153 423 119
SD 16
26
11
37 12
263 252 257 278 307 271
22
0
1 125 265
140 372 107
2 134 282
148 371
89
3 149 304 155 440 136
4 154 292 138 378 86
5 166 328 162 430 102
Mean
146
294
149
398 104
SD 16
24
10
34 20
247 237 291 224 264 253
26
0 1 124 271
2 135 263
3 151 300
4 163 333
5 170 351
Mean
149
304
SD 19
38
147 376 105 128 360 97 149 408 108 170 432 99 181 453 102 155 406 102 21 38 4
252 225 257 269 283 257
22
c00734
W il d l if e In t e r n a t io n a l , ltd
-46APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 2
Experimental
Group
(ppm a.i.)
Bird Day 0
Day5
Change Day 0-5
Day 8
0 1 107 222 115 298
2 126 267 141 360
3 116 203
87 266
4 107 203
96 284
5 111 248 137 348
Mean
113
229
115
311
SD 8 28 24 41
PROJECT NO.: 454-102
Change Day 5-8
76 93 63 81 100 83 15
Total Change
191 234 150 177 237 198
37
0
Group Total
1 2 3 4 5
Mean SD
Mean SD
100 100 102 108 102 102
3
135 24
235 232 233 267 210
235 20
279 42
135 335 132 328 131 317 159 383 108 299 133 332
18 32 144 380 22 53
100 96 84 116 89
97 12
101 16
235 228 215 275 197
230 29
245 34
00735
W il d l if e In t e r n a t io n a l , ltd
-47APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 3
PROJECT NO.: 454-102
Experimental
Group
(ppm a.i.)
Bird Day 0
9.1 1 123
2 118
3 151
4 108
5 119
Mean SD
124 16
Day5
244 231 286 209 231 240
29
Change Day 0-5
Day 8
121 349 113 334 135 399 101 313 112 332 116 345
13 33
Change Day 5-8
105 103 113 104 101 105
5
Total Change (0-8)
226 216 248 205 213 222
17
9.1 1 96 145 49
2 123 250 127
3 127 237 n o
4 112 216 104
5 111 215 104
Mean
114
213
99
SD 12
41
29
Group Total
Mean SD
119 14
226** 36
108** 23
Statistically different from the control group at p < 0.05 (Dunnett's t-test).
146
345
343
316 294
289 83
317* 66
1
95
106
100
79
76 43 91 33
50
222
216
204
183
175 71 198* 55
000736
W il d l if e In t e r n a t io n a l , ltd
-48 -
PROJECT NO.: 454-102
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 4
Experimental
Group
(ppm a.i.)
Bird Day 0
18.3 1 123
2 141
3 150
4 152
5 160
Mean SD
145 14
Day 5
Change Day 0-5
Day 8
261 138 355 297 156 421 270 120 361 286 134 390 294 134 391
282 136 384 16 13 27
Change Day 5-8
94 124 91 104 97 102
13
Total Change
232 280 211 238 231 238
25
18.3
Group Total
1 130 240 110 327
87
2 137 266 129 359
93
3 145 268 123 366
98
4 151 258 107 366 108
5 171 333 162 433 100
Mean
147
273
126
370
97
SD 16 35 22 39
8
Mean
146
277
131
377
100
SD 14
26
18 32
11
197
222
221
215
262
223 24
231 25
C00737
W il d l if e In t e r n a t io n a l , ltd
-49-
PROJECT NO.: 454-102
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 5
Experimental
Group
(ppm a.i.)
Bird Day 0
36.6 1 131
2 132
3 142
4 160
5 179
Mean SD
149 21
Day 5
Change Day 0-5
Day 8
261 130 372 256 124 356 269 127 369 270 110 353 335 156 433 278 129 377
32 17 33
Change Day 5-8
111 100 100 83 98 98
10
Total
241 224 227 193 254 228
23
36.6
Group Total
1 131 230
99 316
86
2 136 287 151 402 115
3 147 263
116 379
116
4 151 255 104 344
89
5 160 324 164 427 103
Mean SD
Mean SD
145 12
147 16
272 36
275 32
127 374 29 44 128 375 22 37
102 14
100 12
185
266
232
193
267
229 39
228 30
000738
W il d l if e In t e r n a t io n a l , ltd
-50-
PROJECT NO.: 454-102
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 6
Experimental
Group
(ppm a.i.)
Bird Day 0
73.2 1 113
2 113
3 142
4 154
5 179
Mean SD
140 28
Day 5
Change Day 0-5
203 90 219 106 286 144 290 136 338 159 267 127
56 28
Day 8
281 288 390 379 480 364
82
Change Day 5-8
78 69 104 89 142 96 29
Total Change
168 175 248 225 301 223 55
73.2
1 109 222
113
2 129 254 125
3 139 243 104
4 147 196
49
5 204 352 148
Mean
146
253
108
SD 36 59 37
Group
Mean
143
260
117*
Total
SD 30 55 33
'Statistically different from the control group at p < 0.05 (Dunnett's t-test).
302
332
340
175
458
321 101 343 90
80
78
97
-21
106
68 51 82 42
193
203
201
28
254
176 86 200* 73
000739
W il d l if e In t e r n a t io n a l , ltd
-51 -
PROJECT NO.: 454-102
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 7
Experimental Group
(ppm a.i.)
Bird Day 0
Day 5
Change Day 0-5
a 5: 00
Change Day 5-8
146
1 108 169
61 236
67
2 138 248 110 330
82
3 151 271
120 375
104
4 151 254 103 341
87
5 164 282 118 380
98
Mean
142
245
102
332
SD 21 44 24 58
88 14
Total Change
128 192 224 190 216 190 38
146
1 115 204
89
2 139 219
80
3 144 229
85
4 159 261 102
5 158 285 127
Group Total
Mean
143
240
97
SD 18 33 19
Mean
143
242
100**
SD 19 37 21
"Statistically different from the control group at p < 0.01 (Dunncn's t-test).
280
287
348
358
374
329 43 331 48
76
68
119
97
89
90 20 89 16
165
148
204
199
216
186 29 188** 32
000740
W il d l if e In t e r n a t io n a l , ltd
-52APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 8
PROJECT NO.: 454-102
Experimental
Group
(ppm a.i.)
Bird Day 0
293 1 115
2 139
3 140
4 155
5 112
Mean SD
132 18
Day 5
Change Day 0-5
Day 8
Change Day 5-8
142 27 179 27
119 -20 165 -20
156 16 217 16
187 32 291
32
134 22 203
22
148 15 211 15 26 21 49 21
Total Change
64 26 77 136 91 79 40
293 1 123
2 120
3 151
4 106 5 124
Mean
125
SD 16
Group Total
Mean SD
129 17
(-) = No data available due to mortality.
205
183
139
158
185
174 26 161** 28
82
63
-12
52
61
49 36 32** 33
289
273
-
223
138
231 68
220** 55
82
63
-12
52
61
49 36 57** 44
166
153
-
117
14
113 69 94** 54
W il d l if e In t e r n a t io n a l , ltd
-53-
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 9
PROJECT NO.: 454-102
Experimental
Group
(ppm a.i.)
Bird Day 0
586 1 122
2 135
3 152
4 154
5 165
Mean SD
146 17
Day 5
Change Day 0-5
Day 8
105 -17 126
143 8 209
160 8 217
129 -25
-
195 30 143
146 1 174 34 22 46
Change Day 5-8
21 66 57
-52 23 54
Total Change
4 74 65
-22 30 47
586 1 105
2 120
3 149
4 153 5 182
Mean
142
SD 30
Group Total
Mean SD
144 23
(-) = No data available due to mortality.
114
130
-
128
128
125 7
137** 27
9
10
-
-25
-54
-15 31 -6** 26
0.01 (Dunnett's t-test).
159
185
-
185
-
176 15
175** 34
45
55
-
57
-
52 6
36** 41
54
65
-
32
-
50 17 39** 36
C0 0 7 4
W il d l if e In t e r n a t io n a l , ltd
-54APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 10
PROJECT NO.: 454-102
Experimental
Group
(ppm a.i.)
Bird Day 0
1171 1 124
2 140
3 145
4 150
5 166
Mean SD
145 15
Day 5
Change Day 0-5
Day 8
88 -39 --
96 -49 118 -32
-100 -40
17 9
Change Day 5-8
-
-
Total Change
-
-
1171
1 128
87 -41
2 110 - -
3 145 132 -13
4 157 - 5 200 154 -46
Mean
148
124
-33
SD 34 34 18
Group
Mean
147
112**
-37**
Total
SD 25 28 13
(-) = No data available due to mortality.
2No mortalities occurred in any of the treatment or control groups after Day 8.
-
-
-
185
185 -
1852
-
-
-
-
31
31
-
312
-
-
-
-15 -15
152
-
000743
W il d l if e In t e r n a t io n a l , ltd
-55APPENDIX VI
PROJECT NO.: 454-102
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 11
Experimental Group
(ppm a.i.)
0
Bird
1 2 3 4 5 Mean SD
Day 8
404 381 415 429 418 409
18
Day 15
721 633 685 692 679 682
32
Change Day 8-15
317 252 270 263 261 273
26
Day 22
895 780 861 822 847 841 43
Change Day 15-22
174 147 176 130 168 159 20
Total Change (8-22)
491 399 446 393 429 432 40
0 1 399 606 207 810 204
2 394
604 210 791
187
3 409 638 229 819 181
4 430 543 113 730 187
5 484 752 268 924 172
Mean
423
629
205
815
186
SD 37 77 57 70 12
411 397 410 300 440
392 54
0
Group Total
1 372 583 211 779 196
2 371 613 242 823 210
3 440 636 196 859 223
4 378 594 216 794 200
5 430 627 197 816 189
Mean
398
611
212
814
204
SD 34
22
19 31
13
Mean
410
640
230
823
183
SD 30 56 47 49 24
407 452 419 416 386 416
24
413 42
000744
W il d l if e In t e r n a t io n a l , ltd
-56-
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 12
PROJECT NO.: 454-102
Experimental Group
(PPm a i >
9.1
Bird
1 2 3 4 5 Mean SD
Day 8
349 334 399 313 332 345*
33
Day 15
Change Day 8-15
Day 22
602 253 821 549 215 730 616 217 802 562 249 767 546 214 744 575 230 773 32 20 38
Change
219 181 186 205 198 198
15
Total Change
472 396 403 454 412 427
34
Experimental
Group (PPm a i ~)
Change
Change
Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22
18.3 1 355 611 256
2 421 693 272
3 361
578 217
4 390 626 238
5 391 618 231
Mean
384
625
242
SD 27 42 22
Statistically different from the control group at p < 0.05 (Dunnett's t-test).
811 938 713 794 799
811 81
200 245 135 168 181
186 41
Total Change
465 517 352 404 408 427 62
0 0 0 7 -:'5
W il d l if e In te r n a tio n a l , ltd
-57-
PROJECT NO.: 454-102
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 13
Experimental
Group
(PPm ad.)
Bird Day 8
36.6 1 372
2 356
3 369
4 353
5 433
Mean SD
377 33
Day 15
Change Day 8-15
Day 22
Change Day 15-22
677 305 942 265 607 251 817 210 613 244 793 180 559 206 746 187 642 209 840 198 620 243 828 208 44 40 73 34
Total Change
570 461 424 393 407 451
71
Experimental Group
.. (PP, n a i )
73.2
B ird
1 2 3 4 5 Mean SD
Day 8
281 288 390 379 480 364
82
D ay 15
Change Day 8-15
Day 22
Change D ay 15-22
482 201 648 166 480 192 646 166 636 246 864 228 599 220 796 197 699 219 955 256
579 216 782 203 97 21 135 39
Total Change
367 358 474 417 475 418
56
C00746
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-58-
PROJECT NO.: 454-102
APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 14
Experimental Group
Change
Total
146 1 236 475 239 659 184
2 330 56 9 239 739 170
3 375 631 256 738 107
4 341 570 229 754 184
5 380 576
196 550
-26
Mean
332*
564
232
688* 124*
SD 58 56 22 86 90
423 409 363 413 170 356 106
Experimental
Group
Change
Change
(ppm a.i.)
Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22
293 1 179 447 268
2 165 415 250
3 217 526 309
4 291 516 225
5 203 430 227
Mean SD
211** 49
467** 51
256 35
^Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statisticaily different from the control group at p < 0.01 (Dunnett's t-test).
731 646 748 704 676
701* 41
284 231 222 188 246
234 35
Total Change
552 481 531 413 473 490
54
C00747
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-59APPENDIX VI
Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 14
PROJECT NO.: 454-102
Experimental
Group
Change
Change
(ppm a.i.)
Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22
586 1 126 308 182 525 217 2 209 469 260 706 237 3 217 460 243 675 215
Total Change
399 497 458
5 143 340 197 546 206
Mean
174
394
221
613 219
SD 46 82 37 91 13
403
439 47
Experimental Group
(ppm a.i.)
Bird Day 8
Day 15
Change Day 8-15 Day 22
Change Day 15-22
1171 1 - - 2
J
4
5 185 383 198
Mean
18?
38?
SD - - -
^n=l, could not be evaluated statistically using Dunnett's t-test.
--
634 251 _p
63? --
Total Change
-
449 4?^
-
C0 0 7 4 8
W il d l if e In t e r n a t io n a l , ltd
-60APPENDIX VII
PROJECT NO.: 454-102
Feed Consumption (g/bird/day) by Pen from a Mallard 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 2 3 4 5 6 Mean SD
109 92 85 93 82 94 92 10
145 140 117 132 100 116 125 17
000749
W il d l if e Internatio nal, ltd
-61 APPENDIX VII
PROJECT NO.: 454-102
Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 2
Experimental Group (ppm a.i.)
Pen
Exposure Period Day 0-5
Post-Exposure Period Day 6-8
9.1 1 73
2 72
Mean
73
130 104 117
18.3 1 90
2 91
Mean
91
135 128 132
36.6 1 103
2 85
Mean
94
129 120 125
73.2 1 79
2 74
Mean
77
109 94 101
146 1 89
2 121
Mean
105
102 217 159
293 1 28
2 59
Mean
44
61 65 63
586 1 40
2 32
Mean
36
43 67 55
1171 1 23
2 22
Mean
22
25 26 25
C00750
W il d l if e In t e r n a t io n a l , ltd
PROJECT NO.: 454-102
-62APPENDIX VII
Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 3
Experimental Group (ppm a.i.)
Pen
Control
1 2
3
Mean SD
Exposure Period Day 8-15
185 161 166 171 13
Post-Exposure Period Day 15-22
159 182 199 180 20
C00751
W il d l if e In te r n a tio n a l , ltd
-63 APPENDIX VII
PROJECT NO.: 454-102
Feed Consumption (g/bircEday) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 4
Experimental Group (ppm a.i.)
Pen
Exposure Period Day 8-15
Post-Exposure Period Day 15-22
9.1 1 172
198
18.3 1 186
204
36.6 1 165
179
73.2 1 148
173
146 1 159
164
293 1 109
132
586 1 114
143
1171 1 106
154
CCO7 5 2
W il d l if e In t e r n a t io n a l , LTD
-64APPENDIX VIII
PROJECT NO.: 454-102
CHANGES TO PROTOCOL
This study was conducted in accordance with the approved Protocol with the following changes:
1. The protocol was amended to add an additional test concentration of 10 ppm a. i..
2. 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.
3. The protocol was amended to change the test concentrations from 0, 10, 20, 40, 80, 160, 320, 640 and 1280 ppm a.i., to 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. Test concentrations were changed to reflect the test substance purity given in the new certificate of analysis.
4. Individually numbered wing bands were used to uniquely identify each duckling. The protocol required leg bands to be used to identify the birds.
5. 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.
6. The final temperature from one brooder unit on Day 8 and all brooder units on Day 22 were not recorded.
7. The afternoon observations were inadvertently not recorded for birds in a single brooder unit on May 8, 1999.8
8. Observations for control birds from two brooder levels were inadvertently not recorded on Day 6 o f the test. Temperatures for these brooder levels were also not recorded on this day. Observations for control birds from one brooder level were inadvertently not recorded on Day 22 of the test.
C00753
W il d l if e In ter n a tio n a l, l t d
-65 APPENDIX IX
PERSONNEL INVOLVED IN THE STUDY
PROJECT NO.: 454-102
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 B. Nixon, Ph.D., Manager, Analytical Chemistry (6) Tim Kendall, Supervisor, Analytical Chemistry (7) Raymond L. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist
060754
W il d l if e In t e r n a t io n a l , l t d
-66APPENDIX X
REVISED STUDY SUMMARY
PROJECT NO.: 454-102
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 purity 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
000755
W il d l if e In t e r n a t io n a l , l t d
PROJECT NO.: 454-102
-67-
REVISED STUDY SUMMARY BASED ON A PURITY OF 86.9%
SPONSOR:
SPONSOR'SREPPRESENTAT1VE:
LOCATIONOFSTUDY, RAW DATAANDACOPYOFTHE FINALREPORT:
3M Corporation Ms. Susan A. Beach
Wildlife International Ltd. Easton, Maryland 21601
WILDLIFEINTERNATIONAL LTD. PROJECTNUMBER: TESTSUBSTANCE: STUDY: NOMINALTEST CONCENTRATIONS: TESTDATES:
LENGTHOFTEST:
454-102 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Dietary LC50 Study with the Mallard 0,8.8,17.6,35.1,70.3,141,281, 562 and 1125 ppm a.i.
Experimental Start - April 22, 1999 Biological Termination - May 1 4 ,1999 Experimental Termination - May 14, 1999 22 days
TESTORGANISM: SOURCEOFTESTORGANISMS:
AGE OF TEST ORGANISMS:
Mallard (A n u s p la ty r h y n c h o s )
Whistling Wings, Inc. Hanover, IL 61041-3512 10 daysat test initiation
LC50:
95% CONFIDENCELIMITS:
NOMORTALITY CONCENTRATION:
NO-OBSERVED-EFFECTCONCENTRATION:
603 ppma.i. 430 and 919 ppma.i. 141 ppma.i. 35.1 ppma.i.
AMENDED
000756
W il d l if e In t e r n a t io n a l , l t d _________
-68-
PROJECT NO.: 454-102
INTERIM CERTIFICATE OF ANALYSIS
Jtsvbton 3
Centre Analytical LaboratoriuCOA Reference Ft 0234II8A 3M Preduct: PFOS,Lo<217
Reference #: SD-018
T refH i
Parity: 86.%
SpccUIm Hmm
------------- SSSR-------------
Parity*
86.9%
Ayfwanace Identification
NMR
While Cryetelline Powder
Conform Porilive
llllliil
Total % Impurity
"
(U >M S)
Total % Imparity
(G C /M S)
Related Compounds -
POAA Residual Solvent* tjQ A )
Purity by DSC
Inorganic Aniona (IC)
1. Chloride 2. Fluoride
3. Bromide
4. >81n>e
J. Nitrite 6. Phocpbate
7. Sulfate*
Organic Acida * (1C)
1. TFA 2. PFPA
3. HFBA 4. NFPA
Elemental Analypu1:
1
1. Carbon
2. Hydrogen 3. Nitrogen 4. Suitor 5. Fluorine
1. Theoretical Value - 17.8% 2. Theoretical Value - 0% 3. Theoretical Value - 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60%
1. 0.005 w t/w t.% 2. 0.001 w t/w t* 3. 1.439 w t/w t.% 4. 6.849 wt/wt.% 5. <0.001 wt/wt.% 6. 0.005 wt/wt.% 7. <0.001 wL/wt.%
1.91 w t/w t.% M l wtSwt.%
N ose Detected
0.J3 wt/wt.% None Detected Wot A pplicable'
1. <0.015 w t/wt.% 2. 0.59 wt/wt.% 3. <0.040 w t/w t% 4. <0.009 w t/w t% 5. <0.006 wt/wt.% 6. <0.007 w t/w t% 7. 8.76 wt/wt.%
J. <0.1 n l / w t K 2. <0.1 w tJ w tH 3. O .lO w tM K 4 . 0 .2 8 wL/wL%
1. 12.48 wt/w t.% 2. 0.244 wt/wt.% 3. 1.74 w t/w t.% 4. 8.84 w t/wt.% J . 54.1 w t/w t.%
C O A 0U -01SA
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AMENDED
000757
0-00758
C00759
C0760
600761
