Document jBwvDymKaRdgLyMqyLa8L4KaN
24) OECD 205-OPPTS 850.2200, Acute avian toxicity (Mallard), 454-1 13
T-7485: A DIETARY LC50 STUDY WITH THE MALLARD
SANITIZED
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454- 1 13 3M LAB PROJECT NO.: E00-1429
DEC 0 9 2003
U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft)
OPPTS Number 850.2200
OECD Guideline 205
AUTHORS:
Sean P. Gallagher Jennie Grimes Joann B..Beavers Raymond L. VanHoven
STUDY INITIATION: June 4,2002
STUDY COMPLETION: January 9,2003
1
SUBMITTED TO
3M Corporation Environmental Laboratory
Building 2-3E-09
935 Bush Avenue St. Paul, Minnesota 55106
W iId1ife Intemational, Ltd.
8598 Commerce Drive Easton, Maryland 2 1601
(410) 822-8600
Page 1 of 53
--
Wildlife International, Ltd.
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Project Number 454- 1 13
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation TITLE: T-7485: A Dietary LC50 Study with the Mallard WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-113 3M LAB PROJECT NO.: E00-1429 STUDY COMPLETION: January 9,2003
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; which is basically in compliance with OECD Principles of Good Laboratory Practice, (ENVMCKHEM (98) 17), Paris 1998; and Japan MAFF, 11 NohSan Notification No. 6283, Agricultural Production Bureau, 1 October 1999.
STUDY DIRECTOR:
f * &L Sean P. Gallagher
Senior Biologist, Avian Toxicology
1
DATE
SPONSOR APPROVAL:
Wildlife International, Ltd.
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Project Number 454-1 13
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, (ENV/MC/CHEM (98) 17), Paris 1998; and Japan MAFF, 11 NohSan Notification No. 6283, Agricultural Production Bureau, 1 October 1999. 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
Test Substance Preparation and Analytical Sample Preparation
June 6,2002
Matrix Fortifications
June 7,2002
Body Weight
Sample Collection
Analytical Data & Draft Report
June 11,2002
June 28,2002
1
August 2, 5, & 6, 2002
Biological Data & Draft Report
Final Report
August 6 & 7,2002 January 9,2003,
DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT
June 7,2002 June 7,2002 June 11,2002 June 28,2002
August 6, 2002
August 8,2002 January 9,2003
June 18,2002 June 12,2002 June 13,2002 August 5,2002
August 6,2002
August 13,2002 January 9,2003
e study based unless otherwise noted.
Robert N. McGee, B.S. Quality Assurance Representative
DATE<.,,
9 -4 .2 -->w 3
wild1ife Internationa1, Ltd.
-4REPORT APPROVAL
Project Number 454-1 13
SPONSOR: 3M Corporation TITLE: T-7485: A Dietary LC50 Study with the Mallard WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-1 13 3M LAB PROJECT NO.: E00-1429
STUDY DIRECTOR:
Sean P. Gallagher
I
Date
Senior Biologist, Avian Toxicology
CHEMISTRY PRINCIPAL INVESTIGATOR: 1
R a d d d . VanHoven, Ph.D. Scientist, Chemistry
REPORT APPROVED BY:
Joann B. beavers Director of Avian Toxicology
Date
1 I4!01 Date
Willard B. Nixon, P d D . Director of Chemistry
.
wiId I if e InternationaI. 1;td.
-5TABLE OF CONTENTS
Project Numbcr 454-1 13
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 9 Page 9 Page 9
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 9 Page 9 Page 10 Page 10 Page 10 Page 11 Page 11 Page 11 Page 13 Page 14 Page 14 Page 14 Page 14
RESULTS ............................................................................................................................. Page 15
Diet Analysis ...................................................................................................................... Mortalities and Clinical Observations ................................................................................
Body Weight and Feed Consumption ................................................................................. Gross Necropsy ................................................................................................................... Liver Weight .......................................................................................................................
Page 15 Page 15 Page 15 Page 16 Page 16
CONCLUSION..................................................................................................................... Page 16
REFERENCES ..................................................................................................................... Page 17
Wildlife International, Ltd.
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TABLES AND APPENDICES
Projcct Number 454- 1 I3
TABLE 1: TABLE 2: TABLE 3: TABLE 4:
Cumulative Mortality from a Mallard Dietary
Toxicity Study with T-7485 ......................................................................... Page 18
Mean Body Weight (g) from a Mallard Dietary
Toxicity Study with T-7485 ......................................................................... Page 19
Mean Feed Consumption (ghirdday) from a Mallard
Dietary Toxicity Study with T-7485 ............................................................ Page 20
Mean Liver Weight (g) and Mean Relative Liver Weight
(Liver Weight (&/Body Weight (g))
from a Mallard Dietary Toxicity Study with T-7485 ...................................
Page 2 1
APPENDIX I:
1
Certificate of Analysis ...........................................................................
Page 22
APPENDIX 11: Diet Formulation.................................................................................... Page 26
APPENDIX 111: Diet Preparation .._............,........._......._.._........_....................._.........Page 27
APPENDIX IV: Analytical Methods and Results ............................................................ Page 28
APPENDIX V: Cumulative Mortality by Pen from a Mallard Dietary
Toxicity Study with T-7485..................... .............................................. Page 4 1
APPENDIX VI: Individual Body Weights (g) from a Mallard Dietary Toxicity Study with T-7485................................................................... Page 42
APPENDIX VII: Feed Consumption (ghirdday) by Pen from a Mallard Dietary Toxicity Study with T-7485 ........................................ Page 49
APPENDIX VIII: Individual Liver Weights (g) from a Mallard Dietary
Toxicity Study with T-7485 ...................................................................
Page 5 1
APPENDIX IX: Changes to Protocol ............................................................................... Page 52
APPENDIX X: Personnel Involved in the Study ............................................................ Page 53
wildlife International, Ltd.
-7SUMMARY
Project Numbcr 454- 1 13
SPONSOR: 3M Corporation
TEST SUBSTANCE: T-7485
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-1 13
STUDY: T-7485: A Dietary LC50 Study with the Mallard
RESULTS:
Thedietary M 5 0 value for mallard exposed to T-7485 was determined to be greater than 10,OOQ ppm a.i., the highest concentration tested. No mortalities occured at any of the concentrations tested. The no mortality concentration was 10,000 ppm a.i.
There consu
were mptio
n n
o o or
v
e l
rt iv
Tsi s of
er weig
tox hts
i
ci at
t
y or any
treatment-related of the concen
effects trations
o
n te
feed sted.
Additionally, there were no apparent treatment-related effects on body
weight among birds in the 1000, 1780, 3160 and 5620 ppm a.i. treatment
groups. However, there' was a treatment-related reduction in body weight
gain among birds in the 10,000 pprn a.i. treatment group during the
exposure period. Based upon a reduction in body weight gain at the
10,000 pprn a.i. test concentration, the nqobserved-effect concentration
was 5620 ppm a.i,,
TEST DATES:
Hatch -May 29,2002 Acclimation - May 31 2002 to June 6,2002 Experimental Start (EPA) -June 6,2002 Experimental Start (OECD) -June 6,2002 Experimental Termination - July 19, 2002
NOMINAL TEST CONCENTRATIONS:
0, 1000, 1780,3160,5620 and 10,000 ppm a.i.
Ml'ildlge Intemationul, Ltd.
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SUMMARY (contined)
Project Numbcr 454-1 13
TEST ANIMALS:
Mallard (Anusplatyrhynchas)
AGE TEST ANIMALS: Approximately 8 days of age at test initiation
SOURCE TEST ANIMALS: Whistling Wings 113 Washington Street Hanover, Illinois 61041
STUDY COMPLETION: January 9,2003
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Projcct Numbcr 454-1 13
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 June 6, 2002 to June 28, 2002. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-1 13 in archives located on the Wildlife International, Ltd. site.
OBJECTIVE The objective` of this study was to evaluate the toxicity of T-7485 to the mallard (Anm 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); OECD Guideline 205, Guideline forhesting of Chemicals, Avian Dietary Toxicity Test (2); and upon ASTM Standard E857-87, "Standard Practice for Conducting SubDietary Toxicity Tests with Avian Species" (3).
Test Substance
The test substance was received from 3M Corporation on March 27, 2000 and was assigned
Wildlife International, Ltd. Identification Number 52 16 upon receipt. The test substance was white
powder identified as: Potassium Perfluorobutane Sulfonate;
; Lot No.: 2; CAS No.
29420-49-3. The reported purity of the test substance was 97.3%, with an expiration date of January
17, 2007 (Appendix I). The test substance was stored under ambient conditions.
Treatment Groups The test consisted of a geometric series of five test concentrations and a control group. Thirty
mallard ducklings were assigned to the control group and twelve mallard ducklings 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 or six ducklings each. Nominal dietary concentrations used
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Project Numbcr 454-1 13
in this study were 0, 1000, 1780, 3 160, 5620 and 10,000 parts per million active ingredient (ppm a.i.) of T-7485. 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 subjected to gross necropsy. Livers were weighed and liver and blood samples were also collected for potential analysis. The remaining birds were maintained on basal ration until Day 22. On Day 22, these birds were euthanized, liver weights were recorded, and samples of liver tissue and blood were collected.
Duration of the Test
The primary phases of this test and their approximate durations were:
1. Acclimation - 6 days.
2.
Exposure - 5 days.
1
3 . Post-exposure observation - 3 or 17 days
Test Birds All mallard (Anus plutyrhynchos) were approximately eight days of age and appeared to be in
good health at initiation of the test. The birds were obtained from Whistling Wings, Hanover, IL and were hatched on May 29, 2002. Birds ranged in weight from 79 to 138 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex. 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 receipt 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). 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.
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Project Number 454-1 13
Diet Preparation The test substance was mixed directly in basal ration into which corn oil had been added. The
concentration of corn oil in the control and treated diets was 2%. Mixing was done with a Hobart mixer (Model Number AS200T). All dietary test concentrations were adjusted to 100% active ingredient 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 1000, 1780, 3160, 5620 and 10,000 ppm a.i. (Appendix 111).
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 1000 ppm a.i. a.i. test diet and six samples from the 10,000 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 group diet and two verificatid samples were collected from each remaining treatment diet at preparation on Day 0. At the end of the exposure period (Day 5), one sample was collected from the control diet and two samples were collected from each treatment diet 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. chemistry laboratory.
Analytical Method A liquid chromatography mass spectrometry (HPLC/MS/MS) based method was developed at
Wildlife International, Ltd. for the analysis of the T-7485 in avian diet. The analytical method consistcd of sample weighing, extraction with methanol, filtration, dilution with methanol and microfiltration. Dilutions into the calibration range of the HPLC/MS/MS methodology werc perfoimed with a solution of 50% methanol and 50% NANOpure@water. Samples were then analyzed by direct injection. A method flowchart is provided in Appendix IV, Figure 1.
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Project Numbcr 454- 1 I3
Concentrations of T-7485 in extracts of the samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (50 mm x 2.0 mm, 3-pm particle size) fitted with a Keystone Javelin C I 8guard column (20 m x 2.0 mm i.d.). The instrument parameters are summarized in Appendix IV, Table 1.
All primary and secondary stock preparations were adjusted for the punty of the test substance (97.3%). A 1.00 mg a.i./mL primary stock solution of T-7485 in methanol was prepared by weighing 0.1027 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions (100 and 10.0 mg a.i./L) of T-7485 in methanol were prepared by serial volumetric dilution of the primary stock.
Calibration standards were prepared in a solution of 50% methanol and 50% NANOpure@water
by appropriate dilutions of the 100 mg a.i./L dst ck solution of T-7485 in methanol. Calibration
standards of T-7485, ranging in concentration from 0.100 to 0.500 mg a.i./L, were analyzed with each sample set. The calibration standard series was injected at the beginning and end of the run, and one standard was injected, at a minimum, after every five samples, Linear regression equations were generated using peak area responses versus the respective concentrations of the calibration standards. A representative calibration curve is presented in Appendix IV, Figure 2. The concentration of T7485 in the samples was determined by substituting the peak area responses of the samples into the applicable linear regression equation. Representative ion chromatograms of low and high-level calibration standards are presented in Appendix IV, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix IV, Table 2.
The instrument limit of detection (LOD) was set based upon the injection volume (5.00 pL) and
the lowest standard concentration 0.100 mg a.i./L. The LOD was set at 0.500 ng on-column. The method limit of quantitation (LOQ) for these analyses was set at 150 ppin a.i. based upon the lowest matrix fortification level analyzed concurrently with the samples. The 0.100 mg a.i./L T-7485 standard was equivalent to a calculated value of 40 ppm a.i. T-7485 in the matrix blank extract. Measured values greater than or equal to the ppm a.i.equivalent were reported.
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Project Number 454- 1 13
Along with the sample analyses, three matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the ppm a.i. equivalent of the lowest standard during the sample analyses (Appendix IVYTable 3). A representative ion chromatogram of a matrix blank sample is presented in Appendix IV, Figure 5 .
Avian diet samples were fortified at 150,3000, and 12000 ppm a.i. and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 87.7%, .99.0% and 98.2% (Appendix IVYTable 3). Sample measured concentrations were not corrected for the mean procedural recoveries. A representative ion chromatogram of a matrix fortification sample is presented in Appendix IV, Figure 6.
Housing and Environmental Conditions During acclimation and testing, all birds were housed indoors in batteries of thermostatically
controlled brooding pens manufactured by Safeguard Products, Inc. (Model No. 5355). Each pen had floor space that measured approximately 62 X 9 2 cm. Ceiling height was approximately 25.5 cm. External walls, ceilings and floors were constructed of vinyl-coated wire grid. 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 uniquely numbered wing bands.
Through Day 11 of the test, the average temperature in the brooding compartment of the pens was 3 1C k1"C (SD). Brooding was discontinued on Day 11,when test birds were of sufficient size to therimregulate. Average ambient room temperature for this study was 24.9"C i 0.7"C (SD) with an averasc relative humidity of 74% f 5% (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 196 lux of illumination. Housing and husbandry practices were based on guidelines established by the National Research Council (4).
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Projcct Numbcr 454-1 13
Observations During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or
physical injury were not used for the study. 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 Weightsmeed Consumption Individual body weights were measured at the initiation of the test, on Days 5 and 8, and
Days 15 and 22 for al1,remainingbirds. 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 815 and 16-22 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.
1
Gross Necropsy On Day 8 of the test, half of the birds in each treatment group and half of the control birds
were euthanized and subjected to gross necropsy. On Day 22 of the test, all remaining birds were
euthanized and subjected to gross necropsy. Prior to euthanasia, blood was collected from each bird when possible. During necropsy, the liver was removed and weighed. Blood and liver samples were transferred to the sponsor for analysis if deemed appropriate.
Statistical Analyses Thcre were no treatment related mortalities observed in this study, therefore, it was not
possible to perform the calculation (5,6,7) of an LC50 value using the computer program of Stephan (8). The LC50 value was estimated to be greater than 10,000 ppm a.i., the highest concentration tested. Differences in the mean body weights, body weight change, absolute liver weights and relative liver weights between the control group and the treatment groups were determined by an Analysis of Variance (ANOVA). Bonferoni's t-Test, as run with Toxstat Software (9,lO) was used to compare the five treatment group means with the control group mean and assessed the statistical significance of the differences observed.
Wildlife International, Ltd.
- 15RESULTS
Projwt Ntiinbcr 454-1 13
Diet Analysis Diet samples collected from the 1000 and 10000 ppm a.i. test concentrations were analyzed to
evaluate the homogeneity of the test substance in the diet. The mean and standard deviation for the
1000 and 10000 ppm a.i. test concentrations were 938 * 60.6 pprn a.i. and 9440 rt 627 ppm a i ,
respectively. The corresponding coefficients of variation were 6.46% and 6.65%, respectively
(Appendix IV,Table 4). A typical ion chromatogram of a test sample is shown in Appendix IV,
Figure 7.
Samples collected during Day 0 of the test to verify test substance concentrations for the 1000, 1780, 3160, 5620, and 10000 ppm a.i. diets had mean measured concentrations of 938, 1700, 2940, 5420, and 9440 ppm a i , respectively. These values represented 93.8, 95.5, 93.0, 96.4, and 94.4% of
nominal concentrations, respectively (Appendix IV,Table 5). Analysis of diet samples collected
from feeders after being held at ambient temperature for 5 days averaged 106%, 92.9%, 101%, 102%,
and 108% of the Day 0 values for the 1000, b 1780, 160, 5620 and 10000ppm a.i. test concentrations,
respectively (Appendix IV, Table 5).
Mortalities and Clinical Observations
There were no mortalities in the control group and all control birds were normal in appearance and behavior throughout the test (Table 1 and Appendix V). In addition, there were no mortalities or
overt signs of toxicity at the 1000, 1780, 3160, 5620 and 10,000 ppm a.i. test concentrations. All birds at all test concentrations were normal in appearance and behavior throughout the test.
Body Weight and Feed Consumption Whcn coinpared to the control group, there were no apparent treatment-relatcd effects on body
weight among birds in the 1000, 1780, 3160 or 5620 ppm a i treatment groups during the exposure period (Days 0-5). Any differences between the control group and the 1000, 1780, 3160 and 5620 ppm a.i. treatment groups were not statistically significant. However, there was a statistically significant (p<O.Ol) reduction in body weight gain among birds in the 10,000 ppm a.i. treatment group during the exposure period that was considered treatment related (Table 2 and Appendix VI).
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Project Number 454-1 13
When compared to the control group, there was a statistically significant (p<0.05) reduction in body weight gain among birds in the 3160 ppm a.i. treatment group from Days 15 to 22 of the post exposure period. However, the reduction was not concentration responsive. Additionally, a similar reduction was not noted during the exposure period or during the subsequent post-exposure intervals (Day 5 to 8 and Day 8 to 15) at this treatment level. Therefore, this reduction was not considered treatment-related.
When compared to the control group, there were no apparent treatment-related effects on feed consumption at any of the concentrations tested (Table 3 and Appendix VII).
Gross Necropsy Gross necropsy revealed one bird in the control group with a subcapsular hematoma on the right
lobe of the liver. Another control bird was noted with a pale and mottled spleen. The necropsy results for all other birds were not remarkable.
Liver Weight
1
When compared to the control group, there were no apparent treatment-related effects on mean absolute liver weight or mean relative liver weight (liver weight as a percentage of body weight) for any of the concentrations tested. Any differences between the control and each treatment group were not statistically significant. Mean absolute liver weights and mean relative liver weights are presented in Table 4. Individual liver weights are presented in Appendix VIII.
CONCLUSION The dietary LC50 value for mallard exposed to T-7485 was determined to be greater than 10,000ppm a.i., the highest concentration tested. No mortalities occured at any of the concentrations tested. The no mortality concentration was 10,000 pprn a.i.
There were no overt signs of toxicity or treatment-related effects on feed consumption or liver weights at any of the concentrations tested. Additionally, there were no apparent treatment-related
effects on body weight among birds in the 1000, 1780, 3160 and 5620 ppm a.i. treatment groups. However, there was a treatment-related reduction in body weight gain among birds in the 10,000 ppm
a.i. treatment group during the exposure period. Based upon a reduction in body weight gain at the 10,000 ppm a.i. test concentration, the no-observed-effect concentration was 5620 pprn a.i.
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REFERENCES
Prvjcct Numbcr 454- 1 13
1 U.S. Environmental Protection Agency, 1996. Series 850 - Ecological Effects Test Guidelines
(draft), OPPTS Number 850.2200: Avian Dietaiy Toxicity Test.
2. Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing of Chemicals. Guideline 205. Paris.
3. 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.
4. National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, D.C. National Academy Press. 125 pp.
5. 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.
/ 6. Finney, D.J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London.
7. Thompson, W.R. 1947. Bacteriological Reviews, Vol2, No.2: 115-145.
8. Stephan, C.E. 1978. U.S. EPA, Environmeiital Research Laboratory, Duluth, MN. Personal Communication
9. Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments
with a Control. Jour. Amer. Statis. Assoc. 50:1096:1121
10. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University of Wyoming.
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TABLE I Cumulative Mortality from a Mallard Dietary Toxicity Study with T-7485
Project Number 454-I 13
Espetiincnliil
Group
(ppm a.i.)
Day0
No. Dead Per NO. Exposed Exposure Period
Day1 Day2 Day3 Day4
Day5
. No. Dead Per No. Exposed Post-Exposure Period
Day6 Day7 D a y s ' Day22'
Control 0
Treatment IO00 1780 3160 5620 10.000
0130 0130. 0130 0130 0130 0130
\
0112 0/12 0112 0112 0112 0112 0112 Of12 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112
013 0
013 0
0/12
0112
0112
0112
0112
0112
0112
0112
0112
0112
The LC50 value \+'asestimated to be greater than 10,000 ppin a i , the highest concentration tested.
' - t l a l f o f ~ h ebirds were euthanized on Day 8 for necropsy and tissue collection.
' - N o mortalities occui-red in any of the control or treatment groups from Day 8 to Day 22.
0130
0115
011 2
016
0112
016
0112
016
0112
016
0112
016
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TABLE 2
Mean Body Weight (6) from a Mallard Dietary Toxicity Study with T-7485
Experimental Group
(ppm a.i.)
Exposure Period Day0 Day5 Change'
~~
Day 8
Post-Exposure Period Change' Day 15 Change' Day22
Total Change' Change
Control 0
Mean 105 SD 14
Treatment
1000
Mean 1 I O SD 16
1780
Mean 109 SD 14
212 107
36
24
232 122
32
18
222 113
30
24
299
87
529 226
728 199
622
45
12
50
13
48
23
39
-327
95
548 223
727 178
615
44
14
76
27
79
31
65
309
88
528 213
740 212
630
44
17
44
17
62
30
58
3160 5620 10,000
Mean 108 SD 17
Mean I09 SD 17
Mean I OS SD 17
223 115
34
22
203
94
32
20
192
84**
40
26
320
97
40
13
295
92
48
22
287
95
51
16
551 228
35
24
553 271 86 47
520 230
39
26
710 159*
602
35
10
24
737 184
629
72
38
64
730 209
62 1
33
36
31
* - Signilicantly different thin the conlrol at pi0.05 (Bonferroni's t-Test).
* * - Significantly diffcrcnt rrom thc control at p 4 . 0 1 (Bonferroni's I-Test).
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TABLE 3
Mean Feed Consumption (ghirdday) from a Mallard Dietary Toxicity Study with T-7485
Experimental Group
(ppm a.i)
Exposure Period Days 0-5
Days 6-8
Post-Exposure Period Days 9-15
Control
Mean' . . 73
SD'
10
Treatment
112
163
14
11
1000
Mean
88
1780
Mean
79
128
153
132
161
3160
Mean
72
5620
Mean
61
1142
183
109
129
10000
Mean
60
1 b2
141
I Mean values calculated using Excel in fill1 precision mode. Manual calculation may vary.
~
Days 16-22 226 20
191 228 247 173 181
Wildlife International, Ltd.
Project Numbcr 454- 1 13
-21 TABLE 4 Mean Liver Weight and Relative Liver Weight' from a Mallard Dietary Toxicity Study with T-7485
Experimental Group
Liver
Day 8 Relative
Liver
Day 22 Relative
Control 0
Treatment 1000 1780 3160 5620 10000
Mean 14.315 ' SD 2.588
Mean 17.577 SD 3.640
Mean 15.727 SD 2.533
Mean 15.576 SD 3.992
Mean 15.138 SD 1.790
Mean 14.828 SD 3.115
4.833 0.624
5.332 0.599
5.224 0.646
I4.88
0.584
4.918 0.40 1
5.242 0.544
25.807 4.672
27.449 3.870
25.964 2.087
24.694 1.658
27.071 1.847
28.035 4.041
3.533 0.498
3.781 0.41 1
3.516 0.244
3.478 0.202
3.694 0.330
3.837 0.498
'Relative Liver M'eight = Liver Weight (g)/Body Weight (g) X 100. Differencrs between the control and each experimental group were not significant ( p 0 . 0 5 . Bonferroni's t-Test).
WildlifeInternational, Ltd.
- 22 -
APPENDIX I Certificate of Analysis
Projcct Nutnbcr 454-1 13
' Wildlife Interrintionnl, Ltd.
Project Numbcr 454- I 13
- 23 -
Centre Analytical Laboratories. Inc.
3048 Rsssarch Orwe Stele College. PA 16801
www.cenlrelab.ccrn
Phons: (814) 231-8032
Fax(814) 231-1253or(814)231-1580
PUrlW'
INTERIM CERTIFICATE OF ANAL YSIS
Revision 2 ( I M 7/01)
Centre Analytical Laboratories COA Reference #: 023-032
3M Product: PFBS,Lot 2
Test Control Refcrence #: TCR-00017-71
Purity: 97.3%
Test Nimc
1
Spcclncltlallr
Rault
973%
-- Conforms
NMR.
Mcfals (ICPMS)
1. Calcium
2. Magnesium
3. Sodium'
4. Patusium'
5. Nickel
6. Iron
1. M ~ I I ~ ~ C S C
Teal% Impurity (MAR)
Total Y. Impurity (Lc/MS)
Related Compounds (POA.4)
Total % Impurity (GUMS)
Residual Solvcnts (TGA)
Funty by DSC
I
I
Inorganic Anions (IC)
I . Chloride
2. Fluoride
3. Bromide
4. Nitrate
5 . Nitrite
6. Phosphate
Positive
1. O.O03wtlwt'/l 2. 0.001 wtlwl%
3. 0.768 wrlw.% 4. 10.718 wthvt.% 5 . <0.001 wtlwt% 6. 0.002 wtlwt.%
7. <O.oOl w t h . % 2.07 w/wt.% 5.56 wt/wt%
. None Qumndtid
None Ouantificd None Detected Not Applicable
1. 0.016 wtJwt.% 2. e0.005 vRfwt.% 3 . co.o40wtfwt.% 4. <0.009wtJW.?4 5. 4.006wtlWt.% 6. s0.030 wtlwt% 7. C0.040 wl&t.*A
1. <O.l wtfwt.% 2. 4.1w f w t %
3. co.1 W ~ * A 4 . <0.25 wt./W.%
I Carbon 2 Hydrogen
i
4 Sulfur 5 Fluonnc
Theoretical Value = 14.2% Theoretical Vduc = 0.0% Thcoretical Value = 0.0%
Theoretical Value = 9 5% nicorctic.4 Value = 50 6%
1 . 1 1.o WrfW.%
2. 0.282 wfWr.% 3 . 0.165 wtfwr% 4 . 11.2 w.t..vl% 3. 48.2 w./w.%
COA023032.71 Revision 2.doc
Page I of 3
W i l d 1ife Internntional, Ltd.
Projcct Number 454- I 13
-24-
CEntre Analytical Laboratories, Inc.
3048 Research Drive Stare College, PA 16801
www.cenlrelab.com
Phonw(814)231-8032
Fax: (814) 231-1253 of (814) 231-1580
INTERIM CERTIFICATE OF ANALYSIS
Revlsion 2
Centre Annlylical Laboretorles COA Reference #: 023-032 3M Product: PFBS, Lot 2
Test Control Reference #: TCR-00017-71
Date of Last Analysis: 01/17/01
FIxpudlion Dale: 01/17/07
Storagc Conditions: < -10 'C
Re-assessment Date: 0 1/17/07
- - 'Purity- 100% (Total NMR impurities. 2.07% + Total LC/MS impurities, (5.56%
4.99%') +Total Metal impurities. 0.006%+ Total inorganic anions, 0.016%)
Total impurity tium all tests = 2.662%
P U ~ Y= 100% - 2.662% = 97.3%
* 1.93?'0of the N M R total impurity value is due to the presence of
impYy is also observed in the LCNS analysis at
4.99%. The impurity value is taken fro the N M R analysis as it is deemed the more
quantitative,tcchniquc in the abscncc of standards (See impurity profile on page 3).
'Potassium and sodium arc present as coupterions and thus are excluded &om the purity
calculation.
3.
*Theoretical value cilculations based on the empirical formula, C,FgSO,X' ( W s 3 3 8 . 2 )
COA023032-7 I Rcvwon 2 . d ~
Page 2 of 3
W i l d 1ye InternntionnI, L td.
Project Numbcr 454- I 13
- 25 -
CentrE Anahtical Laboratories. Inc.
3048 Research Drive State College, PA 16801
www.centmlab.com
(814) 231-8032
FEZ: (814) 231-1253 or (814)231.i580
INTERIM CERTIFICATE OF ANALYSIS
Revision 2 Centre Analytical Laboratories COA Reference #: 023-032
3iM Product: PFBS, Lot 2 Test Control Reference #: TCR-00017-71
LCMS Purity Profile:
Peak # 1 2 3
Total
Retentio-n -Ti-me (min) [
1
Mas+) I ldentity
~ ~ 1 6 . yI PFBS
I -
Area
186918 , 1629660 30835100
326.51678
% Impurity '
0.57
4.99
5.56
NMR Purity Profile:
I CF~-CF~-..CF~. -CF~-SOJ-,
2f8.9
97.9
t- I 0.05 I
1.93
0.13
I 0.01
Total impurity
2.07
This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160).
Prepared By: <
L ,
Date
Sc.ientisr,Centre Analytical Laboratories
oL ,f$&f Reviewed By:
/;1
,474
sdh. Flaheny
Date
/1-rrboratoryManager, Centre Analytical Laborntories
COA021032-71 Rcvisian2.doc
Page 3 of 3
wildlife International, Ltd.
Project Numbcr 454- 1 13
-26-
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 (%)
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 D, Vitamin A Riboflavin
Niacin
Pantothenic Acid Vitamin Biz Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite)
Mangancse
Zinc coppcs Iodinc Iron Seie n i u i m
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 brains
102 grams
47 grams 6.8 grams 1.5 grams
51 grams 182 ings
'The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of5% crudc fiber. 'Fermentation By-products (Source of Unidentified Growth Factors).
wildlife International, Ltd.
Projcct Number 454-1 13
- 27 -
APPENDIX 111 Diet Preparation
Weight and volume of constituents used to prepare test diets:
Nominal Concentrations
(ppm a.i.)
0 1000 .1780 3160 5620 10000
Test Substance
(8)
9.2496 16.4645 29.229 1 5 1.9837 92.4971
iBasal Ration
8820.0 8810.8 8803.5 8790.8 8768.0 8727.5
Corn Oil
0
195 195 195 195 195 195
Diets were prepared as follows:
5000.0 g of basal ration was weighed into a tared Hobart mixing bowl.
195 mL of corn oil was added to the ration and the bowl contents were mixed for approximately 5
minutes on a Hobart mixer.
1
The test substance was weighed in a tared weigh boat.
Approximately 100 g of basal ration was removed from the mixing bowl and placed in a Waring@ blender.
The test substance was added to the blender, the weigh boat was rinsed with additional ration from the mixing bowl, with the rinse also being placed in the blender.
The blender contents were blended for approximately 1 minute and transferred to the mixing bowl. The blender was rinsed with uncontaminated ration from the mixing bowl, with the rinse also being returned to the mixing bowl.
The bowl was placed on a Hobart mixer and the contents were mixed for approximately five minutes. The remaining ration was weighed and addcd to thc bowl and the contents were mixed for approximately 10 more minutes.
The diet was transfcrred to a labeled paper feed bag.
The control diet was prepared by weighing 5000.0 g of basal ration into a tared Hobart mixing bowl. 195 mL of corn oil was added and the bowl was placed on the Hobart mixer and the contents wcre mised for approximately five minutes. The remaining ration was weighed and added to the bowl. The contents of the bowl were mixed for approximately tcn minutes. Diet was transferred to a labeled paper feed bag.
wildlife International, Ltd.
Project Number 454-1 13
-28APPENDIX IV Analytical Methods and Results
Wildlife International, Ltd.
Project Numbur 454-1 13
-29Appendix IV
INSTRUMENT:
Table 1
Typical HPLCMSMS Operational Parameters
Hewlett-Packard Model 1 100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode.
ANALYTICAL COLUMN:
Keystone Prism RP column (50 mm x 2 mm I.D., 3-p-1)
GUARD COLUMN:
Keystone Javelin ClScolumn (20 mm x 2 mm I.D.)
OVEN TEMPERATURE:
40C
STOP TIME: FLOW RATE: MOBILE PHASE:
5.00 minutes
200 h PL inute
10% NANOpure Water@with 0.1% Ammonium Formate: 90% Methanol
INJECTION VOLUME:
5.08 p L
T-7485 RETENTION TIME:
Approximately 2.1 minutes
T-7485 hIONITORED MASS:
299.0 amu
wiId1ife InternntionaI, Ltd.
Project Numbcr 454-1 13
-30-
Appendix IV
Table 2
Example Calculations
Sample number 454-1 12,113-7, nominal concentration of 1000 ppm a.i. in avian feed
Peak Area = 1157259
Y-intercept = 287382.5938
Slope = 3571005
Primary Dilution:
Initial Weight (Wi) = dO.0 g
Original Final Volume (VI)= 200 mL
Secondary Dilution:
Initial Volume (Vi) = 0.0500 mL
Final Volume (Vf) = 10.0 mL
Overall Dilution Factor (VJWi x Vfnri) = 4000 mL/g = 4000 L/Kg
'eak area - y-intercept)
T-7485 at instrument (mg a.i./L) =
slope
- (1 157299 - 287382.59382
-
3571005
= 0.24359mg a.i./L
T-7485 in sample (mg a.i./Kg) = T-7485 at instrument (mg a.i./L) x overall dilution factor (L/Kg)
T-7485 in sample (mg a.i./Kg) = 0.24359 mg a i X- 4000 L
L
Kg
= 974 mg a.i./Kg = 974 ppm a.i.
Percent of Nominal Concentration =
';L( T-74S5 [
T-7485
m a.i.) in Sam le
a.i.)
x 100
--974 x 100 = 97.4% - 1000
Instrument Software: MacQuan, version 1.6.
Wildlife InternationaI,Ltd.
Project Number 454-1 13
-31 -
Appendix Iv
Table 3
Matrix Blank and Fortifications Analyzed Concurrently with the Samples
Number (454-1 12,113)
Sample Type
interval
concentration of T-7485 (ppin a i )
Fortified
Measured'.'
Percent Recovery'
Mean Percent of Nominal
MAB-I
Matrix Blank
Day 0
0
<40
MAS- 1
Matrix Fortification
Day 0
150
137
91.1
87.7
MAS-?
Matrix Fortification
Day 0
3000
2600
86.7
MAS-3
Matrix Fortification . Day 0
12000
10200
85.2
MAB-2
Matrix Blank
Day 5
u,
0
<40
MAS-4
Matrix Fortification
Day 5
150
157
105
99.0
MAS-5
Matrix Fortification
Day 5
3000
2810
93.5
MAS-6
Matrix Fortification
Day 5
12000
I1800
98.6
MAB-3'
Matrix Blank
Day 0
0
<40
MAS-73
Matrix Fortification
Day 0
150
143
95.6
98.2
MAS$
Matrix Fortification
Day 0
3000
2990
99.6
lvl A S-9
Matrix Fortification
Day 0
12000
11900
99.4
'The lowest level analytical standard was equivalent to a calculated value of 40 ppin a.i. in the matrix blank extract. Measured valucs greater than or equal to the ppin a.i. equivalent are reported. 'MeasuIcd and ptYCKll1 1-ecoveryvalues calculated using MacQuan ver. 1.6 and rounded to three significant figures. Manual calculations may vary slightly. 'Qualily control samples analyzed concurrent with re-extracted sample 454-1 12,113-8 (Table 5).
wiId1ife Internatio12aI, Ltd.
- 32 -
Appendix IV Table 4
Homogeneity of T-7485 in Avian Diet
Project Number 454-1 13
Nominal Concentration
(ppin a.i.) 1000
Sample
I.D. Number (454- 1 12,113)
2 3 4 5 6 7
Location Sampled In Mixing Vessel
Top Left Top Right Middle Left Middle Right Bottom Left, Bottom Right
T-7485 Concentration
Measured'
(ppm a.i.)
829
908 954
995
\
966
974
IO000
14
Top Left
15
Top Right
16
Middle Left
17
Middle Right
18
Bottom Left
19
Bottom Right
10000 9230 10300 8720 8850 9510
'Measured values were not coi~ccletlfor procedural recoveries based on sample sets (Table 3).
Mean (8
Standard Deviation (SD) Coefficient of Variation (CV)
% 938 SD =60.6 CV = 6.46
Mean Percent of Nominal
93.8
9440
94.4
SD = 627
CV = 6.65
w i l d 1ife Internationa1, Ltd.
Project Number 454-1 13
Nominal Concentration
(ppin a.i.)
- 33 -
Appendix IV
Table 5
Ambient Stability of T-7485 in Avian Diet During the Mallard Dietary LC50 Study
T-7485Concentration
Sample Number (454-1 12,l13)
Week I , Day 0
Measured' (ppm a.i.)
Mean Measured (ppin a i )
Mean Percent of Nominal
Sample Number (454-113-)
Week 1, Day 5
Measured2 (ppm a i )
Mean Measured (ppm ai.)
Mean Percent of
Day 0
0
1
< 40'
31
1000
2-7'
938'
93.8
32
k
33
1780
8
1 7704
I700
95.5
34
9
1630
35
3160
IO
3000
2940
93.0
36
II
2880
37
5620
12
5420
5420
96.4
38
13
5410
39
IO000
14-10'
9440'
94.4
40
41
< 40'
1000 993
1540 1620
2810 31 10
5870 5220
10700 9650
997 1580 2960 5540 10200
106 92.9
101 102
IO8
'Day 0 values are lioin homogeneity samples presented in Table 4. ?Meaw. i.etl values \vcrc not corrected for mean procedural recoveries based on sample sets (Table 3). 'The lo\vcst Ic\,cl analyliciil standard \vas equivalent to a calculated value of40 ppin a.i. in the matrix blank extract. Measured values greater than or equal to the ppin a.i. equivalent are reported. 'Result is the nie;ui of duplicate re-exlraction of original sample. Original recovery was 78.2% of nominal concentration due to procedural error.
Re-extraction duplicate measured values were 1760and I780ppin a.i., representing 99.0% and 99.9% of the nominal concentration, respectively.
WiId1ife Inten1ationa1, ttd.
Project Number 454- I 13
- 34 -
Appendix IV
Analytical Method Flowchart for the Analysis of T-7485 in Avian Diet
Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique.
1
Weigh 10 g samples of the matrix blank, matrix fortification, and test samples into weigh boats and transfer to 8-02. French square glass bottles. Record weights.
1
For each sample, measure 100 m L s of methanol with a graduated cylinder and transfer into the French square bottle.
1
Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm.
L
Filter into a 200-mL volumetric flask using a powder funnel and glass wool. Rinse retained feed 3 times with methanol into filtrate. Bring to volume with methanol.
1
Transfer an aliquot of each sample into a 10-mL B-D syringe fitted with a 0.45 pm Acrodisc@and filter into a scintillation vial.
&
& Prepare appropriate dilution(s) to bring final concentr tion into the calibration range of the LCIMSIMS methodology. Use 50% methanol:50% ANOpureO water dilution solvent. 1 Ampulate and submit samples for HPLC/MS/MS analysis.
Figure 1. Analytical method flowchart for the analysis of "-7485 in avian diet.
Wildlife International, Ltd.
- 35 -
Appendix IV
Area
Projcct Nurnbcr 454- I 13
Figure 2. Representative calibration curve for T-7485.Slope = 3571005, y-Intercept = 287382.5938, r = 0.99518.
WildlifeInternational, Ltd.
Project Number 454-1 13
- 36 -
Appendix IV
PFBS-1
STD 0.100 mg a.iJL
5292-OOl A-1
4.98 in 1 period
pws No Internal Standard
Use Area
1: 4.97 MRM. 298 scans
299.0. Noise Thres.
100.0
Quant Thres. Min. Width Mult. Width
50.0 12 - 10
Base. Width
35
I
RT Win. ( s e a ) 2 0
Smooth
1
Expected RT
2.28
Area
614522
Height 89890
Start Time
1.98
End Time
2.46
integration Width
0.49
Retention Time Integration Type
2.1 1
A - 88
Tue, Jun 11, 2002 13:43
intensity: 400000 cps
j904
3 4
26 2
1n.I
/"I
I 51 83
180 207
253
04
I
I
I
I
I
I
I
I
I
I
I
I
I
I
41
81 121 161 201 241 281 Scan
0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
Figure 3. Representative ion chromatogram of a low-l$rel T-7485 standard, 0.100 mg a.i./L.
Wi1dlife InternntionnI, L,td.
Project Nuinber 454- I I3
- 37 -
Appendix IV
PFES-5
STD 0.500 mg a.l.lL
5292-001 A-5
4.98 in 1 period FFBS
No Internal Standard
Use Area
299.0-
Noise Thres.
Quanl Thres.
Min. Width Mult. Width Base. Width RT Win. (secs) Smooth
100.0
50.0
12
10
35
20
'
1
Expected RT
2.28
Area
1918626
Height 247205
Start Time
1.98
End Time
2.56
Integralion Width
0.59
Retention Time Integration Type
A
-2.1 1 BB
Tue, Jun 1 1 , 2002 14:07
intensity: 400000 cps
26
4
3
2
1
286
I
41
8 1 121 161 201 241 281 Scan
0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
Figure 4. Representative ion chromatogram of a highfevel T-7485 standard, 0.500 mg a.i./L.
WiId1ife InternLItiona1, Ltd.
Project Nuinbcr 454-1 13
- 38 -
Appendix IV
PFBS-7
MAB-1
454-1 12.1 13-
Tue, Jun 1 1 . 2002 14:19
4.98 in 1 period PWS No internal Standard Use Area
1: 4.97 MRM, 298 scans
299.0
Noise Thres. Quant Thres. Min. Width Mutt. Width Base. Width RT Win. (sea) Smooth
100.0 50.0 12 10
35 ,
20 1
Expected RT
2.28
Area
0
Height
0
Start Time End Tim Integration Width
0.00
0.00 0.00
Retention Time
0.00
Integration Type
1O h
4i 1 3
2010-
51 7 7 107126
intensity: 400000 cps 192 233254 285
Figure 5 . Representative ion chromatogram of a matrix blank sample, (454-1 12,113-MAB-1). The arrow indicates the approximate retenti& time of T-7485. 10-g sample, Overall dilution = 400X.
W i l d 1ife Internationnl, Ltd.
- 39 -
Appendix IV
Project Number 454-1 13
PFSS-8
MAS-1
454-112.113 -
Tue, Jun 1 1 , 2002 14:25
4.98 in 1 period RBS No Internal Standard Use Area
intensity: 400000 cps
1: 4.97 M R M ,298 scans 299.0
Noise 'I nres. Quant Thres.
100.0 50.0
Min. Width
12
Mult. Width
10
Base. Width
35
RTWin. (sea) 20
Smooth
1
Expecled RT
2.28
Area
1507570
Height 20301 7
Star( Time
1.98
End l7n-e
2.56
Integration Width Retention Time Integration Type
0.59 2.11
A - 88
3 'I'
2 1
0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
Figure 6. Representative ion chromatogram of a matrii fortification (454-1 12,113-MAS-1). Nominal concentration 150 ppm ai., 10-g sample, Overall dilution = 400X.
WiZdlifeInternational, Ltd.
Project Number 454-1 13
- 40 -
Appendix IV
PFBS-33
38
454-1 13-
Wed, Jun 12, 2002 1739
m4.98 in 1 period
No Internal Standard
Use Area
1: 4.97 MRM, 298 scans
299.0 Noise Thres. Quant Thres.
100.0 50.0
Min. Width
12
Mult. Width
10
Base. Width
35
RTWin. (secs) 2 0
Smooth
1
Expected RT
2.28
Area
1357166
Height
149848
Start Time
2.01
End Tima
2.60
Integration Width
0.59
Retention Time Integration Type
A
-2.1 8 BB
l00l
9d
4d
3
2 1
intensity: 400000 cps
130
41 0.69
81 1.36
121 2.03
161 2.70
201 3.37
241 4.04
2 8 1 Scan 4.71 Time
Figure 7. Representative ion chromatogram of an avian diet sample on Day 5, 543-1 13-38 Nominal concentration 5620 ppm a i , 10-g sample, Overall dilution = 20000X.
wiId1ife Int e mationaI, XItd.
Project Number 454-1 13
-41 -
APPENDIX V Cuinulative Mortality by Pen froin a Mallard Dietary Toxicity Study with T-7485
Expel-imental
No. Dead Per No. Exposed
Group
Pen
Exposure Period
(ppm xi.)
Day0 Day 1 Day2 Day3 Day4 Day5
Control 0
1
015
015
015
015
015
015
0
2
015
015
015
015
015
015
0
3
015
015
015
015
015
015
0
4
015
015
015
015
015
015
0
5
015
015
015
015
015
015
0
6
015
015
015
015
015
015
Treatment 1000 1000
1
016
016
016
OM,
016
016
2
016
016
016
016
016
016
1780
1
016
016
016
016
016
016
1780
2
016
016
016
016
016
016
3 160 3160
1
016
016
016
016
016
016
2
016
016
016
016
016
016
5620
1
016
016
016
016
016
016
5620
2
016
016
016
016
016
016
10,000 I0,OOO
1
016
016
016
016
016
016
2
016
016
016
016
016
016
No. Dead Per No. Exposed Post-Exposure Period
Day6 Day7 Day8' Day22?
015
015
015
015
015
015
-
015
015
015
015
015
015
015
015
015
015
015
015
0/5
01 5
015
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
'The LC50 value was estimated to be - Half of the birds were euthanized
greater than 10,000 ppin ai.,the highest concentration on Day 8 for necropsy and tissue collection.
tested.
' - N o mortalities occurred in any ol'thc control or treatment groups after Day 8,
Wildlife Internntional, Ltd.
- 42 -
Projuct Numbcr 454-1 13
APPENDIX VI
Individual Body Weights (g) from a Mallard Dietary Toxicity Study with T-7485 Page 1
Experimental Group
(ppm a.i.) Bird
Day 0
Total Day 5 Change Day 8 Change Day 15 Change Day 22 Change Change
0
1
88 160 72 230 70
2
97 188 91 269 81
3
102 212
10 291 79
4
113 24 1
28 346 105
5
125 265 40 367 102
Mean
105 213 108 301 87
SD
14
42
27
56 15
0
1
79 152 73 211 59
2
90 191 101 278 85
3
99 209 110 291 82
4
111 223 112 300 77
5
121 255 134 328 73
Mean
100 206 106 281 75
SD
17
38 22 44 10
0
1
88 185 97 287 102
2
95 186 91 276 90
3
105 229 124 315 86
4
113 20 1
88 280 79
5
120 255 135 346 91
Mean
104 211 107 301 90
SD
13
30 21 30
8
- Indicates bird was euthanized on Day 8 for sampling.
wiId1ife Internationa1, Ltd.
- 43 -
Project Nurnbcr 454-1 13
APPENDIX VI
Individual Body Weights (8) from a Mallard Dietary Toxicity Study with T-7485 Page 2
Experimental
Group
Total
(ppma.i.) Bird D a y 0 Day5 Change Day8 Change Day 15 Change Day22 Change Change
0
1
87 178 91 252 74 468 216 694 226 607
2
94 192 98 274 82 498 221 684 186 590
3 103 220 117 310 90 546 236 774 228 671
4 116 239 123 333 94 546 213 740 194 624
5 120 258 138 367 109 591 224 820 229 700
Mean 104 217 113 307 90 530 223 742 213 638
SD
14 33 19
46 13
48
9
57
21
46
1 0
1
83 121 38 183 62 433 250 642 209 559
2
98 195 97 281 86 499 218 681 182 583
3 104 197 93 294 97 512 218 727 215 623
4 108 208 100 292 84 509 217 700 191 5 92
5 137 290 153 391 101 635 244 789 154 652
Mean 106 202 96 288 86 518 229 708 190 602
SD
20 60 41
74 15
73
16
55
24
36
0
1
92 209 117 296 87 515 219 693 178 60 1
2
98 216 118 307 91 549 242 756 207 658
3 107 208 101 310 102 524 214 741 217 634
4 119 258 139 357 99 577 220 774 197 655
5 126 217 91 298 81 540 242 707 167 58 1
Mean 108 222 113 314 92 541 227 734 193 636
SD
14 21 18
25
9
24
14
34
21
34
Group Total
Mean 105 212 107 299 87 529 226 728 199 622
SD
14 636 24
45 12
49
13
48
23
59
Wildlife International, Ltd.
- 44 -
Project Number 454- I 13
APPENDIX VI
Individual Body Weights (g) from a Mallard Dietary Toxicity Study with T-7485 Page 3
Experimental
Group
Total
(ppma.i.) Bird D a y 0 D a y 5 Change D a y 8 Change Day 15 Change Day22 Change Change
1
1000
2
3
4
5
6
Mean
SD
85 189 104 268 79
97 232 135 321 89
I07 219 112 308 89
112 234 122 324 90
120 266 146 373 107
130 255 125 373 118
109 233 124 328 95
16 27
15
40 14
1
89 177 88 252 75 446 194 663 217 574
1000
2
96 212 16 293 81 485 192 616 131 520
3 109 209 00 308 99 521 213 708 187 599
4 112 250 38 351 101 605 254 808 203 696
5 123 251 28 355 104 593 238 752 159 629
6 138 284 46 395 111 640 245 812 172 674
Mean 111 231 119 326 95 548 223 727 178 615
SD
18 38 22
51 14
76
27
79
31
65
Group Total
Mean 110 232 122 327 95 548 223 727 178 615
SD
16 32
18
44 14
76
27
79
31
65
- Indicates bird x'as euthanized on Day 8 for sampling.
wiId1i,fe Internationa1, I,td.
- 45 -
Project Numbcr 454- I 13
APPENDIX VI Individual Body Weights (g) from a Mallard
Dietary Toxicity Study with T-7485 Page 4
Experimental
Group
Total
(ppma.i.) Bird Day0 Day5 Change Day8 Change Day 15 Change Day22 Change Change
1780
1 2 3 4 5 6 Mean SD
. 89 183 94 93 192 99 105 229 124 109 249 140 122 264 142 127 193 66 108 218 111 15 34 30
256 73 270 78 325 96 360 111 365 101 244 51 303 85 54 22
1780
1 1
90 206 16 295 89 490 195 740 250 650
2
97 184 87 269 85 474 205 645 171 548
3 109 221 12 306 85 544 238 762 218 653
4 117 251 34 348 I 97 550 202 74 1 191 624
5 119 256 137 367 111 595 228 836 241 717
6 130 234 104 307 73 517 210 717 200 587
Mean 110 225 115
3J5 90
528 213
740 212
630
SD
15 27 19
36 13
44
17
62
30
58
Group
Mean 109 222 113 309 88 528 213 740 212 630
Total
SD
14 30 24
44 17
44
17
62
30
58
- Indicates bird was euthanized on Day 8 for sampling.
wiId1ijie Internationa1, Ltd.
- 46 -
Projcct Nuiiibcr 454-1 13
APPENDIX VI
Individual Body Weights (g) from a Mallard Dietary Toxicity Study with T-7485 Page 5
Experimental
Group
Total
(ppm a,;.) Bird Day 0 Day 5 Change Day 8 Change Day 15 Change Day 22 Change Change
3160
1 2 3 4 5 6 Mean SD
80 167 87 94 185 91 108 228 120 118 245 127 122 276 154 125 253 128 108 226 118 18 42 25
250 83 257 72 318 90 344 99 360 84 366 113 316 90 51 14
1
83 189 106 304 / 115 525 221 68 1 156 598
3160
2
90 199 109 286 87 522 236 672 150 582
3 106 230 124 329 99 541 212 700 159 594
4 119 258 139 359 101 619 260 768 149 649
5 122 241 119 353 112 546 193 71 1 165 5 89
6 129 207 78 310 103 554 244 730 176 601
Mean 108 221 113 324 103 551 228 710 159 602
SD
18 27 21
29 10
35
24
35
10
24
Group
Mean 108 223 115 320 97 551 228 710 159 602
Total
SD
17 34 22
40 13
35
24
35
10
24
- Indicates bird was euthanized on Day 8 for sampling.
Wildlife International, Ltd.
- 47 -
Project Number 454- I 13
APPENDIX VI
Individual Body Weights (g) from a Mallard Dietary Toxicity Study with T-7485 Page 6
Experimental
Group
Total
(ppm a.i.) Bird Day 0 Day 5 Change Day 8 Change Day 15 Change Day 22 Change Change
5620
1 2 3 4 5 6 Mean SD
84 181 97
95 203 108
107 219 112
116 217 101
126 229 103
133 246 113
110 216 106
19 22
6
256 75 280 77 306 87 315 98 356 127 339 93 309 93 37 19
1
85 131 46 194 1 63 447 253 661 214 576
5620
2
97 170 73 237 67 482 245 653 171 556
3 102 193 91 272 79 548 276 767 219 665
4 111 197 86 328 131 691 363 819 128 708
5
123 238 115
339 101 591 252
808 217
685
6 - 128 215 87 319 104 558 239 714 156 586
Mean 108 191 83 282 91 553 271 737 184 629
SD
16 37 23
58 26
86
47
72
38
64
~~~
Group
Mean 109 203 94 295 92 553 271 737 184 629
Total
SD
17 32 20
48 22
86
47
72
38
64
- Indicates bird was euthanized on Day 8 for sampling.
Wildlife International, Ltd.
-48 -
Project Number 454-1 13
APPENDIX VI
Individual Body Weights (g) from a Mallard Dietary Toxicity Study with T-7485 Page 7
Experimental Group
(ppm ai.) Bird
~
~~
~~~
Total Day 0 Day 5 Change Day 8 Change Day 15 Change Day 22 Change .Change
10,000
1 2 3 4 5 6 Mean SD
83 118 35 100 167 67 92 173 81 112 219 107 125 237 112 132 218 86 107 189 81 19 44 28
202 84 256 89 249 76 338 119 327 90 327 109 283 95 5 5 16
1
84 151 67 217 1 66 ' 4 5 9 242 680 221 596
10,000
2 100 173 73 264 91 521 257 732 211 632
3 109 163 54 273 -110 521 248 777 256 668
4 1 1 1 207 96 307 100 533 226 750 217 639
5 120 224 104 323 99 508 185 713 205 593
6 130 250 120 360 110 580 220 726 146 596
Mean 109 195 86 291 96 520 230 730 209 621
SD
16 39 25
50 16
39
26
33
36
31
Group
Mean 108 192 84 287 95 520 230 730 209 621
Total
SD
17 40 26
51 16
39
26
33
36
31
- Indicates bird was euthanized on Day S for sampling.
WildlveInternational, Ltd.
-49 -
Project Number 454-1 13
APPENDIX VI1
Feed Consumption (ghirdday) by Pen from a Mallard Dietary Toxicity Study with T-7485 Page 1
Experimental Group
(ppm a.i) Pen
Exposure Period Days 0-5
Days 6-8
Post-Exposure Period Days 9-15
Days 16-22
Control
1
62
0
2
73
3
82
4
70
5
63
6
87
Mean'
73
SD
10
88
118
113
110
153
218
111
159
21 1
b3 1
175
249
1
112
163
226
.*14
11
20
' Mean values calculated using Excel in full-precision mode. Manual calculation may vary.
- Indicates bird was euthanized on Day 8 for sampling.
Wildlife International, Ltd.
- 50 -
Project Numbcr 454-1 13
APPENDIX VI1
Feed Consumption (ghirdday) by Pen from a Mallard Dietary Toxicity Study with T-7485 Page 2
Experimental Group
(ppm a.i.) Pen
Exposure Period Post-Exposure Period Post-Exposure Period Post-Exposure Period
Days 0-5
Days 6-8
Days 9-15
Days 16-22
1000
1
85
133
2
90
123
I53
191
Mean'
88
128
153
191
1780
1
78
128
2
79
137
161
228
Mean'
79
132/
161
228
3160
1
72
114
2
73
169
183
247
Mean'
72
' 142
183
247
5620
1
68
114
2
54
103
129
173
Mean'
61
I09
129
I73
10.000
1
65
104
2
54
101
141
181
Mean'
60
I02
141
181
' Mean values calculated using Excel in full-precision mode. Manual calculation may vary.
- Indicates bird was euthanized on Day 8 for sampling.
Wildlife International, Ltd.
-51 -
Project Number 454-1 13
APPENDIX VI11 Individual Liver Weights (g) from a Mallard Dietary Toxicity Study with T-7485
Replicate
1
2
3
4
5
6
7
8
'
9
10
11
12
13
14
15
Mean'
SD
Replicate
1
2
3
4 5 6 7 8 9
10 11 12
13 14 15 Mean' SD
w
0
10.423 13.533 13.655 15.072 16.034 12.193 14.766 12.346 14.489 13.183 15.682 12.259 15.602 13.696 21.799 14.315 2.588
1000
14.779 16.787 13.522 16.630 23.205 20.538
Test Concentration (Dam a.i.)
1780
3 160
11.612 11.422
14.582 11.892
16.453 14.612
19.088 14.821
17.128 19.441
15.496 21.270
5620
12.335 15.357 14.841 14.766 15.620 17.906
17.577 3.640
15.727 p.533
15.576 3.992
15.138 1.790
~~
10,000 9.866 15.807 12.771 18.895 16.150 15.479
14.828 3.115
0
29.919 22.799
26.250
31.345 37.977 22.717 22.160 22.965 21.348 26.384 21.619 26.076 28.230 26.553 20.764
25.807 4.672
~
Test Concentration (ppm a i )
1000
1780
3160
5620
29.016 26.240 22.878 26.81 1
20.478 24.570 24.640 24.758
26.401 ' 24.662
24.093
30.148
27.167 28.038 25.280 27.828
30.841 28.755 23.643 25.806
30.789 23.519 27.632 27.077
27.449 3.870
25.964 2.087
24.694 1.658
27.071 1.847
d using Excel in full-precision mode. Manual calculation m a w .
~
10,000
22.255 27.412
28.648
29.002 26.268 34.624
28.035 4.041
wildlife International, Ltd.
- 52 -
Appendix IX Changes to Protocol
Projcct Number 454- 113
The study was conducted in accordance with the approved Protocol with the following change.
1. The protocol was amended to indicate blood and liver samples will be analyzed at the Sponsor's discretion and results of tissue analysis will not be reported. Additionally, change the Sponsor's Representative to Sue Beach.
2. The protocol was amended to indicate statistical comparisons of body weight and liver weight data will be made.
3. The protocol was amended to change the Sponsor's Representative to John Newstead.
WiId1i;fe InternationaI, Ltd.
- 53 -
APPENDIX X Personnel Involved in the Study
Project Number 454-1 13
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 of Avian Toxicology ( 3 ) Linda R. Mitchell, Manager of Ecotox Operations (4) Sean P. Gallagher, Senior Biologist, Avian Toxicology ( 5 ) Willard B. Nixon, Ph.D., Director of Chemistry (6) Timothy Z. Kendall, Supervisor, Chemistry (7) Raymond L. Van Hoven, Ph.D.,'Scientist, Chemistry
I