Document NeGBEozavxRvkow9avN5v3Gww

BACK TO MAIN PFOS: A FROG EMBRYO TERATOGENESIS ASSAY -XENOPUS (FETAX) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-116 3M LAB REQUEST NO. U2723 ASTM Method E 1439-91 AUTHORS: Susan J. Palmer Henry O. Krueger, Ph.D. STUDY INITIATION DATE: May 11, 2000 STUDY COMPLETION DATE: April 6, 2001 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 Page 1 o f 181 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . 2- - PROJECT NO.: 454A-116 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Frog Embryo Teratogenesis A ssay-Xenopus (FETAX) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-116 STUDY COMPLETION: April 6, 2001 This study was not conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989. Data collection, record keeping, training and facility records at Wildlife International, Ltd. complied with Good Laboratory Practices, with the following exceptions: The test substance was not characterized in accordance with full GLP compliance prior to the study; 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 has been recharacterized in accordance with GLP on September 7, 2000. The stability of the test substance under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. The phase of the study conducted by University o f Maryland was not conducted in compliance with Good Laboratory Practices. STUDY DIRECTOR: Senior Biologist SPONSOR APPROVAL: BACK TO MAIN W i l d l i f e In t e r n a t i o n a l , Lt d ._______________________ project no .: 454a-i i 6 -3 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 Parts 160 and 792,17 August 1989. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: DATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: Test Substance Preparation May 12, 2000 May 12, 2000 May 18, 2000 Analytical Data and Draft Report July 12 & 13, 2000 July 13, 2000 July 14, 2000 Timothy A. Springer, Ph D. Manager, Regulatory and Technical Support DATE BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -4 REPORT APPROVAL project no.: 454a -i i 6 SPONSOR: 3M Corporation TITLE: PFOS: A Frog Embryo Teratogenesis Assay -Xenopus (FETAX) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-116 STUDY DIRECTOR: MANAGEMENT: Henry O. Krueger, Ph.1 Director, Aquatic Toxicology and Non-Target Plants DATE -o I DATE BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -5 TABLE OF CONTENTS p r o j e c t n o .: 454a - i i 6 Title/Cover Page........................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement....................................................................................................2 Quality Assurance Statement......................................................................................................................................3 Report Approval...........................................................................................................................................................4 Table of Contents.........................................................................................................................................................5 Summary....................................................................................................................................................................... 6 Introduction.................................................................................................................................................................. 8 Objective...................................................................................................................................................................... 8 Experimental Design................................................................................................................................................... 8 Materials and Methods................................................................................................................................................ 9 Results and Discussion.............................................................................................................................................. 10 Conclusions................................................................................................................................................................ 12 References.................................................................................................................................................................. 13 TABLES Table 1 - Summary of Analytical Chemistry D a ta .................................................................................................14 Table 2 - LC50, EC50, MCIG and TI V alues....................................................................................................... 15 APPENDICES Appendix I - Certificate of Analysis..................................................................................................................16 Appendix II - Evaluation of the Toxicity and Teratogenicity o f Chemical U2723 Using the Frog Embryo Teratogenesis Assay - Xenopus (FETAX).......................................19 Appendix III - The Analysis of PFOS in Frog Embryo Teratogenesis A ssay-Xenopus (FETAX) Solutions in Support of Wildlife International, Ltd. Project No.: 454A-11 6 ......................160 Appendix IV - Changes to the Protocol..............................................................................................................180 Appendix V - Personnel Involved in the Study................................................................................................. 181 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -6 - p r o j e c t n o .: 454A-116 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: STUDY: TEST SUBSTANCE: REFERENCE SUBSTANCE: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF EXPOSURE: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: SUMMARY 3M Corporation Rochelle R. Robideau Wildlife International, Ltd. Easton, Maryland 21601 454A-116 PFOS: A Frog Embryo Teratogenesis Assay-Xenopus (FETAX) PFOS (Perfluorooctanesulfonate, Potassium Salt) IUPAC Name: 1-Octanesulfonic acid,1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassium salt; CAS #2795-39-3 6-aminonicotinamide Negative Control, 1.82, 3.07, 5.19, 8.64, 14.4 and 24.0 mg a.i./L and 5.5 and 2500 mg/L 6-aminonicotinamide 1st Assay: Negative Control, 2.00, 2.83,4.73, 7.90, 14.7 and 24.6 mg a.i./L 2ndAssay: Negative Control, 1.91,3.04,4.82, 7.97, 13.3 and 23.1 mg a.i./L 3rd Assay: Negative Control, 1.93, 3.27, 5.25, 8.26, 14.0 and 23.9 mg a.i./L 1st Assay: Experimental Start - May 15, 2000 Biological Termination - May 19, 2000 Experimental Termination - May 22, 2000 2nd and 3rdAssays: Experimental Start - May 22, 2000 Biological Termination - May 26, 2000 Experimental Termination - May 30, 2000 96 Hours South African Clawed Frog (.Xenopus laevis) University of Maryland Wye Research and Education Center Queenstown, Maryland 21658 Embryos BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -7- p r o je c t n o .: 454A-ii6 SUMMARY - Continued - RESULTS (BASED ON MEAN MEASURED CONCENTRATIONS): 1st Assav: 2ndAssav: 3rd Assav: 96-hour l c 50: 13.8 mg a.i./L 17.6 mg a.i./L 15.3 mg a.i./L 95% CONFIDENCE LIMITS: 12.38-15.31 mg a.i./L 15.53 - 19.96 mg a.i./L 1 3 .1 3 - 17.75 mg a.i./L 96-hour e c 50: 12.1 mg a.i./L 17.6 mg a.i./L 16.8 mg a.i./L 95% CONFIDENCE LIMITS: 1 0 .0 0 - 14.58 mg a.i./L 13.49 -2 2 .8 8 mg a.i./L 12.35 - 22.82 mg a.i./L MINIMUM CONCENTRATION TO INHIBIT GROWTH (MCIG): Not calculable 7.97 mg a.i./L 8.26 mg a.i./L TERATOGENIC INDEX (Tl): 1.1 1.0 0.9 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . 8- INTRODUCTION p r o j e c t n o .: 454A-116 This study was conducted in three assays by Wildlife International, Ltd. for 3M Corporation at the University o f Maryland Wye Research and Education Center (UM-WREC) in Queenstown, Maryland and at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f the first assay was conducted from May 15 to May 19, 2000, while the second and third assays were concurrently conducted from May 22 to May 26, 2000. Raw data generated by Wildlife International, Ltd., data obtained from the University o f Maryland, and a copy o f the final report are filed under Project Number 454A -116 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to determine the possible developmental toxicity of PFOS (Perfluorooctanesulfonate, Potassium Salt), also referred to as test substance U2723, on the embryos ofthe South African clawed frog (.Xenopus laevis) during a 96-hour exposure period. EXPERIMENTAL DESIGN Three definitive assays were conducted with PFOS using the frog embryo teratogenesis assay -Xenopus (FETAX). During each assay, Xenopus embryos were exposed to a geometric series of six test concentrations (PFOS in FETAX solution), a negative control (FETAX solution only), and two concentrations of a reference toxicant (6-aminonicotinamide in FETAX solution) for 96 hours. Two replicate test chambers were maintained in each treatment group and four replicate test chambers were maintained in each control group. Each test chamber contained 25 embryos for a total of 50 embryos per treatment group and 100 embryos per control group. An abiotic replicate at the highest concentration was included in each assay for analytical sampling at 96 hours. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an exploratory range finding toxicity test. Nominal test concentrations selected were 1.82,3.07,5.19, 8.64,14.4 and 24.0 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined at Wildlife International, Ltd. from samples of test solution collected from each treatment and the negative control group prior to and at the end of each assay. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -9 - p r o j e c t n o .: 454A-H6 The test solutions were prepared at Wildlife International, Ltd. and were delivered to UM-WREC prior to each assay. At UM-WREC, the embry os were randomly assigned to test chambers containing the appropriate FETAX solution and were observed for 96 hours. The FETAX solutions in the test chambers were renewed every 24 hours, and mortality observations and general water quality measurements were made at each 24-hour renewal period. At the end of the 96-hour exposure period, each surviving embryo was examined for malformations using a dissecting microscope. An LC50 value for mortality and an EC50 value for malformation were calculated at 96 hours for each assay. When possible, the minimum concentration to inhibit growth (MCIG) and the teratogenic index (TI) were calculated for each assay. MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A Frog Embryo Teratogenesis Assay - Xenopus (FETAX)". The protocol was based on procedures outlined in ASTM Standard E l439-91 Standard Guide fo r Conducting the Frog Embryo Teratogenesis Assay - Xenopus (FETAX) (1) and ASTM Standard E729-88a Standard Guide fo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (2). Test and Reference Substances The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot num ber 217 (T-6295). Inform ation provided by the Sponsor indicated a purity of 98.9%, and an expiration date of 2008. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated September 7, 2000 indicated a purity of 86.9% and an expiration date of August 31,2001. The test substance was stored at ambient room temperature. The reference substance was maintained by UM-WREC. It was identified as 6-aminonicotinamide (CAS #329-89-5; Sigma; Lot No. 125H0006). Preparation of Test Concentrations Prior to each assay, FETAX solution was prepared at UM-WREC and delivered to Wildlife International, Ltd. for the preparation of the stock and test solutions. The second and third assays were conducted concurrently using common test solutions. A primary stock solution was prepared in FETAX solution at a concentration of 48 mg a.i./L. The primary stock solution was mixed by sonication and stirring to aid in the solubilization o f the BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 10- p r o je c t n o .: 454A-116 test substance. The primary stock was proportionally diluted with FETAX solution to prepare the six test concentrations. Nominal test concentrations were 1.82,3.07,5.19,8.64,14.4 and 24.0 mg a.i./L, based on a test substance purity of 86.9%. The test solutions were delivered to UM-WREC prior to the start o f each assay. The reference toxicant solutions were prepared at UM-WREC. Analytical Chemistry Samples were collected from each test solution and the negative control solution at preparation, and at the termination of each assay (96 hours) to measure concentrations of the test substance. Samples o f test solution from the abiotic replicates also were collected at 96 hours. The samples were analyzed at Wildlife International, Ltd. Analytical procedures used in the analysis of the samples are provided in Appendix II. In-Life Phase o f Assays Details of the in-life phase o f the three assays conducted at UM-WREC are included in Appendix I. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFOS in FETAX solution samples collected during the three assays are presented in Table 1 and in the analytical chemistry report (Appendix II). Nominal concentrations selected for use in this study were 1.82, 3.07, 5.19, 8.64, 14.4 and 24.0 mg a.i./L. In the first assay, the measured concentrations of PFOS in test solution samples collected prior to initiation o f exposure of the test organisms ranged from 112to 14 l% o fth e nominal concentrations. Samples collected at test termination had a measured concentration range o f 54.7 to 98.6% o f nominal values. The sample from the abiotic 24.0 mg a.i./L treatment group was comparable to samples from the 24.0 mg a.i./L treatment group with the frog embryos present. For the second and third assays, common test solutions were prepared. The measured concentrations of PFOS in test solution samples collected prior to initiation of exposure of the test organisms ranged from 95.8 to 117% of the nominal concentrations. In the second assay, samples collected at test termination had a measured concentration range of 80.7 to 112% of nominal values. The sample from the abiotic 24.0 mg a.i./L treatment group was comparable to samples from the 24.0 mg a.i./L treatment group with the frog embryos present. In the third assay, samples collected at test termination had a measured concentration range of 93.4 to 114% of nominal values. The sample from the abiotic 24.0 mg a.i./L treatment group was comparable to samples from the 24.0 mg a.i./L treatment group with the frog embryos present. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 11 - p r o j e c t n o .: 454A-116 When measured concentrations of the samples analyzed prior to and at the end of each assay were averaged, the mean measured concentrations for the first assay were 2.00, 2.83, 4.73, 7.90, 14.7 and 24.6 mg a.i./L, representing 110,92.2,91.1,91.4,102 and 103% of nominal concentrations, respectively. The mean measured concentrations for the second assay were 1.91,3.04,4.82,7.97,13.3 and 23.1 mg a.i./L, representing 105, 99.0, 92.9, 92.2, 92.4 and 96.3% of nominal concentrations, respectively. The mean measured concentrations for the third assay were 1.93, 3.27, 5.25, 8.26, 14.0 and 23.9 mg a.i./L, representing 106, 107, 101, 95.6, 97.2 and 99.6% of nominal concentrations, respectively. The results of the study were based on the mean measured test concentrations for each assay. Definitive Assay Results The results of the three definitive FETAX assays conducted with PFOS at the UM-WREC are included in Appendix I and are summarized in Table 2. Exposure to PFOS caused significant embryo mortality at the two highest test concentrations (14.4 and 24.0 mg a.i./L nominal concentration) in each o f the three assays. Most of the embryo mortality appeared to be caused by the gut coiling through the body wall at the two highest concentrations. Embryo survival was >90% at all test concentrations up to 8.64 mg a.i./L nominal concentration. The 96-hour LC50 values for PFOS ranged from 13.8 to 17.6 mg a.i./L mean measured concentration in the three assays (Table 2). There was a positive correlation between PFOS exposure and malformations in each of the three assays, with increasing concentrations causing an increase in the incidence of malformed embryos. The 96-hour EC50 values, based on the incidence o f malformed embryos, ranged from 12.1 to 17.6 mg a.i./L mean measured concentration in the three assays (Table 2). The most common types of malformations caused by exposure to PFOS were improper gut coiling, edema, notochord abnormalities and facial abnormalities. Growth of the embryos was not effected by exposure to PFOS in the first assay. In the second and third assays, the minimum concentration inhibiting growth (MCIG) was 8.64 mg a.i./L nominal concentration. This reduction in growth may be attributed more to the increased incidence of malformed embryos in the test concentrations than to an overall decrease in growth. The teratogenic index (TI), defined as the 96-hour LC50 value for mortality divided by the 96-hour EC 50 value for malformed embryos, provides an estimate o f the teratogenic risk associated with PFOS. The TTs in the three assays ranged from 0.9 to 1.1, indicating the PFOS has a low potential to be a developmental hazard. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 12- CONCLUSIONS p r o j e c t n o .: 454A-116 The 96-hour LC50 value for mortality for Xenopus laevis exposed to PFOS in each o f three assays was 13.8, 17.6 and 15.3 mg a.i./L mean measured concentration, respectively. The 96-hour EC50 value for embryo malformation in each o f three assays was 12.1, 17.6 and 16.8 mg a.i./L mean measured concentration, respectively. The MCIG, determined in the second and third assays, was 7.97 and 8.26 mg a.i./L mean measured concentration, respectively. The TTs ranged from 0.9 to 1.1 in the three assays, indicating the PFOS has a low potential to be a developmental hazard. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -13REFERENCES p r o j e c t n o .. 454A-116*12 1 ASTM S tandard E l 439-91. 1998. Standard Guide fo r Conducting the Frog Embryo Teratogenesis Assay - Xenopus (FETAX). American Society for Testing and Materials. Philadelphia, Pennsylvania. 2 ASTM S tandard E729-88a. 1994. Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians, American Society for Testing and Materials. Philadelphia, Pennsylvania. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 14- PROJECTNO.: 454A-116 Table 1 Summary o f Analytical Chem istry D ata Nominal Test Concentration (mg a.i./L) Negative Control Sampling . Time Measured (Day) Concentration (mg a.i./L) O2 <LOQ4 43 <LOQ 1st Assay Mean Measured Concentration (mg a.i./L) <LOQ Percent of Nominal __ Measured Concentration (mg a.i./L) <LOQ <LOQ 2 nd Assay1 Mean Measured Concentration (mg a.i./L) <LOQ Percent of Nominal __ Measured Concentration (mg a.i./L) <LOQ <LOQ 3rd Assay1 Mean Measured Concentration (mg a.i./L) <LOQ Percent of Nominal 1.82 0 2.58 2.00 110 1.77 4 1.42 2.04 1.91 105 1.77 2.08 1.93 106 3.07 0 3.94 2.83 92.2 3.59 3.04 99.0 3.59 3.27 107 4 1.72 2.49 2.94 5.19 0 6.62 4.73 91.1 5.45 4.82 92.9 5.45 5.25 101 4 2.84 4.18 5.05 8.64 0 10.7 7.90 91.4 8.43 7.97 92.2 8.43 8.26 95.6 4 5.09 7.51 8.09 14.4 0 18.5 4 10.8 14.7 102 14.5 12.1 13.3 92.4 14.5 13.5 14.0 97.2 24.0 0 26.9 24.6 103 23.0 23.1 96.3 23.0 23.9 99.6 4 22.3 23.1 24.7 24.0 (abiotic) 4 23.7 - 98.6 23.9 -- 99.6 24.1 - 100 1 The 2nd and 3rd assays were conducted concurrently using common batches of test solution prepared for Day 0. 2 Day 0 samples were collected from the test solutions at preparation prior to transfer to UM-WREC. 3 Day 4 samples were collected from the test solutions at the termination of each assay at UM-WREC and transferred to Wildlife International, Ltd. for analysis. 4 The limit of quantitation (LOQ) was 0.240 mg a.i./L. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 15- p r o je c t n o .: 454A-116 Table 2 LC50, EC50, MCIG and TI V alues1 Parameter 96-Hour LC50 (mg a.i./L): 95% Confidence Limits (mg a.i./L): 1st Assay 13.8 12.38-15.31 2ndAssay 17.6 15.53-19.96 3rd Assay 15.3 13.13-17.75 96-Hour EC50 (mg a.i./L): 95% Confidence Limits (mg a.i./L): 12.1 10.00-14.58 17.6 13.49-22.88 16.8 12.35 -2 2 .8 2 Minimum Concentration Inhibiting Growth (MCIG) (mg a.i./L): _ 2 7.97 8.26 Teratogenic Index (TI): 1.1 1.0 0.9 1 Values are based on the mean measured concentrations for each assay. 2 The MCIG could not be calculated because PFOS had no effect on embryonic growth at any of the test concentrations with surviving embryos in the 1st assay. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 16- APPENDIXI Certificate of Analysis PROJECT NO.: 454A-116 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFO S,L ot217 Reference#: SD-018 Teat Name Specifications Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium3 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (L C /M S ) Total % Impurity (G C /M S ) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulihte4 Organic A cids5 (IC) 1. T FA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis4: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder v, T. : 'iWifS-i?;:;L'/pi&.i ! . < t f f i , ' . : ! [Mi'',ji?i iif&i;; i?P j*:' ;[\P;: s::f i !S:? >:. i^sllUiiSLS.! :- P ' ft. j I:l!- i.;>>.'.*>;L"vP..; : J--K* t'!1 ^?=i h k3 ;r/i:=!> .. ................. v:* ;y : ;7 ,: 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% 86.9% Conforms Positive 1. 0.005 wt./wt.% 2. 0.001 wt./wt.% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 5. <0.001 w tJw t.% 6. 0.005 wt./wt.% 7. <0.001 wt./wt.% 1.93 wt./wt.% 8.41 wt./wt.% None Detected 0.33 wt./wt.% None Detected N ot Applicable5 1. <0.015 wtVwt.% 2. 0.59 wtVwt.% 3. <0.040 w t7w t% 4. <0.009 w tJw t.% 5. <0.006 wtVwt.% 6. <0.007 wt./wt.% 7. 8.76 wt.Avt.% 1. <0.1 wL/wt.% 2. <0.1 w t/w t.% 3. 0.10 w t/w t.% 4. 0.28 w t/w t.% 1. 12.48 w t/w t.% 2. 0.244 w t/w t.% 3. 1.74 w t/w t.% 4. 8.84 w t/w t.% 5. 54.1 w t/w t.% COA023-018A Page 1 of 3 W il d l if e In t e r n a t io n a l , Lt d . - 17- BACK TO MAIN PROJECTNO.: 454A-16 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% JPotassium is expected in this salt form and is therefore not considered an impurity. ^Purity by DSC is generally not applicable to materials o f low purity. N o endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4is not considered animpurity.5 5T F A HFBA NFPA PFPA Trifluoroacetic acid Heptafiuorobutyric add Nonofluoropentanoic arid Pentafluoropropanoic add `Theoretical value calculations based on the empirical formula, C*FI7S0 3 "K+(MW=53 8) This work was conducted under EPA Good Laboratory Practice Standards (40 C F R 160). 1 COA023-018A Page 2 of 3 W il d l if e In t e r n a t io n a l , Lt d . - 18- BACK TO MAIN ' PROJECT NO.: 454A-L6 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: ________________________ ________ ____ ; David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed B y : ________________________ '_______ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of 3 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 19- PROJECT NO.: 454A-116 APPENDIX II Evaluation of the Toxicity and Teratogenicity of Chemical U2723 Using the Frog Embryo Teratogenesis Assay -Xenopus (FETAX) W il d l if e In t e r n a t io n a l , Lt d . -20- BACK TO MAIN PROJECTNO.: 454A-116 Report No. W REC-00-02 EVALUATION OF THE TOXICITY AND TERATOGENICITY OF CHEMICAL U2723 USING THE FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) Final Report Prepared for W ildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 Prepared by Steven D. Turley and Dennis T . Burton University of Maryland W ye Research and Education Center P.O. Box 169 Queenstown, MD 21658 July 15, 2000 Revision Date: March 19, 2001 W il d l if e In t e r n a t io n a l , Lt d . -21 - BACK TO MAIN PROJECT NO.: 454A-116 TABLE OF CONTENTS Page TABLE O F C O N T E N T S ........................................................................................................... ii 1. IN T R O D U C T IO N ..................................................................................................................1-1 2. M ATERIAL AND M E T H O D S .............................................................................................2-1 3. R ESULTS AND DISCUSSIO N ........................................................................................ 3-1 4. REFER ENC ES ............................. 4-1 A PP EN D IX 1. FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CO NDUCTED W ITH C H E M IC A L U 2 7 2 3 (A S S A Y 1 ) .................... A 1-1 A PP EN D IX 2. FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CO NDUCTED W ITH C H E M IC A L U 2 7 2 3 (A S S A Y 2 ) ....................A 2-1 A PP EN D IX 3. FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTED W ITH C H E M IC A L U 2 7 2 3 (A S S A Y 3 ) .................... A3-1 A PP EN D IX 4. RAW DATA SHEETS FOR FROG EMBRYO TERATO G ENESIS ASSAY-XENOPUS (FETAX) ASSAYS 1, 2, AND 3 CONDUCTED ON CHEMICAL U 2723 ............................................................................ A4-1 A PP EN D IX 5. 6-AM INO NICO TINAM IDE REFERENCE TO XIC A N T DATA FO R XENOPUS L A E V IS ....................................................................... A5-1 A PP EN D IX 6. PHOTOGRAPHS OF NORMAL AND MALFORMED EMBRYOS EXPOSED TO CHEMICAL U 2723 ................................ A6-1 ii W il d l if e In t e r n a t io n a l , Lt d . -22- BACK TO MAIN PROJECTNO.: 454A-116 1. INTRODUCTION The evaluation of the potential toxicity and teratogenicity of chemical U 2723 was initiated at the request of Mr. David A. Palmer, W ildlife International, Ltd., Easton, Maryland. The study was conducted by Mr. Steven D. Turley and Dr. Dennis T . Burton of the University of Maryland W ye Research and Education Center, Queenstown, Maryland. The test program was performed in accordance with the "Revised Statem ent of W ork for Evaluating the Potential Teratogenicity of U2723 Using 96-H our Frog Embryo Teratogenesis Assay-Xenopus (FETAX)' , submitted to and approved by W ildlife International, Ltd., March 3 0 ,2 0 0 0 (Turiey, 2000). The potential toxicity and teratogenicity of chemical U 2723 was evaluated by the Frog Embryo Teratogenesis Assay-Xenopus . (FETA X). Three 96-h definitive FETAX assays were conducted on chemical U2723. 1-1 W il d l if e In t e r n a t io n a l , Lt d . -23- BACK TO MAIN PROJECTNO.: 454A-116 2. MATERIALS AND METHODS Three definitive FETAX assays were conducted on chemical U 2723 using the frog embryo teratogenesis assay-Xenopus (FETAX). The assay is a 96-h quantitative developm ental assay used to screen for developmental toxicants in aquatic media. The assays w ere conducted using the static renewal (solutions renewed every 2 4 h) test protocol Designation E 1439-91 of the American Society forTesting and M aterials (ASTM, 1998). Embryolethality (96-h LC50), malformations (96-h EC50), and growth retardation were used as endpoints in the assays. Embryos were obtained from breeding colonies ofXenopus laevis at the University of M aryland W ye Research and Education Center (UM D/W REC). The UM D /W R EC adult colonies w ere maintained in flow-through ( - 4 replacement volumes per day) circular polyethylene aquaria (0.91 m I.D . x 0.36 m high) with a water depth of 10 cm. Each aquarium contained a maximum of 10 adults. UM D/W REC non-chlorinated deep well w ater held at 2 3 .5 0 .5 C served as the culture medium. All frogs were fed every 5 -6 d with commercial beef liver supplemented with liquid vitamins (PolyViSolTM ; Mead-Johnson Nutritionals, Evansville, Indiana). The colony was held under a photoperiod of 16 h light:8 h dark. Mating pairs were bred in the dark in 23.5 0.5 C UM D/W REC non-chlorinated w ater at -7 0 d intervals by injecting 400 and 800 I.U. of human chorionic gonadotropin (H C G ) in the dorsal lymph sac of the males and females, respectively. Amplexus occurred 4 -6 h after injecting HCG; egg deposition occurred 9-12 h following HCG injection. Embryos between normal stage 8 blastulae and normal stage 11 gastrulae were used to initiate the assays (Nieuwkoop and Faber, 1975). Embryos from separate breeding pairs w ere used for each test to check for variability in the sensitivity of individual clutches of embryos. All embryos were examined two times for viability. The embryos w ere de-jellied in a 2% L-cysteine solution (2 g of L-cysteine per 98 mL of FETAX solution). Once de-jellied, the embryos were rinsed and re-suspended in FETAX solution (ASTM , 1998). The embryos w ere assayed in covered 60 mm glass Petri dishes containing 10 mL of solution. All embryos w ere randomly assigned to Hie Petri dishes. Two replicates of 25 embryos/replicate were used for each of six assay treatments; four replicates w ere used for the controls as required by the test protocol (ASTM , 1991). The assays w ere conducted at 24 0.2C under a 12-h light; 12-h dark photoperiod (fluorescent lights; ~75 foot candles at the surface of the test medium) in a constant tem perature environmental chamber. All Petri dishes w ere randomly placed in the environmental chamber used for the assays. W ildlife International, Ltd., personnel prepared and delivered stock solutions of each concentration at the start of each assay. The concentration of each solution was quantified at W ildlife International, Ltd. Two concentrations ofthe reference toxicant, 6-aminonicotinamide (CAS #329-89-5; Sigma; Lot No. 125H 0006), were used at each assay period as outlined in the ASTM (1998) protocol. 2-1 W il d l if e In t e r n a t io n a l , Lt d . -24- BACK TO MAIN PROJECTNO.: 454A-116 M ortality observations and general water quality measurements w ere made at each 24-h renewal period. All dead embryos were removed at each observation period. At the end of the 96-h exposure period, each surviving embryo was examined for malformations using a dissecting microscope. The identification and interpretation of malformations in the embryos at 96 h were made via the atlas of Bantle et al. (1991). The head-tail length of each surviving embryo was determined via Sigma Scan's digitizing software (SPSS, Corte M adera, C alifornia). A random group of normal embryos and embryos with each type of malformation were preserved in 10% buffered formalin (pH 7 ) for photographing. The Trimmed Spearm an-Karber statistical procedure was used to determ ine the 96h LC 50 for mortality and 96-h EC50 for malformations. The minimum concentration to inhibit growth (M C IG ) was determined by Bonferroni's T-Test. All statistical tests were performed using Toxstat (W E S T and Gulley, 1994). A minimum probability level of 0.05 was used. The teratogenic index (T l) was calculated by dividing the LC50 by the EC50. The test acceptability criteria given in the ASTM (1998) test protocol was used for each definitive test. 2-2 W il d l if e In t e r n a t io n a l , Lt d . -25 - BACK TO MAIN PROJECT NO.: 454A-116 3. RESULTS AND DISCUSSION The data for definitive assays 1 ,2 , and 3 are summarized in Appendices 1,2, and 3, respectively. Copies ofthe raw data sheets and 6-aminonicotinamide reference toxicant data for assays 1 ,2 , and 3 are given in Appendices 4 ,5 , and 6, respectively. The nominal concentrations of 1 .9 ,3 .2 ,5 .4 ,9 .0 ,1 5 .0 and 25.0 mg/L on the raw data sheets correspond to the nominal concentrations of 1.82, 3.07, 5.19, 8.64, 14.4 and 2 4.0 mg a .i./L in the report. Photographs of normal and malformed embryos of each type observed in the study are given in Attachment 1. Table 1 is summary of the endpoint parameters established in the three definitive assays. Table 1. Summary of Data for the Three Assays with Chemical U2723* Param eter LC50 (95% C. I.) EC 50 (95% C. I.) M CIG Tl Assay 1 13.8 (12 .3 8 -1 5 .3 1 ) 12.1 (10 .0 0 -1 4 .5 8 ) b 1.1 Assay 2 17.6 (1 5 .5 3 -1 9 .9 6 ) 17.6 (1 3 .4 9 -2 2 .8 8 ) 7 .9 7 1.0 Assay 3 1 5 .3 (1 3 .1 3 -1 7 .7 5 ) 16.8 (12.35 - 22.82) 8 .2 6 0 .9 * The LC50, EC 50, and M C IG values are based on mean measured concentrations of chemical U 2723 (mg a.i./L ) for each assay. bThe M CIG could not be calculated because chemical U 2723 had no effect on embryonic growth at any of the test treatments with surviving embryos (Appendix A; Table A 1- 4). Exposure to chemical U2723 caused significant embryo mortality at the two highest treatm ent concentrations (14.4 and 24.0 mg/L) in each of the three assays. Most of the embryo mortality appeared to be caused by the gut coiling through the body wall at the two highest assay treatments. Embryo survival was 90% at all exposure treatm ents up to 8.64 mg/L. The 96-h LC50s for chemical U2723 ranged from 13.8 to 17.6 mg/L (Table 1). There was a positive correlation between chemical U 2723 exposure and malformations in each of the three assays, with increasing concentrations causing an increase in the incidence of malformed embryos. The 96-h EC50s, based on the incidence of malformed embryos, ranged from 12.1 to 17.6 mg/L (Table 1). The most common types of malformations caused by exposure to chemical U 2723 were: improper gut coiling, 3-1 W il d l if e In t e r n a t io n a l , Lt d . -26- BACK TO MAIN PROJECTNO.: 454A-116 edem a, notochord abnormalities and facial abnormalities. Growth of the embryos was not affected by exposure to chemical U 2723 in assay 1. In assays 2 and 3 , the minimum concentration inhibiting growth (M C IG ) was 7.97 and 8.26 mg/L, respectively. This reduction in growth may be attributed more to the increased incidence of malformed embryos in these test concentrations than an overall decrease in growth. Malformed embryos are generally smaller than normally developing embryos. The teratogenic index (Tl), which by definition is the 96-h LC50 divided by the 96-h EC 50 (malformed embryos), provides an estimate of the teratogenic risk associated with a m aterial (Dumont et a l., 1983). T l values of 1.5 to 2.0 indicate that a m aterial may be a potential teratogen. M aterials with T l values >2.0 should be considered for further teratogenicity testing. The Tl's in the current study ranged from 0 .9 to 1.1 which indicate that chemical U 2723 has a low potential to be a developmental hazard. The three assays met all of the ASTM (1998) protocol test acceptability criteria, with one exception. The reference toxicant (6-aminonicotinamide) high concentration of 2,500 mg/L did not m eet the ASTM protocol test acceptability criterion. A concentration of 2,500 mg/L should kill between 40 and 60% of the embryos in 96 h. All embryos died at 2,500 mg/L in all three reference toxicant tests. All three reference toxicant tests met the protocol test acceptability criteria for the low reference toxicant concentration of 5.5 mg/L which should cause between 40 and 60% malformed embryos after 96 h of exposure. The 5 .5 mg/L reference toxicant tests produced an average of 55.6, 55.3, and 53.1% malformed embryos during assays 1 ,2 , and 3, respectively. The results of the reference toxicant concentrations in the three current tests are consistent and not at variance with previous experience in our laboratory. The survival of control embryos after 96 hours in our laboratory consistently meet the ASTM protocol acceptability criterion of >90% survival. In the current chemical U2723 assays, the average survival of the controls was 99, 99, and 100% , in assays 1 ,2 , and 3, respectively. Th e chain of custody forms for delivery of the assay solutions from W ildlife International, Ltd. to the UM D/W REC were retained by W ildlife International, Ltd. Thus, copies of the chain of custody forms are not included in this report. 3-2 W il d l if e In t e r n a t io n a l , Lt d . -27- BACK TO MAIN PROJECTNO.: 454A-116 4. REFERENCES ASTM . 1998. Standard guide for conducting the frog embryo teratogenesis assayXenopus (FETA X). ASTM Designation E 1439-91. 1998 Annual Book of ASTM Standards Section 11 W ater and Environmental Technology, Amer. Soc. Testing M aterials, Philadelphia, P A Bantle, J.A., J.N. Dumont, R.A. Finch, and G. Linder. 1991. Atlas of abnorm alities. A guide for the performance of FETAX. Oklahoma State Univ., Stillwater, OK. Dumont, J., T . Schultz, M. Buchanan, and G. Kao. 1983. Frog embryo teratogenesis assay-Xenopus (FETAX) - A short-term assay applicable to complex environmental mixtures. Pages 393-405 in: W aters, Sandhu, S.S., J. Lewtas, L. Claxton, N. Chem off, and S. Nesnow, eds. Short-term bioassays in the analysis of complex environmental mixtures III, Plenum, New York, NY. Nieuwkoop, P. D. and J. Faber. 1975. Normal tables of Xenopus laevis (Daudin), 2nded. North Holland, Amsterdam, Netherlands. Turley, S.D. 2000. R evised statem ent of w ork fo r evaluating the potential terato g en icity o f U 2 7 2 3 using 96-hour frog em bryo terato g en esis assayXenopus (F E T A X ). M arch 3 0 ,2 0 0 0 . University of Maryland W ye Research and Education Center, Queenstown, MD. W E S T (W estern Ecosystem Technology, Inc.) and Gulley, D. D. 1994. Toxstat, v. 3.4. W estern Ecosystem Technology, Inc., Cheyenne, W Y. 4-1 W il d l if e In t e r n a t io n a l , Lt d . -28- BACK TO MAIN PROJECTNO.: 454A-116 A P P E N D IX 1 F R O G E M B R Y O T E R A T O G E N E S IS A S S A Y -XEN O PU S (F E T A X ) C O N D U C T E D W IT H C H E M IC A L U 2723 (A S S A Y 1) Test Method: Type of Test: Date: In v e s tig a to r Laboratory: C hem ical: Designation: Source: G eneral W ater Quality: Test Medium: Test Organism: Scientific Name: Age at Start of Test: Source: Experimental Chambers: M aterial: Test Solution Volume: No. Organisms/Replicate: No. Organisms/Treatment: Lighting: Endpoints: Test Temperature: ASTM Designation E 1439-91 ASTM (1998) Static renewal (every 24 h) May 15-19, 2000 S. D. Turley UM D/W REC U2723 W ildlife International, Ltd. See Table A1-1 FETAX solution Xenopus laewis Stage 8 blastula to stage 11 gastrula UM D/W REC culture Glass petri dishes 10 mL 25 Control: 100 Treatment: 50 Fluorescent; 60-85 foot candles Mortality; malformation; growth 24 2#C A1-1 W il d l if e In t e r n a t io n a l , Lt d . -29- BACK TO MAIN PROJECTNO.: 454A-116 Results: Note: For Assay 1, statistical calculations are based on the mean measured concentrations of 2 .0 0 ,2 .8 3 ,4 .7 3 ,7 .9 0 ,1 4 .7 0 and 24.60 mg a.i./L, rather than on the nominal concentrations. M ortality: The 96-h LC50 = 13.8 mg a.i./L (95% confidence limits = 12.38 -1 5 .3 1 ). The mortality data are summarized in Table A1-2. M alform ations: The 96-h EC 50 (malformed embryos) = 12.1 mg a.i./L (95% confidence limits = 1 0 .0 0 -1 4 .5 8 ). The number of malformed embryos are summarized in Table A 1-2. The types of malformations are summarized in Table A1-3. Growth: Chemical U 2723 had no effect on embryonic growth at any of the test treatments with surviving embryos. As a result, the minimum concentration inhibiting growth (M C IG ) could not be calculated. The growth data are summarized in Table A1-4. Teratogenic Index The teratogenic index (T l) = 1.1. A1-2 W il d l if e In t e r n a t io n a l , Lt d . -30- BACK TO MAIN PROJECTNO.: 454A-116 Table A1 -1. Summary of The General W ater Chemistry Data for Chemical U 2723 Assay 1 - Dissolved Oxygen (mg/L) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean M in Max 0 .0 0 8 .3 8 .0 8 .4 7 .7 8 .3 7 .4 8 .3 7 .7 8.1 7 .4 8 .4 1.82 Test Concentrations (mg/L) 3 .0 7 5 .1 9 8 .6 4 14.40 2 4 .0 0 8.4 8.4 8.4 8.4 8.4 8.5 7.8 7.9 7.9 8.0 8.0 7.9 8.4 8.5 8.4 8.5 8.5 8.4 7.7 7.6 7.7 7.8 7.8 7.8 8.3 8.4 8.3 8.2 8.3 8.3 7.5 7.7 7.7 7.8 7.9 7.9 8.2 8.1 8.2 8.1 8.1 8.1 7.7 7.6 7.5 7.7 7.7 7.7 8.0 8.1 8.1 8.1 8.1 8.1 7.5 7.6 7.7 7.8 7.8 7.8 8.4 8.5 8.4 8.5 8.5 8.5 A1-3 W il d l if e In t e r n a t io n a l , Lt d . -31 - BACK TO MAIN PROJECT NO.: 454A-116 T able A1 -1. (Continued) - pH (Standard Units) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H M in Max 0 .0 0 1.82 Test Concentrations (mg/L) 3 .0 7 5 .1 9 8 .6 4 7 .7 4 7 .7 8 7 .7 9 7 .7 8 7 .7 8 7 .2 6 7 .2 4 7 .2 4 7 .4 5 7 .3 0 7 .3 8 7 .3 4 7 .4 3 7 .3 5 7 .4 3 7 .1 6 7 .2 2 7 .1 7 7 .2 5 7 .2 0 7 .2 8 7 .2 3 7 .3 2 7 .2 3 7 .3 4 7.11 7 .1 3 7 .1 3 7 .2 0 7 .1 6 7 .2 3 7 .1 7 7 .2 5 7 .2 5 7 .2 6 7.11 7.11 7 .7 4 7 .1 5 7 .1 3 7 .7 8 7 .1 8 7 .1 6 7 .7 9 7 .1 9 7 .1 7 7 .7 8 7 .1 8 7 .1 8 7 .7 8 1 4 .4 0 7 .7 6 7 .3 4 7 .4 4 7 .2 3 7 .3 5 7 .1 8 7 .2 6 7 .2 0 7 .1 8 7 .7 6 2 4 .0 0 7 .6 2 7 .3 3 7 .3 2 7 .2 5 7 .2 7 6 .9 5 7 .1 8 7 .1 9 6 .9 5 7 .6 2 A1-4 W il d l if e In t e r n a t io n a l , Lt d . - 32- BACK TO MAIN PROJECTNO.: 454A-116 Table A 1-1. (Continued) - Tem perature (C) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean M in Max 0 .0 0 1.82 Test Concentrations (mg/L) 3 .0 7 5 .1 9 8 .6 4 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 1 4 .4 0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 2 4 .0 A1-5 W il d l if e In t e r n a t io n a l , Lt d . -33- BACK TO MAIN PROJECTNO.: 454A-116 Table A1 -2. FETAX Toxicity Test Data - Percent Embryo Mortality and Malformations after 96 Hours of Exposure to Chemical U2723 - Assay 1 Concentration (mg/L) Control Rep 1 2 3 4 Number Embryos Alive 24 25 25 25 Percent Mortality 4.0 0.0 0.0 0.0 Number Embryos Malformed 1 1 1 1 Percent Malformed 4.2 4.0 4.0 4.0 1.82 1 24 4.0 2 8.3 2 25 0.0 2 8.0 3.07 1 24 2 24 4.0 4.0 2 8.3 5 20.8 5.19 1 23 8.0 7 30.4 2 22 12.0 3 13.6 8.64 14.40 1 21 16.0 4 19.0 2 23 8.0 7 30.4 1 16 36.0 9 56.3 2 15 40.0 11 73.3 24.00 10 20 100 100 A1-6 W il d l if e In t e r n a t io n a l , Lt d . -34- BACK TO MAIN PROJECTNO.: 454A-116 Table A1 -3. FETAX Toxicity Test Data - Type and Number of Malformed Embryos after 96 Hours Exposure to Chemical U2723 - Assay 1 Malformation Test Concentration (mg/L) 0 1.82 3.07 5.19 8.64 14.40 24.00 Rep Rep Rep Rep Rep Rep Rep 1 2 3 4 12 1 2 1 2 1 2 1 2 12 Severe 1 Stunted Gut 1 1 1 1 1 1 2 5 7 3 4 5 7 11 Edema: Multiple 22 224 3 Cardiac 1 131 224 1 Abdominal 131 2 13 2 Facial 22 224 2 Cephalic Optic 22 1 12 6 Tal Notochord 11 Fin Face Eye 11 122 1 14 32 3 4 6 11 Brain Hemorrhage Cardiac 12 31 Blisters Other* # Malformed 1 1 1 1 2 2 2 5 7 3 4 7 9 11 * Anal edema. A1-7 W il d l if e In t e r n a t io n a l , Lt d . -35- BACK TO MAIN PROJECTNO.: 454A-116 Table A1 -4. FETAX Toxicity Test Data - Embryonic Growth (Length) after 96 Hours Exposure to Chemical U2723 - Assay 1 Concentration (mg/L) Control 1.82 3.07 5.19 8.64 14.40 24.00 Rep 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 Number Embryos Alive 24 25 25 25 24 . 25 24 24 23 22 21 23 16 15 0 0 Average Length (mm) 9.05 8.40 8.29 8.62 8.44 8.14 8.88 8.72 8.42 8.60 8.83 8.58 8.41 7.74 Mean Average Length (mm) 8.59 8.29 8.80 8.51 8.71 8.08 A1-8 W il d l if e In t e r n a t io n a l , Lt d . -36- BACK TO MAIN PROJECTNO.: 454A-116 APPENDIX 2 FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTED WITH CHEMICAL U2723 (ASSAY 2) Test Method: Type of Test: Date: Investigator Laboratory: Chemical: Designation: Source: General W ater Quality: Test Medium: Test Organism: Scientific Name: Age at Start of Test: Source: Experimental Chambers: Material: Test Solution Volume: No. Organisms/Replicate: No. Organisms/Treatment: Lighting: Endpoints: Test Temperature: ASTM Designation E 1439-91 ASTM (1998) Static renewal (every 24 h) May 22-26,2000 S. D. Turley U M D /W R E C U2723 Wildlife International, Ltd. See Table A2-1 FETAX solution Xenopus laevis Stage 8 blastula to stage 11 gastrula UMD/W REC culture Glass petri dishes 10 mL 25 Control: 100 Treatment: 50 Fluorescent; 60-85 foot candles Mortality; malformation; growth 24 2C A2-1 W il d l if e In t e r n a t io n a l , Lt d . -37- BACK TO MAIN PROJECTNO.: 454A-116 Results: Note: For Assay 2, statistical calculations are based on the mean measured concentrations of 1.91, 3.04, 4.82, 7 .9 7 ,1 3 .3 0 and 23.10 mg a.i./L, rather than on the nominal concentrations. Mortality: The 96-h LC50 = 17.6 mg a.i./L (95% confidence limits = 15.53 -19.96). The mortality data are summarized in Table A2-2. Malformations: The 96-h EC50 (malformed embryos) = 17.6 mg a.i./L (95% confidence limits = 13.49 - 22.88) The number of malformed embryos are summarized in Table A2-2. The types of malformations are summarized in Table A2-3. Growth: The minimum concentration inhibiting growth (MCIG ) =7.97 mg a.i./L. The growth data are summarized in Table A2-4. Teratogenic Index: The teratogenic index (Tl) = 1.0. A2-2 W il d l if e In t e r n a t io n a l , Lt d . -38- BACK TO MAIN PROJECTNO.: 454A-116 Table A 2-1. Summary of The General W ater Chemistry Data for Chemical U2723 Assay 2 - Dissolved Oxygen (mg/L) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max 0.00 8.2 8.3 8.2 7.6 8.1 7.5 8.1 7.6 8.0 7.5 8.3 1.82 Test Concentrations (mg/L) 3.07 5.19 8.64 14.40 24.00 8.2 8.1 8.2 8.1 8.2 8.2 8.3 8.0 8.1 8.1 8.1 8.2 8.2 8.2 8.2 8.1 8.1 8.2 7.8 7.5 7.2 7.2 7.2 7.1 8.1 8.0 8.1 8.0 8.0 8.1 7.6 7.5 7.6 7.7 7.3 7.2 8.2 8.1 8.1 8.0 8.1 8.0 7.6 7.5 7.5 7.6 7.5 7.5 8.0 7.9 7.9 7.9 7.8 7.8 7.6 7.5 7.2 7.2 7.2 7.1 8.3 8.2 8.2 8.1 8.2 8.2 A2-3 W il d l if e In t e r n a t io n a l , Lt d . -39- BACK TO MAIN PROJECT NO.: 454A-116 Table A 2-1. (Continued) - pH (Standard Units) Dav 0 OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Min Max 0.00 1.82 Test Concentrations (mg/L) 3.07 5.19 8.64 7.14 7.24 7.31 7.33 7.37 7.12 7.11 7.17 7.21 7.21 7.28 7.23 7.35 7.24 7.35 7.18 7.15 7.22 7.23 7.23 7.30 7.24 7.37 7.24 7.38 7.20 7.17 7.20 7.21 7.22 7.26 7.25 7.34 7.24 7.36 7.11 7.11 7.20 7.21 7.17 7.24 7.20 7.20 7.31 7.29 7.23 7.37 7.33 7.24 7.38 14.40 7.40 7.26 7.39 7.23 7.41 7.23 7.40 7.39 7.23 7.41 24.00 7.37 7.27 7.34 7.24 7.32 7.25 7.35 7.34 7.24 7.37 A2-4 W il d l if e In t e r n a t io n a l , Lt d . -40- BACK TO MAIN PROJECTNO.: 454A-116 Table A2-1. (Continued) - Temperature (#C) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max 0.00 1.82 Test Concentrations (mg/L) 3.07 5.19 8.64 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 14.40 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.00 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 A2-5 W il d l if e In t e r n a t io n a l , Lt d . -41 - BACK TO MAIN PROJECT NO.: 454A-116 Table A2-2. FETAX Toxicity Test Data - Percent Embryo Mortality and Malformations after 96 Hours of Exposure to Chemical U2723 - Assay 2 Concentration (mg/L) Control Rep 1 2 3 4 Number Embryos Alive 25 25 25 24 Percent Mortality 0.0 0.0 0.0 4.0 Number Embryos Malformed 1 2 0 1 Percent Malformed 4.0 8.0 0.0 4.2 1.82 1 25 0.0 2 8.0 2 25 0.0 2 8.0 3.07 1 23 2 22 8.0 12.0 2 0 8.7 0.0 5.19 1 24 4.0 1 4.2 2 22 12.0 4 18.2 8.64 1 22 12.0 4 18.2 2 23 8.0 5 21.7 14.40 24.00 1 22 12.0 4 18.2 2 13 48.0 9 69.2 1 9 64.0 6 66.7 2 6 76.0 4 66.7 A2-6 W il d l if e In t e r n a t io n a l , Lt d . -42- I BACK TO MAIN projectno.: 454A-116 Table A2-3. FETAX Toxicity Test Data - Type and Number of Malformed Embryos after 96 Hours Exposure to Chemical U2723 - Assay 2 o CM CO 0 : CM : cm Malformation 1 0 Rep Severe Stunted Gut 1 Edema: Multiple Cardiac Abdominal Facial Cephalic Optic Tail Notochord Fin Face 12 Eye Brain Hemorrhage Cardiac 2 Blisters Other* # Malformed 1 Test Concentration (mg/L) 1.82 3.07 5.19 8.64 14.40 24.00 Rep Rep Rep Rep Rep Rep 4 121212 12 12 12 111 2 2 2 2 2 1 12 1 4132664 21 22 1 3 11 13 1 3 4 3 3 13 11 22319 13 122 12 14454 964 A nal edema. A2-7 W il d l if e In t e r n a t io n a l , Lt d . -43 - BACK TO MAIN PROJECT NO.: 454A-116 Table A2-4. FETAX Toxicity Test Data - Embryonic Growth (Length) after 96 Hours Exposure to Chemical U2723 - Assay 2 Concentration (mg/L) Control 1.82 3.07 5.19 8.64 14.40 24.00 Rep 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 Number Embryos Alive 25 25 25 24 25 25 23 22 24 22 22 23 22 13 9 6 Average Length (mm) 8.94 8.81 8.90 8.88 8.39 8.50 8.42 8.71 8.69 8.74 7.94 7.92 7.89 7.13 7.50 7.27 Significantly different at alpha = 0.05 (Bonferroni T-Test) Mean Average Length (mm) 8.88 8.45 8.57 8.72 7.93* 7.51* 7.39* A2-8 W il d l if e In t e r n a t io n a l , Lt d . -44- BACK TO MAIN " PROJECTNO.: 454A-116 APPENDIX 3 FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTED WITH CHEMICAL U2723 (ASSAY 3) Test Method: Type of Test: Date: Investigator: Laboratory: Chemical: Designation: Source: General W ater Quality: Test Medium: Test Organism: Scientific Name: Age at Start of Test: Source: Experimental Chambers: Material: Test Solution Volume: No. Organisms/Replicate: No. Organisms/Treatment: Lighting: Endpoints: Test Temperature: ASTM Designation E 1439-91 ASTM (1998) Static renewal (every 24 h) May 22-26, 2000 S. D. Turley UMD/WREC U2723 Wildlife International, Inc. See Table A3-1 FETAX solution Xenopus laevis Stage 8 blastula to stage 11 gastrula UMD/W REC culture Glass petri dishes 10 mL . 25 Control: 100 Treatment: 50 Fluorescent; 60-85 foot candles Mortality; malformation; growth 24 2C A3-1 W il d l if e In t e r n a t io n a l , Lt d . -45 - BACK TO MAIN ' PROJECT NO.: 454A-116 Results: Note: For Assay 3, statistical calculations are based on the mean measured concentrations of 1.93, 3.27, 5.25, 8 .2 6 ,1 4 .0 0 and 23.90 mg a.i./L, rather than on nominal concentrations. Mortality: The 96-h LC50 = 15.3 mg a.i./L (95% confidence limits = 13.13 -1 7 .7 5 ). The mortality data are summarized in Table A3-2. Malformations: The 96-h EC50 (malformed embryos) = 16.8 mg a.i./L (95% confidence limits = 12.35-22.82). The number of malformed embryos are summarized in Table A3-2. The types of malformations are summarized in Table A3-3. Growth: The minimum concentration inhibiting growth (MCIG) =8.26 mg a.i./L. The growth data are summarized in Table A3-4. Teratogenic Index: The teratogenic index (Tl) = 0.9. A3-2 W il d l if e In t e r n a t io n a l , Lt d . -46- BACK TO MAIN PROJECTNO.: 454A-116 Table A 3-1. Summary of The General W ater Chemistry Data for Chemical U2723 Assay 3 - Dissolved Oxygen (mg/L) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max 0.00 8.2 8.3 8.2 7.4 8.1 7.3 8.1 7.4 7.9 7.3 8.3 1.82 Test Concentrations (mg/L) 3.07 5.19 8.64 14.40 24.00 8.2 8.1 8.2 8.1 8.2 8.2 8.3 8.0 8.2 8.2 8.2 8.3 8.2 8.2 8.2 8.1 8.1 8.2 7.2 7.1 7.0 7.0 7.0 7.0 8.1 8.0 8.1 8.0 8.0 8.1 7.1 7.1 7.0 7.0 7.1 7.0 8.2 8.1 8.1 8.0 8.1 8.0 7.5 7.5 7.5 7.6 7.4 7.4 7.9 7.8 7.8 7.8 7.8 7.8 7.1 7.1 7.0 7.0 7.0 7.0 8.3 8.2 8.2 8.2 8.2 8.3 A3-3 W il d l if e In t e r n a t io n a l , Lt d . -47- BACK TO MAIN PROJECTNO.: 454A-116 Table A 3-1. (Continued) - pH (Standard Units) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Min Max 0.00 1.82 Test Concentrations (mg/L) 3.07 5.19 8.64 7.14 7.24 7.31 7.33 7.37 7.18 7.11 7.19 7.21 7.15 7.28 7.14 7.35 7.15 7.35 7.21 7.15 7.20 7.23 7.18 7.30 7.17 7.37 7.17 7.38 7.23 7.17 7.21 7.21 7.25 7.26 7.26 7.34 7.24 7.36 7.18 7.11 7.23 7.21 7.19 7.24 7.24 7.15 7.31 7.31 7.14 7.37 7.35 7.15 7.38 14.40 7.40 7.14 7.39 7.15 7.41 7.28 7.40 7.40 7.14 7.41 24.00 7.37 7.16 7.34 7.23 7.32 7.25 7.35 7.29 7.16 7.37 A3-4 W il d l if e In t e r n a t io n a l , Lt d . -48- BACK TO MAIN PROJECTNO.: 454A-116 Table A3-1. (Continued) - Temperature (C) DavO OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max 0.00 1.82 Test Concentrations (mg/L) 3.07 5.19 8.64 14.40 24.00 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 A3-5 W il d l if e In t e r n a t io n a l , Lt d . -49- BACK TO MAIN PROJECTNO.: 454A-116 Table A3-2. FETAX Toxicity Test Data - Percent Embryo Mortality and Malformations after 96 Hours of Exposure to Chemical U2723 - Assay 3 Concentration (mg/L) Control Rep 1 2 3 4 Number Embryos Alive 25 25 25 25 Percent Mortality 0.0 0.0 0.0 0.0 Number Embryos Malformed 1 1 0 0 Percent Malformed 4.0 4.0 0.0 0.0 1.82 1 25 0.0 1 4.0 2 25 0.0 2 8.0 3.07 1 25 0.0 1 4.0 2 25 0.0 1 4.0 5.19 1 25 0.0 1 4.0 2 25 0.0 2 8.0 8.64 1 23 8.0 1 4.3 2 20 20.0 5 25.0 14.40 24.00 1 14 44.0 4 28.6 2 14 44.0 7 50.0 1 6 76.0 5 83.3 2 5 80.0 3 60.0 A3-6 W il d l if e In t e r n a t io n a l , Lt d . -50- BACK TO MAIN PROJECTNO.: 454A-116 Table A3-3. FETAX T oxicity T est Data - Type and Number of Malformed Embryos after 96 Hours Exposure to Cnemical U2723 - Assay 3 Malformation 12 0 Rep 3 Severe Stunted Gut 1 Edema: Multiple Cardiac Abdominal Facial Cephalic Optic Tal Notochord Fin < Face Eye 1 Brain Hemorrhage Cardiac BDstere Other* Malformed 110 Test Concentration (mg/L) 1.82 3.07 5.19 8.64 14.40 24.00 Rep Rep Rep Rep Rep Rep 4 12 12 12 12 12 12 21 13 23 1 13 1 1 1 111 1 1 33 1 11 33433 2 1 3131 0 12 1112 154 7 53 * Anal edema. A3-7 W il d l if e In t e r n a t io n a l , Lt d . -51 - BACK TO MAIN PROJECT NO.: 454A-116 Table A3-4. FETAX Toxicity Test Data - Embryonic Growth (Length) after 96 Hours Exposure to Chemical U2723 - Assay 3 Concentration (mg/L) Control 1.82 3.07 5.19 8.64 14.40 24.00 Rep 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 Number Embryos Alive 25 25 25 25 25 25 25 25 25 25 23 20 14 14 6 5 Average Length (mm) 9.54 9.64 9.31 9.40 8.83 9.36 9.11 9.44 9.25 9.30 8.70 8.31 8.08 8.14 8.06 7.53 Significantly different at alpha = 0.05 (Bonferroni T-Test) Mean Average Length (mm) 9.47 9.10 9.28 9.28 8.51* 8.11* 7.80* A3-8 W il d l if e In t e r n a t io n a l , Lt d . -52- BACK TO MAIN PROJECT NO.: 454A-116 APPENDIX 4 ASSAY 1 RAW DATA SHEETS FOR FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) AND 6-AMINONICOTINAMIDE REFERENCE TOXICANT FOR CHEMICAL U2723 Note: The nominal concentrations of 1 .9 ,3 .2 ,5 .4 ,9 .0 ,1 5 .0 and 25.0 mg/L in the raw data correspond to the nominal concentrations of 1.82,3.07, 5 .1 9 ,8.6 4,1 4 .4 0 and 24.00 mg a.i./L in the report. A4-1 FORM 8 D (P a g e 1 e H j : FETAX TOTAL SUMMARY DATA SHEET ----------------- --------------------- ------ ---------------------- . Paje_ I I I i Aquatic Tbxicology l Best Practices for the Culture and Testingof Selected fishand ftog Species . ubora.ocy Manuel W il d l if e In t e r n a t io n a l , Lt d . - 54- BACK TO MAIN p r o je c t n o .: 454A-116 FO RM 8C : FETAX MORTALITY DATA Notebook/Btntkr No._ Test N um berT ^ g-nVlvC U l Test Material U 2 7 Z 3 _ _ _ Petri Dish/ Aquarium ID Concentration Initial No. of Embryos Ol WStCenVolA 2 5 oz %IJCirArlB EST 03 fEiFttCxUC 2S 04 nw(l4olD 2S 05 0. lAnA. A 2S I.VJL 0 25 07 3-1 ,r^A- A 25 08 3.1/nj/i. 25 00 5.4 ra)L A 25" 10 5.4 rmlL 25 II 9.0ft|c A 2 5 11 9.0.3V R 25 13 2 5 H IS^u. B 2 5 15 250p^1l A 25" 18 &0niu El Z S 24 Hr. 0 O O 0 O O O 0 0 0 2 O O 2 O O M ortality 48 71 Hr. Hr. OO O0 OO OO 01 OO 0O 0O 2O O2 20 20 2I 15 2 19 0n Total No. % Total No. 96 Dead Dead Embryos Hr. Embryos Embryos Alive/Fixed 1 14 O0 O 0OO OO O 0 14 000 t i4 1 14 02 8 01 3 2 4- lb 0 28 O 9 3 fe 2 10 4 0 4- 25 100 2 2 5 100 2+ 25 25 25 24 25 24 24 23 22 21 23 lb 15 O 0 " s c "S C " s X 8y^c 8,fT FTOate, 1 O-astei*__DatieL OTatIe16 SjflDate, Reviewed fly. Aquatic Toxicology 8 t Practices fo r the C u lture and Testing of Selected fish and Frog Species Laboratory M anual A 4-3 Sy^ x InOate . s W il d l if e In t e r n a t io n a l , Lt d . -55- PROJECT NO.: 454A -116 B A C K TO M AIN FO RM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA Moicbook/Sinder N q . _ I I I I I 1 1 I 4 I 4 & M 4 i iu ~ U. 5 1 QS 1J I i o c ai i o 3 Oz i I Reviewed 8y_ Aquatic Toxicology 8esc Practices for the Culture and Testing of Selected fish, and Frog Species Laboratory Manual A 4-4 E W il d l if e In t e r n a t io n a l , Lt d . -56- FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION' DATA PROJECT NO.: 454A-116 Noicbook/Binder No._______ ___ B A C K TO M AIN I a i I i 4 4 4 1 By____________________ _____________ Aquatic Toxicology 8est Practices (or the Culture and Testing of Selected fish and Frog Species Laboratory Manual AU-'i W il d l if e In t e r n a t io n a l , Lt d -57- FO R M 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA PROJECT NO.: 454A-116 Notcbuck/Bindcf N o . ______ B A C K TO M AIN 1 I I I 1 1 I I Reviewed 5y I Aquatic Toxicology Best Practices for the Culture and Testing of Seleaed fis h and Frog Species Laboratory Manual A4-6 W il d l if e In t e r n a t io n a l . LTn ' -----------------:----------------------------------------- PROJECT NO.: 454A- -58- I 1 I I d W il d l if e In t e r n a t io n a l , Lt d . -59- PROJECT NO.: 454A-116 B A C K TO M AIN I 1 1 I I I I I I I Aquatic Toxicology Best Practices for the Culture and Testing of Selected fish and Frog Species Laboratory Manual A 4-8 W il d l if e In t e r n a t io n a l , Lt d -60- PROJECT NO.: 454A-116 B A C K TO M AIN I I I 8y Dofr I Aquatic Tbxicoiogy Best Practices for the Culture and Testing of Selected Fish and Frog Species * Laboratory Manual A4-9 W il d l if e In t e r n a t io n a l , Lt d . -61 - PROJECT NO.: 454A-116 B A C K TO M AIN I I I I 1 I I J J l l j J Be Practices for the Culture and Testing of Selected fish and Frog Species Laboratory Manual A 4-10 w rm B A C K TO M AIN W il d l if e In t e r n a t io n a l , Lt d . -62- I I l I I I I I 1 I 1 1 1 1 J i PROJECT NO.: 454A-116 W il d l if e In t e r n a t io n a l , Lt d . 63 - PROJECT NO.: 454A-116 B A C K TO M AIN ] i j j J W il d l if e In t e r n a t io n a l , Lt d -64- PROJECT NO.: 454A-116 FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA 1 rr s o o 1 1a BW-- I IN ^ n 1 Na .1 IINN X -1 1* 1M IN X g oIN * 1 1 .1 * < j ~~~ ~ ~ ~ m 11 J ' J8 S j .1 I_ 1 - vO ' 1 <3 vn V > j n .1 1 .1 Ln .1 _-- IN >< _ ! 1 JS = T2 H _ O >i a 1 1 -L_ -- H II _ K. .i Sc s 1 X .1 ** V ---- ----- -- ---- -- -- ~ ~ "---- -- ~ """-- -- -- 1 5""1 ~~ C _ _> <5 _ >< ----- -- IN __> i i x x~ J*i c l Si l v< X ~~ J* J LC I- SI3 3S 1j ! s S i 1s i s <aIJ I5 i l i y i _ Jl ! u's Ciw3 1a l i 1 ! 3a *53 Oo. h 1 I .5 s 3* ! s m 2 1 t*a cV 1 5 .9 OQ s O J1 Reviewed 8y -- -- -- -- -- -- o a(e_ -- ----- a -------------------------------- j Aquatic Tkicology J Best Practices for the Culture and Testing of Selected Fish and Frog Species . Uboroto-y .Manual A4-13 'ir m I 392 W il d l if e In t e r n a t io n a l , Lt d . 65- PROJECT NO.: 454A-116 B A C K TO M AIN FORM 8 H : FETAX INDIVIDUAL EMBRYO f*xbook/Bindtf N o ._ 1 $ -'S HI 'd 3T Q Jc cO r~ c c\J ro S i Oh3- 1 a _L j_ a f f_ rree _ ! 1 _ X 3 |_ aV j 9* l i am-* 1 r. X 11 V X I *n * _ -- X E X 3 re X 1 s X 1si O X aIi exS_ a _X g X X t*X _ E f"" r-- --r--" ---------- -- n ! -r " ~ _ Page_ ~ -- -- ------ -- -- ~ ' ---- ~~ -- *1 -_ _ X |B re __ x : 1 _V o pa "X 7*~r<717< x X ~r<x- 7< ~ " " ------ ~ ---- ~ _-- <T 3 Q | I 1 1i VI I 1 1 S J i S_ 3 , C2veni/ u3 *3 *2s31 4 3 a3C a O Jh<31-' - .5 M -**s C) z *3o 3 si'VSaI. 2C5O o5S Rtriewtd By_ Aquatic Toxicology 8 e Practice! for the Culture and Testing of Selected fish and Frog Spec.es Laboratory Manual A 4-14 K 3G 3 a W il d l if e In t e r n a t io n a l , Lt d . -66- FORM 8 H : FETAX INDIVIDUAL EMBRYO m PROJECT NO.: 454A-116 B A C K TO M AIN 1 I I I I I I I I 1 1 ! 1 1 Reviewed 8 y_ J Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual J A4-15 W il d l if e In t e r n a t io n a l , Lt d . -67- PROJECT NO.: 454A-116 B A C K TO M AIN 1 I I I I I I ! I I 1 Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish, and Frog Species Laboratory Manual A 4-16 392 W il d l if e In t e r n a t io n a l , Lt d . 68 - PROJECT NO.: 454A-116 B A C K TO MAIN I I I 1 1 I 1 I J J J A4-17 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -69- PROJECT NO.: 454A-116 96 Hour Test Data Effluent/G roundw aterXfoxicanb C .W m ica\ L )?272 3 _________ Pate: I 5 - \ 9 7A7~\ TV&rmVwii, u 1 ________________________ ;_________ __ Analyst: SL*. T ,J ^ Species: ____________________ Age at Start of Test: RlasLAa fefe&.,8-ii) Control Day 0 FETRn Cor\Vot 0H Temperature (C) 24.0 D.O. (mg/L) 8 .3 PH / 7 .7 4 - Conductivity Alkalinity Hardness ' Day 1 Day 2 Day 3 Day 4 Old New Old New Old New Old , 240 24 o -0 8.4 7 2 (o 7 .2 4 2 4 .o in l.\L> 24-0 8 .3 722 1O 2 -4 .0 7 4 8 .3 7.U 7 . i 3 2 4 .0 7 .7 n.w \3cmfi- U 2.723 Temperature ("C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Davo 0H ? 4 ,o 8 .4 7 .7 8 Day 1 Old New 24.0 7 4 .0 7. ft 8 .4 7.24 7.45 Day 2 Day 3 Old New | Old New 74-0 7 ,7 7 .i7 7 4 O b.4vO S 3 7.-S7,2,5 7.13 ' 2 4 .0 8 .2 720 Day 4 Old 24.0 7.7 7.V-S- iULCC|/L U 2123 Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0,H 2 4 -0 84 779 | Day 1 Old New 74-0 Z4.o 7 .9 8.<T 7.30 7 3 8 Day 2 Day 3 Old Ne'.v Old New ? 4 .o 2 4 .0 24.0 2 4 .0 7.6. .4 I 7.7 8 4 7.20 7.28 Tifa 7.2,3 Day 4 | Old 2 4 .0 7.G 7.ig> 1 I A4-18 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 -70- TtV 1 96 Hour Test Data Page JL_of JL. UZ723 Temperature (C) 0 .0 . (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H 24.0 8A 770 Day 1 Day 2 Day 3 Day 4 Old New Old New Old New Old 2-4.0 24-0 1) 8.4 7.54 7 4 3 2 4 0 14.0 24,0 7 .7 0.3 7 . 7 7.23 I 7 .3 2 7 .i7 24.0 24.0 8 .2 7 .S 7 2 S 7.V 8 ]____ 9-Q to/l 1Day 0 0H Temperature (C) D.O. (mg/L) pH Conductivity 2 4 .0 SA 7.13 Alkalinity Hardness Day 1 Old New 24.0 2 4 0 8 . o ff.-sr 7.35 7 4 3 Day 2 Old New 2 4 .0 78 7 .2 3 24.0 8 .2 7 .3 4 Day 3 Day 4 Old New Old 24.0 7.8> 7 25 34,0 24 .0 8 .I 7 .7 7.2<o 7 . 18 \ 5 ,0 .t* O l U Z7Z5 Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH 2 4 .0 8A 7 .7 6 Oay 1 Old New 2 4 .0 24-0 Qo 8 .T 7,34 7 4 4 Day 2 Day 3 Old New Old New 24.0 24.0 Z4.0 3 4 .0 7 8 $ . 3 7.4 A 773 7 3 6 7.i8 7.26 Day 4 Old 3 4 .0 7.7 7.20 I I A4-19 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -71 - PROJECT NO.: 454A-116 96 Hour Test Data Page 3 of 2 2 5 .0 mtilL Temperature (C ) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H 2 4 .0 8.T ~l.<oZ Day 1 Day 2 Old New Old New V \.o 7A-0 1A-o 2 4 .0 8.4 7 . $ 1 3 3 7.3Z 7 .7 S 7 2 7 Day 3 Day 4 Old New Old 2 4 . 0 7 4 -0 24.0 7 .4 8.1 7 7 6 AS" 7. 18 7 . \ 9 Temperature (C ) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H Day 1 Old New Day 2 Old New Day 3 Old New Day 4 Old Temperature (C ) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH - Day 1 Day 2 Day 3 Old New Old New Old N ew Day 4 Old M-20 W il d l if e In t e r n a t io n a l , Lt d . -72- BACK TO MAIN p r o j e c t n o .: 4 5 4 A-1 1 6 96 Hour Test Data Effluent/Groundwater/Toxicant: (o-fm .'w mE/wwA. (u -V \ ) Date: s l i 5 ' | - ,5'|itilto ___________________________________________ ;__________ Analyst: S T _________ Species: __________________ Age at Start of Test: Control Day 0 >5rnq/L (Cj-An^) 0 H Temperature (C) 2 4 .0 D.O. (mg/L) PH Conductivity flA n ^ io Alkalinity Hardness Day 1 Day 2 Day 3 Day 4 Old New Old New Old New Old 7 4.0 'lAr*O 74.0 8.0 8.% 7.) 7 .}\ T i l 7 aA 74.0 83 7.30 747) 7.8 7. 16 24.0 7 .7 0 24.0 7.(o 7.o7 250Q f^ |L Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H 74.0 8.S 7.44- Day 1 Old New 74.0 ?A.o 77 d 7.26> 1.70 Day 2 Old New l4.o 24.0 7,7 8.3 7.14 l.Z> Day 3 Old New 74.0 ?,4o 7.6 8.3 7 AO -74'7 Day 4 Old 34 0 7.4 7.04- *' I1 Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH Day 1 Old New Day 2 Day 3 Old New Old New Day 4 Old A 4-21 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 -73- G hem ical U 2723- D efinitive T e st 1 Embryo Length M easu rem en ts (mm) Page 1 of x 3 C ontrol A C o n tro ls C o n tro le C ontrol D 1 .9 m g /L A 1.9 m g /L B 1 8.8 6 8.66 8.96 8.78 8.76 8.48 2 9 .0 6 8 .0 3 8.83 8.91 9 6.26 3 9 .3 6 8 .2 5 8.83 8.01 9.11 8 .2 3 4 9 .2 5 8.69 8.07 8.63 8.25 8 .2 5 8.93 8.1 8.44 8.08 8.5 8.75 6 9 .3 2 8 .3 5 9.41 8.61 8.82 8.31 7 9.76 8.4 8.05 8.83 7.44 8 .3 8 9 .2 9 7 .7 4 7.51 9.1 8.3 7.83 9 9 .3 7 8 .3 8 7 .8 8 8 .9 4 8.38 7 .9 6 10 9 .5 4 8 .6 8 7.96 9 8.23 8 .5 3 11 9 .3 9 7 .2 9 8.1 8.83 8.93 8 .1 6 12 9 .2 8 8 .2 8 7.96 7.58 8.58 7 .2 7 13 9 .2 9 8 .5 8 9.05 8.01 7.59 8 .5 14 8 .7 6 8.81 8.17 8.33 9.45 8 .7 5 15 8 .2 2 8.36 8.28 8.75 8.51 8.41 16 8.19 8.6 7.45 8.83 8.87 8 .2 5 17 7 .9 3 8.85 7.94 8.4 8.17 8.16 18 9 .5 6 8.72 8.17 8 .1 4 8.77 8 .1 6 19 8 .9 6 8 .3 6 7 .3 4 8 .5 4 8.24 8 .0 3 20 ' 8.51 8 .5 5 10.06 8.78 7.88 8 .4 7 21 9 .1 7 9.02 9.22 8.95 9.05 8.21 22 9 .1 9 8.16 7.47 8.53 8.47 8 .2 7 23 8 .5 8 7 .8 7 7.81 9.46 7.93 7 .6 7 24 9 .4 5 8 .7 3 8.02 8 .2 9 7 .4 2 8 .5 5 25 8 .6 4 8.36 9.12 7 .6 7 M ean Min M ax Stand. Dev. 9 .0 5 0 8 3 3 3 7 .9 3 9 .7 6 0 .4 7 2 3 4 7 7 8 .4 0 4 7 .2 9 9 .0 2 0 .3 8 8 6 1 9 4 8.2936 7 .3 4 10.06 0 .6 6 9 5 9 4 9 8 .6 1 7 2 7.58 9 .4 6 0 .4 2 9 6 2 7 8.44375 7.42 9 .4 5 0 .5 3 2 3 0 2 7 8 .1 3 5 2 6 .2 6 8 .7 5 0 .5 1 9 0 4 6 6 A 4-22 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 -74- C hem ical U 2723- D efinitive T e st 1 Embryo Length M easu rem en ts (mm) Page 2 o fx 3 3.2 m g/L A 3.2 mg/L B 5.4 mg/L A 5.4 m g/L B 9.0 mg/L A 9.0 m g/L B 1 9 .2 4 9.22 5.61 9.7 8.73 9.16 2 9 .1 5 8 .6 6 8.41 8.92 8.89 8 .5 2 3 8.84 9.4 6.85 7.33 7.24 9.18 4 7 .5 3 8 .7 8 6.63 9.14 8.4 6.34 5 8 .9 5 8 .7 4 8.59 8.98 8.37 8 .6 6 9.21 8 .7 6 7.42 8.25 8.44 8 .8 4 7 8.01 9 .0 9 8.98 8.45 9.51 8 .8 9 8 9 .2 5 8 .3 4 8.73 8.37 9.23 7.41 9 9 .5 4 9 .3 7 8.92 6.25 8.66 8 .8 3 10 9.39 8.44 8.6 8.84 8.87 8.73 11 8 .0 7 8 .3 5 8.39 8.75 6.98 9 .2 6 12 10.08 8 .0 3 7.61 9.26 8.32 9 .4 5 13 9 .2 9.04 8.51 7.97 8.83 9 .4 2 14 8 .8 4 9 .1 8 9.35 8.79 9.15 9 .2 2 15 8.9 4 8.76 8.16 8.5 9.5 8.99 16 8 .0 9 8.07 9.1 8.9 3 9.4 5 9.01 17 9 .3 8 7.17 9.41 9.11 9.81 9 .8 2 18 8 .7 8 8 .9 7 8.72 9.76 9.09 9 .4 19 8 .0 8 9 .1 8 8.91 8.72 8.94 6 .9 5 20 9 .6 6 9 .5 8 9.32 8.33 9.6 7 .5 7 21 8 .8 4 8 .2 7 9.57 8.58 9.52 8 .2 4 22 9 .2 8 8.61 8.79 8.27 9 .2 4 23 7 .6 9 8.23 9 6 .3 3 24 9.16 9.04 25 M ean Min Max Stand. Dev. 8 .8 8 3 3 3 3 3 7 .5 3 1 0 .0 8 0 .6 5 0 1 4 8 3 8 .7 2 7 .1 7 9 .5 8 0 .5 4 4 8 6 5 4 8.4165217 5.61 9.57 0 .9 8 3 0 8 2 4 8.6 6.25 9.76 0 .7 5 4 4 0 2 9 8.8347619 6.98 9.81 0 .7 2 3 5 3 7 3 8 .5 8 2 6 0 8 7 6 .3 3 9 .8 2 0 .9 8 9 1 1 4 5 A 4-23 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 -75 - C hem ical U 2723- D efinitive T e st 1 Em bryo Length M easu rem en ts (mm) Page 3 of 3 15mg/LA 15mg/LB 25mg/LA 1 9 6.55 2 9 .57 7.85 3 7.8 7.65 4 7.73 7.76 5 7.79 8 .9 6 9.5 7.28 7 7.5 7.43 8 8.86 8.52 9 7.46 8.2 10 9 .2 8 8.48 11 7 .8 4 6.51 12 10.24 13 8.51 14 7.14 15 8.91 16 7.41 17 18 19 20 21 22 23 24 25 25 mg/L B 6-A m inonicotinam ide 5 .5 m g /L A 5.5 mg/L B 7.95 6 .9 9 8.21 7 .7 7 8.46 7 .9 9 8.3 8.92 9.6 8.4 9.02 7 .8 7.46 9 .1 2 7.59 9 .2 3 7.7 8.11 8.1 7 .5 5 8.16 8 .5 7 8.01 7 .9 6 7 .9 9 8.41 9.11 8 .6 9 8.81 7 .9 5 8.41 8 .1 2 8.16 9 .0 4 7.44 8 .2 7 7.39 7 .8 4 7.8 7.93 8.12 7 .1 5 8.91 7 .3 8 7.65 M ean Min M ax S tand. Dev. 8 .4 0 8 7 5 7 .1 4 1 0 .2 4 0 .9 4 2 9 5 1 9 7 .7 3 9 0 9 0 9 6.51 8 .9 0 .7 7 2 6 8 9 5 8 .1 8 9 1 3 0 4 7.39 9.6 0 .5 8 3 2 5 0 4 8 .1 4 5 6 .9 9 9 .2 3 0 .6 1 4 0 5 5 6 A 4-24 W il d l if e In t e r n a t io n a l , Lt d . 76- B A C K TO MAIN I 1 1 l 1 i i i I a Best Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual A 4-25 E5 W il d l if e In t e r n a t io n a l . Lt d . 77- B AC K TO MAIN FORM 8H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA (*x*bookJin<terNo. 3 OH evi a1 <N in) ro n g I I e rr<e y ? r* * B m s * , I 3' * m I & j r r. I ZjPj YieI V * <3 V7 we x i I w* =y J re y 3 4 =X l (Jt a % T e X 'Pi? X XX - * ss - x X `X > X X X I t .2 > XS X XX X x we >< X i * 1 X XX X cC * X X o re -J XS XX < ( V V? X v xi X X X X Z6 in I MALFORMATIONS: I 1I 3a i >OC- I I 5 S S wt vc3 a O 21w _w o3. i U 1 1 3< ? i 0! O *a 5 ; -<C* 3 .U3 tCha. Si Z 5 oa O 4 er/ewed8y_ Ojte 4 Aquatic Toxicology Be5t Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual A 4-26 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -78- p r o je c t n o .: 4 5 4 A-1 1 6 CRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 U 2 J 2 3 - Ase* ] \ - 1X50 DATE! 5/15/00 TOXICANT : U2723 SPECIES: xenopus RAW DATA: ---------- - C o n c e n tra tio n (mg/1) .00 2.00 2.83 4.73 7.90 14.70 2 4 .60 TEST NUMBER: 1 DURATION: Number Exposed 100 50 50 50 50 50 50 M o rta litie s 1 1 2 5 6 19 50 96 h SPEARMAN-KARBER TRIM: 1.01% SPEARMAN-KARBER ESTIMATES: LC50: 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 13.77 12.38 15.31 NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING. ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION. A4-27 W il d l if e In t e r n a t io n a l , Lt d . -79- BACK TO MAIN PROJECTNO.: 454A-116 TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 UZ1Z3- Assay \ - (ecso) DATE: 5/15/00 TOXICANT : U2723 SPECIES : xenopus TEST NUMBER: 1 DURATION: RAH DATA: ---------- C o n c e n tra tio n (mg/1) .00 2.00 2.83 4.73 7.90 14.70 Number Exposed 99 49 48 45 44 31 M o rta litie s 4 4 7 10 11 20 SPEARMAN-KARBER TRIM: 36.98% SPEARMAN-KARBER ESTIMATES : EC50: 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 12.08 10.00 14.58 96 h NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING. ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION. T i* lc5q/ec5o- B.n/iz.oa - itf- A4-28 W il d l if e In t e r n a t io n a l , Lt d . -80- BACK TO MAIN PROJECTNO.: 454A-116 T itle : F ile : u2723:Assay 1: embryo le n g th d ata (mg) ASSAY1LE. Transform : Shapiro - W ilk 's Test fo r Norm ality D = 0.6683 W = 0.9649 C r i t i c a l W = 0.8250 (alpha = 0.01 , N = 14) W = 0.8740 (alpha = 0 .0 5 , N = 14) NO TRANSFORMATION Data PASS n o r m a lit y t e s t (alpha = 0 .0 1 ) . Continue a n a ly s is . BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -81 - PROJECT NO.: 454A-116 T itle : F ile : u2723:Assay 1: embryo le n g th d a ta (mg) ASSAY1LE. Transform : NO TRANSFORMATION B a r t l e t t ' s T e st f o r Homogeneity o f V arian ce C a lc u la t e d B1 s t a t i s t i c = 2.3953 (p-value = 0.7922) Data PASS B1 homogeneity t e s t a t 0.01 l e v e l . C on tin u e a n a ly s is . C r i t i c a l B = 15.0863 (alpha = 0 .0 1 , d f = 5) = 11.0705 (alpha = 0 .0 5 , d f = 5) U sin g Average Degrees o f Freedom (Based on average r e p lic a t e s iz e o f 2.33) C a lc u la t e d B2 s t a t i s t i c = 2.2190 (p-value = 0.8181) Data PASS B2 homogeneity t e s t a t 0.01 l e v e l . C on tin u e a n a ly s is . A4-30 W il d l if e In t e r n a t io n a l , Lt d . -82- BACK TO MAIN p r o j e c t n o .: 4 5 4 A-1 1 6 T i t l e : U2723:Assay 1: embryo le n g th d ata (mg) File s ASSAY1LE. Transform : ANOVA T a b le NO TRANSFORMATION SOURCE DF SS MS F Between 5 0.7451 0.1490 1.7838 W ithin (Error) 8 0.6683 0.0835 Total 13 1.4134 (p -valu e = 0.2224) C r i t i c a l F = 6.6318 (alpha = 0.01, d f = 5.8) 3.6875 (alpha = 0.05, d f = 5.8) S in c e F < C r i t i c a l F FAIL TO REJECT Ho: A l l eq u a l (alpha = 0.05) A4-31 ; W il d l if e In t e r n a t io n a l , Lt d . -83- BACK TO MAIN PROJECTNO.: 454A-116 T i t l e : U2723:Assay 1: embryo le n g th d ata (mg) F ile : ASSAY1LE. Transform : NO TRANSFORMATION B o n fe r r o n i t - T e s t - TABLE 1 OF 2 Ho: C o n tro K T re a tm e n t GROUP IDENTIFICATION TRANSFORMED MEAN MEAN CALCULATED IN SIG ORIGINAL UNITS t STAT 0.05 1 con 8.5900 8.5900 2 2.00 mg/1 8.2900 8.2900 1.1985 3 2.83 mg/1 8.8000 8.8000 -0.8390 4 4.73 mg/1 8.5100 8.5100 0.3196 5 7.90 mg/1 8.7050 8.7050 -0.4594 6 14.70 mg/1 8.0750 8.0750 2.0575 B o n fe rr o n i t c r i t i c a l v a lu e = 2.8965 (1 T a ile d , a lph a <= 0 .0 5 , d f = 5,8) T i t l e : u2723:Assay 1: embryo le n g th d ata (mg) F ile : ASSAY1LE. Transform : NO TRANSFORMATION B o n fe r r o n i t - T e s t - TABLE 2 OF 2 Ho : C o n tro K T re a tm e n t GROUP IDENTIFICATION NUM OF REPS MIN SIG DIFF % OF DIFFERENCE (IN ORIG. UNITS) CONTROL FROM CONTROL 1 con 4 2 2.00 mg/1 2 0.7250 8.4 0.3000 3 2.83 mg/1 2 0.7250 8.4 -0.2100 4 4.73 mg/1 2 0.7250 8.4 0.0800 5 7.90 mg/1 2 0.7250 8.4 -0.1150 6 14.70 mg/1 2 0.7250 8.4 0.5150 A4-32 W il d l if e In t e r n a t io n a l , Lt d . -84- BACK TO MAIN PROJECT NO.: 454A-116 APPENDIX 5 A SSAY 2 RAW DATA SHEETS FOR FROG EM BRYO TERATO G ENESIS A SSAY-XENOPUS (FETAX) AND 6-A M IN O N IC O TIN AM ID E R EFER ENC E TOXICANT FOR CHEM ICAL U2723 Note: The nominal concentrations of 1 .9 ,3 .2 ,5 .4 ,9 .0 ,1 5 .0 and 2 5.0 mg/L in the ra w data correspond to the nominal concentrations o f 1 .8 2 ,3 .0 7 , 5 .1 9 ,8 .6 4 ,1 4 .4 0 and 2 4 .0 0 mg a.i./L in the report. A5-1 W il d l if e In t e r n a t io n a l , Lt d . -85- PROJECT NO.. 454A-116 FORM 8D (Page 1 et): FETAX TOTAL SUMMARY DATA SHEET NWebooWSuKfcrA*,. < O * o < CQ I I I 4 4 4 Aquatic Tbricologr B t Practice* fo r the Culture and Testing of Selected Fish and Frog Species . Ubora<ory Manual A5-2 W il d l if e In t e r n a t io n a l , Lt d . -86- BACK TO MAIN PROJECTNO.: 454A-116 FO RM 8C : FETAX MORTALITY DATA HotetxxMfonder No._ Page. Tesi Number Test Material V J l T O I M ortality _________________ [ I %Petri Dish/ Aquarium 10 Total 36Initial No. of 24 48 72 No. Dead Dead Total No. Embryos Concentration Embryos Hr. Hr. Hr. Hr. Embryos Embryos Alive/fixed I ot & vs o r\ 0 0 O 0 zs 01 pyfttf*U R 2? o 0 0 o 0 vs os FMMCiJUIO. IS o 0 0 0 o o vs I 04 ) Vi 0 00 1 1 4.0 24 cc l ^ l L A Vf o 00 0 o 0 Xr> I Ok l.ftiv^ll- Pi n<3 0 0 0 0 0 0 _QT 3.Zj/l A 75 o 2L 0 0 a 8 vs 23 OR 3.Zm.Il ft 77 o l 0 2, 3 12. 22 0> 5-4mjIl A 77 6 00i i4 24 10 S,4-i*Jl R 7 7 O II 8.0^/L ft 75 O \ 1 io 2. 07 3 3 12 17 ZL Z2 iZ 9.0 mJl ft i S 1 0 1 2. 8 23 ft ICOr^lL ft i s 1 14 iS.O^lL R 77 o 0 3 0 3 b 3 12 17 48 ZL 13 z\5 S.CWIl A I 0 lb ts- D i, r I S i 13 t \ |l 5 It, (cAr 19 9 fe O'A 19 SStnjIL A zs 0 o i O 1 4 24 z o 5-S.taL ft 25 o 0 i 1 ?. 8 23 21 ZSOOi^Il A 25 0 9 8 8 2S \0 0 O 2Z ZSMWIl 6 2 5 4r ft 8 5 25 loo o I^ | "r ^ 8ysT Oate Date 1VOate 8,5T Oate ByST Oate I __SI22l. . 2 k 1 ^ S\V I Reviewed 8y Oa<e. J Aquatic Toxicology 8est Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual J A 5-3 387 W il d l if e In t e r n a t io n a l , Lt d . -871 PROJECT NO.: 454A-116 B A C K TO M AIN 1 FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA I I X I U" C\. I NottbooH8indcr Nfa._ Page_ I 1 s CJ 1 3 in 1 cJ 1 V4 3 I %s 1 s s? i i Z 1 sx 1: o1 53 1 Reviewed 8y_ I Aquatic Tbncology Be Practices for the Culture and Testing of Selected Fish and Fro^Species Laboratory Manual fl B ri W il d l if e In t e r n a t io n a l , Lt d . -88- PROJECT NO.: 454A-116 B A C K TO M AIN FORM 8H : FETAX INDIVIDUAL EMBRYO 1 O2 H & tb oo k j& n d tr N q . _ / a g e _ I a >< i a* I a a< J Mt* re y 6 3 r< V I sy 3 " yCl mx I j v/0 1. x I I rvJ 2 X ga 3 in vs X aX 1 nx I OJ ce cO CNi a 2 >< o y r. X W S Es si : fg Cl CC w TN c S O X. s s^ re ts X SBS >c A I X -y XX 1 -5 o= I > S 1 2 3 1i 0Z 1i t 1i 30c M O'a j i 1 1 JE0 3< 1 .g 1 j 3 OCL h- iza3 .s 2. 3 -VM*C riE -w0* 3 C .aa* .i* 1 O1 Reviewed8y_ Oate Aquatic Tbxicology Best Practices for the Culture and Testing of Selected fish and Frog Species . laboratory Manual aE 3 a A5-5 W il d l if e In t e r n a t io n a l , Lt d . -89- FORM 8H : FETAX INDIVIDUAL EMBRYO PROJECT NO.: 454A-116 NcxeooV&'nder No. B A C K TO M AIN J J J J J i i Aquatic Toxicology 8est Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual w rm A 5 -6 j W il d l if e In t e r n a t io n a l , Lt d . -90- PROJECT NO.: 454A-116 i i B A C K TO M AIN I I I I I J I J J i Reviewed fly O jte i Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish and Frog Species laboratory Manual J A5-7 In t e r n a t io n a l . Lt d . -91 - i PROJECT NO.: 454A-116 B A C K TO MAIN I I I I I I I I 1 1 1 4 i 4 4 Aquatic Tbxlcftlogj Be Practices for the Culture and Teing ol Selected Fidi and Frog S p e d e tabotaudy Manual A5-8 W il d l if e In t e r n a t io n a l , Lt d . -92- PROJECT NO.: 454A-116 B A C K TO MAIN ( FORM 8H : FETAX INDIVIDUAL EMBRYO ____ MALFORMATION DATA I II 1 X I 3X 1 I 3X I 0 I tEa* R y 1 sx i 1 d Z 8X i I 3 sx i '3 o 7^ 1 I j1 cn -2 S V I ~ -- -- - i fd 2 X I I 42 3 vO = X ~ - I Q a"3 2 X 1 2X CNi X 1 I 3 =X ss g _2$ J sJ c j f PO 11 1I 21 2X ** X X NX -X 1 S _ _ s g _ ' -- ---- _ -- _ ~ --- -- ----- -- w^ l X 1 ~~ . -- i CD j \ " >< ~~ __ X so || _ *x "" -- -- -- -- 25 X~ " -- Ot i z. --t -1e J2 aS 1J ij 95 *1 I i 1 3 II e 1 4w* j o 1 2 "5 S 1 .S3 " M i 2a. U Oaj. H>- "*3 Z .5 s. s *M ' -C i j^m5 s3 c s 1wasna. 4o J 4 Aquatic Toxicology Best Practice* for the Culture and Testing of Selected fish and Frog Species A 5-9 Laboratory .Manual W il d l if e In t e r n a t io n a l , Lt d . -93 - I iI PROJECT NO.: 454A-116 B A C K TO M AIN I 1 I I J J l J J i J J Reviewed B y Oat* J Aquatic Toxicology 8est Practices for the Culture and Testing of Selected Fish and Frog Species L aboratory M an u al J A 5-10 W il d l if e In t e r n a t io n a l , Lt d . -94- PROJECT NO.: 454A-116 B A C K TO M AIN FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA N cx o o k/Binder No. I I I 1 I I I I I I I 1 1 1 Reviewed 8y_ I Aquatic Toxicology 8est Practices for the Culture and Testing of Selected Fish and Frog Species A5-11 laboratory Manual W il d l if e In t e r n a t io n a l , Lt d . -95 - FORM 8 H : FETAX INDIVIDUAL EMBRYO ` MALFORMATION DATA PROJECT NO.: 454A-116 N cxeooE /8i(x/er No. B A C K TO M AIN I I I I I I I I I I I Reviewed 8y i Aquatic Toxicology 8cit Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual A 5-12 W il d l if e In t e r n a t io n a l , Lt d . -96- FORM 8H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA PROJECT NO.: 454A-116 Notebook/Bindcr No. ^3gr. B A C K TO M AIN I I 1 I I I 1 1 1 1 I 4 4 Aquatic Toxicology Be Practices for the Culture and Testing of Selected Fish and Frog Species * Laboratory Manual A 5 -1 3 W il d l if e In t e r n a t io n a l , Lt d . -97- PROJECT NO.: 454A-116 B A C K TO M AIN i ! I I I I I I J J 1 i J i Reviewed B y ________________ _____________________________________ Q jie i Aquatic lexicology Be Practices for the Culture and Testing of Selected Fish and Frog Species A5-14 Laboratory Manual 392 In t e r n a t io n a l , Lt d -98- PROJECTNO.: 454A -116 B AC K TO MAIN FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA NWcfcooVOoderNo. 2o toi a a cO 00 rne y I JoSt3- rne y ? ny ES 1 ore y I at y a y 1 4 a -y i- 'S) T3 1 a y 1 IJ 3 3 Lil we y i sy <1 y cx$ re y 1 4 cO cv =y I o j a 7e j I 3 m" l>S* SaS ? K- y v* * 1i g g a * y I x i y J |eo re -j y yi (M x s X X yyyy X o j NOTREMARKABLE d <s< JS 31S h 11 3 i 9 i a cs O ia '2"SS3. 3 1 'S 1as. Oa. C73 Z*3 .5 -M3a Uw s. .ism Zf ^3 3 eV `V2aI. -O3 4 Ktritwtd8y_ Oate 4 Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish and Frog Species . Laboratory Manual A5-15 W il d l if e In t e r n a t io n a l , Lt d . -99( I B AC K TO MAIN J TOTAL I FORM 8 H : FETAX INDIVIDUAL EMBRYO _________ m a l f o r m a t i o n d a t a r 4 Ia __ ! -- ~ sr 1N _ _ I 04 _ 1C M a _ _ ~ ~ I 1t a * M *s o 5 / CS .-- ~ ~ o st I I/ 2 >< i I$ : s-? 8 i s .< 1 I BLfl T3 ' ' s < ' ~~ ~ <A Q 2 ""5? w >-d Li 2 ^< 2 ^A c sj i'r r ss ~ _ -- s r4 ; oJ ^q 53 es I1 31 j- s.d 2 ?< S --i -- IS i r * > < _ C _____ X __ IN _ > -S - Z - -- <n o^ tX ! I i I Ii f S3 1I 1 t c it _ ------ -- _ S c tin 3 S 3 O >d 3U U . 2g 1 3 3 -S i 1_ ?U. * 0i O ~i " "" -- -- ~ -- _-- -- ---- ~ ---- -- ~ 1" "-- -- -- "" I 1'S S a' J - S 2 & ' . JS 5 S i5 ig X ia * a . s O Reviewed9y Aquatic Toxicology Be Practice* for the Culture and Te*tingof Selected Fi*hand Frog Specie* . Laboratory Manual AS-I6 W il d l if e In t e r n a t io n a l , Lt d . - 100 - PROJECTNO.: 454A-116 B A C K TO M AIN FORM 8 H : FETAX INDIVIDUAL EMBRYO m a l fo r m a t io n data Hofeboak/Binder N o . _ % ~ ~ ~ -- ------------------ 3o - <r S a1 I sAL _re _ 1r n <r"ni 1 ~ I _jIe n re ? _ ~ ~ ~ d I 1Z a re ~~ ~ e 1~ I --8 I rn 8 ~" ~ M9 I I ft} K <3 -.0 n 1 ~ I1 CM '3 cC J (V r- tcLJF C. C ~ 32 J OC o 5 .1 ><e-i 04 >< 1 1 >< 8 _ _ O > i s vCss __-- _ rC 2f - is __ X 1 Vfl _ x! *n _ vs i _ X1 r_ ___ re __ E S >< 5 V ' -- I ' --- r------ X X _ -- -- "" ---- X--~ ---- X ~r >< -- -- 1Z r1 X-- s x M a s 1 2 1 o at 1V- . 13 S_ 5o H 05 [ j 9 ic i 2 IM 5 u T3 1 .2 *o a a * al -C a. Y 1 3 3 - V- 1 J * # 1 m Va W1 1 a1 3 x5 z* Sr 5 O r: it Gate Aquatic Toxicology Best Practices for the Culture and Testing of Selected F.sh and Frog Spec.es . laboratory .Manual A 5-17 W il d l if e In t e r n a t io n a l , Lt d . - 101 - PROJECT NO.: 454A-116 FORM 8H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA ro iS I i NotebookJBinder No._ P jg e _ B A C K TO M AIN 1 I J' & I iP I JS_rS* 3 in I oJ Ig <r J . rv I 1 t 3I Ix 'S ss Vf I V LO O in lw 1 a 4 3I Remwed8y_ OJt*. 4 Aquatic Toxicology 8est Practices for the Culture and Testing of Selected Fish, and Frog Species Laboratory Manual A 5--18 W il d l if e In t e r n a t io n a l , Lt d . 102 PROJECT NO.: 454A-116 B A C K TO M AIN FO RM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA I I I I 1 I 1 I MoiebookJBinder N o . _ 1 J I I I I i Review/ 8y_ Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish. and Frog Species Laboratory Manual A5-19 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 103 - PROJECT NO.: 454A-116 ^^3 96 Hour T est Data . Effluent/Groundwater/Toxicant: f,Wno&ll 1)2723_________ Date: 5l2Z-ZlZiAco D&ftiVwt 2_____________________ ;_________ Analyst: Species: Wfrs___________________ Age at Start of Test: fsUy. 8-l\ Control Day 0 0H Temperature (C ) 24,0 D.O. (mg/L) fi.T pH Conductivity - -7(\4 Alkalinity Hardness Day 1 Day 2 Old New Old New 1AD lA.o 24.0 24.0 ft.3 8 .2 l.(o 8 .\ 1M 7,U 7.15" Day 3 Old New 24.0 24.0 7 .5 8 .| 7.20 7.17 Day 4 Old 24-.0 7.G .7,11 j 02723 Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH 24.0 8.2 7 .2 4 Day 1 Old New 24.0 74,0 8.3 $ .2 717' 7 2 *' Day 2 Day 3 Old New Old New 74-0 ZAO 2 4 0 2 4 0 7 8 8 .1 7.G 8 .2 1.22. 7.23, 7. ZD 7.21 Day 4 Old 74-0 7.G 7.21 EZ 8.2 rrv^L 02723 Temperature (C) D.O. (mg/L) pH Conductivity Day 0 O.H. 24 ..Q 8.1 7.31 Day 1 Old New 24.0 8.0 i.n 74.0 8.2 1,^6 Day 2 Day 3 Day 4 Old New Old New Old 74.0 2 4.0 24.0 2 4 .0 24.0 7 .^ 8 .0 1 -7.S" 8.1-- 7 . T 7 7 3 7.30 7 2 7 7 \7(a 7 .2 0 Alkalinity Hardness A5-20 1 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d .______ - 104- PROJECTNO.: 454A-116 Tfc>V 2. 96 H our T e s t Data Page of 3 1 m lL unz3 Temperature ( aC) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH 24.0 6 .L 7 .3 3 Day 1 Old | New 24.0 Z4,o 04 8 2 7.23 7 3 5 Day 2 Old New 24.0 24.0 7.2- 8.1 724- 7 27 Day 3 Old New 74 o 7.G 725 7 4 -0 84 7 .3 4 Day 4 Old 14 0 7.S- 7.29 1.0 oulL 02723 Temperature (C ) D.O. (mg/L) pH j Conductivity Alkalinity Hardness Day 0 0H 24.o 8 .i 731 Day 1 Old New o $ 84 7x4 24.0 8.1 735 Day 2 Old New 24-0 4.0 7 .2 8.0 734. 738 Day 3 Old New 24.o 74vO 1 .7 8.0 7 2 4 7.3k Day 4 Old 24.0 l.(o 7.33 Day 0 02-723 Temperature ( ' C) D.O. (mg/L) pH Conductivity OH 2.4-0 8.2 740 Alkalinity Hardness Oay 1 Old New 24.0 24.0 8 .\ 8 4 l.Vo Oay 2 Old New 24o 24.0 7 3 ' 8 .o 7 2 3 7.41 Day 3 Old New 34-0 34.0 7 .3 0.1 7 2 3 7.4? Day 4. Old 240 7-5 7.39 , A5-21 .. W il d l if e In t e r n a t io n a l , Lt d . * e- - 105 - . BACK TO MAIN PROJECT NO.: 454A-116 96 H our T est Data Page .V of 3 Z 5.0,^ |u 0 rU 3 Temperature (C ) D.O. (mg/L) pH ' Conductivity Alkalinity Hardness Day 0 OH 4 .o ex. 7 - 3 "7 Day 1 Day 2 Day 3 Day 4 Old New Old New Old New O ld 2 4 -0 9 .2 , 7.27 1 4 .o 2 4 .0 2 4 - 0 7 .| a .\ 7 .5 4 - ~7.Z4 7 . 3 2 2 4 jo 2 4 .0 7,2- 8 .0 7,25- 7 .3 5 24 0 n rs 7 .3 4 - Temperature (C ) D.O. (mg/L) pH Conductivity Alkalinity. Hardness Day 0 OH Day 1 Day 2 Day 3 Old New Old New Old New Day 4 Old Temperature (C ) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH - Day 1 Day 2 Day 3 Old New Old New Old New Day 4 Old A5-22 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 106- p r o je c t n o .: 4 5 4 A-116 . 96 H our T e st Data Effluent/Groundwater/Toxicant: In - Am-Tcr'ycoVi/u^^. ________ Pate: 5|22-5l2CJoo ' _______________________________ ___________ Analyst: ST________ _ Species: Xttnopus, ______ - .___________ Age at Start of Testi f y ft Oay 0 0H Temperature (C ) 24-o D.O. (mg/L) 8 .L pH . 7.T o Conductivity Alkalinity Hardness Day 1 Old New 74.0 24.0 8.0 8.1 7.8 7 . ^ Day 2 Old New 24.0 7 .T TU' 24.0 8.\ -i.n Oay 3 Old New 24 > 24.0 7 .B 8.1 7 .0 <o 7 . i3 . Day 4 Old 24.0 7 .a 7.02 25C O fr^|u (G-AN) Temperature (C ) D.O. (mg/L) pH | Conductivity Alkalinity Hardness Day 0 OH 24.0 6A 7.0 3 Day 1 Day 2 Old New O ld 1 New 14.0 74.0 24 A ~l.i 8 -0 7 .? 14.0 8.0 7.00 7.08 7.07 7. l4 * r * Oay 3 Old New 74;o 24.0 7 ,7 8.0 7 ,0 3 7.07 Day 4 Old 24.0 7 .L <o78 ________ 1 1 Day Q g.H. Temperature (C ) D.O. (mg/L) ! pH ' Conductivity Alkalinity Hardness Oay 1 Old New Day 2 Old New ,I I Day 3 \ Day 4 Old New Old -- -------- ' _____ I 5-23 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 107- C hem ical U 2723- D efinitive T e st 2 Em bryo Length M e a su re m e n ts (mm) Page 1 of 3 C ontrol A C ontrol B Control C C ontrol D 1.9 m g/L A 1.9 m g /L B 1 9 .4 6 8.71 8.71 9.19 7.88 8 .7 3 2 9 .2 3 8.48 8.41 8.99 7.95 8 .8 5 3 9 .5 8 8 .9 4 8.61 9.17 7.97 9 .0 6 4 8 .6 6 9 .5 2 7.68 9.63 8.6 8.69 5 8 .8 3 8 .7 2 9.12 9 .4 2 9.07 8 .6 6 6 8.59 9.28 8.9 9.55 8.5 8.22 7 8.84 8.52 8.9 9.6 8.53 9.03 8 8.58 9.7 8.78 8.63 8.4 4 9.31 9 8 .4 6 8 .6 2 8.81 9 .0 3 8.63 8 .7 6 10 9 .5 7 8 .9 7 8.85 8.81 8.9 9 .0 4 11 8 .6 8 .9 4 9 .0 2 8 .1 3 8 .5 6 8 .4 4 12 9 .5 3 8 .8 7 9.18 8.86 8 .8 4 8 .3 4 13 9.21 8 .6 7 9.16 8.75 8.49 9 .0 6 14 9.2 8.7 8.42 8.34 8.79 8.11 15 8.63 8.98 9.7 9.47 8.16 6 .8 3 16 9.1 8 .9 8 8.93 9 8.56 8.39 17 8 .5 6 8 .8 3 8.53 9.17 8.93 8 .4 9 18 8.57 8.42 8.5 8.84 8.67 7 .8 19 9 .0 2 9 .2 3 9.31 8.48 6 .3 4 8 .8 5 20 9 .3 9 .0 4 8.97 7.88 8.28 7 .7 8 21 9.1 8 .2 2 9.65 9.14 8.5 8.17 22 8 .8 2 8.17 9.28 8.21 7.66 8 .4 3 23 8 .4 9 8 .4 7 8.98 8.49 8.47 6 .9 5 24 8 .3 5 8.53 9.77 8.4 8.13 9.69 25 9.25 8.82 8.44 8.8 8.73 M ean Min M ax Stand. Dev. 8 .9 4 1 2 8 .3 5 9 .5 8 0 .3 8 5 8 8 9 9 8 .8 1 3 2 8 .1 7 9 .7 0 .3 6 7 9 9 8 2 8.9044 7.68 9.77 0.4612039 8.8825 7 .8 8 9.63 0.488211 8.386 6 .3 4 9.07 0 .5 5 3 1 3 5 8 .4 9 6 4 6 .8 3 9 .6 9 0 .6 5 6 2 8 9 3 A 5-24 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 108- C hem ical U 2723- D efinitive 2 Em bryo Length M easu rem en ts (mm) Page 2 of 3 3.2 m g/L A 3. 2 m g/L B 5. 4 mg/L A 5. 4 m g/L B 9. 0 mg/L A 9. 0 m g/L B 1 7 .7 8 .5 4 8.73 7.03 7.4 7 .3 2 8 .9 8 9 .2 3 8.42 9 .0 7 7.64 8 .8 5 3 8 .4 8 9 .1 7 9.03 9.01 8.25 8 .3 9 4 7 .9 5 8 .5 4 8.88 9 .0 4 7.93 8 .0 9 5 9.32 9 8.65 9.81 7.99 8.11 6 8.98 9.09 8.65 8.53 8.19 7.13 7 8.51 8 .4 2 8.42 8 .2 5 7.48 8 .4 8 8 8 .1 3 8.31 7.96 8.95 7 .3 4 7 .8 3 9 8 .7 3 8.61 8.98 8.28 7.73 8.23 10 8 .7 3 8 .6 6 9.24 8.48 7.32 7 .9 7 11 7 .6 6 8 .2 7 9.14 8.21 7.55 8 .1 9 12 7.86 9 8.39 9 .2 6 7.33 7.31 13 8 .3 8 8 .1 2 8.69 9 .1 4 8.11 8 .0 6 14 8 .3 2 8.35 8.26 8.18 7.92 7.51 15 8 .3 8 .4 7 8.53 8.55 8.21 8 .3 6 16 8 .2 4 8 .0 4 8.69 . 7 .7 9 8.3 7 .4 7 17 8.32 8.93 9.32 9.68 8.38 9.67 18 8 .5 4 8 .4 5 8.62 9.65 7.79 8 .3 19 8 .0 6 8 .5 2 8.74 9 .2 2 8.87 7 .3 7 20 8.61 9 .2 4 8.03 8.86 8.35 7 .9 8 21 7 .6 5 9 .1 8 7.94 8.3 8.65 7.04 22 9 .6 3 9 .4 7 9.22 9 .0 7 7.97 7.8 23 8.58 9.46 6 .7 3 24 8.52 25 M ean Min M ax Stand. Dev. 8 .4 2 7 .6 5 9 .6 3 0 .5 0 8 4 2 8 .7 0 9 5 4 5 5 8 .0 4 9 .4 7 0 .4 0 9 3 1 3 9 8.6879167 7.94 9.46 0.4185792 8 .7 4 3 6 3 6 4 7 .0 3 9.81 0 .6 6 0 8 2 5 9 7.9409091 7.32 8.87 0 .4 3 9 3 1 6 7 7 .9 2 0 4 3 4 8 6 .7 3 9 .6 7 0 .6 5 1 0 9 3 3 A 5-25 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 109 - C hem ical U 2723- D efinitive T e st 2 Em bryo Length M e a su re m e n ts (mm) P age 3 of 3 6-A m inonicotinam ide 15 m g/L A 15 mg/L B 25 m g/L A 25 m g/L B 5.5 mg/L A 5.5 m g/L B 1 7 .5 7.58 7.91 6 .8 4 7.34 7 .0 2 2 9.29 6.28 7.54 7.67 7.89 7.16 3 8.34 7.34 7.59 7.13 6.68 6 .8 4 8.99 6.88 8.18 7.31 8.06 6.91 5 8.86 6.66 7.37 7.37 7.73 6.99 6 7 .9 8 7 .0 3 6 .6 4 7 .2 9 7.26 7 .1 6 7 8.31 6 .7 7 6.93 7.59 7 .2 4 8 8.23 7.96 7.67 6.88 7 .3 3 9 7.73 7.86 7.69 7.4 7.09 10 7.08 6.73 7.13 7 .7 7 11 7 .4 8 7 .8 4 7.21 7.91 12 7 .7 7 7.19 7.44 8 .0 9 13 7.96 6.53 7.07 7 .5 14 7.99 7.67 7 .5 5 15 7.75 7.14 7 .3 5 16 6.78 17 7.34 6.98 7.49 7 .2 2 7.61 18 7.59 19 7.22 7.31 7.3 7 .4 2 6 .8 20 7.5 2 1 ., . 7 .5 1 22 8.39 8.1 7.12 7.57 6 .7 6 7 .1 4 7 .3 7 23 7.38 7 .2 6 24 8.1 25 M ean Min M ax Stand. Dev. 7 .8 9 0 4 5 4 5 6 .7 8 9 .2 9 0 .6 2 8 0 8 4 6 7 .1 2 6 9 2 3 1 6 .2 8 7 .9 6 0 .5 5 0 8 6 8 8 7 .5 0 2 2 2 2 2 6 .6 4 8.18 0 .4 7 2 5 6 6 9 7 .2 6 8 3 3 3 3 6 .8 4 7 .6 7 0 .2 7 4 4 0 2 4 7.41 6.68 8.1 0 .3 7 7 6 7 0 2 7 .2 8 0 4 3 4 8 6 .7 6 8 .0 9 0 .3 4 8 2 6 1 6 A5-26 W il d l if e In t e r n a t io n a l , Lt d . - 110- FORM 8 H : FETAX INDIVIDUAL EMBRYO PROJECT NO.: 454A-116 Notebook/Qindcr No. B A C K TO M AIN 1 I I I I I 1 1 I i I A A Reviewed8y_ Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fishjm d Fro^ Species Laboratory Manual re i W il d l if e In t e r n a t io n a l , Ltd - Ill - PROJECT NO.: 454A-116 B A C K TO M AIN I 1 I I I I I I I I I I J ! A j FO RM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA Notebook/Binder N o._ Page. Reviewed 8y. Aquatic Toxicology 8est Practices for the Culture and Testing of Selected fish and Frog Species Laboratory Manual A 5-28 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . -112- PROJECTNO.: 454A-116 TRIMMED SPEARM AN-KARBER METHOD. V ER SIO N 1 . 5 UZ723- p \ s ^ j Z - N \ c A a l'y ( u is S ) DATE: 5 /2 2 /0 0 TOXICANT : U 2 7 2 3 S P E C IE S : XENOPUS T EST NUMBER: 2 D U R A T IO N : 96 H RAW D A T A : ------------------ C o n c e n tra tio n (M G /L) .0 0 1 .9 1 3 .0 4 4 .8 2 7 .9 7 1 3 .3 0 2 3 .1 0 N um ber E xposed 100 50 50 50 50 50 50 M o rta litie 1 0 5 4 5 15 35 SPEARMAN-KARBER T R IM : 3 0 .2 0 % SPEARMAN-KARBER ESTIM A TES : LC50: 95% LOWER CON FID EN CE: 95% U PPER CONFIDENCE: 1 7 .6 1 1 5 .5 3 1 9 .9 6 N O TE: M O RTA LITY PR O PO R TIO N S WERE NOT MONOTONICALLY A DJU STM ENTS WERE MADE P R IO R TO SPEARMAN-KARBER E S T IM A T IO N . A5-29 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 113 - PROJECT NO.: 454A-116 CRIMMED SPEA R M A N -K A RBER M ETHOD. V E R S IO N 1 . 5 D 2 fi2 3 - 2 .- DATE: 5 /2 2 /0 0 TOXICANT : U 2 7 2 3 S P E C IE S : XENOPUS (te s o ) TEST NUMBER: 2 D tU R A T IO N : 96 H RAW D A T A : ------------------ C o n c e n tra tio n (M G /L) .0 0 1 .9 1 3 .0 4 4 .8 2 7 .9 7 1 3 .3 0 2 3 .1 0 N um ber E xposed 99 50 45 46 45 35 15 M o rta litie s 4 4 2 5 9 13 10 SPEARMAN-KARBER T R IM : 3 4 .7 4 % SPEARM AN-KARBER ESTIM A TES : EC50: 95% LOWER C O N FID EN CE: 95% U PPER CONFIDENCE: 1 7 .5 7 1 3 .4 9 22.88 N O T E : M O RTA LITY PR O PO R T IO N S WERE NOT MONOTONICALLY IN C R E A S IN G . ADJUSTM ENTS WERE MADE P R IO R TO SPEARMAN-KARBER E S T IM A T IO N . T3> LQSo/gesa-1.00 I A5-30 W il d l if e In t e r n a t io n a l , Lt d . - 114- BACK TO MAIN PROJECT NO.: 454A-116 T itle : F ile : U2723: embryo le n g th s - assay 2 ASSAY2LE. Transform : Shapiro - W ilk 's Test fo r Norm ality D = 0.3737 W = 0.9179 C r i t i c a l W = 0.8440 (alpha = 0 .0 1 , N = 16) W = 0.8870 (alpha = 0 .0 5 , N = 16) NO TRANSFORMATION Data PASS n o r m a lity t e s t (alpha = 0 .0 1 ). C ontinue a n a ly s is . BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 115 - PROJECT NO.: 454A-116 T itle : F ile : u2723: embryo le n g th s - assay 2 ASSAY2LE. Transform : NO TRANSFORMATION B a r t l e t t 's T e st f o r Homogeneity o f V ariance C a lc u la t e d B1 s t a t i s t i c = 12.7727 (p-value = 0.0468) Data PASS B1 homogeneity t e s t a t 0.01 l e v e l . C ontinue a n a ly s is . C r i t i c a l B = 16.8119 (alpha = 0 .01 , d f = 6) = 12.5916 (alpha = 0 .0 5 , d f = 6) U sin g Average Degrees o f Freedom (Based on average r e p li c a t e s iz e o f 2.29) C a lc u la t e d B2 s t a t i s t i c = 9.8138 (p-value = 0.1327) Data PASS B2 homogeneity t e s t a t 0.01 l e v e l . C ontinue a n a ly s is . A5-32 W il d l if e In t e r n a t io n a l , Lt d . - 116- BACK TO MAIN PROJECT NO.: 454A-116 T itle : F ile : u2723: embryo le n g th s -' assay 2 ASSAY2LE. Transform: ANOVA Ta b le NO TRANSFORMATION SOURCE Between W ithin (Error) Total DF 6 9 15 SS 5.0788 0.3737 5.4525 MS 0.8465 0.0415 F 20.3873 (p -valu e = 0.0001) C r i t i c a l F = 5.8018 (alpha = 0.01, d f = 6,9) = 3.3738 (alpha = 0.05, d f = 6,9) S in c e F > C r i t i c a l F REJECT Ho: A l l equal (alpha = 0.05) A5-33 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 117- PROJECTNO.: 454A-116 T i t l e : u2723: embryo le n g th s - assay 2 F ile : ASSAY2LE. Transform : NO TRANSFORMATION B o n fe rr o n i t - T e s t - TABLE 1 OF 2 Ho: C o n tro K T re a tm e n t GROUP IDENTIFICATION TRANSFORMED MEAN MEAN CALCULATED IN SIG ORIGINAL UNITS t STAT 0.05 1 control 8.8825 8.8825 2 1.91 mg/1 8.4450 8.4450 2.4793 3 3.04 mg/1 8.5650 8.5650 1.7992 4 4.82 mg/1 8.7150 8.7150 0.9492 5 7.97 mg/1 7.9300 7.9300 5.3977 * 6 13.3 mg/1 7.5100 7.5100 7.7778 * 7 23.1 mg/1 7.3850 7.3850 8.4861 * B o n fe rro n i t c r i t i c a l va lu e = 2.9333 (1 T a ile d , a lph a = 0 .0 5 , d f = 6,9) T i t l e : u2723: embryo le n g th s - assay 2 F ile : ASSAY2LE. Transform : NO TRANSFORMATION B o n fe rr o n i t - T e s t - TABLE 2 OF 2 Ho : C o n tro K T re a tm e n t GROUP IDENTIFICATION NUM OF REPS MIN SIG DIFF % OF DIFFERENCE (IN ORIG. UNITS)i CONTROL FROM CONTROL 1 control 4 2 1.91 mg/1 2 0.5176 5.8 0.4375 3 3.04 mg/1 2 0.5176 5.8 0.3175 4 4.82 mg/1 2 0.5176 5.8 0.1675 5 7.97 mg/1 2 0.5176 5.8 0.9525 6 13.3 mg/1 2 0.5176 5.8 1.3725 7 23.1 mg/1 2 0.5176 5.8 1.4975 A5-34 W il d l if e In t e r n a t io n a l , Lt d . - 118- BACK TO MAIN PROJECT NO.: 454A-116 APPENDIX 6 A SSAY 3 RAW DATA SHEETS FOR FROG EM BRYO TERATOGENESIS A SSAY-XENOPUS (FETAX) AND 6-AM INONICOTINAM IDE REFERENCE TOXICANT FOR CHEM ICAL U2723 Note: The nominal concentrations of 1 .9 ,3 .2 ,5 .4 ,9 .0 ,1 5 .0 and 2 5.0 mg/L in the raw d ata correspond to the nominal concentrations of 1.82, 3 .0 7 , 5 .1 9 , 8 .6 4 ,1 4 .4 0 and 2 4 .0 0 mg a.i./L in the report. A6-1 W il d l if e In t e r n a t io n a l , Lt d . - 119- PROJECT NO.: 454A-116 B A C K T O M A IN FORM 8 D (P a g e 1 TO): FETAX TOTAL SUMMARY DATA SHEET * * * * , Page Z -5 '\ ie s- FT 1 ri o T' Cr-ta3 CC o <3' (T Y? 3C O X r' $ seo 3 %a <Hs s-* T c a. *8 !1 S s& si q cJ tr\ CT eTs*- m et os -to ^ -4 Cs - %9 rO $ IT a ec X cc >0 r o J c? c r b >S rO r~: ctoS F t3 * oE 5 5 O o O o *< r Q <? q C^ - <r c st Q <o Q CD ^" 3 00 rO LO (X o s qr tO (S' Xr CT? r\J o 2rA rJ a* 1 3 -- -- O <\S --- oJ -- in r- to CO rn rJ 0 s a -S1 z II 2 fi "3 ^ 1S 33 O to to r i r0s rWO ^r 3ss v9 xs -D o c\i CvS 1 -- / ** 51 O il o 3 C> $ 30 r* Q 0o 8 8 i* J, $4 {/ o o 0 O o o 0 DO {0 8 * -r~j s _J o ri- 8 o I1 % VII- 1 15 o o o o o o o o c ) 0 cJ 0 - := ft o - in in OJ 00 H > - 1 --*!ja 1/; 13 is111$/1 to Vrqi to to to Vj XI Cui LO C\J <_n oJ ucOn aI .2e d .! C "Z* a flC < c o <X (C1<X: re) < ~cC < cC < 1 1 I 1 11' 5 1d 15 4 M -z6a3J1-`. 5J 2 :S 2 Ueca a T c- 3 % a & I I ad j 1n s; : ^ j cC j f(f-0f .-J on x lri fsfr=i>i j c \ i 10 C-c J 5 z1 - - - - -- . . . s = = z f> * 1c \ Kt_ < _jL -4 > 5 kf <Sxf' J"1 \T LT>ir; io =X a 5* > 3 V Reviewed 9y__ -- -- -- -- -- -- -- __ 0te_ * Aquatic Tb.ticoiogy 'C E Be Practices for the Culture and Testing of Selected Fish and Frog Species laboratory Manual 153 Eg - Afi-2 W il d l if e In t e r n a t io n a l , Lt d . - 120- BACK TO MAIN PROJECTNO.: 454A-116 FORM 8C : FETAX MORTALITY DATA Nfebookjindtf No. Pagv_ Test Num ber Ta 3 UZTL3Test Material ______ ______________ [ Petn Dish/ Aquarium 10 Initial No. of Concentration Embryos 24 Hr. M o rta lity 4 72 Hr. Hr. Total %No. Total No. 96 Dead Dead Embryos Hr. Embryos Embryos AJive/Fixed o \ fevQff. CAoi A J S .___ a _ ____ 0 o 0?. ..foqtCJJ P> s 0 00 0 03 f a w & U C - z r 0 0 0 Q o 0 0 0 zs ts 6 A feowfi*U\ D - I T 0 Q 00 0 0 2T - . 0 1 I3 ^ |l ft 2 T (1 0 0 0 00 ?S I C 13m1i B Z T o 0 0 _ 3 ___ 3 .1 * V ft 2 T o 0 0 0 00 o 10 Z^ z< I OS ft ?>T o 0 Cft lsAft>jiL . T "I T T 0 00 0 10 00 o 10 zs zs _ 1 0 ___ k u t ft 2 T l 0 0 0 1~ 7 ) 0 o zs 11 K *ili_ A ?JT 1 0 0 2 1 0 2 13 zz I 11 h . ^ / L B Z T 0 0 5 5 to TO 13 l\.olt ft 2?r 0 0 8 ft 11 4 4 14 i 4 J g f i j l ft ? T 0 0 11 0 11 4 4 14 1 I T |2SO*3L A Zb 05 6 (d 19 To (& \h TS-O^Il Z T 2. i0 16 : 2. 20 80 5 I (?*Atofttuivivd. S Sw^Il A 2 S 0O oo Oo 7S 1 -2.0 55<nilL R IS Z1 2So2 j1l A IS O l00 14 2.4 Z 3 ft 1? 2 3 too O 2Z TSbOmuJlL zs 15 IO 9 2 5 JOO O I j I _____ l- l j 11 r s r B^ r rs r 1-rST | % T B,$C 1 IDate . Oate I1Date Oate Oate I 1 20S2x I sr/2.5 ^24 1 1 dlW i Reviewed fly. D ate . J Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual J A6-3 W il d l if e In t e r n a t io n a l , Lt d . - 121 - PROJECT NO : 454A-116 I B A C K TO M AIN I aI a 8 t Practices for the Culture and Testing of Selected Fish, and Frog Species laboratory Manual A 6 --4 W il d l if e In t e r n a t io n a l , Lt d . - 122- PROJECTNO.: 454A-116 B A C K TO MAIN I I FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA I I I 1 I MtXebookJBi'nder No. I 1 1 I 1 I 1 1 I I fovit*Yttd 8y_ I Aquatic Toxicology Best Practices for the Culture and Testing of Selected fish and Frog Species Laboratory Manual ^2 E a Aft--S W il d l if e In t e r n a t io n a l , Lt d . - 123 - PROJECT NO.: 454A-116 B A C K TO MAIN FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA Narook/S-Vxief a*, _ i '> o a 0g a v. i C-H X in C\] a y 1 <reN X i1 !$ a 1-- K; Mm r _ rr _ -- ~ j 1- y on y 25 f3 j W\ y i \r y 3 _ _ r-- " 1 3 'S hs y Ml y wn >< S S 1 :X - y 1 IL m ss cO n y 1 X f_ PI J o -t _ _ _2 5o --! 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Laboratory Manual J A6-6 W il d l if e In t e r n a t io n a l , Ltd - 124- PROJECT NO.: 454A-116 B A C K TO M AIN FO RM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA a N a e b o o fe /IW e r No. Page !8 I I 1 I H if) I I J <c<~\ 1 of I? 1 II I 3 "4~ m 1 1 3 3 1 <view*d 9y_ I Aquatic Tbxlcology 8est Practices for Che Culture and Testing of Selected Fish and Frog Species Laboratory Manual A6-7 W il d l if e In t e r n a t io n a l , Lt d . - 125 - PROJECT NO.: 454A-116 FORM 8 H : FETAX INDIVIDUAL EMBRYO _________ M ALFORM ATION DATA N<x ebook/8indcr N o . 1 I I I I 1 1 J I i I soe oB K CvJ -- aX X s io OJ X rs 3tear X X QX 3` 8 <r. iT 1 5 u-: X 0 X y VO X 'S X i, s1 zX <-* X ro N X J cG r~ CN ia i I, 5S * -X OX X *** X X *es X & as ig X r-1 < nu <*< X 3 rL_T ---J _ m 1! -C 3 is uj oa 2 t a 1 2C I >- 1 o s8 o 0 1 t l i 33 Mo Z. u H.2i c ea 1 1a1 < "3 31 I z0 3a. O >- J 3 u Ol. m* .S* 1 cu X -3 s cM 5 ots Keviewed 3y_ I Aquatic Tbxicology Best Practices for the Culture and Testing of Selected Fish and Frog Species Laborator, Manual A6--8 G3 W il d l if e In t e r n a t io n a l , Lt d . - 126- FO RM 8 H : FETAX INDIVIDUAL EMBRYO PROJECTNO.: 454A-116 B A C K TO M AIN i I I I I 1 1 I I A*vtwe</ I Aquatic Toxicology Best Practices fo r the C u lture and Teiting of Selected RjK and Frog Species L ab o rato ry M a n u a l A 6-9 W il d l if e In t e r n a t io n a l , Ltd - 127- 1 PROJECT NO.: 454A-116 | | i I I 1 1 1 1 _ i I I * i 1 1 3g m fl M 1 A FO RM 8 H : FETAX INDIVIDUAL EMBRYO . ..._ ----------- ------- M ALFORM ATION DATA ` jg Notebook/Sander No , --_------ 2 q- aK 5y tC cv! a Y 3 ay fn a * c d sX T. = > =X i- IT 1R -3 u : a> a* * i i 1 i 2 B i 1 1 P1 1 1 13 -> - X 1!S =y 1 m2 > n Z. 2f * >t 3 -Y ** ^ 1 g 1 g e _ --~ -- -- -- -- ---- ------------------ ~~ H *V X. 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"3 i 3 2 i i i 33 i J Ouaa. t- Z33 < ;$* StijjjouuH Sm u Cu sao. -CV n <5 5 O Reviewed fly ----------------------- Aquatic Tkicology Best Practices for the Culture and Testing of Selected Fish and Frog Species > laboratory Manual A 6-10 i}jte_ -- Si 23 CEa3 W il d l if e In t e r n a t io n a l , Lt d . - 128- FO R M 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA PROJECT NO.: 454A-116 B A C K TO M AIN I I I I I I I I Aquatic Tbxicology 8est Practices for the Culture and Testing of Selected fish, and Frog Species laboratory Manual A6-1 1 W il d l if e In t e r n a t io n a l , Ltd - 129 - PROJECT NO.: 454A-116 B A C K TO M AIN FO RM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA N cx eb oo k/B in de r N o ._ 31 w 1o ___ --T" ""T"TMr Z: =: -- --T--T-- -- -- ~ aX i M X in evil <n X I 1 i- E aX I ri X 1 i O SI X1 X8 I 3 7 | a X 1 >< J 1XVA 3 R Vi X iLi =X V1 co = X i = bi & ~z S! 1 O* SI 3 v X 53 _! 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Lt d . -131 - PROJECT NO.: 454A-116 I I I I I I I I 1 I 4 4 4 4 f Aquatic Toxicology 8est Practices for the Culture and Testing of Selected fish and Froe Species A6--iS Laboratory Manual sa c i E 3E 3 W il d l if e In t e r n a t io n a l , Lt d . 132 B A C K TO M AIN I 1 I I I 1 J ! 4 I Ii Best Practices for the Culture and Testing of Selected Fish and Frog Spec.es . Laboratory Manual A 6-15 5E 3 W il d l if e In t e r n a t io n a l , Ltd - 133 1 PROJECT NO.: 454A-116 B A C K TO M AIN { FORM 8H : FETAX INDIVIDUAL EMBRYO 1 ------------------- m alf ------------------- o r m a t io n DATA fier-- 1 13o- o 1 li li * S a i 3M es 1 1 c1s 3 I on 1 rr -- 1 0 i l i il y P1fMf 1V I_ - 1'S) ta s l<3 im 2 Mi3 1 * S' X 2y I1 I fl * rO 3 pc' 2y S> i 1 1i I dg a y nUn -- a 1 13 Ii Hs -S IS V 'S Vi s >< E :ae 3 S 'S * | g i _.mt rIS 1 o l -> _ p i J r~ -- -- ~~ -- -- XX ----" yy 1 -- 3< --1 yX i 1^ 9 : -is 5i 5$ tu a) 1 2IK 3 i IOz- 0 1o S 15: <1 s a a Oa sa6 1 1 4*v3!* 5 _"3e3 3 1 1 Ua . 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B A C K TO M AIN I I 1 I 1 I I i Aquatic Toxicology Best Practices for the Culture and Testing of Selected Fish d frag S p e c laboratory Manual W il d l if e In t e r n a t io n a l , Lt d . - 136- FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA PROJECT NO.: 454A-116 Noiebook/Bindtf No.________ B A C K TO M AIN I 1 1 I I I I I I I 1 I i 8y Oj<e I Aquatic Toxicology 8est Practices fo r the Culture and Testing of Selected Fish and Frog Species Laboratory Manual A 6 --19 W il d l if e In t e r n a t io n a l , Lt d . - 137- BACK TO MAIN p r o je c t n o .: 4 5 4 A-1 1 6 96 Hour Test Data Efflu ent/GL roundwater/Toxicant: f.Vwmcnl 02.12?) _ Date: 5 /2? -S/lb/oO D'lfrnA'v* 3hL3__________________ Species:_ . Analyst: S U . T,A . Age at Start of Test: BUuk foy.6-U) Control peTAy. Solution Temperature (C) D.O. (mg/L) PH Conductivity Alkalinity Hardness Day 0 0H Z4.0 8.7. 1.14 Day 1 Old New ZAO 24.0 8.3 l i f t 7.U Day 2 Old New 24-0 7.4 7.21 24.0 8.1 1.15 Day 3 Old New 24.0 7.3 1.23 24-0 8.1 1.17 Day 4 Old 2.4.0 7.4 1.(0 1-9 rrxxiL Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H 24.0 8.2. 7.24 Day 1 Old New 24.0 24.0 8.3 8.2 T H 721 `' Day 2 Old New 24.0 2 40 1.2. 8.1 7.20 7.23 Day 3 Old New 24.0 24.o 7.1 8 .2 7.21 7.21 Day 4 Old 24.0 7.5 72\ 1 3 .2 Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH 240 8-1 7.31 Day 1 Old New 24.0 24-0 8.0 8 .2 745 1.28 Day 2 Old New 24.0 24.0 7.1 8.0 7.18 730 Day 3 Old New 24.0 24.0 I t 8.1 7 25 7.2fc Day 4 Old 24.o 7.5 7.24 A6 -2 0 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 138 - PROJECT NO.: 454A-116 DAn.w<,Tto\ 3 96 H our T e st Data Page 2 of 3 5 4 (rOjlL Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H 24.0 8-1 1 . Day 1 Old New 24.0 24.0 8.1 8.1 1.14- 735 Day 2 Old New 2A-0 24.0 7-0 8.1 1 . n 7.37 Day 3 Old New 24.0 7.0 7.2 24.0 8.1 7.34 Day 4 Old 24.0 75 731 9.0 itojIl Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 0H 24.0 8A 7.37 Day 1 Old New 24.0 2.4.0 8.2 8.\ 7.\5 7.35 Day 2 Old New 24.o 2.4.0 7.0 8.0 747 7.28 Day 3 Old New 24.0 24.0 7.0 8.0 7.24 7.3<o Day 4 Old 24.0 7. 7.35 15.0 Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 OH 24o 8.2 740 Day 1 Old New 2 4o 24.o 8 2 8-\ 7.'.4 739 Day 2 Old New 34.0 24.0. 7.0 8-0 775 7.4l Day 3 Old New 24.0 7. \ 728 24.0 8.1 740 Day 4 Old 24 0 7.4 7.40 A .6-21 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 139 - Dt'r>r\L(,TA 3 96 H our T e st Data Page _3_ of J i _ 2.5.0 ir&lu Temperature (C) D.O. (mg/L) PH Conductivity Alkalinity Hardness Day 0 0H 24-0 &.L l.ll Day 1 Old New 24.o 24-0 83 8 Z IMo 7 3 4 Day 2 Old New 24-0 24.o 1.0 8 .\ 72.2 l o t Day 3 Old New 24.0 24.o 7.0 8.0 7.25 7.35 Day 4 Old 24.0 7.4 7.2? Day 0 0H Temperature (C) D.O. (mg/L) 1 pH Conductivity Alkalinity Hardness Day 1 Old New Day 2 Day 3 Old New Old New Day 4 Old Temperature (C) D.O. (mg/L) pH Conductivity Alkalinity Hardness Day 0 j Day 1 I Day 2 OH Old New Old New - Day 3 Old New Day 4 Old A6-22 . BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 140- C hem ical U 2723- D efinitive T e st 3 Embryo Length M e a su re m e n ts (m m) Page 1 of 3 C ontrol A C ontrol B Control C C ontrol D 1.9 m g/L A 1.9 m g/L B 1 10.02 10.08 9.74 9.45 9.14 9 .3 7 2 9 .7 5 9 .8 8 9.05 9.06 9.31 9 .4 8 3 9 .7 6 '1 0 .0 3 9.31 9.65 9.68 9 .8 8 4 9.85 9.9 8.93 8.4 8.86 9 .78 5 9 .4 7 9 .2 2 8.85 9.46 8.54 9 .1 3 6 9 .5 9 9 .5 9 9.09 8.59 9.57 8.11 7 9 .0 6 9 .4 8 ' 8.87 9.64 8.3 8 .7 8 9 .3 8 9 .8 8 8.92 9.1 8.5 9.4 4 9 9 .3 2 9 .2 8 8.54 9.42 8.25 9 .2 8 10 9 .6 7 9 .8 8 9.62 9.49 8.94 9 .4 2 11 9 .7 9 .0 9 9 8.74 8.79 9.41 12 9.71 9 .8 4 9.44 9.8 9.01 7 .6 8 13 9 .6 4 9 .0 4 9.79 8.73 8.62 8 .6 8 14 9 .5 9 .8 8 9.91 9.73 9.03 8 .9 7 15 9 .5 9 9 .3 8 9.54 9.28 9.14 9 .1 3 16 9 .4 4 9 .9 9 8.47 9.25 8.28 10.1 17 9 .8 8 9 .4 6 9.83 9.16 8.49 9 .3 7 18 9 .3 8 9 .7 4 9.38 9.32 7.82 9 .4 2 19 9 .7 6 9.51 9.82 9.8 8.8 9 .2 4 20 8 .9 8 9.52 10.02 9.17 7.93 10.01 21 9 .3 7 9 .6 7 9.22 10.21 9.26 1 0 .2 2 22 9 .3 8 9 .5 2 9.58 ' 9.94 8.93 9 .8 7 23 9 .6 5 10.23 9.53 9.85 . 9.52 9 .5 5 24 9 .5 9 9 .1 5 9.54 9.68 9.11 9 .6 5 25 8.95 9.8 8.76 10.09 8.83 1 0 .1 4 M ean Min M ax Stand. Dev. 9 .5 3 5 6 8 .9 5 1 0 .0 2 0 .2 6 9 7 3 8 8 9 .6 4 1 6 9 .0 4 10.23 0 .3 2 7 4 3 5 5 9.31 8.47 10.02 0 .4 4 1 4 8 4 2 9 .4 0 0 4 8.4 10.21 0 .4 6 4 0 5 3 5 8.826 7.82 9.68 0 .4 8 4 4 3 2 7 9 .3 6 1 2 7 .6 8 1 0 .2 2 0 .6 0 3 3 7 4 7 A6-23 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 141 - C hem ical U 2723- D efinitive T e st 3 Embryo Length M e a su re m e n ts (mm) Page 2 of 3 3 .2 m g /L A 3 .2 m g /L B 5 .4 m g/L A. 5 .4 m g /L B 9 .0 m g/L A 9 .0 m g /L B 1 9 .3 4 9 .4 9 9.75 9.71 9.44 8 .1 7 2 8 .4 7 9 .3 7 9.09 9.33 8.31 8.61 3 9 .1 5 9.79 9.15 8.93 8.4 8.29 4 9 .3 6 9 .4 7 8.73 8.62 9.1 8 .1 2 5 8 .4 8 9.61 9.29 8.58 8.8 8.22 6 9 .4 8 9.72 9.53 9.37 8.71 8 .2 7 7 7.61 9 .4 4 9.02 9.29 7.97 8 .8 7 8 9 .9 9.18 9.69 9.89 9.08 7 .8 6 9 9 .2 6 8 .9 4 9.36 8 .6 6 8.31 9 .3 8 10 9 .24 9.4 9.28 9.71 6.15 8 .3 3 11 8.91 8.75 9.38 9 .3 7 8.43 7.21 12 8 .5 8 10.02 9.52 9.3 9.33 7.68 13 9 .5 7 9 .8 2 9.01 9.53 9.24 8.31 14 9 .5 9 9 .4 7 9.02 8.98 9.17 9 .2 5 15 9 .0 5 9.37 9.12 9.55 9.03 7 .7 8 16 9 .1 5 9 .1 7 10.11 8.9 9.14 7.76 17 9 .4 8.81 8.9 9.02 8.79 7 .0 4 18 8 .5 9 8.81 9.12 9.23 8.48 8 .9 3 19 9 .4 4 9.8 8.25 9.66 9.3 8.66 20 9 .4 2 9.81 8.85 8.37 8.97 9 .3 6 21 9 .4 6 9 .8 3 8.24 9,7 9.07 22 9 .4 3 9.35 9.67 10 7.56 23 9 .4 7 10.01 9.93 10.03 9.35 24 8 .6 4 9.58 9.97 9.29 25 8 .7 3 9.06 9.28 9.46 M ean Min Max Stand. Dev. 9 .1 0 8 8 7 .6 1 9 .9 ' 0 .5 0 0 8 8 5 2 9 .4 4 2 8 8.75 10.02 0.3709281 9.2504 8.24 10.11 0.4712526 9 .2 9 9 2 8.37 10.03 0 .4 5 1 2 2 9 1 8.7013043 6.15 9.44 0.7362331 8 .3 0 5 7 .0 4 9 .3 8 0 .6 5 3 4 4 8 3 A 6-24 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . PROJECT NO.: 454A-116 - 142- C hem ical U 2723- Definitive T e st 3 Embryo Length M e a su re m e n ts (m m) Page 3 of 3 6-A m inonicotinam ide 1 5 m g /L A 1 5 m g /L B 2 5 m g /L A 25 m g/L B 5 .5 m g /L A 5.5 m g/L B 1 8 .9 7 .1 7 7.99 9 .1 9 8.6 6.8 7 2 8 .2 4 9.05 7.49 6 .1 4 7.79 9 .1 4 3 9.17 8 .2 2 8.23 8 8.1 8.21 4 7 .8 9 7 .4 4 8.17 7 .8 3 9.01 7 .7 5 8 .4 4 8 .1 2 8.03 6.51 7.26 7 .7 6 6 7.56 9 .2 7 8.44 7.99 8 .0 4 7 7.59 7.04 8.14 8 .3 8 7.69 7 .6 2 8.6 7 .6 6 9 8.2' 8 .8 8 8.31 7.51 10 7.64 8.12 8.3 7.43 11 7 .2 3 8 .5 8.01 8 .6 12 8.17 7.98 7.71 8 .14 13 7.89 8 .2 3 7.87 8 .3 6 14 8.5 8.3 8.4 7 .6 7 15 6.98 7 .8 4 16 7.14 7 .5 9 17 8.8 8 .6 4 18 7.77 8.1 19 7.91 8 .3 3 20 8.28 7.21 21 8.49 7 .0 9 22 8.22 7 .6 3 23 7.88 8 .1 5 24 7.57 8 .3 7 25 7.29 M ean Min Max Stand. Dev. 8 .0 7 9 2 8 5 7 7 .2 3 9 .1 7 0 .5 4 5 2 9 4 1 8 .1 3 8 5 7 1 4 7 .0 4 9 .2 7 0 .6 6 5 6 3 8 6 8.0583333 7.49 8.44 0.32127.35 7 .5 3 4 8.0168 6 .1 4 6.98 9.19 9.01 1.2287107 . 0.5144959 7 .9 3 0 8 3 3 3 6 .8 7 9 .1 4 0 .5 3 2 1 9 7 4 A6-25 W il d l if e In t e r n a t io n a l , Lt d . - 143 - ! PROJECT NO.: 454A-116 B A C K TO M AIN I I I I I I I 1 I 1 4 i 4 Aquatic Tb.tlcology Best ractices for the Culture and Testing o i Selected Fish and Frog Species a Laboratory Manual wrrn A6-26 W il d l if e In t e r n a t io n a l , Lt d . - 144 - PROJECT NO : 454A-116 B A C K TO MAIN I I I I I 1 i I < 1 FORM 8 H : FETAX INDIVIDUAL EMBRYO MALFORMATION DATA M t t c b o o k J & n d t r No. 2 oH 0 s s S 3X a y Sy fr Crvi y -a 6 c y sy a y a y s s r-> y u \T) *o X X cO 5C J $I 3 5 3 s GN : X X XX *"* X XX yX X y y XXX X y yX X QX --X Is y s * Xs wX X X y X X X * y Is y X " X J !Cl X X y X X r-- Sl xd su a e3t 0 I 1 i e t h* 1 O Z "3 O il e 1 Vn wea Oa iJ %. "a * T3 Sa e 0 1 3 "3 3O SL 0 .Ha. O !3` i 3 Z J X X X X X X viewed X .'5 M 0 1 JT3 e4* VaSa. -5e Z 5 Aquatic Tkicology Best Practices for the Culture and Testing of Selected Fish and Frog Species Laboratory Manual A6-27 Q BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 145- PROJECTNO.: 454A-116 .'RIM M ED S P E A K M A N -K A R B E R M E T H O D . V E R S IO N 1 . 5 o n 2 3 -Rssaj 3 - rrVteVV) (ixso') DATE: 5 /2 2 /0 0 TOXICANT : U 2 7 2 3 SPE C IE S: x e n o p u s T EST NUMBER: 3 D1U R A T IO N : RAW D A T A : ------------------------- C o n c e n tra tio n (m g/1) .0 0 1 .9 3 3 .2 7 5 .2 5 8 .2 6 1 4 .0 0 2 3 .9 0 N um ber E xposed 100 50 50 50 50 50 50 M o rta litie s 0 0 0 0 7 22 39 SPEARMAN-KARBER T R IM : 2 2 .0 0 % SPEARMAN-KARBER ESTIM A TES : LC50: 95% LOWER CONFIDENCE: 95% U PPER CONFIDENCE: 1 5 .2 6 1 3 .1 3 1 7 .7 5 96 h A6-28 W il d l if e In t e r n a t io n a l , Lt d . - 146- BACK TO MAIN PROJECTNO.: 454A-116 BRIMMED S P E A R M A N -K A R B E R M ETHO D. V E R S IO N 1 . 5 \32 .12.3 - (35CS) DATE: 5 /2 2 /0 0 TOXICANT : U 2 7 2 3 S P E C IE S : XENOPUS TEST NUMBER: 3 D U R A T IO N : 96 H RAW D A T A : ------------------ C o n c e n tra tio n (M G /L ) .0 0 1 .9 3 3 .2 7 5 .2 5 8 .2 6 1 4 .0 0 2 3 .9 0 N um ber E xposed 100 50 50 50 43 28 11 M o rta litie 2 3 2 3 6 11 8 SPEARM AN-KARBER T R IM : 2 7 .8 3 % SPEARM AN-KARBER ESTIM A TES : EC50: 95% LOWER CO N FID EN CE: 95% U PPER CONFIDENCE: 1 6 .7 9 1 2 .3 5 22.82 N O TE: M O RTA LITY PR O PO R TIO N S WERE NOT MONOTONICALLY IN C R E A S IN G . A D JU ST M E N T S WERE MADE P R IO R TO SPEARM AN-KARBER E S T IM A T IO N . T l - LCSo/tCSD =90.91 A6-29 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 147- PROJECTNO.: 454A-116 T itle : F ile : U2723:ASSAY 3: embryo le n g th s (mg) ASSAY3LG. Transform : Shapiro - W ilk 's Test fo r Norm ality D = 0.5007 W = 0.9545 C r i t i c a l W = 0.8440 (alpha = 0.01 , N = 16) W = 0.8870 (alpha = 0.05 , N = 16) NO TRANSFORMATION Data PASS n o r m a lity t e s t (alpha = 0 .0 1 ). C ontinue a n a ly s is . BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 148 - PROJECT NO.: 454A-116 T itle : F ile : U2723: ASSAY 3: embryo le n g th s (mg) ASSAY3LE. Transform : NO TRANSFORMATION B a r t l e t t ' s T e st f o r Homogeneity o f V ariance C a lc u la te d B1 s t a t i s t i c = 5.7971 (p-value = 0.4463) Data PASS B1 homogeneity t e s t a t 0.01 l e v e l . C on tin u e a n a ly s is . C r i t i c a l B = 16.8119 (alpha = 0 .01 , d f = 6) = 12.5916 (alpha = 0 .05 , d f = 6) U sin g Average Degrees o f Freedom (Based on average r e p lic a t e s iz e o f 2.29) C a lc u la t e d B2 s t a t i s t i c = 5.0874 (p-value = 0.5327) Data PASS B2 homogeneity t e s t a t 0.01 l e v e l . C on tin u e a n a ly s is . A6-31 W il d l if e In t e r n a t io n a l , Lt d . - 149- BACK TO MAIN PROJECTNO.: 454A-116 T itle : F ile : U2723:ASSAY 3: embryo le n g th s (mg) ASSAY3LE. Transform: ANOVA Ta b le NO TRANSFORMATION SOURCE DF SS MS F Between 6 5.9602 0.9934 17.8546 W ithin (Error) 9 0.5007 0.0556 Total 15 6.4609 (p -valu e = 0.0002) C r i t i c a l F = 5.8018 (alpha = 0.01, d f = 6,9) = 3.3738 (alpha = 0.05, d f = 6,9) S in c e F > C r i t i c a l F REJECT Ho: A l l equal (alpha = 0.05) A6-32 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 150- PROJECTNO.: 454A-116 T i t l e : U2723:ASSAY 3: embryo len g th s (mg) F ile : ASSAY3LE. Transform : NO TRANSFORMATION B o n fe rr o n i t - T e s t - TABLE 1 OF 2 Ho: C o n tro IcTreatment GROUP 1 2 3 4 5 6 7 IDENTIFICATION CONTROL 1.93 mg/1 3.27 mg/1 5.25 mg/1 8.26 mg/1 14.0 mg/1 23.9 mg/1 TRANSFORMED MEAN 9.4725 9 .1150 9.2750 9.2750 8.5050 8.1100 7.7950 MEAN CALCULATED IN SIG ORIGINAL UNITS t STAT 0.05 9.4725 9.1150 9.2750 9.2750 8.5050 8.1100 7.7950 1.7501 0.9668 0.9668 4.7363 6.6700 8.2121 * * * B o n fe rr o n i t c r i t i c a l v a lu e = 2.9333 (1 T a ile d , a lp h a = 0 .0 5 , d f = 6,9) T i t l e : U2723:ASSAY 3: embryo le n g th s (mg) F ile : ASSAY3LE. Transform : NO TRANSFORMATION B o n fe rr o n i t - T e s t - TABLE 2 OF 2 Ho: Control<Treatment GROUP IDENTIFICATION NUM OF REPS MIN SIG DIFF % OF DIFFERENCE (IN ORIG. UNITS) CONTROL FROM CONTROL 1 CONTROL 4 2 1.93 mg/1 2 0.5992 6.3 0.3575 3 3.27 mg/1 2 0.5992 6.3 0.1975 4 5.25 mg/1 2 0.5992 6.3 0.1975 5 8.26 mg/1 2 0.5992 6.3 0.9675 6 14.0 mg/1 2 0.5992 6.3 1.3625 7 23.9 mg/1 2 0.5992 6.3 1.6775 A6-33 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 151 - p r o je c t n o .: 4 5 4 A-1 1 6 ATTACHMENT 1 PHOTOGRAPHS OF NORMAL AND MALFORMED EMBRYOS EXPOSED TO CHEMICAL U2723 Photo: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Description: Assay 1; FETAX solution exposure Assay 1; FETAX solution exposure Assay 1; 8.64 mg a.i./L (nominal concentration) exposure Assay 1; 8.64 mg a.i./L (nominal concentration) exposure Assay 2; 5.19 mg a.i./L (nominal concentration) exposure Assay 2; 5.19 mg a.i./L (nominal concentration) exposure Assay 2; 24.0 mg a.i./L (nominal concentration) exposure Assay 2; 24.0 mg a.i./L (nominal concentration) exposure Assay 3; 14.4 mg a.i./L (nominal concentration) exposure Assay 3; 14.4 mg a.i./L (nominal concentration) exposure Assay 1; 8.64 mg a.i./L (nominal concentration) exposure Assay 1; 8.64 mg a.i./L (nominal concentration) exposure Assay 1; 14.4 mg a.i./L (nominal concentration) exposure Assay 1; 8.64 mg a.i./L (nominal concentration) exposure W il d l if e In t e r n a t io n a l , Lt d . BACK TO MAIN PROJECT NO.: 454A-116 Photo 1: (7.5x) Normally developing Xenopus laevis embryo at 96 hours. Note- tight coiling of the gut and full head region. Photo 2: (15x) Normally developing Xenopus laevis embryo at 96 hours. W il d l if e In t e r n a t io n a l , Lt d . BACK TO MAIN PROJECT NO.: 454A-116 Photo 3. (7.5x) Malformations observed: Multiple edemas, including gut, cardiac, facial and optic edemas; facial abnormalities and improper gut coiling. Photo 4: (15x) Malformations observed: Multiple edemas, including gut, cardiac, facial and optic edemas; facial abnormalities and improper gut coiling.  BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d .___________________ pr o je c t n o .: 4 5 4 A-116 - 154- Photo 5: (15x) Malformation observed: Improper (loose) gut coiling. Photo 6: (15x) Malformation observed: Improper (loose) gut coiling. W il d l if e In t e r n a t io n a l , Lt d . -155- BACK TO MAIN PROJECT NO.: 454A-116 Photo 7: (7.5x) Malformations observed: Multiple edemas, including gut, cardiac, facial and optic edemas; extreme lack of gut coiling; severe facial abnormalities. Photo 8: (15x) Malformations observed: Multiple edemas, including gut, cardiac, facial and optic edemas; extreme lack of gut coiling; severe facial abnormalities. W il d l if e In t e r n a t io n a l , Lt d . BACK TO MAIN ' PROJECT NO.: 454A-116 Photo 9: (7.5x): Malformations observed: Anal edema; slight facial abnormalities; slightly curved notochord. Photo 10: (15x): Malformations observed: Anal edema; slight facial abnormalities; slightly curved notochord. W il d l if e In t e r n a t io n a l , Lt d . BACK TO MAIN PROJECT NO.: 454A-116 Picture 11: (7.5x) Malformations observed: Severe cardiac edema; slight gut edema; extremely improper gut coiling, curvature of the notochord; slight facial abnormality (caused by cardiac edema). Picture 12: (15x) Malformations observed: Severe cardiac edema; slight gut edema; extremely Improper gut coiling; curvature of the notochord; slight facial abnormality (caused by cardiac edema). W il d l if e In t e r n a t io n a l , Lt d . -158- BACK TO MAIN PROJECT NO.: 454A-116 Picture 13: (15x) Malformations observed: Multiple edemas, including cardiac, gut, facial and optic edemas; severe facial abnormalities; severe lack of gut coiling. Picture 14: (15x) Malformations observed: Multiple edemas, including cardiac, gut, facial and optic edemas; severe facial abnormalities; severe lack of gut coiling. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 159- p r o je c t n o .: 4 5 4 A-1 1 6 ATTACHMENT 2 LETTER FROM STEVEN TURLEY OF UNIVERSITY OF MARYLAND, WYE RESEARCH AND EDUCATION CENTER March 19, 2001 Mrs. Sue Palmer Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 Dear Sue: Enclosed is a copy of the revised final report. All of the suggested questions or corrections were addressed. Although LC50, EC50 and MCIG values changed when actual mean measured concentrations were used, the Tl values were the same for all three tests. Thus, data still suggest that this chemical has little teratogenic potential based on FETAX results. On the reference toxicant question, the 2500 mg/L concentration has consistently caused high mortality at our laboratory. The low reference toxicant concentration has also been very consistent, with low mortality and approximately 50% malformed embryos. I feel that the mortality observed in the high reference toxicant concentration does not compromise the results of these three assays in any way. Other laboratories working with FETAX often have similar high mortality in the 2,500 mg/L concentration. Over the last 5 years our laboratory has performed numerous standard and modified FETAX assays. In each study, there has been consistently high mortality in the 2,500 mg/L reference toxicant exposure, while mortality and malformations have been extremely low in embryos reared in FETAX solution. The best example of this was an extended FETAX study performed last year for the U.S. Fish and Wildlife Service. At the 2,500 mg/L reference toxicant concentration, there was 100% mortality. In the FETAX solution exposures, greater than 90% of the embryos survived (and completed metamorphosis) 140-d post-hatch. I think this example demonstrates the quality of embryos produced by our in-house culture. The lack of a signature in the review space was an oversight on my part. The data was reviewed initially by our former QAQC person at the lab. Since she was no longer a W REC employee, she did not know whether she could legally sign the data sheets. This employee was my wife ( and believe me- there are no harsher critics than a spouse). After she reviewed the data, Dr. Burton and I reviewed the data again. The lack of initials was just an oversight on my part. After receiving the report from you for the revisions, I reviewed the data again and found no mistakes. Please review the revised report, and if you have any corrections let me know. I will make any changes you deem necessary. Sincerely, BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 160- PROJECT NO.: 454A-116 APPENDIX III The Analysis of PFOS in Frog Embryo Teratogenesis Assay -Xenopus (FETAX) Solutions in Support of Wildlife International, Ltd. Project No.: 454A-116 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 161 REPORT APPROVAL projectno.: 454a-ii6 SPONSOR: 3M Corporation TITLE: PFOS: A Frog Embryo Teratogenesis Assay - Xenopus (FETAX) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-116 PRINCIPAL INVESTIGATOR: MANAGEMENT: Director, Analytical Chemistry BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 162- p r o j e c t n o .: 4 5 4 A-1 1 6 Introduction Frog embryo teratogenesis assay-Xenopus (FETAX) solution samples were collected from three definitive assays designed To determine the effects o f PFOS (Perfluorooctanesulfonate, Potassium Salt) to the embryos o f the South African clawed frog (.Xenopus laevis). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-116. The analyses o f these FETAX solution samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Test solutions for the first assay were prepared on May 15, 2000 and samples from these solutions were analyzed on May 15 and 22, 2000. Test solutions for the second and third assay were prepared on May 19, 2000 and samples from these solutions were analyzed on May 19 and 30, 2000. Test Substance and Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. 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 material, was identified as: 1H, 1H, 2FI, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619 97-2. The standard (hereafter referred to as 4HPFOS) was stored under ambient conditions. Analytical Method The method used for the analysis of the FETAX solution samples was based on methodology developed at Wildlife International, Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4FI PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 163 - p r o j e c t n o .: 454a - ii Concentrations o f PFOS in the standards and 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 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Ci analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit of Quantitation 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 0.00480 to 0.0480 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) o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f 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 Figures 3 and 4, respectively. The method limit of quantitation (LOQ) for these analyses was set at 0.240 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor of the matrix blank samples (50). Matrix Blank and Fortification Samples Four matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5. FETAX solution was fortified at 0.480, 6.72 and 28.8 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 164 - PROJECT NO.: 454A-116 for the mean procedural recovery o f 97.2%. A representative ion chromatogram o f a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-116-7, nominal concentration of 24.0 mg a.i./L in FETAX solution. Peak Area Ratio = Analyte Peak Area/Intemal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration o f Internal Standard Internal Standard Concentration: 0.100 mg a.i./L Initial Volume: 0.100 mL Final Volume: 100 mL Dilution Factor: 1000 PFOS Peak Area: 1158987 Internal Standard Peak Area: 2125037 Peak Area Ratio: 0.5454 Calibration curve equation. Slope: 1.9275 Intercept: 0.0260 Curve is weighted (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at Instrument = Slope x Internal Standard Concentration = 0.02695 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at Instrument x Dilution Factor 0.02695 x 1000 = 26.95 BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 165 - PROJECT NO.: 454A-116 PFOS (mg a.i./L) in sample Percent o f Nominal Concentration = PFOS (mg a.i./L) nominal x = 112% RESULTS Sample Analysis FETAX solution samples were collected from each of three definitive FETAX assays prior to test initiation (Day 0) and at test termination (Day 4 or 96 hours). The first assay was initiated on May 15, 2000 and terminated on May 19, 2000. The second and third assays were initiated on May 19, 2000 and terminated on May 23, 2000. In the first assay, the measured concentrations o f PFOS in test solution samples collected prior to initiation o f exposure of the test organisms ranged from 112 to 141% of the nominal concentrations. Samples collected at test termination had a measured concentration range o f 54.7 to 98.6% o f nominal values. The sample from the abiotic 24.0 mg a.i./L treatment group was comparable to samples from the 24.0 mg a.i./L treatment group with the frog embryos present (Table 4). A representative ion chromatogram of a test sample is shown in Figure 7. For the second and third assays, common test solutions were prepared. The measured concentrations o f PFOS in test solution samples collected prior to initiation of exposure o f the test organisms ranged from 95.8 to 117% o f the nominal concentrations (Tables 5 and 6). In the second assay, samples collected at test termination had a measured concentration range of 80.7 to 112% of nominal values. The sample from the abiotic 24.0 mg a.i./L treatment group was comparable to samples from the 24.0 mg a.i./L treatment group with the frog embryos present (Table 5). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 166 - p r o j e c t n o .: 454A -116 In the third assay, samples collected at test termination had a measured concentration range of 93.4 to 114% o f nominal values. The sample from the abiotic 24.0 mg a.i./L treatment group was comparable to samples from the 24.0 mg a.i./L treatment group with the frog embryos present (Table 6). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 167 - p r o je c t n o .: 454a -i i 6 INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil Cis column (50 mm x 2 mm I.D., 3-prn particle size) OVEN TEMPERATURE: STOP TIME: FLOW RATE: 30C 5.00 minutes 220 pL/minute MOBILE PHASE: INJECTION VOLUME: 70.0% Methanol : 30.0% NANOpure Water containing 0.1% Formic Acid 5.0 pL PFOS RETENTION TIME: Approximately 3.2 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.1 minutes PFOS MONITORED MASS: 498.6 amu INTERNAL STANDARD MONITORED MASS: 426.7 amu BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 168 - p r o j e c t n o .: 454a -i i 6 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-116-) MAB-1 Sample Type Matrix Blank Sample Set Day 0 - 1st Assay Measured Concentration ofP F O S 1 (mg a.i./L) < LOQ MAB-2 MAB-3 Matrix Blank Matrix Blank Day 0 - 2nd and 3rd Assay Day 4 - 1st Assay < LOQ < LOQ MAB-4 Matrix Blank Day 4 - 2nd and 3rd Assay < LOQ 1 The limit o f quantitation (LOQ) was 0.240 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0,00480 mg a.i./L) and the dilution factor of the matrix blank samples (50),_______ BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 169 - p r o j e c t n o .: 4 5 4 A-1 1 6 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-116-) MAS-1 MAS-2 MAS-3 Sample Set Day 0 - 1st Assay Concentrations of PFOS (mg a.i./L) Fortified1 Measured1 0.480 6.72 28.8 0.490 7.12 30.2 Percent Recovered2 102 106 105 MAS-4 MAS-5 MAS-6 Day 0 - 2nd and 3rd Assay 0.480 6.72 28.8 0.441 6.32 27.4 91.8 94.0 95.1 MAS-7 MAS-8 MAS-9 Day 4 - 1st Assay 0.480 6.72 28.8 0.443 6.20 27.7 92.3 92.2 96.1 M AS-10 Day 4 - 2nd and 3rd Assay 0.480 0.449 93.5 M AS-11 MAS-12 6.72 6.77 101 28.8 28.1 97.6 Mean = 97.2 Standard Deviation = 5.08 CV = N= 5.22% 12 'Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 170- p r o je c t n o .: 454a -i i 6 Table 4 Measured Concentrations o f PFOS in Frog Embryo Teratogenesis Assay-Xenopus (FETAX) Solution Samples - First Assay Nominal Test Concentration12 (mg a.i./L) 0.0 (Negative Control) 0.0 (Positive Control) Sample Number (4 5 4 A -1 16-) 1 8 9 Sampling Time (Day) 0 4 4 PFOS Measured Concentration1,2 (mg a.i./L) < LOQ < LOQ < LOQ Percent of Nominal3 -- -- - 1.82 2 0 10 4 3.07 3 0 11 4 5.19 4 0 12 4 8.64 5 0 13 4 2.58 141 1.42 77.8 3.94 128 1.72 55.9 6.62 128 2.84 54.7 10.7 124 5.09 58.9 14.4 6 0 14 4 18.5 128 10.8 74.9 24.0 7 0 15 4 26.9 112 22.3 93.0 24.0 16 4 23.7 98.6 (Abiotic) 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 The limit o f quantitation (LOQ) was 0.240 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor o f the matrix blank samples (50). 3 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. ___________ ___________________________________________ BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 171 - p r o j e c t n o .: 4 5 4 A- 1 16 Table 5 Measured Concentrations o f PFOS in Frog Embryo Teratogenesis Assay-Xenopus (FETAX) Solution Samples - Second Assay Nominal Test Concentration1 (m ga.i./L ) 0.0 (Negative Control) Sample Number (454A-116-) 17 24 Sampling Time (Day) 0 4 PFOS Measured Concentration1,2 (mg a.i./L) < LOQ < LOQ Percent of Nominal3 "" 1.82 18 0 26 4 1.77 97.3 2.04 112 3.07 19 0 27 4 3.59 117 2.49 81.0 5.19 20 28 0 4 5.45 105 4.18 80.7 8.64 21 0 29 4 8.43 97.6 7.51 86.9 14.4 22 30 0 4 14.5 100 12.1 84.3 24.0 23 31 0 4 2 3 .0 23.1 95.8 96.4 24.0 32 4 23.9 99.6 (Abiotic Control)_________________ _______________________ _ _ _ _ _ _________________________ 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 The limit of quantitation (LOQ) was 0.240 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor o f the matrix blank samples (50). 3 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes._____________________ _______________ ___________________________ BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 172- p r o j e c t n o .: 4 5 4 A-1 1 6 Table 6 Measured Concentrations o f PFOS in Frog Embryo Teratogenesis Assay-Xenopus (FETAX) Solution Samples - Third Assay Nominal Test Sample Sampling PFOS Measured Percent Concentration1 Number Time Concentration1,2 of (mg a.i./L)_______ (454A -116-)_________ (Day)______________(mg a.i./L)__________ Nominal3 0.0 (Negative Control) 17 33 0 4 < LOQ < LOQ -- -- 1.82 18 0 35 4 1.77 97.3 2.08 114 3.07 19 0 36 4 3.59 117 2.94 95.7 5.19 20 37 0 4 5.45 105 5.05 97.5 8.64 21 38 0 4 8.43 97.6 8.09 93.6 14.4 22 39 0 4 14.5 100 13.5 93.4 24.0 23 40 0 4 23.0 24.7 95.8 103 24.0 (Abiotic Control) 41 4 24.1 100 1 Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 The limit of quantitation (LOQ) was 0.240 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor o f the matrix blank samples (50). 3 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. W il d l if e In t e r n a t io n a l , Lt d . - 173 - BACK TO MAIN PROJECT NO.: 454A-116 M ETHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) SOLUTION Prepare matrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into FETAX solution using gas-tight syringes and Class A volumetric flasks. I Dilute matrix fortification and test samples into the range o f the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the appropriate volume of sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. I Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in frog embryo teratogenesis assayXenopus (FETAX) solution. W il d l if e In t e r n a t io n a l , Lt d . - 174- BACK TO MAIN p r o j e c t n o .: 4 5 4 A-1 1 6 Figure 2. A typical calibration curve for PFOS. Slope = 1.9275; Intercept = 0.0260; r = 0.9973. Curve is weighted (1/x). W il d l if e In t e r n a t io n a l , Lt d . - 175 - BACK TO MAIN PROJECT NO.: 454A-116 intensity: 1 8 0 0 0 0 cp s Figure 3. A representative ion chromatogram of a low-level (0.00480 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , Lt d . - 176 - BACK TO MAIN PROJECT NO.: 454A-116 intensity: 1 8 0 0 0 0 c p s Figure 4. A representative ion chromatogram of a high-level (0.0480 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , Lt d . - in - BACK TO MAIN p r o j e c t n o .: 4 5 4 A-1 1 6 intensity: 1 8 0 0 0 0 c p s Figure 5. A representative ion chromatogram of a matrix blank sample (454A-116-MAB-1). The arrow indicates the retention time of PFOS. W il d l if e In t e r n a t io n a l , Lt d . - 178 - BACK TO MAIN PROJECT NO.: 454A-116 intensity: 1 8 0 0 0 0 c p s Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-116-MAS-l, 0.480 mg a.i./L nominal concentration). W il d l if e In t e r n a t io n a l , Lt d . - 179- BACK TO MAIN PROJECTNO.: 454A-116 intensity: 1 8 0 0 0 0 c p s Figure 7. A representative ion chromatogram of a test sample (454A-116-7, 24.0 mg a.i./L nominal concentration). BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 180- APPENDIXIV Changes to Protocol PROJECT NO.: 454A-116 This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to change the test substance name at the Sponsor's request. 2. The nominal concentrations were recalculated since the Sponsor requested that the purity o f the test substance be changed from 90.49% to 86.9%. BACK TO MAIN W il d l if e In t e r n a t io n a l , Lt d . - 181 - p r o j e c t n o .: 454A-H6 APPENDIX V Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Susan J. Palmer, Senior Biologist 5. Raymond L. VanHoven, Ph D., Scientist