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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 0.Krueger, Ph.D. STUDY INITIATION DATE: May 11,2000 STUDY COMPLETION DATE: April 6,200 1 SUBMITTED TO: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55 144 WildlifeInternational, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 181 WILDLI FE INTER NATIoNAL, LTD. BACK TO MAIN PROJECT NO.: 454A-116 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Frog Embryo TeratogenesisAssay -Xenopus (FETAX) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 116 STUDY COMPLETION: April 6,200 1 This study was not conducted in compliance with Good Laboratory Practice Standards as published by the U.S.Environmental ProtectionAgency in 40 CFR Parts 160 and 792, 17August 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 characterizationwas performed according to 3M StandardOperatingProceduresand Methods, and all raw data are being maintainedin the 3M archives. The test substancehas been recharacterizedin 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 studyconducted by University of Maryland was not conducted in compliancewith Good Laboratory Practices. STUDY DIRECTOR: Susan J. Palmer Senior Biologist r .,_. - . - . SPONSOR APPROVAL: 4 - 4-61 DATE BACK TO MAIN WILDLIFE INTERNATIONALLT, D. -3QUALITY ASSURANCE STATEMENT PROJECT NO.: 454A-116 This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental ProtectionAgency, 40 CFR Parts 160 and 792,17 August 1989. The dates of all inspections and audits and the dates that any findingswere reportedto the Study Director `and LaboratoryManagementwere 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 WILDLIFE INTERNATIONALLT, D. -4REPORT APPROVAL PROJECT NO.: 454A-116 SPONSOR: 3M Corporation TITLE: PFOS: A Frog Embryo Teratogenesis Assay -Xenopus (FETAX) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 116 STUDY DIRECTOR: r Susan J. Palmer Senior Biologist MANAGEMENT : Director, Aquatic Toxicology and Non-Target Plants 4-6 -0I DATE DATE BACK TO MAIN WILDLI FE INTERNATIONALLT, D. -5TABLE OF CONTENTS PROJECT NO.: 454A-116 TitleKover Page................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement.............................................................................................. .2 Quality Assurance Statement............................................................................................................................... 3 Report Approval 4 Table of Contents ................................................................................................................................................ .5 ................................................................................................................................................ 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 Data .................................................................................. . 14 Table 2 - LC50, EC50, MCIG and TI Values ................................................................................................. 15 APPENDICES Appendix I - Certificateof Analysis .............................................................................................. 16 Appendix I1 - Evaluation of the Toxicity and Teratogenicity of Chemical U2723 Using the Frog Embryo TeratogenesisAssay -Xenopus (FETAX) .................................... 19 Appendix I11 - The Analysis of PFOS in Frog Embryo TeratogenesisAssay -Xenopus (FETAX) Solutionsin Support of Wildlife International,Ltd. Project No.: 454A- 116 .......... Appendix IV - Changes to the Protocol ....................................................................................................... 180 Appendix V - Personnel Involved in the Study .................................................................................... 181 BACK TO MAIN WILDLIFE INTERNATIONALL, TD. -6- PROJECT NO.: 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 TeratogenesisAssay -Xenopus (FETAX) PFOS (Perfluorooctanesulfonate, Potassium Salt) IUPAC Name: 1-Octanesulfonicacid,1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassiumsalt; 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 lstAssay: 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 31dAssay: Negative Control, 1.93,3.27,5.25, 8.26, 14.0 and 23.9 mg a.i./L lstAssay: ExperimentalStart - May 15,2000 Biological Termination - May 19, 2000 ExperimentalTermination - May 22,2000 2ndand 3'd Assays: ExperimentalStart - May 22,2000 Biological Termination - May 26: 2000 ExperimentalTermination - 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 WILDLIFE INTERNATIONALL, TD. -7- PROJECT NO.: 454A-116 SUMMARY - Continued - RESULTS (BASED ON MEAN MEASURED CONCENTRATIONS) : 96-HOUR LC50: 1St Assay: 13.8mg a.i./L 2ndAssay: 17.6mg a.i./L 31d Assay: 15.3mg a.i./L 95% CONFIDENCE LIMITS: 12.38- 15.31 mg a.i./L 15.53- 19.96mg a.i./L 13.13 - 17.75mg a.i./L 96-HOUR EC50: 12.1mg a.i./L 17.6mg a.i./L 16.8 mg a.i./L 95% CONFIDENCE LIMITS: 10.00 - 14.58 mg a.i./L 13.49- 22.88 mg a. /L 12.35- 22.82 mg a.i./L MINIMUM CONCENTRATION TO INHlBIT GROWTH (MCIG): TERATOGENIC INDEX (TI): Not calculable 1.1 7.97 mg a.i./L 1.o 8.26 mg a.i./L 0.9 BACK TO MAIN WILDLIFE INTERNATIONALL, TD. -8INTRODUCTION PROJECT NO.: 454A-116 This study was conducted in three assays by Wildlife International, Ltd. for 3M Corporation at the University of 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 of 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 of Maryland, and a copy of 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, onthe embryosofthe South African clawed frog (Xenopus laevis) during a 96-hour exposure period. EXPERIMENTAL DESIGN Three definitive assays were conductedwith 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-aminonicotinamidein FETAX solution) for 96 hours. Tworeplicatetest chamberswere maintainedin each treatment group and four replicate test chambers were maintained in each control group. Each test chamber contained 25 embryos for a total of SO 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 selectedwere 1.82,3.07,5.19, 8.64, 14.4 and 24.0 mg active ingredient (a.i.)/L. Mean measured test concentrationsweredeterminedat 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 WILDLIFE INTERNATIONALL, TD. -9- PROJECT NO.: 454A-116 The test solutions were prepared at Wildlife International, Ltd. andwere deliveredto UM-WREC prior to each assay. At Uh4-WREC, the embryos were randomly assigned to test chambers containing the appropriate FETAX solution and were observed for 96 hours. The FETAX solutions in the test chamberswere 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 LCSO 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 E1439-91 Standard Guidefor Conducting the Frog Embryo Teratogenesis Assay -Xenopus (FETM) (1) and ASTM Standard E729-88a Standard Guide for 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 number 2 17 (T-6295). Information 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 identifiedas 6-aminonicotinamide(CAS #329-89-5; Sigma; Lot No. 12SH0006). Preparation of Test Concentrations Prior to each assay, FETAX solution was prepared at UM-WREC anddeliveredto WildlifeInternational, 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 of the BACK TO MAIN WILDLIFE INTERNATIONLATLD,. - 10- PROJECT NO.: 454A-116 test substance. The primary stock was proportionally diluted with FETAX solution to prepare the six test concentrations. Nominal test concentrations were 1.82,3.07,5.19,8.64,14.4and 24.0 mg a.i./L,basedonatest substance purity of 86.9%. The test solutions were delivered to UM-WREC prior to the start of each assay. The reference toxicant solutions were prepared at UM-WREC. Analytical Chemistw Samples were collectedfrom 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 of 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 11. In-Life Phase of Assays Details of the in-life phase of the three assays conducted at UM-WREC are included in Appendix I. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrationsof PFOS in FETAX solution samples collecteddwingthe three assays are presented in Table 1 and in the analytical chemistry report (Appendix 11). 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 of exposure of the test organisms ranged from 112to 141% of the nominal concentrations. Samplescollected at test termination had a measured concentration range of 54.7 to 98.6% 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. For the second and third assays, common test solutions were prepared. The measured concentrationsof PFOS in test solution samples collectedprior 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 concentrationrange of 93.4to 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 WILDLIFE INTERNATIONALLT, D. - 11 - PROJECT NO.: 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 concentrationsfor the second assay were 1.91,3.04,4.82,7.97, 13.3and 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 concentrationsfor the third assay were 1.93,3.27,5.25, 8.26, 14.0and 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 concentrationsfor each assay. DefinitiveAssav Results The results of the three definitiveFETAX assays conductedwith PFOS at the UM-WREC are included in Appendix I and are summarized in Table 2. Exposure to PFOS caused significantembryo mortality at the two highest test concentrations(14.4 and 24.0 mg a.i./Lnominal concentration)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 concentrations. Embryo survivalwas >90% at all test concentrationsup to 8.64mg a.i./Lnominal concentration. The 96-hour LC values for PFOS ranged from 13.8to 17.6mg a.i./Lmeanmeasuredconcentrationinthethree assays (Table 2). There was a positive correlationbetween PFOS exposureand malformationsin each of the three assays, with increasingconcentrationscausing an increasein the incidence of malformed embryos. The 96-hour EC50 values, based on the incidence of malformed embryos, ranged from 12.1 lo 17.6 rng a.i./L mean measured concentrationin the three assays (Table 2). The most common types of malformationscaused by exposure to PFOS were improper gut coiling, edema, notochord abnormalitiesand facial abnormalities. Growth of the embryos was not effected by exposureto PFOS in the first assay. In the second and third assays, the minimum concentration inhibitinggrowth (MCIG) was 8.64 mg a.i./Lnominal concentration. This reduction in growth may be attributed more to the increased incidence of malformed embryos in the test concentrationsthan to an overall decrease in growth. The teratogenicindex (TI), defined as the 96-hour LC50 value for mortality divided by the 96-hour EC50 value for malformed embryos, provides an estimate of the teratogenicrisk associatedwith PFOS. The TI'Sin the three assays ranged from 0.9 to 1.1,indicatingthe PFOS has a low potential to be a developmental hazard. WILDLIFE INTERNATIONALL, TD. BACK TO MAIN PROJECT NO.: 454A-116 CONCLUSIONS The 96-hour LC50 value for mortality forXenopus luevis exposed to PFOS in each of three assays was 13.8, 17.6 and 15.3mg a.i./L mean measured concentration,respectively. The 96-hour EC50 value for embryo malformation in each of 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./Lmeanmeasured concentration,respectively. The TI'Sranged from 0.9 to 1.1in the three assays, indicatingthe PFOS has a low potential to be a developmental hazard. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. -13REFERENCES PROJECT NO.: 454A-116 1 ASTM Standard E1439-91. 1998. Standard Guidefor Conducting the Frog Embryo Teratogenesis Assay -Xenopus (FETAX). American Society for Testing and Materials. Philadelphia,Pennsylvania. 2 ASTM Standard E729-88a. 1994. Standard Guidefor ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. Philadelphia, Pennsylvania. BACK TO MAIN WILDLIFE INTERNATIONAL,TD. - 14- PROJECT NO.: 45412-116 Table 1 Summary of Analytical Chemistry Data Nominal Test Concentration (mg a.i./L) Negative Control Sampling Time (Day) Measured Concentration (mg a.i.iZ) O2 <LOQ~ 43 dOQ 1st Assay Mean Measured Concentration (mg a.i.:'L) <LoQ Percent of Nominal _- Measured Concentration (mg a.i.iL) <LOQ <LOQ 2ndAssay' Mean Measured Concentration (mg a.i.iL) cLOQ Percent of Nominal __ Measured Concentration (mg a.i./Z) <LOQ <LOQ 31d Assay' Mean Measured Concentration (mg a.i.L) cLOQ Percent of Nominal __ 1.82 0 2.58 2.00 1 10 1.77 1.91 105 1.77 1.93 106 4 1.42 2.04 2.08 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 7.5 1 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 4 22.3 24.0 (abiotic) 4 23.7 24.6 103 23.0 23.1 -_ 98.6 23.9 23.1 96.3 23.0 24.7 _- 99.6 24.1 23.9 99.6 __ 100 I The 2"d and 31dassays were conducted concurrently using common batches of test solution prepared for Day 0. Day 0 samples were collected from the test solutions at preparation prior to transfer to UM-WREC. 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 The limit of quantitation (LOQ) was 0.240 mg a.i.L. BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 15- PROJECT NO.: 454A-116 Table 2 LC50, EC50, MCIG and TI Values ' Parameter 1" Assay ~~ 2ndAssay 96-Hour LC50 (mg a.i./L): 13.8 17.6 95% Confidence Limits (mg a.i./L): 12.38- 15.31 15.53- 19.96 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 Minimum Concentration Inhibiting Growth (MCIG) (mg a.i./L): --2 7.97 3rdAssay 15.3 13.13- 17.75 16.8 12.35- 22.82 8.26 TeratogenicIndex (TI): 1.1 1.0 0.9 1 Values are based on the mean measured concentrationsfor each assay. 2 The MCIG could not be calculated because PFOS had no effect on embryonic growth at any of the test concentrationswith survivingembryos in the 1" assay. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 16- APPENDIX I Certificate of Analysis PROJECT NO.: 454A- 1t6 INTERIM CERTIFICATE OF ANALYSIS Revision 1(Sn/OO) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS, Lot 217 Reference #: SD-018 1. carbod 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine COA023418A 'Zhcorccloal Value = 5.95% Theontical Value = 60% 1. 12.48 wt./wt.% 2. 0.244 wt./wt.% 3. 1.74 wt./wt.% 4. a.84 wt./wt.% 5. 54.1 wt./wt.% Page I of 3 WILDLIFE INTERNATIONALL,TD. - 17- BACK TO MAIN PROJECT NO.: 454A-116 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-lOC Re-assessment Date: 08/31/01 'Purity = lOO?/o - (sum of metal impurities, 1.45% +LC/MSimpurities, 8.4l%+Inorganic Fluoride, 0.59%+Nh4Rimpurities, 1.93%+organicacid impurities, 0.38%+POAA, 0.33%) - Total impurity from all tests = 13.09% Purity = 1W/o 13.09?!= 86.9% 2Potassiumis expected in this salt form and is therefore not considered an impurity. +urity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4Suffir in the sample appearsto be converted to so4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfbr determinationin the elementalanalysis, lending confidenceto this interpretation. Based on the results, the SO4 is not considered an impurity. %A HFBA NFPA PFPA Trifluoroacetic acid Heptafluombutyric acid Nonofluoropentanoic acid Pentafluoropropanoicacid %heoretical value calculationsbased on the empirical formula, CsFnS03~+(MW=538) This work was conducted under EPA Good LaboratoryPractice Standards (40CFR 160). I COA023-018A Page 2 of 3 WILDLIFE INTERNATIONALL, TD. - 18- BACK TO MAIN PROJECT NO.: 454A-1.'16 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LCMS Purity Profile: Impurity c4 cs C6 c7 wtlwt. Yo 1.22 1.33 4.72 1.14 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The CS value was calculated usingthe 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.Bedl Date Scientist,Centre Analytical Laboratorie~ Reviewed By: John Flaherty Date Laboratory Manager, Centre Analytical Laboratories cOA023-0 18A Page 3 of 3 BACK TO MAIN WILDLIFE INTERNATIONALLT, D. - 19- PROJECT NO.: 454A-116 APPENDIX I1 Evaluation of the Toxicity and Teratogenicity of Chemical U2723 Using the Frog Embryo Teratogenesis Assay -Xenopus (FETAX) WILDLI FE INTERNATIONAL, LTD. - 20 - BACK TO MAIN PROJECT NO.: 454A-116 Report No. WREC00-02 EVALUATION OF THE TOXICITY AND TERATOGENICITY OF CHEMICAL U2723 USING THE FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) Final Report Prepared for Wildlife International, Ltd. 8598 Commerce Drive Easton, MD 21601 Prepared by Steven D. Turley and Dennis T. Burton University of Maryland Wye Researchand EducationCenter P.O. Box 169 Queenstown, MD 21658 July 15,2000 Revision Date: March 19,2001 WILDLI FE INTERNATIONAL, LTD. - 21 - BACK TO MAIN PROJECT NO.: 454A-116 TABLE OF CONTENTS Page TABLE OF CONTENTS ................................................ ii 1. INTRODUCTION .................................................. 1-1 2. MATERIALAND METHODS ........................................ .2-1 3. RESULTSAND DISCUSSION ...................................... .3-1 4. REFERENCES .................................................. .4-1 ......... APPENDIX 1. FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTEDWITH CHEMICAL U2723 (ASSAY 1) Al-1 APPENDIX 2. FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTEDWITH CHEMICAL U2723 (ASSAY 2) . . . . . . . . . A2-1 APPENDIX 3. FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTEDWITH CHEMICAL U2723 (ASSAY 3) . . . . . . . . .A3-1 APPENDIX 4. RAW DATA SHEETS FOR FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) ASSAYS 1,2, AND 3 CONDUCTED ON CHEMICAL U2723 .................................. A41 APPENDIX 5. 6-AMINONICOTINAMIDE REFERENCE TOXICANT DATA FORXENOPUSUEVlS ................................ A5-1 APPENDIX 6. PHOTOGRAPHS OF NORMAL AND MALFORMED EMBRYOS EXPOSED TO CHEMICAL U2723 ............... A6-1 ii WILDLIFE INTERNATIONAL, LTD. - 22 - BACK TO MAIN PROJECT NO.: 454A-116 1. INTRODUCTION The evaluation of the potential toxicity and teratogenicity of chemical U2723 was initiated at the request of Mr. David A Palmer, Wildlife International, Ltd., Easton, Maryland. The study was conductedby Mr. Steven D. Turley and Dr. DennisT. Burtonof the Universityof MarylandWye Researchand EducationCenter, Queenstown, Maryland. The test programwas performedin accordancewith the 'Revised Statement of Work for Evaluating the Potential Teratogenicity of U2723 Using %Hour Frog Embryo Teratogenesis Assay-Xenopus (FETAXY, submitted to and approved by Wildlife International,Ltd., March30,2000 (Turley,2000). Thepotentialtoxicity andteratogenicity of chemical U2723 was evaluated by the Frog Embryo Teratogenesis Assay-Xenopus . (FETAX). Three 964 definitive FETAX assays were conducted on chemical U2723. 1-1 WILDLI FE I NTERNATIONAL, LTD. BACK TO MAIN PROJECT NO.: 454A- 116 2. MATERIALS AND METHODS Three definitive FETAX assays were conducted on chemical U2723 using the frog embryo teratogenesis assay-Xenopus (FETAX). The assay is a 96-h quantitative developmental assay used to screen for developmentaltoxicants in aquatic media. The assays were conducted using the static renewal (solutions renewed every 24 h) test protocol DesignationE 1439-91 of the American Societyfor Testingand Materials(ASTM, 1998). Embryolethality(964 LC50), malformations(964 EC50), and growth retardation were used as endpoints in the assays. Embryoswere obtainedfrom breedingcoloniesofXenopuslaevisat the University of MarylandWye Researchand EducationCenter (UMDMIREC). The UMDMlREC adult colonies were 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. UMDWREC non-chlorinated deep well water held at 23.5 f 0.5 'C served as the culture medium. All frogs were fed every 5-6 d with commercialbeef liversupplementedwith liquidvitamins (PolyViSoP; Mead-Johnson Nutritionals, Evansville,Indiana). The colony was heldunder a photoperiodof 16 hlight:8 h dark. Mating pairs were bred in the dark in 23.5 f 0.5 "C UMDMlREC non-chlorinated water at -70 d intervals by injecting 400 and 800 I.U.of human chorionic gonadotropin (HCG) inthe dorsal lymphsacof the malesandfemales, respedively. 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 breedingpairswere usedfor eachtestto checkfor variabilityinthe sensitivityof individual clutches of embryos. All embryos were examined two times for viability. The embryos were dejellied in a 2% Lcysteine solution (2 g of L-cysteine per 98 mL of FETAX solution). Once dejellied, the embryoswere rinsedand re-suspended in FETAXsolution (ASTM. 1998). The embryos were assayed in covered 60 mm glass Petri dishes containing 10mL of solution. All embryoswere randomly assigned to the Petri dishes. Two replicates of 25 embryoslreplicate were used for each of six assay treatments; four replicates were used for the controls as required by the test protocol (ASTM, 1991). The assays were conducted at 24 f 0.2'C under a 1241 light: 1241dark photoperiod (fluorescent lights; -75 foot candles at the surface of the test medium) in a constant temperature environmental chamber. All Petri dishes were randomly placed in the environmental chamber used for the assays. Wildlife 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 Wildlife International, Ltd. Two concentrationsofthe referencetoxicant,6-aminonicotinamide(CASM29-89-5;Sigma; Lot No. 125H0006),were used at each assay periodas outlined inthe ASTM (4998)protocol. 2-1 WILDLIFE INTERNATIONALL, TD. - 24 - BACK TO MAIN PROJECT NO.: 454A- 116 Mortalityobservations andgeneralwater quality measurementswere madeat each 2441renewal period. All dead embryoswere removedat each observation period. At the end of the 961.1exposure period, each surviving embryowas examinedfor malformations using a dissecting microscope. The identificationand interpretationof 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 Madera, California). A random group of normal embryos and embryos with each type of malformation were preserved in 10% buffered formalin (pH 7) for photographing. TheTrimmedSpearman-Karberstatisticalprocedurewas usedtodeterminethe96- h LC50 for mortality and 96-h EC50 for malformations. The minimum concentration to inhibit growth (MCIG) was determined by Bonferroni's T-Test. All statistical tests were performed using Toxstat (WEST and Gulley, 1994). A minimum probability level of 0.05 was used. The teratogenic index (TI)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 WILDLI FE INTERNATIONAL, LTD. - 25 - BACK TO MAIN PROJECT NO.: 454A-116 3. RESULTS AND DISCUSSION The data for definitive assays 1,2, and 3 are summarized inAppendices 1,2, and 3, respectively. Copiesofthe rawdatasheetsand6aminonicotinamidereferencetoxicant datafor assays 1,2, and 3 are given inAppendices4,5, and6, respectively. The nominal concentrationsof 1.9,3.2,5.4,9.0,15.0 and25.0 mg/Lonthe rawdata sheets correspond to the nominal concentrations of 1.82, 3.07, 5.19, 8.64, 14.4 and 24.0 mg a.i./L in the report. Photographsof normaland malformedembryosof eachtype observedinthe study are given inAttachment 1. Table 1is summary of the endpoint parametersestablishedin the three definitive assays. Table 1. Summary of Datafor the Three Assays with Chemical U2723' Parameter LC50 (95% C. I.) EC50 (95% C.I.) MClG Assay 1 -13.8 (12.38 15.31) -12.1 (10.00 14.58) b Assay 2 -17.6 (15.53 19.96) -17.6 (13.49 22.88) 7.97 Assay 3 -15.3 (13.13 17.75) -16.8 (12.35 22.82) 8.26 'The LC50, EC50, and MClG values are based on mean measured concentrations of chemical U2723 (mg a.i./L) for each assay. The MClGcould not be calculatedbecausechemical U2723had no effect on embryonic growth at any of the test treatments with surviving embryos (AppendixA; Table Al- 4). ExposuretochemicalU2723causedsignificantembryo mortalityat the twohighest treatment concentrations (14.4 and 24.0 mg/L) in each of the three assays. Most of the embryo mortalityappearedto becausedbythe gut coilingthrough the bodywall atthe two highest assay treatments. Embryo survival was 290% at all exposure treatments up to 8.64 mg/L. The 9641LC5Osfor chemical U2723rangedfrom 13.8 to 17.6 mg/L (Table 1). There was a positive correlation between chemical U2723 exposure and malformations in each of the three assays, with increasing concentrations causing an increaseinthe incidenceof malformedembryos. The 9641EC50s, basedonthe incidence of malformedembryos, rangedfrom 12.1to 17.6 mg/L (Table1). The mostcommontypes of malformations caused by exposure to chemical U2723 were: improper gut coiling, 3-1 WILDLI FE INTERNATIONAL, LTD. - 26 - BACK TO MAIN PROJECT NO. 454A- 116 edema, notochord abnormalities and facial abnormalities. Growth of the embryos was not affected by exposure to chemical U2723 in assay 1. Inassays 2 and 3 ,the minimumconcentrationinhibitinggrowth (MCIG) was 7.97 and 8.26 m a , respectively. This reduction ingrowthmay be attributedmoreto the increased incidence of malformedembryos inthese test concentrationsthan an overall decrease in growth. Malformed embryos are generally smaller than normally developing embryos. The teratogenicindex (TI),which by definition is the 9641LC50divided by the 9641 . EC50 (malformed embryos), provides an estimateofthe teratogenicrisk associatedwith a material (Dumont et al., 1983). TI values of 1.5 to 2.0 indicate that a material may be a potential teratogen. Materials with TI values ~ 2 . 0should be considered for further teratogenicity testing. The TI'S inthe current study rangedfrom 0.9 to 1.l which indicate that chemical U2723 has a low potential to be a developmental hazard. The threeassaysmetallof theASTM(1998) protocoltest acceptabilitycriteria,with one exception. The referencetoxicant (6aminonicotinamide)highconcentrationof 2,500 mg/Ldid not meetthe ASTM protocoltest acceptability criterion. A concentrationof 2,500 mg/L should kill between40 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 acceptabilitycriteriafor the low referencetoxicant concentrationof 5.5 mg/L which shouldcause between40 and60%malformedembryosafter 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 1W%,in assays 1,2, and 3, respectively. The chain of custody forms for delivery of the assay solutions from Wildlife International, Ltd. to the U M D M E C were retainedby Wildlife International, Ltd. Thus, copies of the chain of custodyforms are not included in this report. 3-2 WILDLI FE INTERNATIONAL, LTD. - 27 - BACK TO MAIN PROJECT NO.: 454A-116 4. REFERENCES ASTM. 1998. Standard guide for conducting the frog embryo teratogenesis assayXenopus (FETAX). ASTM DesignationE 1439-91. 1998 Annual Book of ASTM StandardsSection 11 Water and Environmental Technology, Amer. SOC.Testing Materials, Philadelphia, PA Bantle, J.A, J.N. Dumont, R.A Finch, and G. Linder. 1991. Atlas of abnormalities. A guide for the performance of FETAX. Oklahoma State Univ., Stillwater, O K Dumont, J., T. Schultz, M. Buchanan, and G. b o . 1983. Frog embryo teratogenesis assay-Xenopus (FETAX)-A short-termassay applicableto complexenvironmental mixtures. Pages 393405 in: Waters, Sandhu, S.S.,J. Lewtas, L. Claxton, N. Chernoff, and S.Nesnow, eds. Short-term bioassays in the analysis of complex environmental mixtures 111, Plenum, New Yo% NY. Niewvkoop, P.D. and J. Faber. 1975. Normaltables of Xenopuslaevis (Daudin),2"ded. North Holland, Amsterdam, Netherlands. Turley, S.D. 2000. Revised statement of work for evaluating the potential teratogenicity of U2723 using 96-hour frog embryo teratogenesis assayXenopus (FETAX). March 30,2000. University of MarylandWye Researchand Education Center, Queenstown, MD. WEST (Western Ecosystem Technology, Inc.) and Gulley, D. D. 1994. Toxstat, v. 3.4. Western Ecosystem Technology, Inc., Cheyenne, W. 4-1 WILDLIFE INTERNATIONALL, TD. - 28 - BACK TO MAIN PROJECT NO.: 454A-116 APPENDIX 1 FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) CONDUCTED WITH CHEMICAL U2723 (ASSAY 1) Test Method: Type of Test: Date: Investigator: Laboratory: Chemical: Designation: Source: General Water Quality: Test Medium: Test Organism: Scientific Name: Age at Start of Test: Source: Experimental Chambers: Material: Test SolutionVolume: No. OrganismdReplicate: No. Organismmreatment: Lighting: Endpoints: Test Temperature: ASTM DesignationE 1439-91 ASTM (1998) Static renewal (every 24 h) May 15-19,2000 S. D. Turley UMDMREC U2723 Wildlife International, Ltd. See Table A1-1 FETAX solution Xenopus laevis Stage 8 blastulato stage 11gastrula UMDMREC culture Glass petri dishes 10 mL 25 Control: 100 Treatment: 50 Fluorescent; 6085 foot candles Mortality; malformation; growth 24 f 2C A1 -1 WILDLI FE INTERNATIONAL, LTD. - 29 - BACK TO MAIN PROJECT NO.: 454A-116 Results: Note: For Assay 1,statistical calculationsare based on the mean measured concentrationsof 2.00,2.83,4.73,7.90, 14.70 and 24.60 mg a.i./L, ratherthan on the nominal concentrations. Mortalitv: The 96-h LCW = 13.8 mg a.i./L (95% confidence limits = - 12.38 15.31). The mortalitydata are summarized in Table A1-2. Malformations: - The 964 EC50 (malformed embryos) = 12.1 mg a.i./L (95% confidence limits = 10.00 14.58). The number of malformed embryos are summarized in Table A1-2. The types of malformationsare summarized inTable A1-3. Growth: Chemical U2723 had no effect on embryonic growth at any of the test treatments with surviving embryos. As a result, the minimum concentration inhibiting growth (MCIG) could not be calculated. The growth data are summarized inTable A1-4. Teratoaenic Index The teratogenic index (TI) = 1.I. A1 -2 ~~~ ~ ~ ~ WILDLIFE INTERNATIONALL, TD. - 30 - ~ BACK TO MAIN PROJECT NO.: 454A-116 Table A1-1. - Summary Assay 1 of The General Water Chemistry Dissolved Oxygen (mglL) Data for Chemical U2723 - Dav 0 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 8.3 8.0 8.4 7.7 8.3 7.4 8.3 7.7 8.1 7.4 8.4 Test Concentrations (mgk) 1.82 3.07 5.19 8.64 14.40 24.00 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 7.0 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 7.7 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 WILDLIFE INTERNATIONALL, TD. -31 - BACK TO MAIN PROJECT NO.: 454A-116 Table A1-1. - (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 Test Concentrations (mgll) 0.00 1.82 3.07 5.19 8.64 7.74 7.78 7.79 7.78 7.78 7.26 7.24 7.30 7.34 7.35 7.24 7.45 7.38 7.43 7.43 7.16 7.17 7.20 7.23 7.23 7.22 7.25 7.28 7.32 7.34 7.1 1 7.13 7.16 7.17 7.25 7.13 7.20 7.23 7.25 7.26 7.1 1 7.15 7.18 7.19 7.18 7.1 1 7.13 7.16 7.17 7.18 7.74 7.78 7.79 7.78 7.78 14.40 7.76 7.34 7.44 7.23 7.35 7.18 7.26 7.20 7.18 7.76 24.00 7.62 7.33 7.32 7.25 7.27 6.95 7.18 7.19 6.95 7.62 A1 -4 WILDLI FE I NTERNATIONAL, LTD. - 32 - BACK TO MAIN PROJECT NO.: 454A-116 Table Al-1. - (Continued) Temperature ("C) Dav 0 OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max Test Concentrations(man) 0.00 1.82 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 I 24.00 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 A1 -5 WILDLI FE INTERNATIONAL, LTD. - 33 - BACK TO MAIN PROJECT NO.: 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 (msn) Control Rep Number Embryos Alive 1 24 2 25 3 25 4 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 4.0 2 8.3 2 24 4.0 5 20.8 5.19 1 23 8.0 7 30.4 2 22 12.0 3 13.6 8.64 1 21 16.0 4 19.0 2 23 8.0 7 30.4 14.40 1 16 36.0 9 56.3 2 15 40.0 11 73.3 24.00 1 0 100 2 0 100 A1 -6 WILDLI FE I NTERNATIONAL, LTD. - 34 - BACK TO MAIN PROJECT NO.: 454A-116 Table A I -3. - FETAXToxicity Test Data T pe and Number of Malformed Embryos - after 96 Hours Exposureto demical U2723 Assay I -severe out 0 1.82 3.07 5.19 8.64 14.40 24.00 Rbp Rep Rep Rep Rep RW Rep 1234121212121212 1 1 1 1 1 1 1 2 5 7 3 4 5 7 11 EdWIa: Multipls cardiac 1 22 131 2243 2241 Awomlnal 131 2132 Facial 22 2242 22 1126 11 m FaCO 11 1 2 2 ' 1 1 4 3 2, 3 4 6 EP 11 Brah 1 2 31 #Malformed 1 1 I 1 2 2 2 5 7 3 4 7 9 11 *Analedema. A1 -7 WILDLIFE INTERNATIONAL, LTD. - 35 - BACK TO MAIN PROJECT NO.: 454A-116 - Table A 1 4 FETAX Toxicity Test Data Embryonic Growth (Length) after 96 Hours Exposure to Chemical U2723 - Assay 1 Concentration Rep Number Embryos (mglL) AI ive Control 1 24 2 25 25 25 1.82 24 25 3.07 1 24 2 24 5.19 23 22 8.64 21 23 14.40 16 15 24.00 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 WILDLI FE INTERNATIONAL, LTD. - 36 - BACK TO MAIN PROJECT NO.: 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 Water Quality: Test Medium: Test Organism: Scientific Name: Age at Start of Test: Source: Experimental Chambers: Material: Test Solution Volume: No. OrganismslReplicate: No. Organismsmreatment: Lighting: Endpoints: Test Temperature: ASTM Designation E 1439-91 ASTM (1998) Static renewal (every 24 h) May 22-26,2000 S.D. Turley UMDMREC U2723 Wildlife International, Ltd. See Table A2-1 FETAX solution Xenopus laevis Stage 8 blastulato stage 1 1 gastrula YMDMREC culture Glass petri dishes 10 mL 25 Control: 100 Treatment: 50 Fluorescent; 60-85 foot candles Mortality; malformation; growth 24 f 2C A2-1 WILDLI FE INTERNATIONAL, LTD. - 37 - BACK TO MAIN PROJECT NO.: 454A-116 Results: Note: For Assay 2, statistical calculationsare basedon the mean measured concentrationsof 1.91,3.04,4.82,7.97,13.30and 23.10mg a.i./L, rather than on the nominalconcentrations. Mortality: The 96-h LC50 = 17.6mg 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) = 17.6mg a.i./L (95% confidence limits = 13.49 22.88) The number of malformed embryos are summarized in Table A2-2.The types of malformationsare summarized in Table A2-3. Growth: The minimum concentrationinhibiting growth (MCIG ) =7.97mg a.i./L. The growth data are summarized in Table A 2 4 Teratwenic Index: The teratogenic index (TI) = 1 .O. A2-2 WILDLIFE INTERNATIONALL, TD. - 38 - BACK TO MAIN PROJECT NO.: 454A-116 - Table A2-1. Summary of The GeneralWater ChemistryData for Chemical U2723 Assay 2 - Dissolved Oxygen (mglL) Test Concentrations (mglL) 0.00 1.82 3.07 5.19 8.64 14.40 24.00 Dav 0 OH 8.2 8.2 8.1 8.2 8.1 8.2 8.2 Dav 1 24 H 8.3 8.3 8.0 8.1 8.1 8.1 8.2 OH 8.2 8.2 8.2 8.2 8.1 8.1 8.2 Dav 2 24 H 7.6 7.8 7.5 7.2 7.2 7.2 7.1 OH 8.1 8.1 8.0 8.1 8.0 8.0 8.1 Dav 3 24 H 7.5 7.6 7.5 7.6 7.7 7.3 7.2 OH 8.1 8.2 8.1 8.1 8.0 8.1 8.0 Dav 4 24 H 7.6 7.6 7.5 7.5 7.6 7.5 7.5 Mean 8.0 8.0 7.9 7.9 7.9 7.8 7.8 Min 7.5 7.6 7.5 7.2 7.2 7.2 7.1 Max 8.3 8.3 8.2 8.2 8.1 8.2 8.2 A23 WILDLIFE INTERNATIONALL, TD. - 39 - BACK TO MAIN PROJECT NO.: 454A-116 Table A2-1. (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 Test Concentrations (mdL) 0.00 1.82 3.07 5.19 8.64 7.14 7.24 7.31 7.33 7.37 7.12 7.17 7.21 7.23 7.24 7.11 7.21 7.28 7.35 7.35 7.18 7.22 7.23 7.24 7.24 7.15 7.23 7.30 7.37 7.38 7.20 7.20 7.22 7.25 7.24 7.17 7.21 7.26 7.34 7.36 7.11 7.21 7.20 7.29 7.33 7.11 7.17 7.20 7.23 7.24 7.20 7.24 7.31 7.37 7.30 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 WILDLI FE INTERNATIONAL, LTD. - 40 - BACK TO MAIN PROJECT NO.: 454A-116 Table A2-1. (Continued) -Temperature ("C) Dav 0 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 Test Concentrations (mg/L) 1.82 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 WILDLIFE INTERNATIONALL, TD. - 41 - BACK TO MAIN PROJECT NO. 454A- 116 - Table A2-2. FETAXToxicity Test Data Percent Embryo Mortalityand Malformations - after 96 Hours of Exposureto Chemical U2723 Assay 2 Concentration (mgW Control Rep Number Embryos Alive 1 25 2 25 3 25 4 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 8.0 2 8.7 2 22 12.0 0 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 WILDLIFE INTERNATIONALL, TD. - 42 - BACK TO MAIN PROJECT NO.: 454A-116 Table A2-3. - K - FETAX Toxicity Test Data T pe and Number of Malformed Embryos after 96 Hours Exposure to C emical U2723 Assay 2 Malformation Ted c m c m h h (W) 0 1 .a 3.07 5.19 8.64 14.40 24.00 Rep Rep Rep Rep Rep Rep Rep 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1'2 1 12 1 11 2 1 12 1 4132664 2 1 3 221 4 3 11 3 13 1 3 1 3 11 2231913 2 #Maifomndl Anal edema. 1 2 201222014454964 ~ ~~ A2-7 WILDLIFE INTERNATIONALL, TD. BACK TO MAIN PROJECT NO.: 454A-116 - Table A 2 4 FETAX Toxicity Test Data Embryonic Growth (Length) after 96 Hours - Exposure to Chemical U2723 Assay 2 Concentration Rep Number Embryos (mglL) Alive Control 1 25 2 25 3 25 4 24 1.82 1 25 2 25 3.07 1 23 2 22 5.19 1 24 2 22 8.64 1 22 2 23 14.40 1 22 2 13 24.00 1 9 2 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 Mean Average Length (mm) 8.88 8.45 8.57 8.72 7.93* 7.51" 7.39* Significantlydifferent at alpha = 0.05 (BonferroniT-Test) A2-8 WILDLIFE INTERNATIONALL, TD. - 44 - BACK TO MAIN PROJECT NO.: 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 Water Quality: Test Medium: Test Organism: Scientific Name: Age at Start of Test: Source: Experimental Chambers: Material: Test SolutionVolume: No. OrganismslReplicate: No. Organismsnreatment: Lighting: Endpoints: Test Temperature: ASTM DesignationE 1439-91 ASTM (1998) Static renewal (every 24 h) May 22-26,2000 S. D. Turley UMDMREC U2723 Wildlife International, Inc. See Table A3-1 FETAX solution Xenopus laevis Stage 8 blastula to stage 11 gastrula UMDMREC culture Glass petri dishes 10mL - 25 Control: I00 Treatment: 50 Fluorescent; 60-85 foot candles Mortality; malformation; growth 24 f 2C A3-1 WILDLIFE INTERNATIONALL, TD. - 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, 5.25,8.26, 14.00 and 23.90 mg a.i./L, rather than on nominal concentrations. Mortality: The 964 LC50 = 15.3 mg a.i./L (95% confidence limits = - 13.13 17.75). The mortality data are summarized in Table A3-2. Malformations: - The 96-h EC50 (malformedembryos) = 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. Teratoaenic Index: The teratogenic index (TI) = 0.9. A3-2 WILDLIFE INTERNATIONALL, TD. BACK TO MAIN PROJECT NO.: 454A- 116 - Table A3-1. Summary of The General Water Chemistry Datafor Chemical U2723 Assay 3 Dissolved Oxygen (mg/L) - ~~ Dav 0 OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max Test Concentrations(mglL) 0.00 1.82 3.07 5.19 8.64 14.40 24.00 8.2 8.2 8.1 8.2 8.1 8.2 8.2 8.3 8.3 8.0 8.2 8.2 8.2 8.3 8.2 8.2 8.2 8.2 8.1 8.1 8.2 7.4 7.2 7.1 7.0 7.0 7.0 7.0 8.1 8.1 8.0 8.1 8.0 8.0 8.1 7.3 7.1 7.1 7.0 7.0 7.1 7.0 8.1 8.2 8.1 8.1 8.0 8.1 8.0 7.4 7.5 7.5 7.5 7.6 7.4 7.4 7.9 7.9 7.8 7.8 7.8 7.8 7.8 7.3 7.1 7.1 7.0 7.0 7.0 7.0 8.3 8.3 8.2 8.2 8.2 8.2 8.3 A3-3 WILDLI FE INTERNATIONAL, LTD. - 47 - BACK TO MAIN PROJECT NO.: 454A-116 Table A3-1. - (Continued) pH (StandardUnits) Dav 0 OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Min Max Test Concentrations( m a ) 0.00 1.82 3.07 5.19 8.64 7.14 7.24 7.31 7.33 7.37 7.18 7.19 7.15 7.14 7.15 7.1 1 7.21 7.28 7.35 7.35 7.21 7.20 7.18 7.17 7.17 7.15 7.23 7.30 7.37 7.38 7.23 7.21 7.25 7.26 7.24 7.17 7.21 7.26 7.34 7.36 7.18 7.21 7.24 7.31 7.35 7.1 1 7.19 7.15 7.14 -7.15 7.23 7.24 7.31 7.37 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 A34 t WILDLI FE INTERNATIONAL, LTD. - 48 - BACK TO MAIN PROJECT NO.: 454A-116 Table A3-1. - (Continued) Temperature ("C) Dav 0 OH Dav 1 24 H OH Dav 2 24 H OH Dav 3 24 H OH Dav 4 24 H Mean Min Max Test Concentrations (mglL) 0.00 1.82 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 A3-5 WILDLIFE INTERNATIONALL, TD. -49- BACK TO MAIN PROJECT NO.: 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 (mgn) Control Rep Number Percent Number Percent Embryos Mortality Embryos Malformed Alive Malformed 25 0.0 4.0 25 0.0 4.0 25 0.0 0.0 25 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 WILDLIFE INTERNATIONALL, TD. - 50 - BACK TO MAIN PROJECT NO.: 454A-116 Table A3-3. - i - FETAXToxicity Test Data T pe and Number of Malformed Embryos after 96 Hours Exposure to C emical U2723 Assay 3 0 1.82 3.07 5.19 0.64 14.40 24.00 Rep ReD Rep Rep Rep Rep Rep 1 2 3 4 12 1212 1 21 2 1 2 1 1 2 1 13 23 1 1 3 1 1 1 1 1 33 1 111 11 33433 2 1 3131 A3-7 WILDLIFE INTERNATIONAL,TD. -51 - BACK TO MAIN PROJECT NO.: 454A-116 Table A 3 4 - FETAX Toxicity Test Data Embryonic Growth - Exposure to Chemical U2723 Assay 3 (Length) after 96 Hours Concentration Rep Number Embryos (m9n) Alive Control 1 25 Average Length (mm) 9.54 Mean Average Length (mm) 9.47 2 25 9.64 3 25 9.31 4 25 9.40 1.82 1 25 8.83 9.10 2 25 9.36 3.07 1 25 9.1 1 9.28 2 25 9.44 I 5.19 1 25 9.25 9.28 2 25 9.30 8.64 1 23 8.70 8.51 2 20 8.31 14.40 1 14 8.08 8.1l * 2 14 8.14 24.00 1 6 2 5 8.06 7.80" 7.53 Significantly different at alpha = 0.05 (BonferroniT-Test) A3-8 WILDLIFE INTERNATIONALL, TD. - 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 concentrationsof 1.9,3.2,5.4,9.0,15.0and 25.0 mgR I I in the raw data correspondto the nominal concentrations of 1.82,3.07, 5.19,8.64,14.40 and 24.00 mg a.i./Lin the report. I ! .. A4-1 BACK TO MAIN | Ee ee 5 a ------ . 5= 5 E5A pes ee i sep Em po EE oe | het : z if EE EEE ==EE 3 5 |P| E E E m Ee s E ae E rJ ere E eJ e re s sr n Heh1 E ie re3 diw ||| |: 3 f: cq LES|||| E F E E E re R ee e ee r e ee es e eBereieee|||| fF8E {| E F e aPes et eHe i PE E E F e osr oe eT reteee rr e | e --1--|| 5 t F a e iee e ammme nsa|ff| 4 & 1 1| : i| [iT] YA i z: WILDLIFE INTERNATIONALL, TD. - 54 - FORM 8C: FETAX MORTALITY DATA BACK TO MAIN PROJECT NO.: 454A-116 I Mortality I 1 I 1 I I I BACK TO MAIN . BACK TO MAIN mnI BACK TO MAIN m .r ' 4@ p 2 Y- BACK TO MAIN 5 r c U m T tZ t nn BACK TO MAIN mn BACK TO MAIN f BACK TO MAIN . BACK TO MAIN k3 1 BACK TO MAIN f B i/r BACK TO MAIN i | BN ; em ee mm mm mm g6 5a i i3 iLiH p-- E y--er S rDE e TE) S E EcEnETnTESEe EEE = 3z 2 35 {| E =] ; e A A ] - z 2z T| (E EE EEE a ee | 5S8 :- I | EE EEE EE 28 dg i || EEE ee : 1 : ||| || E E EEe E EH EEEe EE e e E E : e]| 8 Fi{I| || CE E e e E etmtme eee E i = Sm t r ttmt | iy an Il g BACK TO MAIN WE _--. i _- | 2 |} et we I. .2 33 |Hl) : | S F-- R a : Hi HH3 33 Hd '| 2 3| | = PT 0 . a h ma E e3i[2I 3 dol . .HEE BE e BEe IEEES=E | ii I 8 8 BACK TO MAIN f'8VI 0 BACK TO MAIN PaI.3. 2 z. 00 .-.. . .. BACK TO MAIN 52| 3 [13 3 z i i rm dlTha CTR BRIA canStaTh oligome 1S r Z 53 3 3 | ;| i ||| [ EE E ( E lEE i E E E E en EE nr EFeE b e EEnEEEe e EEEEi EEEe e T i52e 3 t550 i||E EEEE ee {||HN |EeE F E oamFeE E e E e E F e e E e e E e e eE e] f 2 C2 {| EE E EEE e e e e . FI 55 IHan | || gg f4i1 :g WILDLIFE INTERNATIONALL, TD. - 69 - BACK TO MAIN PROJECT NO.: 454A-116 Alkalinity Hardness I I I .. I I *I I Alkalinity Hardness ,I I I WILDLIFE INTERNATIONAL, LTD. - 70 - 96 Pag eHZo-uroTf .e3st D a t a BACK TO MAIN PROJECT NO.: 454A-116 PH Conductivity Alkalinity Hardness I I 7.78 7.35 7.43 '7.2.7 17.34-1'7.25 7.26 7.IE IIIII I I I I I1 I I I A4-19 WILDLI FE INTERNATIONAL, LTD. - 71 - 96 Hour Test Data Page of 3_ BACK TO MAIN PROJECT NO.: 454A-116 Alkalinity Hardness I I I I Temperature ( O C) Day 0 OH Day 1 Day 2 Day3 Old New Old New Old New Day4 Old I III III I I Temperature (O C) 0.0. (mg/L) PH Conductivity Alkalinitv Hardness Day 0 OH -. Day 1 Day 2 Day3 Old New Old New Old New Day4 Old .. WILDLIFE INTERNATIONALL, TD. - 72 - BACK TO MAIN PROJECT NO.: 454A-116 96 Hour Test Data Effluent/Groundwaterfloxicant: b - F)rn.w,-,,r&irHmu;t. (i t + I) Date: ~ ~ I S ~ - S ~ I ~ I [ U J Analyst: ' Species: b.,,, . Age at Start of Test: &8 - 6 g , z \ PH Conductivity AIkalinity Hardness .75b 7.31 7 3 7.\q 7.9 84-21 BACK TO MAIN WILDLI FE INTERNATIONAL, LTD. - 73 - PROJECT NO.: 454A-116 Chemical U2723- Definitive Test 1 Embryo Length Measurements (mm) Page 1 of x 3 Control A Control 1 8.86 2 9.06 3 9.36 4 9.25 5 8.93 6 9.32 7 9.76 8 9.29 9 9.37 10 9.54 11 9.39 12 9.28 13 9.29 14 8.76 15 8.22 16 8.19 17 7.93 18 9.56 19 8.96 20 . 8.51 21 9.17 22 9.19 23 8.58 24 9.45 25 8 Control 8.66 8.03 8.25 8.69 8.1 8.35 8.4 7.74 8.38 8.68 7.29 8.28 8.58 8.81 8.36 8.6 8.85 8.72 8.36 8.55 9.02 8.16 7.87 8.73 8.64 C Control 8.96 8.83 8.83 8.07 8.44 9.41 8.05 7.51 7.88 7.96 8.1 7.96 9.05 8.17 8.28 7.45 7.94 8.17 7.34 10.06 9.22 7.47 7.81 8.02 8.36 D 1.9 8.78 8.91 8.01 8.63 8.08 8.61 8.83 9.1 8.94 9 8.83 7.58 8.01 8.33 8.75 8.83 8.4 8.14 8.54 8.78 8.95 8.53 9.46 8.29 9.12 mglL A 1.9 8.76 9 9.1 I 8.25 8.5 8.82 7.44 8.3 8.38 8.23 8.93 8.58 7.59 9.45 8.51 8.87 8.17 8.77 8.24 7.88 9.05 8.47 7.93 7.42 mglL B 8.48 6.26 8.23 8.2 8.75 8.31 8.3 7.83 7.96 8.53 8.16 7.27 8.5 8.75 8.41 8.25 8.16 8.16 8.03 8.47 8.21 8.27 7.67 8.55 7.67 Mean Min Max Stand. Dev. 9.0508333 7.93 9.76 0.4723477 8.404 7.29 9.02 0.38861 94 8.2936 7.34 10.06 0.6695949 8.6172 7.58 9.46' 0.429627 8.44375 7.42 9.45 0.5323027 8.1352 6.26 8.75 0.5190466 84-22 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-116 - 74 - Chemical U2723- Definitive Test 1 Embryo Length Measurements (mm) Page 2 o f x 3 3.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 mglL A 3.2 9.24 9.15 8.84 7.53 8.95 9.21 8.01 9.25 9.54 9.39 8.07 10.08 9.2 8.84 8.94 8.09 9.38 8.78 8.08 9.66 8.84 9.28 7.69 9.16 mglL B 5.4 9.22 8.66 9.4 8.78 8.74 8.76 9.09 8.34 9.37 8.44 8.35 8.03 9.04 9.18 8.76 8.07 7.17 8.97 9.18 9.58 8.27 8.61 8.23 9.04 mglL A 5.4 5.61 8.41 6.85 6.63 8.59 7.42 8.98 8.73 8.92 8.6 8.39 7.61 8.51 9.35 8.16 9.1 9.41 8.72 8.91 9.32 9.57 8.79 9 mglL B 9.0 9.7 8.92 7.33 9.14 8.98 8.25 8.45 8.37 6.25 8.84 8.75 9.26 7.97 8.79 8.5 8.93 9.11 9.76 8.72 8.33 8.58 8.27 mglL A 9.0 8.73 8.89 7.24 8.4 8.37 8.44 9.51 9.23 8.66 8.87 6.98 8.32 8.83 9.15 9.5 9.45 9.81 9.09 8.94 9.6 9.52 mglL B 9.16 8.52 9.18 6.34 8.6 8.84 8.89 7.41 8.83 8.73 9.26 9.45 9.42 9.22 8.99 9.01 9.82 9.4 6.95 7.57 8.24 9.24 6.33 Mean Min Max Stand. Dev. 8.8833333 7.53 10.08 0.6501483 8.72 7.17 9.58 0.5448654 8.4165217 5.61 9.57 0.9830824 8.6 6.25 9.76 0.7544029 8.8347619 6.98 9.81 0.7235373 8.5826087 6.33 9.82 0.9891145 M-23 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-116 - 75 - Chemical U2723- Definitive Test 1 Embryo Length Measurements (mm) Page 3 of 3 15 mglL A 15 mglL B 25 mglL A 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 9.28 8.48 11 7.84 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 mglL B 6-Aminonicotinamide 5.5 mglL A 5.5 mglL B 7.95 6.99 8.21 7.77 8.46 7.99 8.3 8.92 9.6 8.4 9.02 7.8 7.46 9.12 7.59 9.23 7.7 8.1 1 8.1 7.55 8.16 8.57 8.01 7.96 7.99 8.41 9.1 1 8.69 8.81 7.95 8.41 8.12 8.16 9.04 7.44 8.27 7.39 7.84 7.8 7.93 8.12 7.15 8.91 7.38 7.65 Mean Min Max Stand. Dev. 8.40875 7.14 10.24 0.9429519 7.7390909 6.51 8.9 0.7726895 8.1 891 304 7.39 9.6 0.5832504 8.145 6.99 9.23 0.6140556 84-24 LE |I -- BACK TO MAIN i i ii f wraton Br wn DE Tr) iSne Tey } Er CE 2 151 VHBE ieEarsEEmmmmeimn i = i | S-- e 3 Tf Eth Ea EE == ! ee i = = i ee 3] Hmm TT 11 i|= = = -- HEFHEE { ; f= ; S TIT IX Es TT FEE =H Fi = HEHEHE IN | N E| 2|58| f ||| IN 2 : !ol | | ffB] 3: 3 BACK TO MAIN Eda am a om om om ee = 2 a | i if wet eraoretrag : : Es Po ii , Ee Si : ; a = ie n| 5 2 iHS =H hm em em |Ho ; HEpeeo !i e ee e e 3 : : i i me i | E EE jiPE E rhA e emEanH e e tEEE te e aRse e aRae H eEee Es a0 ifl| g: i WILDLIFE INTERNATIONALT, D. BACK TO MAIN PROJECT NO.: 454A-116 CRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 DATE: 5/15/00 TOXICANT : u2723 SPECIES: Xenopus --_ RAW DATA: - Concentration ( w /1) .oo 2.00 2.83 4.73 7.90 14.70 24 60 TEST NUMBER: 1 DURATION: Number Exposed 100 50 50 50 50 50 50 Mortalities 2 5 6 19 50 SPEARMAN-KARBER TRIM: 1.01% SPEARMAN-KARBER ESTIMATES: IC50 : 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 13.77 12.38 15.31 96 h A4-21 WILDLIFE INTERNATIONAL, LTD. - 79 - BACK TO MAIN PROJECT NO.: 454A-116 TRIMMED SPEARMAN-KARBER METHOD. U2723-ASJ \ - VERSION 1.5 DATE: 5/15/00 TOXICANT : U2723 SPECIES: Xenopus TEST NUMBER: 1 DURATION: RAW D-A-T-A-: Concentration ( w /1) .oo 2.00 2.83 4.73 7.90 14.70 Number Exposed 99 49 48 45 44 31 Mortalities 4 4 7 10 11 20 SPEARMAN-KARBER TRIM: 36.98% 96 h SPEARMAN-KARBER ESTIMATES: EC50 : 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 12.08 10.00 14.58 NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING. .........A.D.J.U.S.T.M.E.N.T.S..W.E.R.E..M.A..D.E..P.R.I.O.R..T.O..S.P.E.A.R.M.A.N.-.K.A.R.B..E.R.E..S.T.I.M.A.T.I.O.N............ T I= m/EC!jO= I3.17/12.082 in14 A4-28 WILDLIFE INTERNATIONALL, TD. - 80 - BACK TO MAIN PROJECT NO.: 454A-116 Title: u2723:Assay 1: embryo length data (mg) File: ASSAYlLE. Transform: NO TRANSFORMATION D = 0.6683 W = 0.9649 Critical W = 0.8250 w = 0.8740 (alpha = 0.01 (alpha = 0.05 , , N N = = 14) 14) Data PASS normality test (alpha = 0.01). Continue analysis. A4-29 WILDLI FE INTERNATIONAL, LTD. - 81 - BACK TO MAIN PROJECT NO.: 454A-116 Title: File: u2723:Assay 1: embryo length data (mg) ASSAYlLE Transform: NO TRANSFORMATION Bartlett's Test for Homogeneity of Variance Calculated B1 statistic = 2.3953 (p-value = 0.7922) Data PASS B1 homogeneity test at 0.01 level. Continue analysis. Critical B = 15.0863 (alpha = 0.01, df = 5) = 11.0705 .. (alpha = 0.05, df = 5) ............................................................................ Using Average Degrees of Freedom (Based on average replicate size of 2.33) Calculated B2 statistic = 2.2190 (p-value = 0.8181) Data PASS B2 homogeneity test at 0.01 level. Continue analysis. A4-30 WILDLIFE INTERNATIONALL, TD. - 82 - BACK TO MAIN PROJECT NO.: 454A-116 Title: u2723:Assay 1: embryo length data (mg) File: ASSAYlLE. Transform: NO TRANSFORMATION SOURCE DF ss MS F Between 5 0.7451 0.1490 1.7838 Within (Error) 8 0.6683 0.0835 Total 13' 1.4134 (p-value = 0.2224) Critical F = 6.6318 (alpha = 0.01, df = 5,8) = 3.6875' (alpha = 0.05, df = 5,8) , Since F < Critical F FAIL TO REJECT Ho: All equal (alpha = 0.05) A4-3 1 BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 83 - PROJECT NO.: 454A-116 Title: u2723:Assay 1: embryo length data (mg) File: ASSAYlLE. Transfo m : I Bonferroni t-Test - TABLE 1 OF 2 NO TRANSFORMATION Ho: ControlcTreatment TRANSFORMED MEAN CALCULATED IN SIG G-R-O-U-P- ....I.D..E.N.T.I.F..I.C.A.T.I..O.N ---M--EA-N----- --O-R-I-G-I-N-A-L--U-N-I-T-S--- t--S-T-A-T- 0-.-0-5 1 con 8.5900 8.5900 2 2.00 mg/l 8.2900 8.2900 1.1985 3 2.83 mg/l. 8.8000 8.8000 -0.8390 4 4.73 mg/l 8.5100 8.5100 0.3196 5 7.90 mg/l 8.7050 8.7050 -0.4594 ...6................1.4...7.0...m.g./l .---8-.-0-75-0------------8-.0-7-5-0-------2-.-0-57-5-------------- Bonferroni t critical value = 2.8965 (1 Tailed, alpha = 0.05, df = 5,8) Title: u2723:Assay 1: embryo length data (mg) File: ASSAY 1LE. Transform: NO TRANSFORMATION A4-32 WILDLI FE INTERNATIONAL, LTD. - 84 - BACK TO MAIN PROJECT NO.: 454A-116 APPENDIX 5 ASSAY 2 RAW DATA SHEETS FOR FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) AND 6-AMINONICOTINAMIDE REFERENCETOXICANT FOR CHEMICAL U2723 Note:The nominal concentrationsof 1.9,3.2,5.4,9.0, 15.0 and 25.0 mg/L in the raw data correspond to the nominal concentrations of 1.82,3.07, 5.19,8.64, 14.40 and 24.00 mg a.i./L in the report. A5-1 BACK TO MAIN f|i =5d= Er -- == | ETEEEE | | = Eset : i EE eek | i =E=e m EEE e=-- | EE : iH FatEe = e Hoh = =i= | i Sissies i| Eg sH sniE te= E H=E= E= || | 1 EEariEera LEE cal -- : = cr cr e Se 25 See1 EE Ho rr Sep -- 1 | i: || 7 t - t 111 = 3 ; bof Il :Z WILDLIFE INTERNATIONALLT, D. - 86 - I I 4 1 I 1 I I 1 I 1 1 1 1 8 II 1 1 1 FORM 8C: FETAX MORTALITY DATA 1 Mortality Reviewed .y BACK TO MAIN PROJECT NO.: 454A-116 1 Oatr BACK TO MAIN f meme mmm BACK TO MAIN :| Iif i re Ee neDeTa ot &k oicthet nSlhteeloLsy rer : z: :: ||{ E E Em E e a E e mdgo of| akESisumEsteaaisaIasnss= as : gL 8. 2 s =a ee iI e ee ee n=SE ToPl EEE EE i | EEEe FEARe HHH in Ih_ e BACK TO MAIN B F T: a p t2 1 BACK TO MAIN L&E LE IE BACK TO MAIN ---- g | i : i;|Is,fEr rEe grorr"eDr bEe--Ee e--nSha alln ! i | i| 1 [E =E f E me mI me m e e e H e= Siii:{ ks:f : i f!H x = Ses = Ee = E Mo e ee e 1 iF { E E HiEien nEse csesEe stEstten o . | E ESiitiEse sssaasEFEsRERnEnRnt an ff SSL | BACK TO MAIN oe z a iH : Ee ii r meni 0 eno aDTt ED C cnnSE Ts ; f2E} = - :` i : | EEE - 4H||EB= IE EE e r Eisse ese ae tese tis iiql|; ! he Te Ea es 1 HH Fo HH . | Eee TT HHH HEE = HE f/f : BACK TO MAIN I \D W I BACK TO MAIN I W P I BACK TO MAIN 8 :' sG s5: 3 2 P I E5 BACK TO MAIN BACK TO MAIN tS I EQ BACK TO MAIN bE Be Be Be be fe be Re ee _-- z 6 i ; ii f ---- 12. Dub W22 cSn tnTyn :: 2j ; EE [ETICT eT 7 a iI ii |(= E EEifie Eaeesse aettttnmnanns ii1H2 :5 5:f iAi E he e e e 5 : ! See i! E E SlEse tEaEste eE esmeieeies I | E EE ee eee | un fii `: \ - - - --a --- | " BACK TO MAIN J i |i far v3erane-- s _ F oo R B 238: ee | & EEE=EHi8z| BiH ! E = = -- ET - Hi a F--E_-- cL e T e EgHgz3 |H | = E = = Hi = aEE e E s i Eee SEE 3: SHH EHH= i 0 [1 n Z 39 |::22] dg|g $ 3 BACK TO MAIN oo 25 75 jt i 3g {|i| e E soasnEaJon B wnmaeDniR alTTZo2bETEoT C PocEnTStEe i ETE yP EEE Ea EE 25z2 325 2 i | EE E E hhT l E mk mE --e --E E --e -------- |5_655 : 0o UN |} o|| E E EE EE E E E EE EE E E E Ee2f6 d ii ii ||| || CEE E Emm H E I R EE A EE E EEE EEE E E e H || 3 2> || E E EE F e re E A e E eEe 1 [ T-- EH | 25 an ff il | | 3 BACK TO MAIN E 9 BACK TO MAIN P WILDLIFE INTERNATIONALL, TD. - 103 - ?* \os 3 96 Hour Test Data BACK TO MAIN PROJECT NO.: 454A- 116 \.qI+- W723 .Temperature ( O C ) D.O.(mglC) t PH . Conductivity 1Alkalinity Hardness I Day 0 OH 24.0 Day 1 Old New 24.0 W D Day2 -1 Day3 Old New I Old New 24.0 240 124.0 240 Day 4 Old B.0 , 8'2 J3.3 ,8.2 . %8 18.i 176 8.2 7.6 7.24 7.17' 721 7 2 2 (7.23 17.2.0 721 7.21 ' I I I I I I I I I I I I . .I 1 I .. I I I I I I I I I -- -Temperature ( " c )24.-0. 0.0.(mglL) Q.1 PH Conductivity 7\31 Alkalinity Hardness 24.0 a.0 24.0 24.0 124.0 24.0 Q O 8.i 7.C 8.017.5 8.\ 7.2\ 7.a 73 3 1,3017.~27 2 6 I 24.0. 7,s . 7.20 I h5-20 WILDLIFE INTERNATIONALLT, D. - 104- * &,.A* 2 96 Hour Test Data Page of 3 BACK TO MAIN PROJECT NO.: 45412-116 9.0q1L Oay 0 U2723 0 H TemPerature ( " c ) 24.0 0.0. (mglC) 8.1 fJH 1.37 - Conductivity Alkal.inity Hardness t I I Day 1 Old New 24,o 24.0 I8.i 8.t Il.Zq 7.35 I I I I Day 2 I Old Nsw 1 1 3.024.0 Day3 . Old New Z4,O 7 42 Day4 Old % .O 7.2 18.0 13.7 8.0 7.6 7.24. 17.30 17.24 7.36 7.33 I . I I1I I I I I I I I 1. A!i-21 WILDLI FE I NTERNATI ONAL, LTD. - 105 - 9Pa6gHe_o3u_roTfA est Data BACK TO MAIN PROJECT NO.: 454A-116 (Alkalinity Hardness I I I II I II I D.O. (mg/L) Day 0 OH Day 1 Old I New Day 2 Old I New Day3 Old I New Day4 Old I I I I I I I Temperature ("C) 0.0. (mglL) : PH Conductivity Alkalinity Hardness Day0 OH -. Day1 Old I New _. Day 2 Day3 I Day4 Old 1 New Old I New I Old I I I I I I I I I I I I I I I I '. I As-22 WILDLIFE INTERNATIONAL, LTD. - 106- BACK TO MAIN PROJECT NO.: 454A-116 -2!-I f - I Day0 (b- A 4 IOH Temperature ( " c ) 240 0.0. (rng/L) 8.t PH 7.03 Conductivity Alkalinity Hardness I I I 0ay1 OZYZ Day3 I ~ a y d I I I Old New Old New Old New Old I 24.0 24.0 29.0 24.0 124.0 24.0 M.0 I 7.9 8.017.7 8.0 I7,7 8.0 7.b I 1.00 7.08 7.07 17.14 17,03 7.07 b.98 I' II -1 *I I . 'I I * ' I I II .I Day 0 0.H IDay 1 Old New I Oay 2 Oay 3 I .I Old N w Old New Day 4 Old I Ttmperature ( O C) -. I I 0 0. (mg/L) I \ PH I I Conductivity I 1 AIkalinity I Hardness I1 , I I I I 85-23 BACK TO MAIN WILDLI FE INTERNATIONAL, LTD. PROJECT NO.: 454A- 116 - 107- Chemical U2723- Definitive Test 2 Embryo Length Measurements (mm) Page 1of 3 Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 A Control 9.46 9.23 9.58' 8.66 8.83 8.59 8.84 8.58. 8.46 9.57 8.6 9.53 9.21 9.2 8.63 9.1 8.56 8.57 9.02 9.3 9.1 8.82 8.49 8.35 9.25 B Control 8.71 8.48 8.94 9.52 8.72 9.28 8.52 9.7 8.62 8.97 8.94 8.87 8.67 8.7 8.98 8.98 8.83 8.42 9.23 9.04 8.22 8.17 8.47 8.53 8.82 C Control 8.71 8.41 8.61 7.68 9.12 8.9 8.9 8.78 8.81 8.85 9.02 9.18 9.16 8.42 9.7 8.93 8.53 8.5 9.31 8.97 9.65 9.28 8.98 9.77 8.44 D 1.9 9.19 8.99 9.17 9.63 9.42 9.55 9.6 8.63 9.03 8.81 8.13 8.86 8.75 8.34 9.47 9 9.17 8.84 8.48 7.88 9.14 8.21 8.49 8.4 mglL A 1.9 7.88 7.95 7.97 8.6 9.07 8.5 8.53 8.44 8.63 8.9 8.56 8.84 8.49 8.79 8.16 8.56 8.93 8.67 6.34 8.28 8.5 7.66 8.47 8.13 8.8 mglL B 8.73 8.85 9.06 8.69 8.66 8.22 9.03 9.31 8.76 9.04 8.44 8.34 9.06 8.1 1 6.83 8.39 8.49 7.8 8.85 7.78 8.17 8.43 6.95 I 9.69 8.73 Mean Min Max Stand. Dev. 8.9412 8.35 9.58 0.3858899 8.8132 8.17 9.7 0.3679982 8.9044 7.68 9.77 0.461 2039 8.8825 7.88 9.63 0.48821 1 8.386 6.34 9.07 0.5531 35 8.4964 6.83 9.69 0.6562893 AS-24 BACK TO MAIN WILDLIFE I NTERNATIONAL, LTD. PROJECT NO.: 454A- 116 - 108 - Chemical U2723-Definitive 2 Embryo Length Measurements (mm) Page 2 of 3 3.2mglL A 3.2mglL B 5.4mglL A 5.4mglL 8 9.0mglL A 9.0mg1L B 1 7.7 8.54 8.73 7.03 7.4 7.3 2 8.98 9.23 8.42 9.07 7.64 8.85 3 8.48 9.17 9.03 9.01 8.25 8.39 4 7.95 8.54 8.88 9.04 7.93 8.09 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 8.42 8.42 8.25 7.48 8.48 8 8.13 8.31 7.96 8.95 7.34 7.83 9 8.73 8.61 8.98 8.28 7.73 8.23 10 8.73 8.66 9.24 8.48 7.32 7.97 11 7.66 8.27 9.14 8.21 7.55 8.19 12 7.86 9 8.39 9.26 7.33 7.31 13 8.38 8.12 8.69 9.14 8.1 I 8.06 14 8.32 8.35 8.26 8.18 7.92 7.51 15 8.3 8.47 8.53 8.55 8.21 8.36 16 8.24 8.04 8.69 7.79 8.3 7.47 17 8.32 8.93 9.32 9.68 8.38 9.67 18 8.54 8.45 8.62 9.65 7.79 8.3 19 8.06 8.52 8.74 9.22 8.87 7.37 20 8.61 9.24 8.03 8.86 8.35 7.98 21 7.65 9.18 7.94 8.3 8.65 7.04 22 9.63 9.47 9.22 9.07 7.97 7.8 23 8.58 9.46 6.73 24 8.52 25 Mean Min Max Stand. Dev. 8.42 8.7095455 8.6879167 8.7436364 7.9409091 7.9204348 . 7.65 8.04 7.94 7.03 7.32 6.73 9.63 9.47 9.46 9.81 8.87 9.67 0.50842 0.4093139 0.4185792 0.6608259 0.4393167 0.6510933 115-25 BACK TO MAIN WILDLIFE INTERNATIONALL, TD. PROJECT NO. 454A-116 - 109 - Chemical U2723- Definitive Test 2 Embryo Length Measurements (mm) Page 3 of 3 6-Aminonicotinamide 15 mglL A 15 mglL B 25 mg1L A 25 mglL B 5.5 mglL A 5.5 mglL 8 1 7.5 7.58 7.91 6.84 7.34 7.02 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 7.98 7.03 6.64 7.29 7.26 7.16 7 8.31 6.77 6.93 7.59 7.24 8 8.23 7.96 7.67 6.88 7.33 9 7.73 7.86 7.69 7.4 7.09 10 7.08 6.73 7.13 7.77 11 7.48 7.84 7.21 7.91 12 7.77 7.19 7.44 8.09 13 7.96 6.53 7.07 7.5 14 7.99 7.67 7.55 15 7.75 7.14 7.35 16 6.78 6.98 7.22 17 7.34 7.49 7.61 18 7.59 7.31 7.42 19 7.22 7.3 6.8 20 7.5 8.1 6.76 21. . 7.51 7.12 7.14 22 8.39 7.57 7.37 23 7.38 7.26 24 8.1 25 Mean Min Ma Stand. Dev. 7.8904545 6.78 9.29 0.6280846 7.1269231 6.28 7.96 0.5508688 7.5022222 6.64 8.18 0.4725669 7.2683333 6.84 7.67 0.2744024 7.41 6.68 8.1 0.3776702 7.2804348 6.76 8.09 0.3482616 AS-26 BACK TO MAIN f t I c c 0 I I BACK TO MAIN B 9 P;i I WILDLIFE INTERNATIONALL, TD. - 112- TRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 BACK TO MAIN PROJECT NO.: 454A-116 DATE: 5/22/00 TOXICANT : U2723 SPECIES: XENOPUS TEST NUMBER: 2 DURATION: R-A-W- D-A-T-A-: Concentration (MG/L) .oo 1.91 3.04 4.82 7.97 13.30 23.10 Number Exposed 100 50 50 50 50 50 50 Mortalities 1 0 5 4 5 15 35 SPEARMAN-KARBER TRIM: 30.20% SPEARMAN-KARBER ESTIMATES: LC50 : 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 17.61 15.53 19.96 96 H . .. AS-29 BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 113 - PROJECT NO.: 454A-116 PRIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 U2723-R s 2~-. V I ~ ~ r n a , t mC sE C ~ ) DATE: 5/22/00 TOXICANT : U2723 SPECIES: XENOPUS TEST NUMBER: 2 DURATION: 96 H --- ---- RAW DATA: Concentration Number Mortalities (MG/L) Exposed .oo 99 4 1.91 50 4 I 3.04 45 2 4.82 46 5 7.97 45 9 13.30 35 13 23.10 15 10 SPEARMAN-KARBER TRIM: 34.74% SPEARMAN-KARBER ESTIMATES: EC50 : 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 17.57 13.49 22.88 NOTE: MORTALITY PROPORTIONS WERE NOT MONOTONICALLY INCREASING. ADJUSTMENTS WERE MADE PRIOR TO SPEARMAN-KARBER ESTIMATION. ----------------------.-------------------------------------------------------- AS-30 WILDLI FE INTERNATIONAL, LTD. - 114- Title: u2723: embryo lengths- assay 2 File: ASSAY2LE. Transform: D = 0.3737 W = 0.9179 Critical W = 0.8440 (alpha = 0.01 I N = 16) W = 0.8870 (alpha = 0.05 I N = 16) BACK TO MAIN PROJECT NO.: 454A-116 NO TRANSFORMATION As-3 1 BACK TO MAIN WILDLIFE INTERNATIONALT, D. - 115 - PROJECT NO.: 454A-116 Title: File: u2723: embryo lengths- assay 2 ASSAY2LE. Transfo m : NO TRANSFORMATION Bartlett's Test for Homogeneity of Variance Calculated B1 statistic = 12.7727 (p-value = 0.0468) Data PASS B1 homogeneity test at 0.01 level. Continue analysis. Critical B = 16.8119 (alpha = 0.01, df = 6) = 12.5916 (alpha = 0.05, df = 6) Using Average Degrees of Freedom (Based on average replicate size of 2.29) Calculated B2 statistic = 9.8138 (p-value = 0.1327) Data PASS B2 homogeneity test at 0.01 level. Continue analysis. AS-32 WILDLIFE INTERNATIONALL, TD. - 116 - Title: u2723: embryo lengths- assay 2 File: ASSAY 2LE. Transfo m : BACK TO MAIN PROJECT NO.: 454A-116 NO TRANSFORMATION A5-33 WILDLI FE INTERNATI ONAL, LTD. - 117 - BACK TO MAIN PROJECT NO.: 454A- 116 Title: u2723: embryo lengths- assay 2 File: ASSAY 2 LE. Transfonu: NO TRANSFORMATION ......B.o.n.f.e.r.r.o.n.i..t.-.T.e.s.t.....-....T.A.B.L.E..1..O.F..2..............Ho.:...C.o.n.t.r.o.l.<.T.r.e.a.t..m.e.nt TRANSFORMED MEAN CALCULATED IN SIG G-R-O-U-P- ....I.D.E..N.T.I.F.I..C.A.T.I.O..N ---M-E-A-N----- --O-R-I-G-I-N-A-L--U-N-I-T-S--- t--S-T-A-T- 0-.-0-5 1 control 8.8825 8.8825 2 1.91 mg/l 8.4450 8.4450 2.4793 3 3.04 mg/l 8.5650 8.5650 1.7992 4 5 4.82 mg/l 7.97 mg/l 8.7150 7.9300 8.7150 7.9300 0.9492 5.3977 * 1 6 13.3 mg/l 7.5100 7.5100 7.7778 * ...7................2.3..1...m.g./.l........7...3.8.50................7...3.8.5.0..........8...4.8.6.1. * Bonferroni t critical value E: 2.9333 (1 Tailed, alpha = 0.05, df = 6,9) Title: u2723: embryo lengths- assay 2 File: ASSAY2LE. Transfo m : NO TRANSFORMATION ......B.o.n.f.e.r.r.o.n.i..t.-.T.e.s.t.....-....T.A.B.L.E..2..O.F..2.............H.o.:..C..o.n.t.r.o.l.<.T.r.e.a.t.m.e.n.t NUM OF #IN SIG DIFF % OF DIFFERENCE G-R-O-U-P- ....I.D..E.N.T.I.F..I.C.A.T..I.O.N -R-E-P-S--- -(-I-N-O-R-I-G-.--U-N-I-T-S-)- C-O-N-T-R-O-L- F-R-O-M--C-O-N-T--R-O-L 1 control 4 2 1.91 mg/l 2 0.5176 5.8 0.4375 3 3.04 mg/l 2 0.5176 5.8 0.3175 4 4.82 mg/l 2 0.5176 5.8 0.1675 5 7.97 mg/l 2 0.5176 5.8 0.9525 6 13.3 mg/l 2 0.5176 5.8 1.3725 ...7...............2.3...1...m.g./.l......2..............0...5.1.7.6..........5..8.........1...4.9.75 As-34 WILDLI FE INTERNATIONALLT, D. - 118- BACK TO MAIN PROJECT NO.: 454A- 116 APPENDIX 6 ASSAY 3 RAW DATA SHEETS FOR FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) AND 6-AMINONICOTINAMIDE REFERENCE TOXICANT FOR CHEMICAL U2723 Note: The nominal concentrations of 1.9,3.2,5.4,9.01, 5.0and 25.0 mg/L in the raw data correspond to the nominal concentrations of 1.82,3.07, 5.19, 8.64, 14.40and 24.00mg a.i.R in the report. A6-1 f 9 tZ BACK TO MAIN WILDLI FE INTERNATIONAL, LTD. - 120 - BACK TO MAIN PROJECT NO.: 454A-116 1 1 I i 1 I I 1 I I I 1 Reviewed (ly Oace 1 I BACK TO MAIN mm mmm mm mmm 5 5 i fl cma ee oe 3 z :2 5 Jerre 3 SE SY 53 | EEE 3 g E VE||Ee EeE EeE e E EE ES || |EE E ee EEeeeE E e EEEe] ct |{| E E = HE e eee e e eee + IE EEEEHH . ait | fI i d: |--- mm I _-------- BACK TO MAIN | EEE = Nn = Te ;| jamsteas ths esemB B13 mies || 2; iT Ee" i: me = || | {| = I= BifEiEiChHssEeRsRsEsEss -- = | |ii. = = f= = E---- {i ee a Mf i HEE iHenH e eH e s Cem mmm ;5 BACK TO MAIN A b | E EE mmmE| 3 iq 3 z Is # le re r melDiaAbe TLDRoncSEil lnelTeemi2e5 n z z 3 5 JdE|||E E EEE EE s fT i| || | E E E EE e e ee e e e EEE eee ESSEisEIHSSSCCoREEEsEREsEn . i an folf| 3: BACK TO MAIN 8 BACK TO MAIN RS - iH oo [i| BmaEli e soit SER es || hol ii, l} S B= H Eph EH a Es HEiit| || i = = ee i ii = = } 1_ {| 2 ; = = BE e EeEe en0 EEE=H = Z m=H S s | do i = H HE eH e I : BACK TO MAIN . BACK TO MAIN 4az..? BACK TO MAIN I I h, 00 I ? 8 .G .8 2 4 $ a C D BACK TO MAIN BACK TO MAIN P ii BACK TO MAIN La | - = a. -- -- - 5g 3 " nn dl DEsbeTt3 Feato s]o 2g a Z 8 3 | -- : ==--E iEFellFa]lhe o a| e2.e=]] $32 ||| | -- |C-- er oe = : ; me Fis: 8 | | 5 S SE FL E E Ee 3 i |i -- .. 5 ES = =| H EEi ssEsEE EH e stH = x =i : fl : mm - a _ 5s -- BACK TO MAIN : ]i [-- | pn ited 3: z 2 2 5 i Em Fl =e ot s i3 r Ie m HEE= 3 Fz T i3 ll E Er e g2iz3gz 3 i 1 Ee = "8z i' i i- = = = it = = e SEE = EE he He Ee ain Il a BACK TO MAIN I w w w I BACK TO MAIN 2aE n B w P I t21 BACK TO MAIN F 2 ..0) 3: f.p irit 9 P f 9 1 k t r i Dlrh iYlrl1 No. BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 137 - BACK TO MAIN PROJECT NO.: 454A- 116 96 Hour Test Data Efflu UGroundwaterlToxicant:f , h \ m \ uL723 DTn,hUc,Tist 3 Date: 513 -$!6/m Ana Iy st: Species: Age at Start of Test: I I I I Alkalinity Hardness 86-20 Wi LDLI FE INTERNATIONAL, LTD. - 138 - 96 Hour Test Data Page 2of 3 BACK TO MAIN PROJECT NO.: 454A-116 9.0m?- Day 0 OH IDay 1 Old New 1 I Day 2 Day3 Old New Old New Day4 Old Conductivity Atkalinity Hardness AIkalinity Hardness BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 139 - PROJECT NO.: 454A-116 96 Hour Test Data Page A of A 25.0qIL Temperature ( " C ) 0.0. (mg/L) PH Conductivity AI kal inity Hardness Day 0 0 H 24.0 8.2 1.31 Day 1 Day 2 Old I New Old I New 24.0 24.0 24.0 24.0 2.3 8.2 7.0 8.1 1.16 1.S 7.23 7.32 Day3 Old 1 New 24.0 24.0 7.0 8.0 1.25 1.35 Day4 Old 24.3 7.4 7.8 86-22 BACK TO MAIN WILDLI FE I NTERNATIONAL, LTD. PROJECT NO.: 454A- 116 - 140 - Chemical U2723- Definitive Test 3 Embryo Length Measurements (mm) Page 1 of 3 Control A Control B Control 1 10.02 10.08 2 9.75 9.88 3 9.76 10.03 4 9.85 9.9 5 9.47 9.22 ' 6 9.59 9.59 7 9.06 9.48 8 9.38 9.88 9 9.32 9.28 10 9.67 9.88 11 9.7 9.09 12 9.71 9.84 13 9.64 9.04 14 9.5 9.88 15 9.59 9.38 16 9.44 9.99 17 9.88 9.46 18 9.38 9.74 19 9.76 9.51 20 8.98 9.52 21 9.37 9.67 22 9.38 9.52 23 9.65 10.23 24 9.59 9.15 25 8.95 9.8 C Control 9.74 9.05 9.31 8.93 8.85 9.09 8.87 8.92 8.54 9.62 9 9.44 9.79 9.91 9.54 8.47 9.83 9.38 9.82 10.02 9.22 9.58 ' 9.53 9.54 8.76 D 1.9 9.45 9.06 9.65 8.4 9.46 8.59 9.64 9.1 9.42 9.49 8.74 9.8 8.73 9.73 9.28 9.25 9.16 9.32 9.8 9.17 10.21 9.94 9.85 9.68 10.09 mglL A 1.9 9.14 9.31 9.68 8.86 8.54 9.57 8.3 8.5 8.25 8.94 8.79 9.01 8.62 9.03 9.14 8.28 8.49 7.82 8.8 7.93 9.26 8.93 9.52 9.1 1 8.83 mg/L B 9.37 9.48 9.88 9.78 9.13 8.11 8.7 9.44 9.28 9.42 9.41 7.68 8.68 8.97 9.13 10.1 9.37 9.42 9.24 10.01 10.22 9.87 9.55 9.65 10.14 Mean Min Max Stand. Dev. 9.5356 8.95 10.02 0.2697388 9.6416 9.04 10.23 0.3274355 9.31 8.47 10.02 0.4414842 9.4004 8.4 10.21 0.4640535 8.826 7.82 9.68 0.4844327 9.3612 7.68 10.22 0.6033747 86-23 BACK TO MAIN WILDLIFE INTERNATIONALLT, D. PROJECT NO.: 454A-116 - 141 - Chemical U2723- Definitive Test 3 Embryo Length Measurements (mm) Page 2 of 3 3.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 mglL A 3.2 9.34 8.47 9.15 9.36 8.48 9.48 7.61 9.9 9.26 9.24 8.91 8.58 9.57 9.59 9.05 9.15 9.4 8.59 9.44 9.42 9.46 9.43 9.47 8.64 8.73 mglL 6 5.4 9.49 9.37 9.79 9.47 9.61 9.72 9.44 9.18 8.94 9.4 8.75 10.02 9.82 9.47 9.37 9.17 8.81 8.81 9.8 9.81 9.83 9.35 10.01 9.58 9.06 mglL A. 5.4 9.75 9.09 9.15 8.73 9.29 9.53 9.02 9.69 9.36 9.28 9.38 9.52 9.01 9.02 9.12 10.11 8.9 9.12 8.25 8.85 8.24 9.67 9.93 9.97 9.28 mglL 6 9.0 9.71 9.33 8.93 8.62 8.58 9.37 9.29 9.89 8.66 9.71 9.37 9.3 9.53 8.98 9.55 8.9 9.02 9.23 9.66 8.37 9,7 10 10.03 9.29 9.46 mglL A 9.0m g l i B 9.44 8.17 8.31 8.61 8.4 8.29 9.1 8.12 8.8 8.22 8.71 8.27 7.97 8.87 9.08 7.86 8.31 9.38 6.15 8.33 8.43 7.21 9.33 7.68 9.24 8.31 9.17 9.25 9.03 7.78 9.14 7.76 8.79 7.04 8.48 8.93 9.3 8.66 8.97 9.36 9.07 7.56 9.35 Mean Min Max Stand. Dev. 9.1088 7.61 9.9' 0.5008852 9.4428 8.75 10.02 0.37092M 9.2504 8.24 10.11 0.4712526 9.2992 8.37 10.03 0.451 229 1 8.7013043 6.15 9.44 0.7362331 8.305 7.04 9.38 0.m44a3 A6-24 BACK TO MAIN WILDLI FE INTERNATIONAL, LTD. PROJECT NO.: 454A-116 - 142 - Chemical U2723- Definitive Test 3 Embryo Length Measurements (rnm) Page 3 of 3 6-Arninonicotinarnide 15 mglL A 15 rnglL B 25 mglL A 25 mg/L B 5.5 rnglL A 5.5 mglL B 1 8.9 7.17 7.99 9.19 8.6 6.87 2 8.24 9.05 7.49 6.14 7.79 9.14 3 9.17 8.22 8.23 8 8.1 8.21 4 7.89 7.44 8.17 7.83 9.01 7.7 5 8.44 8.12 8.03 6.51 7.26 7.76 6 7.56 9.27 8.44 7.99 8.04 7 7.59 7.04 8.14 8.3 8 7.69 7.62 9 . 8.2- 8.88 8.6 7.66 8.31 7.51 10 7.64 8.12 8.3 7.43 11 7.23 8.5 8.01 8.6 12 8.17 7.98 7.71 8.14 13 7.89 8.23 7.87 8.36 14 8.5 8.3 8.4 7.67 15 6.98 7.84 16 7.14 7.59 17 8.8 8.64 18 7.77 8.1 19 7.91 8.33 20 8.28 7.21 21 8.49 7.09 22 8.22 7.63 23 7.88 8.15 24 7.57 8.37 25 7.29 Mean Min Max Stand. Dev. 8.0792857 7.23 9.17 0.5452941 8.1385714 7.04 . 9.27 0.6656386 8.0583333 7.49 8.44 0.32127.35 7.534 8.0168 6.14 6.98 9.19 9.01 I.2287107 , 0.5144959 7.9308333 6.87 9.14 0.5321974 66-25 BACK TO MAIN DME ER Ee Ae mmm mm -- -- -- g 5 5A 5 . 3 3 iB H| or ere ernrn TTE rhDEeeTEe AaED Eco EaESdaETen o EeE e iTdepe])IH2z z5 i Ee LEE PDERT beR ee 2 C i ! [E Ee e EH eee38 eee gs o $ d 81 || |||| E E E EE e E EE reee E z PI| | E E E E ee reR et E e e E t e E| {| E t = C = em E H E E H ERmE t e errr | " 1 [EH Ee i if i I g an t BACK TO MAIN i EE dAi : ; i B= 3 - -| - -- a - 5 2 5 A z EERE i 2 2 i --------td____ Dab Tek3 oo aE HH 5 2 | TE Ho5Hz2 F 5 HH FP ! | = == = - Gs 32 | = | E = = D a = i IEEE - eHiE=s H E | J 0 [1 BACK TO MAIN - 145 - .XIMMED SPEARMAN-KARBER METHOD. VERSION 1.5 DATE: 5/22/00 TOXICANT : u2723 SPECIES: Xenopus TEST NUMBER: 3 DURATION: R-A-W- D-A-T-A-: Concentration (ms/l) .oo 1.93 3.27 5.25 8.26 14.00 23.90 Number Exposed 100 50 50 50 50 50 50 Mortalities 0 0 0 0 7 22 39 SPEARMAN-KARBER TRIM: 22.00% SPEARMAN-KARBER ESTIMATES: LC5 0 : 95% L O W E R CONFIDENCE: 95% UPPER CONFIDENCE: 15.26 13.13 17.75 96 h A6-28 WILDLIFE INTERNATIONALT, D. - 146 - BACK TO MAIN PROJECT NO.: 454A- 116 DATE: 5/22/00 TOXICANT : U2723 SPECIES: XENOPUS TEST NUMBER: 3 DURATION: R-A-W- D-A-T-A-: Concentration (MG/L) .oo 1.93 3.27 5.25 8.26 14.00 23.90 Number Exposed 100 50 50 50 43 28 11 Mortalities 2 3 2 3 6 11 8 SPEARMAN-KARBER TRIM: 27.83% SPEARMAN-KARBER ESTIMATES: EC50 : 95% LOWER CONFIDENCE: 95% UPPER CONFIDENCE: 16.79 12.35 22.82 96 H A6-29 WILDLI FE INTERNATIONAL, LTD. - 147 - Title: U2723:ASSAY 3: embryo lengths (mg) File: ASSAY 3LE. Transform: BACK TO MAIN PROJECT NO.: 454A-116 NO TRANSFORMATION D = 0.5007 W = 0.9545 Critical W = 0.8440 (alpha = 0 . 0 1 I N = 16) W = 0.8870 (alpha = 0.05 I N = 16) Data PASS normality test (alpha = 0.01). Continue analysis. -. A6-30 BACK TO MAIN WILD LIFE INTERNATIoNAL, LTD. - 148 - PROJECT NO.: 454A-116 Title: U2723:ASSAY 3: embryo lengths (mg) File: ASSAY3LE. Transfo m : NO TRANSFORMATION ................B.a.r.t.l.e.t.t.'s....T.e.s.t..f.o.r..H.o.m.o.g.e.n.e.i.t.y..o.f...V.a.r.i.a.n.c.e................ Calculated B1 statistic = 5.7971 (p-value = 0.4463) Data PASS B1 homogeneity test at 0.01 level. Continue analysis. Critical B = 16.8119 (alpha = 0.01, df = 6) = 12.5916 (alpha = 0.05, df = 6) Using Average Degrees of Freedom (Based on average replicate size of 2.29) Calculated B2 statistic = 5.0874 (p-value = 0.5327) Data PASS B2 homogeneity test at 0.01 level. Continue analysis. A6-3 1 WILDLIFE INTERNATIONALL, TD. - 149 - BACK TO MAIN PROJECT NO.: 454A- 116 Title: U2723:ASSAY 3: embryo lengths (mg) File.: ASSAY3LE. Transfo m : NO TRANSFORMATION SOURCE DF ss MS F Between 6 5.9602 0.9934 17.8546 Within (Error) 9 0.5007 0.0556 Total 15 6.4609 (p-value = 0.0002) Critical F = 5.8018 (alpha = 0.01, df = 6,9) = 3.3738 (alpha = 0.05, df = 6,9) Since F > Critical F REJECT Ho: All equal (alpha = 0.05) A6-32 BACK TO MAIN WILDLIFE INTERNATIONALT, D. - 150 - PROJECT NO.: 454A-116 Title: U2723:ASSAY 3: embryo lengths (mg) File: ASSAY3LE. Transform: NO TRANSFORMATION ......B.o.n.f.e.r.r.o.n.i..t.-.T.e.s.t.....-....T.A.B.L.E..1..O.F..2.............H.o.:..C..o.n.t.r.o.l.<.T.r.e.a.t.m.e.n.t TRANSFORMED MEAN CALCULATED IN SIG G-R-O-U-P- ....I.D..E.N.T.I.F..I.C.A.T..I.O.N ---M-E-A-N----- --O-R-I-G-I-N-A-L--U-N-I-T-S--- t--S-T-A-T- 0-.-0-5 1 CONTROL 9 4725 9.4725 2 1.93 mg/l 9 1150 9.1150 1.7501 3 3.27 mg/l 9.2750 9.2750 0.9668 4 5 5.25 mg/l 8.26 mg/l 9.2750 8.5050 9.2750 8.5050 0.9668 4.7363 * 6 14.0 mg/l 8.1100 8.1100 6.6700 * ...7...............2.3...9...m.g./.l.......7...7.9.5.0...............7...7.9.5.0..........8...21.2.1... * Bonferroni t critical value = 2.9333 (1 Tailed, alpha = 0.05, df = 6,9) Title: U2723:ASSAY 3: embryo lengths (mg) File: ASSAY3LE Transfo m : NO TRANSFORMATION A6-33 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. - 151 - PROJECT NO.: 454A-116 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; 14.4mg 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; 14.4mg a.i./L (nominal concentration)exposure Assay 1; 8.64 mg a.i./L (nominal concentration)exposure WILDLI FE INTERNATIONLATLD,. BACK TO MAIN PROJECT NO.: 454A-116 Photo 1: ( 7 . 5 ~N) ormally 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. WILDLIFE INTERNATIONAL,TD. BACK TO MAIN PROJECT NO.: 454A-116 Photo 3: (7.5~M)alformations 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 coiIing. WILDLIFE INTERNATIONLATLD, . - 154- BACK TO MAIN PROJECT NO.: 454A-116 Photo 5: (15x) Malformation observed: Improper (loose) gut coiling. Photo 6: (15x) Malformation observed: Improper (loose) gut coiling. WILDLIFE INTERNATIONAL,TD. - 155 - BACK TO MAIN PROJECT NO.: 454A-116 Photo 7: ( 7 . 5 ~ )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. WiLDL~FE INTERNATIONLATLD, . BACK TO MAIN PROJECT NO.: 454A-116 Photo 9: ( 7 . 5 ~ )M:alformationsobserved: Anal edema; slight facial abnormalities; slightly curved notochord. Photo 10: (15x): Malformationsobserved: Anal edema; slight facial abnormalities; slightly curved notochord. WI LDLlFE INTERNATIONAL, LTD. - 157 - BACK TO MAIN PROJECT NO.: 454A- 116 Picture 11: (7.5~M)alformations 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). WILDLIFE INTERNATIONALL,TD. - 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 WILDLIFE INTERNATIONALL,TD. - 159 - PROJECT NO. 454A- 116 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: Enclosedis a copy of the revisedfinal report. All of the suggested questions or correctionswere addressed. Although LC50, EC50 and MClGvalues changed when actual mean measured concentrations were used, the TI 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 referencetoxicant question, the 2500 mglL concentration has consistentlycaused high mortality at our laboratory. The low reference toxicant concentrationhas also beenvery consistent, with low mortalityand approximately 50% malformed embryos. I feel that the mortalityobserved inthe high reference toxicant concentrationdoes not compromisethe results of these three assays in any way. Other laboratoriesworking with FETAXoflen have similar high mortality in the 2,500 mg/L concentration. Over the last 5 years our laboratoryhas performed numerous standardand modifiedFETAXassays. Ineach 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 andWildlife Service. At the 2,500mgk referencetoxicant concentration,there was 100% mortality. In the FETAX solution exposures, greater than 90% of the embryossurvived (andcompletedmetamorphosis)14O-dpost-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 reviewedinitiallyby our former QAQC personat the lab. Since she was no longer a WREC employee, she did not knowwhether 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. Afler 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 correctionslet me know. Iwill make any changes you deem necessary. Sincerely, SteveTurlev A , BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 160 - PROJECT NO.: 454A- 116 APPENDIX I11 The Analysis of PFOS in Frog Embryo TeratogenesisAssay -Xenopus (FETAX) Solutions in Support of Wildlife International,Ltd. Project No.: 454A-116 BACK TO MAIN WILDLIFE INTERNATIONLATLD,. - 161 - REPORT APPROVAL PROJECT NO.: 454A-116 SPONSOR: 3M Corporation TITLE: PFOS: A Frog Embryo Teratogenesis Assay -Xenopus (FETAX) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-116 PRINCIPAL INVESTIGATOR: ScientZ MANAGEMENT: Director, Analytical Chemistry BACK TO MAIN WILDLIFE INTERNATIONALLT, D. - 162 - PROJECT NO.: 454A-116 Introduction Frog embryo teratogenesis assay-Xenopus (FETAX) solution samples were collected from three definitive assays designed to determine the effects of PFOS (Perfluorooctanesulfonate,Potassium Salt) to the embryos of the South African clawed frog (Xenopus laevis). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-116. The analyses of these FETAX solution samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLCMS). 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. identificationnumber 4526 upon receipt. The internal standard, a granular material, was identified as: lH, lH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 2761997-2. 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 of PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix I1 of Wildlife International, Ltd. protocol number 454/0 11299/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 4H 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 WILD LIFE INTER NATIoNAL, LTD. - 163 - PROJECT NO.: 454A-116 Concentrations of 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 CISanalytical 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 Ouantitation 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) of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFOS in the samples was determined by substitutingthe 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 WILDLIFE INTERNATIONALLT, D. - 164 - PROJECT NO.: 454A-116 for the mean procedural recovery of 97.2%. A representative ion chromatogram of 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 FE,TAX solution. Peak Area Ratio = Analyte Peak AredInternal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentrationof Internal Standard Internal Standard Concentration: 0,100 mg a.1.L Initial Volume: 0,100 mL Final Volume: 100 mL Dilution Factor: 1000 PFOS Peak Area: 1158987 Internal Standard Peak Area: 2 125037 Peak Area Ratio: 0.5454 Calibration curve equation. Slope: 1.9275 Intercept: 0.0260 Curve is weighted (l/x) PFOS (mg a.i./L) at Instrument = Peak area raStiloope(Y-intercept' x Internal Standard Concentration - 0.5454 - 0.0260 x 0.100 1.9275 = 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 WILDLIFE INTERNATIONALLT, D. - 165 - PROJECT NO.: 454A-116 PFOS m a.i./L in sample Percent of Nominal Concentration = PFos((:g nominal x 100 ---2264.9.05 x 100 = 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 of PFOS in test solution samples collected prior to initiation of exposure of the test organisms ranged from 112 to 141% of the nominal concentrations. Samples collected at test termination had a measured concentration range of 54.7 to 98.6% of nominal values. The sample from the abiotic 24.0 mg a.i./Ltreatment 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 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 (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 WILDLIFE INTERNATIONALLT, D. - 166 - PROJECT NO.: 454A-116 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 (Table 6). BACK TO MAIN Wi LD LIFE INTER NATIONAL, LTD. - 167 - PROJECT NO.: 454A-116 INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatographwith a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: Keystone Betasil CIScolumn (50 mm x 2 mm I.D., 3-prn particle size) 30C 5.OO minutes 220 pL/minute 70.0% Methanol : 30.0% NANOpure@Water containing 0.1% Formic Acid 5.0 pL 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 WILDLIFE INTERNATIONALLT, D. - 168 - PROJECT NO.: 454A-116 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A- 1 16-) MAB- 1 Sample Type Matrix Blank Sample Set Day 0 - lstAssay Measured Concentration of PFOS' (mg a.i./L) LOQ MAB-2 Matrix Blank Day 0 - 2ndand 31dAssay < LOQ MAB-3 Matrix Blank Day 4 - 1'' Assay < LOQ MAB-4 Matrix Blank Day 4 - 2ndand 31dAssay < LOQ I The limit of quantitation (LOQ) was 0.240 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor of the matrix blank samples (50). BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 169 - PROJECT NO.: 454A-116 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-116-) MAS-I MAS-2 MAS-3 Sample Set Day 0 - lstAssay Concentrations of PFOS (mg a.i./L) Fortified' Measured' 0.480 0.490 6.72 7.12 28.8 30.2 Percent Recovered2 102 106 105 MAS-4 Day 0 - 2ndand 3rdAssay 0.480 0.441 91.8 MAS-5 6.72 6.32 94.0 MAS-6 28.8 27.4 95.1 MAS-7 Day 4 - lstAssay 0.480 0.443 92.3 MAS-8 6.72 6.20 92.2 MAS-9 28.8 27.7 96.1 MAS- 10 Day 4 - 2ndand 3rdAssay 0.480 0.449 93.5 MAS- 11 6.72 6.77 101 MAS-12 28.8 28.1 97.6 Mean = 97.2 Standard Deviation = cv = N= 5.08 5.22% 12 1Concentrations were corrected for change in test substance purity (90.49% to 86.9%) per Certificate of Analysis dated September 7, 2000. *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 reDorting Dumoses BACK TO MAIN WILD LIFE INTER NATIONAL, LTD. - 170 - PROJECT NO.: 454A-116 Table 4 Measured Concentrations of PFOS in Frog Embryo Teratogenesis Assay-Xenopus (FETAX) Solution Samples - First Assay Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) Sample Number (454A- 116-) 1 8 Sampling Time (Day) 0 4 PFOS Measured Concentration''2 (mg a.i./L) < LOQ < LOQ Percent of Nominal3 -- -- 0.0 9 4 (Positive Control) 1.82 2 0 10 4 2.58 141 1.42 77.8 3.07 3 0 11 4 3.94 128 1.72 55.9 5.19 4 0 12 4 6.62 128 2.84 54.7 8.64 5 0 13 4 10.7 124 5.09 58.9 14.4 6 0 14 4 24.0 7 0 15 4 18.5 128 10.8 74.9 26.9 112 22.3 93 .O 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 of quantitation (LOQ) was 0.240 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor of 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 BACK TO MAIN WILDLI FE INTER NATIONAL, LTD. - 171 - PROJECT NO.: 454A- 116 Table 5 Measured Concentrations of PFOS in Frog Embryo Teratogenesis Assay-Xenopus (FETAX) Solution Samples - Second Assay Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-116-) 17 24 Sampling Time (Day) 0 4 PFOS Measured Concentration',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 0 28 4 5.45 105 4.18 80.7 8.64 21 0 29 4 8.43 97.6 7.5 1 86.9 14.4 22 0 30 4 14.5 100 12.1 84.3 24.0 23 0 31 4 23 .O 95.8 23.1 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 of the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor of 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 WILDLI FE INTER NATIONAL, LTD. - 172 - PROJECT NO.: 454A-116 Table 6 Measured Concentrations of PFOS in Frog Embryo Teratogenesis Assay-Xenopus (FETAX) Solution Samples - Third Assay Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-116-) 17 33 Sampling Time (Day) 0 4 PFOS Measured Concentration"* (mg a.i./L) < LOQ LOQ Percent of __ Nominal3 -_ 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 0 37 4 5.45 105 5.05 97.5 8.64 21 0 38 4 8.43 97.6 8.09 93.6 14.4 22 0 39 4 14.5 100 13.5 93.4 24.0 23 0 40 4 23.0 95.8 24.7 103 24.0 41 4 24.1 100 (Abiotic Control) I 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 of the lowest calibration standard analyzed (0.00480 mg a.i./L) and the dilution factor of 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. WILDLIFE INTERNATIONALT, D. - 173 - BACK TO MAIN PROJECT NO.: 454A-116 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX) SOLUTION Prepare matrix fortification samples by spiking the requisite volume of PFOS stock solutions directly into FETAX solution using gas-tight syringes and Class A volumetric flasks. Dilute matrix fortification and test samples into the range of 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 (dv). 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. Ampulate samples and submit for LCMS analysis Figure 1. Analytical method flowchart for the analysis of PFOS in frog embryo teratogenesis assayXenopus (FETAX) solution. WILDLIFE INTERNATIONALLT, D. - 174 - 1.00 7 0.90 1 0.80 1i BACK TO MAIN PROJECT NO.: 454A-116 2 I0.60 +0 n 7 .r( w 0.50 cd 0.40 0.30 7' i 0.20 1 0.10 1 L Figure 2. 0.00 0.10 0.20 0.30 0.40 0.50 Concentration (Ratio) A typical calibration curve for PFOS. Slope = 1.9275; Intercept = 0.0260; r = 0.9973. Curve is weighted (l/x). WILDLIFE INTERNATIONALT, D. - 175 - BACK TO MAIN PROJECT NO.: 454A-116 100 90 80 70 60 50 40 30 20 10 34 01 I I I 86 I I I I 190 263 I 1 1 I Figure 3. A representative ion chromatogram of a low-level (0.00480 mg a.i./L) PFOS standard. WILD LIFE INTER NATIONAL, LTD. - 176 - BACK TO MAIN PROJECT NO.: 454A-116 intensity: 180000 cps 41 0.69 8 1 1 2 1 1 6 1 2 0 1 2 4 1 281 Scan 1.36 2.02 2.69 3.36 4.03 4.70Time Figure 4. A representative ion chromatogram of a high-level (0.0480 mg a.i./L)PFOS standard. WILDLI FE INTER NATIONAL, LTD. - 177 - BACK TO MAIN PROJECT NO.: 454A-116 100- 9 0- 8 0- 7 0- 6 0- 5 0- 4 0- 3 0- 2 0- 1 0- 0 1530 5 3 I I I -I intensity: 180000 cps 108 137 168192 223 I 1 1 1 1 1 1 1 1 272 1 1 'igure 5 . A representative ion chromatogram of a matrix blank sample (454A-116-MAB-1). The arrow indicates the retention time of PFOS. WILDLIFE INTERNATIONAL, LTD. - 178 - BACK TO MAIN PROJECT NO. 454A-116 1009 00 0- 7 0- 6 05 04 03 0- 20- 1 0- o+-I 3 1.I I 192 165 6 8 104 140- I I I I I I I I 247 284 1 I I I 'igure6 . A representative ion chromatogram of a matrix fortification sample (454A-116-MAS-1, 0.48 mg a.i./L nominal concentration). WILDLIFE INTERNATIONALT, D. - 179 - BACK TO MAIN PROJECT NO.: 454A-116 intensity: 180000 cps 41 0.69 81 1.36 121 2.02 161 2.69 201 3.36 241 4.03 2 8 1 Scan 4.70Time 'igure7. A representative ion chromatogram of a test sample (454A-116-7, 24.0 mg a.i./L nominal BACK TO MAIN WILDLI FE INTER NATIONAL, LTD. - 180- APPENDIX IV 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 of the test substance be changed from 90.49%to 86.9%. BACK TO MAIN WILDLI FE INTER NATIONAL , LTD. - 181 - PROJECT NO.: 454A-116 APPENDIX V Personnel Involved in the Study The followingkey Wildlife International, Ltd. personnel were involvec in the conductormanagementof th~s 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