Document 7R8DwaQ6Jq6bGr9JMVqGOz0Na

A R i^ 'O ia &s -L PROTOCOL PFOS: A 7-DAY TOXICITY TEST WITH DUCKWEED (Lemna gibba G3) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines . OPPTS Number 850.4400 3M Lab RequestNo. U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue S t Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 September 17,1999 004532 Wildlife International, ltd -2- PFOS: A 7-DAY TOXICITY TEST W ITH DUCKWEED (Lemna gibba G3) SPONSOR: SPONSOR'S REPRESENTATIVE: S' TESTING FACILITY: STUDY DIRECTOR: 3M Corporation Environmental Laboratory 93S Bush Avenue St. Paul, M innesota 55106 Rochelle R. Robideau W ildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 Ku rtB rotta* Senior Aquatic Biologist LABORATORY MANAGEMENT: Henry O. Krueger, PhJD. Director o f Aquatic Toxicology & Non-Target Plants Proposed Dates: Experimental Start Date: Project No.: Test Concentrations: Test Substance No.: FOR LABORATORY USE ONLY , Experimental Termination Date: /// - Reference Substance No. (if applicable): PROTOCOL APPROVAL STUDY DIRECTOR LAB TORY MANAGEMENT PROTOCOL N O .: 454/091799/LEM 7D/SUB454 004533 3M LAB REQUEST NO. U2723 Wildlife International, ltd -3 - IN T R O D U C T IO N W ildlife International, Ltd. will conduct a seven-day toxicity test with the duckweed, Lemna gibba G3, for the Sponsor at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in the U.S. Environmental Protection Agency Series 850 Ecological Effects Test Guidelines OPPTS Number 850.4400 (1). Raw data for all work performed at W ildlife International, Ltd. and a copy o f the final report will be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in the final report. PURPOSE The purpose o f this study is to detennine the toxicity o f Perfluorooctane Sulfonic A dd, Potassium Salt (hereafter referred to as PFOS) to duckweed, Lemna gibba G3, under static test conditions. EXPERIM ENTAL DESIGN Fronds o f duckweed, Lemna gibba G3, will be exposed to a geometric series o f at least six test concentrations and a negative (culture medium) control for seven days. Nominal test concentrations will be chosen in consultation with the Sponsor based upon information such as rangefinding toxicity data, known toxidty information or physical/chemical properties o f PFO S.. Target concentrations will not exceed 100 mg/L or the solubility limit o f the test substance in water. Generally, each concentration o f PFOS used in the definitive test will be at least 50% o f the next higher treatment, unless information concerning the concentration-effect curve indicates that a different dilution factor would be more appropriate. Uniform, healthy looking plants w ill be removed from the stock culture for testing. Five plants totalling approximately 15 fronds will be added to each test chamber. Care will be taken to ensure that the number of fronds in each test chamber is approximately equal. Three "biological" replicates per treatment will be tested. One or mcxe additional "analytical"replicates will be included in the test, as needed, to provide test solution for concentration verification on Days 3 ,5 and 7 o f the exposure. An abiotic replicate at the highest concentration will be included in die test and will be sampled on Days 3 ,5 and 7 . In order to control bias, the position o f die test chambers will be determined by indiscriminate draw daily during the exposure period. No other potential sources o f bias are expected to affect the results o f the study. The test results will be calculated based on direct counts o f the numbers o f duckweed fronds. These measurements will be taken on Day 3, Day 5 and at the end of die te st Observations o f chlorosis, break-up o f duckweed colonies, changes in color, root destruction and other abnormal appearances will also be documented. An IC10, IC50 and IC90 values (e.g., the toxicant concentration that produces a 10%, 50% PROTOCOL NO.: 454/091799/LEM 7D/SUB454 004534 3M LAB REQUEST NO. U2723 Wildlife International, ltd -4 - or 90% reduction in frond number relative to the controls) will be calculated based on total frond numbers at Days 3 ,5 and 7 o f the exposure period. The no observed adverse effect concentration (NOAEC), which represents the highest concentration that induces no adverse effects on plant and frond production or appearance, will be determined based upon evaluation o f the statistical results, the dose-response pattern, and other observed effects. M ATERIALS AND METHODS TestSuhstance ^. Information on die characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International, Ltd. written verification that the test substance has been characterized according to GLPs prior to its use in the study; If written verification o f GLP test substance characterization is not provided to Wildlife International, Ltd., it will be noted in the compliance statemento f the final report. The attached form IDENTIFICATION O F TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. The Sponsor is responsible far all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end o f the study. Test Solution Preparation The desired concentrations o f PFOS will be obtained by preparing a single stock solution in medium and diluting the appropriate volume of stock solution with medium. The test solutions may also be prepared using secondary dilutions o f die primary stock, based upon die concentration desired. The test substance will be administered to the test organism in culture medium. This route o f administration was selected because it represents the most likely route o f exposure to duckweed, which exist suspended on a water surface with roots extending into the water column. Test Organism The test species will be the duckweed, Lemna gibba G3. This species is representative o f an . important group o forganisms and was selected for use in the test based upon past use and ease o f handling in the laboratory. Original duckweed plants will be obtained from the United States Department o f Agriculture, Washington, D.C. or another suitable supplier.. Stock cultures will be maintained in culture PROTOCOL NO.: 454/091799/LEM 7D/SUB454 004535 3M LAB REQUEST NO. U2723 Wildlife International, ltd -5 - medium at Wildlife International, Ltd. Duckweed used in toxicity tests will be obtained from stock cultures that have been actively growing in this medium for at least 2 weeks. Culture Medium Culture medium prepared according to W ildlife International, Ltd. Standard Operating Procedures will be used as dilution water. The concentrations o f the medium components are presented in Table 1. Stock nutrient solutions will be prepared by adding reagent-grade or better chemicals to W ildlife International, Ltd. well water purified by reverse-osmosis. Appropriate volumes o f the stock nutrient solutions will then be diluted w ith purified well w ater to prepare the medium. The medium will be filter sterilized (0.22pm) or autoclaved prior to use in the te s t The pH o f the medium should be 5.0 0.1. Analyses will be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents o f the well water and results o f die most recent GLP compliant analyses will be summarized in the final report Specifications for acceptable levels o f contaminants have not been established for culture medium. However, there are no levels o f contaminants reasonably expected to be present in the medium that are considered to interfere with the purpose or conduct o f the study. Test Apparatus Test chambers will be 250-ipL Nalgene beakers covered with disposable petri dishes and containing 100 mL o f test solution. The test chambers w ill be indiscriminately positioned on a shelfin an environmental chamber. Test chambers will be labeled w ith the project number, test concentration, and replicate. Environmental Conditions The test will be conducted at 25 2C under continuous warm-white fluorescent lighting at an intensity o f5000 750 lux. Light intensity will be measured at five locations surrounding the test chambers on Day 0 o f the te st Temperature will be measured twice daily in a container o f water adjacent to the test chambers in the environmental chamber using a liquid-in-glass mercury thermometer. -------- The pH d f each treatment and the control will be measured at test initiation and termination using a - Fisher Accumet Model 915 pH meter or equivalent Samples for pH measurements at test initiation will be collected from the individual batches o f test solution prepared for each treatment and control group. A t test termination, pH will be measured in pooled samples o f test solution collected from each o f the three replicates o f each respective treatment and control group. 004536 PROTOCOL NO.: 454/091799/LEM 7D/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd - 6- Bioloeical Measurements Growth, defined as an increase in the total number o f fronds, will be assessed on Day 3, Day 5 and at the end o f the te st Additionally, the total number o f duckweed plants present in each test chamber will be determined at test term ination The numbers o f chlorotic, necrotic and dead fronds will be enumerated. Chlorotic fronds are those possessing areas o f bleached color progressing from green to yellow. Necrotic fronds are those with localized regions o f dead or decaying tissue, usually surrounded by healthy tissue. Fronds with no apparent living tissue, usually all brown, black, white, yellow or clear, will be considered dead. The duckweed plantswill be observed for frte presence o froot destruction (ie., dead or shortened roots in a treatment group compared to the controls) and breakup o f duckweed colonies (i.e., an increased number o f free-floating individual fronds in a treatment group compared to the controls). Any other abnormalities in frond or plant appearance will also be documented. Sampling for Analytical Measurements Samples o f the exposure solutions will be collected on Days 0, 3, 5 and 7 and analyzed for test substance concentrations. The termination day samples may be filtered (0.45 pm), as necessary, to remove plant debris prior to analysis. Day 3 and 5 samples will be collected from the extra replicate test cham bers) included in the test to provide samples for that analysis and from the abiotic replicates. Samples will be placed in an appropriate storage container (e.g., polyethylene, or polypropylene bottle) and will be analyzed immediately, or stored under refrigeration until analyzed. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group Day 0 Day 3 Day 5 Control Solvent Control (if needed) 111 111 Level 1-Low Concentration Level 2 Level 3 Level 4 TLvmVr trCm l\ -JC v Level 6-High Concentration 111 111 111 1 11 1 11 1 2l 21 | Totals 'includes abiotic samples. 8 99 Day 7 1 1 1 1 1 1 1 21______ 1 91 PROTOCOL NO.: 454/091799/LEM 7D/SUB454 004537 3M LAB REQUEST NO. U2723 Wildlife International, ltd -7 - The above numbers o f samples represent those collected from the test and do not include quality control (QC) samples such as m atrix blanks and fortifications prepared and analyzed during the analytical chemistry phase o f the study. Analytical Chemistry Chemical analysis o f the samples will be performed by Wildlife International, L td The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix IL The methodology used to analyze the test samples will be documented in the raw data and summarized in die final report. Statistical Analyses - Percent inhibition will be calculated for each treatment and control group using the following formula: Mean Control - M ean Treatment Percent Inhibition = Frond Nnmher Frond Number X 100 M ean Control Frond Number The Day 7 IC50 value will be determined, when possible, using linear interpolation or other suitable techniques (3) with treatment response (frond number) and exposure concentration data. The percentages o f dead, chlorotic and necrotic fronds will be calculated for each treatment and control group as the number o f dead, chlorotic or necrotic fronds to the total number o f fronds present in each test chamber. The data will be evaluated for normality and homogeneity o fvariances. If these assumptions are met, Dunnett's procedure or the Bonferroni t-test (4) may be used to evaluate the data. If the assumptions o f normality and homogeneity o f variances are not met, non-parametric statistical methods such as the KruskalW allis test (4) may be used to evaluate die data. A no observed adverse effect concentration (NOAEC) will . be determined based upon evaluation o f the statistical results, the dose-response pattern and other observed effects (e.g., percent chlorosis, necrosis, etc.). RECORDS TO BE M AINTAINED Records to be maintained for data generated at W ildlife International, Ltd. will include, but not be limited to: 1. Copy o f signed protocol. 2. Identification and characterization o f the test substance, if provided by Sponsor. 3. Dates o f initiation and term ination o f the te s t r . n /t cr 'i c PROTOCOL N O .: 454/091799/LEM 7D/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, l t d - 8- 4. Source o f plants. 5. Culture conditions. 6. Growth measurements. 7. Calculation and preparation o f test concentrations. 8. Observations. 9. If applicable, die methods used to analyze test substance concentrations and the results o f analytical measurements. 10. Statistical calculations. 11. Test conditions and physical/chemical measurements. 12. Copy o f final report FINAL REPORT A final report o f the results o f the study will be prepared by Wildlife International, Ltd. The report will include, but not be limited to the following, when applicable: 1. Name and address of the facility performing the study. 2. Dates on which the study was initiated and completed. It is the responsibility o f the Sponsor to provide the final date that data are recorded for chemistry pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures stated in the approved protocol, including any changes in the original protocol. 5. The test substance identified by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor. 6. Stability and the solubility o f the test substances under the conditions o f administration, if provided by the Sponsor. 7. A description o f the methods used to conduct the te st 8. A description of the test system, including the source of the test organisms and their scientific name. 9.----- A description o f the preparation o f the test solutions, methods used to allocate organisms to the test --------chambers and begin the test, numbers o f organisms and chambers per treatment, and duration o f the te s t 10. A description o f all circumstances that may have affected the quality or integrity o f the data. 11. The name o f the Study Director, the names o f other scientists or professionals, and the names o f all supervisory personnel, involved in the study. 004539 PROTOCOL N O .: 454/091799/LEM 7D/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd -9 - 12. A description o f the transformations, calculations, or operations performed on the data, a summary . and analysis o f die biological data and analytical chemistry data, and a statement o f the conclusions drawn from these analyses. 13. Statistical methods employed for analyzing the data. 14. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study. 15. The location where all raw data and the final report are to be stored. 16. A statement preparrd by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates o f any findings reported to the Study Director and Management 17. If it is necessary to make corrections or additions to a final report after it has been accepted, such changes shall be in the form o f an amendment issued by the Study Director. The amendment shall clearly identify the part o f the final report that is being amended and the reasons for the alteration. Amendments shall be signed and dated by the Study Director. CHANGING O F PROTOCOL Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendm ent.!will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 and/or Part 792); OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98)17); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International, Ltd. is routinely examined by the W ildlife International, Ltd. Quality Assurance U nit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement o f compliance with Good Laboratory Practices will be prepared for all portions o f the study conducted by W ildlife International, Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at W ildlife International, Ltd. and a copy o f the final report will be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in the final report PROTOCOL NO.: 454/091799/LEM 7D /SUB454 004540 3M LAB REQUEST NO. U2723 Wildlife International, l t d - 10- REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.4400: Aquatic Plant Toxicity Test Using Lem naspp., Tiers ! a n d n . 2 ASTM S tan d ard Guide 1415-91 E. 1991. Standard Guidefo r Conducting Static Toxicity Tests with Lemna gibba G3. 3 N orberg-K ing, T J . 1993. A Linear Interpolation M ethodfo r Sublethal Toxicity: The Inhibition Concentration (ICp) Approach (Version 2.0). U.S. Environmental Protection Agency. Environmental Research Laboratory. Duluth, Minnesota. 4 W est, I n c and D. D. Gulley. 1996. TQXSTATRelease 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming. \ PROTOCOL N O .: 454/091799/LEM 7D/SUB454 004541 3M LAB REQUEST NO. U2723 Wildlife International, ltd - 11TABLE 1 M-HOAGLAND'S MEDIUM W ITHOUT EDTA OR SUCROSE CONSTITUENTS Compound N om inal C oncentration H3BO3 Z nS04*7H20 Na2M o04*2H20 CuS 0 4*5H20 MnCI2-4H20 KH2P 0 4 KN03 Ca(N 03)2*4H20 M gS04*7H20 FeCl3*6H20 Tartaric Acid 2.86 0.22 0.12 0.08 3.62 0.68 1.515 1.180 0.492 5.4 3.0 m g/L m g/L m g/L m g/L m g/L g/L g/L g/L g/L m g/L The pH o f the medium w ill be adjusted, as necessary, to 5.0 0.1 using 0 .1 N NaOH or. 10% HCL. \ PROTOCOL N O .: 454/091799/LEM 7D/SUB454 004542 3M LAB REQUEST NO. U2723 Wildlife International, ltd -12- APPENDKI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completedby Sponsor I. Test Substance Identity (name to be used in the report): PFOS (Perfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A_______________________________ Internal Standard: 1.12.2H.H.HJH Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch Number: Lot 217___________ - . ' - _________ Test Substance Purity(% Active Ingredient): 98.9 Expiration Date: 2008 E TestSubstance Characterization Have the identity,strength,purity andcon^ositkorothercharacteristics vvhkiiappropriatEtydefinette test substance andrefererre standardbeen detenmnedpriortoitsuseinthisstuctyinaccotdancewifhGLPStandards? Yes x No DL Test Substance Storage Conditions Pleaseindicatethe recommendedstorageconditicns atWildlifeInternational, Ltd. Ambient____________________________________________________________ Has the stability o fthe testsubstanceunderthese storageconditions beendeterminedin accordancewith GLP Standards? _____ Yes O tter pertinent stability information; x ' No IV. TestConcentrations: Adjusttestconcentrationto 100% a i x based uixmthe purity givenabove. Do notadjusttest concentrationto 100% a t Testthe materialAS IS. V. Toxicity Infbnnation: Mammalian: RatLD(5500 251 mg/kg Mouse LD50 N/A Aquatic: InvertebrateToxicity (EC/LC50) Fish Toxicity (LC5C Daphnia marna: 27 me/L__________ Rainbow Trout: 11 me/L Daphnia magna: 50 mg/L Fathead Minnow: 38 mg/L -Please see MSDS PROTO COL NO.: 454/091799/LEM 7D/SUB454 004543 3M LAB REQUEST NO. U2723 Wildlife International, l t d -13- APPENDIXII Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination o f Perfluoroocta^e- Sulfonic A d d Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f the above method will be maintained in die raw data. The actual methodology used to analyze the test samples wffl be documented in the raw data and summarized in the final report PROTOCOL NO.: 454/091799/LEM 7D/SUB454 004544 3M LAB REQUEST NO. U2723 Wildlife Internationallto. PROJECT N O .: 454A-111 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 7-DAY TOXICITY TEST WITH DUCKWEED (Lemna gibba G3) PRO TOCOL NO: 454/091799/LEM7D/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PR O JE C T NO.: 454A-111 EFFECTIV E DATE: February 11.2000__________________________ 3M LAB REQUEST NO.: U2723 AMENDMENT: Culture Medium Pape 5 Change: The pH o f the medium should be 5.0 0 .1 . Add: The pH o f the medium should be 7.5 0 .1 . REASON: To change test medium from Hoagland's to 20X AAP medium. AMENDMENT: TABLE 1, Page 11 Remove: Entire table. Add: TABLE 1 20X AAP MEDIUM -- Component M gC l2*6H20 CaCl2*2H20 HaBOj MnCl2*4H20 ZnCl2 FeCi3*6H20 CoC12*6H20 Na2M o04*2H20 CuC12*2H20 N a2EDTA*2H20 NaN03 M gS04*7H20 k 2h p o 4 NaHCOa N om inal Concentration 243.2 mg/L 88.0 mg/L 3.712 mg/L 8.32 mg/L 65.6 pg/L 3.196 mg/L 28.56 Pg^L 145.2 pg/L 0.240 Pg/L 6.00 mg/L 510.0 mg/L 294.0 mg/L 20.88 mg/L 300.0 mg/L The pH o f the medium w ill be adjusted, as necessary, to 7.5 0.1 using 0.1 N NaOH or 10% HC1. 004545 Wildlife International ltd. PROJECT NO. 454A-11I Page 2 o f2 REASON: To change test medium from Hoagland's to 20X AAP medium. 2. STUDY DIRECTOR DA 2 /qU o DATE 3 /3 / d AT ' 004S46 Wildlife International ltd. PROJECT NO.: 454A-111 Page 1 o f 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 7-DAY TOXICITY TEST WITH DUCKWEED (Lemna gibbo G3) PRO TO CO L NO: 454/091799/LEM7D/SUB454 AMENDMENT NO.: 2 SPONSOR: 3M Corporation PRO JECT NO.: 454A-111 EFFECTIV E DATE: February 22.2000__________________________3M LAB REQUEST NO.: U2723 AMENDMENT: Page 2 '*/ Add: Experimental Start Date: M arch 3,2000 Experimental Tennination Date: March 10,2000 Test Concentrations: Negative Control, 1 3 ,25,50, 100,200 and 400 mg a.i./L Test Substance No.: 4675 REASON: The above information was not known when the protocol was signed by the Study Director. STUDY DIRECTOR 3/2,oo DATE 'S/a/so DAT 004547 Wildlife International ltd. PROJECT NO.: 454A -113 Page 1 o f 2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST WITH THE MARINE DIATOM (Skeletonema costatum ) PRO TO CO L NO: 454/091799/SKE96/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PR O JEC T NO.: 454A-113 EFFEC TIV E DATE: February 28.2000__________________________3M LAB REQUEST NO,; U2723 AMENDMENT: Page 2 'Sj Add: Experimental Start Date: M arch 3,2000 Experimental Tem anation Date: M arch 7,2000 Test Concentrations: Negative Control and 4.0 mg a.L/L Test Substance No.: 4675 REASON: The above information was not known when the protocol was signed by the Study Director. AMENDM ENT: Experimental Design. Page 3 Change: The marine diatom, Skeletonema costatum, will be exposed to a geometric series o f six test concentrations and a negative (culture medium) control for 96 hours. To: The marine diatom, Skeletonema costatum, will be exposed to a test concentration at the lim it o f solubility and a negative (culture medium) control for 96 hours. REASON: To change experimental design to a lim it te st AMENDM ENT: Experimental Design. Pape 3 Change: Test solutions will be inoculated with approximately 77,000 cells/mL Test solutions w ill be inoculated with 10,000 cells/mL. To: Test solutions will be inoculated with approximately 77,000 cells/mL. REASON: Typographical error. AMENDM ENT: Experimental Design. Page 3 Change: Three "biological" replicates per experimental group will be prepared for evaluating cell densities. To: Six "biological" replicates per experimental group will be prepared for evaluating cell densities. REASON: To change experimental design to a lim it te st 004548 fWetoed buOA 5 3-l-oo Wildlife International ltd. PROJECT N O .: 454A-113 Page 2 o f2 AM ENDM ENT: Sampling for Analytical Measurements. Page 7 Change: Day 4 samples will be composited solutions from the three replicates remaining at the aid o fthe test To: Day 4 samples will be composited solutions from the six replicates remaining at the end o f the te st REASON: To change analytical sampling based on additional replicates provided in the lim it te st 004549