Document emZz48oxBQD8Kb9wZOMo58x79

/\RJ2C,'018S oes^s PROTOCOL PFOS: A 96-HOUR TOXICITY TEST W ITH TH E M ARINE DIATOM (Skeletonema costatum) U .S. Environm ental Protection Agency / Series 850 - Ecological Effects Test Guidelines OPPTS Num ber 850.5400 3M Lab Request No. U2723 Subm itted to 3M Corporation Environm ental Laboratory 935 Bush Avenue S t Paul, M innesota 55106 Wildlife International, Ltd. 8598 Commerce D rive Easton, M aryland 21601 (410) 822-8600 Septem ber 17,1999 004515 Wild life International, ltd -2- PFOS: A96-HOUR TOXICITY TEST W ITH THE M ARINE DIATOM (Skeletonema costatum) SPONSOR: 3M Corporation Environm ental Laboratory 93S Bush Avenue S t Paul, M innesota 55106 SPONSOR'S REPRESENTATIVE: Rochelle R. Robideau TESTIN G FACILITY: v STUDY DIRECTOR: W ildlife International, Ltd. 8598 Commerce Drive Easton, M aryland 21601 KurtProttar C&sy Senior A quatic Biologist ijiej** LABORATORY MANAGEMENT: Henry 0 . Krueger, Ph.D. D irector o f A quatic Toxicology & N on-Target Plants - FO R LABORATORY USE ONLY Proposed Dates: Experim ental Start D ate: ___________________________ Experim ental Term ination Date: . ___________ Project N o.: T est C o ncentrations:_______ ;____________________________ '_____________________ T est Substance N o .:____________ Reference Substance N o. (if applicable): PROTOCOL APPROVAL H Z ? /O O DATE PROTOCOL NO.: 454/091799/SKE96/SUB454 DATE fd a t e 004516 3M LAB REQUEST NO. U2723 Wild life International, ltd -3 - IN T R O D U C T IO N W ildlife International, Ltd. will conduct a 96-hour toxicity test with the marine diatom, Skeletonema costatum, 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 850Ecological Effects Test Guidelines OPPTS Number 850.5400 (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 * v. * PURPOSE The purpose o f this study is to determine the toxicity o f Perfluorooctane Sulfonic A dd, Potassium Salt (hereafter referred to as PFOS) to the marine diatom, Skeletonema costatum. EXPERIM ENTAL DESIGN 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. Target concentrations will not exceed 100 mg/L or the solubility lim it o f the test substance in water. Generally, the nominal concentration o f each test substance 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. Test solutions will be inoculated with approximately 77,000 cclls/mL Test solutions will be inoculated with 10,000 cells/mL. Three "biological" replicates per experimental group will be prepared for evaluating cell densities. One or more additional "analytical" replicates will be included in the test, as needed, to provide test solution for concentration verification on Day 3 o f the exposure. An abiotic replicate at the highest concentration will be included in the test and will be sampled on Day 3 and Day 4. In order to control bias, the position o f die flasks 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 response o f the algae will be measured in terms o f cell density, biomass expressed as area under the growth curve, and growth rate. An EC50 (i.e., the theoretical test concentration that produces a 50% reduction in the measured param eter) value for cell density will be calculated for each 24 hour interval. EC10, EC50 and EC90 values for cell density, biomass (Et,C) and growth rate (EtC) will be calculated, if possible, at the 72 and 96 hour intervals. The no observed adverse effect concentration (NOAEC), which ........ PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 004S7 Wild life International, ltd -4 - is the highest test concentration that induces no adverse inhibitory effect on growth, will be determined relative to each parameter at 72 and 96 hours based upon evaluation o f the statistical results and the doseresponse pattern. A t die end o f the 96-hour exposure, algistatic effects w ill be differentiated from IgiciHnl effects in those treatments which are maximally inhibited. M ATERIALS AND METHODS Test Substance v Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing W ildlife 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 W ildlife International, Ltd., it will be noted in the compliance statement o f the final report The attached form ID EN TIFICA TIO N O F TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. The Sponsor is responsible for 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 o f stock solution with medium. The test solutions may also be prepared using secondary dilutions o f the primary stock, based upon the concentration desired. PFOS will be administered to the test organism in algal medium. This route o f administration was selected because it represents die m ost likely route o f exposure to algae, which exist suspended in a water column. PROTOCOL NO.: 454/091799/SKE96/SUB454 004518 3M LAB REQUEST NO. U2723 Wildlife International, l t d -5 - Test Organism The test species will be the marine alga, Skeletonema costatum. This species is representative o f an important group o f algae and was selected for use in the test based upon past use and ease o f handling in the laboratory. Stock cultures, obtained from the Culture Collection o f Algae at the University o f Texas at A ustin or another supplier, will be maintained in culture medium under test conditions at W ildlife Interna tional, Ltd. for a minimum o f two weeks prior to use in a toxicity te st Algae used in toxicity tests will be in exponential growth phase, which is defined as the period o f growth when algal cells are dividing at a constant rate. Just prior to beginning the te s t an inoculum o fthe stock culture will be prepared so that each milliliter o f inoculum contains enough cells to provide an initial cell density o f approximately 77,000 cells/mL in each replicate. Culture Medium Culture medium prepared from natural or artificial seawater according to W ildlife International, Ltd. Standard Operating Procedures will be used as dilution water. The concentrations o f the components in the medium are presented in Table 1 (2). Stock nutrient solutions will be prepared by adding reagent-grade chemicals to W ildlife International, Ltd. well w ater purified by reverse-osmosis. Appropriate volumes o f the stock nutrient solutions will then be diluted with artificial or natural seawater to prepare the medium. The medium will be filter sterilized (0.22 pm) or autoclaved prior to use. 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 the m ost recent GLP compliant analyses will be summarized in the final rep o rt 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 sterile 250-mL polycarbonate Erlenmeyer flasks plugged with foam stoppers and containing 100 mL o f test solution. The test chambers will be indiscriminately positioned on a PROTOCOL NO.: 454/091799/SKE96/SUB454 004519 3M LAB REQUEST NO. U2723 Wildlife International, ltd 6- - mechanical shaker table in an environmental chamber and will be shaken continuously at approximately 100 rpm. Test chambers will be labeled w ith the project number, test concentration and replicate. Environmental Conditions Test flasks will be held at 20 2C under 14 hours o f cool-white fluorescent lighting at an intensity o f 4300 430 lux and 10 hours o f darkness. lig h t intensity will be measured a t five locations surrounding the test flasks cm Day 0 o f the te st Temperature will be recorded twice daily in a container o f water adjacent to the test flasks using aliquid-in-glass thermometer. The pH o f each treatm ent and control group will be measured at test initiation and termination using a Fisher Accumet Model 915 pH m eter o r equivalent Samples for pH measurement at test initiation will be collected from the individual batches o f test solution prepared for each treatm ent 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 treatm ent and control group. Biological Measurements Cell densities will be monitored during the test by conducting cell counts using a hemacytometer and a microscope. One sample will be collected from each replicate o f the treatm ent and control groups at approximate 24-hour intervals during the exposure period. The samples will be evaluated immediately or . will be held in the dark at approximately 4C until cell counts can be performed. Each sample -will be diluted using an electrolyte solution (Isotcm), as needed, to maintain counting accuracy. A small amount o f each sample will be loaded onto a hemacytometer and the total number o f cells in 10 grids will be counted. The cell density o f the sample will be calculated based o n th e mean number o f cells per grid. M icroscopic examination o f algae will be m ade to assess changes in gross morphology. Algae will be observed to assess changes in cell shape, cell color, and aggregation (clumping) o f the cells. Observations will be used to, determine whether growth treatm ent-related effects include those changes in gross morphology. PROTOCOL NO.: 454/091799/SKE96/SUB454 004520 3M LAB REQUEST NO. U2723 Wild life International, ltd -7 - Sampling for Analytical Measurements Samples o f the exposure solutions will be collected on Days 0 ,3 and 4 and analyzed for test substance concentrations. Day 0 samples will be taken from each batch o f test solution prior to their distribution amnng the replicate test chambers. Day 3 samples will be collected from the extra replicate test cham bers) mrJuHwf in the test to provide sample for that analysis and from the abiotic replicates. I f more than one replicate is included for the Day 3 sample analysis, then the solutions from each replicate will be composited to provide the sam ple for that treatm ent Day 4 samples will be composited solutions from the three replicates remaining at the a id o f thVe'te s t Samples collected on Days 3 and 4 may be centrifuged or filtered to remove algal cells prior to analysis. Samples will be analyzed immediately or placed in an appropriate storage container (e.g., polypropylene or polyethylene bottle) and stored until analyzed. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group DayO Day 3 C o n tro l Solvent Control (if needed) Level 1-Low Concentration Level 2 Level 3 Level 4 L e v e l5 Level 6-High Concentration 11 11 11 11 11 11 11 1 21 T o ta ls d e lu d e s abiotic samples. 89 Day 4 1 1 1 1 1 1 1 21 9 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 W ildlife International L td The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix EL PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 004521 Wildlife International, ltd 8- - Modifications made to the analytical method will be documented in the raw data and described in the final report Statistical Analyses Biomass expressed as area undo- the growth curve will be calculated for the treatm ent and control groups using the following formula: A = ((Nr Noy2Xti>+<(N,+Nr2Noy2)(t2-t1)+...-K(NI, 1+Nn-2Noy2X V L i) W here: v A = Area under the growth curve N0= M easured number o f cells/mL at to N t =Measured number o f cells/mL at t] N2 =Measured number o f cells/mL a t t2 Na -M easured number o f cells/mL a t t> ti = Time o f first measurement after beginning o f test (hours) t2 = Time o f second measurement after beginning o f test (hours) L = Time o f nth measurement after beginning o f test (hours) - Growth rates (p) will be calculated for the treatm ent and control groups using die following formula: In No-In N0 j. - ---------------- tu to fj, " Average specific growth rate N0 = Measured number o f cells/mL at to Nn= M easured number o f cells/mL at L to " Time o f beginning o f test (hours) L = Time o f nth measurement after beginning o f test (hours) Percent inhibition will be calculated for each treatm ent group based upon mean cell density, biomass and growth rate using the following formula: v M ean Control - M ean Treatment Percent Inhibition = Response______Response M ean Control Response X 100 PROTOCOL NO.: 454/09I799/SKE96/SUB454 004522 3M LAB REQUEST NO. U2723 Wildlife International, ltd -9 - EC values will be determined, when possible, using linear interpolation or other suitable techniques with treatment response (Le. cell densities, biomass and growth rate) and exposure concentration data for each 24-hour interval o f the test. A no observed adverse effect concentration (NOAEC) will be selected based on an evaluation o f the dose-response pattern and the results o f the statistical analysis using individual replicate values o f the cell density, biomass and growth rate. Statistically significant differences between the control and the treatm ent groups will be identified using analysis o f variance (ANOVA) and a test to compare treatm ent mean responses to the control response (e.g., Dunnett's test or Bonferroni's t-test). Data will be assessed for normality and homogeneity o f variances prior to performing die ANOVA. Transformations will be used to correct any condition o f non-normality or unequal error variances, if possible. If a solvent control is used in addition to the negative control, their responses will be compared using Student's t-te st D ata from the two control groups will be pooled far treatm ent level analyses if no statistical differences ex ist Otherwise, all comparisons will be made to the solvent control group. Statistical comparisons for the ANOVA and die comparison o f mean responses will be carried out using TOXSTAT V3.5 software (4) or equivalent RECORDS TO BE MAINTAINED Records to be maintained for data generated at W ildlife International, L td .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 4. Source o f algae. 5. Culture conditions. 6. Growth measurements. 7. Calculation and preparation o f test concentrations. 8. Observations. 9. If applicable, the methods used to analyze test substance concentrations and the results o f analytical measurements. 10. Statistical calculations. 11. Test conditions and physical/chemical measurements. in a c PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd 12. Copy o f final report -10- FINAL REPORT A final report o f the results o f the study will be prepared by W ildlife International, Ltd. The report will include, but not be lim ited to the following, when applicable: 1. Name and address o f the facility performing the study. 2. Dates on which the study w as initiated and com pleted It is the responsibility o f the Sponsor to provide the final date th at data are recorded forchem istiy pathology and/or supporting evaluations that may be generated a t other laboratories. 3. A statem ent 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 die original protocol 5. The test substance identified by name, chemical abstract number or code number, strength*purity, composition, and other characteristics provided by the Sponsor. 6. Stability and solubility o f the test substances under the conditions o f adm inistration, if provided by the Sponsor. 7. A description o f the m ethods used to conduct the test. 8. A description o f the test system, including the source o f 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 treatm ent, and duration o f die test 10. A description o f circumstances th at may have affected the quality or integrity o f the data. 11. The name o f the Study D irector and the names o f other scientists, professionals, and supervisory personnel involved in the study. 12. A description o f the transform ations, calculations, or operations performed on the data, a summary . . and analysis o f the biological data and analytical chemistry data, and a statem ent o f the conclusions \ _ drawn from these analyses. 13. Statistical methods em ployed 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. 004524 PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd - ii- is . The location where raw data and final report are to be stored. 16. . A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audita 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 made 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. s\' ' 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. Amendments will be considered as part of the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by die Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report. GO OD 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 W ildlife International, Ltd. is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statem ent 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 file final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report PROTOCOL NO.: 454/091799/SKE96/SUB454 004525 3M LAB REQUEST NO. U2723 Wildlife International, ltd -12- REFERENCES 1 U.S. Environm ental P rotection A gency. 1996. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II. 2 ASTM Standard G uide 1218-90E. 1990. Standard Guidefo r Conducting Static 96-Hour Toxicity Tests with Microalgae. American Society for Testing and M aterials. Philadelphia, Pennsylvania. 3 N orberg-K ing, T-J* 1993. A LinearInterpolation M ethodfo r Sublethal Toxicity: The Inhibition Concentration (iCft Approach (Version 2.0). U S . Environmental Protection Agency, Environmental Research Laboratory, Duluth, M innesota. 4 W est, I n c and D. D. Gulley. 1996. TOXSTATRelease 3.5. W estern EcoSystems Technology, Inc. Cheyenne, Wyoming. PROTOCOL NO.: 454/091799/SKE96/SUB454 004526 3M LAB REQUEST NO. U2723 Wildlife International, ltd -13TABLE1 SALTWATER ALGAL MEDIUM CONSTITUENTS C om ponent Nom inal C oncentration FeCl3.6H 20 MnCl2.4H 20 ZdS04.7H 20 CuS04.5H 20 CoC12.6H 20 H3BO3 N a2E D T A .2H 20 K3PO4 NaN03 Na2S i0 3.9H 20 Thiamine Hydrochloride Biotin B 12 0.72 mg/L 2.16 mg/L 0.675 mg/L 2.36 pg/L 6.06 pg/L 17.1 mg/L 15.0 mg/L 3.0 mg/L 50.0 mg/L 20.0 mg/L 0.25 mg/L 0.05 pg/L 0.5 pg/L The above components will be added to instant saltwater at a salinity o f approx imately 30 parts per thousand. The pH o f the medium will be adjusted, as necessary, to between 7.8 and 8.1 using 0 .1 N NaOH or 10% HC1. PROTOCOL NO.: 454/091799/SKE96/SUB454 004527 3M LAB REQUEST NO. U2723 Wildlife International, ltd -14- APPENDIXI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Compieteciby Sponsor I. Test Substance Identity (name to be used in the report): PFOS IPerfhioronctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): A nalytical Standard: N/A________________________________ Internal Standard: 1.1.2.2HLHJHLH Perflnomoctane Sulfonic Acid Test Substance Sample Code or B atch Num ber: Lot 217_____________ _______ ________________ Test Substance Purity(% A ctive Ingredient): 98.9_______ Expiration Date: 2008___________ _ D. Test Substance Characterization Have die identity,strength,purity and conqxrsitionor other characteristics which appropriatelydome the test substance andreferencestandardbeen detenmned priorto ite use in this study in aoccxdancewthGLP Standards? ~ Yes x No EL Test Substance Storage Conditions Ptea<y.nvtirate riv. nyfrnrnpririflri stnragRcmdilinns at W ildlife Internationa^ Tiri Ambient________________________________;___________________________ ___________ Has toe stabilityo fthe testsubstance underthese storage conditions been detemnned in acoadanoewthGLP Standards? ______Yes x No Otherpertinentstability information: IV. Test Concentrations: Acusttest concentrationto 100% a i x based upon the purity f%) given above. Do not adjusttest ccncentation to 100% ai. Test mematerialAS IS. V. Taxrdlylhfcnnation: Mammalian- RatLDS(5O0 251 m g/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Daphnia mapna- 27 mp/T,__________ Daphnia m apna: 50 mp/L Fathead Minnow: 38 mg/L OtherToxicityInformationincluding findings o fchronic and subcfaronictests): Please see MSDS_____________________________________________ PROTOCOL NO.: 454/091799/SKE96/SUB454 004528 3M LAB REQUEST NO. U2723 Wild life International, ltd -15- A P P E N D IX II Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by die Sponsor in the following analytical m ethods: 1. Liquid Chromatography M ass Spectrometry (LCM S) M ethod for the Determination o f Perfluorooctane Sulfonic A dd Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f die above method will be maintained in the raw data. The actual methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO.: 454/091799/SKE96/SUB454 004529 3M LAB REQUEST NO. U2723 vv/VA/ VV VO.OO rAA n u 2 0632 WILDLIFE INTERNATIONAL Wild life International ltd. @008 PROJECT NO.: 454A-113 Page 1 o f2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST W ITH THE M ARINE DIATOM (Skektonm a cosiatum ) PR O TO C O L N O : 454/091799/SKE96/SUB454 SPO N SO R: 3M Corporation DRAFT AM EN DM EN T N O .: 1 P R O JE C T N O .: 454A-113 E FF E C T IV E D A TE: February 28.2000___________________________ 3M LA B R E Q U E ST N O .: U2723 AMENDMENT: Page 2 Add: Experim ental Start Date: M arch 3 ,2 0 0 0 Experim ental Term ination Date: M arch 7 ,2 0 0 0 Test Concentrations: N egative Control and 4.0 m g a.i./L Test Substance N o.: 4675 REA SO N : The above inform ation was not known when (he protocol w as signed by the Study Director. A M EN DM EN T: Experim ental Design. Pane 3 Change: The m arine diatom, Skclclonema costatum , w ill be exposed to a geometric series o f six test concentrations and a negative (culture medium) conlrol for 96 hours. To: The m arine diatom, Skeletonema costatum , will be exposed to a test concentration at the lim it o f solubility and a negative (culture medium) control fo r 96 hours. REA SO N ; To change experimental design to a lim it test. A M EN DM EN T: Experim ental Design. Page 3 Change: Test solutions w ill be inoculated w ith approxim ately 77,000 cells/m L T est solutions w ill be inoculated w ith 10,000 cells/mL. To: T est solutions w ill be inoculated with approxim ately 77,000 cells/m L. REA SO N : Typographical error. AM EN DM EN T: Experim ental Design. Pace 3 Change: Three "biological" replicates per experim ental group w ill be prepared for evaluating cell densities. To: Six "biological" replicates per experim ental group w ill be prepared fo r evaluating cell densities. REA SO N : To change experimental design to a lim it te s t 004530 OAA UQJ T T lL U L lffc JU 'U C IW A ilU N A L @009 PROJECT NO.: 454A-113 W i l d l i f e In t e r n a t io n a l l -t-p .____________________ _______________________________________ P as e 2 o f 2 DRAFT A M EN D M EN T: Sam pling for A nalytical M easurements. Page 7 Change: D ay 4 sam ples will be com posited solutions from the three replicates remaining a t the end o f the test To: D ay 4 sam ples will be com posited solutions from the six replicalcs remaining at the end o f the te s t R EA SO N ; To change analytical sam pling based on additional replicates provided m the lim it le st Sf* STUDY D IRECTO R LABORATORY MANAGEMENT DATE DATE DATE / / 004531