Document O15ne4DQLoK8ZJG4XenDe3B6e

/U016-OIS7 0 & 2 'V PROTOCOL PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER DIATOM (Navicula pelliculosa) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue ; S t Paul, M innesota 55106 Wildlife International, Ltd. 8598 CommerceDrive ' Easton, Maryland 21601 (410)822-8600 September 17,1999 004498 Wildlife International, ltd - 2- PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER DIATOM (Navicula pelliculosa) SPONSOR: 3M Corporation Environmental Laboratory 93S Bush Avenue S t Paul, Minnesota SS106 SPONSOR'S REPRESENTATIVE: V* TESTING FACILITY: Rochelle R Robideau W ildlife International, Ltd. 8598 Commerce Drive Easton, M aryland 21601 STUDY DIRECTOR- Kurt-Dfotiag C a s ^i Senior Aquatic Biologist HK LABORATORY MANAGEMENT: Henry O. Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants ___________________________ F O R LABORATORY USE ONLY | Proposed Dates: Experimental Start Date: _________________________ ^ Experimental Termination Date: ' Project No.: / / 2- Test C oncentrations:_______ ' _________________________________ Test Substance N o .:____________Reference Substance No. (if applicable): PROTOCOLAPPROVAL STUDY DIRECTOR DATE i/z e /o o PROTOCOL NO.: 454/091799/NAV96/SUB454 DATE DATE 7 004499 3M LAB REQUEST NO. U2723 Wildlife International, ltd -3 - IN T R O D U C T IO N Wildlife International, Ltd. will conduct a five-day toxicity test with the freshwater diatom, Navicula pelliculosa, for the Sponsor at the W ildlife 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 850.5400 (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 Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. PURPOSE The purpose o f this study is to determine the toxicity o f Perfluorooctane Sulfonic Acid, Potassium Salt (hereafter referred to as PFOS) to the freshwater diatom, Navicula pelliculosa. EXPERIM ENTAL DESIGN The freshwater diatom, N avicula pelliculosa, 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 freed 100 mg/L or the solubility limit 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 10,000 cells/mL. Three "biological" replicates per experimental group will be prepared for evaluating cell densities. One or more additional "analytical" replicates will b e , > 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 sam pled. on Day 3 andD ay4. In order to control bias, the position o fth e 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 fth e study. \ - - 1 The response of the algae w ill 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 parameter) value for cell density will be calculated for each 24 hour interval. EC10, EC50 and EC90 values for cell density, biomass (EbC) and growth rate (E ) will be calculated, if 004500 PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd -4 - possible, at the 72 and 96 hour intervals. The no observed adverse effect concentration (NOAEC), which is the highest test concentration that induces no adverse inhibitory effect bn 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. At the end o f die 96-hour exposure, algistatic effects will be differentiated from algicidal 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 Wildlife International, Ltd. w ritten 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 statement o 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 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. The test substance will be administered to the test organism in algal medium. This route o f administration was selected because it represents the m ost likely route o f exposure to algae, which exist suspended in a water column. Test Organism The test species will be the freshwater diatom, Navicula,pelliculosa. This species is representative o f an important group of 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 PROTOCOL NO.: 454/091799/NAV96/SUB454 004501 3M LAB REQUEST NO. U2723 Wildlife International, ltd -5 - at Austin or another supplier, will be maintained in culture medium at W ildlife International, Ltd. for a minimum o f two weeks prior to use in a toxicity te st Diatoms 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 test, an inoculum of the stock culture will be prepared so that each milliliter o f inoculum contains enough cells to provide an initial cell density o f approximately 10,000 cellsAnL in each replicate. 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 components in the medium are presented in Table 1 (2). 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 with purified well water 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 of selected organic and inorganic constituents o f the well water and results o f the 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 die 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 mechanical shaker table in an environmental chamber and will be shaken continuously at approximately 100 rpm. Test chambers will be labelled with the project number, test concentration andjrepjicate.___ . Environmental Conditions Test flasks will be held at 24 2C under continuous cool-white fluorescent lighting at an intensity o f4300 10% lux. Light intensity will be measured at five locations surrounding the test flasks on Day 0 004502 PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd 6- - o f the test Temperature will be measured twice daily in a container o f water adjacent to the test flasks in the environmental chamber using a calibrated, hand-held mercury thermometer. The pH of each treatment and control group will be measured at test initiation and terminating using a Fisher Accumet Model 91S pH meter or equivalent Samples fra*pH measurement 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. 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 treatment 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 (Isoton), 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 on the mean number o f cells per grid. Microscopic examination of algae will be made 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 treatm ent-related effects include those changes in gross morphology. Sampling for Analytical Measurements Samples o fthe 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 among the replicate test chambers. Day 3 samples will be collected from the extra replicate test cham bers) included in the test to provide sample for that analysis and from the abiotic replicates. If more than one replicate is included for the Day 3 sample analysis, then the solutions from each replicate will be composited to provide the sample for that treatm ent Day 4 samples w ill be composited solutions from the three replicates remaining at the end o f the te st PROTOCOL NO.: 454/091799/NAV96/SUB454 004503 3M LAB REQUEST NO. U2723 Wildlife International, ltd -7 - Samples collected on Days 3 and 4 may be centrifuged or filtered to remove algal cells prim: to analysis. Samples will be analyzed immediately o r 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 Day 0 Day 3 Control Solvent Control (if needed) Level 1-Low Concentration 11 11 11 Level 2 Level 3 Level 4 L ev e ls Level 6-High Concentration 11 11 11 11 1 2l Totals 'includes abiotic samples. 89 Day 4 1 1 1 1 1 1 1 21 9 The above numbers o f samples represent those collected from die 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 w ill be perform ed by W ildlife International Ltd. The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix II. Modifications made to the analytical method will be documented in the raw data and described in the final report \ i .. Statistical Analyses Biomass expressed as area under the growth curve will be calculated for the treatment and control groups using the following formula: 0 0 ^ 504: PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 Wildlife International, ltd -8 - A = ((N,-Noy 2 )(t1>+<(N1+N2-2No)/2)(t2-t1)+...-K(Nn.14-Nn-2Noy2)(tn-t1, 1) ' Whore: A = Area under the growth curve N0= Measured number o f cells/mL at to Ni =Measured number o f cells/mL at ti N2-M easured number o f cells/mL at t2 Ntt -M easured number o f cells/mL at to ti = Time o f first measurement after beginning o f test (hours) t2= Time o f second measurement after beginning o f test (hours) to= Time o fnth measurement after beginning o f test (hours) Growth rates (m) will be calculated for the treatment and control groups using the following formula: In Nn.In N0 fj. = -----------:-- t-tb jj. = Average specific growth rate N0= Measured number o f cells/mL at to Nn= Measured number o f cells/mL at tn to = Time o f beginning o f test (hours) t,, = Time o f nth measurement after beginning o f test (hours) P erm it inhibition will be calculated for each treatment group based upon mean cell density, biomass and growth rate using the following formula: Mean Control - Mean Treatment Percent Inhibition = Response Response Mean Control Response X 100 EC values will be determined, when possible, using linear interpolation or other suitable techniques with treatment response (i.e. cell densities, biomass and growth rate) and exposure concentration data for each 24-hour interval o f the te st 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 die treatment groups will be identified using analysis o f variance (ANOVA) and a test to compare treatment mean PROTOCOLNO.: 454/091799/NAV96/SUB454 004505 3M LAB REQUEST NO. U2723 Wildlife International, ltd -9 - responses to the control response (e.g., Dunnetfs test or Bonferroni's t-test). Data will be assessed for normality and homogeneity o f variances prior to performing theANOVA. 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-test D ata from the two control groups will be pooled for treatment level analyses if no statistical differences e x ist Otherwise, all comparisons will be made to the solvent control group. Statistical comparisons for the ANOVA and the comparison o f mean responvs.e\ s will be carried out using TOXSTAT V3.S software (4) or equivalent RECORDS TO BE MAINTAINED Records to be maintained for data generated at Wildlife 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 termination o f the te st 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. 12. Copy o f final report 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 limited to the following, when applicable: 1. Name and address o f the facility performing the study. 004506 PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 *rV. Wildlife International, ltd -10- 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 i 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 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 administration, if provided by the Sponsor. 7. A description o f the methods used to conduct the te st 8. A description o f 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 die test chambers and begin the test, numbers o f organisms and chambers per treatment, and duration o f die test 10. A description o f circumstances that may have affected the quality or integrity o f the data. 11. The name o f the Study Director and die names o f other scientists, professionals, and supervisory personnel involved in the study. 12. A description o f the transformations, calculations, or operations performed on the data, a summary and analysis o f the 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 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 audits were made and the dates o f any findings reported to the Study Director and M anagem ent 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 PROTOCOL NO.: 454/091799/NAV96/SUB454 004507 3M LAB REQUEST NO. U2723 Wildlife International, ltd -ii- shall clearly identify die 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 die 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 o fth e protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by die v Study Director and filed with the raw data. A ll changes to the protocol will be indicated in the final rep o rt 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 W ildlife International, Ltd. is routinely examined by die W ildlife International, Ltd. Quality Assurance Unit 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 w ill 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 (he W ildlife International, Ltd. site, or at an alternative location to be specified in the final report. PROTOCOL NO.: 454/091799/NAV96/SUB454 004508 3M LAB REQUEST NO. U2723 Wildlife International, ltd -12- REFERENCES 1 U.S. Environm ental P ro tectio n A gency. 1996. Series 850- Ecological Effects Test Guidelines {draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II. 2 ASTM Standard Guide 1218-90E. 1990. Standard Guidefo r Conducting Static 96-Hour Toxicity Tests with M icroalgae. American Society for Testing and M aterials. Philadelphia, Pennsylvania. x> 3 N orberg-K ing, T J . 1993. A L inearinterpolation M ethodfor Sublethal Toxicity: The Inhibition Concentration (ICj) Approach (Version 2.0). U.S. Environmental Protection Agency, Environmental Research Laboratory, D uluth, Minnesota. 4 W est, In c and D. D. G ulley. 1996. TOXSTATRelease 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming. \ PROTOCOL NO.: 454/091799/NAV96/SUB454 004509 3M LAB REQUEST NO. U2723 Wildlife International, ltd -13TABLE1 FRESHWATER ALGAL MEDIUM WITH SILICA CONSTITUENTS Component Nominal C oncentration MgCl2*6H20 CaCl2*2H20 H3BO3 MnCl2*4H20 12.16 mg/L 4.40 mg/L 0.1856 mg/L 0.416 mg/L ZnCl2 3.28 g/L FeCl3-6H20 0.1598 mg/L CoC126H20 Na2M o042H20 CuC12*2H20 Na2EDTA-2H20 1.428 g/L 7.26 g/L 0.012 g/L 0.300 mg/L NaNOj M gS047H20 25.50 mg/L 14.70 mg/L K2H P04 1.044 mg/L NaHCOa N a2Si03*9 H 20 15.00 mg/L 20.0 mg/L Na2Se035 H20 0.010 mg/L The pH o fthe medium will be adjusted, as necessary, to 7.S 0.1 using 0.1 N NaOH or 10% HC1. PROTOCOL NO.: 454/091799/NAV96/SUB454 004510 3M LAB REQUEST NO. U2723 Wildlife International, ltd -14- APPENDIXI 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: 1.1JZ.2HJULH Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch N um ber Lot 217______________ j______ v. Test Substance Purity (% Active Ingredient): 98.9 E v iratio n Date: 2008 ' ______ E TestSubstanceCharacterization _Yes HL TestSubstance Storage Conditions PleaseindicatBfterectTOnendedstrTageooodilions atVfikDifehitemational,Ltd- Ambient____________________________________________________ Has the stabilityo fthe testsubstanceunderthese storageconditions been determinedin aocodancewith CEP Standards? _____ Yes Otherpertinent stability information: x __N o x No IV. TestConcentrations: Adjusttest concentrationto 100% ai. _ _ _ x _ _ _ b a s e d upon the purity (%) givenabove. Donot adjusttestconcentrationto 100% ___________ a l Testthe materialAS IS- V. ToxicityMxmation: Mammalian: RatLDSO 251m e/ke MouseLD50 N/A Aquatic: InvertebrateToxidty (EC/LC50) Fish Toxicity(LC50) Daphnia magna: 27 m e/L__________ Rainbow Trout: l l t n e / L Daphnia maena: 50m e/L__________ Fathead Minnow: 38 me/L Other Toxicity Information (includingfindings o fchronic and subchronictests): Please see MSDS________________________________________________________________ PROTOCOL NO.: 454/091799/NAV96/SUB454 004511 3M LAB REQUEST NO. U2723 Wildlife International, ltd -15- A P P E N D IX n Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: . 1. Liquid Chromatography M ass Spectrometry (LCMS) Method for the Determination o f Perfluorooctane Sulfonic A d d Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f the above method will be maintained in the raw data. The actual methodology used to analyze the test samples win be documented in the raw data and summarized in the final report PROTOCOL NO.: 454/091799/NAV96/SUB454 004512 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd.________________________________ PROJECT NO.: 454A-112 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER DIATOM (Navicula pelliculosa) PRO TO CO L NO: 454/091799/NAV96/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PR O JE C T NO.: 454A-112 EFFECTIV E DATE: February 23.2000__________________________ 3M LAB REQUEST NO.: U2723 AMENDMENT: Page 2 "v Add: Experimental Start Date: February 25,2000 Experimental Termination Date: February 29,2000 Test Concentrations: Negative Control, 70.0,92.5, 125,167,225,300 and 395 mg a.i./L Test Substance No.: 4675 REASON: The above information was not known when the protocol was signed by the Study Director. AMENDMENT: Experimental Design. Page 3 Change: Target concentrations will not exceed 100 mg/L or the solubility limit o f the test substance in water. To: Target concentrations will not exceed 1000 mg/L or the solubility limit ofthe test substance in water. REASON: Nominal test concentrations exceed 100 mg/L. Wildlife International ltd. PROJECT NO.: 454A-112 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER DIATOM (Navcula pelliculosa) PROTOCOL NO: 454/091799/NAV96/SUB454 AM ENDM ENT NO.: 2 SPONSOR: 3M Corporation PR O JE C T NO.: 454A-112 EFFECTIV E DATE: February 28.2000 3M LAB REQ U EST NO.: U2723 AMENDMENT: Biological-Measurements Page 6 Add: A t the end of the test, algistatic effects, which result in the inhibition o f cell growth, will be differentiated from algicidal effects, which result in the death o f cells. Aliquots (0.5 mL) o f test solution will be taken from each replicate o f those treatment groups where growth is maximally inhibited. Maximally inhibited treatments are those in which it cannot be visually determined if live algal cells are present (e.g., absence o f color to the test solution). These aliquots will be pooled by concentration and diluted w ith untreated culture medium to a concentration o f the test substance that theoretically should not affect growth. A negative control will be prepared by diluting a 0.5-mL aliquot from one negative control replicate to 100 mL with culture medium. Growth o f these subcultures will be monitored for a maximum o f 9 days to determine whether inhibition o f growth observed during the test is reversible. Samples will be collected for cell counts at recovery phase initiation, termination and at approximately 3-day intervals between initiation and termination. The recovery phase may be terminated once algal growth is sufficient to indicate that the algal cells collected from die treatment groups have frilly recovered from effects due to the test substance. This will be determined through visual examination o f the recovery test solutions. REASON: To add a recovery phase to the protocol.