Document e5YwBk0xnVLx9QJEjwKOewRR4

A R tL 6 -c m o s s -3 PROTOCOL PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Anabaenaflos-aquae) U.S. Environmental Protection Agency ^'S eries 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 3M Lab Request No. U2723 S ubm ittedto 3M Corporation Environmental Laboratory 935 Bush Avenue S t Paul, Minnesota 55106 Wildlife International, Ltd. t 8598 Commerce Drive Easton, Maryland 21601 (410)822-8600 September 17,1999 G044S2 Wild life International, ltd -2- PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Anabaenaflos-aquae) SPONSOR: 3M Corporation Environmental Laboratory 935 Bush Avenue S t Paul, Minnesota 55106 SPONSOR'S REPRESENTATIVE: Rochelle R Robideau TESTING FACILITY: W ildlife International, Ltd. 8598 Commerce Drive Easton, M aryland 21601 STUDY DIRECTOR LABORATORY MANAGEMENT: Cary Sutherland, Senior Biologist Henry O. Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants __________________________ FO R LABORATORY USE ONLY_________ Proposed Dates: Experimental Start D a te :____________________________ E xperim ental Termination Date: .- P rq jectN o .:______ ( / O ____________________________________________ Test C oncentrations:_______ :________________________________________ Test Substance No.: 4675 Reference Substance No. (if applicable): 4526 PROTOCOL APm VAL STUDY DIRECTOR DATE DATE PROTOCOL NO.: 454/091799/ANA96/SUB454 004483 3M LAB REQUEST NO. U2723 Wild life International, ltd -3 - INTRODUCTTON W ildlife International, Ltd. will conduct a four-day toxicity test w ith the freshwater alga, Anabaena flos-aquae, for the Sponsor at die Wildlife International, L td 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: A lgal Toxicity, Tiers I and I I (draft) (1). Raw data for all work performed at W ildlife International, L td and a copy o f the final report will be filed by project number in archives located on the Wildlife International, L td 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 A cid Potassium Salt (hereafter referred to as PFOS) to the freshwater alga, Anabaenaflos-aquae. EXPERIM ENTAL DESIGN The freshwater alga, Anabaena flos-aquae, 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 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 facto: 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 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 the 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 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 (ErC) will be calculated, if PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 004484 Wild life 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 on growth, will be determined relative to each parameter at 72 and 96 hours based upon evaluation o f the statistical results and die doseresponse pattern. A t the end o f the 96-hour exposure, algistatic effects w ill be differentiated from algicidal effects in those treatments which are maximally inhibited. M ATERIALS AND M ETHODS Test Substance 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 die 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, L td , 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 PFOS will be administered to the test organism in algal medium. This route o f administration was selected because.it represents the most likely route o f exposure to algae, which exist suspended in a water column. .. ......... \ ...... Test Organism The test species will be the freshwater alga, Anabaenaflos-aquae. 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 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U 2 7 ||Q 4 4 5 Wild life International, ltd -5 - Austin or another supplier, will be maintained in culture medium at Wildlife International, Ltd. for a minimum o f two weeks prior to use in a toxicity te s t 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 die test, an inoculum o f 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. V/ 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 l (2). Stock nutrient solutions will be prepared by adding reagent-grade or better chemicals to Wildlife 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 o f selected organic and inorganic constituents o f the well water and results of the m ost 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. T est 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 rp m Test chambers will be labeled with the project number, test concentration, and replicate. Environmental Conditions Test flasks will be held at 24 2C under continuous cool-white fluorescent lighting at an intensity o f 2200 10% lux. Light intensity will be measured at five locations surrounding the test flasks on Day 0 PROTOCOL NO.: 454/091799/ANA96/SUB4S4 3M LAB REQUEST NO. U 2 7 ^ ^ g g Wild life International, ltd 6- - o f the te st Temperature will be measured twice daily in a container o f water adjacent to the test flasks in the environmental chamber using a liquid-in-glass thermometer. The pH o f each treatment and control group will be measured at test initiation and termination using a Fisher Accumet Model 915 pH meter or equivalent Samples for 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 die 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 24hour intervals during the exposure period. The samples will be held 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. Using this technique, the minimum quantifiable cell density is 1000 cells/mL. Samples o f test solutions will be collected from each o f the three replicates per treatment and control group at the end o f the test These samples will be pooled within their respective treatments, and subsamples will be removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Growth o f cells in the flasks also will be assessed for aggregations or flocculations o f cells and adherence o f the cells to the test chamber. A t the end o f 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 with 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 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 Wild life International, ltd -7 - 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 die algal cells collected from the treatment groups have fully recovered from effects due to the test substance. This will be determined through visual examination o f the recovery test solutions. Sampling for Analytical Measurements Samples o f the exposure solutions will be collected cmDays 0,3 and 4 and analyzed for test substance concentrations. Day 0 samples will be taken from each batch o ftest 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 die sample for that treatm ent Day 4 samples will be composited solutions from the three replicates remaining at the end o f die te st 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 Day 0 Day 3 D ty4 Control Solvent Control (if needed) Level 1-Low Concentration Level 2 Level 3 _Level 4 \ Level 5 Level 6-High Concentration 11 1 1 -1 1 11 1 11 1 11 1 1 1. 1 11 1 1 21 2* Totals 'includes abiotic samples. 89 9 0044S8 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 Wild life International, ltd - 8- 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 will be performed by W ildlife International L td The analytical method used will be basednipon methodology provided by the Sponsor and identified in Appendix IL Modifications made to the analytical method w ill be documented in the raw data and described in the final report. Statistical Analyses Biomass expressed as area under the growth curve will be calculated for the treatment and control groups using die following formula: A = ((N1-N0)/2Xt1>+<(N,+N2-2N0)/2)(t2-t1)+...+((NlM+Nn-2N(J)/2)(tn-tt, l) Where: A = Area under the growth curve N0- Measured number o f cells/mL at to Ni Measured number o f cells/m L at ti N2Measured number o f cells/mL a tt2 N,, Measured number o f cells/mL at L ti = Time o f first measurement after beginning o f test (hours) t2 Time o f second measurement after beginning o f test (hours) t,, = Time o f nth measurement after beginning o f test (hours) Growth rates (jx) will be calculated for the treatm ent and control groups using die following formula: ' lnNa-lnNo ^ = ---------------- tn-to n Average specific growth rate Na = Measured number o f cells/mL at to Nn Measured 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) 004439 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 Wild life International, ltd -9 - Percent 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. x ell 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 the treatment groups will be identified using analysis o f variance (ANOVA) and a test to compare treatment"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 the 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-test Data from the two control groups will be pooled for treatment level analyses if no statistical differences exist Otherwise, all comparisons will be made to the solvent control group. Statistical comparisons for the ANOVA and the 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, 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. 004490 PROTOCOL NO.: 454/091799/ANA96/SUB4S4 3M LAB REQUEST NO. U2723 Wild life International, ltd -10- 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. 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 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 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 treatment, and duration o f the te s t -40r- 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 the names o f other scientists, professionals, and supervisory personnel involved in the study. 004491 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 Wild life International, ltd -li- 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 die 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 anagement 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. 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 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 w ith 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 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 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 Wildlife .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. 004492 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 Wild life International, ltd - 12- REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines {draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and n. 2 ASTM S tandard Guide 1218-90E. 1990. Standard Guidefo r Conducting Static 96-Hour T a d d ty Tests with M icroalgae. American Society for Testing and M aterials. Philadelphia, Pennsylvania. 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, Inc. and D. D. Gulley. 1996. TOXSTATRelease 3.5. W estern EcoSystems Technology, Inc. Cheyenne, Wyoming. PROTOCOL NO.: 454/091799/ANA96/SUB454 004493 3M LAB REQUEST NO. U2723 Wild life International, ltd -13TABLE 1 FRESHWATER ALGAL MEDIUM CONSTITUENTS Component Nominal Concentration MgCl26H20 CaClj-MaO H3BO3 M hCl24H20 ZnCl2 FeCl3`6H20 CoC12*6H20 Na2M o04*2H20 CuC12-2H20 N a2EDTA2H20 N aN 03 M gS04*7H20 K2H P04 NaHCOs 12.16 mg/L 4.40 mg/L 0.1856m g/L 0.416 mg/L 3.28 g/L 0 .1598mg/L 1.428 g/L 7.26 g/L 0.012 g/L 0.300 mg/L 25.50 mg/L 14.7 mg/L 1.044 mg/L 15.00 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% HCL PROTOCOL NO.: 454/091799/ANA96/SUB454 004494 3M LAB REQUEST NO. U2723 Wild life International, ltd -14- APPENDIXI IDENTMCATION OF TEST SUBSTANCEBY SPONSOR To be Completedby Sponsor I. Test Substance Identity (name to be used in the report): PFOS flPerfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A_______________________________ Internal Standard: 1.1-2.2H.H.H.H Perfluomnctane Sulfonic Acid Test Substance Sample Code or Batch N um ber Lot 217______________ _______ _ Test Substance Purity (% Active Ingredient): 98.9______ Expiration Date: 2008 IL Test Substao Characterization Havethe identity, strength,parity and compositionorotfagcharacteristics whichappropriatelydefine thetest sub&mceandreference standardbeen determinedpriorto its in this stocfyin accordancewi&GLP Standards? ______Yes x No DL TestSubstance StorageConditions Please indicatethe recommendedstorageconditions atWildlifeInternational, Ltd Ambient ___________________________________________________________ * - Has the stabilityo fthe testsubstanceunderthese storageconditions been determinedin accordancewith GLP Standards? Other pertinent stability information: _____ Yes x No IV. TestConcentrations: -_____ x Adjusttestconcentrationto 100% a i based uponthe purity (%) given above, Do notadjusttest concentrationto 100% ___________ a i TestmematerialAS IS. V. ToxicityInformation: Mammalian: RatLDSO 251 m e/ke Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity(LC50) Daphnia mapna: 97 mg/L__________ Rainbow Trout: 11 mg/L Daphnia maena: 50 me/L__________ Fathead Minnow: 38 mg/L OtherToxicityInfonnation(induding findings ofchronic and subchronictests): - I -- Please see MiSDS ---------------------------------------------- ------- -------------------------- - PROTOCOL NO.: 454/091799/ANA96/SUB454 004495 3M LAB REQUEST NO. U2723 Wild life International, ltd -15- APPENDEXn Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. liq u id Chromatography Mass Spectrometry (LCMS) Method for the Determination o f Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) InFresbwater, Saltwater and Algal Medium A copy o f die above method will be maintained in die raw data. The actual methodology used to analyze the test sanqiles wiH be documented in the raw data and summarized in the final report / \ \ PROTOCOL NO.: 454/091799/ANA96/SUB454 004496 3M LAB REQUEST NO. U2723 Wild life International ltd. PROJECT NO.: 454A-110 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Anabaena fios-aquae) PROTOCOL NO: 454/091799/ANA96/SUB454 AMENDMENT N O.: 1 SPONSOR: 3M Company PR O JE C T N O .: 454A-110 EFFECTIV E DATE: February 23,2000__________________________ 3M LAB REQ U EST NO.: U2723 AMENDMENT: Page 2 Add: Experimental Start Date: February 25,2000 Experimental Termination Date: February 29,2000 Test Concentrations: Negative Control, 3 9 .5 ,6 1 .0 ,9 2 .5 ,1 4 5 ,225.and 345 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 lim it ofthe test substance in water. REASON: Nominal test concentrations exceed 100 mg/L. 2 . RY MANAGEMENT iyfr A'&oo