Document Z4X3nXvLEBdxKb5rdJEnZejZ0

TOXICITY TO AQUATIC PLANTS TEST SUBSTANCE Identity:A mixturecontainingperfluorooetanesulfonamtaey; alsobe referretdo as PFOS, FC-95,or as a component ofFC-203C and L-5440.(1-Octanesulfoniaccid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecpaoftlausosriou-m, saltC,AS # 2795-39-3) Remarks: The 3M productionlotnumber was notnoted.The test sample isFC-203C and isreferretdo by thetestinglaboratoryas "81-1/8". Currentinformatioinndicateistisa mixtureof 1.47% testsubstance,60% water,30% diethyleneglycolbutylether,3% sodium octylsulfate3,% hydroxyfoamer,10% polyoxyethylenemonooetylphenylether,0.12% sodium laurylsulfatea,nd 0.05% benzotdazol. The followingsummary appliesto the testsample as a mixture of the testsubstance in water soludon with incompletelycharacterized concentradon of impurities.Data may not accuratelyrelatetoxicity of the testsample with thatof the tluorochomicalcomponent. METHOD: Method: USEPA - 600/9-78-018and ASTM-E-35.21 DraftNo. 1. Type (testtype): Acute Static GLP: No Year study pedormed: 1981 Species: Selenastrumcapricomutum Source: OriginalolbytainedfromUSEPA-ERL, CorvalliOsr,egon, maintainedinculturemedium atthe3M EnvironmentalLaboratoryS,t. Paul,MN. Element basis: Algalcellcounts(cells/mlc)e,lldry weights. Exposure period: 96 hours StatisticaMlethods: Regression model. Analyticalmonitoring: pH and temperature. Test Conditions: AlgalNutrientMedium: Nutrientniedium per EPA - 600/9-78-018.This nutrienstervedas thediluentforallalgaloperations.The pH ofthis synthefiaclgalmedium was adjustedto7.5priorto use inassays. Stock and testsolutionpreparation:Stock solutionwsere preparedin thealgalstandardtestmedium, however, no furtherdescriptiownas provided. Exposurevessels:250 ml Erlenrneyecrosntainin5g0 mitestsolution. AgitationS:hakencontinuousalty100 rpm. Number ofreplicate3s: Initicaelllloading1:.0X 104 cells/mL Number of concentrations: fiveplusa blankcontrol Lighting:400 foot-candlesfrom continuouscool-whitefluorescent lighting. Water Chemistry: pH range (0-96hours): Not noted. Test temperature range (0-96hours):23 + 2*C (incubator) RESULTS Nominal concentrations: Bk control2,80, 420,650, 1,000,and 1,550 mg/L Element values: 96-hour EC50 Cell-Dryweight: 408 (156 - 995) mg/L 96-hourEC50 AJgalCellCount: 420 (197- 868) mg/L Element values are based on nominal concentrations. Reversibilityof Growth InhibitionA:liquatsofthe 1,000 and 1,550 rng/L testsolutionswere dilutedwithalgalmedium and culturedfor10 days. Based on growth observed inthe recoveryphase, the effecton algal growth was found to be algistatic. Remarks: Testingwas conducted on the mixtureofthetestsubstance as describedinthe testsubstance remarks field.The values reportedapply tothatmixtureand not thefluorochemicalcomponents alone. CONCLUSIONS The testsubstance exhibitsa 96-hourEC5o cellcount value of420 (197 868 mg/L) and a 96-hourEC50 based on celldryweightof408 (156 - 995) mg/L. This testsubstancewas determined tobe algistatc. Submitter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota, 55133 DATA QUALITY ReliabilitKyl:imiscrhankin=g 2.Thisstudymeetsthecriterfioarquality testing.However, the study lacksinformationon purityofthetest substance and itlacksanalyticaclonfirmationofthe amount of fluorochemicalproportioninthe solutionT.here are alsosome errors (presumed typographicaelrrors)inthe report.The TechnicalReportcover sheet indicatesa "40 day exposure" and page 2 indicatesthe algalculture was storedinthe dark "at40OC". REFERENCES Test was conducted by the 3M Company, EnvironmentalLaboratory,St. Paul,MN, Lab Request number 6811 S, 1981. OTHER Last changed: 6/26/00 TECHNI.CAL REPORT. SUMMARY D ate 6/12/81 @O.:."TECtl@ft@CLO-MMUNICATIONS CE NTER - 201-2CN (important-Ifreportisprintedon bothsidesofpaper,sendtwocopiestoTCC.) Division . Environ mental Project Engineering & Pollution control Dept.,Numbw (Envir. Laboratory@ 0535 Proj*ctNumber Commercial Chemicals Report Title 9970012600 Report Number Algal Assay: Bottle Test(AA:BT), Toxicity Testing FC-203C 050 To E. A. Reiner 21-2W LR #6811 s Author(s) M. iTt.Elnabarawy Notebook Reference 2-3E Employee Numberts) 46981 -N-o,-o-f-Togin-caisuding Covershoot 5 SECURITY 10, Open:' Closed 3M CHEMICAL REGISTRY New Chemicals Reported Yes No KEYWORDS: (Selectternu from 3M Thesaurus. Sugpst other applicablte erms.) CURRENT OBJECTIVE: To determine the algal growth responses (EC following a 40-day exposure period to varying concenti2@Tons of IFC-203( followed by a .10-day recovery period. & PC Div. (Env. Lab) Algal Assay Bottle Test Toxicity REPORT ABSTRACT: (200-250words)ThisabstracitnformatioinsdistributbeydtheTechnicaClommunicationsCentert. alert3M'ersto Cormany R&D. The algal acute. 4-day EC -. values obtained for freshwater 50 green algA6 (Selenastrum capr cornutum) following exposure to FC-203C in tr-iplicate experiments were 408 and-420 mg/l.' These values represent the algal grovith response as measured by cell-dry weight and algal cell-count, respectively. No.test.material level. concentration was defined as the no-effect The. algal biomasq,.*moni.toring'measuremeiits -following, a 10-day recovery-period -iiidicated'insignif icaht --algicidal....feefxt.at concentrations tested. - Since the'algal-,cells have.recovered resumed logarithmic growth*when.-resuspended in fresh medmium,* the 4-day,median growth response (EC 50 is interpreted as an-inhibitory.effect. 7 InIddr. l7rK FC-203C/MTE Page Two June 23t 1981 CONCLUSIONS In these triplicated 4-day EC50 algal assays, the calculated values for freshwater green algae (Selenastrum Capricornutum) were 408 and 420 mg/l. These values represent the aigal growth response as measured by cell-dry weight and algal cell count, respectively. No test material concentration was defined as the-no-effect level. The algal biomass monitoring measurements following a 10-day recovery period indicated little or no algicidal effects at concentra-tions--tested. Since the algal cells have recovered-and .resumed logarithmic _qrowth when resuspended in fresh medium, the 4-day median growth response (EC50) is interpreted as an inhibitory effect. INTRODUCTION In testing the possible effects of chemical substances on the aquatic environment, unicellular algae are recommended as a model system for evaluating the influence of chemicals on aquatic primary producers, plants, and phytoplankton. Toxic effects are tested on growing algal cultures that undergo cell-division during the test. Algal growth response include: a) no effect, b) stimulatory, z) inhibitory, d) algistatic, or algicidal. The test substance is a clear amber liquid, soluble in water at ambient room temperature. These bioassays performed on this' material evaluated its potential algal toxicity and the data generated form the basis of this report., 'MATERIAL AND EXPERIMENTAL DESIGN These algal assays consisted of a 4-day exposure period followed by a 10-day recovery period. The test protocol utilized for this study was modified after those described by USEPA 600/9-78-018 and ASTM-E-35.21 Draft No. 1. (1), (2). Test Species: A culture of the freshwater green algae (Chlorophyc6me4, Selenastrum Capricornutum of the order chlorococcales was obtainecl from USEPA - E , Corv., Oregon (February 13, 1981). The test algae are non-motile unicellular cells and having the appearance of a new moon. The algal culture was stored in the dark at 400C until use.. 4 FC-203C/MTE Page Three June 23, 1981 Inoculum: Algae from a 7-day old stock culture were used as inoculum to give a starting optimum inoculum level of green algae Selenastrum Capricornutum 1 x 104 cells/ml. The use of a 7-10 day old stock culture will insure the presence of a sufficient amount of viable.algal cells in the exponential growth phase. The initial algal cell count in the stock culture was determined with the aid of a hemocytometer (370,000 cells/ml). Algal Culture Medium: Standard culture and test medium was prepared as outlined in Attachment #1. This algal culture medium provided all nutrients esseneial for algal growth and also served as the diluent for all algal operations including the preparation of stock solutions. The pH of this synthetic algal medium was adjusted to 7.5 prior to use in assays. Culture Flasks: -Each 250 ml erlenmeyer containing 50 ml of test solution comprised a test flask. Culture flasks and all other glassware were specially prepared as described in Attachment #2. Autoclaved foam plugs were used as flask closures, allowing for free gas exchange. Range-Finding Test-(Exploratory): The exploratory test consisted of determining the 4-day algal biomass in single flasks containing standard culture medium plus the test material at concentrations covering several orders of magnitude: 0.1, 1.0, 10, 100 and 1,000 mg/l.. Full-Scale Test-IDefinitive): All definitive algal assays were carried out in triplicate using 250 ml Erlenmeyer flasks containing.50 ml of te@st solution. The definitive test consisted of two culture stages: a 4-day exposure period followed by a 10-day recovery period. The purpose of the exposure (contact) period was to verify inhibitory effects. The recovery (subculture) stage provided information on' algistatic/algicidal effects., The following FC-203C logarithmic concentration: 280, 420, 650, 1,000 and 1,550 mg/l were used to initiate the 4-day contact period. This dose range of dilutions was selected to bracket 4-day EC50 values predicted from preliminary exploratory testing. Three flasks containing 100% fresh algal culture medium plus algal cells comprised the nontreated controls. The recovery stage was initiated by FC-203C/MTE Page Four June 23, 1981 subculturing (0.5 ml/50 ml) from each of the three replicates of the two highest test concentrations into flasks containing fresh algal medium. These two treatments induced inhibitory effects greater than 80% during the contact period. Fresh stock solutions of the test substance were prepared in the algal standard test medium. See Attachment #3. Test Conditions: All algal operations were carried out under sterile conditions, in order to avoid contamination with bacteria and other algae. Algal cultures were incubated in an environme ntal chamber under the following standard growth conditions" : *Temperature - 23 + 20C (70-770F) *Fluorescent illumination - 400 ft.C. + 10% *iree gas exchange - Continuous Platform Shaking 100 + 10 rpm. Algal Biomass Monitoring: The algal growth response was measured by algal cell-dry weight (mg/1) and by cell count (cells/ml). Both growth measurements were made with.algal cultures after the 4-day exposure period and ofily cell-count was made with the sulcultures after a 10-day recovery period. Both procedures of biomass measurements are briefly described in Attachment #4. EC50 values and 95% confidence limits we're calculated utilizing 3M SIXCUR, a TRAC System for regression models of experimental data. TEST RESULTS The results of biomass measurements of the green algae Selenastrum Capricornutum used in these studies are selt-explyn-atory and-i-re-detailed in data sheets attached. The results of the definitive test are summarized in Table 1. FC-203C/MTE Page Five 'June 23, 1981 Table 1 Definitive Tes@ EC50 valuesp mg/l with Selenastrum Capricornutum Type Test Algal Biomass Monitoring Cell-Dry Weight mg/l Cell Count No./l ml 4-Day Exposure Period 408(156-995)* 420(197-868)* *95% Confidence limits. The data reported herein are based on studies performed by M. T. Elnabakawy. Accompanying data sheets Comprise original data. A copy of this report is filed in the Environmental Laboratory Archive System, Building 2-3E. It should be noted that the reported EC50 values were evaluated on the basis of the calculated initial concentrations of FC-203C in.test solutions. REFERENCES (1)Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. The Selenastrum Capricornutum P.rintz Algal Assay Bottle Test: Experimental design, application, and data interpretation. U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-600/9-78-018. 125 p. (2) ASTM-E-35.21. 1980. Proposed Conducting Toxicity Tests,with Algae. Draft No. 1. Standard Practice For Freshwater and Saltwater ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (AA:BT) TOXICITY TEST DATA SHEET LR Type Test ---- Material Tested 15 C- 0 S'UC ot Physical Description Cieo,,r A-%6t-wr- L!@ .,A Algal Species:Green Aliaeq Scienastrum capricornutum U.C e r A Lo,6 Source (:Ocvakks Date Rec'd YL Age of lnoculurn Aov Test Container:250 mi Erierimeyer flasks with foam plugs Sample-to-Volume Ratio: -190 Initial Coll Loading: 10000 calls/mi Initial pH of Synthetic Algal Madium:.7.5* 0.1 Test Conditions: 0 Temperature - 23* 20C (70-Z7 F) Fluorescent Illumination WO--ft.c. 10 % Free Gas Exchange Continuou!-e &T--tTo-rSma-kin_q_at 100110 Date Started (o Date Cwo feted (p - / S-- F investigator rpm. ALGAL CELL DRY WEIGHT, HEAN VALUE In TYPE TEST TREATMENT EXPOSURE: 4-DAY CONTACT PERIOD RECOVERY: 10-DAY SUBCULTURE PERIOD CONIROL -- "120 1,000 ALIGAL tiK COWT" Nn FFFErTSIST114ULATf)RY/IKHIIUTnR NML -.SE6 COUUI ALCISTATIJ/ALGICIDAL AN VALUE NO.Lal III RREATMENT EXPO .'ONTROL RitS oo-t)oo 000 -2 q 6.@-000 s-o 1,000 1. so,ooo do.-OOO E-C IL ITPIY ALCISTATIC,/ALGICInAL (1) Based on mean values of each triplicate sets of flasks. (2) Method of calculations; ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (AA:BT) .,RECOVERY STAGE DATA TUMMARY ALGAL CELL COUNT. Cellslml (MEAN VALUES) TREATMENT. CONTROL OOC> 2 3 4 5 REF. TOX. INITIAL ALGAL CELL LOAD ING y 4660 700 0 0- FINAL ALGAL CELL COUNT (2) '-290000 40()0 00 C)C)O I (1). Based on 0.5m)/50m] subculture made from 4-day exposure cultures. (2). 10-day recovery cultures.