Document 1Q1V8k7Gb4QGB0jRbDaJNMRwa

TOXICITY TO AQUATIC PLANTS TEST SUBSTANCE Identity:A mixturecontainingperfluorooctanesulfonatmea;y alsobe referredtoas PFOS, FC-95, oras a component ofFC-206C and 81-2/8.(1-Octanesulfoniaccid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafploutoarsos-i,um salt,CAS # 2795-39-3) Remarks: The testsample isFC-206C. Currentinformatioinndicatesit isa mixtureof0.73% PFOS, 76.09% water,15% diethyleneglycolbutyl ether,4% urea,1.55% sodium octylsulfate1,.47% hydroxyfoamer, 1.05% polyoxyethylenemonooctylphenylether,0.06% sodium laurylsulfatea,nd 0.05% benzotriazole. The followingsummary appliesto a mixture with incompletely characterizedconcentration of impudties. Data may not accurately reflectthe toxicityof the fluorochemical compnent of the test sample.. METHOD: Method: USEPA - 600/9-78-018and ASTM-E-35.21 DraftNo. 1. Type (testtype): Acute Static GLP: No Year study performed: 1981 Species: Selenastrumcapricomutum Source: Originalloybtainedfrom USEPA-ERL, CorvallisO,regon, maintainedinculturemedium atthe 3M EnvironmentalLaboratory,St. Paul,MN. Element basis: Algalcellcounts (cells/mlc)e,lldry weights. Exposure period: 96 hours StatisticaMlethods: Regression model. Analyticalmonitoring: Not given. Test Conditions: Algal NutrientMedium: Nutrientmedium per U.S. EPA 600/9-78-018. This nutrienmtedium providedallmineralnutrientesssentialforalgal growth and alsoserved as thediluentforallalgaloperations.The pH of thissyntheticalgalmedium was adjustedto7.5 priorto use inassays. Stock and testsolutionpreparation: Stock solufionswere prepared in thealgalstandardtestmedium, however no furtherdescdpfionwas provided. Exposurevessels2:50 mlEriennieyecrosntaini5n0gmitestsolution. AgitationS:hakencontinuousalty100rpm. Number ofreplicate3s: Initicaelllloading1:.0 X 104 cells/mL Number of concentrations: fiveplusa blank control Lighting:400 foot-candiesfrom continuouscool-whitefluorescent lighbng. Water Chemistry: pH range (0-96 hours): 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: 345 (34- 1,630)mg/L 96-hourEC50 AlgalCellCount: 379 (106- 1,137)mg/L Element valuesare based on nominalconcentrabons. Reversibilitoyf Growth InhibitionA:liquotsofthe 1,000and 1,550 mg/L testsolutonswere dilutedwithalgalmedium and culturedfor10 days. Based on growth observed inthe recoveryphase,the effecton algal growthwas found to be algistatic. Remarks: Tesbng was conducted on the mixtureas describedinthetest substance remarks field.The valuesreportedapplytothatmixtureand notthe fluorochemicalproportionalone. CONCLUSIONS The testsubstance exhibitas 96-hourEC5o cellcount valueof379 (106 1,137) rng/Land a 96-hourEC50 based on celldryweightof345 (341,630)mg/L. Thistestsubstance was determinedto be algistatic. Submitter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota,55133 DATA QUALITY ReliabiliKtlyi:miscrhankin=g2.Thisstudymeetsthecriterfioarquality testing.However, the studylacksinformatioonn purityofthe test substance and itlacksanalyticaclonfirmatioonftheamount of fluorochemicalproportioninthe solution. REFERENCES Testwas conducted by the 3M Company, EnvironmentalLaboratory,St. Paul,MN, Lab Request number 6812S, 1981. OTHER Last changed: 6/26/00 F o rm 6/.47'I-- B TECHNICAL REPORT to: !TEC NICAL COMMUNICATIONS CENTER - 201-2CN SUMMARY Date 6/23/81 (ImportantI-freporitsprinteodn bodisidesofpapewrn,dtmcopiesto TCC.) Division Environmental Project 'laa2mikillw Report Title Algal Ass%ay: To Engineering and Pollution Control (Envir. Lab) %4-Vl Bottle Test (AA:BT), Toxicity Testing FC-206C Dopt. Number 0535 Project Number - 9970012600 Report Number 1051 E. A. Reiner - 21-2W LR 6812 S Author(s) M. T. Elnabarawy - 2-3E Employee Nurnber(s) 46981 Notebook Reference No. of Pages Including Coversheet 5 SECURITY@. Open Closed 3M CHEMICAL REGISTRY New Chemicals Reported 0 Yes No KEYWORDS: (Selectterms from 3M Thesaurus. Suggest other applicable terms.) CURRENT OBJECTIVE: To determine the algal growth responses (EC following a EE & PC Div. 4-day exposure period to varying concentrai?O'Rs followed by a 10-day recovery period. of FC-206C, (Env. Lab) Algal Assay Bottle Test Toxicity . REPORT ABSTRACT: (200-250 words) This abstractinformationisdistributebdy the TechnicalCommunications Center t( alert3M'ers to Company R&D. The algal acute 4-dag EC values obtained.for freshwater 5 green algae (Selenastrum 8apricornutum) following exposure to FC-206C in triplicate experiments were 345 and 379 mg/l. These values represent the-algal growth response as measured by cell-dry weight and algal cell-count, respectively. No test material concentration level. was def ined as the no-ef f ect The algal biomass monitoring measurements following a 10-day recovery period indicated insignificant algicidal effect at concentrations tested. Since the algal cells have.recovered and resumed logarithmic growth when resuspended in fresh medium, the 4-day median growth as an inhibitory effect. response (EC 50 is interprete :nformationLiaison nitials: FC-206C/MTE Page Two June 23, 1981 CONCLUSIONS In these triplicated 4-day EC50 algal assays, the calculated values for freshwater green algae (Selenastrum Capricornutum) were 345 and 379 mg/l. These values represent the algal growth response as measured by cell-dry weight and algal cell count, respectively. No test material concentration was defined as the no-etfect level. The algal biomass monitoring measurements following a lo-day recovery period indicated little or no algicidal effects at concentrations tested. Since the algal cells have recovered-and resumed logarithmic growth 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,_c) 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 (Chlorophyd(--ae), Selenastrum Capricornutum of the order chlorococcales was obtained from USffP-A-- ERL, 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.. FC-206C/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 I x 104 cells/ml. The use of a 7-10 day old stock culture will insure 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 essential 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 (Explorf!tory)-: The exploratory test consisted of determining the 4-day algal biomass in single flasks containing standard culture medium p us the test material at concentrations covering several ers of magnitude: 0.1, 1.0, 10, 100 and 1,000 mg/l. Full-Scale Test (Definitive): All definit.ive algal assays were carried out in triplicate using 250 ml erlenmeyer flasks containing 50 ml of test 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-206C logarithmic concentration: 2.8.014,2-0, 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. algal culture medium nontreated controls. Three flasks containing 100% fresh plus algal'cells comprised the The recovery stage was initiated by FC-206C/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 great6r 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 environmental chamber under the following standard growth conditions: *Temperature 23 + 20C (70-770F) *Fluorescent illumination - 400 ft.C. + 10% *Free 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 only cell-count was made with the sulcultures after a 10-=day recovery period. Both proceduresof biomass measurements are briefly described in Attachment #4. EC50 values and 95%,confidence limits were 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 self-explanatory and are detailed in data sheets attached. The results of the definitive test are summarized in Table 1. FC-206C/MTE Page Five June 23, 1981 Table 1 Definitive Test - ECSO values, mg/l with Selenastrum Capricornutum Type Test Algal Biomass- Monitoring Cell-Dry Weight mg/l Cell Count No./l ml 4-Day Exposure Period 345(34-1630)* 379(106'1137)* *95% Confidence limits. The data reported herein are based on studies performed by M. T. Elnabarawy. 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-206C in test solutions. REFERENCES (1)Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. The Selenastrum Capricornutum Printz Algal Assay Bottle Test: Experimi@nt-al design, ica'tion, and data interpretation. U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-600/9-78-018. 125 P. (2) ASTM-E-35.21; 1980. Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 1. ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (AA:BT) TOXICITY TEST DATA SHEET LR Type Test Material Tested F-C Ex;a.5,ufp- -,._Lot Physical Description Algal Species:Green Algae, Solonast rum ca-or-icornutum u S' i@ L,%k, Sou rce (@@ c 1/4\\,S' A,@ Date Rec'd Age of Inoculum Test Container:250 ml Er)enmeyer flasks with foam plugs 7 Ao, Sample-to-Volume Ratio: ZO tc> -ZS-O Initial Coll Loading: 10000 calls/mi Initial pH of Synthetic Algal Medium: 7.5* 0.1 Test Conditions: Temperature - Z3* 20 c (70-7Z0F) Fluorescent IITLoi-nation - 406 ft.c.:t 10 % Free Gas Exchan" - Continuous platform -shaking at 100210 rpm. Date Started 91 Date CoM leted investigator tA.'T.E'IrNa6 ALGAL CELL DRY WEIGHT, MEAN VALUE In mg/l TYPE TEST TREATMENT EXPOSURE: 4-DAY CONTACT PERIOD @REC RECO0VERY: 10-DAY SUBCULTURE PERIOD CONTR()L @l 2 0 6 1000 7 'g gC50 (2) ALriAL tikuwln FrrFrTt TCREATMELiZN(gITDD TREATMENT CONTROL -2L _ Nn FFFFtTS/qT INNINITNRY ALGISTATIC/ALGICIDAL c gulls nfbn VALUE W. 40.L.1lior TFCT XxPpO(SURE? y FRIM 660 IRF PI:RInn 0-ooo i 'Z0 ,Ooo '0,00 4 /go-oloo 2) qL @kFP4qWT H Nn FFP ULTORY (1) "sed on meanvaluesof eachtriplicatseetsof flasks. (2) Methodof calculations: GICIBAL F TREATMENT CONTROL 100L) 2 3 4 5 REF. TOX. ENVIRONMENTAL LABORATORY ALGAL ASSAY- BOTTLE TEST (AA:BT) LI@ RECOVERY STAGE DATA SUMMARY ALGAL CELL COU04T, fells/ii-,l(MEAN VALUES) 0(0 INITIAL ALGAL CELL LOADING 2) FINAL ALGAL CELL COUNT OC>o (ooo Ll o o o 0 0 '000 Based on 0.5m]/50rnisubculture made from 4-day exposure cultures. 10-day recovery cultures.