Document Moy6mBR8b9m51xJd0vVGQNwxz

TOXICITY TO AQUATIC PLANTS TEST SUBSTANCE Identity:N-ethylperfluorooctanseulfonamidoethanolm;ay alsobe referredto as N-ETFOSE Alcoholor FM-3422. (1-Octanesulfonamide,N-ethyl1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-N-(2-hydroxyethyl@, CAS # 1691-99-2) Remarks: Materialisan off-whitew,axy solidofuncharacterizedpurity:The limitedwater solubiliotfythismaterialprohibiteda definitive determinationofthe aquatictoxicity. METHOD Method Followed: Modified(modeled) afterthose described by USEPA - 600/9-78-018;ASTM-E-35.23 DraftNo. 2; OECD; A.G. Payne. Type (testtype): Acute Staticbioassay GLP: No Year study performed: 1981 Species/Strain/SupplierF:reshwatergreen algae,Selenastrumcapticomutum. Obtainedfrom USEPA - ERL, Corv.,Oregon (July14,1981). Analyticalmonitoring: Algalcellcounts(cells/maln)d temperature. Exposure period: 4,7,10,and 14 days StatisticaMlethods: EC50 valuesand 95% confidencelimitwsere calculatedutilizianglinearregressionmodel. Test Condition Remarks: lnoculum: Algae from a 7-day-oldstockculturewere used as inoculum togivea startinogptimum inoculum levelofgreen algae 1XI 04 cells/ml.The use of a 7 to 10-day-oldstockcultureinsuredthe presence ofa sufficienntumber ofviable algalcellsinthe exponentialgrowth phase. The initialgalcellcount inthe stockculturewas determined usinga hemocytometer (338,000cells/ml). Algal NutrientMedium: Sterilesyntheticalgalnutrienmtedium. This nutrient medium providedallmineralnutrientsessentialforalgalgrowth and alsoserved as the diluentforallalgaloperations.The pH ofthissyntheticalgalmedium Was adjustedto7.50.1priortouse inassays. Culture Flasks: Sterilecultureflasksconsistedof 250 mi Edenmeyers containing50 ml syntheticalgalnutrientmedium. Full-ScaleTest (Definitive): Alldefinitivaelgalassays were carriedout intriplicautseing250 mi Edenmeyer flaskscontaining50 ml oftestsolution.The definitivaessay consistedoffour exposure periods:4, 7, 10,and 14 days. The followingN-ETFOSE Alcohol,Lot716, logarithmiconcentrations:180,320, 560, 1000, and 1800 mg/L were used to initiathee exposure study (individual weights).Three flaskscontaining100% freshalgalnutrienmtedium plusalgal cellscomprised the nontreatedcontrols. 0.5 mi Ethanol/ 1 litearlgalnutrienmtedium was used as a carriersolvent. Test Conditions: Allalgaloperationswere carriedout under asepticconditionsinorderto avoid contaminationwithbacteriaand otheralgae. Algalcultureswere maintainedin an environmentalchamber under the followingstandardgrowth conditions: Temperature: 2320C (70-770F) Fluorescent illumination: 400 ft.candles 10% Free gas exchange: ContinuousPlatformShaking,100 10 rpm. RESULTS Testingended with4,7, 10,and 14-day calculatedEC50 valuesgreaterthanthe solubilitlyimiotfthetestmaterial.EC5o calculatedvalueswere greaterthan 1800 mg/L (based on reductionincellcount). CONCLUSIONS CalculatedEC50 values indicatethismaterialhas insignificatnotxicittyoalgae. DATA QUALITY ReliabilityK:limischranking= 3.This studylacksinformatioonn purityofthe testsubstance and actualmeasurements ofthe amount oftestsubstance in solution.Testingwas conducted atapproximately5 ordersofmagnitude above the solubilitlyimiotfthe testsubstance. REFERENCES 3M TechnicalReport Summary, Multi-PhaseAlgalAssay Test Method, New Methods Development - N-ETFOSE Alcohol,Lot716, Report Number 008, ProjectNumber 9970030000, M. T. Einabarawy,December 30, 1981. OTHER Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul, Minnesota, 55133 Last changed: 5/18/00 Form 6747-11-C TECHNICAL REPORT SUMMARY 0ate 12/30/81 TO: TECHNICAL COMMUNICATIONS CENTER - 201-2Ci,4 (Importan-t ifreporitsPrinteodn bothsidesofpaper,sendtwocopiestoTCC.)See "GuidelinefsorCompletionof TechnicaRleport Summary " availablferomInformationLiaisoonr TCC (3-5545). Division Environmental Project New Methods Agport Title Laboratory Development (EE & PC) - FM-3422, Lot 716 r4ulti-Phase To Algal Assay Test Method Dopf- Number 0535 Project-Nurnber 9970030000 We-P-0-r-t-Wu-Mber 008 D. Bacon/R. Authorts) Bohon/E. M. T. Elnabarawy Notebook Reference Reiner/A. Welter EmployeeNumt)or(s) 46981 No. ofPages IncludingCovarshoot SECURITYPP,, KEYWORDS: IncludeLab Code r-IOpen (6ompany Confidential) 13 Ciosed (SpecialAuthorization) 3M CHEMICAL REGISTRY Now ChemicaRlesported C3 Yes NO No CURRENT OBJECTIVE: 1. To evaluate green algae the algal growth response of freshwater "Selenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical FK-3422, Lot 716. 2. To develop an algal test protocol based on variable exposure or recovery periods to the test compound. EE & PC Div. (Env. Lab) REPORT ABSTRACT: Thisabstracitnformatioinsdistributbeyd theTechnicaClommunicationsCentertoalert3M'ersto Company R&D.itisCompany confidentimaalterial. In these triplicated effects on organisms four-phase assays, the initial following varied exposures to FM-3422 were assessed an indication populations. over several generations. of the possible effects on Algal biomass was assessed Test results algal gave t quaneitatively in terms of algal cell-count (No./ml), and the median growth response EC50 (mg/1) was calculated infoSrImSaL,,on Initi FM-3422/MTE Page Two December 30, 1981 CONr-LUSI@-JNS 1) Materials possessing an EC50 greater than 1,000 mg/l are considered to be insignificantly phytotoxic to algae and aquatic plant species. 2) Results indicated that extended exposures (e.g., >7 days) induced no greater reduction in algal cell counts. SUMMARY A four-phase algal assay was developed to evaluate algal growth response of the freshwater unicellular green algae "Selenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical, Fri-3422. This multiphase algal assay evaluated algal growth responses during four consecutive exposure periods: 4, 7, 10, and s Algal growth response was measured in terms of an increase in biomass as algal cell-count (No./ml). Cell-count results used for the calculation of EC50's (mg/1) were averages of triplicated sets of test culture flasks. In these triplicated multiphase algal assays, the calculated EC50 values (mg/1) for freshwater green algae "Selenastrum capricornutum" are presented in Table 1. These values represent the median growth response (EC50 mg/1) following exposure to the test material for 4, 7, 10, and 14 days. 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 algal growth (aquatic primary producers), aquatic plants, and phytoplankton. Toxic effects are tested on growing algal cultures that undergo cell-division during the test. These growth responses may be: a) stimulatory, b) inhibitory and/or algicidal. MATERIAL AND EXPERIMENTAL DESIGN The test subs4%-.anc(eFM-3422, Lot 716) is an off-white solid, with limited solubility in water at ambient room temperature. The bioassays performed on this material evaluated its potential algal toxicity and the data generated form the basis of this report. The test protocol utilized for this study was modified after those described by USEPA - 600/9-78-018; ASTM-E-35.23 Draft No. 2; OECD; A. G. Payne. (1), (2), (3), (4). FM-3422/MTE Page Three December 30, 1981 Test Species: A bacteria-free culture of the freshwater planktonic green algae (Chlorophyceae), Selenastrum capricornutum of the order chlorococcales was obtained fr USEPA - ERL, Corv.,.Oregon (July 14, 1981). The test algae are non-motile unicellular cells, having the appearance of a new moon. The algal culture was stored in the dark at 40C. Inoculum: Algae from a 7-day-old stock culture were used as inoculum give a starting optimum inoculum level of green algae Selenastrum capricornutum 1 x 104 cells/ml. The use of a to 10-daTz@Tid stock -c-urt-ureinsured the presence of a sufficient number of viable algal cells in the exponential growth phase. The initial algal cell count in the stock culture was determined using a hemocytometer cells/ml). (338,000 to 7 Algal Nutrient Medium: Mineral (inorganic) standard nutrient medium for culturing and testinq algae was prepared as outlined in Attachment #1. This nutrient medium provided all mineral nutrients essential for algal growth and also served as the diluent for all algal operations. The pH of this synthetic algal medium was adjusted to 7.5+.l 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 used as flask closures, permitted free gaseous exchange to occur. Full-Scale Test (Definitive): All definitive algal assays were carried out in triplicate using 250 ml Erlenmeyer flasks containing 50 ml of test solution. The definitive assay consisted of four simultaneous exposure tests: 4, 7, 10 and 14 days. The following FM-3422, Lot 716 logarithmic concentrations: 180, 320, 560, 1,000 and 1,800 mq/1 were used to initiate the exposure study. Three Elasks containing 100% fresh algal nutrient medium plus algal cells comprised the nontreated controls. Procedure and steps for preparation of algal test flasks are outlined in Attachment #3. Fli-3422/MTE Page Four December IJ3, 1981 Test Conditions: All algal operations were carried out under aseptic conditions, in order to avoid contamination with bacteria and other algae. Algal cultures were maintained 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 appraised quantitatively by using algal cell counts (cells/ml). Procedures for algal biomass measurements are briefly described in Attachment #4. EC50 values and 95% confidence limits were calculated utilizing 3M SIXCUR, a TRAC System for reqression models of experimental data. TEST RESULTS The results of biomass measurements of the green algae Selenastrum capricornutum used in these studies are self =exp-lanatory and are detailed in the attached data sheets. Calculated EC50 (mg/1) values indicating algal growth response to FM-3422, Lot 716, following exposure periods of 4, 7, 10, and 14 days are presented in Table 1. EC50 calculated values were greater than 1,800 mg/l, indicating insignificant toxic effects to algae. The data reported herein are based on studie@'developed and performed by M. T. Elnabarawy and R. R. Robideau. Accompanying data sheets comprise original data. It should be noted that all the reported EC values were calculated on the basis of the initial concentrations of FM-3422, Lot 716, in test solutions at the beginning of the bioassay. FM-3422/MTE Page Five December 30, 1981 REFERENCES (1) Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. The Selenastrum Caprico' rnutum Printz Algal Assay Bottle Test: ExperimenEa-1 design, application, and data interpretation. U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-600/9-78-018. 125 P. (2) ASTM-E-35.23. 1981. Proposed Conducting Toxicity Tests with Algae. Draft No. 2. Standard Practice For Freshwater and Saltwater (3) OECD Guidelines for Testing of Chemicals (1981) Section 2, Effects on Biotic Systems, Test 201 "Algae, Growth Inhibition Test," Adopted May 12, 1981. (4) Payne, A. G. and R. H. Hall, 1979. A Method for Measuring Algal Toxicity and Its Application to the Safety Assessment of New Chemicals. ASTM STP #667, p. 171-180. TABLE 1 Based Algal Growtb Response to FM-3422 Lot 7".'o EC_90's (mg/1)( on Reducation In Cell Count(2) Exposure (Contact) Days_ 4 >1,800 7 >1,800 10 >1.1800 14 >1,800 (1) (2) riethod of EC50 calculation: 3M SIXCUR, a TRAC system for regression models of experimental data. Algal cell-count (No./ml) measured in triplicated sets of culture flasks. ATTACHMENT I NUTRIENT MEDIUM FOR FRESHWATER ALGAE A. MACRONUTRIENTS STOCK SOLUTIONS (CONCENTRATED) @Prep-aredseparately with deionized water.) 1) 25-500 gm NANO in I liter Di water. 3 2) 1.044 gm K2 HPO 4 in I liter Di water. 3) 12.159 gm MgCli 6H20 in I liter DI water. 4) 14.700 gm MgSOL@7H20 in I liter Di water. 5) 4.410 gm CaClZ.2H20 in I liter DI water. 6) 15.000 gm NAHCO 3 in liter Di water. B. MICRONUTRIENTS STOCK SOLUTION (CONCENTRATED) (Combined in a single one-liter stock mix.) 1) 185.5 mg H3 B03 2) 415.6 mg MnCi2.4H20 3) 3.27 mg ZnC] 2 4) 1.43 mg COC12-6H20 5) 0.01 mg CuC]2 6) 7.26 mg Na2MO04-2H20 7) 96 mg FeC] 3 8) 300 mg Na2EDTA*2H20 PREPARATION OF SYNTHETIC ALGAL NUTRIENT MEDIUM Add one mi of each macronutrient stock solution plus one ml micronutrients stock mix per I liter of deionized water; sonicate and then filter through 0.22 lim membrane; adjust p.Hto 7.5 t 0.1; store in the dark at 40C. AT -.PCtimEt74 PREPARATION OF ALGAL CLILTURE FLASKS All flasks used in main,taining and testing algae were borosilicate glass (KIMAX). viade of - flasks were brushed inside with a stiff bristle brush; - washed with non-phosphate detergent (MICRO) and rinsed 3 tinies with tap water; - rinsed with a lg/,HCI solution; - rinsed 3 times with tap water and 3 times with 0.1. water; 0 - dried in an oven at 70 C for 2 hours (placed inverted); 0 - and autoclaved with foam plugs inserted at 121 C for 20 minutes. ATTACHMENT 3 ALGAL FLASKS CONTROL PREPARATION OF ALGAL TEST FLASKS IN CHRONOI:OGICAL ORO@ ALGAL MEDIUM V-7M,eA27, @tpa @-(ip CONTENTS + TEST MATERIAL + ALGAL INOCULUM (ml) or LI.M,=JAL + + TOTAL 50 I Ioo + + 50 2 + + 50 3 + + 50 4 1000 mg@p 46,5 + + 50 5 1Soo PAC + + 50 6 + + 50 7 + + 50 1) Determineinitialalgal cellcount in the stock culture. 2) AdjustpH of algal nutrientmedium to 7.5 + 0.1 MI. 3) Make up stock solutionswith algal nutrientmedium as desired. -A) Control flasks,preparations: I) Add lnoculum(volumepredetermined);initialcell-loadingof I x 104 ce IIs/ml; 2) Add algal nutrientmedium to bring volume to 50m]; swirl flasks, and place autoclaved foam plugs. 8) Test flasks: 1) Add algal nutrient medium; 2) Add test material (dissolvedin algal medium if required); 3) Swirl flasks; 4) Add,algat inoculum; 5) Swirl flasks.and place autoclaved foam plugs. ou4uj wF-iiycc@,-:@o..Acez DkpecrL-y iN-to cLuiuRe o.5-Al ILTkW-RXl Ilt7cR ALC-AU MU-M%eXr MfZkUKi U-r*Z Afa CA@K'KKEK 1501-vet4T. Ces-tt4Tt@ :3--S6.4,.):A544t9)z1s; '53-S 1558l(DoCqr--LLS/ -A IALJ lwocuuuak vouut-ie PEJR PW4@sc (I r ATTACHMENT 3 PREPARATION OF ALGAL TEST FLASKS IN CHRONOLOGICAL--ORDER ALGAL FLASKS CONTROL 2 ALGAL MEDIUM (mi) + TEST HATERI (mI) o r + CONTENTS + ALGAL INOCULUM &0*@ (mi) + + + + + TOTAL (0)1) 50 50 50 3 + + 50 4 + + 50 5 + + 50 6 + + 50 7 + + 50 1) Determineinitialalgal call count in the stock culture. 2) Adjust pH of algal nutrientmedium to 7.5 + 0.1 mi. 3) Make up stock solutionswith algal nutrientmedium as desired. A) Controlflasks,preparations: 4 1) Add inor-ulu(mvolumeBredetermineg);initialcelr loadingof I x 10 calls/m); 2) Add algal nutrientmedium to bring volume to 50mi; swirl flasks, and-place autoclaved foam plugs. B) Test flasks: 1) Add algal nutrient medium; 2) Add test material (dissolvedin algal medium if required); 3) Swirl flasks; 4) Add algal inoculum; 5) Swirl flasks.and place autoclaved foam plugs. I&ADNIOUA@J '-OJE144M -PLACEZ IVA-M 14F-mjV-I-rToAC-Mv, CoumT@s -5-S -64 -S(o 4t 2-S :) 3'5-8 ALEtAi,; lwoL"Lu@4 \Ioukme PeR Uksr- (ty1.64MWQX$Otd)- :35SPOC) &kL5/4 ATTACWMENT 4 ALGAL BIOMASS MONITORING A. GRAVIMETRIC CELL DRY WEIGHT (1) The filter recommended is Millipore type BD with an 0.6 micrometer pore size. The method is as follows: - Dry filters for two hours at 700C in an oven; - Cool filters in a desiccator containingdesiccant,for at least two hours before weighing; - Filter a suitable measured aliquot of the culture under a vacuum or pressure not to exceed 8psi; - Rinse the filter funnel with D.I. water; 0 - Dry the filter to constant weight at 70 C, cool in a desiccator for two hours and weigh. Basic instrument used: Analytical Balance; Mettler ME 30. B. SPECTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLL a (2) Basic instrument used: Stectrophotometer; Bausch & Lomb, Spectronic 20. C. IMPROVED NEUBAUER 0.1 mm DEEP HEMOCYTOMETER COUNTING CHAMBER AND OPTICAL MlCROMrLTER (Used to @ieisure diameter of algal MCV). (3) (I)@ Miller, W. E., J. C. Greene, and T. Sh;royama. 1978. Selenastrum capricarnut!LmM- Printz Algal Assay Bottle Test: Experimental Design, Application, and Data InterpretationProtocol. Ecol. Res. Series EPA-600/9-78-Oi8. Corvallis, Oregon. pp. 27. (2). APHA-AWWA-WPCF (1975). "Standard Methods for the Examination of Water and Wastewater,,"(M. Franson, manag. ed.), ]4th edition, pp. 1030-1031. (3). Brite-Line (R), Any.--ricOapntical Corporation,Buffalo, New York.