Document wKDQM1EY4zbekd8DEeamaKJoB

Multi-PhaseE Method 0 L TOXICITY TO AQUATIC PLANTS (E.G.,ALGAE) TEST SUBSTANCE Identity:Perfluorooetanesulfonamtaey; alsobe referredtoas PFOS or FC-95. (1-Octanesulfbniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassiumsaltC,AS # 2795-39-3) Remarks field: The testsubstanceisa whitepowder (3M Lot583)of uncharactedzedpurity. The followinsgummary isabbreviateddue to thefactthatthisstudyhas been superceded by a more recenttest. METHOD Method: Test protocolutilizewdas modifiedafterthosedescribedby USEPA - 600/9-78-018,1978;ASTM-E-35.23,1981; OECD 201, 1979. ASTM STP #667 Type: Semi-chronic GLP: No Year completed: 1981 Species:Selenastrumcapricomutum RESULTS EC50Values,mg/L (CI) Exposure (contact) Cell-DrWyeight Days 4 115(18-65) 7 122 (41-366) 10 128 46-372) 14 1 146(33-350) Cell-Count 82 (NoConf.Limits) 99(19-398) 98(16-431) 95(12-455) Remarks: The statisticparlogram used was questionable. DATA QUALITY ReliabilityK:limischranking= 2. Thisstudysatisfiecdriterifaorquality testingatthetimeperformed,butactualconcentrationwsere not measured. Resultswere based on nominalconcentrationsA.dditonally, sample puritywas notpropedy characterized. REFERENCES This study was conducted by the 3M Company, Environmental Laboratory,1981. OTHER Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota,55133 Last changed: 5/3/00 Fotrr.$747-1 1-C TECHNICAL REPORT TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN SUMMARY Oats L 12/16/81 flmportent- Ifr*POrtispdnted on boM Sidesof paper,nnd two CopiesW SummarY-evoilablefrom InformationLia4wn or TCC (3-5545). oivwon Environm Project ntal Laboratory (EE & PC) -,pIW@,Po+-hnel-q Develor)ment - PC-95, Lot 583 Millti-PhaAe To Exposure /RecoverV Algal Assay TCC.)S@e -OUldelinefsor Test Method Completionof TechnicalReport 040L Nurn 0535 rraiect N umber 9970030000 wo-P_-t-wu-m-wl- 007 Bohon/E. Reiner/A. Welter M. T- iginabarawy Noze*OOIC ptefwenco 46981 NO. Of Pag" Including Coversheat SECURITYIO KEYWORDS: includeLob Code C' )pan C3 Closed (Company Confkiential-).- (SP*dal Autharindon) 3M CHEMICAL REGISTRY Now Chomi=lsRewrted C) Yes EN No CURRENT OBJECTIVE: 1. To evaluate the algal growth response of freshwater green algae "Selenastrum capricornutum" over several generations as they may be aff*ected by exposure to the fluorochemical FC-95, Lot 583..p 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: ThisabstracitnformatiiosndistributheydtheTechnicaClommunicationCsentertoaler3tM'srsto Company R&D. I In these triplicated four-phase exposure/recovery assays, the initial effects on organisms followinq varied exposures to FC-95 were assessed over several generations. Test results gave an indication of the possible effects on 4R.Lgalpopulations. Algal biomass was assessed quantitatively in terms of cell-dry weigbt (mg/1) and cell-coLTnt (No./ml) . The following biomass indicatori were measured and calculated for: A. verification of inhibitory effects 1. Median growth response EC50 (mg/1) from varied exposures. 2. EC10 and EC90 (mg/1) for same exposure periods. 3. No-effect levels. B. Verification of algicidal effects 1. Resumption or absence of logarithmic growth in subcultures during varied periods of recovery. init :e FC-95/MTE Page Two December 16, 1981 CONCLUSIONS 1) Materials possessing an ECSO in the range of 10-100 mg/l are considered to be slightly phytotoxic to algae and aquatic plant species. 2) The algal growth response in most subcultures following varied periods of recovery indicated minimal algicidal effects (deathlof algal cells) at concentrations tested.' The algal cells recovered and resumed logarithmic growth when resuspended in fresh nutrient medium in the absence of the test material. The median growth response (ECSO) was therefore interpreted as basically indicative of an inhibitory effect as opposed to an algicidal effect. In the former case, pliotosynthesis, cell-growth, and cell-division, are reduced whereas an algicidal effect causes direct cell-destruction and cell-death. 3) Results indicated that extended exposures (q.g., >7 days) induced no greater inhibition but slower redovery rates in the subcultures were noted. SUMMARY A four-phase exposure/recoveL-y algal assay was developed to, evaluate algal growth response of the freshwater unicellular green algae uselenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical, FC-95. This multiphase algal assay evaluated algal growth responses during four consecutive exposure/recovery combinatiohp: 4/10, 7/7, 10/4, and 14 days. As is clearly indicated, the duration of exposure periods increased, while respective recovery periods decreased. The combined duration of each exposure/recovery phase was fixed at 14 days. Algal growth response was measured in terms of an increase in biomass as cell-di.-yweight (mg/1) and cell-count (No./ml). Dry-weight and cell-count results used for the calculation of ECSO's (mg/1) were averages of triplicated Sets of test culture flasks. See test schematic: Phase Exposure (Day) Recovery (Day) 1 4 7 10 IV 14 10 7 4 None Total No. (Day) 14 14 14 14 In these triplicated multiphase multiqeneration algal assays, the calculated ECSO values (mg/1) for freshwater green algae "Selenastrum capricornutum" are presented in Table 1. These values represent the median growth response (ECSO mg/1) following exposure to the test material for 4, 7, 10, and 14 days. FC-95/MTE Page Three December 16, 1981 The values of EC10 and EC90 (mg/1) for each of the exposure periods listed earlier were calculated in terms of cell-count and are.presented in Table 2. No test concentrations were defined as a no-effect level. To verify algicidal effects, and to assess Possible resumption of growth in the absence of the test material subcultures were established from each triplicated set of test culture flasks combined at the end of each exposure period. Algal recovery response was evaluated following respective recovery periods of 10, 7, and 4 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, ari6 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 substance (FC-95, Lot 583) is a fine white powder, soluble 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 U@EPA - 600/9-78-018; ASTM-E-35.23 Draft No. 2; OECD; A. G. Payne. (1), (2), (3), (4). Test Species: A bacteria-free culture of the freshwater planktonic green algae (Chloraphyceae), Selenastrum capricornutum of the order chlorococcales was obtained fFom-USYP-A - ERL, Corv., Oregon (February 18, 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 to give a starting optimum inoculum level of green algae Selenastrum capricornutum I x 104 cells/ml. The use of a 7 to io-a-aF=:Fldstock curtu-re insured 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 (300,000 cells/ml). FC-95/MTE Page Four December 16, 1981 Algal Nutrient Medium: Mineral (inorganic) standard nutrient medium for culturing and testing 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 including the preparation of stock solutions. 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. Range-Finding Test (Exploratory): The exploratory test consisted of determining the 4-day algal biomass in triplicated flasks containing standard nutrient medium plus the test material at concentrations covering several orders of magnitude: 1, 5, 10, 50, 100, and 500 mg/l. 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/recovery tests (4/10, 7/7, 1.0/4, and 14/0 days. The purpose of the exposure (contact) stage was to verify inhibitory effects. The recovery (subculture) stage.evaluated the viability of growth-inhibited algae and verified algicidal effects. The following FC-95, Lot 583 logarithmic concentrations: 26, 40, 61, 93, 145, and 225 mg/l were used to initiate the exposure stage. This dose range of dilutions was selected to bracket the 4-day EC50 values predicted from preliminary exploratory testing. Three flasks containing 100% fresh algal nutvient'medium plus algal cells comprised the nontreated controls. A fresh stock solution of the test substance was prepared in the algal nutrient medium immediately prior to testing. The initial pH of this stock Solution was 7.3. Procedure and steps for preparation of algal test flasks are outlined in Attachment 03. Each recovery stage was initiated by subculturing (0-5/50 ml)' in the absence of the test material. Algal cells from each triplicated set of culture flasks were combined and then resuspended into fresh nutrient medium. Subculture methods are briefly described in Attachment #4. FC-95/MTE Page Five December 16, 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 either algal cell-dry weight (mg/1) afid/or by cell counts (cells/ml). Both growth measurements*were made with algal cultures after each of the exposure and recovery stages. Both procedures for algal biomass measurements are briefly described in Attachment #5. ECSO 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 al gae Selenastrum capricornutum used in these studies are self-ex-pla--natoryand are detailed in the attached data sheets. Calculated EC50 (mg/1) values indicating algal growth response to FC-95, Lot 583, following exposure periods of 4, 7l 10, and 14 days are presented in Table 1. The values of EC10 and EC90 for each of the exposure periods listed above were calculated in terms of cell-count and are presented in Table 2. Recovery data and resumption of logarithmic growth in subcultures are summarized in Table 3. To illustrate the action of test material and to give an overview of what has happened in the exposure experiment, toxicity curves are shown in Figures 1 and 2. Utilizing the EC50 values, toxicity curves were constructed against time on semilog plots. The data reported herein are based on studies developed and performed by M. T. Elnabarawy and R. R. Robideau. Accompanying data sheets comprise original data. FC-95/MTE Page Six December 16, 1981 It should be noted that all the reported EC values were calculated on the basis of the initial concentrations of FC-95, Lot 583, in test solutions at tE-e-E-e-g'iTnnoifng the bioassay. REFERENCES (1) Miller,W. E.p J. C. Greene,and T. Shiroyama.1978. The Selenastrum Capricornutum Printz Algal Assay Bottle Test: Expe rime 1 design-,---a-p-pTicatioann,d data interpretation. U.S. ' Environmental Protection Agency,, Corvallis, Oregon. EPA-600/9-78-018. 125 p. (2)ASTM-E-35.23.--, 1981. 'Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 2. (3) OECD Guidelines for Testing of Chemicals (Z98l) Section 2, Effects on Biotic Systems, Test 201 "Algae,*@G.rowth 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 Algal Growth Response to PC-95, Lot 583 ECso'S (mg/1)(1) Exposure (Contact) Days Cell-Dry Weight(2) Cell-Count(3) 4 115(18-65)(4) 82(tioLimits) 7 122(41-366) 10 128(46-372) 99(19-398) 98(16-4il) 14 146(33-350) 95(12-455) (1) (2) (3) (4) Methodof mcscnalculation:3tiSIXCUR,a TRAC systemfor regression models of experimental data. Growth response parameter; cell-dry weight (mg/1), measured in.triplicated sets of culture flasks. Growth response Parameter; cell-count (no. cells/ml), measured in triplicated sets of culture flasks. 95% Confidence Limits. Exposure (Contact) Days 4 7 10 14 .TABLE 2 Algal Growth Response to FC-95, Lot 583 EC10's and EC90's (mg/1) Based on Reduction in Cell-Count ECIO 10 173 18 179 16 179 16 174 TABLE 3 Recovery Stage Data Summary Algal Cell-Count, Cells/ml (KEAN VALUES)(1) Treatment Control Multi Phase Exposure/ covery Phase 1 4/10 Days Phase 11 7/7 DaysiPhase 111 10/4 Days Initial Final Initial Final lniti-aT-Flnal Loading(2) Count(3) Loading Count Loading Count 1,29-9,.. 336pOOO 1,520 3U4,UU----I-,560 176,000 1 26 mg/l 1,047 330,000 1,483 312,0..00 1,380 144,000 2 40 mg/l 967 298,000 1,333 252,000 1,300 124,000 3 61 mg/l 935 266,000 1,060 222,000 1,310 110,000 4 93 mg/l 273 248,000 503 210,000 513 78,000 5 145-mg/l 57 244,000 123 82,-000 97 6,0-00 6,225 mg/l 47 190,000 67 56,000 50 0 (1) Based on 0.5 ml/50 ml subcultures established from each triplicated sets of culture flasks combined. (2) Measured at the end of each exposure period. (3) Measured at the end of each recovery period. ATTACHMENT I NUTRIENT MEDIUM FOR FRESHWATER ALGAE A. MACRONUTRIENTS STOCK SOLUTIONS (CONCENTRATED) (Prepared separately with dolonized water.) 1) 25.500 gm NANO in I literDi water. 3 2) 1.01+4gm K2HP04 In I liter DI water. 3) 12.159 ginmgcii 6H2o In I liter DI water. 4) 14.700 grnMgSOi@7H20 in I IIter DI water. 5), 4.410 gm CaCI2-2"20 In I liter Di watero 6) 15-000 gm NaMC03 In liter 01 water. Be MICRONUTRIENTS STOCK SOLUTION (CONCENTRATED) (Combined in a single one-liter stock mix.) 1) 185.5 mg H3803 2) 415 .6 mg mnc I:f4H2 0 3) 3.27 mg ZnCi 2 4) 1.43 mg COC12-6H20 5) 0.01 mg CuC]2 6) 7.26 mg Na2MoOW 2H20 7) 96 mg FeC13' 8) 300 mg Na2EDTAo2H20 PREPARATION OF SYNTHETIC ALGAL NUTRIENT MEDIUM - Add one ml of each macronutrient stock solution plus one mi micronutrients stock mix per I liter of doicnized water; - sonicate and then filter through 0.22 po membrane; - adjust P."to 7.5 :t0.1; - store In the dark at 40C. ATTACHNENT 2 PREPARATION OF ALGAL CULTURE FLASKS Al I flasks used in maintaining and testing algae were made of barosilicato glass (KIPAX). - flasks were brushed Insidewith a stiff bristle brush; - washed with non-phosphate detergent (NICRO) and rinsed 3 times with tap water-, - rinsed with a 10% HCI solution; - rinsed 3 t,ims with tap water and 3 times with D,I,.water; a dried In an oven at 70 c for 2 hours (placed Inverted); and autocinved with fown plugs Inserted at 1240C for 20 minutes. ATTACHMENT 3 PREPARATION OF ALGAL TEST FLASKS IN CHRONOLOGICAL ORDER_L ALGAL FLASKS CONTROL ALGAL MEDIUM (mll 2 XAO du (to) 45. ZZ, CONTENTS + TEST MATERIAL + ALGAL INOCULUM (mi)or (mg)* (MI) + NONE + 1.1 + Z.oj + 1.,-[ + 08 wl + TOTAL (mi) 50 50 50 3 tolvad C.S.oS) @@5. to + 4.1 "%A + 50 4 + I. 1!5w4 + 50 5 + x5wl + 50 6 225W + + i.ri 50 7 + + 50 I DetermineInitialalgal cell count in the stockculture. 2) Adjust PH of algal nutrientmedium to 7.5 + 0.1 mi. 3) Make up stock solutions,withalgal nutrientmediumas desired. A) Control flaskspreparations: . 104 ce II5/ml; 1) Add inoculum (volumepredetermined)i;nitialcell loadingof I x 2) Add algal nutrient medium to bring volume to 50m]; swirl flasks,and place autoclavedfoam plugs. 6) Testflasks: 1) Add algal nutrient medium; 2) Add test material(dissolvedin algalmedium if required); 3) Swirl flasks; 4) Add algal inoculum; 5) Swirl flasks, and place autoclavedfoam.plugs. 5voci-i5out-ncw: to5o w:SlI.R (IoSo /1000 wl) EaciLIwd IMITIA&J pJA ATTACHMENT 4 SUBCULTURE METHOD IN ALGAL ASSAYS (0-5mi/50 mi) OBJECTIVE: PROCEDURE: To determine algal recovery response in absence of test substance. Transfer 0.5ml from each of the triplicate culture flasks into Corning-45ml sterile centrifuge tubes. Centrifuge at 10,000 rpm for 5 minutes. Decant supernatant liquid and save the precipitated algal cells. Using the same centrifuge tube, resuspend algal cells in algal nutrient medium to a fixed volume: ?5ml. Mix thoroughly and decant the top 10mi of algal call suspension, and transfer the remaining volume (3mi) of algal cell suspension into a clean 250mlErien*yer culture flask. Using the same centrifuge tubes, transfer 45m] of fresh algal nutrient medium Into the above Erlenmeyer culture flask, to brlng total volume to 50 mi. ATTACHMENT 5 ALGAL BIOMASS MONITORING A. GRAVIMETR-IC CELL DRY WEIGHT (1) ThefIIterrecommendeidsMiIIiporetypeBDwithan 0.6micrometer pore stze. The method Is as follows: - Dry f Ilters for two hours at 700C in an oven; - Cool filters In a desiccatorcontainingdesiccant,for at least two hours before weighing; - Fil't-iasrsultable measured aliquot of the culture under a vacuum or pr' essure not to exceed 8pst; - Rinse the filter funnel with D.I. water.; - Dry the filter to constant weight at 760c, cool In a desiccator for two hours and weigh. Basic instrument used: Analytical Balance; Mettler ME 30. B. SPETTROPHOTOMM IC DETERMINATION OF CHLOROPHYLL a (2) Basic Instrument used: Stectrophotmeter; BaU3ch & Lamb, Spectronic 20. C. IMPROVED NEUBAUER 0.1 mm DEEP HEMOCYTOMETER CO UNTING CHAMBER AND OPTICAL MICROMETER (Used to me;sure diameter of algal t4CV-Ii(-3) Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. Salanastrum capricornutum Printz Algal Assay Bottle Test: Experimental Design, Application, and Data InterpretationProtocol. Ecol. Res. Series EPA-600/9-78-018. Corvallis, Oregon. pp. 27. (2). APHA-AWWA-WPCF (1975). '-StandardMethods for the Examination of Water and Wastewaters$$ (M. Franson, manag. ad.), 14th edition, pp. 1030-1031. (3). Brlte-Line (R). American optical Corporation,Buffalo, New York. 13 4 its it N13. 2 24C-L210 CYCLE:EX ClETZGEt4 GRAPki MI-LI313AMITHMIC to DIV$iiloNs PER PAPER ITICH t4 4-. T !L 7 ri t1 Ll DICTZGCN CORPDq.%710N ti $A Ti 7f 01 1 7 rI -II-.I---i I;F 7- 'ji .. ...... I Y-7 . ..... 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