Document N2RdLRRgwyRdjQ9M6Eqa8GRop

TOXICITY TO AQUATIC PLANTS (e.g.A,lgae) TEST SUBSTANCE Identity:Perfluorooctanoiaccid,ammonium salt;may alsobe referredto as PFOA ammonium salt,Ammonium perfluorooctanoateP,FO, FC116, FC-126, FC-1 69,or FC-143. (Octanoicacid,pentadecafiuoro,ammonium salt,CAS # 3825-26-1) Remarks: The 3M productionlotnumber was 37. The testsample isFC143. It'psuritywas notsufficientclhyaracterizedt,hough currentinformation indicatesitisa mixtureof 96.5 -100% testsubstance and 0 - 3.5% C6, C7, and Cg perfluoroanalogue compounds. METHOD: Method Followed: Modified(modeled) afterthose describedby USEPA - 600/9-78-018;ASTM-E-35.23 DraftNo. 2; OECD; A.G. Payne. Type (testtype): Acute Static GLP: No Year study performed: 1981 Species: Selenastrumcapricomutum. Source: USEPA - ERL, Corv.,Oregon (July14,1981). Analyticalmonitoring: Algalcellcounts(cells/mlc)e,lldryweights,and temperature. Exposure period: 4,7, 10,and 14 days StatisticaMlethods: EC50 values and 95% confidencelimitwsere calculatedutilizi3nMg Sixcur,a linearegressionmodel. Test orgamisms laboratoryculture:Algae from a 7-day-oldstockculture Algal NutrientMedium: Sterilesyntheticalgalnutrienmtedium. This nutrienmtedium served as thediluentforallalgaloperations.The pH ofthis syntheticalgalmedium was adjustedto7.50.1priortouse inassays. Stock and Test Solution Preparation: A primarystocksolutionwas preparedinalgalmedium ata concentrationof5 g/L. Aftermixing,the primarystocksolutionwas dilutedwithalgalmedium to preparethe sixtest concentraions. Exposure vessels: Steril2e50 mL Erienmeyer flaskscontaining50 mL of testsolutionand stopperedwithautoclavedfoam plugs. Agitation:Continuous platformshaking at 100 + 10 rpm Number of replicates:three Initiaallgalcellloading: 1.0 X 104 cells/mL Number of concentrations: sixplusnegativecontrol Nominal concentrations: Bk control,100, 180, 320, 560, 1000, and 1800 mg/L Test conditions: Temperature: 2320C (70-770F) Fluorescent illumination: 400 ft.candles 10% RESULTS Algal Growth Response ECw values Exposure (Contact) Cell-DryWeight (1) Cell-Count (2) Days mg/L (95% C.I.) mg/L (95% C.I.) 4 149 (57-340) 49(28-75) 7 70(34-118) 30(21-40) 10 49(15-96) 27(8-50) 14 73(25-147) 43(14-81) (1) Growthresponseparameter;cell-dwreyight(mg/L)m,easured intriplicatsedtsof culturfelasks. (2) Growthresponseparameter;cell-cou(nntumbercell/smL),measured intriplicatsedts ofculturfelasks. Algal Growth Response ECIO and EC90 values based on Cell-Count (number of cells I mL) Exposure (Contact) Days 4 7 10 14 ECio mg/L (95% C.I.) 5.3(3-7) 3.3(2-4) 2.9(1-5) 5(2-8) ECgo mg/L (95% C.I.) 624 (C.In.otcalculated) 283 (150-590) 386 (C.In.otcalculated) 307 (C.I.notcalculated) Element valuesbased on nominal concentrations. CONCLUSIONS Ammonium perfluorooctanoateexhibitsa 14-day EC5o (cellcount)value of43 mg/L witha 95% confidenceintervaolf 14 to81 mg/L. DATA QUALITY ReliabilityK:limischranking= 2.Thisstudy meets thecriterifaorquality testingatthe timeitwas conducted. However, thestudy lacksinformationon purityofthe testsubstance and actualmeasurements oftheamount oftest substance insolution. REFERENCES 3M TechnicalReport Summary, Multi-PhaseExposure /Recovery Algal Assay Test Method, Report Number 006, ProjectNumber 9970030000, M. T. Einabarawy, October 16, 1981. OTHER Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 Last changed: 5/24/00 r.c;rm674 7 11-8 TECHNICAL REPORT SUMMARY TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN timport3nt-Ifreportisprinteodn bothsidesofpaperw,ndt"copies to TCC.) Division Environmental Project Now Methods ReportTi'ge- Laboratory Development EE & PC - 2-3E FC-143, Lot 37 Multi-Phase To Exposure/Recovery Algal Assay D. Bacon/R. -Kuth-or9t) Bohon/ M. T. Elnabarawy NotsbooZ Rofere-nco /A. Welter Test Method Date 16/81 Dept. Numbw 0535 PrNo-iu*mcbtff 9970030000 RWort Numb*r 006 Employ" Number(s) 46981 No. of PftesIncludingCovershoot SECURITY 10- M Open KEYWORDS: (Selecttorrmfrom 3M ThesaurusS.unestother applicawbmlse.) EE & PC-Div. (Env. Lab) 0 Closed (SpeciaAluthorization) 3M CGHIESM!ICAL :;;RECGISTRY Now ChemicalsReported 0 Yes C No CURRENT OBJECTIVE: 1. To evaluate the algal growth response of freshwater green algae "Selenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical FC-143, lot 37. 2. To develop an algal test protocol based on variable exposure or recovery periods to the test compound. REPORT ABSTRACT: (200-250words)ThisabstracitnformatioinsdistributbeydtheTechnicaClommunicationsCenterto. aler3tM'erstoCompany R&D. In these triplicated four-phase exposure/recovery assays, the initial effects on organisms following varied exposures to FC-143 were assessed over several generations. Test results gave an indication of the possible effects on algal populations. Algal biomass was assessed quantitatively in terms of cell-dry weight (mg/1) and cell-count (No./ml). The following biomass indicators 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. Inform& Initial&: FC-143/MTE Page Two November 16, 1981 CONCLUSIONS 1) Materials possessing an EC50 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 all subcultures following varied periods of recovery indicated minimal algicidal effects (death of 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 (EC50) was therefore interpreted as basically indicative of an inhibitory effect as opposed to an algicidal effect. In the former case, photosynthesis, cell-growth, and cell-division, are reduced whereas an algicidal effect causes direct cell-destruction and cell-death. 3) Exposure/recovery results indicated that extended exposures (e.g., >7 days) induced greater inhibition and consequently exhibited slower recovery rates in the subcultures. SUMMARY A four-phase exposure/recovery 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, FC-143. This multiphase algal assay evaluated algal growth responses during four consecutive exposure/recovery combinations: 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-dry weight (mg/1) and cell-counl.-(No./ml). Dry-weigbt and 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 multigeneration 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. The values of ECIO and EC90 (mq/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. -PC-143/MTE Page Three November 16, 1981 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, 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 substance (FC-143, lot 37) 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 USEPA - 600/9-78-018; ASTM-E-35.23 Draft No. 2; OECD; A. C. Payne. (1), (2), (3), (4). Test Species: A bacteria-free culture of the freshwater planktonic green algae (Chlorophyceae), Selenastrum capricornutum of the order chlorococcales was obtained from USEPA - ERL, Co-rv., 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 1 x 104 cells/ml. The use of a 7 to 10-daT-:i@ld stock -cu-rt-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 (277,000 cells/ml). -PC-143/MTE Page Four November 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 determininq.the 4-day algal biomass in triplicated flasks containing standard nutrient medium plus the test material at concentrations covering several orders of magnitude: 100, 250, 500, 750, 1,000, and 1500 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, 10/4, 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-143 logarithmic concentrations: 100, 180, 320, 560, 1000, and 1800 mg/l were used to initiate the exposure stage. This dose range of dilutions was selected to bracket the 4-day ECSO values predicted from preliminary exploratory testing. Three flasks containing 100% fresh algal nutrient 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 7.4. Procedure and steps for preparation of algal test flasks are outlined in Attachment #3. 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. @C-143/MTE Page Five November 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) and/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. EC50 values and 95% confidence limits were calculated utilizing 3M SIXCUR, a TRAC System for regression models of experimenta a a.@@ TEST RESULTS The results of biomass measurements of the green algae Selenastrum capricornutum used in these studies are self-explanatory and arii-detailed in the attached data sheets. Calculated EC50 (mg/1) values indicating algal growth response to FC-143, lot 37, following exposure periods of 4, 7, 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-143/MTE Page Six November 16, 1981 It should be noted that all the reported EC values were calculated on the basis of the initial concentrations of FC-143, lot 37, in test solutions at the beginning of the bioassay. REFERENCES (1)Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. The Selenastrum Capricornutum Printz 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-35.23. 1981. Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 2. (3) OECDGuidelinefsorTestingof Chemicals(1981)Section2, Effects on Biotic Systems, Test 201 "Alqae, 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 Algal Growth Response to FC-143, Lot 37 EC50's (mg/1)(1) Exposure (Contact) Days Cell-Dry Weight(2) Cell-Count(3) 4 149(57-341)(4) 49(28-75) 7 70(34-118) 30(21-40) 10 49(15-96) 14 73(25-147) 27(8-50) 43(14-81) (1) (2) (3) (4) Method of EC50 calculation: 3M SIXCUR, a TRAC system for 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-143, Lot 37 ECIO's and EC90's (mg/1) Based on Cell-Count (no. cells/ml) fslln 5.3(3-7) 3.3(2-4) 2.9(1-5) 5(2-8) EC90 624(No Limits) 283(150-590) 386(No Limits) 307(60-No Limits) TABLE 3 Reqagexly Stage Data Summary Algal Cellj@Wunt-y) Cells/mi (MEAN VALUES)(1) Treatment Initial Algal Cell Loading(2)Final Algal Cell-Count(3) T-days 7 Days lU Days 10 Days 7 Days 4 5a-ys Control 2,017 2,430 2,453 380tOOO 256,000 136,000 1 100 mg/l 643 580 587 372,000 226FOOO 48,000 2 180 mg/l 530 290 280 348,000 204,000 20,000 3 320 mg/l 383 273 380 300,000 214,000 20,000 4 560 mg/l 237 137 290 236,000 200,000 18-1000 5 11000 mg/l 57 60 103 310,000 174,000 18,000 6 1,800 mg/l 73 73 73 182,000 54,000 18,000 (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. 10 L.,-@ oI@i@LLN@,. ,,@i , ,. @, SEMI-LOGARITHMIC 2 CYCLES X 10 DIVISI13NS PER EC5" INCri co IA 0 11 CD . @@,"I I. I ri Tt lilt T-1.T CD- t T-i -w I -7 -4- rT 7T TT !Iliii@l L t-7 ........ 7 -------- -------- -71 !:50 Cuv;@&- A" urrw -M "FC k45 Lo-r51 0 K- Ce-i I C-CA@ (V@a!/YKt) olo wx Lr) Ln 4 TT Lo -1 8 ci io It 15 ATTACHMENT I NUTRIENTMEDIUM FOR FRESHWATER ALGAE A. MACRONUTRIENTS STOCK SOLUTIONS (CONCENTRATED) (Prepared separately with delonized water.) 1) 25.500 gm NANO in I liter Di water. 3 2) i.o44 gm K2Hpo4 In I liter Di water. 3) 12.159 gm MgC]i 6H20 in I liter DI water. 4) 14.700 gm MgSO4!7H20 in I liter Di water. 5) 4.410 gm CaCIZ-2H20 in I liter Di water. 6) 15.000 gm NaHCO3 In liter Di water. B. MICRONUTRIENTS STOCK SOLUTION (CONCENTRATED) (Combined in a single one-liter stock mix.) 1) 185.5 mg H3BO3 2) 415.6 mg mncl274H20 3) 3.27 mg ZnC) 2 4) 1.43 mg COC12-6H20 5) 0.0) mg CUC12 6) 7.26 mg Na2MO04-2H20 7) 96 mg FeC'3 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 deionized water; sonicate and then filter through 0.22 jan membrane; adjust P-Hto 7.5 :t 0. 1; store in the dark at 40C. ATTACHMENT 2 PREPARATION OF ALGAL-CULTURE FLASKS All flasks used in maintaining and testing algae were made of borosilicate glass (KIMAX). - flasks were brushed inside with a stiff bristle brush; - washed with non-phosphate detergent (MICRO) and rinsed 3 times with tap water; - rinsed with a 10% 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); - and autoclaved with foam plugs Inserted at 1210C for 20 minutes. ATTACNt4ENT3 PREPWTIgl At ML TESTFlasKs .LINCHREOLRJICALOltOERI ALGAL FLASKS CONTROL c AL"L- MO IUN + TIST MAYImRaIlA@ + ALGAL IWULUM n to1) or (0)) N ot4 + l.E3 0 1toovwp (5w@5OW4 4 + 1.0 V4 + w TOTAL (M 1) 50 50 2 leow (q + + a 50 3 (itp) 0 + + n 50 4 to + + a 50 5 tooow4f 6 IE@oo (90) InD 7 + C) vvj + + + 1,6 + + n 50 n 50 a 50 1) DetermineInitialalgalcell count In the stockculture. .2) Adjust pH of algal nutrientmedium to 7.5 + 0.1 mi. 3) Make up stock solutionswith algal nutrientmedium as desired. A) Controlflaskspreparations: - 04 1) Add lnoculum(volumepredetermined);Initialcell loadingof I x 1 cells/mi; 2) Add algal nutrientmedium to bring volume to 50m); swirl flasks,and place laved foam plugs. B) Test flas*kUst:oc 1) Add algai nutrient medium; 2) Add test material(dissolvedIn algal medium if required); 3) Swirl flasks; 4) Add algai inoculum; 5) Swirl flasks,and place autoclavedfoam plugs. '5-Tc)c-t-\ F,AC+@ -. 5-s/'Q ATTACHMENT 4 SUBCULTURE 'METHOD IN ALGAL ASSAYS (0.5 mll5o 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. - Contrlfugeat 10,OW rpm for 5 minutes.. Decant supernatantliquidand save the precipitatedalgal cells. Using the same centrifugetube, rosuspendalgal cells In algal nutrient wadium to a fixed volume: 15ml. Mix thoroughly and decant the top ]Oml of algal ce,l] suspension, and transferthe remainingvolume (5m))of algal cell suspension Into a clean 250mi Erienmeyerculture flask. Using the som centrifugetubes, transfer45ml of freshalgal nutrientmedium Into the above Erlermeyerculture flask, to bring total volume to 50 ml. ATTACHMENT 5 ALGAL B-IOMASS MONITORING A. GRAVIMETRIC CELL DRY WEIGHT (1) The filterrecommendeids MilliporteypeBD withan 0.6micrometer pore size. The method is as follows: - Dry filters for two hours at 700C in an oven; - Cool filtersin a desiccator containing desiccant, for at least two hours before weighing; - Filter a suitable measured aliquot of the culture under a vacuumor pressurenot to exceed8psi; - Rinse the filter funnel with D.I. water; - Dry the filter to constantweightat 700C, cool in a desiccator for two hours and weigh. Basic instrumentused: Analytical Balance; Mettler ME 30. B. SPECTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLL a (2) Basic instrumentused: Stectrophotometer;Bausch & Lomb, Spectronic20. C. IMPROVED NEUBAUER 0.1 mm DEEP HEMOCYTOMETER COUNTING CHAMBER AND OPTICAL MICROMETER -CUsedto @iihsurediameter of algal f4CV).(3 (I). Miller,W. E., J. C. Greene,and T. Shiroyama. 1978- Selenastrum caprlcornutumPrintz Algal Assay Bottle Test: Experimental Design,Application,and Data InterpretationProtocol. Ecol. Res. Series EPA-600/9-78-018. Corvallis,Oregon. pp. 27. (2). APtiA-AWWA-WPCF(1975). "Standard Methods for the Examinationof Water and Wastewater," (M. Franson, manag. ed.), 14thedition, pp. 1030-1031. (3). Brite-Line(R),AniericanOpticalCorporation,Buffalo,New York. tRVI$tOWKKTAL LABOItAT(ItY ALGAL ASSAY -. BOTTLE TEST (AA:BT) ALGAL GROWTH RESPONSE TO EC 500 s EXPOSURE (1) NO. kys CELL-ORY WEIGHT CELL COTJNT DAYS .(=/I) (no. cells/ml) 1114-S.-I 4(?. 4- 10 (,5ie-s:,,&ry (2-R,i-1@i a) loq.1 7,-9,1 7 (54, 1- iilq) il4q 4 4, -1- q/,o REC(YVERY (2) CELL-DRY WEIGHT (mg/1) CELL COUNT (no. ce)ls/mi) SEQUENTIAL CGMINATIONS OF EXP6SURE/RECOVERY 4 + 10 7+7 10+ 4 14 + 0 PERIODS (DAYS),- (1) Productivityis based on percent growth reduction;valuesindicate inhibitory response. (2) Productivityis based on percent growth reduction;vaiues indicate recovery response.