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TOXICITY TO AQUATIC PLANTS TEST SUBSTANCE Identity:Perfluorooctanoiaccid,ammonium salt;may alsobe referredto as PFOA ammonium salt,Ammonium perfluorooctanoateP,FO, FC116, FC-126, FC-1 69, FC-143 or as the major component ofFC1015. (Octanoicacid,pentadecafluoro-a,mmonium salt,CAS 3825-26-1) Remarks: The testsample isFC-1015. It'psuritywas notsufficiently characterizedt,hough currentinformationindicatesisa 30% straighctarbon chainversionofFC-143-in80% water. The 3M productlotnumber was "HOGff 205". The followingsummary applies to the testsample as a mlxture of the testsubstance In water solutionwith Incompletely characterized concentrations of impurities.Data may not accurately relatetoxicityof the testsample with thatof the testsubstance. Data was used to comparv toxicityof the branchedlstraightchain ammonium perfluorooctanoatehomologue mixture InFC-143 vs.what Issupposed to be the 1000Astraightcarbon chain ammonium perfluorooctanoate In FC-1015. METHOD: Method: OECD 201, USEPA - TSCA, Buideline797.1050 Type (testtype): Acute Static GLP: No Year study performed: 1996 Species: Selenastrumcapricomutum Source: Originalolbytainedfrom the UniversitoyfTexas atAustinon January 10, 1996, maintainedinculturemedium atT.R. WilburyLaboratories, Inc,Marblehead,Massachusetts. Element basis: Algalcellcounts (cells/mlc)e,lldryweights. Exposure period: 96 hours StatisticaMlethods: Probitanalysis Analyticalmonitoring: pH and temperature. Test Conditions: Algal NutrientMedium: Sterilenrichednutrienmtedium per USEPA 1978 guideline.This nutrientmedium providedallmineralnutrientesssentialfor algalgrowth and alsoserved as the diluentforallalgaloperations.T.he pH of thissyntheticalgalmedium was adjusted-t7o.5 priorto use inassays. Stock and testsolutionpreparation: A 3330 mg/L stocksolutionwas prepared by dilutin3g.33 grams oftestsubstance in1 liteorfwater. Aliquots were then added directltyo dilutiownater intestvesselstocreatetest solutions. Exposure vessels: 250 ml Erienmeyers containing100 ml testsolutionand capped withinvertedglass beakers. Agitation: Shaken continuouslyat 100 rpm. Number of replicates:3 Initiaclellloading: 1.0 X 104 cells/mL Number of concentrations: fiveplusa blankcontrol Lighting:400 foot-candlesfrom continuouscool-whitefluorescentlighting. Water Chemistry: pH range (0-96 h7ours): 7.5 --10.8(controlexposure) 7.4- 7.6 (3,330mg/L exposure) Test temperature range (0-96hours): 24.0- 24.2 OC (incubator) RESULTS Nominal concentrations: Blkcontrol,210, 430, 830,1670, and 3330 mg/L Algal Growth Response EC50 values Exposure (Contact) Hours 24 48 72 96 Cell-Count mg/L (95% C.I.) 2510 (1340->3330) >3330 2040 (1190->3330) 1980 (1710-2360) Average Specific Growth Rate mg/L (95% C.I.) 1700 (673->3300) >3330 >3330 >3330 Element values are based on nominal concentrations. Reversibilityof Growth InhibitionA:liquotsofthe 3330 mg/L testsolution were dilutedwithalgalmedium and culturedfor72 hours. Based on growth observed inthe recoveryphase, the effecton algalgrowth was found tobe algistatic. Remarks: Testingwas conducted on the mixtureofthe testsubstance as describedinthetestsubstance remarks field.The values reportedapplyto thatmixtureand not the testsubstance. CONCLUSIONS FC-1 015 exhibitsa 96-hour EC,50cellcountvalue of 1980 (1710-2360 mg/L) and a 96-hour E C50 growth ratevalue of>3330 mg/L. The 96-hourNo Observed EffectConcentration(NOEC) is210 mg/L forcell count and 430 mg/L forgrowthrate.The Lowest Observed Effect Concentration(LOEC) is430 mg/L forcellcountand 830 mg/L forgrowth rate.Thistestsubstancewas determinedtobe algistatic. Submitter: 3M Company, EnvironmentalLaboratoryP,.O.Box 33331, St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:ilmischranking= 2.Thisstudymeets thecriterifaorquality testing.However, thestudylacksinformatioonn puritoyfthetestsubstance and actualmeasurements oftheamount oftestsubstanceinsolution.Also, no explanationisgivenas towhy the48-hourvalueswere inexcess ofthe24 hour values. REFERENCES Testwas conducted by T.R.WilburyLaboratoriesI,nc.,of Marblehead, Massachusettsatthe requestofthe3M Company, Lab Request number P1624,1996. OTHER Last changed: 5/24/00 study Title Growth and Reproduction Toxicity Test With FC-1015 And The Freshwater Alga, Solonastrum capricornutum Guideline Number U.S. EPA-TSCA, Guideline 797.1050 OECD, Guideline 201 Authors Timothy J. Ward Jacqueline M. Novius Robert L. Boeri Study Completed May 15, 1996 Sponsor 3H Company Environmental Lak>orator-y 935 Bush Building Avenue 2-3E-09 st'. Paul7, Minnesota 55144 Test ng Facility T R -':@iibry' L.bo.ato.ies,,.Inc. 4 0 D k i',-Lan 1. TAZLB Or CO.iKXi SZCTION: 1. Table of Contents ii. Index of Tables and Figures II-I. Summary IV. GenoUl information V. Introduct4-on VIC Methods and Materials VII. Results VIII. References IX. signature Page Appendix A. Water Quality Data from The Toxicity Test PHGE 2 3 4 5 6 6 16 17 18 I r s5 y@ r ii. xxdzx or TABLzs AnD vicuitzs Table 1. Chemical characterization of a representative sample of water used to formulate test media for the toxicity test with the freshwater alga, solonastr-um capricornut=, and rC-1015. Table 2. Test conditions summary for the toxicity test with FC-1015 and the freshwater alga, solonastr-um ca,p,ricornutum. Table 3. cell growth FC-1015 and capricornutum. data from the toxicity test with the freshwater a@ga, selenastrum Table 4. Average specific from the control freshwater alga, FC-1015. growth rate and percent change from the toxicity test with the selennstrum capricornutum, and Table 5. Effective concentrations CECIOS, Ec50m, EC909) from the toxicity test with the freshwater alga, selenastrum capricornutum, and FC-1015. Table A.l. Temperature measured with FC-1015 and the capricorrutum. during the freshwater toxicity test alga, Selenastrum Table A.2. pH values measured during FC-1015 and the freshwater capricarnutlim. the toxicity test alga, selenastrum with Figure 1. Growth of Selenastrum capricornutum to rc-1015 for 96 hours. exposed PAGE 7 9 12 14 15 19 20 ,z.R@;..wilbury Study,N umbe ",029-TH Ill. BUNKALRY The toxicity of FC-1015 to the freshwater alga, Solonamtrum capricornutum, in deacrik>adin this final report. The text was conducted for 3M company for 96 hours from April 19 to 23, 1996 at T.R. wilbury Laboratories, Inc. in Marblehead, Massachusetts. It was conducted according to procedures of the U.S. Environmental Protection Agency (1993) and the OECD (1984). FC-1015 was supplied by the sponsor. The test was performed under static conditions with five concentrations of test substance and a dilution water cont-rol at a temperature of 24 1 2*C. Nominal concentrations of FC-1015 were 0 (control), 210, 430, 830, 1,670, and 3,330 mg/L. The d-ilutionwater was sterile enriched media adjusted to a pH of 7.5. Exposure of algae to the test substance for 96 hours resulted in a median effective concentration (EC50) of 1,980 mg/L FC-1015 (95% confidence interval - 1,710 to 2,360 mg/L) when calculated using th number of calls/ml, and >3,330 mg/L when calculated using the averag: pecific growth rate. The 96 hour no observed effect concentration (NOEC) 8Is 210 mg/L and the lowest observed effect concentration (LOEC) in 430 Mg/L when calculated using the number of cells/ml. The NOEC is 430 mg,'L and the LOEC is 830 when calculated with the average specific growth rate. 6 uav.nLuMmDerI.U XV. GE"SLAL INFORMATION Material Testedt sponsort Test Substance shipped From: Reported Purity: Experimental Start Date: Experimental Date: completion Axchive the Raw Copy of Location for Data and a the Final Report: FC-1015 3H Company Environmental Laboratory 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55106 3H company Environmental Laboratory 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55106 1001 April 19, 1996 - April 23, 1996 T.R. Wilbur-y Laboratories, Marblehead, Massachusetts Inc. ;tudy-_ A,z 029-TH V. iwrxovu=lox This study was sponsored by 3H Company. The objective of the study was to determine the 24, 48, 72, and 96 hour effective concentrations (EclOn, EC50s, EC90s) and no observed effect concentration (NOEC) of the text substance to the freshwater alga, Solonastrum capricornutum, under static conditions. The test was conducted following U.S. EPA (TSCA) and OECD guidelines but at the Sponsor's request it was not conducted according to Good Laboratory Practice (GLP) rules. The report contain. section@ that describe the methods and materials employed in the study and results of the investigation. The report also contains an appendi-x that presents water quality data collected during the test. VI. MZTHOI)S AND MATIMIALS TEST SUBSTANCET was was The saliple of FC-1015 (T.R. Wilbur-y Laboratories sample delivered to T.R. wilbur-y Laboratories on April 4, 1996. delivered in a metal can within a cardboard box Number 604) The samplo at ambient temperature. The test substance was contained in 1 250-ml plastic bottle. The label attached to the bottle contained the following information: -rc-,,- 1015, (LR P1624), 4/2/48-. See KSDS, Susan A. Beach, 2-3E-09, 16l2) 778-7452-, ',v, FC-1015 (a clear liquid) was supplied by 3m Company, St Paul, Minnesota. Prior to use the test substance was stored in the dark at room temperature. The test substance was assumed to have a purity of loo%,. active ingredient and to be stable under storage and testing conditionsU:iused test substance is returned to the sponsor. DILUTION WATER: water used for acclimation of test organisms and for all toxicity, testing was sterile enriched media JU.S. EPA, 197B; T.R. WilbUry standard, operating Procedure number 6) adjusted to a target pEiof 7.5 prior to Characterization of a representative sample of water used to for-mulate' media is presented in Table 1. TEST ORGANISMI I AIlgae @-from a cuitu -@Univernitk Lab@@' to 1 f, used or the re 'originally '@OfTTexast,.- test-(solon&Btrum procured _,at from Austin capricornutum, the culture colloc znd , dalivered,;t x on 0 T.R 1 @as a a,'ii Wil '10'!'1%996. --'The@culture'wa's @ttra@'sf red' itaiii- b ia;-to a t'ea.nd, ma intakin4d-. a the; definiti@e@4.i6,it, t he @'st'rat.@,,o n, v. tifi8,ation*@i*,Of,@,t@'o'cul '.. axonomic key.@;@ Stu d y'Number 1029-TH Table 1. Chemical characterization of a representative sample of water used to formulate tomt media for the toxicity test with the freshwater alga, solonastrum capricornutum, and FC-1015. Parameter' unit of Measurement Detection Limit Measured value Metals Aluminum )Lrsenic Boron cadmium Chromium cobalt Copper Iron i,ead Mercury Nickel Silver Z4.nc Nitrate chloride Fluoride Total Organic carbon Total Phosphorus organochlorine Pesticides Toxophone organophosphorus Pesticides Dimethoate 7EPP Monocrotophos PCBs mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L. mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L as N mg/L mg/L pg/L ;jg/L jjg/'L ;jg/L pg/L p g/L 0.1 0.01 0.5 0.0002 0.01 0.03 0.005 0.03 0.005 0.0003 0.03 0.02 0.02 0.05 1 0.1 I 0.03 0.5 2 0.5,, -2.0 2.0 20 0 5' ND2 ND ND 4 ND ND RD ND 0.03 ND ND ND ND ND ND ND ND ND No tiD IM ND ND ND ND t as- -.I.,,Parametors _dilut on st ng A. f4LP no in acs a t ba o ec@ Pa.coagin am,[ waolc Au cju9 t to 19cl9u5,@ n, ML4DuLy a. N i@@l 29-XH all TOXICITY TEST3 The toxicity toot was performed from April 19 to 23, 1996, according to procedures of the U.S. Environmental Protection Agency (1993@ and the OECD (1984). A nu=ary of the test conditions is presented in Table 2. A screening test was not conducted because nominal concentrations for the definitive test were provided by the sponocr. The test was conducted at a target temperature of 24 1 2*C with five concentrations of test substance and a dilution water control. N-inal concentrations of the test substance were: 0 mg/L (control), 210, 43.0, 830, 1,6.70, and 3,330 mg/L. A 3,330 mg/L stock solution was formulated by adding 3.33 9 of test substance to a 1,000 ml Class A volumetric flask and filling the flask to the mark with sterile algal media. The stock solution was mixed and appropriate &mounts were added directly to dilution water in test vessels to formulate test media. Algae were distributed among three replicates of each treatment at the rate of approximately 10,000 cel'-s/ml. The test was perfor-med in 250 ml glass Erler=eyer flasks that contained 100 ml of test solution. Test vessels were capped with inverted glass beakers and randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator during the test (a random numbers table was used to select the location of each vessel). A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of 400 footcandles. The number of algal cells/ml in each test vessel and the occurrence of relative size differences, unilsual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells wan determined visually by means of direct microscopic examination with a hemocytometer. Cell counts were made and recorded daily during the 96 hour test. The determination of whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test (0.5 mi of media from each 3,330 mg/L vessel were tra-nsferred to a vessel containing 100 ml of fresh media without FC-1015 and incubated for 72 hours under test conditions). Temperature of the incubator was measured and: recorded: daily (thermometer number 3794), and pH (Beckman model pHl 12 motor; insirument-@--tt n,,- r 144 i was determined in each test vessel, at the, beginning and end of the @T Q in a repro aant ativi'v's-@maI'of watei incubate d with the@-teaztvessels was continuously re ord@ai T.R. wilbury@study Number 1029-TH @Pagi A Table 2. Test conditions @,,--ry for the toxicity test with FC-1015 and the freshwater alga, Solonastrum capricornutum. specigst culture Acclimation Pariodt solonastruin capricornutum >14 days Test Duration: 96 hours Test Vessels: 250 ml glass flasks volume of Test Media: 100 ml Number of Replicate Test vesselst 3 Inoc,.ilLim. -10,000 cells per ml oscillation Rate 100 rpm Incu.bator Temperature Range: 24.0-24.2*C Photoperiod: 24 hours light anli 0 hours dark. Light Intensity: 400 footcandles water Quality Measurements: PH in each test vessel at the beginning Incubator and end of the test. temperature daily. Dilution water analysis is summarized in Table 1. Ead Points: Acceptability criteria:, 24, 48, 72, and 96 li-jurECIO, EC50, I and EC90; no observed effect concentration. lo "k garithmic growth at 96 hours a nd acceptable temperature range. 12 1 ly ,studvl, STATISTICILL KMODS, The average specific growth rate was calculated as the natural log of the number of calls/ml at 24, 48, 72, and R6 hours minus the natural lc,gof the number of calls/ml at 0 hours divided by the exposure period. The percent change from the control was calculated by subtracting the treatment average specific growth rate fr= the control average specific grow-.h rate, dividing the difference by the average specific growth rate in the control, and multiplying that value by 100. Results of the toxicity test were interpreted by standard statistical techniques, when possible. The probit method (Stephan, 1983) was used for calculating ECIO, EC50, and EC90 values (the probit method was used despite cautionary statements to values compared to other mothode@. using the number of cello/ml and the because EC50 values compared well All calculations were performed average specific growth rates and .ICZ[Lir,caolncentrations (itcec) w@s determined ()LNOVA) and the number of rc-1015. The no observed effect concc,.itraticn using a parametric one-way analysis of variance of cells/ml and the average specific growth rate in each test vessel at the and of the test. .@-w bury's udy,, N -6@, -or VIZ. PJtSttLTS Insoluble material was not observed in test vessels during the toot The algal . population grow well, resulting in an average of 1,471,000 throughout calls/ml in the control after 96 hours the test was within acceptable limits (Table 3). Water quality Ile.@,Z.t@l. (Appendix A) .' The range' of incubator temperatures was 24.0 to 24.2*C (Table A-1). The pH of, test'. Media was not significantly affected by the test substance at the beginning of the test (Table A.2). Biological data generated by the acute toxicity test with IPC-1015 are presented in Tables 3 and 4. The growth curve for algae exposed to the test substance for 96 hours is presented in irigure 1 (the slope of the dose-response curve is 2.2 based on cells/ml at 96 hours). ' The ECIO,@ EC50, and EC90 vatues for algae exposed to FC-1015 are presented in Table 5. The 96 hour EC50 (and associated 95 percent confidence lixlits) is',., 1,980 mg/L (1,710 and 2,360 mg/L) when c@lculated using the number of calls per ml, and >3,330 mg/L when calculated using the average specific growth rate. No effects (size differences, unusual call shape&, color flocculations, adherence of cells to test containers, or aggregation cells) were noted during the test. The 96 hour NOEC is 210 mg/L and the LOEC is 430 mg/L FC-1015, when calculated using the number calls/ml and the 96 hour NOEC is 430 mg/L and the LOEC is 830 mg/L FC-1015t when calculated using the average specific growth rate. The determination of whether toxic effects were algi3tatic r algicidal was performed at the conclusion of the toxicity test. A 0.5 ml aliquot of media from each 3,330 mg/L vessel was transferred to a'vesse containing fresh media without FC-1015 and incubated for 72 hours un test conditions. Algae increased from <10,000 830,000 cells/ml, indicating that the effect of FC-1015 at concentration was algistatic rather than algicidal. z tot-, @i;tudy Number.@ lo@2"'9@-TH a Table 3. call growth data from the toxicity test with FC-1015 and the freshwat*r alga, solonastrum capricornutum. Nominal Concentration of rc-1015 (mg/L) Replicate 0 (control) 210 430 830 1,670 3,330 1 2 3 mean 1 2 3 mean t control 1 2 3 mean % control 1 2 3 mean t control 1 2 3 mean t control 1 2 3 mean t control, Number of calls/ml x 103 (hour) 0 24 49 72 96 10 32 10 32 10 34 10 33 10 30 10 30 10 34 10 31 100 94 10 30 10 40 10 2B 10 33 100 100 10 30 10 24 10 2B 10 27 100 82 10 20 10 22 10 16 10 19 100 58 10 16 10 12 10 14 10 14 100 42 136 664 146 662 128 696 1.37-,,-, 674 154 724 146 650 144 712 695 108 103 138 620 140 598 148 662 142 627 104 93 156 400 160 484 150 436 155 440 113 65 140 404 142 402 138 @380 140 295 102 59 88 280 84' 232 so 250 84 254 6 1', 38 1,480 1,518 1,416 1,471 1,442 1,496 1,466 1,468 100 1,338 1,280 1,452 1,357 92 1,190 1,096 1,168 1,151 78 798 862 870 843 57 460 454 488 467 '-32 V studi'Number,1029-7H@ Oag*,-12'ot,2^ 10000000 - IDOOOOO u IDOOOO 10000 0 24 48 Hours F--Control 830 m8/L -a-- 110 mc@IL 1,670 mg/L i 72 96 430 mg/L 3,3 30 mg/L 1. Growth of selenastr-umcapricorn um exposed.to'rc-lois for:-@@ 0 WiLburv study,Numbe, Table 4. Average specific growth rate and percent change from the control from the toxicity test with the freshwater alga, Sol*nAStrum capricornutum, and rc-1015. Nominal concentration of rc-iOI5 (mg/L) Average Specific Growth Rate 24hr 48hr 72hr 96hr Percent Change Prom control 24hr 48hr 72hr 96hr 0 (control) 210 430 830 1,670 3,330 0.050 0.055 0.058 0.052 0.047 0.056 0.059 0.052 0.050 0.055 0.057 0.051 0.041 0.057 0.053 0.049 0.027 0.055 0.051 0.046 0.014 0.044 0.045 0.040 6 -2 -2 0 0 0 2 2 18 -4 9 6 46 0 12 12 72 20 22 23 r.ml,@-wilburX@study Number 102?9,7-TH Table 5. Effective concentrations (zclos, xc5on, EC90m) from the toxicity test with the freshwater al(ja, S&IOnastrum capricornutum, and rc-iols. Time EC Value (mg/L) 95 Percent Confidence T-i-its (mg/L) Calculated using the number of C*Ils/wl 24 hours 49 hOLrS 72 hours 96 hours Eclo EC50 EC90 - 524 - 2,510 - >3,330 EC50 >3,330 Eclo Ec50 Ec90 448 2,040 >3 , 3 3 0 Eclo Ec50 EC90 533 1,980 >3,330 <210 - 1,010 1,340 - >3,330 ->3,330 >3,330 <210 814 1,190 - >3,330 >3,330 417 - 643 1,710 - 2,360 >3,330 calculated using the ilverage Specific Growth Rate 24 hours 48 hours.,72 hours 96 ho ECIO EC50 EC90 - 541 - 1,700 - >3,330 EC5.0 - >3, 330 EC10 EC50 EC90 - 1,330 - >3,330 - >3, 330 E 10 - 1 430 @E 50 - >3,330 E 0 ->3.,330 <21() 673 2,260 1,110 >3,330 >3,33J >3, 330 970 1 770 -,,,@>3330@ - '-,@@:;>3 330 'i;3,3 3-0 tudy,;N Vill. PXFRREKCZB OECD. 1981. OECD Guidelines for Testing of Chemicals. Annex 2. OECD Principles of Good Laboratory Practice. Adopted I June 1981. OECD. 1984. OECD Guidelines for Effects on Biotic Systems. Adopted 4 April 1984. Testing Method of chemicals. 201, Alga Growth Section 21 Inhibition Test. Stephan, C.E. 1983. computer Program for Calculation of LCSO values. Personal cc=unication. U.S. EPA. 1978. The selonmatz-um capricornutum Printz Algal Bottle Test EPA-600/9-78-018. Envirorawntal Reek:a-rchLaboratory, Corvallis, Oregon. U.S. EPA. 1993. 40 CFR Part 797. Toxic Substances Control Act Test Guidelinas; Final Rules. section 797.1050. U.S. EPA. 1993. 40 CFR Part 792. Toxic Substances Control Act (TSCA); Good Laboratory Practice standards; Final Rule. sicKATUILE PkGo Jac/qualine M. Oevium study Director Biologist ic Fo@raL.,,Kowalski Aq t c oxicologist Je@anne P. Hagaiu Biologist For; roven M. i@t-6vens B;-OlogiSt, Rob@rt L. 'Boerl -coauthor T tby J. Wdrd or 3t Appendix A: WATER QUJLLITY DATA rRO14 TSE TIOXICITT TTST ZA', p , 'F@k@o -lbury stuay'siu Tabl* A. l. Tomp*rature measured during the toxicity test with TC-1015 and the freshwater alga, solonastrum capricornutum. Hour of Exposure Temperature of Incubator (*C) 0 241.0 24 24.1 48 24.2 72 24.0 96 24.0 ,Stu N ar.@.IOZV H Table A.2. pH values measured during the toxicity test with rC-1015 and the freshwater alga, sel*namtrum capricornutum. NoszLinal Concentration of rc-1015 (mg/L) 0 (control) 210 430 830 1,670 3,330 Replic8t& 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 pH initi@a Final 7.5 7.5 7.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 10.7 10.8 10.8 9.9 9.8 10.0 6.5 6.4 6.4 6.1 7.0 7.4 7.6 7.6 7.4 7.6 7.5 7.6