Document Qeeb9q8qdkMMYk0v8jMpvMNo

TOXICITY TO AQUATIC PLANTS (E.G.,ALGAE) TEST SUBSTANCE Identity:Perfluorooctanoiaccid;may alsobe referredtoas PFOA, FC26, or FX-1001. (Octanoicacid,pentadecafluoro-C,AS # 33567-1) Remarks: The 3M productionlotnumber was 269. The testsubstance isa white powder. The testsample isFC-26 referredtoby thetest laboratoryas N2803-3. The purityofthe sample was not sufficiently characterizeda,lthoughcurrentinformationindicatesitisa mixtureof 96.5 - 100% testsubstance and 0 - 3.5% C6, C7, and Cg perfluorohomologue compounds. Remarks field:The sample preparationdirectiongsiventothe laboratory were todissolvethe testmaterialina 50:50 water:isopropanolsolution.In the protocolamendment, itwas statedthatthesample was combined withisopropanolina 50:50 ratiopriortouse. METHOD Method: U.S. EPA-TSCA Guideline797.1050 Test: Staticacute GLP: Yes Year completed: Study completedin1995. Reportcompletedin1996. Species: Selenastrumcapricomutum Source: Originallfyrom The CultureCollectioonfAlgae attheUniversity ofTexas atAustin,maintainedinculturemedium atT.R. Wilbury,Inc., Marblehead, MA. Element basis:Reported two ways: number ofcells/mLand growth rate. Exposure period:96-hours Year study conducted: 1995 Year study completed: 1996 Analyticalmonitoring: Nominal concentrations.pH and temperature. Test organisms laboratoryculture:Algae cultureswere growing inU.S. EPA-recommended sterileenrichedmedium foratleast14 days priorto testinitiation. Test Conditions: Algal medium: preparedto U.S. EPA recommended concentrationsby spikingdeionizedwater withnutrientstocks.The pH ofthe syntheticalgalmedium at testinitiatiwoans 7.4. Algal medium used forculturinagnd as diluent. Stockand testsolutionpsreparationA: 1,000mg/Lprimary stocksolutionwas preparedinsterileenrichedmedia. Appropriate amounts ofthisstocksolutionwere added directltyodilutiownater toformulatethe testmedia. Exposure vessels: 250 mL glassErienmeyer flaskscontaining50 mL oftestsolution. Agitation:Shaken continuouslyat 100 rpm Number of replicates:three Initiaallgalcellloading: 1.0 X 104 cells/mL Number of concentrations: Fiveplusa negativecontrol Water chemistry: pH range: (0- 96 hours) 7.4- 10.3 (controelxposure) 2.9- 3.0 (1,000mg/L exposure) Test temperature range: (0- 96 hours) 23.5- 24.OOC Lightlevels:(0- 96 hours) -380 ft-cfrom continuouscool-whitfeluorescent lighting Remarks: The pH ofthetestsolutionsforthe250, 500 and 1,000 mg/L exposure concentrationwsere inthe range of2.9-4.0attestinitiatioTnh.is low pH would have adverselyaffectedthe survivaalnd subsequent growth ofthe algae. RESULTS Nominal concentrations: Blank control,63, 125,250, 500, 1,000 mg/L (Testedas 50:50 mixtureoftestsubstanceand isopropanol)T.he concentrationoftestsubstance insolutionwas 32, 63, 130,250 and 500 mg/L. Element value (as-tested): 72-hour EC50 (celldensity) 180 (125-250)mg/L 72-hourErC50 (growthrate) 180 (125-250)mg/L 96-hourECro (celldensity) 180 (125-250)mg/L 96-hourE,C5o(growthrate) 180 (125-250)mg/L 96-hourNOEC: 125 mg/L Element value (based on concentrationoftestsubstance in solution): 72-hourEC5o (celldensity) 90 (63-130)mg/L 72-hourErC50 (growthrate) 90 (63-130)mg/L 96-hourEC50 (celdlensity) 90 (63-130)mg/L 96-hourErC50 (growthrate) 90 (63-130)mg/L 96-hour NOEC: 63 mg/L Allelement values based on nominal concentrations. A toxicittyestconducted previouslywithisopropanolat 1,000 mg/L showed no effecton algalgrowth. Statisticamlethods: Celldensitiesg,rowth ratesand percentinhibition values used toestimatethe EC,o,EC5o,and EC90 valuesand 95% confidencelimitwsere calculatedusingthecomputer softwareof C.E. Stephan. The no observed effectconcentratio(nNOEC) was calculated usingone-way analysisofvariance(ANOVA). Control response: Satisfactory Biologicalobservations after96-hours: Nominal Mean Number Concentration of Cellsper (as-tested), mL mg/L Percent Percent Inhibitivoina Inhibitivoina Density Growth Rate Control 1,671,000 - - 63 1,732,000 -4 -2 125 1,560,000 7 0 250 <10.000 100 100 500 <10,000 100 1,000 <10,000 100 Observations, Algal cellcounts in each testvessel were determined by means ofdirectmicroscope countswitha hemocytometer. After96 hours ofexposure, therewere no signsofaggregation,flocculatioonr adherence ofthe algae totheflasksinthe controlorany testtreatmentgroup. In additiont,herewere no noticeablechanges incellsize,coloror morphology when compared tothe control. Reversibilityof Growth Inhibition:Effectofthetestsubstance was determined tobe algistatibcased on the resultsofthe post-definititveest exposure. CONCLUSIONS The testsample 96-hour EC50 and 95% confidenceintervaflor Selenastrum capricomutum was determinedusingtwo calculation methods. The as-testedtestsubstance 96-hourECso forSelenastrum capricomutum was determined tobe 180 mg/L witha 95% confidence intervaolf125 - 260 mg/L, when calculatedusingeitherthe celldensityor growth rate.The as-testedtestsubstance96-hourno observed effect concentration(NOEC) was 125 mg/L. The 96-hourEC5o forSelenastrum capricomutum based on the concentratioonftestsubstance insolution was determinedto be 90 mg/L witha 95% confidenceintervaolf63 - 130 mg/L usingeitherthe celldensityor growth rate.The 96-hour no observed effectconcentratio(nNOEC) forthe testsubstance insolution was 63 mg/L. No signsofaggregation,flocculatioonr,adherence were noted inany ofthetestsolutionsT.his testsubstance was determinedto be algistatic. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY Reliability:Klimischranking3. The studylacksanalyticamleasurement oftestsubstance concentrationsinthetestsolutionsand sample purityis not sufficientclhyaracterizedA.dditionalltyh,ereappears tobe a discrepancybetween the sample preparationdirectiongsiventothe laboratoryand the procedureconducted by the laboratorytoprepare the testsolutions.Initilaolw pH valuesinthe higherconcentrationsmay have had an adverse effecton the survivaalnd growth ofthe algae. REFERENCES This study was conducted atT.R.WilburyLaboratoriesI,nc.,Marblehead, MA, atthe requestofthe 3M Company. OTHER Last changed: 5/25/00 Study Title Growth and Reproduction ToXicitY Test with X2003-3 and the Preshwater Alga, solonastrum capricarnutum Data R*quiremati Guideline Number U.S. ZPA-TSCA, Guideline 797.1050 Authors Timothy j. ward Jeanne P. Magazu Robert L. Boari Study Completed January 24, 1996 sponsor 3H Company Enviromental Laboratory 935 Bush Avenue Building 2-3z-09 St. Paul, Minnesota 55144 Testing Facility T.R. Wilbury Laboratories, 40 Doaks Lan* Marblehead, Massachusetts inc. 01945 t4 T.R. wllbury Study Number 693-TH Page I OOC)D LAWRMAT PRAMICZ COWLIANCZ STATZMENT This study was conducted according to EPA Good Laboratory PractLc* Regulations (40 CrR 792), with the following exc*ptions. The stability of the test substance under test conditions was assumed but not verified at T.R. wilbury Laboratories. Deviations from the study protocol are discussed in section xxx of this report. Jeanne P. Magaz.,@ Study Director T.R. Wilbury study Number 893-n, Page 2 of 24 11. QUILLITRABSURAWM STATnam Submitted bYl T.It.wilbury Laboratories, Inc. 40 Doak@ Lane Marblehead, Massachusetts cortificationt T.R. Wilbury Laboratories, Inc. study number 893-TB, Growth and Reproduction Toxicity Test with U2803-3 and the Freshwater Alga, solonast=m capricorj3utum, was audited by the Quality Assurance Unit for compli&nc* with the protocol, standard operating procedures, and applicable Good Laboratory Practices (U.S. ZPA, 1993). Quality assurance audits were performed and the findings reported to T.R. Wilbury Laboratories management and the study director an the following date@: Aspect Audited Protocol: In-life: Draft Reportt Final Report: Audit Date S/ 4/95 9/ 8/95 9/29/95 1/24/96 Reported to Study Director S/ 4/95 9/ B/95 9/29/95 1/24/96 Reportee to Management S/ 4/95 1/24/96 1/24/96 1/24/96 P. Paradic* Quality Assurance officer T.R. wilbury study Number 893-TH Page 3 of 24 III. TABLE OF CONTZWZS SECTION: X. Good Laboratory Practice Compliance Statement ii. Quality Assurance Statement 111. Tabl* of Contents IV. index of Tables and rigures V. summary VI. General information VII. introduction VIII. Methods and materials IX. Results X. References Xi. Signature Page XII. Protocol Deviations Appendix JL. Water Quality Data from Toxicity Test PAGE 2 3 4 5 6 7 8 8 13 19 20 21 22 T.R. Wilbury Study Number $93-TH Page 4 of @24.,,,' 7 zv. ZNDXX or TAWAS "D rzonn Table 1. Chemical characterization of a representative N&VIO of toot media and water used to formulate test media for the toxicity test with the freshwater alga, so)on"t=n caprivornutum, and U2803-3 Table 2. Test system X2803-3 and capricornutum summary for the toxicity test with the freshwater alga, solonastr-,- Table 3. C*ll growth data from the toxicity test with M2803-3 and the freshwater capricornutum alga, solen&8tz,,- Table 4. Table S. Historic cell growth data from a toxicity test with isopropyl alcohol and the freshwater alga, solonastrum capricornutum (from Ward, ot.al., 1995) Average specific from the control freshwater alga, U2803 -3 growth rate and percent change from the toxicity text with the Solonastrum capricornutum, and Table 6. Effective concentrations (EC50s) from the toxicity test with the freshwater alga, Solonastrum capricoznutum, and U2803-3 Table A.I. Temperature measured with U2803-3 and the capricornutum during the freshwater toxicity test alga, 562onast:.-m Table A.2. pn values measured during the toxicity test with U2803-3 and the freshwater alga, Solonastzum capricernutum Figure 1. Growth of So2onastrm capricornutum to 92803-3 for 96 hours exposed PAGE 9 11 14 1! 17 is 23 24 16 ?.A. Wilbury study Number 893 -TH : Page 5 of 24 V. stuany The toxicity of N2803-3 to the freshwater alga, solon&strum O&Prlcornutum, is described in this final report. The test was conducted for 3M Company for 96 hours from August B to 12, 1995, at T.R. Wilbury T-m ratories, Inc, in x=blehead, Massachusetts, It was conducted by Jeanne Nagazu, Pot*r rovalski, Jacqueline movium, Xeven Stevens, ftter Movium, Robert B"ri, and Timothy ward according to the protocol developed for T.R. Wilbury study Number 893-TH. N2eO3-3 van supplied by the sponsor. The test was performed under static conditions with five concentrations of test substance and a dilution water control at a temperature of 24 t 2*c. The sample of N2803-3 supplied by the sponsor was combined with isopropanol in a 50:50 ratio prior to use, and this mixture 13 defined as the test substance an-test*d. A toxicity test was previously conducted with 1,000 mg/L isopropanol (ward st.al., 1995) and algal growth was not affected at 1,000 mg/L, indicating t@at the concentration of isopropyl alcohol in the sample of N2803-3 can not, by itself, account for the toxicity of U2803-3 to algae. Results of the toxicity test are reported twice, once based on nominal concentrations of test substance an-tosted and a second time based on concentrations of test substance as-r*coived (N2803-3 without isopropanol). Nominal concentrations of U2803-3 &*tested were 0 (control), 63, 125, 250, 500, and 1,000 mg/L and nominal concentrations of N2803-3 as-received were 0 (Control), 32, 63, 130, 250, and 500 mg/L. The dilution water was sterile enriched media. Exposure of algae to the test substance for 96 hours resulted in a median effective concentration (EC50) of 180 mg/L N2eO3-3, with SS% Confide=* limits of 125 and 260 mg/L, when calculated using either the .I'M r of calla per ml or the average specific growth rate, based on test substance an-tested J50% N2803-3 and 501 isopropanol). The 96 hour no observed effect concentration (NOEC) in 125 mg/L and th* low.4stobserved effect concentration (LOEC) is 250 mg/L, based an test substance astested. The 96 hour EC50 is 90 mg/L H2803-3 (95% confidence limits - 63 and 130 mg/L, when calculated using either the number of cells per ml or the average specific growth rate and the 96 hour NOZC is 63 mg/L and the LOEC in 230 mg/L, based on test substance an-received (N2803-3 without isopropanol). T.R. wilbury Study number 993-TH Page 6 of 24 Vi. OURRAL zwopmzzon Material Tested: sponsors Test Substance Shipped From: Reported Purity: study Initiation Date: Experimental Start Dates Experimental Completion Date: Study Completion Date: Archive Location for the Biological Raw Data and a copy of the Final Reports U2003-3 3M company Environmental Laboratory 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55144 3H company Environmental Laboratory 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55144 100% August 16, 1995 (protocol signed) September 8, 1995 September 12, 1995 January 24, 1996 T.R. Wilbury Laboratories, Inc. Marblehead, Massachusetts @T.R. wilbury study Number e93-TH Page 7 of. 24 Ilk - Viz. IMODUCTION This study was sponsored by 3H Company. The objectiv* of the study was to determine the 24, 49, 72, and 96 hour effective concentrations (ZCIOB, ZCSON, EC90s) and no observed effect concentration (MORC) of the test substance to the freshwater alga, Sflonamtrum caprlcornutum, under s1t9a9t3i)c. conditions. The test protocol was based an U.S. EPA methods (EPAP The report contains sections that describe the methods and materials employed in the study, and results of the investigation. The report also contains an appendix that presents water quality data collected during the test. TXST BUBSTJLKCR2 VIXX. HVMDB AND HATERIALS The sample of M2003-3 (T.R.wilburyLaboratoriesSample Number 521) was delivered to T.R. wilbury Laboratorieson August 14, 1995 in a cardboard box at ambient temperature. approximately50 ml glass jar. The label attacihtedwtaosthceonbottatilneedinicnludaend the following information: "N2803-3". N2803-3 is white sol id) was supplied by 3M Company, St Paul Mro4o-mnnotneomtpear.aPtruiroer. to use the Ample of N2803-3 was stored in the dark at The sample of N2e03-3 supplied by the sponsor was combined with isopropanolin a 50:50 ratio prior to use. mixture was then consider*d to be 1001 test substance dTuhriisng5OtS5h0e performanceof the toxicitytest. All resultsare reported both on the basis of test substanceas-tested (50% N2803-3and 50% isopropanol)and test substanceas-received(N2803-3withoutisopropacol).The M2803-3was assumedto have a purity of 100% activeingredientand to be stable under tshteorsapgoenasnodr.testing conditions. unused test substancewill be returned to DILUTION WATLPRs water used for acclimation of test organisms a:icfior all toxicity testing was sterile enrichedmedia (U.S.EPA, 1978; T.R. wilbury standard operating Procedure number 6) adjusted to a target pH of 7.5 with 0.1 n pDrCe1spernitoerd tion uTsaeb.leC1h.aracterizationof a representativesample of media in Taft ORGAXIBNI from aAcluglateurueseodrifgoirnatlhleytpersotcu(rSeedlfornoamstthreumCcualptruirceorCnoultluemc,tiUoTnEXOf1A6l4g8a)ewaats the University of Texas at Austin and delivered to T.R. Wilbury Laboratories on April 11, 1995. Tte culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. subsample of algae used to inoculate media at the start of the definitTihvee test came from a 10 day old culture. organisms was verified using an appropriatiedteanxtoinfoimcicationof the culture key. T.R. Wilbury Study Number 893-TS Page 8 of 24 Table Chemical characterization of a representative sample of test Media and water used to formulate tsst media for the toxicity toot with the freshwater alga, 501*nastrm caPrIcOrautum, and U2803-3. Parameter' Unit of Measurement Detection LinLit Measured Value PH Total Phosphorus Nitrate chloride Total organic carbon Metals Arsenic cadmium Copper Fluoride Boron Aluminum Chromium cobalt Iron Lead mercury nickel silver zinc Organochlorine Pesticides Plus Toxaphous Organophosphorus Pesticides PCBB pH units Mg/L Mg/L &a N Mg/L mg/L Mg/L mg/L mg/L mg/L Mg/L mg/L mg/L mg/L mg/L mg/L mq/L mg/L Mg/L mg/L pg/L jig/L ug/L ;jg/L -0.03 0.05 1 I 0.01 0.0002 0.005 0.1 0.5 0.1 0.01 0.03 0.03 0.005 0.0003 0.03 0.02 0.02 0.5 2 0.52 0.5 7.4 0.46 0.08 14 UD3 ND 0.0002 ND ND RD ND ND ND 0.08 RD No ND ND NO ND ND ND ND Notes: 1. The pa was measured in a control test vessel at the start of.;, the test. Total organic carbon, total phosphorus, nitrate,, chloride, fluoride, metals, pesticide, and PCB data Werei collicted during February, 1995, an part of routine biannual water quality testing (these analysis were performed by PIC" Xnc., Hampton, Now Hampshire). 2. ND,- not detected at or,above the detection 1 . . .. . ... T.R..Wilbury Study Number 893-TH Pag@19 of -24 TOXICITY TUT, Results concentrations offor atheradnegfeinfiitnidvientgestte.st were used to select the performed from The range finding test was August 2S to 29, 1995. At the conclusion of the test the number of cells/mi equall*d the following percent of the controls 0.050, 0.50, 5.0, and 50 mg/L - >1001 and 500 mg/L - <It, based on test substance an-received. The definitive toxicity test was performed from September 9 to 12, 1995, according to T.R. Wilbur,;Protocol 893-To (Growth and Reproduction Toxicity Test with N2803-3 and the Freshwater Alga, solons8trum capricornutum). The protocol, which was signed by the study director on August 16t 1995t is based on procedures of the U.S. Environmental Protection Agency (1993). A summary of the test system is presented in Table 2. The test was conducted at a target temperature of 24 t 2*c with five concentrations of test substance and a dilution water control. concentrations of the test substance as-tested were: 0 mg/L (controNlo)m,in6a3l, 125, 250, 500, and 1,000 mg/L. A 1#000 mg/L stock solution of test substance an-tested was formulated on September 8. 1995 by adding I.v g U2803-3 an-tested to sterile enriched media and adjusting the final media volume to 1,000 ml. Appropriate amounts of this stock solution were added directly to dilution water to formulate test media. A text was performed from June 26 to 30, 1995 with isopropyl alcohol (Mallinckrodt, Lot 3035), a component that represents 500 of test substance (Ward, et.al., 1995). This test was conducted at 4.4 mg/L and at 1,000 mg/L. A 1,000 mg/L stock solution was formulated on June 26, 1995 by adding 0.5 g isopropyl alcohol to sterile enriched media and adjusting the final media volume to 500 al. Algae were distributed among three replicates of each treatment at the rate of 10,000 calls/ml. The test was performed in 250 ml glass Erlenmeyer flanks that contained 100 ml of test solution. Test vessels were randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator during the toot (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 approximately 380 foatcandles. The of number of algal calls/ml in each test vessel and the occurrence relative size differences, unusual call shapes, colors, flocculations, adherence determined of calls to test containers, or aggregation of cells was visually by means of direct microscopic examination with a hozxmytometer.' 96 hour test. cell counts were made and recorded daily during the T.R. wilbury study Number 893-TH Page 10 of 24 Table 2. Toot system summary for the toxicity test with U2803-3 and the freshwater alga, solonastrum capricarautum. sp*ci*st Culture Acclimation Poriods Test Duration: Test Vessels: volume of Test Media: Number of Replicate Toot Vessels: xnoculum: OXCill&Zion Ratet Incubator Tomp*rature Range: Photoperiodt Light Intensityt Water Quality Measurements: End Pointat Acceptability Criteria: S*Ionastrumampricornutum >14 days 96 hours 250 ml glass flasks 100 mi 3 10,000 cells per ml 100 rpm 23.5-24.00c 24 hours light and 0 hours dark. -380 footcandles pH in each test vessel at the beginning and end of the test. Incubator temperature daily. Dilution water analysis is a--rized in Table 1. 24, 48, 72, and 96 hour Ecio, zc5o, and EC90; no observed effect concentration. Logarithmic growth at 96 hours and acceptable temperature rang*. T.R. Wilbury Study Number 893-TH Pago 11 of 24 The determination of whether toxic effects were algistatic or algicldal was performed at the conclusion of the toxicity test (0.5 al of media from each 250 mg/L vessel was transferred to a vessel containing fresh media without X2803-3 and incubated for 216 hours under test co=Utions). Temperature of the incubator was measured and recorded daily (thermometer number 2960), and ps (Backman model pal 12 owtorl instrument number 224) was determined in each test vessel at the beginning and and of the test. The temperature La a representative vessel of water incubated with the test vessels was continuously recorded. STATISTICAL NXTNODBT The average specific growth rate was calculated an the natural log of the number of cells/ml at 24, 46, 72, and 96 hours minus the natural log of the number of cells/ml at 0 hours divided by the exposure period. The percent change from the control was calculated by subtracting the treatment average specific growth rat* from the control average specific growth rate, dividing the difference by the average specific growth rate in the control, and multiplying tiat value by 100. Results of the toxicity test were interpreted by standard statistical techniques. The binouLial/interpolation method (Stephen, 1984) was used for calculating 72 and 96 hour values. All calculations were performed using the number of calls/ml and the average specific growth rates and nominal concentrations of the test substance. The no observed effect concentration (HOEC) was determined using a parametric one-way analysis of variance (ANOVA) and the number of c*lls/ml and the average specific growth rate in each test vessel at the and of the test. T.R. Wilbury study xumber 093-Ts Page 12 of 24 IX. RRMTS No insolublmeaterialwas noted in any test vessel during the test. The algal populationin the controltest vesselsgrow wall, resultingLa an averageof 1,671,000cells/mlafter 96 hours (Table3). Water quality throughout the test was within acceptablelimits (AppendixA). The range of incubator temperaturesvan 23.5 to 24.OOC (TableA.1). The pa of test media (Table A.2) was affected by the toot substance at the beginning of the test. Test substance concentrationsabove 125 mg/L reduced pa values to 4.0 and lower. Biological data generated by the &cuts toxicity test with U2803-3 are presented in Tables 3 and 4. The growth curve for algae exposed to the test substance for 96 hours is presentedin Figure I (the slope of the dome-r*mponsecurve could not be calculated from this data set). The 72 and 96 hour EC50 values for algae exposed to N2803-3 are pr,@sentedin Table 6. The 96 hour EC50 (and associated 95 percent confidence limits) in 180 mg/L (125 and 260 mg/L), based on test substanc* as-tent*d (50% U2803- 3 and 501 isopropanol) and the 96 hour EC50 (and associated 95 percent confidence limits) is 90 mg/L (63 and 130 mg/L), based on test substance as-recolved (N2803-3without isopropanol). No effects (size differences unusual cell shapes, colors, flocculations,adherence of cells to ton@ containers,or aggregationof cells) were noted. The 96 hour no observed effect concentration (NOEC) is 125 mg/L and the lowest observed effect concentration (LOEC) is 250 mcj/LN2803-3. based on test substance astested and the 96 hour HOEC is 63 mg/L and the lowest observed effect concentration (Lozc) is 130 mg/L U2803-3, based on test substance asreceived. The determinationof whether toxic effects were algixtatic or algicidalwas performed at the conclusionof the toxicitytest. A 0.5 ml aliquot of media from each 250 mg/L (an-tosted)vessel wo-itransferred to a vessel containing fresh media without N2803-3 and incubated for 216 hours under test conditions. Algae increased from <10,000 cells/mL to 4,320,000 cells/mL, indicating that the effect of N2803-3 at this concentrationwas algistaticrather than algicidal. Biological data generated by a previous exposure of alga* to isopropyl alcohol (ward, st.al., 1995) are presented in Table 4. Algal growth was not affected at 1,000mg/L, indicatingthat the concentration of ,isopropyl alcohol toxicity in N2803-3 can not, by itself, account for the of U2803-3 to algae. T.R. WLIbury Study'Numb*r 893-TH Pago 1-3 of 24 Table 3. C*ll growth data from the &cut* toxicity test with X2803-3 and the freshwater alga, sol*n"trum caprlcornutum. nominal Concentration of U2803-3 (Mg/L) as-T*stod as-Rec*iv.ed Rep. 0 (control) 63 32 125 63 250 130 500 250 1,000 Soo 1 2 3 mean t control 1 2 3 mean 1 control 1 2 3 mean 0 control 1 2 3 mean I control 1 2 3 mean t control 1 2 3 mean 0 control Number of Cella/ml x 103 (hour) 0 24 10 10 10 10 -- 10 10 10 20 100 10 10 10 10 100 10 10 10 10 100 10 10 10 10 100 10 10 10 10 100 22 20 22 21 -- 28 26 is 24 114 10 32 22 21 100 <10 <10 <10 <10 <48 <10 <10 <10 <10 <48 <10 <10 <10 <10 <48 48 72 96 168 146 154 156 -- 134 152 160 149 96 92 9c 140 109 70 <10 <10 -CIO <10 <6 <10 <10 <10 <10 <6 <10 <10 -CIO <10 <10 360 1,800 402 1,612 510 1,600 424 1,671 -- -- 532 520 420 487 115 540 480 520 513 121 <20 <10 -clo <10 <2 <10 <10 <10 <10, <2 <10 <io <10 <10 <2 1,720 1,010 1,666 1,732 lo-' 1,620 1,660 1,400 1,560 93 <100 <1 <10 <10 -cl <10 <10 <10 <10 <1 <100 <1 -CIO <1'C0iL T.R. Wilbury Study Number 093-TH Page 14 of 2 Table 4. Historic cell growth data from au &CutO toxicitytest with isOPrOPY1 alcohol and the freshwater alga, ifelonsetrus caprlccrnutum(fromward, *t.al.,1995). Nominal Concentration of loopropyl Alcohol . (Mg/L) rep. 0 (control) 4.4 1,000 1 2 3 mean 1 control 1 2 3 mean t control 1 2 3 mean t control number of cello/al x 103 (hour) 0 24 48 72 ga 10 22 10 20 10 23 10 22 -- -- 10 14 10 32 10 16 13 21 loo 95 10 31 10 26 10 24 10 27 100 123 134 538 1,132 132 568 1,260 114 614 1,356 127 573 1,249 -- -- -- 112 564 1,236 128 554 1,270 144 584 1,064 128 574 1,190 101 loo 95 136 600 1,370 144 580 1,338 106 512 1,334 129 567 1,347 102 99 108 T.R. Wilbury Study number 893-TH Pag* 15 of 24 10000000 E IODODOO 100000 10000 Controls 250 -g/L 24 48 Hoozs --I-63 ffig/L - 500 mgA 72 96 125 rng/L 1.000 v/L Yigure Growth of SolonaBtz= caPricOrnutum laxposedto U2803-3 for 96 hours. T.S. Wilbury Study Number 893-TH P&ge 16 of 24 Table s. Average specific growth rate &ad percent change from the control from the toxicity tent with the freshwater alga, sel*nastrm caprioccautm, and K2803-3. Nominal Concentration of U-2003-3 (mg/L) aS-Tested as-Rsc*iv*d Average specific Growth Rat* 24hr 48hr 72hr 96hr Percent Change From Control 24hr 48hr 72hr 96hr 0 (control) 63 32 125 63 250 130 500 250 1,000 500 0.031 0.057 0.052 0.053 0.036 0.056 0.054 0.054 0.031 0.050 0.055 0.053 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 -- - -16 2 0 12 100 100 100 100 loo 100 -- -- -4 -2 -6 0 100 100 100 100 100 100 T.R. WiLbury study Number 093-TH Page 17 of 24 Table S. Effective conces@trationg 4SC50s) from tb* toxicity test with the freshwater alga, solonamexua ampricornutum, and U2803-3. Time EC Valu* (mg/L) 95 Percent Confidence Limits (29/L) Calculated based an Test Substance an-Tested (300 M2803-3 and 30% loopropanal) 1. calculated using the Number of calls/al 72 hours 96 hours ECSO - 180 EC50 a 180 125 - 250 225 - 250 2.,Calculated Using the Average specific Growth Rate 72 hours 96 hours ECSO - 180 ECSO n 180 125 - 250 125 - 250 Calculated based on Test Substance as-Received isopropanal) (x28O3-3 without 1. Calculated Using the Number of cello/al 72 hours 96 hours ZCSO - 90 EC50 a 90 63 - 130 63 - 130 2. calculated Using the Average Specific Growth Rate 72 hours 96 hours zc50 - 90 ECSO,-. 90 63 - 130 63 - 130 Notes ZC10 and EC90 values this toxicity test. could not be calculated using the data from T.R. wilbury Study Number 893-TB Pago 18 of 24 Bruce, R.D., and Verateog. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environ. Toxicol. and Cbem. Vol-ii. no. 10, PP. 1,485-1,494. StOPh&n,. C.E. 1983. Computer Program for Calculation Of LCSO Values. U.S. EPJL. 1978. The solonastrum capricornutum priatz jllg&lAssay Battle Test. EPA-600/9-78-010. Environmental Research Laboratory, Corvallis, Oregon. U.S. EPA. 1993. 40 CFR Part 797. Toxic Substances Control Act Test Guidelines; Final Rules. section 797.1050. U.S. EPA. 1993. 40 CrR Part 792. Toxic substances Control Act (TrCA), Good Laboratory Practice Standards; Final Rule. Ward, T.J., J.H. Xagazu, and R.L. Boari. 1995. Growth and Reproduction Toxicity Test with L-13492 and the Freshwater Alga, Sol*nastrum capricornutum. Study conducted for 3M Company. Laboratories final report number 841-TH. T.R. wilbury T.it.Wilbury Study Numb*r 893-TH Page 19 of 24 SZCMTUITX PAOX -manne p. KacjAkZj Study Dir*ctoi v Aquatic Toxicologist e!ver L. ROWainki Aquatic Toxicologist *V us Biologist J4iC4UV.L.InMg. N*%#fUS Biologist r A(A, zurs moven M. Biologist R orpt r,@. Coauthor All Tim*.hy i AUto?r T.R. wilbury study Number 893-TH P&gs 20 of 24 Zll- PROTOCOL AXON== AND MIATZ=S The Protocol was amended to specify that the test substance would be combined in a 50:50 ratio with isopropanol and this solution would be considered the test substance at 1001 active ingredient and to list nominal concentrations for the definitive study. amendments were made to the protocol. No other PROTOCOL DEVIATIONST The 24 and 48 hour ECIO, rc5ol and EC90 values were not calculated. The 72 and 96 hour EC10 and tc9o values and the slope of the done response curve could not be calculated with the data from this test. The pu of deviations deviations test media was 7.4 rather did not affect the outcome were made from the protocol. than 7.5 at 0 br. of this test and no These ottar T.R. Wilbury Study Number 893-TH Page 21 of 24 AppendiX AS WATZR QUALITY DATA rRM TOXICITY TMM T.R. wilbury studk Muliber 893-n Pago 22 of 24 Tabl* A. I. Temperature measured during the toxielty test with N2803-3 and the freshwater alga, Solonastrum capricorautum. Hour of Exposure ToWerature of incubator (-c) 0 24.0 24 23.6 48 23.5 72 23.0 96 23.5 T.R. Wilbury study Number 093-TH Page 23 of 24 T able A.2. pH values measured during the toxicity test with U2803-3 and . the fr*shwateralga, Solonastrumcaprivornutum. Hominal Concentration (mg/L) as-Tentod as-R*Ceived Test with U2803-3 0 (control) 63 32 125 63 250 130 500 250 1,000 500 Replicate 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 PH initial Final 7.4 7.4 7.4 6.8 6.8 6.0 6.4 6.3 6.3 4.0 4.0 4.0 3.4 3.4 3.4 3.0 2.9 2.9 10.3 10.3 10.3 10.3 10.3 10.3 10.3 10.3 10.3 4.1 4.1 4.1 3.4 3.4 3.4 3.0 3.0 3.0 T.R. wilbury study Number 893-TH, Peg* 24 of 24