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ACUTE TOXICITY TO FISH TEST SUBSTANCE Identity:Perfluorooctanoiaccid;may also be referredtoas PFOA, FC26, or FX-1001. (Octanoicacid,pentadecafluoro-C,AS # 33567-1) Remarks: The 3M productiolnotnumber was 269. The testsubstance isa whitepowder. The testsample isFC-26 referredto by the test laboratorays 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:isopropanoslolution.In the protocolamendment, itwas statedthatthe sample was combined withisopropanolina 50:50 ratiopriortouse. METHOD Method: U.S. EPA-TSCA Guideline797.1400 Type: Staticacute GLP: Yes Year study conducted: 1995 Year study completed: 1996. Species: Pimephalespromelas Supplier:AquaticBiosystems,FortCollinsC,O Analyticalmonitoring:DO, conductivitpyH, and temperaturewere monitoreddaily. Exposure period: 96-hours Statisticamlethods: LC50 valuescalculatedw,hen possible,by probit analysis,moving average method or binomialprobabilitwyithnon-linear interpolatiounsingthe computer softwareof C.E. Stephan. Test fishage: Not given. Length and weight of fishincontrol:Average totallength= 33.4 mm, average wet weight = 0.35 g. Loading: 0.23 g fish/L Pretreatment: None Test conditions: DilutionWater: Deionizedwateradjustedtoa hardness of40-48 mg/L as CaC03/L DilutiownaterchemistryR:epresentatisvaemplegiveninTable 1 ofthe finalreport. Temperature: 22 + 2-C Lighting: Cool-wki7tfeluorescentbulbswithan intensitoyf31 ft-c. Photopedod of16-hourslight8,-hoursdark witha 15 minute transitiopneriod. Stock and testsolutionspreparation: Appropriateamounts of testsubstance were added directltyothe dilutiownater inthetest vessels.No insolublematerialwas noted duringthe test. Concentrations dosing rate: Once Stabilityof the testchemical solutions:Not noted. Exposure vessels: 20 L glassaquada containingapproximately 15 L oftestsolutionw,ater depth approximately18 cm. Number of replicates:two Number offishper replicatet:en Number of concentrations:fiveplusa negativecontrol Water chemistry during the study: Dissolved oxygen range (0- 96 hours): 6.4- 8.7 mg/L (controelxposure) 5.2- 8.8mg/L (220mg/L exposure) Conductivityrange (0- 48 hours) 120 pmhos/cm (controelxposure) 110 pmhos/cm (220mg/L exposure) pH range (0- 96 hours) 7.1- 7.6 (controelxposure) 6.2- 7.2 (220 mg/L exposure) Test temperature range (0- 96 hours) 21.9- 22.6*C (controelxposure) 21.9 - 22.60C (220 mg/L exposure) Remarks: The pH ofthetestsolutionfsorthe 360, 600 and 1,000mg/L exposure concentrationwsere inthe range of3.0-4.3attestinitiatioTnh.is low pH would have adverselyaffectedthesurvivalofthefatheadminnow. RESULTS Nominal concentrations: Blank control,130,220, 360, 600, 1,000 mg/L (Testedas 50:50mixtureoftestsubstanceand isopropanol)T.he concentrationoftestsubstance insolutionwas 65, 110, 180, 300 and 500 mg/L. Element value (as-tested): 24-hour LC5o = 280 (220 - 360) mg/L 48-hour LC50 = 280 (220 - 360) mg/L 72-hourLC5o = 280 (220 - 360) mg/L 96-hour LC5o = 280 (220 - 360) mg/L 96-hour NOEC = 220 mg/L Element value (based on concentration of testsubstance in solution): 24-hour LC50 = 140 (110 -180) mg/L 48-hour LC50 = 140 (110 -180) mg/L 72-hourLC5o = 140 (110 -180) mg/L 96-hourLC50 = 140 (110 - 180) mg/L 96-hour NOEC = 110 mg/L Allelement valuesbased on nominal concentrations. A toxicittyestconducted previouslywithisopropanolat500 mg/L showed no mortalitoyr sublethaleffects. StatisticaElvaluation of Mortality:Allofthe LC5o valueswere determined by binomialinterpolation. Biologicalobservations after96-hours: Fishinthe controland the 130, and 220 mg/L exposure concentrationasppeared normal. Totalmortality was observed by 24 hours inthe360, 600 and 1,000 mg/L exposure concentrations. Cumulative percent mortality: Nominal AsTested Conc., mg/L 24-hours 48-hours 72-hours 96-hours Control 0 0 0 0 130 0 0 0 0 220 0 0 0 0 360 100 100 100 100 600 100 100 100 100 1 1,000 100 100 100 1 Lowest concentration Mortality of controls: causing None 100% mortality: 360 mg/L CONCLUSIONS The as-testedtestsubstance 96-hour LC50 forfatheadminnow was determined tobe 280 mg/L witha 95% confidenceintervaolf220 - 360 mg/L. The as-testedtestsubstance96-hourno observed effect concentrati(oNnOEC)was 220 mg/L.The 96-hourLC50 forfathead minnow based on the concentrationoftestsubstance insolutionwas determined to be 140 mg/L witha 95% confidenceintervaolf110 - 180 mg/L. The 96-hourno observed effectconcentratio(nNOEC) forthe test substance insolutionwas 110 mg/L. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:limischranking3. The studylacksanalyticamleasurement oftestsubstance concentrationsinthetestsolutionsand sample purityis notsufficientclhyaracterizedA.dditionalltyh,ereappears tobe a discrepancybetween the sample preparationdirectiongsiventothe laboratoryand the procedureconducted by the laboratorytoprepare the testsolutions.The absence ofpartiamlortalitayt intermediatdeoses resultedinthe sharp dose-response curve seen inthisstudy. As a result, the LC50 valuesdetermined inthisstudymay not accuratelyreflectthe truetoxicitoyfthe solution.The low pH values inthe highconcentrations testedmay have had an adverse affecton survival. REFERENCES This studywas conducted atT.R. WilburyLaboratoriesI,nc.,Marblehead, MA, atthe requestofthe 3M Company. OTHER Last changed: 5/25/00 study Title Acute Toxicity of U2803-3 to the rathead Kinnow, pimpb&l" pzomolas Data Requirements Guideline Number U.S. EPA-TSCA, Guideline 797.1400 Authors Timothy j. Ward J6anDe P. Magazu Robert L. Boeri Study Completed January 24, 1996 Sponsor 3H Company E-iromental Laboratory 93S Bush Avenue Building 2-3Z-O9 St. Paul, Minnesota 55244 Testing Facility T.R. Wilbury Laboratories, inc. 40 Doaks Lane. Marblehead, Massachusetts 01945 I. t. WilbUry Study Number 091-To Page I of 25 I- GOOD LAWRATORY FRAMICX COMPLIAUM STATEMM This study was conducted according to EPA cood Laboratory Practice Regulations (40 CFR 792), with the following exception. The stability of the test substance under test conditions was assumed but not verified at T.R. Wilbury Laboratories. Deviations from the study protocol and protocol amendments are presented in section xxx of this report. Jeanne P. Magazu study Director T.R. WLIbury study mumbor'991-TH"@, Page 2 of 25 21. QUALITY ASSURAWCN STATMIMM Submitted by# T.R. Wilbuzy Laboratories, Inc. 40 Doaks Lane Marblehead, Massachusetts Certification: T.R. Wilbury Laboratories, Inc. study number 891-Tit, Acute Toxicity Of N2603-3 to the rathead minnow, P.iAophalso promolas, was audited by the Quality Assurance Unit for compliance with the protocol, standard operating procedures, and applicable Good Laboratory Practices (U.S. EPA, 1993). Quality assurance audits war* performed and the findings reported to T.R. Wilbury Laboratories management and the study director on the following datez: Aspect Audited Protocol: in-life$ Draft Peportt Final Report: Audit Date S/ 4/95 9/ 5/95 9/13/95 1/24/96 Reported to Study Director S/ 4/95 9/ 5/95 9/13/95 1/24/96 Reported to management e/ 4/95 1/24/96 1/24/96 1/24/96 Arthur P. ara dice Quality Assurance Officer T.R. Wilbury Study Number 891-TH Page 3 of 25 111. TANX or cowZX3rgS BZCTION3 PAGZ 1. Good Laboratory Practice Compliance Statement 2 11. Quality Assurance Statement 3 III. Table of Contents 4 IV. Index of Tables and Figures 5 V. summary 6 Vi. General information 7 VII. Introduction VIII. Methods and Materials IX. Results 13 X. References is XX. Signature Page 19 XII. Protocol Amendments and Deviations 20 Appendix A. water Quality Data from Toxicity Test 21 T.R. WLIbury study Number 891-TH Pag* 4 of 25 IV. XKDRI OF TABLU Am riouns Table 1. Table 2. Table 3. Ch*mical characterization sample of dilution water test with fathead and N2803-3. minnows, of a representative used for the toxicity Pimphal" pz@moi", Test system summary for the toxicity test with N2803-3 and fathoad minnows, Ploophal" prmolas. survival toxicity promelas, and sublethal effect data from the test with fathead minnows, Pinephalso and N2803-3. Table 4. Table S. Survival and sublethal effect data from the toxicity test with fathead minnows, Plwphal" promola,g, and isopropanol. median lethal toxicity promelas, test and concentrations (LCSOS) from the with fathead N2803-3. minnows, P"ophales Table A.l. Dissolved oxygen concentration, conductivity, pH, and temperature measured during the toxicity test with fathead minnows, Plmephalos promelas, and N2803-3. Table A.2. Figure 1. Dissolved oxygen concentrationt conductivity, pH, and temperature measured during the toxicity test with fathead minnows, Pinephales prome..!@s,ant! isopropanol. survival of fathoad minnows, Plmophal" exposed to N2803-3 for 96 hours. promelas, p 9 21 14 15 17 22 24 16 T.R. Wilbury Study Number 891-Tif Peg* 5 of 25 V. ,rho acute toxicity of U2803-3 to the f&thead minnow, plmwp"los PrC"la&o 3M C-pany Laboratories, is described in this final report. The for 96 hours from s*ptember I to 5. inc. in Marblehead, Massachusetts. Jeanne Magazu, Jacqu*lino Novius, Peter Kowalski, test was conducted for 1995, at T.R. Wilbury it was conducted by Koven stevens, Peter Novius, Robert Doori, &" for T.R. wilbury study sponsor. Timothy Number ward according to the protocol developed B91-TH. U2803-3 was supplied by the The test was performed under static conditions with five concentrations of test substance and a dilution water control at a temperature of 21.0 to 22.6*C. The dilution water was filtered dsionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 40-48 mg/L as CACO.I. Aeration was initiated at 72 hours to maintain dievolved oxygen concentrations above an acceptable level. The sample of U2803-3 supplied by the sponsor was combined with isopropanol in a 50:50 ratio prior to use, and this mixture is defined an the test substance as-tested. A toxicity test was also conducted with 500 mg/L isopropanol and no mortality or sublethal effects were observed. Results of the toxicity test are reported twice, once based on nominal concentrations concentrations isopropanol). of test substance as-tested and a second time of test substance as-received (N2803-3 Nominal concentrations of U2803-3 as-tested based on without were 0 (control), 130, 220, 360, 600, and 1,000 mg/L and nominal concentrations of N2803-3 as-received were 0 lcontrol)t and nominal. Pathead minnows 65, 110, 100, 300, and 500 mg/L used in the test were procured from a commercial supplier and acclimated to test conditions at T.R. Wilbury Laboratories. After 96 hours of exposure the control fish had an average wet weight (blotted dry) of 0.35 g and an average total length of 33.4 m. All fish were in good condition at the beginning of the study. Exposure of fathead minnows to the teat substaace resulted in a 96 hour median lethal concentration (LCSO) of 2eO mg/L N280.3-3, with a 95% confidence interval of 220 to 360 mcj/L and a 96 hour no observed effect concentration (NoEc) of 220 mg/L, based on test substance an-tested (50% N2803-3 and 50% isoproyanol). 1 The 96 hour LC50 is 140 mg/L U2803-3, with a 95% confidence interval of 110 to 180 mg/L and the 96 hour NOEC is 10 Mg/L, based on test substance isopropanol). an-received (N2803-3 without T.R. wilbury study Number 891-TH Page 6 of 25 Vl- OURVA:6 ZXFOPXRTZON Material Tentods Sponsor: Test Substance shipped rromt Reported Purityt study initiation Date: Experimental start Datet Experimental completion Datol study Completion Datet JLrchiveLocation for the Biological Raw Data and a Copy Reports of the Final U2803-3 3H Company Environmental Laboratory 935 Bush Avenue Building 2-3Z-D9 Gt- Paul, Minnesota 55144 3M Company Environmental To 367 Grove St. ratory Building 53-59-02 st- Paul, Minnesota 55101 100% August 16, 199S (protocol signed) September 2, 1995 September 5, 1995 January 24, 1996 T.R. wilbury Laboratories, H-bleboad, Massachusetts inc. T-it. Wilbury *Study M=ber 891-n Page 7 of 25 VIZ. IWMVJCTIOK This study was sponsored by 3H company, st Paul, Kinnemot&. The Objective of the study was to determine the 24, 48, 72, and 96 hour mdian lethal concentrations (LCSOS) of the test subst-n o to the fathead minaw, Plmophali6apromelas, a freshwater fish, under static conditions. The test protocol was based on U.S. EPA methods (U.S. ZPA, 1993). The report contains sections that describe the method@ 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. TZBT SUBSTAMCBs VXII. MC=ODS AND KLTZRIALS The sample of X2003-3 (T.R. Wilbury Laboratories Sample Number 530) was delivered to T.R. Wilbury Laboratories on August 29, 1995 in a 5 gallon metal bucket within a cardboard box at ambient temperature. it was contained in an approximately 200 ml glass jar. The label attached to the bottle included the following informations IN2003-3, S. Beach, 2-3E09, (612) 778-7452, 8/22/95.* N2803-3 (a white solid) was supplied by 3H company, st Paul, Minnesota. Prior to use the sample of U2803-3 was stored in the dark at room temperature. The sample of M2803-3 supplied by the sponsor was combined with isopropanol in a 50:50 ratio prior to use. mixture was then considered to be 100% test substance This SO:50 during the performance of the toxicity test. All results are reported both an the basis of test substance as-tested (50% W2803-3 and 500 isopropanol) and test substance as-received IN2803-3 without isopropanol). The U2803-3 was assumed to have a purity of 1001 active ingredient and to be stable under storage and testing conditions. unused test substance will be returned to the sponsor. DILUTION WATZR: Water used for acclimation of test organisms and for all toxicity testing van dsionized water collected at T.R. Wilbury Laboratories in Marblehead, Massachusetts. Wat*r was adjusted to a hardness of 40-48 mg/L as CACO, and stored in SOO-gallon polyethylene tanks where it was aerated and continuously passed through a particle filter, ultraviolet sterilizer, and activated carbon. Results of chemical analysis of a representative sample of water are presented in Table 1. TM ORQ=IgDgs Juvenile fathead minamot organisms were from a single 'Finsphales source and promeles, employed were procured from an test Aquatic Sicaystems, Fort Collins,' Colorado an August 10, 1995. The species of the fish was verified using an appropriate taxonomic key. T.R. Wilbury Study Number 691-To Pago 0 of 25 Table 1. chemical characterization of a representative sample of dilution water used for the toxiaity test with fatbsad minnows, vinephalespromolas, and K2803-3. Parameterl Unit of Measuremnt Detection T.I.it Measured value metals Aluminum Arsenic Boron cadmium chromium Cobalt Copper Iron Lead mercury Nickel silver zinc Nitrate chloride Fluoride Total organic carbon Total Phosphorus organochlorine Pesticides Toxaphons Organophosphorus Pesticides Dimethoate TEPP Monmrotophoo PCBB 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 an N Mg/L mg/L pg/L pg/L )jg/L ug/L pg/L pg/L lig/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 I 0.1 I 0.03 0.5 2 0.5 2.0 2.0 2.0 0.5 MD2 ND ND RD ND ND ND 0.03 RD ND RD ND ND ND RD ND RD RD ND ND ND ND ND ND notess 2. Parameters war* measured in deionlz*d water (used to formulate dilution water) that was collected during August, 1995 and analyzed by Pace, Inc. as part of, routine. water quality testing. 2. ND not detected at or above the detection lwt,.,@ T.M. Wilbury study Number 891-TH Page 9 of 25 Prior to testing the fish were maintained under flow through conditions in a 270 liter fiberglass tank. During acclimation fish were not treated for disease, they were free of apparent sickness, injuries, and abnormalities at the beginning of the test, and mortality during the final 48 hours of acclimation was less than 3%. During the 14 day period before the start of the toot the acclimationtemperaturerange was 21.0 to 22.3*C and the dissolved oxygen concentrationwas always at least 8.0 Mg/L. During acclimation fish were fed a dry commercial food (Tetra Kin* staple Foodp Lot t TNOS) daily except during the 48 hours in@eiately preceding the test. rish were not fed during the test. At the conclusion of the test the control fish had an average total length of 33.4 mm and an average wet weight of 0.35 g (loading during the definitive toxicity toot was 0.23 9/L). TOXICITY TZSTING: A static screening test was conducted from August 18 to 22, 1995 with five concentrations of test substance as-roc*ived and a dilution water control. Nominal concentrat-ons of N2803-3 were 0.05, 0.50, 5.0, 50, and 500 mg/L. After 96 hours of expaoure there was 1001 survival -n the control, 90% survival at 0.05 mg/L, 1001 survival at 0.50, 5.0, and 50 mg/L, and Ot survival at 500 mg/L. The definitive toxicity test was performed from September I to 5, 1995, according to T.R. wilbury Protocol 891-TH (Acute Toxicity of N2803-3 To the Fathe&d Minnow, Plmophales promelas). The protocol, which was signed by the study director on August 16, 1995, is based on proc*duroo of the U.S. Enviro=ental Protection Agency (1993). A summary of the test system is presented in Table 2. The test was conducted at a target temperature of 22 t 2*C with five concentrations of test substance and a dilution water concentrations of the test substance an-tost*d were: control. Nominal 0 mg/L (control), 130, 220, 360, 600, and 1,000 mg/L. Appropriate amounts of test substance were added directly to dilution water in test vessels media. to formulate test Twenty fathead minnows were indiscriminately and equally distributed among two replicates of each treatment. The test was performed in 20 liter glass aquaria that contained 15 liters of test solution (water depth was approximately 18 cm). Test vessels were randomly arranged in a water bath during the 96 hour text (a random numbers table was used to select the location of each vess*lj and they were loosely covered. A 16 hour light and 8 hour dark photoperiod with a 15 minute transition period was automatically maintained wit. cool-whit* fluorescent -@lights that provided a light intensity of 31 footcandl*s. Aeration was initiated after the,72 hour dissolved oxygen concentration masurem@tai t6 maintain'@ll dissolved oxygen concentrations above acceptable levels.' T.R. wilbuiy study Number 891-Tif Page 10 of 25 Table2. Test system summary for the toxicity test with N2$03-3 and fathoad minnows, Pinephales prom*las. speciest Ag*/Bizo of Test organisms: Toot Durations Test Vesselat Depth of Test Media: Number of Replicate Toot Vemoolot Organisms per Replicate; Temperature Range: Minimum Dissolved oxygen: Photoperiod: Light Intensity: Water Qui[lity measurements: Ezid Points s Ploophalos promelas Juv*nilea/-0.35 grams, wet weight 96 hours 20 liter glass aquaria Is cm 2 10 21.9-22.6*C 5.2 mg/L 16 hours light and 8 hours dark with a 15 minute transition period. 31 footcandles Dissolved oxygen, pH, temperature, and conductivity daily in each test vessel. Hardness and alkalinity were measured at the start and the end of toot in dilution water. Acidity, Toc and particulate matter were measured in dilution water. Temperature measured at least *very hour in I test vessel. Dilution water analysis is summarized in Table 1. 24, 48, NOEC, 72, and 96 hour LCSO; 96 hour Acceptability Criteria: slot control mortality and acceptable tomperature,rangs. T.R. Wilbury Study Number 891-TH Page 11 of 25 The number of surviving Organisms and the occurrence of sublethal offsets (immobilization, loss of equilibrium, less of reflex, excitability,discoloration,or change in behavior) were determined visually and recorded initially and after 24, 40, 72, and 96 hours (mortalityand sublethaleffects were monitoredtwice each day). Dead test organismswere removedwhen first observed. Dissolvedoxygen (Ysr Model 57 motor; instrument number 1). pH (sockxlan model pUl 12 motor; instrument number 124), conductivity (Cole Parmor motor, instrument number 94), and temperature (Beckman model pHI 12 motor; instrument number 124, probe pUT-4) were measured and recorded daily in each test chamber that contained live animals. The tempa:ature in one test vessel was recorded at least hourly during the test. A static test was conducted from September 29 to October 3, 2995 with 500 mg/L isopropanol (7.5 9 isopropanol added to 15 liters of dilution water) and a dilution water control. The test was established with 2 replicates of each concentration and 10 fish per replicate, and it was conducted in a manner similar to the test with H2803-3. Test vessels were randomly arranged in a water bath and the light intensity was 28 footcandles. STATISTZCAL MZTNODST Results of the toxicity test were interpreted by standard statistical techniques. Computer methods (Stephan, 1983) were used to calculate the mdian lethal concentrations ILC509). All calculations war* performed using nominal concentrations of test substance as tested and the number of live fathead minnows. The no observed effect concentration in the highest concentration of test substance that did not cause any toxicant-related mortality or sublethal effects. T.R. Wilbury study Number 891-TH Peg* 12 of 25,;,,,c@.,@',@@@@., XX. anuLTS water XnBOlubl* material was Dot noted during the test. Biological quality data generated by the acute toxicity test &re an d presented in Table 3 and 4 and Appendix A, respectively. The measured concentrations Of dissolved oxygen ranged from 5.2 to 9.0 mq/L, the temperature ranged from 21.8 to 22.6*C, conductivity ranged from 110 to 370 pmhos/cr, and pH ranged from 3.0 to 7.6 (conductivity and pH were affected by the test substance). one hundred porca-itsurvival occurred in the control exposure and these fathead minnows did not exhibit any sublethal effects. During the toxicity test with 500 mg/L isopropanol no mortality or sublethal effects war* observed and the 96 hour LCSO value is greater than 500 mg/L. The 24, 48, 72, and 96 hour LC509 for fathead minnows exposed to the test substance are presented in Table 5. The 96 hour LCSO for fath*ad minnows exposed to the test substance in 280 mg/L, with a 954 confidence interval of 220 to 360 mg/L and the 96 hour NOEC is 220 mg/L, based an test substance an-tested (501 N2eO3-3 and 501 isopropanol). The 96 hour LC50 is 140 mg/L, with a 95% coniidence interval of 110 to 180 mg/L and the 96 hour NOEC in 110 mg/L, based on test substance an-receiv*d (N28(3-3 without isopropanol). The slope of the 96 hour dose response curve could not be determined with these data. T.R. Wilbury study Number 891-TH Page 13 of 25 Table 3. survival and sublethal effect data from the toxicity test with fath*adminnows,Plmophalsopromolas,and U2803-3. Nominal Concentration of H-2803-3 (mg/L) as-Tested an-Received Rep. Number Aliv* 0 24 48 72 96 hr hr hr hr hr Number Affected 0 24 48 72 96 hr hr hr hr hr 0 (control) 130 65 220 210 360 Igo 600 300 1,000 500 1 10 10 10 10 10 2 10 10 10 10 10 1 10 10 10 10 10 2 10 10 10 10 10 1 10 10 10 10 10 2 10 10 10 10 10 1 10 0 0 0 0 2 10 0 0 0 0 1 10 0 0 0 0 2 10 0 0 0 0 1 10 0 0 0 0 2 10 0 0 0 0 0000 0 0000 o 0000 0 0000 0 0000 0 0000 0 0 -- -- -- -0 -- -- -- -- 6 7 0 0 Notes: 1. All of the fish exposed to 1,000 mg/L test substance as-taste; died immediately and 7 of the 20 fish exposed to 600 mg/L test substance an-tested died immediately. 2. kffected fish were swimming in short, irregular bursts and then floated motionlessto the bottom of the test vessels. T.R. Wilbury Study Number 891-TH fi 4' Page 14 of 25 Table 4. Survival and sublethal effect data from the toxicity test with fathead minnows, Ploophales promolas, and isopropanol. nonlual Concentration of Isopropanol (Mg/L) Rep. Number Alive 0 24 hr hr 48 72 96 hr hr hr Number Affected 0 24 48 72 96 hr hr hr hr hr 0 icontrol) Soo 1 10 ic 10 10 10 2 10 10 10 10 10 1 10 10 10 10 20 2 10 10 10 10 10 0000 0 0 000 0 0 000 0 0 000 0 T.R. WLlbury Study Number 891-TH Page 15 of 25 20 0 c 15 Ai L z 0.1 130 220 360 600 1000 Concentration of Tes i Subs iar@ce (,!,q Figure 1. survival of fathoad minnows, Pinephalem promelas, exposed to 02803-3 for 96 hours. T.R. Wilbury Study Number 891-TH page 16 f 2@:,@-: AL Table S. Median lethal concentrations (LC50s) from the toxicity test with fathead minnows, Vinophales proselas, and U2803-3. Exposure period Lcso 95 percent confidence limit calculation method CalculatOd based On Test substance 00-Tested (501 *2803-3 and 501 Xsopropanol) 24 hours 280 mg/L 220 to 360 mg/L Binomial 48 hours 280 mg/L 220 to 360 mg/L Binomial 72 hours 280 mg/L 220 to 360 mg/L Binomial 96 hours 280 Mg/L 220 to 360 mg/L Binomial calculated based on Test substance as-Received (82803-3 without Xxopropanol) 24 hours 140 mg/L 110 to 180 mg/L Binomial 48 hours 72 hours 140 Mg/L 140 Mg/L 110 to 180 mg/L 110 to 180 mg/L Binomial Binomial 96 hours 140 Mg/L 110 to 180 mg/L Binomial Note: Tb* 24, 48, 72, and 96 hour LC50 values from the toxicity test with isopropanol are greater than 500 mg/L. T.R. Wi2.bury study Number 091-TH Page 17 of,25 X. RzrzRzucxs Stephan, C.Z. 1983. Computer Program for the calculation of LC50 Values. U.S. EPA. Duluth, MU. Personal Communication. U.S. EPA. 1993. Toxic substances control Act Test Guidelines; Final Rules. section 797.1400. U-S. rPA. 1993. 40 CPR Part 792. Toxic Substances Control Act ITSCA); Good Laboratory Practice Standards. Code of Federal Regulations, Title 40, July 1, 1993. T.R. Wilbury Study Number 091-TH Page 28 of 25 Xr. alonATURZ FJLGE Ac ir iianneP. xai&zu@ Study Dir&ctor Aquatic Toxicologist Rok6rt L. Doori Coauthor I //,t Pe r L. Xowalski Aqt.tic Toxicologist j@acqtloiineM. NwVius Biologist For: X*v*n X.'@Stevens Biologist 17 Pot*r C. Novium Biologist TlU thy i J WiLrd Au or T.R. wilbury study Number 891-TH Pag* 19 of 25 m mix. PROTOCOL ANzKDNx3rn AND DZVIATIONS PROTOCOL ANZX=Ctwrs I The protocol was amended to specify that the test substance would be comprised of 50% N2803-3 and 501 isopropanol, to note the nominal concentrations for the definitive test, and to not* that an additional toxicity test "uld be conducted with isopropanol. PROTOCOL DZVIATIONSI Dissolved oxygen concentrations were <601 saturation at 72 hr prior to aeration. Toot vessels were aerated prior to sponsor approval. The slope of the done response curve was not provided. In the isopropanol test, not all fish were between 0.1 and 1.0 9. These deviations did not affect the outcome of the study and no deviations were made from the protocol. other T.R. Wilbury Study Number 891-TH Page 20 of 25 Aypendix A. WATZR QUALITY D&T& rM* TOXICITT wilbury Study Number 891-TH' Page 21 of 25 Table A.I. Dissolved oxygen concentration, conductivity, pH# and temperature measured during the toxicity test with fathead minnows, vl"phalos prceelas, and U2803-3. Nominal Concentration of U2803-3 am-Tested lmg/L) Rep. Dissolved oxygen (mg/L) 0 24 48 72 96 hr hr hr hr hr 0 (control) 1 2 130 1 2 220 1 2 360 1 2 600 1 2 1,000 1 2 0.6 7.5 6.5 6.5 7.4 8.7 7.4 6.5 6.4 7.5 8.7 7.5 6.5 5.8 7.5 8.9 7.5 6.5 5.5 7.5 8.7 7.4 6.5 5.2 7.2 8.8 7.5 6.5 5.3 7.2 9.7 8.0 8.7 8.0 -- -- -- 8.9 8.0 8.9 8.1 -- -- -- 9.0 -9.0 Conductivity fpmhoo/cm) 0 24 48 72 96 hr hr hr hr hr 120 120 110 110 120 120 120 110 120 120 110 110 110 120 120 110 110 110 120 120 210 110 110 120 110 110 110 110 120 110 120 120 120 120 -- -- -- 100 200 200 200 -- -- -- 350 -- -- -- -370 -- -- -- -- T.R. wilbury study Number 891-TH ? Page 22 of 25 Table A.I. Continued. Nominal Concentration Of N2$03-3 &S-T*mtod (mg/L) Rep. PH 0 24 48 72 96 hr hr hr hr hr To " ratur* (OC) 0 24 4B 72 96 hr hr hr hr hr 0 (control) 1 2 130 1 2 220 1 2 360 1 2 600 1 2 1,000 1 2 7.6 7.3 7.2 7.1 7.4 7.6 7.3 7.2 7.1 7.4 6.6 7.2 7.2 7.1 7.4 6.7 7.2 7.2 7.1 7.4 6.3 6.9 6.9 6.9 7.2 6.2 6.8 6.9 6.9 7.2 4.2 4.5 4.3 4.6 -- -- --- -- -- 3.4 3.5 3.4 3.4 3.0 -3.0 22.1 22.6 21.9 22.0 21.9 22.1 22.6 21.9 22.0 21.9 21.9 22.6 21.B 22.0 21.9 21.9 22.6 21.8 22.0 21.9 22.4 22.S 21.9 22.0 21.9 22.0 22.6 21.9 22.0 21.9 22.1 22.5 -- -- -22.1 22.5 22.1 22.6 -- -- -22.1 22.5 -- -- -- 22.5 -22.5 T.R. Wilbury Study Number 891-TH Page 23 of 25 Table A.2. Dissolved oxygen concentration, conductivity, pH, and temperature measured during the toxicity toot with fatho&d mlnums, Plnophalom promlas, and isopropanal. Mcninal concentration of Xxopropanol (Mg/L) R*p. Dissolved oxygen (Mg/L) 0 24 40 72 96 hr hr hr hr hr 0 (control) 1 2 500 1 2 8.6 7.9 7.6 7.1 6.2 8.6 8.0 7.6 7.4 6.8 0.6 8.1 7.8 6.2 5.5 0.6 6.0 7.6 6.3 5.6 Conductivity (whoslem) 0 24 48 72 96 hr hr hr hr hr 140 160 160 160 160 140 160 160 160 160 140' 160 160 160 160 140 160 160 160 160 77 ;.R. Wilbury Study number 091 Pago 24 25"@ T&ble A.2. Continued. Nominal PH Concentr&tLon of isopropanol 0 24 48 72 96 (Mg/L) Rep. hr hr hr hr hr Towerature (OC) 0 24 48 72 96 hr hr hr hr hr 0 (control) 1 2 500 1 2 7.8 7.9 7.7 7.8 7.7 7.8 7.8 1.7 7.8 7.7 7.8 7.6 7.e 7.6 7.6 7.8 7.8 7.8 7.8 7.6 21.4 21.9 21.9 21.S 22.1 21.4 21.9 21.9 21.S 22.1 21.4 21.9 21.9 21.5 22.1 21.4 21.9 21.9 21.S 22.1 T.R. WLIbury Study Number 691-TH Page 25 of 2S