Document npN9mJ55ndaeDmpBBaB3Rp7z

ACUTE TOXICITY TO FISH TEST SUBSTANCE Identity:Perfluorooctanoiacid,ammonium saltm;ay alsobe referretdo as PFOA ammonium saltA,mmonium perfluorooctanoatPeF,O, FC-1 16, FC-126, FC-1 69, FC-143, oras a major component of FX-1003. (Octanoicacid,pentadecafluoro-a,mmonium salt, CAS # 3825-26-1) Remarks: The 3M productionlotnumber was 2327. The testsample isFX-1003. It'psuritywas notsufficientclhyaracterized, though curren_tinformatioinndicateistisa solutionof <45% ammonium perfluo(ooctanoat5e0,%- water,<3% inertperfluodnatecdompound and 1 -2%C5andC7perfluoFo analoguecompounds. The followingsummary appliestothe testsample as a mixture ofthe testsubstance in watersolutionwithincompletelycharacterized concentrationsofimpurities.Data may not accuratelyrelatetoxicity of thetestsample withthatofthe testsubstance. METHOD: Method: OECD 203 Type: Staticacute GLP: Yes Year completed:1990 Species:Pimephalespromelas Supplier:AquaticResearch Organisms,Hampton, New Hampshire Analyticalmonitoring:Temperature,pH, conductivitayn,d dissolved oxygen Exposure period:96-hours Statisticamlethods: Non-linearinterpolatiomno,ving average,and/or probitanalysis Test fishage: Juvenile Length and weight: Average length 3.8cm Average weight 0.45g (wet) Pretreatment:None. Testconditions: Dilutiownater:Hampton,New Hampshirewellwater Dilutiownaterchemistry: pH: 7.4 Conductivity: 1500 pmhos/cm Hardness: 88 mg/L (asCaC03) LightingC:ool-whiftleuorescebnutlbswithan intensiotfy35 uEs-lm-2A. dailyphotoperiodof 16 hours lightand 8 hours darkwas maintained throughoutthe testingperiod. Test conditions remarks: The fishwere fed a commercial fishfood once or twicedailyduringtheacclimationperiod,food beingwithheld throughoutthetestperiod.Fishwere acclimatedtotesttemperaturefor 14 days priortouse inassays. Aerationwas employed after48 hours to maintaindissolvedoxygen concentrationsabove acceptablelevels. Stock and testsolutionpreparation:Test solutionswere createdby directindividuIaweight additions. Conceritrationsdosing rate:Once Stabilityofthe testchemical solutions:Not noted Exposure vessels: 19.6 L glassaquariacontaining15 litertsestsolution at an approximate depth of 17 cm. Number of replicates:two Number offish per replicate:10 Loading rate:0.30 g/L Number of concentrations:fiveplusa blankcontrol Water chemistry during the study: Conductivityrange: (0-96hours) 1200 - 1500 pmhos/cm (controlexposure) 1300 - 1600 pmhos/cm (1,000mg/L exposure pH range (0-96hours): 7.4 - 8.4 (controelxposure 7.8 - 8.2 (1,000mg/L exposure) Temperature range (0-96hours): 21.0- 22.0 OC (controelxposure 21.0- 22.0 -C (1,000mg/L exposure) Dissolved oxygen range (0-96hours): 6.1- 9.2mg/L (controelxposure 6.2- 9.1mg/L (1,000mg/L exposure) RESULTS Nominal concentrations: Bk control,150, 250, 400, 600, and 1,000 mg/L Element value: 96-hour LC50 = >1,000 mg/L Element value based on nominal concentrations Testingwas conducted on the mixtureofthe testsubstance as describedinthe testsubstance remarksfield.The values reportedapply to thatmixtureand notthe testsubstance. CONCLUSIONS The testsample 96-hour LC50 forfatheadminnow was determinedto be >1,000 mg/L. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota,55133 DATA QUALITY ReliabilitKyl:imischranking2. Testingmeets the criterifaorquality testing.However, sample puritywas notproperlycharacterizeadnd it lacksarialyticcaolnfirmationoftestsubstance concentrations. REFERENCES Test was conducted by EnviroSystems,Inc.of Hampton, NH at the requestofthe 3M Company, 1990. The EnviroSystems studynumber was 9014-3 OTHER Last changed: 5/25/00 Study Title Static Acute Toxicity of FI-1003 to the Fathead Minnow, Pinephales provelas Authors Timothy J. Vard Robert L. boeri Study Completed March 15, 1990 Performing Laboratory EnviroSystess Division esou rce Analysts, Incorporated P 0. Box 778 one Lafayette Road ton,'New Hampshire 03942 &aviLro*-ztem$'Stu Y:Null Tf,4,@ f@ OIAZ'T 41 1. GOOD LABORATORY PRACTICI STATMFENT This study was conducted according to OECD Good Laboratory Practice Regulations. Neither the Study Director nor the sponsor are aware of any circumstances that would affect the integrityof this study. Food was not witheld from fish during the 24 hours prior to the test initiation. This deviation is not believed to have affected the study. Ito other deviations existed that significantlyaffectedthe' validityof the study. Robert L. Doeri Coauthor and Study Director TAui thy J. lia %or Jelanne P. nagazfu Aquatic Toxicologist Peler L@ Kowalski -''AquaticToxicologist Z7 @C Envirasys teas Stijdyliumber-9014-3 :Page 2 of 16@ d kMtLFM - I @ft 11. CERTIFICATIONOr GOOD LABORATORYPRACTICES Submitted by: EnviroSystess Division Resource Analysts, Incorporated P.O. Box 778 One Lafayette Road Hampton, New Hampshire 03842 Certification:- Data presented in this report were derived by methods and with materials identified in the section of the report entitled "Methods and Materials.* The test was performed in accordance with EnviroSystems Protocol 9014-3 and the Product Registration Aquatic Toxicology Laboratory Standard Operating Procedures Manli;tl.The toxicity test was performed by Jeanne Magazu, Peter Kowalski, Robert Boeri, and Timothy Vard. The raw data and final repors,will be archived at Resource Analysts Inc. for at least 10 years. All data transcribed from the raw data to the report were checked for accuracy by two persons, and all data were verified by Quality Assurance Auditors. Quality assurance audits were performed (and reported to management) on February 1,1990, February 15, 1990, and March 14,1990(March 14, 1990). 7. ;7, Danette Tekelenburg Quality Assurance Auditor i6 Doreen Newhouse Quality Assurance Officer 'w@ T, virosysteas Study Numbez 9014-3 nIt @3 ';'ROWL" AIK*^ W- SjbMloy 6fMLUPORE iri. TABLZ oircoxtziffs SECTION: Good Laboratory Practice Statement ii. Certification of Good Laboratory Practices -Table of Contents IV. Index of Tables and Figures V. Summary vi. Introduction VII. Methods and Materials VIII. Results TX. References Appendix A. Vater Quality Data from Toxicity Test PACE 2 3 4 5 6 7 7 io 13 14, page of @.16 -t*-4tA Ana^ kv.@31 Id rV. INDZX OF TABLES AND TIGURES Table 1. Chemical characterizationof a representative ample of natural well water used as dilution :ater for toxicitytest Table-.2. Survivaldata from toxicitytest Table 3. Nedi7n lethal concentrations(LC50s) from toxicitytest Table A.l. Conductivity, pH, tesperat-irea,nd dissolved oxygen concentration measured during toxicity test PAGE a 11 12 is Euvirosystemi Study Number 9014-3 s'@!5'of V. SUMMARY The acute toxicity of FX-1003 to the fatbead minnow, Pinephales promelas, is described in this final report. The test was conducted for 3M Company for 96 hours during February 12 to 16. 1990, at the EnviraSystems Division of Resource Analystx, Inc. in Hampton, 'New Hampshire. It was conducted by Jeanne Magazu, Peter Kowalski, Robert Boeri, and Timothy Ward according to the protocol developed for EnviroSystems Study Number 9014-3. The test was performed uiider static conditions with five concentrations -of test substance and a dilution water control at a mean temperature of 21.90C. The dilution water was filtered natural well water collectecf from wells at Hampton, New Hampshire. Aeration was employed to maintain dittolved oxygen concentrations above an acceptable level. Nominal concentrations of FX-1003 were: 0 mg/L fcontrol), 150 ag/L. 250 mg/L, 400 mg/L, 600 m;/L, and 1,000 mg/L. Noainal concentrations were used for all calculations. Fish used in the test were purchased from a commercial supplier (Aquatic Research organisms, Hampton, New Hampshire) and acclimated under test conditions for 63 days. After 96 hours of exposure the control fish had an average wet weight (blotted dry) of 0.45 g (resulting in a loading rate of approximately 0.30 g/L), and an average total length of 3.8 ca. All fish were in good condition at the beginning of the study. Exposure of fathead minnows to the test substance resulted in a 96 hour LC50 of )1,000 mg/L FX-1003, the highest tested concentration. The 96 hour no observed effect concentration is estimated to be )1,000 mg/L. t EnviroSystess Study Number 9014-3 Page 6 of 16 Vt. INTRODUCTION This study was sponsored by 3H Company, St. Paul, Minnesota. The objective of the study was to determine the acute toxicity of FX-1003 to the fathead minnow, a freshwater fish. The report contains sections that describe the methods and materials employed in the study, and the results of the investigation. The report also contains an appendix that presents the water quality data collected during the test. VII. Mri ODS AND NJLTERIALS TEST SUBSTANCE: FX-1003 (EnviroSystems Sample Number 2308E) was delivered to EnviroSystems on January 29, 1990. It was contained in a 500 aL plastic 'bottle that was labelled with the following information: G2$82-1. FX-1003 (a yellow liquid) was supplied by 3M Company, 935 Bush Avenue. St. Paul, Minnesota. Prior to use the test material was stored in the dark at room temperature. A reserve sample (approximately I gram) will be archived at EnviroSystems for a minimum of 10 years. DILUTION WATER: Vater used for acclimation of test organisms and for all toxicity testing was well water collected from wells at EnviraSystess in Hampton, New Hampshire. Water was adjusted to a hardness of 98 mg/L as CACN and stored in 500-gallon polyethylene tanks, where it was aerated. Results of chemical analysis of a representative sample of water are presented in Table 1. TEST ORGANISM: Juvenile fathead minnows employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were purchased from a commercial supplier (Aquatic Research Organisms, Hampton, New Hampshire) and acclimated at the EnviroSysteas facility for 63 days. Control fish were weighed at the conclusion of the toxic4ty test. Prior to testing, fish were maintained in 100% dilution water under flow through conditions in an all glass aquarium.','During acclimation fish were not 'treated for disease and they were free of apparent sickness, injuries, and abnormalities at the beginning ,of the test.,@-Duringthe acclimation period 14 days prior to the test initiation-the temperature..-',, ranged from 21.00 to 23.50C, and the dissolved'oxygen concentration was maintained above 9.0 mg/L. Fish were fed a'@cooneicial@'fisb food (EnviroSysteas lot number TMOI) once or'.tw'cedaily., Envirosystems Study Number 9014-3 -Page 7 of 16 lnr-SuAbdftdkdM L Table 1: Chemical characterization of a representativesample of natural well water used as dilutionwater for toxicitytest Parameter pH Conductivity Hardness Or ganocblorine pesticides Organophosphorus pesticides Polychlorinated biphenyls Unit of Measurement Reporting Limit ps units umbos/cm mg/L as CaCO3 ug/L 0.5 ug/L 0.5 ug/L 0.5 Measured Value 7.4 1500 NDO NDO ND* 2Not detected above the reporting limit. -EnviroS ys@eas Study' Number 9014-3 "Page 8 of 16 R-Ar-*"k W_ &ftdkwydMFM TOXICITY TESTING: A screening test with the test substance was conducted during February 6 - 10, 1990. Nominal concentrations of test substance were 0.1 ov/L, I mg/L, 10 mg/L, 100 mg/L, and 1,000 mg/L. After 96 hours of exposure there was 100% survival at all tested cono:entrations. The definitive toxicity test was performed during February 12 16, 1990, according to EnviroSysteas Test Protocol 9014-3 (Acute Toxicity of FX-1003 To The Fathead Minnow Pinephales pronelas), which was signed by the Study Director on February 6, 1990. It is based on procedures of the OECD (1984). The test was conducted at a target temperature of 23 2*C with five concentrations of test substance and a dilution water control. No stock solution was prepared as test material was added directly to dilution itater contained in the test vessels without the use of a solvent. NomitMl concentrations of the test material were: 0 mg/L (control), 150 mg/L, 250 mg/L, 400 mg/L, 600 mg/L. and 1,000 mg/L. Twenty fish were randomly and equally distributed among two replicates of each treatment. The test was performed in 19.6 liter glass aquaria (approximately 20 ca in width, 40 ca in length, and 25 cm in height) that contained 15 liters of test solution twater depth was approximately 17 cm). Test vessels were randomly arranged in a water bath during the 96 hour test (a random numbers table was used to select the location of each vessel). A 16 hour light and 8 hour dark pbotoperiod was automatically maintained with cool-wbite fluorescent lights that provided a light intensity of 35 uEs-im-2- Aeration was employed after 48 hours to maintain dissolved oxygen concentrations above acceptable levels. Fish were not fed during the test. The number of surviving organisms and the occurrence of sublethal effects (loss of equilibrium, erratic swimming, loss of reflex, excitability, discoloration, or change in behavior) were determined visually and recorded initially and after 24, 48, 72, and 96 hours. Dead test organisms were removed when first observed.'Dissolved oxygen (YSI Model 57 meter; instrument number PRL-3), pg (Beckman mod-I pHl 12 meter; instrument number PRL-4), @'coriductivityflabcoup SCT meter, instrument number PRL-9). and temperature (ASTH mercury thersomet r; thermometer number 2040) were measured and recorded daily in each t:st chamber that contained live aninals.'-. STATISTICAL METHODS: Results of the toxicity.,teit@:-coui@ ..not be interpreted by standard statistical techniques- due ,-,t6.,100%survival at the highest tested concentration EnvireSystess Study Number 90@14-@3 Page 9 of 16 Ane*03 In& Sig,"C-v d K*AFM Vill. RESULTS All test vessels containing TX-1003remained clear throughout the test. Biological and water qualitydata generated by the acute toxicity test are presented in Table 2 and Appendix A, respectively one hundred percent survival occurred in the control exposure. Control fish had an average total length of 3.8 cm, and an average wet weight (blotted dry) of 0.45 q at the end of the test. Loadingrate during the toxicity test was approximately0.30 9/L. The 24, 48, 72, and 96 hour LCSOs for fish exposed to FX-1003 are presented in Table 3. The 96 hour LCSO is )1,000 mg/L FX-1003, the highest---testedconcentration. The estimated 96 hour no observed effect concentratiofiis >1,000 mg/L FX-1003. aa P a 9 0 10. of 6 y Number 9014 .@;;3t Table 2. S"rvI i7al data from toxicitytest Nominal Concentration rep- Number Ilive ---------Ohr 24hr 48hr 72hr 96hr Number Affected Ohr 24hr 48br 72hr 96hr 0 (control I ISO 250 400 600 11000 1 10 10 10 10 2 10 10_ 10 ' 10 10 -1 io 10 10 10 10 2 10 10 10 10 1 10 12 i10o 1100 1100 1100 10 10 1 10 10 10 10 10 2 10 10 10 ()10 1 10 1() 20 10 2 10 10 10 @10 10 10 1 10 @10 10 2 10 10 :10 10 10 10 '0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .0 0 0 0 0 0 0 0 @o 0 -0 0 0 0 00 0 0 -0 N170o.'ttse,:"-fSSuublleet!hhaall-ef!f-ec-t-l--------l-o-s-s--f----.-1-.------------ loss of reflex. ectsiinncclluudede1033of equ excitability, ilibrium, erratic imming. discoloration, and.change in behsw avior ie 4w- z -A mviro'Srstess s tudr Number".90 pago 11 Of 16 -n 7, Table 3. bodian lethal concentrations(Lcsos)from toxicitytest Exposure period LCSO 24 hours )1,000 mg/L 48 hours )1,000 mg/L 72 hours )1,000 mg/L 96 hours )1,000 mg/L 95 percent confidence limits LCSO calculation method RUDY S, a Study@'N"bei 9014 -@3 hwloft kr- &&WdW d MUVK IX. REFII=CZS OECD. 1981. Decision of the Council Concerning the Mutual Acceptance of Data in the Assessment of Chemicals. Annex 2. OECD Principles of Good Laboratory Practice. C (81) 30 (rinaii. OECD. Biotic 1984. 1984. Guidelines for Testing of Chemicals. Section 2: Effects on Systems. Method 203, Fish Acute Toxicity Test. Adopted April 4, Envi.rosyste s@Study Number 9014 -3 Page 13-of 1 6 d D@endi TER OUALITY DATA FROM _MAXICITY TEST ,-,..6sistiiifStudyNumber 14 of 16' ) AL 9014-3 Table A.l. Conductivity, rR. temperature, and dissolved oxygen concentration measured during toxicity test Nominal concentration (mg/L) Rep. Conductivity (unho/cm) 0 24 48 72 96 hr hr hr hr br 0 (control) 1 2 iso 1 2 250 1 2 400 1 2 600 1 2 1,000 1 2 1500 1300 1200 1400 1300 1500 1300 1200 1400 1300 1500 1300 1200 1400 1300 1500 1300 1200 1400 1300 1500 1400 1200 1400 1300 1500 1400 1200 1400 1300 1500 1400 1200 1400 1300 1500 1400 1200 14GO 1300 1500 1400 13DO 1400 1300 1500 1400 1300 1400 1300 1600 1400 1300 1500 1400 1600 1400 1300 1500 1400 PH 0 24 49 72 96 hr hr hr br hr 7.4 7.7 8.0 8.3 8.4 7.4 7.5 9.0 8.3 8.4 7.4 7.6 7.9 8.0 9.3 7-4 7.6 7.9 8.1 8.3 7.4 7-6 7.9 8.1 8.3 7.5 7.6 7.9 8.1 8.3 7.5 7.6 7.9 8.2 8-3 7.5 7.6 7.9 8.1 8.3 7.6 7.7 8.0 8.1 8.2 7.6 7.8 8.0 8.2 8.3 7.8 7.8 8-0 8.1 9.2 7.8 7.9 8.0 8.1 8.2 .-EnviraSysl*easStudy Number 9014-3 page 15 of 16 WitSlb"mdMIU "able A-1- Nominal concentration (mg/L) -Rep. osperature (0c) 0 24 48 72 hr hr hr 1,, 9h6r 0 (control) 1 2 150 1 2 250 1 2 400 1 2 600 2 2 11000 1 2 21-0 22.0 22.0 22.0 21.8 21.0 22.0 22.0 22.0 21.8 21.o 22.0 22.0 22 0 21.9 21.0 22.0 22.0 22.'021.9 21.0 22.0 22.0 22 0 21.8 21.0 22.0 22.0 22.'021.8 21.0 22.0 22.0 22 0 21.8 21.0 22.0 22.0 22' 0 21.8 21.0 22.0 22.0 22 0 21.8 21.0 22.0 22 .0 22 0 21.8 21.0 22.0 22.0 22.o 21.8 21-0 22-0 22.0 22.0 21.8 Dissolved 03cygeii(mg/L) 0 24 48 72 96 hr hr hr hr hr 9'1 9.1 9.2 9-1 9.1 9-1 B'S 8.3 0 a8--1 8.1 8.0 6-1 6.3 6-0 6-5 6.1 6.7 8-6 a.8 8.5 8.7 8.6 8.6 92 9*.2 9.2 9.2 9.0 S.g 9-1 88 .2 6.4 8.6 .8.9 9-1 .1 .4 8.9 9.0 99--11 8a .0 6.8 3.8 9.0 . . 9-0 9.0 9 .1 8.2 6.2 8.9 9.1 9.1 8.2 6. 2 a.9 9.0 __p a I osystems-S 16 of' I t a Y. KIIJILwe@9014-@3 t tv- A@; 9z Oct En7iroSysteas Effective D,t,.StudY Number 9014-3 January, 1990 @-RESOTIRCE AJFALYSTS, INCORPORATED ENVIROSYSTEMS DIVISION PRODUCT RE';ISTRATION LABonroRy TOXZCOLM STUDY PROTOCOL Acute Toxici Vathead M.lanowt,y of FX-1003 TProoateh.eZas THIS IS A CERTIFIF _D TRUE Copy. SIGNATURE DATE@!!. 9 pong@o3 122n_sor @,3N company PO BOX 33428 @St- Paul, Xinnes,t, 55133 Sponsor App'roval of Prot6@oi , Dat F-&Cilitz rac -Lafayetet"'Road ffamPtOu@'INR,'@-03842 s t12 aitieid" stilai S - - W,,es tudy'Di Atiol a -0; -January omplet, a Dat6' Bar caei19go.-Robert Doeri ec.@, ,jtpprovalt p gel Da te of TITLE EnviroSystems.StudY Number 9014-3 Acute Toxicity of FX-1003 to the Fathead Minnow, PiRephales proselss 2.0 PURPOSE To 'dtteraine the 24, 48, 72, (LCSOs),of test substance to fish and 96 hour median lethal concentrations exposed under static conditions. 3.0 TEST MATERIAL 3.1 The test substance and related stability and puritydata will be supplied -by the sponsor. Test material will be stored at 2-40C in the 4D@riginalshipping container, unless alternate storage conditions are specified by the -.eponsor.Handling of the test mat rial will be in accordance with information contained in the sponsor-:upplied Material Safety, D-ita'Sheet and RAI Standard Operating Procedure 01-02-1106. A subsample-of test substance (approximately1 gram) will be removed from the original containerand transferredto a glass vial for archiving.All unused test substance will be returned to the sponsor. 3.2 Calculations are based on nominal concentrations of the test sduiblsuttainocne.waTteesrt wimtatheoruital stock solutions are prepared in deionized or the use of a solvent(carrier)if possible.If a solvent is required, dinetbylfornamide, triethylene glycol, or acetone will be used. The concentrationof solvent will be limited to 100 mg/L. 4.0 TEST SPECIES 4.1 The selection of test species is determined by the sponsor. Healthy juveniles from a single source will be used to initiatethe test.They will be obtained from a commercial supplier. Fish should be 1-3 ca long at the start of the test. The length and wet weight of control organisms will be determined at the end of the test. a4p.p2ropriaItdeetnatxiofniomciactikeoynso.f the @test animals shall be verified using 5.0, PRETEST OBSERVATIONS AND PROCEDURES 5.1 Pretest observation data concernin9 the 'receipt date, source, handling procedures,,,., @of,test animals disease will be treatment@,(ifany),'health, recorded feeding, and mortality y@. and ireported.',,'@, 5 2 -@Fisb will be @@,f.SZ4,--@T@COUditiOD3prior., ma3*.ntained'@rdunp'r-'static,',replacemento,r' flow7.tiro@i to .@test -.@;,',,'-@photoper:iaondd 't i6ifait').o@n-i4ns,the same water and at the"same' vi II be 'fed at mpe'rature @-that@@-will@bi'usedfor testing.,The east once dai y with''commercialfish animals shrimp nauplij during -'accli@ati6n."-'.@,ixcept'for food and/4@r,b@iin'e' the 24 ,bour'@@ period immediately-.preceeding ana yzed to: verif -the ..tet initiation. Each batch of food'i@illbe pesticides. Fish y -that it iSs@,-@@I'Lfree,'@@of-'-me@asurable concentrations'of"@ temperature, will be maintainediiidilut4-onwater at test.@conditi6is' t hardness,'",.anpdbotopiii6d)@foe at'least'22-15days'-prior'to, 'Page 2 of 6 ik , ANOWL br- of EnviroSyst*2s Study Number 9014-3 6.'0 EXPOSURE COIIDI'TIONS 6.? The study will be conductedunder staticconditionsand test media will only be provided at the beginning of the test. The test animals will be added to test 2edia after the toxicant is added to the test media. 6.2 Dilution 'water will be natural ground water. It will have a hardness of 50-250 mg/L as CaCO3, a pH of 6.0-9.5, be free of measurable concentrations of pesticides, and meet the requirements specified in U.S.EPA (1986,1987). Water will be passed through a (20 micron filter and chemically characterized (at least twice yearly analysis at RAI) prior to useaf 6.3 Water temperature-will be 23 + 20C. 6.4 Dissolved oxygen concentration will be maintained above 80% saturation. If aeration is required it will be supplied to all test chambers. Sponsor approval will be obtained prior to aeration. 6.5 Photoperiod will be automatically controlled and adjusted to 16 hours light and 9 hours dark. 6.6 The test vessels will be 19.6 L glass aquaria containing at least Is L of solution. Loading rate will be less than 1.0 g/L. 7.0 STUDY CONDUCT 7.1 Range Finding Investigation 7.1.1 The results of the range finding investigation and/or previous testing data will be used to select the concentration range needed for determination of the definitive LC50s. 7.1.2 If a range finding test is conducted, the animals will be exposed to a series of concentrations of -test substance plus a control (dilution water without test substance-)and a solvent control (if necessary). 7an.1 3 At ','least10 animals will be exposed to each treatment. The exposed in groups 'of 10 animals per ressel@, each .,Containing!@at.-, least 15 L of solution.':,T@he.an'aals will be equally #7a nd randomli.@@assigned to ..the-.test,4esse li"4(arandom n uab ers table.,., -,'used to choose the test vessel foi'@eaebspecimen), and theY" ill@not,@be,fed .,7.2 0 PK7. 2. Thir auimal:i"wil be e or. 96 o ri to a series or at' f-iii-'concentrationsof tes ub t nc .Xi-control and a solvent -control,@1(if':"necessary)under sta ic conditions.@,The highest tested'@*@l*.@ concentration... will @ be 1,, g/L. betteen concentrati6ni'vill be Page 5RMXAM Ai@ kIC@&bicfty d -L5, EnviroSysteas Study Number VUI%-I 7.2.2 animals assigned Each treatment group will per duplicate test vessel. to the test vessels. consist of 20 animals with 10 The animals will be randomly 7 2.3 Dissolved oxygen concentration, PR. temperature, and co* nductivity ill be measured and recorded at the beginning of the test and daily thereafter in all test vessels. as long as livingr animals are present in those vessels. 7.2.4 hours. gardness in the dilution water will be measured at 0 and 96 7.-2.5 Mor_t-ality and sublethal effects (loss of equilibrium, erratic swimming, loss of reflex, excitability,discoloration, or 'Change of behavior) will be recorded at 24 hour intervals during the test. Dead animals will be removed when first observed. 7.3 Data-Analysis and Statistical Methods The 24, 48, 72 and 96-bour LC50 values and their 95% confidence intervals will be calculated by the non-linear interpolation. moving average and/or probit methods. if toxicity occurs the slope of the dose-response curve will be provided. 8.0 QUALITY ASSURANCE MID QUALITY CONTROL 9.1 The test will be repeated if any of the following conditions occur: 8.1.1 Control survival at or if behavioral controls. the end of the test is less than 90%, abnormalities are observed in the 8.1.2 Temperature in any test vessel is outside of the acceptable range or dissolved oxygen concentrations fall below 90% saturation. 8.2 The test-vill be-conducted according to OECD Good Laboratory Practice Standards. 8.3 All critical phases of the test conducted at Resoarce will be audited by the Quality Assurance Unit Analysts Inc. 8 4 The Study.,irector will be responsible for reviewing all data and for r;cor'din any @@'-cbanges to procedures outlinedL,ino'this protoc ol in a protocol amend men t;@Piotocol amendments will be approved by the sponsor. 8 5 Original data'.willbe archived at Resource"A"n'a'lys'Itnsc'. for at least 10 years.or'it'iiilble transferred to the sponsor.for,:'arbc'iving.!,, t":g iKo kv- &A)om)rfofIALL140M EnviroSystess Study Number 9014-3 9!0 REPORT 9.1 Three original reports will be prepared after reviewof a draft report by the sponsor. The final reportwill be signetby the Study Director--and staff that conducted the study and prepared the report, and by the Quality Assurance Unit Representative. 9.2 The final report will consistof at least the followinginformation: 9.2.1 --- Title__page, including the study title,data requirement, author, study completion date, testing facility, and sponsor. 9.2.2 Sp@asor-supplied confidentialityclaims. 9.2.3 Good Laboratory Practice Statement. 9.2.4 Certificationof Good Laboratory Practices. 9.2.5 Table of Contents. 9.2.6 Summary of test procedures and results. 9.2.7 Introduction. 9.2.8 Methods and Materials, including a descriptionof test metbods, organisms, dilution water, and statistical techniques. 9.2.9 Results, including all data from range-finding and definitive tests, and LCSO values. 9.2.10 References. 9.2.11 Appendices, including all water quality data and the test protocol with amendments (if any). 10.0 REFERENCES OECD. 1984. i OECD'.Guidelines for Testing of Chemicals. Section 2: Effects on. Biotic -Systems.' @.Method 203,, Fish Acute.ToxicityTest. AdopteQ J@pril'4,,l,i,i-4,. U.S. EPA IL 8 06.- TR Part7'797.: Toxic Substances Control Act Test Guideli6ii' ina *Ru es.@@-Federal Regis ter,@M,onday, -January 6, 1986. i Section..79171406.1 U.S. EPA. 19 8 7 4 0,7-C,rR Part 797.'@@-"T@ii4@ ub-stan' ro Act Test,, Guidelines;'@Uendments. Federal Register,,Vednesday,'Nay 20, 1987.- Section .797:1400 0 It@ W- Suboda@oifL4XUPM EnviroSysteas Study Number 5014-3 SUMMARY OF TEST CONDITIONS Acceptable Species: Reference: Test Type: Duration: Flow Rate: Dilution vater: .Ave/Size of Test Organisms: Acclimation Period: Temperature: Photopiri6d; Light Intensity: Number of Concentrations: Dilution Factor:Number of Replicates: Number of Organisms/Replicate: Test Vessel Size: Test Vessel Volume: Solvent: Loading Rate: Feeding: Dissolved Oxygen Levels: Effects Measured: Test Concentration Analysis: Va ter Quality Measurements: Acceptability Criteria: Pimepba2es promeles OECD 203 Static 96 Hours Not Applicable Natural Ground Vater Juveniles; 2.0 + 1.0 ca long 12-15 days 23 + 20 16 Hours Ligbt/O Hours Dark Ambient Laboratory Lighting 5 or more Si.8 2 or more 10 19.6 L 15 L Dinetbylforaamide, Trietbylene Glycol, or Acetone (all less than 100 ag/L) (1.0 g/L None During Exposure kgO% saturation Death; note sublethal effects None DO, pH, Temperature and Conductivity at 0, 24, 48, 72, and 96 Hours Hardness in,dilution water at 0 and 96 hours S10% Control Mortality':' Acceptable Temperature Range Acceptable Dissolved Oxygen Concentrations Absence of Behavieral Abnormalities in Controls p age 6.9t 6 '7 ArCkVLW-SubddWdL*150% ALI'