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AR226-3005 i'^S-i'^s^'f'S ....;'); a :ii;%a ^''"a'-i:-3A'"f;^''--".1^'M^! , ^T-'.:.1"^ ^;^'sa '.?f''r-.^;'l^',.^'Usi^ ''i'^i?! .^lifc '6^BNi||laMt:,^ Organisation for 1 Guideline for Testing Chemicals: 202 Autnor KB'rthB.Pierson.Ph.D I ^tudCyompleted 0n I July 6.1994 i P^rforminLa^boratoTV E. i. du P< nt de Nemout^/and Cfimpany Hask^ i Laboratory for Toxicology andHnilustrial Medicine Elkton Road, iPost<3ffiorBb^^O Ne Mark, Delaware 19714 Medical Research Proiect SCACBl inW10'1 toed.OO8noico' com?a"y San" Page 1 of 15 TA 3LE OF CONTENTS DuPontHLR591-92 Page .....................................................3 .....................................................5 1. Purpose.................... .....................................................6 11. Records.................... .....................................................6 III. Materials and Methods.... .....................................................6 A. Tes Dilu t t i Su on b s t a n c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 B. C. Animal Water.......... .....................................................6 Culture......... .....................................................6 D. Test Meth Chemical o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 E. F. Statistical Analysis..... Analysis..... .....................................................7 .....................................................8 IV. Results and Discussion.... .................................................... 8 V. Conclusion................. ....................................................8 VI. Literature Cited............ .....................................................8 VII. r a bCiheesm.i.c.a. .l .C. .h.a. .r.a.c.t.e. r.i.s.t.i.c.s.s.o.f. .H. .a.s.k.e. .ll. .L.a.b. .o.r.a.t.o.r.y. . . . . . . . . . . . . . . . . . . . . . . . . 9 . 9 1. 2. Measured Concentration s of H-19518H------W--eJlLliWn aa te48r-.H..o..u..r.,....... Static, Un- crated Test.. (^^^^^^^"J 10 43.. WPhaytseircCalhaenmdisCtroymoficthael $Cccohnatrraocl taetriTsetiscts SoftaHr-t1..9..51.8.N..--...--...^..^..--..--g.r.|.. 11 Test Solutions........... . ..........................W?^^^~^^^''<.... 12 5-JDbserved Immobility of 3aphnia magna Exposed to H-19518 I------------to9r 48 ho jrs in a Static, Unaerated Test.................. 14 6.Hn95T6nBBBB] Concentrations Causing Immobility to 50% (EC'50) of Daphnia'magna at Specific Time Intervals ............... 15 ^oo^nTSCACBl Coinpa1^ Sanded. Does 2 ------ NG Hi oh Sa a Erdle oo] | `SubstanceTasted|1:4 | -- Synonyms: | [io | | I---- i DuPontHLA591-92 yo T4 1\ a ] | . 6 eu | [0 HaskellNumber. | "Htes18 | rio: || [mm f | cps ertcna TSA Sponsor|: DuPont Chemicals E.lduPontde`Nemours and Company | | Wilmington, Detaware 19898 ! ee ------------------------------ 1 Sanitized. Does not contain TSCA CBS :]w?sp^"a^ DuPontHLR591-92 STATIC, iOSO OF H-19518 magna SUMMABY H-19518 was slightly toxic to Daphnia magna WQIWAQS (less than 24 hours old) in an unaerated, static, 48-hourte<t. Nominal concentrations were 0,2.05. 5.13.12.8,32.0, 80.0.200, and 500 mg/L. Miasured concentrations at 48 nours were 0.45.2.67,4.07, 14.1. 30.3,74.7,172, and 49 3 mg/L the 48-hour EC50. based on measured concentrations at 48 hours a )d immobility, was 71.7 mg/L with a 95% fiducial limit of 52.9 to 98.3 mg/L Work By: /^W^.P^re^ Robert W. Peterson, B. A. Toxicology Associate bl&P ^ -7-^y /99- Mo/Day/Year THIS REPORT IS APPROVE?FOR ISSUE. ^ ^ . P/^^ Keith B. Pierson. Ph.D. Research Toxicologist and Study; Director Environmental Sciences ^ V^y/99^ Mo/Day/Year Company Sanltaed. Does not contain TSCA CBI DuPontHLR59l-92 I. PURPOSE The goal of this study was to assess the acute toxicity of H-19518 to Daphnia magna neonates less than 24 hours old. II. RECORDS All data and the final report will be maintained either at Haskftll Laboratory for Toxicology and Industrial Medicine (Newark, DJaware) or be archived at the DuPont Records Management Center (Wilmington. Delaware). III. MATERIALS AND METHODS A. Test Substance The test substance is described under General Information. A stock solution was prepared by dissolving the test substance in filtered fish tank water (see below) and surring for 30 minutes. Aliquots of the stock solution were added to filtered fish tank water to prepare 1100 mL of composite test solution. The composite was then apportioned among the tour replicate test chambers (262 mL per flask) using a graduated cyiinder. Each of the above solutions was kept in a sealed container with minimal head space to avoid potential loss of the test substance through volatility. The stability and solubility of H-19518 in filtered fish tank water and siorage conditions were not determined. B. Dilution Water Water (""able 1) originated from the Haskell Laboratory well and flowed through aquaria containing fathead minnows, Pimephales promo/as, prior to use in this dap^id test. This water was termed filtend. fish tank water (FFTW). C. Ammai Culture Parent Daphnia magna were reared at Haskell Laboratory sn 250 mL Pyrex beakers (1 per beaker) which contained 200 mL of FFTW or autoclaved FFTW held at approximately 20C. Daphnids were fed two green algae, Selenastrum capricornutum and Ankistrodesmus falcatus. at a rate of 75,000 cells/mL ot each species (lotal 150,000 cells/mL) on Monday, Wednesday, and Friday during water renewal (culture start through day 12). From day 14 on daphnids were fed only Se'enastrum c-apricomutum at a rate of 150,000 cells/mL on Monday, Wednesday, and Friday during water renewal. Neonates (less than 24 hours old) were coiiacted from 20 day old parent daphnids for use in this test. Sickness, injury, and abnormality were not observed. Daphnia magna were identified by labels on the beakers and test vepseis Company Sanitized. Does no* contain TSCA CBI 6 DuPontHLR591-92 ,.- D. Test Methods [1] Nominal test concentrations concentrations and a water control were used. Seven test 32.n, 80.0, 200, and 500 mg/L. were 22..0055..05..1103,,1i2t.-8.u, ,^. .. "~-m"a"x'--im"u"m"ridiiaammeetteerr;; 12.5-cm solution ---i Screw-top Erienmeyer flasks (262 mL; 8~ -cm height)capped with a valve assembly (Miniert)were us~ e filled to the to depth; and IS.R-cm total approximatel2y62 mL of test solution and was volatility. Four Each flask contained space and thus limit loss of test substance from replicate contained five minimize head replicates were used for each test concentration; each of 20 daphnids per test concentration. daphnids (1 daphnid/52.4 mL) for a total control and test beakers using random numbers. Test unaerated. Daphnids were added to the held between 20.3 and 20.7C (mean 2o0f.616C)hoaunrds wligehret (161 to solutions were Daphnids were not fed during the test. eAmpphlooyteodpewriitohdapproximately25 minutes of t2r2a6nsLituioxn) avlelrisguhst (81 htoou3rsLudxa)rkpnreescsedwiansgand following the 16 hour light interval. Observations were made daily. Yellow Springs. OH), (YS! Model 54A, Yellow Springs Instruments, Medfield, MA), and Dissolved oxygen pH (Corning Model 61 OA, Corning Scientific Instruments. Society of Testing Materials mercury thermometer) they temperature (registeredAmerican in each 1100 mL test solution and the control after control and all test were measured at test start equilibrium,in temperature all replicates of the daphnid immobility. (Measurements of reached test concentrations at the end of the.test and/or total could not be made without loss of potentially probes individual replicate concentrations components and because the pH and dissolved oxygen alkalinity (Hach kit, Hach volatile test substance fit through the neck of the Erienmeyer flasks. Total (Hach kit. Hach Chemical would not Loveland, CO), EDTA hardness (YSl model 33, Yellow Springs Cherrrcat Company, Loveland, CO), and conductivity dilution water were measured before Company, Springs, OH) of the continuously-recording Instruments, Yellow oaphnids were addpd at the beginning of the test. A control to check for temperature variation during thermometer was i-ov?d in a dummy the 48-hour test. E. Chemical Analysis Jackson Laboratory at conducted after the test ended at the DuPont standards were completely Analyses were Chambers Works, Deepwater. NJ. Samples and Wickbold Torch. The resulting water was then combusted in a hydrogen/oxygen concentration using an Orion fluoride ion selective either a analyzed for total fiuoride calculated from the standards using Concentrations were then electrode. three or four point calibration curve. C^\ ompany - Sanitized. Does not contain TSCA CB! 7 DuPontHLR591-92 F. Statistical Analysis The 48-hour EC50 was calculated by the SAS Probit method (21. IV. RESULTS AND DISCUSSION conducted in uncovered 250 mL Pyrex glass beakers v. i immobilities A 48-hour rangerinder was and 1,000 mg/L Observed nominal concentrations of 0,0.1,1.0.10,100. were 0,0.0,0.0, and 100%, respectively. definitive studieswere conducted. Data from, the first twforowmertehedtishciradrddeedfinitive Three apparent lack of dose-related immobility. Data because of an study are reported herein. conducted using nominal concentrations of 0,2.05, 5.13, replicate flask at The definitive study was 200, and 500 mg/L. Measured concentrations in one 493 mg/L (Table 2). An 12.8. 32.0, 80.0, 48 hours were 0.45, 2.67, 4.07,14.1c,on3c0e.n3t,r7a4ti.o7n,1s7o2v,ear ntidme occurred (Table 2); a unexpected increase in measured this increase was not determined. It may suggest that the test of test substance specific cause for completely in solution at test start. The presence All chemical and explained. substance was not in the control was noted (Table 2), but could not be (Tables 3 and 4) were within expected physical parameters in the definitive defined test as the inability to swim at least two body lengths The ranges. Immobilization was direction within 15 seconds after gentle prodding with 48 hours a glass rod. was 71.7 mg/L with a in any one 48-hour EC50 based upon measured concentrations at mg/L (Tables 5 and 6). 95% fiducial interval of 52.9 to 98.3 V. CONCLUSION H-13518 showed slight toxicity to Daphnia magna neonates (less than 24 hours old) in an unaerated, static, 3-hour test. VI. LITERATURE CITED Co-Operation and Development (OECD). Guideline for Organisation for Economic 1. Testing Chemicals: 202. 4 April 1984. Probit Analysis, 3rd Ed., Cambridoe University Press 2. Finney, D. J. 1971. 333 p. ^"ysan^ oD. oesn0^^ 8 DuPontHLR591-92 STATIC, ACUTE. 48-HOUR EC50 OF H-19518 TO Daphnia magna TABLE 1 Characteristics of Haskell Laboratory Well Water1 Chemical BOD. mg/L COD, mg/L DOC. mg/'L TOC2, mg/L Total KjeldahlN, mg/L Ammonia N. mg/L Turbidity, NTU Phenolics, mg/L Color, apparent Co/R7 Solids Total suspended, Aluminum, mg/L Antimony, mg/L Arsenic, mg/L Beryllium, mg/L mg/L Boron4, mg/L Cadmium4, mg/L Calcium, mg/L Chloride3- mg/L Chromium, mg/L Cobalt, mg/L Copper, mg/L Cyanide, mg/L Iron3. mg/L Fluoride3, mg/L <2 <10 1.4 1.8 0.8 <0.2 <1.0 <0.050 <5 <4 <0.100 <0.060 <0.010 <0.001 0.020 <0.002 24.3 6.2 <0.010 <0.020 .^0.010 <0.025 0.001 <0.1 Lead, mg/L Magnesium, mg/L MBAS/LAS, mg/L Mercury, mg/L Nickel, mg/L Nitrite3, mg/L Nitrate3, mg/L Phosphate3. mg/L Potassium, mg/L Selenium. mg/L Silver, mg/L Sodium, mg/L Suifate3, mg/L Sulfide, mg/L Zinc, mg/L Volatile priority pollutants Acid extractable priority pollutants Base/neutral priority pollutants Pesiicides/PCBs Organophosphate pesticides5,|ig/L <0.075 3.7 <0.05 <0.00020 <0.020 <0.1 1.9 <0.1 1.7 <0.005 <0.010 8.2 5.0 <0.10 <0.058 ND6 ND6 ND6 ND6 ^^"^^^---.^--.^----.c^ indicated otherwise, analyses performed ^ate of sample collection 26 May 1992 unless Certification Corporation, Edison, New Jersey; ^ate of sample Delaware, at Environmental Testing and performed at DuPont Engineering Test Center, Newark, Chemicals, Jackson collection. 1 July 1992, 10 June 1992, analyses performed at DuPcnt Published Method Detection Limit (BMDL); 3Date of collection New Jersey; ^elow the Madison, Wl; SNone detected; ^nits Laboratory, Deepwater, performed at Hazleton Laboratories, Inc., analyses cobalt/platinum reference. based on 9 ,, DuPontHLR591-92 STATIC. ACUTE, 48-HOUR EC50 OF H-19518 TO Daphnia magna TABLE 2 Measured Concentrations of H-19518 in a 48-Hour, Static. U Nominal Concentration (mg/L) Measured Concentration (mg/L) Start3 Endh Control 2.05 5.13 12.8 32.0 80.0 200 5on 0.21 1.99 3.77 12.0 25.5 65.0 162 456 0.45 2.67 4.07 14.1 30.3 74.7 172 493 ''Analyses were conducted on the composite 1100 mL test solution before it was apportioned among the four replicate flasks. analyses were conducted on one randomly-chosen replic (total four replicate from each test concentration. Co. "^^n;,n..l.li^. 'D>oes c0^^ lot Q4 ^ 10 DuPontHLR591-92 STATIC, ACUTE, 48-HOUR EC50 OF H-19518 TO D;.phna magna TABLE 3 Water Chemistry of the Control at Test S^art Total Alkalinity (mg/L as CaCOa) EDTA Hardness (mg/L as CaCOs) Conductivity (ixmhos/cm) 78 80 170 ^ - ^ . . , .' 0*^-j^ '-s0c4. ce, n DuPontHLR591-92 STATIC, ACUTE. 48-HOUR EC50 OF H-19518 TO Daphnia magna TABLE 4 PDIiSsSsOoIlVv!ed Oxygen. mo/L Control (0.45) 2.67 4.07 14.1 30.3 74.7 172 493 Q.^ -c 8.7b 8.613 8.^ 8.5^ 8.61' B.Gb 8.^ 7.1 7.3 7.4 7.4 7.5 7.3 7.3 7.4 7.2 7.2 7.3 7.3 7.4 7.4 7.4 7.4 7.3 7.2 7.3 7.3 77..13 77..42 77..42 77.23 7.5 7.4 7.4 7.3 &H Cont.ol (0.45) 2.67 4.07 14.1 30.3 74.7 172 493 7.4b 7.4b 7.4b -4b 7'.4b 7.4b 7.4b 7.4b 73 7.3 7.3 7.3 7.3 7.3 7 3 7.3 7.2 7.2 7.3 7.2 7.3 7.3 7.3 7.3 7.2 7.3 7.2 7.3 7.3 7.3 7.3 7.3 7.3 7.4 7.3 7.3 7.3 7.3 7.3 7.3 containing five daphnids each at test 8A, B, C, and D represent replicate test vessels liter of each solution before it was divided into were taken of the 1.1 reading was not made. start; bReadings replicates; sDash (-) indicates c.'^Pann, ysaniti^.^ ^nof 12 co^a'.''^SC4, DuPontHLR591-92 STATIC, ACUTE. 48-HOUR EC50 OF H-19518 TO Daphnia magna TABLE 4, Continued Physical and Chemical Characteristicsof Solutior s H-19518(TM----------Test Tamperature. C _^ 20.6 20.7 20.6 20.7 20.7 20.6 20.7 20.7 20.7 20.7 Control (0 45) 2.67 20.3b 20.3b 20.7 20.7 20.7 20.7 4.07 20.4b 20.313 20.6 20.7 20.7 20.7 20.7 20.6 20.7 20.7 20.7 14.1 30.3 20.3b 20.7 20.7 20.7 20.6 74.7 20.3b 20-4b 20.6 20.7 20.7 20.7 20.7 172 493 20.3b TXW daphnidseach > test represent replicate test vessels containing five solution before it was divided into ^A, B, C, and D taken of the 1.1 liter of each ere^prlti;cahtReesa; d^ina^sshw(e-^reindicates reading was not made. o c< ft^ Sana ^ecr oes ^e,^/i'm ^CAi 13 DuPontHLR591-92 ACUTE. 48-HOUR EC50 OF STATIC. TO Daphnia magma H-19518 TABLE 5 Immobile of Daphnia magna Exposed to Unaerated Test Observed -*"*"^--------br 48 Hours in a Static, H-19511 Observed Immobility in Percent Measured Concentrations (mg/L) Control (0.45; 2.67 4.07 14.1 30.3 74.7 172 4t09^3 8A, B, C and D start. 0 0 0 0 0 0 0 0 represent 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 40 replicate test 0 0 0 0 0 0 0 0 vessels 0 0 0 0 20 40 60 100 containing five 0 0 0 0 0 0 0 0 40 20 40 100 60 80 100 100 daphnid.-isx e^-airc-rhi u 0 0 0 20 40 100 00 ;a>tt test C, ofl^,,iy^","f^ ^oes o/. '^TS 14 DuPontHLR591-92 ACUTE, 48-HOUR EC50 OF STATIC. TO Daphnia magna H-19518 TABLE 6 Immobility to 50% l^oncentrationCsausing Intervals ta magna at SpecificTime concentrations. calculated because of insufficient measured values could not be "faBNaAsemdeaunpsonnot available; i. e.. imrnobilities at 24 hours. c^ 1ys^ 'Pa/,, tf. o"o, es ^Of ^'fa^ 15