Document 2Njz5DkBaj5GKjZ5vqR35daDL
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Study Title H-25914: Influence on Growth and Growth Rateofthe Green Alga Selenastrum capricornutum
AUTHORS: Robert A. Hoke, Ph.D. Barbra D. Ferrell, B.S.
STUDY COMPLETED ON: August 18, 2003
PERFORMING LABORATORY: E.L du Pont de Nemours and Company
Haskell Laboratory for Health and Environmental Sciences
Elkton Road, P.O. Box 50
--
Newark, Delaware 19714-0050
LABORATORY PROJECT ID: DuPont-13348
CompanySaitama.Dons no contain TSCA Ca
H-25914: Influence on Growth and Growth Rate ofthe Green Alga Selenastrum capricormutum --_
DuPont-13348
CERTIFICATION `We, the undersigned, declare that this report provides an accurate evaluationofdata obtained from this study.
WorkComply: __ApnbiDDa isoy
.
BAasrsborcaiDa\tFeeSrcrieeln,i5s.t5.
AugDate 203
Tesuedby StadyDirector:
sur 3 Robert A.Hee, PRD.
8 Ftalocd
~~
SeniorResearch Ecotoxicologinsdt Manages
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STUDY INFORMATION
substan Tec RNY|
Synonyms/Codes: + H-25914 3
Submiter's Notebook Numbers: [SSNIT)
Haskell Number: 25914
cas Registry Number [HNN
pure JET)
Known Inputs; Pa
pDuurronsriesssas
Physical Characteristics: Amber liquid
Stability: The test substance appeared to be stable under the
conditions ofthe study; no evidenceof instability was observed.
USA. Sponsor: E.I: du Pont de Nemours and Company
*
`Wilmington, Delaware 19898
Study Initiated/Completed: July 22, 2003/ (sce report cover page)
-_-- J-- ----"
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SUMMARY
AgresetnudaylgwaaSsecloenndauscttreudm tcoapdreitceorrmniunteumt.he" eTffheectalogfaHe-w2e5r9e1e4xopnostehde gtoronwotmhinaanldcgornocwentthrraattieoonfstohfe 0.1,11,10, and 100 mg H-25914/Literofnutrient medium (ppm).
`The algae were exposed for 72 hours (3 days) without test medium renewal. The effect was edexnpsrietsys)erdelaastpievercteontthienbhlibaintkio(ncuilntugrreomwtehdibuams)edcoonntrhoelalftohrytcheell72c-ohuonutr(a(ldsaoyr3e)feirnrteedrvtaoloasfctehlel test
"The 72-hour ECso, and the 95% confidence interval (C.L) based on nominal concentrationsisas
follows:
Healthy Cell Count ECw
117 mg/L. (95% C1. = 0.75 to 1.59 mg H-25914/L)
H-25914 exhibited medium concern under TSCA and was "toxic" to aquatic organisms
E(RU5D1i)r,ecctliavsesiifniaed7a2s-hdoaunrg,earcouutse
ftoerstthuesienngvitrheongmreenetn,
and
alga
Saeslseignnaesdtrtuhme
csaypmrbioclor"nNut"uumn.der
the
--~
-- ba TAlhseoEkCnyowins daesfiPnreeduadoktihrechenffeerciteilvaescuobnccaepntirtaataion" produci5n0%g inbibition of growth relative to the control
_-- mm
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MATERIALS AND METHODS
A. TestSystem
Selenastrum study.
capricornutum,
a
freshwater,
unicellular,
non-motile,
green
alga,
was
used
in
this
U"TnhieveorrsiigtinyoaflTceulxtaurse astouArucsetiwnas- AtuhsetiDne,paTretxmaesn7t8o7f1B3o-t7a64n0y. - Culture Collection ofAlgac - The
t`oThtehceuslttuudrye,mSeetlheonadsftorruSmeclaepnraisctrourmnuctaupmricuclotrunruetsuwmewraesmabiansteadinoendpuunbdleisrhtehdelsiatmeeraetnuvrier."onmPerniotral
cfloansdkisticoonnstauisneidnginaptpherosxtiumdya.telTyhe50ormgLaonifsfmisltweerrede (c=ulftiulrteerd-sitnersitleirzieldi)zesdyn2t5h0etmicLalEgralle-namsseayyer
procedure (AAP) nutrient medium 7 days. The flasks were fitted with
and were aseptically transferred to fresh foam stoppers to permit gas exchange.
medium
every
3
to
B. Test Design
--~ The test design used for the Selenastrum capricormutum study is described below:
_--
Nutrient Flask Volume Solution Volume Volume
Organism
Medium (mL)
(mL)
Ratio
e Selenae strumecr atpricoe rnutum-- ~~ A-- AP --2-- 50 --r--e 50-- ---- ------ El--
C.
AAP Nutrient (Appendix A)
Medium
Preparation
for
Selenastrum
capricornutum
AAP nutrient
solutions and
mediu
1 mL of
m was prepared by adding
the micronutrient stock so
lu1 tmioLnotfoeaapcphrooxfitmhaete6lyma8c0r0onmutLroifenMtisltloick-Q
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each 1 study.
liteof
medium)
were
used
to
prepare
the
larger
volumesof
the
medium
required
for
the
`The medium pH was adjustedtoapproximately 7.5. The medium was filter-sterilized using
Corning" pre-sterilized filtration systems each witah 0.22 pm cellulose acetate filter.
eet Company Sanitized. Does not contain TSCA CBI
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1. Test Solution Preparation
Fusoeudr ninomthiinsasltutdeyst.coAnlcieqnutortastoifontshe(0f.i1l,te1r,-s1t0r,ialinzded1A00AmPgmuHt-ri2e5n9t1m4e/dLiAuAmPwenruteriuesnetdmfeodrituhem)blwaenrke (normal culture medium) control solution.
2. Test Culture Preparation
sFtoerrielaiczehdb2l5a0nkmcLonEtrrollenamnedyteerstfslaoslkustifoint,te2d awliitqhuosttser(il5i0zemdLfoeaacmhs)twopepreersp.laTceodaicnhiseevpaertahteedesired innoimtiiantailon,coannceanptprraotxiiomnoatfeap0p.3r4o0ximmLate(=ly34100,a0L0)0 aSleilqeunoatsotfrualmgcalapirnioccourlmuumtufmrocmelalsl/omgLaraitthtemsitcally growing stock culture was aseptically transferred to cach flask.
D. Experimental Design and Test Conditions
rTeh-epotseisttifolnaesdkseawcehrewoprlkaicnedg odnaya. sAhiarketremtpabelreatiunraecihnatmhebecrhainmbaenronw-assysrteecmoartdiecddceosnitginnuaonudslwyerweith 2a rceopnltiicantueosu.s Ttehmepearlagtaeurweerreecoirndceru.batEeadcfhorblaapnpkrocxointmraotlelaynd72tehsotucrosncweintthroauttiotneswtamsedteisutemdraesnewal ~~ taanrdgceetllencvoiurnotnsmweentraelmpaadrea.meUtleursmianraetdieosncwriabsedsuipnptlhieedfoblylcoowoiln-gwthaibltee. fluorescent tubes. The
Initial Population
Photo- Shaking
Illumination Period Speed (Qumens/m' = ux) _ (hours) _ (rpms)
Temperature Q
Ss 10,000cells/mt L 60001010000S 24 O 100 us2
E. Selenastrum capricornutum Growth Measurement `Sneulmebnearsotfrucemlclasptraikceonrmfurtoummagnraopwptrhomxeiamastuere0m.e2nmtLwassamdpelteerfmrionmedcabcyhvfilsausakllaty acpopurnotxiinmgattheely `7c2ohmopuorusndfrmoimcrsotsucdoypien.itiAantioanl.iqTuhoteocfoeuanctshwsearmeplceonwdausctleodaduesdinigntaohtehema2cwyetlolmseotfetrheand a Ahlelmaceclyltsolmoectaeterdainndt8hesq1u6asrqeusafrresomweearcehcgoruindteardeaan(d16retcootarldesdquaasrehse)alwtehryeorseulnehcetaeldthfyo.r coTuhnetitnogt.al nCeulmlbseoruotfsicdeelltshecsoeun1t6egdriwdasswmeurletinpoltiecdoubnyte1d0,n0o0r0itnoclduedteedrmiinntehethteotnalumnubmebreorf.celOlbsseprevratmiilolinlsitaern.d sctouudnytsroefchoredasl.thTyhaensdtautinshteiacallthcyalcceulllasti(co.ngo.,fdtehfeorEmCesdo,wsacsenbeassceednto,nstmuenatend)hewaelrtehyrceeclolrcdoeudnitns tahned nominal concentrations.
-
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RESULTS sBoalsuetdioonns wveisruealcloebasrearvnadticoonlso,rltehsesbbleafnokrecoandtdriotliosnoolfuatligonalancdelltsh.e 0A.f1i,e1r, th1e0,aaddnidti1o0n0ofmagl/gLaltecsetlls, aptpherotexsitmaantdelblya2n5kcCo.ntTrohlefallagsakes wweerree pilnaccuebdatoend afosrh7a2kehrotuarbsle(3indaaycs)hawmibtehoruwtimthedaituemmpreernaetwuarle.of cUhluammibneartiwoansw6a8s85sulpupmlieends/bmy'co(o=ll-uwxh)i.teThfeluosrheaskcienngtstpubeeesd. wTahse9m8eraevnolliugthitonisntpeenrsimtiyniunttehe(rpm). ``TmheeaspuHremmeeanstusorfemtehnetteosftt hseoluctuilotnurseatmteedstiuinmitwiaatsioanprparnogxeidmaftreolmy 77..551attost7u.d56y,satanrdt.atTtheestpH termination ranged from 7.77 to 8.10.
CONCLUSIONS
`The 72-hour EC, is listed below:
including
the
95%
confidence
interval
(C.L.)*
based
on
nominal
concentrations
Healthy Cell Count
~
EC
117 mg/L (95% C1. =0.75 10 1.59 mg H-25914/L)
H-25914 exhibited medium concern under TSCA and was "toxic" to aquatic organisms
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--~ d a Thee ECy s is define edas tha e effective concentration" producinag 50% inhibitionofgrowth relative tothe control
ms
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REFERENCES
1.
Slob, W., (2002). 298-312,
Dose-response modelingofcontinued endpoints.
Toxicol. Sci., 66,
2. uSnmdrecrheTkS,CJA,:Clmeemtehnotdss, aR.n,dMeovracluoactki,onRo.,fdaantda.RabEenrvti,rWon.m(e1n9t9a3l).ToAxsisceoslsoignyagnecdoRliosgikcal hazard
Assessment, ASTM STP 1179. American Society for Testing
(W.G. Landis, 1.5. Hughes, and Materials, Philadelphia.
and
M.A.
Lewis,
Eds.),
pp
22-39.
3.
Official 52.1.
Journalof
the
European
Communities,
Volume
36,
4
May
1993,
pp.
68,
section
4. MAislslaeyr,BWot.tEl.e;TGerset;enUe.,S.J.CE.;nviSrhoinrmoeynatmaal, PT.rotTehcetiSoenlAegneansctyr;umU.cSa.prGiocvoerrnnutmuemntPrPirnitnztiAnlggaOlffice: Washington, DC, 1978; EPA-600/9-78-013.
5. AComnedruicctainngSoSctiateitcy: 9fo6r-hTeTsotxiincgitayndTeMsattserwiiatlhsM(iAcSrToMal)g.ae"(1i9n90A)S.T"MStAannnduaardl GBuoiodkeoffSotrandards, EI218-90. Vol. 11.04, Philadelphia, PA.
--~
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--~
TABLES
TT
-- TT T--
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TABLE 1
PH MEASUREMENTS OF TEST SOLUTIONS
`Nominal Concentration
(mg/L)
Test Initiation (0 hours) pH
Test Termination (72 hours) pH
Blank Control
7.56
7.77
01
7.53
1
7.53
8.10 7.98
10
7.51
100
7.51
7.78 7.80
r ee e---- re -- p----
--
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TABLE 2
CELL COUNT DATA SUMMARY
Nominal Concentration
Day 0 Healthy Cell Count (cells/mL)
Day 3 Healthy Cell Count (cells/mL)
Blank Control
Replicate | Replicate 2
10,000 10,000
2,600,000 2,740,000
0.0 mg/L,
Replicate 1 Replicate 2
10,000 10,000
2,980,000 2,720,000
1 mg/L
Replicate 1
10,000
Replicate 2
10,000
1,730,000 1,280,000
~~
10 mg/L,
Replicate |
10,000
240,000
Replicate 2
10,000
210,000
100 mg/L.
Replicate | Replicate 2
10,000 10,000
---
230,000 340,000
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--~
FIGURE
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FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE
HEE
1
~~
T now ww wm wn wm wm "
.
ERSTE
o
New
i
--_
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--_
APPENDICES
--
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---
APPENDIX A
AAP Nutrient Medium Constituents
Ll
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----AA AAP NP UTRIN ENTMMEEU DDIIUUMMT CCOONNSSR TTIITTUUI EENNTTSSENT
Stock Component
Solutions Concentration
(/L)
___
Final Component
Prepared Medium Concentration (mg/L)
Macronutrient Stock #1
NaNOy
25.500
Na 11001 No 4200
Macronutrient Stock #2
NaHCO;
15.000
c 21a K 0469
Macronutrient Stock #3
KaHPO;
1.044
P0186
Mg 2904
Macronutrient Stock #4
MgSO:TH,0
14.700
--~
Ca 12m s 1911
Macronutrient Stock #5
MgCl6H,0
12.164
Macronutrient Stock #6
CaCly2H,0
4410
ComponeSntStteocckkSSoolluCtoiinoocnnessnt"r_ ation_ (mg/_ L) _CompFoFininenanaltPP_rreeCppoaanrrceeednMtMreeadtiiouunmm_(ug/- L)
Micronutrient Stock HBO; MnClo+d H,0 ZnCl CoCle6 H:0 CuCLe2H;0 NasMoOs2 H:0 FeClye6 H,0 Na:EDTA-2 H;0 -_--
185.520 415.610 3271 1.428 0012 7.260 160.000 300.000
B 32460 Mn 115374 Zn 1570 Co 0354 Cu 0004 Mo 2878 Fe 33051
~ a IAnsclruedpeosrtmeadginneRseifuemrefnrcoedm.both magnesium sulfite and magnesium chloride
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--_
APPENDIX B
72-Hour Healthy Cell Count Statistics
--~ ----
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Dupont13348
72-HOUR HEALTHY CELL COUNT STATISTICS
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Exact Permutation BebmoBe ertoitd
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SKPI-PGVLNAIILFTUESR1E3SAUR5LETnSTLOARERE71FF5oOnCRObRoBACmUTPeEEnDRRToEELSDnDEWIHTHSCONTFINUINY CORRECTION ACTOR
Jonckheore Trend Test mTo E sm abi
on Dose 0 o.0mxmmiioirs
+
Lowest 4 xenon
Doses thew 00 soiowr sos?
pe
Joncknesze --5
Trend Test 2.295a59o3
on Dose 0 0.0xkvmeaire
+
Lowest 3 Doses thea 10. worHRstow cos;
pe
Jonckheere Trend Test on Dose 0 + Lowest 2 Doses thru 5 an TS nsLoor obmeaser wer! stow cos3
Thus, the 0EC-1 pom.
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5050 Batimatation OBSERVED PERCENT CHANGE FROM CONTROL MEAN
obs cone mew perme on
312
0o.i0 2286..5700 10 Isls
2o aaa
22 2
i5 wwolo 220ss gIg s;2
OBSERVED & FPOFRROEMRDUMIULCLATALEL=DFCPReE1OR1MC_E(-- CJNoNTunCtH?-- ANG/ F10R-- 0O0M00CONTROL MEAN
Obs CONC N MEW PRED poTCANG PREDPCICHNG
21 3
00.10 32 102
22865700 155
2336.52927120 1sie034
70 44
3
00 2 2025 00s
-60 -is
5 1000 2 208 oloeo i"g5s2
"-110000
CovarianceECSM0atruisxinagnMdetShtoadn=dNaArRdQUEArRrDoTr, ofOECPDarMaomdeetler2Estimates
~
Er
vo
scs0
se
vEo50 00..0881196352 -00..008415634383 00..2910259115
Nodel 2 Parameter Estimates, C50 and 95% Confidence Bounds
vo
ses
ws
vs
28.2210 1.16872 0.7521 1.5e703
Hodel 2 Bvaluation Measures
SSREG SSERR SSTONC sstow souame
3195.43 21.5787 150.621 3217.07 0.85673
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--~
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