Document 37JXv22KRKGm7ag8B8x5wbvbJ
AR226- 3259
441
TRADE SECRET
#R226 - 3257
Dupont 11410
Study Title H-25434: Influence on Growth and Growth Rateofthe Green
`Alga Selenastrum capricornutum
LaboratoryProject ID: DuPont-11410
AUTHOR: Terry Lee Sloman, B.S.
_
STUDY COMPLETED ON: October 4, 2002
PERFORMING LABORATORY: E.L du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.0. Box 50 Newark, Delaware 19714-0050
work Request Nuvzer: [IIR
stupy Cove Numser:JF
Rete `CompanySaniized. DoesnotcontTSaCAiCanl
Hof2t5h4e3G4r:eenIAnflluaenSceleonnsGtrrcowathparndiGcroorwnRae --_
Dupont11410
CERTIFICATION
`We, the undersigned,
from this study.
declare
that
this
report
provides
an
accurate
evaluationofdata
obtained
WorkCompt: __PolaoaSsombIr>a2bei oe Souti
Issued by Study Director:
doTeomycLoaxSeSaeniB35.
4octgBuben2002. 4OctoDbee 200%
e-- r --I-- eerie -- -- Company Sanitized. Doss not contain TSCA a
Hof:t2h5e43Gr4eeInnAfllugeancSeeloennGasrtorwutmhcaandprGriocwothrnRate
STUDY INFORMATION
Substance Tested: {NENA
Svnonyms/Codes: + H-25434
Haskell Number: 25434
Dupont 11410
-
KnownImpurities: WER Physical Characteristics: Off-white, milky semi-opaque aqueous dispersion
Stability: The test substance appeared to be stable under the conditionsofthe studs; no evidence of instability was observed.
`Sponsor: E.L du Pont de Nemours and Company Wilmington, Delaware 19898 USA.
Study Initiated/Completed: September 24, 2002/ (see report cover page) In-Life Initiated/Completed: September 24, 2002 / September 27, 2002
eH Company Saniized. Doesnotcontain TSCA CBI
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SUMMARY
gAresetnudaylgwaaSsecloenndaustcrtuemd tcoapdreitceorrmniunteumt.he TefhfeecatlogfaeH-we2r5e43e4xpoonsethdetgornoowmtihnaanldcognrcoewntthrartaitoeonsfotfhe 0.01, 0.1,1, 10, 100, and 1000 mg H-25434/Literof nutrient mediurn (ppm).
e`Txhpereaslsgeade awserpeerecxepnotsiendhifboirti7o2nhionugrrsow(3thdabyass)ewdiotnhohuetaltetshtymceeldlicuomunrten(ealwsaol.rTefherereedftfoeactswcaesll density) relative to the blank (culture medium) control for the 72-hour (day 3) intervalofthe test. c`Tohnece7n2t-rhaotiuornEsCasroe,aisncflouldilnogwtshe:95% confidence intervals (C.L.),* based on nominal
Healthy Cell Count EC
36.2 mg/L (95% C.L not calculated)
H-25434 exhibited medium concem*underTSCA and classified "harmful" to aquatic organisms (RS5el2e)nausnidreurm ctahperEicUorDniurteucmt.ive in a 72-hour, acute test using the green alga
2 cTohnetrEoClyis defined as the "effective concentration" producing a S0% inhibition ofgrowth relative to the
b CMBchBCaNuplmlSaoonfgthaw,nadLrePH.Parlao,ndudLcoNtnesdlodCneo.rrp,,L.(A1.99(81)9.89)A.nGReSn/er1alRiPzeLdLpirnoecaerduMroedeclosn,du2ctiendgitWiionl,laCmhsa'ptteesrt,anpdppr3o2b5i.tanalysis,
4 SUnnievdeercsoirt,yGP.rWes.,nAdmeCso.chran, W.G. (1967). StatisticalMethods, 6 dition, Table A4, pp 469. The lowa State
Smmertchhoedks,aJn,dCelvaelmueantsi,onRo,fdMaotrac.ocEkn,viRro.namnednRtaableTrotx,icWo.lo(g1y9a93n).d RAissksAesssseisnsgmcecnolto,giAcSalThMaSzTarPdu1n17d9e,rTSCA:
--~
P(hWi.lGa.deLpahnidai.s, JS. Hughes, and M.A. Lewis, Ed), pp 22:39. American Society for Testing and Materials,
f Offical Journal ofthe European Communit, Volume 36, May 1993, pp. 8, secton 5.2.1
HE
i
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MATERIALS AND METHODS
A TestSystem
Selenastrum study.
capricornutum,
a
freshwater,
unicellular,
non-motile,
green
alga,
was
used
in
this
`The original culture Universityof Texas
source was at Austin -
the Departmentof Botany Austin, Texas 78713-7640.
Culture
Collectionof
Algae
-
The
`oTthehecuslttuudrye,mSeetlheondasftorruSmeclaepnraisctorrunmuctaupmricuclotrunruetsuwmewreasmabiansteadionendpuunbdleisrhtehdelsiatmeeraetnuvrier.o)nmPerinotral
fcloansdkisticoonnstauisneidnginaptpherosxtuidmya.telTyhe50ormgLanoifsmfsilwteerreed (c=ulftiulrteerd-sitnersitleirzieldi)zesdyn2t5h0emtiLc alEgralle-nasmseayyer
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. TestDesign
--~ "The test design used for the Selenastrum capricornutum study is described below:
Organism
Nutrient Medium
FlaskVolume (mL)
SolutionVolume (mL)
Volume Ratio
-- Selenastrum capricor-- utum~~AAP -- 250 -- 50 sit
C. AAP Nutrient Medium Preparation for Selenastrum capricornutum
(Appendix A)
AAP nutrient medium" was prepared by adding 1 mLofeachofthe 6 macronutrient stock
s(odleuitoinoinzseda)nwdat1 emr,Lowiftthhmeixmiincrgonafutterriecnatchstaodcdkitsioolnu.tiTohnetovaoplpurmoexiofmtahteelyme8d0i0ummLwoafs Mbirloluig-hQtto elaicther1lwiitterhoadfdmietidoinualm)Miwleir-Qeusweadtetor.prAeppparroeptrhieatlearpgreropvoorltuiomnesso(1fmthLe omfeedaicuhmstroecqkuisroeldutfioornthfeo.r study.
`The medium pH was adjusted to approximately 7.5. The medium was flter-strilized using
Comning pre-sterilized filtration systems each with a 0.22 pm cellulose acetate filter.
~~
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SSoHLf-t2sh5e e4G3Gr4r:eeleeInnnAfAlle luggeaancSeneloennc aGsrtorwus tmhcaantdprGirrcoowrunRatemcopricomutDuDauPPoonnnttl1_ u14i10o
~
I. Test Solution Preparation
mSiexdinuommi)nwaelrteesutsceodncienntthriastsitoundsy.(0.A0l1i,qu0.o1t,so1,ft10h,e 1f0i0l,tear-nsdter1i0l0iz0edmAgAHP-2n5u4tr3i4e/nLt mAeAdPiunumtrwieernet used for the blank (normal culture medium) control solution.
2. Test Culture Preparation
For each sterilized
blank control and test solution, 2 250 mL Erlenmeyer flasks fitted
awliitqhuosttser(il5i0zemdLfocaacmhs)towpepreersp.laEcaedchinflsaespkarwaatse
labeled
iwniitthialas.stTudoyancuhmibeever,theesdtessuibrsetdannocem,incaolncecnotnrcaetnitorna,tiroenpolfiacaptpernouxmibmeart,eldayte1,0a,0n0d0experimenter's
aSleilqeunoatsotfraulmgaclapirnioccourlnuumtufmrocmelalsl/omgLaraithtemsitcailniltyiagtrioonw,ianng astpopcrkoxciulmtautree0w.a2s08asmepLti(c=al2l0y8 pL)
transferred to each flask.
D. Experimental Design and Test Conditions
eT-hpeosteisttifolnaesdkseawcehrewoprlkaicendg odany.a sAhiarketremtapbelreatiunraecihnatmhbeecrhainmb2enronw-assysrteecmoartdiecddceosnitginnuaonudslwyerweith 2a rceopnltiicnatueosu.s Ttehmepaelrgaateurweerreecoirndceurb.atEeadcfhorblaapnpkrocxoinmtraotlelaynd72tehsotucrosncweintthroauttiotneswtamsedteisutmedraesnewal ~~ taanrdgecetllencvoiurnotnsmweenrtaelmpaadrea.mIetlelrusmairneatdieosncwriabsedsuipnplthiecdfoblylcoowoiln-gwthaibtlee.fluorescent tubes. The
Initial Population 10,000 cells/mL
Photo- Shaking
lumination ~~ Period Speed (Qumens/nr' = lux) _(hours) _ (rpms)
Temperature co
60001010000 24 100 4x2
E. Selenastrum capricornutum Growth Measurement
`Sneulmebnearsotfrucemllcsaptraikceonrnfurtoummagnraopwptrhomxeiamastuere0m.2enmtLwassamdpelteerfmrionmedeabcyhvfilsausaklalty acpopurnotxiinmgattheely
c7o2mhpoouursndfrmoimcrsotsucdoypien.itiAatnioanl.iqTuhoteofceouancths wsearmeplceonwdausctleodaduesdinigntaohtehemahceymtaocmyettoemretaenrdaand
u1n6hgeraildtshyw.erTeheselteotcatledn.umAbllerceolflscellolcsatceodunitnetdhewa1s6 mgruildtsipwleirede bcoyu1n0t,e0d0a0ntdordeectoerrdmeidneasthheeanlutmhybeorr
onfumceblelrs.peOrbsmielrlvilaittiero.nsCaelnldscoouutnstisdeofthheeaselt1h6ygarniddsuwneheraelntohty cceolulnst(eed.gn.,ordeifnocrlmuedde,d isnentehsecteodt,al
stunted) were `mean healthy
recorded in the cell counts and
study records. The statistical nominal concentrations.
calculationofthe
ECso
was
based
on
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RESULTS
tBeasstesdolountivoinssuwaleroebscelrevaartiaonnds,htahdebnloacnoklocrobnetfroolresoaldudtiitoinonaonfdalthgeal0.c0e1ll,s.0.1H,ow1,ev10e,ra,ntdh1e01000m0g/mLg/L
atensdt sbollauntkiocnonwtaroslcllaosukdsy wbeefroerpelaadcdeidtioonnoafsahlagkaelrctealblls.e iAnftaecrhtahmebaedrdiwtiitohnoaftaelmgpaelracetlulrse, othfe test
apUplruomxiniamtaitoenlywa2s5sCu.ppl`iTehde balygaceoowle-rwheiitnecfulbuaotreedscfeonrt7t2ubheos.ursTh(3edmaeysa)nwliitghhtouitntmeensdiityuminrtehneewal.
`cThhaempbHermweaassu7r5e3m4enltuomfetnhs/emc'ul(t=urleuxm).edTihuemswhaaksianpgpsrpoexeidmawtaesly170.05raetvsotluutdiyosntsarpt,erTmhienuptHe (rpm).
measurementsofthe test termination ranged from
solutions a test 6.72 t0 7.05.
initiation
ranged
from
7.29
t0
7.63,
and
at
test
(CONCLUSIONS
`The 72-hour ECso, including the 95% confidence intervals (C.1),* based on nominal
concentrations are as follow>s:
Healthy Cell Count
-
ECs
36.2 mg/L (95% C.L not calculated)
(HR-52254)3u4ndexehritbhieteEdUmeDidrieucmtivceonincear7"2-uhnoduerr,TaScuCteAtaesntducsliansgsitfhieedgr"eheanrmaflugla" to aquatic organisms Selenastrum capricornutum.
a cTohnteroElC.yis defined as the "effective concentration"producinga 50% inhibition ofgrowth relative to the
b (mSeWmtGrh.cohdeLsakenJ,dn,idsC,elv1ea5ml.euanHttuisgo,nhoRe.sf,,dMaaotnrad.coMEc.nkAv,.ir.Lo,enwaminesdn,tREaadblseT)r,t,popW.2s2(-13i999a.3n)c.dAmRoeiArssisklcAeassniosnsSgosgecmcioeeyltnoyigfiAocrSaTTlehsatSziaTnrgPdau1nnd1d7Me9artTerSiCalAs:,
--_
OPfhfiliacdaelphJioau.mal ofthe European Communities, Vohum 36, 4 May 1993, pp. 68, section 5.2.1
essen eer `Company Sanitzed. Does not contain TSCA CBI
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REFERENCES 1. AMislslaeyr,BWo.El:;e TGerste;enUe.,S.J.EC.n;viSrhoinrmoeynatmaal, PTr.otTehcteiSoenlAegneansctyr;umU.cSa.prGiocvorenrumteumntPrPirnitnztiAnlggaOlffice:
Washington, DC, 1978; EPA-600/9-78-015. 2. A`CmoenrdiuccatinnSgoSctiaettiyc 9fo6r-hTeTsotxiincgiatyndTeMsattserwiiatlhsM(iAcSroTaMl)g.ac"(1i9n90A)S.T"MStAannnduaardl BGuoiodkeoffoSrtandards,
E1218-90. Vol. 11.04, Philadelphia, PA.
3. BanBdNprSoobfittwaanraelyPsriosducts Corp, (1998). AnRS/IRPL procedure conducting Williams' test
4.
McCullogh, LP. and Nelder, 1A. (1989). pp 328. Chapman and Hall, London.
Generalized
Linear
Models,
2"
edition,
Chapter
9,
5. PSpne4d6e9c.or,ThGe.WIo.waanSdtaCtoechUrnainve,rWsi.tGy.Pr(e1s9s6,7)A.meSst.atistical Methods, 6" edition, Table Ad,
6. SunmdrecrheTkS,CJA,:Clmeemtehnotds,s aR.n,dMeovracluoactki,onRo.,fdaantda.RabEenrvti,rWon.me(1n9t9a3l).ToAxsisceoslsoignyagnedcoRliogsikcal hazard
PN
Assessment, ASTMSTP 1179. American Society for Testing
a(nWd.MGa.teLrainadlis,s,Ph1.i5l.adHeulgphhieas.,
and
M.A.
Lewis,
Eds.),
pp
22-39.
7.
Official 52.1.
Journalof
the
European
Communities,
Volume
346Ma,y
1993,
pp.
68,
section
--~ -_--
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DDiuPPoonwt-i1n14i10o
TABLES
~~ Trees. `Company Sanitized. Does not contain TSCA C81
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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
7.63
672
0.01
739
693
01
733
6.98
1
7.35
10
732
7.03 7.05
100
7.32
697
1000
729
692
--_
--_
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oH-f2t5h4e3G4r;eeInnfAllugeancSeeloennaGsrtorwutmhcaanpdriGcroorwntuhtuRmate
DuPont 11410
TABLE 2 DATA SUMMARY
Nominal Concentration
Da0y Healthy Cell Count (cells/mL)
Day 3 Healthy Cell Count (cells/mL)
Blank Control Replicate 1 Replicate 2
10,000 10,000
2,640,000 2110000
0.01 mg/L,
Replicate 1
10,000
Replicate 2
10,000
2,820,000 2,800,000
0.1 mg/L,
Replicate 1
10,000
Replicate 2.
10,000
2,810,000 2,580,000
1mgL
~
Replicate 1
10,000
Replicate 2
10,000
2,680,000 2,360,000
10 mg/L.
Replicate |
10,000
Replicate 2
10,000
2,610,000 2,020,000
100 mg/L,
Replicate 1
10,000
Replicate 2
10,000
50,000 240,000
1000 mg/L,
Replicate 1
10,000
0
Replicate 2
10,000
0
ee
---- `Company Sanitized. Does not contain TSCA CBI
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--_
FIGURE
_-- Company Sanitized.DoosnotconTtSaCAiCBnI
H-25434: Influenceon Growth and Growth Rate oftheGreen Alga Selenastrumcapricornutim
DuPont-11410
PN
FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE
72 We Henke Con Count heer
-- ee
ne
...
t
ge
i
--~
hi
\
[
m= `Company Sanitized. Does not contain TSCA CBI
H-25434: Influence on Growth and Growth Rate ofthe Green Alga Selnasirum capricornutun --~
DuPont-11410
APPENDICES
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DuPont-11410
APPENDIX A AAP Nutrient Medium Constituents
-- Te Company Sanitzed. Doesnotcontain TSCA CBI
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Dupont 11410
AAP NUTRIENT MEDIUM CONSTITUENTS ComponenSttock SolutCioonncse"ntration (g/L) CompoFnienanltPreCpoanrceendtMraetdiiounm(mg/L)
MNaacNrOosnutrient Stock #1 25.500 .
NNoa 412.00001
Macronutrient Stock #2. NeHCO;
15.000
c 24 K 0469
Macronutrient Stock #3
KHPO;
1.044
Po 01s
Mg 2.904"
Macronutrient Stock #4 MgSO:THO
14700
Csc o1n20
Macronutrient Stock #5 MgCl6H,0
12.164
Macronutrient Stock #6 CaCla2H0
4410
ComponenStto_c_k _So_luCtoinocnenstration
(mg/L)
__
Final Component
P_reCpoanrceedntMreadtiiounm(ug/L)
Micronutrient Stock HBO;
185.520
B 32460
MaCly+d H:0 ZaCly
415610 3271
Mn Zn
115374 150
CCuoClCyl+y26HH::00
0L041228
CCuo 00305044
NFeeCMloyO6yHr:20HO 71.6206.0000
Mo Fe
2878 33051
NeEDTA2 H:0 300.000
------
-- 4 AInsclruedpeosrtmeadginneRsefiearefncoem2.bth magnesium ule and magnesium chloride.
-_
Te
Company Sanitized. Does not contain TSCA CBI
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DuPont-11410
--~
APPENDIX B
72-Hour Healthy Cell Count Statistics
---
`Company Sanitized. Does not contain TSCA CBI
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72-HOUR HEALTHY CELL COUNT STATISTICS
wRarn.k Dose 99olalUay0l oIO oaaTinoop 3 sonoids
Tae eaEthdy Col-- l Count. (beelierot)
eReasnponse Sstedror Tmeraannstom sTriadnsBfroeremrs nm
Zasiaolmosodooooon sooo
1a`ga0ss0en0n00s mono
gGLiaaadavisaison)s Gadosas
oGoil.ooaubisissseaees 0lowel
333 2
66f.l4aadsd0sermsdnsssa
DTiHsIcSso STseknsoS 3gS5iiso0mu0oesnosssst oaaiicoonesesioassss 333 -ESoi.osss6eeiosesssssse
~ 0l-o0..i34e00n8s08e00s1 3%b..soaeeo3sreesars
Rank eric.
38o rroeam
o1on Faioooesn
~
ORCEnoeemot-asniid8delidoogmStaesrsi]tthe5mat%st.rthaeg3sTat3lo5or%nneaCetLxitoefnno,r20aL:ed.emcazsea=osni1n0g5a1mt0ar0e)n.d+. 0.5), applied to the data.
HIS OIL sss PIS SR CIs OREN
in --_
-
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~
DuPonetndHaEsnkveilrloLnambeonrtaatlorSyiofnorceHsealth
orcas
FROM:
J.C. Maslanka FM
November 4, 2002 pe ffrme)
TEST SUBSTANCE STABILITY OF H:25425
MHHaaessdkkieecllalllRSeRafemesprelaeernccNheuSPmtrboaejnredc:atrNduNmubemrb:er `AAnnaallyyttiiccaall TReesptorCtodNeu:mber NotebookReferences:
Ga22m554%a] A2002.004
Om)
tached is the analytical report for the study identified above, This analysis was fpeorfootrhemredputpoossateissfy protocol requirements for test substance stability but may also be used
arse
-- comeaTM' santizet oes con
`TEST SUBSTANCE STABILITYOFH.25425
HMaiskcelal SRaempslcehNuPmrbeer:t uber:
Analytical Report Number:
[2u502m5 ]
HA2002.094
c TAhseapmeprlm segaotfaHg-e2oo 5f4a2c5tiwvaes sinsgurbemdiietnentd(e4on.1Os)cwtavosbem3rea1s4u,hr2et0d002paonghanaplyoznedd Eiootctahoberr p15g, 200
When (2 sample was submicd.
~
ACKOWLEGEMENTS
Sample preparation by Sheila A. Riley (Chemistry Associate). Sample analysis by Bogdan Szostek (Senior Research Chemist).
SIGNATURE
repority
_GJoanneittC.PMlaasclatnikante, AnsyicalSaftChemist
wtbtsoors
Date
Date Issued:
4 More avons
cB
--_
sesd: 0 naresa!anTSCA
compe!
HA-2002-094 psge2of2
TM
METHODS
Analysis for the percent of ai. in a sample of H-25425 was by high-performance liquid
chromatography (HPLC) with a Gel Permeation Column (GPC) and refractive index detector
saucbcsotradnicneg atondthtehefoalnlaloywtiincgalmestthaondd.rdSosntioccaktsioolnutwiaosn.used to completely dissolve the test
SAMPLE PREPARATION & ANALYSIS
(Al1i0qmuLot)st(o0.g0i4v1e3n,o0m.i0n4a0l7caonndce0n.t0r4at1i2ognsroafms4)13o0f,H:4205704,25anwder4e12d0ispsopl,vedreisnpetcettirvaehlyyd.rofuran CHROMATOGRAPHIC CONDITIONS
Instrument:
Hewlett-Packard Model 1100 HPLC
MCFoolbloiuwlmRenaP:the:ase
S11t000ym%rLa/tgemteirHnlaRhyd4r;o7f.u8ramnm(TxHF3)00mm (GPCcolumn)
CInojleuctminonTeVmopleurmaet:ure: ~~ 3s5opCl
Detection:
Refractive Index.
CALIBRATION & QUANTITATION
rr
msAtaaskneedpaacrradalti(eb9as3ta.im1op7nl%espooulfruetH)i-.o2n5sA4t2sh5oactbkersatscoskluuebttsietodantnohcfeeHtw.eas2ts5s4doe2lsu5itgiionnnastt.eerdPaahesyadktrhoheefaiongrahaltnystwircaoaslmsreerfedpelr1ie0cnactee
dc'ueHrtPveLerCmb/iyGnPeldCe/absRytI-asapqnpualalryyeissnigsroergferesepasesccihtoincv.aelpMieberaaakstuihoreneigdshoctlosunttcioeonntthwreeactraieloiunbssreafdtoirtooneacccuhrov(een,stssaotlcuatrlioiunbawtceiroten
RESULTS
5H.:5285242500c1luemdifnurtoems.thTehHePsLpConsceorlurempnoarsteadrepsuolrvteydpeg withig rreetenhteionsatmipmleoefwaasbosuutbmited.
.
TablIe. The percent ofa. inthe F-o2o54m25Hs2a5m4p3l5e analyzed POerccentt o15b,2e00r. - Nominal Messed Nomiml
21 3
aw0o mwoo osas 0 mo 20
[[SAtvuenrdaagredD%evNioamtiionnal |Coeicentof Variation
0982s 1%
--~ aotcontaiTSCA! cat
company Senized. DO
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