Table Of Content:
OECD/OCDE 221
Adopted
23March2006
OECDGUIDELINESFORTHETESTINGOFCHEMICALS
Lemnasp.GrowthInhibitionTest
INTRODUCTION
t1h.egenusThiLseTmensatG(udiudceklwieneedi)s.desiIgtneisdtboaassesdessontheextoixsitciintgyogfuisdueblsitnaensce(s1t)o(2f)r(e3s)(h4w)a(t5e)r(6a)quabtuitcpilnacnltusdoesf
modificationsofthosemethodstoreflectrecentresearchandconsultationonanumberofkeyissues.The
proposedmethodhasbeenvalidatedbyaninternationalring-test(7).
h2.avebeenTheixsteGnusiidveellyinsetuddeisecdriabnesdtaorxeictihteystuebsjteicntgoufsitnhgeLsetamnndaargdsibrbeafearnreddLteomanbaovme.inoTrh,ebottahxoonfowmhyicohf
Lemnaspp.isdifficult,beingcomplicatedbytheexistenceofawiderangeofphenotypes. Although
goennetthiiscsvoaurriacbeiloiftyvairniatbhielirteystpoonrseecotommtoexnidcaantsspeccainfioccccluornewiftohrLuseemnwait,hthtehriseGauriedceulrirneen.tlIytisnhsouuflfidcibeentnodtaetda
thatthetestisnotconductedaxenicallybutstepsaretakenatstagesduringthetestproceduretokeep
contaminationbyotherorganismstoaminimum.
3. Detailsoftestingwithrenewal(semi-staticandflow-through)andwithoutrenewal(static)ofthe
testsolutionaredescribed. Dependingontheobjectivesofthetestandtheregulatoryrequirements,itis
recommendedtoconsidertheapplicationofsemi-staticandflowthroughmethods,e.g.forsubstancesthat
are rapidly lost from solution as aresult ofvolatilisation, photodegradation, precipitation or
biodegradation.Furtherguidanceisgivenin(8).
4. DefinitionsusedaregiveninAnnex1.
PRINCIPLEOFTHETEST
5. ExponentiallygrowingplantculturesofthegenusLemnaareallowedtogrowasmonoculturesin
differentconcentrationsofthetestsubstanceoveraperiodofsevendays.Theobjectiveofthetestisto
quantifysubstance-relatedeffectsonvegetativegrowthoverthisperiodbasedonassessmentsofselected
measurementvariables. Frondnumberistheprimarymeasurementvariable. Atleastoneother
measurementvariable(totalfrondarea,dryweightorfreshweight)isalsomeasured,sincesome
substancesmayaffectothermeasurementvariablesmuchmorethanfrondnumbers. Toquantify
csasounbtschteeanntEcrCea-tXri(eoel.nag.tberEdiCnge5fi0fn)egctasb,ougtroawstpheciinfietdhext%estinshoilbuittiioonnsofisgrcoowmtphar(ee.dg.w5i0th%)thiastdeotferthmeinceodntarnodlsexapnredsstehde
6. Thetestendpointisinhibitionofgrowth,expressedaslogarithmicincreaseinmeasurement
vraercioarbdleed(ianvearsaegreiesspeocfiftiecstgsroolwuttihonrsa,tet)hdeucroinncgentthreaetxipoonsburriengpienrgioad.bouFtraomsptehceifaiveedrxag%esipnehciibfiitciognroowftghrorawttehs
rate(e.g.50%)isdeterminedandexpressedastheErCx(e.g.ErC50).
7. AnadditionalresponsevariableusedinthisGuidelineisyield,whichmaybeneededtofulfil
specificregulatoryrequirementsinsomecountries.Itisdefinedasmeasurementvariablesattheendofthe
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221 OECD/OCDE
erexcpoorsduerdeipneraiosedrimeisnoufstetshtesmoleuatsiounrse,mtehnetcvoanrcieanbtlreastiaotntbhreinsgtairntgoafbotuhteaexsppoesciufrieedpexri%od.inhFibriotimonthoefyyiieelldd
(e.g.,50%)iscalculatedandexpressedastheEyCx(e.g.EyCso).
8. Inaddition,thelowestobservedeffectconcentration(LOEC)andthenoobservedeffect
concentration(NOEC)maybestatisticallydetermined.
INFORMATIONONTHETESTSUBSTANCE
9. Ananalyticalmethod,withadequatesensitivityforquantificationofthesubstanceinthetest
medium,shouldbeavailable.
tb1ih0oe.dsetgrruacdtauIbrniaflliotrfymo.armtuiWloaant,oepnrurtsihotelyu,tbeiwsltaitsteuyrbassntoadlnubcviealpiwtohyui,rcshptarmbeiaslsyiutrybeeincuaswneaftubeleriaunnsedesdtlaitbgolhtic,sahplicnKuglaat,thKee<,tHwees,ntvrcayop'nosduirLtiaporwnesscsiounnrcseltuaadnnetd,s
whichwillindicateifsignificantlossesofthetestsubstanceduringthetestperiodarelikely. Thiswill
helpindicatewhetherparticularstepstocontrolsuchlossesshouldbetaken. Whereinformationonthe
solubilityandstabilityofthetestsubstanceisuncertain,itisrecommendedthatthesebeassessedunderthe
conditionsofthetest,i.e.growthmedium,temperature,lightingregimetobeusedinthetest.
11. WhenpHcontrolofthetestmediumisparticularlyimportant,e.g.whentestingmetalsor
substanceswhicharehydrolyticallyunstable,theadditionofabuffertothegrowthmediumis
recommended(seeparagraph21). Furtherguidancefortestingsubstanceswithphysical-chemical
propertiesthatmakethemdifficulttotestisprovidedin(8).
VALIDITYOFTHETEST
12. Forthetesttobevalid,thedoublingtimeoffrondnumberinthecontrolmustbelessthan2.5
days(60h),correspondingtoapproximatelyaseven-foldincreaseinsevendaysandanaveragespecific
gberoawttthairnaetdeuosfi0n.g27a5stda’t1i.ctUesstinrgegtihmeeme(5d)i.aIatnidstaelsstocoanntdiictiipoantseddetshcartitbheidsicnrittheirsiGouniwdielllinbee,atchihsiecvraitbelreiounncdaenr
s1e4m.i-staticandflow-throughtestconditions.Calculationofthedoublingtimeisshowninparagraph49.
REFERENCESUBSTANCE
13. Referencesubstance(s),suchas3,5-dichlorophenolusedintheinternationalringtest(7),maybe
testedasameansofcheckingthetestprocedure.Itisadvisabletotestareferencesubstanceatleasttwice
ayearor,wheretestingiscarriedoutatalowerfrequency,inparalleltothedeterminationofthetoxicity
ofatestsubstance.
DESCRIPTIONOFTHEMETHOD
Apparatus
Allequipmentincontactwiththetestmediashouldbemadeofglassorotherchemicallyinert
material.Glasswareusedforculturingandtestingpurposesshouldbecleanedofchemicalcontaminants
thatmightleachintothetestmediumandshouldbesterile.Thetestvesselsshouldbewideenoughforthe
fdmroioennsidsmnouotmfmdvaiotfltfeuerrmeenitfoctfho1el0orn0oiometsLsitinontuehcaehcchtohtneetsrtboolvtevtseossemslseilossfatdtovhigesretode.swtwTvihetsehseocluhsto,iocbveuetrolfaatpempsiitnnvgiesmastuelmtshediseepnntodhtocorffitt2ihc0ealmteasmst.laonnIdgt
astheserequirementsaremet. Glassbeakers,crystallisingdishesorglasspetridishesofappropriate
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OECD/OCDE 221
dimensionshaveallprovedsuitable.Testvesselsmustbecoveredtominimiseevaporationandaccidental
contamination,whileallowingnecessaryairexchange.Suitabletestvessels,andparticularlycovers,must
avoidshadowingorchangesinthespectralcharacteristicsoflight.
15. Theculturesandtestvesselsshouldnotbekepttogether.Thisisbestachievedusingseparate
environmentalgrowthchambers,incubators,orrooms.Illuminationandtemperaturemustbecontrollable
andmaintainedataconstantlevel(seeparagraphs35-36).
Testorganism
16. TheorganismusedforthistestiseitherLemnagibbaorLemnaminor. Shortdescriptionsof
duckweedspeciesthathavebeenusedfortoxicitytestingaregiveninAnnex2. Plantmaterialmaybe
obtainedfromaculturecollection,anotherlaboratoryorfromthefield.Ifcollectedfromthefield,plants
shouldbemaintainedincultureinthesamemediumasusedfortestingforaminimumofeightweeksprior
touse.Fieldsitesusedforcollectingstartingculturesmustbefreeofobvioussourcesofcontamination.If
obtainedfromanotherlaboratoryoraculturecollectiontheyshouldbesimilarlymaintainedfora
minimumofthreeweeks. Thesourceofplantmaterialandthespeciesandclone(ifknown)usedfor
testingshouldalwaysbereported.
17. Monocultures,thatarevisiblyfreefromcontaminationbyotherorganismssuchasalgaeand
protozoa,shouldbeused.HealthyplantsofL.minorwillconsistofcoloniescomprisingbetweentwoand
fivefrondswhilsthealthycoloniesofL.gibbamaycontainuptosevenfronds.
o1u8.tcomeoTfhethqeuatleisttyaannddsuhnoiuflodrmtihteyreoffortehebeplasnetlsecutseeddwifotrhtchaeret.estYwoiullngh,avreapaidsliygngifriocwaintnginpfllaunetnscewiotnhotuhte
viinsciibdleencleesioofnscoolrondiiesscocloormaptriiosnin(gchlaotrolseiasst)tshwooulfdrobndes.useAd.laGrogoednuqumableitryocfulstiunrgelseafrreonidnsdicisatienddibcyataivheigohf
environmentalstress,e.g.nutrientlimitation,andplantmaterialfromsuchculturesshouldnotbeusedfor
testing.
Cultivation
19. Toreducethefrequencyofculturemaintenance(e.g.whennoLemnatestsareplannedfora
period),culturescanbeheldunderreducedilluminationandtemperature(4-10°C).Detailsofculturing
aregiveninAnnex3. Obvioussignsofcontaminationbyalgaeorotherorganismsmayrequiresurface
sterilisationofasub-sampleofLemnafronds,followedbytransfertofreshmedium(seeAnnex3).Inthis
eventualitytheremainingcontaminatedcultureshouldbediscarded.
20. Atleastsevendaysbeforetesting,sufficientcoloniesaretransferredasepticallyintofreshsterile
mediumandculturedfor7-10daysundertheconditionsofthetest.
Testmedium
21. DifferentmediaarerecommendedforLemnaminorandLemnagibba,asdescribedbelow.
CarefulconsiderationshouldbegiventotheinclusionofapHbufferinthetestmedium(MOPS(4-
morpholinepropanesulphonicacid,CASNo:1132-61-2)inL.minormediumandNaHC03inL.gibba
mtoexdiciiutym.)SwtheeinnbeitrigsMseusdpieucmte(d9)thiastailtsmoiagchcteprteaabctlewaisthlotnhgetaesstthseubvasltiadnicteycarnidteirinaflaureenmceett.heexpressionofits
22. AmodificationoftheSwedishstandard(SIS)Lemnagrowthmediumisrecommendedfor
culturingandtestingwithL.minor.ThecompositionofthismediumisgiveninAnnex4.
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221 OECD/OCDE
23. Thegrowthmedium,20X-AAP,asdescribedinAnnex4,isrecommendedforculturingand
testingwithL.gibba.
24. Steinbergmedium,asdescribedinAnnex4,isalsosuitableforL.minor,butmayalsobeused
forL.gibbaaslongasthevaliditycriteriaaremet.
Testsolutions
25. Testsolutionsareusuallypreparedbydilutionofastocksolution. Stocksolutionsofthetest
substancearenormallypreparedbydissolvingthesubstanceingrowthmedium.
26. Thehighesttestedconcentrationofthetestsubstanceshouldnotnormallyexceedthewater
solubilityofthesubstanceunderthetestconditions,itshouldbenotedhoweverthatLemnaspp.floaton
thesurfaceandmaybeexposedtosubstancesthatcollectatthewater-airinterface(e.g.poorlywater-
solubleorhydrophobicsubstancesorsurface-activesubstances).Undersuchcircumstancesexposurewill
resultfrommaterialotherthaninsolutionandtestconcentrationsmay,dependingonthecharacteristicsof
thetestsubstance,exceedwatersolubility. Fortestsubstancesoflowwatersolubilityitmaybenecessary
toprepareaconcentratedstocksolutionordispersionofthesubstanceusinganorganicsolventor
dispersantinordertofacilitatetheadditionofaccuratequantitiesofthetestsubstancetothetestmedium
andaidinitsdispersionanddissolution.Everyeffortshouldbemadetoavoidtheuseofsuchmaterials.
Thereshouldbenophytotoxicityresultingfromtheuseofauxiliarysolventsordispersants.Forexample,
commonlyusedsolventswhichdonotcausephytotoxicityatconcentrationsupto100pL/Linclude
acetoneanddimethylformamide.Ifasolventordispersantisused,itsfinalconcentrationshouldbe
reportedandkepttoaminimum(<100pL/L),andalltreatmentsandcontrolsshouldcontainthesame
concentrationofsolventordispersant.Furtherguidanceontheuseofdispersantsisgivenin(8).
Testandcontrolgroups
27. PriorknowledgeofthetoxicityofthetestsubstancetoLemna,e.g.fromarange-findingtest,will
helpinselectingsuitabletestconcentrations. Inthedefinitivetoxicitytest,thereshouldnormallybeat
leastfivetestconcentrationsarrangedinageometricseries.Preferablytheseparationfactorbetweentest
concentrationsshouldnotexceed3.2,butalargervaluemaybeusedwheretheconcentration-response
curveisflat. Justificationshouldbeprovidediffewerthanfiveconcentrationsareused.Atleastthree
replicatesshouldbeusedateachtestconcentration.
28. Insettingtherangeoftestconcentrations(forrange-findingand/orforthedefinitivetoxicity
test),thefollowingshouldbeconsidered:
• TapoprodpertieartmeinleevealnoEfCcXo,nfteisdtenccoen.centFroartieoxnasmpslheo,ulidfbersatcikmeattintghethEeCEXC5v0a,luethetohiegnhseusrteteasnt
coofntceesnttcroantcieonntrsahtoiuolnds,beasgsroecaitaetredthcaonnftihedeEnCce50ivnatelruvea.lsIfwitlhlebEeCl5a0rvgaelaunedliaesproouptseirdaesosfestshemernatngoef
thestatisticalfitofthemodelmaynotbepossible.
• IftheaimistoestimatetheLOEC/NOEC,thelowesttestconcentrationshouldbelowenough
sothatgrowthisnotsignificantlylessthanthatofthecontrol. Inaddition,thehighesttest
concentrationshouldbehighenoughsothatgrowthissignificantlylowerthanthatinthe
control.Ifthisisnotthecase,thetestwillhavetoberepeatedusingadifferentconcentration
range(unlessthehighestconcentrationisatthelimitofsolubilityorthemaximumrequired
limitconcentration,e.g.100mg/L).
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OECD/OCDE 221
29. Everytestshouldincludecontrolsconsistingofthesamenutrientmedium,numberoffrondsand
colonies,environmentalconditionsandproceduresasthetestvesselsbutwithoutthetestsubstance.Ifan
auxiliarysolventordispersantisused,anadditionalcontroltreatmentwiththesolvent/dispersantpresentat
thesameconcentrationasthatinthevesselswiththetestsubstanceshouldbeincluded.Thenumberof
replicatecontrolvessels(andsolventvessels,ifapplicable)shouldbeatleastequalto,andideallytwice,
thenumberofvesselsusedforeachtestconcentration.
30. IfdeterminationofNOECisnotrequired,thetestdesignmaybealteredtoincreasethenumber
ofconcentrationsandreducethenumberofreplicatesperconcentration.However,thenumberofcontrol
replicatesmustbeatleastthree.
Exposure
31. Coloniesconsistingof2to4visiblefrondsaretransferredfromtheinoculumcultureand
randomlyassignedtothetestvesselsunderasepticconditions.Eachtestvesselshouldcontainatotalof9
to12fronds. Thenumberoffrondsandcoloniesshouldbethesameineachtestvessel. Experience
gainedwiththismethodandring-testdatahaveindicatedthatusingthreereplicatespertreatment,with
eachreplicatecontaining9to12frondsinitially,issufficienttodetectdifferencesingrowthof
approximately4to7%ofinhibitioncalculatedbygrowthrate(10to15%calculatedbyyield)between
treatments(7).
i3n2.fluenceAofrsapantdioamlidsiefdfedreensciegsnifnorlilgohtcaitnitoennsoiftythoertteesmtpevreastseulrse.inAthbeloicnckuebdadteosriigsnroerquriarneddotmormeipnoismiitsioenitnhge
ofthevesselswhenobservationsaremade(orrepositioningmorefrequently)isalsorequired.
(3i3..e.themIefaasuprreeldimcionnacreyntsrtaatbiiolintyfatlelsstbsehloowws8th0at%thoeftetshtesmuebastsaunrceedcionnictieanltrcaotnicoenntcraantnioont)beovmearinttheaitneesdt
duration(7days),asemi-statictestregimeisrecommended.Inthiscase,thecoloniesshouldbeexposed
tofreshlypreparedtestandcontrolsolutionsonatleasttwooccasionsduringthetest(e.g.days3and5).
Thefrequencyofexposuretofreshmediumwilldependonthestabilityofthetestsubstance;ahigher
frequencymaybeneededtomaintainnear-constantconcentrationsofhighlyunstableorvolatile
substances.Insomecircumstances,aflow-throughproceduremayberequired(8)(10).
34. Theexposurescenariothroughafoliarapplication(spray)isnotcoveredinthisGuideline;
instead,see(11).
Incubationconditions
35. Continuouswarmorcoolwhitefluorescentlightingshouldbeusedtoprovidealightintensity
selectedfromtherangeof85-135pEm'V1whenmeasuredinaphotosyntheticallyactiveradiation(400-
700nm)atpointsthesamedistancefromthelightsourceastheLemnafronds(equivalentto6500-10000
lux).Anydifferencesfromtheselectedlightintensityoverthetestareashouldnotexceedtherangeof±
15%. Themethodoflightdetectionandmeasurement,inparticularthetypeofsensor,willaffectthe
measuredvalue.Sphericalsensors(whichrespondtolightfromallanglesaboveandbelowtheplaneof
measurement)and“cosine”sensors(whichrespondtolightfromallanglesabovetheplaneof
measurement)arepreferredtounidirectionalsensors,andwillgivehigherreadingsforamulti-pointlight
sourceofthetypedescribedhere.
36. Thetemperatureinthetestvesselsshouldbe24±2°C.ThepHofthecontrolmediumshould
notincreasebymorethan1.5unitsduringthetest.However,deviationofmorethan1.5unitswouldnot
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221 OECD/OCDE
invalidatethetestwhenitcanbeshownthatvaliditycriteriaaremet.AdditionalcareisneededonpHdrift
inspecialcasessuchaswhentestingunstablesubstancesormetals.See(8)forfurtherguidance.
Duration
37. Thetestisterminated7daysaftertheplantsaretransferredintothetestvessels.
Measurementsandanalyticaldeterminations
38. Atthestartofthetest,frondnumberinthetestvesselsiscountedandrecorded,takingcareto
ensurethatprotruding,distinctlyvisiblefrondsareaccountedfor. Frondnumbersappearingnormalor
abnormal,needtobedeterminedatthebeginningofthetest,atleastonceevery3daysduringtheexposure
period(i.e.onatleast2occasionsduringthe7dayperiod),andattesttermination. Changesinplant
development,e.g.infrondsize,appearance,indicationofnecrosis,chlorosisorgibbosity,colonybreak-up
orlossofbuoyancy,andinrootlengthandappearance,shouldbenoted.Significantfeaturesofthetest
medium(e.g.presenceofundissolvedmaterial,growthofalgaeinthetestvessel)shouldalsobenoted.
39. Inadditiontodeterminationsoffrondnumberduringthetest,effectsofthetestsubstanceonone
(ormore)ofthefollowingmeasurementvariablesarealsoassessed:
(i) totalfrondarea,
(ii) dryweight,
(iii) freshweight.
40. Totalfrondareahasanadvantage,inthatitcanbedeterminedforeachtestandcontrolvesselat
thestart,during,andattheendofthetest.Dryorfreshweightshouldbedeterminedatthestartofthetest
fromasampleoftheinoculumculturerepresentativeofwhatisusedtobeginthetest,andattheendofthe
testwiththeplantmaterialfromeachtestandcontrolvessel.Iffrondareaisnotmeasured,dryweightis
preferredoverfreshweight.
41. Totalfrondarea,dryweightandfreshweightmaybedeterminedasfollows:
(i) Taontaalylsifsr.onAdsairlehao:ueTthteeotfottahletfersotnvdesasreelaaonfdpalllanctoslconainebsemcaapytubreedduestienrgmianeviddeboyciammaegrea
(i.e.byplacingthevesselonalightbox)andtheresultingimagedigitised. By
calibrationwithflatshapesofknownarea,thetotalfrondareainatestvesselmaythen
bedetermined.Careshouldbetakentoexcludeinterferencecausedbytherimofthetest
vessel. Analternativebutmorelaboriousapproachistophotocopytestvesselsand
plants,cutouttheresultingsilhouetteofcoloniesanddeterminetheirareausingaleaf
areaanalyserorgraphpaper. Othertechniques(e.g.paperweightratiobetween
silhouetteareaofcoloniesandunitarea)mayalsobeappropriate.
(ii) Dryweight:Allcoloniesarecollectedfromeachofthetestvesselsandrinsedwith
distilledordeionisedwater.Theyareblottedtoremoveexcesswaterandthendriedat
60°Ctoaconstantweight. Anyrootfragmentsshouldbeincluded. Thedryweight
shouldbeexpressedtoanaccuracyofatleast0.1mg.
(iii) Fmraetserhiawle)igthutb:esAlwlitcholsomnailels(a1remmt)ranhsofleersredinttoheprre-owuenidegdhebdotptoolmyss.tyrTehnee(tourbeosthaerreitnheernt
centrifugedat3000rpmfor10minutesatroomtemperature.Tubes,containingthenow
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OECD/OCDE 221
driedcolonies,arere-weighedandthefreshweightiscalculatedbysubtractingthe
weightoftheemptytube.
Frequencyofmeasurementsandanalyticaldeterminations
4t2h.eendofIfthaesttaestti.ctIefstadseesmiig-nstiastuiscetde,stthdeespiHgnoifseuascehd,trtehaetpmeHntsshhoouullddbbeemmeeaassuurreeddiantetahcehbbeagticnhnionfg‘afnredsha’t
testsolutionpriortoeachrenewalandalsointhecorresponding‘spent’solutions.
43. Lightintensityshouldbemeasuredinthegrowthchamber,incubatororroomatpointsthesame
distancefromthelightsourceastheLemnafronds.Measurementsshouldbemadeatleastonceduringthe
test.Thetemperatureofthemediuminasurrogatevesselheldunderthesameconditionsinthegrowth
chamber,incubatororroomshouldberecordedatleastdaily.
44. Duringthetest,theconcentrationsofthetestsubstancearedeterminedatappropriateintervals.In
statictests,theminimumrequirementistodeterminetheconcentrationsatthebeginningandattheendof
thetest.
4±52.0%ofItnhseemnio-msitnatailcctoenstcsenwthraetrieont,heitcoisncneenctersastairoyntooftahnealtyesstesaullbsftraenschelyisprneoptaerxepdectetsetdstoolurteimoansinawnidthtihne
samesolutionsateachrenewal(seeparagraph33).However,forthosetestswherethemeasuredinitial
cpornocveindterdattioosnhoofwtthheattetshtesiunbitsitaalnccoencisenntortatwiiotnhsinar±e2r0ep%eaotafbnleomainndalstbaubtlew(hi.ee.rewistuhfifnictiheentraenvgiede8n0ce-c1a2n0b%e
oftheinitialconcentration),chemicaldeterminationsmaybecarriedoutononlythehighestandlowesttest
concentrations.Inallcases,determinationoftestsubstanceconcentrationspriortorenewalneedonlybe
performedononereplicatevesselateachtestconcentration(orthecontentsofthevesselspooledby
replicate).
46. Ifaflow-throughtestisused,asimilarsamplingregimetothatdescribedforsemi-statictests,
includinganalysisatthestart,mid-waythroughandattheendofthetest,isappropriate,butmeasurement
of‘spent’solutionsisnotappropriateinthiscase. Inthistypeoftest,theflow-rateofdiluentandtest
substanceortestsubstancestocksolutionshouldbecheckeddaily.
4m7a.intaineIdfwtihtehrieni±se2v0id%enocfetthheatnotmhiencaolncoernmtreaatsiuornedofintihtiealsucbosntcaennctreatbieoinngthtreosutegdhohuatstbheeetnests,atainsaflaycstiosriolyf
theresultscanbebasedonnominalormeasuredinitialvalues. Ifthedeviationfromthenominalor
measuredinitialconcentrationisnotwithin±20%,analysisoftheresultsshouldbebasedonthe
geometricmeanconcentrationduringexposureormodelsdescribingthedeclineoftheconcentrationofthe
testsubstance(8).
Limittest
48. Undersomecircumstances,e.g.whenapreliminarytestindicatesthatthetestsubstancehasno
toxiceffectsatconcentrationsupto100mg/Loruptoitslimitofsolubilityinthetestmedium(whichever
isthelower),alimittestinvolvingacomparisonofresponsesinacontrolgroupandonetreatmentgroup
(100mg/Loraconcentrationequaltothelimitofsolubility),maybeundertaken.Itisstrongly
recommendedthatthisbesupportedbyanalysisoftheexposureconcentration.Allpreviouslydescribed
testconditionsandvaliditycriteriaapplytoalimittest,withtheexceptionthatthenumberoftreatment
replicatesshouldbedoubled. Growthinthecontrolandtreatmentgroupmaybeanalysedusinga
statisticaltesttocomparemeans,e.g.aStudent'st-test.
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221 OECD/OCDE
DATAANDREPORTING
Doublingtime
4t9h.estudyT(poardaegtrearpmhin1e2)t,htehedofuoblllionwgintgimfeor(mTudl)aoifsfursoenddwniutmhbdeartaaonbdtaaidnheedrefnrcoemttohethciosntvraollidvietsyseclrsi:terionby
Td=In2/p
wherepistheaveragespecificgrowthratedeterminedasdescribedinparagraphs54-55.
Responsevariables
50. Thepurposeofthetestistodeterminetheeffectsofthetestsubstanceonthevegetativegrowth
ofLemna. ThisGuidelinedescribestworesponsevariables,asmembercountrieshavedifferent
preferencesandregulatoryneeds.Tnorderforthetestresultstobeacceptableinallmembercountries,the
effectsshouldbeevaluatedusingbothresponsevariables(a)and(b)describedbelow.
(a) Aivnetrhaegelosgpaerciitfihcmsgroofwtfhrornadten:utmhibserrse,spoannsdeivnariaadbdilteiiosn,caolcnultahteedbaosnisthoefbacshiasngoefschianngtehse
logarithmsofanothermeasurementparameter(totalfrondarea,dryweightorfresh
weight)overtime(expressedperday)inthecontrolsandeachtreatmentgroup. Itis
sometimesreferredtoasrelativegrowthrate(12).
(b) Yaidediltdi:otnh,isonretshpeonbsaseivsaorfiacbhlaenigsecsalicnualnatoetdheornmtehaesbuarseimseonftcphaarnagmeestienrf(rtootnadlnfuromnbdera,reaa,nddriyn
weightorfreshweight)inthecontrolsandineachtreatmentgroupuntiltheendofthe
test.
5cut1hop.emopntaersatavebcrlIoatengdseaihntosdiupoeltncdhsiifsbioecdfingftorfhtoeiesrwdetGnhtcuheiradatmteeutlosi(xtniEecrbCietaxyr)revewaicallodlughenegsiersneceeaddrlactwluolh,lyaetdnbeuedeusbhtiiyongghutesthrihenetghmraatenthsehursleeetmssautltowtifsoctabrhlaeessbetpaedossntiu.sspeoEovnfCaryXtiihaveeballldreuese(ssEpayerbCceatxs)inevoidetf
approaches. Thisshouldnotbeinterpretedasadifferenceinsensitivitybetweenthetworesponse
variables,simplythatthevaluesaredifferentmathematically.Theconceptofaveragespecificgrowthrate
isbasedonthegeneralexponentialgrowthpatternofduckweedinnon-limitedcultures,wheretoxicityis
estimatedonthebasisoftheeffectsonthegrowthrate,withoutbeingdependentontheabsolutelevelof
thespecificgrowthrateofthecontrol,slopeoftheconcentration-responsecurveorontestduration. In
cisondterapsetn,derenstulotsnbtahseesdpeucpiofinctgheroywitehldrraetespoofnstehevadruicabklweeaerdesdpeepceinedseunsteudpionneaalclhthteessteaotnhderonvatrhieablmeas.xiEmyuCmx
specificgrowthratethatcanvarybetweenspeciesandevendifferentclones. Thisresponsevariable
shouldnotbeusedforcomparingthesensitivitytotoxicantsamongduckweedspeciesorevendifferent
clones. Whiletheuseofaveragespecificgrowthrateforestimatingtoxicityisscientificallypreferred,
toxicityestimatesbasedonyieldarealsoincludedinthisGuidelinetosatisfycurrentregulatory
requirementsinsomecountries.
52. Toxicityestimatesshouldbebasedonfrondnumberandoneadditionalmeasurementvariable
(totalfrondarea,dryweightorfreshweight),becausesomesubstancesmayaffectothermeasurement
variablesmuchmorethanthefrondnumber. Thiseffectwouldnotbedetectedbycalculatingfrond
numberonly.
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OECD/OCDE 221
53. Thenumberoffrondsaswellasanyotherrecordedmeasurementvariable,i.e.totalfrondarea,
domrneyatshwueerivegamhletuneotsrfoofcrrceatsshhieowinen.idgiShvutid,busaaelrqeureetpnaltbiucdlaaattteaesdaanntaodlgynesotithsecrea.lgwc.iuttlhoatteehsdetimcmeoaantnceesantfLorraOtEeiaCocn,hsNtorOfeaEtthCmeenottresgtErCsouuXbpss.htoaunlcdebfeorbeaascehd
Averagespecificgrowthrate
54. Theaveragespecificgrowthrateforaspecificperiodiscalculatedasthelogarithmicincreasein
thegrowthvariables-frondnumbersandoneothermeasurementvariable(totalfrondarea,dryweightor
freshweight)-usingtheformulabelowforeachreplicateofcontrolandtreatments:
M1-J_ln(Ay)-tln(M>)
where:
tPNNi;j-j::::ammtiveemeaaerssauupgerrereeismmopeedenncttfifrvvioaacmrrgiiiaratbboollweejtihinnrttahhteeettfeerssottmoorrticcmooennttirrootloljvveesssseellaattttiimmeeji
Foreachtreatmentgroupandcontrolgroup,calculateameanvalueforgrowthratealongwithvariance
estimates.
55. Theaveragespecificgrowthrateshouldbecalculatedfortheentiretestperiod(time“i”inthe
aboveformulaisthebeginningofthetestandtime“j”istheendofthetest).Foreachtestconcentration
andcontrol,calculateameanvalueforaveragespecificgrowthratealongwiththevarianceestimates.In
addition,thesection-by-sectiongrowthrateshouldbeassessedinordertoevaluateeffectsofthetest
substanceoccurringduringtheexposureperiod(e.g.byinspectinglog-transformedgrowthcurves).
Substantialdifferencesbetweenthesection-by-sectiongrowthrateandtheaveragegrowthrateindicate
deviationfromconstantexponentialgrowthandthatcloseexaminationofthegrowthcurvesiswarranted.
Inthiscase,aconservativeapproachwouldbetocomparespecificgrowthratesfromtreatedcultures
duringthetimeperiodofmaximuminhibitiontothoseforcontrolsduringthesametimeperiod.
5g567r..oup)accPoerrdcienngttionhtihbeitfioolnloofwignrgofwotrhmuraltae:(Ir)maythenbecalculatedforeachtestconcentration(treatment
where:
pp%cTIr:::mmpeeearacnnenvvtaalliuuneheifbfoiortripponiininntthaheevetcrroaengattermoselpnetcigfriocugprowthrate
Yield
Effectsonyieldaredeterminedonthebasisoftwomeasurementvariables,frondnumberandone
othermeasurementvariable(totalfrondarea,dryweightorfreshweight)presentineachtestvesselatthe
startandattheendofthetest.Fordryweightorfreshweight,thestartingbiomassisdeterminedonthe
basisofasampleoffrondstakenfromthesamebatchusedtoinoculatethetestvessels(seeparagraph20).
Foreachtestconcentrationandcontrol,calculateameanvalueforyieldalongwithvarianceestimates.
Themeanpercentinhibitioninyield(%Iy)maybecalculatedforeachtreatmentgroupasfollows:
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)
221 OECD/OCDE
%/,=(,bcb-ebrx100
where:
58. bb%cTIy:::ffpiiennaarllcebbniitoommreaadssussctmmiiionnnuuissnyssittaaerrlttdiinnggbbiioommaassssfinortthheetcroenattrmoelntgrgoruopup
Plottingconcentration-responsecurves
Ipyloctatledc.ulatCeodncaesntsrhaotiwonn-irnepsapornasgeracpuhrv5e6sorerla5t7i)ngamndeatnhepelrocgenctoangceenitnrhaitbiiotnioonfofthtehetersetsspuobnssteanvacreiasbhloeu(lIdr,boer
ECXestimation
5ma9en.dasuyrieelmdeEsn(ttEiymvCaatxr)ei,sabeolafecth(hteootEfalCwXfhr(ioec.nghd.,asErheCoau,50l)ddsrhyionuwletdiugrbnhet,bbeaosrebdafsrueepsdhonuwpebioognthhtf)a.rvoenrdTahginesusmpibesceibrfeiccaangudrseoownttheherraeadtdeair(teiEortCneasxlt)
substancesthatimpactfrondnumberandothermeasurementvariablesdifferently. Thedesiredtoxicity
po(tarortfaarlmeesfthreowrnesdigaahrrtee)a.t,hderreyfwoereigfhotu,roErCfrXevsahlwueeisghfto)r;eEacyChxin(hfirbointdionnumlbeveerl);xacnaldcuElyaCtexd(:toEtarClxfr(ofnrdonadrenau,mdbreyr)w;eiEghrCt,x
Statisticalprocedures
60. Theaimistoobtainaquantitativeconcentration-responserelationshipbyregressionanalysis.It
ispossibletouseaweightedlinearregressionafterhavingperformedalinearisingtransformationofthe
responsedata,forinstanceintoprobitorlogitorWeibullunits(13),butnon-linearregressionprocedures
arepreferredtechniquesthatbetterhandleunavoidabledatairregularitiesanddeviationsfromsmooth
distributions. Approachingeitherzeroortotalinhibitionsuchirregularitiesmaybemagnifiedbythe
transformation,interferingwiththeanalysis(13). Itshouldbenotedthatstandardmethodsofanalysis
usingprobit,logit,orWeibulltransformsareintendedforuseonquantal(e.g.mortalityorsurvival)data,
andmustbemodifiedtoaccommodategrowthrateoryielddata.Specificproceduresfordeterminationof
ECXvaluesfromcontinuousdatacanbefoundin(14),(15),and(16).
61. Foreachresponsevariabletobeanalysed,usetheconcentration-responserelationshipto
csahlocuulldatbeepdoientteremsitniemda.tesGooofdnEeCsXsvoaflufeist.ofWthheenresppoosnsisbeled,attaheto9t5he%rceognrefsisdieonncemoldimeiltsshfooruledacbheaesssteimsasteed
eithergraphicallyorstatistically. Regressionanalysisshouldbeperformedusingindividualreplicate
responses,nottreatmentgroupmeans.
62. EC50estimatesandconfidencelimitsmayalsobeobtainedusinglinearinterpolationwith
bootstrapping(17),ifavailableregressionmodels/methodsareunsuitableforthedata.
u63s.inganalFyosriseosftivmaartiiaonnceof(AthNeOLVOAE)Cteacnhndiqhueensc.eTthheeNmOeaEnC,foirteiascnheccoenscseanrtyrattoiconommpuasrtethterneabtemecnotmmpeaarnesd
Wwiiltlhiatmhse’tceosnttrmoalymbeeaunsefuuslin(g18a)(n19a)p(p2r0o)p(r2i1a)t.eItmuilstniepcleesscaormyptaoriasssoenssowrhetrtehnedrttehsetAmNetOhVodA.aDsusnunmepttti’osnoorf
homogeneityofvarianceholds.Thisassessmentmaybeperformedgraphicallyorbyaformaltest(22).
SuitabletestsareLevene’sorBartlett’s.Failuretomeettheassumptionofhomogeneityofvariancescan
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