Test No. 232: Collembolan Reproduction Test in Soil [electronic resource]

2009·0.61 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 232
Adopted:
7September2009
OECDGUIDELINESFORTESTINGCHEMICALS
CollcinhoianReproductionTestinSoil
INTRODUCTION
t1h.ecollemTbhoilsanTsesitnGsouiil.deIltiinsebaisseddesoingneexidstfionrgapsrseoscseidnugretshe(1e)ff(e2c)t.sTohfecphaermtihceanlosgeonnettihceFroelpsroodmuicatiCvaendoiudtaputanodf
sexuallyreproducingFolsomiafimetariaaretwoofthemostaccessiblespeciesofCollembola,andtheyare
culturableandcommerciallyavailable.Whenspecifichabitatsnotcoveredbythetwospeciesneedtobe
assessedtheprocedureisextensiblealsotootherspeciesofCollembolaiftheyareabletofulfilthevalidity
criteriaofthetest.
2. Soil-dwelling Collembola are ecologically relevant species forecotoxicological testing.
Collembolansarehexapodswithathinexoskeletonhighlypermeabletoairandwater,andrepresent
arthropodspecieswithadifferentrouteandadifferentrateofexposurecomparedtoearthwormsand
enchytraeids.
3. PopulationdensitiesofCollembolacommonlyreach105m2insoilandleaflitterlayersinmany
tbeiroremsatsrisalaencdosryesstpiermasti(o3n)i(s4).loAwd,uletsstitmyapticeadllbyemtewaeseunre1%0.5an-d55mm%.(t5)h]e.irTchoenitrrimboutsitonitmopotrottaalntsoirlolaenimmaayl
thereforebeaspotentialregulatorsofprocessesthroughmicrobivoryandmicrofaunapredation.Springtails
arepreyanimalsforawidevarietyofendogeicandepigeicinvertebrates,suchasmites,centipedes,
spiders,Carabidaeandrovebeetles.Collembolacontributetodecompositionprocessesinacidicsoils
wheretheymaybethemostimportantsoilinvertebratesbesidesenchytraeids,sinceearthwormsand
diplopodsaretypicallyabsent.
4. F.fimetariahasaworldwidedistributionandiscommoninseveralsoiltypesrangingfromsandy
toloamysoilsandfrommulltomorsoils.Itisaneyeless,unpigmentedcollembolan.Ithasbeenrecorded
inagriculturalsoilsalloverEurope(6).Ithasanomnivorousfeedinghabit,includingfungalhyphae,
bacteria,protozoaanddetritusinitsfood.Itinteractsthroughgrazingwithinfectionsofplantpathogenic
fungi(7)andmayinfluencemycorrhiza.asisknowntobethecaseforF.Candida.Asmostcollembolan
speciesitreproducessexuallyrequiringthepermanentpresenceofmalesforeggfertilization.
5o.ccursinFv.erCyanhdiigdhaniusmablesorsdiisntrhiubumtuesdwroirchldswiitedse..ItAilsthaonuegyhelietsiss,nuontpciogmmemnotnedincmololsetmbnoatluarna.lIstoihlass,iatwoeflteln-
developedfurca(jumpingorgan)andanactiverunningmovementandjumpsreadilyifdisturbed.The
ecologicalroleofF.CandidaissimilartotheroleofF.fimetaria.butthehabitatsaremoreorganicrichsoils.
Itreproducesparthenogenetically.Malesmayoccuratlessthan1perthousand.
©OECD,(2009)
tYpheoerumOiaEsrsCeiDfo,rneefprrtooomvuitsdheeedtOhtihEseCmDsao.tuerricaelifsordupleyrsmoennatli,onneodn.-cAonmymecrocmimaelrcpiuarlpousseesowfitthhiosutmasteeerkiianlgisprsiuobrjcecotnsteonwtriftrtoemn
232 OECD/OCDE
PRINCIPLEOFTHETEST
6c.oncentratSiyonnschorfonthoeusteastdusltub(sFt.anfciememtairxiead)ionrtojuavemnoidliefi(Fe.dCOaEndCiDda)artCioflicliealmbsoolilauasriengexapo5s%edotrogaanircanmgaettoerf
content(oranalternativesoil)(8).Thetestscenariocanbedividedintotwosteps:
-Arange-findingtest,incasenosufficientinformationontoxicityisavailable,inwhichmortalityand
reproductionarethemainendpointsassessedafter2weeksforF.fimetariaand3weeksforF.
Candida
-Adefinitivereproductiontestinwhichthetotalnumberofjuvenilesproducedbyparentanimalsand
thesurvivalofparentanimalsareassessed.Thedurationofthisdefinitivetestis3weeksforF.
fimetariaor4weeksforF.Candida.
Thetoxiceffectofthetestsubstanceonadultmortality'andreproductiveoutputisexpressedasLCXandECX
bcayusfiettxing%thmeordtaatlaittyoaornraepdpurcotpirioanteinmroedperlodbuyctinvoen-louitnpeuatr,rreegsrpeescstiiovneltyo,eosrtiamlatteerntahteivceolyncaesnttrhaetiNonOEthCat/LwoOuElCd
value(9).
INFORMATIONONTHETESTSUBSTANCE
7. Thephysicalproperties,watersolubility,thelogKow,thesoilwaterpartitioncoefficientandthe
vapourpressureofthetestsubstanceshouldpreferablybeknown.Additionalinfonnationonthefateofthe
tCehsetmsiucbasltaindceentiinfiscoaitli,onsuocfhthaesttehsetrsautbesstaonfcpehoatcocloyrsdiisngantodIhyUdProAlCysniosmeanncdlabitoutriec,dCeAgrSa-dnautimobne,r,isbdaetscihr,ablloet.,
structuralformulaandpurity'shouldbedocumentedwhenavailable.
8. ThisGuidelinecanbeusedforwatersolubleorinsolublesubstances.However,themodeof
applicationofthetestsubstancewilldifferaccordingly.TheGuidelineisnotapplicabletovolatile
substances,i.e.substancesforwhichtheHenry'sconstantortheair/waterpartitioncoefficientisgreater
thanone,orsubstancesforwhichthevapourpressureexceeds0.0133Paat25°C.
VALIDITYOFTHETEST
9. Thefollowingcriteriashouldbesatisfiedintheuntreatedcontrolsforatestresulttobe
consideredvalid:
Meanadultmortalityshouldnotexceed20%attheendofthetest;
Themeannumberofjuvenilespervesselshouldbeatleast100attheendofthetest;
Thecoefficientofvariationcalculatedforthenumberofjuvenilesshouldbelessthan30%atthe
endofthedefinitivetest.
REFERENCESUBSTANCE
10. AreferencesubstanceshouldbetestedatitsEC50concentrationforthechosentestsoiltype
eoirtghaenrisatmsreignutlhaertiesnttesryvasltsemorarpeorsessipbloyndiinncglwuidtehdinintheeancohrmteasltlreuvnel.toAvseuriitfayblteharteftehreencreesspuobnssteanocfetihsebotreisct
acid,whichshouldreducereproductionby50%(10)(11)atabout100mg/kgdryweightsoilforboth
species.
©OCDE,(2009) 2
OECD/OCDE 232
DESCRIPTIONOFTHETEST
Testvesselsandequipment
11. Containerscapableofholding30gofmoistsoilaresuitabletestvessels.Thematerialshouldeither
beglassorinertplastic(non-toxic).However,usingplasticcontainersshouldbeavoidedifthetestsubstance
exposureisdecreasedduetosorption.Thetestvesselsshouldhaveacross-sectionalareaallowingtheactual
soildepthwithinthetestvesseltobe2-4cm.Thevesselsshouldhavelids(e.g.glassorpolyethylene)thatare
designedtoreducewaterevaporationwhilstallowinggasexchangebetweenthesoilandtheatmosphere.The
containershouldbeatleastpartlytransparenttoallowlighttransmission.
12. Normallaboratoryequipmentisrequired,specificallythefollowing:
dryingcabinet;
stereomicroscope;
pH-meterandluxmeter;
suitableaccuratebalances;
adequateequipmentfortemperaturecontrol:
adequateequipmentforairhumiditycontrol(notessentialifexposurevesselsarecoveredby
lids);
temperature-controlledincubatororsmallroom;
forcepsoralow'-suctionairflowdevice.
Preparationofthetestsoil
13. AmodifiedOECDartificialsoil(8)isusedwithanorganicmattercontentof5%.Alternativelya
naturalsoilcouldbeused,astheartificialsoildoesnotresemblenaturalsoils.Therecommendedcomposition
oftheartificialsoilisasfollows(basedondryweights,driedtoaconstantweightat105°C):
250%%skpahoalginnucmlapyea(tk,aoaliirn-idtreiecdonatnedntfipnreelfyergarboluynadb(oavpear3t0i%cl)e;sizeof2±1mmisacceptable):
- parpepdrooxmiimnaatnetllyy7fi4ne%saainrd-dwriietdhimnodursetrtihalans5an0d%(odefptehnedpianrgtiocnletshbeeatmwoeuennt5o0faCndaC02300neemdiecdr)o,ns.
Tshhoeuledxaacdtdaumpoutnot75of%s.anddependsontheamountofCaC03(see below), together they
- t<he1.a0m%oucnatlcoifucmaclacribuomnactaerb(oCnaatCe0t3o,pbuelvaedrdiesdedm,aaynaldyetpiecanldgprraidnec)iptaolloybtoaninthaepHoqufa6l.i0ty/±natu0r.e5;
ofthepeat(seeNote1).
Ndeotteerm1i:neTdhbeyammeoausnutrionfgCtaheC0pH3roefqupirree-dinwciullbadteepdemnodisotnstohielcsuobm-psoanmepnltessoifmtmheedisaoitlelsyubbsetrfaotreeatnhedtsehsto.uldbe
Note2:ItisrecommendedtomeasurethepHandoptionallytheC/Nratio.CationExchangeCapacity(CEC)
andorganicmattercontentofthesoilinordertoenableanormalisationatalaterstageandtobetterinterpret
theresults.
Note3:Ifrequired,e.g.forspecifictestingpurposes,naturalsoilsfromunpollutedsitesmayalsoserveas
t(ecsotllaencdt/ioorncsiutlet)u,rpeHs,ubtsetxrtautree.H(poawretivceler,siizfendaitsutrrailbustoiioln)i.suCsEedC,aitndshoorugladnibcemcahtatrearctceornitseendtatanldeaisttsbhyouolrdigbien
freefromanycontamination.Fornaturalsoilitisadvisabletodemonstrateitssuitabilityforatestandfor
achievingthetestvaliditycriteriabeforeusingthesoilinadefinitivetest.
©OCDE,(2009)
232 OECD/OCDE
14. Thedryconstituentsofthesoilaremixedthoroughly(e.g.inalarge-scalelaboratorymixer).The
maximumwaterholdingcapacity(WHC)oftheartificialsoilisdeterminedinaccordancewithprocedures
describedinAnnex5.Themoisturecontentofthetestingsoilshouldbeoptimisedtoattainalooseporous
smoailxismtruumctuWrHeCa.llowingcollembolanstoenterintothepores.Thisisusuallybetween40-60%ofthe
15. Thedryartificialsoilispre-moistenedbyaddingenoughde-ionisedwatertoobtainapproximately
hdsaeolltufetroimfointnhaietnifaoinn1ao:l5fwrpaatHteiroaicmsoinuxtsteeundrte(2ao-cf7csodoraidlyisanngbdetfoo1rAMentnpheoexttae6ss)st.isIutfmartthc,ehilsnooinolrddciesr(mKtoCor1ee)quaoicrliid0bi.rc0a1tteh/Masntactbaihlleicsrieeuqtmuhiecrhaelcdoirdriiadtnyeg.e(,FCoiatrCctlah2ne)
beadjustedbyadditionofanappropriateamountofCaCOj.Ifthesoilistooalkalineitcanbeadjustedbythe
additionofaninorganicacidhannlesstocollembolans.
16. The pre-moistened soil is dividedintoportionscorrespondingtothenumberoftest
concentrations(andreferencesubstancewhereappropriate)andcontrolsusedforthetest.Thetest
compoundsareaddedandthewatercontentisregulatedaccordingtotheparagraph24.
Selectionandpreparationoftestanimals
17. TheparthenogeneticF.Candidaistherecommendedspecies,asintheringtestingofthemethod
i(s1u1s)etdh,isitsspheocuiledsmmeeetttthheevvaalliiddiittyyccrriitteerriiaaofuotrlisnuerdviivnaplamroagrreapohft9e.nAtthatnheF.stfaritmeotfatrhiea.tesItftahneaalntiemranaltsivsehosupledcibees
wellfedandtheagebetween23-26daysforF.fimetariaand9-12daysforF.Candida.Foreachreplicate,the
numberofF.fimetariashouldbe10malesand10females,andforF.Candida10femalesshouldbeused(see
Annex2andAnnex3).Thesynchronousanimalsareselectedrandomlyfromthedishesandtheirhealthand
physicalconditionischeckedforeachbatchaddedtoareplicate.Eachgroupof10/20individualsisaddedtoa
randomlyselectedtestcontainerandthebigfemalesofF.fimetariaareselectedtoensureaproperdistinction
fromtheF.fimetariamales.
Preparationoftestconcentrations
18. Fourmethodsofapplicationofthetestsubstancecanbeused:1)mixingthetestsubstanceinto
thesoilwithwaterasacarrier,2)mixingthetestsubstanceintothesoilwithanorganicsolventasa
carrier,3)mixingthetestsubstanceintothesoilwithsandasacarrier,or4)applicationofthetest
substanceontothesoilsurface.Theselectionoftheappropriatemethoddependsonthecharacteristicof
thecompoundandthepurposeofthetest.Ingeneral,mixingofthetestsubstanceintothesoilis
recommended.However,applicationproceduresthatareconsistentwiththepracticaluseofthetest
substancemayberequired(e.g.sprayingofliquidformulationoruseofspecialpesticideformulationssuch
asgranulesorseeddressings).Thesoilistreatedbeforethecollembolansareadded,exceptwhenthetest
compoundisaddedtothesoilsurfacecollembolansshouldbeallowedtoenterthesoil.
Testsubstancesolubleinwater
19. Asolutionofthetestsubstanceispreparedindeionisedwaterinaquantitysufficientforall
replicatesofonetestconcentration.Eachsolutionoftestsubstanceismixedthoroughlywithonebatchofpre-
moistenedsoilbeforebeingintroducedintothetestvessel.
©OCDE,(2009) 4
OECD/OCDE 232
Testsubstanceinsolubleinwater
20. Forchemicalsinsolubleinwater,butsolubleinorganicsolvents,thetestsubstancecanbedissolved
inthesmallestpossiblevolumeofasuitablesolvent(e.g.acetone)stillensuringpropermixingofthechemical
Winhtehnesaoniloargnadnimcixsionlgveinttwiisthusaedp,oratlliotnesotfctohneceqnutarrattziosnasndanrdeqaunireadd.ditOinolnyalvosloaltvielnetsnoelgvaenttisveshcoountlrdolbeshuosueldd.
containthesameminimumamountofthesolvent.Applicationcontainersshouldbeleftuncoveredfora
certainperiodtoallowthesolventassociatedwiththeapplicationofthetestsubstancetoevaporate,
ensuringnodissipationofthetoxiccompoundduringthistime.
Testsubstancepoorlysolubleinwaterandorganicsolvents
21. Forsubstancesthatarepoorlysolubleinwaterandorganicsolvents,quartzsand,whichshouldbea
partofthetotalsandaddedtothesoil,ismixedwiththequantityoftestsubstancetoobtainthedesiredtest
concentration.Thismixtureofquartzsandandtestsubstanceisaddedtothepre-moistenedsoilandthoroughly
mixedafteraddinganappropriateamountofdeionisedwatertoobtaintherequiredmoisturecontent.Thefinal
mixtureisdividedbetweenthetestvessels.Theprocedureisrepeatedforeachtestconcentrationandan
appropriatecontrolisalsoprepared.
Applicationofthetestsubstanceontothesoilsurface
22. Whenthetestsubstanceisapesticide,itmaybeappropriatetoapplyitontothesoilsurfaceby
spraying.Thesoilistreatedafterthecollembolansareadded.Thetestcontainersarefirstfilledwiththe
moistenedsoilsubstrate,andtheanimalsaddedandthenthetestcontainersareweighted.Inordertoavoid
anydirectexposureoftheanimalswiththetestsubstancebydirectcontact,thetestsubstanceisappliedat
leasthalfanhourafterintroducingtheCollembola.Thetestsubstanceshouldbeappliedtothesurfaceof
thesoilasevenlyaspossibleusingasuitablelaboratory-scalesprayingdevicetosimulatesprayapplication
inthefield.Theapplicationshouldtakeplaceatatemperaturewithin±2°Cofvariationandforaqueous
solutions,emulsionsordispersionsatawaterapplicationrateaccordingtotheriskassessment
recommendations.Therateshouldbeverifiedusinganappropriatecalibrationtechnique. Special
formulationslikegranulesorseeddressingscouldbeappliedinamannerconsistentwithagriculturaluse.
Foodisaddedafterspraying.
PROCEDURE
Testconditions
23. Thetestmeantemperatureshouldbe20±1°Cwithatemperaturerangeof20±2°C.Thetestis
carriedoutundercontrolledlight-darkcycles(preferably12hourslightand12hoursdark)with
illuminationof400to800luxintheareaofthetestvessels.
t2h4.eendofInthoerdteesrt.toWecihgehctktlhosesso>il2h%umiisdrietpyl,etnhieshveedssbeylsthaereadwdeiitgihonedofatdet-heiobneigsiendnwiantge,r.inItthsehomuildddlbeeannodteadt
thatlossofwatercanbereducedbymaintainingahighair-humidity(>80%)inthetestincubator.
2de5f.initiveTtheset.pMHeasshuourledmebnetsmeashsouurleddbaettmhaedbeegiinnnoinnegeaxntdrathceonetnrodlosfabmoptlhetahnedraonngee-efxitnrdainsgatmepslteanodftthhee
treated(allconcentrations)soilsamplespreparedandmaintainedinthesamewayasthetestcultures,but
withoutadditionofthecollembolans.
5 ©OCDE,(2009)
232 OECD/OCDE
Testprocedureandmeasurements
26. Foreachtestconcentration,anamountoftestsoilcorrespondingto30gfreshweightisplaced
intothetestvessel.Watercontrols,withoutthetestsubstance,arealsoprepared.Ifavehicleisusedfor
applicationofthetestsubstance,onecontrolseriescontainingthevehiclealoneshouldberuninaddition
tothetestseries.Thesolventordispersantconcentrationshouldbethesameasthatusedinthetestvessels
containingthetestsubstance.
27. Theindividualspringtailsarecarefullytransferredintoeachtestvessel(allocatedrandomlytothe
testvessels)andplacedontothesurfaceofthesoil.Forefficienttransferoftheanimals,alow-suctionair
flowdevicecanbeused.Thenumberofreplicatesfortestconcentrationsandforcontrolsdependsonthe
testdesignused.Thetestvesselsarepositionedrandomlyinthetestincubatorandthesepositionsarere-
randomizedweekly.
t2e8s.t-vessel.FoOrnthdeaFy.2f1lmceotalrlieambtoelstantsweanrteyeaxdturlatcst,ed10frmoamletsheansdoil10anfdemcaoluenst,ed2.3-F2o6rdFa.yfslmoeldtasrhioautlhdebgeeunsdeedrpaerer
discriminatedbysizeinthesynchronisedanimalbatchusedforthetest.Femalesaredistinctivelylargerthan
themales(SeeAnnex3)
29. FortheF.Candidatest,ten9-12daysoldjuvenilespertestvesselshouldbeused.Onday28,the
collembolansareextractedfromthesoilandcounted.
30. Asasuitablefoodsource,asufficientamount,e.g.2-10mg,ofgranulateddriedbaker'syeast,
commerciallyavailableforhouseholduse,isaddedtoeachcontaineratthebeginningofthetestandafter
about2weeks.
w31e.eks(F.AtCatnhdeiedan)d,ofcotlhleetmebsto,lamonrstaalrietyexatnrdacrteepdrofdurcotmiotnhearteesatssseosisled(.seAeftAernn3ewxee4k)san(Fd.fclomuenttaerdia)(12a)n.dA4
collembolanisrecordedasdeadifnotpresentintheextraction.Theextractionandcountingmethodshould
bevalidated.Thevalidityincludesextractionefficiencyofjuvenilesgreaterthan95%,e.g.byaddinga
knownnumbertosoil.
32. PracticalsummaryandtimetableofthetestprocedurearedescribedinAnnex2.
Testdesign
Range-findingtest
33. Whennecessary,arange-findingtestisconductedwith,forexample,fivetestsubstance
concentrationsof0,1.1.0,10,100,and1000mg/kgdry'weightofsoilandtworeplicatesforeachtreatment
andcontrol.Additionalinformation,fromtestswithsimilarcompoundsorfromliterature,onmortalityor
reproductionofCollembolamayalsobeusefulindecidingontherangeofconcentrationstobeusedinthe
range-findingtest.
34. Thedurationoftherange-findingtestistwoweeksforF.flmetariaand3weeksforF.Candidato
ensureoneclutchofjuvenileshasbeenproduced.Attheendofthetest,mortalityandreproductionofthe
Collembolaareassessed.Thenumberofadultsandtheoccurrenceofjuvenilesshouldberecorded.
©OCDE,(2009) 6
OECD/OCDE 232
Definitivetest
t35w.oreplicFaotresdeftoerrmeiancahtitoenstofcotnhceenEtCraXt(ieo.ng.tErCeiaot,meEnCt5a0)n,dtwseilxvecocnotnrcoelntrreapltiicoantsesshaorueldrebceotmemsetendd.eAdt.lTeahset
spacingfactormayvarydependingonthedose-responsepattern.
36. FordeterminationoftheNOEC/LOEC,atleastfiveconcentrationsinageometricseriesshould
betested.Fourreplicatesforeachtestconcentrationtreatmentpluseightcontrolsarerecommended.The
concentrationsshouldbespacedbyafactornotexceeding1.8.
37. AcombinedapproachallowsfordeterminationofboththeNOEC/LOECandECx.Forthis
combinedapproach,eighttreatmentconcentrationsinageometricseriesshouldbeused.Fourreplicates
foreachtreatmentpluseightcontrolsarerecommended.Theconcentrationsshouldbespacedbyafactor
notexceeding1.8.
38. Ifnoeffectsareobservedatthehighestconcentrationintherange-findingtest(i.e.1000mg/kg),
tchonetrroelp.roAduclitmiiotntteessttwcialnlbperovpiedrefotrhmeedopapsoratulniimtiyttteostd,eumsoinnsgtraatteestthcaotnctheenrteraitsionnoosfta1ti0s0t0icamlgly/ksgignainfdictahnet
effectatthelimitconcentration.Eightreplicatesshouldbeusedforboththetreatedsoilandthecontrol.
DATAANDREPORTING
Treatmentofresults
v3tee9ss.ts,elo)r.WTihlTlehieastmarste’ipsrttoiecsdtau.lc9tani5av%leyscoiousnt,fpeiu.dtge.nisAcetNhiOentVemraAvianlpsroeacnrededpcuoarilencstu,l(aceto.egmd.paftorhreeisnntdurimevbiadetumraelnotftsrjebuayvtemSnetinultedsemneptarnto-std.eustc,edDupnenrettte’sst
40. Thenumberofsurvivingadultsintheuntreatedcontrolsisamajorvaliditycriterionandshould
bedocumented.Asintherange-findingtest,allotherharmfulsignsshouldbereportedinthefinalreport
aswell.
LCxandECx
aK4r1aer.bcearlcmuelEatCthxeod-dv,uasloiurnegss,iampippnlrceolpuirdniitanetgreptoshlteaaittriiosantsi)sc.aolcAimnaetteEdhCoxldosiwse(oreb.gta.anidlnoeugdipsptbeiyrci9onr5sWe%reticibonugnlfalidvfeaunlncuceetilcoinom,rirttsersfipomornmtdehidengSppatreoaamxrem%taenro-,f
thecontrolmeanintotheequationfound.TocomputetheECsooranyotherECx,thecompletedataset
shouldbesubjectedtoregressionanalysis.LC50isusuallyestimatedbyprobitanalysisorsimilaranalysis
thattakesintoaccountthebinomiallydistributedmortalitydata.
NOEC/LOEC
42. IfastatisticalanalysisisintendedtodeterminetheNOEC/LOEC,pcr-vessclstatistics(individual
vtoessOeElsCaDreDcoocnsuimdeenrted5r4eploincatthese)CaurrerneenctesAspaprry.oaAcphpersopirniattheestSattaitsitsitciaclalmeAtnhaolydssisshooufldEcboetouxsiceidtyaccDaotrad:inga
GinuviedsatnigcaetetdouAspipnlgicoante-itoanil(e9)d.hIynpogtehneesrials,teasdtvienrgsaetepff<ec0t.s05o.fthetestsubstancecomparedtothecontrolare
43. Normal distribution and vari7ancehomogeneitycanbetestedusing©aOnCDaEpp,ro(p2r0i0a9t)e
232 OECD/OCDE
Vstaartiisatniccael(teAstN,OeVgA.)thaendShsaupbisreoq-uWeinltkmtuelsttia-ncdompLaerviesnoentteesstt,srceasnpebcetipveelryfo(rpmie0d..05M)u.ltipOlnee-cwomapyarAinsaolnyssi(se.og.f
Dunnctt'stest)orstep-dor™trendtests(e.g.Williams'test)canbeusedtocalculatewhetherthereare
msd(iseegettnlehierofcmitdicisnaonne(teto.hdgfe.iftNBfheeoOrnEefrneCecrcerasoonmnd(imp-etUhn<-edteeL0sdO.t0E5tCa)ecs.tcboeartcdwcieonregdnitnotghHetoolcomOntEorrColDJsonaDcnokdchuetemhreeen-vtTaerr5ip4osut(sr9)at)e.tsrtOetnshduebrtsewtsitas)nec,ceonucolondn-cpbeaenrtaurmsaetetidrointcso
Limittest
44. Ifalimittest(comparisonofcontrolandonetreatmentonly)hasbeenperformedandthe
prerequisitesofparametrictestprocedures(normality,homogeneity)arefulfilled,metricresponsescanbe
evaluatedbytheStudenttest(t-test).Theunequal-variancet-test(Welcht-test)oranonparametrictest,
suchastheMann-Whitney-U-testmaybeused,iftheserequirementsarenotfulfilled.
45. Todeterminesignificantdifferencesbetweenthecontrols(controlandsolventcontrol),the
replicatesofeachcontrolcanbetestedasdescribedforthelimittest.Ifthesetestsdonotdetectsignificant
differences,allcontrolandsolventcontrolreplicatesmaybepooled.Otherwisealltreatmentsshouldbe
comparedwiththesolventcontrol.
Testreport
46. Thetestreportshouldatleastincludethefollowinginformation:
Testsubstance
theidentityofthetestsubstance,batch,lotandCAS-number,purity;
- physico-chemicalpropertiesofthetestsubstance(e.g.logKow,watersolubility,vapourpressure,
Henry’sconstant(H)andpreferablyinfomiationonthefateofthetestsubstanceinsoil)ifavailable;
- theformulationofthetestsubstanceandtheadditivesshouldbespecifiedifnotthepurechemicalis
tested:
Testorganisms
identificationofspeciesandsupplierofthetestorganisms,descriptionofthebreedingconditions
andagerangeoftestorganisms;
Testconditions
descriptionoftheexperimentaldesignandprocedure:
preparationdetailsforthetestsoil;detailedspecificationifnaturalsoilisused(origin,history,particle
sizedistribution,pH.organicmattercontent);
waterholdingcapacityofthesoil;
descriptionofthetechniqueusedtoapplythetestsubstancetothesoil;
testconditions:lightintensity,durationoflight-darkcycles,temperature;
apHdeascnrdipwtaitoenrocfonthteenfteeodfitnhgerseogilimaet,thtehesttayrpteaannddeandmooufnttheoftefsoto(dcounsterdolinantdheetaecsth,treaftmeeendti)n;gdates;
detaileddescriptionoftheextractionmethodandextractionefficiency;
©OCDE,(2009) 8
OECD/OCDE 232
Testresults
thenumberofjuvenilesdeterminedineachtestvesselattheendofthetest;
numberofadultsandtheirmortality(%)ineachtestvesselattheendofthetest;
adescriptionofobviousphysiologicalorpathologicalsymptomsordistinctchangesinbehaviour;
theresultsobtainedwiththereferencetestsubstance;
ETChieo)NOtoEgCet/hLerOEwiCthva9lu5e%s,cLonCfXidfoerncmeoritnatleirtvyalas.ndAEgCrXapfohrorfeptrhoedufictttieodnm(omdoesltluysLedC5f0o,rLCcialoc,ulEaCti5o0n,,anitds
functionequationanditsparameters(See(9));
allinformationandobservationshelpfulfortheinterpretationoftheresults;
poweroftheactualtestifhypothesistestingisdone(9);
deviationsfromproceduresdescribedinthisTestGuidelineandanyunusualoccurrencesduring
thetest;
validityofthetest;
forNOEC,whenestimated,theminimaldetectabledifference.
9 ©OCDE,(2009)
.
232 OECD/OCDE
LITERATURE
(1) WH&ialnSeodsnbsoJ,oALktado.nf,dsCoKhirilcoihgnehvsetPretHreb(ra1t9e98t)oxTiceisttyintgestwsit(hed.thHecLoolklkeembaonldanCsAI.MviVriadins,GeFs.teCla),ndpipd.a1a3n1d-1F5.6.fmJeothanriWai.leIyn
(2) IdeStOer(m1in9a9t9i)onSoiolfQeuffaelcittsyo-nEfrfeepcrtosduocftiosoni.lNpool.lut1a1n2t6s7.onInCteorlnlaetmiobnoallaO(rFgoalnsiosmaitaionCanfodridSat)a:ndMaertdihsoadtiofno,r
Geneve
(3) BurgesAandRawF(Eds)(1967)SoilBiology.AcademicPress.London
(4) PetersenHandLuxtonM(1982)Acomparativeanalysisofsoilfaunapopulationsandtheirrolein
decompositionprocesses.Oikos39:287-388
(5) PetersenH(1994)Areviewofcollembolanecologyinecosystemcontext.ActaZoologicaFennica195:
111-118
(6) H(oIpSkBiNn0-S1P9-(815949078)4.-1B)iologyoftheSpringtails(Insecta:Collembola) OxfordUniversityPress.330pp
(7) UlberB(1983)EinflussvonOnychirurusfimatusGisin(Collembola,Onychiuridae)undFolsomia
fmetariaL.(Collembola,Isotomidae)aufPythiumultimumTrow,einenErregerdesWurzelbrandesder
ZGuc(kEedrsr)i,ibPer.ocIeneNdienwgstroefntdhseiVnI.soiilntBeironaltoigoyna(lLceoblrluonqPuhi.umAnodnrseoiHlMz,oolDoegyM.eLdotusvaAi,n-Glrae-gnociurvee-W(iBbeol,giWuam)u,th3y0
August-2September1982,1Dieu-Brichart,Ottignies-Louvain-la-Neuve,pp.261-268
(8) NOoE.C2D26.(2O00E6)C,DPrGeudiadteloirnyemsiftoer(tHhyepToeasstpiinsg(oGfeCohleameilcapasl)s,acOuEleCiDfe,r)Parreipsroductiontestinsoil,TestGuideline
(9) OOEECCDDs(e2r0i0e6s),onCutersrteinntgaapnpdraosascehsessmienntthNeusmtabteisrti5ca4l,aEnNaVly/sJiMs/oMfOeNcoOt(o2xi0c0i6ty)1da8t,a:OaEgCuDidaPanrciestoapplication.
(10) Scott-FordsmandJJandKroghPH(2005)BackgroundreportonprevalidationofanOECDspringtailtest
guideline.EnvironmentalProjectNr.986.Miljostyrelsen61pp.DanishMinistryfortheEnvironment.
(11) Krogh.PH..2009.ToxicitytestingwiththecollembolansFolsomiafmetariaandFolsomiaCandidaand
theresultsofaringtest.DanishEnvironmentalProtectionAgency,EnvironmentalProjectNo.1256,pp.
66.
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