Earthworms
Eisenia
fetida
affect
the
uptake
of
heavy
metals
by
plants
Vicia
faba
and
Zea
mays
in
metal-contaminated
soils
A.
Lemtiri
a,*
,1,
A.
Liénard
a,1,
T.
Alabi
b,
Y.
Brostaux
c,
D.
Cluzeau
d,
F.
Francis
b,
G.
Colinet
aa
DepartmentofBiosystemsEngineering,Soil–Water–PlantExchanges,GemblouxAgro-BioTech—UniversityofLiège,Belgium
b
DepartmentofAgroBioChem,FunctionalandEvolutionaryEntomology,GemblouxAgro-BioTech—UniversityofLiège,Belgium
c
DepartmentofAgroBioChem,AppliedStatistics,ComputerScienceandMathematics,GemblouxAgro-BioTech—UniversityofLiège,Belgium
dRennesUniversity,CNRSEcoBio,StationBiologiquedePaimpont,France
ARTICLE INFO Articlehistory:
Received31October2014
Receivedinrevisedform15November2015 Accepted18November2015
Availableonline24December2015 Keywords:
Heavymetals-contaminatedsoils Eiseniafetida Viciafaba Zeamays Bioavailability Bioaccumulation Phytoextraction ABSTRACT
Earthwormsincreasetheavailabilityofheavymetalsinsomesituationsandaidinmaintainingthe
structureandqualityofsoil.Theintroductionofearthwormsintometal-contaminatedsoilshasbeen
suggested asan aidforphytoremediationprocesses. In Wallonia,Belgium, acenturyof industrial
metallurgicactivitieshasledtothesubstantialpollutionofsoilsbyheavymetals,includingcopper(Cu),
zinc(Zn),lead(Pb)andcadmium(Cd),duetoatmosphericdusts.Twoplantspecies,ViciafabaandZea
mays,andearthworms(Eiseniafetida)(Savigny,1826)wereexposedtodifferentconcentrationsof
long-term-contaminatedsoilsfor42days.Thesoilsamples,whichwerecollectedfromthelandsurroundinga
formerZn-Pbore-treatmentplant,exhibiteddifferentlevelsofheavymetals.Ouraimwastoevaluatethe
roleofearthwormsE.fetidaontheavailabilityofmetalsinsoilsandtheireffectsonmetaluptakebyV.
fabaandZ.maysplantsatdifferentsoilconcentrations.
Theresultssuggestthatearthwormsandplantsmodifiedtheavailabilityofmetalsincontaminated
soilsafter42daysofexposure.Earthwormlife-cycleparameterswereaffectedbymetalcontamination
and/ortheadditionofplants;cocoonproductionandweightweremoreresponsivetoadverseconditions
thanearthwormsurvivalorweightchange.TheconcentrationsofPbandCd inearthwormtissues
decreasedinthepresenceofplants.Resultsshowedthatmetalaccumulationinplantsdependedonthe
metalelementconsideredandthepresenceofearthworms.However,thepresenceofearthwormsdid
not changethe concentrations of metalsin plants, except for Cd. In the presence or absence of
earthworms,V.fabaaccumulatedhigherconcentrationsofCuandZncomparedwithZ.mays,which
accumulatedhigherconcentrationsofCd.Thesefindingshaverevealedthatearthwormactivitiescan
modifytheavailabilityofheavymetalsforuptakebyplantsincontaminatedsoils.Moreover,thestudy
resultsshowthat theecologicalcontextof phytoremediationshouldbe broadenedbyconsidering
earthworm-plant-soilinteraction,whichinfluenceboththehealthoftheplantandtheuptakeofheavy
metalsbyplants.
ã2015ElsevierB.V.Allrightsreserved.
1.Introduction
Heavy metalsare continuously beingadded to soils through anthropogenicactivitiessuchasindustrialization,mining,smelting, andlandapplicationofsewagesludge(Harlavanetal.,2010).In Wallonia(SouthregionofBelgium)duringthetwolastcenturies, processing of metal-bearing ore has emitted metal-bearing
particulatesladenwithCd,PbandZn(Graitson,2005).Thefallouts in thevicinityofplantshaveled tosignificantcontaminationof topsoil(Liénardetal.,2014).Amongtheheavymetals,copper(Cu), zinc(Zn),lead(Pb),andcadmium(Cd)levelsinsoilarereceiving extensiveattentionbecauseoftheiracuteandchronictoxicological effectsonplantsandanimals(ChengandWong,2002;Li,M.etal., 2009;Li,N.Y.etal.,2009).Whenthesefourelementsco-occurinthe soil,theircombinedeffectsareverycomplex.Mixturetoxicity is difficult to study because metals can interact at various levels (Dicksonetal.,1994)suchastheexposurelevel,theuptakelevel,the target level (Rüdiger and Ralf-Rainer, 2010), and the internal pathway of detoxification(Vijveret al.,2011).Cleaning up soils contaminatedwithheavymetalsusingtraditionaltechniquescanbe
* Correspondingauthorat:DepartmentofBiosystemsEngineering,Soil–Water– PlantExchanges,PassagedesDéportés2,5030Gembloux,Belgium.
Fax:+3281614817.
E-mailaddress:alemtiri@doct.ulg.ac.be(A. Lemtiri).
1
Theseauthorscontributedequallytothiswork. http://dx.doi.org/10.1016/j.apsoil.2015.11.021 0929-1393/ã2015ElsevierB.V.Allrightsreserved.
ContentslistsavailableatScienceDirect
Applied
Soil
Ecology
destructiveto thesoil.Remediationof contaminated soilsusing earthwormsandplantsappearstobecost-effectiveand environ-mentallyfriendly technology.Most of metal-accumulating plantsare characterizedbyslowgrowth,lowbiomassyieldandbroadgrowth requirements.Moreover,theyareoftenincapableofaccumulating highconcentrationsofpotentiallyphytotoxicmetals,suchasCu,Zn, Pb,and Cd,in their above groundbiomass(Kumar et al.,1995; Komarek et al., 2007). Plants vary in response to metals, in mechanismofuptakeandofavoidingdamage.Metaltolerantplants suchasThlaspicaerulesens(Papoyanetal.,2007;Saltetal.,1998)and Brassicajuncea(Brunetetal.,2009)havebeenusedfor phytoex-tractiontestsofleadfromcontaminatedsites.Aplant,whichcanbe usedforphytoextraction,shouldbeabletogrowrapidly,toproduce largebiomassandtoaccumulatehighconcentrationsofmetals. Availabilityofmetalsinsoilhasbeenreportedtobedependenton soil type, metal species, metal kinetics and age of metal contaminationinthesoil(Beeby,1993; vanGesteletal.,1995). The availabilityof heavymetals in soil is determined by their chemical speciation(Babich and Stotzky,1980)and their inter-actionswithorganicmatterandmineralsoilparticles(LiandLi, 2000),whichcanbeinfluencedbysoilorganisms.Therehavebeen manystudiesconcernedwiththecombinedeffectsofheavymetals onearthwormsandplants(Morgan,1988;Brokbartoldetal.,2012; Qiuetal.,2011;Israretal.,2011).
Earthworms improve soil structure, contribute to organic matterdecompositionandnutrientcycling(Lemtirietal.,2014) andplayakeyroleinterrestrialecotoxicologicalriskassessment (Sheppardetal.,1997;Weeksetal.,2004).Thebeneficialroleof earthwormsinsoil,influencingarangeofchemical,physicaland biologicalprocesses,hasbeenreported(Scheu,1987;Edwardsand Bohlen,1996;McCredieandParker,1992;CurryandBaker,1998). Some earthworm species are easy to culture and handle for experiments,and thushavebecome standardtestorganisms in ecotoxicology (Organisation for Economic Co-operation and Development (OECD), 1984, 2004). On the other hand, it is recognizedthattheaccumulationofheavymetalsinearthworms dependsmorestronglyonthefractionofmetalthatisavailablefor uptakeratherthanthetotalmetalconcentration.Daietal.(2004),
SpurgeonandHopkin,(1996)andHobbelenetal.(2006)observed significantcorrelationsbetweentheconcentrationsofheavymetal accumulatedinearthwormsandavailablemetalconcentrationsof fieldsoils.Becauseoftheircapacitytoaccumulateandconcentrate largequantities oforganicandinorganicpollutants, earthworm speciesarewidelyrecognizedassuitable organismsfor biomo-nitoringtheeffectsofheavymetalsincontaminatedsoils(Reddy andRao,2008;PeijnenburgandVijver,2009).Numerousstudies investigated the effects of metals on earthworms in terms of mortality(Neuhauseretal.,1985;Fitzpatricketal.,1996;Spurgeon etal.,1994,2000),lossofweight(e.g.Khaliletal.,1996;Spurgeon andHopkin,1996;Maboetaetal.,2004),cocoonproduction(e.g.
Ma,1988;SpurgeonandHopkin,1996),cocoonviability(e.g.van Gesteletal.,1992;SpurgeonandHopkin,1996)andgrowth(e.g.
vanGesteletal.,1991;Khaliletal.,1996).Mostofthesestudies havebeenshort-termexperiments(14or21days),performedin artificialsoilsorsoilartificiallycontaminatedbytheadditionof metal in solution (Nahmani et al., 2007). Few studies have investigated field-contaminated soils with multiple metals (Weltje,1998;ConderandLanno,2000;Feisthaueretal.,2006). Despitealargebodyofliteratureontheimpactofearthwormson theavailabilityofmetals insoils, onlya fewstudieshavebeen carriedoutaddressingearthworm-facilitatedmetalextractionby plants.
In ordertogainbetterunderstandingofthemetaluptakeby plantsincontaminatedsoils,inthepresenceofearthworms,the aimsofthepresentstudywereto:(1)investigatethechangein metalavailabilityinthesoilgiventhepresenceofearthwormsand
plants;(2)evaluatetheeffectsofmetalconcentrationsonsurvival, bodyweight,cocoonproductionandcocoonweightofearthworms (Eiseniafetida)and(3)assesstheeffectofearthwormsonmetal uptake by plants. Earthwormand plantmetal bioaccumulation studies wereperformed to betterunderstand therelationships betweenavailabilityofmetalsinsoilandtheirbioaccumulationin organisms.
2.Materialsandmethods
2.1.Studyareaandcharacterizationofexperimentalsoils
Thestudyareaconsistsofa3kmradiuscirclesurroundingthe Sclaigneaux calaminary site. The term “calaminary” originates from“Calamine”whichisageneraldescriptionofzinc-oressuchas zinc–silicate,zinc–carbonateortheassemblageofhemimorphite, smithsonite, hydrozincite and willemite (Dejonghe,1998). This areaisknownforitshistoricalsoilcontaminationbyaformerZn– Pboretreatmentplant(Liénardetal.,2014).TheCu,Cd,PbandZn metals originate from historic atmospheric fallout of dusts enrichedin thesefourmetals.Thesoilsusedintheexperiment representoneofthethreemajorsoilstypepresentonthestudy area(Liénardetal.,2011),andarecharacterizedasloamy-stony soilswithgravel(Cambisols(Siltic) (WRB,2014))developed on ancient alluvial gravely terracesof Meuseriver. Three samples werecollectedfromcontaminatedfieldsandacontrolsamplewas collected froman uncontaminated field. Sampling points were locatedaccordingtodistancefromthemetalsource.Fourplotsof about20–25m2insizewereselectedforthestudy.Thestudysites
wereasfollows:C3(mostpolluted),1kmfromtheformerZn–Pb ore treatment plant (source); C2, 2.3km from the source; C1, 3.5kmfromthesource;andC0(thecontrol),asitethathassoil properties comparable to the contaminated sites, but with no history of contamination. For analysis of the physicochemical characteristicsofthesoil(includingheavymetalconcentrations), soilsamplesofapproximately1kgeach,weretakeninApril2014 from four places in each plot and mixed together. Four core samplesrandomlytakenfromeachplottoadepthof20cmwere combined to form a composite sample for each plot. The soil samplesweredriedunder shadeconditions fortwo weeksand sievedat8mmtoremovegravel.Thissievedsoilwasusedforthe potexperiment. For physicochemicalanalyses, thesoil samples weresievedat2.0mmandasubsamplewascrushedto200
m
m. Particle size distribution (clay, silt and sand fractions) was determined by sedimentation using the pipette method (van Ranstetal.,1999).SoilpHwasmeasuredbycreatingaslurryin distilledwater(pHwater)and1NKCl(pHKCl)(w:v2:5ratio).Totalorganiccarbon(TOC)wasdeterminedfollowingtheWalkleyand Blackmethod(WalkleyandBlack,1934)andtotalnitrogen(N)was estimated bymodifiedKjeldahl method (Nelsonand Sommers, 1996).Effectivecationexchangecapacity(CEC)wasdeterminedby usingahexamminecobalttrichloridesolutionfollowingISO23470. Pseudo-totaltraceelement(Cu, Cd,Pb,Zn)concentrationswere determined after aqua regia digestion following ISO 11466 (referredtoasARmetalconcentration).Availablemajor(Ca,Mg, K,P)andtrace(Cu,Cd,Pb,Zn)elementsweredeterminedafter extractionwithCH3COONH4(0.5M)andEDTA(0.02M)atpH4.65
(w:v1:5ratio)andagitationfor30min(referredtoasavailable metalconcentration)(LakanenandErviö,1971).Theconcentration of P in theresultingextract was determined bycolorimetry at 430nm.Theconcentrationsoftheotherelementsinthesolution weremeasuredbyflameatomicabsorptionspectrometry(VARIAN 220,AgilentTechnologies,SantaClara,CA,USA).Allconcentrations werecalculatedonthebasisofdryweight(DW)ofsamples(40C, 24h).Thedetectionlimitsfor AR/availablemetalswere, respec-tively,0.66/0.10mgkg1(Cd),0.99/0.15mgkg1(Cu),3.33/0.50mg
kg1 (Pb) and 0.33/0.05mgkg1 (Zn). To assess the available (soluble)fractionofmetalsinthesoil,CaCl2extractionwasused(at
both the start and the end of the experiment) (McGrath and Cegarra,1992).Extractionswith0.01MCaCl2 havebeenwidely
usedforassessingthemobilityandavailabilityofheavymetalsin soils. As part of the quality control program for the study, a standardreferencematerialwasusedandanalyzedwitheachset ofsamples.Selectedphysicochemicalsoilcharacteristicsandmetal concentrationsinthesoilsarepresentedinTable1.
2.2.Experimentalprocedure
The experimental design involved four factors: (i) metal concentrationrepresentedbyfourdifferentsoilswithan increas-inglevelofcontamination(C0,C1,C2andC3),(ii)plants(Zeamays,
Viciafaba),(iii)earthwormsE.fetida,and(iv)foodforearthworms (Table2a).Tentreatmentswerepreparedfromthesoilsamplesto createasetofsampleswithdifferentcombinationsof(thetwo) plants,earthworms andfoodpresence andeach treatment was replicatedfourtimes,exceptforthecontrolsamplewithfoodonly (CP0E0F1)whichwasreplicatedonce(Table2b).Theexperiment
wasconductedundercontrolledconditions(i.e.16hlightand8h darkat201C)(ReneickeandKriel,1981)during42days.Pots
(16cmdiameter20cmhigh)werepreparedwith2.25kgofdry soil.Atthebeginningoftheexperiment,soilmoisturecontentwas adjustedto18%,correspondingto65%ofthesoil'swaterholding capacity,checkedregularlyandadjustedtothedesiredvalueby addingdeionizedwater.
For eachtreatment withearthworms,20specimens E.fetida weredepositedonthesurfaceofthesoilinpots.Accordingtothe OECDmethods(OECD,2004),earthwormswereacclimatizedforat least 48h prior to exposure, in the experiment conditions (temperature, light, soil moisture, etc.). Healthy earthworms weighing between 200 and 600mg each, and well-developed clitellumwereintroducedsevendaysafterplantseeding.Toensure theearthwormssurvivalduringtheexperimentationperiod,they werefedweeklywithadriedmixtureofhorsemanure(75%)and oatflakes(25%)toprovide0.5gperearthworm.Thetestcontainers were covered with a perforated lid to limit water loss due to evaporation,toallowaerationandpreventtheearthwormsfrom escaping.
Z. mays and V.fabaweretheplant speciesused duringthis experiment. Ten seeds were seeded in pots according to experimental design. Plant growth was held under controlled conditions: 201C and 181C day and night temperatures respectively, with 60%5%relative humidity. Germinationwas determinedbyvisualseedlingemergence(Gongetal.,2001).The plantswerewateredwithdeionizedwater.Thedistributionofthe microcosms in the chamber was randomized and changed biweekly.
2.3.Acquisitionoforganismaldata
After42daysofexposure,earthwormmortalitywaschecked, andthelivingadultswerehand-collected.Theearthwormsand cocoonsineachpotwerecountedusingtheproceduredescribed intheOECDmethod(OECD,2004).After48hcleaningoutofgut contentonmoistfilterpaper,theearthwormswereweighedand checked foranymorphologicalsymptoms. Thefilterpaperwas changed threetimesperdaytopreventcoprophagy.All earth-wormsineachcontainerwereweighedasagroupandrecorded as a datum. The relative growth rate of earthworms was
Table1
Aquaregia,availableandsolublemetalconcentrations(mgkg1DW),available majorelements(mg100g1DW),andotherchemicalparametersmeasuredinthe
fourexperimentalsoils(C0:controlsoil,C1,C2andC3:contaminatedsoils).
Parameters C0 C1 C2 C3 pHwatera 8.20 7.45 7.11 8.08 pHKCla 7.64 6.78 6.61 7.75 TOC(%)b 2.04 1.74 1.67 1.50 N(%)c 0.190 0.160 0.180 0.150 CEC(meq100g1)d 15.2 11.8 10.7 10.5 Clay(%)e 21.0 12.3 13.3 14.6 Silt(%)e 62.5 72.4 59.4 46.1 Sand(%)e 16.5 15.3 27.2 39.3 AquaRegiaf Cd(mgkg1,DW) 0.710 2.48 4.27 9.28 Cu(mgkg1,DW) 15.0 11.7 21.7 19.3 Pb(mgkg1,DW) 29.0 69.0 129 255 Zn(mgkg1,DW) 103 208 384 743 Availableg Cd(mgkg1,DW) 0.550 1.95 2.89 6.07 Cu(mgkg1,DW) 3.59 13.34 4.89 6.97 Pb(mgkg1,DW) 12.4 41.1 71.2 169 Zn(mgkg1,DW) 15.8 61.9 65.6 172 Ca(mg100g1,DW) 479 191 221 554 Mg(mg100g1,DW) 23.2 22.2 11.2 10.6 K(mg100g1,DW) 23.4 37.8 20.4 27.7 P(mg100g1,DW) 13.5 16.1 14.7 16.2 Solubleh Cd(mgkg1,DW) 0.010 0.050 0.100 0.060 Cu(mgkg1,DW) 0.140 0.12 0.11 0.14 Pb(mgkg1,DW) <DL <DL <DL <DL Zn(mgkg1,DW) 0.020 0.370 0.690 0.240 (DL:detectionlimit). apH
waterwithdistilledwater(w:v2:5ratio)andpHKClwith1NKCl(w:v2:5
ratio).
b
WalkleyandBlackmethod(1934).
cModified
Kjeldahlmethod(NelsonandSommers,1996).
d
HexamminecobalttrichloridesolutionfollowingISO23470.
eSizeparticlesbysedimentationusingthepipettemethod(vanRanstetal.,
1999).
f
AquaregiadigestionfollowingISO11466.
g
ExtractionwithCH3COONH4(0.5M)andEDTA(0.02M)atpH4.65(w:v1:5
ratio)(LakanenandErviö,1971).
h
Extractionwith0.01MCaCl2.
Table2
Experimentaldesignforecotoxicitytest:(a)presentationofdifferentfactorsand treatments,(b)descriptionoftreatmentswithdifferentcombinationsoffactors.
(a) Factors Treatments
Concentration C0,C1,C2,C3 Plants Noplant(P0) Viciafaba(P1) Zeamays(P2) Earthworms Presence(E1) Absence(E0) Food Presence(F1) Absence(F0) (b) Treatment Description n 1 C0,1,2&3P0E0F1 4 2 C0,1,2&3P0E0F0 16 3 C0,1,2&3P0E1F0 16 4 C0,1,2&3P0E1F1 16 5 C0,1,2&3P1E0F0 16 6 C0,1,2&3P1E0F1 16 7 C0,1,2&3P1E1F1 16 8 C0,1,2&3P2E0F0 16 9 C0,1,2&3P2E0F1 16 10 C0,1,2&3P2E1F1 16
calculatedaccording tothefollowingequation:relativegrowth rate=(Wt–W0)/(W0)100%,whereW0istheinitialaverageweight
ofearthworms,andWtistheaverageweightafter42days.
Withregardtotheplants,after42daysofexposure,theshoots wereremovedbycuttingtheV.fabaandZ.maysplantsclosetothe soilsurface andweighed. Rootmass was determinedfollowing washingwithDIwateranddryinginpaperbags(40C)forone week.Similarly,andfollowing48hforgutclearing,earthworms wereplacedintoanovenat40Covernight;thedryearthworms
wereweighed(DW).
Samplesofdriedplantandearthwormtissuesweredigestedin amixtureof15mlofnitricacid(HNO3,65%)and15mlofperchloric
acid(HClO4,70%)during16h.Aftercompleteevaporation,5mlof
HCl(10%)was addedand thesampleswerebroughttoa25ml volumewithdistilledwater.Thesesolutionswerestoredat4Cin polyethylenetubes beforeanalysis. Metalconcentrations inthe samplesweremeasuredbyflameatomicabsorptionspectrometry (VARIAN 220, Agilent Technologies, Santa Clara, CA, USA). The detection limits for Cu, Zn, Pb and Cd were 0.6, 0.2, 2 and 0.4mgkg1,respectively.
2.4.Statisticalanalysis
Data normality and homoscedasticity were verified with Shapiro-Wilk’sandBartlett’stests.Inmostcases,parametrictests wererelevant,butinothersequivalentnon-parametrictestswere moreappropriate.
Firstly,theeffectsofsoilmetalconcentration(C0,1,2&3),plants
(P0, P1, P2), earthworms (E0,E1), food (F0, F1), time (T0, T1) and
samplingblock(randomfactor)onmetalavailabilityinsoilwere evaluatedthroughanalysisofvariance(ANOVA)withinageneral linearmodelinMinitab16software(MinitabInc.,StateCollege, PA, USA). Secondly, the effects of soils concentration (C0,1,2&3),
plants (P0, P1, P2), food (F0, F1) and sampling block (random
factor)onearthworm mortality,earthwormweight,earthworm
reproduction(numberandweightofcocoons)andthe concentra-tionofmetalsinearthwormtissueswereevaluated.Finally,the effects ofsoilmetalconcentration(C0,1,2&3),earthworms(E0,E1),
food(F0,F1)andsamplingblock(randomfactor)ontheuptakeof
metals by plants (P1, P2) were investigated. Differences were
consideredsignificantatP0.05.
Whenthedifference wassignificant,acomparison ofmeans was conducted by a post-hoc Tukey test. If the hypothesis of parametersofthatmodelwasnotsatisfied,aKruskal–Wallistest wasusedinsteadtodetecttheeffectsofthevariousfactors. 3.Results
3.1.Effectofearthwormandplantactivitiesontheavailabilityof metalsinsoil
Soilsamplestakenfromthesampletreatments(control,soil withearthworms,soilwithplants,andsoilwithbothearthworms andplants)wereanalyzedtocomparepHwaterandtheavailable metalfraction.Table3givespHvaluesandmetalfraction(0.01M CaCl2) remaining in the soil after 42 days of exposure in the
presenceorabsenceoforganisms(plantsorearthworms). SoilpHvaluesvariedfrom6.3to7.2.Nosignificantdifference wasobservedamongthesixtreatments(P>0.05).The introduc-tion of earthworms, or plants, or the combination between earthwormsandplantsdidnotaffectsoilpHvalues.
Presence of V. faba decreased the availability of Cd in soil (P=0.02), but no significant differences on the availability of metalswereobservedfortheothertreatments.
3.2.Earthwormtraits
ThemeanconcentrationsofCu,Zn, PbandCd(expressedas mgkg1DW) in E.fetida tissues after 42 days of exposure are reportedinFig.1.Significantdifferenceswereobservedbetween
Table3
Mean(s.e)pHwaterandmetalfractionextractableusing0.01MCaCl2procedureinsoil(mgkg1DW)beforeexperiment(Soil)andafter42daysexposure(Treatments)inthe
absence/presenceofplants(Viciafaba/Zeamays)and/orabsence/presenceofearthworms(Eiseniafetida).Differentlettersindicateasignificantdifferenceaccordingto Tukey’s testatP0.05.
Soilparameters Soil Treatments
P0E0F0 P0E1F1 P1E0F0 P2E0F0 P1E1F1 P2E1F1 pH C0 7.25 7.20.17 7.20.16 7.20.18 7.10.18 7.10.14 7.10.21 C1 6.40 6.40.23 6.30.19 6.40.12 6.40.11 4.90.15 6.60.04 C2 6.28 6.40.05 6.40.16 6.30.18 6.40.12 6.40.11 6.60.19 C3 6.67 7.10.13 7.10.13 7.10.18 7.10.15 7.00.15 7.00.15 Cd C0 0.01 0.01 0.01 0.00 0.00 0.00 0.00 C1 0.06 0.04 0.04 0.03 0.03 0.03 0.08 C2 0.10 0.04a 0.07ab 0.07b 0.05ab 0.07ab 0.06ab C3 0.10 0.04 0.04 0.05 0.03 0.06 0.06 Cu C0 0.14 0.01 0.00 0.02 0.04 0.00 0.15 C1 0.12 0.02 0.02 0.01 0.00 0.00 0.02 C2 0.11 0.02 0.00 0.02 0.00 0.08 0.23 C3 0.14 0.00 0.00 0.00 0.00 0.00 0.09 Pb C0 0.00 <DL <DL <DL <DL <DL <DL C1 0.00 <DL <DL <DL <DL <DL <DL C2 0.00 <DL <DL <DL <DL <DL <DL C3 0.00 <DL <DL <DL <DL <DL <DL Zn C0 0.00 <DL <DL <DL <DL <DL <DL C1 0.36 0.05 0.46 0.43 0.38 0.31 0.30 C2 0.69 0.57 0.74 1.05 0.64 0.62 0.65 C3 0.24 0.39 0.22 0.38 0.27 0.40 0.57
the concentrations of elements found in E. fetida for all the contaminated soils, except for copper. Theconcentration of Pb metalin earthworm tissuesclearly increased withexposure to raisedsoilPbconcentration(P<0.001).
Ourresultsshowedthatadditionofplantsaffectedthemetal concentrationsinearthwormtissuesexceptforCu(Fig.1a).Tissue concentration of Zn in E. fetida was significantly higher when plants were added. V. faba plant affected significantly the accumulationof Znbyearthworms(Fig.1b).Pb and Cdmetals concentrationinE.fetidatissuesdecreasedinthepresenceofV. fabaorZ.maysplantsforC2andC3soils(Fig.1candd)
3.2.1.Earthwormmortality
An analysis of variance was performed on the results of earthworm life-cycle parameters in order to evaluate whether there was a change in mortality after 42 days of exposure. Throughouttheexperimentalperiod,noearthwormsdiedinthe control soils (C0). Thus, this observation suggested that the
experimentalconditionswerevalidintermsofprovidingsuitable media for earthworm’s survival. After 42 days of exposure, mortalityratesinthecontaminatedsoilswerenotdifferentfrom the controls (data not shown). In addition, regardless of the concentrationofpollutedsoilinanysample,earthwormmortality wasnotaffectedbytheadditionofplantsV.fabaorZ.mays.Only the“food” factorsignificantlyaffected themortalityofE.fetida (P=0.036).
3.2.2.Earthwormbodyweight
Twenty worms were weighed at the beginning of the experiment and theremainingnumber of earthwormsafter42 dayswas>17forallsamples.Thequestionsinvestigatedwere:was thereageneralchangeinearthwormweightordidthechangevary accordingtowormfeeding,soilcontaminationorthepresenceof plants?
Significant difference in body weight was found between earthwormsin potswithfoodandearthwormsinpotswithout food(Table4)soweperformedaseparateanalysisforpotswith andwithoutafoodsupply.Inthepotswithoutfood,noplantswere cropped.Nosignificantinteractionwasfoundwithtime,whilethe factors“Soil”and “Time”weresignificant (P=0.021)and highly significant(P=0.0001),respectively.Regardingtheeffectof soil contamination,it seemsthat thedifferenceswerelinkedtothe initialweightoftheearthworms.Thechangeintheirbodyweight overtimecanbeconsideredasequivalentforallthesoiltypes,that isameandecreaseof195mgperearthwormafter6weeks.
For pots which received food, significant interactions were foundbetween“PlantandTime”,“BlocandTime”and“Plantand Soil”. We found that the mean weight of earthworms at the beginningoftheexperimentwashigher(40mg)inBloc2thanin theotherthree.Separateanalysesofvariancewereperformedfor thetreatmentswithoutplants,withZ.maysorwithV.faba.The firstpointtobenotedisthattherewasanincreaseofwormweight foreverypotwithoutaplant,andasmallerincreaseforsoilC0,C2
Fig. 1.Mean(s.e)tissueconcentrations(mgkg1drybodyweight)ofCu(a),Zn(b),Pb(c)andCd(d)inEiseniafetida(E1)specimensinthepresenceoffood(F1),after42days
exposuretocontrol(C0)andcontaminated(C1,C2andC3)soils,intheabsence(P0)orpresenceofViciafaba(P1)andZeamays(P2)plants.Differentlettersindicateasignificant
andC3withV.faba.RegardingZ.mays,onlythesoilC1showedan
increaseofthemeanmassoftheworms.Wecanconcludethatthe actionofgivingfoodtowormshadasignificantimpactwhenno plantwascultivated,whilethepresenceofaplantdidfacilitatethe gainofmass.
3.2.3.Earthwormreproduction
Cocoon productionwas calculatedper surviving earthworm.
Table5showstheeffectsofmetalsandtheadditionofplantson reproductiveparametersofE.fetida,includingcocoonproduction perearthwormsandweightpercocooninsoilsafter42daysof exposure.Asignificant(P<0.001)reductionincocoonproduction andcocoonweight was seenfor thecontrol and contaminated soils, when food was unavailable. With the addition of food, earthwormsproducedmorecocoons(6.65perworm)andwitha greatermeanweight(19mgpercocoon)thanintheabsenceof food(1.75perworm;12mgpercocoon).However,nosignificant difference in earthworm reproduction was observed between earthwormskeptincontrolsoilandcontaminatedsoils.
The presenceof plantsalsoenhanced thereproductionof E. fetida. Cocoon production in the treatments with plants was significantly higher than in the treatments without plants,
suggesting that the reproduction response was influenced by thepresenceofplantsinthesoil.ThepresenceofV.fabaorZ.mays significantly increased the reproduction activity of E. fetida in controland contaminatedsoils.Thissuggeststhatreproduction andcocoonweightweresensitivetoenvironmentalchangessuch astheintroductionofplants.
3.3.Earthwormmetalconcentrations
ThemeanconcentrationsofCu,Zn, PbandCd(expressedas mgkg1DW)inE.fetidaafter42daysofexposurearereportedin
Fig.1.Differenceswereobservedbetweentheconcentrationsof elementsfoundinthetissuesofE.fetidaforallthecontaminated soils. The concentration of Pb in earthworm tissues clearly increasedwithexposuretoraisedsoilPbconcentration(P<0.001). Theadditionofplantsalsoaffectedearthwormmetal concen-trations.The concentrationof Pb and Cdin earthworm tissues decreased in the presence of V. faba or Z. mays for C2 and C3
samples. The concentration of Zn in E. fetida tissues was significantlyhigherinthepresenceofplants.ThepresenceofV. fabaaffectedsignificantlytheconcentrationofZninearthworm tissues.
Table5
Totalnumberofcocoons,cocoonproductionrates(numberofcocoons/survivingadultearthworms/month),andmeanweightofcocoons(s.e)collectedafter42days exposureandproducedbyEiseniafetidaexposedtosoilscollectedfromthethreemetal-contaminatedsites(C1,C2andC3)andthecontrolsoil(C0).
Treatments Cocoonsnumber Cocoonsproductionrate(Nb/adult/month) Meancocoonweight(mg)
C0P0E1F0 151 1.350.45 11.561.84 C1P0E1F0 187 1.670.43 10.432.45 C2P0E1F0 97 0.870.32 12.483.38 C3P0E1F0 123 1.101.32 13.433.00 C0P0E1F1 451 4.031.14 17.812.78 C1P0E1F1 436 3.890.81 18.951.92 C2P0E1F1 457 4.082.23 20.161.53 C3P0E1F1 536 4.780.33 18.681.91 C0P1E1F1 539 4.811.13 15.282.70 C1P1E1F1 560 5.000.38 14.801.11 C2P1E1F1 571 5.101.00 17.254.55 C3P1E1F1 608 5.430.97 17.874.25 C0P2E1F1 500 4.461.14 16.953.95 C1P2E1F1 547 4.880.76 18.882.88 C2P2E1F1 505 4.511.25 16.291.33 C3P2E1F1 594 5.300.96 15.771.27
P0:noplant,P1:Viciafaba,P2:Zeamays,E0:noearthworm,E1:earthworms,F0:nofood,F1:food.
Table4
Mean(s.e)weightofearthwormsEiseniafetida(g)atthebeginning(T0)andafter42daysexposure(T1)withdifferentcombinations,inthepresence(F1)andabsence(F0)of
food.(C0:controlsoil,C1–C2–C3:contaminatedsoils,P0:noplant,P1:Viciafaba,P2:Zeamays,&:decreasebetweenT0andT1,%:increasebetweenT0andT1,%%:highincrease
betweenT0andT1).DifferentlettersindicateasignificantdifferenceaccordingtoTukey’s-testatP0.05.
F0 F1 T0 T1 T0 T1 C0P0 22.104.65ab 13.103.20ab & 21.854.20ab 25.455.15bc %% C0P1 22.054.30ab 23.404.95ab % C0P2 21.553.95ab 20.604.45a C1P0 21.604.10ab 12.452.95ab & 22.754.45ab 24.706.35bc % C1P1 21.954.35ab 22.005.15ab C1P2 22.454.50ab 24.755.40bc % C2P0 21.354.25a 11.353.00a & 21.454.15ab 25.806.45bc %% C2P1 21.504.20ab 22.454.35ab % C2P2 22.954.15ab 22.605.95ab C3P0 23.304.00b 12.653.05b & 21.053.85a 27.154.90c %% C3P1 23.104.40b 24.305.10bc % C3P2 22.754.00ab 21.955.30ab
3.4.Plantmetalconcentrations
ThemeanoftheconcentrationsofheavymetalswithinV.faba andZ.maysplants,withandwithoutthepresenceofE.fetida,are giveninFig.2.Incontaminatedsoils,metalconcentrationswere significantly different between the two plants. Cu and Zn concentrations were lower in Z. mays compared with V. faba (Fig.2aandb).PbconcentrationofZ.maysandV.fabaascompared tothecontrol(Fig.2c).CdconcentrationwashigherinZ.maysin comparisonwith V.faba(Fig. 2d). Thedata showed a complex interaction between the metals in the soil solution and their accumulationinplants(bothspeciesandmetaldependant).When theplanttissueconcentrationdatawascomparedtotheavailable concentration,nocorrelationwasobservedforV.fabaandZ.mays. TheadditionofearthwormstosoilscontaminatedbyCu,ZnandPb didnotaffectmetalconcentrationsinplants;onlyforCddidwe observedanincreasedconcentrationinZ.mays.Asthe concentra-tionofCu,ZnandCdinsoilincreased,theconcentrationsofCu,Zn andCdinZ.maysplantsalsoincreased,whileforV.faba,theplants’ metalconcentrationsremainedunchanged.
4.Discussion
4.1.EffectsofearthwormsandplantsonthepHandtheavailabilityof metalsinsoils
ItiswellestablishedthatsoilpHisakeyfactoraffectingthe adsorption–desorptionbehaviorsandhenceavailabilityofheavy
metalsin soil. In thisstudy,itwasfound thatthe presenceor absence oforganisms (earthwormsandplants) didnot change thepHvalues.Ourresults arehoweverinconsistentwithmost previousstudies,whichhaveshownthat,incontaminatedsoils earthwormactivityincreasedsoilpH(Wenetal.,2006;Udovic and Lesta, 2007). The mechanisms by which E. fetida should changepHvaluearestillunclear(Sizmurand Hodson,2009).
Whenthesoiliscontaminatedwithamixtureofmetals,their combinedeffectshaveproventobeverycomplex.After42daysof exposure,followingtheadditionofE.fetida,itcanbeseenthatCd and Zn fractions were less available than other metals. The significant decrease in the available fraction of Cd in soils suggestedthatE.fetidaeitheraccumulatedthosemetalsintheir tissuesorthatearthwormactivitymaychangethechemicalform and availabilityofCdfractionin soils.Thesignificantdifference was observed for the C2 concentration, which represents the
highestCaCl2extractableCdsoilconcentration.Thesefindingsare
inconsistentwithpreviousstudies,whichsuggestedanincreasein the availability of metal fractions as a result of earthworm activities(Wenetal.,2004;Coeurdassieretal.,2007;Udovicand Lesta,2007).Lukkarietal.(2006)howeverreportedanincreaseof Zn fractions in the presence of earthworms, concluding that earthwormstendedtodecreasethemobilityofZninthesoil.In some studies, the addition of earthworms has been shown to decrease or not affect the fraction of metals bound to either organicmatterorcarbonates(Wenetal.,2004;Ruizetal.,2011). Thelackofobserveddifferencesintheavailabilityofothermetals (Cu,Pb,andZn)betweenthecontrolandtheearthworm-inhabited
Fig.2.EffectsofEiseniafetida(E1)onmeanconcentrations(s.e)(mgkg1dryplantweight)ofCu(a),Zn(b),Pb(c)andCd(d)inViciafaba(P1)andZeamays(P2),after42days
soilsmayindicatethatalthoughpassagethroughtheearthworm guthasanimpactonmetalmobility,thisisonlyatemporaryeffect (Lukkarietal.,2006).Theseconflictingresultscouldbemainlydue todifferences in(i)soil characteristics,(ii)pollution mode, (iii) metaltype,and(iv)evenphysiologicalandecologicaldifferences among earthwormspecies. The effect of earthworms onmetal availabilityinsoilshasnotbeenextensivelydocumented(Sizmur andHodson,2009).
Ourfindingsshowedasignificantcorrelationbetween extract-ableZnandpHlevel.ChangesinpHaffecttheavailabilityofZn.The pHlevel,organicmattercontentandavailabilityofheavymetalsin soilarecriticalfactorsfortheheavymetalaccumulationbyboth plantsandanimals(SpurgeonandHopkin,1996;Osteetal.,2001). TheadditionofV.fabaincreasedtheavailablefractionsofCdand Zn.Thisresultcanbeexplainedbythevitalroleofplantroots, which played in altering metal speciation in soils through rhizosphere processes. Plant roots can modify the speciation and availability of metal (Hashimoto et al., 2010). Another possibilityforenhancingmetalavailabilityistheinvolvementof soilmicroorganismsandplantroot-associatedbacteria(Kamnev andvanderLeile,2000;vanderLelie,1998).
4.2.Earthwormtraits
4.2.1.Earthwormadultsurvivalandgrowth
Thehighsurvivalrateobservedfortheearthwormsinthemetal contaminatedsoilsafter42daysofexposureandtheabsenceof visibledamageontheplantsmaybeexplainedbythelowrangeof metal contamination. The stability and lack of mortality for biological organisms observed in old contaminated soils were reportedbySchrecketal.(2011).Theavailabilityofmetalinfield soilsoftendecreasesovertime(LockandJanssen,2003a,b).
ThebodyweightofE.fetidawassignificantlyaffectedbyfood.In thetreatmentswithoutfood,thelossofbodyweightwasmost likelyduetothefactthatorganiccarbonandfoodsupplyavailable inthesesoilswereinsufficienttomaintaintheinitialbodyweight of earthworms. Body weight of the earthworms was not significantly affected by metal contamination. This could be explained by the fairly low of metal concentrations or by the competitionofmetalswithotheressentialelementsinthesoils. Theseeffectssuggestedthatsoilcharacteristicscandecreasethe availabilityof metals and modify theirtoxicity. As reportedby
Ernstetal.(2008),soilpropertiescanaffectmetalbioavailability and their subsequent impacts on earthworm physiology and behavior.Somereportssuggestedthatinsomecases,organisms primarilyrespondedtocertainattributesratherthantothemetal concentration(Changetal.,1997).SoilpHandorganiccarbonhave been claimed to be important factors for affecting metal bioavailability to ecological receptors (Spurgeon and Hopkin, 1996;PeijnenburgandJager,2003;Bastaetal.,2005;Daytonetal., 2006).Inourstudy, soilpHremainedintheneutral toslightly alkaline range. Many other studies have reported the metal’s toxicity to earthworms, tested both in artificial and field soil (Neuhauser et al., 1985; Spurgeon et al., 1994; Spurgeon and Hopkin1995).Theyconcludedthatthetoxiceffectsofmetalswere lesssevereinfieldsoils(SpurgeonandHopkin,1995).Itshouldbe notedthatsimultaneousexposuretoseveralmetalscanalsolead toantagonistic,notnecessarilytoadditiveorsynergisticeffects,as observedbyKhaliletal.(1996).
TheadditionofV.fabaandZ.maysdidnotaffecttheearthworm weightafter42daysinallconcentrations.Wecansupposethat metallic elements adhered quickly on the root surface and penetrated into plant tissues, which explained the inhibited developmentseenafter 28 daysfor both plants, which inturn wouldmakemetalslessavailable(datanotshown).Aconsequence ofthepresenceofearthwormsandplantsinthesamepotscould
have changed resource allocation and create a competition betweentheplantsandtheearthworms.
4.2.2.Cocoonproductionandcocoonweight
Severalstudiesindicatedthatcocoonproductionisoneofthe most sensitive biological responses in toxicity tests (Ma,1984; Spurgeonetal.,1994;SpurgeonandHopkin,1996,1999;Kulaand Larink, 1997;Reineckeetal.,2001;Homaetal.,2003),withastrong decreasein cocoonproductionwithincreasedmetal concentra-tions.
Theintroductionofplants(V.fabaorZ.mays)affectedcocoon production.Thissuggestedthatanimportantamountofenergy hasbeenallocatedtotheproductionofearthwormcocoonsand the rest was allocated to cocoon development. The opposite responseofthetwoendpointsforE.fetidasuggestedatrade-offof energy between cocoon production and cocoon weight. This patternwereverydifficulttoexplain:itispossiblethatE.fetida livinginastressedenvironmentis“forced tochoose”onwhich process to spend its energy. Indeed, it was the availability of sufficient foodthatwas of primeimportance formaintaininga highcocoonproductionandweight(ReineckeandViljoen,1990) andthiscouldhaveexplainedthedecreaseincocoonweightwe observed. Spurgeon and Hopkin (1996) observed that E. fetida produced cocoons that increasedin weight withincreased soil concentrationsofametalmixture,mainlyCu,Zn,PbandCd.Inour study,thecocoonweightwasindependentofthemetal concen-trations.Ourfindings disagreedwithseveral authors(Spurgeon et al., 2000; Ávila et al., 2009) who found negativeeffects on earthwormreproductioninmetalcontaminatedsoils.Thedirect relationship between E. fetida reproduction and soil metal concentrationmustbediscussedwithcautionsinceearthworm reproductiondependsonmanydifferentsoilfactors.Inparticular soilorganicmattercontentplayedanimportantroleinmitigating theeffectsofmetals(Ávilaetal.,2009vanGesteletal.,2011),while earthwormreproductionmayalsobereducedathighsoilpH(van Gesteletal.,1992).
4.3.Metalaccumulationinearthworms
Earthwormsareconsideredtohavetwouptakepathwaysfor heavy metal: dermal and intestinal. According to Lanno et al. (2004),earthwormscantakeupmetalsfromsoileitherthrough directdermalcontactwiththeminsoilsolutionorbyingestion ofbulksoilorspecificsoilfractions.Distributionofmetalsamong soilfractionsisalsoconsideredtobeimportantfortheirtoxicity and bioavailability to earthworms (Becquer et al., 2005). The resultsweobtainedshowedthatZnandCuwereaccumulatedin E.fetidatissueswithoutexceeding80mgkg1and8mgkg1DW, respectively. Numerousauthorshave suggested thatE. fetidais abletoregulateZnbybindingZnintheirchloragogenoustissue (Morgan,1981; MorganandMorris,1982;Morganand Winters, 1982; Cotter-Howells et al., 2005). Since the binding of Zn to metallothioneinsisreversible,itwaslikelythatZn-thioneinsmay regulate the concentrations of metal in the body tissue by allowing rapid elimination of Zn (van Gestel et al., 1993; Marinussenetal.,1997).Indeed, Znisanessentialelementand itsinternallevelisregulatedbyearthworms;theefficiencyofZn accumulationprobablyrelatestoanecessityforastoredpoolof available Zn in anticipation of future physiological demand (Nannoni et al., 2011). Our findings showed that Cu concen-trations were relatively low. This essential metal may also be regulatedbyearthworms.Inaddition,inthepresentexperiment, the metal concentration was measured in earthworm tissues following48hofgutdepuration,whichmayhaveresultedina lossofmetalsfromthetissuesoftheearthworms(Spurgeonand Hopkin,1999).
TheaccumulationofCdandPbbyE.fetidaincreasedwiththe increasing concentrations of metals in the soil. Cd was highly accumulatedbyE.fetida.Thisphenomenoncouldbeexplainedby itshighmobilityinsoilanditschemicalanalogywithZn(Liand Thornton, 2001; Nannoni et al., 2011). Less Pb uptake and accumulationbyE.fetidawasobservedwhich shouldbelinked withloweravailabilityofPb.
Metal toxicity involves three steps: exposure, uptake, and reactionwith the biological target. In E.fetida, the Pb and Cd accumulation increasedin soil withgreater soil metal concen-trations. By contrast, Zn and Cu have a steady level of accumulation.It is likely that Cdand Pb didnot usethesame uptakepathwaysasZnandCu.Lietal.(2010)providedevidence that the uptake of Cd by E. fetida precedes through calcium channels,whereasZnuptakeis carrier-mediatedbyproteinsor othersulfhydryl-containingcompounds,implyingthatthe mech-anismsofCdandZnuptakeinE.fetidaareessentiallydifferent. However,interactionbetweenmetalsshouldalsobeconsidered. Weltjeetal.(1998)compileddataonsublethaltoxicityandtissue concentrations of Cu, Zn, Pb and Cd mixtures in earthworms. Mixturetoxicityshiftedfrommainlyantagonismtowardsnearly concentration-addition when the endpoints were based on extractable metal concentrations instead of total soil concen-trations.
4.4.Earthwormandplantsinteraction
ThispartofthestudyinvestigatedtheeffectofE.fetidaonthe abilityofV.fabaandZ. maystoextractmetals incontaminated soils.Plantsaccumulatedelementstovaryingdegreesdepending onthesoilsandmetals.V.fabaaccumulatedhigherconcentrations of Cu and Znthan Z. mays which accumulated higher concen-trationsofCd.TheseresultssuggestedthatZ.maysandV.fabacan facilitateCdand Cuextractionfromcontaminatedsoils, respec-tively. V. faba is not known as a metal tolerant plant. To the contrary,itssensitivity tometals justifieditsusein ecotoxicity tests(CordovaRosaetal.,2003;Ünyayaretal.,2006)asitseemsto beoneof themostmetalsensitive plantspecies(Rahouietal., 2008). Z. mays plant appeared to be a good candidate for phytoextraction.PreviousreportshaveclassifiedZ.maysasaroot accumulator(Li,N.Y.etal.,2009;MenchandMartin,1991).The difference between the two plants could be explained by the differences in metal absorption mechanisms. Looking at plant accumulation of elements, Pignattelli et al. (2012) also found differences in the influence of the available and total concen-trationsbetweenarsenic and other elements(e.g.,Zn and Cd), whichwereattributedtothedifferenceintheuptakemechanisms ofmetals.Wecanhypothesizethatthemetaluptakebyplantsis notonlycontrolledbytheelementdistributioninthesoilbutalso bytheexposurepathways.TheconcentrationofCuinV.fabaandZ. maysfollowthesametrendwhateverthemetalconcentrationsin soil.ThesefindingsmaybeexplainedbecauseCuisanessential micronutrientforplantnutritionanddeficiencyeffects couldbe mistaken for toxic responses. Concentrations between 5 and 20mgkg1DWplantareconsideredadequatefornormalgrowth, whereasconcentrationshigher than20mgkg1DWare consid-eredtoxic(Adriano,2001).TheanalysisofPbmetalconcentrations in plants did not reveal significant differences. The extent of assimilationofheavymetalsfromsoildependsonwhetherthey are present in a form that can be absorbed by plants. Two mechanisms can explain this result: (i) Pb could be strongly adsorbedbysoilparticles (Smical etal., 2008)and (ii)thelow affinity of plants for this element, which has no role in their metabolism;whileCdelementisrelativelymobileinsoilandcan bemoreeasilyabsorbedbyZ.mays.
The metal uptake of the two types of plant grown in contaminatedsoilunderpresenceofearthwormsvariedaccording totheplantspeciesandthemetal.Thissuggestedthattheseplants havedifferentcapacitiestoabsorbandeliminatetoxicelements, butthedetailedmechanismneedstobefurtherinvestigated.As suggested by Wang and Li (2006), the higher uptake of heavy metalsbyplantsunderearthworminoculationwasprobablydue to the increase in dry matter production stimulated by earth-worms.ThepresenceofearthwormincreasedCdconcentrationin Z.mays,exceptforthecontrol,probablybecausetheamountof availableCdwastoolow.Anincreaseinmetalconcentrationfor plantsinthepresenceofearthwormswasalsoobservedbyWen et al. (2004). Yu et al. (2005) found that earthwormactivities increased Cd uptake and plant growth, thereby improving the phytoextractionefficiencyofmetalhyperaccumulatorsinlowto medium level metal-contaminated soils. As earthworms are known to affect the distribution of microorganisms (Brown, 1995),wehypothesizedthattheeffectsofearthwormsonmetal uptake by plants resulted in part from their impact on soil microorganisms.Microorganismsassociatedwithplant-rootsare knowntobemajordriversofmetalspeciationinsoilsandcould promote a betterefficiency in phytoremediation (Abou-Shanab etal.,2003).Dandanetal.(2007)foundthattheearthwormbodies andearthwormcastsarerichinaminoacidsandproteins,and soil-availablecarbon.Theseorganicmaterialsmayformchelateswith heavymetals,thuscontributingtoenhancethetransportofheavy metalsfromsoiltoplant.
In ourexperiment,whenplants wereinoculated with earth-worms, Cd and Pb accumulation in E. fetida decreased. The presence of Z. mays and V. faba can prevent and reduce the accumulationofCdandPbinearthwormtissues.Probably,plants can accumulate part of the Cd and Pb, which may create competition betweenthetwo organisms. Pb and Cduptake by E.fetidawashigherinthepresenceofZ.mayscomparedwithV. fabaforthetreatmentwiththehighestmetalconcentration(C3).
The effect of the two plants on earthworm metal uptake was different.Actually,byproducingexudates,plantscanmodifymetal speciationandtheirbehaviourinsoil,especiallyintherhizosphere (Chaignonand Hinsinger,2003;Uzuetal.,2009)and hence,as resultsshowed,plantscanmodifymetalaccumulationin earth-worms.
5.Conclusion
ThepresentstudyshowedthatsurvivalandgrowthofE.fetidais insensitivetoCu,Zn,Pband Cdmixtures,and regardlessofthe presence of V. faba or Z. mays. However, negative effects of contaminated soils on cocoon production were observed. The introductionofplantsdidnotaffectsignificantlycocoon produc-tionorweightpercocoon.Theseresultssuggestedthattheadult earthwormsusedhadatoleranceforthetestedconcentrationsof metals.
Thisstudyshowedthatinoculatingmetal-contaminatedsoils withE.fetidadecreasedCdandZnchemicalavailability.Theonly contraryresultwasobservedforconcentrationsofCdandZnin the presence of V. faba. Zn and Cu did accumulate in E. fetida tissues. The concentrations of Cd and Pb in the earthworms increasedwithincreasingconcentrationinthesoil.Thesefindings suggestthatearthwormsarelikelytoemploydifferentpathways fortheuptakeofdifferentmetals.Furthermore,theadditionofZ. mays or V. faba reduced the accumulation of Cd and Pb in earthwormtissues.Thisisprobablybecauseplantscan accumu-late part of the available Cd and Pb concentrations, effectively creatinganuptakecompetitionbetweenthedifferentorganisms. In plants, the accumulation of metals varied between the two species: V. faba plants accumulated higher Cu and Zn
concentrations,whereasZ.maysaccumulatedhigherCu concen-trations.AftertheadditionofE.fetida,higheruptakeofCdbyZ. mayswasobserved, indicatingthat E.fetidaearthworm activity enhancesCduptake forthisplantspecies.
Our study showed that metal accumulation is a complex process that cannot be predicted by measuring the available fraction of metals alone. The final tissue concentrations after exposuretothemetalsdependedonthephysiological character-isticsof the organisms(earthworms orplants), not onlyonits regulationpathwaysbutalsoonitsexposureroutes.Nevertheless, while further research to establish the optimum species and combinationsofthemtouseisneeded,thisstudysuggeststhat improvingphytoextractiontreatmentofindustrialsitespolluted witha mixture of metals by useof earthworms and plants is possible.
Acknowledgements
The author A. Lemtiri is thankful to the Department of BiosystemsEngineering,Soil–Water–PlantExchanges,Gembloux Agro-Bio Tech, University of Liege in Belgium. The present research was founded by the Department of Biosystems Engineering, Soil–Water–Plant Exchanges. The authors thank Prof.CluzeauDanielfromtheStationBiologiquePaimpont,CNRS, Université de Rennes. The authors wish thank Dina for her internshipin theDepartment of BiosystemsEngineering, Soil– Water–PlantExchanges.
References
Abou-Shanab,R.A.,Angle,J.S.,Delorme,T.A.,Chaney,R.L.,vanBerkum,P.,Moawad, H.,2003.Rhizobacterialeffectsonnickelextractionfromsoilanduptakeby Alyssummurale.NewPhytol.158,219–224.
Adriano,D.C.,2001.TraceElementsinTerrestrialEnvironments:Biogeochemistry, Bioavailability,andRiskofMetals,seconded.Springer-Verlang,NewYork. Ávila,G.,Gaete,H.,Sauvé,S.,Neaman,A.,2009.Organicmatterreducescopper
toxicityfortheearthwormEiseniafetidainsoilsfromminingareasinCentral Chile.Chil.J.Agric.Res.69,252–259.
Babich,H.,Stotzky,G.,1980.Environmentalfactorsthatinfluencethetoxicityof heavymetalsandgaseouspollutantstomicroorganisms.Crit.Rev.Microbiol.8, 99–145.
Basta,N.T.,Ryan,J.A.,Chaney,R.L.,2005.Traceelementchemistryin residual-treatedsoil:keyconceptsandmetalbioavailability.J.Environ.Qual.34,49–63. Becquer,T.,Dai,J.,Quantin,C.,Lavelle,P.,2005.Sourceofbioavailabletracemetals forearthwormsfromaZn-,Pb-andCd-contaminatedsoil.SoilBiol.Biochem.37, 1564–1568.
Beeby,A.,1993.Toxicmetaluptakeandessentialmetalregulationinterrestrial invertebrates.In:Dallinger,R.,Rainbow,P.S.(Eds.),EcotoxicologyofMetalsin Invertebrates.LewisPublishers,Chelsea,MI.
Brokbartold,M.,Wischermann,M.,Marschner,B.,2012.Plantavailabilityand uptakeoflead,zincandcadmiuminsoilscontaminatedwithanti-corrosion paintfrompylonsincomparisontoheavymetalcontaminatedurbansoils. WaterAirSoilPollut.223,199–213.
Brown,G.G.,1995.Howdoearthwormsaffectmicrofloraandfaunalcommunity diversity?PlantSoil63,247–269.
Brunet,J.,Repellin,A.,Varrault,G.,Terryn,N.,Zuily-Fodil,Y.,2009.Lead accumulationintherootsofgrasspea(LathyrussativusL.):anovelplantfor phytoremediationsystems?C.R.Biol.331,859–864.
Chaignon,V.,Hinsinger,P.,2003.Abiotestforevaluatingcopperbioavailabilityto plantsinacontaminatedsoil.J.Environ.Qual.32,824–833.
Chang,L.W.,Meier,J.R.,Smith,M.K.,1997.Applicationofplantandearthworm bioassaystoevaluateremediationofalead-contaminatedsoil.Arch.Environ. Contam.Toxicol.32,166–171.
Cheng,J.,Wong,M.H.,2002.EffectsofearthwormsonZnfractionationinsoils.Biol. Fert.Soils36,72–78.
Coeurdassier,M.,Scheifler,R.,deVaufleury,A.,Crini,N.,Saccomani,C.,DuMont,L.S., 2007.Earthwormsinfluencemetaltransferfromsoiltosnails.Appl.SoilEcol. 35,302–310.
Conder,J.M.,Lanno,R.P.,2000.Evaluationofsurrogatemeasuresofcadmium,lead, andzincbioavailabilitytoEiseniafetida.Chemosphere41,1659–1668. CordovaRosa,E.V.,Valgas,C.,Souza-Sierra,M.M.,Correa,A.X.R.,Radetski,C.M.,
2003.Biomassgrowth,micronucleusinduction,andantioxidantstressenzyme responsesinViciafabaexposedtocadmiuminsolution.Environ.Toxicol.Chem. 22,645–649.
Cotter-Howells,J.,Charnock,J.M.,Winters,C.,Kille,P.,Fry,J.C.,Morgan,A.J.,2005. Metalcompartmentationandspeciationinasoilsentinel:theearthworm, Dendrodrilusrubidus.Environ.Sci.Technol.9,7731–7740.
Curry,J.P.,Baker,G.H.,1998.Castproductionandsoilturnoverbyearthwormsinsoil coresfromSouthAustralianpastures.Pedobiologia42,283–287.
Dai,J.,Becquer,T.,Rouiller,J.H.,Reversat,G.,Bernhard-Reversat,F.,Nahmani,J., Lavelle,P.,2004.Heavymetalaccumulationbytwoearthwormspeciesandits relationshiptototalandDTPA-extractablemetalsinsoils.SoilBiol.Biochem.36, 91–98.
Dandan,W.,Huixin,L.,Feng,H.,Xia,W.,2007.Roleofearthworm–strawinteractions onphytoremediationofCucontaminatedsoilbyryegrass.ActaEcol.Sin.27, 1292–1298.
Dayton,E.A.,Basta,N.T.,Payton,M.E.,Bradham,K.D.,Schroder,J.L.,Lanno,R.P.,2006. Evaluatingthecontributionofsoilpropertiestomodifyinglead
phytoavailabilityandphytotoxicity.Environ.Toxicol.Chem.25,719–725. Dickson,K.L.,Giesy,J.P.,Wolfe,L.,1994.Bioavailability:Physical,Chemical,and
BiologicalInteractions.CRC,BocaRaton,FL.
Edwards,C.A.,Bohlen,P.J.,1996.BiologyandEcologyofEarthworms.Chapmanand Hall,London.
Ernst,G.,Zimmermann,S.,Christie,P.,Frey,B.,2008.Mercury,cadmiumandlead concentrationsindifferentecophysiologicalgroupsofearthwormsinforest soils.Environ.Pollut.156,1304–1313.
Feisthauer,N.C.,Stephenson,G.L.,Princz,J.I.,Scroggins,R.P.,2006.Effectsof metal-contaminatedforestsoilsfromtheCanadianshieldtoterrestrialorganisms. Environ.Toxicol.Chem.25,823–835.
Fitzpatrick,L.C.,Muratti-Ortiz,J.F.,Venables,B.J.,Goven,A.J.,1996.Comparative toxicityinearthwormsEiseniafetidaandLumbricusterrestrisexposedto cadmiumnitrateusingartificialsoilandfilterpaperprotocols.Bull.Environ. Contam.Toxicol.57,63–68.
Gong,B.,Wilke,M.,Strozzi,E.,Fleischmann,S.,2001.Evaluationandrefinementofa continuousseedgerminationandearlyseedlinggrowthtestfortheuseinthe ecotoxicologicalassessmentofsoils.Chemosphere44,491–500.
Graitson,E.,2005.InventaireetcaractérisationdessitescalaminairesenRégion wallonne.Nat.Mosana58,83–124.
Harlavan,Y.,Almogi-labin,A.,Herut,B.,2010.TracingnaturalandanthropogenicPb insedimentsalongtheMediterraneanCoastofIsraelusingPbisotopes.Environ. Sci.Technol.44,6576–6582.
Hashimoto,Y.,Takaoka,M.,Shiota,K.,2010.Enhancedtransformationoflead speciationinrhizospheresoilsusingphosphorousamendmentsand phytostabilization:anX-rayabsorptionfinestructurespectroscopy investigation.J.Environ.Qual.40,696–703.
Hobbelen,P.H.F.,Koolhaas,J.E.,vanGestel,C.A.M.,2006.Bioaccumulationofheavy metalsintheearthwormsLumbricusrubellusandApporectodeacaliginosain relationtototalandavailablemetalconcentrationsinfieldsoils.Environ.Pollut. 144,639–646.
Homa,J.,Niklinska,M.,Plytycz,B.,2003.Effectofheavymetalsoncoelomocytesof theearthwormAllolobophorachlorotica.Pedobiologia47,640–645.
Israr,M.,Jewell,A.,Kumar,D.,Sahi,S.V.,2011.Interactiveeffectsoflead,copper, nickelandzincongrowth,metaluptakeandantioxidativemetabolismof Sesbaniadrummondii.J.Hazard.Mater.186,1520–1526.
Kamnev,A.A.,vanderLeile,D.,2000.Chemicalandbiologicalparametersastoolsto evaluateandimproveheavymetalphytoremediation.Biosci.Rep.20(4),239– 258.
Khalil,M.A.,Abdel-Lateif,H.M.,Bayoumi,B.M.,vanStraalen,N.M.,1996.Analysisof separateandcombinedeffectsofheavymetalsonthegrowthofAporrectodea caliginosa(Oligochaeta;Annelida),usingthetoxicunitapproach.Appl.SoilEcol. 4,213–219.
Komarek,M.,Tlustos,P.,Szakova,J.,Chrastny,V.,Ettler,V.,2007.Theuseofmaize andpoplarinchelant-enhancedphytoextractionofleadfromcontaminated agriculturalsoils.Chemosphere67,640–651.
Kula,H.,Larink,O.,1997.Developmentandstandardizationoftestmethodsforthe predictionofsublethaleffectsofchemicalsonearthworms.SoilBiol.Biochem. 29,635–639.
Kumar,P.B.,Dushenkov,V.,Motto,H.,Raskin,I.,1995.Phytoextraction:theuseof plantstoremoveheavymetalsfromsoils.Environ.Sci.Technol.29,1232–1238. Lakanen,E.,Erviö,R.,1971.Acomparisonofeightextractantsforthedetermination
ofplantavailablemicronutrientsinsoils.ActaAgral.Fenn.123,223–232. Lanno,R.,Wells,J.,Conder,J.,Bradham,K.,Basta,N.,2004.Thebioavailabilityof
chemicalsinsoilforearthworm.Ecotoxicol.Environ.Saf.57,39–47. Lemtiri,A.,Colinet,G.,Alabi,T.,Cluzeau,D.,Zirbes,L.,Haubruge,E.,Francis,F.,2014.
Impactsofearthwormsonsoilcomponentsanddynamics:areview.Biotechnol. Agron.Soc.Environ.18,121–133.
Li,L.Y.,Li,R.S.,2000.TheroleofclaymineralsandeffectofH+
ionsonremovalof heavymetal(Pb2+
)fromcontaminatedsoil.Can.Geotech.J.37,296–307. Li,L.Z.,Zhou,D.M.,Peijnenburg,W.J.G.M.,Wang,P.,vanGestel,C.A.M.,Jin,S.Y.,Wang,
Q.Y.,2010.UptakepathwaysandtoxicityofCdandZnintheearthwormEisenia fetida.SoilBiol.Biochem.42,1045–1050.
Li,M.,Liu,Z.T.,Xu,Y.,Cui,Y.B.,Li,D.S.,Kong,Z.M.,2009.ComparativeeffectsofCd andPbonbiochemicalresponseandDNAdamageintheearthwormEisenia fetida(Annelida,Oligochaeta).Chemosphere74,621–625.
Li,N.Y.,Li,Z.A.,Zhuang,P.,Zou,B.,McBride,M.,2009.Cadmiumuptakefromsoil maizewithintercrops.WaterAirSoilPollut.199,45–56.
Li,X.D.,Thornton,I.,2001.Chemicalpartitioningofheavyandmajorelementsin soilscontaminatedbyminingandsmeltingactivities.Appl.Geochem.16,1693– 1706.
Liénard,A.,Bock,L.,Colinet,G.,2011.Intérêtdescartesdessolspourl’élaboration d’unestratégied’échantillonnageensolscontaminésparretombées atmosphériques:applicationàl'étudedel’effetsolsurledevenirdeséléments tracesmétalliques.Biotechnol.Agron.Soc.Environ.15,669–682.
Liénard,A.,Brostaux,Y.,Colinet,G.,2014.SoilcontaminationnearaformerZn–Pb ore-treatmentplant:evaluationofdeterministicfactorsandspatialstructures atthelandscapescale.J.Geochem.Explor.147,107–116.
Lock,K.,Janssen,C.R.,2003a.InfluenceofagingonZnbioavailabilityinsoils. Environ.Pollut.126,371–374.
Lock,K.,Janssen,C.R.,2003b.Influenceofagingoncopperbioavailabilityinsoils. Environ.Toxicol.Chem.22,1162–1166.
Lukkari,T.,Teno,S.,Vaeisaenen,A.,Haimi,J.,2006.Effectsofearthwormson decompositionandmetalavailabilityincontaminatedsoil:microcosmstudies ofpopulationswithdifferentexposurehistories.SoilBiol.Biochem.38,359– 370.
Ma,W.C.,1984.Sublethaltoxiceffectsofcopperongrowth,reproductionand litterbreakdownactivityintheearthwormLumbricusrubellus,with observationsontheinfluenceoftemperatureandsoilpH.Environ.Pollut.33, 207–219.
Ma,W.C.,1988.Toxicityofcoppertolumbricidearthwormsinsandyagricultural soilsamendedwithCu-enrichedorganicwastematerials.Ecol.Bull.39,53– 56.
Maboeta,M.S.,Reinecke,S.A.,Reinecke,A.J.,2004.Therelationshipbetween lysosomalbiomarkerandorganismalresponsesinanacutetoxicitytestwith Eiseniafetida(Oligochaeta)exposedtothefungicidecopperoxychloride. Environ.Res.96,95–101.
Marinussen,M.P.J.C.,vanderZee,S.E.A.T.M.,deHan,F.A.M.,Bouwman,L.M.,Hefting, M.M.,1997.Heavymetal(copper,lead,andzinc)accumulationandexcretionby theearthworm,Dendrobaenaveneta.J.Environ.Qual.26,278–284. McCredie,T.,Parker,L.,1992.TheroleofearthwormsinWesternAustralia
agriculture.J.Agric.WesternAust.33,160–165.
McGrath,S.P.,Cegarra,J.,1992.Chemicalextractabilityofheavymetalsduringand afterlong-termapplicationsofsewagesludgetosoil.J.SoilSci.43,313–321. Mench,M.,Martin,E.,1991.Mobilizationofcadmiumandothermetalsfromtwo
soilsbyrootexudatesofZeamaysL.:NicotianatabacumL.andNicotianarustica L.PlantSoil132,187–196.
Morgan,A.J.,1981.Amorphologicalandelectronmicroprobestudyoftheinorganic compositionofthemineralisedsecretoryproductsofthecalciferousglandand chloragogenoustissueoftheearthwormLumbricusterrestrisL.CellTissueRes. 220,829–844.
Morgan,A.J.,Morris,B.,1982.Theaccumulationandintracellular
compartmentationofcadmium,lead,zinc,andcalciumintwoearthworm species(DendrobaenarubidaandLumbricusrubellus)livinginahighly contaminatedsoil.Histochemistry25,269–285.
Morgan,A.J.,Winters,C.,1982.Theelementalcompositionofthechloragosomesof twoearthwormspeciesLumbricusterrestrisandAllolobophoralongadetermined byelectronprobeX-raymicroanalysisoffreeze-driedcryosections. Histochemistry73,589–598.
Nahmani,J.Y.,Hodson,M.E.,Black,S.,2007.Areviewofstudiesperformedtoassess metaluptakebyearthworms.Environ.Pollut.145,402–424.
Nannoni,F.,Protano,G.,Riccobono,F.,2011.Uptakeandbioaccumulationofheavy elementsbytwoearthwormspeciesfromasmeltercontaminatedareain northernKosovo.SoilBiol.Biochem.43,2359–2367.
Nelson,D.W.,Sommers,L.E.,1996.Totalcarbon,organiccarbon,andorganicmatter. In:Sparks,D.L.,Page,A.L.,Helmke,P.A.,Loeppert,R.H.,Soltanpour,P.N., Tabatabai,M.A.,Johnston,C.T.,Sumner,M.E.(Eds.),MethodsofSoilAnalysisPart 3—ChemicalMethods.SoilScienceSocietyofAmericaInc.,Madison,pp.961– 1010.
Neuhauser,E.F.,Loehr,R.C.,Milligan,D.L.,Malecki,M.R.,1985.Toxicityofmetalsto theearthwormEiseniafetida.Biol.Fertil.Soils1,149–152.
OrganisationforEconomicCo-operationandDevelopment,1984.Earthwormacute toxicitytests.GuidelinefortheTestingofChemicals,No.207.OECD,Paris. OrganisationforEconomicCo-operationandDevelopment,2004.Earthworm
reproductiontests(Eiseniafetida/Eiseniaandrei).GuidelinefortheTestingof Chemicals,No.222.OECD,Paris.
Oste,L.,Dolfing,J.,Ma,W.,Lexmond,T.M.,2001.Cadmiumuptakebyearthwormsas relatedtotheavailabilityinthesoilandtheintestine.Environ.Toxicol.Chem. 20,1785–1791.
Papoyan,A.,Pineros,M.,Kochian,L.V.,2007.PlantCd2+
andZn2+
statuseffectson rootandshootheavymetalaccumulationinThlaspicaerulescens.PlantPhysiol. 175,51–58.
Peijnenburg,W.J.G.M.,Jager,T.,2003.Monitoringapproachestoassess bioaccessibilityofmetals:matrixissues.Ecotoxicol.Environ.Saf.56,63–77. Peijnenburg,W.J.G.M.,Vijver,M.G.,2009.Earthwormsandtheiruseineco(toxico)
logicalmodeling.In:Devillers,J.(Ed.),EcotoxicologyModeling:Emerging TopicsinEcotoxicology,vol2.Springer,NewYork,pp.177–204.
Pignattelli,S.,Colzi,I.,Buccianti,A.,Cecchi,L.,Arnetoli,M.,Monnanni,R.,Gabbrielli, R.,Gonnelli,C.,2012.Exploringelementaccumulationpatternsofametal excluderplantnaturallycolonizingahighlycontaminatedsoil.J.Hazard.Mater. 227,362–369.
Qiu,H.,Vijver,M.G.,Peijnenburg,W.J.G.M.,2011.Interactionsofcadmiumandzinc impacttheirtoxicitytotheearthwormAporrectodeacaliginosa.Environ. Toxicol.Chem.30,2084–2093.
Rahoui,S.,Chaoui,A.,Ferjani,E.,2008.Differentialsensitivitytocadmiumin germinatingseedsofthreecultivarsoffababean(ViciafabaL.).ActaPhysiol. Plant30,451–456.
Reddy,N.C.,Rao,J.V.,2008.Biologicalresponseofearthworm,Eiseniafoetida (Savigny)toanorganophosphorouspesticide.Profenofos.Ecotoxicol.Environ. Saf.71,574–582.
Reinecke,A.J.,Reinecke,S.A.,Maboeta,M.S.,2001.Cocoonproductionandviability asendpointsintoxicitytestingofheavymetalswiththreeearthwormspecies. Pedobiologia45,61–68.
Reinecke,A.J.,Viljoen,S.A.,1990.Theinfluenceofwormdensityongrowthand cocoonproductionofthecompostwormEiseniafetida(Oligochaeta).Rev.Ecol. Biol.Sol27,221–230.
Rüdiger,H.,Ralf-Rainer,M.,2010.CellBiologyofMetalsandNutrients.Springer, Heidelberg,Germany.
Salt,D.E.,Smith,R.D.,Raskin,I.,1998.Phytoremediation.PlantPhysiol.PlantMol. Biol.49,643–668.
Scheu,S.,1987.Theroleofsubstratefeedingearthworms(Lumbricidae)for bioturbationinabeechwoodsoil.Oecologia72,192–196.
Schreck,E.,Foucault,Y.,Geret,F.,Pradere,P.,Dumat,C.,2011.Influenceofsoilageing onbioavailabilityandecotoxicityofleadcarriedbyprocesswastemetallic ultrafineparticles.Chemosphere85,1555–1562.
Sheppard,S.C.,Bembridge,J.D.,Holmstrup,M.,Posthuma,L.,1997.Advancesin earthwormecotoxicology.ProceedingsfromtheSecondInternational WorkshoponEarthwormEcotoxicology,SETAC,Amsterdam. Sizmur,T.,Hodson,M.E.,2009.Doearthwormsimpactmetalmobilityand
availabilityinsoil?Areview.Environ.Pollut.157,1981–1989.
Smical,A.I.,Hotea,V.,Oros,V.,Juhasz,J.,Pop,E.,2008.Studiesontransferand bioaccumulationofheavymetalsfromsoilintolettuce.Environ.Eng.Manag.J. 7,609–615.
Spurgeon,D.J.,Hopkin,S.P.,1995.Extrapolationofthelaboratory-basedOECD earthwormtoxicitytesttometal-contaminatedfieldsites.Ecotoxicology4, 190–205.
Spurgeon,D.J.,Hopkin,S.P., 1996.Effectsofmetal-contaminatedsoilsonthegrowth, sexualdevelopment,andearlycocoonproductionoftheearthwormEisenia fetida,withparticularreferencetozinc.Ecotoxicol.Environ.Saf.35,86–95. Spurgeon,D.J.,Hopkin,S.P.,1999.Tolerancetozincinpopulationsoftheearthworm
Lumbricusrubellusfromuncontaminatedandmetal-contaminatedecosystems. Arch.Environ.Contam.Toxicol.37,332–337.
Spurgeon,D.J.,Hopkin,S.P.,Jones,D.T.,1994.Effectsofcadmium,copper,leadand zincongrowth,reproductionandsurvivaloftheearthwormEiseniafetida (Savigny):assessingtheenvironmentalimpactofpoint-sourcemetal contaminationinterrestrialecosystems.Environ.Pollut.84,123–130. Spurgeon,D.J.,Svendsen,C.,Rimmer,V.R.,Hopkin,S.P.,Weeks,J.M.,2000.Relative
sensitivityoflife-cycleandbiomarkerresponsesinfourearthwormspecies exposedtozinc.Environ.Toxicol.Chem.19,1800–1808.
Udovic,M.,Lesta,n.D.,2007.Theeffectofearthwormsonthefractionationand bioavailabilityofheavymetalsbeforeandaftersoilremediation.Environ. Pollut.148,663–668.
Ünyayar,S.,Celik,A.,Cekic,F.O.,Gozel,A.,2006.Cadmium-inducedgenotoxicity, cytotoxicityandlipidperoxidationinAlliumsativumandViciafaba. Mutagenesis21,77–81.
Uzu,G.,Sobanska,S.,Aliouane,Y.,Pradere,P.,Dumat,C.,2009.Studyof leadphytoavailabilityforatmosphericindustrialmicronicandsub-micronic particlesinrelationwithleadspeciation.Environ.Pollut.157,1178–1185. vanderLelie,D.,1998.Biologicalinteractions:theroleofsoilbacteriainthe
bioremediationofheavymetal-pollutedsoils.In:Vangronsveld,J., Cunningham,S.D.(Eds.),Metal-ContaminatedSoils.InsituInactivationand Phytorestoration.Springer-VerlagBerlinHeidelberg,NewYork,pp.31–50. vanGestel,C.A.M.,Borgman,E.,Verweij,R.A.,DiezOrtiz,M.,2011.Theinfluenceof
soilpropertiesonthetoxicityofmolybdenumtothreespeciesofsoil invertebrates.Ecotoxicol.Environ.Saf.74,1–9.
vanGestel,C.A.M.,DirvenvanBreemen,E.M.,Baerselman,R.,1993.Accumulation andeliminationofcadmium,chromiumandzincandeffectsongrowthand reproductioninEiseniaandrei(Oligochaeta,Annelida).Sci.TotalEnviron.Suppl. 134,585–597.
vanGestel,C.A.M.,Rademaker,M.C.J.,VanStraalen,N.M.,1995.Capacitycontrolling parametersandtheirimpactonmetaltoxicity.In:Salomons,W.,Stigliani,W.M. (Eds.),BiogeodynamicsofPollutantsinSoilsandSediments.Springer,NewYork, pp.171–192.
vanGestel,C.A.M.,vanBreemen,E.M.D.,Baerselman,R.,1992.Influenceof environmentalconditionsonthegrowthandreproductionoftheearthworm Eiseniaandreiinanartificialsoilsubstrate.Pedobiologia36,109–120. vanGestel,M.,vanDis,W.A.,DirvenvanBreemen,E.M.,Sparenburg,P.M.,
Baerselman,R.,1991.Influenceofcadmiumcopperandpentachlorophenolon growthandsexualdevelopmentofEiseniaandrei(Oligochaeta,Annelida).Biol. Fert.Soils12,117–121.
Vijver,M.G.,Elliott,E.G.,Peijnenburg,W.J.G.M.,DeSnoo,G.R.,2011.Response predictionsfororganismswater-exposedtometalmixtures:ameta-analysis. Environ.Toxicol.Chem.30,1482–1487.
Walkley,A.,Black,I.A.,1934.AnexaminationoftheDegtjareffmethodfor determiningsoilorganicmatter,andaproposedmodificationofthechromic acidtitrationmethod.SoilSci.37,29–38.
Wang,D.,Li,H.,2006.Effectofearthwormsonthephytoremediationof zinc-pollutedsoilbyryegrassandIndianmustard.Biol.Fert.Soils43,120–123. Weeks,J.M.,Sorokin,N.,Johnson,I.J.,Whitehouse,P.,Ashton,D.,Spurgeon,D.,
Hankard,P.,Svendsen,C.,2004.Biologicaltestmethodsforassessing contaminatedland.ScienceGroupReportP5-069/TR1.EnvironmentAgency, Bristol,UK.
Weltje,L.,1998.MixturetoxicityandtissueinteractionsofCd,Cu,Pb,andZnin earthworms(Oligochaeta)inlaboratoryandfieldsoils:acriticalevaluationof data.Chemosphere36,2643–2660.
Wen,B.,Hu,X.,Liu,Y.,Wang,W.,Feng,M.,Shan,X.,2004.Theroleofearthworms (Eiseniafetida)ininfluencingbioavailabilityofheavymetalsinsoils.Biol.Fert. Soils40,181–187.
Wen,B.,Liu,Y.,Hu,X.,Shan,X.,2006.Effectofearthworms(Eiseniafetida)onthe fractionationandbioavailabilityofrareearthelementsinnineChinesesoils. Chemosphere63,1179–1186.
WorldReferenceBaseforSoilResources,2014.Internationalsoilclassification systemfornamingsoilsandcreatinglegendsforsoilmaps.WorldSoil ResourcesReportsNo.106.FAO,Rome.
Yu,X.,Cheng,J.,Wong,M.,2005.Earthworm–mycorrhizainteractiononCduptake andgrowthofryegrass.SoilBiol.Biochem.37,195–201.
