Metal Tolerance, Accumulation and Translocation in Poplarand Willow Clones Treated with Cadmium in Hydroponics

Massimo Zacchini & Fabrizio Pietrini &Giuseppe Scarascia Mugnozza & Valentina Iori &Lucia Pietrosanti & Angelo Massacci

Received: 14 February 2008 /Accepted: 22 June 2008 /Published online: 9 July 2008# Springer Science + Business Media B.V. 2008

Abstract To evaluate the phytoremediation capabili-ty of some poplar and willow clones a hydroponicscreening for cadmium tolerance, accumulation andtranslocation was performed. Rooted cuttings wereexposed for 3 weeks to 50 μM cadmium sulphatein a growth chamber and morpho-physiologicalparameters and cadmium content distribution invarious parts of the plant were evaluated. Total leafarea and root characteristics in clones and specieswere affected by cadmium treatment in differentways. Poplar clones showed a remarkable variabilitywhereas willow clones were observed to be morehomogeneous in cadmium accumulation and distri-bution. This behaviour was further confirmed by thecalculation of the bio-concentration factor (BCF) andthe translocation factor (Tf). Mean values of all theclones of the two Salicaceae species showed that

willows had a far greater ability to tolerate cadmiumthan poplars, as indicated by the tolerance index (Ti),calculated on the dry weight of roots and shoots ofplants. As far as the mean values of Tf was concerned,the capacity of willows to translocate was double thatof poplars. On the contrary, the mean values of totalBCF in poplar clones was far higher with respect tothose in willows. The implications of these results inthe selection of Salicaceae clones for phytoremediationpurposes were discussed.

Keywords Bioconcentration factor . Cadmium .

Hydroponic culture . Phytoremediation . Poplar .

Translocation factor .Willow

1 Introduction

The enhanced level of pollutants in soil and water dueto industrialisation is one of the major environmentalproblems on a global scale. In particular, cadmium isconsidered one of the most widespread pollutant havingtoxic effects on plants and animals. Cadmium enters theenvironment from industrial processes, heating systems,urban traffic, phosphate fertilizers and the mineralisationof rocks (Rauser and Muwly 1995). Plants exposed totoxic cadmium concentration undergo a stress condi-tion, revealed by harmful symptoms such as chlorosis,growth inhibition, a reduction in water and nutrientuptake, alteration of enzyme activity and photosynthe-

Water Air Soil Pollut (2009) 197:23–34DOI 10.1007/s11270-008-9788-7

M. Zacchini : F. Pietrini :V. Iori : L. Pietrosanti :A. Massacci (*)Institute of Agro-Environmental and Forest Biology,The National Council of Research,Via Salaria Km 29,300 Monterotondo Scalo,00015 Rome, Italye-mail: angelo.massacci@ibaf.cnr.it

G. Scarascia MugnozzaDISAFRI, University of Tuscia,Via San Camillo De Lellis,01100 Viterbo, Italy

sis impairment (Sanità di Toppi and Gabbrielli 1999;Pietrini et al. 2003). To remove cadmium and otherpollutants from contaminated areas, unconventionaltechniques involving biological processes have beensuccessfully applied. In particular, plants can be usedto remove heavy metals from soil and accumulatethem in the harvestable parts. This technology, calledphytoextraction (Kumar et al. 1995; Raskin et al. 1997;Padmavathiamma and Li 2007), is less expensive andless damaging to the environment than conventionalremediation systems that consist mainly in the excava-tion and incineration of soil (Cunningham and Ow1996). Another advantage of this technology is theproduction of biomass, which can eventually be used inproducing energy and other commodities. The efficien-cy of phytoextraction depends largely on the metalbioavailability present in the contaminated matrix aswell as on several characteristics of the plant such as thecapability to hyperaccumulate essential and unessentialmetals, a fast growth, a deep and extended root systemand the ability to translocate metals to the aerial parts.

Over the last few years, forest trees have beenstudied to assess how potential they are in remediat-ing heavy-metal contaminated sites (Rosselli et al.2003; Pulford and Watson 2003; Unterbrunner et al.2007). With regards to the phytoremediation strategysome aspects of forest tree biology and cultivation,i.e. the large biomass yield that can be used toproduce energy, an extended and deep root apparatus,a low impact on trophic chains and the adaptability ofsome tree species to grow in marginal soils, are allvery interesting. Metal uptake by trees is reported tobe small but, on a hectare scale, the removal of heavymetals from soil could be more effective respect tohyperaccumulating plants due to a greater yield ofbiomass (Greger and Landberg, 1999; Fischerováet al. 2006).

Several studies have focused their attention on thepotentiality of willows and poplars in phytoextraction(Riddell-Black 1994; Punshon and Dickinson 1999;Robinson et al. 2000; Pulford et al. 2002; Laureysenset al. 2004a; Kuzovkina and Quigley 2005). In fact,these Salicaceae are reported not only to be adaptableto growing in severe soil conditions, characteristic ofcontaminated areas, but also to be capable of accumu-lating heavy metals (Pulford and Watson 2003).Cultural management of willows and poplars by meansof short rotation coppice cultures (SRC) is anotherinteresting aspect to be considered in phytoremediation

strategies (Ceulemans et al. 1992; Scarascia-Mugnozzaet al. 1997; Perttu 1999; Rockwood et al. 2004). In thiscontext, Dickinson and Pulford (2005) have reportedthat willow SRC can be utilised as an efficient andcost-effective method in removing cadmium contami-nation from agricultural soil. Moreover, heavy metalaccumulation in poplar and willow clones variedsignificantly (Landberg and Greger 1996; Watson etal. 1999; Laureysens et al. 2004b).

Most of the studies carried out on trees reportedthat the heavy metal accumulation pattern shows apredominant compartmentalisation in the roots and alow translocation to the shoots. This is probably themajor constraint to overcome for a more efficientutilization of these species to rid soils of metalcontamination. Then, it is necessary to screen forestplant material to establish what plants are most adaptto translocate the absorbed metal to the aerial parts,especially to the stem tissues that are not renewablelike foliage and that can be harvested and utilised inenergy production.

Many authors have reported differences betweenwillow and poplar clones in the partitioning of heavymetals within the tree organs (Mills et al. 2000;Robinson et al. 2000, 2005; Lunáčková et al. 2003a;Fischerová et al. 2006; Unterbrunner et al. 2007).Nevertheless, few studies have compared theresponses of willow and poplar clones to the presenceof cadmium in a hydroponic system (Šottníková et al.2003; Lunáčková et al. 2003b; Dos Santos Utmazianet al. 2007). Hydroponic culture is a very useful toolfor selecting from a considerable number of individ-uals. In fact, it reduces not only the period of growthand the length of time of treatment of the plants butalso the space required to carry our the experiment. Inaddition variability due to the environmental factors isalso reduced. In general, data obtained by a hydroponicscreening need to be confirmed by field performancetrials, even if Watson et al. (2003) have pointed out thatresults obtained in hydroponics and in field experi-ments are similar.

This study was aimed at evaluating the response ofcadmium tolerance, accumulation and translocation indifferent poplar and willow clones in a hydroponicculture. The characterisation of several Salicaceaeclones to assess their effectiveness in tolerating andbio-concentrating cadmium is very important inspecifying the potential of these plant species tophytoremediate cadmium-polluted soils.

24 Water Air Soil Pollut (2009) 197:23–34

2 Materials and Methods

2.1 Plant Material and Growth Conditions

Previously rooted stem cuttings (20-cm-long) takenfrom poplar and willow clones (listed in Table 1) weredivided into two stocks to be treated in hydroponicswith 0 (control) or 50 μM of cadmium sulphate(Sigma, St. Louis, USA) for 3 weeks. Particularattention was paid to choose homogenous rootedcuttings to introduce randomly in the experimentaltreatment that consisted in pots filled with third-strengthHoagland’s nutrient solution, pH 6.5 (mol l−1): 1.34×10−3 Ca(NO3)2 4H2O, 1.74×10

−4 NH4H2PO4, 2.01×10−3 KNO3, 0.66×10−3 MgSO4 7H2O, 0.41×10−4

NaOH, 2.97×10−5 EDTA, 2.98×10−5 FeSO4 7H2O,3.22×10−6 H3BO3, 0.67×10−6 MnCl2 4H2O, 1.04×10−7 ZnSO4 7H2O, 0.7×10−7 CuSO4 5H2O, 0.46×10−7 MoO3, 2.86×10

−8 Co(NO3)2 6H2O (Arnon andHoagland 1940). Cuttings were grown in a controlledclimate chamber equipped with metal halide lamps(Powerstar HQI-TS; Osram, Munich, Germany) pro-viding a photon flux density of 300 μmol m−2 s−1 for14 h at 25°C. During the 10 h dark period thetemperature was 20°C. The relative humidity was 70–80%. The nutrient solutions were replaced entirelytwice a week to prevent depletion of metals andnutrients and to expose plants to a constant metalconcentration. An aeration system based on pumps wasused to avoid lack of oxygen. Each treatment groupconsisted of five cuttings from each clone. Thediameters of the cuttings ranged from 1.2 to 1.7 cmand no significant differences among clones wereobserved. At the end of the experimental period,control and treated plants were harvested and washedwith 0.05 M calcium chloride for 30 min in slowagitation without damaging the roots. After leaf androot measurements, plants were separated into aerialparts (leaves, secondary stems and original cutting) androots. The leaf area was measured using a leaf areameter Li 3000 (Licor, Nebraska, USA) and then eachplant part was dried in an oven at 80°C until a constantweight was reached.

2.2 Cadmium Determination

Metal concentration was measured by an atomicabsorption spectrophotometer (Perkin Elmer, Nor-walk, CT, USA) on digested samples of aerial parts

and roots. Dried material was milled to a fine powder(Tecator Cemotec 1090. SampleMill; Tecator, Hoganas,Sweden), then accurately weighed and mineralised.Mineralisation was performed by treating 250 mg ofpowdered samples with 6 ml of concentrated HNO3 and2.5 ml of H2SO4 and by heating (TMD20 HeaterSystem, Velp Scientifica, Milano, Italy) in a two stepprocedure: 120°C for 25 min followed by 250°C for15 min.

2.3 Bio-concentration Factor (BCF), TranslocationFactor (Tf) and Tolerance Index (Ti) Calculation

According to Zayed et al. (1998), the cadmium bio-concentration factor (BCF) of root system and aerialpart (stem+leaves and secondary stems) was calcu-lated as follows:

BCF ¼ cadmium concentration in the harvested plant material mg kg�1� �

cadmium concentration in the solution mg kg�1� �

The translocation factor (Tf) was calculated toevaluate the capability of plant to accumulate themetal, absorbed by roots, in the aerial part:

Tf ¼ cadmium concentration in the aerialparts mg kg�1� �

cadmium concentration in the the roots mg kg�1� � � 100

Table 1 Populus and Salix species tested in the experiment

Clone Species/hybrid Origin

A4A Populus×canadensis Mönch. ItalyLuisa Avanzo Populus×canadensis Mönch. ItalyI-214 Populus×canadensis Mönch. ItalyLux Populus deltoides Bartr. USA11-5 Populus×generosa A. Henry USAPoli Populus nigra L. Italy58-861 Populus nigra L. Italy6K3 Populus alba L. Italy14P11 Populus alba L. ItalyNisqually Populus trichocarpa Torr. & A.Gray USASS5 Salix alba L. ItalySP3 Salix alba L. Italy6-03 Salix alba L. Italy2-03 Salix alba L. ItalyQuirani Salix sp. autochthonous clonea ItalyCretone Salix sp. autochthonous clonea Italy

a Collected near sulphurous springs 30 Km N-E of Rome.

Water Air Soil Pollut (2009) 197:23–34 25

The tolerance index (Ti) was calculated to measurethe ability of the plant to grow in the presence of agiven concentration of metal, according to Wilkins(1978):

Ti ¼ Dry weight of the plants grown in cadmium solution

Dry weight of the plants grown in control solution� 100

Dry weight of plant was referred to roots, secondarystems and leaves.

2.4 Statistical Analysis

The data reported refer to a single typical experimentwith five replicates. Normally distributed data wereprocessed with a two-way analysis of variance(ANOVA) using the SPSS software tool.

3 Results

Effects due to cadmium were detected in all param-eters but not in the mean root number. Differences inall parameters were found both in poplar or willowclones. A significant interaction between poplar orwillow clones and cadmium treatment was found in allthe morpho-physiological parameters analysed but notin the mean root number in willow clones (Table 2).

In Fig. 1 a comparison between representativewillow and poplar plants whether treated or not withcadmium is reported. No chlorosis symptoms wererevealed in both plant species. Shoot and root growthin willows was not particularly affected by cadmiumexposition while a significant reduction of the sameoccurred in poplars.

Damage exerted by cadmium at leaf level is animportant aspect to evaluate in plants screened forphytoremediation. In fact, an efficient photosyntheticapparatus allows plants to maintain an effective

transpiration flux that drives metals from roots toaerial parts. Total leaf area is a parameter sensitive tocadmium presence in the growth medium. Figure 2shows the total leaf area of poplar and willow cloneswhether subjected or not to 21 days of cadmiumtreatment in a hydroponic experiment. All poplarclones revealed a dramatic reduction in total leaf areacaused by cadmium exposure. The heavy metal treat-ment affected especially the clones 11-5, I-214, L.Avanzo and 14P11 that showed a greater reduction inleaf area. A4A and Nisqually, that showed contrastingbehaviour under control condition, revealed less inhibi-tion than the other clones under metal treatment. Inwillows, 6-02 and 2-03 clones resulted particularlyaffected by cadmium treatment, while the other clonesshowed no reduction in total leaf area.

The root system plays a key role in the interactionbetween contaminants and plant. In poplar and willowclones the effect of cadmium on the root system wasanalysed measuring some morphological parameterssuch as the mean number of roots per plant, the meanroot length per plant and total root length. Cadmiumtreatment reduced the mean number of roots per plantonly in five poplar clones while no effect was observedin willow clones (data not shown). The mean rootlength per plant (Fig. 3) was noticeably affected bycadmium treatment. In poplars, eight clones out of tenshowed a significant reduction in length. The greatestreduction resulted in clone 11-5. Clones Lux and I-214 were not affected by metal treatment. In willows,clones SS5 and Quirani showed no inhibition in meanroot length as a result of cadmium treatment. On thecontrary, the mean root length of the other clones wasaffected more or less dramatically by such exposure.The entire root system, expressed as total root length(Fig. 4), was negatively influenced by metal treatmentin six out of ten poplar clones with a dramaticreduction in clones 14P11, 6K3 and 58-861, and a

Table 2 Observed significance levels (P values) for effects of clone, of cadmium treatment and their interaction from ANOVA forsome morpho-physiological parameters in poplar and willow cuttings grown in hydroponic solution

Factor Total leaf area Mean root length Mean root number Total root length

Poplar clone <0.001 <0.001 <0.001 <0.001Cadmium treatment <0.001 <0.001 0.205 <0.001Interaction <0.001 0.004 0.036 0.008Willow clone 0.009 <0.001 <0.001 <0.001Cadmium treatment 0.431 <0.001 0.505 0.018Interaction <0.001 0.049 0.347 0.045

26 Water Air Soil Pollut (2009) 197:23–34

consistent reduction in clones Luisa Avanzo, 11-5 andPoli. Clones Nisqually, A4A, I-214 and Lux werestatistically not affected by cadmium treatment. CloneLux showed the longest root system under metaltreatment among the tested poplar clones. In willowclones (Fig. 4), total root length was negativelyaffected by metal treatment in Cretone, SP-3 and 2-03 while no effect was observed in SS5, Quirani and6-03. SP-3 and 6-03 showed the longest root extensionamong the willow clones grown in cadmium addedsolution.

In Fig. 5 the concentration of cadmium detected inroots and the aerial parts of poplar and willowcuttings exposed for 3 weeks to 50 μM cadmiumsulphate is reported. Metal concentration in controlcuttings was below the threshold of detection. Aconsiderable concentration of cadmium was found inthe roots of each poplar clone, varying greatly onefrom the other. Root accumulation represented ap-proximately 95% of the total cadmium accumulatedby the whole plants. P. nigra (clones 58-861 and Poli)showed the highest metal root concentration while

A4A Lux

L.Avanzo14P11

11-5 6K3

NisquallyPoli58-861

I-214

Tot

al le

af a

rea

(cm

2 )

0

500

1000

1500

2000

2500

3000

Quirani

CretoneSP3

6-032-03

SS5

Poplar clones Willow clonesFig. 2 Total leaf area (cm2)measured at the end of theexperiment on poplar andwillow clones grown in thepresence of 0 (control, blackbars) and 50 μM (greybars) cadmium sulphate.Values are the mean of fivereplicates. Error bars indi-cate standard error

Fig. 1 Morphologicalaspect of willow (a) andpoplar (b) plants exposed to50 μM cadmium sulphate(T) compared to control (C)

Water Air Soil Pollut (2009) 197:23–34 27

clones Lux and 6K3 the lowest. Accumulation ofcadmium in the aerial parts of the plant among poplarclones varied noticeably. Clones Poli and Lux showedthe highest metal concentration while L.Avanzo, 58-861 and 6K3 the lowest.

In willows (Fig. 5), root cadmium concentrationwas very homogeneous among clones with the highestcontent detected in clone 6-03. Root cadmium concen-tration on average was approximately 87% of the totalconcentration measured in the whole plant. On thecontrary, in the aerial part of the plant cadmiumconcentration varied much more with the highest metalaccumulation found in clones 6-03 and Quirani and thelowest in 2-03.

To evaluate the capability of poplar and willowclones to extract and accumulate cadmium in theplant, the bio-concentration factor (BCF) was calcu-lated. In Fig. 6, the BCF of poplar and willow clones,referring to the root system and the aerial part of theplant, is reported. In poplars, the root BCF repre-sented approximately 97% of the whole plant BCF,with the highest root BCF resulting in clones 58-861and Poli and the lowest in 6K3. The aerial part BCFindicated Poli and Lux as the poplar clones with thegreatest capability to accumulate cadmium in leaf andstem tissues. In willows, the clone 6-03 resulted, fromroot BCF, as the most efficient cadmium bio-accumulator among the screened willow clones. The

Mea

n ro

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nght

(cm

)

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60Poplar clones

Quirani

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2-03SS5

Willow clones

A4A Lux

L.Avanzo14P11

11-5 6K3

NisquallyPoli58-861

I-214

Fig. 3 Mean root length(cm) measured at the end ofthe experiment on poplarand willow clones grown inthe presence of 0 (control,black bars) and 50 μM(grey bars) cadmium sul-phate. Values are the meanof five replicates. Error barsindicate standard error

Poplar clones

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t (cm

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Quirani

CretoneSP3 6-03

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L.Avanzo14P11

11-5 6K3

NisquallyPoli58-861

I-214

Fig. 4 Total root length(cm) measured at the end ofthe experiment on poplarand willow clones grown inthe presence of 0 μM (con-trol, black bars) and 50 μM(grey bars) cadmium sul-phate. Values are the meanof five replicates. Error barsindicate standard error

28 Water Air Soil Pollut (2009) 197:23–34

BCF of the aerial part showed that clones Quirani and6-03 were the most efficacious in bio-accumulatingcadmium in the above ground tissues while the clone2-03 was the least efficient.

The capability of poplar and willow clones toaccumulate cadmium in the above ground tissue wasfurther confirmed by calculating the translocationfactor (Tf), that indicated the percentage of theabsorbed metal that reached the aerial part of theplant respect to that present in the roots (Fig. 7). Thispercentage varied greatly among poplar clones. Thehighest Tf values were observed in clones Lux andPoli while the lowest were found in 58-861. Calcu-lation of the Tf in willow plants showed a similar

translocation capability among clones whereas in theclone 2-03 this value was slightly lower.

4 Discussion

The availability of selected plant material is a keyfactor if phytoremediation is to be efficiently appliedto different types of contaminated substrate (soil,water, sludge etc.).

Poplar and willow clones, due to their growth,genetic and cultural characteristics, are potential candi-dates in the remediation of contaminated substrates. Inthis work the behaviour of a significant number of

Cad

miu

m c

once

ntra

tion

(mg

. Kg-1

)

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A4ALux

L.Avanzo14P11

11-5 6K3

NisquallyPoli58-861

I-214Quirani

CretoneSP3

6-032-03

SS5

Poplar clones Willow clonesFig. 5 Cadmium concentra-tion (mg kg−1) in roots(black bars) and aerial parts(grey bars) of poplar andwillow clones grown in hy-droponics for 3 weeks in50 μM cadmium sulphate.Values are the mean of fivereplicates. Error bars indi-cate standard error

Bio

-con

cent

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ctor

(B

CF

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NisquallyPoli58-861

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Quirani

CretoneSP3

6-032-03

SS5

Poplar clones Willow clones

Fig. 6 Bio-concentrationfactor (BCF) in roots (blackbars) and aerial parts (greybars) of poplar and willowclones grown in hydropon-ics for 3 weeks in 50 μMcadmium sulphate. Valuesare the mean of five repli-cates. Error bars indicatestandard error

Water Air Soil Pollut (2009) 197:23–34 29

clones with regards to cadmium tolerance, accumulationand translocation to aerial parts were compared. Thesefundamental aspects should form the criteria to befollowed in screening plants for selection in phytor-emediation. Metal tolerance, and consequently theprotection of the integrity and functionality of theprimary physiological and metabolic processes (Pietriniet al. 2003), is an essential pre-requisite for a plant tobe utilised in phytoremediation. Nevertheless, thischaracteristic should be the result of a combination ofmetal absorption and reduction of damaging effects,and not be merely due to metal exclusion. Tolerance atroot level, that means the preservation of the selectiveproperty of the cell membrane, represents the first stepin metal absorption and loading into the xylem vessels.In the present work, the root system of poplar andwillow clones, analysed by following morphologicalparameters such as mean root length per plant and totalroot length, showed remarkable differences in toleranceto cadmium (Figs. 3 and 4). Total root length did notsignificantly result reduced in 40% of poplar clonesand in 50% of willow clones, and in the latter caseproved to be more tolerant than poplar clones tocadmium at root level.

Absorbed metal can be loaded onto xylem vessels bybinding it to organic acids, thiol and amine compounds(Kramer et al. 1996; Keltjens and van Beusichem 1998;Rauser 1999), to be transported to the shoots. In leaves,cadmium can represent a very toxic agent, since it candestroy thylacoidal membranes and alter enzymeactivities, hindering photosynthesis (Becerril et al.

1988; Pietrini et al. 2005). In the present work, theeffect of cadmium on leaves of poplar and willowclones was evaluated by measuring the total leaf area(Fig. 2). Poplar clones showed, on average, a moreevident reduction of the leaf area than willow clones.In fact, in willows the leaf area of only two clones outof six was negatively affected by the presence of metal,whereas in poplars, contrary to Pilipović et al. (2005),all clones presented a remarkable reduction in thisparameter. A reduction in leaf area in poplar andwillow species following cadmium treatment was alsofound by Lunáčková et al. (2003a). Differencesbetween the two species for what regards cadmiumtolerance was further confirmed by the analysis of thetolerance index, Ti (Table 3). On the basis of the drybiomass of the total plants, the Ti revealed that onaverage willows tolerate cadmium much more thanpoplars. According to the scheme proposed by Luxet al. (2004), willow clones tested in this work can bedefined as highly tolerant (Ti>60) while poplar clonesas moderately tolerant (Ti between 35 and 60). Withrespect to these data, a relatively higher Ti of root andleaves in some willows and poplar clones was reportedby Dos Santos Utmazian et al. (2007) but in that casethe cadmium concentration was far less than that usedin our experiment. Moreover, a remarkable differencein cadmium tolerance among willow clones was foundby Punshon and Dickinson (1999) and Kuzovkinaet al. (2004).

Cadmium accumulation differed greatly in poplarclones while in willows it was more homogeneous

Tra

nslo

catio

n fa

ctor

(T

f)

0

10

20

30

A4ALux

L.Avanzo14P11

11-5 6K3

NisquallyPoli

58-861I-2

14Quirani

CretoneSP3

6-032-03

SS5

Poplar clones Willow clonesFig. 7 Translocation factor(Tf) calculated at the end ofthe experiment on poplarand willow clones grown inthe presence of 50 μM cad-mium sulphate. Data angletransformation (arcsin

ffiffiffiffi%

p)

was performed. Values arethe mean of five replicates.Error bars indicate standarderror

30 Water Air Soil Pollut (2009) 197:23–34

(Fig. 5). On average the amount of cadmium thatpoplar clones were capable of accumulating wasdouble that of willow clones (Table 3) even if it wasalmost entirely confined in roots. On the contrary,willows showed a greater ability than poplars toaccumulate the metal in the aerial part. Data reportedin literature about cadmium concentration and alloca-tion among organs are often contradictory and this ismainly due to the different experimental conditionsadopted. In fact, metal concentration, type and lengthof exposure (whether the metal solution is renewedover time or not) and type of substrate can noticeablymodify metal availability for the plant. Moreover,washing procedures at the end of the experiment canaffect the quantity of metal measured in roots. In thepresent work, root washing was performed withcalcium chloride for a far shorter time with respectto Dos Santos Utmazian et al. (2007) and this couldexplain, together with the different metal concentra-tion used, why the cadmium accumulation differedfrom that found by those authors. Nevertheless, ourdata were in accordance with those previouslyreported by Robinson et al. (2000) and Lunáčkováet al. (2003b), in different experimental trials, show-ing a prominent cadmium accumulation in poplarroots and a more evident capability in willows toallocate metal in the aerial part. In this context, thebioconcentration factor (BCF) can give further valu-able information regarding the capability for theseSalicaceae to extract metal from a contaminatedmatrix. Poplar and willow clones, tested in this work,exhibited very different BCF values in root and aerialpart (Fig. 6) and, on average, poplar clones showeddouble the capability to remove metal from thesolution with respect to willow clones (Table 3),expressed as total BCF. A comparison with otherstudies, as has already been stated above in the caseof cadmium accumulation, is difficult to perform dueto the entirely different conditions in substrate andmetal concentration used. However, the BCF calcu-

lated in the present work was consistently lower thanthat reported in similar hydroponic studies by Ait Aliet al. (2004) and Wang et al. (2008) even if they werecarried out on herbaceous plants with lower cadmiumconcentrations.

The ability to accumulate metal in the aerial partswith respect to roots can be better illustrated bycalculating the translocation factor (Tf). In this workthe Tf in willows was double that in poplars (Table 3).That willows have a better capacity to translocatecadmium (expressed as leaf:root ratio) with respect topoplars in a hydroponic experiment was also reportedby Dos Santos Utmazian et al. (2007). It is worthmentioning that the Tf in Salicaceae clones, measuredin our work, is lower than that found in herbaceousplants cultivated in pots by Mattina et al. (2003) andMarchiol et al. (2004) or in wetlands (Deng et al.2004), though it is difficult to compare the results dueto the different experimental conditions. Evaluation ofthe Ti, BCF and Tf in poplar and willow clones in thepresent experiment confirms that Salicaceae plantshave a considerable potentiality to remove cadmiumfrom a contaminated medium that could be increasedby improving the translocation of the metal to shoots.

The comparison between poplars and willowsregarding cadmium accumulation and distributionrevealed that these two Salicaceae species could eachbe used for different purposes in phytoremediationstrategies. On the one hand, in fact, poplars whichshowed a remarkable ability to bio-concentrate cad-mium in the root system, could be efficiently used inthe remediation of polluted water (rhizofiltration) orcontaminated sites to limit metal percolating to thewater layer (phytostabilisation). On the other hand,willow clones proved to be potentially promising intranslocating and concentrating cadmium in the aboveground organs associated with a great metal tolerance.Hence, this species could be very useful in removingpollutants from the soil to the harvestable parts ofplants (phytoextraction) and, if cultured in SRC

Table 3 Comparison between poplar and willow for tolerance index (Ti), bio-concentration factor (BCF), translocation factor (Tf),cadmium concentration (mg kg−1) in roots and aerial parts of plants

Plant species Ti BCF Tf Cadmium in roots Cadmium in aerial parts

Poplar 45 (±3.03) 159 (±9.08) 10 (±0.45) 9962 (±563) 293 (±19)Willow 73 (±3.19) 80 (±2.92) 23 (±0.68) 4296 (±164) 651 (±36)

Data (± E.S.) refer to mean value of cuttings from all clones grown in hydroponics for 3 weeks in 50 μM cadmium sulphate. For Tiand Tf, data angle transformation (arcsin

ffiffiffiffi%

p) was performed.

Water Air Soil Pollut (2009) 197:23–34 31

management, could yield also biomass for energyproduction, thereby carrying out a double ecologicalservice.

The effective capability of these Salicaceae clonesto accumulate cadmium in the above ground organs iscurrently under evaluation in an outdoor mesocosmsystem covering the whole growing season. Thissystem is a cultural technique more similar to theenvironmental conditions of an open-field thanhydroponics. Preliminary results showed only a slightdecrease in the BCF and Tf values in willow andpoplar plants cultivated in a mesocosm system withrespect to hydroponics (data not shown), confirmingtheir potentiality to extract cadmium from a contam-inated matrix and accumulate it in the aerial part.

Metal translocation to the above ground organs is acrucial biochemical process in an effective utilisationof plants to remediate polluted sites. In fact, a moreefficient mobilisation of metals from root to the aboveground organs could reduce the damaging effectsexerted by these pollutants on root physiology andbiochemistry. This would improve the effectiveness ofplant metal uptake allowing metal removal from thecontaminated substrate over time. Therefore metaltolerance, bioaccumulation and translocation capabil-ity must be considered together to evaluate species,clones or individuals with interesting perspectives inphytoremediation, in order to characterise the bio-chemical and molecular traits involved in theseprocesses. From results obtained in this trial poplarclones showed contrasting behaviour in cadmium bio-concentration and allocation in plant parts. Twoclones of Populus nigra, Poli and 58-861, exhibitedparticular and interesting characteristics to be betterelucidated. In fact, both clones showed the highestlevel of metal accumulation among poplar clones.However, Poli proved to have a remarkable abilitywhereas 58-861 exhibited the lowest ability to bio-concentrate cadmium in the aerial part. Willow cloneswere more homogeneous regarding cadmium toler-ance and accumulation but no clear indications wereobtained since the most efficient cadmium accumula-tor clone, 6-03, was also the most affected bycadmium exposure at leaf level. Interesting responseswere then obtained in the clone SS5 and in theautochthonous clone Quirani. It is worth noting thatthe Quirani clone was collected in a sulphurous areaand cadmium is a well known sulphur-philic metal.Studies are still in progress to characterise poplar and

willow clones for what concerns the biochemical andmolecular processes involved in the accumulation andtranslocation of cadmium to the above ground organs.

Acknowledgements This work was funded by MIUR (Min-istry for Education, University and Research) under PRIN 2005project no. 2005-072892. Authors also wish to thank Prof.Paolo Sequi (CRA-RPS) for research collaboration withinPRAL research project and Antonio Barchetti (CRA-RPS) forvaluable technical assistance.

References

Ait Ali, N., Pilar Bernal, M., & Ater, M. (2004). Tolerance andbioaccumulation of cadmium by Phragmites australisgrown in the presence of elevated concentrations ofcadmium, copper and zinc. Aquatic Botany, 80, 163–176.doi:10.1016/j.aquabot.2004.08.008.

Arnon, D. I., & Hoagland, D. R. (1940). Crop production inartificial culture solutions and in soils with specialreference to factors influencing yields and absorption ofinorganic nutrient. Soil Science, 50, 463–483.

Becerril, J. M., Munoz-Rueda, A., Aparicio-Tejo, P., &Gonzales-Murua, C. (1988). The effects of cadmium andlead on photosynthetic electron transport in clover andlucerne. Plant Physiology and Biochemistry, 26, 357–363.

Ceulemans, R., Scarascia-Mugnozza, G., Wiard, B. M.,Braatne, J. H., Hinckley, T. M., Stettler, R. F., et al.(1992). Production physiology and morphology of Populusspecies and their hybrids grown under short rotation. I.Clonal comparisons of 4-year growth and phenology.Canadian Journal of Forest Research, 22, 1937–1948.

Cunningham, S. D., & Ow, D. W. (1996). Promises and prospectsof phytoremediation. Plant Physiology, 110, 715–719.

Deng, H., Ye, Z. H., & Wong, M. H. (2004). Accumulation oflead, zinc, copper and cadmium by 12 wetland plant speciesthriving in metal-contaminated sites in China. EnvironmentalPollution, 132, 29–40. doi:10.1016/j.envpol.2004.03.030.

Dickinson, N. M., & Pulford, I. D. (2005). Cadmiumphytoextraction using short-rotation coppice Salix: Theevidence trail. Environment International, 31, 609–613.doi:10.1016/j.envint.2004.10.013.

Dos Santos Utmazian, M. N., Wieshammer, G., Vega, R., &Wenzel, W. W. (2007). Hydroponic screening for metalresistance and accumulation of cadmium and zinc in twentyclones of willows and poplars. Environmental Pollution,148, 155–165. doi:10.1016/j.envpol.2006.10.045.

Fischerová, Z., Tlustos, P., Szakova, J., & Sichorova, K. (2006).A comparison of phytoremediation capability of selectedplant species for given trace elements. EnvironmentalPollution, 144, 93–100. doi:10.1016/j.envpol.2006.01.005.

Greger, M., & Landberg, T. (1999). Use of willow inphytoextraction. International Journal of Phytoremediation,1, 115–123. doi:10.1080/15226519908500010.

Keltjens, W. G., & van Beusichem, M. L. (1998). Phytochelatinsas biomarkers for heavy metal stress in maize (Zea mays L.)and wheat (Triticum aestivum L.): Combined effects ofcopper and cadmium. Plant and Soil, 203, 119–126. doi:10.1023/A:1004373700581.

32 Water Air Soil Pollut (2009) 197:23–34

Kramer, U., Cotter-Howells, J. D., Charnock, J. M., Baker, A.J. M., & Smith, J. A. C. (1996). Free histidine as a metalchelator in plants that accumulate nickel. Nature, 379,635–638. doi:10.1038/379635a0.

Kumar, P. B. A. N., Dushenkov, V., Motto, H., & Raskin, I.(1995). Phytoextraction: The use of plants to removeheavy metals from soils. Environmental Science &Technology, 29, 1232–1238. doi:10.1021/es00005a014.

Kuzovkina, Y. A., Knee, M., & Quigley, M. F. (2004). Cadmiumand copper uptake and translocation in five willow (Salix L.)species. International Journal of Phytoremediation, 6, 269–287. doi:10.1080/16226510490496726.

Kuzovkina, Y. A., & Quigley, M. F. (2005). Willows beyondwetlands: Uses of Salix L. species for environmentalprojects. Water, Air, and Soil Pollution, 162, 183–204.doi:10.1007/s11270-005-6272-5.

Landberg, T., & Greger, M. (1996). Differences in uptake andtolerance to heavy metals in Salix from unpolluted andpolluted areas. Applied Geochemistry, 11, 175–180.doi:10.1016/0883-2927(95)00082-8.

Laureysens, I., Blust, R., De Temmerman, L., Lemmens, C., &Ceulemans, R. (2004a). Clonal variation in heavy metalaccumulation and biomass production in a poplar coppiceculture: I. Seasonal variation in leaf, wood and barkconcentrations. Environmental Pollution, 131, 485–494.doi:10.1016/j.envpol.2004.02.009.

Laureysens, I., Bogaert, J., Blust, R., & Ceulemans, R. (2004b).Biomass production of 17 poplar clones in a short-rotationcoppice culture on a waste disposal site and its relation tosoil characteristics. Forest Ecology and Management, 187,295–309. doi:10.1016/j.foreco.2003.07.005.

Lunáčková, L., Masarovičová, E., Králová, K., & Streško, V.(2003a). Response of fast growing woody plants fromfamily Salicaceae to cadmium treatment. Bulletin ofEnvironmental Contamination and Toxicology, 70, 576–585. doi:10.1007/s00128-003-0024-2.

Lunáčková, L., Šottníková, A., Masarovičová, E., Lux, A., &Streško, V. (2003b). Comparison of cadmium effect onwillow and poplar in response to different cultivationconditions. Biologia Plantarum, 47, 403–411. doi:10.1023/B:BIOP.0000023884.54709.09.

Lux, A., Šottníková, A., Opatrná, J., & Greger, M. (2004).Differences in structure of adventitious roots in Salixclones with contrasting characteristics of cadmium accu-mulation and sensitivity. Physiologia Plantarum, 120,537–545. doi:10.1111/j.0031-9317.2004.0275.x.

Marchiol, L., Sacco, P., Assolati, S., & Zerbi, G. (2004).Reclamation of polluted soil: Phytoremediation potential ofcrop-related Brassica species. Water, Air, and Soil Pollution,158, 345–356. doi:10.1023/B:WATE.0000044862.51031.fb.

Mattina, M. J. I., Lannucci-Berger, W., Musante, C., & White,J. C. (2003). Concurrent plant uptake of heavy metals andpersistent organic pollutants from soil. EnvironmentalPollution, 124, 375–378. doi:10.1016/S0269-7491(03)00060-5.

Mills, T. M., Robinson, B. H., Green, S., Clothier, B., Fung, L.E., & Hurst, S. (2000). Difference in Cd uptake anddistribution within poplar and willow species. In Proceed-ings of the 42nd Annual Conference and Expo of the NewZealand Water and Waste Association, Rotorua, NewZealand.

Padmavathiamma, P. K., & Li, L. Y. (2007). Phytoremediationtechnology: Hyper-accumulation metals in plants. Water,Air, and Soil Pollution, 184, 105–126. doi:10.1007/s11270-007-9401-5.

Perttu, K. L. (1999). Environmental and hygienic aspects ofwillow coppice in Sweden. Biomass and Bioenergy, 16,291–297. doi:10.1016/S0961-9534(98)00012-9.

Pietrini, F., Iannelli, M. A., Montanari, R., Bianconi, D., &Massacci, A. (2005). Cadmium interaction with thiols andphotosynthesis in higher plants. In A. Hemantaranjan(Ed.), Advances in plant physiology (pp. 313–326).Jodhpur, India: Scientific Publishers.

Pietrini, F., Iannelli, M. A., Pasqualini, S., & Massacci, A. (2003).Interaction of cadmium with glutathione and photosynthesisin developing leaves and chloroplasts of Phragmitesaustralis (Cav.) Trin. ex strudel. Plant Physiology, 133,829–837. doi:10.1104/pp.103.026518.

Pilipović, A., Nikolić, N., Orlović, S., Petrović, N., & Krstić, B.(2005). Cadmium phytoextraction potential of poplarclones (Populus spp.). Zeitschrift für Naturforschung, 60c,247–251.

Pulford, I. D., Riddell-Black, D., & Stewart, C. (2002). Heavymetal uptake by willow clones from sewage sludge-treatedsoil: The potential for phytoremediation. InternationalJournal of Phytoremediation, 4, 59–72. doi:10.1080/15226510208500073.

Pulford, I. D., & Watson, C. (2003). Phytoremediation of heavymetal-contaminated land by trees – A review. EnvironmentInternational, 29, 529–540. doi:10.1016/S0160-4120(02)00152-6.

Punshon, T., & Dickinson, N. M. (1999). Heavy metalresistance and accumulation characteristics in willows.International Journal of Phytoremediation, 1, 361–385.doi:10.1080/15226519908500025.

Raskin, I., Smith, R. D., & Salt, D. E. (1997). Phytoremediationof metals: Using plants to remove pollutants from theenvironment. Current Opinion in Biotechnology, 2, 221–226. doi:10.1016/S0958-1669(97)80106-1.

Rauser, W. E. (1999). Structure and function of metal chelatorsproduced by plants – The case for organic acids, aminoacids, phytin, and metallothioneins. Cell Biochemistry andBiophysics, 31, 19–48. doi:10.1007/BF02738153.

Rauser, W. E., & Muwly, P. (1995). Retention of cadmium inroots of maize seedlings. Role of complexation byphytochelatins and related thiol peptides. Plant Physiology,109, 195–202. doi:10.1104/pp.109.1.195.

Riddell-Black, D. (1994). Heavy metal uptake by fast growingwillow species. In P. Aronsson, & K. Perttu (Eds.), Willowvegetation filters for municipal wastewaters and sludges.A biological purification system (pp. 145–151). Uppsala,Sweden: Department of Ecology and EnvironmentalResearch, Section of Short Rotation Forestry.

Robinson, B. H., Mills, T. M., Green, S., Chancerel, B., Clothier,B., Fung, L. E., et al. (2005). Trace element accumulationby poplars and willows used for stock fodder. New ZelandJournal of Agriculture Research, 48, 489–497.

Robinson, B. H., Mills, T. M., Petit, D., Fung, L. E., Green, S.,& Clothier, B. (2000). Natural and induced cadmium-accumulation in poplar and willow: Implications forphytoremediation. Plant and Soil, 227, 301–306. doi:10.1023/A:1026515007319.

Water Air Soil Pollut (2009) 197:23–34 33

Rockwood, D. L., Naidu, C. V., Carter, D. R., Rahmani, M.,Spriggs, T. A., Lin, C., et al. (2004). Short-rotation woodycrops and phytoremediation: Opportunities for agroforest-ry? Agroforestry Systems, 61, 51–63. doi:10.1023/B:AGFO.0000028989.72186.e6.

Rosselli, W., Keller, C., & Boschi, K. (2003). Phytoextractioncapacity of trees growing on a metal contaminated soil. Plantand Soil, 256, 265–272. doi:10.1023/A:1026100707797.

Sanità di Toppi, L., & Gabbrielli, R. (1999). Responses tocadmium in higher plants. Environmental and ExperimentalBotany, 41, 105–130. doi:10.1016/S0098-8472(98)00058-6.

Scarascia-Mugnozza, G., Ceulemans, R., Heilman, P. E.,Isebrands, J. G., Stettler, R. F., & Hinckley, T. M.(1997). Production physiology and morphology of Populusspecies and their hybrids grown under short rotation. II.Biomass components and harvest index of hybrid andparental species clones. Canadian Journal of ForestResearch, 27, 285–294.

Šottníková, A., Lunáčková, L., Masarovičová, E., Lux, A., &Streško, V. (2003). Changes in the rooting and growth ofwillow and poplars induced by cadmium. BiologiaPlantarum, 46, 129–131. doi:10.1023/A:1022395118998.

Unterbrunner, R., Puschenreiter,M., Sommer, P.,Wieshammer, G.,Tlustos, P., Zupan, M., et al. (2007). Heavy metal accumu-

lation in tree growing on contaminated sites in CentralEurope. Environmental Pollution, 148, 107–114. doi:10.1016/j.envpol.2006.10.035.

Wang, K. S., Huang, L. C., Lee, H. S., Chen, P. Y., & Chang, S.H. (2008). Phytoextraction of cadmium by Ipomoeaaquatica (water spinach) in hydroponic solution: Effectsof cadmium speciation. Chemosphere, 72, 666–672.doi:10.1016/j.chemosphere.2008.03.034.

Watson, C., Pulford, I. D., & Riddell-Black, D. (1999). Heavymetal toxicity responses of two willow (Salix) varietiesgrown hydroponically: Development of a tolerance screen-ing test. Environmental Geochemistry and Health, 21,359–364. doi:10.1023/A:1006796720300.

Watson, C., Pulford, I. D., & Riddell-Black, D. (2003).Screening of willow species for resistance to heavy metals:Comparison of performance in a hydroponics system andfield trials. International Journal of Phytoremediation, 5,351–365. doi:10.1080/16226510390268748.

Wilkins, D. A. (1978). The measurement of tolerance to edaphicfactors by means of root growth. The New Phytologist, 80,623–633. doi:10.1111/j.1469-8137.1978.tb01595.x.

Zayed, A., Gowthaman, S., & Terry, N. (1998). Phytoaccumu-lation of trace elements by wetlands plants: I. Duckweed.Journal of Environmental Quality, 27, 715–721.

34 Water Air Soil Pollut (2009) 197:23–34