heavy metal tolerance in common fern species

CSIRO PUBLISHING

www.publish.csiro.au/journals/ajb Australian Journal of Botany, 2007, 55, 63–73

Heavy metal tolerance in common fern species

Anthony G. KachenkoA,C, Balwant SinghA and Naveen P. BhatiaB

AFaculty of Agriculture, Food and Natural Resources, The University of Sydney, NSW 2006, Australia.BInstitute for Nuclear Geophysiology, Australian Nuclear Science and Technology Organisation,Menai, NSW 2234, Australia.

CCorresponding author. Email: [email protected]

Abstract. The effects of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn) on the growthand uptake of 10 fern species was investigated under a controlled environment in order to evaluate their suitability forphytoremediation. Fern species included Adiantum aethiopicum, Blechnum cartilagineum, Blechnum nudum, Calochlaenadubia, Dennstaedtia davallioides, Doodia aspera, Hypolepis muelleri, Nephrolepis cordifolia, Pellaea falcata and thearsenic (As) hyperaccumulating Pteris vittata. Ferns were exposed to four levels of metals at concentrations of 0, 50,100 and 500 mg kg−1 for a period of 20 weeks. The response of ferns significantly varied among species and metals. Ingeneral, heavy-metal translocation was limited, with metals being absorbed and held in roots, suggesting an exclusionmechanism as part of the ferns’ tolerance to the applied metals. Similar metal-accumulation patterns were observed for allspecies in that accumulation generally increased with increasing metal treatments; in most cases a sharp increase in metalaccumulation was observed between 100 and 500 mg kg−1 treatments, suggesting a breakdown in tolerance mechanismsand unrestricted metal transport. This was corroborated by enhanced visual toxicity symptoms and a reduction in survivalrates among ferns when exposed to 500 mg kg−1 metal treatments; and to a lesser extent 100 mg kg−1 metal treatments. Ofthe species investigated, N. cordifolia and H. muelleri were identified as possible candidates in phytostabilisation of Cu,Pb, Ni or Zn contaminated soils. Similarly, D. davallioides appeared favourable for use in phytostabilisation of Cu andZn contaminated soils. These species had high survival rates and accumulated high levels of the aforementioned metalsrelative to other ferns investigated. Ferns belonging to the family Blechnaceae (B. nudum, B. cartilagineum and D. aspera)and C. dubia (Family Dicksoniaceae) were least tolerant to most metals, had a low survival rate and were classified asbeing unsuitable for phytoremediation purposes. Metal tolerance was also observed in P. vittata when exposed to Cd, Crand Cu; however, no hyperaccumulation was observed.

Introduction

Soil pollution of metals is a global problem with ramificationsto human, animal and environmental health. An increase inmetal pollution has long been associated with populationgrowth, fuelled by industrial advancement. Activities that havecaused metal contamination include mining and smelting,industrial and manufacturing emissions and application offertilisers and sewage sludge contaminated with metals (Singh2001). Industrialised countries and developing countries areequally affected; however, in the latter there is an increasedpressure to use contaminated soils for food production(Patel et al. 2005).

Cd and Pb contamination have been the focus of numerousstudies and are considered serious environmentally toxicmetallic pollutants (Koeppe 1977; Sanchez-Camazano et al.1994; Liu 2003). Metals including Cu, Cr, Ni and Zn arealso considered serious environmental pollutants and at highconcentrations are often associated with the expression ofphytotoxicities in plants (Chatterjee and Chatterjee 2000;Cuypers et al. 2002; Denkhaus and Salnikow 2002). Cu andZn are essential micronutrients and are required by plantsat low concentrations; beyond the critical toxicity levels ofCu >20–30 µg g−1 and Zn >100–300 µg g−1, phytotoxicity

symptoms are often pronounced (Marschner 1997). There havebeen few studies addressing Cr toxicity in plants; however, itis known that Cr toxicity depends on its valence state, withCr(VI) being highly toxic and mobile compared with Cr(III)(Shanker et al. 2005).

In response to contaminated soils, a variety of physico-chemical remediation methods has been adopted, includingsolidification, electrokinetics and encapsulation (Mulligan et al.2001). In many cases, these strategies have resulted incriticisms in regards to their high cost, energy intensiveness,site destructiveness, associated logistical problems and growingdegree of public dissatisfaction (Rulkens et al. 1998). Theimplementation of alternative strategies that address theseconcerns is critical in effectively removing metallic pollutantsfrom soil. In recent years, significant progress has been madewith the implementation of phytoremediation techniques asalternative technologies capable of remediation and restoringmetal contaminated soils.

An approach to phytoremediation is phytostabilisation, animportant in situ site stabilisation technique that uses plantsas a preventative barrier. The end result is a two-way barrierthat prevents the leaching of heavy metals throughout the soilprofile, while minimising erosion and ecological exposure to

© CSIRO 2007 10.1071/BT06063 0067-1924/07/010063

64 Australian Journal of Botany A. G. Kachenko et al.

the heavy metals. Phytostabilisation is a versatile techniqueand has been successfully applied in the containment ofheavy metals in mine spoils, metalliferous waste and smelters(Smith and Bradshaw 1979; Pierzynski et al. 2002; Stoltz andGreger 2002).

Plants suitable for phytostabilisation need to express a degreeof tolerance that enables them to survive in contaminated soils.Three mechanisms have been suggested by which plants respondto heavy metals. They relate to the metal concentrations foundin plants and the substrates in which they grow (Baker 1981).Plants termed ‘accumulators’ concentrate metals in abovegroundbiomass regardless of the metal concentration in which theyare growing. Such properties have been found in ferns, forexample P. vittata and Pytyrogramma calomelanos for arsenic(As) (Ma et al. 2001; Francesconi et al. 2002). Conversely,plants termed ‘excluders’ maintain low concentrations of metalsin aboveground biomass, compared with their substrate, upto a certain threshold before the mechanism breaks down.Last, plants termed ‘indicators’ are those in which the metalconcentration in the aboveground biomass reflects the substrateconcentration, and are often used in mineral prospecting(Nkoane et al. 2005).

Ferns have often been associated with contaminated soils,particularly those associated with mining operations. A notableexample is the Chinese brake fern (P. vittata) that was identifiedgrowing on an arsenical mine dump in Rhodesia (Wild 1974);however, it was not until 2001 that it earned its classificationas the first As hyperaccumulator (Ma et al. 2001). The silverdust fern (P. calomelanos) is the only fern outside the Pterisgenus that has been identified as an As hyperaccumulator(Francesconi et al. 2002). Lepp (2001) indicated that ferns areregarded as the fourth-most abundant group (in terms of speciesrichness) of plants associated with Cu-enriched substrates andalso reported their dominant presence on serpentine and otherultramafic bodies. Pellaea calomelanos and Cheilanthes hirtaare the only ferns reported on nickeliferous, non-serpentinesoils (Brooks and Malaisse 1995), and aquatic species such asSalvinia and Azolla are the only remaining ferns reported asmetal accumulators (Sela et al. 1989; Outridge and Hutchinson1991). Terrestrial ferns have received less attention than vascularplants in relation to metal tolerance and accumulation andconsequently there are few systematic investigations that haveconsidered the interactions between ferns and heavy-metalsubstrates.

Large stretches of land across Australia have beencontaminated by a variety of heavy metals, mainly because ofintensive agriculture and mining activities (Sproal et al. 2002;Archer and Caldwell 2004; Pietrzak and McPhail 2004; Cooper2005). Owing to strict quarantine regulations on import ofplanting material suitable for phytoremediation, we screenedseveral native Australian ferns for their tolerance to a varietyof heavy metals (Cd, Cr, Cu, Ni, Pb and Zn). None of theseAustralian species has been studied for metal tolerance andaccumulation. The primary objective of this study was todetermine the degree of accumulation and the biomass producedwhen nine of the most common Australian fern species alongwith P. vittata were exposed to elevated levels of metalsunder controlled conditions. This information could be used toselect suitable species available for phytoremediation of metal-contaminated soils within Australia and elsewhere.

Materials and methodsNine Australian native fern species along with a known Ashyperaccumulator P. vittata were screened for their potentialto tolerate Cd, Cr, Cu, Ni, Pb and Zn. Species were chosenon the basis of their hardiness, vigour and distribution patternacross Australia (McCarthy 1998), and included: Adiantumaethiopicum, Blechnum cartilagineum, B. nudum, Calochlaenadubia, Dennstaedtia davallioides, Doodia aspera, Hypolepismuelleri, Nephrolepis cordifolia, Pellaea falcata. With theexception of N. cordifolia, all ferns were procured from aspecialist fern nursery (Sonters Fern Nurseries, Winmalee, NSW,Australia), and were ∼3–4 months old. Nephrolepis cordifoliawas propagated from samples obtained from the wild (GarigalNational Park, Roseville, NSW, Australia).

Potting mix (Debco� general container mix) was used inthe experiment and amended with 10% coarse perlite mix forimproved aeration and drainage. Approximately 800 g of air-dried potting mix was weighed into each plastic pot (diameterof 14 cm) and a plastic saucer placed under each pot to collectany possible leachate, hence maintaining a closed system. Metalsalts (AR grade) were applied as 0, 50, 100 and 500 mg kg−1

dry weight of potting mix. Cd was added as Cd(NO3)2·4H2O,Cr as K2Cr2O7, Pb as Pb(NO3)2, Ni as NiSO4·6H2O, Cu asCuSO4·5H2O and Zn as ZnSO4·7H2O. Ferns were arranged ina completely randomised experimental design and there werethree replicates for each treatment. After 1 week of incubation,one fern (3–4-frond stage) was transplanted into each pot (Tuand Ma Lena 2002). Plants were grown in a glasshouse for20 weeks, watered daily with deionised water (to 60–70%water-holding capacity) and no fertiliser was applied during theexperimental period.

Ferns were harvested after 20 weeks and separated into rootsand fronds. Harvested material was thoroughly washed by usinga three step-washing sequence consisting of dilute 0.01% HCl,followed by tap water and last with deionised water (Reuteret al. 1988). Samples were then air-dried, weighed and placedin an oven at ∼80◦C for 48–72 h. Dried samples were weighed,mechanically ground (<1 mm) and digested in a mixture of nitricacid (HNO3) and perchloric acid (HClO4) (Miller 1998).

With each batch of digestions, a sample blank and twocertified reference plant materials, pine needles (1575) and peachleaves (1547), were included for quality control. For all metalsthere was >95% recovery.

Extracts were analysed for various elements with a VistaVarian 220FS flame atomic absorption spectrometer. Sampleswith concentrations below detection limit of flame atomicabsorption spectroscopy (namely Cd, Cr, Ni and Pb) wereanalysed with a Vista Varian Spectra 220Z graphite furnaceatomic absorption spectrometer.

Statistical analysisAnalysis of variance (two-way ANOVA) was carried outto test for significant differences with GENSTAT version 8(Payne 2005). The parameters analysed included frond androot biomass, metal concentration and metal accumulation.Data were log-transformed before analysis and differences wereconsidered significant if P ≤ 0.05. An unbalanced treatmentdesign was used as some species lost replicates duringexperimentation.

Heavy metal tolerance in common Australian fern species Australian Journal of Botany 65

Results

Frond and root biomass

For all metals, frond and root biomass differed significantlyamong species and the effects of Cd and Pb treatments onfrond biomass were also significant (Table 1). In general, frondbiomass was greater than root biomass for species excludingA. aethiopicum, B. cartilagineum, C. dubia and D. aspera(Figs 1, 2). For all species, frond and root biomass waslowest when the plants were exposed to Cd and exhibited adose-dependent decrease with increasing Cd applied (Figs 1a,2a). Survival data indicated a higher percentage of deaths inCd-treated ferns than in those treated with other metals (Table 2).Among all ferns, N. cordifolia responded most favourably tometal treatments, with no reported deaths (Table 2) and highfrond biomass across all metal treatments except Cd (Fig. 1a).Phytotoxicity symptoms appeared in N. cordifolia at highertreatment levels than in other species when exposed to Zn(stunted growth and chlorotic new growth) and Cr (chloroticnew growth). D. aspera also survived the exposure of mostmetals except Pb (Table 2); however, lower frond biomass wasrecorded for D. aspera than for N. cordifolia. Hypolepis muellerihad the highest frond biomass across all Cr, Cu, Pb and Zntreatment levels (Fig. 1) and a high survival rate when exposed toall metals except Cd (Table 2). B. nudum, B. cartilagineum andC. dubia grew least favourably (Table 2), with low frond biomassacross most metal treatments (Fig. 1). Among these species,phototoxicities were more pronounced and symptoms includedinterveinal yellowing, chlorotic, stunted growth and necroticpinnae tips. In general, frond biomass in ferns exposed to Zntreatments increased with increasing Zn levels (Fig. 1f ) and ahigh survival rate was observed among all species (Table 2).

Heavy-metal concentrations in fronds and roots

For all metals, frond and root concentrations differedsignificantly among species and treatments (Table 1).

Table 1. Results of two-way ANOVA showing probability (P) for biomass, metal concentration and accumulationin ferns between populations, treatments and interaction between species and treatments

Measurement SignificanceCd Cr Cu Pb Ni Zn

BiomassFronds Species <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Treatment 0.005 0.564 0.861 0.038 0.127 0.143Species × treatment 0.060 0.045 0.002 0.152 0.004 0.018

Roots Species <0.001 <0.001 <0.001 <0.001 <0.001 <0.001Treatment 0.010 0.792 0.167 0.196 0.154 0.586Species × treatment 0.044 0.025 0.001 0.294 0.009 0.019

Metal concentrationFronds Species <0.001 <0.001 <0.001 0.005 0.016 <0.001

Treatment <0.001 <0.001 <0.001 <0.001 <0.001 <0.001Species × treatment <0.001 <0.001 0.001 0.010 0.108 0.003

Roots Species <0.001 <0.001 <0.001 <0.001 <0.001 <0.001Treatment <0.001 <0.001 <0.001 <0.001 <0.001 <0.001Species × treatment 0.225 <0.001 0.581 <0.001 <0.001 0.001

AccumulationSpecies <0.001 <0.001 <0.001 <0.001 <0.001 <0.001Treatment <0.001 <0.001 <0.001 <0.001 <0.001 <0.001Species × treatment 0.018 <0.001 <0.001 0.025 <0.001 0.273

Concentrations of Cd, Pb and Ni remained uniform across 50and 100 mg kg−1 treatments and increased at the 500 mg kg−1

treatment. The highest concentration of Cd was observedin fronds of D. davallioides (386 µg g−1 dry weight (DW)),suggesting that D. davallioides may be able to hyperaccumulateCd in the aboveground biomass (Fig. 3a). For the remainingspecies, the concentrations of Cd ranged from 4.1 (N. cordifolia)to 87.9 µg g−1 DW (P. falcata). For Pb (Fig. 3e), the highestconcentration was observed in the fronds of P. falcata(62 µg g−1 DW) and at both the 50 and 100 mg kg−1 treatments,the concentration of Pb in most cases was below 6 µg g−1,with the exception of B. nudum at the 50 mg kg−1 treatment(21 µg g−1 DW). The highest Ni concentration in fronds wasobserved in D. aspera (161 µg g−1 DW) and high concentrationswere also observed in B. cartilagineum (121 µg g−1 DW). Forthe remaining species, the concentration of Ni ranged from 0.8(P. vittata) to 68 µg g−1 DW (P. falcata).

For all species exposed to Cd, Ni and Pb treatments, theconcentrations were higher in the roots than in the fronds and,in general, increased with increasing concentrations of metals.Concentrations of Cd in the roots were up to 7-fold higher thanthose in the fronds (Fig. 4a), whereas concentrations of Ni andPb were up to 36-fold higher in the roots than in the fronds(Fig. 4d, e).

Concentrations of Cr in the fronds ranged from 0.07(N. cordifolia) to 33 µg g−1 DW (P. falcata) among all speciesand similar concentrations were also observed in the frondsof ferns exposed to Cu, ranging from 4.12 (C. dubia) to32 µg g−1 DW (D. davallioides) (Fig. 3c). For all treatmentlevels, fronds generally contained much lower concentrationsof Cr and Cu than did roots (Fig. 4b, c) and for all species,the highest concentrations were observed at the 500 mg kg−1

treatment level. Among all species, concentrations of Cr in theroots were up to 178-fold higher than those in the fronds andconcentrations of Cu were up to 18-fold higher in the roots thanin the fronds.


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Concentrations of Zn in the fronds increased withincreasing concentrations of Zn, especially between the 100and 500 mg kg−1 levels (Fig. 3f ). The highest concentrationof Zn was recorded in B. nudum (216 µg g−1 DW)at the 500 mg kg−1 treatment level, followed closely byB. cartilagineum (202 µg g−1 DW). Across 50 and 100 mg kg−1

Zn treatments, the mean concentration of Zn in fronds rangedfrom 18 (D. aspera) to 107 µg g−1 DW (P. falcata). Theconcentration of Zn in the roots followed a pattern similar tothat in fronds (Fig. 4f ), except that the concentrations were upto 6-fold higher in the roots than in the fronds.

Heavy-metal accumulation

Heavy-metal accumulation was calculated by multiplying thetotal amount of biomass produced (frond and root) with the

respective concentrations of metal in the fronds and roots, togive an indication of the efficiency of metal accumulation foreach species (per plant basis). For all 10 ferns investigated inthis study, the level of metal accumulation varied significantlyamong species and treatment levels in response to metalsapplied (Table 1). The pattern of Cd (Fig. 5a) and Pb (Fig. 5e)accumulation remained uniform across 50 and 100 mg kg−1

treatment levels and increased at the 500 mg kg−1 treatmentlevel. The highest accumulation of Cd was 168 µg plant−1

and was found in D. davallioides; for the remaining species,accumulation of Cd was less than 90 µg plant−1. Among speciestreated with Pb, the highest accumulation occurred at the500 mg kg−1 treatment level in D. aspera (469 µg plant−1).

The highest accumulation of Cu was observed at the500 mg kg−1 treatment level in H. muelleri, followed by


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Table 2. Survival rate (%) of the 10 ferns across all treatments ofvarious metals after 20 weeks of growth

Fern species Survival rate (%)Cd Cr Cu Pb Ni Zn

Adiantum aethiopicum 67 100 100 100 89 100Blechnum cartilagineum 11 100 100 44 89 67Blechnum nudum 44 44 67 56 56 89Calochlaena dubia 78 56 67 67 89 89Dennstaedtia davallioides 89 44 100 44 78 89Doodia aspera 100 100 100 89 100 100Hypolepis muelleri 56 100 100 78 100 89Nephrolepis cordifolia 100 100 100 100 100 100Pellaea falcata 67 67 89 89 89 89Pteris vittata 78 67 67 67 78 89

D. davallioides, with 210 and 195 µg plant−1, respectively forthe two species (Fig. 5c). The highest accumulation of Znwas observed in A. aethiopicum (655 µg plant−1) followedby D. davallioides (377 µg plant−1). H. muelleri recorded thehighest accumulation of Zn at both the 50 and 100 mg kg−1

treatments, with values of 233 and 248 µg plant−1, respectively(Fig. 5f ). Across all Cu treatments, the accumulation of Cuin most species was below 65 µg plant−1 and for Zn below240 µg plant−1.

In general, the level of Ni accumulation (Fig. 5d ) increasedwith increasing concentration of Ni. The highest level of Niaccumulation was found in P. falcata (196 µg plant−1), followedby B. cartilagineum and N. cordifolia (both 131 µg plant−1).Nickel accumulation at the 50 and 100 mg kg−1 treatment levels


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700Adiantum aethiopicum Blechnum cartilagineumBlechnum nudum Calochlaena dubia Dennstaedtia davallioides Doodia asperaHypolepis muelleri Nephrolepis cordifoliaPellaea falcata Pteris vittata

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Fig. 3. Concentrations (µg g−1 DW) of (a) Cd, (b) Cr, (c) Cu, (d ) Ni, (e) Pb and ( f ) Zn in the fronds of different fern species after 20 weeks of growth.Log-transformed data have been back-transformed. Data points and vertical bars are least square means and ± s.e., respectively (n = 3).

ranged from 3.54 (P. falcata) to 62 µg plant−1 (D. aspera).Accumulation of Cr (Fig. 5b) was generally similar across alltreatments, the highest accumulation observed in P. vittata(102 µg plant−1) at the 500 mg kg−1 treatment level.

Metal translocation by ferns

Translocation factor (TF) is the ratio of metal concentration inthe shoot to the metal concentration in the root and providesan indication of internal metal transportation. Translocationfactors for each species are presented in Table 3. The dataindicate that metals accumulated by the ferns were largelyretained in roots, as shown by mean TFs < 1. Higher TFs did

not correlate with greater metal accumulation in harvestablebiomass. For example, in response to Zn, the mean TF forD. davallioides was 0.91 which is more than four times greaterthan the 0.25 mean TF for H. muelleri (Table 3); however,accumulation of Zn in harvestable biomass was greater inH. muelleri than in D. davallioides (data not shown). Trendswere observed among species and metals. For example, whenconcentrations of Cu, Ni and Pb increased, TFs decreased.Conversely, TFs increased when concentrations of Cd and Crin the treatments increased, but remained relatively constantacross Zn treatments (data not shown). Among all species,TFs for Cu were the lowest whereas those for Zn werethe highest.


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Fig. 4. Concentrations (µg g−1 DW) of (a) Cd, (b) Cr, (c) Cu (d ) Ni, (e) Pb and ( f ) Zn in the roots of different fern species after 20 weeks of growth.Log-transformed data have been back-transformed. Data points and vertical bars are least square means and ± s.e., respectively (n = 3).

Discussion

The results obtained from this study demonstrate the degree ofphysiological response of ferns to various heavy metals. The fernspecies investigated in this study differed widely in their abilityto accumulate heavy metals. In general, significantly greatermetal accumulation occurred in roots than fronds, suggestinga limited mobility and translocation of metals once absorbed byferns. Sequestration of metals in roots enables plants to continuegrowing uninhibited and it is an important mechanism of heavymetal tolerance (Ernst et al. 1992). This commonly observedphenomenon, in which plants can restrict translocation of heavymetals from roots to shoots, has been reported in numerousrecent studies. For example, Deng et al. (2004) found greater

concentration of Pb, Zn, Cu and Cd in the roots of 12 wetlandspecies growing in metal-contaminated environments. Recently,Srivastava and Ma (2005) screened 11 fern species for Seaccumulation and found higher concentrations of Se in the rootsof all but three species. Weng et al. (2005) investigated theeffect of Cu on four Elsholtzia ecotypes and observed stimulatedroot growth in ecotype E. splendens when treated with higherconcentrations of applied Cu. Furthermore, the authors notedthe absence of toxicity symptoms and proposed that an increasein root-to-shoot ratio with the addition of Cu could be a possiblestrategy leading to increased tolerance in E. splendens. In ourstudy, A. aethiopicum, B. cartilagineum and D. aspera speciesexposed to Cu had a root biomass greater than frond biomass


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and, like E. splendens, it is possible that this may be a strategyleading to Cu tolerance. These same species also had an increasein root-to-shoot ratio with increasing levels of Pb and, like Cu, itis possible that Pb sequestration in the roots increased toleranceto Pb in these species.

The growth response of ferns investigated in this studysuggests the exclusion mechanism of tolerance. This mechanismis effective up to a threshold value (metal concentration)above which the mechanism breaks down and results inunrestricted metal uptake and eventual death (Baker 1981).Indeed, this mechanism observed in the fern species investigatedmay be a constitutive property expressed when these plantsare exposed to high concentrations and inimical levels ofmetals. With the exception of D. davallioides, no species

were considered hyperaccumulators because concentrationsof heavy metals in fronds remained below the criterionused to define hyperaccumulating species, i.e. plants withconcentration of Cd > 0.01% DW, concentrations of Cu, Cr,Ni or Pb > 0.1% and concentration of Zn > 1% DW (Bakerand Brooks 1989; Baker et al. 2000). Two replicates ofD. davallioides demonstrated hyperaccumulating characteristicswhen treated with 500 mg kg−1 of Cd; however, severe stunting,chlorotic growth and necrotic margins were observed. Similarsymptoms also occurred in this species when exposed to 50 and100 mg kg−1 of Cd and across all treatments, concentrations ofCd were higher in the roots than in the fronds. These observationssuggest a likely breakdown of the tolerance mechanism, whichis further evident with a reduction in frond biomass of all


Table 3. Mean metal translocation factors across all treatment levels (TF; concentration ratio of metalin the frond to root) of the 10 fern species after 20 weeks of growth

The degree of metal uptake in harvestable biomass for each species is ranked according to its mean frond metaluptake across the 50 and 100 mg kg−1 levels as denoted in parentheses

Fern species Translocation factorCd Cr Cu Pb Ni Zn

Adiantum aethiopicum 0.38 (5) 0.15 (7) 0.26 (6) 0.27 (7) 0.34 (5) 0.53 (4)Blechnum cartilagineum 0.21 (6) 0.23 (8) 0.12 (7) 0.26 (2) 0.40 (9) 0.44 (7)Blechnum nudum 0.60 (10) 0.76 (5) 0.16 (8) 0.15 (5) 0.61 (8) 0.61 (10)Calochlaena dubia 0.53 (3) 0.44 (6) 0.18 (10) 0.33 (10) 0.26 (10) 0.41 (8)Dennstaedtia davallioides 0.88 (2) 0.97 (2) 0.27 (3) 0.43 (3) 0.74 (1) 0.91 (3)Doodia aspera 0.30 (9) 0.11 (9) 0.13 (9) 0.22 (9) 0.48 (6) 0.39 (9)Hypolepis muelleri 0.44 (7) 0.48 (4) 0.12 (1) 0.15 (1) 0.16 (2) 0.25 (1)Nephrolepis cordifolia 0.35 (8) 0.08 (10) 0.20 (4) 0.30 (4) 0.51 (4) 0.86 (2)Pellaea falcata 0.38 (1) 0.32 (1) 0.20 (5) 0.25 (6) 0.47 (3) 0.84 (5)Pteris vittata 0.19 (4) 0.42 (3) 0.15 (2) 0.30 (8) 0.32 (7) 0.69 (6)

surviving plants (Fig. 1a) and indicates that D. davallioidesis not a Cd hyperaccumulator. An earlier study by Tam andSingh (2004) identified N. cordifolia as a Cu hyperaccumulator(2324 µg g−1) growing naturally on a heavy metal-contaminatedsoil; however, in our study the mean concentration of Cu inthe fronds of N. cordifolia was below 9 µg g−1. Furthermore,a greater concentration of Cu was observed in their roots.

In the present study, the degree of accumulation of allmetals was generally uniform across the 50 and 100 mg kg−1

metal treatments (Fig. 5), thereafter increasing sharply whenplants were exposed to 500 mg kg−1 metal treatments. Thispattern of metal uptake is commonly observed in excluderspecies and further suggests a possible breakdown in the ferns’tolerance mechanism, resulting in unrestricted uptake. Activetransport of metals may have resulted in elevated levels infronds of ferns treated at the 500 mg kg−1 level, supported by thepresence of necrotic lesions and in some cases death. The speciesinvestigated have been subsequently ranked according to theirmetal-accumulation efficiencies and hence phytoremediationpotentials by taking the overall mean metal accumulation inharvestable biomass at the 50 and 100 mg kg−1 metal treatments(Table 3). The 500 mg kg−1 treatment was omitted owing to theonset of severe phytotoxicity and death of some replicates. Theseresults indicate restricted movement of metals into harvestablefrond biomass as further seen with TFs < 1 (Table 3). This,coupled with the absence of hyperaccumulation, indicates thatthese ferns are unsuitable for phytoextraction of metals.

However, these species may be suitable candidates forphytostabilisation of contaminated soils. Good phytostabilisingplants should tolerate high concentrations of metals andimmobilise contaminants in the roots, with minimaltranslocation to harvestable biomass (Salt et al. 1995). Thisenables harvestable biomass to continue growing uninhibitedby metals and further reduces the possibility of metalspassing into the food chain through the activity of herbivores(McIntyre 2003). These properties together with survival data(Table 2) are of paramount importance when consideringspecies for phytostabilising purposes. The results from thisstudy suggest N. cordifolia and H. muelleri may be suitable inphytostabilisation of Cu-, Pb-, Ni- or Zn-contaminated soils.With respect to each metal, these species grew successfully

across a wide range of concentrations, yielded higher quantitiesof frond biomass and accumulated higher concentrations ofmetals in fronds than the other investigated species. Bothspecies are rhizomatous, thus enabling them to readily coloniselarge volumes of contaminated soils and further restrict thepossibility of metals movement through leaching and airbornespread. N. cordifolia is a particularly robust species, commonthroughout rainforest areas, displaying epiphytic characteristicsat times and often considered a garden escape (McCarthy 1998).Similarly, H. muelleri is also considered robust, preferringmoist shaded conditions in sclerophyll forest, but being alsoassociated with open country (McCarthy 1998). Dennstaedtiadavallioides, also a rhizomatous species, showed promise inphytostabilisation of soils contaminated with Cu and Zn, andexpressed similar growth characteristics to those observed inN. cordifolia and H. muelleri. D. davallioides is associatedwith moist conditions in rainforest and eucalypt forests and isalso regarded as a naturalised species (McCarthy 1998). Thesuitability of these species for phytostabilisation was furtherconfirmed with minimal phytotoxicity symptoms observed whenexposed to metal treatments up to 500 mg kg−1.

Both H. muelleri and D. davallioides belong to theDennstaedtiaceae family, which suggests that this fern familymay be tolerant to heavy metals. Previous studies have reportedheavy metal tolerance and uptake across several specieswithin a family. For example, screening of As tolerance andaccumulation in ferns by Meharg (2003) indicated that fernsin the family Pteridaceae could tolerate up to 100 mg As kg−1

in growing medium and could subsequently accumulatebetween 17 and 2493 µg As g−1. Further investigation isneeded to confirm whether additional species belonging tothe family Dennstaedtiaceae exhibit heavy metal tolerance orhyperaccumulation.

Plants expressing tolerance to heavy metals have evolveddifferent mechanisms, including chelation, trafficking andsequestration, in order to survive heavy metal-rich growingconditions (Clemens 2001). We suggest cellular and subcellularsequestration of metals in roots among the ferns studied, inparticularly N. cordifolia and H. muelleri, which may furtherexplain their tolerance when exposed to a variety of heavy metals.Lin et al. (2003) reported that nearly 60% of the total Cu in the


roots of Helianthus annuus L. was bound to the cell-wall fractionand the cell-wall plasma membrane. Similarly, MacFarlane andBurchett (2000) reported higher concentrations of Cu, Zn andPb in epidermal, cortical parenchyma and endodermal cell-walltissues of Avicennia marina roots. These studies suggest thatrestriction of metals in the roots through sequestration is animportant mechanism which results in a reduction of metaluptake and a similar mechanism may have contributed to thetolerance observed in the ferns studied. This aspect warrantsfurther consideration to elucidate if sequestration plays a role intolerance of heave metals in ferns.

Among all species investigated, C. dubia (Dicksoniaceae)did not exhibit any favourable growth characteristics, such astolerance to heavy metals (Table 2) and metal accumulation(Fig. 5) for most of the heavy metals. Similarly, poor growthcharacteristics were observed in B. cartilagineum and B. nudum,both members of the Blechnaceae family. In both species, weobserved low survival rates (Table 2) and metal accumulation(Fig. 5) relative to the other species investigated. Doodiaaspera, also a member of the Blechnaceae family, grewpoorly in response to metal treatments; however, this specieshad a higher survival rate than did both B. cartilagineumand B. nudum (Table 2). In the surviving replicates fromall three aforementioned species, we observed a significantdegree of phytotoxicity, particularly with respect to Cd andNi treatments. The symptoms included stunted new growth,necrotic frond margins, chlorosis and interveinal yellowing.These findings suggest that the aforementioned species areless suitable for phytoremediation purposes. P. vittata, whichhas been previously identified as an As hyperaccumulator(Ma et al. 2001), demonstrated relative tolerance to Cd, Crand Cu. This species displayed higher accumulation and frondbiomass production for all three metals than did several ofthe other species investigated. These results indicate thatP. vittata is not only tolerant to As, but could also be used inphytostabilisation of Cd-, Cr- and Cu-contaminated soils. Inaddition, a recent study by An et al. (2006) demonstrated thatP. vittata had a high tolerance to Zn and reported concentrationsof up to 271 µg g−1 when exposed to 2000 mg Zn kg−1.However, when exposed to 500 mg Zn kg−1, concentrations were∼70 µg g−1 which are in agreement with the results observed inthe present study (73 µg g−1).

Conclusion

The present investigation shows that many fern species canabsorb a wide range of metals (Cd, Cr, Cu, Ni, Pb and Zn)and may be used to colonise and remediate metal-contaminatedsoils in a phytostabilisation system. The exclusion of heavymetals appeared the principal physiological response enablingthe survival of these ferns when exposed to metal-rich growingconditions. The accumulation response of most ferns was similarup to 100 mg kg−1 and generally followed a linear increase.Thereafter, it appeared as though the exclusion mechanismenabling tolerance had deteriorated, with the onset of visualtoxicity symptoms becoming more evident, coupled with a sharpincrease in accumulation and a reduction in total biomass andsurvival rates. Moreover, restricted uptake and translocationof heavy metals was limited, with roots containing higher

concentrations of metals in most cases. The growth andmetal-accumulation characteristics of both N. cordifolia andH. muelleri could prove useful in phytostabilisation of Cu-, Pb-,Ni- or Zn-contaminated environments, viz. abandoned mines,smelters, industrial and urban sites. Similarly, D. davallioidesmay also be suitable for phytostabilisation of Cu- andZn-contaminated soils. All three species are perennials,rhizomatous and their evasive nature would ensure fastcolonisation and establishment to act as a vegetation cover inorder to prevent erosion and minimise the spread of metals.Conversely, species belonging to the family Blechnaceae showedpoor growth characteristics and would be unsuitable for sucha purpose. Field-based studies of metal-contaminated sites arewarranted to address the scope and application of these species.These studies should address environmental pressures such astemperature and light intensity and soil factors such as moisturecontent, metal bioavailability and multiple-metal systems.

Acknowledgements

The authors thank George and Valerie Sonter for supplying plant material andproviding advice. Anthony acknowledges the financial assistance providedby The Faculty of Agriculture, Food and Natural Resources, The Universityof Sydney and the Commonwealth of Australia through an AustralianPostgraduate Award scholarship.

References

An Z-Z, Huang Z-C, Lei M, Liao X-Y, Zheng Y-M, Chen T-B (2006) Zinctolerance and accumulation in Pteris vittata L. and its potential forphytoremediation of Zn- and As-contaminated soil. Chemosphere 62,796–802. doi: 10.1016/j.chemosphere.2005.04.084

Archer MJG, Caldwell RA (2004) Response of six Australian plant species toheavy metal contamination at an abandoned mine site. Water, Air, and SoilPollution 157, 257–267. doi: 10.1023/B:WATE.0000038900.66771.bf

Baker AJM (1981) Accumulators and excluders – strategies in the responseof plants to heavy metals. Journal of Plant Nutrition 3, 643–654.

Baker AJM, Brooks RR (1989) Terrestrial higher plants whichhyperaccumulate metallic elements: a review of their distribution,ecology and phytochemistry. Biorecovery 1, 81–126.

Baker AJM, McGrath SP, Reeves RD, Smith JAC (2000) Metalhyperaccumulator plants: a review of the ecology and physiology ofa biological resource for phytoremediation of metal-polluted soils.In ‘Phytoremediation of contaminated soil and water’. (Eds N Terry,G Banuelos) pp. 85–108. (CRC Press: Boca Raton, FL)

Brooks R, Malaisse F (1995) ‘The heavy metal-tolerant flora of southcentralAfrica.’ (Balkema: Rotterdam, The Netherlands)

Chatterjee J, Chatterjee C (2000) Phytotoxicity of cobalt, chromiumand copper in cauliflower. Environmental Pollution 109, 69–74.doi: 10.1016/S0269-7491(99)00238-9

Clemens S (2001) Molecular mechanisms of plant metal tolerance andhomeostasis. Planta 212, 475–486. doi: 10.1007/s004250000458

Cooper JL (2005) The effect of biosolids on cereals in central New SouthWales, Australia. 2. Soil levels and plant uptake of heavy metals andpesticides. Australian Journal of Experimental Agriculture 45, 445–451.doi: 10.1071/EA03100

Cuypers A, Vangronsveld J, Clijsters H (2002) Peroxidases in roots andprimary leaves of Phaseolus vulgaris copper and zinc phytotoxicity:a comparison. Journal of Plant Physiology 159, 869–876.doi: 10.1078/0176-1617-00676

Deng H, Ye ZH, Wong MH (2004) Accumulation of lead, zinc, copper andcadmium by 12 wetland plant species thriving in metal-contaminatedsites in China. Environmental Pollution (Barking, Essex : 1987) 132,29–40. doi: 10.1016/j.envpol.2004.03.030


Denkhaus E, Salnikow K (2002) Nickel essentiality, toxicity, andcarcinogenicity. Critical Reviews in Oncology/Hematology 42, 35–56.doi: 10.1016/S1040-8428(01)00214-1

Ernst WHO, Verkleij JAC, Schat H (1992) Metal tolerance in plants. ActaBotanica Neerlandica 41, 229–248.

Francesconi K, Visoottiviseth P, Sridokchan W, Goessler W (2002)Arsenic species in an arsenic hyperaccumulating fern, Pityrogrammacalomelanos: a potential phytoremediator of arsenic-contaminated soils.Science of the Total Environment 284, 27–35.doi: 10.1016/S0048-9697(01)00854-3

Koeppe DE (1977) The uptake, distribution, and effect of cadmium and leadin plants. Science of the Total Environment 7, 197–206.doi: 10.1016/0048-9697(77)90043-2

Lepp NW (2001) Bryophytes and Pteridophytes. In ‘Metals in theenvironment’. (Ed. MNV Prasad) pp. 159–170. (Marcel Dekker:New York)

Lin J, Jiang W, Liu D (2003) Accumulation of copper by roots, hypocotyls,cotyledons and leaves of sunflower (Helianthus annuus L.). BioresourceTechnology 86, 151–155. doi: 10.1016/S0960-8524(02)00152-9

Liu ZP (2003) Lead poisoning combined with cadmium in sheep andhorses in the vicinity of non-ferrous metal smelters. Science of the TotalEnvironment 309, 117–126. doi: 10.1016/S0048-9697(03)00011-1

Ma LQ, Komar KM, Tu C, Zhang W, Cai Y, Kennelley ED (2001) A fern thathyperaccumulates arsenic. Nature 409, 579. doi: 10.1038/35054664

MacFarlane GR, Burchett MD (2000) Cellular distribution of copper, leadand zinc in the grey mangrove, Avicennia marina (Forsk.) Vierh. AquaticBotany 68, 45–59. doi: 10.1016/S0304-3770(00)00105-4

Marschner H (1997) ‘Mineral nutrition of higher plants.’ (Academic Press:London)

McCarthy PM (1998) ‘Flora of Australia. Vol. 48: ferns, gymnosperms andallied groups.’ (CSIRO: Canberra)

McIntyre T (2003) Phytoremediation of heavy metals fromsoils. In ‘Advances in biochemical engineering biotechnology.Phytoremediation’. (Ed. D Tsao) pp. 97–123. (Springer: Berlin)

Meharg AA (2003) Variation in arsenic accumulation – hyperaccumulationin ferns and their allies. New Phytologist 157, 25–31.doi: 10.1046/j.1469-8137.2003.00541.x

Miller RO (1998) Nitric-perchloric acid wet digestion in an open vessel.In ‘Handbook of reference methods for plant analysis’. (Ed. Y Kalra)pp. 57–61. (CRC Press: Boca Raton, FL)

Mulligan CN, Yong RN, Gibbs BF (2001) Remediation technologies formetal-contaminated soils and groundwater: an evaluation. EngineeringGeology 60, 193–207. doi: 10.1016/S0013-7952(00)00101-0

Nkoane BBM, Sawula GM, Wibetoe G, Lund W (2005) Identification of Cuand Ni indicator plants from mineralised locations in Botswana. Journalof Geochemical Exploration 86, 130–142.doi: 10.1016/j.gexplo.2005.03.003

Outridge PM, Hutchinson TC (1991) Induction of cadmium tolerance byacclimation transferred between ramets of the clonal fern Salviniaminima Baker. New Phytologist 117, 597–605.doi: 10.1111/j.1469-8137.1991.tb00964.x

Patel KS, Shrivas K, Brandt R, Jakubowski N, Corns W, Hoffmann P (2005)Arsenic contamination in water, soil, sediment and rice of central India.Environmental Geochemistry and Health 27, 131–145.doi: 10.1007/s10653-005-0120-9

Payne RW (2005) ‘Genstat release 8 reference manual.’ (VSN International:Hamperdeen, UK)

Pierzynski GM, Schnoor JL, Youngman A, Licht L, Erickson LE (2002)Poplar trees for phytostabilization of abandoned zinc-lead smelter.Practice Periodical of Hazardous, Toxic, and Radioactive WasteManagement 6, 177–183.doi: 10.1061/(ASCE)1090-025X(2002)6:3(177)

Pietrzak U, McPhail DC (2004) Copper accumulation, distribution andfractionation in vineyard soils of Victoria, Australia. Geoderma 122,151–166. doi: 10.1016/j.geoderma.2004.01.005

Reuter DJ, Robinson JB, Peverill KI, Price GH (1988) Guidelines forcollecting, handling and analyzing plant material. In ‘Plant analysis aninterpretation manual’. (Eds DJ Reuter, JB Robinson) pp. 20–30. (InkataPress: Melbourne)

Rulkens W, Tichy R, Grotenhuis J (1998) Remediation of polluted soil andsediment: perspectives and failures. Water Science and Technology 37,27–35. doi: 10.1016/S0273-1223(98)00232-7

Salt DE, Blaylock M, Kumar NP, Dushenkov V, Ensley BD, Chet I, Raskin I(1995) Phytoremediation: a novel strategy for the removal of toxicmetals from the environment using plants. Bio/Technology 13, 468–474.doi: 10.1038/nbt0595-468

Sanchez-Camazano M, Sanchez-Martin MJ, Lorenzo LF (1994) Leadand cadmium in soils and vegetables from urban gardens ofSalamanca (Spain). Science of the Total Environment 146–147,163–168. doi: 10.1016/0048-9697(94)90233-X

Sela M, Garty J, Tel-Or E (1989) The accumulation and the effect of heavymetals on the water fern Azolla filiculoides. New Phytologist 112, 7–12.doi: 10.1111/j.1469-8137.1989.tb00302.x

Shanker AK, Cervantes C, Loza-Tavera H, Avudainayagam S (2005)Chromium toxicity in plants. Environment International 31, 739–753.doi: 10.1016/j.envint.2005.02.003

Singh B (2001) Heavy metals in soils: sources, chemical reaction andforms. In ‘GeoEnvironment: proceedings of the 2nd Australia andNew Zealand conference on environmental geotechnics’. Newcastle,New South Wales. (Eds D Smith, S Fityus, M Allman) (AustralianGeochemical Society: Newcastle, NSW)

Smith RAH, Bradshaw AD (1979) The use of metal tolerant plantpopulations for the reclemation of metalliferous wastes. Journal ofApplied Ecology 16, 595–612. doi: 10.2307/2402534

Sproal R, Turoczy NJ, Stagnitti F (2002) Chemical and physical speciation ofarsenic in a small pond receiving gold mine waste effluent. Ecotoxicologyand Environmental Safety 53, 370–375.doi: 10.1016/S0147-6513(02)00057-X

Srivastava M, Ma LQ (2005) Uptake and distribution of selenium in differentfern species. International Journal of Phytoremediation 7, 33–42.

Stoltz E, Greger M (2002) Accumulation properties of As, Cd, Cu, Pb andZn by four wetland plant species growing on submerged mine tailings.Environmental and Experimental Botany 47, 271–280.doi: 10.1016/S0098-8472(02)00002-3

Tam Y, Singh B (2004) Heavy metals availability at industrially contaminatedsoils in NSW, Australia. In ‘Waste management’. (Eds AL Juhasz,G Magesan, R Naidu) pp. 97–120. (Science Publishers: Plymouth, UK)

Tu C, Ma Lena Q (2002) Effects of arsenic concentrations and formson arsenic uptake by the hyperaccumulator ladder brake. Journal ofEnvironmental Quality 31, 641–647.

Weng G, Wu L, Wang Z, Luo Y, Christie P (2005) Copper uptake by fourElsholtzia ecotypes supplied with varying levels of copper in solutionculture. Environment International 31, 880–884.doi: 10.1016/j.envint.2005.05.032

Wild H (1974) Arsenic tolerant plant species established on arsenical minedumps in Rhodesia. Kirkia 9, 265–278.

Manuscript received 27 March 2006, accepted 23 October 2006

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