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Effects of Amendments on Copper,Cadmium, and Lead Phytoextractionby Lolium Perenne from Multiple-MetalContaminated SolutionB. Gunawardana a , N. Singhal a & A. Johnson aa Department of Civil & Environmental Engineering, University ofAuckland, Auckland, New ZealandVersion of record first published: 15 Jan 2011.

To cite this article: B. Gunawardana , N. Singhal & A. Johnson (2011): Effects of Amendments onCopper, Cadmium, and Lead Phytoextraction by Lolium Perenne from Multiple-Metal ContaminatedSolution, International Journal of Phytoremediation, 13:3, 215-232

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International Journal of Phytoremediation, 13:215–232, 2011Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226510903567448

EFFECTS OF AMENDMENTS ON COPPER, CADMIUM,AND LEAD PHYTOEXTRACTION BY LOLIUM PERENNEFROM MULTIPLE-METAL CONTAMINATED SOLUTION

B. Gunawardana, N. Singhal, and A. JohnsonDepartment of Civil & Environmental Engineering, University of Auckland,Auckland, New Zealand

Chemical amendments can increase metal uptake by plant roots and translocation to shoots,however their effectiveness can be influenced by the presence of other amendments andmetal ions in a multiple-metal environment. A range of amendments and combinations weretested to explore their effect on phytoextraction of Cu, Cd, and Pb by perennial ryegrass(Lolium perenne) from solutions containing one or more of these metals. The amendmentsstudied included EDDS (an aminopolycarboxylic acid), histidine (an amino acid), citric acid(an organic acid), rhamnolipid (a biosurfactant) and sulfate (an inorganic ligand). For allamendment treatments, the presence of multiple metals in solution reduced shoot concentra-tions of Cd and Cu, while Pb levels in shoots were generally enhanced by the presence of Cu.Although slightly toxic to the plants, EDDS (1 mM) was the most effective individual amend-ment for enhancing shoot metal uptake and translocation from solution without significantlyreducing biomass yield. The combination Rhm+Cit+EDDS resulted in the highest shootmetal concentrations of all the treatments but also caused severe phytotoxicity. Amendmentcombinations Rhm+His and Sulf+Cit were less toxic for plant growth while moderatelyenhancing metal mass accumulation in shoots and thus could be considered as alternativetreatments for enhanced phytoextraction.

KEYWORDS: metal, chelate, organic acid, amino acid, surfactant, combination, ryegrass

INTRODUCTION

The presence of heavy metals at high concentrations in soil can limit future landuse options as metals are not degradable and therefore will persist for an indefinite period.Elevated concentrations of copper (Cu), cadmium (Cd), and lead (Pb) can be toxic to manyplant species. Selection of an appropriate treatment method for a heavy metal pollutedsite depends on the characteristics and concentrations of metals present. Phytoextractionis a promising treatment method for remediation of metal contaminated soils and water(Salt et al., 1998). This method uses high biomass producing plants to remove contaminantsvia accumulation in harvestable tissues such as stems and leaves (Pilon-Smits, 2005). Manyof the phytoextraction studies have been based on hydroponic studies and have contributedto increased understanding of metal uptake and translocation to the aboveground parts by

Address correspondence to N. Singhal, Department of Civil and Environmental Engineering, University ofAuckland, Private Bag 92019, Auckland, New Zealand. E-mail: [email protected]

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216 B. GUNAWARDANA ET AL.

various plant species such as hyperaccumulators and metal tolerant plants (Brown et al.,1994, Lasat, 2002, Weng et al., 2005, Johnson et al., 2009). However, the efficiency of metaluptake by plants is associated with the bioavailability of metals of concern for root uptake.Chemically enhanced phytoextraction uses natural and synthetic chelators to overcome thelimitations of natural phytoextraction where metals may remain bound to soil particles.By enhancing the solubility of the metals in contaminated media, metals have increasedavailability for plant root uptake and translocation to the shoots.

Metal contamination of soil and water often consists of several metals at high con-centrations rather than a single metal (Marchiol et al., 2004). Although this situation willbe commonly encountered in site remediation, limited research work has been conducted toinvestigate the phytoextraction capabilities of plants in these conditions (Luo et al., 2006;Quartacci et al., 2006, 2007). The chemically-enhanced phytoextraction process dependson high rates of metal extraction by plant roots and an increase in the transfer of metalsto shoots (do Nascimento and Xing, 2006). It is known that the uptake and translocationprocesses of a metal can be affected by the presence of other metals in the growing medium;soil or solution (Luo and Rimmer, 1995; Blaylock and Huang, 2000). Competition betweenmetal ions can significantly influence metal uptake in a multi-component system (Kaliset al., 2006) due to the independent, synergistic (additive) or antagonistic interactionsbetween metals (Kahle, 1993; Kabata-Pendias and Pendias, 2001). In binary mixtures ofheavy metals, the synergistic or antagonistic response of plants seems to be a concentrationdependent effect that is less metal-specific at high metal concentrations. The joint effect ofbinary mixtures of metals has been found to be synergistic only when one element exceedssome critical level of toxicity (Kabata-Pendias and Pendias, 2001).

Over the last decade, various chelating agents have been used for different purposes,including as soil extractants and for maintaining the solubility of micronutrients in hydro-ponic solutions. A wide range of amendments including organic acids (Boominathan andDoran, 2003; Quartacci et al., 2005), chelating agents (Chen and Cutright, 2001; Alkortaet al., 2004; Meers et al., 2005) and inorganics (Bolan and Duraisamy, 2003) have beenused to enhance metal bioavailability for effective uptake from the roots. Many phytoex-traction studies have been carried out using synthetic chelates such as ethylenediaminete-traacetic acid (EDTA) and nitrilotriacetic acid (NTA) in order to artificially enhance metalsolubility, resulting in a marked increase in metal uptake by plants (Vassil et al., 1998;Turgut et al., 2004; Johnson et al., 2009). As an alternative to these chelating agents,[S,S]-ethylenediamine-N,N ′-disuccinic acid (EDDS), a biodegradable chelator with lowertoxicity, has been trialled in phytoextraction studies. It is regarded as an environmentallysafe chelating agent for phytoextraction of Cu and other metals from multiple metal con-taminated soils (Quartacci et al., 2007). EDDS has been shown to enhance the uptakeof heavy metals such as Cu, Cd, Pb, zinc (Zn), and nickel (Ni) by various plant species(Grcman et al., 2003, Kos et al., 2003, Kos and Lestan, 2003, Luo et al., 2005, Meerset al., 2005, Johnson et al., 2009). Histidine is an important naturally-occurring amino acidinvolved in phytoextraction (Callahan et al., 2006). With its ability to act as a tridentateligand via its carboxylate, amine, and imidazole functions, histidine has been shown tohave a role in metal hyperaccumulation (Kramer et al., 1996, Kerkeb and Kramer, 2003,Callahan et al., 2006). A study conducted to explore the role of free histidine in xylemloading of nickel in Brassica juncea L. cv Vitasso found a 75% increase in Ni translocationfrom roots to shoots with equimolar addition of L-histidine to a root medium that con-tained 300 µM nickel (Kerkeb and Kramer, 2003). Citric acid is a naturally occurring lowmolecular weight organic acid that is present in high concentrations in the cell vacuoles of

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EFFECT OF COMBINED METALS AND AMENDMENTS ON PHYTOEXTRACTION 217

photosynthetic plant tissues. It is known to be released by plant roots for the mobilization ofmetals from soil and to form complexes with metal ions (Marschner, 1995). Enhanced phy-toextraction of metals with citric acid has been observed in various studies (Huang et al.,1998, Elkhatib et al., 2001, Chen et al., 2003, Quartacci et al., 2005, Evangelou et al.,2006, Wu et al., 2007). The anionic biosurfactant rhamnolipid, produced by Pseudomonasaeruginosa, has been extensively studied for phytoextraction of heavy metals (Jordan et al.,2002, Johnson et al., 2009) and remediation of heavy metal contaminated soils (Mulligan,2005, 2007). The mechanisms involved in enhanced phytoextraction with inorganic amend-ments are different from those with organic amendments (Schmidt, 2003). Solubilizationof heavy metals with inorganic agents relies mainly on desorption (Brummer et al., 1986).The application of sulfur amendments and sulfate salts has been studied for phytoextractionof heavy metals (Kayser et al., 2000, Puschenreiter et al., 2001).

The enhancement of phytoextraction by chemical amendments seems to be plantand metal specific. In addition, amendments have varying chemical affinities for differ-ent metals, and the presence of multiple metals might hinder the beneficial effects ofapplied amendments (do Nascimento et al., 2006). Thus, chemically enhanced phytoex-traction may encounter limitations with multiple-metal contaminated sites as the effectof amendments on metal complex formation would vary depending on the nature of thesolution. Chemical amendments with sufficiently differing stability constants for the metalspresent in solution are necessary for selective complexation of one metal in the presence ofanother.

The application of chemical amendments aims to enhance metal phytoextractionfirstly by facilitating the release of metals from soil, and secondly by increasing the uptakeof metals by plants. Hydroponic studies are used in order to elucidate the mechanisms ofchemically-enhanced phytoextraction without the confounding effects of soil. This studyaims to explore the effects of amendments and their combinations on copper uptake fromsolution with binary mixtures of metals by a relatively metal-tolerant model species (Loliumperenne). Specifically, this study investigates metal mixtures; Cu with Cd (Cu+Cd), Cuwith Pb (Cu+Pb) and Cu with Cd and Pb (Cu+Cd+Pb) to study the influence of multiplemetals on metal uptake. EDDS (an aminopolycarboxylic acid), histidine (an amino acid),citric acid (an organic acid), rhamnolipid (a biosurfactant) and sulfate (an inorganic ligand)were chosen for their different mechanisms for enhancing metal solubility and mobilizationin solution.

EXPERIMENTAL METHODOLOGY

Hydroponic experiments were carried out in the presence of Cu only, Cu with Cd,Cu with Pb, and Cu with both Cd and Pb in the solution with and without amendments.Amendments were applied individually and in combinations over short term (2 and 10 day)and long term (30 day) exposure durations. Perennial ryegrass (Lolium perenne SR4500)seeds were first germinated in washed glass beads (ABR T-170, Syntech Distributors)supplemented with Hoagland’s nutrient solution modified by supplying iron as ferric am-monium citrate instead of ferric tartrate. The plants were grown for 30 days with a 12-hourphotoperiod. The average ambient temperature ranges were 20±2◦C and 16±2◦C duringthe day and night, respectively. The 30-day old plants were then transplanted into vialswith metals and the amendments. Ten plants were suspended in each vial using PTFE tape.Controls, with Cu and metal mixtures (Cu+Cd, Cu+Pb, Cu+Cd+Pb) were included (with-out amendments) in each experiment. Metal treatments included either 10 mg/L copper as

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Cu(NO3)2.2.5 H2O, 1 mg/L cadmium as CdCl2 or 5 mg/L of Pb as Pb(NO3)2 added to thenutrient solution individually and in combinations. Amendments studied included EDDS(Fluka), histidine (Ajax Finechem), citric acid (Baker), JBR425 rhamnolipid biosurfactant(Jeneil Biosurfactant Company) and sulfate as Na2SO4 (BDH). Experiments were per-formed in triplicate. The pH of the initial solutions was adjusted to 6, which is a favourablelevel for plant growth and metal solubility. With long-term experiments, treatment solu-tions were replaced weekly to maintain constant concentrations over the study period.Table 1 summarizes the concentrations of metals and amendments used in the experiments.The concentrations of metals and amendments were chosen following preliminary toxicitystudies and speciation modelling.

Following the 2-, 10-, or 30-day exposure times, plants were removed from treatmentsolutions for analysis, with roots rinsed with deionized water to remove any metals looselyadhering to root surfaces. Roots and shoots (stem + leaves) were sectioned and oven driedat 104◦C for 24 hours with dry weights of plant parts recorded. Dried plant materialswere ashed at 550◦C for 4 hours in a muffle furnace with the ash then dissolved in 2 mLof 2 M nitric acid. Acid digested samples were diluted appropriately with deionized wa-ter and analyzed using graphite furnace atomic absorption spectrophotometry (GF-AAS)(GTA-110/SpectrAA50, Varian, Inc.).

Statistical analysis of the data was performed using SPSS Statistics v17.0. Multivari-ate and univariate ANOVA was used to determine significant effects and interactions of thevarious amendment and metal treatments. Multiple regression analysis was used to deter-mine the influence of each metal and amendment on tissue mass and metal concentrations.Significance was determined at the p < 0.05 level. Chemical speciation modelling was car-ried out for EDDS, citric acid, sulfate and their combinations using Visual MINTEQ version2.60 (obtained from www.lwr.kth.se/english/oursoftware/vminteq) to give an insight intothe speciation of the hydroponic solutions.

Table 1 Concentrations of metals and amendments in nutrient solutions

Treatment Concentration (mM)

Cu+2 0.158 (10 mg/L)Cd+2 0.009 (1 mg/L)Pb+2 0.024 (5 mg/L)Cu+2 + Cd+2 0.158 Cu+2 + 0.009 Cd+2

Cu+2 + Pb+2 0.158 Cu+2 + 0.024 Pb+2

EDDS 1Histidine (His) 1.5Citric acid (Cit) 2Rhamnolipid (Rhm) 0.15 (1.7 × CMC∗)Sulfate (Sulf) 3Rhm + EDDS 0.15 Rhm + 1 EDDSRhm + His 0.15 Rhm + 1.5 HisRhm + Cit 0.15 Rhm + 2 CitRhm + Sulf 0.15 Rhm + 3 SulfRhm + Cit + EDDS 0.15 Rhm + 2 Cit + 1 EDDSSulf + Cit 3 Sulf + 2 Cit

∗CMC represents the critical micelle concentration, which for Jeneil rhamnolipid is 50 mg/Lwith a molecular weight of 567 g (Jeneil Biosurfactant Co. LLC, 2001; Neilson et al., 2003).

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RESULTS AND DISCUSSION

The results of the present study were analyzed based on the visual observationsof toxicity symptoms, root and shoot biomass yield, uptake of metals by roots, metalconcentrations in shoot tissues and degree of metal transfer from roots to shoots.

Effects of Amendment and Metal Combinations on Plant Growth

In some treatments, plants showed symptoms of toxicity including chlorosis, necrosis,reduced growth and leaf abscission. The severity of toxicity was visually assessed withrespect to the relevant metal-only controls and is summarized in Table 2. When appliedseparately, EDDS and rhamnolipid did not severely reduce plant health, however, whenapplied in combination, their detrimental effects were significantly increased. Plants treatedwith metals and the combinations Rhm+Cit+EDDS and Rhm+EDDS suffered the mostsevere phytotoxicity of all the treatments with plant growth visibly stunted and plants almostdead after 30 days. The chlorotic symptoms observed could be due to the additive effectsof multiple metals which exceed the capacity of plants to activate their defence systems(Navari-Izzo and Quartacci, 2001). Cu toxicity has been considered as one of the limitingfactors in the phytoremediation of multiple metal contaminated soils (Lombi et al., 2001).The influence of sulfate on plant growth appeared to be positive in metal mixtures, andimproved plant health when applied in combination with citric acid. Other amendments andcombinations appeared to have only a small effect, or had no consistent observable effect onplant growth. Figure 1 shows the root and shoot dry mass yields of hydroponically-grownperennial ryegrass from solutions containing one or more of Cu, Cd, and Pb relative to thecontrols after 30 days. In the current study, plants grown in unamended control solutionswith Cu+Cd produced the least root and shoot biomass than plants with other metals andcombinations alone. With individual metals in control solutions, root and shoot biomassproduction decreased in the order Pb>Cd>Cu (data not shown), while concentrations of themetals decreased in the order Cu>Pb>Cd (Table 1). This indicates that the concentrationof each metal is not directly proportional to the biomass change observed. In controlplants, a similar level of root and shoot biomass production was observed with Cu, Cu+Pband Cu+Cd+Pb treatments, which was approximately 30% less than that seen in the Pbtreatment.

Root Growth

Individual application of EDDS and citric acid with metal mixtures significantlyenhanced root growth, with the beneficial effect greater than that seen for plants with Cualone. When applied with single or multiple metals, sulfate increased the root biomass overthat of the corresponding controls. The influence of EDDS, citric acid and sulfate with themetal mixtures appears to be favourable for root growth and may be due to reduced toxicity.EDDS has previously been observed to alleviate metal toxicity in both roots and shootswhen applied at low concentrations (0.5 mM) (Tandy et al., 2006). Speciation modellingindicates that free metal concentrations in solution are reduced when these amendments areadded. Histidine did not significantly affect root growth with the metal mixtures (Figure 1).When applied to metal mixtures, rhamnolipid decreased root biomass yield by up to 32%,with root growth suppressed compared to that with Cu alone. The detrimental effects ob-served may be due to surfactant molecules damaging cell membranes, causing phytotoxicity

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Table 2 Effects of amendments on key parameters (relative to the respective metal-only controls) for perennialryegrass grown with single and multiple metals over 30 days

Toxicity Metal Mass Uptake

Root ShootShoot

Amendment Metal Observed Biomass Cu Cd Pb Cu Cd Pb

EDDS Single Metal − na na − + +++ ++ +Cu + Cd − na na + ++ −Cu + Pb − na na + ++ ++Cu +Cd + Pb na + − ++ ++ ++ − +

His Single Metal + + + + − +++ +++ −Cu + Cd − na na + + +Cu + Pb − na − na na ++Cu +Cd + Pb na + + ++ ++ ++ na +

Cit Single Metal na na + na na ++ ++ naCu + Cd + + ++ ++ ++ +Cu + Pb na na + ++ na ++Cu +Cd + Pb na + + na ++ ++ − ++

Rhm Single Metal − na + − − + ++ −Cu + Cd na na na + na naCu + Pb na na na − ++ −Cu +Cd + Pb − − na − − − − −

Sulf Single Metal − na ++ na + ++ ++ −Cu + Cd + + + ++ na +Cu + Pb + + + + + −Cu +Cd + Pb + na + na na na na −

Rhm + EDDS Single Metal — − na − − +++ ++ naCu + Cd – na − − ++ naCu + Pb – − − − ++ ++Cu +Cd + Pb − − − − + ++ na +

Rhm + His Single Metal + na na na na ++ ++ naCu + Cd na na + ++ ++ naCu + Pb na na na na ++ +Cu +Cd + Pb − − + na na ++ − na

Rhm + Cit Single Metal − na ++ na ++ ++ ++ +Cu + Cd na na + ++ + +Cu + Pb na na + ++ ++ +Cu +Cd + Pb − − + na ++ + na +

Rhm + Sulf Single Metal − na + − na ++ ++ naCu + Cd − na + − + +Cu + Pb + na na na ++ +Cu +Cd + Pb − − + − na + na na

Rhm + Cit + EDDS Single Metal — − + − − +++ ++ +Cu + Cd — − na − ++ −Cu + Pb — − − na ++ ++Cu +Cd + Pb − − − − + ++ + ++

Sulf + Cit Single Metal ++ na ++ − ++ ++ + +Cu + Cd + na ++ +++ ++ +Cu + Pb + na ++ ++ ++ +Cu +Cd + Pb na na ++ + ++ + na +Legend: % Change Detriment Improvement

0–20% na na20–100% − +100–500% −− ++>500% −−− +++

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Figure 1 Effect of chemical amendments and combinations on dry root and shoots mass after 30 days (relative tocontrol treatments). The significance of differences between the means was evaluated by the Dunnett (two-sided)post-hoc test compared to respective (metal-only) control treatments [∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001].

and impeded root growth. With single and multiple metals, the amendment combinationsRhm+EDDS, Rhm+Sulf and Rhm+Cit+EDDS significantly decreased root growth, withdecreases in Cd treatments of 65%, 41% and 77% respectively. The Sulf+Cit treatmentwas beneficial for root growth, with increases of up to 73% observed (Figure 1).

Shoot Growth

Individual amendments with metal mixtures either enhanced, or did not significantlyaffect shoot biomass yield suggesting that single amendments were not detrimental togrowth. Furthermore, when applied to metal mixtures, histidine, citric acid and sulfatewere generally beneficial to shoot biomass after 30 days (Figure 1). In general, except forSulf+Cit, amendment combinations were detrimental to shoot biomass or had no significanteffect (Figure 1). The effect with Rhm+Cit+EDDS was significant with 36% reduction inshoot biomass yield after 30 days for all metal treatments.

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The reduction in shoot biomass yield observed in the current study could be attributedpartly to the combination of heavy metal concentration and chemical amendment additionwhich may change the chemical components in the solution (Chen and Cutright, 2001). Adecrease in shoot dry biomass of Indian mustard has been observed with 5 mMkg−1 NTAapplied to multiple-metal contaminated soil. This was linked to the high uptake of Cd,Cu, Pb and Zn in shoot tissues (Quartacci et al., 2006). Once chemical amendments areintroduced, they tend to form complexes with the metals present in the solution to varyingdegrees, depending on their chemical affinities to each metal (Salt et al., 1998). Simulta-neous competition of cations for amendments may affect the free metal and amendmentconcentrations in the solution. This may create an imbalance of chemical components inthe solution leading to various toxicities as the free metal ions and unbound amendmentsare known to be more toxic to plants (Salt et al., 1998, Vassil et al., 1998).

EFFECTS OF AMENDMENT AND METAL COMBINATIONS ON METAL

CONCENTRATIONS IN PLANT TISSUE

Effects of amendments on Cu, Cd and Pb concentrations in roots and shoots areshown in Figures 2, 3 and 4. Concentrations of Cu, Cd and Pb in shoot tissue of unamended(control) treatments after 30 days were 40, 10, and 46 µg/g-dry weight respectively in thesingle metal treatments, and 97, 33, and 32 µg/g-dry weight respectively in the Cu+Cd+Pbtreatments. In the Cu+Cd control treatment, Cu and Cd concentrations in shoot tissue were208 and 57 µg/g-dry weight respectively, while Cu and Pb concentrations were 120 and35 µg/g-dry weight respectively in the Cu+Pb treatment. In general, shoot Cu and Cdconcentrations were higher when these metals were applied singly, rather than in a metalmixture, while the presence of other metals enhanced Pb concentrations in shoot tissues.With metal mixtures, individual amendments were less effective for increasing shoot metalconcentrations than when applied with single metals (Figure 2).

The effects of metals and amendments on key uptake parameters for all treatmentsafter 30 days are summarized in Table 2. The concentrations of Cd in shoot tissues were sig-nificantly reduced by the presence of Cu and Cu+Pb in all treatments other than Sulf+Cit.In all amendment treatments other than citric acid and rhamnolipid, the presence of Cd andPb decreased Cu accumulation in shoots (Table 2) indicating antagonistic interactions of Cdand Pb with amendments on Cu uptake and translocation. Chemical speciation modellingwas carried out using Visual MINTEQ version 2.60 to give an insight into the speciationof the hydroponic solutions with a single metal and multiple metals. Results show that thepresence of multiple metals would not greatly alter the concentration of free Cu+2, Cd+2 orPb+2 ions in control solutions, compared to when these metals were applied individually. Inthe presence of EDDS and sulfate, modelling indicates little competition between Cu, Cdand Pb for ligand availability. In treatments where citric acid was used, the degree of com-plexation of Cu was not significantly influenced by the presence of Cd and Pb. However,the concentrations of complexed Cd and Pb were significantly decreased by the presenceof other heavy metals. Citric acid appeared to dominate complexation in all amendmenttreatments where it was included. It has been evidenced, by both physiological and molec-ular studies, that metal transporters in plants have relatively poor selectivity (Reid 2001).Hence, in multiple-metal environments, metal ions and their complexes would effectivelycompete for uptake, potentially hindering Cu uptake and movement in shoots while encour-aging uptake of other metals (Figure 3). It has previously been found that complexation

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Figure 2 Effect of chemical amendments and combinations on Cu concentrations in roots and shoots after 30days (relative to control treatments). The significance of differences between the means was evaluated by theDunnett (two-sided) post-hoc test compared to respective (metal-only) control treatments [∗P < 0.05, ∗∗P < 0.01and ∗∗∗P < 0.001].

of Cd with rhamnolipid is rapid and stable (Tan et al., 1994). Competition between metalcomplexes and free metals ions for root sorption sites may also increase metal uptake toshoots by reducing the sorption and retention of heavy metal ions in root tissues.

The influence of EDDS was significant and resulted in the highest shoot Cu concen-tration among all the individual amendments, though this effect was reduced to nearly onefifth by the presence of other metals in the solution (Figure 2). EDDS also caused the rootCu concentration to be significantly lower than the Cu control, indicating higher Cu translo-cation to the shoots. EDDS has previously been shown to be effective in the enhancementof shoot Cu uptake in plants grown on multiple metal contaminated soils (Luo et al., 2005,Meers et al., 2005, Luo et al., 2006, Quartacci et al., 2007). The application of 5 mM/kgEDDS to a soil contaminated with Cu, Cd, As, Pb and Zn enhanced the Cu concentration

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Figure 3 Effect of chemical amendments and combinations on Cd concentrations in roots and shoots after 30days (relative to control treatments). The significance of differences between the means was evaluated by theDunnett (two-sided) post-hoc test compared to respective (metal-only) control treatments [∗P < 0.05, ∗∗P < 0.01and ∗∗∗P < 0.001].

in Brassica carinata shoots 4-fold compared to control plants one week after treatment(Quartacci et al., 2007). The effectiveness of EDDS for increasing metal concentrations inshoots was dependent on the metals applied, and decreased in the order Cu>Cd>Pb. In thepresence of other metals, the influence of EDDS on Cu and Cd uptake generally lessened.In many treatments, the retention of Cd in root tissue was significantly increased by thepresence of Cu (Figure 3). However, in some treatments this effect was diminished by thesimultaneous presence of Pb, perhaps due to the displacement of Cd from sorption sitesby Pb cations, as Pb retention in roots was generally increased in multi-metal treatments(Figure 4).

The rhamnolipid biosurfactant was only effective for increasing metal concentra-tions in shoot tissue when used in combination with other amendments (Figure 2). By

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Figure 4 Effect of chemical amendments and combinations on Pb concentrations in roots and shoots after 30days (relative to control treatments). The significance of differences between the means was evaluated by theDunnett (two-sided) post-hoc test compared to respective (metal-only) control treatments [∗P < 0.05, ∗∗P < 0.01and ∗∗∗P < 0.001].

itself, rhamnolipid is unlikely to facilitate increased transport to shoots as the surfactantmolecules and complexes formed are large and intact micelles formed would not be likelyto traverse effectively through the cell membranes (Kvesitadze et al., 2005). Rhamnolipidhas previously been found to be less effective than the chelator EDTA for complexing andremoving Cu, Pb and Zn from a multiple metal contaminated soil (Jordan et al., 2002).

In general, amendment combinations markedly increased shoot Cu, Cd, and Pbconcentrations in single and multiple metal environments. In particular, when applied tosingle and multiple metal solutions, the Rhm+Cit+EDDS treatment significantly increasedshoot Cu, Cd, and Pb concentrations after 30 days, although severe phytotoxicity was alsoobserved (Table 2). Interestingly, when applied to binary metal mixtures, Rhm+His andSulf+Cit treatments significantly enhanced Cu accumulation in shoots without decreasingbiomass yield (Table 2), and therefore could be considered as viable amendments forenhancing Cu phytoextraction from multiple metal environments. Rhamnolipid, EDDS andcitric acid appeared to have synergistic effects with Cu for increasing Pb concentrations in

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Table 3 Values for standardized coefficients (β) and model regression coefficients (R2) from multiple regressionanalysis

Tissue Mass Root Concentration Shoot Concentration

Treatment Shoot Root Cu Cd Pb Cu Cd Pb

EDDS −0.322 −0.323 −0.445 −0.45 0.215 0.867 ns† 0.622Histidine ns ns ns 0.199 0.217 0.224 ns 0.161Citric acid ns 0.232 0.243 ns 0.556 0.242 0.194 0.428Sulfate ns ns 0.364 ns 0.323 0.103 ns nsRhamnolipid −0.399 −0.359 0.406 −0.151 −0.165 0.127 ns 0.166Cu −0.461 −0.44 na∗ −0.287 −0.429 na ns 0.132Cd −0.224 −0.217 ns na −0.369 ns na −0.163Pb ns ns ns −0.383 na −0.256 −0.432 naR2 0.617 0.561 0.546 0.596 0.679 0.777 0.277 0.599

†ns = not significant and ∗na = not applicable.

shoot tissues, with 4-fold and 9-fold increases in the Pb concentrations in the Rhm+EDDSand Rhm+Cit+EDDS treatments respectively.

Multiple regression analysis was used to investigate the influence of amendment andmetal treatments on plant biomass and tissue metal concentrations. In general, high regres-sion coefficients were found, indicating that models were able to represent the experimentaldata. The magnitude of the individual standardized coefficients (given in Table 3) indicatesthe relative influence of each factor in the model. The only factor to show a beneficial effectfor plant biomass was citric acid, while EDDS, rhamnolipid, Cu and Cd were detrimentalfor both root and shoot growth. Histidine, citric acid and sulfate tended to increase metalconcentrations in both roots and shoots. Antagonistic interactions between Cd and Pb re-sulted in reduced levels of these metals in root and shoot tissue when both metals werepresent in solution. The presence of Pb also lowered Cu concentrations in shoot tissue. Thepresence of EDDS, rhamnolipid and Cu generally increased shoot metal concentrations,but reduced metal concentrations in root tissue.

ANOVA was used to establish the main effects and interactions of the different ex-perimental factors influencing biomass yield (root and shoot growth) and metal uptake.For amendments, the factors considered were the presence of EDDS, histidine, citric acid,rhamnolipid and sulfate, while the factors for metals were Cu, Cd, and Pb. Significantinteractions (p < 0.05) were determined from between-subjects effects. For biomass accu-mulation, the main effects of both rhamnolipid and Cu were modified by the presence ofEDDS. Two-way Cu-rhamnolipid and Cu-sulfate interactions were modified by the pres-ence of citric acid. The presence of EDDS modified the main effect of Cu and Pb on metalconcentrations in shoot tissue, with Cd also modifying the effect of Pb. The main effect ofEDDS on root metal concentrations was modified by the presence of citric acid, and themetals Cu and Pb. Significant interactions also occurred between sulfate and all metals,in addition to rhamnolipid. The significant two-way interaction between Cd and Pb wasmodified by the presence of either sulfate or histidine.

Enhanced phytoextraction of metals using combined treatments has been previouslyreported (Elless and Blaylock 2000, Luo et al., 2006). Combined application of 3.33 mM/kgEDTA with 1.67 mM/kg EDDS (2:1) showed the highest efficiency in increasing the Cu, Pb,

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Zn and Cd concentrations in Zea mays shoots (Luo et al., 2006). The results of the presentstudy have shown generally higher metal accumulation with EDDS when applied singlyand in combinations. This implies that EDDS in single and multiple metal contaminatedsolutions may help overcome specific exclusion mechanisms that prevent transport of Cuto above-ground tissues. Plants are known to vary in their ability to exclude metals fromphotosynthetic tissues (Weis et al., 2004). Unbound EDDS may bind to essential cationsin root cells resulting in the breakdown of exclusion mechanisms in plants (Luo et al.,2006). Metal complexes may also enter the root through breaks in the root endodermis andCasparian strip and be rapidly transported to the shoots (Bell et al., 1991). Furthermore, ithas been shown that Cu can be taken up and transported within plant parts as Cu-EDDScomplexes (Luo et al., 2005).

Translocation

Metal translocation indicates the ability of amendments to affect metal transfer fromroots to shoots and is expressed as the ratio of metal in the shoots to that in the roots. Figure 5shows the effect of chemical amendments on metal translocation from roots into shoottissues after 30 days in single and multiple metal solutions. Copper translocation fromroots to shoots was affected by the amendments and simultaneous presence of Cd or Pb(Figure 5). In general, compared to the metal mixtures, amendments and their combinationscaused a dramatic increase in Cu translocation when only Cu was applied to the solution.In the presence of other metals, the translocation of Cd was reduced by approximately oneorder of magnitude for all amendment treatments. However, the translocation of Pb wasincreased further by the presence of Cu than by any amendment or combination. This wasmost obvious in treatments with histidine and Rhm+Cit+EDDS where translocation ofPb was increased by factors of 3.7 and 3.2 respectively due to the presence of Cu in theexposure solution.

When applied to metal mixtures, EDDS significantly enhanced Cu translocation.EDDS has been shown to significantly increase the shoot:root concentration ratios of Cu,Pb, Zn and Cd from a multiple-metal contaminated soil (Luo et al., 2005). However, inthe current study, the enhancement with EDDS in metal mixtures was approximately threetimes less than that with EDDS and Cu only in the solution (Figure 5). Thus, interactions ofCd and Pb with EDDS resulted in reduced Cu transport through the plant parts. The metal-succinate complexes simultaneously present in the solution would effectively compete foruptake hindering the Cu transport rate to the shoots (Luo et al., 2005). The comparativelylow metal translocation observed with sulfate in the metal mixtures could be attributed tothe relatively small differences between the stability constants of heavy metal-amendmentcomplexes of sulfate with Cu, Cd, and Pb (Table 4). This would cause strong competitionamong Cu, Cd, and Pb for complexation resulting in higher free metal ion concentration inthe solution (Wang et al., 2007).

When applied to metal mixtures, the amendment combinations Rhm+EDDS andRhm+Cit+EDDS generally enhanced metal translocation from roots to shoots, with theeffect greater for Cu than Cd and Pb. This enhancement effect could be attributed to theinfluence of EDDS. The application of other amendment combinations with Cu+Cd didnot cause an improvement in Cu translocation but increased Cu translocation when appliedto Cu+Pb (Figure 5). Due to the more negative charge on the complex ion than the metal

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Figure 5 Effect of chemical amendments on translocation of Cu, Cd and Pb from root to shoot tissues after 30days (relative to control treatments).

cation itself, metal complexation by ligands such as EDDS is likely to reduce metal sorptionto cation exchange sites in plant tissues (Wenger et al., 2003). This will have the effectof reducing metal retention in root tissues, allowing greater translocation of metals to theshoots.

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Table 4 Properties of amendments included in the experiments

Log K∗

Chemical name Molar Mass (g) Molecular Formula M:L† Ratio Cu Cd Pb

EDDS 292.2 C10H13N2O8 1:1 18.4 10.8 12.7Histidine 155.2 C6H9N3O2 1:2 18.1 9.7 9.0Citric acid 192.1 C6H8O7 1:1 5.9 4.5 8.9Sulfate 50.0 SO−2

4 1:1 2.4 2.5 2.8Rhamnolipid 504–650 C26H48O9, C32H58O13 1:2 9.3 6.9 8.6

†M = Metal and L = Ligand. ∗Stability constants are given for the most stable form with metal at an ionicstrength of 0.1 and a temperature of 25◦C (with the exception of citric acid at 20◦C). Data for EDDS, histidine,citric acid and sulfate compiled from (Martell and Smith, 1974) and for rhamnolipid from (Ochoa-Loza et al.,2001).

CONCLUSIONS

Chemically assisted uptake and accumulation of metals in plants has received muchattention in phytoextraction. However, the effect of chemical amendments could be affectedby the simultaneous presence of various metals in the solution. Thus, phytoextraction studieson multiple metal contaminated solutions may face difficulties in choosing the appropriateplant species, chemicals and mechanisms for enhanced efficiency of extraction of metal inconcern.

The results of the current study have shown that EDDS was generally the most effec-tive among the amendments tested for enhancing shoot metal concentrations. Regardlessof the amendment treatment, metal concentrations in shoot tissues were decreased by thepresence of other heavy metals in the exposure solutions, with the notable exception of Pb,for which concentrations were enhanced by the presence of Cu. For Cd in particular, thepresence of other metals in solution clearly negated the beneficial effects of amendments forenhancing metal concentrations in shoots. This may indicate competition between metalsand their complexes for uptake pathways in the plant, which may be limiting. Histidinewas only effective for increasing Cu and Cd concentrations in shoots when these metalswere applied individually, although it also slightly increased Pb levels in the presence ofCu. Citric acid enhanced shoot Cd accumulation when applied to Cd-only treatments butthe effect for Pb was not significant unless Cu was also present in solution. Rhamnolipidand sulfate were not effective for improving Pb concentrations in shoots, but significantlyenhanced Cd levels in shoots in Cd-only treatments. Metal translocation to shoot tissue wasenhanced by the presence of amendments, however, the effectiveness of amendments andcombinations in metal mixtures was less compared to that with only a single metal in thesolution. Amendment combinations with metal mixtures increased the mass accumulationof Cu in shoot tissue. With rhamnolipid, the mass uptake of Cu to shoots was greaterfrom the Cu+Pb mixture than that with Cu only in the solution. Combined application ofRhm+Cit+EDDS resulted in the highest metal translocation and shoot concentrations ofall the treatments, although dramatic decreases in shoot biomass yield were also observed.Thus, EDDS was superior to Rhm+Cit+EDDS for increasing metal phytoextraction frommetal mixtures without affecting shoot biomass yield. Interestingly, when applied to metalmixtures, Rhm+His and Sulf+Cit treatments significantly enhanced the Cu accumulationin shoots without decreasing biomass yield and therefore thus could be considered viable

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alternatives for metal phytoextraction from multiple metal environments. Other amendmentcombinations impeded plant growth showing limited potential as effective treatments forphytoextraction from multiple metal contaminated solutions over an extended period.

ACKNOWLEDGMENTS

The authors would like to acknowledge the constructive feedback from the anonymousreviewers and the advice regarding statistical analysis provided by Dr. M. Blumenstein andDr. D. Scott (University of Auckland).

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effects of amendments on copper, cadmium, and lead phytoextraction by lolium perenne ...

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