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Published in J. Environ. Qual. 32:1669-1676 (2003).
© 2003 ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Heavy Metals in the Environment

The Role of Nitrilotriacetate in Copper Uptake by Tobacco

K. Wenger*,a,c, S. K. Guptaa, G. Furrerb and R. Schulinb

a Swiss Federal Research Station for Agroecology and Agriculture, Liebefeld, Schwarzenburgstrasse 155, CH-3003 Bern, Switzerland
b Institute of Terrestrial Ecology (ITO), ETH Zurich, Grabenstrasse 3, CH-8952 Schlieren, Switzerland
c Institute of Terrestrial Ecology (ITO), ETH Zürich, Grabenstrasse 3, CH-8952 Schlieren, Switzerland

* Corresponding author (wenger{at}ito.umnw.ethz.ch).

Received for publication February 1, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In growth chamber experiments we studied the effect of nitrilotriacetate (NTA) on Cu uptake by tobacco (Nicotiana tabacum L.). Plants were exposed for 6 d to 126 µM Cu and 500 µM NTA in nutrient solutions without and with 10 g L-1 montmorillonite. Approximately seven times less Cu was dissolved in the montmorillonite solutions than in the nutrient solutions alone. In the absence of NTA, montmorillonite effectively competed with plant roots for Cu, although Cu remained bound to the roots. Nitrilotriacetate increased Cu uptake and translocation into shoots of tobacco by a factor of 3.5 from the nutrient solution and by a factor of 26 from the montmorillonite nutrient solution. Neither growth reduction nor any other visible sign of Cu toxicity was found in the presence of NTA with Cu concentrations of 190 mg kg-1 in the shoots. In the absence of NTA, high Cu concentrations in root samples led to a brownish discoloration of the roots.

Abbreviations: EDTA, ethylendiaminetetraacetate • NTA, nitrilotriacetate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN RECENT YEARS, phytoextraction of heavy metals has been proposed as a novel cleanup technology (Jørgensen, 1993; Cunningham et al., 1995; McGrath et al., 1998). Unfortunately, efficient metal uptake by remediation plants is often limited by the availability of the metals for uptake, in particular in neutral or alkaline soils.

One promising strategy to increase the phytoavailability of heavy metals in soil is to amend soils with organic ligands that form water-soluble complexes. Many authors, including Jørgensen (1993), Blaylock et al. (1997), Huang et al. (1997), Epstein et al. (1999), and Wu et al. (1999), have shown that metal uptake by plants can be increased by the addition of ethylenediaminetetraacetate (EDTA). Other authors report that the use of chelating substances such as diethylenetriaminepentaacetate (DTPA) and EDTA did not enhance (Athalye et al., 1995) and in some cases reduced (Robinson, 1997) heavy metal uptake by plants. Considerable debate exists on whether heavy metals were taken up as metal complexes or only after first being split from the chelate. In general, a lower rate of uptake of metal cations from metal–organic complexes is reported than for free cations (Jarvis, 1987). Chaney et al. (1972) demonstrated that Fe is taken up by plants only after first being split from the Fe–chelate complex. However, Vassil et al. (1998) and Epstein et al. (1999) observed Pb–EDTA in the xylem sap of Indian mustard [Brassica juncea (L.) Czern.], suggesting that some plants may be able to take up metals in this form. They hypothesized that physiological stress induced uptake of the intact Pb–EDTA complex. Extended X-ray absorption fine structure (EXAFS) analysis indicated that in primary leaves of bean plants 60 to 70% of Pb is present as Pb–EDTA (Sarret et al., 2001), implying that about 30 to 40% of the complex has split before uptake.

Few studies (Kayser et al., 2000; Kulli et al., 1999) have been performed with NTA as the ligand to assist phytoextraction as far as we know. Although NTA is a weaker chelator than EDTA, it is strong compared with low-molecular organic acids such as citrate and oxalate (Smith and Martell, 1976). Nitrilotriacetate is particularly interesting for chelate-assisted phytoextraction because it combines high biodegradability (Bolton et al., 1996; Tiedje and Mason, 1974) with chelating strength. In contrast, EDTA is poorly degraded in soils (Kari and Giger, 1996).

This study examined whether NTA enhanced Cu uptake and translocation within tobacco from nutrient solutions. To simulate competitive sorption by mineral soil surfaces, the clay mineral montmorillonite was added to the nutrient solution as an additional factor in one series of experiments. Montmorillonite is an important clay mineral in soils with a high sorption capacity. Tobacco has previously been used in other investigations of metal uptake by high-biomass-producing plants (Mench and Martin, 1991; Guadagnini, 2000; Kayser et al., 2000). Copper was chosen as the pollutant because it is particularly toxic to many plant species (Pahlsson, 1989) and because of its relevance as a soil pollutant. In Switzerland, for example, about 50000 ha of land show elevated Cu concentrations (Studer et al., 1995). Excessive Cu induces a decrease in microbial biomass and therefore poses a risk to soil quality. Although possible adverse effects of Cu on human health are not considered significant, the sensitivity of sheep to Cu is well known (Bundesamt für Umwelt, Wald und Landschaft, 1997). Furthermore, Cu was a primary contaminant on the field site where the effects of NTA amendments were studied by Kayser et al. (2000).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Nutrient Solution without Minerals
To avoid competition between EDTA, NTA, and micronutrients we modified a Hoagland solution by eliminating the micronutrients and by replacing NaFe(III)EDTA with FeSO4·7H2O (Table 1). The nutrient solutions were buffered with 2 mM MES (2-morpholinoethanesulfonic acid monohydrate). With the exception of MES, obtained from Fluka (Buchs, Switzerland), all other chemicals were obtained from Merck (Darmstadt, Germany). All solutions were made with high-purity 18 M{Omega} cm-1 water (Millipore, Bedford, MA).


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  		Table 1. Composition of the modified Hoagland solution used in this study.

 
Four treatments (control, NTA, Cu, and Cu + NTA) were performed in two experiments (Table 2). The pH of all solutions was adjusted with NaOH to 6 before the plant uptake experiments.


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  		Table 2. Nitrilotriacetate (NTA) and copper concentrations in the nutrient solutions used in the various treatments.

 
Nutrient Solution with Montmorillonite
Montmorillonite (SWy-2) was obtained from the Clay Minerals Society, Columbia, MO. Suspension experiments were analogous to the nutrient solution treatments except for the addition of 10 g L-1 montmorillonite to the solutions defined in Table 2. The suspension pH was adjusted to 6 with NaOH in all treatments. The vessels containing the suspensions were placed on a rotary shaker for 24 h at 125 rpm. Before the plant uptake experiments, pH was readjusted to 6.

Plant Uptake in Nutrient Solution Experiments
The experiments were performed in a growth chamber on a 16 h (25°C)–8 h (15°C) day–night cycle. Plants were grown in 1.4-L pots (diameter = 10 cm) with a cover to protect plant roots against light. Nutrient solution experiments without montmorillonite were performed with ‘Burley 92’ tobacco, whereas suspension experiments were performed with ‘Badischer Geudertheimer’ tobacco due to a shortage of plant material. To compare the two cultivars, control and Cu treatment were performed with both in the experiments without montmorillonite. There were replicate pots containing one tobacco plant per treatment. After 6 d, the tobacco plants were harvested and rinsed with deionized water. Roots and shoots were divided and dried at 70°C to a constant weight. The oven-dried plant material was ground in a titanium mill. Samples of 500 mg were then microwave-digested in a mixture of 5 mL HNO3 (65%) and 3 mL H2O2 (30%). The digested samples were then diluted to 25 mL in high-purity 18 M{Omega} cm-1 water.

Copper, Ca, Mg, K, and Fe concentrations in plant extract and filtered nutrient solutions were determined by use of inductively coupled plasma–atomic emission spectrometry (ICP–AES; Model 2000; PerkinElmer, Wellesley, MA). Nitrilotriacetate solution concentrations were analyzed with a Dionex (Sunnyvale, CA) DX 500 ion chromatograph coupled to a CD 20 conductivity detector, an AS 11 column with AG 11 precolumn, and an ASRS 1 suppressor.

Modeling
Chemical speciation calculations were performed with the program ChemEQL (Müller, 1996). Stability constants were taken from Smith and Martell (1976). The log K values of the surface species of montmorillonite were taken from Bradbury and Baeyens (1997) and Neubauer et al. (2000). Two types of surface hydroxyl groups ({equiv}SsOH with high affinity and {equiv}SwOH with low affinity) as considered by Lothenbach et al. (1997) and Bradbury and Baeyens (1997) were chosen. In addition, cation exchange sites ({equiv}X-) were taken into account. The values of the model parameters used in the calculations are listed in Table 3.


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  		Table 3. Conditional stability constants (temperature = 25°C, ionic strength = 0.003) and surface site concentrations used for modeling sorption of Cu to montmorillonite, and aqueous speciation calculations.

 
Statistical Analysis
The metal concentrations in plant tissue were log-transformed to normalize the frequency distributions. Analysis of variance (ANOVA) was performed with the general linear model procedure of SAS Version 6.12 (SAS Institute, 1997). If the F value indicated significant differences (P < 0.05), post hoc pairwise comparisons were performed with Tukey and Bonferroni adjustment of probabilities.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Metal Speciation in Mineral-Free Nutrient Solution
Table 4 shows that only 10 to 20% of the added Fe was dissolved in the control and Cu treatments. Furthermore, only 30% of the added Cu was dissolved in the Cu treatment. According to the speciation calculations, the addition of NTA led to an almost 100% complexation of Fe and Cu, while only 3 to 5% of the Ca and Mg formed NTA complexes. Without NTA, 95 to 100% of the Cu was present as a free metal cation.


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  		Table 4. Concentrations (µM) and aqueous speciation{dagger} (%) of nutrients and nitrilotriacetate (NTA) as affected by treatments for nutrient solution without minerals.

 
Metal Speciation in the Presence of Montmorillonite
In the absence of NTA, montmorillonite removed between 30 and 50% of the Fe and almost all of the Cu from the solution, while 60 to 70% of the Mg and approximately 55% of the Ca in the Cu treatment remained in solution. In the presence of NTA, sorption of any metal cation by montmorillonite was negligible (Table 5). In the NTA treatment even more Ca and Fe were observed in solution than originally added. The speciation calculations were similar to those described in the absence of montmorillonite (Table 4).


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  		Table 5. Concentrations (µM) and aqueous speciation{dagger} (%) of nutrients and nitrilotriacetate (NTA) as affected by treatments for nutrient solution with montmorillonite.

 
The model calculations predicted that about 80% of the added Cu in the Cu treatment was adsorbed on montmorillonite, while a Cu sorption of 96% was experimentally determined. According to the model, the main fraction of adsorbed Cu was bound to the low-affinity sites of the surface hydroxyl groups of montmorillonite (Table 6).


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  		Table 6. Sorption of Cu to montmorillonite as determined by measurement and modeling (conditions: 126 µM Cu, 10 g L-1 montmorillonite).

 
Plant Growth and Metal Uptake in Plants from Mineral-Free Nutrient Solutions
The Cu treatment led to a 26-times-greater Cu concentration in the tobacco (cv. Burley 92) root samples than the combined Cu + NTA treatment, while in the shoots highest Cu concentrations were found in the Cu + NTA treatment. Here the Cu concentrations were 3.5 times greater than in the Cu treatment, amounting to 143 mg kg-1 plant material (Fig. 1) . Both tobacco cultivars showed similar effects on Cu uptake, although the cultivar Badischer Geudertheimer accumulated more Cu in both shoots and roots than the cultivar Burley 92 in the Cu treatment (data not shown). The treatments had no significant effect on Fe uptake in either plant.



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  		Fig. 1. Copper and iron concentrations (mg kg-1) in shoots and roots and biomass production (g plant-1) of tobacco (cv. Burley 92) in different treatments for the nutrient solution experiment without montmorillonite. Columns with no common index are significantly different at P <= 0.05. NTA, nitrilotriacetate.

 
Despite the strong effects on copper uptake, the treatments had no significant effect on the growth of the plants (Fig. 1), despite the fact that in the Cu treatment, tobacco (cv. Badischer Geudertheimer) plants showed a brownish discoloration of the roots as a result of the Cu exposure.

Plant Growth and Metal Uptake in Plants from Nutrient Solution with Montmorillonite
In Cu-enriched suspensions, NTA increased Cu uptake into shoots by a factor of 26, while it decreased Cu concentrations in the root samples by a factor of 3.6 (Fig. 2) . Approximately seven times less Cu was dissolved in the montmorillonite suspensions than in the nutrient solutions (Tables 4 and 5). Accordingly, less Cu was bound by plant roots from the montmorillonite suspensions than from the nutrient solutions (Fig. 1 and 2; Table 5).



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  		Fig. 2. Copper and iron concentrations (mg kg-1) in shoots and roots and biomass production (g plant-1) of tobacco (cv. Badischer Geudertheimer) in different treatments for the nutrient solution experiment with montmorillonite. Columns with no common index are significantly different at P <= 0.05. NTA, nitrilotriacetate.

 
In the presence of montmorillonite, NTA significantly increased Fe uptake into the roots and in the combined Cu + NTA treatment into the shoots (Fig. 2). No significant treatment effects on plant growth were observed (Fig. 2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nutrient Solution without Minerals
According to our speciation calculations, precipitation of Ca10(OH)2(PO4)6 (hydroxyapatite), Fe(OH)3, FePO4(H2O)2 (strengite), and Cu(PO4)2(H2O)2 should have occurred at pH > 5. In this study, pH was adjusted to a value of 6, which is favorable for the formation of metal hydroxides. In the presence of NTA, more Fe and Cu were kept in solution than without, due to NTA–Fe or NTA–Cu complex formation. However, according to the speciation calculations, the solubility product of all of the before-mentioned phosphates was still exceeded. Due to the addition of NTA in excess of the total Cu and Fe added, a near 100% complexation of these two metals resulted. The formation of Cu–NTA complexes is known as a comparatively fast reaction (Morel, 1983) and precipitation of metal phosphates may not have occurred due to the kinetics of the system.

Nutrient Solution with Montmorillonite
In the absence of NTA, Cu was strongly adsorbed by montmorillonite while the addition of NTA kept the metal in solution due to the formation of stable negatively charged Cu–NTA complexes. This agrees with findings of Neubauer et al. (2000), who showed that NTA prevented sorption of Cu, Zn, and Cd to montmorillonite and kaolinite over a pH range from 4 to 10. Differences in the modeled and measured sorption of Cu to montmorillonite (17%) may be due to differences in experimental conditions between this study and that of Neubauer et al. (2000). Calculations were made with the log K values for Cu sorption determined by Neubauer et al. (2000). A Cu to montmorillonite ratio of 12.6 µM Cu per g montmorillonite was obtained in our experiments, whereas Neubauer et al. (2000) derived a value of 84.5 µM Cu per g montmorillonite. A higher Cu sorption onto montmorillonite was to be expected in our experiment due to the lower Cu to montmorillonite ratio used.

In the presence of NTA, Ca and Fe concentrations in solution were greater than the amount originally added. A batch experiment with 500 µM NTA and 10 g L-1 montmorillonite led to considerable amounts of Ca, Fe, K, and Mg in solution (data not shown). This finding indicates that the additional amounts of Ca and Fe in the treatments probably came from the montmorillonite.

Metal Uptake by Plants
The NTA effect on Cu uptake can be explained by its influence on Cu speciation in the solution. In the Cu treatments without and with montmorillonite the Cu2+ was the dominant species, according to the speciation calculations, while in the Cu + NTA treament only the Cu–NTA complex was present.

Due to its high content of carboxylic groups, the apoplasm (the cell wall continuum of roots and other plant tissue) acts as an effective cation exchanger. Cations therefore tend to be attracted to the intercellular space of the root cortex, whereas anions are "repelled." Copper might even be specifically bound in a nonionic form to nitrogen-containing groups of either glycoproteins or proteins of ectoenzymes in the cell wall (Van Cutsem and Gillet, 1982). This binding of cations in the cell wall is considered to be one mechanism that might play a role in copper tolerance (Turner, 1970). Due to the positive charge of the Cu2+ species in the Cu treatments, they were bound in the cell wall of the roots. In an ultrastructural study using transmission electron microscopy, Jarvis and Leung (2001) observed retention of unchelated Pb mainly in cell walls of roots, particularly around intercellular spaces, and also in bacteroids and mitochondria. They speculated that the membranes of both bacteroids and mitochondria have an affinity for lead due to the presence of cation exchange sites.

Although Cu concentrations of about 9000 mg kg-1 in the roots of tobacco (cv. Badischer Geudertheimer) were observed in the experiments without montmorillonite, the capacity of this mechanism is limited (Woolhouse, 1983). Although we did not observe a significant decrease in root growth in our short-term experiments, others reported severe toxic effects of Cu on plant growth in nutrient solution experiments (Ebbs and Kochian, 1997; Jung, 2000). Ebbs and Kochian (1997) observed in a 14-d experiment a decrease of 70 to 80% in root biomass in Indian mustard at concentrations of 5 µM Cu in nutrient solutions, while Jung (2000) found a decrease in root biomass in white lupine (Lupinus albus L.) of 80% in treatments with 62 µM Cu after 40 d of experimental time.

In the Cu + NTA treatment, the formation of negatively charged Cu–NTA complexes prevented the Cu from binding to the cation exchange sites in the cell walls of the roots. The Cu–NTA complex might be taken up and translocated within the plant as an anion and thus overcome the control mechanism of the plants. According to Marschner (1995), the critical toxicity level of Cu in the shoots of crop plants is greater than 20 to 30 mg kg-1. No growth reduction or any other visible sign of Cu toxicity was found in the presence of NTA at shoot Cu concentrations of 190 mg kg-1. The most plausible hypothesis is that the accumulated Cu was taken up in the form of Cu–NTA complexes, which remained stable and thus biochemically inactive in the plant. Vassil et al. (1998) directly measured Pb–EDTA complexes in the xylem sap of Indian mustard and reported no health effects at an equimolar (0.5 mM) Pb and EDTA treatment, while phytotoxicity was observed with EDTA added in excess to the metal. Vassil et al. (1998) assumed that free protonated EDTA in leaves displaced essential divalent cations, disrupting the biochemistry of the leaf cells. He also speculated that the high uptake of Pb–EDTA was due to destruction of the physiological barrier(s) in the roots. While similar effects were described by Collins et al. (2002) for Zn–EDTA uptake by Indian mustard, a different pattern was observed for barley (Hordeum vulgare L.) and potato (Solanum tuberosum L.), suggesting that Zn–EDTA might be passively taken up by these two species via an apoplastic pathway. Collins et al. (2002) also observed little effect of high concentrations of EDTA on the transpiration of barley and potato and only low xylem Zn–EDTA concentrations compared with those detected in Indian mustard, supporting the hypothesis of an apoplastic uptake of barley and potato.

In our study, we did not observe phytotoxic effects of NTA, although we added NTA in excess to Cu and 60% of the added NTA was present as HNTA2- in the nutrient solution according to the speciation calculations. At the root apex, the Casparian band is not yet fully developed and thus is a zone allowing apoplasmic transport into the stele (Huang and van Steveninck, 1989). Furthermore, Crowdy and Tanton (1970) illustrated that the uptake of Pb–EDTA was restricted to the region between 3 and 140 mm from the root tip, where the suberization of the cell walls had not yet occurred. This pathway would allow metal–chelates to circumnavigate the impermeable Casparian band and proceed an apoplastic uptake of an intact complex. Based on the literature review, it was hypothesized that the uptake of Cu–NTA complex by tobacco was via an apoplastic pathway (a passive extracellular transport into the xylem).

The addition of montmorillonite did not change the effect of NTA on the Cu uptake by the plants. In the absence of NTA, the montmorillonite effectively outcompeted the plant roots for the added Cu and Fe. Only small amounts of Cu and Fe were available for plant root uptake and thus Cu and Fe concentrations in the roots were considerably smaller than in the experiments without montmorillonite.

The addition of NTA effectively prevented Cu sorption to montmorillonite by forming strong water-soluble negatively charged complexes. Additionally, NTA facilitated uptake and translocation of Cu within the tobacco plants and mitigated toxic effects of Cu on the plants as previously described. All these effects are advantageous in using NTA as a soil amendment to assist phytoextraction of this metal in the remediation of contaminated sites.


   ACKNOWLEDGMENTS
 
This study was financially supported by the Swiss National Science Foundation, Swiss Priority Program Environment. We greatly thank M. Guadagnini, A. Kayser, and W. Stauffer for providing tobacco seedlings for the nutrient solution experiment. We are grateful to H.J. Bachmann, R. Hort, C. Dähler, B. Schüpbach, and O. Wyss for their consistent help and to Susan Tandy for proofreading the English.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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This Issue in Journal of Environmental Quality

JEQ 2003 32: 1577-1582. [Full Text]  




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