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Journal of Environmental Quality 32:500-506 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Heavy Metals in the Environment

Ethylenediaminedissuccinate as a New Chelate for Environmentally Safe Enhanced Lead Phytoextraction

H. Grcman, D. Vodnik, S. Velikonja-Bolta and D. Lestan*

Agronomy Dep., Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

* Corresponding author (domen.lestan{at}bf.uni-lj.si)

Received for publication May 9, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Using a soil column experiment, we compared the effect of a single dose and weekly additions of ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedissuccinate (EDDS) on the uptake of Pb, Zn, and Cd by Chinese cabbage [Brassica rapa L. subsp. pekinensis (Lour.) Hanelt], and on the leaching of heavy metals through the soil profile. The analysis of plant material revealed that both chelates increased the concentrations of Pb and, to a lesser extent, also of Zn and Cd in the leaves of the test plant. The most effective applications were single doses of 10 mmol EDTA and EDDS kg-1 soil, which caused the concentrations of Pb in the shoots to increase 94.2- and 102.3-fold, respectively, relative to the control. The same dose of EDTA increased the concentration of Zn and Cd in the leaves 4.3- and 3.8-fold and of EDDS 4.7- and 3.5-fold, respectively. In treatments with weekly additions and lower concentrations of both chelates, EDTA was more effective than EDDS in increasing the plant uptake of Pb. In soil columns treated with weekly additions of 10 mmol kg-1 EDTA, on average 22.7, 7.0, and 39.8% of initial total Pb, Zn, and Cd in the soil were leached through the soil profile. The same amount of EDDS caused much lower leaching of Pb and Cd—only 0.8 and 1.5% of initial total concentrations. Leaching of Zn, 6.2% of the total concentration, was comparable with the EDTA treatment. A biotest with red clover (Trifolium pratense L.) indicated a greater phytotoxic effect of EDTA than EDDS addition. EDDS was also less toxic to soil fungi, as determined by phospholipid fatty acid (PLFA) analysis, and caused less stress to soil microorganisms, as indicated by the trans to cis PLFA ratio. Chelate addition did not prevent the development of arbuscular mycorrhiza on red clover.

Abbreviations: EDTA, ethylenediaminetetraacetic acid • EDDS, ethylenediaminedissuccinate • HM, heavy metal • PLFA, phospholipid fatty acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
HEAVY METAL (HM) contamination of soils has become a serious problem in areas of intensive industry and agriculture. Widespread low to medium pollution of agricultural land is a particular problem. The remediation of large areas of agricultural land by conventional technologies intended for small areas of heavily contaminated sites is not economically feasible. However, if remedial action is not undertaken, the availability of arable land for cultivation will decrease, because of stricter environmental laws limiting food production on contaminated lands. Toxic HMs, especially Pb contamination of agricultural soils, poses a major environmental and health problem. These soils are still in need of a cost effective and environmentally safe technological solution.

Recently, HM phytoextraction has emerged as a promising, cost-effective alternative to conventional engineering-based remediation methods (Salt et al., 1995). Secondary benefits include wide public acceptance due to its aesthetic value, while the cultivation of plants preserves and enhances all biological components of the polluted medium. The goal of successful phytoextraction is to reduce concentrations of HMs in contaminated soil to acceptable levels within a reasonable time frame. To achieve this, plants must accumulate high levels of HMs and produce high amounts of biomass. Early phytoextraction research focused on hyperaccumulating plants, which have the capacity to concentrate high amounts of HMs in their plant tissues (Baker et al., 1994). However, hyperaccumulators often accumulate only a specific element, and are generally slow-growing, low-biomass-producing plants, with few known agronomic characteristics. Moreover, there is no real hyperaccumulating plant for Pb, one of the most widespread and toxic metal pollutants in soil.

Many hydroponic studies have revealed that the uptake and translocation of HMs in plants are enhanced by increasing the HM concentration in the nutrient solution (Huang et al., 1997). The bioavailability of HMs in the soil is, therefore, of paramount importance for successful phytoremediation. Lead has limited solubility in soil solution and, hence, limited bioavailability due to formation of complexes with organic and inorganic soil colloids, sorption on oxides and clays, and precipitation as carbonates, hydroxides, and phosphates (Ruby et al., 1999). Successful phytoremediation of Pb must therefore include mobilization of HMs into soil solution in direct contact with plant roots. In most soils capable of supporting plant growth, the phytoavailable levels of HMs, particularly Pb, are low and do not allow substantial plant uptake if chelates are not applied.

Chelates have been shown to desorb heavy metals from the soil matrix into the soil solution, facilitate Pb transport into xylem, and increase Pb translocation from roots to shoots of several fast-growing, high-biomass-producing plants (Huang et al., 1997). The literature to date reports a number of chelates that have been used for chelate-induced hyperaccumulation. These include EDTA, CDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaaceticacid), EGTA [ethyleneglycol-bis(ß-aminoethyl ether), N,N,N',N-tetraacetic acid], EDDHA [etylenediamine-di (o-hydroxyphenylacetic acid)], HEDTA (N-hydroxyethylenediaminetriacetic acid), HEIDA [N-(2-hydroxyethyl)iminodiacetic acid], and NTA (nitrilotriacetic acid) (Cooper et al., 1999; Wu et al., 1999; Blaylock et al., 1997). These chelates, however, are not specific to HMs and are subject to numerous interferences with other cations present in soil at much higher concentrations. Many synthetic chelates and their complexes with HMs are toxic (Dirilgen, 1998; Sillanpaa and Oikari, 1996) and poorly photo-, chemo-, and biodegradable in soil environments (Nörtemann, 1999). A combination of widespread use in fertilizers and slow decomposition has led to background concentrations of EDTA in European surface waters in the range of 10 to 50 mg L-1 (Kari et al., 1995). Authors investigating chelate-enhanced phytoextraction have also pointed out the risk of possible transference of Pb and other HMs from soil to ground water and promotion of off-site migration (Cooper et al., 1999; Huang and Cunningham, 1996; Kulli et al., 1999). All the potential risks when using chelates for phytoextraction should therefore be thoroughly evaluated before steps toward further development and commercialization of this remediation technology are taken.

In the present study, we compare the efficiency of two chelating agents: EDTA as one of the more efficient and widely tested chelates (Wu et al., 1999; Huang et al., 1997; Blaylock et al., 1997), and a biodegradable chelate, ethylenediaminedissucinate (EDDS). EDDS was reported by Jones and Williams (2001) as biodegradable, and strongly complexing with transition metals and radionuclides. Jaworska et al. (1999) assessed environmental risks for its use in detergent applications. Mineralization of EDDS in sludge-amended soil was rapid and complete in 28 d. The reported calculated half-life was 2.5 d. No recalcitrant metabolites were found in the degradation profile of EDDS. The toxicity to fish and daphnia was low (EC50 > 1000 mg L-1). EDDS has substituted traditional chelates such as EDTA in a number of commercial products, for example, industrial detergents. The current price for 1 Mg of EDDS is approximately GB£5000 (US$7800).

Using soil column experiments, we studied the effects of different amounts and modes of EDTA and EDDS application into the soil on Pb, Zn, and Cd uptake by the test plant Chinese cabbage. We also studied the leaching of HMs through the soil profile and the phytotoxicity and toxicity of chelate addition to arbuscular mycorrhiza formation and other soil microorganisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil Preparation and Experimental Setup
Soil samples were collected from a 0- to 30-cm surface layer at an industrial site of a former Pb and Zn smelting plant in Slovenia. The pH (CaCl2) of the samples was 6.8. Organic matter (determined with the Walkley–Black method) was 5.2%; total N (Kjeldahl digestion) was 0.25%. The particle-size distribution (sedimentation method) was 55.4% sand, 12.0% coarse silt, 18.9% fine silt, and 13.7% clay. Available P2O5 and K2O (extraction with 0.04 mol L-1 acetic acid and 0.1 mol L-1 ammonium lactate) were 373 mg kg-1 and 92 mg kg-1 respectively. Amounts of heavy metals in soil (AAS [atomic absorption spectroscopy] after digestion with aqua regia) included 1100 mg Pb kg-1, 800 mg Zn kg-1, and 5.5 mg Cd kg-1. The soil texture was sandy loam. The soil field water capacity was 24.6 g 100 g-1, determined in accordance with ISO Standard 11274 (International Organization for Standardization, 1998). After being air-dried, the soil was passed through a 4-mm sieve.

The influence of EDTA trisodium salt (Fluka, Steinheim, Germany) and EDDS trisodium salt (Octel, Cheshire, UK) on Pb, Zn, and Cd plant uptake and leaching was tested in a soil column experiment with five replicates for each treatment. Air-dried soil (3755 g) was placed in 18-cm-high, 15-cm-diameter columns that were equipped with trapping devices (polypropylene jars and tubes) for leachate collection. Plastic mesh (diameter = 0.2 mm) was placed in the bottom of the columns to retain the soil. Soils were fertilized in all treatments with 150 mg kg-1 N and K as (NH4)2SO4 and K2SO4, respectively. Three-week-old seedlings of Chinese cabbage (‘Nagaoka F1’) were transplanted into the columns and grown for one week to adapt, before chelate application on Day 1 of the experiment. The experiment was conducted in a greenhouse at ambient temperature (15–30°C) illuminated with natural light. The EDTA and EDDS were applied in 100 mL of deionized water in four partial-weekly additions of 5 and 10 mmol kg-1 (total) on Days 1, 8, 15, and 22 of the experiment, or in a single dose of 3, 5, and 10 mmol kg-1 on Day 22 of the experiment. The plants were watered three times a week with 400 mL of tap water. Aboveground tissues were harvested by cutting the stem 1 cm above the soil surface. Biomass was determined after the tissues were dried to a constant weight at 60°C. The leachates were sampled on Days 7, 14, 21, and 28 of the experiment, filtered through Whatman (Maidstone, UK) no. 1 filter paper and stored in cold storage before further analysis. Total amounts of water leached were 760, 800, 1290, 1300, and 1270 mL for the control, 5, and 10 mmol kg-1 weekly additions of EDDS and 5 and 10 mmol kg-1 weekly additions of EDTA, respectively.

Heavy Metals Determination
For the analysis of metals content, the soil samples were ground in an agate mill for 10 min and then passed through a 150-µm sieve. After the digestion of soils in aqua regia, AAS was used for the determination of HM concentrations.

Shoot tissues were collected and thoroughly washed with deionized water. Plant samples were dried at 60°C to constant weight and ground in a titanium centrifugal mill. Metal concentrations in plants were determined after an acid dissolution technique with microwave heating. Plant tissue samples (250–300 mg dry weight) were treated with 65% HNO3 and analyzed by flame AAS (Pb and Zn) and by electrothermic AAS (Cd). Heavy metal concentrations in leachates were determined by inductively coupled plasma–atomic emission spectroscopy (ICP–AES). Controls of the analytical procedure were performed with blanks and reference materials (BCR 60 and BCR 141R, Community Bureau of Reference, Brussels, Belgium; for plant and soil) treated in the same way as the experimental samples. Two determinations of the concentration of HMs were made per sample.

Estimation of Arbuscular Mycorrhizal Inoculum Potential
The total mycorrhizal inoculum potential of soils after chelate application was determined by growing bait plants (red clover) in intact cores of pre-treated soils to measure the rate of mycorrhiza formation. One 150-cm3 intact soil core was sampled from pots with weekly addition of EDTA and EDDS, after the aboveground portions of the cabbage plants were harvested. Each core was sown with 30 seeds of red clover. The pots were placed in a greenhouse. The plants were harvested three months later, the dry weight of the shoots was determined, and the roots were stained with trypan blue (Phillips and Hayman, 1970) and examined for mycorrhizal infection (Trouvelot et al., 1986).

Phospholipid Extraction and Determination
The toxicity of EDTA and EDDS additions on the microbial population in contaminated soil was tested in a 10-week growth chamber experiment with three replicates for each treatment and for the control. Two-hundred grams of air-dried contaminated soil (prepared as described above) was placed in a 1-L glass jar and kept at 24°C and 85% relative humidity. The soil moisture was maintained at 70% field water capacity with deionized water. After three weeks of stabilization, 10 mmol kg-1 EDTA and EDDS were applied in 20 mL of deionized water. Five grams of soil from the upper 1-cm layer of each pot was sampled on Days 1 and 56 after chelate addition and the structure of microbial populations was assessed with the phospholipid fatty acids technique (PLFA). The modified procedures of Frostegård et al. (1991) and Guckert et al. (1986) were used for lipid extraction, fractionation, and mild alkaline methanolysis. Fatty acids methyl esters were quantified with gas chromatography–mass spectrometry (GC–MS) with 19:0 methyl ester as an internal standard. Fatty acids were designated with the nomenclature described by Frostegård and Bååth (1996). The sum of the following fatty acids was used to represent bacteria: i15:0, a15:0, 15:0, i16:0. 16:1{omega}9c, i17:0, a17:0, 17:0, cy17:0, and cy 19:0 (Frostegård and Bååth, 1996). Fungi were represented by 18:2{omega}6 (Vestal and White, 1989) and actinomycetes by 10Me 18:0 (Klamer and Bååth, 1998). The ratio between trans 18:1{omega}9 and cis 18:1{omega}9 was used to monitor stress levels to microorganisms (Guckert et al., 1986). The structure of microbial groups in soil was presented in relative shares of microbial groups, determined as mol % of PLFAs indicative for particular microbial groups against total PLFAs. It must be kept in mind that the development of different groups of microorganisms inferred from the changes in the PLFA pattern does not give the absolute amounts of biomass of different groups, since conversion factors from the microorganism groups to actual biomass are lacking.

Statistical Analyses
The data were statistically evaluated with analysis of variance. Lead and Zn concentrations in plant tissue and cabbage biomass were square root–transformed before analysis to stabilize the variance. The Tukey multiple range test was used to determine the significance (P <= 0.05) between all possible pairs.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Heavy Metal Uptake by Plants
Because the test plant uptake of HMs provides an estimate of the amount of HM that can be removed from a contaminated soil, it is an important criterion for chelate selection in phytoextraction remediation. Since Chinese cabbage has shown substantial Pb and Zn phytoextraction potential (Xian, 1989), we used it as a test plant in our experiment. The analysis of plant material indicated that both chelates applied to the soil increased the concentrations of Pb in the leaves of the test plant (Fig. 1) . For Pb phytoextraction, single and weekly additions of all concentrations of EDTA were more effective than EDDS, except at the highest single dose of chelate applied (10 mmol kg-1) where the effects of EDTA and EDDS were similar; Pb concentrations in the shoots increased 94.2- and 102.3-fold relative to the control. The same amount of EDTA and EDDS applied in four weekly additions was significantly less effective and resulted in 59.7- and 10.3-fold increases of Pb in leaves. All other single or weekly additions of lower chelate concentrations were less effective. Weekly additions of EDDS on Pb plant uptake were less effective, probably because of the rapid EDDS and Pb–EDDS complex biodegradability reported by Jaworska et al. (1999). Consequently, the availability of mobilized Pb species in the soil was shorter term than in EDTA treatments. This hypothesis, however, should be tested in additional experiments and with chemical analyses of chelate degradability in soil. Witschel and Egli (1998) reported that cometabolism of EDDS was initiated by a carbon–nitrogen lyase catalyzing the nonhydrolytic cleavage of the C–N bond between the ethlylenediamine part of the molecule and one of the succinyl residues.



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  		Fig. 1. Concentrations of Pb, Cd, and Zn in leaves of Chinese cabbage grown on contaminated soil in response to different EDTA (black) and EDDS (white) addition: 3 mmol kg-1 single dose addition (3S), 5 mmol kg-1 single dose addition (5S), 5 mmol kg-1 weekly additions (5W), 10 mmol kg-1 single dose addition (10S), 10 mmol kg-1 weekly additions (10W), and control soil with no EDTA addition. Means of five replicates are presented; error bars represent standard deviation. Statistically different treatments according to the Tukey test are labeled with different letters.

 
Our results are comparable with experiments by Epstein at al. (1999) in which the addition of 5 and 10 mmol EDTA kg-1 to Pb-contaminated soil increased the shoot Pb concentrations in Indian mustard [Brassica juncea (L.) Czern.] 26-fold and more than 100-fold, respectively. However, other authors have reported much higher plant Pb concentrations induced by chelate application. Blaylock et al. (1997) tested five chelating agents. They used 3-week-old seedlings and measured more than 15 000 mg Pb kg-1 (300-fold increase) in the dry weight of shoots of Indian mustard after a 10 mmol kg-1 EDTA addition. Huang et al. (1997) determined 8960 mg kg-1 (111-fold increase) and 2410 mg Pb kg-1 (57-fold increase) in two-week-old pea (Pisum sativum L.) and corn (Zea mays L.) shoots transplanted into a soil substrate pre-treated with 1.5 mmol EDTA kg-1. The possible reasons for higher Pb concentrations reported by Blaylock et al. (1997) and Huang et al. (1997) compared with Epstein et al. (1999) and our results include the use of very young plants, favorable speciation of HMs in the soil, and the experimental setup, in which no losses of the chelate–Pb complex due to leaching of the substrate occurred. In a field lysimeter study, Huang et al. (1997) measured 785 mg kg-1 (28-fold increase) of Pb in Indian mustard grown in soil after treatment with 5 mmol EDTA kg-1.

In our study, both EDTA and EDDS were similarly effective in enhancement of Cd and Zn plant uptake; the addition of 10 mmol kg-1 of EDTA in a single dose increased the concentrations of Zn and Cd in leaves 4.3- and 3.8-fold compared with the control treatment (Fig. 1). EDDS addition caused 4.7- and 3.5-times-higher concentrations of Zn and Cd in plant tissues, respectively. Other treatments were less effective and increased the concentrations of Cd and Zn in the shoots by less than three and four times compared with the control. In other studies in which other chelates were tested, the results were comparable with ours. Kayser et al. (2000) examined NTA, a biodegradable synthetic chelate (Ward, 1986). The addition of 8.4 mmol kg-1 to the soil increased the plant tissue concentrations of Zn and Cd by a factor of two to three. Kulli et al. (1999) also tested the effect of NTA and urea on Cd and Zn accumulation in Italian ryegrass (Lolium multiflorum Lam.) and lettuce (Lactuca sativa L.). Only the highest NTA concentration tested (20 mmol kg-1 soil) increased shoot Cd and Zn concentrations by a factor greater than three relative to the control.

The ability of EDTA to enhance Pb plant uptake more than Zn and Cd (Fig. 1) and other HMs was also reported by Blaylock et al. (1997). The logarithms of stability constants for Pb–, Zn–, and Cd–EDTA complexes are 17.88, 16.44, and 16.36, respectively (Bucheli-Witschel and Egli, 2001). EDDS enhances Pb plant uptake more than Zn and Cd plant uptake (Fig. 1). The logarithms of stability constants for Pb–, Zn–, and Cd–EDDS complexes are 12.7, 13.49, and 10.8 (Bucheli-Witschel and Egli, 2001). The presumed reason for the higher effectiveness of both chelates for Pb plant uptake compared with Zn and Cd is the inherently higher phytoavailability of Zn and Cd in soil, as indicated by the significant uptake of Zn and Cd into the test plant Chinese cabbage in control treatments (Fig. 1).

Heavy Metal Leaching
The dynamics of Pb, Zn, and Cd leaching from soils treated with weekly additions of EDTA and EDDS are presented in Fig. 2 . In control treatments, the concentrations of Pb and Cd in leachates were below the detection limits of 0.04 and 0.005 mg L-1, respectively. The amount of Zn leached was always below 0.02 mg kg-1 of soil. As expected, EDTA application had a pronounced influence on HM leaching. In columns treated with 10 mmol kg-1 EDTA, 22.7, 7.0, and 39.8% of initial total Pb, Zn, and Cd in the soil was leached down the soil profile during the four weeks of the experiment. EDDS caused significantly less leaching of Pb and Cd than EDTA. The same amount of EDDS leached 0.8, 6.2, and 1.5% of initial total Pb, Zn, and Cd. Leaching of Zn was comparable in both chelate treatments. As reported by Vandevivere et al. (2001), Pb– and Cd–EDDS complexes are readily biodegradable, while Zn–EDDS was biodegraded only after an extended lag phase. The biodegradability of EDDS–HM complexes is a plausible explanation for 28.1- and 23.7-times lesser amounts of Pb and Cd leached from the EDDS treatment compared with the EDTA treatment. The mass balance of HMs leached and extracted into the harvestable parts of plants is shown in Table 1.



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  		Fig. 2. The effects of 5 and 10 mmol kg-1 weekly additions of EDDS and EDTA on Pb, Zn, and Cd leaching from soil during the phytoextraction experiment. The means of five replicates are presented; error bars represent standard deviation.

 

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  		Table 1. The mass balance of heavy metals in percentages of initial total heavy metals in soil. Results show heavy metals leached and extracted into the harvestable parts of plants in treatments with weekly addition of EDTA or EDDS and control treatments. Results are presented as means of five replicates ± standard deviation.

 
As mentioned in the introduction of this paper, several authors have emphasized the risks associated with leaching of HMs from the root zone due to chelate application, and the importance of assessing this risk. However, there are no reports in the literature on direct measurements of HM leaching during a phytoextraction experiment. Cooper et al. (1999) estimated the risk of leaching using a Toxicity Characteristic Leaching Procedure (TCLP) at the end of a phytoextraction experiment with three different chelating agents (CDTA, DTPA, and HEDTA). All chelates increased the leaching of Pb compared with the control. As shown by TCLP, the leaching of Pb in soils treated with high rates (20 mmol kg-1) of CDTA and DTPA tested greatly exceeded the USEPA regulatory limits (5 mg L-1).

Phytotoxicity
In all treatments in which chelates were applied, visual symptoms of toxicity were observed as necrotic lesions on cabbage leaves. The symptoms were more prominent on older leaves. Single and weekly additions of 10 mmol kg-1 EDTA resulted in rapid senescence of the plant shoots and lowered the yield of cabbage biomass (Table 2) compared with the control. Plant growth was most strongly inhibited by the highest single dose of EDTA. EDDS was less phytotoxic than EDTA, and showed no statistically significant reduction (P <= 0.05) in cabbage growth compared with the control treatment. This could be explained either by an inherently lower phytotoxicity of EDDS or as a result of EDDS rapid biodegradation in soil, as reported by Jaworska et al. (1999).


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  		Table 2. Chinese cabbage biomass yield in treatments with different EDTA and EDDS additions. Results are presented as means of five replicates ± standard deviation.

 
To evaluate the post-treatment toxicity of the remediated soil, a bioassay with red clover was performed. The growth of red clover in the bioassay experiment was strongly dependent on the chelate pre-treatment of the soil substrate. The total biomass of the shoots per pot revealed a strong negative effect of 5 and 10 mmol kg-1 weekly EDTA additions (Fig. 3) . No negative effect of EDDS was found in the bioassay experiment, regardless of the concentration (Fig. 3). Mycorrhizal infection was present in all clover plants examined, including with the EDTA treatment, regardless of their poor growth. Host plants seemed to be more sensitive to chelate and HM treatment than arbuscular mycorrhizal fungi.



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  		Fig. 3. Red clover shoot dry weight in response to 5 mmol kg-1 (5W) and 10 mmol kg-1 (10W) weekly additions of EDDS or EDTA and control soil with no chelate addition. Means of four replicates are presented; error bars represent standard error.

 
Influence of Chelate Addition on Soil Microorganisms
Phytoextraction is a long-term technology. It is therefore imperative to sustain the quality of the soil and to enable vigorous growth of the phytoextracting plants. Soil microorganisms are critically important for normal functioning of soils and are common indicators of soil quality. The toxicity of EDDS and EDTA addition on soil bacteria, actinomycetes, and fungi was studied with PLFA. The phospholipid fatty acid technique is a relatively new tool in environmental microbiology and enables an insight into the structure of microbial populations in natural media (Vestal and White, 1989; White et al., 1998). We detected 32 different PLFAs in our treatments, and identified 23 of them. In total, these marker PLFAs represented 43 to 58% of total PLFAs.

The shifts in the structure of microbial communities as the result of chelate application were determined with marker PLFAs expressed as mol %. As shown in Fig. 4 , the mol % of PLFAs representing fungi was significantly lower than in the control treatment on Days 1 and 56 after EDTA addition. In contrast, no statistically significant (P <= 0.05) effect of EDDS addition on fungal PLFA markers was observed. Neither PLFA markers for bacteria nor those for actinomycetes changed significantly because of EDTA or EDDS additions (Fig. 4). The share (mol %) of fungal biomass, which is dominant in most soils (Thorn, 1997), seems to be underrated in Fig. 4, since conversion factors from marker PLFAs of microbial groups to actual biomass are lacking.



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  		Fig. 4. The structure of microbial groups (fungal, bacterial, and actinomycetes) determined as mol % of PLFAs in soil after 1 and 56 d of chelate addition in a single dose of 10 mmol kg-1 soil. Means of three replicates are presented; error bars represent standard deviation. Statistically different treatments according to the Tukey test are labeled with different letters.

 
EDTA addition significantly increased the trans to cis ratio of PLFAs on Days 1 and 56 after chelate application (Fig. 5) . The trans to cis ratio is associated with starved or stressed microorganisms in natural environments (Guckert et al., 1986). EDDS addition had a statistically significant effect (P <= 0.05) only 56 d after the application, compared with the control treatment (Fig. 5).



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  		Fig. 5. Stress index (trans to cis PLFA ratio) of microbial populations in soil after 1 and 56 d of chelate addition in a single dose of 10 mmol kg-1 soil. Means of three replicates are presented; error bars represent standard deviation. Statistically different treatments according to the Tukey test are labeled with different letters.

 
The results are in accordance with phytotoxicity tests. EDDS addition was less toxic to soil fungi than EDTA and caused less stress to soil microorganisms. The PLFA results indicate that soil fungi are more sensitive to chelate addition than other soil microbial groups. This is in accordance with the results of Dahlin et al. (1997). They reported that the effect of HMs on the PLFA pattern was small, except for 18:2{omega}6 PLFA, which decreased in sludge-amended Cd-, Cr-, Cu-, Pb-, and Zn-contaminated soil. This specific PLFA is indicative for fungi (Frostegård et al., 1993). EDDS is a naturally occurring substance and this is the likely reason for its low toxicity. It was first isolated from a culture filtrate of the actinomycete Amycolatopsis orientalis. It was detected in an antibiotic screening program due to its ability to inhibit Zn2+–dependent phospholipase (Nishikiori et al., 1984).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The results of this study support EDDS as a promising new chelate for enhanced, environmentally safe phytoextraction of soils contaminated principally with Pb. Measurements of Pb in leaves of Chinese cabbage revealed that high single doses of EDDS and EDTA, as one of the most tested chelates, were the most, and equally, effective. In contrast to EDTA, however, EDDS addition caused only minor leaching of Pb, and was significantly less toxic to plants and soil microorganisms.

Even the highest concentrations of HMs in harvestable plant tissues achieved in this study were still far from the concentrations required for efficient phytoextraction procedures. A plant Pb concentration of more than 1% in the dry biomass is required for a phytoextraction technology capable of reducing the Pb concentration in soil by 500 mg kg-1 and below the limits set by the European Council Directive on Protection of the Environment (86/278/EEC) in a reasonable time span of 20 to 25 yr (European Community, 1986). In addition to new biodegradable chelates, new techniques of chelate application need to be developed to safely increase the bioavailability of HMs in soils (i.e., controlled release of chelate adjusted to the physiological characteristics of plants), together with transgenic plants with high biomass yield and improved Pb accumulation potential in the harvestable plant parts.


   ACKNOWLEDGMENTS
 
This work was supported by the Slovenian Ministry for Science and Technology, Grant no. J4-0694-0486-98. We thank Mr. Bostjan Kos and Mr. Klavdij Bajc for technical assistance. We also thank Dr. Joanna Jaworska from Procter & Gamble for the gift of EDDS.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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This Issue in Journal of Environmental Quality

JEQ 2003 32: 377-382. [Full Text]  




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The SCI Journals Agronomy Journal Crop Science
Journal of Natural Resources
and Life Sciences Education		 Vadose Zone Journal
Soil Science Society of America Journal Journal of Plant Registrations The Plant Genome