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EDTA enhanced heavy metal phytoextraction metal accumulation leaching and toxicity
Plant and Soil 235: 105–114, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 105 EDTA enhanced heavy metal phytoextraction: metal accumulation, leaching and toxicity H. Gr ˇ cman, Š. Velikonja-Bolta, D. Vodnik, B. Kos & D. Leštan 1 Agronomy Department, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. 1 Corresponding author ∗ Received 7 November 2000. Accepted in revised form 25 May 2001 Key words: cadmium, contaminated soil, EDTA, lead, phytoextraction, zinc Abstract Synthetic chelates such as ethylene diamine tetraacetic acid (EDTA) have been shown to enhance phytoextraction of some heavy metals from contaminated soil. In a soil column study, we examined the effect of EDTA on the uptake of Pb, Zn and Cd by Chinese cabbage (Brassica rapa), mobilization and leaching of heavy metals and the toxicity effects of EDTA additions on plants. The most effective was a single dose of 10 mmol EDTA kg −1 soil where we detected Pb, Zn and Cd concentrations that were 104.6, 3.2 and 2.3-times higher in the aboveground plant biomass compared to the control treatments. The same EDTA addition decreased the concentration of Pb, Zn and Cd in roots of tested plants by 41, 71 and 69%, respectively compared to concentrations in the roots of control plants. In columns treated with 10 mmol kg −1 EDTA, up to 37.9, 10.4 and 56.3% of initial total Pb, Zn and Cd in soil were leached down the soil profile, suggesting high solubility of heavy metals-EDTA complexes. EDTA treatment had a strong phytotoxic effect on the red clover (Trifolium pratense) in bioassay experiment. Moreover, the high dose EDTA additions inhibited the development of arbuscular mycorrhiza. The results of phospholipid fatty acid analyses indicated toxic effects of EDTA on soil fungi and increased environmental stress of soil microfauna. Abbreviations: HM – heavy metal; PLFA – phospholipid fatty acid; DGFA – diglyceride fatty acid Introduction Heavy metal (HM) contamination of soils has be- come a serious problem in areas of intense industry and agriculture. HMs are deposited in soils by at- mospheric input and the use of mineral fertilizers or compost, and sewage sludge disposal. Soils polluted with HMs pose a health hazard to humans as well as plants and animals, often requiring soil remediation practices. Conventional remediation methods usually involve excavation and removal of contaminated soil layer, physical stabilization (mixing of soil with ce- ment, lime, apatite etc.), or washing of contaminated soils with strong acids or HM chelators (Berti et al., 1998; Steele and Pichtel, 1998). ∗ FAX NO: +386-01-423-1088; E-mail: domen.lestan@bf.uni- lj.si Wide-spread low to medium level pollution of agri- cultural land represents a specific problem. In Europe, though the extent of areas that are affected has not been accurately determined, the polluted agricultural lands likely encompass several million of ha (Flath- man and Lanza, 1998). European Union Council dir- ective (1986) limits values for concentrations of HMs in arable soils to 3 mg kg −1 for Cd, 140 mg kg −1 for Cu, 75 mg kg −1 for Ni, 300 mg kg −1 for Pb, 300 mg kg −1 for Zn and 1.5 mg kg −1 for Hg. The remediation of large areas of agricultural land by conventional technologies used for small areas of heavily contam- inated sites is not feasible economically. However, if no remediation action is undertaken, the availability of arable land for cultivation will decrease, because of stricter environmental laws limiting food production on contaminated lands. 106 Recently, heavy metal phytoextraction has emerged as a promising, cost-effective alternative to the conventional engineering-based remediation methods (Salt et al., 1995). Early phytoextraction research fo- cused on hyperaccumulating plants which have the ability to concentrate high amounts of HMs in their plant tissues. However, hyperaccumulators often ac- cumulate only a specific element, and are as a rule slow growing, low biomass producing plants with little known agronomic characteristics (Cunningham et al., 1995). This constrains their practical use for phytore- mediation, since the total metal extraction is the product of plant biomass and HM tissue concentration. Recent research has shown that chemical amend- ments, such as synthetic organic chelates, can enhance phytoextraction by increasing HMs bioavailability in soil thus enhancing plant uptake, and translocation of HMs from the roots to the green parts of tested plants (Epstein et al., 1999; Huang et al., 1997). Of the che- lates tested, ethylene diamine tetraacetic acid (EDTA) was often found to be the most effective (Blaylock et al., 1997). Restrictions apply, however, to the use of EDTA and other chelating agents. EDTA and EDTA-HM complexes are toxic (Dirilgen, 1998; Sillanpaeae et al., 1996) and poorly photo-, chemo- and biodegrad- able in soil (Nörtemann, 1999). In situ application of chelating agents can cause groundwater pollution by uncontrolled metal dissolution and leaching. There- fore, the potential risks of use of EDTA or other chelators for phytoextraction should be thoroughly evaluated before steps towards further development and commercialization of this remediation technology are attempted. In the present study, soil column experiments were used to evaluate the effects of different amounts and modes of EDTA application on Pb, Zn and Cd uptake by test plant Brassica rapa. We monitored the leach- ing of HMs and EDTA through the soil profile. We also tested phytotoxicity and toxicity of EDTA addi- tion to arbuscular mycorrhiza formation and other soil microorganisms. Materials and methods Soil preparation and experimental set up Soil samples were collected from 0–30 cm surface layer at an industrial site of a former Pb and Zn smelter in Slovenia. The following soil properties were de- termined: pH (CaCl 2 ) 6.8, organic matter 5.2%, total N 0.25%, sand 55.4%, coarse silt 12.0%, fine silt 18.9%, clay 13.7%, P (as P 2 O 5 ) 37.3 mg 100 g −1 , K(asK 2 O) 9.2 mg 100 g −1 , Pb 1100 mg kg −1 ,Zn 800 mg kg −1 ,Cd5.5mgkg −1 .Soiltexturewas sandy loam. After being air-dried, the soil was passed through a 4-mM sieve. The influence of EDTA (Fluka, Steinheim) on Pb, Zn and Cd plant uptake, leaching, and toxicity was tested in soil column experiment with four replicates for each treatment. We placed 3755 g of air dried soil into 18 cm high 15 cm diameter columns which were equipped with trapping devices for leachete col- lection. Plastic mesh (D=0.2 mm) was placed to the bottom of the columns to retain the soil. We fertil- ized the soils in all treatments with 150 mg kg −1 N and K as (NH 4 ) 2 SO 4 and K 2 SO 4 , respectively. Three weeks old seedlings of Brassica rapa L. var. pekinen- sis (Nagaoka F1) were transplanted into columns and were grown for 4 weeks. In some treatments, EDTA was applied in 100 ml of deionised water in four partial-weekly additions (1, 8, 15, 22 day of culture). In others, EDTA was added in a single dose of total of 3, 5 and 10 mmol kg −1 EDTA on the 22th day of cultivation. We used three different watering regimes (Table 1). We harvested the abovegroundtissues on the 28th day of cultivation, by cutting the stem 1 cm above soil surface. We determined biomass after the tissues dried at 60 ◦ C reached a constant weight. We sampled leachates on 6th, 13th, 20th and 27th day of cultivation. They were filtered through What- manNo.1filterpaperandstoredincoldstoragefor further analysis. 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 diges- tion of soils in aqua regia, AAS was used for the determination of HM concentrations. Shoot tissues were collected and thoroughly washed with deionized water. Roots were carefully but vigorously washed with running water to remove soil particles. This procedure presumably removed dead plant roots. Plant samples were dried at 60 ◦ C to con- stant weight and ground in a titanium centrifugal mill. Metal concentrations in plant tissue samples (250– 300 mg dry weight) were determined using acid (65% HNO 3 ) dissolution technique with microwave heating and analysed by Flame-AAS or at low concentration of Cd and Pb by Electrothermal-AAS. HMs concen- 107 Table 1. Amounts of water added in three watering regimes during the course of phytoextraction Regime Water added (mL) Time of experiment (day) 13581012151719222426Total A 200 200 200 200 200 200 300 300 300 300 300 300 3000 B 300 300 300 300 300 300 400 400 400 400 400 400 4200 C 200 200 500 200 200 500 300 300 600 300 300 600 4200 trations in leachates were determined by Flame-AAS. Controls of the analytical procedure were performed using blanks and references materials (BCR 60 and BCR 141R, Community Bureau of Reference, for plant and soil) treated in the same way as experimental samples. Two determinations of the concentration of HMs was realized per sample. EDTA determination EDTA in leachate was determined spectrophotomet- ricaly according to the procedure of Hamano et al. (1993). Estimation of arbuscular mycorrhizal inoculum potential The total mycorrhizal inoculum potential of soils from different EDTA treatments (watering regime A only – Table 1) was determined by growing bait plants Tri- folium pratense in intact cores of pre-treated soils to measure the rate of mycorrhiza formation. One 150 ml intact soil core was sampled from each pot after the above ground portions of cabbage plants were harvested. Each core was sown by 30 seeds of red clover Trifolium pratense. In a previous experiment, we found that rhizobia were present in the substrate, therefore, no rhizobia was added to the pots. The pots were placed in the greenhouse. The plants were har- vested after 3 months. Shoots were oven dried and dry weight was determined. The roots were washed, cleared with 10% KOH at 90 ◦ C for 75 min, rinsed in tap water and stained with trypan blue in lactoglycerol for 15 min at 90 ◦ C. The staining method was mod- ified from Phillips and Hayman (1970). Mycorrhizal infection was estimated according to Trouvelot et al. (1986). Mycorrhizal frequency (F%) was calculated. Phospholipid extraction and determination Structure and activity of microbial populations was as- sessed using phospholipid and diglyceride fatty acids techniques (PLFA and DGFA). At the end of the phytoextraction experiment, 5 g of soil from the up- per layer of each column with single EDTA additions were sampled. Lipids were extracted with one phase mixture (chloroform, methanol, citric buffer pH 4), diglicerides separated from phospholipids and glycol- ipids on SPE-Si columns, subjected to mild alkaline methanolysis, and methy esters quantified with GC- MS according to Frostegård et al. (1991) and White et al. (1998). 19:0 methyl ester was used as internal standard. The ratio between dead and viable microbial bio- mass was calculated by dividing total diglycerides (DGFA) by total phospholipids (PLFA) (Ringelberg et al., 1997). The structure of microbial groups in soil was presented in relative shares of microbial groups, de- termined as mol% of PLFAs indicative for particular microbial groups against total PLFAs. One must bear in mind that the development of different groups of mi- croorganisms inferred from the changes in the PLFA pattern does not give absolute amounts of biomass of different groups, since conversion factors from the microorganism groups to actual biomass are lacking. Fatty acids were designated using the nomenclature described by Frostegård and Bååth (1996). Statistical analyses The data were statistically evaluated with analysis of variance. HM concentrations in plant tissue were square-root transformed before analysis to stabilize the variance. Tukey’s multiple range test was used to determine the significance (P=0.05) between all possible pairs. 108 Figure 1. Pb, Cd and Zn concentrations in roots and leaves of Brassica rapa grown on contaminated soil (watering re- gime A) in response to the 3 mmol kg −1 single dose addition (EDTA/3S), 5 mmol kg −1 single dose addition (EDTA/5S), 5 mmol kg −1 weekly additions (EDTA/5W), 10 mmol kg −1 single dose ad- dition (EDTA/10S), 10 mmol kg −1 weekly additions (EDTA/10W) of EDTA and control soil with no EDTA addition. Means of four replicates are presented, error bars represent standard deviation. Results Heavy metals plant uptake and leaching Cabbage (Brassica rapa) was selected as a test plant due to its substantial Pb and Zn phytoextraction po- tential (Xian, 1989). The analysis of plant material indicated that the addition of HM complexing agent EDTA to the soil increased the concentrations of Pb, Zn and Cd in the leaves of the test plant (Figure 1). Plant uptake of Pb was particularly enhanced. Even at the lowest tested single dose addition of EDTA (3 mmol kg −1 soil), the concentrations of Pb in leaves were 16.6-times higher than those in control plants. When 10 mmol kg −1 EDTA was added in single dose, Pb concentration in the leaves increased for 104.6- times. The same amount of EDTA applied in four weekly additions resulted in 44-fold increase of Pb in leaves. It was significantly less effective than a single dose and statistically comparable to weekly additions of the total 5 mmol kg −1 EDTA. The effect of EDTA additions on Cd and Zn plant uptake was less prominent. In the treatment with the highest EDTA addition (10 mmol kg −1 in single dose), the concentration of Cd and Zn in leaves increased for 2.3 and 3.2-fold, respectively, compared to the control treatment (Figure 1). Other single or weekly EDTA additions increased Cd and Zn content in plant tissues for less than 2-times compared to control and had no statistically different effects. The increase watering re- gime (watering regime B,C) slightly decrease the HMs concentration in plant tissue. As shown in Figure 1, a single addition of 10 mmol kg −1 EDTA significantly (P=0.05) decreased the con- centration of Zn and Cd in roots of tested plants by 71 and 69% compared to concentrations in the roots of control plants. The decrease of Pb concentration in roots by 41% compared to the control was not statist- ically significant (P=0.05). Dead roots, which could influence the results of HM analysis, were removed during sample preparation. The analysis of leachates, collected from control treatments and treatments with weekly additions of EDTA, suggested that EDTA mobilized heavy metals and caused significant leaching. The dynamic of Pb, Zn and Cd leaching is presented in Figure 2. The concentrations of Pb and Cd in leachates of control treatments were bellow the detection limits of instru- ment (0.4 mg L −1 and 0.025 mg L −1 , respectively). The amount of Zn leached was bellow 0.02 mg kg −1 of soil in all control treatments. As expected, the water- ing regime and the amount of water applied had strong influence on HM leaching. In columns watered with lesser amounts of water (regime A) a constant increase of leachate HM concentration was observed during the experiment. In regimes (B and C) with more abund- ant watering, the concentration of metals in leachate ceased to increase at the end of experiment, presum- ably because most of the metals had been leached by then. The mass balance of HMs leached and extracted into the harvastable parts of plants is shown in Table 2. Thirty six and 40% of total applied EDTA was leached through the soil profile in columns with 5 and 10 mmol kg −1 of applied EDTA (weekly additions, regime C), respectively (Figure 3). 109 Figure 2. The influence of different watering regimes (A, B, C) on Pb, Zn and Cd leaching from soil in treatments with weekly additions of 5 and 10 mmol kg −1 of EDTA during phytoextraction experiment. The means of four replicates are presented, error bars represent standard deviation. Figure 3. EDTA content in soil column leachate (watering regime C) in response to 5 mmol kg −1 weekly additions and to 10 mmol kg −1 weekly additions of EDTA. The means of four replicates are presented, error bars represent standard deviation. Phytotoxicity In all treatments where EDTA was applied, visual symptoms (necrotic lesions on the leaves of Chinese cabbage) of HM or EDTA toxicity were observed. The symptoms were more prominent on older leaves. Single dose 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 3). The growth of red clover in the bioassay exper- iment depended strongly on the substrate. Both the number of plants developed (not shown) and total biomass of the shoots per pot (Figure 4) revealed a sig- nificant negative impact of EDTA treatment on growth of test plants. The effect was more pronounced in treatments where high EDTA amounts were added in single application. No mycorrhizal infection was found in plants growing in soil treated with 5 and 10 mmol kg −1 EDTA in a single addition. If the same cumulative amount of EDTA was applied in sequential weekly additions, arbuscular mycorrhiza developed, but its 110 Table 2. The mass balance of HMs in percentages of initial total HMs in soil. HMs leached and extracted into the harvastable parts of plants in treatments with weekly addition of EDTA and control treatments according to watering regimes A,B,C are shown. Results are presented as means of four replicates±s.d. Treatment Pb Zn Cd % Leached Control A ND 0.002±0.000 ND B ND 0.005±0.001 ND C ND 0.004±0.001 ND 5 mmol EDTA/kg soil A 6.3±2.0 3.0±0.8 18.7±5.2 B 19.1±1.8 7.0±0.6 43.1±8.3 C 17.3±3.8 6.6±1.0 40.9± 5.1 10 mmol EDTA/kg soil A 19.2±5.9 6.2±1.7 32.8±8.7 B 37.9±1.6 10.4±0.3 51.0±1.3 C 34.6±3.4 10.1±0.7 56.3±5.5 % Extracted Control A <0.002 0.028±0.007 0.141±0.018 B <0.002 0.022±0.005 0.115±0.019 C <0.002 0.020±0.001 0.105±0.011 5 mmol EDTA/kg soil A 0.072±0.013 0.036±0.005 0.191±0.022 B 0.046±0.009 0.024±0.002 0.132±0.013 C 0.039±0.010 0.026±0.004 0.113±0.009 10 mmol EDTA/kg soil A 0.061±0.031 0.031±0.010 0.129±0.006 B 0.038±0.008 0.026±0.005 0.120±0.024 C 0.057±0.014 0.026±0.005 0.113±0.004 ND not detected. frequency (F%) was lower compared to the control treatment. Despite the negative influence of 3 mmol kg −1 EDTA on the growth of red clover (Figure 4), heavy mycorrhizal infection was present in all developed plants (Figure 4). The effect of EDTA addition on soil microorganisms Phospholipid and diglyceride fatty acids analyses (PLFA and DGFA) were used to determine the ef- fect of a single EDTA additions on soil microflora at the end of phytoextraction experiment. In total 50 different PLFAs were detected, and 27 of these were identified. PLFAs can be used to identify microbial groups. PLFAs used to indicate bacteria were (i15:0, a15:0, 15:0, i16:0, i17:0, a17:0, cy17:0), PLFA used as actinomycetes marker was 10Me-18:0 and PLFA used as marker for fungi was 18:2w6,9 (Vestal and White, 1989). Figure 4. Red clover (Trifolium pratense) shoot dry weight and ar- buscular mycorrhizae frequency in response to the 3 mmol kg −1 single dose addition (EDTA/3S), 5 mmol kg −1 single dose addition (EDTA/5S), 5 mmol kg −1 weekly additions (EDTA/5W), 10 mmol kg −1 single dose addition (EDTA/10S), 10 mmol kg −1 weekly additions (EDTA/10W) of EDTA and control soil with no EDTA addition. Means of four replicates are presented, error bars represent standard error. The major shifts in the structure of microbial com- munities as the result of different EDTA additions was 111 Figure 5. The structure of microbial groups (bacterial, fungal and actinomycetes) determined as mol% of PLFAs in soil treated with different additions of EDTA. The results are the means of two replicates. Figure 6. Stress index (trans/cis PLFA) of microbial popula- tions and the ratio between dead and viable microbial biomass (DGFA/PLFA) in soil treated with different additions of EDTA. The results are means of two replicates. determined using marker PLFAs expressed as mol%. In total, these marker PLFAs represented 23–28% of total PLFA. The PLFAs representing fungi decreased with increasing concentrations of EDTA in soil while neither of the PLFA markers of bacteria or actinomy- cetes changed significantly at higher doses of EDTA (Figure 5). However, the changes of the PLFA pat- tern does not give an absolute amount of biomass for different groups, since conversion factors from the mi- croorganism group to actual biomass are still not avail- able. Especially the share of fungal biomass, which is dominant in most soils (Thorn, 1997) seemed to be underrated in Figure 5. The ratio between dead and vi- able biomass (DGFA/PLFA) increased dramatically in soil treatments with higher EDTA concentration (Fig- ure 6). The dead fungal biomass presumably accounts for the increase of DGFA. Also, the trans/cis ratio Table 3. Chinese cabbage biomass yield in treatments with dif- ferent EDTA additions. Results are presented as means of four replicates±s.d. Treatment Dry matter (g) Control 12.7±1.1 a EDTA 3 mmol/kg, S 11.9±0.9 a,b EDTA 5 mmol/kg, S 11.2±1.6 a,b,c EDTA 5 mmol/kg, W 11.1±1.2 a,b,c EDTA 10 mmol/kg, S 7.9±1.2 c EDTA 10 mmol/kg, W 8.2±2.3 c a,b,c Statistically different treatments according to Tukey test, P=0.05. S, single dose addition of EDTA. W, weekly additions of EDTA. of PLFA increased at higher EDTA concentrations (Figure 6). The increased fatty acids trans/cis ratio is associated with starved or stressed microorganisms in natural environments (Guckert et al., 1986). Discussion The goal of successful phytoextraction is to reduce HM levels 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. Many hydroponic studies revealed that the uptake and translocation of HM in plants are enhanced by increasing HM concentration in the nutrient solution (Huang et al., 1997). The bioavailability of HMs in the soil is, therefore, of para- mount importance for successful phytoremediation. Pb, as one of the most widespread metal pollutants in soil, has limited solubility in soil solution and bioavail- ability due to complexation with organic and inorganic soil colloides, sorption on oxides and clays and pre- cipitation as carbonates, hydroxides and phosphates (Ruby et al., 1999). Therefore, a successful phytore- mediation must include mobilization of HMs into the soil solution that is in direct contact with plant roots. Results of our study indicated up to 104.6, 2.3 and 3.2-fold increase of Pb, Cd and Zn concentra- tion, respectively, in leaves of Chinese cabbage grown on EDTA (10 mmol kg −1 ) treated soil. No statistic- ally significant difference in Cd and Zn plant uptake was observed when single dose and weekly 5 mmol kg −1 EDTA additions were compared (Figure 1). The greater ability of EDTA to enhance Pb plant uptake above Zn and Cd and other HMs was also reported earlier (Blaylock et al., 1997) and appears to be related 112 to the binding capacity of EDTA for different metals. Formation of Pb-EDTA complex is expected to be the dominant metal-EDTA complex in most soils between pH 5.2 and 7.7 (Sommers and Lindsay, 1979). At higher doses (10 mmol kg −1 EDTA), a single dose addition of chelate was much more effective than weekly additions. It seems that high concentrations of EDTA caused desorption of less available species of HMs which are more strongly bound to the soil particles. Interestingly, the concentration of Zn and Cd in the roots of plants grown on EDTA treated soil was lower than in the roots of plants grown on control soil (Figure 1). However the concentration of Pb in roots was not statistically significant lower compared to control plants. Lead retention in the roots is based on Pb binding to ion exchangeable sites on the cell wall and extracellular precipitation in the form of Pb carbonate deposited in the cell walls (Cunningham et al., 1995). Our observations only partially confirm that EDTA effectively prevents cell wall retention of HM and influenced not only HM uptake but also enhanced HM translocation in the plant (Blaylock et al., 1997). It is well documented that the primary target of HM toxicity and in particular lead toxicity (Godbold, 1994) is the root and not the shoot. Hence, lower exposure of roots to HM could be crucial for the plant performance and consequently also for the successful remediation process. The use of chelates as soil amendments to increase the bioavailability of HM has raised some concern over the potential increased mobility of the metal- chelate complex in the soil. Several authors have emphasized the possibility of HM groundwater con- tamination or other off site migrations (Copper et al., 1999; Huang and Cunningham, 1996). While EDTA and other chelates are commonly used additives for remediation of HM contaminated soil in ex situ soil washing techniques (Mark et al., 1998), no data on EDTA promoted metal leaching during phytoextrac- tion were found. High concentrations of Pb, Zn and Cd and EDTA found in soil column leachates (Fig- ures 2 and 3) suggest high water solubility of Pb, Zn and Cd-EDTA complexes. These results suggest that phytoextraction using chelates must be designed properly to prevent migration of soluble HMs. We are currently investigating some managing practices to accomplish this. In general, there is little known about the im- pact of different phytoremediation practices on soil microorganisms. Recent studies with hyperaccumu- lating plants revealed a great impact on the quantity and species composition of arbuscular mycorrhizal propagules as well as on mycorrhiza function during long-term metal-remediation treatments (Pawlowska et al., 2000). There is a great need to assess the poten- tial influence of phytoremediation on soil microflora, especially when organic chelators are applied. We designed a bioassay experiment with red clover in order to evaluate the post treatment toxicity of soils used in the EDTA treatment experiment. The ana- lysis of plant growth revealed a strong inhibitory effect of EDTA on the Chinese cabbage (Table 3) and the red clover (Figure 4). Both direct adverse action of EDTA and the increased bioavailability of soil HMs could influence plant performance negatively. Very high total and shoot HM concentrations were meas- ured in the treatments where the most adverse effects of the EDTA were observed (10 mmol kg −1 EDTA). Hence, it is possible that HMs were affecting physiolo- gical processes even in the above ground parts of the plants. On the other hand, several studies suggest that the toxicity of different metals can also be mitigated by EDTA binding (Postma et al., 2000; Sillanpaeae and Oikari, 1996). In our case, further experiments would help us to evaluate the adverse effects of HM and EDTA separately. Both the presence of EDTA and HMs could in- fluence the development of red clover arbuscular my- corrhiza as it is known that they can be the factors influencing photosynthetic activity of the host and car- bon allocation to the roots can mediate mycorrhizal association in terms of quantity (the rate of mycor- rhizal infection) and quality (physiological interac- tions between the symbionts) (Smith and Read, 1997). The adverse effects of HMs on the occurrence of ar- buscular mycorrhizal fungi, HM tolerance in these micro-organisms,and their effects on metal uptake and transfer to plants are well documented (Leyval et al., 1997). There is, however, very little informationon the direct effects of EDTA on arbuscular mycorrhiza (Ez- awa et al., 1995). Although the results of mycorrhizal bioassay experiments vary with the use of different bait plants and environmental conditions, they provide a relative measure of mycorrhizal fungus inoculum (Brundrett et al., 1996). In our experiment, the es- timation of the mycorrhizal colonization of red clover revealed a similar EDTA dose-dependent response of mycorrhizae as it was found for the plant growth (Figure 4). However, 3 mmol kg −1 EDTA treatment did not negatively influence mycorrhiza formation al- though it strongly inhibited plant growth. This sug- gests a higher sensitivity of plants to the presence of 113 EDTA or bioavailable HMs compared to arbuscular fungi. More detailed studies would be needed to con- firm this presumption and to evaluate the influence of EDTA treatment on mycorrhizal development. The toxicity of EDTA on soil bacteria, actinomy- cetes and fungi was studied with PLFA and DGFA methods. PLFA and DGFA are relatively new tools in environmental microbiology and enable the insight into the structure of microbial populations in complex substrates, and give an indication of environmental stress inflicted on microbial populations (Vestal and White, 1989). The results are in accord with phyto- toxicity and arbuscular mycorrhize tests. Increasing doses of EDTA increased the cultural stress (DGFA analysis, trans/cis ratio of PLFA methyl esters) of soil microflora (Figure 6). The PLFA results indic- ated that soil fungi are more sensitive to EDTA or to EDTA mediated increase of HMs bioavailability than are soil bacteria and actinomycetes (Figure 5). This can be partly explained by a very diverse bacterial metabolism which enables bacterial species to adjust to different environmental conditions. Our data are also in accord with results of Dahlin et al. (1997). They reported that the effect of HMs on the PLFA pattern was small, except for 18:2u6 PLFA, which decreased in sludge amended, Cd, Cr, Cu, Pb and Zn contamin- ated soil, compared to the control soil. This specific PLFA is an indicator the amount of fungi in the soil (Frostegard et al., 1993). Conclusion The results of this study muddy the waters regarding the possible use of EDTA for in situ phytoextraction of HM contaminated soils. The addition of EDTA en- hanced accumulation of HMs in green parts of the test plant. 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