EDTA enhanced heavy metal phytoextraction metal accumulation leaching and toxicity

EDTA enhanced heavy metal phytoextraction metal accumulation leaching and toxicity

© 2001 Kluwer Academic Publishers Printed in the Netherlands.

EDTA enhanced heavy metal phytoextraction: metal accumulation,

leaching and toxicity

H Grˇcman, Š Velikonja-Bolta, D Vodnik, B Kos & D Leštan1

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

1Corresponding 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−1for Cd, 140 mg kg−1for

Cu, 75 mg kg−1for Ni, 300 mg kg−1for Pb, 300 mg

kg−1for Zn and 1.5 mg kg−1for 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

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 formaaddi-tion 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 ) 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 P2O5) 37.3 mg 100 g−1,

K (as K2O) 9.2 mg 100 g−1, Pb 1100 mg kg−1, Zn

800 mg kg−1, Cd 5.5 mg kg−1 Soil texture was

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 (NH4)2SO4and K2SO4, 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−1EDTA on the 22th day of

cultivation We used three different watering regimes (Table 1) We harvested the aboveground tissues 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-man No 1 filter paper and stored in cold storage for 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% HNO3) dissolution technique with microwave heating and analysed by Flame-AAS or at low concentration

of Cd and Pb by Electrothermal-AAS HMs

concen-Table 1 Amounts of water added in three watering regimes during the course of phytoextraction

Time of experiment (day)

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

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 −1single dose addition (EDTA/5S), 5 mmol

kg −1weekly additions (EDTA/5W), 10 mmol kg−1single dose

ad-dition (EDTA/10S), 10 mmol kg −1weekly 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−1EDTA 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−1EDTA.

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−1in 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 rere-gime B,C) slightly decrease the HMs concentration in plant tissue

As shown in Figure 1, a single addition of 10 mmol

kg−1EDTA 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−1of

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)

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 −1of 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 −1weekly additions and to 10 mmol

kg −1weekly 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

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.

% Leached

% Extracted

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 −1single dose addition (EDTA/5S), 5 mmol kg −1weekly 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

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.

EDTA 3 mmol/kg, S 11.9 ±0.9a,b

EDTA 5 mmol/kg, S 11.2 ±1.6a,b,c

EDTA 5 mmol/kg, W 11.1 ±1.2a,b,c

a,b,cStatistically 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−1EDTA 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

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 concentraphytoextrac-tions 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 information on 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

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 However, EDTA addition also caused leaching

of Pb, Zn and Cd through the soil profile and had toxic

effects on test plants and soil microorganisms The

results, therefore, emphasize the importance of EDTA

risk assessment for each specific soil and

phytoextrac-tion condiphytoextrac-tions New non-toxic chelates, and methods

to prevent the leaching of the HMs-chelate complex

down the soil profile need to be evaluated

Acknowledgements

This work was supported by the Slovenian Ministry

for Science and Technology, grant No

J4-0694-0486-98 We thank Mr Klavdij Bajc, Mrs Zalka Ilc, Mrs

Ana Zorˇc for technical assistance and Dr Nataša J Vidic and Glenn S Jaecks, M Sc for correcting the English

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Section editor: P Ryan