EDTA and HEDTA printed - Các phức EDTA và HEDTA, vai trò của chúng trong môi trường

Các phức EDTA và HEDTA, vai trò của chúng trong môi trường

CHEMOSPHERE

www.elsevier.com/locate/chemosphere

EDTA and HEDTA effects on Cd, Cr, and Ni uptake by

Helianthus annuus

Hong Chen, Teresa Cutright *

Department of Civil Engineering, The University of Akron, Akron, OH 44325-3905, USA Received 26 October 2000; received in revised form 16 January 2001; accepted 18 January 2001

Abstract

Phytoremediation has shown great potential as an alternative treatment for the remediation of heavy-metal-con- taminated soils and groundwater However, the lack of a clear understanding pertaining to metal uptake/translocation mechanisms, enhancement amendments, and external effects on phytoremediation has hindered its full-scale applica- tion The objective of this research was to investigate the ability of synthetic chelators for enhancing the phytoreme-

diation of cadmium-, chromium- and nickel-contaminated soil Ethylenediaminetriacetic acid (EDTA) and

N-(2-hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA) were applied to the soil at various dosages to elevate metal

mobility Uptake into and translocation within Helianthus annuus was determined It was found that EDTA at a rate of

0.5 g/kg significantly increased the shoot concentrations of Cd and Ni from 34 and 15 to 115 and 117 mg/kg, re- spectively The total removal efficiency for EDTA was 59 ug/plant HEDTA at the same application rate resulted in a total metal uptake of 42 ug/plant These research demonstrated that chelator enhancement is plant- and metal-specific and is subjective to inhibition when multiple heavy metals are present Results also showed that chelator toxicity re- duced the plant’s biomass, thereby decreasing the amount of metal accumulation © 2001 Elsevier Science Ltd All

rights reserved

Keywords: Chelators; EDTA; HEDTA; Helianthus annuus; Phytoremediation; Metals

1 Introduction

Phytoremediation is a process that uses living green

plants for the in situ risk reduction for contaminated

soil, sludge, sediments, and groundwater through con-

(Anonymous, 1998) It has been shown to be more ad-

vantageous than conventional technologies for remedi-

ating heavy-metal-contaminated soils The advantages

include large-scale application; growing plants is rela-

tively inexpensive; plants provide an aesthetic value to

the landscape of contaminated sites, and concentrated

” Corresponding author Tel.: +1-330-972-4935; fax: +1-330-

972-6020

E-mail address: tcutright@uakron.edu (T Cutright)

hazardous wastes require smaller disposal facilities, and the potential exists to recover metals from the biomass

(Saxena and KrishnaRaj, 1999)

At sites contaminated with heavy metals, phyto- remediation can be applied as different strategies based

on the specific site condition They may include phy-

toextraction, where metals are transported from the soil

into the harvestable shoots (Salt and Blaylock, 1995),

rhizofiltration, where plant roots or seedlings grown in

aerated water precipitate and concentrate toxic metals (Raskin et al., 1997), phytovolatilization, in which plants extract volatile metals (e.g., Hg and Se) from soil and

volatilize them from the foliage (Salt and Blaylock,

1995), and phytostabilization, 11 which metal-tolerant plants are used to reduce the mobility of heavy metals

(Raskin et al., 1997) For sites contaminated with both

heavy metals and toxic organics, phytoremediation 1s 0045-6535/01/$ - see front matter © 2001 Elsevier Science Ltd All rights reserved

PII: S0045-6535(01)00031-5

still applicable (Saxena and KrishnaRaj, 1999) because

the rhizosphere association between plants and soil mi-

croorganisms can be utilized to degrade or transform

complex organic—metal mixtures This process has been

called phytotransformation or phytodegradation

All plants have the potential to absorb a wide variety

of metals from the soil For the most part, plants tend

to only absorb those metals that are essential for their

survival and growth The most remarkable exception to

this general rule is a small group of plants that can

tolerate, uptake, and translocate high levels of certain

heavy metals that would be toxic to any other known

organism Such plants are termed “‘hyperaccumula-

tors’ According to Brown et al (1995), hyperaccu-

mulator species are those plants whose leaves may

contain >100 mg/kg Cd, >1000 mg/kg Ni and Cu, or

>10000 mg/kg Zn and Mn (dry weight) when grown in

metal-rich soils With this extraordinary ability, these

plants can be used in future environmental remediation

activities, however, full-scale applications have yet to be

achieved One important reason for this les with the

lack of thorough knowledge on the biological processes

involved in metal acquisition, transport, and shoot

accumulation

Salt et al (1998) proposed that in the process of

acquiring metal ions from soil, plants have evolved

several strategies for increasing the metal bioavailability

due to the high binding capacity for metallic micronu-

trients by soil particles The first strategy is the plants’

ability to produce metal-chelating compounds (phytos-

iderophores) such as mugenic and avenic acids to

mobilize metal compounds from soil (Vonwiren et al.,

1996) The second approach involves the solubilization

of metals by exuding protons from roots to acidify the

rhizosphere soil (Crowley et al., 1991) Alloway (1995)

further suggested that the roots possess a significant

CEC due to the presence of carboxyl groups, which

might help to move ions through the outer part of the

root to the plasmalemma where active absorption

occurs

In addition to natural plant adaptations, the addi-

tion of synthetic chelators, soil acidifiers, or commer-

cial nutrients can enhance phytoremediation Several

studies have documented the success of pH adjustments

for mobilizing metals (Salt et al., 1998; Entry et al.,

1996; Chaney et al., 1997; Huang et al., 1997) Al-

though soil acidification increased metal mobility, it

decreased the microbial activity of the surrounding

area (Cornish et al., 1995; Salt et al., 1998; Chen,

2000) Only the addition of synthetic chelators has been

shown to increase both the metal mobility within the

soil as well as the uptake (and translocation) through

the plant tissue without being irreversibly toxic to mi-

crobial activity For instance, Huang and Cunningham

acetic acid (HEDTA) on Pb accumulation enhance-

ment and found that 1 week after transplanting, the shoot Pb concentration was increased from 40 to

10600 mg/kg In addition to shoot concentration, the

shoot to root Pb content was increased from 0.2 to 1.2 Blaylock (1997) showed that chelator supplements in-

creased the uptake of Pb, Cd, Cu, Ni, and Zn Huang

et al (1997) further reported that among the five che- lators, Ethylenediaminetriacetic acid (EDTA) was the most efficient in increasing shoot Pb concentration in

both pea and corn, followed by HEDTA They found the order of the effectiveness in increasing Pb accu- mulation to be EDTA > HEDTA > diethylenetrinitril- opentacetic acid (DTPA)> ethylenegluatarotriacetic

(EDDTA) Dushenkovy et al (1999) demonstrated that

to increase '*’Cs bioavailability, of the 20 amendments

tested ammonium salts had the greatest effect

Although chelators may increase the effectiveness of phytoremediation by means of increasing the removable metal concentrations, not all studies agree Robinson

et al (1999) reported that in their study, neither calctum

and magnesium carbonates, nor the addition of syn-

thetic chelating agents were effective in increasing metal uptake by Berkheya coddii on serpentine soils Bennett

et al (1998) also found that their attempt to enhance nickel uptake in B coddii by adding EDTA and citric acid to the substrates actually caused a decrease in nickel uptake, despite causing an increase in the concentration

of soluble nickel

The objective of this research was to investigate the ability of synthetic chelators to enhance the phyto-

remediation of cadmium-, chromium- and nickel-con-

HEDTA) and dosage required to elevate metal mobility and subsequent uptake and translocation within the

plant tissues were determined

2 Experimental methods 2.1 Soil sources and characterization

An agricultural soil was collected from a clean resi-

dential garden center in northeastern Ohio The soil was air-dried under room temperature and mixed daily until

an 8% water content was reached Soil was characterized

for soil texture, soil pH, field capacity, cation-exchange capacity (CEC), organic matter content (OM), and contaminant background concentrations Soil texture,

pH, and field capacity were measured by the procedures described by Tan (1995) CEC was determined by the method proposed by Gillman (Nedelkoska and Doran, 2000) OM and TOC were analyzed with a Shimadzu

total organic carbon analyzer (TOC-5000) equipped

with a solid sample module (SSM-S000A) The back- ground concentrations of total sorbed Cd, Cr, and Ni

were determined with EPA method 3050 (Dramer et al.,

1996) (Table 1)

2.2 Soil preparation

To initiate the experiments, air-dried soil was

weighed and loaded into 21 Al pans (0.32 m x 0.25

m x 0.04 m) Each pan contained 1.5 kg soil DW per

pan The soil was then rehydrated with a standard nu-

trient solution containing 250 mg N (NH,NO;), 60 mg

Mg (MgSO¿), 109 mg P (KH;PO/¿), and 207 mg K

(KH;PO¿,+K;SO¿) per kg soi DW (Senden et al.,

1990) Two days later, the appropriate metal solution

was spiked into the soil in Al pans and mixed thor-

oughly For the first experimental set, the solution

contained 50 ppm Cd’*, (as CdCl, -2.5H,O), 50 ppm

Cr”* (CrC1; - 6H;O and 50 ppm N* (NiSO/¿ -6H;O) for

a total metal concentration of 150 mg/kg The metal

concentration corresponds to the individual metal ele-

ment content and not the overall compound The second

experimental set had a reduced concentration of 30 mg/

kg for each metal After the metal solution was added,

the soil was allowed to equilibrate for a period of 10

days in the greenhouse The equilibration involved un-

dergoing three cycles of saturation with DI water and air

drying, before being remixed and vegetated (Muller and

Kordel, 1993) At day 12, pans were amended with ei-

ther EDTA or HEDTA (Sigma Chemical) at a concen-

tration of 1 or 2 g/kg Chelator selection was based on

the previous work by Huang et al (1997)

A control and blank pan were also prepared with

supplemental nutrients and/or metals and were subjected

to the saturation cycles as outlined above The control

contained non-metal-spiked-vegetated soil for the in-

tention of obtaining data related to background activi-

Table 1

Physical and chemical characteristics of agricultural soil used in

this study

soil

loam

* Mean + SE (each analysis was performed in duplicate)

ties, such as the plant accumulation of background heavy metals and biomass growth in uncontaminated soil The four blanks consisted of the same levels of

spiked metal concentrations as treatments with an ex- ception that no plants were grown in the soil The pur- pose of the blanks was to determine the vegetation effect

on metal mobility in the contaminated soil and to ensure

accuracy and precision in the analyses

2.3 Cultivar source and seedling preparation Cultivar selection was based on the plant’s ability to

achieve hyperaccumulator status for at least one metal

The dwarf sunspot sunflower, Helianthus annuus, has

been proven to be effective at removing heavy metals

and is capable of extracting higher than average amounts of several radionuclides (Cooney, 1996; Gal- lego et al., 1996; Dushenkov et al., 1997; Gouthu et al., 1997; Sun and Shi, 1998; Chen, 2000; Zavoda et al.,

2001)

Seeds of H annuus were obtained from USDA/ARS

Plant Introduction Station of Iowa State University

They were initially sown in commercial potting soil

(SCHULTZ Professional Potting Soil Plus, SCHULTZ

Company) in a greenhouse illuminated with natural light Supplementary light was provided for maintaining 15-h photo-period daily Greenhouse temperature was 28°C in the daytime and 15°C at night After 2 weeks of growth in the potting soil, seedlings with similar biomass

were transferred to the metal-spiked soil and the ex- periment was initiated Nine seedlings were used per pan Unless otherwise specified, seedlings were harvested

4 weeks later

2.4 Plant harvest and analysis

During harvest, plants were gently removed from soil

and washed until free of soil Roots, leaves, and stems were further separated with scissors and dried in a convection oven at 70°C for 3 days (Page, 1982) Tissues were milled with mortar and pestle and digested fol- lowing the procedure outlined by Zheljazkov and Er- ickson (1996) One g of milled plant matter was soaked

in 20 ml of concentrated nitric acid After 6 h, the mixture was boiled to 50% of its original volume Then,

4 ml of perchloric acid was added and the mixture ref-

luxed for 90 min The solution was finally diluted with

DI water to 25 ml of total volume and analyzed with flame atomic absorption spectroscopy (Buck 200 AA) 2.5 Analysis of total metal and mobile metal fractions in

the soil For this manuscript, the mobile metal fraction is

defined as the fraction that is not tightly bound to soil

and is mobile without the addition of chelators The

total metal concentration is the summation of the bound

and mobile fractions In order to differentiate between

the mobile and sorbed fractions, two different extraction

methods were used The concentration of the total Cd,

Cr, and Ni was determined via an EPA acid digestion

method 3050 (Carter, 1993)

Approximately 10 ml of 1:1 HNO; was added to 2 g

of air-dried soil (<1 mm) in a 500-ml ball-shaped flask

and heated at 95°C for 15 min Five ml concentrated

HNO, was added and the solution was refluxed for an

additional 30 min at 95°C This was repeated once and

the final solution obtained was reduced to 5 ml Once

cooled, approximately 25 ml of 30% H,O, was added to

the solution in 1-ml increments, followed by the addition

of 5 ml of concentrated HCI The digestate was filtered

through a Whatman® No 42 filter paper and the solu-

tion was diluted to 50 ml with DI water The solution

was analyzed by FAAS (Buck 200 AA)

To extract the mobile metal fraction in the soil, a

procedure proposed by Maiz et al (1997) was followed

Two grams of air-dried soil sample was transferred into

a capped 40 ml heavy-duty PRYEX centrifuge tube,

mixed with 20 ml 0.01 M CaCl, solution, and agitated in

a rotary shaker at 200 rpm for 2 h After 2 h, the soil

suspension was centrifuged at 2500 rpm for 15 min and

the supernatant was collected for FAAS analysis

2.6 Statistical analysis

The experiments were designed as a two-stage nested

design with two types of chelators as the primary factors

For each factor, two different concentrations were used

The difference between specific pairs of means was

(P < 0.05) Statistical analysis of the data was performed

by using SigmaStat 2.0 (SPSS Science, Chicago, IL)

2.7 Results and discussion

2.7.1 Chelator effect on plant growth

Adding HEDTA and EDTA led to a severe yield

reduction in the biomass across the treatments In the

first experimental set with higher metal concentration

and chelator (1 and 2 g/kg) doses, plants appeared to be

chlorotic and showed signs of wilting 1 day after the

experiment was initiated Within | week, all plants were

dead Therefore, the metal concentration was lowered

to 30 mg/kg per metal and the chelator additions low-

ered to 1.0 and 0.5 g/kg for the next set of experiments

Lowering both metal concentrations and chelator ad-

ditions extended plant growth to some degree but a

large number of plants still died within 2 weeks Plants

grown in 0.5 g/kg EDTA-treated soil exhibited better

growth rate and higher biomass was obtained This was

supported by the visual observations where more than

half of the plants grown in the soil amended with

0.5 g/kg EDTA maintained vigorous growth through- out 4 weeks However, growth was still severely

retarded in comparison to non-chelator treatments For example, plants subjected to 30 mg/kg per metal with- out chelators had less than a 10% reduction in biomass,

and none of the plants died Furthermore, control pans containing only chelator additions (i.e., no metals pre-

sent) did not exhibit a severe biomass reduction Therefore, the severe reduction in growth was attrib- uted to the combination of heavy metal concentration and chelator addition

As compared with the control plants, the average

shoot biomass of the treatment plants decreased by more than 75% for the 150-ppm contaminated soil (Fig I(a)) and more than 50% for the 90-ppm soil (Fig 1(b)) Plants in HEDTA-amended soil exhibited approxi- mately the trend in biomass reduction This indicated

that the levels of HEDTA added or the metal-HEDTA

compounds formed in soil were already too high and therefore, toxic to the plants Addition of EDTA ap-

peared to be less toxic to plants compared to HEDTA

as shown by a higher biomass However, the yields be-

tween EDTA and HEDTA were not statistically differ- ent A possible reason was due to the different toxicity

of the two chelators and/or their metal—chelator com- pounds formed As a whole, this study demonstrated

that synthetic chelator addition had a significant adverse effect on plant growth

© L

Fig 1 Effect of adding chelators on shoot biomass of 9 plants grown in heavy-metal-contaminated soil (a) Cd, Cr, and Ni at

50 mg/kg of each, (b) Cd, Cr, Ni spiked at 30 mg/kg of each Bars marked with (*) are statistically different with the control (P < 0.05) Error bars represent +SE of (n = 3)

2.7.2 Effects of chelators on mobile fractions of Cd, Cr,

and Ni in soil

As anticipated, chelator addition significantly in-

creased the mobile fractions of Cd, Cr, and Ni as com-

pared with control (Fig 2) Cr had the greatest increase

as its mobile fraction was raised by approximately 40-

fold in HEDTA-treated soil and 60-fold in EDTA-

amended soil (Fig 2(b)) Cd and Ni were also increased

by more than 4- and 2-fold, respectively (Figs 2(a) and

(c))

The mobile fractions of Cd and Ni were shown to

increase with increasing levels of HEDTA and EDTA

added to the soil (Fig 2) For chromium, however, in-

creasing of mobile fraction was more strongly dependent

on chelator species than on chelator concentration

Since its mobile fraction did not increase when chelator

levels were increased to 1.0 g/kg, it may indicate that the

chelator level of 0.5 g/kg was high enough to elevate the

bioavailable Cr to the maximum level Compared with

HEDTA, EDTA had approximately the same capability

Š 25

=

& 304

a 15 +

= 10 3

5 +

25

20 +

A

Chelator Treatment (g/kg) Fig 2 Effects of HEDTA and EDTA additions on the mobile

fractions of (a) Cd, (b) Cr, and (c) Ni in soil for individual metal

concentrations of 30 mg/kg For comparing chelator treatments

with the control, bars marked with a (*) are statistically dif-

ferent (P < 0.05) For comparing different chelator treatments,

the mean value followed by different capital letters are statis-

tically different (P < 0.05) Error bars represent +SE of (n = 3)

to increase the mobile fraction of Cd and Ni while it was

more efficient at mobilizing Cr than HEDTA The general order of bioavailable metal concentrations as a

result of chelator addition in each treatment was Cr>Cd = Ni

While enhanced metal mobility can increase the up- take into plants, the potential for movement into the groundwater is also increased An increase in metal

migration to the groundwater would have a detrimental impact on the environment Therefore, care should be

taken when selecting the final chelator addition for field applications The dosage must be high enough to mo-

bilize the metals to the root zone without being too high

to cause toxicity or elevated groundwater concentra- tions

2.7.3 Impact of chelator amendments on metal accumu- lation in plants

Fig 3 contains the shoot and/or root tissue accu-

mulation for each metal that resulted from the different

chelator amendments Adding chelators significantly

140.00

Ea shoot

,S

= 100.00

:

+ op

Sẽ

pe

œ

li

(a)

70.00

*

= 50.00 | Mroot

a=

2 @ 2000:

|

a

4

(b) 0.0 | | 0.5 | | 1.0 | | 05 |

| 7

200.00

180.00 Ì Eishoot

7

š 140.00 |

Mroot

& Sp 80.00

proms

Chelator Treatment (g/kg soil)

Fig 3 Effect of adding HEDTA and EDTA on the tissue concentrations of the dwarf sunspot sunflower (a) Cd, (b) Cr, and (c) Ni with individual spiked concentration of 30 mg/Kg For a given plant tissue, bars denoted with (*) are statistically different from the control Error bars represent +SE of (n = 3)

enhanced shoot concentrations of Cd and Ni (Figs 3(a)

and (c)) The shoot content of Cd and Ni were increased

by more than 2-fold and 4-fold as a result of the in-

creased mobile fractions of Cd and N1 in soil In contrast

to shoot concentrations, root levels of Cd and Ni were

decreased by a small fraction as compared to the con-

trol Therefore, they may be translocated to the shoot to

a greater extent than the non-chelated complexes As a

result, the root concentrations of Cd and Ni were

slightly lowered

Fig 3(b) indicates that chelator additions, regardless

of the source or concentration, did not increase the

shoot concentration of Cr This was surprising since the

mobile fraction of Cr surged from 0.47 to over 15 mg/kg

as shown in Fig 2(b) In contrast, the root content of Cr

was enhanced The root concentration increase was ap-

parently due to the increase of bioavailable Cr in soil

The Cr-chelator compound may have different physio-

chemical properties as compared with Cd- and Ni-

complexes, therefore, it could not be translocated to

shoots

Analysis of Fig 3 indicated that the 0.5 g/kg

HEDTA dose had the best performance in enhancing

the concentrations of Cd and Ni in shoot and the

concentration of Cr in root tissue However, it should

be noted that the enhanced high tissue concentration as

a result of chemical amendment might not necessarily

produce a high removal efficiency for the target metal

contaminant since biomass change is another deter-

mining factor

Some researchers (Huang and Cunningham, 1996;

Blaylock, 1997; Huang et al., 1997) have reported that

chelators such as HEDTA and EDTA may enhance the

shoot concentration of Pb by more than 100-fold

However, in this study, these chelators demonstrated

only limited capability to improve the shoot accumula-

tions of Cd, Cr, and Ni This is because most of the

current chelator studies focus on single metals like Pb

Therefore inhibition from other metals would not im-

pede uptake and translocation Moreover, different

plant species have been used in their studies As a result,

it is believed that chelator enhancement is plant- and

metal-specific and is also subject to the interaction and

subsequent inhibitory effects when multiple heavy metals

are present

2.7.4 Effect of chelator addition on total metal accumu-

lation

As compared with the control, the addition of che-

lators decreased heavy metal accumulation by plants

(Fig 4) This was due to the severe biomass reduction

If phytoremediation enhancement with chelators is go-

ing to succeed, a strategy that may protect plant bio-

mass from heavy loss is necessary In this study, EDTA

at 0.5 g/kg appeared to be the best addition of the four

treatments with a total removal rate of 59 ug/plant (535

800

600 3

500 3

400 3

1 L] TL] il [la

80

60 3

50 3

40 3

203

400

300 4

250 3

200

150

100 4

Chelator Treatment (g/kg)

Fig 4 Effect of adding HEDTA and EDTA on the total metals accumulated by nine plants (a) total Cd accumulation, (b) total

Cr accumulation and, (c) total Ni accumulated Error bars represent +SE of (n = 3)

ug/pan), even though it caused a decrease in compari-

son with the control which was 103 pg/plant (927 pg/

pan) HEDTA at the application rate of 0.5 g/kg had

the highest metal concentration increase, yet it only

resulted in a total metal uptake of 42 pg/plant (376.7 ug/pan) These results indicate that the 0.5 g/kg chelator dosage may be still be too high

2.8 Conclusions and recommendations EDTA and HEDTA both significantly enhanced the

metal concentration in plant tissues, however, they re- sulted in a severe biomass loss of more than 50% As a

result, the total amount of metals removed by plants was decreased The study also determined that the effect of synthetic chelators on phytoremediation is subject to the

influence of multiple metal interactions and _ specific plant species With decreasing biomass aside, the che- lator additions resulted in bioavailable metal order of Cr>Cd = Ni

For this study, the 0.5 g/kg EDTA application

achieved the best results However, at this application rate the use of chelators may not be economically

competitive with other technologies Future studies will

focus on identifying the lowest, cost-effective chelator

addition that will enhance metal mobility and uptake

without posing a detrimental impact on groundwater

quality

Acknowledgements

This work was conducted under the funding of

University of Akron Faculty Research Grant #1425

The authors wish to extend their appreciation to Dr

Randy Mitchell of the Department of Biology in Uni-

versity of Akron who provided greenhouse space for our

phytoremediation studies

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