effects of lead and chelators on growth, photosynthetic activity

Environmental Pollution 144 (2006) 11e18www.elsevier.com/locate/envpol

Effects of lead and chelators on growth, photosynthetic activityand Pb uptake in Sesbania drummondii grown in soil

Adam T. Ruley a, Nilesh C. Sharma a, Shivendra V. Sahi a,*, Shree R. Singh b,Kenneth S. Sajwan c

a Department of Biology, Western Kentucky University, 1906 College Heights Blvd 11080, Bowling Green, KY 42101-1080, USAb Alabama State University, 915 S. Jackson Street, Montgomery, AL 36104, USA

c Department of Natural Sciences and Mathematics, Savannah State University, Savannah, GA 31404, USA

Received 1 June 2005; accepted 8 December 2005

Sesbania drummondii tolerates and accumulates high concentrations of Pb.

Abstract

Effects of lead (Pb) and chelators, such as EDTA, HEDTA, DTPA, NTA and citric acid, were studied to evaluate the growth potential ofSesbania drummondii in soils contaminated with high concentrations of Pb. S. drummondii seedlings were grown in soil containing 7.5 gPb(NO3)2 and 0e10 mmol chelators/kg soil for a period of 2 and 4 weeks and assessed for growth profile (length of root and shoot),chlorophyll a fluorescence kinetics (Fv/Fm and Fv/Fo) and Pb accumulations in root and shoot. Growth of plants in the presence of Pb þchelators was significantly higher (P < 0.05) than the controls grown in the presence of Pb alone. Fv/Fm and Fv/Fo values of treated seedlingsremained unaffected, indicating normal photosynthetic efficiency and strength of plants in the presence of chelators. On application of chelators,while root uptake of Pb increased four-five folds, shoot accumulations increased up to 40-folds as compared to controls (Pb only) depending onthe type of chelator used. Shoot accumulations of Pb varied from 0.1 to 0.42% (dry weight) depending on the concentration of chelators used.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Sesbania drummondii; Pb-remediation; Chelators; Pb accumulation

1. Introduction

Lead (Pb) contamination in soil is a widespread phenome-non and originates from automobiles, metal smelting plants,mines, lead-contaminated sewage sludge, industrial wastes,etc. (Zakrzewski, 1991). Pb exposure to plants causes effectssuch as the disturbance in mitosis (Liu et al., 1994; Wierz-bicka, 1994), induction of leaf chlorosis (Johnson and Proctor,1977), depression of photosynthetic rate, (Bazzaz et al., 1974),inhibition in root and shoot growth (Fargasova, 1994; Liuet al., 1994), and inhibition and activation of enzymaticactivities (Van Assche and Cliisters, 1990). Severe Pb

* Corresponding author. Tel.: þ1 270 745 6012; fax: þ1 270 745 6856.

E-mail address: [email protected] (S.V. Sahi).

0269-7491/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2006.01.016

contamination in soils may lead to a variety of environmentalproblems e loss of vegetation, ground water contamination,and ultimately Pb toxicity to animals and humans (Bodyet al., 1991). Thus, there is an urgent need for remediationof contaminated sites using an effective and environment-friendly technology such as phytoremediation.

In recent years, phytoremediation has emerged as a viablebiotechnology to decontaminate the heavily polluted sites(Blaylock et al., 1997; Huang et al., 1997; Kirkham, 2000;Sharma et al., 2004). This strategy makes use of hyperaccumu-lator plants, which have the inherent potential to survive andaccumulate excessive amounts of metal ions in their biomasswithout incurring damage to basic metabolic functions(Cunningham et al., 1997). With successive cropping and har-vesting of accumulator crops, the levels of contaminants canbe reduced substantially. For a plant species to be efficient

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12 A.T. Ruley et al. / Environmental Pollution 144 (2006) 11e18

in lead phytoextraction it should accumulate metal concentra-tion >0.1% of shoot dry weight, besides having high biomassproductivity (Kirkham, 2000). A balance between metal accu-mulation and plant biomass productivity is critical for a plant-species to be used in Pb phytoextraction (Huang andCunningham, 1996). From this standpoint, plant species suchas Indian mustard, pea, and corn were focused recently forPb phytoremediation research. These species accumulatehigh amounts of lead, and produce satisfactory biomass(Huang et al., 1997; Blaylock et al., 1997; Epstein et al.,1999). Another interesting Pb accumulator is Sesbania drum-mondii, a perennial large bushy plant with greater biomassproductivity than the above plant species (Ruley, 2004). S.drummondii grows naturally in seasonally wet places of thesouthern coastal plains of the United States and tolerateshigh concentrations of soil Pb. It demonstrated a unique poten-tial of Pb accumulation in aerial parts from an aqueous solu-tion (Sahi et al., 2002).

To compensate for the relatively low metal accumulationcapacities of Indian mustard, corn, pea and other potentialplant species, chelates such as ethylenedinitrilotetraaceticacid (EDTA), N-(2-hydroxyethyl)ethylenediaminetriaceticacid (HEDTA), diethylene trinitrilopentaacetic acid (DTPA),trans-1,2-cyclohexylenedinitrilotetraacetic acid (CDTA) andethylenebis [oxyethylenetrinitrilo] tetraacetic acid (EGTA)were supplemented to the Pb-contaminated soils (Blaylocket al., 1997; Huang et al., 1997; Epstein et al., 1999;Kirkham, 2000; Sarret et al., 2001). Application of chelatorsinduces metal desorption from minerals and boosts transloca-tion of Pb from root to shoot. A chelate-assisted increase of100 to 200 folds in shoot Pb accumulation was noticed in In-dian mustard (Blaylock et al., 1997; Epstein et al., 1999) whileseveral-fold increases were observed in pea and corn (Huanget al., 1997). Kirkham (2000) reported a significant increasein shoot Pb when sunflower plants were grown in soils con-taminated with sewage sludge. Chelators not only facilitatePb uptake and translocation, but also protect plants from oxi-dative stress that is produced as a result of heavy metal (Pb)exposure, as reported in Sesbania seedlings grown in vitro(Ruley et al., 2004). Studies show how exposure of Pb or otherheavy metals affect growth and photosynthetic activities inplants (Xiong, 1997; KrishnaRaj et al., 2000). Chlorophylla fluorescence, a non-destructive marker of the photosyntheticapparatus, has been used extensively in screening for abioticstresses, such as heat, chilling, drought, salinity and heavymetal stresses (Becerril et al., 1988; Krause, 1991; KrishnaRajet al., 2000; MacFarlane, 2003). In the present investigation,we have utilized chlorophyll a fluorescence parameters asa quantitative marker to assess and compare the tolerance ofSesbania sp. when exposed to Pb and different chelators.

Therefore, in order to understand the effects of highconcentrations of Pb and chelators, this study was focused todetermine 1) growth profile, 2) chlorophyll a fluorescencekinetics [Fv/Fm and Fv/Fo], and 3) Pb accumulation in Sesbaniadrummondii seedlings grown in soils contaminated with a highconcentration of Pb in the presence or absence of syntheticchelators, such as EDTA, DTPA, HEDTA, NTA and citric

acid. Comparing the efficacy of different chelators on Pbaccumulation by Sesbania was also aimed in this study.

2. Materials and methods

2.1. Preparation of seed bed and pot plants

Seeds of Sesbania drummondii were scarified in 85% H2SO4 for 35 min,

rinsed for 30 min, sterilized in 0.1% HgCl2, and rinsed for 10 min. After

sterilization, seeds were germinated into trays containing peat moss and

vermiculite (Sahi et al., 2002). Three week-old seedlings of similar growth

(8e10 cm long shoots and 6e10 cm long roots) were selected and transferred

to individual pots filled with 2.0 kg of soil (three parts soil and one part sand

passed through 2 mm sieve). The soil used in this experiment belonged to

Pembroke series e dark brown silt loam and neutral to slightly alkaline e having

characteristics of Mollic epipedon (80e100; 700; 180; 15 g/kg sand, silt, clay

and organic matter, respectively). The soil was spiked, 6 weeks before planting,

with different concentrations of Pb(NO3)2 and chelators as described below.

After transplantation into individual pots, plants were maintained in greenhouse

under 16 h light/8 h dark regime, and watered as needed until harvest.

2.2. Treatment of Pb þ chelators

Seedlings were grown in the presence of Pb and the chelators (EDTA,

DTPA, HEDTA, NTA, citric acid). Each experimental group consisted of 15

seedlings grown individually in pots containing 7.5 g Pb(NO3)2 and 1.25e

10 mmol chelators/kg soil. Lead nitrate was dissolved in sufficient amount

of water and applied in soil 6 weeks prior to planting. Different solutions of

chelators were applied to individual pots after a week of planting. For each

experimental group, controls were set up without Pb(NO3)2 and containing

the same concentration of synthetic chelators. Controls were also set up

with 0 or only 7.5 g Pb(NO3)2/kg soil. Plants were harvested after 2 and

4 weeks, separated into roots and shoots, and then measured for growth by

means of shoot and root length.

2.3. Estimation of photosynthetic activities

Before each harvest, seedlings were analyzed for photosynthetic activities

by measuring chlorophyll a fluorescence parameters (Ruley et al., 2004). This

was performed using the Handy-PEA instrument (Hansatech Instruments,

UK). Plants were dark-adapted for 30 min and then given a 1 s pulse of red

light. The following fluorescent parameters were measured: Fo, the minimum

chlorophyll a fluorescence after the dark-adaptation, and Fm, the maximum

fluorescence after the pulse of red light. From these two measurements the

Fv (the variable fluorescence calculated as the difference between the minimal

and maximal fluorescence), Fv/Fm (the ratio of variable to maximal fluorescence)

and Fv/Fo (the ratio of variable to minimal fluorescence) values were determined.

2.4. Pb analysis

Roots and shoots were dried at 60 �C (2 d) for Pb analysis by ICP-MS

(Sahi et al., 2002). Samples were weighed and placed into a 15 ml screw cap-

ped Teflon beaker. Concentrated HNO3 (3 ml) was added to the sample, and

the beaker was placed on a hot plate at a temperature of 100 �C overnight,

and the contents were then evaporated to dryness. Samples were allowed to

cool and mixed gravimetrically with 2% HNO3 to a volume of 20 ml. The

ICP-MS analysis was carried out using external calibration procedure, and

Y (0.1 ppm) was used as an internal standard to correct for drift and matrix

effect (Sahi et al., 2002).

2.5. Statistical analysis

All statistical analyses were performed using SYSTAT 9 for Windows 95.

Growth and photosynthetic measurements were the means of 6 samples taken

from 2 experiments; 3 replicates were taken in each experiment. Four samples

13A.T. Ruley et al. / Environmental Pollution 144 (2006) 11e18

each treatment were processed for Pb accumulation. The analysis of variance

(ANOVA) appropriate for the design was carried out to detect the significance

of differences (P < 0.05) among the treatment and control means and Tukey

HSD post hoc test was performed to compare among the groups for significant

differences.

3. Results

3.1. Effects of chelated Pb on plant growth

Fig. 1 depicts the effect of Pb þ chelators or chelators(only) on plant growth, as shown by the shoot length. Forboth lengths of time, Pb þ DTPA, Pb þ NTA or Pb þ citricacid treatments resulted in the shoot growth not significantlydifferent (P < 0.05) than controls (grown in the presence ofchelators only), with an exception of plants grown atPb þ 2.5 mmol citric acid/kg soil (Fig. 1A,B). At the sametime, plants had longer shoots (P < 0.05) as a result of thesetreatments, particularly after 4-weeks, relative to the plantsgrown in the presence of Pb only. However, significantly re-duced shoot length (P < 0.05) was observed in case of plantsgrown in the presence of Pb þ EDTA (10 mmol) or HEDTA.Control plants had also reduced shoots at 10 mmol HEDTA/kg soil (Fig. 1B). Fig. 2 compares root length of Sesbaniaplants grown in the presence of Pb þ chelators or chelatorsonly. Roots of plants grown in soil containing Pb þ 5,10 mmol citric acid or EDTA were significantly greater(P < 0.05) than those of plants grown in the presence of

chelators only, or Pb only (Fig. 2A,B). Growth of plant rootsin the presence of Pb þ DTPA or HEDTA did not differ signif-icantly from controls (P > 0.05) during both 2- and 4-weektreatments. However, plants grown in the presence ofPb þ NTA (2.5 and 10 mmol/kg soil), and citric acid (1.25and 2.5 mmol/kg soil) had significantly reduced roots(P < 0.05) relative to the controls (Fig. 2A,B).

3.2. Effects of chelated Pb on photosynthesis

Fv/Fm ratios of S. drummondii seedlings grown in the pres-ence of Pb þ chelators or Pb alone are depicted in Fig. 3A,B.Fv/Fm ratios of plants exposed to Pb þ chelators were not sig-nificantly different (P > 0.05) than controls. The notable ex-ceptions to this pattern occurred at two weeks in plantsexposed to Pb þ 10 mmol HEDTA or NTA/kg soil; however,these plant groups showed normal Fv/Fm ratios (>0.8) atfour weeks (Fig. 3A). On the other hand, control plants grownin the presence of chelators demonstrated a different pattern ofFv/Fm ratios (Fig. 3B). Plants grown in the presence of EDTAalone were the most severely affected, not surviving until4 weeks at a concentration of 5 or 10 mmol/kg. Also, plantsgrown in the presence of 10 mmol HEDTA/kg soil (alone) sur-vived less than 2 weeks (Fig. 3B).

Fig. 4A,B illustrate the effect of Pb and synthetic chelatorson photosynthetic activities of plants, as measured by Fv/Fo ra-tios. In most of the treatments, Fv/Fo ratios in Sesbania plants

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Fig. 1. Effects of 7.5 g Pb(NO3)2 þ 0e10 mmol/kg EDTA (E), DTPA (D), HEDTA (H), NTA (N) and citric acid (C) on Sesbania drummondii shoot length: (A)

Shoot length of plants grown in the presence of Pb þ chelators for 2 and 4 weeks. (B) Shoot length of control plants grown in the presence of chelators (alone) for 2

and 4 weeks. Values represents mean � S.E., where n ¼ 6.


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Fig. 2. Effects of 7.5 g Pb(NO3)2 þ 0e10 mmol/kg EDTA (E), DTPA (D), HEDTA (H), NTA (N) and citric acid (C) on Sesbania drummondii root length: (A) Root

length of plants grown in the presence of Pb þ chelators for 2 and 4 weeks. (B) Root length of control plants grown in the presence of chelators (alone) for 2 and

4 weeks. Values represents mean � S.E., where n ¼ 6.

were at or above 4.0; the only group of plants that demon-strated a differential pattern was those grown at 10 mmolHEDTA/kg soil (two weeks), though these plants also recov-ered showing Fv/Fo ratios at 5.0 by week four (Fig. 4A). How-ever, Fv/Fo ratios in control plants (grown in the presence ofchelators alone) had a different pattern (Fig. 4B), with plantsgrown in the presence of 5 or 10 mmol EDTA/kg soil not sur-viving until 4 weeks and those grown in the presence of10 mmol HEDTA/kg soil surviving less than 2 weeks.

3.3. Uptake of chelated Pb

Fig. 5A compares Pb concentrations in Sesbania shootsgrown in the presence of Pb þ chelators or Pb alone. Applica-tion of a chelator, at any concentration, resulted in a rapid in-crease in shoot Pb, as compared to the shoot Pb of plantsgrown in the presence of Pb alone. The effect of chelatorson shoot accumulations of Pb was concentration-dependent,except in citric acid treatments, where shoot Pb was maximumat the lowest concentration of the chelate (Fig. 5A). The typeof chelator had also a pronounced effect on Pb accumulationin shoots. It was observed that chelators increased Pb uptakein the order EDTA > HEDTA > DTPA > NTA > citric acid(Fig. 5A). Fig. 5B shows root Pb concentrations in plantsgrown in the presence of Pb þ synthetic chelators or Pb alone.It was observed that in the presence of any of the chelatorstested, at any concentration, Pb absorption in roots was signif-icantly higher than in plants grown in the presence of Pb with-out chelators (Fig. 5B). It was also noticed that in respect of

HEDTA and NTA treatments, the concentration of Pb in rootsincreased with an increase in chelator concentration.

4. Discussion

4.1. Growth

Results show that growth of Sesbania plants in the presenceof Pb þ chelators was either significantly higher (P < 0.05)than the plants grown in Pb-contaminated soils or not signifi-cantly different (P > 0.05) than controls, grown in the pres-ence of chelators only. Growth in the presence of Pb þchelators resulted in a significantly decreased (P < 0.05) shootlength only in the case of 5 or 10 mmol HEDTA/kg soil. At thesame time, it is also apparent that 10 mmol HEDTA/kg soil(alone) resulted in the significantly reduced (P < 0.05) shootlength relative to normal plants, grown without Pb or chelator(Fig. 1B), proving the point that higher concentration ofHEDTA was itself toxic to Sesbania plants. A seeminglydifferent trend emerged in plant roots, where the root lengthwas affected by Pb þ lower concentrations of citric acid(1.25 or 2.5 mmol/kg) and Pb þ higher concentrations ofDTPA or HEDTA (Fig. 2A,B). This is interesting to notethat higher concentrations of citric acid (5, 10 mmol/kg) favorplant growth, particularly root growth, while lower concentra-tions affect root growth. As binding affinity of citric acid islow for Pb, only the high concentrations of this chelator willeffectively bind and form a complex with Pbþþ, reducingthe toxic effects on plants. Measurements of plant biomass


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Fig. 3. Effects of 7.5 g Pb(NO3)2 þ 0e10 mmol/kg EDTA (E), DTPA (D), HEDTA (H), NTA (N) and citric acid (C) on chlorophyll a fluorescence kinetics (Fv/Fm

values) of Sesbania drummondii: (A) Fv/Fm of plants grown in the presence of Pb þ chelators for 2 and 4 weeks. (B) Fv/Fm of control plants grown in the presence

of chelators (alone) for 2 and 4 weeks. Values represents mean � S.E., where n ¼ 6.

reflected the same trend. Plant shoot and root weights, in mostof the Pb þ chelator treatments, were significantly higher thanthose of plants grown in Pb alone or chelators alone (data notpresented). These observations suggest a protective role forchelating agents against Pb toxicity in S. drummondii. Studieson mechanisms of Pb toxicity suggest that Pb2þ binds tonucleic acids and causes aggregation and condensation ofchromatin, as well as stabilization of DNA double helix inhib-iting the processes of replication, transcription and ultimatelythe cell division and plant growth (Johnson, 1998). Chelatorsapplied to the Pb-contaminated soil may form complexeswith Pb2þ thus inactivating and minimizing the cytologicalimpacts of free metal ions. Heavy metal toxicity in plantsalso occurs with the induction of oxidative stress at cellularlevel following production of reactive oxygen species (Dixitet al., 2001; Geebelen et al., 2002). Application of chelatorshas been reported to mitigate Pb-induced oxidative stress bymodulating antioxidative enzyme activities in Sesbania seed-lings (Ruley et al., 2004), and this may also be one of the rea-sons for better growth of Sesbania plants in the presence ofa chelator.

In this study, Sesbania seedlings were grown in the soilcontaminated with 7.5 g Pb(NO3)2/kg soil, in the presence orabsence of chelators, as these plants were observed to grow

healthier at this concentration of soil Pb in a preliminary study.Though plants grew even at the higher concentrations of Pb,but stunting or dwarfing of shoots was a marked feature, par-ticularly, at a concentration of 10 g/kg soil, and those grown inthe presence of 15 g Pb/kg soil could not survive until 4 weeks(data not shown).

4.2. Photosynthetic activity

The Fv/Fm value is an indicator of the photosyntheticefficiency of plants, while Fv/Fo value indicates the size andnumber of active photosynthetic centers in the chloroplast,and thus the photosynthetic strength of the plant. An Fv/Fm

value of 0.8 or higher in all the treatments (Fig. 3A) similarto controls (Fig. 3B) indicates that the plant is healthy andnot suffering photosynthetic stress as a result of Pb uptake. Ex-ception to this was found only in the plants grown inPb þ 10 mmol HEDTA/kg soil, where Fv/Fm value wasaround 0.7 at the second week, the value picking up to a levelof 0.8 in the fourth week. Notably, plant growth was also af-fected when plants were grown at this treatment (Figs. 1A,Band 2A,B). The another notable feature was recorded in thecontrols grown in the presence of 5 or 10 mmol EDTA/kgsoil (alone), where Fv/Fm values showed a sharp decline in


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Fig. 4. Effects of 7.5 g Pb(NO3)2 þ 0e10 mmol/kg EDTA (E), DTPA (D), HEDTA (H), NTA (N) and citric acid (C) on chlorophyll a fluorescence kinetics (Fv/Fo

values) of Sesbania drummondii: (A) Fv/Fo of plants grown in the presence of Pb þ chelators for 2 and 4 weeks. (B) Fv/Fo of control plants grown in the presence

of chelators (alone) for 2 and 4 weeks. Values represents mean � S.E., where n ¼ 6.

the second week, plants not surviving until the fourth week,while those grown in Pb þ EDTA exhibited a normal trend. Asimilar trend of depressed photosynthetic activity was reportedwhen Sesbania seedlings were grown in the solution culturecontaining EDTA or HEDTA alone (Ruley et al., 2004). TheFv/Fo values of all the treatments were also normal, 4 orgreater, except at Pb þ 10 mmol HEDTA/kg soil (Fig. 4A).At the same time, controls grown in the presence of10 mmol HEDTA/kg soil (alone) died before 2 weeks. Similarto Fv/Fm values, Fv/Fo values were significantly reduced incontrols (5 or 10 mmol EDTA/kg soil, alone). Growth of thesecontrols was also severely affected as seen earlier. Another in-teresting observation that was recorded in this study was theoccurrence of similar normal Fv/Fm or Fv/Fo values for boththe groups of plants grown in the presence or absence of Pb(Figs. 3A,B and 4A,B). It is therefore reasonable to concludethat exposure to Pb, either alone or in combination with che-lators, does not affect the photosynthetic machinery of Sesba-nia drummondii. These results are in agreement with the reporton Pelargonium sp., where Fv/Fm and Fv/Fo values were notsignificantly affected by Pb accumulation (KrishnaRaj et al.,2000). However, observations in Sesbania sp. differed fromthose in Avicennia marina, where heavy metal (Zn) causeddepression of photosynthetic activity in a dose-dependent

manner (MacFarlane, 2003). Lead affects chlorophyll synthesisthrough inhibition of d-aminolevulinic acid dehydratase, which,in turn, depresses photosynthetic activity of plants througha reduction in chlorophyll content (Geebelen et al., 2002). Itis believed that a Pb accumulating plant if maintains photosyn-thetic activity while accumulating Pb, it will survive and toler-ate toxic concentrations of Pb (KrishnaRaj et al., 2000).

4.3. Pb accumulation

Results show that application of a chelator increased con-centrations of Pb in roots as well as shoots of Sesbania byseveral folds, relative to the plants grown in Pb only. Whileroot uptake of Pb increased four-five folds, shoot accumula-tions increased up to 40-folds as compared to controls (Pbonly) depending on the type of chelator used. Shoot accumu-lations of Pb varied from 0.1 to 0.42% (dry weight) dependingon the concentration of chelators (EDTA, DTPA, HEDTA)used. The type of chelator also influenced Pb accumulationin Sesbania shoots significantly. It was noticed that chelatorsincreased Pb transport in Sesbania in the order EDTA >HEDTA > DTPA > NTA > citric acid. This pattern of effec-tiveness of chelators (in Sesbania) is consistent with the earlierreport on maize (Huang et al., 1997). The most likely


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Fig. 5. Effects of 7.5 g Pb(NO3)2 þ 0e10 mmol/kg EDTA (E), DTPA (D), HEDTA (H), NTA (N) and citric acid (C) on Pb accumulations in Sesbania drummondii:

(A) Shoot Pb after 2 weeks of growth (B) Root Pb after 2 weeks of growth. Values represents mean � S.E., where n ¼ 4.

explanation for chelate-stimulated Pb transport lies in the en-hanced solubility of Pb in soil on application of a chelator likeEDTA (Huang et al., 1997; Epstein et al., 1999). It has alsobeen shown in a number of studies that application of chelat-ing agents results in the stimulated translocation of Pb fromplant roots to shoots (Blaylock et al., 1997; Huang et al.,1997; Epstein et al., 1999). EDTA and HEDTA were shownto have increased Pb translocation from root to shoot by200-folds in maize and pea, and translocation was highly spe-cific to the plant species and genotype (Huang and Cunningham,1996; Huang et al., 1997). In Indian mustard, several fold-in-crease in shoot concentrations of Pb and other heavy metalswas recorded as a result of EDTA application in Pb-contami-nated soils (Blaylock et al., 1997). Recent findings suggest thatPb is transported in the plant shoot as the Pb-EDTA complexand thus increasing the concentration of Pb þ chelate may re-sult in maximizing Pb accumulation in shoots (Epstein et al.,1999; Sarret et al., 2001).

It was also noted during this study that plants in some con-trol groups exposed only to chelators (EDTA, HEDTA) experi-enced chlorosis and distortion of leaves, stunted growth, anddecreased survival. These symptoms are consistent with defi-ciencies of magnesium, copper and possibly molybdenum(Hopkins, 1999). Furthermore, as effects of chelators (controls)were dose-dependent and time-dependent, it may be inferredthat application of chelators alone resulted in the removal of es-sential metal nutrients from soil, leading to deficiencies in theplants. Geebelen et al. (2002) observed a similar effect.

The findings related to the above physiological parametersindicate that Sesbania drummondii may be a probable candi-date for its use in phytoremediation of Pb. However, its Pb re-moval capability needs to be tested in the real soil conditionsof Pb contamination, as Pb availability may be generally highin spiked soils. When the feasibility of Sesbania-mediated re-mediation remains unclear at this stage, what makes this plantattractive is its seemingly unaffected growth in the presence ofhigh concentrations of Pb and chelators. The significance ofthis species is further enhanced by the large biomass thatthis plant, being a bushy shrub, can generate in natural condi-tions. On the other hand, maize, Indian mustard or pea thoughaccumulate greater concentrations of Pb, but have potentialdisadvantage of being crop species. Due to centuries of selec-tive breeding, crop plants have been developed that are notonly easy for humans to consume, but are also easier for otheranimal species to consume. Once Pb enters the food chain, thiscan be a serious environmental concern (Robinson et al.,2003). This concern may be minimal by incorporating Sesba-nia drummondii in a Pb remediation strategy, as this taxon isnot a food crop and, in its natural state, is toxic to a varietyof animal species (Banton et al., 1989).

5. Conclusions

Results demonstrate that Sesbania drummondii thrives ona high concentration of Pb (7.5 g/kg soil) in the presence ofdifferent concentrations of chelators such as EDTA, HEDTA,


DTPA, NTA and citric acid. Photosynthetic efficiency andstrength as reflected by chlorophyll a fluorescence parameters(Fv/Fm and Fv/Fo) remains unaffected in the presence ofPb þ chelators. In the presence of chelators, shoot accumula-tions of Pb vary from 0.1 to 0.42% (dry weight) depending onthe type and concentration of a chelator. It was noticed thatchelators increased Pb transport in Sesbania in the orderEDTA > HEDTA > DTPA > NTA > citric acid. This studyprovides evidence for a protective role of a chelator againstPb toxicity in Sesbania drummondii. The most important ad-vantage of using Sesbania drummondii in a phytoextractionscheme may be its large biomass and easy cultivation.

Acknowledgements

The authors thank the Applied Research and TechnologyProgram of the Ogden College of Science and Engineeringand the Department of Biology, Western Kentucky Universityfor supporting the research.

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