Introduction

Heavy metals occur naturally as elemental components in the Earth’s crust (Demirbas 2008; Chopra et al. 2009). Some heavy metal such as copper (Cu), cobalt (Co), iron (Fe), manganese (Mn), nickel (Ni), and zinc (Zn) are essentially required by all living organisms in trace amount for biological metabolism and growth. In contrast, many other heavy metals such as cadmium (Cd), mercury (Hg), lead (Pb), and tin (St) have no essential biological function and can be freely bioaccumulated through the food chain (Prasad and Strzałka 1999; Kabata-Pendias 2010).

For years, heavy metal soil contamination has been a global environmental issue as human activities have continuously released these pollutants into the surroundings via agrochemical leaching, disposal of toxic wastes and effluents, and the atmospheric deposition from industrial activities (Bradl 2005; Meuser 2010; Hasanuzzaman and Fujita 2013). Long-term exposure via direct respiration (inhalation), drinking water, and/or ingestion of food contaminated with heavy metals may be adversely harmful to both environmental health (living ecosystem) and human well-being when the tolerance levels are exceeded (Järup 2003; Duruibe et al. 2007).

Various types of soil remediation including physical (dig-and-dump, thermal desorption, fracturing, and soil washing), chemical (solidification-stabilization, reduction-oxidation, etc.), and biological (biosorption, bioleaching, and biofiltration) techniques for heavy metal removal reported over the past decades (Mulligan et al. 2001; van Deuren et al. 2002; Sherameti and Varma 2010; Anjum et al. 2012). Nonetheless, most of these remediation technologies are considerably complicated and cost ineffective and are technically difficult to conduct. As a result, phytoremediation has turned out to be the most viable strategy using plants to clean up heavy metals in contaminated soil. Garbisu and Alkorta (2001), McIntyre (2003), and Ali et al. (2013) suggested that the application of phytoremediation would be esthetically non-destructive to the surrounding, environmentally pleasing, and often required minimum cost for operation and maintenance.

Among numerous types of plants tested for phytoremediation, Vetiver grass, Vetiveria zizanioides (Linn.) Nash proven to be an effective species with quick growth, deep fibrous root system, and high adaptability and tolerance to many extreme environmental stresses including the elevated high concentration levels of heavy metals (Truong et al. 2008; Danh et al. 2009; Truong and Danh 2015; Ng et al. 2017, 2018). To further enhance the accumulation of heavy metals in plants, assorted enrichment materials for instance, disodium ethylene-diamine-tetra-acetate (EDTA) were expansively used as an effective low-cost metal chelating agent for phytoremediation purposes (Han et al. 2004; Luo et al. 2005; Hovsepyan and Greipsson 2005; Seth et al. 2011; Ng et al. 2016). Due to its high efficiency to solubilize metals and metalloids in the soils, EDTA is extensively used to facilitate heavy metal phytoremediation (Shahid et al. 2014; Suthar et al. 2014; Luo et al. 2016a; Jiang et al. 2019). Nevertheless, recent studies (Han et al. 2004; Sinegani et al. 2015; Özkan et al. 2016; Vargas et al. 2016; Chen et al. 2018; Luo et al. 2018) indicated that synthetically designed chemical chelators including EDTA able to enhance metal accumulation and translocation in different plant parts for single heavy metal soil contamination.

However, at present, there is still a lack of information on EDTA-enhanced phytoremediation of mixed heavy metal–polluted soils with Vetiver grass. Most of the past studies emphasized solely on the application of EDTA to improve the heavy metal accumulation without considering the influences of single and mixed contaminations of heavy metal uptake by different plant parts (lower root, upper root, lower tiller, and upper tiller) in Vetiver grass (Andra et al. 2011; Luo et al. 2016a; Anning and Akoto 2018; Wasino et al. 2019). To address these uncertainties, this study was conducted to analyze the growth performance, accumulation trend, and capability of metal uptake from both single and mixed Cd-, Pb-, Cu-, and Zn-contaminated soils enhanced with EDTA using Vetiver grass.

Materials and methods

Site location and experimental setup

The greenhouse pot experiments were carried out at the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur. Vetiver grass, Vetiveria zizanioides (Linn.) Nash was selected for this experiment and placed under nine different types of single and mixed heavy metal enhanced treatments (Table 1). All treatments were conducted with triplicates (n = 3) under the completely randomized design (CRD).

Table 1 Greenhouse design with treatment variables
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Soil management and sampling preparation

Top soil (0–20 cm) was collected from the field site (3° 7′ N latitude; 101° 39′ E longitude) situated within the University of Malaya, Kuala Lumpur, for planting purposes. The collected soil underwent preliminary physico-chemical soil characterization (Table 2). Soil was air-dried for a week followed with passing through < 4 mm sieve to eliminate large non-soil components and gravels. The dull reddish brown soil composed of 84.6% sand, 10.5% silt, and 4.9% clay.

Table 2 Physico-chemical properties of selected soils
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Vetiver grass seedlings were purchased from Humibox Malaysia whereby fresh plantlets with a uniform height (20–25 cm) were selected for this study. Each Vetiver grass was carefully grown in a plastic pot (0.18 m diameter × 0.16 m depth) filled with 2 kg of soil for all treatments. All treatments were watered uniformly by using a 50 mL glass beaker of tap water once a day. Plant growth performances such as tiller number, height, and percentage plant survivor rate were continuously recorded all over the entire 60 days of experiment.

The artificially spiked single and mixed heavy metal soils were adjusted using cadmium nitrate tetrahydrate (Cd(NO3)2·4H2O), copper(II) sulfate (CuSO4), lead(II) nitrate (Pb(NO3)2), and zinc sulfate heptahydrate (ZnSO4·7H2O) salt compounds as well as the disodium ethylene-diamine-tetra-acetate, C10H14N2Na2O8·2H2O (EDTA). The concentration of single and mixed heavy metal soils were determined based on the maximum allowable naturally occurring levels set by the Canadian Council of Ministers of Environment (CCME 1999), Department of Environment, Malaysia (DOE 2009), and European Union (Lado et al. 2008) soil contamination guidelines.

In terms of soil amendment, although the possible outcomes for heavy metal phytoaccumulation may increase with EDTA, a standard composition of 10 mmol EDTA/kg was selected in this study based on the research findings obtained in Grčman et al. (2001) and Ng et al. (2016). The artificially spiked soil was then repeatedly stirred and incubated for a fortnight to achieve the homogeneity of the desired single and mixed heavy metal soils are obtained.

Sample and chemical analyses

At the end of 60-day experimental period, all Vetiver treatments were harvested and pre-washed in running tap and filter water, followed by deionized water to eliminate all forms of soil adhering material before separating the Vetiver grass into four different parts (lower and upper sections of roots and tillers) (Fig. 1). All plant samples were oven-dried for 72 h until a constant dry weight were obtained in order to determine the dry matter content (g/m2) of the plant samples before it was homogenized using mortar and pestle.

Fig. 1
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Plant cross-section between the roots and shoots (tillers) of Vetiver grass

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Approximately, 0.5 g of the homogenized dried plant and soil samples underwent acid digestion with hydrogen peroxide (H2O2), nitric acid (HNO3), and hydrochloric acid (HCl) as accordance to Method 3050B (US EPA 1996) and subsequently with Method 7000B (US EPA 2007) using a Perkin-Elmer AAnalyst 400 flame atomic absorption spectrometer (F-AAS) for the total recoverable elemental analysis. All chemicals used were of analytical reagent standard or of the best grade available. The German Federal Institute for Materials Research and Testing (BRM#12-mixed sandy soil) certified reference material was utilized to control the highly precision techniques of chemical analysis with an average metal recovery rate for Cd (96.1%), Pb (106.9%), Cu (102.9%), and Zn (96.8%), respectively.

Data calculation and statistical analyses

The growth performance of Vetiver grass was assessed using the tolerance index (TI) and root-tiller (R/T) quotient while the ability for metal translocation and accumulation were evaluated by the biological concentration factor (BCF), biological accumulation coefficient (BAC), translocation factor (TF), and percentage of metal uptake efficacy (Kabata-Pendias 2010; Alloway 2013; Ali et al. 2013; Ng et al. 2016) as follows:

  • R/T quotient = dry matter content in roots/dry matter content in tillers

  • TI = total dry matter content in heavy metal treatments/total dry matter content in control

  • TF = heavy metals concentration in tillers/heavy metals concentration in roots

  • BCF = heavy metals concentration in roots/heavy metals concentration in soil

  • BAC = heavy metals concentration in tillers/heavy metals concentration in soil

  • Metal uptake efficacy (%) = (heavy metals concentration in tillers/total heavy metals concentration accumulated in Vetiver grass) × 100

All recorded data were analyzed by using the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) tests for significant differences among treatment means at the 95% level of confidence with by employing the Microsoft Excel Office 365 versions 2016 software.

Results

Plant growth performance

The initial soil pH varied from 4.19 to 6.17 where the Cd + Pb + Cu + Zn + EDTA treatment recorded the lowest pH of 4.19 while the highest pH of 6.17 was observed in the control (Fig. 2). Upon harvesting, Cd + EDTA, Pb + EDTA, Cu + EDTA, Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments showed an increased in pH ranging from 4.70 to 5.54, where the highest pH increment (+ 0.98 pH units) was observed in the Cd + Pb + Cu + Zn + EDTA treatment. The soil pH levels in all single and mixed heavy metal treatments were significantly (p < 0.05) affected compared to the control. The application of both single and mixed heavy metals substantially influenced the overall change in soil pH in all treatments.

Fig. 2
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Changes in soil pH of Vetiver grass in single and mixed heavy metal treatments. Vertical bars represented standard deviation while the same letters indicated no significant difference among all treatments at 0.05 probability level

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Table 3 shows significant differences (p < 0.05) in tiller number, plant height, and percentage of survival of Vetiver grass among all single and mixed heavy metal treatments. All treatments with the exception of EDTA (27.0) and Pb + EDTA (27.7) treatments exhibited significantly lower (p < 0.05) tiller number compared with the control. Both of the mixed Cu + Zn + EDTA (12.8) and Cd + Pb + Cu + Zn + EDTA (13.5) treatments recorded the lowest tiller number among all the treatments, respectively. Similarly, all treatments with the exception of Pb + EDTA (56.02 cm) displayed significantly lower (p < 0.05) plant height as compared with the control. Control plant height (69.74 cm) was 52.3% higher than the Cd + Pb + Cu + Zn + EDTA treatment which recorded the lowest plant height of 33.25 cm. On the other hand, EDTA (96.7%) and Pb + EDTA (77.3%) treatments showed no significant difference (p > 0.05) of percentage survivor rate with the control. Conversely, the percentage of survival among all the other single and mixed heavy metal treatments (69.3–74.7%) were significantly affected (p < 0.05) compared to the control, with Cu + Zn + EDTA treatment recording the lowest (67.3%) percentage survivor rate.

Table 3 Tiller number, plant height (cm), and plant survivor rate (%) of Vetiver grass in single and mixed heavy metal treatments
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The dry matter contents of tiller and total Vetiver grass in all treatments were significantly lower (p < 0.05) compared to the control (Table 4). The Cu + EDTA treatment displayed the lowest total dry matter content (9.67 ± 0.11 g/m2) with an average of 41.2% reduction compared to the control. The single metal treatments recorded comparatively higher dry matter contents than the mixed heavy metal treatments. In contrast, no significant difference (p > 0.05) was found in the root-tiller (R/T) quotient, tolerance index (TI), and dry matter content in the roots of Vetiver grass in all the treatments.

Table 4 Dry matter content (g/m2), root-tiller quotient (R/T), and tolerance index (TI) of Vetiver grass in single and mixed heavy metal treatments
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Accumulation of heavy metals

The concentration of Cd, Pb, Cu, and Zn accumulations in the roots, tillers, and overall plant of Vetiver grass for all single and mixed heavy metal treatments are shown in Tables 5, 6, 7, and 8. The accumulation of all four heavy metals in the lower and upper parts of both roots and tillers for all treatments was comparatively variable.

Table 5 Concentration of Cd (mg/kg) in both lower and upper roots and tillers of Vetiver grass in single and mixed heavy metal treatments
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Table 6 Concentration of Pb (mg/kg) in both lower and upper roots and tillers of Vetiver grass in single and mixed heavy metal treatments
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Table 7 Concentration of Cu (mg/kg) in both lower and upper roots and tillers of Vetiver grass in single and mixed heavy metal treatments
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Table 8 Concentration of Zn (mg/kg) in both lower and upper roots and tillers of Vetiver grass in single and mixed heavy metal treatments
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With regard to Cd accumulation, all the Cd + EDTA, Cd + Pb + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments recorded significantly higher (p < 0.05) Cd in both lower and upper roots and tillers of Vetiver grass compared to the control (Table 5). Similarly, the total roots, total tillers, and overall total accumulation for Cd + EDTA, Cd + Pb + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments exhibited significantly greater (p < 0.05) Cd among all other treatments. The highest accumulation of Cd was found in the upper tillers of Cd + Pb + Cu + Zn + EDTA (128.03 ± 17.95 mg/kg) followed by the lower roots of Cd + EDTA (119.60 ± 20.43 mg/kg) treatments. Between roots and tillers, unlike Pb, Cu, and Zn, the accumulation of Cd was noticeably higher in the tillers than in the roots with the exception of the Cd + EDTA treatment and the control. A relatively higher Cd accumulation was demonstrated in the upper roots and upper tillers of the Cd + Pb + EDTA treatment compared with its lower plant parts, respectively. In contrast, the accumulation of Cd was appreciably higher in the lower roots and lower tillers in the Cd + EDTA treatment compared to its upper plant parts. Nonetheless, the order of Cd accumulation among single and mixed Cd treatments was in the order of Cd + Pb + EDTA > Cd + Pb + Cu + Zn + EDTA > Cd + EDTA >> other treatments.

Similarly, with regard to Pb accumulation, the Pb + EDTA, Cd + Pb + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments recorded significantly higher (p < 0.05) Pb in both lower and upper roots and tillers of Vetiver grass compared to the control (Table 6). A significantly higher (p < 0.05) amounts of Pb accumulation was observed in the total roots, total tillers, and overall total accumulation for Pb + EDTA, Cd + Pb + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments among all other treatments. The upper tillers for Cd + Pb + Cu + Zn + EDTA (531.67 ± 36.19 mg/kg) and Cd + Pb + EDTA (368.80 ± 15.09 mg/kg) treatments recorded the highest accumulation of Pb among all treatments. Between roots and tillers, the accumulation of Pb was remarkably higher in the tillers than in the roots among all treatments. The upper roots and upper tillers for Cd + Pb + EDTA treatment as well as the upper tillers for Pb + EDTA and Cd + Pb + Cu + Zn + EDTA treatments accumulated considerably higher Pb compared with different plant parts. The accumulation trend for Pb among the different treatments was in the following order: Cd + Pb + Cu + Zn + EDTA > Cd + Pb + EDTA > Pb + EDTA >> other treatments.

A significantly higher (p < 0.05) Cu accumulation was found in both lower and upper roots and tillers of Vetiver grass for Cu + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments compared to the control (Table 7). Similarly, the total roots, total tillers, and overall total Cu accumulation for Cu + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments demonstrated significantly higher (p < 0.05) Cu among all the treatments. The upper tillers for Cd + Pb + Cu + Zn + EDTA (862.40 ± 231.34 mg/kg) and Cu + Zn + EDTA (538.97 ± 41.88 mg/kg) recorded the highest accumulation of Cu. Between roots and tillers, the accumulation of Cu was substantially higher in the tillers than in the roots among all treatments. The accumulation of Cu in the lower roots and upper tillers for Cd + Pb + Cu + Zn + EDTA and Cu + Zn + EDTA treatments were reasonably greater than other plant parts, respectively. Among the different Cu treatments, the accumulation of Cu was in the order of Cd + Pb + Cu + Zn + EDTA > Cu + Zn + EDTA > Cu + EDTA >> other treatments.

The accumulation of Zn was significantly higher (p < 0.05) in the Zn + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments in both lower and upper roots and tillers of Vetiver grass than the control (Table 8). The Zn + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments exhibited a significantly higher (p < 0.05) Zn in the total roots, total tillers, and overall total Zn accumulation among all other treatments. The upper tillers of Cd + Pb + Cu + Zn + EDTA (3504.80 ± 353.40 mg/kg) and Zn + EDTA (3399.87 ± 485.06 mg/kg) recorded the highest accumulation of Zn among all the treatments. Between roots and tillers, the accumulation of Zn was markedly greater in the tillers than the roots for all treatments. The lower roots and upper tillers for Zn + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments accumulated substantially higher amounts of Zn compared to the other plant parts. The accumulation trend for Zn was in the following order: Zn + EDTA > Cd + Pb + Cu + Zn + EDTA > Cu + Zn + EDTA >> other treatments.

Between single and mixed metal treatments, mixed Cd + Pb + Cu + Zn + EDTA treatment exhibited appreciably higher accumulation of Cu and Pb while mixed Cd + Pb + EDTA treatment was higher in Cd accumulation than the single metal treatments. On the other hand, Zn + EDTA was the only single metal treatment that showed higher accumulation of Zn than the other mixed metal treatments. The findings obtained indicated that the single Zn + EDTA treatment accumulated the highest overall Zn (8068.13 ± 407.35 mg/kg) while the highest accumulation of Cu (1977.47 ± 293.68 mg/kg) and Pb (1096.57 ± 75.60 mg/kg) was recorded in the mixed Cd + Pb + Cu + Zn + EDTA treatment, respectively. The mixed Cd + Pb + EDTA treatment demonstrated the highest overall total amount for Cd (302.97 ± 29.44 mg/kg). Generally, the trend of heavy metal accumulation for all treatments were in the order of Zn >>> Cu > Pb >> Cd regardless of the total amount of heavy metal put into the soil.

Heavy metal uptake and translocation

Tables 9, 10, 11, 12, and 13 show the association of soil-plant accumulation for all single and mixed metal treatments in terms of translocation factors (TF), biological concentration factors (BCF), biological accumulation coefficients (BAC), and percentages of metal uptake efficacy.

Table 9 Biological concentration factor (BCF) of Cd, Pb, Cu, and Zn accumulations in the lower and upper root of Vetiver grass in single and mixed heavy metal treatments
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Table 10 Biological accumulation coefficient (BAC), translocation factor (TF), and metal uptake efficacy (%) of Cd accumulation in the lower and upper tiller of Vetiver grass in single and mixed heavy metal treatments
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Table 11 Biological accumulation coefficient (BAC), translocation factor (TF), and metal uptake efficacy (%) of Pb accumulation in the lower and upper tiller of Vetiver grass in single and mixed heavy metal treatments
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Table 12 Biological accumulation coefficient (BAC), translocation factor (TF), and metal uptake efficacy (%) of Cu accumulation in the lower and upper tiller of Vetiver grass in single and mixed heavy metal treatments
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Table 13 Biological accumulation coefficient (BAC), translocation factor (TF), and metal uptake efficacy (%) of Zn accumulation in the lower and upper tiller of Vetiver grass in single and mixed heavy metal treatments
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The ability for translocation of heavy metal from soil to root in plant is assessed using the BCF coefficient. Both lower (7.116–12.008) and upper (4.733–6.529) roots for single and mixed Zn treatments showed significantly higher (p < 0.05) BCF values than the other treatments. Despite the tolerably higher accumulation of heavy metals in tillers than roots, both lower and upper roots for the single and mixed Cd (1.837–5.980), Cu (1.376–3.931), and Zn (4.733–12.008) treatments recorded relatively high BCF values > 1, suggesting that uptake of Cd, Cu, and Zn from soil to roots was substantially greater and the roots acted as the sink for accumulation of these heavy metals.

Nevertheless, the BAC, TF, and percentages of metal efficacy were employed to evaluate the capabilities and competency of heavy metal uptake and transport from roots to tillers. Similarly, the lower and upper tillers in all the specified single and mixed heavy metal treatments showed appreciably BAC values > 1 compared with the other treatments. The BAC values > 1 in lower and upper tillers for single and mixed Cd (1.503–6.402), Pb (0.671–2.658), Zn (7.764–16.999), and Cu (3.515–8.624) treatments indicated that the tillers acted as the sink for their accumulation due to the fairly effective translocation of these heavy metals from roots to tillers.

On the other hand, despite the relatively higher accumulation of heavy metal in tillers than roots, TF values < 1 were recorded in the lower and upper tillers for all the single and mixed heavy metal treatments. However, the mixed Cd + Pb + Cu + Zn + EDTA treatment exhibited TF values > 1 in both lower (1.503) and upper (2.356) tillers for Pb accumulation compared to the other treatments.

In terms of percentages of metal efficacy, the upper tillers for mixed Cd + Pb + Cu + Zn + EDTA treatment exhibited the highest metal efficacy for Cd (49.1%) and Pb (48.5%) among all treatments. In contrast, the lower tillers of mixed Cu + Zn + EDTA (38.6%) followed by the single Cu + EDTA (35.8%) treatment recorded the highest metal efficacy for Zn and Cu, respectively. Between single and mixed treatments, the mixed Cd + Pb + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments recorded considerably higher percentages of metal efficacy for Cd, Pb, and Zn than the single metal treatments. However, the lower tillers of single Cu + EDTA (35.769%) treatment demonstrated reasonably higher percentage of Cu efficacy than the mixed metal treatments. Generally, the percentages of metal efficacy were remarkably higher in the upper tillers than the lower tillers for accumulation of all heavy metals.

Discussion

The accumulations of all heavy metals were found in both lower and upper parts of roots and tillers in the single and mixed heavy metal treatments. Besides, similar effects of EDTA application were reported in Chen et al. (2004c), Zhao et al. (2011), and Ali and Chaudhury (2016) who observed the accumulation trends of heavy metals in both roots and tillers of the plants.

EDTA was generally used as a common chelating agent for phytoremediation to enhance the bioavailability of heavy metals for uptake by plants in the soil (Meers et al. 2009; Shahid et al. 2014; Bloem et al. 2017). As a result, the accumulation trends responded differently when EDTA was applied in all the single and mixed heavy metal treatments. The presence of EDTA molecules enhance the extraction of metals from exchangeable sites and subsequently formed soluble metal-EDTA complexes (Hadi et al. 2010; Leleyter et al. 2012; Jean-Soro et al. 2012; Dipu et al. 2012). This indicated that the application of EDTA as soil amendment managed to enhance overall accumulation of Cd, Pb, Cu, and Zn by 1.21- to 2.79-fold from both single and mixed heavy metal contaminated soil. In contrast, Lai and Chen (2004) found no significant influence with the application of EDTA at 5 and 10 mmol/kg soil for both Zn and Pb accumulations in Vetiver grass. However, the application of 5 and 25 mmol/kg of EDTA by Chen et al. (2004c) and Ng et al. (2016) recorded reasonably higher concentrations of Pb and Cd in Vetiver grass, respectively.

Moreover, the findings of this study also showed that soil pH became more acidic in all the single and mixed heavy metal treatments when EDTA was added as compared to the control. This condition could affect the bioavailability of metals as a change in soil pH could conceivably affect the capability of EDTA to form complexes (Peng et al. 2009; Bennedsen et al. 2012). This was suggested by Sommers and Lindsay (1979) and Shahid et al. (2014) that metal-EDTA complexes were predominantly formed between pH 5.2 and 7.7 in most soil conditions due to soil acidification.

In addition to the single and mixed heavy metal treatments, this study included and tested separately the response of sole EDTA treatment by comparing with the control. However, the results showed no major significant findings in terms metal accumulation with the sole EDTA treatment compared to the control. Nevertheless, the single Zn + EDTA treatment accumulated highest Zn compared to the other mixed metal treatments. Furthermore, the single Zn + EDTA as well as mixed Cu + Zn + EDTA and Cd + Pb + Cu + Zn + EDTA treatments recorded > 1000 mg/kg of Zn accumulation in almost all of the lower and upper parts of both roots and tillers.

Recent studies by Antiochia et al. (2007), Danh et al. (2009), and Aksorn and Chitsomboon (2013) reported that Vetiver grass was both Pb and Zn hyperaccumulator plants. However, despite the high accumulation of Zn in both roots and tillers, the results of this study suggested that Vetiver grass may be a Cd hyperaccumulator plant due to its high phytoaccumulation ability in the upper tillers for both mixed Cd + Pb + EDTA and Cd + Pb + Cu + Zn + EDTA treatments. The fundamental characteristics of hyperaccumulator plants (Baker and Brooks 1989; Van der Ent et al. 2013) were that plant species are capable of growing and bioaccumulate under extremely high concentrations of heavy metals greater than 100 mg/kg of Cd; or 1000 mg/kg of Pb and Cu; or 10,000 mg/kg of Zn in its plant tissues.

Similarly, Vetiver grass may be regarded as both competent phytostabilizers and phytoextractors due to its BCF and BAC values being > 1, as well as the high accumulation in the lower and upper parts of roots and tillers for all types of heavy metals. This study demonstrated that the roots and tillers acted as the sink for the accumulation of all heavy metals in the presence of EDTA as a chelator agent to enhance the phytoremediation process in Vetiver grass irrespective of single and/or mixed metal treatments.

Previous studies by Lai and Chen (2005), Wuana et al. (2016), and Luo et al. (2016a, b) used different types of plant species such as rainbow pink (Dianthus chinensis), castor (Ricinus communis), and chickpea (Cicer arietinum) demonstrated similar findings on the enhancement of EDTA. Correspondingly, this study further expanded to cover separate parts of the lower and upper roots and tillers of Vetiver grass in order to provide an extensive phytoevaluation of the translocation of heavy metals upwards from the lower roots through the top of the plant’s tillers.

Furthermore, the direct use of Malaysian garden soil spiked with metal salts in pot trial experiments instead of in situ site experiments for this study would inevitably incur unfavorable effects such as additional increase of heavy metal accumulation due to various biotic and abiotic conditions that may influence the overall results of phytoremediation. Consequently, it cannot be ruled out that the experimental design employed with the application of spiked treatments using pot assays may elevate the phytoaccumulation of heavy metals in both the soil-to-roots and roots-to-tillers of Vetiver grass.

Despite its strong phytoaccumulation ability to enhance metal contaminants in plants, both EDTA and metal-EDTA complexes have its drawbacks as they are poorly biodegradable with high toxicity and are extremely persistent in the soils (Oviedo and Rodríguez 2003; European Chemicals Bureau 2004; Goel and Gautam 2010; Zhao et al. 2010; Mühlbachová 2011; Bloem et al. 2017). Additionally, this study also demonstrated that there was a major significant reduction in terms of tiller number, plant height, plant survivor rate, and dry matter content of Vetiver grass when EDTA was applied, irrespective of both single and mixed metal treatments. Thus, it is crucial to note that the application of EDTA could inhibit plant growth performance, as reported by Chen and Cutright (2001) and Chen et al. (2004a, b). As a result, the appropriate management of the use of EDTA concentrations was ultimately vital to optimize metal phytoaccumulation in Vetiver grass as well as to reduce its toxicity, metal leaching, and other potential risks to the environment.

Conclusions

This study revealed that mixed Cd + Pb + EDTA, Cu + Zn + EDTA, and Cd + Pb + Cu + Zn + EDTA treatments were adequately effective to accumulate higher concentration for Cd, Pb, and Cu than the single metal treatments. In contrast, single Zn + EDTA treatment demonstrated the opposite trend, whereby a higher accumulation for Zn was observed among mixed heavy metal treatments. Predominantly, the inclination of heavy metal accumulation in Vetiver grass for all treatments were in the following order of Zn >>> Cu > Pb >> Cd. In terms of different plant parts, both upper roots and tillers of Vetiver grass showed high tendency for the uptake of substantially larger amounts of all heavy metals, regardless of single and/or mixed metal treatments. As a result of the comparably higher concentration in tillers than roots and with BAC values > 1, Vetiver grass may be recommended as a potential phytoextractor for all heavy metals, whereby its tillers acted as the sink for heavy metal accumulation in the presence of EDTA in all treatments.