Effects of chelates on soil microbial properties, plant growth and heavy metal accumulation in plants

Ecological Engineering 73 (2014) 386–394

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Effects of chelates on soil microbial properties, plant growth and heavy metal accumulation in plants Junghun Lee, Kijune Sung * Department of Ecological Engineering, Pukyong National University, Busan 608-737, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 2 May 2014 Received in revised form 18 August 2014 Accepted 13 September 2014 Available online 8 October 2014

Although chelates can enhance remediation efficiency in phytoremediation, they can cause adverse ecosystem effects. The purpose of this research was to investigate the potential effects of chelating agents on plant and soil ecosystems. The effects of ethylene diamine tetraacetic acid (EDTA), ethylene diamine disuccinate (EDDS), and humic acids (HAs) on soil microbial properties, plant biomass, and metal accumulation were investigated in soils contaminated with Cd, Cu, Pb, Zn, and Ni. Four herbaceous plants were used for experimental purposes: Brassica juncea,Brassica campestris, Sorghum bicolor, and Helianthus annuus. EDTA was most effective for heavy metal uptake by plants, but had significant effects on plants and soils. Although EDDS addition can increase metal uptake, its effects are restricted to some plants and metal species. Plant selection can therefore be a more important factor than use of EDDS. HAs had limited influence on increases in heavy metal uptake but had favorable effects on plants and soil. Decreased plant biomass was strongly related to decreased chlorophyll content caused by increased metal concentrations in plant shoots. Results suggest that more efficient chelates may have higher toxicity. Long-term monitoring is therefore necessary to assess soil quality changes after the addition of chelating agents. Furthermore, the effects of the latter on ecosystems, as well as their metal removal efficiency, should be considered when selecting chelating agents for improved phytoremediation. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Herbaceous plant Multiple metal species EDTA EDDS Humic acids

1. Introduction Heavy metals such as lead (Pb), zinc (Zn), copper (Cu), cadmium (Cd), and nickel (Ni) are released from various sources, including mine wastes, fossil fuel combustion, fertilization, insecticides, and industrial wastewater (Lombi et al., 2001; Sun et al., 2010). After combining with soil particles, heavy metals can persist in soils, with harmful effects on biota (Brown, 1979). Phytoremediation is a cost-effective alternative to conventional physicochemical remediation of soils contaminated with heavy metals (Garbisu and Alkorta, 2001; Salt et al., 1998). However, remediation efficiency can be low when the uptake potential of heavy metals by plants is restricted. Chelating agents that help desorb heavy metals from soil, increase the plant-available fraction and uptake amounts, and transport metals to aboveground parts, have been used in phytoremediation to enhance remediation efficiency (Cooper et al., 1999; Huang et al., 1997; Shen et al., 2002). However, the addition of chelating agents can directly or indirectly affect plant

* Corresponding author. Tel.: +82 51 629 6544; fax: +82 51 629 6538. E-mail address: [email protected] (K. Sung). http://dx.doi.org/10.1016/j.ecoleng.2014.09.053 0925-8574/ ã 2014 Elsevier B.V. All rights reserved.

health, soil microbial properties, and heavy metal concentrations in soils and plants (Fig. 1). EDTA (ethylene diamine tetraacetic acid) has been widely used but is known to be persistent and toxic in nature and can cause disturbance to ecosystems (Gr9 cman et al., 2001; Saifullah et al., 2009). It can also cause secondary groundwater contamination if heavy metals are not taken up by plants and enter groundwater (Sarkar et al., 2008; Wu et al., 2004). There has therefore been increased interest in the use of biodegradable chelating agents that are not persistent and that are thus less harmful to the environment. EDDS (ethylene diamine disuccinate), a degradable natural aminopolycarboxylic acid, can compensate for shortcomings of EDTA and increase plant-available metals and mobility (Evangelou et al., 2007a). Some studies suggest that EDDS is more effective at Cu and Zn accumulation than EDTA (Luo et al., 2005; Tandy et al., 2004). Humic acids (HAs) are also known to increase plant-available metal forms and decrease soluble and extractable forms of heavy metals (Halim et al., 2003; Lagier et al., 2000) but there have been few studies on their effects on microorganisms and plants (Park et al., 2011; Sung et al., 2013). Two problems can be encountered when applying chelates for phytoremediation to soils contaminated with heavy metals. First, if the increase in heavy metal concentrations in plants resulting from

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Table 1 Physicochemical characteristics of the soil used in the study. Parameters

Values

pH Organic matter (%) EC(mS/cm) Texture

7.83  0.1 2.47  0.2 0.76  0.02 67.12 19.75 13.13 31.97  0.4 149.13  4.8 112.12  1.1

CEC (meq/100 g) TN (mg/kg) P2O5 (mg/kg)

Fig. 1. Potential effects of chelates on plants, soil ecosystems, and heavy metals during phytoremediation.

chelate addition is not sufficiently high, phytoremediation is not feasible. Instead, contaminated plants can adversely affect ecosystem health through the food chain (Sung et al., 2001; Peralta-videa et al., 2009). Second, when phytoremediation removes heavy metals from soil but soil quality is degraded by the toxicity of persistent chelates, future use of the soil can be limited (Bucheli-Witscheli and Egli, 2001). Potential threats to the environment should therefore be considered before employing chelates for phytoremediation. The objectives of this study were: (1) to investigate chelate (EDTA, EDDS, and HAs) effects on soil microbial properties in soil contaminated with multiple metal species; (2) to identify chelate effects on plant growth and health using four herbaceous plants that are edible or produce biomass, and (3) to evaluate chelate effects on heavy metal uptake by plants.

Sand (%) Silt (%) Clay (%)

Loamy sand

water (with conductivity of 4 mS/cm) was added. Soil samples were collected after 1, 2, 4, and 8 days; dehydrogenase activity and total microbial numbers were analyzed. 2.1.2. Chelate effects on heavy metal accumulation and biomass of herbaceous plants Four herbaceous plants, Brassica juncea, Brassica campestris, Sorghum bicolor, and Helianthus annuus, were used to investigate chelate effects on heavy metal accumulation and biomass change. B. juncea and H. annuus have been used in phytoremediation but

2. Materials and methods 2.1. Experimental setup 2.1.1. Chelate effects on soil microbial properties Experimental soil was collected from a flower garden in Busan, Korea (35
80 300 N and 129
60 2500 E). The loamy sand soil was airdried, passed through a 2 mm sieve, and contaminated using a homogenizer with PbCl2 (Kanto, Japan), CuCl2 (Acros, Belgium), CdCl2 (Kanto, Japan), NiSO4 (Kanto, Japan), and ZnSO4 (Junsei, Japan). The level of contamination was determined on the basis of the soil contamination warning levels established for residential areas of Korea, namely 400 mg/kg for Pb, 500 mg/kg for Cu, 10 mg/ kg for Cd, 200 mg/kg for Ni, and 600 mg/kg for Zn. For purposes of the experiment, 100 g of soil were placed in a glass beaker, with 3 replicates, and stored for a 4-week aging period. Chelator levels were selected taking into account the results of other published research. Various concentration ranges of chelating agents have been applied in other studies: 0.13–15 mmol/kg of EDTA (Andra et al., 2009; Jiang et al., 2003), 0.5–10 mmol/kg of EDDS (Gr9cman et al., 2003; Luo et al., 2005), and 0.1–2% HAs (Halim et al., 2003; Park et al., 2013; Wang et al., 2010). In this study, 5 mmol/kg mixtures of EDTA (Sigma–Aldrich, USA) and EDDS (Sigma–Aldrich, USA), and 0.5% HAs were prepared with distilled water, and 25 mL of chelate-containing water were applied to assess chelate effects on heavy metals. To simulate the top application method under field conditions, chelates were sprayed onto topsoil once. The experiment was conducted over 8 days in a greenhouse with a daylight period of 16 h, light intensity of 3500  800 lux, humidity of 30–40%, and a temperature of 25–28
C. Contaminated soils without chelator (control), used for determining the effects of chelates on microbial properties, were also prepared, and the same amount of distilled

Fig. 2. Changes in dehydrogenase activity and microbial numbers in soils contaminated by heavy metals during the experiment. Values are means of three replicates. Bars represent standard deviation. Small letters (a, b, c) in the figures show significant differences with time for the same chelates, and capital letters (A, B, C) show significant differences between treatments at day 8, respectively, (p < 0.05).

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some parts of B. juncea and H. annuus are edible (January et al., 2008; Singh and Sinha, 2005) and B. campestris and S. biocolor can be used to produce biodiesel (Ahmad et al., 2012; Lal, 2005; Zhao et al., 2009). The same soil used for the experiments investigating change in microbial properties was used, but with different heavy metal concentrations at soil contamination warning levels established for the forests of Korea, namely 200 mg/kg for Pb, 150 mg/kg for Cu, 4 mg/kg for Cd, 100 mg/kg for Ni, and 300 mg/kg for Zn. For purposes of the experiment, 900 g of soil were placed in a glass beaker with 3 replicates and stored for a 4-week aging period. Stainless pots (14.5 cm height; 10.5 cm diameter) were filled with the soil; ten seeds of B. juncea and B. campestris and seven seeds of S. bicolor and H. annuus were sown, taking into account plant size. Twenty days after seeding, 50 mL of water containing 5 mmol/kg of EDTA and EDDS and 0.5% HAs were applied to the tops of the pots. Two kinds of control, with uncontaminated soil (UNC) and contaminated soil without chelate additions (control), were prepared for each plant species and the same amount of distilled water was applied to the controls. The experiment was conducted over 35 days in a greenhouse, with a daylight period of 16 h, light intensity of 3500  800 lux, humidity of 30–40%, and a temperature of 25–28
C. 2.2. Methods of analysis 2.2.1. Biophysicochemical soil properties Soil pH and EC were measured using an electrode method in a 1:1 (w/v) soil-water paste (Thomas, 1996). The organic matter content was measured using the loss-on-ignition method (Nelson and Sommers, 1996). Cation exchange capacity (CEC) was measured using the 1 N acetic acid replacement methods (NAAS, 1988). The fractions of sand, silt, and clay were determined using the pipette method and the soil was classified as loamy sand

according to the USDA textural classification. Total nitrogen was analyzed using a Kjedahl digestion and distillation system (Buchi, Switzerland). Available phosphorous was analyzed using the molybdenum blue method. Table 1 shows the physicochemical soil properties used in the experiment. Dehydrogenase activity was measured colorimetrically using the reduction of 2, 3, 5-triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) (Baligar et al., 1991). Soil microbial numbers were analyzed using PetrifilmTM (3 M, USA) after inoculation, with spreading on plates with one ml of diluted samples, and incubation at 37
C for 24 h. EC: electrical conductivity, CEC: cation exchange capacity, TN: total nitrogen Heavy metals in soil were extracted using the US EPA 3050B method with HNO3–H2O2 digestion (US EPA, 1996) and measured using an atomic absorption spectrometer (AAnalyst 800, PerkinElmer, USA). SRM 2711a, Montana II soil, and SRM 1570a trace elements in spinach (NIST, USA) as standard reference materials, were added and analyzed. Quantities of 96.1%, 97.4%, 97.7%, 98.1%, and 97.3% of Pb, Cu, Zn, Cd, and Ni, respectively, were recovered from SRM 2711a. Additionally, 98.2%, 99.1%, 95.4%, and 97.9% of Cu, Zn, Cd, and Ni, respectively, were recovered from SRM 1570a. Heavy metal concentrations in the soil used in the study were 14.87  5.1 mg/kg, 11.75  4.3 mg/kg, 49.19  1.2 mg/kg, 4.39  0.5 mg/kg, and 0.34  0.1 mg/kg for Pb, Cu, Zn, Ni, and Cd, respectively. 2.2.2. Effects on chlorophyll content, biomass and heavy metal accumulation in herbaceous plants Changes in the relative chlorophyll content were measured using a SPAD 502 chlorophyll meter (Konica Minolta, Japan). Dry biomass and heavy metal concentrations in plants were measured after 20 days. Plants were harvested, washed carefully with

Fig. 3. Root and shoot biomass in contaminated soil during experiments with (a) Brassica juncea, (b) Brassica campestris, (c) Sorghum bicolor, and (d) Helianthus annuus. UNC: uncontaminated soil. Values are means of three replicates except for UNC and control. Bars represent standard deviation. Small letters (a, b, c) in the figures show significant differences between treatments at p < 0.05.

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distilled water, separated into roots and shoots, dried at 80
C in an oven, ground using a mortar, and weighed for measurement of dry biomass. The heavy metals in the plants were then extracted using the US EPA 3050B method with HNO3–H2O2 digestion (US EPA, 1996) and measured using an atomic absorption spectrometer (AAnalyst 800, PerkinElmer, USA). To compare plant accumulations of heavy metals, a bioconcentration factor (BCF) of root and shoot for each treatment was calculated using the following equations;   C root (1) BCFr ¼ C soil   C shoot BCFs ¼ C soil

(2)

where Cshoot and Croot are heavy metal concentrations in shoots and roots (mg/kg dw), respectively. Csoil represents heavy metal concentrations in soil (mg/kg). 2.2.3. Statistical analysis To determine the effects of chelate type on soil microbial properties, heavy metal accumulations, and changes in plant chlorophyll content, analysis of variance (ANOVA) with Duncan multiple comparison tests was used to establish the presence of significant differences between treatments. Pearson correlation

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coefficients were also determined between factors related to plant health and metal concentrations in plants. Effects of chelates and plants on heavy metal concentrations in soils and plants were analyzed using a two-way ANOVA. All statistical tests were performed using SAS 9.1 (SAS Inc., USA) at the 95% significance level.

3. Results and discussion 3.1. Effects on soil microbial properties Soil dehydrogenase activity (DHA) increased over time except in EDTA-added soil; the latter had the lowest value of 28.51  4.55 mg-TPF/kg dry soil/day (Fig. 2a). The highest DHA of 69.08  6.21 mg-TPF/kg-dry soil/day was found in the EDDS-added soil on day 8 and EDDS could improve overall microbiological activity in soils contaminated with multiple species of heavy metals. Epelde et al. (2008) found that EDDS was rapidly dissipated and less toxic to the soil microbial community than EDTA. The authors also found that EDTA showed lower DHA (325.9  22.0 mg INTF/kg dry sol/h) than EDDS (441.9  69.9 mg INTF/kg dry sol/h) at 2500 mg/kg in Pb-contaminated soil. Although these results are hard to compare directly with our data, because experimental conditions (such as species, concentrations of heavy metals, and chemicals used for DHA analysis – iodonitrotetrazolium chloride,

Fig. 4. Effects of chelates on plant chlorophyll content in (a) Brassica juncea, (b) Brassica campestris, (c) Sorghum bicolor, and (d) Helianthus annuus. UNC: uncontaminated soil. Values are means of three replicates. Bars represent standard deviation. Small letters (a, b, c) in the figures show significant differences at p < 0.05.

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Table 2 Pearson correlation coefficients (r) for decrease in chlorophyll content (SPAD) and plant biomass, and concentration increases in roots and shoots. (%) Decrease

Concentration increase in root (%)

(%) Decrease

SPAD

Biomass

Cd

Cu

Pb

Zn

Ni

Cd

Cu

Pb

Zn

Ni

SPAD Biomass

1.000 0.741**

0.741** 1.000

0.647** 0.599*

0.720** 0.764**

0.200 0.329

0.554* 0.461

0.479 0.393

0.77** 0.819**

0.642** 0.562*

0.513* 0.369

0.571* 0.468

0.722** 0.722**

* **

Concentration increase in shoot (%)

(p < 0.05). (p < 0.01).

INT and 2,3,5-triphenyltetrazolium chloride, TTC) were different, these suggest that EDTA addition could have more harmful effects on overall microbial activity than EDDS. Kos and Leštan (2003) found increased substrate-induced respiration in soil with 5 mmol/ kg of EDDS concentration and showed that EDDS could help to increase overall microbial activity. DHA also increased in HA-added soil and its effect appeared to be slow compared to EDDS-added

soil. This was likely because EDDS was utilized more rapidly by microorganisms than HAs. The soil microbial number before the addition of chelating agents was 2.63  0.36  107 CFU/g. After the addition of chelators, the microbial number decreased to 1.93  0.32  107 CFU/g in EDTA-added soil, with values of 2.54  0.55  107 CFU/g and 2.53  0.33  107 CFU/g in EDDS- and HA-added soils, respectively

Fig. 5. Bioconcentration factors of Cd, Cu, Pb, Zn, and Ni in the roots of four plants. CONT: control. Values are means of three replicates except for control. Bars represent standard deviation. Small letters (a, b, c) in the figures show significant differences between plant treatments at p < 0.05.

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(Fig. 2b). The results showed that EDTA effects on soil microbial number were more rapid than those of other chelating agents. Although microbial numbers increased after day 2 in all treatments, EDTA-added soil had the lowest values of all treatments on day 8 (Fig. 2b). EDDS- and HA-added soils had microbial number values of 7.33  1.27  107 (CFU/g) and 8.55  0.30  107 (CFU/g), respectively, with these values being 1.93 and 2.24 times higher than those of EDTA-added soil. The lower level of microbial activity in EDTA-added soil than in the control (5.41  0.81 107) suggests that EDTA can restrain microbial activities in soils contaminated with heavy metals. The changes in microbial number with time, like those for DHA, suggest that EDDS can be easily utilized by microorganisms and that HAs have a subsequent,

391

favorable influence on microbial number and activity. Soil microbial properties can be used to assess soil quality as biological indicators (Gómez-Sagasti et al., 2012). Decreased microbial properties suggest that soil quality is degraded after the addition of chelating agents, even if more heavy metals are removed by plants. Because EDTA is known to be persistent in soil, microbial activity is likely restrained for longer than when using other chelates. However, soil microbial processes can be affected by chelates and the addition of chelating agents can also lead to increased heavy metal concentrations in soil solutions. A more efficient chelate may have higher toxicity (Mühlbachova, 2011). Therefore, long term monitoring is needed following the use of chelating agents to assess soil quality changes.

Fig. 6. Bioconcentration factors of Cd, Cu, Pb, Zn, and Ni in shoots of four plants. CONT: control. Values are means of three replicates except for control. Bars represent standard deviation. Small letters (a, b, c) in the figures show significant differences between plant treatments at p < 0.05.

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3.2. Plant biomass and leaf chlorophyll content Fig. 3 shows the root and shoot biomass and the root-to-shoot ratios of the plants used. H. annuus produced the highest total biomass (165.0–205.7 mg) and S. bicolor had the highest root-toshoot ratios (0.39–0.62) of plants used in the study. Effects of heavy metals on plant growth differed with species. B. juncea and S. bicolor biomass decreased by 40.6% and 47.3%, respectively, due to heavy metal contamination when compared to plant biomass growth in uncontaminated conditions. In the case of S. bicolor, roots were more severely affected by heavy metals than shoots. However, B. campestris biomass increased by 24.8% even with heavy metal presence, and this species exhibited metal tolerance. H. annuus was likewise not significantly affected by heavy metal contamination (p < 0.05). Root and shoot biomass were also influenced following the addition of chelating agents, except in the case of H. annuus (p < 0.05). HAs produced positive effects on both root and shoot growth in soils contaminated with heavy metals, especially in B. juncea and B. campestris. Angin et al. (2008) reported that HAs addition increased Pb availability and enhanced Pb accumulation without decreasing the yield of Vetiveria zizanioides; this species had shown decreasing yields in soils contaminated with high levels of Pb and in the absence of HAs. HAs can facilitate plant growth by providing nutrients, enhancing fertility and physicochemical properties of soil (Ibrahim and Goh, 2004; Sung et al., 2013), and making soil conditions favorable for plant growth. A possible enhancing factor is that plants in HA-added soil have higher plant biomass. In contrast to HAs, EDTA produced negative effects on plant biomass. EDTA addition led to decreased plant biomass in B. campestris, B. juncea, and S. bicolor. However, EDDS addition did not show significant biomass reduction in comparison to the controls. In contrast to the results of this study, other research suggested that EDDS could be more harmful to some plants than EDTA. EDDS reduced biomass of beans, corn, and cardoon plants to a greater extent than EDTA (Epelde et al., 2008; Luo et al., 2005). EDDS was also reported to be more toxic to Nicotiana tabacum than EDTA (Evangelou et al., 2007b). Chelate effects on plant growth can differ with heavy metal concentrations in soil and can be more significant when heavy metal concentrations are low. When heavy metal concentrations are high, plant growth is affected more by heavy metals than by chelates (Jiang et al., 2003). Plant growth could be affected by chelate toxicity and by toxicity from increased heavy metal concentrations in soil solution and in the plant. As plants accumulate more heavy metals, photosynthesis and biomass production could be reduced (Clijsters and Assche, 1985). Investigation of chlorophyll content may indicate whether

photosynthesis is affected by chelates or by heavy metal accumulation. Heavy metals, as well as chelates, affected plant chlorophyll content in this study, with all leaves affected except for those of H. annuus (Fig. 4). H. annuus was not affected by heavy metal contamination and decreased only in EDDS-added soil. Murillo et al. (1999) found no decrease of aboveground biomass of H. annuus grown in soil contaminated with Cd, Cu, Mn, Pb, Sb, Tl, and Zn. In general, plants have cellular mechanisms for metal detoxification (Hall, 2002; Schützendübel and Polle, 2002). They can store heavy metals in less harmful parts such as cell walls and vacuoles and have protection and repair system for plasma membranes. The results suggest that H. annuus could have such efficient heavy metal detoxification mechanisms or that it retains more heavy metal in roots than in shoots. Chlorophyll content decreased noticeably after EDTA addition in the case of B. juncea. Effects of EDDS on plant leaves varied among plants, with a positive effect on B. campestris but a negative effect on H. annuus. Chlorophyll content in HA-added soil suggested that HAs alleviated toxic effects of heavy metals on B. juncea and B. campestris leaves. The decrease in chlorophyll content may result in decrease in plant biomass. Correlation analysis showed a 0.741 correlation coefficient (r) between percentage decreases of chlorophyll content and plant biomass at p < 0.01. Decreased chlorophyll content was more strongly related to increased metal concentrations in shoots rather than roots (Table 2). Biomass decrease showed fewer significant correlations with plant concentration increases than with chlorophyll content. The results suggest that increased metal concentrations in shoots had an effect on the decrease in chlorophyll content in plants, consequently leading to reduced plant biomass. 3.3. Heavy metal accumulation and distribution within plants The bioconcentration factors of roots (BCFr) and shoots (BCFs) were different for each metal and plant species and varied with different chelate additions (Figs. 5 and 6). All chelates, except for HAs, increased the BCFr of all metals and plants. However, HAs decreased the BCFr of B. juncea for Cd, Cu, Pb, and Zn and had the least significant effects of the chelates used in the study. EDTA was more influential for increases of BCFr in all plants except H. annuus. Other studies suggested that EDDS was more efficient for Cu uptake by H. annuus than EDTA (Meer et al., 2005) but similar BCFr values for Cu were observed with both EDTA and EDDS in this study. The BCFr value was highest for Cd but lowest for Pb. The BCFr for Cd increased as follows: H. annuus < S. bicolor < B. campestris < B. juncea. BCFs were lower than BCFr because heavy metal concentrations in roots were higher than those in shoots in all plants. EDTA and

Table 3 Results of a two-way ANOVA showing significance of the effect of plants and chelates in chelator-addition phytoremediation. Factors

Treatment

DF

Zn

Ni

Concentration in soil

Plant Chelate Plant  Chelate

3 3 9

0.87 1.94 0.62

1.18 5.33** 1.41

0.02 33.74** 0.50

2.36*** 44.85** 2.97*

0.25 12.30** 0.33

Root bioconcentration factor (BCFr)

Plant Chelate Plant  Chelate

3 3 9

28.95** 15.97** 4.36**

8.70** 8.60** 3.39**

15.61** 37.83** 7.15**

26.38** 26.29** 6.73**

14.19** 14.21** 2.87*

Shoot bioconcentration factor (BCFs)

Plant Chelate Plant  Chelate

3 3 9

25.28** 14.23** 5.95**

4.10* 3.64* 1.93***

55.37** 138.04** 34.34**

8.45** 22.54** 4.33**

19.90** 14.96** 4.14**

F-values Cd

* **

(p < 0.05). (p < 0.01). (p < 0.1).

***

Cu

Pb

J. Lee, K. Sung / Ecological Engineering 73 (2014) 386–394

EDDS additions increased the BCFs of all plants and metals except in the case of Cd for H. annuus. However, HAs reduced the BCFs of B. juncea (Cd and Pb), and B. campestris and H. annuus (Cd). EDTA was most effective for increasing BCFr as well as BCFs of all plants (Fig. 6c). Stimulated translocation from roots to shoots in B. juncea was observed with EDTA addition (Jiang et al., 2003). The results showed that EDDS addition can increase metal uptake but that its effects are restricted to some plant and metal species. Therefore, plant selection can be a more important factor than use of EDDS. The effects of HAs on heavy metal uptake could vary due to their complex structure (Shahid et al., 2012). Although the effects of HAs on heavy metal uptake were lowest amongst the chelates investigated in this study, HAs application can be more effective when soil has an elevated toxicity level due to combined pollution with metals and organic chemicals or in long duration experiments, because soil with HAs can increase the degradation of organic contaminants, reduce toxicity, and chelate more metals than soil without HAs. As observed from the effects of chelates on microbial properties, effects of HAs appeared slowly and longer exposure may increase heavy metal accumulation in plants. Increased BCFr and BCFs for Cd, Cu, Pb, and Ni in B. campestris and H. annuus were observed during the 60-day experiment in which the soil was also contaminated with 2000 mg/kg of TPHs (Park et al., 2013). The effects of plant species on metal concentrations in soil were only different in the case of Zn (p < 0.1), while the effects of chelate type differed for Cu, Pb, Zn, and Ni concentrations in soil (p < 0.01) (Table 3). However, bioconcentration factors of plant roots and shoots for all metals were affected by all plant species and chelates. The results suggest that chelate type can be more influential in metal remediation efficiency than plant selection. 4. Conclusion Chelating agents affected metal uptake, distribution in plants, and bioconcentration factors, but differed with metals, plant species, and chelates. Plant growth could be affected by chelate toxicity and by toxicity from increased heavy metal concentrations in soil solution and in the plant. Plant biomass was strongly related to decreased chlorophyll content caused by increased metal concentrations in plant shoots. EDTA was most efficient for phytoremediation of soil contaminated with heavy metals, but its adverse effects on plants and soils cannot be neglected, and thus plant management and soil quality improvement may be needed during its use. Although EDDS addition can increase metal uptake, its effects are restricted to some plant and metal species. Therefore, plant selection can be a more important factor when using EDDS in soil contaminated with multiple metal species. The effects of HAs on heavy metal uptake increase were the least significant in this study but the effects on plants and soil were favorable and showed increased soil quality and plant growth. If heavy metal concentrations are not relatively elevated and site management is needed for a longer period of time, use of HAs may be a good alternative to other chelates. Acknowledgement “This work was supported by the Pukyong National University Research Abroad Fund in 2012 (PS-2012-0914)”. References Ahmad, M., Sadia, H., Zafar, M., Sultana, S., Khan, M.A., Khan, Z., 2012. The production and quality assessment of mustard oil biodiesel a cultivated potential oil seed crop. Energy Sources Part A 34, 1480–1490.

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