Chelator complexes enhanced Amaranthus hypochondriacus L. phytoremediation efficiency in Cd-contaminated soils

Chemosphere 237 (2019) 124480

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Chelator complexes enhanced Amaranthus hypochondriacus L. phytoremediation efficiency in Cd-contaminated soils Kai Wang a, 1, Yonghong Liu b, 1, Zhengguo Song c, *, Di Wang a, Weiwen Qiu d a

Agro-Environmental Protection Institute, Ministry of Agriculture of China, Tianjin, 300191, China College of Science, Huazhong Agricultural University, Wuhan, 430070, China c Department of Civil and Environmental Engineering, Shantou University, Shantou, 515063, China d The New Zealand Institute for Plant and Food Research Limited, Private Bag, 4704, Christchurch, 8140, New Zealand b

h i g h l i g h t s
GLDA and NTA combination can enhance the phytoextraction efficiency of Amaranthus hypochondriacus L.
The addition of chelating agent can effectively promote Cd bioavailability in soil.
Most chelating agents can increase Cd accumulation by Amaranthus hypochondriacus L.
3 mM GLDA þ2 mM NTA combination has the highest amount of extracted Cd in all treatments.
GLDA and NTA combined treatment significantly boost soil enzyme activity.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 May 2019 Received in revised form 18 July 2019 Accepted 28 July 2019 Available online 29 July 2019

The use of degradable chelating agent to enhance phytoextraction is a promising and low-cost method for remediation of heavy metals-polluted soil. However, very limited information is available regarding the effect of chelating agent combinations on plant growth and its capacity to extract metals. In this study, a pot experiment was conducted to evaluate the applicability of [N, N]-bis glutamic acid (GLDA), nitrilotriacetic acid (NTA), [S, S]- ethylenediamine disuccinic acid (EDDS), and citric acid (CA) alone and in combination to enhance the phytoextraction efficiency of amaranth (Amaranthus hypochondriacus L.) in two Cd-contaminated agricultural soils (S1 soil 2.12 mg/kg and S2 soil 2.89 mg/kg; the environmental standard value of Cd in agricultural soils in China is lower than 0.8 mg/kg). The results showed that, except for EDDS, other treatments had no obvious effect on plant biomass, and even promoted biomass increase to reach 1.06 (S1), 2.07 (S2) g/pot. The increase in total Cd extraction amount by 5 mM of single chelators GLDA and NTA reached 3.87 and 2.81 (S1), and 3.28 and 2.50 (S2) times that of the control group, respectively. For complexed chelating agents, G-N (GLDA þ NTA) combinations (GLDA ¼ 3 mM, NTA ¼ 2 mM) extracted the highest amount of Cd compared with other treatments, reaching 0.36 and 0.52 mg/pot (4.50 and 3.71 times that of the control group), respectively. The order of extraction amount was G-N > GLDA > NTA > G-E (GLDA þ EDDS) > G-C (GLDA þ CA) > CA (5 mM total Cd concentration). Moreover, soil enzyme activity of G-N treatment increased significantly compared to that of the control group, indicating the great application potential of a composite chelating agent relative to a single chelating agent. Therefore, degradable chelators, especially the G-N combination, can effectively increase the available Cd content and greatly enhance the ability of plants to absorb and transport Cd in soils. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: T Cutright Keywords: Degradable chelator Phytoextraction Amaranthus hypochondriacus L. Bioavailable Cd Soil enzyme activity

1. Introduction

* Corresponding author. E-mail address: [email protected] (Z. Song). 1 Kai Wang and Yonghong Liu contributed equally to the work and should be regarded as co-first authors. https://doi.org/10.1016/j.chemosphere.2019.124480 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

Heavy metal pollution in agricultural soils is one of the most concerning environmental issues in modern times; it is caused by both geological and anthropogenic activities including sedimentation, magmation, farming, and especially, industrial production (Wang et al., 2018a, 2018b, 2018c). In China, the total area of

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polluted land has reached 2.88  106 ha and grows at a rate of 46,700 ha annually (Quinton and Catt, 2007; Li et al., 2014). Among heavy metals, high concentration of cadmium (Cd) is recognized as a significant environmental pollutant for living organisms as it is highly toxic and can persist in soil for a long time under natural conditions (Li et al., 2014; Hu et al., 2017). Hence, soil heavy metal decontamination is becoming a hot topic all over the world. Phytoremediation is a low cost, green, and aesthetically appealing technique suitable for low to medium level heavy metals-polluted soils that does not damage the soil structure or environment (Zaier et al., 2010; Luo et al., 2017). Recent studies have evaluated many accumulator plant species such as Chinese cabbage (Wang et al., 2018b), Brassica napus (Rossi et al., 2019),
ne et al., 2009), Pteris vittata (Zeng et al., Indian mustard (Duque 2019), Helianthus annuus (Farid et al., 2018a), Solanum nigrum (Luo et al., 2011), Brassica campestris (Yang et al., 2011), and Sedum alfredii (Sun et al., 2009) for their uses in the phytoremediation of contaminated soil. Selection of a species suitable for the specific soil conditions being treated is the most critical decision in determining rska Socha et al., 2017). Amaranthus hypoproject success (Nadgo chondriacus L. was selected to remediate Cd-contaminated soil, and it obtained preferable result due to its strong accumulation ability to target metals and high economic benefits (Ding et al., 2013). Conventional phytoremediation techniques have limited practical application (Wang et al., 2019). More importantly, heavy metal extraction by plants is a relatively time-consuming process, especially in the case of heavy metals with low availability (Evangelou et al., 2007;Wang et al., 2018a, 2018b, 2018c). Previous studies have shown that the use of organic chelating agents to enhance the extraction ability of hyperaccumulators is more beneficial for the extraction of heavy metals than traditional phytoremediation methods, also known as induced phytoextraction or chelatingassisted phytoextraction (Clabeaux et al., 2013). Aminopolycarboxylic acids (APCAs) chelating agents such as ethylenediaminetetraacetic acid (EDTA), [S, S]- ethylenediamine disuccinic acid (EDDS), nitrilotriacetic acid (NTA), and [N, N]-bis glutamic acid (GLDA), as well as low molecular weight organic acids (LMWOAs) chelating agents, such as citric acid (CA), oxalic acid (OA), and tartaric acid (TA) have shown great performance in mobilizing soil heavy metals due to their multi-ligand structure, which tends to form stable compounds with metals (Wang et al., 2010; Guo et al., 2019). Some chelating agents with high application rates, such as EDTA, have strong chelating abilities on copper (Cu), zinc (Zn), and cadmium (Cd) (Jiang et al., 2019). However, due to their structural characteristics, the chelating agents can persist in the soil for a long time, causing secondary pollution and safety hazards (Zaier et al., 2010). Therefore, finding a new environmentally friendly chelating agent that can be combined with plant extraction is key to solving this problem (Kos and Lestan, 2004a; Wei et al., 2012). GLDA is a generation of green chelating agents with high heavy metal extraction efficiency (Wu et al., 2015). Research by Wang et al. (2016) showed that the removal efficiency of cadmium, lead, and zinc reached 70.62, 74.45, and 34.43%, respectively, at a 75 mM concentration, a pH of 4.0, and a contact time of 60 min. The biodegradation tests showed that more than 90% of GLDA degraded within 20 days. As a degradable substitute for EDTA, EDDS is widely used in plant extraction experiments (Meers et al., 2005). NTA is also a highly effective chelating agent with a half-life degradation period of approximately 7 days (Hu et al., 2017). In addition to the above-mentioned APCAs chelating agents, CA, as a representative LMWOAs, has also received more attention. The soil heavy metal ion mobilization ability of single chelating agents is often limited (Guo et al., 2019). Meers et al. (2005) showed that the chelation effect of EDDS on Cd extraction in agricultural soil was not as good as that on Cu and Zn. Therefore,

chelator complexes can theoretically increase plant heavy metal uptake from polluted soil, compared to single chelating agents. In the process of plant extraction, due to the limitations of the structure and the properties of the chelating agent, it is easy to cause heavy metal dissolution and secondary pollution (Parisien et al., 2016). Therefore, to evaluate the feasibility of an extraction method, not only the amount of extracted heavy metal and extraction efficiency must be evaluated, but also heavy metal mobility after extraction (Guo et al., 2018). The modified BCR method categorizes soil heavy metals into different forms (Rauret et al., 1999). According to the morphological changes in heavy metals before and after treatment, the effect of a chelator on heavy metal mobility can be more intuitively reflected (Cappuyns et al., 2007). The aim of this study were to: 1) compare the efficiency of different degradable chelators and their combinations in enhancing Cd uptake using amaranth grain (Amaranthus hypochondriacus L.) in two different level of Cd-contaminated soils; 2) investigate phytoremediation indicators including plant biomass, translocation factor, bioaccumulation factor, and total Cd removal; 3) evaluate the modified BCR sequential extraction method feasibility based on the morphological distribution of Cd and soil enzyme activities before and after treatment using a pot experiment with two different level of Cd-contaminated soils. 2. Materials and methods 2.1. Soil samples and chelating agents Two types of Cd-contaminated agricultural soils were collected from surface soil (0e20 cm) in two farms in Xinxiang city (Henan Province, China). The collected soil was air dried, sieved through a 3 mm sieve, then pulverized to a fine powder. The sieved soil was mixed evenly once a week for three weeks. All soil samples were analyzed in triplicate for total heavy metal content and soil physical and chemical properties (Table 1). EDDS (analytical reagent grade) was purchased from Ruitaibio Chemical Reagent Co., Ltd. GLDA (molecular weight ¼ 351.13 g/mol, solid content  45%, and density ¼ 1.35 g/cm3) was developed by Cool Chemistry Co., Ltd. CA and NTA (analytical reagent grade) were purchased from Macklin Chemical Reagent Co., Ltd. Soil enzyme kits were purchased from Suzhou Keming Biological Co., Ltd. 2.2. Plant material The seeds of Amaranthus hypochondriacus L. were obtained from the Chinese Academy of Agricultural Sciences. 2.3. Pot experiment design Pot experiments were performed in triplicate. A series of 25 cm high and 15 cm diameter pots containing air-dried Cd-contaminated soil (2.0 kg) were prepared, and the soil in each pot was equilibrated for two weeks. The soil moisture content was adjusted to 75% of its water holding capacity by weighing the pot weekly and adding deionized water to compensate for weight loss by evaporation. Nitrogen (CO(NH2)2), phosphate (Ca(H2PO4)2), and potassium (KCl) fertilizers (0.049, 0.150, and 0.038% of soil weight, respectively) were added to each pot. The pot bottoms were sealed to eliminate mobilized Cd leaching. The seeds were sterilized in H2O2 solution (2%, v/v) for 15 min, washed repeatedly with tap water, soaked in deionized water overnight, then germinated in a seedling tray containing vermiculite. Seedlings were transplanted after one week, with one surviving seedling per pot. The plants were grown in the greenhouse for 3 months, then the chelating

K. Wang et al. / Chemosphere 237 (2019) 124480

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Table 1 Basic physical and chemical properties and heavy metals content of the two soils. parameters

Low Cd-contaminated soil（S1）

High Cd- contaminated soil（S2）

Soil texture

Brown sandy soil

Brown clay soil

CEC(cmol/kg) pH Organic matter content％ Available N（mg/kg） Available P（mg/kg） Available K（mg/kg） Total Cd（mg/kg） Total Pb（mg/kg） Total Cu（mg/kg） Total Zn（mg/kg）

8.8 8.5 6.4 75.0 84.8 169.0 2.12 15.67 13.45 76.83

20.1 8.3 10.6 92.8 87.2 238.2 2.89 17.36 15.47 63.14

agent (The single chelating agent treatment included two concentrations of 3 mM and 5 mM, and the total concentration of the complex chelating agent was 5 mM) was uniformly added two weeks before plants harvest. The treatment plan for the experimental group is shown in Table S1. The control groups were treated with equal volume of deionized water instead of chelating agent solution. At harvest, the plants were separated into aerial and underground parts after removal from the soil, and were washed three times each with tap water and distilled water, respectively. The plants were dried in an oven at 75  C for 48 h, and the plant dry weight (DW) was recorded after a constant weight was reached. At the same time, a uniformly mixed soil sample from each pot was collected. The air-dried soil and the treated crop samples were separately ground into powders and analyzed. 2.4. Plant heavy metal analysis Plant sample treatment was carried out using the HNO3/HClO4 digestion method. The plant samples (0.1 g) were digested, then the digested solution was washed in a 25 mL flask and made up to the volume of 25 mL with deionized water. Plant Cd concentration was determined using ICP-MS.

morphological distribution of Cd before and after the addition of chelating agents to the soils (Rauret et al., 1999). The specific steps and morphology definitions are shown in Table 2. The sequential extraction experiment was performed using a 0.5 g soil sample. After each extraction step, the extracted suspension was centrifuged at a speed of 4000 g for 20 min, and the residual supernatant was filtered through a 0.45 mm membrane filter. The residue is washed with deionized water three times before the next extraction step. The obtained filtrate was transferred to a 50 mL centrifuge tube stored at 4  C for further analysis. 2.7. Soil enzyme activity analysis Catalase, invertase, and urease activity were evaluated in this study. Catalase was measured using the triphenyl tetrazolium chloride (TTC) reduction colorimetric method (Watanabe et al., 1993). The activity unit was expressed in terms of the amount of TTF produced. The 3,5-dinitrosalicylic acid colorimetric method was employed to evaluate the activity of acid invertase; the activity unit was expressed in terms of the amount of glucose produced (Goncalves et al., 2010). Urease was measured via the sodium phenolate-sodium hypochlorite colorimetric method; the activity unit was represented in terms of the amount of NH3eN produced (Willis et al., 1996). All tests were repeated three times.

2.5. Calculation of potentially toxic metal distribution The biological concentration factor (BCF) gives an index of the ability of a plant to absorb toxic metals from the soil into the shoot and root system. It was calculated as follows:

BCF ¼

Cp Cs

where Cp (mg/kg) represented the Cd concentration in dry shoots at harvest and Cs was the initial soil Cd concentration. The translocation factor (TF) gives an index of the ability of a plant to translocate the metals from roots to shoots. This parameter was calculated as:

TF ¼

Ca Cr

where Ca (mg/kg) and Cr (mg/kg) represented the metal concentration in plant shoots and roots, respectively. 2.6. Morphological Cd analysis To evaluate the effect of various chelating agents on the concentration of bioavailable Cd in the soil, the modified BCR sequential extraction method was applied to measure the

2.8. Statistical analysis Statistical analysis was performed using the SPSS statistical package (version 10.0). All the values reported in this work were means of at least three independent replications. Data were tested at a significance level of P < 0.05. 3. Results and discussion 3.1. Experimental soil characteristics Table 1 shows the basic physical and chemical properties of the two soils (S1: brown sandy, S2:brown clay) and the content of four heavy metals. Through the comparison of S1 and S2 soils, it was found that S2 had higher cation exchange capacity (CEC), organic matter (OM), available N, available P and available K values than S1, which were more conducive to plant growth. The Cd content of the two soils was 2.12 and 2.89 mg/kg, respectively (dry soil basis), and clearly did not meet the agricultural use requirements for agricultural available soil environmental quality standards in China (the Cd concentration in soils that can be used for agricultural production is fixed as follows: for rice field 0.3 (pH  5.5), 0.4 (5.5 < pH  6.5), 0.6 (6.5 < pH  7.5), 0.8 (pH > 7.5) mg/kg, other land use types 0.3 (pH  5.5), 0.4 (5.5 < pH  6.5), 0.5

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Table 2 Chemical reagents and analytical conditions for the modified BCR sequential extraction method.

Step 1 Step 2 Step 3 Step 4 Step 5

Fraction

Operation

Water-soluble state Acid-soluble state Reducible state

20 mL of deionized water was added and shaken for 16 h to obtain the supernatant. 20 mL of HOAc (0.11 M, pH 2.8) was added and shaken for 16 h to obtain the supernatant. (The soil samples need to be washed with deionized water after each step) 20 mL NH2OH$HCl (0.5 M, pH 2) was added and shaken for 16 h to obtain the supernatant.

Oxidizable state 10 mL H2O2 was added to the sampes and the samples were heated at 85  C for 1 h; 5 mL H2O2 (30%, pH 2) was added again and the samples were heated at 85  C for 1 h，then NH4OAc (1 M, pH 2) was added and shaken for 16 h to obtain the supernatant. Residual state Digestion with HCleHNO3eHFeHClO4 for 7 h.

The other chemical reagents used in this experiment are of analytical grade.

(6.5 < pH  7.5), 0.6 (pH > 7.5) mg/kg). High Cd accumulation in soil S1 and S2 may have been caused by the excessive use of heavy metal-containing pesticides and fertilizers, as well as the impact of  et al., 2006). Cd-related mining (Mico 3.2. Plant growth and biomass production There were no obvious signs of toxicity in the control or treatment groups, such as yellow leaves or dysplasia. Amaranth shoot and root biomass production under different treatments are shown in Table 3. The biomass of shoots and roots were between 8.6711.61, 2.25e2.64 (S1) and 10.64e13.67, 2.73e3.42 (S2) g/pot, respectively. The addition of GLDA and NTA promoted amaranth biomass production and was more obvious with increased concentration. This is mainly because of the rapid degradation characteristics of GLDA and NTA, which only persist in soil for a short time and have a limited impact on the soil environment. Moreover, NH3, which is required for plant growth, is their main by-product. These results were corroborated by those of Guo et al. (2018). The addition of EDDS in both soils showed a significant inhibition in the biomass of amaranth grain as the concentration increased; these results are supported by those of Hseu et al. (2013). The heavy metal-EDDS complex can enter the roots via the Casparian strip, where it is quickly transported to the shoots; the toxic effects of EDDS may damage the physiological root barriers and cause a decrease in plant biomass (Wang et al., 2009). Luo et al. (2006) found that in soil with the concentration of 0.45 mg/kg Cd, 5.0 mmol/kg EDDS reduced the stem dry weight of maize and

Table 3 Dry matter weight (g/pot) of the root and shoot of amaranth grain of different treatments in the two soils. Treatment

CK GLDA（a） GLDA（b） NTA（a） NTA（b） CA（a） CA（b） EDDS（a） EDDS（b） GLDA þ NTA GLDA þ CA GLDA þ EDDS

Low-contaminated soil （S1）

High- contaminated soil（S2）

shoot (g/pot)

root (g/pot)

shoot (g/pot)

root (g/pot)

10.81 ± 0.09c 11.35 ± 0.03b 11.61 ± 0.43b 10.73 ± 0.05c 11.29 ± 0.05b 9.85 ± 0.07d 9.91 ± 0.07d 8.91 ± 0.02f 8.67 ± 0.16f 11.87 ± 0.09a 10.02 ± 0.16d 9.52 ± 0.04e

2.44 ± 0.03c 2.56 ± 0.06 ab 2.64 ± 0.05a 2.58 ± 0.07 ab 2.60 ± 0.06 ab 2.51 ± 0.05b 2.56 ± 0.05 ab 2.35 ± 0.03cd 2.25 ± 0.04d 2.63 ± 0.06a 2.55 ± 0.08 ab 2.27 ± 0.08d

11.98 ± 0.12 h 12.25 ± 0.10f 13.67 ± 0.06b 11.78 ± 0.05i 13.25 ± 0.05c 12.02 ± 0.12gh 12.68 ± 0.06e 12.15 ± 0.12 fg 10.64 ± 0.18k 14.05 ± 0.11a 12.85 ± 0.06d 10.98 ± 0.13j

3.09 ± 0.06bc 3.26 ± 0.06 ab 3.35 ± 0.05 ab 3.25 ± 0.04 ab 3.42 ± 0.07a 3.14 ± 0.04abc 3.21 ± 0.54cd 3.03 ± 0.06bcd 2.73 ± 0.08d 3.42 ± 0.04a 3.32 ± 0.06 ab 2.75 ± 0.14d

a: at the concentration of 3 mM, b: at the concentration of 5 mM. The total concentration of the complex chelating agent is 5 mM, and the ratio is 3:2. Numerical values followed by different letters in the same column were significantly different at P < 0.05. The same as follows.

soybean to 52% and 61% of the control plants, respectively. CA, an organic acid secreted by plant root system, has a limited effect on the growth of plant biomass at a low concentration, which is consistent with the results of our study. At a concentration of 3 mM, CA has no significant effect on plant growth, while at a concentration of 5 mM, it only showed a slight promoting effect. The G-N (GLDA þ NTA) combination treatment enhanced biomass production and reached 1.06 (shoot) and 0.19 (root) g/pot in S1, and 2.07 (shoot) and 0.56 (root) g/pot in S2, respectively. The G-E combination (GLDA þ EDDS) reduced biomass production down to 0.79 (shoot) and 0.14 (root) g/pot in S1, and 1.01 (shoot) and 0.34 (root) g/pot in S2, respectively. Structurally, although both GLDA and EDDS are degradable chelating agents, the complex formed by the combination of GLDA and EDDS is non-degradable. When added to the soil, it affects the plant growth environment and decreases plant biomass. The G-C (GLDA þ CA) combination increased biomass production compared to the single CA treatment (5 mM), and inhibited biomass production compared to the single GLDA (5 mM) treatment. This indicated that GLDA played the primary role in terms of increasing the biomass in the G-C combination. Plant growth in soils with chelating agents may be affected by a variety of factors, including soil fertility levels, soil structure, and the presence of toxic metal chelate complexes (Begum et al., 2012). Studies have shown that the addition of most chelating agents to the soil will cause an increase the concentration of heavy metal ions in the soil solution that will inhibit plant growth and reduce biomass (Quartacci et al., 2007). According to Quartacci et al. (2007), the dry weight of Brassica juncea was 33.5% lower than that of the control group after adding 5 mM NTA to soil contaminated with the rate of 40 mg/kg Cd. Given the fact that high concentrations of EDDS can be toxic to plant roots and affect plant nutrient absorption, except for the strong inhibitory effect of EDDS treatment on plant biomass, most of the treatments in this experiment showed plant growth promotion (Hseu et al., 2013). This is mainly because the content of total heavy metals in agricultural soil is generally lower than that in industrial sludge, and heavy metal and nutrient elements in agricultural soil can be more tightly  ska, bound to the soil surface through the aging effect (Kołodyn 2010). 3.3. Cd uptake by plants and total Cd removal in soils Different chelating agents and chelating agent combinations resulted in significantly different Cd concentrations in the shoots and roots of amaranth grain, as shown in Fig. 1. In this experiment, the Cd concentration in all treatment groups was much higher than the general toxicity level of normal growing plants. Compared to the control, Cd concentration in the shoot and root increased after the addition of a chelating agent to the soil. This is mainly because

K. Wang et al. / Chemosphere 237 (2019) 124480

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Fig. 1. Cd concentration in shoots and roots.

chelating agents contain negatively charged hydroxyl or carboxyl groups, and can form stable chelating compounds with positively charged Cd that is conducive to the absorption and accumulation of Cd by plants, further increasing the concentration of Cd in shoots and roots of plants (Farid et al., 2018b). In all treatments in the two soils, the Cd concentrations of shoots and roots were between 10.31-28.64 and 13.52e23.56 mg/ kg (S1), and 13.85e33.52 and 10.56e24.42 mg/kg (S2), respectively. The maximum concentrations appeared in the G-N treatment, which were 4.10 and 3.92 (S1), and 3.42 and 2.60 (S2) fold higher than that of the control. The lowest concentrations appeared in the CA treatment, which were 1.43 and 2.25 (S1), and 1.39 and 1.25 (S2) fold higher than that of the control, respectively. This is mainly related to the chelating capacity of chelating agents and the stability of chelating agents and heavy metal complexes. In addition to the G-C combination, the chelator combinations were more advantageous in Cd accumulation in the shoots and roots of plants than those of a single chelating agent, which contributes to the addictive effect between two chelating agents (Farid et al., 2017a). Moreover, the Cd concentration in shoots was slightly higher than that in roots, which is in line with the findings of Hseu et al. (2013). As heavy metal phytoextraction is based on defense and resistance mechanisms, on one hand the roots inhibit heavy metal absorption while promoting heavy metal transport, on the

other hand these mechanisms reduce toxicity. Generally, in multileaf plants, it is more conducive to Cd transport owing to the influence of transpiration (Wei et al., 2012b). The amount of total Cd extracted from the two soils is shown in Fig. 2. In all treatments, after the addition of the chelating agent, the amount of extracted total Cd increased to different degrees compared to the control group, and the highest (G-N) reached 0.36 (S1) and 0.52 (S2) mg, which were 4.5 and 3.71 times that of the control group, respectively. The order of total Cd extraction amount was G-N > GLDA > NTA > G-E > G-C > CA (5 mM total concentration). Compared with the single CA treatment, the G-C combination had a limited improvement on the amount of total Cd removal. Although the biomass of shoots and roots was inhibited to varying degrees, the addition of EDDS increased total Cd removal by 0.17 (S1) and 0.23 mg (S2), compared to that of the control group. This indicated that there was no necessary connection between plant biomass and total Cd removal for amaranth grain, and that the addition of a chelating agent can cause plant biomass deficiency (Evangelou et al., 2007). In the single NTA and EDDS treatments, the total Cd removal decreased with increased chelating agent concentration, which was consistent with the result of Hseu et al. (2013). The main reason may be that some chelating agents, such as EDDS and NTA, accumulate easily around plant roots and even precipitate due to their inherent dispersibility in the soil, affecting

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K. Wang et al. / Chemosphere 237 (2019) 124480

chelating agent combinations, the G-N combination had a stronger effect on Cd removal, and the BCF value and the TF values reached 13.36 and 1.26 (S1), and 10.90 and 1.35 (S2), respectively. In both Cd-contimated soils, the increase of BCF value was significant after the addition of the chelating agent, implying that the chelating agent combination has great application potential in chelatorenhanced phytoextraction. 3.5. Morphological distribution of Cd before and after treatment

Fig. 2. The amount of total Cd removal by amaranth grain.

plants normal physiological processes (Freitas and Do Nascimento, 2009). 3.4. Cd phytoremediation efficiency by amaranth grain Amaranthus hypochondriacus L. has been recommended as a potential macrophyte for Cd-contaminated agricultural soil phytoremediation because it is Cd-tolerent, with a large biomass and biological concentration factor (BCF) for Cd (Del Carmen RamırezMedeles et al., 2003). The values for amaranth grain BCF and translocation factor (TF) in different treatment and control groups are listed in Table 4. After chelation treatment, the order of plant Cd accumulation and transport ability was as follows: GN > GLDA z NTA > EDDS > G-E > G-C > CA > CK. The BCF and TF values for Cd in the control group were generally low, but a significant improvement was obtained after the addition of chelating agents to the soil, indicating that the chelating agents enhance the ability to accumulate and transport Cd and assist with soil heavy metal phytoremediation (Wei et al., 2012b). In S1, the TF values were all greater than 1.00 (except for the CA treatment), and the maximum value reached 1.44, indicating that the CA treatment had a limited mobilization effect on soil Cd. When the concentration reached 3 mM, increased chelating agent concentration had a limited increase on the accumulation and transport abilities of Cd, consistent with the research by Hu et al. (2014). In the case of the

Table 4 The BCF and TF values of different treatment in the two soils. Treatment

CK GLDA(a) GLDA(b) NTA(a) NTA(b) CA(a) CA(b) EDDS(a) EDDS(b) GLDA þ NTA GLDA þ CA GLDA þ EDDS

Low-contaminated soil （S1）

High- contaminated soil （S2）

BCF

TF

BCF

TF

2.94 ± 0.06 h 10.15 ± 0.11c 11.54 ± 0.04b 11.90 ± 1.23b 8.27 ± 0.06e 4.58 ± 0.07g 5.02 ± 0.20g 10.01 ± 0.17c 8.80 ± 0.11de 13.36 ± 0.05a 6.90 ± 0.02f 9.18 ± 0.03d

0.76 ± 0.04e 1.22 ± 0.08b 1.25 ± 0.04b 1.44 ± 0.05a 1.12 ± 0.05c 0.80 ± 0.04e 0.84 ± 0.05e 1.04 ± 0.09cd 1.11 ± 0.05c 1.26 ± 0.05b 0.95 ± 0.06d 0.95 ± 0.03d

3.19 ± 0.09j 7.68 ± 0.06e 8.78 ± 0.07c 9.52 ± 0.07b 7.21 ± 0.04f 4.99 ± 0.10i 5.83 ± 0.05 h 8.06 ± 0.15d 6.58 ± 0.07g 10.90 ± 0.60a 6.43 ± 0.06g 6.96 ± 0.11f

0.88 ± 0.07cd ́ 1.18 ± 0.09abc 1.36 ± 0.05 ab 1.35 ± 0.01 ab 1.20 ± 0.11abc 1.13 ± 0.12abc 1.23 ± 0.04abc 1.42 ± 0.07a 1.30 ± 0.03 ab 1.35 ± 0.04 ab 1.05 ± 0.11bc 1.01 ± 0.60d

The effect on the forms of soil Cd before and after the addition of the chelating agents is shown in Fig. 3. The modified BCR method divides the heavy metals in the soil into five forms, in which the water soluble and acid-soluble forms have strong bioavailability and are more easily absorbed by plants than the reducible and residual forms. Under natural conditions, the order of the various morphological contents is reducible z oxidizable > residual > acidsoluble > water soluble forms (Rauret et al., 1999). After the chelating agent addition, the percentage of available Cd in the two soils increased to different degrees, reaching 15.3e70.4% (S1) and 14.5e61.4% (S2), respectively. The maximum content of bioavailable Cd occurred in the G-N combination, and increased by 5.83 and 3.75 times compared to the control group respectively. Unlike the other treatments, the Cd phytoextraction ability of the CA treatment was significantly weaker which was mainly related to the structural and chemical properties of CA itself. More carboxyl groups in the other three chelating agents and chelator combinations would promote the formation of ligand-metal ion complexes and metal-chelator complexes, and indirectly lead to soil-Cd dissolution (Wang et al., 2018a, 2018b, 2018c). A study by Farid et al. (2017) showed that at the same concentration, the mobilization ability of LMOWAs chelators for heavy metals was generally much lower than that of APCAs chelators. Under natural conditions, most of the water-soluble and acid-soluble Cd would be transformed into more stable forms, such as reducible and residual states through the aging effect. Wang et al. (2016) showed that with the addition of a chelating agent, a large amount of acid-soluble Cd, and a part of the oxidizable/reducible Cd were released into the soil, which is beneficial for plant Cd absorption and accumulation. 3.6. Soil enzyme activities The changes in catalase, urease, and invertase activities in S1 and S2 before and after plant extraction are shown in Table 5. Different treatments had different effects on the activities of these three enzymes in the soil. After GLDA treatment, the activities of catalase and urease in the two soils increased significantly by 1.70 and 2.02 (S1), and 1.32 and 1.24 times (S2), respectively, compared to the control group, and the activity was further promoted as the concentration of GLDA increased. This was in line with the results of Guo et al. (2019). However, urease activity decreased. Unlike in the GLDA treatment, the activity of the three enzymes was inhibited in the soil treated by EDDS, and catalase inhibition was the most obvious, decreasing by 39.5% (S1) and 26.8% (S2), respectively, as compared to the control groups. Studies have shown that free EDDS in the soil may destroy the physiological barrier of root systems by removing Fe2þ, Ca2þ, and other divalent cations from the plasma membrane; this causes irreversible effects on the soil environment and results in the overall decline of some enzyme activities such as catalase and superoxide dismutase (Attinti et al., 2017). Compared with the other treatments, the NTA and CA treatments showed great differences in enzyme activity but there was no obvious regularity. These treatments had a certain relationship with the soil type and the chelating agent. In the two soils treated with G-N, catalase, urease, and invertase

K. Wang et al. / Chemosphere 237 (2019) 124480

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Fig. 3. Species distribution of Cd before and after the treatment. (The total concentration of the chelating agent is 5 mM; the ratio in the chelator combinations is 3:2).

Table 5 Enzyme activity analysis of rhizosphere soil before and after plant extraction. Treatment

Low Cd-contaminated soil（S1）

High Cd-contaminated soil（S2）

CAT (mg/g)

UE (mg/g)

SC (mg/g)

CAT (mg/g)

UE (mg/g)

SC (mg/g)

CK GLDA(a) GLDA(b) NTA(a) NTA(b) CA(a) CA(b) EDDS(a) EDDS(b) GLDA þ NTA GLDA þ CA GLDA þ EDDS

0.41 ± 0.03cd 0.62 ± 0.02 ab 0.70 ± 0.04a 0.50 ± 0.04c 0.54 ± 0.01bc 0.50 ± 0.02c 0.43 ± 0.03cd 0.32 ± 0.03de 0.25 ± 0.03e 0.63 ± 0.06 ab 0.50 ± 0.03c 0.39 ± 0.02cd

0.60 ± 0.03bc 0.49 ± 0.05cd 0.42 ± 0.03def 0.68 ± 0.06 ab 0.75 ± 0.06a 0.54 ± 0.02bcd 0.46 ± 0.04de 0.43 ± 0.02def 0.30 ± 0.01f 0.62 ± 0.04abc 0.46 ± 0.03d 0.32 ± 0.02ef

6.86 ± 0.45d 7.47 ± 0.53c 8.42 ± .0.87a 5.41 ± 0.10f 5.01 ± 0.20g 7.02 ± 0.42d 7.61 ± 0.51c 6.01 ± 0.40e 5.87 ± 0.21e 7.48 ± 0.35c 8.02 ± 0.46b 7.05 ± 0.21d

0.37 ± 0.04cde 0.45 ± 0.03bc 0.75 ± 0.04a 0.40 ± 0.05bcd 0.38 ± 0.01cde 0.42 ± 0.02bcd 0.48 ± 0.04bc 0.28 ± 0.01de 0.22 ± 0.03e 0.53 ± 0.02b 0.42 ± 0.03bc 0.39 ± 0.04ce

2.24 ± 0.21abc 2.02 ± 0.30cd 1.87 ± 0.10ef 2.11 ± 0.09bcd 2.32 ± 0.30 ab 2.36 ± 0.22 ab 2.40 ± 0.27a 1.92 ± 0.24de 1.75 ± 0.18ef 2.36 ± 0.15 ab 2.18 ± 0.22abc 1.62 ± 0.13f

9.66 ± 0.87d 10.42 ± 0.64c 12.07 ± 0.96b 10.61 ± 0.72c 12.41 ± 0.64a 9.62 ± 0.62d 9.05 ± 0.35e 8.20 ± 0.21f 6.42 ± 0.72g 10.36 ± 0.53c 8.45 ± 0.71f 6.44 ± 0.11g

activities increased to different degrees, reaching 1.53, 1.03 and 1.09 times (S1), and 1.40, 1.05 and 1.07 times (S2) that of the control groups. This indicated that the G-N combination had a higher applicability than other treatments in the two soils, and the effects of the G-C and G-N combinations on the three enzyme activities differed greatly. Studies have shown that the enzyme activity in soil has a close relationship with the concentration of bioavailable Cd in the soil, and a certain concentration of Cd stress can promote the activity of specific enzymes in the soil (Singh and Ghoshal, 2013). When the concentration of Cd is further increased, this effect will change from promotion to inhibition. Since the enzyme acts as a relatively stable protein, heavy metal ions can be used as an auxiliary group to promote the formation of a coordination bond between the enzyme center and the substrate, thereby maintaining a certain obligate structure between the active centers of the enzyme (Wu et al., 2015). Therefore, the addition of a chelating agent will change the concentration of available Cd in the soil to different extents, further affecting soil enzyme activity. Moreover, since the chelating agents used in this experiment are all degradable chelating agents, the degradation products are mostly NH3. This would have a positive impact, improving soil fertility, increasing in the number of soil microorganisms, and increasing the soil enzyme activity. 4. Conclusion The use of biodegradable chelating agents to enhance the phytoextraction of heavy metals in soil can achieve sustainable and environmental-friendly remediation, which is a relatively new concept and has attracted wide attention. Various treatments

including bare control, single chelator addition and their combination were designed to investigate the effect of chelating agent on the efficiency of phytoextraction. The results are listed as follows: 1) there was no significant inhibition on biomass growth of Amaranthus hypochondriacus L. at low Cd concentrations in different biodegradable chelating agent treatments, except for EDDS treatment. 2) The addition of chelators can significantly enhance the ability of the plants to absorb and transport Cd to varying degree that could cause plant biomass deficiencies. 3) The G-N combination treatment removed the largest amount of Cd, showing the great application potential of complex chelators in heavy metals phytoremediation. 4) The addition of chelating agents can alter the presence and bioavailability of heavy metals in the soil, further increasing the uptake of Cd by the plants. 5) Most biodegradable chelating agents have limited effects on soil enzyme activity due to their short duration in the soil environment, and degradation of chelating agents even promotes the activity of some soil enzymes. Acknowledgments The authors are grateful for financial support from the National Key Research and Development Program of China (No. 2016YFD0800806), the National Natural Science Foundation of China (41771525) and STU scientific research foundation for talents (NTF19025). Appendix A. Supplementary data Supplementary data to this article can be found online at

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