immobilization and phytotoxicity of chromium in contaminated soil remediated by cmc-stabilized nzvi

Accepted Manuscript

Title: Immobilization and Phytotoxicity of Chromium inContaminated Soil Remediated by CMC-Stabilized nZVI

Author: Yu Wang Zhanqiang Fang Yuan Kang Eric PokeungTsang

PII: S0304-3894(14)00325-2DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2014.04.056Reference: HAZMAT 15897

To appear in: Journal of Hazardous Materials

Received date: 6-2-2014Revised date: 22-4-2014Accepted date: 23-4-2014

Please cite this article as: Y. Wang, Z. Fang, Y. Kang, E.P. Tsang,Immobilization and Phytotoxicity of Chromium in Contaminated SoilRemediated by CMC-Stabilized nZVI, Journal of Hazardous Materials (2014),http://dx.doi.org/10.1016/j.jhazmat.2014.04.056

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http://dx.doi.org/doi:10.1016/j.jhazmat.2014.04.056

http://dx.doi.org/10.1016/j.jhazmat.2014.04.056

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Immobilization and Phytotoxicity of Chromium in Contaminated

Soil Remediated by CMC-Stabilized nZVI

Yu Wanga,b, Zhanqiang Fanga,b*, Yuan Kanga,b，Eric Pokeung Tsangb,c

a. School of Chemistry and Environment, South China Normal University, Guangzhou,

Guangdong, China, 510006

b. Guangdong Technology Research Center for Ecological Management and Remediation of

Urban Water Systems, Guangzhou, Guangdong, China, 510006

c Department of Science and Environmental Studies, Hong Kong Institute of Education, Hong

Kong, 00852, China

* Corresponding author, Tel: +86-20-39313279; Fax: +86-20-39313279;

E-mail address: [email protected] (Z. Fang).

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Abstract

The toxic effect of Cr(VI)-contaminated soil remediated by sodium carboxymethyl

cellulose stabilized nanoscale zero-valent iron (CMC-stabilized nZVI) was assessed

through in vitro toxicity and phytotoxicity tests. In vitro tests showed that 0.09 g L-1 of

Fe0 nanoparticles (soil-to-solution ratio was 1 g:10 mL) significantly reduced the

toxicity characteristic leaching procedure (TCLP) leachability and physiological based

extraction test (PBET) bioaccessibility of Cr by 82% and 58%, respectively.

Sequential extraction procedures (SEP) revealed that exchangeable (EX) Cr was

completely converted to Fe-Mn oxides (OX) and organic matter (OM). Accordingly,

phytotoxicity tests indicated that after 72-h remediation, Cr uptakes by edible rape and

Chinese cabbage were suppressed by 61% and 36%, respectively. Moreover, no

significant increase in Cr uptake was observed for either species after a 1-month static

period for the amended soil. Regarding Fe absorption, germination and seedling

growth, both plant species were significantly affected by CMC-nZVI-exposed soils.

However, similar phytotoxicity tests conducted after 1 month showed an improvement

in cultivation for both plants. Overall, this study demonstrated that CMC-nZVI could

significantly enhance Cr immobilization, which reduced its leachability, bioavailability

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and bioaccumulation by plants. From a detoxification perspective, such remediation is

technologically feasible and shows great potential in field applications.

Keywords: Cr(VI)-contaminated soil, nanoscale zero-valent iron (nZVI), remediation,

ecotoxicological effects, plants

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1. Introduction

Chromium is widely used in electroplating, leather tanning and wood processing

due to its outstanding corrosion resistance and bright metallic lustre [1, 2]. However,

the disposal of chromium residual, released by atmospheric deposition or irresponsible

discharge, gives rise to serious soil contamination [3]. It has been estimated that the

storage of untreated Cr slag in China has exceeded 400 million tons [4]. Recently, the

use of nanoscale zero-valent iron (nZVI) as a reactive media for the in situ remediation

of chromium in solid waste has been extensively investigated [1] [5-7].

In a previous study, we tested the feasibility of using CMC-stabilized nZVI (CMC-

nZVI), which was synthesized using steel pickling waste liquor, for the in situ

remediation of Cr(VI)-contaminated soil. The results demonstrated that 0.09 g L-1 of

nanoparticles at a soil-to-solution ratio of 1 g:10 mL could reduce 80% of pre-loaded

Cr(VI). Compared with Cr(VI), the resultant Cr species in the forms of Cr(OH)3 or

Cr(III)/Fe(III)(oxy)hydroxide were more stable and approximately 10-100 times less

toxic [8]. Nevertheless, although such remediation could transform soil metal into its

less toxic form, it could not actually remove the metal from the soil. Consequently,

concerns have been raised about the ecotoxicity of the retained chromium, limiting the

engineering applications of nZVI technology. Therefore, research on the fate and

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ecotoxicity of Cr species in remediated soil is urgently needed before field applications

can be popularised.

To date, several investigators have demonstrated that nZVI is capable of

significantly reducing the Cr leachability and subsequent toxicity by one of the

chemical extraction methods (e.g. TCLP or the California Waste Extraction Test) [1]

[7]. However, studies showing differences between Cr leachability rates cannot be the

whole story. Compared with the previous investigations, this study used not only

TCLP to test the Cr leachability but also PBET to assess its bioaccessibility. Moreover,

SEP was used to identify the cause of the enhanced stability of Cr.

CMC-nZVI was intended for use in soil. Due to water movement, higher plants are

more or less likely to be exposed to nZVI and its reaction products. Nowadays, some

useful insights into biotoxicology of nZVI have begun to emerge [9-11]. Meanwhile,

some researches have been conducted on the interaction between nanomaterials and

cocontaminants in agroecosystems [12, 13]. However, the phytotoxicity of Cr(VI)-

contaminated soil remediated by nZVI is still unknown. It is necessary to determine

whether such remediation effectively reduces the bioaccumulation and translocation of

Cr in plants, and whether the introduction of CMC-nZVI causes a secondary

environmental problem. Moreover, it is important to confirm that such remediation is

capable of preserving or restoring soil quality before such soil can be reused for

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planting. Therefore, in this study we first investigated the toxic effects of CMC-nZVI

amended soil, CMC-nZVI and Cr(VI)-contaminated soil on two common crops: rape

(Brassica campestris L.) and Chinese cabbage (Brassica pekinensis).

The specific objectives were to 1) explore the immobilization and bioaccumulation

of Cr in after-amended soil through in vitro tests and plant-based tests; 2) determine

whether such remediation would cause some harmful effects on Fe absorption by

plants, plants germination and seedling growth; 3) confirm whether such remediation

could preserve or restore soil quality before such soil can be reused for planting.

2. Materials and methods

2.1 Materials

The CMC-nZVI used in this study was prepared by sodium borohydride reduction

method using steel pickling waste liquor, which was reclaimed from the Jinlai steel

plant in Guangzhou. Some of the physical and chemical properties and the details

regarding the synthesis of CMC-nZVI can be found in our previous study [14, 15]. The

concentrations of Fe0 and CMC used in this study were 0.3 g L-1 and 0.1% (w w-1),

respectively. The size of these nanoparticles ranged from 10 to 100 nm with a

Brunauer–Emmett–Teller (BET) surface area of 40 ± 2 m2 g-1. Edible rape (Brassica

campestris L.) and Chinese cabbage (Brassica pekinensis) seeds were purchased from

the Vegetable Research Institute at Guangdong Academy of Agriculture Sciences

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(Guangzhou, China). The seeds were sterilized for 10 min with a 0.5% (v v-1) sodium

hypochlorite solution and then rinsed copiously with deionized (DI) water.

2.2 Remediation of Cr(VI)-contaminated soil

The chromium-free soil sample was collected from the Higher Education Mega

Centre in Guangzhou, China. Cr(VI)-spiked soil samples were prepared using the

following procedure: 10 mL of K2Cr2O4 solution at a concentration of 200 mg L-1 was

mixed with 10 g of air-dried soil and stirred until the mixture was air dried to a

constant weight. The resulted Crtotal and Cr(VI) concentrations were 200 and 102 mg

kg-1, respectively. The remediation was initiated by amending the Cr(VI)-spiked soil

with the CMC-nZVI suspension at a soil-to-solution ratio of 1g:5 mL. The mixtures

were placed on an end-to-end rotator with 30 rpm to react for 72 h and then used for in

vitro toxicity or phytotoxicity tests.

2.3 In vitro toxicity tests

To estimate the effect of CMC-nZVI on Cr(VI) immobilization, TCLP (EPA

Method 1311 [16]) were performed by comparing the leachability of Cr in the soil

before and after the nanoparticle treatment. Moreover, a more biochemically oriented

method, PBET [17], was used to evaluate the in vitro bioaccessibility of soil-bound Cr.

The sequential extraction procedures (SEP) developed by Tessier et al. [18] were used

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to quantify the fraction of various operationally defined Cr species. All of the tests

were duplicated to ensure data quality.

2.4 Phytotoxicity tests

The phytotoxicity tests were conducted in a set of 120 mm petri dishes covered with

double-layer filter papers. To provide enough nutrients for plant growth, each of petri

dishes received 5 g of soil sample and 25 mL of nanoparticle suspension or DI water.

Then, 15 seeds were placed inside each petri dish and the seeded dishes were placed in

a growth chamber (GP-01, Huangshi Hengfeng Medical Instrument Co, China) with

day and night temperatures of 25°C (16 h) and 20°C (8 h), respectively. The 16-h

photoperiod had an illumination intensity of 1200 Lux. During plants growth, seeds

germination was recorded and they were watered with DI water daily to maintain the

original moisture. After eight days, the seedlings were harvested and the root portions

were washed thoroughly with DI water. The clean plant samples were then separated

into three parts (roots, stems and leaves) and the length of each part was measured.

Then, the separated plant samples were dried in an oven at 80°C for 48 h and the

weights of the dried tissues were recorded. For comparison, seven soil samples were

used for phytotoxicity tests: S1. fresh soil (control); S2. Cr(VI)-contaminated soil; S3.

fresh soil mixed with CMC-nZVI; S4. fresh soil mixed with CMC-nZVI for 72 h; S5.

Cr(VI)-contaminated soil mixed with CMC-nZVI; S6. Cr(VI)-contaminated soil

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remediated by CMC-nZVI for 72 h; and S7. Cr(VI)-contaminated soil remediated by

CMC-nZVI and then kept static for 1 month. The experiments were performed twice,

in triplicate each time.

2.5 Fe and Cr determination in vegetables

The samples of dried roots, stems and leaves were ashed in a muffle furnace at

550°C for 6 h to pre-concentrate the Cr. The ashes were then digested by 5 mL

concentrated nitric acid in a hot plate to less than 1 mL and diluted to 5 mL with 1%

hydrochloric acid. The digested samples were filtered using 0.22 um hydrophilic

membranes and analyzed for the total Fe and Cr content by flame atomic-absorption

spectrophotometer (FAAS, TAS-986, Beijing Purkinje General Instrument Co, Ltd,

China).

2.6 Fe(II) analysis

To determine changes in the concentration of ferrous ions (Fe(II)) during plant

growth, a suspension was extracted from the area around the root zone daily. Samples

of this mixture were centrifuged at 5000 rpm for 10 min and then the supernatant was

reserved for Fe(II) determination. Fe(II) was measured using the phenanthroline

spectrophotometric method [19] with a visible spectrophotometer (722S, Shanghai,

China) at 510 nm in a 1-cm-long glass cell with a detection limit of 0.03 mg L-1.

2.7 Statistical analysis

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A one-way ANOVA, followed by a Student-Newman-Keuls (SNK) multiple

comparison test (p＜0.05), was used to determine the significant differences among

different groups.

3. Results and Discussion

3.1 In vitro toxicity tests

3.1.1 Leachability of soil-bound Cr

Fig. 1 illustrates the TCLP leachability of soil-bound Cr for the soils treated with

CMC-nZVI at various treatment times. The untreated soil displayed the greatest Cr

(both Crtotal and Cr(VI)) leachability, as described previously [7]. When the soil was

treated with CMC-nZVI for 0.25 h, the leachability of Cr(VI) decreased rapidly from

its original 31% to below the detection limit (4 ug L-1) and then remained unchanged.

In contrast, the TCLP leachability of Crtotal for untreated soil was 52%. When the soil

was amended with CMC-nZVI for 0.25 h, the Crtotal decreased sharply to 22%.

Moreover, after 0.25 h, the treatment time effect became less conspicuous. For the

treated soil, the leachability of Crtotal was reduced by only 12% from 0.25 h to 10 h.

The difference in the leachability of Crtotal between the untreated and the 10-h-treated

soil was the equivalent of the Cr(VI) leachability of the untreated soil. These results

indicated that most of the reduced Cr presented in a stable form that could not be

easily extracted by acid solution. After a 72 h reaction time, the TCLP leachability of

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Crtotal unceasingly decreased to 9.5%, suggesting that the addition of CMC-nZVI also

decreased Cr(III) leachability. As for the concentration of extracted Cr, Crtotal in the

TCLP fluid for the untreated soil was 5.16 mg L-1, which was slightly above the EPA

TCLP limit (5 mg L-1) for identifying hazardous wastes [16]. After the CMC-nZVI

treatment, the Cr concentrations (include Cr(VI) and Crtotal) in the TCLP extracts were

far below the regulated limit.

3.1.2 Bioaccessibility of soil-bound Cr

Compared with the TCLP, the PBET procedure uses a much more aggressive

extracting agent (pH 2.3) at a much higher liquid-to-soil ratio (100:1) [20]. The

method used in this study was designed to assess the in vitro bioaccessibility of the

resultant Cr in the amended soil for humans. Similar to the TCLP leachability, the

bioaccessibility of Cr was decreased with CMC-nZVI remediation (Fig. 2). For Cr(VI),

over 20% of the pre-loaded Cr in the untreated soil was bioaccessible. When CMC-

nZVI was present, the Cr(VI) concentration in the PBET extracts remained

undetectable throughout the treatment times, suggesting that all of the PBET-leached

Cr for the treated soil was in the less toxic form of Cr(III). For Crtotal, the

bioaccessibility decreased from 21.3% for the untreated soil to 13% at 0.25h, and then

steadily declined to 9% at 72 h, a reduction of 58%. However, the difference in

extracted Crtotal between the untreated and the treated soils was much lower than the

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extracted Cr(VI) of the untreated soil, indicating that the bioaccessibility of Cr in the

treated soil was contributed by both the original and reduced Cr(III). These

observations revealed that the accessibility of the resultant Cr depended on the acidity

of the extractant. The Cr(VI) and Crtotal concentration in the PBET extracts for

untreated soils were 0.42 and 0.4 mg L-1, respectively. After 72-h remediation, all of

the extracted Cr was present in trivalent form and subsequently reduced to 0.18 mg L-1.

3.1.3 Speciation of soil-bound Cr

Several authors have evaluated sequential extraction procedures for identifying the

relative availability/leachability of soil-bound heavy metals by partitioning the

particulate trace metals into five fractions [20-22]. The five species have been defined

as exchangeable (EX), bound to carbonates (CB), Fe-Mn oxides-bound (OX), organic

matter-bound (OM) and residual (RS). The relative availability obeys the following

order: EX＞CB＞OX＞OM＞RS. Fig. 3 shows the changes in the fractions of the five

Cr species in the untreated and treated soils. The primary Cr species for the untreated

soil were split among EX (27.8%), CB (15.7%), OX (40.5%) and OM (16%). After

0.25-h remediation, the EX fraction was completely converted to OX and OM, which

were increased by 19% and 5.4%, respectively. Nevertheless, prolonging the reaction

time did not significantly influence the alteration of the Cr species. The major Cr

species for the 72-h amended soil were split among CB (19%), OX (61%) and OM

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(20%). This distinctive alteration in Cr speciation, especially the substantial increase in

OX fraction, accounted for the reduced leachability and bioaccessibility of Cr in the

previous study. According to previous investigations, the elevated OX fraction might

be largely attributed to the precipitation of Cr(OH)3 or

Cr(III)/Fe(III)oxides/hydroxides (CrxFe1-xOOH and (CrxFe1-x)(OH)3) during the CMC-

nZVI treatment [23, 24]. Overall, the conversion of more easily available Cr (EX) to

the less available OX and OM species was evident, and thus was held responsible for

the enhanced stability of Cr following the nanoparticle treatment.

3.2 Phytotoxicity tests

3.2.1 Accumulation and translocation of Cr

Immobilization of Cr in soil, which reduced its uptake and translocation in plants,

was significant for Cr(VI)-contaminated soil remediation. To further validate the

immobilization efficiency of soil-bound Cr using CMC-nZVI, the Cr content of all the

exposed plants was measured using FAAS to determine the accumulation amount.

Table 1 shows that addition of CMC-nZVI to Cr(VI)-contaminated soil significantly

decreased the Cr content in rape tissues. Although the leaf Cr levels in all of the

treatments were relatively low, ranging from 9 to 74 mg kg-1, the reduction of Cr

accumulation in these tissues was most obvious compared to that of others (decreased

by 73%, 89% and 88% for S5, S6 and S7, respectively). In addition, this trend was

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particularly evident in the stem tissue, the Cr content of which decreased from 132 to

50 mg kg-1 for S6 (a reduction of 62%). Although less dramatic than that of the shoots,

the Cr content of the root tissue also tended to decrease with CMC-nZVI remediation

(48% for S6). For the whole plant (Fig. 4), a remarkable decrease of Cr content (a

34% reduction) was observed following the CMC-nZVI injection, and another 27%

reduction was obtained after the completion of 72-h remediation. Thus, the reduction

of Cr(VI) in soil by CMC-nZVI was extremely fast and the resultant Cr species was

low in bio-availability for rape. As expected, after keeping the amended soil static over

1 month, little increase in Cr uptake was observed, confirming the stability of Cr(III)

in soil.

The total Cr content of the Chinese cabbage tissues and whole plants are shown in

Table 1 and Fig. 4, respectively. Similar to the Cr content of rape, that of Chinese

cabbage showed a pronounced decrease after CMC-nZVI remediation. The leaf Cr

content of S2 was 50 mg kg-1. After the introduction of CMC-nZVI, the Cr content was

significantly reduced by 36% (S3), and prolonging the reaction time generally resulted

in a lower Cr concentration. The stem Cr content of S2 was 116 mg kg-1. Promptly, the

introduction of CMC-nZVI initiated an obvious suppression of Cr uptake (55%), and

furthermore, complement of a 72-h remediation resulted in a 71% reduction. The

pattern of Cr uptake in roots among the different treatments was identical to that of the

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leaves, although the maximal reduction rate was only 16% in the 72-h amended soil.

Overall, the whole plant Cr content of S2 was 401 mg kg-1, with the transient contact

between CMC-nZVI and the contaminated soil resulting in a 29% reduction in Cr

accumulation. However, a further reduction (7%) obtained after 72-h remediation had

no statistical significance. Furthermore, a 1-month static period for the amended soil

also had no effect on Cr uptake in the different tissues or the whole plant.

Comparing the Cr content in rape with that in Chinese cabbage revealed some

interesting trends. First, the maximum quantity of Cr was always accumulated in the

roots of all of the exposed plants, and unfortunately the suppression of Cr uptake in this

tissue was lowest. The enhanced accumulation of chromium in root tissue might result

from the presence of organic acids in the root exudates, which formed chromium

complexes that facilitated chromium uptake [25]. Moreover, the accumulated Cr was

immobilized in the vacuoles of the root cells, resulting in poor translocation to the

shoots [26]. Second, although rape and Chinese cabbage had the same level of whole

plant Cr content, CMC-nZVI remediation yielded significantly greater suppression of

Cr uptake in rape (34-63%) than in Chinese cabbage (29-37%), suggesting that

different plant species have distinct capabilities when it comes to accumulating Cr that

are also influenced by Cr species. Finally, no significant increase in Cr uptake was

observed in S7 (1-month static sample) for either species, indicating that the resultant

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Cr was presented in a stable form not easily re-oxidized by air.

To better understand the effect of CMC-nZVI remediation on the translocation

capability of Cr for the two plants, the CF and TF were calculated, respectively, and

the results are shown in Table 2. The CF and TF were defined as the ratio of Cr

amount in plant leaves to that in soils, and Cr amount in leaves to that in roots,

respectively [27]. Notably, the CF and TF values for both plant types had declined

significantly after CMC-nZVI remediation, whereas those with slight increases in S7

(1-month static sample) were rare instances of statistical significance. Furthermore,

rape had a more dramatic decline in both CF and TF values than Chinese cabbage.

The general reduction in Cr uptake observed in the presence of CMC-nZVI sheds

new light on the remediation of Cr(VI)-contaminated soil, although the responsible

mechanism for this phenomenon has not been validated. According to our previous

work, 80% of the pre-loaded Cr(VI) that was reduced to Cr(III) after a 72-h

remediation might explain the poor bioaccumulation. Zeyed et al. observed that, in 7

out of 10 analyzed crops, more Cr accumulated when plants were grown with Cr(VI)

than with Cr(III) [28]. Meanwhile, López-Luna et al. noted that the low observed

adverse effect concentration (LOAEC) for Cr(VI) was much lower than that for Cr(III)

towards wheat, oat and sorghum [29]. Consequently, such remediation can

significantly decrease the bioavailability and bioaccumulation of Cr by plants through

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converting Cr(VI) to Cr(III), subsequently reducing its phytotoxicity. The present

results indicate that such remediation is technologically feasible from the

detoxification perspective.

3.2.2 Fe absorption

Iron is an important nutritional ingredient for plant growth and development, as it is

involved in many vital enzymatic reactions for nitrogen fixation, DNA synthesis

(ribonucleotide reductase) and hormone synthesis (lipoxygenase and ACC oxidase)

(ACC, 1-aminocyclopropane-1-carboxyllicacid) [30-32]. However, information

concerning the effect of nZVI application on plants’ Fe acquisition, assimilation and

storage is limited. To test the effect of CMC-nZVI remediation on Fe absorption, the

Fe content of all harvested plants in this study was analysed. As Table 3 shows, the Fe

content of all rape tissues tested decreased from underground to aerial, and the trend of

Fe absorption by different tissues was similar to that of the total element presented.

For ease of comparison, the Fe content analysis results below were based on the whole

plant level (Fig. 5). The total Fe content of the control plant was 6170 mg kg-1.

However, Fe absorption was significantly suppressed in the presence of Cr(VI). When

fresh CMC-nZVI was injected, the whole plant Fe content was increased to 8317 mg

kg-1 (1.3 times that of the control plant). Conversely, exposure to the 72-h aged CMC-

nZVI resulted in a reductive absorption of 4997 mg kg-1. Interestingly, the plants

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exposed to fresh nanomaterials and contaminants contained 1.7 times more Fe than

that of the control plants, yet Fe absorption in the 72-h amended soil was restored to

5377 mg kg-1 and the slight increase observed in the 1-month static sample was

insignificant. Overall, the fresh CMC-nZVI significantly accelerated Fe absorption by

rape while the aged CMC-nZVI was likely to result in the suppression of Fe absorption.

The Fe content of Chinese cabbage tissues and whole plant values are shown in

Table 3 and Fig. 5, respectively. Similar to the case of rape, most of the assimilated Fe

was stored in the roots and less in the shoots, and the trends of Fe content in S2

observed in rape was also evident for Chinese cabbage. Accordingly, the fresh CMC-

nZVI facilitated the Fe absorption and the 72-h aged counterpart had the opposite

effect. However, it is interesting to note that the addition of fresh CMC-nZVI to the

Cr(VI)-contaminated soil yielded significantly less Fe absorption than that of the

control (decreased by 34%), suggesting that while the fresh nZVI may lead to more Fe

uptake, the effect of the co-exposure of nanomaterials and contamination on Fe

absorption was complicated and may depend on plant species. Moreover, following

remediation completion, Fe absorption gradually recovered up to the normal level.

Taken together, the results presented above demonstrated that the remediation of

Cr(VI)-contaminated soil by CMC-nZVI had some effect on Fe absorption. To be

specific, Fe absorption might be accelerated (suppressed) by fresh (aged) CMC-nZVI.

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Recent research has shown that nZVI might be taken up by plant root cells and then

deposited within cell walls and inside the cells [11]. Indeed, similar bioaccumulation

has been published for many nanomaterials including iron oxide nanoparticles (Fe3O4)

[33], fullerene C70 [34], silica nanoparticles [35], AuNPs [36] and CuNPs [37]. Even

though the importance of plants’ uptake and accumulation of nanoparticles has

become increasingly recognized, the mechanism of such accumulation remains unclear.

There are several possible mechanisms by which fresh nZVI could enhance Fe uptake

into plants. One possibility is that they penetrate the seed coat and are assimilated by

the seed embryo. As Khodakovskaya et al. [38] suggested, nanotubes were able to

penetrate the seed coat while supporting and allowing water uptake inside the seeds.

Another way for nZVI to enter the plant is via root epidermal cells by endocytosis [35,

36, 39]. According to Liu et al. [40], the half time of nZVI in soil after injection was

from 90 to 180 d. Because the seeds used in this study were allowed to germinate for

only 8 days, it is conceivable that some of the nZVI were internalized this way.

3.2.3 Effects on germination and growth

To determine the effect of the amended soil and the CMC-nZVI suspension on plant

growth, some toxicological endpoints such as seed germination percent, seedling

growth and biomass production were measured during the cultivation. The stimulation

of Cr(VI) in the plant growth of rape and Chinese cabbage was obvious, as indicated

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by Han et al. [27]. However, plants exposed to nZVI would cause toxic effects. For

rape, the most obvious symptom was suppression in germination, which presented as a

lower germination rate and germination retardation (Supplementary Material, Fig. S.

1(a)). Moreover, CMC-nZVI exposure significantly decreased root biomass but

increased plant leaves biomass in all treatments (Supplementary Material, Table S.

1). No statistically significant inhibitory effect was observed in plant elongation except

for S5, which was reduced by 61% compared with control plants (Fig. 6(a)). Overall,

the fresh CMC-nZVI led to a more pronounced phytotoxicity in rape than the 72-h-

aged CMC-nZVI. For Chinese cabbage, CMC-nZVI addition had somewhat similar

adverse effects including lower germination rate, growth retardation, yellowing of

leaves, root stunting and lower root biomass (Supplementary Material, Figs. S. 1(b),

6(b) and Table S. 1, respectively). Nevertheless, it was interesting to note that the 72-

h-aged CMC-nZVI had a more serious negative effect on rape than the fresh

counterpart, especially for germination and biomass production. This is opposite to

what has previously been observed in rape, indicating that the extent of inhibition is

plant species and nanoparticle property dependent. Finally, similar phytotoxicity tests

conducted after 1 month showed an improvement in cultivation for both plant types,

suggesting that such remediation gradually restored soil quality enough to permit its

reuse in planting.

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The results indicated that the soil amended by CMC-nZVI affected the plants’

cultivation more or less, which was in agreement with the literature that investigated

other organisms [10, 41, 42]. Ma et al. noted that exposure to nZVI significantly

reduced the transpiration and growth of hybrid poplars [11]. In this study, it is difficult

to discern whether the phytotoxic response was induced by nZVI or the reduced Cr

species (e.g. Cr(OH)3 and Cr(III)/Fe(III)(oxy)hydroxide). However, the results

obtained from the above comparison tests suggested that CMC-nZVI may play an

important role in these toxic effects. Several hypotheses could be developed

concerning the phytotoxicity such as Fe(Ⅱ) toxicity, oxidants formation (e.g. OH·,

Fe(IV)) during Fe0/Fe(Ⅱ) oxidation [42-44], internal reactive oxygen species (ROS)

production [9, 10] and other NP-specific toxicity [43]. The hypothesis regarding Fe(II)

toxicity was directly supported by the change of Fe(II) concentration in the cultivated

soil solution, which decreased from 20 mg L-1 to below the detection limit after 8-day

post planting (Supplementary Material, Fig. S. 2). According to previous research,

higher concentrations of Fe(II) entering into plants have reacted with reduced forms of

oxygen to catalyze the production of free radical species such as hydroxyl radicals,

which might attack plant cells, i.e. damaging membranes, breaking up cellular

integrity, nicking DNA, inactivating enzymes and proteins, etc. [30, 31]. Moreover,

the introduction of associated chloride and sulphate from steel pickling waste liquor

may contribute to the phytotoxicity of nZVI. Finally, the adverse effects were also

observed in a timely manner, probably due to the oxidation of Fe0/ Fe(II) during nZVI

aging in soil whose surface was in contact with air. To avoid this secondary pollution,

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the purification of the steel pickling waste liquor before the synthesis of CMC-nZVI

and the complete oxidation of nZVI after remediation should be considered in field

applications.

4. Conclusion

The present study demonstrated, for the first time, that applying CMC-nZVI to

remove Cr(VI) from contaminated soil could significantly enhance Cr immobilization,

thus reducing its leachability, bioavailability and bioaccumulation by plants – although

it had some effect on Fe absorption. The results also suggested that such remediation

exerted an inhibitory effect on plant growth, which might be ascribed to the specific

physicochemical properties of nZVI. Moreover, a restoration in cultivation within a

short time-span highlighted that the complete depletion of nZVI after remediation

must be considered. After all, from a detoxification perspective, such remediation is

technologically feasible and shows promising potential in field applications. Under the

premise of efficiently stabilizing Cr in soil, further work should focus on reducing the

negative effects of soil remediation by CMC-nZVI.

Acknowledgements

This work was supported by the Guangdong Technology Research Center for

Ecological Management and Remediation of Urban Water Systems (2012gczxA005),

Guangdong Technology Research Center for Ecological Management and

Remediation of Water System.

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Tables

Table 1 Cr distribution in leaves, stems and roots of rape and Chinese cabbage grown in

different chrome-containing soil samplesa

Plant S2 S5 S6 S7 rape leaf 74 ± 3 a 20 ± 1 b 8 ± 2 c 9 ± 1c stem 132 ± 10 a 101 ± 7 b 50 ± 5 c 42 ± 5 c root 182 ± 17 a 136 ± 7 b 94 ± 4 c 92 ± 2c

Chinese cabbage

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leaf 50 ± 7 a 32 ± 4 b 27 ± 3 b 29 ± 2 b stem 116 ± 6 a 52 ± 8 b 34 ± 6 c 31 ± 4 c root 235 ± 14 a 200 ± 4 b 197 ± 3 b 193 ± 3 b

a Results are calculated based on mg Cr per kg dry plant tissue. Within a row, the values followed by

different letters are significantly different (one-way ANOVA with an SNK multiple comparison test at p＜

0.05).

Table 2 Concentration factors (CF) and transfer factors (TF) of Cr for rape and Chinese

cabbage grown in different chrome-containing soil samples

Plant species Soil samples CF TF S2 0.725 0.407 S5 0.196 0.147 S6 0.078 0.085

rape

S7 0.088 0.098 S2 0.490 0.213 S5 0.314 0.160 S6 0.265 0.137

Chinese cabbage

S7 0.284 0.150

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Table 3 Fe distribution in the leaves, stems and roots of rape and Chinese cabbage grown in different soil samplesa

Plant S1 S2 S3 S4 S5 S6 S7 rape leaf 339 ± 4 e 810 ± 10 a 684 ± 12 b 315 ± 5 f 383 ± 20 d 402 ± 10 d 583 ± 18 c stem 1271 ± 52 c 478 ± 19 g 1356 ± 24 b 565 ± 3 f 1670 ± 11 a 798 ± 22 e 940 ± 19 d root 4560 ± 120 c 2345 ± 43 e 6277 ± 198 b 4117 ± 194 d 8333 ± 197 a 4177 ± 202 d 4212 ± 52 d

Chinese cabbage leaf 499 ± 13 cg 765 ± 15 e 1523 ± 46 b 694 ± 8 f 905 ± 30 d 1941 ± 71 a 1236 ± 17 c stem 2431 ± 118 b 1833 ± 36 c 2659 ± 128 a 635 ± 7 e 924 ± 4 d 305 ± 5 f 949 ± 8 d root 4696 ± 165 c 4245 ± 75 d 6709 ± 210 a 3344 ± 125 e 3200 ± 79 e 4270 ± 50 d 5220 ± 20 b

a Results are calculated based on mg Fe per kg of dry plant tissue. Within a row, the values followed by different letters are significantly different (one-way ANOVA

with an SNK multiple comparison test at p＜0.05).

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Figure Captions

Fig. 1 Reduction of TCLP-based leachability of soil-bound Crtotal and Cr(VI) for soils

amended with CMC-nZVI at various treatment times.

Fig. 2 Reduction of PBET-based bioaccessibility of soil-bound Crtotal and Cr(VI) for

soils amended with CMC-nZVI at various treatment times.

Fig. 3 Changes of Cr speciation in soils amended with CMC-nZVI at various treatment

times.

Fig. 4 Total plant Cr content of rape or Chinese cabbage grown in different chrome-

containing soil samples. Within a plant species, different letters indicate significant

difference (p＜0.05).

Fig. 5 Total plant Fe content of rape or Chinese cabbage grown in different soil

samples. Within a plant species, the values followed by different letters are

significantly different (one-way ANOVA with an SNK multiple comparison test at p

＜0.05).

Fig. 6 The root and shoot elongation of (a) Rape and (b) Chinese cabbage grown in

different soil samples. Within the same plant tissue, the values followed by different

letters are significantly different (one-way ANOVA with an SNK multiple comparison

test at p＜0.05).

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Figures

Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Highlights

Fate and ecotoxicity of Cr species in nZVI-amended soil are reported originally.

CMC-nZVI can significantly reduce the leachability and bioavailability of Cr.

Fe sorption by plants can be enhanced by fresh CMC-nZVI but inhibited by aged ones.

Complete oxidation of nZVI after use is proposed to improve plants cultivation.

immobilization and phytotoxicity of chromium in contaminated soil remediated by cmc-stabilized nzvi

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