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Toxicity and bioavailability of antimony in edible amaranth (Amaranthus tricolor Linn.) cultivated in two agricultural soil types
Journal Pre-proof Toxicity and bioavailability of antimony in edible amaranth (Amaranthus tricolor Linn.) cultivated in two agricultural soil types Qianyun Zhong, Congli Ma, Jianwen Chu, Xiaolin Wang, Xitao Liu, Wei Ouyang, Chunye Lin, Mengchang He PII:
S0269-7491(19)35282-0
DOI:
https://doi.org/10.1016/j.envpol.2019.113642
Reference:
ENPO 113642
To appear in:
Environmental Pollution
Received Date: 15 September 2019 Revised Date:
31 October 2019
Accepted Date: 16 November 2019
Please cite this article as: Zhong, Q., Ma, C., Chu, J., Wang, X., Liu, X., Ouyang, W., Lin, C., He, M., Toxicity and bioavailability of antimony in edible amaranth (Amaranthus tricolor Linn.) cultivated in two agricultural soil types, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113642. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphical abstract
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Toxicity and bioavailability of antimony in edible amaranth
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(Amaranthus tricolor Linn.) cultivated in two agricultural soil
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types
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Qianyun Zhong, Congli Ma, Jianwen Chu, Xiaolin Wang, Xitao Liu, Wei Ouyang, Chunye
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Lin, Mengchang He*
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State Key Laboratory of Water Environment Simulation, School of Environment, Beijing
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Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, China
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*Corresponding author:
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Mengchang He
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State Key Laboratory of Water Environment Simulation
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School of Environment
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Beijing Normal University
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Beijing, 100875, China
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Tel & fax: +86-10-5880 7172
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E-mail: [email protected]
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Abstract
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Although elevated levels of antimony (Sb) in agricultural soil and plant systems can have
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harmful effects on human health and ecosystems, little is known about the toxicity of Sb to
22
plants and its mechanism. The assessment of Sb bioavailability is essential for understanding
23
its potential risks and toxicity. In this study, we used pot experiments with two agricultural
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soil types spiked with Sb to investigate the dose-effect relationship between exposure to Sb
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and toxic effects (growth and bioaccumulation) on edible amaranth (Amaranthus tricolor
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Linn). Soil solution (pore water) and seven single extractants were used to assess the
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bioavailability of Sb. Different toxic effects of Sb to amaranth cultivated in two types of soils
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(alkaline and acid soil) were observed. In alkaline soil (chestnut soil, pH 8.39), antimony is
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more easily absorbed by root and transported to shoot by plants, leading to more adverse
30
effects, than in acid soil (pH 4.91) under the same exposure level. Our findings also highlight
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the need for more attention on asymptomatic accumulation of Sb in plants, especially for
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agricultural products cultivated in contaminated areas. The extraction efficiency of Sb was
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various in different extractants and soil types, Mehlich 3, NaHCO3 and Na2HPO4 for Sb were
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more efficient than other extractants in both tested alkaline and acid soil. Based on the
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extractability and correlation coefficients of toxic effects on amaranth and extractable Sb, we
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found that 0.1 M Na2HPO4 is the best extractant to predict the bioavailability of Sb in soil,
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and M3 is a suitable alternative. Antimony concentration in soil solution can also be used as
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an alternative indicator of the bioavailability of Sb.
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Capsule: Toxicity and bioavailability of antimony in edible amaranth in soils
41 42
Keywords: antimony; toxicity; bioavailability; extraction; soil solution (pore water).
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2
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1. Introduction
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Antimony (Sb) and its compounds are toxic to humans with potential carcinogen effects
46
(Filella et al., 2009; Friberg et al., 2005; Gebel et al., 1997; Hammel et al., 2000). A large
47
amount of Sb have been released into the environment due to several activities related to Sb,
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including its mining and smelting, and the production and use of antimony-based products
49
(Chu et al., 2019; He et al., 2012). As a natural trace element, Sb is not necessary for plants
50
(Ainsworth et al., 1991). Nevertheless, it can be absorbed from the soil by plants through the
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root system and cause toxic effects (Ainsworth et al., 1991; He and Yang, 1999; Ma et al.,
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2019; Shtangeeva et al., 2011; Tschan et al., 2009; Wang et al., 2018). Therefore, plants
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growing in contaminated areas can accumulate Sb (Okkenhaug et al., 2011; Qi et al., 2011;
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Telford et al., 2009; Wu et al., 2019). Such enrichment of Sb in food crops, which might be
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asymptomatic, is potentially deleterious to ecosystems and humans (Cai et al., 2016; Corrales
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et al., 2014; Feng et al., 2013; Wu et al., 2011). For people living in the vicinity of Sb mines
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in China, the dietary intake of Sb (554 µg/day) is 1.5-fold higher than the tolerable daily
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intake (TDI, 360 µg/day) (Wu et al., 2011). This increased Sb intake was attributed to the
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consumption of rice, vegetables (especially leaf vegetables), drinking water and meat (Wu et
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al., 2011). However, the mechanisms of Sb uptake, translocation, and toxicity to plants, as
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well as any dose-effect relationship, remain unclear (Ji et al., 2017; Wang et al., 2018).
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Therefore, we need to pay extra attention on the environment quality on Sb contaminated soil,
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especially agricultural soil.
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The bioavailability of metal(loid)s in the soil is defined as the amount of a given metal(loid)
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that can be absorbed and have an active effect on organisms, and it depends on its species in
66
environment (He et al., 2019; Shahid et al., 2012; Wilson et al., 2010). In the soil samples
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collected from Sb mining area (Xikuangshan, China), Sb(V) is the dominate fraction of
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bioavailable Sb at a high level (6.3–748 mg kg-1) (Okkenhaug et al., 2011). Microbial
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communities were affected by extractable Sb and pH, and various in different utilization types
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of soil (Sun et al., 2019). The measurement of the bioavailability of several contaminants in 3
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ecosystems can provide an accurate ecological risk assessment. The bioavailability of Sb in
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soil is commonly assessed using traditional chemical extraction methods, including
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single-step extraction and sequential extraction protocols (Ettler et al., 2007; He, 2007).
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Among other extraction solutions, Mehlich 3 (M3) is a combination of chemicals widely used
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to extract several micro and macronutrients from different soil types (Mehlich, 1978, 2008;
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Sims et al., 2002). Diffusive gradients in thin films (DGT) have also been used to detect
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bioavailable Sb (Wang et al., 2018). In addition to the extraction of metal(loid)s from soil
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samples, the direct assessment of metals in soil solution (pore water) might be more sensitive
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than extractions in laboratory and thus more relevant to the assessment of ecological risks
80
(Moreno-Jimenez et al., 2011).
81
In this study, we designed pot experiments using two different types of farmland soil spiked
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with Sb. The aims were: (1) to investigate the toxic effects (growth and bioaccumulation) of
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Sb on edible amaranth grown in two agricultural soil types, and (2) to assess the
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bioavailability of Sb in different types of farmland soil analyzing soil solution and soil
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extracts produced by several extraction protocols.
86 87
2. Materials and methods
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2.1. Pot experiments
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2.1.1. Soil types and treatments
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Two Chinese agricultural soils were collected at a depth of 0–20 cm from Datong in Shanxi
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Province (“chestnut soil”, calcareous alkaline soil, S1) and Jiangmen in Guangdong Province
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(“red earth”, acid soil, S2). Physical and chemical properties of the soils are shown in Table 1.
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Soil samples were air-dried and sieved, and different levels of analytical grade potassium
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antimony tartrate (0, 100, 200, 500, 1,000, 1,500 mg kg-1) were added to the soils. After aging
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for 14 days under 70% of the field water capacity, soil samples were air-dried and passed 4
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through a 2 mm sieve prior to pot experiments. Aged soil (700 g) was put into the pots, which
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were set up as three replicates for each experimental condition in a randomized block design.
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We tested the isolated effects of tartrate and potassium ions (up to 2,000 mg kg-1) in a set of
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preliminary experiments and found no effect of these ions on plant growth.
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2.2.2. Plants
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Several antimony-sensitive species were screened from common vegetables in a set of
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preliminary experiments on germination and root elongation. Edible amaranth (Amaranthus
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tricolor Linn.) was chosen considering its adaptability to a wide range of soil properties and
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tolerance to high humidity. Edible amaranth seeds were purchased from the Institute of
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Vegetables (Chinese Academy of Agricultural Sciences), immersed in 2% H2O2 for 30 min,
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and washed with ultrapure water. Then, seeds were transferred to a petri dish covered with
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wet filter paper and incubated at 4 °C for 24 h for pregermination. Soil samples in the pots
108
were hydrated to approximately 70% of the field water capacity and incubated for 2 d. Basic
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fertilizer (0.429 g CO(NH2)2, 0.263 g KH2PO4, and 0.42 g KCl per kg of soil) was
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incorporated into soil with irrigation. Ten seeds with the germ not exceeding 2 mm were
111
sowed in each pot. Pots were placed in a climate chamber (PQX-600P, Saifu, China) with
112
preset conditions (12 h photoperiod with light intensity 11,000 lux, day/night temperature of
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25 and 20 °C, and 50% relative humidity). The water content of the soil was controlled by
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weighting the pots and water was added to the bottom cup of each pot to maintain 70% of the
115
field water capacity. The positions of the pots in the climate chamber were changed randomly
116
every day. After each seedling produced 2 leaves, the number of seedlings in each pot was
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reduced to 6, which were at the same growth stage. 5
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Whole plants were harvested after 6 weeks of growth, washed with deionized water, and
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separated into shoot and root. Fresh weights of roots and shoots were obtained with an
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electronic balance after drying the samples’ surface water using absorbent paper. Main root
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length and plant height of edible amaranth were measured with caliper. Soil samples were
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collected after harvesting plants, then air-dried and sieved (2 mm mesh) before extraction
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procedures.
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2.2. Chemical extraction and soil solution (pore water) sampling
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2.2.1. Chemical extraction
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Seven single-step extractants were used to assess the availability of Sb and compare results
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with previous studies (Ettler et al., 2007; Flynn et al., 2003; He, 2007; Mehlich, 2008). They
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were: ultrapure water, 0.01M CaCl2, DTPA (0.005 M DTPA + 0.01 M CaCl2 + 0.1 M TEA,
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pH 7.3), 0.1 M Na2HPO4, 1 M NH4H2PO4, 0.5 M NaHCO3, and M3 (0.2 M CH3COOH + 0.25
130
M NH4NO3 + 0.015 M NH4F + 0.013 M HNO3 + 0.001 M EDTA). The extractions were based
131
on a solid liquid ratio of 1:10 under agitation (200 rpm) for 2 h at 20 ± 2 °C. Then, soil
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samples were centrifuged at 2,000 × g for 15 min. The supernatant was filtered through a
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membrane filter (0.45 µm) and had its pH adjusted prior to Sb detection.
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2.2.2. Soil solution (pore water) sampling
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Soil solution was sampled using Rhizon MOM soil moisture samplers (Rhizosphere
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Research Products, Wageningen, The Netherlands). This sampler consists of a porous plastic
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tube (5 cm in length, 2.5 mm in diameter, and an average aperture of 0.15 µm), capped with a
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colorless transparent extension tube (5 cm in length), and a female luer lock at the other end.
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Soil solution was collected one week before harvesting the plants. The soil moisture samplers
140
were placed in the pots 48 h before sampling. Soil solution samples were filtered through
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membrane filters (0.45 µm) immediately after sampling for Sb detection.
6
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2.3. Antimony content in samples
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The levels of Sb were measured in crude soil samples, soil extracts, soil solution, and plant
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samples. Total Sb in soil samples were determined using atomic fluorescence spectroscopy
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(HG-AFS, AFS 9700, Titan Instrument, China) after digesting soil samples (0.2 g passed
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through a 100 mesh sieve) in microwave-assisted digestion (MARS, CEM, USA) with
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HCl+HNO3 (3:1) (Zhang et al., 2018). The samples from single-step extractions and soil
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solution collection were tested using HG-AFS after filtration. Plant samples were oven-dried
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at 80 °C, ground, and subjected to microwave-assisted digestion with HNO3. Then, the
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digestion products were analyzed in a inductively coupled plasma mass spectrometer
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(ICP-MS, NexION 300X, PerkinElmer, Waltham, MA, USA).
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All procedures of solid digestion were carried out in triplicate and with 2 blanks. Two
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standard soil reference materials (GBW07402, GBW07406), and a standard plant reference
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material (bush leaves, GSV-2), were used for quality control of the digestion and analytical
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procedures. A standard solution containing a mixture of As and Sb (GBW(E)130540) was
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used in the calibration for the atomic fluorescence spectroscopy analyses. All reference
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materials were purchased from the National Institute of Metrology (China). Antimony levels
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measured in GBW07402, GBW07406, and GSV-2 were 1.1 ± 0.1 mg kg-1, 66 ± 2 mg kg-1,
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and 0.108 ± 0.012 mg kg-1, which agreed with their expected values (1.3 ± 0.2 mg kg-1, 60 ± 7
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mg kg-1, and 0.095 ± 0.014 mg kg-1), respectively.
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2.4. Statistical analysis
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Data were analyzed using ANOVA (Duncan’s test was used for multiple comparisons) and
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Pearson correlation coefficient (r, two-tailed) in SPSS 21.0 (IBM, Armonk, NY, USA). The
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calculations of EC50 and regression (Logistic), as well as the production of all figures, were
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performed using Origin(Pro) 9 (OriginLab, Northampton, MA, USA).
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7
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3. Results and discussion
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3.1. Effects of Sb on growth of edible amaranth
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The effects of Sb on the fresh weight of roots and shoots, root length, and height of
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edible amaranth exposed to different Sb concentrations in two agricultural soils are shown in
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Figure 1. Although almost all plants survived in soil S1, a toxic inhibition effect was observed
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under different Sb exposure levels (Figs. 1a and b). Root length and height of the seedlings
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tended to decrease but no statistical difference was detected. Fresh weight of roots (P = 0.016,
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F = 4.451) and shoots (P = 0.034, F = 3.545) decreased significantly when Sb concentration in
175
soil was 1,402 mg kg-1. Conversely, amaranth cultivated in soil S2 were not significantly
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affected by Sb exposure. Biomass and growth of seedlings planted in S2 were promoted under
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low Sb levels and inhibited at high Sb levels, but these trends were not statistical significant
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(Figs. 1c and d). Root elongation was not significantly affected by any treatment both in soil
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S1 and S2. Noteworthy, the large standard error values associated with root length
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measurements might be the result of damage to seedling roots during harvesting. Therefore,
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further studies are needed to investigate whether root length can be an indicator of Sb toxicity.
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Overall, the results above indicated that the toxic effects of Sb on plants differed between
183
calcareous alkaline soil (S1) and acidic soil (S2). Unfortunately, the half maximum effective
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concentration (EC50) of Sb versus biomass could not be calculated since we could not achieve
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an adequate curve fit and the standard error was large.
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3.2. Accumulation and translocation of Sb in edible amaranth
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Expectedly, accumulation of Sb in root and shoot of edible amaranth increased as Sb
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concentration in the soils increased regardless of soil type (Fig. 2). For S1, when Sb
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concentration in soil exceeded 440 mg kg-1, Sb content in shoots (P < 0.01, F = 49.353) and
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roots (P < 0.01, F = 228.939) increased significantly. The maximum concentration of Sb in
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shoots and roots were 348 ± 81 mg kg-1 dry weight and 803 ± 62 mg kg-1 DW in plants
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exposed to 1,402 mg kg-1 Sb (Fig. 2a). In the case of soil S2, when Sb concentration in the
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soil exceeded 370 mg kg-1, Sb concentration increased significantly in shoots (P < 0.01, F = 8
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26.123) and roots (P < 0.01, F = 12.683) compared with the control group (Fig. 2b). Moreover,
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the highest concentration of Sb in the shoots was 34.3 ± 2.0 mg kg-1 DW at the Sb level of
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1,308 mg kg-1, and in roots it was 208 ± 85 mg kg-1 DW at the Sb level of 1,171 mg kg-1 in
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soil S2.
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We calculated two factors to better understand the accumulation and distribution of Sb in
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amaranth plants. The bioaccumulation factor (BAF) was calculated comparing the
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concentration of Sb in plant tissues with that in the soil. The translocation factor (TF), which
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is the ratio between Sb concentration in shoots and that in roots, was used to evaluate the
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ability of plants to translocate Sb from roots to shoots. There were no clear patterns in BAF
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and TF of edible amaranth exposed to Sb in the two soil types (Table 2). The values for BAF
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in shoot and root of plants maintained in soil S1 were 0.17–0.33 and 0.31–0.57 respectively.
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These values were higher than those for plants maintained in S2 soil, which were 0.03–0.05
206
and 0.09–0.26. For both soils, the BAFs of roots were greater than those of shoots. Seedlings
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in soil S1 (0.43–0.90) and S2 (0.16–0.38) had TF values below 1, suggesting that Sb tends to
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be stored in roots of edible amaranth. Overall, the findings indicate that the bioaccumulation
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of Sb was promoted in soil S1 compared with S2 and roots accumulated more Sb than shoots.
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These results are consistent with those obtained in maize exposed to antimony (Zhang et al.,
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2018).
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Such accumulation of Sb in amaranth is similar to that observed in sunflower (Helianthus
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annuus) cultivated in antimony-contaminated agricultural soil (Tschan et al., 2010) and
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Eucalyptus michaeliana cultivated in soils supplemented with nutrients and lime (Wilson et
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al., 2013). Since the biomass of amaranth grown in S2 (acid soil) was not significantly
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affected by Sb, the accumulation of Sb in amaranth without evident toxic symptoms might
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allow the inadvertent Sb intake through the food chain, representing a potential risk to human
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health (Corrales et al., 2014). Roots accumulated more Sb than shoots of amaranth in both
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soils, which is generally consistent with previous findings (Shtangeeva et al., 2011; Wilson et
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al., 2013; Zhang et al., 2018). This might have deleterious effects since antimony 9
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accumulation in roots affects the distribution of nutrients and trace elements in plants
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(Shtangeeva et al., 2011).
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The bioaccumulation of Sb in amaranth can reflect the status of Sb pollution in soil to some
224
extent. The Pearson’s correlation analysis showed an extremely significant correlation
225
between Sb concentrations in tissues of amaranth and total Sb in soil (Table 5). Based on our
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findings, the accumulation of Sb in tissues of amaranth might be a suitable indicator to
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evaluate the ecological risk of Sb contamination in agricultural soil. Linear regression,
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nonlinear regression-logistic were used to fit the dose-response relationship. For plants in soil
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S1, the R2 values of linear regression tended to be higher than that of other curves. The
230
equations used to quantify the relationship between total Sb concentration in tissues of
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amaranth and soils are shown in Table 3 (equations 1–4). In Table 3, “Cshoot” and “Croot” in the
232
equations refer to the concentration of Sb in plant tissues, “x” means Sb concentration in soil
233
(equations 1–4), soil solution samples (equations 4–8), and soil extracts using 0.1 M Na2HPO4
234
(equations 9–12).
235 236
3.3. Bioavailability assessment of Sb in soil
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3.3.1. Bioavailability assessment of Sb in different soil extracts
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The ability of seven single extractants to extract Sb from different two soil types are shown
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in Table 4. The extractability of Sb by the different methods can be ranked, from the highest
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to the lowest, as: M3 > NaHCO3 > Na2HPO4 > NH4H2PO4 > water > CaCl2 > DTPA for S1,
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and M3 > NaHCO3 > Na2HPO4 > water > DTPA > NH4H2PO4 > CaCl2 for S2. And the
242
extraction of Sb from soil strongly depends on the total Sb amount in soil (Ettler et al., 2007).
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With the exception of DTPA and water, more Sb could be extracted from soil S1 than from S2.
244
The extraction rates of Na2HPO4 in S1 and S2 were 13.05–25.78% (mean 19.61%) and 5.54–
245
19.68% (mean 13.37%) respectively. The extraction efficiencies of water in soil S1 and S2
246
were 3.69–9.68% (mean 6.92%) and 2.46–17.55% (mean 10.25%) respectively, which were
247
higher than the ratio of water (0.5–2.9%) in previous studies (Pierart et al., 2015). Antimony 10
248
extraction is affected by soil characteristics (such as pH, organic matter, total organic carbon,
249
and cation exchange capacity), and differences among extractants have been reported in
250
previous studies (Ettler et al., 2007; Nakamaru and Martín Peinado, 2017). Some previous
251
work reported relatively low Sb extraction percentage of Na2HPO4 (Tan et al., 2018; Zhang et
252
al., 2018) and another report (Ettler et al., 2007), whereas, the results in our study indicated a
253
much higher Sb extractability of Na2HPO4. That might be related to the characteristic and
254
properties of the tested spiked soils. More Sb was successfully extracted in antimony-spiked
255
soil than contaminated soil (Filella et al., 2009; Hansen and Pergantis, 2008; Pierart et al.,
256
2015). The condition of extraction (the particle size of the soil, temperature, time etc.) are also
257
needed to take into consideration.
258
The relationship between Sb content measured by different means and biotoxication on
259
amaranth was analyzed using the Pearson correlation coefficient (Table 5). Regardless of the
260
extractant used, Sb content in the two tested soils were significantly correlated to the total Sb
261
in soil, indicating that extracted Sb can generally be used as proxies of Sb contamination. For
262
soil S1, no relationship was found between fresh weight or root length and Sb content in soil
263
solution or Sb content in soil extract. The top three correlation coefficients between plant
264
height of amaranth and extractions protocols followed the order: DTPA = NH4H2PO4 >
265
Na2HPO4 > NaHCO3 (P < 0.05). Antimony content in shoots of amaranth was significantly (P
266
< 0.01) correlated with Sb concentration measured in all soil extracts and soil solution; the top
267
three correlation coefficients followed the order: NaHCO3 = Na2HPO4 > NH4H2PO4 > M3. A
268
similar correlation was reported between extracted Sb in the soil and Sb in leaves of spinach
269
(Hammel et al., 2000). The correlation coefficients between Sb yield by single extractants and
270
Sb concentration in roots ranked from the largest to the lowest were DTPA, NaHCO3, M3,
271
and Na2HPO4. For soil S1, Sb extracted by 0.1 M Na2HPO4, 0.5 M NaHCO3 and M3 are the
272
most relevant extractions to assess the biological effects of Sb on plants. In the case of soil S2,
273
no relationship was found between biomass or growth and soil solution or single extractant.
274
Antimony content in both shoots and roots of amaranth was significantly (P < 0.01) correlated 11
275
with all single extractants and with soil solution Sb levels (P < 0.05) as well. The top three
276
correlation coefficients for shoots were Na2HPO4 > NH4H2PO4 > M3, and for roots they were
277
water > Na2HPO4 > NH4H2PO4, which were different from the results of potted maize (Zhang
278
et al., 2018). That might be related to the properties of soil and species of plant.
279
In summary, for both soil S1 and S2, the most efficient extractants were M3, NaHCO3, and
280
Na2HPO4. According to Pearson correlation coefficient, the seven single extraction methods
281
and soil solution can be used to predict bioavailability of Sb. Considering the extractability
282
and correlation coefficients between biological effects and extractable Sb, NaHCO3, Na2HPO4,
283
and M3 might effectively predict the bioavailability of Sb in alkaline soil, whereas Na2HPO4,
284
NH4H2PO4, and M3 might do so in acid soil. Therefore, we suggest the use of 0.1 M Na2HPO4
285
and M3 as extractants to appropriately predict the bioavailability of Sb in soil.
286
3.3.2. Bioavailability assessment of Sb in soil solution
287
Antimony content in the spiked soils (S1 and S2) released into soil solution were
288
determined to assess its bioavailability (Fig. 3). The pH of soil S1 and S2 treated by Sb and its
289
soil solution (pH 7.95–8.39, 5.90–6.29) did not change significantly. For both soil types, Sb
290
content in soil solutions increased significantly and was correlated with total Sb in soil;
291
correlation coefficients between total Sb in soil and Sb in soil solution were 0.946 (P < 0.01)
292
and 0.963 (P < 0.01) for S1 and S2 respectively. In soil S1, when Sb concentration exceeded
293
440 mg kg-1, Sb released into soil solution increased significantly (P < 0.01, F = 180.022). In
294
soil S2, at Sb levels higher than 200 mg kg-1, total Sb content in soil solutions increased with
295
the increasing addition of Sb; this trend was also observed for Sb concentration in seedlings.
296
More Sb was released into soil solution in alkaline soil (0–58.78 mg L-1) than that in acid soil
297
(0.02–17.84 mg L-1). The fraction of Sb released into soil solution in soil S1 and S2 were
298
1.95%–4.19% and 0.12%–2.04%, respectively. Such observation is similar as the study of
299
Wilson and colleagues (Wilson et al., 2013). One reason to explain that is the effect of soil pH
300
on Sb release into pore water (Nakamaru and Martín Peinado, 2017). Indeed, previous
301
findings reported that the biogeochemical behavior of Sb in the environment greatly depends 12
302
on environmental factors such as soil pH and redox potential (Eh), which can impact the
303
adsorption and deposition of Sb (Wilson et al., 2010; Wilson et al., 2014). For instance, the
304
adsorption of Sb(V) by Fe and Al(OH)x is inhibited under increased soil pH values, leading to
305
higher Sb levels in soil solution (Nakamaru et al., 2006). Another reason might be that Sb
306
solubility is controlled by the presence of Ca[Sb(OH)6]2 (Okkenhaug et al., 2011), while S1 is
307
kind of calcareous alkaline soil with high level of calcium carbonate content ( >95%).
308
The concentration of Sb released into soil solution of both soils was significantly correlated
309
with levels of Sb accumulation in amaranth (Table 5), which might be that the absorption of
310
Sb by plants is proportional to the concentration of soluble Sb in soil (Tschan et al., 2009).
311
For soil S1, this correlation extremely significantly (P < 0.01) for amaranth seedlings (both
312
roots and shoots). In the case of S2, Sb content in soil solution was positively correlated with
313
Sb concentration in roots (P < 0.05) and shoots (P < 0.01) of amaranth. Although the transport
314
mechanism of Sb(V) in plants is still unknown (Tschan et al., 2009), we observed a
315
dose-effect relationship. Our results support the use of Sb levels in soil solution to predict the
316
accumulation of Sb in plants. The equations for Sb accumulation in amaranth and Sb in soil
317
solution are shown in Table 3 (equations 5–8). Moreover, our findings suggest that Sb in
318
alkaline soil is more easily absorbed by root and transported to shoot by plants, causing more
319
severe effects than those of Sb in acid soil even at the same Sb exposure level.
320
3.3.3. Prediction of Sb bioavailability in soil
321
Although significant correlations were detected between Sb levels in soil solution and Sb
322
accumulation in plants, their coefficients were slightly lower than those between some soil
323
extracts and Sb content in plants. In that regard, the analysis of soil solution might not the
324
most convenient indicator of Sb levels in soil because of possible errors during sampling
325
(such as soil moisture) and the relatively high costs of samplers. Nevertheless, knowing the
326
characteristics of soil solution, a medium in direct contact with ions in soil and plant roots,
327
can provide relevant information about soil conditions. Conversely, single extraction
328
protocols are effective tools for predicting bioavailability. In general they have lower costs 13
329
and simpler procedures than those of soil solution analyses. Still, there are key differences
330
between extraction protocols. For example, the composition and preparation of the M3
331
extractant are relatively complex, whereas those of 0.1 M Na2HPO4 extractant is simple and
332
convenient. Therefore, we suggest 0.1 M Na2HPO4 to be used to assess mobile Sb content in
333
both alkaline and acid soil. The equations describing the relationship between Sb
334
concentration in shoot and root of amaranth and Sb extracted using 0.1 M Na2HPO4 are
335
shown in Table 3 (equations 9–12).
336
4. Conclusion
337
We observed different toxic effects of Sb to amaranth between two types of soils (alkaline
338
and acid). In alkaline soil, Sb was more easily taken up by root and transported to shoot by
339
plants, causing more adverse toxic effects than in acid soil. Antimony in alkaline soil
340
significantly impacted the fresh weight of roots and shoots, decreasing the biomass of both
341
tissues at the highest concentration. Conversely, growth and biomass were not significantly
342
affected by Sb in acid soil, underscoring the need to watch for asymptomatic accumulation of
343
Sb by plants in contaminated agricultural soils.
344
Antimony extracted by single extractants and in soil solution could be used as good
345
predictors of Sb bioavailability. The Sb extraction efficiency differed among extractants and
346
soil types. For both alkaline and acid soils, M3, NaHCO3 and Na2HPO4 were the most
347
efficient extractants. In alkaline soils, NaHCO3, Na2HPO4, and M3 might be more appropriate
348
to predict the bioavailability of Sb, whereas Na2HPO4, NH4H2PO4, and M3 are suitable for
349
acid soil analyses. We suggest 0.1 M Na2HPO4 as a general extractant for assessing the
350
bioavailability of Sb regardless of soil type. However, if Sb and nutrients need to be evaluated
351
simultaneously, M3 is a good choice. Moreover, relevant environmental information about
352
contaminated soil can be provided by the assessment of soil solution. Lastly, considering that
353
this study was based on two kinds of spiked soils, further studies on different types of natural
354
soils in contaminated sites are needed. The standardization of the extraction processes and the
355
better understanding of the mechanisms underlying antimony toxicity in contaminated soil 14
356
warrant further research.
357 358
Acknowledgements
359
This work is part of the project “Research on Migration/Transformation and Safety
360
Threshold of Heavy Metals in Farmland Systems” (2016YFD0800405), which was supported
361
by National Key Research and Development Program.
362 363
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364
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19
472
Table 1. Physical and chemical properties of S1 and S2 soils. Sb Soil
Soil type
CEC
CaCO3
Amorphous Fe
Amorphous Mn
Particle composition (%)
(g/kg)
(cmol/kg)
(g/kg)
(g/kg)
(g/kg)
2–0.05 mm
0.05–0.002 mm
< 0.002 mm
pH -1
(mg kg )
473 474
OM
S1
Chestnut soil
1.50
8.39
21.63
17.28
96.56
1.43
0.36
27.64
55.67
16.69
S2
Red earth
2.73
4.91
46.10
18.00
0.80
7.87
0.12
14.98
45.08
39.94
OM, organic matter. CEC, cation-exchange capacity.
20
475
Table 2. Bioaccumulation factor (BAF) and translocation factor (TF) in edible amaranth (Amaranthus
476
tricolor Linn.) seedlings exposed to different antimony concentrations in alkaline (S1) and acid (S2)
477
soils. Total Sb Shoot BAF
Experimental
Root BAF
TF
(mg kg-1) group S1
S2
S1
S2
S1
S2
S1
S2
Control
1.50
2.73
0.33
0.05
0.52
0.24
0.64
0.20
1
71.30
102.00
0.32
0.03
0.39
0.09
0.83
0.38
2
218.00
193.00
0.17
0.05
0.19
0.20
0.90
0.23
3
440.00
370.00
0.33
0.05
0.39
0.26
0.85
0.20
1165.0
1171.0 0.20
0.03
0.31
0.14
0.64
0.20
0.25
0.03
0.57
0.16
0.43
0.16
0.27
0.04
0.39
0.18
0.72
0.23
4 0
0
1402.0
1308.0
5
Mean
0
0
–
–
478
21
479
Table 3. Equations used to evaluate the relationship between Sb accumulation in plant tissues and Sb
480
concentration in the two soils (1–4), soil solution samples (4–8), and soil extracts using 0.1 M
481
Na2HPO4 (9–12). Soil
R2
Equation number
Cshoot = 3.935 + 0.2340x
0.939
(1)
Croot = 0.5099x - 42.56
0.921
(2)
Cshoot = (30.60 - 30.28) / (1 + (x / 294.8)2.177)
0.889
(3)
Croot = (210.2 - 212.7) / (1 + (x / 419.6)1.787)
0.772
(4)
Cshoot = 829.0 + (2.146 - 829.0) / (1 + (x / 83.51)0.847)
0.938
(5)
Croot = 2.297 + 13.5x
0.988
(6)
Cshoot = 30.30+(2.27-30.30)/(1+(x/2.02)3.41)
0.798
(7)
Croot = 10.10 + 30.9x
0.780
(8)
Cshoot = 1.23x - 19.59
0.895
(9)
Croot = 2.53x - 75.98
0.779
(10)
Cshoot = 29.42 - 24.34 / (1 + (x / 67.99)7.66)
0.862
(11)
Croot = 1.29x - 1.27
0.865
(12)
Equation
S1
S2
S1
S2
S1
S2
482
22
483
Table 4. Antimony extraction efficiency (%) from soils S1 and S2 using single extractants. Extractant Soil
484
Water
CaCl2
DTPA
NaHCO3
NH4H2PO4
Na2HPO4
M3
S1-1
9.25
7.40
1.46
37.37
25.13
25.78
59.63
S1-2
9.68
7.51
6.58
27.56
23.34
20.87
53.87
S1-3
7.98
7.97
4.22
28.33
21.02
22.18
55.99
S1-4
3.99
3.43
2.47
18.61
11.68
13.05
23.36
S1-5
3.69
3.86
6.69
22.69
14.65
16.18
36.28
Mean
6.92
6.03
4.29
26.91
19.16
19.61
45.83
S2-1
2.46
0.35
9.36
6.35
3.35
5.54
7.90
S2-2
12.15
3.32
10.10
18.03
7.97
18.34
21.53
S2-3
17.55
7.24
10.15
21.20
8.25
19.68
20.62
S2-4
11.35
4.32
9.78
11.12
4.63
10.26
11.66
S2-5
7.72
4.96
9.89
16.87
5.81
13.03
14.93
Mean
10.25
4.04
9.86
14.71
6.00
13.37
15.33
M3, Mehlich 3 extraction solution.
23
485
Table 5. Linear correlation coefficients (r) between plant parameters and Sb content in soil, soil
486
solution, or different soil extracts from S1 and S2 soils. Extractant
Soil Parameter
Soil Sb solution Sb
Water
CaCl2
DTPA
NaHCO3
NH4H2PO4
Na2HPO4
M3
S1 soil Fresh Weightroot
-0.262
-0.4
-0.206
-0.211
-0.524
-0.359
-0.372
-0.354
-0.35
Fresh Weightshoot
-0.687
-0.698
-0.768
-0.75
-0.758
-0.761
-0.79
-0.769
-0.757
Root Length
-0.088
-0.136
-0.043
0.007
-0.214
-0.134
-0.137
-0.12
-0.088
Stem Height
-0.759
-0.792
-0.773
-0.772
-0.843*
-0.824*
-0.843*
-0.828*
-0.809
Sb-Shoot
0.980**
0.969**
0.938**
0.964**
0.918**
0.990**
0.985**
0.990**
0.978**
Sb-Root
0.937**
0.999**
0.840*
0.889*
0.979**
0.972**
0.963**
0.969**
0.972**
Sb-soil
–
0.946**
0.937**
0.936**
0.873*
0.982**
0.970**
0.976**
0.934**
Fresh Weightroot
-0.608
-0.573
-0.657
-0.613
-0.606
-0.574
-0.597
-0.597
-0.593
Fresh Weightshoot
-0.537
-0.505
-0.596
-0.545
-0.535
-0.506
-0.53
-0.531
-0.526
Root Length
-0.079
-0.219
-0.19
-0.209
-0.084
-0.159
-0.141
-0.16
-0.134
Stem Height
-0.56
-0.533
-0.552
-0.567
-0.557
-0.519
-0.52
-0.516
-0.515
Sb-Shoot
0.971**
0.977**
0.968**
0.989**
0.973**
0.986**
0.997**
0.998**
0.995**
Sb-Root
0.944**
0.900*
0.999**
0.936**
0.944**
0.921**
0.962**
0.961**
0.958**
Sb-soil
–
0.963**
0.937**
0.981**
1.000**
0.980**
0.985**
0.979**
0.985**
S2 soil
487
*Correlation is significant at the 0.01 level (two-tailed).
488
**Correlation is significant at the 0.05 level (two-tailed). 24
489
Figure captions
490
Figure 1. Biomass and length of edible amaranth (Amaranthus tricolor Linn.) exposed to different
491
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth). Fresh
492
weight of root (black columns) and shoot (red columns) of plants grown in S1 (a) and S2 (c) soils.
493
Length of root (black columns) and height (red columns) of plants grown in S1 (b) and S2 (d)
494
soils. Different letters (e.g. a, b, c) indicate statistically significant differences at P<0.05.
495 496
Figure 2. Antimony uptake by edible amaranth (Amaranthus tricolor Linn.) exposed to different
497
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth).
498
Antimony concentration (mg kg-1 dry weight) in root (black columns) and shoot (red columns) of
499
plants grown in S1 (a) and S2 (b) soils. Different letters indicate statistically significant
500
differences at P<0.05.
501 502
Figure 3. Antimony concentration in soil solutions collected from (a) alkaline (S1, chestnut soil) and
503
(b) acidic soil (S2, red earth). Different letters indicate statistically significant differences at
504
P<0.05.
25
505 80
2.5 a
2.0
70
b
Root Shoot
a a
a
ab 60
ab
Length (mm)
Fresh weight (g)
a
Root Shoot
ab
1.5 b
b
1.0
ab
ab b
50
40
a
a
ab
0.5
ab
ab a
ab
ab
a
c
b
30
c
0.0 1.50
71.3
440
218
1,165
20
1,402
1.50
71.3
Sb in soil S1 (mg kg-1)
506
c
Root Shoot
6
Length (mm)
Fresh weight (g)
ab a
ab
0
2.73
ab
102
ab
193
370
a
a a
a
a
ab
60
bc
bc a
b
c
40
1,171 1,308
2.73
-1
507
a
b a
d
a
80
ab
ab
1,402
Root Shoot a
2
1,165
100
a 4
440
218
Sb in soil S1 (mg/kg)
102
193
370
1,171
1,308
-1
Sb in soil S2 (mg kg )
Sb in soil S2 (mg kg )
508 509
Figure 1. Biomass and length of edible amaranth (Amaranthus tricolor Linn.) exposed to different
510
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth). Fresh weight
511
of root (black columns) and shoot (red columns) of plants grown in S1 (a) and S2 (c) soils. Length of
512
root (black columns) and height (red columns) of plants grown in S1 (b) and S2 (d) soils. Different
513
letters (e.g. a, b, c) indicate statistically significant differences at P<0.05.
26
515
1000
a
Shoot Root
800
Sb concentration in amaranth (mg kg-1 DW)
Sb concentration in amaranth (mg kg-1 DW)
514
d
600
400
c
d
c 200
0
b a
a
1.50
a
a
71.3
a
218
b
a
440
1,165
1,402
300
b
Shoot Root
250
cd 200
cd 150
bc
100 50 0
ab a
2.73
a
a
a
102
a
193
b
370
c
c
1,171
1,308
Sb in soil S2 (mg kg-1)
Sb in soil S1 (mg kg-1)
516
Figure 2. Antimony uptake by edible amaranth (Amaranthus tricolor Linn.) exposed to different
517
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth). Antimony
518
concentration (mg kg-1 dry weight) in root (black columns) and shoot (red columns) of plants grown in
519
S1 (a) and S2 (b) soils. Different letters indicate statistically significant differences at P<0.05.
27
521
a
100
Sb concentration in soil solution (mg/L)
Sb concentration in soil solution (mg/L)
520
80 d
60
40 c 20
0
b a
a
1.50
71.3
a
218
440
1,165
1,402
Sb in soil S1(mg/kg)
b
25
20
d
15
cd 10
bc 5
ab 0
a
a
2.73
102
193
370
1,171
1,308
Sb in soil S2 (mg/kg)
522 523
Figure 3. Antimony concentration in soil solutions collected from (a) alkaline (S1, chestnut soil) and
524
(b) acidic soil (S2, red earth). Different letters indicate statistically significant differences at P<0.05.
525
28
Highlights: 1. Antimony in alkaline and acid soil caused different toxic effects to amaranth. 2. More attention should be paid on asymptomatic accumulation of Sb in plants. 3. Soil solution and single extraction were used to assess the bioavailability of Sb. 4. 0.1 M Na2HPO4 is the best extractant to predict the bioavailability of Sb in soil. 5. Mehlich 3 and soil solution are alternative to assess the bioavailability of Sb.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0269-7491(19)35282-0
DOI:
https://doi.org/10.1016/j.envpol.2019.113642
Reference:
ENPO 113642
To appear in:
Environmental Pollution
Received Date: 15 September 2019 Revised Date:
31 October 2019
Accepted Date: 16 November 2019
Please cite this article as: Zhong, Q., Ma, C., Chu, J., Wang, X., Liu, X., Ouyang, W., Lin, C., He, M., Toxicity and bioavailability of antimony in edible amaranth (Amaranthus tricolor Linn.) cultivated in two agricultural soil types, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113642. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphical abstract
1
Toxicity and bioavailability of antimony in edible amaranth
2
(Amaranthus tricolor Linn.) cultivated in two agricultural soil
3
types
4 5
Qianyun Zhong, Congli Ma, Jianwen Chu, Xiaolin Wang, Xitao Liu, Wei Ouyang, Chunye
6
Lin, Mengchang He*
7 8
State Key Laboratory of Water Environment Simulation, School of Environment, Beijing
9
Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, China
10 11
*Corresponding author:
12
Mengchang He
13
State Key Laboratory of Water Environment Simulation
14
School of Environment
15
Beijing Normal University
16
Beijing, 100875, China
17
Tel & fax: +86-10-5880 7172
18
E-mail: [email protected]
1
19
Abstract
20
Although elevated levels of antimony (Sb) in agricultural soil and plant systems can have
21
harmful effects on human health and ecosystems, little is known about the toxicity of Sb to
22
plants and its mechanism. The assessment of Sb bioavailability is essential for understanding
23
its potential risks and toxicity. In this study, we used pot experiments with two agricultural
24
soil types spiked with Sb to investigate the dose-effect relationship between exposure to Sb
25
and toxic effects (growth and bioaccumulation) on edible amaranth (Amaranthus tricolor
26
Linn). Soil solution (pore water) and seven single extractants were used to assess the
27
bioavailability of Sb. Different toxic effects of Sb to amaranth cultivated in two types of soils
28
(alkaline and acid soil) were observed. In alkaline soil (chestnut soil, pH 8.39), antimony is
29
more easily absorbed by root and transported to shoot by plants, leading to more adverse
30
effects, than in acid soil (pH 4.91) under the same exposure level. Our findings also highlight
31
the need for more attention on asymptomatic accumulation of Sb in plants, especially for
32
agricultural products cultivated in contaminated areas. The extraction efficiency of Sb was
33
various in different extractants and soil types, Mehlich 3, NaHCO3 and Na2HPO4 for Sb were
34
more efficient than other extractants in both tested alkaline and acid soil. Based on the
35
extractability and correlation coefficients of toxic effects on amaranth and extractable Sb, we
36
found that 0.1 M Na2HPO4 is the best extractant to predict the bioavailability of Sb in soil,
37
and M3 is a suitable alternative. Antimony concentration in soil solution can also be used as
38
an alternative indicator of the bioavailability of Sb.
39 40
Capsule: Toxicity and bioavailability of antimony in edible amaranth in soils
41 42
Keywords: antimony; toxicity; bioavailability; extraction; soil solution (pore water).
43
2
44
1. Introduction
45
Antimony (Sb) and its compounds are toxic to humans with potential carcinogen effects
46
(Filella et al., 2009; Friberg et al., 2005; Gebel et al., 1997; Hammel et al., 2000). A large
47
amount of Sb have been released into the environment due to several activities related to Sb,
48
including its mining and smelting, and the production and use of antimony-based products
49
(Chu et al., 2019; He et al., 2012). As a natural trace element, Sb is not necessary for plants
50
(Ainsworth et al., 1991). Nevertheless, it can be absorbed from the soil by plants through the
51
root system and cause toxic effects (Ainsworth et al., 1991; He and Yang, 1999; Ma et al.,
52
2019; Shtangeeva et al., 2011; Tschan et al., 2009; Wang et al., 2018). Therefore, plants
53
growing in contaminated areas can accumulate Sb (Okkenhaug et al., 2011; Qi et al., 2011;
54
Telford et al., 2009; Wu et al., 2019). Such enrichment of Sb in food crops, which might be
55
asymptomatic, is potentially deleterious to ecosystems and humans (Cai et al., 2016; Corrales
56
et al., 2014; Feng et al., 2013; Wu et al., 2011). For people living in the vicinity of Sb mines
57
in China, the dietary intake of Sb (554 µg/day) is 1.5-fold higher than the tolerable daily
58
intake (TDI, 360 µg/day) (Wu et al., 2011). This increased Sb intake was attributed to the
59
consumption of rice, vegetables (especially leaf vegetables), drinking water and meat (Wu et
60
al., 2011). However, the mechanisms of Sb uptake, translocation, and toxicity to plants, as
61
well as any dose-effect relationship, remain unclear (Ji et al., 2017; Wang et al., 2018).
62
Therefore, we need to pay extra attention on the environment quality on Sb contaminated soil,
63
especially agricultural soil.
64
The bioavailability of metal(loid)s in the soil is defined as the amount of a given metal(loid)
65
that can be absorbed and have an active effect on organisms, and it depends on its species in
66
environment (He et al., 2019; Shahid et al., 2012; Wilson et al., 2010). In the soil samples
67
collected from Sb mining area (Xikuangshan, China), Sb(V) is the dominate fraction of
68
bioavailable Sb at a high level (6.3–748 mg kg-1) (Okkenhaug et al., 2011). Microbial
69
communities were affected by extractable Sb and pH, and various in different utilization types
70
of soil (Sun et al., 2019). The measurement of the bioavailability of several contaminants in 3
71
ecosystems can provide an accurate ecological risk assessment. The bioavailability of Sb in
72
soil is commonly assessed using traditional chemical extraction methods, including
73
single-step extraction and sequential extraction protocols (Ettler et al., 2007; He, 2007).
74
Among other extraction solutions, Mehlich 3 (M3) is a combination of chemicals widely used
75
to extract several micro and macronutrients from different soil types (Mehlich, 1978, 2008;
76
Sims et al., 2002). Diffusive gradients in thin films (DGT) have also been used to detect
77
bioavailable Sb (Wang et al., 2018). In addition to the extraction of metal(loid)s from soil
78
samples, the direct assessment of metals in soil solution (pore water) might be more sensitive
79
than extractions in laboratory and thus more relevant to the assessment of ecological risks
80
(Moreno-Jimenez et al., 2011).
81
In this study, we designed pot experiments using two different types of farmland soil spiked
82
with Sb. The aims were: (1) to investigate the toxic effects (growth and bioaccumulation) of
83
Sb on edible amaranth grown in two agricultural soil types, and (2) to assess the
84
bioavailability of Sb in different types of farmland soil analyzing soil solution and soil
85
extracts produced by several extraction protocols.
86 87
2. Materials and methods
88
2.1. Pot experiments
89
2.1.1. Soil types and treatments
90
Two Chinese agricultural soils were collected at a depth of 0–20 cm from Datong in Shanxi
91
Province (“chestnut soil”, calcareous alkaline soil, S1) and Jiangmen in Guangdong Province
92
(“red earth”, acid soil, S2). Physical and chemical properties of the soils are shown in Table 1.
93
Soil samples were air-dried and sieved, and different levels of analytical grade potassium
94
antimony tartrate (0, 100, 200, 500, 1,000, 1,500 mg kg-1) were added to the soils. After aging
95
for 14 days under 70% of the field water capacity, soil samples were air-dried and passed 4
96
through a 2 mm sieve prior to pot experiments. Aged soil (700 g) was put into the pots, which
97
were set up as three replicates for each experimental condition in a randomized block design.
98
We tested the isolated effects of tartrate and potassium ions (up to 2,000 mg kg-1) in a set of
99
preliminary experiments and found no effect of these ions on plant growth.
100
2.2.2. Plants
101
Several antimony-sensitive species were screened from common vegetables in a set of
102
preliminary experiments on germination and root elongation. Edible amaranth (Amaranthus
103
tricolor Linn.) was chosen considering its adaptability to a wide range of soil properties and
104
tolerance to high humidity. Edible amaranth seeds were purchased from the Institute of
105
Vegetables (Chinese Academy of Agricultural Sciences), immersed in 2% H2O2 for 30 min,
106
and washed with ultrapure water. Then, seeds were transferred to a petri dish covered with
107
wet filter paper and incubated at 4 °C for 24 h for pregermination. Soil samples in the pots
108
were hydrated to approximately 70% of the field water capacity and incubated for 2 d. Basic
109
fertilizer (0.429 g CO(NH2)2, 0.263 g KH2PO4, and 0.42 g KCl per kg of soil) was
110
incorporated into soil with irrigation. Ten seeds with the germ not exceeding 2 mm were
111
sowed in each pot. Pots were placed in a climate chamber (PQX-600P, Saifu, China) with
112
preset conditions (12 h photoperiod with light intensity 11,000 lux, day/night temperature of
113
25 and 20 °C, and 50% relative humidity). The water content of the soil was controlled by
114
weighting the pots and water was added to the bottom cup of each pot to maintain 70% of the
115
field water capacity. The positions of the pots in the climate chamber were changed randomly
116
every day. After each seedling produced 2 leaves, the number of seedlings in each pot was
117
reduced to 6, which were at the same growth stage. 5
118
Whole plants were harvested after 6 weeks of growth, washed with deionized water, and
119
separated into shoot and root. Fresh weights of roots and shoots were obtained with an
120
electronic balance after drying the samples’ surface water using absorbent paper. Main root
121
length and plant height of edible amaranth were measured with caliper. Soil samples were
122
collected after harvesting plants, then air-dried and sieved (2 mm mesh) before extraction
123
procedures.
124
2.2. Chemical extraction and soil solution (pore water) sampling
125
2.2.1. Chemical extraction
126
Seven single-step extractants were used to assess the availability of Sb and compare results
127
with previous studies (Ettler et al., 2007; Flynn et al., 2003; He, 2007; Mehlich, 2008). They
128
were: ultrapure water, 0.01M CaCl2, DTPA (0.005 M DTPA + 0.01 M CaCl2 + 0.1 M TEA,
129
pH 7.3), 0.1 M Na2HPO4, 1 M NH4H2PO4, 0.5 M NaHCO3, and M3 (0.2 M CH3COOH + 0.25
130
M NH4NO3 + 0.015 M NH4F + 0.013 M HNO3 + 0.001 M EDTA). The extractions were based
131
on a solid liquid ratio of 1:10 under agitation (200 rpm) for 2 h at 20 ± 2 °C. Then, soil
132
samples were centrifuged at 2,000 × g for 15 min. The supernatant was filtered through a
133
membrane filter (0.45 µm) and had its pH adjusted prior to Sb detection.
134
2.2.2. Soil solution (pore water) sampling
135
Soil solution was sampled using Rhizon MOM soil moisture samplers (Rhizosphere
136
Research Products, Wageningen, The Netherlands). This sampler consists of a porous plastic
137
tube (5 cm in length, 2.5 mm in diameter, and an average aperture of 0.15 µm), capped with a
138
colorless transparent extension tube (5 cm in length), and a female luer lock at the other end.
139
Soil solution was collected one week before harvesting the plants. The soil moisture samplers
140
were placed in the pots 48 h before sampling. Soil solution samples were filtered through
141
membrane filters (0.45 µm) immediately after sampling for Sb detection.
6
142
2.3. Antimony content in samples
143
The levels of Sb were measured in crude soil samples, soil extracts, soil solution, and plant
144
samples. Total Sb in soil samples were determined using atomic fluorescence spectroscopy
145
(HG-AFS, AFS 9700, Titan Instrument, China) after digesting soil samples (0.2 g passed
146
through a 100 mesh sieve) in microwave-assisted digestion (MARS, CEM, USA) with
147
HCl+HNO3 (3:1) (Zhang et al., 2018). The samples from single-step extractions and soil
148
solution collection were tested using HG-AFS after filtration. Plant samples were oven-dried
149
at 80 °C, ground, and subjected to microwave-assisted digestion with HNO3. Then, the
150
digestion products were analyzed in a inductively coupled plasma mass spectrometer
151
(ICP-MS, NexION 300X, PerkinElmer, Waltham, MA, USA).
152
All procedures of solid digestion were carried out in triplicate and with 2 blanks. Two
153
standard soil reference materials (GBW07402, GBW07406), and a standard plant reference
154
material (bush leaves, GSV-2), were used for quality control of the digestion and analytical
155
procedures. A standard solution containing a mixture of As and Sb (GBW(E)130540) was
156
used in the calibration for the atomic fluorescence spectroscopy analyses. All reference
157
materials were purchased from the National Institute of Metrology (China). Antimony levels
158
measured in GBW07402, GBW07406, and GSV-2 were 1.1 ± 0.1 mg kg-1, 66 ± 2 mg kg-1,
159
and 0.108 ± 0.012 mg kg-1, which agreed with their expected values (1.3 ± 0.2 mg kg-1, 60 ± 7
160
mg kg-1, and 0.095 ± 0.014 mg kg-1), respectively.
161
2.4. Statistical analysis
162
Data were analyzed using ANOVA (Duncan’s test was used for multiple comparisons) and
163
Pearson correlation coefficient (r, two-tailed) in SPSS 21.0 (IBM, Armonk, NY, USA). The
164
calculations of EC50 and regression (Logistic), as well as the production of all figures, were
165
performed using Origin(Pro) 9 (OriginLab, Northampton, MA, USA).
166
7
167
3. Results and discussion
168
3.1. Effects of Sb on growth of edible amaranth
169
The effects of Sb on the fresh weight of roots and shoots, root length, and height of
170
edible amaranth exposed to different Sb concentrations in two agricultural soils are shown in
171
Figure 1. Although almost all plants survived in soil S1, a toxic inhibition effect was observed
172
under different Sb exposure levels (Figs. 1a and b). Root length and height of the seedlings
173
tended to decrease but no statistical difference was detected. Fresh weight of roots (P = 0.016,
174
F = 4.451) and shoots (P = 0.034, F = 3.545) decreased significantly when Sb concentration in
175
soil was 1,402 mg kg-1. Conversely, amaranth cultivated in soil S2 were not significantly
176
affected by Sb exposure. Biomass and growth of seedlings planted in S2 were promoted under
177
low Sb levels and inhibited at high Sb levels, but these trends were not statistical significant
178
(Figs. 1c and d). Root elongation was not significantly affected by any treatment both in soil
179
S1 and S2. Noteworthy, the large standard error values associated with root length
180
measurements might be the result of damage to seedling roots during harvesting. Therefore,
181
further studies are needed to investigate whether root length can be an indicator of Sb toxicity.
182
Overall, the results above indicated that the toxic effects of Sb on plants differed between
183
calcareous alkaline soil (S1) and acidic soil (S2). Unfortunately, the half maximum effective
184
concentration (EC50) of Sb versus biomass could not be calculated since we could not achieve
185
an adequate curve fit and the standard error was large.
186
3.2. Accumulation and translocation of Sb in edible amaranth
187
Expectedly, accumulation of Sb in root and shoot of edible amaranth increased as Sb
188
concentration in the soils increased regardless of soil type (Fig. 2). For S1, when Sb
189
concentration in soil exceeded 440 mg kg-1, Sb content in shoots (P < 0.01, F = 49.353) and
190
roots (P < 0.01, F = 228.939) increased significantly. The maximum concentration of Sb in
191
shoots and roots were 348 ± 81 mg kg-1 dry weight and 803 ± 62 mg kg-1 DW in plants
192
exposed to 1,402 mg kg-1 Sb (Fig. 2a). In the case of soil S2, when Sb concentration in the
193
soil exceeded 370 mg kg-1, Sb concentration increased significantly in shoots (P < 0.01, F = 8
194
26.123) and roots (P < 0.01, F = 12.683) compared with the control group (Fig. 2b). Moreover,
195
the highest concentration of Sb in the shoots was 34.3 ± 2.0 mg kg-1 DW at the Sb level of
196
1,308 mg kg-1, and in roots it was 208 ± 85 mg kg-1 DW at the Sb level of 1,171 mg kg-1 in
197
soil S2.
198
We calculated two factors to better understand the accumulation and distribution of Sb in
199
amaranth plants. The bioaccumulation factor (BAF) was calculated comparing the
200
concentration of Sb in plant tissues with that in the soil. The translocation factor (TF), which
201
is the ratio between Sb concentration in shoots and that in roots, was used to evaluate the
202
ability of plants to translocate Sb from roots to shoots. There were no clear patterns in BAF
203
and TF of edible amaranth exposed to Sb in the two soil types (Table 2). The values for BAF
204
in shoot and root of plants maintained in soil S1 were 0.17–0.33 and 0.31–0.57 respectively.
205
These values were higher than those for plants maintained in S2 soil, which were 0.03–0.05
206
and 0.09–0.26. For both soils, the BAFs of roots were greater than those of shoots. Seedlings
207
in soil S1 (0.43–0.90) and S2 (0.16–0.38) had TF values below 1, suggesting that Sb tends to
208
be stored in roots of edible amaranth. Overall, the findings indicate that the bioaccumulation
209
of Sb was promoted in soil S1 compared with S2 and roots accumulated more Sb than shoots.
210
These results are consistent with those obtained in maize exposed to antimony (Zhang et al.,
211
2018).
212
Such accumulation of Sb in amaranth is similar to that observed in sunflower (Helianthus
213
annuus) cultivated in antimony-contaminated agricultural soil (Tschan et al., 2010) and
214
Eucalyptus michaeliana cultivated in soils supplemented with nutrients and lime (Wilson et
215
al., 2013). Since the biomass of amaranth grown in S2 (acid soil) was not significantly
216
affected by Sb, the accumulation of Sb in amaranth without evident toxic symptoms might
217
allow the inadvertent Sb intake through the food chain, representing a potential risk to human
218
health (Corrales et al., 2014). Roots accumulated more Sb than shoots of amaranth in both
219
soils, which is generally consistent with previous findings (Shtangeeva et al., 2011; Wilson et
220
al., 2013; Zhang et al., 2018). This might have deleterious effects since antimony 9
221
accumulation in roots affects the distribution of nutrients and trace elements in plants
222
(Shtangeeva et al., 2011).
223
The bioaccumulation of Sb in amaranth can reflect the status of Sb pollution in soil to some
224
extent. The Pearson’s correlation analysis showed an extremely significant correlation
225
between Sb concentrations in tissues of amaranth and total Sb in soil (Table 5). Based on our
226
findings, the accumulation of Sb in tissues of amaranth might be a suitable indicator to
227
evaluate the ecological risk of Sb contamination in agricultural soil. Linear regression,
228
nonlinear regression-logistic were used to fit the dose-response relationship. For plants in soil
229
S1, the R2 values of linear regression tended to be higher than that of other curves. The
230
equations used to quantify the relationship between total Sb concentration in tissues of
231
amaranth and soils are shown in Table 3 (equations 1–4). In Table 3, “Cshoot” and “Croot” in the
232
equations refer to the concentration of Sb in plant tissues, “x” means Sb concentration in soil
233
(equations 1–4), soil solution samples (equations 4–8), and soil extracts using 0.1 M Na2HPO4
234
(equations 9–12).
235 236
3.3. Bioavailability assessment of Sb in soil
237
3.3.1. Bioavailability assessment of Sb in different soil extracts
238
The ability of seven single extractants to extract Sb from different two soil types are shown
239
in Table 4. The extractability of Sb by the different methods can be ranked, from the highest
240
to the lowest, as: M3 > NaHCO3 > Na2HPO4 > NH4H2PO4 > water > CaCl2 > DTPA for S1,
241
and M3 > NaHCO3 > Na2HPO4 > water > DTPA > NH4H2PO4 > CaCl2 for S2. And the
242
extraction of Sb from soil strongly depends on the total Sb amount in soil (Ettler et al., 2007).
243
With the exception of DTPA and water, more Sb could be extracted from soil S1 than from S2.
244
The extraction rates of Na2HPO4 in S1 and S2 were 13.05–25.78% (mean 19.61%) and 5.54–
245
19.68% (mean 13.37%) respectively. The extraction efficiencies of water in soil S1 and S2
246
were 3.69–9.68% (mean 6.92%) and 2.46–17.55% (mean 10.25%) respectively, which were
247
higher than the ratio of water (0.5–2.9%) in previous studies (Pierart et al., 2015). Antimony 10
248
extraction is affected by soil characteristics (such as pH, organic matter, total organic carbon,
249
and cation exchange capacity), and differences among extractants have been reported in
250
previous studies (Ettler et al., 2007; Nakamaru and Martín Peinado, 2017). Some previous
251
work reported relatively low Sb extraction percentage of Na2HPO4 (Tan et al., 2018; Zhang et
252
al., 2018) and another report (Ettler et al., 2007), whereas, the results in our study indicated a
253
much higher Sb extractability of Na2HPO4. That might be related to the characteristic and
254
properties of the tested spiked soils. More Sb was successfully extracted in antimony-spiked
255
soil than contaminated soil (Filella et al., 2009; Hansen and Pergantis, 2008; Pierart et al.,
256
2015). The condition of extraction (the particle size of the soil, temperature, time etc.) are also
257
needed to take into consideration.
258
The relationship between Sb content measured by different means and biotoxication on
259
amaranth was analyzed using the Pearson correlation coefficient (Table 5). Regardless of the
260
extractant used, Sb content in the two tested soils were significantly correlated to the total Sb
261
in soil, indicating that extracted Sb can generally be used as proxies of Sb contamination. For
262
soil S1, no relationship was found between fresh weight or root length and Sb content in soil
263
solution or Sb content in soil extract. The top three correlation coefficients between plant
264
height of amaranth and extractions protocols followed the order: DTPA = NH4H2PO4 >
265
Na2HPO4 > NaHCO3 (P < 0.05). Antimony content in shoots of amaranth was significantly (P
266
< 0.01) correlated with Sb concentration measured in all soil extracts and soil solution; the top
267
three correlation coefficients followed the order: NaHCO3 = Na2HPO4 > NH4H2PO4 > M3. A
268
similar correlation was reported between extracted Sb in the soil and Sb in leaves of spinach
269
(Hammel et al., 2000). The correlation coefficients between Sb yield by single extractants and
270
Sb concentration in roots ranked from the largest to the lowest were DTPA, NaHCO3, M3,
271
and Na2HPO4. For soil S1, Sb extracted by 0.1 M Na2HPO4, 0.5 M NaHCO3 and M3 are the
272
most relevant extractions to assess the biological effects of Sb on plants. In the case of soil S2,
273
no relationship was found between biomass or growth and soil solution or single extractant.
274
Antimony content in both shoots and roots of amaranth was significantly (P < 0.01) correlated 11
275
with all single extractants and with soil solution Sb levels (P < 0.05) as well. The top three
276
correlation coefficients for shoots were Na2HPO4 > NH4H2PO4 > M3, and for roots they were
277
water > Na2HPO4 > NH4H2PO4, which were different from the results of potted maize (Zhang
278
et al., 2018). That might be related to the properties of soil and species of plant.
279
In summary, for both soil S1 and S2, the most efficient extractants were M3, NaHCO3, and
280
Na2HPO4. According to Pearson correlation coefficient, the seven single extraction methods
281
and soil solution can be used to predict bioavailability of Sb. Considering the extractability
282
and correlation coefficients between biological effects and extractable Sb, NaHCO3, Na2HPO4,
283
and M3 might effectively predict the bioavailability of Sb in alkaline soil, whereas Na2HPO4,
284
NH4H2PO4, and M3 might do so in acid soil. Therefore, we suggest the use of 0.1 M Na2HPO4
285
and M3 as extractants to appropriately predict the bioavailability of Sb in soil.
286
3.3.2. Bioavailability assessment of Sb in soil solution
287
Antimony content in the spiked soils (S1 and S2) released into soil solution were
288
determined to assess its bioavailability (Fig. 3). The pH of soil S1 and S2 treated by Sb and its
289
soil solution (pH 7.95–8.39, 5.90–6.29) did not change significantly. For both soil types, Sb
290
content in soil solutions increased significantly and was correlated with total Sb in soil;
291
correlation coefficients between total Sb in soil and Sb in soil solution were 0.946 (P < 0.01)
292
and 0.963 (P < 0.01) for S1 and S2 respectively. In soil S1, when Sb concentration exceeded
293
440 mg kg-1, Sb released into soil solution increased significantly (P < 0.01, F = 180.022). In
294
soil S2, at Sb levels higher than 200 mg kg-1, total Sb content in soil solutions increased with
295
the increasing addition of Sb; this trend was also observed for Sb concentration in seedlings.
296
More Sb was released into soil solution in alkaline soil (0–58.78 mg L-1) than that in acid soil
297
(0.02–17.84 mg L-1). The fraction of Sb released into soil solution in soil S1 and S2 were
298
1.95%–4.19% and 0.12%–2.04%, respectively. Such observation is similar as the study of
299
Wilson and colleagues (Wilson et al., 2013). One reason to explain that is the effect of soil pH
300
on Sb release into pore water (Nakamaru and Martín Peinado, 2017). Indeed, previous
301
findings reported that the biogeochemical behavior of Sb in the environment greatly depends 12
302
on environmental factors such as soil pH and redox potential (Eh), which can impact the
303
adsorption and deposition of Sb (Wilson et al., 2010; Wilson et al., 2014). For instance, the
304
adsorption of Sb(V) by Fe and Al(OH)x is inhibited under increased soil pH values, leading to
305
higher Sb levels in soil solution (Nakamaru et al., 2006). Another reason might be that Sb
306
solubility is controlled by the presence of Ca[Sb(OH)6]2 (Okkenhaug et al., 2011), while S1 is
307
kind of calcareous alkaline soil with high level of calcium carbonate content ( >95%).
308
The concentration of Sb released into soil solution of both soils was significantly correlated
309
with levels of Sb accumulation in amaranth (Table 5), which might be that the absorption of
310
Sb by plants is proportional to the concentration of soluble Sb in soil (Tschan et al., 2009).
311
For soil S1, this correlation extremely significantly (P < 0.01) for amaranth seedlings (both
312
roots and shoots). In the case of S2, Sb content in soil solution was positively correlated with
313
Sb concentration in roots (P < 0.05) and shoots (P < 0.01) of amaranth. Although the transport
314
mechanism of Sb(V) in plants is still unknown (Tschan et al., 2009), we observed a
315
dose-effect relationship. Our results support the use of Sb levels in soil solution to predict the
316
accumulation of Sb in plants. The equations for Sb accumulation in amaranth and Sb in soil
317
solution are shown in Table 3 (equations 5–8). Moreover, our findings suggest that Sb in
318
alkaline soil is more easily absorbed by root and transported to shoot by plants, causing more
319
severe effects than those of Sb in acid soil even at the same Sb exposure level.
320
3.3.3. Prediction of Sb bioavailability in soil
321
Although significant correlations were detected between Sb levels in soil solution and Sb
322
accumulation in plants, their coefficients were slightly lower than those between some soil
323
extracts and Sb content in plants. In that regard, the analysis of soil solution might not the
324
most convenient indicator of Sb levels in soil because of possible errors during sampling
325
(such as soil moisture) and the relatively high costs of samplers. Nevertheless, knowing the
326
characteristics of soil solution, a medium in direct contact with ions in soil and plant roots,
327
can provide relevant information about soil conditions. Conversely, single extraction
328
protocols are effective tools for predicting bioavailability. In general they have lower costs 13
329
and simpler procedures than those of soil solution analyses. Still, there are key differences
330
between extraction protocols. For example, the composition and preparation of the M3
331
extractant are relatively complex, whereas those of 0.1 M Na2HPO4 extractant is simple and
332
convenient. Therefore, we suggest 0.1 M Na2HPO4 to be used to assess mobile Sb content in
333
both alkaline and acid soil. The equations describing the relationship between Sb
334
concentration in shoot and root of amaranth and Sb extracted using 0.1 M Na2HPO4 are
335
shown in Table 3 (equations 9–12).
336
4. Conclusion
337
We observed different toxic effects of Sb to amaranth between two types of soils (alkaline
338
and acid). In alkaline soil, Sb was more easily taken up by root and transported to shoot by
339
plants, causing more adverse toxic effects than in acid soil. Antimony in alkaline soil
340
significantly impacted the fresh weight of roots and shoots, decreasing the biomass of both
341
tissues at the highest concentration. Conversely, growth and biomass were not significantly
342
affected by Sb in acid soil, underscoring the need to watch for asymptomatic accumulation of
343
Sb by plants in contaminated agricultural soils.
344
Antimony extracted by single extractants and in soil solution could be used as good
345
predictors of Sb bioavailability. The Sb extraction efficiency differed among extractants and
346
soil types. For both alkaline and acid soils, M3, NaHCO3 and Na2HPO4 were the most
347
efficient extractants. In alkaline soils, NaHCO3, Na2HPO4, and M3 might be more appropriate
348
to predict the bioavailability of Sb, whereas Na2HPO4, NH4H2PO4, and M3 are suitable for
349
acid soil analyses. We suggest 0.1 M Na2HPO4 as a general extractant for assessing the
350
bioavailability of Sb regardless of soil type. However, if Sb and nutrients need to be evaluated
351
simultaneously, M3 is a good choice. Moreover, relevant environmental information about
352
contaminated soil can be provided by the assessment of soil solution. Lastly, considering that
353
this study was based on two kinds of spiked soils, further studies on different types of natural
354
soils in contaminated sites are needed. The standardization of the extraction processes and the
355
better understanding of the mechanisms underlying antimony toxicity in contaminated soil 14
356
warrant further research.
357 358
Acknowledgements
359
This work is part of the project “Research on Migration/Transformation and Safety
360
Threshold of Heavy Metals in Farmland Systems” (2016YFD0800405), which was supported
361
by National Key Research and Development Program.
362 363
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19
472
Table 1. Physical and chemical properties of S1 and S2 soils. Sb Soil
Soil type
CEC
CaCO3
Amorphous Fe
Amorphous Mn
Particle composition (%)
(g/kg)
(cmol/kg)
(g/kg)
(g/kg)
(g/kg)
2–0.05 mm
0.05–0.002 mm
< 0.002 mm
pH -1
(mg kg )
473 474
OM
S1
Chestnut soil
1.50
8.39
21.63
17.28
96.56
1.43
0.36
27.64
55.67
16.69
S2
Red earth
2.73
4.91
46.10
18.00
0.80
7.87
0.12
14.98
45.08
39.94
OM, organic matter. CEC, cation-exchange capacity.
20
475
Table 2. Bioaccumulation factor (BAF) and translocation factor (TF) in edible amaranth (Amaranthus
476
tricolor Linn.) seedlings exposed to different antimony concentrations in alkaline (S1) and acid (S2)
477
soils. Total Sb Shoot BAF
Experimental
Root BAF
TF
(mg kg-1) group S1
S2
S1
S2
S1
S2
S1
S2
Control
1.50
2.73
0.33
0.05
0.52
0.24
0.64
0.20
1
71.30
102.00
0.32
0.03
0.39
0.09
0.83
0.38
2
218.00
193.00
0.17
0.05
0.19
0.20
0.90
0.23
3
440.00
370.00
0.33
0.05
0.39
0.26
0.85
0.20
1165.0
1171.0 0.20
0.03
0.31
0.14
0.64
0.20
0.25
0.03
0.57
0.16
0.43
0.16
0.27
0.04
0.39
0.18
0.72
0.23
4 0
0
1402.0
1308.0
5
Mean
0
0
–
–
478
21
479
Table 3. Equations used to evaluate the relationship between Sb accumulation in plant tissues and Sb
480
concentration in the two soils (1–4), soil solution samples (4–8), and soil extracts using 0.1 M
481
Na2HPO4 (9–12). Soil
R2
Equation number
Cshoot = 3.935 + 0.2340x
0.939
(1)
Croot = 0.5099x - 42.56
0.921
(2)
Cshoot = (30.60 - 30.28) / (1 + (x / 294.8)2.177)
0.889
(3)
Croot = (210.2 - 212.7) / (1 + (x / 419.6)1.787)
0.772
(4)
Cshoot = 829.0 + (2.146 - 829.0) / (1 + (x / 83.51)0.847)
0.938
(5)
Croot = 2.297 + 13.5x
0.988
(6)
Cshoot = 30.30+(2.27-30.30)/(1+(x/2.02)3.41)
0.798
(7)
Croot = 10.10 + 30.9x
0.780
(8)
Cshoot = 1.23x - 19.59
0.895
(9)
Croot = 2.53x - 75.98
0.779
(10)
Cshoot = 29.42 - 24.34 / (1 + (x / 67.99)7.66)
0.862
(11)
Croot = 1.29x - 1.27
0.865
(12)
Equation
S1
S2
S1
S2
S1
S2
482
22
483
Table 4. Antimony extraction efficiency (%) from soils S1 and S2 using single extractants. Extractant Soil
484
Water
CaCl2
DTPA
NaHCO3
NH4H2PO4
Na2HPO4
M3
S1-1
9.25
7.40
1.46
37.37
25.13
25.78
59.63
S1-2
9.68
7.51
6.58
27.56
23.34
20.87
53.87
S1-3
7.98
7.97
4.22
28.33
21.02
22.18
55.99
S1-4
3.99
3.43
2.47
18.61
11.68
13.05
23.36
S1-5
3.69
3.86
6.69
22.69
14.65
16.18
36.28
Mean
6.92
6.03
4.29
26.91
19.16
19.61
45.83
S2-1
2.46
0.35
9.36
6.35
3.35
5.54
7.90
S2-2
12.15
3.32
10.10
18.03
7.97
18.34
21.53
S2-3
17.55
7.24
10.15
21.20
8.25
19.68
20.62
S2-4
11.35
4.32
9.78
11.12
4.63
10.26
11.66
S2-5
7.72
4.96
9.89
16.87
5.81
13.03
14.93
Mean
10.25
4.04
9.86
14.71
6.00
13.37
15.33
M3, Mehlich 3 extraction solution.
23
485
Table 5. Linear correlation coefficients (r) between plant parameters and Sb content in soil, soil
486
solution, or different soil extracts from S1 and S2 soils. Extractant
Soil Parameter
Soil Sb solution Sb
Water
CaCl2
DTPA
NaHCO3
NH4H2PO4
Na2HPO4
M3
S1 soil Fresh Weightroot
-0.262
-0.4
-0.206
-0.211
-0.524
-0.359
-0.372
-0.354
-0.35
Fresh Weightshoot
-0.687
-0.698
-0.768
-0.75
-0.758
-0.761
-0.79
-0.769
-0.757
Root Length
-0.088
-0.136
-0.043
0.007
-0.214
-0.134
-0.137
-0.12
-0.088
Stem Height
-0.759
-0.792
-0.773
-0.772
-0.843*
-0.824*
-0.843*
-0.828*
-0.809
Sb-Shoot
0.980**
0.969**
0.938**
0.964**
0.918**
0.990**
0.985**
0.990**
0.978**
Sb-Root
0.937**
0.999**
0.840*
0.889*
0.979**
0.972**
0.963**
0.969**
0.972**
Sb-soil
–
0.946**
0.937**
0.936**
0.873*
0.982**
0.970**
0.976**
0.934**
Fresh Weightroot
-0.608
-0.573
-0.657
-0.613
-0.606
-0.574
-0.597
-0.597
-0.593
Fresh Weightshoot
-0.537
-0.505
-0.596
-0.545
-0.535
-0.506
-0.53
-0.531
-0.526
Root Length
-0.079
-0.219
-0.19
-0.209
-0.084
-0.159
-0.141
-0.16
-0.134
Stem Height
-0.56
-0.533
-0.552
-0.567
-0.557
-0.519
-0.52
-0.516
-0.515
Sb-Shoot
0.971**
0.977**
0.968**
0.989**
0.973**
0.986**
0.997**
0.998**
0.995**
Sb-Root
0.944**
0.900*
0.999**
0.936**
0.944**
0.921**
0.962**
0.961**
0.958**
Sb-soil
–
0.963**
0.937**
0.981**
1.000**
0.980**
0.985**
0.979**
0.985**
S2 soil
487
*Correlation is significant at the 0.01 level (two-tailed).
488
**Correlation is significant at the 0.05 level (two-tailed). 24
489
Figure captions
490
Figure 1. Biomass and length of edible amaranth (Amaranthus tricolor Linn.) exposed to different
491
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth). Fresh
492
weight of root (black columns) and shoot (red columns) of plants grown in S1 (a) and S2 (c) soils.
493
Length of root (black columns) and height (red columns) of plants grown in S1 (b) and S2 (d)
494
soils. Different letters (e.g. a, b, c) indicate statistically significant differences at P<0.05.
495 496
Figure 2. Antimony uptake by edible amaranth (Amaranthus tricolor Linn.) exposed to different
497
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth).
498
Antimony concentration (mg kg-1 dry weight) in root (black columns) and shoot (red columns) of
499
plants grown in S1 (a) and S2 (b) soils. Different letters indicate statistically significant
500
differences at P<0.05.
501 502
Figure 3. Antimony concentration in soil solutions collected from (a) alkaline (S1, chestnut soil) and
503
(b) acidic soil (S2, red earth). Different letters indicate statistically significant differences at
504
P<0.05.
25
505 80
2.5 a
2.0
70
b
Root Shoot
a a
a
ab 60
ab
Length (mm)
Fresh weight (g)
a
Root Shoot
ab
1.5 b
b
1.0
ab
ab b
50
40
a
a
ab
0.5
ab
ab a
ab
ab
a
c
b
30
c
0.0 1.50
71.3
440
218
1,165
20
1,402
1.50
71.3
Sb in soil S1 (mg kg-1)
506
c
Root Shoot
6
Length (mm)
Fresh weight (g)
ab a
ab
0
2.73
ab
102
ab
193
370
a
a a
a
a
ab
60
bc
bc a
b
c
40
1,171 1,308
2.73
-1
507
a
b a
d
a
80
ab
ab
1,402
Root Shoot a
2
1,165
100
a 4
440
218
Sb in soil S1 (mg/kg)
102
193
370
1,171
1,308
-1
Sb in soil S2 (mg kg )
Sb in soil S2 (mg kg )
508 509
Figure 1. Biomass and length of edible amaranth (Amaranthus tricolor Linn.) exposed to different
510
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth). Fresh weight
511
of root (black columns) and shoot (red columns) of plants grown in S1 (a) and S2 (c) soils. Length of
512
root (black columns) and height (red columns) of plants grown in S1 (b) and S2 (d) soils. Different
513
letters (e.g. a, b, c) indicate statistically significant differences at P<0.05.
26
515
1000
a
Shoot Root
800
Sb concentration in amaranth (mg kg-1 DW)
Sb concentration in amaranth (mg kg-1 DW)
514
d
600
400
c
d
c 200
0
b a
a
1.50
a
a
71.3
a
218
b
a
440
1,165
1,402
300
b
Shoot Root
250
cd 200
cd 150
bc
100 50 0
ab a
2.73
a
a
a
102
a
193
b
370
c
c
1,171
1,308
Sb in soil S2 (mg kg-1)
Sb in soil S1 (mg kg-1)
516
Figure 2. Antimony uptake by edible amaranth (Amaranthus tricolor Linn.) exposed to different
517
concentrations of antimony in alkaline (S1, chestnut soil) and acidic soil (S2, red earth). Antimony
518
concentration (mg kg-1 dry weight) in root (black columns) and shoot (red columns) of plants grown in
519
S1 (a) and S2 (b) soils. Different letters indicate statistically significant differences at P<0.05.
27
521
a
100
Sb concentration in soil solution (mg/L)
Sb concentration in soil solution (mg/L)
520
80 d
60
40 c 20
0
b a
a
1.50
71.3
a
218
440
1,165
1,402
Sb in soil S1(mg/kg)
b
25
20
d
15
cd 10
bc 5
ab 0
a
a
2.73
102
193
370
1,171
1,308
Sb in soil S2 (mg/kg)
522 523
Figure 3. Antimony concentration in soil solutions collected from (a) alkaline (S1, chestnut soil) and
524
(b) acidic soil (S2, red earth). Different letters indicate statistically significant differences at P<0.05.
525
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Highlights: 1. Antimony in alkaline and acid soil caused different toxic effects to amaranth. 2. More attention should be paid on asymptomatic accumulation of Sb in plants. 3. Soil solution and single extraction were used to assess the bioavailability of Sb. 4. 0.1 M Na2HPO4 is the best extractant to predict the bioavailability of Sb in soil. 5. Mehlich 3 and soil solution are alternative to assess the bioavailability of Sb.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: