Mercury accumulation plant Cyrtomium macrophyllum and its potential for phytoremediation of mercury polluted sites

Accepted Manuscript Mercury accumulation plant Cyrtomium macrophyllum and its potential for phytoremediation of mercury polluted sites Yu Xun, Liu Feng, Youdan Li, Haochen Dong PII:

S0045-6535(17)31470-4

DOI:

10.1016/j.chemosphere.2017.09.055

Reference:

CHEM 19926

To appear in:

ECSN

Received Date: 10 April 2017 Revised Date:

12 September 2017

Accepted Date: 12 September 2017

Please cite this article as: Xun, Y., Feng, L., Li, Y., Dong, H., Mercury accumulation plant Cyrtomium macrophyllum and its potential for phytoremediation of mercury polluted sites, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.09.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Mercury Accumulation Plant Cyrtomium macrophyllum and Its

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Potential for Phytoremediation of Mercury Polluted Sites Yu Xun, Liu Feng*, Youdan Li, Haochen Dong

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Department of Environmental Engineering and Sciences, Beijing University of Chemical Technology,

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Beijing, 100029, P R China

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Abstract

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Cyrtomium macrophyllum naturally grown in 225.73 mg kg-1 of soil mercury in

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mining area was found to be a potential mercury accumulator plant with the

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translocation factor of 2.62 and the high mercury concentration of 36.44 mg kg-1

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accumulated in its aerial parts. Pot experiments indicated that Cyrtomium

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macrophyllum could even grow in 500 mg kg-1 of soil mercury with observed

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inhibition on growth but no obvious toxic effects, and showed excellent mercury

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accumulation and translocation abilities with both translocation and bioconcentration

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factors greater than 1 when exposed to 200 mg kg-1 and lower soil mercury, indicating

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that it could be considered as a great mercury accumulating species. Furthermore, the

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leaf tissue of Cyrtomium macrophyllum showed high resistance to mercury stress

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because of both the increased superoxide dismutase activity and the accumulation of

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glutathione and proline induced by mercury stress, which favorited mercury

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translocation from the roots to the aerial parts, revealing the possible reason for

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Cyrtomium macrophyllum to tolerate high concentration of soil mercury. In sum, due

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to its excellent mercury accumulation and translocation abilities as well as its high

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resistance to mercury stress, the use of Cyrtomium macrophyllum should be a

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ACCEPTED MANUSCRIPT promising approach to remediating mercury polluted soils.

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Keywords: soil, Cyrtomium macrophyllum, mercury, phytoremediation

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1. Introduction

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Soil mercury pollution has become a worldwide environmental issue. It is serious in

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mercury mining areas, chlorine industrial plants, and artisanal gold smelting areas,

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etc., resulting in many mercury pollution sites (Driscoll et al., 2013). These mercury

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polluted sites are widely distributed throughout the world, for example, Wuchuan and

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Wanshan mercury mining areas in Guizhou Province, China (Qiu et al., 2006; Li et

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al., 2008), abandoned gold mining and smelting area in Dehua, Fujian Province,

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China, and the Almadén mercury mining area in the middle of Spain (Higueras et al.,

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2006), the Amazon region where small-scaled and artisanal gold mining activities

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have been conducted (UNEP, 2013) and so on, causing great risks to ecological

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environment and human health.

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The remediation of mercury polluted sites is an urgent but difficult task. Although

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various traditional remediation technologies such as mechanical removal and

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chemical engineering are available, they are often expensive and incompatible to soil

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structure and fertility (Padmavathiamma and Li, 2007; Singh et al., 2016). Hence,

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some

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phytoremediation, are being investigated as suitable alternatives (Arthur et al., 2005;

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Mani and Kumar, 2014).

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Phytoremediation, as its name implies, basically refers to the use of plants and

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associated soil microbes to reduce the concentrations or toxic effects of contaminants

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novel,

and

more

environmental

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friendly

technologies,

including

ACCEPTED MANUSCRIPT in the environments (Cunningham and Berti, 1993; Ali et al., 2013). It comprises of a

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group of processes, and phytoextraction which was firstly raised by Chaney et al.

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(1983) is considered the most promising phytoremediation process nowadays.

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According to Garbisu and Alkorta (2001), in the phytoremediation context of

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contaminated sites, the ideal plants for phytoextraction should be tolerant to high

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levels of metals, accumulate high levels of the metals in their harvestable parts, and

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have a rapid growth rate, a profuse root system as well as the potential to produce a

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high biomass in the sites. So far, phytoextraction is mainly accomplished by means of

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hyperaccumulators which typically have the basic features of high metal accumulation

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capacity and tolerance to contaminant (Baker and Brooks, 1989; Kumar et al., 1995;

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Rascio and Navari-Izzo, 2011; Zeng et al., 2011; Van der Ent et al., 2013; Vilas Boas

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et al., 2014; Pandey et al., 2015). More than 500 plant taxa have been cited in the

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literatures to date as hyperaccumulators for many metals, like nickel, cadmium, zinc,

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copper, and arsenic (Bauddh et al., 2015), however, only few mercury accumulator

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plants were reported (Marrugo-Negrete et al., 2016), let alone mercury

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hyperaccumulators.

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As an accumulator plant, not only can it have the capacity to concentrate metals, but

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also can protect its sensitive cellular activity and structures from excessive levels of

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metals, especially toxic metals like mercury. Plants exposed to unfavorable

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environmental conditions can cause oxidative stress with the increase of reactive

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oxygen species (ROS), including hydrogen peroxide (H2O2), hydroxyl radicals (OH·)

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and superoxide anions (O2−), which lead to lipid peroxidation of cell membranes and

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ACCEPTED MANUSCRIPT the damage of individual organelles resulting in the death of plants (Pisoschi and Pop,

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2015; Chakraborty et al., 2016). In response to the oxidative stress, plants can

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counteract the overproduction of ROS by their antioxidant systems, which includes

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the increase of antioxidant enzymes (such as superoxide dismutase, SOD), sugar,

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peptides (such as glutathione, GSH) and amino acid contents (such as proline),

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variation in the lipid composition, content of soluble proteins and gene expression

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(Biczak, 2016; Park et al., 2017).

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Owing to natural selection, there are some metal hyperaccumulators or tolerant plants

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in highly metals polluted sites, such as the mining areas. The idea of using plants to

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remove metals from soils just came from the discovery of these wild plants, often

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endemic to naturally mineralized soil, that accumulate high concentrations of metals

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in their foliage (Baker and Brooks, 1989; Raskin et al., 1994). Because of the mining

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and smelting of mercury and gold mines, there were many highly mercury polluted

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sites in China, where a lot of mercury tolerant plants grew well with high biomass, to

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provide abundant resources for the screening and application of mercury accumulator

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plants (Rothenberg et al., 2012; Ran et al., 2017). Due to the biodiversity of

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autochthonous plants in the mercury polluted sites of mining areas in China and even

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in the world, the phytoremediation processes employed by local species is of great

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significance for the remediation of the mercury polluted sites.

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In present study, several wild plants were sampled from the selected mercury polluted

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sites to evaluate their abilities of mercury accumulation, and after preliminary

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screening, the wild plant Cyrtomium macrophyllum, which belongs to terrestrial fern,

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ACCEPTED MANUSCRIPT and is a perennial herbaceous plant and widely distributed over China, was found to

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have the potential to extract mercury from soil. Accordingly, the aim of the present

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study was to investigate the phytoremediation potential of Cyrtomium macrophyllum

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using artificially mercury polluted soils based on the actual soil mercury

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concentrations in mining areas. Afterwards, the mercury uptake, translocation and

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bioconcentration abilities of Cyrtomium macrophyllum over time (60 days) were

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studied. Besides, the physiological response of Cyrtomium macrophyllum including

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SOD activity and the accumulation of GSH and proline were investigated for

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mechanism study of mercury stress.

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2. Materials and Methods

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2.1 Sampling of wild plants and soils

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Through field survey, two highly mercury polluted sites were selected. One site is

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located at 117°55′-118°32′E and 25°23′-25°56′N in Dehua mountain area, Fujian

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Province, where the highest concentration of mercury in soil was found to be up to

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69.1mg kg-1 due to historical gold mining and smelting (Zheng et al., 2015). The other

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site is located at 109°11′-109°14′E and 27°30′-27°32′N in Wanshan, Guizhou

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province, where the mercury enrichment in local plant was observed, with mercury

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concentration in different tissues of native plants ranging from 0.47 mg kg-1 to 331.4

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mg kg-1 (Ding et al., 2004).

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Plant species growing well with high abundance were selected based on biomass per

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plant at each site, and three plants of each plant species were randomly collected

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using disposable gloves and stainless-steel spade. Meanwhile, around the rhizosphere

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ACCEPTED MANUSCRIPT of each plant sample, one composite soil sample (0-30 cm in depth) was collected

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through the sampling and mixing of three soil sub-samples (about 500 g) collected in

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the same area where the plant sample was located. During on-site sampling, each

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plant sample and its associated soil sample were placed in a labelled sealable HDPE

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bag individually, and then stored in a portable refrigerator to keep cool and moist. All

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plant and soil samples were transported to the laboratory as soon as possible.

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Once in the laboratory, plant species was identified in accordance with the Flora of

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China (Wu et al., 2004). All plant samples were thoroughly washed with tap water

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and rinsed with deionized water to remove any sediment particles attached to the plant

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surface. After air-dried, each plant sample was sorted by species and separated into

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roots and aerial parts, and then was stored separately in labelled sealable HDPE bags

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in a cool dark and dry environment for mercury analysis. Meantime, each soil sample

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was air-dried at room temperature, ground to 0.149 mm and less, and then stored in a

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labelled sealable HDPE bag in a refrigerator for mercury analysis.

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2.2 Pot experiment

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2.2.1 Preparation of soils for the pot experiment

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The raw soil used for the pot experiment was collected from the surface layer (0~20

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cm) of soils in the farmland of the suburbs in Beijing, China. The soil pH was 7.46 ±

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0.08 measured by a pH-meter with 1:1 (soil: water ratio, w/v) suspension (McLean,

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1982), organic matter (OM) content was 0.87 ± 0.05% measured by the method of

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loss on ignition (Heiri et al., 2001) by which the soil was burnt at 500 °C for 5 h, and

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mercury in the soil was 0.045 ± 0.025 mg kg-1.

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ACCEPTED MANUSCRIPT Nine gradients of mercury concentrations, including control, 5, 10, 20, 50, 100, 200,

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500 and 1000 mg kg-1 based on preliminary experiment and actual soil mercury

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concentrations in relevant mine area, were set, and accordingly the different polluted

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soils were prepared with the raw soil by adding HgCl2 solution diluted by stock

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solution (1000 mg L-1). In order to stabilize the newly introduced mercury in the soil,

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resulting in a similar distribution of mercury in long-term polluted soils (Ma et al.,

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2015), all these polluted soils were incubated for five weeks before use for the pot

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experiment.

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2.2.2 Preparation of Cyrtomium macrophyllum seedlings for the pot experiment

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Cyrtomium macrophyllum for the pot experiment was collected in the form of

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seedling in the Fanjingshan National Nature Reserve (FNNR, at 108°45′-108°48′E

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and 27°49′-28°01′N). FNNR is a place in Guizhou Province that has barely been

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affected by anthropogenic activities, where the soil is free of pollution by mercury and

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other heavy metals.

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Cyrtomium macrophyllum seedlings with the same size were collected entirely

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inclusive of the soil around the roots and transported to the laboratory. Hydroponic

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units were constructed to cultivate these seedlings, ensuring normal conditions,

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adjustment and stabilization before use (Bonfranceschi et al., 2009). Each unit

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consisted of 10 glass jars (8 cm in diameter and 14 cm in height) containing Hoagland

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medium as nutrient solution (Hoagland and Arnon, 1950), with pH adjusted to 6.0.

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Plastic tripods (6 cm in diameter and 7 cm in height) with openings at the top were

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placed inside the jars to hold the seedlings. All seedlings were cultivated for at least

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ACCEPTED MANUSCRIPT one week before use in the hydroponic units with a density of 5 individuals in a jar.

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The mercury concentration in these seedlings was detected to be lower than the limit

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of detection (LOD, 20 µg kg-1 biomass for plant samples).

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2.2.3 Experiment design

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About 500 g of polluted soil (moistened to 50% the saturation point with deionized

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water) was transferred to a φ22.5 cm × 15 cm pot, and two uniform Cyrtomium

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macrophyllum seedlings were transplanted into the polluted soil at each pot. Three

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replicates were prepared per polluted soil, and the blank control was set up with the

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raw soil. Considering mercury concentration gradients, the number of harvesting and

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replicates, 108 pots (9 × 4 × 3) were prepared in total. All these pots were randomly

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placed in a greenhouse located at Beijing University of Chemical Technology, in

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Beijing, China, where the atmospheric mercury concentration was as low as 3.23 ng

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m−3 (Zhang et al., 2013). The pot experiment was conducted between September and

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November 2016. During this period, the average air temperature was stable at about

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20 °C, and the sunshine time was about 550 h. In order to eliminate the interference of

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air temperature, light and atmospheric mercury deposition on the experiment, the

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greenhouse has a certain degree of isolation from the outside space with adequate

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ventilation. Soil moisture was controlled by weighing pots and adjusting the weight

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with deionized water every second day.

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Cyrtomium macrophyllum was harvested every 15 days from the 15th day after

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transplanting, and the pot experiment lasted for 60 days. During harvesting, two

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plants of Cyrtomium macrophyllum in a pot (3 replicates) at each soil mercury

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ACCEPTED MANUSCRIPT concentration were harvested and rinsed 3 times with deionized water to remove

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adhering soil particles. Among them, one was used for the determination of Hg

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concentration, which was divided into roots and aerial parts, while the other one was

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used for the assay of physiological indexes. At the same time, soil samples were

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collected for the determination of total mercury concentration.

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2.3 Determination of mercury concentration in soil samples

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After air-dried and ground to 0.149 mm and less, each soil sample was digested using

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aqua regia for 2 h at 96 °C water bath (Liu, 2005).

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Mercury concentration in the digestions was measured by the atomic fluorescence

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spectrophotometry using a double-channel atomic fluorescence spectrometer (Beijing

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Haiguang Instruments, AFS3100), following the recommendations by US EPA

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Method 7474 (US EPA, 2007). Specific operating parameters of the spectrometer read

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as follows: alkali 2% KBH4 solution as a reductant with a flow rate of 4.80 mL min-1,

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5% HCl solution as a carrier liquid with a flow rate of 2.50 mL min-1, and high-purity

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argon as a carrier gas with a flow rate of 300 mL min-1.

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All analyses were performed for 3 individual replicates per soil sample. Analytical

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quality of the measurements was evaluated in triplicate with the certificated soil

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material GBW07404 (0.59 ± 0.05 mg kg-1, supplied by the Institute of Geophysical

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and Geochemical Exploration, China), and the percentage of recovery was 98.7%.

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The LOD of the measurements was determined to be 35 µg kg-1 for soil samples,

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calculated as the mean plus three times the standard deviation (Buccolieri et al.,

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2006).

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ACCEPTED MANUSCRIPT 2.4 Plant analysis

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2.4.1 Determination of biomass and mercury concentration

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Plant samples were dried in an oven at 60 °C to a constant weight and dry weights

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were recorded. For mercury analysis, these plant samples were further digested with

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concentrated HNO3 and 30% H2O2 in the presence of 400 W microwave for 1 hour

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using a fully automatic microwave digestion system (XH-800C, Beijing Xianghu

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Science and Technology Development Co. Ltd.) (Rodushkin et al., 1999).

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Following the same method and procedure for the test of mercury concentration in

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soil digestions mentioned above, the mercury concentration in plant extracts was

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measured. Similarly, the certificated plant material of Citrus leaf (GBW10020, 150 ±

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20 µg kg-1, supplied by the Institute of Geophysical and Geochemical Exploration,

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China) was used to evaluate the quality of the measurements with a percentage

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recovery of 97.4%, and the LOD of the measurements was determined to be 20 µg

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kg-1 biomass (dry weight) for plant samples.

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2.4.2 Assay of physiological indexes

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Three physiological indexes were involved in this assay, as SOD, GSH and proline.

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Fresh matter samples were used in this assay, which were taken from the fully

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matured intact leaves (third leaves from the top of the shoot) of the Cyrtomium

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macrophyllum collected from each pot.

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SOD activity assay: fresh leaf samples (500 mg) were homogenized in 5 mL 100 mM

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potassium phosphate buffer (pH7.6) containing 1 mM EDTA-Na2 and 0.5 mM

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ascorbate, and then centrifugated at 10,000 rpm for 15 min at 4 °C to produce the

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ACCEPTED MANUSCRIPT supernatant as crude enzyme extracts. SOD was assayed by the nitro-blue tetrazolium

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(NBT) method as described by Beauchamp and Fridovich (1971). The 3 mL of

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reaction mixture containing 50 mM K-phosphate buffer (pH 7.3), 13 mM methionine,

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75 µM NBT, 0.1 mM EDTA, 4 µM riboflavin and enzyme extract (0.2 mL) was

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exposed to illumination to start reduction of NBT to blue formazan, which was

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measured as the increase in absorbance at 560 nm. One unit of SOD was defined as

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the amount of enzyme required to inhibit 50% of the NBT photoreduction in

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comparison with controls under the assay conditions. The specific SOD activity was

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expressed as enzyme units per gram fresh weight of tissue from soil.

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GSH content assay: leaf samples (500 mg) were homogenized in 5% trichloroacetic

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acid and then centrifugated at 15,000 rpm for 10 min at 4 °C to give the supernatant

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as crude extracts. GSH content was assayed according to the method described by

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Smith et al. (1988). 2.6 mL 150 mM NaH2PO4 buffer (pH7.7) and 0.2 mL

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5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB, 75.3 mg DTNB dissolving in 30 mL 100

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mM pH6.8 phosphate buffer) were added to 0.2 mL of the supernatant to form the

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reaction mixture. After well mixing and maintaining at 30 °C for 5 min, the

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absorbance was recorded at 412 nm against phosphate solution blank. The GSH

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content was determined from a standard curve and expressed on a fresh weight basis.

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Proline content assay: leaf samples (500 mg) were homogenized with 80% alcohol

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(v/v) and silica sand, extracted for 20 min with 80% alcohol at water bath to obtain

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proline extracts. After purifying with zeolite and activated carbon and filtration,

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proline content in the filtrates was determined by the ninhydrin colorimetric method

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ACCEPTED MANUSCRIPT described by Troll and Lindsley (1955). Glacial acetic acid and ninhydrin reagent (1

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mL each) were added to 1 mL of the supernatant to form the reaction mixture. The

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closed test tubes with the reaction mixture were incubated at 96 °C for 1 h in a hot

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water bath and after incubation 2 ml of toluene was added to each tube followed by

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mixing. The absorbance of the pink red upper phase was recorded at 520 nm against

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toluene blank. The proline concentration was determined from a standard curve and

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expressed on a fresh weight basis.

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All assays were performed 3 individual replicates per plant sample.

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2.5 Data analysis

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The translocation factor (TF) and the bioconcentration factor (BCF) were estimated

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using the mercury concentrations determined. TF for a plant was calculated as the

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ratio mercury concentration in the aerial parts to the roots, while BCF was expressed

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as the ratio mercury in the roots to that in soil.

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The results were presented as the mean ± standard deviation of triplicate

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determinations. After evaluating the normality by Ryan–Joiner test and the

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homogeneity of variance by Bartlett test, the data were subjected to statistical analysis

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of variance (ANOVA) with SPSS 20.0, and a significance of 0.05 was selected. The

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calculations and graphic work were performed using Excel 2010 software.

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3 Results and Discussion

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3.1 Mercury accumulation and translocation abilities of wild plants

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It was found that many wild plants grew in clusters at two selected sites, but plant

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ACCEPTED MANUSCRIPT species was relatively simple, dominated by clumps of herbage. Total 14 dominant

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wild plants were collected and recorded, including Cyrtomium macrophyllum, conyza

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canadensis and so on (Table 1). All these plants were growing with high biomass, and

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most showed high resistance to mercury toxicity.

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The soils in Dehua site are slightly acidic with pH ranging from 6.07 to 6.27, while

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the soils in Wanshan site are weakly alkaline with pH ranging between 8.04 and 8.22.

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Soils in both sites contain moderate amounts of organic matter, with the contents

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ranging from 2.92% to 3.44% and 2.75% to 3.24%, respectively.

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The mercury concentration in the rhizosphere soil ranged from 99.87 ± 1.13 mg kg-1

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to 237.81 ± 6.12 mg kg-1, revealing great risk to environment and human health.

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Mercury concentrations in these wild plants were different from each other

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significantly (Table 1), and Cyrtomium macrophyllum was found to accumulate the

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highest concentration of mercury, with 13.90 mg kg-1 in its root and 36.44 mg kg-1 in

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its aerial parts. With TFs being less than 1, more than half of these wild plants

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accumulated mercury in their roots rather than in their aerial parts, indicating their

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relatively poor translocation abilities. Conversely, the rest plants could better transfer

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mercury accumulated in their roots into their aerial parts, with TFs ranging between

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1.32 ± 0.09 and 2.62 ± 0.18, and the highest TF was observed for Cyrtomium

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macrophyllum, which is comparable to those of some reported mercury accumulator

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plants, like 0.96 for Rumex induratus (Moreno-Jiménez et al., 2006), 1.19 for

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Capsicum annuum and 0.62 for Jatropha curcas (Marrugo-Negrete et al., 2016). It

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should be considered that TFs sometimes don’t represent the plants’ ability to

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ACCEPTED MANUSCRIPT translocate mercury, because plants can also assimilate atmospheric Hg by their leaves,

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and even more so when there may be high concentrations of Hg in air in the

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environment where they grow. This can give wrong TFs, because the plant does not

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take Hg from the ground (Leonard et al., 1998; Ms et al., 2003; Marrugo-Negrete et

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al., 2016).

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Relatively low BCFs were observed for these wild plants, ranging between 0.007 ±

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0.005 for xanthium sibiricum patrin ex widder and 0.174 ± 0.058 for Commelina

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communis from Dehua, which implied that their mercury accumulation capacity

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remained to be low. However, for plants growing naturally in the soils heavily

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polluted by mercury for long term, BCFs sometimes do not truly reflect their mercury

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accumulation capacities. This may be due to that mercury presents in the long-term

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polluted soils mainly in the form of residual fractions, and the proportion of

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bioavailable mercury that can be easily absorbed by plants is often very low (Martin

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and Griswold, 2009; Dong et al., 2017). On the other hand, even if these wild plants

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had low BCFs, the absolute concentration of mercury accumulated in aerial parts of

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most plants was higher than 10 mg kg-1, which was of great concern to

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phytoremediation. The highest concentration of 36.44 mg kg-1 was found in the aerial

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parts of Cyrtomium macrophyllum, it was comparable to and even higher than the

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reported accumulation level in aerial parts of other discovered plants to date, such as

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Jatropha curcas (Marrugo-Negrete et al., 2015), Trifolium repens (Liu and Wang,

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2014), Rumex induratus and Marrubium vulgare (Moreno-Jiménez et al., 2006),

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Typha domingensis Per (Millán et al., 2014) and so on.

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ACCEPTED MANUSCRIPT The concentration of mercury in common plants (including herbs) usually ranges

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from 1 µg kg-1 to 100 µg kg-1, and the lowest observed effect concentration of

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mercury recommended by US EPA for phytotoxicity is 0.3 mg kg-1 (US EPA, 1996),

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this means that Cyrtomium macrophyllum can accumulate mercury at least 100-folds

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higher than the common plants do. In this respect, with a TF of 2.62, although its BCF

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was relatively low to 0.061, Cyrtomium macrophyllum could be regarded as a

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potential mercury accumulator.

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3.2 Cyrtomium macrophyllum growth and biomass during the pot experiment

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During the experiments, Cyrtomium macrophyllum grew healthy and exhibited high

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levels of biomass production, except for exposure to 1000 mg kg-1 (in which slight

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toxic effects such as chlorosis and necrosis were observed with small leaves and

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stunted growth). After growing for 60 days in pots, the average height of Cyrtomium

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macrophyllum reached 45 cm.

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The growth behavior of Cyrtomium macrophyllum was determined by measuring the

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biomass accumulation (Fig.1). The biomass accumulation decreased for each

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exposure time along with the increased soil mercury concentrations, however, this

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decrease was not evident when exposed to 100 mg kg-1 and below. A significant final

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decrease of 37.1% compared with the control was found due to the observed toxic

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effects when exposed to 1000 mg kg-1. However, for those plants growing healthy

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with no observed toxic effects, growth inhibition was also found, with a relatively

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high final biomass reduction of 20.2% observed for the plants exposed to 500 mg kg-1.

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Growth inhibition and reduction of biomass production are frequently observed

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ACCEPTED MANUSCRIPT phenomena in plants exposed toxic levels of mercury (Patra and Sharma, 2000;

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Xylander et al., 1998). In fact, several studies have suggested that the growth

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inhibition of plants exposed to high levels of mercury produces significant toxic

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damage to cells (Ortega‐Villasante et al., 2007; Zhou et al., 2007). Similarly,

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reductions in biomass may occur because extra energy is required to counter mercury

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stress on tissues. On the other hand, because the roots are subjected to direct contact

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with the contaminant and consequently suffer damage from mercury exposure,

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inhibition of root development is the first symptom of plant stress (Zornoza et al.,

338

2010), and therefore, the homeostasis of water and nutrient transport from the roots

339

may be affected, leading to biomass reduction.

340

3.3 Mercury concentration in Cyrtomium macrophyllum during the pot experiment

341

The concentration of mercury in the roots and aerial parts of Cyrtomium

342

macrophyllum were shown in Table 2. The amount of mercury accumulated in the

343

roots rose along with the increased soil mercury concentration and exposure time, and

344

the highest concentrations of mercury in the roots were observed after 60 days of

345

exposure to each soil mercury concentration. This showed that increasing exposure

346

time favored an increase of mercury concentrations in the roots that might be

347

associated with their direct exposure to mercury in soil.

348

The mercury amount accumulated in the aerial parts continued to rise with the

349

exposure time when exposed to 50 mg kg-1 and below, and the highest concentration

350

of 397.23 mg kg-1 was found after 60 days of exposure to 25 mg kg-1 of mercury in

351

soil. However, a peak concentration was observed during early exposure followed by

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ACCEPTED MANUSCRIPT continuous decline when exposed to 100 mg kg-1 and above, especially, the mercury

353

concentration in the aerial parts continued to decrease during whole exposure to 1000

354

mg kg-1 of mercury in soil, with the lowest concentration of 37.87 mg kg-1 after 60

355

days of exposure.

356

Further analysis showed that the decrease of mercury concentrations in the aerial parts

357

was closely related to the increase of mercury amount accumulated in the roots, and

358

mercury accumulation in the roots gradually slowed down along with the increased

359

exposure time and soil mercury concentration, especially, the mercury amount

360

accumulated in the roots increased a little during whole exposure time when exposed

361

to 1000 mg kg-1 of soil mercury. As several studies have demonstrated (Ortega‐

362

Villasante et al., 2007; Zhou et al., 2007; Zornoza et al., 2010), it could be concluded

363

from above results that either exposure to extremely high concentration (e.g. 1000 mg

364

kg-1) of mercury or prolonged exposure to relatively high concentration (e.g. 100 mg

365

kg-1, 200 mg kg-1 and 500 mg kg-1) might produce stress on Cyrtomium macrophyllum

366

tissues, especially the roots, and therefore induce resistance mechanisms of

367

Cyrtomium macrophyllum to restrict mercury uptake and accumulate them in the roots,

368

and accordingly to prevent the aerial parts from toxic effects (necrosis and chlorosis).

369

3.4 Translocation and bioconcentration abilities of Cyrtomium macrophyllum

370

The mercury concentration in polluted soils after plant cultivation at different

371

exposure time was presented in Table 3. Mercury mass balance at each treatment was

372

made, which indicated the sum of the mercury absorbed by the plant and the

373

remaining mercury in the soil is close to the initial amount of mercury in the soil at all

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17

ACCEPTED MANUSCRIPT cases (See MR in Table 3). At the same time, it was not detectable for the mercury

375

concentration in the leaf tissues of plant growing in non-polluted soil (Table 2).

376

Therefore, it can be reasonably inferred that the mercury in the Cyrtomium

377

macrophyllum comes from the polluted soil through plant uptake.

378

Accordingly, the TFs were calculated (Table 4). In general, the TFs decreased with the

379

increased soil mercury concentration. When exposed to 200 mg kg-1 and below, the

380

TFs during whole exposure time were higher than 1, a value that is characteristic of

381

accumulator plants (Baker, 1981). Nevertheless, when exposed to 500 mg kg-1,

382

especially to 1000 mg kg-1, the TFs decreased greatly from 0.78 to 0.14, indicating

383

that when TF is close to 0.1, exclusion of metal from plant tissues occurs (Khan et al.,

384

2009). As discussed above, these low TFs might indicate a resistance to the

385

contaminant by Cyrtomium macrophyllum to avoid toxic effects on the aerial parts,

386

therefore, mercury decreased its translocation.

387

The BCFs decreased with increased soil mercury concentration but gradually

388

increased with increased exposure time (Table 4). In addition, the BCFs were higher

389

than 1, a threshold value indicating mercury accumulation behavior in plants (Melgar

390

et al., 2009) during whole exposure period when exposed to 100 mg kg-1 and below,

391

however, the BCFs were lower than 1 during whole exposure period when exposed to

392

500 mg kg-1 and above. As described above, these lower BCFs less than 1 might

393

ascribed to the tolerance mechanisms of Cyrtomium macrophyllum when exposed to

394

extremely high toxic levels of mercury.

395

In summary, combined with the discussions on biomass accumulation, although

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ACCEPTED MANUSCRIPT Cyrtomium macrophyllum could grow without observed toxic effects when exposed to

397

500 mg kg-1 of soil mercury, extremely high levels of soil mercury exposure (e.g. 500

398

mg kg-1 and 1000 mg kg-1 in this study) could induce the tolerance mechanisms of

399

Cyrtomium macrophyllum to restrict its mercury accumulation and translocation.

400

However, Cyrtomium macrophyllum could be still considered a great mercury

401

accumulating species due to its excellent accumulation and translocation abilities in

402

the highly mercury polluted soils (e.g. soil mercury concentration up to 200 mg kg-1 in

403

this study).

404

3.5 Tolerance of Cyrtomium macrophyllum leaf tissue to mercury stress

405

As described above, the ability of Cyrtomium macrophyllum to tolerate high mercury

406

concentrations in the soil might be due to restriction in mercury uptake and excellent

407

translocation toward the aerial parts. Moreno et al. (2005) have indicated that the

408

leaves are the final mercury recipient of the plant and have higher mercury levels than

409

stems. Hence, the resistance of leaf tissue to mercury stress will be of great

410

importance to the translocation of mercury from the roots to the aerial parts. For this

411

reason, such physiological indexes for leaf tissue as the SOD activity and the contents

412

of GSH and proline, as well as their changes during the pot experiment were observed

413

to evaluate the resistance of Cyrtomium macrophyllum leaf tissue to mercury stress.

414

SOD, an important antioxidative enzyme, together with the reduced GSH, one of the

415

important intracellular antioxidants, can effectively eliminate reactive oxygen species

416

(ROS) induced by metal stress for preventing membrane lipid from overoxidation

417

(Blasco et al., 2015; Chrysargyris et al., 2017). When exposed to 100 mg kg-1 and

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19

ACCEPTED MANUSCRIPT lower levels of soil mercury, the statistically significant increases (p<0.05) in both the

419

SOD activity and the GSH content in Cyrtomium macrophyllum were observed

420

compared to the control (Fig.2a and Fig.2b) during whole exposure period, indicating

421

that mercury stress could induce increased SOD activity and GSH content to improve

422

the resistance of Cyrtomium macrophyllum to ROS damage. In addition, the SOD

423

activity increased first and reduced later with the increased soil mercury concentration

424

during each exposure period, and the initial soil mercury concentration associated

425

with decreased SOD activity decreased with the prolonged exposure time, indicating

426

that low-concentration mercury stress could induce increased SOD activity to

427

eliminate ROS and alleviate the damage to Cyrtomium macrophyllum, whereas

428

high-concentration mercury stress might damage the antioxidant system of

429

Cyrtomium macrophyllum, making SOD activity inhibited and thus leading to

430

decreased resistance to free radicals and peroxides. Other researchers have observed

431

similar results, they found low concentration of mercury could induce increased SOD

432

activity and high concentration of mercury led to decreased SOD activities (Li et al.,

433

2013; Yan et al., 2017); yet the decreased SOD activities were not observed in some

434

study (Smolinska and Szczodrowska, 2017) which may be due to that the

435

concentration of mercury did not reach the tolerance limit of the experimental plant.

436

However, different from the change trend in SOD activity, GSH content continued to

437

rise with the increased soil mercury concentration during whole exposure time. This

438

implied that GSH should possibly play an important role in resistance to oxidation

439

damage rather than SOD in the case of high concentration of mercury stress.

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ACCEPTED MANUSCRIPT Besides being an important antioxidant, the reduced GSH was a precursor to

441

phytochelatins, and itself could directly chelate with heavy metal ions to reduce their

442

toxicity (Wójcik and Tukiendorf, 2011; Samuilov et al., 2016). Hence, the mercury

443

stress on Cyrtomium macrophyllum might not only induce increased GSH to assist the

444

antioxidant system in eliminating ROS, but also possibly induce the formation of

445

phytochelatins (Manara, 2012; Szalai et al., 2013). These formed phytochelatins

446

together with GSH itself could chelate with mercury ion to reduce the toxicity of

447

mercury to Cyrtomium macrophyllum (Dago et al., 2014). So, the increase of GSH

448

content was favorable when suffered mercury stress, indicating the better resistance of

449

Cyrtomium macrophyllum to mercury stress.

450

Proline is an important intracellular substance for osmotic adjustment. Many studies

451

have demonstrated that low concentration of metal stress will inhibit but high

452

concentration of metal stress will promote proline accumulation (Szafrańska et al.,

453

2011; John et al., 2009; Fidalgo et al., 2013). Similar results were also observed in

454

this study. The proline content in Cyrtomium macrophyllum was statistically

455

significantly (p<0.05) reduced compared with the control at the beginning and at low

456

soil mercury concentration, and showed a trend of increasing with prolonged exposure

457

time and/or increased soil mercury concentration (Fig.2c). This meant that mercury

458

stress might inhibit water absorption by cells in Cyrtomium macrophyllum and

459

accordingly influence

460

accumulation could regulate this disturbed homeostasis and thus protect the structural

461

and functional integrity of cells (Yilmaz and Parlak, 2011). Besides, proline had the

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cellular

osmotic

21

homeostasis,

the

increased

proline

ACCEPTED MANUSCRIPT capability of eliminating ROS for preventing membrane lipid from overoxidation.

463

Therefore, the significant increase in proline content might be also ascribed to its

464

involvement in eliminating ROS induced by mercury stress on Cyrtomium

465

macrophyllum. In short, the increase in proline was helpful, signifying that

466

Cyrtomium macrophyllum had relatively high resistance to mercury stress.

467

To sum up, due to the increased SOD activity and the accumulation of GSH and

468

proline induced by mercury stress, the leaf tissue of Cyrtomium macrophyllum

469

showed high resistance to mercury stress. Through the removal of ROS by the

470

combined actions of SOD, GSH and proline for preventing membrane lipid from

471

overoxidation, the formation of mercury chelates with GSH or its possible successor

472

phytochelatin for reduced mercury toxicity, and the adjustment of cellular osmotic

473

homeostasis by proline, Cyrtomium macrophyllum could maintain metabolic

474

homoeostasis and grow normally without observed toxic effects when exposed to high

475

soil mercury concentration.

476

4. Conclusion

477

Some wild plants were selected and evaluated for their mercury accumulation and

478

translocation abilities. Among them, Cyrtomium macrophyllum was found to have the

479

phytoremediation potential for highly mercury polluted sites.

480

Pot experiments indicated that Cyrtomium macrophyllum could grow with no

481

observed toxic effects when exposed to 500 mg kg-1 and lower levels of soil mercury,

482

but the inhibition on growth was observed with the highest reduction of 20.2% in

483

biomass compared to the control.

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22

ACCEPTED MANUSCRIPT Although extremely high concentration of soil mercury exposure could restrict

485

mercury accumulation and translocation in Cyrtomium macrophyllum, when exposed

486

to 200 mg kg-1 and lower levels of soil mercury, Cyrtomium macrophyllum still

487

showed excellent mercury accumulation and translocation abilities with both TF and

488

BCF greater than 1, indicating that it could be considered as a great mercury

489

accumulating species.

490

With the aid of both the increased SOD activity and the accumulation of GSH and

491

proline induced by mercury stress, the leaf tissue of Cyrtomium macrophyllum

492

showed high resistance to mercury stress, which favorited mercury translocation from

493

the roots to the aerial parts. This was probably the reason for Cyrtomium

494

macrophyllum to tolerate high concentration of soil mercury.

495

In summary, due to its excellent mercury accumulation and translocation abilities as

496

well as its high resistance to mercury stress, Cyrtomium macrophyllum should be a

497

good candidate for phytoremediation of mercury polluted sites.

498

Acknowledgements

499

The authors gratefully acknowledge the financial support for this work by the

500

Ministry of Land and Resources of P. R. China (Grant No. 201411089).

501

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carbaryl stress in Caenorhabditis elegans. Pesticide Biochemistry & Physiology 138, 43. Yilmaz, D.D., Parlak, K.U., 2011. Nickel-induced changes in lipid peroxidation, antioxidative enzymes, and metal accumulation in Lemna gibba. International Journal of Phytoremediation 13, 805-817.

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Zeng, X.-W., Ma, L.Q., Qiu, R.-L., Tang, Y.-T., 2011. Effects of Zn on plant tolerance and non-protein thiol accumulation in Zn hyperaccumulator Arabis paniculata Franch. Environmental and Experimental Botany 70, 227-232.

Zhang, L., Wang, S.X., Wang, L., Hao, J.M., 2013. Atmospheric mercury concentration and chemical speciation at a rural site in Beijing, China: implications of mercury emission sources. Atmospheric Chemistry & Physics Discussions 13, 10505-10516. Zheng, M., Feng, L., He, J., Chen, M., Zhang, J., Zhang, M., Wang, J., 2015. Delayed geochemical hazard: A tool for risk assessment of heavy metal polluted sites and case study. Journal of Hazardous Materials 287, 197-206. Zhou, Z.S., Huang, S.Q., Guo, K., Mehta, S.K., Zhang, P.C., Yang, Z.M., 2007. Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago sativa L. Journal of Inorganic Biochemistry 101, 1-9. 27

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Zornoza, P., Millán, R., Sierra, M.J., Seco, A., Esteban, E., 2010. Efficiency of white lupin in the removal of mercury from contaminated soils: Soil and hydroponic experiments. Journal of Environmental Sciences 22, 421-427.

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Table 1 Mercury accumulation and translocation abilities of the collected wild plants in two mining sites

225.73 ± 4.06 190.99 ± 2.14 226.47 ± 4.32 99.87 ± 1.13 105.76 ± 2.49 234.70 ± 5.01 178.89 ± 1.69 193.04 ± 1.97 237.81 ± 6.12 158.44 ± 3.89 122.63 ± 2.57 209.31 ± 5.03 156.98 ± 4.92 102.46 ± 3.55 187.43 ± 1.57 173.44 ± 3.72

In root 13.90 ± 0.46 5.73 ± 0.13 16.70 ± 0.35 12.22 ± 0.61 8.47 ± 0.25 12.83 ± 0.17 4.50 ± 0.12 7.61 ± 0.74 13.74 ± 0.82 1.28 ± 0.09 17.19 ± 0.29 37.17 ± 0.71 13.48 ± 0.35 13.02 ± 0.23 4.77 ± 0.15 11.97 ± 0.42

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Cyrtomium macrohyllum a Woodwardia unigemmata a Dennstaedtia hirsute a Ageratum conyzoides b Sonchus arvensis b Sonchus arvensis a Artemisia argyi a Ixeris denticulata a Conyza canadensis a Xanthium sibiricum patrin ex widder a Commelina communis b Commelina communis a Scirpus triqueter b Cyperus rotundus b Calathodes oxycarpa a Imperata cylindrica b

Mercury concentration in wild plants (mg kg-1) In aerial part

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36.44 ± 0.96 8.94 ± 0.75 14.62 ± 0.49 11.49 ± 0.36 11.40 ± 0.19 17.31 ± 0.33 5.96 ± 0.17 12.27 ± 0.31 9.96 ± 0.24 1.89 ± 0.13 6.01 ± 0.52 8.38 ± 0.34 6.81 ± 0.18 2.48 ± 0.26 3.32 ± 0.15 10.93 ± 0.31

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Mercury concentration in rhizosphere soil (mg kg-1)

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BCF

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0.061 ± 0.032 0.032 ± 0.011 0.068 ± 0.023 0.138 ± 0.035 0.082 ± 0.056 0.052 ± 0.037 0.023 ± 0.041 0.037 ± 0.052 0.056 ± 0.017 0.007 ± 0.005 0.143 ± 0.065 0.174 ± 0.058 0.080 ± 0.032 0.105 ± 0.023 0.029 ± 0.016 0.066 ± 0.037

2.62 ± 0.18 1.56 ± 0.21 0.88 ± 0.16 0.94 ± 0.31 1.34 ± 0.22 1.35 ± 0.13 1.32 ± 0.09 1.61 ± 0.11 0.72 ± 0.07 1.48 ± 0.15 0.35 ± 0.09 0.23 ± 0.07 0.50 ± 0.13 0.19 ± 0.08 0.70 ± 0.13 0.91 ± 0.21

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Low-case letters a and b referred to that the wild plants were collected in the sites of Wanshan and Dehua respectively.

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Table 2 Mercury concentration in Cyrtomium macrohyllum growing in mercury-polluted soils at different exposure time In aerial parts g

24.53±3.15 48.83±5.09f 121.18±3.67e 153.65±2.18d 177.52±4.79c 184.59±5.21c 225.62±2.66b 259.18±1.98a ND

g

64.76±5.46 131.35±6.58e 285.73±5.58b 289.41±4.79b 302.41±3.45a 237.93±4.38c 176.39±2.35d 92.05±2.04f ND

In root

45 days of exposure

In aerial parts g

31.64±4.78 61.18±6.01f 124.28±3.96e 160.41±4.67d 181.64±5.21c 195.35±5.11c 236.04±4.81b 268.32±3.07a ND

g

77.92±5.23 157.28±7.32f 318.16±4.26c 341.74±5.38b 376.48±4.91a 275.97±4.21d 206.31±3.76e 58.09±2.89h ND

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94.61±3.47 188.04±2.99e 377.48±7.12a 344.57±5.35b 280.65±4.23c 250.26±4.58d 154.15±5.21f 40.73±4.12h ND

Mercury concentration in different parts of Cyrtomium macrohyllum is expressed in mg kg-1 dry mass. Different lower-case letters in each column indicates significant difference (p<0.05) between treatments. ND: not detectable. The detection limit for mercury was 20 µg kg-1 dry weight.

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37.88±3.42 70.68±4.13g 148.84±5.15f 175.52±6.12e 190.42±5.34d 210.96±7.25c 244.19±9.89b 271.36±5.87a ND

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44.38±6.02 89.03±5.01e 157.64±3.19d 191.46±3.91c 197.48±4.67c 228.31±7.34b 261.55±3.27a 273.88±2.98a ND

107.42±4.79f 223.47±7.56d 397.23±5.13a 369.57±3.45b 243.44±5.23c 232.74±5.76d 119.47±5.01e 37.87±3.19g ND

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Table 3: Mercury concentration in polluted soils and its recovery at different exposure time after plant cultivation 45 days of exposure

Concentration

MR (%)

Concentration

MR (%)

4.39±0.41 8.80±0.69 22.32±2.75 47.13±3.66 97.01±2.90 196.37±5.21 495.47±4.79 996.84±10.35

99.81±8.23 100.11±7.12 99.86±6.48 100.01±6.33 98.99±3.92 99.55±3.19 99.78±1.89 99.89±1.57

3.53±0.35 7.08±0.61 19.15±2.49 43.35±3.61 92.67±3.79 193.42±4.39 494.75±9.67 993.77±12.58

99.99±7.34 99.99±6.38 100.03±5.29 99.96±5.28 100.01±4.11 99.70±2.67 99.42±2.51 99.71 ±2.18

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60 days of exposure

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MR (%)

Concentration

MR (%)

2.42±0.33 5.04±0.47 14.97±1.38 40.34±2.93 91.54±4.05 191.78±4.99 493.33±7.48 986.68±14.38

99.97±7.68 100.03±5.92 99.98±4.28 100.06±4.39 100.19±3.17 99.87±2.89 100.99±2.08 100.92±2.05

1.67±0.27 3.20±0.32 13.03±1.42 38.08±2.84 91.00±3.87 190.26±5.38 493.49±8.39 978.14±13.29

99.96±5.92 100.00±4.87 99.96±7.12 100.09±4.89 100.10±4.13 99.63±3.17 100.07±1.99 98.26±2.38

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Mercury concentration in polluted soils is expressed in mg kg-1 dry soil. MR defined as the ratio of the sum of the mercury absorbed by the plant and the remaining mercury in the soil to the initial amount of mercury in the soil.

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BCF

TF

BCF

2.64 2.69 2.36 1.88 1.70 1.29 0.78 0.36

5.58 5.55 5.43 3.26 1.83 0.94 0.45 0.26

2.46 2.57 2.56 2.13 2.07 1.41 0.87 0.22

8.97 8.64 6.49 3.70 1.96 1.01 0.48 0.27

45 days of exposure

60 days of exposure

TF

BCF

TF

BCF

15.65 14.02 9.94 4.35 2.08 1.10 0.49 0.27

2.42 2.51 2.52 1.93 1.23 1.02 0.46 0.14

27.06 27.82 12.10 5.03 2.17 1.20 0.53 0.28

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Table 4 Translocation and bioconcentration factors of Cytromium macrohyllum determined by the pot experiments

2.50 2.66 2.54 1.96 1.47 1.19 0.63 0.15

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Fig.1 Biomass accumulation of Cytromium macrohyllum in mercury-polluted soils at different exposure time (Significant differences among data are indicated with different lower-case letters, p<0.05)

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（c） a: SOD activity b: GSH content c: proline content Fig.2 SOD activity and accumulation of GSH and proline in leaf tissue of Cyrtomium macrohyllum changed with soil mercury concentration and exposure time (Significant differences among data are indicated with different lower-case letters, p<0.05)

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Highlights • Cyrtomium macrophyllum was screened out to be a potential mercury accumulator plant.

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• Mercury accumulation and translocation abilities of Cyrtomium macrophyllum were tested by pot experiment. • Cyrtomium macrophyllum shows capability of tolerating mercury stress.

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• Cyrtomium macrophyllum is a promising species for the phytoremediation of mercury polluted sites.