Enhancement of phytoextraction efficiency coupling Pteris vittata with low-dose biochar in arsenic-contaminated soil

Abstract Phytoremediation of arsenic (As) by Pteris vittata (P. vittata) is a cost-effective and environmentally friendly method for restoring As-contaminated sites. However, the phytoextraction efficiency is low in some cases, such as clay soil, thus biochar was applied to enhance the efficiency of As extraction. The paper investigated the effect of biochar on soil characteristic, As mobility, and As uptake in P. vittata with a 90-day greenhouse experiment. Biochar derived from rice straw was added at rates of 0.5, 1.5, and 4% (w/w). The results showed that, under biochar amendment, soil pH raised from 5.24 to 6.03 and 4.91 to 5.85, soil dissolved organic carbon (DOC) increased 11.1–46.1% and 2.8–11.2%, respectively, in rhizosphere and bulk soils. Biochar also increased soil catalase (CAT) activity significantly, especially for the rhizosphere soil. Besides, biochar increased the labile As in the soils and transfer coefficient from roots to aboveground, thereby enhancing As accumulation by P. vittata tissues. The accumulation of As in fronds of P. vittata was up to 350 mg kg−1 in 1.5% biochar, which was more than twice the control and far beyond other biochar treatments. The results indicate that biochar addition is favorable to improve phytoremediation of P. vittata in As-contaminated soil and 1.5% (w/w) biochar may be a reasonable application ratio, thus providing an effective solution to enhance the efficiency of As phytoextraction. NOVELTY STATEMENT Biochar increased soil catalase activity in the rhizosphere of P. vittata. Biochar increased the labile concentration of arsenic in soil and arsenic accumulation in P. vittata significantly. Combining biochar and P. vittata reduced arsenic in soil. Biochar amendment was favorable for phytoremediation of P. vittata in arsenic-contaminated soil.


Introduction
The sources of arsenic (As) on earth include both natural and anthropogenic, and the release from As-enriched minerals is the main source in the environment (Singh R et al. 2015).However, the primary sources of As contamination in soil are human behaviors, such as coal combustion, industrial processes, the application of pesticides and fertilizers containing As, and especially mining and smelting.Arsenic often occurs in sulfidic ores such as realgar (As 4 S 4 ) (Seidl et al. 2019).Shimen realgar mine in central south China (Hunan province) is the largest realgar mine in Asia, with more than 1,500 years mining histories (Tang et al. 2016).The long and wide As mining and smelting industry lead to highly As-contaminated water and As-accumulated farmland, even after the mine was abandoned in 2010 (Yang F et al. 2018).Arsenic concentrations in adjacent rivers can be up to 14.5 mg L À1 , and average As concentration in farmland soils is as high as 99.51 mg kg À1 (Zhu et al. 2015), far exceeding Chinese National Environmental Quality Standards for agriculture soil, which is 30 mg kg À1 As in flooded soils (GB15618-1995).The As-contamination in soils is one of the considerable concern with respect to environment and health risks.To overcome these problems, a great deal of research has been done to gain knowledge about the safe and economic remediation of arsenic-contaminated soils (Singh D et al. 2017;Singh R et al. 2021).The massive residents in As-contaminated area and its adjacent area suffered from As exposure and risks via the food chain, from soil to human body.Therefore, to protect animal and human health, the As-contaminated soils urgently need to be restored.
So far, there are many studies on the promising development of phytoremediation of heavy metal contaminated soil (Seth et al. 2012).For example, Spirodela polyrrhiza L. was one of the potential phytoremediator species in aquatic environments, presenting high capacity of metals extraction (Seth et al. 2007).Helianthus annuus L. was found to be resistant to lead (Pb) and having the capability of accumulating a large amount of Pb in the tissues (Seth et al. 2011).Compared with physical and chemical remediation, phytoremediation of As-contaminated soils offers an environmentally friendly and more cost-effective method for soil remediation (Wan et al. 2016).A brake fern, P. vittata, had been discovered as As hyperaccumulator, because it is extremely efficient in accumulating As from soils and translocating to above-ground biomass (Ma et al. 2001).It can accumulate up to 1,442-7,526 mg As in per kg frond from As-contaminated soils (Singh R et al. 2015).The hyperaccumulation mechanism of As by P. vittata is associated with rhizosphere characteristics and specific uptake mechanisms, including physiological and molecular mechanisms (Fitz et al. 2003;Singh R et al. 2015).As an example, root exudates and rhizobacterial activities could increase As dissolution and thus enhance plant uptake (Tu S et al. 2004;Liu et al. 2016;Han et al. 2023).
However, the efficiency of As extraction by P. vittata is low in some cases, such as in clay soil, As accumulation is lower than loam or sandy soils (Liao et al. 2004).So we need explore some approaches to enhance the ability of P. vittata in As uptake, translocation, and accumulation.Various soil amendments have been used to assist phytoextraction by P. vittata for As-contaminated soils, such as compost and phosphate.Application of carbon-rich composts in soils had shown the ability to increase As mobility in soil and thus aid plant uptake/accumulation (Cao et al. 2003).Biochar is also carbon-enriched materials produced via slow pyrolysis of organic biomass at relatively low temperatures (<700 C) under limited oxygen condition (Yin et al. 2016), having shown its ability to reduce metals mobility and phyto-toxic effects.Rice straw-derived biochar can decrease Cd, Pb and Zn concentration in rice plants by reducing the solubility (Zheng et al. 2012).Beesley and Marmiroli (2011) also detected that biochar is able to immobilize and retain Cd, Zn and As on biochar's surface.The high surface area and functional groups (such as carboxylic, carbonyl and hydroxyl moieties) in biochar enables enhanced metals sorption to their surfaces, thus immobilize contaminant when applying polluted soils (Beesley et al. 2011).Unlike cationic metals, As mobility is increased with increasing pH in soil and binds to anion exchange sites on soils such as Fe and Mn oxides and oxyhydroxides.It has been reported that biochar derived from rice-straw increased As uptake by rice root through enhancing its solubility (Yin et al. 2017).Therefore, biochar is a potential material to promote As transfer from soil to plants and increase the phytoremediation efficiency.
We speculated that biochar could promote P. vittata growth and enhance its uptake with As.Here, biochar and P. vittata were coupled in the present study to improve the growth of P. vittata and As extraction for higher phytoremediation efficiency from As-contaminated soil.The specific objectives of the paper were: (1) to investigate the effects of biochar on As uptake by P. vittata; (2) to identify possible mechanisms responsible for As uptake after biochar application.

Soil sample collection and biochar production
Soil samples contaminated with high As were collected from a rice paddy near Shimen realgar mine in Hunan province (Lat/Long: 29 39'26"N, 111 02'37"E), the largest realgar deposit in Asia.Surface soil samples (0-20 cm) were collected, air-dried and sieved through a 2 mm sieve.Soil properties, including texture, pH, DOC, total C, total N, total S and total metals were determined.Details are shown in Table S1.
Biochar was produced by slowly pyrolyzing rice straw at 450 C for 1 h under anaerobic condition with a continuous N 2 flow in a box furnace (Thermo Scientific, BF51732BPMC-1, USA).The detailed procedures had been described by Yin et al. (2016).The biochar product is highly alkaline (pH, 10.7) and relatively higher cation exchange capacity (CEC) (33.4 cmol kg À1 ).
Pot trial for soil amended with biochar and grown P. vittata Pot experiments were conducted using As contaminated soil under greenhouse condition with photoperiods of 16 h light/8 h dark, relative humidity of 70%, and mean temperature of 25 C.In preliminary experiments, we compared the difference of As solubility in the soil with various biochar addition ratios and four treatments (0, 0.5, 1.5 and 4% (w/w) biochar addition) were applied in this experiment.Biochar and soil were thoroughly mixed before being placed into PVC pots (18 Â 20 cm dxh).Each treatment had three repetitions, with 2.5 kg soil in each pot.All treatments were maintained 50% water holding capacity (WHC) with deionized water to pre-equilibrate for 1 month before plant growth.Then established P. vittata plants with 5-6 fronds were planted in a nylon mesh bag with 20 lm pore size (12 cm high and 7.5 cm diameter), which was settled in the center of each pot.This bag was used to keep the growing roots within it, forming a separation of root/rhizosphere compartment from a bulk soil compartment, while nutrients were allowed to pass freely for plant uptake (Lu Y et al. 2007).All pots were randomized inside the greenhouse and rearranged every 3 days.The ferns were watered using deionized water to hold 50% WHC every 3 days during the whole growing period.

Plant sampling and analysis
After 90 d growth, the intact P. vittata plants were carefully dug out from soil and separated into root, stem and frond.These tissues were adequately washed and freeze-dried at À80 C for 2 days, and then ground before digestion with HNO 3 /H 2 O 2 (EPA 3050b).As concentration was then analyzed using a quadrupole inductively coupled plasma mass spectrometry (ICP-MS, NexION 300X, PerkinElmer).

Soil sampling and analysis
Soil samples were collected from rhizosphere (inside root bag) and bulk area (outside root bag).One half fresh soils were reserved for catalase (CAT) activity analysis and the rest were freeze-dried for other determination, including pH, DOC concentration, diffusive gradients in thin-films (DGT) -available As and digestion to analysis the total As concentration.The detailed procedure of CAT activity analysis was as follows: The fresh soils were homogenized in 5 mL 100 mM potassium phosphate buffer (pH 7.6).The homogenized samples were centrifuged at 10,000Âg for 5 min.The supernatant was used as a crude enzyme extract in soil CAT activity analyses and the soil CAT was determined as a decrease in absorbance at 240 nm for 1 min following the decomposition of H 2 O 2 (Gunes et al. 2009).The reaction mixture (3 mL) contained 15 mM H 2 O 2 , 50mM phosphate buffer (pH 7.0), and 50 mL of crude enzyme extract at 25 C. Total As concentration were analyzed as same as plant samples.See below for DGT analysis procedure.

The DGT deployment to the rhizosphere and bulk soil
The technique of diffusion gradient in thin films (DGT) was used to assess bioavailable As in rhizosphere soils.Precipitated Zirconia (PZ) DGT gels and diffusive gels were prepared following the procedure outline by Guan et al. (2015) and Zhang H and Davison (1995), respectively.The DGT-holding devices were based on a simple tight-fitting piston design, consisting of a backing cylinder and a front cap with a window of 2.54 cm 2 area.The PZ binding gel, diffusive gel and 100-mm-thick, 0.45-mm pore size hydrophilic polyethersulfone membrane were assembled following the procedure outlined by Zhang H et al. (1998).
Rhizosphere and bulk soils were sieved to <2 mm and placed in individual acid-washed plastic specimen cup (20 g for each subsample).Appropriate amounts of MQ water were added to obtain 60% moisture content and equilibrated at 22 C for 2 days.The moisture content of the soils was then raised to 100% to obtain a soil slurry and incubated for 24 h prior to DGT deployment.Soil and water were mixed thoroughly using a plastic rod when adjusting the humidity.Thereafter, DGT devices were deployed to the equilibrated soils at 22 C for 24 h, ensuring good contact between the soil and the window of DGT device.After retrieval, DGT devices were taken out and rinsed with MQ water to remove soil particles and then disassembled.The PZ gel was retrieved and immersed in 10 ml of 0.5 mol L À1 NaOH for 24 h to elute As.The eluate was analyzed by ICP-MS.The concentration of C DGT (mg L À1 ) was calculated based on the published method (Luo et al. 2018).
The bioaccumulation factor (BAF) and translocation factor (TF) The BAF and TF of P. vittata were calculated to analyze the plant/soil relationships.BAF is the plant/soil As concentration ratio.These two factors are calculated as follows:

Statistical analysis
All data were present as the mean of replicates with standard deviation.Difference significance was performed using one-way analysis of variance (ANOVA).Duncan's multiple range test (p < 0.05) was implemented using the IBM SPSS Statistics 22 software for the calculations.
Quality assurance and quality control (QA/QC) The experiments in this study were set up in a completely randomized factorial design with three replicates of each treatment.The quality assurance and quality control procedure were conducted by using standard reference materials, including standard reference soil GBW07403 (GSS-3) or standard reference solution (SLRS-6), provided by the National Research Center for Certified Reference Materials of China.

Results and discussion
Effects of biochar on pH and DOC in P. vittata rhizosphere Effects of biochar on pH and DOC in P. vittata rhizosphere and bulk zone are shown in Table 1.Both in the rhizosphere soil and bulk soil, the amendment of biochar showed a significant liming effect as the pH raised from 5.24 to 6.03 and 4.91 to 5.85, probably due to the dissolution of metal oxides, hydroxides and carbonates induced by biochar (Houben and Sonnet 2015) and the high mineral ash content in biochar (Yin et al. 2016).Besides, the increase in soil pH was observed in the rhizosphere, which were 0.13-0.58units higher than bulk areas.Similar to a previous study reported by Gonzaga et al. who observed significantly higher pH in P. vittata rhizosphere than bulk soil (Gonzaga et al. 2006).
The high rhizosphere pH is likely caused by the low buffer capacity and ion balance caused by high excretion of hydroxyl groups (Xu et al. 2014).Root exudates released from roots is able to acidify rhizosphere soil to release As.The uptake of oxyanions-As (HAsO 4 2À and H 2 AsO 4À ) by plants may also release OH À into the rhizosphere and thus increasing rhizosphere pH (Gonzaga et al. 2009).
Biochar amendment increased soil DOC by 11.1-46.1% and 2.8-11.2%,respectively, in rhizosphere and bulk soils (Table 1).High concentration of carbon in biochar might be one of the causes.It had been reported that biochar could promote soil-derived organic carbon solubility, likely associated with pH changes (Zhang M et al. 2013).Besides, higher DOC concentrations in rhizosphere than bulk soils were also observed.This might result from root exudation, such as phytic and oxalic acids, which increased the carbon source in the rhizosphere (Kalbitz et al. 2000;Tu S et al. 2004).

Effect of biochar on rhizosphere soil catalase (CAT) activity
Plants have well-organized enzymatic and non-enzymatic antioxidant defense systems (Shahid et al. 2017).Rhizosphere soil CAT activity was quantified to assess whether the biochar addition caused a change in the relative environment toxicity.CAT can be found in all aerobic microorganisms, plant and animal cells and it can split hydrogen peroxide into molecular oxygen and water and thus prevent cells from damage by reactive oxygen species (ROS) (Kim et al. 2011).CAT has been considered as a general index for evaluating soil microbial activity (Rodriguez-Kabana and Truelove 1982;Stpniewska et al. 2009).In the present study, biochar amendment enhanced CAT activity in rhizosphere soil (Figure 1).The activities of CAT were increased 18.4-35.7%with biochar amendment relative to the control, which was similar with previous study that CAT activities enhanced significantly with an increasing application dose of Eichornia biochar (Masto et al. 2013).In the present study, the maximum CAT activity was observed with 1.5% biochar addition and the untreated rhizosphere soil showed very low CAT activities.The results indicate that biochar can greatly increase the oxidative capacity of soil microorganisms.
It had been reported that biochar has a great labile fraction of carbon, which can be used as an energy source by soil microorganisms (Lehmann et al. 2011).The mineralization of soil organic matter (SOM) is an considerable microbially-mediate process by which carbon and other nutrients are converted from organic into inorganic forms (Nannipieri et al. 2012).Besides, pH increase induced by biochar addition is another factor, increasing proton amounts, and thus increase microbial biomass and bacteria abundance (Lehmann et al. 2011).Soil pH is one of the main factors driving changes in soil enzyme activity (Zhang D et al. 2022).Significant positive correlation was observed between pH and CAT activities in the rhizosphere (p < 0.05, Table S2).It had been found that the changes in environmental pH greatly affect the respiration and metabolism (Srinivasan and Mahadevan 2010).CAT activity is an important indicator of soil redox capability associated with soil biochemical processes, such as soil energy and nutrient transformation (Zhang G et al. 2020), which may alter As behavior in soil and uptake by plants (Yang X et al. 2022).

Effect of biochar on labile arsenic in the soils
The DGT method had been used as a predictive tool to assess potential metal(loid)s availability in the soil (Zhang H et al. 2001;Cattani et al. 2009).After P. vittata harvest, biochar application increased DGT-As concentration, especially in the rhizosphere soils (Figure 2a).The highest growth rates with 4% biochar up to 906.7% in the rhizosphere and 728.6% in the bulk area.This was most probably resulted from the high pH and DOC after biochar addition (Table 1).
Arsenic bioavailability in soil increased with increasing the soil pH and DOC (Mensah et al. 2022).Generally, the increase in pH would be expected to increase As mobility because the mobility of arsenic oxyanion increases.Arsenate (AsV) is the major As species in aerobic soils and it tends to be negatively charged (Yin et al. 2016).As a result, As is released from positively charged soil surfaces when the soil pH is increased, thus tends to mobile (Gomez-Eyles et al. 2013).The enhancement of rhizosphere pH may increase rate of HAsO 4 2À in soils and increase negative surfaces charges of soil minerals (iron and aluminum oxides) (Gonzaga et al. 2006).Hartley et al. (2009) noted an increase in pore water As after biochar addition, whilst Lomaglio et al. (2017) also found biochar addition increased the available DGT-As concentration.The release of As was found to be influenced by DOC dynamics from As-DOC complexes (Williams et al. 2011).Biochar is recognized as a potential DOC source and has been proved to increase DOC concentrations in soils (Table 1).The concentration of DOC in rhizosphere significantly affects nutrient, contaminant mobility, microbial activity and soil properties (Kaiser et al. 2002).DOC, as the most labile organic ligands in the soil, can compete with oxyanionic As for adsorption sites on the surface of aluminum/iron oxides, and ultimately increase the mobility of oxyanions in soil (Jeon et al. 2018).
Besides, the enhancement of CAT activity with biochar application raises microorganism activity, which can also affect the redox conditions of the soils and thus impact As speciation.Positive correlations were found between CAT activities in rhizosphere soil and the amounts of As uptake by stems or fronds (Figure 3).That might suggest microbes also promote As absorption by P. vittata.Microbes can fuel to reduction of As(V) to As(III).Labile DOM could provide energy sources for some reducing micro-organisms, for instance Fe reducing bacteria, leading to reduction of Fe oxides, thus resulting in the release of As bound to precipitated Fe oxides (Balasoiu et al. 2001;Mladenov et al. 2010).The difference of DGT-As between rhizosphere and bulk  area also was observed.The labile As in rhizosphere has 6.4-29.3%higher than bulk soils, and the rate increase with the ratio.This indicates root growth has great impact on labile As and it could be due to rhizosphere acidification and DOC source enhanced by root exudates.Rhizodeposition not only promoted root exudation but also enhanced soil organic matters mineralization (Obeidy et al. 2016), thus affecting As mobility.P. vittata could exude high amounts of organic carbon including phytic and oxalic acids, thus effectively dissolve As in both As-contained minerals and As-contaminated soils (Tu S et al. 2004).Fitz et al. (2003) also found a significantly higher DOC concentration in P. vittata rhizosphere than in the bulk soil after growing in a soil containing 2,270 mg kg À1 As with higher pH and higher clay content.
Biochar increases arsenic uptake in P. vittata tissue which decreases arsenic concentrations in soils Mean As concentrations in different tissues of P. vittata followed the order fronds > stems > roots, which is consistent with previous study (Fitz et al. 2003).The fronds accumulated over 150 mg of As kg À1 whereas the concentration in roots was below 50 mg of As kg À1 (Figure 4).It was reported that P. vittata can survive in a soil containing up to 500 mg kg À1 As and accumulate up to 5,600 mg As kg À1 dry frond (Tu C et al. 2002).In the present study, P. vittata grew well in the soil with 114 mg As kg À1 and uptake more than 150-350 mg As in per kg frond.
Transfer coefficient, defined as the As concentration ratio between aboveground biomasses (stem and frond) and underground biomasses (root), is used to determine the effectiveness of P. vittata in translocating As from root to aboveground parts.The concentration of As found in the roots was relatively lower, compared to stem and frond.The transfer coefficients of As significantly increased with biochar application (p < 0.01), with 1.5% rate having the highest transfer coefficient (Table 2).The 1.5% treatment was the most effective in increasing As uptake in P. vittata biomass.As expected, the combined amendment of biochar with P. vittata for three months reduced soil As concentration by 4.5-11.5% in rhizosphere soils and 0.9-8.9% in bulk soil (Figure 2).The results proved that biochar was effective in increasing As uptake by P. vittata and decrease As concentration in soils (Oliveira et al. 2017).
The biochar treatments significantly enhanced As uptake by P. vittata, with stem and frond concentrations increasing by 62.9-213.2%and 56.3-116.2%,respectively, when compared to the control (Figure 4).It is similar with previous results that biochar application significantly increased As concentration in ryegrass shoots with 60 t ha À1 treatment (equivalent to 13.3% of the upper 30 cm layer) (Gregory et al. 2014).This could be attributed to the increase in soil alkalinity and DOC caused by biochar addition (Table 1), resulting in the enhancement of As mobility in the soil (Gonzaga et al. 2006;Williams et al. 2011;Jeon et al. 2018).The data from DGT test have found biochar increased evidently available As in the soil, and thus enhanced its uptake by plants.
There exist significantly positive correlations between DGT available As in rhizosphere soil and total As in root (p < 0.05, Figure 4, Table S2).Biochar application increases available As in the rhizosphere by replacing adsorbed As, thus resulting in elevated As uptake.When just consider the low ratio treatments (0.5%, and 1.5%), the correlation coefficients are much higher than all treatments between labile As in soil and accumulated As by P. vittata.This suggested the application ratio for biochar should keep below 1.5%, and higher ratio (such as 4%) may not provide more advantages and cause resource waste instead.
It is not unexpected that the great DGT available As with 4% biochar unable adequately translate to the plant.A high application rate for biochar seems to be harmful to plant growth through decreasing nutrient availability or creating adverse nutrient ratios in soil.Biochar has a very high C/N ratio, which could cause N immobilization and nutritional deficiencies for plants (Lehmann et al. 2003).The rich organic carbon in biochar impacted soil water retention capability and limited the amount of P uptake, influencing root growth (Gaur and Adholeya 2000).

Conclusion
This study investigated the impact of biochar amendment on As availability in soil and uptake by P. vittata.The results showed that the tandem use of biochar and phytoremediation is an effective method for As-contamination remediation.Biochar enhanced phytoextraction by increasing labile As and stimulating soil enzyme activity.The application of biochar increased arsenic mobility in soil and arsenic accumulation in P. vittata.Also, soil properties, including soil pH, DOC, and CAT activity, were improved.CAT activity in soil increased significantly with the addition of biochar, especially in rhizosphere soils.Besides, the results showed that increasing the addition rate of biochar was beneficial to increase the efficiency of As extraction by P. vittata.Biochar increased the labile concentration of arsenic in the soil, which resulted in a significant increase in arsenic accumulation in P. vittata in biochar-amended soils.This enlightens that supplementing the soil with the appropriate rate of biochar can be applied to the phytoremediation of arsenic contamination.Our results indicated that the reasonable application ratio for biochar should be below 1.5% when restoring As-contaminated soil with P. vittata, as a consequence of the highest As uptake efficiency by plants and As remove from soil.However, the enhanced mobility of As may also pose a risk to the surrounding environment.
To have a comprehensive picture about biochar role in phytoremediation in As-contaminated soil, further experiments should include long-term application on different types/rate biochar in various soils.

Figure 3 .
Figure 3. Relationship between rhizosphere soil characteristics (pH_R, DOC_R, S_CAT, and DGT_R) and As concentrations (mg kg À1 ) in P. vittata tissues (root, stem, and frond).Notes: pH_R, DOC_R and DGT_R indicate pH, DOC and DGT value in the rhizosphere of PV.S_CAT is CAT activities of soil.The blue lines are the correlation fitting lines for all treatments (the BC ratio are CK, 0.5%, 1.5% and 4%).

Table 1 .
The variation of pH and DOC in soils amended with different BC ratio.
Notes: values are given as mean ± SD for three measurements.Different letter indicates significant difference among treatments (p < 0.05).
BC ratioAs-rhizosphere soil As-aboveground (mg kg À1 ) As-underground (mg kg À1 ) Notes: values are given as mean ± SD for three measurements.