Microwave Soil Treatment along with Biochar Application Alleviates Arsenic Phytotoxicity and Reduces Rice Grain Arsenic Concentration

Rice grain arsenic (As) is a major pathway of human dietary As exposure. This study was conducted to reduce rice grain As concentration through microwave (MW) and biochar soil treatment. Collected soils were spiked to five levels of As concentration (As-0, As-20, As-40, As-60, and As-80 mg kg−1) prior to applying three levels of biochar (BC-0, BC-10, and BC-20 t ha−1) and three levels of MW treatment (MW-0, MW-3, and MW-6 min). The results revealed that MW soil treatment alleviates As phytotoxicity as rice plant growth and grain yield increase significantly and facilitate less grain As concentration compared with the control. For instance, the highest grain As concentration (912.90 µg kg−1) was recorded in the control while it was significantly lower (442.40 µg kg−1) in the MW-6 treatment at As-80. Although the BC-10 treatment had some positive effects, unexpectedly, BC-20 had a negative effect on plant growth, grain yield, and grain As concentration. The combination of BC-10 and MW-6 treatment was found to reduce grain As concentration (498.00 µg kg−1) compared with the control (913.7 µg kg−1). Thus, either MW-6 soil treatment alone or in combination with the BC-10 treatment can be used to reduce dietary As exposure through rice consumption. Nevertheless, further study is needed to explore the effectiveness and economic feasibility of this novel technique in field conditions.


Introduction
Arsenic (As) is a toxic metalloid, which is ubiquitously present in the environment. It has raised serious global concern because of its adverse effect on human health and the ecosystem [1]. The sources of As in soils are mostly geogenic. Weathering of different As containing minerals is considered a major geogenic process to release As in the environment [2]. Anthropogenic sources such as mining, some industrial manufacturing processes, energy and fuel production, preservation of timber, application of different As containing pesticides, fertilizers, and municipal waste to the land are responsible for soil and water As contamination [3]. Although As contaminated groundwater, which is used for drinking purposes, is probably the major pathway of human exposure [4], the use of this As polluted groundwater for crop irrigation gives rise to high deposition of As in the topsoil and ultimately results in the As uptake by crops [5], which augments As exposure through the food chain and threatens human health. Excessively high As pollution in water, soil, and crops has already been identified in many countries [6][7][8].
It has been demonstrated that rice can accumulate 10 times more As than other cereal crops (e.g., wheat and barley) [9]. As a consequence, a considerable amount of As has already been reported in rice grains throughout the world, such as <0.01-2.05 µg g −1 for Bangladesh, 0.31-0.70 µg g −1 for China, 0.03-0.044 µg g −1 for India, and 0.11-0.66 µg g −1 Although several studies have been conducted to investigate As immobilization efficiency of different biochars, biochar produced by MW-assisted pyrolysis has not been well studied. Microwave-assisted pyrolysis has been substantiated as a viable alternative to conventional pyrolysis because of its higher heating rate, and lower requirement for pre-processing of the feedstock, which enhances the yield and quality of the char [37][38][39]. Furthermore, no published study has so far reported a combination of MW soil heating and biochar treatment for soil As remediation. Hence, in As contaminated soil the MW and biochar treatment could be a novel technique to reduce the As uptake by plants and ultimately reduce As accumulation in rice grain, which will decrease the human health risk through dietary exposure. Therefore, this study aimed to investigate the potential impacts of MW soil heating and MW pyrolyzed biochar on the alleviation of As phytotoxicity and accumulation of As in rice grain.

Soil Collection and Preparation
Soils were collected from a crop production paddock of the Dookie campus of the University of Melbourne (Paddock H12, 36 • 23 51 S; 145 • 43 17 E) at a depth of 0-15 cm. The soil was a grey to grey-brown clay and was classified as a Congupna clay [40] or a Grey Vertosol [41]. The soil collection site was selected based on its availability on campus and its suitable soil texture since rice cultivation needs clay types of soil. The soil was collected in December 2017 (summer season). Some important soil properties are given in Table 1. The collected soil was dried and sieved through a 4 mm mesh to minimize the undesired effects of stones, sticks, and clods. This operation did not reflect the true field situation where the distribution of coarse material is highly irregular. However, this was essential to ensure a uniform experimental condition for MW soil heating. After sieving, a soil lot of 8.5 kg soil (with 15% moisture) was thoroughly mixed and shifted into unperforated pots (diameter 27 cm and height 30 cm), to prevent the loss of water-soluble As from the pots [42].

Physicochemical Properties of Experimental Soil
The physicochemical properties of the soil were analyzed to ascertain the levels of nutrients as well as other elements present, following the standard method of analysis. For analysis of the soil properties, a composite soil sample was sent to the Nutrient advantage Laboratory, a NATA accredited laboratory in Australia (Lab number: 11958, ISO/IEC 17025). The physicochemical properties of the pre and post-microwave treated soil are presented in Table 1.

Arsenic Application
The soils were spiked at five different levels of As concentration (0, 20, 40, 60 and 80 mg kg −1 dry soil) using sodium arsenate heptahydrate (Na 2 HAsO 4 .7H 2 O) [43]. Respective amounts of sodium arsenate were mixed with deionized water to prepare the As solutions. The As solution was mixed with the soil by spraying and homogenizing thoroughly by hand mixing. Since the initial As concentration of the soils, prior to treatment, was <0.01 mg kg −1 (Table 1), there was no chance of further As being available from the soil. To establish an equilibrium condition between soil and applied As, soil moisture was maintained at field capacity for two weeks as a waiting period before applying biochar and MW treatment.

Biochar Preparation, Characterization and Application
Three different levels of sawdust derived biochar (0, 10, and 20 t ha −1 ) were applied in previously As spiked soils. Since several studies have been conducted on As immobilization using biochar, there are several rates of biochar application, based on the soil contamination and biochar types. In this study, the most common rate of biochar, which was used, was proposed by other researchers [44,45]. Biochar was prepared from pine sawdust by an MW assist pyrolysis technique at around 650-700 • C temperature [46]. A MW chamber, consisting of six magnetrons (1 kW each), operating at a frequency of 2.45 GHz, was used to prepare the biochar (Figure 1a; Custom designed and built microwave system). A high temperature (1450 • C) tolerant quartz crucible (Height 30 cm, diameter 10 cm) with a lid was used to prepare the biochar. The lid was placed on the full crucible to limit oxygen availability during the pyrolysis process in the MW chamber. An infrared camera (FLIR T1050SC model; FLIR Systems Inc., Orlando, FL, USA) was used to measure the pyrolysis temperature by capturing thermal images immediately after the MW heating ( Figure 1b). The yield, total ash content, and volatile matter, of biochar, were calculated based on the following Equations (1) [47], (2) [48], and (3) [47], respectively.
where, W 1 is the dry weight of the sawdust sample before pyrolysis, and W 2 is the final biochar weight.
Total ash (%) = ( where, W c is the weight of the crucible, W 1 is the weight of the sawdust sample and crucible and W 2 is the weight of the ash and the crucible Volatile matter (%) = ( where, A is the weight of dried sample and crucible, B is the weight of crucible, and C is the weight of residue and crucible after ignition. The specific surface area (BET), pore size, and total volume of pores were measured using a Autosorb iQ3 gas adsorption analyzer (Quantachrome, Beach, FL, USA). Before gas sorption analyses, samples were degassed overnight at room temperature and then incubated for 1 h at 250 °C. The properties of sawdust biochar are given in Table 2 and the scanning electron microscopic (SEM) structure of sawdust biochar is given in the supplementary figure ( Figure S1).  The specific surface area (BET), pore size, and total volume of pores were measured using a Autosorb iQ3 gas adsorption analyzer (Quantachrome, Beach, FL, USA). Before gas sorption analyses, samples were degassed overnight at room temperature and then incubated for 1 h at 250 • C. The properties of sawdust biochar are given in Table 2 and the scanning electron microscopic (SEM) structure of sawdust biochar is given in the Supplementary Figure (Figure S1).

Microwave Soil Heating
One week after the biochar application, three levels of MW energy (0, 127.06, and 254.12 kJ kg −1 soil) were applied for 0, 3 and 6 min to treat the soil to attain soil temperatures around room temperature, 60 and 90 • C, respectively (Figure 1c,d). The duration of MW irradiation to heat the soil to the desired temperature was determined by following the method of previous research work [53]. A MW chamber, consisting of six magnetrons (1 kW each), operating at a frequency of 2.45 GHz, was used for soil treatment (Figure 1a). The soil temperature was measured for each MW treatment at a depth of 10-15 cm, immediately after MW energy exposure, by using liquid-in glass thermometers [54]. An infrared camera (FLIR T1050SC model) was also used for taking thermal images to show the energy dissipated and temperature distribution across the MW treated soil. Due to the very high dependence of the dielectric properties on moisture content [55], the moisture content in the soil will greatly affect the heating effect of MW energy dissipated in the soil. In this experiment, the moisture content was maintained at around 15% (w/w) at the time of MW soil treatment.

Experiment Setup
After the application of As, biochar and MW treatments, along with the control treatments and each with four replicates (total of 180 pots) the pots were placed in the glasshouse, following a completely randomized design (CRD). To describe the treatment combination more conveniently, abbreviated forms were used for As treatments (As-0, As-20, As-40, As-60 and As-80), biochar treatments (BC-0, BC-10 and BC-20) and MW treatments (MW-0, MW-3 and MW-6). To supply adequate nutrients for proper plant growth, diammonium phosphate (DAP) for N and P and Potassium Sulphate (K 2 SO 4 ) for K and S were applied to each pot as a basal dose, as per standard practices for Australian rice cultivation [56], prior to seed sowing. The rest of the calculated N was supplied as urea in two split doses, one at the tillering stage and another one at the panicle initiation stage of plant growth. The application rate of N, P, K and S was 200, 30, 18, and 23 kg ha −1 , respectively. Twenty seeds of the YRM_70 variety (Oryza sativa L.) were sown per pot on the 15th of January 2018. At the three-leaf stage, extra seedlings were removed, leaving 12 seedlings per pot. Insects and diseases were controlled as per standard methods of rice cultivation [56], and weeds were removed by hand when needed. Tap water was used for irrigation purposes. This water source contained As below the detection limit (<0.01 µg L −1 ); therefore, there were no possibilities of As addition from the tap water to the potting soil. After seed sowing, soil moisture was maintained at field capacity up to the three-leaf stage of plant growth. Flooding irrigation was started at the three-leaf stage and maintained at a predetermined level (10cm) in the pot throughout the growing period and stopped 10 days before harvesting the plants [56,57]. After 150 days of the growing period, at the physiological maturity stage, the crop was harvested on the 14th of June 2018.

Recording of Agronomic Data
Leaf chlorophyll content was measured at the tillering stage as a SPAD value using the Chlorophyll Meter-SPAD-502Plus (Soil-Plant Analysis Development) [58]. At the tillering stage, the total number of tillers per pot was counted prior to collecting plant samples (3 hills per pot) for shoot biomass measurement. At the physiological maturity stage, rice grains were collected from the panicle by hand threshing and yield (g pot −1 ) was calculated as described previously [59]. Total filled grain and sterile spikelets were counted to calculate the spikelet sterility percentage. The fresh weight of all plant samples was recorded prior to drying at 60 • C in a dehydrating oven (Thermoline Scientific, TD-500F) for 48 h to determine the dry weight.

Grain Total Arsenic Analysis
Grain total As analysis was performed as per the method described in the user manual of atomic fluorescence spectrometry (AFS; PSA 10.055 Millennium Excalibur, 2009, USA) [60]. Since the method is generalized for solid materials, some modifications were made for the wheat grain As analysis. The modifications were (i) a 0.5 g sample used for analysis instead of 0.25 g because generally rice grain As concentration is lower than in soil; (ii) heating time was extended up to 90-100 min until a clear solution appeared (as an indication of good digestion), whereas 40 min was suggested in the original method; and (iii) digested liquid was filtered with Whatman 42 (ashless, 2.7 µm) filter paper, as it is better than the 541 (ashless, 20-25 µm) and is usually used in heavy metal analyses.

Statistical Analysis
For the statistical analysis of data, R software (version: 3.6.2, R core team, Vienna, Austria) [61] was used. Normality and homogeneity of variance of the data were tested. The analysis of variance (ANOVA) test was performed to determine the significance of tested treatments on variables. The Least Significant Difference (LSD) test was used to compare the treatment means at the 5% level of significance. Pearson correlation test was performed to determine the correlation coefficient among the variables. Thermal images were captured with an infrared camera (FLIR T1050SC, FLIR Systems Inc., Orlando, FL, USA) and post-processed in MATLAB software (version: R2015b, MathWorks, Natick, MA, USA) [62].

Results
This section is divided according to the various parameters that were assessed during the experiment.

Plant Growth and Grain Yield
The results revealed that plant growth and grain yield decreased significantly with increasing soil As concentration, while it increased significantly in the MW-3 and MW-6 treatments, compared with MW-0 treatment across all the soil As concentration. Biochar had both positive and negative effects, based on the application rate and soil As concentration. In terms of the combination of MW and biochar treatments, the highest plant growth and grain yield were observed in MW-6 with BC-10 treatment combination. To describe the plant growth, leaf chlorophyll content, tiller number and shoot biomass were recorded. The detailed results are given below.

Leaf Chlorophyll Content (as SPAD Value)
Leaf chlorophyll content (SPAD value) decreased significantly (p = 0.029) with increasing soil As concentration while, it increased significantly (p < 0.001) with MW treatments (MW-3 and MW-6) compare with MW-0. The lowest chlorophyll content was observed at the highest As treatment (As-80) in MW-0, whereas the value at MW-6 was significantly higher. Unexpectedly, the biochar had a significant (p < 0.001) negative effect on chlorophyll content, especially at BC-20 (Table 3). The combined effect of MW and biochar was non-significant (Table S1). Values having different superscript letters indicate significant differences among the treatments. Least significant difference (LSD) test was performed at 5% level of significance to determine the difference between the treatments.

Tiller Number
Tiller number reduced significantly (p < 0.001) with increasing soil As concentration. However, a significantly (p < 0.001) higher number of tillers was found in the MW treated pots. The MW-6 had higher tiller numbers than the MW-0 and the MW-3 treatments (Figure 2a). Significantly (p = 0.039) higher tiller numbers were also observed in the biochar treatments (BC-10 and BC-20) compared with BC-0 ( Figure 2b). The combined effect of MW and biochar was non-significant ( Figure S2).  Values having different superscript letters indicate significant differences among the treatments. Least significant difference (LSD) test was performed at 5% level of significance to determine the difference between the treatments.

Tiller Number
Tiller number reduced significantly (p < 0.001) with increasing soil As concentration. However, a significantly (p < 0.001) higher number of tillers was found in the MW treated pots. The MW-6 had higher tiller numbers than the MW-0 and the MW-3 treatments (Figure 2a). Significantly (p = 0.039) higher tiller numbers were also observed in the biochar treatments (BC-10 and BC-20) compared with BC-0 ( Figure 2b). The combined effect of MW and biochar was non-significant ( Figure S2).

Shoot Biomass
Shoot biomass was collected at two different growth stages, at the tillering stage and at physiological crop maturity. At the tillering stage, shoot biomass reduced significantly The Least Significant Difference Test (LSD) was performed at 5% level of significance. In boxplot, values having different superscript letters indicate significant differences among the treatments.

Shoot Biomass
Shoot biomass was collected at two different growth stages, at the tillering stage and at physiological crop maturity. At the tillering stage, shoot biomass reduced significantly (p < 0.001) with increasing soil As concentration, while in the MW treated pots, significantly (p < 0.001) higher biomass was recorded. In view of the MW treatments, higher biomass was harvested from the MW-6 treatment compared with the MW-3 and MW-0 treatments. The highest biomass was observed in MW-6 at As-0 and lowest in the MW-0 at As-80 ( Figure 3a). The effect of biochar and the combined effect of MW and biochar were non-significant. At physiological maturity, shoot biomass reduced significantly (p < 0.001) in response of soil As concentration, although significantly (p < 0.001) higher biomass was recorded in the MW treatments (MW-3 and MW-6) irrespective of soil As concentration compared with MW-0. Especially higher biomass was harvested in the MW-6 treatment compared with the MW-0 and MW-3 treatments (Figure 3b). The effect of biochar application showed strong decreasing trends (p = 0.089) with increasing biochar rates. However, a combination of biochar and MW treatment (MW-6 and BC-10 treatment) had the highest shoot biomass at As-0.
In view of the MW treatments, higher biomass was harvested from the MW-6 treatment compared with the MW-3 and MW-0 treatments. The highest biomass was observed in MW-6 at As-0 and lowest in the MW-0 at As-80 ( Figure 3a). The effect of biochar and the combined effect of MW and biochar were non-significant. At physiological maturity, shoot biomass reduced significantly (p < 0.001) in response of soil As concentration, although significantly (p < 0.001) higher biomass was recorded in the MW treatments (MW-3 and MW-6) irrespective of soil As concentration compared with MW-0. Especially higher biomass was harvested in the MW-6 treatment compared with the MW-0 and MW-3 treatments (Figure 3b). The effect of biochar application showed strong decreasing trends (p = 0.089) with increasing biochar rates. However, a combination of biochar and MW treatment (MW-6 and BC-10 treatment) had the highest shoot biomass at As-0.

Spikelet Sterility (%)
Rice spikelet sterility increased significantly (p < 0.001) with increasing soil As concentration while MW soil treatment significantly (p < 0.001) reduced the spikelet sterility across all the soil As concentrations. For example, the highest spikelet sterility was observed at As-80 in MW-0 while, the lowest value was recorded at As-0 in MW-6 ( Figure  4a). In response to biochar application, spikelet sterility reduced significantly (p < 0.001) in biochar treatments compared with BC-0 ( Figure 4b). The highest sterility was observed at As-80 with BC-0 while, the lowest sterility was observed at As-0 in BC-20. The combined effect of MW and biochar was found to be non-significant.

Spikelet Sterility (%)
Rice spikelet sterility increased significantly (p < 0.001) with increasing soil As concentration while MW soil treatment significantly (p < 0.001) reduced the spikelet sterility across all the soil As concentrations. For example, the highest spikelet sterility was observed at As-80 in MW-0 while, the lowest value was recorded at As-0 in MW-6 ( Figure 4a). In response to biochar application, spikelet sterility reduced significantly (p < 0.001) in biochar treatments compared with BC-0 ( Figure 4b). The highest sterility was observed at As-80 with BC-0 while, the lowest sterility was observed at As-0 in BC-20. The combined effect of MW and biochar was found to be non-significant.
In view of the MW treatments, higher biomass was harvested from the MW-6 treatment compared with the MW-3 and MW-0 treatments. The highest biomass was observed in MW-6 at As-0 and lowest in the MW-0 at As-80 (Figure 3a). The effect of biochar and the combined effect of MW and biochar were non-significant. At physiological maturity, shoot biomass reduced significantly (p < 0.001) in response of soil As concentration, although significantly (p < 0.001) higher biomass was recorded in the MW treatments (MW-3 and MW-6) irrespective of soil As concentration compared with MW-0. Especially higher biomass was harvested in the MW-6 treatment compared with the MW-0 and MW-3 treatments (Figure 3b). The effect of biochar application showed strong decreasing trends (p = 0.089) with increasing biochar rates. However, a combination of biochar and MW treatment (MW-6 and BC-10 treatment) had the highest shoot biomass at As-0.

Spikelet Sterility (%)
Rice spikelet sterility increased significantly (p < 0.001) with increasing soil As concentration while MW soil treatment significantly (p < 0.001) reduced the spikelet sterility across all the soil As concentrations. For example, the highest spikelet sterility was observed at As-80 in MW-0 while, the lowest value was recorded at As-0 in MW-6 ( Figure  4a). In response to biochar application, spikelet sterility reduced significantly (p < 0.001) in biochar treatments compared with BC-0 ( Figure 4b). The highest sterility was observed at As-80 with BC-0 while, the lowest sterility was observed at As-0 in BC-20. The combined effect of MW and biochar was found to be non-significant.

Grain Yield
Rice grain yield per pot reduced significantly (p < 0.001) in response of soil As concentration increases, while significantly (p < 0.001) higher grain yield was recorded in MW treatments compared with MW-0 irrespective of soil As concentration. Significantly higher grain yield was harvested in the MW-6 treatment compared with the MW-0 and MW-3. The lowest yield was recorded at As-80 in MW-0 and the highest yield was recorded at As-0 in MW-6 ( Figure 5a). The highest yield reduction (82.36%) was recorded at As-80 in comparison to the control treatment. However, less reduction was observed under MW treatment. For example, only 32.86% reduction was found when MW-6 treatment was applied at the same As-80 treatment. The MW-6 treatment increased yield by 92.59% at As-0, compared with the control. Microwave treatment increased rice grain yield up to As-60 (2.78%) while, a yield decline was observed at As-20 with no MW treatments ( Figure 6). The grain yield increased significantly (p = 0.014) in the biochar treatments compared with the BC-0 (Figure 5b). The combined effect of MW and biochar was non-significant ( Figure S3).  Replicated mean values are shown in the boxplot. The different letter indicates the significant difference (LSD test at 5% level of significance) among the treatments.

Grain Yield
Rice grain yield per pot reduced significantly (p < 0.001) in response of soil As concentration increases, while significantly (p < 0.001) higher grain yield was recorded in MW treatments compared with MW-0 irrespective of soil As concentration. Significantly higher grain yield was harvested in the MW-6 treatment compared with the MW-0 and MW-3. The lowest yield was recorded at As-80 in MW-0 and the highest yield was recorded at As-0 in MW-6 ( Figure 5a). The highest yield reduction (82.36%) was recorded at As-80 in comparison to the control treatment. However, less reduction was observed under MW treatment. For example, only 32.86% reduction was found when MW-6 treatment was applied at the same As-80 treatment. The MW-6 treatment increased yield by 92.59% at As-0, compared with the control. Microwave treatment increased rice grain yield up to As-60 (2.78%) while, a yield decline was observed at As-20 with no MW treatments ( Figure  6). The grain yield increased significantly (p = 0.014) in the biochar treatments compared with the BC-0 (Figure 5b). The combined effect of MW and biochar was non-significant ( Figure S3).     Replicated mean values are shown in the boxplot. The different letter indicates the significant difference (LSD test at 5% level of significance) among the treatments.

Grain Yield
Rice grain yield per pot reduced significantly (p < 0.001) in response of soil As concentration increases, while significantly (p < 0.001) higher grain yield was recorded in MW treatments compared with MW-0 irrespective of soil As concentration. Significantly higher grain yield was harvested in the MW-6 treatment compared with the MW-0 and MW-3. The lowest yield was recorded at As-80 in MW-0 and the highest yield was recorded at As-0 in MW-6 ( Figure 5a). The highest yield reduction (82.36%) was recorded at As-80 in comparison to the control treatment. However, less reduction was observed under MW treatment. For example, only 32.86% reduction was found when MW-6 treatment was applied at the same As-80 treatment. The MW-6 treatment increased yield by 92.59% at As-0, compared with the control. Microwave treatment increased rice grain yield up to As-60 (2.78%) while, a yield decline was observed at As-20 with no MW treatments ( Figure  6). The grain yield increased significantly (p = 0.014) in the biochar treatments compared with the BC-0 (Figure 5b). The combined effect of MW and biochar was non-significant ( Figure S3).

Grain Total Arsenic Concentration
Grain As concentration increased significantly (p < 0.001) with increasing soil As concentrations, whereas it was significantly (p < 0.001) lower in the MW treatments, compared with the MW-0 across all the soil As concentration. Grain As concentration was highest (912.9 µg kg −1 ) at As-80 without MW treatment while it was significantly lower (442.4 µg kg −1 ) in MW-6 at the same As treatment (As-80). The lowest grain As concentra-Energies 2021, 14, 8140 11 of 20 tion was observed at As-20 with MW-6 treatment (Figure 7a). Thus, the reduction of As concentration was significantly higher in MW treated pots compared with the MW-0 treatment throughout the soil As concentration (Figure 8a). Furthermore, biochar application also had a significant (p < 0.001) effect on grain As concentrations (Figure 7b). The BC-10 treatment recorded less grain As concentration at As-20 and As-40 treatments whereas, biochar could not reduce grain As concentration at higher soil As treatment (As-60 and As-80) (Figure 8b). On the other hand, grain As concentration was higher in the BC-20 treatment compared with BC-0 and BC-10 at all soil As concentration (Figure 7b). The combined effect of biochar and MW was non-significant ( Figure S4).

Grain Total Arsenic Concentration
Grain As concentration increased significantly (p < 0.001) with increasing soil As concentrations, whereas it was significantly (p < 0.001) lower in the MW treatments, compared with the MW-0 across all the soil As concentration. Grain As concentration was highest (912.9 µg kg −1 ) at As-80 without MW treatment while it was significantly lower (442.4 µg kg −1 ) in MW-6 at the same As treatment (As-80). The lowest grain As concentration was observed at As-20 with MW-6 treatment (Figure 7a). Thus, the reduction of As concentration was significantly higher in MW treated pots compared with the MW-0 treatment throughout the soil As concentration (Figure 8a). Furthermore, biochar application also had a significant (p < 0.001) effect on grain As concentrations (Figure 7b). The BC-10 treatment recorded less grain As concentration at As-20 and As-40 treatments whereas, biochar could not reduce grain As concentration at higher soil As treatment (As-60 and As-80) (Figure 8b). On the other hand, grain As concentration was higher in the BC-20 treatment compared with BC-0 and BC-10 at all soil As concentration (Figure 7b). The combined effect of biochar and MW was non-significant ( Figure S4).

Correlation of Plant Growth and Yield Parameter with Grain Total Arsenic Concentration
Pearson's correlation coefficient (r value) showed that all the plant growth parameters were positively correlated with the yield parameters and all the growth and yield parameters were negatively correlated with grain As concentration. Two-sided tests of

Grain Total Arsenic Concentration
Grain As concentration increased significantly (p < 0.001) with increasing soil As concentrations, whereas it was significantly (p < 0.001) lower in the MW treatments, compared with the MW-0 across all the soil As concentration. Grain As concentration was highest (912.9 µg kg −1 ) at As-80 without MW treatment while it was significantly lower (442.4 µg kg −1 ) in MW-6 at the same As treatment (As-80). The lowest grain As concentration was observed at As-20 with MW-6 treatment (Figure 7a). Thus, the reduction of As concentration was significantly higher in MW treated pots compared with the MW-0 treatment throughout the soil As concentration (Figure 8a). Furthermore, biochar application also had a significant (p < 0.001) effect on grain As concentrations (Figure 7b). The BC-10 treatment recorded less grain As concentration at As-20 and As-40 treatments whereas, biochar could not reduce grain As concentration at higher soil As treatment (As-60 and As-80) (Figure 8b). On the other hand, grain As concentration was higher in the BC-20 treatment compared with BC-0 and BC-10 at all soil As concentration (Figure 7b). The combined effect of biochar and MW was non-significant ( Figure S4).

Correlation of Plant Growth and Yield Parameter with Grain Total Arsenic Concentration
Pearson's correlation coefficient (r value) showed that all the plant growth parameters were positively correlated with the yield parameters and all the growth and yield parameters were negatively correlated with grain As concentration. Two-sided tests of

Correlation of Plant Growth and Yield Parameter with Grain Total Arsenic Concentration
Pearson's correlation coefficient (r value) showed that all the plant growth parameters were positively correlated with the yield parameters and all the growth and yield parameters were negatively correlated with grain As concentration. Two-sided tests of correlation difference showed that all the correlation coefficients (r value) were statistically significant except the correlation (r = −0.1048 ns ) between leaf chlorophyll content and grain sterility (Table 4). Table 4. Pearson's correlation matrix of different plant growth and yield parameter with grain arsenic (As) concentration.

Effect of Soil Arsenic on Plant Growth, Grain Yield and Grain Arsenic Concentration
It is evident from this study that rice plant growth and grain yield is reduced significantly with increasing soil As concentration from 20 to 80 mg kg −1 soil. A higher concentration of As is toxic to most plants, which can interfere with the plants' metabolic processes and inhibit plant growth and development through As induced phytotoxicity. Previous research revealed that As can significantly decrease the rice plant growth and grain yield along with reducing the seed germination rate, plant height, panicle number, filled grain and total grain weight when grown in As contaminated soil [63][64][65]. Furthermore, reduced leaf number, length, and area were also reported due to As phytotoxicity [66]. Even, higher soil As concentrations led to plant death [59]. These findings undoubtedly show that the reduction of plant growth was ultimately the result of As phytotoxicity, which agrees with the results of this experiment. Straighthead is the most common disease in rice, which is due to fewer filled grains, and the panicle remains upright when plants are exposed to a high concentrations of soil As [67]. Straighthead disease was observed (data not provided) in this experiment at the higher concentrations of soil As treatments. Therefore, higher spikelet sterility was recorded at higher soil As concentration (Figure 4), which results in fewer filled grains and ultimately low grain yield in As treated pots.
In addition, higher soil As concentration can lead to a lower photosynthesis rate by decreasing the chlorophyll content, which reduces plant growth and grain yield [59]. Reductions in protein and chlorophyll content [68], and reductions in photosynthetic rate [59] of rice plants grown in As contaminated soil were reported. The results of the present experiment reveal that leaf chlorophyll content decreased with increasing soil As concentration (Table 3), which agrees with the above statement. From Pearson's correlation analysis, it is also evident that the correlation between chlorophyll content and growth parameters was significantly positive (Table 4), which also supports the above discussion.
From Figure 7 it was evident that rice grain As concentration increased significantly with increasing soil As concentration. The uptake of As by plants is dependent on several factors such as plant species or variety, soil As concentration, soil pH, soil redox potential (e.g., oxidized or reduced condition in the soil), soil texture, other ions in the soil solution, and the chemical form of As (i.e., As speciation) [69,70]. Among all these factors, soil As concentration is an important factor. Several studies assuredly concluded that rice grain As concentration increased when plants were exposed to higher soil As [57,71]. Thus, rice grains can accumulate more As when grown in highly As contaminated soil, which agrees with the current findings from this experiment.

Effect of Microwave Soil Treatment on Plant Growth, Grain Yield and Grain Arsenic Concentration
Although the rice plant growth and grain yield were reduced significantly with increasing soil As concentration, MW soil treatment showed a significantly beneficial effect on rice plants' growth and grain yield across all the soil As concentrations. Particularly in the MW-6 treatment, plant growth and grain yield were significantly higher compared with the MW-3 and MW-0 treatment. Recent studies on MW soil treatment showed a significant increase in rice plant fresh biomass (50.90%) and dry biomass (42.40%), higher tiller numbers (387 m −2 ) compared with the control plot (268 m −2 ), and higher grain yield (7.80 t ha −1 ) than the control plots (5.60 t ha −1 ) [72]. A field study reported 1.20-1.50 t ha −1 extra rice grain yield in MW treated plots where a 2 kW MW generator, operating at 2.45 GHz, was used for 60s to treat the field soil. This achieved a soil temperature of about 70-75 • C in top soil layer (0-5 cm) [73]. These findings agree with the results of this current experiment.
The possible reasons for increased crop growth and grain yield of rice in MW-treated soil can be explained by a couple of changes in soil after MW heating and its impact on other related circumstances. One of the possible reasons could be the increased soil nutrient (e.g., N, P, and S) availability in MW treated soil (Table 1), which could enhance the plant growth and grain yields. Similar findings were reported in the previous research where, MW treatment increased the availability of some soil nutrient (e.g., N, S, and P) [19,73,74]. To investigate the MW heating effect on soil N availability Khan et al. [53] designed an experiment, using wheat as their test species, where they imply that MW soil treatment mineralized soil indigenous N, which resulted in a significant increase in crop biomass by 175% and grain yield by 92% compared with the control. A recent study revealed increased inorganic P (+1.2-fold compared with the control), and nitrate-N content in soil [75].
A study reported that MW irradiation induced disintegration of microbial cells, which can release the intracellular and extracellular macromolecules, may increase the soluble OM and organic-N (org-N) mineralization in the soil [76]. Previous research reported three pathways of org-N transformation: (1) microorganisms based org-N mineralization to ammonium, (2) release of org-N due to cell lysis, and (3) ammonium excreted from the bacterial grazing on soil fauna [77]. Research also showed that the org-N mineralization following MW irradiation of soil is of microbial origin [78]. However, MW heating impacts on soil microbes need to be further investigated for a better understanding of its role in crop growth and yield.
In terms of grain As concentration of the current study, MW soil treatment significantly reduced the As concentration in rice grains across all the soil As concentrations (Figure 7). Several factors could contribute to reducing the As concentration in the rice grain in MW treatments. One factor could be the dilution effect in which plants could uptake the same amount of As from the soil, but this could be diluted after translocating into the grain because of the higher grain yield in the MW treatment. Besides the enhancement of crop growth and grain yield, the increased soil P and Si concentration after MW soil heating in this current study (Table 1) could explain the reduced grain As concentration. PO 4 3− and Si are known to be analogous to As(V) and As(III), respectively [79]. Therefore, increased PO 4 3− and Si availability in the soil results in enhanced competition for adsorption sites on soil particle surfaces and for plant uptake because of the similar uptake mechanism of PO 4 3− and As(V) through PO 4 3− transporter and Si and As(III) through aquaporin channels present in the plant root [80,81]. Thus, the increased soil P and Si concentration after MW soil heating could compete with As(V) and As(III) for plant uptake and reduce the accumulation in the grain. However, some different results are also reported after MW soil heating, like a decline in P concentration [78], and no significant effect on total N, P, K and S concentrations [54]. Thus, a further detailed investigation is needed, in controlled conditions, to understand the MW heating effect on soil physicochemical properties at different temperature levels, because the soil temperature of around 75-85 • C neither strongly modifies the soil properties [82] nor totally disinfects the soil [83].
Another important change in the soil after MW heating is the alteration of the physical and chemical properties of SOM, particularly the molecular composition (C, H, O and N), chemical structure, and enhanced humification of SOM [84,85]. The formation of humic and fulvic acids, the more easily degradable forms of recalcitrant humic substances due to the thermal degradation induced by MW soil irradiation, are the possible mechanism of SOM humification. This would have enhanced the amount of carbon and free amino acids for turnover in the carbon and NH 4 + pool, which may favor soil health [82,84]. However, the mechanism behind this assumption is still in question. It has also been reported that MW soil heating can increase soil organic carbon [86], macromolecular organic substances that possess a higher number of functional groups [84], and synthesis of organometallic and coordination compounds [87]. These organic substances can retain, decrease mobility, reduce the bioavailability and adsorb soil heavy metals [88]. Hur et al. reported that MW soil heating can enhance the binding efficiency of hydrophobic organic containments with the more humified SOM [85]. Therefore, there were possibilities of As adsorption and less accumulation into the grain. Thus, the above changes in SOM could enhance the adsorption of As and reduce plant uptake. Further detailed investigations are needed to understand As behavior in the soil after MW treatment.

Effect of Biochar on Plant Growth, Grain Yield and Grain Arsenic Concentration
The addition of sawdust biochar at 10 t ha −1 soil significantly increased plant growth and grain yield compared with the control treatment, while it decreased again at 20 t ha −1 of biochar application irrespective of As and MW treatment. Several studies showed that biochar application in soil has the potential to increase rice crop growth and grain yield. For example, Khan et al. conducted a pot experiment with sewage sludge derived biochar and reported a significant increase in rice shoot biomass, grain yield, and total number of tillers [34]. They used 5 and 10% (w/w) biochar and found a 71.3 and 92.2% increase in shoot biomass compared with the control soil, respectively. Another experiment also revealed enhanced rice crop growth and grain yield using wheat straw biochar in a field experiment [89]. The results of the current experiment support these findings. Furthermore, in the view of grain As concentration, biochar application showed both negative and positive effects based on the application rate. At BC-10, lower grain As was observed in As-20 and As-40 soil As concentration compared with the BC-0 (Figure 8b). On the other hand, grain As concentration increased in the BC-20 treatment compared with the BC-0 across all the soil As concentrations.
Numerous researchers confirmed the use of biochar as an effective material for As remediation, due to its tremendous ability to adsorb As with subsequent alleviation of As phytotoxicity [90,91]. For example, a significant reduction in the concentrations of As(III), As(V), and DMA by 72, 62, and 74%, respectively, in rice were reported using sewage sludge biochar (2%, 5% and 10% on a dry weight basis) [92]. However, both mobilization and immobilization of As by biochar application in soil have been reported. For example, biochar can decrease the As content in plant tissues by retaining As on its surface [33,90]. Even though surface adsorption and complexation are identified to play significant roles in the interaction among biochar and As [35,93], the mechanisms of As immobilization by biochar are usually complex and differ from soil properties and types of biochar [94,95]. The enhanced bioavailability of soil P for plant uptake after the addition of biochar has been reported [96,97]. Being a PO 4 3− analog, increased P can reduce As(V) uptake and accumulation in rice grain. In addition, the application of biochar was found to increase the soil S content and its availability (13-16 fold), which could have interacted with As and reduced its uptake by the rice plants [98]. Furthermore, increased Si concentration in the soil solution, after biochar application, has also been reported, which may participate in As immobilization via the formation of silicate precipitates and may reduce As uptake by competing with As(III) for the same uptake transporter [99]. On the other hand, biochar application can result in As mobilization in rice soils under anaerobic conditions [100]. Zheng et al. [91] also reported an increase (327%) in As accumulation in rice grain due to biochar application prepared from rice residues. Some other studies also reported the increased availability of As after biochar application [36,45] resulting in the high As toxicity to rice plants [101]. Arsenic can mobilize due to reactions with ionizable functional groups of biochar and interaction with DOC release due to biochar application. As(V) can reduce to As(III) by interacting with DOC could be serving as electron donors [100,102], which enhances As mobility [103,104]. It was reported that biochar was sometimes unable to adsorb As(III) [105] and is more challenging to immobilize than As(V) because of its high mobility [106].
Moreover, it is widely reported that the addition of biochar to soils has resulted in pH increases [107,108]. During the pyrolysis process, at high temperature, cations are transformed into oxides, hydroxides or carbonates in the biochar and [109] dissolution of these alkaline substances increase the soil pH [110]. In the soil solution, As is mainly present as hydro anion, and increased soil pH after biochar application can reduce As sorption capacity by decreasing the positively charged sites on soil minerals, which increases As mobilization and release from the soil [111]. Khan et al. [34] reported the mobilization of As after application of sewage sludge biochar into soil due to a rise in soil pH. Hartley et al. [112] also reported the mobility of As with biochar results from the rise in soil pH. The pH of the current experimental soil also increased after the sawdust biochar application ( Figure S5). At BC-10 and BC-20 treatment the average soil pH was 8.15 and 9.00, respectively, while 7.73 was observed in the BC-0 treatment. Therefore, based on the above discussion, higher soil pH at BC-20 could enhance the availability of As in the soil solution and ultimately higher rice grain As accumulation was observed. Thus, reduction or increase of As mobility, bioavailability, and plant uptake depends on the application rate of biochar. Therefore, it requires precise studies in terms of As binding, transformation, and release into the soil after adding of sawdust biochar.

Conclusions
Microwave soil treatment had the potential to alleviate As phytotoxicity and reduce the grain As concentration. Especially, the MW-6 treatment was found to be more effective than the MW-3 compared with MW-0. Thus, MW soil treatment could be used as a novel technique for As remediation. Application of sawdust biochar at the rate of 10 t ha −1 in low to moderate As contaminated soils (20-40 mg kg −1 ) could alleviate As phytotoxicity in rice but the higher application rate (BC-20) could increase As phytotoxicity and grain As concentration. The combine treatment of MW-6 with BC-10 could be an option for As remediation. However, for a better understanding of the enhanced plant growth in MW treated soil, in combination with biochar, further validation experiments are needed, especially in the field condition.