Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic Concentration

*Mohammad Humayun Kabir, Graham Brodie, Dorin Gupta and Alexis Pang*

#### **Abstract**

Arsenic (As) contamination in soil and accumulation in food crops has raised much concern worldwide due to its phytotoxicity and possible human health risk. This study was conducted to determine whether microwave (MW) soil treatment could alleviate As phytotoxicity and reduce wheat grain As concentration or not. Experimental soils were spiked to five levels of As concentration (As-0, As-20, As-40, As-60, and As-80 mg kg–<sup>1</sup> ) prior to applying three levels of MW treatment (MW-0, MW-3, and MW-6 minute). Significantly higher plant growth and grain yield and lower grain As concentration was recorded in MW treatments compared with the control treatment. For instance, significantly higher grain yield (28.95 g pot–<sup>1</sup> ) and lower grain As concentration (572.03 μg kg–<sup>1</sup> ) were recorded in MW-6 treatment compared with MW-0 (22.03 g pot–<sup>1</sup> and 710.45 μg kg–<sup>1</sup> , respectively) at the same soil As concentration. Hence, MW soil treatment has the potential to alleviate As phytotoxicity and to reduce the grain As concentration. Ultimately, MW soil treatment will reduce As bioaccumulation in the human body even if wheat is grown in As contaminated soil. Nevertheless, further validation experiments are needed to explore the effectiveness of MW treatment in field conditions.

**Keywords:** microwave, soil heating, arsenic mitigation, wheat, grain arsenic

#### **1. Introduction**

Arsenic (As) is the most devastatingly toxic heavy metal and is raising global concerns for sustainable agriculture and human health, due to its ultimately toxic effect, persistence in nature, and ability to bio-accumulate in the ecosystem [1]. Inorganic As is considered to be a Group I human carcinogen and responsible for different types of cancer [2]. It is estimated that 220 million people, worldwide, are exposed to elevated concentrations of As in drinking water, which are above the World Health Organization (WHO) standard limit (10 mg l–<sup>1</sup> ) [3]. In addition to geogenic sources of As, which mainly contaminate drinking water, it can build up in soil because of long-term excessive use of As contaminated irrigation water and ultimately results in As uptake by crops [4]. Thus, the presence of As in the food chain, through the water-soil-crop pathway is triggering concerns about human health [1]. Excessively high As pollution in water, soil, and crops has already been identified in many countries [5–8]. Even though rice is a good accumulator of As, concerns are mounting about the amount of As being found in other crops, like vegetables, tubers, fruits, and even wheat [9].

Although there is no worldwide standard safe limit of As in food grains, the European Commission, recently (January 2016), set the maximum limits for As in milled rice (polished or white rice) as 200 μg kg–<sup>1</sup> [10]. A wheat field experiment with a 12.00 mg kg–<sup>1</sup> soil As concentration, reported 2.00–17.00 μg kg–<sup>1</sup> of As in grain samples [11]. While 5.00–285.00 μg kg–<sup>1</sup> [12], 4.00–362.00 μg kg–<sup>1</sup> [13], and 1.00–500.00 μg kg–<sup>1</sup> of As were also reported in wheat grain collected from an As contaminated site, where soil As concentration ranged from 3.00 to 201.00 mg kg–<sup>1</sup> [14]. Thus, besides rice, wheat could be a major source of dietary As. Wheat is the second most-produced (771.72 million tonnes) cereal crop throughout the world, with the highest harvested area being 218.54 million ha [15]. Therefore, to feed the rising global population, wheat will stay as a vital component of human nutrition. Hence, increasing its quality of production, free from toxic heavy metals, is an important requirement for sustainable agriculture and food security. Therefore, As remediation techniques, not only for drinking water but also for soil, are crucial to avoid food As contamination through crop uptake.

Different physical, chemical, and biological techniques are being used for remediation of As contamination in soil. These remediation methods include vitrification, electrokinetic treatment, soil flushing and solidification, phytoremediation by hyper accumulative plants, etc. [16]. Hitherto, these methods have been frequently revealed to be ineffective, costly, or too lengthy, with usage being restricted to smaller-scale operations with lower efficiency, selectivity, and disposal of materials after remediation [17]. Thus, alternative options or combinations of technologies, for reducing soil As pollution, are required.

Recent research has revealed that pre-sowing microwave (MW) soil heating application in agricultural systems is a promising technique, which not only has potential to control weeds by deactivating the soil weed seed bank [18, 19], but it can also significantly increase crop growth and yield of rice and wheat by increasing some soil nutrients (N, P, K, and S) availability as a result of soil humification processes and nutrient recovery from dead microorganisms after exposure to MW heating [20–23].

Microwave energy is a form of electromagnetic radiation, with wavelengths ranging from 1 m to 1 mm and frequencies between 300 MHz to 300 GHz, which can induce the rotation of the dipoles of polar molecules (e.g. water), due to the oscillating electromagnetic field, which results in the generation of heat by intermolecular friction [24]. This produces a fast heating rate, since soil moisture is considered to be an efficient absorber of MW radiation [25]. Thus, MW heating has major advantages over other heating processes. These advantages include short start-up, selective heating, precise control, no direct contact with heated materials, and volumetric heating [26, 27]. Because of these advantages, MW has been used in diversified fields including removal of organic contaminants [28] and immobilization of some toxic metals (Cu, Mn, Th, Zn, Ni, Cd, Cr, and Pb) in soil [29] and solid sediments [30, 31]. However, no study has been found to address soil As immobilization using MW heating to alleviate As phytotoxicity in wheat. Therefore, this study was designed to

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

explore whether MW soil treatment could alleviate As phytotoxicity and reduce wheat grain As concentration or not. Thus, the hypotheses of this study were (i) microwave soil treatment increases the soil organic matter, increases soil organic carbon, nitrogen mineralization, and nutrients availability that might be favorable for better plant growth and development, (ii) better plant growth and higher grain yield as the result of MW soil treatment will reduce the grain As concentration by means of dilution effect and (iii) microwave soil treatment synthesized the macromolecular organic substance that possesses a higher number of functional groups and organometallic and coordination compounds that are able to retain As, decrease mobility, and reduce bioavailability by adsorbing As.

#### **2. Materials and methods**

#### **2.1 Experimental site, soil collection, and preparation**

Experimental soils were collected from a wheat production paddock of the Dookie agricultural farm (36°37<sup>0</sup> S; 145°70<sup>0</sup> E) at a depth of 0–15 cm. The soil was a brownishgray loam and classified as a Major Clay Loam [32] or a red mesotrophic-haplic dermosol [33]. Some important soil properties are given in **Table 1**. The collected soils were 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, it was essential to ensure a uniform experimental condition for MW soil heating. After sieving, 8.5 kg of soil was thoroughly mixed and shifted into pots (diameter 27 cm and height 30 cm). Unperforated pots were used to prevent the loss of water-soluble As from the pots [34].

#### **2.2 Physicochemical properties of soil**

Soil samples were sent to the Nutrient Advantage Laboratory, a NATA (National Association of Testing Authorities, Australia) accredited laboratory (Lab number: 11958, ISO/IEC 17025), for analysis of soil properties. The physicochemical properties of the soil are presented in **Table 1**.

#### **2.3 Arsenic application**

Five different levels of As concentration (0, 20, 40, 60, and 80 mg kg– <sup>1</sup> soil) as sodium arsenate heptahydrate (Na2HAsO4�7H2O) [35] were mixed with the initial soil. Respective amounts of sodium arsenate were mixed with deionized water to prepare the As solutions. Then, the As solution was mixed with the soil by spraying and homogenizing thoroughly by hand mixing. The background As concentration in soil varies depending on the extent of As pollution in that area. For example, in highly naturally contaminated agricultural soil, the concentration of As in Bangladesh is 20– 83 mg kg–<sup>1</sup> [36], in China it is 40–70 mg kg–<sup>1</sup> [37], and in India, it is 9–105 mg kg–<sup>1</sup> [38]. Therefore, the As treatments in this study represented the different extent of contamination from several countries. To establish an equilibrium condition between soil and applied As, soil moisture was maintained at field capacity for 2 weeks prior to applying the MW treatment.


#### **Table 1.**

*Physicochemical properties of pre and post microwave (MW) treated soils before sowing.*

#### **2.4 Microwave application**

Three levels of MW energy were applied for 0, 3, and 6 min to attain soil temperatures of around room temperature, 60 and 90°C, respectively. The duration of MW irradiation to heat the soil at the desired temperature was determined by following the method of previous research work [23, 39]. Soil heating at around 90°C has been found to be effective for controlling weed infestations and destroying weed seed banks in the topsoil without significantly changing the soil properties [39]. Therefore, the same soil heating temperature was also used in this experiment to explore the potentiality in As phytotoxicity alleviation. Soil heating at around 60°C was included as an intermediate treatment between 90°C and control.

An MW chamber, consisting of six magnetrons (1 kW each), operating at a frequency of 2.45 GHz, was used for soil treatment (**Figure 1**). Energy dissipated in the soil sample after MW treatment (**Figure 1b**), and chamber electric field (**Figure 1c**)

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

#### **Figure 1.**

*(a) Schematic diagram of 6-kW microwave (MW) chamber (internal capacity of approximately 1.0 m<sup>3</sup> ) [23, 40], (b) energy dissipated in the soil sample after MW treatment, (c) chamber electric field, and (d) thermal images captured with an infrared camera (FLIR C3) after 3 min of MW irradiation of soil showing the temperature of 60 5°C.*

was modeled. The modeling was done using XFdtd software version 7.9 produced by Remcom (USA). **Figure 1(b)** illustrates the microwave's electric field distribution in the soil sample only, which **Figure 1(c)** illustrates the microwave's electric field distribution throughout the whole chamber. Both illustrations are on a vertical plane that passes through the center of the soil sample and the soil sample was in the center of the chamber, resting on the floor of the chamber. 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 [41]. An infrared camera 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 [25], the moisture content in the soil will greatly affect the heating effect of MW energy on the soil. In this experiment, the moisture content was maintained at around 15% (w/w) at the time of MW soil treatment.

#### **2.5 Experiment setup**

The experiment was conducted in a glasshouse, at Dookie campus, The University of Melbourne, Australia, by following a completely randomized design (CRD) with four replications. To describe the treatment combination more conveniently, abbreviated forms have been used for As treatments (As-0, As-20, As-40, As-60, and As-80) and MW treatments (MW-0, MW-3, and MW-6). Before the seed sowing, mono ammonium phosphate (MAP) fertilizer was applied (equivalent to 150 kg ha–<sup>1</sup> ) to each pot as a basal dose, as per standard practices for Australian wheat cultivation. The rest of the N requirement was calculated (based on a total 150 kg N ha–<sup>1</sup> ) and applied, as urea, in two split doses viz. at early stem elongation (GS30-32) stage and booting (GS45-49) stage. Twelve seeds of the EGA Gregory wheat variety (*Triticum aestivum* L.) were sown per pot on the 6th of June 2017. Tap water was used for crop irrigation purposes. This water source contained As below the detection limit (<0.01 μg l<sup>1</sup> ); thus, there were no possibilities of As addition from the tap water to the pot soil. After 180 days of the growing period, at the physiological maturity stage, the crop was harvested on the 5th of December 2017.

#### **2.6 Recording of crop agronomic data**

The plant height was measured as a distance from the soil surface to the top of a plant using a measuring scale. Plant vigor data was recorded by an ordinal scale ranging from 1 (low vigor) to 9 (high vigor). Leaf chlorophyll content was measured as SPAD (Soil-Plant Analysis Development) value using the Chlorophyll Meter-SPAD-502Plus [42] at the tillering stage. To get the plant height, plant vigor, and leaf chlorophyll content data, five plants within a pot were selected randomly and data recorded as the mean value of these five plants. At the tillering stage, plant samples (3 hills per pot) were collected to determine the shoot biomass and measure the leaf area, width, and length of the last fully expanded leaf by using a leaf area meter (LASER Leaf Area Meter, CI-202, CID Bio-Science, USA). At the physiological maturity stage, the crop was harvested, and shoot biomass, total number of spikes, root biomass, and grain yield were recorded. Both the shoot and root samples were dried at 60°C in an oven for 48 h to determine the dry biomass.

#### **2.7 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) [43]. 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 wheat 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;

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

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 usually used in heavy metal analysis.

#### *2.7.1 Sample preparation*

The whole grain sample was oven-dried at 105°C for 24 h prior to grinding and homogenizing with the ultra-centrifugal mill (RETSCH, ZM 200) [44]. The powdered sample was stored in a polypropylene pot for further analysis. Sample digestion was performed by hydrochloric-nitric (HCl:HNO3 = 3:1) di-acid (aqua regia) with a block digester (VELP Scientifica, DK-42). For pre-digestion, a 0.5 g powdered sample was taken into a 100 ml digestion tube (Ø 26 mm). After that, 12 ml of concentrated HCl (37%, 12 M) and 4 ml of concentrated HNO3 (70%, 15.8 M) were added and left overnight to allow the vigorous initial reaction to subside. Excessive foaming was reduced by adding 2–3 drops of n-dodecane into the mixture. The mixture was heated at 140°C for 90–100 min until the appearance of a clear solution. After cooling the tube, the liquid was filtered with Whatman 42 filter paper.

#### *2.7.2 Atomic fluorescence spectrometry*

Total As analysis was performed using atomic fluorescence spectrometry (AFS; PSA 10.055 Millennium Excalibur) [43]. Prior to total As determination, all the samples were pre-reduced with potassium iodide (1% m/v) and ascorbic acid (0.2% m/v) to reduce As(V) to As(III). For analysis standard preparation, 1000 2 mg l–<sup>1</sup> CRM (certified reference material) As standard supplied in 2% HNO3 (Sigma-Aldrich) was used as a standard stock solution. A working standard solution of 10 mg l–<sup>1</sup> was prepared weekly from the stock solution and used to prepare calibration standards (0– 10 μg l–<sup>1</sup> ). The standards and samples were prepared by following the same analytical matrix of 25% (v/v) HCl, 1% (m/v) potassium iodide, and 0.2% (m/v) ascorbic acid.


#### **Table 2.**

*Operating environment of atomic fluorescence spectrometer (AFS) for total arsenic analysis [43].*

Sodium tetra hydroborate (0.7% m/v in 0.1 mol l–<sup>1</sup> NaOH) was continuously added to the sample during the analysis to produce gaseous arsine (AsH3), which was atomized using a hydrogen diffusion flame. The overall reactions are represented in the following Eqs. (1)–(4) [45]. Atomic fluorescence was measured after excitation using an As boosted discharge hollow cathode lamp (Photron) [46]. The operating states of AFS for As determination are given below in **Table 2**.

$$\text{Sample} + \text{KI} + \text{Asororic acid} + \text{HCl} \rightarrow \text{KCl} + 2\text{HI} \rightarrow 2\text{H}^+ + 2\text{I}^- + 2\text{e}^- \tag{1}$$

$$\text{AsO}\_4\text{}^{3-} + 2\text{H}^+ + 2\text{I}^- + 2\text{e}^- \rightarrow \text{AsO}\_3\text{}^{3-} + \text{I}\_2 + \text{H}\_2\text{O} \tag{2}$$

$$\text{NaBH}\_4 + \text{HCl} \rightarrow \text{NaCl} + \text{H}\_3\text{BO}\_3 + 8\text{H} \tag{3}$$

$$\rm AsO\_4^{3-} + H \rightarrow AsH\_3 + H\_2 \tag{4}$$

#### *2.7.3 Quality assurance of arsenic analysis*

For quality control, the appropriate procedures and safety measures were taken to ensure the consistency of the results by following the techniques described by Thompson and Walsh [47]. Samples were handled carefully to avoid cross-contamination. All glassware was cleaned with the laboratory dishwashing machine followed by a 10% HNO3 solution and rinsing with deionized water. High purity analytical grade chemicals and gases (99.99% pure) were used for the analysis to ensure the minimal blank concentration value. Deionized water was used for all dilutions and preparation of chemicals during the analysis. To ensure good recovery of sample As, a 1568b rice flour standard reference material (SRM), from NIST (National Institute of Standard and Technology), was used at the time of digestion. Therefore, the block digestion set consisted of one blank, one SRM, one duplicate and with the remaining 39 tubes being the main samples with three replications each. Data was deemed to be acceptable if recovery of SRM As was �10% and the calculated relative standard deviation (RSD) of duplicate samples was no greater than 5%. To provide measurement clarification regarding the response of the Millennium Excalibur, the background equivalent concentration (BEC) was calculated using Eq. (5) to determine the performance of the instrument.

$$\text{BEC} = \frac{\text{Background value}}{\text{Peak height}} \times \text{standard concentration} \tag{5}$$

The lower the BEC value the more sensitive the instrument. If the BEC value was below 0.5, the instrument was considered to be operating correctly.

#### **2.8 Statistical analysis**

Statistical analysis of recorded data was performed using GenStat (16th Edition, VSN International) software. Normality and homogeneity of variance of the experimental 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 performed to compare the treatments' means at a 5% level of significance. The Pearson correlation test was performed to determine the correlation coefficient among the variables. For grain As concentration data, Grubb's test was performed to identify outlier values, which were replaced by the other replicates' average values, if found. After MW soil heating, thermal images were captured with an infrared camera (FLIR C3) and post-processed in MATLAB (MathWorks, Inc., USA) software.

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

#### **3. Results**

#### **3.1 Plant growth and grain yield**

The results revealed that the addition of As to the soil had a significant negative impact on plant growth and grain yield, and MW soil treatments provided a beneficial effect compared with non-MW treated soils, irrespective of soil As concentration. To describe the plant growth some growth parameter results are given below.

#### *3.1.1 Plant height*

Plant height decreased significantly (*p* < 0.001) with increasing soil As concentration. This trend was observed up to 60 days after sowing (DAS) of plant growth. After that, the effect of As on plant height was not statistically significant. Plant height increased significantly (*p* < 0.001) in MW treatments irrespective of soil As concentration throughout the growing period. Greater plant height was recorded in MW-6 compared with MW-3 and MW-0 treatment (**Table 3**).

#### *3.1.2 Plant vigor*

With the increase of soil As concentration, plant vigor decreased significantly (*p* < 0.001), while significantly (*p* < 0.001) higher plant vigor was found in the MW treatments. For instance, at As-80 the plant vigor was lowest (4.00) in MW-0, whereas significantly higher plant vigor was observed in the MW-6 (7.00) treatment (**Figure 2a**).

#### *3.1.3 Leaf chlorophyll content*

Leaf chlorophyll content increased significantly (*p* < 0.001) in the MW treatments compare with the control. For example, at As-80 significantly higher leaf chlorophyll content (45.88) was recorded in the MW-6 treatment compared with the MW-0 treatment (39.10). In the MW-6 treatment, no significant changes were observed in


*Mean data with superscript same letter are not significantly different. Least Significant Difference (LSD) test performed at a 5% level of significance to determine the difference between the arsenic (As) and microwave (MW) treatments. DAS (Days after sowing) indicates the sampling time.*

#### **Table 3.**

*Mean plant height (cm) in response to microwave (MW) soil heating and soil arsenic (As) treatments.*

#### **Figure 2.**

*Effect of microwave (MW) soil treatment on wheat plant growth in arsenic (As) contaminated soils. (a) Plant vigor, (b) leaf chlorophyll content, (c) tiller number, (d) shoot biomass at tillering stage, (e) shoot biomass at crop harvest, and (f) root biomass. Bar represents the mean value with standard error and different letters indicate the significant difference (LSD at p = 0.05) among the treatments.*

chlorophyll content across all soil As concentrations. Although the effect of soil As concentration on the leaf chlorophyll content was not significant (*p* = 0.187), a decreasing trend was observed in the MW-0 treatment (**Figure 2b**).

#### *3.1.4 Tiller number*

Tiller number reduced significantly (*p* < 0.001) across the treatments with increasing soil As concentration. However, a significantly (*p* = 0.005) higher tiller number was obtained in the MW treatments. This was especially so in the MW-6 treatment where the tiller number was higher than the MW-0 and the MW-3 treatment. The highest tiller number (26.25) was recorded in the MW-6 treatment at As-20 soil As concentration, while it was 21.75 and 18.75 in MW-3 and MW-0 treatment, respectively (**Figure 2c**).

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

#### *3.1.5 Plant biomass*

At the tillering stage, shoot biomass was 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 (**Figure 2d**). At crop harvest stage, shoot biomass was reduced significantly (*p* < 0.008) in response to increased soil As concentration, whereas significantly (*p* < 0.001) higher biomass was recorded in the MW treatments. In the MW-6 treatment, higher biomass was harvested compared with the MW-0 and MW-3 treatment (**Figure 2e**). Like shoot biomass, similar results were observed for root biomass (**Figure 2f**).

#### *3.1.6 Leaf area, width, and length*

With increasing soil As concentration, leaf area, width, and length were reduced, although the effect was not statistically significant. On the other hand, leaf area (*p* < 0.001), width (*p* < 0.001), and length (*p* = 0.048) increased significantly in MW soil treatments. The lowest value for all leaf parameters was found at the highest As concentration with no MW treatment, while the highest value was found in the MW-6 treatment, irrespective of soil As concentration (**Table 4**).

#### *3.1.7 Total number of spikes*

There was no significant (*p* = 0.064) effect of As on total spike number, but a significantly (*p* < 0.001) higher number of spikes was found in the MW treatments. The highest spike number (17.00) was found in the MW-6 treatment, while it was 12.00 and 9.00 in MW-3 and MW-0 treatment respectively at As-20 treatment (**Figure 3a**).

#### *3.1.8 Grain yield*

The wheat grain yield increased significantly (*p* < 0.001) in the MW treated pots, while the effect of As on grain yield was not significant (*p* = 0.210). Higher grain yield


*Mean data with superscript same letter are not significantly different. Least Significant Difference (LSD) test performed at a 5% level of significance to determine the difference between the arsenic (As) and microwave (MW) treatments. # Data recorded at tillering stage.*

#### **Table 4.**

*Effect of microwave (MW) treatment on leaf area, length, and width at different soil arsenic (As) concentration.#*

#### **Figure 3.**

*Effect of microwave (MW) soil treatment on (a) spike number, (b) grain yield, (c) grain arsenic (As) concentration in response to different soil As concentration, and (d) grain As concentration reduction in MW treatments. Bar represents the mean value with standard error and different letters indicate the significant difference (LSD at p = 0.05) among the treatments.*

was found in the MW-6 treatment compared with the MW-0 and MW-3 treatments. For instance, a significantly higher grain yield (28.95 g pot–<sup>1</sup> ) was recorded in the MW-6 treatment compared with the MW-3 (23.21 g pot–<sup>1</sup> ) and MW-0 (22.03 g pot–<sup>1</sup> ) treatments at As-40 treatment (**Figure 3b**).

#### *3.1.9 Grain total arsenic concentration*

Grain As concentration increased significantly (*p* < 0.001) with increasing soil As concentration while it was significantly (*p* < 0.001) lower in the MW treatments compare with the control (**Figure 3c**). At As-80 the highest grain As concentration (710.45 μg kg–<sup>1</sup> ) was recorded in MW-0 while, it was significantly lower (572.03 μg kg–<sup>1</sup> ) in the MW-6 treatment. The highest grain As concentration reduction (37.98%) was observed in the MW-6 treatment at As-60 followed by the MW-3 treatment (32.20%) compared with the MW-0 treatment (**Figure 3d**).

#### **3.2 Grain mineral content**

Grain P (*p* < 0.001) content decreased significantly, and K (*p* = 0.008) and Na (*p* < 0.001) content increased significantly with the increase of soil As treatment, while there was no significant effect of MW treatment on grain P, K and Na content. On the other hand, Mn (*p* = 0.012) and Zn (*p* < 0.001) content decreased significantly with the increasing soil As concentration, while Mn (*p* < 0.001) and Zn (*p* < 0.001)


**5.** *macronutrient content in response to microwave (MW) soil treatment at different soil arsenic (As) concentration.*

**Table**  *Grain* 

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*



**Table**

*Grain*  *Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*


*Significance at p <sup>&</sup>lt; 0.05. \*\*Significance at p <sup>&</sup>lt; 0.01. \*\*\*Significance at p <sup>&</sup>lt; 0.001.*

#### **Table 7.**

*Pearson's correlation matrix of different growth and yield parameters with grain arsenic concentration and accumulation.*


#### **Table 8.**

*Pearson's correlation matrix of different grain minerals with grain arsenic concentration and accumulation.*

content increased significantly in the MW treated soil compared with the control. The effect of As on grain Ca, Mg, Fe, and Cu was statistically non-significant. However, grain Mg (*p* = 0.012), Fe (*p* < 0.001), and Cu (*p* = 0.003) content increased significantly while, Ca (*p* < 0.001) content decreased significantly in the MW treated soil compared with the control treatment (**Tables 5** and **6**).

#### **3.3 Correlation of grain arsenic with plant growth and yield parameters and grain mineral content**

Pearson's correlation coefficient (*r* value) showed that all the growth parameters were positively correlated with the yield parameters, and all the growth and yield parameters were negatively correlated with grain As concentration. Although the grain yield was negatively correlated with grain As concentration (*r* = 0.1511), the correlation coefficient was statistically non-significant (**Table 7**). Also, Pearson's correlation coefficient (*r* value) showed that, grain K (*r* = 0.46\*\*\*) and Na (*r* = 0.48\*\*\*) were positively correlated, while grain P (*<sup>r</sup>* <sup>=</sup> 0.86\*\*\*), S (*<sup>r</sup>* <sup>=</sup> 0.34\*\*), Cu (*<sup>r</sup>* <sup>=</sup> 0.30\* ), Mn (*<sup>r</sup>* <sup>=</sup> 0.29\* ), and Zn (*<sup>r</sup>* <sup>=</sup> 0.62\*\*\*) were negatively correlated with the grain As concentration (**Table 8**).

#### **4. Discussion**

It is well known that soil As has adverse effects on plant growth and development. Previous research revealed that plant growth traits such as plant height, tiller number, and, the number of grains per spike can decrease significantly with increasing soil As concentration [48]. Pigna et al. [49] reported a 60% plant biomass and 83.6% root biomass reduction of wheat in As contaminated soil. Also, several other experiments reported that the reduction of plant growth was ultimately the result of As phytotoxicity at high soil As concentrations [50–52]. This experiment also found a significant reduction in plant growth represented by plant height, plant vigor, tiller number, shoot, and root biomass with increasing soil As concentration, which agrees with these other studies. Like other growth parameters, similar results were observed in leaf chlorophyll content (measured as SPAD), leaf area, leaf width, and length, which also reflect the lower plant growth at higher soil As concentration. Chlorophyll consists of mainly N, as a core component, and its content in the leaf represents the chlorophyll content. Higher soil As can reduce the N content in various crops [53, 54]. Thus, it was anticipated that higher soil As concentrations may also decrease N content in wheat plants, which may lead to a decrease in chlorophyll content. The results of the present experiment revealed that higher soil As concentrations decreased the chlorophyll content, therefore leading to a lower photosynthesis rate, which might have reduced plant growth and grain yield.

In relation to grain yield, a decreasing trend was observed as As concentration in the soil increased, although it was not statistically significant. From the results, it is clear that, at the early growth stage, As had more effect on the plant growth, whereas, at the mature stage As had less effect (**Figure 2**), which can be correlated with the final grain yield (**Figure 3b**). A similar result was observed in the previous experiment conducted on wheat grown in As contaminated soil (0–40 mg kg–<sup>1</sup> ), by Asaduzzaman et al., where less impact of As was reported on grain yield [48]. Some other previous experiments also revealed a greater effect of As on the shoots but showed less effect on *Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

the yield and yield contributing characters [48, 52], which is similar to the current results of this study.

On the other hand, the results showed that the MW soil treatment had a significantly beneficial effect on wheat plant growth and grain yield irrespective of soil As concentration (**Figures 2** and 3**b**). A previous study by Khan et al. reported a 33.1% increase in plant dry biomass and a 39.2% increase in grain yield in MW treated soil [55]. Similarly, some other studies demonstrated that increased plant growth and grain yield resulted from MW treatment of soil as well [18, 19]. The above findings agree with the results obtained in this experiment. One of the possible reasons for the increased growth and yield of wheat is the higher availability of nutrients for the plants in MW-treated soil. Increased N and S availability in soil was reported after application of MW [56]. Speir et al. reported increased N levels in MW treated soil compared with the control [57]. Additionally, another previous study showed increased indigenous soil N after MW soil heating [21]. A similar result was observed in this present study where, N, P, and S increased after MW soil heating (**Table 1**). Using SPAD as a method for leaf chlorophyll measurement, a higher value (58–64) was reported in MW treated soil as compared with the control (42–56) pots [58]. In this present study, leaf chlorophyll content was significantly higher in the MW treated pots which could correlate with the higher photosynthesis rate and ultimately contributed to the higher crop growth and grain yield.

Alternatively, MW also has negative impacts on the microbial community in the soil. By generating heat, MW energy would kill certain microbes. As a result, MW irradiation-induced disintegration of the cell walls can release the intracellular and extracellular macromolecules, which may increase the soluble OM in the soil and release some nutrients [27]. Previous research reported three pathways of organic N (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 bacteria grazing on soil fauna [59]. Research has shown that the org-N mineralization following MW irradiation of soil is of microbial origin [57]. Furthermore, nurturing and/or shaping the soil microbial communities was reported as the result of humification and thermal denaturation of soil organic compounds after soil heating [20, 60]. These microbial communities accelerate the availability of different soil nutrients that could be responsible for better plant growth and grain yield [22].

From **Figure 3c**, it was evident that the grain As concentration increased significantly with increased soil As concentration. Several studies described a higher concentration of wheat grain As when cultivated in soil containing higher As [48, 61]. A significantly high concentration of As in wheat grain (220.00–620.00 μg kg–<sup>1</sup> ), grown in soil containing 2.00–70.00 mg kg–<sup>1</sup> As, compared with those (0–50.00 μg kg–<sup>1</sup> ) grown in 1.00–13.40 mg kg–<sup>1</sup> soil As has been reported [62]. A similar trend was observed in this current experiment where, the grain As concentration was lower at low soil As concentrations, whereas it was higher at high soil As concentrations. However, MW soil treatment significantly reduced the As concentration in wheat grain (**Figure 3c**). This reduced grain As concentration could be explained by a couple of changes after MW soil heating. One of the possible reasons could be increased plant biomass and grain yield in MW treated soil, where there was a dilution effect on As concentration in the plants grown in the MW treated soil. From the Pearson's correlation study (**Table 7**) it is evident that the grain As concentration (*r =* 0.1511ns) was negatively correlated with grain yield but non-significantly. This also explains the diminishing effect of As on grain yield. Another reason could be the increased soil P and Si concentration after MW soil heating (**Table 1**). The PO4 <sup>3</sup> level in the soil is

known to control plant growth and development, and As(V) is a PO4 <sup>3</sup> analog [63]. Therefore, increasing PO4 <sup>3</sup> in the soil results in enhanced competition between PO4 <sup>3</sup> and As(V) for sorption sites on soil particle surfaces and for plant uptake because of the similar uptake mechanism of PO4 <sup>3</sup> and As(V) through PO4 <sup>3</sup> transporter present in the plant root [63]. Furthermore, Si can compete with As(III) for plant uptake due to a similar uptake mechanism through aquaglyceroporins the more likely nodulin 26-like intrinsic protein (NIP) class of aquaporin channels [64, 65]. Thus, the increased soil P and Si concentration after MW soil heating could complete with As(V) and As(III) for plant uptake and reduce the accumulation in the grain.

However, according to the Steindorf-Rebhun-Sheintuch equation, ligand exchange theory, and a share charge hypothesis, PO4 <sup>3</sup> has more probability to replace As(V) from soil adsorption site depending on the concentration of PO4 <sup>3</sup> and As(V). Nevertheless, PO4 <sup>3</sup> might also be desorbed by As(V) due to a mass action effect of a high ratio of As(V):P concentrations in the soil solution [66, 67]. Therefore, PO4 <sup>3</sup> and As (V) interaction needs to be considered for applying PO4 <sup>3</sup> amendment as As remediation technique. Some researchers reported that, at low soil As concentration, displacement of soil PO4 <sup>3</sup> by As(V) increased the availability of PO4 <sup>3</sup> to the plant, which resulted in the increase of plant growth parameters [49, 68]. In this experiment, it was also found that shoot biomass at both tillering stage and final crop harvest, root biomass, and spike number increased at low soil As concentrations (As-20 and As-40). However, all these traits decreased again at higher As concentrations (As-60 and As-80). Previous researches also reported plant growth and yield increases due to small additions of As in tomato, potato, rye, corn, and wheat [69–71] which agrees with the findings of this present study in wheat. Although, As is not an essential element for plants, small amounts of As can stimulate plant growth and increase plant biomass by releasing some P from soil adsorption sites and making it available for plant uptake [72].

Furthermore, As concentration in wheat grain also depends on the genetic differences of different varieties. Previous research reported that different wheat varieties, grown in the same soil As concentration, can tolerate, accumulate and translocate different concentrations of As due to phytoextraction or phyto-morphological potential of the varieties [73, 74]. The wheat variety used in this study could accumulate less As due to genetic constituents. However, further experiments are needed with different wheat varieties to explore the varietal effect on As accumulation. In addition, it has been demonstrated that MW soil heating markedly altered the physical and chemical properties of SOM [75] and enhanced the humification of SOM [76]. It has also increased the soil organic carbon and N mineralization [77], macromolecular organic substances that possess a higher number of functional groups [76], and syntheses of organometallic and coordination compounds [78]. These organic substances can retain, decrease mobility, reduce bioavailability, and adsorb soil heavy metals [79]. Therefore, more As could be adsorbed by the adsorption sites in the soil and become unavailable for plant uptake which ultimately could reduce the grain As concentration.

From **Table 5**, it was evident that grain P content decreased significantly, while Na content increased significantly with increasing soil As concentration. A similar result was found in another study [80] where, P uptake decreased in As treatments. Since, sodium arsenate was used in this study to artificially contaminate the soil, the addition of this Na might contribute to the higher plant uptake in higher As treated soil and ultimately more accumulation of Na in the grain. The result also shows that grain Mn, Zn, and Cu content decreased significantly with increasing soil As concentration,

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

while they increased significantly in the MW treatments compared with the control. Addition of As can reduce Mn content in shoots and roots [80], which results in lower Mn translocation to the grain. By contrast, the opposite findings have also been reported [81] where increased Mn content with increased As was observed. It is known that divalent Mn is absorbed by facilitated diffusion across the plasmalemma [82]. It is possible that As phytotoxicity may hamper the activity of the root plasmalemma and reduce Mn2+ absorption. Similarly, decreased Zn and Cu content in shoots and roots [80] can facilitate the lower translocation to the grain. Similar findings were reported in another study [83], where an antagonistic relation between As and Zn was described. Another study also reported lower Zn content in rice grain, where higher As was present in the soil [84]. However, MW soil treatment can increase the grain Zn and Cu by reducing the As phytotoxicity. From the Pearson's correlation (**Table 8**) it was also evident that grain K and Na content were significantly positively correlated with grain As concentration and significantly negatively correlated with grain P, Cu, Mn, and Zn content.

#### **5. Conclusions**

Although the elevated concentration of soil As can reduce the plant growth and grain yield of wheat due to the As phytotoxicity, MW soil treatment can mitigate this As phytotoxicity. The MW-6 treatment showed a better influence than the MW-0 and MW-3 treatments. Furthermore, MW soil treatment can reduce grain As concentration. Although the soil remains contaminated after MW treatment, wheat grain As concentration was lower in the MW treated pots, which results in lower As accumulation in humans through wheat consumption. Nevertheless, further experiments are needed to explore the effectiveness of MW treatment with different types of soils in field conditions.

#### **Acknowledgements**

We thank Dr. Ravneet Kaur Jhajj, laboratory manager for her assistance with chemical analysis in the laboratory.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Mohammad Humayun Kabir1,2, Graham Brodie1 \*, Dorin Gupta<sup>1</sup> and Alexis Pang<sup>1</sup>

1 Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Dookie, Australia

2 Department of Soil Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

\*Address all correspondence to: grahamb@unimelb.edu.au

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Microwave Soil Treatment Alleviates Arsenic Phytotoxicity and Reduces Wheat Grain Arsenic… DOI: http://dx.doi.org/10.5772/intechopen.102730*

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Section 4
