Zinc oxide nanoparticles in combination with biochar alleviates arsenic accumulation in field grown rice crop

doi:10.21203/rs.3.rs-2221512/v1

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Zinc oxide nanoparticles in combination with biochar alleviates arsenic accumulation in field grown rice crop

https://doi.org/10.21203/rs.3.rs-2221512/v1

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Plants require zinc (Zn) as an essential micronutrient since Zn plays important roles in metabolism. Arsenic (As) is a toxic metalloid whose accumulation in plants hampers growth of plants. Rice crops are grown under flooded conditions that make them vulnerable to double jeopardy of Zn deficiency andAs stress. The goal of this study was to observe the effect of ZnO nanoparticle (NPs) and ZnO-NPs + biochar on As and Zn accumulation in and growth of rice plants grown under flooded conditions. The study area was Chausa block in Buxar district, Bihar, India. Rice crops were treated with 80 mg L^-1 of ZnO-NPs and biochar was added to soil at rate of 10 t ha^-1. ZnO-NPs were sprayed at heading and jointing stage of crop development. The results showed a significant increase in plant biomass and total chlorophyll content in ZnO-NPs and ZnO-NPs + biochar with respect to control. The decline in electrolytic leakage and malondialdihyde (MDA) content was observed to be 48-62% and 14-55%, respectively in treated plants as compared to control. Further, As accumulation was reduced in rice tissues and grains while Zn accumulation was increased. The enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT) showed higher activity in treated crop plants than in control plants. The findings confirm that ZnO-NPs and their combined use with biochar in flooded regions can help to improve growth, yield and reduced As accumulation in rice plant.

Antioxidant enzymes

Chlorophyll

Electrolytic leakage

Malondialdehyde

Zinc (Zn) is known as the second most abundant transition metal found in organisms. Zn is an essential micronutrient element for plant growth and development. Its coordination geometry is mainly responsible for its reaction with enzyme (Broadley et al., 2007). Zinc oxides, sulfides, sulfates, carbonates, phosphate and silicates are some minerals which are the main sources of Zn in soils (Sturikova et al., 2018). Plants avail Zn in the form of Zn^+ 2 from soil. Zn bioavailability to plants varies greatly, and soil pH is one of the prime factors responsible for this. The soils with Zn deficiency or with low Zn bioavailability are increasing. Further, it is known that the bioavailability of Zn is reduced due to huge application of phosphorus fertilizers for achieving better growth and yield of agricultural crops (Cakmak et al., 2010). Some Zn deficiency symptoms, which are reported are impaired stem elongation, root apex necrosis, interveinal chlorosis, and bronzing (Marshner, 1995). Therefore, an adequate amount of Zn as an external amendment is essential for alleviating the deficiency of Zn in plants and thereafter in humans (Cakmak et al., 2017; 2018).

Arsenic (As) is a toxic carcinogenic metalloid ubiquitously present throughout the earth. It is not a necessary element for fauna and flora, but it is accumulated in plants and enters the food chain. Long time exposure to As through contaminated drinking water, cereals and vegetables can lead to serious health risks to humans (Kumari et al., 2018). The cultivation method of rice under flooded and anaerobic conditions promotes the translocation and accumulation of As in edible parts of rice (Srivastava et al., 2022). Although rice is one of the most potent As accumulating crops, it is cultivated in the highly As contaminated regions (Upadhyay et al., 2021; Yadav et al., 2021). Several practices have been researched for reducing the accumulation of As in rice grains, viz nutrient and fertilizer management (nitrogen, silica, zinc, iron), microbial supplementation and water management (Li et al., 2019; Chauhan et al., 2017; Awasthi et al., 2018; Huhmann et al.,2019; Seyfferth et al., 2019; Srivastava et al., 2019). Some studies have shown that engineered NPs such as ZnO, cerium oxide (CeO₂) and copper oxide (CuO) NPs can significantly reduce the accumulation of As in rice tissue when applied in hydroponic conditions (Wang et al.,2018; 2019).

Nanotechnology is an emerging technology which is garnering attention due to its specificity and application potential in agriculture and different fields (Liu and Lal 2015; Rizwan et al., 2017).Nanoparticles interact with plants depending on their properties causing many morphological and physiological changes. The chemical composition, size, reactivity and dose are the main characteristics of NPs which make them beneficial to plants (Garcia-Gomez et al.,2017; Khodakovskaya et al., 2012). Application of nanotechnology as nano-fertilizers has great potential to enhance yield of crop plants (Adisa et al., 2019; Madzokere et al.,2021; Beig et al.,2022) When the proper concentration of NPs is applied to plants, it shows a fruitful effect on seed germination, growth, yield and tolerance (Nair et al., 2010; DeRosa et al., 2010; Faizan et al., 2021). ZnO-NPs have been found to improve plant growth, photosynthetic pigment and total soluble protein content, and alleviate oxidative stress in plants. Foliar application of ZnO-NPs showed better absorption than bulk form of Zn (ZnSO₄, bulk ZnO) (Li et al., 2018).

Biochar is known as conditioner for soil where it acts as an efficient carbon sink. Chemical composition, pH, particle size, and surface chemistry are the key properties which d etermine the effectiveness of biochar in soil. Due to having high porosity, pH, surface area and cation exchange capacity, biochar potentially binds to the trace metals and makes them less available to plant (O’Connor et al., 2018). Use of biochar reduced the oxidative stress in several plants (Rehman et al.,2016; Abbas et al., 2017; Ali et al., 2018). In this background, the goal of the present study was to know the effects of solo or combined application of ZnO-NPs and biochar on rice plants grown in flooded conditions. It was hypothesized that soil quality enhancement through biochar and interaction of biochar and NPs would reduce As accumulation in rice grains.

The present experiment was conducted to evaluate the effects of ZnO-NPs alone and in combination with biochar to reduce the accumulation of As and increase the uptake of Zn in rice grains, which is consumed by major part of world population. Arsenic is a non-essential element for plant, so its stress can inhibit the growth and yield of crops.

2.1 Study area

The present field experiment was conducted on an agricultural field in Buxar, Chausa block (latitude 25.5084 N, longitude 83.8925 E) in Bihar state of India. Three treatments were used in the field trial, each of which was delivered in three repetitions in a randomized full block design (RCBD). Each experimental plot was chosen to be 8100 cm² (90 × 90 cm) in size. The treatments included control, ZnO-NPs (80 mg L^− 1), ZnO-NPs (80 mg L^− 1) + biochar (10 t ha^− 1). The selected level of NPs was based on the previous studies (Adrees et al., 2020). The solution of ZnO-NPs (Himedia) was made in of deionized water and then sonicated for 30 minutes, and required volume was maintain before applying in block. At initial growing stage 500 ml volume was required for a block but as plant growth increase the requirement reached 1 L for a block. The biochar was produced at 500^oC for 4 hours with the feedstock of bamboo straw as per the method given by, He at al. (2021). The pH of biochar was 9, and EC value was 2.76 dS m^− 1 and 24% of organic carbon. pH and EC were calculated for biochar in the ratio (1:10) biochar/ water by using waterproof Oaklon multi-parameter tester (TCSTestrTM-35 series) and organic carbon was estimated by Olsen (1954) method. For this, 1 g of biochar was used, followed by the addition of K₂Cr₂O₇, H₂SO₄, orthophosphoric acid, and diphenylamine indicator, which was then titrated with ferrous ammonium sulphate. The biochar was applied to soil at rate of 10 t ha^− 1. The fertilizers NPK were applied at a given rate of 40–60–20 kg ha^− 1 respectively as per local farmers practice. The application of nitrogen and phosphate fertilizer as urea and DAP respectively was performed in two stages: during field preparation and tillering stage (between 35 and 45 days after planting). The first foliar spray of NPs with 0.1% (v/v) Tween 20 (0.1ml in 1L solution) was applied on 45th day, second on 65th day when there was inflorescence, and third was applied on 90th day at the time of grain filling. Tween-20 was applied as surfactant to enhance the uptake of foliar applied NPs (Liu et al., 2016).

2.2 Soil sampling and analysis of physicochemical properties

Soil sampling was performed at ploughing zone depth (0–20 cm) at the time of transplantation in July, 2021. The collected soil samples were air dried, grounded and sieved with 2.0 mm sieve and homogenized for the analysis of different physicochemical parameters. The organic matter in soil was estimated as per protocol given by Walkley and Black (1934). Soil pH and electrical conductivity (EC) was analyzed by waterproof Oaklon multi-parameter tester (TCSTestrTM-35 series). The procedure for organic carbon, soil pH and EC was followed as same as for biochar. Further, available phosphorus was estimated by following the method of Olsen (1954). This was accomplished using 2.5 g of dry soil, charcoal, and Olsen reagents. It was filtered, then ammonium molybdate and SnCl₂ were added, and at 660 nm, the intensity of the blue colour was measured. Soil protease was estimated according to the method of Ladd and Butler (1972). Alkaline and acidic phosphomonoesterase and diesterase were estimated by following the methods of Tabatabai and Bremner (1969) and Eivazi and Tabatabai (1977), respectively.

2.3 Harvesting, growth and yield measurement

The plant sampling was performed at two durations in the whole cultivation period. Firstly, the samples were collected at 85 days after transplanting (DAT) at milking stage of cultivation. Root and shoot lengths were measured at this stage only with the help of manual metric scale. The biomass data was collected by the use of digital balance. Leaf samples were collected for the estimation of chlorophyll content and other biochemical assays. The second sampling was performed at 135 DAT when rice crops were considered to be mature to harvest. The root and shoot length were measured, spikes were separated, and fertile spikelets per spike were counted. The grains were separated and dried, and then 1000 grains weight was taken for the yield estimation.

2.4 Measurement of electrolytic leakage and lipid peroxidation

For electrolytic leakage (EL) analysis, protocol of Dionnisio-Sese and Tobita (1998) was followed. The fresh samples (20 leaf disc) were submerged in 40 ml of deionized water and after 10 min, electrical conductivity (EC₁) was recorded. The solution was then kept in water bath at temperature 50–60 ⁰C for 25 min, and EC₂ value was measured. In the end, solution was boiled at 100 ⁰C for 10 min, and the electrical conductivity (EC₃) was measured. Electrolytic leakage was calculated using the following formula.

Electrolytic leakage (%) = (EC₂-EC₁) *100/EC₃

Lipid peroxidation was measured by estimating the production of MDA by following the protocol given by Heath and Packer (1968). For the estimation of MDA, 100 mg of plant sample was homogenized in 1.4 ml of 0.5% of thiobarbituric acid (TBA) in 20% trichloroacetic acid (TCA) and then centrifuged at 10,000 g for 15 min. Supernatant was taken and incubated at 95 ^oC for 45 min and again centrifuged at 10,000 g for 15 min. The absorbance of the supernatant was taken at 532 and 600 nm. MDA level was calculated by using the extinction coefficient of 155 mM^− 1cm^− 1.

2.5 Sample digestion and instrumental analysis

The plant samples were separated into different parts (root, flag leaves, grains). These parts were cleaned by gentle shaking and kept into beaker for oven-drying. Dried samples were collected, and grain samples were dehusked to get the edible grain samples and crushed into powdered form. The samples were digested with HNO₃ (70% Suprapur Merck; Darmstadt-Germany) in heating test tube block digester at 180 ^oC for 1 hour and then at 200 ^oC for 45–60 minute till the sample turned in white crystalline appearance (Srivastava et al., 2014). After proper digestion, final volume for each sample was maintained up to 10 ml with ultrapure (Type I) water (Milli-Q) and filtered with 0.22 µm pore size filter paper. Finally, the Zn and As contents were measured through (HA-AAS). For hydride generation, analytical standard sodium borohydride (3%; Merck), sodium hydroxide (2.5%; Merck) and hydrochloric acid (6 M; Merck) were used. The precision of the analysis was checked by certified standard reference materials (SRMs) (NIST, USA) such as 1568a rice flour and 1573a tomato leaf as per details given in Khanam et al. (2022) The certified As concentration of 1568a rice flour and 1573a tomato leaf are given in (Supplementary Table S1). The quality control was performed by triplicate analysis and by the measurement of recovery of spiked digested samples. The recoveries varied from 95 to 98% for all the tested samples.

2.6 translocation factor

Translocation factor of As and Zn was calculated by taking the ratio of As/Zn concentration of shoot (shoot _As/Zn) to As and Zn concentration of root (Root _As/Zn) and ratio of (grain _As/Zn) to As and Zn concentration of shoot (Shoot _As/Zn).

TF _{Root to Shoot} =Shoot _As/Zn / Root _As/Zn

TF _{shoot to grain}= Grain _As/Zn/ Shoot _As/Zn

2.7 Measurement of pigments

Fresh leaf samples were homogenized in 80% chilled acetone and centrifuged at 10,000 g for 10 min, and the absorbance of supernatant was taken at 480 nm, 510 nm, 645 nm and 663 nm for the estimation of total chlorophyll and carotenoids (Arnon, 1949 and Duxbury and Yentsch 1956).

2.8 Antioxidant enzymes assay

Protein content of the supernatant was measured following Bradford’s method (Bradford, 1976). For enzyme assays, samples were homogenized in potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 1% polyvinyl pyrrolidone, 1% glycerol and 0.05 g sodium ascorbate, under chilled conditions. Homogenate was centrifuged at 12,000 x g for 15 min at 4°C. The supernatant was used for the assay of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX). The activity of SOD was measured by the method of Beauchump and Fridovich (1971). The activity of SOD was defined as the amount of enzyme required to cause 50% inhibition of nitroblue tetrazolium (NBT) reduction measured at 560 nm. Activity of CAT enzyme was estimated by following the method proposed by Aebi (1974). 1 ml of reaction mixture comprised 50 mM of potassium phosphate buffer (pH 7.5), 20 mM of H₂O₂ and 20 µl of enzyme extract. H₂O₂ consumption which cause decrease in absorbance was measured at 240 nm (extinction coefficient 39.4 mM^− 1 cm^− 1). APX activity was estimated by the method given by Nakano and Asada (1981). The activity of APX was measured by estimating the rate of ascorbate oxidation (extinction coefficient 2.8 mM^− 1cm^− 1) at 290 nm. Activity of GPX was assayed according to the method of Hemeda and Klein (1990).

2.9. Statistical analysis

The experiment was arranged in a Randomized Completed Block Design (RCBD) with three replications. The statistical analysis was done by using Graphpad Prism which included unpaired t-test to analyse the significance of changes between different treatments at probability levels of 0.05.

3.1 Topsoil physicochemical parameters and element concentrations

Electrical conductance (EC) and the pH of soil were recorded in the range of 84.9-102.33 µS cm^− 1 and 7.66–7.73, respectively (Table 1). The mean organic carbon and available phosphorus of soil were 1.5–1.76% and 75.87–89.62 kg ha^− 1. The soil protease activity was estimated to be 5.06–6.09 µg tyrosine g^− 1 dw 2 h^− 1. Alkaline phosphomonoesterase, acid phosphomonoesterase and diesterase activities was estimated to be 119.47-147.85 p-Nitrophenol (µg g^− 1dw h^− 1), 91.22-120.63 p-Nitrophenol (µg g^− 1dw h^− 1) and 20.82–49.23 p-Nitrophenol (µg g^− 1dw h^− 1), respectively. The level of As and Zn ranged from 3.65–3.83 µg g^− 1 and 27.26–50.34 µg g^− 1, respectively.

Table 1

Soil physicochemical properties (pH, EC, Organic carbon, available phosphorus, soil enzymes and element concertation (Zn, As) of selected agricultural top soil from study area.
S. No.	Soil parameter	Control	ZnO	ZnO + Biochar
1	pH	7.66 ± 0.15	7.66 ± 0.057	7.73 ± 0.057
2	EC (µS cm^− 1)	84.9 ± 4.070	102.33 ± 2.51	97.16 ± 2.84
3	Organic carbon (%)	1.76 ± 0.5	1.6 ± 0.76	1.5 ± 0.5
4	Available phosphorus (kg ha^− 1)	89.62 ± 3.7	80.48 ± 1.9	75.87 ± 0.79
5	Protease (µg tyrosine g-¹ dw 2 h^− 1)	6.09 ± 0.49	5.81 ± 0.87	5.06 ± 0.45
6	Alkaline monophosphoesterase (p-nitrophenol (µg g^− 1 dw h^− 1)	147.85 ± 1.28	121.54 ± 2.16	119.47 ± 1.03
7	Acidic monophosphoesterase (p-nitrophenol (µg g^− 1 dw h^− 1)	120.63 ± 2.69	117.41 ± 12.76	91.22 ± 8.48
8	Diesterase (p-nitrophenol (µg g^− 1 dw h^− 1)	20.82 ± 0.24	38.9 ± 1.27	49.23 ± 0.04
9	Zn (µg g^− 1)	50.34 ± 5.03	27.26 ± 1.30	29.63 ± 3.2
10	As (µg g^− 1)	3.83 ± 0.06	3.65 ± 0.09	3.75 ± 0.023

3.2 Growth and grain yield

ZnO-NPs and ZnO-NPs + biochar treated plants showed better growth and yield in all aspects (root, shoot length, biomass, penicle length, number of fertile spikelets and 1000 grains weight) as compared to that of control plants. The root and shoot length of ZnO-NPs and ZnO-NPs + biochar treated plants were 4% and 2%, and 6% and 8% higher than control, respectively (Fig. 2A). Fresh biomass of plant was observed to be higher in ZnO-NPs and ZnO-NPs + biochar treated plants by 14% and 26% respectively than that of control plants (Fig. 2B). Grain yield is the end product, which decides the suitability of an amendment and it depends on various physiological processes occurring during the plant growth and development. ZnO-NPs alone as well as in combination with biochar showed better grain yield than control, 1000 grains weight was higher by 30% and 33% in ZnO-NPs treated and ZnO-NPs + biochar treated plants respectively (Fig. 2E). Correspondingly, the number of fertile spikelets was found to be higher by 12% and 16% as compared to control (Fig. 2D).

3.3 Electrolytic leakage and lipid peroxidation

ZnO-NPs alone as well in combination with biochar showed reduction in leaf electrolytic leakage than that of control (Fig. 3A). It was found to be lowered by 48% and 62% in ZnO-NPs and ZnO-NPs + biochar, respectively. The damage in the lipid bilayer was estimated by MDA content. The MDA in root and leaves was lowered by the application of ZnO-NPs by 14% and 34% in respect to control (Fig. 3B). In combined use of biochar and ZnO-NPs, it was lowered by 55% and 42% in root and leaves, respectively (Fig. 3B).

3.4 Pigment content and Protein

Total chlorophyll, carotenoid and protein contents were higher in ZnO-NPs and ZnO- NPs + biochar applied plants as compared to the control plants. Total chlorophyll and carotenoids were estimated to be 44% and 28%, and 37% and 22% higher in ZnO-NPs and ZnO- NPs + biochar applied plants, respectively as compared to control ones (Fig. 3C). Protein content was increased in ZnO-NPs and ZnO-NPs + biochar treated plants but it was not significant.

3.5 Antioxidant enzyme activities

APX was recorded to be increased by 127% and 64% in root of ZnO-NPs and ZnO- NPs + biochar combination respectively while in shoot it was higher by 45% and 71% in respective treatments. Which is statistically significant compared to control (Fig. 4A). GPX was estimated to be higher in roots by 152% and 123% while in shoot it was higher by 126% and 129% in respective treatment (Fig. 4B). CAT activity was higher by 351% and 458% in root of ZnO-NPs and ZnO-NPs + biochar treatments, in shoot it was higher by 128% and 154% respectively (Fig. 4C). The maximum SOD activity was detected in ZnO-NPs + biochar treated plants followed by ZnO-NPs alone treated ones. SOD activity was found to increase in root and shoot by 15 and 2%, respectively when treated with ZnO-NPs alone and in combination with biochar it was increased by 12% and 15% respectively which is not statistically significant (Fig. 4D).

3.6 Accumulation of Zn and As in different parts of plant

At maturity, Zn accumulation was recorded to be higher in ZnO-NPs and ZnO-NPs + biochar treated roots by 23% and 17%, in shoot by 67% and 69%, and in grains by 51% and 50%, respectively as compared to control (Fig. 5C). Arsenic accumulation was found to be lower in plant tissues and grains of ZnO-NPs and biochar treatments. It was found to be lower in root by 68% and 66% in shoot by 92% and 91% while in grain it showed lowest accumulation which was lowered by 91% and 94% respectively in comparison to control (Fig. 5D). Accumulation of As in rice tissue was in following sequence root > shoot > grain and sequence of comparative accumulation of As in control and treated rice grains was control > ZnO-NPs ≥ ZnO- NPs + biochar.

In harvested tissue samples, the translocation factor of As from root to shoot was recorded to be the highest (0.83) in control followed by ZnO-NPs treated plants (0.22) and then in ZnO-NPs + biochar treated plants (0.20). Zinc translocation factor from root to shoot was found to be the highest in ZnO-NPs + biochar treatment, which was 0.86, followed by that in ZnO- NPs treated plants (0.66). Translocation factor of As from shoot to grain varied from 0.18 to 0.21 in various treatments. Translocation factor of Zn from shoot to grain was 1.94 in control, 1.29 in ZnO-NPs and 1.18 in ZnO-NPs + biochar treated plants.

3.7 Correlation analysis

A strong correlation was observed in plant growth (root length, shoot length, biomass) and yield parameters (number of fertile spikelets, panicle length, tiller number, thousand grain weight) with antioxidant enzymes (APX, GPX, CAT, SOD) and Zn concentration in different tissues meanwhile a moderate positive correlation with chlorophyll a (R² = 0.66), chlorophyll b (R² = 0.32) and carotenoid (R² = 0.52) was observed. Plant overall growth, yield and Zn concentration showed a negative correlation with As concentration (R²=-0.67, -0.61, -0.91 and − 0.74) as well as MDA root, shoot and EL. APX, GPX, CAT and SOD showed strong correlation R² = 0.49, 0.9,0.81,0.83) with each other while negative correlation with MDA (R²=-0.73), EL (R²= -0.75) and As (R²=-0.96) concentration. MDA showed strong correlation with EL (R² = 0.26) and As concentration (R² = 0.89 in rice tissue. Figure 6 is showing correlation matrix plot of control and treated sample.

Both Zn deficiency and As accumulation in edible parts of crop plants are serious problems, and their co-occurrence in same plant can exacerbate the situation. The need exists not only to reduce As accumulation in crops but also enhance the level of essential elements like Zn (Khan et al., 2022; Bienert et al., 2017). Zn is essential for plant growth and its deficiency causes growth and yield reduction of crops. The availability of zinc is impacted by soil pH levels over 7. High levels of organic matter and bicarbonate are other significant factors that prevent rice plants from absorbing zinc. Instead of being in soil solution, the majority of the zinc is associated with hydrous oxide and clay surfaces. As a result, plants have limited access to zinc (Armour et al., 1990)

The application of Zn as a fertilizer or as ZnO-NPs can act as a source of Zn in plants to accelerate their metabolic and enzymatic processes (Li et al., 2018, 2019). It has been reported that ZnO-NPs improve plant growth at optimum concentration (Mahajan et al., 2011; Soliman et al., 2015) by providing Zn^+ 2 as micronutrient (Faizan et al., 2018; Liu and Lal 2015). ZnO-NPs can be applied as foliar spray or in soil. However, in field, soil application can affect the soil properties as well as a greater amount of NPs would be needed while foliar application needs lesser amount and also reduces chances of major changes in soil chemistry. In our finding, it has been observed that plant growth, yield and Zn accumulation were enhanced while As accumulation was reduced by application of ZnO-NPs alone and biochar combination (Fig. 2, 5). Addition of ZnO-NPs and biochar enhanced the biomass of plants which diluted the As concentration in rice tissue. It can be considered as an indirect consequence of ZnO-NPs application.

Biochar is known to support the growth by creating a competition between metal and nutrients at the root surface as well as immobilizing metals in soil (Abbas et al., 2017; Ali at al 2019). Arsenic shows antagonistic relationship with Zn when it is in soil (Ma et al.,2020; Gong et al., 2020) so, it may be possible that with the increased accumulation of Zn by foliar application, absorption of As reduced. Improved growth of plants upon application of ZnO-NPs has been reported in many plants like cucumber, alfalfa, tomato and maize (de la Rosa et al., 2013, Khan et al., 2022). It depicts that ZnO-NPs serves as growth regulator in normal as well as stressed conditions. The better growth was noticed in terms of root-shoot length, biomass, tiller numbers, thousand grain weight (TGW), panicle length, and number of fertile spikelets in ZnO-NPs and ZnO-NPs + biochar treated plants in respect to control (Fig. 2). In Leucaena leucocephala seedlings ZnO-NPs application enhanced the growth in Cd and Pb metal stress condition (Venkatachalam et al., 2017). Application of ZnO-NPs and biofertilizer in maize plant under the 50 and 100 ppm of As stress helped plants to cope up the stress and showed significant increment in plant growth and biomass. ZnO-NPs help plants to maintain the nutrient and mineral supply and increase leaf biomass and Zn content in perilla (Perilla frutescens var. crispa f. purpurea) (Salachna et al.,2021). The improved growth was noticed to be even greater in biochar and NPs combination, which might be due to biochar mediated immobilization of As while augmenting soil conditions for rice plant growth.

Carotenoids act as free radical scavenger and protect plants from photooxidation (Roychoudhury et al., 2021). In our experiment increased carotenoid and photosynthetic pigment were also noticed in the treatment of ZnO-NPs and biochar (Fig. 3c). Zn plays an important role in chloroplast development and biosynthesis of chlorophyll (Rai-Kalal and Jajoo, 2021). In maize plant application of ZnO-NPs supported better growth, biomass and chlorophyll contents (Adrees et al., 2021). It might be due to supplement of Zn which supports the activation of magnesium and manganese for the biosynthesis of pigments (Ghafariyan et al., 2013). Enhanced level of photosynthetic pigment in pearl millet by supplement of ZnO-NPs (Tarafdar et al., 2014) as well as several plants in metalloid stress condition (Venkatachalam et al., 2017; Hussain et al., 2018; Pullagurala et al., 2018b; Rizwan et al., 2019) has been demonstrated. The observed growth improvement in the present study is apparently linked to ZnO-NPs mediated changes in photosynthetic pigments. While the increase in carotenoids presumably played a role in stress tolerance.

Arsenic accumulation and Zn deficiency cause stress which was noticed in terms of membrane damage and electrolytic leakage. However, a lower level of MDA and electrolytic leakage was recorded in foliar application of ZnO-NPs and biochar (Fig. 4a, c). Earlier research on wheat is in corroboration to our results (Rizwan et al., 2019). Membrane permeability is altered by the application of ZnO-NPs which hinders the uptake of As in cell and protects the membrane damage (Rajput et al., 2012; Khan et al., 2022. In cotton (Gossypium hirsutum) application of ZnO-NPs reduced MDA content in comparison to control plant, and promoted the plant growth and enzymatic activity (Priyanka and Venkatachalam, 2016). It may be due to the fact that Zn can maintain the structural integrity and stability of cells (Singh et al., 2016a, b). Due to maintained membrane structure the antioxidant enzymes APX, GPX, SOD and CAT were found to increase (Rizwan et al., 2016, 2017). As well as Zn induced production of ROS scavenging proteins (Faizan et al., 2018) help plants in avoiding oxidative stress. In mustered plants, application of ZnO-NPs positively regulated the growth and activity of antioxidant enzyme (Rao and Shekhawat, 2014). Plants have a systematic mechanism to detoxify the ROS. CAT and SOD are key antioxidant enzyme that scavenge O₂⁻ free radicals and H₂O₂ from cells (Zhang et al., 2014). The increased level of these enzymes depicts that there was coordination in enzyme activity, and they are preventing the oxidative stress by the application of ZnO-NPs and biochar.

The growth of rice crops in flooded field, which caused Zn deficiency and As accumulation in crops. However, the growth retardation was recovered by amendment of ZnO-NPs and biochar because of direct uptake of Zn through NPs as well as via biochar mediated improvement of soil properties. The level of As in rice plants including grains was reduced while that of Zn was increased. The growth improvement was attributable to poor growth reduction and higher level of photosynthetic pigments. Antioxidant enzymes played an important role in reducing stress in plants. The present study delineates that the application of ZnO-NPs and biochar are beneficial to plants. It shows better yield in rice plant as well as help plant to cope up with stress conditions. It can be a better source to enhance the yield and overcome the deficiency of Zn and reduced uptake of As in plants and subsequently in humans.

Acknowledgements: The researcher would like to express their gratitude to the anonymous reviewers for their efforts to improve the quality of this paper.

Author contribution: Kavita Shukla performed the field work, laboratory experiments and prepared original draft of the manuscript. Rubina Khanam and Jayanta Kumar Biswas performed AAS for arsenic and zinc analysis. Sudhakar Srivastava planned and supervised the whole work as well as revised and finalized the manuscript for communication.

Funding: Kavita Shukla is thankful to CSIR for Junior Research Fellowship (09/013(0818)/2018-EMR-I). Sudhakar Srivastava is thankful to Institute of Eminence, Banaras Hindu University for the research grant (Scheme No. 6031).

Data availability: All data generated or analysed during this study are included in this paper.

Ethical approval: The manuscript does not have potential conflicts of interest. The research does not involve human participants or animals.

Consent to participate: Not applicable

Consent for publication: Not applicable

Conflict of interest: The authors declare no conflict of interest.

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Zinc oxide nanoparticles in combination with biochar alleviates arsenic accumulation in field grown rice crop

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Study area

2.2 Soil sampling and analysis of physicochemical properties

2.3 Harvesting, growth and yield measurement

2.4 Measurement of electrolytic leakage and lipid peroxidation

2.5 Sample digestion and instrumental analysis

2.6 translocation factor

2.7 Measurement of pigments

2.8 Antioxidant enzymes assay

2.9. Statistical analysis

3. Results

3.1 Topsoil physicochemical parameters and element concentrations

3.2 Growth and grain yield

3.3 Electrolytic leakage and lipid peroxidation

3.4 Pigment content and Protein

3.5 Antioxidant enzyme activities

3.6 Accumulation of Zn and As in different parts of plant

3.7 Correlation analysis

4. Discussion

5. Conclusions

Declarations

References

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