Foliage application of selenium and silicon nanoparticles alleviates Cd and Pb toxicity in rice (Oryza sativa L.)

Journal Pre-proof Foliage application of selenium and silicon nanoparticles alleviates Cd and Pb toxicity in rice (Oryza sativa L.)

Bilal Hussain, Qiang Lin, Yasir Hamid, Muhammad Sanaullah, Liu Di, Muhammad Laeeq ur Rehman Hashmi, Muhammad Bilal Khan, Zhenli He, Xiaoe Yang PII:

S0048-9697(20)30005-X

DOI:

https://doi.org/10.1016/j.scitotenv.2020.136497

Reference:

STOTEN 136497

To appear in:

Science of the Total Environment

Received date:

15 November 2019

Revised date:

30 December 2019

Accepted date:

1 January 2020

Please cite this article as: B. Hussain, Q. Lin, Y. Hamid, et al., Foliage application of selenium and silicon nanoparticles alleviates Cd and Pb toxicity in rice (Oryza sativa L.), Science of the Total Environment (2018), https://doi.org/10.1016/j.scitotenv.2020.136497

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof Foliage application of selenium and silicon nanoparticles alleviates Cd and Pb toxicity in rice (Oryza Sativa L.)

Bilal Hussain1, Qiang Lin1, Yasir Hamid1, Muhammad Sanaullah2, Liu Di3, Muhammad Laeeq ur Rehman Hashmi1, Muhammad Bilal Khan1, Zhenli He4, Xiaoe Yang1*

Ministry of Education (MOE) Key Laboratory of Environmental Remediation and

of

1

Ecosystem Health, College of Environmental and Resources Science, Zhejiang

Institute of Soil and Environmental Sciences, University of Agriculture, 38080

-p

2

ro

University, Hangzhou 310058, People’s Republic of China

re

Faisalabad, Pakistan 3

lP

Jiangxi Yangte River Economic Zone Research Institute, Jiujiang University, Jiujiang,

4

na

China

Indian River Research and Education Center, Institute of Food and Agricultural

Jo ur

Sciences, University of Florida, Fort Pierce, Florida 34945, USA

*Corresponding author

Professor Xiao e Yang College of Environmental and Resource Sciences, Zhejiang University Hangzhou 310058, P.R.China Tel: +86-13858085377, Fax: +86-571-88982907 14 E-mail: [email protected]

Abstract 1

Journal Pre-proof

Direct discharge of untreated industrial waste water in water bodies and then irrigation from these sources has increased trace metals contamination in paddy fields of southern China. Among trace metals, cadmium (Cd) and lead (Pb) are classified as most harmful contaminants in farmland to many organisms including plants, animals and humans. Rice is a staple food which is consumed by half population of the world;

of

due to longer growth period it can easily absorb and accumulate the trace metals from soil. The objective of study was to check the efficacy of Se and Si NPs (nanoparticles)

ro

alone or in combination on metals accumulation and Se-fortified rice (Oryza sative L.)

-p

production as their efficiency remained untested. Alone as well as combined

re

application of Se- and Si-NPs (5, 10 and 20 mg L-1) was achieved along with CK. All

lP

the treatments significantly reduced the Cd and Pb contents in brown rice, except CK,

na

Se3, Si1 and Se1Si3. Combined application of Se and Si (Se3Si2) was more effective in reducing the Cd and Pb contents by 62 and 52 %, respectively. In addition, foliar

Jo ur

application of both NPs improved the rice growth and quality by increasing the grain yield, rice biomass, and Se contents in brown rice. Highest concentration of Se (1.35 mg kg-1) in brown rice was observed with combined application of Se- and Si-Nps (Se3Si2). Selenium speciation revealed the presence of organic species (74%) in brown rice. The combinations of different doses of Se- and Si-Nps are the main determining factor for total concentration of metals in grains. These results demonstrate that foliage supplementation of Se and Si-Nps alleviate the Cd and Pb toxicity by reducing the metals’ concentration in brown rice. Additionally foliage supplementation improved the nutritional quality by reducing the phytic acid contents 2

Journal Pre-proof

in rice grains. Key

words:

Selenium;

Silicon;

Nanoparticles;

Selenium

speciation;

Bio-fortification; Soil contamination 1. Introduction Trace metals contamination in agricultural soils results in increasing environmental

of

issues, agricultural production and jeopardizing food security risk. According to United States Environmental Protection Agency (US-EPA) among trace metals,

ro

cadmium (Cd) and lead (Pb) are classified as most harmful contaminants in farmland

-p

to many organisms including plants, animals and humans (Hamid et al., 2019a;

re

Kelepertzis, 2014). Trace metals enter in soil environment by haphazard dumping

lP

practices and numerous anthropogenic actions like mining wastes, phosphate

na

fertilizers application, sewage sludge, industrial waste and pesticide residues (Di et al., 2017; Hamid et al., 2019c) and estimated biological half-life of these metals’ is nearly

Jo ur

30 years (Phillip, 1980). In China, recent remarkable urbanization and development in industry has increased the contamination about 20% of the cultivated farmland with trace metals (Yu et al., 2006; Wang et al., 2015, Hamid et al., 2020). Plants can uptake trace metals from soil along with other essential nutrients while growing in contaminated soil (Yu et al., 2017). The uptake of Cd and Pb in plants and accumulation in edible parts are considered as serious risk to human beings as they are being consumed in body (Tsukahara et al., 2003). Rice is the dominant cereal crop in Asian countries and is considered as a staple food for 60% of Chinese population. Rice is much prone to uptake and accumulation 3

Journal Pre-proof

of trace metals compared to other grain crops (Williams et al., 2007). Unfortunately, trace metals contamination arises extensively in paddy fields of China, increasing the threat to rice safety and rice derived products (Zhu et al., 2016; Xie et al., 2017). In southern China, rice grains contamination exceeds the limit of allowable Cd and Pb concentration (0.2 mg kg-1, GB2726-2012) provided by China National Food Security

of

Standards. As China have huge population with limited land resources, so it is very important to counteract the accumulation of metals in rice and reduce the risk to

ro

human health.

-p

Selenium (Se) is a well thought-out essential micronutrient for humans and animals

re

(Mao et al., 2016). About 15 % of the global population is Se deficient and according

lP

to the WHO reports, China is one among the 40 Se-deficient countries in the world

na

(Tan et al., 2016). The latest survey revealed that 72 % China mainland soil is Se deficient (Dinh et al., 2018). Moreover, silicon (Si) is found as the second most

Jo ur

abundant element in the earth crust. Appropriate supply of Si plays a vital role in plant growth and indirectly counteracts the biotic and abiotic stresses (Aziz et al., 2015). It is believed that Si may reduce metals toxicity by ion adsorption (Greger et al., 2016), or via improving the antioxidant system (Shi et al., 2005). The application of chemical regulator/mediator is considered as a more practicable and cost effective approach for safer food production. A number of chemical mediators have been used to improve growth and yield of crops grown on metal contaminated site such as silicon, zinc, iron and selenium (Yu et al., 2018; Yin et al., 2019). Among these substances, Se and Si were shown as potential regulators in 4

Journal Pre-proof

minimizing the accumulation of trace metals in food chain (Gao et al., 2018). Previously, many methods have been elaborated for exogenous application of Se and Si which includes seed coating and priming or combining with fertilizer and foliar application and among these, foliar application is considered as a most convenient, easy applicable and acceptable practice. It has been stated that NPs have high mobility

of

and poses prominent bioactivity and biosafety properties due to their greater surface to volume ratio as compared to soluble inorganic salts of Se and Si (Xia et al., 2017;

ro

Djanaguiraman et al., 2018). Therefore, NPs could be exploited as a better solution

-p

for the detrimental effects of trace metals on rice.

re

In the present study, we synthesized Se-NPs and evaluated their impact alone and in

lP

combination with commercially available Si-NPs on biomass and yield parameters of

na

rice grown on metal contaminated soil. Our study further aimed to investigate the role of NPs on Cd and Pb accumulation and translocation from root to shoot and grains.

Jo ur

Additionally, the positive effects of NPs were assessed on enhancing quality parameters and Se concentration and speciation in brown rice. 2. Materials and Methods 2.1. Synthesis of Se-NPs Selenium NPs were synthesized using a chemical reduction method described by Lin and wang (2005) with some modifications. Briefly, 0.01 M sodium dodecylsulfate (SDS) (C12H25O4SNa, 99%; Acros) was prepared that the final SDS concentration well above for sufficient suspending capacity. Selenous acid was prepared by dissolving the selenium dioxide (SeO2, 98%; Sigma). Reducing agent sodium 5

Journal Pre-proof

thio-sulfate pentahydrate (Na2S2O3·5H2O; Riedel-deHaen) was prepared in 20 mL of SDS solution to a final concentration of 156 mM. The solution containing selenous acid was slowly added (drop by drop) in to the reducing agent and the suspension solution after reaction was preceded for sufficient time to complete reduction. During the reduction reaction, solution was continuously mixed well with magnetic stirrer.

of

While Si-NP is commercially available (qingdaojiyida Si chemical company, China)

2.2. Characterization of Se NPs and Si NPs

ro

and was applied for reduced metals accumulation in food chain.

-p

Particle sizes of Se- and Si-NPs dispersions were measured by using transmission

re

electron microscopy (TEM, TecnaiTM Spirit TEM, FEI Company, Hillsboro, OR,

lP

USA). The colloids were firstly pre-treated through a flocculation to remove the

na

excessive compounds such as SDS. The energy dispersive X-ray (EDS, X’Pert PRO, PANalytical, Almelo, the Netherlands) analysis was performed to verify the elemental

Jo ur

composition of the various colloids (Shoaib et al., 2018). The zeta potential of the Se- and Si-NPs was measured by using NanoPlus “Particle Size & Zeta Potential Analyzer” (Particulate Systems a division of Micromeritics, 4356 Communications Drive, Norcross GA, 30093, USA). Briefly, four replicates of each sample were diluted with milliQ water and sonicated for 2 min for better scattering of particles. Samples were taken in the electrode cell and measurement was done at a scattering angle of 90o at 25° C. 2.3. Field site description The experiment was conducted in a Cd and Pb contaminated paddy field located in 6

Journal Pre-proof the west of Zhejiang province, China (Latitude 28° 58′ 49″ N and longitude 118° 57′ 33″ E). The soil was contaminated due to the excessive use of phosphate fertilizers, agrochemicals application and irrigation from contaminated water channels (Cao et al., 2020) having Cd and Pb concentrations 0.84 and 86.12 mg kg-1 soil, respectively. These total metal contents exceed the Chinese Environmental Quality

of

Standards for the soil. The soil chemical properties were analyzed according to the

2.4. Experimental design and treatments

ro

methods described by (Bao, 2008; Li, 2000) and presented in Table 1.

-p

Field was prepared in a randomized complete block design (RCBD) with each plot

re

size of 20 m2 (4 m x 5 m) having three random replications. In this study there were 7

lP

single treatments as, Se and Si was applied at rate of 5, 10 and 20 mg L-1 along with

na

CK (control). This experiment also contains 9 combined treatments including Se1Si1 (5:5 mg L-1), Se1Si2 (5:10 mg L-1), Se1Si3 (5:20 mg L-1), Se2Si1 (10:5 mg L-1),

Jo ur

Se2Si2 (10:10 mg L-1), Se2Si3 (10:20 mg L-1), Se3Si1 (20:5 mg L-1), Se3Si2 (20:10 mg L-1), and Se3Si3 (20:20 mg L-1). To increase the efficiency of the foliar treatments Tween 80 was used as surfactant in all the treatments including CK. First foliar application was applied at late tillering (20th august) followed by 2nd application on heading stage (14th September). Late rice cultivar (Yongyu-8050) seedlings were cultured in last week of May, and transplanted after 28 days of growth. Rice plants were harvested on 15th November at maturity stage. All cultural practices and fertilizers applications (N-P2O5-K2O: 145-60-165 kg ha-1) were kept same for each plot throughout the experiment. Yield attributes (1 m2/ plot) were measured in field at 7

Journal Pre-proof

harvesting and plants were rinsed with deionized water and divided into grains, husk, shoots and roots for further analysis in the lab. 2.5. Plant analysis Plant samples were dried at 65° C until constant weight for total metal determination in various parts. The metals concentration in grounded roots, shoots, husk and grains

of

was analyzed by digesting 0.20 g plant sample with HNO3 and HClO4 at 170° C until brown fumes disappears, cooled and diluted to 25 mL with ultra-pure water. Blank

ro

and standard referenced soil (GSS-5) and plant (GBW (E) 080684) were digested and

-p

analyzed with each batch to check the accuracy of results. The digested plant samples

re

were subjected to ICP-MS for Cd, Pb and micro-nutrients determination (Bao, 2008:

na

2.6. Selenium analysis

lP

Hamid et al., 2018).

Total Se concentration in brown rice was measured by digesting 0.30 g ground sample

Jo ur

with acid mixture of HNO3-HCl (3:1) at 60° C for 30 min and continued for another 60 min at 100° C. After that 2.5 mL of acid mixture was added another time and temperature was raised to 150° C until the brown fog disappears and white fog appears in digestion tubes. Digested tubes were cooled to 100° C, and additionally 2.5 mL of 6 mM HCl was added to reduce SeO42- to SeO32-, continue heating at 100° C until brown fog disappears. Make the volume up to mark and subjected to double channel hydride atomic fluorescence photometer (Dong et al., 2017). For determination of organic and inorganic Se concentrations, brown rice flour (1 g) was weighed in an Erlenmeyer flask and make volume to 30 mL by adding ultra-pure 8

Journal Pre-proof

water. After 30 min of sonication, the extractant was centrifuged for 10 min at 5000 rpm. After that, cyclohexane (5mL) was added in supernatant and water phase was collected 4h later in a beaker. These solutions containing inorganic Se were heated 2-3 min and HCl (6 mol L-1) was added to reduce SeO42- to SeO32-. The solution was subjected to atomic fluorescence photometer after diluting the supernatant to 10 mL

of

with ultra-pure water. Organic Se was noted by subtracting the inorganic Se from total Se. Total soil Se was measured with same methodology used for plant samples except

ro

HNO3-HClO4 (3:2) was used for digestion.

-p

2.7. Determination of phytic acid and protein contents

re

Phytic acid was determined by following the method of Dai et al. (2007) with some

lP

modifications. Briefly 0.5 g rice flour was extracted with 0.2 M HCl (10 mL), shaken

na

for 2 hours and centrifuged to get the solution. The resulting solution (2.5 mL) was mixed thoroughly with 0.2 % FeCl3 (2 mL) and heated on water bath (30 min), cooled

Jo ur

and centrifuged (15 min). The supernatant was then discarded and washed three times with ultra-pure water. After that, 3 mL of 1.5 M NaOH was added in the solution and centrifuged again for 10 min. Again, supernatant was discarded and remaining solution was mixed with 0.5 M HCl (3 mL) to dissolve the residues. Finally, the solution was diluted with ultra-pure water to make final volume (10 mL) and subjected to ICP-MS (Agilent 7500a, Agilent Technologies, CA, USA) for the determination of Fe. The phytic acid content was calculated by multiplying Fe content by the factor 4.2. The Kjeldhal method was followed to determine protein in rice samples by total 9

Journal Pre-proof

nitrogen. Briefly, rice sample (0.5 g) was digested by 20 mL H2SO4, and then distilled in KjelFlex K-360 (Buchi, Flawil, Switzerland) with

NaOH (40% w/v) and boric

acid (2% w/v) (methyl red and bromocresol green as an indicator solution), then titrated with 0.02 mM H2SO4. The protein contents were subsequently calculated by multiplying nitrogen content by a conversion factor of 5.95 (Ohtsubo et al., 2005).

of

2.8. In vitro digestion Selenium bio-accessibility in the rice grain was checked following by an in vitro

ro

gastrointestinal digestion procedure explained by Cámara-Martos et al. (2007) with

-p

some modifications. Briefly, 5.0 g of rice grain powder from each sample was soaked

re

in saline buffer solution (15 mL) and pH was adjusted to 2 with 6 M HCl. While for

lP

the gastric phase of digestion, 0.5 mL pepsin solution (pH-5) was added and samples

na

were incubated at 37° C for 2h in a shaking water bath. Prior to gastrointestinal digestion pH was adjusted to 5 with 1 M NaHCO3, following the addition of

Jo ur

pancreatin bile solution (2.5 mL), the samples were further incubated for 2h and pH was adjusted to 7.2 with 0.5 M NaOH. The digested samples were centrifuged (3500 x g) at 4° C for 1h. The supernatant was collected and the concentration of Se was determined by the atomic fluorescence photometer and computed by using the following equation. 𝐵𝑖𝑜𝑎𝑣𝑎𝑖𝑙𝑎𝑏𝑖𝑡𝑦 (%) =

𝐵𝑖𝑜𝑎𝑐𝑐𝑒𝑠𝑠𝑖𝑏𝑙𝑒 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑒 𝑖𝑛 𝑔𝑟𝑎𝑖𝑛 𝑥 100 (𝑆𝑒 𝐶𝑜𝑛𝑐. )𝑎𝑐𝑖𝑑 𝑑𝑖𝑔𝑒𝑠𝑡

2.9. Translocation factor for Cd and Pb Translocation of Cd and Pb from roots-shoots-grains was determined by using the formula described by Hamid et al., 2019b. 10

Journal Pre-proof

𝑇𝑟𝑎𝑛𝑠𝑙𝑜𝑐𝑎𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑓𝑜𝑟 𝐶𝑑/𝑃𝑏 (%𝑇𝐹) =

𝐶𝑑/𝑃𝑏 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑠𝑕𝑜𝑜𝑡𝑠 𝑋 100 𝐶𝑑/𝑃𝑏 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑟𝑜𝑜𝑡𝑠

𝑇𝑟𝑎𝑛𝑠𝑙𝑜𝑐𝑎𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑓𝑜𝑟 𝐶𝑑/𝑃𝑏 (%𝑇𝐹) =

𝐶𝑑/𝑃𝑏 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑔𝑟𝑎𝑖𝑛𝑠 𝑋 100 𝐶𝑑/𝑃𝑏 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑠𝑕𝑜𝑜𝑡𝑠

2.10.

Statistical analysis:

The presented data is means of three replicates (± SE) and was analyzed using Excel and treatments means were differentiated by analysis of variance (ANOVA) using

of

LSD’s and Tukey’s test (P< 0.05). Graphical presentations were completed by using

ro

Origin Pro 8.5 (OriginLab Corp, Northampton, MA, USA). Correlation coefficients

-p

and principal component analysis (PCA) were performed by using the SPSS 20.0.

re

3. Results and Discussion

lP

3.1. Characterization of Nano particles

na

Nano particles synthesis by SDS system provides fine control over the particles size. Sodium thiosulfate was enough strong to ensure the complete conversion of precursor

Jo ur

molecules to nano-Se particles. Transmission electron microscopy (TEM) images (Figure 1 A, B) shows the mean diameter of NPs for Se and Si, respectively. We prepared good sized control NPs of Se and Si having mean diameters of 12.26±2, 18.04±3 nm respectively. The SEM concomitant and EDX elemental dots map of the NPs are illustrated in Figure 1. The results of EDX map showed the major elements as C, O, Ca, Mg, S and Se in the suspension solution. The intensity of dots showed the percentage of elements present in suspension. 3.2. Zeta potential The zeta potential degree illustrates the electrostatic repulsion degree between the 11

Journal Pre-proof

adjacent and similar charged particles in dispersion. Nano emulsion with ZP of +30 mV to −30 mV is considered a very stable form (Jiang et al., 2009). Zeta potential of all Se-NPs and Si-NPs ranges from -19 to 38 mV. Zeta potential is considered excellent technique for particles surface properties and describes the stability of nano emulsion. The colloidal system with low zeta potentials are unstable and high zeta

of

potentials are electrically stabilized (Morga et al., 2013). In our study, the zeta

ro

potential of all NPs was more than -19 mV, signifying the stable nature of both NPs. 3.3. Grains and biomass yield of rice

-p

Grains yield of rice was significantly increased with treatments combination by 27, 24

re

and 18% with Se3Si2, Si3 and Se2Si3 respectively as compared to CK (Figure 2).

lP

While, foliage treatment (Se3Si2, Si3) improved the biomass yield by 50, 47%

na

followed by Se2Si3 (38%) when compared with CK. Metal stress can inhibit plant growth which results in leaf chlorosis, impedance of plant metabolism and production

Jo ur

of excess reactive oxygen species (Clemens et al., 2013). It is widely reported that the application of Se can alleviate the oxidative stress and increase biological and grain yield (Boldrin et al., 2013).

Our results showed a good correlation of grain Se with grain yield (0.369**), and biological yield (0.370**) (Figure 6) and PCA findings (Figure 7) also confirms the positive effect of foliar application on mitigating the toxic effect of trace metals. The principal components PC1 and PC2 respectively accounts for 59.34 % and 10.81% of the variation. In present study, we also found that the application of Si alone and coupled with Se increased biological yield as well as grains yield of rice indicating the 12

Journal Pre-proof

enhancement of plant resistance to metals stress. 3.4. Cd uptake and accumulation in rice parts There was a clear difference in Cd accumulation in brown rice, and uptake by roots and shoots with influence of foliar treatments (Figure 3). Cd concentrations were significantly reduced in grains (56, 37 and 23%), husk (57, 37 and 35), and shoots (72,

of

73 and 44%) with treatments Se3Si2, Si3 and Se2Si3 respectively as compared to CK. While, when compared with CK, the root Cd contents were decreased by 35, 34 and

ro

27% with Si3, Se2Si2 and Se2Si3 foliar treatments. In present study, single

-p

application of Si-NPs at high dose and combination of Se-NPs remarkably reduced

re

the Cd concentration in shoots, husk and grains of late rice. While foliar application of

lP

Se3 (20 mg L-1) increased the Cd accumulation in grains as compared to CK (Figure

na

3B). Similar findings were also reported by (Gao et al., 2018) where Se application was not helpful in reducing grains Cd concentration. Foliar application of Se2, Si3

Jo ur

and Se3Si2 significantly decreased the Cd concentration in brown rice below the MPCC (0.2 mg kg-1: maximum permissible concentration in China). Previous studies revealed that Cd stress could annoy the cellular redox balance by production and accumulation of ROS which can damage the plant cells (Dinh et al., 2018). Selenium application can alleviate the Cd toxicity by decreasing the oxidative stress, as Se decreased the ROS level by 9.7 fold in rice suspension cells as compared to control (Cui et al., 2018). The Cd transporters (NRAMP5, OsHMA3, OsHMA2 (Cui et al., 2017) plays a significant role in determining metal concentration in rice, which might be influenced by changes in the environmental factors e.g. foliar spraying with Se or 13

Journal Pre-proof

Si (Gao et al., 2018). The positive effect of Si mainly depends upon the deposition in plant tissues which can enhance the strength and rigidity to improve the ability against adverse stresses (Ma and Yamaji, 2006). Si is taken up by rice plant as monosilic acid and stored in plant leaves by forming a double cuticle layer (Mitanai et al., 2005) and this layer can chunk the passage of Cd in rice. Meanwhile, another study revealed that

of

rice suspension cell when exposed to Cd solution can bound or accumulates the Cd in cell wall with hemicellulose (a form of Si) having a net negative charge which is

ro

likely to reduce the Cd uptake (Ma et al., 2015). Furthermore, Si have ability to

-p

regulate the specific genes which are responsible for uptake and accumulation of

re

metals, for instance, genes expression related to Cd transport in shoot (OsLCT1 and

lP

OsNramp5) was down regulated by the application of nano-silica (Cui et al., 2017).

na

On the other hand, the application of Se at high dose (20 mg L-1) increased the Cd accumulation (9%) in grains, which is attributed to dependency of Cd accumulation

Jo ur

on dosage of Se and Si. In short, our results revealed the significant reduction in Cd contents in various parts of rice with Se and Si combined treatments. Moreover, the interaction between Cd conc. and Se conc. in grains showed the negative correlation (Figure 6) (-0.338**) and PCA results also validated the findings (Figure 7). Our findings are in line with Lin et al. (2012) that the negative correlation between grain Cd and grain Se concentration might be owed to the supplementation of Se or Si in reducing the Cd contents in plant edible parts (Gao et al., 2018). 3.5. Pb uptake and accumulation in rice parts Lead concentration in plant parts followed the order root > shoot > husk > grains. 14

Journal Pre-proof

Foliar application of Se or/and Si combinations significantly reduced the Pb concentration in grains by 52, 50 and 46% with Se3Si2, Se3Si1, and Se2Si1, respectively (Figure 4). Meanwhile, shoot Pb was reduced by 57, 56 and 49% in Se3Si1, Se3Si2 and Se2Si3 treated plots respectively as compared to CK. Total Pb accumulation in rice plant was observed highest in CK while lowest in Se3Si2. Pb

of

contents in grains (Figure 4B) were lower than MPCC in all treatments except CK, Si1, Se1Si3 and Se2Si1. The presented data showed that the proper supplementation

ro

of Se or/and Si decreased the accumulation of Pb in grains and shoots of rice.

-p

As reported earlier, excess amount of Pb affect the activity defensive system of

re

enzymes and ROS (reactive oxygen species) system in plants as, the ROS production

lP

prompts a protective mechanism which could serve as protective barrier for Pb

na

accumulation (Li et al., 2019). Excessive Pb can also increase the oxidative stress by enhancing the production of ROS, H2O2 and hydroxyl radicals (Alyemni et al., 2018).

Jo ur

Selenium application can decrease the Pb accumulation by lowering the oxidative stress. He et al. (2004) reported that application of Se decreased the Pb concentration of Chinese cabbage and lettuce by 20% through the inhibition of free radicals and H2O2 production. Similar results were reported by Fargasova et al. (2006) that supplementation of Se in white mustard reduced Pb contents to 84.9% by reducing the oxidative stress. The application of Se significantly reduced the Pb concentration in oil seed rape (Zhilin et al., 2013). The potential role of Se supplementation in alleviation of toxic metals is also reported (Saidi et al., 2014). Foliage applications of Se coupled with Si reduced the accumulation of Pb by decreasing oxidative stress 15

Journal Pre-proof

(Alyemeni et al., 2018). 3.6. Phytic acid and protein content in rice grains Data regarding phytic acid and protein content in rice grains as affected by the foliar application is presented in Table 2. Phtytic acid in grains was significantly reduced by the foliar application except Si1. Phytic acid content ranged from 2.80 mg g-1 in the

of

CK to 2.09 mg g-1 in Se3Si2. Foliar application of Se and Si alone or in combination decreased the phytic acid content with maximum decrease (25%) in Se3Si2 and

ro

minimum in Se1 (5%). Foliar spraying decreased the phytic acid concentration by 16%

-p

each with Si2, Se1Si1 and Se3Si3 treatment as compared to CK. Present results

re

showed that application of Se or Si alone or in combination decreased the phytic acid

lP

contents in rice grains, but the maximum reduction was observed with combined

na

application. Reducing the phytic acid (phytate) contents are important regarding the bio accessibility of micronutrients especially in cereals. A reduction in phytic acid

Jo ur

contents leads to increased absorption of Zn and Fe (Egli et al. 2014: Stmopen et al. 2013). In another study, Hurrell et al. (2003) reported that low concentration of phytic acid increases the absorption of Zn and Fe in wheat. Protein contents in rice grains showed a significant increasing trend by foliar applications except Se1 and Se3 (Table 2). Lowest and highest grains protein contents (8.77 and 10.17%) were found in the CK and Se3Si2 respectively. Foliar application of Se or Si alone or in combination increased protein content in rice grains. A negative correlation was observed between grain Se and phytic acid (-0.369**) (Figure 6) and similar observations were noted when all the data was subjected to PCA (Figure 7). 16

Journal Pre-proof

3.7. Se concentration, speciation and bio-availability Foliar treatment of Se increased the brown rice Se concentration significantly in a dose dependent order (Figure 5). The Se concentration in brown rice was ranged from 1.35 mg kg-1 (Se3Si2) to 0.042 mg kg-1 in CK. Organic Se was found as protein, starch and lipids bound, which were difficult to extract with water (Figure 5b). Total

of

content of organic and inorganic Se was increased by increasing the Se concentration in foliar treatments. Approximately, 74% of Se absorbed by the plants was stowed in

ro

organic form except CK. Furthermore, Si1, Si2 and Si3 also showed a reduction in

-p

organic Se contents as compared to other treatments. Our results depicted an increase

re

in inorganic Se contents with an increase in Se fertilization. The increase in inorganic

lP

Se contents was observed in treatment Se3 (35%) but still lower than the CK (40%).

na

Se species in the fertilizers are main contributor to the enrichment of Se in plants (Deng et al., 2017). Foliar application of Se is more effective in biotransformation

Jo ur

from inorganic to organic Se where proper supplementation at heading stage increased the Se contents in wheat and rice (Deng et al., 2017; Huang et al., 2017). The main portion of Se assimilated from soil to plant remained as inorganic in non-supplemented rice (Fang et al., 2009). The maximum bioavailability of Se was observed in Se3Si2 (83%) followed by Si2 (65%) with minimum in CK (Table 2). Foliar application of Se alone or in combination of Si significantly changes the bioavailability of Se. The variance in bioavailability may be related to other micronutrients availability. Similar results were stated by Egli et al. (2014) who reported that foliar supplementation of Se or Si 17

Journal Pre-proof

increased the availability of Fe and Zn. 3.8. Metals translocation in grains The translocation (TF) values for Cd and Pb following foliar treatments are presented in Table 2. Highest translocation of both metals (TFroot-shoot and shoot-grain) was found in CK followed by Se3 as compared to other treatments. Foliar treatment Se3Si2 and Si3

of

decreased the Cd translocation by 35 and 26% as compared to CK. While translocation for of Pb (TF root-shoot and shoot-grain) was highest in Se1Si1 (38.13 %) and

ro

Si1 (8.48%) treatments respectively. But there was no significant difference in TF for

-p

Pb between foliar treatments and CK. In present study, foliar spray treatments resulted

re

in alleviation of Cd uptake and accumulation in brown rice. Previous studies reported

lP

that transporters play a vital role in Cd uptake and accumulation such as OsHMA3

na

and OsHMA2, NRAMP5 genes that regulate the expression of these transporters but their efficiency may differ with genotypes or may be influenced by the environmental

Jo ur

factors such as foliar treatments (Sasaki et al., 2012: Luo et al., 2018). Moreover, Cui et al. (2017) reported that the expression of genes associated with Cd uptake was subdued by the application of silica nano particles. 4. Conclusion Cd and Pb accumulation in paddy fields have been grown as threatening tool for environment and human health. Foliar spraying with Se and Si-NPs provides an effective approach to minimize the accumulation of metals in brown rice grown on contaminated soils. But the Cd and Pb accumulation depends upon the application rate as well as different combinations. Application of Se-NPs at high rates (20 mg L-1) 18

Journal Pre-proof

improved the Cd accumulation in brown rice while the same rate when subjected with Si @ 10 mg L-1 decreased the Cd accumulation by 62%. Our results depicted that the highest decrease in metal accumulation in brown rice was observed with foliar spraying of Si3 (20 mg L-1) and Se3Si2 (20:10 mg L-1). Furthermore, foliar treatments improved the grain yield and quality of brown rice by reducing metals accumulation.

of

Se bio-fortification (1.35 mg kg-1 brown rice) was achieved with foliar application of Se (Se3Si2) and in vitro digestion confirms the Se bioavailability by 86% in grains.

ro

Foliar application also reduced the production of phytic acid and improved the protein

-p

contents in rice grains. Results of this study showed a negative correlation between Se

re

contents, phytic acid and Cd or Pb concentration. Finally, we concluded that foliar

lP

application of Si-NPs with higher Se-NPs concentration is feasible tool to reduce

Acknowledgments

na

metals accumulation when planting rice in multi element polluted paddy soils.

Jo ur

This study was supported by Shanghai Agricultural and Rural Affairs Commission (#201902080008F01134), Zhejiang Science and Technology Bureau (#2018C02029), the Key Projects from Ministry of Science and Technology of China (#2016YFD0800805), and the Fundamental Research Funds for the Central Universities. We are very grateful for their financial support. References Alyemeni, M.N., Ahanger, M.A., Wijaya, L., Alam, P., Bhardwaj, R., Ahmad, P., 2018. Selenium mitigates cadmium-induced oxidative stress in tomato (Solanum lycopersicum L.) plants by modulating chlorophyll fluorescence, osmolyte 19

Journal Pre-proof

accumulation, and antioxidant system. Protoplasma. 255, 459–469. Aziz, R., Rafiq, M.T., Li, T., Liu, D., He, Z., Stoffella, P.J., et al., 2015. Uptake of cadmium by rice grown on contaminated soils and its bioavailability/toxicity in human cell lines (Caco-2/HL-7702). J. Agric. Food Chem. 63, 3599–3608. Bao, S.D., 2008. Soil Agricultural Chemistry Analysis Method, third ed. China

of

Agriculture Press, Beijing, China (in Chinese). Boldrin, P.F., Faquin, V., Ramos, S.J., Boldrin, K.V.F., Avila, F.W., 2013. Guilherme

-p

Compos. Anal. 31, 238–244.

ro

LRG. Soil and foliar application of selenium in rice biofortification. Food

re

Cámara-Martos, F., Barbera, R., Amaro, M.A., Farre, R., 2007. Calcium, iron, zinc

lP

and copper transport and uptake by Caco-2 cells in school meals: Influence of

na

protein and mineral interactions. . Food Chem. 100, 1085-1092. Cao, X., Wang, X., Tong, W.B., b, Gurajala, H.M., Lu, M., Hamid, Y., Feng, Y., He,

Jo ur

Z.L., Yang, X., 2019. Distribution, availability and translocation of heavy metals in soil oilseed rape (Brassica napus L.) system related to soil properties. .Environ. Pollut. 252, 733-741.

Clemens, S., Aarts, M.G., Thomine, S., Verbruggen, N., 2013. Plant science: the key to preventing slow cadmium poisoning. Trends Plant Sci. 18 (2), 92–99. Cui, J., Liu, T., Li, F., et al., 2017. Silica nanoparticles alleviate cadmium toxicity in rice cells: mechanisms and size effects. Environ. Pollut. 228, 363–369. Cui, J., Liu, T., Li, Y., Li, F., 2018. Selenium reduces cadmium uptake into rice suspension cells by regulating the expression of lignin synthesis and 20

Journal Pre-proof

cadmium-related genes. Sci. Total Environ. 644, 602–610. Dai, F., Wang, J., Zhang, S., Xu, Z., Zhang, G., 2007. Genotypic and environmental variation in phytic acid content and its relation to protein content and malt quality in barley. Food Chem. 105: 606–611. Deng, X., Liu, K., Li, M., Zhang, W., Zhao, X., Zhao, Z., Liu, X., 2017. Difference of

of

selenium uptake and distribution in the plant and selenium form in the grains of rice with foliar spray of selenite or selenate at different stages. Field Crop.

ro

Res. 211:165–171.

-p

Di, P.M., Cheng, R.R., Lieberman, A.E., et al., 2017. De novo prediction of human

re

chromosome structures: epigenetic marking patterns encode genome architecture.

lP

Proc. Natl. Acad. Sci. 114 (46), 121–126.

na

Dinh, Q.T., Cui, Z., Huang, J., Tran, T.A.T., Wang, D., Yang, W., Zhou, F., Wang, M., Yu, D., Liang, D., 2018. Selenium distribution in the Chinese environment and its

Jo ur

relationship with human health: A review. Environ. international, ISSN: 1873-6750, Vol: 112, 294-309. Djanaguiraman, M., Belliraj, N., Bossmann, S.H., Vara Prasad, P.V., 2018. High-Temperature stress alleviation by selenium nanoparticle treatment in grain sorghum. ACS Omega. 3, 2479-2491. Dong, Z., Tianyu, D., Jun, Y., Zhenan, H., 2017. Selenium accumulation in wheat (Triticum aestivum L) as affected by coapplication of either selenite or selenate with phosphorus. Soil sci.plant nut.63 (1), 37-44. Egli, I., Davidsson, L., Zeder, C., Walczyk, T., Hurrell, R., 2014. Dephytinization of 21

Journal Pre-proof

a complementary food based on wheat and soy increases zinc, but not copper, apparent absorption in adults. J Nutr. 134:1077–1081. Fang, Y., Zhang, Y., Catron, B., Chan, Q., Hu, Q., Caruso, J.A., 2009. Identification of selenium

compounds

using

HPLC-ICPMS

and

nano-ESI-MS

in

selenium-enriched rice via foliar application. J. Anal. At. Spectrom. 24, 1657–

of

1664. Fargasova, A., Pastierova, J., Svetkova, K., 2006. Effect of Se-metal pair

ro

combinations (Cd, Zn, Cu, Pb) on photosynthetic pigments production and metal

-p

accumulation in Sinapis alba L. seedlings. Plant Soil Environ. 52:8–15.

re

Gao, M., Zhou, j., Li, H., Zhang, W., Hu, Y., Liang, J., Zhou, J., 2018. Foliar spraying

lP

with silicon and selenium reduces cadmium uptake and mitigates cadmium

na

toxicity in rice. Sci. of the Total Environ. 631–632, 1100–1108. Greger, M., Kabir, A.H., Landberg, T., Maity, P.J., Lindberg, S., 2016. Silicate reduces

Jo ur

cadmium uptake into cells of wheat. Environ. Pollut. 211, 90–97. Hamid, Y., Lin, T., Muhammad. Y., Bilal, H., Afsheen, Z., Muhammad, Z. A., Zhen-li, H., Xiaoe, Y., 2019 a. Comparative efficacy of organic and inorganic amendments for cadmium and lead immobilization in contaminated soil under rice wheat cropping system. Chemosphere. 214, 259-268. Hamid, Y., Tang, L., Hussain, B., Usman, M., Lin, Q., Rashid, M.S., He, Z.L., Yang, X., 2020. Organic soil additives for the remediation of cadmium contaminated soils and their impact on the soil-plant system: A review. Sci. Total Environ. 707, 136121. 22

Journal Pre-proof

Hamid, Y., Tang, l., Irfan, S.M., Cao, X., Hussain, B., Zahir, A.M., Usman, M., He, Z.L.,Yang, X., 2019c. An explanation of soil amendments to reduce cadmium phytoavailability and transfer to food chain. Sci. of the Total Environ. 660, 80– 96. Hamid, Y., Tang, L., Lu, M., Hussain, B., Zehra, A., Bilal, K.M., He, Z.L., Kumar,

of

G.H., Yang, X., 2019b. Assessing the immobilization efficiency of organic and inorganic amendments for cadmium phytoavailability to wheat. J. Soils and

ro

Sediments. 19: 3708.

-p

Hamid, Y., Tang, L., Wang, X., Hussain, B., Yaseen, M., Zahir, A. M., Yang, X., 2018.

re

Immobilization of cadmium and lead in contaminated paddy field using inorganic

lP

and organic additives. Sci. Reports. 8:17839.

na

He, P.P., Lv, X.Z., Wang, G.Y., 2004. Effects of Se and Zn supplementation on the antagonism against Pb and Cd in vegetables. Environ. Int. 30:167–172.

Jo ur

Huang, Q.Q., Wang, Q., Wan, Y.N., Yu, Y., Jiang, R.F., Li, H.F., 2017. Application of X-ray absorption near edge spectroscopy to the study of the effect of sulphur on selenium uptake and assimilation in wheat seedlings. Physiol. Plant. 61, 726– 732. Hurrell, R.F., Reddy, M.B., Juillerat, M.A., Cook, J.D., 2003. Degradation of phytic acid in cereal porridges improves iron absorption by human subjects. American Society for Clinical Nutrition. 77, 1213-1219 Jiang, J., Oberdörster, G., Biswas, P., 2009. Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies J. 23

Journal Pre-proof

Nanoparticle Res. 11, 77-89. Kelepertzis, E., 2014. Accumulation of heavy metals in agricultural soils of Mediterranean: insights from Argolida basin, Peloponnese, Greece. Geoderma 221, 82-90. Li, H.S., 2000. Principle and Technology of Plant Physiological and Biochemical

of

Experiment. Higher Education Press, Beijing,China (in Chinese). Li, X., Ma, L., Li, Y., Wang, L., Zhang, L., 2019. Endophyte infection enhances

-p

Ecotoxicol. Environ. Saf. 174, pp. 255-262.

ro

accumulation of organic acids and minerals in rice under Pb2+ stress conditions.

re

Lin, L., Zhou, W., Dai, H., Cao, F., Zhang, G., Wu, F., 2012. Selenium reduces

lP

cadmium uptake and mitigates cadmium toxicity in rice. J. Hazard. Mater. 235,

na

343–351.

Luo, J.S., Huang, J., Zeng, D.L., Peng, J.S., Zhang, G.B., Ma, H.L., et al., 2018. A

9, 645.

Jo ur

defensin-like protein drives cadmium efflux and allocation in rice. Nat. Commun.

Ma, J., Cai, H., He, C., Zhang, W., Wang, L., 2015. A hemicellulose-bound form of silicon inhibits cadmium ion uptake in rice (Oryza sativa) cells. New Phytol. 206, 1063–1074. Ma, J.F., Yamaji, N., 2006. Silicon uptake and accumulation in higher plants. Trends Plant Sci. 11, 392–397. Mao. J.Y., Pop, V.J., Bath, S.C., Vader, H.L., Redman, C.W.G., Rayman, M.P., 2016. Effect of low-dose selenium on thyroid autoimmunity and thyroid function in UK 24

Journal Pre-proof

pregnant women with mild-to-moderate iodine deficiency. Eur. J. Nutr. 55(1), 55-61. Mitani, N., Ma, J.F., Iwashita, T., 2005. Identification of the silicon form in xylem sap of rice (Oryza sativa L.). Plant Cell Physiol. 46, 279–283. Morga, M., Adamczyk, Z., Oćwieja, M., 2013. Stability of silver nanoparticle

of

monolayers determined by in situ streaming potential measurements. J. Nanopart Res. 15(11): 2076.

ro

Ohtsubo, K., Suzuki, K., Yasui, Y., Kasumi, T., 2005. Bio-functional components in

re

Compos. Anal. 18: 303–316.

-p

the processed pre-germinated brown rice by a twin-screw extruder. J. Food

lP

Phillipp, R., 1980. Cadmium in the environment and cancer registration. J. Epidemiol.

na

Community Health. 34, 151–151.

Saidi, I., Chtourou, Y., Djebali, W., 2014. Selenium alleviates cadmium toxicity by

Jo ur

preventing oxidative stress in sunflower (Helianthus annuus) seedlings. J. Plant Physiol. 171, 85–91.

Sasaki, A., Yamaji, N., Yokosho, K., Ma, J.F., 2012. Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice. The Plant Cell. 24, 2155– 2167. Shi, Q., Bao, Z., Zhu, Z., He, Y., Qian, Q., Yu, J., 2005. Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase. Phytochemistry. 66, 1551–1559. Shoaib, A., Waqas, M., Elabasy, A., Cheng, X., Zhang, Q.Q., Shi, Z., 2018. 25

Journal Pre-proof

Preparation and characterization of emamectin benzoate nanoformulations based on colloidal delivery systems and use in controlling Plutella xylostella (L.) (Lepidoptera: Plutellidae). RSC Adv. 8, 15687–15697. Stmopen, 2013. Phytic acid content in different parts of grain and its correlation with rice quality traits. Adv. J. Food Sci. Technol. 2: 328-334.

of

Tan. L.C., Nancharaiah, Y.V., van, E.D., Hullebusch, Lens, P.N.L., 2016. Selenium: environmental significance, pollution, and biological treatment technologies.

ro

Biotechnol. Adv. 34, 886-907

-p

Tsukahara, T., Ezak,i T., Moriguchi, J., Furuki, K., Shimbo, S., Matsuda-Inoguchi,

re

N., Ikeda, M., 2003. Rice as the most influential source of cadmium intake

lP

among general Japanese population. Sci. Total Environ. 305, 41–51.

na

Wang, S., Wang, F., Gao, S., 2015. Foliar application with nano-silicon alleviates Cd toxicity in rice seedlings. Environ. Sci. Pollut. Res. 22 (4), 2837–2845.

Jo ur

Williams, P.N., Villada, A., Raab, A., Figuerola, J., Green, A.J., Feldmann, J., Meharg, A.A., 2007. Greatly enhanced arsenic shoot assimilation in rice leads to elevated grain levels compared to wheat and barley. Environ. Sci. Technol. 41:6854–6859. Xia, X., Ling, L., Zhang, W.X., 2017. Genesis of pure Se (0) nano- and micro-structures in wastewater with nanoscale zero-valent iron (nZVI). Environ. Sci. Nano 4, 52-59. Xie, L.H., Tang, S.Q., Wei, X.J., Shao, G.N., Jiao, G.A., Sheng, Z.H., et al., 2017. The cadmium and lead content of the grain produced by leading Chinese rice cultivars. Food Chem. 217, 217–224. 26

Journal Pre-proof

Yin, H., Qi, Z., Li, M., Ahammed, G.J., Chu, X., Zhou, J., 2019. Selenium forms and methods of application differentially modulate plant growth, photosynthesis, stress tolerance, selenium content and speciation in Oryza sativa L. Ecotoxicol. Environ. Saf. 169, 911-917. Yu, H., Wang, J., Fang, W., Yuan, J., Yang, Z., 2006. Cadmium accumulation in

of

different rice cultivars and screening for pollution-safe cultivars of rice. Sci. Total Environ. 370, 302-309.

ro

Yu, J., Han, J., Kim, Y.J., et al., 2017. Two rice receptor-like kinases maintain male

-p

fertility under changing temperatures. Proc. Natl. Acad. Sci. U. S. A. 114 (46),

re

12327.

lP

Yu, Y., Wang, A., Li, X., Kou, M., Wang, W., Chen, X., Xu, T., Zhu, M., Ma, D., Li, Z.,

na

Sun, J., 2018. Melatonin-stimulated triacylglycerol breakdown and energy turnover under salinity stress contributes to the maintenance of plasma membrane

256.

Jo ur

HþeATPase activity and Kþ/Naþ homeostasis in sweet potato. Front. Plant Sci. 9,

Zhilin, Wu., Xuebin, Y., Gary, S., Bañuelos, Z-Q., Ying, L., Linxi, Y., 2016. Indications of Selenium Protection against Cadmium and Lead Toxicity in Oilseed Rape (Brassica napus L.). Front. Plant Sci. 7:1875. Zhu, H., Chen, C., Xu, C., Zhu, Q., Huang, D., 2016. Effects of soil acidification and liming on the phytoavailability of cadmium in paddy soils of central subtropical China. Environ. Pollut. 219, 99–106.

27

Journal Pre-proof

Conflict of Interest The authors declare no conflict of interest. Figure 1. Transmission electron microscopy (TEM) images of NPs particles (A) Selenium (B) Silicon suspension and corresponding energy dispersive X-ray spectra (EDS) representing the percentage of element (C-I) in final suspension Figure 2. Biological and grain yield of late rice following foliar treatments. Presented

-p

ro

of

data are mean values of three replicates while error bar represents the standard error. Different letters represent the significant difference between treatments at p< 0.05 level and same letters represent not significant between treatments at the p< 0.05 level. Whereas Se1:Si1=5mg L-1, Se2:Si2= 10 mg L-1 and Se3: Si3 =20mg L-1

lP

re

Figure 3. Cadmium concentration in the Root, shoot, Husk and brown rice following foliar treatments. Presented data are mean values of three replicates while error bar represents the standard error. Different letters represent the significant difference between treatments at p< 0.05 level and same letters represent not significant between treatments at the p< 0.05 level. Whereas Se1:Si1=5mg L-1, Se2:Si2= 10 mg L-1 and Se3: Si3= 20 mg L-1. Maximum permissible concentration in China (MPCC)

na

Figure 4. Lead concentration in the Root, shoot, Husk and brown rice following foliar

Jo ur

treatments. Presented data are mean values of three replicates while error bar represents the standard error. Different letters represent the significant difference between treatments at p< 0.05 level and same letters represent not significant between treatments at the p< 0.05 level. Whereas Se1:Si1=5mg L-1, Se2:Si2= 10 mg L-1 and Se3: Si3= 20 mg L-1. Maximum permissible concentration in China (MPCC) Figure 5. Selenium concentration and speciation in brown rice following foliar

treatments. Presented data are mean values of three replicates while error bar represents the standard error. Different letters represent the significant difference between treatments at p< 0.05 level and same letters represent not significant between treatments at the p< 0.05 level. Whereas Se1:Si1=5mg L-1, Se2:Si2= 10 mg L-1 and Se3: Si3= 20 mg L-1 Figure 6. Linear correlation between Rice Se Concentration and Cd, Pb, grain and

biological yield, phytic acid and protein content Figure 7. Principal Component analysis of 48 samples corresponding to 16 treatments

to the first (PC1) and second (PC2) principal components.

28

Journal Pre-proof

Table 1. Basic physico-chemical properties of the soil used for the experiment. Properties

Values Red earth

pH

5.65 ± 0.07

Organic matter (%)

7.25 ± 0.03

Total N (g kg-1)

1.17 ± 0.09

of

Soil type

Available P (mg kg-1)

ro

68.48 ± 2.26

Available K (mg kg-1)

-p

169.73 ± 6.89

re

Total Cd (mg kg-1)

Available Pb (mg kg-1)

0.34 ± 0.01 106.46 ± 4.43 22.09 ± 1.92 0.28± 0.06

Jo ur

Total Se (mg kg-1)

na

Total Pb (mg kg-1)

lP

Available Cd (mg kg-1)

0.85 ± 0.06

29

Journal Pre-proof

Table 2. Phytic acid and protein content in brown rice following foliar treatments. Presented data are means values of three replicates with the standard error. Different letters with in same column represent the significant difference between treatments at p< 0.05 level and same letters with in same column represent not significant between treatments at the p< 0.05 level. Bioavailability for selenium and translocation factor for Cd and Pb are presented in form of percentage. acid

Protein %

-1

(mg g )

Selenium

Translocation factor Cd (%)

Translocation factor Pb (%)

bioavailability

Root-shoot

Root-shoot

Shoot-grains

(%)

Se1

2.64 ±0.055

8.77 ±0.032 j

65±2

19.81

27.82

6.84

20.04

16.75

38.08

6.15

31.98

8.67

25.65

6.74

36.77

18.39

20.79

6.33

69.5±5

32.71

9.41

21.15

8.48

9.54 ±0.032 e

66±2

35.04

13.10

33.68

6.64

9.63 ±0.026 d

76±2

15.26

16.80

25.50

5.80

9.23 ±0.027 gh

71±4

22.86

12.68

38.13

6.28

9.06 ±0.026 i

71±4

9.22 ±0.026 h

81±3.5

b Se2

2.59 ±0.039 b

b Si1

2.71 ±0.023 a b

Si2

2.33 ±0.029 d

Se1Si1

2.20 ±0.025 e

9.34 ±0.030 f

Jo ur

Si3

9.00 ±0.035 i

2.34 ±0.033

78±2

lP

2.64 ±0.026

na

Se3

Shoot-grains

36.63

-p

2.80 ±0.101 a

re

CK

of

Phytic

ro

Treatments

d

Se1Si2

2.45 ±0.032 c

9.32 ±0.026 fg

73±2.5

35.96

9.17

30.48

7.00

Se1Si3

2.63 ±0.026

9.48 ±0.023 e

69±6

34.40

9.59

34.62

6.79

b

Se2Si1

2.46 ±0.028 c

9.86 ±0.027 b

71±7

28.72

8.70

30.86

6.38

Se2Si2

2.38 ±0.015 c

9.73 ±0.024 c

79±3.6

34.44

12.01

23.42

6.72

d Se2Si3

2.20 ±0.026 e

9.67 ±0.023 cd

79±2.

28.19

12.83

32.63

7.25

Se3Si1

2.21 ±0.009 e

9.93 ±0.034 b

79±4

22.99

13.21

21.91

7.95

Se3Si2

2.09 ±0.017 e

10.17 ±0.048 a

86±2.4

10.21

14.63

27.35

7.56

Se3Si3

2.33 ±0.017

9.86 ±0.024 b

78±3

31.17

8.46

36.96

6.71

d

30

Journal Pre-proof

Highlights



Foliar Se and Si-NPs has been regarded for decreased metals accumulation in rice grains. Se and Si NPs improved the rice quality and Se biofortification.



Se application @ 20 mg L-1 did not stop metals translocation between the plant

of



parts.

ro

Combinations of Se- and Si-Nps are the main determining factor for metals

na

lP

re

-p

accumulation.

Jo ur



31

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7