Effect of water management and silicon on germination, growth, phosphorus and arsenic uptake in rice

Ecotoxicology and Environmental Safety 144 (2017) 11–18

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Effect of water management and silicon on germination, growth, phosphorus and arsenic uptake in rice

MARK

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Zahida Ziaa, Hafiz Faiq Bakhata, Zulfiqar Ahmad Saqibb, Ghulam Mustafa Shaha, Shah Fahadc, , Muhammad Rizwan Ashrafd, Hafiz Mohkum Hammada, Wajid Naseema, Muhammad Shahida a

Department of Environmental Sciences, COMSATS Institute of Information Technology Vehari, Pakistan Saline Agriculture Research Center (SARC), Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad, Pakistan c College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China d Department of Entomology, University of Agriculture, Faisalabad, Sub-campus Burewala/Vehari, Pakistan b

A R T I C L E I N F O

A B S T R A C T

Keywords: Germination Aerobic Anaerobic cultivation As uptake

Silicon (Si) is the 2nd most abundant element in soil which is known to enhance stress tolerance in wide variety of crops. Arsenic (As), a toxic metalloid enters into the human food chain through contaminated water and food or feed. To alleviate the deleterious effect of As on human health, it is a need of time to find out an effective strategy to reduce the As accumulation in the food chain. The experiments were conducted during SeptemberDecember 2014, and 2016 to optimize Si concentration for rice (Oryza sativa L.) exposed to As stress. Further experiment were carried out to evaluate the effect of optimum Si on rice seed germination, seedling growth, phosphorus and As uptake in rice plant. During laboratory experiment, rice seeds were exposed to 150 and 300 µM As with and without 3 mM Si supplementation. Results revealed that As application, decreased the germination up to 40–50% as compared to control treatment. Arsenic stress also significantly (P < 0.05) reduced the seedling length but Si supplementation enhanced the seedlings length. Maximum seedling length (4.94 cm) was recorded for 3 mM Si treatment while, minimum seedling length (0.60 cm) was observed at day7 by the application of 300 µM As. Silicon application resulted in 10% higher seedling length than the control treatment. In soil culture experiment, plants were exposed to same concentrations of As and Si under aerobic and anaerobic conditions. Irrigation water management, significantly (P˂0.05) affected the plant growth, Si and As concentrations in the plant. Arsenic uptake was relatively less under aerobic conditions. The maximum As concentration (9.34 and 27.70 mg kg DW−1 in shoot and root, respectively) was found in plant treated with 300 µM As in absence of Si under anaerobic condition. Similarly, anaerobic condition resulted in higher As uptake in the plants. The study demonstrated that aerobic cultivation is suitable to decrease the As uptake and in rice exogenous Si supply is beneficial to decrease As uptake under both anaerobic and aerobic conditions.

1. Introduction Rice is one of the major food crops in Southern Asia where groundwater contaminated with As is used for the irrigation of paddy fields (Dixit et al., 2016). It has been estimated that 1000 t of As per year are added to the agricultural soils as a result of applying As loaded ground water for irrigation purposes (Duxbury and Panaullah, 2007). Bioavailability of As to plant is governed by biological, chemical and physical processes and their interactions altering metal speciation and behavior in soil-plant systems (Bakhat et al., 2017). Concentration of As in plants depends on plants root ability to uptake and transport it from soil to roots/shoots. The most widely adopted conditions to cultivate the rice in field are water submerged conditions. Anaerobic conditions

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of the paddy fields facilitate the reductive dissolution and release of the adsorbed arsenate (AsV) in the soil stoma water (Bakhat et al., 2017). Furthermore, anaerobic conditions in paddy soils usually lead to the reduction of AsV into more mobile arsenite (AsIII) (Takahashi et al., 2004; Punshon et al., 2016). Rice is an efficient crop in As uptake in comparison to other cereal crops (Bhattacharya et al., 2009; Su et al., 2009). Studies showed that As concentrations in rice plant depends on As presence in soil and/or irrigated groundwater in addition to other factors governing the As mobility and uptake in plant-rhizosphere. In rice, As is taken up by plant roots using macro-nutrient transporters; AsV via the phosphate while AsIII through Si transporters (Ma et al., 2008). Arsenate is a chemical analog of phosphate and shares the uptake pathway in rice

Corresponding author. E-mail addresses: [email protected] (H.F. Bakhat), [email protected], [email protected] (S. Fahad).

http://dx.doi.org/10.1016/j.ecoenv.2017.06.004 Received 24 February 2017; Received in revised form 31 May 2017; Accepted 2 June 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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with the same transporters (Chen et al., 2017). Therefore, antagonistic effects of phosphate on AsV uptake and vice versa are probable in rice plant. Silicon can influence AsIII uptake competitively as AsIII shares the same uptake pathway as Si (Sanglard et al., 2016). The rice Si transporter LSi1 is permeable to AsIII and it acts as AsIII influx transporter. Silicon transporter LSi2 is an efflux transporter of Si which transports Si/AsIII from exodermis to endodermis and towards the stele of the root cells. Redox status (governed by soil moisture contents) of paddy fields is another important factor controlling the As bioavailability to rice plant. Changing soil redox status through water management in paddy fields has been supposed to be one of sustainable solution to reduce As accumulation in rice. A remarkable effect of aerobic conditions turns the speciation pattern towards AsV in comparison to AsIII which has prominently higher solubility, plant availability, and toxicity (Takahashi et al., 2004). Therefore to eliminate the As associated risks, integrated approaches are needed to cultivate the rice especially in those areas that are facing higher contamination of As in ground water or in soil. Among these strategies aerobic rice production with judicious fertilization of nutrient can be a solution to tackle this specific problem (Bakhat et al., 2017). Aerobic rice cultivation is a revolutionary way of rice production and requires only 50% of the water required for irrigated rice production for achieving yield levels of 4–6 Mg t ha−1 (Anil et al., 2014). In addition to water saving, uptake of As can be decreased by plant through aerobic rice production. Understanding of the combined effects of rice cultivation under aerobic condition with exogenous Si is critical strategy to eliminate the elevated uptake of the toxic metalloid. Therefore, present study was conducted; 1) to investigate the effect of two rice cultivation method on As uptake in rice and, 2) to determine the effect of optimum Si on rice germination, phosphorus and As uptake in rice.

Table 1 Physicochemical properties of soil used for the experiment. Values are the means of three replicates ± Standard Error. Characteristics

Values

Soil Texture Electrical conductivity (dS m−1) pH-H2O extract Organic matter (%) Available phosphorous (mg kg−1 soil) Available potassium (mg kg−1 soil) Saturation percentage

Silt loam 1.88 ± 0.06 7.78 ± 0.04 0.60 ± 0.05 6.44 ± 0.10 123.34 ± 9.79 37.27 ± 1.47

2.2. Laboratory experiment to determine effect of Si supplementation on seed germination and seedling growth In this experiment, seeds in the petri plates lined with filter paper were soaked with the solution of 0 mM Si +0 µM As, 0 mM Si+150 µM As, 0 mM Si+300 µM As, 3 mM Si +0 µM As, 3 mM Si +150 µM As, 3 mM Si+300 µM As. Total six treatments with three replicates were performed during the experiment. In this experiment, germination percentage, root and shoot length were recorded. 2.3. Pot trail and experimental set up Rice variety KSK-133 nursery was grown in pots. After the establishment, the seedlings were transplanted to soil pots. Soil was taken from the research farm of the COMSATS Institute of Information Technology, Vehari campus. The soil was thoroughly mixed and sieved using a 4 mm mesh to remove plant parts and other debris. Soil was processed for physico-chemical properties (Table 1). The soil was alkaline in nature with low in available phosphorus and organic matter contents. Afterwards, 7 kg of soil were filled in pots and basal doses of fertilizers nitrogen, phosphorus, and potassium @170 kg, 90 kg and 60 kg per hectare, respectively were added. Nitrogen was added in three split doses (At 1st day of nursery transplantation (DAP), 30 DAP and 45 DAP) while full dose of potassium and phosphorus was added at the pot filling stage. The treatment were arranged as completely randomized design with four repeats.

2. Materials and methods Highly pure analytical-grade reagents and chemicals were purchased and used for the solutions preparation. Standard stock solution of As was prepared by dissolving sodium arsenite (Na-AsO2, M.W. 129.91, BDH, England) and sodium arsenate (Na2HAsO4·7H2O; MW: 312.01, BDH, England) in ultra-pure water while solutions of required concentrations were prepared by further dilution of this stock solution. Silicon solution was prepared using sodium trisilicate solution (Sigma Aldrich).

2.3.1. Seedlings transplantation In each pot, five seedlings were transplanted and thinned in to two after establishment of the seedlings. After thinning, the pots were divided into two groups. Twenty four pots were kept under flooded conditions to maintain anaerobic environment with treatments; T1Si0As0, T2-Si0As150, T3-Si0As300, T4-Si3As0, T5-Si3As150 and T6Si3As300. Other twenty four pots were kept moist with lesser amount of water to maintain aerobic conditions with the Si and As treatments; T7Si0As0, T8-Si0As150, T9-Si0As300, T10-Si3As0, T11-Si3As150 and T12Si3As300.

2.1. Optimization experiment Optimization experiments were performed to determine the growth response of rice variety KSK-133 against As stress in presence of various levels of Si. Sand prewashed with acid (growing media) was filled in the pots and seedlings were transferred. Nutrient solution was applied as described by Zhu et al. (2009) to fulfill the nutritional requirements. After one week of transplantation, to make most effective use of the situation or resources, four sets of experiments were arranged in a completely randomized design with different concentrations of Si and As. These treatments were as: Set 1) 0 mM Si (control), 0.25 mM Si, 1 mM Si, 2 mM Si, 3 mM Si; Set 2) 0 µM As (Control), 50 µM As, 100 µM As, 150 µM As, 300 µM As; Set 3) 0 mM Si+ 0 µM As (Control), 0.5 mM Si +150 µM As, 1 mM Si + 150 µM As, 3 mM Si + 150 µM As; Set 4) 0 mM Si+ 0 µM As (Control), 0.5 mM Si + 300 µM As, 1 mM Si + 300 µM As, 3 mM Si + 300 µM As. The nutrient solution was applied fortnightly to compensate the nutrient depletion in the growth medium. After two months of treatments application, the plants were harvested and washed with deionized water. Afterward, these were separated into root and shoot and fresh weights of the fractions were recorded using analytical balance (AS 220R Radwag, Europe).

2.3.2. Plants harvesting and growth attributes measurements Three months after the treatments application, plant height and number of tillers per plant were recorded. Plants were harvested, washed with deionized water and separated into roots and shoots. The fresh weights of roots and shoots were measured by using weighing balance. The samples were kept in oven at 78 °C till constant weight and then the dry weights were recorded. 2.3.3. Chemical analysis The plant samples (root and shoot) were acid digested with nitric and perchloric acid as reported by Miller (1998). Briefly, one gram plant sample was taken in conical flask, kept overnight after adding 5 mL concentrated HNO3 and 5 mL HClO4. Next day again 5 mL of concentrated HNO3 were added and plant material was digested on hot plate by increasing the temperature slowly to 120 °C for 2 h and 12

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plant−1). A distinct effect of Si to alleviate the As toxicity was observed for both low and high As treatments (As 150 µM and 300 µM). Increasing Si supplementation under 150 µM and 300 µM As exposure resulted a gradual increase in shoot growth; however, the effects were more pronounced with higher Si supply (3 mM). For As 150 µM, there was about 159% increase in shoot biomass with 3 mM Si as compared to its lower concentration 0.5 mM. Similarly, an increase of 140% (5.75 Vs 13.84) was observed for higher Si as compared to lower Si treatment under higher As (300 µM) exposure condition. From the results of the optimization experiment, it can be concluded that 1 mM Si supply is optimum under no-As-stress while 3 mM Si supply yielded maximum plant biomass under As stress conditions. Therefore, for further experimentation, 3 mM Si was used as the optimum concentration for alleviation of As toxicity in rice plants (Fig. 1).

afterwards the temperature was increased to 180 °C for an hour. After digestion, plant material was cooled and made the volume 50 mL with distilled water (Miller, 1998). Digest was filtered with Whatman filter paper No.42 and proceeded for As determination through hydride generation atomic absorption spectroscopy (Talukder et al., 2012). Specific As uptake (SAU) was calculated using the formulae given in Eq. (1) (Zhiyan et al., 2008).

Root − As Concentration×RootBiomass + Shoot SAU = − As Concentration×SootBiomass RootBiomass

(1)

Vanadate molybdate method using UV–visible spectrophotometer was used to determine phosphorus concentration in plant samples (Chapman and Pratt, 1961). To prepare ammonium vanadate-molybdate reagent, ammonium heptamolybdate [(HH4)6MoO24·4H2O] (22.5 g) was dissolved in 400 mL distilled water and 1.25 g of ammonium metavanadate (NH4VO3) in 300 mL hot distilled water. These solutions were mixed in 1 L volumetric flask and allowed the mixture to cool at room temperature. Concentrated HNO3 (250 mL) was added to this mixture, cooled at room temperature and made volume 1 L with distilled water. Five mL of each of the filtered digested sample and ammonium vanadomolybdate were taken into 50 mL volumetric flask, diluted to 50 mL volume with distilled water and kept for half an hour for the stabilization of the developed blue color. Phosphorus was measured with spectrophotometer Model UV Winlab Lambda 25 (Perkin-Elmer Instruments, USA). Instrument was calibrated with a series of P standard solutions (0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 mg L−1) and standard curve was drawn. The absorbance of light by the test samples was recorded at 410 nm wavelength and an actual concentration was obtained by comparing with standard calibration curve. For the quantification of Si, plant material was digested in 3 mL of 65% HNO3 and 2 mL 30% H2O2. The Teflon tubes were put on hot plate. After first step 10 mL of 20%NaOH were added and material was heated for 1 h. The digested material was filtered and sample diluted to 100 mL. The filtrate was used to determine Si by color method. One mL of sample was taken in 50 mL flask added with 0.26 N HCl (1.60 mL), (NH4)6Mo7O24 (10%) (1 mL), 10% tartaric acid (1.60 mL), and reducing agent (0.80 mL). Color development was completed within 1 h and absorbance was measured at 600 nm at spectrophotometer. The reducing agent was prepared by dissolving 250 mg Na2SO3, 125 mg 1amino-2-naphthol-4-sulfonic acid, and 7.5 g NaHSO3 in 50 mL of distilled water (Elliott and Snyder, 1991).

3.2. Effect of silicon supplementation on seed germination and seedling growth of rice under arsenic stress Germination of seeds was recorded to see the effect of Si on rice seeds subjected to As stress (Fig. 2A). Increasing concentration of As resulted in a continuous decrease in germination percentage in comparison to control. Arsenic exposure at both concentrations (As 150 µM and As 300 µM), germination of seeds was decreased about 40–50% in comparison to control. At higher As level (As 300 µM), the germination was lowest (almost 30% in comparison to control treatment). Although the effect of Si application was not significant (P < 0.05) but Si supplementation raised the germination of seeds almost 8–10% as compared to control (Fig. 2A). Silicon application showed maximum germination at the beginning but later its effect was lowered as compared to control. In addition, Si application increased the germination of seeds notably in presence of As in comparison to only seed subjected to As stress. Seedling length was measured to check the effect of Si and As application on rice seeds (Fig. 2B). Si-supplementation improved the seedlings length throughout the experimental period as compared to control. Addition of As inhibited the shoot length. In comparison to control, As stress resulted in a decrease of 3 and 6 folds in seedlings length. Root growth decreased with increasing concentration of As. In the absence of Si supplementation (0 mM Si), decrease in root growth was almost 95% for 150 µM As as compared to control. Silicon supplementation (3 mM Si) increased the root growth about 32% as compared to control. There was no positive effect of Si application on root growth under As stress (Table 2). 3.3. Plant growth attributes

2.4. Statistical analysis

Results showed that two cultivation systems markedly differ for the number of tillers plant−1. Maximum numbers of tillers were observed when plants were grown under anaerobic conditions with additional Si supply. While, As stress inhibited the number of tillers in both treatments (0 mM Si and 3 mM Si) at both concentrations (150 µM As and 300 µM As) under anaerobic conditions. Under aerobic conditions, the effects of As stress were less pronounced. No positive effects of Si were observed on numbers of tillers upon Si-supplementation in aerobic cultivations. Plant height was significantly affected by As application in both cultivation systems. Maximum plant height (57.00 cm) was observed when plants were grown with exogenous Si supply (3 mM Si) under anaerobic conditions. At lower level, As stress significantly reduced the plant height in anaerobic conditions. While under aerobic conditions, the maximum plant height (39.63 cm) was observed in control treatment supplied with Si (Silicon 3 mM+ Arsenic 0 µM) and minimum height (32.13 cm) was observed when plants were treated with higher level of As and Si (Silicon 3 mM + Arsenic 300 µM). There was no positive effect of Si on height of plant under As stress in both cultivation systems (Table 3).

Completely randomized design was used to analyze the obtained experimental data. The experimental data were subjected to analysis of variance using SAS (2004) (SAS/STAT 9.1). Multiple comparisons were done using least significant difference test. For all analyses, a P-value of less than 5% (P < 0.05) was interpreted as statistically significant. 3. Results 3.1. Optimization of silicon for rice growth under arsenic stress Silicon improved the plant growth as measured in terms of shoot growth. Shoot growth increased gradually by increasing Si concentration up-to 1 mM, while a decreasing trend in growth was observed beyond 1 mM Si concentration (up-to 3 mM). Maximum shoot weight (18.68 g plant−1) was observed at 1 mM Si supply in comparison to 10.81 g plant−1 observed in control treatment (Fig. 1). A gradual decrease in shoot growth was recorded with increasing concentration of As (0 µM to 300 µM). Minimum shoot growth was measured at 300 µM As (2.73 g plant−1) nearly 75% lower as compared to control (10.81 g 13

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Fig. 1. Response of rice variety KSK-133 to silicon, arsenic and the combination. Four set of treatments were applied are represented as: A) 0 mM Silicon (control), 0.25 mM Silicon, 1 mM Silicon, 2 mM Silicon and 3 mM Silicon; B) 0 µM Arsenic (Control), 50 µM Arsenic, 100 µM Arsenic, 150 µM Arsenic and 300 µM Arsenic; C) 0 mM Silicon + 0 µM Arsenic (Control), 0.5 mM Silicon +150 µM Arsenic, 1 mM Silicon + 150 µM Arsenic, 3 mM Silicon + 150 µM Arsenic; and D) 0 mM Silicon + 0 µM Arsenic (Control), 0.5 mM Silicon + 300 µM Arsenic, 1 mM Silicon + 300 µM Arsenic, 3 mM Silicon + 300 µM Arsenic. The completely randomized design was used for analysis of variance and mean values were compared using least significant difference at 5% level of probability. Bars with in a single plot sharing same letter did not differ significantly from each other. Values are the means of three replicates ± SE.

Fig. 2. Effect of silicon and arsenic application seed germination percentage (A) and seedling length (B). Seeds of rice variety KSK-133 were socked in distilled water for 36 h and were put on filter paper moistened with different concentration of Si and As. Data was recorded at 3rd day for germination and at 6th day of seed socking. The completely randomized design with factorial arrangements was used for analysis of variance and mean values were compared using least significant difference at 5% level of probability. Germination percentage and seedling lengths were significantly affected by time (p = 0.0001 and 0.0001, respectively) and treatment (p =0.0001and 0.0001, respectively) while interaction between treatment and time was non-significant for germination percentage and seedling lengths with p values 1.0 and 0.26, respectively. Values are the means 3 replicates ± SE.

As stress did not show any positive effects on plant fresh biomass in both cultivation systems. The aerobic cultivations system results in markedly less plant dry biomass as compared to anaerobic cultivation system (Table 3). Under anaerobic conditions, exogenous Si application resulted in maximum dry weight. Arsenic presence in the root medium sharply declined the plant dry mass at both levels in comparison to control treatment under anaerobic conditions. While, under aerobic conditions maximum plant dry biomass was observed in control treatment. There was a decreasing

Maximum plant fresh biomass was observed when plants were supplied with exogenous Si supply (Silicon 3 mM + Arsenic 300 µM). Under anaerobic conditions As application in growth medium resulted in a sharp decline in plant fresh biomass and the two levels of As (150 and 300 µM) were statistically non-significant to each other. In contrast to anaerobic condition, different levels of As showed a gradual decreasing effect on the fresh biomass in aerobic cultivation. There was a continuous decrease in fresh shoot biomass with increasing concentration of As in presence or absence of Si. Silicon application under 14

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Table 2 Effect of silicon and arsenic addition in growth medium on root length. Seeds of rice variety KSK-133 were socked in filtered water for 36 h and were put on filter paper moistened with different concentration of silicon and arsenic. Data was recorded at 9th day of seed socking. Values are the means of three replicates ± SE. Means sharing same letter did not differ significantly from each other at 5% level of probability. Silicon and arsenic addition to the germination medium 0 mM Si Days th

9 10th

3 mM Si

0 µM As

150 µM As

300 µM As

0 µM As

150 µM As

300 µM As

270.48 ± 40.19 b 308.10 ± 45.43 b

12.86 ± 8.93 c 14.76 ± 9.64 c

1.90 ± 0.91 c 6.67 ± 1.67 c

356.19 ± 45.61 a 399.05 ± 47.40 a

6.19 ± 4.24 c 7.14 ± 4.13 c

8.10 ± 3.34 c 12.86 ± 1.65 c

LSD for 9th day = 77.34. LSD for 10th day = 83.87.

significant effect of Si and As addition to the growth medium. Silicon application markedly increased the concentration of phosphorus in shoot tissues. The maximum concentration (3.25 mg g−1 DW) was observed in 3 mM Si treated plants, whereas the minimum concentration (1.60 mg g−1 DW) was observed when plants were treated with Silicon 3 mM + Arsenic 300 µM under aerobic conditions. Arsenic presence in root medium significantly decreased the phosphorus concentration in both cultivation systems. At intermediate stress level (150 µM As), Si application showed some positive effects to mitigate the negative effects of As on phosphorus concentration. While at higher dose of As (300 µM As), Si did not produced any significant effect on shoot phosphorus concentrations (Table 4). Plant grown under anaerobic conditions showed an increase in phosphorus concentration in roots with the increasing concentration of As. Maximum concentration of phosphorus (2.37 mg g−1 DW) was recorded when plants were stressed with higher dose of As (300 µM As) while the minimum concentration (1.00 mg g−1 DW) was observed in As (150 µM As) treatment under aerobic cultivation. Under anaerobic condition plant showed an increasing trend with the increasing concentration of As in the growth medium. There was a significant effect of Si application on phosphorus concentration in rice roots under both cultivation systems. Under aerobic conditions, maximum phosphorus concentration (2.44 mg g−1 DW) was recorded when plants were supplied with Si under no As-stress conditions. In aerobic conditions, a gradual significant decreasing trend

trend in dry weight with increasing concentration of As in the presence or absence of Si under aerobic conditions. There was no positive effect of Si on dry weight of plants under As stress in both aerobic and anaerobic cultivation conditions. Fresh weight of rice roots was recorded to evaluate the effect of Si and As application on roots weight under aerobic and anaerobic conditions. Maximum root fresh weight was observed in plant supplied with Si under control conditions, while between cultivations systems, anaerobic system produced higher biomass as compared to aerobic conditions. A continuous decrease in weight was observed with the increasing concentration of As in the root medium in both cultivation systems. There was no positive effect of Si application under As stress in both water systems as compared to controlled treatments (Table 3). There was a clear decrease in dry weight under aerobic conditions as compared to anaerobic conditions. In both water systems, low root weights were measured as compared to control, while Si application showed a positive effect in root weight only under controlled conditions. There was no remediation by Si application under As stress in both water systems. A continuous decrease in weight was measured with increasing concentration of As. 3.4. Phosphorus, silicon and arsenic concentration in shoots and roots Data recorded for phosphorus concentration in shoots showed a

Table 3 Effect of silicon and arsenic application on number of tillers plant−1, plant height, root and biomass accumulation cultivated under anaerobic and aerobic conditions. Rice plant were grown for 3 months in pots filled with soil treated with different concentrations of As and Si. The completely randomized design was used for analysis of variance and mean values were compared using least significant difference at 5% level of probability. Bars with in a single plot sharing same letter did not differ significantly from each other. Values are the means of 4 replicates ± SE. Treatment Code

T1 T2 T3 T4 T5 T6 Mean T7 T8 T9 T10 T11 T12 Mean

Interaction (WM × Si × As)

Anaerobic-Si0As0 Si0As150 Si0As300 Si3As0 Si3As150 Si3As300 Aerobic-Si0As0 Si0As150 Si0As300 Si3As0 Si3As150 Si3As300

Shoot parameters

Root parameters

No. of tillers

Plant height (cm)

Shoot Fresh weight (g plant−1)

Shoot Dry weight (g plant−1)

Root Fresh weight (g plant−1)

Root Dry weight (g plant−1)

17.00 ± 1.19 ab 18.88 ± 1.01ab 15.38 ± 0.80 bc 21.38 ± 0.47 a 14.63 ± 0.67 bc 13.13 ± 1.44 de 16.73 ± 0.77 9.50 ± 0.64 f 10.25 ± 0.72 ef 8.25 ± 1.87 f 9.50 ± 0.45 f 8.63 ± 0.48 f 7.13 ± 0.48 f 8.88 ± 0.93

56.13 ± 0.66 a 56.63 ± 0.72 a 52.38 ± 2.17 ab 57.00 ± 1.51 a 47.63 ± 2.77 b 50.25 ± 2.46 b 52.93 ± 1.87 38.25 ± 2.15 c 37.13 ± 0.94 cd 35.25 ± 1.51 cd 39.63 ± 2.25 c 35.50 ± 1.26 cd 32.13 ± 1.55 d 36.31 ± 1.61

73.46 ± 4.51 b 51.09 ± 3.89 c 47.37 ± 5.55 c 88.02 ± 8.04 a 49.32 ± 2.87 c 46.86 ± 4.18 c 59.35 ± 4.84 21.90 ± 1.59 d 18.76 ± 1.84 d 16.67 ± 1.88 d 21.16 ± 1.11 d 17.70 ± 1.13 d 15.47 ± 1.59 d 18.61 ± 1.52

30.37 ± 2.47 b 21.55 ± 3.96 c 17.54 ± 5.27 c 41.46 ± 4.32 a 16.74 ± 1.27 c 19.36 ± 1.50 c 24.50 ± 3.13 8.97 ± 0.80 d 7.31 ± 0.59 d 6.40 ± 0.46 d 8.14 ± 0.36 d 7.16 ± 0.53 d 6.33 ± 1.04 d 7.38 ± 0.63

32.88 ± 2.64 b 32.31 ± 5.62 b 24.17 ± 3.50 c 51.17 ± 3.73 a 35.37 ± 8.75 b 31.47 ± 7.51 b 34.56 ± 5.29 14.24 ± 0.97 d 12.61 ± 0.99 d 8.28 ± 1.71 d 14.85 ± 1.72 d 10.80 ± 1.33 d 8.49 ± 1.27 d 11.54 ± 1.33

5.35 ± 0.63 b 5.16 ± 0.58ab 4.24 ± 0.45 ab 8.09 ± 0.69 a 4.97 ± 0.87 ab 5.35 ± 1.40 b 5.52 ± 0.77 1.94 ± 0.09 c 1.87 ± 0.14 c 1.53 ± 0.39 c 2.16 ± 0.04 c 1.87 ± 0.10 c 1.21 ± 0.16 c 1.76 ± 0.15

Anaerobic cultivation: (T1-T6) T1= Silicon 0 mM+ Arsenic 0 mM, T2 = Silicon 0 mM+ Arsenic 150 µM, T3= Silicon 0 mM+ Arsenic 300 µM, T4= Silicon 3 mM+ Arsenic 0 mM, T5= Silicon 3 mM+ Arsenic 150 µM and T6= Silicon 3 mM+ Arsenic 300 µM Aerobic cultivation: (T7-T12) T7= Silicon 0 mM+ Arsenic 0 µM, T8= Silicon 0 mM+ Arsenic 150 µM, T9= Silicon 0 mM+ Arsenic 300 µM, T10= Silicon 3 mM+ Arsenic 0 µM, T11= Silicon 3 mM+ Arsenic 150 µM and T12= Silicon 3 mM+ Arsenic 300 µM

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Table 4 Effect of silicon and water management on phosphorus, silicon and arsenic concentrations in shoots and roots of the rice plant. Rice Varity KSK-133 was grown for 3 months under aerobic and anaerobic conditions in pots filled with soil and treated with different concentrations of As and Si. The completely randomized design was used for analysis of variance and mean values were compared using least significant difference at 5% level of probability. Bars with in a single plot sharing same letter did not differ significantly from each other. Values are the means of 4 replicates ± SE. Treatment Code

T1 T2 T3 T4 T5 T6 Mean T7 T8 T9 T10 T11 T12 Mean

Interaction (WM × Si × As)

Anaerobic-Si0As0 Si0As150 Si0As300 Si3As0 Si3As150 Si3As300 Aerobic-Si0As0 Si0As150 Si0As300 Si3As0 Si3As150 Si3As300

P Conc. (µg g plant DW−1)

Si Conc. (mg g plantDW−1)

As Conc. (µg g plant DW−1)

Shoot

Root

Shoot

Root

Shoot

Root

2.53 ± 0.04 cd 2.22 ± 0.06 ef 1.95 ± 0.06 fh 3.25 ± 0.17 a 2.61 ± 0.10 c 1.92 ± 0.11 fh 2.41 ± 0.09 2.12 ± 0.04 eg 1.85 ± 0.05 gi 1.69 ± 0.03 hi 2.91 ± 0.05 b 2.30 ± 0.10 de 1.60 ± 0.17 i 2.08 ± 0.07

1.55 ± 0.07 cd 1.94 ± 0.10 c 2.20 ± 0.07 b 1.48 ± 0.08 de 2.37 ± 0.12 ab 2.03 ± 0.10 b 1.90 ± 0.09 1.28 ± 0.07 e 1.00 ± 0.05 f 1.05 ± 0.06 f 2.44 ± 0.05 d 1.43 ± 0.07 de 1.32 ± 0.04 e 1.42 ± 0.06

20.84 ± 0.65 d 12.85 ± 0.44 f 12.07 ± 0.42 f 35.55 ± 0.97 a 28.66 ± 0.64 b 25.04 ± 0.60 c 22.50 ± 0.65 20.54 ± 0.60 d 17.55 ± 0.67 e 16.04 ± 0.36 e 30.74 ± 0.78 b 25.64 ± 1.46 c 24.23 ± 0.52 c 22.46 ± 0.73

15.46 ± 0.51 e 10.79 ± 0.61 gh 9.11 ± 0.41 h 29.04 ± 0.62 d 25.97 ± 0.57 b 22.17 ± 0.81 cd 18.76 ± 0.59 13.98 ± 0.56 ef 12.92 ± 1.10 fg 11.24 ± 0.77 gh 22.17 ± 1.54 cd 22.55 ± 0.48 c 19.90 ± 0.85 d 17.13 ± 0.88

0.19 ± 0.14 g 6.37 ± 0.40 c 9.34 ± 0.55 a 0.24 ± 0.11 g 5.11 ± 0.24 de 6.87 ± 0.46 b 4.69 ± 0.32 0.34 ± 0.14 g 3.91 ± 0.39 e 6.06 ± 0.52 cd 0.32 ± 0.16 g 2.50 ± 0.26 f 4.90 ± 0.44 de 3.00 ± 0.32

1.50 ± 0.28 h 18.60 ± 0.87 c 27.70 ± 1.06 a 1.33 ± 0.28 h 13.08 ± 0.88 e 22.20 ± 1.05 b 14.07 ± 0.77 1.08 ± 0.39 h 10.25 ± 0.91 f 16.00 ± 0.63 d 1.15 ± 0.14 h 5.78 ± 0.46 g 9.28 ± 0.51 f 7.26 ± 0.51

Anaerobic cultivation: (T1-T6) T1= Silicon 0 mM+ Arsenic 0 mM, T2 = Silicon 0 mM+ Arsenic 150 µM, T3= Silicon 0 mM+ Arsenic 300 µM, T4= Silicon 3 mM+ Arsenic 0 mM, T5= Silicon 3 mM+ Arsenic 150 µM and T6= Silicon 3 mM+ Arsenic 300 µM. Aerobic cultivation: (T7-T12) T7= Silicon 0 mM+ Arsenic 0 µM, T8= Silicon 0 mM+ Arsenic 150 µM, T9= Silicon 0 mM+ Arsenic 300 µM, T10= Silicon 3 mM+ Arsenic 0 µM, T11= Silicon 3 mM+ Arsenic 150 µM and T12= Silicon 3 mM+ Arsenic 300 µM.

concentration of As in root medium in both cultivated conditions. Maximum concentration was measured for T3 (Silicon 0 mM+ Arsenic 300 µM; anaerobic conditions), that is about 90% higher than the control treatment and minimum concentration was recorded for control. Silicon supplementation (3 mM Si) significantly decreased As in roots at both As concentration (150 µM, 300 µM As) as compared to 0 mM Si under both cultivated conditions. The comparison of two the cultivation methods showed that As concentration was relatively lower under aerobic conditions than anaerobic conditions.

in phosphorus concentration was observed with the increasing concentration of As in the growth medium. It was also observed that the concentration of phosphorus was affected by increasing the concentration of As from 150 µM to 300 µM in the growth medium in the absence of Si, but there was a clear decrease in phosphorus concentration with increasing concentration of As when it is supplemented with 3 mM Si (Table 4). Results indicated that plant supplied with exogenous Si had higher concentration of Si in shoot tissues. Maximum shoot Si concentration (35.55 mg g−1 DW) was observed when plants were grown with Si supplementation under no As-stress. A gradual decreasing trend in shoot Si concentration was observed with the increasing concentration of As in the root medium. While, under aerobic conditions exogenous Si supply had no-significant effect on Si concentration with increasing concentration of As. Data for Si concentration in root showed that plants grown with additional Si supply has higher amount of Si in comparison to no-Sireplete plants. Under anaerobic conditions, Si concentration decreased by the increasing concentration of As. Among treatments, maximum concentration of Si was measured for Si-treated plants (29.04 mg g−1 DW) and minimum was measured for Silicon 0 mM +Arsenic 300 µM (9.11 mg g−1 DW). Under aerobic conditions, there is a slight decrease in root Si concentration with the increasing concentration of As. Maximum concentration was measured for Si supplied plant under no stress conditions (22.55 mg g−1 DW) and minimum for As stressed (11.24 mg g−1 DW). Silicon supplementation (3 Mm Si) increased (36–43%) the concentration of Si in roots as compared to 0 mM Si. In the absence of Si supplementation, maximum concentration of Si was measured for control (13.98 mg g−1 DW), and minimum was observed for As300 µM treated plants. Arsenic concentration increased in shoots with increasing concentration of As in root medium. Increase in concentration was significant in all treatments under both aerobic and anaerobic conditions. Maximum concentration was recorded for T3 under anaerobic conditions, which is (9.34 µg g−1 DW) about 90% greater than control (0.19 µg g−1 DW). Silicon supplementation significantly decreased the As concentration under anaerobic conditions, while a significant decrease was also measured (for 150 µM As) under aerobic conditions. In rice roots, an increasing trend of As was recorded with increasing

4. Discussion 4.1. Effect of silicon supplementation on germination of rice seeds subjected to arsenic stress During a preliminary experiment, rice seeds were exposed to different concentrations of As and Si to determine their effects on germination of seeds. Germination of seeds proved to be very sensitive to As contamination (Liu et al., 2005). Decreased germination rate in response to As exposure in rice has been reported by Shri et al. (2009). These effects might be due to of As interaction with the enzymes that are responsible for starch metabolism hence decreasing the germination of rice seeds. In plants, α-amylase, β-amylase and starch phosphorylase are major starch hydrolyzing enzymes (Yang et al., 2001). Energy for germination of seeds and for growth of roots and shoots is provided by sugars metabolism and for this purpose α-amylase converts endospermic stored starch into metabolizable sugars (Kaneko et al., 2002). Similarly a significant reduction in seed germination has been reported in wheat due to decreased amylolytic activities of the αamylase, β-amylase under As exposure (Jha and Dubey, 2005; Liu et al., 2005). In addition, Sharma (2012) concluded that poor seed germination due to As was attributed to poor cell wall metabolism and hormonal signaling. While, an improvement in seedlings length was observed when Si was supplemented as compared to As alone at both low and high concentrations. Although the effects were minor but these might be attributed due to improvement in defense mechanism and antioxidant systems of seedlings by Si supplementation (Tripathi et al., 2013). 16

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rhizosphere from oxidized (aerobic) to reduced (anaerobic) conditions markedly decrease the As contents in various parts of rice plant (Talukder, 2012). On the other hand, AsV is a dominant form of As under aerobic conditions, its less availability to plants due to its negative charge and its binding with other minerals explains the low concentration of As in rice roots and shoots (Meharg, 2004). Silicon significantly decreased the As concentration in both cultivated conditions. Silicon and AsIII (a dominant form of As under anaerobic conditions) compete for the uptake site at root surface. Transporters, Nodulin 26 Intrinsic Proteins (NIPs) are responsible for AsIII uptake in rice roots. As uptake via LSi1 and its efflux via LSi2 towards xylem and shoots is affected by increase in Si concentration (Ma et al., 2008). In addition, Si nutrition increases the oxidation power of rice roots, hence creation favorable conditions for As immobilization through AsIII conversation into AsV (Fleck et al., 2011). Arsenic presence in root medium significantly reduced the concentration of Si in rice roots and shoots under anaerobic conditions and non-significantly under aerobic conditions. The decreased Si concentration in plant tissues may be due to antagonistic effect of AsIII under anaerobic conditions, while the non-significant decrease in roots As concentration may be due to the presence of AsV in rhizosphere which did not have any effect on Si transportation. Another possible reason of the decreased Si concentration under aerobic conditions is the less availability of Si due to less amount of water as the Si is taken up in rice roots by aquaporin channels (LSi1) (Ma et al., 2008). In shoots, concentration of Si decreased with increasing concentration of As in both aerobic and anaerobic conditions. The LSi2 translocate the Si from roots to shoots, which is also an efflux transporter for AsIII. In both cultivated conditions AsIII affected the Si translocation and this decrease may be due to the effect of AsIII on Si translocation from roots to shoots. Majority of the As which is taken up by plant is reduced into AsIII as Xu et al. (2008) reported that AsIII accounts for 92–99% of the As in roots of rice and tomato. Most of the plants have high capacity to reduce AsV into AsIII through arsenic reductase using glutathione as reductant (Dixit et al., 2016). Arsenite have high affinity for glutathione and phytochelatins which is an important mechanism for As detoxification. So, a similar antagonistic effect may affect the Si translocation in shoots under aerobic conditions as well. A continuous increase in concentration of phosphorus was observed in rice roots under anaerobic condition with increasing concentration of As. The increased phosphorus concentration in rice roots may be due to an increased concentration of As in root medium that may have increased the competition between As and phosphorus for sorption sites in soil. So it could be speculated that the increase in As concentration may cause the release of phosphorus and increase its bioavailability to roots. Another possible reason of an increase in phosphorus concentration in rice roots with increasing concentration of As is its speciation under two different water regimes. Under anaerobic conditions, AsIII has no antagonistic effect on phosphorus concentration. Phosphorus concentration increased with increasing concentration of As demonstrated that its uptake was not restricted due to the presence of AsIII. While under aerobic conditions, concentration of phosphorus is decreased in rice roots. Arsenate is the analog of phosphorus as both AsV and phosphate use same uptake transporter in rice plants. Arsenate use phosphate transport system to pass through plasma membrane (Dixon, 1997). So, a decrease in phosphorus concentration may be due to increasing competition between AsV and phosphate for transportation in rice roots (Geng et al., 2005). Increase in phosphate nutrition cause the suppression of high affinity arsenate/phosphate transport system (Dixon, 1997). Similarly, phosphate concentration can decrease the increasing concentration of As. Aerobic conditions also promotes the oxidation of substances like iron, manganese and hydrogen sulfide around rhizosphere. Additionally, aerobic condition around rhizosphere promotes the iron oxide growth that forms a plaque around the roots (Reddy and DeLaune, 2008). This plaque formation under aerobic condition may be a possible cause of phosphorus decrease in roots with

4.2. Effect of different cultivated conditions (aerobic and anaerobic) on plant physical and chemical attributes Plant physiological parameters i.e plant height and biomass reduction with increasing concentration of As in both cultivation systems indicates As toxicity in rice plants as reported in many other studies (Islam, 1999; Geng et al., 2005). Among the cultivation systems, anaerobic rice cultivation produced higher biomass and the possible reason of this increased growth is the availability of ample amount of water as rice is water loving plant. However, the effects of As stress were more prominent under anaerobic conditions than aerobic conditions. The possible reason of the effects of As toxicity under anaerobic conditions may be due to higher availability of As to rice plants under anaerobic conditions as AsIII is a dominant As species under reduced conditions (Talukder et al., 2012). Moreover. AsIII is more mobile in the environment over wide range of pH. Alternatively, the decreased As toxicity under aerobic condition might be due to conditions favoring the binding of AsV with mineral oxides and organic materials and less mobility in the soil. Arsenate has a strong affinity for soil minerals like Fe-hydroxides which leads to decreased As solubility and hence bioavailability to plants (Takahashi et al., 2004; Xu et al., 2008). Arsenate becomes less liable then AsIII, as it is negatively charged over most of the pH ranges so it sorbs on aluminum and iron oxide (WHO, Environmental Health Criteria, 2001). Therefore, under aerobic conditions As availability has been decreased and ultimately uptake also decreased (Fig. 3). Rice plants responded to the exogenous application of Si as its concentration in roots and shoots increased with higher level of Si in root medium. Beneficial effects of Si application on rice growth (Fig. 1) are in line with the findings of other studies as reported by Alvarez and Datnoff (2001). Increase in plant growth may be attributed to the changes in physiological and morphological characteristics which are facilitated by the presence of Si. Silicon mediated in morphological and biochemical changes has been suggested as a possible reason of increased plant growth (Epstein, 1999). Kaufman et al. (1979) proposed a “Window-hypothesis” by which Si deposition acts as a window in leaf epidermal cells and enhances the light transmission to mesophyll cells which takes part in photosynthesis. In this way the exogenous Si application can facilitate the CO2 fixation in rice plants (Ma et al., 2002). Silicon functioning as a balancing element for other minerals nutrients may also be the possible reason of its beneficial effects on plants under no-stress conditions (Marschner et al., 1990). Comparison of two cultivated conditions showed that the uptake of As is relatively less under aerobic condition than anaerobic condition in rice roots and shoots. It is evident from many studies that AsIII, the dominant species under anaerobic conditions, is the major form taken up by the plants as neutral form (As (OH)3) through Si influx transporters (Xu et al., 2008; Su et al., 2009). Therefore, changing

Fig. 3. Specific arsenic uptake in rice plants variety 133-KSK. Rice Variety KSK-133 was grown for 3 months in pots filled with soil amended with different concentrations of As and Si cultivated under anaerobic and aerobic conditions. The completely randomized design was used for analysis of experimental data and mean values were compared using least significant difference at p 5%. Bars showing different letter differ significantly from each other. Values are the means of four replicates ± SE.

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Aquat. Bot. 83, 321–331. Islam, M.S., 1999. Arsenic Toxicity Remediation in Rice Plants (MSc. Thesis). Department of Soil Sciences, University of Dhaka, Bangladesh, pp. 3–120. Jha, A.B., Dubey, R.S., 2005. Effect of arsenic on behavior of enzymes of sugar metabolism in germinating rice seeds. Acta Physiol. Plant. 27, 341–347. Kaneko, M., Itoh, H., Ueguchi, -Tanaka, M., Ashikari, M., Matsuoka, M., 2002. The αamylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium. Plant Physiol. 128, 1264–1270. Kaufman, P.B., Takeoka, Y., Carlson, T.J., Bigelow, W.C., Jones, J.D., Moore, P.H., Ghosheh, N.S., 1979. Studies on silica deposition in sugarcane (Saccharum spp.) using scanning electron microscopy, energy-dispersive X-ray analysis, neutron activation analysis, and light microscopy. Phytomorphology 29 (2), 185–193. Kim, Y.H., Khan, A.L., Hamayun, M., Kang, S.M., Beom, Y.J., Lee, I.J., 2011. 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increasing concentration of As. Phosphorus and As have high affinities for these Fe-oxides, so and immobilization of these minerals by Fe(III)oxides may also occurred (Meharg, 2004). Silicon supplementation increased the concentration of phosphorus in roots and shoots under no As stress. Silicon is taken up by the plants as non-essential elements unlike phosphorus, potassium and nitrogen. However, Si helps to improve the plant growth and mitigates environmental stresses (Epstein, 1999; Kim et al., 2011). Increase in Si concentration in root medium may increase the uptake of phosphorus in rice roots. It may be due to desorption of phosphorus from adsorption sites within the soils. 5. Conclusion It is concluded that the aerobic conditions are more favorable to decrease arsenic uptake in rice. Aerobic conditions decrease arsenic stress on rice growth as compared to anaerobic conditions. Growth attributes were negatively affected when there was less availability of water. Silicon supplementation proved beneficial for growth attributes in the absence of arsenic stress, but under arsenic stress, there was no significant effect of silicon on plant growth attributes. However, silicon supplementation significantly decrease the arsenic uptake especially in plants grown under aerobic conditions. Arsenic stress decreased phosphorus uptake in the rice plant while silicon application partially reduced the effect of arsenic on phosphorus in rice shoot. References Alvarez, J., Datnoff, L.E., 2001. The economic potential of silicon for integrated management and sustainable rice production. Crop Prot. 20, 43–48. Anil, K., Yakadri, M., Jayasree, G., 2014. Influence of nitrogen levels and times of application on growth parameters of aerobic rice. Int. J. Plant, Anim. Environ. Sci. 4, 231–234. Bakhat, H.F., Zia, Z., Fahad, S., Abbas, S., Hammad, H.M., Shahzad, A.N., Abbas, F., Alharby, H., Shahid, M., 2017. Arsenic uptake, accumulation and toxicity in rice plants: possible remedies for its detoxification: a review. Environ. Sci. Pollut. Res. http://dx.doi.org/10.1007/s11356-017-8462-2. Bhattacharya, P., Samal, A.C., Majumdar, J., Santra, S.C., 2009. Transfer of arsenic from groundwater and paddy soil to rice plant (Oryza sativa L.): a micro level study in West Bengal, India. World J. Agric. Sci. 5, 425–431. Chapman, H.D., Pratt, F.P., 1961. Ammonium vandate-molybdate method for determination of phosphorus. Methods Anal. Soils, Plants Water 1, 184–203. Chen, Y., Han, Y.H., Cao, Y., Zhu, Y.G., Rathinasabapathi, B., Ma, L.Q., 2017. Arsenic Transport in Rice and Biological Solutions to Reduce Arsenic Risk from Rice. Front. Plant Sci. 8, 268. http://dx.doi.org/10.3389/fpls.2017.00268. Dixit, G., Singh, A.P., Kumar, A., Mishra, S., Dwivedi, S., Kumar, S., 2016. Reduced arsenic accumulation in rice (Oryza sativa L.) shoot involves sulfur mediated improved thiol metabolism, antioxidant system and altered arsenic transporters. Plant Physiol. Biochem. 99, 86–96. Dixon, H.B.F., 1997. The biochemical action of arsenic acids especially as phosphate analogues. Adv. Inorg. Chem. 44, 191–227. Duxbury, J.M., Panaullah, G., 2007. Remediation of Arsenic for Agriculture Sustainability, Food Security and Health in Bangladesh; FAO Water Working Paper. FAO, Rome, pp. 28. Elliott, C.L., Snyder, G.H., 1991. Autoclave-induced digestion for the colorimetric determination of silicon in rice straw. J. Agric. Food Chem. 39, 1118–1119. Epstein, E., 1999. Silicon Annual Review of Plant Physiology and Plant. J. Mol. Biol. 50, 64–664. Fleck, A.T., Nye, T., Repenning, C., Stahl, F., Zahn, M., Schenk, M.K., 2011. Silicon enhances suberization and lignification in roots of rice (Oryza sativa L). J. Exp. Bot. 62, 2001–2011. Geng, C.N., Zhu, Y.G., Liu, W.J., Smith, S.E., 2005. Arsenic uptake and translocation in seedlings of two genotypes of rice is affected by external phosphate concentrations.

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