**2.3 Chemical analysis**

Home-yard ponds and HTW waters were collected in iodized bottles and stored in ice box immediately and then in the laboratory deep freeze. Water quality of the pond and HTW waters is given in **Figure 2**. Whole plants of *Azolla* and *Spirodela*, roots, shoots, and grain samples of rice plant were collected separately and dried at 70°C for 3 days, grounded, and kept in poly-bags till analysis. Arsenic was determined by atomic absorption spectrometer (Shimadzu AA-680/G V-3) after digestion with nitric and perchloric acids at 5:1 ratio in a closed system. Arsenic concentration in brown-rice grains was cross-checked by neutron activation analysis

**Figure 2.**

*Water quality of pond (control) and STW (installed 3 years ago) used for growing BR-28 rice in fields. Unit for tAs is μg/l and for others as mg/l. After [43]. n = 5; vertical bars are standard deviations.*

**131**

350 m<sup>2</sup>

*Protecting Rice Grains from Arsenic Toxicity through Cultural Management...*

(NAA) using 3 MW Research Reactor at Atomic Energy Research Establishment, Saver, Dhaka. PO4-P in the culture medium was determined following [37].

a ratio of 1:3 in volumetric flasks with reflex condenser under a closed system.

phosphate, and muriate of potash in a solution at the rate of 40, 20, and 10 kg/ha/ day, respectively [29]. The fertilizer solution was mixed with the pond water every day just after 10% harvest before noon time. The production was 1 ton/ha/day and

Mini-scale bioremediation was carried out in 50 L capacity RCC (cemented) tubs lined with polythene [29]. The tubs were set in the open area of a house having sufficient natural light. Arsenic-contaminated HTW (installed 25 years ago) having 475.5 ± 10.6 μg/l and 6.30 mg/l iron was used. Fresh DW from the cultivation pond was spread onto the tub water to form a complete cover. The amount per tub added was weighed, and the same amount was added after every 24 hours for 6 days. 100 ml surface water, 20 g fresh DW, and sediment from each tub bottom were

Large-scale bioremediation experiment was carried out in about 1 meter deep,

 pond situated near to the experimental rice field [29]. It was filled with arsenic (495 ± 10 μg As/l was considered as 500 μg As/l)-contaminated STW water which appeared reddish brown in color. The STW water had 8.85 mg/l iron. About 350 kg DW was spread over the stored STW water in the pond and divided into eight blocks by bamboos for keeping the plants equally spread for As absorption. After every 2 days, 1.0 kg fresh DW, 100 ml water, and 50 g sediment were collected from each block for chemical analysis and for using as feed for broiler and Bengal goat. All the floating DW was then harvested (kept in a shallow pit for decomposition and used in biogas production). Fresh DW was again spread completely

Effects of arsenic on the arsenic toxicity of winter-rice grain of variety BR28 were studied in a farmer's field having silt clay containing sand 4.31%, silt 45.56%, clay 50.13%, pH 6.35, EC 200 μS/cm, iron 0.87%, arsenic 3.21 mg/kg soil, and organic matter 1.85%, in Silmondi and Narailbag soil series at Meghna floodplain [29]. Research plots were 19 × 19 m in five replications arranged in complete randomized block design. NPK fertilizers from urea, TSP, and MP, respectively, were applied at 45, 15, and 40 kg/ha, respectively, to all plots at the time of land preparation other than N. The N was applied at 25 kg/ha 15 days after transplanting (DAT) and 20 kg/ha before flowering. "Boro" rice var. BR28 seedlings of 10 days old

**2.4 Cultivation of** *S. polyrhiza* **in a production pond**

water.

**2.5 Bioremediation of arsenic-contaminated HTW water**

The DW was cultivated in a 400 m<sup>2</sup>

collected for analysis of As in the samples.

1 kg DW covers about 1 m<sup>2</sup>

covering the water surface.

**2.6 Cultivation of rice**

Soil samples were collected from a depth of 0–5 cm in five replications from each plot on the basis of composite sampling method in [38]. Determinations of various fractions were carried out as described: EC by EC meter in [39]; pH by a microprocessor pH-meter, soil to water ratio was 1:2.5; organic carbon by wet oxidation method; organic matter by multiplying the percentage of organic carbon with conventional Van Bemmelen's factor of 1.74 in [40]; textural classes and particle size distribution by hydrometer method as described in [41, 42], respectively; and As by AAS (Perkin Elmer M-3110, USA) following low temperature sample digestion with HNO3-HCl mixture at

pond adding urea nitrogen, triple super

*DOI: http://dx.doi.org/10.5772/intechopen.85909*

*Protecting Rice Grains from Arsenic Toxicity through Cultural Management... DOI: http://dx.doi.org/10.5772/intechopen.85909*

*Protecting Rice Grains in the Post-Genomic Era*

tions: continuous light flux of 120 μE/m2

**2.2 Determination of nitrogenase activity**

**2.3 Chemical analysis**

116 and *S. punctata* Dh 117, Lemnaceae) were considered to select efficient As absorbing floating plants [9] by growing them in the growth room under controlled condi-

relative humidity at 30 ± 1°C. Liquid inorganic nutrient medium Chu 10D-N [30], a modified version of [31], with double the strength of P (3.56 mg/l) and K (9.45 mg/l) for *Azolla* strains, and IRRI medium with NH4NO3 as nitrogen source following [32] for species of *Spirodela* were grown in conical flasks of 150 ml capacity containing 50 ml medium adding arsenic trioxide as inorganic As (iAs) source before autoclaving. *S. polyrhiza* and *S. punctata* were grown at much higher arsenic concentrations as the plant is often found to grow in polluted waters and stressed environment [33]. The plants were grown at pH 6.00 [34, 35]. All plant biomasses after 3 days growth were oven

Nitrogenase activity in the presence of arsenic was determined by acetylene reduction assay (ARA) technique of [36] and details of the experiment given in [9].

Home-yard ponds and HTW waters were collected in iodized bottles and stored in ice box immediately and then in the laboratory deep freeze. Water quality of the pond and HTW waters is given in **Figure 2**. Whole plants of *Azolla* and *Spirodela*, roots, shoots, and grain samples of rice plant were collected separately and dried at 70°C for 3 days, grounded, and kept in poly-bags till analysis. Arsenic was determined by atomic absorption spectrometer (Shimadzu AA-680/G V-3) after digestion with nitric and perchloric acids at 5:1 ratio in a closed system. Arsenic concentration in brown-rice grains was cross-checked by neutron activation analysis

*Water quality of pond (control) and STW (installed 3 years ago) used for growing BR-28 rice in fields. Unit for* 

*tAs is μg/l and for others as mg/l. After [43]. n = 5; vertical bars are standard deviations.*

dried at 90°C for 24 hour for determining growth and As absorbed [9].

/s from daylight fluorescent tubes and 70–90%

**130**

**Figure 2.**

(NAA) using 3 MW Research Reactor at Atomic Energy Research Establishment, Saver, Dhaka. PO4-P in the culture medium was determined following [37].

Soil samples were collected from a depth of 0–5 cm in five replications from each plot on the basis of composite sampling method in [38]. Determinations of various fractions were carried out as described: EC by EC meter in [39]; pH by a microprocessor pH-meter, soil to water ratio was 1:2.5; organic carbon by wet oxidation method; organic matter by multiplying the percentage of organic carbon with conventional Van Bemmelen's factor of 1.74 in [40]; textural classes and particle size distribution by hydrometer method as described in [41, 42], respectively; and As by AAS (Perkin Elmer M-3110, USA) following low temperature sample digestion with HNO3-HCl mixture at a ratio of 1:3 in volumetric flasks with reflex condenser under a closed system.

### **2.4 Cultivation of** *S. polyrhiza* **in a production pond**

The DW was cultivated in a 400 m<sup>2</sup> pond adding urea nitrogen, triple super phosphate, and muriate of potash in a solution at the rate of 40, 20, and 10 kg/ha/ day, respectively [29]. The fertilizer solution was mixed with the pond water every day just after 10% harvest before noon time. The production was 1 ton/ha/day and 1 kg DW covers about 1 m<sup>2</sup> water.

### **2.5 Bioremediation of arsenic-contaminated HTW water**

Mini-scale bioremediation was carried out in 50 L capacity RCC (cemented) tubs lined with polythene [29]. The tubs were set in the open area of a house having sufficient natural light. Arsenic-contaminated HTW (installed 25 years ago) having 475.5 ± 10.6 μg/l and 6.30 mg/l iron was used. Fresh DW from the cultivation pond was spread onto the tub water to form a complete cover. The amount per tub added was weighed, and the same amount was added after every 24 hours for 6 days. 100 ml surface water, 20 g fresh DW, and sediment from each tub bottom were collected for analysis of As in the samples.

Large-scale bioremediation experiment was carried out in about 1 meter deep, 350 m<sup>2</sup> pond situated near to the experimental rice field [29]. It was filled with arsenic (495 ± 10 μg As/l was considered as 500 μg As/l)-contaminated STW water which appeared reddish brown in color. The STW water had 8.85 mg/l iron. About 350 kg DW was spread over the stored STW water in the pond and divided into eight blocks by bamboos for keeping the plants equally spread for As absorption. After every 2 days, 1.0 kg fresh DW, 100 ml water, and 50 g sediment were collected from each block for chemical analysis and for using as feed for broiler and Bengal goat. All the floating DW was then harvested (kept in a shallow pit for decomposition and used in biogas production). Fresh DW was again spread completely covering the water surface.

### **2.6 Cultivation of rice**

Effects of arsenic on the arsenic toxicity of winter-rice grain of variety BR28 were studied in a farmer's field having silt clay containing sand 4.31%, silt 45.56%, clay 50.13%, pH 6.35, EC 200 μS/cm, iron 0.87%, arsenic 3.21 mg/kg soil, and organic matter 1.85%, in Silmondi and Narailbag soil series at Meghna floodplain [29]. Research plots were 19 × 19 m in five replications arranged in complete randomized block design. NPK fertilizers from urea, TSP, and MP, respectively, were applied at 45, 15, and 40 kg/ha, respectively, to all plots at the time of land preparation other than N. The N was applied at 25 kg/ha 15 days after transplanting (DAT) and 20 kg/ha before flowering. "Boro" rice var. BR28 seedlings of 10 days old were transplanted and irrigated once in a week with pond water as control and 500 µg As/l contaminated STW water that dries up by the end of that week and watered again for the next one week [29].
