Pot experiment to investigate the As accumulation in rice
To investigate the As accumulation in different parts of rice, a pot experiment was conducted at University of Chittagong campus using a popular rice variety BR-28 and four treatments such as 0.0, 25.0, 50.0 and 75.0 mg/L As containing irrigation water. The experimental site had subtropical and humid climate with adequate sunshine. From the seedbed, seedlings of 35 days old were uprooted carefully in the morning and on the same day, five seedlings were transplanted on each plastic pot (having no leakage) with three replications. The seedlings which died within first week of transplantation were discarded and replaced with new seedlings. Bio-fertilizers were applied in appropriate amount to provide the necessary nutrients. Throughout the growth period, 3–4 cm water above soil level was maintained in each treatment and the irrigation was continued before 10 days of harvest. At the maturity stage, the full-grown rice plants were carefully uprooted and the rice grains were harvested. Thereafter, the collected root, straw, husk and grain samples were washed thoroughly with As-free tape water followed by several rinsing with de-ionized water to remove soil and other contaminants. After drying the washed samples in the hot air oven at 60 °C for 72 h, the samples were stored at room temperature in airtight polyethylene bags having proper labeling. Finally, the samples were digested separately according to heating block digestion procedure [14] and As concentrations were measured by Flow Injection Hydride Generation Atomic Absorption Spectrophotometer, FI-HG-AAS (iCE 3300 AA system, Thermoscientific, China) at BCSIR Laboratory, Chittagong.
Animals and treatment
Twenty female Wistar albino rats, weighing between 160 and 170 g were collected from animal house of Jahangirnagar University, Dhaka and were allowed free access to laboratory rodent diet and water ad-libitum throughout the experimental period. Our institutional and national guidelines were followed for the care and use of laboratory animals throughout the experiment. For the experimental treatment, the animals were randomly divided into four groups (I, II, III and IV) containing five rats in each group. The control group (Group-I) was fed with normal laboratory pellets while groups II, III, and IV received normal laboratory pellets supplemented with arsenic contaminated rice, C. olitorius leaf powder (4 %) and arsenic contaminated rice plus C. olitorius leaf powder (4 %) respectively, for a period of 150 days.
Preparation of C. olitorius leaf powder
Fresh young C. olitorius leaves were collected from local market and later identified by Dr. Sheikh Bokhtear Uddin, a taxonomist (Department of Botany, University of Chittagong, Bangladesh). The leaves were washed thoroughly with distilled water, sun-dried and then powdered by grinding. After that, C. olitorius leaf powder (4 % wt/wt) was mixed with respective pellet diet of rat as aforementioned and used throughout the experiment.
Preparation of As contaminated rice powder
A popular rice variety of Bangladesh, BR-28, was collected from the local market and tested for background As concentration by FI-HG-AAS according to Rahman et al. [14]. No As was detected in that rice sample. After that, the rice was soaked in 200 mg/L sodium arsenite solution for 36 h and again tested for As concentration, and the amount of As accumulated in rice grain was found 46.33 ± 0.01 mg/kg. The As contaminated rice was dried, blended and mixed with the respective pellet diet and used throughout the experiment.
Collection of blood and separation of serum
On 150th day, rats were fasted overnight and sacrificed next morning by light ether anesthesia. Blood was collected through cardiac puncture. For each rat, an aliquot of blood samples was taken in a heparinated tube for haematological examination while the remaining blood sample was collected in another test tube and allowed to clot formation at room temperature for 20 min. Then, the tubes were centrifuged at 3000 r.p.m for 10 min. After centrifugation, serum samples were pipetted out & collected into pre-labeled wintrobe tubes. From collected blood and serum samples, haematological and biochemical analyses were carried out.
Collection and preservation of different organs
After opening chest and abdomen of the rats, the liver, heart, spleen and both kidneys were carefully removed, washed in normal saline water and then immersed separately into pre-labeled 10 % formalin containing specimen container for histopathological investigations. Some portions of liver, heart, spleen and kidneys were preserved at -20 °C for detection of As.
Haemato-biochemical assay
Using Auto-Haematology Analyzer (Beckmann, USA), different haematological indices such as total white blood cell (WBC) count, total red blood cell (RBC) count, haemoglobin (Hb) concentration and platelet count were estimated. In addition, different serum indices such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, urea, total protein, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were measured by using the kits from Human Gmb H (Germany) and the analyzer (CHEM-5V3, Erba, Mannheim, Germany). Concentration of low density lipoprotein cholesterol (LDL-C) in serum was also calculated. To calculate the mean values, all the samples were analyzed in triplicate.
Determination of As in different tissues
The concentration of As in different organs (liver, kidney, spleen, heart) was determined using FI-HG-AAS method [15]. From each organ, 0.25 g sample was weighed and taken in beaker. The samples were digested with a mixture of HClO4-HNO3 solution (ratio 1:3 v/v) at 130 °C. After removal of HNO3 by evaporation, the digested samples were diluted with deionized water up to 100 ml. The concentrations of As in digested samples were measured at 193.7 nm wave length and 10 mA current using Atomic Absorption Spectrophotometer equipped with As lamp. Vapour generation accessory (VGA) was used to produce hydride vapours using 0.6 % sodium borohydride and 10 Mm HCl.
Histopathological study
At first, gross section of liver, kidney, spleen and heart tissues (preserved in 10 % formalin containing specimen container) were taken. Then the tissues were cut in longitudinal and transverse pieces, passed through ascending series of ethanol baths, cleared in toluene and embedded in paraffin. Tissues were sectioned at 5 μm and stained with Haematoxylin and Eosin (H&E). Stained sections were then mounted on glass slides with DPX and covered with a cover slip. Finally, histopathological changes were examined by light microscope and photographed using a digital camera.
Statistical analysis
Statistical analysis was performed with SPSS for Windows V.22. All data were analyzed by using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMART) with a p-value < 0.05 considered to be statistically significant. All the values are expressed as mean ± SEM.
