Pot experiment was conducted in the micronutrient experimental field near shade house, Soil Science Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur Gazipur on 22 March, 2020. For potting soil, surface soil samples (0–20 cm) were collected from an abandoned plot in shade house, Soil Science Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur Gazipur. The collected soil samples were air dried, ground and sieved to pass through a 2mm sieve to remove debris and stones. Representative portion of the prepared soil sample was analysed for its physical and chemical parameters.
Microbes and biochar preparation:
Water hyacinth, barnyard grass, and fern were collected from the field and air-dried at room temperature. Instead, they are cut into small pieces. They are then placed in biochar making devices (made in the Division of Soil Science, BARI) and pyrolyzed under oxygen-limited conditions. The temperature of pyrolysis was elevated to 650°C at a rate of about 20°C per minute and kept constant for 1 h (Lehmann and Joseph, 2009; Park et al., 2011). The biochar was then allowed to cool down to room temperature and ground to pass a sieve of 0.25 mm. Rhizobium, Azotobacter and phosphate solubilizing bacterial (PSB) strains were collected from Soil Microbiology Laboratory which were previously cultured by YEMA, Jensens’s and Pikovskaya’s media, respectively. Peat based Rhizobium, Azotobacter and PSB bacterial inoculum were used containing 108 cells g-1 inoculum. Before sowing, seeds were mixed thoroughly with the peat based inoculum at the rate of 50 g inoculum kg-1 seed and 6 seed were sown per pot.
Treatment combination:
Apart from microbes, soil samples were mixed by adding biochar materials at a rate of 2 g kg-1 soil. The treatments for the microbes were performed as mentioned above. Seven treatments consisting of three types of microbes and three types of biochar along with a contaminated control. The treatments were: (i) Contaminated soil, no amendment i.e. control, (ii) contaminated soil + Amendment with Rhizobium sp, (iii) contaminated soil + amendment with Azotobacter sp, (iv) contaminated soil + amendment with phosphorus solubilizing bacteria, (v) contaminated soil + amendment with water hyacinth biochar, (vi) contaminated soil + amendment with barnyard grass biochar, (vii) contaminated soil + amendment with fern plant biochar.
Experimental setup:
The experiment consisted of a total of 21 pots, each pot of 28 cm deep and 27 cm diameter plastic pot, containing 10 kg air dried soil. All the pots were fertilized two days before sowing with N: 90 mg kg-1 soil, P: 75 mg kg-1 soil, K: 140 mg kg-1 soil, S: 30 mg kg-1 soil, Zn: 2 mg kg-1 soil, B: 1 mg kg-1 soil. Urea, triple supper phosphate (TSP), muriate of potash (MoP), gypsum, zinc sulphate monohydrate (ZnSO4, H2O) and boric acid were used as a source of N, K, S, Zn and B, respectively. Nitrogen was added in three splits, the first split during final land preparation and the remaining splits at 30 days and 50 days after sowing. Arsenic was added to the pots at the rate of 4 mg kg-1 soil. Arsenic was added as As2O3 in solutions and all fertilizers were added separately as solutions and thoroughly mixed with the soil.
Maize (Zea mays var. BARI hybrid Maize-7) seeds were sown directly in pots at a density of 6 seeds per pot. Twelve days after sowing the seedlings were thinned to 2 plants per pot. Water requirement was monitored visually. Intercultural operations such as weeding and irrigation were done whenever required. The crop was harvested at 62 days following seeding of maize, when it had attained reproductive maturity (before flowering). Soil was removed from the roots by careful and repeated washing in water. Shoots and roots were washed with tap water and then rinsed twice with deionized water.
Preparation and preservation:
The crops were harvested and separated into roots, stems and leaves. Then they were rinsed firstly with tap water followed by distilled water to eliminate dust, dirt, possible parasites or their eggs and then were again washed with deionized water. The clean samples were air-dried and placed in an electric oven at 65°C for 72–96 h depending on the sample size. The dries samples were homogenized by grinding using a ceramic coated grinder. The final samples were kept in labeled polypropylene containers at ambient temperature before analysis.
Digestion and determination of Arsenic:
The total plant As concentration was determined digesting the plant sample (1 g) with concentrate HNO3 of 10 ml and H2O2 having a ratio of 2:1. After cooling, the solution was made up to a final volume (50 ml) with distilled water in a volumetric flask. Reduction with KI and ascorbic acid in presence of NaBH4 and NaOH. Total As concentration of extract was determined by Hydride Generation Atomic Absorption Spectroscopy. Analysis of each sample was carried out three times to obtain representative results and the data are reported in µg kg-1 (on a dry matter basis).
Statistical analysis:
The experiment was planned with 7 treatments and three replications in complete randomized form (CRD) and the data of the study was presented as mean value and standard deviation. Significant differences were tested among treatments by one-way ANOVA and via Tukey HSD tests for multiple comparisons at a 5% significance level. All statistical analyses were carried out using STATISTIX-10. The data was graphed and calculate linear correlation coefficients using Microsoft Office Excel 2007.