Soil Sampling:
Polluted soil was collected for experiment from Kalakoir, Konabari, Gazipur. The side was irrigated by water from the Turag river which is highly contaminated with industrial effluents, sewage sludge, municipal waste water and urban runoff. Six composite topsoil samples (0-20 cm depth) from six farmers' fields were collected at random (10 individual samples per field). The collected soil was air-dried and passed through a 2 mm sieve to obtain homogeneous particle size. The physical and chemical properties of the initial soil which was used in pot are presented in Table 1.
Biochar preparation:
Water hyacinth, barnyard grass, and fern were collected from the field and air-dried at room temperature for one week. Instead of grinding, 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 limited oxygen 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.
Microbes preparation:
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, maize 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:
Soil samples were mixed by adding biochar materials at the rate of 2 g kg-1 soil, except microbes. The microbe treatments were carried out as stated in the above. There were seven treatments composing three types of microbes and three types of biochar along with a 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 plastic pots, each containing 10 kg soil. Pots were placed in a completely randomized design with three replications per treatment at a shade house of Soil Science Division, BARI, Joydebpur, Gazipur. Maize (Zea mays var. BARI hybrid Maize-7) seeds were sown directly in pots at a density of 6 seeds per pot on 22 March, 2020. Twelve days after sowing the seedlings were thinned to 2 plants per pot. 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 super phosphate, muriate of potash, gypsum, zinc sulphate monohydrate (ZnSO4. H2O) and boric acid were used as a source of N, P, K, S, Zn and B, respectively. Nitrogen was applied in two equal splits, the first split before sowing and the remaining splits at 6-8 leaf of plants after sowing. Wetting cycles (at field capacity) and air-drying every week were performed, during a period of about three months.
The plant was harvested at 62 days following from seeding, when it had attained reproductive maturity (before flowering). Soil was removed from the roots by careful and plants were washed with tap water followed by deionized water.
Preparation and preservation:
The clean plant samples were air-dried and placed in an electric oven, dried at 85 °C for 72 h, weighed for dry biomass. The dried plant samples were homogenized by grinding using a ceramic coated grinder and used for metal analysis. Samples of contaminated soils were spread on plastic trays and allowed to dry at ambient temperature for 8 days. The dried samples of soils were ground with a ceramic coated grinder and sieved through a nylon sieve. The final samples were kept in labeled polypropylene containers at ambient temperature before analysis.
Digestion and Analytical Procedure:
One gram of each sample was weighed into 50-ml beakers, followed by the addition of 10 ml mixture of analytical grade acids HNO3: HCIO4 in the ratio 5:1, and left overnight for complete contact of material. Next day, the digestion was performed at a temperature of about 190ºC for 1.5 h. After cooling, the samples were transferred into 100 ml volumetric flask and solution was made up to a final volume raised up to the mark with distilled water. The metal concentrations were determined by atomic absorption spectrometry using a VARIAN model AA2407 Atomic Absorption Spectrophotometer (AAS). Analysis of each sample was carried out three times to obtain representative results and the data reported in µg g-1 (on a dry matter basis).
Determination of Transfer Factor (TF):
The transfer coefficient was calculated by dividing the concentration (Mirecki et al., 2015). The transfer coefficient was calculated by dividing the concentration of heavy metals in vegetables by the total heavy metals concentration in the soil. TF = Cplant / Csoil
where, Cplant = metal concentration in plant tissue, mg kg-1 dry weight and Csoil = metal concentration in soil, mg kg-1 dry weight.
Statistical analysis:
The experiment was designed in completely randomized (CRD) with 7 treatments and three replications. Treatment effects were determined by analysis of variance with the help of statistical package STATISTIX-10 and mean separation was tested by Tukey HSD.