2.1.3. Plant Samples

Since there was no crop grown at the time of sampling in the agricultural field, the plant samples (leaves) of three wildly growing plant species viz. *Cannabis sativa* L. (Cannabaceae), *Chenopodium album* L. (Amaranthaceae), and *Ricinus communis* L. (Euphorbiaceae) on the boundaries of agricultural fields in the vicinity of industries were collected to explore their heavy metal bioaccumulation potential. The leaves were thoroughly washed using tap water followed by distilled water, oven-dried at 70 ◦C, grounded to a fine powder by pestle mortar, and stored in airtight polyethylene bags at 4 ◦C until further analysis.

#### *2.2. Physico-Chemical Characteristics of Industrial Effluents and Soil*

The soil extract (1:5 *w*/*v*) was prepared by adding 20 g of the collected soil sample in 100 mL of distilled water. This solution was kept in a mechanical shaker for 12 hours at room temperature and filtered through Whatman No. 1 filter paper [31]. The filtrate was termed soil extract and was used for further analysis of the physico-chemical parameters (pH, electrical conductivity, calcium, sodium, and magnesium). Total organic carbon of the soil was estimated using the dry combustion method [32]. A core measuring cylinder (100 ML) was used for bulk density (BD) estimation [33]. Soil texture was determined by the sieving and sedimentation method [34]. On the basis of size, different particles of soil were grouped as: sand: 0.5–2.00 mm; silt: 0.002–0.5 mm; clay: <0.002 mm. The analysis of the physico-chemical parameters (pH, temperature, total solids (TS), total dissolved solids (TDS), total suspended solids (TSS), total hardness, alkalinity, calcium, chloride, magnesium, sodium, and phosphate) of effluent samples was carried out following the standard protocols of the American Public Health Association [35,36]. The sodium content of both effluents and soil samples was measured using a Flame Photometer (Model-128; Make: Systronics). The pH of each effluent was measured using a pH meter (Model: μ pH system 361; Make: Systronics).

## *2.3. Heavy Metal Estimation*

Heavy metal contents in collected samples were determined using the flame atomic absorption spectrophotometer (AAS) (Agilent 240 FS AA model), at variable/recommended wavelength of 228.80 nm for cadmium, 240.70 nm for chromium, 357.90 nm for cobalt, 324.80 nm for copper, 217.0 nm for lead, and 213.90 nm for zinc. Limits of detection (μg/L) for different metals were cadmium (1.5), cobalt (3), chromium (5), copper (1.2), lead (7), and zinc (1.6). The airflow rate was maintained at 13.50 L/min for all heavy metal determinations. The acetylene flow rate was set at 2.00 L/min for Cd, Co, Cu, Pb, and Zn, and at 2.90 L/min for Cr estimations while the lamp currents were set at 4.00 mA, 7.00 mA, 7.00 mA, 4.00 mA, 10.00 mA, and 5.00 mA for determination of Cd, Co, Cr, Cu, Pb, and Zn, respectively. All the glassware was thoroughly washed and oven-dried before use. Double distilled water and analytical grade reagents were used during the whole experiment. The standard solutions (1000 mg/L) of Agilent made for different metals were used to prepare solutions of varying concentrations as 0.5, 1, 1.5 (mg/L) for cadmium and zinc; 5, 10, 15 (mg/L) for chromium, lead, nickel, and cobalt; and 1, 3, 5 (mg/L) for copper using the serial dilution method. The accuracy (>95%) of the instrument was maintained throughout

the experiment by thorough washing. For which, after every 10 sample readings, the standards were run to observe the accuracy of the instrument. Soil samples were digested using aqua regia, that is, a mixture of one part concentrated nitric (HNO3) and three parts hydrochloric acid (HCl) following the method described by the authors of [37] with minor modifications. For this purpose, 1 g of finely ground soil sample was digested slowly with aqua regia on a hot plate in a fume hood till white fumes appeared, indicating the complete digestion of the soil sample. Plant sample digestion was carried out using a tri-acid mixture, that is, five parts of nitric acid (HNO3) and one part of both perchloric (HClO4) and sulfuric acid (H2SO4) as prescribed by Allen [38]. Only concentrated acids were used for both types of digestion. The digested soil and plant samples were filtered using Whatman No.1 filter paper and diluted with double distilled water up to a final volume of 50 mL.

#### *2.4. Metal Bioaccumulation Factor (BAF)*

In order to assess the accumulation of heavy metals from the soil in the agricultural fields in the vicinity of the industries into the three plant species (*Cannabis sativa* L., *Chenopodium album* L. and *Ricinus communis* L.), the bioaccumulation factor (BAF) was calculated. The bioaccumulation factor is commonly used to study the fate of different environmental contaminants in plants [39]. Ali et al. [40] documented that BAF is the ratio of the concentration of heavy metals in the crop to that in the soil. Accordingly, BAF was calculated using the following equation.

$$\text{BAF} = \mathbb{C}\_{\text{plant}} / \mathbb{C}\_{\text{soil}} \tag{1}$$

where, Cplant stands for concentrations of heavy metal in plant leaves and Csoil stands for concentrations of heavy metal in soil.
