**2. Methodology**

This report involves an experimental qualitative design which included heavy metals laboratory testing and statistical analysis.

### **2.1 Identification of soil and plant sampling sites**

To identify sampling sites, several climatic conditions were investigated. This included the physical conditions of the study area such as the geology of the environment, type of soil as well as rainfall. Sampled sites were evenly distributed to allow the determination of possible pollution sites and to know the degree of heavy metal pollution.

Other considered factors are:


Considering the relationship between the parent rocks and non-point source pollution, it is believed that 32 samples for four metals will be sufficient to represent the study area and perform statistical and geostatistical evaluation.

### **2.2 Soil sampling**

According to Branquiho et al. [60], spatial dust effects are localized and it ranges less than 1 km from the source pollutant. To allow even distribution of soil samples, soil samples were taken at 500 m and 1000 m away from the gold mining site in various directions – north, northeast, northwest, south, southwest, southeast, east, and west. At each distance, two samples were taken using an auger at a depth of 0–10 cm (topsoil) and 10–2- cm (subsurface). These depths were used in previous studies [61, 62] and they suggest the disparity in land contamination at varied distances [61].

A site devoid of any physical human activity was selected to collect a soil sample referred to as the control or background sample. This Background sample was taken 10 km away from the study area (260 15′ 08" S 250 11′ 32″ E) at the same depth used for other collected samples. This was essential to have an uncompromised Background sample. The climatic and physical conditions of the study area and the background site were identical.

Three samples above 1 kg were collected at each of the sampled site for precision purposes. A total of 28 soil samples were collected, stored in a plastic bag, and taken to the laboratory for further analysis. Each of the soil samples coordinate were recorded using a handheld Garmin GPSMAP 65 s Global Positioning System, manufactured by Garmin, South Africa.

### **2.3 Plant sampling**

Plant samples were collected at the same spots where soil samples were taken. A total of four plant samples were collected at the northern and eastern location of the mine at same spot where soil samples were taken. At other sampled location, the plant of interest was not found at the specified distance, 500 m, and 1000 m. Plants rely on the physical and chemical conditions of the topsoil, hence, they are disturbed by these circumstances.

In this study, a specific plant species, *E. hypnoides,* was collected. Collected plant samples were thoroughly cleaned with tap water, followed by 0.1 mol/L of hydrochloric acid (HCl) and deionized water. The washing of the plant samples was important to remove every soil and dust particles from the sample. If the plant samples were left unwashed, the soil and dust particle will influence the determined concentration of the studied heavy metals in the sample.

In line with the method of Ma et al. [63], plant samples were placed inside an oven at 70°C for three days. This was done to remove moisture content of the plant sample. After drying, all the plant samples were then pulverized in a mill and packed in a well labeled sealable plastic bag before proceeding to aqua regia digestion of the samples.

### **2.4 Determination of heavy metals**

The analysis of cadmium (Cd), lead (Pb), zinc (Zn), and mercury (Hg) were done using the inductively coupled plasma mass spectrometry (ICP-MS) for both the collected soil and plant samples. These selected heavy metals are toxic to humans, animals, and plants. They have been reportedly found at high concentrations within the environment of a gold mining and processing.

Before using the ICP-MS to determine the concentrations of the studied heavy metals, the collected and labeled plants and soil samples were digested using 0.6 mL of concentrated sulfuric acid (H2SO4), 0.6 mL of concentrated nitric acid (HNO3) and 1.8 mL of concentrated HCl for two hours at 95°C.

After digestion and the cooling of the machine, each sample volume was brought up to 10 mL by adding deionized water. Each sample was then arranged in the ICP-MS machine accordingly to determine the concentration of the studied heavy metal in each sample. This procedure has been used in the study of Kamunda et al. [64]. The detection limit of Zn, Cd, Pb and As using the ICP-MS are 1.173 mg/kg, 0.006 mg/kg, 0.045 mg/kg and 0.026 mg/kg respectively.
