## 2 Materials and Methods  
### Soil Washing Experiments Using Chitosan  
For the laboratory washing experiment using chitosan, a soil sample from 10-90 cm depth was collected from a pasture located about 800 m downstream of the \"Larga de Sus\" abandoned mining perimeter (GPS coordinates: 46\({}^{\circ}\)07\({}^{\prime}\)55.2\({}^{\prime\prime}\) N, 23\({}^{\circ}\)09\({}^{\prime}\)09.2\({}^{\prime\prime}\) E) from Zlatna (Alba County, Romania). The physicochemical properties of the soil sample used in the washing experiments can be found in our previously published studies [41; 42].  
The soil washing experiments were performed in order to determine the factors affecting the decontamination efficiency of the collected soil sample using chitosan as washing agent.  
A commercial chitosan sample extracted from shrimp shells was investigated as a potential extracting agent for Cu and Pb found in the collected soil. In soil washing experiments, the investigated washing agent was used its natural state and mixed with distilled water.  
The decontamination experiments were conducted at laboratory scale on 5 g polluted soil samples in a 100 mL capacity glass flasks in a stirrer with continuous orbital rotation-oscillation stirring at 100 oscillations/min, at different washing solution concentrations (2% and 5%), S/L ratios (0.2; 0.125; 0.1 and 0.0625 dec. fr.) and durations of stirring (0-40 h).  
The concentration of copper and lead in the soil before and after washing with chitosan was determined by atomic adsorption spectrometry (AAS). Prior to the AAS analysis, the polluted and decontaminated soil samples were processed as follows: dried at 95 \({}^{\circ}\)C for about 10 h, homogenized, crumbled, and milled to pass through a 250 \(\upmu\)m sieve. After that, 3 g of previously processed sample, 1 mL of distilled water, 21 mL of concentrated HCI (Hydrochloric Acid), and 7 mL of concentrated HNO\({}_{3}\) (Nitric Acid) were combined in a 100 mL glass flask which was heated in a sand bath for about 2 h. The mixture was then filtered through a 0.45 \(\upmu\)m pore size filter into a 100 mL volumetric flask, filled to the mark with distilled water, and analysed for Cu and Pb concentration through AAS.  
The removal efficiency of Cu and Pb (\(\upmu\)) from the collected heavy metal-contaminated soil was determined using the following equation [43]:  
\[\upmu\left(\%\right)=\frac{m_{e}}{m_{i}}*100 \tag{1}\]  
where \(m_{e}\) is the extracted pollutant concentration (mgkg\({}^{-1}\)) and \(m_{i}\) is the initial pollutant concentration present in the soil (mgkg\({}^{-1}\)).  
The experimental data obtained according to the above methodology which served to develop the mathematical models are presented in Tables 1 and 2.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline  & & \multicolumn{2}{c}{**Cu**} & & \multicolumn{2}{c}{**Pb**} \\ \cline{2-7}
**n** & **X1** & **X2** & **Y** & **X1** & **X2** & **Y** \\  & **(dec. fr.)** & **(h)** & **(\%)** & **(dec. fr.)** & **(h)** & **(\%)** \\ \hline
1 & 0.2 & 2 & 21.53 & 0.2 & 2 & 3  
All of the experiments and analyses were performed in duplicate at 25 \({}^{\circ}\)C, and the average values were used to develop the mathematical model. All reagents were of analytical grade or ultra-pure. The water was distilled using a Technosklo water distiller.