**5.2. Analytical methods**

Determination of heavy metal concentration, in both plants and soil underneath, was done by Inductively Coupled Plasma - Atomic Emission Spectrometry method (ICP-AES). For analyze, the samples were mineralized in Berghof microwave digester, plants by mixture with 10 ml of nitric acid concentrated 65% and 2 ml of hydrogen peroxide, and soil in mixture 1:1 with nitric acid (according with Berghof method). The advantage of this method is the multiele‐ mental detection, which give the possibility, in one shot, to read a wide range of elements [46]. For this research, analyzes were conducted with Liberty 110 spectrometer of Varian brand. The minimal detection limits of device range according to the analyzed element and is 0.4 mg/ kg for Zn, Mn and Cu; 0.5 mg/kg for Cr and Co; 0.6 mg/kg for Sn, Ni and Pb. The concentrations values for analyzed metals were expressed in milligrams of metal per kilogram of dry soil or plants (mg/kg).

The soil pH was determined with a portable pH-meter, WTW 3110 SET 2, with precision of 0.01 units. For pH analyzes, 5 g of each soil sample were mixed with 50 ml KCl 0.1N, F 1000, Tt 0.0056 g/ml and homogenized for 15 minutes with a magnetic stirrer.

The deposition on electrodes was evaluated by microscopy and quantitative assessed by Energy Dispersive X-Ray Fluorescence method (EDXRF) [47], using a PW4025 – MiniPal – Panalytical type EDXRF Spectrometer. The XRF determinations were conducted in Helium atmosphere, excited for 300 s, without any filter, at 16 kV voltage. The current intensity was automatically adjusted by the use of a 3.6 µm Mylar tissue [48]. The surface of electrodes was evaluated for heavy metal concentration before and after electrolysis.

#### **5.3. Data analysis**

The bioaccumulation factor (BF) for studied plants was calculated as the ratio between metal concentration in plants and metal concentration in soil:

$$BF = \frac{\mathcal{C}\_{plant}}{\mathcal{C}\_{soil}} \tag{1}$$

where: Cplant represents the metal concentration in plants and Csoil represents the metal concentration in soil.

To calculate the percentage of metal concentration by drying, the metal content of dry matter was reported to the element content of the fresh biomass and to calculate the percentage of metal concentration by incineration, the metal content of ash was reported to the element content of the dry matter.

Percentage of metal extraction into solution (%) was calculated as ration between the metal content of 400 ml solution (filtered leachate) and the metal content of 10 g of ash:

$$\text{Percentage of metal extraction into solution} \left( \% \right) = \frac{\text{solution concentration} \star 400/1000}{\text{ash concentration} \star 10/1000} \tag{2}$$

The quantity of metal recovered by electrolysis was calculated according with the mass of metal deposition on cathode (0.7 mg), percentage of ash from the fresh biomass (5.1%) and the mass of fresh plants that can be harvested from one hectare (40 t):

$$\text{Recovered metal} \left( \mu \text{g/} 5 \text{g ash} \right) = \frac{\text{metal concentration in cathode deposit} \left( \text{mg/kg} \right)}{1000} \text{\*} 0.7 \tag{3}$$

From 40 t of fresh biomass of *Lolium perenne* that can be harvested from one hectare, with a percentage of ash of 5.1%, can be obtained 2.04 t of ash.

$$\text{Recovered metal} \left(\text{g per ha}\right) = \frac{\text{recovered metal} \left(\text{mg}/5\,\text{gash}\right)}{5} \ast 2.04 \tag{4}$$
