## 2 Materials and Methods
### Analysis of Plant and Soil  
The contents of Cu and Zn in plant and soil samples were assessed separately by using individual methods. In the case of plants, after they were dried at 60 \({}^{\circ}\)C, they were ground and ashed in a furnace at 450 \({}^{\circ}\)C for 6 h according to the method described by [PERSON] et al. [23]. Briefly, ash was dissolved in 5 mL of 6 mol-dm3 HCl and diluted to a constant volume (15 mL) with distilled water. In the case of soil, the total and bioavailable amounts were determined by using the _aqua regia_ procedure [24] and DTPA complexing solution [17], respectively. The concentrations of metals in an ionic form in obtained extracts of plants and soil were determined by flame atomic absorption spectrometry (FAAS) by using a Varian Spectra AA 220 FS apparatus. All the assays identifying the amounts of nutrients in the tested samples were performed in three replications.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Parameter** & **Soil** & **Compost** & **Fly Ash** \\ \hline pH & 7.0 & 6.8 & 13.7 \\ CuTOT (mg\(\cdot\)kg\({}^{-1}\)) & 200 * & 33 & 44 \\ ZnTOT (mg\(\cdot\)kg\({}^{-1}\)) & 100 * & 15 & 20 \\ \hline \hline \end{tabular}  
* According to [22], the permissible content for Cu is up to 140 mg\(\cdot\)kg\({}^{-1}\) and for Zn up to 300 mg\(\cdot\)kg\({}^{-1}\).  
\end{table}
Table 1: Basis properties of soil, compost and fly ash (data for composite samples).  
On the basis of Cu and Zn, total (\(\text{C}\text{U}_{\text{TOT}}\), \(\text{Z}\text{T}_{\text{TOT}}\)) and bioavailable amounts (\(\text{C}\text{U}_{\text{DTPA}}\), \(\text{Z}\text{T}_{\text{DTPA}}\)) in the soil and, in cultivated plants, two separate bioconcentration factors were calculated according to the ratio of metal concentration in plant seeds (only plant seeds are included because they are processed for consumption as stated above) and total or bioavailable amounts of metals as follows [11]:  
\[\text{BCF}_{\text{T}}=\frac{\text{metal in plant seeds}}{\text{total metal content}}\]  
\[\text{BCF}_{\text{B}}=\frac{\text{metal in plant seeds}}{\text{bioavailable metal amount}}.\]  
The estimated daily intake (EDI) and the health risk index (HRI) were calculated. For the estimated daily intake (EDI) of metals the following equation was used [25]:  
\[\text{EDI}\left[\text{$\mathrm{\SIUnitSymbolMicro g}$\cdot$\mathrm{kg}$}^{-1} \right]=\frac{\text{metal in plant seeds $\cdot$ average daily consumption$*$}}{\text{body weight}**},\]  
where \(*\) is based on WHO data [26] on adults in Europe who have an average daily consumption of 2.0 g oat per person per day. Due to the fact that WHO does not give the daily consumption of amelina oil, considering that this plant is an oil crop, the same dose as provided by WHO for rapeseed was adopted, i.e., 7.3 g per person per day. Furthermore, \(**\) is based on Polish statistics where the average body weight of adult women is 65 kg and that of men is 83 kg.  
The health risk index (HRI) was calculated by using the following equation given by [PERSON] et al. [27], cited after [20]:  
\[\text{HRI}=\frac{\text{EDI}}{\text{RfD}*},\]  
where \(*\) RfD is the reference oral dose; the values for Cu and Zn amounted to 0.04 and 0.30 (mg/kg/day), respectively (values given according to US-EPA [28]).