## 3 Results
### Heavy Metals in Harvested Biomass  
ICP-MS results for the three subsamples (randomized blocks) for each treatment regarding Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Cd and Pb showed about 1% of outlier data. The normality of distribution of the different combinations of factors was confirmed for 95% of treatments, but homoscedasticity was not confirmed by the vast majority of samples (\(\alpha\) = 0.05). Table 2 summarizes the mean values and standard deviations for each heavy metal among the different samples.  
Figure 1: Experimental field photographed from the air during the period of this study. The letters represent the different species used: _A. donax_ (Ad); switchgrass (Pv) and mixed meadow (MM). Each block has the dimensions of 10 m \(\times\) 10 m with a distance of 10 m between the plots. The grass between the plots was regularly mown, to avoid growth of surrounding weeds.  
Figure 2: Estimated biomass yield of the treatments, each column representing each harvest operation under the different combination of year (I, II and III), species (Arundo—Ad, switchgrass—Pv and mixed meadows—MM) and harvest times (October and February). Error bars represent SD and letters a, b and c label statistically homogeneous subgroups (\(p\) < 0.05).  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline
**Y** & **S** & **HT** & **Pb** & **Cd** & **Mo** & **As** & **Zn** & **Cu** & **Ni** & **Co** & **Fe** & **Ma** & **Cr** & **Al** \\ \hline \multirow{3}{*}{**I**} & **A** & October & 0.43 \(\pm\) 0.1 & 0.06 \(\pm\) 0.01 & 1.39 \(\pm\) 0.2  
Considering a three-way interaction design for year (Y), species (S) and harvest time (HT) factors, 99% of combinations were statistically significant (\(p<0.05\)). The analysis of variance designed with a two-way interaction design highlighted a significant effect of factor Y as main effect and/or in its interaction with S and HT, for all heavy metals considered in this experiment (\(p<0.05\)), except Zn. By pairwise comparisons among Y levels on main and combined effects, significant differences were highlighted when comparing the first year with second and third years, for all heavy metals (\(p<0.05\)), representing 81% of significant differences caused by this factor.  
In almost all cases, metals were less accumulated in the biomasses of the first year than in the other two years, since the plants had not reached maturity.  
Considering these results, the first year was excluded from the following calculations to consistently reduce type 2 errors in the analysis.  
PCA analysis picked out Zn, Fe and Al as the three most relevant components explaining almost all the variance among samples in the second and third years. The results highlighted that treatments were clustering by HT and S factors, whereas Y appeared to influence only the scattering within the clusters (Figure 3).  
Figure 3: PCA biplot (**A**) and clustering analysis (**B**) based on the accumulation of 12 metals (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Cd and Pb) in the biomass of _A.__dmax_ L. (Ad), switchgrass (Pv) and mixed meadow (MM), considering the year of cultivation, second (_II) and third (_III), and the harvest time (October: _O and February: _F). In PCA analysis the first two axes (PC1 and PC2) explain 86.56% of the total variance.  
For the last two years of the experiments, the factor Y maintained an effect on heavy metals' uptake only for Co, As, Cd and Pb--elements sharing low accumulation in the harvested biomass, rarely exceeding 1 mg/kg\({}^{-1}\) (Table 2). By multivariate analysis of variances, different effects were highlighted in the second and third years, with species (S) and harvest time (HT) factors and/or their interaction being significant (\(p<0.05\)) for all metals excluding As and Zn. Samples' contents of the 12 heavy metals are plotted in Figure 4.  
Figure 4: _Cont_.