## 4 Discussion
We decided not to consider the data obtained in the first year of experimentation--although it is reported in Table 2--given that in the first year of planting both _Arundo_ and switchgrass were not in full production and had not fully developed either their rhizomes or the root systems. This decision was supported by pairwise comparisons among Y levels on main and combined effects, where significant differences were observed when comparing the first year with second and third years, for all heavy metals accumulated. In fact, when considering the data reported in Figure 3, concerning the biplot PCA and clustering analysis obtained for the 12 heavy metals considered in this study, the same accumulation pattern was found in the second and third years. The results shown in Table 2 and Figure 4, regarding the accumulation of heavy metals in the biomasses of the three different species, are in agreement with previous work reporting the bioaccumulation factor (BAF = C plant/C soil) considering only the biomass harvested of Ad [54], Pv [55] and MM [56]. The highest value of phytoextraction was found for Zn (Figure 4). Zn is an essential micronutrient for plant growth; hence, it is not surprising that it was present in the harvested biomass in higher concentrations in comparison to the other heavy metals such as Cu, Co, Fe, Ni, Mn and Mo that can also be removed from soils via phytoextraction [57]. For the heavy metals that lack a known biological function (Al, Cr, Cd and Pp), but represent an important environmental issue, significant uptake has been reported [58; 59; 60]. The metals accumulated in plant biomass reflect the metal contents present in the soil. Each species has its own accumulation profile (we could say a chemical barcoding or chemotype) as shown in the PCA (Figure 3), where the analyses of the various biomasses cluster by species (Ad, Pv and MM), year (II and III) and harvest period (October and February). One of the main factors that can explain the differences observed among different species lies in the different capacities that plants have to release organic compounds (e.g., chelators) via root apparatus capable of modifying the rhizosphere and the pH of the soil. Of course, these chemical changes affecting the soil around the root system increase the solubility of metals [61]. Another aspect to take into consideration lies in the different physiology of the plants under study: Ad and Pv are monocotyledons, whereas MM consisted mainly of two dicotyledonous species (_Medicago sativa_ and _Chenopodium album_). Furthermore, Ad and Pv are rhizomatous plants, but the rhizomatous apparatus of arundo is much larger, and Pv is able to produce seeds, as is MM [62]. Hence, by taking together all these different characteristics, we can hypothesize, starting from the results reported in Figure 3, that: (i) In the case of Ad, the difference in the metals accumulated between the biomass collected in February and October can be explained by the translocation of the nutrients present in the epigeal part of the plant towards the rhizomes (statistically significant difference in the case of Zn, Figure 4). (ii) In the case of Pv there are no major differences between the two types of biomass (harvested in October and February) probably due to the small rhizomatous apparatus, unable to translocate large quantities of nutrients. However, one statistically significant difference was observed in the case of Cd, of which more wasaccumulated in the biomass collected in October (Figure 4). In this case we can hypothesize that the Cd accumulated in the seeds still present on the plant in October was lost in February where the seeds would have fallen to the ground. The fact is well known that Cd is accumulated in plant seeds, representing a big issue for agriculture conducted on soils polluted by this metal [63]. (iii) For what concerns MM, the explanation regarding the differences observed for the metals accumulated in the two different harvest times could be more complex since it is a polyphite crop.