**3. Results**

#### **3.1 Growth, physiological and biochemical parameters**

A negative effect on growth was found, expressed in a decrease in total biomass, as in **Figure 1A** and **B**. This result varied approximately 82 and 59% between the control (0 ppm) and the maximum concentration of Zn(II) (3000 ppm) and Cu(II) (1500 ppm), respectively. The dry weight of the root and the aerial part decreased by 82% for Zn(II), whereas 62 and 56% for Cu(II), respectively. A significant reduction was observed from the lowest concentration of Zn(II) (1000 ppm) while for Cu(II), this decrease was observed from the middle concentration (1000 ppm). The reduction of biomass, both shoot and root, shows the same pattern, as the metal concentration increases, the decrease of biomass becomes greater (**Figure 1A** and **B**).

**Figure 1A** and **B** represent chlorophyll and carotenes concentration in Zn (II) and Cu(II) systems, respectively. A significant decreased of chlorophyll and carotenes concentration was observed in Cu(II) treatment (1500 ppm) compared with the control (**Figure 1B**). This difference was approximately 47 and 16% for chlorophyll and carotenes content, respectively. However, chlorphyll and carotens concentration in Zn(II) systems (**Figure 1A**) were not affected.

**Figure 3** shows the relativity conductivity (RC) percentage in roots and leaves in Zn(II) (A) and Cu(II) (B) systems. A gradual increase of relativity conductivity (RC) in roots with increasing Zn(II) and Cu(II) concentrations was noted. On the other hand, the RC in leaves biomass was not affected by Zn(II) and Cu(II) concentrations (**Figure 3A** and **B**).

**Figure 4A** and **B** represent malondialdehyde (MDA) content in the roots and leaves of *Canna indica* plants in Zn(II) (A) and Cu(II) (B) systems, respectively. As observed in **Figure 4A** and **B**, malondialdehyde (MDA) content in leaves had significant differences at maximum concentrations of Zn(II) and Cu(II)

compared with the control. However, statistically significant increase of malondialdehyde (MDA) content was only detected in roots at 1500 ppm Cu(II) system (**Figure 4B**).

The soluble protein content in leaves and roots is shown in **Figure 5**. In general, it was determine there are not statistically significant differences of soluble protein content in roots for Zn(II) and Cu(II) systems, whereas the soluble protein content in leaves biomass decreased about 26% compared with the Cu(II) maximum concentration and the control (**Figure 5A** and **B**).

**Figure 6** represents proline content in leaves and roots for Zn(II) and Cu(II) systems. The proline content in leaves increased with the increase of Zn(II) and Cu(II) concentrations, but statistically significant differences were determine only in the maximum concentrations for both metals compared with control system (**Figure 6A** and **B**).

#### **3.2 Bioaccumulation and extraction of Zn(II) and Cu(II)**

**Figure 7A** and **B** show the mean bioaccumulation values for Zn(II) and Cu(II) in shoot, roots, and total biomass of *Canna indica*, respectively. A higher bioaccumulation of Zn(II) and Cu(II) in the root than in the aerial part was observed. The results demonstrated that *C. indica* bioaccumulated 872.99 694.68 mg Zn(II) kg<sup>1</sup> dry weight (DW) of total biomass (SD), almost 77 times higher than the control (withouth heavy metal) (**Figure 7A**). The maximum concentration of Cu(II) in total biomass was 1432.15 91.13 mg Cu(II) kg<sup>1</sup> DW (SD) (**Figure 7B**).

On the other hand, the bioavailability (BAI), accumulation (AI), translocation (TI), and bioaccumulation (BI) indexes were calculated with the results mentioned above (**Table 1**). It was determined that BAI, AI, and BI indexes ˃ 1 for Zn(II) and Cu (II) system. These results mean *C. indica* plant was efficient in extracting Zn(II) and Cu(II) from the substrate. However, *C. indica* plant did not translocate Zn(II) and Cu (II) to the aerial part as TI index was ˂ 1 (**Table 1**).
