*3.2. nCeO2 in Plant Fractions*

Before setting up the experiment on the entire vegetative cycle of *S. flos-cuculi*, some preliminary observations were carried out to demonstrate that *n*CeO2 was assimilated by the roots of plants and subsequently translocated to the upper plant parts. They were evidently necessary to set up the subsequent experiment illustrated in this paper. At first, a test was carried out to demonstrate the entry of *n*CeO2 within germinating seeds of *S. flos-cuculi* seeds [32]. Subsequently, under the same conditions as the main experiment, we wanted to verify that even in the presence of a complex matrix (that is, the potting soil compared to the very simple conditions of the germination test) the roots of *S. flos-cuculi* were able to take up the *n*CeO2.

The results reported in Figure 3A,B clearly show that the *n*CeO2 was absorbed by the roots of *S. flos-cuculi*, and then moved upwards to reach the leaf tissues. The magnitude of pulses quantitatively represents the presence of *n*CeO2 in plant tissue; after the *n*CeO2 root absorption, only about 25% of nanoparticles moved to the plant leaves (Figure 3B). The mean size of *n*CeO2 was 33 ± 2 nm and 31 ± 1.5 nm in the roots and leaves, respectively (Table S2, Supplementary Materials), meaning that after being assimilated, *n*CeO2 did not undergo relevant aggregation. In the plant extract sample, the spICP-MS analysis also provided the concentration of the ionic form of an element dissolved from a nanostructure. In our case, the dissolved ionic Ce was very low, and equal to 4.86 ± 0.4 <sup>μ</sup>g kg−<sup>1</sup> in the roots and 0.08 ± 0.03 <sup>μ</sup>g kg−<sup>1</sup> in the leaves of *S. flos-cuculi*, respectively (Table S2, Supplementary Materials).

**Figure 3.** Particle size distribution of *n*CeO2 extracted after enzymatic digestion procedure from (**A**) roots and (**B**) leaves of *S. flos-cuculi*.

#### *3.3. Plant Growth*

An overall view of the experimental data relating to plant growth is showed in Table S3, Supplementary Materials, containing the results of a two–way ANOVA. In particular, the table reports the *p*-values testing the statistically significant effects of the *n*CeO2 dose (D1, D2 and D3), *n*CeO2 concentration (20 and 200 mg kg−1), and their interaction on biometric variables of *S. flos-cuculi*.

In broad terms, only the root dry weight (*p* = 0.0009 \*\*\*) responded in a statistically significant way to the dose of *n*CeO2, while this did not happen in the case of the other plant fractions and the leaf area. The factor "concentration" determined statistically significant effects in the case of root dry matter (*p* = 0.0281 \*), stem dry matter (*p* = 0.0000 \*\*\*), and total plant dry weight (*p* = 0.0000 \*\*\*), as well. Finally, because the root apparatus was directly exposed to the soil matrix, as expected we recorded a statistically significant interaction of "dose X concentration" for root dry matter (*p* = 0.0018 \*) (Table S3, Supplementary Materials).

Carefully observing the effects of treatments on the vegetative development of *S. flos-cuculi*, some aspects of particular interest can be highlighted. As already mentioned, the root biomass dry weight, being the plant fraction directly exposed to the treatments, showed to be particularly sensitive to the experimental conditions. The development of the root apparatus responded positively to D1 (single dose of *n*CeO2 provided to pot soil before seed germination). At both concentrations of *n*CeO2, an increase of 29% (at 20 mg kg−1) and 9% (at 200 mg kg−1) in root biomass compared to the control was observed (Figure 4).

At the lowest concentration, the higher doses of *n*CeO2 (D2 and D3) did not promote the same effect detected for D1. The weight of the root biomass returned to a level very close to that of the control plants. This also occurred for D2 at the highest concentration (200 mg kg<sup>−</sup>1), while the response to D3 was a reduction of about 27% in root development compared to the control (Figure 4). Additionally, a statistical analysis was performed by isolating the concentration factor, i.e., testing the effect of single and repeated administration of *n*CeO2 to plants within the same concentration level. In this case, considering D1 as the reference within each concentration, we evaluated the effect of the additional doses of *n*CeO2 on the plant root biomass (Figure 4). Whether the single *n*CeO2 dose stimulated the production of root biomass, the second additional dose (D3), even though supplied to the plants at a late vegetative stage, resulted in a reduction in the root biomass. Compared to D1, we recorded a reduction in root dry matter of *S. flos-cuculi* by approximately 21% and 33%, for *n*CeO2 20 mg kg−<sup>1</sup> and 200 mg kg<sup>−</sup>1, respectively (Figure 4).

44

**Figure 4.** Root dry mass of *S. flos cuculi*. Comparison of effects based on single (D1) and repeated (D2, D3) applications of 20 and 200 mg kg−<sup>1</sup> *n*CeO2, respectively. Letters indicate statistically significant difference between treatments (*<sup>p</sup>* <sup>≤</sup> 0.05) using one-way ANOVA followed by Tukey's test. † One-way ANOVA *p*-value within each concentration: asterisks indicate the statistically significant difference of dose factor at \* 0.05 ≥ *p* ≥ 0.01; \*\*\**p* ≥ 0.001, respectively.

As reported in Table S3 Supplementary Materials, some other biometric variables were observed in plants. In particular, on the aboveground plant biomass, the number of stems and leaves for each plant were counted. The total leaf area per plant and the leaf dry matter were recorded as well. For these variables, the statistical analysis did not reveal significant effects of the treatments, whereas there was a very evident negative effect of *n*CeO2 on dry matter accumulation in the stems of *S. flos-cuculi* (Figure 5). Regardless of the concentration and dose of *n*CeO2, the negative effect of the treatment determined an average reduction of 75.5% in dry matter accumulation in the stems compared to the control.

As reported in Supplementary Materials (Figures S1–S4), the response to treatments of other biometric variables did not confirm either the stimulating effect highlighted on the case of root biomass or the negative effect on the dry matter accumulation on the stems *S. flos-cuculi*. Indeed, although the biomass of the stems was reduced, the architecture of the plants was not affected; the number of stems in the treated plants was no different from that of the control plants (Figure S1, Supplementary Materials). Even the number of leaves per plant, the leaf area per plant and the accumulation of dry matter in the leaves themselves were not affected by the treatments (Figures S2–S4, Supplementary Materials).

Figure 6 reports the plants' total dry matter. Aggregating the different effects observed on the plant fractions could hide the impact of *n*CeO2 treatments. However, in our case this did not happen. Albeit to a lesser extent than that observed for the weight of the stems, the effect of *n*CeO2 on plant development is also visible on total biomass production. The negative effect of the treatments on the growth of *S. flos-cuculi* is statistically significant (*p* = 0.00000 \*\*\*), regardless of the *n*CeO2 dose and even at the lower concentration of nanoparticles (Figure 6).

**Figure 5.** Stem dry mass of *S. flos cuculi*. Comparison of effects based on single (D1) and repeated applications (D2, D3) of 20 and 200 mg kg−<sup>1</sup> *n*CeO2, respectively. Letters indicate statistically significant difference between treatments (*p* ≤ 0.05) using one-way ANOVA followed by Tukey's test. † One-way ANOVA *p*-value within each concentration.

**Figure 6.** Plant dry mass of *S. flos cuculi*. Comparison of effects based on single (D1) and repeated applications (D2, D3) of 20 and 200 mg kg−<sup>1</sup> *n*CeO2, respectively. Letters indicate statistically significant difference between treatments (*p* ≤ 0.05) using one-way ANOVA followed by Tukey's test. † One-way ANOVA *p*-value within each concentration.

After C fixation, the plant biomass was allocated according to species-specific patterns that are also influenced by environmental conditions as well as biotic and abiotic stress. Data regarding the dry weight of the plant fractions and the leaf area per plant were used to calculate new parameters (see Table S4, Supplementary Materials) that allowed us to evaluate the effects of *n*CeO2 treatments with a more accurate perspective. Additionally, in

this case we can appreciate an overview of the effects of the experimental factors through the results of the two-way ANOVA (Table S5, Supplementary Materials). The effect of the "dose" factor was statistically significant only in the case of the root mass fraction (RMF) and the S/R ratio, while the response to the "concentration" factor was much more evident: only for specific leaf area (SLA) was the effect not statistically significant in the ANOVA. The interaction between the experimental factors was statistically significant for the RMF and the SLA (Table S5, Supplementary Materials). One-way ANOVA was used to evaluate the effects of treatments compared to the control and within the same concentration of *n*CeO2.

Compared to the control and regardless of the *n*CeO2 concentration, the RMF was enhanced by D1, whereas D2 and D3 determined a subsequent drop of RMF. At the lowest concentration of *n*CeO2 concerning D1, we observed an almost-equal reduction in RMF in response to D2 and D3 (−33%). Additionally, at the highest concentration of *n*CeO2, the response to D2 and D3 was negative, although in this case it was gradual, with the reduction in RMF concerning D1 equal to −17% and −33%, for D2 and D3, respectively. However, the RMF of D2 and D3 treated plants was always higher than the control plants (Table 1).

**Table 1.** Root mass fraction (RMF), shoot to root ratio (S/R ratio), and specific leaf area (SLA) ± standard deviation of *S. flos-cuculi* grown in presence of different inputs of 20–200 mg kg−<sup>1</sup> *<sup>n</sup>*CeO2. Statistically significant differences (*<sup>p</sup>* <sup>≤</sup> 0.05) are indicated by the letters using one-way ANOVA followed by Tukey's test. Dashed box indicate ANOVA *p*-values (*p* ≤ 0.05) within the *n*CeO2 concentration. ns: not significant at *p* ≤ 0.05; \* and \*\* significant at *p* ≤ 0.05 and *p* ≤ 0.01.


SLA did not respond to the single experimental factors; however, ANOVA revealed a statistically significant effect for the interaction "dose X concentration" (*p* = 0.0243 \*). Regarding the effects of the treatments, we observed a possible SLA stimulating effect of *n*CeO2 20 mg kg−<sup>1</sup> D1 and D2. At the same time, a certain variability prevented this empirical evidence from being statistically verified, whereas we observed a significant reduction in SLA in plants that received D3 compared to the controls (Table 1). In plants of *S. flos-cuculi* treated with *n*CeO2 200 mg kg<sup>−</sup>1, SLA responded differently (*p* = 0.0243 \*). Indeed, a slight reduction in SLA compared to the control due to treatment D1 (−4.7%) is associated with an evident increase in this parameter in response to treatments D2 and D3 (+10.7% and +18.6% greater than the control, respectively) (Table 1). Further ratios calculated from biometric variables (Stem mass fraction SMF, Leaf mass fraction LMF, Shoot to root ratio Shoot/Root and Leaf area ratio LAR) are reported in Supplementary Materials (Figures S5–S8).
