*3.4. Cerium Concentration in Plant Fractions*

A general view of the Ce uptake and accumulation in plant tissues as affected by experimental factors is given in Table S6, Supplementary Materials. The factor "dose" result was statistically significant only for Ce concentration in plant stems (*p* = 0.0000 \*\*\*), while the Ce accumulation in each plant tissue, as expected, increased responding to the

factor "concentration". A statistically significant interaction "dose x concentration" was observed in the roots (*p* = 0.0313 \*) and stems (*p* = 0.0021 \*\*).

Table 2 reports data regarding the Ce concentrations in plant fractions. At first glance, the data indicate that plant Ce uptake was not very high compared to the treatments. Concerning the plant fractions, as expected, Ce in the roots was higher than the others.

**Table 2.** Ce concentration in plant fraction and Ce translocation factor in *S. flos cuculi* grown in the presence of different inputs of *<sup>n</sup>*CeO2 (20 and 200 mg kg−1.) Data are mean <sup>±</sup> standard deviation. Statistically significant differences (*p* ≤ 0.05) are indicated by the letters using one-way ANOVA follow by Tukey's test. Dashed boxes indicate ANOVA *p*-values (*p* ≤ 0.05) within *n*CeO2 concentration. ns: not significant at *p* ≤ 0.05; \*\* significant at *p* ≤ 0.01.


On average, the treatment concentration of Ce in the root tissues (3074 μg kg−1) was four times higher than that of the stems (779 μg kg−1) and two times higher than that found in the leaves (1594 μg kg−1), respectively. However, we do not observe a clear and statistically significant response to the *n*CeO2 doses regardless of the treatment concentration. However, the statistically significant interaction "dose X concentration" is explained by the different response in terms of Ce accumulation in roots after dose D3 *n*CeO2 200 mg kg−<sup>1</sup> that was about 49% higher than the average D1–D2. (Table 2).

After being taken up by the roots, a fraction of Ce moved towards the aerial plant fractions to be allocated in the stems. A statistically significant effect of the dose factor and of the interaction is visible by observing the concentration of Ce in the stems (Table 2). Here, although the highest Ce concentration was detected at D3 *n*CeO2 20 mg kg−1, the most evident effect of the "dose" factor can be appreciated for plants exposed to *n*CeO2 200 mg kg−<sup>1</sup> (*p* = 0.0015 \*\*; Table 2). Finally, the leaves represent the final allocation of Ce in plant aerial biomass. Here, Ce accumulation was higher in than in stems; however, due to a certain variability, it was not possible to statistically verify a significant effect of the experimental treatments (Table 2)

#### **4. Discussion**

Only in 2012 were the effects of ENMs over the whole plant cycle studied [33]. In soybeans (*Glycine max* L.), it was demonstrated that Ce concentrations in the roots and the concentration of *n*CeO2 in soil were correlated. Nanoceria negatively influenced the yield of soybean and N2-fixation by affecting the efficiency of the symbiotic system established with *Bradyrhizobium*: a dramatic example of the influence on cultivated plants and wild species' ecological services, as well.

A large body of literature reports negative responses observed at different plant growth stages. When germinating seeds are exposed to *n*CeO2, other effects could be verified, basically depending on particle size and concentration. Additionally, statistically significant species-specific responses were reported, regarding root elongation being more sensitive to *n*CeO2 than germination [34–36]. Other studies explored the physiological implications of the *n*CeO2 plant uptake, concluding that plants responded to the treatments increasing the antioxidant enzyme activities. However, the oxidative stress induced by high concentrations of *n*CeO2 cannot be attenuated by the antioxidant system [37–40].

The growth of *S. flos-cuculi* was negatively affected by *n*CeO2. Suppose the root apparatus development in plants treated at the lowest *n*CeO2 concentration has not undergone apparent alterations at the highest concentration; in this case, the effect is evident and progressively increases as the *n*CeO2 dose increases. The impact of *n*CeO2 on plant growth was much more apparent in the biomass of plant stems. We observed a slowdown in plant growth. The number of plant stems did not change, but they were shorter than the controls'. No statistically significant evidence was found regarding the effects of treatments on leaf biomass (evaluated by counting the number of leaves per plant, the leaf area, and the leaf dry weight). However, likely the relevant data variability detected in the treated plants compared to that of the control plants was an early signal of plant stress.

SLA is a very informative parameter in plant ecology. The total leaf area ratio to total leaf dry mass correlates with whole-plant growth linking C gain and water loss [41]. Even though we calculated the SLA using data from a single biomass sampling at the end of the growth cycle of *S. flos-cuculi*, the response of SLA to the *n*CeO2 treatments allowed us to interpret the experimental data more effectively. In particular, the increase in SLA responded to the dose of *n*CeO2 received by the plant. Moreover, this could be a consequence of the slowing of the vegetative growth rate and could lead us to conclude that the *n*CeO2 negatively affects the C accumulation by leaf tissues. Our data do not allow us to identify the specific cause precisely. However, this observation corroborates the literature evidence regarding the slowing of the plant growth cycle [42] and photosynthesis, both in terrestrials and aquatic plants [43,44].

The growing number of nanotechnology applications in various fields inevitably results in the release of nanomaterials into the environment. Models demonstrated that wastewater and sewage sludge are the primary vectors by which ENMs end up in the environment [45]. Apart from the quantitative aspect, nanomaterials' flows can occur differently concerning the position of the target to the source (e.g., a single massive event or events repeated over time). Literature papers concerning the effects of ENMs on plants always report experiments where the nanomaterials were applied in a single concentration, whereas a more realistic exposure scenario involves repeated pulses.

In our study, plants of *S. flos-cuculi* were grown in soil amended with *n*CeO2. The experimental design was conceived assuming that the soil could receive different *n*CeO2 pulses over time, thereby obtaining three different doses of *n*CeO2 supplied at different growth stages of *S. flos-cuculi*. At the moment, we cannot compare our data with other works having the same experimental approach. We have already cited a paper reporting Ag and Cu nanoparticles' effects on seedlings of *Pinus sylvestris* and *Quercus robur*. A single dose of nanomaterials was administered to plants by three subsequent foliar applications in that study, whereas in our experiment were provided three amounts of *n*CeO2. However, in both experiments, the experimental factor "dose" or merely the phenological stage at which plants received the treatments showed some influence on the consequences of the treatment. Therefore, this early indication suggests that this type of study should be further developed. Other studies of soil ecology have used the same approach. In particular, it was demonstrated that soil enzyme activity is differently affected by repeated ENM doses, indicating that additive effects occur [46,47]. It will be necessary to compile these different works to achieve a complete evaluation of the effects of ENMs on the soil–plant system.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2079-4 991/11//229/s1: Figure S1. Number of stems per plant in specimens 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 S2. Number of leaves per plant in specimens 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 S3. Total leaf area in plants 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 S4. Leaf dry matter in plants 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 (*<sup>p</sup>* <sup>≤</sup> 0.05) using one-way ANOVA followed by Tukey's test. † One-way ANOVA *<sup>p</sup>*-value within each concentration. Figure S5. Stem mass fraction 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 S6. Leaf mass fraction 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 (*<sup>p</sup>* <sup>≤</sup> 0.05) using one-way ANOVA followed by Tukey's test. † One-way ANOVA *<sup>p</sup>*-value within each concentration. Figure S7. Shoot to root ratio in *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 S8. Leaf area ratio 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 (*<sup>p</sup>* <sup>≤</sup> 0.05) using one-way ANOVA followed by Tukey's test. † One-way ANOVA *p*-value within each concentration. Table S1. Average, PDI and ζ-potentials of *n*CeO2 25 nm. Table S2. Most frequent particle size, mean particle size, number of pulses and concentration of dissolved Ce determined by sp-ICP-MS analysis after enzymatic extraction from roots and leaves of Silene flos-cuculi. Table S3. Two-way ANOVA *p*-values testing the statistically significant effects of dose and concentration and their interaction of *n*CeO2 on biometric variables of *S. flos-cuculi*. Table S4. Biomass allocation variables calculated from plant measurements (Poorter et al., 2011). Table S5. Two-way ANOVA *p*-values testing the statistically significant effects of dose and concentration and their interaction of *n*CeO2 on growth indices of *S. flos-cuculi*. Table S6. Two-way ANOVA *p*-values testing the statistically significant effects of dose and concentration and their interaction on Ce concentrations in fractions of *S. flos-cuculi* and Ce translocation factor.

**Author Contributions:** Conceptualization, D.L. and L.M.; formal analysis, D.L. and L.M.; funding acquisition, L.M.; investigation, D.L.; methodology, A.M., B.P. and E.G.; supervision, L.M.; Writing— Original draft, L.M.; Writing—Review and editing, G.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Regione Friuli Venezia Giulia, General Directorate of Environment, project "Nanomateriali 2017–2020".

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors are grateful to PhD course in "Environmental Life Sciences" jointly offered by the Universities of Trieste (Italy) and University of Udine (Italy). L.M. is grateful to Regione Friuli Venezia Giulia, General Directorate of Environment, for the financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.

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