*2.3. Experimental Setup*

Seeds of *S. flos-cuculi* were purchased from SemeNostrum (Udine, Italy). The soil used for this experiment was Compo Sana organic potting mix containing forest products, compost, perlite, and fertilizer (soil pH = 6.8–7.2). The potting substrate, having a Ce concentration of 17.3 mg kg−1, was amended with water suspensions of *n*CeO2 of 25 nm in order to reach a final substrate concentration of 20 and 200 mg kg−<sup>1</sup> *n*CeO2. Tap water was used as the control. Before sowing, *n*CeO2 suspensions were stirred and sonicated for 30 min to avoid the aggregation, and the first addition of *n*CeO2 to the substrate occurred in one dose through irrigation. The amended substrate was carefully mixed in order to obtain the prearranged concentrations. The *n*CeO2 amended substrates were stored in the dark at 15 ◦C for three days for conditioning before planting seeds. After soil equilibration, the pots were filled with 500 g soil. Repeated applications of *n*CeO2 were performed after 20 and 40 days from seedling emergence (DSE) in separate sets of pots. The experimental setup is showed in Figure 1. More precisely, D1 refers to the pots that received only the *n*CeO2 dose before sowing; D2 refers to the pots that received a second adjustment 20 DSE; and D3 refers to the pots that underwent three applications (the last one occurred 40 DSE). The additional treatments were carried out via irrigation with the solutions containing the same initial *n*CeO2 concentration (20 or 200 mg L−1). This was to attempt to recreate a situation of chronic "contamination". The experiment was carried out in a semi-controlled greenhouse facility at the experimental farm of the University of Udine (Italy).

The trial was set up in a randomized experimental design, focusing the attention in particular on repeated treatments. Each treatment was replicated four times. Seeds were put about 2.5 cm deep in the soil and pots were placed in full sunlight, at 18–27 ◦C (night/day) with a relative humidity of around 60%. Two weeks after seed planting, the seedlings were thinned to two seedlings per pot. During the trial, pots were irrigated every three days and randomly reallocated every week. After 60 days, control and treated plants were harvested. Fresh plant biomass was separated into roots, stems, and leaves, and then weighed. Then, the plant fractions were thoroughly washed in tap water and rinsed three times with distilled water. In addition, roots were washed in 400 mL of 0.01 M of nitric acid in a shaker bath at 300 rpm for 5 min to remove metal ions adsorbed at the surface. Leaf area was measured using an LI-3100C Area Meter (Li-Cor Corporation, Lincoln, NE, USA). After that, the plant fractions were oven dried at 60 ◦C for three days, and weighed.

**Figure 1.** (**A**) Experimental setup showing the combination of treatments: control, single-, double-, and triple-dosed plants (Ctrl, D1, D2, and D3, respectively); (**B**) *S. flos-cuculi* plants at 40 DSE.
