**1. Introduction**

Nanoscience and nanotechnology are rapidly developing in different applications, having the potential to considerably improve human life. Much progress has been made in applying the application of engineered nanomaterials (ENMs) and nano-enabled products in medicine, energy, electronics, innovative materials and many more fields [1]. On the other hand, the increase in the industrial production of ENMs inevitably leads to their release into the environment [2].

Once released in terrestrial ecosystems, ENMs enter watercourses and soils reaching the biota [3]. Since 2006, the Organization for Economic Cooperation and Development (OECD) has developed toxicity test guidelines for ENMs [4]. Currently, the endpoints/targets of such tests are the green algae *Raphidocelis subcapitata*, the daphnid *Daphnia magna*, the fish *Danio rerio*, the sediment organism *Lumbriculus variegatus*, soil microflora and terrestrial invertebrates *Enchytraeus crypticus* and *Eisenia fetida* [5].

The global biomass on Earth is dominated by plants, which are the primary producers in terrestrial and water ecosystems and represent about 80% of the biota [6]. Their life cycle is strongly dependent on their relationships with air, soil, and water. However, for that very reason, they constitute the first biological target of ENMs, and are not considered among the environmental targets of ENMs by the OECD guidelines. It would be advisable to evaluate the impact of ENMs and consider the consequences concerning the ecosystem services that plants provide [7].

The literature lacks systematic knowledge regarding the effects of ENMs on vascular plants. In part, this is due to the very high pace of research and development on nanomate-

**Citation:** Lizzi, D.; Mattiello, A.; Piani, B.; Gava, E.; Fellet, G.; Marchiol, L. Single and Repeated Applications of Cerium Oxide Nanoparticles Differently Affect the Growth and Biomass Accumulation of *Silene flos-cuculi* L. (*Caryophyllaceae*). *Nanomaterials* **2021**, *11*, 229. https:// doi.org/10.3390/nano11010229

Received: 16 December 2020 Accepted: 13 January 2021 Published: 16 January 2021

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rials. However, the most important reason concerns the fact that the discussion regarding the most appropriate experimental strategies is still open [8,9]. The studies carried out on crops [10–12] have been far more numerous compared to those on spontaneous plant species, and between the latter the papers on aquatic species largely prevail over those on terrestrial plants. According to the last "State of the World's Plants and Fungi" release [13], the number of vascular plants species currently known is about 391,000. Only about 150 species have a significant commercial value, and 20% of them account for more than half of the plants eaten by humans [14,15]. Therefore, we optimistically assume that the relationships between ENMs and vascular plants have been studied much less than 0.05% of higher plant species, so far. Practical gap-filling actions are expected on this issue in the next future.

Cerium oxide nanoparticles (*n*CeO2) are a rare earth nanomaterial with several engineering and biological applications due to their catalytic, electrochemical, and optical properties [16]. With an estimated annual global production of 100–1000 tons per year, *n*CeO2 is among the most widely utilized metal oxide nanoparticle in Europe [17]. The Organization for Economic Cooperation and Development included *n*CeO2 in the list of ENMs for immediate priority testing [18].

As previously mentioned, the existing body of literature regarding the relationships between ENMs and plants is mostly focused on agricultural plant species. While this is justified concerning the potential risks of human exposure to nanomaterials through food consumption, in a broader ecological context, the impacts of ENMs on the whole primary producers should not be underrated in a broader ecological context. From this perspective, more aquatic [19–21] and wetland species [22–24] have been studied than terrestrial varieties. Concerning terrestrial ecosystems, to the best of our knowledge, *Pinus sylvestris* L. and *Quercus robur* L. are the only non-food terrestrial plant species to have been investigated for the exposure to ENMs [25].

The fate and effects of ENMs in the soil-plant system are always studied by supplying plants with ENMs at different concentrations, sizes, and shapes, and structured with several capping molecules in a single dose and at given time [26]. What remains is whether and how ENMs affect plant metabolism and plant growth under realistic conditions. Regardless of the ENMs source, plants are likely exposed to ENMs over a much longer time, at relatively lower concentrations but repeated pulses of ENMs; it is this last aspect about which we developed our experiment. The main goal of this study was to evaluate and compare the effects of a single dose and two and three repeated applications of *n*CeO2 at different concentrations on the growth of *Silene flos-cuculi* (L.).
