2. Physico-Chemical Properties of Nanomaterials and Implications for Plant Toxicology

Nanomaterials are defined as materials with at least one dimension in the nanoscale (1–100 nm). If all their external dimensions do not differ significantly within the nanoscale, the term nanoparticle (NP) is preferred to distinguish these objects from others like nanofibers or nanoplates. At the nanoscale, materials behave very differently compared to larger scales, and they often display unique chemical and physical properties. This makes the nanotechnological transformation of matter a promising field to develop new products and processes. On the counterpart, their unique properties allow novel materials to interact unexpectedly with biological systems ^[6].

The main distinction can be established between two different kinds of NPs: (1) naturally occurring, which are mostly amorphous and chemically and physically highly variable particles of nanometer dimensions, and (2) custom man-made artificial nanoparticles with highly reproducible physico-chemical properties. Natural NPs are usually generated in an uncontrolled way by natural processes (i.e., volcanic activity), or they are undesirable by-products of human activities processes. On the other hand, synthetic NPs are engineered materials with well-defined dimensions and controlled physical and chemical properties, which are the major factors driving their industrial applications.

Among the fundamental physical properties of NPs that have to be considered are overall size, surface area, and mechanical properties ^[6]. The overall size of NPs impacts the mechanism by which NPs can spread through the plant vascular system. Uptake of NPs by plant roots and translocation to upper tissues has been demonstrated for different species when fed with a suspension of NPs in synthetic media. Both apoplastic and symplastic transport of NPs may also occur under natural growth conditions, with a size limit for symplastic transport in most plants ^[4][7][8]. NPs with a diameter below 5 nm can translocate through the pores of the cell wall, 8–20 nm particles can move preferably between cells through plasmodesmata, and larger >50 nm particles can be internalized by the endocytosis ^[3]. Other factors affecting the amplitude of nanotoxicity for plants are the shape and surface area of the NP, which have been studied using crop plant models ^[9].

Regarding chemical properties, a large variety of chemical compositions and surface coating exist ^[6]. Metallic NPs are prepared from silver (Ag), gold (Au), copper (Cu), or metal oxides like TiO₂. Carbonaceous nanofibers include single-well and multi-well carbon nanotubes (CNTs). Semiconductors such as silicon and ceramic are also used to make NPs. Polymeric NPs, on the other hand, are mostly colloid solids produced from polycaprolactone, polyacrylate, alginate, etc. The chemical composition of NPs, as well as their coating, can further regulate the chemical stability (e.g., changes in the redox state), overall reactivity, covalent attachment, and persistent binding to biomolecules. In most toxicological studies, metallic NPs exhibit the highest toxicity for living organisms, independently of taxa, and this has been strongly connected to the release of metal ions that induce the formation of reactive oxygen species (ROS) ^[10][11][12]. On the other side, carbonaceous and organic NPs show very low toxicity, or moreover, they might exert positive effects on plant growth or resistance to other environmental stress conditions. Nanomaterials can be made from different chemical compositions, in order to deliver faintly available nutrients and growth-promoting compounds, exhibiting beneficial rather than toxicological effects for plants ^{[13][14][15][16]}.

Aside from the physico-chemical properties of individualized NPs, the formation of NP clusters and their aggregation induced by the medium or the environment can further impact the overall nanotoxicology. An aggregation process can dramatically increase the overall size of NP clusters and show a varying biological behavior of organisms. Overall, NP aggregates have been shown to induce decreased toxicity compared to individualized NPs in plants ^[17][18]. NP in the environment undergoes aging processes such as chemical transformation, aggregation, and disaggregation. The interplay between these processes and the NP transport determines the fate and ultimately the phyto-toxicological potential of NPs (Figure 1A).

3. Applications, Production, and Release to the Environment

Engineered nanomaterials represent a fast-growing market, as they have a high potential for product adoption in a variety of industries including aerospace and vehicle production, construction, chemical catalysis, or agri-food sectors ^[15][16][19]. Rapid developments in biomedical technology and pharmaceutical research are also expected to augment the industrial production of NPs in the years to come ^[20][21]. These materials are increasing their presence in consumer products and domestic devices, mostly as coatings to improve mechanical, optical, or antibacterial properties. As examples, NPs are now used in the manufacture of crack-resistant paints, scratchproof eyeglasses, transparent sunscreens, ceramic coatings for solar cells, or self-cleaning fabrics. The use of nano-scaled particles instead of their counterpart bulk materials provides advantages and increases the competitiveness of several market products. Nanoparticles of titanium oxide used in sunscreens, for example, have the same chemical composition as the larger white titanium oxide particles used in conventional products for decades, but nanoscale titanium oxide is transparent. Silver nanoparticles continuously discharge Ag⁺ ions, and they are used in clothing to kill the bacteria known to cause undesirable odors. Silica (SiO₂) is a part of the normal mix in conventional concrete, but the use of its nano-form improves particle packing and mechanical properties of the concrete. Cerium oxide (CeO₂) nanoparticles can switch from oxidation to reduction catalysts, and they have emerged as fascinating and lucrative material in biological fields such as biomedicine, drug delivery, and bio scaffolding ^[22].

As a result of the increasing demand by the market, the production and consequent release of NPs to the environment is growing more and more every year. For very frequently produced NPs like CeO₂-, SiO₂- and Ag-NPs, recent surveys estimated the annual global production volumes over 100,000, 1000–10,000, and 100–1000 t/a, respectively ^[20]. Probabilistic modeling has been used to predict the flow of released NPs to environmental compartments in the next years and to quantify their amounts in different environmental sinks ^[20][23]. Although for most environmental compartments NPs pose a relatively low risk of toxicity, organisms residing near NP “point sources”, like production plant outfalls and waste treatment plants, may be at increased risk. The model also indicates that the concentrations of NPs in soil and sediments will be higher than those in water or air. In agreement with existing measurements, modeling of NP dispersion, transport and fate predict that soils will be the final sink and major contaminant source of NPs released into the environment. Taking into account NPs life cycle, as well as the differences in their transport and stability, predicted concentrations of NPs in agricultural soils yield up to 10 μg/kg for 2050 ^[20]. For sludge-treated soil areas, for example, predicted concentrations might be as much as 40-fold higher.

The investigations of NP production volumes, release, and persistence into different environmental compartments, as well as their toxicological effects, increased the perception of the risk that novel nanomaterials represent for environmental and human health ^[24][25]. As a consequence, more research was prompted to define ecotoxicological limits of exposure to NPs.

4. The Scales of Ecotoxicity for Nanomaterials Using Plant Assays

The investigation of potential risks for living organisms has resulted in the development of an ecotoxicological scale for nanomaterials. NPs are included in the list of chemical substances for which environmental effects are monitored and regulated by different countries. By using green algae and plant assays, nanomaterials have been classified in both aquatic and terrestrial ecotoxicity categories, where they rank from harmful to very highly toxic ^[26]. In aquatic environments, Ag, Au and Fe NPs result in being very highly toxic with EC₅₀ values of <0.1 mg/L, followed by other metallic (Zn, Cu) NPs with EC₅₀ = 0.1–1 mg/L (highly toxic), Cd and Ti NPs (moderately toxic, EC₅₀ = 1–10 mg/L) and Graphene/Carbon NTs (slightly toxic, EC₅₀ > 10–100 mg/L). In terrestrial ecosystems, only Ag NPs are classified as toxic in plant assays, having effects at EC₅₀ > 10–100 mg/kg dry-weight soil.

This classification only takes into account the chemical composition of NPs, without discrimination among sizes in the nanoscale or surface properties. As expressed before, these properties are important factors that govern NP stability and mobility as a colloidal suspension, and likely they will influence interactions with algae in natural aquatic systems or with rhizosphere and plant roots in terrestrial environments ^[27]. Thus, the phytotoxicity of nanomaterials in natural environments could largely differ from what is expected from standard ecotoxicological assays.

5. Presence and Generational Transmission of Nanomaterials in Crop Plants

An early study that stimulated further research about the genetic effects and potential toxicity of nanomaterials for plants was performed by Lin et al. ^[28]. These researchers used transmission electron microscopy (TEM) to demonstrate the uptake and transportation of NPs in crop plants. Rice plants were seeded in a suspension of natural organic matter to mimic freshwater ecosystems, and different concentrations of CNTs were added to the suspension. Seeds were kept in this germination medium for two weeks until they were transplanted to soil pots without further NP treatment. Tissues of plants at various developmental stages were sampled for TEM monitoring to evidence the translocation of CNTs from roots to stem, and from stem to leaves. However, more interestingly, the study showed that nanomaterials that were accumulated in the first generation of exposed plants could be transmitted to the second generation through seeds. Since rice provides food crops of over half the world’s population, this work suggested the potential impact of disposed nanomaterials on the food chain and raised important concerns about the genetic consequences of plant–NPs interactions. Subsequently, efforts to investigate these consequences using genome-wide technologies were pushed forward in other crop plants like tomato ^[29] or the model species Arabidopsis thaliana.

6. Transcriptomics Studies in Plants to Evaluate Nanoimpact

A powerful approach to determine how an organism responds to a particular environmental challenge is to determine how it changes the expression of its genome ^[30]. In this line, transcriptome studies to approach nanoimpact have been performed in several photosynthetic species, from unicellular green algae ^[31] to higher vascular plants like tomato ^[29], rice ^[32], or A. thaliana ^{[33][34][35][36][37][38][39][40][41]}. Thus, transcriptional data covering a range of species for which genomics tools are fully developed are available to assess for nanoimpact on plants. Omics technologies have the power to shift the research on plant-NP interactions from low-throughput, single end-point bioassays to high-throughput discovery ^[42].

While information on the transcriptional effects of NP exposure is available, the findings are somewhat contradictory. Several studies show a strong effect on the transcription of stress-related genes and suggest high toxicity for the plant, whereas some others do not find significant transcriptional changes and have concluded that NPs are unlikely to produce any adverse effect for the plant. In parallel, physiological and biochemical changes observed upon NP exposure include both significant reduction and significant promotion of plant growth, elongation or shortening of plant roots, the formation of ROS, or no indications at all for oxidative stress. One reason for these apparently contradictory conclusions is that many of these studies are focused on specific effects for a given, chemically defined type of NP, and other factors like size-dependent effects are not taken into account. Moreover, a very small fraction of plant genes is used as markers for conventional toxicological studies, and they are mostly included in predefined functional categories, such as oxidative stress response, which are not very informative at the morphological level. As a consequence, there is a missing node to relativize the toxic effects of NPs.