**2. Materials and methods**

#### **2.1. Nanoparticle characterization**

Titanium (IV) oxide anatase nanopowder having a minimal average particle size of 25 nm was purchased from Sigma‐Aldrich (product ID 637254). Titanium nanoparticle (*n*TiO2 ) characterization was carried out at the Facility for Environmental Nanoscience Analysis and Characterization (FENAC), University of Birmingham (UK). Further details on analytical methods are provided by Marchiol et al. [14].

#### **2.2. Laboratory experiment**

#### *2.2.1. Seed germination and root elongation*

Ten seeds of spring barley (*Hordeum vulgare* L. var. Tunika) were transferred in sterile condi‐ tions into each Petri dishes soaked with distilled water (Ctrl), 500, 1000, and 2000 mg l−1 *n*TiO2 suspensions; each treatment was replicated three times. The Petri dishes were taped and placed in the dark at 21°C for 3 days. The germination percentage was calculated as the ratio of ger‐ minated seeds out of the total seeds. The seedlings obtained were used for the measurements of their total root length with ImageJ [15]. Root elongation was calculated as the average length and the sum of all roots emerged from each seed.

#### *2.2.2. Mitotic index*

electronics, cosmetics, sporting equipment, wastewater treatment, medicine, and more recently in agriculture and the food industry [2]. The agri‐food was the last sector in terms of succession to be interested by this technological revolution but at the same time it would be far reaching in the next years [1]. In fact, the nanotechnology has recently emerged as the technological advancement to develop and transform the entire agri‐food sector, in terms of increasing global food produc‐ tion and nutritional value, quality and safety of food [3]. The type of nanoparticles (NPs) or nano‐ materials (NMs) used in plant science are quite wide, but they could be clustered in two principal groups: the carbon nanomaterial (CBNMs) and the metal‐based nanomaterials (MBNMs) [4]. In

nanoparticles (*n*TiO2

[6–9]. More recently, Dehkourdi and Mosavi [10] used nano‐anatase to treat parsley seeds, which resulted in an increase in the percentage of germination, the germination rate index, the root and shoot length, the fresh weight, the vigor index, and the chlorophyll content of the seedlings. Also, Feizi et al. [11] observed that the germination rate of *Salvia officinalis* improved when the seeds

etative growth, for example, Hong et al. [7] demonstrate an acceleration in the rate of evolution of oxygen by chloroplasts in spinach plants. Another experiment on spinach demonstrated a gain in the photosynthetic carbon reaction in treated plants [9]. More recently, Qi et al. [12] treated tomato

in the photosynthetic rate with respect to the control ones. Currently, the application of nano‐ materials in the field of primary production is still under investigation, and therefore, it may take many years before specific nanoproducts for agriculture are commercialized worldwide [1]. Since the studies performed up to now have been conducted in a very simplified experimental condition, we still lack accurate information on what is happening in the soil. Further research is

Titanium (IV) oxide anatase nanopowder having a minimal average particle size of 25 nm was purchased from Sigma‐Aldrich (product ID 637254). Titanium nanoparticle (*n*TiO2

characterization was carried out at the Facility for Environmental Nanoscience Analysis and Characterization (FENAC), University of Birmingham (UK). Further details on analytical

Ten seeds of spring barley (*Hordeum vulgare* L. var. Tunika) were transferred in sterile condi‐ tions into each Petri dishes soaked with distilled water (Ctrl), 500, 1000, and 2000 mg l−1 *n*TiO2

required to ensure complete success for these applications of nanotechnologies [13].

terial between the MBNMs. Several papers demonstrate the positive effects of *n*TiO2

, CeO2

. Previous studies demonstrate a positive effect also during the plant veg‐

and put them in a mild heat stress, the plants resulted to have an improvement

, Fe3 O4

, ZnO, and AgNO3

) that represent the most used nanoma‐

[5]. Our

on plants

)

the group of MBNMs, the most common NPs are TiO<sup>2</sup>

experiments were focused on TiO2

**2. Materials and methods**

**2.1. Nanoparticle characterization**

**2.2. Laboratory experiment**

methods are provided by Marchiol et al. [14].

*2.2.1. Seed germination and root elongation*

were exposed to *n*TiO2

24 Application of Titanium Dioxide

plants with *n*TiO2

Seeds of barley were sterilized and transferred in sterile conditions into Petri dishes soaked with distilled water. After 3 days, the germinated seedlings with actively growing roots (at least 2.5 cm in length) were placed in the *n*TiO2 suspensions (0, 500, 1000, 2000 mgl−1) for 24 h. Ten root tips per each treatment were studied to evaluate possible genotoxic effects of *n*TiO2 . The samples prepared were evaluated for a total of about 10,000 cell observations per treatment. The mitotic index was recorded in Feulgen‐stained preparations as the per‐ centage of dividing cells out of the total number of cells scored.

### *2.2.3. Titanium uptake*

The treated barley seedlings were rinsed three times with MilliQ water. Subsequently, they were divided into three portions: roots, seeds, and coleoptiles. The seedlings portions were dried at 70°C for 24 h, and they were acid‐digested (10 ml HNO<sup>3</sup> 65%) in a microwave oven according to USEPA method 3052. Plant extracts were filtered (0.45 mm PTFE), diluted with MilliQ water, and analyzed by ICP‐OES.

#### **2.3. Greenhouse experiment**

#### *2.3.1. Soil characterization and nTiO<sup>2</sup> amendment*

The possible effects of *n*TiO2 to barley were also evaluated along their entire life cycle; for this purpose, seeds of barley were grown in soil contaminated with *n*TiO2 at 500 and 1000 mg kg−1. The soil characterization data were reported by Marchiol et al. [14]. According to Priester et al. [16], the soil was amended with *n*TiO2 powder before sowing plants reaching the final concen‐ tration of 500 and 1000 mg kg−1 of *n*TiO2 .

#### *2.3.2. Plant growth*

In a semi‐sealed greenhouse, seeds of spring barley were sown in pots containing the *n*TiO2 ‐ enriched soils. After 2 weeks, seedlings were thinned to two seedlings per pot. During the plant growth, the pots were watered twice a week to maintain soil at 60% WHC. Phenological stages were monitored by adapting the Decimal Growth Scale [17] throughout the growth cycle and were based on 50% of plants within the treatments at each stage. Plants were har‐ vested at physiological maturity. Plant shoots were severed at the collar and separated into stems, leaves, spikes, and grains. Leaf area was measured using a LI‐3100C Area Meter. The plant fractions were oven dried at 105°C for 24 h and weighed.

#### *2.3.3. Spectroscopy analysis*

Plant fractions were acid‐digested in a microwave oven according to USEPA method 3052. Titanium concentration in plant fractions, such as roots, stems, and leaves, was determined by an ICP‐OES, whereas Ti concentration in kernels was determined by an ICP‐MS.

#### *2.3.4. TEM observations*

Serial ultrathin sections from each species were cut with a diamond knife, mounted on cop‐ per grids, stained in uranyl acetate and lead citrate, and then observed under a Philips CM 10 transmission electron microscope (TEM) operating at 80 kV.

### *2.3.5. Macronutrient and micronutrient concentrations in kernels*

Total B, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, P, and Zn contents were determined by an ICP‐OES with an internal standard solution of Y. Total Ce and Ti contents were determined by an ICP‐ MS with an internal standard solution of 72Ge and <sup>89</sup>Y. Total N and S content were determined through an Elemental CHNS Analyzer using up to 2.5 mg of finely ground samples.

### *2.3.6. Amino acids in kernels*

Amino acids analysis was performed using a LC 200 Perkin Elmer. More technical details about amino acids analysis were provided by Pošćić et al. [18].

#### *2.3.7. Data analysis*

The experiments were carried out in a completely randomized factorial design. Analysis of variance was conducted with a one‐way ANOVA. Tukey's Multiple Comparison test at 0.05 p level was used to compare means. Statistical analysis was performed using the SPSS program (SPSS Inc. Chicago, IL, USA, ver. 17).
