**3.1. Characterization of** *n***TiO2**

The *n*TiO2 powder measured with BET has a specific surface area equal to 61.6 m<sup>2</sup> g−1 that aver‐ age value is slightly higher than the product specifications which declares a specific surface area comprehended between 45 and 55 m2 g−1. The height distribution measured with AFM of the *n*TiO2 powder results 41.8 ± 24.3 nm on average (**Figure 1A**). The *n*TiO2 suspension was characterized at dimensional level also by TEM (**Figure 1B**). In this case, the instrument allows to measure the diameters of nanoparticles and the average size result equal to 24.09 ± 7.22 nm. The DLS instrument gives information about nanoparticles at three different levels: (i) at size level, in fact the instrument displays the zeta average size of nanoparticles, which correspond to the average diameter; (ii) at stability level by measuring the zeta potential of nanoparticles,

Application of Nanotechnology in Agriculture: Assessment of TiO2 Nanoparticle Effects on Barley http://dx.doi.org/10.5772/intechopen.68710 27

**Figure 1.** Characterization of: (A) height classes (nm) of *n*TiO2 in powder form by AFM. (B) Diameter classes (nm) of *n*TiO2 suspension obtained by TEM.

this index gives information about the electric potential in the interfacial double layer, if this value is comprised between −30 and +30 mV, the suspension tends to flocculate; and (iii) at heterogeneity level by the Polydispersity Index (PDI). The *n*TiO2 suspensions result to have a zeta average size, zeta potential, and PDI equal to 925 ± 105 nm (**Figure 2A**), 19.9 ± 0.55 mV (**Figure 2B**), and 0.84 ± 0.17 nm, respectively. These values indicate a suspension made by big nanoparticles which tend to aggregate along time, and this brings to a wide‐size distribution.

#### **3.2. Seed germination experiments**

*2.3.3. Spectroscopy analysis*

26 Application of Titanium Dioxide

*2.3.4. TEM observations*

*2.3.6. Amino acids in kernels*

(SPSS Inc. Chicago, IL, USA, ver. 17).

**3.1. Characterization of** *n***TiO2**

area comprehended between 45 and 55 m2

*2.3.7. Data analysis*

**3. Results**

The *n*TiO2

the *n*TiO2

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

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

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

Amino acids analysis was performed using a LC 200 Perkin Elmer. More technical details

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

powder measured with BET has a specific surface area equal to 61.6 m<sup>2</sup>

powder results 41.8 ± 24.3 nm on average (**Figure 1A**). The *n*TiO2

age value is slightly higher than the product specifications which declares a specific surface

characterized at dimensional level also by TEM (**Figure 1B**). In this case, the instrument allows to measure the diameters of nanoparticles and the average size result equal to 24.09 ± 7.22 nm. The DLS instrument gives information about nanoparticles at three different levels: (i) at size level, in fact the instrument displays the zeta average size of nanoparticles, which correspond to the average diameter; (ii) at stability level by measuring the zeta potential of nanoparticles,

g−1 that aver‐

suspension was

g−1. The height distribution measured with AFM of

through an Elemental CHNS Analyzer using up to 2.5 mg of finely ground samples.

an ICP‐OES, whereas Ti concentration in kernels was determined by an ICP‐MS.

transmission electron microscope (TEM) operating at 80 kV.

*2.3.5. Macronutrient and micronutrient concentrations in kernels*

about amino acids analysis were provided by Pošćić et al. [18].

#### *3.2.1. Germination and root development*

After 3 days, the treated seeds (**Figure 3A**) were used for the calculation of germination per‐ centage (**Figure 3B**). The root elongation does not seem affected by *n*TiO2 treatments at 1000 and 2000 mg l‐1, in fact, the average values are quite comparable with the germinated control seeds, whereas the average total roots value seems to slightly increase with increasing concen‐ trations. The statistical analysis, however, shows there are not significant differences between treatments. The germinated seeds treated for 7 days were used for measuring their root elonga‐ tion (**Figure 3C**). The root elongation does not seem affected by *n*TiO2 treatments at 1000 and 2000 mg l−1; in fact, the average values are quite comparable with the germinated control seeds,

**Figure 2.** DLS data of *n*TiO2 suspension: (A) *n*TiO2 zeta average size distribution. (B) *n*TiO2 zeta potential.

rather the average total roots value seems to slightly increase with increasing concentrations. Conversely, the seeds treated with *n*TiO2 at 500 mg l−1 seemed to be affected in a negative way with respect to the other treatments. The statistical analysis has put on evidence a significant negative effect of *n*TiO2 at 500 mg l−1 for the root elongation, instead there are no significant differences between the other treatments.

Application of Nanotechnology in Agriculture: Assessment of TiO2 Nanoparticle Effects on Barley http://dx.doi.org/10.5772/intechopen.68710 29

**Figure 3.** (A) Petri dishes with treated barley seedlings; (B) germination percentage of seeds (mean ± SE; *n* = 3); and (C) total root length in barley seedlings treated with *n*TiO2 suspension at 0, 500, 1000, and 2000 mg l−1.

#### *3.2.2. Mitotic index*

rather the average total roots value seems to slightly increase with increasing concentrations.

with respect to the other treatments. The statistical analysis has put on evidence a significant

at 500 mg l−1 seemed to be affected in a negative way

zeta potential.

at 500 mg l−1 for the root elongation, instead there are no significant

zeta average size distribution. (B) *n*TiO2

Conversely, the seeds treated with *n*TiO2

suspension: (A) *n*TiO2

differences between the other treatments.

negative effect of *n*TiO2

**Figure 2.** DLS data of *n*TiO2

28 Application of Titanium Dioxide

**Figure 4A** reports data of mitotic index (MI), which was used as a sensor of genotoxicity. As plant roots grow, the cell division is usually very fast in the apical meristem of root tips. In our case, the control seedlings have a MI lower than the seedlings treated with *n*TiO2 suspension at 500 mg l−1 but comparable with the other treatments. Like the germination percentage also this parameter results not significantly affected by treatments.

#### *3.2.3. Titanium seedlings uptake*

The concentration of total titanium in different portions of barley seedlings is shown in **Figure 4B**. A dose‐response was recorded in the accumulation of titanium since the titanium concentration in the seedling fractions increased with the increase of *n*TiO2 exposure concen‐ tration. In particular, the seedlings grown in the presence of *n*TiO2 at 500 mg l−1 did not uptake and translocate the titanium in other seedling portions. Instead, the seedlings grown in the presence of *n*TiO2 at 1000 mg l−1 showed an uptake and a translocation of titanium in each por‐ tion. This trend is confirmed by the seedlings grown in the presence of *n*TiO2 at 2000 mg l−1; in fact, these seedling portions have the highest concentrations of titanium with respect to the seedling portions of the other treatments. The roots are the most interested area of accumula‐ tion; in fact, this portion recorded the highest concentrations of total titanium than the other portions for each treatment. This is particularly evident in the seedlings grown with *n*TiO2 at 1000 mg l−1 where the concentration results significantly different from the other seedling por‐ tions, whereas it is not like that in seedlings grown in the solution with 2000 mg l−1.

**Figure 4.** Effects of *n*TiO2 on seedlings of *Hordeum vulgare*. (A) Mitotic index (%) (mean ± SE; *n* = 3) observed in root tips of *n*TiO2 ‐treated seedlings. (B) Concentration of Ti in seeds, roots, and shoots (mean ± SE; *n* = 3) of *n*TiO2 barley‐treated seedlings. Different letters indicate statistical difference between treatments at Tukey's test (*p* < 0.05).

#### **3.3. Life cycle study**

#### *3.3.1. Plant growth*

The data obtained from the phenological observation are shown in **Figure 5**. The barley plants grown in soil spiked with *n*TiO2 at 500 and 1000 mg kg−1 are in delay with respect to the con‐ trol barley plants in reaching each physiological maturity; this delay is already appreciable from the second leaf stage. At the end of the physiological maturity, the barley plants were used for measuring the representative parameters of plant growth in particular, the plant height, number of tillers, total leaves area, and grain yield (**Figure 6**). The plant height was not affected in a significant way by the *n*TiO2 treatments; however, there is a gradual increment of plant height with the increase of *n*TiO2 concentration in the soil. The number of tillers, like the plant height parameter, increases at the increase of *n*TiO2 into the soil. Differently from the previous parameter, the average number of tillers of barley plants grown in the soil spiked with *n*TiO2 at 2000 mg l−1 show almost significant difference from the other treatments. The total leaf area parameter has the same trend of number of tillers parameter, also in this case there is an increase of leaves surface at the increase of *n*TiO2 in the soil with a slightly sig‐ nificant difference in the average value obtained for the plants, which were grown in the soil spiked with 1000 mg kg−1 of *n*TiO2 . The last parameter took into account has been the plant yield. This parameter was affected by the treatments in a different way with respect to the other ones; in fact, the control barley plants did not significantly differ from the barley plants treated with 1000 mg kg−1 of *n*TiO2 except for the barley plants grown in the soil spiked with 500 mg kg−1 of *n*TiO2 resulted significantly affected.

#### *3.3.2. Spectroscopy analysis*

The spiked soil and the barley plant portions were analyzed by ICP‐OES and ICP‐MS in order to check the total concentration of Ti (**Table 1**). The soils spiked with *n*TiO2 at 500 and 1000 mg l −1 have a significant difference from the soil without *n*TiO2 , though the soil spiked with *n*TiO2 at 500 mg l−1 results slightly different from the control soil. These results confirm that the soil spiking was performed in a correct way. The analyses of the barley plant portions show there Application of Nanotechnology in Agriculture: Assessment of TiO2 Nanoparticle Effects on Barley http://dx.doi.org/10.5772/intechopen.68710 31

**Figure 5.** Duration of vegetative and reproductive phenological phases of *Hordeum vulgare* grown in control soil and *n*TiO2 ‐spiked soil. Asterisk denotes significant differences between control and treated plants (*p* ≤ 0.05).

are no significant differences in the Ti concentrations both between the treatments and between treatments and control. The only exception is the total Ti concentration in the stem portion of the plants treated with *n*TiO2 at 1000 mg kg−1 which results slightly different from the same por‐ tion of the other plants.

#### *3.3.3. TEM observations*

**3.3. Life cycle study**

**Figure 4.** Effects of *n*TiO2

30 Application of Titanium Dioxide

of *n*TiO2

grown in soil spiked with *n*TiO2

affected in a significant way by the *n*TiO2

of plant height with the increase of *n*TiO2

spiked with 1000 mg kg−1 of *n*TiO2

treated with 1000 mg kg−1 of *n*TiO2

500 mg kg−1 of *n*TiO2

*3.3.2. Spectroscopy analysis*

the plant height parameter, increases at the increase of *n*TiO2

there is an increase of leaves surface at the increase of *n*TiO2

resulted significantly affected.

check the total concentration of Ti (**Table 1**). The soils spiked with *n*TiO2

have a significant difference from the soil without *n*TiO2

The data obtained from the phenological observation are shown in **Figure 5**. The barley plants

‐treated seedlings. (B) Concentration of Ti in seeds, roots, and shoots (mean ± SE; *n* = 3) of *n*TiO2

seedlings. Different letters indicate statistical difference between treatments at Tukey's test (*p* < 0.05).

trol barley plants in reaching each physiological maturity; this delay is already appreciable from the second leaf stage. At the end of the physiological maturity, the barley plants were used for measuring the representative parameters of plant growth in particular, the plant height, number of tillers, total leaves area, and grain yield (**Figure 6**). The plant height was not

the previous parameter, the average number of tillers of barley plants grown in the soil spiked

total leaf area parameter has the same trend of number of tillers parameter, also in this case

nificant difference in the average value obtained for the plants, which were grown in the soil

yield. This parameter was affected by the treatments in a different way with respect to the other ones; in fact, the control barley plants did not significantly differ from the barley plants

The spiked soil and the barley plant portions were analyzed by ICP‐OES and ICP‐MS in order to

at 500 mg l−1 results slightly different from the control soil. These results confirm that the soil spiking was performed in a correct way. The analyses of the barley plant portions show there

at 2000 mg l−1 show almost significant difference from the other treatments. The

at 500 and 1000 mg kg−1 are in delay with respect to the con‐

on seedlings of *Hordeum vulgare*. (A) Mitotic index (%) (mean ± SE; *n* = 3) observed in root tips

treatments; however, there is a gradual increment

concentration in the soil. The number of tillers, like

. The last parameter took into account has been the plant

except for the barley plants grown in the soil spiked with

into the soil. Differently from

barley‐treated

in the soil with a slightly sig‐

at 500 and 1000 mg l

, though the soil spiked with *n*TiO2

−1

*3.3.1. Plant growth*

with *n*TiO2

To verify the uptake and subsequent translocations of *n*TiO2 from roots to aerial plant frac‐ tions, ultrastructural analyses on plant leaf tissues were carried out. Rare clusters of nanopar‐ ticles were found in leaves sampled from plants grown in soil enriched with the different combinations of *n*TiO2 , at both concentrations (**Figure 7**). *n*TiO2 were observed in leaf cells and, in particular, in the stroma of the chloroplast and in the vacuoles. Despite the treatment, the chloroplast ultrastructure appeared normal (**Figure 7B**).

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

The accumulation of macronutrients in barley kernels is shown in **Table 2**. Both N and S concentrations increase at the increment of *n*TiO2 , whereas for Ca there was not the same behavior. Apparently, K, P, and Mg concentrations in kernels did not respond to the treat‐ ment. **Table 3** reports the concentrations of micronutrients in kernels. The *n*TiO2 treatments determined an increase in Fe, Mn, and Zn concentrations in barley kernels, whereas B and Cu concentrations were not influenced by the treatments.

**Figure 6.** Biometric variables of *Hordeum vulgare* observed in plants grown in control soil and *n*TiO2 ‐spiked soils. Variables are respectively: (A) plant height, (B) number of tillers per plant, (C) total leaf area per plant, and (D) grain yield per plant. Bars are mean standard error (*n* = 5). Different letters indicate statistical difference between treatments at Tukey's test (*p* < 0.05).


**Table 1.** Ti concentration observed in soil, roots, stems, leaves, kernels of primary and secondary spikes of barley plants grown in control (Ctrl) and *n*TiO2 ‐spiked soil.

#### *3.3.5. Amino acids in kernels*

The effects of *n*TiO2 treatments on amino acid concentrations in kernels are shown in **Table 4**. Overall, Glu and Pro are the most abundant amino acids in kernels with concentration ranges of 31–43 and 15–21 mg·g−1, respectively. The *n*TiO2 treatments did not significantly modify concentrations of Ala, Arg, Asp, His, Ser, and Trp. On the opposite, the concentration of Cys, Glu, Gly, Ile, Leu, Lys, Phen, Pro, Tyr, and Val in kernels significantly increased in response to the *n*TiO2 treatments. In the case of Thr, the response to the treatment was less evident. At last, only in the case of Met contradictory results were recorded.

Application of Nanotechnology in Agriculture: Assessment of TiO2 Nanoparticle Effects on Barley http://dx.doi.org/10.5772/intechopen.68710 33

**Figure 7.** Representative TEM micrograph of leaf tissues of *Hordeum vulgare* plants grown in (A) control soil and (B) *n*TiO2 1000 mg kg−1‐spiked soils. Clusters of Ti nanoparticles are visible in the stroma of the chloroplasts of *n*TiO2 ‐treated plants (B).


**Table 2.** Nitrogen percentage and concentration of macronutrients in barley kernels at ripening from main shoot grown in control soil (Ctrl) and *n*TiO2 ‐spiked soil.


*3.3.5. Amino acids in kernels*

grown in control (Ctrl) and *n*TiO2

of 31–43 and 15–21 mg·g−1, respectively. The *n*TiO2

last, only in the case of Met contradictory results were recorded.

‐spiked soil.

treatments on amino acid concentrations in kernels are shown in **Table 4**.

treatments. In the case of Thr, the response to the treatment was less evident. At

treatments did not significantly modify

‐spiked soils.

Overall, Glu and Pro are the most abundant amino acids in kernels with concentration ranges

**Figure 6.** Biometric variables of *Hordeum vulgare* observed in plants grown in control soil and *n*TiO2

Variables are respectively: (A) plant height, (B) number of tillers per plant, (C) total leaf area per plant, and (D) grain yield per plant. Bars are mean standard error (*n* = 5). Different letters indicate statistical difference between treatments

**Treatment Soil (mg kg−1) Roots (mg kg−1) Stems (mg kg−1) Leaves (mg kg−1) Spike (μg kg−1)** Ctrl 1797 ± 119 b 77 ± 3.19 a 0.26 ± 0.04 ab 1.03 ± 0.06 a 2.19 ± 1.2 a Ti 500 2153 ± 119 ab 66.7 ± 7.49 a 0.28 ± 0.03 ab 1.39 ± 0.35 a 1.71 ± 0.53 a Ti 1000 2537 ± 56.3 a 81.7 ± 4.96 a 0.39 ± 0.06 a 0.96 ± 0.09 a 1.39 ± 0.27 a

Values are mean ± SE (*n* = 5). Same letters indicated no statistical difference between treatments at Tukey's test (*p* ≤ 0.05).

**Table 1.** Ti concentration observed in soil, roots, stems, leaves, kernels of primary and secondary spikes of barley plants

concentrations of Ala, Arg, Asp, His, Ser, and Trp. On the opposite, the concentration of Cys, Glu, Gly, Ile, Leu, Lys, Phen, Pro, Tyr, and Val in kernels significantly increased in response

The effects of *n*TiO2

at Tukey's test (*p* < 0.05).

32 Application of Titanium Dioxide

to the *n*TiO2

Values are mean ± SE (*n* = 5). Same letters indicated no statistical difference between treatments at Tukey's test (*p* ≤ 0.05).

**Table 3.** Concentration of micronutrients in barley kernels at ripening from main shoot grown in control soil (Ctrl) and *n*TiO2 ‐spiked soil.


**Table 4.** Amino acid (mg·g−1) concentration in barley kernels at ripening from main shoot grown in soil spiked with none (Control), 500 mg *n*TiO2 ·kg−1, and 1000 mg *n*TiO2 ·kg−1.

### **4. Discussion**

The *n*TiO2 suspensions did not affect germination of *H. vulgare*. Our results are in agreement with the observations carried out, respectively, on rice [19], lettuce, radish, and cucumber [20], tomato [21], and pea [22]. According to Ref. [11], we demonstrated that *n*TiO2 treatment did not affect root elongation of seedlings. Other authors published opposite results. In fact, Mushtaq [23] showed an inhibitory effect of *n*TiO2 on root elongation in cucumber, whereas Fan et al. [22] verified decrease in the number of secondary lateral roots in pea. The *n*TiO2 treatments did not influence the mitotic index. That is in contrast with Moreno‐Olivas et al. [24] which observed a *n*TiO2 ‐induced genotoxicity in hydroponically cultivated zucchini. Although the size of *n*TiO2 used in that experiment is comparable to ours, those experiments were carried out in different conditions than ours. This can result in different experimental conditions, with particular regard to the *n*TiO2 traits (e.g., different grade of agglomeration due to different z‐average size and zeta potential). On the other hand, the results obtained by ICP‐OES analyses seem to indicate a *n*TiO2 uptake by root tissue and a subsequent trans‐ location in the other seedling tissues. This result could be an indication of a real uptake and translocation of *n*TiO2 . A second hypothesis is that, despite the use of appropriate analytical protocols, the analysis may have been disturbed by sample contamination. In that case, the element concentration in the plant tissues could be significantly overestimated due to a frac‐ tion of metal simply adsorbed onto the external sample surface.

With regard to the effects along the entire life cycle, the response of plant phenology was in accordance with previous studies [25, 26]. In fact, the barley plants treated with *n*TiO2 result to have a longer vegetative phase. During this phase, the plants keep growing and the leaves continue their photosynthetic activity and consequently the production of photo‐ synthates [27]. Taking into account such evidences, a higher biomass production and grain yield in treated plants respect the control ones it is expected. The analyses of biometric parameters confirm in part the expected results. Except for the plant height and grain yield per plants the other parameters result positively affected. Our results confirm other experi‐ mental evidences. In particular, studies carried out on *Spinacia oleracea* have demonstrated that *n*TiO2 promotes plant photosynthesis increasing light absorbance and transformation of light energy and enhancing Rubisco activity [28, 29]. The positive effects of *n*TiO2 treatments were evidenced also at the grain level. The grains obtained from treated plants result to have a higher content of macro (Na and Ca) and micronutrients (Fe, Mn, and Zn); moreover, a positive effect of *n*TiO2 treatment was also observed for several amino acids. The increase of macro/micronutrient and amino acid concentrations in kernels could be an effect related with the longest vegetative phase caused by the *n*TiO2 treatments. In order to find the relationship between the effects and treatment, the material obtained at the end of the experiment was analyzed by ICP‐OES and observed by TEM. The ICP‐OES analyses and the TEM observa‐ tions were carried out in order to know if the *n*TiO2 can enter into plant tissues and subse‐ quently cause the observed effects on plants. The ICP‐OES results did not put on evidence an effective uptake of titanium by the plants, but the TEM observations show the presence of *n*TiO2 in the stroma of the chloroplast and in the vacuoles of leaf cells. This discrepancy in results could be related with the agglomeration tendency of *n*TiO2 in water, previously evi‐ denced by *n*TiO2 characterization analyses. The agglomeration makes the *n*TiO2 less available because their increased dimension makes difficult the passage of them through the cell wall, this means only the *n*TiO2 with the smallest size can pass this plant barrier and consequently small amount of titanium could be uptaken.

### **5. Conclusions**

**4. Discussion**

(Control), 500 mg *n*TiO2

suspensions did not affect germination of *H. vulgare*. Our results are in agreement

treatment

on root elongation in cucumber, whereas

traits (e.g., different grade of agglomeration

‐induced genotoxicity in hydroponically cultivated zucchini.

 **500 mg kg−1** *n***TiO2**

 **1000 mg kg−1**

used in that experiment is comparable to ours, those experiments

with the observations carried out, respectively, on rice [19], lettuce, radish, and cucumber

**Table 4.** Amino acid (mg·g−1) concentration in barley kernels at ripening from main shoot grown in soil spiked with none

did not affect root elongation of seedlings. Other authors published opposite results. In fact,

Fan et al. [22] verified decrease in the number of secondary lateral roots in pea. The *n*TiO2 treatments did not influence the mitotic index. That is in contrast with Moreno‐Olivas et al.

were carried out in different conditions than ours. This can result in different experimental

due to different z‐average size and zeta potential). On the other hand, the results obtained

[20], tomato [21], and pea [22]. According to Ref. [11], we demonstrated that *n*TiO2

·kg−1.

Mushtaq [23] showed an inhibitory effect of *n*TiO2

·kg−1, and 1000 mg *n*TiO2

**Amino acid Ctrl** *n***TiO2**

34 Application of Titanium Dioxide

Alanine (Ala) 5.65 ± 0.51 a 7.35 ± 1.05 a 6.75 ± 0.16 a Arginine (Arg) 7.55 ± 1.32 a 9.26 ± 0.56 a 9.12 ± 0.83 a Aspartic acid (Asp) 7.18 ± 0.67 a 8.58 ± 0.65 a 9.09 ± 0.49 a Cysteine (Cys) 6.85 ± 0.13 b 8.07 ± 0.01 a 8.42 ± 0.36 a Glutamic acid (Glu) 31.2 ± 3.56 b 40.7 ± 3.73 a 43 ± 1.83 a Glycine (Gly) 5.98 ± 0.45 b 7.74 ± 0.29 a 8.01 ± 0.42 a Histidine (His) 3.14 ± 0.51 a 3.66 ± 0.18 a 3.89 ± 0.18 a Isoleucine (Ile) 5.29 ± 0.47 b 6.42 ± 0.36 a 6.77 ± 0.25 a Leucine (Leu) 9.4 ± 0.74 b 11.2 ± 0.81 a 11.7 ± 0.42 a Lysine (Lys) 3.67 ± 0.31 b 5.85 ± 0.33 a 5.98 ± 0.45 a Methionine (Met) 2.39 ± 0.13 b 3.08 ± 0.01 a 3 ± 0.20 b Phenylalanine (Phe) 7.48 ± 0.94 b 9.12 ± 0.65 a 9.37 ± 0.45 a Proline (Pro) 14.8 ± 1.68 b 20.4 ± 3.04 a 21.4 ± 1.41 a Serine (Ser) 5.84 ± 0.54 a 6.78 ± 0.36 a 6.84 ± 0.18 a Threonine (Thr) 4.61 ± 0.31 a 5.11 ± 0.36 ab 5.35 ± 0.18 ab Tryptophan (Trp) 1.15 ± 0.67 a 0.53 ± 0.01 a 0.75 ± 0.18 a Tyrosine (Tyr) 3.36 ± 0.42 b 4.34 ± 0.20 a 4.22 ± 0.36 a Valine (Val) 7.04 ± 0.49 b 8.29 ± 0.65 a 8.68 ± 0.40 a

conditions, with particular regard to the *n*TiO2

[24] which observed a *n*TiO2

Although the size of *n*TiO2

The *n*TiO2

The amount of products containing nanoparticles will increase in the future years; this will bring an increase of their presence in the environment. In the last years, the majority of lit‐ erature was focused to investigate the potential negative impact of this new kind of material on human, animals, and plants, but in our study, we put on evidence the potential beneficial effects. At first, we demonstrate the absence of negative impact during the early development stages of barley plants; in fact, each *n*TiO2 concentration did not affect the germination per‐ centage and root elongation, except for the lowest concentration (*n*TiO2 500 mg kg−1) which significantly affects in a negative way the last parameter. The analysis focus moves to evalu‐ ation of the possible effects at genetic level; for this purpose, the mitotic index was analyzed. The results also show, in this case, the absence of an effect for this parameter. The AFM and DLS analyses give information about the tendency of *n*TiO2 to form big agglomerate once dissolved in MilliQ water, this makes the *n*TiO2 less available for the seeds/seedlings, and the absence of effects could be related to the incapacity of *n*TiO2 to cross the cell wall. However, the ICP‐OES and ICP‐MS analyses demonstrate the capacity of seeds/seedlings to uptake the titanium, then the absence of effects in the early developmental stages is not due to the absence of titanium in the plant tissues but to this unharmful effect. The experiment set up to evaluate the possible effect of *n*TiO2 along the entire barley life cycle demonstrates the posi‐ tive dose‐response effect on vegetative growth, and this has a direct effect on the composition and nutritional value of barley grains.
