Physiological response of culture media-grown barley (Hordeum vulgare L.) to titanium oxide nanoparticles

ABSTRACT

Since the fate of nanoparticles after their release in the environment and their possible transfer in plants and subsequent impacts is still largely unknown, this paper evaluates the potential phytotoxic effects of up to 20% w/w TiO₂ nanoparticles (nTiO₂) on barley cultivated in hydroponics and agar media. The X-ray diffraction analysis confirmed that nTiO₂ powder corresponds to anatase phase. On agar medium only high concentrations of nTiO₂ (10% and 20% w/w) induced significant inhibition of shoot growth. However, hydroponics treatment with nTiO₂ up to 1000 mg L⁻¹ did not show any adverse effect on the shoot growth. In both experiments, (i) root growth inhibition effects became visible with increasing concentration of nTiO₂, (ii) plants treated with nTiO₂ showed no change in chlorophyll a and b content, even though the plants absorbed nTiO₂, (iii) weight of biomass treated with nTiO₂ was not significantly different compared to control. Therefore, we assume that transport of nTiO₂ into the aerial parts is limited due to the presence of effective mechanical or physiological barriers in roots. Overall, it appears that early root growth is a relevant indicator of potential effects of nTiO₂ exposure. Our results also indicate that synthesized nTiO₂ are not significantly toxic to the barley when applied at the concentrations used in this work, even though plants absorb titanium.

KEYWORDS:

• Barley
• growth medium
• roots
• shoots
• TiO₂ nanoparticles

Introduction

How toxic or beneficial the nanoparticles really are depends on many factors, such as how they are being made, what kind of chemicals have been used in the production, what type of coating nanoparticles possess, their crystallinity, size and morphology, specific surface area and reactivity, 3D spatial structure, aggregation/agglomeration state, etc. (Madhusudan Reddy et al. 2003; Yang et al. 2008). There are many ways to produce nanoparticles; manufactured nanoparticles may even differ from one preparation method and company to another. Titanium dioxide particles are produced and used in varying particle size fractions including fine (approximately 0.1–2.5 μm) and nanosize (<0.1 μm, primary particles) (Dankovic et al. 2007). Titanium dioxide nanoparticles (nTiO₂) are manufactured worldwide in large quantities for use in a wide range of applications; they have been used in paints, coatings, plastics, papers, inks, medicines, pharmaceuticals, food products, cosmetics, toothpaste, sportswear, and others (Wolf et al. 2003; Wang et al. 2007; Skocaj et al. 2011; Larue et al. 2014). nTiO₂ possess different physicochemical properties compared to their fine particle analogs, which might alter their bioactivity (Shi et al. 2013). Traditionally, TiO₂ fine particles have been considered as poorly soluble, low-toxic (ACGIH 1992), and chemically inert. nTiO₂ were considered medically harmless until recently. However, studies have revealed that nTiO₂ are more toxic than fine particles (Zhao et al. 2009). People have encountered them for over one hundred years in a massive scale in the form of white pigment commonly used in paints. It is known that no burn occurs when the unprotected living tissue is exposed to an upper paint layer containing a high amount of the photocatalytically active particles, but so far it is not clear how the nTiO₂ behave in the human body when penetrating it another way, for example, by inhalation or food. Nanoparticles from consumer products usually end up in the sewage sludge of wastewater treatment plants. Several million tons of dried-out sludge are subsequently mixed into agricultural soil each year. Therefore it is extremely important to know how much of these substances end up in plants, and what kind of transformations they subject to. Production of cereals has hundreds years of tradition in Slovakia and even today cereals are the most important crops in society; according to available data, in 2015, estimated spring barley (Hordeum vulgare L.) yield was 3.46 t/ha (Klikušovská et al. 2015). World production of barley is soon expected to climb to a 144.6Mt high (USDA 2015). Since the fate of nanoparticles after their release in the environment is still largely unknown and their possible transfer in plants and subsequent impacts have not been studied in detail (Larue et al. 2014), this paper specifically focuses on the potential toxic effect of nTiO₂ on growth and germination of one of the world’s most widespread crops.

Materials and methods

Preparation of nTiO₂

The thermal hydrolysis process was carried out using TiOSO₄ (>29% Ti (as TiO₂) basis, technical, Sigma–Aldrich), analytical grade ammonia solution (SLAVUS Ltd, Bratislava, Slovakia), and distilled water. In a standard experiment, 10 g of TiOSO₄ was dissolved in 150 mL of distilled water at room temperature. The solution was then heated at 80°C for 1 h under constant stirring. Titanyl sulfate was precipitated by adding concentrated ammonia; drops of ammonia were added to adjust the pH to 7.0, the solution was then left under stirring for 1 h. The resulting white precipitate was washed three to four times with deionized water to remove impurities, dried at 60°C for 24 h, and obtained powder was then calcined in air at 300°C for 3 h (Ngamta et al. 2013).

Characterization of TiO₂ nanoparticles

TEM

Before starting the experiment, the characterization of the morphology and particle size distribution of TiO₂ phase crystals was conducted using a transmission electron microscope (TEM) JEOL 1200 EX (JEOL Ltd, Tokyo, Japan), operating at 120 kV. Powder samples were stirred in distilled water, then an appropriate amount of specimen was put on a carbon grid.

SEM

For the characterization of TiO₂ in an aggregate form, a scanning electron microscope (SEM) QUANTA 450 FEG (FEI Company, USA) was used. The parameters for the optimized measurement were set at accelerating voltage 15 kV.

X-ray analysis

X-ray diffraction (XRD) measurements were carried out using Bruker D8 DISCOVER diffractometer equipped with X-ray tube with rotating Cu anode operating at 12 kW (40 kV, 300 mA). All measurements were performed in parallel beam geometry with parabolic Goebel mirror in the primary beam. The diffraction patterns in the angular range 20–80° of 2θ were recorded in grazing incidence set-up with the angle of incidence α = 1.5°. Parallel plate collimator with the angular acceptance 0.35° was inserted in the diffracted beam.

Preparation of nTiO₂-amended culture medium – type I and barley cultivation

In all, 300 mg of agar and specified amount of nTiO₂ was added to 25 mL of distilled water, to prepare 0–20% (m/m) nTiO₂ suspensions. These suspensions were sterilized at 121°C for 15 min, and after removal from autoclave immediately poured into culture vessels and allowed to cool. Then, seven barley seeds per dish with different nTiO₂ concentrations were transferred to culture vessels. During the first 24 h, the seeds were kept in the dark, then cultured for another five days in a 16 h light/8 h dark cycle at 25°C.

After completion of the cultivation process, plants were washed in distilled water, the lengths of the shoots and the longest root developed were measured and dry weight of plant biomass was recorded. Chlorophyll in leaves was extracted with N,N-dimethylformamide (Centralchem, Slovensko). The absorbance was read at 649 and 665 nm with a spectrophotometer (Optima SP-300). The total contents of chlorophyll a and b were calculated using the extinction coefficients and the equations by Ritchie (2006).

Preparation of nTiO₂-amended culture medium – type II and barley cultivation

The culture medium was prepared by mixing 200 mL of distilled water with 200 mL of Hoagland solution (Hoagland & Arnon 1950). Then, 350 mg of agar (Agar Agar Type I, HiMedia) was added; the solution was boiled and allowed to cool. Prepared nTiO₂ at concentrations of 0, 100, 150, 200, 400, 600, and 1000 mg L⁻¹ were then added to the medium. The suspension was sonicated for 30 min to ensure the uniform distribution of nanoparticles. The control experimental medium contained no nanoparticles.

Barley seeds, supplied by the Sempol, Bratislava, were sterilized in 10% sodium hypochlorite solution and germinated for three days in Petri dishes wrapped in plastic foil. After three days, seven seedlings (for each nTiO₂ concentration) were embedded in a polystyrene holder and placed in a beaker, with roots immersed directly into the culture medium. Air was blown into the solution through a plastic tube with diameter of 0.5 mm connected with a compact laboratory peristaltic pump, type PLP-330 (Behr Labor-Technik, Germany), at a flow rate of 100 mL min⁻¹. Besides providing air delivery to plant roots, the constant airflow prevented nanoparticles from settling at the bottom of the beaker.

Plants were cultured for another seven days in a 16 h light/8 h dark cycle at 25°C. After completion of the cultivation process, plants were washed in distilled water, the lengths of the shoots and the longest root developed were measured and both the wet and dry weights of plant biomass were recorded. Chlorophyll in leaves was extracted with N,N-dimethylformamide (Centralchem, Slovakia). The absorbance was read at 649 and 665 nm with a spectrophotometer (Optima SP-300). The total contents of chlorophyll a and b were calculated as mentioned previously.

Determination of titanium content

Ti was determined using ICP-MS (Perkin-Elmer Sciex Elan 6000) equipped with a cross-flow nebulizer and a Scott double-pass spray chamber. Isotope ⁵⁰Ti was used for data acquisition; ¹⁰³Rh was used as the internal standard. Plasma power: 1200 W.

Results and discussion

Characterization of nTiO₂

TiO₂ exists in a large number of stable and metastable polymorphs, such as rutil, anatase, brookite, etc. Anatase and brookite, both metastable phases, transform into rutile, a stable phase, upon heating (Tobaldi 2012). XRD measurements reveal that the powder heated at 300°C corresponds to anatase (Figure 1). According to SEM analysis, the synthesized anatase phase (Figure 2) forms particles in a wide range of size and morphology. The nanoparticles tend to aggregate; the diameters of TiO₂ aggregates vary from several nanometers to several micrometers (50 µm), with a mean size of 5–10 nm (Figure 3). Anatase-type TiO₂ has a crystalline structure that corresponds to the tetragonal system with dipyramidal habit; it may also occur as rounded spherical grains (Figure 2). Besides, aggregates also exhibit various types of overgrowth. TiO₂ that occurs as aggregated material has its surface covered by adsorbed individual nanoparticles.

Figure 1. XRD patterns of anatase powder with tetragonal symmetry.

Figure 2. SEM image of anatase-type TiO₂ nanopowder: TiO₂ particles form aggregates varying in size from several nanometers up to 50 µm.

Figure 3. TEM image of anatase-type TiO₂: particle size ranges from 5 to 25 nm with a mean size of 5–10 nm. There is no evidence that nTiO₂ have a dominant habit; individual grains have a tendency to form a spherical shape.

nTiO₂-amended culture medium – type I

The results depicted in Figure 4 illustrate a statistically significant effect on the root growth even at the lowest nTiO₂ concentrations (IC₅₀ = 1.51%). The above-ground parts growth was significantly inhibited by 10% content nTiO₂ (IC₅₀ = 28.1%). Since the shoot system and the root system develop more or less parallel and they exert mutual influence, it is unlikely that a reduction of the aerial parts may only be attributed to cytotoxic effect of transported titanium. It is rather a physiological response to lower nutrient supply caused by the reduction in root length. The presence of an effective mechanical or physiological barrier is also confirmed by the analyses of physiological parameters – the contents of chlorophyll a and b in the aerial plant parts. Results in Table 1 show that even at high concentrations, no significant effect of nTiO₂ was detected. While the average weight values of dry shoot plant biomass in treatments with nTiO₂ concentrations over 2.5 g L⁻¹ were lower compared to the control experiment, the statistically significant adverse effects at the level of 0.05 manifested only at the highest concentration tested (Table 2(a)).

Figure 4. Average length of roots and shoots of barley (Hordeum vulgare L.) after six-day cultivation on agar medium (type I) under different nTiO₂ concentrations (%). The lengths of both roots and shoots diminish with rising nTiO₂ concentration. A significant change was observed at concentrations higher than 0.5% and 2.5% for roots and shoots, respectively. The error bars represent standard deviation from seven measurements (* statistically significant difference from control on significance level α = 0.05, ** statistically significant difference from control on significance level α = 0.01, *** statistically significant difference from control on significance level α = 0.001).

Table 1. Content of chlorophyll a and b, total and a:b ratio of leaf sections from the top of the first and second leaves of barley (Hordeum vulgare L.) after cultivation in agar medium (type I) or in hydroponics (type II).

	nTiO2 concentration in culture medium – type I (g L^−1)
	0	5	10	25	100	200
Chl a (µg mg^−1)	0.78 ± 0.09	0.78 ± 0.19	0.92 ± 0.24	0.71 ± 0.29	0.76 ± 0.13	0.82 ± 0.12
Chl b (µg mg^−1)	0.31 ± 0.02	0.27 ± 0.04	0.30 ± 0.07	0.24 ± 0.09	0.31 ± 0.06	0.31 ± 0.04
Chl total	1.09 ± 0.10	1.05 ± 0.23	1.22 ± 0.31	0.95 ± 0.15	1.07 ± 0.18	1.12 ± 0.16
a:b	2.52 ± 0.22	2.86 ± 0.43	3.02 ± 0.28*	2.90 ± 0.23	2.51 ± 0.33	2.64 ± 0.30
	nTiO2 concentration in culture medium – type II (mg L^−1)
	0	100	150	200	400	600	1000
Chl a (µg mg^−1)	1.05 ± 0.22	1.28 ± 0.13*	1.12 ± 0.12	1.40 ± 0.38	1.04 ± 0.19	1.04 ± 0.23	1.14 ± 0.24
Chl b (µg mg^−1)	0.25 ± 0.07	0.30 ± 0.04	0.29 ± 0.04	0.30 ± 0.09	0.24 ± 0.05	0.25 ± 0.08	0.25 ± 0.06
Chl total	1.30 ± 0.29	1.58 ± 0.16	1.41 ± 0.16	1.70 ± 0.50	1.28 ± 0.23	1.29 ± 0.31	1.39 ± 0.29
a:b	4.34 ± 0.44	4.35 ± 0.37	3.93 ± 0.24	4.71 ± 0.36	4.37 ± 0.44	4.36 ± 0.84	4.47 ± 0.10

Note: Standard deviation value was determined from seven measurements.

*Statistically significant difference from control on significance level α = 0.05.

Table 2. The dry weight of shoot biomass of barley (Hordeum vulgare L.) after cultivation in (a) agar medium (type I) or in (b) hydroponics (type II).

	nTiO2 concentration in culture medium – type I (g L^−1)
(a)	0	0.5	1	2.5	10	20
d.wt. (mg)	7.6 ± 0.9	8.5 ± 1.1*	9.6 ± 1.2***	7.08 ± 1.8	6.67 ± 1.76	5.5 ± 2.1
	nTiO2 concentration in culture medium – type II (mg L^−1)
(b)	0	100	150	200	400	600	1000
d.wt. (mg)	19.6 ± 3.0	14.7 ± 2.7**	22.7 ± 6.7	13.9 ± 4.2*	18.8 ± 3.9	18.7 ± 5.0	17.4 ± 4.8

Notes: Standard deviation value was determined from seven measurements.

*Statistically significant difference from control on significance level α = 0.05.

**Statistically significant difference from control on significance level α = 0.01.

***statistically significant difference from control on significance level α = 0.001.

This experiment ought to be perceived as preliminary, conducted with a purpose to determine a concentration required to achieve 50% inhibition (IC₅₀). From the collected data it was found that concentration of nTiO₂ required to be effective in inhibiting growth and selected physiological functions was higher than expected. The release of nTiO₂ was most probably hindered by the dense agar medium significantly affecting the particle transport to the plants. Assuming these extremely high concentration levels (≥10 g L⁻¹) of nTiO₂ are normally not common (have not been reported) in the environment, the concentration range up to 1000 mg L⁻¹, which is usually applied in ecotoxicological studies of nanoparticles’ adverse effects on cultural plants, was selected for further evaluation in our hydroponic experiment. In consequence, in culture media – type I, the plant biomass was not further analyzed for Ti content by ICP-MS.

nTiO₂-amended culture medium – type II

Many crop species are commonly used for phytotoxicity testing. Studies evaluating the effects of nanoparticles on plants have been mainly performed by root exposure on plants grown in hydroponics. There were some reported positive effects (Landa et al. 2012; Singh et al. 2012; Feizi et al. 2013; Song et al. 2013), negative effects (Asli & Neumann 2009; Ruffini Castiglione et al. 2011), or no effect (Seeger et al. 2009; Landa et al. 2012; Larue et al. 2012; Song et al. 2013) of nTiO₂ on plant development.

For the purpose of our experiment, barley was grown in hydroponic culture for seven days, the plants were then removed from the beaker, weighted, and the lengths of roots and shoots were measured. Roots and shoots were analyzed for titanium content, and shoots were analyzed for total chlorophyll. Plants were then air-dried for one week and weighted again.

From the measured values, it is obvious that nTiO₂ at the studied concentration range have no effect on the growth of aerial plant parts. ICP-MS analysis confirmed that there was no enhanced Ti translocation into aerial plant parts under nanoparticle treatments under 400 mg nTiO₂L⁻¹ compared to the control (Table 3). On the contrary, the average roots’ length was less with the increase in nTiO₂ concentrations. The roots at nTiO₂ concentration of 400 mg L⁻¹ reached only half of the length of roots in the control sample (Figure 5). Elevated nTiO₂ concentration did not significantly affect the plant weight. The contents of chlorophyll a and chlorophyll b under nTiO₂ treatment are set out in Table 1. There were no significant changes for total chlorophyll in groups treated with different concentrations (Student’s t-test, at P ≤ .05). The ratio of chlorophyll a:b was relatively constant, varying from 3.91 to 4.63. The proportion of chlorophyll a was larger than that of chlorophyll b.

Figure 5. Average lengths of roots and shoots of barley (Hordeum vulgare L.) after seven-day cultivation in hydroponics (type II) under different nTiO₂ concentrations (mg L⁻¹). Only root length is affected when concentration of nTiO₂ is above 150 mg L⁻¹. The error bars represent standard deviation from seven measurements (* statistically significant difference from control on significance level α = 0.05, ** statistically significant difference from control on significance level α = 0.01, *** statistically significant difference from control on significance level α = 0.001).

Table 3. Titanium concentrations (mg kg⁻¹ d wt.) in roots and shoots of barley (Hordeum vulgare L.) cultivated for seven days in nTiO₂-amended hydroponics (type II) and determined by ICP-MS.

nTiO2	Ti content
(mg L^−1)	Roots	Shoots
0	26.3	5.4
100	1030	7.4
150	166	5.8
200	949	5.6
400	1490	24.1
600	418	14.1
1000	508	11.5

Findings of the present study correspond with some of the results found in literature. The effect of nTiO₂ on root growth was observed in several agronomic plant species at similar or lower nTiO₂ concentrations. Andersen et al. (2016) exposed 10 agronomic plant species to different concentrations of nTiO₂ (0, 250, 500, and 1000 µg mL⁻¹) to examine potential effects on germination and early seedling development. Six of ten species responded to nTiO₂ through a change in average root length, with four of the six species showing decreased and two species showing increased root length. Regarding barley-related plants (Poaceae family), nTiO₂ decreased average root length in oats, while the root length remained unaffected in ryegrass. Use of low concentrations (20–80 mg L⁻¹) of nTiO₂ enhanced fennel seed germination (Feizi et al. 2013) and caused an increase of root elongation during the first stages of development of wheat (50–100 mg L⁻¹, Larue et al. 2012). Some other nanoparticles of metal oxides such as ZnO and Al₂O₃ behave in a similar way to nTiO₂. There is therefore reason to believe that the photocatalytic activity occurring on the surface of metal oxides’ nanoparticles, including the production of reactive oxygen species (Larue et al. 2012; Andersen et al. 2016), can be one of the important processes that make these nanoparticles toxic. This fact was observed also by Ghosh et al. (2010) who reported an interaction between nTiO₂ and both plant and human cells where nTiO₂ produced superoxide radicals inducing oxidative stress and lipid peroxidation. By accumulating in the cell wall surfaces of primary roots and reduction of cell wall pore size, nTiO₂ may also interfere with water transport to some extent (Asli & Neumann 2009).

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Lucia Kořenkova completed her Masters´ and PhD degree at the Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia, where she currently works as a researcher at the Institute of Laboratory Research on Geomaterials. Her research interests cover the areas of soil mapping and classification, crop-soil interactions, environmental investigation and nanoparticle research. She has published several papers in scientific journals and has given presentations at scientific and professional meetings.

Martin Šebesta is currently a PhD student at the Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. He researches nanoparticle transport in soils and their transport to plants and other soil organisms.

Martin Urík is a researcher at the Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. He has published several research articles on environmental geochemistry of potentially toxic metals and metalloids.

Marek Kolenčík is an associate professor at the Department of Geology and Soil Science, Faculty of Agrobiology and Food Resources at Slovak University of Agriculture in Nitra. He has published more than 20 research articles with more than 100 SCI citations on bioleaching, biohydrometallurgy and bionanotechnology research.

Gabriela Kratošová is a senior researcher at VŠB - Technical Univerzity of Ostrava, Nanotechnology Centre, Czech Republic. She is an electron microscopy specialist. She has published several articles on biosynthesis of nanoparticles, their characterization and application in catalysis and disinfection.

Marek Bujdoš earned his PhD degree in analytical chemistry. He is a researcher at the Institute of Laboratory Research on Geomaterials, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. He works on developing new methods of atomic and mass spectrometry.

Ivo Vávra is currently a senior researcher at the department of Microelectronics and Sensors, Institute of Electrical Engineering of Slovak Academy of Sciences. His expertise lies in the analytical approach for the characterization of nanoparticles examined via transmission electron microscopy analysis. He has published many excellent articles on bio-nano-materials and their unique physical and chemical properties.

Edmund Dobročka is currently a senior scientist at the Institute of Electrical Engineering of Slovak Academy of Sciences. His research interest is focused on X-ray diffraction analyses of the structural properties of thin films with many relevant published articles on this topic.

ORCID

Martin Urík http://orcid.org/0000-0001-7998-7992

Additional information

Funding

This work was supported by the Scientific Grant Agency of the Slovak Republic Ministry of Education and the Slovak Academy of Sciences under VEGA contract No. 1/0203/14 and SGS project No. SP2016/72.