CC BY
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Section 10. Agricultural sciences
Turov Volodymyr Vsevolodovich, Corresponding Member of NAS of Ukraine, Chuiko Institute of Surface Chemistry of NAS of Ukraine, Kyiv, Ukraine E-mail: [email protected]
Yukhymenko Elena Viktorovna, Chuiko Institute of Surface Chemistry E-mail: [email protected]
Krupskaya Tetiana Vasilievna, PhD, Chuiko Institute of Surface Chemistry E-mail: [email protected]
Suvorova Lyudmyla Anatolievna, Macrosorb. LT, Vilnius, Lithuania E-mail: [email protected]
Influence of nanosilicas on seeds germination parameters and state of water in nanocomposites “ekostim” and partially dehydrated roots of wheat
Abstract: It is shown that nanosilica A-300 can stimulate the germination of wheat seeds like protective and stimulating mixtures based on methylated silica and mineral fertilizers. The state of water in partially dehydrated wheat roots at different stages of germination is investigated. It is found that the residual water in biomaterials is in the cluster state and is revealed as two signals with different values of the chemical shift in 1H NMR spectra 5 and 1 ppm for strongly and weakly associated water, respectively. Nanosilica locating in the zone of germination significantly alter the concentrations of different forms of water. An assumption was made that bioactivity of nanosilica A-300 is due to the fact that it stabilizes weakly associated form of water.
Keywords: Н NMR spectroscopy, strongly- and weakly bound water, water clusters, composite system, roots of wheat, trace elements, major elements.
Application of dust-like coatings, created on the basis of nanoparticles of silicas or their mixtures which actively influence water balance of germinative seeds and provide plants with local nutrition (the protective and stimulating mixtures — PSM), may become a promising direction of application of nanotechnologies for pre-treatment of seeds before sowing. Such protective pre-treatment can effectively solve the problems which arise in case of winter and early spring sowing for many types of crops [1-3]. Nanostructural forms of trace elements are being intensively sought at present time [4-6]. As the crystal nanostructures possess considerably bigger surface energy than the bulk ones, dissolution of them in soil moisture and transition to the germination zone may be performed with a significantly lower energy use.
At the Chuiko Institute of Surface Chemistry of NAS of Ukraine novel protective and stimulating compounds for pretreatment of crop seeds before sowing, which provide an increase in germination capacity, decrease in number of affected seedlings and, eventually, increase in crop yield, were developed. They are nanocomposites, to the structure of which, if necessary, may be added a full range of essential major elements (nitrogen, phosphorus, potassium), trace elements (B, Mg, Mn, Zn, Cu, Mo, Co, etc.), crop protection agents, growth
stimulators, ameliorative agents, organic or microbiological fertilizers and adhesive-carriers [7].
The influence of silica nanoparticles on seed grain during early stages of germination has still not been fully investigated. In [8; 9] an assumption is made that nanoparticles may influence a state of water in the germination zone, and the method of low-temperature 1H NMR spectroscopy is applied to demonstrate the capability ofweakly associated water, whose molecules take part in formation of less than two hydrogen bonds for each molecule, to form at the interface of nanosilica particles or layers of PSM. A large amount of weakly associated water was found as well in a number of weakly hydrated biological objects, including seeds of wheat [8].
The purpose of this paper is to study the state of water bonded nanocomposites “Ecosim” and in roots ofwheat during early stages of germination, and how it is influenced by the presence of particles of hydrophilic nanosilica or protective and stimulating mixtures, prepared on the basis of methylated (hydrophobic) silica and mineral fertilizers in a zone of contact between seeds and water medium.
Experimental part
A finely dispersed silica A-300 with a specific surface area of 300 m2/g, produced by Kalush Test Experimental Plant
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Influenceofnanosilicasonseedsgerminationparametersandstateofwaterinnanocomposites“ekostim”andpartiallydehydratedrootsofwheat
(Ukraine) and synthesized by high temperature hydrolysis of SiCl4 in a flame of an oxyhydrogen torch, was used. Methyl silica AM-1 (Kalush, Ukraine) is synthesized by treating aerosil A-300 with methylchlorosilane. PSM was produced by mecha-nochemical activation of methylaerosil AM-1 with a specific surface area of 300 m2/g in a ball mill in the presence of mixture (1:1:1) of potassic, phosphoric and nitric fertilizers.
Seeds of winter wheat (cultivar Kiyanka), which were couched in the Petri dishes at (22-25) оС in ordinary drinking water (control 1), in the same amount ofwater containing 1 wt % of finely dispersed silica (2), and pre-treated protective and stimulating mixtures (3), were investigated. On the bottom of cups there was a filter paper. Filter paper was moistened daily, i. e. seeds of wheat were constantly moist.
After three days of germination half of all seeds was taken, and calculation of germination energy was made for it; germination capacity, length and mass of roots and seedlings were estimated. On the eighth days the same observations were made for the rest of seedlings and roots.
The NMR spectra were recorded using a NMR spectrometer of high-resolution Varian Mercury (operating frequency 400 MHz). The 90° probe pulse with a duration of 3 ps was
used. The temperature was controlled by Bruker VT-1000 device with relative mean errors ± 1 K. The signal intensity was determined by measurement of area of peaks by using procedure of decomposition of signal into its constituents in the assumption of Gaussian shape of a signal and optimization of its zero line and phase with relative mean errors not lower than 5 % for well-resolved signals, and ± 10 % for the overlapping signals. To prevent supercooling of water in the studied objects, the measurements of the concentration of unfrozen water were carried out on heating of samples preliminarily cooled to 210 K. Concentration of unfrozen water in samples was calculated by comparison of signal intensity of water in the sample, containing an unknown amount of water, with signal intensity of water, determined by thermogravimetric analysis. The initial humidity of root samples was 7 wt %. An NMR technique for measurements and determination of thermodynamic characteristics and radii of clusters of interfacial water is described in detail in [8].
Results and discussion
Values of biometric parameters of germinated seeds, which were grown under different conditions, are given in Table 1.
Table 1. - Biometric parameters of wheat germination in the presence of nanosilica A-300 and PSM in comparison with control
Parameter Control 1 % SiO2 PSM
Germination energy, % 67 75(+8%) 88(+21 %)
Germination capacity, % 74 81(+7 %) 95 (+21 %)
Seedling length, cm. 7.5 10,7 (+42 %) 11.3 (+50 %)
Wet weight of seedlings, g. 1.05 1.65 (+57 %) 1.66 (+58 %)
100 seedlings mass, g. 1.54 2.21 (+43 %) 1.9 (+23 %)
Root length, cm. 4.5 3.4 (-24 %) 8.4 (+86 %)
Wet weight of roots, g. 1.1 1.23 (+12 %) 2.0 (+82 %)
Amount of roots per plant, pcs. 2.6 3.7 (+42 %) 4.2 (+61 %)
As is clear from the Table, both nanomaterials may have a considerable stimulating influence on biometric parameters of germinated wheat. There is a significant increase of length and mass of seedlings, as well as amount of roots per plant. PSM has more influence on germination capacity and wet weight than the finely dispersed silica A-300 has. Thus, however, one should consider that PSM consists of the complex of mineral fertilizers, whose localization at the seed surface is provided by capsulation of them by the hydrophobic membrane consisting of particles of a methyl-silica, while A-300 may have a stimulating influence only due to the interaction of nanoparticles with biosystems of plant in a germination zone.
On the surface of most seed types there are hydrophobic areas which provide sensitivity to the hydrophobic component of product «Ekostim» (nanoparticles of methyl silica AM-1). Therefore hydrated powders «Ekostim» in the medium of low-polarity organic solvent may serve as a fine model of examination of interaction between particles of product «Ekostim» and water. For this purpose we chose deuterochloroform, which in 1H NMR spectra produces only small signal for the residual protons of CHCl3 with chemical shift §H = 7.26 ppm.
Recorded at different temperatures 1H NMR spectra of water in suspended in deuterochloroform medium powder «Ekostim», which contains 12 wt % of water, are given in Figure 1. As is clear from Fig. 1, water is observed in the form of two signals (1 and 2) which have chemical shifts 5 and 1.2 ppm respectively. Density ratio of signals 1 and 2 is 2:1. According to the above-mentioned classification signal 1 relates to the protons of strongly associated water (SAW) regulated by hydrogen bonding network, and signal 2 — to weakly or non-associated water (WAW), in which the number of hydrogen bonds per one water molecule is less than unity.
Considering that salts which are present in product «Ekostim» possess significant hydrophilic characteristics, one may suppose that the signal of strongly associated water is determined by the water, adsorbed on the surface of salt microcrystals, distributed across the surface of hydrophobic silica nanoparticles. Dominating in spectra at low temperatures signal of weakly associated water may be related to the water on the phase boundary of product «Ekostim» and hydrophobic medium. The water on the boundary of product «Ekostim» and seed surface may possess the same characteristics.
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Section 10. Agricultural sciences
Fig. 1. Influence of temperature on the form of 1H NMR spectra of water in suspension of the product «Ekostim», which contains 12 wt % of water, in deuterochloroform medium
Thus the composition of the product «Ekostim» is quite complex (besides hydrophobic silica АМ-1 it includes very soluble in water substances — urea, superphosphate and KCl) we chose one of the components, in particular urea — (NH2)2CO, to study water forms which may appear on the
WAW
a)
SAW
ö (ppm)
c)
phase boundary of nanodisperse hydrophobic-hydrophilic solid objects and low-polarity organic medium. In high-resolution spectra protons of NH2 groups are recorded only in solutions, since in solid objects due to low molecular mobility the NMR signal is very wide [10].
Recorded at different temperatures 1H NMR spectra of powders «Ekostim» which contain different amounts ofwa-ter are given in Figure 2. Composites were received by mech-ano-chemical activation of methyl silica AM-1 and urea. Concentration ratio AM-1: (NH2)2CO for all samples remained constant and was 1:4. The measurements were taken in low-polarity medium of deuterochloroform.
In conditions of sample minimum humidity, which corresponds to its water saturation from air under normal conditions (СН2 О = 1 wt %) several proton signals are recorded in deuterochloroform medium (Fig. 2-a). Signals of chloroform and tetramethylsilane (SH = 7.26 и 0 ppm. respectively) relate to dispersion medium. The dominating in spectra signal is the signal ofWAW. It consists of two components, which differ in width and slightly differ in chemical shift. It may be assumed that narrower signal with weak intensity relates to water, dissolved in phase of liquid chloroform. Strongly associated water appears as well in the form of 2 signals, which may be related to signals of urea solution and water, adsorbed on interphase boundaries of heterogenic system.
SAW
~l--'----1---'----1---1---r---’----1---’----1
8 6 4 2 0 -2
й (ppm)
b)
SAW
d)
Fig. 2. Recorded at different temperatures 1H NMR spectra of powders АМ-1 adsorptively modified by urea in medium CDCl3 under: a) — СН2 О=1 wt %; b) — СН2 О=3.4 wt %; c) — СН2 О=11 wt %; d) — СН2 О=11 wt % after additional mechano-chemical activation in the presence of water
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Influenceofnanosilicasonseedsgerminationparametersandstateofwaterinnanocomposites“ekostim”andpartiallydehydratedrootsofwheat
As the temperature decreases, the intensity of the signal of SAW2 decreases much greater, which gives reason to consider it as conditioned by less concentrated urea solution or integrated large water polyassociates [8].
As the concentration of water in sample increases, the amount of weakly associated water, related to the interphase boundary of nanocomposite and hydrophobic medium, decreases (Fig. 2b-d). In spectra the signal of NH2-groups with chemical shif §H = 5.6 ppm appears. These data indicate that nearly all strongly associated water relates to liquid film of urea solution, forming on the surface of microcrystals of solid urea, which is a component of nanocomposite.
Recorded at different temperatures 1H NMR spectra of water in partially dehydrated roots of the control sample of germinated wheat in air and in the low-polarity organic solvent — deuterochloroform (CDCl3) — are given in Figure 3. The deuterated analogue was used to prevent introduction of intensive signal of solvent protons in spectra. In air residual water is observed in spectra in the form of two stretching signals, whose chemical shifts are about 1 and 5 ppm.
According to the classification given in [8] they should be referred to weakly and strongly associated water (WAW and SAW respectively). The low-polarity medium leads to considerable decrease of the width of water signals, and SAW is recorded in the form of two signals — SAWa и SAWb , which differ in width and chemical shift. Thus the residual signal CH of protons of non-deuterated component of chloroform and CH3-groups of tetramethylsilane (TMS) added to chloroform as the standard are also observed in spectra. Considerable decrease of the width of signals in spectra should be referred to decrease in inhomogeneous broadening related to big difference between magnetic susceptibilities of air and biomaterial [10]. Signals of protons of glycan biopolymer chains, which form the basis of dehydrated roots, aren’t observed in spectra considering very short (up to 10-6) transverse relaxation time of protons in solids [10]. For other samples of roots (whose spectra aren’t given) the spectra remained similar to those given in Figure 4 and changed only due to some redistribution of intensities of strong and weakly associated water signals.
Fig. 3. Recorded at different temperatures 1H NMR spectra of water in wheat roots at their humidity of 7 wt % in air (a, b) and in the medium of CDCl3 (c, d) in case of germination of them within 3 (a, c) and 7 (b, d) days
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Section 10. Agricultural sciences
Fig. 4. Temperature dependences of signal intensity of different forms of interfacial water recorded in air and in the medium of CDCl3 in partially dehydrated wheat roots at various terms of germination
It follows from Fig. 3 a, b, that with decrease in temperature the signal intensity of SAW decreases much more strongly, than WAW i. e. weakly associated water is mainly strongly bound water (SBW, which is capable of freezing at T < 250 K [8]). The low-polarity medium of chloroform reduces the capacity of WAW for freezing even more (Fig. 3 c, d). The correlation of intensities of weakly and strongly associated water may significantly change with the increase in time of germination, and this effect depends on the measurement medium. So, in air (Fig. 3 a, b) after 3 days of germination slightly stronger signal intensity of WAW is recorded, and after 7 days of germination slightly stronger signal intensity of SAW is recorded. On the contrary, in the medium of CDCl3 (Fig. 3 c, d) along with the increase in time of germination the amount of SAW increases drastically. This implies, that even the hydrophobic organic
medium isn’t inert to the water localized in the internal cavities cased in a biopolymer (cellulose) matrix of material of dehydrated roots, and is capable of influencing correlation of amount of different water forms, as well as energetic parameters of water bounding with inner phase boundaries.
Temperature dependences of signal intensity of different forms of interfacial water for partially dehydrated roots couched in the presence of nanosilica A-300 and PSM in comparison with control are given in Figure 4. For recorded in air spectra only total amounts of strongly and weakly associated water (CSAW+WAW) were calculated, since the division of the broad, closely located signals led to considerable miscalculation. In the medium of CDCl3, when possible, concentrations of two types of strongly associated water (CSAW (a) CSAW (b)) and weakly associated water (CWAW) were calculated. Vertical
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Influenceofnanosilicasonseedsgerminationparametersandstateofwaterinnanocomposites“ekostim”andpartiallydehydratedrootsofwheat
line at T = 250 K allows to estimate the content of strongly and weakly bound water (WBW which freezes at T > 250 K) for each studied system.
From the results shown in Fig. 4 it follows that in air (Fig. 4 a, b) in samples there is a great amount of weakly and strongly bound water. The contribution from WAW decreases for the sample couched in the presence of A-300. In the presence of PSM after 3 days of germination the correlation WBW/SBW slightly changes, and after 7 days the amount of WBW increases a little.
In the medium of CDCl3 the major part of strongly associated water, recorded in spectra as a signal (b), is weakly bound (Fig. 4 c, d). At the same time almost all water, which is observed as a signal (b), belongs to the strongly associated water. After 3 days of germination in the medium of A-300, in the layer of SAW, the part of weakly associated water decreases, and after 7 days — increases.
In a layer of weakly associated water (Fig. 4 e, f) after 3 days of germination in the presence of both nanomaterials the decrease of total amount of WAW is observed, and the minimal values of CWAW are recorded in the presence of finely dispersed silica A-300, however for this sample after 7 days of germination CWAW becomes maximal and is more than 60 %
of the total content of water in biomaterial. Over half of total amount of weakly associated water should be referred to strongly bound water.
The conclusion
Thus the nanostructured highly dispersed silica A-300 as well as PSM on the basis of methylated silica may have an essential influence on biometric parameters of germination of wheat, increasing the volume and mass of forming seedlings and roots. It is discovered that water in partially dehydrated wheat roots at different stages of germination is in a cluster state and in strongly and weakly associated forms which are registered in 1H NMR spectra in the form of separate signals differing in size of chemical shift.
Germination of seeds in the presence of nanomaterials changes a correlation of concentrations of different water forms. The size of the effect depends on the type of the used material and period of germination. In the presence of finely dispersed silica A-300 after 7 days from the beginning of germination in samples of roots the maximal amount of weakly associated water is recorded. Apparently it’s one of the main factors defining high biological activity of this nanosilica, which is capable of stimulating development of plants even in the absence of nutrients introduced from the outside.
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