Chemical, Biological and Mechanical Characterization of Wood Treated with Propolis Extract and Silicon Compounds

: The development of new bio-friendly alternatives for wood conservation is of great interest and necessary for environmental protection. In this paper, the preparations based on the propolis extract and silicon compounds were used as green wood preservatives. The wood was treated with 15% propolis extract (EEP) and two propolis-silane preparations, namely, EEP-VTMOS / TEOS (EEP with vinyltrimethoxysilane and tetraethyl orthosilicate) and EEP-MPTMOS / TEOS (EEP with 3-(trimethoxysilyl) propyl methacrylate and tetraethyl orthosilicate). The aim of the research was to determine the properties of treated wood, which was characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), atomic absorption spectroscopy (AAS), X-ray ﬂuorescence (XRF), and scanning electron microscopy (SEM). Moreover, the resistance against brown-rot fungus Coniophora puteana and the mechanical properties of treated wood were also determined. The analysis of phenolic compounds concentration in treated wood indicated that phenols were in greater extent leached from wood treated with the propolis extract than from wood impregnated with the propolis-silane preparations. The presence of silicon in treated wood both before and after leaching was conﬁrmed by CP MAS NMR measurements. In turn, AAS and XRF analyses indicated that the degree of Si leaching from wood impregnated with EEP-VTMOS / TEOS was approximately two times lower than from EEP-MPTMOS / TEOS treated wood. The results of chemical analyses conﬁrmed that the constituents of the propolis-silane preparations formed permanent bonds with wood. In turn, the results of the antifungal e ﬃ cacy against C. puteana showed that the propolis extract and the propolis-silane preparations limited the fungus activity, even the wood was subjected to leaching procedure. The treated wood showed an increase in bending strength and a decrease in the modulus of elasticity compared to untreated wood. The obtained results indicate that the propolis-silane preparations can be promising green wood preservatives, harmless for the natural environment.

Forests 2020, 11, x FOR PEER REVIEW 3 of 17 scanning electron microscopy (SEM). The resistance of the treated wood against brown-rot fungus C. puteana was also assessed. Moreover, the mechanical properties, namely, bending strength and modulus of elasticity of treated wood, were determined.

Wood Treatment
The investigated material was Scots pine (Pinus sylvestris L.) sapwood without knots or other growth inhomogeneity. The wood samples with dimensions of 5(T) × 10(R) × 40(L) mm 3 were used for biological and chemical analyses. The wood samples with dimensions of 5(T) × 10(R) × 150(L) mm 3 were applied in the determination of mechanical parameters. Before impregnation process, the specimens were conditioned at 65 ± 5% relative humidity (RH) and 20 ± 2°C to attain equilibrium moisture content of approximately 12%. Next, all the wood samples were impregnated with EEP, EEP-VTMOS/TEOS and EEP-MPTMOS/TEOS using the vacuum method, according to EN 113: 1996 [43]. The samples underwent 15 min under vacuum conditions-0.8 kPa and 2 h under atmospheric pressure. After impregnation, all the samples were removed from the impregnating solutions and weighed to determine the uptake of the solutions. The wood sample retention (kg/m 3 ) was calculated as the following equation: where Ma-the wood mass before treatment (g); Mb-the wood mass after treatment (g); cconcentration of propolis extract or propolis-silane preparations constituents (%); v-the volume of the wood sample (cm 3 ). After impregnation, all the wood samples were cured for four weeks in room conditions (RH = 65 ± 5%; T = 20 ± 2 °C).

Accelerated Aging of Wood-Leaching Procedure
The aim of the artificial ageing (leaching in water) was to determine anti-leaching stability of the treatment preparations constituents from the treated wood. The leaching procedure of the treated

Wood Treatment
The investigated material was Scots pine (Pinus sylvestris L.) sapwood without knots or other growth inhomogeneity. The wood samples with dimensions of 5(T) × 10(R) × 40(L) mm 3 were used for biological and chemical analyses. The wood samples with dimensions of 5(T) × 10(R) × 150(L) mm 3 were applied in the determination of mechanical parameters. Before impregnation process, the specimens were conditioned at 65 ± 5% relative humidity (RH) and 20 ± 2 • C to attain equilibrium moisture content of approximately 12%. Next, all the wood samples were impregnated with EEP, EEP-VTMOS/TEOS and EEP-MPTMOS/TEOS using the vacuum method, according to EN 113: 1996 [43]. The samples underwent 15 min under vacuum conditions-0.8 kPa and 2 h under atmospheric pressure. After impregnation, all the samples were removed from the impregnating solutions and weighed to determine the uptake of the solutions. The wood sample retention (kg/m 3 ) was calculated as the following equation: where M a -the wood mass before treatment (g); M b -the wood mass after treatment (g); c-concentration of propolis extract or propolis-silane preparations constituents (%); v-the volume of the wood sample (cm 3 ). After impregnation, all the wood samples were cured for four weeks in room conditions (RH = 65 ± 5%; T = 20 ± 2 • C).

Accelerated Aging of Wood-Leaching Procedure
The aim of the artificial ageing (leaching in water) was to determine anti-leaching stability of the treatment preparations constituents from the treated wood. The leaching procedure of the treated wood samples for the outdoor application was performed according to EN 84:2000 [44]. The wood samples (used for biological test and chemical analyses) were impregnated with deionized water by vacuum method (20 min) and next soaked in deionized water for 14 days. The water was exchanged 10 times for all duration of leaching procedure.

Decay Resistance Test
The decay resistance of the control and treated wood samples with the propolis extract and the propolis-silane preparations took place before and after the leaching procedure wood samples against brown-rot fungus-Coniophora puteana (Schumacher ex Fries) Karsten BAM 112 (BAM Ebw. 15). This was carried out in accordance with the modified EN-113:1996 [43]. Scots pine (Pinus sylvestris L.) sapwood with dimensions of 5(T) × 10(R) × 40(L) mm 3 was used in this study. The control and treated wood samples (five replicates of each impregnation variant, before and after leaching) were placed into Petri dishes and exposed to decay fungi at 22 ± 2 • C and relative humidity of 70 ± 5% for 8 weeks. One control sample and treated sample were placed in each Petri dish. After exposure to C. puteana, the fungus mycelium was removed from each wood sample, and the samples were weighed in order to determine the mass loss of each wood sample caused by fungi.

Mechanical Properties
Bending strength and modulus of elasticity were measured according to PN-77/D-04103 [45] and PN-63/D-0411 [46], respectively, using ZWICK ZO50TH wood testing machine (Zwick/Roell, Ulm, Germany). Determination of mechanical parameters was carried out on wood samples with dimensions of 5(T) × 10(R) × 150(L) mm 3 . This size of the samples was chosen to ensure that even penetration of treatment preparations had occurred throughout the wood sample. The distance between supports during the experiment was 120 mm. The load was applied in the midway of the sample, in the tangential direction. Before measurements, all wood samples were conditioned at 20 ± 2 • C and relative humidity of 52 ± 2% to constant weight. In accordance with ISO 13061-2 [47], the density of the conditioned samples was determined by a stereometric method, using an analytical balance accurate to 0.001 g (Sartorius GmbH, Göttingen, Germany) to measure the mass of samples and a digital caliper with an accuracy of up to 0.01 mm to determine their dimensions. The density was calculated as the ratio of the mass to the volume. Wood moisture content (MC) was determined by a gravimetric method, according to ISO 13061-1 [48]. All determination was carried out in ten replicates.

Chemical Characterization of Wood
For chemical analyses, the control and treated wood samples with dimensions of 5(T) × 10(R) × 40(L) mm 3 were used. In chemical analyses (except for SEM measurements), the treated wood after the leaching procedure (according to EN 84:2000) was also used to determine the durability of the bond between treatment preparations constituents and wood. Six samples from each treatment variant (before and after leaching procedure) were used in XRF analysis. Then, the six samples of each treatment variant were ground on a laboratory mill (Ika Werke, Staufen, Germany) and in the form of sawdust were used in the FTIR, AAS, NMR, and phenolic content analyses.

Fourier Transform Infrared Spectroscopy (FTIR)
The ground untreated and treated wood samples were mixed with KBr (Sigma Aldrich, Darmstadt, Germany) at a 1/200 mg ratio, and in the form of a pellet were analyzed using the Nicolete iS5 spectrophotometer with Fourier transform (Thermo Fisher Scientific, Waltham, MA, USA). The spectra of wood samples were registered, at a range of 500-4000 cm −1 , at a resolution of 4 cm −1 , registering 32 scans.

Phenolic Concentration Analysis
The ground treated wood samples (0.2 g) before and after leaching in water were extracted with 10 mL methanol (Avantor Performance Materials, Gliwice, Poland) for 24 h at a room temperature using a PSU-10 orbital shaker (SIA Biosan, Riga, Latvia). After extraction, the samples were filtered, and supernatants were used to determine the total phenolic content using Folin-Ciocalteu reagent, according to the method described by Singleton et al. [49]. The supernatants (0.1 mL) were placed in a test tube, followed by the addition of 0.25 mL Folin-Ciocalteu reagent (Sigma Aldrich, Darmstadt, Germany) and 2.65 mL deionized water. After 5 min, 3 mL of a 10% sodium carbonate solution (Avantor Performance Materials, Gliwice, Poland) were added to each tube. The solutions were shaken and incubated for 40 min at room temperature. After this time, the absorbance was measured at λ = 760 nm using a UV-VIS spectrophotometer Carry-300BI (Agilent Technologies, Santa Clara, CA, USA). The results were expressed as mg of gallic acid equivalents/mL of solutions. All determinations were carried out in triplicate.

29 Si Nuclear Magnetic Resonance (NMR)
The solid-state cross-polarization magic angle spinning (CP MAS) NMR experiments were performed on a BRUKER Avance III 400 spectrometer (Billerica, MA, USA) operating at 400.13 for 1 H and 79.495 MHz for 29 Si and equipped with a MAS probe head using 4-mm ZrO 2 rotors. A sample of Q8M8 was used for setting the Hartmann-Hahn condition and as a chemical shift reference (δ = 0.00 ppm). The spectra of treated wood samples were recorded with a MAS frequency of 8000 Hz and proton 90 • pulse of 6.0 µs in length and a contact time of 5 ms. The repetition delay was 4 s, and the spectral width was 48.0 kHz. The FIDs (free induction decay) were accumulated with a time domain size of 2 K data points with SPINAL16 decoupling sequence during the acquisition time.

X-Ray Fluorescence (XRF)
The samples of treated wood with dimensions of 5(T) × 10(R) × 40(L) mm 3 were used in this study. The surfaces of treated wood were analyzed using X-ray fluorescence spectrometer Bruker Tracer III-SD (Billerica, MA, USA). Each sample was scanned in five points using a collimator with a 5 × 5 mm 2 screen. The time of one measurement was 30 s. Quantitative values of silicon on the treated wood surface were determined using the MajMudRock calibration method.

Flame Atomic Absorption Spectrometry (FAAS)
The ground treated wood samples (0.5 g) were mineralized with 8 mL of nitric acid (Sigma Aldrich, Darmstadt, Germany) in the mineralization system (CEM Corporations, Matthews, NC, USA). The digested solutions were filtered and diluted to 50 mL with deionized water. The concentration of silicon in the samples was determined by a flame atomic absorption spectrometry (FAAS) using an AA280FS spectrometer (Agilent Technologies, Santa Clara, CA, USA). The correctness of the method was verified by analysis of the certified reference material NCS DC 73350-leaves of poplar (NACIS, Shanghai, China). The results were expressed as the average values in triplicate measurements.

Scanning Electron Microscopy (SEM)
The surface morphologies of treated wood samples were examined by a Zeiss EVO 40 scanning electron microscope (Carl Zeiss AG, Oberkochen, Germany), which used an electron acceleration voltage of 10 keV. Before microscope analysis, small wood samples (10 mm square) were trimmed from treated wood and next coated with a layer of gold using a Balzers SCD00 sputter coater (BalTec Maschinenbau AG, Pfäffikon, Switzerland).

Statistical Analysis
The results were analyzed using a one-way analysis of variance (ANOVA) applying Tukey's Honest Significant Differences (THSD) Test. Statistical significance was defined as p < 0.05. All the statistical analyses were performed using the TIBCO Software Inc. Statistica version 13.1 (Palo Alto, CA, USA).

Chemical Characterization of Treated Wood
In the first stage of the research, the chemical interaction between the components of the propolis-silane preparations and wood was determined using instrumental methods. The wood was treated with 15% extract of Polish propolis and two propolis-silane preparations, namely, EEP-VTMOS/TEOS and EEP-MPTMOS/TEOS. The concentration of the propolis extract was chosen based on our previous studies, which indicated that Scots pine wood impregnated with ethanolic propolis extract above 12% concentration limited fungal decay [33]. Figure 2 presents the FTIR spectrum of the propolis extract, and Table 1 shows the most important bands of this spectrum, characterized according to literature data [50][51][52][53].

Statistical Analysis
The results were analyzed using a one-way analysis of variance (ANOVA) applying Tukey's Honest Significant Differences (THSD) Test. Statistical significance was defined as p < 0.05. All the statistical analyses were performed using the TIBCO Software Inc. Statistica version 13.1 (Palo Alto, CA, USA).

Chemical Characterization of Treated Wood
In the first stage of the research, the chemical interaction between the components of the propolis-silane preparations and wood was determined using instrumental methods. The wood was treated with 15% extract of Polish propolis and two propolis-silane preparations, namely, EEP-VTMOS/TEOS and EEP-MPTMOS/TEOS. The concentration of the propolis extract was chosen based on our previous studies, which indicated that Scots pine wood impregnated with ethanolic propolis extract above 12% concentration limited fungal decay [33]. Figure 2 presents the FTIR spectrum of the propolis extract, and Table 1 shows the most important bands of this spectrum, characterized according to literature data [50][51][52][53]. In the spectrum of the propolis extract, the bands assigned to the vibrations of C=O, C=C, N-H and C-H bonds derived from phenolic compounds present in propolis were observed. The bands in the spectrum were observed at: 2926 and 2849 cm −1 , which are connected with C-H stretching vibrations and confirm the presence of long-chain alkyl compounds in the propolis extract. The observed stretching and bending bands at 1636, 1514 and 1450 cm −1 correspond mainly to aromatic rings of phenolic compounds specific for propolis extracts. In addition, the wide band with a maximum at 3420 cm −1 described to the stretching vibrations of O-H band also confirms the presence of phenolic compounds in the extract.  In the spectrum of the propolis extract, the bands assigned to the vibrations of C=O, C=C, N-H and C-H bonds derived from phenolic compounds present in propolis were observed. The bands in the spectrum were observed at: 2926 and 2849 cm −1 , which are connected with C-H stretching vibrations and confirm the presence of long-chain alkyl compounds in the propolis extract. The observed stretching and bending bands at 1636, 1514 and 1450 cm −1 correspond mainly to aromatic rings of phenolic compounds specific for propolis extracts. In addition, the wide band with a maximum at 3420 cm −1 described to the stretching vibrations of O-H band also confirms the presence of phenolic compounds in the extract. In Figure 3, the spectra of wood treated with the propolis extract before and after leaching with water are shown. In Figure 3, the spectra of wood treated with the propolis extract before and after leaching with water are shown. The wide band of O-H stretching vibration at 3445 cm −1 observed in the spectrum of untreated wood narrowed in the spectra of treated wood, which may indicate that the hydroxyl group of the wood formed hydrogen bonds with propolis constituents. In the spectra of treated wood, there is the observed loss of band at 1735 cm −1 , assigned to C=O stretching vibration of carboxyl and acetyl groups in wood, which is visible in the spectrum of untreated wood. In the spectra of propolis treated wood appeared the new band at 1637 cm −1 associated with C=O, C=C and N-H vibrations from propolis constituents, namely, flavonoids and amino acids. The band at 1456 cm −1 for skeletal C=C aromatic rings of flavonoids was observed in the spectra of treated wood. The bands at 1085 and 1035 cm −1 responsible for C-C, C-OH and C-O-C vibrations originating from flavonoids and alcohols in propolis were observed in the spectra of treated wood both before and after leaching. The changes in the FTIR spectra of untreated and propolis treated wood indicated that constituents of the propolis extract formed chemical bonds with the wood components. However, the changes in the intensity of the bands mainly at 2850, 2920 and in the range 850-600 cm −1 in the spectra of treated wood before and after leaching suggest that water may leach part of the propolis components from the treated wood structure.

FTIR Characterization
The spectra of wood treated with the propolis-silane preparations, namely, EEP-VTMOS/TEOS and EEP-MPTMOS/TEOS, are presented in Figures 4 and 5, respectively. The wide band of O-H stretching vibration at 3445 cm −1 observed in the spectrum of untreated wood narrowed in the spectra of treated wood, which may indicate that the hydroxyl group of the wood formed hydrogen bonds with propolis constituents. In the spectra of treated wood, there is the observed loss of band at 1735 cm −1 , assigned to C=O stretching vibration of carboxyl and acetyl groups in wood, which is visible in the spectrum of untreated wood. In the spectra of propolis treated wood appeared the new band at 1637 cm −1 associated with C=O, C=C and N-H vibrations from propolis constituents, namely, flavonoids and amino acids. The band at 1456 cm −1 for skeletal C=C aromatic rings of flavonoids was observed in the spectra of treated wood. The bands at 1085 and 1035 cm −1 responsible for C-C, C-OH and C-O-C vibrations originating from flavonoids and alcohols in propolis were observed in the spectra of treated wood both before and after leaching. The changes in the FTIR spectra of untreated and propolis treated wood indicated that constituents of the propolis extract formed chemical bonds with the wood components. However, the changes in the intensity of the bands mainly at 2850, 2920 and in the range 850-600 cm −1 in the spectra of treated wood before and after leaching suggest that water may leach part of the propolis components from the treated wood structure.
The changes observed in the spectra of wood treated with the propolis-silane preparations both before and after leaching with water indicated that the constituents of the preparations formed permanent bonds with the wood components.  Table 2 presents the results of the leaching of phenolic compounds, which are the main bioactive components of propolis, from wood treated with the propolis extract and the propolis-silane preparations.  The most important changes in the spectra of wood treated with the propolis-silane preparations compared to the spectrum of untreated wood were as follow: narrowing of the wide band in the range 3200-3500 cm −1 for O-H stretching vibrations, losing of the band at 1735 cm −1 , assigned to C=O stretching vibration of carboxyl and acetyl groups in wood, appearing the new band at 1640 cm −1 associated with C=O, C=C and N-H vibrations derived from propolis constituents, namely, flavonoids and amino acids and the new band at 1456 cm −1 for skeletal C=C aromatic rings of flavonoids. In addition, in the spectra of wood treated with EPP-VTMOS/TEOS, the bands at 1270 and 1160 cm −1 assigned to vibrations of SiOCH3 and SiCH3 bonds and the bands in the range of 1095-1085 cm −1 indicating vibrations of SiOCH3 group were observed. The bands at 833, 770 and 685 cm −1 attributed to the vibrations of Si-C and Si-O bands were also visible in the spectra of wood treated with EEP-VTMOS/TEOS both before and after leaching [52,[54][55][56]. In turn, in the spectra of wood treated with EEP-MPTMOS/TEOS, new bands at: 833, 765, and 696 cm −1 attributed to vibrations of Si-C and Si-O bands were observed [52,54,55].

Leaching of Treatment Preparations Constituents from Treated Wood
The changes observed in the spectra of wood treated with the propolis-silane preparations both before and after leaching with water indicated that the constituents of the preparations formed permanent bonds with the wood components.  Table 2 presents the results of the leaching of phenolic compounds, which are the main bioactive components of propolis, from wood treated with the propolis extract and the propolis-silane preparations. The changes observed in the spectra of wood treated with the propolis-silane preparations both before and after leaching with water indicated that the constituents of the preparations formed permanent bonds with the wood components. Table 2 presents the results of the leaching of phenolic compounds, which are the main bioactive components of propolis, from wood treated with the propolis extract and the propolis-silane preparations. In order to determine the degree of PC leaching from treated wood, the total content of phenolic compounds in the treated wood before and after leaching procedure with water was analyzed. The degree of PC leaching from the treated wood was in the range of 3.7-18.1%, which indicate that phenolic compounds were scarcely leached from the wood structure. Akcay et al. [9] stated that propolis was not leached from wood treated with the extract of Turkish propolis. The authors did not detect phenolic compounds in the leachate after 16 h of leaching wood impregnated with the propolis extract [9]. In turn, the obtained results indicated that phenolic compounds were in greater extent leached from wood treated with the propolis extract than from wood treated with the propolis-silane preparations. The lowest degree of phenolic compounds leaching was observed for wood treated with EEP-VTMOS/TEOS, which was only 3.7%. The results show that used silicon compounds with hydrophobic properties as constituents of treatment preparations limited the leaching of phenolic compounds from impregnated wood. The silicon compounds have previously used in wood protection to reduce leaching of the bioactive component from the wood structure. Literature data indicated that silicon compounds were able to limit the leaching of boron and caffeine from treated wood [36,42].

Leaching of Treatment Preparations Constituents from Treated Wood
In the next stage of the research process, NMR spectra of the treated wood were performed in order to determine the presence of silicon in the wood structure. The 29 Si CP MAS NMR spectra of wood impregnated with the propolis-silane preparations before and after leaching procedure are presented in Figure 6.  In order to determine the degree of PC leaching from treated wood, the total content of phenolic compounds in the treated wood before and after leaching procedure with water was analyzed. The degree of PC leaching from the treated wood was in the range of 3.7-18.1%, which indicate that phenolic compounds were scarcely leached from the wood structure. Akcay et al. [9] stated that propolis was not leached from wood treated with the extract of Turkish propolis. The authors did not detect phenolic compounds in the leachate after 16 h of leaching wood impregnated with the propolis extract [9]. In turn, the obtained results indicated that phenolic compounds were in greater extent leached from wood treated with the propolis extract than from wood treated with the propolis-silane preparations. The lowest degree of phenolic compounds leaching was observed for wood treated with EEP-VTMOS/TEOS, which was only 3.7%. The results show that used silicon compounds with hydrophobic properties as constituents of treatment preparations limited the leaching of phenolic compounds from impregnated wood. The silicon compounds have previously used in wood protection to reduce leaching of the bioactive component from the wood structure. Literature data indicated that silicon compounds were able to limit the leaching of boron and caffeine from treated wood [36,42].
In the next stage of the research process, NMR spectra of the treated wood were performed in order to determine the presence of silicon in the wood structure. The 29 Si CP MAS NMR spectra of wood impregnated with the propolis-silane preparations before and after leaching procedure are presented in Figure 6. In the spectra of EEP-VTMOS/TEOS treated wood ( Figure 6A), three well-defined signals were observed at −73 ppm, −81 ppm and −102 ppm, which were assigned to T 2 structure, T 3 structure and Q 3 free silanols structure, respectively [56][57][58][59][60]. In the spectrum of treated wood after leaching ( Figure  6B), three signals were also observed with the same shifts as in the spectrum of treated wood before leaching. However, the intensity of these signals was slightly lower compared to the intensity of the signals in the spectrum of unleached wood, suggesting that the Si concentration in the leached wood In the spectra of EEP-VTMOS/TEOS treated wood ( Figure 6A), three well-defined signals were observed at −73 ppm, −81 ppm and −102 ppm, which were assigned to T 2 structure, T 3 structure and Q 3 free silanols structure, respectively [56][57][58][59][60]. In the spectrum of treated wood after leaching ( Figure 6B), three signals were also observed with the same shifts as in the spectrum of treated wood before leaching. However, the intensity of these signals was slightly lower compared to the intensity of the signals in the spectrum of unleached wood, suggesting that the Si concentration in the leached wood was lower than in wood before leaching. In the spectrum of wood treated with EEP-MPTMOS/TEOS ( Figure 6C), two low-intensity signals were found at -60 ppm and -100 ppm, which were assigned to T 2 and Q 3 structures [57][58][59][60]. In the spectrum of treated wood after leaching procedure ( Figure 6D), these signals were shifted and observed at −59 ppm and −103 ppm. The low signal-to-noise ration observed in the spectra of wood treated with EEP-MPTMOS/TEOS indicates a relatively small number of silicon atoms in the wood structure.
In order to determine the degree of silicon compounds leaching from treated wood, the silicon concentration determination in the samples was performed using atomic absorption spectroscopy (AAS) and X-ray fluorescence spectroscopy (XRF). The Si concentration results in the wood samples presented in Table 3 indicated that the wood treated with EEP-VTMOS/TEOS contained higher silicon concentration than wood treated with EEP-MPTMOS/TEOS which is in agreement with the results of NMR measurements. The degree of Si leaching from wood impregnated with EEP-VTMOS/TEOS was more than two times lower than the degree of Si leaching from wood treated with EEP-MPTMOS/TEOS. The degree of Si leaching from wood treated with both preparations was lower than reported in the literature. The degree of Si leaching from pine wood treated with [3-(2-aminoethylamino)propyl]-trimethoxysilane was 53%, and the degree of Si leaching from wood treated with the propolis-caffeine-silane preparation was 24% [36,59]. In turn, the degree of Si leaching from wood treated with preparation consisted of the propolis extract and silanes (methyltrimethoxysilane and 3-(trimethoxysilyl)propyl methacrylate) was 10%, which indicate that the VTMOS-TEOS and MPTMOS-TEOS silanes used in this study formed permanent bonds with wood [61]. Due to the fact that the analysis of Si concentration in wood by atomic absorption spectroscopy is a destructive method, in research the Si concentration on the wood surface was also determined by X-ray fluorescence diffraction, which is a non-destructive method. The XRF method was previously used in the analysis of different element concentration in the wood [62][63][64]. The silicon concentration in treated wood and the degree of Si leaching determined by XRF are shown in Table 4. Table 4. The silicon concentration in treated wood and the degree of Si leaching determined by X-ray fluorescence spectroscopy (XRF).

Treatment Preparations
Silicon Comparing the results of Si concentration in impregnated wood obtained by atomic absorption spectroscopy (AAS) and X-ray fluorescence spectroscopy (XRF), it is noticeable that the results obtained by the XRF method are two orders of magnitude smaller than the AAS results, which is associated with the measurement method. The Si concentration in wood samples determined by AAS is associated with the analysis of the element concentration in the entire sample volume. In turn, the  Table 4 indicated that surface of wood treated with EEP-MPTMOS/TEOS characterized by higher concentration of this element compared to wood treated with EEP-VTMOS/TEOS. On the other hand, the results of the AAS analysis (Table 3) indicated that wood treated with EEP-VTMOS/TEOS contained higher Si concentration than wood impregnated with EEP-MPTMOS/TEOS, which may be associated with longer chain lengths of MPTMOS, which is a component of EEP-MPTMOS/TEOS preparation. The MPTMOS could have been deposited on the wood surface because the silicon compounds with long chains penetrated the cell wall worse than silanes with shorter chains [39]. It should be noted, however, that the degree of Si leaching obtained based on the XRF results is comparable to the degree of Si leaching obtained from the AAS results, which indicate that the non-destructive XRF method can be used to determine various elements that are components of wood preservatives in treated wood.

The Resistance of Treated Wood Against C. puteana
The results of the antifungal efficacy against C. puteana, expressed as average mass loss of wood samples treated with the propolis extract and the propolis-silane preparations, are presented in Table 5. The results of the biological test show that the action of the fungus caused the mass loss of treated wood in the range of 2.9% to 4.8%, and untreated control wood samples in the range of 44.7% to 49.2%. The protective activity of the propolis extract and the propolis-silane preparations can be seen compared to the mass loss of treated and untreated wood samples. The mass loss values of each treated wood were statistically similar, except for the value of propolis treated wood after leaching, which was statistically higher than the others. In the case of wood treated with the propolis extract, an increase in the mass loss of wood after the leaching procedure was noticeable, which was associated with partial leaching of phenolic compounds from the wood structure (Table 2). In turn, the mass losses of wood treated with the propolis-silane preparations before and after leaching procedure were statistically similar, which indicates that the components of impregnating preparations not leached from the wood structure and effectively protected it against the degradation action of C. puteana. The wood treated with EEP-MPTMOS/TEOS exhibited the lowest value of mass loss both before and after leaching with water.
The literature data indicated that the extract of Turkish propolis at a 7% concentration protected wood against T. versicolor and N. lepideus, while the extract of Polish propolis at a concentration above 12% protected wood against fungal decay caused by C. puteana [9,33]. The mass loss of pine wood treated with the extract of Polish propolis at a concentration of 12% and 18.9% after exposure to C. puteana was 3.3% and 2.3%, respectively [33]. In turn, the wood treated with soda-based propolis solution after leaching procedure did not show resistance against C. puteana [65]. The mass losses of wood treated with the propolis-silane preparations both before and after leaching procedure were similar, indicating that the constituents of these preparations did not leach from the wood structure, and the wood after leaching with water still showed resistance against the destructive action of fungus.
The previous work of the authors indicated that wood treated with the propolis extract with silanes (methyltrimethoxysilane and 3-(trimethoxysilyl)propyl methacrylate) exhibited resistance against C. puteana both before and after leaching procedure [61]. The preparation consisted of the propolis extract, caffeine, and silanes (methyltrimethoxysilane and octyltriethoxysilane) inhibited the growth of C. puteana on wood samples-even the wood was subjected to leaching procedure [36].

Bending Strength and Modulus of Elasticity of Treated Wood
In accordance with the fact that the impregnation of wood with chemical preservatives may have an effect on its mechanical properties in Table 6, there are presented the results of bending strength and modulus of elasticity determined for wood treated with the propolis extract and the propolis-silane preparations. The wood treated with the propolis extract and the propolis-silane preparations was characterized by a 3-4% increase in the equilibrium moisture content compared to untreated samples, which was associated with higher hydrophobic properties of treated wood, which was previously described by the authors [66]. The modulus of elasticity determined for treated wood was statistically lower compared to the modulus elasticity of untreated control samples. The modulus of elasticity determined for wood protected with the propolis extract and the propolis-silane preparations was about 7% lower than for unprotected wood. In turn, the bending strength values of wood impregnated with the propolis extract and the propolis-silane preparations were statistically higher than the value for untreated wood samples. The value of bending strength determined for EEP treated wood was about 4% and wood impregnated with EEP-MPTMOS/TEOS and treated with EEP-VTMOS/TEOS was about 6% higher than for untreated, control samples. The increase in the bending strength of treated wood compared to untreated samples may be associated with the deposition of impregnating agents, and thus, with an increase in the density of treated wood. Moreover, according to the SEM imagines presented in Figure 7, the silicon compounds filled the cell lumen. In turn, the SEM imagine of wood treated with the propolis extract showed that the wood cells were separated from each other, which can be associated with the action of aqueous ethanol used as a propolis solvent. Literature data indicated that ethanol-water mixtures have an influence on the wood structure causing dissolving a main part of lignin in compound middle lamellae (CML) and releasing individual cells at sectioning [67,68]. However, the ethanolic solution does not affect on the cell wall S2 layer, which is one of the main factors determining the strength properties of wood [69].
to the SEM imagines presented in Figure 7, the silicon compounds filled the cell lumen. In turn, the SEM imagine of wood treated with the propolis extract showed that the wood cells were separated from each other, which can be associated with the action of aqueous ethanol used as a propolis solvent. Literature data indicated that ethanol-water mixtures have an influence on the wood structure causing dissolving a main part of lignin in compound middle lamellae (CML) and releasing individual cells at sectioning [67,68]. However, the ethanolic solution does not affect on the cell wall S2 layer, which is one of the main factors determining the strength properties of wood [69].  The impregnation of wood with chemical preservatives caused changes in the mechanical properties of wood [70][71][72]. The pine wood impregnated with ionic liquids showed a lower value of bending strength and modulus of elasticity compared to untreated wood [70]. The wood treated with chitosan solution exhibited an increase of the modulus of elasticity and no significant changes in the modulus of rupture compared to untreated wood [73]. The pine wood modified with methyl-etherified melamine formaldehyde resin, a low molecular weight phenol-formaldehyde resin, a higher molecular weight phenol-formaldehyde resin and dimethylol dihydroxyethyleneurea showed significantly lower bending strength values than control specimens [71]. In turn, the treatment with tetraethoxysilane showed no effect on the mechanical properties of pine wood compared to untreated samples [72]. The full explanation of the influence of impregnation with the propolis extract and the propolis-silane preparations on the mechanical properties of treated wood requires further investigations.

Conclusions
The paper presents the results of chemical, biological and mechanical characterization of wood treated with the propolis extract and the propolis-silane preparations. The results of FTIR analysis and determination of phenolic compounds concentration in wood treated with the propolis extract indicated that part of propolis constituents was leached by water from the wood structure. In turn, the changes in the FTIR spectra of wood impregnated with the propolis-silane preparations showed that the constituents of the preparations formed permanent bonds with the wood components. The analysis of phenolic compounds concentration in treated wood indicated that phenols were in greater extent leached from propolis treated wood than from wood impregnated with the propolis-silane preparations. The lowest degree of phenols leaching was observed for wood treated with EEP-VTMOS/TEOS, which was only 3.7%. The results show that used silicon compounds with hydrophobic properties as a component of treatment preparations limited the leaching of phenolic compounds from impregnated wood. The presence of silicon in wood treated with the propolis-silane preparations both before and after leaching was confirmed by 29 Si CP MAS NMR measurements. In turn, AAS and XRF analyses indicated that the degree of Si leaching from wood impregnated with EEP-VTMOS/TEOS was approximately two times lower than from wood treated with EEP-MPTMOS/TEOS. The results of chemical analyses confirmed that the constituents of the propolis-silane preparations formed permanent bonds with wood. The results of the antifungal efficacy against C. puteana show that the propolis extract and the propolis-silane preparations limited the fungus activity, even the wood was subjected to leaching procedure. The most effective protection against tested fungus was observed for wood treated with EEP-MPTMOS/TEOS. The mass loss of EEP-MPTMOS/TEOS treated wood before and after leaching was 2.9% and 3.2%, respectively. However, the mass loss values of each treated wood were statistically similar, except for the value of propolis treated wood after leaching, which was statistically higher than the others. The protective activity of the propolis extract was lower when the wood was subjected to leaching, which was associated with partial leaching of phenolic compounds