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

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

Magdalena Wo´zniak 1,*, Patrycja Kwa´sniewska-Sip 2,3 , Michał Krueger 4, Edward Roszyk 5 and Izabela Ratajczak 1

1 Department of Chemistry, Faculty of Wood Technology, PoznańUniversity of Life Sciences, Wojska Polskiego 75, 60625 Poznań,Poland; [email protected] 2 Air Quality Investigation Department, Łukasiewicz Research Network-Wood Technology Institute, Winiarska 1, 60654 Poznań,Poland; [email protected] 3 Institute of Chemical Wood Technology, Faculty of Wood Technology, PoznańUniversity of Life Sciences, Wojska Polskiego 38/42, 60637 Poznań,Poland 4 Department of the Ancient Civilisations of the Mediterranean, Faculty of Archaeology, Adam Mickiewicz University in Poznań,Uniwersytetu Poznańskiego7, 61614 Poznań,Poland; [email protected] 5 Department of Wood Science and Thermal Techniques, Faculty of Wood Technology, PoznańUniversity of Life Sciences, Wojska Polskiego 38/42, 60637 Poznań,Poland; [email protected] * Correspondence: [email protected]

Received: 7 July 2020; Accepted: 18 August 2020; Published: 20 August 2020

Abstract: 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 efficacy 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.

Keywords: propolis; silicon compounds; Coniophora puteana; mechanical properties; natural preservatives; NMR; XRF; FAAS

Forests 2020, 11, 907; doi:10.3390/f11090907 www.mdpi.com/journal/forests Forests 2020, 11, 907 2 of 17

1. Introduction Wood, as a natural renewable resource, is susceptible to decay and biodegradation by fungi and, to a lesser extent, by bacteria. Therefore, the durability and service life of wooden products is increased by using various methods of wood protection, including thermal modification or treatment with numerous chemicals, such as silicon compounds, titanium dioxide nanoparticles, or silver nanoparticles [1–5]. Nowadays, wood protection employs also natural compounds with low or no toxicity to humans and the environment, including chitosan, natural oils or propolis [6–9]. Essential oils and their components (e.g., citral, thymol, eugenol, cinnamaldehyde and carvacrol) were also applied in wood protection, increasing the wood durability against molds and decay fungi [10–13]. The important source of natural wood preservatives is plant and wood extractives, which contain a mixture of many components with various biological activity. The extracts of Cupressus sempervirens and Thuja occidentalis demonstrated activity against wood decay fungi—Ganoderma lucidum and Hexagonia apiaria [14]. In turn, wood treated with Pinus rigida heartwood extract showed good inhibition to the growth of molds, such as Alteria alternata, Chaetomium globosum and Trichoderma viride [15]. The bioactive components of wood extracts, such as phenolic compounds, play an important role in the natural durability of the wood. Latorraca et al. [16] reported lower durability of juvenile heartwood of Robinia pseudoacacia to Coniophora puteana and Trametes versicolor, due to lower total concentrations of phenolic compounds and flavonoids than in the mature heartwood. According to Sirmah et al. [17], the natural durability of Prosopis juliflora wood can be attributed to flavonoid—mesquital. In turn, latifolin and 4-methoxydalbergione isolated from Dalbergia latifolia heartwood showed activity against T. versicolor [18]. Propolis is a resinous material collected by Apis mellifera from the buds of various trees and plant exudates [19,20]. The chemical composition of propolis is very complex, mainly due to the variability of plant sources growing around hives. More than 300 chemical constituents have been identified in propolis from different geographical regions [20]. The main class of chemical components present in propolis are phenolic compounds, including flavonoids, phenolic acids and their esters [21–24]. According to literature, the phenol fraction of propolis is indicated as the main fraction responsible for its biological activity [19,25]. Moreover, terpenes, minerals or amino acids have also been identified in propolis samples [20,26,27]. The extracts of propolis showed biological properties, including antioxidant, anticancer, antiviral, and antibacterial activity [20,21,23,24,28,29]. Propolis also exhibited antifungal activity against molds and yeasts [21,23,30,31]. In addition, the extract of Argentine propolis showed activity against xylophagous fungi isolated from decayed wood: Ganoderma applanatum, Lenzites elegans and Schizophyllum commune [32]. Due to biological properties, mainly antifungal activity, propolis has been applied in wood protection. The wood treated with the extract of Turkish propolis showed resistance against T. versicolor and Neolentinus lepideus, and treated with the extract of Polish propolis against C. puteana [9,33]. The wood treated with the extract of Spanish propolis limited decay of wood caused by T. versicolor [34]. The propolis extract was also a component of preparations applied to protect wood. The wood impregnated with the preparation consisted of chitosan, and the propolis extract showed higher resistance against T. versicolor compared to untreated wood samples [35]. In turn, the preparation based on propolis extract, caffeine, and silicon compounds inhibited the growth of C. puteana on treated wood samples [36]. The silicon compounds are used in various applications, such as buildings, paper, textiles, as well as wood impregnation [37]. The wood treated with silicon compounds is characterized by the improvement of hydrophobic and antifungal properties [3,38–40]. However, the silanes with amino groups showed higher antifungal effect than the silicon compounds without –NH2 groups [38]. Therefore, silicon compounds have been often used as a component of wood preservatives, whose purpose is to support the action of active substances or to prevent their leaching from the wood structure [36,41,42]. The aim of the research was to determine the properties of wood treated with bio-friendly preservatives based on the propolis extract and silicon compounds. The paper presents the results of chemical analysis of treated wood, including Fourier transform infrared spectroscopy (FTIR), nuclear ForestsForests2020 2020, 11, 11, 907, x FOR PEER REVIEW 3 of3 of 17 17

scanning electron microscopy (SEM). The resistance of the treated wood against brown-rot fungus C. magneticputeana was resonance also assessed. (NMR), atomicMoreover, absorption the mechanical spectroscopy properties, (AAS), namely, X-ray ﬂuorescence bending strength (XRF) and and scanningmodulus electron of elasticity microscopy of treated (SEM). wood, The were resistance determined. of the treated wood against brown-rot fungus C. puteana was also assessed. Moreover, the mechanical properties, namely, bending strength and modulus2. Materials of elasticity and Methods of treated wood, were determined.

2.2.1. Materials Propolis-Silane and Methods Preparations

2.1. Propolis-SilaneIn this study, Preparations the 15% ethanolic extract of Polish propolis prepared in 70% ethanol and two propolis-silane preparations were used for wood impregnation. The first preparation (EEP- In this study, the 15% ethanolic extract of Polish propolis prepared in 70% ethanol and two VTMOS/TEOS) consisted of 15% ethanolic extract of propolis (EEP) and silanes: propolis-silane preparations were used for wood impregnation. The first preparation (EEP-VTMOS/TEOS) vinyltrimethoxysilane (VTMOS) and tetraethyl orthosilicate (TEOS) at 5% concentration. EEP (15%), consisted of 15% ethanolic extract of propolis (EEP) and silanes: vinyltrimethoxysilane (VTMOS) and 3-(trimethoxysilyl) propyl methacrylate (MPTMOS) and tetraethyl orthosilicate (TEOS) at 5% tetraethyl orthosilicate (TEOS) at 5% concentration. EEP (15%), 3-(trimethoxysilyl) propyl methacrylate concentration were the components of the second preparation (EEP-MPTMOS/TEOS). The silicon (MPTMOS) and tetraethyl orthosilicate (TEOS) at 5% concentration were the components of the second compounds (Figure 1) were purchased from Sigma Aldrich (Darmstadt, Germany) and the propolis preparation (EEP-MPTMOS/TEOS). The silicon compounds (Figure1) were purchased from Sigma Aldrich extract from PROP-MAD (Poznań, Poland). (Darmstadt, Germany) and the propolis extract from PROP-MAD (Pozna´n, Poland).

TEOS VTMOS MPTMOS

FigureFigure 1. 1.The The chemical chemical structures structures of of silicon silicon compounds. compounds. TEOS, TEOS, tetraethyl tetraethyl orthosilicate; orthosilicate; VTMOS, VTMOS, vinyltrimethoxysilane;vinyltrimethoxysilane; MPTMOS, MPTMOS, 3-(trimethoxysilyl)propyl 3-(trimethoxysilyl)propyl methacrylate. methacrylate.

2.2.2.2. Wood Wood Treatment Treatment TheThe investigated investigated material material was was Scots Scots pine pine ( Pinus(Pinus sylvestris sylvestrisL.) L.) sapwood sapwood without without knots knots or or other other 3 growthgrowth inhomogeneity. inhomogeneity. The The wood wood samples samples with with dimensions dimensions of of 5(T) 5(T) ×10(R) 10(R) ×40(L) 40(L) mm mmwere3 were used used × × forfor biological biological and and chemicalchemical analyses.analyses. The The wood wood samples samples with with dimensions dimensions of of5(T) 5(T) × 10(R)10(R) × 150(L)150(L) mm3 × × mmwere3 were applied applied in the in the determination determination of of mechanical mechanical parameters. parameters. Before Before impregnationimpregnation process,process, the the specimensspecimens were were conditioned conditioned at at 65 65 ±5% 5% relative relative humidity humidity (RH) (RH) and and 20 20 ±2 ◦2°CC to to attain attain equilibrium equilibrium ± ± moisturemoisture content content of of approximately approximately 12%. 12%. Next,Next, allall thethe woodwood samplessamples werewere impregnatedimpregnated withwith EEP,EEP, EEP-VTMOSEEP-VTMOS/TEOS/TEOS and and EEP-MPTMOS EEP-MPTMOS/TEOS/TEOS using using the vacuumthe vacuum method, method, according according to EN to 113: EN 1996 113: [431996]. The[43]. samples The samples underwent underwent 15 min under15 min vacuum under vacuum conditions—0.8 conditions—0.8 kPa and 2kPa h under and 2 atmospheric h under atmospheric pressure. Afterpressure. impregnation, After impregnation, all the samples all the were samples removed were from removed the impregnating from the impregnating solutions and solutions weighed and 3 toweighed determine to determine the uptake the of uptake the solutions. of the so Thelutions. wood The sample wood retention sample retention (kg/m ) was(kg/m calculated3) was calculated as the followingas the following equation: equation: 3 (Mb Ma) c 10 R kg/m = −− ·∙∙10· (1) kg/m = v (1) where Ma—the wood mass before treatment (g); Mb—the wood mass after treatment (g); c—concentrationwhere Ma—the ofwood propolis mass extract before or propolis-silanetreatment (g); preparationsMb—the wood constituents mass after (%); treatmentv—the volume (g); ofc— theconcentration wood sample of (cmpropolis3). extract or propolis-silane preparations constituents (%); v—the volume of 3 the Afterwood impregnation,sample (cm ). all the wood samples were cured for four weeks in room conditions After impregnation, all the wood samples were cured for four weeks in room conditions (RH = (RH = 65 5%; T = 20 2 ◦C). 65 ± 5%;± T = 20 ± 2 °C).± 2.3. Accelerated Aging of Wood—Leaching Procedure 2.3. Accelerated Aging of Wood—Leaching Procedure The aim of the artiﬁcial ageing (leaching in water) was to determine anti-leaching stability of the treatmentThe preparationsaim of the artificial constituents ageing (leaching from the treatedin water) wood. was to The determine leaching anti-leaching procedure of stability the treated of the treatment preparations constituents from the treated wood. The leaching procedure of the treated

Forests 2020, 11, 907 4 of 17 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.

2.4. 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) mm3 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.

2.5. 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) mm3. 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.

2.6. Chemical Characterization of Wood For chemical analyses, the control and treated wood samples with dimensions of 5(T) 10(R) 40(L) × × mm3 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.

2.6.1. 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 1 1 of wood samples were registered, at a range of 500–4000 cm− , at a resolution of 4 cm− , registering 32 scans. Forests 2020, 11, 907 5 of 17

2.6.2. 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.

2.6.3. 29Si 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 1H 29 and 79.495 MHz for Si and equipped with a MAS probe head using 4-mm ZrO2 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.

2.6.4. X-Ray Fluorescence (XRF) The samples of treated wood with dimensions of 5(T) 10(R) 40(L) mm3 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 mm2 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.

2.6.5. 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.

2.6.6. 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). Forests 2020, 11, 907 6 of 17 Forests 2020, 11, x FOR PEER REVIEW 6 of 17

2.7.2.7. StatisticalStatistical AnalysisAnalysis TheThe resultsresults werewere analyzedanalyzed usingusing a one-way anal analysisysis of of variance variance (ANOVA) (ANOVA) applying applying Tukey’s Tukey’s HonestHonest SignificantSignificant DiDifferencesfferences (THSD)(THSD) Test.Test. StatisticalStatistical significancesignificance was was defined defined as as pp << 0.05.0.05. All All the the statisticalstatistical analysesanalyses werewere performedperformed usingusing the TIBCO Software Inc. Inc. Statistica Statistica version version 13.1 13.1 (Palo (Palo Alto, Alto, CA,CA, USA). USA).

3.3. ResultsResults andand DiscussionDiscussion

3.1.3.1. ChemicalChemical CharacterizationCharacterization ofof TreatedTreated WoodWood InIn thethe ﬁrstfirst stagestage ofof thethe research,research, thethe chemicalchemical interaction between the the components components of of the the propolis-silanepropolis-silane preparationspreparations and and wood wood was was determ determinedined using using instrumental instrumental methods. methods. The wood The wood was wastreated treated with with 15% 15%extract extract of Polish of Polish propolis propolis and two and propolis-silane two propolis-silane preparations, preparations, namely, namely, EEP- EEP-VTMOSVTMOS/TEOS/TEOS and EEP-MPTMOS/TEOS. and EEP-MPTMOS/TEOS. The concentrat The concentrationion of the ofpropolis the propolis extractextract was chosen was chosenbased basedon our on previous our previous studies, studies, which indicated which indicated that Scots that pine Scots wood pine impregnated wood impregnated with ethanolic with ethanolicpropolis propolisextract above extract 12% above concentration 12% concentration limited fungal limited decay fungal [33]. decay [33].

3.1.1.3.1.1. FTIRFTIR CharacterizationCharacterization FigureFigure2 presents2 presents the the FTIR FTIR spectrum spectrum of the of propolis the propolis extract, extract, and Table and1 showsTable the1 shows most importantthe most bandsimportant of this bands spectrum, of thischaracterized spectrum, characterized according toaccording literature to data literature [50–53 data]. [50–53].

FigureFigure 2.2. The FTIR spectrum of the propolis extract. extract.

InIn the the spectrum spectrum ofof thethe propolispropolis extract,extract, thethe bandsbands assignedassigned toto thethe vibrationsvibrations of CC=O,=O, C=C, C=C, N–H N–H andand C–H C–H bonds bonds derived derived from from phenolic phenolic compounds compounds present present in in propolis propolis were were observed. observed. The The bands bands in thein 1 spectrumthe spectrum were observedwere observed at: 2926 at: and 2926 2849 and cm 2849− , which cm−1, arewhich connected are connected with C–H with stretching C–H stretching vibrations andvibrations confirm and the confirm presence the of presence long-chain of long-chain alkyl compounds alkyl compounds in the propolis in the extract. propolis The extract. observed The 1 stretchingobserved stretching and bending and bands bending at 1636,bands 1514 at 1636, and 1514 1450 and cm− 1450correspond cm−1 correspond mainly tomainly aromatic to aromatic rings of phenolicrings of compoundsphenolic compounds specific for specific propolis for extracts. propolis In extracts. addition, In the addition, wide band the withwide aband maximum with a at 1 3420maximum cm− described at 3420 cm to−1 thedescribed stretching to the vibrations stretching of vibrations O–H band of also O–H confirms band also the confirms presence the of presence phenolic compoundsof phenolic incompounds the extract. in the extract.

Table 1. The characteristic absorption bands of the propolis extract.

Wavenumber [cm−1] Type of Bands Main Attribution 3420 O–H, N–H stretching O–H and N–H bands 2926 C–H stretching C–H of methyl and methylene group

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Table 1. The characteristic absorption bands of the propolis extract.

1 Wavenumber [cm− ] Type of Bands Main Attribution Forests 2020, 113420, x FOR PEER REVIEW O–H, N–H stretching O–H and N–H bands 7 of 17 2926 C–H stretching C–H of methyl and methylene group 28492849 C–H C–H stretching stretching C–H C–H of hydrocarbon of hydrocarbon 17011701 C=O C =O stretching stretching C=O Cof= Olipids of lipids and andflavonoids flavonoids 16361636 C=O, C C=C,=O, C N–H=C, N–H skeletalskeletal aromatic aromatic rings rings of flavonoids of flavonoids and and amino amino acids acids 15141514 aromatic aromatic skeletal skeletal C=C Caromatic=C aromatic rings rings of flavonoids of flavonoids 1450 C–H bending C–H of flavonoids and aromatic rings 1450 C–H bending C–H of flavonoids and aromatic rings 1374 C–H bending C–H2 of flavonoids 13741271 C–H C-O bending C–H stretching2 of flavonoids C-O 12711161 C-O C-O, C-OH stretching bands stretching of lipids C-O and tertiary alcohols stretching bands of flavonoids and 11611089 C-O, C-C,C-OH C-OH stretching bands of lipids and tertiary alcohols 1089 C-C, C-OH stretching bands of flavonoidssecondary and alcohols secondary alcohols

InIn FigureFigure3 3,, the the spectra spectra ofof woodwood treatedtreated withwith thethe propolis extract before and and after after leaching leaching with with waterwater areare shown.shown.

FigureFigure 3.3. The FTIR spectra of Scots pine wood wood (A), (A), w woodood treated treated with with the the propolis propolis extract extract (B) (B )and and woodwood treatedtreated withwith thethe propolispropolis extract after leaching (C). (C). The wide band of O–H stretching vibration at 3445 cm 1 observed in the spectrum of untreated 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 narrowed in the spectra of treated wood, which may indicate that the hydroxyl group of the woodwood formedformed hydrogen bonds bonds with with propolis propolis constituen constituents.ts. In the In thespectra spectra of treated of treated wood, wood, there there is the is 1 theobserved observed loss lossof band of band at 1735 at 1735cm−1,cm assigned− , assigned to C=O to stretching C=O stretching vibration vibration of carboxyl of carboxyl and acetyl and groups acetyl groupsin wood, in which wood, is which visible is in visible the spectrum in the spectrum of untrea ofted untreated wood. In wood. the spectra In the spectraof propolis of propolis treated wood treated 1 woodappeared appeared the new the band new at band 1637 at cm 1637−1 associated cm− associated with C=O, with C=C C =andO, CN–H=C andvibrations N–Hvibrations from propolis from 1 propolisconstituents, constituents, namely, flavonoids namely, flavonoids and amino and acids. amino The acids. bandThe at 1456 band cm at−11456 for skeletal cm− for C=C skeletal aromatic C=C aromaticrings of flavonoids rings of flavonoids was observed was observedin the spectra in the of spectratreated ofwood. treated The wood. bands Theat 1085 bands andat 1035 1085 cm and−1 1 1035responsible cm− responsible for C-C, C-OH for C-C, and C-O-C C-OH andvibrations C-O-C orig vibrationsinating from originating flavonoids from and flavonoids alcohols andin propolis alcohols inwere propolis observed were in observed the spectra in theof treated spectra wood of treated both before wood and both after before leaching. and after The leaching. changes Thein the changes FTIR inspectra the FTIR of untreated spectra of and untreated propolis and treated propolis wood treated indi woodcated indicatedthat constituents that constituents of the propolis of the propolisextract extractformed formed chemical chemical bonds bondswith the with wood the woodcomponents. components. However, However, the changes the changes in the inintensity the intensity of the of 1 thebands bands mainly mainly at 2850, at 2850, 2920 2920 and andin the in range the range 850–600 850–600 cm−1 cmin −thein spectra the spectra of treated of treated wood wood before before and andafter after leaching leaching suggest suggest that water that water may leach may leachpart of part the ofpropolis the propolis components components from the from treated the treatedwood woodstructure. structure. TheThe spectraspectra ofof woodwood treated with the propolis-silane preparations, preparations, namely, namely, EEP-VTMOS/TEOS EEP-VTMOS/TEOS andand EEP-MPTMOSEEP-MPTMOS/TEOS,/TEOS, areare presentedpresented in Figures 44 andand5 5,, respectively. respectively.

Forests 2020, 11, x FOR PEER REVIEW 8 of 17

Figure 4. The FTIR spectra of Scots pine wood (A), wood treated with EEP-VTMOS/TEOS (B) and wood treated with EEP-VTMOS/TEOS after leaching (C). Forests 2020, 11, 907 8 of 17

ForestsThe 2020 most, 11, x importantFOR PEER REVIEW changes in the spectra of wood treated with the propolis-silane preparations8 of 17 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 [52,54–56]. 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]. The changes observed in the spectra of wood treated with the propolis-silane preparations both FigureFigure 4.4. TheThe FTIRFTIR spectraspectra of Scots pine wood (A), (A), wood treated with with EEP-VTMOS/TEOS EEP-VTMOS/TEOS (B) (B )and and before and after leaching with water indicated that the constituents of the preparations formed woodwood treated treated withwith EEP-VTMOSEEP-VTMOS//TEOSTEOS afterafter leachingleaching ((C).C). permanent bonds with the wood components. 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 [52,54–56]. In turn, in the spectra of wood treatedFigureFigure with 5.5. EEP-MPTMOS/TEOS,The FTIR spectra of Scots new pine bands wood wood at:(A), (A 833,), wood wood 765, treated treated and 696 with with cm EEP-MPTMOS/TEOS EEP-MPTMOS−1 attributed to/TEOS vibrations (B) (B and) and of Si- C andwoodwood Si-O treatedtreated bands withwith were EEP-MPTMOSEEP-MPTMOS observed [52,54,55].//TEOSTEOS after leaching (C). (C). The changes observed in the spectra of wood treated with the propolis-silane preparations both 3.1.2.beforeThe Leaching and most after important of Treatmentleaching changes with Preparations water in the indicated spectra Constituents of th woodat thefrom treated constituents Treated with Wood the of propolis-silane the preparations preparations formed compared to the spectrum of untreated wood were as follow: narrowing of the wide band in the permanentTable 2 bonds presents with the the results wood of components. the leaching of phenolic compounds, which are the main bioactive range 3200–3500 cm 1 for O–H stretching vibrations, losing of the band at 1735 cm 1, assigned components of propolis,− from wood treated with the propolis extract and the propolis-silane− to C=O stretching vibration of carboxyl and acetyl groups in wood, appearing the new band at preparations.1 1640 cm− associated with C=O, C=C and N–H vibrations derived from propolis constituents, namely, 1 ﬂavonoids and amino acids and the new band at 1456 cm− for skeletal C=C aromatic rings of ﬂavonoids. In addition, in the spectra of wood treated with EPP-VTMOS/TEOS, the bands at 1270 1 and 1160 cm− assigned to vibrations of SiOCH3 and SiCH3 bonds and the bands in the range of 1095–1085 cm 1 indicating vibrations of SiOCH group were observed. The bands at 833, 770 and − 3 1 685 cm− 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 [52,54–56]. In turn, in the spectra of 1 wood treated with EEP-MPTMOS/TEOS, new bands at: 833, 765, and 696 cm− attributed to vibrations of Si-C and Si-O bands were observed [52,54,55]. TheFigure changes 5. The FTIR observed spectra in of theScots spectra pine wood of wood(A), wood treated treated with with the EEP-MPTMOS/TEOS propolis-silane preparations(B) and both beforewood treated and after with leaching EEP-MPTMOS with/TEOS water after indicated leaching that (C). the constituents of the preparations formed permanent bonds with the wood components. 3.1.2. Leaching of Treatment Preparations Constituents from Treated Wood 3.1.2. Leaching of Treatment Preparations Constituents from Treated Wood Table 2 presents the results of the leaching of phenolic compounds, which are the main bioactive componentsTable2 presents of propolis, the results from of wood the leaching treated of with phenolic the propolis compounds, extract which and are the the propolis-silane main bioactive componentspreparations. of propolis, from wood treated with the propolis extract and the propolis-silane preparations.

Forests 2020, 11, x FOR PEER REVIEW 9 of 17 Forests 2020, 11, 907 9 of 17

Table 2. The content of total phenolic compounds (PC) in treated wood and the degree of PC leaching. Table 2. The content of total phenolic compounds (PC) in treated wood and the degree of PC leaching. Total Phenolic Compounds Treatment Total Phenolic CompoundsConcentration Concentration The Degree of PC Leaching TreatmentPreparations Preparations [mg[mg GAE GAE/mL]/mL] The Degree of PC[%] Leaching [%] ULUL L L EEPEEP 8.19 8.190.18 ± 0.18 6.71 6.710.08 ± 0.08 18.1 18.1 ± ± EEP-VTMOS/TEOS 8.41 0.11 8.10 0.16 3.7 EEP-VTMOS/TEOS 8.41± ± 0.11 8.10± ± 0.16 3.7 EEP-MPTMOS/TEOS 8.38 0.07 7.70 0.27 8.1 EEP-MPTMOS/TEOS 8.38± ± 0.07 7.70± ± 0.27 8.1 UL—unleached wood; L—leached wood (acc. EN 84). Expressed as average standard deviations. UL—unleached wood; L—leached wood (acc. EN 84). Expressed as average± ± standard deviations.

InIn order order to to determine determine the the degree degree of of PC PC leaching leaching from from treated treated wood, wood, the the total total content content of of phenolic phenolic compoundscompounds in in the the treatedtreated woodwood before and and after after leaching leaching procedure procedure with with water water was was analyzed. analyzed. The Thedegree degree of ofPC PC leaching leaching from from the the treated treated wood wood was in thethe rangerange ofof 3.7–18.1%, 3.7–18.1%, which which indicate indicate that that phenolicphenolic compounds compounds were were scarcely scarcely leached leached from from the the wood wood structure. structure. Akcay Akcay et et al. al. [9 ][9] stated stated that that propolispropolis was was not not leached leached from from wood wood treated treated with with the th extracte extract of of Turkish Turkish propolis. propolis. The The authors authors did did not not detectdetect phenolic phenolic compounds compounds in in the the leachate leachate after after 16 16 h ofh of leaching leaching wood wood impregnated impregnated with with the the propolis propolis extractextract [9 ].[9]. In In turn, turn, the the obtained obtained results results indicated indicated that that phenolic phenolic compounds compounds were were in in greater greater extent extent leachedleached from from wood wood treated treated with with the the propolis propolis extract extract than than from from wood wood treated treated with with the the propolis-silane propolis-silane preparations.preparations. The The lowest lowest degree degree of of phenolic phenolic compounds compounds leaching leaching was was observed observed for for wood wood treated treated withwith EEP-VTMOS EEP-VTMOS/TEOS,/TEOS, which which was was only only 3.7%. 3.7%. The The results results show show that that used used silicon silicon compounds compounds with with hydrophobichydrophobic properties properties as as constituents constituents of of treatment treatmen preparationst preparations limited limited the the leaching leaching of of phenolic phenolic compoundscompounds from from impregnated impregnated wood. wood. The silicon The silicon compounds compounds have previously have previously used in wood used protection in wood toprotection reduce leaching to reduce of the leaching bioactive of componentthe bioactive from component the wood from structure. the wood Literature structure. data indicatedLiterature that data siliconindicated compounds that silicon were compounds able to limit were the leachingable to limit of boron the leaching and caﬀ eineof boron from and treated caffeine wood from [36,42 treated]. woodIn the[36,42]. next stage of the research process, NMR spectra of the treated wood were performed in order toIn determinethe next stage the presenceof the research of silicon process, in the NMR wood spectra structure. of the The treated29Si wood CP MAS were NMR performed spectra in oforder wood to impregnated determine the with presence the propolis-silane of silicon in preparationsthe wood structure. before andThe after29Si CP leaching MAS NMR procedure spectra are of presentedwood impregnated in Figure6. with the propolis-silane preparations before and after leaching procedure are presented in Figure 6.

Figure 6. The 29Si CP MAS NMR spectra of wood treated with EEP-VTMOS/TEOS (A), wood treated 29 withFigure EEP-VTMOS 6. The Si/TEOS CP MAS after NMR leaching spectra (B), of wood wood treated treated with with EEP-MPTMOSEEP-VTMOS/TEOS/TEOS (A (C), )wood and wood treated treatedwith EEP-VTMOS/TEOS with EEP-MPTMOS /afterTEOS leaching after leaching (B), wood (D). treated with EEP-MPTMOS/TEOS (C) and wood treated with EEP-MPTMOS/TEOS after leaching (D). In the spectra of EEP-VTMOS/TEOS treated wood (Figure6A), three well-deﬁned signals were observedIn the at spectra73 ppm, of EEP-VTMOS/TEOS81 ppm and 102 ppm,treated which wood were (Figure assigned 6A), three to T 2well-definedstructure, T 3signalsstructure were − − − 2 3 andobserved Q3 free at silanols −73 ppm, structure, −81 ppm respectively and −102 ppm, [56–60 which]. In thewere spectrum assigned of to treated T structure, wood T after structure leaching and (FigureQ3 free6B), silanols three signalsstructure, were respectively also observed [56–60]. with In the the same spectrum shifts of as treated in thespectrum wood after of leaching treated wood (Figure before6B), three leaching. signals However, were also the observed intensity ofwith these the signals same shifts was slightly as in the lower spectrum compared of treated to the intensitywood before of theleaching. signals in However, the spectrum the intensity of unleached of these wood, signals suggesting was slightly that the lower Si concentration compared into thethe leachedintensity wood of the signals in the spectrum of unleached wood, suggesting that the Si concentration in the leached wood

Forests 2020, 11, 907 10 of 17 was lower than in wood before leaching. In the spectrum of wood treated with EEP-MPTMOS/TEOS (Figure6C), two low-intensity signals were found at –60 ppm and –100 ppm, which were assigned to T2 and Q3 structures [57–60]. In the spectrum of treated wood after leaching procedure (Figure6D), 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 Table3 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].

Table 3. The silicon concentration in treated wood and the degree of Si leaching determined by atomic absorption spectroscopy (AAS).

Silicon Concentration Treatment Preparations [ppm] The Degree of Si Leaching [%] UL L EEP-VTMOS/TEOS 779.90 1.34 756.10 6.30 3.1 ± ± EEP-MPTMOS/TEOS 596.48 8.94 551.15 7.14 7.6 ± ± UL—unleached wood; L—leached wood (acc. EN 84). Expressed as average standard deviations. ±

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 [62–64]. The silicon concentration in treated wood and the degree of Si leaching determined by XRF are shown in Table4.

Table 4. The silicon concentration in treated wood and the degree of Si leaching determined by X-ray ﬂuorescence spectroscopy (XRF).

Silicon Concentration Treatment Preparations [ppm] The Degree of Si Leaching [%] UL L EEP-VTMOS/TEOS 5.54 0.11 5.33 0.14 3.8 ± ± EEP-MPTMOS/TEOS 7.61 0.34 7.06 0.18 7.2 ± ± UL—unleached wood; L—leached wood (acc. EN 84). Expressed as average standard deviations. ±

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 Forests 2020, 11, 907 11 of 17 determination of Si concentration in wood samples using XRF took place only on the wood surface. Moreover, the Si concentration results presented in Table4 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 (Table3) 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.

3.2. 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 Table5.

Table 5. Retention and mass loss of treated wood after exposure to C. puteana.

Mass Loss of Mass Loss of Treatment Retention Leaching Test Treated Wood Control Wood Preparations [kg/m3] [%] [%] - 96.9 0.4 3.1 b 0.3 49.2 3.6 EEP ± ± ± EN 84 95.3 0.3 4.8 a 0.3 48.2 2.0 ± ± ± - 159.4 0.8 3.3 b 0.2 48.1 1.8 EEP-VTMOS/TEOS ± ± ± EN 84 158.4 0.5 3.5 b 0.1 48.1 2.6 ± ± ± - 164.6 0.4 2.9 b 0.1 48.0 4.3 EEP-MPTMOS/TEOS ± ± ± EN 84 161.8 0.6 3.2 b 0.2 44.7 4.2 ± ± ± Expressed as average standard deviations. Values denoted with identical letters do not diﬀer signiﬁcantly. ±

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 (Table2). 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. Forests 2020, 11, 907 12 of 17

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].

3.3. 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 Table6, 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.

Table 6. Moisture content, density, bending strength and modulus of elasticity of treated wood.

Treatment Moisture Content Density Modulus of Elasticity Bending Strength Preparations [%] [kg/m3] [MPa] [MPa] Control samples 10 623 16 17129 a 684 150.9 b 4.7 ± ± ± EEP 7 713 14 15945 b 461 158.0 a 4.8 ± ± ± EEP-VTMOS/TEOS 6 736 12 15848 b 552 160.8 a 3.9 ± ± ± EEP-MPTMOS/TEOS 6 741 13 15924 b 516 161.8 a 4.1 ± ± ± Expressed as average standard deviations. Values denoted with identical letters do not diﬀer signiﬁcantly. ±

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 Figure7, 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]. Forests 2020, 11, x FOR PEER REVIEW 12 of 17

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].

3.3. 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.

Table 6. Moisture content, density, bending strength and modulus of elasticity of treated wood.

Moisture Content Density Modulus of Elasticity Bending Strength Treatment Preparations [%] [kg/m3] [MPa] [MPa] Control samples 10 623 ± 16 17129 a ± 684 150.9 b ± 4.7 EEP 7 713 ± 14 15945 b ± 461 158.0 a ± 4.8 EEP-VTMOS/TEOS 6 736 ± 12 15848 b ± 552 160.8 a ± 3.9 EEP-MPTMOS/TEOS 6 741 ± 13 15924 b ± 516 161.8 a ± 4.1 Expressed as average ± standard deviations. Values denoted with identical letters do not differ significantly. 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 Forestsindividual2020, 11 ,cells 907 at sectioning [67,68]. However, the ethanolic solution does not affect on the cell wall13 of 17 S2 layer, which is one of the main factors determining the strength properties of wood [69].

(a) (b) (c)

FigureFigure 7. 7.The The SEMSEM imagines of wood wood treated treated with with EEP EEP (a (),a ),wood wood treated treated with with EEP-VTMOS/TEOS EEP-VTMOS/TEOS (b), ( b), andand wood wood treated treated withwith EEP-MPTMOSEEP-MPTMOS/TEOS/TEOS ( (cc).).

The impregnation of wood with chemical preservatives caused changes in the mechanical properties of wood [70–72]. 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.

4. 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 29Si 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 Forests 2020, 11, 907 14 of 17 from the wood structure. The treated wood showed an increase in bending strength and a decrease in the modulus of elasticity compared to untreated wood. In summary, based on the obtained results, it can be concluded that the propolis-silane preparations can be promising green wood preservatives, harmless for the natural environment. The bio-friendly preservatives can be used for the treatment of wood both in indoor and outdoor applications. However, the further investigation on the effect of the propolis-silane preparations and their single components (propolis extract, silicon compounds and the solvent) on the mechanical properties of treated wood should be performed in the future.

Author Contributions: Conceptualization. M.W. and I.R.; investigation. M.W., P.K.-S., and M.K.; writing—original draft preparation. M.W.; writing—review and editing. P.K.-S., M.K., E.R. and I.R.; supervision. I.R. All authors have read and agreed to the published version of the manuscript. Funding: The research was partially funded by National Science Centre, grant number 2019/03/X/NZ9/01800, and by a statutory activity from the Polish Ministry of Science and Higher Education (506.472.02.00). The article was co-financed within the Ministry of Science and Higher Education programme—“Regional Initiative Excellence” 2019–2022, project No. 005/RID/2018/19. Conflicts of Interest: The authors declare no conflict of interest.

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