Influence of Heat Treatment and Tannin Impregnation on Boron Depletion and Wood Durability

Article Inﬂuence of Heat Treatment and Tannin Impregnation on Boron Depletion and Wood Durability

Dercilio Junior Verly Lopes *, H. Michael Barnes and Gabrielly dos Santos Bobadilha

Department of Sustainable Bioproducts, Starkville, MS 39759, USA; [email protected] (H.M.B.); [email protected] (G.d.S.B.) * Correspondence: [email protected]

Received: 13 January 2020; Accepted: 4 February 2020; Published: 11 February 2020

Abstract: Heat treatment (HT) of a mixture of tannins and inorganic boron compounds showed effective results against wood decay organisms. Boron compounds play a critical role in the preservation of wood against wood decay organisms. The use of tannins and HT represents a relatively new environmentally friendly approach to the wood preservation industry. The aim of this study was to investigate the effect of tannin impregnation and HT on boron depletion, and termite and fungi resistance. Southern yellow pine (SYP) and yellow-poplar (YP) cube samples were used for this study. A mixture of condensed tannins from the Quebracho tree and disodium octaborate tetrahydrate (DOT) was injected into the specimens using a vacuum/pressure cycle, and the specimens were then heat-treated under N2 atmosphere for four hours at 190 ◦C to investigate both the tannin’s fixative ability to reduce boron leaching and the performance of the mixture against brown and white-rot fungi and termites. Tannins restricted boron leaching in 46% and 34% for SYP and YP, respectively, and also significantly increased the resistance against white-rot fungi for YP. Tannins and HT showed unpredictably good performance against termites. Tannins may be acting to denature proteins; in that case, fungal enzymes would be inhibited. This study revealed the importance of HT on a mixture of boron and tannins to decrease boron leaching and increase the durability of preservative systems.

Keywords: heat treatment; DOT; tannins; leaching; termites; fungi

1. Introduction In the last few years, the interest in heat treated wood has increased due to the decline in production of naturally durable wood, the increase in demand for more environmentally benign wood preservative techniques, and governmental regulations. According to [1], heat treatment (HT) is a process performed between the temperatures of 160 ◦C and 260 ◦C for a period of 15 min to 24 h. While lower temperatures only slightly affect wood properties, higher temperatures result in cell wall degradation. Heat treatment of wood specifically improves durability against wood decay fungi, decreases moisture absorption, decreases equilibrium moisture content, and increases dimensional stability. It also changes the appearance of the wood; it becomes darker. Heat treatment also increases weathering resistance and decreases both wettability and mechanical properties [2–5]. According to [6], a future aim of HT is improved decay resistance. This development will likely be based on a combination of boron impregnation with HT due to the proven efficacy of boron against termites and wood decay fungi and its low cost. Borates have been used for the pressure treatment of several wood products. Borate-treated wood products accept finishes and paints and are environmentally friendly. Although borates have many protective characteristics, their use in wood

Forests 2020, 11, 201; doi:10.3390/f11020201 www.mdpi.com/journal/forests Forests 2020, 11, 201 2 of 12 products is limited because of boron’s natural solubility in water, which leads to a rapid depletion of treated wood in outdoor exposed conditions [7–12]. Research has been conducted to address boron depletion. The most common strategies for reducing boron leaching are surface and envelope treatments, wood bulking, the use of water repellant, organo-boron compounds, a combination of biocides and non-biocidal agents, metallo-borates, stabilized boro-esters, protein borates, in situ polymerization and boron silicates [13–19]. Previous research has shown the potential of boron fixation by tannin auto condensation. The authors in [20] described a mechanism in which boric acid was partly fixed to a condensed tannin network with boron mobility being maintained. The authors in [21] leached a mixture of tannins and boron for 80 h. After the leaching period, a 30% loss of boron occurred. The trend of increased leaching continued as the experiment progressed. The effectiveness of tannins toward fixing boron under high temperatures and for longer leaching periods has not yet been investigated. In addition, little research has been done regarding boron’s efficacy on multiple wood species. Broadening this spectrum is relevant in order to improve durability across the widely varying range of species and characteristics of wood [22–24]. In this study, a solution of disodium octaborate tetrahydrate (DOT) and condensed tannins were pressure and heat-treated into southern yellow pine and yellow-poplar with the aim of reducing boron leaching. In addition, performance against wood decay organisms was evaluated with the goal of assessing the role of thermal modification. The specific objectives of the work were to:

(1) Test boron fixation through heat treatment and tannin impregnation. (2) Investigate wood decay after leaching against brown-rot fungus Gloeophyllum trabeum in southern yellow pine and white-rot fungus Trametes versicolor in yellow-poplar; (3) Determine eastern subterranean termite (Reticulitermes flavipes) attack on heat-treated southern yellow pine modified with tannins/borates.

2. Materials and Methods

2.1. Wood Samples Southern yellow pine (Pinus spp., specific gravity = 0.56 0.2) and yellow-poplar (Liriodendrum ± tulipifera, specific gravity = 0.47 0.3) (hereafter referred to as SYP and YP, respectively) blocks (19 ± × 19 19 mm) (radial, tangential, longitudinal), free of visible defects and heartwood were chosen for × the experiments. For the termite test, sapwood of SYP samples (25 mm 25 mm 6 mm) (r, t, l) (Pinus × × spp.), also free of visible defects, were chosen. The specimens were oven-dried at 103 2 C until they ± ◦ reached a constant weight. To minimize the effects of sample variation, 10 groups (5 treatments and two leaching periods) of samples with equal weight distributions were assigned. Heat-treated, heat-treated with DOT, heat-treated with tannins, and heat-treated with tannins and DOT (hereafter called HT, HT DOT, HT Tannin, and HT T/DOT, respectively) treatment levels were tested.

2.2. Tannin Characterization The tannin solution was prepared by dissolving 80 g of tannins into 800 mL of mechanically stirred deionized water heated at 60 ◦C. For the treatments that contained boron, 12 g of DOT was dissolved in the same manner as the tannin solution. Tannins came from the Quebracho tree (Aspidosperma spp.), acquired from Shellac Inc. (Napa, CA, USA) with 74–77% guaranteed purity. The impurities were water, amino acids, and monomeric and oligomeric carbohydrates. DOT was supplied by US Borax (Valencia, CA, USA). The pH and viscosity of the treating solutions are shown in Table1. Viscosity measurements were performed at 60 rpm with spindle number 61 at 60 ◦C. Forests 2020, 11, 201 3 of 12

Table 1. pH and viscosity of treating solutions. HT: heat treatment; T/DOT: tannins/disodium octaborate tetrahydrate.

Treatment pH Viscosity (cp) HT Tannin 5.83 2.8 HT T/DOT 6.14 2.6

The formulations were impregnated into wood using a full cell process with full vacuum (550+ mm. Hg) for 30 min to remove the majority of the air trapped inside the cell wall. The solution was injected into the retort under vacuum. The vacuum was reduced to atmosphere pressure, then 2 pressure was increased to 0.88 MPa (9 kgf cm− ) for 30 min. After impregnation, the surfaces of the samples were wiped oﬀ to remove the excess of solution and retention was calculated by weight gain. The net tannin retention was calculated by multiplying the gross retention by 77%, which was the tannin purity. Samples were air dried for two weeks at ambient temperature (approximately 20 ◦C and 65% relative humidity) to complete DOT and tannin stabilization. After the air-drying period, samples were oven dried until a constant weight at 102 3 C and their anhydrous weight and volume after ± ◦ impregnation were recorded.

2.3. Thermal Modification Thermal modification was performed in a 20 L chamber under a nitrogen atmosphere with pressure slightly above atmospheric pressure. Temperature at 190 ◦C was held for four hours for both species. The temperature was increased by 15 ◦C/min from ambient temperature. A total of 110 samples were heat treated (55 per species). The weight of the samples after thermal modification was obtained and recorded. The percentage mass loss (ML) of thermally modified samples was calculated using Equation (1). ! m m ML (%) = 100 1 − 2 , (1) × m1 where: m1 = initial oven-dry mass after impregnation, g; m2 = weight of samples after thermal modification, g.

2.4. Leaching Method Leaching was performed on SYP and YP following the American Wood Protection Association—AWPA E10 (AWPA 2017) protocol with a modiﬁcation that a leaching period of 15 days was used with removals taking place at 6 h, 24 h, 24 h, 24 h and 48 h, and thereafter at each 24 h interval until completion (15 days). The SYP and YP leachates from HT DOT and HT T/DOT were saved for analysis. In total, 60 leachates were assessed for the presence of boron in a 50 mL plastic bottle (30 leachates for species and 15 per treatment). Leachates were microwave digested with a MARSXpress (CEM, Charlotte, NC, USA) and analyzed with an Agilent 7900 Inductively Coupled Plasma-Mass Spectrometry (Agilent, Pato Alto, CA, USA). The amount of boron present in the leachate was recorded in parts per million (ppm). The initial parts per million (ppmi) was obtained by Equation (2). ! ! Wa f ter imp. Wod ppmi = 1000 − C (2) × 0.8 ∗ where:

Wafter imp. = weight after impregnation, g; Wod = oven-dry weight before impregnation, g; Forests 2020, 11, 201 4 of 12

C = concentration of the solution; 0.8 = volume of the solution, L. Forests 2020, 11, x FOR PEER REVIEW 4 of 12 Leaching was also performed on termite samples in the same manner. For the HT Tannin treatment, Leaching was also performed on termite samples in the same manner. For the HT Tannin weight loss due to leaching (WLleach) was calculated according to Equation (3). treatment, weight loss due to leaching (WLleach) was calculated according to Equation (3). m m 푚22..− 푚3 3 WLWLleachleach= =100 100 x ( − ) (3(3)) × 푚m2 2 wherewhere:: m2 == ooven-dryven-dry weight weight before before leaching, leaching, g. g. m3 == ooven-dryven-dry weight weight after after leaching, leaching, g. g.

2.5. Fungal Fungal Resistance Resistance Samples from the treatments were tested to to evaluate their their resistance resistance against against biological biological attack. attack. First, the the samples samples were were leached leached according according to Section to Section 2.4, then 2.4, the then procedures the procedures for the fungi for th teste fungi followed test followedAWPA E10 AWPA [25]. E10 The [ test25]. wasThe test performed was performed for 12 weeks. for 12 Asweeks. AWPA As AWPA E10 recommends E10 recommends and to and obtain to obtaindiscriminant discriminant results, results, samples samples from SYP from were SYP subjected were subjected to brown-rot to brown fungus-rot ( Gloeophyllum fungus (Gloeophyllum trabeum), trabeumand YP) samples, and YP weresamples exposed were toexposed white-rot to white fungus-rot (Trametes fungus ( versicolorTrametes). versicolor The weight). The loss weight due to loss fungal due toattack fungal was attack the response was the variableresponse selected variable to selected quantify to the quantify eﬃcacy the of efficacy the treatments. of the treatments. The following The followingdeviations deviation from the standards from the were standard employed: were for employed samples: containing for samples DOT, containing a thin, rigid, DOT perforated, a thin, rigid, grid perforatedwas placed grid over was the placed mycelium over to the prevent mycelium undesirable to prevent boron undesirable leaching boron when leaching in contact when with in moisture contact withfrom moisture the feeder from strips the (Figure feeder1), strips and the (Figure blocks 1) were, and addedthe blocks to the were test added only after to the total test colonization only after total and colonizationcoverage of the and feeder coverage strip of and the mesh. feeder The strip grid and was mesh. sterilized The grid to thewas same sterilized conditions to the recommendedsame conditions in recommendedAWPA E10 [25 ].in AWPA E10 [25].

Figure 1. ExperimentalExperimental setup setup for soil/block soil/block test. 2.6. Termite Resistance 2.6. Termite Resistance The purpose of this study was to determine the termite resistance of SYP (treated and untreated, The purpose of this study was to determine the termite resistance of SYP (treated and untreated, leached and unleached) when exposed to the Eastern subterranean termite (Reticulitermes ﬂavipes) in a leached and unleached) when exposed to the Eastern subterranean termite (Reticulitermes flavipes) in no-choice feeding test according to AWPA E1 [26] with the following noted modiﬁcations: a no-choice feeding test according to AWPA E1 [26] with the following noted modifications: 1. OmittedOmitted AWPA AWPA E1 E1sectionssections 6.4.2 6.4.2 and and 8.1. 8.1.4–8.164–8.16 pertaining pertaining to to termite termite mortality mortality following following testing. testing. TunnelingTunneling presen presence,ce, majority majority termite termite position, position, and and termite termite mortality mortality were approximated as per AmericanAmerican Society Society for for Testing Testing and Materials Materials—ASTMD3345 – ASTMD3345 [27] sectionssections 12.2.2–12.2.4.12.2.2–12.2.4. 2. Omitted the moisture content portion of AWPA E1 6.3.1 and 6.3.2 as bottles maintained in the chamber do not require this procedure. This allowed us to avoid disrupting termite activity during the course of the study. Termites were collected near Starkville, MS and kept under favorable conditions until testing was initiated. The termites used from this colony consisted of approximately 6% soldiers. At the end

Forests 2020, 11, 201 5 of 12

2. Omitted the moisture content portion of AWPA E1 6.3.1 and 6.3.2 as bottles maintained in the chamber do not require this procedure. This allowed us to avoid disrupting termite activity during the course of the study.

Termites were collected near Starkville, MS and kept under favorable conditions until testing was initiated. The termites used from this colony consisted of approximately 6% soldiers. At the end of the test, the weight loss and visual rating due to termite attack were calculated to assess the eﬃcacy of the treatments.

2.7. Statistical Analysis For the analysis of weight loss due to Gloeophyllum trabeum, Trametes versicolor, and termite attack, a visual rating scale was performed in an A B factorial arrangement of treatments in a completely × randomized design, where A was designed as the treatments and B was assigned as unleached and leached period. The mass loss due to heat treatment was analyzed as a completely randomized design where the sources of variation were treatment levels, namely, HT Tannin, HT, HT DOT and HT T/DOT. The error term, εijk, was assumed to be normally distributed with a mean of 0, variance of 2 2 σ , and that it was independently and identically distributed (εijk~N (0, σ )) for all experiments. In addition, least square difference (LSD) at the 0.05 significance level was performed as a post hoc test in cases where either the interactions or factors were detected as significant by the F-test (p 0.05) in ≤ the analysis of variance (ANOVA). All data was generated using SASTM software version 9.4 (SAS Institute, Cary, NC, USA).

3. Results and Discussion

3.1. Preservative Retention Preservative retention level and penetration are two methods to quantify the impregnation of a treatment. Even with an eﬀective preservative system, poor retention and penetration may lead to wood decay. Table2 shows the retention levels of DOT and tannins for SYP and YP.

Table 2. Initial and remaining DOT retention after leaching.

Southern Yellow Pine 3 3 Initial Retention (kg m− ) Remaining Retention (kg m− ) Treatment Leaching Period DOT Tannin DOT Tannin Heat-treated DOT 15 days 7.4 - 0.2 - Heat-treated Tannin 15 days - 39.7 - 38.3 Heat-treated T/DOT 15 days 8.1 42.1 4.6 39.2 Yellow-Poplar 3 3 Initial Retention (kg m− ) Remaining Retention (kg m− ) Treatment Leaching Period DOT Tannin DOT Tannin Heat-treated DOT 15 days 8.6 - 1.7 - Heat-treated Tannin 15 days - 42.6 - 41.2 Heat-treated T/DOT 15 days 8.3 42.9 4.0 40.1

The AWPA U1–Commodity section A: Sawn products [28] recommends a minimum preservative 3 3 retention of 4.5 kg/m and 2.7 kg/m of B2O3 where Coptotermes formosanus and Reticulitermes flavipes are active, respectively. The remaining retentions of 4.6 kg/m3 and 4 kg/m3 DOT (Table2) for SYP and 3 3 YP, respectively, can be converted into 3.1 kg/m and 2.7 kg/m of B2O3, which has been shown to protect the wood only against Reticulitermes flavipes [29]. Table2 shows that initial retention levels for both HT DOT and HT T /DOT treatments were higher for YP. Several anatomical factors may have played key roles in the treatability characteristics, hence Forests 2020, 11, 201 6 of 12 Forests 2020, 11, x FOR PEER REVIEW 6 of 12 intracheid the preservative-wall bordered retention. pit pairs. For There hardwoods, are also effective most of thebut flowlimited path flow mechanisms paths through are ray through tracheids the vesselsto longitudinal when they tracheids are not and blocked through by tyloses.resin canals; In addition, however, fibers these and are ray of secondary parenchyma importance. contribute The to woodpresence permeability; of earlywood however, and latewood these are can less also influential. affect the Longitudinal flow of fluid raysinto arewood. not The easily earlywood penetrated of and,dried as softwood such, their is contribution usually less is permeable minimal. thanA major the flow latewood, path mechanism while in hardwoods for softwoods, the is reverse through is tracheid-wallgenerally true bordered due to larger pit pairs. earlywood There arevessels also e[30ffective]. but limited flow paths through ray tracheids to longitudinal tracheids and through resin canals; however, these are of secondary importance. The3.2. presenceMass Loss of earlywood and latewood can also affect the flow of fluid into wood. The earlywood of driedFigure softwood 2 shows is usuallythat SYP less was permeable more resistant than to the thermal latewood, degradation while in hardwoods,than YP with the mass reverse loss isof generally1.2% and true9.4%, due respectively. to larger earlywood These results vessels align [30 with]. previous research [31]. The highest mass loss was observed on HT DOT for both species. This may be due to the possible conversion of DOT into 3.2. Mass Loss boric acid at high temperatures, leading to the acidic degradation of cell wall components. FigureIn YP 2samples shows, thatthe mass SYP was loss morevaried resistant from 1.5% to thermal (HT Tannin) degradation to 13.7% than (HT YP DOT). with This mass variation loss of 1.2%can be and explained 9.4%, respectively. by the complex These reactions results align taking with place previous during research heat treatment, [31]. The such highest as the mass destruction loss was observedof hemicellulose on HT DOT and fordegradation both species. of the This amorphous may be due region to the of possible cellulose. conversion These changes of DOT lead into boricto an acidincrease at high in lignin temperatures, percentage leading content, to the waxes, acidic general degradation carbohydrates, of cell wall resins, components. and terpenes [32–34].

(a) (b)

FigureFigure 2.2. MassMass lossloss duedue toto heatheat treatment,treatment, ((aa)) southern southern yellowyellow pinepine and and ( b(b)) yellow-poplar. yellow-poplar.1 1 MeansMeans withwith thethe samesame letterletter areare notnotstatistically statisticallydi differentﬀerent at at the the 0.05 0.05 signiﬁcance significance level. level.

InThe YP mass samples, loss of the samples mass loss impregnated varied from with 1.5% tannins (HT Tannin) was somewhat to 13.7% (HT similar. DOT). The This heat variation treatment can beresulted explained in 1.5% by the and complex 3.9% of reactionsmass loss taking for YP place and duringSYP, respectively. heat treatment, This such result as may thedestruction be due to the of hemicellulosecomplexation andof tannin degradations with ofwood the amorphouscomponents, region which of would cellulose. increase These the changes thermal lead stability to an increase of cell inwall lignin components percentage or content,tannins at waxes, 190 °C general. In fact, carbohydrates, the authors in resins, [35] found and terpenes that condensed [32–34]. tannins have a maximumThe mass thermal loss of stability samples at impregnated 258 °C due to with the tannins presence was of somewhataromatic structures. similar. The heat treatment resulted in 1.5% and 3.9% of mass loss for YP and SYP, respectively. This result may be due to the complexation3.3. Boron Leaching of tannins Control with wood components, which would increase the thermal stability of cell wall components or tannins at 190 C. In fact, the authors in [35] found that condensed tannins have a For both species, it is evident◦ that tannins were able to prevent boron leaching (Figure 3). maximum thermal stability at 258 C due to the presence of aromatic structures. Somewhat better results were found◦ for SYP. The total leaching for the SYP HT DOT treatment was 3.3.462.68 Boron ppm Leaching, whereas Control HT T/DOT was 247.2 ppm. In the case of YP, HT DOT leached 456 ppm and HT T/DOT leached 298.41 ppm. Furthermore, the reductions in boron leaching when tannins were used wereFor 46.5% both and species, 34.5%, it isrespectively. evident that tannins were able to prevent boron leaching (Figure3). Somewhat better results were found for SYP. The total leaching for the SYP HT DOT treatment was 462.68 ppm, whereas HT T/DOT was 247.2 ppm. In the case of YP, HT DOT leached 456 ppm and HT T/DOT leached 298.41 ppm. Furthermore, the reductions in boron leaching when tannins were used were 46.5% and 34.5%, respectively.

Forests 2020, 11, 201 7 of 12 Forests 2020, 11, x FOR PEER REVIEW 7 of 12

(a) (b)

Figure 3. Cumulative boron leaching in southern yellow pine (a)) andand yellow-poplaryellow-poplar ((bb).).

There waswas aa steep steep and and typical typical loss loss of boronof boron in thein earlythe early stages stages of the of leaching the leaching [36,37 [].36 The,37] flat. The curve flat aftercurve 120 after h is120 due h tois due undetectable to undetectable levels oflevels boron of inboron the leachate.in the leachate. The detectable The detectable threshold threshold for boron for wasboron 4 ppm.was 4 In ppm. this case,In this most case, of themost boron of the leaching boron occurredleaching inoccurred the first in five the days first forfive both days treatments for both andtreatments species. and For species. HT T/DOT, For the HT leaching T/DOT, might the leaching have occurred might have due to occurred an incomplete due to polymerization an incomplete ofpolymerization the tannins [21 of]. the For tannin HT DOT,s [21]. leaching For HT DOT, might leaching have occurred might have because occu ofrred unfixed because or of poorly unfixed fixed or preservativespoorly fixed preservatives near the wood near surface the wood [38]. surface [38]. The eefficacyfficacy of tanninstannins inin preventing preventing boron boron leachingleaching may be explained by the fact that after impregnationimpregnation and heat treatment,treatment, boron waswas mixedmixed withwith tanninstannins and complexedcomplexed deeperdeeper in thethe cellcell wall.wall. In that case, boron would leach less. Also, research has indicated that tannins are able to chelate withwith severalseveral metalsmetals andand formform metal–tanninmetal–tannin complexes.complexes. Such metals were aluminum, iron, iron, copper, chromiumchromium andand the metalloid boron [[3939].]. Fur Furthermore,thermore, boron would react through OH groups present inin thethe B-ringB-ring ofof thethe tannintannin moleculemolecule byby generatinggenerating aa tannin–borontannin–boron complexcomplex resistantresistant toto leaching.leaching.

3.4. Fungi Resistance Figure4 4 shows shows the the average average weight weight loss loss (%) (%) for for SYP SYP and and YP. YP. The The interaction interaction between between treatment treatment and leachingand leaching period period for Trametes for Trametes versicolor versicolorwas notwas significant not significant (p > 0.005).(p > 0.005). Thus, Thus, only only the main the main effect effect was analyzed.was analyzed. This may This be may explained be explained by the limitedby the limitedenzymatic enzymatic activity in activityT. versicolor in T. after versicolor heat treatment,after heat leadingForeststreatment, 2020 to, 1 smallleading1, x FOR di ffPEERtoerences small REVIEW differences in weight loss in w beforeeight andloss afterbefore leaching. and after leaching. 8 of 12 Beech (Fagus sylvatica) heat treated at 240 °C for 8 h was found to undergo weight loss due to T. versicolor attack of approximately 5% [40]. Enzymatic activities were detected only in the first 30 days of exposure and ceased after this period. Practically no cellobiohydrolase activity were detected. Heat treatment seemed to disrupt the enzymatic activities involved in wood degradation, leading to low weight loss as in the case of this work. However, the weight loss of 20.6% for HT Tannin in YP may be explained by the ability of white-rot fungi to degrade lignin by producing ligninolytic enzymes [41].

(a) (b)

Figure 4. Weight loss caused by Gloeophyllum trabeum ((aa)) and and TrametesTrametes versicolor ((bb)) on on SYP SYP and and YP, YP, respectively. 11 MMeanseans with the same letter were not statistically signiﬁcantsignificant didifferentﬀerent atat αα == 0.05.

BeechFigure (4aFagus shows sylvatica that there) heat was treated a significant at 240 ◦ Cdecrease for 8 h in was weight found loss to for undergo HT pine weight when loss compared due to T.to versicolorthe controlattack; however of approximately, averages still 5% [ indicate40]. Enzymatic high degradation. activities were In contrast, detected onlyHT yellow in the- ﬁrstpoplar 30 (Figure 4b) was highly resistant to fungal attack (with statistical difference) when compared to control samples. This durability improvement may be associated with alterations in the cell wall and wood porosity during thermal modification, which may have hindered fungal activity [42]. Tannins performed efficiently against brown-rot fungi for both leached and unleached treatment levels (Figure 4a). In the case of HT T/DOT, durability improvement was approximately twice as high as HT DOT after leaching. It was even better when tannins were tested alone, where the mass loss was 2% after leaching. Condensed tannins most likely acted as inhibitors of enzymes such as cellulases and lignases through complexation after thermal modification [43]. This inhibitory performance was seen only for SYP, as the weight loss in YP samples was relatively high for HT Tannin (20.6%). The intense white- rot fungal degradation on YP may be due to the increase of laccase activity, which is a phenoloxidase enzyme produced by white-rot fungi [44–46]. In fact, phenol rings are present in the chemical composition of tannin molecules [47]. Furthermore, such composition favored the development of the fungi by catalyzing the oxidation of phenol rings and thereby increasing weight loss 3.5 times. Table 3 shows the least square difference after the AWPA E1 protocol. This table should be carefully analyzed as there is no practical meaning in analyzing leaching in control and samples that were heat-treated for 15 days.

Table 3. Weight loss caused by Reticulitermes flavipes.

Weight Loss (%) Time Treatment Mortality 2 Tunneling 3 Positioning 4 Mean (Days) 26.76 A 1 Control 15 s + u 25.84 A Control 0 s + u/d 20.62 B HT DOT 15 s + u 13.08 C HT T/DOT 15 x + N/A 10.95 C Heat-treated 15 x + N/A 10.41 CD Heat-treated 0 x + N/A 6.20 ED HT Tannin 15 x + N/A 3.02 E HT Tannin 0 x + N/A 2.66 E HT DOT 0 x − N/A 1.55 E HT T/DOT 0 x − N/A 1 means with the same letters were not statistically significant different at the 0.05 significance level. 2 mortality: ‘s’ = small (0–33%); ‘x’ = complete. 3 tunneling: ‘+’ = yes; ‘-’ = no. 4 positioning: ‘u’ = beneath surface; ‘d’ = on surface; ‘N/A’ = not applicable.

Forests 2020, 11, 201 8 of 12 days of exposure and ceased after this period. Practically no cellobiohydrolase activity were detected. Heat treatment seemed to disrupt the enzymatic activities involved in wood degradation, leading to low weight loss as in the case of this work. However, the weight loss of 20.6% for HT Tannin in YP may be explained by the ability of white-rot fungi to degrade lignin by producing ligninolytic enzymes [41]. Figure4a shows that there was a significant decrease in weight loss for HT pine when compared to the control; however, averages still indicate high degradation. In contrast, HT yellow-poplar (Figure4b) was highly resistant to fungal attack (with statistical difference) when compared to control samples. This durability improvement may be associated with alterations in the cell wall and wood porosity during thermal modification, which may have hindered fungal activity [42]. Tannins performed efficiently against brown-rot fungi for both leached and unleached treatment levels (Figure4a). In the case of HT T /DOT, durability improvement was approximately twice as high as HT DOT after leaching. It was even better when tannins were tested alone, where the mass loss was 2% after leaching. Condensed tannins most likely acted as inhibitors of enzymes such as cellulases and lignases through complexation after thermal modification [43]. This inhibitory performance was seen only for SYP, as the weight loss in YP samples was relatively high for HT Tannin (20.6%). The intense white-rot fungal degradation on YP may be due to the increase of laccase activity, which is a phenoloxidase enzyme produced by white-rot fungi [44–46]. In fact, phenol rings are present in the chemical composition of tannin molecules [47]. Furthermore, such composition favored the development of the fungi by catalyzing the oxidation of phenol rings and thereby increasing weight loss 3.5 times. Table3 shows the least square di fference after the AWPA E1 protocol. This table should be carefully analyzed as there is no practical meaning in analyzing leaching in control and samples that were heat-treated for 15 days.

Table 3. Weight loss caused by Reticulitermes ﬂavipes.

Weight Loss (%) Mean Treatment Time (Days) Mortality 2 Tunneling 3 Positioning 4 26.76 A 1 Control 15 s + u 25.84 A Control 0 s + u/d 20.62 B HT DOT 15 s + u 13.08 C HT T/DOT 15 x + N/A 10.95 C Heat-treated 15 x + N/A 10.41 CD Heat-treated 0 x + N/A 6.20 ED HT Tannin 15 x + N/A 3.02 E HT Tannin 0 x + N/A 2.66 E HT DOT 0 x N/A − 1.55 E HT T/DOT 0 x N/A − 1 means with the same letters were not statistically significant different at the 0.05 significance level. 2 mortality: ‘s’ = small (0–33%); ‘x’ = complete. 3 tunneling: ‘+’ = yes; ‘ ’ = no. 4 positioning: ‘u’ = beneath surface; ‘d’ = on surface; ‘N/A’ = not applicable. −

The weight loss for heat-treated specimens was approximately 2.5 times smaller on average than on controls. The opposite is found in the literature [48,49]. Values up to three times higher for modified wood compared to controls have been reported in [48] for Pinus sylvestris L. The increase in durability of heat-treated wood against termites may be associated with the change in the termite diet when compared to control samples. The flagellate species in the termite gut seem to be nutritionally specialized, each filling a specific niche in the digestion of cell wall components [48]. Thus, when the chemical structure of the wood is changed by heat treatment, it also changes how flagellate enzymes phagocytose and digest the wood, leading to starvation, although termites could continue to feed themselves from the wood. Surprisingly, HT Tannin treatment was effective against termites. There was no statistical Forests 2020, 11, 201 9 of 12 difference between unleached and leached treatments. This finding may be explained by the deep penetration of tannins into the cell wall with further stabilization after heat treatment with reduced (Figureleaching5 ).(Figure Another 5). Another theory is theory that the is that impregnation the impregnation of wood of withwood tannins with tannin increaseds increased the cell the wall cell wallresistance resistance to the to mechanicalthe mechanical and and enzymatic enzymatic attack attack of of termites. termites. In In research research by by [[5050],], JapaneseJapanese cedar (Cryptomeria japonica)japonica) waswas impregnatedimpregnated with with chemically chemically modiﬁed modified mimosa mimosa tannins tannin ands and then then exposed exposed to C.to formosanusC. formosanus. The. The results results indicated indicated a weight a weight loss loss of approximately of approximately 10%, 10%, which which was higherwas higher than whatthan waswhat found was found in this in study. this study.

Figure 5. Weight loss due to leaching in termite samples. (x = mean; horizontal line = median, whisker Figure 5. Weight loss due to leaching in termite samples. (x = mean; horizontal line = median, whisker = range). = range). The interaction between treatments and leaching period for the visual grading scale was significant (p The0.001). interaction As shown between in Figure treatments6, there was and no leaching statistical period di ff erence for the in visual the visual grading rating scale scale was of ≤ significantunleached ( HTp ≤ T0.001)./DOT As compared shown in to Figure HT DOT, 6, there where was termites no statistical caused difference only a light in the attack visual on rating both. Thescale e ffofectiveness unleached of HT tannins T/DOT in compared restraining to boron HT DOT, leaching where impacted termites the caused durability only ofa light the HT attack T/DOT on both.leached The treatment, effectiveness as at theof tannins end of thein restrain leachinging procedure, boron leaching the change impacted in visual the rating durabilit is cleary of (varying the HT T/DOTfrom 2.4 leached to 7, whichtreatment, corresponds as at the end to approximately of the leaching threeprocedure times, the better change performance). in visual rating In general, is clear (thevarying attack from upon 2.4 control to 7, samples which corresponds was intense and to approximately statistically significant three times as compared better performance to HT samples,). In Forestswhereingeneral, 2020 the , 11 ,attack x FOR PEERupon was REVIEW moderate. control samples was intense and statistically significant as compared 10to of HT 12 samples, wherein the attack was moderate.

Figure 6. 6. VisualVisual rating rating scale scale of specimens of specimens after the after American the American Wood Protection Wood P Associationrotection A EIssociation experiment. EI experiment.Values followed Values by the followed same letter by the were same not statistically letter were di notfferent statisti bycally Least differentSquare Di byfference Least multiple Square Dcomparisonifference multiple test at the comparison 0.05 significance test at the level. 0.05 significance level.

4. Conclusions Preservative retention after 15 days of water leaching was higher in SYP specimens than in YP, with both being protected against attack from Reticulitermes flavipes. Impregnation of SYP and YP with tannins and further heat treatment were able to prevent boron leaching by 46.5% and 34.5%, respectively, which is higher than previous research has found. Heat-treated wood showed intermediate-low durability against G. trabeum and high durability against T. versicolor. In contrast, HT Tannin was effective in hindering brown-rot fungus with weight loss as low as 2.0%. Surprisingly, treatment with tannins consistently performed well independent of leaching period, reaching a visual rating score of 7. As a whole, our findings demonstrate that condensed tannins contribute to boron stabilization. This additive has the potential to be included in a series of preservative systems due to its low price and environmental profile.

Author Contributions: Conceptualization, Methodology, Validation, Data Curation and Analysis, Writing: D.J.V.L., H.M.B., G.d.S.B. Supervision: H.M.B. Funding acquisition: H.M.B. Review: H.M.B.

Funding: This research was funded, in part, by the USDA National Institute of Food and Agriculture McIntire- Stennis project 1009413 and is a contribution of the Forest and Wildlife Research Center, Mississippi State University. It is under SBP 968 designation.

Conflicts of Interest: The authors declare no conflict of interest.

References

Forests 2020, 11, 201 10 of 12

4. Conclusions Preservative retention after 15 days of water leaching was higher in SYP specimens than in YP, with both being protected against attack from Reticulitermes flavipes. Impregnation of SYP and YP with tannins and further heat treatment were able to prevent boron leaching by 46.5% and 34.5%, respectively, which is higher than previous research has found. Heat-treated wood showed intermediate-low durability against G. trabeum and high durability against T. versicolor. In contrast, HT Tannin was effective in hindering brown-rot fungus with weight loss as low as 2.0%. Surprisingly, treatment with tannins consistently performed well independent of leaching period, reaching a visual rating score of 7. As a whole, our findings demonstrate that condensed tannins contribute to boron stabilization. This additive has the potential to be included in a series of preservative systems due to its low price and environmental profile.

Author Contributions: Conceptualization, Methodology, Validation, Data Curation and Analysis, Writing: D.J.V.L., H.M.B., G.d.S.B. Supervision: H.M.B. Funding acquisition: H.M.B. Review: H.M.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded, in part, by the USDA National Institute of Food and Agriculture McIntire-Stennis project 1009413 and is a contribution of the Forest and Wildlife Research Center, Mississippi State University. It is under SBP 968 designation. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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