Biogenic Synthesis of Copper and Zinc Oxide from Eucalyptus dunnii Leaves for Pinus elliottii Wood Preservation

Abstract

The present study aims to evaluate the mechanical properties, colorimetric characteristics, and decay resistance of Pinus elliottii woods treated with oxides synthesized via green chemistry. For this purpose, an aqueous extract from Eucalyptus dunnii leaves was used to synthesize particles based on copper- and zinc-based oxides, as well as a binary oxide system (CuO/ZnO). Sodium polyacrylate was employed as a dispersant, impregnating the oxides into the wood through a horizontal autoclave using a modified Bethell process, assisted by a compressor, applying a pressure of 0.8 MPa for 30 min. The exposure to weathering aging did not significantly alter the mechanical properties of the samples, but it caused the leaching of particles from the treated wood surface, as shown by colorimetric results. Regarding the decay resistance, the copper-based oxide proved to be the most effective treatment against Trametes versicolor (a white-rot fungus), reducing mass loss down to 1.2%. The CuO/ZnO formulation reduced the mass loss caused by Gloeophyllum trabeum to 1.1%, while the zinc oxide showed minimal efficacy. Thus, oxides synthesized via green chemistry using aqueous leaf extracts and mild thermal conditions for synthesis and calcination proved effective in enhancing the wood resistance against biotic deterioration agents.

1. Introduction

Pinus elliottii is a commercially important species in Brazil, particularly in the southern region, where it is widely employed in construction and furniture manufacturing [1,2,3]. Pinus species account for about 18% of Brazil’s 9.55 million hectares of planted forests, emphasizing their strategic role in the national forestry industry [4]. Its features, including mechanical strength, renewability, and ease of handling, make it a versatile and competitive raw material from both technical and economic perspectives [2].

When used in outdoor environments, wood materials are exposed to abiotic agents, such as ultraviolet solar radiation and leaching by rain, which cause significant damage to external wood applications [5,6]. These agents trigger the material’s deterioration, leading to the discoloration characteristics of weathering. Weathering not only alters the wood’s surface appearance but also affects its wettability, surface layer resistance, chemical composition, microscopic structure, and the dislodgement of poorly adhered surface treatments [7,8].

Besides abiotic agents, such as UV radiation and moisture, wood is also vulnerable to biotic degradation, primarily caused by xylophagous fungi and insects. Among the most destructive fungal species are Trametes versicolor (white rot) and Gloeophyllum trabeum (brown rot). White-rot fungi, such as T. versicolor, possess enzymatic systems capable of degrading all major wood components, including lignin, cellulose, and hemicelluloses, through oxidative and hydrolytic mechanisms [9]. In contrast, brown-rot fungi, like G. trabeum, primarily depolymerize cellulose and hemicelluloses, leaving behind a lignin-rich, brittle matrix that compromises the mechanical strength of the wood [9,10]. These fungi are responsible for rapid mass loss and significant structural deterioration, especially under conditions of high humidity and moderate temperatures, which are common in many service environments.

To address these challenges, various preservative treatments have been employed, including chemical treatments, thermal modification, and technologies utilizing nanoscale particles [11,12]. One of the most common treatments, chromated copper arsenate (CCA), has been widely applied to pressure-treated wood since the 1930s. This preservative is effective in protecting wood from insect damage and microbial decay. However, the use of CCA has been increasingly restricted in some regions due to its potential environmental impacts and health risks, primarily due to the presence of arsenic in its composition [13,14].

The search for CCA alternatives has driven the development of more sustainable wood treatments, utilizing green synthesis principles and nanoscale particles. Recent studies, such as that by Evans et al. [15], highlight the potential of novel methods, such as nanopreservatives and plant extracts, which combine a low environmental impact with reduced costs. These alternatives leverage biological compounds, including polyphenols, enzymes, and terpenoids, which help stabilize free radicals and enhance the wood’s resistance to decay [16,17,18]. Zinc and copper oxides, for instance, have proven effective in protecting against fungi and providing photoprotection, while also being more resistant to leaching [19,20,21,22,23].

Furthermore, several Eucalyptus species have been widely explored for the green synthesis of metal oxide nanoparticles due to their rich phytochemical profiles and high bioreducing capacity [24,25,26]. In particular, Eucalyptus globulus, Eucalyptus radiata, and Eucalyptus grandis were successfully employed to obtain nanostructured zinc oxide through eco-friendly synthesis methods, often yielding materials with effective antimicrobial properties and enhanced morphological control. For instance, Obeizi et al. [27] demonstrated the synthesis of ZnO nanoparticles using E. globulus essential oil, highlighting its potent antimicrobial and antibiofilm activities against a broad spectrum of pathogenic strains, including Staphylococcus aureus and Pseudomonas aeruginosa. Similarly, Droepenu et al. [28] explored the use of Eucalyptus radiata leaf extracts to mediate the formation of zinc oxide nanostructures with distinct morphological features (spherical and rod-like) and observed that the calcination temperature significantly affected the particle size and bioactivity. Additionally, Houissa et al. [29] reported the biosynthesis of ZnO nanoparticles using Eucalyptus grandis, where the phytoconstituents present in the aqueous extracts facilitated the nucleation and stabilization of nanostructured oxides with a promising antifungal performance.

The aqueous extract of Eucalyptus dunnii leaves contains a complex mixture of bioactive compounds, including polyphenols, flavonoids, terpenoids, and various organic acids, which are commonly reported in the phytochemical profile of Eucalyptus species [27,28]. These compounds act as both reducing and stabilizing agents during green synthesis, which may facilitate the transformation of metal salts, such as copper and zinc acetates, into their corresponding oxides. Specifically, the hydroxyl and carbonyl functional groups present in phenolic compounds can chelate metal ions and promote their thermal decomposition into oxide forms under mild calcination conditions [29].

Green synthesis routes, particularly those using plant extracts, are known to influence the structural, morphological, and stereochemical properties of metal oxides when compared to conventional synthesis methods. Bioactive compounds, such as phenolics, flavonoids, and organic acids in the extracts, act as reducing and capping agents during the synthesis process, controlling crystal nucleation and growth and leading to differences in the particle size, crystallinity, surface area, and even electronic properties of the resulting material [30]. For example, zinc oxide nanoparticles synthesized from bio-based extracts may display smaller particle sizes and a higher surface reactivity than those obtained via chemical routes, due to the complex interactions between phytochemicals and metal ions during synthesis [31]. Such structural modifications are relevant because they may enhance the material’s interaction with wood polymers and its performance in biological or environmental applications [32].

Therefore, a biogenic route not only supports oxide formation but also influences particle morphology and stability. A comparison between the thermal decomposition of metal acetates with and without the plant extract would help elucidate the catalytic role of the phytoconstituents. Without the extract, salt calcination would require higher temperatures or result in less controlled nucleation and growth, while the presence of the extract promotes oxide formation at lower temperatures, highlighting the eco-friendly advantage of the green chemistry approach. This study aims to evaluate the mechanical and color properties, as well as weathering and decay resistances, of Pinus elliottii wood treated with oxides produced via green chemistry methods. The hypothesis is that this oxide treatment will enhance the wood’s short- and long-term performance, providing an effective and sustainable alternative to conventional treatments involving toxic compounds.

2. Materials and Methods

At a private property in Morro Redondo, Brazil (31°34′59” S; 52°39′08” W), leaves of Eucalyptus dunnii Maiden were collected from 5 trees which were approximately 6 years old. All trees were in the same solar exposure, and the collection was conducted in October 2023. For the green synthesis of our oxides, copper(II) acetate monohydrate (≥98% purity, Cu(CH₃COO)₂·H₂O; Dinâmica Química Contemporânea, Indaiatuba, Brazil) and zinc acetate dihydrate (≥98% purity, Zn(CH₃COO)₂·2H₂O; Synth, Diadema, Brazil) were used as precursor salts. Poly(acrylic acid sodium salt) (PAA/Na), with a molecular weight of approximately 5100 g·mol⁻¹ and ≥99% purity (Sigma-Aldrich, St. Louis, MO, USA), was used as a dispersant to maintain the oxides suspended in distilled water.

2.1. Obtaining and Processing Leaves and EXTRACT Preparation

The collected leaves were dehydrated in a forced-air oven 36 (±3) °C for two days until reaching a constant mass. Subsequently, these dried leaves were ground in a Willey knife mill. The resulting powder was not sieved, and all particle sizes were used in the extraction process. To preserve their properties, the powder was stored at 4 (±1) °C and protected from light. In the aqueous extraction process, 10 g of the powder derived from the leaves of E. dunnii was incorporated into an Erlenmeyer flask containing 100 mL of deionized water. Extraction was carried out in a thermostatic bath 80 (±3) °C for 75 min. Afterward, the solutions were centrifuged at 3200 rpm for 10 min and vacuum-filtered using a cellulose paper filter. The obtained extracts were placed in screw-cap bottles and stored at 4 (±1) °C, protected from light, until their application.

The ethanolic extract of the Eucalyptus dunnii leaves used in this study was previously shown to contain phenolic compounds, carboxylic acids, alcohols, amines, and other oxygenated organics, as identified by FTIR [33]. It is important to note that the composition of the plant extract may vary depending on factors such as the season, geographic location, and the individual performing the extraction. Although care was taken to describe the process in detail, we acknowledge that the lack of chemical profiling of the extract limits reproducibility. Therefore, we recommend that future studies include standardization protocols and analytical characterization (e.g., HPLC or FTIR) to ensure greater consistency in the extract composition.

2.2. Green Synthesis of CuO and ZnO Oxides

In Erlenmeyer flasks with a capacity of 100 mL, 1 g of the precursor salt and 20 mL of the extract were added. The solution was carefully poured and subjected to vigorous magnetic stirring (80 ± 3 °C) for a period of 20 min. After the stipulated time, the solution was transferred to evaporation capsules and placed in a muffle furnace without the need for preheating. The furnace was programmed to increase the temperature at a rate of 12 °C per min, and the samples remained in the furnace for 2 h after reaching temperatures of 100, 200, 300, 400, and 500 °C, respectively. After calcination, the samples were dispersed using a porcelain mortar and pestle. The prepared samples were stored for future characterization.

In the preparation of the CuO/ZnO formulation, 5 g of each salt precursor was added to 200 mL of the corresponding extract in an Erlenmeyer flask. The subsequent phases of the synthesis were conducted according to the instructions mentioned previously. The structural characterization of the synthesized oxides by FTIR and XRD was reported in detail elsewhere for the same precursor salts and Eucalyptus dunnii leaf extract [33]. A future study comparing the calcination of precursor salts with and without the plant extract is recommended to better understand the catalytic role of the phytochemicals in oxide formation.

2.3. Fabrication of Test Specimens and Impregnation

The samples were produced using boards from Pinus elliottii wood, which was harvested from young trees, approximately 25 years old. Five test specimens were used for each treatment, with dimensions of 5 × 2.5 × 1.5 cm (longitudinal × tangential × radial), subsequently placed in a climate-controlled chamber under temperature 20 (±3 °C) and relative humidity (65 ± 3%) conditions controlled for one week for stabilization.

The preservative suspension based on oxides was prepared with a concentration of 2 wt%. The concentration of 2 wt% for the oxide suspension was selected based on preliminary tests that demonstrated sufficient impregnation into the wood without causing excessive surface deposition or particle agglomeration. This concentration is also in line with previous studies involving nanoparticle-based wood treatments, where similar loadings provided effective results without compromising wood structure or handling properties [34,35,36]. Thus, in 1350 g of distilled water, 27 g of each oxide treatment and 1.35 g of PAA/Na were added. The treatment with only PAA/Na underwent the same analyses as the rest, aiming to determine the possible influence of the polymer on the results. To carry out the impregnation of the preservative suspension into the wood, a laboratory-scale horizontal autoclave was used, through the full-cell process (Bethell). In the first step, using a vacuum pump, an initial vacuum of −0.1 MPa was applied for 15 min. In the second step, the preservative suspension was introduced into the chamber through the differential pressure between the internal (autoclave chamber) and external (ambient) environments. With the use of a compressor, the content inside the cylinder was subjected to a pressure of 0.8 MPa for 30 min. After treatment, the samples were again placed in a climate-controlled room under the same conditions mentioned earlier, allowing the fixation of the preservative inside the wood. Descriptions of the samples produced and acronyms used throughout the work are described in

Table 1. The adopted nomenclatures and acronyms for the samples in this study.

Code	Description
W	Wood
W-PAA/Na	Wood with poly(acrylic acid sodium salt)
W-CuO	Wood with PAA/Na and CuO
W-ZnO	Wood with PAA/Na and ZnO
W-CuO/ZnO	Wood with PAA/Na and CuO/ZnO
BW	Before weathering
AW	After weathering

.

2.4. Weathering Aging

The accelerated aging test was employed as a method to simulate the degradation process induced by natural weathering, which includes exposure to solar radiation and precipitation. The tests were conducted using UUV-STD-SPRAY-4400 equipment (Bass Equipamentos, Barueri, Brazil). The device was equipped with 40 W UV fluorescent lamps (non-visible range), which are typically rated by UV irradiance rather than luminous flux. The procedure involved the application of an operational cycle consisting of 8 h of ultraviolet (UV) radiation exposure, followed by 3 h and 45 min of condensation and, finally, 15 min of water spray. This complete cycle was repeated 240 times, resulting in an accumulated exposure of 720 h during the test, based on previous studies [37,38].

2.5. Decay Attack

The analysis of biodegradation by fungi was carried out by adapting the BS EN 113 (2020) standard. The fungi used were Trametes versicolor LPF-108 (white rot) and Gloeophyllum trabeum LPF-203 (brown rot). Glass bottles were initially autoclaved (121 °C, 111 kPa, 20 min) containing 60 mL of malt agar culture medium (50 g of concentrated malt extract, 25 g of bacteriological agar, and 1000 mL of distilled water). After 5 days in a BOD chamber (65–75% humidity, 23 °C) with no growth of contaminating microorganisms, the flasks were inoculated with xylophagous fungi. Before being placed in the glasses, the wood was previously dried (105 °C), weighed, and sterilized (121 °C, 111 kPa) for 20 min and, after 24 h, for another 10 min. After the fungi occupied the entire area of the cultivation medium, the wood was inserted into the flasks, remaining in that medium for 16 weeks. At the end of the period, the wood was cleaned, dried, and weighed again.

2.6. The Characterization of the Woods

The colorimetric variations caused by the treatments and weathering were evaluated using a Konica Minolta CR 400 colorimeter, with a 10° viewing angle and CIELab system [32]. Three readings per sample were taken, evaluating brightness (L*), green–red chromatic coordinate (a*), blue–yellow chromatic coordinate (b*), and total color variation (ΔE). All parameters were analyzed before and after subjecting the samples to accelerated aging. The total color variation (ΔE) was calculated based on the differences observed in L*, a*, and b* before and after the treatments. This parameter represents how much the color of the sample changed overall, considering not only how light or dark it became, but also any shifts toward red or green (a*) and toward yellow or blue (b*).

The apparent density and static bending properties were determined following recommendations and adaptations of American Society for Testing and Materials standards—ASTM D2395 [39] and ASTM D143 [40], respectively—after and before weathering. For apparent density, the samples were dried in an oven (100 ± 3 °C) until reaching a constant weight. The static bending test was conducted to measure the modulus of elasticity and modulus of rupture of the materials, performed on a universal testing machine EMIC DL 300, with a 3 kN load cell and computerized data acquisition system.

Additionally, the mass gain after impregnation of the particles in the woods was evaluated using a Shimadzu ATX224 analytical balance (Shimadzu, Kyoto, Japan). Similarly, the mass loss after weathering and fungal attack tests was investigated.

2.7. Statistical Analysis

All tests were carried out with at least 6 reproduction samples per composition. The obtained data were analyzed for normality using the Shapiro–Wilk test, for homoscedasticity using the Hartley test, and for independence of residuals through graphical analysis. Since it was a parametric analysis, the results were subjected to analysis of variance using the F-test (p ≤ 0.05). In case of statistical significance in the analysis of variance, the means of each variable were compared through mean tests. The treatments were compared to each other using the Tukey test (p ≤ 0.05) and individually to the control using the Dunnett test (p ≤ 0.05), while weathering situations were compared using the t-test (p ≤ 0.05).

3. Results and Discussion

3.1. Colorimetry

Figure 1 shows the visual appearance of all wood samples immediately after treatment. The untreated wood (W) and PAA/Na-only treated sample (W-PAA/Na) retained their natural light color, while all oxide-treated samples exhibited visible darkening. The W-CuO/ZnO group exhibited the darkest tone, followed by W-CuO and W-ZnO, respectively. The variation in coloration reflects the nature and distribution of the impregnated oxides. A clear heterogeneity in the deposition is visible, especially in the W-CuO/ZnO and W-CuO groups, where darker and lighter patches are present within some samples. This non-uniformity may be attributed to the physical instability of the suspension and the agglomeration of the particles due to a lack of size control during synthesis.

From a chemical perspective, this uneven surface coverage may be influenced by the wood anatomy, lignin distribution, and differential absorption pathways along the grain. The wood surface is inherently heterogeneous, and the local concentration of lignin and other biomolecules may affect not only the deposition of metal oxides but also subsequent reactions, such as furfural formation or the secondary transformations of the organic compounds. According to Varga et al. [6] and Jirouš-Rajković and Miklečić [7], such variability is common in treatments involving particles that are not strongly reactive with wood polymers, as leaching and agglomeration become more pronounced.

Figure 2 quantifies the color variation (ΔE) for each group before (ΔEBW) and after aging (ΔEAW). Among all treatments, W-CuO/ZnO showed the highest ΔEBW, reaching 48.01, which is approximately 57% higher than W-CuO (30.6) and 87% higher than W-ZnO (25.7). The W-PAA/Na group displayed the lowest ΔEBW (6.2), confirming its negligible impact on coloration. The significant increase in ΔE for the dual-oxide treatment (CuO/ZnO) correlates with the more intense and heterogeneous darkening observed in Figure 1. These color shifts are partially retained after weathering (ΔEAW), indicating partial leaching. For instance, W-CuO/ZnO lost ~42% of its ΔE signal, suggesting some detachment of the surface-bound oxides. This behavior is consistent with the hypothesis that weak bonding between biogenically synthesized oxides and the wood surface leads to poor weather resistance, as discussed by Bejo et al. [8]. Even so, the remaining ΔEAW values (e.g., 27.8 for W-CuO/ZnO) still exceed the visual threshold for noticeable change, implying partial retention. Furfurylated woods typically present ΔE values of 35–39, while thermally modified woods reach 43–220 depending on the temperature [41]. Therefore, the results obtained for W-CuO/ZnO indicate that the color change effect is on par with established industrial treatments.

Figure 3 presents the L* values of each treatment before and after weathering. The W-CuO/ZnO group exhibited the lowest initial L* (33.17), contrasting sharply with the control (77.29) and W-PAA/Na (76.50). After aging, the L* of W-CuO/ZnO increased to 49.65, a 49.7% rise, evidencing a substantial removal of surface oxides. Interestingly, W-ZnO also exhibited an increase in L* (from 45.21 to 56.60, +25.2%), while W-CuO remained practically unchanged (48.81 to 47.44, −2.8%), suggesting a higher resistance to UV-triggered leaching or more consistent adhesion to the wood. These trends reinforce the interpretation that CuO oxides—although dark—are more resilient to leaching than ZnO or the mixed oxides (CuO/ZnO). This behavior is consistent with weak physicochemical interactions, such as hydrogen bonding or van der Waals forces, rather than covalent bonding to lignocellulosic components. This aligns with data from Humar et al. [42] and Humar and Žlindra [43], who observed higher leaching rates for copper-based compounds when not chemically fixed to wood.

Despite the loss in pigmentation, visual inspections (as shown in Figure 4) confirm that dark zones remain, suggesting that some oxides penetrate deeper into the wood or become partially immobilized within surface cavities. In general, wood tends to darken when exposed to UV radiation, as it is capable of degrading components such as lignin and non-cellulosic polysaccharides, in addition to leaching extractives [44]. The studied treated woods, however, were already dark before aging, and the lightening is caused by the removal of the particle layer covering the test specimens by the water action, as can be observed in the changes in Figure 4. It is also possible to conclude that the particles were not chemically bonded to the wood, making them susceptible to leaching. Following this idea, the retention of the compounds in the wood was not ideal, which were probably leached during the dew stages in the weathering chamber, according to the colorimetric variations recorded. Even preservatives with a recognized effectiveness, such as CCA and creosote, are widely used to protect timber, but their effectiveness can be compromised if not sufficiently retained. CCA’s efficacy depends on factors such as the leach resistance, penetration depth, and persistence within treated wood [45]. However, arsenic is the least stable element in wood tissue, while copper is most affected by leaching [41], which aligns with the findings of the present study.

Although W-CuO remained with the average of L* unchanged (Figure 3), alongside W-CuO/ZnO it showed the highest values of ΔEAW, meaning they were the treatments that had the greatest color variation due to aging. W-ZnO was the least affected, having the lowest ΔEAW. These observations corroborate what is observed in Figure 3. Despite visually observing the removal of a layer of particles from the wood surface, this is not complete, as the presence of the material remains apparent. To determine the exact loss of the treatment, it would be necessary to carry out leaching tests with a quantification of the metals. With the impossibility of conducting this test, the leaching was approximated by the loss of mass after 30 days in the accelerated aging chamber.

3.2. Physical Properties

Figure 5 presents the apparent density (in g·cm⁻³) of the treated woods. Among the treatments, only W-ZnO exhibited a statistically significant higher density (0.534 g·cm⁻³), representing a 22.2% rise compared to the control. Other treatments, including W-CuO and W-CuO/ZnO, did not differ significantly. This result suggests that ZnO was more effectively retained within or on the wood matrix compared to the other oxides, at least in terms of mass. However, it is important to consider that this increased density may reflect surface deposition rather than deep penetration, as discussed by Gallio et al. [41] for Al₂O₃-based treatments. The minimal density change observed in W-CuO and W-CuO/ZnO may be related to either (i) a less effective impregnation of the particles or (ii) the formation of lightweight agglomerates that do not significantly alter the bulk mass. The wood anatomy also plays a key role, as the vessel size and pit distribution influence particle migration and retention.

Figure 6 displays the weight gain (WG; %) after impregnation and the weight loss (WL; %) after accelerated aging, highlighting the leaching behavior of each treatment. The W-ZnO and W-CuO/ZnO groups had the highest initial mass gain (~5.2% and 5.3%, respectively), while W-CuO showed the lowest (~3.2%). After weathering, mass loss was most evident in W-PAA/Na (−5.9%) and W-ZnO (−3.4%). The W-CuO and W-CuO/ZnO groups retained more material, losing only 1.4% and 1.5%, respectively. These values suggest that CuO-based systems are more resistant to water-induced leaching than ZnO alone, possibly due to the higher affinity or partial fixation to lignocellulosic compounds. In particular, ZnO may interact more superficially with wood polymers due to its lower reactivity with the lignin and hemicellulose functional groups. By comparing the retained mass (initial gain minus loss), it was noted that W-CuO/ZnO maintained ~2.01%, W-CuO ~1.76%, and W-ZnO ~1.80% of their initial added mass—which is very similar among oxides. These findings are consistent with prior work by Bejo et al. [8], who showed that metal oxides tend to remain partially fixed even after an extended exposure to simulated rainfall.

The slight discrepancy between W-ZnO’s greater weight gain and equal retention may be explained by the presence of larger or loosely bound agglomerates, which contribute to the initial mass but are easily leached. Conversely, CuO might have formed finer or better-integrated deposits. These outcomes highlight the importance of optimizing not only the oxide chemistry but also the particle morphology and dispersion techniques, to improve both the initial uptake and long-term retention.

3.3. Mechanical Properties

Figure 7 presents the MOE values (in MPa) for all wood treatments before and after the exposure to UV and moisture cycles. Initially, the control (W) showed a typical modulus of 6320 MPa. Among the treated samples, W-PAA/Na and W-CuO had slightly higher MOEs (~7300 MPa), while W-ZnO was comparable to the control. Notably, W-CuO/ZnO exhibited a significantly lower MOE of 4485 MPa (−29% compared to the control), indicating a potential stiffening interference from the CuO/ZnO formulation. After accelerated aging, most treatments converged statistically, with all MOE values ranging from ~5100 to ~6700 MPa. Interestingly, the W-CuO/ZnO group exhibited a 25% increase post-aging, while W-CuO experienced a 31% reduction. This inversion suggests that part of the stiffness reduction initially observed in W-CuO/ZnO may be due to the superficial interference from the particle layer, which partially leached off during weathering.

This behavior supports the idea that leaching can “recover” mechanical performance by removing brittle or poorly bonded external layers. According to Acosta et al. [38], particle coatings on wood may either enhance or impair performance depending on their integration with the surface. The greater variability after aging (CV from 19.4% to 32.3%) reinforces the effect of aging in creating uneven degradation paths in each treatment. From a theoretical standpoint, CuO particles may promote stiffer bonding when integrated into the wood matrix, whereas when present only on the surface, they can behave as flaws, reducing the overall elasticity.

Figure 8 displays the MOR results (in MPa), showing no statistically significant difference between the treatments and control, either before or after aging. The control (W) exhibited 62.6 MPa initially and decreased slightly to 57.8 MPa post-aging. The W-CuO/ZnO group had the lowest initial MOR (44.3 MPa), which then increased to 56.4 MPa—a 27.3% gain. All other treatments, including W-CuO and W-ZnO, experienced slight decreases post-aging, which is within the margin of experimental variation. This increase in strength for W-CuO/ZnO reinforces the interpretation from the MOE results: the removal of poorly adhered external layers during weathering may expose more cohesive internal regions, effectively improving the mechanical integrity. Alternatively, the initial impairment could have come from the presence of loosely bound surface clusters disrupting the stress transfer.

Studies such as Holy et al. [46] observed similar effects when using nanoparticle coatings on Pinus sylvestris wood—where the mechanical properties were sensitive to the surface adhesion and aging behavior of the coatings. From a theoretical viewpoint, the MOR is typically less sensitive to surface effects than the MOE, as rupture depends more on internal flaws and fiber bridging. The observed recovery in W-CuO/ZnO after aging further suggests that the mechanical compromise was superficial and not intrinsic to the wood material.

3.4. Decay Resistance

Figure 9 shows the percentage of mass loss after 16 weeks of exposure to fungal degradation. The untreated control wood (W) showed a high susceptibility to both fungi, with a 21.3% loss for T. versicolor and 22.9% for G. trabeum. These values classify Pinus elliottii as moderately resistant to white rot and non-resistant to brown rot under the tested conditions. Among the treatments, W-CuO was the most effective against T. versicolor, reducing the mass loss to 1.2%—a 94.6% reduction compared to the control. The W-CuO/ZnO sample performed best against G. trabeum, limiting the degradation to just 1.1% (−95.2% relative to the control). W-ZnO and W-PAA/Na did not statistically differ from the untreated wood, with mass losses of 11.9–22.1%, indicating a limited antifungal protection.

These results suggest a selective antifungal efficacy depending on the fungal type and oxide used: CuO is particularly active against white rot, while the CuO/ZnO system shows synergy in combating brown rot. This supports findings by Phiwdang et al. [47], who reported that CuO/ZnO oxides had enhanced antifungal and antioxidant activity compared to individual oxides. The performance of the combined oxide treatment may be attributed to the combined mechanisms of action. CuO generates reactive oxygen species (ROS) that damage fungal cell walls and DNA, while ZnO disrupts the membrane integrity and ion homeostasis [48]. Their co-presence could lead to complementary stress responses in fungi, increasing toxicity.

Interestingly, despite ZnO’s known antifungal properties, the W-ZnO treatment proved ineffective in wood forms under the tested conditions. This limited performance may be attributed to the weak adhesion to the wood substrate and/or superficial deposition, which likely facilitated leaching prior to fungal colonization. Such behavior aligns with the findings of Holy et al. [46], who reported the variable antifungal efficacy of ZnO depending on its fixation and the wood species. In contrast, in Petri dish assays, the CuO/ZnO combination demonstrated a superior inhibition of Trichoderma sp. compared to individual oxides, indicating a possible synergistic mechanism. This result supports the rationale for investigating the dual-oxide formulation and not only the individual oxides. Moreover, it underscores the importance of evaluating the oxide behavior in different application contexts—whether in vitro or within lignocellulosic matrices. These findings are further supported by Bak and Németh [47], who demonstrated the antifungal activity of metal oxides under different formulations and wood treatments. The observed discrepancies with prior studies are thus likely related to synthesis methods, wood species, and oxide retention characteristics.

The biological results also align with weight retention data (Figure 5), where W-CuO/ZnO and W-CuO had the lowest mass loss due to weathering, suggesting a better retention of active compounds and, hence, greater protection over time. From a practical perspective, the results confirm that biogenically synthesized CuO and CuO/ZnO can act as eco-friendly alternatives to conventional wood preservatives, offering a protection comparable to chromated compounds but without toxic residues.

Shiny et al. [49] observed the effectiveness of CuO NPs against white- and brown-rot fungi, depending on the concentration used. The inhibition of fungal growth was more effective against Trametes hirsuta (white rot) than against Oligoporus placenta (brown rot), with values of 100% and 34.44%, respectively. Other authors have also demonstrated the efficiency of various oxides against mass loss due to attacks by xylophagous fungi, such as ZnO, CuO, TiO2, Al2O3, and MgO [46,50,51].

It is established that inhibiting the fungal growth in culture environments with a direct exposure to the antifungal agent is different from preventing mass loss in treated woods due to fungal attacks. Shiny et al. [49], who had achieved a 100% inhibition of Trametes hirsuta fungal growth in a culture medium using CuO nanoparticles, exposed woods impregnated with the same particles to the same fungus, resulting in a mass loss of 15.01%. The same behavior was observed for the fungus O. placenta: CuO nanoparticles inhibited 34.44% of the growth, while the mass loss of the treated wood was 23.12%.

The CuO/ZnO formulation was effective in inhibiting the growth of Trichoderma sp. fungus in Petri dishes, being more efficient than CuO and ZnO alone. The antioxidant activity of the binary oxide system was also superior to the individual oxides [47,52]. As observed, the synergy of the CuO and ZnO oxides can be exploited in various applications. The individual oxides seem to be more efficient against white-rot fungi, while brown-rot fungi have shown to be more resistant and cause greater damage to the wood [48,50,53].

The fungal decay test was conducted in bottles, with each bottle containing a sample from the control group and a treated sample, thus confirming that the fungus was metabolically active and capable of efficiently consuming the wood compounds. The mass loss results for both fungi are shown in Figure 9. The W-PAA/Na and W-ZnO treatments did not show significant differences from the control for either fungus, indicating that they were not effective in preventing mass loss.

Furthermore, in line with results from other authors, the woods exposed to the brown-rot fungus had a higher mass loss in treatments W, W-PAA/Na, W-CuO, and W-ZnO, demonstrating that Gloeophyllum trabeum was more aggressive than Trametes versicolor. The W-CuO was significantly different from the control against both fungi, but it was more effective against white rot, with a mass loss of 1.16%, whereas for brown rot, it was nearly 8%.

The dual-oxide treatment had the opposite effect to W-CuO, being more efficient against brown rot, where the mass loss was only 1.13%, while in white rot it was 5.24%. W-CuO/ZnO was the material that showed the best overall efficiency, capable of providing protection to the wood against both tested fungi. The results obtained by the CuO/ZnO-based impregnation against Gloeophyllum trabeum may indicate that the effect of the oxides is enhanced when used together, that is, there is a synergistic effect that gives the wood a greater antifungal capacity.

The highest mass loss of the untreated wood by T. versicolor was 32.91%, and against Gloeophyllum trabeum it was 51.02%. These results alone would classify pine wood as moderately resistant and non-resistant, respectively. At the end of the 16-week period during which the samples remained in contact with the fungus, most of them were completely covered by their growth. In brown rot, no treatment was able to completely prevent the growth of the fungus around the wood; however, for white rot, the mixed oxides inhibited the fungal growth, which ended up only taking place at the lower end of the sample.

Figure 10 shows the visual appearance of the samples after the exposure to the decay tests with fungi. Notably, the control wood (W) and the W-PAA/Na treatment exhibited a severe surface deterioration, darkening, and a loss of structural integrity—typical signs of fungal colonization and degradation, particularly in untreated or insufficiently protected wood. Meanwhile, the oxide-treated samples revealed varying degrees of surface preservation, confirming the differential effectiveness of the treatments.

The W-CuO specimen (Figure 10) retained a relatively uniform and darkened surface, with minimal visible fungal growth, indicating successful protection against decay. This observation is consistent with its low mass loss and strong resistance to T. versicolor, reported in Figure 9. Conversely, W-ZnO exhibited visible fungal colonization, discoloration, and material breakdown, suggesting that ZnO was less effective under these conditions. The dual-oxide treated sample (W-CuO/ZnO) showed a well-preserved surface, with limited signs of degradation, particularly against G. trabeum, supporting the observed biological resistance data.

These visual differences reinforce the idea that while individual oxides may confer partial protection, their combined use appears more effective against specific decay fungi. The physical appearance of the samples after testing aligns with the quantitative mass loss data and highlights the biological durability of CuO and CuO/ZnO formulations. In contrast, the W and W-PAA/Na treatments failed to prevent fungal growth, as evidenced by the clear visual degradation.

This figure also supports the interpretation that biological resistance is closely tied to the interaction of oxide particles with the wood matrix. In CuO and CuO/ZnO treatments, lignin-rich regions and vessel walls may have promoted the partial entrapment or surface adsorption of the active particles, reducing the fungal enzymatic access. These findings are in line with studies such as those by Mantanis et al. [53] and Bak and Németh [50], who observed a significant resistance using metal oxides, although variations are expected depending on the synthesis route and oxide characteristics.

Despite ZnO’s known antifungal properties, W-ZnO was not effective in wood form in this study, likely due to poor adhesion and leaching. This aligns with observations by Holy et al. [46] and Terzi et al. [54], who reported a variable effectiveness depending on the oxide form and fungal strain. While CuO showed a strong protection against white rot, the CuO/ZnO combination was more effective against brown rot—confirming the potential for synergistic interactions when both oxides are combined.

Importantly, no previous studies were found that examined the co-application of copper and zinc oxides for wood protection. Thus, the promising results obtained here with the CuO/ZnO formulation highlight its potential as a novel, biogenic-based wood preservative, deserving of further evaluation in diverse fungal and environmental contexts. Despite these limitations, the results highlight potential interaction mechanisms between CuO/ZnO and the wood matrix, where lignin-rich zones and vessel walls may promote the partial entrapment or adsorption of the particles, contributing to the observed biological resistance.

4. Conclusions

This study demonstrated the potential of copper and zinc oxides synthesized via green chemistry from Eucalyptus dunnii leaf extracts as environmentally friendly wood preservatives. The treated Pinus elliottii samples exhibited significant modifications in visual their appearance, physical properties, mechanical behavior, and durability. Among the tested groups, W-CuO achieved the highest protection against Trametes versicolor (white rot), reducing the mass loss to 1.16%, which represents a 94.6% reduction compared to the untreated control (21.3%). W-CuO/ZnO was the most effective against Gloeophyllum trabeum (brown rot), with only a 1.13% mass loss, a 95.2% reduction from the control (22.9%). In contrast, W-ZnO resulted in mass losses of 11.86% and 22.07%, respectively, showing a limited protection likely due to superficial deposition and higher leachability. Overall, the CuO/ZnO formulation provided the best broad-spectrum decay resistance among the tested groups. In terms of visual appearance, W-CuO/ZnO exhibited the most intense total color change (ΔE = 48.01), indicating a significant surface darkening and heterogeneity after treatment. After artificial aging, this value decreased to ΔE = 27.8, corresponding to a 42% reduction, which is consistent with the partial leaching of oxides. The W-CuO treatment retained a more uniform dark appearance, with a smaller change in L* (from 48.81 to 47.44, a 2.8% decrease), confirming better photostability and particle retention. Meanwhile, W-ZnO showed a more significant L* increase (from 45.21 to 56.60, a 25.2% rise), suggesting greater oxide loss. The mass variation analysis supported these findings. The W-CuO and W-CuO/ZnO groups had the lowest mass losses after weathering (1.4% and 1.5%, respectively), compared to 3.4% for W-ZnO and 5.9% for W-PAA/Na. In terms of retention (initial gain minus post-aging loss), W-CuO/ZnO retained 2.01%, W-CuO retained 1.76%, and W-ZnO retained 1.80% of the impregnated mass. In the mechanical performance, the untreated wood exhibited a modulus of elasticity (MOE) of 6320 MPa before aging. W-CuO/ZnO initially showed the lowest MOE (4485 MPa) but increased to 5587 MPa post-aging—a 25% gain. W-CuO, conversely, decreased from 7362 MPa to 5064 MPa, a 31% reduction. In terms of the modulus of rupture (MOR), W-CuO/ZnO increased from 44.25 MPa to 56.43 MPa, while the control decreased slightly from 62.62 MPa to 57.76 MPa. These findings suggest that surface-bound oxides can initially impair the stiffness and strength but may detach under environmental exposure, revealing more cohesive internal structures and recovering performance. The results indicate that green-synthesized CuO/ZnO formulations exhibit dual functionality: they provide a significant resistance to fungal decay while demonstrating a moderate resistance to weathering-induced degradation. Although ZnO alone was ineffective in protecting the wood, its combination with CuO improved the antifungal performance—especially against brown rot. However, both ZnO and the CuO/ZnO formulation showed limitations in long-term durability due to partial leaching.

Therefore, future work is explicitly suggested to focus on improving the oxide retention in the wood structure, particularly through chemical bonding strategies or the incorporation of stabilizing binders. Additionally, to better understand the morphological distribution and anchoring of particles on the wood surface, future studies should include scanning electron microscopy (SEM) images, which are essential to clarify the spatial deposition and potential penetration of the oxides. Furthermore, comparisons with oxides synthesized via conventional wet chemistry methods should be pursued to evaluate the influence of the synthesis route on performance and stability. These enhancements could enable more durable applications of biogenic metal oxides in sustainable wood preservation.