Ammoniacal Zinc Borate for Wood Protection against Fungi and Insects

: The organic nature of wood makes it susceptible to abiotic and biotic degradation. Impregnation with chemical products is the most common method of protection. Only inorganic borates combine the unique set of properties that make them well suited for wood protection: they are insecticidal, fungicidal and ﬂame retardants. In this work, zinc borate is suspended in an ammonia solution and applied in depth to wood. Its resistance to leaching is evaluated. The product is tested against rot fungi ( Gloeophyllum trabeum and Trametes versicolor ) and a xylophagous insect ( Anobium punctatum ). The results verify its effectiveness as a wood protector, while leaching less than other borate-based products.


Introduction
Wood is a renewable, easy-to-use and inexpensive material, widely used for its physicochemical properties in construction, furniture and various other applications. However, its organic nature makes it susceptible to abiotic and biotic degradation [1]. UV radiation from the sun is the most common abiotic agent in wood weathering. It causes progressive surface damage changing, the appearance of the wood [2]. However, biotic agents such as fungi and insects bring about the most severe damage and the greatest economic losses.
Decay fungi are able to reduce the wood mass by significant percentages. Their classification depends on which structural polymers they are able to degrade, which shows on the appearance of the wood. All decay fungi are able to depolymerize and metabolize cellulose and hemicellulose, but differ in their capacity to degrade lignin. White, fibrous wood is characteristic of white rot fungi; they can completely mineralize lignin, making the wood lose all of its mechanical properties and up to 97% of its mass. Soft rot fungi, most present in wood in contact with the soil, also degrade lignin, but not as much as white rot fungi [3]. On the other hand, wood attacked by brown rot fungi is characterized by a distinctive brown color. Instead of degrading lignin, brown rot fungi only chemically modify it, after which it quickly repolymerizes [2,3]. However, the wood still loses its mechanical properties.
Among insects, the most damaging are termites and the powder-post beetle. Termites are ubiquitous insects that degrade the wood from the inside; the humidity content of In this work, ammoniacal ZnB is applied to three wood species-two hardwoods and one conifer-and tested against white and brown rot fungi, and against Anobium punctatum larvae. E. grandis and P. taeda are the most common commercial species in Uruguay while Populus sp. was chosen for its availability, its treatability and its low natural durability against fungi and insects. The first study in our laboratory verifies the general effectiveness against fungi of ammoniacal zinc borate applied by the vacuum-vacuum (V-V) method by evaluating the susceptibility of the treated wood to fungal decay [39]. Herein, we analyze whether application by vacuum-pressure-vacuum (V-P-V) improves the fungicidal properties of ammoniacal ZnB. In the case of A. punctatum larvae, the insecticidal effectiveness of the product was tested by comparing the two application methods (V-V vs. V-P-V).

Strains and Culture Conditions
Two strains of fungi were used: Trametes versicolor (BW001) (white rot) and Gloeophyllum trabeum (BB001) (brown rot), both from the fungal collection of the Forestry Laboratory of Sede Tacuarembó, Universidad de la República del Uruguay. The strains were maintained on a malt extract (12.5 g L −1 ) and agar (20 g L −1 ) medium (MA) previously sterilized by autoclaving at 121 • C for 15 min. Both the malt extract and agar were from Oxoid Ltd. (Basingstoke, UK).

Wood Decay Test: Conditioning and Impregnation
Eucalyptus grandis Hill ex Maiden, Populus spp. and Pinus taeda L. sapwood specimens (110 per species) of dimensions 50 ± 2.0 [L] × 25 ± 2.0 [T] × 15 ± 2.0 [R] mm 3 , free of defects and with no visible evidence of fungal infection or insect damage, were used. The specimens were oven-dried at 103 ± 2 • C until they reached a constant mass, and then weighed (0.001 g) to register their initial dry mass. Anhydrous density was determined; specimens with a density that did not differ from the mean density value by more than 15% were selected (50 per species). Selected specimens were treated with the ammoniacal ZnB suspension by the Bethell method (30 min initial vacuum at −0.6 bar, 60 min pressure at 10 bar and 30 min final vacuum at −0.6 bar) in a laboratory autoclave of 100 cm in length and 20 cm in diameter. The retention was determined for all specimens by the Equation (1): where: R is the retention of impregnating treatment (kg/m 3 ); w1 is the initial anhydrous weight of each sample (kg); w2 is the weight of each treated sample after treatment (kg); C is the ZnB concentration (%) and v is the volume of each sample (m 3 ). The treated specimens were stored at 30 ± 2 • C for seven days to allow fixation. 10 specimens of each species were not treated further and were used as controls.

Leaching Test
Half of the treated specimens (20 per species) were subjected to leaching tests according to EN 84 [40], an accelerated aging test which simulates long-term use conditions. The specimens were placed in a desiccator at 40 mbar vacuum for 20 min; then, five times their volume of deionized water was allowed into the desiccator. The vacuum was released, the specimens remained under water for 2 h and then the water was changed. For the next 14 days the water would be changed daily. Finally, all specimens (leached and non-leached) were maintained at 22 ± 2 • C and 65 ± 2% relative humidity for four weeks and then tested for resistance to fungal decay according to the EN113-1 standard [41].
Basidiomycetes were first inoculated in an MA medium in 600 ml flasks. After mycelia had completely covered the bottom of the flask (c. 3 weeks), two wood specimens, one untreated control and one treated with ammoniacal zinc borate, both previously sterilized with steam (20 min of steam, cool at room temperature for 24 h, 10 min of steam), were placed in the flasks. The flasks were incubated in the dark for 16 weeks at 23 ± 2 • C and 60%-70% RH. Following incubation, the specimens were weighed, oven-dried at 103 ± 2 • C and weighed again to register final dry mass.
The fungicidal effectiveness was determined through the average % weight loss (initial dry mass − final dry mass/initial dry mass) of treated specimens after 16 weeks of exposure to fungi.

Quantification of Leached Zinc
The amount of zinc leached from the wood in water was quantified with an atomic absorption equipment with flame oven (900T PinAAcle) (PerkinElmer, Beaconsfield, UK) [42].

Insect Larvae
A. punctatum larvae were collected from infested timbers in the Estación Experimental Bernado Rosengurt of the Universidad de la República del Uruguay. They were kept in storage blocks in a culture chamber for two months until needed. The average weight of the larvae was approximately 3.5 mg; larvae in good conditions and not infested with mites were selected and inoculated in the test specimens according to the BS EN 48: 2005 standard [43]. The treated specimens were stored at 30 ± 2 • C for 7 days to allow fixation. Then, they were conditioned at 20 ± 2 • C and 65 ± 2% RH for 2 weeks. Before testing, six cylindrical holes approximately 5 mm deep were drilled in the two transverse cross-sections of each specimen, creating a pattern of two lines of three holes 10 mm from the large faces of the specimen; the distance between the holes within a row was 15 mm, and the distance between the rows was 10 mm. The remaining untreated specimens (6 per wood species) were used as controls.

Resistance Test to Anobium punctatum
The selected A. punctatum larvae were placed in the 12 holes of each specimen. The holes were sealed with a glass plate and fixed with adhesive tape. The test specimens were kept in the culture chamber at 20 ± 2 • C and 80 ± 2% RH, and placed horizontally on their cross-section faces. After the first 2 weeks, the glass plate was removed; dead larvae or larvae that had not started to bore into the wood were replaced, and the cross-section was sealed with paraffin wax. All specimens were kept in the culture chamber for 14 weeks. The results are considered valid when at least 70% of the larvae retrieved from the control specimens were alive and showed a satisfactory level of activity. Results are expressed as the number of larvae retrieved from test specimens, separating dead or dying larvae from living larvae, along with mortality rates.

SEM Analysis
The distribution of impregnated ammoniacal ZnB was observed using a JEOL JCM-6000: Neoscope II Benchtop scanning electron microscope (JEOL, Tokio, Japan), used in the high vacuum mode with an accelerating voltage of 10 kV and SE detector.

Statistical Analysis
The mass losses by fungal activity were statistically tested by the One-Way Anova test. The significance (p-value < 0.05) found in the Anova test between treatments was compared afterward with the Tukey test.
For the larvae mortality rates, a generalized linear mixed model was adjusted, since the response variable was binomially distributed, with a logit link function, considering dead larvae as successes. Four sources of variation were considered: genus (P. taeda and Populus sp.), treatment (Control, VPV and VV), genus by treatment interaction and wood sample, this last one being the only random effect in the model. For the distance of larvae from the nearest face-length of gallery (LG) in longitudinal direction-a linear mixed model was adjusted, accounting for the same sources of variations, as well as fixed and random effects, of the previous model. The significance of fixed effects was assessed using the likelihood ratio test (LRT) and the p-values of random effects were obtained by parametric bootstrap methods, using 10,000 replicates [44]. Significant differences identified while comparing sources of variation were evaluated for larvae mortality through odds ratios (OR) and for LG throughout mean difference tests.

ZnB in the Wood
The chemical retention values of ZnB in the specimens is one of the parameters that indicates the efficiency of the impregnation process, and is presented in Table 1. The retention values do not differ between species, and are generally close to [50] or higher than [37] reported in the literature for analogous treatments on other pinus. Differences in anatomy and density between the used hardwood and the pinus result in lower retention values on the former [35] and with lower ammonia content [39]. However, the ammonia seems to improve retentions, explaining the results. Table 1 also shows significant differences in retention levels between the specimens of different sizes. When wood is impregnated by vacuum-pressure-vacuum, the ammonia in the impregnating suspension causes the E. grandis wood to swell to twice its volume and deform when dried. The high pH (8.5) could have also contributed to this. Neither Populus sp. nor P. taeda were affected by the ammonia. The damage the ammonia causes to the more sensitive E. grandis wood would have been more noticeable in the bigger specimens needed for insect tests. It is for this reason the species was not used at this stage. Figure 1 shows SEM microphotographs of the treated specimens. It can be observed that ZnB, easily carried by the ammonia, distributes uniformly and homogeneously inside the wood. ZnB occupies not only empty lumens, but also the insides of the cell wall; no areas of preferential salt accumulation can be observed. Preservatives distribute in such a way if some of its particles are smaller than 25,000 nm. This uniform distribution of ammoniacal ZnB inside the wood has been previously determined by EDS analysis [51]. According to Freeman and McIntyre [23], bigger particles would obstruct the tracheids and prevent further preservative from going into the wood.  Figure 1 shows SEM microphotographs of the treated specimens. It can be observed that ZnB, easily carried by the ammonia, distributes uniformly and homogeneously inside the wood. ZnB occupies not only empty lumens, but also the insides of the cell wall; no areas of preferential salt accumulation can be observed. Preservatives distribute in such a way if some of its particles are smaller than 25,000 nm. This uniform distribution of ammoniacal ZnB inside the wood has been previously determined by EDS analysis [51]. According to Freeman and McIntyre [23], bigger particles would obstruct the tracheids and prevent further preservative from going into the wood.  Since boron compounds and the structural polymers of the wood only form weak links [22,52], high environmental humidity causes boron to leach. Studying its impact on the effectiveness of the preservative is critical; decay fungi start to deteriorate the wood at a humidity content of 30%, which goes up as the decay process goes on [2], in turn favoring further leaching. This is not the case for powder-post beetles such as Anobium punctatum, which attacks wood with humidity content as low as 8%-20% [4]. Figure 2 shows the % of ZnB lost in leaching water during the first 9 days of the leaching test. On the first day, P. taeda loses 50% less ZnB than the other species. There is no literature about the specific interaction between zinc borate and wood components. However, it is known that other boron compounds react rather quickly with the polysaccharides and slowly with the lignin [22]. The reactions are still slow with low kinetic constants at 20 • C. Therefore, the comparatively low leaching of the preservative from P taeda wood on day 1 could be attributed to its higher lignin content, which is higher than in hardwoods. Although ZnB retention values are analogous between species, the more complex anatomy of the hardwoods prevents the preservative from reaching the ultrastructure of the wood and thus leaches more easily on day 1. the effectiveness of the preservative is critical; decay fungi start to deteriorate the wood at a humidity content of 30%, which goes up as the decay process goes on [2], in turn favoring further leaching. This is not the case for powder-post beetles such as Anobium punctatum, which attacks wood with humidity content as low as 8%-20% [4]. Figure 2 shows the % of ZnB lost in leaching water during the first 9 days of the leaching test. On the first day, P. taeda loses 50% less ZnB than the other species. There is no literature about the specific interaction between zinc borate and wood components. However, it is known that other boron compounds react rather quickly with the polysaccharides and slowly with the lignin [22]. The reactions are still slow with low kinetic constants at 20 °C. Therefore, the comparatively low leaching of the preservative from P taeda wood on day 1 could be attributed to its higher lignin content, which is higher than in hardwoods. Although ZnB retention values are analogous between species, the more complex anatomy of the hardwoods prevents the preservative from reaching the ultrastructure of the wood and thus leaches more easily on day 1.

Wood Decay Test
The decay capacity of the fungi is validated according to EN 113-1 [41] by the average 20% mass loss of the untreated control specimens of all wood species, as shown in Figure  3.

Wood Decay Test
The decay capacity of the fungi is validated according to EN 113-1 [41] by the average 20% mass loss of the untreated control specimens of all wood species, as shown in Figure 3. a humidity content of 30%, which goes up as the decay process goes on [2], in turn favoring further leaching. This is not the case for powder-post beetles such as Anobium punctatum, which attacks wood with humidity content as low as 8%-20% [4]. Figure 2 shows the % of ZnB lost in leaching water during the first 9 days of the leaching test. On the first day, P. taeda loses 50% less ZnB than the other species. There is no literature about the specific interaction between zinc borate and wood components. However, it is known that other boron compounds react rather quickly with the polysaccharides and slowly with the lignin [22]. The reactions are still slow with low kinetic constants at 20 °C. Therefore, the comparatively low leaching of the preservative from P taeda wood on day 1 could be attributed to its higher lignin content, which is higher than in hardwoods. Although ZnB retention values are analogous between species, the more complex anatomy of the hardwoods prevents the preservative from reaching the ultrastructure of the wood and thus leaches more easily on day 1.

Wood Decay Test
The decay capacity of the fungi is validated according to EN 113-1 [41] by the average 20% mass loss of the untreated control specimens of all wood species, as shown in Figure  3.  Mass losses of treated, non-leached specimens are significantly lower than the untreated regardless of the species, for a significance level of 5%. Hence, ammoniacal ZnB is effective against the tested fungi in dry environments (i.e., not leached). Effectivity in nonleached specimens is similar between the three wood species, with Pinus taeda performing slightly better.
When leached, the effectivity is affected differently depending on the wood species: effectivity in Pinus taeda is the least affected, while Eucalyptus grandis is the most affected. The vacuum-pressure-vacuum impregnation of Populus sp., despite reaching greater retention, does not result in a better performance against fungi when compared with vacuum-vacuum impregnation [39]. Ammoniacal ZnB applied by vacuum-vacuum to P. taeda and E grandis wood is effective against xylophagous fungi.

Anobium punctatum Resistance Test
At the end of the test, the percentage of recovered larvae, percentage of live larvae and mortality rate (proportion of dead or dying larvae after the incubation period) were analyzed. As 100% of the inoculated larvae were recovered from the control specimens at the end of the test, and over 75% were alive, the results are considered valid. The mortality rate is lower in P. taeda specimens because of the natural preference of A. punctatum larvae for coniferous wood [53,54].

Larvae Mortality Analysis
The significance test of the components of the model for larvae mortality, which considered genus, treatments and genus by treatment interaction as fixed effects and wood sample as a random effect, shows that all were statistically significant at the 5% level (Table 2); therefore, genus by treatment interaction needs to be analyzed in more detail and interpretation of the principal effects is not adequate. The interaction analysis implies grouping the results by each one of the effects that are involved in the interaction. Firstly, grouping by genus shows that in P. taeda, no differences are found between the treatments. However, in Populus sp., all treatments are statistically different among each other, VV being the most effective one with a percentage of 92.1% of death larvae, compared with the 64.3% generated by VPV treatment (Figures 4 and 5).     The odd ratios (OR) that sustain previous interpretations for both interaction analysis are presented in Table 3, with section A being grouped by genus analysis and section B the one that implied grouping by treatments. Table 3. Odd ratios for comparison among levels for larvae mortality, grouping by genus (A) and by treatments (B).  The odd ratios (OR) that sustain previous interpretations for both interaction analysis are presented in Table 3, with section A being grouped by genus analysis and section B the one that implied grouping by treatments. Table 3. Odd ratios for comparison among levels for larvae mortality, grouping by genus (A) and by treatments (B).

Length of the Gallery in Longitudinal Direction Analysis
Similarly, for larvae mortality analysis, the Anova of the model proposed was performed and once again, all factors, including the interaction between genus and treatments, are statistically significant at the 5% level (Table 4). Hence, the interaction analysis was carried out.
When grouping by genus, the results show that for P. taeda, only VPV is statistically different to the control, with an estimated mean difference of 1.47 mm (Figure 6). On the other hand, for Populus sp., VPV and VV are similar in terms of effectiveness and both are different to the control, with an estimated distance reduction of 2.06 mm and 2.75 mm, respectively. Similarly, for larvae mortality analysis, the Anova of the model proposed was performed and once again, all factors, including the interaction between genus and treatments, are statistically significant at the 5% level (Table 4). Hence, the interaction analysis was carried out. When grouping by genus, the results show that for P. taeda, only VPV is statistically different to the control, with an estimated mean difference of 1.47 mm (Figure 6). On the other hand, for Populus sp., VPV and VV are similar in terms of effectiveness and both are different to the control, with an estimated distance reduction of 2.06 mm and 2.75 mm, respectively. Finally, the analysis of the interaction by grouping according to treatments shows the exact same results as for larvae mortality, there are only differences between both genera considered in VV treatment (Figure 7). Finally, the analysis of the interaction by grouping according to treatments shows the exact same results as for larvae mortality, there are only differences between both genera considered in VV treatment (Figure 7). Similarly, as for larvae mortality, the figures shown in Table 5 support the results, but in this case, a mean difference test was performed, keeping the structure that in section A the interaction was analyzed when grouping by genus, and section B when grouping by treatments.  Similarly, as for larvae mortality, the figures shown in Table 5 support the results, but in this case, a mean difference test was performed, keeping the structure that in section A the interaction was analyzed when grouping by genus, and section B when grouping by treatments. The analysis above indicates that ammoniacal zinc borate applied by vacuum-vacuum is the most effective treatment against the larvae in Populus sp. specimens, presenting the highest mortality rate at 92.1%. The application by vacuum-pressure-vacuum leads to better retention levels, but mortality is only 64.3%. This indicates that an increase of ZnB inside the wood does not influence the normal development of A. punctatum larvae. Moreover, larval tunnels in these specimens had an average length of 1.39 mm (VPV) and 0.71 mm (VV), with a reduction of 2.06 mm and 2.75 mm compared to control, respectively.
In P. taeda, larvae mortality did not differ between application methods. Larval tunnels of treated specimens are shorter than those of untreated specimens: for vacuum-pressurevacuum, tunnels are reduced by an average 1.47 mm, while for vacuum-vacuum, tunnels are reduced by an average 1.19 mm.
It must be noted that the different behavior depending on the wood species seems to respond to the nutritional patterns of the larvae rather than to the amount of ZnB inside the wood.
The results show that the larvicidal effects of ZnB are not instantaneous. Nonetheless, the mortality rates and length of the tunnels show significant improvements over untreated wood (control samples) This test verifies the lethal effect of ammoniacal zinc borate on a population of larvae of A. punctatum already inside the test specimens, which hinders their advance inside the wood. Future tests should evaluate its effectiveness (and of other borates) on oviposition and hatching. Other than the ovicidal properties of boric acid [55], no background is found in this regard, or on the effects of ZnB or other borates on the adult insect.

Conclusions
Ammoniacal zinc borate is an effective fungicide for Pinus taeda, Eucalyptus grandis and Populus sp. wood.
It is the most effective when applied by a vacuum-pressure-vacuum.
ZnB proves to be effective as an indoor insecticide against Anobium punctatum larvae in both Pinus taeda and Populus sp. when applied by a vacuum-pressure-vacuum in an autoclave. However, Populus sp. has a better performance when impregnated by vacuumvacuum. This result is influenced by the nutritional pattern of the larvae, which prefer Pinus wood.
The ammoniacal zinc borate has a larvicidal effect on Anobium punctatum. E. grandis wood is affected by the high pH of the suspension, and it is the species that leaches the easiest.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.