Copper-lignin with laccase-mediated polymerization fixation for wood preservation: determination of durability in marine environment using novel technologies

Copper-lignin with laccase-mediated polymerization fixation for wood preservation: determination of durability in marine environment using novel technologies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Copper-lignin with laccase-mediated polymerization fixation for wood preservation: determination of durability in marine environment using novel technologies Cristian Bolaño Losada, Mari Carmen Fernández-Costas, Diego Moldes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8347225/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract In the marine environment, the durability of wood is heavily compromised by harsh conditions and the presence of marine xylophages. Biocide treatments are often employed to prolong the lifetime of wood, however, its leaching into the environment motivates the development of alternative treatments due to environmental concerns. In this study, Scots pine samples were treated with a solution based on copper and polymerized Kraft lignin via laccase enzyme was tested. In this treatment, Kraft lignin was intended to work as adsorbent of copper while laccase-assisted polymerization was intended for fixation to the wood. To discern the contribution of each component, a set of samples were prepared with different combinations of the three components (presence/absence) and compared to untreated controls. The wood samples were immersed for one year in Vigo estuary (Spain). Biofouling coverage was monitored along the immersion time and the final mass accumulated was measured. Copper retention and distribution were studied by ICP-OES and XRF, respectively. Marine xylophage attack was evaluated quantitatively using X-ray computerized tomography analysis. The treatments containing copper were less affected by biofouling and marine xylophages. Treatments containing lignin limited the impregnation depth and consequently the copper content was lower than the samples with just copper. However, XRF analysis showed that in the parts where Kraft lignin was highly present, copper was better retained than parts that not. Unfortunately, due to the previously indicated impregnation limitation, the role of laccase in lignin polymerization could not be assessed properly. Additionally, in this study a set of novel technologies for the study of wood for marine use was used. These techniques included the use of X-ray CT and the Itrax Core Scanner, a device that allows the acquisition of high-quality image, energy dispersive X-ray fluorescence, and X-ray radiography. Marine xylophages biofouling wood preservatives Itrax Core Scanner. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Wood is a resource that has been used by diverse civilizations for land and marine uses due to its mechanical resistance, low cost, easy production, renewability and sustainability. However, wood is not exempt from deterioration due to abiotic and biotic factors. Therefore, wood treatments for preservation have been developed throughout human history according to the end use. Particularly, the marine environment can be considered one of the most hostile due to stressful factors such as saline water, corrosion, biofouling, marine xylophages, etc (Khademibami & Bobadilha, 2022). Marine biofouling results from the adhesion of diverse organisms such as barnacles, algae, and microbial biofilms to the material surface. Biofouling represents several problems for underwater structures, such increase in weight, increase in frictional forces in ships, corrosion, etc. From the first moment that a material is immersed, a series of events begins, starting from the adsorption of organic molecules, followed by the development of microbial films, and the subsequent adhesion of bigger organisms (algae, invertebrates, etc.). This process is like an ecological succession that eventually achieves a stable and complex biological community (M. Callow & Callow, 2002). Treatments to avoid biofouling are especially important in paint formulations for ship hulls due to the impact on operative costs (maintenance, fuel consumption, etc.) (Schultz et al., 2011). On the other hand, wood is also susceptible to marine xylophages, which in contrast to biofouling, actively compromises its structural integrity by boring into and feeding on it. Typical marine xylophages are boring bivalves (teredos) and crustaceans (limnoriids). There are three reported species of limnoriids in European sea waters (Borges, Merckelbach, & Cragg, 2014). They display a boring activity following a pattern of parallel channels near to the wood surface (Barroso Lopes et al., 2014; Cragg et al., 2007) digesting wood components with putative cellulases and modifying lignin by redox reactions promoted by their hemocyanin (Besser et al., 2018; King et al., 2010; Malyon, 2011). In addition, limnoriids tolerate creosote and copper-based wood treatments (Borges, Merckelbach, & Cragg, 2014; Tupper et al., 2000). On the other side, there are seventeen species of teredo reported in European marine waters (Borges, Merckelbach, Sampaio, et al., 2014). Teredos, or shipworms, use their shelves to deeply bore into wood. They utilize wood to sustain their gut microbiota; however, it has been suggested that their primary nutritional intake comes from debris filtration (Paalvast & van der Velde, 2013). This implies that they use wood more as shelter than a primary food source. Shipworms were one of the most feared species in naval shipping and caused panic and important changes in the construction of Dutch dykes in the past (Nelson, 2016). Several international organisms have defined standards, recommendations, categories and treatments for wood according to the end use, including the marine environment. To give an instance, the American Wood Protection Association defined categories for wood treatments according to a criterion, being the UC5 the one for marine use (AWPA, 2018). To prevent and reduce these hazards, creosote, resins or CCA (chrome, copper and arsenic) treatments are often applied by impregnation. In the case of CCA, chromium acts as a fixative, which by means of redox reactions, forms insoluble complexes with copper and arsenic, thereby precipitating and fixating them in the wood. This is a requirement since otherwise the water-borne nature of these biocides would make them highly susceptible to leaching to the environment, when in contact with water. However, the application of CCA treatments has been limited for several end uses. In marine environments, it is known that certain amounts of metals are leachate (Borges et al., 2004), raising concerns about their environmental effects (Brown & Eaton, 2000). Similarly, in Europe, the wood preservation with creosote has been proposed to be reduced or substituted by the European Chemical Agency with specific restrictions marked by the Regulation EU 2022/1950 (Commission Implementing Regulation (EU), 2022). Reducing leachability is crucial to both avoid potential environmental impacts and extend the lifetime of preservative treatments. Nonetheless, the search for environmentally friendly preservative treatments for wood is field with a lot to explore (Cragg et al., 1999). In previous studies, Fernández-Costas et al. (Fernández-Costas et al., 2017a, 2017b) developed a wood treatment consisting of impregnation with Kraft lignin and copper followed by an enzymatic cross-linking via laccase for its fixation. Lignin is one of the polymers that constitute wood and is also one of the most abundant residues generated worldwide, primarily by the pulp and paper industry (Bajwa et al., 2019). During the Kraft pulping, lignin is solubilized and separated from the other wood polymers as result of its breakdown and functionalization with polar groups such as hydroxyl and carboxyl groups (Evstigneyev & Shevchenko, 2019). Indeed, this functionalization of Kraft lignin makes it a good adsorbent of heavy metal cations following a cooperative adsorption mechanism of these groups (Chen et al., 2018), resulting in a better capacity than pulp or wood itself (Sciban & Klasnja, 2004). On the other hand, laccases are enzymes often used by lignocellulolytic organisms with the aim to break down lignin through the generation of radicals and access the glucose held in cellulose (Martínez et al., 2009). However, these radicals in lignin do not always promote breakdown, but also the crosslinking with other units, a feature that was exploited for free-adhesive composite applications (Gouveia et al., 2018; Nasir et al., 2015). Similarly, Fernández-Costas et al. (Fernández-Costas et al., 2017b) demonstrated effective laccase-assisted crosslinking of Kraft lignin with adsorbed copper into wood. In fact, this treatment reduced copper leachability while keeping biocide activity against wood rot fungi and eventually led to the Spanish patent ES2639137B1 (Fernández-Costas et al., 2018). In this work, we used the same approach as in our previous publication (Fernández-Costas et al., 2017b) but applied in the marine environment. Scots pine ( Pinus sylvestris ) treated and un-treated samples were submerged for one year on the Galician coast (Vigo, Spain). The evolution of biofouling, attack of marine xylophages and copper retention were evaluated. Advance methodologies such as X-ray computerized tomography (CT X-ray) or Itrax Core Scanner for simultaneous multiple test acquisition (high-resolution RGB image, X-ray radiography, and energy-dispersive X-ray fluorescence (ED-XRF) were applied. Materials and methods Wood samples and other materials Scots pine (Pinus sylvestris ) was chosen due to be non-durable and non-toxic wood species, contrasting with other known naturally durable species in marine environments (Şen et al., 2019). Blocks of 80 x 80 x 10 mm size (longitudinal x tangential x radial directions) with an average of 30 g dry weight were provided by the Wood Center of Innovation and Technological Services (CIS-madeira, San Cibrao das Viñas, Spain). Each piece was drilled with a hole (from the transversal plane) in the middle of each side with a separation of 5 mm from the edge (Supplementary information 1, Fig. S1). The Kraft lignin was obtained from eucalyptus black liquor kindly provided by Ence (Pontevedra, Spain) by acid precipitation following the procedure of Gouveia et al. (Gouveia et al., 2012). The laccase employed was the commercial product Novozym© 51003 kindly provided by Novozymes (Denmark) and derived from Myceliophthora thermophila . This enzyme product was previously used for lignin polymerization and molecule-grafting for wood functionalization (Bolaño et al., 2021; Gouveia et al., 2012; Schneider et al., 2019). All the rest of chemicals were purchased from Merck (Germany). Wood impregnation and treatments The main treatment involved the simultaneous application of three components: copper, Kraft lignin and laccase. To evaluate the contribution of each component to the results, additional treatments using individual or binary combinations of the components were prepared. The names of the treatments and component combinations are summarized in Table 1. Table 1. Wood treatments and the corresponding combinations of the treatment solution components. Kraft lignin was initially solubilized with NaOH 0.1 M solution at a ratio of 1:20 (w/v) and mixed by magnetic stirring for 1h. Water and/or other solutions were added sequentially and timely according to the treatment and impregnation procedure while always keeping the final lignin concentration at 20 g/L. Then, H 2 KPO 4 /HK 2 PO 4 amounts were added to reach pH 7 and 0.1 M. In the corresponding treatments, copper (CuCl 2 ) was prepared as a concentrated solution in distilled water (20-fold) with pH adjusted to 7 and added to the treatment solution resulting in a final concentration of 25 mM. The treatment with just copper was performed without the phosphate buffer since coagulation was observed when both were mixed. In contrast, treatments containing Kraft lignin and copper did not show coagulation in the presence of phosphates, therefore phosphate buffer was preferred in these cases (Supplementary information 1, Fig. S1). When corresponding, laccase was loaded to a final concentration of 50 U/g dry weight of wood. The activity of laccase was determined by ABTS method according to Gouveia et al. (Gouveia et al., 2013). The impregnation of wood samples was performed based on EN113 with several modifications (European Committee for Standarization, 1996). A Pyrex glass mini-reactor vessel of 2 L with a frosted glass joint lid and four inlet (AFORA, Spain) was employed (Supplementary information 1, Fig. S1). One inlet was connected to a vacuum pump; another was kept for introduction of solutions and the two left were kept hermetically closed. Four wood samples were placed inside the vessel with plastic grids positioned to avoid the contact between wood pieces or with the vessel walls, since it can reduce the exposed surface. On top of the pieces a plastic mesh and a weight was placed to ensure total immersion in the solution. Vacuum was applied at a value of 0.2 bar for 15 min and right after, 1.25 L of treatment solution was let in, and vacuum was applied 10 min more. Then, the vacuum was stopped, and the inlets were opened suddenly to bring the treatment back to atmospheric pressure. Later, the treatment was incubated for 2 h at 50°C, the optimal temperature for laccase activity. Finally, the samples were left on top of a metallic mesh to let the exceeding solution run out for some minutes, then weighed, and left hold in the air in a rack for air drying for two weeks. The wet weight was used to calculate the treatment solution retention. Air drying was preferred over oven drying because this condition was previously reported to be better for lignin crosslinking assisted by laccase (Fernández-Costas et al., 2017a). Three pieces of treated wood were used for the immersion test, while one piece was kept as a pre-immersion reference for analysis. Immersion structure and location A rectangular structure of 700 x 350 mm with total weight of 2.5 kg made of steel for marine use was constructed. This structure had two eyebolts in the top part and one in the bottom. All the borders were provided with several holes to attach the corresponding blocks of wood by use of plastic zip tie gaffers. The triplicates of each treatment were placed by columns along the rectangular structure. The structure with the samples was immersed in a recreational dock at Bouzas (Vigo, Spain) in a slot belonging to the Center of Marine Research from the University of Vigo (CIM-UVigo) at 42°13'31.66"N, 8°45'38.25"W coordinates (Fig. 1). Two ropes were used to hold the structure by the two upper eyebolts, while a ballast was added to the bottom eyebolt to maintain the structure vertically. The structure was immersed on 11th of July 2016 and maintained for one year. Twice a month, the structure was raised briefly to take pictures and evaluate the evolution of biofouling coverage. At the completion, the structure was retired for sample acquisition and further analysis. Water variables (temperature, salinity, dissolved oxygen, pH, etc.) were acquired from public historical records of the oceanographic sampling station “EF” (42°14.10′N, 8°46.80′W) from the Technologic Institute for the Galician Marine Environment Control (INTECMAR). Biofouling coverage, impact and biomass. For the study of biofouling coverage impact, the methodology of Bresy et al. (Bressy et al., 2014) was used with slight modifications. Four main organism groups were evaluated and assigned a “severity factor” (SF): Biofilm and slime (group 1; SF = 1), macroalgae (group 2, SF = 2), non-encrusting macro-organisms (ascidians, hydroids, sponges, etc.) (group 3, SF = 3), and encrusting macro-organisms (barnacles, bryozoans, tube worms, bivalves, etc.) (group 4, SF = 6). The coverage of each organism group was associated with an “Intensity factor” (IF) according to its percentage respectively: no presence (IF = 0); 1 to < 10% (IF = 1); 10 to < 20% (IF = 2); 20 to < 40% (IF = 3); 40 to < 60% (IF = 4); 60 to < 80% (IF = 5); and 80 to < 100% (IF = 6). To reflect the impact of biofouling, an “antifouling efficiency” ( N ) parameter was obtained from the Eq. 1. The higher N value, the worst performance against biofouling. $$\:\text{N}=\text{Ʃ}\left(\text{S}\text{F}\:\text{x}\:\text{I}\text{F}\right)\:\:\:\:\:\:\:\:\:\:\:Equation\:1$$ The coverage was determined from the regular pictures acquired throughout the year using Inkscape software for metric determination. Six timepoint were considered for analysis which reflected the more relevant changes along the immersion time. Examples of biofouling organisms found in this study are shown in Supplementary information 1, Fig. S2. After the completion of the immersion period, the biofouling biomass was scrapped and separately storage according to the wood sample face (light and non-light exposed). Dry weight (55°C for 4 days and 105°C for 24 h) and ash content (450°C for 4 h) were determined for each sample of biofouling. Itrax Core Scanner analysis (ED-XRF, X-ray radiography and high-resolution image) After removal of biofouling and drying, wood samples were analyzed with an Itrax Core Scanner (Cox Analytical Systems, Sweden) for sequential acquisition of high-resolution RGB image, X-ray radiography, and ED-XRF analysis. The instrument was controlled by the CoreScanner 8.6.3 software, the evaluation of XRF spectra with Q-spec 8.6.0, and post-processing and data analysis of RGB images and grey-scale radiographies with Redicore 8.6.0 software. First, a surface topography scan of high resolution was acquired and used to keep sample-detector distance constant, especially important for EDXRF. High resolution RGB images were obtained with resolution at a step size of 500 µm. The ED-XRF acquisition was done with a molybdenum anode with 30 kV, 50 mA, step size 1000 µm and acquisition time of 10 s. The X-ray radiography scan was one with 45 kV, 10 mA, step size of 1000 µm and acquisition time of 200 ms. Scans were acquired with a width of 2 cm and in the middle section of the piece from axial direction. Marine xylophage impact The surface of the wood samples after biofouling removal was explored for the presence of limnoriids tunnels and images were acquired with a USB digital magnifying lens. Presence of teredo was detected by X-ray radiographies from Itrax Core Scanner. Further analyses were done with CT X-ray analysis using a CBCT i-CAT® (DEXIS, United States). CT X-ray images were acquired in the 3 spatial planes (sagittal, transversal and frontal) with a resolution of 0.2 mm in DICOM format. Volume loss was determined by analysis of the CT X-ray images using the software Medical Imaging Interaction Toolkit (MITK) (Dinkelacker et al., 2025). The images were processed to determine the volume of the bored holes, calcareous depositions, valves and bodies of teredos. First, each piece of wood was cropped from the whole image set. Then, an appropriate grey scale for the piece to make enough contrast of the different elements (empty space, calcium carbonate, organic biomass of teredo body, etc.) was selected. 2D segmentation was prepared using the segmentation tool Region growing 3D . This tool works defining a grey scale threshold and placing a seed point in a selected structure visualized creating selection that grows according to the grey scale threshold selected. The 3D segmentation was converted to a smoothed polygonal model for visualization, and the volume of the model was obtained. The percentage of wood lost was determined by summing up all the cavities’ volumes over the total volume of the wood piece. The 3D polynomial segmentation models were exported for visualization as digital image in TIFF format. Determination of copper content and retention ED-XRF was acquired by Itrax Core Scanner from the immersed and non-immersed reference wood pieces as previously mentioned. This non-destructive technique was used for semi-quantitative comparison of copper amounts and distribution between samples. It is noteworthy that the acquisition with this technique has limited penetration, in the sample depending on different factors (sample matrix, excitation energy, detector, atomic number, etc.), which often ranges approximately from 10 to 100 µm. The total content of copper was determined by inductively coupled plasma with optical emission spectrometry (ICP-OES) analysis. This analysis was done after biofouling removal and Itrax analysis, since it requires the total combustion of the sample. Additionally, the non-immersed reference wood pieces were used to account for the initial copper content value. First, the samples were placed in a muffle furnace at 550°C for 4 h. The ashes were re-dissolved in a volume of 2% (w/w) nitric acid solution. The copper results were expressed as kg/m 3 of wood. Results and discussion Copper content and retention The copper content of the treated samples was calculated theoretically using the solution uptake results and the concentration of copper in the solution, while empirically values were obtained from ICP-OES analysis. The results are shown in Table 2 . Table 2 Solution uptake, theoretical and measured (ICP-OES) retention of copper, and leaching percentage for each treatment. Leaching percentage was calculated from the measured copper values of pieces before (t 0 ) and after (t 1 ) immersion in the sea for one year. Average density of the Scots pine wood pieces was 480 kg/m 3 . Uptake Theoretical retention Measured retention ICP-OES Treatment solution (kg/m 3 ) Cu (kg/m 3 ) Cu t 0 (kg/m 3 ) Cu t 1 (kg/m 3 ) Leaching % LAC 555 ± 15 - - - - LAC + KL 458 ± 69 - - - - CU 547 ± 14 0.868 ± 0.023 0.830 0.143 ± 0.018 82.7 ± 2.156 KL + CU 477 ± 51 0.757 ± 0.082 0.377 0.027 ± 0.001 92.8 ± 0.243 LAC + KL + CU 489 ± 43 0.776 ± 0.069 0.328 0.019 ± 0.005 94.2 ± 1.564 The solution uptake values were similar than in other studies treating Scots pine samples for marine use (Humar & Lesar, 2013). Theoretically, all the treatments with copper should have a retention of around 0.75–0.86 kg/m 3 . However, there was a discrepancy between theoretical and measured copper retentions in the KL + CU and LAC + KL + CU treatments. The measured copper retention in KL + CU and LAC + KL + CU treatments was almost 50% lower than expected by the theoretical calculations. In these treatments, the presence of lignin influenced the retention of copper. A sample of each treatment was cut along the axial direction and visually observed. In the LAC + KL, KL + CU and LAC + KL + CU treatments it was observed that a main accumulation of lignin in the axial edges followed the direction of floema and xylem. Lignin was concentrated mainly in the first 3 mm in the axial direction (Supplementary information 1: Fig. S3 ). Similar problems were reported in other impregnation treatments using lignin as preservative (Borrega, 2022). This might indicate that the pores were progressively clogged by lignin. The saturated lignin solution, the limited differential vacuum pressure of impregnation (≈ 1 atm) and possible coagulation interactions of polymeric lignin and divalent copper might explain these observations. Therefore, possible effect of lignin alone remained mainly limited to the surface. Therefore, copper retention was likely affected by the presence of lignin. After one year of immersion, all the copper containing treatments lost between 80–95% of their copper content. This highlights the importance of using fixatives; for example, chromium in CCA. The LAC + KL + CU treatment was based on the study of Fernandez-Costas (Fernández-Costas et al., 2017b), which resulted in good results of preservation against wood rot fungi even after a laboratory leachability test. However, several points must be noted: 1) the primary purpose of the treatment was for land use, and the leachability test was done with soaking the pieces for a relative short period; 2) the pieces were much smaller than in this study and the relative penetration along the pieces was higher; 3) the salinity of sea water increase leachability by creating complexes with copper, e.g. chloride – copper (Hingston et al., 2001; Lebow et al., 1999); 4) attack of xylophages increases the contact surface with water. The analysis of copper by means of ED-XRF by Itrax Core Scanner indicated higher content and homogeneous distribution of copper along the pieces of pre-immersed CU treatment. Whereas the pre-immersed KL + CU and LAC + KL + CU pieces had slightly lower values of copper overall, while regarding its distribution, copper was slightly higher in the edges (Fig. 2 ). This aligns with the previously mentioned differences in copper retention and the fact that lignin penetration was mainly present in the first couple of millimeters in the pieces. After one year of immersion, copper values were reduced significantly, especially in KL + CU and LAC + KL + CU treatments, although conserving the same pattern of copper distribution. These suggest that, where lignin was highly present, the copper was retained efficiently. The ED-XRF data and X-ray radiographies from all sample replicates are fully shown in Supplementary information 2: Fig. S1 and Fig. S2 . Considering these results, several modifications of the treatment should be made in future: increase of biocidal content, lignin solution concentration and filtration, increase of vacuum-pressure conditions of impregnation, etc. Therefore, improvement of impregnation conditions should be explored in the future to avoid clogging of the wood cell lumen. Additionally, the Itrax Core Scanner has the potential to be calibrated for quantitative data (Kelloway et al., 2014), which in this case could be conducted by impregnation of the same type of wood with different copper concentrations and determined by external analysis, e.g. ICP-OES. Biofouling evolution and impact The evolution and impact of biofouling coverage was reflected by the antifouling efficiency ( N ) value shown in Fig. 3 a. As reminder, the higher the N value, the worse antifouling performance. Firstly, it is worthwhile noting that there was a sharp change in the timepoint of February-March 2017, in terms of percentages of the fouling groups and total biofouling coverage. During February 2017, an Atlantic squall reached the Galician coast generating an extreme wave and promoting red alerts in all the region 1 . The storms dumped enormous amounts of rain, which was evidenced by a sharp decrease in the water salinity of the Vigo estuary (Supplementary information 1: Fig. S4). The shear forces created during this event impacted the biofouling coverage, which in some cases removed part of the accumulated biofouling mass. On the other hand, the control pieces showed a faster progression of colonization and higher N values than the rest of treatments, until the extreme wave event of February 2017. On the other hand, the treatments CU and LAC + KL + CU had similar progression and the lowest N values among all the treatments. LAC, LAC + KL and KL + CU treatments had also similar patterns of progression, but N values were more like the control samples. It must be highlighted that the biofouling area coverage does not always reflect clearly the impact in terms of mass accumulation. Therefore, the interpretation of the N results was complemented with the values of the biomass accumulated at the end of the immersion test (Fig. 3 b). For all treatments, the biomass accumulated was mainly from calcareous organisms (mussels, bryozoans, tube worms, etc.) as reflected by the small difference between dry weight and ash content. The treatments containing copper were the most effective, lowering the amount of accumulated biomass, following the order CU > LAC + KL + CU > KL + CU. Whereas, a significant difference was found between the control and the treatments LAC + KL and LAC, with higher values for these last ones. This observation suggested that the impregnation process, the laccase presence, and incubation (2 h at 50°C) might have removed extractives (wax, resins, terpenes, etc.) that could affect the colonization pattern. Several studies have reported that laccases could also play a role in removal of lipophilic compounds, through a laccase-mediator mechanism (Gutiérrez et al., 2009). Indeed, a reduction in hydrophobicity, determined by the water contact angle, was detected in these samples (Supplementary information 1, Fig. S5). Hydrophobicity plays an important role in adhesion of settlement and adhesion of algae (J. A. Callow & Callow, 2006; M. E. Callow & Callow, 2000) while opposite for mussels and barnacles (Wiegemann, 2005). However, it was not possible to determine the separate effects of laccase and incubation because no treatment included water impregnation followed by incubation alone. Additionally, the removal of certain extractives might not only affect settlement due to hydrophobicity but also potential biocidal or inhibitory effects. Lastly, by comparing LAC and LAC + KL treatments, Kraft lignin itself seemed to not have efficiency avoiding biofouling settlement. Biofouling progression by organism group The coverage of biofouling was evaluated along the marine trial time for all the samples. Generally, there were clear differences in biofouling coverage between the face of the wood samples independently from the treatment (Fig. 4 ). This was caused by differences in light exposure: one side of the wood samples faced the area beneath the floating dock shadow, while the other sides did not (Fig. 1 b). The total biofouling coverage was higher and reached total surface coverage earlier in the face with more light exposition. The colonization by different organism groups was also different. As expected, algae had more presence in the light exposed face, with small contributions in the non-light exposed face, mainly attributed to red algae. Light is a relevant factor for the colonization of growth of photosynthetic algae, while red algae are adapted to lower intensities of light due to their pigment adaptations (Lebret et al., 2009). Additionally, a higher coverage of algae correlated with lower colonization of non-encrusting and encrusting fouling macro-organisms as can be noted comparing light and non-light exposed faces. In biofouling colonization, there are interactions between organisms that benefits the colonization but also competition (Rao et al., 2007). In this study, it seemed that between algae and encrusting and non-encrusting fouling macro-organisms, there was some type of competitive interaction. On the other hand, non-encrusting macro-organisms had the lowest presence and coverage in all the treatments and sides. This group was represented mainly by ascidians and sponges. Colonization of ascidians is dependent on the roughness, porosity and chemistry of the materials, that needs to be favorable for the settlement of the larva settlement, which has limited natatorial abilities (Chase et al., 2016). The exposed surfaces of the wood were quite smooth, which can explain the low presence of this group in the biofouling coverage, in comparison with other materials such as concrete. Differences were also detected between samples that contained copper and those that did not. The treatments with copper slowed down the progression of biofouling coverage, especially in the non-light exposed faces. The biofouling group that was delayed the most in these treatments was the encrusting macro-organisms at all timepoints and sides. The most efficient treatment reducing the total coverage and the most impactful biofouling groups was CU, followed by LAC + KL + CU and KL + CU. On the other hand, both LAC and LAC + KL differed from the control samples in terms of total coverage and individual biofouling group coverage. In comparison, control samples were covered by fouling faster, especially by algae and encrusting fouling. Marine xylophage presence During the immersion time, the water temperature and salinity ranged from 12–19°C and 29.3–35.7 PSU, respectively (Supplementary information 1: Fig. S4). These conditions were in the range that allows correct spawn, growth, and boring and feeding activity of marine xylophages (Bersoza Hernández & Angelini, 2019; Borges et al., 2009). The CT X-ray analysis revealed the infestation of marine xylophages in the wood samples (Fig. 5 ). The only treatment that showed no-attack of marine xylophages in any of the replicates was the CU treatment, while the rest presented signs of attack of different types and magnitude. The KL + CU and LAC + KL + CU treatments presented attack of limnoriids but not teredos. This agrees with the reported tolerance of limnoriids to copper, which they accumulate as granules in the digestive system, and hypothetically use it for their enzymes and blood pigments (Tupper et al., 2000). On the other hand, LAC, LAC + KL and control presented attack of both teredos and limnoriids. Based on the presence of teredo valves, most of the pieces were colonized by only one or two big teredos, and in some cases, additional young specimens starting infestation were detected in one LAC replicate and the control. Overall, the range of volume loss for the treatments was between 0 and 8.4%, with the corresponding volume loss in descending order: LAC, LAC + KL, control, CU + KL, LAC + KL + CU and CU. In addition, considering the ratings from EN275 (European Committe for Standarization, 1992) to determine marine xylophage attack, the pieces containing copper could be classified as “no attack” while the rest of treatments presented slightly or moderate attack (Table 3 ). However, it is worth noting that the EN275 standard procedure is intended for an immersion time of five years as minimum. To provide context and facilitate the comparison of our results, hereafter some studies on marine immersion of pine are introduced. For example, in the work of Romano et al. (Romano et al., 2013), samples of pine were immersed for one year in the Blanes Canyon (coast of Gerona, Spain) and an adjacent open slope at 900–1500 m depth. The canyon environment was much richer in debris than the open slope resulting in ten times bigger infestation (15 individuals/dm 3 vs 200 − 100 individuals/dm 3 ). In the work of Palanti et al. (Palanti et al., 2015) and Humar and Lesar (Humar & Lesar, 2013), Scots pine samples were immersed in the Adriatic Sea at harbors at 6 m depth, near the sediment, which after 6–10 months were almost completely degraded by marine xylophages. Similarly, a work immersing different types of wood at intertidal zones in Florida estuaries for 3–6 months found volume loss ranged from 0 to 8%, with higher values found when the wood was closer to the sediment (Bersoza Hernández & Angelini, 2019). In this study, the experiment was immersed in a floating dock at Atlantic Sea, at a shallow depth (0.5 m), exposed to light, separated from the sediment, and at a location not particularly rich in debris. The light and shallow position could affect colonization of limnoriids since they have negative phototaxis and limited natatory capabilities (Borges et al., 2009), but also the competition with the strong colonization of biofouling. A negative correlation has been reported between biofouling coverage and marine xylophage presence (López et al., 2024), in agreement with the results of this study. Finally, low levels of debris might have affected the presence of teredo, as they primarily feed filtrating debris (Paalvast & van der Velde, 2013), in contrast to limnoriids that rely on wood digestion (King et al., 2010). Table 3 Rating values according EN275 to define attack of limnoriids and teredo in the wood pieces tested after one year of exposition. Limnoriid rating Teredo rating Overall rating LAC 0.7 1.0 Slight LAC + KL 1.0 1.0 Moderate CU 0.0 0.0 No attack KL + CU 0.7 0.0 No attack LAC + KL + CU 0.3 0.0 No attack CONTROL 1.0 1.0 Moderate Separately, we have shown a modern approach to quantify the attack of marine xylophages by means of CT X-ray analysis. This analysis, at last in our experience, was cheaper than 2D X-ray acquisition providing not only quantitative data on volume loss but also acquisition of details such as number, morphology and size of teredinids. Additionally, the CT X-ray analysis was conducted in MITK, an open-source software that is freely available and user friendly. Therefore, this approach is superior to the established in the standard EN275 (European Committe for Standarization, 1992). For further details, the short videos of the 3D reconstructions of each treatment are included in Supplementary information 3. Thus, pictures of surface with details of limnoriids and teredo boring sites are shown in Supplementary information 2: Fig. S3 . Conclusions A wood treatment combining enzymatic polymerized Kraft lignin and copper was explored for marine use in this study. Although this treatment was previously probed to be efficient for land use against wood root fungi with reduced leachability, it was not efficient enough for marine use. Copper was effective bioactive compound to reduce biofouling and marine xylophage attack, although higher concentrations and retention should be improved in the treatment. Kraft lignin by itself did not show clear effects on biofouling or marine xylophage attack prevention. In addition, although Kraft lignin impregnation was complicated, copper was retained better where its presence was high. Improvements of impregnation of lignin should be a crucial step for further development. Separately, impregnation and incubation for enzymatic polymerization removed partially some extractives (wax, resins, etc.) affecting the mass and composition of biofouling. On the other hand, this study has employed a modern and comprehensive methodology to evaluate biofouling and marine xylophage impact. The use of CT X-ray analysis proved to be superior and cheaper than the approach of EN275 by standard X-ray radiographies. The Itrax Core Scanner has demonstrated to be an excellent tool for automated multi-acquisition of X-ray radiography, ED-XRF and RGB images, perfectly fitting with the purposes of this study. Indeed, there are other models such as the Itrax Multiscanner (COX analytical systems, Sweden) made specifically for wood analysis with much higher XRF resolution, allowing even analysis of changes in minerals in the growth rings. We consider that these methods can bring inspiration and new applications in the field of wood preservative treatments. Finally, this study has provided data for biofouling coverage and marine xylophage attack in Scots pine exposed in the Atlantic Sea at shallow depth (0.5 m) depth and separated from the sediment. These results might serve as reference for future work. Declarations Conflict of interest The authors declare no competing interests. Funding The study was funded by the Secretaría Xeral de Universidades, Xunta de Galicia, through the projects EDC431C2017-GRC and GRC-ED431C 2025/47. Author Contribution Conceptualization: CBL, MCFC, and DM. Methodology: CBL and MCFC. Formal analysis: CBL. Visualization: CBL. Resources: DM. Writing original draft: CBL. Writing, reviewing and editing: CBL, MCFC, and DM. All authors have read and agreed on the published manuscript. Acknowledgments The authors would like to thank the CACTI (University of Vigo, Spain), especially to Jorge Millos Alfeirán, for the assistance in the acquisition of Itrax Core Scanner results and ICP-OES; the RIAIDT (University of Santiago de Compostela, Spain) for assistance in acquisition of CT X-ray data; the CIM (University of Vigo, Spain) for assistance and allowance for the placement of the immersion experiment setup; and the INTECMAR (Xunta de Galicia, Spain) for the public available oceanographic data. Data Availability All datasets generated for this study are available in the Zenodo repository upon reasonable request from the authors: https://doi.org/10.5281/zenodo.17835546 References AWPA. (2018). Use category system: user specification for treated wood. American Wood Protection Association Standard . Bajwa, D. 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H., Bolanõ Losada, C., Moldes, D., Fontana, R. C., De Siqueira, F. G., Prieto, A., Martínez, M. J., Martínez, Á. T., Dillon, A. J. P., & Camassola, M. (2019). A sustainable approach of enzymatic grafting on eucalyptus globulus wood by laccase from the newly isolated white-rot basidiomycete marasmiellus palmivorus VE111. ACS Sustainable Chemistry and Engineering , 7 (15), 13418–13424. https://doi.org/10.1021/acssuschemeng.9b02770 Schultz, M. P., Bendick, J. a, Holm, E. R., & Hertel, W. M. (2011). Economic impact of biofouling on a naval surface ship. Biofouling , 27 (1), 87–98. https://doi.org/10.1080/08927014.2010.542809 Sciban, M., & Klasnja, M. (2004). Study of the Adsorption of Copper (II) Ions from Water onto Wood Sawdust, Pulp and Lignin. Adsorption Science & Technology , 22 (3), 195–206. Şen, S., Sivrikaya, H., & Yalçin, M. (2019). Natural durability of heartwoods from European and tropical Africa trees exposed to marine conditions. African Journal of Wood Science and Forestry , 7 (1), 1–008. www.internationalscholarsjournals.org Tupper, B. C., Pitman, A. J., & Cragg, S. M. (2000). Copper accumulation in the digestive caecae of Limnoria quadripunctata Holthius (Isopoda: Crustacea) tunnelling CCA-treated wood in laboratory cultures. Holzforschung , 54 , 570–576. Wiegemann, M. (2005). Adhesion in blue mussels (Mytilus edulis) and barnacles (genus Balanus): Mechanisms and technical applications. In Aquatic Sciences (Vol. 67, Issue 2, pp. 166–176). https://doi.org/10.1007/s00027-005-0758-5 Footnotes https://www.laopinioncoruna.es/galicia/2017/02/01/entrado-primer-temporal-galicia-24336617.html Additional Declarations No competing interests reported. Supplementary Files 20251022Supplementaryinformation1.pdf 20251027Supplementaryinformation2.pdf 20251202Supplementaryinformation3.rar Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviews received at journal 16 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviews received at journal 15 Feb, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 26 Jan, 2026 Editor assigned by journal 26 Jan, 2026 Submission checks completed at journal 13 Dec, 2025 First submitted to journal 12 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8347225","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591569318,"identity":"b12e0807-848f-494b-b6af-ba393bc8b7be","order_by":0,"name":"Cristian Bolaño Losada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFACxgYgYZHAwN588AGQxcNHpBaJBAaeY8kGIC1sRFoF1CKRoyYBYhLUwj/7cPPHLzUSefwMOWyVX3PsZNgYmB8+uoHP+HOJbdIyxySKJRvOHrstuy0Z6DA2Y+McPFoMeBjbmCXYJBI3HOxLuy25jRmohYdNmoCW5s8S/4BaDvOYFUtuqydKS4PkxzaglmM8Zowftx0mrEXiDGObNGMf0C89bMnSjNuO87AxE/ALfw/7448/vtnk8cs/Pvjx57Zqe3725oeP8WkBAWYeFAYzAeUgwPgDnTEKRsEoGAWjABkAAIw5RBa8dBFxAAAAAElFTkSuQmCC","orcid":"","institution":"CINTECX, University of Vigo","correspondingAuthor":true,"prefix":"","firstName":"Cristian","middleName":"Bolaño","lastName":"Losada","suffix":""},{"id":591569319,"identity":"c3baa7fd-83f4-4c18-970c-1fa81fe6e2e0","order_by":1,"name":"Mari Carmen Fernández-Costas","email":"","orcid":"","institution":"CINTECX, University of Vigo","correspondingAuthor":false,"prefix":"","firstName":"Mari","middleName":"Carmen","lastName":"Fernández-Costas","suffix":""},{"id":591569320,"identity":"d4c0bdb9-cb4d-406a-a76e-d8a0b14c03b5","order_by":2,"name":"Diego Moldes","email":"","orcid":"","institution":"CINTECX, University of Vigo","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Moldes","suffix":""}],"badges":[],"createdAt":"2025-12-12 15:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8347225/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8347225/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104166655,"identity":"efb578fc-b309-4f95-991e-d507305a38be","added_by":"auto","created_at":"2026-03-08 14:18:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":227173,"visible":true,"origin":"","legend":"\u003cp\u003eA) Vigo estuary with the location of the immersion test marked with a red pin and the oceanographic station from Intecmar marked with a yellow star. B) Structure with the wood treated pieces placed at the floating dock of Bouzas (Vigo, Spain). Maps reproduced from OpenStreetMap under the terms of Open Database License (ODbL) v1.0.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/692a38e18786d3dc502bcc4b.jpg"},{"id":104404553,"identity":"8a57ca68-29d3-4ecc-8e29-170cc56eeaff","added_by":"auto","created_at":"2026-03-11 12:20:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92028,"visible":true,"origin":"","legend":"\u003cp\u003eItrax Core Scanner results for copper contained treatments (CU, KL+CU and LAC+KL+CU) and control. RGB high quality image with overlapped X-ray radiography is displayed with the corresponding copper XRF values in kilo counts per second (Kcps) units at the bottom. For comparison, pre-immersion results are displayed on the top and corresponding post-immersion results on the bottom.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/1238e1100acff7e4ffbac80d.jpg"},{"id":104166656,"identity":"017c6f42-7560-4324-a852-c45134a05a5a","added_by":"auto","created_at":"2026-03-08 14:18:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126279,"visible":true,"origin":"","legend":"\u003cp\u003eA) The evolution of the average (both faces) antifouling efficiency (\u003cem\u003eN\u003c/em\u003e) along with the immersion time for each wood treatment. B) Total biofouling dry weight and ash content in the different wood treatments at the end of the immersion time. Error bars display the standard deviation of three independent sample replicates.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/c508e9893b3c7b900a0097ac.jpg"},{"id":104166658,"identity":"07e5ffbe-e068-4ada-ac90-ad3c3927b212","added_by":"auto","created_at":"2026-03-08 14:18:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":325611,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of biofouling coverage of each fouling organisms’ group. G1 (slime, biofilm, etc.): orange; G2 (algae): green, G3 (non-encrusting macro-organisms): blue; G4 (encrusting macro-organisms): grey; for each treatment and side (light, non-light exposed, and both/averaged).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/a46d5f6e0e04210a4e7f6d38.jpg"},{"id":104403820,"identity":"be054668-9662-43ae-a991-e0649b8cc0bf","added_by":"auto","created_at":"2026-03-11 12:19:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285109,"visible":true,"origin":"","legend":"\u003cp\u003eCT X-ray results of control, laccase (LAC); laccase and Kraft lignin (LAC+KL); copper (CU); Kraft lignin and copper (KL+CU); laccase, Kraft lignin and copper (LAC+KL+CU) wood treated samples. Replicates of the same treatment are organized by columns. A grey scale slide of the wood piece is shown as background, tunnels created by teredos in blue and by limnoriids in violet, teredo valves in orange, and residual and dried soft bodies of teredos in green. The total volume loss of each sample is presented in percentage values in red color.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/3c57457db9a6d19a987c94b3.jpg"},{"id":104779618,"identity":"c43a3ae5-8f7c-41a7-9776-8bea0cd0e8a3","added_by":"auto","created_at":"2026-03-17 07:43:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1936949,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/30343c68-93d3-43ad-b78e-2cd834ffa1cd.pdf"},{"id":104166661,"identity":"85dc5e2e-6166-4bf5-ab9b-94260fd6090f","added_by":"auto","created_at":"2026-03-08 14:18:53","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":677853,"visible":true,"origin":"","legend":"","description":"","filename":"20251022Supplementaryinformation1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/e0552fe68a1bd769fe055d09.pdf"},{"id":104166660,"identity":"9a839001-339a-4190-b66e-b3fd335e1794","added_by":"auto","created_at":"2026-03-08 14:18:53","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":696896,"visible":true,"origin":"","legend":"","description":"","filename":"20251027Supplementaryinformation2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/94a7ca2a2bd2c997932bbd68.pdf"},{"id":104166662,"identity":"4043924d-f481-4210-a835-ce3598af065a","added_by":"auto","created_at":"2026-03-08 14:18:53","extension":"rar","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9522167,"visible":true,"origin":"","legend":"","description":"","filename":"20251202Supplementaryinformation3.rar","url":"https://assets-eu.researchsquare.com/files/rs-8347225/v1/d2a8b9d2c578408fa86d463d.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Copper-lignin with laccase-mediated polymerization fixation for wood preservation: determination of durability in marine environment using novel technologies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWood is a resource that has been used by diverse civilizations for land and marine uses due to its mechanical resistance, low cost, easy production, renewability and sustainability. However, wood is not exempt from deterioration due to abiotic and biotic factors. Therefore, wood treatments for preservation have been developed throughout human history according to the end use. Particularly, the marine environment can be considered one of the most hostile due to stressful factors such as saline water, corrosion, biofouling, marine xylophages, etc (Khademibami \u0026amp; Bobadilha, 2022).\u003c/p\u003e \u003cp\u003eMarine biofouling results from the adhesion of diverse organisms such as barnacles, algae, and microbial biofilms to the material surface. Biofouling represents several problems for underwater structures, such increase in weight, increase in frictional forces in ships, corrosion, etc. From the first moment that a material is immersed, a series of events begins, starting from the adsorption of organic molecules, followed by the development of microbial films, and the subsequent adhesion of bigger organisms (algae, invertebrates, etc.). This process is like an ecological succession that eventually achieves a stable and complex biological community (M. Callow \u0026amp; Callow, 2002). Treatments to avoid biofouling are especially important in paint formulations for ship hulls due to the impact on operative costs (maintenance, fuel consumption, etc.) (Schultz et al., 2011). On the other hand, wood is also susceptible to marine xylophages, which in contrast to biofouling, actively compromises its structural integrity by boring into and feeding on it. Typical marine xylophages are boring bivalves (teredos) and crustaceans (limnoriids). There are three reported species of limnoriids in European sea waters (Borges, Merckelbach, \u0026amp; Cragg, 2014). They display a boring activity following a pattern of parallel channels near to the wood surface (Barroso Lopes et al., 2014; Cragg et al., 2007) digesting wood components with putative cellulases and modifying lignin by redox reactions promoted by their hemocyanin (Besser et al., 2018; King et al., 2010; Malyon, 2011). In addition, limnoriids tolerate creosote and copper-based wood treatments (Borges, Merckelbach, \u0026amp; Cragg, 2014; Tupper et al., 2000). On the other side, there are seventeen species of teredo reported in European marine waters (Borges, Merckelbach, Sampaio, et al., 2014). Teredos, or shipworms, use their shelves to deeply bore into wood. They utilize wood to sustain their gut microbiota; however, it has been suggested that their primary nutritional intake comes from debris filtration (Paalvast \u0026amp; van der Velde, 2013). This implies that they use wood more as shelter than a primary food source. Shipworms were one of the most feared species in naval shipping and caused panic and important changes in the construction of Dutch dykes in the past (Nelson, 2016).\u003c/p\u003e \u003cp\u003eSeveral international organisms have defined standards, recommendations, categories and treatments for wood according to the end use, including the marine environment. To give an instance, the American Wood Protection Association defined categories for wood treatments according to a criterion, being the UC5 the one for marine use (AWPA, 2018). To prevent and reduce these hazards, creosote, resins or CCA (chrome, copper and arsenic) treatments are often applied by impregnation. In the case of CCA, chromium acts as a fixative, which by means of redox reactions, forms insoluble complexes with copper and arsenic, thereby precipitating and fixating them in the wood. This is a requirement since otherwise the water-borne nature of these biocides would make them highly susceptible to leaching to the environment, when in contact with water. However, the application of CCA treatments has been limited for several end uses. In marine environments, it is known that certain amounts of metals are leachate (Borges et al., 2004), raising concerns about their environmental effects (Brown \u0026amp; Eaton, 2000). Similarly, in Europe, the wood preservation with creosote has been proposed to be reduced or substituted by the European Chemical Agency with specific restrictions marked by the Regulation EU 2022/1950 (Commission Implementing Regulation (EU), 2022). Reducing leachability is crucial to both avoid potential environmental impacts and extend the lifetime of preservative treatments. Nonetheless, the search for environmentally friendly preservative treatments for wood is field with a lot to explore (Cragg et al., 1999).\u003c/p\u003e \u003cp\u003eIn previous studies, Fern\u0026aacute;ndez-Costas et al. (Fern\u0026aacute;ndez-Costas et al., 2017a, 2017b) developed a wood treatment consisting of impregnation with Kraft lignin and copper followed by an enzymatic cross-linking via laccase for its fixation. Lignin is one of the polymers that constitute wood and is also one of the most abundant residues generated worldwide, primarily by the pulp and paper industry (Bajwa et al., 2019). During the Kraft pulping, lignin is solubilized and separated from the other wood polymers as result of its breakdown and functionalization with polar groups such as hydroxyl and carboxyl groups (Evstigneyev \u0026amp; Shevchenko, 2019). Indeed, this functionalization of Kraft lignin makes it a good adsorbent of heavy metal cations following a cooperative adsorption mechanism of these groups (Chen et al., 2018), resulting in a better capacity than pulp or wood itself (Sciban \u0026amp; Klasnja, 2004). On the other hand, laccases are enzymes often used by lignocellulolytic organisms with the aim to break down lignin through the generation of radicals and access the glucose held in cellulose (Mart\u0026iacute;nez et al., 2009). However, these radicals in lignin do not always promote breakdown, but also the crosslinking with other units, a feature that was exploited for free-adhesive composite applications (Gouveia et al., 2018; Nasir et al., 2015). Similarly, Fern\u0026aacute;ndez-Costas et al. (Fern\u0026aacute;ndez-Costas et al., 2017b) demonstrated effective laccase-assisted crosslinking of Kraft lignin with adsorbed copper into wood. In fact, this treatment reduced copper leachability while keeping biocide activity against wood rot fungi and eventually led to the Spanish patent ES2639137B1 (Fern\u0026aacute;ndez-Costas et al., 2018).\u003c/p\u003e \u003cp\u003eIn this work, we used the same approach as in our previous publication (Fern\u0026aacute;ndez-Costas et al., 2017b) but applied in the marine environment. Scots pine (\u003cem\u003ePinus sylvestris\u003c/em\u003e) treated and un-treated samples were submerged for one year on the Galician coast (Vigo, Spain). The evolution of biofouling, attack of marine xylophages and copper retention were evaluated. Advance methodologies such as X-ray computerized tomography (CT X-ray) or Itrax Core Scanner for simultaneous multiple test acquisition (high-resolution RGB image, X-ray radiography, and energy-dispersive X-ray fluorescence (ED-XRF) were applied.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eWood samples and other materials\u003c/h2\u003e\n \u003cp\u003eScots pine \u003cem\u003e(Pinus sylvestris\u003c/em\u003e) was chosen due to be non-durable and non-toxic wood species, contrasting with other known naturally durable species in marine environments (Şen et al., 2019). Blocks of 80 x 80 x 10 mm size (longitudinal x tangential x radial directions) with an average of 30 g dry weight were provided by the Wood Center of Innovation and Technological Services (CIS-madeira, San Cibrao das Vi\u0026ntilde;as, Spain). Each piece was drilled with a hole (from the transversal plane) in the middle of each side with a separation of 5 mm from the edge (Supplementary information 1, Fig. S1).\u003c/p\u003e\n \u003cp\u003eThe Kraft lignin was obtained from eucalyptus black liquor kindly provided by Ence (Pontevedra, Spain) by acid precipitation following the procedure of Gouveia et al. (Gouveia et al., 2012).\u003c/p\u003e\n \u003cp\u003eThe laccase employed was the commercial product Novozym\u0026copy; 51003 kindly provided by Novozymes (Denmark) and derived from \u003cem\u003eMyceliophthora thermophila\u003c/em\u003e. This enzyme product was previously used for lignin polymerization and molecule-grafting for wood functionalization (Bola\u0026ntilde;o et al., 2021; Gouveia et al., 2012; Schneider et al., 2019).\u003c/p\u003e\n \u003cp\u003eAll the rest of chemicals were purchased from Merck (Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eWood impregnation and treatments\u003c/h3\u003e\n\u003cp\u003eThe main treatment involved the simultaneous application of three components: copper, Kraft lignin and laccase. To evaluate the contribution of each component to the results, additional treatments using individual or binary combinations of the components were prepared. The names of the treatments and component combinations are summarized in Table\u0026nbsp;1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. \u0026nbsp;\u003c/strong\u003eWood treatments and the corresponding combinations of the treatment solution components.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1772694235.png\" style=\"width: 601px;\"\u003e\u003c/p\u003e\n\u003cp\u003eKraft lignin was initially solubilized with NaOH 0.1 M solution at a ratio of 1:20 (w/v) and mixed by magnetic stirring for 1h. Water and/or other solutions were added sequentially and timely according to the treatment and impregnation procedure while always keeping the final lignin concentration at 20 g/L. Then, H\u003csub\u003e2\u003c/sub\u003eKPO\u003csub\u003e4\u003c/sub\u003e/HK\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e amounts were added to reach pH 7 and 0.1 M. In the corresponding treatments, copper (CuCl\u003csub\u003e2\u003c/sub\u003e) was prepared as a concentrated solution in distilled water (20-fold) with pH adjusted to 7 and added to the treatment solution resulting in a final concentration of 25 mM. The treatment with just copper was performed without the phosphate buffer since coagulation was observed when both were mixed. In contrast, treatments containing Kraft lignin and copper did not show coagulation in the presence of phosphates, therefore phosphate buffer was preferred in these cases (Supplementary information 1, Fig. S1). When corresponding, laccase was loaded to a final concentration of 50 U/g dry weight of wood. The activity of laccase was determined by ABTS method according to Gouveia et al. (Gouveia et al., 2013).\u003c/p\u003e\n\u003cp\u003eThe impregnation of wood samples was performed based on EN113 with several modifications (European Committee for Standarization, 1996). A Pyrex glass mini-reactor vessel of 2 L with a frosted glass joint lid and four inlet (AFORA, Spain) was employed (Supplementary information 1, Fig. S1). One inlet was connected to a vacuum pump; another was kept for introduction of solutions and the two left were kept hermetically closed. Four wood samples were placed inside the vessel with plastic grids positioned to avoid the contact between wood pieces or with the vessel walls, since it can reduce the exposed surface. On top of the pieces a plastic mesh and a weight was placed to ensure total immersion in the solution. Vacuum was applied at a value of 0.2 bar for 15 min and right after, 1.25 L of treatment solution was let in, and vacuum was applied 10 min more. Then, the vacuum was stopped, and the inlets were opened suddenly to bring the treatment back to atmospheric pressure. Later, the treatment was incubated for 2 h at 50\u0026deg;C, the optimal temperature for laccase activity. Finally, the samples were left on top of a metallic mesh to let the exceeding solution run out for some minutes, then weighed, and left hold in the air in a rack for air drying for two weeks. The wet weight was used to calculate the treatment solution retention. Air drying was preferred over oven drying because this condition was previously reported to be better for lignin crosslinking assisted by laccase (Fern\u0026aacute;ndez-Costas et al., 2017a). Three pieces of treated wood were used for the immersion test, while one piece was kept as a pre-immersion reference for analysis.\u003c/p\u003e\n\u003ch3\u003eImmersion structure and location\u003c/h3\u003e\n\u003cp\u003eA rectangular structure of 700 x 350 mm with total weight of 2.5 kg made of steel for marine use was constructed. This structure had two eyebolts in the top part and one in the bottom. All the borders were provided with several holes to attach the corresponding blocks of wood by use of plastic zip tie gaffers. The triplicates of each treatment were placed by columns along the rectangular structure.\u003c/p\u003e\n\u003cp\u003eThe structure with the samples was immersed in a recreational dock at Bouzas (Vigo, Spain) in a slot belonging to the Center of Marine Research from the University of Vigo (CIM-UVigo) at 42\u0026deg;13\u0026apos;31.66\u0026quot;N, 8\u0026deg;45\u0026apos;38.25\u0026quot;W coordinates (Fig.\u0026nbsp;1). Two ropes were used to hold the structure by the two upper eyebolts, while a ballast was added to the bottom eyebolt to maintain the structure vertically. The structure was immersed on 11th of July 2016 and maintained for one year. Twice a month, the structure was raised briefly to take pictures and evaluate the evolution of biofouling coverage. At the completion, the structure was retired for sample acquisition and further analysis.\u003c/p\u003e\n\u003cp\u003eWater variables (temperature, salinity, dissolved oxygen, pH, etc.) were acquired from public historical records of the oceanographic sampling station \u0026ldquo;EF\u0026rdquo; (42\u0026deg;14.10\u0026prime;N, 8\u0026deg;46.80\u0026prime;W) from the Technologic Institute for the Galician Marine Environment Control (INTECMAR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofouling coverage, impact and biomass.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the study of biofouling coverage impact, the methodology of Bresy et al. (Bressy et al., 2014) was used with slight modifications. Four main organism groups were evaluated and assigned a \u0026ldquo;severity factor\u0026rdquo; (SF): Biofilm and slime (group 1; SF\u0026thinsp;=\u0026thinsp;1), macroalgae (group 2, SF\u0026thinsp;=\u0026thinsp;2), non-encrusting macro-organisms (ascidians, hydroids, sponges, etc.) (group 3, SF\u0026thinsp;=\u0026thinsp;3), and encrusting macro-organisms (barnacles, bryozoans, tube worms, bivalves, etc.) (group 4, SF\u0026thinsp;=\u0026thinsp;6). The coverage of each organism group was associated with an \u0026ldquo;Intensity factor\u0026rdquo; (IF) according to its percentage respectively: no presence (IF\u0026thinsp;=\u0026thinsp;0); 1 to \u0026lt;\u0026thinsp;10% (IF\u0026thinsp;=\u0026thinsp;1); 10 to \u0026lt;\u0026thinsp;20% (IF\u0026thinsp;=\u0026thinsp;2); 20 to \u0026lt;\u0026thinsp;40% (IF\u0026thinsp;=\u0026thinsp;3); 40 to \u0026lt;\u0026thinsp;60% (IF\u0026thinsp;=\u0026thinsp;4); 60 to \u0026lt;\u0026thinsp;80% (IF\u0026thinsp;=\u0026thinsp;5); and 80 to \u0026lt;\u0026thinsp;100% (IF\u0026thinsp;=\u0026thinsp;6). To reflect the impact of biofouling, an \u0026ldquo;antifouling efficiency\u0026rdquo; (\u003cem\u003eN\u003c/em\u003e) parameter was obtained from the Eq.\u0026nbsp;1. The higher \u003cem\u003eN\u003c/em\u003e value, the worst performance against biofouling.\u003c/p\u003e\n\u003cdiv id=\"Equa\"\u003e\n \u003cdiv id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\text{N}=\\text{Ʃ}\\left(\\text{S}\\text{F}\\:\\text{x}\\:\\text{I}\\text{F}\\right)\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:Equation\\:1$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe coverage was determined from the regular pictures acquired throughout the year using Inkscape software for metric determination. Six timepoint were considered for analysis which reflected the more relevant changes along the immersion time. Examples of biofouling organisms found in this study are shown in Supplementary information 1, Fig. S2.\u003c/p\u003e\n\u003cp\u003eAfter the completion of the immersion period, the biofouling biomass was scrapped and separately storage according to the wood sample face (light and non-light exposed). Dry weight (55\u0026deg;C for 4 days and 105\u0026deg;C for 24 h) and ash content (450\u0026deg;C for 4 h) were determined for each sample of biofouling.\u003c/p\u003e\n\u003ch3\u003eItrax Core Scanner analysis (ED-XRF, X-ray radiography and high-resolution image)\u003c/h3\u003e\n\u003cp\u003eAfter removal of biofouling and drying, wood samples were analyzed with an Itrax Core Scanner (Cox Analytical Systems, Sweden) for sequential acquisition of high-resolution RGB image, X-ray radiography, and ED-XRF analysis. The instrument was controlled by the CoreScanner 8.6.3 software, the evaluation of XRF spectra with Q-spec 8.6.0, and post-processing and data analysis of RGB images and grey-scale radiographies with Redicore 8.6.0 software. First, a surface topography scan of high resolution was acquired and used to keep sample-detector distance constant, especially important for EDXRF. High resolution RGB images were obtained with resolution at a step size of 500 \u0026micro;m. The ED-XRF acquisition was done with a molybdenum anode with 30 kV, 50 mA, step size 1000 \u0026micro;m and acquisition time of 10 s. The X-ray radiography scan was one with 45 kV, 10 mA, step size of 1000 \u0026micro;m and acquisition time of 200 ms. Scans were acquired with a width of 2 cm and in the middle section of the piece from axial direction.\u003c/p\u003e\n\u003ch3\u003eMarine xylophage impact\u003c/h3\u003e\n\u003cp\u003eThe surface of the wood samples after biofouling removal was explored for the presence of limnoriids tunnels and images were acquired with a USB digital magnifying lens. Presence of teredo was detected by X-ray radiographies from Itrax Core Scanner. Further analyses were done with CT X-ray analysis using a CBCT i-CAT\u0026reg; (DEXIS, United States). CT X-ray images were acquired in the 3 spatial planes (sagittal, transversal and frontal) with a resolution of 0.2 mm in DICOM format. Volume loss was determined by analysis of the CT X-ray images using the software Medical Imaging Interaction Toolkit (MITK) (Dinkelacker et al., 2025). The images were processed to determine the volume of the bored holes, calcareous depositions, valves and bodies of teredos. First, each piece of wood was cropped from the whole image set. Then, an appropriate grey scale for the piece to make enough contrast of the different elements (empty space, calcium carbonate, organic biomass of teredo body, etc.) was selected. 2D segmentation was prepared using the segmentation tool \u003cem\u003eRegion growing 3D\u003c/em\u003e. This tool works defining a grey scale threshold and placing a seed point in a selected structure visualized creating selection that grows according to the grey scale threshold selected. The 3D segmentation was converted to a smoothed polygonal model for visualization, and the volume of the model was obtained. The percentage of wood lost was determined by summing up all the cavities\u0026rsquo; volumes over the total volume of the wood piece. The 3D polynomial segmentation models were exported for visualization as digital image in TIFF format.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eDetermination of copper content and retention\u003c/h2\u003e\n \u003cp\u003eED-XRF was acquired by Itrax Core Scanner from the immersed and non-immersed reference wood pieces as previously mentioned. This non-destructive technique was used for semi-quantitative comparison of copper amounts and distribution between samples. It is noteworthy that the acquisition with this technique has limited penetration, in the sample depending on different factors (sample matrix, excitation energy, detector, atomic number, etc.), which often ranges approximately from 10 to 100 \u0026micro;m.\u003c/p\u003e\n \u003cp\u003eThe total content of copper was determined by inductively coupled plasma with optical emission spectrometry (ICP-OES) analysis. This analysis was done after biofouling removal and Itrax analysis, since it requires the total combustion of the sample. Additionally, the non-immersed reference wood pieces were used to account for the initial copper content value. First, the samples were placed in a muffle furnace at 550\u0026deg;C for 4 h. The ashes were re-dissolved in a volume of 2% (w/w) nitric acid solution. The copper results were expressed as kg/m\u003csup\u003e3\u003c/sup\u003e of wood.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCopper content and retention\u003c/h2\u003e \u003cp\u003eThe copper content of the treated samples was calculated theoretically using the solution uptake results and the concentration of copper in the solution, while empirically values were obtained from ICP-OES analysis. The results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSolution uptake, theoretical and measured (ICP-OES) retention of copper, and leaching percentage for each treatment. Leaching percentage was calculated from the measured copper values of pieces before (t\u003csub\u003e0\u003c/sub\u003e) and after (t\u003csub\u003e1\u003c/sub\u003e) immersion in the sea for one year. Average density of the Scots pine wood pieces was 480 kg/m\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUptake\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTheoretical retention\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eMeasured retention\u003c/p\u003e \u003cp\u003eICP-OES\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003esolution\u003c/p\u003e \u003cp\u003e(kg/m\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCu\u003c/p\u003e \u003cp\u003e(kg/m\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCu t\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(kg/m\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCu t\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(kg/m\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeaching\u003c/p\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLAC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e555\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLAC\u0026thinsp;+\u0026thinsp;KL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e458\u0026thinsp;\u0026plusmn;\u0026thinsp;69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e547\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.868\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.143\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e82.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKL\u0026thinsp;+\u0026thinsp;CU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e477\u0026thinsp;\u0026plusmn;\u0026thinsp;51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.757\u0026thinsp;\u0026plusmn;\u0026thinsp;0.082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.243\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e489\u0026thinsp;\u0026plusmn;\u0026thinsp;43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.776\u0026thinsp;\u0026plusmn;\u0026thinsp;0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.564\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe solution uptake values were similar than in other studies treating Scots pine samples for marine use (Humar \u0026amp; Lesar, 2013). Theoretically, all the treatments with copper should have a retention of around 0.75\u0026ndash;0.86 kg/m\u003csup\u003e3\u003c/sup\u003e. However, there was a discrepancy between theoretical and measured copper retentions in the KL\u0026thinsp;+\u0026thinsp;CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU treatments. The measured copper retention in KL\u0026thinsp;+\u0026thinsp;CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU treatments was almost 50% lower than expected by the theoretical calculations. In these treatments, the presence of lignin influenced the retention of copper. A sample of each treatment was cut along the axial direction and visually observed. In the LAC\u0026thinsp;+\u0026thinsp;KL, KL\u0026thinsp;+\u0026thinsp;CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU treatments it was observed that a main accumulation of lignin in the axial edges followed the direction of floema and xylem. Lignin was concentrated mainly in the first 3 mm in the axial direction (Supplementary information 1: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Similar problems were reported in other impregnation treatments using lignin as preservative (Borrega, 2022). This might indicate that the pores were progressively clogged by lignin. The saturated lignin solution, the limited differential vacuum pressure of impregnation (\u0026asymp;\u0026thinsp;1 atm) and possible coagulation interactions of polymeric lignin and divalent copper might explain these observations. Therefore, possible effect of lignin alone remained mainly limited to the surface. Therefore, copper retention was likely affected by the presence of lignin.\u003c/p\u003e \u003cp\u003eAfter one year of immersion, all the copper containing treatments lost between 80\u0026ndash;95% of their copper content. This highlights the importance of using fixatives; for example, chromium in CCA. The LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU treatment was based on the study of Fernandez-Costas (Fern\u0026aacute;ndez-Costas et al., 2017b), which resulted in good results of preservation against wood rot fungi even after a laboratory leachability test. However, several points must be noted: 1) the primary purpose of the treatment was for land use, and the leachability test was done with soaking the pieces for a relative short period; 2) the pieces were much smaller than in this study and the relative penetration along the pieces was higher; 3) the salinity of sea water increase leachability by creating complexes with copper, e.g. chloride \u0026ndash; copper (Hingston et al., 2001; Lebow et al., 1999); 4) attack of xylophages increases the contact surface with water.\u003c/p\u003e \u003cp\u003eThe analysis of copper by means of ED-XRF by Itrax Core Scanner indicated higher content and homogeneous distribution of copper along the pieces of pre-immersed CU treatment. Whereas the pre-immersed KL\u0026thinsp;+\u0026thinsp;CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU pieces had slightly lower values of copper overall, while regarding its distribution, copper was slightly higher in the edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This aligns with the previously mentioned differences in copper retention and the fact that lignin penetration was mainly present in the first couple of millimeters in the pieces. After one year of immersion, copper values were reduced significantly, especially in KL\u0026thinsp;+\u0026thinsp;CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU treatments, although conserving the same pattern of copper distribution. These suggest that, where lignin was highly present, the copper was retained efficiently.\u003c/p\u003e \u003cp\u003eThe ED-XRF data and X-ray radiographies from all sample replicates are fully shown in Supplementary information 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering these results, several modifications of the treatment should be made in future: increase of biocidal content, lignin solution concentration and filtration, increase of vacuum-pressure conditions of impregnation, etc. Therefore, improvement of impregnation conditions should be explored in the future to avoid clogging of the wood cell lumen.\u003c/p\u003e \u003cp\u003eAdditionally, the Itrax Core Scanner has the potential to be calibrated for quantitative data (Kelloway et al., 2014), which in this case could be conducted by impregnation of the same type of wood with different copper concentrations and determined by external analysis, e.g. ICP-OES.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiofouling evolution and impact\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe evolution and impact of biofouling coverage was reflected by the antifouling efficiency (\u003cem\u003eN\u003c/em\u003e) value shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. As reminder, the higher the \u003cem\u003eN\u003c/em\u003e value, the worse antifouling performance.\u003c/p\u003e \u003cp\u003eFirstly, it is worthwhile noting that there was a sharp change in the timepoint of February-March 2017, in terms of percentages of the fouling groups and total biofouling coverage. During February 2017, an Atlantic squall reached the Galician coast generating an extreme wave and promoting red alerts in all the region\u003csup\u003e1\u003c/sup\u003e. The storms dumped enormous amounts of rain, which was evidenced by a sharp decrease in the water salinity of the Vigo estuary (Supplementary information 1: Fig. S4). The shear forces created during this event impacted the biofouling coverage, which in some cases removed part of the accumulated biofouling mass.\u003c/p\u003e \u003cp\u003eOn the other hand, the control pieces showed a faster progression of colonization and higher \u003cem\u003eN\u003c/em\u003e values than the rest of treatments, until the extreme wave event of February 2017. On the other hand, the treatments CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU had similar progression and the lowest \u003cem\u003eN\u003c/em\u003e values among all the treatments. LAC, LAC\u0026thinsp;+\u0026thinsp;KL and KL\u0026thinsp;+\u0026thinsp;CU treatments had also similar patterns of progression, but \u003cem\u003eN\u003c/em\u003e values were more like the control samples.\u003c/p\u003e \u003cp\u003eIt must be highlighted that the biofouling area coverage does not always reflect clearly the impact in terms of mass accumulation. Therefore, the interpretation of the \u003cem\u003eN\u003c/em\u003e results was complemented with the values of the biomass accumulated at the end of the immersion test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). For all treatments, the biomass accumulated was mainly from calcareous organisms (mussels, bryozoans, tube worms, etc.) as reflected by the small difference between dry weight and ash content. The treatments containing copper were the most effective, lowering the amount of accumulated biomass, following the order CU\u0026thinsp;\u0026gt;\u0026thinsp;LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU\u0026thinsp;\u0026gt;\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU. Whereas, a significant difference was found between the control and the treatments LAC\u0026thinsp;+\u0026thinsp;KL and LAC, with higher values for these last ones. This observation suggested that the impregnation process, the laccase presence, and incubation (2 h at 50\u0026deg;C) might have removed extractives (wax, resins, terpenes, etc.) that could affect the colonization pattern. Several studies have reported that laccases could also play a role in removal of lipophilic compounds, through a laccase-mediator mechanism (Guti\u0026eacute;rrez et al., 2009). Indeed, a reduction in hydrophobicity, determined by the water contact angle, was detected in these samples (Supplementary information 1, Fig. S5). Hydrophobicity plays an important role in adhesion of settlement and adhesion of algae (J. A. Callow \u0026amp; Callow, 2006; M. E. Callow \u0026amp; Callow, 2000) while opposite for mussels and barnacles (Wiegemann, 2005). However, it was not possible to determine the separate effects of laccase and incubation because no treatment included water impregnation followed by incubation alone. Additionally, the removal of certain extractives might not only affect settlement due to hydrophobicity but also potential biocidal or inhibitory effects.\u003c/p\u003e \u003cp\u003eLastly, by comparing LAC and LAC\u0026thinsp;+\u0026thinsp;KL treatments, Kraft lignin itself seemed to not have efficiency avoiding biofouling settlement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiofouling progression by organism group\u003c/h2\u003e \u003cp\u003eThe coverage of biofouling was evaluated along the marine trial time for all the samples. Generally, there were clear differences in biofouling coverage between the face of the wood samples independently from the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This was caused by differences in light exposure: one side of the wood samples faced the area beneath the floating dock shadow, while the other sides did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The total biofouling coverage was higher and reached total surface coverage earlier in the face with more light exposition. The colonization by different organism groups was also different. As expected, algae had more presence in the light exposed face, with small contributions in the non-light exposed face, mainly attributed to red algae. Light is a relevant factor for the colonization of growth of photosynthetic algae, while red algae are adapted to lower intensities of light due to their pigment adaptations (Lebret et al., 2009). Additionally, a higher coverage of algae correlated with lower colonization of non-encrusting and encrusting fouling macro-organisms as can be noted comparing light and non-light exposed faces. In biofouling colonization, there are interactions between organisms that benefits the colonization but also competition (Rao et al., 2007). In this study, it seemed that between algae and encrusting and non-encrusting fouling macro-organisms, there was some type of competitive interaction. On the other hand, non-encrusting macro-organisms had the lowest presence and coverage in all the treatments and sides. This group was represented mainly by ascidians and sponges. Colonization of ascidians is dependent on the roughness, porosity and chemistry of the materials, that needs to be favorable for the settlement of the larva settlement, which has limited natatorial abilities (Chase et al., 2016). The exposed surfaces of the wood were quite smooth, which can explain the low presence of this group in the biofouling coverage, in comparison with other materials such as concrete.\u003c/p\u003e \u003cp\u003eDifferences were also detected between samples that contained copper and those that did not. The treatments with copper slowed down the progression of biofouling coverage, especially in the non-light exposed faces. The biofouling group that was delayed the most in these treatments was the encrusting macro-organisms at all timepoints and sides. The most efficient treatment reducing the total coverage and the most impactful biofouling groups was CU, followed by LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU and KL\u0026thinsp;+\u0026thinsp;CU. On the other hand, both LAC and LAC\u0026thinsp;+\u0026thinsp;KL differed from the control samples in terms of total coverage and individual biofouling group coverage. In comparison, control samples were covered by fouling faster, especially by algae and encrusting fouling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMarine xylophage presence\u003c/h2\u003e \u003cp\u003eDuring the immersion time, the water temperature and salinity ranged from 12\u0026ndash;19\u0026deg;C and 29.3\u0026ndash;35.7 PSU, respectively (Supplementary information 1: Fig. S4). These conditions were in the range that allows correct spawn, growth, and boring and feeding activity of marine xylophages (Bersoza Hern\u0026aacute;ndez \u0026amp; Angelini, 2019; Borges et al., 2009).\u003c/p\u003e \u003cp\u003eThe CT X-ray analysis revealed the infestation of marine xylophages in the wood samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The only treatment that showed no-attack of marine xylophages in any of the replicates was the CU treatment, while the rest presented signs of attack of different types and magnitude. The KL\u0026thinsp;+\u0026thinsp;CU and LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU treatments presented attack of limnoriids but not teredos. This agrees with the reported tolerance of limnoriids to copper, which they accumulate as granules in the digestive system, and hypothetically use it for their enzymes and blood pigments (Tupper et al., 2000). On the other hand, LAC, LAC\u0026thinsp;+\u0026thinsp;KL and control presented attack of both teredos and limnoriids.\u003c/p\u003e \u003cp\u003eBased on the presence of teredo valves, most of the pieces were colonized by only one or two big teredos, and in some cases, additional young specimens starting infestation were detected in one LAC replicate and the control. Overall, the range of volume loss for the treatments was between 0 and 8.4%, with the corresponding volume loss in descending order: LAC, LAC\u0026thinsp;+\u0026thinsp;KL, control, CU\u0026thinsp;+\u0026thinsp;KL, LAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU and CU. In addition, considering the ratings from EN275 (European Committe for Standarization, 1992) to determine marine xylophage attack, the pieces containing copper could be classified as \u0026ldquo;no attack\u0026rdquo; while the rest of treatments presented slightly or moderate attack (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, it is worth noting that the EN275 standard procedure is intended for an immersion time of five years as minimum.\u003c/p\u003e \u003cp\u003eTo provide context and facilitate the comparison of our results, hereafter some studies on marine immersion of pine are introduced. For example, in the work of Romano et al. (Romano et al., 2013), samples of pine were immersed for one year in the Blanes Canyon (coast of Gerona, Spain) and an adjacent open slope at 900\u0026ndash;1500 m depth. The canyon environment was much richer in debris than the open slope resulting in ten times bigger infestation (15 individuals/dm\u003csup\u003e3\u003c/sup\u003e \u003cem\u003evs\u003c/em\u003e 200\u0026thinsp;\u0026minus;\u0026thinsp;100 individuals/dm\u003csup\u003e3\u003c/sup\u003e). In the work of Palanti et al. (Palanti et al., 2015) and Humar and Lesar (Humar \u0026amp; Lesar, 2013), Scots pine samples were immersed in the Adriatic Sea at harbors at 6 m depth, near the sediment, which after 6\u0026ndash;10 months were almost completely degraded by marine xylophages. Similarly, a work immersing different types of wood at intertidal zones in Florida estuaries for 3\u0026ndash;6 months found volume loss ranged from 0 to 8%, with higher values found when the wood was closer to the sediment (Bersoza Hern\u0026aacute;ndez \u0026amp; Angelini, 2019).\u003c/p\u003e \u003cp\u003eIn this study, the experiment was immersed in a floating dock at Atlantic Sea, at a shallow depth (0.5 m), exposed to light, separated from the sediment, and at a location not particularly rich in debris. The light and shallow position could affect colonization of limnoriids since they have negative phototaxis and limited natatory capabilities (Borges et al., 2009), but also the competition with the strong colonization of biofouling. A negative correlation has been reported between biofouling coverage and marine xylophage presence (L\u0026oacute;pez et al., 2024), in agreement with the results of this study. Finally, low levels of debris might have affected the presence of teredo, as they primarily feed filtrating debris (Paalvast \u0026amp; van der Velde, 2013), in contrast to limnoriids that rely on wood digestion (King et al., 2010).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRating values according EN275 to define attack of limnoriids and teredo in the wood pieces tested after one year of exposition.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLimnoriid rating\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTeredo rating\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOverall rating\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLAC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSlight\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLAC\u0026thinsp;+\u0026thinsp;KL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo attack\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKL\u0026thinsp;+\u0026thinsp;CU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo attack\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLAC\u0026thinsp;+\u0026thinsp;KL\u0026thinsp;+\u0026thinsp;CU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo attack\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCONTROL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSeparately, we have shown a modern approach to quantify the attack of marine xylophages by means of CT X-ray analysis. This analysis, at last in our experience, was cheaper than 2D X-ray acquisition providing not only quantitative data on volume loss but also acquisition of details such as number, morphology and size of teredinids. Additionally, the CT X-ray analysis was conducted in MITK, an open-source software that is freely available and user friendly. Therefore, this approach is superior to the established in the standard EN275 (European Committe for Standarization, 1992). For further details, the short videos of the 3D reconstructions of each treatment are included in Supplementary information 3. Thus, pictures of surface with details of limnoriids and teredo boring sites are shown in Supplementary information 2: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA wood treatment combining enzymatic polymerized Kraft lignin and copper was explored for marine use in this study. Although this treatment was previously probed to be efficient for land use against wood root fungi with reduced leachability, it was not efficient enough for marine use.\u003c/p\u003e \u003cp\u003eCopper was effective bioactive compound to reduce biofouling and marine xylophage attack, although higher concentrations and retention should be improved in the treatment. Kraft lignin by itself did not show clear effects on biofouling or marine xylophage attack prevention. In addition, although Kraft lignin impregnation was complicated, copper was retained better where its presence was high. Improvements of impregnation of lignin should be a crucial step for further development. Separately, impregnation and incubation for enzymatic polymerization removed partially some extractives (wax, resins, etc.) affecting the mass and composition of biofouling.\u003c/p\u003e \u003cp\u003eOn the other hand, this study has employed a modern and comprehensive methodology to evaluate biofouling and marine xylophage impact. The use of CT X-ray analysis proved to be superior and cheaper than the approach of EN275 by standard X-ray radiographies. The Itrax Core Scanner has demonstrated to be an excellent tool for automated multi-acquisition of X-ray radiography, ED-XRF and RGB images, perfectly fitting with the purposes of this study. Indeed, there are other models such as the Itrax Multiscanner (COX analytical systems, Sweden) made specifically for wood analysis with much higher XRF resolution, allowing even analysis of changes in minerals in the growth rings. We consider that these methods can bring inspiration and new applications in the field of wood preservative treatments.\u003c/p\u003e \u003cp\u003eFinally, this study has provided data for biofouling coverage and marine xylophage attack in Scots pine exposed in the Atlantic Sea at shallow depth (0.5 m) depth and separated from the sediment. These results might serve as reference for future work.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was funded by the Secretar\u0026iacute;a Xeral de Universidades, Xunta de Galicia, through the projects EDC431C2017-GRC and GRC-ED431C 2025/47.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: CBL, MCFC, and DM. Methodology: CBL and MCFC. Formal analysis: CBL. Visualization: CBL. Resources: DM. Writing original draft: CBL. Writing, reviewing and editing: CBL, MCFC, and DM. All authors have read and agreed on the published manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors would like to thank the CACTI (University of Vigo, Spain), especially to Jorge Millos Alfeir\u0026aacute;n, for the assistance in the acquisition of Itrax Core Scanner results and ICP-OES; the RIAIDT (University of Santiago de Compostela, Spain) for assistance in acquisition of CT X-ray data; the CIM (University of Vigo, Spain) for assistance and allowance for the placement of the immersion experiment setup; and the INTECMAR (Xunta de Galicia, Spain) for the public available oceanographic data.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll datasets generated for this study are available in the Zenodo repository upon reasonable request from the authors: https://doi.org/10.5281/zenodo.17835546\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAWPA. (2018). 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Springer Berlin Heidelberg. https://doi.org/10.1007/s00226-018-1059-1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-Costas, C., Palanti, S., Sanrom\u0026aacute;n, M. \u0026Aacute;., \u0026amp; Moldes, D. (2017a). Enzymatic grafting of kraft lignin as a wood bio-protection strategy. Part 1: Factors affecting the process. \u003cem\u003eHolzforschung\u003c/em\u003e, \u003cem\u003e71\u003c/em\u003e(9), 681\u0026ndash;687. https://doi.org/10.1515/hf-2016-0109\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-Costas, C., Palanti, S., Sanrom\u0026aacute;n, M. \u0026Aacute;., \u0026amp; Moldes, D. (2017b). Enzymatic grafting of kraft lignin as a wood bio-protection strategy. Part 2: Effectiveness against wood destroying basidiomycetes. 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Enzymatic delignification of plant cell wall: from nature to mill. \u003cem\u003eCurrent Opinion in Biotechnology\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(3), 348\u0026ndash;357. https://doi.org/10.1016/j.copbio.2009.05.002\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNasir, M., Hashim, R., Sulaiman, O., Nordin, N. A., Lamaming, J., \u0026amp; Asim, M. (2015). Laccase, an Emerging Tool to Fabricate Green Composites: A Review. \u003cem\u003eBioResources\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(10), 6262\u0026ndash;6284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson, D. L. (2016). The Ravages of Teredo: The Rise and Fall of Shipworm in US History, 1860\u0026ndash;1940. In \u003cem\u003eEnvironmental History\u003c/em\u003e (Vol. 21, Issue 1, pp. 100\u0026ndash;124). Oxford University Press. https://doi.org/10.1093/envhis/emv118\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaalvast, P., \u0026amp; van der Velde, G. (2013). What is the main food source of the shipworm (Teredo navalis)? A stable isotope approach. \u003cem\u003eJournal of Sea Research\u003c/em\u003e, \u003cem\u003e80\u003c/em\u003e, 58\u0026ndash;60. https://doi.org/10.1016/j.seares.2013.03.003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalanti, S., Feci, E., \u0026amp; Anichini, M. (2015). Comparison between four tropical wood species for their resistance to marine borers (Teredo spp and Limnoria spp) in the Strait of Messina. \u003cem\u003eInternational Biodeterioration and Biodegradation\u003c/em\u003e, \u003cem\u003e104\u003c/em\u003e, 472\u0026ndash;476. https://doi.org/10.1016/j.ibiod.2015.07.013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao, D., Webb, J. S., Holmstr\u0026ouml;m, C., Case, R., Low, A., Steinberg, P., \u0026amp; Kjelleberg, S. (2007). Low densities of epiphytic bacteria from the marine alga Ulva australis inhibit settlement of fouling organisms. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e, \u003cem\u003e73\u003c/em\u003e(24), 7844\u0026ndash;7852. https://doi.org/10.1128/AEM.01543-07\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomano, C., Voight, J. R., Company, J. B., Plyuscheva, M., \u0026amp; Martin, D. (2013). Submarine canyons as the preferred habitat for wood-boring species of Xylophaga (Mollusca, Bivalvia). \u003cem\u003eProgress in Oceanography\u003c/em\u003e, \u003cem\u003e118\u003c/em\u003e, 175\u0026ndash;187. https://doi.org/10.1016/j.pocean.2013.07.028\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider, W. D. H., Bolan\u0026otilde; Losada, C., Moldes, D., Fontana, R. C., De Siqueira, F. G., Prieto, A., Mart\u0026iacute;nez, M. J., Mart\u0026iacute;nez, \u0026Aacute;. T., Dillon, A. J. P., \u0026amp; Camassola, M. (2019). A sustainable approach of enzymatic grafting on eucalyptus globulus wood by laccase from the newly isolated white-rot basidiomycete marasmiellus palmivorus VE111. \u003cem\u003eACS Sustainable Chemistry and Engineering\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(15), 13418\u0026ndash;13424. https://doi.org/10.1021/acssuschemeng.9b02770\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz, M. P., Bendick, J. a, Holm, E. R., \u0026amp; Hertel, W. M. (2011). Economic impact of biofouling on a naval surface ship. \u003cem\u003eBiofouling\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(1), 87\u0026ndash;98. https://doi.org/10.1080/08927014.2010.542809\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSciban, M., \u0026amp; Klasnja, M. (2004). Study of the Adsorption of Copper (II) Ions from Water onto Wood Sawdust, Pulp and Lignin. \u003cem\u003eAdsorption Science \u0026amp; Technology\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(3), 195\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŞen, S., Sivrikaya, H., \u0026amp; Yal\u0026ccedil;in, M. (2019). Natural durability of heartwoods from European and tropical Africa trees exposed to marine conditions. \u003cem\u003eAfrican Journal of Wood Science and Forestry\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(1), 1\u0026ndash;008. www.internationalscholarsjournals.org\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTupper, B. C., Pitman, A. J., \u0026amp; Cragg, S. M. (2000). Copper accumulation in the digestive caecae of Limnoria quadripunctata Holthius (Isopoda: Crustacea) tunnelling CCA-treated wood in laboratory cultures. \u003cem\u003eHolzforschung\u003c/em\u003e, \u003cem\u003e54\u003c/em\u003e, 570\u0026ndash;576.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiegemann, M. (2005). Adhesion in blue mussels (Mytilus edulis) and barnacles (genus Balanus): Mechanisms and technical applications. In \u003cem\u003eAquatic Sciences\u003c/em\u003e (Vol. 67, Issue 2, pp. 166\u0026ndash;176). https://doi.org/10.1007/s00027-005-0758-5\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Footnotes","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.laopinioncoruna.es/galicia/2017/02/01/entrado-primer-temporal-galicia-24336617.html\u003c/span\u003e\u003cspan address=\"https://www.laopinioncoruna.es/galicia/2017/02/01/entrado-primer-temporal-galicia-24336617.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"wood-science-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wsat","sideBox":"Learn more about [Wood Science and Technology](http://link.springer.com/journal/226)","snPcode":"226","submissionUrl":"https://submission.nature.com/new-submission/226/3","title":"Wood Science and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Marine xylophages, biofouling, wood preservatives, Itrax Core Scanner.","lastPublishedDoi":"10.21203/rs.3.rs-8347225/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8347225/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the marine environment, the durability of wood is heavily compromised by harsh conditions and the presence of marine xylophages. Biocide treatments are often employed to prolong the lifetime of wood, however, its leaching into the environment motivates the development of alternative treatments due to environmental concerns. In this study, Scots pine samples were treated with a solution based on copper and polymerized Kraft lignin via laccase enzyme was tested. In this treatment, Kraft lignin was intended to work as adsorbent of copper while laccase-assisted polymerization was intended for fixation to the wood. To discern the contribution of each component, a set of samples were prepared with different combinations of the three components (presence/absence) and compared to untreated controls. The wood samples were immersed for one year in Vigo estuary (Spain). Biofouling coverage was monitored along the immersion time and the final mass accumulated was measured. Copper retention and distribution were studied by ICP-OES and XRF, respectively. Marine xylophage attack was evaluated quantitatively using X-ray computerized tomography analysis.\u003c/p\u003e \u003cp\u003eThe treatments containing copper were less affected by biofouling and marine xylophages. Treatments containing lignin limited the impregnation depth and consequently the copper content was lower than the samples with just copper. However, XRF analysis showed that in the parts where Kraft lignin was highly present, copper was better retained than parts that not. Unfortunately, due to the previously indicated impregnation limitation, the role of laccase in lignin polymerization could not be assessed properly.\u003c/p\u003e \u003cp\u003eAdditionally, in this study a set of novel technologies for the study of wood for marine use was used. These techniques included the use of X-ray CT and the Itrax Core Scanner, a device that allows the acquisition of high-quality image, energy dispersive X-ray fluorescence, and X-ray radiography.\u003c/p\u003e","manuscriptTitle":"Copper-lignin with laccase-mediated polymerization fixation for wood preservation: determination of durability in marine environment using novel technologies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 14:18:48","doi":"10.21203/rs.3.rs-8347225/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T14:48:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-19T21:51:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T19:53:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83899005100244448862271703147676453962","date":"2026-03-12T18:02:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-15T08:17:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285366937897328393522658696174644149059","date":"2026-01-26T17:54:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169869344059877531204043857709928482364","date":"2026-01-26T16:23:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-26T14:48:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-26T14:47:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-13T11:44:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Wood Science and Technology","date":"2025-12-12T15:24:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"wood-science-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wsat","sideBox":"Learn more about [Wood Science and Technology](http://link.springer.com/journal/226)","snPcode":"226","submissionUrl":"https://submission.nature.com/new-submission/226/3","title":"Wood Science and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"591188f8-f1c1-49a3-b948-8752578a6ec3","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T13:45:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 14:18:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8347225","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8347225","identity":"rs-8347225","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}