Copper-treated Wood Research Articles

Revealing the structural and chemical properties of copper-based nanoparticles released from copper treated wood.

Copper-based preservatives consisting of micronized and nanoscale copper particles have been widely used in applications for wood protection. The widespread use of these preservatives along with the potential release of copper-containing nanoparticles (Cu NPs) during the life cycle of treated wood, has raised concerns over the impacts on the environment and occupational exposure. Along with assessing the potential hazards of these materials, a critical step is determining the chemical and morphological characteristics of the copper species released from copper-treated wood. Therefore, a combination of scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS) was utilized to characterize and differentiate the released copper-containing particles based on their structures, sizing, and chemical properties. Airborne wood dust samples were collected during the abrasion and sawing of micronized copper (MC) treated wood in a laboratory testing system. Based on the signature Cu L2,3 edge of EEL spectra, three different copper species (i.e., basic copper carbonate, copper, and copper–wood complex) were identified as major components of the embedded particles in wood dust. In addition, two types of individual Cu NPs consisting of basic copper carbonate and copper were identified. The variation of morphologies and chemical properties of copper-containing particles indicates the importance of copper–wood interactions to determine the formation and distribution of copper species in wood components. Our findings will advance the fundamental understanding of their released forms, potential transformation, and environmental fate during the life cycle.

Gene Expression Analysis of Three Putative Copper-Transporting ATPases in Copper-Tolerant Fibroporia radiculosa.

Copper tolerance of brown-rot basidiomycete decay fungi can lessen the efficacy of copper-containing wood preservatives for wood products in-service. The purpose of this study was to evaluate wood mass loss and differential expression of three genes that have putative annotations for copper-transporting ATPase pumps (FIBRA_00974, FIBRA_04716, and FIBRA_01430). Untreated southern pine (SP) and SP treated with three concentrations of ammoniacal copper citrate (CC, 0.6, 1.2, and 2.4%) were exposed to two copper-tolerant Fibroporia radiculosa isolates (FP-90848-T and L-9414-SP) and copper-sensitive Gloeophyllum trabeum isolate (MAD 617) in a 4-week-long standard decay test (AWPA E10-19). Decay of copper-treated wood was inhibited by G. trabeum (p = 0.001); however, there was no inhibition of decay with increasing copper concentrations by both F. radiculosa isolates. Initially, G. trabeum and one F. radiculosa isolate (L-9414-SP) highly upregulated FIBRA_00974 and FIBRA_04716 on copper-treated wood at week 1 (p = 0.005), but subsequent expression was either not detected or was similar to expression on untreated wood (p = 0.471). The other F. radiculosa isolate (FP-90848-T) downregulated FIBRA_00974 (p = 0.301) and FIBRA_04716 (p = 0.004) on copper-treated wood. FIBRA_01430 expression by G. trabeum was not detected, but was upregulated by both F. radiculosa FP-90848-T (p = 0.481) and L-9414-SP (p = 0.392). Results from this study suggest that all three test fungi utilized different mechanisms when decaying copper-treated wood. Additionally, results from this study do not provide support for the involvement of these putative gene annotations for copper-transporting ATPase pumps in the mechanism of copper-tolerance.

Differential Expression of a Putative Copper Homeostasis CutC Gene in Fibroporia radiculosa During Initial Decay of Copper-Treated Wood

Abstract Living organisms require copper for several cellular processes. Yet intracellular concentrations of copper must be regulated to avoid toxicity. Not much is known about mechanisms of copper regulation in wood decay fungi. However, one putative annotation for a copper homeostasis CutC gene (FIBRA_00129), found in other brown-rot wood decay fungi, has been annotated in Fibroporia radiculosa. The aim of this study was to evaluate wood mass loss and differential expression of FIBRA_00129 during initial decay of untreated and copper-treated wood by two copper-tolerant F. radiculosa isolates (FP-90848-T and L-9414-SP) compared with copper-sensitive Gloeophyllum trabeum. Untreated southern pine (Pinus spp.) and ammoniacal copper citrate treated southern pine at three concentrations (0.6%, 1.2%, and 2.4%) were used in a 4-week-long standard decay test. Results showed G. trabeum was unable to decay copper-treated wood while both F. radiculosa isolates successfully decayed southern pine at all copper concentrations. G. trabeum and F. radiculosa L-9414-SP showed no detectable FIBRA_00129 expression over the course of this study. F. radiculosa FP-90848-T showed greater FIBRA_00129 downregulation on copper-treated wood than on untreated wood (P = 0.003). Additionally, there was greater FIBRA_00129 downregulation in F. radiculosa FP-90848-T at week 3 compared with other weeks (P = 0.015). Future studies are needed to further evaluate FIBRA_00129 during the decay process to determine its potential role in copper-tolerance.

Method for characterizing extracellular proteins from the cell wall proteome of the copper tolerant fungus Phialophora malorum

Phialophora malorum is a well-known copper tolerant fungus causing soft rot decay of copper-treated wood in-service in ground contact situations worldwide. The aim of the present work was to develop a method that allowed studies of the mechanism(s) behind extracellular copper tolerance by examining the profile of proteins expressed by P. malorum in the cell wall/slime (subproteome) environment in mycelia grown in Cu-supplemented media. The study involves development of a novel non-destructive approach using mild extraction, concentration, separation (SDS-PAGE electrophoresis) and characterization (MALDI-TOF MS/MS) of the proteins present. Studies revealed differential protein profiles and expression in Cu-supplemented (0.1% CuSO4) vs control shake cultures after 10 days growth. Twelve proteins including several hydrolytic (i.e. glucosidases, pectinase, polygalacturonase) enzymes were either unique or over-expressed in Cu-cultures compared to controls. The cell wall location of the proteins was consistent with a role in copper tolerance and detoxification by binding Cu-ions extracellularly. Several of the proteins (e.g. hydrolases, pectinase) are also involved in wood cell wall biomineralization. It is proposed that the concentration of metabolites (organic acids) slowly released in close vicinity of hyphal surfaces by weakly bound enzymes over-expressed through Cu-stress may function to reduce levels of free non-chelated Cu (II) ions entering the cell cytoplasm and therefore provide a first level of defense against copper toxicity.

Micronized copper-treated wood: copper remobilization into spores from the copper-tolerant wood-destroying fungus Rhodonia placenta

Life cycle of Rhodonia placenta showing the uptake and speciation of copper in basidiospores.

Assessment of the bioaccessibility of micronized copper wood in synthetic stomach fluid

The widespread use of copper in treated lumber may result in a potential for human exposure. Due to a lack of information concerning the release of copper from treated wood particles following oral ingestion, the in vitro bioaccessibility of copper from copper-treated wood dust in synthetic stomach fluid (SSF) and DI water was investigated. Copper-containing particles ranging in size from nano-scale to micron-scale were observed by transmission electron microscopy (TEM) in thin sections of these micronized copper-treated wood products. Three copper-treated wood products (liquid alkali copper quaternary and two micronized copper quarternary products) from different manufacturers were incubated in the extraction media. The released copper was then fractionated by centrifugation and filtration through 0.45μm and 10kDa filters, respectively. Soluble copper released into isolated fractions was measured using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Total copper from each wood product was also determined using microwave-assisted acid digestion of dried wood samples and ICP-OES. The bioaccessible copper released into SSF was between 83 and 90% for all wood types. However, the percent of copper released in DI water was between 14 and 25% for all wood products. These data suggest that copper is highly bioaccessible at the low pH values present in the stomach and may pose a potential exposure risk upon ingestion.

Molecular identification and phylogenic analysis by sequencing the rDNA of copper-tolerant soft-rot Phialophora spp.

The genus Phialophora includes some of the most copper-tolerant and frequently isolated fungi causing soft rot of copper-treated wood in service. The aim of this work was to conduct a phylogenic analysis of different Phialophora species and strains isolated from treated wood, evaluate strain compatibility, and examine their copper-tolerance variability under liquid- and solid-culture conditions. Phylogenic analysis of Phialophora malorum (four strains), Phialophora mutabilis (two strains), Lecythophora mutabilis (one strain), and Phialophora sp. A. (three strains) originating from different parts of the world showed clustering into three major clades. Phialophora sp. A. refers to a number of fungal isolates identified as Phialophora spp. previously isolated by our laboratory from preservative-treated stakes and poles in Sweden. P. mutabilis strains fell into a single cluster together with Phialophora lignicola and Phialophora hoffmanii; P. malorum strains clustered in their own group (93% bootstrap value) and Phialophora sp. A. clustered together with Phialophora botulispora. Compatibility studies with the different Phialophora species/strains showed discriminative behavior. All Phialophora species showed mutual inhibition (incompatibility), as did strains of P. malorum and P. mutabilis. Phialophora sp. A. strains showed mutual intermingling but incompatibility with P. botulispora. Species/strain variability regarding copper tolerance was evaluated using in-vitro liquid and solid media containing different CuSO4 concentrations. P. malorum 211-C-15-1 showed the highest copper-tolerance growing on 6.4% CuSO4 supplemented agar and 3.2% CuSO4 in liquid cultures, followed by P. malorum ATCC 66716 (3.2% w/v CuSO4-agar). P. mutabilis strains and L. mutabilis showed growth on 0.64% CuSO4 incorporated in liquid and agar cultures and Phialophora sp. A. (25M3) at 0.32% w/v CuSO4 on agar. The results emphasize that copper tolerance varies greatly within and between species of Phialophora.

Quantification of mobilized copper(II) levels in micronized copper-treated wood by electron paramagnetic resonance (EPR) spectroscopy

Abstract Sapwood sawdust from southern pine was treated with micronized copper (MC) under various conditions and the mobilized copper(II) (Cumob) concentrations were determined in the treated wood by electron paramagnetic resonance (EPR) spectroscopy. The spectral parameters for the copper sulfate (CuSO4)-treated sapwood and those of the MC-treated sapwood were very similar. A linear correlation was found between the intensities of copper (Cu) EPR spectra and those of Cu energy-dispersive X-ray fluorescence spectroscopy in a series of CuSO4-treated sapwood reference samples. Thus, the EPR signal intensities could be reliably correlated to the mass of reacted Cu present using this calibration curve. The amount of the Cumob in sawdust treated by MC suspensions increased during the first 2–3 days after the initial treatment and then reached a maximum during the 7-day monitoring period. In the case of the treatment with MC alone or MC azole, an increased MC concentration led to an elevated amount of Cu (to a maximum of ∼0.23% Cu) solubilized by the sapwood. If the wood was treated with MC quat, the Cumob initially increased, but at higher concentrations the Cumob content decreased, due to the interference by the quat cobiocide on the acid reaction between the wood and the basic Cu carbonate. An examination of commercially-treated wood confirmed the laboratory observations.

Choice tests and neighbor effects during fungal brown rot of copper- and non-treated wood

In previous work, a ‘choice’ test was used to test wood durability in mixtures of wood types. Results demonstrated that a filamentous fungus (Gloeophyllum trabeum) made a ‘choice,’ degrading white pine significantly less when less-durable woods were readily available. For this experiment, we substituted white pine with copper-treated pine and used three fungi (G. trabeum, Serpula himantioides, and Wolfiporia cocos) representing a range of copper tolerances. Southern pine blocks treated commercially with micronized copper azole or treated in-house using copper ethanolamine were used as treated wood substrates. Trials were soil-block tests, using two-block combinations to include non-treated, non-durable pine or as single-block treatments. After 12 weeks of decay, copper tolerance by S. himantioides and W. cocos was evident while G. trabeum was not copper tolerant. Availability of non-durable pine blocks, however, had no effect on decay in copper-treated wood. This contrasts with our past results using white pine. The reason for the discrepancy may relate to fungal mechanisms of copper tolerance (e.g., constitutive oxalic acid secretion) which may differ from mechanisms to overcome extractives in white pine. More fungal research using choice trials to assess these ‘neighbor effects’ is logical, given increased use of mixed, composite, and hybrid materials.

Bioavailability and form of copper in wood treated with copper-based preservative

The biological performance of copper-treated wood is related to the amount and form of copper available in treated wood. The amount of copper biologically available in wood treated with copper-based wood preservatives such as micronized copper azole, micronized copper quat, alkaline copper quat, copper chrome arsenic and copper azole was estimated by using non-sequential chemical extraction method. DI water, KNO3, KF, Na4P2O7, EDTA disodium salt and HNO3 solutions were used to extract copper from treated wood in an ultrasonic bath. The extracts and water washings were collected, centrifuged and analyzed to determine the amount of copper. High amount of copper was extracted with EDTA disodium salt and HNO3 solutions followed by sodium pyrophosphate, while that extracted with KNO3, KF and DI water was low. EDTA disodium salt and sodium pyrophosphate are known as good complexing/chelating agents, capable of chemically reacting with copper in treated wood, to form water-soluble copper salts. HNO3 was capable of extracting almost 100% of copper from copper-treated wood because of its low pH capable of solubilizing solid, unfixed and fixed copper in wood. KNO3 reacted with exchangeable form of copper, while KF is responsible for the extraction of physically adsorbed copper. DI water mostly removed soluble form of copper from treated wood. The amount of copper bioavailability from copper-treated wood can be estimated by the amount of copper extracted using DI water, KNO3 and KF solutions under given environmental conditions. The amount of bioavailable copper extracted from micronized copper–treated wood using DI water, KNO3 and KF solutions, although found to be low as compared with that extracted from ACQ, CCA and CA wood, was sufficient to prevent the growth of wood-destroying fungi.

Assessing the relative bioavailability of copper to fungi degrading treated wood

Copper is ubiquitous as a biocide component in wood preservatives. Some fungi detoxify copper by immobilizing copper ions with oxalate, decreasing its physiological availability (bioavailability). Decreases in copper bioavailability may also occur during wood treatment. To date, however, copper retention in wood has been measured as overall weight-to-volume concentration without an estimate of its bioavailability and without assessment of its relative contribution to preservative efficacy. Here, we gauge the bioavailability of copper ions in treated wood by using oxalate to pre-treat wood prior to colonization by a moderately copper-tolerant fungus. Copper-treated wood was treated with a gradient of sodium oxalate concentrations, rinsed thoroughly, and exposed in soil-block jars to an isolate of Serpula himantioides. Wood treated with copper ethanolamine was extremely effective in preventing decay by S. himantioides, but toxicity could repeatedly be overcome above a threshold level of oxalate pretreatment. In agar plates, copper-treated wood stimulated oxalate production by S. himantioides, but levels were less than those needed (>10 mM) to overcome copper in soil-block jars. This capacity to overcome copper using an oxalate pretreatment was absent in commercially available samples treated with co-biocide(s). Results demonstrate a useful relative measure of copper bioavailability, with potential to be modified for specific quantification.

Effects of copper amine treatment on mechanical properties of PVC/wood‐flour composites

AbstractCopper amine–treated wood flour was added to PVC [poly(vinyl chloride)] matrix in order to manufacture PVC/wood‐flour composites. Effects of copper treatments on the mechanical properties of PVC‐wood composites were evaluated. Unnotched impact strength, flexural strength, and flexural toughness of the composites were significantly improved by the wood‐flour copper treatment. The optimum copper concentration range was 0.2 to 0.6 wt% of wood flour. Fractured surfaces were examined by using scanning electron microscopy (SEM) combined with energy‐dispersive spectroscopy (EDS). PVC/wood interfacial debonding was the main fracture mode of untreated wood‐flour composites, whereas wood‐particle pullout and breakage dominating the fractured surfaces of copper‐treated wood‐flour composites. On the fractured surfaces, more PVC could be found on the exposed copper‐treated wood particles than on untreated wood, a result suggesting improved PVC‐wood interfacial adhesion after copper treatments. J. Vinyl Addit. Technol. 10:70–78, 2004. © 2004 Society of Plastics Engineers.

Correlation between oxalic acid production and copper tolerance in Wolfiporia cocos

The increased interest in copper-based wood preservatives has hastened the need for understanding why some fungi are able to attack copper-treated wood. Due in part to accumulation of oxalic acid by brown-rot fungi and visualization of copper oxalate crystals in wood decayed by known copper-tolerant decay fungi, oxalic acid has been implicated in copper tolerance by the formation of copper oxalate crystals. Nineteen isolates of the brown-rot fungus Wolfiporia cocos were evaluated for oxalic acid production and weight loss on wood treated with 1.2% copper citrate. Twelve of 19 isolates that caused moderate to high weight losses in copper citrate-treated wood produced low oxalic acid in liquid culture, whereas isolates with high oxalic acid production had low weight losses in treated wood. Seven W. cocos isolates demonstrated enhanced weight loss in Cu-treated wood. Wood weight loss was unaffected by the presence of copper citrate for two W. cocos isolates and weight loss was lower for 10 isolates compared to weight losses in untreated wood. Citrate did not significantly influence oxalic acid production in liquid culture. Previous hypotheses linking oxalic acid and copper tolerance were based upon observations of single isolates of Postia and Tyromyces. Although most isolates produced more oxalic acid in copper citrate-treated wood than in untreated wood, we found no statistical relationship between the amount of oxalic acid production in liquid culture or wood and copper tolerance in W. cocos. Production of oxalic acid does not seem to be the factor controlling copper tolerance in W. cocos. The diversity seen within W. cocos demonstrates that caution should be used when reporting results, so that generalizations are not based on the behavior of a single isolate.