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Research Collection
Doctoral Thesis
Assessing the effectiveness and environmental risk ofnanocopper-based wood preservatives
Author(s): Civardi, Chiara
Publication Date: 2016
Permanent Link: https://doi.org/10.3929/ethz-a-010811851
Rights / License: In Copyright - Non-Commercial Use Permitted
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DISS ETH NO. 23783
ASSESSING THE EFFECTIVENESS AND ENVIRONMENTAL RISK OF NANOCOPPER-
BASED WOOD PRESERVATIVES
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
Presented by
Chiara Civardi
MSc, University of Southampton
Born on 7th March 1988
Citizen of
Italy
Accepted on the recommendation of
Prof. Dr. Ingo Burgert, ETH Zurich, examiner
Prof. Dr. Francis W.M.R. Schwarze, Empa St. Gallen, co-examiner
Dr. Peter Wick, Empa St. Gallen, co-examiner
Prof. Dr. Siegfried Fink, University of Freiburg, co-examiner
2016
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To Aunt Gina and Tenia
Because I miss you so much
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Abstract Nanotechnology is revolutionizing our lives, from consumer products to medicine, electronics,
and building materials. In fact, nanoparticles are recognized to have properties that differ from their bulk
counterparts. Despite the potentials of nanoparticles, their innovative properties may also cause specific
nano risks. These occur under two circumstances: if nanoparticles are hazardous, and if humans and the
environment are exposed to them.
In wood protection, nanomaterials have recently been developed to outperform conventional
wood preservatives and lengthen the service life of timber structures. These nanoparticles-based wood
preservatives are best known as “micronized copper”, which are composed of basic copper carbonate
nanoparticles and a second organic biocide.
Copper-based nanoparticles are frequently associated with adverse health effects, however the
environmental fate of micronized copper -which is already present in the market- is not clear yet. In this
thesis, we investigate whether the use of nanoparticles in micronized copper used for wood protection has
an added value, and we monitor, quantify, and visualize the fate of copper from its wood preservative
formulation stage, to the impregnation of wood, and ageing of the structures. The majority of the studies
on wood preservatives remobilization in the environment deal with the release of copper in water and soil.
Here, we mostly focus on the air compartment. In fact, lungs are considered as the main entry portal for
nanoparticles and inhalation of copper-based nanoparticles from wood preservatives may cause adverse
health effects. In particular, two possible release pathways were assessed: wood dust produced by
mechanical abrasion of treated wood surfaces, and release via spores of copper-tolerant wood-destroying
basidiomycete fungi that colonize treated wood. In addition, preliminary in vitro cytotoxicity tests were
conducted on lung epithelial cells and macrophages, representing a simplified human lung, to assess the
potential acute hazard of wood dust from micronized copper-treated wood, and compare it with wood dust
generated from wood treated with a conventional wood preservative.
On the basis of the experimental results, micronized copper effectively protected the wood from
soilborne fungi and wood-destroying basidiomycetes, and homogeneously penetrated into easily treatable
wood species. However, the treatment is not effective against copper-tolerant wood-destroying fungi, and
refractory wood species are only poorly treated. Copper can be released in the air both via wood dust and
spores, but not in a nanoparticulate form. Therefore, micronized copper does not pose a specific nano risk.
Furthermore, the preliminary cytotoxicity assessment did not reveal a specific nano hazard. Further, the
possibility of adverse health effects related to copper is likely only a risk for woodworkers, which are
continuously exposed to wood dust.
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Sommario Le nanotecnologie stanno rivoluzionando le nostre vite, dai prodotti di consumo a medicina,
elettronica e materiali da costruzione. Infatti, le nanoparticelle hanno proprietà che differiscono rispetto ai
loro corrispettivi macroscopici. Nonostante le potenzialità delle nanoparticelle, le loro proprietà innovative
possono anche causare specifici nano rischi. Questi possono verificarsi qualora le nanoparticelle fossero
tossiche e l’uomo o l’ambiente fossero esposti a queste
Nel campo della protezione del legno, nanomateriali sono stati recentemente sviluppati al fine di
migliorare i preservanti convenzionali presenti e garantire una maggior durata delle strutture lignee.
Questi preservanti a base di nanoparticelle sono meglio conosciuti come “rame micronizzato” e sono
composti da particelle di carbonato di rame associate ad un secondo biocida organico.
Le nanoparticelle a base di rame sono spesso associate a esiti clinici avversi, tuttavia il destino
ambientale del rame micronizzato -già presente nel mercato- non è ancora chiaro. In questa tesi viene
valutato se l’uso di nanoparticelle contenute nel rame micronizzato ha un valore aggiunto al fine di
proteggere il legno e viene monitorata, quantificata e visualizzata la sorte del rame a partire dallo stadio di
formulazione come preservante, all’impregnazione del materiale ligneo, fino all’invecchiamento del
materiale. La maggior parte degli studi sulla remobilizzazione dei preservanti nell’ambiente considera
solamente l’emissione di rame nell’acqua e nel suolo. Qui viene considerato principalmente il rilascio
nell’aria. Infatti i polmoni sono considerati come il principale portale per l’ingresso di nanoparticelle e
l’inalazione di nanoparticelle a base di rame a causa dell’uso di rame micronizzato puó avere conseguenze
negative sulla salute. In particolare, due possibili scenari per l’emissione di nanoparticelle sono stati
considerati: tramite polvere di legno generata dall’abrasione meccanica di superfici lignee trattate e
tramite spore di basidiomiceti rame-tolleranti in grado di colonizzare il legno trattato. In aggiunta, studi
citotossicologici preliminari sono stati condotti in vitro su cellule epiteliali e macrofagi, al fine di
riprodurre in maniera simplificata il polmone umano e valutare la potenziale tossicità acuta della polvere
di legno generata da legno trattato con rame micronizzato e compararla con quella proveniente da legno
trattato con preservanti convenzionali.
Sulla base dei dati sperimentali, il rame micronizzato protegge in maniera efficace da carie soffici
e basidiomiceti, inoltre penetra in modo omogeneo in legni facilmente trattatabili. Tuttavia, il trattamento
non risulta efficace contro le carie del legno rame-tolleranti. Pergiunta, specie legnose refrattarie non
risultano facilmente trattabili. Il rame puó essere remobilizzato nell’aria tramite polvere del legno e spore,
ma non in forma nanoparticellare. Di conseguenza, il rame micronizzato non pone un nano rischio
specifico. Inoltre, è probabie che gli effetti negativi sulla salute si possano verificare solamente in soggetti
esposti in maniera prolungata alla polvere di legno, come falegnami.
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Acknowledgments This work was financially supported by the Swiss National Research Foundation (Grant No.
406440_141618) and the Transnational Access to Research Infrastructures activity in the 7th Framework
Program of the EC under the Trees4Future project (Trees4Future, 284181).
It would not have been possible to complete this project without the help of a number of people,
to whom I am grateful. First of all, I would like to thank my supervisors and examiners from ETH, Empa,
and Freiburg University: Prof. Dr. Ingo Burgert, Prof. Dr. Francis Schwarze, Dr. Peter Wick, and Prof. Dr.
Siegfried Fink, as well as my former supervisor Prof. Dr. Ottmar Holdenrieder. Their expertise, support
and trust were fundamental.
More in general, I would like to express my sincere thanks to all the (past and current) members
of the Wood Materials Science, Applied Wood Materials and Particles-Biology Interactions Groups. A
special mention goes to Dr. Mark Schubert, Javier Ribera Regal, Andrè Hach, Maribel Butron, Mirjam
Jordi, Dr. Cordula Hirsch, Dr. Jean-Pierre Kaiser, Liliane Diener, Joel Benz, and Prof. Dr. Bernd Nowack.
I am also thankful to Dr. Lukas Schlagenhauf, former PhD student in the Analytical Chemistry and
Functional Polymers Empa Groups, for the fruitful collaboration on the abrasion of treated wood. In
addition, I am indebted to Yeon Bahk, Elisabeth Michel, Adrian Wichser, and He Xu.
In addition, I am grateful to Prof. Dr. Joris Van Acker, Dr. Jan Van den Bulcke and the whole
Woodlab team from the Ghent University. They warmly welcomed me and their guidance was extremely
appreciated.
Further mention goes to Dr. Daniel Grolimund and the microXAS staff, as well as to Prof. Dr.
Maarten Nachtegaal, the SuperXAS and Cook’n’Look staff and attendants from ETH the Paul Scherrer
Institute.
Prof. Dr. Claudio Mucchino from the Chemistry Department at the Università degli Studi di
Parma, and David Kistler from Eawag also deserve to be thanked for the ICP-OES and ICP-MS analysis
performed.
All in all, I owe a lot to all the people I met and got to know during these three years, from Empa,
ETH, Ghent, Dortmund, NRP 64 (plus 62 and 66), the IRG meetings, the SUN and ECONANOSORB
communities, WCS Brazil/Pantanal/Cerrado, Telejob, United Academics, the Sundari Yoga Teacher
Training, the Zurich Wednesday Evening Writing group, Capoeira Angola Palmares and casa Cotignina.
I am indebted to my parents and my relatives, which always want the best for me and spur me to
do better. Finally, a big thank is due to Michele and Tenia, which shared with me the highs and lows and
kept me strong with their love.
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Table of contents
List of Abbreviations .................................................................................................................................... ix
1. Introduction ............................................................................................................................................. 11
1.1 Research question .............................................................................................................................. 11
1.2 Thesis overview ................................................................................................................................. 14
1.3 Publications and author contributions ............................................................................................... 14
1.4 References of Chapter 1 .................................................................................................................... 15
2. Background & literature review .............................................................................................................. 20
2.1 Wood ................................................................................................................................................. 20
2.2 Wood-decay fungi ............................................................................................................................. 23
2.3 Wood preservatives ........................................................................................................................... 27
2.4 Release of Cu from wood into the environment ................................................................................ 29
2.5 References of Chapters 2.1-2.4 ......................................................................................................... 30
2.6 Spore compartmentalization of Cu .................................................................................................... 37
3. Materials & methods ............................................................................................................................... 54
3.1 Wood and wood preservatives........................................................................................................... 54
3.1.1 Wood species .............................................................................................................................. 54
3.1.2 Micronized copper ...................................................................................................................... 54
3.2 Wood preservative effectiveness ....................................................................................................... 54
3.2.1 Wood impregnation .................................................................................................................... 55
3.2.2 Effectiveness against soft rot fungi-ENV 807 ............................................................................ 55
3.2.3 Effectiveness against wood-destroying basidiomycetes-EN 113 ............................................... 55
3.3 Mechanical abrasion .......................................................................................................................... 56
3.4 X-ray techniques ................................................................................................................................ 56
3.4.1 X-ray computed tomography ...................................................................................................... 57
3.4.2 X-ray absorption spectroscopy ................................................................................................... 57
3.5 Ion-coupled plasma-mass spectrometry and optical emission spectrometry ..................................... 58
3.6 Acute toxicity for human lungs ......................................................................................................... 59
3.7 References of Chapter 3 .................................................................................................................... 59
4. Results & discussion ............................................................................................................................... 62
4.1 Wood preservative effectiveness ....................................................................................................... 62
4.1.1 Micronized copper wood preservatives: efficacy of ion, nano, and bulk copper against the
brown rot fungus Rhodonia placenta .................................................................................................. 62
4.1.2 Effectiveness of micronized copper azole against soft rot fungi ................................................ 84
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4.2 Penetration and distribution of Cu in wood ....................................................................................... 92
4.3 Mechanical abrasion of treated wood .............................................................................................. 110
4.4 Spore compartmentalization of Cu .................................................................................................. 133
5. Discussion & conclusions ..................................................................................................................... 149
5.1 General discussion ........................................................................................................................... 149
5.1.1 Hypothesis 1: Within MC particle size range, Cu-based NPs, rather than Cu-based
microparticles, are the main responsible for wood protection against wood decomposing fungi ..... 149
5.1.2 Hypothesis 2: Cu-tolerant wood-decomposing basidiomycetes exhibit the same tolerance
mechanisms towards Cu, independently from the form of Cu .......................................................... 151
5.1.3 Hypothesis 3: Similarly to mycorrhizal fungi, Cu-tolerant wood-destroying basidiomycetes can
accumulate Cu in their spores ............................................................................................................ 153
5.1.4 Hypothesis 4: Abrasion of MC-pressure-treated wood may lead to further dissemination of Cu-
based NPs in the environment ........................................................................................................... 154
5.1.5 Hypothesis 5: Cu-based NPs from MC wood preservative formulations, if inhaled, can cause
adverse effects due to specific nano effects....................................................................................... 155
5.1.6 Further comments ..................................................................................................................... 155
5.2. Conclusions & outlook ................................................................................................................... 156
5.3. References of Chapter 5 ................................................................................................................. 158
6. Curriculum vitae .................................................................................................................................... 164
7. Declaration of personal contribution ..................................................................................................... 166
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List of Abbreviations % w/w Mass Percent
ζ Zeta
ABTS 2,2-Azino-Bis(3-ethylbenzoThiazoline-6-Sulfonic acid)
ACQ Alkaline Copper Quaternary
ANOVA ANalysis Of Variance
APS Aerodynamic Particle Sizer
CC Chromated Copper
CCA Chromated Copper Arsenate
CdSO4 Cadmium Sulfate
CO2 Carbon Dioxide
CPC Condensation Particle Counter
CrO3 Chromium Trioxide
CT Computed Tomography
Cu Copper
Cu2+ Divalent Copper
CuCl2 Copper Chloride
CuCO3∙Cu(OH)2 Basic Copper Carbonate
CuSO4 Copper Sulfate
CuSO4∙5H2O Copper Sulfate Pentahydrate
Cys Cysteine
DCF 2',7'-DiChloroFluorescein
DMA Differential Mobility Analyzer
Empa Eidgenössische MaterialPrüfungsAnstalt
(Swiss Federal Laboratories for Materials and Technology)
EN ***, ENV *** European Standard, followed by number
ELISA Enzyme-Linked ImmunoSorbent Assay
ETH Eidgenössische Technische Hochschule
(Swiss Federal Institute of Technology)
EXAFS Extended X-ray Absorption Fine Structure
FCS Fetal Calf Serum
Fig. Figure
HBSS Hank’s Balanced Salt Solution
H2DCF-DA 2’,7’-DiChlorodiHydroFluorescein-DiAcetate
HNO3 Nitric Acid
H2O2 Hydrogen PerOxyde
HSD Honestly Significant Difference
ICP Ion-Coupled Plasma
K2Cr2O7 Potassium Dichromate
LCA Life Cycle Assessment
LCIA Life Cycle Impact Assessment
LPS LipoPolySaccharides
MC Micronized Copper
MCA Micronized Copper Azole
MCA_HTBA Micronized Copper Azole with high amount of Tebuconzole
MCA_LTBA Micronized Copper Azole with low amount of Tebuconzole
MS Mass Spectrometry
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MTS 3-(4,5-diMethylThiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-
Sulfophenyl)-2H
MWCNT Multi-Walled Carbon NanoTube
NTA Nanoparticle Tracking Analysis
NP Nanoparticle
OES Optical Emission Spectrometry
PM Particulate Matter
pH Potential Hydrogenium
RH Relative Humidity
RPMI Roswell Park Memorial Institute
ROS Reactive Oxygen Species
S1 Outer Secondary Wall
S2 Central Secondary Wall
S3 Inner Secondary Wall
SDS-PAGE Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis
SEM Scanning Electron Microscopy
SMPS Scanning Mobility Particle Sizer
SYP Southern Yellow Pine
TBA Tebuconazole
TEM Transmission Electron Microscopy
TNF-α Tumor Necrosis Factor Alpha
TTM Ammonium TetraThioMolybdate
UV UltraViolet
XAS X-ray Absorption Spectroscopy
XANES X-ray Absorption Near Edge Structure
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1. Introduction
1.1 Research question The following dissertation focuses on the wood preservative “micronized copper” (MC): its
effectiveness and possible remobilization in the air compartment.
Copper is an essential biocide for wood in contact with the soil (EN 335: use class 4) [1], due to
its unique ability to inhibit soft rot and other soilborne fungi, thus it is considered to be a mainstay in
wood protection [2]. However, the field of wood preservation is constantly developing new formulations,
in order to overcome different issues: homogeneous penetration of wood preservatives into the wood
structure, especially in poorly permeable (refractory) wood species; protection against wood-destroying
fungi, especially if tolerant to Cu or azoles; reduced toxicity and risk for human health and the
environment. The latest innovation in wood preservative formulation is best known as MC.
MC wood preservatives are composed of basic Cu carbonate (CuCO3·Cu(OH)2) particles with
sizes that range from 1 nm to 250 µm and an organic co-biocide (either azoles or quaternary ammonium
compounds) [3, 4]. MC is believed to have an added value compared to conventional wood preservatives,
due to its novel impregnation chemistry, which should facilitate the impregnation of refractory wood
species, e.g. Norway spruce, White fir, and Douglas fir, and provide a long-term protection due to the
presence of a Cu reservoir, that constantly releases bioavailable Cu2+ ions in wood against wood-
destroying fungi [5].
Despite the misleading name and the broad particle size range, as measured in different studies [6,
7], MC is actually a nanomaterial according to the definition from the European Commission [8]
recommendation that describes it as “a natural, incidental or manufactured material containing particles,
in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles
in the number size distribution, one or more external dimensions is in the size range 1 nm - 100 nm. In
specific cases and where warranted by concerns for the environment, health, safety or competitiveness the
number size distribution threshold of 50 % may be replaced by a threshold between 1 and 50 %.”. In
addition, within the biological sciences larger particles can exert similar effects and are therefore also
classified as nanomaterials [9].
Since its introduction in 2006, MC has developed into the largest end uses for wood products all
over the world [10]: it is currently used in more than 75% of residential lumbers in the US [11] and a total
of approximately 20 million m3 of wood is treated worldwide [12]. This makes MC one of the largest
scale commercial applications for nanotechnologies [10]. At present, MC systems are not used for wood
protection in Europe, however, wood preservative companies are currently testing MC formulations to
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seek approval in different European countries, e.g. France, Germany and Switzerland. The North
American and European wood decking market greatly differ in term of wood species used. While the first
mostly utilizes Southern yellow pine (SYP, like Pinus caribaea, Pinus echinata, Pinus palustris, and
Pinus taeda), Central Europe mainly uses Norway spruce (Picea abies) and White fir (Abies alba), which
are refractory to treatment. Thus, as the penetration and efficacy of MC in SYP or Norway spruce are not
likely to be comparable, the investigation of these two aspects is fundamental.
Nanoparticles (NPs) and MC among them, are not dangerous per se, but despite their potential,
the current knowledge on the human and environmental risks is limited. This can emerge if both exposure
and hazard are observed. As the development and use of MC for wood protection advances, an increase in
MC production and worldwide diffusion is likely to lead to an increase in the intentional and/or
unintentional release of Cu-based NPs in the environment, and a subsequent increase in human and
environmental exposure is a realistic scenario. NP risk is a function of NP hazard and NP exposure [13],
and there is evidence for Cu- based NPs toxicity towards aquatic organisms [14–19], terrestrial plants
[20], mammals [21-26], and humans [27–32]. Therefore, it is extremely important to determine if and how
much humans and the environment could be exposed to Cu- based NPs if they are released from MC-
treated wood. In particular, the respiratory tract is considered as the most sensitive entry portal for
nanoparticles into the human organism [13, 33]. To date, the environmental fate of conventional wood
preservatives and MC has mainly focused on leaching into the soil and water compartments, neglecting
possible releases in the air. Due to these two facts, the current research monitored the fate of Cu-based
NPs from MC formulation, to their penetration and interaction with wood, and finally their possible
release into the air compartment. Two possible release pathways were investigated: the release during
mechanical abrasion of treated wood, and copper uptake and dissemination by fungal spores. Human
exposure pathway to copper and other toxic metals through abrasion of wood treated with conventional
wood preservatives was already recognized by Townsend et al. [34]. Therefore, the abrasion of wood
treated with MC or improper disposal of its debris may pose a further environmental and human health
risk via the release of dust or wear debris containing Cu-based NPs. In addition, interactions between Cu-
based NPs and wood-destroying Cu-tolerant fungi are poorly understood. Studies [35-37] revealed a
mechanism of Cu deposition into mycorrhizal and ascomycete fungal spores as a consequence of exposure
to Cu, which corresponds to an uptake of up to 40% w/w [38]. Fungal spores can account for up to 4-11%
of the fine particle (<2.5 μm diameter) mass in rural and urban air [39, 40]; with 64% of these spores
coming from basidiomycetes [41]. Therefore, the uptake of Cu due to inhalation of Cu-loaded spores may
take place in large amounts.
The aim of this thesis was to determine whether MC is superior to conventional wood
preservatives, especially for wood species that are commonly used for the European wood decking
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market. This implies that the penetration into refractory wood species is facilitated, the efficacy of the
treatment against wood-destroying fungi is enhanced, and that the formulation does not constitute a health
and environmental risk. The focus was set on specific stages of MC’s life cycle. In accordance with what
discussed during the international workshop “Lifecycle impacts of copper nanomaterials released from
timber preserving impregnations”, held on the 22nd January 2016 at Ca’ Foscari University of Venice and
organized by the EU FP7 SUN and ECONANOSORB projects, we suppose that the compounding,
formulation and disposal phases are the least dispersive ones, due to the industrial control, and they are not
discussed here. Therefore, we mainly assess the impregnation stage, which is fundamental for the future
particle modification, and the use stage, which is believed to be widely dispersive. In order to fulfil the
main aim and research question, the following objectives were set:
1. Particle characterization, and effectiveness of MC towards different wood-destroying fungi,
namely soilborne fungi and basidiomycetes
2. Discrimination of ionic, nano and bulk Cu effects that could lead to a better MC performance
against wood-destroying fungi
3. Visualization of MC penetration, interaction, and particle speciation into easily treatable and
refractory wood species
4. Life cycle studies of Cu-tolerant wood-destroying fungi exposed to MC to determine if Cu
can be released via spores and in which form
5. Characterization of the particles released from MC-treated wood during an abrasive process,
in terms of size, morphology, and chemical composition
6. Preliminary lung toxicity assessment of MC and particles released from MC-treated wood
The work here proposed was carried out within the framework of the following working
hypotheses:
1. Within MC particle size range, Cu-based NPs, rather than Cu-based microparticles, are the
main responsible for wood protection against wood decomposing fungi
2. Cu-tolerant wood-decomposing basidiomycetes exhibit the same tolerance mechanisms
towards Cu, independently from the form of Cu
3. Similarly to mycorrhizal fungi, Cu-tolerant wood-destroying basidiomycetes can accumulate
Cu in their spores
4. Abrasion of MC-pressure-treated wood may lead to further dissemination of Cu-based NPs in
the environment
5. Cu-based NPs from MC wood preservative formulations, if inhaled, can cause adverse effects
due to specific nano effects.
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1.2 Thesis overview This thesis is structured in six chapters. Chapter 1 provides an introduction into motivation and
objectives the research project. Chapter 2 provides an introduction and background information based on
the existing literature available on the topic, and it contains a review article published in a peer-reviewed
journal, and excerpts from the paper proposed for oral presentation at the 46th Annual Meeting of the
International Research Group on Wood Protection. Chapter 3 highlights and explains the main materials
and methods used to unravel the research question. Chapter 4 presents the main experimental findings and
consists of 3 research articles, either published or under peer-review, and 2 additional studies. This chapter
assesses the effectiveness of the MC treatment against soft rot and other soilborne fungi and wood-
destroying basidiomycetes, its ability to penetrate refractory wood species, the particles released by
mechanical abrasion of treated wood surfaces and their relative cytotoxicity towards lung cells, and the
spore compartmentalization as a Cu release pathway. The discussion and interpretation of the results,
followed by drawing conclusions and an outlook are presented in Chapter 5. The articles here proposed
are reprinted with the permission of the respective publishers.
1.3 Publications and author contributions 1. Civardi C, Schlagenhauf L, Benz J, Hirsch C, Van den Bulcke J, Schubert M, Van
Acker J, Wick P, Schwarze FWMR. Environmental fate of micronized copper.
International Research Group on Wood Protection IRG/WP 15-50310, 9 pp. (Excerpts)
Chiara Civardi summarized the results and the existing literature, and wrote the manuscript.
Mark Schubert, Francis W.M.R. Schwarze, and Peter Wick provided expertise, guidance and
critical reading. All authors contributed to finalize the manuscript.
2. Civardi C, Schwarze FWMR, Wick P. Micronized copper wood preservatives: An
efficiency and potential health risk assessment for copper-based nanoparticles.
Environmental Pollution 2015; 200: 126–132.
Chiara Civardi summarized the existing literature and wrote the manuscript. Francis W.M.R.
Schwarze, and Peter Wick provided expertise, guidance and critical reading. All authors
contributed to finalize the manuscript.
3. Civardi C, Schubert M, Fey A, Wick P, Schwarze FWMR. Micronized copper wood
preservatives: Efficacy of ion, nano, and bulk copper against the brown rot fungus
Rhodonia placenta. PLoS ONE 2015; 10(6): e0128623.
Chiara Civardi and Mark Schubert conceived and designed the experimental plan. Chiara
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Civardi, Mark Schubert, and Angelika Fey performed the experiments. Chiara Civardi and
Mark Schubert analyzed the data. Chiara Civardi summarized the results and wrote the
manuscript. Mark Schubert, Francis W.M.R. Schwarze, and Peter Wick provided expertise,
guidance and critical reading. All authors contributed to finalize the manuscript.
4. Civardi C, Van den Bulcke J, Schubert M, Michel E, Butron MI, Boone MN, Dierick M,
Van Acker J, Wick P, Schwarze FWMR. Penetration and effectiveness of micronized
copper in refractory wood species. PLoS ONE 2016; 11(9): e0163124.
Chiara Civardi and Jan Van den Bulcke conceived and designed the experimental plan.
Chiara Civardi, Jan Van den Bulcke, Mark Schubert, Elisabeth Michel, and Maria Isabel
Butron performed the experiments. Chiara Civardi, Jan Van den Bulcke, Mark Schubert,
Matthieu N. Boone, and Manuel Dierick analyzed the data. Chiara Civardi and Jan Van den
Bulcke summarized the results and wrote the manuscript. Mark Schubert, Matthieu N. Boone,
Manuel Dierick, Joris Van Acker, Francis W.M.R. Schwarze, and Peter Wick provided
expertise, guidance and critical reading. All authors contributed to finalize the manuscript.
5. Civardi C, Schlagenhauf L, Kaiser J-P, Hirsch C, Mucchino C, Wichser A, Wick P,
Schwarze FWMR. Release of Copper-Amended Particles from Micronized Copper-
Pressure-Treated Wood during Mechanical Abrasion. Accepted, Journal of
Nanobiotechnology 2016.
Chiara Civardi, Lukas Schagenhauf, Jean-Pierre Kaiser, and Cordula Hirsch conceived and
designed the experimental plan. Chiara Civardi, Lukas Schagenhauf, Jean-Pierre Kaiser,
Cordula Hirsch, Claudio Mucchino, and Adrian Wichser performed the experiments. Chiara
Civardi, Lukas Schagenhauf, Jean-Pierre Kaiser, and Cordula Hirsch analyzed the data.
Chiara Civardi summarized the results and wrote the manuscript. Francis W.M.R. Schwarze,
and Peter Wick provided expertise, guidance and critical reading. All authors contributed to
finalize the manuscript.
1.4 References of Chapter 1 1. European Committee for Standardization. EN 335 Durability of wood and wood-based products-
Use classes: definitions, application to solid wood and wood-based products. Brussels, BE; 2013.
2. Hughes A. The tools at our disposal. In: Bravery AF, Peek RD. Environmental optimisation of
wood protection. Proceedings of the Final Conference; 2004 Mar 22-23; Estoril, P; Brussel, BE:
Publications Office of the European Union; 2006.
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3. Leach RM, Zhang J. Micronized wood preservative formulations comprising metal compounds
and organic biocides. World patent 2004091875, 32 pp; 2004.
4. Leach RM, Zhang J. Micronized wood preservative formulation. World patent 2005104841, 26
pp.; 2005.
5. Xue W, Kennepohl P, Ruddick J. Chemistry of copper preservative treated wood. IRG/WP 14-
30651. Proceedings IRG Annual Meeting; 2014 May 11-15; St. George, UT. Stockholm, S:
International Research Group on Wood Protection; 2014.
6. Platten WE, Luxton TP, Gerke T, Harmon S, Sylvest N, Bradham K, Rogers K. Release of
micronized copper particles from pressure-treated wood. U.S. Environmental Protection Agency,
Washington DC; 2014. EPA Report No. EPA/600/R-14/365. Available:
http://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=P100LJWI.txt [Accessed: 09 June 2015].
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platyurus. Chemosphere 2008 Apr; 71(7): 1308–1316
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46(3): 1819–1827.
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and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells
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rapid in vivo screening method for nanotoxicity. Toxicol. Appl. Pharmacol. 2008, 232(2): 292–
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34. Townsend T, Solo-Gabriele H, Tolaymat T, Stook K, Hosein N. Chromium, copper, and arsenic
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2. Background & literature review In order to comment the potentials and pitfalls of MC wood preservatives, it is necessary to
discuss first basic concepts of wood science. Wood is a natural product that has been used for centuries as
a building material. In order to understand its potential applications and use it at its best, it is fundamental
to maximize its service life, reduce any form of decay, and apply suitable protective systems. Therefore, it
is essential to know about wood anatomy and composition, how decay can occur, and the wood
preservative tools at our disposal. Further, it is necessary to know how wood preservatives can be
remobilized and end up in the environment. All these aspects are discussed below.
2.1 Wood Wood, or xylem, is defined as the secondary and permanent tissue of vascular plants and it mainly
fulfils structural as well as water and nutrient transport functions. It is surrounded by the cambium and an
inner and outer bark (phloem). Two different wood types can be identified: softwood and hardwood,
depending on whether they belong to gymnosperms or angiosperms, respectively. The studies presented in
this thesis only involve softwood species, therefore the discussion in this chapter will be limited to it.
Softwood is predominantly composed of longitudinal tracheids, and to a minor fraction of xylem
ray tissue, longitudinal parenchyma, and, in some genera, resin canals lined with epithelial cells. Every
wood cell is composed of a cavity, called lumen, surrounded by a cell wall structure that can be further
subdivided into the outermost layer, the middle lamella, the primary wall, and the innermost secondary
wall; in some cases, a warty layer next to the lumen can be identified. Three layers can be then
distinguished within the secondary wall: the outer secondary wall (S1), the central secondary wall (S2),
and the inner secondary wall (S3). The cellulose-free middle lamella is located between adjacent cells [1,
2]. For cell connection, three kinds of pits can be identified: simple pits, bordered pits, and cross-field pits.
These connect two parenchyma cells, two tracheids, and parenchyma cells in rays with axial tracheids,
respectively. The dominating type are the bordered pits, which are important for transport and exchange of
substances between the lumina of contiguous dead cells [3]. These are cavities in the lignified cell walls,
and their porous pit membrane lies in the center of each pit. Since pits are responsible for the passage of
liquids (plant water transport) and the halt of gas voids (prevent from embolism) from one lumen to
another, they provide resistance to the liquid flow [3], which then adds to the resistance encountered in the
lumen. Therefore pits largely affect the permeability to liquids. In particular, when wood is dried, the
bordered pit’s torus (central thickening) tends to move across the pit chamber and seal off one of the pit
apertures. This phenomenon is called pit aspiration, and the degree of pit aspiration accounts for
treatability of a certain wood species [4]. For instance, while the percentage of unaspirated pits in Norway
spruce latewood is approximately 20-25%, it is up to 50% in Scots pine [5]. Further, the incrustation or
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occlusion of pits due to extractives can heavily affect the liquid flow through pits. Furthermore, the
number and morphology of cross-field pits heavily affect wood permeability [5]. While in easily treatable
Scots pine, they are fenestrate and with a large thin membrane, in Norway spruce they are of the piceoid
type, with smaller membrane and smaller dimensions [6, 7]. Resin canals, if present, are important
pathways for liquids, too [8]. In particular, longitudinal resin ducts sometimes intersect with rays by
means of smaller radial resin passages. In terms of size, for conifer wood the conduction of liquids occurs
in the hollow tracheids (longitudinal) of 20-40 μm in diameter, parenchymal rays and their pits for what
constitutes the radial direction [12-14]. From either of these, liquids can then penetrate into the cell
through the pits, whose average diameter in conifers is 300 nm [12]. Wood cell wall capillary network
allows the path flow of particles with diameters up to 10 nm [13-17]. In fact, whether fluids or particles
can enter into the cell wall depends on their size. In addition, liquid polarity, viscosity, and surface tension
affect the permeability, i.e. non-polar [18], low-viscosity [19], and high surface tension [20] liquids can
penetrate more easily in wood.
Another factor influencing the penetration of liquids in wood is the amount of earlywood versus
latewood. Softwood cells formed early in the growth periods have larger lumina and thinner walls,
elements that favor the transport of liquids. These cells are known as earlywood tracheids. The rate of
growth reduces later in the growth period, and the cells, densely packed, are characterized by a smaller
diameter and thicker walls (latewood tracheids). The mechanical strength of wood is mainly provided by
the thick-walled latewood cells. This last feature is the reason why latewood is generally more resistant to
liquid flow than earlywood. Further, wood can be divided into sapwood (i.e. xylem tissue composed of
dead cells that transport water) and heartwood, which is formed by the innermost layer of sapwood during
the growth cycle. When correlated to their structures, it appears clear that sapwood is more permeable to
liquids, while heartwood is more resistant to the passage of fluids. In conifers, this is mainly due to the
changes that occur in the bordered pits. However, to which extent heartwood is less permeable than
sapwood heavily depends on the wood species.
From a chemical perspective, wood is mainly composed of cellulose, hemicelluloses and lignin
macromolecules, which altogether represent over 90% of the wood structure. These are not
homogeneously distributed in the cell wall. Beside these major components, the remaining up to 10% of
wood is composed of a percentage of non-structural constituents with low molecular weights, called
extractives, like vegetable waxes and fats (1.4%), pectic substances (0.2-2%), proteins, terpenes, phenols,
alcohols, organic acids, monosaccharides and disaccharides. Some of these, like certain terpenoids and
phenolic compounds, are toxic against microorganisms or insects, thus play a role in the “the inherent
resistance of wood to attack by wood-destroying organisms.” [21], or natural durability of wood [22, 23].
Whereas, pectic substances are responsible for the flexibility of the primary cell wall [24].
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Cellulose is the major component of wood cell walls, and it provides strength to the cell wall. It
forms crystalline fibrils that are aligned parallel and arranged to form separate layers in the secondary
wall. This molecule is characterized by linear sequences of cellobiose, a disaccharide obtained by the
reaction of two D-anhydroglucopyranose molecules. On the surface of the cellulose molecules, over the
median plane of the pyranosic ring, there lie hydroxylic groups [C(1)-OH] that account for the linearity of
the chains, and the formation of intra- and inter-chain hydrogen bonds with other cellulose molecules and
with water. While a fraction of cellulose is present in amorphous state, another fraction results in highly
ordered arrangements (monocline symmetry) of cellulose chains that then form crystal lattices. Therefore,
cellulose can be defined as a paracrystalline polymer [25]. Cellulose chains are generally differentiated on
the basis of their size: microfibrils of approx. 2.5 nm [26], and fibril aggregates (approx. 20-25 nm).
Hemicelluloses, which surround cellulose microfibrils and fibril aggregates, and being part of the
latter, are relatively short (degree of polymerization <200), non-linear, heterogeneous polysaccharides.
They act as a matrix, and are mainly formed by glucose, mannose, arabinose, and xylose. Because of the
length of their chains, the presence of side branches, their non-crystalline structure, and the presence of
many carboxylic groups, hemicelluloses are more soluble in water and more reactive than cellulose. There
are several kinds of hemicellulose and the most common in softwoods is (galacto)glucomannan, a polymer
that contains glucose and mannose units in the backbone structure, and galactose in side branches. The
second most common hemicellulose in softwood is arabinoglucuronoxylan.
Lignin is an amorphous, hydrophobic and aromatic three-dimensional polymer that is mostly
found in the middle lamella. This molecule is an encrusting substance responsible for the cells’
compressive strength, thus it is responsible for the transverse rigidity of the tracheids. Within the cell wall,
lignin creates a three-dimensional network that is partly linked with hemicelluloses by covalent bonds.
The chemical composition of lignin is variable and mainly depends on the plant species; however, the
macromolecule can be described as a polymer of phenylpropene units and substantially formed by three
cinnamic acids that react randomly: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These
three compounds are the precursors of p-hydroxyphenil, guaiacyl, and syringyl units, respectively.
In terms of chemical elements, the main constituents of wood are carbon (49-51%), oxygen (43-
44%), hydrogen (6-7%) and nitrogen (0.1-0.3%). Other minor cations are magnesium, calcium, potassium,
manganese, iron, sodium and aluminum; anions are present as sulfates, carbonates, phosphates and
chlorides.
The main features that influence the physical properties and behavior of wood are its moisture
content, capillary absorption ability and density. Moisture content refers to the quantity of water absorbed
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by the inner wood substrate, present as liquid free water within the cell cavities, or as bound water within
the cell walls, and is defined as follows:
𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (%) =𝑀𝑎𝑠𝑠 𝑜𝑓 𝑚𝑜𝑖𝑠𝑡 𝑤𝑜𝑜𝑑 (𝑔) − 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑑𝑟𝑦 𝑤𝑜𝑜𝑑 (𝑔)
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑑𝑟𝑦 𝑤𝑜𝑜𝑑 (𝑔)× 100
The situation where the wood cell wall is fully saturated is called the fiber saturation point. Any
change to less than this moisture content will affect the mechanical properties of the material, whereas
variations above this point have little or no effect. The capillary absorption ability mainly depends on the
permeability of the cell structure and is strictly linked to the moisture content.
Finally, density is defined as the ratio of the mass and volume of a substance. Because wood is
variable in its composition and is a porous material, able to contain gaseous, liquid or solid matter, inter-
and intraspecific differences in wood, and moisture content are the main factors influencing density.
Density also plays a role in the permeability of wood to liquids. However, since moisture content can
affect it, different definitions of wood density are available. In particular, two types of densities can be
identified: oven-dry density, defined as the oven-dry mass (drying to constant weight at 103°C) per oven-
dry unit volume; and air-dry (or raw) density, calculated as the mass of wood in equilibrium with
atmospheric conditions per volume of wood in equilibrium with atmospheric conditions. In general, low
density wood is more treatable, due to the higher porosity. In fact, the porosity of wood determines the
maximum amount of liquid that can enter into the structure [27].
2.2 Wood-decay fungi Wood is susceptible to deterioration and decay, in particular from biological origins. One of the
major cause of decay is attack by fungi. Fungi, or Eumycota, are eukaryotic and heterotrophic organisms
characterized by the presence of a rigid cell wall that is mainly composed of chitin. They are subdivided
into five phyla: Chytridiomycota, Zygomicota, Glomeromycota, Ascomycota and Basidiomycota [28].
Fungi are the organisms most frequently involved in biodeterioration, because they are able to decompose
a vast number of materials of different origins. They can directly interact with organic matter, which is
used for trophic purposes, and can also exert their action indirectly on inorganic materials that can,
however, contain organic fractions to support fungal growth. Moreover, many species are saprophytes and
are generally characterized by adaptive capabilities that allow them to grow and live efficiently in various
environments. Although the Kingdom of Eumycota is extremely heterogeneous, there are characteristics
that are associated with the organisms belonging to this taxon: fungi are heterotrophic organisms, a feature
that generally manifests as forms of parasitism, saprophytism or symbiosis; furthermore, fungal species
are not photosynthetic and, like animals, the reserve/stored substance used to obtain energy is glycogen;
finally, the cell wall is primarily composed of chitin.
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Fungi are characterized by different developmental stages (spore, hypha, mycelium, fruiting
body) that greatly differ in their morphology. Spores are small, reproductive cells of different shapes and
colors, either unicellular or multicellular. Hyphae are rod-shaped eukaryotic cells with protoplasm and
sometimes vacuoles; other features include mitochondria, ribosomes and endoplasmic reticulum. At
another level, there is the mycelium, or thallus, which is formed by several hyphae [29] to enable
maximization of the surface area in contact with the environment for uptake of raw materials. The
mycelial structure forms a mat that can have a variety of morphological appearances (cottony, woolly,
plumose, leathery etc.) and can grow indefinitely. Finally, the fruiting body originates from the mycelium
and is responsible for the development and release of sexual spores. The characteristic feature of all
basidiomycetes (or macrofungi) is the basidium, in which meiosis occurs. This process results in the
formation of basidiospores (four for each basidium), which are sexual spores produced on sterigmata. The
mycelium changes from monokaryotic into dikaryotic, as there are two different haploid nuclei within one
hyphal compartment. In addition, the hyphae of basidiomycetes generally have dolipore septa to prevent
the nuclei migrating between the different compartments.
The first stage in the life cycle of the basidiomycetes is dissemination of the basidiospores into
the environment and their germination. Each of these spores contains a single haploid nucleus. Under
suitable conditions, a spore will produce a filament, the germ tube, which repeatedly branches in order to
create a monokaryotic primary mycelium. In this phase, the monokaryon can produce oidia that can either
germinate to produce other monokaryotic colonies or work as fertilizers. Next, a combination of different
monokaryotic mycelia of different mating types fuse together in a process called plasmogamy and
dikaryotic (i.e. with two nuclei) hyphae develop. This phase is accompanied by an increase in the hyphal
diameter and the formation of clamp connections. The function of the clamps is to ensure that both the
original and daughter cells contain two genetically distinct nuclei. The projecting hooks snare one of the
new nuclei produced and then two septa are formed: one between them and the original cells and the
second between the old and new cells; subsequently, the clamps fuse with the new cell, releasing the
nucleus. The dikaryon can then grow and produce a network of hyphae and eventually, under suitable
environmental conditions, the fungus produces a fruiting body, in some cases referred to as a mushroom
or bracket, in which the mycelium becomes more dense and firm. Afterwards, within the margins of the
new structure, the spore-bearing layer (or hymenium) develops and is characterized by hyphae that form
the basidia (Fig. 1). At the same time, the spore-producing layer becomes folded and forms either pores
(Fig. 1) or lamellae. On the end of the basidia, four basidiospores develop after karyogamy and meiosis
(Fig. 1). Finally, they are released to the environment and a new cycle begins.
Some fungal species are lignolytic, thus able to digest wood components, causing decay, referred
to as rot. Fungi often require a minimum moisture content of 20% to start colonizing wood and then most
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species require a level between 35% and 60% relative humidity (RH) to continue growing. However, this
strictly depends on the species involved, e.g. Serpula lacrymans initially requires a wood moisture content
of 30-40%, but then is able to grow at RH <20% and so its decay is commonly referred to as ‘dry rot’ [30,
31]. In any case, water is required as it is the indispensable medium for life systems, it is a reactant in the
hydrolytic reactions involved in cell wall digestion, it is the solvent and diffusion medium for the
decomposing enzymes released by the hyphae, and it acts as a swelling agent, facilitating the penetration
of the hyphae and the digestive enzymes into the woody cell wall.
Temperature, as well as pH, directly affect fungal enzymatic and metabolic reactions, and
subsequently the organism’s ability to directly colonize the woody substrate. Although interspecific
variations occur, the optimum temperature for fungal growth is in the range of 20-35°C, and the best pH
ranges from 3 up to 6. Light, on the other hand, does not particularly affect the vegetative development of
the fungus: the mycelium usually grows in the dark, but basidiomycetes’ fruiting bodies only develop
under direct daylight. The quantity of light required, however, is highly variable.
Fig. 1. Section from a fruiting body of the basidiomycete fungus R. placenta. Pores, basidia, and
spores are highlighted.
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Fungi are aerobic organisms and as such, require free oxygen to carry out their normal metabolic
processes; even so, the quantity of oxygen required depends on the species involved. Organic and
inorganic compounds are necessary nutrients, the intake of which is required to synthesize proteins and
enzymes, chitin and other cell components. In order to fulfil this function, fungi require carbon (from the
cellulose and hemicelluloses in wood), nitrogen, a series of essential cations (such as calcium, phosphor,
potassium, sulfur, magnesium), vitamins (B1 and B6) and minor amounts of metals (iron, copper, zinc,
boron, manganese) [32]. When fungi colonize the woody substrate, they modify the material in different
ways: wood-destroying fungi frequently cause a high mass loss, with a subsequent reduction in strength,
whereas stains and molds are only responsible for pitting, foxing and superficial damage. Wood
decomposition occurs as fungi excrete enzymes that are able to decompose the structural elements of the
woody cells in order to obtain nutrients. In 1874, Hartig [33] introduced the terms ‘brown’ and ‘white’ rot,
in order to classify the different types of damage to wood caused by fungi, which can turn wood either
darker or lighter in color. Later on, the term ‘soft rot’ was invented to define softening of the woody
material. These different types of damage depend on the enzymes produced by the fungal species to
convert cellulose, and hemicelluloses into simple sugars [34].
White rot is primarily caused by ascomycetes and basidiomycetes that start degrading lignin and
then continue depolymerizing cellulose and hemicelluloses [35]; in this case, the decay is referred to as
selective delignification. The decomposition of holocellulose and lignin can occur contemporaneously,
which is referred to as simultaneous rot. In the first case, lignin is decomposed by hyphae growing on the
inner secondary wall and then depolymerization proceeds through the secondary wall towards the middle
lamella; in simultaneous rot, however, the erosion of the cell wall occurs in close proximity to the hyphae
[35].
Brown rot fungi largely belong to the Basidiomycota and mainly break down the polysaccharidic
component of wood (e.g. cellulose and hemicelluloses), leaving lignin almost unaltered, though
sometimes modified by demethylation or oxidation processes [36]. This inability to attack lignin can be
explained from an evolutionary point of view: brown rot fungi evolved from white rot fungi [37], and their
main feature developed from simplification of the mechanisms to initially decompose cellulose, which
allowed extinction of the energetically disadvantageous lignocellulose degrading apparatus [38]. It is
noteworthy that brown rot fungi depolymerize holocellulose, generating degradation products faster than
their rate of usage [39]. Decomposition is believed to occur via a Fenton system, although it is still unclear
whether other mechanisms are involved [40]. In the first stage, degradation is conducted by hyphae
growing on the cell wall and the enzymes then diffuse through the S3 layer until the target S2 region,
which has a high content of cellulose, is reached. The result is a rapid degradation of the S2 layer, while
the S3 and middle lamella remain almost intact [35]. Although the major target of brown rot decay is
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conifer wood, this should not be seen as a more favorable substrate, because the correlation lies in the
predominant distribution of brown rot species in northern regions where conifers are abundant [41]. Most
of the Cu-tolerant fungi belong to this classification, e.g. Poria and Antrodia spp. [42], which is explained
in evolutionary terms by their ability to produce more oxalic acid than white rot fungi: the latter secrete
oxalic acid-decarboxylase enzymes that do not allow the accumulation of acids, resulting in less ability to
develop resistance mechanisms and survive effectively at lower pH values [43].
Compared with the other two types of decay, the fungi causing soft rot have a relatively simple
and slow mode of wood degradation. These fungi, which mainly belong to the Ascomycotina and
Basidiomycotina Phyla and to mitosporic fungi [44, 45], are able to grow on damp or wet wood and their
moisture tolerance frequently ranges from dry dormancy to active decomposition at levels of almost
complete saturation [46]. The soft rot action is mainly exerted on the outer layers of wood and the attack
begins from the wood surface, where the fungus enters via the xylem rays; the hyphae then penetrate
depressions in the cell wall, from where they penetrate into the secondary wall to reach the cellulose-rich
S2 layer, branching and secreting cellulase enzymes to create longitudinally aligned rhomboidal cavities
(soft rot Type 1). Soft rot Type 2 fungi are also able to grow within the lumen, causing erosion through the
cell [47]. Lignin is decomposed by soft rot only if of syringyl origin, whereas guaiacyl-rich layers are
often resistant. In addition, soft rot fungi commonly require high amounts of nitrogen, either from wood or
from the environment.
2.3 Wood preservatives Wood preservatives aim to increase the durability of wood and lengthen its service life.
Therefore, wood preservatives should satisfy a series of criteria, the main one being product performance.
This means that the wood preservative can uniformly penetrate into the wood structure [48], which occurs
according to the transport mechanisms described in Chapter 2.1, and that the product can effectively
protect wood from decay. Besides, wood preservatives should guarantee low production cost (in terms of
raw material extraction, material processing, and product manufacture), health and environmental safety.
From a life cycle assessment (LCA) and life cycle impact assessment (LCIA) perspective, it means that
the use of treated wood should be advantageous to the use of untreated wood.
Industrial pre-treatment of timber with wood preservatives can be conducted via different carriers,
namely tar oils, solvents, or water (either with or without metals). Their different properties makes them
suitable for different wood applications, therefore wood preservatives can be distinguished on the basis of
wood uses. According to the European regulations, 5 end use (or hazard) classes can be distinguished [49],
and wood preservatives should prevent from the most common type of biological attack for each end use.
These are listed below:
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1. Interior, dry wood; mainly susceptible to biological attack by insects
2. Interior, damp; subject to decay from insects and decay fungi
3. Exterior, protected or unprotected; exposed to insects, decay fungi, and bluestain (disfiguring)
fungi
4. In-ground or fresh water; where deterioration can be caused by insects, decay fungi, bluestain
(disfiguring) fungi, and soft rot fungi
5. Marine; whose decay is caused by insects, decay fungi, bluestain (disfiguring) fungi, soft rot
fungi, and marine borers.
For in-ground timber structures (hazard class 4), Cu is essential for its protection, as it has the
unique ability to inhibit soft rot [50] and most white rot fungi [51]. The fungicidal activity of Cu and its
potential in wood protection have long been known. The first account of chemical wood preservatives
dates back to the 17th century, when Cu salts were used for the first time as phytomedicines against wood-
decay fungi, together with mercury chloride and arsenates [52]. However, the so-called first generation of
Cu-based wood preservatives arrived only two centuries ago, with the development of creosote, chromated
Cu arsenate (CCA) in the 1930s, and then penta (pentachlorophenol) in the 1950s [53]. Of these, the
features of CCA enabled it to become the predominant product in the market for the preservation of a
wide variety of wooden objects for industrial and residential purposes. It is a low-cost, easy to use
formulation and, being waterborne, CCA does not have a tang odor, and all of its components (Cu,
chromium and arsenic) contribute actively to the effective and long-lasting preservation of wood.
Following the same path of CCA, ammoniacal Cu arsenate and ammoniacal Cu zinc arsenate waterborne
systems were developed to deal with refractory species in particular. Despite the advantages of these
products and their wide usage, health and environmental concerns arose in the 1970s regarding air and
water pollution caused by chromium and arsenic, and this resulted in a US review of these pesticides
between 1978 and 1980, when they were classified as restricted use. In 2004 registration of CCA for
residential applications was withdrawn [54], and from 1998 the use of CCA was forbidden in situations
where human contact could occur in Europe [55]. Consequently, a new generation of preservatives was
developed, as CCA disposal began. The second generation of products for wood protection was
characterized by boron- or Cu-rich waterborne systems, combined with an organic co-biocide to act
against Cu-tolerant fungi. Although boron is mainly in the form of boric acid or derivatives (borates), the
Cu successors of CCA can be further distinguished into metal complexes or systems with uncomplexed
Cu. This group of wood-preservative systems commonly has higher concentrations of Cu and does not
contain either arsenic or chromium. However, in these preservatives the absence of chromium causes
major leaching and corrosion issues [56, 57], resulting in ineffective preservation and chemical dispersal
in the environment. These concerns lead to the development of third- and fourth-generation products, in
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which totally organic systems or biocontrol strategies are used [57]. The chemicals used in these systems
are azoles, mainly triazoles such as tebuconazole (TBA), chlorotalonil (2, 4, 5, 6-
tetrachloroisophthalonitrile), and betaine. Even these products can lead to environmental problems, as not
only do they leach but they can also evaporate or be depleted by chemical, biological and photo reactions
[57, 58]. The non-biocidal preservation systems and biocontrol mechanisms, in contrast, have the potential
to be environmentally safe, but are only at preliminary stages of development. MC consists of
CuCO3·Cu(OH)2 particles with a size range of 1 nm-250 µm, according to manufacturers, combined with
a second organic biocide that provides protection against basidiomycetes, either azole (MCA) or
quaternary ammonium compounds (MCQ) [59, 60]. Although MC still contains Cu as main biocide, it has
an impregnation chemistry that differs from conventional Cu-based wood preservatives: part of
CuCO3·Cu(OH)2 immediately solubilizes during the impregnation process and is complexed by wood
organic macromolecules, while another fraction does not react and acts as reservoir, i.e. Cu is solubilized
afterwards [61]. Therefore, the issues related to leaching and corrosion are reduced [62], and a continuous
protection over time is hypothesized [63].
2.4 Release of Cu from wood into the
environment The loss of wood preservative active ingredients from wood does not only result in the loss of
protection against biodeterioration and biodegradation, but it also have a negative impact on human health
and the environment. Due to the intrinsic nature of wood preservatives, pressure-treated-wood contains
organic and inorganic components that can be toxic to non-target organisms, if released into the
environment. More precisely, these components may be released in air, soil, or water as a consequence to
the conditions where pressure-treated wood is located. The main substance of concern in (pressure-
treated) in-ground timber structure is Cu.
One of the major cause of Cu migration is leaching, which can release Cu in soil, fresh and
marine waters, with their relative proportions depending on the precise location. The speciation of Cu
from leachates also depends on the characteristics of the environment where pressure-treated wood is
located.
Soil in the vicinity of treated wood, similarly to agricultural soils [64], is a repository of Cu and
the metal is likely to deposit in the upper part, close to treated timber structures exposed to the soil. Here
Cu2+ ions and CuCO3·Cu(OH)2 particles leached from wood treated with MC or conventional wood
preservatives can undergo speciation due to soil pH, organic and inorganic matter. These parameters play
a key role in determining whether Cu would be mobile and/or bioavailable. In particular, soils high in pH
or organic matter would complex free Cu2+ ions [65] or precipitate the residual Cu carbonate nanoparticles
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[66, 67], which are likely to aggregate/agglomerate into bigger and stable CuCO3·Cu(OH)2 particles. In
acid soils poor in organic matter CuCO3·Cu(OH)2 would dissolve and result in mobile, free, Cu2+ ions.
If Cu is leached into water, the physicochemical properties of the water in which Cu is transported
play a key role in determining the Cu speciation and transport. Still, it is possible to draw some general
conclusions. In seawater, freshwater and brackish water, Cu mainly interacts with dissolved components,
principally with organic ligands that reduce Cu bioavailability. Cu is seldom found free (Cu2+ ions),
inorganically complexed, or adsorbed to particulate matter. At the sediment-water interface the amount of
bioavailable Cu can significantly increase, due to different oxygen levels and organic matter
decomposition.
Cu could also be remobilized in the air compartment. The main cause of it is believed to be via
production of wood dust that contains Cu [68]. Since wood dust can cause per se adverse health effects
[69-73], in particular diseases to the respiratory tract, the presence of Cu may exacerbate the stress
response.
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31. Low GA. An investigation of selected effects of environment on the dry rot fungus Serpula
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32. Zabel RA, Morrell JJ. Wood microbiology: decay and its prevention. 1st ed. London, UK:
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33. Hartig R. Wichtige Krankheiten der Waldbäume. 1st ed. Berlin, DE: Springer; 1874.
34. Rayner ADM, Boddy L. Fungal decomposition of wood: its biology and ecology. 1st ed.
Chichester, UK: John Wiley and Sons; 1988.
35. Schwarze FWMR. Wood decay under the microscope. Fungal Biol. Rev. 2007; 21(4): 133–170.
36. Green F III, Highley TL. Mechanism of brown-rot decay : paradigm or paradox. Int. Biodeterior.
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37. Hibbett DS, Donoghue MJ. Analysis of character correlations among wood decay mechanisms,
mating systems, and substrate ranges in homobasidiomycetes. Syst. Biol. 2001; 50(2): 215–242.
38. Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M, Kubicek CP, et al. Genome,
transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique
mechanisms of lignocellulose conversion. Proc. Natl. Acad. Sci. 2009; 106(6): 1954–1959.
39. Cowling EB. Comparative biochemistry o f the decay of sweetgum sapwood by white-rot and
brown-rot fungi. U.S. Department of Agriculture, Washington DC; 1961. Tech. Bull. No. 1258.
40. Arantes V, Milagres AMF, Filley TR, Goodell B. Lignocellulosic polysaccharides and lignin
degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions. J. Ind.
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41. Watling R. Taxonomic status and ecological identity in the basidiomycetes. In: Frankland JC,
Hedger JN, Swift MJ. Decomposer basidiomycetes: their biology and ecology-British
Mycological Society Symposium 4. Cambridge, UK: Cambridge University Press; 1982.
42. Köse C, Kartal SN. Tolerance of brown-rot and dry-rot fungi to CCA and ACQ wood
preservatives. Turkish J. Agric. For. 2010; 34: 181–190.
43. Rypáček V. Biologie holzzerstörender Pilze. 1st ed. Jena, DE: VEB Gustav Fischer Verlag; 1966.
44. Savory JG. Breakdown of timber by ascomycetes and fungi imperfecti. Ann. Appl. Biol. 1954;
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45. Duncan CG, Eslyn WE. Wood-decaying ascomycetes and fungi imperfecti. Mycologia. 1966 Jul-
Aug; 58(4): 642–645.
46. Liese W, Ammer U. Über den Befall von Buchenholz durch Moderfäulepilze in Abhängigkeit von
der Holzfeuchtigkeit. Holzforschung. 1964; 18: 97–102.
47. Hale MD, Eaton RA. The ultrastructure of soft rot fungi. II. Cavity-forming hyphae in wood cell
walls. Mycologia 1985; 77(4): 594–605.
48. Arsenault RD. Factors influencing the effectiveness of preservative systems. In: Nicholas DD.
Wood deterioration and its prevention by preservative treatments. Vol. 2, 121–278. New York,
NY: Syracuse University Press; 1973.
49. European Committee for Standardization. EN 335 Durability of wood and wood-based products-
Use classes: definitions, application to solid wood and wood-based products. Brussels, BE; 2013.
50. Hughes A. The tools at our disposal. In: Bravery AF, Peek RD. Environmental optimisation of
wood protection. Proceedings of the Final Conference; 2004 Mar 22-23; Estoril, P; Brussel, BE:
Publications Office of the European Union; 2006.
51. Illman B, Highley TL. Fungal degradation of wood treated with metal-based preservatives: 1.
Fungal tolerance. IRG/WP 96-10163. Proceedings IRG Annual Meeting; 1996 May 19-24;
Guadaloupe, FA. Stockholm, S: International Research Group on Wood Protection; 1996.
52. Schiessl U. Historischer Überblick über die Werkstoffe der schadlingsbekiimpfenden und
festigkeitserhohenden Holzkonservierung. Maltechnik Restauro 1984; 90(2) :9-40.
53. Richardson BA. Wood preservation. 2nd ed. London: Routledge; 1993.
54. U.S. Environmental Protection Agency. Notice of receipt of requests to cancel certain chromated
copper arsenate (CCA) wood preservative products and amend to terminate certain uses of CCA
products. Federal Register 2002; 67(36):8244–8246.
55. European Commission. Directive 98/8/EC of the European Parliament and of the Council of 16
February 1998 concerning the placing of biocidal products on the market. Official Journal of the
European Commission 1998; L 123: 1–63.
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56. Schultz TP, Nicholas DD. Introduction to developing wood preservative systems and molds in
homes. In: Schultz TP, Militz H, Freeman MH, Goodell B, Nicholas DD. Development of
commercial wood preservatives: efficacy, environmental, and health issues. Washington DC:
American Chemical Society Symposium Series; 2008. p. 2–8.
57. Freeman MH, Nicholas DD, Schultz TP. Nonarsenical wood protection: alternatives for
chromated copper arsenate, creosote and pentachlorophenol. In: Townsend TG, Solo-Gabriele H.
Environmental impacts of preservative treated wood. CRC Press, Taylor & Francis; 2010. p. 19–
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58. Wang X, Wang X, Zhang H, Wu C, Wang X, Xu H, et al. Enantioselective degradation of
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59. Leach RM, Zhang J. Micronized wood preservative formulations comprising metal compounds
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63. Freeman M, McIntyre C. Micronized copper wood preservatives: strong indications of the
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64. Kiaune L, Singhasemanon N. Pesticidal copper (I) oxide: environmental fate and aquatic toxicity.
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65. Temminghoff EJ, Van der Zee SE, de Haan FA. Copper mobility in a copper contaminated sandy
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66. Ponizovsky AA, Allen HE, Ackerman AJ. Copper activity in soil solutions of calcareous soils.
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67. Rodriguez‐Rubio P, Morillo E, Madrid L, Undabeytia T, Maqueda C. Retention of copper by a
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68. Decker P, Cohen B, Butala JH, Gordon T. Exposure to wood dust and heavy metals in worker
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69. Barcenas CH, Delclos GL, El‐Zein R, Tortolero‐Luna G, Whitehead LW, Spitz MR. Wood dust
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from North America. Int. J. Occup. Environ. Med. 1997; 39(2): 148–156.
71. Carosso A, Ruffino C, Bugiani M. Respiratory diseases in wood workers. Br. J. Ind. Med. 1987;
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72. Demers PA, Teschke K, Kennedy SM. What to do about softwood? A review of respiratory
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2.6 Spore compartmentalization of Cu Environmental Pollution 200 (2015) 126-132
Review
Micronized copper wood preservatives: An efficiency and potential health risk assessment for
copper-based nanoparticles
Chiara Civardia, b
Francis W.M.R. Schwarzea
Peter Wickc, ∗
[email protected]
aEmpa Swiss Federal Laboratories for Material Testing and Research, Wood Laboratory,
Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland
bInstitute for Building Materials, ETH Zurich, Wolfgang-Pauli Strasse 10, CH-8093 Zurich, Switzerland
cEmpa Swiss Federal Laboratories for Material Testing and Research, Materials-Biology Interactions
Laboratory, Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland
*Corresponding author.
Abstract
Copper (Cu) is an essential biocide for wood protection, but fails to protect wood against Cu-tolerant
wood-destroying fungi. Recently Cu particles (size range: 1 nm–25 μm) were introduced to the wood
preservation market. The new generation of preservatives with Cu-based nanoparticles (Cu-based NPs) is
reputedly more efficient against wood-destroying fungi than conventional formulations. Therefore, it has
the potential to become one of the largest end uses for wood products worldwide. However, during
decomposition of treated wood Cu-based NPs and/or their derivate may accumulate in the mycelium of
Cu-tolerant fungi and end up in their spores that are dispersed into the environment. Inhaled Cu-loaded
spores can cause harm and could become a potential risk for human health. We collected evidence and
discuss the implications of the release of Cu-based NPs by wood-destroying fungi and highlight the
exposure pathways and subsequent magnitude of the health impact.
We assess the fungicidal activity of particulate copper wood preservatives and their possible release in the
air by Cu-tolerant wood-destroying fungi.
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Keywords: Micronized copper; Copper-tolerant fungi; Nanocopper exposure; Wood-destroying fungi;
Wood preservatives
1 Introduction
Wood was one of the first materials used by humans for a wide variety of applications. Moreover,
this natural product is still used because of its exceptional density–strength properties, as well as being
economically advantageous, sustainable, and carbon dioxide neutral. Hence, it is believed to be one of the
most preferred building materials in the future (Buchanan and Levine, 1999).
Despite its positive features, wood is susceptible to different factors that cause its decay, and
various biodeterioration organisms such as fungi, cyanobacteria, bacteria, protists, and insects have the
capacity to decompose wood. In particular, fungi, insects, and bacteria are considered to be the principal
pests of timber. Therefore, methods to improve the durability of wood have been developed since early
times (Unger et al., 2001) and the chemical preservation of wood products appears to be the most common
and developed strategy, with three generations' history. Health and environmental concerns with the older
1st-generation preservative systems (arsenic, penta) led to rapid and profound changes on a worldwide
basis as we moved to the 2nd and 3rd generation Cu rich preservatives.
Nanotechnology, which is currently one of the most investigated areas of research, has influenced
the utilization of wood preservatives because it has enabled the development of innovative metal-based
biocides (Clausen, 2007; Dorau et al., 2004; Green and Arango, 2007; Kartal et al., 2009; Kim and Kim,
2006; Akhtari et al., 2013a).
More precisely, the wood preservation industry has begun to profit from formulations using
particulate Cu systems, also known as micronized Cu, that contain a considerable amount of nanosized Cu
(Mcintyre, 2010), which can readily penetrate into the wood (Geers et al., 2014; Matsunaga et al., 2007).
Since 2006 more than 11,800,000 m3 of wood treated with micronized Cu has been sold (Griffin, 2011),
which corresponds to an annual use of 79,000 tonnes of Cu systems (Evans et al., 2008), greatly exceeding
the quantities estimated for titanium dioxide NPs (Gottschalk et al., 2009).
In this review, the term Cu-based NPs will be used to indicate formulations containing a Cu
nanosized fraction, according to the current nanomaterials definition (Commission Recommendation,
2011) and to recent findings (Mcintyre, 2010; Geers et al., 2014; Matsunaga et al., 2007). Cu-based NPs
can be manufactured from metallic Cu or Cu compounds (e.g. Cu carbonate). Although wood
preservatives based on Cu-based NPs are currently available, surprisingly there is little information on
their hazard potential and the interactions between this biocide and wood-destroying Cu-tolerant fungi are
poorly understood.
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The objectives of this review are the assessment of (i) the effectiveness and the risk of wood
preservatives based on Cu-based NPs, (ii) to discuss the probability of Cu-tolerant fungi accumulating,
and re-mobilizing Cu during their life cycle, and (iii) subsequently the magnitude of the health impact.
2 Wood preservative treatments
To date, the main fungicide used for treating wood in contact with the soil is Cu (Cu carbonate,
Cu citrate), and currently there is no satisfactory alternative yet: Cu compounds are the only biocides that
show a high efficiency against soft rot fungi and other soilborne fungi, which cause the greatest damage to
wood products in contact with the soil (Hughes, 2004).
Cu is generally required at low concentrations by every organism in order to sustain metabolic
processes for its survival, but at higher concentrations it causes serious, in some cases irreversible
alterations in metabolic activities (Gadd, 1993). Furthermore, despite its excellent properties as fungicide,
Cu is considered to be a toxicant with minimal effect on mammals (Lebow, 1996), although the effect on
aquatic communities is relevant (Eisler, 1998; Roales and Perlmutter, 1980). The features of Cu allow it to
act as a biocide, or as a growth or reproduction inhibitor for biodeteriogents. In addition, Cu is also able to
cause genetic perturbation or mutation.
2.1 Potential of Cu-based NPs formulations as wood preservatives
We are currently experiencing a development of particulate-based wood preservatives, in terms of
composition and size range of the particles, in order to maximize the biocidal effect and effectively protect
wood in contact with the soil from biodeterioration. As discussed above, the Cu-based NPs formulations
currently available have a mean particle size that is evidently nanoscale, as indicated in Fig. 1 that shows
the particles from a Cu-based NPs preservative formulation.
Fig. 1 Waterborne micronized copper formulations for wood preservatives: impregnation of wood samples
and TEM micrograph on particles from a micronized copper.
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The use of Cu-based NPs instead of bulk Cu reputedly improves the durability of wood against
fungal decomposition (Kartal et al., 2009; Cookson et al., 2010; McIntyre and Freeman, 2009; Akhtari et
al., 2013b) because of the following properties of NPs: (i) ability to penetrate bordered pits because they
are smaller than the mean opening (300 nm), (ii) increase in the effective surface area of Cu, and enhanced
dispersion stability; (iii) less viscous formulations than bulk ones, and (iv) presence of a reservoir effect
that allows a continuous protection over time (Xue et al., 2014; Freeman and Mcintyre, 2013). These
properties, which enable easier impregnation and deeper and more homogeneous uptake of the biocide
into the wood, can be further improved by selecting specific supporting systems (e.g. surfactants) (Green
and Arango, 2007). Moreover, the presence of chromium and arsenic is no longer necessary; however,
leaching of the nanometal greatly depends on treatment procedure and product formulation (Kartal et al.,
2009; Preston et al., 2008; Ding et al., 2013).
Finally, there is only little evidence to suggest that Cu-based NPs are more efficient against
soilborne fungi or some Cu-tolerant wood-destroying fungi (Kartal et al., 2009; Cookson et al., 2010;
Tang et al., 2013).
2.2 Fungicidal mechanisms of Cu-based NPs
Although the literature on the fungicidal properties of Cu-based NPs is sparse and fragmented,
several studies investigated the toxic mechanisms of Cu ions.
In addition, the nanoparticles commercially available are generally poorly characterized (Altes,
2008). Furthermore, there is a substantial lack of knowledge in the underlying mechanisms on how Cu-
based NPs wood preservatives function, namely whether the toxicity is exerted by the nanoparticles
themselves or by the release of ions, or only in combination with secondary biocides. Thus at present the
use of Cu-based NPs for wood protection is an insufficiently understood system, in regards to both the
effectiveness of the treatment and its safety.
What is generally known is that the principal mode of action of Cu-based NPs can be identified as
highly reactive due to their larger specific surface area (Chen et al., 2006; Oberdürster, 2000), which
results in the production of reactive oxygen species (ROS), mainly peroxides, which induce a series of
chain reactions and oxidative stress to the exposed organism (Heinlaan et al., 2008; Saliba et al., 2006)
and cause DNA damages. As demonstrated for white rot fungi, these reactions may eventually result in the
in vitro inhibition of lignocellulose-degrading enzymes (Shah et al., 2010). In addition, particles may
interact with proteins and mitochondria damaging or disrupting them (Chang et al., 2012). Moreover, a
strong ability to interact with the cell wall has been suggested (Heinlaan et al., 2008; Shah et al., 2010)
and homeostatic processes may be impaired by Cu dissolution (Chang et al., 2012). These lethal
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interactions mentioned above are described in Fig. 2, where the toxic mechanisms for fungi colonising
wood treated with Cu-based NPs are shown at the cellular level.
Fig. 2 Cu-based NPs and their uptake and the toxic mechanisms for non Cu-tolerant fungi (left) and Cu
detoxification mechanisms for Cu-tolerant fungi (right) colonizing wood treated with Cu-based NPs. In
non Cu-tolerant fungi the Cu-based NPs can enter the cell (1) and form ROS (2) or have disruptive effects
on mitochondria, proteins and DNA (2, 3); otherwise the Cu-based NPs may undergo dissolution (1) and
interfere with cell homeostatic processes (2). In Cu-tolerant fungi, Cu-based NPs taken up by the cell can
be complexed and precipitated (1) or sequestrated in vacuoles (1). To avoid further Cu penetration, the Cu
uptake (both as NPs or as ions) from the surrounding environment is reduced (2) and the cell wall is able
to bind Cu (2). By taking up glucose (1) to produce oxaloacetate (2), the production of oxalate (3) and its
extracellular release (4) allow the complexation of Cu, forming non-bioavailable Cu oxalate (5).
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Due to these features, Cu-based NPs may require lower concentrations of the metal with similar
or even better efficacy than water based and dissolved conventional Cu formulations (Kartal et al., 2009;
Mahapatra and Karak, 2009).
2.3 Fungal Cu-tolerance mechanisms
The continuous improvement of Cu-based formulations is mainly driven by the need to improve
the efficiency of Cu biocides against Cu-tolerant fungi. Several brown rot fungi have the ability to grow
and survive at Cu2+concentrations up to 1.6 mM (Hughes, 2004) or 100 mg/kg (Gadd et al., 2007),
because of their resistance and tolerance mechanisms, which are depicted in Fig. 2, where the tolerance
mechanisms for fungi colonising wood treated with Cu-based NPs are shown at the cellular level:
- Cu complexes and/or precipitates in the presence of extracellular products, which detoxify Cu by altering
it, i.e. in presence of oxalate Cu2+ ions are precipitated as non-bioavailable Cu oxalate (Clausen et al.,
2000)
- metal binding to the cell wall, to avoid Cu penetration through the cell
- intracellular Cu complexes/precipitates, or modification of the Cu2+ ions, to shift Cu in a non-
bioavailable form
- vacuolar compartmentalization, which isolates Cu from the living organelles in the cell
- reduction in Cu uptake by fungi, which prevents Cu from entering the cell
- spore compartmentalization (Figs. 3 and 4) (Cornejo et al., 2013), to translocate Cu away from the cell
Fig. 3 Claroideoglomus claroideum spores from the rhizosphere of Imperata condensate growing for 6
months in a 450 mg Cu kg−1 spiked-soil (A–C) or in a non-spiked soil (D) (Reproduced from Cornejo et
al., 2013 with permission from Elsevier).
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Fig. 4 Influence of time and temperature on the uptake of Cu (initial concentration 19 μg/mL) by
Neurospora crassa spores (100 million/mL) (Reproduced from Somers, 1963 with permission from
Wiley).
All these mechanisms, which allow fungi to grow and develop fruit bodies and spores, have
different degrees of efficacy on the basis of the substrate composition (Gharieb et al., 2004), and the Cu
compounds against which the fungi are tested (De Groot and Woodward, 1999; Köse and Kartal, 2010;
Green and Clausen, 2005). In addition, inter (De Groot and Woodward, 1999; Köse and Kartal, 2010;
Guillén et al., 2009; Sierra-Alvarez, 2007) and intraspecific (Clausen et al., 2000; De Groot and
Woodward, 1999; Sierra-Alvarez, 2007; Collet, 1992; Duncan, 1958; Green and Clausen, 2003)
differences have been observed, suggesting that individual survival strategies enable adaptation to Cu-
polluted environments. As a result, Cu-tolerant fungi are one of the main causes for early failures of in-
ground timber structures in the US and Europe (Lebow et al., 2003; Bollmus et al., 2012). Thus, in
Germany 4.800.000 wood poles are in service and annually 1% (48.000) early failures are caused by Cu-
tolerant fungi. The total annual expenses for replacing wood poles are estimated EUR 36.000.000 (BASF
Wolman, personal communication).
3 Risk is defined by hazard and exposure
According to Oberdorster et al. (Oberdörster et al., 2005), there is a NPs risk if there is evidence
for both exposure and hazard. Cu-based NPs-based wood preservatives are used in increasingly higher
retentions, so potential exposure to humans and the environment is evident. However, precise estimations
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on how much and in which form Cu is released are not possible because of the lack of quantitative data. In
the case of NP hazards, additional aspects, compared with conventional chemicals, have to be taken into
account: (i) uptake and bio-distribution are dissimilar to bulk or ionic forms, (ii) surface effects influence
cellular metabolism and signaling cascades, and (iii) material properties differ, e.g. size, composition,
degree of crystallinity, surface, modification of NPs after contact with the environment and the
consequences of such alterations (Joner et al., 2008).
With regard to Cu-based NPs, studies of different formulations confirm their cytotoxic activity,
but a thorough exposure assessment is still not possible, because of the lack of quantitative release data.
Thus, at the moment the uncertainties do not enable precise predictions on the risk, and therefore a
suitable risk management is not possible (Som et al., 2012).
3.1 Hazard identification and characterization
The hazard of Cu-based NPs for organisms belonging to different phyla has been highlighted in
several studies, as the toxicity exerted is not species-specific and results in non-target effects.
Although part of the Cu-based NPs in the preservative formulation reacts with wood, forming
complexes, a fraction of Cu-based NPs does not react and remains as insoluble basic copper carbonate
particles (Xue et al., 2014).
What has generally been noticed is that non-dissolved NPs easily interact with the cell membrane
(Oberdürster, 2000) and eventually penetrate it (Geiser et al., 2005), in contrast to bulk Cu, via a Trojan-
horse-like mechanism (Limbach et al., 2009). Once inside the cell, depending on the formulation, the Cu-
based NPs can either generate ROS via Fenton's reaction or go through intracellular dissolution (Studer et
al., 2010) and cause cell damage via the creation of “hot spots” of Cu ions (Limbach et al., 2009; Karlsson
et al., 2009; Midander et al., 2009).
Moreover, because of their dimensions, NPs can be inhaled (Geiser and Kreyling, 2010), may
cross biological barriers and are transported via lymphatic and circulatory systems to different tissues and
organs, where accumulation can result in severe injuries and damage to living cells (Buzea et al., 2007).
First indications of the adverse effects of Cu-based NPs on the brain (Sharma and Sharma, 2007), the
gastrointestinal (Altes, 2008; Meng et al., 2007) and respiratory tracts of mammals (Chen et al., 2006;
Limbach et al., 2005; Stearns et al., 1994; Vallyathan and Gwinn, 2006), as well as ecotoxicity in aquatic
ecosystems have been reported (Shah et al., 2010; Shaw et al., 2012; Shi et al., 2011).
Karlsson et al. (Karlsson et al., 2008a, 2008b) and Zhang et al. (Zhang et al., 2012) found Cu
oxide NPs to be highly cytotoxic (MTS and ATP logEC50 = 1.38 ± 0.03 μg/mL for human bronchial
epithelial cells, MTS logEC50 = 1.12 ± 0.04 μg/mL and ATP 1.16 ± 0.03 μg/mL for rat
alveolarmacrophage cells) (Zhang et al., 2012), causing severe damage to DNA (concentration of 20
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μg/cm2). There are only a few studies on the effect of Cu-based NPs on the cardiac system, although the
effects caused by ultrafine particles have been widely investigated (De Hartog et al., 2003; Pope et al.,
2004) and similar patterns of disease can be assumed. In addition, there is evidence that NPs (including
Cu) cannot be removed easily by phagocytosis (Karlsson et al., 2008b).
However the main studies conducted were focused on the determination of acute toxicity at single
dose, literature concerning low dose but chronic exposure and long-term disease is lacking to date
(Limbach et al., 2009).
In addition to these negative effects, since fungal spores can cause adverse health effects when
inhaled (Denning et al., 2006), their presence in concomitance with Cu-based NPs may cause additional
stress responses.
3.2 Potential exposure scenario
At present, no research has been conducted to verify human or environmental exposure to Cu-
based NPs from treated wood as a potential source, therefore it is impossible to predict and estimate the
risk properly. From the risk assessment perspective, as long as there is a lack of accurate and quantitative
data on the release and effects of Cu-based NPs in the environment, the precautionary principle should be
applied. However, the presence in the market of wood preservatives based on Cu-based NPs acts against
this principle; even more concerning is the fact that over 75% of the residential lumber produced in the
USA is now treated with such formulations (Freeman and Mcintyre, 2013), implying that domestic
exposure could be already occurring, potentially.
Although it has been argued that the quantity of Cu disseminated in the environment should not
exceed threshold concentrations (Velleux et al., 2012), the possibility of Cu-based NPs being released
either alone or absorbed by fungal spores has not been considered so far. Spore uptake has already been
reported for bulk Cu by Cornejo (Cornejo et al., 2013) et al. and Somers (Somers, 1963), and subsequent
spread by this vehicle is evident. Cu-based NPs are hazardous when inhaled, and will mainly exert their
toxicity on the respiratory tract of humans, the most sensitive entry portal (Krug and Wick, 2011).
The aspect of spores as a vector of Cu-based NPs should not be underestimated, because fungal
spores can account for up to 4–11% of the fine particle (<2.5 μm diameter) mass in rural and urban air
(Womiloju et al., 2003); 64% of these spores belong to basidiomycetes in which a range of Cu-tolerant
wood-decay fungi are classified (Fröhlich-Nowoisky et al., 2009). The concentration of fungal spores is
estimated to be approximately 103–104 m−3 on continental land surfaces, for a global emission of 5 × 1010
kg/year (Elbert et al., 2007), which implies that fungal spore production is one of the largest sources of
organic aerosols.
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As these formulations are already being used (e.g. the US market) a careful monitoring and
fundamental understanding of the underlying mechanism how these Cu-based NPs behave is necessary.
This knowledge could also be used to design or control their function and fate in order to create a safer
product with similar efficiency.
4 Conclusions and recommendations
The current knowledge provides circumstantial evidence for the release of Cu-based NPs from
treated wood into the environment. In addition to leaching (Ding et al., 2013), one likely release route may
be during the colonization of treated wood by copper-tolerant wood-destroying fungi. The accumulation
of Cu in spores of mitosporic fungi has already been identified for standard Cu salts and compounds.
The environmental fate of Cu-based NPs from wood preservatives is only poorly understood.
Despite broad knowledge of Cu-based NPs toxicity, to date the assessment of the potential risk related
with the introduction of Cu-based NPs for wood protection (thousands of tons annually) is impossible to
make. Therefore, the quantification of accumulation rate and release of Cu is mandatory. This issue can be
solved only if the nanoscience and the wood protection communities interact and collaborate, as already
stated by Evans et al. (Evans et al., 2008) years ago. More specific, nanosafety experts and wood
specialists have the skills, analytics and understanding in order to explore and assess the environmental
fate and health impact of Cu-based NPs in wood preservatives.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
Acknowledgments
The work is financially supported by the Swiss National Research Foundation (Grant No.
406440_141618). We are indebited to Liliane Diener for the TEM investigation, to Philipp Rogenmoser
and Urs Bünter for the imaging technical support. The authors acknowledge Prof. Ingo Burgert and Prof.
Ottmar Holdenrieder for proofreading.
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6723–6732.
Womiloju T.O., Miller J.D., Mayer P.M. and Brook J.R., Methods to determine the biological composition
of particulate matter collected from outdoor air, Atmos. Environ. 37, 2003, 4335–4344.
Xue W., Kennepohl P. and Ruddick J.N.R., Chemistry of Copper Preservative Treated Wood, 2014,
International Research Group on Wood Protection, 7, IRG/WP 14-30651.
Zhang H., Ji Z., Xia T., Meng H., Low-kam C., Liu R. and Pokhrel S., Use of metal oxide nanoparticle
band gap to develop a predictive paradigm for oxidative stress and acute pulmonary
inflammation, Am. Chem. Soc. Nano 6, 2012,4349–4368.
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3. Materials & methods In this chapter only the main techniques are described, in order to explain why a certain method
was used. Specific details about material and methods can be found within the compiled papers.
3.1 Wood and wood preservatives 3.1.1 Wood species
The studies were conducted on defect-free sapwood and heartwood specimens from two different
wood species, Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.). The first one
provides a good example of an easily treatable wood species, widely used in North America, and standard
in wood preservative effectiveness tests; whereas Norway spruce is a refractory wood species commonly
used in Central Europe, due to its abundance [1]. The assessment of MC penetration in both species gives
a clear overview on the suitability of the treatment for the two different wood markets, as well as highlight
potential major differences in the impregnation chemistry.
3.1.2 Micronized copper
Two commercially available MC formulations were tested. Both contained tebuconazole (TBA)
as co-biocide, defining them as MC azole (MCA) formulations.
The MC particles present in both formulations were comparable in terms of size, morphology,
and zeta (ζ)-potential, according to both the manufacturer’s descriptions and the characterization
conducted. The techniques used to characterize the nanoparticles mostly consisted in NP tracking analysis
and transmission electron microscopy (TEM). The difference between the two formulations was the
concentration of TBA, which accounted to 0.4% w/w or 5% w/w. The size range of the MC particles
according to the manufacturer was between 1 nm and 250 µm, however the analyses conducted revealed
narrower size distributions for both formulations: the mean diameters were 104 ±1.7 nm (mode: 87±2.2
nm) for one formulation and 174±5.9 nm (mode: 150±8.2 nm) for the other one.
The chromated Cu (CC) formulation used as reference adheres to the ENV 807 [2] guidelines and
it is composed of 50% w/w copper sulfate pentahydrate (CuSO4∙5H2O), 48% w/w potassium dichromate
(K2Cr2O7), 2% w/w chromium trioxide (CrO3).
3.2 Wood preservative effectiveness The effectiveness of MC-treated wood against fungal decay was assessed according to the
guidelines provided by the European Standards EN 113 [3] and ENV 807 [2], tests that focus on the wood
preservative effectiveness against wood-destroying basidiomycetes and soft rot fungi, respectively. The
main outcome of these tests is the wood mass loss, which is the percentage change between the wood
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mass before and after fungal exposure. These tests allow to correlate wood mass losses with preservative
effectiveness. The wood preservative treatment (or its dilution) is considered effective against wood-
destroying fungi when the wood mass losses are below 3%, otherwise the treatment is considered
ineffective.
3.2.1 Wood impregnation
Wood block dimensions, initial mass, and dry mass (oven dried at 103 °C for minimum 18 h)
were recorded, oven dry and raw densities were calculated based on these measurements.
The wood samples were subsequently immersed in different dilutions of the wood preservative
and stored under vacuum for 20 minutes, shaking the container from time to time to avoid particle
deposition, and at normal conditions for 2 hours. Afterwards, the samples were removed from the
solutions, the excessive liquid on the wood block surface was removed with filter paper. Wood samples
mass after impregnation was measured, solution uptake was calculated as the difference between the wood
block mass after impregnation and its initial oven dried mass, preservative retention was calculated as
follows:
𝑃𝑟𝑒𝑠𝑒𝑟𝑣𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 (𝑘𝑔
𝑚3) =
𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑢𝑝𝑡𝑎𝑘𝑒 (𝑘𝑔) × 𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑉𝑜𝑙𝑢𝑚𝑒 (𝑚3)
The samples were then stored in a conditioning chamber avoiding contact between specimens for
8 weeks, according to the indications provided by the MC manufacturers. The container was kept covered
for 1 week, and then slowly opened within the 8 week drying period.
3.2.2 Effectiveness against soft rot fungi-ENV 807
Natural top soil without any agro-chemicals, with pH between 6 and 8, and a water holding
capacity between 25% w/w and 60% w/w was used. The soil was distributed in test vessels, wetted with
water to reach the 95% of its water holding capacity, thoroughly mixed, and the masses was recorded.
Small wood stakes (100 x 10 x 5 mm) untreated, MCA- and CC-treated were exposed to leaching
accelerated ageing procedure (EN 84) [4] and were then planted into the soil with 20 mm in length
protruding and with 20 mm space in between the samples. The vessels were stored at 28°C and 85% RH
for 32 weeks. After 16 and 32 weeks incubation, replicate sets of test specimens were removed, brushed
free of soil particles, and weighted. The samples were subsequently oven dried (103 °C). The percentage
of wood mass loss was calculated from the dry mass before and after soil plantation.
3.2.3 Effectiveness against wood-destroying basidiomycetes-EN 113
Fungal mycelia were grown in 9 cm diameter Petri dishes with 25 mL solid medium (autoclave
sterilized) containing 4% (w/v) malt extract and 2.5% (w/v) agar at 22°C and 70% RH. After min. 2 weeks
(time required for the fungus to colonize the whole Petri dish) mycelia plugs of 9 mm in diameter were
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placed in Kolle flasks with 75 mL solid medium (autoclave sterilized) containing 4% (w/v) malt extract
and 2.5% (w/v) agar. The fungi were allowed to grow in the Kolle flasks for 2 weeks at 22°C and 70% RH
(time required for the fungus to colonize the whole Kolle flask). Afterwards a glass structure for support
and the wood samples (50 x 25 x 15 mm) were placed into the Kolle flasks. The Kolle flasks were then
stored at 22°C and 70% RH for 16 weeks. After incubation, wood blocks were removed from the Kolle
flasks, brushed free of mycelium, weighted, and oven dried at 103 °C. The percentage of mass loss was
calculated from the dry mass before and after fungal exposure. The wood mass loss values were then
corrected by subtracting the wood mass loss values obtained from sterile control wood samples.
3.3 Mechanical abrasion Different woodworking activities, such as sawing, wiping, or milling, are responsible for the
generation and release of airborne wood dust. To measure the release of particles during the mechanical
abrasion of wood, we used a Taber Abraser. This is a widely used device to simulate sanding processes
and to study abrasion resistances of materials and coatings, and is recognized by various international
standards; e.g. EN 13696 [5] and EN 14354 [6]. It provides a reproducible continuous abrasion process
under defined conditions. Several studies have used the Taber Abraser to generate particles from
nanocomposites, to measure the particle size distribution, and to check if nanoparticles were released [7-
11]. An abrasion process generally releases particles in the size range from a few nm to several µm. The
size, morphology, and Cu content of the wooden abraded particles were assessed. Based on lung
penetration [12], asbestos-like pathogenicity [13, 14], and heavy metal accumulation [15], the parameters
above mentioned would provide an indication on the likelihood of adverse health effect in case of
inhalation of the airborne particles released. The full size range of the wood dust particles released was
characterized by aerosol measurement devices directly after the abrasion process using a scanning
mobility particle sizer (SMPS), to detect particles below 1 µm, and an aerodynamic particle sizer (APS),
for the measurement of bigger particles (up to 10 µm). After collection on grids, the morphology of the
abraded particles was analyzed by scanning electron microscopy (SEM). Finally, elemental analysis to
quantify the Cu content in the wood dust was performed by means of ion-coupled plasma (ICP)-mass
spectrometry (MS) and ICP-optical emission spectrometry (OES).
3.4 X-ray techniques X-rays are electromagnetic radiations with energies in the range between 100 eV and 100 KeV.
These allow the X-rays to interact with atoms’ core electrons and provide indications on the elements
being hit by them; therefore they can be applied for qualitative and semi-quantitative analytical purposes.
The interactions between the X-rays and matter can be ascribed to three basic phenomena: absorption,
scatter, and fluorescence. These constitute the underlying principles behind X-ray methods.
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The X-ray analysis performed originated from both laboratory-scale and synchrotron X-ray
sources. X-ray computed tomography (CT) allowed to non-invasively investigate the distribution of
copper in the wood, as well as the fungal decomposition pathways. Whereas X-ray absorption
spectroscopy, (XAS) enabled the assessment of copper speciation and distribution in fungal structures
(mycelia and spores).
3.4.1 X-ray computed tomography
X-ray CT allows the reconstruction of the internal structure of a material (or a portion of it)
without physical cutting of the sections for the examination. This is achieved in a relatively short time,
under ambient conditions, and almost no sample preparation is required. In X-ray CT imaging, X-rays are
directed at an object from multiple orientations and the beam intensity is monitored. Changes in the
intensity are caused by X-ray energy, X-ray beam path length, and the materials composing the sample
(material linear attenuation coefficient). Afterwards, algorithms are used to reconstruct the distribution of
X-ray intensities in the sample volume.
Large wood samples were analyzed by means of the multi-resolution micro-CT scanner
Nanowood [16, 17] built at the Ghent University Centre for X-ray Tomography (Ghent University,
Belgium). These samples were selected to assess the overall distribution of Cu in wood or to illustrate
fungal decay patterns. The samples were scanned using a closed type microfocus X-ray tube at 100 kV
and 80 µA, 1000 projections and 1000 ms exposure time per projection. Reconstructions were performed
with the Octopus Reconstruction software package [18], a tomography reconstruction package for parallel,
cone-beam and helical geometry licensed by InsideMatters (www.insidematters.be), resulting in
reconstructed data with an approximated voxel pitch of 31 μm. The reconstructed volumes were analyzed
using Octopus Analysis, previously known as Morpho+ [19] and also licensed by InsideMatters. For the
assessment of Cu penetration, the entire wood block was segmented and Cu was separated and quantified
within the block. Volumes were also rendered in 3D using VGStudio Max software.
Small wood samples were used to investigate the Cu distribution or fungal decay in wood at the
cellular level. The samples were scanned using an open type nanofocus X-ray tube at 80 kV and 45 µA,
1000 projections and 1500 ms exposure time per projection. Reconstructions were also performed with the
Octopus Reconstruction software package, resulting in volumes with an approximate voxel pitch of 0.8
μm.
3.4.2 X-ray absorption spectroscopy
XAS is an element-specific method that provides information on the near atomic environment (2-
3 closest neighboring atoms’ shells) of the absorbing atoms from the selected element. This is achieved by
characterizing the element’s electronic transitions and the photoelectron emissions. It is possible to
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distinguish three sections in X-ray absorption spectra: the pre-edge region, the X-ray absorption near edge
structure (XANES), and the extended X-ray absorption fine structure (EXAFS).
The speciation of Cu was examined using the microXAS beamline at the Swiss Light Source. The
samples were placed on a sample holder with Kapton foil window. The materials were analyzed at room
temperature. Energy calibration of the Si111 double crystal monochromator was obtained by
measurements of a metallic Cu reference foil (in transmission mode) prior and after taking experimental.
First inflection point of the metallic copper K-edge spectrum was set to 8979 eV. Due to low Cu
concentrations, XANES spectra of the samples were collected in fluorescence mode using a single
element Silicon Drift Diode detector (SDD, KETEK GmbH). The beamsize was defocused resulting in a
700 µm diameter beam. In addition to the wood samples, several reference materials were analyzed at
room temperature. To examine the possibility of beam-induced damage, spectra were collected at least
twice from the same location and the spectra were compared to determine if the beam had caused a change
in speciation. The XAS data processing software Athena was used to analyze the XAS spectra [20]. For
each individual sample, multiple spectra were collected and merged. The XANES spectra obtained were
used to compare the Cu speciation. The linear combination fitting tool provided by the software Athena
was used to determine the speciation of Cu.
3.5 Ion-coupled plasma-mass spectrometry and
optical emission spectrometry ICP-MS and ICP- OES are commonly used techniques to determine the total content of selected
elements in a sample. The samples are introduced as liquids -if solid, acid digestion prior to the elemental
analysis is performed- and turned by means of a nebulizer into an aerosol, which is sprayed into the center
of the plasma. The latter vaporizes, atomizes, ionizes, excites and subsequently relases the aerosol’s
particles, which are then detected and quantified by optical emission spectrometers or mass spectrometers.
OES uses the emission spectrum of the material analyzed, whose wavelength is element-specific, while
MS separates the ions based on their mass-to-charge ratio. Both MS and OES provide information on the
concentration of the element of interest based on the signal intensity detected.
These two techniques were employed to quantify the amount of copper contained in wood, wood
dust, cell eluates, and fungal structures. The solid samples underwent acid digestions before being
analyzed. The detailed procedures differed for each samples and are summarized in the research articles
within Chapter 4.
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3.6 Acute toxicity for human lungs Basic acute toxicity tests on a biological model representing the lungs were performed to obtain
preliminary indications on the potential hazard of wood dust from MC-pressure-treated wood. The
biological model consisted of A549 lung epithelial and human acute monocytic leukaemia THP-1 cell
lines. The oxidative stress paradigm [21] was employed and the following parameters were assessed:
1. Reactive oxygen species (ROS) formation: overproduction of ROS can cause cell oxidative
stress and damages to the cell structures. Therefore, the concentration of ROS within a cell is
a good indicator of cellular fitness
2. Cell viability: to assess if the cells are undergoing apoptosis
3. Inflammatory response: release of the pro-inflammatory tumor necrosis factor TNF-α.
3.7 References of Chapter 3 1. Abegg M, Brändli U-B, Cioldi F, Fischer C, Herold-Bonardi A, Huber M, et al. Fourth national
forest inventory - result tables and maps on the Internet for the NFI 2009-2013 (NFI4b).
Eidgenossische Forschungsanstalt WSL, Birmensdorf, CH; 2014. Available:
http://www.lfi.ch/resultate/ [Accessed: 02.12.2015].
2. European Committee for Standardization. ENV 807 Wood preservatives- Determination of the
effectiveness against soft rot micro-fungi and other soil inhabiting microorganisms. Brussel, BE;
2001.
3. European Committee for Standardization. EN 113 Wood preservatives- Test method for
determining the protective effectiveness against wood destroying basidiomycetes. Determination
of toxic values. Brussel, BE; 1997.
4. European Committee for Standardization. EN 84 Wood preservatives- Accelerated ageing of
treated wood prior to biological testing. Leaching procedure. Brussel, BE; 1997.
5. European Committee for Standardization. EN 13696 Wood flooring- Test methods to determine
elasticity and resistance to wear and impact resistance. Brussel, BE; 2008.
6. European Committee for Standardization. EN 14354 Wood-based panels- Wood veneer floor
covering. Brussel, BE; 2004.
7. Vorbau M, Hillemann L, Stintz M. Method for the characterization of the abrasion induced
nanoparticle release into air from surface coatings. J. Aerosol Sci. 2009 Mar; 40(3): 209–217.
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8. Wohlleben W, Brill S, Meier MW, Mertler M, Cox G, Hirth S, et al. On the lifecycle of
nanocomposites: comparing released fragments and their in-vivo hazards from three release
mechanisms and four nanocomposites. Small 2011 Aug 22; 7(16): 2384–2395.
9. Golanski L, Gaborieau A, Guiot A, Uzu G, Chatenet J, Tardif F. Characterization of abrasion-
induced nanoparticle release from paints into liquids and air. J. Phys. Conf. Series 2011 Jul;
304(1): 012062.
10. Schlagenhauf L, Chu BTT, Buha J, Nüesch F, Wang J. Release of carbon nanotubes from an
epoxy-based nanocomposite during an abrasion process. Environ. Sci. Technol. 2012 Jun 4; 46:
7366–7372.
11. Wohlleben W, Meier MW, Vogel S, Landsiedel R, Cox G, Hirth S, et al. Elastic CNT-
polyurethane nanocomposite: synthesis, performance and assessment of fragments released during
use. Nanoscale 2013 Jan 7; 5(1): 369–380.
12. Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: An Emerging Discipline Evolving
from Studies of Ultrafine Particles. Environ. Health Perspect. 2005 Mar 22; 113(7): 823–839.
13. Palomaki J, Valimaki E, Sund J, Vippola M, Clausen PA, Jensen KA, et al. Long, needle- like
carbon nanotubes and asbestos activate the NLRP3 inflammasome through a similar mechanism.
ACS Nano 2011 Sep 27; 5(9): 6861–6870.
14. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WAH, Seaton A, et al. Carbon nanotubes
introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study.
Nat. Nanotechnol. 2008 Jul; 3(7): 423–428.
15. Gordon T, Spanier J, Butala JH, Li P, Rossman TG. In vitro bioavailability of heavy metals in
pressure-treated wood dust. Toxicol. Sci. 2002; 67(1): 32–37.
16. Dierick M, Van Loo D, Masschaele B, Van den Bulcke J, Van Acker J, Cnudde V, et al. Recent
micro-CT scanner developments at UGCT. Nucl. Instum. Meth. B 2014 Apr 1; 324: 35–40.
17. Dierick M, Van Loo D, Masschaele B, Boone M, Van Hoorebeke L. A LabVIEW® based generic
CT scanner control software platform. J. X-ray Sci. Technol. 2010 Oct 29; 18(4): 451–461.
18. Vlassenbroeck J, Dierick M, Masschaele B, Cnudde V, Van Hoorebeke L, Jacobs P. Software
tools for quantification of X-ray microtomography at the UGCT. Nucl. Instrum. Meth. Phys. Res.
A 2007 Sep 21; 580(1): 442–445.
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19. Brabant L, Vlassenbroeck J, De Witte Y, Cnudde V, Boone MN, Dewanckele J, et al. Three-
dimensional analysis of high-resolution X-ray computed tomography data with Morpho+.
Microsc. Microanal. 2011 Apr; 17(2): 252–263.
20. Ravel B, Newville M. ATHENA and ARTEMIS: interactive graphical data analysis using
IFEFFIT. Phys. Scripta 2005; 2005(T115): 1007.
21. Nel A, Xia T, Mädler L, Li N Toxic potential of materials at the nanolevel. Science 2006;
311(5761): 622-627.
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4. Results & discussion
4.1 Wood preservative effectiveness 4.1.1 Micronized copper wood preservatives: efficacy of ion, nano, and bulk
copper against the brown rot fungus Rhodonia placenta
PLoS ONE10 (2015) e0128623
Micronized copper wood preservatives: efficacy of ion, nano, and bulk copper against the brown rot
fungus Rhodonia placenta
Short title: Ionic, nano and bulk copper against Rhodonia placenta
Chiara Civardi1,2*, Mark Schubert1, Angelika Fey1, Peter Wick3, Francis W.M.R. Schwarze1*
1Empa, Applied Wood Materials, Dübendorf/ St. Gallen, Switzerland
2ETH, Institute for Building Materials, Zürich, Switzerland
3Empa, Particles- Biology Interactions, St. Gallen, Switzerland
*Corresponding authors
Email: [email protected] (CC), [email protected] (FS)
Keywords: Copper tolerance, Micronized copper azole, Nanoparticles, Wood decay
Abstract
Recently introduced micronized copper (MC) formulations, consisting of a nanosized fraction of basic
copper (Cu) carbonate (CuCO3·Cu(OH)2) nanoparticles (NPs), were introduced to the market for wood
protection. Cu NPs may presumably be more effective against wood-destroying fungi than bulk or ionic
Cu compounds. In particular, Cu- tolerant wood-destroying fungi may not recognize NPs, which may
penetrate into fungal cell walls and membranes and exert their impact. The objective of this study was to
assess if MC wood preservative formulations have a superior efficacy against Cu-tolerant wood-
destroying fungi due to nano effects than conventional Cu biocides. After screening a range of wood-
destroying fungi for their resistance to Cu, we investigated fungal growth of the Cu-tolerant fungus
Rhodonia placenta in solid and liquid media and on wood treated with MC azole (MCA). In liquid
cultures we evaluated the fungal response to ion, nano and bulk Cu distinguishing the ionic and particle
effects by means of the Cu2+ chelator ammonium tetrathiomolybdate (TTM) and measuring fungal
biomass, oxalic acid production and laccase activity of R. placenta.
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Our results do not support the presence of particular nano effects of MCA against R. placenta that would
account for an increased antifungal efficacy, but provide evidence that attribute the main effectiveness of
MCA to azoles.
Introduction
Copper (Cu) has long been known for its fungicidal properties and it is an essential biocide for wood in
contact with the soil, as it is the only active substance that hitherto successfully inhibits wood
decomposition by soft rot fungi [1]. The efficacy of Cu- based wood preservatives against wood-
destroying fungi is mainly exerted by Cu in its soluble form, as Cu2+ ions [2–4]. However, an increased
copper efficacy may be achieved at the nanoscale: several nanoparticles (NPs) have been shown to be
more toxic to prokaryotes and eukaryotes than larger particles of the same chemical composition [5, 6] or
dissolved ions [7-9]. In fungi, it is believed that NPs may enter the fungal cell through endocytosis [10] or,
if smaller than the pores, across the cell walls eluding barrier and entering the plasma membrane [11, 12].
Afterwards, the NPs may be able to cross the cell membrane, enter into the fungal cell and get in direct
contact with cell components. However, to date there is a lack of understanding of their specific mode of
action in fungi, despite their key role in wood decomposition of treated wood or bioremediation of
contaminated soils. Recently, basic CuCO3·Cu(OH)2 particulate systems for wood protection were
introduced to the US market [13]. These wood preservatives, commonly known as micronized copper
(MC) formulations, contain a considerable amount of nanosized CuCO3·Cu(OH)2 with low water
solubility, and in some cases purely consist of nanoparticles [14].
When wood is treated with MC, some Cu reacts with wood and part of it remains in the form of unreacted
Cu particles that provide a reservoir effect [15]. Most of the studies [16] on Cu bioavailability of
micronized Cu have focused on the release of Cu2+ ions [17–21]. However, the superior efficacy of the
treatment [22–25] may be partially due to insolubilized persistent CuCO3·Cu(OH)2 NPs that diffuse
through the fungal cell wall and its membrane and exert their toxic effects, also described as the Trojan
horse mechanism [26].
In Cu- tolerant (brown rot) fungi the threshold for Cu- NPs may be lower than the threshold for Cu2+ ions,
due to nano formulation, and subsequently fungi may not be able to trigger Cu- tolerance mechanisms in
presence of small amounts of MC. In this case, the main wood degradation mechanisms for brown rot
fungi, i.e. free hydroxyl radical production via Fenton reaction, may be impaired. Thus, the production of
mediators for the Fenton reaction, which also implicates Cu oxidases [27, 28], may not be stimulated by
Cu- tolerant fungi, resulting in a biochemical activity pattern dissimilar from the array that occurs when
Cu- tolerant fungi are exposed to bulk or ionic Cu. In particular, the production of oxalic acid [29] or
laccase [30] may be reduced. Although Tang et al. [27] provided a thorough investigation on the gene
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expression of fungi exposed to MC, so far no comparison with conventional Cu-based wood preservatives
has been made.
Therefore the objective of this study was to determine if MC is more effective than standard Cu
compounds against wood-destroying fungi due to specific nano formulation. For this purpose we first
investigated the growth of different wood-destroying fungi in different MC-amended solid media and
selected the most Cu-tolerant strain for further investigation. Subsequently, we assessed growth and
enzyme activities of the selected Cu- tolerant fungus Rhodonia placenta (Fr.) Niemelä, Larss. & Schigel
(= P. placenta) in liquid cultures amended with Cu2+ ions from Cu sulfate (CuSO4), MC (nano), bulk
CuCO3·Cu(OH)2, with and without ammonium tetrathiomolybdate (TTM) a well-known Cu2+ ion chelator
used to treat Cu poisoning and Wilson’s disease in human and animals [31-34]. In fungi, a high dose of
TTM (IC50 1.0±0.2 µM) can inhibit the production of tyrosinase [35], an enzyme responsible for melanin
synthesis, but low dosages are well tolerated. The ligand binds selectively to Cu2+ ions, allowing us to
investigate whether Cu2+ ions derived from solubilized Cu-NPs and is responsible for the toxicity [36] in
Cu- amended fungal culture media. The impact of MC co-biocide, tebuconazole (TBA), was also
assessed.
Two mediators involved in the Fenton reaction were measured to assess different fungal responses to Cu:
oxalic acid and laccase. Oxalic acid is believed to be a key component in Cu detoxification due to its
ability to bind Cu, whereas laccase is produced by R. placenta during wood colonization [29].
Subsequently, wood decay caused by R. placenta on wood samples treated with MC, bulk
CuCO3·Cu(OH)2 and TBA was assessed according to EN 113 [37] to determine the effectiveness of the
different components in a more natural setting.
Materials and Methods
Materials
2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), bulk CuCO3·Cu(OH)2, CuSO4, TBA and
TTM were purchased from Sigma Aldrich, while agar, malt extract and potassium chloride from VWR
(Oxoid, Darmstadt, Germany).
The oxalic acid assay EnzytecTM oxalic acid was purchased from R-Biopharm AG. The silver stain kit,
DodecaTM Silver Stain was obtained from BIO-RAD.
Two commercial aqueous suspensions of MCA were investigated. The two MCA formulations contain
comparable amounts of Cu particles but differ in the amount of TBA: MCA_HTBA contained 5% w/w
and MCA_LTBA 0.4% w/w TBA.
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NP characterization
Cu particles in the MCA formulations were characterized prior to fungal exposure. Particle morphology
was assessed by transmission electron microscopy (TEM) with a Zeiss 900 microscope (Zeiss SMT,
Oberkochen, Germany). TEM grids (400 mesh) coated with 8 nm of carbon were incubated for 20 s on a
10 µl droplet of MCA diluted with nanopure water. The excess suspension fluid was drawn off with filter
paper.
Particle size distribution was measured by nanoparticle tracking analysis (NTA) using a NanoSight LM20
(NanoSight Ltd., UK) on MCA diluted with Milli-Q water. Data analysis was performed with NTA 2.3.5
software (NanoSight Ltd., UK). Particle diameters are reported as average and standard deviation of seven
video recording of the sample. Zeta potential measurements were carried out on MCA diluted with Milli-
Q water using a Zetasizer NanoZS (Malvern Instruments, UK).
Screening forCu-tolerance
All fungi used in the solid media study were wood-destroying basidiomycetes commonly used in EN 113
tests: Antrodia serialis (Fr.) Donk isolate 43, Coniophora puteana (Schumach.) Karst isolate 62,
Gloeophyllum trabeum (Pers.) Murrill isolate 100, R. placenta isolate 45, Trametes versicolor (L.) Lloyd
isolate 159 from the Empa culture collection. Fungal mycelia (9 mm in diameter) were grown in 9 cm
Petri dishes with 25 mL solid medium (autoclave sterilized) containing 4% (w/v) malt extract and 2.5%
(w/v) agar. The media were amended with either 0.01% (w/v) or 0.05% (w/v) of MCA_HTBA or
MCA_LTBA. Three replicates were prepared for each condition. Cultures were stored at 22 °C and 70%
RH. The cultures were inspected regularly, and their 4 cardinal points were marked to determine the
growth radii until the colonies reached the edges of the Petri dishes. Fungal growth rate for each colony
(in mm per day) was determined dividing the mean value of the latest growth radii minus that of the
earliest by the number of days elapsed between the measurements.
Fungal response to Cu2+ ions and particles
The fungus used in the liquid culture study was R. placenta isolate 45 from the Empa culture collection.
Fungal mycelia were grown in 500 mL Erlenmeyer flasks with 250 mL liquid culture medium (autoclave
sterilized) containing 1% (w/v) malt extract and 0.6% (w/v) potassium chloride.
To understand the effect of ion, nano or bulk Cu, the following materials were added respectively: CuSO4,
MCA, CuCO3·Cu(OH)2. The concentration of CuCO3·Cu(OH)2, and CuSO4 was 0.02 mM. The quantity
of MCA was calculated based on the equivalent 0.02 mM of total Cu. The interference caused by the azole
biocide on the fungus was assessed by adding TBA. The amount of TBA, alone or with Cu, was calculated
as the amount of TBA content in MCA (5% w/w). TTM (0.02 mM) was used to separate the Cu-based
particles from the Cu2+ ions. All these materials were added to the liquid cultures as indicated in Table 1.
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Table 1- Scheme of the liquid cultures used.
Liquid culture media
Without TTM With 0.02 mM TTM
Control Control
0.02 mM MCA 0.02 mM MCA
0.02 mM CuCO3·Cu(OH)2 0.02 mM CuCO3·Cu(OH)2
0.02 mM CuCO3·Cu(OH)2 + 5% w/w TBA 0.02 mM CuCO3·Cu(OH)2 + 5% w/w TBA
0.02 mM CuSO4
5% w/w TBA
0.02 mM CuSO4
---
TTM= ammonium tetrathiomolybdate; MCA= micronized copper azole; CuCO3= Cu carbonate;
TBA= 5 tebuconazole; CuSO4= Cu sulfate
Each treatment was repeated in triplicates. Each flask was inoculated with 1 disc (8 mm in diameter) of
the strain pre-cultured in solid medium and incubated in an orbital shaker (100 rpm) at 22°C for 9 weeks.
The pH of the liquid culture was between 4.5 and 5, assuming that some Cu particles would not solubilize
but would remain suspended in the liquid media. After incubation, the biomass was harvested by filtration
and oven dried at 107°C for 24 hours. Fungal growth was estimated as wet and dry biomass weight.
Laccase activity
Laccase activity in R. placenta 45 was measured after 16 weeks incubation in untreated, MCA_LTBA and
MCA_HTBA-treated wood colonized by R. placenta 45 (see protective effectiveness of MCA active
ingredients) and after 2, 4, and 9 weeks in liquid cultures. For detection of laccase in wood, the colonized
samples were treated according to Wei et al. [30]. The grounded wood was stirred overnight at 4C° in dist.
H2O containing 1M NaCl to extract extracellular proteins. The liquid phase was separated from the solids
by vacuum filtration through Whatman no 1 paper and concentrated in an ultrafiltration cell (Ultracel,
Millipore) fitted with 10-kDa cutoff membrane.
Laccase activity was measured as initial velocity of the oxidation of ABTS (2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid), 3 mM) to its cation radical at room temperature (22-25°C) and at
pH 4.5 (citrate buffer 100 mM). Changes in absorbance (∆A) at 420 nm were recorded with UV-visible
spectrophotometer (Genesys 10S UV-vis, Thermo Scientific Inc., Waltham, MA, USA). One volumetric
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activity unit (U) was defined as the amount of enzyme transforming 1 µmol of ABTS per min and the
volumetric activities were calculated using an extinction coefficient (ε) of 36000 mol-1 L cm-1[38, 39].
Oxalic acid assay
After incubation, 0.5 mL of liquid media were taken from each culture medium and oxalate concentration
in the samples was analyzed with a spectrophotometer (Genesys 10S UV-vis, Thermo Scientific Inc.,
Waltham, MA, USA) at 590 nm as specified by the instruction manual (EnzytecTM Oxalic acid, R-
Biopharm AG). Samples were diluted 100-fold with distilled water before being used in the assay as they
showed very high activities.
To determine any further changes in the protein secretions of R. placenta 45 exposed to the different
amended media after incubation, 0.2 mL of liquid media was taken from each culture medium. Protein
extracts from the control, CuCO3·Cu(OH)2, CuSO4, MCA liquid media -with and without TTM- plus
markers (BIO-RAD) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) using 10% gels loaded with 5 μL of each liquid culture sample and the marker. Gels were silver
stained with the DodecaTM Silver Stain kit according to the instruction manual (BIO-RAD).
Efficacy of MCA active substances
Scots pine (Pinus sylvestris L.) sapwood blocks (50 x 25 x 15 mm) were pressure treated according to the
European standard EN 113 [37] with different concentrations of MCA (2%, 1.6%, 1.33%, 1.07%, 0.8%,
0%). Scots pine sapwood blocks (50 x 25 x 15 mm) were also impregnated with 2% and 1.6% equivalent
concentrations of CuCO3·Cu(OH)2 or TBA. No permits were required for the described study, which
complied with all relevant regulations. No endangered or protected species were involved. After drying,
the samples were exposed to R. placenta 45 at 22 °C and 70% RH. Test procedures were performed
according to the European standard EN 113 [37]. After incubation, wood blocks were removed from the
culture vessels, brushed free of mycelium and oven dried at 103±1 °C. The percentage of weight loss was
calculated from the dry weight before and after the test.
Statistical analysis
Growth data and oxalic acid concentrations from fungi growing on solid and liquid media and on wood
were log-transformed and data expressed as percentages, such as mass loss, were arcsine-transformed
prior to analysis (ANOVA) and back-transformed to numerical values for visualization. Means were
separated using Tukey’s-HSD (Honestly Significant Difference) test at significance level p<0.05. The
statistical package used for all analyses was SPSS® (Version 17.0, SPSS Inc., Chicago, IL, USA).
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Results
NP characterization
Fig. 1. Characterization of Cu particles in the micronized Cu azole (MCA) formulations assessed.
TEM micrographs of (a) MCA_HT [40] and (b) MCA_LT; average particle size distribution of (c)
MCA_HT and (d) MCA_LT. Data represented as mean ± standard deviation of seven repetitions. The
MCA_HTBA formulation contains high amount of tebuconazole (TBA) (5% w/w), whereas MCA_LTBA
contains low amount of TBA (0.4% w/w).
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The Cu particles in the two MCA formulations were comparable and appeared heterogeneous in size and
morphology, as shown in the TEM micrographs (Figs.1a, b). The size distributions were also similar for
the MCA formulations (Figs. 1 c, d). The mean diameter was 104 ±1.7 nm (mode: 87±2.2 nm) for
MCA_HT and 174±5.9 nm (mode: 150±8.2 nm). Therefore, the Cu particles were solely in the nano-
range. The Cu particles in diluted MCA_HT and MCA_LT had an average ζ-potential of -21.0±0.4 mV
and -16.5±1.4 mV respectively, indicating suspensions that tend to aggregate.
Screening for Cu-tolerance
We evaluated the growth of different wood-destroying fungi in MCA-amended media to identify the most
Cu-tolerant strain for subsequent studies. Fig. 2 shows the mean growth rate for A. serialis 43, C. puteana
62, G. trabeum 100, R. placenta 45, and T. versicolor 159 in the different media.
Both concentrations of the MCA formulations caused appreciable reductions in fungal growth rate
compared to the controls (p-value < 0.001). C. puteana 62 was not able to grow in any of the amended
media, showing no tolerance to Cu or TBA, whereas A. serialis 43 and G. trabeum 100 effectively grew
only in 0.01% MCA_LTBA. Differences in overall fungal growth rates were more evident at 0.01% for
MCA_LTBA and MCA_HTBA, as distinct patterns were apparent (p-value < 0.001). Media with 0.05%
MCA similarly inhibited fungal growth. Minor growth rates at such concentrations were recorded only for
T. versicolor 159 and R. placenta 45. These two strains also outperformed the other fungi at lower
concentrations (p-value < 0.001).In particular, mean growth of R. placenta 45 was overall the highest,
which indicated a high Cu-tolerance. Therefore, this strain was selected for the subsequent tests.
Fungal response to Cu2+ ions and particles
We assessed the response of R. placenta 45 to Cu ions, NPs or bulk material to determine if nano effects
may account for a superior efficacy of MCA. The effect of Cu2+ from dissolution of CuSO4, nano Cu from
MCA, bulk CuCO3·Cu(OH)2, and TBA on fungal growth is shown in Fig. 3 as mean fungal wet biomass
values (dried biomass are in accordance and are not shown).
The differences observed between the tested groups were significant, as indicated by Tukey’s test on the
wet biomass production. The addition of TTM to liquid cultures substantially reduced biomass production.
What emerged is that TBA strongly suppresses growth of R. placenta 45. When TBA was associated with
Cu (in MCA or with CuCO3·Cu(OH)2) its inhibition was reduced as follows: TBA > TBA +
CuCO3·Cu(OH)2 > MCA.
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Fig. 2. Influence of different concentrations and formulations of micronized copper azole (MCA) on
the daily growth rate of A. serialis 43, C. puteana 62, G. trabeum 100, R. placenta 45, and T.
versicolor 159. The MCA_HTBA formulation contains high amount of TBA (5% w/w), whereas
MCA_LTBA contains low amount of TBA (0.4% w/w). Data represented as mean ± standard deviation of
three repetitions.
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Fig. 3. Influence of different forms of Cu on the fungal wet biomass produced by R. placenta 45 in
liquid cultures. TTM= 0.02 mM ammonium tetrathiomolybdate; MCA= 0.02 mM of free Cu2+ ions in
micronized copper azole; CuCO3= 0.02 mM Cu carbonate; TBA= 5% w/w tebuconazole; CuSO4= 0.02
mM Cu sulfate. Data represented as mean ± standard deviation of three repetitions. Shared letters indicate
no significant difference in wet biomass production, different letters denote significant differences in wet
biomass production after the Tukey’s HSD test.
Laccase activity
Laccase was detected in both MCA- treated (MCA_LTBA and MCA_HTBA) and untreated wood. The
amount detected was minor (approx. 1U/L). On the other hand, monitoring of the enzymatic reaction of
laccase in liquid cultures by spectrophotometry did not indicate the production of laccase by R. placenta
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45 in the growth media after 2, 4 and 9 weeks incubation period (data available through the ETH Data
Archive at: http://doi.org/10.5905/ethz-1007-21).
Oxalic acid production
Fig. 4 shows the mean values of oxalic acid produced by R. placenta 45 at different conditions.
The amount of oxalic acid measured represents only soluble free acid and salts, but it does not take into
account the copper oxalate and/or calcium oxalate water insoluble precipitates. The differences observed
between the different groups were significant (Tukey’s test). Similarly to the results highlighted in the
fungal biomass tests, TBA heavily suppressed the production of oxalic acid by R. placenta 45. Oxalic acid
production in cultures with MCA, CuCO3·Cu(OH)2 , CuCO3·Cu(OH)2+TBA was lower than in the
controls. The addition of TTM to the Cu- amended liquid cultures resulted in an increase in the oxalic acid
production from MCA and CuCO3·Cu(OH)2, both containing CuCO3·Cu(OH)2, whereas it did not cause
any major difference in CuCO3·Cu(OH)2+TBA and CuSO4. The highest oxalic acid concentration was
measured in cultures exposed to MCA + TTM.
The protein profiles obtained by SDS-PAGE showed no difference in the protein expression profiles
involved for R. placenta 45 under different growth conditions (data available through the ETH Data
Archive at: http://doi.org/10.5905/ethz-1007-21), therefore it is evident that laccase and oxalic acid are
the main contributors and no further protein analysis to determine different behavior due to Cu exposure
was performed.
Efficacy of MCA active substances
We assessed the contribution of TBA and CuCO3·Cu(OH)2 in MCA-treated wood. Wood mass losses for
the different treatments are presented in Fig. 5.
MCA_HTBA, MCA_LTBA and TBA effectively protected wood against fungal colonization and
degradation. In particular, MCA_HTBA and MCA_LTBA significantly inhibited fungal growth even at
the lowest concentration of 0.8% compared to the controls (p-value < 0.001 in both cases, data available
through the ETH Data Archive at: http://doi.org/10.5905/ethz-1007-21). Wood samples treated with
CuCO3·Cu(OH)2 did not reduce mass losses (p-value = 1 for both concentrations). The effects of
MCA_HTBA and MCA_LTBA were significantly different at concentrations ≥ 1.33% (data available
through the ETH Data Archive at: http://doi.org/10.5905/ethz-1007-21): while the mass losses of
MCA_HTBA-treated wood samples were < 3%, mean mass losses of MCA_LTBA-treated wood samples
decreased by 9.1%. At concentrations of 1.6% the effect of MCA_LTBA was comparable to the
equivalent amount of TBA alone (p-value = 0.997), whereas at concentrations of 2% MCA_LTBA
showed a better performance (p-value < 0.001).
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Fig. 4. Influence of different forms of Cu on the oxalic acid production by R. placenta 45 in liquid
cultures. TTM= 0.02 mM ammonium tetrathiomolybdate; MCA= 0.02 mM of free Cu2+ ions in
micronized copper azole; CuCO3= 0.02 mM copper carbonate; TBA= 5% w/w tebuconazole; CuSO4=
0.02 mM Cu sulfate. Data represented as mean ± standard deviation of three repetitions. Shared letters
indicate treatments that were not significantly different, different letters denote significant differences in
treatments after the Tukey’s HSD test.
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Fig. 5. Assessment of micronized Cu azole (MCA) formulations, associated mass losses and the role
of each active substance for wood protection against R. placenta 45. CuCO3= Cu carbonate; TBA=
tebuconazole. The MCA_HTBA formulation contains a high amount of TBA (5% w/w), whereas
MCA_LTBA contains a low amount of TBA (0.4% w/w). Data are represented as mean ± standard
deviation of four repetitions. Shared letters indicate treatments that were not significantly different,
different letters denote significant differences in treatments after the Tukey’s HSD test.
Discussion
Cu is currently used to protect wood from fungal decomposition due to its antifungal properties. In
particular, Cu is responsible for interference with homeostatic processes and cell membrane functions
[41], protein and enzyme damage and precipitation [42], production of reactive oxygen species [43, 44]
and DNA disruption [45]. When Cu is available as NPs these effects may be enhanced. We investigated
here in a systematic approach which formulation (ionic, nano or bulk) of Cu is the most effective against
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Cu-tolerant basidiomycetes. We discriminated between the effects caused by the particles themselves and
those caused by their dissolution into Cu2+ ions using TTM, a chelator for Cu2+ ions.
Our results showed that T. versicolor 159 and R. placenta 45 were the two strains that were less
influenced by the MCA formulations. We mainly attributed this behavior to TBA-tolerance mechanisms
for the white rot fungus T. versicolor 159 [46], and to Cu-tolerance mechanisms for the brown rot fungus
R. placenta 45 [47]. The main mode of action for TBA consists in fungal cell membrane disruption by
inhibition of ergosterol formation [48], whereas Cu exerts its toxic effects on fungal cells by disrupting
different basic metabolic processes. Therefore, R. placenta 45 was selected for the subsequent studies, as
it would provide an indication for possible nano effects exerted by MCA on highly Cu-tolerant fungi that
would reduce the Cu threshold level and would result in effective protection of wood at lower Cu
concentration than the Cu2+ ion or bulk counterpart.
The liquid culture study confirmed the suitability of TTM to discriminate between Cu2+ ionic and particle
effects. In TTM only-amended cultures, biomass and oxalic acid production were lower than in the control
cultures, indicating that TTM bound to essential Cu2+. Therefore, the study shows that TTM can be
effectively employed for studies on Cu-based NPs, for instance in the field of nanotoxicology, where a
similar approach has been developed for zinc oxide NPs by Bürki-Thurnherr et al. [49].
It was not possible to identify the presence of laccase in liquid cultures, although this was clearly detected
on wood in the EN 113 study. In addition, the amount of laccase detected in untreated and MCA-treated
wood was similar. These two findings indicate that laccase is probably not the principal mechanism for Cu
detoxification in R. placenta. Furthermore, we have evidence that supports the fundamental role played by
laccase in the Fenton reaction. The Fenton reaction is used by brown and white rot fungi to initialize the
attack of wood, as it allows the depolymerization of polysaccharides and lignin by generating radicals
[50], whereas in artificial media sugars are readily available, hence radicals are not required. Fungal
biomass and oxalic production measurements provided a clear picture on fungal response to Cu in its
various forms and TBA. The lower oxalic acid concentrations found in MCA, CuCO3·Cu(OH)2 ,
CuCO3·Cu(OH)2+TBA cultures is in good agreement with Green and Clausen findings [51], which
revealed that the oxalic acid production of two R. placenta strains was reduced in Cu-treated wood. The
higher oxalic acid concentrations in CuSO4 (Cu2+ ions)-amended cultures can be related to the increased
biomass produced. In addition, the oxalic acid production was stimulated in Cu+TTM-amended cultures,
therefore confirming that free Cu can reduce oxalic acid production. For both fungal biomass and oxalic
acid production, the major inhibiting agent was TBA, however the effects were reduced in the presence of
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Cu, especially for MCA. Therefore, we hypothesize that Cu, here at sub-lethal concentrations, can
stimulate growth and enzyme production of R. placenta, as indicated in former studies [52, 53]. In
addition, for MCA other chemicals in the formulation may have influenced fungal growth by providing
additional nutrients. Thus, for the concentrations used, there was no indication of a Cu+TBA additive or
synergistic effects against Cu-tolerant fungi. Although there is a lack of scientific literature on Cu and
TBA additive, synergistic or antagonist effects, Sun et al. [54] showed a similar behavior for a range of
moulds that can biotransform TBA [55, 56], hence effectiveness of pure Cu was higher than Cu combined
with TBA. In any case, we found no evidence for a specific nano effect against R. placenta and the main
active substance against R. placenta was clearly TBA. This means that the fungus is able to recognize Cu
also as MCA NPs and can trigger the same Cu-tolerance mechanisms typically shown in the presence of
bulk Cu or Cu ions. No additional protein expression patterns were evident in SDS-PAGE analysis,
therefore oxalic acid and laccase were valid parameters for determining fungal response to Cu.
Finally, to complete the study, we investigated fungal growth on treated wood i.e. a more natural setting.
In this case, the EN 113 guidelines were applied and mass losses of wood treated with bulk
CuCO3·Cu(OH)2, TBA and two MCA formulations differing in TBA content were compared. This test
provides indications on the expected short term effectiveness of the wood treatments.
The recorded mass losses were in good agreement with our findings on fungal growth in liquid cultures.
Even in treated wood TBA is largely responsible for the effectiveness of MCA, although high
concentrations of Cu also affect the performance. In particular, for the formulations and wood decay fungi
assessed, we propose a 1.6% MCA < Cu threshold ≤ 2% MCA. The low effectiveness of CuCO3·Cu(OH)2
is mainly attributed to the poor penetration into the wood: the wood samples did not show any color
change towards green/blue due to the presence of Cu, and unreacted CuCO3·Cu(OH)2 only appeared as
fine dust unbound on the sample surfaces.
In conclusion, the NPs in the MCA formulations assessed did not provide additional protection against R.
placenta and the main effectiveness has to be attributed to TBA. Therefore, considering the antifungal
properties, the efficacy of the MCA formulations tested are not better than conventional Cu azole
formulations that do not employ nanotechnologies. MCA-treated wood will still be susceptible to
biodegradation by Cu- or TBA-tolerant fungi. From a life cycle assessment perspective, MCA is less eco-
efficient than Cu azole, due to the higher energy consumption during the milling process of MC [57].
However, this would also imply no additional risk for the microbial community in vicinity of MCA-
treated wood.
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Further studies with other wood-destroying fungi and different MC formulations are required to provide a
more comprehensive picture on MC NPs effects on wood-destroying fungi. In addition, field studies are
required to confirm our lab-scale findings and assess the long term performance. In particular, TTM could
not be used in the EN 113 test, due to the absence of a liquid environment that would allow chelation of
Cu2 + ion. Therefore, tests with wood samples immersed in liquid cultures and TTM, in a setting similar to
the one suggested by the EN 275 [58] guidelines, may provide further details on the fungal response to
Cu2+ ions and particles. Future studies should focus on the fungal gene pathways that are involved for
tolerance mechanisms against TBA and Cu.
Acknowledgments
We are indebted to Daniel Heer for the technical assistance, to Liliane Diener for the TEM investigation
and to Xu He for the ζ-potential measurements. The study was financially supported by the Swiss National
Research Foundation (NRP 64, Grant No. 406440_141618).
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39. Lomascolo A, Record E, Herpoël-Gimbert I, Delattre M, Robert JL, Georis J, et al. Overproduction
of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer. Appl
Environ Microbiol. 2003 Apr; 94(4): 618–624.
40. Civardi C, Schwarze FWMR, Wick P. Micronized copper wood preservatives: An efficiency and
potential health risk assessment for copper-based nanoparticles. Env Poll. 2015 May; 200:126-132.
41. Ohsumi Y, Kitamoto K, Anraku Y. Changes induced in the permeability barrier of the yeast
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42. Kim JH, Cho H, Ryu SE, Choi MU. Effects of metal ions on the activity of protein tyrosine
phosphatase VHR: highly potent and reversible oxidative inactivation by Cu 2+ ion. Arch Biochem
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43. Krumova EZ, Pashova SB, Dolashka-Angelova PA, Stefanova T, Angelova, MB. Biomarkers of
oxidative stress in the fungal strain Humicola lutea under copper exposure. Process Biochem.2009
Mar; 44(3): 288-295.
44. Sharma SS, Dietz KJ. The relationship between metal toxicity and cellular redox imbalance.
Trends Plant Sci. 2009 Jan; 14(1): 43-50.
45. Sagripanti JL, Goering PL, Lamanna A. Interaction of copper with DNA and antagonism by other
metals. Toxicol Appl Pharm. 1991 Sep 15; 110(3): 477-485.
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46. Woo C, Daniels B, Stirling R, Morris P. Tebuconazole and propiconazole tolerance and possible
degradation by Basidiomycetes: A wood-based bioassay. Int Biodeter Biodegradation 2010 Aug;
64(5): 403-408.
47. Sutter HP, Jones EBG, Walchli O. The mechanism of copper tolerance in Poria placenta (Fr.)
Cke. and Poria vaillantii (Pers.) Fr. Mater Organismen 1983; 18: 241-262.
48. Joseph-Horne T, Hollomon D, Loeffler RST, Kelly SL. Altered P450 activity associated with
direct selection for fungal azole resistance. FEBS J. 1995 Oct 30; 374(2): 174-178.
49. Buerki-Thurnherr T, Xiao L, Diener L, Arslan O, Hirsch C, Maeder-Althaus X, et al. In vitro
mechanistic study towards a better understanding of ZnO nanoparticle toxicity. Nanotoxicology
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50. Arantes V, Milagres AM, Filley TR, Goodell B. Lignocellulosic polysaccharides and lignin
degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions. J Ind
Microbiol Biotechnol. 2011 Apr; 38(4): 541-555.
51. Green III F, Clausen CA. Copper tolerance of brown-rot fungi: time course of oxalic acid
production. Int Biodeter Biodegradation 2003 Mar; 51(2): 145-149.
52. Englander CM, Corden ME. Stimulation of mycelial growth of Endothia parasitica by heavy
metals. Appl Microbiol. 1971 Dec; 22(6): 1012-1016.
53. Wazny J, Thornton JD. Comparative laboratory testing of strains of the dry rot fungus Serpula
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54. Sun F, Bao B, Ma L, Chen A, Duan X. Mould-resistance of bamboo treated with the compound of
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mutants of Trichoderma viride. Acta Phytopathol Entomol Hung. 1999 Dec 10; 34(1-2): 27-34.
56. Obanda DN. Biotransformation of organic wood preservatives by micro-organisms [dissertation].
Baton Rouge (LA): Louisiana State University; 2008.
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57. Habicht J. Mikronisiertes Kupfer im Holzschutz. Bewertung der Einsatzmöglichkeiten unter
europäischen Bedingungen. In: Universität Göttingen, Burckhardt-Institut, Abteilung Holzbiologie
und Holzprodukte, editors. 26. Holzschutz-Tagung. Neue Normen, neue Erkenntnisse; 2010 Apr
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58. European Committee for Standardization. EN 275 Wood preservatives. Determination of the
protective effectiveness against marine borers. Brussel, BE; 1992.
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4.1.2 Effectiveness of micronized copper azole against soft rot fungi
Unpublished report
Keywords: Micronized copper azole, Pressure-treated wood, Soilborne fungi, X-ray computed
tomography
Abstract
The protective efficacy of micronized copper azole (MCA) against soft rot fungi was assessed by
combining the European Standard ENV 807 [1], which relates wood decay with wood mass loss, and X-
ray computed tomography (CT) to visualize the decay at low and high resolutions. The results indicate
that in a laboratory setting MCA can protect wood against soft rot fungi and other soilborne
microorganisms.
Introduction
Copper (Cu) is essential for the protection of wood in ground contact (use class 4, European Standard EN
335 [2]), as it has the potential to prevent decay by soft rot fungi. Recently, an innovative wood
preservative treatment based on particulate Cu was developed. This is best known as “micronized copper”
(MC) and is mainly composed of basic copper carbonate particles, whose sizes range between 1 nm and
250 μm, and an organic co-biocide, either azole (MCA) or quaternary ammonium compounds (MCQ) [3,
4]. Since its introduction to the wood protection market in 2006 [5], issues have been raised concerning its
effectiveness against soft-rot fungi [6]. In order to answer these questions, a few studies investigated these
aspects. Zhang and Ziobro [7] assessed the performance of MCA- and MCQ-pressure-treated P. radiata
and alpine ash (E. delegatensis), wood species uncommon in Europe, against soft rot fungi and soilborne
microorganisms in laboratory and field settings. Their tests highlight how MCA performed equally or
better than conventional or MCQ wood preservatives. Ray et al. [8] investigated the effectiveness of
MCQ-pressure-treated beech (F. sylvatica) and Scots pine (P. sylvestris), which are European wood
species, against soft rot fungi applying the European Standard ENV 807 [1]. Their results indicate that
MCQ and conventional preservatives provide equivalent performance in beech, but the conventional wood
preservatives performed better in pine. This paper aims at filling the gaps investigating the effectiveness
of MCA for the European Scots pine against soft rot fungi. In this way, we provide a direct comparison
between the performance of MCA and MCQ, as well as the influence of the wood species on the
treatment. We combined the laboratory soil block tests with a visual assessment by means of X-ray
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computed tomography (CT) to get an insight into the effective mechanisms, and on the type and
development of decay.
Material and methods
Wood and wood preservatives
Small wood stakes (100 x 10 x 5 mm) excised from the of Scots pine (Pinus sylvestris L.) sapwood were
pressure-treated according to the European standard ENV 807 [1] with different concentrations (2.00%,
1.60%, 1.33%, 1.07%, 0.80%, 0.50%, 0.00%) of a commercial aqueous MCA formulation and with two
different concentrations (2.00%, 1.60%) of a conventional CC wood preservative prepared according to
the ENV 807 [1]. Leaching of all test wood samples was performed according to the European standard
EN 84 [9].
Effectiveness against soft rot fungi
After drying, the ENV 807 wood samples were inserted and buried into boxes filled with unsterile
conditioned natural top soil for 32 weeks at 28 °C and 85% RH. Test procedures were performed
according to the European standard ENV 807 [1]. After incubation, wood blocks were removed from the
test vessels, brushed free of soil and oven dried at 103±1 °C for a minimum of 18 h. The percentage of
mass loss was calculated from the dry mass before and after the test.
Visualization of wood decay
Treated and untreated, colonized and uncolonized ENV 807 wood blocks and small subsamples (approx. 5
x 0.5 x 0.5 mm) extracted from the larger blocks were scanned with the multi-resolution micro-CT
scanner Nanowood [10, 11] built at the Ghent University Centre for X-ray Tomography (Ghent
University, Belgium). The large wood blocks were visualized to assess the overall wood decay. The
samples were scanned using a closed type microfocus X-ray tube at 100 kV and 80 µA, 1000 projections
and 1000 ms exposure time per projection. Reconstructions were performed with the Octopus
Reconstruction software package [12], a tomography reconstruction package for parallel, cone-beam and
helical geometry licensed by InsideMatters (www.insidematters.be), resulting in reconstructed data with
an approximated voxel pitch of 31 μm. The reconstructed volumes were analyzed using Octopus Analysis,
previously known as Morpho+ [13] and also licensed by InsideMatters.
The small subsamples were used to investigate fungal decay in wood at the cellular level. The samples
were scanned using an open type nanofocus X-ray tube at 80 kV and 45 µA, 1000 projections and 1500
ms exposure time per projection. Reconstructions were also performed with the Octopus Reconstruction
software package, resulting in volumes with an approximate voxel pitch of 0.8 μm.
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Statistical analysis
Wood mass losses, which were expressed as percentages, were arcsine-transformed prior to analysis
(ANOVA) to obtain a normal distribution, and back-transformed to numerical values for graphical
display. Means were separated using Tukey’s-HSD (Honestly Significant Difference) test at significance
level p<0.05. The statistical package used for all analyses was SPSS® (Version 17.0, SPSS Inc., Chicago,
IL, USA).
Results
Effectiveness against soft rot fungi
Wood mass losses for the different MCA- and CC-pressure-treated samples after 32 weeks are reported in
Fig. 1.
Generally, a cut-off at 3% is applied to determine if a preservative treatment is effective or not. The soft
rot activity in the test environment, as measured in the control samples, was not high (mean mass loss
below 15%). For industry purpose, these wood mass losses would invalidate the test, however these data
are valid for research purposes. For the ENV 807 to be valid, the mass loss differences between the
control and water-treated (0.00%) samples shouldn’t be significant, to prove that there was no issue with
the impregnation process. In our case, the mass losses from the control and water-treated samples were
comparable, indicating that the impregnation was conducted correctly. MCA significantly inhibited soft
rot growth on wood blocks even at the low concentration of 0.80%, with mass losses as low as
1.92±0.86%. However even at the lowest concentration of 0.50% wood decay was considerably reduced
(average wood mass loss: 3.1±1.5%). No significant difference was observed between the 0.80%, 1.07%,
and 1.33% MCA-pressure-treated wood samples. Whereas the mass losses in 2.00% and 1.60% MCA-
pressure-treated wood were considerably low (and comparable). Even 2.00% and 1.60% CC-pressure-
treated wood blocks showed mass changes below |3|%. However, these samples were characterized by a
slight mass increase, which could probably be explained by the interaction of CC with wood and/or soil.
Therefore, the results of MCA and CC were not comparable, as suggested by the output from Tukey’s
HSD test, and it is not possible to determine if CC- or MCA-pressure-treated wood had a better
performance against soft rot fungi. Wood cell-wall treatments are more effective than cell-lumen ones
against soft rot fungi [14] and are less prone to leaching [15]. The performance of MCA against soft rot
fungi was positive, despite the leaching procedure.
Visualization of wood decay
X-ray CT provided a useful tool to confirm the presence and type of decay on ENV 807 wood blocks.
Both high and low resolution scans of the ENV 807 wood blocks showed how the fungi were active only
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in the outer layers of wood, which is typical for soft rot decay (Fig. 2a). The low resolution scans revealed
that decay occurred more abundantly in the earlywood. This may be caused by the larger openings in
Fig. 1. Assessment of micronized Cu azole (MCA) and Cu/chromium (CC) effectiveness against soft
rot fungi. Data are represented as mean ± standard deviation of four repetitions. Shared letters indicate
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treatments that were not significantly different, different letters denote significant differences in treatments
after the Tukey’s HSD test.
Fig. 2. Reconstructed slices of MCA-pressure-treated Scots pine at (a) low and (b, c) high
resolutions. The decay occurs from the outer surfaces towards the inner layers. Soft rot decay is more
abundant in earlywood. The arrows in (b) indicate the soft rot decay. In (c) the arrow point to a high-
density spot that may be attributed to Cu.
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earlywood compared to latewood, which allow the soilborne fungi to penetrate more easily into the wood
structure.
Another factor could be the presence of higher amount of Cu in latewood after pressure-treatment, as
indicated by Evans et al. [16]: although earlywood’s openings and pits could account for an easier
penetration of Cu, latewood has a higher number of carboxylic acid reaction sites that could promote the
solubilization and complexation of Cu by wood components. No fungal structure was visible in the high
resolution scans (Fig. 2b). This agrees with the type of fungal attack expected, as soft rot fungi do not
have hyphal structures like basidiomycetes, therefore we can confirm that the wood decay was caused by
soft rot and other soilborne microorganisms, without any interference from other organisms. In both
reconstructed samples it was also difficult to locate bright spots that would account for obvious particles
or accumulation of high-density Cu spots, which appear rarely across the reconstructions (Fig. 2c). This
could indicate that Cu is distributed within the wood structure as fine particles (or ions) and that the co-
biocide tebuconazole (not visible by X-ray CT) could have played a significant role against soft rot fungi.
Discussion
The aim of this study was to assess whether MCA can protect wood from soft rot fungi inhabiting the soil
and outperform conventional wood preservatives. We combined the European Standard test ENV 807 [1]
with X-ray CT imaging in order to thoroughly assess the effectiveness of a MCA wood preservative and
the decay caused to the wood blocks in soil. Our results are in good agreement with Zhang and Ziobro [7]
and Ray et al. [8], and indicate that MCA is a valid wood preservative against soft rot fungi. In particular,
the concentrations required to effectively prevent fungal decay were as low as 0.80%. Based on the good
performance, we hypothesize that leaching of MCA-pressure-treated wood is likely to result in wood cell-
wall Cu treatment. However tebuconazole may contribute to the effectiveness against soft rot fungi before
its possible biodegradation by microorganisms inhabiting the soil [17]. Long-term stake test studies
according to EN 252 [18] with MCA-pressure-treated Scots pine should be performed in order to confirm
the validity of the laboratory test, and for comparison with laboratory and field tests on MCQ-pressure-
treated wood [19-21]. Further, investigations on the contribution of tebuconazole in MCA towards soft rot
fungi should be carried out.
References of Chapter 4.1.2
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1. European Committee for Standardization. ENV 807 Wood preservatives – Determination of the
effectiveness against soft rotting micro-fungi and other soil inhabiting microorganisms. Brussels,
BE; 2001.
2. European Committee for Standardization. EN 335 Durability of wood and wood-based products-
Use classes: definitions, application to solid wood and wood-based products. Brussels, BE; 2013.
3. Leach RM, Zhang J. Micronized wood preservative formulations comprising metal compounds
and organic biocides. World patent 2004091875, 32 pp; 2004.
4. Leach RM, Zhang J. Micronized wood preservative formulation. World patent 2005104841, 26
pp.; 2005.
5. Osmose. Consumer safety and product performance of micronized copper technology confirmed.
Osmose press release, Griffin (GA); 2009. Available: http://www.prnewswire.com/news-
releases/consumer-safety-and-product-performance-of-micronized-copper-technology-confirmed-
65722682.html [Accessed: 12 June 2015]
6. Archer K. Cell wall penetration of particulate copper wood preservative system in southern pine.
Unpublished oral presentation at 38th Annual Meeting; 2007 May 20-24; Jackson Lake, WY.
International Research Group on Wood Protection; 2007.
7. Zhang J, Ziobro RJ. Laboratory and field studies on the efficacy of micronized copper azole
(MCA) preservative. CWPA Proceedings; 2009; pp. 69-75.
8. Ray M, Dickinson D, Archer K. A comparison of the performance of related copper based
preservatives against soft rot. IRG/WP 10-30540. Proceedings IRG Annual Meeting; 2010 May 9-
13; Biarritz, FR. Stockholm, SE: International Research Group on Wood Protection; 2010.
9. European Committee for Standardization. EN 84 Wood preservatives – Accelerated ageing of
treated wood prior to biological testing-Leaching procedure. Brussels, BE; 1997.
10. Dierick M, Van Loo D, Masschaele B, Van den Bulcke J, Van Acker J, Cnudde V, et al. Recent
micro-CT scanner developments at UGCT. Nucl. Instum. Meth. B 2014 Apr 1; 324: 35-40.
11. Dierick M, Van Loo D, Masschaele B, Boone M, Van Hoorebeke L. A LabVIEW® based generic
CT scanner control software platform. J. X-ray Sci. Technol. 2010 Oct 29; 18(4):451-461.
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12. Vlassenbroeck J, Dierick M, Masschaele B, Cnudde V, Van Hoorebeke L, Jacobs P. Software
tools for quantification of X-ray microtomography at the UGCT. Nucl. Instrum. Meth. Phys. Res.
A 2007 Sep 21; 580(1): 442-445.
13. Brabant L, Vlassenbroeck J, De Witte Y, Cnudde V, Boone MN, Dewanckele J, et al. Three-
dimensional analysis of high-resolution X-ray computed tomography data with Morpho+.
Microsc. Microanal. 2011 Apr; 17(2): 252-263.
14. Hale MD, Eaton RA. Soft-rot cavity formation in five preservative-treated hardwood species.
Trans. Br. Mycol. Soc. 1986 Jun; 86(4): 585-590.
15. Arsenault RD. Factors influencing the effectiveness of preservative systems. In: Nicholas DD.
Wood deterioration and its prevention by preservative treatments. Vol. 2, 121-278. New York,
NY: Syracuse University Press; 1973.
16. Evans P, Limaye A, Averdunk H, Turner M, Senden TJ, Knackstedt MA. Use of X-Ray Micro-
Computed Tomography to Visualize Copper in Preservative Treated Wood. IRG/WP 12-20488.
Proceedings IRG Annual Meeting; 2012 May 6-10; Kuala Lumpur, MY. Stockholm, SE:
International Research Group on Wood Protection; 2012.
17. Obanda DN, Shupe TF. Biotransformation of tebuconazole by microorganisms: evidence of a
common mechanism. Wood Fiber Sci. 2009Apr; 41(2): 1-11.
18. European Committee for Standardization. EN 252 Field test method for determining the relative
protective effectiveness of a wood preservative in ground contact. Brussels, BE; 2014.
19. Cooper P, Ung Y. Comparison of laboratory and natural exposure leaching of copper from wood
treated with three wood preservatives. IRG/WP 08-50258. Proceedings IRG Annual Meeting;
2008 May 25-29; Istanbul, TR. Stockholm, SE: International Research Group on Wood
Protection; 2008.
20. Lebow S, Hatfield C, Crawford D, Woodward B. Long-Term Stake Evaluations of Waterborne
Copper Systems. Proceedings of the Ninety-Ninth Annual Meeting of the American Wood-
Preservers' Association; 2003.
21. Preston A, Jin L, Nicholas D, Zahora A, Walcheski P, Archer K, Schultz T. Field Stake Tests with
Copper-based Preservatives. IRG/WP 08-30459. Proceedings IRG Annual Meeting; 2008 May
25-29; Istanbul, TR. Stockholm, SE: International Research Group on Wood Protection; 2008.
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4.2 Penetration and distribution of Cu in wood PLoS ONE 11 (2016) e0163124
Penetration and effectiveness of micronized copper in refractory wood species
Short title: Can micronized copper protect refractory wood?
Chiara Civardi1,2*, Jan Van den Bulcke3, Mark Schubert2, Elisabeth Michel4, Maria Isabel Butron2,
Matthieu N. Boone5, Manuel Dierick5, Joris Van Acker3, Peter Wick6, Francis WMR Schwarze2*
1ETH, Institute for Building Materials, Zürich, Switzerland
2Empa, Applied Wood Materials, Dübendorf/ St. Gallen, Switzerland
3UGCT - Woodlab-UGent, Laboratory of Wood Technology, Department of Forest and Water
Management, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
4Empa, Advanced Fibers, St. Gallen, Switzerland
5UGCT - Radiation Physics, Department of Physics and Astronomy, Proeftuinstraat 86/N12, Ghent
University, 9000 Ghent, Belgium
6Empa, Particles-Biology Interactions, St. Gallen, Switzerland
*Corresponding authors
Email: [email protected] (CC), [email protected] (FS)
Keywords: Copper-tolerant wood-destroying fungi, Copper uptake, Micronized copper azole, Norway
spruce, Pressure-treated wood, Wood durability, X-ray computed tomography
Abstract
The North American wood decking market mostly relies on easily treatable Southern yellow pine (SYP),
which is being impregnated with micronized copper (MC) wood preservatives since 2006. These
formulations are composed of copper (Cu) carbonate particles (CuCO3·Cu(OH)2), with sizes ranging from
1 nm to 250 µm, according to manufacturers. MC-treated SYP wood is protected against decay by
solubilized Cu2+ ions and unreacted CuCO3·Cu(OH)2 particles that successively release Cu2+ ions
(reservoir effect). The wood species used for the European wood decking market differ from the North
American SYP. One of the most common species is Norway spruce wood, which is poorly treatable i.e.
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refractory due to the anatomical properties, like pore size and structure, and chemical composition, like pit
membrane components or presence of wood extractives. Therefore, MC formulations may not be suitable
for refractory wood species common in the European market, despite their good performance in SYP. We
evaluated the penetration effectiveness of MC azole (MCA) in easily treatable Scots pine and in refractory
Norway spruce wood. We assessed the effectiveness against the Cu-tolerant wood-destroying fungus
Rhodonia placenta. Our findings show that MCA cannot easily penetrate refractory wood species and
could not confirm the presence of a reservoir effect.
Introduction
Wood is a widely used building material and one of the reasons is its availability in various countries. This
also results in a geographic-dependent decking market, as each country mostly utilizes the most accessible
wood species. For instance, the North American market mostly uses SYP [1], whereas Norway spruce is
the most used species in Central Europe [2].
The choice of a certain wood species for building applications results in clearly defined performance and
service life, which can range from a few to many years. The specific anatomical and compositional
features of wood are species-specific and cause major differences in the natural durability and the
permeability to wood preservatives (or treatability), resulting in different wood species-based durability
classes. According to the EN 350-2 [3] treatability is defined as the ease at which wood can be penetrated
by liquids, e.g. wood preservatives. This mainly depends on the wood openings, the size and structure of
tracheids, fibers, vessels, bordered- and simple pits. Due to the large size of the simple pits SYPs (Pinus
caribaea, Pinus echinata, Pinus palustris, and Pinus taeda) are considered as easily treatable species,
especially their sapwood [3]; whereas Norway spruce sapwood and particularly heartwood [4] are
refractory to wood preservatives due to closure of the bordered pits [5, 6]. Therefore, wood preservative
treatments developed for the easily treatable wood of the North American market may not be suitable for
refractory wood species, like European widely used Norway spruce [2], or they may require additional
treatments prior to impregnation, e.g. wood incising. This process facilitates drying of refractory species
[7], and improves the penetration and retention of preservatives [8, 9].
One of the most common and at the same time most demanding building applications for wood is for
structures in contact with the soil, defined as use class 4 by the European Standard EN 335 [10]. The latter
wood products have to be treated with copper (Cu)-based wood preservatives to avoid decay by soil-borne
microorganisms, especially soft-rot fungi [11]. The latest advance in Cu-based wood preservation is
known as “micronized copper” (MC), and consists of basic Cu carbonate (CuCO3·Cu(OH)2) particles with
a size range of 1 nm-250 µm, according to manufacturers, combined with a second organic biocide that
provides protection against basidiomycetes, either azole (MCA) or quaternary ammonium compounds
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(MCQ) [12,13]. MC reputedly has a special impregnation chemistry that differs from conventional wood
preservatives [14], i.e. part of CuCO3·Cu(OH)2 immediately solubilizes during the impregnation process
and is complexed by wood organic macromolecules, while another fraction does not react and acts as
reservoir, i.e. Cu is solubilized afterwards [14] and provides a continuous protection against wood-
destroying fungi [15].
The entire literature on Cu distribution and speciation of this wood preservative in wood deals with easily
treatable SYP [15-22], Scots pine wood [23], or red pine [17], and hypotheses on the possible penetration
ability of MC in refractory wood species have been proposed [24]. However, to our knowledge there are
no studies available that demonstrate the benefits of MC formulations for refractory wood species, either
from North America or from Europe. Thus actual data on the Cu distribution from MC-treated refractory
wood, as well as on its resistance against wood-destroying basidiomycetes are missing.
We hypothesize that the properties of MC by themselves cannot guarantee a homogeneous wood
preservative penetration into refractory wood species, similarly to conventional wood preservatives. In
order to verify that, in this paper we assess and compare the penetration of copper from MCA in Scots
pine sapwood, Norway spruce sapwood and heartwood without any prior incision of wood. The
penetration of Cu was assessed directly by means of X-ray computed tomography (CT) and ion-coupled
plasma-optical emission spectroscopy (ICP-OES), and by indirect methods provided by the European
standard EN 113 guidelines [25]. In addition, we compared MCA penetration effectiveness with its
protective effectiveness against the Cu-tolerant [26] wood-destroying fungus Rhodonia placenta, which
we previously used to test the ionic, nano, and bulk Cu effects of MCA [27]. By using a Cu-tolerant
basidiomycete we could get an insight into the mechanisms behind MC superior effectiveness compared
to conventional wood preservatives, as the fungus would not immediately succumb due to the presence of
Cu, even if minimal, as it would happen with soft rot fungi.
Material and methods
Materials
A commercial aqueous suspension of MCA was investigated. The formulation tested here coincides with
the formulation with high amount of tebuconazole MCA_HTBA we used in our previous investigation
[27]. The latter study also provides a full characterization of the Cu particles in the MCA formulation. In
brief, the measured particle size distribution of MCA was 104±1.7 nm with an average zeta potential of -
21±0.4 mV.
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Wood blocks (50 x 25 x 15 mm) were excised from Scots pine (Pinus sylvestris L.) sapwood, Norway
spruce (Picea abies (L.) Karst.) heartwood and sapwood. Norway spruce sapwood and heartwood were
localized by visual inspection, the heartwood was selected from an area as close as possible to the center
of the trunk, while sapwood was selected from the outer region. The wood samples were pressure-treated
according to the European standard EN 113 [25] with different concentrations of a commercial MCA
aqueous suspension (100.00%, 2.00%, 1.60%, 1.33%, 1.07%, 0.80%, 0.00%). Three repetitions of
100.00% MCA-pressure-treated wood samples and six replicates for the diluted MCA-pressure-treated
wood specimens were prepared. Small needle-shaped wood specimens (5 x 0.5 x 0.5 mm) from Scots pine
sapwood, Norway spruce sapwood and heartwood were cut from the outer surface of EN 113 untreated
blocks and were subsequently treated with MCA by dipping them into 100.00% MCA. No permits were
required for the described study, which complied with all relevant regulations. No endangered or protected
species were involved.
Cu penetration, uptake and distribution in wood
Preservative retention
Preservative retention in kg/m3 was calculated following the guidelines from the European standard EN
113 [25] with the following formula:
𝑃𝑟𝑒𝑠𝑒𝑟𝑣𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 =𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑢𝑝𝑡𝑎𝑘𝑒∗𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑤𝑜𝑜𝑑 𝑠𝑎𝑚𝑝𝑙𝑒∗ 1000
The solution uptake was calculated as the difference between the wood samples’ wet mass after
impregnation and the oven dried (103±1 °C for 18 h minimum) initial mass prior impregnation, according
to the European standard EN 113 [25].
Quantification of Cu in wood
Cu in wood was quantified by ICP-OES (Perkin-Elmer OPTIMA 3000, detection limit: 0.005 mg/L). For
Scots pine sapwood, Norway spruce sapwood and heartwood EN 113 wood blocks untreated and pressure-
treated with 100% MCA were ground into sawdust. Samples from the inner wood were selected cutting
the first 15 mm off of the samples off and grinding the inner surface obtained. In addition samples from
the outer edge (first 15 mm) of Scots pine sapwood were collected. In this way, a penetration gradient
from the easily treatable wood species could be assessed. Digestion of the samples prior to ICP-OES was
conducted according to Platten et al. [28]. Three replicates of each sawdust sample were digested with 3
mL of HNO3 (65%) and 1 mL of H2O2 (30%) (MLS 1200 MEGA digestion system). Cu plasma standard
solutions (100 mg/L) were used for calibration.
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Visualization of Cu in wood
Before and after treatment, EN 113 wood blocks and small needle-shaped wood specimens from the three
wood materials considered (Scots pine sapwood, Norway spruce sapwood and heartwood) pressure-
treated with 100.00% MCA were analyzed by means of the multi-resolution micro-CT scanner Nanowood
[29, 30] built at the Ghent University Centre for X-ray Tomography (Ghent University, Belgium). The EN
113 blocks were visualized to assess the overall penetration of Cu. The samples were scanned using a
closed type microfocus X-ray tube at 100 kV and 80 µA, 1000 projections and 1000 ms exposure time per
projection. Reconstructions were performed with the Octopus Reconstruction software package [31], a
tomography reconstruction package for parallel, cone-beam and helical geometry licensed by
InsideMatters (www.insidematters.be), resulting in reconstructed data with an approximated voxel pitch of
31 μm. The reconstructed volumes were analyzed using Octopus Analysis, previously known as Morpho+
[32] and also licensed by InsideMatters, to approximately visualize Cu distribution. Therefore, the
reconstructed volumes were bilateral filtered to remove noise with edge preservation. Subsequently the
wood was separated from the surrounding air such that further calculations were only performed within
the wood block. Finally, Cu was segmented based on thresholding. The threshold level of the latter
segmentation was chosen conservatively such that no wood was selected, which was based on scans of
untreated wood blocks scanned with identical settings. Volumes were also rendered in 3D using VGStudio
Max software.
The smaller needle-shaped specimens were used to investigate the Cu distribution in wood at the cellular
level, and possible presence of nanoparticles attached or in the wood cells. The samples were scanned
using an open type nanofocus X-ray tube at 80 kV and 45 µA, 1000 projections and 1500 ms exposure
time per projection. Reconstructions were also performed with the Octopus Reconstruction software
package, resulting in volumes with an approximate voxel pitch of 0.8 μm. Phase contrast effects were
reduced using the Paganin method [33], also significantly improving the signal-to-noise ratio [34]. Due to
the violation of the homogeneous object assumption in this method, additional smoothing around the MC
is however introduced [35]. Obtaining quantitative results from these data is therefore not directly
possible.
For both the low and high resolution scans, approximate detection limits on X-ray CT scans of Cu in wood
were calculated, using the NIST XCOM database [36].
Effectiveness against Cu-tolerant basidiomycetes
After drying, the 2.00%, 1.60%, 1.33%, 1.07%, 0.80%, and 0% MCA-pressure-treated wood samples
were exposed for 16 weeks at 22 °C and 70% RH to the Cu-tolerant wood-destroying basidiomycete R.
placenta isolate 45 from the Empa culture collection. Test procedures were performed according to the
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European standard EN 113 [25]. After incubation, wood blocks were removed from the culture vessels,
brushed free of mycelium and oven dried at 103±1 °C for a minimum of 18 h. The percentage of mass loss
was calculated from the dry weight before and after the test.
Some of the results from MCA-pressure-treated Scots pine wood were formerly published in a previous
study [27], and we integrated it here to provide a complete overview on the effectiveness of MCA in
different wood species.
Statistical analysis
Preservative retention data were log-transformed and data expressed as percentages (Cu amounts in wood
and wood mass losses) were arcsine-transformed prior to analysis (ANOVA) and back-transformed to
numerical values for visualization. Means were separated using Tukey’s-HSD (Honestly Significant
Difference) test at significance level p<0.05. The statistical package used for all analyses was SPSS®
(Version 17.0, SPSS Inc., Chicago, IL, USA).
Results
Cu penetration, uptake and distribution in wood
Preservative retention
According to the European standard EN 113 [25], we gathered indications on the expected MCA retention
in easily treatable Scots pine sapwood and refractory Norway spruce sapwood and heartwood (Fig. 1). The
three wood materials considered share the same preservative retention pattern (p-value=0.137) across all
diluted MCA-pressure-treated wood samples (2.00%, 1.60%, 1.33%, 1.07%, 0.80%), which decreases
with the MCA concentration applied. Therefore, comparable amounts of Cu could be expected in different
wood species pressure-treated with the same MCA concentration, independently of their permeability.
This pattern was not applicable to the 100% MCA-pressure-treated wood samples, where the preservative
retentions were certainly lower, even though the expected amount of Cu was higher. Moreover, a
difference between the refractory Norway spruce heartwood and the more accessible Norway spruce
sapwood and the easily treatable Scots pine sapwood was visible.
Quantification of Cu in wood
We quantified the amount of Cu in both easily treatable Scots pine (sapwood) and refractory Norway
spruce (sapwood and heartwood). Fig. 2 summarizes the weight percentages of Cu detected by ICP-OES
in untreated and 100.00% MCA-pressure-treated Scots pine sapwood (inner and outer regions), Norway
spruce sapwood (inner), and Norway spruce heartwood (inner).
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Fig. 1. Preservative retention in Scots pine sapwood, Norway spruce sapwood, and Norway spruce
heartwood calculated according to the EN 113 guidelines [25]. Data are represented as mean ±
standard deviation of three replicates.
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Fig. 2. Percentage of Cu in Scots pine sapwood (inner and outer surface), Norway spruce sapwood
(inner), and Norway spruce heartwood (inner) measured by ion-coupled plasma-optical emission
spectroscopy (ICP-OES). Data are represented as mean ± standard deviation of three repetitions. Shared
letters indicate treatments that were not significantly different, different letters denote significant
differences in treatments after the Tukey’s HSD test.
When untreated, both Scots pine and Norway spruce wood contain comparable (p-value=1) negligible
amounts of Cu (below detection limit). In 100.00% MCA-pressure-treated wood samples, Scots pine
sapwood (outer regions) had the highest amount of Cu. This percentage decreased in the interior of Scots
pine sapwood, but the values remained higher than in Norway spruce sapwood or heartwood. The latter
contained the lowest Cu amount, thus Norway spruce heartwood was the most refractory wood treated
here. The Tukey’s HSD test indicates that the Cu amount in 100.00% MCA-pressure-treated Norway
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spruce sapwood was not significantly different from the one in 100.00% MCA-pressure-treated Norway
spruce heartwood (p value=0.908) and the interior part of Scots pine sapwood (p-value=0.489).
Visualization of Cu in wood
X-ray CT and subsequent analysis allowed qualitative and semi-quantitative determination of Cu in MCA-
pressure-treated Scots pine sapwood, Norway spruce sapwood, and Norway spruce heartwood.
Thresholding of the EN 113 wood blocks was performed to distinguish Cu in the tomographic
reconstructions. The same conservative threshold was used for all EN113 wood samples.
In Fig. 3 the threshold-based Cu penetrations (red) in the three wood materials considered are visible. The
Cu thresholding on the 31 μm resolution scans of the 100.00% MCA-pressure-treated Norway spruce
samples accounted for only 1% (heartwood) or up to 2% (sapwood) of the volume, which was
significantly lower than the percentages detected by ICP-OES.
In Scots pine and Norway spruce sapwood blocks it was clearly visible that the penetration of Cu occurred
predominantly via resin canals and was more abundant in the latewood. In Scots pine Cu was also present
within the rays. It was furthermore calculated that the detection limit of Cu would be of the order of 0.1 µg
per voxel for the low resolution scans.
At the cellular level, the greyscale patterns in Scots pine and Norway spruce wood coincide, with bright
spots (high-density Cu) around the cell wall or filling the cell lumina, as indicated in Fig. 4. In the three
wood materials considered no major difference in the Cu uptake by ray parenchyma and ray tracheids was
observed. The localization of Cu in the wood ultrastructure and the form of Cu, however, remain
uncertain. In particular, even with the maximum intensity projections, it is unclear whether Cu is into or
on the cell wall, and if ions, nanoparticles, or aggregates/agglomerates are responsible for the larger bright
areas within the cell wall. It was furthermore calculated that the detection limit of Cu would be of the
order of 2 pg per voxel for the high resolution scans.
Fig. 3. Three-dimensional reconstruction of Cu penetration (red) in (a) Scots pine sapwood, (b)
Norway spruce sapwood, and (c) Norway spruce heartwood.
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Fig. 4. Reconstructed slices before (above), after (middle) MCA-pressure-treatment, and maximum
intensity projections after MCA-pressure-treatment (below) of Scots pine sapwood (a, d, g), Norway
spruce sapwood (b, e, h), and Norway spruce heartwood (c, f, i). The brighter spots indicate areas
containing high-density elements (Cu).
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Fig. 5. Assessment of micronized Cu azole (MCA) concentrations against R. placenta 45 and
associated mass losses in Scots pine sapwood, Norway spruce sapwood, and Norway spruce
heartwood. Data are represented as mean ± standard deviation of six replicates. Plain font was used for
Scots pine sapwood, underline for Norway spruce sapwood, and italics for Norway spruce heartwood.
Effectiveness against Cu-tolerant basidiomycetes
We assessed if the difference in Cu penetration in the three MCA-pressure-treated wood materials
considered affected the wood protection effectiveness against the Cu-tolerant fungus R. placenta 45.
Wood mass losses for the different wood species and MCA concentrations are reported in Fig. 5.
Tukey’s HSD test showed no differences between the mass losses from 0.00% MCA and control wood
samples for each of the three wood materials considered. Among the different wood species, the mass
losses in control samples were slightly lower for Scots pine sapwood (p-value < 0.001), while they were
equivalent for Norway spruce sapwood and heartwood (p-value=0.456). Differences between the sapwood
and heartwood from Norway spruce were recorded after treatment with MCA (p-value < 0.001). All MCA
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concentrations prevented R. placenta 45 from colonizing Norway spruce heartwood, with mass losses well
below 3.0%. On the contrary, despite a significant reduction in mass losses when compared to the 0.00%
MCA and control, even concentrations of 1.60% MCA were not sufficient to protect Norway spruce
sapwood from degradation (mass loss: 10.7±2.1%). At concentrations of 2.00% MCA decay results were
more variable, with recorded mass losses between 0.3% and 13.5%. Mass losses from Scots pine control
samples were not comparable to the ones from Norway spruce sapwood and heartwood (p-value < 0.001).
Scots pine MCA-pressure-treated samples appeared significantly decayed below concentrations of 2.00%
MCA (above 3%), while the latter caused mass losses of 2.75±0.975%.
Discussion
The aim of this study was to assess if MC could penetrate refractory wood species without pre-treatment,
i.e. incising, and consequently provide an added value than conventional wood preservatives. We
compared the pressure-treatment penetration effectiveness of Cu from an MCA formulation in easily
treatable Scots pine sapwood and refractory Norway spruce sapwood and heartwood. The comparison was
carried out using three different techniques: the indirect calculation of wood preservative retention after
impregnation, as indicated by the EN 113 guidelines [25], the quantification of Cu by ICP-OES, and the
density-based greyscale thresholding on X-ray CT reconstructions. We also aimed to correlate MCA
penetration with protective effectiveness against the wood-destroying fungus R. placenta 45.
The nature of the EN 113 preservative retention formula [25] does not consider the treatability of the
wood species. In the present study this resulted in an equal amount of expected MCA penetrating in the
Scots pine and Norway spruce wood blocks at a given MCA concentration, and a linear correlation
between the MCA concentration and the preservative retention, independent of the wood species. This
calculation appears to diverge from the directly measured amount of Cu in the three wood materials
considered. The ICP-OES analysis revealed that the background level of Cu present in untreated wood is
negligible, as its concentrations in both Scots pine and Norway spruce (heartwood and sapwood) were
below the instrument’s detection limit. Therefore, the amount of Cu detected in MCA-pressure-treated
wood can be attributed solely to the wood preservative. Our results from the ICP-OES measurements on
MCA-pressure-treated wood indicate that Cu was more abundant in Scots pine sapwood, especially on the
surface, and only half of the Cu percentage found in Scots pine was detected in Norway spruce heartwood,
the most refractory wood in this study. These results clearly showed that the amount of Cu penetrating
into the wood heavily depends on the wood species and on the presence of sapwood (more accessible) or
heartwood (less accessible) [4]. In addition, X-ray CT scanning and subsequent analysis enabled Cu
distribution visualization in wood based on thresholding of the images. It should be noted that this results
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in semi-quantitative data, since it is not trivial at all to derive quantitative data from X-ray CT scans.
Furthermore, comparison with other methods for quantification of Cu in wood, is not straightforward as
well since the thresholding applied, results in a percentage of voxels containing Cu, and does not relate to
the exact amount of Cu present within those voxels. Although regions containing very low amounts of Cu
could be overlooked, a detection limit of approximately 0.1 µg is small enough such that, if present, it
would be visible.
For Scots pine sapwood 7% of the Scots pine wood block volume was found to be Cu. We applied the
same thresholding on Norway spruce sapwood and heartwood reconstructions. While Cu was rather
homogeneous across the whole Scots pine wood section, similarly to what is observed in other easily
treatable species [16-23], in Norway spruce wood it was mostly located on the surface. In accordance with
Evans et al. [18] and in good agreement with findings on western hemlock by Xue et al. [37], Cu was
more abundant in the latewood and mainly distributed in rays and resin canals. Further, the Cu distribution
pattern coincides with the one highlighted from MC by Evans et al. [16], which differs from the Cu
distribution pattern from conventional amine Cu wood preservatives [16]. The threshold of Norway spruce
sapwood and heartwood resulted in lower Cu percentages amounts than those detected by ICP-OES. This
indicates that most of Cu is not present as particles, aggregates or agglomerates with size detectable at 31
μm, i.e. the resolution of the scans. This was confirmed by the scans at higher resolution, where Cu was
visible at the cell wall level. Although regions containing very low amounts of Cu could be overlooked, a
detection limit of approximately 2 pg is small enough such that, if present, it would be visible. The
submicron resolution, however, did not allow to precisely locate Cu in the cell wall, i.e. if Cu diffuses into
(cell-wall treatment) or only onto it (cell-lumen treatment). This issue has previously been hypothesized
[38], and the difference is critical for wood protection because wood cell-wall treatments are certainly
more effective against white and soft rot fungi [39, 40] and also more resistant to leaching [41]. In
addition, it was not possible to determine if the Cu present was solubilized or available as unreacted -
single or aggregated/agglomerated- particles responsible for a reservoir effect. Cu appeared equally
distributed in ray parenchyma and ray tracheids, similarly to what Olsson et al. [42] observed. This
confirms that despite the innovative impregnation chemistry of MC, the wood preservative fluid flow is
still subject to the same resistance, i.e. the pits between ray parenchyma and tracheids. In fact, while in
easily treatable Scots pine, these cross-field pits are fenestrate and with a large thin membrane, in Norway
spruce they are of the piceoid type, with smaller membrane and smaller dimensions [43, 44].
The effectiveness of MCA against the Cu-tolerant fungus R. placenta greatly differed for Scots pine
sapwood, Norway spruce sapwood and heartwood. Despite the valid performance of MCA-pressure-
treated Norway spruce heartwood even at the lowest MCA concentration (0.80%), the protection of Scots
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pine wood appeared to be more difficult. The different patterns cannot be explained by differences in
fungal virulence or wood natural durability, as the mass losses for Scots pine sapwood control samples
were slightly lower than those of Norway spruce sapwood or heartwood. One explanation may be related
to the Cu-tolerance of R. placenta and the interactions between Cu and tebuconazole within the MCA
formulation. As previously demonstrated [27], tebuconazole plays a major role as active ingredient against
R. placenta, however sub-lethal concentrations of Cu in tebuconazole-amended media can actually
stimulate the growth of the fungus. In the present study Norway spruce heartwood samples contained the
lowest amount of Cu, and most of it was located at the surface. The same is likely to apply for the co-
biocide tebuconazole, whose low amount could still suffice to exert an antifungal effect. Hence the fungus
has almost no Cu that would help it surviving in a tebuconazole-amended media. In Norway spruce
sapwood the amount of Cu measured by ICP-OES increased slightly, providing R. placenta the conditions
to survive despite the presence of tebuconazole. Finally, in Scots pine wood the amounts of Cu were even
higher, providing even more resources, until at high MCA concentrations Cu becomes lethal together with
tebuconazole (threshold concentration). Another possible explanation involves the amount of reacted Cu.
Most of Cu contained in Norway spruce heartwood is likely to be in the form of fine particles (below 31
µm) or ions, as indicated by the low resolution X-ray CT scan and the thresholding coupled with the ICP-
OES analysis, whereas Cu appears in larger clusters in Norway spruce sapwood, and even larger in Scots
pine wood. This could result in a reduction in the ratio between solubilized bioavailable Cu2+ and
unreacted CuCO3·Cu(OH)2 in more easily treatable wood. Therefore, in the short-term the MC reservoir
effect, i.e. unreacted CuCO3·Cu(OH)2 particles slowly solubilizing in wood, may be counterproductive. In
fact, the short-term nature of the EN 113 studies, which last only 16 weeks, would not allow to assess the
long-term protection provided by a reservoir effect, which may be visible after several months or years.
Despite their different nature, the two possible explanations, i.e. Cu-tebuconazole interaction or reacted-
unreacted Cu ratio, share a common conclusion: the CuCO3·Cu(OH)2 particles in MCA formulations do
not contribute to a better short-term performance because they either support the growth of the wood-
destroying fungus, or are not bioavailable and cannot exert an antifungal effect.
In the long-term, further mechanisms beside the reservoir effect should be considered. Cu from MCA-
pressure-treated Norway spruce can be released in the environment, similarly to what is observed for
MCA-pressure-treated SYP [284], thus the environmental impact should be considered.
In conclusion, our hypothesis that MC cannot readily penetrate refractory wood species, which are
commonly used in Central Europe, was confirmed. Therefore, from a wood penetration perspective, MC
performance is comparable to conventional wood preservatives, and the adoption of MC for refractory
wood species in the European market would still require pretreatment such as incising. However the MCA
formulations succeeded in protecting refractory wood species against R. placenta, and the treated
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refractory wood was destroyed less than easily treatable MCA-pressure-treated wood. Nevertheless in the
short-term CuCO3·Cu(OH)2 particles do not provide an added value for the wood preservative
formulation. Future studies should focus on MC’s Cu speciation in wood and its interaction with wood
ultrastructure. In this way, the presence of a reservoir effect, of cell-wall or cell-lumen treatments, and the
basis of MCA effectiveness could be thoroughly understood.
Acknowledgments
We are indebted to Daniel Heer for the technical assistance. The work is financially supported by the
Swiss National Research Foundation (Grant No. 406440_141618) and the Transnational Access to
Research Infrastructures activity in the 7th Framework Program of the EC under the Trees4Future project
(Trees4Future, 284181).
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4.3 Mechanical abrasion of treated wood Accepted by: Journal of Nanobiotechnology
Release of Copper-Amended Particles from
Micronized Copper-Pressure-Treated Wood during
Mechanical Abrasion
Chiara Civardi1,2‡*, Lukas Schlagenhauf3,4,5‡, Jean-Pierre Kaiser6, Cordula Hirsch6, Claudio Mucchino7,
Adrian Wichser4,6, Peter Wick6, Francis W.M.R. Schwarze1*.
1Empa, Applied Wood Materials, St. Gallen, Switzerland
2ETH, Institute for Building Materials, Zürich, Switzerland
3Empa, Functional Polymers, Dübendorf, Switzerland
4Empa, Analytical Chemistry, Dübendorf, Switzerland
5ETH, Institute for Environmental Engineering, Zürich, Switzerland
6 Empa, Particles- Biology Interactions, St. Gallen, Switzerland
7 Università degli Studi di Parma, Dipartimento di Chimica, Parma, Italy
*Corresponding Authors: Chiara Civardi, ETH, Institute for Building Materials, Stefano-Franscini-Platz 3,
8093 Zürich, Switzerland. Email: [email protected] and Prof. Dr. Francis W.M.R. Schwarze,
Empa, Laboratory for Applied Wood Materials, Lerchenfeldstrasse 5, CH- 9014 St. Gallen. Phone: +41 58
765 72 47. Fax: +41 582747694. E-mail: [email protected]
‡These authors contributed equally.
KEYWORDS: Cytotoxicity, Copper particles, Debris, Exposure, Inhalation, Wood dust
ABSTRACT
Background: We investigated the particles released due to abrasion of wood surfaces pressure-treated with
micronized copper azole (MCA) wood preservative and we gathered preliminary data on its in vitro
cytotoxicity for lung cells. The data were compared with particles released after abrasion of untreated,
water (0% MCA)-pressure-treated, chromated copper (CC)-pressure-treated wood, and varnished wood.
Size, morphology, and composition of the released particles were analyzed.
Results: Our results indicate that the abrasion of MCA- pressure-treated wood does not cause an additional
release of nanoparticles from the unreacted copper (Cu) carbonate nanoparticles from of the MCA
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formulation. However, a small amount of released Cu was detected in the nanosized fraction of wood dust,
which could penetrate the deep lungs. The acute cytotoxicity studies were performed on a human lung
epithelial cell line and human macrophages derived from a monocytic cell line. These cell types are likely
to encounter the released wood particles after inhalation.
Conclusions: Our findings indicate that under the experimental conditions chosen, MCA does not pose a
specific additional nano-risk, i.e. there is no additional release of nanoparticles (NPs) and no specific
nano-toxicity for lung epithelial cells and macrophages.
INTRODUCTION
Thousands of tons of wood chips and sawdust are being generated each day by industry, domestic
environment, or improper disposal of debris. Further, the presence of wood preservatives may pose an
environmental and human health risk due to release of toxic metals like arsenic and copper (Cu). Such an
exposure pathway has already been recognized for various preservatives, in particular for chromated Cu
arsenate (CCA) [1, 2].
We are currently experiencing an increased use of particulate Cu wood preservatives in order to
effectively protect wood from decay and lengthen its service life. More specifically, basic Cu carbonate
particulate systems with a size range between 1 nm and 25 μm were introduced for wood protection in the
US market in 2006 [3]. This has resulted in more than 11,800,000 m3 of wood treated with micronized Cu
(MC) formulations [4], which corresponds to over 75% of residential lumbers produced in the US [4].
MC wood preservatives include a nanosized fraction of basic Cu carbonate, which may be of high
concern: there is a strong indication that different Cu- based nanoparticles (NPs) have a high toxicity for
aquatic organisms [5-10], terrestrial plants [11], mammals [12-17], and humans [18-23].
To date, the environmental fate of Cu carbonate particles from MC- pressure-treated wood has mostly
assessed their leachability [24-28]. However particles generated by abrasion of MC-treated wood may be
more hazardous than wood dust untreated or treated with conventional wood preservatives, due to the
presence of Cu-based NPs. Platten et al. [29] and Santiago-Rodríguez et al. [30] recently assessed how
exposure to Cu from wood dust originated from MC-pressure-treated wood can occur via dermal transfer
or oral ingestion. Therefore, it is extremely important to determine the dust composition that can be
inhaled after exposure –occupational or not– to abraded particles from MC-pressure-treated wood and its
hazard to human lungs.
The current study characterizes the particles released from MC azole (MCA)-pressure-treated wood and
compares them with particles generated from wood untreated, pressure-treated with the conventional
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wood preservative chromated Cu (CC), and with varnished untreated and MCA-pressure-treated wood.
Subsequently, it assesses acute cytotoxic reactions of MCA, its components tebuconazole and Cu2+, as
well as particles abraded from MCA-, CC-pressure treated wood and untreated wood to lung epithelial
cells and macrophages.
MATERIALS AND METHODS
MC characterization
We used a commercially available MC azole (MCA) formulation. This is the same as the formulation with
high amount of tebuconazole MCA_HTBA we used in a previous investigation [31]. A full
characterization of the Cu particles in the MCA formulation is available from the latter study. To
summarize briefly, the measured particle size distribution of MCA was 104±1.7 nm with an average zeta
potential of -21±0.4 mV.
Wood sample preparation
Octagonal specimens of Scots pine (Pinus sylvestris L.) sapwood (90 mm diameter x 20 mm height) were
used for the abrasion study. The specimens were prepared and pressure-treated with 2% aqueous
suspensions of MCA or CC reference preservative, prepared according to the European standard ENV 807
[32]. After an 8-week drying procedure, some of the MCA-pressure-treated samples were coated three
times with intervals of 24 hours with a primer, i.e. solution of deck lacquer (90%) and white spirit (10%).
The control materials were composed of: untreated wood samples and samples pressure-treated with a 0%
MCA solution in distilled water, varnished untreated wood samples.
Abrasion setup
The experimental setup has been described by Schlagenhauf et al. [33]. To simulate the abrasive process, a
Taber Abraser (Model 5135, Taber, North Tonawanda, NY) was used. While the wood sample rotates, the
Taber Abraser uses one abrasive wheel that abrades the sample continuously at the point of contact. The
sample rotates 60 times per minute and the weight applied on the wheel is 0.75 kg. The samples were
abraded with S-42 sandpaper strips (Taber) mounted on a CS-0 (Taber) rubber wheel. A conductive
silicone tube (TSI) with a rectangular inlet at the tube entrance with a 4.8 mm2 suction area was placed
directly behind the abrasion area to collect the particles. The air flow was driven by a pump
(N816.1.2KN.18, KNF, Germany). Devices for aerosol characterization and particle collection were
included in the tubing system.
Wood dust characterization
The generated particles were characterized in triplicates both in the aerosol form by particle size
distribution measurements with an aerodynamic particle sizer (APS, Model 3321, TSI, Shoreview, MN)
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and a scanning mobility particle sizer (SMPS) consisting of a differential mobility analyzer (DMA)
equipped with a long DMA column (Model 3080, TSI) and a condensation particle counter (CPC) (Model
3775, TSI). During each measurement, three particle size distributions were recorded. The recording time
for each distribution was 195 s. The background distribution (without abrasive processes) of each
experiment was measured three times. The experimental setup was verified by means of an atomizer
aerosol generator (Model 3079, TSI). The particle size distributions obtained were processed as described
by Schlagenhauf et al. [33]. In addition, the particles were collected on stubs and analyzed by means of
scanning electron microscopy (SEM, Hitachi S-4800; Hitachi High-Technologies US and Canada, Illinois,
USA). The stubs were plasma gold-sputtered (Polaron Equipment, SEM coating Unit E5100, Kontron AG,
Switzerland; 5 mA, 1 mbar) prior to image acquisition.
The presence of Cu in the generated particles was assessed in the collected particles through ICP-MS
(PerkinElmer Elan 6100, detection limit: 0.004 µg/L) and two distinct ICP-OES (Perkin-Elmer OPTIMA
3000, Jobin-Yvon HORIBA Ultima 2, detection limit for both instruments: 0.005 mg/L) instruments. In
this way, we could benefit from the two different detection limits, as well as identify any effect of the
instrumentation and –especially– of sample preparation on the detected amount of Cu. Analyses were
carried out on the whole size range of abraded particles and on particles <1μm collected on Nucleopore
track-etch membrane filter (111106, pore size 0.2 μm, Whatman, UK). For ICP-MS and Perkin-Elmer
OPTIMA 3000 ICP-OES Cu content analysis, the collected particles were dissolved nitric acid (HNO3,
65%, Supra Pure) and hydrogen peroxide (H2O2, 30%, Supra Pure) and subsequently underwent
microwave digestion (MLS 1200 MEGA, Milestone, Leutkirch, Germany). Cu plasma standard solutions
(1 g/L) were used for calibration. For Jobin-Yvon HORIBA Ultima 2 ICP-OES analysis a similar
procedure was used, but without the addition of hydrogen peroxide. The detector voltage was set using a
100 mg/L standard solution, while a 7 levels calibration curve was employed for quantification.
Cell culture
The human alveolar epithelial cell line A549 (ATCC: CCL-185) was grown in Roswell Park Memorial
Institute (RPMI-1640) medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Lonza),
2 mM L-glutamine (Gibco), 50 µg/mL penicillin (Gibco), 50 µg/mL streptomycin (Gibco), and 100
µg/mL neomycin (Gibco) at 37°C in a humidified atmosphere containing 5% carbon dioxide (CO2,
hereafter referred to as complete cell culture medium and standard growth conditions, respectively). Cells
were subcultured at approximately 80-90% confluency once a week using 0.5% Trypsin-EDTA (Sigma-
Aldrich).
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Formation of reactive oxygen species (ROS)
The formation of ROS in A549 cells was determined using the 2’, 7’-dichlorodihydrofluorescein-diacetate
assay (H2DCF-DA), as described by Roesslein et al. [34]. For experimental details see supplementary
information.
Cell viability
To assess mitochondrial activity as a measure of cell viability/cell death in A549 cells Cell Titer96®
Aqueous One Solution (Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-
2-(4-sulfophenyl)-2H (MTS) as a water-soluble tetrazolium compound was used according to the
manufacturer’s protocol. In brief, 1.5 x 104 A549 cells were seeded in 200 µL complete cell culture
medium in a 96-well plate and grown over night under standard growth conditions. Thereafter medium
was removed and cells were incubated for 3 h or 24 h in 200 µL complete cell culture medium containing
the respective stimuli (abraded particles from MCA-, CC-pressure-treated wood or untreated wood, or
eluates derived thereof as described below). Cadmium sulfate (CdSO4) in different concentrations served
as positive control, untreated cells as negative control. After appropriate incubation times (3 h, 24 h)
medium was replaced by 120 µL of MTS working solution (composed of 20 µL MTS plus 100 µL of
phenol-red-free RPMI-1640 w/o supplements) per well and cells were incubated for 60 min at standard
growth conditions. Absorption was detected at 490 nm using an ELx800 microplate reader (BioTEK
Instruments).
Data processing
Blank samples treated exactly the same way but containing no cells were run with every cell-based assay.
Values given in the graphs are blank-corrected and subsequently normalized to the untreated sample. The
mean of at least three independent experiments (each run with technical triplicates) and the corresponding
standard deviations are shown.
Sample preparation for cytotoxicity analysis
Cytotoxicity was assessed in two different scenarios: i) Abraded particles released from MCA-, CC-
pressure-treated as well as untreated wood were diluted in appropriate media and directly applied to
cultured cells. ii) Eluates from the same abraded particles were used to assess the cytotoxicity of active
soluble components contained in and released from the wood. Therefore, 4 mg of abraded wood particles
per mL elution medium were incubated for 24 h at 37°C on a rotating platform. Supernatant was collected
after centrifugation at 500g for 5 min. Elution medium for ROS detection was Hank’s Balanced Salt
Solution (HBSS; for experimental details see supplementary information). For cell viability assessment
and cytokine detection (see supplementary information) eluates were produced in phenol-red free RPMI
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(without supplements) which was supplemented after centrifugation with 10% FCS, 2 mM L-glutamine,
50 µg/mL penicillin, 50 µg/mL streptomycin, and 100 µg/mL neomycin. The HBSS supernatant as well as
the supplemented RPMI supernatant contain the highest possible amount of released components and were
labeled “100% eluate”. Serial 1:2 dilutions were performed in the respective media and concordantly
termed “50%, 25%, etc. eluates”.
Determination of Cu content in eluates
The Cu content in eluates of the abraded particles from untreated, CC- and MCA-pressure-treated wood
was determined by ICP-MS (Sector Field SF-ICP-MS Element 2 from Thermo Finnigan, detection limit:
0.004 µg/L). Prior to analysis, the specimens were acidified with nitric acid (HNO3, 65%, Supra Pure) and
hydrogen peroxide (H2O2, 30%, Supra Pure) and subsequently underwent microwave digestion (MLS
1200 MEGA, Milestone, Leutkirch, Germany). Cu plasma standard solutions (1 g/L) were used for
calibration.
Production of cytokines. The release of the pro-inflammatory cytokine TNF-α was assessed in
macrophages derived from the monocytic cell line THP-1 (ATCC: TIB-202) using the Ready-SET-Go!®
Elisa kit (eBioscience) according to the manufacturer’s protocol. For cell culture conditions and
experimental details see supplementary information.
RESULTS AND DISCUSSION
Wood dust particle size
The particle size distributions for the different wood samples (untreated, 2% MCA- pressure-treated, 2%
CC- pressure-treated, 0% MCA- pressure-treated, varnished, 2% MCA-pressure-treated and varnished) are
shown in Figure 1 (a, b). More specific, Figure 1a represents the particle size distributions measured by
SMPS below 1 μm, while Figure 1b presents the distributions measured by APS above 1 μm. All the
samples show a similar pattern below 1 μm, with peaks at about 400 nm; while two different outlines are
visible above 1 μm: one for the abraded particles from varnished samples, and another for the abraded
particles of unvarnished samples. In the first case the peak is between 700 nm and 1.3 μm, while in the
second one it is around 2.3 μm. Therefore, the set up maximizes the release of coarse (PM10), fine
(PM2.5) and ultrafine particles (generally defined as smaller than 100 nm). These three particle size
fractions are commonly associated with adverse health effects in humans, as demonstrated by Schwartz et
al. [35], Raaschou-Nielsen et al. [36], and Oberdörster et al. [37]. In addition, the setting fitted the purpose
of detecting any variation in the generated wood dust at the nanoscale, which may have occurred due to
the presence of Cu carbonate NPs. In any case, no additional release of a nanosized fraction was observed
for the 2% MCA-treated wood.
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We could observe how the application of varnishes influences the particle size released increasing the
average dimensions, reducing the exposure to ultrafine particles.
The APS results on the aerodynamic particle diameter are in good agreement with the study from Thorpe
and Brown [38], in which the wood dust size distribution after different sanding processes was assessed.
The mean particle diameter was comprised between 1.52 μm and 2.65 μm. However, further different
abrasive processes, e.g. cutting, grinding, welding, may cause the release of wood dust with different
particle size distributions. Despite that, as our abrasive set up maximizes the release of coarse, fine and
ultrafine particles, we can suppose that different abrasive processes would not release more nanoparticles
than our system.
Our tests focused on Scots pine only, however the different wood species could play a role may release
particles that differ in the size distribution, due to the wood properties, as demonstrated by Lehmann and
Fröhlich [39] and Ratnasingam et al. [40]. In the case of MCA-treated wood, the wood species features
may also influence the amount of Cu carbonate particles present in the wood after impregnation.
In terms of human exposure, our results indicate that a fraction of the abraded particles produced by the
different wood samples could penetrate the lower airways (tracheo-bronchiolar regions or even the
alveolar sacs), due to their small size. The application of varnishes alter the size distribution of the abraded
particles and by that would shift the particle deposition to the nasopharyngeal and tracheo-bronchiolar
regions [41]. However, the broad size range of the particles does not allow a precise quantification of
particle deposition in the respiratory tract.
Wood dust particle morphology
The generated particles from untreated, CC-, and MCA-pressure-treated wood were morphologically
assessed by SEM (Figure 1 c, d, e). Visual inspection of all the SEM images collected confirmed the
presence of particles below 10 μm, as well as the presence of bigger particles (102 μm), beyond the APS
and SMPS detection limits adopted. In addition, no difference between the different wood samples (Figure
1 d, e) was encountered, indicating no mechanical alteration due to the wood treatments, in accordance
with the APS and SMPS results. In all cases, the generated particles appeared mostly fibrous, although
irregular and heterogeneous in shape and size. The surfaces were not always flat.
Various studies reported similar features from SEM investigations on wood dust from various wood
species [42, 43]. In particular, Mazzoli and Favoni [44] reported no difference in wood dust particle size
and morphology from different wood species, suggesting no dissimilarity for in vitro cytotoxicity.
However, wood species that are documented to be carcinogenic, e.g. beech [45], were not assessed. In that
case, different structures responsible for the increased adverse effects may be observed. In addition, the
abrasive process may also generate wood dust particles that differ in size and morphology.
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Cu content in wood dust
By means of ICP-OES and ICP-MS analyses we could assess the different concentrations of Cu in wood
dust from untreated and MCA- pressure-treated wood samples, as shown in Table 1. Combining the ICP-
OES and ICP-MS results, which are concordant, we determined a baseline amount of Cu in untreated
wood at 0.01±0.02 mg/g. Similarly, when the wood was varnished the baseline amount was found at
0.02±0.01 mg/g. When MCA- pressure-treated wood was abraded, the amount of Cu released was
2.02±0.09 mg/g, corresponding to 0.20% w/w of the total amount of treated wood, and it drastically
reduced when varnish was applied (0.23±0.01 mg/g). This difference may be due to the higher release of
varnish instead of wood, therefore implying that varnishes may prevent release of Cu during mechanical
abrasion of treated wood. The amount of Cu release was almost double in CC-pressure-treated wood
(4.26±0.01 mg/g). This is due to differences in the formulations: in fact, the amount of Cu in the initial CC
formulation doubles the amount in MCA. Since 2% is an economically feasible concentration, generally
used in the timber industry, the result indicate that at similar dilutions (2%) MCA-pressure-treated wood
would release less Cu due to mechanical abrasion. The percentage of Cu released from MCA- pressure-
treated wood is in good agreement with studies on indoor sawing of CCA-treated wood: Decker et al. [46]
reported 0.3% Cu in wood dust, while Nygren et al. [47] 0.1%. In addition, a comparison can be made
between our results and the ones from the less invasive wiping experiment reported in the EPA report
[24]. In fact, in the latter, the amount of Cu released from MCA- pressure-treated wood was lower and
comprised between 0.0135 mg and 0.072 mg.
The amount of Cu detected in the wood dust nanosized fraction was below the Cu concentration in the
whole wood dust, both from untreated and MCA-pressure-treated wood. In particular the concentration of
Cu in the nanosized dust generated by MCA-pressure-treated wood was1.50±0.30 mg/g (0.15% w/w).
Therefore, combining these data with the SMPS results we can conclude that most of the Cu released was
bound to the larger wood particles, however a small amount of Cu bound to the nanosized fraction would
deposit in the deep lungs, if inhaled. Therefore, toxicological studies are required to fully assess the hazard
on human health.
Cytotoxicity assessment
The most critical exposure route for sawdust particles is the lung. Therefore, we focused our in vitro study
on the lung epithelial cell line A549 and macrophages differentiated from the monocytic cell line THP-1.
Both cell types are likely to be among the first cell types getting in touch with inhaled particles. We
investigated potential adverse effects of sawdust particles abraded from untreated wood, MCA-pressure
treated wood and CC-pressure treated wood. Furthermore, to assess the effects caused by soluble
compounds, rather than by wood dust per se, eluates from these three types of wood particles were
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included in the cytotoxicity evaluation. These results were compared to the toxicity induced by direct
treatment of lung epithelial cells with MCA and its active components tebuconazole and Cu2+ ions from
copper sulfate pentahydrate (CuSO4∙5H2O).
According to the ROS paradigm [34] the interaction of (nano) particles with cells is likely to induce
elevated cellular levels of ROS. Subsequent oxidative stress reactions can then cause severe damage to
biomolecules (proteins, lipids and nucleic acids), induce inflammatory reactions and finally lead to cell
death. Therefore we initially assessed the overproduction of ROS using the DCF assay. As shown in
Supplementary Figure S1, only the positive controls Sin-1 and MWCNT led to a considerable increase of
ROS levels in A549 cells. All eluates and abrasion particles tested did not elevate ROS formation.
However, cell death can also be triggered by ROS independent pathways. We therefore investigated cell
viability of A549 lung epithelial cells using the MTS assay. The assay internal positive control CdSO4
induces cell death in a dose-dependent manner (Figure 2a) thus indicating that toxicity can be reliably
detected under the experimental conditions.
The cytotoxicity of MCA itself was determined up to a concentration of 2% (v/v) in cell culture medium.
In parallel, its active compounds tebuconazole and Cu2+ were analyzed in equivalent amounts (Figure 2b
and supplementary information). Our results reveal a toxicity ranking of tebuconazole < Cu2+ < MCA,
which indicates an additive effect of tebuconazole and Cu2+. Further, our results suggest that the
cytotoxicity of MCA is likely to be caused by Cu2+ ions than nanoparticles.
The highest, technically feasible, concentration of abraded particles that could be applied to A549 cells
was 80 µg/mL equaling to a growth area of 47 µg/cm2. For all three types of sawdust particles no
cytotoxicity could be detected up to this concentration and over an incubation period of 24 h (Figure 2c).
According to Table 1 the highest amount of 80 µg particles from MCA- or CC-pressure-treated wood
contain 0.16 µg Cu2+ or 0.34 µg Cu2+, respectively. Measurements of eluates of the respective abraded
particles revealed that only a fraction of 4.4% of Cu2+ is released into the medium over a period of 24 h
(Table 1). Therefore we do not expect concentrations above 0.007 µg/mL or 0.015 µg/mL Cu2+ for the two
samples, respectively. In relation to Figure 2b, were Cu2+ ion cytotoxicity starts above 5 µg/mL (=0.01%),
these values appear very low. However, the following considerations will relate the chosen in vitro doses
to an inhalation scenario for wood workers. If we consider an inhalation volume of 1.9 L per breath and
roughly 26 breathes per min during heavy exercise [48] we can assume a total volume of 24 m3 air to be
inhaled during an 8 h working day. According to Decker et al. [46], wood dust concentrations in air may
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range from 0.6 mg/m3 (sampled at outdoor working sites over a period of 229 min) to a maximum of 49
mg/m3 (sampled during indoor sanding operations over a period of 127 min). With these data a total
amount of 3.8 mg to 555 mg inhaled particles per working day can be estimated. Considering 102 m2 of
total lung surface area [49] and assuming all the wood dust particles to be deposited in the lung we can
estimate a total deposited amount of wood dust particles of 0.004 µg/cm2 to 0.545 µg/cm2. In this scenario
the 47 µg/cm2 in vitro dose is a rather high concentration mimicking a repeated exposure over at least 17
weeks (indoor) to a whole lifetime (49 working years; outdoor). Nevertheless, spatially restricted effects
due to particle deposition, cellular uptake of particles and potential intracellular Cu2+ release cannot be
addressed, neither by in vitro toxicity tests nor by the above demonstrated exposure calculations. In
summary the doses chosen in the present study adequately reflect a worst case exposure scenario for wood
workers.
Furthermore, we analyzed eluates produced from the three types of abraded wood particles and assessed
the cytotoxicity of soluble factors released from the sawdust on A549 cells. As shown in Figure 2d no
cytotoxicity could be detected after 24 h of incubation with eluates from untreated as well as MCA-
pressure-treated wood. Eluates from CC-pressure-treated wood particles reduced cell viability at the
highest concentration tested to 63% viable cells compared to untreated control cultures. This highest
eluate concentration (Table 1) contained only 0.8 µg/mL Cu2+. As Cu2+ ion cytotoxicity started at
concentrations beyond 5 µg/mL (=0.01%) (Figure 2b) Cu2+ is most likely not the main reason for the
observed effect, but rather chromium. Further investigations are necessary to prove a real human hazard
from CC-pressure-treated wood, which was not the scope of the present study. Besides that, our results
clearly indicate that there is no additional nano-specific effect, as abraded particles from MCA-pressure-
treated wood as well as eluates thereof did not induce any cytotoxicity under the experimental conditions
tested. This provides further evidence to the hypothesis that Cu2+ ions rather than nanoparticles are
responsible for any adverse effects.
Besides cell viability, inflammatory reactions at sublethal concentrations can be an indication for non-
acute but nevertheless relevant adverse effects. Therefore we assessed the release of the pro-inflammatory
cytokine TNF-α from immune responsive cells in vitro using the enzyme-linked immunosorbent assay
(ELISA) technique. We used macrophages differentiated from THP-1 monocytes as the model cell line.
Initially, cell viability was investigated to assure sublethal concentrations were applied for subsequent
cytokine release experiments. THP-1 macrophages were exposed to the respective stimuli for 8 h and cell
viability was assessed using the MTS assay. For technical details see supplementary information. CdSO4
served again as the assay internal positive control and induced cytotoxicity in a dose-dependent manner
(Supplementary Figure S2a). Following the same experimental design as described for A549 cells MCA
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and its active components tebuconazole and Cu2+ were applied in equivalent amounts (Supplementary
Figure S2b). In this case, the effects of Cu2+ and MCA were comparable, therefore even in this case the
effects from MCA appear to be caused by Cu2+ ions rather than nanoparticles. Cell viability was affected
at concentrations above 0.05% MCA in a dose-dependent manner. All three abraded wood particle types
(up to 80 µg/mL) as well as eluates thereof did not induce an adverse response (Supplementary Figure
S2c, d) in THP-1 macrophages. Accordingly, for cytokine release measurements MCA, tebuconazole and
Cu2+ were used at concentrations below 0.05% MCA-equivalents and abraded wood particles were used
up to 80 µg/mL. Lower eluate concentrations (6.25% to 25.00%) showed an increase in cell viability
rather than a decrease. Therefore we used concentrations below 25.00% for ELISA experiments.
Treatment with the positive control lipopolysaccharides (LPS) led to a 16-fold and 25-fold increase in
TNF-α release at 10 and 100 ng/mL LPS, respectively (Supplementary Figure S3). However, no
significant release of TNF-α could be observed after treatment with MCA, its active components, abraded
wood particles or eluates thereof at any of the concentrations tested (Supplementary Figure S3). Thus,
even in this case no specific nano effect was observed.
In summary our findings on the cytotoxicity reveal (1) a toxicity ranking of tebuconazole < Cu2+ < MCA
(2) no induction of cytotoxicity for abraded particles up to 80 µg/mL (3) only a minor toxicity was found
for the highest concentration of eluates resulting from CC-pressure-treated wood, which was only
observed for A549 lung epithelial cells, and it is likely due to the presence of chromium in the
formulation; most importantly (4) no additional nano hazard (caused by the presence of Cu-based NPs per
se) was identified. Furthermore, our cytotoxicity study indicates low adverse effects for low-frequency
consumer exposure. However, woodworkers can be continuously exposed to wood dust, in particular since
dust-exposed woodworkers do not always wear appropriate respirators approved for wood dust [50]. The
wood being processed may have been pressure-treated with Cu-based formulations, and the particles
released can increase the adverse effects due to the presence of Cu. However, MCA is likely to be the
safest alternative: no nano hazard was evidenced, and the amount of Cu, especially easily bioavailable Cu,
in CC was double the amount in MCA. Furthermore, both types of human cells tested showed lower
adverse effects (higher cell viability) when compared to cells exposed to CC. In conclusion, the abrasion
of MCA-pressure-treated wood does not constitute a nano-specific risk. Nonetheless, further more
advanced toxicity studies on tissues and in vivo are required.
Table 1. Cu content in sawdust particles and eluates thereof.
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wood treatment µg Cu/mg abraded particles µg Cu/mL medium (eluates*)
[release in %]
untreated 0.01±0.02 0.01±0.01
MCA-pressure treated 2.02±0.09 0.36±0.01 [4.4%]
CC-pressure treated 4.26±0.01 0.75±0.01 [4.4%]
RPMI medium (w/o wood) n.a. 0.00±0.01
*4 mg abraded particles were incubated in 1 mL phenolred free RPMI for 24 h at 37°C on a rotating
platform; after centrifugation at 500 g for 5 min supernatants were further processed for ICP-MS
measurements.
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FIGURES
Figure 1. Characterization of the abraded particles. (a) Particle size distributions of untreated wood
(control), water-treated wood (0% MCA), MCA-treated wood (2% MCA), and CC-treated wood (CC)
measured by SMPS. Most of the abraded particles had a diameter of 400 nm. Data represented as mean of
three repetitions. (b) Particle size distributions of untreated wood (control), water-treated wood (0%
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MCA), varnished wood (varnished), CC-treated wood (CC), MCA-treated wood (2% MCA), and
varnished MCA-treated wood (2% MCA varnished) measured by APS. Most of the abraded particles had
a diameter of about 1 μm. When varnish is applied, the average diameter shifts towards 2.3 μm. Data
represented as mean of three repetitions. (c, d) SEM images of wood dust generated by the abrasion
process on 2% MCA-treated wood. (e) SEM image of wood dust generated by the abrasion process on
untreated wood (control).
Figure 2. Cell viability assessment in A549 lung epithelial cells. Cells were treated for 24 h with the
indicated concentrations of (a) CdSO4 as the positive control (b) MCA, tebuconazole and Cu (c) abraded
sawdust particles from untreated, MCA-pressure treated and CC-pressure treated wood (d) eluates of the
respective wood particles. Cell viability was assessed using the MTS assay. *Tebuconazole and Cu2+ were
applied in the respective amounts present in MCA as described in supplementary information.
ASSOCIATED CONTENT
Supporting information. Concentration considerations of MCA components; Formation of reactive
oxygen species (ROS); Culture conditions and cell viability assessment of THP-1 cells; Production of
cytokines.
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Concentration considerations of MCA components. The active components of MCA are tebuconazole
and Cu-carbonate (CuCO3Cu(OH)2) in its nanoparticulate form. MCA itself is commonly applied to wood
as a 2% solution in water. For cell viability testing we assumed a worst case scenario with human
exposures at a maximum of 2%. Tebuconazole as well as Cu concentrations were calculated accordingly
to match the respective amount present in e.g. 2% MCA. CuSO4∙5H2O was used as a soluble Cu2+ source.
Formation of reactive oxygen species (ROS). A549 cells were seeded into 96-well plates at a density of
2 x 104 cells per well in a volume of 200 µL complete cell culture medium one day prior to treatment.
Cells were incubated with 50 µM H2DCF-DA (Molecular Probes) in HBSS at standard growth conditions.
After two washing steps with pre-warmed HBSS cells were treated with abraded wood particles (MCA-,
CC-pressure-treated and untreated) and eluates thereof. The nitrite oxide donor 3-morpholinosydnonimine
hydrochloride (Sin-1; 50 µM) as well as multi-walled carbon nanotubes (MWCNT; Baytubes, Bayer
Technologies; 10 µg/mL and 20 µg/mL) were used as positive controls. Fluorescence intensity was
measured after 1, 2, 3 and 4 h from the same plate using a Mithras2 plate reader (Berthold Technologies)
at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Only values from the 3 h
measurement are shown. The mean of at least three independent experiments (each run with technical
triplicates) and the corresponding standard deviations are shown.
Culture conditions and cell viability assessment of THP-1 cells. The human monocytic cell line THP-1
(ATCC: TIB-202) was grown as cell suspension in complete cell culture medium under standard growth
conditions in complete cell culture medium (RPMI-1640 supplemented with 10% FCS (Lonza), 2 mM L-
glutamine (Gibco), 50 µg/mL penicillin (Gibco), 50 µg/mL streptomycin (Gibco), and 100 µg/mL
neomycin (Gibco)). Subculturing was performed by replacement of medium when the cell density reached
8 x 105 cells/mL. Cell concentrations were not allowed to exceed 106 cells/mL. To determine sublethal
concentrations of abraded particles, eluates thereof as well as MCA components (MCA itself,
tebuconazole, Cu2+) cell viability was assessed using the Cell Titer96® AQueous One Solution (Promega)
as described in the main text with the following exceptions. 8 x 104 THP-1 monocytes were seeded in 200
µl complete cell culture medium containing 200 nM PMA (12-myristate-13-acetate) per 96-well. Cells
were differentiated into adherently growing macrophages in the presence of PMA for 72 h under standard
growth conditions. After one washing step with pre-warmed phosphate buffered saline (PBS) cells were
treated with the compounds of interest in complete cell culture medium (w/o PMA) for 8 h.
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Figure S1. None of the particles and eluates tested enhanced ROS production in A549 cells. ROS
formation was measured in A549 cells after three hours of incubation with indicated concentrations of
abraded wood particles as well as eluates thereof. Sin-1 (50 µM) as well as MWCNT served as the
positive controls. Sin-1: 3-morpholinosydnonimine hydrochloride.
Thereafter medium was replaced by 120 µL of MTS working solution, cells were incubated for 60 min at
standard growth conditions and absorption was read at 490 nm using an ELx800 microplate reader
(BioTEK Instruments).
Production of cytokines. 4 x 104 THP-1 monocytes were seeded in 200 µL complete cell culture medium
per well of a 96-well plate and were differentiated into macrophages over period of 72 h in the presence of
200 nM as described above. After one washing step with pre-warmed PBS cells were treated with the
MCA compounds, abraded particles and eluates thereof for 8 h under standard growth conditions (w/o
PMA). Cells treated with 10 ng/mL and 100 ng/mL lipopolysaccharide (LPS) served as positive control
samples, untreated cells as negative control samples. Cell-free supernatants were harvested by
centrifugation (500g, 5 min) and frozen at -80°C until subsequent cytokine analysis. TNF-α concentrations
were determined using the Ready-SET-Go!® Elisa kit (eBioscience) according to the manufacturer’s
protocol. Absorbance values were measured at 630 nm using a Mithras2 plate reader (Berthold
Technologies) and normalized to the untreated control sample. The mean of three independent
experiments (each run with technical duplicates) and the corresponding standard deviations are shown.
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Figure S2. No acute cytotoxicity was detected after treatment of THP-1 cells with abraded wood
particles and eluates thereof. MCA as well as tebuconazole and Cu2+ induce a dose dependent toxicity
in THP-1 cells. *Tebuconazole and Cu2+ were applied in the respective amounts present in MCA as
described above.
Figure S3. None of the substances tested induced the release of the pro-inflammatory cytokine TNF-α.
*Tebuconazole and Cu2+ were applied in the respective amounts present in MCA as described above.
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AUTHOR INFORMATION
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
Funding sources
The study was financially supported by the Swiss National Research Foundation (NRP 64, Grant No.
406440_141618). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Acknowledgments
The work was financially supported by the Swiss National Research Foundation (Grant No.
406440_141618). We are indebted to Alexandra Rippl for the cytokine analysis and the maintenance of
the cell cultures, to Daniel Heer for the technical support, to He Xu for the zeta potential measurements,
and to David Kistler for the ICP-MS analysis.
Abbreviations
A549, human lung epithelial cells; APS, aerodynamic particle sizer; CC, chromated copper; CCA,
chromated copper arsenate; CdSO4, cadmium sulfate; CPC, condensation particle counter; Cu, copper;
CuSO4∙5H2O, copper sulfate pentahydrate; DMA, differential mobility analyzer; FCS, fetal calf serum;
HBSS, Hank buffered salt solution; HRP, horseradish peroxidase; H2DCF-DA, 2’,7’-
dichlorodihydrofluorescein-diacetate assay; ICP-MS, inductively coupled plasma mass spectrometry; ICP-
OES, inductively coupled plasma optical emission spectrometry; IL-8, interleukin 8; LPS,
lipopolysaccharides; MC, micronized copper; MCA, micronized copper azole; MTS, formazan compound;
MWCNT, multiwalled carbon nanotube; NP, nanoparticle; ROS, reactive oxygen species; PBS, phosphate
buffered saline; PMA, phorbol-12-myristate-13-acetate; PSN, penicillin-streptomycin-neomycin
antibiotic; RPMI, Roswell Park Memorial Institute; SEM, scanning electron microscopy; Sin-1, 3-
morpholinosydnonimine hydrochloride; SMPS, scanning mobility particle sizer; THP-1, human acute
monocytic leukaemia cells; TNF-α, tumor necrosis factor.
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4.4 Spore compartmentalization of Cu Micronized copper-treated wood: copper remobilization into spores from the copper-tolerant wood-
destroying fungus Rhodonia placenta
Unpublished report
Keywords: Copper speciation, Micronized copper azole, Pressure-treated wood, Basidiospore
compartmentalization, X-ray absorption spectroscopy
Abstract
Different brown rot wood-destroying fungi have the ability to develop Cu-tolerance mechanisms in order
to effectively grow and decompose wood treated with copper (Cu), compulsory biocide for in ground
timber structures. We analyzed if the Cu-tolerant wood-destroying basidiomycete R. placenta is able to
compartmentalize Cu in its basidiospores. In addition, we assessed if the use of micronized copper (MC)
wood preservatives formulations, composed of basic Cu carbonate particles with sizes ranging from 1 nm
to 250 µm, could lead to basidiospores loaded with Cu-based nanoparticles (NPs) that may be inhaled and
lead to adverse health effect, due to specific nano effects In our study we combined elemental analysis and
visual inspection to quantify the amount of Cu in basidiospores, its distribution and the Cu species present.
Our results indicate that basidiospores from R. placenta can accumulate Cu, however the initial Cu-based
NPs undergo speciation. Therefore no specific nano-specific risk was highlighted.
Introduction
Copper (Cu) is widely used as fungicide, e.g. in agriculture [1] or wood protection [2]. However, certain
fungal strains developed Cu-tolerance mechanisms that allow them to effectively colonize Cu-polluted
environments. Within the field of wood protection, Cu-tolerant brown rot (basidiomycete) fungi are
responsible for most early failures in timber structures worldwide [3]. The latter include wooden
applications in contact with the soil (use class 4) [4], like poles, fences, playgrounds, or decks. The
detoxification mechanisms developed by Cu-tolerant wood-destroying fungi are mainly based upon
complexation of Cu2+ ions by fungal secretions, like oxalic acid [5], or compartmentalization of Cu within
cell structures, e.g. vacuoles [6]. Fungal cell wall mainly acts as cation exchanger, due to the negative
charge of its functional groups, for example Cu2+ ions can bind or get complexed by carboxylic,
phosphate, amine or sulfhydryl groups [6]. Direct uptake of Cu by fungal spores that would exert an
antifungal effect was described by Somers [7], while the uptake of Cu by mycorrhizal fungi and
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subsequent compartmentalization of Cu in spores has been reported in different studies [8-10]. In these
studies, Cu deposition in spores accounts for fungal survival. However, no indication on a similar trend for
Cu-tolerant wood-decaying basidiomycetes is available. In particular, if nanoparticle (NP)-based
formulations, like the newly developed micronized copper (MC) wood preservatives, are used for in
ground wood applications, Cu-based NPs may end up in the basidiospores. Despite the literature is still
fragmented and unclear, airborne spores can cause acute phase responses, and some basidiospores, can
exacerbate respiratory tract illnesses [11-16]. In our commentary [17], we hypothesized the likelihood of
such mechanisms and the possible nano-specific risks for human health in the event that basidiospores are
inhaled. Here, we hypothesize that R. placenta can compartmentalize Cu in its basidiospores (Hypothesis
1) and that the release of Cu-based NPs via basidiospores is likely to occur (Hypothesis 2). To test our
hypotheses we assessed the amount and form of Cu remobilized from MC-pressure-treated wood into
fruiting bodies and basidiospores of the Cu-tolerant [18] wood-destroying basidiomycete R. placenta. This
fungus was selected due to its high Cu-tolerance, as indicated by a previous screening study we conducted
[19], and its abundance in decayed timber structures [20, 21]. Visual inspections with light microscopy
and transmission electron microscopy (TEM) were combined with elemental analysis by means of ion-
coupled plasma mass spectroscopy (ICP-MS) and synchrotron-based X-ray absorption spectroscopy
(XAS) to determine the amount, distribution and movement across fungal structures, and Cu species in
basidiospores. In addition, we assessed whether basidiospores produced in Cu-rich environments were as
viable as control basidiospores or not.
Material and methods
Materials
Two commercial aqueous suspensions of MCA were investigated. The two MCA formulations coincide
with the ones used in our previous investigation [19]. These contain comparable amounts of Cu particles
but differ in the amount of tebuconazole (TBA): MCA_HTBA contained 5% w/w and MCA_LTBA 0.4%
w/w TBA. The above mentioned previous study also provides a full characterization of the
CuCO3∙Cu(OH)2 particles in the MCA formulation. In brief, the measured particle size distribution of MC
and MCA was 104±1.7 nm and 174±5.9 nm with an average zeta potential of -21±0.4 mV and -16.5±1.4
mV, respectively.
Scots pine (Pinus sylvestris L.) sapwood blocks (50 x 25 x 15 mm) were pressure-treated according to the
European standard EN 113 [22] with 2% MCA_LTBA or MCA_HTBA (MCA concentrations relevant for
the wood protection market). After drying, untreated and treated samples were grinded into sawdust.
Sporulation of R. placenta 45 was stimulated growing the fungus at 22 °C and 70% RH under cool white
fluorescent light, as suggested by Croan [23], for 6 hours a day on 20 g sawdust amended with 100 mL
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Fig. 1. R. placenta 45 producing fruiting bodies and releasing basidiospores. The fungus was grown
on sawdust produced from untreated and MCA-pressure-treated wood.
solid medium (autoclave sterilized) composed of 4% (w/v) malt extract and 2.5% (w/v) agar. R. placenta
45 fruiting bodies developed on Petri dishes external circumferences and released the basidiospores,
which deposited on an aluminum foil placed underneath, as shown in Fig. 1. In this way, we could be
certain that the basidiospores would not come directly in contact with Cu and that the
compartmentalization would have taken place due to Cu uptake by other fungal structures.
Quantification of Cu in fungal structures
Basidiospores and fruiting bodies produced by R. placenta 45 grown on sawdust from untreated,
MCA_HTBA-, and MCA_LTBA-pressure-treated wood were collected from the aluminum foil to
perform elemental analysis. The presence of Cu in basidiospores was assessed through Quadrupole Q-
ICP-MS Elan6100 (Perkin Elmer), while the amount of Cu in fruiting bodies was measured by Sector
Field SF-ICP-MS Element 2 (Thermo Finnigan). Detection limit for both instruments was 0.004 µg/L.
Digestion of the samples prior to ICP-OES was conducted according to Platten et al. [24]. Prior to
analysis, three replicates of each specimen were acidified with nitric acid (HNO3, 65%, Supra Pure) and
hydrogen peroxide (H2O2, 30%, Supra Pure) and subsequently underwent microwave digestion (MLS
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1200 MEGA, Milestone, Leutkirch, Germany). Cu plasma standard solutions (100 mg/L) were used for
calibration.
Speciation of Cu in fungal structures
The speciation of Cu in basidiospores was examined using the microXAS beamline at the Swiss Light
Source. The collected basidiospores were pressed as pellets into a sample holder with Kapton tape
window. The materials were analyzed at room temperature. Energy calibration of the Si111 double crystal
monochromator was obtained by measurements of a metallic Cu reference foil (in transmission mode)
prior and after experimental analysis. First inflection point of the metallic copper K-edge spectrum was set
to 8979 eV. Due to low Cu concentrations, XANES spectra of the experimental samples were collected in
fluorescence mode using a single element Silicon Drift Diode detector (SDD, KETEK GmbH). The
beamsize was defocused resulting in a 700 µm diameter beam. In addition to the fungal samples, several
reference materials, purchased as Cu salts (Cu carbonate (CuCO3∙Cu(OH)2), metallic Cu (reduced (or
zero-valent) Cu), Cu sulfate (CuSO4)) or prepared by mixing Cu with different ligands (Cu chloride
(CuCl2) aqueous, Cu oxalate, Cu-cysteine (CuCys)) were analyzed at room temperature. To examine the
possibility of beam-induced damage, spectra were collected at least twice from the same location and the
spectra compared to determine if the beam had caused a change in speciation. The XAS data processing
software Athena was used to analyze the XAS spectra [25]. For each individual sample, multiple spectra
were collected and merged. The XANES spectra obtained were used to compare the Cu speciation in
basidiospores produced by R. placenta 45 on sawdust from untreated and MCA-pressure-treated wood.
The linear combination fitting tool provided by the software Athena was used to determine the speciation
of Cu in the two types of spores.
Spore visualization
Basidiospores by R. placenta 45 grown on sawdust from untreated and MCA_LTBA-pressure-treated
wood were investigated by TEM. They were fixed by diluting them with 3% glutaraldehyde in 0.1 M
sodium cacodylate puffer and then sucked up into a capillary tube (Leica-Microsystems, Heerbrugg,
Switzerland). After a post-fixation step in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer, spores
were dehydrated through a graded ethanol series followed by acetone and embedded in Epon resin
(Sigma-Aldrich). Ultrathin sections were contrasted with 2% uranyl acetate and lead citrate before
observation in a Zeiss EM 900 (Carl Zeiss Microscopy, GmbH, Germany) at 80 kV.
Spore viability
Basidiospores produced by R. placenta 45 grown on sawdust from untreated and MCA_LTBA-pressure-
treated wood were collected and cultivated in 9 cm Petri dishes with 25 mL solid medium (autoclave
sterilized) containing 4% (w/v) malt extract and 2.5% (w/v) agar. Part of the media was amended with
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0.01% (w/v) MCA_LTBA. Three replicates were prepared for each condition. Cultures were stored at 22
°C and 70% RH. The cultures were inspected regularly, and their 4 cardinal points were marked to
determine the growth radii until the colonies reached the edges of the Petri dishes.
Results
Quantification of Cu in fungal structures
ICP-MS analysis allowed the detection and quantification of Cu in fruiting bodies and basidiospores from
R. placenta 45. The findings are summarized in Table 1.
While the amount of Cu in fungal structures cultivated on untreated sawdust was minimal, the presence of
Cu increased when the fungi were exposed to MCA-pressure-treated wood. What emerged is that the
amount of Cu taken up by fruiting bodies in MCA_HTBA-pressure-treated wood doubled the one from
fruiting bodies exposed to MCA_LTBA, whereas the same trend was not encountered in basidiospores,
whose Cu uptake in MCA_HTBA and MCA_LTBA environments appeared similar. Since no major
change between the spores produced in MCA_LTBA and MCA_HTBA environments, only MCA_LTBA
was used for the subsequent analyses, in order to minimize any effect from TBA. Based on these results,
we proved that Cu is accumulated in R. placenta 45 basidiospores. However this analysis provided no
indication on whether Cu was present in ionic, nano, or bulk form.
Speciation of Cu in fungal structures
Basidiospores from R. placenta 45 exposed to sawdust from untreated and MCA_LTBA-pressure-treated
sawdust were examined with XAS. The multiple spectra collected from the same location confirmed that
no or minimal radiation damage occurred during the analysis. Maintaining the exact same experimental
geometry, XRF measurements revealed different count rates for Cu fluorescence in the two type of
basidiospores (90’000 counts for Cu in basidiospores produced by R. placenta 45 grown on sawdust from
MCA_LTBA-pressure-treated wood, less than 30’000 counts for Cu in basidiospores produced on
Table 1- Amount of Cu detected in fungal structures by means of ICP-MS
Structures Cu content
Control MCA_LTBA MCA_HTBA
Fruiting bodies 16±1 μg/L 2768±98 μg/L 7018±205 μg/L
Spores 17±13 μg/g 239±201 μg/g 184±64 μg/g
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untreated wood). Furthermore, even when normalized (Fig. 2), the spectra differed, indicating dissimilar
Cu speciation.
In particular, the spectra differed in the near-edge (XANES) features around 8985 eV, on the edge
(between 8990 eV and 9000 eV), and in the fine structure (Fig. 3). The fine structure (EXAFS) reveals the
possibility of different neighboring atoms, and thus a different coordination for Cu in the two types of
basidiospores.
The results of the linear combination fitting performed on the spectra from the basidiospores using
CuCO3∙Cu(OH)2, CuSO4, reduced (or zero-valent) Cu, CuCl2 aqueous, Cu oxalate, CuCys as standards is
shown in Table 2.
Fig. 2. XANES spectra of R. placenta 45 basidiospores grown on sawdust produced from
MCA_LTBA-pressure-treated wood (blue) and untreated wood (red). The near-edge features differ,
indicating differences in the Cu speciation.
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Fig. 3. EXAFS spectra of R. placenta 45 basidiospores grown on sawdust produced from
MCA_LTBA-pressure-treated wood (blue) and untreated wood (red). The fine structure features
differ, indicating differences in the Cu speciation.
Table 2- Results of the linear combination fitting for the two types of basidiospores
Standards Weights
Control MCA_LTBA
Cu oxalate 9±12% 2±5%
CuCO3∙Cu(OH)2 0±11% 0±4%
CuSO4 0±11% 1±4%
Reduced (or zero-valent) Cu 29±3% 0±1%
CuCl2 aqueous 20±9% 13±4%
CuCys 41±21% 83±9%
R-factor 0.0086632 0.0017728
Chi-square 0.19225 0.03298
Reduced Chi-square 0.0016021 0.0002703
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In both cases, the major component in the speciation of Cu seems to be CuCys. However, while this
accounts for 41±21% in spores produced on untreated sawdust, the percentage heavily increases in
basidiospores produced in the MCA_LTBA environment (83±9%). Reduced (or zero-valent) Cu weights
29±3% in the linear combination fitting from the spectra of basidiospores generated by R. placenta grown
on sawdust from untreated wood, whereas it doesn’t contribute to the linear combination fitting from the
spectra of basidiospores generated by R. placenta grown on sawdust from MCA_LTBA-pressure-treated
wood. The other major component in both cases is CuCl2 aqueous. The weight of the other standards in
the linear combination fitting from both basidiospores, i.e. CuCO3∙Cu(OH)2, CuSO4, and Cu oxalate, was
null or extremely low. To confirm the absence of Cu particles accumulating in basidiospores we
investigated them by means of TEM.
Spore visualization
TEM investigations of R. placenta 45 basidiospores (Fig. 4) did not reveal any major difference between
spores grown on the different type of sawdust. In both cases, the spores appear ellipsoidal, whose sizes
range between 1 and 5 µm in length and 2 µm in width. No accumulation of electron-dense material
attributable to Cu was visible in both types of basidiospores. This indicates that Cu is likely present as fine
particles or as ions, and not as big clusters. Furthermore, no deformation of the cell organelles was visible.
We proceeded by assessing basidiospore viability to determine whether the accumulation of Cu results in
non-viable basidiospores.
Spore viability
Basidiospores of R. placenta 45 produced in sawdust cultures from untreated and MCA_LTBA-pressure-
treated wood were cultivated on artificial media, amended with MCA_LTBA or not (control). After 2
weeks incubation, both basidiospore types equally grew in control artificial media, covering the whole
Petri dish surface. Nonetheless, differences in the growth were visible once the basidiospores were
produced on MCA_LTBA-amended artificial media. While the basidiospores produced by R. placenta 45
on untreated sawdust did not germinate on MCA_LTBA-amended artificial media, basidiospores from
MCA_LTBA-pressure-treated wood sawdust could germinate and grow effectively. The growth speed of
these latter was slower on MCA_LTBA-amended artificial media than on controls: the mean growths after
2 weeks were 6.6±0.6 cm and 8.5±0.0 cm, respectively.
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Fig. 4. TEM micrograph of R. placenta 45 basidiospores grown on sawdust produced from MCA-
pressure-treated wood. The basidiospores appear ellipsoidal, whose lengths range between 1 and 5 µm
and widths of 2 µm. Neither deformation of the cell organelles nor accumulation of electron-dense
material attributable to Cu were visible.
Discussion
The aim of this study was to determine whether Cu-tolerant wood-destroying basidiomycetes exhibit
mechanisms of Cu compartmentalization in basidiospores as survival strategy while colonizing treated
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wood, if the Cu uptake in basidiospores is direct or occurs via different fungal structures, and in which
form Cu occurs in basidiospores. In particular, we aimed to understand if Cu-based NPs contained in MC
wood preservative formulations could be remobilized and released into the environment.
To investigate these aspects we used the highly Cu-tolerant [18] wood-destroying fungus R. placenta and
stimulated the production of basidiospores when growing on sawdust from MCA-pressure-treated and
untreated wood. The basidiospores were released in a Cu-free environment and subsequently analyzed to
determine if Cu was present, in which form, and whether the basidiospores were viable or not. The
method here developed to stimulate the sporulation of R. placenta proved to be sound and reproducible.
ICP-MS and XRF analyses highlighted a major difference in the amount of Cu contained in the two kinds
of basidiospores, with basidiospores generated by R. placenta grown on sawdust from MCA_LTBA-
pressure-treated wood containing up to 10 times more Cu. Therefore, our study revealed the presence of
spore compartmentalization mechanisms by the Cu-tolerant wood-destroying basidiomycete tested here.
This is the first time such a compartmentalization strategy is shown in non-mycorrhizal fungi. If we
compare the amount of Cu found in basidiospores with the amount of Cu in wood dust, as reported by
different studies [26, 27], we can see how they differ by a factor of 10: while wood dust from wood
treated with Cu-based wood preservatives contains between 0.1% [26] and 0.3% [27] Cu, only 0.02% Cu
was found in R. placenta basidiospores. In addition, the amount of Cu detected in the basidiospores
seemed to reach a threshold level. The concentration of Cu in the fruiting bodies growing in contact with
the MCA formulation with the highest fungicidal properties due to the presence of high amount of
tebuconazole (MCA_HTBA) doubled the one detected in fruiting bodies exposed to MCA_LTBA;
however the amount of Cu in the respective spores was comparable. Therefore, we can consider the
amount of Cu detected as a maximum concentration. These findings suggest that if present in equal
amount, Cu-loaded wood dust is more likely to be a threat than basidiospores. Further, the size of the
basidiospores, as evaluated by TEM analysis, was comprised between 1 µm and 5 µm. In terms of
respiratory tract uptake, this finding suggests that, if inhaled, the basidiospores would likely deposit in the
middle respiratory tract [28-30], independently on the presence of Cu. Despite that, sensitive subjects that
already experience adverse effects due to basidiospore inhalation, may face an exacerbation of their
reactions [11-16].
Beside the differences in the amount of Cu, dissimilarities in the speciation of Cu could be emphasized by
XAS analysis, both in the XANES and EXAFS regions. In fact, the amount of Cu bound to cysteine in
basidiospores heavily increases in the presence of MCA_LTBA-pressure-treated-wood, doubling the
CuCys amount observed under normal conditions (basidiospores generated by R. placenta grown on
sawdust from untreated wood). Difference in the coordination of Cu, in terms of neighboring atoms or
coordination structures, was also suggested by the mismatching EXAFS spectra. Similar findings on
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biological material were obtained by Kopittke et al. [31] in the cowpea root system, and by Fomina et al.
[32] in ectomycorrhizal fungi. Similarly to the latter study, the increased importance of CuCys in the
linear combination fitting in basidiospores produced in presence of MCA_LTBA is likely to indicate the
presence of Cu resistance mechanisms [33, 34]. By contrast, the amount of reduced Cu or Cu2+ free ions
complexed in water in basidiospores decreased when R. placenta was exposed to Cu (sawdust from
MCA_LTBA-pressure-treated-wood). In fact, Cu tolerance mechanisms convert bioavailable Cu2+ (as free
ions or loosely bound) into non-bioavailable (thus not toxic) Cu complexes or precipitates, like CuCys.
The XAS finding that Cu is mostly bound to cysteine or present as free ions complexed in water was also
confirmed by TEM investigation. No electron-dense material was visible in the micrographs, especially in
the ones from basidiospores produced in a Cu-rich environment (generated by R. placenta grown on
sawdust from MCA_LTBA-pressure-treated wood). This is in good agreement with the finding that
metallic Cu is likely present only in basidiospores generated by R. placenta grown on sawdust from
untreated wood, and that neither CuCO3∙Cu(OH)2 nor CuSO4 contributed to the XAS spectra from both
kinds of basidiospores. This indicates that the initial CuCO3∙Cu(OH)2 particles contained in the MCA
wood preservative formulation are unlikely to be remobilized and end up in the basidiospores, instead they
undergo a change in speciation. Therefore, the Cu compartmentalization mechanism here highlighted is
not likely to depend on the wood preservative system, but it is likely to apply to different ones, i.e.
conventional wood preservative as well as different MC formulations. Oxalate is believed to be implicated
in metal tolerance for brown rot fungi [35]. Therefore, it was surprising to find that, Cu oxalate did not
contribute too. This, combined with the observations that all basidiospores were still viable, and
basidiospores generated in a Cu-rich environment subsequently germinated more effectively in Cu-
amended artificial media, indicates that Cu-tolerance mechanisms for R. placenta basidiospores must be
involved, but they do not rely on Cu complexation by oxalic acid to form non-bioavailable Cu oxalate.
Our results on basidiospore viability are not in contrast with previous studies [36] showing that the
germination of R. placenta basidiospores is inhibited by MC if the fungus was not previously exposed to
Cu. However, exposure to Cu prior to the production of spores by R. placenta is likely to trigger the
basidiospores’ ability to adapt to Cu, and germinate in Cu-rich environments. Therefore, we can suppose
that Cu detoxification mechanisms in spores can be inherited.
For the first time the mechanism of Cu compartmentalization in basidiospores is shown to exist in wood-
destroying basidiomycetes. Therefore, Hypothesis 1 was verified. However, the Cu taken up undergoes
changes in speciation, with no CuCO3∙Cu(OH)2 particle identifiable. Therefore, Hypothesis 2 was
falsified, and our findings reveal that the fungal remobilization of Cu from MC wood preservatives does
not pose a nano-specific risk. However, the compartmentalization mechanisms highlighted are likely to be
valid for conventional wood preservatives too. Therefore, a more detailed assessment on the human and
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environmental risk is required. In particular, a quantitative assessment on the exposure to Cu-loaded
spores and on their toxicity should be conducted.
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severe asthma: a summary of the evidence. Eur. Respir. J. 2006 Mar; 27(3): 615–626.
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15. Lopez M, Voigtlander JR, Lehrer SB, Salvaggio JE. Bronchoprovocation studies in basidiospore-
sensitive allergic subjects with asthma. J. Allergy Clin. Immunol. 1989; 84(2): 242–246.
16. Santilli Jr J, Rockwell WJ, Collins RP. (1985). The significance of the spores of the
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17. Civardi C, Schwarze FWMR, Wick P. Micronized copper wood preservatives: an efficiency and
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18. Sutter H, Jones EBG, Walchi O. The mechanism of copper tolerance in Poria placenta (Fr.) Cke.
and Poria vaillantii (Pers.) Fr. Mater. Organismen 1983; 18: 241–262.
19. Civardi C, Schubert M, Fey A, Wick P, Schwarze FWMR. Micronized copper wood
preservatives: efficacy of ion, nano, and bulk copper against the brown rot fungus Rhodonia
placenta. PLoS ONE 2015 Nov 10: e0142578.
20. Schmidt O. Indoor wood-decay basidiomycetes: Damage, causal fungi, physiology, identification
and characterization, prevention and control. Mycol. Progress 2007 Nov; 6: 261–279.
21. Eslyn WE. Utility pole decay. Part II: basidiomycetes associated with decay in poles. Wood Sci.
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22. European Committee for Standardization. EN 113 Wood preservatives- Test method for
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of toxic values. Brussel, BE; 1997.
23. Croan S. Basidiosporgenesis by brown-rot basidiomycetes in vitro. IRG/WP 95-10126.
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27. Nygren O, Nilsson CA, Lindahl R. Occupational exposure to chromium, copper and arsenic
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31. Kopittke PM, Menzies NW, de Jonge MD, McKenna BA, Donner E, Webb RI, et al. In situ
distribution and speciation of toxic copper, nickel, and zinc in hydrated roots of cowpea. Plant
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32. Fomina M, Charnock J, Bowen AD, Gadd GM. X-ray absorption spectroscopy (XAS) of toxic
metal mineral transformations by fungi. Environ. Microbiol. 2007 Feb; 9(2): 308–321.
33. Jaeckel P, Krauss G, Menge S, Schierhorn A, Rücknagel P, Krauss, GJ. Cadmium induces a novel
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35. Tang JD, Parker LA, Perkins AD, Sonstegard TS, Schroeder SG, Nicholas DD, et al. Gene
expression analysis of copper tolerance and wood decay in the brown rot fungus Fibroporia
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36. Stirling R, Drummond J, Morris PI. Inhibition of basidiospore germination by copper from MCQ,
ACQ and CCA leachates. IRG/WP 12-10778. Proceedings IRG Annual Meeting; 2012 May 6-10;
Kuala Lumpur, MY. Stockholm, SE: International Research Group on Wood Preservation; 2012.
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5. Discussion & conclusions
5.1 General discussion This dissertation focused on a pressing question for the wood protection and nanosafety
communities: whether Cu-based NPs-containing-MC is superior to conventional wood preservatives.
More precisely, whether the penetration into refractory wood species is facilitated, the efficacy of the
treatment against wood-destroying fungi is enhanced, and that the formulation does not constitute a health
and environmental risk. In terms of possible risks, we investigated whether there is an airborne release of
Cu-based NPs caused by MC-pressure-treated wood during its service life. The main aims and objectives,
and the research hypothesis are listed in Chapter 1.1, page 13. In order to thoroughly answer the question,
the penetration and protective effectiveness of MC were assessed first (Hypotheses 1, 2). In fact, these
aspects are essential to monitor the fate and effectiveness of Cu from MC formulations. Subsequently, two
major release pathways were identified and investigated: the compartmentalization of Cu in spores due to
wood colonization by Cu-tolerant wood-destroying basidiomycetes (Hypotheses 2, 3), and the production
of wood dust by mechanical abrasion (Hypothesis 4). Further, a preliminary assessment of the possible
adverse health effects caused by inhalation of wood dust from MC-pressure-treated wood was conducted
(Hypothesis 5).
5.1.1 Hypothesis 1: Within MC particle size range, Cu-based NPs, rather than
Cu-based microparticles, are the main responsible for wood protection against
wood decomposing fungi
The thorough particle characterization conducted on MCA formulations aimed at describing the
Cu-based particles main properties, i.e. size, morphology, ζ. The full characterization on the MCA
formulations tested is available in Chapter 4.1.1. This provided striking evidence on the presence of Cu-
based particles solely in the nm range, therefore confirming the major role of NPs in MC-treated wood, in
terms of both impregnation abilities and potential effectiveness against wood-decomposing fungi. Our
results are in good agreement with other studies on MC formulations [1-3], indicating that different MC
formulations are likely to fall under the European definition of nanomaterials [4]. In particular, the two
MCA formulations here assessed had average sizes of 104±1.7 nm (mode: 87±2.2 nm) for MCA_HTBA
and 174±5.9 nm (mode: 150±8.2 nm) for MCA_LTBA. These findings provide support to Hypothesis 1.
The first speciation of Cu-based NPs particles from MC occurs once the impregnation of wood
takes place. In fact, this is the basis of the postulated innovative impregnation chemistry of MC
formulations. Once wood is treated with MC, a fraction of CuCO3·Cu(OH)2 solubilizes and reacts with
wood components, while part of it remains in the form of unreacted CuCO3·Cu(OH)2 particles that slowly
solubilize with time, providing a reservoir effect [5] responsible for a continuous protection against wood-
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decomposing fungi [6]. Already from this first phase, our studies demonstrate the importance and the
challenges of NP environmental fate monitoring. As a matter of fact, different methods with increasing
detection limits were required in order to keep track of Cu transport and speciation. However, even the
high resolution techniques didn’t show detection limits low enough to detect single NPs in the biological
systems. First, an investigation on the penetration of Cu from MCA in wood was conducted. More
precisely, we assessed the output of MCA-pressure-treatments of easily treatable Scots pine sapwood and
of refractory Norway spruce sapwood and heartwood. In this way, we could infer if the innovative
impregnation chemistry and the presence of NPs could lead to a better penetration in wood species
commonly used in the European market –in particular the refractory ones– without the need for pre-
treatments prior impregnation, e.g. incision. This would provide an added value to MC compared to
conventional wood preservatives. By combining ICP-OES and X-ray CT we could conclude that while Cu
could easily and homogeneously penetrate into Scots pine; it mainly lies on the surface of Norway spruce
(both sapwood and heartwood). Superficial treatments of wood are not effective, as they provide a shield
that can be penetrated by wood-destroying fungi once cracks occur on the wood surface, due to abiotic or
biotic agents. Furthermore, the analyses confirmed a small, though significantly different, higher amount
of Cu in Norway spruce sapwood than in the heartwood. Besides yielding different types of information,
the diverse analytical techniques sometimes provided results that –without being in disagreement– were
however not easily comparable, due to the nature of the analyses or their sensibilities. In fact, both the
ICP-OES and the X-ray CT analyses provided evidence for a different penetration gradient in easily
treatable and refractory wood species, however the precise Cu quantifications differed. This is a common
issue in the detection of NPs in biological or environmental matrixes [7]. In addition, the detection limits
of X-ray CT did not allow to determine whether Cu in wood cells was present as ions or particles below
0.8 µm, therefore impeding to clearly define whether single particles responsible for the reservoir effect
were present. Therefore, the presence of a reservoir effect, which would make MC an innovative wood
preservative couldn’t be confirmed or disputed. Despite the issues encountered, we could confidently
conclude that although the distribution of Cu from MCA differs than the one from conventional wood
preservatives like alkaline copper quaternary (ACQ) [8], with Cu from MC mostly present in the latewood
rather than in earlywood [8]. Pre-treatments to facilitate the penetration of wood preservatives in
refractory wood would still be necessary, as the penetration of Cu in Norway spruce without any pre-
treatment was not sufficient. Therefore, to determine which wood preservative treatment works best, a
LCA/LCIA approach should be applied, where variables like material inputs, environmental outputs,
transportation requirements from raw material extraction up through the point of use should be taken into
account. In the scientific literature, it is possible to find contradicting findings: while some studies indicate
how the LCA outputs from MC and conventional Cu amine wood preservatives do not greatly differ, due
to energy consumption related to milling [9, 10], other LCA and LCIA researches have shown how MCA
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and MCQ are environmentally preferable to ACQ [11, 12], and similar conclusions can be drawn for Cu
azole wood preservatives. The reason behind the mismatching LCA/LCIA outputs lies in the particulate
matter formation, i.e. different methods used to mill the NPs. Therefore, MC is as efficient as or more
efficient than conventional wood preservatives. In addition, compared to conventional wood preservatives,
MC does not require solvent during compounding, which also impacts the transportation requirements,
and lower amounts are require to effectively protect wood. Further, lower leaching and corrosion potential
were highlighted by Tsang et al. [11]. Pre-treatment (incising) of Norway spruce would be required for
both MC and conventional wood preservatives, therefore since the penetration of MC and conventional
wood preservatives is comparable, as proven in this thesis, the evaluation of the best system to use should
shift towards the LCA/LCIA outputs, which are more favorable for MC. Thus, we can conclude that the
energy efficiency of MC will still outperform conventional formulations in Europe. From a LCA/LCIA
perspective, our results combined with the existing literature on MC’s LCA/LCIA lead to the conclusion
that MC would be more preferable to ACQ even for European refractory wood species.
5.1.2 Hypothesis 2: Cu-tolerant wood-decomposing basidiomycetes exhibit the
same tolerance mechanisms towards Cu, independently from the form of Cu
After assessing the microdistribution of Cu in wood, we tested the performance against soft rot
fungi inhabiting the soil and the Cu-tolerant wood-destroying basidiomycete R. placenta. The wood
preservative effectiveness of MC was assessed according to the European guidelines ENV 807 [13] and
EN 113 [14]. These tests, which correlate the wood mass losses with the ability of the fungus to overcome
the presence of the biocides in wood, would enable to understand the antifungal performance of MC. Even
though standardized tests were used, it is always challenging to predict the long-term performance of
wood preservatives, in particular when innovative systems with different impregnation chemistries are
developed. Further, the behavior of fungi can radically change between artificial laboratory conditions and
natural setting, as well as vary within different artificial media. Short-term laboratory tests provide quickly
and at reduced costs an initial idea on the performance of wood preservatives. They are necessary, but not
sufficient. Therefore, the short-term laboratory studies conducted provided an indication on the possible
efficacy, but they should be associated with long-term field stake tests, e.g. EN 252 [15]. These would
provide a more realistic scenario on the service life of pressure-treated wood. The short-term efficacy of
MCA_HTBA against soft rot and other soilborne fungi was confirmed, even at relatively low
MCA_HTBA concentrations. More precisely, the recorded wood mass losses were below 3% for wood
stakes treated with MCA_HTBA concentrations as low as 0.80%. However, the duration and nature of the
ENV 807 test cannot precisely discriminate between the effects of Cu and TBA. In fact, in the long-term
the latter is likely to undergo degradation or decomposition, either abiotic [16] or biotic [17,18], leaving
Cu as the only antifungal agent. This would promote the survival of Cu-tolerant species only or the
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development of Cu-tolerance and resistance mechanisms in the microbial communities in close proximity
to the MC-pressure-treated timber structures [19]. These communities are not likely to be affected by the
presence of Cu-based NPs, according to our studies on R. placenta. We assessed different parameters for
fungal fitness and Cu-tolerance in R. placenta exposed to Cu2+ ions, MCA and CuCO3·Cu(OH)2: fungal
biomass, oxalic acid and laccase production, and wood mass losses. In addition, we could discriminate any
effect caused by particle solubilization and release of Cu2+ ions by means of the Cu2+ ion selective chelator
TTM. What emerged is that the undissolved NP fraction from MCA does not exert specific Cu nano
effects towards the Cu-tolerant fungus, which exhibits the same Cu-tolerance mechanisms independently
on the form of Cu. Therefore Hypothesis 2 is supported. Instead, the importance of using NPs rather than
bulk material was confirmed by the EN 113 results from MCA_LTBA- and bulk CuCO3·Cu(OH)2-
pressure-treated wood: the latter performed poorly against fungal attack because the impregnation was not
successful due to the size of the particles, thus unreacted CuCO3·Cu(OH)2 appeared as unbound dust on
the wood sample surfaces. What also emerged from the fungal fitness study is that the presence of Cu in
sub-lethal concentrations actually helps the fungus to overcome the issue of TBA, both in artificial media
and wood. Even more, TBA in both MCA_LTBA and MCA_HTBA did not prevent R. placenta from
sporulation, even at high concentrations (2.00% MCA-pressure-treated wood) and in laboratory condition,
confuting the belief that tebuconazole inhibits the sporulation of wood-destroying fungi [20]. Hence, the
combination of Cu and TBA does not result in a synergistic effect against R. placenta. This can also
explain the different efficacy exhibited by MCA_HTBA in pressure-treated Scots pine sapwood, Norway
spruce sapwood and heartwood. In Norway spruce heartwood, the most refractory wood according to ICP-
OES and X-ray CT analyses, the low amount of TBA is sufficient to inhibit fungal colonization and the
low amount of Cu is not sufficient to provide support to the fungus to overcome the TBA obstacle,
whereas in the more accessible Norway spruce sapwood the amount of Cu reaches concentrations useful
for R. placenta, until it reaches the threshold concentration (easily treatable Scots pine) and start exerting a
toxic effect on the fungus, which is no longer able to colonize the wood substrate. To summarize, the
results on the antifungal effectiveness indicate that MCA can perform as good as conventional wood
preservatives on the short-run. Combining the LCA/LCIA outputs from the scientific literature previously
discussed together with the antifungal performance here tested, we can conclude that MC is superior to
conventional wood preservatives, as MC shows a higher energy efficiency that results in a lower impact
on the environment. This conclusion would be valid also within the European decking market and its
refractory wood. Therefore it should be preferred for in-ground timber applications.
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5.1.3 Hypothesis 3: Similarly to mycorrhizal fungi, Cu-tolerant wood-
destroying basidiomycetes can accumulate Cu in their spores
As long as Cu remains within the timber structure, independently on its size, concentration, or
species, it does not pose a risk, as no exposure can occur. However, issues arises once Cu is remobilized.
Studies on Cu leachability from MC-pressure treated wood indicate low leaching potential for MC [1, 11,
21-24]. However, between 42% and 88% of the Cu leached was present as CuCO3·Cu(OH)2
microparticles or NPs [1], resulting in a release of Cu-based NPs in the soil and water compartments. The
research here conducted characterized the airborne particles released from MCA-pressure-treated wood
due to abrasive processes or spore compartmentalization mechanisms by the Cu-tolerant fungus R.
placenta. These two scenarios can occur during the service life of MC-pressure-treated wood or its
disposal in landfills. In these studies, easily treatable Scots pine sapwood was selected as the only type of
wood to use, in this way we could achieve a full impregnation of wood and maximize the potential release
that would result in a worst case scenario. The main focus was on fine and ultrafine particles, which can
reach the deep lungs and exert adverse health effects on the lungs [25-27]. In particular, our goal was to
assess if the use of MC could lead to an additional nano-release due to the presence of unreacted Cu-based
NPs from the initial MC wood preservative formulation.
Despite the positive performance of MCA in wood, the Cu-tolerant wood-destroying
basidiomycete R. placenta was still able to colonize both MCA_LTBA and MCA_HTBA-pressure-treated
wood, due to the presence of Cu-tolerance mechanisms. A setting to stimulate the formation of fruiting
bodies and basidiospores in R. placenta during the colonization of wood was developed. The experimental
conditions adopted allowed the fungus to grow and colonize wood that was pressure-treated with
concentrations of MCA_LTBA and MCA_HTBA relevant for the wood protection market (2.00%).
Further, the setting did not allow the basidiospores to get directly in contact with MCA-pressure-treated
wood, so that the presence of Cu in these structures could be attributed entirely to Cu uptake and
remobilization by the fungus, and not to direct Cu uptake by basidiospores. Elemental analysis, namely
ICP-OES and XAS, revealed a high uptake of Cu by R. placenta basidiospores produced in presence of
MCA-pressure-treated wood. Further, the amount of Cu did not depend on the content of TBA, as similar
Cu concentrations were detected in basidiospores produced in presence of MCA_LTBA and
MCA_HTBA. In addition, Cu was also detected in fruiting bodies, confirming the presence of a Cu
compartmentalization mechanism and providing support to Hypothesis 3. The examination of the average
Cu species in basidiospores produced in normal conditions (untreated wood) and MCA-pressure-treated
wood highlighted a difference in the Cu speciation. Comparing the XAS spectra from both spores and
reference materials, we could observe how the amount of unreacted CuCO3·Cu(OH)2 particles released via
basidiospores, even in presence of MCA, is likely to be negligible. In fact, the TEM micrographs showed
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that Cu was not present as precipitates, agglomerates, aggregates, or deposits of electron-dense material,
therefore indicating that Cu was likely present as fine particles or as free or bound Cu2+. Further, the
comparison of XAS spectra from basidiospores and reference materials indicates that Cu is likely to be
bound to organic compounds, in particular to Cys. The formation of CuCys complexes in fungal structures
was already observed in previous studies [28, 29] and was attributed to Cu-tolerance mechanisms. More
precisely, Cys is abundant in metallothioneins, which play a major role in heavy metal homeostasis and
sequestration [30, 31]. Despite the high level of Cu, the TEM micrographs indicate that basidiospores
from MCA-pressure-treated wood did not show any deformation and appeared viable. Even more, the
spore viability was assessed in control and MCA-amended artificial media by assessing the radial growth
in Petri dishes. What emerged is that Cu-loaded basidiospores could colonize the artificial media
containing Cu more effectively than basidiospores produced in presence of untreated wood. This indicates
that, beside hyphae and mycelia, also spores may have acquired tolerance for Cu. This is the first time the
acquisition of Cu-tolerance mechanisms by spores is highlighted. In fact, previous studies assessed the
germination of spores from Cu-tolerant fungi that were not exposed to Cu in the sporulation phase [32].
5.1.4 Hypothesis 4: Abrasion of MC-pressure-treated wood may lead to further
dissemination of Cu-based NPs in the environment
Besides Cu release via spore, we investigated the outcome of mechanical abrasion of MCA-
pressure-treated wood. For untreated, MCA-, and conventional CC-pressure-treated wood, we
characterized the wood dust particle size (APS and SMPS), morphology (SEM), and the Cu content (ICP-
OES). In terms of size distribution, the wood dust produced did not differ significantly from untreated
wood or CC-pressure-treated wood. In particular, it did not result in an increase in the nanosized fraction
of wood dust released. Furthermore, the elemental analysis performed on the whole wood dust and on the
nanosized fraction, indicated that Cu is mostly released with the bigger wood dust particles, above 1 µm,
with an increase from 0.15 % Cu in the nanosized fraction to 0.20% in the whole wood dust. This is in
accordance with previous findings from Platten et al. [3], which characterized the wood dust released by
wiping MCA-pressure-treated wood and observed Cu to be mostly associated with large cellulose or
lignin particles. Therefore, we can conclude that MC is not a source of additional exposure to
nanoparticles, indicating that Hypothesis 4 is not supported. Furthermore, the application of varnishes on
untreated or treated wood surfaces can increase the wood dust particle size, which was also evidenced by
Nowack et al. [33] on sieved fragmented products from CuO-acryl antimicrobial wood coatings.
Therefore, varnishes could be used to further reduce the risk. We can hypothesize a minimal contact for
consumers, whose exposure to MCA-pressure-treated wood and its dust is occasional, whereas exposure
to wood dust mostly occurs among woodworkers [34-36], thus it could be considered as a substance of
concern for professional users, as occupational exposure occurs more frequently.
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5.1.5 Hypothesis 5: Cu-based NPs from MC wood preservative formulations, if
inhaled, can cause adverse effects due to specific nano effects
The amount of Cu detected in wood dust greatly exceed the ones found in spores (10-folds
higher), and the chances of releasing particles below 1 µm –which can reach the deep lungs [25-27] if
inhaled– is likely to occur only due to mechanical abrasion, as R. placenta spores sizes ranged from 1 µm
to 5 µm. Therefore, a preliminary in vitro hazard assessment to determine eventual adverse health effects
was conducted solely on the abraded particles. To simulate the lung environment we used lung epithelial
cell lines and macrophages, and we assessed the potential adverse effects of dust from MCA-pressure-
treated wood by monitoring the oxidative stress, cell viability, and cellular inflammation. Both the MCA
wood preservative formulation, reference materials (TBA, Cu2+ ions, positive control CdSO4), wood dust
from untreated, MCA-, and CC-pressure-treated wood, as well as eluates from the same wood dust were
assessed. The findings from the in vitro citotoxicological studies indicate that MCA and dust from MCA-
pressure-treated wood did not induced a nano-specific toxicity in lung epithelial cells and macrophages,
and it exerted fewer adverse effects than wood dust from CC-pressure-treated wood. Therefore, despite
dust from MCA-pressure-treated wood may pose a hazard, this is not related to specific nano effects. This
indicates that Hypothesis 5 is not supported under the experimental conditions adopted.
5.1.6 Further comments
Our results suggest that the release of Cu in the air compartment due to mechanical abrasion or
spore compartmentalization does not depend on the presence of Cu-based NPs. More precisely, the
abrasion of MC-pressure-treated wood does not produce a higher fraction of wood dust in the nano range,
and the cytotoxicological studies revealed no additional toxicity from wood dust generated by abrasion of
MC-pressure-treated wood dust compared to CC-pressure-treated wood, therefore indicating that CU-
based NPs do not pose an additional risk. In addition, via spores, Cu is not likely to be released as
CuCO3·Cu(OH)2 NPs, as it undergoes speciation. Therefore, the release mechanisms here assessed are
likely common to any Cu-based wood preservative formulations. Furthermore, when these findings are
combined with the results from Platten et al. [1] results, and integrated in a mass balance, we can conclude
that only the water and soil compartments are exposed to Cu-based NPs, although only to a limited extent.
If we combine this finding with the fact that MC is generally needed in lower amount than conventional
wood preservatives to effectively protect wood [11], we can hypothesize that the release of Cu from MC
via spores is a best case scenario, while conventional wood preservatives may cause a higher release of Cu
via spores. Besides, a major percentage of Cu is released via particles (wood dust or spores) with sizes
above 1 µm, which cannot easily reach the lower respiratory tract. However, other fungi, more
widespread, with smaller spores and allergenic, e.g. Aspergillus spp. or Penicillium spp. may pose a higher
risk [37]. Further, asthmatic or allergic individuals, which would suffer from phase reaction due to the
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contact with spores [38-43], could experience an exacerbation of the symptoms. In addition, the nanosized
fraction, which can be generated by mechanical abrasion of pressure-treated wood, showed less adverse
effects in lung epithelial cells and macrophages than conventional CC. The scientific literature on release
of Cu from MC-pressure-treated wood in the water and soil compartments showed that the amount of Cu
detected in the two environmental compartments, mainly due to leaching, is lower than what measured
from wood pressure-treated with conventional wood preservatives [1, 11, 21-24]. When the evidence on
the possible release in the air compartment, as measured in this thesis, is combined with the studies that
assessed the release in water and soil just mentioned, it clearly emerges how MC outperforms
conventional wood preservatives by overall releasing lower amounts of Cu in all the different
environmental compartments.
5.2. Conclusions & outlook This thesis examined the fate and behavior of Cu-based NPs from MC preservatives during the
wood impregnation and service life of in ground timber structures and focused on the release in the air
compartment. Beside particle sizes and elemental composition, transformation and alteration processes of
the initial nanomaterial were considered, according to the current paradigm for risk assessment of
nanomaterials [44]. The research project was carried out within the framework of the following working
hypotheses, which were supported or confuted as follows:
1. Hypothesis 1: supported
2. Hypothesis 2: supported
3. Hypothesis 3: supported
4. Hypothesis 4: not supported
5. Hypothesis 5: not supported.
In summary, our findings reveal that MC is a valid alternative to conventional wood
preservatives, in terms of effectiveness, and environmental and health risks. In particular, in the air
compartment, MC does not pose any additional nano-specific risks due to the NPs present in the
formulation. An airborne release of Cu was observed when MC-pressure-treated wood is subjected to
mechanical abrasion or colonization by Cu-tolerant wood-destroying fungi, however the particle sizes, the
Cu concentrations, and the hazard assessment do not provide evidence for higher health and
environmental risks than the ones posed by conventional wood preservatives. Even more, when compared
to other wood preservatives, MC actually constitutes a safer alternative. This is not only valid for the air
compartment, but for Cu from MC released in water and soil too [1, 40-43]. Cu is a mainstay for in-
ground timber applications, and currently there is no satisfactory alternative [45]. Therefore, based on our
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findings, it is here suggested the use of MC over conventional wood preservatives, as it outperforms them
on different levels.
The analytical methodologies used in the current study proved to be capable and comprehensive
tools to address the issues of wood preservative effectiveness, estimation of emissions, and hazard for
human lungs of Cu-based NPs from MC in a realistic manner. However, there are still a few open points
that need to be addressed for a better monitoring of Cu-based NPs used for wood protection, in terms of
flows and concentrations, as well as hazard assessment. More precisely:
1. Additional studies on the speciation of Cu in the different stages of MC wood preservatives
could provide further information on the mechanisms behind their effectiveness and lead to
improvements in the NPs and in the formulation that could lead to better impregnation,
enhancement of the reservoir effect, reduction of preservative release (e.g. leaching) and
corrosion potential. Moreover, they could help to better define the environmental fate of MC
in the different environmental compartments and during the whole life cycle of MC and MC-
pressure-treated wood
2. X-ray CT at different resolutions allowed to determine the macro- and microdistribution of
Cu in wood, and provided indication on the possible form of Cu. However, the laboratory-
scale instruments couldn’t visualize single NPs. Nanoscale ptychography [46] combined with
3D tomographic reconstruction may be able to resolve the interaction between Cu and a
single wood cell at an unprecedented scale. Further, when these techniques are applied to
decayed wood cells, the understanding of wood decomposition mechanisms would provide a
useful tool for wood protection advances, e.g. by the development of more tailor-made wood
preservatives.
3. Despite the risk related to MC-pressure-treated wood appear to be low, a quantitative
assessment of the amount of Cu-loaded spores released in the air can lead to a better risk
characterization. In particular, establishing which Cu-tolerant fungi –either basidiomycetes or
not– are able to compartmentalize Cu in their spores is essential for a precise emission
assessment. This would not only be relevant for Cu from pressure-treated wood, but from
other Cu sources too, like Cu polluted environments where Cu-tolerant fungal communities
can be found [47]
4. Beside the qualitative and quantitative evaluation on Cu-loaded spores, an assessment of
spores transport and mobility is here suggested. This would enable to determine whether other
human and environmental exposure can occur, e.g. if the Cu- loaded spores deposit on the
ground, and dermal transfer or ingestion by humans or animals can take place
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5. Further studies should investigate more in depth the hazard of wood dust from MC-pressure-
treated wood or Cu-loaded spores, with more advanced models mimicking the lungs, e.g.
utilizing ex vivo or in vivo approaches [48]
6. Similarly, the toxicity of Cu-loaded spores from wood-destroying fungi belonging to different
phyla should be evaluated. In fact, while the evaluation here conducted is mainly based on the
physical properties of Cu-loaded spores (size, form of Cu), they may anyway exacerbate
respiratory diseases or induce allergic reactions due to the spores themselves [35, 49], which
may be enhanced by the presence of Cu
7. The currently available LCA/LCIA and material flow models on MC can be implemented
with the recent findings, in order to provide a more realistic overview.
To conclude, the current work provided a useful approach to monitor NPs in different media, as
well as an insight into MC, its potentials and pitfalls. However, “wood science is not rocket science… it’s
much more complicate!” [50], and the same applies to nanoscience. For the science behind MC and its
Cu-based NPs “there’s still plenty of room at the bottom” [51].
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6. Curriculum vitae
Chiara Civardi
PERSONAL INFORMATION
Date of birth: 07. 03. 1988
Place of birth: Codogno (MI), Italy
Citizenship: Italian
EXPERIENCE
05.2016-10.2016 Project Drawdown Sausalito, US
Volunteer Researcher
04.2016-04.2016 Wildlife Conservation Society do
Brazil/Pantanal/Cerrado
Campo Grande, BR
Wildlife Conservation Intern
10.2013-present United Academics Amsterdam, NL
Science Blogger
08.2013-01.2015 Telejob Zurich, CH
Seminar & Event Organizer
10.2012-03.2016 Empa St. Gallen, CH
PhD Student
03.2012-09.2012 The Mary Rose Trust Portsmouth, UK
Conservation Intern & STEM Assistant
10.2010-06.2011 Università degli Studi di Parma Parma, IT
Chemistry Intern
EDUCATION
10.2012-11.2016
(expected)
ETH Zurich, CH
PhD in Civil Engineering
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Pending result, Thesis: “Effectiveness and environmental risk of nanocopper-based wood
preservatives”
09.2011-09.2012 University of Southampton Southampton, UK
MSc in Maritime Archaeology
Pass with Distinction, Thesis: “Conservation of Mary Rose’s cordage: diagnosis and treatments”
10.2007-06.2011 Università degli Studi di Parma Parma, IT
BSc in Science and Technology for the Conservation and Restoration of Cultural Heritage
110/110 cum laude, Thesis: “Wood preservative treatments based on metal cations and
nanoparticles vehiculated by hybrid organic-inorganic polymers”
LIST OF PUBLICATIONS
Schubert, M., Fey, A., Ihssen, J., Civardi, C., Schwarze, F.W.M.R., Mourad, S. (2015). Prediction
and optimization of the laccase-mediated synthesis of the antimicrobial compound iodine (I2).
Journal of biotechnology, 193, 134-136.
Civardi, C., Schwarze, F.W.M.R., Wick, P. (2015). Micronized copper wood preservatives: An
efficiency and potential health risk assessment for copper-based nanoparticles. Environmental
Pollution, 200, 126-132.
Civardi, C., Schlagenhauf, L., Benz, J., Hirsch, C., Van den Bulcke, J., Schubert, M., Van Acker,
J., Wick, P., Schwarze, F.W.M.R. (2015). Environmental fate of micronized copper. International
Research Group on Wood Protection IRG/WP 15-50310, 9 pp.
Schubert, M., Ruedin, P., Civardi, C., Richter, M., Hach, A., Christen, H. (2015). Laccase-
catalyzed surface modification of thermo-mechanical pulp (TMP) for the production of wood
fiber insulation boards using industrial process water. PloS ONE, 10(11), e0142578.
Civardi, C., Schubert, M., Fey, A., Wick, P., Schwarze, F.W.M.R. (2015). Micronized copper
wood preservatives: Efficacy of ion, nano, and bulk copper against the brown rot fungus
Rhodonia placenta. PloS ONE, 10(6), e0128623.
Civardi, C., Schubert, M., Wick, P., Schwarze, F.W.M.R. (2016). Copper nanoparticles for wood
protection: effectiveness and remobilisation in the air. Lifecycle impacts of copper nanomaterials
released from timber preserving impregnations workshop proceedings. Venice, 2016 Jan 22.
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7. Declaration of personal contribution
