Transformations of Nanoenabled Copper Formulations Govern

Article pubs.acs.org/est

Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Transformations of Nanoenabled Copper Formulations Govern Release, Antifungal Effectiveness, and Sustainability throughout the Wood Protection Lifecycle Daniele Pantano,† Nicole Neubauer,‡ Jana Navratilova,§,# Lorette Scifo,⊥ Chiara Civardi,¶,∇ Vicki Stone,† Frank von der Kammer,§ Philipp Müller,‡ Marcos Sanles Sobrido,⊥ Bernard Angeletti,⊥ Jerome Rose,⊥ and Wendel Wohlleben*,‡ †

Nano Safety Research Group, Heriot-Watt University, Edinburgh EH14 4AS, United Kingdom Material Physics, RAA/OR and RAA/OS, BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany § Deepartment of Environmental Geosciences, University of Vienna, 1090 Vienna, Austria ⊥ CNRS-IRD-Collège de France - INRA, CEREGE Marseille University, 13545 Aix-en-Provence, France ¶ Empa, Swiss Federal Laboratories for Materials Science and Technology, 9014 St. Gallen, Switzerland ∇ ETH, Institute for Building Materials, 8049 Zurich, Switzerland ‡

S Supporting Information *

ABSTRACT: Here we compare the standard European benchmark of wood treatment by molecularly dissolved copper amine (Cu−amine), also referred to as aqueous copper amine (ACA), against two nanoenabled formulations: copper(II)oxide nanoparticles (CuO NPs) in an acrylic paint to concentrate Cu as a barrier on the wood surface, and a suspension of micronized basic copper carbonate (CuCO3·Cu(OH)2) for wood pressure treatment. After characterizing the properties of the (nano)materials and their formulations, we assessed their effects in vitro against three fungal species: Coniophora puteana, Gloeophyllum trabeum, and Trametes versicolor, finding them to be mediated only partially by ionic transformation. To assess the use phase, we quantify both release rate and form. Cu leaching rates for the two types of impregnated wood (conventional and nanoenabled) are not significantly different at 172 ± 6 mg/m2, with Cu being released predominantly in ionic form. Various simulations of outdoor aging with release sampling by runoff, during condensation, by different levels of mechanical shear, all resulted in comparable form and rate of release from the nanoenabled or the molecular impregnated woods. Because of dissolving transformations, the nanoenabled impregnation does not introduce additional concern over and above that associated with the traditional impregnation. In contrast, Cu released from wood coated with the CuO acrylate contained particles, but the rate was at least 100-fold lower. In the same ranking, the effectiveness to protect against the wood-decaying basidiomycete Coniophora puteana was significant with both impregnation technologies but remained insignificant for untreated wood and wood coated by the acrylic CuO. Accordingly, a lifecycle-based sustainability analysis indicates that the CuO acrylic coating is less sustainable than the technological alternatives, and should not be developed into a commercial product.

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toxicity against aquatic communities.3,4 Nonetheless, not all fungi are sensitive to copper, in fact, several brown rot fungi can grow at ionic concentrations of up to 100 mg/kg.1,5 This

INTRODUCTION Copper (Cu) is the most widely used fungicide for treating wood in contact with the soil, with no satisfactory alternative available since it is the only biocide that shows significant effects against soft rot fungi and other soil borne fungi.1 Cu is compulsory for inground timber structures (use class 4) as preferred antimicrobial wood preservative for its small effect on mammals, including humans2 even if it shows a relatively high © XXXX American Chemical Society

Received: August 23, 2017 Revised: November 1, 2017 Accepted: January 4, 2018

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DOI: 10.1021/acs.est.7b04130 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

Figure 1. Characterization of copper formulations. TEM images of micronized Cu (A), Cu−amine (B), pristine CuO NPs (C) and CuO NPs in acrylate with TiO2 (D). Analytical ultracentrifugation (E, compare SI) describes micronized Cu by a median diameter of 124 nm in mass metrics (black line, as relevant for performance), and by a median diameter of 34 nm in number metrics (red line, as relevant for identification as nanomaterial for regulatory purposes by the European Commission definition). (F) photograph of wood specimen, showing the typical undesired greenish color of Cu−amine (ACA) treated wood vs the natural appearance of micronized-Cu treated wood. See Tables SI_1, SI_2, SI_3 for a summary of properties.

peculiar category of fungi produces high economic damage since they are the main cause for the early failure of in-ground timber structures in the US and Europe.6,7 The high resistance shown by these fungi is one of the key factors driving the continuous development of copper-based formulations and their alternatives. In fact, these new formulations have shown the ability to bypass some of these resistance mechanisms,8 and elicit a toxic effect considered lower in humans if compared to microorganisms,9 even if recent studies show that copper oxide nanoparticles (CuO NPs) are still highly toxic against human cell lines.10,11 While the toxic mechanism of copper ions is known, there is still not sufficient information about the exact mechanism of Cu NPs12 and little evidence whether nanoforms of copper without transformation can be more efficient than copper salt against soil borne fungi or Cu-tolerant wood-destroying fungi.13−15 Furthermore, mechanistic interpretation is very challenging because several forms of copper could transform by oxidation to copper oxide (CuO) or sulfidation to copper sulfide (CuS), concomitantly with dissolution and release of Cu ions that may undergo further reaction.16 In the past decade, formulations of CuCO3·Cu(OH)2 (or micronized copper) were developed in size distributions that contain a considerable fraction of nanoparticles,17 from whence the term “nano-enabled” formulations. Because of the size distribution, these formulations are not only able to penetrate into the wood during pressure treatment18,19 but are also reported to improve the durability of wood against fungal decomposition.14,15,20,21

In our study, two radically different nanoenabled copper formulations were analyzed: CuO NPs in an acrylic paint to concentrate Cu at 15 kg/m3 as barrier on the wood surface, and a suspension of micronized basic copper carbonate (CuCO3· Cu(OH)2) for pressure treatment at 1 kg/m3 throughout the bulk wood (see Figure 1). The unusual barrier of Cu in the acrylic coating, with no Cu concentration within the wood, has the potential to reduce regular consumption from 1 kg/m3 to 0.01 kg/m3, depending on outer dimension of the wood (see Supporting Information (SI) for details). We benchmark the two nanoenabled formulations against conventional wood treatment by impregnation with molecularly dissolved Cu− amine (also known as ACA). First, we characterized the properties of pristine (nano)material formulations and assessed their antifungal effects on a specifically developed in vitro model. Then, we characterized the release of particulate and ionic species during simulated leaching and UV/rain weathering of treated wood and reassessed the effects against the fungus that showed the highest resistance to the original formulations. Finally, our results were placed into a sustainability context for decision-making using the LICARA nanoSCAN.

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MATERIALS AND METHODS CuO NPs were supplied by PlasmaChem (Germany) within the Sustainable Nanotechnologies (SUN) project, with a primary particle size of 15−20 nm and a surface area of 47 m2/g. A surface modification of CuO_ascorbate22 developed by ISTEC−CNR (Italy) was compared to the pristine NPs (see the SI for details). Pristine CuO was formulated as 1.5% B


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an accelerated aging test of pine specimens treated with copperbased formulations for simulating exposure to water. Initially, 4 kPa vacuum-atmospheric pressure cycle with leaching medium deionized water (DIW) was applied. The wood blocks were maintained in DIW (5 volumes of DIW to 1 volume of wood) for 14 days with nine water changes at 20 °C and 65% of relative humidity. The leaching waters were subjected to ICPMS (PerkinElmer Nexion 300) analysis for the total copper content determination and single-particle-ICPMS (spICPMS) for CuO acrylic paint-treated wood samples. For the leaching experiment, three independent replicates of the treated and untreated woods were used. For the ICPMS analysis from each specimen three subsamples were taken and 10× diluted using 10% HNO3 (v/v) prior ICPMS analysis. Laboratory simulation of weathering was adapted from EN 927-6.25 Wood samples were irradiated in a Sunstet XLS+ climate chamber (Atlas Material Testing Solutions, Germany) using a Xe lamp (solar spectrum) at a UV intensity of 65 W/m2 for a duration up to three months. Dry/wet cycles of 1365 min/ 15 min were applied; deionized water was used. Irradiation generated a blackbody standard temperature of 60 °C but the temperature of the wood samples was lower since the wood was not black. Additionally, a 24 h condensation phase using deionized water was performed in a homemade setup once a week.26 Run-off water was sampled during aging by placing the wood blocks on slightly inclined holders in beakers (Figure SI_6). Cu release was quantified using ICP-MS (PerkinElmer Nexion 300) after acidification of the runoff waters at 2.5% HNO3 (Ultrapure NORMATOM 67%). For delayed sampling, the wood specimens were aged up to 3 months resulting in a radiant exposure of 465 MJ/m2 (UV dose integrated 300 to 400 nm) and then exposed to two levels of shear stress, that is, immersion shaking and bath sonication. Finally, releases were analyzed as described by the prevalidated NanoRelease protocol.27 Specifically, the ISO 4892 aging conditions, used previously28 to study weathering of copper− amine treated wood, were adapted to match UV intensity and rain intervals of the direct sampling method. To complement quantification by UV−vis spectrometry and ICP-MS (optionally after filtration by 0.02 μm PVDF) and size distribution by analytical ultracentrifugation (AUC), immersion liquids were dried on ultrathin carbon grids for TEM analysis on a Tecnai G2-F20ST (FEI Company, Hillsboro, USA) microscope operated at 200 keV under bright-field (BF) conditions. Energy dispersive X-ray spectroscopy (EDXS) was applied to determine chemical composition at distinct spots using an EDXi-detection system with an energy resolution of 131 eV at Mn Ka (EDAX, Mahwah, USA). Images and spectroscopy data were evaluated using the Olympus (Tokyo, Japan) iTEM 5.2 (Build 3554) and FEI TIA 4.1.202 software packages. Information on the crystal structure of small sample regions was collected by selected area electron diffraction (SAD), an operation mode of TEM. We employed the LICARA nanoSCAN, a modular webbased tool that supports producers in assessing benefits and risks associated with new or existing nanoenabled products. ITo minimize the ambiguity from the uncertainty of input parameters, we chose the acrylic coating with and without the biocidal CuO additive as a test case. Input parameter details are documented in the SI.

additive into a high-gloss acrylic wood coating containing 43% white pigment TiO2 (non-nano) passivated with an alumina coating as well (Figure 1 and Tables SI_1, SI_2). The acrylic coating was applied to Cu contents of 1.7 g/m2 equaling 0.16 kg/m3 to pine wood specimen. The predispersed formulation contained 54% micronized copper (which is an elemental content of 30% Cu), with a hydrodynamic size median of 124 nm in mass metrics and 34 nm in number metrics, corresponding to an ensemble surface area of 16 m2/g. Platelets dominated the particulate shape, and about 0.5% of the Cu remained after hard centrifugation, identified as Cu ions. X-ray and electron diffraction both confirmed the composition as Malachite crystallinity (Figure 1, Figure SI_1 and Table SI_2). The concentrate formulation of Cu−amine contained 10% Cu. TEM, X-ray, and electron diffraction confirmed the absence of particulate species, and the entire Cu content remains unchanged after hard centrifugation (Figure 1 and Table SI_2). Impregnated wood specimens had 1 and 2 kg Cu per m3 pine wood. In total, about 250 pine wood specimens were prepared, including untreated wood and wood coated by the acrylic coating without CuO as controls (Table SI_3). The total Cu content as detailed in Table SI_3 was 1 kg/m3 for the impregnation technologies, but 15 kg/m3 localized in the barrier technology. For the relatively small wood specimen tested here, the barrier technology corresponded to a nominal content of 0.16 kg/m3, and scaled with the wood specimen dimensions, potentially reaching 0.01 kg/m3 for large wooden installations (see SI for details). Three fungal species suggested in the EN 11323 were used to assess the antifungal efficacy of copper formulations (before wood treatment). Coniophora puteana (Schumacher: Fries) P. Karsten (MUCL 20565), Gloeophyllum trabeum (Persoon: Fries) Murrill (MUCL 11353), and Trametes versicolor (Linnaeus: Fries) Pilát (MUCL 11665) were obtained from the Mycothèque de l′Université Catholique de Louvain and exposed to the same Cu equivalent. Copper sulfate was used as ion-releasing control (CuSO4, Sigma 451657). An inoculum from each fungal culture was independently transferred in freshly made dispersions of malt extract agar (MEA, Sigma 70145) containing either 200 or 100 μg of nominal copper from each treatment and incubated at 20 °C for a week. Two experiments were performed in triplicate for all three species of fungi. The antifungal activity was assessed by measuring the colony diameter in centimeters, and the data analyzed using the One-way ANOVA followed by Dunnett’s multiple comparisons with GraphPad Prism ver. 6.0 (GraphPad Software, USA). In addition, a preliminary screening on the effectiveness of the preservatives against wood-destroying basidiomycetes was determined after treating wood. Untreated (control) and treated Scots pine (Pinus sylvestris L.) sapwood blocks (50 × 25 × 15 mm) were exposed to C. puteana isolate 62 obtained from EMPA (the Swiss Federal Laboratories for Materials Science and Technology) culture collection at 22 °C and 70% relative humidity (RH). Test procedures were performed according to the European standard EN 113.23 After incubation, wood blocks were 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. See Figure SI_13 for details. According to the European norm EN 84,24 leaching studies were performed to evaluate the fixation of copper-based formulations into the wood matrix. This standard describes C


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Figure 2. Comparison between the least and the most copper sensitive species, respectively Coniophora puteana (blue columns) and Gloeophyllum trabeum (red columns), after 1 week exposure. The Y axis represents the diameter of each fungal colony measured in centimeters, being the size of the original fungal plug 1 cm (green basal line). Bars represent the mean data from two repetitions (n = 2), each one conducted in triplicate. Statistical significance expressed as the difference between fungal growth on treated agar versus corresponding control (p ≤ 0.05, 0.01, 0.001 and 0.0001). Data analyzed with GraphPad Prism version 6.0, one-way ANOVA.

Figure 3. Pictures of C. puteana exposed to pristine and safe by design CuO NPs. The same picture was taken from above (A) and below (B). Pigmentation is visible below the fungus treated with the modified CuO NPs (red arrows).

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RESULTS AND DISCUSSION

suggested by the European Centre for Standardization for analyzing antifungal property of wood preservatives,23 were obtained from the Mycothèque de l′Université Catholique de Louvain. Coniophora puteana, also known as cellar fungus, is considered a common element of brown rot wood decay for both soft and hard wood and it is widely accepted to be a relatively Cu-tolerant species.29 The results generated confirmed this observation in that it was the only species to show higher tolerance against all Cu materials tested. A significant inhibition of growth was recorded only at the highest exposure concentration of micronized Cu, Cu−amine, CuSO4, and the modified CuO NPs (ascorbate-coated), while no significant effect was observed using the pristine CuO NPs (Figure 2). Imaging of the C. puteana revealed that all treatments induced pigmentation of the fungal growth. This coloration was visible from both sides of the Petri-dish (Figure 3 and SI_2) for all treatments except the modified CuO NPs (ascorbate-

In the synthesis and formulation phase of the lifecycle, the differences in TEM structures (Figure 1) and XRD crystallinity (Figure SI_1) highlight that the CuO acrylic coating and the micronized copper are nanoenabled formulations, whereas Cu− amine (ACA) is a molecularly dissolved non-nanoform of Cu (Tables SI_1, SI_2). To differentiate the potency of actives released from the biocidal formulation (Table SI_2) and from the treated article (wood, Table SI_3), we first tested the antifungal effectiveness of the (nano)material formulations. For this purpose, we replaced wood by agar as a fungal growth medium and developed a protocol for assessing antifungal properties, with significant improvement (reduced uncertainty) over the current standard procedures performed directly on wood specimen−i.e. the EN 113. Three different species of filamentous rot fungi, D


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Figure 4. Leaching from Cu treated woods based on EN84 standardized sampling. (A) Release rates: wood impregnated with Cu−amine (blue triangles) or micronized Cu (green dots); coated by acrylate (gray diamonds) and by CuO acrylate (black boxes). (B) TEM analysis of a particulate found in leachate from CuO acrylate coated the wood. In the boxed scan area, EDXS identifies the elements Cu, Si, S, C.

the modulation by an adsorbed ascorbate coating indicate that the mode of action of the formulated materials includes action as particles. As the time to complete dissolution is longer than the fungi exposure, particles represent most of the dose, and much fewer ions are available than from copper-amine. However, also newly generated particulate species may contribute, and also an antifungal effect of desorbed ascorbate via its ability to generate hydroxyl radicals through Fenton reaction37 is possible. We then tested the nanoenabled final producttreated wood specimenfor Cu release. Differences were expected between the high surface barrier concentration of CuO acrylate coating and the high bulk concentration of Cu−amine or micronized Cu impregnated woods (Table SI_3 and Figure 1). Also the CuO modified with ascorbate was formulated into an acrylic coating, but the increased tendency to remain agglomerated in the acrylic formulation prevented any further testing. Standardized leaching tests were performed according to the European standards (CEN, 1997) to evaluate the fixation of the different Cu formulations. The leaching results demonstrate that Cu leached (mg/m2) from wood treated either with Cu− amine or micronized Cu have a similar trend (Figure 4, blue vs green symbols). The highest leaching rate was observed at the beginning (first two sampling points) of the experiment and then reached a plateau. This fact can be explained by the release of free Cu on the wood surface. The average leaching rate was 165 mg/m2 for Cu−amine and 178 mg/m2 for micronized Cu. The pH of the leachates for the micronized Cu varied throughout the experiment from 6.3 to 6.8 and for the Cu− amine from 6.6 to 7.5. In comparison, the release from wood coated with CuO acrylate was about 3 orders of magnitude lower, but at 0.40 ± 0.07 mg/m2 was significant against the acrylate-coated wood at 0.1 ± 0.1 mg/m2 (Figure 4, black vs gray symbols). Further analysis by sp-ICPMS indicated that the Cu release is predominantly ionic (>99% considering the time traces, and the lower detection limit), whereas particulate fragments containing Cu were found in leachates of CuO acrylate (Figure SI_6). The morphology and composition of such particulates (Figure 4B) are not identical with pristine CuO, but rather with organic−inorganic precipitation. Preaging of the CuO coated woods (UV/rain, ISO 4892, 465MJ/m2), diminished the particulate releases (Figure SI_6). The total Cu releases increased by 21% after preaging but also increased from

coated), for which the pigmentation was only visible beneath the colony. For this treatment, the colony surface developed a complex structure of air mycelium suggesting a vertical increase of growth (or primordium) rather than horizontal growth (Figure SI_3). A clear decrease of growth together with a strong pigmentation and a morphological change of the culture shape was observable in samples treated with micronized copper (Figure SI_2 C, D) and CuSO4 (Figure SI_2 E, F). Changes in pigmentation, such as melanization, are a wellknown defensive mechanism against the toxic effect of heavy metals,30 and brown rot fungi have been shown to accumulate metals during decay processes.31 The fungus Trametes versicolor, commonly known as turkey tail, is a white rot fungus able to produce oxalate crystals in the presence of a high concentration of metals.32 This fungus demonstrated a dense growth near the original plug with a more diffuse/lighter growth at the periphery. For this reason, the results are presented as internal and external diameter, respectively. The copper-amine solution showed the most potent effect on the growth of T. versicolor (Figure SI_4) while according to the statistical analysis, the other copper containing treatments inhibited the growth of the filamentous fungus significantly when compared to the control. No pigmentation nor morphological changes were observed. Gloeophyllum trabeum is a common brown rot copper-sensitive fungus prevalent in coniferous forest. It was extremely sensitive to almost every treatment tested, even at lower concentrations (Figure 2). Cu−amine showed the highest potency followed by micronized Cu, and the other copper materials, resulting not only in a complete inhibition of growth but also the death of the fungal plug at higher doses. To complete the testing of formulations, we assessed the extent of transformation during the antifungal effectiveness testing and analyzed ion release at pH 5.4 and duration of fungal effectiveness testing. We employed flow-through dissolution testing33,34 to enhance the relevance of Cu nanomaterial transformations by unsaturated conditions.35 For compatibility as eluent, agar gel had to be replaced by a simpler medium, selecting NaNO3 as counterions due to high Cu(NO3)2 solubility, but adjusted to the agar pH of 5.4, because pH was expected to be the most critical parameter despite possible modulation by further medium components.22,36 We found that both particulate formulations (CuO and micronized Cu) release Cu ions, but do not dissolve completely (Figure SI_5), in accord with previous calculations and measurements of solubility.16 The limited dissolution and E


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condensation phase, up to a radiant exposure of 465 MJ/m2. Then, followed by immersion sampling with two levels of shear stress and analysis of releases by the prevalidated NanoRelease protocol.27 We found total particulate releases in the range 400 to 1100 mg/m3 (Table 1). The mass-median size of all fragments was above 1 μm. Nonaged Cu−amine impregnated woods and acrylate coated woods were observed to release small fragments significant in number metrics (Table SI_4 and Figure SI_12). The particulate release, as monitored by either UV−Vis or AUC analysis of the immersion waters, increased with UV/rain aging for the acrylate coatings (Figure 6a), but was largely unrelated to UV/rain aging for the Cu−amine or micronized Cu impregnated woods (Figure 6a, Table 1). TEM scans of the immersion waters highlighted the complex morphology of released fragments, which for the CuO acrylate coated woods consistently found Ti- and Cu-containing particles (Figure 6b) and occasionally allowed identification of the pristine particle TiO2 morphology (Figure_SI 8). The particulate morphologies were similar between immersion waters of woods impregnated with Cu amine (Figure 6c) and those treated with micronized Cu (Figure 6d). In immersion waters from woods freshly impregnated with micronized Cu (nonweathered), particles with a morphology and size similar to the micronized Cu particles were observed (inset Figure 6d). To further elucidate the transformation of the pristine formulation, the Cu and Ti content was determined in total (to include ions and particulate structures, with acid digestion) and after 20 nm filtration (to include only ions). For immersion waters from CuO-coated woods, filtration reduced Ti to baseline (except for one outlier), and reduced the already low Cu to about half or baseline (Table 1). This result indicated that Ti was present in particulate form whereas Cu from CuO acrylate was at least partially present as ions, partially in particulate form, both in accord with TEM. In contrast, for immersion water from impregnated woods, filtration had no significant effect on Cu ICPMS results−both for impregnation by Cu amine or micronized Cu. Traces of nonparticulate Cu above the EDXS detection threshold of 0.2% were identified in the vicinity of organic material (cellulose, bacteria, or both) or inorganic amorphous salts/particles. The high amounts of Cu detected by ICPMS in the Cu amine and micronized Cu impregnated wood samples correlate with high amounts of nonparticulate Cu detected by TEM/EDXS (Table 1, Table SI_4). See the SI for an extensive TEM gallery (Figures SI_8 to SI_11) and additional analysis by low shear sampling in Table SI_4. The susceptibility to shear stress was weak for the pressure-treated woods, as the difference between the NanoRelease successive samplings (shaker, then sonication) is below 20%. The total particulate release from woods (mostly organics) is high compared to the release from plastics by the identical protocol.40 Taken together, both our weathering assays observed a trend of initial increase of release from both impregnated kinds of wood, then a rather constant release from micronized Cu impregnated woods compared to a slightly decreasing trend from Cu−amine impregnated wood. The dependence on UV aging is weak if present at all. The maximum UV dose of 465 MJ/m2 corresponds to several years of outdoor use, but the exact acceleration factor depends on local climate and material.28,41 Sampling methods are critical, evidenced by the difference between the release of 178 mg/m2 Cu by immersed leaching vs 9.4 mg/m2 by runoff sampling during UV/rain weathering vs 169 mg/m2 by condensation sampling, all from

the CuO-free acrylate coated wood. We noted that pH of the leachates of nonaged woods was around 6, in contrast, the pH of the leachates from the aged woods was around 5. To rationalize the reduction of particulate release after aging, one may speculate that the degradation of the acrylate matrix favors dissolution via lowered pH. We then proceeded to synergistic stresses and tested releases25 weathering with humidity condensation, accelerated UV aging and rain intervals with direct runoff sampling. We observed similar release magnitude from woods that were impregnated with either Cu−amine or micronized Cu: across the entire data set an average Cu release of 12.2 ± 10 mg/m2 and 9.4 ± 9.6 mg/m2, respectively, per sampling. While the long-term release tends to increase from woods impregnated with micronized Cu minimally, the trend was constant or decreasing from woods impregnated with Cu amine. The impact of doubled concentration of micronized Cu was low. Very markedly, the UV+rain release from CuO acrylate coated wood was about 10-to 100-fold lower, and in fact not different from the CuO-free acrylate coated wood. The release during the condensation phases also confirmed, at much lower scatter than the runoff waters, that the release from nanoenabled wood impregnated by micronized Cu (an average of 169 ± 114 mg/ m2 per sampling, 2400 mg/m2 cumulated) is not significantly different from conventional wood impregnated by Cu−amine (144 ± 90 mg/m2 per sampling, 2600 mg/m2 cumulated). The condensation phase generates most of the Cu releases, attributed to chemical and especially physical reactions, such as change in water saturation, capillary forces, cracking. We noted that the fluctuations between sampling intervals are correlated between Cu−amine and micronized-Cu woods. Removing the fluctuations would further reduce the statistical error on (the absence of) difference of release between the nanoenabled and conventional impregnation technologies. For synergistic stresses with delayed mechanical (sonication shear) stress sampling, the wood specimens were first aged according to the modification developed from the ISO 489238 which is equivalent to EN927-639 except for a missing

Figure 5. Cumulative Cu release during water condensation phases (diamonds) and UV+rain phases (circles) of the adapted EN927-6 protocol with ICP−MS analysis: (black): CuO acrylate coated wood; (gray) acrylate coated wood; (blue) Cu−amine impregnated wood; (green) micronized Cu impregnated wood. Error bars (smaller than symbol size) represent the standard deviation between 2 replicates for CuO_Acryl series and systematic error for the rest of the data. Micronized Cu data correspond to an average between samples at 1 kg/m3 and 2 kg/m3 for the condensation step as they were placed in the same reactor vessel, but separate for rain phases. Experimental scheme in Figure SI_7. F


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Environmental Science & Technology Table 1. Release after Ageing by NanoRelease Protocol with High Shear Sampling Wood

acrylate coated wood

CuO acrylate coated wood

Cu amine impregnated wood

micronized Cu impregnated wood

a

UV−vis UV/rain weather, MJ/m2

at 360 nm

0 155 310 465 0 155 310 465 0 155 310 465 0 155 310 465

0.07 0.08 0.3 0.85 0.02 0.18 0.26 0.12 2.37 2.19 2.25 1.55 0.33 1.59 1.84 1.54

ICPMS, Cu, ppm total 0.1 0.2 0.4 1.5 0.1 0.3 0.5 0.4 145

50 27

70

CuO_ascorbate > CuO 2. Cu ions (at agar pH): Cu−amine ∼ CuSO4 ≫ Cu(OH)2CO3 ∼ CuO 3. Effectiveness (treated wood): Cu−amine ∼ Cu(OH)2CO3 ≫ CuO > control 4. Cu ion (released from treated wood): Cu−amine ∼ Cu(OH)2CO3 ≫ CuO > control On the analytical side, several methods (filtration-ICPMS, TEM/EDXS, sp-ICPMS) agree on the transformation between synthesis and release: the nanoenabled micronized Cu formulation releases only ionic Cu and does that at a rate that is nearly identical to the conventional Cu−amine formulation. The release in ionic form is attributed to dissolution of micronized Cu which was observed at relevant pH (Figure SI_5). The bulk of the wood imposes transformations by acidic pH, but also releases particulate and molecular species, and thus contributes significantly to H


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Environmental Science & Technology transformations throughout the lifecycle. Platten et al.42 investigated a micronized Cu pressure-treated wood “MCA-1” at very similar loading (0.8 kg/m3) and very comparable size distribution and found only 42 ± 3% as Cu carbonate, but 58 ± 2% as Cu-organics inside the wood. Additionally to acidic pH, Cu has been shown to be susceptible to ligand-assisted dissolution.16 Ligands (amines) enable the conventional Cu− amine formulation, and natural ligands in wood presumably contribute to the transformation inside the wood pores. As novel and complementary evidence, analysis by AUC at 250 000g observed in wood immersion waters (for Cu−amine and micronized Cu), a fraction containing a highly reproducible hydrodynamic size of 0.35 ± 0.03 nm, matching the expected signal of Cu ions (Table SI_4). In accord between sp-ICPMS, TEM, AUC, and filtration-ICPMS (Figures 4, 5, and 6, Table 1, and SI), all evidence supports the complete transformation of micronized Cu during the use phase to Cu ions as released species. Using the same set of techniques, nanoparticle, nonnanoparticle, and ionic releases were detectable from a different class of copper-containing materials, namely antifouling ship vessel paints.45 Matsunaga et al.46 speculated that the acidic pH inside wood would contribute to micronized Cu transformation once applied by impregnation or pressure treatment, and Xue et al.47 found such reactions to remain incomplete. The complete transformation under nonequilibrium conditions w as observed similarly both in highly controlled flow cells33 and in mesocosms,35 and was employed for fate modeling.48 Platten et al. observed both copper carbonate and transformed Cuorganics inside the wood, but concluded on leachates with filtration down to 10 kDa that “the lack of particulate copper likely means that when the copper is released or very soon after it is released, it dissolves and does not persist as a particle”,42 very consistent with our results for woods treated with the same Cu species. Further, our results do not contradict findings by different sampling processes, which assessed Cu releases via fungal spores,49 by wipe testing,50 and by sanding.51 Wipe testing and sanding are relevant for occupational exposures, whereas here we focus on environmental releases. We finally performed a sustainability analysis based on our results that environmental risks during the use phase are dominated by the release of Cu ions for both micronized copper and copper-amine, whereas particulate release contributes for CuO acrylic coating. We further assume that occupational hazards are dominated by inhalation. To further reduce the uncertainty in the estimates and assumptions, identical CuO and micronized Cu formulation were tested in vivo by short-term oral screening (STOS),52 and identical pristine and modified CuO NPs were tested in vivo by ShortTerm Inhalation Study (STIS).53 Furthermore, identical CuO, micronized Cu formulation and Cu−amine formulation were subjected to standard soil eco-toxicity tests on Enchytraeus crypticus, soil microorganisms and fish cell lines (to be published independently). The CuO additive in acrylate coating has no significant benefit. The behavior during the use phase determines both user benefit and environmental risk. The formulation determines human occupational risk. On the basis of the results available to date, we performed a LICARA nanoSCAN estimation of the CuO coating sustainability compared to the conventional acrylic coating. The LICARA nanoSCAN indicates that significantly increased risks and uncertainties by CuO manufacturing are not balanced by user benefits but are in fact exacerbated by lower performance of

CuO acrylic coating compared to the conventional coating (Figure 7 b,c). In conclusion, we found no evidence that woods impregnated with micronized Cu release any other Cu species than ions. At equal Cu loading per cubic meter of wood, form and rate of release are comparable to wood impregnated with molecular Cu−amine but slightly more stable throughout aging; this finding is attributed to the transformations during the wood use. In contrast, wood coatings with a high barrier concentration of CuO release both particulate and ionic species, but a lower percentage of initial Cu, and are not effective for wood protection.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b04130. Additional methods and results for physicochemical characteristic of pristine and ascorbate coated CuO NPs, micronized Cu and Cu-amine formulation together with the results from the X-ray and selected area diffraction analysis; pictures regarding the growth of C. puteana and T. versicolor after 1-week incubation, ion dissolution from CuO and micronized copper at pH 5.4; sp-ICPMS analysis of leachates from acrylate-coated wood, schematics of the EN927-6 protocol, TEM images from NanoRelease protocols of woods treated with copper materials, AUC size distribution of NanoRelease liquids and photograph of effectiveness testing; detailed inputs for the LICARA nanoSCAN (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wendel Wohlleben: 0000-0003-2094-3260 Present Address

# J.N.: U.S. National Research Council Associate, Environmental Protection Agency, Research Triangle Park, NC, USA

Notes

The authors declare the following competing financial interest(s): NN, PM, WW are employees of a company that develops and commercializes nanomatrerials.

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ACKNOWLEDGMENTS We sincerely thank Bernd Nowack (EMPA) for discussions during data evaluation and comments on the manuscript, and Anna Costa (ISTEC) for the provision of characterization data on the ascorbate coating. This work was supported by the project on Sustainable Nanotechnologies (SUN) that receives funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant No. 604305.

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