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Chapter 18

Downloaded by NORTH CAROLINA STATE UNIV on August 4, 2012 | http://pubs.acs.org Publication Date: April 2, 2008 | doi: 10.1021/bk-2008-0982.ch018

The Role of Non-Decay Microorganisms in the Degradation of Organic Wood Preservatives Derek F. Wallace, Steven R. Cook, and David J. Dickinson Division of Biology, Faculty of Natural Sciences, Timber Technology, Sir Alexander Fleming Building, Imperial College, London SW7 2AZ, United Kingdom

Since the implementation of the Biocide Products Directive there has been increasing pressure on the wood preservation industry to utilise environmentally sensitive organic biocides. It has been reported that organic biocides are degraded in wood exposed to the full range of microorganisms resulting in the timber becoming susceptible to fungal decay by organisms otherwise controlled by the biocides. Biodetoxification was found to be mediated by a range of bacteria, with Gram negative proteobacteria often associated with biocide degradation. In particular, strains of Pseudomonas have been reported to degrade a number of QACs, IPBC, chlorothalonil and an oxathiazine derivative, although the mechanisms have not been elucidated. Despite the observed biodegradation of many organic wood preservatives, many of these compounds have been successfully used for a number of years. This chapter reviews the biodegradation of organic biocides that are used to protect wood.

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© 2008 American Chemical Society

In Development of Commercial Wood Preservatives; Schultz, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Biocide Products Directive In recent years the timber preservation industry has responded to environmental, legislative and economic pressures to utilise more environmentally sensitive formulations. The potential hazards posed by wood preservatives have long been highlighted by regulatory bodies, which culminated in the Biocidal Products Directive (BPD) being implemented by the European Parliament in 1998 (Directive 98/8/EC) to monitor placement of biocides and their formulations on the market. Quantification of the toxicity and persistence of these biocidal compounds was one of the principle aims of this legislation. The directive requires manufacturers to detail the environmental hazards of the active chemicals, with particular attention being paid to the ultimate fate of the compounds. Ideally, a candidate biocide should be completely biodegradable, leaving no toxic residues and having minimal environmental impact. It is clear that a number of the chemicals used prior to 1998 as wood-preservatives needed to be replaced and that this represented a significant challenge and opportunity for the industry. The main problem associated with the development of environmentally friendly preservatives based on organic compounds, is the observed biotransformation of these biocides (7, 2, 3, 4). Activity against the causal decay organisms and resistance to physical losses are no longer the sole performance criteria that need to be understood when developing a new wood preservative. Despite these drawbacks, several modern organic biocides (examples of which are discussed to later) have found an important role in wood treatments. However, i f they are to be utilised in the long term in high hazard situations, such as soil contact, it will be necessary to fully understand their biotransformation and attempt to control it.

Propiconazole Propiconazole (cis-trans-1 -[2-(2,4-dichlorophenyl)4-propyl-1,3-dioxolan-2ylmethyl]-l//-l,2,4-triazole) is a triazole antifungal agent which inhibits ergosterol biosynthesis and causes electrolyte leakage and ultrastructural damage to cells (5, 6). Developed for agricultural use, propiconazole has been shown to be effective in the control of sapstain, and both brown rot and white rot Basidiomycetes, with toxic values of between 0.2-0.5 kg m" (7, 8). These toxic values were not, however, found to be adequate to protect wood blocks from fungal attack in a soil burial test, where the wood showed decay after 8 weeks (7, 9). Although resistant fiingi appeared to play a role in biocide depletion, it was clear that there was an additional biological factor in soil which contributed to propiconazole degradation, most likely members of the bacterial community (7, 10, 77). Subsequent studies isolated a number of bacterial strains which showed 3


314 the ability to degrade propiconazole, illustrating the role of bacteria in the degradation of wood preservatives (7, 72). However, studies of radiolabeled propiconazole revealed that the biocide is not utilized as a carbon source, with only 3-10% of the biocide being converted to C 0 in a single year (75, 14). Typically, propiconazole was found to degrade slowly in soil through hydroxylation of the propyl-side chain on the dioxilane ring; see Figure 1 (75, 14). Downloaded by NORTH CAROLINA STATE UNIV on August 4, 2012 | http://pubs.acs.org Publication Date: April 2, 2008 | doi: 10.1021/bk-2008-0982.ch018

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Chlorothalonil Chlorothalonil is a foliar, non-systemic, broad-spectrum, chlorinated fungicide, highly effective against pathogens that infect vegetables, fruits and ornamentals. Chlorothalonil mediates an anti-fungal effect by reacting with sulfhydryl groups and glutathione present in proteins or in cofactors (75). Most reports have shown that chlorothalonil was rapidly degraded by soil microorganisms in 5-36 days, under aerobic conditions (16,17). Katayama et al. (16, 18) demonstrated that bacteria of various taxonomic groups were capable of degrading chlorothalonil in pure culture. In total, 37 soil-inhabiting bacteria were tested for their ability to degrade the biocide, and it was found that strains of Gram negative bacteria, including Acinetobacter, Agrobacterium, Azomonas, Pseudomonas and Xanthomonas, could all degrade 0.5 mg L ' chlorothalonil. A number of Gram positive bacteria, including Bacillus, Corynebacterium and Nocardia strains, were also found to be able to mediate clorothaloniPs degradation, although these bacteria were less tolerant to chlorothalonil than the Gram negative isolates. It was considered that common enzymes ubiquitous in bacteria were involved in the degradation of chlorothalonil by cometabolism (16, 17,18). There was also some evidence that fungi were capable of mediating chlorothalonil degradation (19,20). There are reportedly two major metabolic pathways involved in chlorothalonil degradation. The first involves the displacement of one chlorine atom by a hydroxyl group, potentially via reductive dechlorination, to generate 4-hydroxy-2,5,6-trichloroisophthalonitrile (Mt-1), as shown in Figure 2 (75, 77, 18, 21, 22). A second mechanism suggested was the oxidation/hydration of one cyano group to a corresponding amide and organic acid, to generate Mt-2 and subsequently Mt-3 of Figure 2 (75). Two months after chlorothalonil application to soil, Mt-1 was found to represent 37% of the applied chlorothalonil, whilst 22% had been degraded to l,3-dicarbpmoyl-2,4,5,6-tetrachlorobenzene, represented by the intermediate between Mt-2 and Mt-3 (23). Only 3-14% of the radiolabeled chlorothalonil applied to soil was degraded to C 0 after 90 days, which illustrated that complete mineralisation was not the major metabolic pathway of chlorothalonil degradation (27). 1

1 4

2


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Propiconazole

Figure 1. Proposed degradation pathway of propiconazole (reproduced with permission from reference 13. Copyright 2002 Kluwer Academic Publishers).

K i m et al. (24) recently reported the isolation of an Ochrobacterium anthropi strain capable of efficiently biotransforming the fungicide chlorothalonil from soil. A gene responsible for the chlorothalonil biotransformation was cloned into E. coli and subsequently identified as the open reading frame for glutathione-S-transferase (GST). It has been reported that GST catalyses the conjugation of the glutathione sulfur atom to a large variety of electrophillic compounds of both endobiotic and xenobiotic origin, resulting in detoxification (25, 26). Glutathione-5-transferases are a very broad group of enzymes which have the cosubstrate glutathione in common. The enzymes' specificities are very broad, and the glutathione thiolate ion has been shown to attack the nitrogen of organic nitrite esters, the sulfur of organic thiocyanates and on the oxygen of organic peroxides (26). In a study of the mechanism of reaction with 4-substituted chlorobenzenes, it was concluded that the determining factor was the electrophilicity of the carbon atom attacked (25). This illustrates that there a number of different potential metabolic pathways by which a biocide can be microbially detoxified.



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Mt-3

Figure 2. The degradation pathway of Chlorothalonil (scheme adapted from reference 15 by permission of The Royal Society of Chemistry).

Quaternary Ammonium Compounds Quaternary ammonium compounds (QACs), defined by their quaternary nitrogen atom carrying hydrophobic alkyl chains, have been used extensively as disinfectants, fabric softeners, hair rinses and dispersion agents (27). QACs have also found use as wood preservatives, where they have been found to be highly effective at protecting timber from mould, wood-decaying and sapstaining fungi. Specifically, didecyldimethylammonium chloride (DDAC) was found to perform as well as copper chromic arsenate (CCA) in laboratory tests against wood decay fungi (28, 29). Currently, products containing D D A C account for approximatley 95% of the Canadian sapstain control market (30). The degradation of QACs in wood products has not been studied in detail, however Q A C degradation in wastewater has been widely described. It has been reported that hexadecyltrimethyl ammonium bromide and decyltrimethyl



317 ammonium bromide were both degraded by a co-culture of Xanthamonas and Pseudomonas, through the oxidation of the terminal carbon of the alkyl chain, resulting in the sequential cleavage of acetyl units by β-oxidation (57). Similarly, a strain of Pseudomonas fluor escens, isolated from activated sludge, showed the ability to utilise D D A C as a carbon source, with degradation mediated through two N-dealkylation steps. The initial dealkylation step was mediated by a mono-oxygenase, producing decyldimethylamine, and decanoic acid as the alkyl co-product, with the former compound being further degraded by a second inducible N-dealkylating enzyme (27). Pseudomonas strains clearly have the ability to degrade a wide range of QACs, having been reported to also degrade laurypyridinium chloride, cetyltrimethyl ammonium bromide, hexadecyltrimethylammonium chloride, didecyldimethylammonium chloride and benzalkonium chloride (27, 52, 55, 34). A number of mould fungi have also been isolated which demonstrated the ability to tolerate and degrade QACs, including strains of Pénicillium, Trichoderma, VerticilliumlAcremonium and Gliocladium (55, 36). It was considered that the activity of these fiingi may result in Q A C degradation, through the hydroxylation of the Q A C near the end of the alkyl chain, resulting in the susceptibility of the timber to decay (57). Notably these studies did not take into account bacterial degradation, and it is highly probable that a large bacterial QAC-degrading community would have been present in the wood.

IPBC 3-Iodoprop-2-ynyl-N-butylcarbamate (IPBC) is an organoiodine carbamate fungicide commonly used to control the defacement of softwood timber by sapstaining fimgi, in place of the environmentally persistent pentachlorophenol (38). It has been reported that IPBC is degraded in vitro, resulting in the loss of fungal toxicity (2, 5). Nine strains of bacteria, belonging to the genera Alcaligenes, Enterobacter, Microbacterium and Pseudomonas, were isolated, and it was found that these isolates mediated the dehalogenation of IPBC to prop-2-ynyl-W-butylcarbamate (PBC) and iodine at a 1:1 stiochiometric ratio. The isolates were found to be unable to utilise IPBC as a carbon source and degradation was not mediated by either an extracellular moiety nor through acid hydrolysis (39). It was concluded that IPBC degradation occured via a reductive dehalogenation mediated by a cytosolic moiety which utilised N A D H (and other reductive coenzymes) as a cofactor (39). To overcome the limitations of using a readily degraded compound as a wood preservative, IPBC was combined with QACs such as D D A C (40). Since D D A C has a strong bacteriacidal activity, by disrupting the bacterial cell membrane (41), coformulation has proved to be an effective technique by which to prevent the bacterial-mediated degradation of an organic wood preservative.


318 The potential for bacterial degradation of this coformulation has not yet been ascertained.


Developmental Biocide 3-Benzo[b]thien-2-yl-5,6-dihydro-l,4,2-oxathiazine-4-oxide is a metal-free and halogen-free fongicide of a new chemical class. It is being developed for the control of stains, moulds and algae in paints, woodstains, wood preservatives and coatings. A study conducted by Forster et al. (42) revealed the effectiveness of this chemical against a variety of soft rot fungi and indicated its potential as a biocide to protect wood exposed in ground contact in the absence of heavy metals, which is likely to the next big challenge for the industry A study undertaken to provide detailed information regarding the microbial degradation of the oxathiazine derivative resulted in the isolation of a number of proteobacteria that were able to mediate biocide detoxification (43, 44). Using molecular techniques, the isolates were identified as Pseudomonas, Alcaligenes, Achromobacter, Tetrathiobacter, Ralstonia, Stenotrophomonas, Variovorax and Serratia. The isolates were found to show a range of tolerance to the biocide (see Figure 3 and Table I). It was found that the pseudomonads, Ralstonia and Stenotrophomonas, were able to grow rapidly in Luria Bertani medium containing even saturated quantities of the biocide, whilst the Alcaligenes, Achromobacter and Serratia isolates had an I C at a concentration just below that of the biocide's aqueous solubility (45). In contrast, Tetrathiobacter, Variovorax and Alcaligenes defragrans showed a low tolerance, and were inhibited by concentrations of 150-200 μΜ. However, the observation that these bacterial isolates degrade the oxathiazine derivative in vitro is not evidence, in itself, that the activity of these organisms will mediate its degradation in the timber product. It is therefore critical that these isolates are studied further to determine their modes of resistance and degradation mechanisms. 50

The potential for biocide degradation in treated timber is dependent on the particular application of the wood product (defined as hazard classes; 46). In this study, treated woodblocks were exposed to soil (hazard class IV), which is the most severe environment in terms of potential for fongal degradation and bacterial activity. The potential for bacterial degradation of organic biocides in other hazard categories, such as interior timbers or external joinery, have not been assessed, but are likely to be much reduced.

Conclusions It is clear that the proteobacteria are unparalleled in their ability to degrade organic wood preservatives, having been shown to degrade other biocides (2, 3,


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