Biotransformation of the Polycyclic Musks HHCB and AHTN and

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Environ. Sci. Technol. 2007, 41, 5395-5402

Biotransformation of the Polycyclic Musks HHCB and AHTN and Metabolite Formation by Fungi Occurring in Freshwater Environments CLAUDIA MARTIN,† MONIKA MOEDER,‡ XAVIER DANIEL,§ GUDRUN KRAUSS,† AND D I E T M A R S C H L O S S E R * ,† UFZ, Department of Environmental Microbiology, and UFZ, Department of Analytical Chemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, D-04318 Leipzig, Germany, and Faculte´ des Sciences 1, University of Orle´ans, Rue de Chartres, BP 6759, 45067 Orle´ans cedex 2, France

Micropollutants found in aquatic environments have increasingly raised concerns with respect to their uncertain environmental fate and potentially adverse effects on human health and the environment. The biodegradability of two major representatives of the polycyclic musk fragrances, Galaxolide (HHCB) and Tonalide (AHTN), and the formation of biotransformation metabolites, were investigated with Myrioconium sp. strain UHH 1-13-18-4 and Clavariopsis aquatica, two mitosporic fungi derived from freshwater environments. A particular focus was to assess the effects of extracellular oxidoreductases such as laccases, which are produced by the investigated fungi under certain conditions, on HHCB and AHTN. The fungi converted HHCB and AHTN into various products via initial hydroxylation at different carbon positions. Further metabolism resulted in the subsequent formation of diketone, peroxide, and O-methylated derivatives. Isolated laccases of the investigated fungi were able to oxidize HHCB and AHTN and catalyzed the formation of the metabolite HHCBlactone from HHCB. At particular environmental situations also specified within the present study, biotransformations catalyzed by fungi occurring in freshwater environments may be considered when addressing the fate of polycyclic musks in freshwater systems and potential biological effects of their degradation metabolites.

Introduction The synthetic musk compounds 1,3,4,6,7,8-hexahydro4,6,6,7,8,8-hexamethylcyclopenta[g]-2-benzopyran (HHCB, Galaxolide) and 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydronaphthalene (AHTN, Tonalide) (Table 1) are two main representatives of the polycyclic musk fragrances used in many kinds of personal care products (1-3). Sewage treatment plant (STP) effluents were identified as the major source * Corresponding author phone: +49-341-235-3254; fax: +49-341235-2247; e-mail: [email protected]. † UFZ, Department of Environmental Microbiology. ‡ UFZ, Department of Analytical Chemistry. § University of Orle ´ ans. 10.1021/es0711462 CCC: $37.00 Published on Web 06/27/2007

 2007 American Chemical Society

of these micropollutants in surface waters (1, 3, 4). Due to their hydrophobic nature, HHCB and AHTN tend to sorb onto particles, bioaccumulate in fish, mussels, water birds, and aquatic mammals, and have also been found in human adipose tissue (1-3, 5, 6). Even low concentrations of these musk compounds have been reported to inhibit multixenobiotic resistance (mxr) mechanisms in mussels (7), to cause cell type-dependent anti-estrogenic effects (8), and to inhibit larval development in marine copepods (9). In STPs, HHCB and AHTN removal of approximately 50 to more than 90% mainly caused by sorption onto sludge particles has been reported (10-12). The biodegradability of HHCB and AHTN was assessed with several modified OECD tests and soil- and sediment-containing microcosms, but mineralization was only rarely observed (3). No single bacterial strain acting on HHCB or AHTN has been described so far, whereas pure cultures of terrestrial fungi were shown to convert these musk compounds without mineralizing them (3). The available information about possible organic biotransformation products of HHCB and AHTN is very limited. The HHCB metabolite 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[g]-2-benzopyran-1-one (HHCB-lactone, Table 1) is contained as a byproduct in technical HHCB formulations and may account for up to 10% (11). Its environmental occurrence was demonstrated for STPs, river water, and fish (2, 4, 11, 13). The biological oxidation of HHCB into HHCB-lactone was implicated for wastewater treatment processes (4) and fish (14). However, no enzyme catalyzing the formation of HHCB-lactone from HHCB has been described as yet. More information about the biotransformation metabolites of HHCB and AHTN and the organisms and enzymes involved is expected to foster the assessment of potential risks associated with these compounds. Biotransformation metabolites of numerous organic environmental contaminants have been reported to be more persistent, increasingly prone to long-range transport, or even more toxic than their respective parent compound (15). Strictly aquatic fungi such as aquatic hyphomycetes (Ingoldian fungi) and other environmentally ubiquitous mitosporic fungi occurring in freshwater ecosystems have already been shown to act on the endocrine-disrupting chemical nonylphenol (16). Laccases are extracellular multicopper oxidases most frequently described in white-rot basidiomycetes, which unspecifically oxidize certain lignin constituents and also many xenobiotic compounds (17). Small molecules acting as redox mediators enable the substrate range of laccases to be extended considerably (17). Laccases were also described in non-basidiomycete freshwater fungi and have been implicated in contributing to biotransformation of nonylphenol by these organisms (16). The aim of the present study was to obtain more insights into microbial degradation mechanisms that could affect the environmental fate of potentially hazardous polycyclic musk compounds such as HHCB and AHTN. For this, we focused on musk compound biotransformation and metabolite formation by fungi derived from freshwater environments. We have also assessed the effects of laccases, which were derived from the investigated aquatic fungi, on HHCB and AHTN.

Experimental Section Materials. Further information on HHCB, AHTN, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), vanillic acid, a penicillin-streptomycin formulation, Sylon BTZ, other chemicals, and laccase from Trametes versicolor used within the present study is contained in the Supporting Information. VOL. 41, NO. 15, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. GC-MS Data of Musk Compounds and Their Metabolites Detected in Fungal Culturesa

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TABLE 1. (Continued)

a Fungal cultures were analyzed after 28 (Myrioconium sp.) and 35 days (C. aquatica) of exposure to musk compounds, corresponding to a final fungal dry weight of 3.2 ( 0.4 (mean ( standard deviation for triplicate cultures) g/L for Myrioconium sp. and 3.8 (0.4 g/L for C. aquatica. No significant differences between HHCB- and AHTN-containing cultures were observed. b Retention times of metabolites were expressed in relation to the respective parent compound retention time, due to slightly varying retention times in repeated separations. Typical retention times were 15.60 min for HHCB and 15.69 min for AHTN. c Integrated areas of metabolite peaks in TIC chromatograms in relation to that of the respective parent compound. d Trimethylsilyl derivatives.

Fungal Strains. We tested two different strains of mitosporic aquatic fungi for their ability to metabolize HHCB and AHTN in pure culture experiments. The isolation of Myrioconium sp. strain UHH 1-13-18-4 and the aquatic hyphomycete Clavariopsis aquatica de Wild. strain WD(A)00-1 from freshwater environments and their identification and maintenance has been described previously (in (18) and (16), respectively). Coordinates and further characteristics of the isolation sites (site WD(A) for C. aquatica and site UHH 1 for Myrioconium sp.) have already been reported (18). Musk concentrations in river water of the site UHH 1 (determined upon GC-MS following solid-phase microextraction as previously described (19)) of 87.5-193.8 ng/L (HHCB) and of 49.9-252.7 ng/L (AHTN) were detected during sampling campaigns in February 2002, March 2003, and

September 2003. Concomitantly, nonylphenol was present at 154-260 ng/L (16). Biotransformation of Musks in Fungal Cultures. Liquid culture experiments with pure fungal cultures were conducted in 125 mL Erlenmeyer flasks containing 37.5 mL of a 1% (w/v) malt extract medium (pH 5.6-5.8), thus providing cometabolic conditions. Flasks were inoculated as described previously (16). Fungal cultures were incubated under agitation at 120 rpm and 14 °C in the dark. HHCB and AHTN were aseptically added to fungal cultures pregrown for 3 (Myrioconium sp.) and 5 days (C. aquatica) from 50 mM stock solutions in methanol, to give a final concentration of 250 µM (final methanol concentration 1% [v/v]), respectively. A methanol concentration of 1% did not impede growth of aquatic fungi and biotransformation of nonylphenol in a VOL. 41, NO. 15, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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previous study (16). To improve the solubility of the musk compounds, 0.1% (w/v) Tween 80 was added. Cultures inactivated by 0.5 g/L NaN3, which was added together with the respective musk compound, as well as cultures omitting the polycyclic musks served as controls. For determination of fungal biomass, fungal cultures were harvested at the time points indicated in the text, and fungal dry weights were gravimetrically determined as described previously (16). Dissipation half-life times (DT50 values) of the polycyclic musks were estimated upon determination of their first-order elimination rate constants in active and NaN3-inactivated fungal liquid cultures. For the overall reaction in active fungal cultures, the following equation was considered:

- dC/dt ) kobsC ) kbC + kaC ) (kb + ka)C

(1)

where C is the musk concentration, kobs (d-1) is the observed first-order elimination rate constant, kb is the first-order rate constant (d-1) accounting for biological musk elimination, and ka is the first-order rate constant (d-1) accounting for abiotic musk removal. Accordingly, kb was calculated as

kb ) kobs - ka

(2)

where kobs was determined upon linear regression of the plots ln[musk compound] vs incubation time in active Myrioconium sp. cultures and ka was derived from the corresponding NaN3-inactivated control cultures. DT50 values were calculated as DT50 ) ln(2)/kb. Linear regression of the kinetic data on biodegradation by C. aquatica was not sufficient; hence DT50 values were not determined for this fungus. Biotransformation of Musks by Isolated Laccases. Laccase-containing concentrated crude culture supernatants of Myrioconium sp. and C. aquatica served as enzyme sources in experiments addressing the degradability of HHCB and AHTN by laccases. Further information about laccase formation in Myrioconium sp. and C. aquatica and the concentration of cell-free crude culture supernatants is contained in the Supporting Information. A commercially available laccase from the white-rot fungus T. versicolor was included for comparison. Laccase activities were determined with ABTS as a substrate (16) and are expressed as units, where 1 U corresponds to 1 µmol of product formed per minute. The absence of manganese peroxidase, manganeseindependent peroxidase, and lignin peroxidase was verified as described earlier (16). Reaction mixtures contained 0.2 U laccase/mL, HHCB and AHTN at concentrations decribed in the text, and 0.1% Tween 80, in 100 mM sodium citrate buffer (pH 4.0). The artificial redox mediator ABTS was additionally included at 1 mM, where indicated. Controls contained heat-inactivated laccase preparations (autoclaving at 120 °C for 20 min). Enzymatic incubations were carried out under agitation at 120 rpm and 25 °C in the dark. Analysis of Musk Compounds and Biotransformation Products. HHCB and AHTN concentrations in fungal culture supernatants and enzymatic degradation experiments were routinely determined by HPLC (see the Supporting Information for details). For GC-MS analysis of HHCB and AHTN biotransformation products fungal cultures were harvested directly after the musks were added (day 0) and after 28 days (Myrioconium sp.) or 35 days (C. aquatica) of incubation in the presence of musks, respectively. Further details of the ethyl acetate extraction procedure, sample processing, and GC-MS analysis are contained in the Supporting Information. Reaction mixtures containing isolated laccases were harvested after 6 days of incubation and further processed as described in the Supporting Information. 5398

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FIGURE 1. Time courses of (a) HHCB and (b) AHTN concentrations in active (solid symbols) and NaN3-inactivated (open symbols) liquid cultures of Myrioconium sp. Symbols represent means ( standard deviations for triplicate cultures (smaller than symbol size where not visible). Correlation coefficients of linear regressions used to determine k values from ln[musk compound] vs time were as follows: r 2 > 0.93 for both HHCB- and AHTN-containing active cultures, r 2 ) 0.70 for inactive AHTN-containing cultures, and r 2 ) 0.37 for HHCB-containing inactive cultures. First-order elimination rate constants of musks ( standard errors for active (kobs) and NaN3-inactivated cultures (ka) and corresponding P values (t test for the significance of slopes smaller than zero) are also shown. For HHCB, an obtained ka ) 0.0008 ( 0.0006 d-1 (P ) 0.27) was considered as negligible (ka ≈ 0). Active cultures finally displayed a fungal dry weight of 3.2 ( 0.4 g/L (mean ( standard deviation for triplicate cultures), with no significant differences between HHCBand AHTN-containing cultures. The structure of the HHCB metabolite HHCB-lactone was confirmed by total reflection IR spectroscopy and GC-MS/ MS experiments (see the Supporting Information for details).

Results and Discussion Removal of HHCB and AHTN by Fungal Cultures. In active Myrioconium sp. cultures HHCB concentrations had decreased to 24.3 ( 4.4 µM (mean ( standard deviation from triplicate cultures) and AHTN concentrations had decreased to 10.6 ( 0.4 µM by the end of the experiment (Figure 1). In NaN3-inactivated control cultures, final HHCB and AHTN concentrations were 161.8 ( 14.2 and 177.8 ( 3.7 µM, respectively. These corresponded to recoveries (relative to the 250 µM initially added) of approximately 65% for HHCB and 71% for AHTN. In active C. aquatica cultures, HHCB and AHTN concentrations had decreased to 60.8 ( 7.4 and 31.5 ( 1.4 µM after 40 days of cultivation, respectively. About 78% of HHCB (corresponding to 195.9 ( 20 µM) and approximately 71% of AHTN (corresponding to 178.4 ( 6.2 µM) were finally recovered from the corresponding inactivated controls. The time course of musk concentrations in active Myrioconium sp. cultures and corresponding controls and the determined first-order elimination rate constants are reported in Figure 1. HHCB removal was negligible in the inactive control cultures. In contrast, AHTN concentrations in the inactive controls were decreased to a certain extent, possibly due to photodegradation (20) during sampling and sample processing where light was not excluded. AHTN is consider-

ably more susceptible to photolysis than HHCB (20). From the kobs and ka values shown in Figure 1, first-order rate constants for biological musk elimination (kb values) of 0.075 and 0.101 d-1 were calculated for HHCB and AHTN, respectively. The corresponding DT50 values were about 9.2 days for HHCB and 6.9 days for AHTN. Hence, HHCB is more resistant to Myrioconium sp. than AHTN. The same effect has been described for higher eukaryotes such as bluegill sunfish, where first-order elimination rate constants of 0.337-0.577 d-1 and 0.215-0.261 d-1 were obtained for AHTN and HHCB, respectively (3). In contrast, a faster HHCB removal was observed in pure cultures of the terrestrial whiterot fungus Phanerochaete chrysosporium, where HHCB and AHTN disappeared within 3 and 6 days, respectively (3). Detection of Biotransformation Products in Fungal Cultures. Upon GC-MS analysis, a compound with a molecular ion (M+) at m/z 272 already present in untreated HHCB matched reported mass spectral characteristics of the HHCB metabolite HHCB-lactone (11) (Table 1). Its initial concentration accounted for 4% of that of HHCB (based on the peak areas of HHCB-lactone and HHCB in TIC chromatograms). The identity of HHCB-lactone was confirmed by total reflection IR spectroscopy and GC-MS/MS experiments, where a typical lactone IR signal at 1743 cm-1 was in agreement with the mass spectral data (see Figure 1S in the Supporting Information for the proposed main fragmentation pathways). In NaN3-inactivated and HHCB-containing Myrioconium sp. cultures, the initial HHCB-lactone concentration increased to 253.6 ( 65.3% (means ( absolute deviations for duplicate cultures) after 28 days following HHCB addition (calculated from the peak areas of the molecular ion of HHCBlactone at m/z 272), suggesting a certain degree of autoxidative formation from HHCB as was implicated in a previous study (2). In active Myrioconium sp. cultures, the final HHCBlactone concentration was 2.2 ( 0.2% of that detected in inactivated controls and would point to a fungal attack on HHCB-lactone. In contrast, the final HHCB-lactone concentration in active C. aquatica cultures was 114.5 ( 8.2% of that found in inactivated controls. This suggests that either C. aquatica does not act on HHCB-lactone or that the detected HHCB-lactone concentrations reflect equilibrium between biochemical reactions catalyzing its formation and those catalyzing its degradation. HHCB-lactone has been described as a HHCB degradation metabolite in pure cultures of terrestrial fungi such as Cladosporidium cladosporiodes and P. chrysosporium, where C. cladosporiodes had converted 19% of HHCB into HHCB-lactone within 4 weeks (3). The transformation of HHCB into HHCB-lactone and its corresponding hydroxy acid was also reported for microcosm experiments with activated sludge (3). GC-MS characteristics of compounds newly appearing in HHCB- and AHTN-containing cultures and possible metabolite structures are summarized in Table 1. These compounds were not observed in the untreated parent musks or in fungal controls omitting the musks and thus prove cometabolic biotransformation of HHCB and AHTN. More detailed rationales for the structure proposals shown in Table 1 and possible pathways of their biological formation are provided in the Supporting Information. Besides HHCBlactone, HHCB metabolites with higher polarity than that of the parent compound have been detected in pure cultures of terrestrial fungi and in microcosms employing different soil types and activated sludge but no structures were proposed (3). The temporary presence of AHTN biotransformation metabolites with one and two additional oxygen atoms has been described for the white-rot fungus P. chrysosporium but again no structure proposals were given (3). The formation of metabolites E, G, and K has already been suggested for photo-oxidation of AHTN (21, 22), which is in support of a possible minor contribution of photodeg-

radation to the AHTN removal observed in Myrioconium sp. cultures (Figure 1). However, metabolites G and K may also be formed biologically (see the Supporting Information for details). A reduction of the keto group in the ethanone moiety of AHTN proposed for the major AHTN metabolite M, which was found in C. aquatica cultures (Table 1), was also described for the terrestrial fungus Aureobasidium pullulans (3). No attempts were made to explain the structures of metabolites N and L. The potential biological effects of HHCB and AHTN metabolites remain to be elucidated. Removal of HHCB and AHTN by Isolated Laccases. Laccase-containing concentrated crude culture supernatants of Myrioconium sp. and C. aquatica, and a commercial laccase from T. versicolor were used to study laccase effects on HHCB and AHTN which have not been described before. Laccase formation in Myrioconium sp. and C. aquatica is described in the Supporting Information. HHCB and AHTN concentrations at the beginning and after 6 days of enzymatic incubation and their respective percentages of changes in concentrations are summarized in Table 2. Most efficient removal of the musks was observed for the T. versicolor laccase, followed by the laccase from Myrioconium sp. The C. aquatica laccase was less effective. This behavior may reflect different redox potentials of the investigated enzymes known to control the rate-limiting step in laccase catalysis (23). The influence of the artificial redox mediator ABTS on the musk oxidation by the laccases from Myrioconium sp. and C. aquatica was also assessed (Table 2). ABTS clearly enhanced HHCB conversion by the C. aquatica laccase but decreased its removal by the laccase from Myrioconium sp. (confirming data of additional experiments not shown). Simultaneous reactions of HHCB and its primary oxidation product(s) competing for ABTS-derived radicals could be an explanation. In contrast, ABTS enhanced AHTN conversion by the Myrioconium laccase but did not affect its resistance toward the laccase from C. aquatica. All together, these results indicate that redox mediators may enhance laccase-catalyzed oxidation of HHCB and AHTN under certain conditions. Natural redox mediators have been described for white-rot basidiomycetes (24). Detection of Biotransformation Products during Laccase Reactions by GC-MS. In HHCB-containing enzymatic assays omitting ABTS the concentrations of HHCB-lactone increased to approximately 780, 353, and 198% (in relation to respective controls containing heat-inactivated enzymes) upon treatment with laccases from T. versicolor, Myrioconium sp., and C. aquatica, respectively (Table 3). This behavior correlates with the observed efficiencies of the three enzymes in removing HHCB in the absence of ABTS (Table 2). Furthermore, it indicates that these laccases convert HHCB into HHCB-lactone, thereby also first demonstrating an enzyme catalyzing this reaction. Abiotic formation of HHCB-lactone and its occurrence in aquatic environments has been attributed to autoxidation of HHCB (2, 21). We suggest a reaction mechanism where laccase catalyzes the formation of a radical from HHCB by one-electron-abstraction and concomitant proton release (Figure 2), similar to laccase oxidation of phenolic substrates (23). This enzymatic step will enable abiotic follow-up reactions such as those already proposed by other authors (21), finally leading to HHCBlactone (Figure 2). A clearly less increase in the HHCB-lactone concentration was observed for the Myrioconium laccase in the presence of ABTS, which may reflect the less efficient HHCB oxidation under these conditions (Table 2). Essentially no differences in the HHCB-lactone concentrations between assays containing active C. aquatica laccase and ABTS and those containing the heat-inactivated enzyme were monitored (Table 3), whereas HHCB was clearly removed by the active VOL. 41, NO. 15, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. HHCB and AHTN Concentrationsa in Laccase-Containing Reaction Mixtures at the Beginning and After 6 Days of Incubation and Percentages of Changes in Concentrations compound, laccase source, and reaction conditionsb HHCB + Myrioconium laccase active enzyme active enzyme + ABTS heat-inactivated enzyme HHCB + C. aquatica laccase active enzyme active enzyme + ABTS heat-inactivated enzyme HHCB + T. versicolor laccase active enzyme heat-inactivated enzyme AHTN + Myrioconium laccase active enzyme active enzyme + ABTS heat-inactivated enzyme AHTN + C. aquatica laccase active enzyme active enzyme + ABTS heat-inactivated enzyme AHTN + T. versicolor laccase active enzyme heat-inactivated enzyme

initial concn. (µM)

final concn. (µM)

concn. change (%)c

230.6 ( 22.6 243.9 ( 5.4 235.8 ( 18.5

160.3 ( 2.2 210.8 ( 16.6 230.3 ( 25.4

-30.5d -16.3d -2.4

192.6 ( 12.1 200.4 ( 14.0 203.9 ( 5.5

172.5 ( 11.3 133.6 ( 6.4 218.0 ( 8.2

-10.4 -33.3d +6.9

205.6 ( 14.0 208.7 ( 3.6

87.7 ( 1.2 220.4 ( 19.4

-57.3d +5.6

210.9 ( 14.6 209.2 ( 2.8 217.0 ( 2.3

185.0 ( 16.5 152.6 ( 2.8 204.4 ( 3.5

-12.3 -27.1d -5.8

181.4 ( 3.1 217.2 ( 4.5 187.0 ( 3.1

179.5 ( 6.3 219.5 ( 6.9 184.8 ( 0.2

-1.0 +1.1 -1.2

197.1 ( 6.1 225.6 ( 5.1

45.8 ( 18.3 206.3 ( 18.3

-76.8d -9.4

a Data represent means ( standard deviations for triplicate experiments. b ABTS was not used in reaction mixtures containing T. versicolor laccase. c Calculated from the final in relation to the initial musk compound concentrations, where (-) indicates a decreasing and (+) indicates an increasing concentration. d Significant concentration change according to Student’s t test (P < 0.05).

TABLE 3. Relative HHCB-Lactone Concentrationsa in Reaction Mixtures Containing HHCB and Fungal Laccases After 6 Days of Incubation laccase source and reaction conditionsb

relative HHCB-lactone concentration (%)

Myrioconium sp. laccase Myrioconium sp. laccase + ABTS C. aquatica laccase C. aquatica laccase + ABTS T. versicolor laccase

353 ( 30 150 ( 20 198 ( 14 96 ( 14 780 ( 51

a In relation to HHCB-lactone concentrations in reaction mixtures containing HHCB and the respective heat-inactivated laccase, which represent 100%. Calculations were based on peak areas of molecular ions of HHCB-lactone detected at m/z 272. Data represent means ( absolute deviations for duplicate assays. The initial HHCB-lactone concentration was 4% of that of HHCB (based on the peak areas of HHCB-lactone and HHCB in TIC chromatograms) and did not significantly increase during the incubation period in heat-inactivated controls. b ABTS was not used in reaction mixtures containing T. versicolor laccase.

enzyme (Table 2). This suggests that products other than HHCB-lactone might also be formed, for instance in further reactions between ABTS radicals and primary HHCB oxidation products. Both quinones and oxidative coupling products are formed during laccase-catalyzed oxidation of PAHs in the presence of redox mediators, and laccase-redox mediator systems were shown to further oxidize quinones resulting from PAH oxidation (25). No metabolites were detected upon laccase treatment of AHTN. Oxidative coupling products potentially formed upon AHTN and HHCB oxidation by the laccases might have been inaccessible with the applied GCMS method, covering a mass range of 50 to 500 amu. In addition to intracellular reactions catalyzed by as yet unknown enzymes (Table 1), laccases may contribute to fungal biotransformation of HHCB and AHTN when produced in sufficient amounts. In Myrioconium sp. excreting laccase under the biotransformation conditions applied within the present study (see Figure 2S in the Supporting Information), laccase reactions might have been interfered 5400

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FIGURE 2. Proposed HHCB-lactone formation pathway by fungal laccases. with reactions of other enzymes involved. Laccases are increasingly described also for bacteria (26), which extends their potential relevance for environmental processes to microorganisms beyond fungi. Laccases may also serve as biocatalysts in environmental biotechnology (17).

Implications of Fungal Musk Biotransformation for Aquatic Processes. Fungi are considered important decomposers of plant-derived organic matter in lotic (27) and lentic freshwater systems (28). Such organisms may also act as cometabolic micropollutant degraders (16) under conditions where organic matter sufficiently supports fungal growth and sufficient oxygen is still available, e.g., in top layers of river and stream bottom sediments, wetlands, and littoral zones of ponds and lakes. Findlay and co-workers (27) determined the fungal biomass content of coarse (leaves, small wood) and fine benthic organic matter associated with streambed surface and subsurface sediments (2-5 cm below the surface) from 9 U.S. headwater streams. Overall, fungal dry weight concentrations (calculated by doubling the fungal carbon contents given in (27) according to (29)) of approximately 15, 9, 1, and 0.4 g per kg dry organic matter (determined as loss on ignition at 550 °C in (27)) were reported for leaves, wood, surface, and subsurface sediments, respectively. Fungi clearly dominate the larger size classes of particulate matter and are less abundant on fine-particulate matter of bottom sediments than bacteria (27). Sedimentation was identified as a major process lowering HHCB and AHTN concentrations in water bodies (20, 30, 31). Considering that upper layers of bottom sediments represent preferred habitats of aquatic fungi, sedimentation spatially links musk compounds to such potential degraders. The higher sensitivity of fungi toward different organic matter types, as compared to bacteria, and their preferential colonization of coarse-particulate organic matter (27) suggests that the fungal influence on the biodegradation of musk compounds in aquatic ecosystems may vary largely, depending on a particular environmental situation. Based on the first-order rate constants for biological musk elimination (kb values) observed with Myrioconium sp., we have roughly estimated HHCB and AHTN half-lives that could be expected for fungal elimination of musks in hypothetical freshwater sediment systems (top 5 cm of bottom sediments including both coarse- and fine-particulate organic matter) (see the Supporting Information for details). Although these estimates may be biased by uncertainties with respect to the actual amount of fungal biomass involved in musk biotransformation and unknown fungal musk conversion rates presumably varying among individual members of the contributing fungal communities, they should enable obtaining an impression about the possible orders of magnitude to be expected. Under our chosen conditions, half-lives ranging from approximately 2567 days for HHCB and 1873 days at low total fungal biomass and activity to about 58 days for HHCB and 38 days for AHTN at higher fungal activity were obtained (see Figure 4S in the Supporting Information). A half-life of HHCB of 79 days was observed in microcosm studies with river sediment but the involved microorganisms were not identified (30). From microcosm experiments employing sludge-amended soil, forest soil, and agricultural soil, HHCB half-lives of 105, 95, and 239 days, respectively, were described (30). In experiments monitoring HHCB and AHTN dissipation in different sludge-amended soils, the musk concentrations remaining after 1 year ranged from zero to about 9% for HHCB and from approximately 40 to 86% for AHTN (32). The processes accounting for removal have not been elucidated further but biological transformation was considered as a possibility. The aforementioned studies are in support of substantial bioconversion of HHCB and AHTN by environmental sediment and soil microorganisms within 1 year or even shorter, a time span which is also covered by our estimated HHCB and AHTN half-lives for fungal degradation in hypothetical sediment systems (Figure 4S). Low environmental concentrations of HHCB and AHTN (ng/L range for surface waters (1, 3, 4) and up to the 3-digit µg/kg range for river sediments (1, 30)) may prevent their

substantial biodegradation, as far as musk utilization as a growth substrate for potentially degrading microorganisms is concerned. However, cometabolic degraders do not depend on the use of a contaminant as a carbon source. Considering fungi feeding on plant-derived organic matter, the local musk concentrations of fungal substrates, i.e., leaf litter, woody debris, and organic domains of fine-particulate sediment fractions should be relevant. Hydrophobic interactions with organic matter largely contribute to the sorption of HHCB and AHTN (12). Higher local musk concentrations in the immediate vicinity of active fungal biomass resulting from sorption would promote mass transfer fluxes to fungal cells and thereby diminish potential kinetic limitations. In sewage sludge showing a high content of organic carbon (for instance about 35% in (12)), HHCB and AHTN concentrations in the 2-digit mg/kg range have frequently been reported (1, 3, 12). No literature data exist for musk concentrations of leaf litter or woody debris. With respect to fine-particulate sediment matter, musk concentrations of roughly 1-2 orders of magnitude higher than those derived from analysis of bulk sediments (up to the 3-digit µg/kg range (1, 30)) could be assumed for the organic domains of sediments when considering an organic carbon content in a range of 1-10%. Hence, musk concentrations of up to the mg/kg range seem reasonable for such organic domains. Under particular conditions, biotransformations catalyzed by freshwater fungi may be relevant when addressing the environmental fate of polycyclic musks and potential biological effects of their degradation metabolites. The degradative capabilities of aquatic fungi may also offer new biotechnological perspectives for the removal of micropollutants from wastewaters.

Acknowledgments We thank the German Research Foundation (DFG) for funding of C.M. within the DFG graduate college 416. We also gratefully acknowledge the help of K. Smith (UFZ) in linguistic improvement of the manuscript.

Supporting Information Available Information about chemicals and other materials, determination of HHCB and AHTN concentrations by HPLC, GCMS analysis of HHCB and AHTN biotransformation products, procedures and instrumentation used for total reflection IR spectroscopy and GC-MS/MS analysis of HHCB-lactone, the proposed main mass fragmentation pathways of HHCBlactone (Figure 1S) and the corresponding rationale, rationales for the proposed structures of musk metabolites other than HHCB-lactone and possible pathways of their biological formation, laccase formation in Myrioconium sp. (Figures 2S and 3S) and C. aquatica and the concentration of cell-free crude culture supernatants, and the procedure used to estimate half-lives of HHCB and AHTN for fungal biotransformation in hypothetical freshwater sediment systems (Figure 4S). This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review May 15, 2007. Accepted May 23, 2007. ES0711462