Role of dissolved humic acids in the biodegradation of a single isomer of nonylphenol by Sphingomonas sp.

Chemosphere 68 (2007) 2172–2180 www.elsevier.com/locate/chemosphere

Role of dissolved humic acids in the biodegradation of a single isomer of nonylphenol by Sphingomonas sp. Chengliang Li

a,b

, Rong Ji c, Ralph Vinken b, Gregor Hommes b, Marko Bertmer d, Andreas Scha¨ffer b,e, Philippe F.X. Corvini b,e,*

a Institute of Soil Science, Chinese Academy of Sciences, 210008 Nanjing, China Biology 5-Environmental Biology and Chemodynamics, RWTH Aachen University, D-52056 Aachen, Germany State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 210093 Nanjing, China d Macromolecular Chemistry, RWTH Aachen University, D-52056 Aachen, Germany e Fraunhofer IME, D-57392 Schmallenberg, Germany b

c

Received 23 October 2006; received in revised form 22 January 2007; accepted 30 January 2007 Available online 23 March 2007

Abstract This study shows the important role of humic acids in the degradation of 14C and 13C labeled isomer of NP by Sphingomonas sp. strain TTNP3 and the detoxification of the resulting metabolites. Due to the association of NP with humic acids, its solubility in the medium was enhanced and the extent of mineralization of nonylphenol increased from 20% to above 35%. This was accompanied by the formation of significant amounts of NP residues bound to the humic acids, which also occurred via abiotic reactions of the major NP metabolite hydroquinone with the humic acids. Gel permeation chromatography showed a non-homogenous distribution of NP residues with humic acids molecules, with preference towards molecules with high-molecular-weight. Solid state 13C nuclear magnetic resonance spectroscopy indicated that the nonextractable residues resulted exclusively from the metabolites. The chemical shifts of the labeled carbon indicated the possible covalent binding of hydroquinone to the humic acids via ester and possibly ether bonds, and the incorporation of degradation products of hydroquinone into the humic acids. This study provided evidences for the mediatory role of humic acids in the fate of NP as a sink for bacterial degradation intermediates of this compound.  2007 Elsevier Ltd. All rights reserved. Keywords:

13

C-labelled NP; Nonextractable residues; Hydroquinone; Bacteria; Detoxification

1. Introduction Humic substances originate from degradation and transformation processes of organic materials and represent by far the most abundant organic materials in the environment (Hayes and Clapp, 2001). Based on their solubility, humic

*

Corresponding author. Address: Biology 5-Environmental Biology and Chemodynamics, RWTH Aachen University, D-52056 Aachen, Germany. Tel.: +49 241 80 27260; fax: +49 241 80 22182. E-mail address: [email protected] (P.F.X. Corvini). 0045-6535/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.01.080

substances can be classified into alkali-insoluble humin, alkali-soluble and acid-insoluble humic acids, and acid-soluble fulvic acids (Stevenson, 1994; Xie et al., 1997; Conzonno and Cirelli, 1998). Humic acids consist of high amounts of aromatic structures and are considered as a hydrophobic material (Christl et al., 2000; Ussiri and Johnson, 2003). They are likely to bind organic pollutants, especially hydrophobic chemicals, via mechanisms ranging from weak interactions like hydrogen bonding and van der Waals forces to strong chemical bonds such as covalent bonding (Bollag et al., 1992; Hayes and Graham, 2000; Hayes and Clapp, 2001; Senesi and Loffredo, 2001). Due to the interactions with humic acids, the mobility, bioaccumulation, and

C. Li et al. / Chemosphere 68 (2007) 2172–2180

biodegradation of the pollutants is affected in environmental compartments (Ka¨stner and Mahro, 1996; Oesterreich et al., 1999; Akkanen and Kukkonen, 2003; Mc Carthy et al., 2003). Among the micropollutants of high concern, nonylphenol (NP) is being intensively studied because of its toxicity, persistence, bioaccumulation in aquatic species and adverse effects on the endocrine system (Granmo et al., 1989; Ekelund et al., 1990; Soto et al., 1991). In fact NP is found as a technical mixture (usually abbreviated tNP), which consists of more than 20 branched isomers of the nonyl chain and is used for the production of the worldwide-used surfactants nonylphenol polyethoxylates (NPnEO) (Wheeler et al., 1997; Thiele et al., 2004). As a consequence of the discharge in the environment and biodegradation processes, tNP is recovered as persistent degradation product of NPnEO (Giger et al., 1987; Tanghe et al., 1998, 1999). Contrarily to the linear alkyl chain NP (4-n-NP), which is widely used as model substance, tNP is recalcitrant (Corvini et al., 2006). More than 85% of the isomers of tNP possess a quaternary a-carbon on the branched alkyl chain, which is resistant to microbial degradation processes such as xand b-oxidation (Van Ginkel, 1996; Wheeler et al., 1997). Furthermore, studies on the fate of NP are impaired by the fact that t-NP is a complex mixture of isomers, which gives rise to a complex metabolic pattern. Except for a study reporting the degradation of tNP into 4-nonyl-2-nitrophenol in mixtures of sandy loam soil/ sludge, almost no information exists concerning the degradation pathways of NP in environmental samples (Telscher et al., 2005), while metabolism of NP by axenic cultures of bacteria has been well documented. Several bacterial strains of the genera Sphingomonas, Sphingobium and Pseudomonas are able to degrade branched isomers of NP (Tanghe et al., 1999; de Vries et al., 2001; Fujii et al., 2001; Soares et al., 2003; Ushiba et al., 2003; Gabriel et al., 2005a,b). NP-degradation pathways in Sphingomonads presents similarities, i.e. the corresponding alcohols of the alkyl side-chains of NP (nonanols) are metabolites produced by these bacteria (Fujii et al., 2000; Tanghe et al., 2000; Gabriel et al., 2005a,b). The initial degradation step involves a hydroxylation at the carbon C4 of the phenol moiety. In the case of Sphingomonas xenophaga strain Bayram the degradation pathway occurs via the formation of alkyloxyphenol and p-benzoquinone (Gabriel et al., 2005a,b). Biodegradation of NP by Sphingomonas sp. strain TTNP3 involves hydroquinone as the central metabolite resulting from a type II ipso-substitution and the hydroquinone is further degraded into succinate, 3,4-dihydroxy butanoic acid, and several unidentified compound(s) (Corvini et al., 2006). Apart from studies on the fate of NP and NPnEO in environmental samples reporting on association constants between NP and organic or mineral material as well as on the formation of nonextractable residues (bound residues) of NP (During et al., 2002; Yamamoto et al., 2003; Telscher et al., 2005), the influence of humic acids on microbial transformation of NP is sparsely documented.

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Due to its high hydrophobicity, NP is likely to associate with humic acids (Log KDOC = 4.09, water solubility 5 mg/l) (Vinken et al., 2004) and to sorb on sediments (Ying et al., 2003). While some authors stated that the hydrophobicity of NP could decrease its bioavailability (Tanghe et al., 1998; Ying et al., 2003), we showed that the association of one NP isomer 4-[1-ethyl-1,3-dimethylpentyl]phenol (p353NP) with brown coal derived humic acids had no effects on the degradation kinetics of p353NP by Sphingomonas sp. TTNP3 (Vinken et al., 2004). It was therefore assumed, that the association between nonylphenol and humic acids occurs rapidly and is reversible (Vinken et al., 2004). Because this study was based only on the determination of the decrease of NP concentration, it did not allow a deep insight into the biological and physico-chemical processes occurring in such complex systems containing humic substances. In the present study, using 13C and 14C labeled p353NP, we report for the first time in detail the fate of NP in cultures of Sphingomonas sp. strain TTNP3 in the presence of dissolved humic acids derived from brown coal and the interactions between NP residues and this humic material.

2. Materials and methods 2.1. Synthesis of substrates and preparation of humic material Non-labeled p353NP isomer was synthesized in large scale by Friedel–Crafts alkylation from phenol and 3,5dimethyl-3-heptanol in presence of boron trifluoride as catalyst (Vinken et al., 2002). 14C-U-ring labeled p353NP and 13 C-labeled p353NP were synthesized from 14C-U-ring labeled phenol and 13C-C1 labeled phenol according to the same method. For the synthesis of 14C-U-ring labeled NP, 14C-U-ring labeled phenol (37 MBq, 2.22 GBq mmol1, Hartmann Analytic, Braunschweig, Germany) was mixed with 10 mg of non-labeled phenol and 31.8 mg of 3,5-dimethyl-3-heptanol in presence of BF3. A total of 17 mg of 14C-p353NP was obtained and redissolved in methanol at a concentration of 5 mg ml1. The specific radioactivity was 0.30 GBq mmol1 and the radiochemical purity was 97.2%. 13C-labeled nonylphenol was obtained by reacting 123 mg of 13C-C1 labeled phenol (Sigma Aldrich, min. 99% purity) directly with 270 mg of nonanol. A total of 122.1 mg 13C-labeled p353NP (97.72% purity) was obtained and was redissolved in petroleum ether at a final concentration of 100 mg ml1. Dissolved humic acid solution was prepared by dissolving brown coal derived humic acids of commercial source (sodium salt; Sigma-Aldrich, Steinheim, Germany) in 0.02 M NaOH solution. The solution was neutralized prior to dialysis at 1 kDa cut-off (Vinken et al., 2002). The humic acid stock solution had a concentration of dissolved organic carbon (DOC) of 2.5 g l1.

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2.2. Microorganism and culture conditions Precultures used for inoculation of the cultures in mineral medium or for resting cell experiments were prepared by inoculating Standard I medium (Merck, Darmstadt, Germany) with a frozen glycerol stock solution of Sphingomonas sp. strain TTNP3 (Vinken et al., 2004). Flasks were incubated aerobically on a rotary shaker (70 rpm) at 28 C for 48 h until the optical density of the cell suspension was 7 at 550 nm (3.8 g cell dry-weight l1). For degradation experiments in mineral salt medium, inoculum was prepared by diluting 100 ll of cell preculture to 2 ml with sterile mineral medium MMO (Stanier et al., 1966). For experiments with resting cells suspensions of Sphingomonas sp. strain TTNP3, cultures of the microorganism in Standard I medium were processed by washing and resuspending the cells to 5.5 g cell dry weight l1 in 50 mM potassium phosphate buffer (pH 7) as described by Corvini et al. (2006). 2.3. Degradation of 14C-U-ring labeled NP by Sphingomonas sp. strain TTNP3 in the presence of humic acids Sphingomonas sp. strain TTNP3 was incubated with C-U-ring labeled NP in the presence and absence of humic acids. The degradation experiments were performed in 20 ml glass flasks hermetically closed by rubber stoppers. Prior to application, 14C labeled NP was diluted with non radioactive NP in CH2Cl2 at a final concentration of 0.5 g l1 with a radioactivity of 30.9 KBq ml1. 100 ll of this NP solution was firstly applied to the empty culture vessels and CH2Cl2 was gently evaporated under sterile conditions. Then 5 ml of mineral medium or mineral medium containing humic acids (1 g l1) was added into the corresponding vessels. Flasks were pre-incubated in the dark at 28 C on a rotary shaker (70 rpm) for 23 h in order to allow the formation of NP-humic acids complexes (Vinken et al., 2004). The biodegradation experiments were started by inoculating the medium with the diluted preculture (2%, V/V). Flasks were incubated under the same conditions as for preincubation over a period of 41 h, before NP was completely degraded (Vinken et al., 2004). Filter papers saturated with 0.2 ml of 1 M NaOH solution were fixed on the bottom of stoppers by means of a needle to trap 14CO2 produced by Sphingomonas sp. strain TTNP3. Abiotic controls (without bacteria) were incubated in parallel under the same conditions with and without humic acid. All assays were carried out in triplicate. 14

2.4. Determination of CO2 and volatile compounds After incubation, the filter papers containing the 14CO2 and 14C-NP trapped were transferred into 20 ml glass vials containing 4 ml of 1 M NaOH solution. After vigorous

shaking and removal of NaOH, the filter papers were further extracted twice with 1 M NaOH. The NaOH extracts were combined together and aliquots were taken for liquid scintillation counting (LSC) analysis (see below). For CO2 indirect determination in triplicate, 0.5 ml of this NaOH was pipetted into three LSC vials and 100 ll of 6 M HCl was added into each vial to remove 14CO2 over night. The quantity of CO2 was calculated as the difference of radioactivity in the NaOH extract before and after acidification. Control experiment, i.e. acidification of a NPcontaining NaOH solution, was carried out in order to calculate the part of NP, which was evaporated during the acidification procedure. NP evaporated from cultures was determined by working out 8 ml of the 12 ml of NaOH extract by means of three extraction steps with 8 ml ethyl acetate each. Ethyl acetate fractions were dried over Na2SO4, filtrated and concentrated to 1 ml by means of a rotary evaporator, and the radioactivity was determined by means of LSC in triplicate. The rubber stoppers were placed in 100 ml vessel and shaken with 40 ml of ethyl acetate for 45 min in order to extract the volatilized NP. After measurement of the radioactivity in the first sample (1 ml), flask was placed again on the shaker and one milliliter aliquots were taken for LSC measurements over a period of four days until the portion of desorbed NP remained constant.

2.5. Determination of radioactivity adsorbed on vessels and in the fractionated medium Aliquots of 100 ll of homogeneous culture were sampled to measure radioactivity by means of LSC. The medium was transferred in other vials and was extracted by ethyl acetate three times (each 10 ml). The empty culture vessel was washed with 3 ml of ethyl acetate and the radioactivity in the rinse was determined by LSC. The extracts of the culture were combined and rotary evaporated to a volume of 1 ml. Aliquots of the concentrated solution were sampled for LSC, HPLC-LSC and GCMS analyses. The culture after ethyl acetate extraction was homogenized by shaking and 0.5 ml aliquots were taken for LSC analyses (triplicate). The extracted culture was pooled into a thin graduated test tube to allow pellets set down on the bottom of the tube. The radioactivity was determined for 0.5 ml of the pellet and the supernatant by means of LSC measurement in triplicate. In the case of the incubation in the presence of humic acids, 0.5 ml of the cell free supernatant was transferred to microtubes and acidified with 6 M HCl to pH 1 in order to precipitate the humic acids. These microtubes were agitated for 10 min on a shaker and centrifuged 15 min at 13 000g. The radioactivity of 0.5 ml of the humic acids-free supernatant was determined by LSC (triplicate measurement). The humic acids pellet was redissolved in 1 ml of O2-free 0.1 M NaOH solution for further LSC and HPLC-GPC analyses.

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2.6. Degradation of 13C and 14C labeled NP by Sphingomonas sp. strain TTNP3 The experiments aimed at obtaining NP residues bound to humic acids for nuclear magnetic resonance spectrometry (NMR) analysis. The degradation experiments were performed in five 1 l glass bottles containing 250 ml of mineral medium and humic acids (at a final concentration of 270 mg l1). Mixture of 2.5 mg of 13C-labeled NP and 44 lg of the radioactive NP-containing solution (total of 52.4 KBq per bottle) was added into each bottle. All other procedures were similar to aforementioned experiments for degradation of 14C-U-ring labeled NP, except for the methods for trapping of 14CO2 and stripped NP and for the precipitation of the humic acids. Trapping of CO2 and volatilized NP was achieved by fitting a glass funnel, filled with polyurethane foam plugs, glass wool, and soda lime pellets, on each culture vessel. The humic acids were precipitated by acidifying the solution with 6 M HCl to pH 1. After separation by centrifugation (10 000g), the humic acids were freeze-dried prior to NMR analysis. 2.7. Abiotic formation of NP residues bound to humic acids in the supernatant of resting cells of Sphingomonas sp. strain TTNP3 100 ml of resting cell suspension of Sphingomonas sp. strain TTNP3 were supplied with a mixture of 10 mg of 13 C-labeled NP and 111 lg of 14C labeled NP (132 KBq total radioactivity applied) and mixed with 250 ml of phosphate buffer medium (pH 7). Incubation was carried out for 90 min at 37 C with magnetic stirring under a hood. An aliquot of 0.5 ml of the reaction mixture was mixed with 0.5 ml methanol and centrifuged for 5 min at 13 000g. The supernatant was taken for HPLC analysis. The remaining volume of resting cell assay was centrifuged at 13 000g for 15 min. It was previously reported that under these conditions NP was completely degraded and the products hydroquinone and other organic acids originating from the phenol moiety of NP were accumulated in the reaction medium (Corvini et al., 2006). The supernatant containing NP metabolites was incubated with mineral medium containing humic acids (final concentration 270 mg l1) in closed flask in the dark at 28 C on a rotary shaker (70 rpm) over a period of 41 h. At the end of the incubation, humic acid suspension containing bound residues of NP was prepared as described in the previous paragraph. 2.8. Analytical techniques 2.8.1. LSC analyses Appropriate volumes of aliquots from the fraction were added to 10 ml of a Lumasafe Plus scintillation cocktail (Lumac*LSC BV, Netherlands) in 20-ml vials, and then determined by Beckman LS 5000TD liquid scintillation counter. Quench was corrected with external standards

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and no chemiluminescence due to the cocktails was observed. In NaOH containing samples chemoluminescence was avoided using an appropriate volume ratio of sample and cocktail. 2.8.2. GC-MS analyses Before GC-MS analyses, 50 ll aliquots of the concentrated samples were dried under a gentle stream of nitrogen and derivatized with 50 ll of N,O-bis-(trimethylsilyl)-trifuoracetamide (BSTFA, Merck, Germany) at 70 C for 15 min. The GC-MS studies were carried out with an HP 5890 Series II gas chromatograph (Agilent Technologies) equipped with an FS-SE-54-NB-0.5 column (25 m · 0.25 mm, 0.46 lm film thickness; CS Chromatographie Service, Germany), coupled to an HP 5971 A mass selective detector (Agilent Technologies) as described previously (Vinken et al., 2002). 2.8.3. High performance radio gel permeation chromatography (HP-14C-GPC) Prior to analysis, samples were centrifuged at 13 000g for 15 min and 100 ll of supernatant was injected onto a GPC ˚ , 8 mm · 300 mm; Polymer column (PSS MCX 1000 A Standards Service GmbH, Mainz, Germany), which separated molecules with molecular weights-range from 200 to 110 000 Da, calibrated with molecular weight standards polystyrene sulfonate sodium salts. The column was eluted with 0.6% of K2CO3 (pH 11, HPLC-grade Roth, Germany) at a rate of 0.1 ml min1 for 150 min at 40 C on the HPLC Series 1100 system equipped with an auto-injector, a degasser, a diode array detector, and an online liquid scintillation radio flow detector (Ramona Star; Raytest, Straubenhardt, Germany) with a cell volume of 1300 ll. The flow rate of the scintillation cocktail (Quicksafe Flow 2; Zinsser Analytic GmbH, Frankfurt, Germany) for the radio detector was 0.5 ml min1. The absorbance signals of humic acids were measured at 360 and 450 nm. 2.8.4. Reversed phase HPLC analysis with radiodetector (RP-HPLC-LSC) Prior to analysis, samples were centrifuged at 13 000g for 15 min, and 50 ll of supernatant was injected onto a Nucleosil column (Agilent Technologies; 4.6 · 150 mm, 5-lm particle size) connected on the same HPLC system as for HP-14C-GPC with online liquid scintillation radio flow detector. The UV signal was measured at 270 nm. The compounds were eluted with a gradient of water and methanol (both HPLC-grade; Roth, Germany) as follows: 90:10 (water:methanol/v:v), 0–7 min; 0:100, 15 min; 0:100, 15– 20 min; 90:10, 30 min; 90:10, 30–33 min at a flow rate of 1 ml min1 at 35 C. 2.8.5. Nuclear magnetic resonance (NMR) spectroscopy Solid state 13C NMR was performed using a Bruker DMX300 spectrometer (Bruker; Rheinstetten, Germany) equipped with a Bruker 4-mm double-resonance probe. 13 C cross polarization magic angle spinning NMR (13C

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CP MAS NMR) spectra were obtained with a recycle time of 1 s, and a contact time of 1 ms. Quantitative 13C direct polarization MAS NMR (13C DP MAS NMR) spectra were obtained with a 5 s recycle time and a 90 13C pulse length of 1.5 ls. In both measurements the spinning speed was 10 kHz. About 16 000 scans were acquired in both measurements. 3. Results 3.1. Degradation of NP by Sphingomonas sp. strain TTNP3 in the presence of humic acids Table 1 summarizes the radioactivity recovered from various fractions of the culture, showing the total recoveries of radioactivity ranged from 89.5% to 104.4%. The gaps in the radioactivity recovery were mainly attributed to the adsorption processes of volatilized NP on and in the rubber stopper. When the stoppers were submerged in ethyl acetate, a slow and continuous release of radioactive NP in the solvent was observed over several days. The presence of humic acids had significant effect on the distribution of radioactivity between the various fractions. In the absence of humic acids, due to its high hydrophobicity and volatility, larger proportions of NP were recovered from the rubber stopper than when humic acids were added (Table 1). Significant amounts of NP were recovered from the wall of culture vessels, but were lower than in assays containing humic acids. About 20% of the NP applied initially was mineralized by Sphingomonas sp. TTNP3 and 42% of NP was volatilized and therefore not available for the bacteria. The reason for the recovery of large amounts of NP in the filter was that the dissociation of NP in the alkaline solution (CO2 trap) contributed trapping NP. However, in the presence of humic acids, not only the volatilization of NP was strongly reduced, but also the extent of mineralization of NP was strongly increased. Furthermore, the amounts of residues which were not extracted by means of ethyl acetate and remained in the aqueous phase increased from 10% to 25% (Table 1).

Further analysis of the radioactivity in the cultures showed that the radioactivity was not predominantly associated to the cells and cell debris, but remained in the cellfree medium (Table 2). The precipitation of humic acids from cell-free medium showed that the increased radioactivity in the medium in the presence of humic acids was mainly localized in the humic acids (Table 2, fraction of organic pellets). However, in the abiotic system (incubation without bacteria), the radioactivity recovered from the humic acids fraction was very low. Incorporation of NP into the bacterial cells was found negligible. Partial cell lysis during the extraction with ethyl acetate may explain the low radioactivity associated to the cell fraction. 3.2. Formation of NP bound residues in culture of Sphingomonas sp. strain TTNP3 In order to gain qualitative information on this residual radioactivity associated to humic acid pellets, Sphingomonas sp. strain TTNP3 was incubated with 14C- and 13 C-labeled NP as substrate. At the end of the incubation, Table 2 Fractionation of radioactivity in the ethyl acetate-extracted culture of Sphingomonas sp. strain TTNP3 and sterile control, in the presence and absence of humic acids Fraction

Assay Inoculated Sterile Inoculated + humic Sterile + humic acids acids

Cell Cell-free medium Organic-free supernatant Organic pellets

1.4 ± 0.4 6.9 ± 1.4

ND ND

0.8 ± 0.3 31.9 ± 0.5

0.1 ± 0.0 1.7 ± 0.3

3.2 ± 0.3

ND

4.7 ± 0.2

0.0 ± 0.0

2.0 ± 0.9

ND

25.5 ± 2.0

1.6 ± 0.3

Values represent the percentage of the radioactivity initially applied. Cell: radioactivity associated to the decanted cells; Cell-free medium: radioactivity remaining in the medium after removal of cell and cell debris; Organic-free Supernatant: radioactivity remaining in the cell-free medium after acidification; Organic pellets: radioactivity associated to the pellets after acidification. ND: not determined.

Table 1 Distribution and recovery (%) of radioactivity after degradation of 14C-U-ring labeled NP by Sphingomonas sp. strain TTNP3 in the presence and absence of humic acids and in sterile control experiment Fraction *

Sum of CO2 and volatilized NP NP CO2 Medium: EtOAc Aqueous phase Culture vessel Vessel stopper Sum

Assay Inoculated

Sterile

Inoculated + humic acids

Sterile + humic acids

65.9 ± 3.6 42.2 ± 0.6 20.4 ± 3.0 14.6 ± 1.4 4.7 ± 1.6 10.5 ± 2.4 3.7 ± 0.3 5.3 ± 0.9 89.5 ± 4.0

59.0 ± 5.3 56.7 ± 3.7 0.0 ± 0.0 20.2 ± 1.4 19.3 ± 1.9 0.1 ± 0.0 6.7 ± 0.2 5.9 ± 0.8 91.8 ± 4.4

52.5 ± 4.4 12.5 ± 0.8 35.6 ± 3.8 32.5 ± 2.0 6.1 ± 0.7 24.6 ± 1.4 5.8 ± 1.0 2.6 ± 0.4 93.4 ± 5.3

16.5 ±3.3 15.4 ± 1.6 0.0 ± 0.0 74.5 ± 6.5 68.6 ± 6.1 2.0 ± 0.4 11.0 ± 1.6 2.4 ± 0.6 104.4 ± 4.0

Values represent the percentage of the radioactivity initially applied ± standard deviation (n = 3). * The sum of CO2 and volatilized NP represents the radioactivity recovered on the filter paper.

C. Li et al. / Chemosphere 68 (2007) 2172–2180

800

30

600

20

400

10

200

14

40

0

UV 360 nm and 450 nm (mV)

C (cps)

the radioactivity remaining in the incubation medium was 52.0 ± 4.9% (n = 5) of the initially applied amount and was mainly associated to three fractions: ethyl acetate extract (23%), cell material (4%), and humic acids (29%). Only 1% of the initial radioactivity was recovered from the humic acids-free medium. Concerning mineralization, 27% of initially applied radioactivity was recovered in pooled soda lime pellets. Approximately 15% of the initial radioactivity was sorbed on the glass funnel material, glass wool and stoppers and corresponded to the volatilized NP. HPLC-LSC analyses showed the radioactivity in the ethyl acetate extract of the culture was almost assigned to original NP, less than 0.01% of the original 14C was recovered as hydroquinone and other hydrophilic compounds. The molecular size distribution of NP residues bound to humic acids was analyzed by HP-14C-GPC and is shown in

10

1

Fig. 1. The results indicate that the residues of NP were not homogenously distributed within the humic acids. The humic acids, to which the NP residues were bound, had a higher dominant molecular weight (6–15 kDa) than the total humic acids (2.5 kDa) and 80% of the NP residues was distributed between humic acids with molecular weight ranging from 300 Da to 40 kDa (value was calculated according to the area beneath the curve). The humic acids containing NP residues were analyzed by solid state 13C NMR (Fig. 2, HA + NP + Sphingomonas). In comparison to the equivalent amount of control humic acid, in both 13C CP MAS NMR and 13C DP MAS NMR spectra, humic acids containing 13C-NP residues had a relatively higher intensity (the area beneath the curve) in the range of 110–200 ppm (aromatic carbons, and carboxyl/carbonyl carbons) than in the range of 0–110 ppm (aliphatic carbons). An additional shoulder signal around 154 ppm in the curve, attributed to aromatic carbons (usually aromatic with –O(H) substitution, such as in resorcinol), occurred in the 13C DP MAS NMR spectrum of the humic acid fraction containing bound residues of NP, indicating that either NP itself or its metabolites was bound to the humic acids. 3.3. Abiotic formation of NP bound residues with humic acids

0

100

2177

0.1

Molecular weight (kDa)

Fig. 1. HP-14C-GPC chromatogram of NP residues bound to humic acids recovered from culture of Sphingomonas sp. strain TTNP3 incubated with 13 C and 14C labeled NP for 48 h; : radioactivity in counts per seconds; 3: UV signal at 360 nm; - - - -: Vis signal at 450 nm.

a

In order to further characterize the bound residues of NP, and to elucidate the origin of the peak at 154 ppm, a solution containing only degradation products of NP was prepared by incubating 13C- and 14C-labeled NP with resting cells of Sphingomonas sp. TTNP3. After one and half hours of incubation with the resting cells, 68% of the initial radioactivity was recovered in the supernatant of the culture. HPLC-LSC and GC-MS analyses of this supernatant showed that NP was completely degraded and that this supernatant contained

b 154 53

154

53 HA+Metabolites

HA+NP+Sphingomonas

HA Control 250

150

50

ppm

-50

250

150

50

-50

ppm

Fig. 2. 13C cross polarization magic angle spinning (CP MAS) NMR (a) and 13C direct polarization magic angle spinning (DP MAS) NMR (b) of humic acids recovered from culture of Sphingomonas sp. strain TTNP3 incubated with [1-13C]-labeled NP as substrate in the presence of humic acids (middle), incubation of resting cells supernatants with humic acids (top), and without substrate (control, bottom).

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hydroquinone and polar organic acids, amounting to 83 and 17% of the injected radioactivity, respectively. After abiotic incubation of this supernatant with humic acids, 9% of the total radioactivity was recovered from the ethyl acetate extract, 10% from the humic acids-free aqueous phase, and 42% from humic acids fraction. HPLC analysis showed 55% and 45% of the radioactivity in the ethyl acetate extract were recovered as hydroquinone and organic acids, respectively, while only organic acids were detected in the aqueous phase. HP-14C-GPC analysis showed that the distribution of bound residues of the NP metabolites within the humic acids was similar to that of NP incubated directly in the presence of humic acids as shown in Fig. 1. 13 C NMR spectra of the humic acids (Fig. 2, HA+Metabolite) showed the same tendency that relatively more 13C signals for aromatic carbon and carboxy/carbonyl carbons (110–200 ppm) in both 13C CP MAS NMR and 13C DP MAS NMR spectra occurred than the control humic acids. However, spectra of HA+Metabolite showed clearly signals at 154 ppm and 53 ppm (Fig. 2, HA+Metabolite), indicating the incorporation of intact aromatic ring of original NP and its ring cleavage products (aliphatic) into the humic acids. 4. Discussion The present study showed that in the presence of humic acids, the mineralization of NP by Sphingomonas sp. strain TTNP3 was doubled, accompanied by the formation of nonextractable residues of NP associated to the humic acids after a short incubation period. The fact that the radioactivity was only localized in the HA fraction in biotic assays clearly shows the dependency of the coupling or ‘‘associating’’ processes by microbial or enzymatic activity. Resting cell experiments with 13C-C1 labeled NP showed that the labeled carbon in the bound residues was present as aromatic state (at 154 ppm) and aliphatic state (at 53 ppm) as identified by solid-state 13C NMR. To the best of our knowledge, this study provided for the first time evidences for the mediatory role of humic acids in the fate of NP as a sink for bacterial degradation intermediates of this compound. The stimulated mineralization of NP in the presence of humic acids was probably related to the higher concentration of NP remaining in the medium through its association with humic acids. In fact, the net effect seen in mineralization might reflect the fact that NP was not volatilized before being degraded, because it was associated with humic acids due to hydrophobic interaction (Vinken et al., 2004) and slowly released in the time-course of biodegradation. Even though it has been assumed that the association of NP with organic matter can reduce the degradation of NP in sludge (Tanghe et al., 1998, 1999), positive effect of humic acids on the biodegradation of xenobiotics has also been documented e.g., for phenantrene byPseudomonas fluorescens (Ortega-Calvo and Saiz-Jime-

nez, 1998). The observation that humic acids had no effect on the degradation of NP was only based on the elimination of NP, but not on the mineralization (Vinken et al., 2004). In addition, the experimental setup used for this previous study did not include the volatility of NP, which was strongly affected by humic acids as shown in the present study (Table 1). The large amounts of NP found in the CO2 traps (Table 1) were in agreement with the high volatility of NP, reported previously by other studies (Dachs et al., 1999; Xie et al., 2004; Telscher et al., 2005). This fact stresses out the importance of making the difference between real mineralization and volatilization during NP degradation studies. The non-homogenous distribution of NP residues within humic acids (Fig. 1) suggested that NP residues were preferentially associated to some active sites or functional groups of the humic acids. The preferential binding to humic substances has also been proposed for the oxidized metabolites of 1,1,1-trichloro-2-2-bis(p-chlorophenyl)ethane (DDT) such as bis(p-chlorophenyl)acetate (DDA), and was likely attributed to enhanced interactions of this metabolite with the organic material in a bound state (Schwarzbauer et al., 2003). Although no chemical degradation method was applied, the nonextractability of NP residues relied probably on covalent bonding, because the samples were treated with ethyl acetate prior to GPC analysis. The extraction of NP from humic acids solution is almost complete with this method and humic acids-NP associate are fully reversible (Vinken et al., 2004). The covalent binding of NP residues is also supported by the 13 C NMR analysis of the residues (Fig. 2). The new signal at 154 ppm in the spectra of the humic acids containing NP bound residues was close to the chemical shift of the labeled C1 of NP (152.6 ppm) (Vinken et al., 2002), indicating the incorporation of the whole aromatic ring of NP metabolites into the humic acids. Because hydroquinone was the sole detectable aromatic compounds after incubation with resting cells, it can therefore be assumed that the bound residues originated from hydroquinone. Hydroquinone is a very reactive compound and tends to form free radicals at the hydroxyls (C1 and C4), which are amenable substrates for binding to humic substances (McCarthy et al., 1997). Assuming that the hydroquinone was bound to humic acids via ester or ether bondings, calculation of the chemical shift of the C1 using software ChemOffice 2004 would give 155 ppm (C atom with free hydroxyl) and 144 ppm (C atom involved in the ester bond) in the case of ester bonding, and 150 ppm for both C atoms in the case of ether bonding. The further examination of the peak at 154 ppm in the 13C-DPMAS-NMR spectrum showed that it does not belong to a single signal but to a group of peaks. Due to the degradation pathways via hydroquinone, metabolites of 13C-C1 labeled NP such as succinate and 3,4-dihydroxy butanoic acid should be labeled at their C1 or C4 atoms. The signal at 173 ppm in the NMR spectra

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(Fig. 2) is typical for carboxyl carbon and could be attributed to the ester binding of these metabolites to the humic acids via their carboxyl groups (C1). Signals at 50–100 ppm of 13C NMR spectrum can be assigned to N- or O-alkyl carbons (Haider et al., 2000). The occurrence of signal at 53 ppm (Fig. 2) suggests the cleavage of aromatic ring of NP followed by incorporation of the cleaved products into the humic acids. The chemical structure of the products attributed to this signal at 53 ppm is unclear. Labeled O alkyl carbons cannot be formed from succinate but only from 3,4-dihydroxy butanoic acid. Nevertheless, C4 of 3,4-dihydroxy butanoic acid would give rise to signal of about 77 ppm, if it is involved in an ether bond or ester bond with another carboxylic acid of humic acids. Thus, it may be reasonable here to assume that this signal can also result from the binding of other non identified small polar degradation products of NP. The formation of hydroquinone was the key step for the immobilization of NP residues on humic acids, this was supported not only by the 13C NMR analyses (Fig. 2) but also by the drastic decrease of radioactivity in the aqueous phase after the removal of humic acids (Table 2). Furthermore, no low-molecular weight polymers of hydroquinone could be detected under the applied conditions. Similar role of microbial oxidations in the covalent binding of metabolites to humic substances has also been found for phenantrene and anthracene (Ka¨stner et al., 1999; Ka¨cker et al., 2002). Hydroquinone derivates are reactive and can be easily transformed into semiquinones and benzoquinones, which lead to the production of superoxides reacting with biological material (McCarthy et al., 1997; Launen et al., 2000). Except for a report describing the binding of hydroquinone on Suwannee humic acids, the formation of hydroquinone bound residues to natural humic acids resulting from the degradation of xenobiotic has never been described, although dihydroxylated aromatic ring compounds such as catechol and resorcinol can bind covalently to humic acids and are precursors of natural and synthetic humic material (Kappler and Haderlein, 2003; Vinodgopal et al., 2004; Perminova et al., 2005; Vinken et al., 2005). The formation of bound residues to humic acids may reduce the toxicity of hydroquinone as long as the latter remains immobilized. Humic substances are ubiquitous in the environment; the degradation of NP and its metabolites by Sphingomonas sp. strain TTNP3 in the presence of humic acids suggests an important role of humic acids in the degradation and detoxification of organic pollutants with phenolic structure.

Acknowledgements We thank Prof. Willy Verstraete and Dr. Nico Boon (LabMet, University Ghent, Belgium) for providing Sphingomonas sp. strain TTNP3. We acknowledge the European Commission for funding AQUAbase under the Human Resources and Mobility Activity within the 6th

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