Environ. Sci. Technol. 2005, 39, 7896-7900
Occurrence of a Nitro Metabolite of a Defined Nonylphenol Isomer in Soil/Sewage Sludge Mixtures M A R K U S J . H . T E L S C H E R , * ,† ULRIKE SCHULLER,† BURKHARD SCHMIDT,† AND A N D R E A S S C H A¨ F F E R † , ‡ RWTH Aachen University, Institute for Environmental Research (Biology V), Worringerweg 1, 52056 Aachen, Germany and Fraunhofer Institute of Molecular Biology and Applied Ecology, 57392 Schmallenberg-Grafschaft, Germany
Uniformly [14C]-ring-labeled 4-(3,5-dimethyl-3-heptyl)phenol (353-nonylphenol) is a highly relevant isomer of the technical nonylphenol mixture. We studied the sorption, desorption, and degradation of the synthesized isomer in an agricultural sandy loam at various soil/sewage sludge ratios. Sorption of 353-nonylphenol was high and differed with the amount of suspended soil in water. log Koc values, which are used to assess the risk of nonylphenol, ranged from 3.80 to 5.75. Desorption was slow and low and resulted in constant concentrations of about 15 ng/L 353nonylphenol in water after several desorption steps. In degradation studies up to 6% of the applied 353-nonylphenol in soil was volatilized; we consider this an important source of nonylphenol in the environment. With increasing amounts of sewage sludge in the soil/sewage sludge mixtures, 353-nonylphenol was stabilized, probably because of the lack of oxygen in sludge aggregates even under oxic conditions in flow-through systems. Unexpectedly, a lesspolar metabolite was detected in amounts up to 40% of the applied nonylphenol after 135 days of incubation. This novel metabolite was identified as 4-(3,5-dimethyl-3-heptyl)2-nitrophenol. This product formation might indicate the existence of novel metabolic pathways of nonylphenol in the environment.
Introduction Nonylphenol is an important industrial chemical with an estimated worldwide market of 340 000 Mg in 2002 (Hager, C. D. SASOL, Marl, Germany, personal communication). Nonylphenol is mostly used for the production of alkylphenol ethoxylates and resins, which have a wide range of applications, and fibers, pesticides, and paints. Nonylphenol is therefore ubiquitous in the environment, even though it is reported to be degraded rapidly in soil (1). For example, Gu ¨nther et al. (2) detected nonylphenol in nearly every food sample analyzed, and a daily intake of 7.5 µg for a German adult was calculated. Nonylphenol has also been detected in the atmosphere (3) and in groundwaters close to agricultural and industrial areas (4). * Corresponding author phone: +49-(0)241-8026686; fax: +49(0)241-8022182; e-mail:
[email protected]. † RWTH Aachen University. ‡ Fraunhofer Institute of Molecular Biology and Applied Ecology. 7896
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Nonylphenol is enriched to 8-4000 µg/g dry wt in sewage sludge during wastewater treatment processes (5-9). Nonylphenol emerges mainly as a degradation product of nonylphenol ethoxylates (10). In the United States, France, Germany, and Denmark sewage sludge is used as an organic fertilizer. Therefore, the persistence of nonylphenol in the environment is of public interest. Nonylphenol in soil is rapidly mineralized with half-lives of 2-10 days (1,11). Various authors have studied the degradation of nonylphenol in various soil/sewage sludge mixtures (12-15). For example, Gejlsbjerg et al. (12) found that 50% of the nonylphenol at different soil/sewage sludge ratios was mineralized in 7.010.6 days. In contrast, Hesselsoe et al. (14) detected nnonylphenol in anaerobic sludge cores even after 3 months of incubation. A number of studies have used technical nonylphenol, a mixture of at least 21 isomers of p-nonylphenol (16-18), whereas only a few have used defined nonylphenol isomers of the technical mixtures (19, 20). Here we studied the degradation of the most relevant isomer of nonylphenol, 4-(3,5-dimethyl-3-heptyl)phenol (353-nonylphenol), which comprises about 20% of two technical mixtures (18). We synthesized uniformly [14C]-ring-labeled ([U-ring-14C]-labeled) 353-nonylphenol and investigated its sorption and desorption in dialysis chambers. Degradation of 353-nonylphenol was studied in a flow-through system using a sandy loam soil mixed with various amounts of sewage sludge.
Experimental Section Materials. 353-Nonylphenol was synthesized via a FriedelCrafts alkylation as described by Vinken et al. (21) using [U-ring-14C]-labeled phenol and 3,5-dimethyl-3-heptanol. The yield was 60% with a chemical purity >97% (gas chromatography-electron impact mass spectrometry; GCEIMS), a radiochemical purity >97% (high-pressure liquid chromatography; HPLC), and a specific radioactivity of 224.22 MBq/mmol. The structure of the compound was verified using nuclear magnetic resonance spectroscopy (NMR). The chemical was used in the experiments without further purification. The soil used in the experiments was an agricultural sandy loam from Bruchko¨bel (Germany). The soil consisted of 61.7% sand, 33.2% silt, and 5.1% clay with a pH (KCl) of 5.50 and a Corg of 0.60%. The soil was sieved to 2 mm, air-dried, and adjusted to 60% maximum water-holding capacity. Sewage sludge processed for use as an organic fertilizer was obtained from the public wastewater treatment plant of Aachen (Germany). The sludge was processed by digestion for 28 days under anoxic conditions, followed by removal of water in a thickener and in a centrifuge. The resulting dry matter content was about 30%. The sewage sludge was used in the degradation experiments without further treatment. Sorption and Desorption Studies. Sorption and desorption experiments were carried out in small-scale dialysis chambers, which have been described in detail (22). Two polytetrafluorethene (Teflon) half-cells, each with a volume of approximately 9 mL, were separated by a dialysis membrane with a molecular weight cutoff of 1000 (Roth, Germany). One half-cell was filled with 8 mL of soil suspension in 0.01 M CaCl2 in purified water (Milli-Q, Millipore GmbH, Schwalbach, Germany), and the other half-cell was filled with 8 mL of 0.01 M CaCl2 in purified water containing [U-ring-14C]labeled 353-nonylphenol. To inhibit microbial activity NaN3 (0.2%) was added to the solutions. Diffusion of solutes was supported by rotating the closed cells (360°) at 10-12 rpm. 10.1021/es050551v CCC: $30.25
2005 American Chemical Society Published on Web 09/07/2005
Samples (500 µL) were taken from the water half-cell after the equilibrium of diffusion was reached within 72 h. After sorption reached equilibrium, desorption was studied by replacing the contaminated water (0.01 M CaCl2) of the water half-cell with uncontaminated purified water (0.01 M CaCl2). The water (0.01 M CaCl2) was replaced15 times. The amount of [U-ring-14C]-labeled nonylphenol in each half-cell was determined by liquid scintillation counting (LSC). Lack of degradation of 353-nonylphenol was verified by thin-layer chromatography (n-hexane:diethyl ether:acetic acid; 50:50:1; by vol). Each experiment was carried out in duplicate. Degradation Studies. Degradation of [U-ring-14C]-labeled 353-nonylphenol was studied in a controlled flow-through system at 20 °C. Water-saturated air was pumped through the chambers. Each assay contained 10 g (dry wt) of soil or soil/sewage sludge mixtures; the composition of soil/sewage sludge mixtures calculated in percent was related to mass. The soil/sewage sludge mixtures were agitated with a glass bar; the average soil/sewage sludge aggregates after agitation were 5 mm in diameter. 353-Nonylphenol was added to a final concentration of 1.53 mg/kg (dry wt of the solids). Volatile compounds were captured in monoethylene glycol and CO2 in 2 M NaOH. Degradation assays were carried out for 135 days. At the end of the incubation period the complete assays were transferred into glass centrifuge tubes (VWR International, Darmstadt, Germany) and extracted sequentially with 20 mL (each) of n-hexane, methanol (twice), methanol/H2O (9:1, v/v), and acetone. For each extraction the samples were shaken for 30 min and centrifuged (25 min, 4,000g). Aliquots (500 µL) of the supernatant were analyzed using LSC. The n-hexane extract was dried over Na2SO4 and then evaporated to dryness. The residue was dissolved in HPLC-grade methanol prior to HPLC and GCEIMS analyses. The methanol, methanol/H2O, and acetone extracts were concentrated by evaporation before analysis. Analytical Analyses. An LS 5000 Td analyzer (Beckman, Germany) with automatic quench correction (external standard) and Lumasafe Plus (for monoethylene glycol samples), Hionic (for 2M NaOH samples), and Carbamax Plus (Canberra-Packard; for combusted samples) scintillation cocktails were used for LSC. The Hewlett-Packard 1100 system (Agilent, Waldbronn, Germany) used for HPLC analyses consisted of a programmable solvent module, a diode array detector module, and a radioisotope detector (Raytest; Straubenhardt, Germany) with a 3139 quartz cell (glass, 32-45 µm; i.d. ) 4 mm; vol. ) 0.20 mL). A reversed-phase column (CC250/4.6 Nucleosil 100-5 C18 HD; CS-Chromatographie Service, Langerwehe, Germany) was used at 35 °C with a flow rate of 1 mL min-1 and solvent A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile) with the following elution profile: A/B (65/35, v/v) for 5 min, then a linear gradient to 100% B in 30 min, isocratic 100% B for 5 min, and a linear gradient to A/B (65/35, v/v) in 5 min. Analyses were terminated by equilibrating the column with A/B (65/35 v/v) for 5 min. Nonlabeled 353-nonylphenol was detected at 280 nm. Samples were concentrated under a stream of N2 prior to GC-EIMS analysis on a Hewlett-Packard 5890 Series II gas chromatograph (Agilent, Waldbronn, Germany) equipped with an FS-SE-54-NB-0.5 column (25 m × 0.25 mm; film thickness ) 0.46 µm; CS Chromatographie Service, Langerwehe, Germany). Helium 5.0 was the carrier gas; the injection volume was 1 µL. The gas chromatograph was connected to a Hewlett-Packard 5971 A MSD mass-selective detector, operated in the scan mode (mass range m/z 50-480) with an electron energy of 70 eV and the following temperature program: isothermal at 85 °C for 5 min, 85-280 °C at 10 °C min-1, and isothermal at 280 °C for 3 min. The injector and interface temperatures were 250 and 280 °C, respectively.
FIGURE 1. Sorption of 353-nonylphenol to soil (sorption coefficients Kd and log Koc) with various amounts of soil suspended in water.
Results and Discussion Sorption of 353-Nonylphenol to Sandy Loam Soil. The binding of 353-nonylphenol to sandy loam soil was studied in dialysis chambers. Aqueous solutions of 353-nonylphenol (c0 ) 726 ng/L) were dialyzed against aqueous suspensions of soil ranging between [ads] ) 7.8 and 187.5 g/L. Dialysis continued until the concentration of 353-nonylphenol in the two half-cells was constant, which usually occurred within 48 h. Sorption of 353-nonylphenol to soil was expressed as the distribution coefficient Kd, which was calculated according to the following equation
Kd )
cads csusp - cw 1 ) ‚ cw cw [ads]
where cads ) concentration of adsorbed 353-nonylphenol (ng/ kg adsorbent), cw ) concentration of 353-nonylphenol in solution (ng/ L), csusp ) total concentration of 353-nonylphenol in the halfcell containing adsorbent (ng/L), and [ads] ) total concentration of the adsorbent (kg/L). Kd values were normalized to the organic carbon content of the soil to obtain the log Koc values. Sorption of 353-nonylphenol to sandy loam soil was significantly correlated with the amount of soil suspended in water (Figure 1). Kd values ranged from 36.1 ( 0.0 to 462.0 ( 4.4 L/kg. After normalization to the organic carbon content, the range was even larger, i.e., log Koc ranged from 3.80 ( 0.04 to 5.75 ( 0.05. Such a result is generally expected for a surface-active chemical such as 353-nonylphenol. The range of sorption coefficients for nonylphenol reported by different authors was as a whole consistent with the values we obtained using different amounts of suspended soil in water. Kollmann et al. (23) determined a Kd value of 45.3 L/kg for four n-nonylphenol (silt loam) using a batch equilibrium method. Du ¨ ring et al. (24) obtained Kd values ranging from 8.5 to 321.8 L/kg for 50 agricultural soils. Ho ¨ llriglRosta et al. (22) reported a Koc value of 9.140. Yamamoto et al. (25) found log Kdoc values of 4.96 for Suwannee River humic acids and 4.70 for Suwannee River fulvic acids. The previous and present results suggest that the concentration of VOL. 39, NO. 20, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Distribution of Radioactivity (% of applied radioactivity) after 135 Days of Incubation of [U-ring-14C]-Labeled 353-Nonylphenol in Soil, Soil Mixed with Different Amounts of Sewage Sludge, and Sewage Sludge soil/sewage sludge ratio fraction
100%:0%
99%:1%
90%:10%
50%:50%
0%:100%
mineralized volatile extractable nonextractable recovered radioactivity
21.8% ( 0.1 6.2% ( 0.1 29.5% ( 0.2 42.0% ( 2.4 99.5% ( 7.2
29.5% ( 0.0 4.8% ( 0.1 28.7% ( 0.3 48.7% ( 3.8 111.6% ( 6.2
23.0% ( 0.1 4.8% ( 0.2 52.3% ( 2.9 29.6% ( 1.7 109.6% ( 8.6
20.3% ( 0.0 1.8% ( 0.0 51.6% ( 1.2 27.5% ( 1.0 96.7% ( 1.2
20.8% ( 0.0 1.7% ( 0.0 49.6% ( 0.7 25.8% ( 1.0 101.2% ( 1.3
adsorbent for surface-active compounds such as nonylphenol is an important parameter that should be taken into account when sorption coefficients are used for risk assessment. We determined the sorption of [U-ring-14C]-labeled 353nonylphenol to the wall of the Teflon chamber and to the dialysis membrane. The chamber wall was extracted with ethyl acetate, and the extract was analyzed by LSC. The amount of [U-ring-14C]-labeled 353-nonylphenol sorbed to the membrane was determined by combustion of the membrane (sample oxidizer). The amount of [U-ring-14C]labeled 353-nonylphenol sorbed to soil was measured by extracting the soil with acetone and analyzing the nonextractable fractions by combustion. Most of the applied nonylphenol was sorbed to soil, and only
