Aerobic Biodegradation Behavior of Nonylphenol Polyethoxylates and

Stock solutions of the standard mixtures were prepared by dis solving a known .... Numerical characters (1−11) at the rear indicate “n” of the N...
0 downloads 0 Views 420KB Size
Download PDF
Environ. Sci. Technol. 2005, 39, 5626-5633

Aerobic Biodegradation Behavior of Nonylphenol Polyethoxylates and Their Metabolites in the Presence of Organic Matter S H I N Y A H A Y A S H I , † S H I G E O S A I T O , * ,‡ JU-HYUN KIM,‡ OSAMU NISHIMURA,† AND RYUICHI SUDO‡ Graduate School of Engineering, Tohoku University, Aoba 06, Sendai 980-8579, Japan, and Center for Environmental Science in Saitama, Kamitanadare 914, Kisaimachi, Saitama 347-0115, Japan

In this paper, the aerobic biodegradation behavior of nonylphenol polyethoxylates (NPnEOs) with ethoxy (EO) units of specific lengths, which were fractionated using highperformance liquid chromatography (HPLC) with photodiode array detection, was studied in the presence of different types of organic materials. NPnEOs and their related metabolites under a modified OECD 301E biodegradation test were monitored using liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS). Biodegradation tests in the presence of organic matters, such as methanol, glucose, and yeast extract, showed the formation of the corresponding nonylphenol polyethoxy carboxylates by the oxidation of the terminal alcoholic group. However, aerobic biodegradation tests without organic matter revealed that NP2EO and NP3EO were predominant metabolites of the long-chainoligomer precursor system which undergo fast and complete shortening. Degradation rates were higher for the longchain oligomers than for shorter ones. The degradation pathway of NPnEOs was greatly influenced by the presence or absence of organic matter. Organic materials such as those given above apparently play a significant role in the formation of the carboxylated metabolites of NPnEOs.

Introduction Nonylphenol polyethoxylates (NPnEOs, where n indicates the number of ethoxy units) are very effective nonionic surfactants, and are widely used in various industries and agriculture. In Japan, their domestic consumption reached approximately 5000 tons in 2000 for use in various products such as detergents, pesticides, and cleaning, textile, and metal working fluids (1). Although the concentration of the parent NPnEOs is reduced through biological degradation, various metabolites are formed and released into the aquatic environment via sewage treatment plants (2-7). Many investigations have clarified that NPnEOs and their various metabolic residues, such as short-chain polyethoxylates, nonylphenol polyethoxy carboxylates (NPmECs, where m indicates the number of ethoxy units plus one terminal * Corresponding author phone: +81-480-73-8369; +81-480-70-2031; e-mail: [email protected]. † Tohoku University. ‡ Center for Environmental Science in Saitama. 5626

9

fax:

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 15, 2005

CH2COOH unit), and nonylphenol (NP), are detected in river water and sediments (8-10). Short-chain polyethoxylates among regenerated metabolites are more toxic to aquatic organisms than their intact precursors (11). In addition, NP, nonylphenol diethoxylate (NP2EO), and nonylphenoxyacetic acid (NP1EC) are suspected to have weak endocrine activities (12). Consequently, concerns regarding NPnEOs and their degradation products in view of risk assessments for natural ecosystems have recently increased. Although acid metabolites such as NPmEC are not as toxic as short-chain polyethoxylates to aquatic biota, another problem associated with trihalomethane formation potential arose from the standpoint of drinking water supply because of chloridizing for water intake from rivers polluted by acid metabolites (13). Thus, knowledge of the biodegradation behavior and transformation route of NPnEOs is necessary for an accurate analysis of the environmental fate of NPnEOs in view of water environment preservation including water utilization. Thus far, many aerobic biodegradation studies have been carried out using natural river water or wastewater as sample and an NPnEO mixture as substrate (14-16). Consequently, it has been difficult to completely monitor the fate of each NPnEO precursor in detail. Aerobic biodegradation tests using a pure polyoxyethylene lauryl ether (C12EO8) greatly contributed to the clarification of the degradation route (17). Recently, the separation and quantitative determination of NPnEOs have also become possible using high-performance liquid chromatography (HPLC), gas chromatography/ mass spectrometry (GC/MS), and liquid chromatography/ mass spectrometry (LC/MS). In particular, LC/MS and LC-MS/MS analyses revealed new types of metabolites, such as NPmECs and metabolites with both chains oxidized (CNPECs) (4, 18, 19). Studies on the pathway of the aerobic biodegradation of NPnEOs showed that it initially proceeds by the shortening of the EO chain, followed by the oxidation of the shortened NPnEOs to their corresponding NPmECs (4, 14, 16, 20). More recently, Jonkers et al., however, have proposed another mechanism (18), which in brief proceeds as follows: fast ω-oxidation of parent NPnEO to the corresponding NPmEC, a slow shortening of the EO chain of the NPmEC produced, and a slow oxidation of the terminal carbon of the branched nonyl chain to form CNPECs. In addition, they assume that NP2EO found in the environment is derived from aerobic degradation processes. However, the main parameters determining the degradation route of NPnEOs are still largely unknown (21). Among such parameters, water temperature and dissolved oxygen (DO) particularly play a crucial role in the degradation. The former controls the degradation rate of a precursor (8) and the latter NP production under nearly zero conditions. To date, there are only several reports on the quantitative determination of NPmECs and CNPECs in comparison with NPnEOs in real environmental water (22). Nonetheless, various related metabolites of NPnEOs have been found in effluents from sewage treatment plants, compared with those found by standardized biodegradation tests (2-7). Recent surveys conducted in Japan on aquatic environments showed the occurrence of long-chain carboxylates (∼10) in relatively polluted river water, suggesting the involvement of BOD constituents (23). These studies phenomenally presented that BOD constituents may have something to do with determining the degradation route of NPnEOs. The objective of this work is to qualitatively investigate the effect of organic matter on the biodegradation route of NPnEOs and their metabolites under an aerobic condition. To achieve this, we perform biodegradation tests by using 10.1021/es048857+ CCC: $30.25

 2005 American Chemical Society Published on Web 06/22/2005

FIGURE 1. Normal-phase chromatogram of an NPnEO mixture being fractionated. The arrow pointing to both sides denotes the collected fraction. pure individual NPnEO oligomers and a filtrate from the secondary effluent of the sewage work as inoculum in the presence or absence of three organic materials according to the modified OECD 301E protocol. In addition, the difference in biotransformation characteristics among NP3EO, NP5EO, and NP7EO is also evaluated.

Experimental Section Reagents and Chemicals. NPnEO surfactant mixtures with average oligomer lengths of 2, 5, 7.5, and 10 EO units, commercially referred to as polyethylene glycol mono-4nonylphenyl ether (n ≈ 2, 5, 7.5, and 10, respectively) were purchased from Tokyo Chemical Industries (Tokyo, Japan). The commercial mixture of NPmECs from m ) 1 to m ) 10 was purchased from Hayashi Pure Chemical Industries (Osaka, Japan). A calibrated mixture of NPnEOs (n ) 1-20) was kindly supplied by the National Institute of Public Health of Japan. A combination of UV and LC/MS methods was used to determine the concentration of each oligomer in the mixture. The UV method is based on the assumption that the sensitivity of each oligomer with n > 2 is constant. Stock solutions of the standard mixtures were prepared by dissolving a known amount of each standard in acetonitrile. Working standard solutions were obtained by further diluting the stock solutions with acetonitrile. For solid-phase extraction and LC analysis, distilled water was further purified using a MilliQ system (Nihon Millipore, Tokyo, Japan). For use as internal standard, fluoranthened10 was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Acetonitrile, methanol, n-hexane, and 2-propanol were of HPLC grade (Wako Pure Chemical Industries, Tokyo, Japan). All other reagents used were of analytical grade. Oligomer Fractionation. Individual NPnEOs with 210 EO units were obtained using normal-phase HPLC by modifying the previously reported procedure (3). The HPLC system consists of an L-7455 photodiode array detector, an L-7200 autosampler, an L-7100 gradient pump, and an L-7300 column oven (Hitachi Ltd., Tokyo, Japan). Normal-phase chromatographic separation was performed using a column packed with Lichrosorb NH2 (244 mm × 4 mm i.d., 7 µm particle size, Kanto Chemical Co. Inc., Tokyo, Japan) at 40 °C. Elution was carried out in the gradient mode at a flow rate of 1.5 mL/min using n-hexane/2-propanol (90:10, v/v) (A) and 2-propanol/water (90:10, v/v) (B). The initial con-

centration (99%) of A in the mobile phase was linearly decreased to 23% in 40 min. The NPnEO mixtures for HPLC were prepared by dissolving each type of polyethylene glycol mono-4-nonylphenyl ether in an n-hexane and 2-propanol (90:10, v/v) mixture. Each fraction was obtained using an SF-2120 fraction collector (Advantec, Tokyo, Japan) as shown in Figure 1. Each fraction containing a purified oligomer was taken for the period shown by the right and left arrow in the chromatogram and then evaporated to dryness in a rotary evaporator, and the residue was redissolved in methanol to prepare a stock solution. The purities of the fractionated polyethoxylates NP2EO, NP3EO, NP5EO, NP7EO, and NP10EO were 62, 92, 98, 95, and 95 wt %, respectively. A rather low purity of NP2EO can be attributable to contaminations caused by a distortion from the original state as the run goes on. Biodegradation Tests. Aerobic biodegradation tests were carried out almost according to a modified OECD 301E biodegradation test protocol (21). The tests were conducted under a continuous-stirring condition in the dark at 23 ( 2 °C. Pure NPnEO oligomers fractionated as described above were used as target molecules. In the tests without organic matter, after the NP3EO, NP5EO, NP7EO, or NP10EO oligomer solution in 2-3 mL of methanol was poured into a 3 L beaker, the solution was allowed to stand at room temperature to completely evaporate methanol. A 2 L sample of distilled water was added to the beaker, and the mixture was subjected to ultrasonication. Methanol, glucose, and yeast extract, which is a commercial product, as organic source were fortified at a concentration of about 88 mg/L in the tests in the presence of organic matter, and NP2EO, NP3EO, NP5EO, and NP10EO oligomers, which include methanol as solvent, were evaporated to dryness as described above. These three organic matters were chosen as commonplace ones because of confirming how they work on the biodegradation. Then 200 mL of distilled water was added, and the residues were completely dissolved in water by ultrasonication and stirring. The secondary effluent collected at Arakawa STP in the area of Saitama Prefecture was passed through a membrane filter of 5 µm pore size, and the filtrate was used as inoculum. Inorganic salts and organic nutrients were also added according to the OECD protocol. The nominal concentrations of NPnEO in both test systems with and without organic matter were 5000 and 7500 nM, VOL. 39, NO. 15, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5627

FIGURE 2. Time course of NP10EO biodegradation in the presence of methanol. Numerical characters (2-11) at the rear indicate “n” of NPnEO, while (1-10) of the front correspond to “m” of NPmEC. Abbreviations and definitions are as given in the text. respectively. A 5 mL portion of the former and 40 mL of the latter were withdrawn as samples at suitable time intervals. The latter required a much higher volume due to NP1EO measurement as well as NPnEO (n ) 2-10) and NPmEC (m ) 1-10). Dissolved organic carbon (DOC) was determined using the total organic carbon analyzer TOC-5000 (Shimadzu Corp., Kyoto, Japan). The concentrations of methanol, glucose, and yeast extract fortified were about 88 mg/L in DOC. The control system without organic matter was also the same as the spiked one. Although the control system contained a small amount of yeast extract as vitamins in the medium according to the 301E OECD protocol, it should be considered as an organicmatter-free system because of the very low concentration of 0.09 mg/L in DOC. DO concentration was also monitored with a DO meter (YSI Corporate, Ohio) to confirm whether an aerobic condition was maintained. It ranged from 6.8 to 8.3 mg/L during the experiment. A fluorescent pigment of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution was in use for the detection of microorganisms using a fluorescent microscope (24). Sample Preparation. Solid-phase extraction was conducted as previously reported (25). Briefly, the sample was passed through a glass microfiber filter graded as GF/C (1.2 µm pore) using an all-glass filtration apparatus and then acidified to approximately pH 2 with 1 M HNO3 before extraction. The cartridges Sep-Pak tC18 (Waters, Milford, MA) were preconditioned with 10 mL of methanol followed by 10 mL of distilled water. After the passage of the sample through the cartridge at 15 mL/min and the separation of the aqueous phase by centrifugation for drying, the organics were eluted by passing 10 mL of methanol followed by 10 mL of dichloromethane. The pooled 20 mL of extract was split into aliquots of 0.5 mL for LC/MS and 19.5 mL for GC/MS, which were both evaporated to dryness in a water bath at 48 °C under a gentle stream of nitrogen. The former was redissolved in 1 mL of a mixture of 0.1% acetic acid and acetonitrile (50:50, v/v) for LC/MS. The latter was substituted for 0.5 mL of ethyl acetate containing an internal standard for GC/MS. 5628

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 15, 2005

LC/MS. The HPLC system consisted of a Waters 2690 separation module (Waters, Milford, MA). Chromatographic separation was performed using a reversed-phase analytical column (Mspak GF-310 4D) of 150 × 4.6 mm from Shodex (Tokyo, Japan). NPnEOs (n ) 2-11) were separated using the mobile phase acetonitrile (A) and 10 mM ammonium acetate (B). The elution gradient started with 80% B, linearly decreased to 10% B in 14 min, and was kept isocratic for 6 min. The column temperature and flow rate were kept constant at 50 °C and 0.4 mL/min, respectively. NPmECs (m ) 1-10) were separated using a mixture of acetonitrile (A) and 0.1% acetic acid (B) as mobile phase. The following solvent program was used: an initial B concentration of 60%, linearly decreased to 25% B in 6 min, linearly decreased to 0% B in 14 min, and kept isocratic for 3 min. The solvent flow rate and column temperature were kept constant at 0.35 mL/min and 45 °C, respectively. A single-quadrupole Waters Micromass ZMD mass spectrometer, equipped with an electrospray (ESI) ion source was used. Data acquisition, data processing, and instrument control were performed using Microsoft Windows NT (v4.0)-based Masslynx software (Micromass). The following operation parameters were used: source temperature, 130°C; desolvation temperature, 320°C; desolvation gas flow rate, 355 L/h; cone gas flow rate, 30 L/h; ESI capillary voltage, 2.8 kV. Quantitative analysis was accomplished using the selected ion monitoring (SIM) mode by external calibration. NPnEOs were identified by confirming the characteristic pattern showing the [M + NH4]+ ion in the positive mode, while NPmECs were identified by their characteristic pattern showing [M - H]- ions in the negative mode with m/z values from 277.4 to 673.9, increasing by 44.05. Calibration curves were generated using linear regression analysis within the concentration range investigated (0.1-4 µg/mL) and gave good fits, that is, r2 ) 0.99370.9991 for NPmECs (m ) 1-10) and r2 ) 0.9974-0.9999 for NPnEOs (n ) 2-11). The molar concentrations of each of the NPnEO and NPmEC oligomers were determined by multiplying the calculated concentration by the molecular weight of the corresponding oligomer. GC/MS. GC/MS for only NP1EO was carried out on a Thermo Quest TRACE MS & TRACE GC 2000 system (Thermo

FIGURE 3. Time course of NP10EO biodegradation in the presence of glucose. Numerical characters are the same as in Figure 2. Quest, Tokyo, Japan), due to the low response factor of NP1EO compared with the other oligomers. The gas chromatograph was equipped with an AS2000 autosampler and a 30 m (0.32 mm i.d.) fused silica capillary column coated with PTE-5 (SUPELCO, Bellefonte, PA), which was connected directly to the mass spectrometer. The injection volume of the extract was 2 µL using splitless injection. The temperature program used was 70 °C isothermal for 1 min and then increased to 300 °C at a rate of 10 °C/min. The carrier gas (helium) flow rate was held constant at 1 mL/m. Ions at m/z 179, 193, and 212 were monitored for the quantitative determination of NP1EO.

NP10EC formed remains unclear. Nearly complete degradation of the added organic matters was observed. Biodegradation tests in the presence of methanol using NP2EO, NP3EO, and NP5EO as precursors were performed simultaneously (data not shown). These precursors disappeared about 4 days faster than NP10EO, especially in NP10EO, with NP10EC forming 85% of the original concentration (molar basis) on the 15th day, and then their concentrations gradually decreased. Only in the case of NP3EO did the parent material decrease at a very slow rate of about 60 nM/day. Consequently, this slow transformation process caused a delay in the formation of NP3EC.

Results and Discussion

Glucose and yeast extract were chosen to investigate whether organic matters other than methanol involve the oxidative process because they are an easily decomposable carbohydrate and a frequently used culture medium. Figures 3 and 4 show the test systems using glucose and yeast extract, presenting microbial attacks similar to those in methanol, with a slight amount of NP9EC being formed.

Figure 1 shows the normal-phase chromatogram of mixtures containing NPnEO with average oligomer lengths of n ) 2, 5, 7.5, and 10 EO units with an injection volume of 50 µL. As shown in the chromatogram, individual NPnEOs with various polyethoxylated components (n ) 1-15) could be completely fractionated and used as a precursor material. Test System in the Presence of Organic Matters. At first, the biodegradation processes of NP10EO in the presence of methanol as organic matter were executed as shown in Figure 2. Only the terminal alcoholic group of an NP10EO-spiked system was oxidized to the corresponding NP10EC without shortening of the EO chain apparently. This phenomenon was observed in all the test systems used, suggesting that oxidation is independent of EO chain length. The amount of NP10EC formed slowly decreased subsequently without selfshortening. Probably the transformation to CNPECs, which is extremely persistent, has occurred (4). Unfortunately, due to the short sampling period of 24 days, the final fate of the

The half-lives of the organic matters added were less than 5 days. In particular, the amount of yeast extract decreased more rapidly than that of the other two organic matters, indicating a preferable carbon source. The concentrations of DOC on the 11th day from the test start were almost less than 10 mg/L. The total number of bacteria was chased only in the methanol-spiked test systems, with 3.4 × 107/mL at the beginning and 3.0 × 108/mL on the seventh day. Between the 7th and 14th days, a slight bacterial growth was observed, and the total bacterial population was 3.5 × 108/mL on the 14th day. The above results show that NPmECs may be VOL. 39, NO. 15, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5629

FIGURE 4. Time course of NP10EO biodegradation in the presence of yeast extract. Numerical characters are the same as in Figure 2.

FIGURE 5. Time course of NP10EO biodegradation without organic matter. Numerical characters (1-11) at the rear indicate “n” of the NPnEO, while (1-10) of the front correspond to “m” of the NPmEC. generated by cometabolism in the oxidation process of methanol. Test System without Organic Matter. The biodegradation of NP10EO without organic matter is shown in Figure 5. Unexpectedly, a stepwise EO chain shortening from NP10EO to shorter chain polyethoxylates was not observed (26). This might be attributed to the very high biotransformation rate. 5630

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 15, 2005

Alternatively, NP10EO might be directly converted to shortchain polyethoxylates, mainly producing NP2EO by releasing a long polyethylene glycol. Two major metabolites from NP10EO, namely, NP2EO and NP3EO, were formed, accounting for approximately 48% and 18% (molar basis) of the initial NP10EO concentration on the 25th day, respectively. The results found are in agreement with the NPnEO

FIGURE 6. Time course of NP3EO biodegradation without organic matter. Numerical characters are the same as in Figure 5.

FIGURE 7. Time course of NP5EO biodegradation without organic matter. Numerical characters are the same as in Figure 5. metabolic pathway evidenced by other authors (4, 14, 15, 19). No significant amount of NP1EO was detected as described in some studies (4, 15). A small amount of NP2EC was formed after the degradation of the precursor as previously reported (18, 19). The results of the biodegradation tests using NP3EO, NP5EO, and NP7EO showed trends similar to that of the NP10EO-spiked system, as seen in Figures 6-8. Moreover, each corresponding NPmEC was not found unlike in the organic-matter-spiked system.

Figure 9 shows the biodegradation profile of each NPnEO precursor. There was an increase for NP5EO found between the beginnning of the experiment and the fourth day, with other NPnEOs showing a concurrent slight increase. Presumably at first these starting materials were temporally adsorbing on the glass reservoir and then redissolved in the solution with time. Long-chain polyethoxylates exhibited a trend of rapid breakdown. In the tests with Triton N-101 (a nonylphenol ethoxylate in which the average number of EO units is 9.5), it completely disappeared 84 h after the start VOL. 39, NO. 15, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5631

FIGURE 8. Time course of NP7EO biodegradation without organic matter. Numerical characters are the same as in Figure 5. (26). It seems likely that the coexistence of a carbon source, such as methanol, glucose, and yeast extract, helps to act rather in an oxidative manner on the linked EO chain. However, the threshold level of organic matter is unknown as yet. It somehow seems likely that bacteria build a most effective degradation route to survive under different environments.

Acknowledgments We thank Y. Ito (METOCEAN Environment Inc.) for providing the analytical method (LC/MS) for NPnEOs. Special thanks are due to H. Yamane (Kao Corp.) for advice on HPLC. We thank J. Hiratsuka and N. Kishida (Waseda University), H. Hoshizaki (Rissho University), and Y. Sato (Toyo University) for their valuable help in sampling and N. Chiba, T. Sakamaki, and S. Hamanaka (Tohoku University) for helpful discussions. FIGURE 9. Biodegradation profiles of individual NPnEOs without organic matter. (26). The biodegradation rate of 1136 nM/day for NP5EO was approximately one order of magnitude higher than that for NP3EO (101 nM/day). NP7EO and NP10EO precursors were completely degraded between the 10th and 14th days, whereas the parent NP3EO was partially degraded after the 25th day, indicating the comparatively recalcitrant nature of the oligomer. These results show a tendency similar to that of the report that NP20EO was broken down more rapidly than NP10EO (27). The lag times of NP7EO and NP10EO observed were similar to those reported previously (14). It is presumed that bacterial communities have longer acclimation times for longer chain polyethoxylates. The NP10EO-spiked system had a prolonged lag time of up to 7-14 days in comparison with other test systems. Once a new type of enzyme capable of metabolizing the targeting material is formed, it degrades the target drastically. Figure 5 is the typical pattern. The biodegradation tests with the same inoculum confirmed the existence of two biodegradation pathways. Alkylphenol ethoxylate-degrading bacteria attack the bulky nonyl group, resulting in metabolization of the EO chain 5632

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 15, 2005

Literature Cited (1) Surfactants Statistic Annual Report; Japan Surfactant Industry Association: Tokyo, 2001. (2) Prats, D.; Ruiz, F.; Vazquez, B.; Rodriguez-Pastor, M. Removal of Anionic and Nonionic Surfactants in a Wastewater Treatment Plant with Anaerobic Digestion. A Comparative Study. Water Res. 1997, 31, 1925-1930. (3) Ahel, M.; Giger, W. Determination of Nonionic Surfactants of the Alkylphenol Polyethoxylate Type by High-Performance Liquid Chromatography. Anal. Chem. 1985, 57, 2584-2590. (4) Di Corcia, A.; Costantino, A; Crescenzi, C.; Marinoni, E.; Samperi, R. Characterization of Recalcitrant Intermediates from Biotransformation of the Branched Alkyl Side Chain of Nonylphenol Ethoxylate Surfactants. Environ. Sci. Technol. 1998, 32, 24012409. (5) Di Corcia, A.; Cavallo, R.; Crescenzi, C.; Nazzari, M. Occurrence and Abundance of Dicarboxylated Metabolites of Nonylphenol Polyethoxylate Surfactants in Treated Sewages. Environ. Sci. Technol. 2000, 34, 3914-3919. (6) Fujita, M.; Ike, M.; Mori, K.; Kaku, H.; Sakaguchi, Y.; Asano, M.; Maki, H.; Nishihara, T. Behaviour of Nonylphenol Ethoxylates in Sewage Treatment Plants in JapansBiotransformation and Ecotoxicity. Water Sci. Technol. 2000, 42, 23-30. (7) Hing-Biu, L.; Thomas, E.Occurrence and Elimination of Nonylphenol Ethoxylates and Metabolites in Municipal Wastewater and Effluents. Water Qual. Res. J. Can. 1998, 33, 389-402.

(8) Maruyama, K.; Yuan, M.; Otsuki, A. Seasonal Changes in Ethylene Oxide Chain Length of Poly(oxyethylene)alkylphenol Ether Nonionic Surfactants in Three Main Rivers in Tokyo. Environ. Sci. Technol. 2000, 34, 343-348. (9) Naylor, C. G.; Mieure, J. P.; Adams, W. J.; Weeks, J. A.; Castaldi, F. J.; Ogle, L. D.; Romano, R. R. Alkylphenol Ethoxylates in the Environment. J. Am. Oil Chem. Soc. 1992, 69, 695-703. (10) Ahel, M.; Giger, W.; Schaffner, C. Behaviour of Alkylphenol Polyethoxylate Surfactants in the Aquatic EnvironmentsII. Occurrence and Transformation in Rivers. Water Res. 1994, 28, 1143-1152. (11) Yoshimura, K. Biodegradation and Fish Toxicity of Nonionic Surfactants. J. Am. Oil Chem. Soc. 1986, 63, 1590-1596. (12) Jobling, S.; Sheahan, D.; Osborne, J. A.; Matthiessen, P.; Sumpter, J. P. Inhibition of Testicular Growth in Rainbow Trout Exposed to Estrogenic Alkylphenolic Chemicals. Environ. Toxicol. Chem. 1996, 15, 194-202. (13) Imai, A.; Matsushige, K.; Fukushima, T.; Kim, Y, H. Trihalomethanes Formation Potential of Dissolved Organic Matter in Lake Kasumigaura. Annual Meeting of Japan Society on Water Environment, Tokyo, March 2000 (in Japanese). (14) Mann, R.M.; Boddy, M.R. Biodegradation of a Nonylphenol Ethoxylate by the Autochthonous Microflora in Lake Water with Observations on the Influence of Light. Chemosphere 2000, 41, 1361-1369. (15) Kvestak, R.; Ahel, M. Biotransformation of Nonylphenol Polyethoxylate Surfactants by Estuarine Mixed Bacterial Cultures. Arch. Environ. Contam. Toxicol. 1995, 29, 551-556. (16) Potter, T. L.; Simmons, K.; Wu, J.; Sanchez-Olvera, M.; Kostecki, P.; Calabrese, E. Static Die-away of a Nonylphenol Ethoxylate Surfactant in Estuarine Water Samples. Environ. Sci. Technol. 1999, 33, 113-118. (17) Marcomini, A.; Zanette, M.; Pojana, G.; Suter, M. J.-F. Behavior of Aliphatic Alcohol Polyethoxylates and their Metabolites under Standardized Aerobic Biodegradation Conditions. Environ. Toxicol. Chem. 2000, 19, 549-554. (18) Jonkers, N.; Knepper, T. P.; De Voogt, P. Aerobic Biodegradation Studies of Nonylphenol Ethoxylates in River Water Using Liquid

(19) (20) (21) (22)

(23)

(24) (25)

(26) (27)

Chromatography-Electrospray Tandem Mass Spectrometry. Environ. Sci. Technol. 2001, 35, 335-340. Manzano, M. A.; Perales, J. A.; Sales, D.; Quiroga, J. M. The Effect of Temperature on the Biodegradation of a Nonylphenol Polyethoxylate in River Water. Water Res. 1999, 33, 2593-2600. Renner, R. European Bans on Surfactant Trigger Transatlantic Debate. Environ. Sci. Technol. 1997, 31, 316A-320A. Wakabayashi, A. Chemical material and ecological toxicity; Japan Environmental Management Association for Industry: Tokyo, Japan, 2000 (in Japanese). Field, J.; Reed, R. Nonylphenol Polyethoxy Carboxylate Metabolites of Nonionic Surfactants in U.S. Paper Mill Effluents, Municipal Sewage Treatment Plant Effluents, and River Waters. Environ. Sci. Technol. 1996, 30, 3544-3550. Ito, Y.; Takahashi, M.; Mizukoshi, A.; Asami, M.; Aizawa, T. Analytical Methods for Nonylphenol Ethoxylates and Their Degradation Products. Annual Meeting of Japan Society on Water Environment, Tokyo, March 2002 (in Japanese). Schnedl, W.; Mikelsaar, A. V.; Breitenbach, M.; Dann, O. DIPI and DAPI: Fluorescence Banding with Only Negligible Fading. Hum. Genet. 1977, 36, 167. Utsunomiya, A. Trace Analysis of Nonylphenol Polyethoxylates and Their Degradation Products in the Aquatic Environment; JSWE Symposium Series; Japan Society on Water Environment: Tokyo, June 2001; pp 15-23 (in Japanese). Maki, H.; Masuda, N.; Fujiwara, Y.; Ike, M.; Fujita, M. Degradation of Alkylphenol Ethoxylates by Pseudomonas sp. Strain TR01. Appl. Environ. Microbiol. 1994, 60, 2265-2271. Quiroga, J. M.; Manzano, M. A.; Sales, D.; Perales, J. A. Biodegradation of a Nonylphenol Polyethoxylate in River Water. Spec. Publ. R. Soc. Chem. 1996, 190, 417-425.

Received for review July 23, 2004. Revised manuscript received April 4, 2005. Accepted April 20, 2005. ES048857+

VOL. 39, NO. 15, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5633