Seasonal Changes in Ethylene Oxide Chain Length of Poly

5-7, 4-Chome, Konan, Minato-ku, Tokyo 108-8477, Japan. Although poly(oxyethylene)alkylphenyl ether (APE) nonionic surfactants with longer ethylene oxi...
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Environ. Sci. Technol. 2000, 34, 343-348

Seasonal Changes in Ethylene Oxide Chain Length of Poly(oxyethylene)alkylphenyl Ether Nonionic Surfactants in Three Main Rivers in Tokyo KAZUKI MARUYAMA, MOUCUN YUAN, AND AKIRA OTSUKI* Department of Ocean Sciences, Tokyo University of Fisheries, 5-7, 4-Chome, Konan, Minato-ku, Tokyo 108-8477, Japan

Although poly(oxyethylene)alkylphenyl ether (APE) nonionic surfactants with longer ethylene oxide (EO) chain than 2 can be the important pollutants in natural aquatic environments as the potential precursor of alkylphenol, an endocrine disrupting chemical, little attention has been paid to those. Water samples were seasonally collected at five sites of each of the three main rivers in Tokyo. APE with different alkyl chains were separated and fractionated using a reversed-phase adsorption enrichment technique with gradient elution in high performance liquid chromatography (HPLC). After the concentration of each fraction, EO chain length (polymerization degree of EO) in APE was determined by electrospray ionization mass spectrometry (ESI/MS). The results indicated that, in three main river waters in Tokyo, poly(oxyethylene)nonylphenyl ether (NPE) was the dominant pollutant among APE, total NPE concentrations with different EO chain lengths were 2-6 nM in summer and 10-35 nM in winter, and its peak in the abundance of NPE components with different EO chain lengths was at 5-8 EO units in winter and shifted to 2-5 units in summer. Laboratory biodegradation experiments using filtered and NPE-spiked river water confirmed that seasonal changes in the abundance of NPE components with different EO chain lengths would be mainly caused by increased bacterial activity due to high water temperature, producing mainly the persistent nonylphenol diethoxylate.

Introduction Poly(oxyethylene or oxypropylene)alkylphenyl ether (APE) nonionic surfactants (Figure 1) are widely used as dispersing agents, detergents, emulsifiers, solubilizers, and wetting agents (1). The production of APE nonionic surfactants in Japan (48 000 tons in 1997) is reported to be still expanding into new markets due to their special property (2), while the use of APE was banned or restricted in some countries in Europe because the breakdown products of APE are more toxic to aquatic organisms than their intact precursors are (3). Although many surfactants find a way into the natural aquatic environment through treated and untreated wastewater discharges, limited papers report the concentrations * Corresponding author phone: +81-3-5463-0451; fax:+81-3-54630398; e-mail: [email protected]. 10.1021/es990563e CCC: $19.00 Published on Web 12/10/1999

 2000 American Chemical Society

FIGURE 1. Structure and acronyms of poly(oxyethylene)alkylphenyl ether (APE) studied. of APE with longer ethylene oxide (EO) chain lengths than 3 in river waters and sediments (4-6), which were shown to be much higher than those of nonylphenol and nonylphenol mono- and diethoxylates (5, 6). On the other hand, there are many papers on the concentrations of nonylphenol, nonylphenol monoethoxylate, and their carboxylates in river water samples and biodegradation of APE in secondary treatment processes (7-18, 26), because the biodegradation of APE nonionic surfactants was reported to lead to the formation of more persistent and toxic intermediates such as alkylphenol, alkylphenol mono- or diethoxylates, and their carboxylates in wastewater treatment processes (11-13). Since APE with longer EO chain lengths than 5 are the major products for industrial use (1), they can be the important pollutants in natural aquatic environments as the potential precursor of alkylphenol as an endocrine disrupting chemical, leading to disturbance of aquatic ecosystems (1922). However, little attention has been paid to EO chain length in APE and seasonal changes in the abundance of APE components with different EO chain lengths (polymerization degree of EO) in river waters. The reason for this may be that it was not easy to determine these components with high molecular masses and low volatility using gas chromatography/mass spectrometry (23). The main purposes of the present study were to examine seasonal changes in the abundance of APE components with different EO chain lengths in nonionic surfactants as the potential precursor of alkylphenol in three main rivers in Tokyo and to get information to predict the behavior of APE nonionic surfactants in river and estuary. In the present paper, alkylphenolate having an EO chain length more than 3 is called APE, and that having 1 or 2 EO units is called alkylphenol monoethoxylate or diethoxylate.

Experimental Section Sampling. Water samples were seasonally collected at five sites in the Tama, Sumida, and Ara Rivers which run in the middle of the Tokyo Metropolitan area and enter Tokyo Bay (December 14 and 26, 1997, April 21 or May 21, and August 11 and 18, 1998) (Figure 2). River water temperature was 12 °C in December and 28 °C in August. The water samples were taken from the surface in the middle of each sampling site using a stainless steel bucket precleaned with methanol, transferred into three precleaned glass containers (1.0-1.2 L), acidified with 9.5 M hydrochloric acid to pH 2, and brought back to the laboratory. All samples were filtered through a Whatman GF/C filter precombusted at 450 °C and stored at 10 °C until analysis within 48 h. Dissolved organic carbon (DOC) concentrations in these river waters in April or May and August 1998 were determined using a Shimadzu TOC 5000A (Kyoto, Japan). Reagents. HPLC-grade methanol was purchased from Wako Chemicals (Tokyo, Japan). All other reagents used were of analytical grade except two reference compounds, poly(oxyethylene) p-octylphenyl ether (OPE) and p-nonylphenyl ether (NPE), that were of technical grade and purchased from VOL. 34, NO. 2, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Sampling sites in three rivers of Tokyo. Tokyo Chemical Industry (Tokyo, Japan). The OPE and NPE are described by the supplier to have nominally average 10 EO units per 1 molecule of alkylphenol. Their 100 mg L-1 stock solutions were prepared in methanol. Pure water was prepared by a Milli-Q System (Millipore, MD) and used for all experiments. Analytical Procedure. Filtered samples (1.0-1.2 L) were passed through a 10 mL bed volume column of Amberlite XAD-16 resin at a flow rate of 10 mL min-1 to adsorb nonionic surfactants. After the column was rinsed with 100 mL of pure water, adsorbed nonionic surfactants were eluted with 150 mL of methanol. Methanol in the eluent was removed by evaporation to dryness using a vacuum rotary evaporator, and the residue was dissolved in 20 mL of a 50%:50% (v/v) ethanol-water solution. The solution was then passed through a 5-mL bed volume column of strong anion-exchange resin (Dowex 1 × 2) at a flow rate of 2 mL min-1 to remove anionic organic compounds. After the column was rinsed with 20 mL of the 50%:50% (v/v) ethanol-water solution, the rinsing solution was combined with the eluate. The combined solution containing APE was concentrated to dryness using a vacuum rotary evaporator. For their separation by HPLC, the residue was dissolved in 1 mL of methanol, and different amounts of the concentrate were injected into HPLC for fractionation and quantification from peak area. The peak fractions containing APE were collected for the ESI mass spectrometric determination of EO chain length, producing repeated peaks at the intervals of m/z 44 due to EO unit (4). After the collected fractions were concentrated to about 50 µL by nitrogen gas stream, 30 µL of 10 mM ammonium acetate solution and 10 µL of 10 mM potassium chloride solution were added to the concentrate that was finally adjusted to 100 µL and infused into ESI/MS. Quantitative limit of this method (defined as three times of detection limit) was 1 nM as the sum of all NPE components with different EO chain length called as total NPE concentrations, when 1 L of sample was taken and 20 µL of the concentrate was injected into HPLC. The recovery was 91 ( 5% at 150 nM level and 80 ( 15% at 10 nM (each n ) 3). Each total NPE concentration was an average value in triplicate samples at one sampling site. HPLC System and Operating Conditions. Separation and measurement of APE nonionic surfactants were performed using a Hitachi L-6200 multisolvent delivery system and a Shimadzu SPD-6A absorbance detector. The UV detector was set at a wavelength of 280 nm. Twenty microliters of the concentrate was injected to a ODS column (3.9 mm in ID × 344

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150 mm in length, µ-Bondasphere C-18, Waters, USA) through an injector and held at a 50%:50% (v/v) methanol-water mobile phase for 5 min at a flow rate of 1 mL min-1. While all interfering compounds with a shorter alkyl chain than the propyl group are passed through the ODS column and removed, targeted compounds with a longer alkyl chain than the butyl group are adsorbed onto the column (25). After a 5 min holding to remove interfering compounds, a linear gradient elution was performed from a 50%:50% (v/v) methanol-water mobile phase to 100% methanol for 10 min. Depending upon the alkyl chain length, first OPE and second NPE were successively eluted with a mobile phase of 100% methanol (4). Mass Spectrometry. Mass spectra were obtained with a TSQ-700 triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI/MS) with a 20 kV dynode (Finnigan-MAT, San Jose, CA). Full-scan mass spectra were acquired with Q1 from m/z (mass-to-charge ratio) 200-1200 at 1.2 s scan-1 and averaged over 20 s to obtain the final spectra. The nebulization gas consisted of nitrogen at 1.034 × 105 N m-2 flowing at 40 mL min-1, and electrospray needle voltage was operated at -4.2 kV. Massto-charge ratio calibration was done using a myoglobin solution (from horse skeletal muscle, M-0630, Sigma Chemicals, St. Louis, MO). The concentrate containing APE was continuously infused into the ESI source at a flow rate of 3 µL min-1 using a Harvard Apparatus Model 22 syringe pump (South Natick, MA). No attempt to determine alkylphenol and alkylphenol monoethoxylate was made because of their extremely low ionization efficiency due to relatively strong hydrophobic property in the ESI process. Calibration. Although the total concentrations of each APE with different alkyl chain could be determined by the area of each peak separated by gradient elution using a reversed-phase adsorption enrichment technique in HPLC (4), the quantification of each component with different chain lengths of EO in each APE was made using ESI/MS as follows. The proportion of the peak height of potassium adduct ion of each component with the difference of m/z 44 to the sum of peak height of all components with different EO chain lengths was calculated, multiplied by the injected amount into HPLC, and divided by each molecular mass with the difference of m/z 44 to express each component as nM. A linear calibration line with the same slope between the amount (nM) of each NPE component with different EO chain lengths and each peak height was obtained, suggesting that the ionization efficiency of potassium adduct ion of each NPE component with different EO chain lengths in ESI/MS was identical. The purity of the NPE mainly used was estimated to be 77.5%, containing 8.0% of OPE and 8.7% of poly(oxyethylene) decylphenyl ether (DPE) as impurities, based on peak heights with the difference of ( m/z 14 due to the methylene group in the ESI/MS spectrum (see Figure 3) (4). Biodegradation Experiments. River water was collected at site 14 of the Tama River on November 10, 1998 and immediately carried back to the laboratory. After the GF/C filtration, each 1 L of the filtrate was transferred into six glass containers. NPE reference compound was spiked to all containers to be 1 mg NPE L-1 and, after inoculation of 1 mL of raw Tama river water, two triplicate containers were incubated with continuous shaking by magnetic stirrer in the dark in the air-conditioned rooms of 20 ( 2 and 12 ( 2 °C, respectively. A 25 mL subsample from each container was taken at different intervals of 12, 24, 48, 72, 96, and 192 h, and analyzed in the same way mentioned above for each NPE component with different EO chain lengths in each peak fraction.

TABLE 2. Composition of Poly(oxyethylene) p-Nonylphenyl Ether with Different EO Chain Length Used as a Reference Compound no. of EO unit per 1 molecule of nonylphenol

FIGURE 3. ESI mass spectrum of potassium adduct ions of NPE used as a reference compound. (See Calibration in Experimental Section.)

TABLE 1. Dissolved Organic Carbon Concentrations in Three River Waters (mg C L-1)a sampling site

a

April or May 1998

August 1998

no. 1 2 3 4 5

Ara River 5.07 4.15 4.56 4.75 4.57

3.41 3.67 3.58 3.85 3.28

no. 6 7 8 9 10

Sumida River 5.35 4.96 5.86 5.42 5.03

3.03 3.08 3.92 3.83 3.36

no. 11 12 13 14 15

Tama River 2.29 2.46 3.76 2.77 2.53

2.20 2.41 3.14 2.91 3.56

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

relative composition (RC:%) ESI/MSa HPLCb 0 0.4 1.2 2.6 6.1 11.3 13.6 14.8 14.2 11.2 9.0 5.8 4.0 2.4 1.4 0.8 0.6 0.4

0.3 1.4 4.0 6.7 9.4 11.8 13.5 13.5 12.2 10.1 7.6 4.7 3.1 1.6

aRCn ) 100 × Hn/Ht. Hn: peak height at each EO unit, Ht: sum of each peak height from EO 2 to 19 in ESI/MS. b Calculated with the formula: RCn ) 100 × An/At, where RCn is the relative composition of each NPE component with different EO units. An: peak area at each EO unit, At: sum of each peak area from EO 2 to 15 separated by HPLC using a carbon column (24).

See Figure 1 for sampling sites.

Results and Discussion To give some idea as to the extent of organic pollution in the three main rivers of Tokyo, DOC concentrations in river waters were measured (Table 1), showing that DOC concentrations in the Sumida and the Ara River waters, which run through the industrial area of the northeast side of Tokyo, were higher than those in the Tama River waters, which mainly runs through the residential area. ESI mass spectrum of the NPE reference compound indicates that it was composed of NPE components having the normal distribution with the central NPE component with an EO chain length of 9 (EO polymerization degree of 9) as shown by the normal distribution of potassium adduct ion peaks with an interval of m/z 44 due to the EO unit with the maximum peak at m/z 655 which would be due to the potassium adduct ion of a NPE component with an EO chain length of 9 (Figure 3). Since each potassium adduct ion did not give any fragment ion in the ESI collision-induced dissociation MS/MS spectrum, we confirmed the formation of potassium adduct ion peaks by the increased peak shift of m/z 21 from ammonium adduct ion peaks to potassium adduct ion peaks by means of addition of potassium chloride solution to the same sample solution after measurement of ammonium adduct ions. Shang et al. (6) successfully applied a sensitive and selective ESI/MS for determination of NPE

FIGURE 4. Typical example of HPLC separation of OPE and NPE in Ara River water sample by linear gradient elution using the enrichment technique. Sampling date and site: December 14, 1997 and no. 3. components with different EO chain lengths in sediments (6). However, they used sodium adduct ion for selected ion mode detection. The composition of NPE with different EO chain lengths used as the NPE reference compound indicates that the components with EO units of 5-15 per 1 molecule of nonylphenol occupied more than 95% (Table 2) (24). An EO chain length of more than 5 is necessary to increase water solubility of APE, and APE with an EO chain length of 5-15 is known to be major products for industrial uses (1, 2). A typical gradient elution chromatogram of the concentrate fractionated from the Ara River sample is shown in Figure 4, demonstrating the presence of OPE and NPE having different retention times of 13.3 and 13.9 min, respectively. The frontal large peaks would be mainly due to the presence of the aromatic compounds that were not adsorbed on the ODS column under the present HPLC condition and had shorter alkyl chain than propyl groups (25). An unknown peak at 16.4 min always appeared regardless of samples by the linear gradient elution, suggesting that it would be due VOL. 34, NO. 2, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. ESI mass spectrum of the NPE fraction from Ara River water sample. Sampling date and site: December 14, 1997 and no. 3.

FIGURE 7. A typical example of seasonal changes in the abundance of NPE components with different EO chain lengths in Ara River water samples. Sampling site: no. 1.

FIGURE 6. Seasonal changes in the NPE concentrations at all sampling sites of three main rivers in Tokyo. to impurity in Milli-Q water and/or methanol used, but no attempt was made to identify it. Figure 5 shows a typical example of ESI mass spectrum of the NPE fraction. The ESI mass spectrum from the Ara River sample in winter indicates that the concentration of a NPE component with an EO chain length of 6 was the highest, and the distribution pattern in the abundance of NPE components with different EO chain lengths was one-sided into shorter EO chain lengths, compared with the normal distribution which would be the original (see Figure 3). Seasonal changes in NPE concentrations at all sampling sites of three rivers indicate that their concentrations, ranging 346

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from 2 to 6 nM in summer to 10-35 nM in winter, were the highest in winter and lowest in summer (Figure 6), suggesting that river water temperature can be an important factor in determining residence time and concentration of NPE in river waters and might be closely related to bacterial cleavage of EO chain in NPE nonionic surfactants. Although NPE was always found in all sampling sites of three rivers in Tokyo, the peak of OPE in HPLC chromatogram was found only at all sampling sites of the Ara River in winter, and the DPE, based on the difference of m/z 14 due to methylene group on the ESI mass spectra, was detected only at two sampling sites of the Sumida River in winter. Since total OPE concentrations were always less than one-fifth of the total NPE concentrations and total DPE concentrations were less than one-tenth, the EO chain length in NPE was mainly examined. Seasonal changes in the abundance of components with different EO chain lengths in the Ara River water samples indicate that the maximum in the distribution of EO chain lengths was at 5-8 units in winter and shifted into shorter EO chain lengths of 2-5 in summer (Figure 7). The seasonal changes in their abundance were similar to those in all sampling sites of three rivers. The present results suggest that high water temperature can cause faster cleavage of EO chains in NPE probably due to increased bacterial activity, resulting in NPE concentrations with shorter EO chain lengths in river waters. It seems that these results are supported by Isobe et al. (26), reporting that the concentrations of nonylphenol in Tama and Sumida River waters, ranging from 0.05 to 1.08 µg L-1 (0.5-4.9 nM), were low in winter and high in summer. That is, NPE would be easily decomposed to nonylphenol in summer, resulting in low NPE and high nonylphenol concentrations, and slowly in winter, resulting in high NPE and low nonylphenol concentrations due to bacterial activity.

FIGURE 8. Relative changes in the abundance of each component with different EO units in biodegradation experiments of spiked NPE in Tama River water filtrate. Conditions: 12 ( 2 °C; (a) 48 h, (b) 96 h, and (c) 192 h, and 20 ( 2 °C; (d) 48 h, (e) 72 h, and (f) 96 h. Laboratory biodegradation experiments using filtered and NPE-spiked river water under two water temperatures of 12 °C (correspondent to early winter) and 20 °C (late spring or fall), respectively, confirmed that the seasonal changes in the abundance of NPE components with different EO chain length in these river waters would be mainly caused by the increased bacterial activity (Figure 8). Although no significant decreases in total NPE concentrations were observed after 48 h under both temperatures in the dark, the maximum peak at 9-10 in the abundance of NPE components with different EO chain length was clearly shifted to 8, but no nonylphenol diethoxylate was detected as the intermediate in the early stage of bacterial cleavage of EO chain. However, after 72 h under 20 °C and 192 h under 12 °C, nonylphenol diethoxylate occupied more than 50% in the remaining NPE. These results suggest that the cleavage of EO chain length in NPE was due to bacterial action, rather than photolysis, and bacterial cleavage of EO chain in NPE occurred via nonylphenol diethoxylate as the persistent intermediate. These results also suggest that, in the early stage of bacterial cleavage of EO chain, terminal EO units in NPE with longer EO chain lengths than 9 would be preferentially cloven, resulting in the peaks at EO chain lengths of 6-8, and, in the

late stage, nonylphenol diethoxylate would be accumulated in water with the disappearance of NPE with EO chain lengths of 6-8 as the bacterial cleavage intermediate, resulting in precipitation by adsorption to suspended materials into sediments by the increased hydrophobicity as found by Nayler et al. (5). Most recently almost the same results on the effect of temperature on biodegradation of NPE in river water were reported using HPLC (27). It may be noteworthy to note that, in the ESI mass spectra of the concentrates without the use of strong anion-exchange resin column as pretreatment, we could not detect any peaks based upon carboxylates that might be derived from nonylphenol mono- and/or diethoxylates in river water samples and sample waters under the laboratory biodegradation experiments. In conclusion, these results proved that this reversedphase adsorption enrichment technique using an ODS column was useful for the isolation of APE with different alkyl chain lengths, making it easy to interpret ESI mass spectra of each fraction containing APE with relatively few peaks of interfering compounds (see Figure 4). The present study indicated that NPE concentration in river waters of Tokyo would be low in summer and high in winter, and the VOL. 34, NO. 2, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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abundance of NPE components with different EO chain lengths would seasonally change; that is, the maximum peak in the distribution pattern of EO chain lengths would be at 5-8 EO units in winter and shift into 2-5 in summer. These changes would be mainly caused by bacterial cleavage of terminal EO chains in NPE. As a result, the present study suggested that field survey of NPE concentrations in environmental waters should be conducted seasonally.

Acknowledgments This research was supported in part by the Tokyu Foundation for Better Environment (no. 1998-31).

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(12) Reinhard, M.; Goodman, N.; Mortelmans, K. E. Environ. Sci. Technol. 1982, 16, 351-362. (13) Giger, W.; Brunner, P. H.; Schaffner, C. Science 1984, 225, 623625. (14) Maki, H.; Okamura, H.; Aoyama, I.; Fujita, M. Environ. Toxicol. Chem. 1998, 17, 650-654. (15) Ahel, M.; Thomas, T.; Giger, W. Environ. Toxicol. Chem. 1987, 21, 697-703. (16) Ahel, M.; Giger, W.; Koch, M. Water Res. 1994, 28, 1131-1142. (17) Scarlett, M. J.; Fisher, J. A.; Zhang, H.; Ronan, M. Water Res. 1994, 28, 2109-2116. (18) Corcia, A. D.; Samperi, R.; Marcomini, A. Environ. Sci. Technol. 1994, 28, 850-860. (19) Jobling, S.; Sumpter, J. P. Aquatic Toxicol. 1993, 27, 361-372. (20) Purdom, C. E. Chem. Ecol. 1994, 8, 275-285. (21) Korach, K. S.; Davis, V. L.; Curtis, S. W.; Bocchinfuso, W. P. In Endocrine Toxicology, 2nd ed.; Thomas J. A., Colby, H. D., Eds.; Taylor & Fransis: London, U.K., 1997; pp 181-211. (22) Liber, K.; Knuth, M. L.; Stay, F. S. Environ. Toxicol. Chem. 1999, 18, 357-362. (23) Ventura, F.; Caixach, J.; Figueras, A.; Espalder, I.; Fraisse, D.; Rivera, J. Water Res. 1989, 23, 1191-1203. (24) Yamagishi, T.; Hashimoto, S.; Otsuki, A. Environ. Toxicol. Chem. 1998, 17, 670-674. (25) Otsuki, A. J. Chromatogr. 1977, 133, 402-407. (26) Isobe, T.; Sattoh, M.; Ogura, N.; Takada, H. J. Jpn. Soc. Water Environ. 1999, 22, 118-126 (in Japanese with English abstract). (27) Manzano, M. A.; Perales, J. A.; Sales, D.; Quiroga, J. M. Water Res. 1999, 33, 2593-2600.

Received for review May 17, 1999. Revised manuscript received October 28, 1999. Accepted November 3, 1999. ES990563E