Environ. Sci. Technol. 1999, 33, 2782-2787
Subcritical (Hot) Water/Ethanol Extraction of Nonylphenol Polyethoxy Carboxylates from Industrial and Municipal Sludges JENNIFER A. FIELD* AND RALPH L. REED Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331
Subcritical (hot) water with ethanol as modifier was used to extract nonylphenol polyethoxy carboxylates (NPECs) with 1-4 ethoxy groups from sludge samples. Quantitative recovery of native NPECs from sludge was accomplished by extracting 0.25 g samples for 20 min with 30% (v/v) ethanol in water at 75 °C and 150 bar. NPECs in the water/ ethanol extract were concentrated by a strong anion exchange (SAX) Empore disk. The NPECs were simultaneously eluted and derivatized to their methyl esters in an autosampler vial. Although carbon dioxide, hot water, and methanol-modified hot water were evaluated for NPEC recovery, ethanol-modified hot water yielded the highest recovery of native (unspiked) NPEC from sludge. Samples analyzed for this study included an anaerobicallydigested municipal sludge, a commercial product containing anaerobically-digested municipal sludge and yard waste, and secondary clarifier sludge obtained from a paper mill. NPEC concentrations ranged from 27 to 113 µg/g with NP2EC as the most abundant oligomer. Samples of anaerobically-digested sludge had ortho-to-para isomer ratios g 1, which indicated the depletion of para NPEC isomers relative to ortho isomers during anaerobic sludge treatment. In contrast, secondary clarifier sludge from a paper mill that had not undergone anaerobic treatment contained only para NPEC isomers.
Introduction Alkylphenol polyethoxylate surfactants (APEOs) are a widely used class of nonionic surfactants with an estimated annual worldwide production of 500 ktons (1). As a result, recent research has focused on identifying all potential biodegradation products and determining their fate during waste treatment and upon entering the environment. A complete understanding of the fate of the degradation products is required since some of the products pose greater risks as toxicants and estrogen mimics than does the parent surfactant (2, 3). The identified degradation products of APEOs include octyl- and nonylphenol, short-ethoxylate chain alkylphenol polyethoxylates (APEOs), alkylphenol polyethoxy carboxylates (APECs) (4, 5), brominated and chlorinated analogues of APEOs and APECs (6, 7), hydroxylated nitrogencontaining products (3), and dicarboxylated products (2, 8). Although octylphenol and nonylphenol and the short ethyoxylate-chain APEOs are more lipophilic, the more water* Corresponding author phone: (541)737-2265; fax: (541)737-0497; e-mail:
[email protected]. 2782
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soluble acid degradation products may be readily transported and are slower to degrade (2, 9, 10). For example, nonylphenol polyethoxy carboxylates (NPECs) were the most abundant relative to short ethoxylate-chain NPEOs and NP in river water and sewage effluents (11-13). Recently, the dicarboxylated degradation products of APEOs were found to be of greater abundance than APECs followed by short-chain APEOs. (2). Of the NPECs detected in effluents and drinking water, the most abundant forms are those containing two ethoxy groups (OP2EC and NP2EC) (2, 14, 15). Some concern over the occurrence and distribution of NPEC results from their characterization as weakly estrogenic toward fish (1618). Because NPECs can potentially biodegrade to NP, NPECs might act as potential sources of NP in the environment. Although a growing body of data exists on the occurrence of NPECs in surface waters and effluents, very few reports exist that document the occurrence of NPECs in sludge (19). As a result, it is generally accepted that the sorption of NPECs to sludge is negligible, and, hence, their occurrence in sludge is considered inconsequential. This view is, in part, due to the lack of analytical methods for the analysis of NPECs in sludge. Although several methods exist for the extraction of NP and NPEO from sludge, including liquid extraction (20), steam distillation (21, 22), and supercritical fluid CO2 with online acetylation (23), to the best of our knowledge, a single method exists for the extraction of NPECs from sludge (19). Lee et al. (19) extracted NP1EC and NP2EC from municipal sewage sludge by adding 2 mL of water/g sludge and extracting with CO2 at 80 °C and 350 bar. Unfortunately, the recovery of NP3EC or NP4EC by this method was not evaluated. The extracted NPECs were derivatized to their methyl esters by adding 2 mL of BF3 in methanol to the extract and heating at 85 °C for 30 min. In this paper, we describe an alternative method for the determination of NPECs in municipal and industrial sludges. Extraction is performed using subcritical (hot) water extraction with ethanol as a modifier. Subcritical (hot) water extraction was evaluated in this study because it has been successfully demonstrated for the extraction of polar and nonpolar analytes from soils and sediments (24-27). The NPECs are recovered from the water/ethanol sludge extract using strong anion exchange Empore disks. The acids analytes are simultaneously eluted from the disk and derivatized to their methyl esters for analysis by CI GC/MS. Finally, the method is demonstrated in the analysis of anaerobicallydigested sewage sludge, a composted mixture of anaerobically-digested sewage sludge and yard waste, and secondary clarifier sludge from a paper mill.
Experimental Section Standards and Reagents. Standards of 4-nonylphenoxyacetic acid (NP1EC, 90% purity), 4-nonylphenol ethoxyacetic acid (NP2EC, 45% purity), 4-nonylphenol diethoxyacetic acid (NP3EC, 80% purity), and 4-nonylphenol triethoxyacetic acid (NP4EC, 60% purity) were prepared as their free acids by Pierre Varineau of Union Carbide (27). The standards were created by reacting chloroacetic acid in the presence of sodium hydroxide with nonylphenol and with nonylphenol mono-, di-, and triethoxylates according to the methods of Marcomini et al. (28). The surrogate standard, 4-bromophenylacetic acid (98%), and the internal standard, 2-chlorolepidine, were purchased from Aldrich Chemical (Milwaukee, WI). Acetonitrile was Burdick and Jackson GC grade (VWR, Bridgeport, NJ). The surrogate standard, 4-bromophen10.1021/es990038f CCC: $18.00
1999 American Chemical Society Published on Web 07/08/1999
ylacetic acid, was added to samples just prior to extraction and was used for the quantitation of NPECs. The internal standard was added just prior to derivatization and was used to determine the absolute recovery of the 4-bromophenylacetic acid surrogate standard. Sample Collection and Preparation. A composite (24-h) sample of sludge that was anaerobically-digested for 23-25 days at 35 °C was collected at the municipal wastewater treatment plant in Corvallis, OR. A sample of commercial fertilizer made from composted anaerobically-digested sewage sludge that has been mixed with yard waste was obtained from Dr. Carter Naylor (Huntsman Corp., Austin, TX). The anaerobically-digested sewage sludge in this material underwent 10 weeks of anaerobic digestion prior to being mixed with yard waste and then was composted for 2 months. A sample of secondary clarifier sludge from a paper mill was provided by Dave Schmedding (NCASI, Corvallis, OR). All samples were air-dried at room temperature for 48 h and then ground to a fine powder in a Model BC-1752 coffee grinder (High Performance Appliances, Danbury, CT). Subcritical (Hot) Water/Ethanol Extraction. A 2.5 mL ISCO stainless steel SFE extraction cartridge (ISCO, Lincoln, NE) was assembled with a 0.2 µm Supor-200 membrane filter (Gelman Sciences, Ann Arbor, MI) covering the sample cartridge exit frit followed by a Whatman GD1UM glass fiber filter. Dried sludge (0.25 g) was mixed with 5 g of Filter Aid 400 glass beads (3M Corporation, St. Paul, MN) and placed into the extraction cartridge on top of the stacked filters. The surrogate standard, 4-bromophenylacetic acid (1 µg) in acetonitrile, was then added to the cell. The cartridge end cap threads were wrapped with three wraps of Teflon tape, after which the end caps were attached and tightened with pliers. The extraction cell was placed into the extractor (ISCO model SFX 2-10) that was heated to 75 °C and equipped with a heated variable-flow restrictor; the restrictor capillary and valve were set at 60 and 80 °C, respectively. The extractor was fitted with a model 100 DX syringe pump that was filled with 30% ethanol (95% purity, McCormick Distilling Co., Weston, MO) in deaerated, deionized water and operated at a pressure of 150 bar. A flow rate of 1.5 and 2 mL/min was used for each 20 min extraction. The water/ethanol extract was collected in a 40 mL glass vial and set aside for solidphase extraction. Solid-Phase Extraction and Derivatization. Strong anion exchange (SAX) Empore disks (25 mm; Varian, Sugarland, TX) were used to extract NPECs from the water/ethanol extract. The SAX disks are composed of 8 µm particles embedded in a Teflon matrix membrane; the particles are composed of a styrene-divinyl benzene polymer with quaternary amine functional groups that have chloride as the counterion. Prior to use the disks were soaked overnight in a solution of 12 mM HCl in acetonitrile and then transferred to acetonitrile and soaked for 5 min in order to remove disk impurities including benzoic acid, perfluorooctanoic acid, and 2-ethylhexyl phthalic acid. After assembling the SAX disks in 25 mm polypropylene filter holders, 5 mL of acetonitrile was passed through the SAX disk, followed by 350 mL of deionized water in order to remove the HCl from the SAX disk. The ethanol/water extract was applied directly to the SAX disk using a vacuum of 5-10 mmHg. The extract vial and the reservoir were rinsed with distilled water (5 mL) and passed through the disk. Once the sample passed through the disk, the disk was dried under full vacuum (25 mmHg) for 30 min. When dry, the disk was removed and placed directly into a 2 mL gas chromatograph autosampler vial. Acetonitrile (1.2 mL) was added to the autosampler vial together with 200 µL of methyl iodide and 1 µg of the 2-chlorolepidine internal standard. The autosampler vial was then tightly capped and
heated at 80 °C for 1 h. Once cooled, the autosampler vials were then analyzed without further manipulation. Caution: Derivatization reactions with methyl iodide should be carried out in a fume hood, and caution should be taken when piercing the vial septum if manual injections are performed since the vial contents are under pressure. Gas Chromatography/Mass Spectroscopy. The identification and quantification of NPECs in sludges was performed by gas chromatography with positive chemical ionization mass spectrometry using ammonia as reagent gas, as described by Field and Reed (14). All NPECs produced molecular ion adducts with ammonia to give base peaks that corresponded to the [M + NH4]+ ions. The mass spectrometer was operated in multiple ion detection mode with m/z 178, 246, 310, 354, 398, and 442 used to detect 2-chlorolepidine and the methyl esters of 4-bromophenylacetic acid, NP1EC, NP2EC, NP3EC, and NP4EC, respectively. Calibration curves were prepared for each group of samples prior to analysis, by derivatizing each acid standard to its methyl ester with diazomethane. Calibration curves were constructed by adding 0.05-50 µg of the methyl ester NPEC standards and 1 µg each of the methyl ester of the 4-bromophenylacetic acid surrogate standard to 1 mL of acetonitrile along with 200 µL of methyl iodide. The addition of methyl iodide to the prederivatized quantitation standards was found to improve the precision and accuracy of the GCMS analysis. A calibration curve was constructed for the quantitation of methylated surrogate (0.2-5 µg) with 1 µg of the 2-chlorolepidine internal standard.
Results and Discussion Solvent-Modified CO2 Extraction. The first phase of method development was to determine if CO2 or solvent-modified CO2 could be used to recover NPECs that had been spiked onto an inert glass bead matrix (e.g., Filter Aid) and blank soil. Filter Aid 400 (5 g; 3M, St. Paul, MN) or blank soil (2 g) was placed in an extraction cell and spiked with 10 µg of each standard NPEC acid and 1 µg of 4-bromophenylacetic acid. The sample was extracted with CO2 at 200 bar and 50 °C at a flow rate of 1.5 mL/min for 20 min. The CO2 extract was collected in 8 mL of acetonitrile. Water was added to this extract, and the acetonitrile was removed by evaporation in a stream of nitrogen. The acid analytes were extracted from the resulting solution using a SAX disk, as described in the methods section. The NPECs and the 4-bromophenylacetic acid surrogate standard were not recovered from either Filter Aid or the blank soil under these conditions. This indicated that CO2 alone was ineffective in solubilizing NPECs. The static addition of 1.5 mL of either methanol or acetonitrile to the extraction cell also failed to extract NPECs and yielded only 1-4% recovery of the surrogate standard, 4-bromophenylacetic acid. Extractions also were performed with static additions of water to the extraction cell containing spiked blank soil and extracted at 80 °C and 350 bar CO2, first under static conditions (10 min) followed by a dynamic extraction of 15 min (19). Although the recovery of the surrogate standard was high (97%), NPEC recovery was low and variable and averaged 37% for NP1EC, 28% NP2EC, 20% NP3EC, and 19% for NP4EC. Continuous pumping of CO2 (350 bar) modified with 10-40% (v/v) methanol at 80 °C also was attempted for the extraction of NPECs from spiked blank soil. For a given NPEC, increasing the percent methanol in CO2 increased the relative recovery (Figure 2). For example, for NP1EC, the recovery increased from 62% for 10% (v/v) methanol to 100% for 40% (v/v) methanol. However, even at 40% (v/v) methanol, the recovery decreased with an increasing number of ethoxylate groups such that only 7% of NP4EC was recovered with 40% (v/v) methanol in CO2. Due to the variable and low recovery associated with solvent-modified CO2, subcritical VOL. 33, NO. 16, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Recovery of NPEC spiked onto soil by methanol-modified CO2 at 350 bar and 80 °C.
FIGURE 3. NPEC recovery from spiked soil at 150 bar and 75 °C with subcritical (hot) water with 20 and 30% (v/v) methanol and ethanol.
FIGURE 1. Typical CI-GC/MS chromatograms for (a) NPEC standards, (b) anaerobically-digested municipal sludge, and (c) secondary clarifier sludge from a paper mill. (hot) water extraction became the focus of the method development. Subcritical (Hot) Water Extraction. Unlike solventmodified CO2, preliminary extraction of NPECs spiked onto Filter Aid and extracted using subcritical (hot) water over a range of temperatures from 25 to 100 °C at a pressure of 350 bar gave good recoveries of NPECs (90-108%) and 4-bromophenylacetic acid (100%). Quantitative recovery of NPECs from the inert Filter Aid matrix demonstrated that (1) the NPECs exhibit good solubility in water over a range of temperatures, (2) the analytes are quantitatively swept from 2784
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the extraction cell, (3) no degradation of the NPECs took place under the extraction conditions, and (4) the NPECs are quantitatively recovered from water extracts using SAX disks. In contrast, poor recoveries of NP1EC (56%) and NP2ECNP4EC ( 2 mL/min did not result in additional yield. We attempted to perform the extractions with the SAX disk inside the extraction cell in a manner similar to that used by Field et al. (27) to determine if the SAX disk could be placed directly inside the extraction cell. The SAX disk and also the eluate that passed through the disk and collected external to the extractor were analyzed for NPECs. Complete breakthrough of the surrogate standard was observed at temperatures ranging from 25 to 75 °C. Only 10% of the NPECs was retained on the disk under the elevated temperature conditions with 90% passing through the disk and recovered in the eluate. For these reasons, NPECs were extracted from the subcritical (hot) water/ethanol extracts using SAX disks external to the extractor. Detection and Quantitation. Since samples of blank sewage sludge were not available, spike and recovery experiments using sludge were not performed to determine the detection limit of the method. As an alternative, the detection and quantitation limits of the method for extracting NPECs from river water, municipal sewage effluent, and paper mill effluent using SAX disks were assumed to apply to the hotwater/ethanol soil extracts. The quantitation limits for that method, defined as the mass of individual NPECs in the 1.2 mL final volume of the autosampler vial needed to produce a signal-to-noise (S/N) of 10:1, were 0.05 µg for NP1EC, 0.1 µg for NP2EC, and 0.5 µg for both NP3EC and NP4EC (14). Assuming that this minimum amount of mass could be extracted from 0.25 g sludge samples, the estimated quantitation limit of the overall method is 0.2 µg/g for NP1EC, 0.4 µg/g for NP2EC, and 2.0 µg/g for both NP3EC and NP4EC. Application to Environmental Sludge Samples. Three environmental sludge samples of various origins were analyzed to demonstrate the potential utility of the method. Replicate extractions were performed on samples of an anaerobically-digested municipal sewage sludge, a composted mixture of anaerobically-digested municipal sewage sludge and yard waste that is sold commercially as a soil amendment, and a secondary clarifier sludge from a paper mill. Replicate analyses were performed on these three sample VOL. 33, NO. 16, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. NPEC Para and Ortho Isomer Concentrations in Various Types of Sludge Samplesa sample
NP1EC (µg/g)
NP2EC (µg/g)
NP3EC (µg/g)
NP4EC (µg/g)
total NPEC (µg/g)
para ortho total
17.9 ( 2.0 (11.2%) 1.2 ( 0.2 (16.7%) 19.1 ( 2.0 (10.7%)
Anaerobically-Digested Sludge (n ) 6) 40.9 ( 3.4 (8.3%) 4.5 ( 0.3 (6.7%) ND 42.0 ( 4.3 (10.2%) 6.3 ( 0.9 (15.9%) ND 82.9 ( 7.1 (8.6%) 10.8 ( 1.2 (11.5%) ND
para ortho total
6.6 ( 0.4 (6.1%) 5.1 ( 0.3 (5.9%) 11.7 ( 0.7 (5.9%)
Composted Sludge/Yard Waste Mixture (n ) 5) 2.1 ( 0.1 (4.8%) ND ND 13.9 ( 0.8 (5.8%) ND ND 16.0 ( 7.1 (5.9%) ND ND
8.7 ( 0.5 (5.8%) 19.0 ( 1.1 (5.8%) 27.7 ( 1.6 (5.6%)
para ortho total
15.0 ( 1.5 (10.0%) ND 15.0 ( 1.5 (10.0%)
Paper Mill Secondary Clarifier Sludge (n ) 5) 40.4 ( 5.0 (12.4%) 19.4 ( 2.4 (12.4%) 17.1 ( 2.9 (17.0%) ND ND ND 40.4 ( 5.0 (12.4%) 19.4 ( 2.4 (12.4%) 17.1 ( 2.9 (17.0%)
91.9 ( 10.2 (11.1%) ND 91.9 ( 10.2 (11.1%)
a
63.2 ( 4.9 (7.8%) 49.5 ( 4.3 (8.7%) 112.7 ( 9.0 (8.0%)
Relative standard deviations are given in parentheses. ND indicates not detected.
types to determine their NPEC composition as well as the accuracy and precision of the analytical method. The precision of the method, indicated by the relative standard deviation (RSD), ranged from 4.8 to 17.0%. The completeness of extraction was checked by air-drying samples that had been extracted and then re-extracting them by the same method. The percent of total NPEC recovered in the second extraction ranged from 0 to 3.1%, and only NP1EC and NP2EC were found in the second extraction. For all samples, chromatograms contained clusters of peaks that corresponded with those of the synthesized NPEC standards, which are >99% para-substituted isomers (Figure 1). The individual NPECs produce peak clusters due to the large number of branched nonyl group isomers. In some cases, an additional peak cluster was observed 1 min before the peak cluster that matched the synthesized NPEC standards (Figure 1). The mass spectra of the early-eluting peaks acquired under full-scan CI-GC/MS mode contained the same characteristic ions as those of para isomer NPEC standards, although differences existed between their relative intensities. Based on their retention times and similar mass spectra, the clusters of early-eluting peaks are most likely the ortho-NPEC isomers. Unfortunately, authentic standards of ortho isomer NPECs were not available for confirmatory analyses. Others have reported the presence of early-eluting ortho isomers of nonylphenol and nonylphenol polyethoxylates with 1-3 ethoxylate units in wastewater treatment plant influents and effluents (20). Samples also were examined for the presence of the chlorinated and brominated NPEC analogues; however, none were found. A sample of anaerobically-digested sewage sludge from Corvallis, OR contained a total NPEC concentration of 112.7 ( 9.0 µg/g (Table 1), which is higher than the range of concentrations (25-65 µg/g) reported for anaerobicallydigested municipal sewage sludges by Lee et al. (19). The dominant oligomer was NP2EC with 73.6% of the total NPEC detected, while 16.9% and 9.5% consisted of NP1EC and NP3EC, respectively. Ortho isomers composed greater than 50% of NP2EC and NP3EC (Table 1), while only 6.3% of NP1EC occurred as the ortho isomer. Overall, the ortho isomers comprised 43.9% of the total NPEC detected in the anaerobically-digested sewage sludge. Lee et al. (19) also found that NP2EC was the major oligomer in anaerobically-digested sewage sludge; however, only NP1EC and NP2EC were determined in their study, and no ortho isomers were reported for any of the NPECs. The anaerobically-digested sewage sludge and yard waste mixture contained a total of 27.7 ( 1.6 µg/g NPEC (Table 1). Only NP1EC and NP2EC were detected and comprised 42.2 and 57.8%, respectively, of the total NPECs detected (Table 1). Of the NP1EC and NP2EC detected, 43.6 and 86.9%, 2786
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respectively, were present as ortho isomers to give an overall composition that was 69% ortho isomers. The secondary clarifier sludge from a paper mill, which has undergone only aerobic treatment, contained a total concentration of 91.9 ( 10.2 µg/g NPEC (Table 1). The composition of this sample was 16.3% NP1EC, 44.0% NP2EC, 21.1% NP3EC, and 18.6% NP4EC (Table 1). No ortho isomers were detected in this sample which has undergone only aerobic treatment. The ratio of ortho to para NP2EC and NP3EC isomers was g1 for the two samples containing anaerobically-digested sewage sludge. The higher percentage of ortho isomers in the anaerobically treated sludge suggests that the sludge digestion process results in the more rapid biodegradation of the para isomers, hence the enrichment of ortho isomers relative to para isomers, since commercial mixtures of nonylphenol polyethoxylates contain only a minor amount (
