Environ. Sci. Technol. 2001, 35, 4655-4659
Concerted Chemical and Microbial Degradation of Sulfophthalimides Formed from Sulfophthalocyanine Dyes by White-Rot Fungi THORSTEN REEMTSMA* AND JUTTA JAKOBS Department of Water Quality Control, Technical University of Berlin, Sekr. KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany
3- and 4-sulfophthalimide (SPI) have recently been shown to be the major product formed by white-rot fungi upon decolorization of sulfophthalocyanine (SPC) textile dyes. To make use of this metabolic potential in textile wastewater treatment, the fungal breakdown products should be degradable by activated sludge. Here, the aerobic degradation of SPI was studied in die-away tests, and biodegradation intermediates and degradation products were analyzed by liquid chromatography-electrospray ionizationmass spectrometry. The degradation of SPI is initiated by a chemical hydrolysis to sulfophthalamic acid (SPAA) with half-lives of 8 and 40 h at pH 6.5 for 4- and 3-SPI, respectively. Then, 4-SPAA can be mineralized by aerobic mixed cultures, while 3-SPAA remained stable throughout the experiment (35 d). Analogously, the potential intermediate in 4-SPAA degradation, the 4-sulfophthalic acid, but not its 3-isomer, can be completely mineralized aerobically by mixed cultures. In all chemical and microbial transformations of these aromatic sulfonates the 4-sulfo-isomer is more reactive than the 3-isomer. The triade of fission of the SPCsystem by white-rot fungi to SPI, chemical hydrolysis of SPI to SPAA, and microbial degradation of SPAA offers a pathway to mineralize the major part of the SPC system of textile dyes, whether in a respective effluent treatment system or in the aquatic environment. More general, these results illustrate on a molecular level how white-rot fungi and bacteria may cooperate in mineralizing structurally complex colored substances.
Introduction The removal of dyes from textile wastewater prior to its discharge or reuse is an important task of industrial wastewater treatment and biological as well as physicochemical means are being used for this purpose (1). Among those dyes that are resistant to bacterial degradation are sulfonated phthalocyanine (SPC) dyes. In the last years, white-rot fungi have been shown to decolorize SPC dyes (2-4) using different peroxidases (5), and only recently sulfophthalimide (SPI) was identified as the major breakdown product (6). However, decolorization by breaking up the chromatogenous system can only be a first step in textile effluent treatment; the reuse of industrial wastewater as well as its * Corresponding author phone: +49-30-31426429; fax: +49-3031423850; e-mail:
[email protected]. 10.1021/es010106+ CCC: $20.00 Published on Web 11/02/2001
2001 American Chemical Society
discharge into receiving waters requires a reduction of the organic load. Following a SPC dye treatment with white-rot fungi it would, thus, be essential to remove the fission product SPI, preferably by activated sludge treatment. Data on the aerobic biodegradability of this sulfonated aromatic compound are, however, not available. The aerobic degradability of aromatic sulfonated compounds is not easily predictable (7). For steric and electronic reasons the degradability tends to decrease with increasing degree of sulfonation, but the position of the sulfonate moiety in the aromatic system also has a strong influence, as shown for substituted benzene sulfonates (8) and for naphthalene sulfonates (9). As reviewed recently liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) is the most powerful technique to detect polar organic compounds in water (10), among them aromatic sulfonates (11, 12). Additionally, LC-MS allows for the detection of transformation products, also from polar sulfonated aromatic compounds (13). The use of LC-MS to study degradation processes may, thus, improve the understanding of natural as well as technical transformation processes of environmental chemicals. In this paper, the aerobic biodegradation of SPI by mixed cultures is studied in order to clarify whether the decolorization of SPC dyes by white-rot fungi would be a useful approach to diminish the wastewater problems related to the use of SPC dyes, e.g. in textile finishing. LC-MS was utilized to detect biodegradation intermediates as well as remaining degradation products.
Experimental Section Reference Materials and Stock Solutions. Sulfophthalic acid triammonium salt (technical grade, approximately 75% 4-SPA, 25% 3-SPA) was purchased from Aldrich (Milwaukee, USA), and phthalimide (PI; >99% purity) was received from Merck (Darmstadt, Germany). Sulfophthalimide (SPI) was synthesized by heating sulfophthalic acid triammonium salt (14) and cleaned by recrystallization as described elsewhere (6). Sulfophthalamic acid (SPAA) was prepared from the SPImixture according to instructions for synthesizing the nonsulfonated analogue by hydrolysis with 10% KOH at room temperature within 30 min (15, 16). The solution was neutralized, filled to a predefined volume, and used without any isolation of the products. All stock solutions used for the quantitation were prepared from mixtures of the respective 3- and 4-sulfo-isomers. Biodegradation Experiments. Aerobic biodegradation was investigated using a test procedure for “ultimate biodegradability” (17) in a climatic chamber at 20 °C and at a pH of 7.4 in the dark with activated sludge of a municipal wastewater treatment plant receiving both sanitary and industrial wastewater. Initial batch volume was 1.5 L at a substrate concentration of about 20 mg/L dissolved organic carbon (DOC). The following variants were investigated: SPI as sole carbon source, SPI poissoned (HgCl2), aniline as a reference substrate, PI as a second reference, and SPI together with PI (with about 40 mg/L of DOC). PI was used as it is the nonsulfonated analogue of SPI and is known to be readily degradable (18). In a second set of experiments, the biodegradation of SPA was tested with PI as a reference under the same experimental conditions as given above. Hydrolysis Experiments. Aqueous solutions of SPI with a concentration of 10 mg/L were adjusted to pH 3, pH 5, pH 6.5, and pH 9 with either H3PO4 or KOH and were filled in 1.5 mL HPLC vials and kept at room temperature. These VOL. 35, NO. 23, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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solutions were repeatedly injected into the HPLC-UV system over a period of 9.5 h, and a final analysis was made after 24 h. The vial containing the pH 3 sample was reanalyzed after 4 weeks. Sample Preparation and Bulk Parameters. Samples of the biodegradation experiments were filtered over 0.45 µm membrane filters (cellulose acetate; Macherey & Nagel, Du ¨ ren, Germany) and stored frozen until analyzed. The UVabsorbance at 254 nm was detected directly after filtration with a Lambda-2 spectrophotometer (Perkin-Elmer, U ¨ berlingen, Germany). The dissolved organic carbon content was determined by UV-persulfate oxidation with a high TOC analyzer (Elementar, Hanau, Germany) as nonpurgeable organic carbon (NPOC). HPLC-UV. Quantitative analyses of the biodegradation experiments were performed by HPLC with UV-detection. Calibration was performed with dilutions from stock solutions of SPI, SPAA, and SPA, assuming equal response for the respective 3- and 4-isomers. The concentration of the SPIsolution was corrected for the impurity of SPAA after SPAA was calibrated. A HP 1100 HPLC system equipped with a solvent degaser, a binary high pressure mixing pump, a column thermostate, an autosampler, and a UV-detector (all from Hewlett-Packard) was used. Separation was performed with a 125 × 3 mm column of Supersphere-100 RP18 endcapped 4 µm material (Knauer, Berlin, Germany) and a 5 × 3 mm precolumn. Eluent A was H2O/MeOH 80/20, and eluent B was H2O/MeOH 30/70, both with 5 mM tributylamine (TrBA, Fluka) and 5 mM acetic acid (HPLC quality; Mallinckrodt Baker, Deventer, Netherlands). Elution at 40 °C started with a linear gradient from 5% B to 90% B in 20 min, followed by 5 min isocratic elution and a return to the initial conditions at 26 min at a flow rate of 0.5 mL/min. Under these conditions, 3- and 4-SPI were not separated. A 10 µL volume of the undiluted samples was injected onto the column. HPLC-ESI-MS/MS. For the qualitative and further quantitative analyses of SPI, SPAA, and SPA with all their isomers a HPLC-ESI-MS/MS method was used (13). The LC-MS system consisted of a HP1100 HPLC system coupled to a Micromass Quattro LC triple quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped with a Z-spray atmospheric pressure ionization interface and an electrospray probe. For analyte separation a 150 × 2 mm 3 µm material phenylhexyl column (Phenomenex, Aschaffenburg, Germany) was used, and the analytes were detected by timeprogrammed multiple-reaction monitoring (MRM). Several transitions were selected for the selective and quantitative detection of all substances and isomers in the negative mode: 4-SPI (226>162, 226>118), 3-SPI (226>182; 226>162, 226>118), 4-SPAA1 (244>227, 244>200, 244>163), 4-SPAA2 (244>200, 244>157), 3-SPAA1 (244>227, 244>183), 3-SPAA2 (244>200), 4-SPA (245>201, 245>157), and 3-SPA (245>227, 245>183). The fragmentations underlying these transitions and further experimental details are given elsewhere (13). Samples of the degradation experiments were diluted 1/20, and a 5 µL-volume was injected for analysis. The LC-MS method allowed to also detect the sulfophthalamides (SPAM). However, SPAM were not detected in any of the experiments performed in this study and are, thus, not mentioned further. SPAM formation from SPI requires elevated ammonium concentrations such as in concentrated ammonium hydroxide (19).
Results and Discussion Aerobic Degradability of SPI. In the test on “ultimate degradability” with aerated activated sludge the DOC of the SPI solution remained stable for about 14 days, while the nonsulfonated phthalimide (PI) was mineralized within 3 days (Figure 1a). After day 14, however, the DOC of the SPI 4656
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FIGURE 1. (a) DOC content (mg/L) and (b) UV-absorbance at 254 nm (1/cm) of the aerobic die-away test of sulfophthalimide (SPI), the abiotic control (SPI+HgCl2), and phthalimide (PI). solution was rapidly diminished to a final 5 mg/L, corresponding to a 70% mineralization. This remaining 5 mg/L of DOC were, however, significantly higher than the residual DOC in the PI degradation (0.8 mg/L). In the abiotic control the DOC remains constant. When degrading a mixture of PI and SPI, we observed a rapid inital DOC diminution due to the mineralization of PI; then the DOC content followed that of the SPI batch with a final DOC of 4.1 mg/L. Despite the constant DOC-content during the first 2 weeks of the experiment some transformation occurred during the first 5 days, as indicated by the increasing UV-absorbance (+ 50% at 254 nm) in the biotic (SPI) as well as in the abiotic (SPI/HgCl2) batch (Figure 1b). While the UV-absorbance then remained stable in the abiotic control, it decreased parallel to the DOC in the biotic SPI batch. These results suggest that the long lag-phase observed for SPI-degradation was due to two processes: (a) an initial abiotic alteration reflected in an increasing UV-absorbance during the first 5 days of the experiments and (b) a period of adaptation required for the mixed culture to allow for significant mineralization of these abiotic transformation products of SPI. The initial abiotic transformation of SPI likely is a hydrolysis to sulfopthalamic acid (SPAA). Though literature on this transformation was not available, the parallel hydrolysis of the nonsulfonated PI to form phthalamic acid in diluted base at room temperature is well-known (15). Indeed, also the PI solution shows a slight initial increase in its UVabsorbance between day 0 and day 1 prior to the rapid mineralization (Figure 1b). When an aqueous SPI-solution was treated with diluted base, its UV-absorbance increased by a factor of 2. This suggests that SPAA exhibits a higher specific UV-absorbance than SPI. The inital increase of the UV-absorbance in the biodegradation experiment, then, reflects the hydrolysis of SPI to SPAA. Analysis of Degradation Products. To obtain detailed insight into these transformation processes a LC-ESI-MS/ MS method was used that was developed for SPI and a range of suspected and potential metabolites and which allowed for distinguishing between the structural isomers (13). Chromatograms obtained by MRM-detection of samples taken at day 0, day 5, and day 35 of the degradation experiment of SPI are displayed in Figure 2. Obviously, the starting material was not pure SPI but contained some SPAA (Figure 2a) from which four isomers exist (13). The SPAA present at the beginning indicates an incomplete synthesis or cleanup. At day 5 of the experiment (Figure 2b), all 4-SPI
FIGURE 3. Concentration of SPI and SPAA in the aerobic batch degradation experiment: (a) SPI poissoned with HgCl2 and (b) SPI with mixed cultures. Concentration of 3-SPAA1 and SPA not shown, since it remained below 10% of the total concentration throughout the experiment.
TABLE 1. Molar and Organic Carbon (DOC) Balance of the Transformation of Sulfophthalimide (SPI) in the Biotic and Abiotic (HgCl2) Batch
biotic
FIGURE 2. SPI degradation as detected by LC-MS with MRMdetection: (a) initial SPI mixture at 0 d, the SPAA already present was an impurity of the SPI; (b) at day 5, showing complete hydrolysis of 4-SPI to 4-SPAA; (c) at day 35, with 3-SPAA2 as major component; (d) SPI mixture after a 55 d abiotic experiment, showing complete hydrolysis of 4- and 3-SPI (for MRM transitions of the compounds refer to Experimental Section). was hydrolyzed, and the signals of the two 4-SPAA isomers markedly increased: hydrolysis of 3-SPI was still incomplete. After 35 days (Figure 2c) all 4-sulfo-isomers (4-SPI and 4-SPAA) were removed, but the corresponding 3-isomers (3-SPI, 3-SPAA) and some 3-SPA were still present. The time concentration curves of SPI and its major metabolites as obtained by HPLC-UV analyses are shown in Figure 3. The abiotic control (Figure 3a) confirms that the hydrolysis of SPI to 4-SPAA and 3-SPAA was a chemical process. Hydrolysis of 4-SPI was completed after 8 days, while the formation of 3-SPAA by hydrolysis of 3-SPI was still incomplete at day 35. In an independent experiment 3-SPI was completely hydrolyzed within 55 days (Figure 2d). Two SPAA-isomers can be formed from each of the two SPI-isomers (13), the 1-carboxyl-2-carboxamide-benzenesulfonate (here denoted as SPAA1) and the 1-carboxamide2-carboxyl-benzenesulfonate (SPAA2). Interestingly, the basecatalyzed hydrolysis of 4- and 3-SPI differs not only in its speed (see below) but also in the product spectrum. 4-SPI hydrolysis leads to a molar ratio of 55/45 for 4-SPAA1/ 4-SPAA2 (Figure 2b, Figure 3), indicating that the sulfonate moiety has only little influence on the mechanism of hydrolysis. This is consistent with the large distance between the sulfo-group and the reaction center. On the contrary, a sulfonate group in 3-position not only slows down the rate of hydrolysis but also strongly directs the base to the carboxylate carbon in β-position, yielding almost exclusively
abiotic
day
SPI (mmol/L)
metabolitesa (mmol/L)
DOC calcd (mg/L)
DOC detect. (mg/L)
0 5 35 0 5 35
0.12 0.03 0.01 0.08 0.04 0.01
0 0.10 0.03 0 0.07 0.09
19 21 4 16 18 17
17 17 5 17 16 15
a Σ (4-SPAA, 3-SPAA, 3-SPA), initial concentrations of SPAA subtracted.
the 3-SPAA2 isomer (Figure 2b-d): in all experiments a molar ratio of 5/95 for 3-SPAA1/3-SPAA2 was found. The structure of the SPAA-isomers was deduced from their fragmentation patterns observed in LC-MS/MS experiments (13). Apart from this first hydrolysis of SPI no further transformation was observable in the abiotic control (Figure 3a), indicating that 4-SPAA and 3-SPAA are stable under the experimental conditions. With the mixed culture (Figure 3b), however, the two 4-SPAA isomers were mineralized. We have not obtained any indication for a degradation of the 3-SPAA, though we cannot rule out that slow biodegradation of these isomers may also take place. The balance of the detected degradation products (Table 1) shows a reasonable agreement between the initial concentration of SPI (0.12 mmol/L and 0.08 mmol/L, respectively at day 0) and the intermediate sum of metabolites formed (0.10 and 0.07 mmol/L, day 5) as well as between the calculated and the measured dissolved organic carbon contents of the samples (Table 1). These data outline that no significant abiotic or biotic transformation product escaped from HPLC-UV or HPLC-MS analyses. This whole reaction sequence and further results discussed below are graphically depicted in Figure 4. Hydrolysis of SPI. Since the hydrolysis of SPI to SPAA appears to be a key step in this mineralization scheme and since this step may be essential for the speed of removal of SPI from an environmental compartment, the kinetic of this VOL. 35, NO. 23, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Degradation scheme starting from SPC-dyes (6) and leading to the mineralization of the 4-SPI, whereas 3-SPI remains as 3-SPAA2. The formation of 4-SPA as intermediate in 4-SPAA degradation is hypothetic.
TABLE 2. Hydrolysis Rates and Half-Lives of 3-SPI and 4-SPI at Different pH-Values According to a Pseudo-First-Order Kinetic Model pH 5
k
[s-1]
3-SPI nda 4-SPI 1.7 × 10-6 b
pH 6.5
t1/2 [h] nda 120b
k
[s-1]
5.0 × 10-6 2.5 × 10-5
pH 9
t1/2 [h] 40 8
k
[s-1]
2.3 × 10-5 1.8 × 10-4
t1/2 [h] 8 1
a nd: not detectable; at pH 3 3-SPI and 4-SPI remained stable over 1 month. b Rough estimate due to a short observation period of 24 h.
FIGURE 5. Concentration of 4-SPI during hydrolysis at different pH values in pure water. process and its pH-dependency in the range of pH 3 to pH 9 was studied separately. The hydrolysis rate was strongly influenced by the pH of the solution. The best graphical fit was obtained by a pseudo-first-order kinetic model, as shown in Figure 5 for 4-SPI. Generally, 4-SPI hydrolyzes 5-10 times faster than 3-SPI, and its half-lives range from 1 h at pH 9 to about 120 h at pH 5 (Table 2), while half-lives of 8 h at pH 9 to 40 h at pH 6.5 were detected for 3-SPI. At a pH of 3, no hydrolysis was observable for any of the two isomers over a time period of 4 weeks. This pH-dependency points out that the hydrolysis of both 3-SPI and 4-SPI is basecatalyzed. These kinetic results agree with the rate of hydrolysis observed in the degradation experiment (Figure 1). It is noteworthy that the 3-isomer of SPI is the less reactive one toward chemical hydrolysis and the 3-isomer of SPAA is the more stable one toward microbial degradation as compared to the respective 4-isomers. 4658
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Degradation of SPA. The pathway along which 4-SPAA is mineralized is unknown, and no degradation intermediates could be detected by HPLC with either UV- or MS-detection. We hypothesize that the degradation of 4-SPAA proceeds through a microbially catalyzed hydrolysis of 4-SPAA to 4-SPA, which may then be degradable by the mixed culture. A pure chemical hydrolysis at ambient pH can be excluded from the experimental results (Figure 2d). This parallels the behavior of the nonsulfonated analogue. It was previously shown that the hydrolysis of phthalamic acid is intramolecularly catalyzed by the (protonated) carboxylic acid moiety (16) and an acidic pH below the pKa of phthalamic acid (pKa ) 3.8) is thus required for a rapid hydrolysis. Consequently, phthalamic acid was found to be chemically stable at neutral pH, at which the carboxylate anion predominates (16). If the degradation of 4-SPAA would occur via 4-SPA, the latter compound must be easily degradable by mixed cultures as, otherwise, larger concentrations of 4-SPA had to be found in the biotic batches (Figure 2a-c). Thus, the degradation of a mixture of 3- and 4-SPA by mixed cultures was studied
TABLE 3. DOC-Content (mg/L) and UV-Absorbance at 254 nm (1/cm) for the Degradation of a Mixture of 3- and 4-Sulfophthalic Acid (SPA) and the Reference Compound Phthalimide (PI) SPA
PI
DOC (mg/L) abs254 (1/cm) DOC (mg/L) abs254 (1/cm) 0-8 days 19-55 days removal (%)
18.4 5.5 70
0.47 0.11 77
17.6 0.8 95
0.13 0.01 89
opens a pathway along which the major portion of SPC compounds, that have long been recognized as being persistent, can be mineralized (Figure 4). This is especially remarkable concerning the fact that SPC dyes have been shown to persist even in advanced oxidation procedures in which hydroxyl radicals are generated (20). These results illustrate on a molecular level the potential benefits of a combined wastewater treatment by white-rot fungi and bacteria, in which the white-rot fungi break up complex chemical structures (3, 6), and the bacteria then mineralize a substantial portion of the breakdown products. A first example of the benefits was recently provided by Knapp and co-workers (21), who observed an improved removal of bulk organic matter by activated sludge from a bleachery effluent, after pretreatment with a wood rotting fungus. Due to the difficulties of incorporating a fungal treatment into a continuous effluent treatment system (22) it may appear questionable, whether this triade or other sequential treatment processes with white-rot fungi and bacteria can be successfully implemented into a technical process. Nevertheless, these results have implications for the environmental fate of many colored and structurally complex compounds such as SPC that are not mineralized in degradation tests or wastewater treatments with bacteria but which may ultimately be mineralized by the concerted action of white-rot fungi and bacteria.
Acknowledgments FIGURE 6. Degradation of a mixture of 3- and 4-SPA in a batch experiment with mixed cultures monitored by LC-ESI-MS/MS analyses (13): 4-SPA (RT 14.36 min) detected by MRM transitions 245 > 184 and 245 > 201 (left axis); 3-SPA (RT 14.54 min) detected by the transitions 245 > 227 and 245 > 157 (right axis). in a batch experiment over 55 days. After an acclimation period of 8 days, 70% of the DOC and 77% of the UVabsorbance were removed until day 19; then all parameters remained stable until the end of the experiment (Table 3). The LC-MS analyses revealed that the 4-SPA was degraded between day 8 and day 19 (Figure 6), while the 3-SPA remained until day 55 (Figure 6). As the SPA mixture contains approximately 25% of the 3-SPA isomer according to the manufacturer, the 70% removal of the DOC and the results of the LC-MS analyses agree well. It is, thus, possible that 4-SPAA is degraded along a microbial hydrolysis followed by mineralization of 4-SPA as illustrated in Figure 4. For 3-SPAA, neither a microbially mediated hydrolysis to 3-SPA took place, as the 3-SPAA concentrations remained stable in the degradation experiment (see above), nor would this have resulted in mineralization, as 3-SPA was also stable during the time period of this biodegradation experiments. Thus, the portion of the 3-sulfo-isomer of SPI or SPAA, originating from the nonselective sulfonation of phthalocyanine to convert it into SPC, appears not to be degradable along the hydrolysis and mineralization pathway available for the 4-isomer (Figure 4). If this was true the 3-isomers should prevail in the environment; monitoring data are, however, not available. Perspective for the Mineralization of SPC Dyes and Other Structurally Complex Compounds. The whole sequence of abiotic transformation of SPI to SPAA and bacterial hydrolysis and mineralization of 4-SPAA has its starting point in the degradation of phthalocyanine dyes by white-rot fungi (3), by which SPI is formed (6). The triad of fungal fission, chemical hydrolysis, and aerobic bacterial mineralization
We gratefully acknowledge financial support by the German Research Council (DFG, Bonn) through SFB 193 ‘Biological Treatment of Industrial Wastewater’ project A14.
Literature Cited (1) Vandevivere, P. C.; Bianchi, R.; Verstraete, W. J. Chem. Technol. Biotechnol. 1998, 72, 289-302. (2) Knapp, J. S.; Newby, P. S.; Reece, L. P. Enzyme Microb. Technol. 1995, 17, 664-668. (3) Heinfling, A.; Bergbauer, M.; Szewzyk, U. Appl. Microbiol. Biotechnol. 1997, 48, 261-266. (4) Young, L.; Yu, J. Water Res. 1997, 31, 1187-1193. (5) Heinfling, A.; Martinez, M. J.; Martinez, A. T.; Bergbauer, M.; Szewzyk, U. FEMS Microbiol. Lett. 1998, 165, 43-50. (6) Heinfling-Weidtmann, A.; Reemtsma, T.; Storm, T.; Szewzyk, U. FEMS Microbiol. Lett. 2001, 203, 179-183. (7) Laue, A. M. Cook. H.; Junker, F. FEMS Microb. Rev. 1999, 22, 399-419. (8) Turnheer, T.; Cook, A. M.; Leisinger, T. Appl. Microbiol. Biotechnol. 1988, 29, 605-609. (9) Altenbach, B.; Giger, W. Anal. Chem. 1995, 67, 2325-2333. (10) Reemtsma, T. Trends Anal. Chem. 2001, 20, 500-517. (11) Storm, T.; Reemtsma, T.; Jekel, M. J. Chromatogr. A 1999, 854, 175-185. (12) Suter, M. J.-F.; Riediker, S.; Giger, W. Anal. Chem. 1999, 71, 897-904. (13) Reemtsma, T. J. Chromatogr. A 2001, 919, 289-297. (14) Ree´, A. Justus Liebigs Ann. Chem. 1886, 233, 226-240. (15) Aschan, O. Ber. Dtsch. Chem. Ges. 1886, 19, 1401-1404. (16) Bender, M. L.; Chow, Y.-L.; Chloupek, F. J. Am. Chem. Soc. 1958, 80, 5380-5384. (17) EN ISO 7827. In German Standard Methods for the Determination of Water, Wastewater and Sludge; Beuth Verlag: Berlin, 1995. (18) Pitter, P. Water Res. 1976, 10, 231-235. (19) Aschan, O. Ber. Dtsch. Chem. Ges. 1886, 19, 1398-1401. (20) Arslan, I.; Balcioglu, I. A. Dyes Pigm. 1999, 43, 95-108. (21) Zhang, F.-M.; Knapp, J.; Tapley, K. N. Water Res. 1999, 33, 919928. (22) Kapdan, I. K.; Kargi, F.; McMullan, G.; Marchant, R. Environ. Technol. 2000, 21, 231-236.
Received for review April 17, 2001. Revised manuscript received September 4, 2001. Accepted September 19, 2001. ES010106+
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