Environ. Sci. Technol. 1999, 33, 2566-2570
Effect of 2,4,6-Trinitrotoluene and Its Metabolites on Human Monocytes D. BRUNS-NAGEL,* S. SCHEFFER, B. CASPER, H. GARN, O. DRZYZGA, E. VON L O ¨ W, A N D D . G E M S A Institute of Immunology and Environmental Hygiene, Pilgrimstein 2, D-35037 Marburg, Germany
A lucigenin-dependent chemiluminescence (CL) assay was developed in which primary human monocytes were used as test cells. 2,4,6-Trinitrotoluene (TNT) and a number of its metabolites were subjected to this test system. A dose-dependent inhibition of CL was observed for most compounds. The following EC50 values were determined: TNT ) 8 µg/mL; 2,4-DANT ) 5 µg/mL; 4-ADNT ) 38 µg/mL; 2-ADNT ) >50 µg/mL; 4-acetyl-2,6-DNT) 50 µg/mL, and 2-acetyl-4,6-DNT ) >50 µg/mL. The CL assay was also applied to aqueous extracts of TNT-contaminated soil before and after bioremediation and to soil extracts free of any nitroaromatics. Extracts of non-contaminated soil reduced the CL by about 60%, whereas an extract of contaminated soil was 100% suppressive. Leachates of bioremediated soil caused a similar response as compost free of any nitroaromatics. The results demonstrate the usefulness of a primary human monocyte culture system for testing environmentally toxic compounds.
Introduction Nitroaromatics are nowadays ubiquitous contaminants of the environment (1). They are educts in drug, dye, and polyurethane foam production and are common compounds in perfumes. A special group of nitroaromatics are explosives. In particular, 2,4,6-trinitrotoluene (TNT) and precursor substances from TNT production contaminate the soil and groundwater of former manufacturing sites. TNT and related compounds have been tested in a number of investigations to assess their toxicity. It is kown that these substances are toxic toward bacteria, fish, plants, and mammalian cells (26). Furthermore, some of the compounds are mutagenic in bacterial tests (6, 7) and are also carcinogenic (8). Because of their toxicity, a cleanup of contaminated sites is needed. Biological techniques are of great interest for this purpose. They are cost-efficient and less destructive to the soil than incineration (9-13). Recently, an anaerobic/aerobic composting system was developed to bioremediate TNTcontaminated soil (14). Investigations with [14C]TNT-spiked soil indicated a stable incorporation of the xenobiotics into humic constituents of the soil (13). The use of 15N-labeled TNT and 15N solid-state NMR analyses revealed that more than 50% of the non-extractable residues were bound in a heterocyclic manner. Furthermore, aniline derivatives were detected (15). Risk assessment of contaminated sites and remediated soil is a complicated and controversial task (8, 16). One major * Corresponding author telephone: 0049-6421-285495; fax: 00496421-282309; E-mail:
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problem is the lack of data concerning the impact of these xenobiotics on humans since most available toxicological data deal with effects on animals, plants, and microorganisms, and it remains questionable whether results of these tests can be extrapolated to probable effects in human beings. To overcome this severe lack, we used primary human monocytes to develop a micro-test for the evaluation of immunotoxic effects of TNT and metabolites on human cells. For toxicological in vitro assessment studies, primary cells are more appropriate than cultured cell lines since the latter are frequently immortal tumor cell lines with a markedly altered responsiveness toward exogenous stimuli. We chose primary monocytes because of two major reasons. First, they play an important role in the human immune system. Negative effects on these cells most certainly are also detrimental to the whole organism. Second, the cells release energy as light during phagocytic activity. This energy emission can be amplified by chemicals such as lucigenin (N-methylacridinium nitrate) and then be measured with specially designed luminometers (17). Lymphocytes, which are important for the antigenspecific immune response of an organism, do not have the ability to emit light and are not suitable for the chosen test system. The principles of the lucigenin-dependent chemiluminescence (CL) are described briefly in the following. Phagocytic cells such as monocytes generate reactive oxygen derivatives and other highly reactive molecules during the respiratory burst. In this process, superoxide is generated by an NADPH-dependent oxidase. Thereafter, superoxide dismutase catalyzes the formation of H2O2, or it is generated spontaneously from superoxide. H2O2 is the substrate for myeloperoxidase, which synthesizes highly toxic metabolites such as hypohalogenides. During the reaction process, instable intermediates occur that emit light of a defined wavelength. After an amplification of this light emission by luciginin, for example, it can easily be measured. In the study presented here, TNT and different microbial TNT transformation products were subjected to this human monocyte-based test system. Furthermore, the test was applied to aqueous leachates of TNT-contaminated soil, TNTcontaminated soil that was bioremediated by anaerobic/ aerobic composting, and different soils free of nitroaromatic contaminations.
Materials and Methods Abbreviations of Test Substances. TNT (2,4,6-trinitrotoluene), 4-ADNT (4-amino-2,6-dinitrotoluene), 2-ADNT (2amino-4,6-dinitrotoluene), 2,4-DANT (2,4-diamino-6-nitrotoluene), 4-acetyl-2,6-DNT (4-acetylamino-2,6-dinitrotoluene), 2-acetyl-4,6-DNT (2-acetylamino-4,6-dinitrotoluene), 4,4′Az (4,4′-azoxy-2,2′,6,6′-tetranitrotoluene), and 2,2′-Az (2,2′azoxy-4,4′,6,6′-tetranitrotoluene) are used. Chemicals. TNT was purchased from Fluka (Buchs, Switzerland). It was recrystallized five times in ethanol before being used. 2,4-DANT was purchased from Promochem (Wesel, Germany). All other test chemicals were provided by the Department of Chemistry, Philipps University, Marburg (Germany). The purity of all chemicals used as test substances was 99% or more. The structures of the test chemicals are given in Table 1. RPMISuppl Buffer. A total of 500 mL RPMI 1640 medium (Seromed Biochrom KG, Berlin, Germany) was supplemented with 5 mL of each of the following solutions: 10 000 U/mL penicillin and 10 000 µg/mL streptomycin (Seromed Biochrom); L-glutamine 200 mM (Gibco BRL/Life Technologies); sodium pyruvate 100 mM (Seromed Biochrom); HEPES buffer 10.1021/es9813414 CCC: $18.00
1999 American Chemical Society Published on Web 06/11/1999
TABLE 1. Chemical Structures of TNT and Different Transformation Products That Were Tested for Immunotoxic Effects toward Human Monocytes
1 M (Seromed Biochrom); and non-essential amino acids (100-fold) (Seromed Biochrom). Preparation of Monocytes. Human monocytes were prepared from buffy coats of healthy blood donors. The mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. Thereafter, the monocytes were enriched by elutriation to a purity of >90% as determined by nonspecific esterase staining or fluorescence-activated cell sorting analysis using fluorescein isothiocyanate-labeled anti-CD-14 as previously described in detail by Sprenger et al. (18). Finally, the monocytes were suspended to a cell density of 6.25 × 106 cells/mL in RPMISuppl. Preparation of Test Solutions. TNT and Metabolites. The substances were dissolved in phosphate-buffered saline (PBS, without Mg2+and Ca2+, Seromed Biochrom). Stock solutions of a concentration of 100 mg/L were prepared for TNT, 4and 2-ADNT, 2,4-DANT, 2-acetyl-4,6-DNT, and 4-acetyl-2,6DNT. The concentration was checked by HPLC analyses. Furthermore, two azoxynitrotoluenes were dissolved. The solubility of these substances was very poor. Analyses showed that only about 0.5 mg/L 2,2′-Az and 2 mg/L 4,4′-Az could be dissolved. All test solutions were stored in darkness at 4 °C until being used. Soil Leachates. TNT-contaminated surface soil was collected on the ground of the former ammunition plant “Tanne” near Clausthal-Zellerfeld, Germany. HPLC analyses of methanolic soil extracts showed that the soil was contaminated with an average of 350 ( 60 mg of TNT, 5.6 ( 0.4 mg of 4-ADNT, and 4.9 ( 0.6 mg of 2-ADNT/kg of dry soil (mean ( standard deviation of five samples). No azoxynitrotoluenes or acetylated TNT metabolites were detectable. Bioremediated soil from the site was provided by the company Plambeck ContraCon (Cuxhaven, Germany). The soil was treated in a 50-L bioreactor (19) in a similar way as described by Winterberg et al. (14). The bioremediated soil contained
about 2.7 ( 1 mg of TNT, 0.7 ( 0.2 mg of 4-ADNT, and 0.7 ( 0.1 mg of 2-ADNT/kg of dry soil soil (mean ( standard deviation of five samples). No azoxynitrotoluenes or acetylated TNT metabolites were detectable. Soils free of nitroaromatics were provided by the botanical gardens in Marburg, Germany. Soil samples were air-dried and sieved (mesh size 50 µg/ VOL. 33, NO. 15, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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A
A
B B
C FIGURE 1. Dose-dependent inhibition of the lucigenin-dependent chemiluminescence response of primary human monocytes by TNT and its transformation products. Each data point represents the mean of nine parallel measurements.The bars indicate the standard deviation. (A) Effect of TNT and reduction products. (B) Effect of acetylated TNT reduction products. mL; 4-acetyl-2,6-DNT ) 50 µg/mL, and 2-acetyl-4,6-DNT ) >50 µg/mL. Inhibition of Monocyte CL by Soil Extracts. To evaluate whether the monocyte CL test is applicable to environmental samples, aqueous extracts of TNT-contaminated soil, bioremediated TNT-containing soil, and different soils with no history of nitroaromatic exposure were tested. Figure 2A,B shows that extracts of soil samples, free of any nitroaromatic contaminations, had already a significant effect on human monocytes. A 1:2 dilution caused an average CL inhibiton of about 60%. Sandy soil was only ineffective after a 128-fold dilution (Figure 2A). Clayey soil and garden soil had to be diluted 256-fold to show the same effect (Figure 2A,B). In the case of compost, even a dilution of 1:256 caused an inhibition of the luminiscence of about 40-50% (Figure 2B). In striking contrast, the TNT-contaminated soil quenched 100% of the CL in a 1:2 dilution (Figure 2C). A 256-fold dilution of the extract caused an inhibition of about 40% of the luminescence. Most interestingly, the plot of the extract of the bioremediated soil is very similar to the one of the compost (Figure 2B,C). The LC inhibition initially had a value of about 60% and a dilution up to 1:256 did not lead to a significant reduction of CL inhibition.
Discussion It is a well-known fact that nitroaromatics and some of their transformation products are toxic. In particular, the explosive TNT and its derivatives have been investigated extensively during the last years (2-7). However, to our knowledge, no 2568
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FIGURE 2. Dose-dependent inhibition of the lucigenin-dependent chemiluminescence response of primary human monocytes by different aqueous soil extracts. Each data point represents the mean of nine parallel measurements.The bars indicate the standard deviation. (A) Effect of sandy and clayey soil. (B) Effect of compost and garden soil. (C) Effect of TNT contaminated and bioremediated soil. investigations were performed concerning the effect of these chemicals on the human immune system. In this study, we show for the first time that TNT and some of its metabolites inhibit the production of activated oxygen species in human monocytes, which indicates that the phagocytic activity of the cells was impeded by these chemicals. In the lowest concentration tested, 2,4-DANT was even more effective than TNT itself (Figure 1A). ADNTs and acetylated metabolites, however, displayed much less CL inhibition (Figure 1A,B). Our results show clearly that the CL assay, as presented here, is a helpful tool to evaluate immunotoxic effects of xenobiotics to humans. To our knowledge, a similar test system has only been used yet with spleen mice cells to test reduction products of TNT and dinitrotoluene (DNT) (20).
not tested not tested no effect not tested not tested no effect not tested not tested 0.047 (0.28 for 2,6-DANT) 6.9 5.2 3.3
TA 98: +/+ TA 100: -/-
+
0.1 11.8 3
mutagenic + not mutagenic -
mutagenic + not mutagenic -
LC50 (µg/mL) EC50 (µg/mL) SR15 (µg/mL)
15.1 4.5 2.8
+ + not tested +
4 24 TA 98: +/+ TA 100: +/+ LC50 (µg/mL) LC50 (µg/mL) mutagenic + not mutagenic -
+
TA 98: -/TA 100 +/+ TA 98: -/TA 100: -/TA 98: -/TA 100: -/TA 98: -/TA 100: -/+
15.22-39.52 EC50 (µg/mL)
TA 98: -/+ TA 100: +/+
13 14 not tested 4 3 not tested
2.39-3.59 EC50 (µg/mL)
18 >250 TA 98: +/+ TA 100: +/+
66 >250 TA 98: +/+ TA 100: +/+
>250 >250 TA 98: not tested/TA 100: not tested/-
not tested not tested
8 EC50 (µg/mL)
18.73->49.29
39.43->49.29
≈20.90-.20.90
not tested not tested 16.18-21.17 >75
5 38 >50
47.99->75
no effect no effect
2,2′-Az 4,4′-Az 2,4-ADNT 4-ADNT 2-ADNT
inhibition of phagocytosis of primary human monocytes (this study) inhibition of bioluminescence Vibrio fischeri (30, 60, and 90 min) (3) inhibition of cell proliferation, different cell lines (ICP-81, ICP-RIA336D, NB4, and EL-4) (4) cytotoxicity, Reuber H35 H411E rat hepatoma cells (6) cytotoxicity, Chinese hamster ovary-K1 cells (6) mutagenicity assay, Ames test, Salmonella typhimurium TA 98 and TA 100 with S9/without S9 (24) mutagenicity assay, Ames test, Salmonella typhimurium TA 98 and TA 100 with S9/without S9 (6) mutagenicity assay, Ames test, Salmonella typhimurium TA 100 without S9 (25) acute toxicity, fathead minnows (21) acute toxicity (48 h), Daphnia magna (26) reduction to 15 offspring/surviving female, Ceriodaphnia dubia (27)
TNT effect applied test system and ref
TABLE 2. Summary of Some Important Toxicological Effects of TNT and Some of Its Metabolites
The authors found that aminotoluenes (triaminotoluene and diaminotoluene) inhibited CL much more effectively than aminonitrotoluenes. Nitrotoluenes such as TNT and DNT were not tested. In our study, we could demonstrate that TNT and 2,4-DANT strongly effeced the CL of human monocytes, which are the most important phagocytic cells to initiate an immune response. Krass (4) tested the effect of 50 different nitroaromatics, aromatic amines, and aromatics on the proliferation of four different cell lines (ICP-81, ICP-RIA336D, NB4, and EL-4). The EC50 values for TNT, 4- and 2-ADNT, and 2,4-DANT determined in this study are compiled in Table 2. Most interestingly, our test system was more sensitive toward TNT and 2,4-DANT than the one used by Krass (4). Drzyzga et al. (3) tested numerous explosives and metabolites with the luminescence inhibition test using Vibrio fisheri as test organism. Some of their results are also summarized in Table 2. As in the test performed by Krass (4), 2,4-DANT showed a much higher toxic effect toward monocytes than to the bacterium V. fischeri. Supporting the results of Drzyzga et al. (3), other authors found a reciprocal relationship between the toxicity of the TNT metabolites and the number of reduced nitro groups for different cell lines (4, 6) and fish (21) (Table 2). Honneycutt et al. (6) proved this finding in cytotoxicity tests with Reuber H35 H4IIE rat hepatoma cells and Chinese hamster ovary-K1 (CHO) cells (Table 2). The rat hepatoma cells were more sensitive to TNT and 2-ADNT than human monocytes. To our knowledge, no data have been published about the effect of TNT and metabolites toward human hepatocytes. Butterworth et al. (22) used primary cultures of human and rat hepatocytes to test 25 chemicals for their genotoxic activity. They found that 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and 2-nitrobenzyl alcohol were not genotoxic for both cell types, whereas 2,4-diaminotoluene was genotoxic for both cells, and 2,6-diaminotoluene elicited a DNA repair response only in human cells. TNT and metabolites were not tested. The results of Butterworth et al. (22) underline the importance of risk assessment studies with human cells. Even though in most cases the results of the rat and human cells are similar, there are exceptions possible. Honeycutt et al. (6) reported results for 4,4′- and 2,2′-Az that differ from our data. In their study, LC50 values of 3 and 14 µg/mL, respectively, were determined (Table 2). In contrast, these substances showed no effect in our CL assay. The low water solubility of these compounds is most probably responsible for the absence of an effect. Other researchers overcame this problem by using solvents such as DMSO as solubilizer. In the case of the monocytes, this is not possible since these cells are extremely sensitive toward solvents. For these reasons, the immunotoxic effect of azoxy compounds on human monocytes remains unclear at present. At this point, it should be mentioned that we did not determine LC values, but EC values. The suppression of CL was not caused by overt toxicity or killing of the cells, which was confirmed by LDH release and the MTT test (data not shown) (23). Furthermore, Honneycutt et al. (6) found that 2,4-DANT was not genotoxic in the Ames test, whereas TNT showed a positive response. The reduction products 2- and 4-ADNT were genotoxic in some cases and not in others (6, 24, 25). An explanation why 2,4-DANT showed such a strong effect in our CL assay cannot be provided at this time. Further investigations are needed to understand this phenomenon. Examinations with different water fleas also showed that a reduction of TNT can cause an increase of toxicity (Table 2) (26, 27). In the case of Ceriodaphnia dubia, 2,4-DANT was also signifcantly more toxic than TNT or ADNT (27). Acetylated TNT metabolites were tested in this study because several authors reported the generation of such TNT
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transformation products by microorganisms (12, 28, 29). In these studies, however, only acetylamino aminonitrotoluenes were analyzed and no acetylamino dinitrotoluenes. Unfortunately, only acetylamino dinitrotoluenes were available to us in sufficient amounts to perform toxicological tests. The results of the tests show that these substances have a suppressive effect on human monocytes, but they were much less potent than TNT. A feature of toxicological test systems should be their applicability to real samples. To investigate this, soil extracts of TNT-contaminated and bioremediated soil and extracts of soils free of any nitroaromatics were subjected to our CL assay. The results of these tests indicate a correlation between the amount of organic material and the inhibition of the CL response. Both soils, compost and the bioremediated soil, were rich in organic matter. The latter was because the soil was remediated by an anaerobic/aerobic composting process, which includes an addition of 20% (w/w) organic material to the contaminated soil. Most likely, the CL suppressive effect of the remediated soil is not due to residues of any xenobiotic compounds. It might be caused by organic material that also occurs in common compost. The data presented in this study show that the use of human monocytes in a CL assay represents a very promising in vitro test system to obtain information on immunomodulating effects of xenobiotics for humans. In our study, we used TNT and different metabolites as test chemicals. It most certainly would have been of greatest interest to examine other environmental pollutants such as polycyclic aromatic hydrocarbons, halogenated organics, heavy metals, etc. At present, a disadvantage of our test system is that we used primary human monocytes. The system would be more effective with cultured cell lines which, however, should maintain the properties of primary cells. The test could be performed much faster, and the variability of cells from different blood donors could be overcome. For this reason, future studies are directed toward the utilization of monocytic cell lines, which could be particularly suitable for CL testing of potentially hazardous compounds for humans.
Acknowledgments This work was supported by the Bundesministerium fu ¨r Forschung, Wissenschaft, Bildung und Technik (BMBF), by the state Lower Saxony, and by the Industrieverwaltungsgesellschaft AG (IVG), Bonn, Germany. We also thank Dr. T. C. Schmidt (EAWAG/ETH, Zu ¨ rich) for helpful discussions and critically reading this manuscript.
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(5) Layton, D.; Mallon, B.; Mitechel, W.; Hall, L.; Fish, R.; Perry, L.; Snyder, G.; Bogen, K.; Malloch, W.; Ham, C.; Dowd, P. Conventional weapons demilitarization: A health and environmental effects database assessmen. Explosives and their cocontaminants. Final Report, Phase II; U.S. Army Medical Research and Development Command: Fort Detrick, MD, December 1987; UCRL-21109. (6) Honeycutt, M. E.; Jarvis, A. S.; McFarland, V. A. Ecotoxicol. Environ. Saf. 1996, 35, 282-287. (7) Tan, E.; Ho, C. H.; Griest, W. H.; Tyndall, R. L. J. Toxicol. Environ. Health 1992, 36, 165-172. (8) Neumann, H. G. Food Chem. Toxicol. 1996, 34, 1045-1051. (9) Gorontzy, T.; Drzyzga, O.; Kahl, M. W.; Bruns-Nagel, D.; Breitung, J.; von Lo¨w, E.; Blotevogel, K.-H. Crit. Rev. Microbiol. 1994, 20, 265-284. (10) Spain, J. C., Ed. Biodegradation of nitroaromatic compounds; Plenum Press: New York, 1995. (11) Breitung, J.; Bruns-Nagel, D.; Steinbach, K.; Kaminski, L.; Gemsa, D.; von Lo¨w, E. Appl. Microbiol. Biotechnol. 1996, 44, 795-800. (12) Bruns-Nagel, D.; Breitung, J.; von Lo¨w, E.; Steinbach, K.; Gorontzy, T.; Kahl, M.; Blotevogel, K.-H.; Gemsa, D. Appl. Environ. Microbiol. 1996, 62, 2651-2656. (13) Drzyzga, O.; Bruns-Nagel, D.; Gorontzy, T.; Blotevogel, K.-H.; Gemsa, D.; von Lo¨w, E Environ. Sci. Technol. 1998, 32, 35293535. (14) Winterberg, W.; von Lo¨w, E.; Held, T. TerraTech 1998, 3, 39-41. (15) Knicker, H.; Bruns-Nagel, D.; Drzyzga, O.; v. Lo¨w, E.; Steinbach, K. Environ. Sci. Technol. 1999, 33, 343-349. (16) Schneider, K.; Hassauer, M.; Kalberlah, F. UWSF-Z. Umweltchem. O ¨ kotoxicol. 1994, 6, 333-340. (17) Thomas, V. L.; Sanford, B. A.; Driscoll, M. S.; Casto, D. T.; Ramaurthy, R. S. J. Immunol. Methods 1988, 111, 227-232. (18) Sprenger, H.; Krause, A.; Kaufmann, A.; Priem, S.; Fabian, D.; Burmester, G. R.; Gemsa, D.; Rittig, M. G. Infect. Immunol. 1997, 65, 4384-4388. (19) Stoffers, H.; Winterberg, R.; Breitung, J.; Bruns-Nagel, D.; von Lo¨w, E.; Fischer, M. Poster at the Fifth International KfK/TNO Conference on Contaminated Soil, Maastricht, 1995. (20) Thierfelder, W.; Masihi, N. Int. J. Immunopharmacol. 1995, 17, 453-456. (21) Bailey, H. C.; Spanggord, R. J. In Aquatic Toxicology and Hazard Assessment: Sixth Symposium, ASTM STP 802; Bishop, W. E., Cardwell, R. D., Heidolph, B. B., Eds.; American Society for Testing and Material: Philadelphia, 1983; pp 98-107. (22) Butterworth, B. E.; Smith-Oliver, T.; Earle, L.; Loury, D. J.; White, R. D.; Doolittle, D. J.; Working, P. K.; Cattley R. C.; Jirtle, R.; Michalopoulos, G.; Strom, S. Cancer Res. 1989, 49, 1075-1084. (23) Garn, H.; Krause, H.; Enzmann, V.; Drossler, K. J. Immunol. Methods 1994, 168, 253-256. (24) Tan, E. L.; Ho, C. H.; Griest, W. H.; Tyndall, R. L. J. Toxicol. Environ. Health 1992, 36, 165-175. (25) Spanggord, R. J.; Stewart, K. R.; Riccio, E. S. Mutat. Res. 1995, 335, 207-211. (26) Pearson, J. G.; Glennon, J. P.; Barkley J. J.; Highfill J. W. In Aquatic tocicology; Kimmerle L. L., Ed.; ASTM STP 667; Ameerican Society for Testing Materials: Philadelphia, 1979; p 284. (27) Stewart, A. J. Oak Ridge National Laboratory, personal communication, 1999. (28) Gilcrease, P. C.; Murphy, V. G. Appl. Environ. Microbiol. 1995, 61, 4209-4214. (29) Bruns-Nagel, D.; Drzyzga, O.; Steinbach, K.; Schmidt, T. C.; von Lo¨w, E.; Gorontzy, T.; Blotevogel, K.-H.; Gemsa, D. Environ. Sci. Technol. 1998, 32, 1676-1679.
Received for review December 23, 1998. Revised manuscript received May 7, 1999. Accepted May 11, 1999. ES9813414
