Biological Treatment of TNT-Contaminated Soil. 2. Biologically

Flow sheet of the biological anaerobic/aerobic treatment of explosives-contaminated soil in technical scale. FIGURE 5. Time course of the load of the ...
0 downloads 16 Views 182KB Size
Download PDF
Environ. Sci. Technol. 1998, 32, 1964-1971

Biological Treatment of TNT-Contaminated Soil. 2. Biologically Induced Immobilization of the Contaminants and Full-Scale Application HILTRUD LENKE,† JU ¨ RGEN WARRELMANN,‡ G R E G O R D A U N , †,§ K E R S T I N H U N D , | UTE SIEGLEN,† ULRICH WALTER,‡ AND H A N S - J O A C H I M K N A C K M U S S * ,† Fraunhofer-Institut fu ¨ r Grenzfla¨chen und Bioverfahrenstechnik, Nobelstrasse 12, 70569 Stuttgart, Germany, Umweltschutz Nord GmbH & Co., Industriepark 6, 27767 Ganderkesee, Germany, and Fraunhofer Institut fu ¨r Umweltchemie und O ¨ kotoxikologie, Auf dem Aberg 1, 57392 Schmallenberg-Grafschaft, Germany

Anaerobic treatment of originally contaminated soil from a former ammunition plant was carried out in a laboratory slurry reactor. While fermenting glucose to ethanol, acetate, and propionate, the anaerobic bacteria completely reduced the nitro groups of 2,4,6-trinitrotoluene (TNT) and aminodinitrotoluenes, which led to a complete and irreversible binding of the reduced products to the soil. 2,4Dinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine were also reduced in the soil slurry and were no longer detectable after the anaerobic treatment. To mineralize the fermentation products, a subsequent aerobic treatment was necessary to complete the bioremediation process. This bioremediation process was tested in a technical scale at Hessisch Lichtenau-Hirschhagen, Germany. A sludge reactor (Terranox system) was filled with 18 m3 of contaminated soil (main contaminants were TNT, 2,4dinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine) and 10 m3 of water. The anaerobic stage was carried out by periodical feeding of sucrose. The sludge was subsequently dewatered and treated aerobically. Chemical analysis revealed an overall reduction of more than 99% of the contaminants. Ecotoxicological tests performed with various aquatic systems (luminescent bacteria, daphnids, algae) and terrestrial systems (respiring bacteria, nitrifying bacteria, cress plants, earth worms) showed that residual toxicity could not be detected after the anaerobic/aerobic treatment.

Introduction Because of the environmental contamination caused by the significance of 2,4,6-trinitrotoluene (TNT), an increasing * Corresponding author, phone: +49-711-685-5487; fax: +49711-685-5725; e-mail: [email protected]. † Fraunhofer-Institut fu ¨ r Grenzfla¨chen und Bioverfahrenstechnik. ‡ Umweltschutz Nord GmbH & Co. § Present address: BASF AG, ZET/ZH, 67056 Ludwigshafen, Germany. | Fraunhofer Institut fu ¨ r Umweltchemie und O ¨ kotoxikologie. 1964

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 13, 1998

number of reports deal with the microbial degradation of this compound especially under anaerobic conditions. Thus, Funk et al. (1) reported the conversion of TNT via 2,4,6trihydroxytoluene to o-cresol by a Clostridium strain. Boothpathy et al. (2) claimed that TNT is metabolized to toluene by sulfate-reducing organisms. In both cases the formation of 2,4,6-triaminotoluene (TAT) as a metabolite was postulated, but up to now evidences for these degradative pathways were still lacking. That complete reduction of TNT leading to TAT, reported by Preuss et al. (3) in a sulfatereducing organism, is a widely found activity in nature could be confirmed by the fact that an anaerobic sludge cometabolically reduces TNT completely to TAT as shown in the accompanying paper (4). Recently, a Clostridium bifermentans was shown to reduce TNT to TAT (5). Phenolic products as a result of TAT hydrolysis and the formation of an adduct of TAT with methyl glyoxal have been described (5). Although further microbial conversion of TAT leading to hitherto unknown metabolites was also observed with the TNTreducing anaerobic sludge (4), this catabolic activity is rather slow and not applicable for the microbial degradation of TNT in the presence of soil components. Anaerobic treatment of artificially contaminated montmorillonitic clay or humic acids led to a complete removal of TNT and its metabolites from the solution. Especially TAT and 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6dinitrotoluene (2-HADNT/4-HADNT) showed strong sorptive interactions with soil components. Therefore, the complete removal of TNT through cometabolic reduction may be explained by irreversible binding of both 2-HADNT/4HADNT and TAT. To test the biologically induced immobilization of TNT as an option of remediation, contaminated soil material from a former ammunition plant at Hessisch Lichtenau-Hirschhagen (Germany) was treated by an anaerobic/aerobic process. The area near Kassel was one of the largest armament plants in Germany with a total production of 135 000 t of TNT between 1938 and 1945. The area comprised 230 ha with 400 buildings, e.g., production sites, filling, and pressing facilities. Half a century after the end of the production, high amounts of TNT and its byproducts are still detectable in soil and groundwater. Because this ground is still used as an industrial and residential area, first measures at this site were to deposit safely some highly contaminated soil and to purify the groundwater with activated carbon filters. On the long run, however, remediation of the contaminated soil is necessary. To evaluate the possibility to bioremediate the TNT-contaminated soil the biologically induced immobilization of TNT was tested in the present study at a technical scale. To evaluate the treated soil material, ecotoxicological tests in aqueous soil extracts and in the soil itself were carried out.

Experimental Section Organisms. For experiments in laboratory scale, the anaerobic mixed culture that was able to reduce TNT while growing on glucose was used (4). For the full-scale experiment, an anaerobic sludge was collected from the ground of a water reservoir at the former production site Hessisch LichtenauHirschhagen, which contained wash water contaminated with TNT and related compounds. Chemicals. Highly pure TNT was generously supplied by T. Rosendorfer (MBB Deutsche Aerospace, Schrobenhausen, Germany) while 2-amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino6-nitrotoluene (2,4-DANT), and 2,4,6-triaminotoluene triS0013-936X(97)00950-4 CCC: $15.00

 1998 American Chemical Society Published on Web 05/21/1998

hydrochloride (TAT) were obtained from Promochem (Wesel, Germany). A mixture of 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene (2-HADNT/ 4-HADNT) was enzymatically produced (4). 4,4′,6,6′-Tetranitro-2,2′-azoxytoluene and 2,2′,4,4′-tetranitro-4,4′-azoxytoluene were kindly supplied by J. Michels (DECHEMA, Frankfurt am Main, Germany) and D. Bruns-Nagel (Philipps University, Marburg, Germany). All other chemicals were obtained from commercial sources. Soil. The contaminated soil used in the present study was obtained from a former TNT production site at Hessisch Lichtenau-Hirschhagen near Kassel (Germany). The soil had the following characteristics: Solid fraction part of 89.1% (dry substance according to ref 6), pH of 7.7 (according to ref 7), organic matter 21 mg of Corg/g dry substance (according to ref 7), and a silt and clay content of 47%. Laboratory-Scale Experiment. A slurry of 850 g of contaminated soil and 850 mL of mineral medium (4) in a 1.3-L bioreactor with an initial glucose concentration of 6 mM was inoculated with the anaerobic mixed culture described (4). The soil slurry was stirred with a helical ribbon impeller (200 rpm). The temperature of the bioreactor was kept at 30 °C, and the pH value was controlled at 7.0 by adding a 10 M NaOH solution. The reaction mixture was protected from the access of air, and the gas exchange with the environment was minimized to establish anaerobic conditions. Glucose was added 10 times a day at an average rate of 0.9 mM glucose/day (every 2.4 h). Due to the oxidation of glucose by the autochthonous soil microorganisms, the redox potential, which was measured with a platinum electrode connected with a Ag/AgCl reference system (Ingold Pt-4805-S7, Steinbach, Germany), dropped from Eh +300 mV at the beginning to -100 mV within 5 days and finally to e-200 mV after 20 days. Samples of 5-10 mL of the soil slurry were centrifuged for 20 min at 3200g to measure the concentration of the contaminants and their metabolites and the fermentation products of glucose. The remaining soil pellet of each sample was extracted two times for 1 h with methanol and dried at 105 °C to refer the amount of sorbed and extractable contaminants to the dry mass. Full-Scale Experiment. The anaerobic treatment process was carried out in the Terranox reactor, which was developed for anaerobic treatments and consists of a trough that is built in segments (5.0 m long, 1.8 m wide, and 2.0 m high) to a length up to 50 m. The reactor is closed with a detachable steel cover to minimize the exchange of gas between the content of the reactor and the outside atmosphere. Each segment is equipped with two sampling points in order to check the status of degradation as well as biological parameters. The reactor is equipped with a stirring device with maintenance-free cutters to homogenize the soil slurry and to mix in nutrients. The number of revolutions of the stirring aggregate (up to 150 revolutions/min) depends on the requirements of the single activities (homogenization or nutrient feeding) and is controlled by a speed governor. For the anaerobic treatment of TNT-contaminated soil, the reactor was filled with 10 m3 of water and 18 m3 of TNTcontaminated soil. To keep the temperature at 15-20 °C, the reactor was encased with an insulating cover foil (polyethylene) that isolated the outside of the reactor and allowed the walls of the reactor to heat by blowing warm air through a perforated pipe. At the beginning of the experiment, anaerobic sludge (0.1 m3), sucrose (80 kg), ammonium chloride (10 kg), and hydrogen phosphate/dihydrogen phosphate (22 kg) were supplemented. The soil suspension was homogenized twice weekly, sucrose was fed at different experimental times, and the pH value was controlled after about 2 days of feeding sucrose and set to a pH of 6.5-7.5 by adding CaO. The anaerobic process was stopped by adding 6 m3 of freshwater and 0.6 m3 of H2O2 (30% w/v). The

resulting 31.5 m3 of soil slurry was treated with a solution of a polymeric flocculent (2.5 m3) and dehydrated with a screen head extruder. After adding bark (3.5 m3), and compost (5.5 m3) as organic structural material to the dehydrated soil (18 m3) the aerobic treatment was carried out as soil windrow. The soil mixture was turned over weekly by a front loader. Soil Sampling. To determine the initial load of the contaminated soil 6 × 5 g of soil (untreated) and 6 × 5 g of soil (air-dried and homogenized by sieving and crushing with a pestle and mortar) were extracted twice for 1 h with methanol at 30 °C. To refer the amount of sorbed and extractable contaminants to the dry mass, the soil samples were dried at 105 °C after extraction. At regular intervals of the anaerobic treatment process, eight soil slurry samples (1 L each) were collected per sampling time from the different segments of the Terranox reactor. After homogenizing and combining the 8 samples 2 × 9-10 mL of the soil suspension were centrifuged for 20 min at 5000 rpm (approximately 3200g) to measure the concentrations of contaminants and their metabolites in the supernatant. The remaining soil pellet was extracted twice for 1 h with methanol at 30 °C and dried at 105 °C to refer the amount of sorbed and extractable contaminants to the dry mass. To quantify the fermentation products by GC 98% formic acid was added to the aqueous supernatant at a ratio of 1:10. During the aerobic treatment process 8 soil samples (1 kg each) were taken at regular intervals from different areas, homogenized, combined and 2 × 4-5 g soil (6 × 4-5 g soil of the last sampling, respectively) were extracted with methanol as described above. To improve the detection limit of the contaminants and metabolites the methanolic extracts were evaporated in a vacuum to 10% of the initial volume. Various desorption experiments were carried out with the biologically treated soil after the anaerobic phase as well as after the subsequent aerobic phase. The soil samples (7-8 g each, the soil samples after the anaerobic treatment were centrifuged for 20 min at 5000 rpm, approximately 3200g) were extracted with 35-40 mL of 2 M NaOH or 2 M HCl for 24 h at 30 °C. Soil Samples taken after 1, 5, and 24 h were centrifuged, neutralized, and analyzed by HPLC. Additionally, soil samples (7-8 g each) were extracted with 35-40 mL of 5 M NaOH/methanol (1: 2, v/v) for 2 h at 90 °C. Samples were taken at 1 and 2 h, and the liquid-phase was analyzed by HPLC after separation of the soil by centrifugation. Analytical Methods. Concentrations of nitrotoluenes and their metabolites in soil slurries were determined by reversedphase high-pressure liquid chromatography (HPLC) with a Lichrocart 250-4 column, filled with 5 µm particles of Lichrospher 100 RP-8 (Merck, Darmstadt, Germany). 2,4Diaminotoluene (2,4-DAT), 2,6-diamino-4-nitrotoluene (2,6DANT), 2,4-diamino-6-nitrotoluene (2,4-DANT), hexahydro1,3,5-trinitro-1,3,5-triazine (RDX), 4-amino-2-nitrotoluene (4A-2-NT), 2-amino-4-nitrotoluene (2-A-4-NT), 2,4,6-trinitrotoluene (TNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2-amino-4,6-dinitrotoluene (2-ADNT), and 2,4-dinitrotoluene (2,4-DNT) in supernatants or methanolic extracts of soil slurry samples were separated by the following gradient system: The program started with 30% methanol and 70% H2O, changed after 10 min within 0.1 min to 40% methanol and 60% H2O, and remained at this composition for another 26.4 min. Then the ratio was set to 50% methanol and 50% H2O between 0.1 min remaining at this composition for at least 9.4 min. To equilibrate the column for the next run, the composition for the last 3.9 min of the run was set back to 30% methanol and 70% H2O. The flow rate was 1 mL/min, and the detection was carried out at 220 nm. 4,4′,6,6′Tetranitro-2,2′-azoxytoluene and 2,2′,4,4′-tetranitro-4,4′azoxytoluene were analyzed by using a mobile phase of 70/ 30 (v/v) methanol/H2O. TAT was analyzed as described VOL. 32, NO. 13, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1965

in the accompanying paper (4). The contaminants and metabolites were identified by their UV-visible spectra measured in situ by a diode array detector and their retention volumes. Ethanol, acetate, propionate, and butyrate were determined with a HP 5890 series II gas chromatograph (HewlettPackard, Palo Alto, CA) with a FFAP-CB column filled with poly(ethylene glycol) nitroterephthalate (25 m × 0.32 mm, i.d., 0.3 µm film thickness, Chrompack, Frankfurt, Germany). The gas chromatograph was equipped with a flame ionization detector, and the detection was carried out at 250 °C. Conditions for analysis on the FFAP-CB column were 85 °C for 5 min, programmed at 2 °C/min to 107 °C, at 10 °C/min to 140 °C, and finally at 20 °C/min up to 250 °C. The samples were injected into the gas chromatograph using a split/ splitless injection system (split of 85 mL/min) at an injection temperature of 250 °C. Helium at a flow rate of 1 mL/min was used as carrier gas. Ecotoxicological Tests in Aqueous Soil Eluates. For the preparation of the aqueous eluates, soil samples were shaken with deionized water (soil, dry weight/water ) 1:2.5; the water content of the soil was considered) for 24 h at room temperature in the dark. For the ecotoxicological tests, the aqueous phase obtained after centrifugation (20.000g; 20 min) was used. The eluates were stored at 4 °C up to 7 days. In case the tests could not be performed within this period, the eluates were frozen (-20 °C). The standardized tests with luminescent bacteria, algae, and daphnids were carried out following the guidelines in refs 8, 9 (luminescence inhibition test with Vibrio fischeri), in refs 10 and 11 (algae, growth inhibition test) as well as in refs 12 and 13 (Daphnia magna: acute immobilization test). To increase the sensitivity of the test with daphnids, some modifications were done. The incubation period was prolonged from 24 to 48 h (14); additionally 20 daphnids per concentration were used. The results are presented as GL, GA, and GD values (GL, GA: lowest value for the dilution factor of the eluate which exhibits less than 20% effect; GD: lowest value for the dilution factor of the eluate which exhibits less than 10% effect). According to ref 14, the samples with GA, D > 4 and GL > 8 were classified as toxic. Ecotoxicological Tests in the Soil. Substrate-induced microbial respiration activity was tested according to the guideline in ref 15. The soil was adjusted to 50% of the maximum water holding capacity (WHCmax), well mixed with glucose (0.4%), and incubated at 22 °C. Ammonium oxidation activity (nitrification) was determined according to Berg and Roswall (16) and modified according to Kandeler (17). The test on mortality of earthworms was performed according to refs 14 and 18. According to ref 14, samples were classified as toxic for a respiration activity of 32 1 1 1

given given low low

a According to ref 9; G , rate of dilution, when the effect is less than 20%; classification as toxic when G > 8 (14). b According to ref 13; G , L L D rate of dilution, when the effect is less than 10%; classification as toxic when GD > 4 (14). c According to ref 11; GA, rate of dilution, when the effect d is less than 20%; classification as toxic when GA > 4 (14). Evaluation according to ref 14.

TABLE 3. Microbial Activities after Anaerobic/Aerobic Treatment Process of TNT-Contaminated Soil test

measurement

min demand of turnovera

unit

microbial respiration activity (O2 uptake) microbial nitrification (rate of nitrite formation)

4.5 1514

0.3 100

mg of O2/(h × 100 g dry soil) ng of N/(5 h × g dry soil)

a

According to ref 14.

are given as G values (Table 2). The higher the G value, the more toxic is the sample. Whereas the untreated soil, the soil eluate, and the supernatant after the anaerobic treatment process showed toxic effects, no toxicity was detectable in the soil eluate after the subsequent aerobic treatment. This indicates that the aerobic treatment is indispensable for complete bioremediation. The results of the tests with the soil eluates clearly showed a different sensitivity of the organisms at the beginning and during the bioremediation process. This result supports the data obtained from chemical analyses where only very low concentrations of TNT and 2,4-DNT were detected. With regard to the EC50 values determined for these substances (TNT: 1.8 mg/L [alga, unpublished]; 2.3 mg/L [luminescent bacteria; unpublished]; 11.7 mg/L [daphnids, 27]; 2,4-DNT: 2.5 mg/L [alga, unpublished]; 2 mg/L [luminescent bacteria; unpublished]; 26.2 mg/L [daphnids, 28]) the concentrations determined by chemical analysis were too low to cause a pronounced effect on the aquatic test systems. Terrestrial tests were performed only at the end of the bioremediation process. The results obtained in the tests with earthworms and cress indicate a sufficient habitat function of the treated soil: No mortality was observed for earthworms. The plant test with cress (Lepidum sativum) showed no toxic effects of the treated soil. Both the overground growth of the cress plants and the degree of rooting were not inhibited and comparable to the control. Furthermore, the microbial respiration activity as well as the nitrification activity considerably exceeded the defined minimum activities (Table 3). Generally microbial activity in natural soils varies considerably according to soil type, its use, and the climate conditions (29-31). For reuse of remediated soils, it is essential that the nutrient cycles can proceed. As during bioremediation, inorganic nutrients or organic substrates are usually added to the contaminated soil; these are not comparable to soils in the environment; and the microbial activities measured in the substrate at the end of bioremediation are difficult to assess. To have a criterion for the estimation, minimum values were defined that have to be reached by remediated soils (14). The microbial activities measured in the substrate at the end of the described remediation procedure exceeded the defined values by a factor of 15. Insam (32) determined the microbial biomass in 21 agricultural soils with the substrate-induced respiration method. The respiration activity referring to the soil with the highest microbial biomass is about a factor of 3 lower than the respiration activity determined in the present 1970

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 13, 1998

study. This may be due to the high content of bark and compost added before the aerobic treatment. Evaluation of the Biologically Induced Immobilization of TNT. The chemical and ecotoxicological analyses have shown that biologically induced immobilization of TNT and related compounds in soil offers a simple technique for soil remediation. Due to the fact that the reduced contaminants, particularly hydroxylaminodinitrotoluenes and TAT, are bound to the soil and subject to some kind of humification, further investigations are necessary on the long-term stability of these bound metabolites. Soil columns with radiolabeled immobilized contaminants are subjected to different time lapse leaching conditions in order to mimic natural catabolic processes in soil. Taking into account that hydroxylaminodinitrotoluenes are often described as initial TNT metabolites in various microbial systems under anaerobic or aerobic conditions (4, 5, 33-39), irreversible binding of these compounds to soil components especially to humic material may play a major role in TNT bioremediation processes. In some cases the condensation of the hydroxylaminodinitrotoluenes with related nitroso compounds also led to an accumulation of azoxy compounds (40) which, however, were not observed at the end of the present bioremediation. An interesting question is the relative stability of the covalent binding of reduced TNT species to soil organic matter. Whereas slightly reducing conditions would favor univalent binding of hydroxylaminodinitrotoluenes, extensive reduction could activate three nitrogen functions of TNT for covalent binding to humic material. Since low molecular weight reductants such as reduced iron and sulfur species or natural organic compounds (41) are present under anoxic conditions, even the nitro groups of bound or sequestered residues may be subject to further reduction. As a consequence, strict anaerobic conditions should generate a higher number of reactive groups per mole of TNT (e.g., TAT as the final reduction product) and, particularly under reaeration of the soil, should effect stronger multivalent chemisorption to soil components. Soil bound residues of pesticides can be analyzed by 13C or 15N NMR spectroscopy after silylation of soil or humic material and organic solvent extraction as recently demonstrated by Haider et al. (42, 43). Using this technique, Dec et al. (44) could distinguish between sequestered and covalent bound cyprodinil or fragments of cyprodinil in soil. We currently carried out a biological anaerobic/aerobic treatment of TNT-contaminated soil in the presence of [14C]TNT and [15N]TNT. The soil humus dissolved in organic solvents upon

silylation will be analyzed at different stages of the biological treatment by radiocounting, 15N NMR spectroscopy, size exclusion, and high pressure liquid chromatography to get information about the chemical structure of the bound TNT metabolites, mechanism of binding, and molecular size of the radioactive fractions. This technique should make clear whether slightly reducing conditions generating aminodinitro- or diaminonitrotoluenes, as postulated for compost systems (45), guarantee sufficiently irreversible binding (46) or multifunctional binding through a two step anaerobic/ aerobic process is necessary to generate an ecologically safe remediation product.

(18) (19) (20) (21) (22) (23)

Acknowledgments

(24)

We gratefully acknowledge Frank Desiere for performing some of the experiments as part of his diploma thesis and Evelyn Herkner for contributing some data on dinitrotoluene (Ph.D. thesis). This research was made possible by a grant from the Federal Ministry of Education and Research (BMBF) for a project with Umweltschutz Nord GmbH & Co.

(25) (26) (27)

Literature Cited (1) Funk, S. B.; Roberts, D. J.; Crawford, D. L.; Crawford, R. L. Appl. Environ. Microbiol. 1993, 59, 2171-2177. (2) Boopathy, R.; Kulpa, C. F.; Wilson, M. Appl. Microbiol. Biotechnol. 1993, 39, 270-275. (3) Preuss, A.; Fimpel, J.; Diekert, G. Arch. Microbiol. 1993, 159, 345-353. (4) Daun, G.; Lenke, H.; Reuss, M.; Knackmuss, H.-J. Environ. Sci. Technol. 1998, 32, 1956-1963. (5) Lewis, T. A.; Goszczynski, S.; Crawford, R. L.; Korus, R. A.; Admassu, W. Appl. Environ. Microbiol. 1996, 62, 4669-4674. (6) DIN 38414-S2: Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung: Schlamm und Sedimente (Gruppe S)sBestimmung des Wassergehalts und des Trockenru ¨ ckstandes bzw. der Trockensubstanz (S 2). 1985. (7) DIN 19684-T1, T3: Bodenuntersuchungsverfahren im landwirtschaftlichen Wasserbau: Chemische Laboruntersuchungen. 1977. (8) EN ISO 11348-3: Water quality: Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (luminescent bacteria test), Part 3: Method using freeze-dried bacteria. 1996. (9) DIN 38412 part 34 and part 341: Testverfahren mit Wasserorganismen (Gruppe L): Bestimmung der Hemmwirkung von Abwasser auf die Lichtemission von Photobacterium phosphoreumsLeuchtbakteriensAbwassertest mit konservierten Bakterien (L34). 1991. (10) OECD 201 1984: Algae, growth inhibition test. (11) DIN 38412 part 33: Testverfahren mit Wasserorganismen (Gruppe L): Bestimmung der nicht giftigen Wirkung von Abwasser gegenu ¨ ber Gru ¨ nalgen (Scenedesmus-ChlorophyllFluoreszenz-Test) u ¨ ber Verdu ¨ nnungsstufen. 1991. (12) OECD 202: Daphnia sp., acute immobilization test and reproduction test. 1984. (13) DIN 38412 part 30: Testverfahren mit Wasserorganismen (Gruppe L): Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenu ¨ ber Daphnien u ¨ ber Verdu ¨ nnungsstufen (L30). 1989. (14) DECHEMA. Bioassays for Soils; Ad-hoc-Committee Methods for Toxicological/ Ecotoxicological Assessment of Soils; Kreysa, G., Wiesner, J., Eds.; DECHEMA: Frankfurt am Main, 1995. (15) ISO/DIS 14240-1: Soil qualitysDetermination of soil microbial biomass - Part 1: Substrate-induced respiration method. 1997. (16) Berg, P.; Rosswall, T. Biol. Fertil. Soils 1985, 1, 131-140. (17) Kandeler, E. Bestimmung der aktuellen und potentiellen Nitrifikation. In Bodenbiologische Arbeitsmethoden; Schinner F.,

(28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40)

(41) (42) (43) (44) (45) (46)

O ¨ hlinger, R., Kandeler, E., Margesin R., Eds.; Springer-Verlag: Heidelberg, 1993. ISO 11268-1: Soil qualitysEffects of pullutants on earthworms (Eisenia fetida)sPart 1: Determination of acute toxicity using artificial soil substrate. 1993. Bundesgu ¨ tegemeinschaft Kompost e. V. Methodenbuch zur Analyse von Kompost; BDK, Ed.; Ko¨ln, Germany, 1994; pp 5758. Boopathy, R.; Kulpa, C. F. Can. J. Microbiol. 1993, 39, 430-433. Noguera D. R.; Freedman, D. L. Appl. Environ. Microbiol. 1996, 62, 2257-2263. McCormick, N. G.; Cornell, J. H.; Kaplan, A. M. Appl. Environ. Microbiol. 1981, 42, 817-823. Kitts, C. L.; Cunningham, D. P.; Unkefer, P. J. Appl. Environ. Microbiol. 1994, 60, 4608-4711. Myrick, G.; Zappi, M.; Fredrickson, H. Abstr. 96th Annu. Meet. Am. Soc. Microbiol. 1996, Abstr. Q-374, p 451. Funk, S. B.; Crawford, D. L.; Crawford, R. L.; Mead, G.; DavisHoover, W. Appl. Biochem. Biotechnol. 1995, 51/52, 625-633. Nendza, M.; Wenzel, A. Sci. Total Environ. 1993, Suppl. 14591470. Pearson, J. G.; Glennon, J. P.; Barkley, J. J.; Highfil, J. W. An approach to the toxicological evaluation of a complex industrial wastewater. In Aquatic Toxicology; Marking, L. L., Kimmerle, R. A., Eds.; ASTM STP 667; ASTM: Philadelphia, 1979; pp 284301. Randall, T. L.; Knopp, P. V. J. Water. Poullut. Control Fed. 1980, 52, 2117-2130. Suttner, T.; Alef, K. Zentralbl. Mikrobiol. 1988, 143, 569-573. Kandeler, E.; Margesin, R.; O ¨ hlinger, R.; Schinner, F. Die Bodenkultur 1993, 44, 357-377. Kandeler, E.; Bo¨hm, K. E. Appl. Soil Ecol. 1996, 4, 181-191. Insam, H. Soil Biol. Biochem. 1990, 4, 525-532. Bumpus, J. A.; Tatarko, M. Curr. Microbiol. 1994, 28, 185-190. Michels, J.; Gottschalk, G. Appl. Environ. Microbiol. 1994, 60, 187-194. Vanderberg, L. A.; Perry, J. J.; Unkefer, P. J. Appl. Microbiol. Biotechnol. 1995, 43, 937-945. HaI¨dour, A.; Ramos, J. L. Environ. Sci. Technol. 1996, 30, 23652370. Fiorella, P. D.; Spain., J. C. Appl. Environ. Microbiol. 1997, 63, 2007-2015. Khan, T. A.; Bhadra, R.; Hughes, J. Ind. Microbiol. Biotechnol. 1997, 18, 198-203. Vorbeck, C.; Lenke, H.; Fischer, P.; Spain, J. C. Appl Environ. Microbiol. 1998, 64, 246-252. Kaplan, D. L. Biotransformation pathways of hazardous energetic organo-nitro compounds. In Biotechnology and Biodegradation, Advances in Applied Biotechnology Series; Kamely, D., Charkrabarty, A., Omenn, G. S., Eds.; Gulf Publishing Company: Houston, 1989; pp 154-181. Dunnivant, F. M.; Schwarzenbach, R. P. Environ. Sci. Technol. 1992, 26, 2133-2141. Haider, K.; Spiteller, M.; Wais, A.; Fild, M. Int. J. Environ. Anal. Chem. 1992, 46, 201-211. Haider, K.; Spiteller, M.; Wais, A.; Fild, M. Int. J. Environ. Anal. Chem. 1993, 53, 125-137. Dec, J.; Haider, K.; Scha¨ffer, A.; Fernandes, E.; Bollag, J.-M. Environ. Sci. Technol. 1997, 31, 2991-2997. Breitung, J.; Bruns-Nagel, D.; Steinbach, K.; Kaminski, L.; Gemsa, D.; von Lo¨w, E. Appl. Microbiol. Biotechnol. 1996, 44, 795-800. Dawel, G.; Ka¨stner, M.; Michels, J.; Poppitz, W.; Gu ¨ nther, W.; Fritsche, W. Appl. Environ. Microbiol. 1997, 63, 2560-2665.

Received for review October 30, 1997. Revised manuscript received March 23, 1998. Accepted April 2, 1998. ES970950T

VOL. 32, NO. 13, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1971