Environ. Sci. Technol. 2009, 43, 516–521
Identification of Metabolites Involved in the Biodegradation of the Ionic Liquid 1-Butyl-3-methylpyridinium Bromide by Activated Sludge Microorganisms THI PHUONG THUY PHAM,† CHUL-WOONG CHO,† CHE-OK JEON,‡ YUN-JO CHUNG,§ MIN-WOO LEE,| AND Y E O U N G - S A N G Y U N * ,†,⊥ Department of Bioprocess Engineering, Chonbuk National University, Chonbuk 561-756, Environmental Biotechnology National Core Research Center, Division of Environmental Biotechnology, Gyeongsang National University, Gyeongsang 660-701, Center for University-wide Research Facilities, Chonbuk National University, Chonbuk 561-756, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, and Division of Environmental and Chemical Engineering and Research Institute of Industrial Technology, Chonbuk National University, Chonbuk 561-756, Republic of Korea
Received December 2, 2007. Revised manuscript received September 25, 2008. Accepted October 30, 2008.
Ionic liquids (ILs) are low melting organic salts that potentially comprise wide application due to their fascinating properties and have emerged as promising “green” replacements for volatile organic solvents. Despite their nonmeasurable vapor pressure, some quantities of ILs will soon be present in effluent discharges since they do have significant solubility in water. Recently, the toxiceffectsofILstowardaquaticcommunitieshavebeenintensively investigated, but little information is available concerning the biodegradable properties of these compounds. The objective of this study was to identify the metabolites generated during thebiotransformationof1-butyl-3-methylpyridiniumbymicroorganisms in aerobic activated sludge. The obtained results revealed that the alkylpyridinium salt was metabolized through the sequential oxidization in different positions of the alkyl side chains. Highperformanceliquidchromatographyandmass-spectrometryanalyses demonstrated that this biodegradation led to the formation of 1-hydroxybutyl-3-methylpyridinium, 1-(2-hydroxybutal)-3-methylpyridinium, 1-(2-hydroxyethyl)-3-methylpyridinium, and methylpyridine. Onthebasisoftheseintermediateproducts,biodegradationpathways were also suggested. These findings provide the basic information * Corresponding author phone: (82)63-270-2308; fax: (82)63-2702306; e-mail:
[email protected]. † Department of Bioprocess Engineering, Chonbuk National University. ‡ Environment Biotechnology National Core Research Center. § Center for University-wide Research Facilities. | Department of Chemical Engineering, Pohang University of Science and Technology. ⊥ Division of Environmental and Chemical Engineering and Research Institute of Industrial Technology, Chonbuk National University. 516
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that might be useful for assessing the factors related to the environmental fate and behavior of this commonly used pyridinium IL.
Introduction Room temperature ionic liquids (ILs) are a new class of solvents, most easily thought of as low-melting salts. Although they were synthesized in the early twentieth century (1), there was not much interest in them until the 1990s since some of the early generations of ILs were extremely sensitive in the presence of air and water, which has most probably limited their range of applications (2). However, recently developed ILs, including salts composed of asymmetrically substituted nitrogen-containing cations (e.g., imidazole, pyridine, pyrrolidine) with inorganic anions such as Br-, BF4-, CF3SO3-, and so forth, are water- and air-insensitive and possess remarkably high thermal stability. At present, considerable attention is being shown in the use of these materials as solvents in a wide range of applications. There is an obvious advantage in performing many reactions using ILs because reaction rates are enhanced, selectivity is improved, and catalysts can be more easily reused (3). In addition, ILs are often considered to be green solvents or green materials. The “greenness” has often been claimed to be an important property in that ILs have no vapor pressure, and therefore are not released into the environment by evaporation. All of these properties make them an alternative as greener or more environmentally friendly media for organic synthesis, catalysis, and analytical applications, where until now volatile organic solvents have been in use (1, 3-5). However, these green credentials are currently unproven in the aquatic communities since solubility of ILs in water is small, but far from negligible (6-8) and the release of ILs into aquatic environments may lead to water pollution and related risks. Recently, more and more data on the biological effects of ILs are becoming available. The published literature include data on toxicity of ILs to different organisms, considering the aquatic and terrestrial compartment as well as different trophic levels, e.g., cells, bacteria, algae, aquatic and terrestrial plants, invertebrates and vertebrates (9). Unlike toxicological studies, biodegradable aspects of ILs have hardly been considered so far. In fact, the properties that make them attractive to industry (i.e., stable to a wide range of chemicals, stable to high temperature, and nonvolatile) suggest potential problems with degradation or persistence in the environment. Initial attempts were made to overcome this drawback as well as to gain information about the behavior of ILs in the environment. Gorman-Lewis and Fein (10) pointed out that the adsorption of ILs onto a range of surfaces, meant to represent those commonly found in the near-surface environment, was minimal, which would result in unimpeded transport of the chemical through subsurface groundwater. Additionally, when different advanced oxidation processes were carried out in order to verify future possibilities of the cleanup of ILs, it was found that these neoteric solvents were relatively resistant to photodegradation (11). Gathergood and co-workers (12-15) reported the first investigation on the biodegradability of dialkylimidazolium ILs incorporated with metabolizable side chain moieties and different counter-anions. Although the results indicated that 1-propoxycarbonyl-3-methylimidazolium and 1-pentoxycarbonyl-3-methylimidazolium with octyl sulfate anion were readily biodegradable in the CO2 headspace test, their degradation pathways and intermediates have not been addressed. Recently, Docherty et al. (16) 10.1021/es703004h CCC: $40.75
2009 American Chemical Society
Published on Web 12/12/2008
examined the biodegradability of imidazolium and pyridinium ILs with different alkyl-chain length, using standard Organization for Economic Cooperation and Development (OECD) dissolved organic carbon (DOC) Die-Away Test. The data showed that biodegradation rates increased with longer alkyl-chain length, and only hexyl, octyl substituted pyridinium-based ILs could be fully mineralized. Nonetheless, intermediate chemical products and biodegradation pathways were not investigated in their study. Kumar et al. (17) reported on the biodegradation of 1-butyl-3-methylimidazolium tetrafluoroborate with experimental results. They identified some degradation products using GC-MS and confirmed some of the products postulated by Jastorff et al. (18). However, the metabolic pathway for degradation of pyridinium has not been reported until now. In the present study, we aim to understand the fate of ILs through their life cycle in the aquatic milieu using microorganisms from activated sludge. Degradation products were analyzed and tentatively identified by means of liquid chromatography/mass spectrometry (LC/MS). The pyridinium moiety, i.e., 1-butyl-3-methylpyridinium bromide, was selected in this research since pyridinium compounds are an important class of chemicals used widely as biocides, cationic surfactants, drugs, and herbicides (19). To the best of our knowledge, this is the first report on biodegradation intermediates and the pathway of pyridinium ILs.
Experimental Section Chemical. 1-butyl-3-methylpyridinium bromide [BMPy] [Br] was supplied by C-tri Co., Korea. The tested IL was obtained at 98% of purity since analytical quality is not feasible for technical applications. In this study, [BMPy] [Br] was used as supplied without any pretreatment. Activated Sludge. The activated sludge was collected from the aeration tank of a municipal wastewater treatment plant (Jeonju, Korea). The inoculum was used as an activated sludge microbial community for the aim of studying the biodegradability under real conditions. After removal of coarse particles by filtration through a fine sieve, the sludge was centrifuged at 3000 × g for 5 min and washed with mineral nutrient medium described in the modified OECD screening test (OECD 301E) (20). This process was repeated three times to ensure thorough washing of the sludge. The final supernatant was decanted and the remaining washed activated sludge was resuspended in mineral medium to yield a concentration of 5 g SS/L and aerated thereafter. Biodegradation Study. Estimation of pyridinium cation was performed using the modified OECD screening test (OECD 301E) as an experimental protocol (20). At the onset of the experiment, stock solution of pyridinium salt was freshly prepared by dissolving it in deionized water. The concentration of applied pyridinium cation was 5.6 mg C/L () 46.7 µM), since higher concentrations may cause toxic effects to the exposed organisms. The previously prepared sludge and the test compound as sole source of organic carbon were inoculated into 100 mL Erlenmeyer flasks containing 40 mL of mineral medium at pH 7.3 ( 0.2. For the abiotic degradation test, the inoculated activated sludge was poisoned by adding 1% solution containing 5% HCl and 0.25 M HgCl2 to the flasks. Test chambers were capped with air-permeable cotton bungs and placed in the incubator maintained at 70 rpm under dark condition at 25 ( 2 °C. During 28-day incubation, samples were withdrawn every three or four days for HPLC-MS. For MS/MS analyses, only samples after 28 days of incubation were investigated. Prior to the analysis, samples were filtered through 0.45 µm poresize membrane syringe filters (Gelman Sciences, Ann Arbor, Michigan) to remove the solid biomass. All experiments were repeated twice.
Analytical Methods. High-performance liquid chromatography. [BMPy] [Br] degradation fragments were separated using an acetonitrile-water gradient method. A reversedphase column (symmetry Xterra C18, 5 µm, 4.6 by 150 mm; Waters) was connected to an Agilent 1100 binary HPLC pump system equipped with an Agilent 1100 series autosampler and a PDA detector. The detector wavelength was set at 264 nm. Acetonitrile was purchased from J.T. Baker, whereas formic acid was provided by Acros, USA. All solvents were degassed and filtered through 0.45-µm-pore-size membrane syringe filters (Gelman Sciences, Ann Arbor, Michigan). The elution gradient used consisted of a combined flow rate of 1 mL/min of 97% A (water containing 0.1% formic acid) and 3% B (acetonitrile containing 0.1% formic acid) for 3 min, which then decreased to 40% A and 60% B for the following 27 min. Liquid chromatography/mass-spectrometry. The LC/MS experiments were performed using an Agilent 1100 Series LC/MSD Trap SL ion trap mass spectrometer coupled to an Agilent 1100 Series capillary LC system. The complete system was fully controlled by Agilent ChemStation software. The column, gradient program, and solvents were exactly the same as that described above for the HPLC analysis. All samples were loaded onto a column using a 10-µL sample loop. The ion trap mass spectrometer was operated with the electrospray (ESI) source in positive ion mode with a standard mass range of 50-250 m/z and 150 m/z was used as the target mass. Tandem mass spectrometry (MS/MS) was conducted on specified parent ions using helium for fragmentation. The fragmentation energy was 0.5 Amplitude. Automated data-dependent MS/MS with active exclusion and maximum resolution scan (MaxResScan) was used to maximize the MS information acquired from a single chromatographic run.
Results Initial attempts to ascertain the biodegradation of pyridinium salts were performed using HPLC with 0.1% formic acid as the ion pair agent. No significant change in peak area, corresponding to IL concentration, was observed in abiotic control (data not shown) indicating that abiotic degradation of IL did not occur under our test conditions. For biodegradation test, the samples were withdrawn every three or four days and used for analysis of remained [BMPy] cation using HPLC-MS (Figure 1). It was found that after 21 days of incubation, microorganisms from activated sludge were able to break down [BMPy] [Br] (Figure 2). Figures 1 and 2 marked a significant change in the IL concentration at the estimation period of 21 days, though slight variation in concentration was observed during the time before. Over a 28-day period, the biotransformation resulted in nearly complete use of [BMPy] and the purported metabolites generated from it, with the final products being the hypothesized methylpyridine. To determine the mass and to deduce the structures of the intermediate compounds, samples containing mixtures of [BMPy] [Br] and its degradation products were directly injected into the MS. Samples taken after 28 days of [BMPy] [Br] degradation yielded data as shown in Figure 3. All ions with different mass in range of 50-250 m/z were individually isolated and were fragmented with helium gas in mass spectrometer. According to their fragmentation patterns observed in these steps, we were able to extrapolate their structures and find three types of ions that can be demonstrative of intermediate (Figure 4). Among the ions, the fragmentation pattern of the ion shown in Figure 4 (A) has the same fragment (m/z 94.1) as that of original chemical. However, the rest was not the same pattern because of the low resolution of the MS spectrometer due to a lack of target ion amount in the sample. Thus, they were interpreted with the procedure stated by McLafferty and VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. HPLC-MS chromatograms showing the biodegradation of 1-butyl-3-methylpyridinium cation after 18, 21, and 28 days of incubation with activated sludge.
FIGURE 2. Time course on the biodegradation of 1-butyl-3methylpyridinium cation. Turecˇek (21). The mass spectrum (m/z 166) (Figure 4A) was consistent with a fragment of 1-hydroxybutyl-3-methylpyridinium containing a hydroxyl group which is located in an unknown position between C1 and C4 of the butyl chain. A second metabolite (Figure 4B) was tentatively identified as 1-(2-hydroxybutal)-3-methylpyridinium with the m/z value of 180. The ion at m/z 138 (Figure 4C) represent a fragment 1-(2-hydroxyethyl)-3-methylpyridinium, which results from the loss of 2 C atoms in the butyl chain. The ultimate product with m/z 94 (Figure 4A) corresponded to methylpyridine. However, as is shown in Figure 5, a small amount of methylpyridine was also detected in the sample of [BMPy] [Br] for LC/MS analysis. This suggests that methylpyridine might account for an impurity in the [BMPy] [Br] stock solution and/or the final biodegradation product. Therefore, further study is needed to confirm if methylpyridine is actual final product. On the basis of the data obtained, the biodegradation pathways were suggested as illustrated in Figure 6.
Discussion The degradation of [BMPy] [Br] by means of DOC Die-Away Test (OECD 301 A) was reported in the literature (16), where this compound was found not to be metabolized by the activated sludge microbial community. However, we found that [BMPy] [Br] was degraded with the production of some intermediate metabolites. This is in contrast to our results. We believe that the major reasons why our results were different from Docherty’s results were as follows: First, the concentration level used in this study was different from that by Docherty et al (16). The injected IL amount in our experiment was 5.60 mg C/L () 46.7 µM); whereas that reported in Docherty et al. was 40 mg C/L () 333 µM). A high 518
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concentration of IL would affect the microorganisms. According to our preliminary study on the toxicity of [BMPy][Br] on the respiration activity of activated sludge microorganisms, the EC50 value was estimated to be 3162 µM (data not shown). At 46.7 µM, the inhibition to microbial respiration was ∼25%. The 25% inhibition is slightly higher than standard value (EC 20), suggested by OECD guideline 301E. In case of experiments performed by Docherty et al., they injected high concentration (333 µM) that would result in about 37% inhibition and longer adaptation time. In addition, Docherty et al. measured the dissolved organic carbon (DOC) as an indication of biodegradation. However, the intermediate compounds could be measured as DOC unless they would be completely decomposed to inorganic carbon like CO2. It is likely that a part of [BMPy][Br] was biodegraded but not detected through the DOC method. From the theoretical prediction of metabolisms (18) and metabolite analysis, it was observed that the metabolism of [BMPy] [Br] by activated sludge appeared to undergo oxidation reactions catalyzed by cytochrome P450 located in the endoplasmatic reticulum of cells. Cytochrome P450 enzymatic action is based on an oxidative mechanism that renders hydrophobic compounds more water-soluble, and hence they are more readily removed via the urine (22). In the first stage, the HEME system of P450 enzymes is activated by the dioxygen molecule iron complex, which can react with the carbon-hydrogen bond via a radical mechanism. In the report of Jastorff and co-workers (18), the products of a cytochrome P450-catalyzed hydroxylation of the 1-butyl3-methylimidazolium cation were proposed according to a widely accepted theoretical model involving a so-called “oxygen rebound” step. Regarding this mechanism, an “ironoxo” species reacts by abstracting a hydrogen atom from the substrate to yield a radical intermediate. This radical then reacts with the iron hydroxide species via a hemolytic substitution reaction (23). Thus, the IL cation can be oxidized at different positions in the alkyl side chains. This mechanism might be applied for elucidating the biotransformation of pyridinium salts since the reaction steps only relevant to the alkyl chain and the final degradation product still contained pyridinium core as being shown in the data obtained. In this respect, 1-butyl-3-methylpyridinium compound was converted to 1-hydroxybutyl-3-methylpyridinium with the addition of a hydroxyl functional group. It is likely that subsequent oxidation of the hydroxyl group at C4 of the butyl chain into carbonyl group concomitant with the addition of a hydroxyl group into C2 lead to the formation of 1-(2hydroxybutal)-3-methylpyridinium, which then decomposes to 1-(2-hydroxyethyl)-3-methylpyridinium and acetaldehyde (Figure 6, pathway I). On a simple approach, hydroxyl functional group might be added into [BMPy] salt at C2 position of the butyl chain. In this way, the generated 1-(2hydroxybutyl)-3-methylpyridinium was degraded to produce 1-(2-hydroxyethyl)-3-methylpyridinium and ethane (Figure 6, pathway II). The methylpyridine detected during [BMPy]
FIGURE 3. Mass spectra of the putative metabolites generated from activated sludge cultures incubated with 1-butyl-3methylpyridinium IL after 28 days.
FIGURE 4. Identification of 1-hydroxybutyl-3-methylpyridinium and methylpyridine (A), 1-(2-hydroxybutyl)-3-methylpyridinium (B), and 1-(2-hydroxyethyl)-3-methylpyridinium (C) as metabolic intermediates produced from 1-butyl-3-methylpyridinium moiety by microorganisms from activated sludge by MS/MS. [Br] degradation may be generated via the elimination of ethanol from 1-(2-hydroxyethyl)-3-methylpyridinium or just
present as an impurity due to the existence of this compound in the standard stock solution. Our study identified until VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Methylpyridine detected by mass-spectrometry analysis as an impurity present in the IL stock solution.
FIGURE 6. Biodegradation pathways of 1-butyl-3-methylpyridinium entity by microorganisms in activated sludge. The intermediates shown in brackets were not detected or confirmed. methylpyridine and further degradation products could not be measured. Taken as a whole, the data suggest that microorganisms in activated sludge might degrade [BMPy] cation by two pathways, one of which is mediated by the formation of 1-(2-hydroxybutal)-3-methylpyridinium and the other deals with the generation of 1-(2-hydroxyethyl)-3methylpyridinium directly from 1-(2-hydroxybutyl)-3-methylpyridinium. The biodegradation data should be interpreted with caution, taking into account the fact that toxic effects of ILs and metabolites may also have a negative impact on their biodegradation. Many quaternary ammonium salts are potential biocides and could inhibit growth of the microorganisms capable of degrading them (24, 25). Additionally, pyridinium salts have been previously reported as possessing similar activity to the carcinogenic pesticide paraquot (1,1′dimethyl-4,4′-bipyridinium chloride), which attacks lipids in cytoplasmic membranes (26). Therefore, the biodegradation results found could be related to the inhibitory effects of ILs on the microorganism populations. Nonetheless, Jastorff et al. (27) pointed out that the introduction of polar functional groups into an alkyl chain yields a reduction in cytotoxicity. Also, in the study on the toxicity of pyridinium chlorides and their biodegradation intermediates, Grabin ´skaSota and Kalka (28) revealed that intermediates obtained as a result of biodegradation process did not cause any harmful effects in addition to organisms tested (i.e., algae, crustacean, and fish). Thus, whether there is an adverse effect of the 520
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pyridinium entity on activated sludge as well as to what extent the sludge microorganisms degraded this IL is the aim of our future research. The present study is the first report to demonstrate the biodegradation of pyridinium IL together with an identification of resulting metabolites. The lack of readily accessible biodegradation data is a big gap in our knowledge when considering the employment of ILs on a pilot or manufacturing scale. Thus, the present results on the metabolic fate of [BMPy] [Br] may be useful in alleviating the environmental impacts related to the introduction of this commonly used IL into the environment. Further experiments about transformation pathways and kinetics of ILs under different environmental conditions and within organisms are highly recommended.
Acknowledgments This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2007-521-D00106), and in part by the Korea Science and Engineering Foundation (KOSEF) NRL Program grant funded by the Korean Government (MEST) (No. R0A-2008-000-20117-0).
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