Environ. Sci. Technol. 2009, 43, 2569–2574
Degradation and Elimination of Various Sulfonamides during Anaerobic Fermentation: A Promising Step on the Way to Sustainable Pharmacy? SIEGRUN A. I. MOHRING,† INA STRZYSCH,‡ MARCOS REIS FERNANDES,† THEKLA K. KIFFMEYER,‡ JOCHEN TUERK,‡ AND G E R D H A M S C H E R * ,† University of Veterinary Medicine Hannover, Institute for Food Toxicology and Analytical Chemistry, Bischofsholer Damm 15, D-30173 Hannover, Germany and Institut fu ¨r Energie- und Umwelttechnik e.V. (IUTA), Bliersheimer Strasse 60, D-47229 Duisburg, Germany
Received July 23, 2008. Revised manuscript received January 28, 2009. Accepted February 3, 2009.
Antibiotics, most notably sulfonamides and tetracyclines, are frequently used veterinary pharmaceuticals in animal husbandry. A new field of application for animal manure is in biogas plants for generating environmentally friendly energy. As a result, antibiotics contained in manure may still reach the environment as fermentation residues are also used on agricultural fields as fertilizers. Therefore, in fermentation tests seven sulfonamides and trimethoprim were investigated regarding their elimination behavior during a five-week fermentation process. Sulfadiazine, sulfamerazine, sulfamethoxazole, sulfadimethoxine, and trimethoprim were nearly completely eliminated while sulfathiazole, sulfamethazine, and sulfamethoxypyridazine showed persistence. For sulfadiazine it was possible by means of mass spectrometry to identify and partly quantify a metabolite, emerging from a hydroxylation at the pyrimidine ring, 4-OH-sulfadiazine. Furthermore, a microbial inhibition test showed a substantial reduction in the antimicrobial activity of the metabolite compared to the parent compound. Thus, the fermentation process may be an efficient way to reduce the load of selected veterinary antibiotics finding their way into the environment. Degradable drugs such as sulfadiazine may therefore, at least in the aspect of residual antibiotic activity of metabolites, be considered as environmentally friendly drugs.
Introduction Antibiotics are widely used in the prevention and treatment of diseases in humans as well as in veterinary medicine. Together with parasiticides, antibiotics are the most important group of veterinary pharmaceuticals. In 1999 as much as 5,000 tons of antibiotics was used for veterinary medicine in the EU, of which 3,500 tons were used for therapeutic * Corresponding author e-mail:
[email protected]. † University of Veterinary Medicine Hannover. ‡ Institut fu ¨r Energie- und Umwelttechnik e.V. (IUTA), Bliersheimer Strasse 60, D-47229 Duisburg, Germany 10.1021/es802042d CCC: $40.75
Published on Web 02/23/2009
2009 American Chemical Society
purposes. Among the most prescribed antibiotics in veterinary medicine is the group of sulfonamides, with sulfadiazine and sulfamethazine being the most important compounds (1, 2). Most antibiotics are excreted unmetabolized via swine and cattle manure. As much as 30-90% can be excreted, this having been poorly absorbed or metabolized (1). In the case of N-acetyl-sulfonamides even the metabolized fraction can be transformed back to its parent compounds in manure (3, 4). Therefore, manure is the most important vehicle for transporting veterinary antibiotics into the environment. In individual manures peak concentrations up to 235 mg/kg sulfadiazine and up to 167 mg/kg sulfamethazine have been detected (5). Thus, substantial amounts reach agricultural soils through manuring and grazing livestock. In soils and adjacent environmental compartments such as surface and groundwater, residues of some antibiotics have been found (6). The potential risks associated with these findings have been the focus of discussion in recent years. On the one hand, there is the concern that new strains of bacteria resistant to antibiotics arise as an aftermath of subtherapeutic concentration over a long period of time. Furthermore, the effects of possible accumulation of certain antibiotics in the environment are unknown (7). In recent years a new area of scientific interest, Green Chemistry, arose (8-11). Green Chemistry, being one of the concepts for sustainability, provides a set of principles that aim to reduce and eliminate hazardous substances in the design, manufacture, and application of chemical products (12). But how do we deal with compounds already brought onto the market and that are indispensable in our modern world? One approach in this aspect is the knowledge of (bio)degradability of pharmaceuticals such as antibiotics. Especially Ku ¨ mmerer et al. investigated the behavior of antibiotics in different test systems, such as Closed Bottle Test (CBT) or Zahn-Wellens test, regarding their degradability (13-17). Most substances (including sulfamethoxazole) investigated in those test systems could not be characterized as readily biodegradable although elimination by other processes might occur. Only Penicillin G was degradable to a degree of 27% within 28 days. In contrast to those findings, in sewage treatment plants sulfonamides are eliminated after an adaptation of the system to similar sulfonamides (18, 19). However, no metabolites could be identified. Manure is also used as a substrate for biogas plants. In biogas plants the process of anaerobic digestion converts organic waste, such as agricultural waste, into biogas which is environmentally friendly generation of energy. Through manure, sulfonamides, besides other substances, are carried into the fermentors. Here, on the one hand, they might influence the process of fermentation. On the other hand, the fermentation process might have an effect on the antibiotics. For some antibiotics the effect on anaerobic fermentation processes has been tested (20-24). Yet, a possible degradation of antibiotics in biogas plants during fermentation has not been investigated in detail. Only Arikan et al. revealed that a removal of 60% of oxytetracycline can be achieved by a 64-day anaerobic digestion (25). Again, the authors could not identify any degradation products. Degradation of aromatic compounds by microorganisms has also become an important issue in the research community. For many years it has been well-known that bacteria have adapted their metabolism to degrade these compounds as a new carbon source (26). It is likely that in biogas plants bacteria also metabolize biologically active compounds such as antibiotics. Therefore, the indirect way of manure through VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Chemical structures, abbreviations, and molecular weights (MW) of seven investigated sulfonamides and trimethoprim.
TABLE 1. Recoveries, Standard Deviation (SD), and Relative Standard Deviation (RSD) for Seven Sulfonamides and Trimethoprim in Fermentation Substratea substance
recoveryb mean ( SD [%]
RSD [%]
sulfadiazine sulfathiazole sulfamerazine sulfamethazine sulfamethoxypyridazine sulfamethoxazole sulfadimethoxine trimethoprim
73.1 ( 11.3 68.4 ( 16.9 72.8 ( 11.1 88.0 ( 10.4 70.9 ( 10.2 95.0 ( 6.3 75.7 ( 13.3 26.6 ( 4.4
15.5 24.7 15.2 11.8 14.3 6.6 17.6 16.7
a Fermentation product from blank fermentation experiments was spiked at four concentrations (50, 100, 500, and 5000 µg/kg) with seven sulfonamides and trimethoprim; three replicates of each concentration were prepared and measured with HPLC-ESI-MS/MS. Recoveries were calculated on the basis of external standards thus representing the overall process efficiency of the analytical method. b n ) 12 (four concentrations, three replicates each).
biogas plants to the fields might allow a reduced impact of pharmaceuticals to the environment. A pilot experiment revealed that sulfadiazine might degrade during the fermentation process in a biogas plant (27). To confirm these preliminary findings, in the present study seven sulfonamides and trimethoprim (Figure 1) were tested for their behavior during anaerobic fermentation. Fermentation tests were carried out in laboratory scale fermentors to investigate possible structure-dependent degradation.
Experimental Section Reagents. Sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine, trimethoprim, and isocytosine were purchased from Sigma (Munich, Germany). All compounds were of at least analytical grade. Water with a resistance of >18.0 MΩ/cm was prepared in-house with a Milli-Q-System (Millipore, Eschborn, Germany). All other chemicals were of analytical or HPLC grade. Stock solutions of all standards, stable for at least six months, were prepared in methanol and stored at -20 °C. Methanolic working solutions were prepared fresh on the day of analysis. 2570
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Fermentation Tests. To study the behavior of the seven sulfonamides during a 34-day fermentation process a batchfermentation system based on the German VDI 4630 guideline was used. This guideline provides rules for assessing the fermentability of organic materials and the necessary equipment and apparatus required for the corresponding test setups. Fermentation test apparatus using the batch procedure is also specified in DIN 38414 Part 8. These tests provide concentration-dependent information on the inhibitory effect of the material under investigation. The normal test procedure lasts 28 days. In up-scale biogas plants, depending on substrate quality and plant design, residence times are between 20 and 60 days. The biogas plant (Loick, DorstenLembeck, Germany), from which the inoculum for the laboratories was obtained, is working with residence times between 25 and 30 days. 5-L Fermentor Studies. In the present study, in two 5-L fermentors (Bigatec, Rheinberg, Germany) 1.89 kg of swine manure from a local biogas plant (Loick, Dorsten-Lembeck, Germany) was used as main substrate. This biogas plant was started as a project of the Fraunhofer Institute, UMSICHT (Oberhausen, Germany). It is run as a regular biogas plant using ca. 4000 m3 manure per year which makes up about one-third of the feed. Thus it is adapted to the slightly higher pH of manure. The obtained manure exhibited relatively high organic matter of 15.2% and to receive comparable manures it was diluted with water (1:1). Fermentation product (0.42 kg) obtained from the same biogas plant was added as inoculum to start the fermentation process. In fermentor A the mixture of manure, water, and fermentation product was spiked with 2 mg of each of the seven sulfonamides and 2.8 mg of trimethoprim per kg of manure. In the same way fermentor B was spiked with 10 mg of each sulfonamide and 14 mg of trimethoprim per kg of manure. Trimethropin is commonly used as a synergist to sulfonamides in veterinary medicine in a ratio of 1:5 to the total quantity of sulfonamides. The sulfonamide concentrations were chosen to represent the median range of manure samples investigated in a field study carried out in 1999/2000 and 2001/2002 in the region of Weser-Ems (Germany). In this study a total of 345 manure samples (swine) was tested for sulfadiazine, sulfamethazine, and three tetracyclines (5). Also, both experiments were performed far below the inhibitory concentration of 500 mg/ kg (results of concentration-dependent fermentation tests for the determination of this inhibition concentration are not shown). The sulfonamides were deliberately tested as a
TABLE 2. Elimination of Seven Sulfonamides and Trimethoprim, and Increase in the Sulfadiazine Metabolite at Two Concentrations during a Fermentation Process of 34 Days as Percentage of Initial Measured Concentration (Mean of Two/Fivea Experiments Is Given)b SDZ start day 8 day 14 day 21 day 28 day 34
SDZ-MB a
100.0 77.0 2.9 0.6 0.6 0.1a
a
8.0 25.9 89.1 87.8 94.2 100.0a
STZ
SMR a
100.0 93.3 90.6 76.9 79.9 103.3a
SMZ a
100.0 64.1 0.9 0.1 0.1 0.0a
SMDP a
100.0 76.2 72.6 63.0 63.7 100.0a
a
100.0 62.6 51.9 39.5 35.2 27.7a
SMX
SDM a
100.0 1.3 1.5 0.2 0.4 0.1a
TMP a
100.0 29.1 12.5 3.4 1.6 0.4a
100.0a 1.1 0.1 0.0 0.0 0.0a
a Mean of five samples (three experiments in 500-mL flasks and two experiments in two 5-L fermentors). b SDZ ) sulfadiazine, SDZ-MB ) sulfadiazine metabolite, STZ ) sulfathiazole, SMR ) sulfamerazine, SMZ ) sulfamethazine, SMDP ) sulfamethoxypyridazine, SMX ) sulfamethoxazole, SDM ) sulfadimethoxine, and TMP ) trimethoprim.
mixture in order to provide an identical experimental setup for all compounds under investigation. Every week samples of about 10 g were drawn from the fermentors and immediately frozen (-18 °C) prior to analysis via LC-MS/MS. Both fermentors were equipped with a milligascounter (Dr. Ing. Ritter Apparatebau GmbH, Bochum, Germany), a methane gas sensor integrated in a BACCom multiplexer box (BlueSens GmbH, Herten, Germany), to monitor the fermentation process. Data were acquired with the BACVis software (BlueSens GmbH, Herten, Germany). The process temperature was preset at 37 °C. Control Studies. Accompanying control experiments were carried out in four 500 mL flasks equipped with eudiometer tubes and gas bags for the measurement of the gas formed during fermentation. In three of the flasks antibiotics were added in amounts equivalent to the main experiments in the 5-L fermentors whereas in one flask no antibiotics were added (blank testing). Due to the small substrate amounts in these containers sampling during the fermentation process was not possible. Samples were drawn only before and after fermentation. Sample Preparation Procedure for MS Analysis. Under consideration of previously published methods a slightly changed liquid-liquid extraction procedure, regarding pH of the extraction buffer, was developed for fermentation substrate samples (6). Briefly, 1 g of substrate was vortexed for 1 min with 1.2 mL of citrate buffer (pH 5) and subsequently extracted twice with ethyl acetate. The dried ethyl acetate phase (rotary evaporation) was redissolved in 1 mL of 90% acetonitrile and 10% 100 mM ammonium acetate in water. The samples were kept at 10 °C in the auto sampler prior to analysis. All glassware used was heated for 2 h at 450 °C, cooled, and rinsed with 2.5 mL of a saturated methanolic EDTA-solution. Liquid Chromatography/Tandem Mass Spectrometry. Mass spectrometry was carried out using an LCQ ion trap with an electrospray ionization source (Thermo Finnigan, San Jose, CA). Here, trimethoprim was included in the existing method previously published (6) and for all eight substances recovery studies in fermentation matrix were performed. The samples were spiked with methanolic stock solutions of the compounds at the 50-5000 µg/kg-level in triplicates. After spiking, the samples were allowed to equilibrate for 10 min at room temperature and then processesed as decribed above. To obtain the mass spectrometric parameters a standard solution (1 ng/µL) was infused through an integrated syringe pump at a flow rate of 10 µL/min. The substance-specific parameters for trimethoprim were m/z 291.3 as the precursor ion, an isolation width of 1.0 m/z, and collision energy (CE) of 47%. For the quantification the following product ions were used: 123.2, 230.2, 258.2, 261.3, 275.3, 276.1. The mass spectrometer was tuned and optimized for all substances, leading to the following general settings: The source polarity was set positive and the spray needle voltage was 5 kV. Drying
gas was nitrogen generated from pressurized air in an Eco inert 2 ESP nitrogen generator (DWT-GmbH, Gelsenkirchen, Germany). The sheath gas flow was set to 100 units, auxiliary gas was turned off, and the capillary temperature was preset at 150 °C. The HPLC system employed was a gradient system consisting of a P4000 pump, an AS3000 auto sampler (ThermoQuest, San Jose, CA), and a Puresil C18 column (5 µm, 4.6 × 150 mm; Waters, Milford, MA) operated at 23 °C. The flow of 1 mL/min was split 1:10 before entering the mass spectrometer. This conventional setting makes the system robust against impurities left in the sample after a quite simple sample clean up. A split of 1:10 reduces the amount of matrix transferred into the mass spectrometer, reducing signal suppression or enhancement, and may be applied without losing sensitivity as ESI-sources act as concentration-sensitive devices. The mobile phase consisted of 0.5% formic acid in water with 1 mM ammonium acetate (solvent A) and acetonitrile (solvent B). After 1 min 100% A the gradient run was 0-25% B in 9 min rising to 50% within 1 min, then lasting for 3.5 min. After each run the column was rinsed for 2 min with 99% acetonitrile and re-equilibrated for 8 min with solvent A. Trimethoprim was analyzed in a second independent run, changing only the MS/MS parameters. All samples were prepared and analyzed in duplicate. Purification, Identification, and Characterization of SDZ-Metabolite. Extracts prepared as described above were chromatographed and the fraction eluting between 7.0 and 7.3 min was collected manually from the split flow of about 60 runs. The pooled fractions were concentrated in a rotary vacuum concentrator (Alpha RVC, Christ, Osterode, Germany) and redissolved in 1 mL of methanol. The metabolite was quantified in this solution via UV-spectrometry at 254 nm on the basis of a sulfadiazine standard solution assuming a similar extinction coefficient. Based on these experiments the metabolite was split into portions of 2 µg, vacuum concentrated, and stored at -30 °C prior to further identification and characterization. Determination of the exact mass was carried out by an Unique HT ESI-ToF (LECO Corporation, Saint Joseph, MI). The sample was infused into the MS-system via an Agilent 1200 SL HPLC (Agilent Technologies, Waldbronn, Germany). Isocratic separation was performed on an Agilent Zorbax SB-C18 column (1.8 µm, 2 × 50 mm) at 30 °C using a mixture of 92:8 (v/v) of 0.1% formic acid in methanol (v/v) and 0.1% formic acid in water (v/v). Post column addition of Agilent TOF Tune Mix (Agilent Technologies, Waldbronn, Germany) followed by single point reference mass correction (622.0291 u) was used to generate accurate mass data. Using a T-type connector 0.5 µL/min of Tune Mix was infused into the total flow of 0.5 mL/min. Further identification was carried out according to Lamsho¨ft et al. (28). The MS/MS spectrum of the SDZmetabolite contained an m/z 112 fragment which was fragmented again (MS3; CE: 25%) (28). By employing this VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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technique, one can differentiate between 4-OH- and 5-OHsulfadiazine. The metabolite was dissolved in methanol and infused into the MS-System via a syringe pump. For comparison also isocytosine, representing the pyrimidine ring on the fourth position hydroxylated sulfadiazine, was infused into the MS-system as methanolic solution. MS/MS data were compared to the MS3-data of the metabolite. For the semiquantification of the antimicrobial activity a BRT MRL-Screening Test (AiM, Munich, Germany) was carried out. The MRL-Screening Test (Geobac. stearothermophilus var. calidolactis C953) is a microbiological test system for detecting inhibitors or residual anti-infectives such as sulfonamides in milk. On a 96-well plate six concentrations (50-2500 µg/L) of both the metabolite and the parent compound were compared to a negative (resuspended inhibitor free milk) and positive control (resuspended Penicillin G Standard, 4 ng/mL milk) integrated in the test system and tested in duplicate. Stock solutions of the metabolite and sulfadiazine were prepared in 0.01 mol/L NaOH. Since the test system was developed and validated for milk, twelve standard solutions were prepared in inhibitorfree milk using the prepared stock solutions. One hundred µL of each concentration, the positive and the negative control were pipetted into single cavities. The test plate was incubated in a thermo-block at 65 °C until the negative control turned yellow (2 h 30 ( 15 min). After incubation the milk was discarded and the plate was rinsed with tap water prior to a visual and photometric evaluation. The photometric measurements were carried out with an Infinite 200 (Tecan, Tecan Group Ltd., Switzerland). Wavelengths for measurement and reference were 450 and 620 nm according to instructions. Synthesis of 5-hHydroxysulfadiazine. 5-Hydroxysulfadiazine was synthesized following the procedure of Pfeifer et al. (29). No further isolation was carried out because only a qualitative analysis of the hydroxylated compound was necessary. One mg of the synthesized substance was weighed in and prepared according to the sample preparation for fermentation substrate.
Results and Discussion Analytical Method. For quantifying sulfonamides in the samples an existing method was adapted for fermentation substrate as matrix. The laboratory is accredited as a test laboratory according to ISO/IEC/EN 17025:2005 from AKS Staatliche Akkreditierungsstelle Hannover, Germany (AKSPL-20337). Regular participation in laboratory proficiency tests (FAPAS, series 0257, 0264, 0294, 02113) for sulfonamide standard mixtures provided by Central Science Laboratory (CSL, York, UK) resulted in excellent z-scores between -0.3 and 0.3. The fermentation product from a blank fermentation experiment always carried out for comparison of the gas production was spiked at four concentrations with seven sulfonamides. Mean recoveries are shown in Table 1. Here the overall process efficiency including any signal suppression or enhancement effects is referred to as recovery. Additional experiments on matrix effects showed no significant signal suppression or enhancement. One reason for this may be the relatively low load of matrix reaching the ESI-source, using a 4.6-mm HPLC column, and splitting the eluent before entering the mass spectrometer. In recent years this conventional approach has been successfully applied to various environmental and food matrixes (6, 30-34). Fermentor Performance. Within all experiments (two 5-L fermentors (fermentor A and B) and four 500-mL flasks) no significant differences of the biogas volume could be observed indicating a normal process. The pH slightly decreased from 7.93 to 7.80 for fermentor A during the 34-day fermentation, and from 7.89 to 7.77 for fermentor B. 2572
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Unfortunately the methane gas sensor had a defect and thus no continuous methane concentration was reported for fermentors A and B. However, since these tests were carried out to especially detect degradation products and to represent realistic concentrations of sulfonamides in manure, and all other parameters were as supposed, this fact can be neglected. In previous experiments (data not shown) dose-response experiments showed that the chosen concentrations were far beyond inhibitory concentrations. The fermentation tests carried out here were discontinuous. Since biogas plants usually are fed continuously and no defined residence time can be achieved like in the laboratory scale fermentors, it is obvious that a complete transferability is not given. Therefore, additional investigations in regular biogas plants have to confirm these findings. Degradation/Elimination. Table 2 shows the elimination of seven different sulfonamides and trimethoprim as percentage of the initial measured concentration during the 34-day fermentation. Four sulfonamides are successively eliminated during the 5-week-fermentation. While sulfadiazine and sulfamerazine are nearly completely eliminated after 2 weeks, trimethoprim and sulfamethoxazole are immediately eliminated within 1 week. Sulfamethoxypyridazine shows a minor elimination to a detectable residue of about 30%. On the other hand, 100% of the initially measured concentration of sulfathiazole and sulfamethazine can still be detected after the 34-day fermentation period. However, the absolute values of sulfathiazole and sulfamethoxazole dropped initially to 25% (SMZ) and 60% (STZ) of the added concentration. These low amounts found at the beginning of the fermentation might be due to an immediate sorption of the compounds to fermentation particles after spiking (35, 36). Applying MS/MS it was possible to partly quantify the metabolite based on the signal of an external standard solution of sulfadiazine. During the fermentation process an increase in metabolite could be found, although the absolute amount could not be determined exactly since the metabolite is not as efficiently ionizable as the parent compound. Derived from the purification experiments of the metabolite an approximate ratio MS/MS to UV response of 1:3 was calculated. This might explain the gap between the disappearance of sulfadiazine and the formation of 4-OHsulfadiazine. Identification of the Sulfadiazine Metabolite. In several experiments a metabolite of sulfadiazine could be characterized and identified. First, the molecular mass found for an unknown peak equals the mass and empirical formula for sulfadiazine plus 16 u leading to the assumption of an occurred hydroxylation. The determined exact mass of the protonated molecular ion was 267.0545 u. Under consideration of the mass accuracy of -0.49 ppm (theoretical mass 267.05463 u) the expected empirical formula C10H10N4O3S was confirmed. It has been shown previously that anaerobic bacteria are able to oxidize organic substances. For example, Chakraborty and Coates describe the anaerobic nitratedependent metabolism of benzene (37). For identification and characterization of the metabolite, an amount of approximately 22 µg could be isolated. MS/MS spectra of the sulfadiazine metabolite (SDZ-MB m/z 267) directly infused into the mass spectrometer revealed an m/z 112 fragment (Figure 2A). According to Lamsho¨ft et al. this indicates the presence of a 4-OH or a 5-OH SDZ-MB hydroxylated at the pyrimidine-ring (28). Further investigation (MS3) of this fragment proved the metabolite to be 4-OHSDZ in the presence of an m/z 70 fragment (Figure 2C) (28). Furthermore, the comparison with isocytosine, which represents the pyrimidine part of the molecule oxidized at fourth position, indicates an MS/MS-spectrum that matches the MS3-spectrum of the metabolite (Figure 2 B and C). The
FIGURE 2. MS/MS (A) and MS3 (C) spectra of 4-OH-sulfadiazine and synthesized 5-OH-SDZ does not generate an m/z 70 fragment (mass spectrum not shown). Furthermore, the 5-OH-SDZ elutes at a different time from the HPLC column. In conclusion the molecular structure of the metabolite as 4-OHsulfadiazine has been confirmed. Antibiotic Activity of 4-OH-SDZ against Geobac. stearothermophilus var. calidolactis C953. A BRT MRL-Screening Test designed for detecting inhibitors or residual antiinfectives in milk was carried out. Employing this test system, a drastically reduced antimicrobial activity of the isolated metabolite could be shownsless than 10% of the parent substance. This tallies well with the findings of Nouws (38) which also confirms the OH-metabolite regarding its residual antibiotic activity. In summary, despite a similar basic structure (Figure 1) there is a probably structure-dependent elimination of four of the seven sulfonamides under investigation. While sulfadiazine, having no substituent at the pyrimidine ring, can be hydroxylated and thus degradated, sulfamethazine, having two substituents, can not be eliminated. Sulfamerazine, exhibiting one substituent, could be completely eliminated. But, in the present study no degradation products for this compound could be identified. Detailed MS/MS investigations in both fermentation experiments revealed that hydroxylation of sulfamerazine probably does not take place at the pyrimidine ring. It could not be clarified conclusively whether another degradation process or a sorption process is responsible for the observed elimination. Sulfamethoxazole is eliminated to a degree of more than 50% even before the fermentation process has been started. Here also the fate of the drug could not be clarified in the present study. The fate of sulfonamides, especially of sulfamethoxazole, a sulfonamide mainly applied in human medicine, is widely discussed. Yet, no degradation products in soil have been found (39). Most studies suggest a strong sorption of sulfonamides in soil (2, 6, 40-42). Chander et al. revealed that tetracycline and tylosin although soil-adsorbed retain their antimicrobial properties (43, 44). In this context antibiotic resistance induced by persisting and probable accumulation of antibiotics in the environment might be an increasing problem in public health (2, 7). Antibiotics are still essential in the treatment of livestock. Through sickness and death of animals not treated, farmers would suffer from huge economic losses. Besides tetracyclines, the group of sulfonamides, still effective against typical diseases, remains important in animal husbandry. Through manure the antibiotics reach the environment and may influence microbial life (44-46). Similar to manure, fermentation residues can also be used as fertilizers on agricultural fields. A possibly reduced antimicrobial activity,
MS/MS spectrum of isocytosine (B). as presented in this study, may in consequence reduce the ecotoxicological impact of these drugs on the environment and lower the risk of antibiotic resistance. Thus, the results of the present study may lead to the identification of environmentally friendly drugs and be a contribution to “green” chemistry (8). Further investigations are underway to identify and characterize other possible degradable veterinary pharmaceuticals.
Acknowledgments This study was supported by the Federal Ministry of Economics and Technology (BMWi), initiated by the German Federation of Industrial Cooperative Research Associations (AiF-FV 185 Z). Many thanks go to Ansgar Adriany (AiM, Munich, Germany) for coaching in the use of and providing test kits (MRL-Screening Test). We are grateful to Mike Duisken (LECO Instrumente GmbH, Mo¨nchengladbach Germany) for performing the exact mass measurements and LECO Instrumente GmbH for the possibility to use their equipment. We thank Marion Schro¨der (University of Veterinary Medicine Hannover) for her excellent technical help, Bettina Sayder and Ute Merrettig-Bruns (Fraunhofer UMSICHT, Oberhausen, Germany) for fruitful discussions and providing supporting information on fermentation tests, the biogas plant Loick (Dorsten-Lembeck, Germany) for providing samples, and Frances Sherwood-Brock for carefully proofreading of the manuscript. Many thanks for helpful comments from three anonymous reviewers.
Note Added after ASAP Publication Due to a production error, a mistake was made to a value in Table 2 of the version published on February 23, 2009. The corrected version was published on March 4, 2009.
Supporting Information Available Photometrically determined inhibition of sulfadiazine (SDZ) and 4-OH-sulfadiazine (SDZ-MB). This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Sarmah, A. K.; Meyer, M. T.; Boxall, A. B. A. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65 (5), 725–759. (2) Tolls, J. Sorption of veterinary pharmaceuticals in soils: A review. Environ. Sci. Technol. 2001, 35 (17), 3397–3406. (3) Langhammer, J.-P. Untersuchungen zum Verbleib antibmikrobiell wirksamer Arzneistoffe als Ru ¨ cksta¨nde in Gu ¨ lle und im Landwirtschaftlichen Umfeld. PhD-Thesis. Rheinische FriedrichWilhelms-Universita¨t, Bonn, Germany, 1989. VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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