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Behaviour of 14C-sulfadiazine and 14C-difloxacin during manure storage
Science of the Total Environment 408 (2010) 1563–1568
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Behaviour of
14
C-sulfadiazine and
14
C-difloxacin during manure storage
Marc Lamshöft, Premasis Sukul, Sebastian Zühlke, Michael Spiteller ⁎ Institute of Environmental Research (INFU), TU Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany
a r t i c l e
i n f o
Article history: Received 7 July 2009 Received in revised form 23 November 2009 Accepted 6 December 2009 Available online 21 December 2009 Keywords: Sulfonamide Fluoroquinolone Metabolites Deacetylation LC-MS/MS Radioactivity balance
a b s t r a c t The persistence of sulfadiazine, difloxacin, and their metabolites has been investigated in stored manure. The manure collected from sulfadiazine (14C-SDZ) and difloxacin (14C-DIF) treated pigs contained N-acetylsulfadiazine (Ac-SDZ), 4-hydroxy-SDZ (4-OH-SDZ), and sarafloxacin (SARA) as the main metabolites, respectively along with their parent compounds. Manures were stored separately at 10 °C and 20 °C at various moisture levels. About 96–99% of the radioactivity remained in extractable parent compounds and their metabolites after 150 d of storage. The formation of non-extractable residue and the rate of mineralization were both negligible in manure containing SDZ and DIF. During storage SDZ concentration increased as a result of the deacetylation of Ac-SDZ, whose concentration decreased proportionally. Hence the environmental effects may be underestimated if the parent compound alone is considered for environmental risk assessment. About 11% and 14% of 4-OH-SDZ were lost after 20 and 40 d of storage; thereafter its concentration increased relatively, highlighting hydroxylation of SDZ. DIF degraded very slowly (7% loss after 150 d) during the storage of manure; in contrast the concentration of SARA decreased rapidly (72–90% loss after 150 d). Dilution of manure and storage at higher temperatures for a reasonable period of time enhanced the rate of reactions of SDZ, DIF and their related metabolites. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Veterinary antibiotics are widely administered to animals to treat diseases and promote growth in agricultural industry (Hassouan et al., 2007). The major route through which veterinary antibiotics enter the environment is the excretion of faeces and urine from animals medicated in livestock and poultry farming, and the subsequent application of contaminated manure as fertilizer into agricultural land (Boxall et al., 2003). After application of the manure the antibiotics and their metabolites may interact with different soil constituents, may enter the food chain by plant uptake, may leach into groundwater, or may occur in surface water via runoff (Boxall et al., 2002; Thiele-Bruhn, 2003; Sukul and Spiteller, 2006). Numerous studies have shown that as much as ∼ 40 to 96% of the administered drugs are excreted by medicated animals (Halling-Sorensen et al., 2001; Lamshöft et al., 2007; Sukul et al., 2009). Elevated antibiotic concentration may affect the structural and functional microbial diversity in soils (Kotzerke et al., 2008) and promote the formation and increase the risk of spreading of resistance genes in the environment (Heuer and Smalla, 2007; Heuer et al., 2008). The antimicrobial activities of many antibiotics' metabolites, with a special reference to sulfonamides and fluoroquinolones, have been reported (Marengo et al., 1997; Wetzstein et al., 2000). Thus, before studying
⁎ Corresponding author. Tel.: +49 231 7554080; fax: +49 231 7554085. E-mail address: [email protected] (M. Spiteller). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2009.12.010
different aspects of environmental risk assessment for antibiotics, it becomes essential to understand the fate of parent compounds and their bio-transformed metabolites in manure originating from medicated animals under realistic storage conditions. The widely used sulfonamide sulfadiazine [SDZ, 4-amino-N-(2pyrimidinyl)benzene sulfonamide (Fig. 1)] has been investigated in numerous studies (Thiele-Bruhn, 2003; Thiele-Bruhn et al., 2004; Kreuzig and Höltge, 2005; Förster et al., 2007; Sukul et al., 2008a,b). SDZ is generally applied as an antibiotic to pigs and calves. It competes with p-aminobenzolic acid in the enzymatic synthesis of dihydrofolic acid and thus inhibits the growth and reproduction of bacteria. From applied SDZ to animals, about 44% are excreted as parent compound, about 26% as acetyl conjugate and 19% as hydroxylated compound (Lamshöft et al., 2007). The N-acetyl derivatives of sulfonamide antibiotics are reported to be deacetylated to the parent compound (Grote et al., 2004). Difloxacin [DIF, 6-fluoro-1-(4-fluorophenyl)-1,4-dihydro-7-(4methyl-1-piperazinyl)-4-oxo-3-quinolonecarboxylic acid (Fig. 1)] belongs to a large group of structurally related antibiotics, fluoroquinolones. DIF exhibits a broad-spectrum activity against Gram-positive and -negative bacteria (Eliopoulos et al., 1985; Stamm et al., 1986), and mycoplasmas (Kenny et al., 1989). It is proposed that it interferes with bacterial DNA metabolism by inhibiting bacterial topoisomeraseII (in Gram-negative bacteria) and topoisomerase-IV (in Grampositive bacteria), preventing DNA synthesis, and thus disrupts bacterial cell duplication (Hooper and Wolfson, 1993; Drlica and Zhao, 1997). DIF differs in particular from other fluoroquinolones on
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2.2. Pig manure The manure from SDZ and DIF treated pigs were collected from Bayer Crop Science AG, Monheim, Germany, and NOTOX Safety and Environmental Research B.V.,'s-Hertogenbosch, The Netherlands, respectively. The details of the administration of antibiotics to the pigs, the doses used, and sample collection are reported in previous publications (Lamshöft et al., 2007; Sukul et al., 2009). Manure properties are listed in Table 1. Control pig manure was free from any antibiotics, since it was obtained from non-treated animals. However, control slurry was extracted and analyzed by LC-MS/MS to confirm the absence of sulfadiazine (Lamshöft et al., 2007) and DIF (Sukul et al., 2009). After collection, pig manure was stored at 4 °C in a polyethylene bucket for 2 weeks. Prior to the experimental set-up, the manure was allowed to attain room temperature.
Fig. 1. Chemical structure of sulfadiazine, difloxacin and their major metabolites.
account of its p-fluorophenyl ring at position N-1 of the quinolone nucleus, which reportedly gives its enhanced activity against Grampositive bacteria (Walker, 2000). However, in some species it is demethylated to its primary metabolite, sarafloxacin (SARA), which also displays potent antibacterial activity (Granneman et al., 1986) and in some species DIF is primarily metabolized by glucuronidation (Chu et al., 1985). Experiments with radio-labelled compounds showed that difloxacin accounted for 95.9% of the radioactivity excreted in pigs (Sukul et al., 2009). Several studies have been performed to investigate the degradation of antibiotics in spiked manure (Kuhne et al., 2000; Teeter and Meyerhoff, 2003; Wang et al., 2006a), soil (Wang et al., 2006b; Heuer et al., 2008) and water (Alexy et al., 2004; Boreen et al., 2005) with a focus on eliminating the contamination caused by veterinary drugs. Additionally, it was always found that aerobic conditions were appropriate to the degradation of the drugs in manure and water (Kuhne et al., 2000; Ingerslev et al., 2001). No information is to our knowledge available on the fate and behaviour of SDZ and DIF and their metabolites in manure and on the effects of various environmental factors. It is normal practice to store manures and slurry for a considerable period of time after collection from livestock farms and before application to soils. The storage time of slurries in the U.K. varies from 0 to 50 months, with an average of 9 months (WRc-NSF, 2000). Thus, storage time should also play a significant role in the dissipation of veterinary drugs. Against this background, attempts have been made in the present investigation to study the kinetics of SDZ, DIF, and their respective metabolites in pig manure, to establish its mass balance and to identify all detectable metabolites after oral administration of selected veterinary drugs to pigs. 2. Materials and methods 2.1. Chemicals The analytical standard grade SDZ (99%) was purchased from Fluka, Seelze, Germany. 14C-SDZ (99%) labelled at the 2-pyrimidine position with a specific radioactivity of 8.6 MBq/mg was obtained from Bayer AG, Wuppertal. The internal standard 13C-SDZ (99%) was obtained from the Department Bio V, University of Aachen, Germany. All six-carbon atoms of the aniline ring were 13C-labelled. DIF (99%) was purchased from Chemos GmbH, Germany. 14C-DIF (99.1%) with a specific radioactivity of 2.15 GBq/mmol and labelled at the 2-pyridine position was obtained from GE Healthcare UK limited, UK. The internal standard 13C15N-DIF was synthesized and purified (N99%, checked by LC/MS) at the Department of Biology V, University of Aachen, Germany. SARA (99%) was purchased from Fluka GmbH, Switzerland. All of the solvents and chemicals used were of analytical grade.
2.3. Storage Manure (50 g) was kept in 300 mL Erlenmeyer flasks fitted with trap attachments. The trap attachments were filled with soda–lime to absorb released 14CO2. The flasks were stored in the dark at two temperatures (10 °C, 20 °C) in incubation chambers. At each sampling point the pH (Scientific Instruments IQ240, Colorado USA) and the redox-potential (WTW ph530, Weilheim Germany) of the manure were measured. All experiments were carried out in triplicate. Dilutions were performed with fresh manure from the control treatment, which did not contain any antibiotic residues. 2.4. Sample extraction Manure samples were extracted immediately after sampling. For SDZ and its metabolites samples of homogenized manure (1 g) were transferred into 15 mL screw-topped; polyethylene centrifuge tubes, and 4 mL of McIlvaine buffer (1 M citric acid: 1 M Na2HPO4, 18.15 mL: 81.85 mL; adjusted to pH 7) and 500 ng of the internal standard 13C6SDZ were added. For DIF and its metabolites 4 mL of acetonitrile (+0.1% formic acid) and 1000 ng of internal standard 13C/15N-DIF were added to 1 g manure. The contents were shaken for 30 s with a vortex mixer, treated for 15 min in an ultrasonic bath and centrifuged for 15 min at 15,000 rpm in a model Avanti J-25 centrifuge (Beckman, Fullerton, CA, USA), equipped with a JLA-16.250 rotor. The supernatants were transferred into glass test tubes. The extraction was repeated with 5 mL McIlvaine buffer for SDZ and its metabolites; while for DIF and its metabolites extraction was repeated with 5 mL acetonitrile–water (20/ 80). The respective resulting extracts were pooled and were directly measured in the HPLC-MS/MS without further treatment or preparation. 2.5. Quantitative determination by LC-MS/MS Quantitation of the administered veterinary drugs and their main metabolites by HPLC-MS/MS was performed by means of previously Table 1 Physicochemical properties of the test manures. Properties
Experiment-1
Experiment-2
Control Manure containing Control Manure containing SDZ and its DIF and its metabolites metabolites Dry mass (%) Total carbon, g/kg pH Biological oxygen demand (BOD)5, g/L Chemical oxygen demand (COD), g/L Electrical conductivity, µS/cm
5.8 31.1 7.7 50
6.0 34.4 7.8 39.8
3.3 16.3 8.2 41
3.5 16.8 8.1 35
79.6
60
35
24
35,500
33,600
18,280
15,970
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described analytical methods (Lamshöft et al., 2007; Sukul et al., 2009).
Table 2 Radioactivity distribution in manure at different sampling periods at 20 °C. Days
2.6. Radioactivity measurements The radioactively labelled extracts were analyzed for radioactivity content by liquid scintillation counting (Beckmann LS6500, Fullerton, CA, USA) using Quicksafe A (Zinsser, Frankfurt, Germany). Insoluble residues were subjected to combustion analysis using an OX500 biological oxidizer (Zinsser, Frankfurt, Germany) and the released 14 CO2 was measured by liquid scintillation counting (LSC) using Oxysolve C-400 (Zinsser, Frankfurt, Germany) as scintillation cocktail. For the determination of 14CO2 from the trap attachments, the soda– lime (10 g) of the trap was dissolved with drop-wise addition of 18% HCl (∼ 50–60 mL) under a gentle stream of nitrogen in a suitable glass apparatus (Sukul et al., 2008a). The released 14CO2 was absorbed by a series of three vials containing 15 mL of an ice-cooled cocktail of Oxysolve C-400. 3. Results and discussion
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parent compound + metabolite(s) mg/L
% recovered radioactivity Non-extractable residue
14
Manure containing SDZ and its metabolites 1 156.0 ± 4.2 98.9 3 151.4 ± 3.3 98.9 7 151.4 ± 4.0 98.2 15 146.0 ± 3.5 99.6 30 148.4 ± 2.6 99.9 60 150.8 ± 2.7 99.7 100 152.8 ± 3.8 99.2 150 151.8 ± 2.9 99.0
0.3 0.3 0.2 0.2 0.4 0.7 0.8 0.9
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2
99.2 ± 0.5 99.2 ± 0.9 98.4 ± 0.8 99.8 ± 0.7 100.3 ± 0.9 100.4 ± 0.8 100.0 ± 0.7 100.1 ± 0.6
Manure 1 3 7 15 30 60 100 150
0.2 0.2 0.2 0.4 0.5 0.7 1.2 4.2
0.0 0.0 0.0 0.0 0.0 0.1 0.5 0.5
106.2 ± 0.9 101.2 ± 0.9 99.7 ± 0.8 101. 4 ± 0.7 98.0 ± 0.8 97.8 ± 0.7 96.7 ± 0.7 95.7 ± 0.7
Extractable residue
containing DIF and its metabolite 17.6 ± 0.7 106.0 16.3 ± 0.6 101.0 16.6 ± 0.5 99.5 16.2 ± 0.6 101.0 15.9 ± 0.6 97.5 15.7 ± 0.6 97.0 15.3 ± 0.5 95.0 15.1 ± 0.4 91.0
CO2
Total
3.1. Radioactivity distribution A radioactivity balance of SDZ and DIF in manure stored at 20 °C was established on the basis of LSC data (Table 2). 0.9% and 4.2% radioactivities were found in the form of non-extractable residue in manure containing SDZ and DIF, respectively after 150 d of storage. The total amount of extractable radioactivity declined. However, the formation of non-extractable residue was negligible. In each case, 14 CO2 was detected in low quantities at all sampling intervals, which indicated that a mineralization was not a major route of antibiotic dissipation during manure storage. Perhaps, this is due to low biological activity (Table 1) and existence of anaerobicity, as evidenced from the redox-potential (Table 3), in the manure system. As a realistic approach the manure was not subjected to any additional aeration from external source. However, it is known that aerobic conditions are appropriate to the degradation of the drugs in manure and water (Kuhne et al., 2000; Ingerslev et al., 2001). Interestingly, a little change in the absolute amount of extracted antibiotic and its metabolites was observed, which correlated with little change in extractable radioactivity, non-extractable radioactivity and mineralization (Table 2). A similar trend was also observed when manure containing 14C-SDZ and 14C-DIF along with their metabolites was stored separately at 10 °C. To our knowledge no information is available on the fate of SDZ and DIF contained in pig manure during storage. However, fate of antibiotics in spiked manure and manure amended soil is available (Kreuzig et al., 2003; Kreuzig and Höltge, 2005; Heise et al., 2006; Schmidt et al., 2008; Wang et al., 2006a, b). A rapid formation of the non-extractable residues was observed as the predominant process in bovine manure spiked with 14C-SDZ (Kreuzig and Höltge, 2005). Results showed that the amount of extractable 14C-SDZ residues continuously dropped from 70% to 5% of the initial applied radioactivity, which differs from our results. This may be due to the difference in the nature of studied manure, route of administration of antibiotics in the manure and analytical methods used. 3.2. Behaviour of drugs under various conditions of storage The redox-potential and the pH of the manure system were monitored at each sampling time (Table 3). No significant change in pH was noticed. For the manure containing SDZ and its metabolites, and for the manure containing DIF and SARA, the pH ranges were 7.8– 8.1, and 8.1–8.3, respectively. The redox-potentials lay between −280 mV and −329 mV for both manures, indicating anaerobic conditions. In order to simulate the farmers' practice we did not purge the incubation vessels with air. However, aerobic conditions were found to be appropriate to the degradation of the drugs in manure and
20 times 1 3 7 15 30 60 100 150
diluted manure containing SDZ and its metabolites 8.2 ± 0.2 99.9 0.3 7.2 ± 0.2 99.9 0.3 8.2 ± 0.2 99.2 0.2 7.7 ± 0.2 99.6 0.2 8.0 ± 0.2 99.1 0.4 8.1 ± 0.3 99.0 0.7 8.1 ± 0.2 98.8 0.8 8.2 ± 0.3 98.7 0.9
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1
100.2 ± 0.8 100.2 ± 0.9 99.4 ± 0.7 99.8 ± 0.9 99.5 ± 0.9 99.7 ± 0.8 99.6 ± 0.8 99.6 ± 0.7
20 times 1 3 7 15 30 60 100 150
diluted manure containing DIF and its metabolite 0.9 ± 0.1 100.5 0.2 0.9 ± 0.1 100.1 0.2 0.8 ± 0.1 99.9 0.2 0.8 ± 0.1 99.5 0.4 0.8 ± 0.1 98.0 0.5 0.8 ± 0.1 97.2 0.7 0.7 ± 0.1 96.0 1.3 0.7 ± 0.1 92.2 3.9
0.0 0.0 0.0 0.0 0.0 0.1 0.4 0.5
100.7 ± 0.9 100.3 ± 0.8 100.1 ± 0.8 99.9 ± 0.7 98.5 ± 0.9 98.0 ± 0.8 97.4 ± 0.7 96.6 ± 0.7
water (Kuhne et al., 2000; Ingerslev et al., 2001; Wang et al., 2006a). Manure containing 51.6 kBq/mL radioactivity for SDZ (74.7 mg/L) and its major metabolites (Ac-SDZ, 36.5 mg/L; 4-OH-SDZ, 44.7 mg/L) were incubated for the storage experiments at 10 °C and 20 °C. The
Table 3 Redox-potential and pH of manure under storage condition at different time intervals. Days
10 °C pH
20 °C Redox-potential (mV)
pH
Redox-potential (mV)
Manure containing SDZ and its metabolites 0 7.8 −322 10 7.8 −312 20 7.9 −320 40 7.9 −310 60 7.9 −299 90 7.9 −280 150 8.0 −285
7.8 7.8 7.9 7.9 7.9 8.0 8.1
− 314 − 301 − 311 − 302 − 320 − 333 − 329
Manure containing DIF and SARA 0 8.1 −310 1 8.1 −315 7 8.1 −301 15 8.1 −302 30 8.1 −300 60 8.2 −305 100 8.2 −280 150 8.2 −281
8.1 8.1 8.1 8.1 8.1 8.2 8.2 8.3
− 300 − 298 − 290 − 285 − 290 − 285 − 284 − 280
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concentration profiles of SDZ and its two main metabolites at different intervals are presented in Fig. 2. The SDZ concentration was found to have increased at all times over its concentration (0 d). Although the increase did not follow a definite pattern at 10 °C, there was a significant and continuous increase at 20 °C. By the end of the experiment (150 d) 7.8% and 35.6% increase in SDZ concentration was measured at 10 °C and 20 °C, respectively (Table 4); indicating a marked influence of temperature. A significant decline in Ac-SDZ was associated with the increase in SDZ concentration. There were 36.7% and 79.1% losses of Ac-SDZ at 10 °C and 20 °C, respectively after 150 d of manure storage also confirming the effect of temperature on the degradation of Ac-SDZ. Although the N-acetyl derivative of sulfonamide antibiotics itself is not antimicrobially active, it is reported to be deacetylated to the parent compound by hydrolysis (Kreuzig and Höltge, 2005; Grote et al., 2004). This highlights the fact that the consideration of the fate of antibiotics' metabolites is urgently required in order to estimate correctly the environmental effect of a veterinary drug in risk assessment procedures. At both temperatures the maximum loss of 4-OH-SDZ was observed after 20 and 40 d of storage; thereafter its concentration increased. This increase highlights the hydroxylation of SDZ during manure storage. In contrast to our results, in a separate experimental set-up where manure was spiked with the antibiotic, the concentration of sulfadimethoxine, another compound of sulfonamide class of antibiotics, was reduced very rapidly with a half-life value of 3 d (Wang et al., 2006a). This difference may be accounted for by the use of a different compound and a different manure, which vary in their respective physicochemical properties. Additionally, in our experiment the manure was not spiked, on the contrary it was obtained from antibiotic treated pigs and it contained SDZ, Ac-SDZ and 4-OH-SDZ. Spiking of the active drug to the manure does not provide answers on the metabolites generated in an animal system. Furthermore, the physicochemical environment of spiked manure and manure containing antibiotics after feeding antibiotics to the animals are quite different. Hence, the realistic approach
Table 4 Percent change of SDZ and its metabolites in manure against their initial concentrations during storage at different conditions. Days of incubation
10 °C % change SDZ
20 °C % change Ac-SDZ
% change 4-OH-SDZ
% change SDZ
% change Ac-SDZ
% change 4-OH-SDZ
– − 14.2 − 25.8 − 32.5 − 35.2 − 34.0 − 36.7
– − 7.7 − 20.7 − 17.9 − 8.9 − 9.7 − 8.7
– + 12.9 + 22.6 + 30.3 + 33.2 + 34.6 + 35.6
– − 26.0 −40.0 − 65.3 − 75.0 − 75.6 − 79.1
– − 10.6 − 11.5 − 14.3 − 6.0 − 3.4 − 4.4
10 times diluted manure 0 10 20 40 60 90 150
– + 15.2 + 21.7 + 27.7 + 30.3 + 32.9 + 35.3
– − 48.8 − 76.0 − 94.3 − 99.3 − 99.7 − 99.8
– − 2.7 −0.7 − 6.2 −8.1 − 7.9 − 7.8
20 times diluted manure 0 10 20 40 60 90 150
– + 9.7 + 27.2 + 30.8 + 31.5 + 32.7 + 33.4
– − 60.4 − 74.4 − 96.8 − 99.1 − 99.1 − 99.3
– − 12.4 + 9.5 + 2.1 + 14.3 + 18.9 + 21.6
Original manure 0 – 10 + 7.1 20 + 6.2 40 + 10.2 60 + 4.4 90 + 6.1 150 + 7.8
should be to treat the animals with active drugs and to study the fate and behaviour of these in manure. Rates of reactions, either in terms of percent gain of SDZ or percent loss of Ac-SDZ, were increased when incubation temperature was
Fig. 2. Concentration profile of SDZ and its major metabolites in manure under storage condition at 10 °C (A) and 20 °C (B), and at dilution of 1/10 (C) and 1/20 (D) of manure.
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increased from 10 °C to 20 °C (Table 4). On the basis of the Arrhenius equation the temperature has a direct influence on the reaction rate constants; an increase in temperature accelerates a reaction by increasing the rate constant. Our results conforms the earlier report (Wang et al., 2006a). Hence, storing manure under warm condition might be a good strategy for reducing antibiotic contamination in the agricultural environment. However, specifically for manure containing SDZ and its metabolites, the situation is entirely different and complex because of the reversible conversion of Ac-SDZ to SDZ. Storing manure at higher temperature increases conversion rate of Ac-SDZ to SDZ; while at a lower temperature this conversion occurs at a comparatively slow rate, SDZ does not dissipate. Dilution of manure, as such does not have any effect on percent increase of SDZ (Table 4). At 20 °C original manure, manure diluted 10 times and 20 times showed 35.6, 35.3 and 33.4% increment relative to its initial concentration, respectively. However, dilution of manure showed a marked effect in percent loss of Ac-SDZ. Nearly 100% of AcSDZ was transformed in diluted manure while 79% loss was evidenced
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Table 5 Percent change of DIF and SARA in manure during storage at different conditions. Days of incubation
10 °C % change DIF
20 °C % change SARA
% change DIF
% change SARA
– − 38.9 − 60.4 − 62.5 − 63.2 − 66.0 − 68.1 − 68.1 − 72.2
– − 2.0 − 3.0 − 2.9 − 3.1 − 3.6 − 5.2 − 6.8 − 7.3
– − 47.9 − 58.3 − 64.6 − 66.0 − 75.7 − 72.9 − 87.5 − 89.6
10 times diluted manure 0 1 3 7 15 30 60 100 150
– 0.0 − 7.3 − 2.4 − 8.5 − 6.1 − 2.4 − 19.5 − 19.5
– − 25.0 − 37.5 − 62.5 − 50.0 −62.5 −62.5 −87.5 −93.8
20 times diluted manure 0 1 3 7 15 30 60 100 150
– − 1.8 − 4.1 − 8.8 −6.5 − 10.6 − 8.2 − 21.2 − 20.6
– − 18.8 −68.8 −75.0 − 68.8 − 87.5 − 93.8 − 93.8 −93.8
Original manure 0 – 1 − 0.2 3 − 1.2 7 − 4.6 15 − 4.1 30 − 3.6 60 − 5.5 100 − 5.6 150 − 7.4
in original manure. At 20 °C, manure diluted 20 times showed a gain in 4-OH-SDZ concentration, relative to its initial concentration. Hence it can be concluded that under dilution a reasonable amount of AcSDZ was converted into SDZ, a portion of which in turn again converted into 4-OH-SDZ. This also establishes the degradation of SDZ. Perhaps a longer storage time would further increase SDZ degradation. This needs to be further investigated. Manure containing 4.3 kBq/mL radioactivity for DIF (16.2 mg/L) and SARA (1.4 mg/L) was stored at 10 °C and 20 °C. DIF showed a persistent behaviour in manure during its storage. About 7% loss of DIF was found after 150 d of manure storage at both 10 °C and 20 °C (Fig. 3, Table 5). However, nearly 72% and 90% losses of SARA were observed after storage for the same period at 10 °C and 20 °C, respectively showing a marked influence of temperature. Dilution of manure also enhanced the dissipation of DIF and SARA. About 20% DIF and 94% SARA were lost after 150 d of storage under both 10 and 20 times dilution of manure at 20 °C. Although the dilution of contaminated manure with manure from control treatment free of antibiotics increases the number of available sorption sites in manure, the observed enhanced dissipation of DIF and SARA during dilution is not due to increased sorption. Non-extractable radioactivity did not increase in diluted manure (Table 2). Control manure without any contamination from antibiotics contained more BOD value than the treated manure (Table 1). Perhaps, addition of control manure to the treated manure for dilution increased the biological activity in the system, which possibly caused the increased rate of reaction. 4. Conclusion
Fig. 3. Concentration profile of DIF and SARA in manure under storage condition at 20 °C (A) and 10 °C (B), and at different dilutions (C) of manure.
Pharmaceutical antibiotics enter agricultural soils and adjacent environmental compartments through the use of contaminated manure and sludge as fertilizer. The present study revealed that in addition to the parent compound, the fate and effect of metabolites
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should also be investigated. Though the parent compound is generally more potent than its metabolites, the latter may still be biological active and its impact may therefore be underestimated if only the parent compound is considered. It is even more important to consider the metabolites, when they are retransformed into the more active parent compound. An experimental design using manure merely spiked with veterinary drugs is not appropriate because it neglects the fate and effect of metabolites originating in the animal. Our experimental results also demonstrated that storing manure contaminated with DIF and SARA contributed little to the degradation of DIF, but showed a pronounced effect in reducing SARA concentration. Furthermore, mixing of contaminated manure with fresh control manure led to a comparatively higher rate of degradation of DIF and SARA. Therefore, it may be a good strategy to mix manure contaminated with DIF and SARA with fresh control manure for reducing their contamination in the environment. However, for manure containing SDZ and its metabolites, the reversible conversion of Ac-SDZ to SDZ made the situation more complex, leading to an accumulation of SDZ in manure. Therefore, frequent fertilization of soil by manure contaminated with SDZ and its metabolites may lead to environmental contamination. Acknowledgements This study has been performed within the frame of the research project “Veterinary medicines in soils: basic research for risk analysis”, funded by the German Science Foundation, Deutsche Forschungsgemeinschaft (DFG), to whom the authors are deeply grateful. Jürgen Storp, Cornelia Stolle and Jennifer Hardes are gratefully acknowledged for their technical support. References Alexy R, Kumpel T, Kummerer K. Assessment of degradation of 18 antibiotics in the closed bottle test. Chemosphere 2004;57:505–12. Boreen AL, Arnold WA, McNeill K. Triplet-sensitized photodegradation of sulfa drugs containing six-membered heterocyclic groups: identification of an SO2 extrusion photoproduct. Environ Sci Technol 2005;39:3630–8. Boxall ABA, Blackwell PA, Cavallo R, Kay P, Tolls J. The sorption and transport of a sulfonamide antibiotic in soil system. Toxicol Lett 2002;131:19–28. Boxall ABA, Blackwell PA, Boleas S, Halling-Sørensen B, Ingerslev F, Jacobsen AM, et al. Environmental risk assessment of veterinary medicines in slurry. Cranfield University, UK (Final Report of the EU ERAVMIS Project No. EVK1-CT-1999-00003); 2003. Chu DTW, Granneman GR, Fernandes PB. ABOTT-56619. Drugs Future 1985;10:543–5. Drlica K, Zhao X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev 1997;61:377–92. Eliopoulos GM, Moellering AE, Reiszner E, Moellering Jr RC. In vitro activities of the quinolone antimicrobial agents A-56619 and A-56620. Antimicrob Agents Chemother 1985;28:514–20. Förster M, Laabs V, Lamshöft M, Pütz T, Amelung W. Analysis of aged sulfadiazine residues in soils using microwave extraction and liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 2007;391:1029–38. Granneman GR, Snyder KM, Shu VS. Difloxacin metabolism and pharmacokinetics in human after single oral dose. Antimicrob Agents Chemother 1986;30:689–93. Grote M, Vockel A, Schwarze D, Mehlich A, Freitag M. Fate of antibiotics in food chain and environment originating from pig fattening. Fresenius Environ Bull 2004;13:1214–6. Halling-Sorensen B, Jsensen J, Tjornelund J, Montforts MHMM. Worst-case estimation of predicted environmental soil concentration (PEC) of selected veterinary antibiotics and residues used in Danish agriculture. In: Kummerer K, editor. Pharmaceuticals in the Environment. Berlin: Springer; 2001.
Hassouan M, Ballesteros J, Vilchez J, Zafra A, Navalόn A. Simple multiresidue determination of fluoroquinolones in bovine milk by liquid chromatography with fluorescence detection. Anal Lett 2007;40:779–91. Heise J, Höltge S, Schrader S, Kreuzig R. Chemical and biological characterization of nonextractable sulfonamide residues in soil. Chemosphere 2006;65:2352–7. Heuer H, Smalla K. Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environ Microbiol 2007;9:657–66. Heuer H, Focks A, Lamshöft M, Smalla K, Matthies M, Spiteller M. Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil. Soil Biol Biochem 2008;40:1892–900. Hooper DC, Wolfson JS. Mechanism of quinolone action and bacterial killing. In: Hooper DC, Wolfson JS, editors. Quinolone antibacterial agents. Washington, DC: American Society for Microbiology; 1993. p. 53–75. Ingerslev F, Torang L, Loke M-L, Halling-Sorensen B, Nyholm N. Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems. Chemosphere 2001;44:865–72. Kenny GE, Hooton TM, Roberts MC, Cartwright FD, Hoyt J. Susceptibilities of genital mycoplasmas to the newer quinolones as determined by the agar dilution method. Antimicrob Agents Chemother 1989;33:103–7. Kotzerke A, Sharma S, Schauss K, Heuer H, Thiele-Bruhn S, Smalla K, et al. Alterations in soil microbial activity and N-transformation processes due to sulfadiazine loads in pig-manure. Environ Pollut 2008;153:315–22. Kreuzig R, Höltge S. Investigations of the fate of sulfadiazine in manured soil: laboratory experiments and test plot studies. Environ Toxicol Chem 2005;24:771–6. Kreuzig R, Kullmer C, Matthies B, Höltge S, Dieckmann H. Fate and behaviour of pharmaceutical residues in soils. Fresenius Environ Bull 2003;12:550–8. Kuhne M, Ihnen D, Möller G, Agthe O. Stability of tetracycline in water and liquid manure. J Vet Med A 2000;47:379–84. Lamshöft M, Sukul P, Zühlke S, Spiteller M. Metabolism of 14C-labeled and non-labeled sulfadiazine after administration to pigs. Anal Bioanal Chem 2007;388:1733–45. Marengo JR, Kok RA, O'Brian K, Velagaleti RR, Stamm JM. Aerobic biodegradation of (14C)-Sarafloxacin hydrochloride in soil. Environ Toxicol Chem 1997;16:462–71. Schmidt B, Ebert J, Lamshöft M, Thirde B, Schumacher-Buffel R, Ji R, et al. Fate in soil of 14 C-sulfadiazine residues contained in the manure of young pigs treated with a veterinary antibiotic. J Environ Sci Health Part B 2008;43:8-20. Stamm JM, Hanson CW, Chu DTW, Bailer R, Vojtko C, Fernandes PB. In vitro evaluation of A-56619 (Difloxacin) and A-56620: new aryl-fluoroquinolones. Antimicrob Agents Chemother 1986;29:193–200. Sukul P, Spiteller M. Sulfonamides in the environment as veterinary drugs. Rev Environ Contam Toxicol 2006;187:67-101. Sukul P, Lamshöft M, Zühlke S, Spiteller M. Photolysis of 14C-sulfadiazine in water and manure. Chemosphere 2008a;71:717–25. Sukul P, Lamshöft M, Zühlke S, Spiteller M. Sorption and desorption of sulfadiazine in soil and soil-manure systems. Chemosphere 2008b;73:1344–50. Sukul P, Lamshöft M, Kusari S, Zühlke S, Spiteller M. Metabolism and excretion kinetics of 14C-labeled and non-labeled difloxacin in pigs after oral administration, antimicrobial activity of manure containing difloxacin and its metabolites. Environ Res 2009;109:225–31. Teeter JS, Meyerhoff RD. Aerobic degradation of tylosin in cattle, chicken, and swine excreta. Environ Res 2003;93:45–51. Thiele-Bruhn S. Pharmaceutical antibiotic compounds in soils — a review. J Plant Nutr Soil Sci 2003;166:145–67. Thiele-Bruhn S, Seibicke T, Schulten H-R, Leinweber P. Sorption of sulfonamide pharmaceutical antibiotics on whole soils and particle-size fractions. J Environ Qual 2004;33:1331–42. Walker RD. Fluoroquinolones. In: Prescot JF, Baggot JD, Walker RD, editors. Antimicrobial therapy in veterinary medicine. 3rd ed. Ames, IA: Iowa State University Press; 2000. Wang Q-Q, Bradford SA, Zheng W, Yates SR. Sulfadimethoxine degradation kinetics in manure as affected by initial concentration, moisture, and temperature. J Environ Qual 2006a;35:2162–9. Wang Q-Q, Guo M-X, Yates SR. Degradation kinetics of manure-derived sulfadimethoxine in amended soil. J Agric Food Chem 2006b;54:157–63. Wetzstein H-G, Karl W, Hallenbach W, Himmler T, Petersen U. Residual antibacterial activity of metabolites derived from the veterinary fluoroquinolone enrofloxacin. 100th General Meeting of the American Society for Microbiology, Los Angeles, CA; 2000. WRc-NSF. The development of a model for estimating the environmental concentrations (PECs) of veterinary medicines in soil following manure spreading (Project code VM0295). Final Project Report to MAFF; 2000.
Contents lists available at ScienceDirect
Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Behaviour of
14
C-sulfadiazine and
14
C-difloxacin during manure storage
Marc Lamshöft, Premasis Sukul, Sebastian Zühlke, Michael Spiteller ⁎ Institute of Environmental Research (INFU), TU Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany
a r t i c l e
i n f o
Article history: Received 7 July 2009 Received in revised form 23 November 2009 Accepted 6 December 2009 Available online 21 December 2009 Keywords: Sulfonamide Fluoroquinolone Metabolites Deacetylation LC-MS/MS Radioactivity balance
a b s t r a c t The persistence of sulfadiazine, difloxacin, and their metabolites has been investigated in stored manure. The manure collected from sulfadiazine (14C-SDZ) and difloxacin (14C-DIF) treated pigs contained N-acetylsulfadiazine (Ac-SDZ), 4-hydroxy-SDZ (4-OH-SDZ), and sarafloxacin (SARA) as the main metabolites, respectively along with their parent compounds. Manures were stored separately at 10 °C and 20 °C at various moisture levels. About 96–99% of the radioactivity remained in extractable parent compounds and their metabolites after 150 d of storage. The formation of non-extractable residue and the rate of mineralization were both negligible in manure containing SDZ and DIF. During storage SDZ concentration increased as a result of the deacetylation of Ac-SDZ, whose concentration decreased proportionally. Hence the environmental effects may be underestimated if the parent compound alone is considered for environmental risk assessment. About 11% and 14% of 4-OH-SDZ were lost after 20 and 40 d of storage; thereafter its concentration increased relatively, highlighting hydroxylation of SDZ. DIF degraded very slowly (7% loss after 150 d) during the storage of manure; in contrast the concentration of SARA decreased rapidly (72–90% loss after 150 d). Dilution of manure and storage at higher temperatures for a reasonable period of time enhanced the rate of reactions of SDZ, DIF and their related metabolites. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Veterinary antibiotics are widely administered to animals to treat diseases and promote growth in agricultural industry (Hassouan et al., 2007). The major route through which veterinary antibiotics enter the environment is the excretion of faeces and urine from animals medicated in livestock and poultry farming, and the subsequent application of contaminated manure as fertilizer into agricultural land (Boxall et al., 2003). After application of the manure the antibiotics and their metabolites may interact with different soil constituents, may enter the food chain by plant uptake, may leach into groundwater, or may occur in surface water via runoff (Boxall et al., 2002; Thiele-Bruhn, 2003; Sukul and Spiteller, 2006). Numerous studies have shown that as much as ∼ 40 to 96% of the administered drugs are excreted by medicated animals (Halling-Sorensen et al., 2001; Lamshöft et al., 2007; Sukul et al., 2009). Elevated antibiotic concentration may affect the structural and functional microbial diversity in soils (Kotzerke et al., 2008) and promote the formation and increase the risk of spreading of resistance genes in the environment (Heuer and Smalla, 2007; Heuer et al., 2008). The antimicrobial activities of many antibiotics' metabolites, with a special reference to sulfonamides and fluoroquinolones, have been reported (Marengo et al., 1997; Wetzstein et al., 2000). Thus, before studying
⁎ Corresponding author. Tel.: +49 231 7554080; fax: +49 231 7554085. E-mail address: [email protected] (M. Spiteller). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2009.12.010
different aspects of environmental risk assessment for antibiotics, it becomes essential to understand the fate of parent compounds and their bio-transformed metabolites in manure originating from medicated animals under realistic storage conditions. The widely used sulfonamide sulfadiazine [SDZ, 4-amino-N-(2pyrimidinyl)benzene sulfonamide (Fig. 1)] has been investigated in numerous studies (Thiele-Bruhn, 2003; Thiele-Bruhn et al., 2004; Kreuzig and Höltge, 2005; Förster et al., 2007; Sukul et al., 2008a,b). SDZ is generally applied as an antibiotic to pigs and calves. It competes with p-aminobenzolic acid in the enzymatic synthesis of dihydrofolic acid and thus inhibits the growth and reproduction of bacteria. From applied SDZ to animals, about 44% are excreted as parent compound, about 26% as acetyl conjugate and 19% as hydroxylated compound (Lamshöft et al., 2007). The N-acetyl derivatives of sulfonamide antibiotics are reported to be deacetylated to the parent compound (Grote et al., 2004). Difloxacin [DIF, 6-fluoro-1-(4-fluorophenyl)-1,4-dihydro-7-(4methyl-1-piperazinyl)-4-oxo-3-quinolonecarboxylic acid (Fig. 1)] belongs to a large group of structurally related antibiotics, fluoroquinolones. DIF exhibits a broad-spectrum activity against Gram-positive and -negative bacteria (Eliopoulos et al., 1985; Stamm et al., 1986), and mycoplasmas (Kenny et al., 1989). It is proposed that it interferes with bacterial DNA metabolism by inhibiting bacterial topoisomeraseII (in Gram-negative bacteria) and topoisomerase-IV (in Grampositive bacteria), preventing DNA synthesis, and thus disrupts bacterial cell duplication (Hooper and Wolfson, 1993; Drlica and Zhao, 1997). DIF differs in particular from other fluoroquinolones on
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2.2. Pig manure The manure from SDZ and DIF treated pigs were collected from Bayer Crop Science AG, Monheim, Germany, and NOTOX Safety and Environmental Research B.V.,'s-Hertogenbosch, The Netherlands, respectively. The details of the administration of antibiotics to the pigs, the doses used, and sample collection are reported in previous publications (Lamshöft et al., 2007; Sukul et al., 2009). Manure properties are listed in Table 1. Control pig manure was free from any antibiotics, since it was obtained from non-treated animals. However, control slurry was extracted and analyzed by LC-MS/MS to confirm the absence of sulfadiazine (Lamshöft et al., 2007) and DIF (Sukul et al., 2009). After collection, pig manure was stored at 4 °C in a polyethylene bucket for 2 weeks. Prior to the experimental set-up, the manure was allowed to attain room temperature.
Fig. 1. Chemical structure of sulfadiazine, difloxacin and their major metabolites.
account of its p-fluorophenyl ring at position N-1 of the quinolone nucleus, which reportedly gives its enhanced activity against Grampositive bacteria (Walker, 2000). However, in some species it is demethylated to its primary metabolite, sarafloxacin (SARA), which also displays potent antibacterial activity (Granneman et al., 1986) and in some species DIF is primarily metabolized by glucuronidation (Chu et al., 1985). Experiments with radio-labelled compounds showed that difloxacin accounted for 95.9% of the radioactivity excreted in pigs (Sukul et al., 2009). Several studies have been performed to investigate the degradation of antibiotics in spiked manure (Kuhne et al., 2000; Teeter and Meyerhoff, 2003; Wang et al., 2006a), soil (Wang et al., 2006b; Heuer et al., 2008) and water (Alexy et al., 2004; Boreen et al., 2005) with a focus on eliminating the contamination caused by veterinary drugs. Additionally, it was always found that aerobic conditions were appropriate to the degradation of the drugs in manure and water (Kuhne et al., 2000; Ingerslev et al., 2001). No information is to our knowledge available on the fate and behaviour of SDZ and DIF and their metabolites in manure and on the effects of various environmental factors. It is normal practice to store manures and slurry for a considerable period of time after collection from livestock farms and before application to soils. The storage time of slurries in the U.K. varies from 0 to 50 months, with an average of 9 months (WRc-NSF, 2000). Thus, storage time should also play a significant role in the dissipation of veterinary drugs. Against this background, attempts have been made in the present investigation to study the kinetics of SDZ, DIF, and their respective metabolites in pig manure, to establish its mass balance and to identify all detectable metabolites after oral administration of selected veterinary drugs to pigs. 2. Materials and methods 2.1. Chemicals The analytical standard grade SDZ (99%) was purchased from Fluka, Seelze, Germany. 14C-SDZ (99%) labelled at the 2-pyrimidine position with a specific radioactivity of 8.6 MBq/mg was obtained from Bayer AG, Wuppertal. The internal standard 13C-SDZ (99%) was obtained from the Department Bio V, University of Aachen, Germany. All six-carbon atoms of the aniline ring were 13C-labelled. DIF (99%) was purchased from Chemos GmbH, Germany. 14C-DIF (99.1%) with a specific radioactivity of 2.15 GBq/mmol and labelled at the 2-pyridine position was obtained from GE Healthcare UK limited, UK. The internal standard 13C15N-DIF was synthesized and purified (N99%, checked by LC/MS) at the Department of Biology V, University of Aachen, Germany. SARA (99%) was purchased from Fluka GmbH, Switzerland. All of the solvents and chemicals used were of analytical grade.
2.3. Storage Manure (50 g) was kept in 300 mL Erlenmeyer flasks fitted with trap attachments. The trap attachments were filled with soda–lime to absorb released 14CO2. The flasks were stored in the dark at two temperatures (10 °C, 20 °C) in incubation chambers. At each sampling point the pH (Scientific Instruments IQ240, Colorado USA) and the redox-potential (WTW ph530, Weilheim Germany) of the manure were measured. All experiments were carried out in triplicate. Dilutions were performed with fresh manure from the control treatment, which did not contain any antibiotic residues. 2.4. Sample extraction Manure samples were extracted immediately after sampling. For SDZ and its metabolites samples of homogenized manure (1 g) were transferred into 15 mL screw-topped; polyethylene centrifuge tubes, and 4 mL of McIlvaine buffer (1 M citric acid: 1 M Na2HPO4, 18.15 mL: 81.85 mL; adjusted to pH 7) and 500 ng of the internal standard 13C6SDZ were added. For DIF and its metabolites 4 mL of acetonitrile (+0.1% formic acid) and 1000 ng of internal standard 13C/15N-DIF were added to 1 g manure. The contents were shaken for 30 s with a vortex mixer, treated for 15 min in an ultrasonic bath and centrifuged for 15 min at 15,000 rpm in a model Avanti J-25 centrifuge (Beckman, Fullerton, CA, USA), equipped with a JLA-16.250 rotor. The supernatants were transferred into glass test tubes. The extraction was repeated with 5 mL McIlvaine buffer for SDZ and its metabolites; while for DIF and its metabolites extraction was repeated with 5 mL acetonitrile–water (20/ 80). The respective resulting extracts were pooled and were directly measured in the HPLC-MS/MS without further treatment or preparation. 2.5. Quantitative determination by LC-MS/MS Quantitation of the administered veterinary drugs and their main metabolites by HPLC-MS/MS was performed by means of previously Table 1 Physicochemical properties of the test manures. Properties
Experiment-1
Experiment-2
Control Manure containing Control Manure containing SDZ and its DIF and its metabolites metabolites Dry mass (%) Total carbon, g/kg pH Biological oxygen demand (BOD)5, g/L Chemical oxygen demand (COD), g/L Electrical conductivity, µS/cm
5.8 31.1 7.7 50
6.0 34.4 7.8 39.8
3.3 16.3 8.2 41
3.5 16.8 8.1 35
79.6
60
35
24
35,500
33,600
18,280
15,970
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described analytical methods (Lamshöft et al., 2007; Sukul et al., 2009).
Table 2 Radioactivity distribution in manure at different sampling periods at 20 °C. Days
2.6. Radioactivity measurements The radioactively labelled extracts were analyzed for radioactivity content by liquid scintillation counting (Beckmann LS6500, Fullerton, CA, USA) using Quicksafe A (Zinsser, Frankfurt, Germany). Insoluble residues were subjected to combustion analysis using an OX500 biological oxidizer (Zinsser, Frankfurt, Germany) and the released 14 CO2 was measured by liquid scintillation counting (LSC) using Oxysolve C-400 (Zinsser, Frankfurt, Germany) as scintillation cocktail. For the determination of 14CO2 from the trap attachments, the soda– lime (10 g) of the trap was dissolved with drop-wise addition of 18% HCl (∼ 50–60 mL) under a gentle stream of nitrogen in a suitable glass apparatus (Sukul et al., 2008a). The released 14CO2 was absorbed by a series of three vials containing 15 mL of an ice-cooled cocktail of Oxysolve C-400. 3. Results and discussion
1565
parent compound + metabolite(s) mg/L
% recovered radioactivity Non-extractable residue
14
Manure containing SDZ and its metabolites 1 156.0 ± 4.2 98.9 3 151.4 ± 3.3 98.9 7 151.4 ± 4.0 98.2 15 146.0 ± 3.5 99.6 30 148.4 ± 2.6 99.9 60 150.8 ± 2.7 99.7 100 152.8 ± 3.8 99.2 150 151.8 ± 2.9 99.0
0.3 0.3 0.2 0.2 0.4 0.7 0.8 0.9
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2
99.2 ± 0.5 99.2 ± 0.9 98.4 ± 0.8 99.8 ± 0.7 100.3 ± 0.9 100.4 ± 0.8 100.0 ± 0.7 100.1 ± 0.6
Manure 1 3 7 15 30 60 100 150
0.2 0.2 0.2 0.4 0.5 0.7 1.2 4.2
0.0 0.0 0.0 0.0 0.0 0.1 0.5 0.5
106.2 ± 0.9 101.2 ± 0.9 99.7 ± 0.8 101. 4 ± 0.7 98.0 ± 0.8 97.8 ± 0.7 96.7 ± 0.7 95.7 ± 0.7
Extractable residue
containing DIF and its metabolite 17.6 ± 0.7 106.0 16.3 ± 0.6 101.0 16.6 ± 0.5 99.5 16.2 ± 0.6 101.0 15.9 ± 0.6 97.5 15.7 ± 0.6 97.0 15.3 ± 0.5 95.0 15.1 ± 0.4 91.0
CO2
Total
3.1. Radioactivity distribution A radioactivity balance of SDZ and DIF in manure stored at 20 °C was established on the basis of LSC data (Table 2). 0.9% and 4.2% radioactivities were found in the form of non-extractable residue in manure containing SDZ and DIF, respectively after 150 d of storage. The total amount of extractable radioactivity declined. However, the formation of non-extractable residue was negligible. In each case, 14 CO2 was detected in low quantities at all sampling intervals, which indicated that a mineralization was not a major route of antibiotic dissipation during manure storage. Perhaps, this is due to low biological activity (Table 1) and existence of anaerobicity, as evidenced from the redox-potential (Table 3), in the manure system. As a realistic approach the manure was not subjected to any additional aeration from external source. However, it is known that aerobic conditions are appropriate to the degradation of the drugs in manure and water (Kuhne et al., 2000; Ingerslev et al., 2001). Interestingly, a little change in the absolute amount of extracted antibiotic and its metabolites was observed, which correlated with little change in extractable radioactivity, non-extractable radioactivity and mineralization (Table 2). A similar trend was also observed when manure containing 14C-SDZ and 14C-DIF along with their metabolites was stored separately at 10 °C. To our knowledge no information is available on the fate of SDZ and DIF contained in pig manure during storage. However, fate of antibiotics in spiked manure and manure amended soil is available (Kreuzig et al., 2003; Kreuzig and Höltge, 2005; Heise et al., 2006; Schmidt et al., 2008; Wang et al., 2006a, b). A rapid formation of the non-extractable residues was observed as the predominant process in bovine manure spiked with 14C-SDZ (Kreuzig and Höltge, 2005). Results showed that the amount of extractable 14C-SDZ residues continuously dropped from 70% to 5% of the initial applied radioactivity, which differs from our results. This may be due to the difference in the nature of studied manure, route of administration of antibiotics in the manure and analytical methods used. 3.2. Behaviour of drugs under various conditions of storage The redox-potential and the pH of the manure system were monitored at each sampling time (Table 3). No significant change in pH was noticed. For the manure containing SDZ and its metabolites, and for the manure containing DIF and SARA, the pH ranges were 7.8– 8.1, and 8.1–8.3, respectively. The redox-potentials lay between −280 mV and −329 mV for both manures, indicating anaerobic conditions. In order to simulate the farmers' practice we did not purge the incubation vessels with air. However, aerobic conditions were found to be appropriate to the degradation of the drugs in manure and
20 times 1 3 7 15 30 60 100 150
diluted manure containing SDZ and its metabolites 8.2 ± 0.2 99.9 0.3 7.2 ± 0.2 99.9 0.3 8.2 ± 0.2 99.2 0.2 7.7 ± 0.2 99.6 0.2 8.0 ± 0.2 99.1 0.4 8.1 ± 0.3 99.0 0.7 8.1 ± 0.2 98.8 0.8 8.2 ± 0.3 98.7 0.9
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1
100.2 ± 0.8 100.2 ± 0.9 99.4 ± 0.7 99.8 ± 0.9 99.5 ± 0.9 99.7 ± 0.8 99.6 ± 0.8 99.6 ± 0.7
20 times 1 3 7 15 30 60 100 150
diluted manure containing DIF and its metabolite 0.9 ± 0.1 100.5 0.2 0.9 ± 0.1 100.1 0.2 0.8 ± 0.1 99.9 0.2 0.8 ± 0.1 99.5 0.4 0.8 ± 0.1 98.0 0.5 0.8 ± 0.1 97.2 0.7 0.7 ± 0.1 96.0 1.3 0.7 ± 0.1 92.2 3.9
0.0 0.0 0.0 0.0 0.0 0.1 0.4 0.5
100.7 ± 0.9 100.3 ± 0.8 100.1 ± 0.8 99.9 ± 0.7 98.5 ± 0.9 98.0 ± 0.8 97.4 ± 0.7 96.6 ± 0.7
water (Kuhne et al., 2000; Ingerslev et al., 2001; Wang et al., 2006a). Manure containing 51.6 kBq/mL radioactivity for SDZ (74.7 mg/L) and its major metabolites (Ac-SDZ, 36.5 mg/L; 4-OH-SDZ, 44.7 mg/L) were incubated for the storage experiments at 10 °C and 20 °C. The
Table 3 Redox-potential and pH of manure under storage condition at different time intervals. Days
10 °C pH
20 °C Redox-potential (mV)
pH
Redox-potential (mV)
Manure containing SDZ and its metabolites 0 7.8 −322 10 7.8 −312 20 7.9 −320 40 7.9 −310 60 7.9 −299 90 7.9 −280 150 8.0 −285
7.8 7.8 7.9 7.9 7.9 8.0 8.1
− 314 − 301 − 311 − 302 − 320 − 333 − 329
Manure containing DIF and SARA 0 8.1 −310 1 8.1 −315 7 8.1 −301 15 8.1 −302 30 8.1 −300 60 8.2 −305 100 8.2 −280 150 8.2 −281
8.1 8.1 8.1 8.1 8.1 8.2 8.2 8.3
− 300 − 298 − 290 − 285 − 290 − 285 − 284 − 280
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concentration profiles of SDZ and its two main metabolites at different intervals are presented in Fig. 2. The SDZ concentration was found to have increased at all times over its concentration (0 d). Although the increase did not follow a definite pattern at 10 °C, there was a significant and continuous increase at 20 °C. By the end of the experiment (150 d) 7.8% and 35.6% increase in SDZ concentration was measured at 10 °C and 20 °C, respectively (Table 4); indicating a marked influence of temperature. A significant decline in Ac-SDZ was associated with the increase in SDZ concentration. There were 36.7% and 79.1% losses of Ac-SDZ at 10 °C and 20 °C, respectively after 150 d of manure storage also confirming the effect of temperature on the degradation of Ac-SDZ. Although the N-acetyl derivative of sulfonamide antibiotics itself is not antimicrobially active, it is reported to be deacetylated to the parent compound by hydrolysis (Kreuzig and Höltge, 2005; Grote et al., 2004). This highlights the fact that the consideration of the fate of antibiotics' metabolites is urgently required in order to estimate correctly the environmental effect of a veterinary drug in risk assessment procedures. At both temperatures the maximum loss of 4-OH-SDZ was observed after 20 and 40 d of storage; thereafter its concentration increased. This increase highlights the hydroxylation of SDZ during manure storage. In contrast to our results, in a separate experimental set-up where manure was spiked with the antibiotic, the concentration of sulfadimethoxine, another compound of sulfonamide class of antibiotics, was reduced very rapidly with a half-life value of 3 d (Wang et al., 2006a). This difference may be accounted for by the use of a different compound and a different manure, which vary in their respective physicochemical properties. Additionally, in our experiment the manure was not spiked, on the contrary it was obtained from antibiotic treated pigs and it contained SDZ, Ac-SDZ and 4-OH-SDZ. Spiking of the active drug to the manure does not provide answers on the metabolites generated in an animal system. Furthermore, the physicochemical environment of spiked manure and manure containing antibiotics after feeding antibiotics to the animals are quite different. Hence, the realistic approach
Table 4 Percent change of SDZ and its metabolites in manure against their initial concentrations during storage at different conditions. Days of incubation
10 °C % change SDZ
20 °C % change Ac-SDZ
% change 4-OH-SDZ
% change SDZ
% change Ac-SDZ
% change 4-OH-SDZ
– − 14.2 − 25.8 − 32.5 − 35.2 − 34.0 − 36.7
– − 7.7 − 20.7 − 17.9 − 8.9 − 9.7 − 8.7
– + 12.9 + 22.6 + 30.3 + 33.2 + 34.6 + 35.6
– − 26.0 −40.0 − 65.3 − 75.0 − 75.6 − 79.1
– − 10.6 − 11.5 − 14.3 − 6.0 − 3.4 − 4.4
10 times diluted manure 0 10 20 40 60 90 150
– + 15.2 + 21.7 + 27.7 + 30.3 + 32.9 + 35.3
– − 48.8 − 76.0 − 94.3 − 99.3 − 99.7 − 99.8
– − 2.7 −0.7 − 6.2 −8.1 − 7.9 − 7.8
20 times diluted manure 0 10 20 40 60 90 150
– + 9.7 + 27.2 + 30.8 + 31.5 + 32.7 + 33.4
– − 60.4 − 74.4 − 96.8 − 99.1 − 99.1 − 99.3
– − 12.4 + 9.5 + 2.1 + 14.3 + 18.9 + 21.6
Original manure 0 – 10 + 7.1 20 + 6.2 40 + 10.2 60 + 4.4 90 + 6.1 150 + 7.8
should be to treat the animals with active drugs and to study the fate and behaviour of these in manure. Rates of reactions, either in terms of percent gain of SDZ or percent loss of Ac-SDZ, were increased when incubation temperature was
Fig. 2. Concentration profile of SDZ and its major metabolites in manure under storage condition at 10 °C (A) and 20 °C (B), and at dilution of 1/10 (C) and 1/20 (D) of manure.
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increased from 10 °C to 20 °C (Table 4). On the basis of the Arrhenius equation the temperature has a direct influence on the reaction rate constants; an increase in temperature accelerates a reaction by increasing the rate constant. Our results conforms the earlier report (Wang et al., 2006a). Hence, storing manure under warm condition might be a good strategy for reducing antibiotic contamination in the agricultural environment. However, specifically for manure containing SDZ and its metabolites, the situation is entirely different and complex because of the reversible conversion of Ac-SDZ to SDZ. Storing manure at higher temperature increases conversion rate of Ac-SDZ to SDZ; while at a lower temperature this conversion occurs at a comparatively slow rate, SDZ does not dissipate. Dilution of manure, as such does not have any effect on percent increase of SDZ (Table 4). At 20 °C original manure, manure diluted 10 times and 20 times showed 35.6, 35.3 and 33.4% increment relative to its initial concentration, respectively. However, dilution of manure showed a marked effect in percent loss of Ac-SDZ. Nearly 100% of AcSDZ was transformed in diluted manure while 79% loss was evidenced
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Table 5 Percent change of DIF and SARA in manure during storage at different conditions. Days of incubation
10 °C % change DIF
20 °C % change SARA
% change DIF
% change SARA
– − 38.9 − 60.4 − 62.5 − 63.2 − 66.0 − 68.1 − 68.1 − 72.2
– − 2.0 − 3.0 − 2.9 − 3.1 − 3.6 − 5.2 − 6.8 − 7.3
– − 47.9 − 58.3 − 64.6 − 66.0 − 75.7 − 72.9 − 87.5 − 89.6
10 times diluted manure 0 1 3 7 15 30 60 100 150
– 0.0 − 7.3 − 2.4 − 8.5 − 6.1 − 2.4 − 19.5 − 19.5
– − 25.0 − 37.5 − 62.5 − 50.0 −62.5 −62.5 −87.5 −93.8
20 times diluted manure 0 1 3 7 15 30 60 100 150
– − 1.8 − 4.1 − 8.8 −6.5 − 10.6 − 8.2 − 21.2 − 20.6
– − 18.8 −68.8 −75.0 − 68.8 − 87.5 − 93.8 − 93.8 −93.8
Original manure 0 – 1 − 0.2 3 − 1.2 7 − 4.6 15 − 4.1 30 − 3.6 60 − 5.5 100 − 5.6 150 − 7.4
in original manure. At 20 °C, manure diluted 20 times showed a gain in 4-OH-SDZ concentration, relative to its initial concentration. Hence it can be concluded that under dilution a reasonable amount of AcSDZ was converted into SDZ, a portion of which in turn again converted into 4-OH-SDZ. This also establishes the degradation of SDZ. Perhaps a longer storage time would further increase SDZ degradation. This needs to be further investigated. Manure containing 4.3 kBq/mL radioactivity for DIF (16.2 mg/L) and SARA (1.4 mg/L) was stored at 10 °C and 20 °C. DIF showed a persistent behaviour in manure during its storage. About 7% loss of DIF was found after 150 d of manure storage at both 10 °C and 20 °C (Fig. 3, Table 5). However, nearly 72% and 90% losses of SARA were observed after storage for the same period at 10 °C and 20 °C, respectively showing a marked influence of temperature. Dilution of manure also enhanced the dissipation of DIF and SARA. About 20% DIF and 94% SARA were lost after 150 d of storage under both 10 and 20 times dilution of manure at 20 °C. Although the dilution of contaminated manure with manure from control treatment free of antibiotics increases the number of available sorption sites in manure, the observed enhanced dissipation of DIF and SARA during dilution is not due to increased sorption. Non-extractable radioactivity did not increase in diluted manure (Table 2). Control manure without any contamination from antibiotics contained more BOD value than the treated manure (Table 1). Perhaps, addition of control manure to the treated manure for dilution increased the biological activity in the system, which possibly caused the increased rate of reaction. 4. Conclusion
Fig. 3. Concentration profile of DIF and SARA in manure under storage condition at 20 °C (A) and 10 °C (B), and at different dilutions (C) of manure.
Pharmaceutical antibiotics enter agricultural soils and adjacent environmental compartments through the use of contaminated manure and sludge as fertilizer. The present study revealed that in addition to the parent compound, the fate and effect of metabolites
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should also be investigated. Though the parent compound is generally more potent than its metabolites, the latter may still be biological active and its impact may therefore be underestimated if only the parent compound is considered. It is even more important to consider the metabolites, when they are retransformed into the more active parent compound. An experimental design using manure merely spiked with veterinary drugs is not appropriate because it neglects the fate and effect of metabolites originating in the animal. Our experimental results also demonstrated that storing manure contaminated with DIF and SARA contributed little to the degradation of DIF, but showed a pronounced effect in reducing SARA concentration. Furthermore, mixing of contaminated manure with fresh control manure led to a comparatively higher rate of degradation of DIF and SARA. Therefore, it may be a good strategy to mix manure contaminated with DIF and SARA with fresh control manure for reducing their contamination in the environment. However, for manure containing SDZ and its metabolites, the reversible conversion of Ac-SDZ to SDZ made the situation more complex, leading to an accumulation of SDZ in manure. Therefore, frequent fertilization of soil by manure contaminated with SDZ and its metabolites may lead to environmental contamination. Acknowledgements This study has been performed within the frame of the research project “Veterinary medicines in soils: basic research for risk analysis”, funded by the German Science Foundation, Deutsche Forschungsgemeinschaft (DFG), to whom the authors are deeply grateful. Jürgen Storp, Cornelia Stolle and Jennifer Hardes are gratefully acknowledged for their technical support. References Alexy R, Kumpel T, Kummerer K. Assessment of degradation of 18 antibiotics in the closed bottle test. Chemosphere 2004;57:505–12. Boreen AL, Arnold WA, McNeill K. Triplet-sensitized photodegradation of sulfa drugs containing six-membered heterocyclic groups: identification of an SO2 extrusion photoproduct. Environ Sci Technol 2005;39:3630–8. Boxall ABA, Blackwell PA, Cavallo R, Kay P, Tolls J. The sorption and transport of a sulfonamide antibiotic in soil system. Toxicol Lett 2002;131:19–28. Boxall ABA, Blackwell PA, Boleas S, Halling-Sørensen B, Ingerslev F, Jacobsen AM, et al. Environmental risk assessment of veterinary medicines in slurry. Cranfield University, UK (Final Report of the EU ERAVMIS Project No. EVK1-CT-1999-00003); 2003. Chu DTW, Granneman GR, Fernandes PB. ABOTT-56619. Drugs Future 1985;10:543–5. Drlica K, Zhao X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev 1997;61:377–92. Eliopoulos GM, Moellering AE, Reiszner E, Moellering Jr RC. In vitro activities of the quinolone antimicrobial agents A-56619 and A-56620. Antimicrob Agents Chemother 1985;28:514–20. Förster M, Laabs V, Lamshöft M, Pütz T, Amelung W. Analysis of aged sulfadiazine residues in soils using microwave extraction and liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 2007;391:1029–38. Granneman GR, Snyder KM, Shu VS. Difloxacin metabolism and pharmacokinetics in human after single oral dose. Antimicrob Agents Chemother 1986;30:689–93. Grote M, Vockel A, Schwarze D, Mehlich A, Freitag M. Fate of antibiotics in food chain and environment originating from pig fattening. Fresenius Environ Bull 2004;13:1214–6. Halling-Sorensen B, Jsensen J, Tjornelund J, Montforts MHMM. Worst-case estimation of predicted environmental soil concentration (PEC) of selected veterinary antibiotics and residues used in Danish agriculture. In: Kummerer K, editor. Pharmaceuticals in the Environment. Berlin: Springer; 2001.
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