From the application of antibiotics to antibiotic residues in liquid manures and digestates: A screening study in one European center of conventional pig husbandry

Journal of Environmental Management 177 (2016) 129e137

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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman

Research article

From the application of antibiotics to antibiotic residues in liquid manures and digestates: A screening study in one European center of conventional pig husbandry Arum Widyasari-Mehta, Susen Hartung, Robert Kreuzig* € €t Braunschweig, Institut für Okologische Technische Universita und Nachhaltige Chemie, Hagenring 30, 38106, Braunschweig, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 November 2015 Received in revised form 31 March 2016 Accepted 6 April 2016

In conventional pig husbandry, antibiotics are frequently applied. Together with excreta, antibiotic residues enter liquid manures finally used as organic soil fertilizers or input materials for biogas plants. Therefore, this first screening study was performed to survey the application patterns of antibiotics from fall 2011 until spring 2013. Manures and digestates were then analyzed for selected antibiotic residues from spring 2012 to 2013. The data analysis of veterinary drug application documents revealed the use of 34 different antibiotics belonging to 11 substance classes at 21 farms under study. Antibiotics, particularly tetracyclines, frequently administered to larger pig groups were detected in manure samples up to higher mg kg
1 dry weight (DW) concentrations. Antibiotic residues in digestates, furthermore, show that a full removal capacity cannot be guaranteed through the anaerobic digestion process in biogas plants. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Antibiotic Conventional pig husbandry Liquid pig manure Biogas plant Digestate

1. Introduction The conventional pig production in Europe is concentrated in Belgium, Denmark, France, Germany, Italy, Spain and the line, 2009). The German center is Netherlands (Bernet and Be mainly located in Lower Saxony where 8.8 million pigs (4.3 million fattening pigs, 0.5 million breeding pigs, 2.2 million piglets, 1.8 million young pigs up to 50 kg body weight) of 28.3 million pigs in Germany are kept often in large-scale farms (German Federal Office of Statistics, 2014). The conventional animal husbandry is connected with the administration of antibiotics. Thus, the German Federal Office of Consumer Protection and Food Safety (BVL, 2015) amounted the quantities of antibiotics delivered in German veterinary medicine in 2014 to a total of 1238 tonnes. In Lower Saxony, up to 600 tonnes were delivered. After the administration, antibiotics are partly excreted by the pigs as unchanged parent compounds and/or formed metabolites. Excretion rates of up to 90% were revealed for, e.g., tetracyclines frequently applied until today (Kumar et al., 2005). Together with excreta, antibiotic residues enter manures stored in cellars, tanks or lagoons resulting in concentrations of >700 mg kg
1 dry weight

* Corresponding author. E-mail address: [email protected] (R. Kreuzig). http://dx.doi.org/10.1016/j.jenvman.2016.04.012 0301-4797/© 2016 Elsevier Ltd. All rights reserved.

(DW), e.g., found for chlortetracycline and oxytetracycline (Pan et al., 2011; Gans et al., 2010). The simultaneous occurrence of antibiotic resistance genes and mobile genetic elements, often found at high abundances, contributes to the contamination of these manures (Binh et al., 2008; Joy et al., 2014; Wolters et al., 2015). Antibiotics, not degraded during manure storage, and antibiotic resistance genes enter soils via manure application (Jechalke et al., 2014). There, the antibiotic residues may be persistent resulting in concentrations of 14e499 mg kg
1 dry soil, e.g., found for doxycycline in a 0e20-cm soil layer (Chen et al., 2012; Zhou et al., 2013a, b). Furthermore, these residues may enhance the abundance of antibiotic resistance genes up to 2 months after their entry into soils (Heuer et al., 2011). Besides transport in soil via runoff or leaching, antibiotics may be taken up by plants (Grote et al., 2007; Du and Liu, 2012). Thus, doxycycline, demeclocycline, chlortetracycline and iso-chlortetracycline were detected in wheat, barley and triticale in concentrations of 30e95 mg kg
1 fresh weight (FW) (Freitag et al., 2008). Besides the gene transfer in topsoils, the distribution of antibiotic resistance genes may be an additional environmental risk. Thus, tetracycline-resistant bacteria were found in aquifers adjacent to manure storage lagoons of pig husbandry farms (Koike et al., 2007). The conventional pig husbandry in the European member states annually produces 295 million tonnes of liquid manures. For

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Germany, 49 million tonnes are estimated (Holm-Nielsen et al., 2009). According to the 33-% contingent of pigs kept in Lower Saxony, there 16 million tonnes are produced. To avoid overfertilization by high-volume applications of manures, a sophisticated manure management is required, focused on supra-regional shipment and recycling of surplus nutrients. The approach realized best, particularly in Germany, is the use of liquid pig manures as co-substrates together with energy crops in biogas plants for the production of renewable energy (Weiland, 2010). Added values are the improved fertilizer quality of the digestates and the reduction of odor and pathogen emissions (Holm-Nielsen et al., 2009). Due to antibiotic loads of liquid manures introduced into biogas plants, current research activities focused on the inhibitory effect or on the removal of antibiotics within the anaerobic digestion process (Chen et al., 2008; Gans et al., 2010; Ratsak et al., 2013; Spielmeyer et al., 2014, 2015). Within those studies, antibiotics were detected up to mg kg
1 DW concentrations in liquid pig manures and digestates. These findings, however, were reported unlinked from any antibiotic application patterns because these data are usually not available. Hence, the consequences of the antibiotic application on the antibiotic contamination of farm fertilizers cannot be assessed. Therefore, this screening study represents the first approach to investigate the relationship between the farm-specific application patterns and frequencies of antibiotics, the number of treated pigs and the occurrence of antibiotic residues in organic fertilizers of farms without and with biogas plants. Farm operating and antibiotic application data were thus surveyed at 21 farms as comprehensively as possible. In spring and fall 2012 and in spring 2013, samples were taken from the manure cellars, silos or lagoons of the farms and from the digestate silos of the biogas plants and analyzed for selected antibiotics.

2.2. Sampling activities According to the application periods of farm fertilizers, the liquid manure samples of pig fattening and pig breeding farms were taken in spring and fall 2012 and in spring 2013. Samples from manure cellars were taken using a probe sampler (2 m length, 53 mm ID) which was introduced 2e4 times into the pre-pits of the cellars to take 8-L manure samples (Hoeksma et al., 1995). These samples were thoroughly stirred up and 300-mL aliquots were transferred into polyethylene bottles. Finally, the sample bottles were transported in cooling boxes to the laboratory for analysis. Before sampling, the manures were thoroughly stirred up in the cellars. In case of stirring up could cause considerable emissions in the animal houses of some farms, unstirred manures were sampled to meet the farm-specific practices. Additionally, manure samples from cellars were taken during pumping-out with the vacuum tanker before field application. For this purpose, a bypass sampler was introduced into the vacuum tubing or samples were taken in backflush mode from the tanker (Derikx et al., 1997). Both sampling techniques were also applied to take manure samples from silos and lagoons. In order to check for the sampling quality, these different sampling techniques were applied at one farm taking samples from the cellar and the silo with probe and bypass sampler. Furthermore, the pumping-out of a lagoon during one day was accompanied by sampling in backflush mode from the vacuum tanker in the morning, at noon and in the evening. Samples from pig breeding farms with farm-own biogas plants were also taken in spring and fall 2012 and in spring 2013. Biogas farms supplied with different input materials from different farms were selectively involved in fall 2012 and in spring 2013. At the biogas plants, the manure samples were taken as described before. Digestates in closed tanks were thoroughly stirred up and samples were collected via the tank outlet valves. At open silos, digestates were sampled, transferred into sample bottles and transported to the laboratory as already described for manure samples.

2. Material and methods 2.3. Target compound analysis 2.1. Survey of farm operating data and antibiotic application patterns For this screening study, 21 farms of different pig husbandry, manure storage and manure treatment systems were selected in Lower Saxony, Germany. The involved farmers cooperated on the condition that farm specific data, including the locations of the farms, were only used in anonymized form. Thus, 8 pig fattening and 8 pig breeding farms were surveyed for the farm-specific antibiotic application patterns and investigated for the antibiotic residues in liquid manures. These farms differed in number of pigs, antibiotic application patterns and manure storage systems. The 5 pig breeding farms with farm-own biogas plants, mainly fed with pig manure and maize silage, differed in construction, size and electrical power of the biogas plants. Moreover, 4 biogas plants supplied with different input materials, e.g., liquid bovine manure, dry chicken manure, whole-plant silages, from different farms, were investigated to achieve more insights about the occurrence of antibiotic residues in digestates. The farm operating data of pig husbandry and manure management as well as biogas plant systems were compiled in cooperation with the farmers. Additionally, the veterinary drug application documents were qualitatively data analyzed from fall 2011 until spring 2013 in order to identify the application patterns and frequencies of antibiotics as well as the number of treated pigs. By means of the available application data, however, mass balances for the farms under study could not be set up.

2.3.1. Target compounds, extraction and clean-up of samples The liquid manure and digestate samples were analyzed for 19 selected target compounds belonging to the substance classes of sulfonamides, diaminopyrimidines, tetracyclines, fluoroquinolones, macrolides and pleuromutilines. Target compounds are detailed in the supplementary material (see Table S1). The antibiotics were purchased from Dr. Ehrenstorfer GmbH, Augsburg, Germany, while the tetracycline epimers were purchased from the European Directorate for the Quality of Medicines and Healthcare, Strasbourg, France. All reference chemicals (purity >94%) were individually dissolved in methanol, acetonitrile or in a mixture of acetonitrile/water (4/1, v/v) (all solvents: HPLC grade; VWR Chemicals Prolabo, Fontenay-sons-Bois, France) to prepare stock standard solutions (1 mg mL
1) and finally diluted to mixed working standard solutions. Before use, all glassware was rinsed with the saturated solution of ethylene diamine tetraacetic acid in methanol to minimize sorption losses of tetracyclines. The samples under study were first analyzed for pH values using a pH meter (Microprocessor pH Meter 535 Multical with pHelectrode SenTTix 61, WTW, Weilheim, Germany). Additionally, the dry weights of 5e7-g fresh samples were determined in duplicates using a moisture analyzer (DLB-A; Kern and Sohn GmbH, Balingen, Germany). Sample preparation was performed in accordance with the methods of Jacobsen and Halling-Sørensen (2006) and Zhou et al. (2012). The details of extraction and clean-up procedure of manure and digestate samples within the current study

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were described by Widyasari-Mehta et al. (2016). Thus, 25-g aliquots of fresh samples were pre-treated using ethylene diamine tetraacetic acid solution, citrate buffer and hydrochloric acid to adjust pH 3.0. After lyophilization, the solids were extracted and rinsed using methanol/ethyl acetate mixture (1/1, v/v). The concentrated raw extracts were cleaned up using n-hexane/water mixture (1/1, v/v) and solid phase extraction (500 mg hydrophilic lipophilic balance; OASIS, Waters GmbH, Eschborn, Germany). After rotary evaporation, the analytes were finally re-dissolved in 5 mL acetonitrile/water mixture (1/1, v/v) with 0.1% formic acid and micro-filtered (0.2 mm, 25 mm diameter, Chromafil, MachereyNagel GmbH, Düren, Germany). The analytical solutions were transferred into 1.5-mL amber autosampler vials and directly analyzed by means of LC-MS/MS. Remaining solutions were stored in 8-mL amber glass vials at
20  C for quality control measurements. 2.3.2. LC-MS/MS analysis Target compound analysis was performed using liquid chromatography coupled to a 4000 QTRAP tandem mass spectrometry (LC-MS/MS; Agilent Technologies, Waldbronn, Germany and AB Sciex, Darmstadt, Germany). The details of the analytical measurement were described in Widyasari-Mehta et al. (2016). Target compounds were detected in multiple reaction monitoring (MRM) considering the analyte-specific retention times, precursor and 2 or 3 product ions to reach the 4 identification points required by the European Commission Decision 2002/657/EC (EC, 2002). The identification points of the target compounds and corresponding LC-MS/MS parameters are listed in Table S1. Ion source parameters were curtain gas: 276 kPa, ion spray voltage: 4 kV, source temperature: 600  C, nebulizing gas: 414 kPa, desolvation gas: 414 kPa, collision gas: medium, interface heater: ON and entrance potential: 10 V. The concentrations of the target compounds were quantified using single-point standard addition in order to compensate matrix effects typical for electrospray ionization (ESI) (Renew and Huang, 2004). To disclose the effects of matrices on the ionization efficiency, two selected blank matrices, i.e., manure and digestate, were prepared. The blank extracts were diluted 1:10 and fortified at 100 pg mL
1. Each was analyzed in duplicates and the responses of the target compounds in both matrices were compared to those in the reconstitution solvent. First, the samples were initially screened. If necessary, the samples were then consecutively diluted 1:2 up to 1:2500 using a water/acetonitrile mixture (1/1, v/v) with 0.1% formic acid to go below 100 pg mL
1. Afterwards, 20 mL of the standard mixture of 1.0 ng mL
1 were fortified into the already measured samples which were subsequently re-analyzed. Due to the epimerization of tetracyclines, the sums of oxytetracycline and 4-epi-oxytetracycline, tetracycline and 4-epi-tetracycline, chlortetracycline and 4-epi-chlortetracycline as well as doxycycline and 6-epi-doxycycline were used for quantitation (Ratsak et al., 2013; Spielmeyer et al., 2015; Widyasari-Mehta et al., 2016). 2.3.3. Analytical quality assurance For analytical quality assurance, mixed working standards were analyzed to record 6-point calibration curves and to determine the instrument detection limits (IDLs). Every sample preparation step, i.e., n-hexane clean-up, solid phase extraction, evaporation and micro-filtration of extracts, was checked for analyte losses. Additionally, fortification experiments (n ¼ 4) were conducted at 0.2 and 10 mg kg
1 DW concentrations to determine the method quantitation limits (MQLs). Intra- and interday precision was determined by repeated injections (n ¼ 2) of standard mixtures at

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50 and 100 pg mL
1, and both matrix samples at 10 mg kg
1 DW on 2 consecutive days. Finally, the analytical precision was checked for real samples by analyzing one manure and one digestate sample each in 4 replicates. One pair of these replicates each was freshly analyzed, while the other pair was analyzed after 12-month storage at
20  C. 3. Results and discussion 3.1. Farm operating data and antibiotic application patterns Within this screening study, 8 pig fattening farms with 300e1545 pigs, 8 pig breeding farms with 100e840 sows and 650e1200 fattening pigs were investigated. There, the liquid pig manures were stored under the animal houses in cellars (storage capacity: 300e3000 m3), in silos (300e2500 m3) or in lagoons (1000e1250 m3). After pumping-out using vacuum tankers, the liquid manures were spread to arable land during spring and fall. Additionally, 5 pig breeding farms with 265e600 sows and 550e2100 fattening pigs using the liquid pig manures as cosubstrates in farm-own biogas plants (electrical power: 250e716 kW) and 4 biogas plants (500e1800 kW) supplied with different input materials from different farms were studied as well. A total overview about the antibiotic application patterns and frequencies of the pig fattening and breeding farms without and with farm-own biogas plants under study is given in Table 1, where the numbers of antibiotic applications and the numbers from smallest to largest pig groups treated are listed. Thus, 34 different antibiotics belonging to 11 different substance classes were applied from fall 2011 until spring 2013. Independent on the pig husbandry systems, broad-spectrum antibiotics, i.e., benzathinbenzylpenicillin/benzylpenicillin-procaine/dihydrostreptomycin, benzylpenicillin-procaine and amoxicillin, were still frequently applied. Here, lowest application frequencies were recorded for the combination antibiotics lincomycin/spectinomycin (1 application/ 300 treated pigs) and ampicillin/cloxacillin (3/5) as well as for apramycin (1/50) applied each at one farm type. In addition to Table 1, the consideration of the farm specific use of antibiotics supplied supplementary information about application patterns and frequencies. At the 8 pig fattening farms, 16 single or combination antibiotics with major focus on the first-generation antibiotics, i.e., b-lactams, sulfonamides and tetracyclines, were applied. Thus, amoxicillin and tetracycline, partly substituted by doxycycline or chlortetracycline, were predominantly administered at 7 of 8 farms. Highest application frequencies were found at a farm keeping 1000 pigs. Here 140e850 and 140e288 pigs were treated for 18 and 20 times with amoxicillin and tetracycline, respectively. At 1 of 8 pig fattening farms with 380 fattening pigs, there was not any use of tetracyclines. Here, lower application frequencies were recorded for amoxicillin (3/80e340), colistin (1/ 150), lincomycin/spectinomycin (1/300), tiamulin (1/16) and tylosin (5/80e340). Broader application patterns and higher application frequencies were recorded at the pig breeding farms with conventional manure management. Here, 26 single or combination antibiotics were administered to individual pigs or groups of up to 1600 pigs. Besides the broad-spectrum b-lactams, sulfonamides, tetracyclines, fluoroquinolones and macrolides were applied. Highest application frequencies of amoxicillin (21 times) and colistin (14) to treat groups up to 500 pigs were recorded at a farm keeping 220 sows and producing 6000 piglets per year. At the farms with farm-own biogas plants, 24 single or combination antibiotics were applied with special consideration of amoxicillin, colistin, doxycycline, enrofloxacin and tulathromycin. Highest application frequencies were recorded at a farm keeping

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Table 1 Antibiotic application patterns at pig fattening and pig breeding farms without and with biogas plants from fall 2011 to spring 2013. Antibiotic classes

Antibiotics

Pig fattening farms

a

Pig breeding farms

b

Number of applications (nA)/number of treated pigs (nP)

Aminoglycosides

Amphenicoles b-lactams

Fluoroquinolones

Lincosamides Macrolides

Pleuromutilines Polymyxines Sulfonamides h

Diaminopyrimidine Tetracyclines

Apramycin dihydrostreptomycin e gentamicin neomycin f florfenicol amoxicillin ampicillin cloxacillin benzylpenicillin g cefquinom ceftiofur danofloxacin enrofloxacin marbofloxacin lincomycin spectinomycin erythromycin tildipirosin tilmicosin tulathromycin tylosin tiamulin colistin sulfadiazine sulfadimethoxine sulfadimidine sulfadoxine sulfamerazine sulfamethoxypyridazine trimethoprim chlortetracycline doxycycline oxytetracycline tetracycline

Pig breeding farms with biogas plants c

d

nA

nP

nA

nP

nA

nP

e 12 e e e 42 e e 15 8 e e 2 e 1 1 e e e 1 28 1 1 5 e e e e e

e 5e248 e e e 30e850 e e 5e152 2e10 e e 30e150 e 300 300 e e e 50 10e800 16 150 135e580 e e e e e

e 87 8 e 20 78 3 3 15 60 6 11 41 e e

e 10e1500 2e100 e 5e30 3e1050 5 5 2e100 2e400 250 2e34 3e270 e e

1 18 15 9 e 103 e e 65 46 16 e 68 48 e

50 20e2400 60e400 200e700 e 2e2000 e e 4e200 3e18 100e400 e 3e300 5e150 e

1 n.s. e 52 24 5 51 14 4 53 9 e e

n.s. n.s. e 100e1000 1e200 35e100 100e1000 36e500 1e1421 3e90 2e4 e e

2 10 1 30 7 e 40 3 e e 12 7 1

80e150 50e300 2000 100e1200 10e500 e 72e892 22e800 e e 6e8 10e17 90

5 8 5 e 50

135e580 580e820 120e365 e 100e385

80 14 20 28 7

1e1421 35e715 30e1600 1e60 380e1400

23 4 87 1 e

6e800 10e17 83e2000 5 e

Detailed information on numbers of treated pigs and antibiotic application patterns and frequencies could be gathered at. a 8 pig fattening. b 7 pig breeding and. c 4 pig breeding farms with biogas plants. For 1 pig breeding farm and 1 farm with biogas plant, only the application patterns were available. d Smallest to largest pig group treated is given. e Applied as benzathin-benzylpenicillin/benzylpenicillin-procaine/dihydrostreptomycin. f Applied as neomycin/benzylpenicillin/benzylpenicillin-procaine. g Applied as benzylpenicillin-procaine, n.s.: not specified, —: not applied. h The sulfonamides were applied together with the diaminopyrimidine antibiotic trimethoprim.

265 sows and 2100 fattening pigs. There were 55 amoxicillin applications to 18e400 pigs, 75 doxycycline applications to 83e700 pigs and 41 enrofloxacin applications to 3e300 pigs. Hence, doxycycline belonged to one of the most frequently applied antibiotics at these farms. The comparison of the application data from the 3 farm management systems indicated the tendency that, the higher the numbers of kept pigs, the higher are the frequencies of antibiotic applications. However, the validation of this observation demands a spatiotemporal registration of the antibiotic applications at a representative number of pig husbandry farms for a comprehensive data collection and statistical analysis. This is exactly the aim of the determination of nationwide indicators for the therapy frequency from production animals in Germany (BVL, 2015).

3.2. Validation of the analytical method 3.2.1. Quality of residue analysis Even though 34 antibiotics from 11 different antibiotic classes

were identified within the survey on the application patterns, the target compound analysis could only focus on the determination of sulfonamides, diaminopyrimidines, tetracyclines, fluoroquinolones, macrolides and pleuromutilines antibiotics (Table S1). Other antibiotics from the substance classes of the blactams, macrolides etc. could not be involved in the same analytical method because of their different chemical behavior during the sample preparation procedure. This target compound selection met the current standard of antibiotic analysis (Zhou et al., 2013a, b; Li et al., 2013; Ratsak et al., 2013; Spielmeyer et al., 2014, 2015). In future, analytical progress can be expected so far as the multimethod of Berendsen et al. (2015), developed for the analysis of antibiotics in bovine and pig feces, may be also established for manure and digestate analysis. For the quality check of LC-MS/MS analysis, first, 6-point calibration curves from 2 to 100 pg mL
1 were recorded with a linearity of >0.990 for all target compounds. IDLs were 10e50 pg absolute or 2e10 pg mL
1. Intra- and inter-day precisions of standard solutions were <9% and <20%, respectively. Detailed precision values for the

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analysis of manure and digestate samples, ranging from 1 to 21%, were given in Table S2. This precision could not be reached for tylosin. Here, relative standard deviations up to 27% occurred within measurements of standard solutions. Constant extraction recoveries could also not be achieved in fortification tests. This analyte specific restriction has been already reported by Hamscher et al. (2002) and Jacobsen and Halling-Sørensen (2006). In accordance with the technical feasibility, the antibiotics and selected metabolites under study could be simultaneously analyzed in liquid pig manure and digestate samples with recoveries of tetracyclines ranging from 71 ± 2% to 104 ± 28% and 51 ± 5% to 92 ± 8% for manure and digestate samples, respectively (Table S2). The recoveries of sulfonamides and fluoroquinolones were lower and often showed higher standard deviations. This may partly lead to an underestimation of sulfonamide residues in manures and digestates. Due to lower application frequencies of sulfonamides compared to those of tetracyclines, however, this is of minor relevance in this screening study. These analyte losses may be caused by extraction inefficiencies due to high matrix affinities of sulfonamides and fluoroquinolones. Losses within the single sample preparation steps, i.e., clean-up with n-hexane partition (average recoveries: 93 ± 13%), solid phase extraction (100 ± 12%), concentration (96 ± 4%) and micro-filtration of extracts (96 ± 5%), could be excluded. By this methodological check, relevant losses of tetracyclines in the autosampler vials could also be excluded. This was also confirmed by the intra- and interday precision tests for standard and sample analyses (Table S2). The analysis of one real manure and one digestate sample in 4 replicates finally confirmed the analytical quality even though one pair of replicates each was analyzed fresh, the other pairs after a 12-month storage at
20  C. Thus, average concentrations of doxycycline found were 11.5 ± 1.9 mg kg
1 DW manure and 4.2 ± 1.0 mg kg
1 DW digestate indicating no relevant alteration of analyte concentrations through sample storage. This fact was also confirmed by DW contents and pH values of manure (3.3 ± 0.2%; 8.2 ± 0.2) and digestate replicates (4.6 ± 0.8%; 8.2 ± 0.3). Matrix effects are well known for LC-MS/MS with ESI resulting in signal enhancement or signal suppression. As shown in Table S2, signal suppression down to
31% could be observed for tetracyclines, while sulfonamides experienced both signal enhancement and signal suppression in manure samples. Due to the higher complexity of digestate samples, the matrix effects on the ionization of all target compounds were shown relatively higher in comparison to those in manure samples. Matrix effects, however, may also interfere the liquid chromatography by lowering S/N ratios. Therefore, MQLs were experimentally set at 0.2 mg kg
1 DW to guarantee S/N ratios >10. To compensate these matrix effects, single-point standard addition was successfully applied. This compensation was additionally supported by the consequent sample dilution to go below <100 pg mL
1. Finally, memory effects were excluded by alternating sample and solvent blank analyses. 3.2.2. Quality of different sampling techniques For analyzing antibiotic residues in liquid pig manures and digestates, different sampling techniques and strategies were applied taking farm-specific practices into special account. Therefore, sampling quality was checked at one farm. Thus, liquid manure samples were taken after stirring up and during pumpingout from the silo using vacuum tanker and bypass sampler. The analysis of different replicates led to matching results of 164 ± 18.9 mg tetracycline kg
1 DW even though liquid pig manures belong to one of the most heterogeneous and complex sample matrices. Using the probe sampler of 2 m length, only 126 mg kg
1 DW were found. Due to the high affinity of tetracycline to biosolids (Hamscher et al., 2002; Prado et al., 2009), here it

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became obvious that the settings at the ground of the silo at 3-m depth could not be reached. In the liquid manure from the cellar, 191 mg tetracycline kg
1 DW were found when the probe sampler was used. This contamination level was confirmed 19 d later, when additional samples were taken with the bypass sampler during the manure transfer from cellar to silo via vacuum tanker. Residues found were 204 ± 4.0 mg tetracycline kg
1 DW. The intraday variability of sampling was additionally checked at one farm during the 1-d pumping-out of a 1250-m3 lagoon. Replicates of liquid manure samples were taken in the morning, at noon and in the evening. Within this sampling sequence, chlortetracycline and tetracycline were found at 36.4 ± 9.7 and 37.7 ± 6.7 mg kg
1 DW, respectively. Despite of permanent stirring during the pumping-out period, major amounts of manure solids were finally left behind on the ground of the lagoon. Considering again the high affinity of tetracyclines to manure solids, these results consistently reflected the manure contamination with chlortetracycline and tetracycline. 3.3. Antibiotic residues in farms with different manure management systems 3.3.1. Farms with conventional manure management An overview about the findings of antibiotics in liquid pig manures is given in Table 2. Residues of antibiotics frequently applied for larger pig groups were determined in the manure samples up to higher mg kg
1 DW concentrations. At the pig fattening farms, tetracycline was thus detected in 20 samples from 1.5 to 300 mg kg
1 DW with a median value of 152 mg kg
1 DW. The next highest concentrations were analyzed for chlortetracycline and doxycycline. Median values were 26.9 and 20.3 mg kg
1 DW, respectively. Corresponding to the application patterns recorded, sulfadiazine was found only in one sample at 0.7 mg kg
1 DW. Within the cluster of breeding farms, the findings were spread to a broader spectrum of applied antibiotics. Here, doxycycline was most commonly detected ranging between 5.0 and 101 mg kg
1 DW with a median value of 19.8 mg kg
1 DW. Highest concentrations were found for oxytetracycline and tetracycline at >200 mg kg
1 DW. More frequent applications of sulfadimidine (synonym: sulfamethazine) to smaller number of pigs resulted in its detection in 5 samples at maximum of 23.0 mg kg
1 DW. In contrast, sulfadimethoxine was found only in 1 sample at 0.5 mg kg
1 DW. Trimethoprim, administered as a synergist of sulfonamides, was detected only in one manure sample. This fact has been already reported by Haller et al. (2002). This was the same for enrofloxacin and tiamulin. In contrast to the positive findings of sulfonamide and fluoroquinolone antibiotics mentioned in Table 2, the other target compounds under study, i.e., sulfamerazine, sulfamethoxypyridazine, danofloxacin and marbofloxacin were not detected in any manure sample from pig fattening and breeding farms above MQLs. These concentrations of antibiotics found in liquid pig manures matched the data published for other centers of pig husbandry, e.g., in China and Austria. There, highest concentrations of 764 mg chlortetracycline kg
1 DW and 770 mg oxytetracycline kg
1 DW were reported by Pan et al. (2011) and Gans et al. (2010), respectively. The minimum and maximum concentrations of the other antibiotics additionally listed in Table 2 are in compliance with the data of this screening study. 3.3.2. Farms with biogas plants The intensity of the pig husbandry systems applied at the farms with farm-own biogas plants was reflected by the highest application frequencies of amoxicillin, enrofloxacin and doxycycline (Table 2). Particularly, the application of doxycycline was not

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Table 2 Residues of antibiotics in liquid manures of 8 pig fattening and 8 pig breeding farms from spring 2012 to spring 2013 in comparison to literature data. Sample

SDZ

SDM

SDX

Liquid manures of pig fattening farms [mg kg
1 DW]: Median e e e MIN n.d. n.d. n.d. MAX 0.7 0.6 n.d. n 1 1 0 Liquid manures of pig breeding farms [mg kg
1 DW]: Median e 2.2 e MIN n.d. 0.8 n.d. MAX n.d. 23.0 n.d. n 0 5 0 Literature data [mg kg
1 DW]: 1 1 0.01 0.13 MIN 0.01 MAX 35.32 1672 32.73

SMDX

TMP

e n.d. n.d. 0

e n.d. n.d. 0

e n.d. 0.5 1 0.34 26.44

CTC

DOXY

OXY

TC

ENF

TYL

TIA

26.9 1.7 46.3 7

20.3 11.0 28.9 3

e n.d. 6.2 1

152 1.5 300 20

e n.d. n.d. 0

e n.d. n.d. 0

e n.d. n.d. 0

e n.d. 0.2 1

37.4 15.8 55.1 4

19.8 5.0 101 12

13.6 0.6 211 5

16.5 1.5 227 6

e n.d. 1.3 1

e n.d. n.d. 0

e n.d. 1.4 1

n.d.5 0.31

0.016 7647

0.15 7708

n.d.6 98.29

0.021 2.210

0.210 1.910

n.s. n.s.

0.016 59.84

SDZ: sulfadiazine, SDM: sulfadimidine, SDX: sulfadoxine, SMDX: sulfadimethoxine, TMP: trimethoprim, CTC: chlortetracycline, DOXY: doxycycline, OXY: oxytetracycline, TC: tetracycline, ENF: enrofloxacin, TYL: tylosin, TIA: tiamulin. Method quantitation limits: 0.2 mg kg
1 DW manure. MIN: minimum, MAX: maximum, DW: dry weight, n.d.: not detected, n.s.: not specified, n: number of positive findings, —: not defined. References: 1: Zhou et al. (2013b), 2: Winckler et al. (2004), 3: Hu et al. (2010), 4: Hu et al. (2008), 5: Zhou et al. (2013a), 6: Qiao et al. (2012), 7: Pan et al. (2011), 8: Gans et al. (2010), 9: Chen et al. (2012), 10: Li et al. (2013).

corresponding phase II-metabolite acetyl-sulfadiazine were found in the manure at 7.3 and 5.5 mg kg
1 DW, respectively. Even though sulfadiazine was classified to be readily degradable in laboratory digestion tests (Mohring et al., 2009), a residue of 0.9 mg kg
1 DW was found in the digestate of this biogas plant. In the manure and digestate samples of fall 2012 and spring 2013, a sulfadiazine contamination could not be found. These findings matched those of Spielmeyer et al. (2014, 2015) who also found sulfadiazine in manure and digestate samples at 1.1 and 1.7 mg kg
1 DW and 0.1 and 0.2 mg kg
1 FW, respectively. At other farms, they also detected sulfadimidine at 201 mg kg
1 DW and 20.7 mg kg
1 FW manure as well as 76.2 mg kg
1 DW and 9.2 mg kg
1 FW digestate, respectively. The decreasing concentrations of the antibiotics from the liquid manures to the digestates may be partly caused by anaerobic biotransformation or irreversible sorption to digester and postdigester materials. In principle, a significant dilution factor has to be taken into account because the biogas plants were normally fed with 60% maize silage and 40% liquid pig manure. Furthermore, it has to be considered that manure and digestate samples were taken

reported in previous studies (Gans et al., 2010; Ratsak et al., 2013; Spielmeyer et al., 2014, 2015). Thus, doxycycline was determined in 16 liquid pig manure samples at 1.7e381 mg kg
1 DW with the median value of 27.4 mg kg
1 DW (Table 3). The 5 findings of enrofloxacin at 0.8e4.7 mg kg
1 DW reflected the higher application frequencies in comparison to the other farms. In the sample with the highest enrofloxacin contamination, the corresponding metabolite ciprofloxacin was also detected at 0.8 mg kg
1 DW. Since this fluoroquinolone antibiotic is rather applied to treat individual pigs, it is normally not detectable above MQL because of high dilution factors. Here, however, groups of up to 300 pigs were treated. Furthermore, digestates were also analyzed to assess the removal potential of the anaerobic digestion process for antibiotic residues introduced into the biogas plants via contaminated liquid manures. As summarized in Table 3, those antibiotics found in the liquid manures were also detected in the digestates. Thus, doxycycline was detected in 14 digestate samples at 1.3e10.5 mg kg
1 DW with a median value of 6.2 mg kg
1 DW. In accordance with a single treatment of 800 pigs, the sulfonamide sulfadiazine and its

Table 3 Residues of antibiotics in liquid manures and digestates of 5 pig fattening and breeding farms with biogas plants from spring 2012 to spring 2013 in comparison to literature data. Sample

SDZ

SDM

SDX

Pig manures [mg kg
1 DW] Median e e e MIN n.d. n.d. n.d. MAX 7.3 n.d. n.d. n 1 0 0 Literature data: Pig manures [mg kg
1 DW] MIN 0.71 7.01 e MAX 1.12 2012 e
1 Digestates [mg kg DW] Median e e e MIN n.d. n.d. n.d. MAX 0.9 n.d. n.d. N 1 0 0
1 Literature data: Digestates [mg kg DW] MIN 1.72 0.34 n.s. MAX 6.31 76.22 n.s.

SDMX

TMP

CTC

DOXY

OXY

e n.d. n.d. 0

e n.d. n.d. 0

e n.d. 1.0 1

27.4 1.7 381 16

e n.d. n.d. 0

e e

0.051 0.483

3.52 36.52

n.d.2 e

e n.d. n.d. 0

e n.d. n.d. 0

e n.d. 0.9 1

n.s. n.s.

n.s. n.s.

n.d.2 3.72

TC

ENF

TYL

TIA

1.5 0.7 5.9 4

1.4 0.8 4.7 5

e n.d. 6.4 1

e n.d. n.d. 0

0.13 7703

1.53 6.62

0.023 1.43

e e

e e

6.2 1.3 10.5 14

e n.d. n.d. 0

1.5 0.9 2.1 4

0.2 0.2 0.3 2

e n.d. n.d. 0

e n.d. n.d. 0

n.d.2 n.s.

0.23 24.03

1.04 17.01

1.11 2.83

n.s. n.s.

n.s. n.s.

SDZ: sulfadiazine, SDM: sulfadimidine, SDX: sulfadoxine, SMDX: sulfadimethoxine, TMP: trimethoprim, CTC: chlortetracycline, DOXY: doxycycline, OXY: oxytetracycline, TC: tetracycline, ENF: enrofloxacin, TYL: tylosin, TIA: tiamulin. Method quantitation limits: 0.2 mg kg
1 DW manure or digestate. MIN: minimum, MAX: maximum, DW: dry weight, n.d.: not detected, n: number of positive findings, n.s.: not specified, —: not defined. References: 1: Ratsak et al. (2013), 2: Spielmeyer et al. (2014), 3: Gans et al. (2010), 4: Gans et al. (2008).

A. Widyasari-Mehta et al. / Journal of Environmental Management 177 (2016) 129e137

135

Table 4 Tetracycline application patterns from fall 2011 to spring 2013 and tetracycline residues in liquid pig manures taken in spring and fall 2012 as well as in spring 2013 from the manure silo of a pig fattening farm. Application period

Number of applications/treated pigs

Sampling

Tetracycline [mg kg
1 DW]

09/2011e02/2012 02/2012e09/2012 09/2012e03/2013

6/140-288 8/140-208 6/144-176

02/15/2012 09/04/2012 02/22/2013

179 300 265

DW: dry weight. Method quantitation limit: 0.2 mg tetracycline kg
1 DW manure.

during the same sampling day, even though there are 60-d to 100d residence times between the manure entry into the digesters, post-digesters and the transfer to the digestate silos. Hence, setting up mass balances for antibiotics demands anaerobic digestion tests at laboratory scale preferably applying the test substances as 14Clabeled radiotracers (Kreuzig, 2010). Nevertheless, these findings clearly show that antibiotics, introduced into biogas plants via contaminated liquid manures, cannot be completely removed by the anaerobic digestion process. The biogas plants supplied with different input materials of different farms were screened once in fall 2012 and in spring 2013. In the digestates of 2 biogas plants fed with liquid pig or bovine manure, tetracycline was found at 6.4 and 0.41 mg kg
1 DW. These residues corresponded to 14.4 and 0.65 mg kg
1 DW of pig and bovine manure, respectively. In the digestates of 2 other biogas plants supplied with solid manures and/or whole plant silages from different farms, antibiotic residues under study were not found above MQLs. 3.4. Farm-specific antibiotic applications and antibiotic residues At some farms, the antibiotic residues detected in liquid pig manures were closely assigned to their application patterns. Thus, at one pig fattening farm, larger pig groups were frequently treated with tetracycline from fall 2011 to spring 2013. With the increase of the therapy frequency from the first to the second application period, the concentration of tetracycline increased from 179 to 300 mg kg
1 DW, followed by the decrease to 265 mg kg
1 DW (Table 4). The close relationship between antibiotics applied and antibiotic residues found can be only identified at farm scale when antibiotic applications, pig excretions, manure transfer from animal house compartments or cellars to pits or silos and sampling activities are matching just in time. In case that sampling is performed from the silo, e.g., before the cellar manure is transferred to the silo, the actual contamination of the farm manure cannot be detected. These uncertainties can be only compensated within farm specific monitoring studies with higher time-resolved sampling intervals. Similar tendencies were found at 2 pig breeding farms with

farm-own biogas plants (Table 5). At biogas plant A, 280 pigs were treated with doxycycline directly before the sampling in spring 2012 resulting in residues in liquid manure of 24.7 mg kg
1 DW. At this sampling date, doxycycline was not detectable in the digestate. After the next treatment, doxycycline concentrations in liquid manure and digestate increased to 166 and 2.1 mg kg
1 DW, respectively. Without any additional doxycycline treatment, residues in manure samples declined while those in the digestates slightly increased. At biogas plant B, there were frequent doxycycline treatments of larger pig groups resulting in varying doxycycline concentrations in the liquid pig manures. Thus, highest concentrations of 110 mg kg
1 DW were found during fall 2012. In the digestates, doxycycline residues seemed to level off at 10 mg kg
1 DW. These findings were principally supported by the field study of Spielmeyer et al. (2015) who investigated residues of sulfonamide and tetracycline antibiotics in 2 biogas plants within a 1-year course. They found higher concentrations of sulfadiazine, sulfadimidine, chlortetracycline and tetracycline in the input than in the output materials. They described varying concentrations over time. Disadvantageously, there the antibiotic residues could not be linked to any farm specific application patterns. They, therefore, concluded that biogas plants may be sinks of antibiotics, even though substance specific elimination rates cannot be calculated based on their data set. However, the detection of antibiotic residues in digestates reported here as well as in those studies of Gans et al. (2010), Ratsak et al. (2013) and Spielmeyer et al. (2014, 2015) rather reflected an incomplete removal of antibiotics through the anaerobic digestion process. In order to clearly identify the removal capacity of biogas plants for the broad spectrum of antibiotics applied today, additional monitoring is necessary starting with the registration of the farm specific antibiotic applications, the development of multimethods for antibiotic analysis from manure to digestate samples and the evaluation of the impact of retention times of antibiotics in the biogas plants after their entry with contaminated manures.

Table 5 Doxycycline application patterns from fall 2011 to spring 2013 and doxycycline residues in liquid pig manures and digestates of 2 pig breeding farms with farm-own biogas plants taken in spring and fall 2012 as well as in spring 2013. Application period

Number of applications/treated pigs

Sampling

Manure

Digestate

Doxycycline [mg kg
1 DW] Biogas plant A 01/2012e03/2012 03/2012e09/2012 09/2012e02/2013 Biogas plant B 09/2011e03/2012 03/2012e09/2012 09/2012e02/2013

1/280 3/200e300 e

03/28/2012 09/19/2012 02/27/2013

24.7 166 13.7

n.d. 2.1 4.8

28/140e700 21/150e400 26/83e400

03/28/2012 09/20/2012 02/28/2013

32.1 110 21.4

4.9 10.1 10.2

DW: dry weight, n.d.: not detected, —: not applied. Method quantitation limits: 0.2 mg doxycycline kg
1 DW manure and digestate.

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4. Conclusions This screening study shows that antibiotics frequently applied in conventional pig husbandry to larger pig groups are detectable in liquid manures at mg kg
1 dry weight concentrations. This environmentally critical aspect gains importance because the occurrence of antibiotics is accompanied by antibiotic resistance genes and mobile genetic elements often found at high abundances. At biogas plants, antibiotics were also found in the digestates. These residues reflected an incomplete removal of antibiotics through the anaerobic digestion process. Hence, the contamination of farm fertilizers can only be reduced by minimizing the use of antibiotics in conventional pig husbandry. Such a comprehensive minimization is pursued by the 16th Amendment to the German Medicines Act enabling the authorities to control farm specific application patterns of antibiotics in order to reach a higher protection against antibiotic resistance. Acknowledgments The authors gratefully acknowledge the financial support for this research project from the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for Agriculture and Food (BLE), Bonn, Germany (grant number 2810HS032). Special thanks deserve to Dr. G. Steffens, T. Eiler, Dr. A. Freitag and Dr. K. Lacü, Chamber of Agriculture, Oldenburg and Braunschweig, Germany, and the cooperating farmers who substantially support these research activities. Many thanks also deserve to Ms. S. Lenz for her sophisticated technical assistance within the analysis of antibiotic €ltge for proofreading this manuscript. residues and to Dr. S. Ho Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jenvman.2016.04.012. References Berendsen, B.J.A., Wegh, R.S., Memelink, J., Zuidema, T., Stolker, L.A.M., 2015. The analysis of animal faeces as a tool to monitor antibiotic usage. Talanta 132, 258e268. line, F., 2009. Challenges and innovations on biological treatment of Bernet, N., Be livestock effluents. Bioresour. Technol. 100, 5431e5436. Binh, C.T.T., Heuer, H., Kaupenjohann, M., Smalla, K., 2008. Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. FEMS Microbiol. Ecol. 66, 25e37. BVL, German Federal Office of Consumer Protection and Food Safety, 2015. Second Data Ascertainment on the Delivery of Antibiotics in Veterinary Medicine (access: 30 March 2016). http://www.bvl.bund.de. Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol. 99, 4044e4064. Chen, Y.S., Zhang, H.B., Lou, Y.M., Song, J., 2012. Occurrence and assessment of veterinary antibiotics in swine manures: a case study in East China. Chin. Sci. Bull. 57, 606e614. Derikx, P.J.L., Ogink, N.W.M., Hoeksma, P., 1997. Comparison of sampling methods for animal manure. Neth. J. Agric. Sci. 45, 65e79. Du, L., Liu, W., 2012. Occurrence, fate, and ecotoxicity of antibiotics in agroecosystems. A review. Agron. Sustain. Dev. 32, 309e327. EC, 2002. European commission decision 2002/657/EC implementing council directive 96/23/EC, concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Commun. 8e36. Freitag, M., Yolcu, D.H., Hayen, H., Betsche, T., Grote, M., 2008. Screening zum Antibiotika-Transfer aus dem Boden in Getreide in Regionen Nordrhein€nden. J. Verbr. Leb. 3, 174e184. Westfalens mit großen Viehbesta Gans, O., Weiss, S., Sitka, A., Pfundtner, E., Scheffknecht, C., Scharf, S., 2008. Determination of selected veterinary antibiotics and quaternary ammonium compounds in digestates of biogas plants in Austria. In: Proceedings of the International Congress CODIS 2008, Compost and Digestate: Sustainability, Benefits, Impacts for the Environment and for Plant Production, ISBN 978-303736-016-3, pp. 67e71. Gans, O., Pfundtner, E., Winckler, Ch, Bauer, A., 2010. Antibiotika in Biogasanlagen. Umweltbundesamt Wien, Austria, ISBN 978-3-99004-088-1, pp. 1e48. Report REP-0287.

German Federal Office of Statistics, 2014. Land- und Forstwirtschaft, Fischerei. Viehbestand. Fachserie 3, Reihe 4.1. https://destatis.de (accessed 30.03.16.). Grote, M., Schwake-Anduschus, C., Michel, R., Stevens, H., Heyser, W., €mpfer, G., Betsche, T., Freitag, M., 2007. Incorporation of veterinary Langenka €lkenrode 57, 25e32. antibiotics into crop from manured soil. Landbauforsch. Vo Haller, M.Y., Müller, S.R., McArdell, C.S., Adler, A.C., Suter, M.J.F., 2002. Quantification of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid-chromatography-mass spectrometry. J. Chromatogr. A 952, 111e120. € per, H., Nau, H., 2002. Determination of persistent Hamscher, G., Sczesny, S., Ho tetracycline residues in soil fertilized with liquid manure by high-performance chromatography with electrospray ionization tandem mass spectrometry. Anal. Chem. 74, 1509e1518. Heuer, H., Solehati, Q., Zimmerling, U., Kleineidam, K., Schloter, M., Müller, T., Focks, A., Thiele-Bruhn, S., Smalla, K., 2011. Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine. Appl. Environ. Microbiol. 77, 2527e2530. Hoeksma, P., Ognk, N.W.M., Eriks, P.J.L., Groot Roessink, G., 1995. Bemonstering von Varkens- en rondveedrijfmest in silo's. IMAG-DLO-III. Rapport/Dienst Landbouwkundig Onderzoek. Institut voor Milieu in Agritechnik, Wageningen, NL, 95e18. Holm-Nielsen, J.B., Al Seadi, T., Oleskowicz-Popiel, P., 2009. The future of anaerobic digestion and biogas utilization. Bioresour. Technol. 100, 5478e5484. Hu, X.-G., Lou, Y., Zhou, Q.-X., Xu, L., 2008. Determination of thirteen antibiotics residues in manure by solid phase extraction and high performance liquid chromatography. Chin. J. Anal. Chem. 36, 1162e1166. Hu, X.-G., Zhou, Q., Luo, Y., 2010. Occurrence and source analysis of typical veterinary antibiotics in manure, soil, vegetables and groundwater from organic vegetable bases, northern China. Environ. Pollut. 158, 2992e2998. Jacobsen, A.M., Halling-Sørensen, B., 2006. Multi-component analysis of tetracyclines, sulfonamides and tylosin in swine manure by liquid chromatographytandem mass spectrometry. Anal. Bioanal. Chem. 384, 1164e1174. Jechalke, S., Heuer, H., Siemens, J., Amelung, W., Smalla, K., 2014. Fate and effects of veterinary antibiotics in soil. Trends Microbiol. 22, 536e545. Joy, S.R., Li, V., Snow, D.D., Gilley, J.E., Woodbury, B., Bartelt-Hunt, S.L., 2014. Fate of antimicrobials and antimicrobial resistance genes in simulated swine manure storage. Sci. Total Environ. 481, 69e74. Koike, S., Krapac, G., Oliver, H.D., Yannarell, A.C., Chee-Sanford, J.C., Aminov, R.I., Mackie, R.I., 2007. Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period. Appl. Environ. Microbiol. 73, 4813e4823. Kreuzig, R., 2010. The reference manure concept for transformation tests of veterinary medicines and biocides in liquid bovine and pig manures. Clean 38, 697e705. Kumar, K., Gupta, S.C., Chander, Y., Singh, A.K., 2005. Antibiotic use in agriculture and its impact on the terrestrial environment. Adv. Agron. 87, 1e54. Li, Y.-X., Zhang, X.-l., Li, W., Lu, X.-f., Liu, B., Wang, J., 2013. The residues and environmental risks of multiple veterinary antibiotics in animal feces. Environ. Monit. Assess. 185, 2211e2220. Mohring, S.A.I., Strzysch, I., Fernandes, M.R., Kiffmeyer, T.K., Tuerk, J., Hamscher, G., 2009. Degradation and elimination of various sulfonamides during anaerobic fermentation: a promising step on the way to sustainable pharmacy? Environ. Sci. Technol. 43, 2569e2574. Pan, X., Qiang, W., Ben, W., Chen, M., 2011. Residual veterinary antibiotics in swine manure from concentrated animal feeding operations in Shanding Province, China. Chemosphere 84, 695e700. Prado, N., Ochoa, J., Amrane, A., 2009. Biodegradation and biosorption of tetracycline and tylosin antibiotics in activated sludge system. Process Biochem. 44, 1302e1306. Qiao, M., Chen, W., Su, J., Zhang, B., Zhang, C., 2012. Fate of tetracyclines in swine manure of three selected swine farms in China. J. Environ. Sci. 24, 1047e1052. €nde Ratsak, C., Guhl, B., Zühlke, S., Delschen, T., 2013. Veterin€ arantibiotika-Rücksta €rresten aus Nordhein-Westfalen. Environ. Sci. Eur. 25, 1e11. in Gülle und Ga Renew, J.E., Huang, C.-H., 2004. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography-electrospray mass spectrometry. J. Chromatogr. A 1042, 113e121. Spielmeyer, A., Ahlborn, J., Hamscher, G., 2014. Simultaneous determination of 14 sulfonamides and tetracyclines in biogas plants by liquid-liquid extraction and liquid chromatography tandem mass spectrometry. Anal. Bioanal. Chem. 406, 2513e2524. Spielmeyer, A., Breier, B., Groißmeier, K., Hamscher, G., 2015. Elimination patterns of worldwide used sulfonamides and tetracyclines during anaerobic fermentation. Bioresour. Technol. 193, 307e314. Weiland, P., 2010. Biogas production: current state and perspectives. Appl. Microbiol. Biotechnol. 85, 849e860. €gerrecklenfort, E., Kreuzig, R., Smalla, K., 2015. Wolters, B., Kyselkov a, M., Kro Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1 group. Front. Microbiol. 5, 1e11. http://dx.doi.org/10.3389/ fmicb.2014.00765. Widyasari-Mehta, A., Suwito, H.R.K.A., Kreuzig, R., 2016. Laboratory testing on the anaerobic biotransformation of the veterinary antibiotic doxycycline during liquid pig manure and digestate storage. Chemosphere 149, 154e160. Winckler, C., Engels, H., Hund-Rinke, K., Luckow, T., Simon, M., Steffens, G., 2004. €rantibiotika in WirtVerhalten von Tetracyclinen und anderen Veterina schaftsdünger und Boden. UBA-Texte 44/04, ISSN 0722e186X.

A. Widyasari-Mehta et al. / Journal of Environmental Management 177 (2016) 129e137 Umweltbundesamt, Dessau-Roßlau, 1e157. http://www.umweltdaten.de/ publikationen/pdf-l/2812.pdf (accessed 30.03.16). Zhou, L.-J., Ying, G.-G., Liu, S., Zhao, J.-J., Chen, F., Zhang, R.-Q., Peng, F.-Q., Zhang, Q.Q., 2012. Simultaneous determination of human and veterinary antibiotics in various environmental matrices by rapid resolution liquid chromatographyelectrospray ionization tandem mass spectrometry. J. Chromatogr. A 1244, 123e138.

137

Zhou, L.-J., Ying, G.-G., Zhang, R.-Q., Liu, S., Lai, H.-J., Chen, Z.-F., Yang, B., Zhao, J.-L., 2013a. Use patterns, excretion masses and contamination profiles of antibiotics in a typical swine farm, south China. Environ. Sci. Process. Impacts 15, 802e813. Zhou, L.-J., Ying, G.-G., Liu, S., Zhang, R.-Q., Lai, H.-J., Chen, Z.-F., Pan, C.-G., 2013b. Excretion masses and environmental occurrence of antibiotics in typical swine and dairy cattle in China. Sci. Total Environ. 444, 183e195.