Article pubs.acs.org/est
Biodegradation of Veterinary Ionophore Antibiotics in Broiler Litter and Soil Microcosms Peizhe Sun,† Miguel L. Cabrera,‡ Ching-Hua Huang,† and Spyros G. Pavlostathis*,† †
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States Department of Crop and Soil Sciences, University of Georgia, Athens, Georgia 30602, United States
‡
S Supporting Information *
ABSTRACT: Ionophore antibiotics (IPAs) are polyether compounds used in broiler feed to promote growth and control coccidiosis. Most of the ingested IPAs are excreted into broiler litter (BL), a mixture of excreta and bedding material. BL is considered a major source of IPAs released into the environment as BL is commonly used to fertilize agricultural fields. This study investigated IPA biodegradation in BL and soil microcosms, as a process affecting the fate of IPAs in the environment. The study focused on the most widely used IPAs, monensin (MON), salinomycin (SAL), and narasin (NAR). MON was stable in BL microcosms at 24−72% water content (water/wet litter, w/w) and 35−60 °C, whereas SAL and NAR degraded under certain conditions. Factor analysis was conducted to delineate the interaction of water and temperature on SAL and NAR degradation in the BL. A major transformation product of SAL and NAR was identified. Abiotic reaction(s) were primarily responsible for the degradation of MON and SAL in nonfertilized soil microcosms, whereas biodegradation contributed significantly in BL-fertilized soil microcosms. SAL biotransformation in soil microcosms yielded the same product as in the BL microcosms. A new primary biotransformation product of MON was identified in soil microcosms. A field study showed that MON and SAL were stable during BL stacking, whereas MON degraded after BL was applied to grassland. The biotransformation product of MON was also detected in the top soil layer where BL was applied.
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INTRODUCTION Ionophore antibiotics (IPAs) are antimicrobial agents widely used by the livestock industry.1,2 Annual sales of IPAs in the U.S. increased from 3.7 million kg in 2009 to over 4.1 million kg in 2011, making them the second top-selling antimicrobial group used for meat-producing animals, after tetracyclines.3−5 Monensin (MON), salinomycin (SAL) and narasin (NAR) (Supporting Information (SI) Table S1) are the most widely used IPAs. They are polyether compounds which inhibit the growth of coccidia, and are cidal agents to Gram-positive bacteria, algae, and protozoa.1,6−11 Capleton et al. assessed 83 veterinary pharmaceuticals based on usage, potential to reach the environment, and toxicity, and ranked IPAs as one of the highest priority groups to be investigated because of the lack of a comprehensive understanding of their environmental occurrence and fate.12 U.S. poultry production consumes 4.7 million kg/year of antimicrobials, compared to 4.7, 1.7, and 1.4 million kg used in the swine and cattle industries, and by humans, respectively.13 In broiler production, it is estimated that one kg of chicken feed contains around 300 mg of IPAs (MON, SAL and NAR combined).14 However, because IPAs are poorly absorbed and broken-down in the animals’ gut, more than 80% of the administered IPAs may be excreted,15 and found in the broiler litter (BL) at 0.2−20 mg/kg.16 After stacking (i.e., a process of © 2014 American Chemical Society
stockpiling waste litter), the BL is almost always used to fertilize agricultural fields at a rate of at least 5 t/hectare/application,17 which may result in 1−100 g of IPAs/hectare/application. Thus, BL is likely a major source of IPA release into the environment. Assessing the fate and degradation of IPAs in BL and BL-fertilized soil will provide critical information on the quantity of IPAs ultimately released into the environment, as well as their role in microbial selection, resulting in the proliferation of (micro)organisms resistant to antimicrobial agents.18 To date, little information is available regarding the degradation of IPAs in BL, though several studies have investigated IPA degradation during animal manure composting. MON and SAL half-lives from less than 5 days to greater than 10 weeks have been reported, depending on the type of animal manure and composting conditions.19−22 Dolliver et al. reported a half-life of 17 days for MON in turkey litter composting.19 SAL in stored pig manure under anaerobic conditions at 20 °C degraded with a half-life of 5 days.21 Received: Revised: Accepted: Published: 2724
October 15, 2013 January 29, 2014 February 4, 2014 February 4, 2014 dx.doi.org/10.1021/es404619q | Environ. Sci. Technol. 2014, 48, 2724−2731
Environmental Science & Technology
Article
under aerobic/microaerophilic conditions. IPAs were not added because the BL used contained a significant amount of IPAs (MON, SAL, and NAR; Table 1). To distinguish biotic from abiotic degradation, autoclaved microcosms were set up and monitored in parallel with nonautoclaved ones. Preliminary tests showed that IPAs were stable during autoclaving (21.5 psi, 121 °C, 30 min). The water content of fresh BL is typically 16−46% (water/ wet litter, w/w)26 and its water holding capacity is around 70%. In order to investigate the effect of water content on IPA degradation, water levels at 24, 40, 57, and 72% water/wet litter (i.e., 32, 67, 133, and 257% water/dry litter; see SI Text S2) were tested. The 24 and 72% water content levels were chosen to represent BL in situ and very high water content conditions, respectively. After addition of water, the tubes were vortexed to uniformly distribute water. All microcosms were kept in a 35 °C constant temperature room. Temperature is another factor expected to significantly affect microbial activity and thus possible degradation of IPAs in BL. During stacking, the BL temperature gradually increases to above ambient temperature and ranges from 35 to 60 °C, depending on aeration conditions and depth of the stacking piles.27 Thus, for this assay temperature values at 35, 45, and 60 °C were selected to simulate conditions during litter stacking. In order to expand the temperature/water content combinations, assays were also conducted at 35 °C with 64% water content, and at 53 °C with 64 and 72% water content. Soil Microcosms. Surface soil samples (top 0−10 cm) were collected from an experimental plot with Bermuda grass/tall fescue that has been receiving BL for more than 10 years and from another plot that has not received BL or any organic fertilizer. The plots are located at the Central Research and Education Center of the University of Georgia (33° 24′ N, 83° 29′ W, elevation 150 m). The soil in the area is dominated by Cecil series (fine, kaolinitic, thermic Typic Kanhapludults). Soil characteristics are shown in Table 1. The soil microcosms were set up in a similar fashion as the BL microcosms. One gram of soil was transferred into glass serum tubes, which were then sealed with rubber stoppers and aluminum caps. Three microcosm series were prepared: soil, soil with sterilized water (1/1 w/w), and soil with sterilized BL water extract (1/1 w/w). The BL water extract was prepared by mixing water and BL (20/1 w/w) for 12 h, followed by centrifugation (3000 rpm, 10 min) and then filter-sterilized (0.2 μm filters). Because the soil used in this study did not contain any measurable amounts of IPAs, MON, and SAL at 1000 μg/kg each were added to each soil tube. Control series were set up with autoclaved soil samples to distinguish between abiotic and biotic reaction(s). The tubes were incubated at room temperature (20−22 °C). Field Study. The field study included BL stacking, followed by application of BL to an experimental grass plot, and rainfall simulations. A summary description of the field study is included in SI Text S3. Analytical Methods. The BL water content was measured gravimetrically after drying at 105 °C for 12 h and its water potential was measured with a WP4C dewpoint potentiometer (Decagon Devices, Pullman, WA). The BL and soil organic carbon content was measured gravimetrically after combustion.28 Ammonia, nitrate, and phosphate in BL and soil extracts were measured following procedures outlined in Standard Methods.29 Headspace gas composition (O2, CO2) was measured by gas chromatography (GC) with thermal conductivity detection.
Decrease of IPAs in soil has also been reported. The half-life of MON applied to agricultural soil at 300 μg/kg was 3.3 and 3.8 days with and without manure amendment, respectively.23 The MON concentration decreased over 10 days in nonsterilized soils; in contrast, after an initial decrease of 40% within 5 days, MON was stable for over 40 days in sterilized soil samples.24 SAL-amended soil incubated under aerobic conditions resulted in its disappearance with a 5-day half-life.25 Although half-lives of IPAs in animal waste and soil have been reported, information related to IPA degradation potential and biotransformation products is limited. The objective of this study was to investigate the biodegradation of IPAs under conditions typically encountered in BL and soil. Microcosms were set up to assess the effect of water content and temperature on the degradation of IPAs in BL, two parameters that are significant for the BL stacking process. Degradation of IPAs was also assessed in soil microcosms set up with BLfertilized and nonfertilized soil samples, in which the effects of water content, carbon source amendment, and initial IPA concentration were investigated. Several primary biotransformation products of IPAs were detected. Additionally, BL and soil samples from a field study were analyzed to assess the fate and potential degradation of IPAs during litter stacking and subsequent application of the BL to agricultural fields, and the results were compared with those obtained in the laboratory study.
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MATERIALS AND METHODS Chemicals. Information on sources of chemicals and reagents is provided in SI Text S1. Broiler Litter Microcosms. The BL used in this study was a mixture of litters obtained from several broiler farms across Georgia, U.S. The fresh litter was stored at 4 °C without any treatment. The characteristics of BL and BL-water extract are shown in Table 1. Table 1. Characteristics of Broiler Litter (BL), NonFertilized Soil, and BL-Fertilized Soil property
BL
foc (kg OC/kg solid)a MON (mg/kg) SAL (mg/kg) NAR (mg/kg)
0.275 0.2 3.9 0.18
pH (units) NH4+ (mM) NO3− (mM) PO43‑ (mM)
7.2 70 0.01 4.72
nonfertilized soil 0.024
