Biodegradation of Veterinary Ionophore Antibiotics in Broiler Litterand Soil MicrocosmsPeizhe 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 polyethercompounds used in broiler feed to promote growth andcontrol coccidiosis. Most of the ingested IPAs are excreted intobroiler litter (BL), a mixture of excreta and bedding material.BL is considered a major source of IPAs released into theenvironment as BL is commonly used to fertilize agriculturalfields. This study investigated IPA biodegradation in BL andsoil microcosms, as a process affecting the fate of IPAs in theenvironment. The study focused on the most widely usedIPAs, monensin (MON), salinomycin (SAL), and narasin(NAR). MON was stable in BL microcosms at 24−72% watercontent (water/wet litter, w/w) and 35−60 °C, whereas SALand NAR degraded under certain conditions. Factor analysis was conducted to delineate the interaction of water and temperatureon 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 biodegradationcontributed significantly in BL-fertilized soil microcosms. SAL biotransformation in soil microcosms yielded the same product asin the BL microcosms. A new primary biotransformation product of MON was identified in soil microcosms. A field studyshowed that MON and SAL were stable during BL stacking, whereas MON degraded after BL was applied to grassland. Thebiotransformation product of MON was also detected in the top soil layer where BL was applied.

■ INTRODUCTION

Ionophore antibiotics (IPAs) are antimicrobial agents widelyused by the livestock industry.1,2 Annual sales of IPAs in theU.S. increased from 3.7 million kg in 2009 to over 4.1 millionkg in 2011, making them the second top-selling antimicrobialgroup used for meat-producing animals, after tetracyclines.3−5

Monensin (MON), salinomycin (SAL) and narasin (NAR)(Supporting Information (SI) Table S1) are the most widelyused IPAs. They are polyether compounds which inhibit thegrowth of coccidia, and are cidal agents to Gram-positivebacteria, algae, and protozoa.1,6−11 Capleton et al. assessed 83veterinary pharmaceuticals based on usage, potential to reachthe environment, and toxicity, and ranked IPAs as one of thehighest priority groups to be investigated because of the lack ofa comprehensive understanding of their environmentaloccurrence and fate.12

U.S. poultry production consumes 4.7 million kg/year ofantimicrobials, compared to 4.7, 1.7, and 1.4 million kg used inthe swine and cattle industries, and by humans, respectively.13

In broiler production, it is estimated that one kg of chicken feedcontains around 300 mg of IPAs (MON, SAL and NARcombined).14 However, because IPAs are poorly absorbed andbroken-down in the animals’ gut, more than 80% of theadministered IPAs may be excreted,15 and found in the broilerlitter (BL) at 0.2−20 mg/kg.16 After stacking (i.e., a process of

stockpiling waste litter), the BL is almost always used to fertilizeagricultural 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 theenvironment. Assessing the fate and degradation of IPAs in BLand BL-fertilized soil will provide critical information on thequantity of IPAs ultimately released into the environment, aswell as their role in microbial selection, resulting in theproliferation of (micro)organisms resistant to antimicrobialagents.18

To date, little information is available regarding thedegradation of IPAs in BL, though several studies haveinvestigated IPA degradation during animal manure compost-ing. MON and SAL half-lives from less than 5 days to greaterthan 10 weeks have been reported, depending on the type ofanimal manure and composting conditions.19−22 Dolliver et al.reported a half-life of 17 days for MON in turkey littercomposting.19 SAL in stored pig manure under anaerobicconditions at 20 °C degraded with a half-life of 5 days.21

Received: October 15, 2013Revised: January 29, 2014Accepted: February 4, 2014Published: February 4, 2014

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Decrease of IPAs in soil has also been reported. The half-lifeof MON applied to agricultural soil at 300 μg/kg was 3.3 and3.8 days with and without manure amendment, respectively.23

The MON concentration decreased over 10 days in non-sterilized soils; in contrast, after an initial decrease of 40%within 5 days, MON was stable for over 40 days in sterilizedsoil samples.24 SAL-amended soil incubated under aerobicconditions resulted in its disappearance with a 5-day half-life.25

Although half-lives of IPAs in animal waste and soil havebeen reported, information related to IPA degradation potentialand biotransformation products is limited. The objective of thisstudy was to investigate the biodegradation of IPAs underconditions typically encountered in BL and soil. Microcosmswere set up to assess the effect of water content andtemperature on the degradation of IPAs in BL, two parametersthat are significant for the BL stacking process. Degradation ofIPAs was also assessed in soil microcosms set up with BL-fertilized and nonfertilized soil samples, in which the effects ofwater content, carbon source amendment, and initial IPAconcentration were investigated. Several primary biotransfor-mation products of IPAs were detected. Additionally, BL andsoil samples from a field study were analyzed to assess the fateand potential degradation of IPAs during litter stacking andsubsequent application of the BL to agricultural fields, and theresults were compared with those obtained in the laboratorystudy.

■ MATERIALS AND METHODSChemicals. 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 acrossGeorgia, U.S. The fresh litter was stored at 4 °C without anytreatment. The characteristics of BL and BL-water extract areshown in Table 1.

To assess the biodegradation potential of IPAs in BL,laboratory microcosms were established by adding 1 g of litterinto 20 mL glass serum tubes, which were then sealed withrubber stoppers and aluminum caps in order to periodicallymonitor the headspace gas composition as an indicator ofmicrobial activity. The tube headspace was refreshed everyother day. Therefore, the BL microcosms were maintained

under aerobic/microaerophilic conditions. IPAs were not addedbecause the BL used contained a significant amount of IPAs(MON, SAL, and NAR; Table 1). To distinguish biotic fromabiotic degradation, autoclaved microcosms were set up andmonitored in parallel with nonautoclaved ones. Preliminarytests 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 IPAdegradation, 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 chosento represent BL in situ and very high water content conditions,respectively. After addition of water, the tubes were vortexed touniformly distribute water. All microcosms were kept in a 35 °Cconstant 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 toabove ambient temperature and ranges from 35 to 60 °C,depending on aeration conditions and depth of the stackingpiles.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 combina-tions, assays were also conducted at 35 °C with 64% watercontent, and at 53 °C with 64 and 72% water content.

Soil Microcosms. Surface soil samples (top 0−10 cm) werecollected from an experimental plot with Bermuda grass/tallfescue that has been receiving BL for more than 10 years andfrom another plot that has not received BL or any organicfertilizer. The plots are located at the Central Research andEducation Center of the University of Georgia (33° 24′ N, 83°29′ W, elevation 150 m). The soil in the area is dominated byCecil series (fine, kaolinitic, thermic Typic Kanhapludults). Soilcharacteristics are shown in Table 1. The soil microcosms wereset up in a similar fashion as the BL microcosms. One gram ofsoil was transferred into glass serum tubes, which were thensealed with rubber stoppers and aluminum caps. Threemicrocosm series were prepared: soil, soil with sterilizedwater (1/1 w/w), and soil with sterilized BL water extract (1/1w/w). The BL water extract was prepared by mixing water andBL (20/1 w/w) for 12 h, followed by centrifugation (3000 rpm,10 min) and then filter-sterilized (0.2 μm filters). Because thesoil used in this study did not contain any measurable amountsof IPAs, MON, and SAL at 1000 μg/kg each were added toeach soil tube. Control series were set up with autoclaved soilsamples 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, followedby application of BL to an experimental grass plot, and rainfallsimulations. A summary description of the field study isincluded in SI Text S3.

Analytical Methods. The BL water content was measuredgravimetrically after drying at 105 °C for 12 h and its waterpotential was measured with a WP4C dewpoint potentiometer(Decagon Devices, Pullman, WA). The BL and soil organiccarbon content was measured gravimetrically after combus-tion.28 Ammonia, nitrate, and phosphate in BL and soil extractswere measured following procedures outlined in StandardMethods.29 Headspace gas composition (O2, CO2) wasmeasured by gas chromatography (GC) with thermalconductivity detection.

Table 1. Characteristics of Broiler Litter (BL), Non-Fertilized Soil, and BL-Fertilized Soil

property BL nonfertilized soil BL-fertilized soil

foc (kg OC/kg solid)a 0.275 0.024 0.030MON (mg/kg) 0.2 <MDLb 0.005SAL (mg/kg) 3.9 <MDL <MDLNAR (mg/kg) 0.18 <MDL <MDL

Water Extractc

pH (units) 7.2 5.7 6.3NH4

+ (mM) 70 4.4 1.6NO3

− (mM) 0.01 12.3 13.0PO4

3‑ (mM) 4.72 0.3 4.6aOC, organic carbon. bMDL, method detection limit. MDL values are0.001, 0.002, and 0.002 mg/kg for MON, SAL and NAR, respectively.cWater extract prepared by mixing deionized water with solid at waterto solid ratios (w/w) of 1/1 for soil, and 20/1 for BL. After shaking for3 h for soil, and 12 h for BL, water extracts were passed through 0.2μm filters.

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Quantification of IPAs in BL and soil microcosms wasachieved by extracting sacrificial tubes, followed by LC/MSanalysis. Samples were spiked with nigericin, used as a surrogatestandard, then extracted with a mixture of hexane and anaqueous buffer. After volume reduction of the solvent extract,the final analytes were reconstituted in 1 mL mixture ofmethanol and 10 mM Na2HPO4 solution (50/50 v/v). Detailedsample preparation procedures have been previously reportedby Sun et al.16 Extracted IPAs were routinely analyzed with anAgilent 1100 Series LC/MSD system (Agilent Technologies,Palo Alto, CA) equipped with a reversed-phase column (2.1 ×150 mm, 3 μm Ascentis RP-amide column; Supelco, Bellefonte,PA). The mobile phase consisted of (A) a mixture of HPLC-grade water and formic acid (99.9/0.1 v/v) with 25%acetonitrile, and (B) HPLC-grade methanol and acetonitrile(50/50 v/v). The mass spectrometer was set at positiveelectron-spray ionization mode (ESI+). The sodium adducts ofIPAs with m/z of [M+23] were used for quantification (SITable S1). A full-scan mode from 100 to 1700 m/z was appliedfor searching for transformation products. Detailed setup hasbeen previously reported by Sun et al.16 A LC/MS/MS unit(Agilent 1260 Infinity LC system, 6410 Triple Quad MSD,Agilent Technologies, Palo Alto, CA) was used for structuralidentification of IPA transformation products. The LC unit wasset at the same conditions as those stated above for the LC/MS. The MS parameters were 135 V fragmentation voltage, and70 eV collision-induced-dissociation (CID) energy.

■ RESULTS AND DISCUSSIONBroiler Litter and Soil Characteristics. The BL was rich

in organic carbon and contained significant amounts ofammoniacal N and phosphate (Table 1). All three IPAs weredetected in BL, and SAL was the highest at 3.9 mg/kg.Degradation of IPAs was not observed in BL kept at 4 °C overthe duration of the study. Nutrients were also available in thetwo soil samples used, with higher organic carbon andphosphate content in the BL-fertilized soil than in thenonfertilized soil. IPAs in both soil samples were below themethod detection limits (MDLs; Table 1).IPA Degradation in Broiler Litter Microcosms. Effect of

Water Content on Microbial Activity. The change ofheadspace O2 and CO2 content was used as an indicator ofmicrobial activity in all BL microcosms incubated at 35 °C. Inall autoclaved BL microcosms, the headspace gas compositiondid not change over 14 days (data not shown). O2consumption and CO2 production profiles suggest lowmicrobial activity in BL microcosms with 24 and 40% watercontent, whereas active respiration occurred in the 57 and 72%series (SI Figure S1). The highest microbial activity wasobserved in litter microcosms with 72% water content, in whichthe headspace O2 was completely consumed within 2 daysinitially, and within 1 day subsequently, suggesting increasedmicrobial activity over time. In the 57% water contentmicrocosms, O2 was initially depleted in 4 days and thenwithin 2 days. Overall, addition of water resulted in increasedmicrobial activity in the BL microcosms incubated at 35 °C.It has been previously shown that microbial activity is

strongly correlated with moisture content in animal waste andwood litters.30−33 Because BL is a mixture of chicken excretaand bedding materials, which usually contain wood shavings,the BL water content is expected to have a strong impact onmicrobial activity. The litter water content tested in this assayranged from 24 to 72%, which corresponds to water potential

values from −14.9 to −0.66 MPa (SI Figure S2). It wasreported that water potential lower than −10 MPa greatlyinhibited bacterial activity in soil.34 Thus, microcosms with awater content of 24% represent a condition of severely limitedwater availability.

Effect of Water Content on IPA Degradation. Degradationof parent IPAs was not observed in BL microcosms with 24 and40% water content over 14 days (Figure 1), which is consistent

with the above-discussed low microbial activity (low O2consumption). However, SAL was almost depleted in BLmicrocosms with 57 and 72% water content, whereas NAR wasdepleted in microcosms with 72% water content after 14 daysof incubation. MON, on the other hand, was stable in all BLmicrocosms over the 14-day incubation. The structures of SALand NAR are very similar, except for a methyl group difference(see SI Table S1). Thus, the degradation of SAL and NAR wasexpected to follow a similar trend. IPAs were not degraded inall autoclaved BL (data not shown), suggesting the loss of IPAsin nonautoclaved microcosms was due to biological activity.Overall, degradation of IPAs at 35 °C was accelerated with anincrease in the litter water content.Our data clearly show that the water content is a limiting

factor to microbial activity in BL at 35 °C. Because the BLwater content is typically 16−46%,26 the results of this studyshow that minimal biodegradation of IPAs is expected duringthe litter stacking process. However, after litter is applied to thefield, its water content may increase (e.g., after rainfall), whichwill likely enhance microbial activity, thus creating conditionsfavorable for the degradation of IPAs.

Effect of Water Content and Temperature on MicrobialActivity and IPA Degradation. The O2 consumption and CO2production in BL microcosms with 24% water contentincreased as the temperature increased from 35 to 60 °C (SIFigure S3), indicating increased microbial activity withincreasing temperature. However, the microbial activity in theBL microcosms with a water content of 72% was significantlylower at 60 °C than at 35 or 45 °C. Correlated with microbialactivity, SAL and NAR degraded faster in series with higher O2consumption and CO2 production rates (Figure 2 and SI FigureS3). However, MON was stable under all conditions tested. Inall autoclaved BL microcosms, neither gas composition changein the headspace nor loss of IPAs was detected (data not

Figure 1. Normalized IPA content in BL microcosms after 14-dayincubation at 35 °C and a range of BL water content. Error barsrepresent one standard deviation of the means (n = 2).

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shown), suggesting that SAL and NAR degradation innonautoclaved microcosms was due to biological activity.Combined Effect of Water Content-Temperature on IPA

Degradation. The results from the water content andtemperature effect assays show that water content andtemperature significantly affect both microbial activity andIPA removal in BL microcosms. Therefore, attempts were madeto directly correlate IPA removal to microbial activity. UsingCO2 production as a measure of microbial activity in the BLmicrocosms, good linear correlations were obtained betweenthe removal of both SAL and NAR and cumulative CO2production as shown in SI Figure S4, suggesting the increasein microbial activity led to a higher extent and rate ofdegradation of IPAs.Our results also showed that the effect of BL water content

on IPA degradation depends on temperature and vice versa.Therefore, to quantitatively evaluate the effect of both watercontent and temperature on IPA degradation in BL micro-cosms, factor analysis was conducted to evaluate the interactiveeffects of water content and temperature on the degradation ofIPAs. Given the very low concentration of IPAs in the BL, thedegradation of IPAs is assumed to follow nongrowth, pseudofirst-order kinetics as was previously reported for SAL.22 Factoranalysis was conducted based on the following equation:

α α α α= + · + · + · ·k W T W T0 1 2 1,2 (1)

where k is the pseudo first-order IPA degradation rate constant(d−1);W is the water content (%, water/wet BL, w/w); T is theincubation temperature (°C); α0 (d

−1) is a constant; α1 (d−1),

α2 (°C−1·d−1) and α1,2 (°C−1·d−1) are coefficients whichevaluate the weighted effect from the change of W, T, and theW−T interaction, respectively, on the degradation of IPAs.Equation 1 is valid forW and T values at which k ≥ 0 (i.e., k < 0means no degradation).Applying eq 1 for 35 ≤ T ≤ 60 °C and 24 ≤ W ≤ 72% to

SAL and NAR degradation rate data (SI Text S4) using theMatLab curve fitting toolbox (SI Figure S5), the followingexpressions were obtained:

= − + + − ·

=

k W T W T0.864 0.023 0.0146 0.00039

(R 0.974)SAL

2(2)

= − + + − ·

=

k W T W T

R

1.140 0.023 0.0193 0.00039

( 0.867)NAR

2 (3)

Analysis of variance (ANOVA, SI Text S4) shows p-value<0.0001, which suggests eq 1 captures the effects of watercontent and temperature on IPA degradation. Furthermore, theplot of estimated k values using eq 2 and 3 versus thoseexperimentally measured shows a very high correlation (R2 >0.943; SI Figure S6). Therefore, eqs 2 and 3 are suitable inexpressing IPA degradation in BL microcosms within theconditions examined in the present study.Several IPAs degradation features could be obtained from the

above expressions. First, the coefficient values in eqs 2 and 3 areclose, suggesting that the effect of water content andtemperature on the degradation of SAL is similar to that ofNAR. Second, the negative values of coefficient α1,2 indicatethat the interaction of water content and temperature have anegative effect on SAL and NAR degradation, which is mainlydue to reduced oxygen solubility and transfer with increasedtemperature at a relatively high water content. In the case of BLwith a water content of 72%, a water/litter slurry was formed asthe water content exceeded the water holding capacity of theBL and thus oxygen transfer to microorganisms through theaqueous phase was likely limited because of the lower oxygensolubility at a higher temperature (e.g., 60 °C). In contrast, inBL microcosms with 24% water content, the litter interstitialspaces were filled with air, which allowed faster oxygen transfer.Third, eqs 2 and 3 can be used to obtain the water content-temperature domains (SI Figure S7) within which SAL andNAR degradation did not occur (i.e., k = 0). Therefore, in orderto achieve degradation of IPAs in BL, both the water contentand temperature should be adjusted and maintained at valuesoutside these water content-temperature domains. Althoughthe domains may vary for different BL samples, eq 1 providesvaluable insights into the management of BL in order to reducetheir IPA content. As previously mentioned, the littertemperature during stacking is typically in the range of 35−60 °C,27 suggesting that addition of water up to the waterholding capacity of the BL (∼70% water/wet litter, w/w) willresult in enhanced IPA degradation.

IPAs Biotransformation Products in BL Microcosms. Onesignificant new peak (m/z 531) was observed on LC/MS andnamed TP-SAL. Addition of SAL to BL microcosms on day 14resulted in a sharp increase of TP-SAL production (Figure 3),confirming that TP-SAL was a SAL degradation product.Previous studies have shown products with m/z of 531 ([M+Na+]) formed as a result of either abiotic or biotic SALtransformation, albeit with different structures (SI FigureS8).21,35−38 In this study, LC/MS/MS was used to confirmthe structure of TP-SAL. Compared to the SAL MS/MSspectrum, the fragmentation pattern of TP-SAL clearly showsthat rings B, C, D, and E are retained in the product (SI FiguresS9, S10), which supports the structure of the previouslyidentified product resulting from SAL biodegradation.37 Thestructure of TP-SAL also implies that biodegradation of NARyields the same product, because the difference between SALand NAR occurs on the ring A moiety (SI Table S1).TP-SAL accumulated in BL microcosms during the 14-day

incubation (Figure 3), showing that TP-SAL was stable under

Figure 2. Normalized IPA concentration in BL microcosms after 14-day incubation at 35, 45, and 60 °C with 24% (A) and 72% (B) watercontent. Error bars represent one standard deviation of the means (n =2).

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the conditions of the BL microcosm assay. To assess possibleTP-SAL degradation in soil, selected samples were then mixedwith nonfertilized soil at 1/1 BL/soil (w/w). After anincubation period of 10 days, no degradation of TP-SAL wasobserved, indicating that TP-SAL is not likely degraded afterBL is applied to the field. Although TP-SAL may be persistentin the environment, a previous study showed that it does notform a complex with either sodium or potassium ions.37 Thus,this product may not possess the antibiotic properties ofionophores which are associated with their capacity to formcomplexes with cations.IPA Degradation in Soil Microcosms. About 20% of

MON was degraded in the control (i.e., autoclaved) non-fertilized soil microcosms under all three conditions (Figure 4A-1), suggesting that abiotic degradation of MON occurred.Significant abiotic degradation (∼20%) of MON was observedin the autoclaved BL-fertilized soil microcosms without wateror filter-sterilized BL extract amendment (Figure 4 A-2).Degradation of MON was observed in nonautoclaved soilmicrocosms amended with water or BL extract regardless ofwhether the soil was fertilized or not. In contrast, minimalbiodegradation of MON was observed in soil microcosmswithout water or BL extract amendment (Figure 4 A-1 and A-2).Significant degradation (∼80%) of SAL was observed in the

nonfertilized soil microcosms without any amendment after a7-day incubation period, whereas only 20−30% of SALdegraded in soil microcosms amended with water or BL extract(Figure 4 B-1). Unlike MON, the loss of SAL after a 7-dayincubation period was comparable to that in the autoclaved soilmicrocosms under all three conditions (Figure 4 B-1),indicating that the decrease of SAL in nonfertilized soilmicrocosms was primarily due to abiotic reactions. About 25%SAL was degraded in autoclaved BL-fertilized soil microcosmsunder all three conditions (Figure 4 B-2). However, the loss ofSAL was significant after a 7-day incubation period innonautoclaved soil microcosms where 50−60% of SAL wasbiodegraded.Assuming pseudo first-order kinetics, the half-lives of IPAs

under the best conditions, that is, soil with amendments, arecomparable to previously reported values, between 3.3 and 5

days.23−25 The observed degradation of MON and SAL inautoclaved soil microcosms suggests that abiotic reactionscontributed to the loss of the parent IPAs in the soil. However,caution is warranted when comparing autoclaved and non-autoclaved soil microcosms used in the present study as the soilstructure and mineral properties may have been altered duringautoclaving. Although no studies have directly investigated theabiotic transformation of IPAs in soil, one study indicated thatabiotic degradation of MON in soil may be significant afterobserving a 40% loss of parent MON in 60Co-irradiated soilsamples incubated for 30 days in the dark.24 IPAs are known toundergo acid-catalyzed transformations.36 Given that the soilused in this study was slightly acidic (Table 1), IPAs likelyunderwent acid-catalyzed transformation. Though hydrolysisand some unknown products were observed in autoclaved soilsamples, further identification of abiotic transformationproducts was beyond the scope of this study.Compared to the autoclaved soil microcosms, MON was

biodegraded in microcosms set up with both nonfertilized andBL-fertilized soil samples, which suggests MON degraders maybe widely present in soil. Indeed, MON transformation by thesoil bacterium Sebekia benihana has been reported.39 Incontrast, SAL was only biodegraded in the BL-fertilized soil

Figure 3. Transformation product of SAL (TP-SAL) in the BLmicrocosms with 72% water content incubated at 35 °C. At day 14,replicate microcosms were amended with nonfertilized soil (1/1, w/w)or 50 mg/kg SAL. Error bars represent one standard deviation of themeans (n = 3).

Figure 4. MON (A) and SAL (B) normalized to initial IPAconcentration (1 mg IPA/kg soil) in microcosms set up withnonfertilized (1) and BL-fertilized soil (2) after 7-day incubation.Abiotic controls were prepared with autoclaved soil. Error barsrepresent one standard deviation of the means (n = 3).

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microcosms. Minimal SAL biodegradation observed in non-fertilized soil microcosms was not due to lack of nutrientsbecause the BL extract is rich in carbon, nitrogen andphosphorus (Table 1). Therefore, microorganisms which candegrade SAL may not be widely present in the soilenvironment. In the BL microcosm assay, SAL readilydegraded, whereas MON was persistent during the 14-dayincubation. The above combined results imply that themicroorganisms responsible for SAL degradation are likelyintroduced to soil with BL application.IPAs Biotransformation Products in Soil Microcosms.

Degradation of SAL in microcosms set up with BL-fertilizedsoil samples yielded one major product peak with m/z of 531on LC/MS, which was identified by LC/MS/MS as the samebiotransformation product as that observed in the BLmicrocosms (i.e., TP-SAL). Therefore, our efforts focused onthe identification of MON biotransformation products in soilmicrocosms.One product peak, named TP-MON (m/z 707, an increase

of 14 m/z units compared to MON), was observed on LC/MSin soil samples incubated for 7 days. Analyses of autoclaved soiland reference soil (i.e., soil without MON amendment)confirmed that the new peak represented a product of MONbiotransformation. To further investigate the biotransformationof MON by soil bacteria, assays were conducted with BL-fertilized soil samples at 50% water content with addition ofMON at 0.2, 1, and 10 mg MON/kg soil. As shown in Figure5A, the MON concentration decreased over the incubationperiod with half-lives of 4−6 days, which is consistent withpreviously reported half-lives of MON in soil.23,24 It isnoteworthy that MON degradation accelerated after the firsttwo days, which may be due to an increase in the populationsize of MON degraders or the level of enzyme(s) involved inMON degradation in soil bacteria.As the MON concentration decreased, the TP-MON peak

area initially increased and then decreased (Figure 5B),indicating TP-MON is likely biodegraded by soil bacteria.Assuming a similar MS signal response factor for MON andTP-MON, the sum of the peak areas of MON and TP-MONremained stable for the first 5 days in the samples with an initial10 mg MON/kg soil, indicating that TP-MON is the majorproduct resulting from the initial MON biotransformation (SIFigure S11). Indeed, about 40% of the parent MON wasbiotransformed to TP-MON in the first 5 days. However,samples amended with 0.2 and 1 mg MON/kg soil, the sum ofpeak areas decreased overtime, which may be due to the factthat less TP-MON was generated and was efficiently removedby soil bacteria.Product Structural Identification.Molecular weight increase

by 14 Da is common in biotransformation of organicsubstances, such as bioconversion of primary carbon of anhydrocarbon to an aldehyde, and oxidation of a −OH to−COOH. Based on the University of Minnesota-Biocatalyst/Biodegradation Database (UM-BBD), the ‘very-likely’ aerobicMON biotransformation pathway results in ring E opening andformation of a structure with a carboxyl group at the end(Figure 6A). The molecular weight is increased by 14 Da due tothe oxidation of the primary hydroxyl carbon. The first step inthe biotransformation of ring E is a ring-opening reactionpossibly via acid-catalyzed hydrolysis. However, at pH above 5,the ring E moiety is stable under abiotic conditions,36 indicatingthat ring E is opened by an enzyme-facilitated hydrolysis.Similarly, the spiral-ketal carbon connecting rings A and B is

also known to undergo acid-catalyzed hydrolysis.36 Thus, it ispossible that ring A and/or B on TP-MON are also opened.Therefore, a TP-MON structure with both rings A and E openis proposed (Figure 6B).The proposed transformation product is supported by the

LC/MS/MS results (SI Figures S12, S13). The m/z 693.5 and707.5 ions were selected for MON and TP-MON, respectively,from the full-scan analysis of products and subjected to collisionactivated dissociation. The mass spectrum of TP-MON wassimilar to that of MON, which strongly suggested that the m/z707.5 peak represents a MON transformation product. Basedon a number of studies which have proposed the MS/MSfragmentation pattern of MON, interpretation of the MS/MSresults was made to further elucidate the molecular structure ofTP-MON. MON and TP-MON shared the common fragmentsat m/z 443.3, 461.3, 479.3, and 501.3, suggesting that oxygenwas added to the portion of the molecule that included fromthe middle part of ring D to ring E.40 Three additionalstructural clues were derived from the TP-MON spectrum: (1)the lack of m/z 617.3 fragment suggested that the C−O bondwas opened in ring E; (2) the new fragment of m/z 605.3resulted from a cleavage on the opened ring E; and (3) the newfragment of m/z 507.3 confirmed that ring A was also opened.Other fragment structures are also proposed and shown in SITable S2. Overall, based on the UM-BBD prediction and MS/MS results, the molecular structure of TP-MON is tentativelyproposed as shown in Figure 6. However, use of otheranalytical techniques, such as high mass resolution MS and

Figure 5. Normalized MON (A) and MON product (TP-MON) (B)in BL-fertilized soil microcosms set up at a range of initial MONconcentration (0.2−10 mg MON/kg soil). Error bars represent onestandard deviation of the means (n = 2).

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NMR, is needed in order to further confirm the TP-MONstructure.Field Study. Significant degradation of MON and SAL was

not observed over 57 days during BL stacking (SI Figure S14).Based on the results of the BL microcosm assay, MON isexpected to be stable during the stacking process. The BL usedin the stacking study had an initial water content of 33% anddecreased to 29% by day 57. At these water content values,based on eq 2, SAL can only be degraded when thetemperature is above 57 °C (SI Figure S4). The litter stacktemperature ranged from 30 to 50 °C depending on the stackdepth, and remained at 45 °C in the center of the stack pile(data not shown). Thus, it is expected that SAL would be stableduring the BL stacking process.After stacking, the litter was applied to an experimental field.

IPAs were stable over four weeks under dry conditions (i.e.,before the simulated rainfall; data not shown). Degradation ofMON occurred after the simulated rainfall (SI Figure S15).MON degradation and TP-MON production was observed inthe top soil in the first week after rainfall application, followedby a decrease of both MON and TP-MON in the subsequenttwo weeks resulting in a MON half-life of 1.5 weeks. SAL wasstable in the top soil over three weeks. These observations areconsistent with the results obtained with the soil microcosmassays, which suggest that MON degraders are likely widelypresent in soils.Environmental Implications. This study assessed the

degradation potential of MON, SAL and NAR in BL and soilmicrocosms under various water content and temperature

conditions. Degradation of IPAs does not likely occur duringthe BL stacking process due to low water availability, thoughSAL and NAR degraders are present in BL. Becausetemperature is not usually controlled during the litter stackingprocess, in order to enhance IPAs degradation, water may beadded to reach a desirable litter water content. From thestandpoint of the fate and degradation of IPAs, SAL and NARare recommended over MON as veterinary IPAs, because theycan be degraded during litter stacking under optimal temper-ature and water content conditions, thus minimizing the releaseof IPAs into the environment. After BL is applied to the field,IPAs can potentially be degraded in the soil, althoughbiodegradation of SAL was only observed in soil which hadbeen receiving BL for years. Primary biotransformationproducts of IPAs in BL and soil were identified in this study.However, these products may be of less environmental concern,because they are either readily biodegraded in the environmentor have weaker to no antibiotic properties compared to theparent ionophores, which are typically associated with theircapacity to form complexes with cations.

■ ASSOCIATED CONTENT

*S Supporting InformationText S1−S4, Tables S1−S2 and Figures S1−S15. This materialis available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Phone: 404-894-9367; fax: 404-894-8266; e-mail: spyros.pavlostathis@ce.gatech.edu.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study was supported by the U.S. Department ofAgriculture Grant 2009-65102-05843. We thank Dr. AaronThompson, Dr. Dorcas Franklin, Sarah Doydora, and JohnRema at the University of Georgia, Athens, GA for assistance incollecting BL and soil samples and comments on this work.

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