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Degradation and Metabolite Production of Tylosin in Anaerobic and Aerobic Swine-Manure Lagoons A. C. Kolz, T. B. Moorman, S. K. Ong, K. D. Scoggin, E. A. Douglass Watershed contamination from antibiotics is becoming Degradation half-lives for tylosin reported in the literature average a critical issue because of increased numbers of confined animal-feeding 4 to 8 days in swine, calf, and chicken manure; 2 to 8 days in aqueous operations and the use of antibiotics in animal production. To understand the manure and soil–manure mixtures; and 10 to 40 days in surface- fate of tylosin in manure before it is land-applied, degradation in manure water simulation systems (De Liguoro et al., 2003; Ingerslev and lagoon slurries at 228C was studied. Tylosin disappearance followed a Halling-Sørensen, 2001; Ingerslev et al., 2001; Loke et al., 2000; biphasic pattern, where rapid initial loss was followed by a slow removal Teeter and Meyerhoff, 2003). However, most of these studies phase. The 90% disappearance times for tylosin, relomycin (tylosin D), and focused on the degradation of tylosin A in aerobic systems and fresh desmycosin (tylosin B) in anaerobically incubated slurries were 30 to 130hours. Aerating the slurries reduced the 90% disappearance times to between manure. Tylosin degradation in an aqueous manure mixture, 12 and 26 hours. Biodegradation and abiotic degradation occur, but strong incubated under methanogenic conditions, produced several identi- sorption to slurry solids was probably the primary mechanism of tylosin fied degradates; however, analysis was hampered by interference disappearance. Dihydrodesmycosin and an unknown degradate with from components of the mixture (Loke et al., 2000). Degradation molecular mass of m/z 934.5 were detected. Residual tylosin remained in rates for tylosin A, in these systems, do not provide adequate slurry after eight months of incubation, indicating that degradation in information about the persistence of other forms of tylosin or the lagoons is incomplete and that residues will enter agricultural fields. Water appearance of degradates in lagoon slurry, where waste may reside Environ. Res., 77, 49 (2005).
for six months before land application. Therefore, it is of interest to tylosin, antibiotic degradation, anaerobic, aerobic, swine understand the fate of common veterinary drugs, such as tylosin, in lagoon storage, before the manure slurry is land-applied.
Typical anaerobic lagoons may have, as their design objectives, a reduction in solids, nutrients, odor, and sludge volume (Zhang, 2001). Retention times for the lagoons will vary with the production Antibiotic residues and increased numbers of antibiotic-resistant phase of the facility and season. In some cases, anaerobic lagoons bacteria have been reported near confined animal feeding operations may be modified to include mechanical aeration, or an aerated (CAFOs) and in agricultural watersheds (Campagnolo et al., 2002; lagoon may follow as an additional stage for treatment of lagoon Chee-Sanford et al., 2001; Haller et al., 2002; Kolpin et al., 2002).
effluent. It is of interest to determine how aeration of lagoons, A major route for entry of veterinary pharmaceuticals into water- a common modification, may change degradation rates.
sheds is through land application of animal biosolids and spills of The objectives for the current research were to investigate the animal waste at facilities using these drugs (Boxall et al., 2001; disappearance of tylosin and to determine the production of degra- Daughton and Ternes, 1999). Swine CAFOs often use antibiotics dates in lagoon slurry. Lagoon slurries were collected from two for therapeutic or growth-promoting purposes. Manure generated at swine CAFOs, amended with tylosin and incubated under aerobic CAFOs, containing excreted residues, is commonly stored in and anaerobic conditions. The persistence of tylosin was also studied earthen lagoons for several months before land application. Incom- by incubating one of the spiked slurries anaerobically for eight plete degradation of pharmaceuticals, in vivo and during manure months. Sodium-azide amended slurry and filtered lagoon liquid storage before biosolids are land-applied, could be a contributing were included to distinguish biological from abiotic degradation or factor to the presence of these drugs in waterways.
from sorption during the studies. Evaluation of solvent and solid- In the year 2000, 92% of swine CAFOs reported using antibiotics phase extraction systems was also done to optimize analyte recovery in a nationwide survey, and the most commonly administered drug for liquid chromatography-tandem mass spectrometry analysis.
was tylosin, a macrolide antibiotic (USDA, 2002). Between 4 and 5million pounds of tylosin and other macrolide antibiotics were soldannually in 2001 and 2000 (Vansickle, 2002). After oral administra- Materials and Methods tion of tylosin at a dose rate of 110 mg/kg, swine were found to Slurry Collection and Characterization.
excrete up to 40% of the tylosin in antibiotically potent forms (Sieck identified as an open anaerobic lagoon (OL) and covered anaerobic et al., 1978). These forms include (in order of prominence) relo- lagoon (CL), were collected from two swine CAFO lagoons, an mycin (tylosin D), tylosin (tylosin A), dihydrodesmycosin, des- open lagoon, and a covered anaerobic lagoon, respectively. The OL mycosin (tylosin B), macrosin (tylosin C), and at least 10 other slurry was a combination of settled solids and lagoon liquid col- degradates in smaller quantities (Sieck et al, 1978; Teeter and lected from below the liquid surface of the lagoon, while CL was Meyerhoff, 2003). Different forms of tylosin are shown in Figure 1.
collected from a pump outlet of the covered lagoon. Collection took January/February 2005 microorganisms in the broth was observed after this period for signsof tylosin toxicity.
To assess the effectiveness of azide on microbial inhibition, 50 g/L of sodium azide was added to CL slurryand incubated for 96 hours. Aliquots of 0.5 mL were plated ontoLuria Bertani agar to determine microbial growth.
Anaerobic Studies.
Anaerobic degradation experiments were conducted by transferring 20 mL of either CL or OL slurries toamber glass vials with Teflon-lined caps. Before the slurries weretransferred to the amber vials, the slurries were shaken for 10minutes on a reciprocating shaker. For both OL and CL slurries, twosets of vials (15 to 18 vials per set) were prepared. For one set ofvials, 400 lg of tylosin tartrate from aqueous stock solution wereadded to each vial giving a concentration of 20 mg/L. After additionof tylosin, the vials were capped, vortexed for 10 seconds, and the Figure 1—Structure of various tylosin forms.
vial headspaces were immediately evacuated and filled with heliumgas (99.99% pure). The second set of vials was spiked with 1 g ofsodium azide (50 g/L) to inhibit biodegradation. After the addition place in the summer, when the average temperature in the lagoons of azide, the vials were vortexed and allowed to rest for 30 minutes was approximately 258C. The slurries were collected in 68-L at 228C. This was followed by spiking the vials with 400 lg of (18-gal) rubber tubs, which were kept on ice during transport.
tylosin and evacuating the headspaces of the vials. In addition, a set The slurries were homogenized and stored in 1.9-L (2-qt) glass of vials were prepared with OL slurry, but spiked with 3900 lg of jars at 48C until use.
tylosin (195 mg/L) to observe differences in disappearance rates and Characterization tests were conducted to determine the pH, solids, products at this higher concentration.
mineral, and carbon content. Dissolved organic carbon in the super- A separate experiment using OL slurry was prepared under natant was determined using UV-persulfate digestion with infrared anaerobic conditions to study the persistence of residuals under long detection. Organic and inorganic carbon solids content were deter- term incubation (eight months). In this study, 240 lg of tylosin mined by combustion and infrared spectrometry. Total solids were tartrate was added (12 mg/L) to the vials. To inhibit biological determined by weight difference after drying an aliquot of slurry at activity, 0.08 g of azide per 20 mL of slurry (4 g/L) was added to 1058C. The oxidation–reduction potential (ORP) and pH of the another set of vials. All vials were incubated at 22 6 18C. Triplicate slurries were measured before and after incubation for the degradation vials were sacrificed at each sampling event and measured for experiments. All measurements were taken at 22 6 18C. The ORP tylosin and degradates.
was measured using a platinum-tipped ORP probe filled with a 3-M Tylosin was added to slurries rather than studying the disappear- silver–silver chloride solution (Thermo Electron, Mississauga, ance rates of tylosin residuals already present in slurries, for several Canada). A hydrogen standard 1424 mV (mV EH) solution was reasons. First, the detected tylosin residuals were too low to signifi- used as a reference for the probe. The background concentrations of cantly determine disappearance rates. Second, tylosin B and D were tylosin in each source material were analyzed by liquid chromatog- the persistent forms of tylosin found in the lagoons, which were not raphy with tandem mass spectrometry (LC–MS–MS).
consistent with the forms excreted by swine, which were primarily Tylosin Stability and Toxicity.
The stability of tylosin was tylosin D and A.
monitored in several sterile matrices: (a) filtered lagoon liquids with Aerobic Studies.
Aerated degradation experiments were pre- 50 g/L sodium azide, (b) Milli-Q purified water (Millipore, Billerica, pared in a similar manner to the anaerobic experiments, except that Massachusetts) with 50 g/L of sodium azide, and (c) Milli-Q water at immediately after spiking the manure slurry with tylosin the vials pH 7 and 9.2. The filtered lagoon liquids were obtained by centri- were capped and a 16-gauge Teflon tube inserted and compressed fuging the OL and CL slurries at 12 500 3 g for 30 minutes. The air (breathing-air grade) bubbled at a continuous rate. A 22-gauge lagoon liquids were decanted, and 50 g/L of sodium azide was added needle was also inserted through the cap as a gas vent. Samples before filtration. Milli-Q water with 50 g/L of sodium azide and were incubated at room temperature (22 6 18C).
Milli-Q water without azide were adjusted with 0.1-M potassium Tylosin Extraction.
Three extraction solvent combinations hydroxide (KOH) to pH 9.2, for comparison with the filtered lagoon were evaluated for tylosin recovery and stability in CL slurry 24 liquids. All matrices were filter-sterilized with sterile 0.2-lm pore- hours after spiking. The solvent combinations tested were methanol size cellulose-acetate filters and dispensed into autoclaved glassware, (Rabølle and Spliid, 2000), methanol–acetonitrile–0.1 M ascorbic under a sterile laminar flow hood. Tylosin tartrate was spiked in all acid (45:45:10, v:v:v) (Teeter and Meyerhoff, 2003), and acetoni- matrices to a concentration of 20 mg/L. The proportion of forms in trile–isopropyl alcohol (95:5, v:v) (Shang et. al., 2001). Extraction aqueous tylosin tartrate stock solution was approximately 93% A, recoveries, reported by the researchers, for the above extraction 5% D, 2% B, and 0.3% C. All containers were incubated in the dark solvent combinations, from soil or manure-soil mixtures, ranged at 22 6 18C, and aliquots were taken from the solutions periodically, between 61% and 96%. Additional testing was conducted to deter- under sterile conditions.
mine if the acetonitrile–isopropyl alcohol combination amended The toxicity of tylosin to slurry microbes was assessed by spiking with 5-M KOH (95:5:0.1, v:v:v) would be appropriate for use, CL slurry with 20 mg/L of tylosin. After 72 hours of incubation at because higher pH is often used to extract macrolides from tissues 228C, a 0.5-mL aliquot of slurry from the CL slurry assay was (Fedeniuk and Shand, 1998). Adjusting the solvent to above pH 9.4 inoculated to 100 mL of a minimal salts broth. The broth was was based on the assumption that tylosin's nonionized form may be incubated at 258C for seven days, and the growth of the slurry easier to extract and occurs at pH above its pKa of 7.4 (O'Neil et al., Water Environment Research, Volume 77, Number 1 2001). To evaluate each solvent combination, triplicate vials 50-min run. Column temperature was maintained at 408C. A cali- containing 20 mL of CL slurries were spiked with 400 lg tylosin bration curve for tylosin tartrate was prepared, showing a strong tartrate (20 mg/L) and 1 g sodium azide (50 g/L) and incubated at linear area response (R2 5 0.9997). Calibrations for tylosin A, B, C, 228C for 24 hours. The extraction procedure consisted of two cycles and D were made by assuming an equal-area-concentration of solvent extraction with sonication and shaking. To examine response for all forms (Teeter and Meyerhoff, 2003). Retention tylosin stability in the solvent, 400 lg tylosin tartrate and 1 g sodium times for tylosin forms B, D, C, and A were 21.2, 21.5, 21.8, and azide were added to 20 mL of each solvent combination, without 22.3 min, respectively. The detection limit for tylosin, with UV, manure slurry, and were subjected to two cycles of sonication under these settings, was approximately 500 lg/L. For the and shaking.
degradation studies, HPLC analysis was not sufficient to determine For the degradation studies, the slurry in each sacrificed vial was tylosin C in the slurry assays. Liquid chromatograph-mass transferred to a 50-mL polypropylene centrifuge tube. The amber spectrometry was used to verify the presence of tylosin A, B, C, vial was rinsed with 10 mL of Milli-Q purified water, and the rinse D, and dihydrodesmycosin, and to determine the molecular mass of water was transferred to the centrifuge tube. The slurry was centri- degradates observed in some samples. The limit of detection for fuged at 12 500 3 g for 30 minutes, resulting in a pellet weighing tylosin A, B, C, and D forms were each approximately 4 lg/L. Mass approximately 0.5 to 1 g and 20 to 30 mL of dark brown, trans- spectra of tylosin forms and degradates were acquired by positive lucent, aqueous supernatant. The supernatant, containing very small and negative ion electrospray on an Agilent 1100 HPLC configured amounts of particulate material, was decanted, measured, and as above, with the addition of 0.1% acetic acid to acetonitrile, retained. Then, 10 mL of acetonitrile–isopropyl alcohol (95:5 v:v) coupled to a 1100 mass selective detector (MSD) ion trap LC–MS– was added to each tube. The tubes were vortexed for 1 minute, MS (Agilent, Palo Alto, California). The drying gas was operated at sonicated (Bransonics Model 5200, Branson Ultrasonics Corp., a flowrate of 12 mL/min at 3508C. The nebulizer pressure was 50 Danbury, Connecticut) for 30 minutes, shaken for 1 hour at 270 psig, scanning mass from 50 to 1200 m/z. Quantification was based cycles per minute on a reciprocating shaker, and then centrifuged at on an external 15-point calibration from 8 to 800 mg/L of the base 12 500 3 g for 15 minutes. The solvent was decanted and retained peak ion (protonated adduct of the molecular ion) of the analyte. For separately from the aqueous supernatant. The procedure was re- each compound, the protonated molecular ion, [M 1 H]1, and at peated by adding another 10 mL of fresh solvent to the pellet and least one confirming ion was acquired, based on MS–MS of the the extract combined with the first 10 mL of solvent extract in an base-peak and fragment ratios formed from the standards and amber vial. The combined solvent extract was dried down to less confirmed by published spectra (Van Poucke et al., 2003 and 2004).
than 5 mL by passing compressed air over the solvent at room Ion suppression tests for the elution of four typical samples showed temperature. The concentrated solvent extract was then transferred no interference of tylosin A at the time window of interest.
to a 250-mL glass beaker, including 20 mL of Milli-Q (deionized) Disappearance Rate Modeling.
A two-compartment, first- water and 1 mL methanol rinse. The solvent extract was further order model was used to describe the total tylosin disappearance.
diluted to 140 mL with Milli-Q water, and the pH was adjusted This model assumes a pool of rapidly degrading tylosin (C1) and above pH 9.4 with 0.5-M KOH. Likewise, 2-mL of the decanted a pool of more persistent tylosin (C2).
aqueous supernatant was transferred to a 100-mL glass beaker anddiluted with 80 mL Milli-Q water. The pH of the dilution was raised Ct ¼ C1ek1t þ C2ek2t above 9.4 with 5-M KOH.
Both the decanted aqueous supernatant and the diluted solvent extract were extracted separately using solid-phase extraction (SPE),under vacuum, at a flowrate less than 5 mL/min. Oasis Hydrophilic- Ct 5 tylosin concentration (% of added) at time t; Lipophilic Balance (HLB) cartridges (200 mg) (Waters, Milford, C1 5 initial tylosin concentration (% of added) in pool 1; Massachusetts) were used and primed with 5 mL methanol, followed C2 5 initial tylosin concentration (% of added) in pool 2 (C1 1 by 4 mL 0.5-M KOH. Glass fiber filters (Fisher Scientific, Hampton, New Hampshire) (1-lm pore-size) were inserted to prevent the SPE k1 5 first-order rate constant (hour21) for pool 1; frit from clogging. The cartridges were rinsed with three 1-mL k2 5 first-order rate constant (hour21) for pool 2; and aliquots of methanol–water–ammonium hydroxide (60:38:2, v:v:v) to remove organic coextracted materials. Tylosin was eluted with The model was fit to the experiment data using nonlinear four 0.5-mL aliquots of acetonitrile–glacial acetic acid (98:2. v:v).
regression with SAS software (SAS, Cary, North Carolina). Note The tylosin-containing acetonitrile eluants were dried down that C2 was set as equal to the total added (100%) minus C1. If C2 or completely, under nitrogen gas at 228C, and reconstituted in 0.5 k2 is not significantly different from zero, the model becomes a mL of 0.01-M ammonium acetate (pH 6.8) to improve tylosin simple first-order model.
stability before analysis. The tylosin recovered from the supernatantand the pellet was summed for each slurry sample.
Tylosin Analysis.
Tylosin was analyzed using an Agilent 1100 Results and Discussion Series high-pressure liquid chromatograph (HPLC) (Agilent Tech- The characteristics of the two nologies, Palo Alto, California). Injection volume of samples was manure slurries are presented in Table 1. The pH for both manure 25 lL, and UV detection wavelength for all tylosin forms was slurries were similar, ranging between 8.5 to 9.1, before incubation.
284.8 nm. A ZORBAX SB-C18 4.6 3 250 mm column was used Background concentrations of tylosin B and D were approximately (Agilent Technologies). The eluants used were 0.01-M ammonium 50 and 15lg/L in OL, respectively, and 1700 and 270 lg/L in the acetate–glacial acetic acid buffer (pH 4.6) and acetonitrile, at a CL slurry, respectively. The OL slurries had higher total solids, constant flowrate of 0.5 mL per minute, starting with 10% acetonitrile organic carbon, and dissolved organic carbon as compared to CL increasing to 100% acetonitrile between 6 and 35 minutes for each slurry, but the total carbon values were similar.
January/February 2005 Table 1—Swine manure characteristics from an openanaerobic lagoon (OL) and a covered anaerobic lagoon(CL).
Tylosin B, D (lg/L) Total solids (g/kg) Organic carbon (g/kg) Phosphorus (g/kg) Tylosin Stability and Toxicity.
Tylosin tartrate was stable for at least one month when stored in Milli-Q water at pH 5.7 to 6.7at 22 6 18C. However, approximately 10% of added tylosin wasdegraded within the first 200 hours in Milli-Q water at pH 9.2.
Figure 3—Tylosin recovered (% of added, sum of tylosinA, B, and D) from (a) sterile OL lagoon liquid, (b)anaerobic OL slurry, and (c) aerated OL slurry. Error barsare 6 1 standard deviation. Initial spiked concentrationwas 20 mg/L as tylosin tartrate.
In the case of azide-amended Milli-Q water at pH 9.3, the amountdegraded was also approximately 10%.
Approximately 20 and 5% of added tylosin was degraded within the first 72 hours for the CL and OL 0.2-lm filtered lagoon liquids,respectively (see Figures 2a and 3a). In azide-amended and un-amended Milli-Q water samples and both filtered lagoon liquids, anunknown degradate with molecular mass of m/z 934.5 appeared inan apparently base-catalyzed reaction (O'Neil et al., 2001; Paesenet al., 1995). The addition of sodium azide in the assays did notappear to influence tylosin stability.
The tylosin toxicity test showed that tylosin was not effective in suppressing microbial viability at 20 mg/L. The minimal salts brothbecame turbid after one week of incubation, and actinomycetes,amoebae, fungi, and bacteria were observed by light microscopy.
Figure 2—Tylosin recovered (% of added, sum of tylosin This is consistent with previous studies, where tylosin was found to A, B, and D) from (a) sterile CL lagoon liquid, (b) be active against gram-positive bacteria, but was only marginally anaerobic CL slurry, and (c) aerated CL slurry. Error bars active on gram-negative bacteria (Prescott, 2000).
are 6 1 standard deviation. Initial spiked concentration Sodium azide was found to be effective at was 20 mg/L as tylosin tartrate.
inhibiting microbial growth in the CL slurry, at a concentration of Water Environment Research, Volume 77, Number 1 Table 2—Two-compartment model of tylosin disappearance in two manure slurries (CL and OL) under aerobic oranaerobic conditions, with or without sodium azide. Initial tylosin concentrations were 20 mg/L, except for OL anaerobichigh treatment, which received 195 mg/L of tylosin. Rate constants are given in hours with 95% confidence intervals.
Manure or treatment OL anaerobic high a k2 not significantly different from zero.
b C2 percentage tylosin not significantly different from zero.
50 g/L. Colonies or growth were absent from plates incubated for 72 hours incubation. Percent recoveries of tylosin (sum of forms 96 hours with the azide-amended slurry.
A, B, and D) with time for CL and OL slurries spiked with 20 mg/L Tylosin concentrations are reported as of tylosin are presented in Figures 2b and 3b, respectively. In both the sum of the A, B, and D forms recovered from the separate CL and OL slurries, there was a rapid disappearance of tylosin (60 analysis of slurry supernatant and slurry solid phase. Recoveries are to 85%) within the initial 24 hours, after which the disappearance expressed as a percentage of the tylosin added to the samples, after of tylosin slowed in both the CL and OL slurries.
correction for tylosin concentrations present in the slurry at the time The two-compartment model (eq 1) was used to model the of collection. The average recoveries from CL and OL slurry using disappearance of tylosin in the OL and CL slurries. A first-order acetonitrile–isopropyl alcohol were 99% (8% RSD, n 5 12) and 93% model was initially used, but was found to overestimate the amount (13% RSD, n 5 12), respectively, for vials sacrificed within 0.5 of tylosin in anaerobic samples during the first 24 hours and hours of spiking. Recoveries for acetonitrile–isopropyl alcohol underestimate the concentrations after 24 hours. The estimated averaged approximately 41% after 24 hours in CL. Methanol and tylosin disappearance rate constants, k1 and k2 and C1 and C2 for the acidified methanol each gave an average 38% recovery after 24 anaerobic studies using OL and CL slurries, are summarized in hours. Adding KOH to acetonitrile–isopropyl alcohol produced a Table 2. The k1 rate constants for the azide-amended and un- higher proportion of tylosin B to tylosin A in the extracts. Because amended OL and CL slurries were not significantly different, at a tylosin stability was desirable during extraction, the addition of KOH confidence interval of 95%. This implies that the rapid disappear- may not be appropriate. Of the three extraction solvents, acetonitrile– ance of tylosin within the first 24 hours may be because of sorption isopropyl alcohol (Shang et al., 2001) was chosen as the extractant of tylosin to the solids. Substantial sorption of tylosin (90 to 99%) also because of its use in the LC–MS–MS analysis. In addition, the has been reported within 1 to 6 hours after spiking in soil and SPE method outlined above for extracting tylosin forms and manure mixtures (Ingerslev and Halling-Sørensen, 2000).
degradates was found to produce enhanced HPLC-baseline resolu- The k1 rate constants for the OL slurries were two to three times tion as compared to other similar methods, which used acidic or non- larger than those for the CL slurries, indicating that tylosin pH modified rinses (Kolpin et al., 2002; Teeter and Meyerhoff, disappearance was more rapid and residues were lower in OL slurry.
2003). Oasis HLB cartridges provided recoveries of 96 to 105% from Hydrophobic binding behavior has been related to sorption of tylosin aqueous tylosin tartrate solutions and were resistant to pH changes (Loke et al., 2002; Tolls, 2001). The OL slurry had higher total solids up to pH 14, necessary to optimize the tylosin SPE procedure.
and organic carbon content than CL, which supports sorption as Anaerobic Studies.
After 72 hours of incubation, the pH of the a process contributing to tylosin disappearance. Teeter and Meyerh- slurries changed from 8.5 to 9.1 to approximately 8.2 to 8.5 and off (2003) reported approximately 30% of the 14C-labeled tylosin dropped further to pH 7.5 to 7.8 after eight months of incubation.
accumulated as bound (nonextractable) residues in manure.
The addition of azide decreased pH of slurries by approximately 0.2 In the azide-amended CL slurry, the k2 rate constant was 0.003 pH units. The ORP in slurries was between 210 and 280 mV EH h21, and the estimated C2 value was 56%, showing that the tylosin after 72 hours of incubation. The addition of azide resulted in a continued to slowly disappear with time. For the unamended CL further decrease of the ORP to between 290 and 2160 mV EH after slurry, the k2 rate constant was 0.004 h21, with a lower C2 value of January/February 2005 making the disappearance follow a first-order model. The estimatedk1 rate constants and C1 and C2 values are presented in Table 2. Forthe azide-amended and unamended OL slurries, the k1 rate constantswere not significantly different. A similar assessment can be madefor the k1 rate constants for the CL slurries. However, the k1 rateconstants for OL were approximately 2 to 3 times larger than the CLslurries. Unlike the anaerobic studies, there was a lower amount ofresidual tylosin remaining at the end of the 72 hours. Less than 1%of the tylosin added remained after 12 days of aeration in OL slurry.
The 90% disappearance time for the OL aerated slurries was 12hours, as compared to 40 hours for the OL anaerobic slurries. ForCL, the 90% disappearance times were 26 and 310 hours for theaerobic and anaerobic slurries, respectively.
Disappearance of tylosin in manure slurries can be attributed to biotic and abiotic degradation and to irreversible sorption (i.e., theformation of nonextractable bound residues). Assuming that theazide was effective in inhibiting microbial activity, the similar k1rate constants for azide-amended and unamended slurries indicatethat the portion of the initial tylosin disappearance attributed tobiodegradation was quite small. In addition, the magnitude ofabiotic degradation was small, with only 5 to 20% of the tylosindegraded in sterile filtered lagoon liquids. Therefore, much of thetylosin disappearance may be because of sorption, particularly in theanaerobically incubated slurries. Aeration increased slurry pH to 9.2 Figure 4—Tylosin forms A, B, D, and unknown degradate to 9.3, which may have accelerated a base-catalyzed reaction and (m/z 934.5) recovered at various times for OL lagoon resulted in lower residuals in aerated slurries. Faster disappearance slurry spiked with 195 mg/L tylosin and incubated rates in aerated samples correspond well to findings by other anaerobically. Average recovery is expressed as the researchers in aerated and anaerobic surface water simulations and percent of tylosin added. Degradate recovery is based soil-manure slurries studies (Ingerslev and Halling-Sørensen, 2001; on the UV peak response for tylosin.
Ingerslev et al., 2001). In these systems, aeration decreased tylosin'shalf-life by 30 days when compared with anaerobic incubation andeliminated detectable residuals in soil-manure slurries within 12 to 39%. The difference in the estimated C2 values for the two CL slurries treatments indicated that there was less residual tylosin in the Tylosin Degradates.
The proportions of the different forms of unamended CL slurry and may imply that biodegradation was tylosin shifted through the tests, and some changes occurred occurring in the unamended CL slurry. The OL slurries behaved in a immediately. The proportion of tylosin forms added to the lagoon similar manner to the CL slurries, with respect to the azide treatment, slurries at the initiation of the experiments was approximately 93% but the estimates of C2 were less in the OL slurry than in the CL.
A, 5% D, 2% B, and 0.3% C. Within 0.5 hours after spiking, the Half-lives may be used to compare the differences in the various average proportion of forms A, D, B, and C in OL slurry spiked at treatments; however, because the disappearance of tylosin was very 195 mg/L was 77% A, 13% D, 9% B, and 0.2% C (Figure 4).
rapid, the time for 90% disappearance of tylosin was chosen as a Similar changes in the proportions of tylosin forms were also noted comparison. The estimated time necessary for 90% tylosin dis- in OL and CL samples after spiking 20 mg/L of tylosin. Tylosin A appearance was 40 and 310 hours for unamended OL and CL decreased more rapidly than D, and both decreased more rapidly slurries, respectively. The 90% disappearance times for OL and CL than tylosin B. Tylosin B concentrations decreased in most slurries, azide-amended slurries were 90 and 500 hours, respectively, except in anaerobically incubated CL slurry, where concentrations indicating that faster disappearance occurred in the unamended of B doubled during the first 72 hours. Tylosin B was the most predominant form in anaerobically incubated OL slurry, but this For the anaerobic OL slurry treated with 195 mg/L tylosin, the k1 appears to be because of its increased relative persistence, as all rate constant and the percent recovery were similar to that with forms decreased in concentration. The production of tylosin B was 20 mg/L of tylosin. The estimated 90% disappearance time was reported in aqueous anaerobically incubated manure mixtures and also similar to that with 20 mg/L tylosin.
aerated soil manure slurries (Ingerslev and Halling-Sørensen, 2001; Aerobic Studies.
The pH in the aerated slurries increased from Loke et al., 2000). Tylosin D was also reported in these studies, but 9.1 to 9.3 after 24 hours of aerobic incubation and remained above to a lesser extent than tylosin B. The highest relative proportion of pH 9.0 after 12 days of aeration. Addition of sodium azide tylosin D in the current research was in aerated CL slurry incubated decreased the slurry pH by approximately 0.1 to 0.2 pH units. After for 72 hours, with 36% A, 36% D, and 27% B. Aerobically in- aeration, the ORP was approximately 1340 mV EH. The ORP cubated OL slurry produced the smallest shifts in proportion of decreased by approximately 10 mV EH in the aerated samples when forms, with tylosin present as 93% A, 3% D, 4% B, and 0% C. After azide was added.
72 hours, tylosin C was not detected in any of the samples.
As in the anaerobic studies, there was a rapid disappearance of Dihydrodesmycosin was not detected in slurry-source materials, but tylosin. The disappearance of tylosin was almost completely increased to trace amounts during the first 72 hours of aerated and described by the first term of the two-compartment model (eq 1), anaerobic incubation. Dihydrodesmycosin eluted at 20.2 minutes on Water Environment Research, Volume 77, Number 1 spectral fragmentation pattern for the degradate is compared withthat of tylosin A in Figure 5. The degradate also responded undernegative ionization mode, indicating that the compound hadamphoteric properties. The mass spectral fragmentation patternand peak UV absorbance of the degradate (285 to 290 nm) arestrongly suggestive of a relation to tylosin A plus m/z 18.
Studies in manure-lagoon slurries indicated that the majority of tylosin was rapidly sorbed and degraded, with 90% disappearanceoccurring in less than five days. Both abiotic and biotic degradationwere apparent. However, disappearance in anaerobic slurries slowedafter 24 hours, and a residual pool of tylosin remained through theextended-anaerobic incubation. Aerating slurries significantly re-duced tylosin residuals, leaving less than 1% of the added tylosinafter 12 days. Tylosin B and D, which retain approximately 35 to80% of the antibiotic activity of tylosin A (Teeter and Meyerhoff,2003), were detected in slurry-source materials. Tylosin B and Dwere produced during the studies and remained after eight monthsof anaerobic incubation, in conventional-lagoon slurry. Dihydro-desmycosin and a degradate with undetermined antibiotic activityalso formed during the experiments and persisted for eight monthsafter tylosin addition. Based on these findings, residual tylosindegradates, with antibiotic activity, may be applied to agricultural Figure 5—Liquid chromatography with tandem mass fields with slurries and contribute to the detection of antibiotic spectrometry fragmentation patterns of (a) tylosin A residues near CAFOs.
(m/z 916.5), (b) unknown degradate (m/z 934.5), and(c) UV chromatograms of tylosin in CL slurry at 0.5 and 72 hours [in milliAbsorbance units (mAu)].
Funding for this work was provided by a grant from the Iowa State Water Resources Research Institute, Ames, Iowa.
Mention of trade names or commercial products herein is solely for chromatographs. Azide amendment did not significantly change the the purpose of providing specific information and does not imply proportion of tylosin forms recovered in samples during short-term recommendation or endorsement by the U.S. Department of Agriculture (Washington, D.C.).
In the long-term anaerobic tests using OL slurries, approximately Angela C. Kolz is a research assistant and Say Kee 1% of the tylosin remained after eight months incubation. The Ong is an associate professor in the Department of Civil, tylosin forms detected after eight months were generally tylosin B Construction and Environmental Engineering, Iowa State University, and D. Comparison of the azide-amended and unamended slurries Ames, Iowa. Thomas B. Moorman is a microbiologist, Kenwood D.
indicated that there was a greater amount of tylosin B, D, Scoggin is a research technician, and Elizabeth A. Douglass is dihydrodesmycosin, and an unknown degradate (described below) a research technician at the USDA-Agricultural Research Service, in the azide-amended slurry than in the unamended slurry.
National Soil Tilth Laboratory, Ames, Iowa. Correspondence should A degradate, eluting at 20.6 mi- be addressed to Say Kee Ong, 394 Town Engineering Building, Iowa nutes, appeared within 12 hours after spiking in all unamended and State University, Ames, Iowa 50011; e-mail: [email protected].
azide-amended anaerobic and aerobically incubated assays. Neither Submitted for publication May 4, 2004; revised manuscript aeration nor sodium azide affected the amount of degradate submitted November 1, 2004; accepted for publication November 1, production. The degradate compound was not detected in slurry- source materials before tylosin addition or in tylosin tartrate The deadline to submit Discussions of this paper is May 15, standards at pH 7.0, but it did appear in sterile lagoon liquids and water at pH 9.2. Recovery of tylosin forms A, D, B, and thedegradate are shown in Figure 4. Recovery of the degradate is basedon an assumption of equal UV area response as tylosin. Typical UV chromatographs at 0.5 and 72 hours show the transformation of Boxall, A. B. A.; Fogg, L.; Blackwell, P. A.; Kay, P.; Pemberton, E. (2001) tylosin A, D, and B, and production of this degradate over time Review of Veterinary Medicines in the Environment; Environment Agency Report P6-012/8TR; U.K. Environment Agency: Bristol, The base peak in the spectrum of the degradate was m/z 934.5, United Kingdom.
Campagnolo, E. R.; Johnson, K. R.; Karpati, A.; Rubin, C. S.; Kolpin, and a major fragment ion was found at m/z 772.5 when analyzed D. W.; Meyer, M. T.; Esteban, J. E.; Currier, R. W.; Smith, K.; Thu, with LC–MS–MS, under positive ion mode. A low-abundance K. M.; McGeehin, M. (2002) Antimicrobial Residues in Animal Waste fragment ion, at m/z 916.5, was also detected. Tylosin A's and Water Resources Proximal to Large-Scale Swine and Poultry characteristic fragment ion of its 916.5 [M 1 H]1 mass under Feeding Operations. Sci. Total Environ., 299, 89–95.
positive ionization mode was 772.5 [M 1 H 2 C7H12O3]1, Chee-Sanford, J. C.; Aminov, R. I.; Krapac, I. J.; Garrigues-Jeanjean, N.; indicating the loss of the mycarose sugar. The MS–MS mass Mackie, R. I. (2001) Occurrence and Diversity of Tetracycline January/February 2005 Resistance Genes in Lagoons and Groundwater Underlying Two Swine Prescott, J. F. (2000) Lincosamides, Macrolides, and Pleuromutilins. In Production Facilities. Appl. Environ. Microbiol., 67, 1494–1502.
Antimicrobial Therapy in Veterinary Medicine, 3rd Ed; Prescott, J. F.; Daughton, C. G.; Ternes, T. A. (1999) Pharmaceuticals and Personal Care Baggot, J. D.; Walker, R. D. (Eds.); Iowa State University Press: Ames, Products in the Environment: Agents of Subtle Change? Environ.
Iowa, 238–244.
Health Persp., 107 (Supplement 6), 907–938.
Rabølle, M.; Spliid, N. H. (2000) Sorption and Mobility of Metronidazole, De Liguoro, M.; Cibin, V.; Capolongo, F.; Halling-Sørensen, B.; Olaquindox, Oxytetracycline and Tylosin in Soil. Chemosphere, 40, Montesissa, C. (2003) Use of Oxytetracycline and Tylosin in Intensive Calf Farming: Evaluation of Transfer to Manure and Soil. Chemo- Shang, D.; Dyck, M.; Dassie, N.; Gibbons, N.; Alleyne, C.; Nicolidakis, H.; sphere, 52, 203–212.
Jia, X. (2001) Rapid Determination of Tylosin and Virginiamycin in Fedeniuk, R. W.; Shand, P. J. (1998) Theory and Methodology of Antibiotic Swine Manure by APcI LC/MS/MS. Proceedings of the 49th Extraction from Biomatrices. J. Chromatogr., A, 812, 3–15.
Conference on Mass Spectrometry and Allied Topics, Chicago, Illinois, Haller, M. Y.; Mu¨ller, S. R.; McArdell, C. S.; Alder, A. C.; Suter, M. J.-F.
May 27—31; American Society for Mass Spectrometry: Santa Fe, New (2002) Quantification of Veterinary Antibiotics (Sulfonamides and Trimethoprim) in Animal Manure by Liquid Chromatography-Mass 15 July 2004).
Spectrometry. J. Chromatogr., A, 952, 111–120.
Sieck, R. F.; Graper, L. K; Giera, D. D.; Herberg, R. J.; Hamill, R. L. (1978) Ingerslev, F.; Halling-Sørensen, B. (2001) Biodegradability of Metronida- 14C Tylosin Tissue Residue Study in Swine. Unpublished report dated zole, Olaquindox, and Tylosin and Formation of Tylosin Degradation November 1978 from Agricultural Biochemistry, Lilly Research Products in Aerobic Soil-Manure Slurries. Ecotoxicol. Environ. Safety., Laboratories, Greenfield, Indiana. Submitted to WHO by Lilly Research 48, 311–320.
Centre Ltd., Windlesham, Surrey, England; http://www.inchem.org/ Ingerslev, F. T.; Loke, M.-L.; Halling-Sørensen, B.; Nyholm, N. (2001) documents/jecfa/jecmono/v29je08.htm (accessed 15 July 2004).
Primary Biodegradation of Veterinary Antibiotics in Aerobic and Teeter, J. S.; Meyerhoff, R. D. (2003) Aerobic Degradation of Tylosin in Anaerobic Surface Water Simulation Systems. Chemosphere, 44, Cattle, Chicken, and Swine Excreta. Environ. Res., 93, 45–51.
Tolls, J. (2001) Sorption of Veterinary Pharmaceuticals in Soils: A Review.
Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Environ. Sci. Technol., 35, 3397–3406.
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filelibrary/5049/678.pdf (accessed 15 July 2004).
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