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The effect of composting on the persistence of four ionophores in dairy manure and poultry litter
Waste Management xxx (2016) xxx–xxx
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The effect of composting on the persistence of four ionophores in dairy manure and poultry litter Osman A. Arikan a,b,⇑, Walter Mulbry a, Clifford Rice a a b
USDA-ARS, Beltsville Agricultural Research Center, Sustainable Agricultural Systems Laboratory, Beltsville, MD 20705, USA Istanbul Technical University, Department of Environmental Engineering, Istanbul 34469, Turkey
a r t i c l e
i n f o
Article history: Received 29 September 2015 Revised 27 April 2016 Accepted 28 April 2016 Available online xxxx Keywords: Ionophore Composting Temperature Dairy manure Poultry litter
a b s t r a c t Manure composting is a well-described approach for stabilization of nutrients and reduction of pathogens and odors. Although composting studies have shown that thermophilic temperatures and aerobic conditions can increase removal rates of selected antibiotics, comparable information is lacking for many other compounds in untreated or composted manure. The objective of this study was to determine the relative effectiveness of composting conditions to reduce concentrations of four widely used ionophore feed supplements in dairy manure and poultry litter. Replicate aliquots of fresh poultry litter and dairy manure were amended with monensin, lasalocid, salinomycin, or amprolium to 10 mg kg 1 DW. Nonamended and amended dairy manure and poultry litter aliquots were incubated at 22, 45, 55, or 65 °C under moist, aerobic conditions. Residue concentrations were determined from aliquots removed after 1, 2, 4, 6, 8, and 12 weeks. Results suggest that the effectiveness of composting for contaminant reduction is compound and matrix specific. Composting temperatures were not any more effective than ambient temperature in increasing the rate or extent of monensin removal in either poultry litter or dairy manure. Composting was effective for lasalocid removal in poultry litter, but is likely to be too slow to be useful in practice (8–12 weeks at 65 °C for >90% residue removal). Composting was effective for amprolium removal from poultry litter and salinomycin in dairy manure but both required 4–6 weeks for >90% removal. However, composting did not increase the removal rates or salinomycin in poultry litter or the removal rates of lasalocid or amprolium in dairy manure. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Ionophores are commonly used in livestock production as coccidiostats as well as for growth promotion (Duffield and Bagg, 2000; Callaway et al., 2003; Sassman and Lee, 2007; Clarke et al., 2014). Ionophores are toxic to both prokaryotic and eukaryotic cells because these compounds interfere with the formation of ion gradients across cell membranes (reviewed in Hansen et al., 2009a). Ionophores are toxic to human cells and are therefore not used except for veterinary purposes. According to a Food and Drug Administration (FDA) report, >4500 tons of ionophores were sold for use in livestock in the United States in 2012 (FDA, 2014). Since ionophores aren’t used in humans, land application of ionophore-containing manure does not pose a potential threat to public health by influencing the development of antibiotic resistance in human pathogens. Many ruminal bacteria are ionophore⇑ Corresponding author at: Istanbul Technical University, Department of Environmental Engineering, Istanbul 34469 Turkey. E-mail address: [email protected] (O.A. Arikan).
resistant but there is little evidence that ionophore resistance can be spread from one bacterium to another (Russell and Houlihan, 2003). Ionophore use is unlikely to lead to resistance in people as this resistance tends to develop more slowly, and may well be a reversible change (O’Neill, 2015). However, these compounds inhibit the activity of many bacteria and eukaryotic cells and can affect soil invertebrates and aquatic organisms (Hansen et al., 2009b; Zˇizˇek et al., 2011). Development of resistance against ionophores and cross-resistance in bacteria to more than one ionophore have also been observed (VKM, 2015). Results for raw vegetables indicate that coccidiostats can be incorporated into vegetables from the soil although detected concentrations are relatively low (Peteghema et al., 2012). Therefore, application of manure with ionophores is a considerable concern. Ionophores have been primarily studied in manure (Donoho, 1984; Schlüsener et al., 2003; Sassman and Lee, 2007; Storteboom et al., 2007; Watanabe et al., 2008; Varel et al., 2012) poultry litter (Webb and Fontenot, 1975; Furtula et al., 2009; Ramaswamy et al.; 2010; Biswas et al., 2012; Bak et al., 2013; Zˇizˇek et al., 2015) and soil (Sassman and Lee, 2007; Bak et al.,
http://dx.doi.org/10.1016/j.wasman.2016.04.032 0956-053X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
2013; Zˇizˇek et al., 2015). Although reported half-lives for these compounds in soils are very short (typically 2–4 days) (Sassman and Lee, 2007; Zˇizˇek et al., 2015) ionophores have been detected in surface water (Cha et al., 2005; Kim and Carlson, 2006; Hao et al., 2006, 2008; Song et al., 2007; Kurwadkar et al., 2013; Bak et al., 2013), groundwater (Watanabe et al., 2008) and sediment (Kim and Carlson, 2006). Hansen et al. (2009a) suggest that ionophores constitute an environmental risk because predicted environmental concentrations of ionophores and their measured concentrations in sediments could be above predicted no-effect levels. Treatment of ionophore-containing manure prior to land application is one possible means of reducing the amount of these compounds that is released into the environment. Manure composting is a treatment process that is widely used for stabilization of nutrients and reduction of pathogens and odors (US Composting Council, 2000). The major factors that affect composting are oxygen, moisture, temperature, carbon and nitrogen (Epstein, 2011). Mean temperatures between 45 and 65 °C can be achieved during composting by applying different intensities of composting practices (Arikan et al., 2009a; Dolliver et al., 2008). Previous laboratory and field-scale composting studies (Arikan et al., 2009a,b; Dolliver et al., 2008) have shown that thermophilic temperatures (50–60 °C) and aerobic conditions rapidly reduce chlortetracycline levels in dairy manure and turkey litter. However, there is limited information on the effect of composting on ionophores, especially among the range of temperatures expected within minimally managed compost piles. The specific objective of this study was to determine the relative effectiveness of composting temperatures to reduce the concentrations of four widely used ionophores (monensin, lasalocid, salinomycin, amprolium) (Mellon et al., 2001). Laboratoryscale incubations were conducted using both dairy manure and poultry litter to determine the influence of the composting matrix on removal rates. Replicate aliquots of fresh poultry litter and dairy manure were amended with monensin, lasalocid, salinomycin, or amprolium to 10 mg kg 1 DW. Non-amended and amended dairy manure and poultry litter aliquots were incubated for 12 weeks under moist, aerobic conditions at 22 °C or at three elevated temperatures (45, 55, or 65 °C) that represent temperatures achievable at different levels of composting intensity. Based on our previous results with tetracyclines and from results from other studies with monensin, we predicted that temperature would influence ionophore removal rates with higher temperatures leading to higher removal rates and extent of removal. In addition, we predicted that removal rates would vary between ionophores and between poultry litter and dairy manure. We also predicted that the influence of temperature on removal rates would be similar in both matrices. The results show that ionophore removal rates were influenced by the specific ionophore, temperature, and matrix. However, in some instances, the influence of temperature on ionophore removal varied between dairy manure and poultry litter samples. 2. Materials and methods 2.1. Chemicals Monensin sodium salt, lasalocid A sodium salt, salinomycin mono-sodium salt hydrate and amprolium hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Structures of these compounds are shown in Fig. 1. HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Pittsburg, PA). All reagents used in this study were analytical grade. Water used for extractions was purified using reverse osmosis and activated carbon. Stock solutions of the ionophore standards for extraction and analysis were prepared monthly by dissolving each
Monensin (Mw= 671 g mol-1, pKa = 6.7, log Kow = 2.8-4.2)
Lasalocid (Mw= 591 g mol-1, pKa = 4.4, log Kow = 1.4-2.8)
Salinomycin (Mw= 751 g mol-1, pKa = 4.5, 6.4, log Kow = 5.2)
Amprolium hydrochloride (Mw= 278 g mol-1, log Kow = -2.5) Fig. 1. Structures (adapted from Clarke et al., 2014) and selected physicochemical properties (adapted from Hansen et al., 2009b; Ding, 2011) of ionophores used in this study.
compound in methanol at a concentration of 100 mg L stored at 20 °C in the dark.
1
and
2.2. Dairy manure and poultry litter Fresh dairy manure was collected from the USDA’s Beltsville Dairy Research Unit (Beltsville, Maryland). Manure was air-dried in a fume hood for 48 h to a moisture content of 6%. The dried manure was ground using a hammer mill to pass a 4.7 mm sieve and stored at 4 °C in a covered container prior to use. Poultry litter was obtained from a covered storage pile at the University of Maryland Eastern Shore’s broiler production facility (Princess Anne, Maryland). The moisture content of the litter as received was 16%. The litter was sieved to remove feathers and large debris. Material that passed a 4.7 mm sieve was collected and stored in a covered container at 4 °C prior to use. The collected dairy manure and poultry litter samples were analyzed for ionophores using the method below and found to contain some of the studied ionophores (shown in Table 3).
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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2.3. Incubation experiment Individual stock solutions of monensin, lasalocid, and amprolium for amending dairy manure and poultry litter were prepared in methanol at 1 g L 1. Amended batches of dairy manure or poultry litter were individually prepared by diluting 1.5 mL of stock solution (containing 1.5 mg of ionophore) with 50 mL of ethyl acetate and stirring the diluted mixture into 150 g DW batches of dairy manure or poultry litter in 4 L glass beakers. Amended batches of manure or litter were stored in open metal trays in a fume hood overnight in order to volatilize ethyl acetate and methanol. During composting, the rate of microbial activity decreases when the moisture level in compost is below 40% (Epstein, 2011). Typical poultry litter is generally dry and water is added at the beginning of poultry litter composting. It is possible that microbial populations in the amended dairy manure may have been reduced by drying and/or exposure to the methanol/ethyl acetate mixture in the steps described above. In order to mitigate this possibility, batches of dried non-amended and ionophoreamended dairy manure were brought up to a moisture content of 65–70% by the addition of fresh solids-separated dairy manure effluent. For the same reason, batches of non-amended and ionophore-amended poultry litter were brought up to a moisture content of 65–70% by the addition of an aqueous extract of fresh poultry litter (100 g litter mixed with 1 L distilled water). A stock solution of salinomycin was prepared in dimethyl sulfoxide at 1 g L 1. A 1.5 mL aliquot of the stock solution (containing 1.5 mg salinomycin) was diluted with 360 mL of solids-separated dairy manure liquid and this mixture was used to amend a 150 g DW batch of air-dried dairy manure. Similarly, a 1.5 mL aliquot of the salinomycin stock solution was diluted with 270 mL of a poultry litter extract and was used to amend a 150 g DW batch of poultry litter. Aliquots of amended and non-amended manure and poultry litter (14.5 g WW, corresponding to 5 g DW) were secured within 15 15 cm squares of color coded nylon mesh using plastic wire ties. Two samples from each treatment were randomly chosen as untreated control samples and were stored at 20 °C prior to extraction and analysis. The remaining mesh bags were placed in sealed 4 L glass jars that were placed in incubators set at 22, 42, 55, or 65 °C. Oxygen and moisture were controlled during the study. Jars were periodically flushed with air to maintain aerobic conditions (flushed five minutes each day for the first two weeks, flushed every two days for weeks 3–4, and flushed every 3 days for weeks 4–12). Sample moisture was maintained by adding water to the jars. Samples (single amended samples and triplicate non-amended samples for each treatment at each time-point) were removed after 1, 2, 4, 6, 8 and 12 weeks, weighed, and stored at 20 °C prior to extraction and analysis. Sample dry weights were determined prior to extraction for calculation of losses of compost mass and ionophore masses during the incubations. Dairy manure and poultry litter characteristics at the beginning of the incubation are shown in Table 1.
Table 1 Dairy manure and poultry litter characteristics at the beginning of the incubation period.a
a
Constituent
Dairy manure
Poultry litter
pH Moisture content, (%) Volatile solids, (% of dry matter) C, (%, wet basis) N, (%, wet basis) C/N
8.6 ± 0.1 71.7 ± 1.4 89.1 ± 0.8 12.1 ± 0.7 0.6 ± 0.1 20.2
8.9 ± 0.1 66.6 ± 4.0 80.9 ± 3.0 12.6 ± 2.0 1.6 ± 0.3 7.9
Values are means ± standard deviation of triplicate samples.
Removal of ionophores was assumed to follow first-order kinetics. A rate constant, k, was determined as the slope of the curve calculated by linear regression. The half-life, t1/2, was then calculated as t1/2 = ln(2)/k. 2.4. Extraction of compounds Frozen samples were dried by lyophilization for 72 h at 50 °C prior to extraction. Aliquots (0.5 g) of lyophilized samples were moistened by the addition of 200 ll of de-ionized water and equilibrated for 10 min. Moistened samples were extracted two times with 12 mL methanol by vortexing for 15 s followed by sonication for 15 min in a sonication bath (Elma, E100H, Elmasonic, Singen, Germany). After each extraction, the extracts were subjected to centrifugation (1200g, 5 min). The resulting supernatants were pooled, brought up to a total volume of 25 mL with methanol, and mixed for 30 s prior to transferring an aliquot to amber autosampler vials for analysis by LC/MS/MS. 2.5. LC/MS/MS analysis The LC instrument was a Waters 2690 XE (Waters Corp., Milford, MA) separations module with an XBridge C18 column (150 mm 2.1 mm i.d., 5 lm) (Waters Corp., Milford, MA) in conjunction with an XBridge C18 guard column at 50 °C. The injection volume was 10 ll. A gradient separation was utilized involving a mixture of solvent A (70:30, 0.5% formic acid:acetonitrile) mixed with solvent B (100% de-ionized water) and solvent C (100% acetonitrile). The gradient program was as follows: initial 2 min. hold with 70:30, A:B at a flow of 0.2 mL min 1; then the flow is increased to 0.3 mL min 1 and a non-linear gradient was started with a setting of 8 transitioning to 2:98, A:C. This setting was maintained for 12 min. The column was returned to initial conditions in 1 min and stabilized for 8 min before the next run is started. Atmospheric pressure ionization-tandem mass spectrometry was performed on a benchtop triple mass spectrometer (LC Quattro Ultima, Waters Corp.) operated in electrospray positive ionization mode. The source parameters were as follows: capillary voltage was set at 3.5 kV and extractor voltage was set at 3 V, respectively; rf lens at 0.1 V; source and desolvation temperatures were 150 and 450 °C. Liquid nitrogen was used to supply the nebulizer and desolvation gas (flow rates were approximately 80 and 400 L h 1, respectively). Argon was used as collision-induced decomposition gas to fragment the parent ions; the typical pressure was 2.6 10 3 mbar. Both high and low mass resolutions were set at 12.0 for both quadrupoles. Acquisition was performed using the multiple-reaction monitoring mode (MRM) in electrospray positive (ES+). The parent and daughter ions used for compound identification and quantitation are listed in Table 2 along with the optimum cone voltages and collision energies used. Optimization was performed by infusion of the standards from a syringe pump (10 ll/min) mixed with the LC effluent (100% A; 200 ll min 1), with high- and low-mass resolution set at 15.0. The detector was a photomultiplier set at 650 V. Analyte concentrations were calculated by the external standard method using Table 2 Parent and daughter ions used for quantitation of ionophores and MS parameters used to produce them. Compound
Parent ion, Daughter ion, Retention time, Cone, Collision, Da Da min V eV
Monensin 693.7 Lasalocid 613 Salinomycin 773.5 Amprolium 150
479.8 377 431 81
10.4 12.5 13.1 2
124 115 115 63
58 45 61 26
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
five point standard calibration curves (fits with > 0.9 r2 values). Peak integration and quantitation were performed automatically using MassLynx 4.0 software (Waters Corp.). 3. Results and discussion
Sample mass loss was slightly influenced by incubation temperature but was not influenced by ionophore amendment (not shown). Mass loss values (30–35% DW loss after 12 weeks) were in the range of values reported for composted samples (Arikan et al., 2007). Ionophore levels were corrected for sample mass loss and are shown in terms of mass rather than concentration.
3.1. Sample amendment, recoveries and compost mass loss
Matrix
Lasalocid
Salinomycin
Amprolium
Dairy manure, non-amended (mg kg 1 DW) 1.0 ± 0.5 (lg) in 5 g (DW) 4.1 ± 2.3 sample
3.3 ± 0.5 14.2 ± 2.3
<0.1 <0.5
<0.1 <0.5
Dairy manure, amendedb (mg kg 1 DW) 6.8 (lg) in 5 g (DW) 29.0 sample
10.3 45.2
Dairy manure, net (mg kg 1 DW) (lg) in 5 g (DW) sample
5.8 24.9
7.0 31.0
10 43.3
3.6 15.8
58
70
100
36
Dairy manure, recovery (%)
Monensin
10.0 43.3
3.6 15.8
Poultry litter, non-amended (mg kg 1 DW) 0.9 ± 0.1 (lg) in 5 g (DW) 5.1 ± 0.2 sample
<0.1 <0.5
Poultry litter, amendedb (mg kg 1 DW) 3.2 (lg) in 5 g (DW) 17.9 sample
9.8 55.5
Poultry litter, net (mg kg 1 DW) (lg) in 5 g (DW) sample
2.3 12.8
9.8 55.5
7.9 44.9
2.6 14.0
23
98
79
26
Poultry litter, recovery (%)
5.3 ± 2.2 28.6 ± 10.7
13.2 73.5
<0.1 <0.5
2.6 14.0
3.2.1. Monensin Results show that ambient temperature treatment was as or more effective than composting temperatures in reducing
Monensin (% of initial mass)
150
22 oC
A
45 oC 55 oC
100
65 oC
50
0
Monensin (% of initial mass)
150
2
4
6
8
10
12
2
4
6
8
10
12
2
4
6
8
10
12
4
6
8
10
12
B
100
50
0
400
Monensin (% of initial mass)
Table 3 Initial concentrations and mass of ionophores in non-amended and amended dairy manure and poultry litter.a
3.2. Effect of time, temperature, and matrix on levels of ionophore residues
350
C
300 250 200 150 100 50 0 400
Monensin (% of initial mass)
All four compounds were added at a dose of 10 mg kg 1 DW so that potential losses of >90% could be determined using existing analytical methods. Background levels of monensin and lasalocid were present in the dairy manure prior to amendment at concentrations of 1.0 ± 0.5 and 3.3 ± 0.5 mg kg 1 DW, respectively (Table 3). Monensin and salinomycin were present in the poultry litter prior to amendment at concentrations of 0.9 ± 0.1 and 5.3 ± 2.2 mg kg 1 DW, respectively (Table 3). Standard deviations of the ionophore concentrations were high due to difficulty of obtaining homogeneous samples from dairy manure and poultry litter. In addition to determining the fate of ionophores in amended samples, removal of these existing residues was also followed by extraction and analysis of incubated non-amended dairy and poultry samples. Initial extractable concentrations of ionophores in nonamended and amended dairy manure and poultry litter show that recoveries of monensin, lasalocid and salinomycin were >58% in each of these matrices (except monensin in poultry litter, 23%). However, recoveries of amprolium were low in both matrices (36% in dairy manure and 26% in poultry litter). Low extraction recovery of amprolium from soil (53%) was also reported (Song et al., 2010). Ionophore concentrations reported in this study were not corrected for recoveries.
350
D
300 250 200 150 100 50 0
a Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Limit of detection is 0.1 mg kg 1 DW and 0.5 lg in 5 g (DW) sample. b Monensin, lasalocid, amprolium and salinomycin were amended at a dose of 10 mg kg 1 DW.
2
Time (weeks) Fig. 2. Monensin in non-amended and amended dairy manure and poultry litter as a function of time and incubation temperature. Panel A, non-amended dairy manure. Panel B, dairy manure amended with 10 mg kg 1 DW monensin. Panel C, non-amended poultry litter. Panel D, poultry litter amended with 10 mg kg 1 DW monensin. Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Initial mass of monensin in non-amended and amended dairy manure and poultry litter are shown in Table 3.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
Table 4 Half-lives of ionophores in non-amended and amended dairy manure and poultry litter (weeks).
Monensin Dairy manure Poultry litter Lasalocid Dairy manure Poultry litter Salinomycin Dairy manure Poultry litter Amprolium Dairy manure Poultry litter
Non-amended
Amended
22 °C
45 °C
55 °C
65 °C
22 °C
45 °C
55 °C
65 °C
1
5
6
4
2
9
10
8
9
–a
–a
7
7
–a
–a
4
8
8
8
6
2
6
4
3
–b
–b
–b
–b
10
–a
–a
3
b
b
–
b
–
–
b
1.5
1.5
2
3.2.2. Lasalocid Results suggest that higher temperatures promote faster lasalocid removal in poultry litter, but there is no similar temperature effect in dairy manure. In non-amended and lasalocid-amended dairy manure, lasalocid levels decreased at all temperatures to 25–50% of initial levels within 6–8 weeks and to 5–40% of initial levels within 12 weeks (Fig. 3a and b). There was no apparent difference among the temperature treatments in terms of the rate or
Lasalocid (% of initial mass)
150
22 oC
A
45 oC 55 oC
100
65 oC
50
0 150
2
4
6
8
2
4
6
8
4
6
8
10
12
B
100
50
0
–
1.5
7
3
5
1
1.5
6
1.5
3
150
Lasalocid (% of initial mass)
Matrix
Comparable ambient temperature incubations of monensincontaining manure were not included in previous composting studies. However, levels of monensin in soils appear to decrease rapidly at ambient temperatures relative to removal rates in untreated or composted manure. Sassman and Lee (2007) reported a very short half-life (2 days) for monensin in moist soils at 23 °C. Using 60Co-sterilized soils, they found significant abiotic removal of monensin in both soils (approximately 40% loss within 5 days). Donoho (1984) reported a 20% decline in monensin levels within 12 days after incorporation of monensin into a field soil plot. Zˇizˇek et al. (2011) recently reported much longer half-life values of monensin in soil (ranging from 3 to 3 and 36 weeks) in toxicity experiments using two types of invertebrates.
Lasalocid (% of initial mass)
monensin levels in both non-amended and monensin-amended dairy manure and poultry litter. In dairy manure, levels of monensin residues (hereafter levels given in the text referred to extractable levels) decreased faster and to a greater extent in incubations at 22 °C relative to levels in samples incubated at composting temperatures (45–65 °C) (Fig. 2a and b). Under the best conditions (incubation at 22 °C), levels decreased with a half-life of approximately 1–2 weeks and decreased to 5–10% of initial levels after 8–12 weeks in dairy manure samples (Fig. 2a,b and Table 4). In samples held at composting temperatures, monensin levels decreased more slowly (with 2–6-week lag period at 65 °C) and ranged from 15 to 30% of initial levels after 12 weeks. Levels of monensin residues in incubated poultry litter decreased more slowly than in dairy manure, with an approximate half-life of 4–9 weeks (Table 4). The influence of temperature on monensin removal was not linear, with slower removal rates at 45 and 55 °C relative to those at 22 and 65 °C (Fig. 2c and d). Levels of residues generally increased within the first 2–4 weeks at all but the highest incubation temperature (65 °C), then remained constant (45 °C) or decreased (22 and 55 °C) during the remaining incubation period. After 8 weeks of incubation, levels of monensin were similar in flasks incubated at 22 and 65 °C (20–45% of initial levels). Our results for monensin removal from dairy and poultry litter at composting temperatures are in general agreement with results from previous studies. Donoho (1984) reported a half-life of 5 weeks for monensin in stockpiled cattle manure and 10 weeks in cattle manure that was incubated at 37 °C. More recently, Storteboom et al. (2007) reported half-life values ranging from 2 to 4 weeks, for monensin in horse and cattle manure that was composted at temperatures ranging from 35 to 55 °C. Dolliver et al. (2008) showed that levels of monensin increased during the first 1–2 weeks of composting of turkey litter, then gradually declined over the 40 day experiment. They calculated half-life values of approximately 2–3 weeks, depending on composting intensity. However, these values may be overestimated since >50% of initial monensin was still present after 6 weeks of composting.
10
12
C
100
50
0 2
10
12
Time (weeks) –b
–b
–b
–b
0.5
1
2
2
–b
–b
–b
–b
–a
5
3
2
a Half-lives of ionophores could not be determined because concentrations of ionophores did not decrease to 50% of the initial levels during the study. b Half-lives of ionophores in non-amended samples could not be determined because initial concentrations of ionophores were <0.1 mg kg 1 DW.
Fig. 3. Lasalocid in non-amended and amended dairy manure and amended poultry litter as a function of time and incubation temperature. Panel A, non-amended dairy manure. Panel B, dairy manure amended with 10 mg kg 1 DW lasalocid. Panel C, poultry litter amended with 10 mg kg 1 DW lasalocid. Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Initial mass of lasalocid in non-amended and amended dairy manure and poultry litter are shown in Table 3.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
extent of removal after 12 weeks. In contrast, results from incubations of lasalocid-amended poultry litter showed that levels of lasalocid were lowest after incubation at 65 °C. At that temperature, levels declined 40% within the first week, followed by a much slower decline over the remaining incubation period (Fig. 3c) with an estimated half-life value of approximately 3 weeks (Table 4). At other temperatures, levels of lasalocid did not decline (45 and 55 °C) or declined after a 6-week lag period (22 °C). These poultry litter incubation results agree with those from a recent study by Zˇizˇek et al. (2015) who determined lasalocid removal rates in composted and untreated chicken manure. Lasalocid levels in a composted wood chip/chicken manure mixture declined 50% within 3 weeks and to levels below 10 lg kg 1 after 7 weeks (corresponding to a calculated half-life of 18 days). In chicken manure stored at ambient temperature, lasalocid levels also decreased 50% within the first three weeks but did not decline further during the 7 week experiment. 3.2.3. Salinomycin Results suggest that higher temperatures promote faster initial salinomycin removal in dairy manure and poultry litter but that treatment differences largely disappear after 8 weeks. After
22 oC
A
45 oC 55 oC
100
65 oC
50
0
2
4
6
8
10
12
B
150
100
50
0
2
Salinomycin (% of initial mass)
150
4
6
8
10
12
Amprolium (% of initial mass)
Salinomycin (% of initial mass)
150
3.2.4. Amprolium Results suggest that although initial removal rates in both dairy manure and poultry are influenced by temperature, the patterns of removal are matrix specific. In amprolium-amended dairy manure, ambient temperatures promoted more rapid removal of amprolium residues compared to treatment at higher temperatures (Fig 5a). However, any temperature effect disappeared within 8 weeks of incubation and all treatments showed similar levels of amprolium (10% of initial levels). In amprolium-amended poultry litter, initial removal rates increased with increased temperature but differences among the three highest temperatures disappeared after 6 weeks (Fig. 5b). At these temperatures, levels of residues ranged from 5 to 15% of initial levels after 6–8 weeks. In contrast to results with dairy manure, levels of amprolium decreased only slightly over time in flasks incubated at 22 °C and remained at 60% of initial values after 12 weeks. We found only one related study examining the fate of amprolium in poultry litter. Dijk and Keukens (2000) included amprolium in residue removal experiments of four veterinary compounds. Their results showed a decrease of 20–30% in amprolium levels after an 8 day composting period. Amprolium levels did not decline further in the composted material during a 3 months storage period.
22 oC
A
45 oC 55 oC
100
65 oC
50
0
C
2 150
100
50
0
2
4
6
8
10
12
Time (weeks) Fig. 4. Salinomycin in amended dairy manure and non-amended and amended poultry litter as a function of time and incubation temperature. Panel A, dairy manure amended with 10 mg kg 1 DW salinomycin. Panel B, non-amended poultry litter. Panel C, poultry litter amended with 10 mg kg 1 DW salinomycin. Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Initial mass of salinomycin in non-amended and amended dairy manure and poultry litter are shown in Table 3.
Amprolium (% of initial mass)
Salinomycin (% of initial mass)
150
8 weeks, levels in salinomycin-amended dairy manure ranged from 5 to 30% of initial levels at all temperatures (Fig. 4a). In non-amended and salinomycin-amended poultry litter, results were highly variable within the first 2–4 weeks. However, levels of salinomycin generally decreased to 10–25% after 6 weeks and <10% after 12 weeks at all temperatures (Fig. 4b and c). These removal rates are slower than those reported by other studies. Ramaswamy et al. (2010) reported >90% removal of salinomycin residues in poultry litter composted at 40–65 °C within 6 days. Although the rate and extent of salinomycin increased with increasing compost temperature in that study, there was no corresponding ambient temperature treatment for comparison.
4
6
8
10
12
4
6
8
10
12
B
100
50
0
2
Time (weeks) Fig. 5. Amprolium in amended dairy manure and poultry litter as a function of time and incubation temperature. Panel A, dairy manure amended with 10 mg kg 1 DW amprolium. Panel B, poultry litter amended with 10 mg kg 1 DW amprolium. Values are from measurement of single samples. Initial mass of amprolium in nonamended and amended dairy manure and poultry litter are shown in Table 3.
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
One limitation of our study was that we did not attempt to examine the possible mechanisms of residue removal. In this regard, inclusion of 60Co sterilized soils and composting substrates in xenobiotic fate studies has been a relatively simple but successful method for determining the relative importance of abiotic binding of ionophores in soil (Sassman and Lee (2007) and tetracyclines in composts (Arikan et al., 2007, 2009b). Comparable studies of ionophores in litter and dairy manure would be useful in understanding their fates in these substrates. In addition, determination of the metabolites would also help to understand the removal mechanism and overall effects. A second limitation is that we do not know if our results from samples maintained at a high moisture content (60–70% moisture) are applicable to real world conditions where unmanaged manure or poultry litter are likely to become very dry over time. It is possible that ionophore removal rates in manure and litter will be significantly influenced by substrate moisture content. Although microbial degradation processes are likely to slow as substrate moisture contents fall below 40%, it is possible that abiotic binding of residues to litter or manure may increase as substrates dry. 4. Conclusions Ionophore removal rates from this study fall within the range of values from other studies that have determined removal rates at composting temperatures. However, inclusion of a range of composting temperatures and ambient temperature treatment in this study complements and extends those previous results and allows for practical decisions of whether composting efforts are likely to reduce specific ionophore levels below those in unmanaged manure or poultry stockpiles. Of the four ionophores tested in dairy manure, only salinomycin was removed more quickly at composting temperatures relative to ambient temperature. In poultry litter, the highest composting temperature (65 °C) increased the removal rates of two of the four ionophores (lasalocid and amprolium) relative to the ambient temperature treatment. However, amprolium removal rates may be too slow to be useful in practice (8–12 weeks at 65 °C for >90% residue removal). Acknowledgments The authors gratefully acknowledge Krystyna M. Bialek Kalinski for her assistance in extraction of ionophores. References Arikan, O., Sikora, L.J., Mulbry, W., Khan, S.U., Foster, G.D., 2007. Composting rapidly reduces levels of extractable oxytetracycline in manure from therapeutically treated beef calves. Bioresour. Technol. 98, 169–176. Arikan, O., Mulbry, W., Ingram, D., Millner, P., 2009a. Minimally managed composting of beef manure at the pilot scale: effect of manure pile construction on pile temperature profiles and on the fate of oxytetracycline and chlorotetracycline. Bioresour. Technol. 100, 4447–4453. Arikan, O., Mulbry, W., Rice, C., 2009b. Management of antibiotic residues from agricultural sources: use of composting to reduce chlortetracycline residues in beef manure from treated animals. J. Hazard. Mater. 164, 483–489. Bak, S.A., Hansen, M., Krogh, K., Brandt, A., Halling-Sørensen, B., Björklund, E., 2013. Development and validation of an SPE methodology combined with LCMS/MS for the determination of four ionophores in aqueous environmental matrices. Int. J. Environ. Anal. Chem. 93, 1500–1512. Biswas, S., McGrath, J.M., Sapkota, A., 2012. Quantification of ionophores in aged poultry litter using liquid chromatography tandem mass spectrometry. J. Environ. Sci. Health, Part B 47, 959–966. Callaway, T.R., Edrington, T.S., Rychlik, J.L., Genovese, K.J., Poole, T.L., Jung, Y.S., Bischoff, K.M., Anderson, R.C., Nisbet, D.J., 2003. Ionophores: their use as ruminant growth promotants and impact on food safety. Curr. Issues Intest. Microbiol. 4, 43–51. Cha, J.M., Yang, S., Carlson, K.H., 2005. Rapid analysis of trace levels of antibiotic polyether ionophores in surface water by solid phase extraction and liquid chromatography with ion trap tandem mass spectrometric detection. J. Chromatogr. A. 1065, 187–198.
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Industrial Composting: Environmental Engineering and Facilities Management. CRC Press, Boca Raton, FL. FDA, 2014. Summary Report on Antimicrobials Sold or Distributed for Use in FoodProducing Animals. Department of Health and Human Services. Furtula, V., Huang, L., Chambers, P.A., 2009. Determination of veterinary pharmaceuticals in poultry litter and soil by methanol extraction and liquid chromatography-tandem mass spectrometry. J. Environ. Sci. Health, Part B 44, 717–723. Hansen, M., Krogh, K.A., Bjöklund, E., Brandt, A., Halling-Sorensen, B., 2009a. Environmental risk assessment of ionophores. Trends Anal. Chem. 28, 534– 542. Hansen, M., Krogh, K.A., Brandt, A., Christensen, J.H., Halling-Sorensen, B., 2009b. Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products. Environ. Pollut. 157, 474–480. Hao, C., Lissemore, L., Nguyen, B., Kleywegt, S., Yang, P., Solomon, K., 2006. 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Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
The effect of composting on the persistence of four ionophores in dairy manure and poultry litter Osman A. Arikan a,b,⇑, Walter Mulbry a, Clifford Rice a a b
USDA-ARS, Beltsville Agricultural Research Center, Sustainable Agricultural Systems Laboratory, Beltsville, MD 20705, USA Istanbul Technical University, Department of Environmental Engineering, Istanbul 34469, Turkey
a r t i c l e
i n f o
Article history: Received 29 September 2015 Revised 27 April 2016 Accepted 28 April 2016 Available online xxxx Keywords: Ionophore Composting Temperature Dairy manure Poultry litter
a b s t r a c t Manure composting is a well-described approach for stabilization of nutrients and reduction of pathogens and odors. Although composting studies have shown that thermophilic temperatures and aerobic conditions can increase removal rates of selected antibiotics, comparable information is lacking for many other compounds in untreated or composted manure. The objective of this study was to determine the relative effectiveness of composting conditions to reduce concentrations of four widely used ionophore feed supplements in dairy manure and poultry litter. Replicate aliquots of fresh poultry litter and dairy manure were amended with monensin, lasalocid, salinomycin, or amprolium to 10 mg kg 1 DW. Nonamended and amended dairy manure and poultry litter aliquots were incubated at 22, 45, 55, or 65 °C under moist, aerobic conditions. Residue concentrations were determined from aliquots removed after 1, 2, 4, 6, 8, and 12 weeks. Results suggest that the effectiveness of composting for contaminant reduction is compound and matrix specific. Composting temperatures were not any more effective than ambient temperature in increasing the rate or extent of monensin removal in either poultry litter or dairy manure. Composting was effective for lasalocid removal in poultry litter, but is likely to be too slow to be useful in practice (8–12 weeks at 65 °C for >90% residue removal). Composting was effective for amprolium removal from poultry litter and salinomycin in dairy manure but both required 4–6 weeks for >90% removal. However, composting did not increase the removal rates or salinomycin in poultry litter or the removal rates of lasalocid or amprolium in dairy manure. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Ionophores are commonly used in livestock production as coccidiostats as well as for growth promotion (Duffield and Bagg, 2000; Callaway et al., 2003; Sassman and Lee, 2007; Clarke et al., 2014). Ionophores are toxic to both prokaryotic and eukaryotic cells because these compounds interfere with the formation of ion gradients across cell membranes (reviewed in Hansen et al., 2009a). Ionophores are toxic to human cells and are therefore not used except for veterinary purposes. According to a Food and Drug Administration (FDA) report, >4500 tons of ionophores were sold for use in livestock in the United States in 2012 (FDA, 2014). Since ionophores aren’t used in humans, land application of ionophore-containing manure does not pose a potential threat to public health by influencing the development of antibiotic resistance in human pathogens. Many ruminal bacteria are ionophore⇑ Corresponding author at: Istanbul Technical University, Department of Environmental Engineering, Istanbul 34469 Turkey. E-mail address: [email protected] (O.A. Arikan).
resistant but there is little evidence that ionophore resistance can be spread from one bacterium to another (Russell and Houlihan, 2003). Ionophore use is unlikely to lead to resistance in people as this resistance tends to develop more slowly, and may well be a reversible change (O’Neill, 2015). However, these compounds inhibit the activity of many bacteria and eukaryotic cells and can affect soil invertebrates and aquatic organisms (Hansen et al., 2009b; Zˇizˇek et al., 2011). Development of resistance against ionophores and cross-resistance in bacteria to more than one ionophore have also been observed (VKM, 2015). Results for raw vegetables indicate that coccidiostats can be incorporated into vegetables from the soil although detected concentrations are relatively low (Peteghema et al., 2012). Therefore, application of manure with ionophores is a considerable concern. Ionophores have been primarily studied in manure (Donoho, 1984; Schlüsener et al., 2003; Sassman and Lee, 2007; Storteboom et al., 2007; Watanabe et al., 2008; Varel et al., 2012) poultry litter (Webb and Fontenot, 1975; Furtula et al., 2009; Ramaswamy et al.; 2010; Biswas et al., 2012; Bak et al., 2013; Zˇizˇek et al., 2015) and soil (Sassman and Lee, 2007; Bak et al.,
http://dx.doi.org/10.1016/j.wasman.2016.04.032 0956-053X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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2013; Zˇizˇek et al., 2015). Although reported half-lives for these compounds in soils are very short (typically 2–4 days) (Sassman and Lee, 2007; Zˇizˇek et al., 2015) ionophores have been detected in surface water (Cha et al., 2005; Kim and Carlson, 2006; Hao et al., 2006, 2008; Song et al., 2007; Kurwadkar et al., 2013; Bak et al., 2013), groundwater (Watanabe et al., 2008) and sediment (Kim and Carlson, 2006). Hansen et al. (2009a) suggest that ionophores constitute an environmental risk because predicted environmental concentrations of ionophores and their measured concentrations in sediments could be above predicted no-effect levels. Treatment of ionophore-containing manure prior to land application is one possible means of reducing the amount of these compounds that is released into the environment. Manure composting is a treatment process that is widely used for stabilization of nutrients and reduction of pathogens and odors (US Composting Council, 2000). The major factors that affect composting are oxygen, moisture, temperature, carbon and nitrogen (Epstein, 2011). Mean temperatures between 45 and 65 °C can be achieved during composting by applying different intensities of composting practices (Arikan et al., 2009a; Dolliver et al., 2008). Previous laboratory and field-scale composting studies (Arikan et al., 2009a,b; Dolliver et al., 2008) have shown that thermophilic temperatures (50–60 °C) and aerobic conditions rapidly reduce chlortetracycline levels in dairy manure and turkey litter. However, there is limited information on the effect of composting on ionophores, especially among the range of temperatures expected within minimally managed compost piles. The specific objective of this study was to determine the relative effectiveness of composting temperatures to reduce the concentrations of four widely used ionophores (monensin, lasalocid, salinomycin, amprolium) (Mellon et al., 2001). Laboratoryscale incubations were conducted using both dairy manure and poultry litter to determine the influence of the composting matrix on removal rates. Replicate aliquots of fresh poultry litter and dairy manure were amended with monensin, lasalocid, salinomycin, or amprolium to 10 mg kg 1 DW. Non-amended and amended dairy manure and poultry litter aliquots were incubated for 12 weeks under moist, aerobic conditions at 22 °C or at three elevated temperatures (45, 55, or 65 °C) that represent temperatures achievable at different levels of composting intensity. Based on our previous results with tetracyclines and from results from other studies with monensin, we predicted that temperature would influence ionophore removal rates with higher temperatures leading to higher removal rates and extent of removal. In addition, we predicted that removal rates would vary between ionophores and between poultry litter and dairy manure. We also predicted that the influence of temperature on removal rates would be similar in both matrices. The results show that ionophore removal rates were influenced by the specific ionophore, temperature, and matrix. However, in some instances, the influence of temperature on ionophore removal varied between dairy manure and poultry litter samples. 2. Materials and methods 2.1. Chemicals Monensin sodium salt, lasalocid A sodium salt, salinomycin mono-sodium salt hydrate and amprolium hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Structures of these compounds are shown in Fig. 1. HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Pittsburg, PA). All reagents used in this study were analytical grade. Water used for extractions was purified using reverse osmosis and activated carbon. Stock solutions of the ionophore standards for extraction and analysis were prepared monthly by dissolving each
Monensin (Mw= 671 g mol-1, pKa = 6.7, log Kow = 2.8-4.2)
Lasalocid (Mw= 591 g mol-1, pKa = 4.4, log Kow = 1.4-2.8)
Salinomycin (Mw= 751 g mol-1, pKa = 4.5, 6.4, log Kow = 5.2)
Amprolium hydrochloride (Mw= 278 g mol-1, log Kow = -2.5) Fig. 1. Structures (adapted from Clarke et al., 2014) and selected physicochemical properties (adapted from Hansen et al., 2009b; Ding, 2011) of ionophores used in this study.
compound in methanol at a concentration of 100 mg L stored at 20 °C in the dark.
1
and
2.2. Dairy manure and poultry litter Fresh dairy manure was collected from the USDA’s Beltsville Dairy Research Unit (Beltsville, Maryland). Manure was air-dried in a fume hood for 48 h to a moisture content of 6%. The dried manure was ground using a hammer mill to pass a 4.7 mm sieve and stored at 4 °C in a covered container prior to use. Poultry litter was obtained from a covered storage pile at the University of Maryland Eastern Shore’s broiler production facility (Princess Anne, Maryland). The moisture content of the litter as received was 16%. The litter was sieved to remove feathers and large debris. Material that passed a 4.7 mm sieve was collected and stored in a covered container at 4 °C prior to use. The collected dairy manure and poultry litter samples were analyzed for ionophores using the method below and found to contain some of the studied ionophores (shown in Table 3).
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2.3. Incubation experiment Individual stock solutions of monensin, lasalocid, and amprolium for amending dairy manure and poultry litter were prepared in methanol at 1 g L 1. Amended batches of dairy manure or poultry litter were individually prepared by diluting 1.5 mL of stock solution (containing 1.5 mg of ionophore) with 50 mL of ethyl acetate and stirring the diluted mixture into 150 g DW batches of dairy manure or poultry litter in 4 L glass beakers. Amended batches of manure or litter were stored in open metal trays in a fume hood overnight in order to volatilize ethyl acetate and methanol. During composting, the rate of microbial activity decreases when the moisture level in compost is below 40% (Epstein, 2011). Typical poultry litter is generally dry and water is added at the beginning of poultry litter composting. It is possible that microbial populations in the amended dairy manure may have been reduced by drying and/or exposure to the methanol/ethyl acetate mixture in the steps described above. In order to mitigate this possibility, batches of dried non-amended and ionophoreamended dairy manure were brought up to a moisture content of 65–70% by the addition of fresh solids-separated dairy manure effluent. For the same reason, batches of non-amended and ionophore-amended poultry litter were brought up to a moisture content of 65–70% by the addition of an aqueous extract of fresh poultry litter (100 g litter mixed with 1 L distilled water). A stock solution of salinomycin was prepared in dimethyl sulfoxide at 1 g L 1. A 1.5 mL aliquot of the stock solution (containing 1.5 mg salinomycin) was diluted with 360 mL of solids-separated dairy manure liquid and this mixture was used to amend a 150 g DW batch of air-dried dairy manure. Similarly, a 1.5 mL aliquot of the salinomycin stock solution was diluted with 270 mL of a poultry litter extract and was used to amend a 150 g DW batch of poultry litter. Aliquots of amended and non-amended manure and poultry litter (14.5 g WW, corresponding to 5 g DW) were secured within 15 15 cm squares of color coded nylon mesh using plastic wire ties. Two samples from each treatment were randomly chosen as untreated control samples and were stored at 20 °C prior to extraction and analysis. The remaining mesh bags were placed in sealed 4 L glass jars that were placed in incubators set at 22, 42, 55, or 65 °C. Oxygen and moisture were controlled during the study. Jars were periodically flushed with air to maintain aerobic conditions (flushed five minutes each day for the first two weeks, flushed every two days for weeks 3–4, and flushed every 3 days for weeks 4–12). Sample moisture was maintained by adding water to the jars. Samples (single amended samples and triplicate non-amended samples for each treatment at each time-point) were removed after 1, 2, 4, 6, 8 and 12 weeks, weighed, and stored at 20 °C prior to extraction and analysis. Sample dry weights were determined prior to extraction for calculation of losses of compost mass and ionophore masses during the incubations. Dairy manure and poultry litter characteristics at the beginning of the incubation are shown in Table 1.
Table 1 Dairy manure and poultry litter characteristics at the beginning of the incubation period.a
a
Constituent
Dairy manure
Poultry litter
pH Moisture content, (%) Volatile solids, (% of dry matter) C, (%, wet basis) N, (%, wet basis) C/N
8.6 ± 0.1 71.7 ± 1.4 89.1 ± 0.8 12.1 ± 0.7 0.6 ± 0.1 20.2
8.9 ± 0.1 66.6 ± 4.0 80.9 ± 3.0 12.6 ± 2.0 1.6 ± 0.3 7.9
Values are means ± standard deviation of triplicate samples.
Removal of ionophores was assumed to follow first-order kinetics. A rate constant, k, was determined as the slope of the curve calculated by linear regression. The half-life, t1/2, was then calculated as t1/2 = ln(2)/k. 2.4. Extraction of compounds Frozen samples were dried by lyophilization for 72 h at 50 °C prior to extraction. Aliquots (0.5 g) of lyophilized samples were moistened by the addition of 200 ll of de-ionized water and equilibrated for 10 min. Moistened samples were extracted two times with 12 mL methanol by vortexing for 15 s followed by sonication for 15 min in a sonication bath (Elma, E100H, Elmasonic, Singen, Germany). After each extraction, the extracts were subjected to centrifugation (1200g, 5 min). The resulting supernatants were pooled, brought up to a total volume of 25 mL with methanol, and mixed for 30 s prior to transferring an aliquot to amber autosampler vials for analysis by LC/MS/MS. 2.5. LC/MS/MS analysis The LC instrument was a Waters 2690 XE (Waters Corp., Milford, MA) separations module with an XBridge C18 column (150 mm 2.1 mm i.d., 5 lm) (Waters Corp., Milford, MA) in conjunction with an XBridge C18 guard column at 50 °C. The injection volume was 10 ll. A gradient separation was utilized involving a mixture of solvent A (70:30, 0.5% formic acid:acetonitrile) mixed with solvent B (100% de-ionized water) and solvent C (100% acetonitrile). The gradient program was as follows: initial 2 min. hold with 70:30, A:B at a flow of 0.2 mL min 1; then the flow is increased to 0.3 mL min 1 and a non-linear gradient was started with a setting of 8 transitioning to 2:98, A:C. This setting was maintained for 12 min. The column was returned to initial conditions in 1 min and stabilized for 8 min before the next run is started. Atmospheric pressure ionization-tandem mass spectrometry was performed on a benchtop triple mass spectrometer (LC Quattro Ultima, Waters Corp.) operated in electrospray positive ionization mode. The source parameters were as follows: capillary voltage was set at 3.5 kV and extractor voltage was set at 3 V, respectively; rf lens at 0.1 V; source and desolvation temperatures were 150 and 450 °C. Liquid nitrogen was used to supply the nebulizer and desolvation gas (flow rates were approximately 80 and 400 L h 1, respectively). Argon was used as collision-induced decomposition gas to fragment the parent ions; the typical pressure was 2.6 10 3 mbar. Both high and low mass resolutions were set at 12.0 for both quadrupoles. Acquisition was performed using the multiple-reaction monitoring mode (MRM) in electrospray positive (ES+). The parent and daughter ions used for compound identification and quantitation are listed in Table 2 along with the optimum cone voltages and collision energies used. Optimization was performed by infusion of the standards from a syringe pump (10 ll/min) mixed with the LC effluent (100% A; 200 ll min 1), with high- and low-mass resolution set at 15.0. The detector was a photomultiplier set at 650 V. Analyte concentrations were calculated by the external standard method using Table 2 Parent and daughter ions used for quantitation of ionophores and MS parameters used to produce them. Compound
Parent ion, Daughter ion, Retention time, Cone, Collision, Da Da min V eV
Monensin 693.7 Lasalocid 613 Salinomycin 773.5 Amprolium 150
479.8 377 431 81
10.4 12.5 13.1 2
124 115 115 63
58 45 61 26
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five point standard calibration curves (fits with > 0.9 r2 values). Peak integration and quantitation were performed automatically using MassLynx 4.0 software (Waters Corp.). 3. Results and discussion
Sample mass loss was slightly influenced by incubation temperature but was not influenced by ionophore amendment (not shown). Mass loss values (30–35% DW loss after 12 weeks) were in the range of values reported for composted samples (Arikan et al., 2007). Ionophore levels were corrected for sample mass loss and are shown in terms of mass rather than concentration.
3.1. Sample amendment, recoveries and compost mass loss
Matrix
Lasalocid
Salinomycin
Amprolium
Dairy manure, non-amended (mg kg 1 DW) 1.0 ± 0.5 (lg) in 5 g (DW) 4.1 ± 2.3 sample
3.3 ± 0.5 14.2 ± 2.3
<0.1 <0.5
<0.1 <0.5
Dairy manure, amendedb (mg kg 1 DW) 6.8 (lg) in 5 g (DW) 29.0 sample
10.3 45.2
Dairy manure, net (mg kg 1 DW) (lg) in 5 g (DW) sample
5.8 24.9
7.0 31.0
10 43.3
3.6 15.8
58
70
100
36
Dairy manure, recovery (%)
Monensin
10.0 43.3
3.6 15.8
Poultry litter, non-amended (mg kg 1 DW) 0.9 ± 0.1 (lg) in 5 g (DW) 5.1 ± 0.2 sample
<0.1 <0.5
Poultry litter, amendedb (mg kg 1 DW) 3.2 (lg) in 5 g (DW) 17.9 sample
9.8 55.5
Poultry litter, net (mg kg 1 DW) (lg) in 5 g (DW) sample
2.3 12.8
9.8 55.5
7.9 44.9
2.6 14.0
23
98
79
26
Poultry litter, recovery (%)
5.3 ± 2.2 28.6 ± 10.7
13.2 73.5
<0.1 <0.5
2.6 14.0
3.2.1. Monensin Results show that ambient temperature treatment was as or more effective than composting temperatures in reducing
Monensin (% of initial mass)
150
22 oC
A
45 oC 55 oC
100
65 oC
50
0
Monensin (% of initial mass)
150
2
4
6
8
10
12
2
4
6
8
10
12
2
4
6
8
10
12
4
6
8
10
12
B
100
50
0
400
Monensin (% of initial mass)
Table 3 Initial concentrations and mass of ionophores in non-amended and amended dairy manure and poultry litter.a
3.2. Effect of time, temperature, and matrix on levels of ionophore residues
350
C
300 250 200 150 100 50 0 400
Monensin (% of initial mass)
All four compounds were added at a dose of 10 mg kg 1 DW so that potential losses of >90% could be determined using existing analytical methods. Background levels of monensin and lasalocid were present in the dairy manure prior to amendment at concentrations of 1.0 ± 0.5 and 3.3 ± 0.5 mg kg 1 DW, respectively (Table 3). Monensin and salinomycin were present in the poultry litter prior to amendment at concentrations of 0.9 ± 0.1 and 5.3 ± 2.2 mg kg 1 DW, respectively (Table 3). Standard deviations of the ionophore concentrations were high due to difficulty of obtaining homogeneous samples from dairy manure and poultry litter. In addition to determining the fate of ionophores in amended samples, removal of these existing residues was also followed by extraction and analysis of incubated non-amended dairy and poultry samples. Initial extractable concentrations of ionophores in nonamended and amended dairy manure and poultry litter show that recoveries of monensin, lasalocid and salinomycin were >58% in each of these matrices (except monensin in poultry litter, 23%). However, recoveries of amprolium were low in both matrices (36% in dairy manure and 26% in poultry litter). Low extraction recovery of amprolium from soil (53%) was also reported (Song et al., 2010). Ionophore concentrations reported in this study were not corrected for recoveries.
350
D
300 250 200 150 100 50 0
a Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Limit of detection is 0.1 mg kg 1 DW and 0.5 lg in 5 g (DW) sample. b Monensin, lasalocid, amprolium and salinomycin were amended at a dose of 10 mg kg 1 DW.
2
Time (weeks) Fig. 2. Monensin in non-amended and amended dairy manure and poultry litter as a function of time and incubation temperature. Panel A, non-amended dairy manure. Panel B, dairy manure amended with 10 mg kg 1 DW monensin. Panel C, non-amended poultry litter. Panel D, poultry litter amended with 10 mg kg 1 DW monensin. Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Initial mass of monensin in non-amended and amended dairy manure and poultry litter are shown in Table 3.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
Table 4 Half-lives of ionophores in non-amended and amended dairy manure and poultry litter (weeks).
Monensin Dairy manure Poultry litter Lasalocid Dairy manure Poultry litter Salinomycin Dairy manure Poultry litter Amprolium Dairy manure Poultry litter
Non-amended
Amended
22 °C
45 °C
55 °C
65 °C
22 °C
45 °C
55 °C
65 °C
1
5
6
4
2
9
10
8
9
–a
–a
7
7
–a
–a
4
8
8
8
6
2
6
4
3
–b
–b
–b
–b
10
–a
–a
3
b
b
–
b
–
–
b
1.5
1.5
2
3.2.2. Lasalocid Results suggest that higher temperatures promote faster lasalocid removal in poultry litter, but there is no similar temperature effect in dairy manure. In non-amended and lasalocid-amended dairy manure, lasalocid levels decreased at all temperatures to 25–50% of initial levels within 6–8 weeks and to 5–40% of initial levels within 12 weeks (Fig. 3a and b). There was no apparent difference among the temperature treatments in terms of the rate or
Lasalocid (% of initial mass)
150
22 oC
A
45 oC 55 oC
100
65 oC
50
0 150
2
4
6
8
2
4
6
8
4
6
8
10
12
B
100
50
0
–
1.5
7
3
5
1
1.5
6
1.5
3
150
Lasalocid (% of initial mass)
Matrix
Comparable ambient temperature incubations of monensincontaining manure were not included in previous composting studies. However, levels of monensin in soils appear to decrease rapidly at ambient temperatures relative to removal rates in untreated or composted manure. Sassman and Lee (2007) reported a very short half-life (2 days) for monensin in moist soils at 23 °C. Using 60Co-sterilized soils, they found significant abiotic removal of monensin in both soils (approximately 40% loss within 5 days). Donoho (1984) reported a 20% decline in monensin levels within 12 days after incorporation of monensin into a field soil plot. Zˇizˇek et al. (2011) recently reported much longer half-life values of monensin in soil (ranging from 3 to 3 and 36 weeks) in toxicity experiments using two types of invertebrates.
Lasalocid (% of initial mass)
monensin levels in both non-amended and monensin-amended dairy manure and poultry litter. In dairy manure, levels of monensin residues (hereafter levels given in the text referred to extractable levels) decreased faster and to a greater extent in incubations at 22 °C relative to levels in samples incubated at composting temperatures (45–65 °C) (Fig. 2a and b). Under the best conditions (incubation at 22 °C), levels decreased with a half-life of approximately 1–2 weeks and decreased to 5–10% of initial levels after 8–12 weeks in dairy manure samples (Fig. 2a,b and Table 4). In samples held at composting temperatures, monensin levels decreased more slowly (with 2–6-week lag period at 65 °C) and ranged from 15 to 30% of initial levels after 12 weeks. Levels of monensin residues in incubated poultry litter decreased more slowly than in dairy manure, with an approximate half-life of 4–9 weeks (Table 4). The influence of temperature on monensin removal was not linear, with slower removal rates at 45 and 55 °C relative to those at 22 and 65 °C (Fig. 2c and d). Levels of residues generally increased within the first 2–4 weeks at all but the highest incubation temperature (65 °C), then remained constant (45 °C) or decreased (22 and 55 °C) during the remaining incubation period. After 8 weeks of incubation, levels of monensin were similar in flasks incubated at 22 and 65 °C (20–45% of initial levels). Our results for monensin removal from dairy and poultry litter at composting temperatures are in general agreement with results from previous studies. Donoho (1984) reported a half-life of 5 weeks for monensin in stockpiled cattle manure and 10 weeks in cattle manure that was incubated at 37 °C. More recently, Storteboom et al. (2007) reported half-life values ranging from 2 to 4 weeks, for monensin in horse and cattle manure that was composted at temperatures ranging from 35 to 55 °C. Dolliver et al. (2008) showed that levels of monensin increased during the first 1–2 weeks of composting of turkey litter, then gradually declined over the 40 day experiment. They calculated half-life values of approximately 2–3 weeks, depending on composting intensity. However, these values may be overestimated since >50% of initial monensin was still present after 6 weeks of composting.
10
12
C
100
50
0 2
10
12
Time (weeks) –b
–b
–b
–b
0.5
1
2
2
–b
–b
–b
–b
–a
5
3
2
a Half-lives of ionophores could not be determined because concentrations of ionophores did not decrease to 50% of the initial levels during the study. b Half-lives of ionophores in non-amended samples could not be determined because initial concentrations of ionophores were <0.1 mg kg 1 DW.
Fig. 3. Lasalocid in non-amended and amended dairy manure and amended poultry litter as a function of time and incubation temperature. Panel A, non-amended dairy manure. Panel B, dairy manure amended with 10 mg kg 1 DW lasalocid. Panel C, poultry litter amended with 10 mg kg 1 DW lasalocid. Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Initial mass of lasalocid in non-amended and amended dairy manure and poultry litter are shown in Table 3.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
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O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
extent of removal after 12 weeks. In contrast, results from incubations of lasalocid-amended poultry litter showed that levels of lasalocid were lowest after incubation at 65 °C. At that temperature, levels declined 40% within the first week, followed by a much slower decline over the remaining incubation period (Fig. 3c) with an estimated half-life value of approximately 3 weeks (Table 4). At other temperatures, levels of lasalocid did not decline (45 and 55 °C) or declined after a 6-week lag period (22 °C). These poultry litter incubation results agree with those from a recent study by Zˇizˇek et al. (2015) who determined lasalocid removal rates in composted and untreated chicken manure. Lasalocid levels in a composted wood chip/chicken manure mixture declined 50% within 3 weeks and to levels below 10 lg kg 1 after 7 weeks (corresponding to a calculated half-life of 18 days). In chicken manure stored at ambient temperature, lasalocid levels also decreased 50% within the first three weeks but did not decline further during the 7 week experiment. 3.2.3. Salinomycin Results suggest that higher temperatures promote faster initial salinomycin removal in dairy manure and poultry litter but that treatment differences largely disappear after 8 weeks. After
22 oC
A
45 oC 55 oC
100
65 oC
50
0
2
4
6
8
10
12
B
150
100
50
0
2
Salinomycin (% of initial mass)
150
4
6
8
10
12
Amprolium (% of initial mass)
Salinomycin (% of initial mass)
150
3.2.4. Amprolium Results suggest that although initial removal rates in both dairy manure and poultry are influenced by temperature, the patterns of removal are matrix specific. In amprolium-amended dairy manure, ambient temperatures promoted more rapid removal of amprolium residues compared to treatment at higher temperatures (Fig 5a). However, any temperature effect disappeared within 8 weeks of incubation and all treatments showed similar levels of amprolium (10% of initial levels). In amprolium-amended poultry litter, initial removal rates increased with increased temperature but differences among the three highest temperatures disappeared after 6 weeks (Fig. 5b). At these temperatures, levels of residues ranged from 5 to 15% of initial levels after 6–8 weeks. In contrast to results with dairy manure, levels of amprolium decreased only slightly over time in flasks incubated at 22 °C and remained at 60% of initial values after 12 weeks. We found only one related study examining the fate of amprolium in poultry litter. Dijk and Keukens (2000) included amprolium in residue removal experiments of four veterinary compounds. Their results showed a decrease of 20–30% in amprolium levels after an 8 day composting period. Amprolium levels did not decline further in the composted material during a 3 months storage period.
22 oC
A
45 oC 55 oC
100
65 oC
50
0
C
2 150
100
50
0
2
4
6
8
10
12
Time (weeks) Fig. 4. Salinomycin in amended dairy manure and non-amended and amended poultry litter as a function of time and incubation temperature. Panel A, dairy manure amended with 10 mg kg 1 DW salinomycin. Panel B, non-amended poultry litter. Panel C, poultry litter amended with 10 mg kg 1 DW salinomycin. Values from non-amended samples are means and standard deviation from triplicate samples. Values from amended samples are from measurement of single samples. Initial mass of salinomycin in non-amended and amended dairy manure and poultry litter are shown in Table 3.
Amprolium (% of initial mass)
Salinomycin (% of initial mass)
150
8 weeks, levels in salinomycin-amended dairy manure ranged from 5 to 30% of initial levels at all temperatures (Fig. 4a). In non-amended and salinomycin-amended poultry litter, results were highly variable within the first 2–4 weeks. However, levels of salinomycin generally decreased to 10–25% after 6 weeks and <10% after 12 weeks at all temperatures (Fig. 4b and c). These removal rates are slower than those reported by other studies. Ramaswamy et al. (2010) reported >90% removal of salinomycin residues in poultry litter composted at 40–65 °C within 6 days. Although the rate and extent of salinomycin increased with increasing compost temperature in that study, there was no corresponding ambient temperature treatment for comparison.
4
6
8
10
12
4
6
8
10
12
B
100
50
0
2
Time (weeks) Fig. 5. Amprolium in amended dairy manure and poultry litter as a function of time and incubation temperature. Panel A, dairy manure amended with 10 mg kg 1 DW amprolium. Panel B, poultry litter amended with 10 mg kg 1 DW amprolium. Values are from measurement of single samples. Initial mass of amprolium in nonamended and amended dairy manure and poultry litter are shown in Table 3.
Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032
O.A. Arikan et al. / Waste Management xxx (2016) xxx–xxx
One limitation of our study was that we did not attempt to examine the possible mechanisms of residue removal. In this regard, inclusion of 60Co sterilized soils and composting substrates in xenobiotic fate studies has been a relatively simple but successful method for determining the relative importance of abiotic binding of ionophores in soil (Sassman and Lee (2007) and tetracyclines in composts (Arikan et al., 2007, 2009b). Comparable studies of ionophores in litter and dairy manure would be useful in understanding their fates in these substrates. In addition, determination of the metabolites would also help to understand the removal mechanism and overall effects. A second limitation is that we do not know if our results from samples maintained at a high moisture content (60–70% moisture) are applicable to real world conditions where unmanaged manure or poultry litter are likely to become very dry over time. It is possible that ionophore removal rates in manure and litter will be significantly influenced by substrate moisture content. Although microbial degradation processes are likely to slow as substrate moisture contents fall below 40%, it is possible that abiotic binding of residues to litter or manure may increase as substrates dry. 4. Conclusions Ionophore removal rates from this study fall within the range of values from other studies that have determined removal rates at composting temperatures. However, inclusion of a range of composting temperatures and ambient temperature treatment in this study complements and extends those previous results and allows for practical decisions of whether composting efforts are likely to reduce specific ionophore levels below those in unmanaged manure or poultry stockpiles. Of the four ionophores tested in dairy manure, only salinomycin was removed more quickly at composting temperatures relative to ambient temperature. In poultry litter, the highest composting temperature (65 °C) increased the removal rates of two of the four ionophores (lasalocid and amprolium) relative to the ambient temperature treatment. However, amprolium removal rates may be too slow to be useful in practice (8–12 weeks at 65 °C for >90% residue removal). Acknowledgments The authors gratefully acknowledge Krystyna M. Bialek Kalinski for her assistance in extraction of ionophores. References Arikan, O., Sikora, L.J., Mulbry, W., Khan, S.U., Foster, G.D., 2007. Composting rapidly reduces levels of extractable oxytetracycline in manure from therapeutically treated beef calves. Bioresour. Technol. 98, 169–176. Arikan, O., Mulbry, W., Ingram, D., Millner, P., 2009a. Minimally managed composting of beef manure at the pilot scale: effect of manure pile construction on pile temperature profiles and on the fate of oxytetracycline and chlorotetracycline. Bioresour. Technol. 100, 4447–4453. Arikan, O., Mulbry, W., Rice, C., 2009b. Management of antibiotic residues from agricultural sources: use of composting to reduce chlortetracycline residues in beef manure from treated animals. J. Hazard. Mater. 164, 483–489. Bak, S.A., Hansen, M., Krogh, K., Brandt, A., Halling-Sørensen, B., Björklund, E., 2013. Development and validation of an SPE methodology combined with LCMS/MS for the determination of four ionophores in aqueous environmental matrices. Int. J. Environ. Anal. Chem. 93, 1500–1512. Biswas, S., McGrath, J.M., Sapkota, A., 2012. Quantification of ionophores in aged poultry litter using liquid chromatography tandem mass spectrometry. J. Environ. Sci. Health, Part B 47, 959–966. Callaway, T.R., Edrington, T.S., Rychlik, J.L., Genovese, K.J., Poole, T.L., Jung, Y.S., Bischoff, K.M., Anderson, R.C., Nisbet, D.J., 2003. Ionophores: their use as ruminant growth promotants and impact on food safety. Curr. Issues Intest. Microbiol. 4, 43–51. Cha, J.M., Yang, S., Carlson, K.H., 2005. Rapid analysis of trace levels of antibiotic polyether ionophores in surface water by solid phase extraction and liquid chromatography with ion trap tandem mass spectrometric detection. J. Chromatogr. A. 1065, 187–198.
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Please cite this article in press as: Arikan, O.A., et al. The effect of composting on the persistence of four ionophores in dairy manure and poultry litter. Waste Management (2016), http://dx.doi.org/10.1016/j.wasman.2016.04.032