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Degradation and dissipation of the veterinary ionophore lasalocid in manure and soil
Chemosphere 138 (2015) 947–951
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Degradation and dissipation of the veterinary ionophore lasalocid in manure and soil Suzana Zˇizˇek a,⇑, Martin Dobeic b, Štefan Pintaricˇ b, Primozˇ Zidar c, Silvestra Kobal d, Matej Vidrih e a
Institute of Pathology, Foresnic and Administrative Veterinary Medicine, Veterinary Faculty, University of Ljubljana, Gerbicˇeva 60, SI-1000 Ljubljana, Slovenia Institute for Environmental and Animal Hygiene with Ethology, Veterinary Faculty, University of Ljubljana, Gerbicˇeva 60, SI-1000 Ljubljana, Slovenia c Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecˇna pot 111, SI-1000 Ljubljana, Slovenia d Institute for Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbicˇeva 60, SI-1000 Ljubljana, Slovenia e Department of Agronomy, Biotechnial Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia b
h i g h l i g h t s The fate of lasalocid in poultry manure and on arable land is largely unknown. We measured lasalocid degradation in manure and compost and dissipation on land. Degradation in compost is faster than in manure. Half-life of lasalocid in soil was 3.1 ± 0.6 d. PEC/PNEC in soil could exceed 1 in a worst-case scenario if manure is not aged.
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Article history: Received 14 May 2014 Received in revised form 4 December 2014 Accepted 8 December 2014 Available online 30 December 2014 Handling Editor: Klaus Kümmerer Keywords: Lasalocid Degradation Dissipation Environmental risk assessment
a b s t r a c t Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It is excreted from the treated animals mostly in its active form and enters the environment with the use of contaminated manure on agricultural land. To properly assess the risk that lasalocid poses to the environment, it is necessary to know its environmental concentrations as well as the rates of its degradation in manure and dissipation in soil. These values are still largely unknown. A research was undertaken to ascertain the rate of lasalocid degradation in manure under different storage conditions (aging in a pile or composting) and on agricultural soil after using lasalocid-contaminated manure. The results have shown that there is considerable difference in lasalocid degradation between aging manure with no treatment (t1/2 = 61.8 ± 1.7 d) and composting (t1/2 = 17.5 ± 0.8 d). Half-lives in soil are much shorter (on average 3.1 ± 0.4 d). On the basis of the measured concentrations of lasalocid in soil after manure application, we can conclude that it can potentially be harmful to soil organisms (PEC/PNEC ratio of 1.18), but only in a worst-case scenario of using the maximum permissible amount of manure and immediately after application. To make certain that no harmful effects occur, composting is recommended. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Extensive use of manure burdens the environment not only with large amounts of nitrogen and metals, but also with residues of veterinary pharmaceuticals and feed additives (Halling-Sørensen et al., 1998). Manure from treated animals in farms usually contains numerous pharmaceuticals and their metabolites. Their introduc-
⇑ Corresponding author at: Laboratory for Environmental Research, University of Nova Gorica, Vipavska 13, SI-5000 Nova Gorica, Slovenia. Tel.: +386 5 3315 388. E-mail address: [email protected] (S. Zˇizˇek). http://dx.doi.org/10.1016/j.chemosphere.2014.12.032 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
tion into the environment is not controlled, a problem that has been neglected for decades (Boxall, 2004). Coccidiosis is a rapidly spreading and often fatal protozoal infection in poultry that can cause serious economic losses. In the European Union, coccidiostats are authorised as feed additives and of the estimated 40.65 million tonnes of feed produced, about 18.33 million tonnes contain coccidiostats (EC, 2008). Broilers and turkeys are treated with coccidiostats almost their entire life. Lasalocid, one of the most frequently used coccidiostats, is a natural ionophore antibiotic produced by the bacterium Streptomyces lasaliensis. In treated animals lasalocid is only partially metabolised and is excreted at least partially in the active form (EFSA, 2004, 2010).
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EFSA (2004) reported that 74–83% of lasalocid in broiler excreta is in the active form, while another study on turkey litter (EFSA, 2010) found only 10% of the administered dose as the parent compound. A large number of compounds were detected in chicken and turkeys excreta with only two metabolites accounting for more than 5% of 14C. Lasalocid degradation in manure has only been reported in one study (EFSA, 2004), which stated a loss greater than 75% after 30 d. When in the environment, lasalocid undergoes mostly biotic degradation (Vertesy et al., 1987; Sassman and Lee, 2007; Hansen et al., 2009a, 2012). The rate of decay, which is much slower in anaerobic conditions, depends on soil properties such as organic content, moisture, temperature and pH (Sassman and Lee, 2007). The reported half-lives of lasalocid in soil were from 0.6 to 14.2 d (EFSA, 2004; Sassman and Lee, 2007), but all were obtained under laboratory conditions. However, no information is available in literature on the degradation of lasalocid in field conditions or in manure before application to soil. Sassman and Lee (2007) studied its degradation in different types of soil under laboratory conditions by adding lasalocid in aqueous solution. They obtained half-lives of approximately 4 d in soil rich in organic carbon (OC) and 1.5 d in low-OC soil. Manure amendment did not change the degradation rate. No degradation of lasalocid was observed in sterilised soil. Hansen et al. (2009b) highlighted this lack of available data and stated that a reliable risk assessment of lasalocid cannot be calculated without further investigation. Degradation of monensin and other structurally similar ionophores has been studied in more detail. Donoho (1984) found that monensin is biodegradable in cattle manure and soil and declines relatively slowly in incubated faeces under relatively anaerobic conditions, but is degraded fairly rapidly in a manure pile under natural weathering conditions. Its half-life in soil was approximately 13 d. Schlüsener et al. (2006) found a half-life of salinomycin in livestock manure of 6 d. Average monensin half-life in spiked turkey litter (Dolliver et al., 2008) was 17 d and depended on treatment of manure (22 d in manure pile, 19 d in manure with weekly water additions and mixing and 11 d when using vessel composting). Half-life of monensin in soil was between 22.7 d in low-OC soil and 4.2 d in higher-OC soil and no degradation was observed in dried soil (Yoshida et al., 2010). Salinomycin degrades rapidly with a half-life of 1.3 d in broiler compost and 4.0 d in manure with no treatment (Ramaswamy et al., 2010). Experiments on salinomycin in lab-scale soil bioreactors (Hansen et al., 2012) showed rapid degradation in aerobic conditions (degraded after 68 h) but very limited degradation under anaerobic conditions. Sun et al. (2014) studied the degradation in broil litter and soil of monensin, salinomycin and narasin. They found that degradation does not occur in broiler litter with low moisture content (24% and 40% moisture). Narasin and salinomycin degraded in broiler litter with higher water content (72%), but monensin remained stable during the 14-day incubation. In soil microcosms, the half-lives of the studied ionophores were between 3.3 and 5 d. The present study was therefore undertaken in order to measure the degradation rates of lasalocid in manure and in soil and to ascertain the risk lasalocid-contaminated manure represents to soil-dwelling organisms and to agricultural ecosystems in general.
2. Materials and methods 2.1. Degradation of lasalocid in manure and compost Chicken manure with no coccidiostats was obtained from a farm in Pivka, Slovenia. It was divided in two piles, one of which was mixed with wood shavings to obtain a C/N ratio of approximately
Table 1 Parameters measured in compost and manure at the beginning and end of the degradation experiments.
Manure Compost
Day Day Day Day
0 84 0 77
pH
% dry matter
% organic matter
% nitrogen
8.8 8.5 8.8 8.7
40.2 66.6 38.7 39.0
9.7 18.0 3.8 11.7
1.81 2.69 0.76 1.23
30:1. The initial concentration of lasalocid in manure was 10.6 mg kg 1 dry weight. For easier sampling during the experiment, subsamples of manure were put into nylon mesh bags (mesh size 1 mm) containing approximately 30 g of sample. The bags were put in the middle of the manure/compost pile. The experiments were performed in 1 m3 polypropylene bioreactors with perforated bottoms and an inlet for aeration (Supplementary online material, Fig. S1). One bioreactor was used per treatment. Temperature was monitored throughout the experiment. Compost was constantly aerated and moisture was adjusted weekly to approximately 60%. It was mixed when a drop in temperature below 50 °C was recorded. The manure treatment was left with no aeration or moisture adjustments and no turning. Samples in nylon mesh bags were taken at 2-day intervals for the first week and in 4-day intervals thereafter. They were stored at 20 °C until analyses. Lasalocid concentrations were expressed on a dry weight basis. Moisture content of the samples was determined by drying at 105 °C for 24 h and their organic matter was estimated by loss on ignition. Nitrogen content was determined by the Kjeldahl method and pH was measured in a 1:2 manure/water slurry (see Table 1). 2.2. Degradation in soil For the soil degradation experiments, the same manure was used as for the experiments described above, after six months of aging. The concentration of lasalocid was 3.01 mg kg 1 dry weight. Manure was used on a 5 m 10 m field where potatoes were previously grown. The mechanical and chemical soil properties are listed in Table 2. The field was divided into three plots and manure was applied at three different concentrations, corresponding to 10, 20 and 30 tonnes of manure per hectare. Before and after application, the field was cultivated with a rotary harrow down to 8– 10 cm depth. The field had been mouldboard ploughed for many years prior to the start of the experiment. Temperature and precipitation were monitored throughout the experiment at a nearby weather station (approximately 200 m from the test field). Sampling was performed using core samplers at five locations in each plot. Each sample was subdivided according to depth (0–6 cm and 6–12 cm) and the five subsamples from each depth were combined into a cumulative sample. Sampling took place every day for the first three days and at three to fourday intervals thereafter. Samples were homogenised by mixing and stored at 20 °C until analyses. The moisture content of the samples was determined by drying at 105 °C for 20 h. 2.3. Chemical analyses For lasalocid extraction, 0.5 g of fresh manure/compost or 2.5 g of soil were mixed with 5 mL isooctane-ethylacetate (9:1 v/v), shaken for 20 min, and centrifuged at 3000 rpm for 5 min. The supernatant was decanted and the samples were re-extracted with further 5 mL of isooctane-ethylacetate (9:1 v/v). The supernatants were combined and cleaned on SPE silica cartridges (JT Baker, 500 mg, 3 mL). Lasalocid was eluted with 5 mL dichloromethanemethanol (9:1 v/v). The samples were evaporated in a water bath at 45 °C under the flow of nitrogen and re-dissolved in methanol.
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Table 2 Physical and chemical properties of the soil in the experimental fields at 0–20 cm depth.
10 t ha 20 t ha 30 t ha
1 1 1
Sand (%)
Coarse silt (%)
Fine silt (%)
Total silt (%)
Clay (%)
Texture class
pH in CaCl2
P2 O5 (mg/100 g)
K2O (mg/100 g)
Org. matter (%)
C (%)
22.4 19.8 15.8
13.3 14.0 17.2
35.7 35.6 372
49.0 49.6 54.4
28.6 30.6 29.8
Silty clay–clay loam Clay loam–silty clay loam Silty clay loam
7.1 7.1 7.1
9.1 7.9 8.3
15.6 16.1 15.4
4.1 4.1 4.6
2.4 2.4 2.7
All samples were measured in triplicates. Extraction recoveries were determined on the basis of standard addition of 1, 5 or 10 mg lasalocid per gram of sample to a blank sample, followed by extraction as described above. Recoveries ranged between 82.0% and 93.8% for soil, 84.2% and 93.6% for manure, and 82.5% and 93.9% for compost. Detailed extraction recoveries are shown in Supplementary online material (Table S1). Results were corrected for average recovery of each sample matrix. Lasalocid was measured on an HPLC system Varian ProStar 363 (Varian, Walnut Creek, CA, USA) with a Phenomenax column, 5 lm, 15 4.60 mm (Phenomenex, Torrance, CA, USA) and Varian 360 fluorescent detector. The mobile phase flow rate was 1.0 mL min 1, the injection volume was 50 lL and the column was kept at 35 °C. The mobile phase consisted of 150 mL of 0.01 M acetic buffer at pH 4.0 and 850 mL of methanol. The detection was carried out at k = 420 nm, the retention time was around 12.3 min. 2.4. Data analyses The half-life of lasalocid in manure and compost was estimated using the Gustafson-Holden bi-phasic kinetic model Ct = C0(1 + bt) a where Ct and C0 are concentrations at time t and at the beginning, respectively, and a and b are the parameters of the gamma probability density function of the degradation constants. The half-life of lasalocid was calculated as (0.5 (1/a)–1)/b (Gustafson and Holden, 1990). 3. Results and discussion
Fig. 1. Degradation of lasalocid in manure (A) and compost (B). Mean concentrations and standard deviations of three independent samples are shown as black squares, temperatures in manure/compost as grey lines. Red curves represent the Gustafson–Holden bi-phasic kinetic model.
3.1. Degradation in manure and compost Unlike what Dolliver et al. (2008) observed in the case of monensin degradation, there were marked differences between the two treatments. Lasalocid in manure (Fig. 1a) degraded with a half-life of 61.8 d and did not fall below 45% of the initial concentration even after 84 d, whereas its half-life in compost (Fig. 1b) was 17.5 d and the concentrations fell below the limit of detection (10 ng g 1) after 80 d. In the compost treatment, we observed a plateau from days 14 to 32. This could possibly be ascribed to desorption of lasalocid from the matrix, but further studies would be required to confirm this assumption. It is likely that the observed decline in lasalocid concentrations is due to degradation and not to a formation of a non-extractable residue, but to prove this, a radiolabelled material should be used, which would enable the calculation of a mass balance. As has been demonstrated by Sassman and Lee (2007), lasalocid degradation only occurs in the presence of microbes. The conditions in manure during the experiment could be expected to greatly reduce microbial activity, especially because of desiccation. Water content in compost was regulated weekly and maintained at approximately 60%, while the water content of manure fell from the initial 59.8% to 33.4% at the end of the experiment (Fig. 2). The drop in moisture content in manure coincided with the observed lag in lasalocid degradation. This is in accordance with the findings reported in EFSA (2004), where no decrease in lasalo-
Fig. 2. Water content of manure and compost during the lasalocid degradation experiment.
cid concentrations was observed in samples that had dried out. Our results suggest that aging manure in a pile with no mixing and no moisture adjustment would only reduce lasalocid levels to approximately one half of the initial concentrations and when manure dries out, the degradation of lasalocid is stopped.
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Lasalocid dissipation rates in soil are shown in Fig. 3. The weather conditions during the experiment (temperature and precipitation) are shown in Fig. 4. The dissipation half-lives of lasalocid were similar regardless of the treatment and soil depth and were 3.1 ± 0.6 d. The dissipation half-lives in soil are much shorter than the degradation half-lives in manure and compost. However, losses of a chemical in soil are due not only to degradation, but also to leaching and volatilisation. The rapid decline in lasalocid concentrations in soil on the third and fourth day of the experiment coincides with precipitation events on those days. Similar was also seen on days nine and fourteen. As can also be seen in the 10 and 20 t ha 1 treatments, lasalocid concentrations rose in the lower soil layer on the ninth day of the experiment. We can therefore conclude that at least some of the losses of lasalocid can be attributed to removal with water in either dissolved or particulate form. Since lasalocid has a high affinity of sorption to soil particles (Sassman and Lee,
20
Temperature (°C)
3.2. Dissipation in soil
Percipitation Temperature
80 70 60 50
15
40 10
30 20
5 0
Precipitation (mm)
950
10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
0
Duration of experiment (days) Fig. 4. Mean daily temperature and precipitation during the field experiment with lasalocid.
2007), its removal in particulate form is more likely. The dynamics of lasalocid followed a similar pattern regardless of the initial concentration of applied manure and was much more rapid compared to degradation in manure or compost. The initial harrowing of the experimental field mixed the soil with lasalocid-containing manure down to the depth of approximately 10 cm. Lasalocid was therefore present in the lower sampling depth from the very beginning of the experiment. Since lasalocid is susceptible to photolysis (Bohn et al., 2013), we would expect it to degrade more rapidly in the top layer of soil than in the 6–12 cm layer. This was partially the case, as in the 10 and 30 t ha 1 plots, the concentrations in the two layers equalised after three and two days, respectively, despite the initially higher values in the 0–6 cm layer. In the 20 t ha 1 plot, lasalocid levels in the top soil layer were constantly higher almost until the end of the experiment. Further studies would be required, such as comparing lasalocid degradation on shaded and non-shaded areas, in order to ascertain the role of photolysis in its environmental fate. Solubility of lasalocid in water is relatively low, about 10 mg L 1 at pH 7 (Bak et al., 2013), its Koc value is 1140 (EFSA, 2010) and, as demonstrated, it is relatively short-lived in soil, so contamination of surface and ground waters with lasalocid from broiler manure is not very likely. 3.3. Environmental risk assessment of lasalocid
Fig. 3. Lasalocid concentrations in soil fertilised with broiler manure at 10 t ha 1 (A), 20t ha 1 (B) and 30t ha 1 (C). Each measurement was made in triplicates, average values and standard deviations are given, concentrations are on a dry weight basis. Red curves represent the Gustafson–Holden bi-phasic kinetic model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
With the obtained results on lasalocid degradation in manure, compost and on agricultural soil, it is now possible to calculate a more accurate risk assessment of lasalocid on agricultural soil. Broiler manure is usually aged for approximately three months before application on land. In most cases on Slovenian farms, it is simply left in a pile with no additional treatment such as aeration, moistening or mixing with plant material. We can therefore expect that about one half of the initial lasalocid levels reach the environment. In their estimates of predicted environmental concentrations (PECs) of lasalocid in soil, Hansen et al. (2009a) calculated a potential concentration of 63.4 lg kg 1 soil, stressing that no actual measured environmental concentrations are available for lasalocid. The results of our experiments on degradation in soil are within the same order (the obtained concentrations in soil after application of manure were 31.8, 57.8 and 121.7 lg kg 1 soil in plots corresponding to 10, 20 and 30 tonnes of manure per hectare, respectively). The quantity of manure applied to agricultural land is regulated in the EU by the Nitrates directive (EC, 1991), which states that in vulnerable zones annual nitrogen emissions from agricultural sources should not exceed 170 kg N ha 1. Locally, the maximum permitted amount of nitrogen is 320 kg ha 1 year 1 for grasslands that are mowed four times per year. Chicken manure
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contains high amounts of nitrogen (approximately 18 mg kg 1), so 10 tonnes of manure per hectare corresponds roughly to 170 kg N ha 1 and 20 tonnes per hectare to 320 kg N ha 1. The maximum concentration of lasalocid that can therefore realistically be expected on agricultural land is 57.8 lg kg 1 (concentration of lasalocid after application on the 20 t ha 1 experimental plot). According to the EC Technical Guidance Document (EC, 2003), the predicted no-effect concentration (PNEC) of a chemical in soil is derived from the lowest available L(E)C50 or NOEC value divided by an assessment factor. EFSA (2010) reported several toxicity studies including plant seedling emergence and growth (lowest EC50 for growth was 87.8 mg lasalocid kg 1 and NOEC was 10 mg kg 1), earthworm acute toxicity (LC50 was 71.8 mg kg 1, NOEC for weight change was 75 mg kg 1), earthworm reproduction (NOEC 41.2 mg kg 1), microbial respiration and nitrification in soil (NOEC > 5 mg kg 1). The lowest EC50 value reported for soil organisms was 4.9 mg kg 1 soil for isopod avoidance (Zˇizˇek and Zidar, 2013). EC10 in this case was 0.54 mg kg 1. Using this value and an assessment factor of 10, the PNEC for lasalocid in soil would be 54 lg kg 1. In a worst case scenario (using the maximum permitted amount of manure), the risk quotient (PEC/PNEC) for lasalocid would therefore be 1.07. This means that we can potentially expect environmental concentrations of lasalocid to be slightly higher than the no-effect levels, but only in situations where the maximum permitted amount on manure is used on land. Since manure is always aged before application on soil, lasalocid is at least partially degraded before it enters the environment. This would decrease the risk quotient below 1. Continual use of lasalocid-contaminated manure on the same area could potentially increase the risk of lasalocid. However, one year after the end of the experiment, no more lasalocid could be detected in soil. There might potentially be a greater risk in arid areas where soil is more likely to dry out, which would slow down the biotic degradation of lasalocid. Since lasalocid is photodegradable, a suitable measure of reducing its risk would be to have a delay of several days between manure application and ploughing in order to enable partial photodegradation. Acknowledgement This study was funded by the Slovenian Research Agency and the Ministry of Agriculture and the Environment of the Republic of Slovenia in the framework of the project V4-1105. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.12.032.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Degradation and dissipation of the veterinary ionophore lasalocid in manure and soil Suzana Zˇizˇek a,⇑, Martin Dobeic b, Štefan Pintaricˇ b, Primozˇ Zidar c, Silvestra Kobal d, Matej Vidrih e a
Institute of Pathology, Foresnic and Administrative Veterinary Medicine, Veterinary Faculty, University of Ljubljana, Gerbicˇeva 60, SI-1000 Ljubljana, Slovenia Institute for Environmental and Animal Hygiene with Ethology, Veterinary Faculty, University of Ljubljana, Gerbicˇeva 60, SI-1000 Ljubljana, Slovenia c Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecˇna pot 111, SI-1000 Ljubljana, Slovenia d Institute for Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbicˇeva 60, SI-1000 Ljubljana, Slovenia e Department of Agronomy, Biotechnial Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia b
h i g h l i g h t s The fate of lasalocid in poultry manure and on arable land is largely unknown. We measured lasalocid degradation in manure and compost and dissipation on land. Degradation in compost is faster than in manure. Half-life of lasalocid in soil was 3.1 ± 0.6 d. PEC/PNEC in soil could exceed 1 in a worst-case scenario if manure is not aged.
a r t i c l e
i n f o
Article history: Received 14 May 2014 Received in revised form 4 December 2014 Accepted 8 December 2014 Available online 30 December 2014 Handling Editor: Klaus Kümmerer Keywords: Lasalocid Degradation Dissipation Environmental risk assessment
a b s t r a c t Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It is excreted from the treated animals mostly in its active form and enters the environment with the use of contaminated manure on agricultural land. To properly assess the risk that lasalocid poses to the environment, it is necessary to know its environmental concentrations as well as the rates of its degradation in manure and dissipation in soil. These values are still largely unknown. A research was undertaken to ascertain the rate of lasalocid degradation in manure under different storage conditions (aging in a pile or composting) and on agricultural soil after using lasalocid-contaminated manure. The results have shown that there is considerable difference in lasalocid degradation between aging manure with no treatment (t1/2 = 61.8 ± 1.7 d) and composting (t1/2 = 17.5 ± 0.8 d). Half-lives in soil are much shorter (on average 3.1 ± 0.4 d). On the basis of the measured concentrations of lasalocid in soil after manure application, we can conclude that it can potentially be harmful to soil organisms (PEC/PNEC ratio of 1.18), but only in a worst-case scenario of using the maximum permissible amount of manure and immediately after application. To make certain that no harmful effects occur, composting is recommended. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Extensive use of manure burdens the environment not only with large amounts of nitrogen and metals, but also with residues of veterinary pharmaceuticals and feed additives (Halling-Sørensen et al., 1998). Manure from treated animals in farms usually contains numerous pharmaceuticals and their metabolites. Their introduc-
⇑ Corresponding author at: Laboratory for Environmental Research, University of Nova Gorica, Vipavska 13, SI-5000 Nova Gorica, Slovenia. Tel.: +386 5 3315 388. E-mail address: [email protected] (S. Zˇizˇek). http://dx.doi.org/10.1016/j.chemosphere.2014.12.032 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
tion into the environment is not controlled, a problem that has been neglected for decades (Boxall, 2004). Coccidiosis is a rapidly spreading and often fatal protozoal infection in poultry that can cause serious economic losses. In the European Union, coccidiostats are authorised as feed additives and of the estimated 40.65 million tonnes of feed produced, about 18.33 million tonnes contain coccidiostats (EC, 2008). Broilers and turkeys are treated with coccidiostats almost their entire life. Lasalocid, one of the most frequently used coccidiostats, is a natural ionophore antibiotic produced by the bacterium Streptomyces lasaliensis. In treated animals lasalocid is only partially metabolised and is excreted at least partially in the active form (EFSA, 2004, 2010).
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EFSA (2004) reported that 74–83% of lasalocid in broiler excreta is in the active form, while another study on turkey litter (EFSA, 2010) found only 10% of the administered dose as the parent compound. A large number of compounds were detected in chicken and turkeys excreta with only two metabolites accounting for more than 5% of 14C. Lasalocid degradation in manure has only been reported in one study (EFSA, 2004), which stated a loss greater than 75% after 30 d. When in the environment, lasalocid undergoes mostly biotic degradation (Vertesy et al., 1987; Sassman and Lee, 2007; Hansen et al., 2009a, 2012). The rate of decay, which is much slower in anaerobic conditions, depends on soil properties such as organic content, moisture, temperature and pH (Sassman and Lee, 2007). The reported half-lives of lasalocid in soil were from 0.6 to 14.2 d (EFSA, 2004; Sassman and Lee, 2007), but all were obtained under laboratory conditions. However, no information is available in literature on the degradation of lasalocid in field conditions or in manure before application to soil. Sassman and Lee (2007) studied its degradation in different types of soil under laboratory conditions by adding lasalocid in aqueous solution. They obtained half-lives of approximately 4 d in soil rich in organic carbon (OC) and 1.5 d in low-OC soil. Manure amendment did not change the degradation rate. No degradation of lasalocid was observed in sterilised soil. Hansen et al. (2009b) highlighted this lack of available data and stated that a reliable risk assessment of lasalocid cannot be calculated without further investigation. Degradation of monensin and other structurally similar ionophores has been studied in more detail. Donoho (1984) found that monensin is biodegradable in cattle manure and soil and declines relatively slowly in incubated faeces under relatively anaerobic conditions, but is degraded fairly rapidly in a manure pile under natural weathering conditions. Its half-life in soil was approximately 13 d. Schlüsener et al. (2006) found a half-life of salinomycin in livestock manure of 6 d. Average monensin half-life in spiked turkey litter (Dolliver et al., 2008) was 17 d and depended on treatment of manure (22 d in manure pile, 19 d in manure with weekly water additions and mixing and 11 d when using vessel composting). Half-life of monensin in soil was between 22.7 d in low-OC soil and 4.2 d in higher-OC soil and no degradation was observed in dried soil (Yoshida et al., 2010). Salinomycin degrades rapidly with a half-life of 1.3 d in broiler compost and 4.0 d in manure with no treatment (Ramaswamy et al., 2010). Experiments on salinomycin in lab-scale soil bioreactors (Hansen et al., 2012) showed rapid degradation in aerobic conditions (degraded after 68 h) but very limited degradation under anaerobic conditions. Sun et al. (2014) studied the degradation in broil litter and soil of monensin, salinomycin and narasin. They found that degradation does not occur in broiler litter with low moisture content (24% and 40% moisture). Narasin and salinomycin degraded in broiler litter with higher water content (72%), but monensin remained stable during the 14-day incubation. In soil microcosms, the half-lives of the studied ionophores were between 3.3 and 5 d. The present study was therefore undertaken in order to measure the degradation rates of lasalocid in manure and in soil and to ascertain the risk lasalocid-contaminated manure represents to soil-dwelling organisms and to agricultural ecosystems in general.
2. Materials and methods 2.1. Degradation of lasalocid in manure and compost Chicken manure with no coccidiostats was obtained from a farm in Pivka, Slovenia. It was divided in two piles, one of which was mixed with wood shavings to obtain a C/N ratio of approximately
Table 1 Parameters measured in compost and manure at the beginning and end of the degradation experiments.
Manure Compost
Day Day Day Day
0 84 0 77
pH
% dry matter
% organic matter
% nitrogen
8.8 8.5 8.8 8.7
40.2 66.6 38.7 39.0
9.7 18.0 3.8 11.7
1.81 2.69 0.76 1.23
30:1. The initial concentration of lasalocid in manure was 10.6 mg kg 1 dry weight. For easier sampling during the experiment, subsamples of manure were put into nylon mesh bags (mesh size 1 mm) containing approximately 30 g of sample. The bags were put in the middle of the manure/compost pile. The experiments were performed in 1 m3 polypropylene bioreactors with perforated bottoms and an inlet for aeration (Supplementary online material, Fig. S1). One bioreactor was used per treatment. Temperature was monitored throughout the experiment. Compost was constantly aerated and moisture was adjusted weekly to approximately 60%. It was mixed when a drop in temperature below 50 °C was recorded. The manure treatment was left with no aeration or moisture adjustments and no turning. Samples in nylon mesh bags were taken at 2-day intervals for the first week and in 4-day intervals thereafter. They were stored at 20 °C until analyses. Lasalocid concentrations were expressed on a dry weight basis. Moisture content of the samples was determined by drying at 105 °C for 24 h and their organic matter was estimated by loss on ignition. Nitrogen content was determined by the Kjeldahl method and pH was measured in a 1:2 manure/water slurry (see Table 1). 2.2. Degradation in soil For the soil degradation experiments, the same manure was used as for the experiments described above, after six months of aging. The concentration of lasalocid was 3.01 mg kg 1 dry weight. Manure was used on a 5 m 10 m field where potatoes were previously grown. The mechanical and chemical soil properties are listed in Table 2. The field was divided into three plots and manure was applied at three different concentrations, corresponding to 10, 20 and 30 tonnes of manure per hectare. Before and after application, the field was cultivated with a rotary harrow down to 8– 10 cm depth. The field had been mouldboard ploughed for many years prior to the start of the experiment. Temperature and precipitation were monitored throughout the experiment at a nearby weather station (approximately 200 m from the test field). Sampling was performed using core samplers at five locations in each plot. Each sample was subdivided according to depth (0–6 cm and 6–12 cm) and the five subsamples from each depth were combined into a cumulative sample. Sampling took place every day for the first three days and at three to fourday intervals thereafter. Samples were homogenised by mixing and stored at 20 °C until analyses. The moisture content of the samples was determined by drying at 105 °C for 20 h. 2.3. Chemical analyses For lasalocid extraction, 0.5 g of fresh manure/compost or 2.5 g of soil were mixed with 5 mL isooctane-ethylacetate (9:1 v/v), shaken for 20 min, and centrifuged at 3000 rpm for 5 min. The supernatant was decanted and the samples were re-extracted with further 5 mL of isooctane-ethylacetate (9:1 v/v). The supernatants were combined and cleaned on SPE silica cartridges (JT Baker, 500 mg, 3 mL). Lasalocid was eluted with 5 mL dichloromethanemethanol (9:1 v/v). The samples were evaporated in a water bath at 45 °C under the flow of nitrogen and re-dissolved in methanol.
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Table 2 Physical and chemical properties of the soil in the experimental fields at 0–20 cm depth.
10 t ha 20 t ha 30 t ha
1 1 1
Sand (%)
Coarse silt (%)
Fine silt (%)
Total silt (%)
Clay (%)
Texture class
pH in CaCl2
P2 O5 (mg/100 g)
K2O (mg/100 g)
Org. matter (%)
C (%)
22.4 19.8 15.8
13.3 14.0 17.2
35.7 35.6 372
49.0 49.6 54.4
28.6 30.6 29.8
Silty clay–clay loam Clay loam–silty clay loam Silty clay loam
7.1 7.1 7.1
9.1 7.9 8.3
15.6 16.1 15.4
4.1 4.1 4.6
2.4 2.4 2.7
All samples were measured in triplicates. Extraction recoveries were determined on the basis of standard addition of 1, 5 or 10 mg lasalocid per gram of sample to a blank sample, followed by extraction as described above. Recoveries ranged between 82.0% and 93.8% for soil, 84.2% and 93.6% for manure, and 82.5% and 93.9% for compost. Detailed extraction recoveries are shown in Supplementary online material (Table S1). Results were corrected for average recovery of each sample matrix. Lasalocid was measured on an HPLC system Varian ProStar 363 (Varian, Walnut Creek, CA, USA) with a Phenomenax column, 5 lm, 15 4.60 mm (Phenomenex, Torrance, CA, USA) and Varian 360 fluorescent detector. The mobile phase flow rate was 1.0 mL min 1, the injection volume was 50 lL and the column was kept at 35 °C. The mobile phase consisted of 150 mL of 0.01 M acetic buffer at pH 4.0 and 850 mL of methanol. The detection was carried out at k = 420 nm, the retention time was around 12.3 min. 2.4. Data analyses The half-life of lasalocid in manure and compost was estimated using the Gustafson-Holden bi-phasic kinetic model Ct = C0(1 + bt) a where Ct and C0 are concentrations at time t and at the beginning, respectively, and a and b are the parameters of the gamma probability density function of the degradation constants. The half-life of lasalocid was calculated as (0.5 (1/a)–1)/b (Gustafson and Holden, 1990). 3. Results and discussion
Fig. 1. Degradation of lasalocid in manure (A) and compost (B). Mean concentrations and standard deviations of three independent samples are shown as black squares, temperatures in manure/compost as grey lines. Red curves represent the Gustafson–Holden bi-phasic kinetic model.
3.1. Degradation in manure and compost Unlike what Dolliver et al. (2008) observed in the case of monensin degradation, there were marked differences between the two treatments. Lasalocid in manure (Fig. 1a) degraded with a half-life of 61.8 d and did not fall below 45% of the initial concentration even after 84 d, whereas its half-life in compost (Fig. 1b) was 17.5 d and the concentrations fell below the limit of detection (10 ng g 1) after 80 d. In the compost treatment, we observed a plateau from days 14 to 32. This could possibly be ascribed to desorption of lasalocid from the matrix, but further studies would be required to confirm this assumption. It is likely that the observed decline in lasalocid concentrations is due to degradation and not to a formation of a non-extractable residue, but to prove this, a radiolabelled material should be used, which would enable the calculation of a mass balance. As has been demonstrated by Sassman and Lee (2007), lasalocid degradation only occurs in the presence of microbes. The conditions in manure during the experiment could be expected to greatly reduce microbial activity, especially because of desiccation. Water content in compost was regulated weekly and maintained at approximately 60%, while the water content of manure fell from the initial 59.8% to 33.4% at the end of the experiment (Fig. 2). The drop in moisture content in manure coincided with the observed lag in lasalocid degradation. This is in accordance with the findings reported in EFSA (2004), where no decrease in lasalo-
Fig. 2. Water content of manure and compost during the lasalocid degradation experiment.
cid concentrations was observed in samples that had dried out. Our results suggest that aging manure in a pile with no mixing and no moisture adjustment would only reduce lasalocid levels to approximately one half of the initial concentrations and when manure dries out, the degradation of lasalocid is stopped.
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Lasalocid dissipation rates in soil are shown in Fig. 3. The weather conditions during the experiment (temperature and precipitation) are shown in Fig. 4. The dissipation half-lives of lasalocid were similar regardless of the treatment and soil depth and were 3.1 ± 0.6 d. The dissipation half-lives in soil are much shorter than the degradation half-lives in manure and compost. However, losses of a chemical in soil are due not only to degradation, but also to leaching and volatilisation. The rapid decline in lasalocid concentrations in soil on the third and fourth day of the experiment coincides with precipitation events on those days. Similar was also seen on days nine and fourteen. As can also be seen in the 10 and 20 t ha 1 treatments, lasalocid concentrations rose in the lower soil layer on the ninth day of the experiment. We can therefore conclude that at least some of the losses of lasalocid can be attributed to removal with water in either dissolved or particulate form. Since lasalocid has a high affinity of sorption to soil particles (Sassman and Lee,
20
Temperature (°C)
3.2. Dissipation in soil
Percipitation Temperature
80 70 60 50
15
40 10
30 20
5 0
Precipitation (mm)
950
10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
0
Duration of experiment (days) Fig. 4. Mean daily temperature and precipitation during the field experiment with lasalocid.
2007), its removal in particulate form is more likely. The dynamics of lasalocid followed a similar pattern regardless of the initial concentration of applied manure and was much more rapid compared to degradation in manure or compost. The initial harrowing of the experimental field mixed the soil with lasalocid-containing manure down to the depth of approximately 10 cm. Lasalocid was therefore present in the lower sampling depth from the very beginning of the experiment. Since lasalocid is susceptible to photolysis (Bohn et al., 2013), we would expect it to degrade more rapidly in the top layer of soil than in the 6–12 cm layer. This was partially the case, as in the 10 and 30 t ha 1 plots, the concentrations in the two layers equalised after three and two days, respectively, despite the initially higher values in the 0–6 cm layer. In the 20 t ha 1 plot, lasalocid levels in the top soil layer were constantly higher almost until the end of the experiment. Further studies would be required, such as comparing lasalocid degradation on shaded and non-shaded areas, in order to ascertain the role of photolysis in its environmental fate. Solubility of lasalocid in water is relatively low, about 10 mg L 1 at pH 7 (Bak et al., 2013), its Koc value is 1140 (EFSA, 2010) and, as demonstrated, it is relatively short-lived in soil, so contamination of surface and ground waters with lasalocid from broiler manure is not very likely. 3.3. Environmental risk assessment of lasalocid
Fig. 3. Lasalocid concentrations in soil fertilised with broiler manure at 10 t ha 1 (A), 20t ha 1 (B) and 30t ha 1 (C). Each measurement was made in triplicates, average values and standard deviations are given, concentrations are on a dry weight basis. Red curves represent the Gustafson–Holden bi-phasic kinetic model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
With the obtained results on lasalocid degradation in manure, compost and on agricultural soil, it is now possible to calculate a more accurate risk assessment of lasalocid on agricultural soil. Broiler manure is usually aged for approximately three months before application on land. In most cases on Slovenian farms, it is simply left in a pile with no additional treatment such as aeration, moistening or mixing with plant material. We can therefore expect that about one half of the initial lasalocid levels reach the environment. In their estimates of predicted environmental concentrations (PECs) of lasalocid in soil, Hansen et al. (2009a) calculated a potential concentration of 63.4 lg kg 1 soil, stressing that no actual measured environmental concentrations are available for lasalocid. The results of our experiments on degradation in soil are within the same order (the obtained concentrations in soil after application of manure were 31.8, 57.8 and 121.7 lg kg 1 soil in plots corresponding to 10, 20 and 30 tonnes of manure per hectare, respectively). The quantity of manure applied to agricultural land is regulated in the EU by the Nitrates directive (EC, 1991), which states that in vulnerable zones annual nitrogen emissions from agricultural sources should not exceed 170 kg N ha 1. Locally, the maximum permitted amount of nitrogen is 320 kg ha 1 year 1 for grasslands that are mowed four times per year. Chicken manure
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contains high amounts of nitrogen (approximately 18 mg kg 1), so 10 tonnes of manure per hectare corresponds roughly to 170 kg N ha 1 and 20 tonnes per hectare to 320 kg N ha 1. The maximum concentration of lasalocid that can therefore realistically be expected on agricultural land is 57.8 lg kg 1 (concentration of lasalocid after application on the 20 t ha 1 experimental plot). According to the EC Technical Guidance Document (EC, 2003), the predicted no-effect concentration (PNEC) of a chemical in soil is derived from the lowest available L(E)C50 or NOEC value divided by an assessment factor. EFSA (2010) reported several toxicity studies including plant seedling emergence and growth (lowest EC50 for growth was 87.8 mg lasalocid kg 1 and NOEC was 10 mg kg 1), earthworm acute toxicity (LC50 was 71.8 mg kg 1, NOEC for weight change was 75 mg kg 1), earthworm reproduction (NOEC 41.2 mg kg 1), microbial respiration and nitrification in soil (NOEC > 5 mg kg 1). The lowest EC50 value reported for soil organisms was 4.9 mg kg 1 soil for isopod avoidance (Zˇizˇek and Zidar, 2013). EC10 in this case was 0.54 mg kg 1. Using this value and an assessment factor of 10, the PNEC for lasalocid in soil would be 54 lg kg 1. In a worst case scenario (using the maximum permitted amount of manure), the risk quotient (PEC/PNEC) for lasalocid would therefore be 1.07. This means that we can potentially expect environmental concentrations of lasalocid to be slightly higher than the no-effect levels, but only in situations where the maximum permitted amount on manure is used on land. Since manure is always aged before application on soil, lasalocid is at least partially degraded before it enters the environment. This would decrease the risk quotient below 1. Continual use of lasalocid-contaminated manure on the same area could potentially increase the risk of lasalocid. However, one year after the end of the experiment, no more lasalocid could be detected in soil. There might potentially be a greater risk in arid areas where soil is more likely to dry out, which would slow down the biotic degradation of lasalocid. Since lasalocid is photodegradable, a suitable measure of reducing its risk would be to have a delay of several days between manure application and ploughing in order to enable partial photodegradation. Acknowledgement This study was funded by the Slovenian Research Agency and the Ministry of Agriculture and the Environment of the Republic of Slovenia in the framework of the project V4-1105. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.12.032.
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