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Environmental risk assessment of ionophores
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Trends in Analytical Chemistry, Vol. 28, No. 5, 2009
Environmental risk assessment of ionophores Martin Hansen, Kristine A. Krogh, Erland Bjo¨rklund, Asbjørn Brandt, Bent Halling-Sørensen Following European guidelines, this article presents an environmental risk assessment for four ionophores (monensin, salinomycin, narasin and lasalocid) based on predicted environmental exposure from broiler production in two scenarios. In a third scenario, measured occurrence data are used to characterize the potential risk. Findings show that predicted environmental concentrations in all environmental compartments and measured environmental concentrations in sediments are above predicted no-effect concentrations, so ionophores might pose an environmental risk. The toxicological effect data reviewed revealed that very limited data exist and that long-term effect data and chronic effect data are not available. ª 2009 Elsevier Ltd. All rights reserved. Keywords: Anticoccidial; Coccidiostat; Eco-toxicology; Environmental exposure; Lasalocid; Monensin; Narasin; Predicted environmental concentration; Predicted no-effect concentration; Salinomycin
1. Introduction Martin Hansen*, Kristine A. Krogh, Erland Bjo¨rklund, Bent Halling-Sørensen Section of Toxicology and Environmental Chemistry, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Asbjørn Brandt Section of Veterinary Medicines, The Danish Medicines Agency, Axel Heides Gade 1, DK-2300 Copenhagen, Denmark
*
Corresponding author. Tel.: +45 35 33 62 65; Fax: +45 35 30 60 13; E-mail: [email protected]
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Ionophores are a sub-group of anticoccidials used extensively in livestock production (i.e. poultry, cattle, swine, sheep and rabbit) and, as a result, they are considered to be emerging environmental contaminants [1–3]. For these compounds, only a few studies report data on occurrence [4–8], fate [9] and eco-toxicological effects [10–13]. No complete assessment is available that reviews the environmental impact of ionophores. In another article in this issue [3], we outlined the status of the current development of analytical methods targeted to environmental samples and reported that the few measured occurrence data on ionophores were found at lg/kg, ng/L and lg/kg levels in manure, surface waters and sediments, respectively. At present, concentration levels of ionophores in the soil compartment have not been reported. Moreover, the available occurrence data could reflect a possible build up of ionophores in lipophilic compartments (e.g., soils and sediments [3]). Unfortunately,
only limited information on consumption data of ionophores is available and many countries do not monitor their usage. Table 1 shows the available consumption data from poultry, cattle, swine, sheep, and rabbit production, revealing that ionophores are widely and heavily used, and that salinomycin, monensin, narasin and lasalocid are the most frequently applied [14–17]. Ionophores act as antiporters (mode of action) by entrapping cations (preferably sodium or potassium), thereby generating neutral zwitterions. These complexes are transported across prokaryotic and eukaryotic cell membranes in exchange for protons, and, accordingly, disrupt ion gradients and, ultimately, cause energy reduction and cell death [12,18–22]. In the European Union (EU), ionophores and other anticoccidial agents are regulated in accordance with the directives on feed additives and may not be put on the market unless a proper authorization has been given by the European Commission based on a scientific evaluation by the European Food Safety Authority (EFSA). Market authorization depends on demonstrating that the ionophores have no harmful effects on human and animal health and the environment [23,24]. These scientific evaluations are performed by the EFSA panel on additives and products or substances used in animal feed [25]. In this article, environmental risk assessment (ERA) is based on an approach suggested by the EFSA panel [26] following three harmonized guidance documents: phase I – exposure assessment [27]; phase II – risk assessment [28]; and, additional guidance [29].
0165-9936/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.02.015
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Table 1. Consumption data on ionophores used in livestock production and predicted worst-case concentrations based on arable area Region Republic of Korea
Denmark
Norway
Ionophore Salinomycin Monensin Lasalocid Salinomycin Narasin Monensin Lasalocid Narasin Monensin Lasalocid
Amount (kg) a)
639,170 136,140a) 66,061a) 10,070b) 1625b) 840b) 243b) 5615c) 889c) 13c)
Arable area
PECsoil (lg/kg)
9,819,000
16.58%
2004
4,239,400
52.59%
2006
30,744,200
2.70%
131 27.9 13.5 1.51 0.24 0.13 0.04 2.25 0.36 0.01
Year
Area (ha)
2006
PECsoil based on 0.2 m ploughing depth. a) Estimated active substance from product sales [16]. b) [17]. c) [14].
The EFSA guidance document [26] on ERA of feed additives follows a tiered approach starting with a decision tree, resulting in an initial worst-case-scenario calculation to give a predicted environmental concentration (PEC) for ionophores in soil, soil-pore water, groundwater, surface water and sediment. If these PECs are above pre-defined trigger values (10 lg/kg soil and 0.1 lg/L in groundwater), they are compared to predicted no-effect concentrations (PNECs) derived from eco-toxicological effect data adjusted with appropriate assessment factors (AFs). Where the PEC/PNEC ratio is
>1, the PEC is refined taking into account losses of the ionophore (metabolism and environmental dissipation mechanisms) and, finally, compared to the PNEC again to indicate if the ionophore poses a potential environmental risk. Fig. 1 outlines this entire ERA procedure. The first aim of this article is to give an overview of the published eco-toxicological data on four ionophores (viz monensin, salinomycin, narasin and lasalocid). The second aim is to present a tiered ERA based on PEC and compare that with existing effect data to assess the potential environmental risk. ERA is also performed by comparing measured environmental concentrations (MECs) with the same effect data. PEC estimations are based on the amount of the individual compounds applied in the EU and only ionophores that are approved for use in poultry production, according to existing EU regulations. The ERA of ionophores comprises three superior scenarios: Scenario I is a worst-case scenario, which predicts the environmental exposure of ionophores in agricultural soils amended with manure from broilers treated with ionophores in agreement with the European regulations; Scenario II is a refinement of Scenario I, where metabolism in the broilers and other environmental dissipation mechanisms are taken into account, leading to a refined PEC; and, Scenario III assesses the potential environmental risk on the basis of previously published occurrence data (MECs) reported in the scientific literature.
2. Materials and methods Figure 1. Overview of the environmental risk-assessment approach.
This section outlines the subsequent results and discussion sections in the ERA of ionophores. Sub-section 2.1. reports on the eco-toxicological effect data obtained so
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Table 2. Acute effect studies on ionophores in the soil environment Test organism
Test method
Effect concentration
Ref.
Monensin
Folsomia fimetaria (springtail) Enchytraeus crypticus (enchytraeid) Eisenia foetida andrei (earthworm) Raphanus sativus (radish) Soil micro-organisms
ISO 11267 (reproduction, 21 days) ISO 16387 (reproduction, 21 days) OECD 207 (mortality, 14 days) Non-standardized (emergence, 14 days) OECD 216 (nitrification) OECD 217 (respiration)
EC50 591 [254–927] mg/kg EC50 356 [95–617] mg/kg LC50 56 mg/kga) LC50 9.8 mg/kg NOEC >5 mg/kg
[12] [12] [37] [36] [37]
Salinomycin
Raphanus sativus (radish) Eisenia foetida andrei (earthworm) Soil micro-organisms
Non-standardized (growth rate, 14 days) OECD 207 (mortality, 14 days) OECD 216 (nitrification) OECD 217 (respiration)
LC50 1.3 mg/kg LC50 71 mg/kg NOEC >2.3 mg/kg
[46] [46] [46]
Narasin
Raphanus sativus (radish) Eisenia foetida andrei (earthworm) Soil micro-organisms
OECD OECD OECD OECD
208 207 216 217
(emergence) (mortality, 14 days) (nitrification) (respiration)
LC50 5.07 mg/kg LC50 46.4 mg/kg NOEC 17.4 mg/kg
[39] [39] [39]
Lasalocid
Lolium perenne (perennial ryegrass) Eisenia foetida andrei (earthworm) Soil micro-organisms
OECD OECD OECD OECD
208 207 216 217
(emergence, 18 days) (mortality, 14 days) (nitrification) (respiration)
EC50 87.8 mg/kg LC50 71.8 mg/kg NOEC >5 mg/kg
[40] [40] [40]
ISO, Guideline number from the International Organization for Standardization; OECD, Guideline number from the Organization for Economic Co-operation and Development; EC50, Effect concentration where half of population is affected; LC50, Concentration where half of population is killed; NOEC, No observed effect concentration. a) Normalized to 5% organic carbon content. 95%-confidential intervals given in brackets.
Table 3. Acute effect studies on ionophores in the aquatic environment Test organism Monensin
Salinomycin
Test method
Effect concentration
Ref.
Lemna gibba (macrophyte, floating) Myriophyllum spicatum (macrophyte, submersed) Selenastrum subspicatus (algae)
ASTM guideline [47] (growth, 7 days) Mesocosms (growth rate, 35 days)
EC50 0.998 [0.955–1.042] mg/L EC50 0.197 [0.042–0.353] mg/L
[10] [13]
OECD 201 (growth rate and biomass)
[36]
Pseudokirchneriella subcapitata (algae)
OECD 201 (growth rate and biomass)
Daphnia magna (crustacean) Oncorhynchus mykiss (fish)
OECD 202 (immobility) OECD 203 (mortality, 96 h)
EC50 0.98 mg/L (growth rate) EC50 4.3 mg/L (biomass) EC50 3.41 mg/L (growth rate) EC50 1.73 mg/L (biomass) EC50 7.29 mg/L LC50 1.88 mg/L
Selenastrum capricornutum (algae)
OECD 201 (growth rate and biomass, 72 h) OECD 202 (immobility, 48 h) OECD 203 (mortality, 96 h)
EC50 EC50 EC50 LC50
3.01 mg/L (growth rate) 2.09 mg/L (biomass) 13.3 mg/L 1.14 mg/L
[46]
EC50 EC50 EC50 LC50 EC50 EC50 EC50 LC50
2.91 mg/L (growth rate) 0.77 mg/L (biomass) 20.6 mg/L 2.23 mg/L 3.1 mg/L (growth rate) 2.0 mg/L (biomass) 5.4 mg/L 2.5 mg/L
[39]
Daphnia magna (crustacean) Oncorhynchus mykiss (fish) Narasin
Selenastrum capricornutum (algae)
Lasalocid
Daphnia magna (crustacean) Oncorhynchus mykiss (fish) Selenastrum subspicatus (algae)
OECD 201 (growth rate and biomass, 72 h) OECD 202 (immobility, 48 h) OECD 203 (mortality, 96 h) OECD 201 (growth rate and biomass)
Daphnia magna (crustacean) Brachydanio rerio (fish)
OECD 202 (immobility, 48 h) OECD 203 (mortality, 96 h)
[37] [37] [37]
[46] [38]
[39] [39] [40] [40] [40]
ASTM, American Society for Testing and Materials guidelines; OECD, Guideline number from the Organization for Economic Co-operation and Development. 95%-confidenc intervals given in brackets. ISO, Guideline number from the International Organization for Standardization; EC50, Effect concentration where half of population is affected; LC50, Concentration where half population is killed.
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far, while sub-section 2.2. describes in detail the methodologies applied in the three scenarios. 2.1. Eco-toxicological effect data The available eco-toxicological data for the ionophores studied are very limited, as reported in Tables 2 and 3, which show data for organisms living in soil and water, respectively. Only short-term effect data are presented, since no chronic effect data are available. Few peer-reviewed eco-toxicological studies have been performed, and only with monensin [10–13]. Two of these studies were mesocosm studies that attempted to mimic minor eco-systemic effects [11,13]. Other sources of eco-toxicological studies were scientific interpretations or opinions reported by the EFSA panel [25]. Their reports were based on effect studies of ionophores provided by the feed-additive manufacturers. Some of the studies followed standardized tests, and this section includes the usable information extracted from these reports. For test organisms living in soil, the acute toxicological level was in the range 5.07–591 mg/kg, with radish being the most sensitive species and springtails the most insensitive of the species tested. No-observed-effect concentrations (NOECs) on respiration and nitrification tests were obtained on soil micro-organisms and found to be in the range of 2–17 mg/kg [25]. Lethal-effect concentrations for the classic soil-test organisms, earthworms, were 46.4–71.8 mg/kg for the four compounds. No effect data exist on dung-dwelling organisms, which might be vulnerable, as was observed for other antiparasiticides (e.g., ivermectin [30]). Similarly, no data was available for sediment-dwelling organisms. The acute toxicological level for aquatic living organisms (e.g., algae, crustaceans and fish) was in the range 0.77–20.6 mg/L for all four compounds. In general, algae were the most sensitive species, while crustaceans were the least sensitive. Monensin was tested towards macrophytes, showing effect concentrations in the range 0.197–0.998 mg/L for these sensitive aquatic organisms.
2.1.1. Predicted no-effect concentrations. The PNECs were derived applying an assessment factor (AF) to the short-term effect data [26]. For the terrestrial compartment, three taxonomic groups (plants, soil-dwelling organisms and micro-organisms) were tested, so that the environmental risk could be established. The most sensitive species were plants (radish) for monensin, salinomycin and narasin, while lasalocid was found to be most sensitive towards earthworms (Table 4). By using an AF of 100, as recommended by the European Medicines Agency [28], the PNEC value was found. Similarly, three taxonomic groups (algae, crustaceans and fish) represented the aquatic compartment, and the fish species were found to give the lowest PNECs using an AF of 1000 [28]. Tables 4 and 5 list the PNEC values derived from results of the assessment. For estimation of the potential risk of the sediment compartment, the PNECsediment values were derived from the PNECaquatic, as suggested by the European Medicines Agency [29], since no effect data were available for sediment-dwelling organisms. The calculations were based on equilibrium partitioning, so they depended on Koc (Equation 9, Supplementary material available in the web version). Values of PNECsediment were in the range 3.44–36.7 lg/kg of wet weight (wwt) sediment (Table 4). 2.2. Environmental risk-assessment scenarios Scenario I predicts the worst-case environmental exposure on the basis of doses applied in European broiler production. Scenario II is a refinement of Scenario I taking into account elimination of the parent ionophore. Scenario III utilizes published environmental occurrence data (MECs). In all scenarios, the risk is characterized using a risk quotient (RQ) as the ratio between environmental concentrations obtained (PECs or MECs) to the PNECs [26]. 2.2.1. Basic set of parameters. Broilers have a life cycle of 35–45 days with an average life of 41 days (this value
Table 4. Scenario I with predicted environmental concentrations (PECs) for ionophores related to predicted no-effect concentrations (PNECs) giving risk quotients (RQs) for soil, surface water and sediment
Monensin Salinomycin Narasin Lasalocid
Dose (mg/kg feed)*
PECsoil (lg/kg)
PECgw (lg/L)
PECsw (lg/L)
PECsed (lg/kg)*
125 70 70 125
650 364 364 650
280 157 157 50
28 16 16 5.0
67 38 38 70
Terrestrial
Aquatic
L(E)C50 (mg/kg)
AF
PNEC (lg/kg)
RQ
L(E)C50 (mg/L)
9.8 1.3 5.07 71.8
100 100 100 100
98 13 51 718
6.6 28 7.2 0.9
1.88 1.14 2.23 2.5
AF 1000 1000 1000 1000
Sediment
PNEC (lg/L)
RQ
PNEC (lg/kg)*
RQ
1.88 1.14 2.23 2.5
15 14 7.0 2.0
5.68 3.44 6.73 36.7
12 11 5.6 1.9
Model parameters given in Supplementary material available in web version: Koc 125 L/kg for monensin, salinomycin and narasin; Koc 732 L/kg for lasalocid; PECgw, PEC in groundwater (equals PECporewater); PECsw, PEC in surface water; PECsed, PEC in sediment. Other model parameters are also given in Supplementary material: AF, Assessment factor; RQ, Risk quotient (PEC/PNEC ratio). * Value given on wet weight (wwt) basis. PECsoil is the steady-state concentration obtained after the sixth application of manure (at day 410).
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Table 5. Scenario II with refined predicted environmental concentrations (PECs) after six consecutive applications of manure (every 82 days) to the same agricultural soil (taking into account metabolism, manure and soil-dissipation mechanisms)
Monensin Salinomycin Narasin Lasalocid
PECsoil (lg/kg)
PECgw (lg/L)
PECsw (lg/L)
PECsed (lg/kg)*
63.4 35.5 35.5 63.4
27.3 15.3 15.3 4.9
2.7 1.5 1.5 0.49
6.6 3.7 3.7 6.8
Terrestrial
Aquatic
Sediment
PNEC (lg/kg)
RQ
PNEC (lg/L)
RQ
PNEC (lg/kg)*
RQ
98 13 51 718
0.6 2.7 0.7 0.1
1.88 1.14 2.23 2.5
1.5 1.3 0.7 0.2
5.68 3.44 6.73 36.7
1.2 1.1 0.5 0.2
Model conditions as in Table 4, but with 81% excretion of parent compound and dissipation half-lives in manure of 22 days with manure dissipating (for 82 days) before adding further manure to the soil. Also, dissipation in soil considered with a half-life of 49 days. * Value given on wet weight (wwt) basis.
is used in the following) [29,31], and, throughout their life, they are constantly treated with ionophores (dose range varied between ionophores and was in the range 50–125 mg/kg of dry weight (dwt) feed) [25] with a withdrawal period of 1–5 days before slaughter [25]. We presumed that the broilers received the maximum allowed dose of the specific ionophore and were treated their entire life cycle (i.e. the withdrawal period of ionophores before slaughter is excluded). On average, the feed intake was 29 kg dwt feed/broiler/year (i.e. 3.26 kg dwt feed per broiler) and the excretion rate was 0.36 kg N/broiler/year [26]. Supplementary material, available in the web version, contains the range of baseset model parameters and equations to give PECs, as defined in the EFSA guidance document [26]. 2.2.2. Scenario I – Worst-case exposure. Worst-case PECs were calculated by assuming that all ingested ionophores were excreted as parent compound and that broiler manure (170 kg N/ha/year) was applied to the agricultural soil in one application [26]. PECsoil was calculated based on the dose of the ionophores and allowed nitrogen levels. The concentration in pore water (PECporewater) was calculated using sorption-distribution coefficient Kd (calculated via Koc) between soil and water. The concentration in groundwater (PECgroundwater) was defined as the value of pore water [26,29,32]. PECsurfacewater was calculated using a dilution factor of 10 compared to PECporewater [26,32]. However, other guidelines recommended a dilution factor of 3 [29]. PECsediment was calculated on the partitioning between surface water and sediment (estimated from Koc) [26,32]. 2.2.3. Scenario II – Refined exposure. In this scenario, the PECs from Scenario I were refined by considering the metabolism of ionophores in the broiler and dissipation in manure, soil and water. Observing two succeeding batches of broilers from the same barn, the first batch of broilers had a life cycle from day 0 to day 41, and the second batch of broilers had a life cycle from day 41 to day 82. When being produced, the manure was not re538
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moved from the barn throughout the life cycle of the broiler. At day 41, the first batch of broilers was slaughtered and the manure was collected and stored in piles or tanks, and the life cycle of the second batch broilers started. At day 82, the manure from the second batch was removed (and broilers were slaughtered), mixed with the manure from first batch and spread onto agricultural land. As a result, the broiler manure was applied every 82 days to the same agricultural soil with an average of 38 kg N/ha, thereby resulting in a total of 170 kg N/ha/year. Dissipation half-lives in manure were demonstrated to be 5 days for salinomycin in pig manure [33]. However, pig manure differs from broiler manure [34]. In another study, monensin has a half-life of 22 days in turkey manure [35]. This latter first-order decay value was used. The EFSA panel has evaluated the metabolism of ionophores in broilers. Total excreted amounts of parent ionophore relative to dose showed great variations (i.e. 10–81% for monensin [36,37], 8–67% for salinomycin [38], 30% for narasin [39] and 74–77% for lasalocid [40]). In Scenario II, we chose to fix the excretion of all ionophores at 81% of the initial dose. For the terrestrial compartment, the reported half-lives of ionophores were in the range 1.3–49 days [3,25], but, with no consensus, and so the 49-day value was used. PECmanure was calculated taking into account dissipation during storage (in the barn and in storage). This was done by subdividing the total amount of ionophores into daily segments and by considering dissipation (halflife 22 days) of the ionophores in each segment (day). Finally, this resulted in a cumulative amount of ionophores after 82 days (Equation 12, Supplementary material available in the web version). PECsoil followed the same cumulative approach with dissipation half-lives of 49 days in soil and in segments of 82 days (Equations 13 and 14, Supplementary material available in the web version). The upper level (PECsoil) after the sixth manure application (at day 410) was used for the subsequent ERA. We also calculated the concentration in manure and soil when variations in dissipation half-lives and storage
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time (broiler life cycle) were taken into account. Monensin and lasalocid gave similar curves, as they were given in same dose, which also applied for salinomycin and narasin. 2.2.4. Scenario III – Measured environmental concentrations. Risk characterization in this scenario was based on MECs or occurrence data, as reviewed by Hansen and co-workers [3]. The available MECs were, primarily, from the USA, where ionophores can be applied for other livestock besides poultry.
3. Results 3.1. Scenario I – Worst-case exposure Results from this scenario yielded PECmanure in the broiler manure of 6428 mg/kg N for salinomycin and narasin, and 11,479 mg/kg N for monensin and lasalocid. After application of manure onto the soil, PECsoil values were estimated to be 364–650 lg/kg and PECs in water compartments 50–280 lg/L (PECporewater and PECgroundwater), 5.0–28 lg/L (PECsurfacewater) and 38– 70 lg/kg wwt (PECsediment). A risk characterization based on these PECs gave RQs for the terrestrial compartment of 6.6, 28, 7.2 and 0.9 for monensin, salinomycin, narasin and lasalocid, respectively. Similarly, RQs were 2.0–15 for the aquatic environmental and 1.9–12 for the sediment compartment. Table 4 summarizes Scenario I. 3.2. Scenario II – Refined exposure The refinements were performed as follows: (1) by considering metabolism for correction of the amount of ionophores excreted; (2) removal by dissipation in the manure during storage; (3) dissipation in the soil after manure amendment; and, (4) dissipation in water and sediment. When considering metabolism (81% excreted) and dissipation (DT50 22 days) in manure, the concentration in mixed manure from two batches of broilers after 82 days reached 1915 mg/kg N for salinomycin and narasin, and 3420 mg/kg N for monensin and lasalocid (black cross, Fig. 2). The cumulative concentrations of monensin and lasalocid in manure are displayed in Fig. 2 (black and red lines). Similar curves could be obtained for salinomycin and narasin, but these were given in lower doses and therefore reached lower concentrations. Fig. 2 also shows the concentrations for broiler life-cycles (storage time) of 35 days or 45 days and for DT50, manure of 5.5 days and 11 days. The graphical data for PECsoil comprise dissipation curves giving actual soil concentrations (black line), while dashed lines show the upper concentration level
Figure 2. Cumulative concentration of ionophores (monensin and lasalocid) in broiler manure when considering dissipation. Two batches of broilers were studied, first batch day 0–41 (black line) and second batch day 41–82 (red line). Manure from first batch was stored day 41–82 (black line). At day 82, manure from both batches was mixed (the resulting concentration is marked with a black cross) and applied to agricultural soil. Dissipation half-lives: 22 days (black and red), 11 days (green) and 5.5 days (blue). Dashed black lines depict the effect when broiler life cycle is changed to 35 days or 45 days.
(Fig. 3). The steady state value (upper concentration) for PECsoil on the sixth manure application (Fig. 3), taking into account soil dissipation (DT50, soil 49 days), was found to be 63.4 lg/kg for monensin and lasalocid, and 35.5 lg/kg for salinomycin and narasin. The effects of dissipation half-lives were also investigated for the soil compartment, and Fig. 3 shows the concentrations depicted with dashed lines. Groundwater concentrations were in the range 4.9–27.3 lg/L, surface water 0.5–2.7 lg/L and sediments 3.7–6.8 lg/kg wwt. RQs were above 1 for salinomycin in all cases and for monensin in the aquatic and sediment compartments. Narasin and lasalocid were in all cases below 1. Table 5 summarizes Scenario II.
Figure 3. Cumulative soil concentration of ionophores (monensin and lasalocid) after six consecutive additions of manure to the same soil every 82 days (black line). Effect of dissipation half-lives: 10 days (blue), 25 days (green) and 49 days (black). Dashed lines depict upper steady-state concentration level.
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Table 6. Scenario III with maximum measured environmental concentrations (MECs, adapted from [3]) related to predicted no-effect concentrations (PNECs) giving risk quotients (RQs) for soil, surface water and sediment
Monensin Salinomycin Narasin Lasalocid
MECsoil (lg/kg)
MECgw (lg/L)
MECsw (lg/L)
MECsed (lg/kg)*
NA NA NA NA
0.390 NA NA NA
0.220 0.040 0.060 0.028
12.1 11.6 6.3 NA
Terrestrial
Aquatic
Sediment
PNEC (lg/kg)
RQ
PNEC (lg/L)
RQ
PNEC (lg/kg)*
RQ
98 13 51 718
U U U U
1.88 1.14 2.23 2.5
0.12 0.04 0.03 0.01
5.68 3.44 6.73 36.7
2.1 3.4 0.9 U
NA, Not analyzed; U, Unknown. Value given on wet weight (wwt) basis.
*
3.3. Scenario III – Measured environmental concentrations This scenario is based on comparing the published occurrence data (MEC) previously reviewed [3] with the PNECs derived. The existing MEC of salinomycin from pig manure (11 lg/kg, [6]) is very low in relation to the predicted values for broilers. No values of MEC are available for soil, so, this compartment cannot be properly evaluated in this scenario. The measurements from the aquatic compartment that exist were mostly obtained from the USA, where the substances are applied for other species. The four ionophores were found in the range 0.001–0.390 lg/L [3]. A few measurements from river sediments (0.9– 31.5 lg/kg dwt [5]) were also available. MECsediment was converted into wet-weight basis for comparability and yielded 0.3–12.1 lg/kg wwt. Consequently, this gave RQs of 0.9 for narasin, 2.1 for monensin and 3.4 for salinomycin (no data on lasalocid). Table 6 summarizes Scenario III.
4. Discussion 4.1. Scenario I – Worst-case exposure All worst-case PEC values were above the trigger values of 10 lg/kg for soil and 0.1 lg/L for surface water (Fig. 1), so studies on environmental fate and effects of the compounds are mandatory to perform risk characterization [26]. This risk assessment with respect to the terrestrial compartment showed that monensin, salinomycin and narasin pose a risk, as they have RQs >1 (Table 4). Lasalocid has an RQ of 0.9. The lower potency of lasalocid relative to the other three ionophores is not easily explained and might reflect the need for more effect studies. For the aquatic environment, risk characterization showed that all ionophores pose a risk with RQ values of 2.0–15 (Table 4). The resulting RQs for sediment were found to be in of same order of magnitude as those for 540
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the aquatic compartment. These estimations indicated that ionophores might pose a risk to organisms dwelling in the sediment compartment. It must be stressed that effects on sediment-living organisms have not been investigated and this might be of concern. Refinement of exposure in all environmental compartments must be performed, as outlined in the guideline [26], because ionophores yield environmental RQs >1 (apart from lasalocid in soil). This refinement of exposure was done in Scenario II. 4.2. Scenario II – Refined exposure Refinement of PECs from Scenario I were performed taking into account an average metabolism rate of 81% for all ionophores in the broilers and dissipation of the parent compound in the environment [26,32]. One study elucidated the toxicity of a transformation product of salinomycin, which was found to be ‘‘toxicologically harmless’’ to mice [41]. However, no other investigations have been performed on the eco-toxicological effect of transformation products from ionophores. The dissipation half-life in broiler manure was set to 22 days, due to literature findings. A manure storage time of 3 months was proposed [26], but the more common value of 41 days was used, as storage capacity is typically restricted [29], combined with mixing of fresh manure (from second-batch broilers) at day 82. Fig. 2 shows the effect of broiler life cycle (or storage time) and DT50 for monensin and lasalocid. These graphical predictions show that the dissipation half-life in manure was the most important variable relative to the broiler life cycle (or the cumulative storage time of the manure). As in manure, the dissipation rate in soil was found to be an important parameter in reducing ionophore concentration. By this refinement, soil concentrations were reduced by a factor of 10 relative to worst-case Scenario I, so the environmental risk persists (Table 5). No dissipation has been reported for the aquatic compartment. In conclusion, we cannot exclude the possibility that ionophores pose an environmental risk.
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4.3. Scenario III – Measured environmental concentrations Few occurrence data exist, so Scenario III can only be considered a preliminary approximation to the true problem. Using MEC values, RQs of 0.01–0.12 (0.21 for groundwater) were obtained for the aquatic compartment, so the ionophores occur in a range that causes minor concern based on the current short-term toxicological data. Comparing MEC and refined PECsurfacewater showed that MECs were a factor of 12–38 lower than the predicted values. This might mean that the proposed guidelines are sufficiently conservative when predicting the aquatic compartment concentrations of ionophores. In addition, the available MEC data are primarily from the USA and these measurements might include a fraction of ionophores used in, e.g., beef production, so the MEC/PEC ratio might be even lower. In contrast to the aquatic compartment, ionophores might pose an environmental risk to sediment-dwelling organisms (RQ 0.9–3.4, Table 6), so there is cause for concern when looking at the sediment compartment based on current occurrence data. 4.4. General discussion and outlook Reported total amounts of ionophores used in different countries (Table 1) give worst-case concentrations up to 131 lg/kg soil (no dissipation) for salinomycin when diluted into total arable area in the Republic of Korea. As a result, there is cause for concern, as these agents are applied in such high quantities in animal production. Available toxicological effect data are mainly obtained from second-hand reporting. The effect data presented are the only data available from the literature and databases. Despite this, the short-term effect data are in the ranges low to high mg/kg for soil and sub-mg/L to low mg/L in the aquatic environment. Chief concerns regarding effect data are that no chronic exposure studies have been demonstrated and that no exposure data are obtainable from sediment-dwelling organisms. There is therefore a need to perform effect testing especially for sediment-dwelling organisms. Moreover, it was found that sorption (Koc) of the ionophores was a crucial model parameter for predicted concentrations in waters, as these calculations are based on partitioning between solids and water. A single research study evaluated sorption of monensin and lasalocid to eight different soils [42] and found Koc values of 125–5700 L/kg and 732–15,700 L/kg, respectively. The Koc values for salinomycin and narasin were defined as the same as those for monensin, due to a comparable structural relationship and unclear, contradictory data reported elsewhere [25]. The refinement of exposure (PEC), as in Scenario II and recommended in guidelines [26,28,29], is acceptable if metabolites are less potent than the parent com-
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pound. However, there are examples of transformation products or metabolites that have a similar or more toxic effect than the parent compound [2,43]. In addition, in broiler production, anticoccidial agents are typically given in shuttle programs (i.e. shifting between ionophores and synthetics between broiler production batches). It has been demonstrated that ionophores might have synergistic (more than additive) effects when combined with other biologically active compounds. When macrolide antibiotics were added to feed containing nontoxic levels of monensin, this caused lethal effects in cattle [44]. In a recent study, narasin was combined with the synthetic compound nicarbazin and the combined mixture was five times more toxic than predicted from additive effects [2]. The risk of adverse effect might therefore be enhanced when manure from ionophoretreated broilers is mixed in fields or in manure storage with manure containing other antibiotics. As PNECs are often based on one data set or a few data sets, this might cause concern. Generally, there is therefore a need to perform more toxicological effect studies, and to include effects of mixing ionophores with other anticoccidial agents, growth promoters and veterinary medicines and transformation products thereof. In the USA, ionophores can be given in combination with other agents {e.g., a product containing monensin, melengestrol (growth hormone), ractopamine (growth promoter) and tylosin (broad spectrum antibiotic) [45]}. There are few MECs from the EU, so there is need to analyze ionophores in the environment where the primary source is from broiler production. Similarly, there is need to investigate leaching from agricultural soils and to conduct more studies on dissipation and metabolism.
5. Conclusions Predictions based on European broiler production demonstrated that, in the worst case (Scenario I), ionophores cause a risk in the terrestrial, aquatic and sediment environments, as RQs are as high as 28. When exposures were refined (Scenario II) by adjusting for metabolism and dissipation mechanisms, it was found that ionophores might still pose an environmental risk (RQs up to 2.7). Occurrence data are only available from groundwaters, surface waters and sediment matrices, so the risk for the terrestrial compartment could not be characterized. From data available, it was found that ionophores possibly pose a risk for sediment-dwelling organisms, as RQs were up to 3.4. This assessment concluded that ionophores might be an environmental risk to organisms living in soils, waters and sediments. There are few effect data, so more effect studies, especially long-term and chronic, are needed to clarify environmental and biological effects of http://www.elsevier.com/locate/trac
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ionophores. In addition, there is a need for more detailed fate profiles (e.g., potential for leaching from agricultural fields). Acknowledgements This research was supported by the Drug Research Academy (Faculty of Pharmaceutical Sciences, University of Copenhagen). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.trac.2009. 02.015. References [1] A.B.A. Boxall, D.W. Kolpin, B. Halling-Sorensen, J. Tolls, Environ. Sci. Technol. 37 (2003) 286A. [2] M. Hansen, K.A. Krogh, A. Brandt, J.H. Christensen, B. HallingSørensen, Environ. Pollut. 157 (2009) 474. [3] M. Hansen, E. Bjo¨rklund, K.A. Krogh, B. Halling-Sørensen, Trends Anal. Chem. 28 (2009), doi:10.1016/j.trac.2009.01.008. [4] J.M. Cha, S. Yang, K.H. Carlson, J. Chromatogr., A 1065 (2005) 187. [5] S.C. Kim, K. Carlson, Water Res. 40 (2006) 2549. [6] M.P. Schlusener, K. Bester, M. Spiteller, Anal. Bioanal. Chem. 375 (2003) 942. [7] W. Song, M. Huang, W. Rumbeiha, H. Li, Rapid Commun. Mass Spectrom. 21 (2007) 1944. [8] N. Watanabe, T.H. Harter, B.A. Bergamaschi, J. Environ. Qual. 37 (2008) S-78. [9] J.G. Davis, C.C. Truman, S.C. Kim, J.C. Ascough, K. Carlson, J. Environ. Qual. 35 (2006) 2250. [10] R.A. Brain, D.J. Johnson, S.M. Richards, H. Sanderson, P.K. Sibley, K.R. Solomon, Environ. Toxicol. Chem. 23 (2004) 371. [11] D.G. Hillis, L. Lissemore, P.K. Sibley, K.R. Solomon, Environ. Sci. Technol. 41 (2007) 6620. [12] J. Jensen, X. Diao, A.D. Hansen, Environ. Toxicol. Chem. 28 (2009) 316. [13] E.B. McGregor, K.R. Solomon, M.L. Hanson, Arch. Environ. Contam. Toxicol. 53 (2007) 541. [14] NORM/NORM-VET 2006, Usage of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Norway, Tromsø / Oslo, Norway, 2007. [15] B. Bengtsson, C. Greko, U.G. Andersson, SVARM 2007: Swedish Veterinary Antimicrobial Resistance Monitoring, The National Veterinary Institute (SVA), Uppsala, Sweden, 2008. [16] Y. Kim, J. Jung, M. Kim, J. Park, A.B.A. Boxall, K. Choi, Environ. Toxicol. Pharmacol. 26 (2008) 167. [17] Danish Veterinary and Food Administration, Danish Medicines Agency, Danish Institute for Food and Veterinary Research, DANMAP 2004 - Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark, Statens Serum Institut, Copenhagen, Denmark, 2004. [18] P. Butaye, L.A. Devriese, F. Haesebrouck, Clin. Microbiol. Rev. 16 (2003) 175. [19] R.K. McGuffey, L.F. Richardson, J.I.D. Wilkinson, J. Dairy Sci. 84 (2001) E194.
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[20] H.H. Mollenhauer, D.J. Morre, L.D. Rowe, Biochim. Biophys. Acta 1031 (1990) 225. [21] B.C. Pressman, Annu. Rev. Biochem. 45 (1976) 501. [22] B.C. Pressman, M. Fahim, Annu. Rev. Pharmacol. Toxicol. 22 (1982) 465. [23] European Union, Off. J. Euro. Comm. L31 (2002) 1. [24] European Union, Off. J. Euro. Comm. L268 (2003) 29. [25] European Food Safety Authority, Panel on additives and products or substances used in animal feed, 2008 (http://www.efsa. europa.eu/EFSA/ScientificPanels/efsa_locale-1178620753812_ FEEDAP.htm). [26] European Food Safety Authority, EFSA J. 529 (2007) 1. [27] VICH, Environmental Impact Assessment (EIAs) for Veterinary Medicinal Products (VMPs) - Phase I, VICH, c/o International Federation for Animal Health, Brussels, Belgium, 2000. [28] VICH, Environmental Impact Assessment for Veterinary Medicinal Products - Phase II Guidance, VICH, c/o International Federation for Animal Health, Brussels, Belgium, 2004. [29] European Medicines Agency (EMA), Committee for Medicinal Products for Veterinary Use (CVMP), Revised guideline on environmental impact assessment for veterinary medicinal products in support of the VICH guidelines GL6 and GL38, EMEA/ CVMP/ERA/418282/2005-Rev.1 Consultation, EMA, London, UK, 2008. [30] J. Ro¨mbke, K.D. Floate, R. Jochman, M.A. Scha¨fer, N. Puniamoorthy, S. Kna¨be, J. Lehmhus, B. Rosenkranz, A. Scheffczyk, T. Schmidt, A. Sharples, W.U. Blanckenhorn, (submitted to Environ. Toxicol. Chem. (2009). [31] Det Danske Fjerkræraad, God produktionspraksis ved produktion af slagtekyllinger (in Danish), 2003. [32] M.H.M.M. Montforts, RIVM - Environmental risk assessment for veterinary medicinal products. Part 1. Other than GMO-containing and immunological products, First update., (1999), RIVM Report 601300 002, RIVM, Bilthoven, The Netherlands, 2000. [33] M.P. Schlusener, M.A. von Arb, K. Bester, Arch. Environ. Contam. Toxicol. 51 (2006) 21. [34] P.L.A. van Vlaardingen, J.A. de Knecht, P.A.H. Janssen, Degradation of veterinary drugs in manure, in: R. Luttik, M.T.M. Van Raaij (Editors), Factsheets for the (Eco)toxicological Risk Assessment Strategy of the National Institute of Public Health and the Environment (RIVM), RIVM, Bilthoven, The Netherlands, 2000, pp. 95–102. [35] H. Dolliver, S. Gupta, S. Noll, J. Environ. Qual. 37 (2008) 1245. [36] EFSA, EFSA J. 42 (2004) 1. [37] EFSA, EFSA J. 283 (2005) 1. [38] EFSA, EFSA J. 75 (2004) 1. [39] EFSA, EFSA J. 90 (2004) 1. [40] EFSA, EFSA J. 53 (2004) 1. [41] L. Vertesy, K. Heil, H.W. Fehlhaber, W. Ziegler, J. Antibiot. (Tokyo) 40 (1987) 388. [42] S.A. Sassman, L.S. Lee, Environ. Toxicol. Chem. 26 (2007) 1614. [43] B. Halling-Sorensen, G. Sengelov, J. Tjornelund, Arch. Environ. Contam Toxicol. 42 (2002) 263. [44] R.J. Basaraba, F.W. Oehme, M.W. Vorhies, G.L. Stokka, J. Vet. Diagn. Investig. 11 (1999) 79. [45] U.S. Food and Drug Administration (FDA), Animal Drugs @ FDA, US FDA, Washington, DC, 2009 (http://www.accessdata.fda.gov/ scripts/animaldrugsatfda/). [46] EFSA, EFSA J. 63 (2004) 1. [47] American Society for Testing and Materials, Standard guide for conducting static toxicity tests with Lemna Gibba G3, 1998.
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Environmental risk assessment of ionophores Martin Hansen, Kristine A. Krogh, Erland Bjo¨rklund, Asbjørn Brandt, Bent Halling-Sørensen Following European guidelines, this article presents an environmental risk assessment for four ionophores (monensin, salinomycin, narasin and lasalocid) based on predicted environmental exposure from broiler production in two scenarios. In a third scenario, measured occurrence data are used to characterize the potential risk. Findings show that predicted environmental concentrations in all environmental compartments and measured environmental concentrations in sediments are above predicted no-effect concentrations, so ionophores might pose an environmental risk. The toxicological effect data reviewed revealed that very limited data exist and that long-term effect data and chronic effect data are not available. ª 2009 Elsevier Ltd. All rights reserved. Keywords: Anticoccidial; Coccidiostat; Eco-toxicology; Environmental exposure; Lasalocid; Monensin; Narasin; Predicted environmental concentration; Predicted no-effect concentration; Salinomycin
1. Introduction Martin Hansen*, Kristine A. Krogh, Erland Bjo¨rklund, Bent Halling-Sørensen Section of Toxicology and Environmental Chemistry, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Asbjørn Brandt Section of Veterinary Medicines, The Danish Medicines Agency, Axel Heides Gade 1, DK-2300 Copenhagen, Denmark
*
Corresponding author. Tel.: +45 35 33 62 65; Fax: +45 35 30 60 13; E-mail: [email protected]
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Ionophores are a sub-group of anticoccidials used extensively in livestock production (i.e. poultry, cattle, swine, sheep and rabbit) and, as a result, they are considered to be emerging environmental contaminants [1–3]. For these compounds, only a few studies report data on occurrence [4–8], fate [9] and eco-toxicological effects [10–13]. No complete assessment is available that reviews the environmental impact of ionophores. In another article in this issue [3], we outlined the status of the current development of analytical methods targeted to environmental samples and reported that the few measured occurrence data on ionophores were found at lg/kg, ng/L and lg/kg levels in manure, surface waters and sediments, respectively. At present, concentration levels of ionophores in the soil compartment have not been reported. Moreover, the available occurrence data could reflect a possible build up of ionophores in lipophilic compartments (e.g., soils and sediments [3]). Unfortunately,
only limited information on consumption data of ionophores is available and many countries do not monitor their usage. Table 1 shows the available consumption data from poultry, cattle, swine, sheep, and rabbit production, revealing that ionophores are widely and heavily used, and that salinomycin, monensin, narasin and lasalocid are the most frequently applied [14–17]. Ionophores act as antiporters (mode of action) by entrapping cations (preferably sodium or potassium), thereby generating neutral zwitterions. These complexes are transported across prokaryotic and eukaryotic cell membranes in exchange for protons, and, accordingly, disrupt ion gradients and, ultimately, cause energy reduction and cell death [12,18–22]. In the European Union (EU), ionophores and other anticoccidial agents are regulated in accordance with the directives on feed additives and may not be put on the market unless a proper authorization has been given by the European Commission based on a scientific evaluation by the European Food Safety Authority (EFSA). Market authorization depends on demonstrating that the ionophores have no harmful effects on human and animal health and the environment [23,24]. These scientific evaluations are performed by the EFSA panel on additives and products or substances used in animal feed [25]. In this article, environmental risk assessment (ERA) is based on an approach suggested by the EFSA panel [26] following three harmonized guidance documents: phase I – exposure assessment [27]; phase II – risk assessment [28]; and, additional guidance [29].
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Table 1. Consumption data on ionophores used in livestock production and predicted worst-case concentrations based on arable area Region Republic of Korea
Denmark
Norway
Ionophore Salinomycin Monensin Lasalocid Salinomycin Narasin Monensin Lasalocid Narasin Monensin Lasalocid
Amount (kg) a)
639,170 136,140a) 66,061a) 10,070b) 1625b) 840b) 243b) 5615c) 889c) 13c)
Arable area
PECsoil (lg/kg)
9,819,000
16.58%
2004
4,239,400
52.59%
2006
30,744,200
2.70%
131 27.9 13.5 1.51 0.24 0.13 0.04 2.25 0.36 0.01
Year
Area (ha)
2006
PECsoil based on 0.2 m ploughing depth. a) Estimated active substance from product sales [16]. b) [17]. c) [14].
The EFSA guidance document [26] on ERA of feed additives follows a tiered approach starting with a decision tree, resulting in an initial worst-case-scenario calculation to give a predicted environmental concentration (PEC) for ionophores in soil, soil-pore water, groundwater, surface water and sediment. If these PECs are above pre-defined trigger values (10 lg/kg soil and 0.1 lg/L in groundwater), they are compared to predicted no-effect concentrations (PNECs) derived from eco-toxicological effect data adjusted with appropriate assessment factors (AFs). Where the PEC/PNEC ratio is
>1, the PEC is refined taking into account losses of the ionophore (metabolism and environmental dissipation mechanisms) and, finally, compared to the PNEC again to indicate if the ionophore poses a potential environmental risk. Fig. 1 outlines this entire ERA procedure. The first aim of this article is to give an overview of the published eco-toxicological data on four ionophores (viz monensin, salinomycin, narasin and lasalocid). The second aim is to present a tiered ERA based on PEC and compare that with existing effect data to assess the potential environmental risk. ERA is also performed by comparing measured environmental concentrations (MECs) with the same effect data. PEC estimations are based on the amount of the individual compounds applied in the EU and only ionophores that are approved for use in poultry production, according to existing EU regulations. The ERA of ionophores comprises three superior scenarios: Scenario I is a worst-case scenario, which predicts the environmental exposure of ionophores in agricultural soils amended with manure from broilers treated with ionophores in agreement with the European regulations; Scenario II is a refinement of Scenario I, where metabolism in the broilers and other environmental dissipation mechanisms are taken into account, leading to a refined PEC; and, Scenario III assesses the potential environmental risk on the basis of previously published occurrence data (MECs) reported in the scientific literature.
2. Materials and methods Figure 1. Overview of the environmental risk-assessment approach.
This section outlines the subsequent results and discussion sections in the ERA of ionophores. Sub-section 2.1. reports on the eco-toxicological effect data obtained so
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Table 2. Acute effect studies on ionophores in the soil environment Test organism
Test method
Effect concentration
Ref.
Monensin
Folsomia fimetaria (springtail) Enchytraeus crypticus (enchytraeid) Eisenia foetida andrei (earthworm) Raphanus sativus (radish) Soil micro-organisms
ISO 11267 (reproduction, 21 days) ISO 16387 (reproduction, 21 days) OECD 207 (mortality, 14 days) Non-standardized (emergence, 14 days) OECD 216 (nitrification) OECD 217 (respiration)
EC50 591 [254–927] mg/kg EC50 356 [95–617] mg/kg LC50 56 mg/kga) LC50 9.8 mg/kg NOEC >5 mg/kg
[12] [12] [37] [36] [37]
Salinomycin
Raphanus sativus (radish) Eisenia foetida andrei (earthworm) Soil micro-organisms
Non-standardized (growth rate, 14 days) OECD 207 (mortality, 14 days) OECD 216 (nitrification) OECD 217 (respiration)
LC50 1.3 mg/kg LC50 71 mg/kg NOEC >2.3 mg/kg
[46] [46] [46]
Narasin
Raphanus sativus (radish) Eisenia foetida andrei (earthworm) Soil micro-organisms
OECD OECD OECD OECD
208 207 216 217
(emergence) (mortality, 14 days) (nitrification) (respiration)
LC50 5.07 mg/kg LC50 46.4 mg/kg NOEC 17.4 mg/kg
[39] [39] [39]
Lasalocid
Lolium perenne (perennial ryegrass) Eisenia foetida andrei (earthworm) Soil micro-organisms
OECD OECD OECD OECD
208 207 216 217
(emergence, 18 days) (mortality, 14 days) (nitrification) (respiration)
EC50 87.8 mg/kg LC50 71.8 mg/kg NOEC >5 mg/kg
[40] [40] [40]
ISO, Guideline number from the International Organization for Standardization; OECD, Guideline number from the Organization for Economic Co-operation and Development; EC50, Effect concentration where half of population is affected; LC50, Concentration where half of population is killed; NOEC, No observed effect concentration. a) Normalized to 5% organic carbon content. 95%-confidential intervals given in brackets.
Table 3. Acute effect studies on ionophores in the aquatic environment Test organism Monensin
Salinomycin
Test method
Effect concentration
Ref.
Lemna gibba (macrophyte, floating) Myriophyllum spicatum (macrophyte, submersed) Selenastrum subspicatus (algae)
ASTM guideline [47] (growth, 7 days) Mesocosms (growth rate, 35 days)
EC50 0.998 [0.955–1.042] mg/L EC50 0.197 [0.042–0.353] mg/L
[10] [13]
OECD 201 (growth rate and biomass)
[36]
Pseudokirchneriella subcapitata (algae)
OECD 201 (growth rate and biomass)
Daphnia magna (crustacean) Oncorhynchus mykiss (fish)
OECD 202 (immobility) OECD 203 (mortality, 96 h)
EC50 0.98 mg/L (growth rate) EC50 4.3 mg/L (biomass) EC50 3.41 mg/L (growth rate) EC50 1.73 mg/L (biomass) EC50 7.29 mg/L LC50 1.88 mg/L
Selenastrum capricornutum (algae)
OECD 201 (growth rate and biomass, 72 h) OECD 202 (immobility, 48 h) OECD 203 (mortality, 96 h)
EC50 EC50 EC50 LC50
3.01 mg/L (growth rate) 2.09 mg/L (biomass) 13.3 mg/L 1.14 mg/L
[46]
EC50 EC50 EC50 LC50 EC50 EC50 EC50 LC50
2.91 mg/L (growth rate) 0.77 mg/L (biomass) 20.6 mg/L 2.23 mg/L 3.1 mg/L (growth rate) 2.0 mg/L (biomass) 5.4 mg/L 2.5 mg/L
[39]
Daphnia magna (crustacean) Oncorhynchus mykiss (fish) Narasin
Selenastrum capricornutum (algae)
Lasalocid
Daphnia magna (crustacean) Oncorhynchus mykiss (fish) Selenastrum subspicatus (algae)
OECD 201 (growth rate and biomass, 72 h) OECD 202 (immobility, 48 h) OECD 203 (mortality, 96 h) OECD 201 (growth rate and biomass)
Daphnia magna (crustacean) Brachydanio rerio (fish)
OECD 202 (immobility, 48 h) OECD 203 (mortality, 96 h)
[37] [37] [37]
[46] [38]
[39] [39] [40] [40] [40]
ASTM, American Society for Testing and Materials guidelines; OECD, Guideline number from the Organization for Economic Co-operation and Development. 95%-confidenc intervals given in brackets. ISO, Guideline number from the International Organization for Standardization; EC50, Effect concentration where half of population is affected; LC50, Concentration where half population is killed.
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far, while sub-section 2.2. describes in detail the methodologies applied in the three scenarios. 2.1. Eco-toxicological effect data The available eco-toxicological data for the ionophores studied are very limited, as reported in Tables 2 and 3, which show data for organisms living in soil and water, respectively. Only short-term effect data are presented, since no chronic effect data are available. Few peer-reviewed eco-toxicological studies have been performed, and only with monensin [10–13]. Two of these studies were mesocosm studies that attempted to mimic minor eco-systemic effects [11,13]. Other sources of eco-toxicological studies were scientific interpretations or opinions reported by the EFSA panel [25]. Their reports were based on effect studies of ionophores provided by the feed-additive manufacturers. Some of the studies followed standardized tests, and this section includes the usable information extracted from these reports. For test organisms living in soil, the acute toxicological level was in the range 5.07–591 mg/kg, with radish being the most sensitive species and springtails the most insensitive of the species tested. No-observed-effect concentrations (NOECs) on respiration and nitrification tests were obtained on soil micro-organisms and found to be in the range of 2–17 mg/kg [25]. Lethal-effect concentrations for the classic soil-test organisms, earthworms, were 46.4–71.8 mg/kg for the four compounds. No effect data exist on dung-dwelling organisms, which might be vulnerable, as was observed for other antiparasiticides (e.g., ivermectin [30]). Similarly, no data was available for sediment-dwelling organisms. The acute toxicological level for aquatic living organisms (e.g., algae, crustaceans and fish) was in the range 0.77–20.6 mg/L for all four compounds. In general, algae were the most sensitive species, while crustaceans were the least sensitive. Monensin was tested towards macrophytes, showing effect concentrations in the range 0.197–0.998 mg/L for these sensitive aquatic organisms.
2.1.1. Predicted no-effect concentrations. The PNECs were derived applying an assessment factor (AF) to the short-term effect data [26]. For the terrestrial compartment, three taxonomic groups (plants, soil-dwelling organisms and micro-organisms) were tested, so that the environmental risk could be established. The most sensitive species were plants (radish) for monensin, salinomycin and narasin, while lasalocid was found to be most sensitive towards earthworms (Table 4). By using an AF of 100, as recommended by the European Medicines Agency [28], the PNEC value was found. Similarly, three taxonomic groups (algae, crustaceans and fish) represented the aquatic compartment, and the fish species were found to give the lowest PNECs using an AF of 1000 [28]. Tables 4 and 5 list the PNEC values derived from results of the assessment. For estimation of the potential risk of the sediment compartment, the PNECsediment values were derived from the PNECaquatic, as suggested by the European Medicines Agency [29], since no effect data were available for sediment-dwelling organisms. The calculations were based on equilibrium partitioning, so they depended on Koc (Equation 9, Supplementary material available in the web version). Values of PNECsediment were in the range 3.44–36.7 lg/kg of wet weight (wwt) sediment (Table 4). 2.2. Environmental risk-assessment scenarios Scenario I predicts the worst-case environmental exposure on the basis of doses applied in European broiler production. Scenario II is a refinement of Scenario I taking into account elimination of the parent ionophore. Scenario III utilizes published environmental occurrence data (MECs). In all scenarios, the risk is characterized using a risk quotient (RQ) as the ratio between environmental concentrations obtained (PECs or MECs) to the PNECs [26]. 2.2.1. Basic set of parameters. Broilers have a life cycle of 35–45 days with an average life of 41 days (this value
Table 4. Scenario I with predicted environmental concentrations (PECs) for ionophores related to predicted no-effect concentrations (PNECs) giving risk quotients (RQs) for soil, surface water and sediment
Monensin Salinomycin Narasin Lasalocid
Dose (mg/kg feed)*
PECsoil (lg/kg)
PECgw (lg/L)
PECsw (lg/L)
PECsed (lg/kg)*
125 70 70 125
650 364 364 650
280 157 157 50
28 16 16 5.0
67 38 38 70
Terrestrial
Aquatic
L(E)C50 (mg/kg)
AF
PNEC (lg/kg)
RQ
L(E)C50 (mg/L)
9.8 1.3 5.07 71.8
100 100 100 100
98 13 51 718
6.6 28 7.2 0.9
1.88 1.14 2.23 2.5
AF 1000 1000 1000 1000
Sediment
PNEC (lg/L)
RQ
PNEC (lg/kg)*
RQ
1.88 1.14 2.23 2.5
15 14 7.0 2.0
5.68 3.44 6.73 36.7
12 11 5.6 1.9
Model parameters given in Supplementary material available in web version: Koc 125 L/kg for monensin, salinomycin and narasin; Koc 732 L/kg for lasalocid; PECgw, PEC in groundwater (equals PECporewater); PECsw, PEC in surface water; PECsed, PEC in sediment. Other model parameters are also given in Supplementary material: AF, Assessment factor; RQ, Risk quotient (PEC/PNEC ratio). * Value given on wet weight (wwt) basis. PECsoil is the steady-state concentration obtained after the sixth application of manure (at day 410).
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Table 5. Scenario II with refined predicted environmental concentrations (PECs) after six consecutive applications of manure (every 82 days) to the same agricultural soil (taking into account metabolism, manure and soil-dissipation mechanisms)
Monensin Salinomycin Narasin Lasalocid
PECsoil (lg/kg)
PECgw (lg/L)
PECsw (lg/L)
PECsed (lg/kg)*
63.4 35.5 35.5 63.4
27.3 15.3 15.3 4.9
2.7 1.5 1.5 0.49
6.6 3.7 3.7 6.8
Terrestrial
Aquatic
Sediment
PNEC (lg/kg)
RQ
PNEC (lg/L)
RQ
PNEC (lg/kg)*
RQ
98 13 51 718
0.6 2.7 0.7 0.1
1.88 1.14 2.23 2.5
1.5 1.3 0.7 0.2
5.68 3.44 6.73 36.7
1.2 1.1 0.5 0.2
Model conditions as in Table 4, but with 81% excretion of parent compound and dissipation half-lives in manure of 22 days with manure dissipating (for 82 days) before adding further manure to the soil. Also, dissipation in soil considered with a half-life of 49 days. * Value given on wet weight (wwt) basis.
is used in the following) [29,31], and, throughout their life, they are constantly treated with ionophores (dose range varied between ionophores and was in the range 50–125 mg/kg of dry weight (dwt) feed) [25] with a withdrawal period of 1–5 days before slaughter [25]. We presumed that the broilers received the maximum allowed dose of the specific ionophore and were treated their entire life cycle (i.e. the withdrawal period of ionophores before slaughter is excluded). On average, the feed intake was 29 kg dwt feed/broiler/year (i.e. 3.26 kg dwt feed per broiler) and the excretion rate was 0.36 kg N/broiler/year [26]. Supplementary material, available in the web version, contains the range of baseset model parameters and equations to give PECs, as defined in the EFSA guidance document [26]. 2.2.2. Scenario I – Worst-case exposure. Worst-case PECs were calculated by assuming that all ingested ionophores were excreted as parent compound and that broiler manure (170 kg N/ha/year) was applied to the agricultural soil in one application [26]. PECsoil was calculated based on the dose of the ionophores and allowed nitrogen levels. The concentration in pore water (PECporewater) was calculated using sorption-distribution coefficient Kd (calculated via Koc) between soil and water. The concentration in groundwater (PECgroundwater) was defined as the value of pore water [26,29,32]. PECsurfacewater was calculated using a dilution factor of 10 compared to PECporewater [26,32]. However, other guidelines recommended a dilution factor of 3 [29]. PECsediment was calculated on the partitioning between surface water and sediment (estimated from Koc) [26,32]. 2.2.3. Scenario II – Refined exposure. In this scenario, the PECs from Scenario I were refined by considering the metabolism of ionophores in the broiler and dissipation in manure, soil and water. Observing two succeeding batches of broilers from the same barn, the first batch of broilers had a life cycle from day 0 to day 41, and the second batch of broilers had a life cycle from day 41 to day 82. When being produced, the manure was not re538
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moved from the barn throughout the life cycle of the broiler. At day 41, the first batch of broilers was slaughtered and the manure was collected and stored in piles or tanks, and the life cycle of the second batch broilers started. At day 82, the manure from the second batch was removed (and broilers were slaughtered), mixed with the manure from first batch and spread onto agricultural land. As a result, the broiler manure was applied every 82 days to the same agricultural soil with an average of 38 kg N/ha, thereby resulting in a total of 170 kg N/ha/year. Dissipation half-lives in manure were demonstrated to be 5 days for salinomycin in pig manure [33]. However, pig manure differs from broiler manure [34]. In another study, monensin has a half-life of 22 days in turkey manure [35]. This latter first-order decay value was used. The EFSA panel has evaluated the metabolism of ionophores in broilers. Total excreted amounts of parent ionophore relative to dose showed great variations (i.e. 10–81% for monensin [36,37], 8–67% for salinomycin [38], 30% for narasin [39] and 74–77% for lasalocid [40]). In Scenario II, we chose to fix the excretion of all ionophores at 81% of the initial dose. For the terrestrial compartment, the reported half-lives of ionophores were in the range 1.3–49 days [3,25], but, with no consensus, and so the 49-day value was used. PECmanure was calculated taking into account dissipation during storage (in the barn and in storage). This was done by subdividing the total amount of ionophores into daily segments and by considering dissipation (halflife 22 days) of the ionophores in each segment (day). Finally, this resulted in a cumulative amount of ionophores after 82 days (Equation 12, Supplementary material available in the web version). PECsoil followed the same cumulative approach with dissipation half-lives of 49 days in soil and in segments of 82 days (Equations 13 and 14, Supplementary material available in the web version). The upper level (PECsoil) after the sixth manure application (at day 410) was used for the subsequent ERA. We also calculated the concentration in manure and soil when variations in dissipation half-lives and storage
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time (broiler life cycle) were taken into account. Monensin and lasalocid gave similar curves, as they were given in same dose, which also applied for salinomycin and narasin. 2.2.4. Scenario III – Measured environmental concentrations. Risk characterization in this scenario was based on MECs or occurrence data, as reviewed by Hansen and co-workers [3]. The available MECs were, primarily, from the USA, where ionophores can be applied for other livestock besides poultry.
3. Results 3.1. Scenario I – Worst-case exposure Results from this scenario yielded PECmanure in the broiler manure of 6428 mg/kg N for salinomycin and narasin, and 11,479 mg/kg N for monensin and lasalocid. After application of manure onto the soil, PECsoil values were estimated to be 364–650 lg/kg and PECs in water compartments 50–280 lg/L (PECporewater and PECgroundwater), 5.0–28 lg/L (PECsurfacewater) and 38– 70 lg/kg wwt (PECsediment). A risk characterization based on these PECs gave RQs for the terrestrial compartment of 6.6, 28, 7.2 and 0.9 for monensin, salinomycin, narasin and lasalocid, respectively. Similarly, RQs were 2.0–15 for the aquatic environmental and 1.9–12 for the sediment compartment. Table 4 summarizes Scenario I. 3.2. Scenario II – Refined exposure The refinements were performed as follows: (1) by considering metabolism for correction of the amount of ionophores excreted; (2) removal by dissipation in the manure during storage; (3) dissipation in the soil after manure amendment; and, (4) dissipation in water and sediment. When considering metabolism (81% excreted) and dissipation (DT50 22 days) in manure, the concentration in mixed manure from two batches of broilers after 82 days reached 1915 mg/kg N for salinomycin and narasin, and 3420 mg/kg N for monensin and lasalocid (black cross, Fig. 2). The cumulative concentrations of monensin and lasalocid in manure are displayed in Fig. 2 (black and red lines). Similar curves could be obtained for salinomycin and narasin, but these were given in lower doses and therefore reached lower concentrations. Fig. 2 also shows the concentrations for broiler life-cycles (storage time) of 35 days or 45 days and for DT50, manure of 5.5 days and 11 days. The graphical data for PECsoil comprise dissipation curves giving actual soil concentrations (black line), while dashed lines show the upper concentration level
Figure 2. Cumulative concentration of ionophores (monensin and lasalocid) in broiler manure when considering dissipation. Two batches of broilers were studied, first batch day 0–41 (black line) and second batch day 41–82 (red line). Manure from first batch was stored day 41–82 (black line). At day 82, manure from both batches was mixed (the resulting concentration is marked with a black cross) and applied to agricultural soil. Dissipation half-lives: 22 days (black and red), 11 days (green) and 5.5 days (blue). Dashed black lines depict the effect when broiler life cycle is changed to 35 days or 45 days.
(Fig. 3). The steady state value (upper concentration) for PECsoil on the sixth manure application (Fig. 3), taking into account soil dissipation (DT50, soil 49 days), was found to be 63.4 lg/kg for monensin and lasalocid, and 35.5 lg/kg for salinomycin and narasin. The effects of dissipation half-lives were also investigated for the soil compartment, and Fig. 3 shows the concentrations depicted with dashed lines. Groundwater concentrations were in the range 4.9–27.3 lg/L, surface water 0.5–2.7 lg/L and sediments 3.7–6.8 lg/kg wwt. RQs were above 1 for salinomycin in all cases and for monensin in the aquatic and sediment compartments. Narasin and lasalocid were in all cases below 1. Table 5 summarizes Scenario II.
Figure 3. Cumulative soil concentration of ionophores (monensin and lasalocid) after six consecutive additions of manure to the same soil every 82 days (black line). Effect of dissipation half-lives: 10 days (blue), 25 days (green) and 49 days (black). Dashed lines depict upper steady-state concentration level.
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Table 6. Scenario III with maximum measured environmental concentrations (MECs, adapted from [3]) related to predicted no-effect concentrations (PNECs) giving risk quotients (RQs) for soil, surface water and sediment
Monensin Salinomycin Narasin Lasalocid
MECsoil (lg/kg)
MECgw (lg/L)
MECsw (lg/L)
MECsed (lg/kg)*
NA NA NA NA
0.390 NA NA NA
0.220 0.040 0.060 0.028
12.1 11.6 6.3 NA
Terrestrial
Aquatic
Sediment
PNEC (lg/kg)
RQ
PNEC (lg/L)
RQ
PNEC (lg/kg)*
RQ
98 13 51 718
U U U U
1.88 1.14 2.23 2.5
0.12 0.04 0.03 0.01
5.68 3.44 6.73 36.7
2.1 3.4 0.9 U
NA, Not analyzed; U, Unknown. Value given on wet weight (wwt) basis.
*
3.3. Scenario III – Measured environmental concentrations This scenario is based on comparing the published occurrence data (MEC) previously reviewed [3] with the PNECs derived. The existing MEC of salinomycin from pig manure (11 lg/kg, [6]) is very low in relation to the predicted values for broilers. No values of MEC are available for soil, so, this compartment cannot be properly evaluated in this scenario. The measurements from the aquatic compartment that exist were mostly obtained from the USA, where the substances are applied for other species. The four ionophores were found in the range 0.001–0.390 lg/L [3]. A few measurements from river sediments (0.9– 31.5 lg/kg dwt [5]) were also available. MECsediment was converted into wet-weight basis for comparability and yielded 0.3–12.1 lg/kg wwt. Consequently, this gave RQs of 0.9 for narasin, 2.1 for monensin and 3.4 for salinomycin (no data on lasalocid). Table 6 summarizes Scenario III.
4. Discussion 4.1. Scenario I – Worst-case exposure All worst-case PEC values were above the trigger values of 10 lg/kg for soil and 0.1 lg/L for surface water (Fig. 1), so studies on environmental fate and effects of the compounds are mandatory to perform risk characterization [26]. This risk assessment with respect to the terrestrial compartment showed that monensin, salinomycin and narasin pose a risk, as they have RQs >1 (Table 4). Lasalocid has an RQ of 0.9. The lower potency of lasalocid relative to the other three ionophores is not easily explained and might reflect the need for more effect studies. For the aquatic environment, risk characterization showed that all ionophores pose a risk with RQ values of 2.0–15 (Table 4). The resulting RQs for sediment were found to be in of same order of magnitude as those for 540
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the aquatic compartment. These estimations indicated that ionophores might pose a risk to organisms dwelling in the sediment compartment. It must be stressed that effects on sediment-living organisms have not been investigated and this might be of concern. Refinement of exposure in all environmental compartments must be performed, as outlined in the guideline [26], because ionophores yield environmental RQs >1 (apart from lasalocid in soil). This refinement of exposure was done in Scenario II. 4.2. Scenario II – Refined exposure Refinement of PECs from Scenario I were performed taking into account an average metabolism rate of 81% for all ionophores in the broilers and dissipation of the parent compound in the environment [26,32]. One study elucidated the toxicity of a transformation product of salinomycin, which was found to be ‘‘toxicologically harmless’’ to mice [41]. However, no other investigations have been performed on the eco-toxicological effect of transformation products from ionophores. The dissipation half-life in broiler manure was set to 22 days, due to literature findings. A manure storage time of 3 months was proposed [26], but the more common value of 41 days was used, as storage capacity is typically restricted [29], combined with mixing of fresh manure (from second-batch broilers) at day 82. Fig. 2 shows the effect of broiler life cycle (or storage time) and DT50 for monensin and lasalocid. These graphical predictions show that the dissipation half-life in manure was the most important variable relative to the broiler life cycle (or the cumulative storage time of the manure). As in manure, the dissipation rate in soil was found to be an important parameter in reducing ionophore concentration. By this refinement, soil concentrations were reduced by a factor of 10 relative to worst-case Scenario I, so the environmental risk persists (Table 5). No dissipation has been reported for the aquatic compartment. In conclusion, we cannot exclude the possibility that ionophores pose an environmental risk.
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4.3. Scenario III – Measured environmental concentrations Few occurrence data exist, so Scenario III can only be considered a preliminary approximation to the true problem. Using MEC values, RQs of 0.01–0.12 (0.21 for groundwater) were obtained for the aquatic compartment, so the ionophores occur in a range that causes minor concern based on the current short-term toxicological data. Comparing MEC and refined PECsurfacewater showed that MECs were a factor of 12–38 lower than the predicted values. This might mean that the proposed guidelines are sufficiently conservative when predicting the aquatic compartment concentrations of ionophores. In addition, the available MEC data are primarily from the USA and these measurements might include a fraction of ionophores used in, e.g., beef production, so the MEC/PEC ratio might be even lower. In contrast to the aquatic compartment, ionophores might pose an environmental risk to sediment-dwelling organisms (RQ 0.9–3.4, Table 6), so there is cause for concern when looking at the sediment compartment based on current occurrence data. 4.4. General discussion and outlook Reported total amounts of ionophores used in different countries (Table 1) give worst-case concentrations up to 131 lg/kg soil (no dissipation) for salinomycin when diluted into total arable area in the Republic of Korea. As a result, there is cause for concern, as these agents are applied in such high quantities in animal production. Available toxicological effect data are mainly obtained from second-hand reporting. The effect data presented are the only data available from the literature and databases. Despite this, the short-term effect data are in the ranges low to high mg/kg for soil and sub-mg/L to low mg/L in the aquatic environment. Chief concerns regarding effect data are that no chronic exposure studies have been demonstrated and that no exposure data are obtainable from sediment-dwelling organisms. There is therefore a need to perform effect testing especially for sediment-dwelling organisms. Moreover, it was found that sorption (Koc) of the ionophores was a crucial model parameter for predicted concentrations in waters, as these calculations are based on partitioning between solids and water. A single research study evaluated sorption of monensin and lasalocid to eight different soils [42] and found Koc values of 125–5700 L/kg and 732–15,700 L/kg, respectively. The Koc values for salinomycin and narasin were defined as the same as those for monensin, due to a comparable structural relationship and unclear, contradictory data reported elsewhere [25]. The refinement of exposure (PEC), as in Scenario II and recommended in guidelines [26,28,29], is acceptable if metabolites are less potent than the parent com-
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pound. However, there are examples of transformation products or metabolites that have a similar or more toxic effect than the parent compound [2,43]. In addition, in broiler production, anticoccidial agents are typically given in shuttle programs (i.e. shifting between ionophores and synthetics between broiler production batches). It has been demonstrated that ionophores might have synergistic (more than additive) effects when combined with other biologically active compounds. When macrolide antibiotics were added to feed containing nontoxic levels of monensin, this caused lethal effects in cattle [44]. In a recent study, narasin was combined with the synthetic compound nicarbazin and the combined mixture was five times more toxic than predicted from additive effects [2]. The risk of adverse effect might therefore be enhanced when manure from ionophoretreated broilers is mixed in fields or in manure storage with manure containing other antibiotics. As PNECs are often based on one data set or a few data sets, this might cause concern. Generally, there is therefore a need to perform more toxicological effect studies, and to include effects of mixing ionophores with other anticoccidial agents, growth promoters and veterinary medicines and transformation products thereof. In the USA, ionophores can be given in combination with other agents {e.g., a product containing monensin, melengestrol (growth hormone), ractopamine (growth promoter) and tylosin (broad spectrum antibiotic) [45]}. There are few MECs from the EU, so there is need to analyze ionophores in the environment where the primary source is from broiler production. Similarly, there is need to investigate leaching from agricultural soils and to conduct more studies on dissipation and metabolism.
5. Conclusions Predictions based on European broiler production demonstrated that, in the worst case (Scenario I), ionophores cause a risk in the terrestrial, aquatic and sediment environments, as RQs are as high as 28. When exposures were refined (Scenario II) by adjusting for metabolism and dissipation mechanisms, it was found that ionophores might still pose an environmental risk (RQs up to 2.7). Occurrence data are only available from groundwaters, surface waters and sediment matrices, so the risk for the terrestrial compartment could not be characterized. From data available, it was found that ionophores possibly pose a risk for sediment-dwelling organisms, as RQs were up to 3.4. This assessment concluded that ionophores might be an environmental risk to organisms living in soils, waters and sediments. There are few effect data, so more effect studies, especially long-term and chronic, are needed to clarify environmental and biological effects of http://www.elsevier.com/locate/trac
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ionophores. In addition, there is a need for more detailed fate profiles (e.g., potential for leaching from agricultural fields). Acknowledgements This research was supported by the Drug Research Academy (Faculty of Pharmaceutical Sciences, University of Copenhagen). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.trac.2009. 02.015. References [1] A.B.A. Boxall, D.W. Kolpin, B. Halling-Sorensen, J. Tolls, Environ. Sci. Technol. 37 (2003) 286A. [2] M. Hansen, K.A. Krogh, A. Brandt, J.H. Christensen, B. HallingSørensen, Environ. Pollut. 157 (2009) 474. [3] M. Hansen, E. Bjo¨rklund, K.A. Krogh, B. Halling-Sørensen, Trends Anal. Chem. 28 (2009), doi:10.1016/j.trac.2009.01.008. [4] J.M. Cha, S. Yang, K.H. Carlson, J. Chromatogr., A 1065 (2005) 187. [5] S.C. Kim, K. Carlson, Water Res. 40 (2006) 2549. [6] M.P. Schlusener, K. Bester, M. Spiteller, Anal. Bioanal. Chem. 375 (2003) 942. [7] W. Song, M. Huang, W. Rumbeiha, H. Li, Rapid Commun. Mass Spectrom. 21 (2007) 1944. [8] N. Watanabe, T.H. Harter, B.A. Bergamaschi, J. Environ. Qual. 37 (2008) S-78. [9] J.G. Davis, C.C. Truman, S.C. Kim, J.C. Ascough, K. Carlson, J. Environ. Qual. 35 (2006) 2250. [10] R.A. Brain, D.J. Johnson, S.M. Richards, H. Sanderson, P.K. Sibley, K.R. Solomon, Environ. Toxicol. Chem. 23 (2004) 371. [11] D.G. Hillis, L. Lissemore, P.K. Sibley, K.R. Solomon, Environ. Sci. Technol. 41 (2007) 6620. [12] J. Jensen, X. Diao, A.D. Hansen, Environ. Toxicol. Chem. 28 (2009) 316. [13] E.B. McGregor, K.R. Solomon, M.L. Hanson, Arch. Environ. Contam. Toxicol. 53 (2007) 541. [14] NORM/NORM-VET 2006, Usage of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Norway, Tromsø / Oslo, Norway, 2007. [15] B. Bengtsson, C. Greko, U.G. Andersson, SVARM 2007: Swedish Veterinary Antimicrobial Resistance Monitoring, The National Veterinary Institute (SVA), Uppsala, Sweden, 2008. [16] Y. Kim, J. Jung, M. Kim, J. Park, A.B.A. Boxall, K. Choi, Environ. Toxicol. Pharmacol. 26 (2008) 167. [17] Danish Veterinary and Food Administration, Danish Medicines Agency, Danish Institute for Food and Veterinary Research, DANMAP 2004 - Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark, Statens Serum Institut, Copenhagen, Denmark, 2004. [18] P. Butaye, L.A. Devriese, F. Haesebrouck, Clin. Microbiol. Rev. 16 (2003) 175. [19] R.K. McGuffey, L.F. Richardson, J.I.D. Wilkinson, J. Dairy Sci. 84 (2001) E194.
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