environmental risk assessment of ionophores

Trends Trends in Analytical Chemistry, Vol. 28, No. 5, 2009

Environmental risk assessmentof ionophoresMartin Hansen, Kristine A. Krogh, Erland Bjorklund, Asbjørn Brandt,

Bent Halling-Sørensen

Following European guidelines, this article presents an environmental risk

assessment for four ionophores (monensin, salinomycin, narasin and lasalo-

cid) 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 environ-

mental concentrations in sediments are above predicted no-effect concen-

trations, 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; Lasa-

locid; Monensin; Narasin; Predicted environmental concentration; Predicted no-effect

concentration; Salinomycin

Martin Hansen*,

Kristine A. Krogh,

Erland Bjorklund,

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]

534

1. Introduction

Ionophores are a sub-group of anticocci-dials used extensively in livestock pro-duction (i.e. poultry, cattle, swine, sheepand rabbit) and, as a result, they areconsidered to be emerging environmentalcontaminants [1–3]. For these com-pounds, only a few studies report data onoccurrence [4–8], fate [9] and eco-toxico-logical effects [10–13]. No completeassessment is available that reviews theenvironmental impact of ionophores. Inanother article in this issue [3], we out-lined the status of the current develop-ment of analytical methods targeted toenvironmental samples and reported thatthe few measured occurrence data onionophores were found at lg/kg, ng/L andlg/kg levels in manure, surface watersand sediments, respectively. At present,concentration levels of ionophores in thesoil compartment have not been reported.Moreover, the available occurrence datacould reflect a possible build up of iono-phores in lipophilic compartments (e.g.,soils and sediments [3]). Unfortunately,

0165-9936/$ - see front matter ª 2009 El

only limited information on consumptiondata of ionophores is available and manycountries do not monitor their usage. Ta-ble 1 shows the available consumptiondata from poultry, cattle, swine, sheep,and rabbit production, revealing thationophores are widely and heavily used,and that salinomycin, monensin, narasinand lasalocid are the most frequently ap-plied [14–17].

Ionophores act as antiporters (mode ofaction) by entrapping cations (preferablysodium or potassium), thereby generatingneutral zwitterions. These complexes aretransported across prokaryotic andeukaryotic cell membranes in exchangefor protons, and, accordingly, disrupt iongradients and, ultimately, cause energyreduction and cell death [12,18–22].

In the European Union (EU), ionophoresand other anticoccidial agents are regu-lated in accordance with the directives onfeed additives and may not be put on themarket unless a proper authorization hasbeen given by the European Commissionbased on a scientific evaluation by theEuropean Food Safety Authority (EFSA).Market authorization depends on demon-strating that the ionophores have noharmful effects on human and animalhealth and the environment [23,24].These scientific evaluations are performedby the EFSA panel on additives and prod-ucts or substances used in animal feed[25]. In this article, environmental riskassessment (ERA) is based on an approachsuggested by the EFSA panel [26] follow-ing three harmonized guidance docu-ments:� phase I – exposure assessment [27];� phase II – risk assessment [28]; and,� additional guidance [29].

sevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.02.015

Table 1. Consumption data on ionophores used in livestock production and predicted worst-case concentrations based on arable area

Region Ionophore Amount (kg) Year Area (ha) Arable area PECsoil (lg/kg)

Republic of Korea Salinomycin 639,170a) 2006 9,819,000 16.58% 131Monensin 136,140a) 27.9Lasalocid 66,061a) 13.5

Denmark Salinomycin 10,070b) 2004 4,239,400 52.59% 1.51Narasin 1625b) 0.24Monensin 840b) 0.13Lasalocid 243b) 0.04

Norway Narasin 5615c) 2006 30,744,200 2.70% 2.25Monensin 889c) 0.36Lasalocid 13c) 0.01

PECsoil based on 0.2 m ploughing depth.a) Estimated active substance from product sales [16].b) [17].c) [14].

Trends in Analytical Chemistry, Vol. 28, No. 5, 2009 Trends

The EFSA guidance document [26] on ERA of feedadditives follows a tiered approach starting with a deci-sion tree, resulting in an initial worst-case-scenario cal-culation to give a predicted environmental concentration(PEC) for ionophores in soil, soil-pore water, ground-water, surface water and sediment. If these PECs areabove pre-defined trigger values (10 lg/kg soil and0.1 lg/L in groundwater), they are compared to pre-dicted no-effect concentrations (PNECs) derived fromeco-toxicological effect data adjusted with appropriateassessment factors (AFs). Where the PEC/PNEC ratio is

Figure 1. Overview of the environmental risk-assessment ap-proach.

>1, the PEC is refined taking into account losses of theionophore (metabolism and environmental dissipationmechanisms) and, finally, compared to the PNEC againto indicate if the ionophore poses a potential environ-mental risk. Fig. 1 outlines this entire ERA procedure.

The first aim of this article is to give an overview of thepublished eco-toxicological data on four ionophores (vizmonensin, salinomycin, narasin and lasalocid). Thesecond aim is to present a tiered ERA based on PEC andcompare that with existing effect data to assess the po-tential environmental risk. ERA is also performed bycomparing measured environmental concentrations(MECs) with the same effect data. PEC estimations arebased on the amount of the individual compounds ap-plied in the EU and only ionophores that are approvedfor use in poultry production, according to existing EUregulations. The ERA of ionophores comprises threesuperior scenarios:� Scenario I is a worst-case scenario, which predicts the

environmental exposure of ionophores in agriculturalsoils amended with manure from broilers treated withionophores in agreement with the European regula-tions;

� Scenario II is a refinement of Scenario I, where metab-olism in the broilers and other environmental dissipa-tion mechanisms are taken into account, leading to arefined PEC; and,

� Scenario III assesses the potential environmental riskon the basis of previously published occurrence data(MECs) reported in the scientific literature.

2. Materials and methods

This section outlines the subsequent results and discus-sion 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) ISO 11267 (reproduction, 21 days) EC50 591 [254–927] mg/kg [12]Enchytraeus crypticus (enchytraeid) ISO 16387 (reproduction, 21 days) EC50 356 [95–617] mg/kg [12]Eisenia foetida andrei (earthworm) OECD 207 (mortality, 14 days) LC50 56 mg/kga) [37]Raphanus sativus (radish) Non-standardized (emergence, 14 days) LC50 9.8 mg/kg [36]Soil micro-organisms OECD 216 (nitrification) NOEC >5 mg/kg [37]

OECD 217 (respiration)

Salinomycin Raphanus sativus (radish) Non-standardized (growth rate, 14 days) LC50 1.3 mg/kg [46]Eisenia foetida andrei (earthworm) OECD 207 (mortality, 14 days) LC50 71 mg/kg [46]Soil micro-organisms OECD 216 (nitrification) NOEC >2.3 mg/kg [46]


Narasin Raphanus sativus (radish) OECD 208 (emergence) LC50 5.07 mg/kg [39]Eisenia foetida andrei (earthworm) OECD 207 (mortality, 14 days) LC50 46.4 mg/kg [39]Soil micro-organisms OECD 216 (nitrification) NOEC 17.4 mg/kg [39]


Lasalocid Lolium perenne (perennial ryegrass) OECD 208 (emergence, 18 days) EC50 87.8 mg/kg [40]Eisenia foetida andrei (earthworm) OECD 207 (mortality, 14 days) LC50 71.8 mg/kg [40]Soil micro-organisms OECD 216 (nitrification) NOEC >5 mg/kg [40]


ISO, Guideline number from the International Organization for Standardization; OECD, Guideline number from the Organization for EconomicCo-operation and Development; EC50, Effect concentration where half of population is affected; LC50, Concentration where half of population iskilled; 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 Test method Effect concentration Ref.

Monensin Lemna gibba (macrophyte, floating) ASTM guideline [47] (growth, 7 days) EC50 0.998 [0.955–1.042] mg/L [10]Myriophyllum spicatum(macrophyte, submersed)

Mesocosms (growth rate, 35 days) EC50 0.197 [0.042–0.353] mg/L [13]

Selenastrum subspicatus (algae) OECD 201 (growth rate and biomass) EC50 0.98 mg/L (growth rate) EC50

4.3 mg/L (biomass)[36]

Pseudokirchneriella subcapitata (algae) OECD 201 (growth rate and biomass) EC50 3.41 mg/L (growth rate) [37]EC50 1.73 mg/L (biomass)

Daphnia magna (crustacean) OECD 202 (immobility) EC50 7.29 mg/L [37]Oncorhynchus mykiss (fish) OECD 203 (mortality, 96 h) LC50 1.88 mg/L [37]

Salinomycin Selenastrum capricornutum (algae) OECD 201 (growth rate and biomass,72 h)

EC50 3.01 mg/L (growth rate) [46]EC50 2.09 mg/L (biomass)

Daphnia magna (crustacean) OECD 202 (immobility, 48 h) EC50 13.3 mg/L [46]Oncorhynchus mykiss (fish) OECD 203 (mortality, 96 h) LC50 1.14 mg/L [38]

Narasin Selenastrum capricornutum (algae) OECD 201 (growth rate and biomass,72 h)

EC50 2.91 mg/L (growth rate) [39]EC50 0.77 mg/L (biomass)

Daphnia magna (crustacean) OECD 202 (immobility, 48 h) EC50 20.6 mg/L [39]Oncorhynchus mykiss (fish) OECD 203 (mortality, 96 h) LC50 2.23 mg/L [39]

Lasalocid Selenastrum subspicatus (algae) OECD 201 (growth rate and biomass) EC50 3.1 mg/L (growth rate) [40]EC50 2.0 mg/L (biomass)

Daphnia magna (crustacean) OECD 202 (immobility, 48 h) EC50 5.4 mg/L [40]Brachydanio rerio (fish) OECD 203 (mortality, 96 h) LC50 2.5 mg/L [40]

ASTM, American Society for Testing and Materials guidelines; OECD, Guideline number from the Organization for Economic Co-operation andDevelopment. 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 meth-odologies applied in the three scenarios.

2.1. Eco-toxicological effect dataThe available eco-toxicological data for the ionophoresstudied 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 havebeen performed, and only with monensin [10–13]. Twoof these studies were mesocosm studies that attemptedto mimic minor eco-systemic effects [11,13]. Othersources of eco-toxicological studies were scientificinterpretations or opinions reported by the EFSA panel[25]. Their reports were based on effect studies ofionophores provided by the feed-additive manufactur-ers. Some of the studies followed standardized tests, andthis section includes the usable information extractedfrom these reports.

For test organisms living in soil, the acute toxicologi-cal level was in the range 5.07–591 mg/kg, with radishbeing the most sensitive species and springtails the mostinsensitive of the species tested. No-observed-effect con-centrations (NOECs) on respiration and nitrification testswere obtained on soil micro-organisms and found to bein the range of 2–17 mg/kg [25]. Lethal-effect concen-trations for the classic soil-test organisms, earthworms,were 46.4–71.8 mg/kg for the four compounds. No ef-fect data exist on dung-dwelling organisms, which mightbe vulnerable, as was observed for other antiparasiticides(e.g., ivermectin [30]). Similarly, no data was availablefor sediment-dwelling organisms. The acute toxicologicallevel for aquatic living organisms (e.g., algae, crusta-ceans and fish) was in the range 0.77–20.6 mg/L for allfour compounds. In general, algae were the most sensi-tive species, while crustaceans were the least sensitive.Monensin was tested towards macrophytes, showingeffect concentrations in the range 0.197–0.998 mg/L forthese sensitive aquatic organisms.

Table 4. Scenario I with predicted environmental concentrations (PECs) fgiving risk quotients (RQs) for soil, surface water and sediment

Dose(mg/kgfeed)*

PECsoil

(lg/kg)PECgw

(lg/L)PECsw

(lg/L)PECsed

(lg/kg)*T

L(E)C50

(mg/kg)

Monensin 125 650 280 28 67 9.8 1Salinomycin 70 364 157 16 38 1.3 1Narasin 70 364 157 16 38 5.07 1Lasalocid 125 650 50 5.0 70 71.8 1

Model parameters given in Supplementary material available in web versiofor lasalocid; PECgw, PEC in groundwater (equals PECporewater); PECsw, PECare also given in Supplementary material: AF, Assessment factor; RQ, Risk*Value given on wet weight (wwt) basis. PECsoil is the steady-state concen

2.1.1. Predicted no-effect concentrations. The PNECswere derived applying an assessment factor (AF) to theshort-term effect data [26]. For the terrestrial compart-ment, three taxonomic groups (plants, soil-dwellingorganisms and micro-organisms) were tested, so that theenvironmental risk could be established. The most sen-sitive species were plants (radish) for monensin, salino-mycin and narasin, while lasalocid was found to be mostsensitive towards earthworms (Table 4).

By using an AF of 100, as recommended by theEuropean Medicines Agency [28], the PNEC value wasfound. Similarly, three taxonomic groups (algae, crus-taceans and fish) represented the aquatic compartment,and the fish species were found to give the lowest PNECsusing an AF of 1000 [28].

Tables 4 and 5 list the PNEC values derived fromresults of the assessment. For estimation of the potentialrisk of the sediment compartment, the PNECsediment

values were derived from the PNECaquatic, as suggestedby the European Medicines Agency [29], since no effectdata were available for sediment-dwelling organisms.The calculations were based on equilibrium partitioning,so they depended on Koc (Equation 9, Supplementarymaterial available in the web version). Values ofPNECsediment were in the range 3.44–36.7 lg/kg of wetweight (wwt) sediment (Table 4).

2.2. Environmental risk-assessment scenariosScenario I predicts the worst-case environmental expo-sure on the basis of doses applied in European broilerproduction. Scenario II is a refinement of Scenario Itaking into account elimination of the parent ionophore.Scenario III utilizes published environmental occurrencedata (MECs). In all scenarios, the risk is characterizedusing a risk quotient (RQ) as the ratio between envi-ronmental concentrations obtained (PECs or MECs) tothe PNECs [26].

2.2.1. Basic set of parameters. Broilers have a life cycleof 35–45 days with an average life of 41 days (this value

or ionophores related to predicted no-effect concentrations (PNECs)

errestrial Aquatic Sediment

AF PNEC(lg/kg)

RQ L(E)C50

(mg/L)AF PNEC

(lg/L)RQ PNEC

(lg/kg)*RQ

00 98 6.6 1.88 1000 1.88 15 5.68 1200 13 28 1.14 1000 1.14 14 3.44 1100 51 7.2 2.23 1000 2.23 7.0 6.73 5.600 718 0.9 2.5 1000 2.5 2.0 36.7 1.9

n: Koc 125 L/kg for monensin, salinomycin and narasin; Koc 732 L/kgin surface water; PECsed, PEC in sediment. Other model parametersquotient (PEC/PNEC ratio).

tration obtained after the sixth application of manure (at day 410).


Table 5. Scenario II with refined predicted environmental concentrations (PECs) after six consecutive applications of manure (every 82 days) tothe same agricultural soil (taking into account metabolism, manure and soil-dissipation mechanisms)

PECsoil

(lg/kg)PECgw

(lg/L)PECsw

(lg/L)PECsed

(lg/kg)*Terrestrial Aquatic Sediment

PNEC (lg/kg) RQ PNEC (lg/L) RQ PNEC (lg/kg)* RQ

Monensin 63.4 27.3 2.7 6.6 98 0.6 1.88 1.5 5.68 1.2Salinomycin 35.5 15.3 1.5 3.7 13 2.7 1.14 1.3 3.44 1.1Narasin 35.5 15.3 1.5 3.7 51 0.7 2.23 0.7 6.73 0.5Lasalocid 63.4 4.9 0.49 6.8 718 0.1 2.5 0.2 36.7 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 manuredissipating (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 theirlife, they are constantly treated with ionophores (doserange varied between ionophores and was in the range50–125 mg/kg of dry weight (dwt) feed) [25] with awithdrawal period of 1–5 days before slaughter [25]. Wepresumed that the broilers received the maximum al-lowed dose of the specific ionophore and were treatedtheir entire life cycle (i.e. the withdrawal period ofionophores 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 was0.36 kg N/broiler/year [26]. Supplementary material,available in the web version, contains the range of base-set model parameters and equations to give PECs, asdefined in the EFSA guidance document [26].

2.2.2. Scenario I – Worst-case exposure. Worst-casePECs were calculated by assuming that all ingestedionophores were excreted as parent compound and thatbroiler manure (170 kg N/ha/year) was applied to theagricultural soil in one application [26]. PECsoil wascalculated based on the dose of the ionophores and al-lowed nitrogen levels. The concentration in pore water(PECporewater) was calculated using sorption-distributioncoefficient Kd (calculated via Koc) between soil and water.The concentration in groundwater (PECgroundwater) wasdefined as the value of pore water [26,29,32].PECsurfacewater was calculated using a dilution factor of10 compared to PECporewater [26,32]. However, otherguidelines recommended a dilution factor of 3 [29].PECsediment was calculated on the partitioning betweensurface 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 themetabolism of ionophores in the broiler and dissipationin manure, soil and water. Observing two succeedingbatches of broilers from the same barn, the first batch ofbroilers had a life cycle from day 0 to day 41, and thesecond batch of broilers had a life cycle from day 41 today 82. When being produced, the manure was not re-


moved from the barn throughout the life cycle of thebroiler. At day 41, the first batch of broilers wasslaughtered and the manure was collected and stored inpiles or tanks, and the life cycle of the second batchbroilers started. At day 82, the manure from the secondbatch was removed (and broilers were slaughtered),mixed with the manure from first batch and spread ontoagricultural land. As a result, the broiler manure wasapplied every 82 days to the same agricultural soil withan average of 38 kg N/ha, thereby resulting in a total of170 kg N/ha/year.

Dissipation half-lives in manure were demonstrated tobe 5 days for salinomycin in pig manure [33]. However,pig manure differs from broiler manure [34]. In anotherstudy, monensin has a half-life of 22 days in turkeymanure [35]. This latter first-order decay value wasused. The EFSA panel has evaluated the metabolism ofionophores in broilers. Total excreted amounts of parentionophore 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 allionophores at 81% of the initial dose. For the terrestrialcompartment, the reported half-lives of ionophores werein 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 dissi-pation during storage (in the barn and in storage). Thiswas done by subdividing the total amount of ionophoresinto daily segments and by considering dissipation (half-life 22 days) of the ionophores in each segment (day).Finally, this resulted in a cumulative amount of iono-phores after 82 days (Equation 12, Supplementarymaterial available in the web version).

PECsoil followed the same cumulative approach withdissipation half-lives of 49 days in soil and in segments of82 days (Equations 13 and 14, Supplementary materialavailable in the web version). The upper level (PECsoil)after the sixth manure application (at day 410) was usedfor the subsequent ERA.

We also calculated the concentration in manure andsoil when variations in dissipation half-lives and storage


time (broiler life cycle) were taken into account.Monensin and lasalocid gave similar curves, as theywere given in same dose, which also applied for salino-mycin and narasin.

2.2.4. Scenario III – Measured environmental concentra-tions. Risk characterization in this scenario was basedon MECs or occurrence data, as reviewed by Hansen andco-workers [3]. The available MECs were, primarily,from the USA, where ionophores can be applied for otherlivestock besides poultry.

Figure 2. Cumulative concentration of ionophores (monensin andlasalocid) in broiler manure when considering dissipation. Twobatches of broilers were studied, first batch day 0–41 (black line)and second batch day 41–82 (red line). Manure from first batchwas stored day 41–82 (black line). At day 82, manure from bothbatches was mixed (the resulting concentration is marked with ablack 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 chan-ged to 35 days or 45 days.

Figure 3. Cumulative soil concentration of ionophores (monensinand lasalocid) after six consecutive additions of manure to the samesoil every 82 days (black line). Effect of dissipation half-lives: 10days (blue), 25 days (green) and 49 days (black). Dashed lines de-pict upper steady-state concentration level.

3. Results

3.1. Scenario I – Worst-case exposureResults from this scenario yielded PECmanure in thebroiler manure of 6428 mg/kg N for salinomycin andnarasin, and 11,479 mg/kg N for monensin and lasalo-cid. After application of manure onto the soil, PECsoil

values were estimated to be 364–650 lg/kg and PECs inwater compartments 50–280 lg/L (PECporewater andPECgroundwater), 5.0–28 lg/L (PECsurfacewater) and 38–70 lg/kg wwt (PECsediment).

A risk characterization based on these PECs gave RQsfor the terrestrial compartment of 6.6, 28, 7.2 and 0.9for monensin, salinomycin, narasin and lasalocid,respectively. Similarly, RQs were 2.0–15 for the aquaticenvironmental and 1.9–12 for the sediment compart-ment. Table 4 summarizes Scenario I.

3.2. Scenario II – Refined exposureThe 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 stor-

age;(3) dissipation in the soil after manure amendment;

and,(4) dissipation in water and sediment.

When considering metabolism (81% excreted) anddissipation (DT50 22 days) in manure, the concentrationin mixed manure from two batches of broilers after 82days reached 1915 mg/kg N for salinomycin and nara-sin, and 3420 mg/kg N for monensin and lasalocid(black cross, Fig. 2). The cumulative concentrations ofmonensin and lasalocid in manure are displayed in Fig. 2(black and red lines). Similar curves could be obtainedfor salinomycin and narasin, but these were given inlower 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

(Fig. 3). The steady state value (upper concentration) forPECsoil on the sixth manure application (Fig. 3), takinginto account soil dissipation (DT50, soil 49 days), wasfound to be 63.4 lg/kg for monensin and lasalocid, and35.5 lg/kg for salinomycin and narasin.

The effects of dissipation half-lives were also investi-gated for the soil compartment, and Fig. 3 shows theconcentrations depicted with dashed lines. Groundwaterconcentrations were in the range 4.9–27.3 lg/L, surfacewater 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 formonensin in the aquatic and sediment compartments.Narasin and lasalocid were in all cases below 1. Table 5summarizes Scenario II.


Table 6. Scenario III with maximum measured environmental concentrations (MECs, adapted from [3]) related to predicted no-effect concen-trations (PNECs) giving risk quotients (RQs) for soil, surface water and sediment

MECsoil

(lg/kg)MECgw

(lg/L)MECsw

(lg/L)MECsed

(lg/kg)*Terrestrial Aquatic Sediment

PNEC (lg/kg) RQ PNEC (lg/L) RQ PNEC (lg/kg)* RQ

Monensin NA 0.390 0.220 12.1 98 U 1.88 0.12 5.68 2.1Salinomycin NA NA 0.040 11.6 13 U 1.14 0.04 3.44 3.4Narasin NA NA 0.060 6.3 51 U 2.23 0.03 6.73 0.9Lasalocid NA NA 0.028 NA 718 U 2.5 0.01 36.7 U

NA, Not analyzed; U, Unknown.*Value given on wet weight (wwt) basis.


3.3. Scenario III – Measured environmental concentra-tionsThis scenario is based on comparing the publishedoccurrence data (MEC) previously reviewed [3] with thePNECs derived.

The existing MEC of salinomycin from pig manure(11 lg/kg, [6]) is very low in relation to the predictedvalues for broilers. No values of MEC are available forsoil, so, this compartment cannot be properly evaluatedin this scenario.

The measurements from the aquatic compartmentthat exist were mostly obtained from the USA, where thesubstances are applied for other species. The four iono-phores 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 wasconverted into wet-weight basis for comparability andyielded 0.3–12.1 lg/kg wwt. Consequently, this gaveRQs of 0.9 for narasin, 2.1 for monensin and 3.4 forsalinomycin (no data on lasalocid). Table 6 summarizesScenario III.

4. Discussion

4.1. Scenario I – Worst-case exposureAll worst-case PEC values were above the trigger valuesof 10 lg/kg for soil and 0.1 lg/L for surface water(Fig. 1), so studies on environmental fate and effects ofthe compounds are mandatory to perform risk charac-terization [26].

This risk assessment with respect to the terrestrialcompartment showed that monensin, salinomycin andnarasin pose a risk, as they have RQs >1 (Table 4).Lasalocid has an RQ of 0.9. The lower potency of lasa-locid relative to the other three ionophores is not easilyexplained and might reflect the need for more effectstudies.

For the aquatic environment, risk characterizationshowed that all ionophores pose a risk with RQ values of2.0–15 (Table 4). The resulting RQs for sediment werefound to be in of same order of magnitude as those for


the aquatic compartment. These estimations indicatedthat ionophores might pose a risk to organisms dwellingin the sediment compartment. It must be stressed thateffects on sediment-living organisms have not beeninvestigated and this might be of concern. Refinement ofexposure in all environmental compartments must beperformed, as outlined in the guideline [26], becauseionophores yield environmental RQs >1 (apart fromlasalocid in soil). This refinement of exposure was donein Scenario II.

4.2. Scenario II – Refined exposureRefinement of PECs from Scenario I were performedtaking into account an average metabolism rate of 81%for all ionophores in the broilers and dissipation of theparent compound in the environment [26,32].

One study elucidated the toxicity of a transformationproduct of salinomycin, which was found to be ‘‘toxi-cologically harmless’’ to mice [41]. However, no otherinvestigations have been performed on the eco-toxico-logical effect of transformation products from iono-phores.

The dissipation half-life in broiler manure was set to22 days, due to literature findings. A manure storagetime of 3 months was proposed [26], but the morecommon value of 41 days was used, as storage capacityis typically restricted [29], combined with mixing of freshmanure (from second-batch broilers) at day 82. Fig. 2shows the effect of broiler life cycle (or storage time) andDT50 for monensin and lasalocid. These graphical pre-dictions show that the dissipation half-life in manurewas the most important variable relative to the broilerlife cycle (or the cumulative storage time of the manure).

As in manure, the dissipation rate in soil was found tobe an important parameter in reducing ionophore con-centration. By this refinement, soil concentrations werereduced by a factor of 10 relative to worst-case ScenarioI, so the environmental risk persists (Table 5).

No dissipation has been reported for the aquaticcompartment. In conclusion, we cannot exclude thepossibility that ionophores pose an environmentalrisk.


4.3. Scenario III – Measured environmental concentra-tionsFew occurrence data exist, so Scenario III can only beconsidered a preliminary approximation to the trueproblem. Using MEC values, RQs of 0.01–0.12 (0.21 forgroundwater) were obtained for the aquatic compart-ment, so the ionophores occur in a range that causesminor concern based on the current short-term toxico-logical data.

Comparing MEC and refined PECsurfacewater showedthat MECs were a factor of 12–38 lower than the pre-dicted values. This might mean that the proposedguidelines are sufficiently conservative when predictingthe aquatic compartment concentrations of ionophores.In addition, the available MEC data are primarily fromthe USA and these measurements might include a frac-tion of ionophores used in, e.g., beef production, so theMEC/PEC ratio might be even lower.

In contrast to the aquatic compartment, ionophoresmight pose an environmental risk to sediment-dwellingorganisms (RQ 0.9–3.4, Table 6), so there is cause forconcern when looking at the sediment compartmentbased on current occurrence data.

4.4. General discussion and outlookReported total amounts of ionophores used in differentcountries (Table 1) give worst-case concentrations up to131 lg/kg soil (no dissipation) for salinomycin whendiluted into total arable area in the Republic of Korea. Asa result, there is cause for concern, as these agents areapplied in such high quantities in animal production.

Available toxicological effect data are mainly obtainedfrom second-hand reporting. The effect data presentedare the only data available from the literature and dat-abases. Despite this, the short-term effect data are in theranges low to high mg/kg for soil and sub-mg/L to lowmg/L in the aquatic environment. Chief concernsregarding effect data are that no chronic exposurestudies have been demonstrated and that no exposuredata are obtainable from sediment-dwelling organisms.There is therefore a need to perform effect testing espe-cially for sediment-dwelling organisms.

Moreover, it was found that sorption (Koc) of theionophores was a crucial model parameter for predictedconcentrations in waters, as these calculations are basedon partitioning between solids and water. A single re-search study evaluated sorption of monensin and lasa-locid to eight different soils [42] and found Koc values of125–5700 L/kg and 732–15,700 L/kg, respectively. TheKoc values for salinomycin and narasin were defined asthe same as those for monensin, due to a comparablestructural relationship and unclear, contradictory datareported elsewhere [25].

The refinement of exposure (PEC), as in Scenario IIand recommended in guidelines [26,28,29], is accept-able if metabolites are less potent than the parent com-

pound. However, there are examples of transformationproducts or metabolites that have a similar or more toxiceffect than the parent compound [2,43]. In addition, inbroiler production, anticoccidial agents are typically gi-ven in shuttle programs (i.e. shifting between ionophoresand synthetics between broiler production batches). Ithas been demonstrated that ionophores might havesynergistic (more than additive) effects when combinedwith other biologically active compounds. When mac-rolide antibiotics were added to feed containing non-toxic levels of monensin, this caused lethal effects incattle [44]. In a recent study, narasin was combinedwith the synthetic compound nicarbazin and the com-bined mixture was five times more toxic than predictedfrom additive effects [2]. The risk of adverse effect mighttherefore be enhanced when manure from ionophore-treated broilers is mixed in fields or in manure storagewith manure containing other antibiotics.

As PNECs are often based on one data set or a few datasets, this might cause concern. Generally, there istherefore a need to perform more toxicological effectstudies, and to include effects of mixing ionophores withother anticoccidial agents, growth promoters and vet-erinary medicines and transformation products thereof.

In the USA, ionophores can be given in combinationwith other agents {e.g., a product containing monensin,melengestrol (growth hormone), ractopamine (growthpromoter) and tylosin (broad spectrum antibiotic) [45]}.There are few MECs from the EU, so there is need toanalyze ionophores in the environment where the pri-mary source is from broiler production. Similarly, thereis need to investigate leaching from agricultural soils andto conduct more studies on dissipation and metabolism.

5. Conclusions

Predictions based on European broiler production dem-onstrated that, in the worst case (Scenario I), ionophorescause a risk in the terrestrial, aquatic and sedimentenvironments, as RQs are as high as 28. When expo-sures were refined (Scenario II) by adjusting for metab-olism and dissipation mechanisms, it was found thationophores might still pose an environmental risk (RQsup to 2.7).

Occurrence data are only available from ground-waters, surface waters and sediment matrices, so the riskfor the terrestrial compartment could not be character-ized. From data available, it was found that ionophorespossibly pose a risk for sediment-dwelling organisms, asRQs were up to 3.4.

This assessment concluded that ionophores might bean environmental risk to organisms living in soils, wa-ters and sediments. There are few effect data, so moreeffect studies, especially long-term and chronic, areneeded to clarify environmental and biological effects of



ionophores. In addition, there is a need for more detailedfate profiles (e.g., potential for leaching from agriculturalfields).

AcknowledgementsThis research was supported by the Drug ResearchAcademy (Faculty of Pharmaceutical Sciences, Univer-sity of Copenhagen).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.trac.2009.02.015.

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environmental risk assessment of ionophores

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