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Aquatic Toxicology 85 (2007) 229–240

Assessment of the environmental fate and effects of ivermectinin aquatic mesocosms

Hans Sanderson a,∗, Brian Laird b, Louise Pope c, Richard Brain d, Christian Wilson e,David Johnson e, Gareth Bryning c, Andrew S. Peregrine f, Alistair Boxall c, Keith Solomon g

a National Environmental Research Institute, Department of Policy Analysis, Aarhus University, Frederiksborgvej 399, Post Box 358,DK-4000 Roskilde, Denmark

b University of Saskatchewan, Department of Soil Science, Saskatoon, Saskatchewan S7N 5A8, Canadac York University, Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom

d Center for Reservoir and Aquatic Systems Research, Department of Environmental Studies, Baylor University, Waco, TX 76798, USAe Canadian Food Inspection Agency, Ottawa, Ontario K1A 0Y9, Canada

f University of Guelph, Ontario Veterinary College, Department of Pathobiology, Guelph, Ontario N1G 2W1, Canadag University of Guelph, Toxicology, Department of Environmental Biology, Guelph, Ontario N1G 2W1, Canada

Received 9 August 2007; received in revised form 29 August 2007; accepted 31 August 2007

bstract

Pharmaceuticals in the environment have been subject to increasing public concern and scientific investigation over the past years. Morehan 100 active pharmaceutical ingredients have been detected in surface waters worldwide at the ng to �g L−1 range. At these low levels it isommonly assumed that only chronic and/or mixture toxic effects will be discernible in aquatic ecosystems and that there are orders of magnitudeetween exposure and effect concentrations. Assessment of potential ecosystem risk of pharmaceuticals are recommended but rarely performed inesocosms, so for most risk assessments the final tier to reduce extrapolation uncertainty is missing. This paper describes the fate and effects of the

nthelmintic drug ivermectin for a 265-day period following treatment (nominal concentration levels of 0, 30, 100, 1000 ng L−1 (or parts per trillionppt)) in fifteen 12,000 L outdoor aquatic mesocosms. Although it is established that ivermectin is highly toxic towards invertebrates, it has beenelieved that ivermectin does not present notable risks to aquatic systems due to the rapid dissipation of the compound and binding to the sediment.ence, fate and exchange of ivermectin between water and sediment were evaluated in this study. The ivermectin DT50aqueous in water was found

o be 3–5 days, but concentrations increased and appeared to be stabile in the sediment at 20–30 ng kg−1 with no assessable DT50sed. Acute effectsfirst week) following ivermectin exposure were identified and cladocerans were particularly sensitive (nom. 100 ppt). Chronic responses (<day

7) were observed for the ecosystem structure and function (nom. 30 ppt). Long-term effects (>229 days) were identified for more sediment-activerganisms (e.g. Chydoriae and Ephemeroptera) (nom. 1000 ppt). This is the first study to demonstrate the potential environmental risk of ivermectint or below the predicted environmental concentration using a standardized test methodology (mesocosm) with minimal extrapolation uncertainty.

2007 Elsevier B.V. All rights reserved.

eaht

eywords: Mesocosm; Ivermectin; Risk assessment; Sediment; Zooplankton

. Introduction

Pharmaceuticals have received increasing environmentalcrutiny and attention over the past years following, among oth-

rs, seminal papers by Daughton and Ternes (1999) and Kolpint al. (2002) detailing the presence of pharmaceuticals in sur-ace waters at low concentrations. However, the environmental

∗ Corresponding author. Tel.: +45 4630 1822; fax: +45 4630 1200.E-mail address: hasa@dmu.dk (H. Sanderson).

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166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2007.08.011

ffects and subtle changes associated with pharmaceuticals inquatic ecosystems remain relatively unknown. Other papersave analyzed the probability of hazard (Sanderson et al., 2003),he magnitude of toxicity for individual and classes of com-ounds (Sanderson et al., 2004a, b), and the ecosystem effectsollowing treatment with high concentrations of high tonnagentibiotics (e.g. four tetracyclines as a mixture) (Wilson et al.,

004) in mesocosms, in an attempt to elucidate the potentialnvironmental impacts of pharmaceuticals in surface waters.espite the high toxicity (EC50 < 1 mg L−1) of many com-ounds (Sanderson et al., 2004a) it has yet to be convincingly

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emonstrated that individual pharmaceutical compounds exhibitignificant environmental risks at the highest tier of testingWilson et al., 2004).

In the European Union (EU), antibiotics and anthelminticsantiparasitic drugs) are the most important groups of veteri-ary pharmaceuticals, both with a market volume greater than00 million Euros in 1999 (Tolls, 2001). Ivermectin is one of theost widely used macrocyclic lactones and has been known as a

otent, effective and safe antiparasitic drug since 1981. The com-ound is commonly used for treatment and prevention of internalnd external parasites of cattle, horses, and other animals on pas-ure (e.g. gastrointestinal and respiratory tract nematodes, flies,rubs, ticks, lice, and mites). Moreover, in Africa it is admin-stered annually to at least 18 million people as a preventativegainst river blindness (onchocerciasis) (Laffont et al., 2002),nd can be used similarly for lymphatic filariasis (Wuchereriaancrofti and Brugia species). Ivermectin is the highest soldndoparasiticide in North America, approximately 50% of theattle population is treated with an anthelmintic per year andpproximately 50% of these treatments are ivermectin, withtandard dose is 0.2 mg kg−1 bodyweight (Boxall et al., 2004).vermectin is also used as a treatment against ectoparasitic cope-ods (Lepeophtherirus salmonis Krøyer and Caligus elongatesordmann), commonly known as Sea Lice in the aquaculture

ndustry. However, the safety margins of using ivermectin inquaculture are very narrow, hence the compound is officiallyot commonly used for this purpose today, however, monitor-ng the dosing level in this usage is of particular importance.he typical oral dose is 0.05 mg kg−1 fish twice weekly forweeks, resulting in predicted levels of 2.2–6.6 mg m−2 iver-ectin reaching the seabed below aquaculture facilities (Collier

nd Pinn, 1998).The first significant considerations concerning the ecologi-

al fate and effects of ivermectin were reported in 1987, whenstudy at Bristol University, United Kingdom showed thathen the drug was given to calves at the recommended dose,

ts insecticidal effect in dung killed beneficial dung-degradingnsects (Coleoptera sp. and Scarabaeidae sp.). As a result, itas argued that this impact could potentially slow nutrient

ycling, and increase dung accumulation in pastures, ultimatelyesulting in decreased pasture productivity (Wall and Strong,987). Subsequently, the United States Food and Drug Admin-stration (USFDA) performed an environmental risk assessmentf ivermectin in 1996 (USFDA, 1996; Halley et al., 1989;essel et al., 1989). The environmental assessment found thataphnia magna was the most sensitive species with a 48 hC50 = 25 ng L−1, and a no observed effect level (NOEL) at10 ng L−1. However, due to high sorption (Koc > 12.600) to

ediments and rapid dissipation in water (DT50aqueous = 12–39 h)he assessment concluded that less than 2 ng L−1 ivermectinould reach surface waters. Consequently, it was concluded

hat the compound did not represent a significant ecologicalisk (USFDA, 1996). In 2003, a European group also reported

n the toxicity of ivermectin and produced a species-sensitivityistribution comprising 17 different species of crustaceans andollusks (Montforts et al., 2003). Ivermectin was very potent,ith significant toxicity exhibited towards non-target organ-

((m1

icology 85 (2007) 229–240

sms by inhibition of signal transmission at GABA-gated andlutamate-gated chloride channels. It is an agonist for theeurotransmitter gamma-aminobutyric acid (GABA), alteringhloride ion exchange in GABA regulated chloride channels,ausing hyperpolarization of the neuronal cells, and flaccidaralysis in arthropods and nematodes. The target speciesecome paralyzed and die as a result of inhibition of inter-neuralnd neuromuscular transmission (MSD, 1988). Despite this, itas concluded that ivermectin would not represent a significantazard due to its physio-chemical properties, which controlsts environmental fate and exposure potential. However, thesessumptions are based on laboratory and modeled predictions,nd have not been verified in replicated mesocosm studies, whichere recently suggested to be a useful tool for the risk assess-ent of veterinary medicinal products (Van den Brink et al.,

005).The aim of this paper was to test these assumptions under

uasi-natural conditions in large outdoor aquatic mesocosms,hereby minimizing extrapolation uncertainty from the labora-ory to the field. The specific assumptions/hypotheses evaluatedan be summarized as follows: Aquatic and sediment half-ife DT50 = 39 h and ∼45 days (USFDA, 1996), respectively;cute and chronic ecological effect concentrations = 25 and0 ng L−1 (Montforts et al., 2004), respectively; ivermectin,hen used as an anthelmintic in cattle does not represent a risk

o aquatic/littoral ecosystem structure and function, assumingealistic worst-case exposure scenarios (25 ng L−1) (Montfortst al., 2004).

In conducting this experiment, we used ivermectin as aorst-case model compound (high toxicity, and high use) to

larify potential impacts of pharmaceuticals on aquatic envi-onments. If ivermectin is not able to produce significant,tandardized/traditional and discernible risks it would suggesthat potential impacts of this compound and most other pharma-euticals on aquatic ecosystems indeed most likely are subtle andifficult to quantify with significant extrapolation uncertaintyithin present environmental risk assessment methodologies,ith hormones as a possible exception. In this study we only

onsider exposure of ivermectin as a result of its use in cattle,nd not due to other exposure sources (pesticide (e.g. in cit-us plantations), small ruminants, pigs, aquaculture, companionnimals (pets) or human usage).

. Materials and methods

.1. Compound

Ivermectin is a semi-synthetic derivative of an avermectin,group of macrocyclic lactones produced by the soil bac-

erium Streptomyces avermitilis. CAS numbers; 70288-86-7ivermectin mixture); 70161-11-4 (component B1a); 70209-81-(component B1b). Ivermectin is a mixture of 80% ivermectin

omponent B1a (5-O-demethyl-22, 23 dihydroavermectin)

C48H74O14; MW = 875.1), and 20% ivermectin component B1b5-O-demethyl-25-de(1-methylpropyl)-22, 23-dihydro-25-(1-ethylethyl) avermectin) (C47H72O14; MW = 861.1) (USFDA,

996; Halley et al., 1989). The octanol/water partitioning coeffi-

H. Sanderson et al. / Aquatic Tox

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ient (Kow) is 1651 (3.2 log Kow), sorption partition coefficientsetween water and organic carbon (Koc) range from 12,600 to5,700 L kg−1 (4.1–4.2 log Koc) (depending on soil characteris-ics), and Kom values range from 4500 to 5500 L kg−1 (USFDA,996; Halley et al., 1989) and the Kd is 333 in sandy loam (Boxallt al., 2004). The structure–activity relationship (SAR) predictedbioconcentration factor (BCF) = 60 (=1.8 log BCF) (BCFWIN.14), with a log Kow of 3.2. We purchased a total of 18 g ofvermectin (CAS # 70288-86-7) from Sigma-Aldrich (96.7%urity) for method development and treatment of the mesocosmsFig. 1).

.2. Mesocosms

The studies were conducted during the summer/fall (treat-ent occurred in the third week of August) 2004 at theniversity of Guelph Mesocosm Facility located at the Guelphurfgrass Institute, Guelph, Ontario, Canada (latitude 43.5◦ N).he facility consists of 30 mesocosms that are sunken into theround and designed to replicate natural pond systems. Theyre constructed according to expert recommendations to controlariability (SETAC, 1991; Sibley, 2000). The mesocosms arepproximately 1.2 m deep with a water depth of 1 m, a diam-ter of 3.9 m and a surface area of 11.95 m2 with a volume ofpproximately 12,000 L of water. The mesocosms are lined withlack PVC, which before use was pressure washed with pondater without the use of detergents and left in the heat of the

un for 7 days. The mesocosm facility is located in an open areaithout differentiated shading or wind exposure. As the top of

ach mesocosm is elevated 15 cm above ground level there iso run-off to or from the mesocosms. Approximately 50% ofhe mesocosm bottom is covered with 5 cm natural soil sedi-

ent containing natural bacterial communities. The mesocosmsere filled with water from a more than one decade old 0.1 ha,m deep adjacent spring-fed man-made pond. Water was cir-ulated between the mesocosms and the pond for four weeks,nd through the circulation with the 0.1 ha pond, mesocosmsere seeded with natural populations of bacteria, phytoplank-

on, and zooplankton. The mesocosms were also seeded withinsed submerged macrophytes (Myriophyllum sibiricum) fromhe adjacent pond, which prior to treatment populated approx-mately 50% of the bottom area. Fish were not introduced to

cect

icology 85 (2007) 229–240 231

he mesocosms. Amphibians and birds did have access to theesocosms, however, the whole facility is enfenced so that

arger mammals do not have access. The mesocosms were lefto stabilize over a period of 12 months after circulation ofater between them for 3 weeks was terminated. There wereo statistically significant differences between the control meso-osms and potential treated mesocosms prior to treatment (day 1,mean > 0.85) for any biotic or abiotic endpoint. The mesocosm

est was performed according to OECD (2006a).

.3. Treatment and residue sampling protocols

The range of ivermectin concentrations that were used washosen for the following reasons; (1) to approach the highestorst case predicted direct environmental exposure concentra-

ions of 1000 ng L−1 (USFDA, 1996); (2) to include the realisticredicted environmental concentration (PEC) of 25–60 ng L−1

alculated in accordance to (Montforts et al., 2004); and (3) topproach the D. magna 48-h LC50 of 25 ng L−1 (Halley et al.,989).

Our treatment regime was hence in triplicate (n = 3): Controls:0; 100; 300; and 1000 ng L−1 nominal concentrations. The con-rols were positive controls (co-solvent acetone at 0.00001%,/v).

The treatment occurred over 4 days (in the first week ofugust 2004) as the majority and highest concentration of iver-ectin in dung occurs with in the first 4 days of being passed in

o the environment (Lumaret et al., 2005). The season for exper-mentation was chosen to mimic the most frequent treatmenteriod before cattle are taken off pasture (USFDA, 1996). Afteray 4, treatment was terminated and the degradation and fatef the parent compound was monitored. Samples were initiallyollected daily for 10 days, thereafter samples were collectedeekly until day 39, then biweekly until day 68, and finallyonthly, until ice covered the mesocosms shortly after day 97.fter the ice cleared in the spring (day 229) sampling on aiweekly basis was resumed.

A 940 mg L−1 ivermectin/acetone stock solution was pre-ared by weighing 0.1000 g of ivermectin (94% purity B1a) intosilanized 100 mL volumetric flask using an analytical balance

Mettler Toledo AB104) and filling to mark with acetone (Fisher,ptima, CAS 67-64-1). The volume of this stock solution to bedded to each mesocosm, resulting in nominal concentrationsf: 30; 100; 300; and 1000 ng L−1 was: 0.319; 1.064; 3.191; and0.638 mL, respectively. The per-area nominal concentrationsere: 83; 25; 8.3; and 2.5 ng m−2 on each of the four treat-ent days. The mesocosms were treated in serial dilution in

riplicate from the stock solution. These volumes were addedo previously labeled 100 mL wide-mouth polypropylene bot-les wrapped with aluminum foil. Acetone was added to eachontainer by mass, producing final volumes of 100 mL (acetoneensity = 79 g 100 mL−1). Acetone was chosen as a co-solventrimarily due to its low toxicity to D. magna, and the total

oncentration in all mesocosms was nominally 0.01 mL L−1:quivalent to ten times less than maximum allowable co-solventoncentrations according to OECD test guideline TG 211 forhe 21-day chronic D. magna laboratory reproduction test.

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Treatment solutions were slowly poured into three locationsf each mesocosm and mixed with a cordless paint stirrer appliedo the water surface at each location for 30–60 s. After all solu-ion had been added, the container was filled with mesocosmater from the treated pond and poured back into the meso-

osm with an additional 10–30 s of mixing. Control mesocosmsere treated with 100 mL of acetone applied using the describedixing technique. The treatments were applied in ascending

reatment concentration on four successive mornings.Water samples for residue analysis were taken with a 2.5 m

ong depth integrated water sampler (Solomon et al., 1982) at 9.m. on every sampling day from various sites around the meso-osms. The total combined volume was 4 L from each mesocosmn each sampling day, and from this a sub-sample of 250 mL wasaken for residue analysis for the controls and those treated with000 and 300 ng L−1, and 300 mL was taken from the meso-osms treated at 100 and 30 ng L−1, due to the lower dosage.emaining excess water was returned to the sampled meso-osm. The residue water samples were transferred to 330 mLmber glass bottles for solid phase extraction the same day.

Sediment was collected from three random locations fromach mesocosm. It was sampled following all other measure-ents to minimize the influence of disturbing the water-sediment

nterface on zooplankton, chlorophyll a, and water residues.ach sample contained sediment from three trays pooled fortotal of 75 mL. Excess water was poured back into the pond.hese samples were immediately frozen following collectionnd maintained frozen until extraction was performed.

.3.1. Solid-phase extraction (SPE)Water residue analysis procedures were modified from an

xisting protocol developed by the United States Geologicalurvey (USGS) for tetracyclines in groundwater (Lindsey et al.,001) and adapted for ivermectin (Loffler and Ternes, 2003).educed light conditions were maintained throughout the anal-sis due to the photosensitivity of ivermectin. SPE cartridgesere conditioned with successive treatments of methanol (3 mL;ptima grade; Fisher; CAS 67-56-1), 0.5 N HCl (3 mL), andltrafiltered (UF) deionized water (3 mL). Experiments varyingxtraction resin and eluant identity and volume were conductedo evaluate the optimal extraction protocol. Optimal efficiencyas attained using hydrophilic-lipophilic balance (HLB) 6 cc

artridges (Waters; product #WAT106202) and methylene chlo-ide (7 mL; optima grade; Fisher; CAS 75-09-2), polypropyleneottles and silanized glassware.

Each water sample was passed through a labelled HLBartridge via TFE tubing (Supelco) and a 12-port vacuum man-fold (Supelco Visiprep; CAT#5-7030) at 8 to 12 mL min−1, notxceeding 20 mmHg. Each cartridge was then rinsed by eluting–10 mL of UF water transferred with 9 in. silanized Pasteuripettes. After passing air through the cartridges for 10–12 min,mL of methylene chloride was transferred to each cartridge.he solvent and ivermectin residues were then eluted at the

reviously stated rate and pressure into 15 mL polypropyleneottles.

Methylene chloride was evaporated in a heat bath (40–60 ◦Cor 2–4 h). Following methylene chloride evaporation each sam-

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icology 85 (2007) 229–240

le was reconstituted in acetonitrile (1 mL; optima; Fisher; CAS5-05-8). The samples were then homogenized by sonicationFisher FS20H) for 5 min then vortexed for 2 min. A 250 �Lliquot of each sample was transferred into amber HPLC vialsontaining polypropylene inserts (Fisher CAT#03-922-3) withilanized 9 in. Pasteur pipettes; the remainder of the extract wasiscarded. The amber vials were immediately capped, crimped,nd placed in a −10 ◦C freezer in the dark. The recoveriesere >90% (Loffler and Ternes, 2003), hence concentrationsere not adjusted. The samples were subsequently shipped by

ourier within 48 h to the Central Science Laboratory (CSL) inhe United Kingdom and analysed by HPLC with fluorescenceetection. Whole frozen sediment samples were also sent to CSLor both extraction and analysis.

.3.2. Analysis of water samplesThe water extracts and calibration standards (100 �L) were

erivatised by adding 100 �L of N-methylimidazole solution1:1 N-methylimidazole/acetonitrile) followed by 150 �L ofrifluoroacetic anhydride solution (1:2 trifluoroacetic anhy-ride/acetonitrile) (De Montigny et al., 1990).

HPLC analysis was performed on an Agilent 1100 seriessing a Sigma Discovery C18 Column (15 cm × 4.6 nm, 5 �m)nd a Phenomenex C18 security guard (4 mm × 3 mm), at 30 ◦C.he mobile phase composition was 5% water and 95% ace-

onitrile at 5 min, with the proportion of acetonitrile increasingo 100% at 20 min. Flow rate was 1 mL min−1 and the injec-ion volume was 50 �L. The fluorescence detector was set to anxcitation wavelength of 365 nm with an emission wavelengthf 475 nm, and the retention time of the ivermectin deriva-ive was approximately 17 min. The sample and HPLC limitsf detection for the water analyses were 1 and 0.3 ng mL−1,espectively.

.3.3. Extraction of sediment samplesSediment samples were defrosted and excess water drained

nd discarded. Approximately 10 g (wet weight) of homoge-ized sediment was weighed into 50 mL Teflon centrifuge tubes.ifteen microlitres acetonitrile was added and the tubes brieflyortex mixed. The tubes were then placed on an end-over-endhaker for 1 h followed by centrifugation at 4000 rpm for 10 min.he supernatant was decanted and stored in amber glass vessels.he samples were re-extracted with a further 15 mL acetoni-

rile and the supernatants combined. Extracts were stored below0 ◦C prior to sample clean up and analysis.

.3.4. Sediment sample clean upSamples were cleaned up prior to HPLC analysis using

everse phase solid phase extraction (SPE) using Strata 100 mg18 SPE cartridges from Phenomenex. Ten millilitre portionsf the extracts were diluted with 35 mL water to which 50 �Lriethylamine was added (Kolar et al., 2004). The SPE car-ridges were activated with 2 mL acetonitrile and conditioned

ith 2 mL acetonitrile/water (30:70). The diluted samples were

oaded using polypropylene reservoirs above the cartridges. Theartridges were washed with 2 mL acetonitrile/water (50:50) andluted with 4 mL acetonitrile into glass centrifuge tubes. The

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ig. 2. Model considering degradation in the water and sediment and degrada-ion in both compartments.

luates were dried under nitrogen at approximately 30 ◦C andeconstituted into 200 �L acetonitrile. Samples and standards200 �L) were derivatized and analysed by HPLC as describedbove but using an injection volume of 75 �L. To determine theediment moisture content approximately 10 g of homogenizedediment sample was weighed, heated in an oven at 108 ◦C untilconstant weight was achieved and the loss in weight recorded.

The recovery of the method was tested by spiking controlediment at three fortification levels with three replicates at eachevel. Mean recoveries were: 93.1% (±1.3); 90.1% (±1.4); and9.8% (±1.1) for: 1; 10; and 100 �g kg−1, respectively. Theample and HPLC limits of detection for the sediment analysesere 0.4 �g kg−1 (wet weight) and 0.7 �g mL−1, respectively.

.3.5. Modeling the water-sediment systemA model that considers single first-order degradation in the

ater phase, sediment phase and exchange between the twohases was fitted to water and sediment data simultaneously.he kinetic model was fitted to the data using the ModelMakeroftware (version 4.0) with the least squares method used todentify the model parameters that fitted the data best (Fig. 2).

One mesocosm from the treatment level of 1000 ng L−1 wasodeled. Concentrations were converted to percentages of the

pplied dose (over 4 days), assuming a sediment depth of 5 cmnd a bulk density of 0.8 g cm3. However, in one case the sumf ivermectin in the water and sediment was over 100% of theheoretically applied dose (132%). This is probably due to theariability in concentrations across the depth of the sediment.

.3.6. Estimation of water dissipation ratesThe dissipation half-live (DT50aqueous) of ivermectin in

he water phase of the mesocosm was calculated assumingrst order kinetics using the software and guidelines recom-ended by the FOCUS work group on degradation kinetics

http://viso.jrc.it/focus/dk/). The concentrations determined inhe three mesocosms of each treatment level were used as thehree replicates in the data analysis.

.4. Biotic and abiotic sampling and measurementrotocols

Zooplankton was sampled using custom-built activity trapsSibley, 2000), where they were trapped during their diurnal

igration cycles. Two traps each with three funnels (each with

0 cm diameter), were placed in each mesocosm approximately–5 cm above the sediment for 20 h. Each funnel ended in a50 mL glass jar, where the organisms were captured. The jars

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(

icology 85 (2007) 229–240 233

ere pre-filled with filtered mesocosm water before being placedn the mesocosm. The samples were filtered through a 30 �mesh to concentrate the captured organisms and then preserved

sing a 37% formalin solution. This sampling yields a volume,urface area, and time integrated sample, with minimal sam-ling disturbance. The identification and grouping of speciesere according to Wilson et al. (2004). The total species rich-ess and abundance was determined for each trophospeciesroup (Yodzis, 1988), as well as ratio of juvenile to adult foropepods.

Chlorophyll a samples were taken with the 2.5 m long depthntegrated water sampler (Solomon et al., 1982) at 9 a.m.n every sampling day from various sites around the meso-osms. A total of 4 L were taken and combined. This wastirred and depending on the apparent density of algae in theonds, a 500 (high densities) to 1000 ml (low densities) aliquotas filtered. The water was pre-filtered through a nitex screen

50 �m mesh) on the top of the filter apparatus to remove thelamentous algae. The water was filtered using a vacuum suc-

ion pump (General Electric Duoseal vacuum pump, model400), set at a pressure of ca. 15 mmHg, though glass fiberlter (1.2 �m Whatman GF/C, 47 mm diameter). The filtersere stored frozen (−20 ◦C) until analysis (within 1 week).hlorophyll contained on the filters was extracted using a 90%cetone solution in which the filters were steeped at 4 ◦C in theark for 20 h, the sample was sonicated for 5–10 min (FisherS20H) then poured into a syringe packed with Glass woolver a 30 mL glass scintillation vial also wrapped with alu-inum foil. The centrifuge tube was rinsed with acetone, whichas also poured into the syringe. The plunger of the syringeas depressed and the filtrate was collected. The total volumef filtrate was measured and then 3–7 ml was poured into alass 13 mm test tube. The concentration was then measureduorometrically on a Turner Designs model TD 700 Fluorom-ter (Sunnyvale CA, USA) to determine chlorophyll a contentccording to USEPA Standard Test Guideline 445.0 (USEPA,997).

Dissolved oxygen and temperature were measured with YSIodel 55 (YSI, Yellow Springs, Ohio), and for pH a Fisherccumet Research AR 20 (Fisher Scientific, USA) was used.ll three assessments were conducted on- site.

.5. Statistical analysis

All abundance data were Log transformed (+1 to eliminateny zero counts) to secure normality of data in Excel 2000TM.pecies richness, abiotic parameters and chlorophyll a data wereot transformed. Analysis of variance (ANOVA) was performedn SASTM, version 8.2, using the general linear model procedureGLM Proc) analysis of least squares means. Adjustment forultiple comparisons was conducted via Dunnett’s test.

.6. Predicted environmental concentration/predicted no

ffect concentration calculation

Calculation of the predicted environmental concentrationPEC) is complex and dependent upon model assumptions

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Table 1Water-sediment model parameter

Sediment, k (day−1) 0.0001a

34 H. Sanderson et al. / Aquati

Montforts et al., 1999), which reflect inherent policies relativeo the regulatory jurisdiction. These assumptions are not harmo-ized globally. The methods used here are based on the Europeangency for the Evaluation of Medicinal products (EMEA) mod-

ls under EU Directive 2004/28/EC (Montforts et al., 2004).Ivermectin is rather persistent in soil with degradation

alf-lives DT50sed (determined at 22 ◦C under laboratory con-itions) of 93–240 days (mean = 187 days, n = 4) (Halley etl., 1989), DT50 in manure of >45 days (Boxall et al., 2004),nd DT50aqueous in water of 0.5 days (Halley et al., 1989).ransport of ivermectin by drainage is considered negligibleince this route is not assessed in the EMEA model at Koc500 L kg−1 (Montforts et al., 2004). In a study on run-off fromattle feedlots in the United States, trace amounts of ivermectin3.2–4.4 ng L−1) were detected in surface water (Nessel et al.,989).

Manure from stabled animals (pigs, cattle) treated withvermectin that is spread on agricultural land will result inpproximately 35 �g kg−1 PECsoil (0.2 mg/kg bodyweight (bw);8% excreted) at Koc = 8500 based on data and scenario fromontforts et al. (1999, 2004). Based on the EMEA model for

orewater to surfacewater partitioning (Montforts et al., 2004)his would yield PECsurface water concentrations of approximately0 ng L−1. This results is only valid if one neglects the EMEA-ule that substances with Koc > 500 L kg−1 (Koschorreck et al.,002) should not be assessed. However, the use of this rules inappropriate for ivermectin (and other similar compoundss well) because the variation in partitioning for many veteri-ary pharmaceuticals in different soils can be up to a factor0, which cannot be explained by normalization to the organicarbon content of the soils. Thus, to arrive at a realistic assess-ent of the availability of these compounds for transport through

he soil, the Koc may not be the appropriate measure to useTolls, 2001; Boxall et al., 2004; Kay et al., 2004). Transport ofarticle-associated compounds from soil to surface waters haseen demonstrated (Kay et al., 2004). Moreover, most veterinaryharmaceuticals have Koc values of 8000–36,000, dependingn soil type (Koschorreck et al., 2002), rendering few analysesequired. Preferential flow exposure via sorption to suspendedolids and transport in, e.g., tile drains is likely for certain veteri-ary pharmaceuticals, such as oxytetracycline (Kay et al., 2004),hat have high Koc values (42,500) and a soil–water distribu-ion coefficient (Kd) (680) in sandy loam (Boxall et al., 2004;ones et al., 2005). Ivermectin has the potential for preferentialows and a PECsurface water of 60 ng ivermectin L−1 followinganure amendment on arable land is both a reasonable and con-

ervative estimate according to EU regulations in EU Directive004/28/EC (Koschorreck et al., 2002; Montforts et al., 2004)ollowing spreading of manure from treated animals on arableand.

For ivermectin administered to grazing animals the EMEAodel assumes that 1% of the administered dose per hectare

nds up in a 100 m long and 1 m wide ditch/waterbody, yielding

PECsurface water of 25 ng L−1 (Montforts et al., 2004). How-

ver, considering a different usage and smaller waterbody theECsurface water according to EU methods could range from 25

o 60 ng L−1 (M. Montforts, personal communication 2005). In

WSW

ig. 3. Measured and modeled ivermectin concentrations in the water (filled tri-ngles and black line) and sediment (open circles and dashed line) of mesocosmreated with 1000 ng L−1 ivermectin.

ontrast, USFDA (1996) calculated a “back-of-the-envelope”ECsurface water of 0.012–46 ng L−1, which was close to factorf 1000 less than based on the EMEA model. In this analysise use the PEC of 25 ng L−1 for the PEC/predicted no effect

oncentration (PNEC) comparison.The PNEC was derived by first determining the lowest

bserved effect concentration (LOEC) where there was atatistically significant difference between treated and con-rol mesocosms (OECD, 2006b). Since the effect sizes (Δ)ere consistently larger than 50%, and the highest treatedesocosms consistently were the most impacted in this

tudy, the NOEC = LOEC/10 (Sijm et al., 2002), yield-ng PNEC = NOEC/1 assuming an assessment factor of oneSolomon et al., 2008).

. Results

.1. Dissipation of ivermectin

Ivermectin rapidly dissipated from the water compartmentf the mesocosm and was sorbed to the sediment (Fig. 3). Thisas attributed to the high Koc reported for ivermectin in soil,2,600–15,700 (Halley et al., 1989). However, modeling of theater-sediment system demonstrated that the loss in water com-artment cannot be explained by partitioning to the sedimentlone (Table 1). There is likely to have been some degradationn the water phase, probably due to photolysis. Halley et al.

ater, k (day−1) 0.0620301ediment → water, r (day−1) 0.0001a

ater → sediment, r (day−1) 0.077583

a Fixed values.

H. Sanderson et al. / Aquatic Toxicology 85 (2007) 229–240 235

(sum

sa

Dmsl

maodwt(c

3

3

wctt

retvsd

3

vvttvtc

3

3

Fig. 4. (A–D) Dissipation rates calculated for ivermectin in the water phase

eems likely that ivermectin will accumulate in the sediment ofnatural system after repeated exposure.

Dissipation was not discernible in the sediment, hence aT50sed was not calculated. We observed a build-up of iver-ectin over the first 3–4 weeks following treatment, which then

tabilized between 20 and 30 �g kg−1 for the highest treatmentevel (Fig. 3).

Fig. 4A–D shows the dissipation rates calculated for iver-ectin in the water phase, the sum of partitioning to the sediment

nd degradation in the water. The mean DT50aqueous calculatedver all treatment levels was 4 days. Loffler et al. (2005) reportedissipation half-lives of 2.9 ± 0.4 days for ivermectin in theater phase in a laboratory water/sediment system. However,

he systems used to study dissipation are clearly very differenta controlled laboratory study and a semi-field study) as was theomposition of the sediments used in the two systems.

.2. Background variables

.2.1. Chlorophyll a and temperatureChlorophyll a was not directly affected by the ivermectin and

as not a limiting factor to the zooplankton population over theourse of the experiment. There was no dose–response, quali-ative trends, or statistically significant differences between thereatments. Random cell-counts and visual observations did not

t1d2

of partitioning to sediment and degradation) for the four treatment levels.

eveal any shift in the algae population dominance towards lessdible species, e.g., blue-green algae, during the study. Wateremperature in aquatic mesocosms, typically a well-bufferedariable, did not demonstrate any treatment related effects ofignificance. However, the highest treatment level consistentlyemonstrated the highest temperature.

.2.2. Dissolved oxygen and pHDissolved oxygen (DO) like temperature, is a well-buffered

ariable nonetheless statistically significant and consistent ele-ation of DO from 25 to 30% was observed at the highestreatment from day 10 to 68. Additionally, we observed sta-istically significant and consistent trends for pH with elevatedalues for the highest treatment level (10–12% increase), withhe highest treatment level having the highest pH levels and theontrols having the lowest from day 10 to 97 (Fig. 5A,B).

.3. Zooplankton

.3.1. CladoceransThe total abundance of cladocerans decreased two days post-

reatment with effective elimination of the organisms from day0 to 32 at all treatment levels. The cladoceran communityemonstrated full recovery in the following spring beyond day29. A similar but statistically stronger and more prolonged

236 H. Sanderson et al. / Aquatic Toxicology 85 (2007) 229–240

issolv

aFbcCwat6diovtt

3

1dcarf

cwr

3

ta

3

wea(eow

3

Fig. 5. (A and B) D

dverse response was observed for cladoceran species richness.ull recovery was observed after day 229. There appeared toe a concentration-dependent shift in the dominance of clado-erans species within the group immediately following dosing.ontrol ponds were dominated by a species of Chydorus genus,hich is closely associated with the sediment. Treated ponds

ll shifted to more littoral and pelagic species. These popula-ions showed signs of recovery in the treated mesocosms after8 days, however, full recovery was not observed in the Chy-orus sp. abundance even in the following spring. The decreasen cladoceran abundance within the first 40 days is a functionf onset of fall and consequently relatively high inter-replicateariability. Abundances in the spring (April) were generally low,here was no ivermectin water concentration dependence withhe spring abundances (Fig. 6A,B).

.3.2. CopepodsWe observed acute effects, most pronounced at the

000 ng L−1 treatment concentration on both copepod abun-ance (Fig. 7A) and species richness (Fig. 7B). However, the

opepods appear to recover more rapidly than the cladocer-ns with a treatment-related increase of up to 300% in theatio of juvenile to adult compared to the untreated mesocosmsrom day 39–54 (Fig. 7C). In contrast to Cladocera, no signifi-

lHn

Fig. 6. (A and B) Cladoceran abun

ed O2 L−1 and pH.

ant concentration-dependent copepod species dominance shiftas observed. Furthermore, the copepod community was fully

ecovered after day 229.

.3.3. OstracodaThe abundance of ostracoda was depressed at the highest

reatment level after day 10 and fully recovered in the springfter day 229 (Fig. 8).

.3.4. Total abundance and total species richnessSignificant reductions in total zooplankton species richness

ere observed ten days following ivermectin treatment. How-ver, there were no significant or clear responses in the totalbundance due to shifts in species dominance (from largercladocerans) to smaller (rotifers (primarily Keratella sp.)) graz-rs. The controls had the highest species richness from day fournward; however, the differences were not significant after theinter (day 97) (Fig. 9A,B).

.3.5. Macroinvertebrates

The macroinvertebrate Ephemeroptera sp. was the only

ager invertebrate species (e.g., Chironomidae, Coleoptera,eteroptera, Odonata, Nematoda) that exhibited clear and sig-ificant reductions. After 265 days the highest treatment level

dance and species richness.

H. Sanderson et al. / Aquatic Toxicology 85 (2007) 229–240 237

F

hHait

3

ta

s3atcp1sR

Eietmcaa

faAdcacaime

4

t

ig. 7. (A–C) Copepod abundance, species richness and juvenile percentage.

ad still not recovered fully compared to the controls (Fig. 10).owever, it is worth noting that Odonata sp. was occasion-

lly observed in untreated control mesocosms. In contrast, thismportant predatory macroinvertebrate was not observed in anyreated mesocosm throughout the duration of the study.

.3.6. Risk comparison (PEC/PNEC)We relied on the PEC = 25 ng L−1 (Montforts et al., 2004) for

his assessment. The study can be spilt into three time phases:cute, day 1–10; chronic, day 10–97; and long term, day 97–265.

b1tD

Fig. 8. Ostracoda abundance.

Acute: Cladoceran abundance and species richness wasignificantly impacted at the lowest treatment level (nom.0 ng L−1), which was measured at approximately 5 ng L−1

round day 10 in the water phase and 10 �g kg−1 inhe sediment, suggestive of an acute risk towards clado-erans (risk quotient (RQ) = 25/5 = 5). Moreover, total zoo-lankton species richness at a nominal concentration of00 ng L−1 would provide an acute risk with a mea-ured exposure concentration at 20 ng L−1, yielding aQ = 1.25.

Chronic: Cladoceran and Copepod species richness,phemeroptera abundance, DO and pH were significantly

mpacted during the chronic phase at nominal concentration lev-ls of 30 ng L−1, and where the measured ivermectin was belowhe detection limit (1 ng L−1) for the water phase and approxi-

ately 25 ng kg−1 in the sediment, suggesting potentially severehronic risks (aquatic RQ = 25) to the zooplankton communitiess well as more indirect ecosystem functional parameters suchs pH and DO.

Long term: The observed chronic effects were still evidentor Ephemeroptera abundance and the cladoceran Chydorous spfter the winter and into the spring on day 229 and day 265.t that time as evident from Fig. 3 ivermectin was no longeretectable in the water-phase but only in the sediment where theoncentration was between 20 and 30 ng kg−1. Ephemeropterand Chydorous sp are relatively more active in the sedimentompartment than most of the other monitored species (Dodsonnd Frey, 1991) and their relative increased long term sensitivitys thus assumed due to a relative elevated exposure via the sedi-

ent. Table 2 summarizes the statistical significance of observedffects relative to nominal PNEC concentrations.

. Discussion and conclusions

We determined the aquatic half-life (DT50) for ivermectino be 3–5 days, roughly twice as long as previously assumed

ased on laboratory tests (Halley et al., 1989; Nessel et al.,989; Loffler et al., 2005) for aquatic systems at our lati-ude (43.5◦ N). We were not able to reproduce the sedimentT50sed of roughly 187 days (Halley et al., 1989); instead, we

238 H. Sanderson et al. / Aquatic Toxicology 85 (2007) 229–240

dance

odeai

(

TP

DDDDDDDDDDDD

Fig. 9. (A and B) Total abun

bserved a stabilization of ivermectin in the sediment with no

iscernible dissipation. This potentially significant prolongedxposure, especially via the sediment, should be taken intoccount in evaluation of the environmental risk assessment ofvermectin. There are two core assumptions in the USFDA

Fig. 10. Ephemeroptera abundance.

lmtoHsbd

kb1Ea4aacWmn

able 2NEC (ng L−1) in nominal concentrations, for measured concentrations see Fig. 4A–

Cladoceran Copepod Ostr

Abundance Sp. richness Abundance Sp. richness Abu

ay 1 NSa NS NS 100 (0.03) NSay 2 1000 (0.04) 300 (0.04) NS NS NSay 4 100 (0.01) 100 (0.03) 1000 (0.002) 1000 (0.004) NSay 10 30 (0.02) 30 (0.009) 1000 (0.006) 1000 (0.002) 100ay 17 NS 30 (0.0001) NS 30 (0.02) NSay 32 1000 (0.0009) 30 (0.0003) 100 (0.02) 1000 (0.04) NSay 39 1000 (0.006) 30 (0.0007) 1000 (0.003) 1000 (0.009) 100ay 54 NS 30 (0.01) NS 30 (0.01) 100ay 68 NS 100 (0.01) NS NS 100ay 97 30 (0.02) 30 (0.01) 1000 (0.02) NS 100ay 229 NS NS NS NS NSay 265 NS NS NS NS NS

a NS: not significant (P > 0.1).b NA: not applicable.

and total species richness.

1996) environmental risk assessment of ivermectin based onaboratory tests; (1) ivermectin dissipates rapidly (12–39 h) pri-

arily via photodegradation, and (2) ivermectin binds stronglyo organic matter (Koc > 12.600), hence a low PECsurface waterf 0.0012–46 ng L−1, and thus low aquatic environmental risk.owever, these findings indicate that ivermectin is persistent in

ediment and that predominately sediment-active organisms cane significantly impacted with no recovery for more than 265ays post treatment as a consequence thereof.

The toxicity of ivermectin towards crustaceans is wellnown with a D. magna LC50 = 25 ng L−1, and an inverte-rate species sensitivity distribution derived PNEC of roughly0 ng L−1 (Montforts et al., 2003). Additionally, Tisler andrzen (2006) reported a D. magna 21-day NOEC for abamectin,nother macrocyclic lactone veterinary anthelmintic drug, at7 ng L−1. A similar toxic mode of action of ivermectin islso found in neurotoxic pesticides which also specificallyffect GABA (�-aminobutyric acid) receptors, including the

yclodiene organochlorine insecticide endosulfan (BPCP, 2006;ardhaugh, 2005). Compared to ivermectin, endosulfan is onlyoderately toxic to adult zooplankton: free-swimming Daph-

ia pulex showed significant lethality at 300 �g L−1 (Barry,

D and P value (brackets)

acoda Total Ephermeoptera Dissolvedoxygen

pH

ndance Sp. richness

NS NS NS NSNS NAb NS NSNS NS NS NS

0 (0.0002) 100 (0.01) 1000 (0.01) 1000 (0.01) 1000 (0.02)30 (0.01) 100 (0.08) NS 1000 (0.01)NS NA 1000 (0.04) 1000 (0.005)

0 (0.0001) NS 30 (0.01) 1000 (0.03) 1000 (0.007)0 (0.0001) 30 (0.009) NA 30 (0.004) 30 (0.007)0 (0.0001) NS 30 (0.001) 30 (0.03) 30 (0.0008)0 (0.001) 300 (0.02) NA NS 30 (0.0002)

NS NA NS NSNS 1000 (0.09) NS NS

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H. Sanderson et al. / Aquati

000). Free swimming D. magna shows a similar sensitivity tondosulfan, as DeLorenzo et al. (2002) calculated a 24-h immo-ilization EC50 of 366 �g L−1. However, endosulfan inducesignificant mortality to D. pulex embryos at concentrations asow as 0.1 �g L−1 with reduced growth rate of the induced

orphs occurring at concentrations ≥0.1 �g L−1 (Barry, 2000).hese concentrations are similar to those calculated here for

vermectin, indicating that anthropogenic chemicals affectingABA receptors appear to pose a particular risk to zooplank-

on. Collier and Pinn (1998) further documented that especiallyhe benthic communities might be at especially elevated risk.herefore, the findings of this study support the conclusions ofollier and Pinn (1998).

Environmental risk assessments at the highest tier involveome degree of interpretational elasticity. Objections could beaised against the PEC of 25 ng L−1 (Montforts et al., 1999),hich would alter the risk equation, e.g., one could claim that

he PEC should equal the highest measured environmental iver-ectin concentration of 4 ng L−1 (Boxall et al., 2004). However,

ven at this PEC there would be risks associated with chronicxposure to ivermectin in aquatic environments. Another objec-ion could be that the peak exposure concentration should besed as the PNEC and not the measured concentration on theiven day where significant effects were observed. Clearly, thebserved effect on any given day is a function of the initial impactn part caused by the peak exposure, the present water concen-ration, and the sediment concentration at the time the effectas observed. Additionally, geometric mean exposure concen-

rations across the entire study period could be used. There is nocientific and regulatory consensus on which method is univer-ally more appropriate, hence, to err on the side of caution weompared the PEC (derived in accordance to European agreedethods (Montforts et al., 2004)) to the measured exposure con-

entration on the sampling day. We did not apply any uncertaintyactor to the assessment, which, depending upon jurisdictionypically ranges from 1 to 5. Regardless of default assumptionsegarding ivermectin exposure and effect concentrations, it islear that one cannot dismiss concerns of both acute and longerm risks of ivermectin to the aquatic environment in Europender realistic conditions and with minimal extrapolation uncer-ainty based on this study.

In this study we chose ivermectin due to its documented highoxicity towards crustaceans and the relative high use comparedo other potentially ecotoxic pharmaceuticals. Additionally, weecided to test the compound with as small an extrapolationncertainty as possible over a long time period. Hence, thisombined high toxicity with relatively high exposure poten-ial thus representing a potential worst-case risk pharmaceuticalo aquatic zooplankton communities. If we were not able toemonstrate any potential risks under this scenario one couldrgue that it would be very difficult to demonstrate risks using atandardized risk assessment methodology (other than relativelyovel sub-lethal endpoints such as hormonal, behavioral, devel-

pmental, feeding, swimming, differential gene expression, etc.)or pharmaceuticals—however, as evident form the data thisull hypothesis could not be not supported. Development andocumentation of novel toxicity methods and consideration of

K

K

icology 85 (2007) 229–240 239

ixture effects (Olmstead and LeBlanc, 2005) are however, verymportant, especially for pharmacodynamic compounds presentt low concentrations in the environment. Additionally, the find-ngs of this study demonstrate the benefits of long term semi-field

esocosm studies, in terms of discerning water-sediment parti-ioning and dissipation of ivermectin over time, multiple specieselative exposure (e.g., via sediment), sensitivity and recoveryime, and finally indirect functional changes in dissolved oxygennd pH following ivermectin exposure.

cknowledgements

Mark H.M.M. Montforts (RIVM) for help with theredicted Environmental Concentration calculation. Bentalling-Sørensen (Copenhagen University) and Maurice Zee-an (USEPA) for comments and suggestions, and two reviewers

or constructive suggestions. This work was funded by the Cana-ian Network of Toxicology Centres (CNTC). The chemicalsnalysis component of the project was funded by the Europeanommission under the EU 6th Framework Project ERAPharmSPI-2003-51135.

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