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Environ Chem Lett (2003) 1:131–134
DOI 10.1007/s10311-003-0032-9
ORIGINAL PAPER
Christian Mougin · Albert Kollmann ·
Jacqueline Dubroca · Paul Henri Ducrot ·
Michel Alvinerie · Pierre Galtier
Fate of the veterinary medicine ivermectin in soil
Accepted: 14 April 2003 / Published online: 12 July 2003
Springer-Verlag 2003
Abstract We investigated the fate of the drug ivermectin
in the soil. We found that ivermectin was transformed
solely by photos, leading to the formation of two
ivermectin isomers. We indeed failed to detect any
biotransformation reaction of the chemical either in the
soil or in fungal liquid cultures. According to its limited
water solubility, the bioavailability of ivermectin was
very low in the soil solution. Here, we show that
ivermectin, transferred to the soil from faeces of drug-
treated cattle, could be stored for long periods in the soil.
Keywords Avermectin · Ivermectin · Soil ·
Transformation · Bioavailability
Introduction
Avermectins are a class of macrocyclic lactone drugs with
insecticidal and anthelmintic properties that have been
developed for the protection of animals (Campbell 1985),
humans (Dadzie et al. 1987) and crops (Putter et al. 1981).
Examples of avermectins used for cattle protection
include ivermectin, doramectin, eprinomectin and mox-
idectin. A recent formulation, the intraruminal-sustained
release bolus, is considered as an efficient means to
control endoparasitic nematodes in ruminants because of
the long-term protection of animals. 80–90% of the drug
dose administered by the bolus is excreted without
transformation in faeces during a 4-month period
(Alvinerie et al. 1999). Faecal excretion of ivermectin
induces toxic effects on non-target organisms, including
key dung-colonising families of insects. The drug has also
been shown to negatively influence the decomposition of
dung organic matter in soil (Sommer et al. 2002), but data
are lacking concerning its impact on soil inhabiting
(micro)-organisms.
The ecotoxicological risk assessment of bolus on
pasture-land ecosystems has not been performed in detail
in the past. Our aim is to provide data concerning possible
side effects of high levels of residues deposited in the soil.
In this paper, we report the results concerning the fate of
ivermectin in soils, as a basis for exposure assessment.
These data will make it possible to design impact studies
of ivermectin on soil fungi. The impact studies will be
presented in a further paper.
Experimental
Chemicals
High-purity ivermectin (22,23-dihydroavermectin B
1
) and
chemical reagents were purchased from Sigma-Aldrich.
8,9- and 10,11-Z isomers of ivermectin were obtained by
photo-transformation of a drug solution layered on glass
plates under sunlight, then identified using mass spec-
trometry,
1
H and
13
C NMR, according to Mrozik et al.
(1988).
Soil characteristics
The soil used in this study was a silt loam collected in the
10–20 cm layer of a meadow in Versailles, France. It
contains 25.5% sand, 55.0% silt and 19.5% clay, and its
organic matter content was 1.65%. The soil pH was 8.1
and the cationic exchange capacity was 10.2 meq 100 g
1
.
The soil was 2 mm sieved then used immediately.
C. Mougin (
)
) · A. Kollmann · J. Dubroca · P. H. Ducrot
Unit de Phytopharmacie et Mdiateurs Chimiques,
INRA, Route de Saint-Cyr, 78026 Versailles, France
e-mail: mougin@versailles.inra.fr
Tel.: +33-130-833102
Fax: +33-130-833119
M. Alvinerie · P. Galtier
Unit de Toxicologie-Pharmacologie,
INRA, 180 Chemin de Tournefeuille, BP 3,
31931 Toulouse, France
Degradation experiments in soils
Incubations were performed on 25 g dry weight soil
samples spiked with 25 g ivermectin (1 mg kg
1
). The
soil was kept at 80% of its moisture-holding capacity by
adding water. Soil samples were incubated either in sterile
or in non-sterile conditions in the dark at 25 C, or under
sunlight at ambient temperature of June, with a mean
value of 20 C).
Bioavailability of ivermectin
The amount of ivermectin bioavailable for soil fungi was
determined using the protocol described by Gaillardon
and Dur (1995). Soil samples (10 g equivalent dry matter)
were placed in 5-cm diameter Petri dishes to give a 3–
4 mm thick layer. Acetonic solutions of ivermectin were
applied to the surface of the soil to obtain a final
concentration of 1 mg kg
1
. The solvent was allowed to
evaporate for 30 min and water was added to ensure that
80% of the moisture-holding capacity of the soil was
maintained. Concentrations of ivermectin in the soil
solution were determined 24 h after treatment. Two
superposed 42.5-mm-diameter glass micro-fibre filters
GF/A (Whatman) were laid on the soil surface and slight
pressure was applied for 10 s to favour wetting of the
filters. The upper filter was then recovered, weighed to
determine the volume of soil solution, and dried overnight
at 40 C.
Degradation experiments in fungal liquid cultures
The ability of two strains of filamentous fungi, Trametes
versicolor (ATCC 32745) and Fusarium solani (isolated
from the soil) was evaluated in liquid cultures. They were
grown on a culture medium (10 mL in 150-mL Erlen-
meyer flasks, Lesage-Meesen et al. 1996) with maltose
and ammonium tartrate as carbon and nitrogen sources,
inoculated with a mycelial mat on agar plug (10-mm
diameter). Cultivation was carried out statically in the
dark at 25 C for 2 days. Then, the medium was
supplemented with 1 mg L
1
ivermectin. Drug transfor-
mation was then checked after 8 days of growth.
Analytical methods
During the soil degradation experiments, residual iver-
mectin and possible transformation products were ex-
tracted every week by shaking the soil (25 g dry weight)
with 50 mL acetonitrile for 1 h. The organic extract was
then centrifuged (5 min, 2,000 g), and aliquots of 6 mL
were mixed with 9 mL distilled water; 10 mL of the
solution was concentrated on an MCH-10 C
18
guard
column (304.6 mm; Varian) with an isocratic high
performance liquid chromatography (HPLC) pump at a
flow rate of 1 mL min
1
. The guard column was fixed to a
ten-way valve. The extracted compounds were then eluted
on a C
18
Nova-Pak analytical column (1504.6 mm;
Waters) with an HPLC pump delivering a solvent system
composed of H
3
PO
4
(0.1% in water), acetonitrile and
methanol (20:57:23, v/v/v). The column eluate was
monitored at 245 nm using a variable wavelength UV-
Vis detector. The detection limit was 80 ng ivermectin.
To analyse bioavailability assays, dry filters were
extracted with 10 mL acetonitrile. The eluate was then
evaporated to dryness and the dry residue was subjected
to the derivation procedure already published (Alvinerie
et al. 1999). Briefly, the dry residue was dissolved in
100 L of an N-methylimidazole solution in acetonitrile.
To initiate the derivatisation, 150 L trifluoroacetic
anhydride solution in acetonitrile was added. After
derivatisation, 100 L of the solution was analysed hy
HPLC. A mobile phase of H
3
PO
4
(0.1% in water),
acetonitrile and methanol (4:66:30, v/v/v) was used.
Fluorescence was detected at an excitation wavelength
level of 377 nm and an emission wavelength of 427 nm.
The detection limit was 1 ng derivatised ivermectin.
Ivermectin in fungal cultures was analysed using the
following procedure: Fungal biomass (700–800 mg dry
weight) from an Erlenmeyer flask was separated from the
medium by filtration on a glass fiber filter after 8 days.
The biomass was then heated in the presence of MeOH
(20 mL per Erlenmeyer flask) at 70 C for 10 min. After
cooling, each extract was concentrated under vacuum and
dissolved in 20 mL diethyl ether. The organic extract was
then pooled with the corresponding culture medium and
shaken for 2 min in a separatory funnel. Finally, the
organic fraction was recovered and dried on MgSO
4
.
These steps were performed in triplicate. The resulting
organic extracts were pooled, evaporated to dryness and
dissolved in 1 mL MeOH for HPLC analysis without
derivatisation. Each experiment was done in triplicate.
Results are expressed as means. The standard deviation
was less than 10% of the mean.
Results and discussion
Transformation of ivermectin in the soil
The fate of ivermectin in soil was studied under various
incubation conditions (Fig. 1). The initial amount of the
ivermectin added (1 mg kg
1
) is commonly found in the
faeces of calves treated with the bolus (Alvinerie et al.
1999), and may represent the maximal amount of
ivermectin in the subsurface of the soil.
The results show that under sterile conditions in the
dark, the drug ivermectin was not degraded (Fig. 1).
Further, under non-sterile conditions, only a slight
decrease of ivermectin concentrations was observed, as
a possible biological effect of endogenous microflora. A
half-life of the chemical of around 240 days was
calculated under these conditions.
By sharp contrast, exposing the soil samples to
sunlight induced in a rapid decrease of ivermecin amounts
132
extracted from the soil, which was stimulated by the
mixing of the soil. The corresponding half-life of the
chemical was 21 days. These results are consistent with
the data previously published (Halley et al. 1990).
Two minor peaks from extracts obtained from soils
incubated under sunlight were detected on the HPLC
chromatograms. They co-eluted with standards of 8,9-
and 10,11-Z isomers of the drug formed through photo-
transformation (Fig. 2), but their low amounts prevented
any structural determination. Formation of these com-
pounds has also been reported previously (Mrozik et al.
1988). No other transformation products could be iden-
tified in organic extracts.
Bioavailability of ivermectin in soil
We measured the amounts of ivermectin in the soil
solution after a 24-h period of sorption of the chemical
onto soil samples. The ivermectin measured by HPLC
amounted to 23.8€13.8 ng (mean € SD of three replicates)
per filter, and corresponded to a concentration of 6 g L
1
soil solution. This value fits with the aqueous solubility of
the highly hydrophobic ivermectin, which could be
approximated to 10
8
M for the design of toxicity
experiments on soil micro-organisms.
The soil used in this study exhibited a moderate
organic carbon content, responsible for the adsorption of
ivermectin, a neutral compound. As a consequence, the
bioavailability of the drug appears essentially governed
by the organic carbon content of the soil rather than its
other properties.
Biotransformation of ivermectin in fungal liquid cultures
We studied the biotransformation of the drug in liquid
cultures of T. versicolor and F. solani (Table 1). The
ability of these strains to transform xenobiotics has been
extensively studied in the laboratory.
After 8 days, ivermectin concentration in the medium
decreased to 70.4% of the initial amount added in controls
flasks non-inoculated with either strain. This decrease
was a result of adsorption of the chemical on glass
surfaces. Ivermectin amounts found after the same period
in the Erlenmeyers flasks inoculated with of T. versicolor
or F. solani were slightly higher, evidencing the lack of
transformation of the drug by the fungal strains.
T. versicolor is a white-rot basidiomycete producing
exocellular oxidases, namely laccases, involved in the
transformation of natural or xenobiotic compounds.
Spiking the cultures with 20 mM xylidine, a well-known
inducer of laccase production (Mougin et al. 2002), did
not enhance ivermectin transformation.
In order to increase the uptake of ivermectin by F.
solani, a fungal strain transforming xenobiotics using
intracellular enzymes, several cultures were treated with
neutral surfactants, namely Triton X100, Tween 20 and
Brij 35, each at two-fold its critical micellar concentra-
tion. As the concentrations of ivermectin globally
increased in the treated cultures, we concluded that the
presence of the surfactants did not have an enhancing
effect on ivermectin transformation by the fungus.
Conclusion
Our data show that that ivermectin can be stored for long
periods in the soil in dark conditions. We were unable to
detect any biotransformation of ivermectin by filamentous
Fig. 2 Photo-isomerization of ivermectin
Table 1 Ivermectin extracted from 8-day liquid cultures submitted
to various incubation conditions
Incubation conditions Extracted ivermectin
(% of initial amount)
Non inoculated control 70.4
T. versicolor alone 81.0
T. versicolor + xylidine 79.7
F. solani alone 74.0
F. solani + Triton X100 71.5
F. solani + Tween 20 80.5
F. solani +Brij 35 84.5
Fig. 1 Transformation of ivermectin in soil under various incuba-
tion conditions
133
fungi, including a powerful white-rot strain. Bioavailabil-
ity of the drug for soil inhabiting organisms was
extremely low in the soil solution, but it may be increased
according to the exposure routes. For example, ivermectin
associated with the surface of particulate matter could
penetrate the gastrointestinal tract of animals. Ivermectin
could also represent a risk for organisms living in aquatic
ecosystems that can be contaminated by the drug strongly
adsorbed onto soil particles or suspended materials.
We know the concentration of the drug in the soil
solution and its main chemical form, the parent com-
pound. It is therefore now possible to design impact
studies of ivermectin on soil fungi.
Acknowledgements This work was supported by the French
program PNETOX. We thank Christine Young (INRA, Jouy en
Josas) for proofreading the manuscript.
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