Fish Physiology and Biochemistry 26: 189–195, 2002.

© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

189

Pharmacokinetics of ivermectin in sea bream, Sparus aurata using a direct

competitive ELISA

P. Katharios1, J. Iliopoulou-Georgudaki1,∗, S. Antimisiaris2, V. Kantzaris1 and M. Pavlidis3

1 University of Patras, Department of Biology, Unit of Pollution and Ecotoxicology, Rio 26500, Greece; 2 University

of Patras, Department of Pharmacy, Rio 26500, Greece; 3 University of Crete, Department of Biology, 71409
Heraklion, Greece; ∗ Author for correspondence (E-mail: j.iliopoulou@upatras.gr)

Accepted: June 6, 2003

Key words: ELISA, ivermectin, Pharmacokinetics, Sparus aurata

Abstract
The pharmacokinetics of ivermectin in the serum of cultured sea bream, Sparus aurata, after a single intraperitoneal
injection of 100 µg kg−1 body weight was studied by the use of direct competitive ELISA. Pharmacokinetic
analysis of the serum concentrations versus time points obtained was performed using non-comparmental analysis
and a compartmental pharmacokinetic model approach. In the latter case a two-compartment open model with a
lag time gave the best fitting. The maximum peak serum concentration was 308.4 ng ml−1 at 2 h post treatment.
The AUC of ivermectin was 10700 ng h ml−1 and the elimination half-life 15.37 h, indicating a rapid uptake, high
bioavailability and fast elimination of the drug by sea bream.

Introduction being used as treatment of these parasitic diseases, its

Ivermectin is a wide spectrum antiparasitic drug,
which belongs to the family of avermectins, naturally
occurring macrocyclic lactones. Its use in aquaculture
has been introduced the past years for the control of
sea lice the most significant disease in salmon farming
(Roth et al. 1993b; Davies and Rodger 2000). It has
been reported to be effective against copepod ectopa-
rasites such as Lepeoptheirus salmonis, Caligus elong-
atus, Ergasilus labracis and Lernaea sp., (Palmer
et al. 1987; Lorio 1989). Although the compound
was initially confined in salmonid culture, today is
extensively used in non-salmonid species worldwide.
Sea bream, Sparus aurata, is the most important
cultured species in the Mediterranean region. During
the past few years the interest of the industry for the
parasitic diseases of sea bream and their treatments has
significantly grown. Parasites identified in sea bream
are the copepod genus Clavela and Hatschekia, the iso-
podes Ceratothoa sargorum, Gnathia maxillaris and
Meinertia italica and the nematodes Contracaecum
fabri, Ascarophis sp., and Raphidascaris sp. (Pa-
poutsoglou 1975). Although ivermectin is currently
pharmacokinetic profile has not been studied in this
species.
For the analysis of ivermectin in the serum of an-
imals, different methods, capable of detecting residues
of the drug in the range of parts per billion, have been
applied. These include HPLC techniques with UV
(Schnitzerling and Nolan, 1985) or fluorescent detec-
tion (Kennedy et al. 1993; Degroodt et al. 1994; Iosi-
fidou et al. 1994), liquid chromatography-electrospray
ionization mass spectrometry (Valenzuela et al. 2000),
radioimmunoassay (Baraka et al. 1996) and ELISA
techniques (Crooks et al. 1998). It is generally ac-
cepted that less expensive and robust immunoassay
procedures are preferred.
The aim of this study was to evaluate the serum
disposition kinetics of ivermectin, after intraperitoneal
administration in sea bream, Sparus aurata.
190
Figure 1. Calibration curve of ivermectin in standard solution and in serum spiked with known concentration.
Materials and methods
Organisms
Sea bream (Sparus aurata; average 100 g B.W.) were
obtained from EKAL S.A. (Fokida, Greece). 30 fish
were placed in a closed re-circulating system con-
taining artificial seawater (S
35±1) and kept for a
conditioning period of 30 days at 22±1 ◦ C prior to the
start of the experiment. During this period, fish were
fed daily with commercial sea bream pellets (LAKY
S.A.), at a ratio of 3% of their B.W. Access to food
was blocked 10 days before ivermectin treatment.
Chemicals
The ivermectin stock solution (1 mg ml−1 Valaneq)
was manufactured by MSD AGVET. The anaesthetic
used was iso-amyl alcohol (Carlos Erba).
Experimental protocol
Fish were randomly selected, weighed, anesthetized
and injected intraperitoneally with 0.5 ml of iver-
mectin solution adjusted at a dose of 100 µg kg−1
fish BW. Fish were allowed to recover from anesthesia
in a well-aerated tank and then placed back in the
experimental tank.
Post injection, nine samples were drawn at 0.5, 1,
2, 4, 8, 24, 96, 144 and 192 h. 3 fish were used for the
first 7 samplings and 4 for the last two. The fish were
anaesthetized and approximately 1 ml of blood was
collected from the caudal vein using syringes and were
sacrificed after sampling. The blood was immediately
centrifuged at 3000 rpm for 15 min and serum was
separated and stored at −20 ◦ C until assayed.
Analytical procedure
A commercially available immunoassay kit was
used for determination of the ivermectin concentra-
tion in fish serum samples. The microtiter based
Enzyme Immuno Assay kit, was purchased from
Euro-Diagnostica, and consists of one 96-well plate
precoated with sheep antibodies to rabbit IgG. In
one incubation step, specific antibody (rabbit anti-
ivermectin), enzyme cojugate (HRP labeled iver-
mectin) and ivermectin standards and samples were
added to the precoated wells. The specific antibodies
bound to the immobilized anti-rabbit antibodies and at
the same time ivermectin (present in the sample and
standards) and enzyme labeled ivermectin competed
for the specific antibody binding sites (competitive
enzyme immunoassay). After an incubation time of
one hour, the non-bound (enzyme labeled) reagents
were removed in a washing step. The amount of
bound enzyme conjugate was visualized by the ad-
 191

dition of substrate chromogen (tetramethylbenzidine,

TMB). Bound enzyme transformed the chromogen
into a colored product. The substrate reaction was
stopped by the addition of sulphuric acid. The color
intensity was measured at 450 nm and was inversely
proportional to the ivermectin concentration in the
standard solution or the sample.
For the calibration curve 6 standard solutions
of ivermectin were used (10, 25, 50, 100, 250,
500 ng ml−1 ). In addition, 4 spiked samples with
ivermectin made in non- ivermectin treated sea
bream serum at concentrations of 12.5, 50, 125 and
250 ng ml−1 were used for a recovery study and 3
serial dilutions of the 250 ng ml−1 sample (40, 20 and
Figure 2. Serum concentration of ivermectin (ng ml−1 ) over time.
10%) for the linearity. The limit of detection of the as-
say was estimated using non treated sea bream serum. Table 1. Recovery of ivermectin from sea bream serum.
All samples and standards were assayed in duplicate. Numbers in parentheses are the standard deviation of the
mean values

Pharmacokinetic analysis Ivermectin added Ivermectin found % recovery

(ng ml−1 ) (ng ml−1 )
The serum concentrations vs. time data were ana-
lyzed using non-linear least squares regression, as 12.5 10.04 (0.17) 80.36
well as the non-compartmental approach. The pharma- 50 59.05 (5.15) 118.11
cokinetic computer program MINSQ II (MicroMath 125 108.5 (5.63) 86.8
250 295.52 (13.95) 118.21
Scientific Software, Utah, USA), was used for the
calculations, in the first case. Initial estimates of
the macroscopic rate constants were obtained by the
method of residuals (Gibaldi and Perrier 1982). The
elimination rate constant K 10 was estimated by sub- of the data points and as the AIC will give the same
jecting the serum concentrations in the terminal phase rankings between models. According to this criterion,
to linear regression analysis. Elimination half-life was the most appropriate model will be that with the largest
calculated by dividing 0.693 by K 10 . The area under MSC.
the serum concentration-time curve from time zero to
the last sampling time as well as time infinity after dos-
ing (AUC 0 → t and 0 → ∝) was calculated using the
combined log-linear trapezoidal rule from time 0h post Results
dose to the time of the last measured concentration,
plus the quotient of the last measured concentration Method of drug determination
divided by K 10 . Cmax and tmax were determined by
visual inspection of the serum concentration vs. time Calibration curves for ivermectin were conducted in
data. buffer as well as in non- ivermectin treated sea bream
From the compartmental models applied (open serum (spiked with the appropriate concentration of
one-compartment and open two compartment mod- ivermectin in each case). As presented in Table 1 and
els, with first order absorption, with and without lag in Figure 1, the calibration curve calculated using
time), the two-compartment open model that allows a the spiked standard solutions was identical with that
lag time, described the observed pharmacokinetic data derived from the standard solutions in buffer, while
best. All diffusion processes were assumed to follow the recovery of ivermectin from serum samples was
first-order kinetics. The model was selected on the 100.87% ± 20.13%. The linearity was investigated in
basis of the residual sum of squares and a modified the range of 10–100 ng ml−1 and the correlation coef-
AIC (Akaike’s model criterion), the Model selection ficient was >0.99. The limit of detection of the assay
criterion (MSC), which is independent of the scaling was 1.6 ng ml−1 .
192
Table 2. Pharmacokinetic parameters derived from the observed pharmacokinetic data after
non-linear least square fitting and non-compartmental analysis. Numbers in parentheses are
the Standard Deviations of the calculated values

Dose: 100 g kg−1 of body weight

Mean Weight: 87 g
Administration: ip
N = 27

Parameter Non-compartmental analysis 2-compartment open model fitting

AUC∝ 8837 10700 (1598)

(ng h ml−1 ) 7795 (log-linear)
AUCt 8677 –
(ng h ml−1 ) 7634 (log linear)
t1/2 (h) 21.21 15.37 (2.30)
K10 (h−1 ) 0.03267 0.0464 (0.0121)
α – 0.3076 (0.0145)
β – 0.0129 (0.0027)
A – 542.6 (17.9)
B – 125.3 (11.6)
C – −667.9 (22.7)
K12 (h−1 ) – 0.1874 (0.0303)
K21 (h−1 ) – 0.0864 (0.0281)
Tlag (h) – 0.1401 (0.0443)
KAB (h−1 ) NC 0.9132 (0.1268)
Cmax (ng ml−1 ) 335.6 (6.738) 308.4 (6.056)
tmax (h) 2 2
Vc (ml) – 0.1814 (0.0094)

NC: Not Calculated.

(log linear) = Values calculated using the log-linear trapezoidal rule.

Pharmacokinetic parameters
The serum concentration-time curve of ivermectin
after ip administration is best described by the two
compartment open model with first-order input, first-
order output and a time lag (Figure 2). The equation
for this two-compartment model is as follows: Cp =
A · e−a·(t −tlag) + B · e−β·(t −tlag) + C · e−KAB ·(t −tlag ) ,
where Cp is the concentration of the drug in serum and
K AB is the first-order absorption rate constant. From
the hybrid constants A, B, C, α and β, the first order
elimination rate constant K 10 , as well as the volume
of the central compartment Vc may be calculated
as described elsewhere (Gibaldi and Perrier 1982).
All pharmacokinetic parameters estimated using the
compartmental and non-compartmental approach are
presented in Table 2.
Figure 2 depicts the time course of ivermectin con-
centration in the blood of sea bream after receiving
100 µg kg−1 of the drug, intraperitoneally. In the same
figure the values obtained after fitting to the above-
mentioned model are also shown (line). This fitting
was the best possible for the pharmacokinetic data
observed, giving an R squared value of 0.934, and a
model selection criteria equal to 1.95.
In addition to the MINSQ least-square fitting pro-
gram used, an additional fitting of the observed phar-
macokinetic data using an older exponential stripping
pharmacokinetic program named ESTRIP (Brown and
Manno 1978), was also performed. In this case the
best fitting (R squared = 0.9055) was achieved when
3 exponentials and a lag time of 0.3065 h were used.
The AUC calculated by the derived poly-exponential
equation was 7224.96 ng h ml−1 , thus a lot closer to
the value calculated using the log-linear transformed
trapezoidal rule.

Discussion
Calibration and recovery
The calibration curve of the standard solutions was
linear at the 10-500 ng ml−1 range giving an almost
identical curve with that obtained from the spiked
standards and the recovery of the substance when
serum samples were applied without any previous ex-
traction treatment was 100.87% ± 20.13% (Table 1,
Figure 1). The limit of detection of the assay was
1.6 ng ml−1 , which is close to the limits of the methods
which use HPLC with UV detection but higher com-
pared to the methods which use fluorescent detection
or mass spectrometry (Schnitzerling and Nolan 1985;
Kennedy et al. 1993; Iosifidou et al. 1994; Valen-
zuela et al. 2001). These facts demonstrate that the
Ivermectin Enzyme Immunoassay KIT is a simple,
sensitive and reliable analytical technique, which is
applicable in such studies. However, care should be
taken for the cross reactivity of the ivermectin anti-
bodies which, according to the manufacturer, is 13.5%
for doramectin, 16.8% for eprinomectin and 13.3%
for abamectin. In addition, there is the possibility of
the cross reactivity of the antibodies with the metabol-
ites of ivermectin (24-OH-H2B1a , 24-OH-H2B1b , 3-
0-Desmethyl-H2B1a and 3-0-Desmethyl-H2B1b ) pre-
viously reported in various animal species (Chiu et al.
1986; Lanusse et al. 1997), though the metabolic
products could not affect the kinetics of the drug in this
study because the peak of the concentration is formed
quite early and it has been shown that in cattle there
are no metabolites in the plasma for at least 14 h (Fink
and Porras 1989).
Pharmacokinetic analysis
Our results demonstrate a rapid absorption of iver-
mectin by sea bream, when the intraperitoneal route
of administration is chosen, giving the maximum
concentration of ivermectin in serum 2 h post treat-
ment. This is in contrast to the results of Hoy et al.
(1990) who found that orally administered ivermectin
is slowly absorbed from the intestine of salmons
reaching maximum concentrations in all organs 4 days
post administration. In Table 3 it is shown that the tmax
of ivermectin differs significantly in human, cattle,
camel and dog. These differences are probably due
to the route of administration, the physiology of each
organism and the diet of the subjects during experi-
mentation (experimental protocol). The bioavailability
of the substance in sea bream is higher compared to
193
the values observed in other animals and this is prob-
ably due to the route of administration, which allows
very rapid absorption (Table 3). It is known that iver-
mectin is a lipophilic substance, and thus, when it is
subcutaneously administered its disposition is highly
affected by the subcutaneous fat pool which acts as
an ivermectin reservoir resulting in a prolongation of
the drug residence time (Lo et al. 1985; Lanusse and
Prichards 1993; Craven et al. 2001). The fat reservoir
in fish is mainly located in the peritoneal cavity and it
is in excess in cultured species. In addition, ivermectin
presents a significant enterohepatic circulation in fish
(Hoy et al. 1990). Therefore, one can logically argue
that in the present situation a longer than normal res-
idence time and lower serum drug availability should
be observed. However, the fish used in this experi-
ment were fastened for 10 days before treatment and
they were not fed during the trial. This experimental
protocol probably resulted to a significant depletion
of the fish lipid content, which explains the fact that
even though the fish received the drug inside their
peritoneal cavity, the absorbance and clearance of the
drug was fast. In addition, Stoffregen et al. (1997)
have shown that the intraperitoneal route of admin-
istration of enrofloxacin in juvenile Atlantic salmon
results in a lower elimination half-life when compared
to the intraarterial, intramuscular and oral routes. Fur-
thermore, the water temperature in the present study
(22 ◦ C) is significantly higher than that used in Hoy
et al. (1990) (7.1 ◦ C) and this may have resulted to the
differences observed in the absorption and elimination
of ivermectin, as the kinetics of xenobiotics in poiki-
lotherm animals is temperature-dependent. Roth et al.
(1993a) investigated the depuration of ivermectin from
the muscle and skin of Atlantic salmon administered
medicated feed (0.05 mg kg−1 ) at weekly intervals
for 9 weeks. These authors showed that the depuration
followed first order kinetics and the elimination half-
life were 120.4 and 188.1◦D for the muscle and skin,
respectively. A similar compound, emamectin ben-
zoate has been studied by Chukwudebe et al. (1996)
in bluegill sunfish. These authors investigated the
bioaccumulation of the compound in whole fish, fillet
and viscera and they concluded that emamectin ben-
zoate neither biomagnifies in the aquatic food chain
nor bioaccumulates in whole fish. The depuration of
emamectin benzoate residues in this study was relative
rapid, with more than 50% clearance occurring after
about 4 days in untreated water. The elimination half
time of ivermectin in the blood of sea bream calculated
in this study is similar to those observed in humans
194
Table 3. Pharmacokinetic parameters of ivermectin following different routes of administration in various animals

Animal Dose Route AUC T1/2 Tmax Cmax Ref.

(µg kg−1 ) (ng h ml−1 ) (h) (h) (ng h ml−1 )

Human (healthy) 150 Oral 3180 36.6 4.9 54.4 Baraka et al. (1996)
Human (patient) 150 Oral 2850 35 5.2 52 Baraka et al. (1996)
Human (patient) 150 Oral 885 22 – – Edwards (1987)
Human (patient) 150 Oral 1543.3 28 – – Edwards et al. (1988)
Cattle 200 SC 4872 141 96 19.9 Lifscitz et al. (1999)
Cattle 200 IM 4512 124 54 22.6 Lifscitz et al. (1999)
Camel 200 SC 722.8 – 295 1.79 Oukessou et al. (1999)
Horses 200 Oral 3184 – – 44 Perez et al. (1999)
Goats 200 SC – 96.7 68.4 6.12 Alvinerie et al. (1993)
Dogs 100 Oral – 44.31 Daurio et al. (1992)
Pigs (Fat) 300 IV 1808 31.8 – – Craven et al. (2001)
Pigs (Thin) 300 IV 1926 28.3 – – Craven et al. (2001)

SC: Subcutaneous.
IM: Intramuscular.
IV: Intravenous.

(healthy/patients) and pigs while the peak concentra-
tion is higher (>300 ng ml−1 ) than those observed
in the experiments summarized in Table 3. The peak
concentration of ivermectin in the serum of sea bream
occurs at 2 h post treatment. In our previous study
(Katharios et al. 2001) it has been shown that the most
important side effects of the drug when using higher
concentrations than those recommended, are the re-
duction of the hematocrit value and the increase of the
hemoglobin content, the number of the white blood
cells, the lymphocytes and monocytes and plasma
glucose. These side effects were more severe at the
first couple of hours post injection, which is in agree-
ment with the results concerning the concentration of
the drug in blood observed in this study.
It can be concluded that based on its pharmacokin-
etic properties, the rapid uptake high biavailability
and fast elimination, ivermectin could be considered
a promising candidate for use in sea bream aquacul-
ture. Further studies should be conducted focused on
the residues of the drug in the flesh of the fish which
must be undetectable when the fish are made available
for human consumption.
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