INTRODUCTION
Halibut (Hippoglossus hippoglossus) and turbot (Scophthalmus
maximus) are the two most important nonsalmonid fish species
now emerging in the Norwegian aquaculture industry. The
production in 1997 was 60 tons/270 000 fry of halibut and 40
tons/300 000 fry of turbot (J. Stoss, personal communication).
The farming of these two species has already encountered
problems with bacterial infections, such as vibrio (Vibrio
anguillarum) and infections with atypical Aeromonas salmonicida.
To be able to apply the right drug and the best dosage regimen
for successful treatment and minimal environmental hazards,
knowledge about the pharmacokinetic and pharmacodynamic
properties of the drug(s) in the actual species are vital.
Treatment with antimicrobial agents in flatfish has mainly
been conducted extrapolating the pharmacokinetic and phar-
macodynamic data known from salmonids (Atlantic salmon,
Salmo salar and rainbow trout, Oncorhynchus mykiss).
Knowledge about the pharmacokinetic properties of anti-
bacterial agents in flatfish, following different routes of
administration, are scarce. One commonly used antimicrobial
agent in flatfish is flumequine. Flumequine is a broad spectrum
synthetic antimicrobial agent belonging to the 4-quinolones and
has the properties of a weak acid.
The aim of this study was to investigate the pharmacokinetic
properties of flumequine in halibut and turbot.
MATERIALS AND METHODS
Formulation of test substance
There are no commercial formulations of flumequine available in
Norway for intravenous (i.v.) or bath administration. Flume-
quine was obtained from Sigma Chemical Co., St. Louis, MO. The
solution for i.v. administration was prepared by dissolving
flumequine in 1.0 M NaOH, regulating the pH to 10.3 with 6 M
HCl. Further lowering of the pH resulted in precipitation of
flumequine. The final volume was adjusted with 0.9% saline to a
concentration of 10 g/L. Flumequine for oral administration was
#1999 Blackwell Science Ltd 122
J. vet. Pharmacol. Therap. 22, 122±126, 1999. PHARMACOKINETICS
Single-dose pharmacokinetics of flumequine in halibut (Hippoglossus
hippoglossus) and turbot (Scophthalmus maximus)
Hansen, M. K., Horsberg, T. E. Single-dose pharmacokinetics of flumequine in
halibut (Hippoglossus hippoglossus) and turbot (Scophthalmus maximus). J. vet.
Pharmacol. Therap. 22, 122±126.
Flumequine was administered to halibut (Hippoglossus hippoglossus) and turbot
(Scophthalmus maximus) intravenously (i.v.) and orally (p.o.) at a dose of 10 mg/
kg bodyweight, and as a bath-treatment at a dose of 10 mg/L water for 2 h,
using identical experimental designs. The study was performed in seawater with
a salinity of 3% and a temperature of 10.3+0.48C (halibut) and 18.0+0.3 8C(turbot). Pharmacokinetic modelling of the data showed that flumequine had
quite similar pharmacokinetic properties in halibut and turbot. Following
intravenous administration, the volumes of distribution at steady state (Vss)
were 2.99 L/kg (halibut) and 3.75 L/kg (turbot). Plasma clearances (Cl) were
0.12 L/kg (halibut) and 0.17 L/h.kg (turbot) and the elimination half-lives
(t�lz) were calculated to be 32 h (halibut) and 34 h (turbot). Mean residence
times (MRT) were 25.1 h (halibut) and 22.2 h (turbot). Following oral
administration, the t�lz were 43 h (halibut) and 42 h (turbot). Maximal plasma
concentrations (tmax) were 1.4 mg/L (halibut) and 1.9 mg/L (turbot), and were
observed 7 h post administration in both species. The oral bioavailabilities (F)
were calculated to 56% (halibut) and 59% (turbot). Following bath adminis-
tration maximal plasma concentrations were 0.08 mg/L (halibut) and 0.14 mg/
L (turbot), and were observed 0 h (halibut) and 3 h (turbot) after the end of the
bath. The bioavailability in halibut following a 2-h bath treatment was 5%.
(Paper received 29 May 1998; accepted for publication 8 December 1998)
M. K. Hansen, Department of Pharmacology, Microbiology and Food Hygiene, Nor-
wegian College of Veterinary Medicine, PO Box 8146 Dep. N-0033 Oslo, Norway.
M. K. HANSEN &
T. E. HORSBERG
Department of Pharmacology, Microbiology
and Food Hygiene, Norwegian College of
Veterinary Medicine, Oslo, Norway
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mixed into a 4 : 3 emulsion of ordinary fishfeed: cod liver oil at a
concentration of 10 g/L (1 mL=1.0076 g, SD=0.0132 g) after
first being dissolved in 1.0 M NaOH. Flumequine for bath
administration was prepared by dissolving flumequine in 1 M
NaOH. The concentrations in the fish tanks were confirmed with
an HPLC-assay.
Test facilities and test fish
The study was conducted at Stolt Seafarm, éye, Norway. The
fish had been raised at the farm, and were held in fibreglass
tanks of 1000 L supplied with running seawater with a salinity
of & 3%. The halibut (Hippoglossus hippoglossus) were & 1-year-
old and weighed 89+15 g (mean+ SD). They were held in
running seawater at the optimal growth temperature of
10.3+0.4 8C. The turbot (Scophthalmus maximus) were & 8-
month-old and weighed 210+55 g (mean+ SD). They were
held in running seawater at the optimal growth temperature of
18.0+0.3 8C.The experimental design was identical for each of the
two species: 90 fish of each species were randomly allocated
into three groups of 30 each, one group for each route
of administration.
Intravenous administration
The fish to be administered flumequine by the i.v. route were
allocated into groups of six, and were administered flumequine
i.v. individually at a dose of 10 mg/kg into the caudal vein. The
i.v. injection was performed with the fish positioned on a damp
cloth, with the bottomside down during injection, after
anaesthetizing with metomidate (10 mg/L water) (HypnodilTM,
Janssen Pharmaceutica, Beerse, Belgium) and weighing. The
flumequine solution was slowly injected using a 1-mL disposable
syringe and a 0.5 6 25 mm needle (Terumo, Leuven, Belgium).
The position of the needle was confirmed by aspiration of blood
before, during and after the injection. Fish in which the needle
dislocated during the injection were discarded and replaced.
Oral administration
The fish-groups to be administered orally (p.o.) were also
allocated into small groups of six, from which the fish
were administered flumequine orally individually at a dose of
10 mg/kg through a stomach tube (Martinsen et al., 1993),
after being lightly sedated with metomidate (10 mg/L water)
before administration.
Bath administrations
The bath administrations were carried out in 1000 L fibreglass
tanks with 100 L static aerated seawater. Flumequine from the
stock solution was diluted with 1 M NaOH and added to the
seawater to a final concentration of 10 mg/L. The fish were kept
in the flumequine bath solution for 2 h, and then transferred to
flow-through water tanks.
Sampling
Blood samples from six fish were collected at 1, 3, 7, 12, 18, 24,
48, 96, 168 and 288 h post administration. In the group given
bath-treatment, the first bloodsample was collected at 0 h post
treatment. The fish were anaesthetized with metomidate (10 mg/
L water) before blood sampling by caudal venipuncture using a
0.5 6 25 mm needle and 1 mL syringe. The blood was sampled
caudal to the injection site in the intravenously administered
group. Each sample consisted of 100 mL blood. The samples were
frozen at ±808C until analysed.
No mortalities were recorded in the experimental fish during
or 1 month after the study.
Analytical procedures
The plasma samples were cleaned by solid-phase extraction on a
column of the Bond Elute type, size 1 mL, with C2 sorbent
material, according to a previously published method (Rasmus-
sen et al., 1989). The concentrations of flumequine in plasma
were determined by means of high-performance liquid chroma-
tography using a fluorescence detector operated at an excitation
wavelength of 325 nm and emission wavelength of 360 nm.
Oxolinic acid was added before cleanup/extraction, and used as
internal standard.
The lower limits of quantitation of the method were 10 mg/L,and it was linear over a tested range of 100±4000 mg/L. Thelinear correlation coefficient was 0.9999. The linearity of the
calibration curve was also tested on a residual plot, revealing no
bias. The recovery of flumequine was from 99% (4000 mg/L) to105% (400 mg/L). Recovery of 105% is probably the result of
some evaporation of organic solvent during elution with vacuum.
Chromatography
The HPLC system used consisted of a Perkin Elmer (Norwalk, CT)
LC 250 pump connected to a Waters (Milford, MA) wisp 710 B
autoinjector and a Perkin Elmer LC 240 fluorescence detector. A
150 6 4.6 mm 5 mm PLRP-S analytic column with a 5.0 6 3.0
mm 36 mm PLRP-S precolumn was used. The integrator was the
Analytic workstation, Omega-2, V2.60, Perkin Elmer. The
system was operated at room temperature (228C) with mobile
phase containing 0.001 M H3PO4: tetrahydrofuran: acetonitrile
(13 : 3 : 4). The flow was 0.7 mL/min.
Pharmacokinetic analysis
Pharmacokinetic evaluation was performed using the computer
program WIN-NONLIN, version 1.1 (Statistical consultants Inc.,
Lexington, KY), in a least square nonlinear regression analysis.
Standard pharmacokinetic parameters were calculated accord-
ing to a noncompartment model. In the i.v. group, the intercept
with the y-axis was calculated by back-extrapolation of the
curve, using the first two data points. In all groups, the curve
was extrapolated to infinity using the lambda-z calculated from
the depletion data, using the algorithm of Dunne (1985).
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 122±126
Pharmacokinetics of flumequine in halibut and turbot 123
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The bioavailabilities of the oral preparations were calculated
comparing the area under the concentration time curve
(AUC)i.v.(0-?) and AUCp.o.(0-?) or AUCbath(0-?), extrapolated to
infinity using the algorithm of Dunne (1985).
RESULTS
The estimated elimination half-life (t�lz) was 34 h (halibut) and
32 h (turbot). The observed volume of distribution (Vss) was 2.99
L/kg (halibut) and 3.75 L/kg (turbot), and the total plasma
clearance (ClT) was, respectively, 0.12 L/h.kg and 0.17 L/h.kg
for halibut and turbot.
Oral administration gave maximal plasma concentrations
(Cmax) of 1.4 mg/L (halibut) and 1.9 mg/L (turbot). The time-
point when maximal plasma concentrations were observed
(tmax) was 7 h post administration. Elimination half-life after oral
administration was calculated to be 43 h (halibut) and 42 h
(turbot). Bioavailability (F) was 56% (halibut) and 59% (turbot).
After bath-administration, Cmax of 0.083 mg/L in halibut and
0.134 mg/L in turbot was detected at 0 h (halibut) and 3 h
(turbot) post treatment (= tmax). The pharmacokinetic para-
meters are listed in Table 1, and the plasma concentration vs.
time of flumequine for halibut is shown in Fig. 1 and for turbot in
Fig. 2.
DISCUSSION
In pharmacokinetic studies in humans and domestic animals,
each individual is normally blood sampled throughout the whole
study period. This experimental protocol is very difficult to use in
studies with fish. In the current study, only small experimental
fish were available (halibut 89 g, turbot 210 g). Frequent blood-
sampling of each fish was considered impossible. This is the
reason why we chose a design in which each fish was blood-
sampled only twice. The small size of the fish also made it difficult
and time consuming to perform intravenous administration.
Anaesthesia was necessary to obtain a good result, when
sampling blood and administering drug by the intravenous
route. The dose of 10 mg/kg for intravenous administration was
chosen by extrapolation of data from pharmacokinetic studies of
flumequine in Atlantic salmon (Rogstad et al., 1993; Elema et al.,
1995). The same dose (and concentration of formulation) was
chosen for the orally administered fish as for the i.v.
administered fish to ensure the comparison between the different
methods of administration was as accurate as possible, and also
for practical reasons regarding preparation and administration.
The amount of cod liver oil used was adjusted to make the
suspension suitable for administration through a stomach tube.
Fish administered flumequine orally were only lightly sedated to
reduce the risk of regurgitation.
The bath administrations were carried out with a dosage of 10
mg/L flumequine in the water. This is the same dosage as used in
the groups given i.v. and oral administration, but with the
amount of flumequine related to the amount of water instead of
bodyweight. The same dosage was chosen to ensure the
comparison between the different methods of administration
was as accurate as possible.
The short duration (2 h) of the bath administration was
chosen because bath administration of long duration, with static
aerated water, is difficult to carry out in a commercial
aquaculture setting.
A noncompartment model was used in the pharmacokinetic
calculations. Other ways of modelling the data were also tested.
The data from the i.v. administered group could be interpreted
using a 1, 2 or 3 compartmental model. Based on the minimal
Akaike's information criterion estimation (Yamaoka et al.,
1978), in which all data were weighted to produce the best
curve fit during the elimination phase, the model which
described the data best was a 3 compartment model. In the 3
compartment model the t�g was estimated to be 50 h in halibut
and 40 h in turbot. In turbot, the last data point (288 h) had to
be omitted for the dataset to be interpreted by the model. The
volume of distribution (Vss) was calculated to be 3.01 L/kg
(halibut) and 2.34 L/kg (turbot). Total body clearance was 0.11
L/h.kg (halibut) and 0.17 L/h.kg (turbot). As expected, t�g was
longer than t�lz. The distribution volume (Vss) and clearance
124 M. K. Hansen & T. E. Horsberg
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 122±126
Table 1. Pharmacokinetic parameters in
halibut and turbot held in running
seawater at 108C and 188C, respectively,following a single dose of 10 mg
flumequine/kg bodyweight intravenously
or orally, or 10 mg flumequine/L in a
seawater bath for 2 h. The parameters
were calculated by a noncompartmental
model
Pharmacokinetic i.v. administration p.o. administration bath administration
Parameter
Halibut Turbot Halibut Turbot Halibut Turbot
ÐÐÐÐÐÐÐÐÐ ÐÐÐÐÐÐÐÐÐ ÐÐÐÐÐÐÐÐÐ
AUC(0-?) (mg.h/L) 83.7 59.3 47.5 35.8 4.5 *
t�lz (h) 32 34 43 42
MRT (h) 25.1 22.2
Vss (L/kg) 2.99 3.75
Cl (L/h.kg) 0.12 0.17
Cmax (mg/L) 1.4 1.9 0.08 0.13
tmax (h) 7 7 0 3
F (%) 56 59 5 *
*(AUC) could not be extrapolated to infinity. AUC (0-?), area under plasma concentration time curve
extrapolated to infinity; t�lz, elimination half life during the elimination phase; MRT, mean residence
time; Vss, volume of distribution at steady state; Cl, plasma clearance; Cmax, maximum plasma
concentration; tmax, time of peak plasma concentration; F, bioavailability.
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are nearly the same in both the noncompartment and the 3
compartment model.
There are few pharmacokinetic studies on halibut and turbot.
Data comparing different ways of administration of flumequine
(or other quinolones) in the two species are not published.
Our results from the i.v. administered group showed that
flumequine had quite similar pharmacokinetic properties in
halibut and turbot.
The elimination half-lives after i.v. administration were
estimated to be 32 and 34 h for halibut and turbot, respectively.
Distribution volume (Vss) was estimated to be 2.99 L/kg (halibut)
and 3.75 L/kg (turbot). This is a large distribution volume,
indicating that flumequine was well distributed to the tissues in
both halibut and turbot. Total body clearance was 0.12 L/h.kg
(halibut) and 0.17 L/h.kg (turbot). Similar results have been
reported for Atlantic salmon (Salmo salar). The studies on
Atlantic salmon were performed at 5±10 8C (Rogstad et al.,
1993; Elema et al., 1995; Martinsen & Horsberg, 1995).
Pharmacokinetic data from oral administration of flumequine
in halibut or turbot are not available. Cmax after oral adminis-
tration in this study was achieved in 7 h at 1.4 mg/L (halibut)
and 1.9 mg/L (turbot). This is a little higher than what has been
found in Atlantic salmon, where Rogstad et al. (1993), Elema et
al. (1995) and Martinsen & Horsberg (1995) reported a Cmax
of 1.42±2.46 mg/L after an oral dose of 25 mg/kg in sea water
(5±10 8C).The bioavailability was calculated to be 56% (halibut) and 59%
(turbot). This is higher than reported from the above mentioned
studies in Atlantic salmon, in which the bioavailability was
calculated to 44.7±46%. The elimination half-life in the orally
administered group was 43 h (halibut) and 42 h (turbot). This is,
as expected, longer than t�lz calculated after i.v. administration
owing to the influence of the absorption process.
The results of the analysis after bath administration showed
that flumequine was poorly absorbed from seawater. This is in
accordance with results obtained earlier, showing decreasing
flumequine absorption in Atlantic salmon with increasing pH
and Ca2+ in the water (O'Grady et al., 1988). Cmax after 2 h bath
treatment with 10 mg/L in our study was only 0.083 mg/L
(halibut) and 0.14 mg/L (turbot). Because of the poor absorp-
tion, flumequine concentrations in plasma very soon reached
values close to the detection limit, reducing accuracy. This is
probably the reason for the apparent small rise in flumequine
concentration from 96 h onwards seen in turbot, which is most
likely an artefact. This made it difficult to interpret the turbot
bath data using the pharmacokinetic model.
Pharmacokinetics of flumequine in halibut and turbot 125
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 122±126
Fig. 1. Mean (+ SEM) plasma concentration
profiles of flumequine in halibut (n=6) after a
single 10 mg/kg dose administered by the
intravenous (i.v.) and oral routes, and a single
10 mg/L dose administered as a bath.
Fig. 2. Mean (+ SEM) plasma concentration
profiles of flumequine in turbot (n=6) after a
single 10 mg/kg dose administered by the
intravenous (i.v.) and oral routes, and a single
10 mg/L dose administered as a bath.
Paper 191 Disc
Samuelsen & Lunestad (1996) studied bath treatment of
halibut with flumequine and oxolinic acid in 3±5 g halibut. They
found a t�lz of 10 h for halibut in muscle tissue after bath
administration of flumequine. This is much shorter than the t�lz
of 34 h in our study. Samuelsen & Lunestad (1996) used fish of
3±5 g whereas our fish had an average weight of 89 g. This, and
the different tissues sampled, are probably the most important
reasons for the differing results. Although flumequine shows a
poor degree of absorption, bath treatment of halibut and turbot is
possible as shown by Samuelsen & Lunestad (1996) and
Samuelsen (1997). To obtain adequate levels of flumequine in
the fish, a high dose has to be administered over a long period of
time. Samuelsen & Lunestad (1996) found a Cmax of 14.2 mg/L
in muscle after a 72 h bath in 150 mg/L flumequine.
Few studies of minimum inhibitory concentration (MIC) of
flumequine for susceptible strains of pathogenic bacteria for
halibut and turbot have been conducted. Samuelsen & Lunestad
(1996) reported MICs for flumequine of two pathogenic vibrios
isolated from halibut to be 0.06 mg/L (Vibrio anguillarum O1 (HI
11341) and 0.015 mg/L (V. anguillarum O2 (HI 11347)). MICs
of susceptible strains of bacteria pathogenic to Atlantic salmon
(Aeromonas salmonicida, Vibrio salmonicida, Vibrio anguillarum,
Yersinia ruckeri) have been reported to range from 0.005 to 0.5
mg/L (for most strains 5 0.1 mg/L) (Barnes et al., 1990;
Martinsen et al., 1992).
V. anguillarum isolated from turbot and atypical A. salmonicida
isolated from halibut have shown large inhibition zones against
flumequine in plate-agar diffusion tests. It is therefore likely that
MICs for these bacteria are in the same range as the bacteria
pathogenic to Atlantic salmon.
Blaser et al. (1987) reported bacterial regrowth in vitro unless
the peak concentration/MIC ratio exceeded 8 : 1 for the
fluoroquinolone enoxacin. If using the same ratio evaluating
the plasma concentrations of flumequine in our study, after oral
administration of 10 mg/kg flumequine, both halibut and turbot
exceed this peak concentration/MIC ratio for most susceptible
strains. The peak concentration and favourable bioavailability
(56% halibut, 59% turbot) makes this flumequine suitable for
oral treatment against bacterial infections in halibut and turbot.
The plasma concentrations from bath administration in 10 mg/L
flumequine for 2 h did not exceed the MICs. Both the dose and
the length of time in the bath must be significantly increased to
achieve a successful bath treatment (peak concentration/MIC
ratio 4 8 for a sufficient time).
This study was performed as a single-dose pharmacokinetic
study, and the results show that flumequine is not suitable for
single-dose administration in the treatment of bacterial diseases.
Although plasma concentrations higher than 8 6 MIC may be
obtained, the flumequine level in plasma will fall below this
concentration too soon for adequate treatment. In order to obtain a
high bioavailability and to minimize the pollution of the environ-
ment with flumequine, the oral route of administration should be
chosen when treating halibut and turbot, whenever possible.
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