4. Discussion
Plasma concentrations of OTC in the fish given the drug by i.v. injection decreased rapidly during the first day after administration. This was probably due to tissue
distribution since the further decline in the later two phases was much slower. The terminal half-life was in the range of 266–327 h, and the total clearance was relatively
Ž
y1 y1
. low 6.3–6.5 ml kg
h . The elimination half-life observed in this study was
considerably longer than those reported for other fish species living at higher water Ž
. temperatures. In carp, the elimination half-life was 169.0 h at 88C Nouws et al., 1992
Ž .
and 139.8 h at 208C Grondel et al., 1987 . In rainbow trout, half-lives of 81.5–130.0 h Ž
. Ž
were found at 108C Black et al., 1991; Nouws et al., 1992 , 94.2 h at 118C Abedini et .
Ž .
Ž al., 1998 , 89.5 h at 128C Grondel et al., 1989 , 60.3 h at 168C Bjorklund and Bylund,
¨
. Ž
. 1991 , and 76.0 h at 198C Nouws et al., 1992 . The elimination half-life was 88.3 h in
Ž .
Ž .
chinook salmon at 118C Abedini et al., 1998 , 52.1 h in ayu at 188C Uno, 1996 , and Ž
. 80.3 h in African catfish at 258C Grondel et al., 1989 . In Atlantic salmon in seawater,
Ž .
half-lives of 50.7 and 12.0 h were calculated at 7–88C Elema et al., 1996 and 158C Ž
. Pye-MacSwain et al., 1992 , respectively. Despite the differences in species and
experimental conditions, these results confirm that water temperature has a major effect on the elimination of OTC in fish. The slower elimination of drugs in fish at low
temperatures compared to high temperatures may partly be due to the lower production Ž
. Ž
. of bile Curtis et al., 1986 and urine Hunn, 1982 at low temperatures.
Comparing the pharmacokinetic parameters calculated at the two dose levels used in the present study, no dose-dependent pharmacokinetics of OTC in Arctic charr after i.v.
Ž .
administration was indicated. Neither the volume of distribution V and V
, the
dŽa rea. dŽss.
Ž .
total body clearance Cl , the MRT nor the distribution and elimination half-lives
T
Ž .
t differed significantly between the two dose groups.
1r2 a , b ,g
Ž .
The apparent volume of distribution V of 2.57–2.90 lrkg indicates that OTC is
dŽa rea.
well-distributed throughout the body, and that the major part of the drug in the body is extravascularly at distribution equilibrium. This is considered an advantage for OTC,
since many of the fish pathogenic bacteria cause abscesses and lesions in the skin and Ž
. muscles, which are poorly vascularized in fish Ferguson, 1989 . The V
is relatively
dŽa rea.
Ž high, considering the low octanolrwater partition coefficient of OTC Coliazzi and
. Ž
Klink, 1969 and the high plasmaprotein binding in salmonids Bjorklund and Bylund,
¨
. 1991 . However, the affinity of tetracyclines for bone tissue and skin may contribute to
Ž .
the high V . The volume of the central compartment V
was approximately three
dŽa rea. c
Ž times larger than the blood volume of salmonids at low temperatures 35 mlrkg;
. Nikinmaa et al., 1981 .
The absorption of OTC in Arctic charr was relatively rapid, but incomplete, for both formulations tested in this study. Although the number of fish in the liposomeralginate
Ž .
group was small n s 3 , it was considered sufficient for the assessment of whether this formulation increased the bioavailability of OTC as compared to the agar formulation.
The mean oral bioavailability of OTC varied between 3.2 and 7.3. Dose and formulation had no significant effect on the bioavailability. Our results are in agreement
with those previously reported. The bioavailability was found to be 1.9–6.9 in Atlantic
Ž .
Ž .
salmon Pye-MacSwain et al., 1992; Elema et al., 1996 , 9.3 in ayu Uno, 1996 ,
Ž .
0.4–0.6 in carp Grondel et al., 1987; Nouws et al., 1992 , and 1.2–5.6 in rainbow Ž
. Ž
. trout Bjorklund and Bylund, 1991; Nouws et al., 1992 . Cravedi et al. 1987 and
¨
Ž .
Rogstad et al. 1991 found that less than 10 of orally administered OTC was absorbed Ž
. in rainbow trout. In a recent publication Abedini et al., 1998 , the bioavailability of
OTC was 24.8 and 30.3 in chinook salmon and rainbow trout, respectively. In the latter study, an oral formulation consisting of gelatin capsules containing a methanolic
solution of OTC was used, and this may be the explanation for the increased bioavail- ability compared with the other studies.
Low oral bioavailability may be due to several factors. Tetracyclines are known to Ž
. form stabile complexes with di- and trivalent cations Ghandour et al., 1992 . These
complexes do not pass biological membranes easily and this may prevent absorption of Ž
. tetracyclines Cravedi et al., 1987; Grondel et al., 1987 . The absorption of OTC may
also be reduced because of unfavourable pH values in the intestine of fish. For instance, Ž
. the intestinal pH of rainbow trout was as high as 9.5 Dauble and Curtis, 1990 , and at
Ž .
this pH, only a small fraction of OTC is non-ionized Stephens et al., 1956 and thereby readily available for absorption. High levels of tetracyclines in liver and bile shortly
Ž after p.o. administration Ingebrigtsen et al., 1985; Plakas et al., 1988; Bjorklund and
¨
. Bylund, 1990 indicate that OTC undergoes significant first pass effect, i.e., extraction
in liver and further elimination into the bile. This process is not directly affected by drug Ž
formulation, and may be the explanation for the relatively low bioavailability 60 or .
Ž less even in warm-blooded animals Fabre et al., 1971; Schifferli et al., 1982; Mevius et
. al., 1986; Dyer, 1989 .
Ž .
At 100 mgrkg, the C was significantly higher p s 0.013 in the group receiving
max
Ž .
Ž OTC in agar 3.93 0.99 mgrml compared to the liposomeralginate group 0.97
. Ž
. 0.33 mgrml . T
also differed significantly p s 0.011 between the two formulations,
max
with mean values of 17.8 and 136.0 h for the agar and liposomeralginate formulation, respectively. These differences could be ascribed to a better disintegration and dissolu-
tion of the drug from the agar formulation compared to the liposomeralginate formula- tion. The absorption of OTC in the liposomeralginate formulation would thereby be
delayed. For comparison, the C
after administration of the same dose in medicated
max
Ž feed was 2.05 mgrml in amago salmon and 1.14 mgrml in rainbow trout Uno et al.,
. 1992 . In the present study, the C
of OTC was 1.51 0.86 mgrml after p.o.
max
administration of 50 mgrkg in agar, while the C was 0.42 mgrml in Atlantic salmon
ma x
Ž .
held in seawater, administered the same dose in medicated feed Elema et al., 1996 . The lower C
value obtained in fish held in seawater andror given medicated feed
max
may be due to complex binding of OTC to di- and trivalent cations, present both in feed and water. Fractional gastric emptying of the drug formulations could explain the large
individual differences observed in C and T
.
max max
Our data indicate that the elimination of OTC from Arctic charr was slower after p.o. administration than after i.v. administration. This may be caused by slow gastric
Ž .
evacuation due to the low temperatures Amundsen and Klemetsen, 1988 and entero- hepatic recycling of the drug, meaning that absorption is the rate-limiting factor for
elimination. Long elimination half-lives of OTC after p.o. administration have also been Ž
. observed in rainbow trout Bjorklund and Bylund, 1990; Rogstad et al., 1991 . Further-
¨
Ž .
more, in a preliminary experiment with Arctic charr n s 4 kept at 18C in freshwater,
the half-life was calculated to be as long as 713 h after p.o. administration of 10 mgrkg, Ž
. encapsulated in liposomeralginate particles unpublished results .
The dorsal aorta cannulation technique made it possible to take blood samples from individual fish over prolonged periods of time, thus allowing the advantage of establish-
ing individual pharmacokinetic profiles. However, several publications have reported that the pharmacokinetics of drugs is different in cannulated and non-cannulated fish
Ž .
Kleinow, 1991; Martinsen et al., 1993; Sohlberg et al., 1996 , and this could limit the value of using this technique. Reduced appetite and swimming activity, which were
Ž .
observed in this study, are indicators of stress. Soivio et al. 1975 and Mazik et al. Ž
. 1994 also reported increase in stress parameters in fish due to cannulation and repeated
Ž blood sampling. Stress, in itself, may increase the metabolic rate in fish Wendelaar
. Bonga, 1997 , and in addition, swimming activity has been shown to affect blood flow
Ž .
Ž .
to the intestines Stevens, 1968 as well as urine flow Hofmann and Butler, 1979 . These factors may have influenced the pharmacokinetics of OTC in this study, but the
significance for the calculated values is uncertain. In the treatment of bacterial fish diseases, antibacterial agents are commonly adminis-
tered in feed. Due to the low oral bioavailability of OTC, a major part of the administered drug would thus enter the environment in an unchanged, active form via
faeces, without having had a therapeutic effect. The fact, that the bioavailability of OTC Ž
. is lower in diseased fish compared to healthy fish Uno, 1996 and that addition of OTC
Ž .
to feed reduces the feed intake in fish Hustvedt et al., 1991 , reduces the efficacy of this Ž
drug even more. Based on its pharmacokinetics and its toxicological Toften and .
Ž .
Ž Jobling, 1996 , environmental Smith et al., 1994 and immunological effects Rijkers et
. al., 1980; Wishkovsky et al., 1987; Siwicki et al., 1989 , the future use of OTC in the
treatment of diseases in farmed fish may be questionable. The pharmacological research in fish should be directed towards alternative antibacterial agents with higher bioavail-
ability, satisfactory distribution and short half-lives, without interfering with normal physiology and immunological defense mechanisms.
5. Conclusion
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