Annual Review of Fish Diseases, Vol. 4, pp. 345-358, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0959~8030194 $24.00 + .OO

0959-8030(94)EOOO2-H

EXPERIMENTAL

METHODS FOR PHARMACOKINETIC STUDIES IN SALMONIDSTor Einar Horsberg

Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep., N-0033 Oslo, Norway

Abstract.

Many aspects of the use of chemical agents to combat diseases in aquaculture should be based on a firm knowledge of their pharmacokinetic behaviour in fish. The environmental conditions (temperature, salinity, pH etc.) under which kinetic studies are conducted, may vary greatly. Pharmacokinetic experiments to determine the rate and magnitude of absorption from water or feed, distribution, qualitative and quantitative metabolism and excretion in fish under various environmental conditions, are important for the determination of correct dosage regimens and withdrawal periods. These studies are often technically very difficult to carry out. Several techniques and experimental designs for different kinetic experiments are described in this review. Techniques requiring considerable manipulation of the fish, such as anaesthesia, catheterisation, cannulation, and immobilisation in metabolism chambers, will subject the fish to significant stress, which in turn may influence the data generated. The parameters reported thus often show considerable divergence. The influence of the experimental design on the results obtained has rarely been studied or addressed in papers describing pharmacokinetic studies in fish. In future studies, more attention should be paid to validation of the experimental methods. Methods, Drugs, Pharmacokinetics, Absorption, Distribution, Elimination, Fish,

Keywords.

Salmonids

INTRODUCTION

Pharmacokinetic studies are essential to establish correct dosage regimens and thereby promote optimal use of drugs in both animals and humans. In food-producing animals, the establishment of correct withdrawal periods is based on firm knowledge of the pharmacokinetic properties of the drug in the species concerned. When reviewing literature on pharmacokinetic studies in fish, one is often struck by the very divergent results, even for the same substance in the same fish species. For example, Hdl, Hustvedt, Salte, and Vassvik (1) and Rogstad, Ellingsen, and Syvertsen (2) both reported several pharmacokinetic parameters for oxolinic acid after IV injection in Atlantic salmon held in sea water. While Rogstad et al. found an elimination half life of 10 hours and a total body clearance of 4.9 l/kg per 24 hours, the corresponding values found by Hustvedt et al. were 60 hours and 0.7 l/kg per 24 hours, respectively. Even when an identical study is done by the same investigator, it can be difficult to reproduce the results. Bjorklund and Bylund studied the pharmacokinetic properties of oxytetracycline given orally to rainbow trout held in fresh water at 16 C in two separate studies. In the first study, the T,,, was observed 1 hour after

administration, and the elimination half-life was calculated to be 115 hours (3). In the second study, the T,,,, was observed at 12 hours after administration, and the elimination half life was 75 hours (4). Much of the divergence between findings may be caused by substantial differences in the experimental designs used by different investigators. The experimental difficulties are much greater than for similar studies in terrestrial animals and humans, and the investigator is faced with methodological problems throughout the study. The first problem is to keep the environmental parameters under control. A number of environmental parameters may affect the data generated, and the pharmacokinetic parameters determined. These include water temperature (3, 5-9), salinity (lo-13), pH (6, 14, 15) hardness (6), oxygen level (16), stocking density, feeding, and management. In addition, endogenous factors, such as size and physiological status (smoltification, sex, sexual maturity, health status of the fish) may be of importance. When the test substance is to be administered, the fish will normally have to be taken out of its natural environment, the water, and anaesthetized, a procedure which may alter the physiological condition of the fish for a long time. The investigator also faces problems when the samples are to be taken. Stressors, which can345

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be difficult to eliminate, may be present during the entire study as a result of the experimental design (e.g. tags, cannulas). Repeated blood sampling from the same individual may be very difficult to carry out, often making it necessary to take samples from different individuals, thereby introducing individual variation as a source of error into the study. The different environmental conditions (salinity, temperature, etc.) under which the studies are conducted further contribute to divergent results. The kinetic parameters generated are, therefore, normally only valid for the specific conditions under which the study has been conducted. Extrapolation of the results (small fish vs. big fish, fresh water vs. sea water, low temperature vs. high temperature, interspecies extrapolation) is difficult, and may often be questionable. This review will attempt to summarize and discuss the experimental methods used by investigators to determine kinetic properties of xenobiotics in salmonid fish, without going into detail about the pharmacokinetic modeling of the results. ANAESTHESIA Most forms of weighing, gavaging, injection, tagging, blood or tissue sampling, and many other necessary procedures, require the fish to be taken out of the water. Suitable anaesthesia is very often necessary to accomplish the task. Some widely-used inhalation anaesthetics, as well as their commonly used dosages, are presented in Table 1. For most purposes, a short-acting anaesthesia, administered via the water, is sufficient. Agents with a poor water solubility are administered from a stock solution (10% benzocaine, or 30% chlorbutanol, in ethanol). Before performing anaesthesia, the fish should be fasted for at least 1 day. The technical procedure is very simple. The anaesthetic agent is added to a small tank of wellaerated water in the desired concentration. It is very important that the temperature and other water

Table

1. Some commonly used anaesthetic agents waterborne administration, and the dosages neccessary to achieve surgical anaesthesia (maintenance) in salmonid fish Dosage 50-100 mg/l(30 mg/l) 50-100 mg/l (30 mg/l) 0.5 ml/l (0.25 ml/I) S-10 mg/l (2.5 mg/l) 300-500 mg/l

for

Agent Tricaine (MS 222) Benzocaine 2-phenoxyethanol Metomidate Chlorbutanol

Ref. 17 18 17 19 20

quality parameters in the anaesthesia tank are the same as in the holding tank. The fish is then gently transferred to the tank. Usual concentrations of the agents will normally produce adequate anaesthesia after l-3 minutes. In homiothermic animals, it is normal to distinguish between four stages of anaesthesia: I analgesia, II delirium, III surgical anaesthesia (several planes), IV collapse. The stages and planes are defined by a subsequentional loss of consciousness, reflexes and life-supporting functions. This scheme has been transferred to the anaesthesia of fish, as described by Klonz and Smith (17). In stage I, a diminished tactile response is observed, as well as some loss of equilibrium. In stage II, exitation is rarely observed, and normally only increasing difficulty in maintaining an upright position is noticed. When CO1 is used for anaesthesia, a distinct exitation stage, lasting about 15 seconds, is often seen. It is unclear whether this exitation occurs before or after loss of consciousness (stage I or II). When stage III is reached, the fish has completely lost its power to maintain equilibrium. In plane 1, there is still some response to external stimuli, such as firm squeezing of the tail (the tail reflex). In plane 2, no such response is observed, and in plane 3, respiratory activity is almost absent. In stage IV, respiration ceases, and a spasmodic overdistension of the opercles (flaring) may be seen. If left in the anaesthetic solution, the fish will die after a few minutes. For procedures requiring deep anaesthesia (e.g. intravascular injections), it may be beneficial to wait for the first flaring. If the task is performed rapidly, the fish will normally recover without problems after being transferred to untreated water. A usual observation is that when the highest recommended concentrations of the anaesthetic agents are used, the fish will become anaesthetized rapidly (stage III within 1 minute) and also recover more rapidly afterwards, compared with anaesthesia at lower concentrations. As regards the following agents, recovery is normally fastest for tricaine and benzocaine, followed by chlorbutanol, 2-phenoxyethanol, and metomidate. Fish can normally not be held in the anaesthetic solution for more than approximately 5-10 minutes at the highest recommended concentrations without risking ventilatory arrest and mortalities. At the latest, it should be removed from the anaesthesia tank immediately once flaring is observed. Special precautions must be taken to keep the fish wet and to protect the mucous layer. The mucous layer is preserved best when the fish is handled with bare hands. Wet latex gloves may be used, but

Methods for kinetic studies in salmonids

347

rubber gloves should be avoided whenever possible. Thin, wet, cotton gloves may, however, be an alternative for bigger fish, as these enable a firm grip without causing much damage to the mucous layer. Another alternative is to wrap the fish in wet chamois leather when handling it. If the operation lasts for more than 1 minute, the anaesthesia should be maintained. The fish may be transferred to a tank containing a lower concentration of the anaesthetic agent (e.g. 30 mg triCaine/l), or the gills should be irrigated by aerated water containing this same concentration of the agent (21). An alternative is to use an injectable anaesthetic. Saffan (0.9% alphaxalone + 0.3% alphadolone acetate), at 1 ml/kg intraperitoneally (22), will produce surgical anaesthesia lasting for about 30 minutes. The gills must be irrigated with water. Mortalities may occur during anaesthesia. They are often the result of ventilatory arrest following an overdose of the anaesthetic agent, or of hypoxia due to insufficient aeration of the anaesthetic solution, or to a combination of these factors. If ventilatory arrest occurs, artificial ventilation may easily be established by irrigation of the buccal cavity with water, e.g. via a plastic tube inserted in the mouth and connected to the water supply. Few studies describing the influence of anaesthesia on the pharmacokinetic properties of other agents are available. The documentation of changes in blood parameters, and other physiological and biochemical functions is, however, extensive (19). The influence of tricaine anaesthesia on hepatic metabolism of xenobiotics has been studied by Farbather (23). It was demonstrated that the activity was impaired for up to 18 hours following anaesthesia. As anaesthesia is often used both when the drug being investigated is administered, and when samples are taken, it cannot be ruled out that the procedure may influence the pharmacokinetic properties exhibited by the drug.BASIC KINETIC STUDIES

Studies are frequently conducted to determine the basic pharmacokinetic properties of a chemical. Such studies are mostly carried out as single dose pharmacokinetic studies, where the drug is administered per OS(PO), intramuscularly (IM), subcutaneously (SC), intraperitoneally (IP), or intravascularly (IV). Alternatively the drug may be administered via the water. Blood or tissue samples are then taken at regular intervals to establish a concentration versus time curve. A number of pharmacokinetic con-

stants may be derived from these studies. These include the absorption rate constant (k,), maximum serum concentration (C,,,), time to maximum serum concentration (T,,,), the area under the curve (AUC), total bioavailability (F), the apparent volume of distribution (I$), the total body clearance (CZr), the elimination rate constant (&), half-lives (f& for absorption (extravascular administration), distribution and elimination, as well as the compartmental model best describing the data. One frequently employed technique is to administer the substance to a group of individuals, take samples from several individuals at each time point, and then use the mean values at the different time points for calculation of the pharmacokinetic parameters. This single individual-single sample approach requires a number of test animals, as the groups at each time point should consist of at least five individuals. An example is given in Figure 1, where the concentration versus time curves are shown for the antibacterial agent florfenicol in Atlantic salmon, after intravascular and oral administration of 10 mg/kg bodyweight in two groups of fish. Ten fish were sampled at each time point (24). Another frequently used technique for single dose studies is to withdraw repeated blood samples from the same individuals, via a permanent cannula in the dorsal aorta, for the determination of individual pharmacokinetic profiles of the drug. This single individual-multiple samples technique also allows cross-over studies to be performed in which, for example, the kinetics associated with both the oral and the intravascular route are studied. Often, the administration of an exact dose in terms of milligrams per kilogram bodyweight is required. The easiest way of determining exact bodyweight is simply to anaesthetize the fish, before placing it on the scales on a wet piece of damp paper or a chamois leather. In some cases, however, anaesthesia is undesirable. The task may then be accomplished by placing a small tank of water on the balance, into which the fish is transferred. Great care must be taken to avoid addition of extra water or spillage of water from the tank during the weighing procedure.Parenteral injections

The administration of drugs by injections is an uncommon practice in commercial fish farming, except for the intraperitoneal administration of vaccines. For experimental purposes, it is, however, of great value to be able to inject the test substance parenterally.

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20

30

40

50

60

70

hours.-

i.v.

p.0. .___Q___

Fig. 1. The concentration-versus-time curves for florfenicol in the plasma of Atlantic salmon held in seawater at 11 C after intravascular or oral administration of 10 mg/kg. The mean and standard deviation of ten fish at each time point are given. Modified after (24).

The administration of an accurate dose of a drug by intramuscular injection is difficult, as the skin will not retract over the injection site. The injected drug is therefore susceptible to reflux through the channel, triggered by swimming movements (19). Nor does the poor vascularization of the muscle tissue facilitate rapid absorption of the drug; instead it is retained and may cause irritation and necrosis at the injection site. In spite of these limitations, this route of administration is occasionally used for pharmacokinetic studies (25-27). A SC injection in the midline just anterior to the dorsal fin will deposit the administered dose between the epaxial muscle packets. The needle is inserted in a cranioventral direction. Reflux is rarely seen from this injection site when the dose is less than 1 ml/kg. As the area in which the dose is distributed is quite large, the possibility of harmful irritation and necrosis is minimized. This injection site is occasionally chosen by investigators to study the kinetics and effect of drugs (28,29). Intraperitoneal injections are more often utilized (25,30-33). The injection needle is normally inserted into the abdominal cavity in the ventral midline just interior to the pelvic fins in a craniodorsal direction. Before deposition of the dose, the needle is

withdrawn slightly, allowing the internal organs to slip away. Some investigators prefer to insert the needle posterior to the pelvic fins to make sure that damage to the spleen is avoided. However, the chance of the needle entering the posterior gut is then greater. Intravascular injections and blood sampling The easiest site for an intravascular injection is probably into the caudal vessels. Several investigators have used this route (4,24,34-36). The fish is deeply anaesthetized, and placed on its back in a v-formed tray. A helper immobilizes the tail. The needle is inserted exactly in the midline just posterior to the anal fin until it stops against the spine, and is kept in this position, or backed off a little in fish heavier than 200 g. The position in the vessel is checked by aspiration of blood into the syringe. This should be done both prior to, during, and after the injection. If the needle translocates during the injection, the fish should be rejected from the study. A success rate of up to 90% may be expected. Investigators using this route of administration should be aware of the fact that blood from the cauda1 vein is distributed back to the heart via the renal portal system. For drugs with a high renal extrac-

Methods for kinetic studies in salmonids

349

tion ratio, this may be of importance, as a certain first-pass effect may occur in the kidney. This route of administration may be looked upon as being analogous to administering drugs via the hepatic portal vein in homoiothermic animals. This fact is hardly ever discussed in the literature. The disadvantage of this method is that normally only mean plasma concentrations at each time point can be used for calculation of kinetic parameters, as blood is not sampled repeatedly from the same individual. On the other hand, repeated handling procedures, which may affect the distribution or clearance of the drug, are avoided (34). An alternative injection site for intravascular administration is the heart (37,38). This intracardiac route is occasionally used in catfish, but rarely in salmon or trout, as the wound in the ventricle may be critical for the fish. The pericardium is often filled with blood clots shortly after the injection. Another possibility is injection into the Ductus Cuvieri, the venous sinus posterior to the atrium of the heart. Lied, Gjerde, and Braekkan (39) described the technique for blood sampling from this site, but it is perfectly possible to use it for injections as well. A spatula is inserted between the fourth and fifth gill arch in anaesthetized fish, and the needle is inserted at the point where a horizontal line drawn from the ventral end of the supracleithrum bone would intercept with a vertical line drawn through the dorsal end of the fifth gill arch. The needle is inserted in a caudoventral direction, and the intravascular position of the needle is checked by aspiration prior to, during, and after injection. This technique is more difficult than admin-

istering the drug into the caudal vein, and the success rate is rarely greater than 50% for fish weighing about 200 g. One of the most popular routes for intravascular administration of drugs in fish is injection into the dorsal aorta via a permanent cannula in this vessel. The same cannula is later used for repeated blood sampling from the same individual. The technique for cannulation of the dorsal aorta was originally described by Schiffman (40), and has later been modified several times (41-44). A number of studies have been carried out utilizing the technique (45-50). The equipment and the cannulation technique described by Soivio, Westman, and Nyholm (42) and Soivio, Nyholm, and Westman (43) has been proven to be very suitable for salmonid fish, and has frequently been used (1,7,9,13,51). The modification described by Soivio et al. (42,43) allows the cannulation procedure to be performed in only 2-3 minutes by a skilled person. No sutures are necessary when a bubble is made with hot air 6-8 cm from the tip of the cannula. This bubble anchors the cannula to its support through the nose. The cannulas may remain functional for 6 months or more. However, a success rate exceeding 50% is rare, due to incorrect positioning of the cannula, clotting problems which may become evident after several weeks, and mortality from other causes. The principle of the method is illustrated in Figure 2. A certain concern about the validity of this method for pharmacokinetic studies has been expressed. Martinsen, Sohlberg, Horsberg, and Burke (51) describe a study in which a group of cannulated Atlantic salmon was given an IV or PO dose of

Cannula

support

Cannula bubble

Fig. 2. Cannulation

of salmon for intravascular

injection

and repeated

blood sampling

from the same individual.

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T.E. Horsberg

sarafloxacin hydrochloride, and a noncannulated group was given the same PO dose. Several deviations from normal physiological conditions were reported. The feed intake and the swimming activity were considerably lower in the cannulated group compared to the noncannulated. In addition, the haematocrit dropped by 40-45% during the 3-week sampling period, an observation also described by Soivio et al. (42). The area under the curve (AUC) and the maximum serum concentration (C,,,,,)for the noncannulated group were considerably lower than for the cannulated group given the drug orally. Similar results were reported by Sohlberg, Aulie, and Sali (9). Blood clots in and around the tip of the cannula, leading to malfunction, are frequently described in connection with the method. Hustvedt et al. (1) gave each fish 450 mg acetylsalicylic acid orally and 150 IU heparine intravascularly through the cannula to prevent these problems. No attempts were made to discuss the possible influence of these substances, or of the technique itself on the pharmacokinetic parameters generated. As stated earlier, these results were markedly different to the figures found for the same parameters by Rogstad et al. (2) in a similar study with uncannulated salmon. The question arises as to whether data generated from this type of study should be transferred to real life situations with uncannulated fish populations and serve as the basis for determination of dosage regimens and estimation of withdrawal times. An uncritical extrapolation of the data is questionable, and further validation of the method seems appropriate. Oral administration of single doses Single, oral doses with an exact amount of the drug according to the bodyweight of the fish, are normally administered by gavage. The actual procedure used differs markedly between investigators. Some prefer administration of the test substance via a stomach tube as an aqueous solution or a suspension in an edible oil (9,52,53), or alternatively as a slurry of medicated feed and water or oil (49,50, 54,55). Others prefer to load the substance into gelatine capsules (25,38,56-59), or to administer the test substance as medicated feed (2,24,27,60). The choice of vehicle for the test substance may greatly affect its absorption from the intestine, and thereby the pharmacokinetic parameters determined. In a study by Martinsen, Horsberg, Sohlberg, and Burke (36), the pharmacokinetic parameters of the fluoroquinolone sarafloxacin in Atlantic salmon was studied. The test substance was administered to the fish in one of four formulations: two differ-

ent types of medicated feed, and two different types of edible oils. The highest bioavailability was found with a suspension of the drug in corn oil (AUC: 12.9 pg*h/ml), while the lowest bioavailability was found with a medicated feed in which the drug was homogenously mixed with the feed components (AUC: 1.9 pg*h/ml). A formulation in capelin oil showed no significant difference in bioavailability compared with feed coated with a capelin oil suspension of the drug (AUC: 3.6 and 4.0 pg*h/ml, respectively). Similar differences in bioavailability between formulations were found by Endo, Onozawa, Hamaguchi, and Kusuda (54) for ultrafine crystals of oxolinic acid (1 pm) versus normal crystals (6.4 pm). The micronized crystals showed a markedly higher bioavailability. These two examples clearly indicate the importance of the drug formulation. Pharmacokinetic parameters, especially those describing the absorption process, cannot be extrapolated uncritically from one formulation to another. When individual doses are administered by gavage, it is very important to check for possible regurgitation of the test substance. This is best accomplished by placing the fish in a small test tank shortly after administration, and following it closely for some minutes. If the test substance-or a part of it -is regurgitated, this usually happens within the first 10 minutes after administration. Regurgitation has, however, been observed as late as two hours after administration of gelatine capsules containing a drug, most probably the result of emetic action of the test substance which became evident when the capsules dissipated in the stomach of the fish. When medicated feed, or a slurry containing feed, is used as the vehicle, regurgitated feed particles are easily seen in the observation tanks. If an edible oil is used, droplets of regurgitated oil may be observed on the water surface. If, however, an aqueous solution is administered, it is not possible to observe regurgitated material, unless some indicator, e.g. red food stain (3), is added. It seems that regurgitation is more likely to occur when the fish is sedated, possibly because the oesophageal sphincter is relaxed during anaesthesia. It is our practice to perform oral gavaging in unanaesthetized fish whenever possible. For fish up to 150 g, a 1 ml tuberculin syringe, from which the anterior delivery part has been cut off and the edge smoothened, is very suitable for oral gavage (Fig. 3). The syringe is loaded with the correct amount of medicated feed just prior to administration. It is also useful for administration of

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351

Fig. 3. Equipment for force-feeding of small fish. The delivery part of a 1 ml tuberculin syringe has been cut off and smoothened to ease insertion into the oesophagus.

substances suspended in other vehicles, e.g. corn oil. The correct dose is then added into a syringe half loaded with unmedicated feed, into which it is allowed to soak for some minutes, before administration. It has been shown that less than 1% of the dose is retained in the syringe (61), and detection of any regurgitation of food particles into which the suspension has soaked, is easy. Oral administration of multiple doses

ble to control the uptake of the medicated diet by individuals. It does, however, simulate commercial use of the substance, and may give valuable information concerning the individual differences in uptake and serum concentration of the drug, as well as the depletion in various tissues. Great care must be taken to avoid unnecessary disturbance of the fish during the administration period, as this may adversely affect the appetite and thereby give misleading results. It is commonly experienced that the best results are achieved in relatively big populations, 500 individuals or more. Feeding regimens should not be changed in connection with the medication period, e.g. from automated feeding to hand feeding. Such changes should, if necessary, be made prior to the administration period. When fish are to be sampled from the tank during the administration period, it is best to do so immediately after the daily administration. If the protocol states that samples are to be taken immediately before start of administration the next day, the fish are merely transferred to a separate tank overnight. Occasionally, this type of protocol is used for more advanced pharmacokinetic and metabolism studies, such as the studies of Cravedi, Choubert, and Delous (66) and Cravedi, Baradat, and Choubert (67). They added an inert tracer, CrzOs to the feed to determine the apparent digestibility. A sophisticated experimental set-up for multiple dose kinetic studies in the same individual after oral administration, was described by Kleinow, Beilfuss, Jarboe, Droy, and Lech (49). The introduction of an indwelling stomach tube and a permanent cannula in the dorsal aorta, permitted the administration of multiple doses. Moreover, repeated blood samples could be drawn from the same individual. Whether or not this experimental set-up influences the parameters determined remains to be clarified. Topical administration Experiments designed to determine the uptake of a drug or another xenobiotic from the water, or to test their pharmacological or toxicological effect on the fish after waterborne administration, are frequently conducted. The major route of absorption will be across the gills, the rate at which this occurs being influenced by the physicochemical properties of the drug (lipid solubility, water solubility, pKavalue), as well as environmental parameters (temperature, salinity, pH, hardness) (68). In salt water, absorption via the intestine is also believed

The kinetic profile of the drug after application of multiple doses may be predicted from parameters derived from single dose kinetic studies. These must, however, be confirmed. Multiple oral doses of test substances are normally administered as medicated feed at a predetermined daily rate, utilizing voluntary uptake of the medicated diet by the fish. This protocol is frequently used for depletion studies conducted to determine withdrawal periods (4,33,62-65). For obvious reasons, it is not feasi-

352

T.E. Horsberg

to be of importance, as fish drink actively, up to 5 ml h-r kg- for a rainbow trout (69). For toxicological studies with aquatic organisms, standard protocols for static, semi-static, and flowthrough tests have been developed by international bodies, such as the Organisation for Economic Cooperation and Development, OECD (70). These protocols give specific recommendations concerning fish species and size, water quality parameters, adaptation, loading, and husbandry. Though pharmacokinetic studies normally aim at describing processes other than those investigated in toxicological studies for which the protocols have been developed, some of the recommendations in the guidelines can be used or modified for the former purpose. Experimental models for determining the movement of drugs and other xenobiotics across the gills of fish have also been established, e.g. the isolated head technique described by Payan and Matty (71) and modified by Part (72). After heparinisation and decapitation, the ventral and dorsal aorta are cannulated, and the ventral aorta cannula connected to a perfusion pump delivering the perfusion medium, the perfusate being collected from the dorsal aorta cannula. The head is placed in a cylindrical

box, and the gills are irrigated with water containing the xenobiotic through a tube inserted into the mouth (Fig. 4). This model is normally used for in vitro determination of the movement of different toxic substances over the gills, but may also be suitable for determination of absorption rate of therapeutics from the water. Such methods have the advantage that several drugs may be studied quite easily in standardized systems. A lot of data concerning relative values between different chemicals may be generated. Direct extrapolation to conditions in intact animals is, however, questionable. In intact animals, the procedure most frequently utilized when performing kinetic studies after waterborne administration, is to use dip or bath exposure in static systems with constant concentrations of the drug. The time of exposure varies from a few seconds to several days. Essential elements of the experimental design for some drugs are outlined in Table 2. The concentration of the active ingredient in the water is only occasionally checked before and/or after the experiment, even with an exposure period of several days. In some cases, this practice is unfavourable, as neither uptake and metabolism by

l-----1I ................ .-

11l-h

Fig. 4. Isolated head technique for studying the movement of xenobiotics over the gills in vitro. The dorsal and ventral aorta are cannulated, and the gills perfused through these vessels. The ventilatory water containing the xenobiotic is passed over the gills at a constant rate through a tube inserted into the mouth. PP = perfusion pump, CP = circulatory pump. Modified after (72).

Methods for kinetic studies in salmonids

353

Table 2. Experimental design for some pharmacokinetic Cone 100 mg I- 1000 mg 1-l 75 mg 1-l 50mg1- 1.1 mg I- 1.6 mg I-

studies in intact salmonids using waterborne administration of various drugs Temp. 3.5-5 C 7-14C 9C 11 C 9C 8-16 C Salinity 0 0 32 %0/O 0 30 %o 0 Time 15 min 12 h 84 h l-4.5 h 60 min 40 min Loading 40 g 1-l 12.5 g I- not reported 28.5 g I- 10 g 1-l not reported Ref. 73 5 10 6 16 8

Drug Tricaine Sulphadimidine Trimethoprim Flumequine Dichlorvos Malachite green

Species Brook trout Rainbow trout Rainbow trout Brown trout Rainbow trout Rainbow trout

the fish, nor breakdown of the active ingredient by light, hydrolysis or other physio-chemical processes are accounted for.DISTRIBUTION AND DEPLETION STUDIES

water molecules. Similar problems may be encountered when 1 is used to label proteins, polysac-

The basic pharmacokinetic studies will indicate how the drug is distributed within the body. An ap-

parent volume of distribution (V,) considerably greater than the intravascular volume will indicate good tissue penetration. A Vdof more than 1 l/kg indicates that the concentration of the drug in the tissues is greater than in the serum. If the profile is best described by a multicompartmental model after intravascular administration, a firm binding of the substance in one or more types of tissues is likely. The exact tissue distribution will, however, have to be determined in special studies. Such studies may be combined with depletion studies to determine the withdrawal periods for the parent molecule and/or its longest retained metabolite, the marker residue. The most commonly used approach for distribution and depletion studies is to sample several tissues at various time points after administration, and to assay them by biological (34,63,74), chemical (4,8,35,59,60,64-66,75,76), radiological (45,47, 67,77), or other methods. When radiolabeled drug molecules are used, only the radioactivity will be registered. One will not be able to determine whether it is associated with the parent drug molecule or a metabolite. In some studies, this is of minor importance, as the kinetic profiles of major metabolites are important for the evaluation of the withdrawal time for a drug. It may, however, also be a disadvantage, as for some xenobiotics, the fragment of the molecule containing the radioatom may join the common intermediary metabolic pool of the body, and be incorporated into normal endogenous substances such as proteins. When tritium (3H) is used to label the drug molecule, it may occasionally be so loosely bound that it is interchanged with hydrogen, mainly in

charides etc. The isotope may be so loosely bound that it detaches from the molecule intended to be labeled. In addition, this relatively big isotope may alter the physiochemical properties of the compound. It is therefore advisable, as far as possible, to ascertain the nature of the compound with which the radioactivity is associated. Another approach to distribution studies is to use the method of whole body autoradiography, as originally described by Ullberg (78). This method is also dependent upon the use of radiolabeled drug molecules. Each fish to be included in the study is administered a dose of 100 &i/kg bodyweight when 14Cis used as the radioisotope. When tritium is used, the dose must be 10 times higher. At predetermined time points, whole fish are sampled, and frozen in an aqueous solution of 1% sodium carboxymethyl cellulose. Sagittal slices of the whole body (20-40 pm) are cut in a cryomicrotome, collected on adhesive tape, freeze-dried and put on X-ray or autoradiographic film. After exposure in a freezer for a considerable period of time (l-3 months), the films are developed and positive pictures are produced. The distribution pattern of the drug in the fish is visualized elegantly by the method, as demonstrated in Figure 5, showing an autoradiogram of 3H- ivermectine in Atlantic salmon 7 days after oral administration. In particular, the distribution of radioactivity to minute organs not suitable for chemical analysis may be studied. Several studies utilizing this method for distribution studies have been published (52,53,56,57,61,79-81). One will face the same problems here as with other studies using radiolabeled substances. The detection method only reveals radioactivity, but does not differentiate between the parent compound, its metabolites, or endogenous compounds into which the radioatom may have been incorporated. Other techniques must be applied to identify the nature of the radioactivity that has been detected.

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The freeze-drying of the sections directly after collection leads to removal of tritium that may have been interchanged with water hydrogen. Evaporation may also be used with other substances to separate metabolites directly on the sections, as demonstrated by Ingebrigtsen, and Skaare (82) for the volatile hexachlorobenzene in rainbow trout. Hexachlorobenzene was evaporated from one parallel series of the sections, leaving only the nonvolatile metabolites behind. Extraction of the sections with different organic and inorganic solvents may also be empolyed. The method is by nature only semi-quantitative. By application of a 2-fold dilution standard of the radioisotope on the film, the relative concentration in several organs may be determined by densitometry (Fig. 5). As only one half of the fish needs to be sectioned for whole body autoradiography, the tissues left can be used for liquid scintillation counting for quantification of radioactivity. The tissues can also be used for chemical separation of the parent compound and its metabolites by high performance liquid chromatography (HPLC) or some other analytical technique, as demonstrated for florfenicol and its metabolites by Horsberg, Martinsen, and Varma (61).METABOLISM AND EXCRETION STUDIES

The use of radiolabeled drug molecules also enables the metabolism of xenobiotics in fish to be studied, and can be combined with several other types of studies, such as basic kinetic or distribution studies. When the main metabolites are known and standards are available, unlabeled substances may be used as well. Many xenobiotics are excreted via the bile, and very high concentrations of the par-

ent molecule and/or its metabolites may often be found in this body fluid. Occationally, there is a need to quantify the excretion of a xenobiotic via various routes (gills, urine, bile, faeces) to establish a mass balance for the drug. The simplest approach for an overall determination of excretion products is simply to give a defined dose of the drug to a small group of fish by gavage or injection, and then to transfer them to a tank with static, clean water. After a predetermined period of time, they are again transferred to a new tank with clean water, while water and faeces particles from the first tank are collected and assayed for the drug and possible metabolites. This procedure may be repeated several times. As the output of bile and faeces is dependant upon feed intake, a daily force feeding may be necessary, e.g. via an indwelling stomach tube, as described by Kleinow et al. (49). At the end of the experimental period, the fish are killed and assayed for the agent/metabolites. It is not possible to differentiate between the various routes by which the drug is excreted, but the sum of excretion products resulting from the unabsorbed fraction, as well as excretion via urine, bile, and over the gills, can be determined. Several techniques have been described for collection of different excretory products. Most commonly utilized is catheterization of the urethra. A thin polyethylene catheter is inserted into the urethra, by probing it dorsally in the vent in well-anaesthetized individuals. It should be pushed gently into the urethra until it meets a constriction, and then secured with a suture in the anterior part of the anal fin. The urine is collected either directly from the catheter using a syringe, or by means of a flexible bag to which

GUT

KIDNEY

LIVER

BILE

BRAIN

Fig. 5. Whole body autoradiogram of an Atlantic salmon 7 days after oral administration of 1 mCi H-ivermectinjkg bodyweight. The picture is produced by placing a 20 pm sagittal section of the whole body on a X-ray film. Bright areas correspond to high levels of radioactivity and thus indicate high concentrations of the drug and/or its metabolites.

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+ water inlet

drain t Port

1

3 catheter

Fig. 6. Metabolism chamber for fish. A rubber diaphragm separates the water circulating over the anterior and the posterior parts of the body. Combined with catheterization of the urethra, the study of excretion via gills, urine, and faeces may be accomplished. Modified after (83).

it is connected (17). By combining this technique with collection of faeces by manual stripping between the ventral fin and vent, as well as assaying of the water, figures for the excretion of the drug via gills (water), kidney (urine), and faeces can be calculated. Some studies have been published in which this technique has been used (77). A procedure for the continuous collection of bile in rainbow trout by catheterization of the bile duct has also been described (17). After opening the abdominal cavity, the short bile duct is located on the fish right side between the gall bladder and the pys loric curvature of the stomach. A 2 mm longitudinal incision is made in the bile duct, and a thin polyethylene catheter is inserted some millimetres in the direction of the liver. The catheter is then fixed with a suture. Its distal end is passed through the pelvic girdle, and fiied to the skin of the abdominal wall with a suture, leaving a sufficient slack in the catheter to prevent dislocation by swimming movements. The abdominal cavity is closed with sutures. This technique has not become very popular in excretion studies, due to the difficulties involved with the catheterization of the bile duct. Another sophisticated approach to metabolism studies was made by Post, Shanks, and Smith (83). The authors described a metabolism chamber for fish, in which the anterior and posterior parts of the fish were separated by a rubber diaphragm (Fig. 6). The water circulating over the anterior part could be assayed for metabolites excreted via the gills, while the water circulating over the posterior part could be assayed for metabolites excreted via the

faeces. Using a urinary catheter, the excretion via urine could also be determined. Klontz and Smith (17) suggested the combination of this technique with the catheterization of the bile duct as described above. Although very elegant, this approach has not been used very much for studies of the excretion of xenobiotics in salmonids, probably due to the practical difficulties involved. It may also be questioned how far the results generated from such an artificial set-up can be applied to real life situations. CONCLUSION The methodological problems involved in pharmacokinetic studies in fish are much greater than for corresponding studies in terrestrial animals, the neccessary techniques involving a considerable degree of manipulation of the fish. As the experimental method becomes more sophisticated, the sources of errors increase, and the extrapolation of the results to situations outside the laboratory becomes questionable. To improve the quality of data obtained from future kinetic studies, investigators are advised to pay more attention to the validation of their experimental methods. REFERENCES1. Hustvedt, S.O., Salte, R., Vassvik, V. (1991). Absorption, distribution and elimination of oxolinic acid in Atlantic salmon (Salmo salar L.) after various routes of administration. Aquaculture 95: 193-199. 2. Rogstad, A., Ellingsen, O.F., Syvertsen, C. (1993). Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administra-

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