Pharmacokinetics, plasma cortisol and effectiveness of benzocaine, MS-222 and isoeugenol measured in individual dorsal aorta-cannulated Atlantic salmon (Salmo salar) following bath administration
Introduction
Anaesthetics are primarily used to immobilize fish and to reduce stress and pain during various procedures such as handling, transport, blood sampling, vaccination and surgery. At low concentrations they are used to reduce activity and metabolic rate, while higher concentrations are routinely specified during procedures that are deemed stressful or painful for the fish. Benzocaine (ethyl para-aminobenzoate) is an effective fish anaesthetic with the desirable characteristics of rapid induction and recovery times and good safety margin for salmonids (Ross and Ross, 2008). During anaesthesia of Atlantic cod (Gadus morhua) however, benzocaine has led to mortalities when the same concentrations as those administered to salmonids have been employed (Mattson and Riple, 1989). The structurally similar tricaine methanesulphonate (Metacaine, MS-222, ethyl meta-aminobenzoate) is the most frequently used anaesthetic for fish. In Norway both benzocaine and MS-222 have a withdrawal time of 21 days.
AQUI-S is a fish anaesthetic/sedative in which the active ingredient is isoeugenol (12% cis- and 88% transisomers). AQUI-S is approved for use in fish in several countries including Australia, New Zealand and Chile, and is pursuing approval in the USA.
While literature on the efficacy of anaesthetics in fish is extensive (Mattson and Riple, 1989, Malmstrom et al., 1993, Taylor and Roberts, 1999, Iversen et al., 2003, Hoskonen and Pirhonen, 2004), little is known about the pharmacokinetic properties of these agents in fish.
The pharmacokinetic properties of benzocaine have been described in rainbow trout (Oncorhynchus mykiss) after both intra-arterial administration and bath exposure (Meinertz et al., 1996, Stehly et al., 1998) and in channel catfish (Ictalurus punctatus) following bath exposure (Hayton et al., 1996). The concentration of isoeugenol has been determined in skin-on fillet of rainbow trout after bath exposure to AQUI-S at different temperatures, durations and concentrations (Meinertz et al., 2006). The accumulation and clearance of isoeugenol have also been studied in edible fillets of silver perch (Bidyanus bidyanus) exposed to varying dosages and temperatures (Kildea et al., 2004). The pharmacokinetic properties of eugenol have recently been studied in rainbow trout held in fresh water at low temperature (4 °C) (Guénette et al., 2007). To our knowledge, there are no complete pharmacokinetic data on MS-222 in fish available in the literature. Plasma levels, metabolism and excretion following exposure have been studied in rainbow trout and in the elasmobranch dogfish (Squalus acanthias) (Hunn et al., 1968, Stenger and Maren, 1974).
In order to establish correct dosage regimes and thereby promote optimal use, data derived from pharmacokinetic investigations are vital. Studies of elimination kinetics are also necessary in order to determine withdrawal times for drugs used in the production of food for human consumption. Additionally, the welfare aspect of confinement and handling must be taken into consideration, whether the use be research, food production or in the case for fish, ornamental.
According to Meinertz et al. (1996), who studied the pharmacokinetic properties of benzocaine in rainbow trout, the initial distribution phase following intra-arterial administration is extremely rapid (about 2 min). This made it difficult to accurately predict the parameters for this part of the curve. In a later study this research group therefore used bath exposure for calculating the pharmacokinetics of benzocaine in rainbow trout at different water temperatures (Stehly et al., 1998). Exposure through bath immersion was chosen in the current study accordingly. However in this study shorter exposure times and higher concentrations than those used by Stehly et al. were utilised in order to better mimic normal aquaculture and field conditions.
The primary aim of the present study was to determine the elimination kinetics of benzocaine, MS-222 and isoeugenol (AQUI-S) in Atlantic salmon following bath administration. The secondary aim was to relate the kinetics to plasma levels of cortisol and variables such as wake-up time, swimming behaviour and respiration depth and frequency. The effect of artificial ventilation during recovery on the elimination kinetics was also studied. This was done in order to reveal a possible negative effect on the recovery by the widespread practise of static water in the recovery tank.
Section snippets
Fish and experimental facilities
Salmo salar (NLA strain — Norwegian breeding programme, 1600 ± 250 g), fed standard diet (Royal AB Redline, T. Skretting AS, Norway), were initially maintained in 1.5 × 1.5 × 1 m standard fibreglass tanks supplied with aerated seawater (9 ± 1.5 °C) and kept under continuous simulated daylight regime at the Institute of Marine Research, Matre Aquaculture Research Station, Norway. Five to ten days prior to cannulation, individual fish were moved into individual experimental tanks (1 × 1 × 0.7 m) and left to
Results
The calibration graph for isoeugenol was found to be linear within the range studied (r = 0.997). The limit of detection (LOD), defined as three times the signal-to-noise ratio was 8 ng ml− 1 for isoeugenol in plasma. The limit of quantification (LQD), defined as ten times the signal to noise ratio, was 25 ng ml− 1.
The plasma concentration-versus-time curves for benzocaine, MS-222 and isoeugenol are shown in Fig. 1A–C, respectively. The mean drug concentrations obtained in eight fish at each
Pharmacokinetic response
For all three compounds tested, ventilation with clean water over the gills following exposure increased the rate of elimination of the drugs (Table 1, Table 2, Table 3). This indicates that elimination via the gills is an important elimination pathway in Atlantic salmon for all three drugs and confirms the findings for benzocaine in rainbow trout and channel catfish (Meinertz et al., 1991, Hayton et al., 1996). Ventilation had the greatest influence on isoeugenol elimination, as shown by the
Acknowledgements
The skilful technical assistance of Victor Steen, IMR with the fish, and of Ulf Nymoen and Magne Kjerulf Hansen, NVH, with the isoeugenol analysis made the study possible. The financial contribution in kind by the Institute of Marine Research, Matre, Norway and the University of Jönköping, Sweden and the PhDgrant to I.H. Zahl from the Research Council of Norway (grant 152898/120) is also greatly appreciated.
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