Abstract

Concentrations of benzocaine (ethyl 4-aminobenzoate) at 40, 60, 80, 90, 100, 110 and 120 mg/1 dissolved in ethanol were used to determine the most effective dose for anesthesia in channel catfish, Ictaluris punctatus, and various ornamental fish including goldfish, C. auratus, Anesthesia was maintained on a recirculation system which is described. The optimum concentration of benzocame for anesthesia was 80 mg/l; concentrations greater than 100 mg/I proved toxic. No detrimental effects were noted on fish held for 7 days after being anesthetized for 10, 20, or 30 minutes at 80 mg/I benzocaine. Various surgical procedures were performed on the fish with uncomplicated recoveries and healing. All water quality parameters tested over 25 days use of the system remained stable. Selected fish were examined histopathologic ally and no abnormalities were noted.

Introduction

Anesthesia in fish is indicated in a variety of situations to reduce the stress of handling, to provide surgical analgesia, and to facilitate experimental research in fish (1). Marking and Meyer have reviewed anesthetics used in aquaculture and emphasize the need for a fish anesthetic which satisfies both efficacy and safety aspects for die animal (2), MS-222^R is the only anesthetic agent licensed for use by the United States Food and Drug Administration on food fish. Figure I gives the chemical identification of both tricaine methane sulfonate and benzocame. MS-222 has an additional methyl sulfonate group which makes the chemical solvent in aqueous solution. Benzocaine, however, is insoluble in aqueous solution, and must first be dissolved in a non-aqueous organic solvent.

Figure 1. Chemical identification of benzocaine and MS-222®

Aldrich Chemical Company, Inc., Milwaukee, Wisconsin 53233
Fort Dodge Laboratories, Fort Dodge, Iowa 50501.

Materials and Methods

Dose response studies were performed by submersing fingerling channel catfish into an anesthetic solution of benzocaine at concentrations of 40, 60, 80, 90, 100, 110, and 120 mg/1. The most effective concentration and the LD50 were determined. The optimum dosage (-)1 80 mg/1 was used to anesthetize four fingerlings at 10, 20, or 30 minutes on the recirculation system after first being induced by direct immersion each week for 3 weeks. The arbitrary 10, 20, and 30 minute times used to anesthetize the fish were chosen to mimic short and long term operation times and to help validate the system. Figure 2 shows the equipment necessary for recirculation anesthesia of fish.

Figure 2. Recirculation system for maintenance of anesthesia. Two 80-liter aquaria are situated so that the bottom of the upper aquarium is at least 40 cm higher than the top of the lower aquarium. Anesthetic solution flows by gravity through plastic tubing of 0.5 cm diameter at 1 liter/min to the mouth of a fish restrained in a cradling device on top of a grid covering the bottom aquarium. A submersible pump in the lower aquarium pumps used anesthetic solution back into the upper aquarium for re-use via a plastic hose of 2 cm diameter. All fish were fasted 24 hours prior to anesthesia. Benzocaine as a stock solution dissolved in ethanol was used to provide the dosages under test. From Brown (3).

In each group, fish were held for 7 days to monitor for delayed mortalities and also randomly sacrificed at 2 and 7 days after anesthetization for gross and histopathological examination. Procedures on selected fish while on the recirculating system included bleeding, skin scrapings and laparotomies. Similar procedures were performed on goldfish, C. auratus, and black moors to prove the safety and efficacy of such a system of anesthesia on common ornamental aquarium fish.

Water quality of the anesthetic solution in the recirculation system was compared with the water entering the laboratory. Parameters tested were Total Ammonia Nitrogen (mg/1), Dissolved Oxygen (mg/1), Hardness (mg/1), Chloride (mg/1), Nitrite (mg/1), Nitrate (mg/1), and pH using Hach Test Kits. Temperature was also monitored. Unionized ammonia (mg/1) was calculated from TAN, pH, and temperature.

Results

Induction, anesthetic response and recovery times of fish anesthetized by direct immersion at 0, 40, 60, 80, 90, 100, 110, and 120 mg/l benzocaine were investigated. Induction is indicated by a tolerance to handling. Recovery is indicated as a return to normal function after being placed in fresh water. The first signs of anesthesia were a loss of rostroventral equilibrium (0 - 40sec) followed by a loss of lateral equilibrium (40sec - 1 min, 45sec). Twitching was observed at all stages of early anesthesia and was more pronounced in the stronger doses along with a "cough" reflex, suggesting some kind of toxic response. Reflex muscle activity could be induced with sharp stimuli during induction. Rapid gill movements happened early in anesthesia while caudal fin undulation occurred in deeper anesthesia. At the lower doses, circling behavior with the head being the pivot point was observed. The most optimal concentration of benzocaine that would quickly give full anesthesia to the fish with rapid recoveries and no deaths was 80 mg/l. The LD50 was calculated to be 107 mg/I benzocaine using the Minitab Statistical Package.

Table 1 shows induction, anesthesia (with apnea), and recovery times (with initial gill movements) of fish anesthetized for 10, 20, and 30 min at 80 mg/I benzocaine. No mortalities occurred at the time of anesthesia or for 7 days afterwards. Gross dissection at 2 and 7 days after anesthesia revealed no abnormalities. Histopathology was performed on the gill, skin, liver, head kidney, and posterior kidney with no abnormalities noted. During anesthesia, all the fish exhibited apnea within 3 minutes. After being placed in fresh water for recovery, gill movements would return usually within 3 minutes followed shortly by full functional recovery. All water parameters tested remained stable and within normal physiological limits for fish health.

Table 1. Mean Induction and Recovery Times (Minutes ± SD) of Fish Maintained at 80 mg/L Benzocaine on a Recirculation Anesthetic System. N - 12

Discussion

Marking and Meyer stated that the ideal anesthetic should permit a reasonable duration of exposure, produce anesthesia within 3 minutes or less, allow recovery within 5 minutes or less, cause no toxicity to fish at treatment levels, present no mammalian safety problems, and leave low tissue residues after a withdrawal time of I hour or less (2). The FDA requires a 21 day withdrawal time on MS-222 because mammalian safety data is unavailable. Benzocaine, on the other hand, is already widely used in human medicine, particularly in throat lozenges where doses of up to 500 mg/day are permitted (4). MS-222 is extremely expensive and licensed supplies are not easily obtainable; therefore, benzocaine is a reasonable alternative which meets all of the above criteria.

Benzocaine can be used at 25 mg/1 to help successfully transport large numbers of live fish by calming the fish (5), thus preventing deteriorative changes in water quality, as well as decreasing physical injuries which would otherwise render them susceptible to secondary bacterial and fungal infections.

Benzocaine obtained as a powder, dissolved in ethanol (most organic solvents will do), and used in a concentration of 80 ppm is very efficacious for quick and smooth anesthesia of fish. The various operations performed on the fish (bleeding, laparotomies, and skin scrapings) indicate that full surgical anesthesia is achieved. By putting the induced fish on the recirculation system, many procedures can be performed for up to 30 minutes (and possibly more) without the fish suffering any deleterious changes or premature recovery during the procedure. Once the working anesthetic solution is made up, it can be used for up to 25 days without significant deterioration in water quality. In this study, the potency of the dissolved benzocaine remained just as effective throughout the 3 weeks.

The apnea observed on the recirculation system is postulated to be due to one or both of the following mechanisms. The oxygenated anesthetic solution being delivered directly over the gills decreases the stimulus for the fish to move its operculum because oxygen is being actively provided, or the stimulus for opercular movement may be centrally depressed by the anesthetic. In either case, the fish is receiving adequately oxygenated water and apparently does not suffer due to the apnea.

The system is easy to equip, assemble, operate, and disassemble. It is convenient and economical for the clinical veterinarian who will encounter an occasional ornamental or pond fish which requires handling, close examination, and possible diagnostic procedures (e.g., skin scrapings for parasites, bacterial cultures, fungal cultures, etc.) that could not otherwise be performed without sedation of the fish.

In conclusion, three objectives were accomplished in this study to establish benzocaine as a safe and effective fish anesthetic. First, dose response studies were employed to determine the optimum dose of benzocaine for reliable and ideal anesthesia, and second, an LD50 was calculated. Finally, fish were anesthetized and recovered to detect any chronic toxic effects. Both channel catfish and ornamental fish used in this study appeared to show no adverse side-effects. The water temperature of the system varied from 18' to 20' C which is considered within normal limits for these fish. A simplified technique for a recirculation system is described. Veterinarians in general practice and curators for large aquarium collections may be interested in using this system for procedures which require prolonged anesthesia of ornamental fish species.

References

1.  Ross, L.G., Ross, B. Principles and practice of fish anesthesia. Assoc Vet Anaesth of G Brit and Ireland 11: 154-189 (1993).

2.  Marking, L.L., Meyer, F.P. Are better anesthetics needed in fisheries? Fisheries 10(6): 2-6 (1985).

3.  Brown, L.A. Recirculation anaesthesia for laboratory fish. In press (1986).

4.  Laird, L.M., Oswald, R.L. A note on the use of benzocaine (ethyl p-aminobenzoate) as a fish anaesthetic. J Inst Fish Mgmt 6(4): 92,93 (1975).

5.  Ferreira, J.T., Schoonbee, H.J. Strut, G.L. The use of benzocaine HCI as an aid in the transport of fish. Aquaculture 42: 169-174 (1984).