Efficacy of tricaine on Peocilia latipinna at different temperatures and concentrations

The present experiment was designed to determine the effects of tricaine methanesulfonate as an anaesthetic for juvenile sailfin silver molly Poecilia latipinna at three different temperatures and anaesthetic concentrations. Silver molly (230 ± 25 mg and 24.07 ± 5.59 mm) were exposed to 150, 200, and 250 mg l tricaine concentrations at 20, 25, and 30°C. Even though, a body of literature exist about the anaesthetic usage on foodfish species, not much information seems to be available on ornamental fish aquaculture. In the experiment, three tricaine methanesulfonate concentrations for each temperature were used to anaesthetize silver molly and recorded their induction, recovery times and survival. The necessary time to make anaesthesia on fish depended on concentration intensity and water temperature rise. When exposed to any of the concentrations, fish achieved a deep state of anaesthesia with induction times of 0.18 1.63 min and recovered fast in 1.53 2.08 min. To maximize safety and reduce fish mortality and stress, the minimum concentration producing desirable anesthetic effects was 150 mg l at all three temperature.


INTRODUCTION
This investigation of the suitability of tricaine methanesulfonate (MS-222) as a fish anaesthetic was prompted by the need for a safe anaesthetic for use in research on the environmental requirements of sailfin silver molly, Poecilia latipinna (Lesueur, 1821).This freshwater species (Cyprinodontiformes: Poeciliidae) is currently under evaluation for ornamental fish.Molly fish is com-mon in two species as P. latipinna and Poecilia sphenops.
In fisheries research and aquacultural operations, anaesthetics are necessary to minimize stress and reduce physical injury of fish during various handling procedures such as weighing, measuring, tagging, sampling, size grading, spawning of broodstock, transporting, vaccinating and surgery (Ross and ross, 1999;Coyle et al., 2004, Mylonas et al., 2005).When choosing an anaesthetic for a particular purpose, several considerations may be taken such as availibility, ease of use, effectiveness, physiological perturbations, cost, nature of the study and safety for the fish, consumers, user and the environment.
MS-222 is chemically called tricaine methanesulfonate and commercially sold as Tricaine-S or Finquel.It looks like a white crystalline powder and dissolved easily in water.But it lowers water pH and caused acidic condition to fish.It should be buffered with baking soda (sodium bicarbonate) or tris-Cl (pH 9) to hold water pH in 7. One of the main handicap to use tricaine is that plasma cortisol level increased in deeply anesthetized fish (Davis and Griffin, 2004;Cho and Heath, 2000;Small, 2003).Induction time is as short as 15 s and is not suggested to use higher than 250 mg l -1 for warm water fish.Recovery ) at 20, 25 and 30°C temperatures to evaluate induction time, recovery time and survival for each concentration.

MATERIALS AND METHODS
The experiments were undertaken, using juvenile sailfin silver molly were grown at Aquarium Unit of Adnan Menderes University Agricultural Faculty, in Aydin, Turkey.Parameters of water source were pH 7.55, DO 6.25 mg l -1 , EC 1316 µs cm -1 , ammonia 0.22 mg l -1 , nitrite 0.07 mg l -1 .alkalinity600 mg l -1 and total hardness 780 mg l -1 .Fish average weight and length (mean ± SD) were 230 ± 25 mg and 24.07 ± 5.5 mm, respectively.Fish had been starved for 24 h prior to the experiment and tricaine methanesulfonate (sigma) stock solution (0.4%, 100 ml) buffered with 1 M Tris-Cl (pH 9.0) and working solution (0.2%, 100 ml) were prepared.Application was done in an aerated 250 ml beaker placed into 9 L aquaria.Fish was exposed to 150, 200, and 250 mg l -1 concentrations of MS-222 at 20, 25, and 30°C water temperatures until anesthesia stage of 4 for induction time and recovery stage of 2 for recovery time.The induction and recovery times were recorded for each temperature and concentration.Following recovery, fish were placed to maintenance aquarium and were observed for 48 h for adverse effects.Experiment was fulfilled on five fish in triplicate (n = 15).
The time of induction was recorded for each fish when fish lost total equilibrium, its operculum rate stopped and fish did not response to pressure on its body (S3).Thereafter, the time of recovery was writen down when fish started swimming in a normal manner (R3) after putting it into the freshwater being identical temperature (Table 1).Anaesthesied each fish was weighed and measured its length.
For statistical analysis, differences between tricaine concentrations and temperatures were examined using analysis of covariance induction time, recovery time, operculum rate, body weight and body length in SAS.Tukey's multiple range was used to compare the means.In all statistical analysis, P < 0.05 was considered significant.

RESULTS
In this study, induction and recovery times of each concentration and temperature were given in Table 2.At 20°C, the induction times of anaesthesic effect in fishes  Figure 2 demonstrated the positive relationship between concentration and recovery time.Fish recovery became long and mortality risk increased when concentration arised.
MS-222-exposed fish had over 100/min operculum rate in all groups.There was no difference among groups.
Anaesthetics are needed for easy handling, sorting, measuring, transporting and surgical procedures in aquaculture and fisheries to facilitate application procedure.The most often used ones include MS-222, 2-phenoxyethanol, quinaldine, benzocaine and metomidate.A suitable anaesthetic may vary.It may depend on stress, environmental factors, fish condition and species.Generally, an ideal anesthetic ougth to induce anesthesia quickly in less than 3 min, permit a fast recovery in 5 min or less, produce no poison to fish and cause no hazard to human and be inexpensive (Mylonas et al., 2005).In this study, 150 mg l -1 of MS-222 was determined ideal concentration for three temperatures and induced anesthesia on fish within 1.63 min and fish regained its equilibrium within 2.08.No fish died during the experiment and researhers incurred no irritation.In fact, trial was conducted in low-cost due to being ornamental fish.Lemm (1993) found that 200 mg l -1 (18°C) of tricain caused 1.86 min of induction time and 200 mg l -1 (23°C) produced 1.83 min of induction time, with recovery times of 5.79 (18°C) and 11.78 (23°C) min, respectively at pH 7.0 -7.8, alkalinity 292 and hardness 342 mg l -1 . In this study, closely related results was observed with 150, 200 and 250 mg -1 concentrations of MS-222 at 20°C when considering induction range although our alkalinity and hardness were two fold of it.However, in recovery range, the same situation was not encountered.At 20°C, 250 mg l -1 of MS-222 displayed a recovery range of 5.05 min.This difference of recovery may be as a result of the fact that molly (230 mg) are quite smaller than striped bass (300 -1500 g) in size.
Even though a number of researchers have recently tried to study in order to replace MS-222 with 2-phenoxyethanol, clove oil, carbon dioxide, Aqui-S, or quinaldine (Davis and Griffin, 2004;Walsh and Pease, 2002;Mylonas et al., 2005;Pirhonen and Schreck, 2003), the anesthetics, MS-222, was more often chosen for applications in aquaculture due to legal use and also because it met the ideal anesthetic requirements (such as short time for induction and recovery, commercial availibity and facileness of preparation).MS-222 was tested on a number of fish species in Table 3.

Conclusion
Various anesthetics are used for aquaculture applications, but MS-222 is the only approved one by the US and UK.There are not much information about anesthetic usage in ornamental fish.Here, it is observed that induction time was significantly different in three temperatures and three concentrations except 200 and 250 mg l -1 at 30°C. it is suggested that the minimum tricain concentration be 150 mg l -1 at the three temperature ( 20 ºC) for safe use.

Figure 1 .Figure 2 .
Figure 1.Mean ± SD of induction time of sailfin silver molly submitted to anaesthesia.

Figure 1
Figure 1 showed a negative relation between concentration and induction time of the anaesthesic effect of MS-222 at all three temperatures.When concentration and temperature increased, induction time reduced.Figure2demonstrated the positive relationship between concentration and recovery time.Fish recovery became long and mortality risk increased when concentration arised.MS-222-exposed fish had over 100/min operculum rate in all groups.There was no difference among groups.

Tricaine conc. (mg l -1 ) Induction time Induction range Recovery time Recovery range
a 1.18 -4.28 100 *Upper case for tricaine concentration.*Lower case for temperature.and equilibrium can be regained in few minutes.MS-222 displays a good safety margin in fish such as effective concentration and maximum safe concentration.Safe margin constricts when temperature arises and becomes smaller for small fish.It is more effective in warm water with low hardness.After exposure, MS-222 is excreted by kidney with urine within 24 h.It is approved for use in aquaculture in the U.S. and U.K. It (MS-222) has

Table 3 .
Anesthetic agent of tricaine methanesufonate used in various fish.