Anaesthetic efficacy of table salt on two live fishes Anabas testudineus and Channa punctatus

Different concentrations of table salt (NaCl) were evaluated for its induction/anaesthetic and physiological effects on two live fish species viz., Anabas testudineus (Koi) and Channa punctatus (Taki). The time taken to immobilize and fully anaesthesize the fishes were quite long, 36-52.83 hours and 37.75-53 hours respectively for Koi, 64.50-71.50 hours and 68.2571.50 hours respectively for Taki, but thereafter a quick mortality occurred in both species. The induction time was negatively related with the concentrations of salt, and independent of the intrinsic factors (total length and total weight) of the fish. No changes in the colours of skin, eye and gill were observed at lower concentrations were observed in A. testudineus, but the eye and gill were found to be affected at low concentrations in C. punctatus, irrespective of the exposure time. Table salt produced a bad smell at all concentrations at longer exposure in both species of fish.


INTRODUCTION
Fishes are subjected to stress everyday due to changes in the culture system, water quality, environment, and their physiology (Koeypudsa & Jongjareanjai, 2011) and other stress factors regarding harvesting, handling and transportation (Woody et al., 2002).Stress disturbs the final internal balance of fish, such as behaviour, growth, reproduction, immune function and disease tolerance (Goos & Consten, 2000;Tanck et al., 2000;Chen et al., 2004;Davis & Griffin, 2004;Morales et al., 2005).To reduce the stress in fishes in culture system, in the research laboratory, handling and transportation several types of anaesthetics have been prescribed by the fisheries scientists.The scientists prefer low euthanization or sedation than full anaesthesia (Wurts, 1995;Davis & Griffin, 2004).However, the degree of anaesthetization varies, for example, for therapeutic use, laboratory research and transportation comparatively longer induction time is preferable, otherwise, a recovery time within 15 minutes in clean water is desirable (Gilderhus & Marking, 1987;Kaiser et al., 2006).
In this context a large number of anaesthetic compounds have been screened for use in aquaculture since a long time.The use of chemical fish anaesthetics sometimes showed hazardous effect in human and other animals when anaesthetized fish were subsequently eaten (Peake, 1998), and some of these compounds, an example, MS222 (tricaine methane sulphonate) is no longer used in some European countries (Kaiser et al., 2006), banned in Canada and restricted in United States (Peake, 1998).As an alternative to these chemical anaesthetics, food salts were introduced in aquaculture, research laboratories, and transportation of fishes and their fingerlings in live condition.Use of sodium bicarbonate was registered in USA as Low Regulatory Priority Compound (Peake, 1998;Bowser, 2001).Along with sodium chloride other food salts like sodium sulfate and sodium bicarbonate have been used to improve the quality of fish during transporting (Wurts, 1995;Gomes et al., 2006;Velasco-Santamaria & Cruz-Casallas, 2008;Koeypudsa & Jongjareanjai, 2011).The United States Food and Drug Administration (FDA) approved table salt or sodium chloride for use as anaesthetic on the food fish (Davis & Griffin, 2004).The present research focused on the anaesthetic effect of table salt (sodium chloride) in two live fishes Anabas testudineus (Bloch) and Channa punctatus (Bloch).

MATERIALS AND METHODS
Selection of fish species: Both Anabas testudineus (Koi) and Channa punctatus (Taki) are hardy fish and can survive out of water for comparatively longer time hence they are marketed in live condition.These two species can live in any type of container with very small volume of water, and also can withstand a wide range of temperature.Both the fishes are popular for their taste.For these reasons they are selected for the present experiment to observe the anaesthetic effect of table salt and the effect on their morphology and behavior.
Collection of fish and acclimatization: Healthy A. testudineus and C. punctatus fishes were collected in live condition from the fish markets of Rajshahi city.After collection the fish were immediately placed in plastic buckets with water, and carried to the Fisheries laboratory of the Department of Zoology, University of Rajshahi.
In the laboratory the fishes were released in aerated aquaria.Two species were kept separately.Tap water was used in these aquaria.Five fishes were released in each aquarium containing 10 liter of water.For the first 24 hours no food was given to the fishes, and afterward rice bran and wheat flour in the form of small ball were given once daily.One third of water of the aquaria was removed daily and fresh water was added.The fishes were acclimatized for week.Feeding was withheld 24-h before the commencement of the experiments.Healthy fishes with strong physique were selected for the experiments.

Concentrations of salt used:
A pilot experiment was run to find out the sublethal concentrations of salt on the test fishes.C. punctatus was found to be more susceptible to salt bath compared to A. testudineus.Based on the pilot experiment, two different sets of salt concentrations were chosen for the two species.The concentrations used against A. testudineus were 30, 40, 50 and 60 mg salt/l of water; and those were against C. punctatus were 5, 10, 15 and 20 mg salt/l of water.
Experimentation: Experimental fishes were collected individually using a hand net to avoid any damage and released separate aquarium containing five liter of fresh tap water.Before releasing the fish the water was mixed with salt and stirred well with a glass rod.Thus three sets of aquaria were used for each concentration of salt, and each fish species.Control batches of both species were set similarly in untreated water.No food and aeration were allowed in the experimental aquaria.The experiments were conducted at room temperature (17-20 o C) for 80 hours.
The time was recorded when each fish was released in the experimental aquarium.The fishes were monitored continuously to observe the effect of salt.First induction to salt was determined as the stage where total loss of mobility became evident (immobilization) and full anaesthetization stage was that when the fish could no longer swim and/or maintain a vertical position in the water.The time taken for immobilization and induction (anaesthetization) of individual fish was recorded.To observe further effect of salt the fishes were kept in the treated water without running the recovery test.The time when death occurred was also recorded.
When the fishes became immobilized, total length (TL) and body weight (TW) of individual fish were measured carefully and quickly then released in the aquarium.
Changes in morphological characters and behavior: Similar sets of experiment were arranged with approximately similar size of fishes as mentioned, to observe the changes of some morphological characters and behavior of each species due to salt bath.Observations were made and the data recorded after 4-, 8-, 12-and 24 hours of exposure.The morphological characters studied included the colors of skin, eye, gills and smell.The swimming behavior of the fishes was observed.
Anaesthetic effect of salt on dissected fish (C.punctatus): Total 40 specimens of C. punctatus of different sizes (total length ranged from 145-160 mm) were immersed in a solution containing 200 g salt in a small quantity of water in a plastic bucket to induce the fish before and during dissection in a practical class.The observation is described in the result.

RESULTS AND DISCUSSION
Anaesthetic effect of salt: A. testudineus was found to be more resistant to the salt induction activity than C. punctatus.A concentrations of 40 mg/l salt immobilized A. testudineus at 52.83 h, whereas, a concentrations of 30 mg/l did not affect the normal behavior of the fish up to 80-h of exposure.The fish became senseless after 53.00 h and died after 71.00 h at 40 mg/l.At highest concentrations (60 mg/l) the time required to immobilize the fish at 36 h; after 37.75 h the fish were fully induced and died after 40.50 h (Table 1a).In case of both the species, the time required for induction stages (motionless to death) were positively related with the extrinsic factor (concentrations of salt), but there was no relationship with the intrinsic factors (total length and total weight) of fish.The differences between induction time at different concentrations were insignificant (Tables 1a, b).

Observation on the dissected Channa punctatus:
The fishes became induced within 10-12 minutes in salt bath.To observe the internal organs the anaesthetized fishes were dissected open from thoracic to abdominal region (up to anus).It took nearly 30-40 minutes to complete the dissection.During this period it was noticed that heart of the dissected fishes were beating rhythmically but at a slight slow rate.Heart beat continued up to the end of the dissection class.Heart beat of these anaesthetized and dissected fishes were observed for 3.30 h (then the specimens were discarded).With all the trauma of incision, clearing some of the body muscles, pinning the abdominal muscles for clear view, the fishes were alive.
then gradually became slow (Table 2b).C. punctatus tried to escape from the salt treated water after 1-2 h at concentrations of 10-20mg/l.
Table salt or sodium chloride is one of the most commonly used drugs in the aquaculture, and fisheries scientists referred it as the 'aspirin of aquaculture' (Swann & Fitzgerald, 1992), as it is a therapeutic against the bacterial diseases in fish (Van Duijn, 1973) and also to treat the ectoparasites of fish, like Costia, Epistylis, Trichodina, Chilodonella, Dactylogyrus and Gyrodactylus (Swann & Fitzgerald, 1992).Fashina-Bombata & Busari (2003) reported that salt treatment is potential to control pathogens in developmental stages of African catfish.
During transportation of live fish a higher percentage of mortality occurs due to the activation of latent disease organisms and osmoregulatory problem (Jensen, 1990) and addition of sodium chloride is able to minimize these stresses (Francis-Floyd, 1995).
During the transportation of live juvenile fish in tropical countries bacterial proliferation occurs, which can be prevented by adding either sodium chloride or methylene blue in the transporting water (Fajardo, 2002).
The anaesthetic effect of table salt was observed on two hardy fishes, A. testudineus and C. punctatus, and found that Anabas can tolerate comparatively higher concentrations of salt than Channa; but the induction time was less in Anabas than that of Channa.Anabas was induced in between 37-53 h, and Channa was induced in between 68-72h, depending on the concentrations.The long time taken for induction could be enhanced by using higher concentrations than those were used in this experiment.Toxicity of salt based on induction time was found to be dependent on the concentration of salt, but not with the total length or total weight of the fishes.The survival time for mosquito fish in salt bath was found to be correlated with the extrinsic factor like salt concentration and intrinsic factor like total weight (Newman & Aplin, 1992).
It was noticed that after total induction both the species died quickly.That means the concentrations used affected the salt level of blood and created osmotic imbalance.Wurts (1995) reported that 10% dehydration occurs in live fish when 10g/l salt was added to the transported water, and resulted in lethality.The author also reported that salt tolerance varies among different species and the temperature.
Both the species suffered salt stress at an exposure of 72h at all concentrations levels.Channa was more stressed than Anabas.The color of skin, eye and gills were affected at higher concentrations and longer exposure in Anabas.At all concentrations levels from 5-20mg/l, skin color changed loosing the brightness and slime secretion was enhanced in C. punctatus, and at longer exposure the scales became loose.The eye color became dull and cloudy and the gills lost the normal deep red color.Salt affected the eye at exposure more than 10h.Normal blood circulation in the gills was affected severely and respiratory stress was detected by discolored gills and rapid opercular rate, along with abnormal and unbalanced slow swimming.However, C. punctatus was alive in anaesthetized condition and tolerated the stress of dissecting trauma for more than three hours.
Besides handling and transportation, other stress factors like chill coma in tropical fish (Sun et al., 1995), physical damage and high nitrate level (Andrews et al., 2002) can also be prevented by the application of salt.Authors also reported the threat of osmoregulatory and other physiological stress occurred due to higher concentration of salt (Andrews et al., 2002;Burgdorf-Moisuk et al., 2011).Again it is observed that the anaesthetic stress in fish can be lowered if sodium chloride is applied along with the anaesthetic (Davis & Griffin, 2004).
Table salt was found to be a good anaesthetic agent which affect the heart rate insignificantly in C. punctatus for more than 3-h in severe traumatic stage and keep the fish alive.The salt concentrations used in the present experiment did not induce both the species quickly, but higher concentrations would be acted as quick inducing agent.Longer exposure to the salt at higher concentrations than the salt content in the fish blood would be fatal, so it is better to use salt in transporting water for juveniles and live fishes and also as therapeutic purpose in pisciculture, rather to use as an anaesthetic.