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Efficiency of clove oil as anesthetic for abalone (Haliotis tuberculata coccinea, revee).

ABSTRACT The efficacy of clove oil as an anesthetic was evaluated in adult Haliotis tuberculata coccinea (shell length, 42-80 mm) and was compared with the commonly used 2-phemoxyethanol. The clove oil was previously mixed at 50% with absolute ethanol, and 4 different concentrations were tested: 0.1, 0.3, 0.5, and 0.7 mL/L. The trial was replicated 3 times. Relaxation and recovery time, muscle condition, and mortality after 1 wk were considered to evaluate the efficiency of anesthetics. The results showed that clove oil at 0.5 mL/L was the lowest concentration that removed all abalone from the rearing shelters in 8.9 [+ or -] 3.5 min, showing 2 [+ or -] 1.5 min of recovery time. Although clove oil is appropriate for handling animals, it was not suitable for pearl culture because it left the abalone muscle hard, making access to the pearl nuclei insertion site difficult. In comparison with 2-phenoxyethanol, clove oil was effective in 10-fold lower doses.

KEY WORDS: anesthetic, clove oil, 2-phenoxyethanol, abalone, Haliotis tuberculata coccinea

INTRODUCTION

In abalone aquaculture, there are many instances requiring some sedation to facilitate handling animals without injuring or stressing them excessively. Abalone is well adapted to hold tightly onto the substratum, and mechanical assistance is necessary to remove them. Dislodgment by mechanical means often results in injury or death because of their slow healing rate, absence of a blood coagulation mechanism, and stress (Armstrong et al. 1971, Genade et al. 1988). Chemical removal, by anesthetizing the animals provides a safer alternative to mechanical removal (White et al. 1996). On the other hand, in pearl culture of abalone, animals require some degree of relaxation to facilitate nucleus implantation.

When choosing an anesthetic, a number of considerations are important, such as efficacy, cost, availability, and ease of use, as well as toxicity to animals, humans, and the environment (Soto & Burhanuddin 1995). Aquilina and Roberts (2000) consider that the anesthetic used for relaxation to allow for the insertion of pearl nuclei in abalone has to have the following characteristics: rapid onset of relaxation, full extension and softening of the muscles for at least 10 min, and rapid recovery. Several anesthetics have been suggested for abalone aquaculture (Sagara & Ninomiya 1970, Prince & Ford 1985, McShane & Smith 1988, Hanh 1989, Tong et al. 1992, White et al. 1996, Aquilina & Roberts 2000, Sharma et al. 2003); however, most of them are chemical products such as magnesium sulfate, 2-phenoxyethanol, pentobarbitone, and benzocaine.

Cloves are the dried flower buds of Eugenia caryophylata, and its active ingredient is eugenol. Aroma chemicals present in natural leaves and flowers have been widely used in aroma therapy since ancient times, suggesting that they have some beneficial health effects. Clove oil has been traditionally used by humans as an oral analgesic, and it has been investigated as sources of antimicrobial agents against oral bacteria (Cai & Wu 1996), it has demonstrated its antifungal (Martini et al. 1996) and antioxidant activity in human food comparable with [alpha]-tocopherol (vitamin E) (Kwang-Geun & Shibamoto 2001), and it is effectively used for asthma and various allergic disorders by oral administration (Kim et al. 1998).

In aquaculture, clove oil is considered superior to a number of anesthetics, such as benzocaine and MS-222 (Munday & Wilson 1997, Keene et al. 1998). Because clove oil is a natural product and is less expensive than other anesthetics used for fish, it has received considerable attention (Endo et al. 1972, Soto & Burhanuddin 1995, Cho & Heath 2000, Wagner et al. 2002, Hoskonen & Pirhonen 2004, Mylonas et al. 2005). However, in mollusc aquaculture, its anesthetic effect has been demonstrated only in the common octopus, Octopus minor (Seol et al. 2007), and in the pearl oyster, Pinctada albina (Norton et al. 1996).

The objective of the present study was to evaluate the use of clove oil as natural anesthetic in Haliotis tuberculata coccinea, comparing its efficacy to the commonly used 2-phenoxyethanol.

MATERIALS AND METHODS

The experiment was undertaken in July 2008, using adults of Haliotis tuberculata coccinea produced at the Institute of Marine Sciences, Gran Canaria Island, Spain. The anesthetics chosen for evaluation were clove oil at 4 different concentrations and 2-phenoxyethanol at 2 mL/L (Table 1).

The trial was conducted in transparent plastic aquaria holding 2 L seawater equipped with a shelter. To aid dissolution, the clove oil dose was shaken at 50% in absolute ethanol, and 2-phenoxyethanol was dispensed directly. Thirty animals per treatment (3 per aquaria), between 42 mm and 80 mm in shell length, previously tagged, were firmly attached to the shelter. The temperature was maintained as natural (22.3[degrees]C) and, pH was measured before and after adding the anesthetics. The animals were kept in the solution for 30 min.

The relaxation time was defined as the length of time between the introduction of the animals into the aquaria and releasing from the shelter. At the conclusion of relaxation, animals were rinsed thoroughly in freshwater before being placed on their shells (dorsal side down) into flowing seawater at the same temperature. Recovery time was defined as the length of time between release and the animal righting itself (turning over) (Aquilina & Roberts 2000). Qualitatively, the effect of each chemical as a muscle relaxant was observed, indicating the muscle condition (soft or contracted) and exudation of mucus.

Mortality was observed the next week. After this period, the trial was replicated 3 times in the following weeks with the same groups of animals.

To evaluate the best clove oil concentration, the results were submitted to a statistical analysis applying a 1-way analysis of variance followed by a Tukey test for multiple comparisons of means, at a 5% significance level (P < 0.05). The dates were transformed to a logarithmic function (Zar 1996). The best clove oil concentration was then compared with the 2-phenoxyethanol result.

RESULTS

The results showed that both 2-phenoxyethanol and clove oil effectively produced anesthesia in H. tuberculata coccinea with no postrecovery mortalities. The reason that 2 animals died at 0.3 ml/L clove oil and 2-phenoxyethanol treatments was a deep knife cut from handling.

Clove oil treatments of 0.1 mL/L and 0.3 mL/L did not anesthetize 1 animal after 30 min (Table 2). Omitting these animals for statistical analyses, the relaxation time of the rest of the animals treated with clove oil decreased with an increase in concentration (855 [+ or -] 287 sec at 0.1 mL/L 786 [+ or -] 248 sec at 0.3 mL/L, 536 [+ or -] 208 sec at 0.5 mL/L, and 472 [+ or -] 139 sec at 0.7 mL/L). Statistical analysis showed significant differences (P < 0.05) in the mean of the high concentrations of clove oil (0.5 mL/L and 0.7 mL/L) compared with low concentrations (0.1 mL/L and 0.3 mL/L; Fig. 1). The recovery time did not show statistical differences (P < 0.05) among different concentrations (181 [+ or -] 89 sec at 0.1 mL/L, 143 [+ or -] 133 sec at 0.3 mL/L, 121 [+ or -] 90 sec at 0.5 mL/L, and 186 [+ or -] 137 sec at 0.7 mL/L; Fig. 2). For this reason, 0.5 mL/L clove oil is the concentration recommended to anesthetize H. tuberculata coccinea that are 42 80 mm in shell length.

The relaxation time observed with 2-phenoxyethanol was less (299 [+ or -] 56 sec), and less dispersal was observed for clove oil at the 0.5-mL/L concentration (536 [+ or -] 208 sec; Fig. 1 and Table 2). However, the recovery time was more (433 [+ or -] 215 sec at 2 mL/L of 2-phenoxyethanol and 121 [+ or -] 90 sec at 0.5 mL/L clove oil), with more dispersal (Fig. 2 and Table 2). Qualitatively, the effect of each chemical as a muscle relaxant was similar, producing a contracted muscle with a little exudation of mucus.

DISCUSSION

The relaxation time recorded for 2-phenoxyethanol (4.9 [+ or -] 0.9 min) was similar to that reported for H. midae (2.9 [+ or -] 2.3 min) by White et al. (1996); however, the recovery time was much shorter (7.2 [+ or -] 3.6 min and 46.0 [+ or -] 24.6 min, respectively).

Based on the results of this study, higher anesthetic doses of clove oil resulted in similar recovery times (P < 0.05). This observation was similar to that found by White et al. (1996) for MgS[O.sub.4] and 2-phenoxyethanol anesthetics in H. midae. However, the expected results were the higher the concentration of clove oil, the longer the recovery time, as reported Seol et al. (2007) in the common octopus (Octopus minor). This may be explained by the fact that higher doses induced anesthesia quicker, thus the animals were removed from the anesthetic bath and placed in clear water sooner than animals exposed to lower doses. Clove oil is highly lipophilic, therefore it adheres to and penetrates the gill epithelium rapidly, and is absorbed by body tissues, such as fat (Stoskopf 1993) and brain (Summerfelt & Smith 1990), once it is in the blood. In this way, Mylonas et al. (2005) considered that the shorter the exposure time to a clove oil bath in the European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata), the smaller the amount of anesthetic absorbed by the body, and the faster its removal from the blood and recovery of the animal when it was placed in clear water. This phenomenon would be similar in abalone.

Clove oil concentrations of 0.1 mL/L and 0.3 mL/L did not anesthetize all animals after 30 min; 0.5 mL/L and 0.7 mL/L did not show statistical differences (P < 0.05) with regard to relaxation time. Thus, the recommended concentration of clove oil as an anesthetic for H. tuberculata coccinea is 0.5 mL/L. Although the relaxation time observed at this concentration was longer than for 2-phenoxyethanol (8.9 [+ or -] 3.5 min and 4.9 [+ or -] 0.9 min, respectively), its effect on animals was more dispersed in time. We consider that this aspect is an advantage in terms of handling abalone during procedures that include enumeration, pathological analysis, and measuring or weighing, because the animals release from their shelters gradually. The recovery time seen with 0.5 mL/L clove oil was less than that of 2-phenoxyethanol (2 [+ or -] 1.5 min and 7.2 [+ or -] 3.6 min, respectively); this is another advantage, because the animals recover faster.

[FIGURE 1 OMITTED]

In pearl culture of abalone, clove oil and 2-phenoxyethanol was not suitable for pearl nuclei insertion at the doses tested, because the recovery time of animals was less than 10 min, and the muscle condition observed with both anesthetics was contracted, making access to the operation site difficult. In the case of clove oil, the muscle condition observed would be caused, in part, by the ethanol used to dissolve it, as observed by Aquilina and Roberts (2000), who used benzocaine in H. iris.

[FIGURE 2 OMITTED]

In conclusion, the results obtained from the current study demonstrate that clove oil can be used as an effective and efficient agent for muscle relaxation of H. tuberculata coccinea. Furthermore, in comparison with the commonly used 2-phenoxyethanol, clove oil was effective in 10-fold lower doses, the odor of clove oil was pleasant and nonirritating in contrast to 2-phenoxyethanol, and, because clove oil is a plant compound, it is expected to decompose readily in the natural environment.

ACKNOWLEDGMENTS

Thanks to Javier Roo for his useful recommendations and for providing clove oil. This study was supported by the JACUMAR (Junta Nacional Asesora de Cultivos Marinos) Project (no. 55172) "Cultivo y Gestion de la Oreja de Mar."

LITERATURE CITED

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AMAIA BILBAO, * GERCENDE COURTOIS DE VICOSE, MARIA DEL PINO VIERA, BEATRIZ SOSA, HIPOLITO FERNANDEZ-PALACIOS AND MARIA DEL CARMEN HERNANDEZ

Grupo de Investigacion en Acuicultura, Universidad de Las Palmas de Gran Canaria e Instituto Canario de Ciencias Marinas, PO Box 56, 35200 Telde, Las Palmas, Canary Islands, Spain.

* Corresponding author. E-mail: abilbao@iccm.rcanaria.es
TABLE 1.
Details of the anesthetic treatments tested as muscle relaxants
for H. tuberculata coccinea.

                                     Final Concentration
Treatment             Supplier             (mL/L)          Final pH

2-phenoxyethanol   Panreac (Spain)            2              8.1
Clove oil *        Guinama (Spain)           0.1             8.1
Clove oil *        Guinama (Spain)           0.3             8.1
Clove oil *        Guinama (Spain)           0.5             8.1
Clove oil *        Guinama (Spain)           0.7             8.1

* Clove oil was mixed with ethanol absolute at 50%.

TABLE 2.
Response of H. tuberculata coccinea to the anesthetics.

                          2-Phenoxyethanol

                               2 mL/L

Relax time (min)         4.9 ([+ or -]) 0.9
Recovery time (min)      7.2 ([+ or -]) 3.6
Mortality after 7 days     1 ([section])

                                     Clove Oil *

                                      0.1 mL/L

Relax time (min)                  >30.00([dagger])
Recovery time (min)      3 ([+ or -]) 1.5 ([double dagger])
Mortality after 7 days                    0

                                      Clove Oil *

                                       0.3 mL/L

Relax time (min)                   >30.00([dagger])
Recovery time (min)      2.4 ([+ or -]) 2.2 ([double dagger])
Mortality after 7 days               1 ([section])

                                           Clove Oil *

                              0.5 mL/L             0.7 mL/L

Relax time (min)         8.9 ([+ or -]) 3.5   7.9 ([+ or -]) 2.3
Recovery time (min)        2 [+ or -] 1.5     3.1 ([+ or -]) 2.3
Mortality after 7 days           0                    0

* Clove oil was mixed with 50% absolute ethanol.

([dagger]) One abalone was still attached to its
shelter after 30 min.

([double dagger]) Omitting the animal that did not
release from its shelter.

([section]) The abalone that died had a deep knife cut from
handling.
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Author:Bilbao, Amaia; De Vicose, Gercende Courtois; Del Pino Viera, Maria; Sosa, Beatriz; Fernandez-Palacio
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2010
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