Received: 4 September 2016 | Revised: 14 February 2017 | Accepted: 27 February 2017 DOI: 10.1111/are.13339 ORIGINAL ARTICLE Anaesthetic efficacy of eugenol on various size classes of angelfish (Pterophyllum scalare Schultze, 1823) Reza Tarkhani | Ahmad Imani Department of Fisheries, Faculty of Natural Resources, Urmia University, Urmia, Iran Correspondence Ahmad Imani, Department of Fisheries, Faculty of Natural Resources, Urmia University, Urmia, Iran. Email: a.imani@urmia.ac.ir | Hadi Jamali | Hamed Ghafari Farsani Abstract Anaesthetic efficacy of eugenol was investigated on Pterophyllum scalare. A total of 130 fish with average weights of 1.0  0.5, 5.0  1.0 and 10.0  1.0 g were subjected to 1.25, 2.5, 4.0, 5.5 and 7.0 mg/L eugenol, and behavioural responses were observed. Induction and recovery times were significantly affected by the interactive effect of eugenol concentration and fish weight (p < .05). Generally, 49.9–128 s after exposure to 1.25–7 mg/L eugenol, fish reached stage 3. Fish entered stage 4 over 55–135 s post exposure to such concentrations. Recovery time was 393.5– 597.7 s in all sizes. Any increase in eugenol concentration led to a significant decrease in the induction time with a subsequent increment of the recovery time. Concentrations of eugenol and fish size along with their interactive effects have significantly contributed to the regression models, with concentration recording the highest beta values for stages 1, 2, 3 and 4 ( 0.903, 0.898, 0.976 and 0.864 respectively) and the product of size and anaesthetic concentration for full recovery in smaller fish (0.647) and eugenol concentration in larger ones (0.967). Recovery time was fitted to induction time to stage 4 via quadratic and linear regression models in smaller and larger fish respectively. Results revealed the minimal eugenol concentration to induce anaesthesia in various size classes of angelfish in less than 3 min was 1.25 mg/L. Our results showed eugenol as an effective and safe anaesthetic; however, it is not advisable for live fish transportation. KEYWORDS anaesthesia, behavioural response, eugenol, Pterophyllum scalare 1 | INTRODUCTION To survive such conditions, application of species-specific concentration and exposure time of an anaesthetic is highly recom- Fish may experience stressful condition during fisheries management mended (Summerfelt & Smith 1990). In this regard, the efficacy of and aquaculture activities including counting, pathological examina- several anaesthetic agents including metomidate, 2-phenoxyethanol, tions, hormonal implants or injections, vaccinations, stripping, trans- quinaldine, tricaine methanesulphonate (MS-222), benzocaine, clove fer and hauling and release (Carmichael & Tomasso 1988; Brown oil and Aqui-STM have previously been examined in different fish 1993; Tarkhani, Imani, Jamali & Sarvi Moghanlou 2016). Such condi- species (Pirhonen & Schreck 2003; Iversen, Finstad, McKinley & tions would result in compromised stamina and cause mortality, Eliassen 2003; Coyle, Durborow & Tidwell 2004; Tarkhani et al. growth reduction and diseases outbreaks in fish population. In addi- 2016). In searching for an appropriate anaesthetic for aquaculture tion, fish response to stressful circumstances via recruiting more industry, various relevant factors such as low expense, high effi- energy reserves which later on would affect individuals’ fitness and ciency, lack of/short withdrawal period, lack of side effects on fish homoeostasis (Park, Hur, Im, Seol, Lee & Park 2008). appetite, blood biochemistry and health, wide toxicity threshold both Aquaculture Research. 2017;1–8. wileyonlinelibrary.com/journal/are © 2017 John Wiley & Sons Ltd | 1 2 | TARKHANI ET AL. to target animal and labours must be considered (Heo and Shin extent that they are referred to as living jewels. Their tranquillity, 2010; Hoseini, Rajabiesterabadi & Tarkhani 2013). Although, MS-222 small and colourful body along with astonishingly eclectic shape and is the only anaesthetic approved by the Food and Drug Administra- behaviour made them popular aquatic pets (Mandal, Mukherjee & tion of the United States of America (USFDA), its application is Banerjee 2010; Johny & Inasun 2016). Being native to Amazon mainly limited due to higher cost and lower efficiency of the chemi- region of South America including Peru, Colombia and Brazil, the cal in controlling plasma cortisol level (Coyle, Dasgupta, Tidwell, Bea- angel fish, P. scalare is considered the most enviable ornamental fish vers, Bright & Yasharian 2005). Moreover, a 21-day withdrawal mainly owing to its attractiveness, reproductive capacity, reasonable period is recommended provided that the fish is intended for human housing and nutritional requirements and adaptability to captivity consumption (Ross & Ross 2008). Particularly, such constraints  mez-Romero 2005; Karayucel, Ak & Karayucel (Garcia-Ulloa & Go encourage using less persistent and natural anaesthetics such as 2006; Froese and Pauly 2014). The species enjoys an insatiable clove oil (Tarkhani et al. 2016). worldwide demand among commercial ornamental fish species fos- Eugenol, 2-methoxy-4-(2-propenyl) phenol, as the major compo- tering its fast growing aquaculture and trade (Chapman, Fitz-Coy, nent of clove oil (70%–90% by weight), is generally regarded as safe Thunberg & Adams 1997). In ornamental fish aquaculture, practice by FDA (Ross & Ross 2008). Lower price and its safety to both handling and also transportation of live fish especially for a long-dis- human and environment have encouraged clove oil application as an tance journey as routine activities impose considerable changes in attractive anaesthetic for fish (Mylonas, Cardinaletti, Sigelaki & Pol- fish physiology and behaviour. Such events may also result in higher zonetti-Magni 2005). rates of mortality and leave the remaining fish susceptible to various Its anaesthetic efficiency has been reported for distinct ornamen- opportunistic fungal or bacterial infections in the one hand and tal fish species including Amphilophus labiatus 9 Amphilophus trimac- ensue economic loss in the other hand. Among various solutions, ulatus (Tarkhani et al. 2016), Pangasius hypophthalmus (Hoseini et al. using anaesthetics to lower stress to fish and decrease physical 2013), Pterophyllum scalare (Mitjana, Bonastre, Insua, Falceto, Este- injury is highly recommended by experts (Johny & Inasu 2016). ban, Josa & Espinosa 2014), Pomacentrus amboinensis (Munday & Therefore, the present study was to assess the anaesthetic efficacy Wilson 1997), Danio rerio (Grush, Noakes & Moccia 2004). With no of eugenol and to establish reliable concentrations for different sizes doubt, one can consider eugenol and isoeugenol as future anaesthet- of angelfish. In the discussion, we shall present appropriate concen- ics of choice in the aquaculture industry due to their efficacy, low trations of eugenol suitable for various management purposes based price, no withdrawal period and lack of side effects on fish appetite on our results and also compare the results of this study with the (Cupp, Hartleb, Fredricks & Gaikowski 2016). It has been shown that results from previous important studies. Aqui-S vet. (iso-eugenol) was able to considerably alleviate the primary and secondary stress responses in European eel, Anguilla anguilla L. and improve animal welfare and survival during and after common aquaculture practices (Iversen, Økland, Thorstad & Finstad 2013). The anaesthetic efficacy of eugenol was the subject of sev- 2 | MATERIALS AND METHODS 2.1 | Fish and experimental conditions eral studies on various fish species such as common carp, Cyprinus A total number of 130 angelfish, P. scalare, with three different size carpio (Hikasa, Takase, Ogasawara and Ogasawara 1986), rabbitfish classes 1.0  0.5, 5.0  1.0 and 10.0  1.0 g were purchased from (Siganus lineatus) (Soto & Burhanuddin 1995), fathead minnow (Pime- a local ornamental fish farm and transferred to laboratory, and each phales promelas Rafinesque, 1820) (Palic, Herolt, Andreasen, Menzel group was separately stocked in 100 L tanks at a density of 10 g/L. & Roth 2006), Common snook (Centropomus undecimalis Bloch, Fish were fed at 1.5% of their body weight per day with a commer-  nior, Nakagome, Mello, Garcia & Amaral Ju  nior 1792) (Bernardes Ju cial diet (Biomar, Nersac, France). All tanks were continuously aer- 2013) and flower horn (Tarkhani et al. 2016). ated, and the daily water exchange rate was 80%. Water quality The literature suggests that the safely effective concentration parameters were measured every other day and all quality criteria and exposure time of eugenol substantially vary depending on fish including pH = 7.0–7.5; temperature = 25.1  1.4°C; N-nitrite = species and size (Hoseini et al. 2013; Cupp et al. 2016). To deter- 0.05  0.1 mg/L; total hardness = 140  12.7 mg/L; DO = 6.3  mine such prerequisites, various stages of anaesthesia were already 0.2 mg/L were within the optimum ranges for freshwater fish described in different fish species according to the behavioural culture. Feeding was given up 24 h before the experiment and responses of an animal including response to stimuli, opercular rate continued a day after recovery from anaesthesia. and fish equilibrium (McFarland 1959; Hikasa et al. 1986; Hoseini et al. 2013). The ideal level of sedation known as deep sedation is indicated by loss of reactivity to external stimuli, decrease in metabolic rate, but maintenance of equilibrium (McFarland 1959) and 2.2 | Anaesthetic preparation and behavioural observations during anaesthetic exposure equals to stage 2 of Anaesthesia as described by Summerfelt and Due to its lower water solubility, a stock solution of eugenol was Smith (1990). prepared by mixing eugenol (Sigma, St. Louis, MO, USA; 99% purity) At the moment, the ornamental fish market is highly competitive and ethanol (Razi, Iran, with 96% purity) with respective volumetric and also demanding with considerably large market values to the ratio of 1:2. Final working solutions containing 1.25, 2.5, 4.0, 5.5 and TARKHANI | ET AL. 7.0 mg/L eugenol were freshly prepared right before experimenta- 3 3 | RESULTS tion. Fish were individually subjected to each anaesthetic solutions (n = 7–10). Plastic 2-L containers with continuous aeration were After 24 hr, no mortalities were observed in all experimental used (Hoseini et al. 2013). Time required to get different stages of groups, and the fish were feeding well within 1 day after treat- anaesthesia was recorded according to fish behaviour (Table 1). Time ment. Two-way required getting complete equilibrium was recorded from transferring between eugenol concentration and average fish body weight with the fish to recovery container (60-L aquaria containing 40 L aerated regard to anaesthetic efficacy of eugenol on angelfish (Table 2). fresh water). Finally, fish were transferred to freshwater aquaria to The fish sequentially entered different anaesthetic stages. In all monitor potential mortality over a 24-h period (Tarkhani et al. 2016). stages, any increase in eugenol concentration led to a significant Table 1 contained behavioural responses of fish at various anaes- decrease in the induction time with a subsequent increment of thetic stages; however, more details were illustrated elsewhere the recovery time (Table 3). Any increases in fish weight simulta- (Hoseini et al. 2013). Our preliminary observations on angelfish neously resulted in a significant increment in the induction and showed that exposure to ethanol did not bring about anaesthesia or recovery time. All size classes showed all anaesthetic stages at any apparent modifications in fish behaviour implying that the con- 1.25 mg/L eugenol. Results showed that 7.50 mg/L eugenol centration of ethanol used had no effects on the fish during the effectively induced stages 3 and 4 very rapidly in comparison to experiment. other concentrations. According to the results, those fish exposed ANOVA revealed a statistically significant interaction to higher concentrations of the anaesthetic agent required longer 2.3 | Statistical analyses time to fully recover and regain their equilibrium (Table 3). Results from stepwise multiple regression analyses yielded the Levene’s test and Kolmogorov–Smirnove were used to evaluate the following regression equations representing the description of time homogeneity of variance of the dependent variables and normality elapsed to reach stage 1, 2, 3, 4 and full recovery from of data set respectively. Two-way ANOVA was used to illustrate anaesthesia; Stage 1 = 103.675–8.633 9 (concentration) + 2.025 9 whether or not there were significant differences among different (size), experimental groups. The general quadratic equation, Z = b0 + b1X Stage 3 = 126.524–10.866 9 (concentration) + 1.674 9 (size) + 0.205 9 2 = 113.788–9.472 9 (concentration) + 2.338 9 (size), Stage + b2Y + b3X2 + b4XY + b5Y2 + ɛ, applied to test for relationships (concentration9size) using polynomial regression; where Z is the response (time to reach tion) + 2.837 9 (size). As distinct sizes of angelfish showed different each anaesthetic stage), X and Y are the independent variables (e.g. responses to various concentrations of eugenol, time elapsed to fish size and eugenol concentration). With the use of a p < .001 cri- fully recover from anaesthesia for smaller fish (1 and 5 g) and the terion for Mahalanobis distance, no outliers were found (Steel, Torrie larger ones (10 g) were separately analysed and reported; full recov- & Dickey 1997; Shanock, Baran, Gentry, Pattison & Heggestad ery 2010). Model validation analysis was also conducted via cross valida- 9 (concentration9size) for smaller fish and full recovery from anaes- tion which requires that the regression model for the training sample thesia = 538.698 + 7.931 9 (concentration) for the larger ones. The replicates the pattern of the full data set (Osborne 2000). All statisti- total variance explained by the model as a whole was 94.4%, F (2, cal analyses were performed using IBM SPSS Statistics for Windows, 127) = 1075.998, p = .000 for stage 1, 94.8%, F (2, 127) = from and Stage 4 = 129.038–9.916 9 (concentra- anaesthesia = 386.364 + 4.387 9 (concentration) + 1.321 Version 20.0 (IBM Corp, Armonk, NY, USA) at the significance level 1163.695, p = .000 for stage 2, 93.5%, F (3, 126) = 606.516, of p < .05. Results were reported as Mean  SE. p = .000 for stage 3 and 92.1%, F (2, 127) = 737.774, p = .000 for stage 4. However, total variance explained by the model was 90.4%, F (2, 91) = 427.640, p = .000 and 93.6%, F (1, 34) = 495.965, p = .000 for full recovery from anaesthesia in smalT A B L E 1 Behavioural response of angelfish to eugenol according to Tarkhani et al. (2016) ler and larger fish respectively. Both independent variables and also their interactive effects at Stage Behaviour of fish least in the case of inducing stage 3 and also full recovery of smaller 0 Normal fish were significantly contributed in the model, with concentration I Relaxation and no response to stimuli: fish were calmed and did not respond to tactile touch 0.898, -0.976 and -0.864 respectively) and the product of size and recording the highest b value for stages 1, 2, 3 and 4 (-0.903, - II Imbalance swimming: fish loss their equilibrium and show imbalance swimming anaesthetic concentration for full recovery in smaller fish (0.647) and III Total loss of equilibrium: fish laid on lateral side, slightly depressed but regular opercular movement was also well fitted to induction time to stage 4 via quadratic and IV Deep anaesthesia: slow and irregular opercular movement V Death: opercular movement ceased Recovery stage Fish regained its equilibrium eugenol concentration in larger angel fish (0.967). Recovery time linear regression models in smaller (32.2%, F (2, 91) = 21.656, p = .001) and larger fish (74.5%, F (1, 34) = 99.160, p = .000) respectively (Fig. 1). In the analysis of the training samples, the relationships between the predictors and the response variables were 4 | TARKHANI ET AL. T A B L E 2 Two-way ANOVA output for anaesthetic efficacy of different concentrations of eugenol (1.25, 2.50, 4.00, 5.50 and 7.50 mg/L) on angelfish with different average body weight (1.00, 5.00 and 10.00 g) Source Dependent Variable Size Stage 1 Type III Sum of Squares 7044.688 Error Total F Sig. 3522.344 213.752 0.000 9432.090 2 4716.045 372.687 0.000 Stage3 10970.879 2 5485.440 458.475 0.000 0.000 14054.297 2 7027.148 643.330 606523.318 2 303261.659 12865.833 0.000 Stage 1 45552.808 4 11388.202 691.088 0.000 Stage 2 54406.657 4 13601.664 1074.875 0.000 Stage3 58741.261 4 14685.315 1227.403 0.000 Stage4 59902.071 4 14975.518 1370.998 0.000 Recovery 41037.252 4 10259.313 435.250 0.000 Recovery Size 9 Concentration Mean Square 2 Stage 2 Stage4 Concentration d.f. Stage 1 695.874 8 86.984 5.279 0.000 Stage 2 1234.435 8 154.304 12.194 0.000 Stage3 2829.828 8 353.729 29.565 0.000 Stage4 3432.788 8 429.099 39.284 0.000 Recovery 3670.648 8 458.831 19.466 0.000 Stage 1 1895.046 115 16.479 Stage 2 1455.231 115 12.654 Stage3 1375.922 115 11.965 Stage4 1256.154 115 10.923 Recovery 2710.675 115 23.571 Stage 1 853460.000 130 Stage 2 1041505.000 130 Stage3 1237336.000 130 Stage4 Recovery 1443671.000 130 28340291.000 130 T A B L E 3 Anaesthetic efficacy (in second) of different concentrations of eugenol (1.25, 2.50, 4.00, 5.50 and 7.50 mg/L) on angelfish with different average body weight (1.00, 5.00 and 10.00 g) Size (g) 1 5 10 Eugenol concentration (mg/L) 1.25 Stage 1 Stage 2 ef 96.500  1.204 * cd 106.000  1.238 de 120.200  0.512 gh Recovery time h 393.500  0.980a ef 394.800  1.306ab 92.900  2.030de 415.400  1.641de 125.300  0.578 78.300  2.017 4.00 73.200  1.919c 79.600  1.916d 5.50 63.200  1.381 b c 7.50 38.800  1.618a 42.600  1.529a 49.000  1.826a 55.600  1.714a 1.25 f g kl i 68.300  1.174 119.780  0.547 91.100  0.936 Stage 4 ij 2.50 103.000  1.528 85.400  1.213 Stage 3 f 84.800  2.097ef 73.300  1.325 126.440  0.603 c 94.700  0.978 79.400  1.352 135.560  0.669 c 408.100  1.952cd 434.900  1.792f 402.000  1.667bc 2.50 93.200  0.929e 101.900  0.948f 111.800  0.712h 122.100  0.781h 417.600  1.875e 4.00 80.700  0.978 d e h g 427.700  1.212f 5.50 65.570  0.612b 73.430  0.719c 88.290  0.565d 451.570  1.587g 7.50 45.250  1.719 a b 59.250  1.319 b 465.630  1.487h 1.25 112.430  0.528g 121.140  0.738g 128.000  0.617l 107.630  0.375 f 116.000  0.655 i 107.430  0.922 f 122.140  0.738 jk 98.380  0.844 ef 4.00 95.290  1.085 e 5.50 74.860  0.911cd 2.50 7.50 60.860  0.508 b 87.800  0.952 52.000  1.813 83.000  1.134de 68.430  0.481 c *Values with different superscripts in each column are significantly different at p < .05. 95.600  0.980 81.140  0.595de b 106.600  0.499 68.380  0.565 134.140  0.800i 127.500  0.707 558.500  1.376j 133.140  1.243 i 572.000  2.127k 90.140  1.262fg 98.860  1.100f cd c 76.290  0.680 547.860  2.613i h 81.860  0.829 582.000  0.690l 597.710  1.658m TARKHANI | ET AL. 5 F I G U R E 1 Relationships between times elapsed to reach anaesthetic stages 4 or recovery in smaller (a) and larger angelfish (b) anaesthetized by eugenol statistically significant; 94.09%, F (2, 97) = 767.553, p = .000 for 4 | DISCUSSION stage 1, 94.67%, F (2, 97) = 845.729, p = .000 for stage 2, 92.93%, F (3, 96) = 418.940, p = .000 for stage 3, 91.78% and F Various anaesthetics have been used in aquaculture industry with (2, 97) = 534.554, p = .000 for stage 4. The validation procedure their own specific merits and demerits. 2-Phenoxyethanol (2-PE) is for full recovery from anaesthesia also showed that the relation- selected over tricaine methane sulphonate (MS-222) due to its lower ships between the predictors and the response variables were sta- cost and the ease of use (Ortuno et al. 2002); however, its usage tistically significant; 89.7% F (2, 68) = 298.217, p = .000 and has been put under a shadow of doubt by inducing a stress response 93.4% F (1, 27) = 382.629, p = .000 for smaller and larger fish in fish (Iwama et al. 1989; Thomas & Robertson 1991; Ortuno et al. respectively. In all stages, the pattern of significance for indepen- 2002). Such increased stress response has also been reported for dent variables in training sample matched the pattern for full data deep anaesthesia induced by MS-222 in fish (Small 2003). Clove oil 2 set. The lack of shrinkage in R obtained for validation sample in as a natural commodity is readily available with competitive prices comparison to R2 of training sample indicated that the regression and also is generally considered as safe (GRAS) compound by the models would be effective in predicting time required to get each U.S. Food and Drug Administration (Summerfelt & Smith 1990). For anaesthetic stage for other experiments on the species using morphological assessments, taking biopsy samples from tissues and eugenol as the anaesthetic agent. organs and also hand stripping of gametes, where long handling 6 | TARKHANI ET AL. periods outside the water are required, the longer recovery time et al. 2013, Tarkhani et al. 2016). Similar to our results on angelfish might be an advantage for clove oil application (Rodri’guez-Gutierrez with 1 g body weight, it has also been shown that 4.0 mg/L etomi- & Esquivel-Herrera 1995). Also, clove oil may be a more appropriate date was able to anaesthetize 0.3-2 g angelfish in 78 s and fish fully anaesthetic for use in commercial aquaculture situations, where recovered in 240 s (Amend, Goven & Elliot 1982). However, in the anaesthetics may be used in large quantities by unskilled labourers present study with eugenol, angelfish required longer time to and directly released into natural water bodies (Javahery, Nekoubin recover from anaesthesia, which may attributable to the nature of & HajiMoradlu 2012). Being the major component of clove oil (70%– anaesthetic agent and its clearance rate from fish tissue. 90% by weight), Small (2003) found that there were not any increase In addition, our results showed that the induction time decreased in blood cortisol concentrations of fish anaesthetized with eugenol. and recovery time increased with any increments in eugenol concen- However, the anaesthetic is only certified by Japan aquaculture tration. In other words, there was a negative correlation between industry. However, narrower safety margin of eugenol compared recovery and induction time. In contrast, investigating the effect of with other common anaesthetic agents has limited effectiveness of eugenol and 2-phenoxyethanol on Dicentrarchus labrax and Sparus eugenol for surgical manipulations due to its quick effect on respira- aurata, Mylonas et al. (2005) found that longer exposure to anaes- tory center of fish (Sladky, Swanson, Stoskopf, Loomis & Lewbart thetic agents led to more drug absorption and subsequently length- 2001; Misawa, Kada & Yoshida 2014). ened the recovery time. However, Roubach et al. (2005) and Stehly As application of inappropriate concentrations of chemical may & Gingerich (1999) could not find noticeable relationships between harm or even kill fish, it is important to determine right anaesthetic body weight and induction or recovery time. Weyl, Kaiser & Hecht concentration for each fish species (Hoseini & Jafar Nodeh 2011). (1996) considered that such contradictory results might be attributa- Previous researchers found that the ideal times for the induction ble to interspecies differences. Moreover, with regard to the efficacy and recovery from anaesthesia were 3 and 5 min respectively (Hseu, of anaesthetic agents in aquatics animal health state and stocking Yeh, Chu & Ting 1998; Marking & Meyer 1985; Gilderhus & Marking density, pH, water temperature, salinity, dissolved oxygen and water 1987). In the present study, all concentrations were efficiently mineral content may also come into effect (Josa, Espinosa, Cruz, Gil, induced anaesthesia within 3 min; however, the time required by Falceto & Lozano 1992; Weyl et al. 1996; Stoskopf & Posner 2008). fish to regain its equilibrium varied from 393.500  0.980 to Nonetheless, it is conceivable that short time exposure to higher 597.710  1.658 s (i.e. 6.5–10 min). It is worth mentioning that concentrations of anaesthetic agents may also lengthen the time according to our preliminary observations on angelfish, time to reach required to regain the normal behaviour. Additionally, Weyl et al. full anaesthesia considerably extended at lower eugenol concentra- (1996) declared that in comparison to exposure time, anaesthetic tions. One must know that defining a suitable time for full anaesthe- concentration is more influential on the recovery time, which was sia and recovery may vary depending on fish species and also reconfirmed by considerably higher b values for eugenol concentra- purpose of inducing anaesthesia (external sampling, fin biopsies, gill tion in the present study. With such speculation, it became apparent biopsies, surgical procedures) require different selection criteria that faster absorption of anaesthetic agents in smaller fish may lead (Stoskopf & Posner 2008). to quick anaesthesia due to relatively larger gill or body surface to Our results also showed that 5.5 mg/L eugenol was the best body volume, and subsequently fish may uptake lower quantity of desired dose for induction of anaesthesia in all three size classes of the chemical via gills via gill epithelia. As a result, one might contem- P. scalare. According to table 1, induction and recovery time at plate that smaller fish would recover from anaesthesia more quickly 5.5 mg/L eugenol were less than those other concentrations, and at than larger ones, as the rate of anaesthetic clearance from fish body 7.5 mg/L, the recovery time was near critical threshold (> 10 min, could be a function of concentration gradient and gill surface area to Marking & Meyer 1985). Various studied sizes of angelfish (1, 5 and body volume ratio (Javahery et al. 2012; Tarkhani et al. 2016), and 10 g) exposed to 5.5 mg/L reached the appropriate stage for han- fish would recover as soon as the concentration of anaesthetic agent dling (stage 3) in 73.3, 81.1 and 90.1 s and for surgery and blood drops to a certain level in their tissues/bodies (Weyl et al. 1996; sampling (stage 4) in 84.6, 95.5 and 106.2 s respectively. Similar to Javahery et al. 2012). Consistent with our results, previous studies our results, when Colossoma macropomum (Tambaqui) juveniles were on Gadus morhua (Zahl, Kiessling, Samuelsen & Hansen 2009), D. sar- exposed to the ideal concentration of clove oil (65 mg/L), they gus and Diplodus puntazzo (Tsantilas, Galatos, Athanassopoulou, required 88.8 s to reach the surgical stage, while time was increased Prassinos & Kousoulaki 2006) and Ictalurus punctatus (Small 2003) to 226.2 s for subadults (Roubach et al. 2005). At ideal concentra- showed that the recovery time was related to body weight or induc- tion of clove oil, Ictalurus punctatus (100 mg/L) required 310.2 s tion time. (Waterstrat 1999), and Salmo salar (30-100 mg/L) required 486 s Regression analyses revealed that fish with different body size (Iversen et al. 2003) to reach the surgical stage. However, the best may differently response to anaesthetic agent, to the extent that for clove oil concentrations for the induction of anaesthesia and han- smaller fish (1 and 5 g) time required to recover from anaesthesia dling of fish were 50-100 mg/L and 10-30 mg/L respectively (Javah- was mainly affected by the interaction of fish body size and eugenol ery, Nekoubin & HajiMoradlu 2012). The findings agree well with concentration, while in larger fish (10 g), it was the concentration of the existing literature on the efficacy of eugenol on different species eugenol which predominantly affected recovery time. Unfortunately, as an anaesthetic (Cunha & Rosa 2006; Park et al. 2008; Hoseini we did not include larger fish individuals (> 10 g) in our study to TARKHANI ET AL. fully picture the fish behaviour to eugenol in larger body sizes. Similarly, it has been shown that fish weight and length may also affect the efficacy of anaesthetic and sedative agents (Ross & Ross 2008). Therefore, it is important to examine the dynamics of eugenol absorption, excretion and/or metabolism in angelfish during the induction time and recovery period to thoroughly characterize the extent, quality and magnitude of induction time, anaesthetic concentration and fish size on recovery time and the anaesthetic efficacy of eugenol. Based on the literature, anaesthesia with eugenol can cause hypoxaemia (75% decrease in arterial oxygen) with subsequent depression to the CNS (Stoskopf & Posner 2008). The situation may lead to medullary collapse and death (i. e., euthanasia) (Kegley, Conlisk & Moses 2010). For instance, mortality due to using high anaesthetic concentrations has been reported by Tsantilas, Galatos, Athanassopoulou, Prassinos & Kousoulaki (2006) and Waterstrat (1999). It is possible that eugenol covers the body and gill surface of fish causing suffocation if used extensively (Sladky et al. 2001). In the present study, no mortality was observed; however, as 7.5 mg/L eugenol may cause angelfish to collapse (with recovery time of near critical threshold of 10 min) and is not suggested to apply to the species. Similarly, no mortality with eugenol was reported on other species such as rainbow trout (Keene et al. 1998), channel catfish (Waterstrat 1999) and kelp grouper (Park et al. 2008). In conclusion, our results suggested eugenol as an effective and safe anaesthetic agent for different sizes of angelfish. 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