International Journal of Zoology

International Journal of Zoology / 2014 / Article

Research Article | Open Access

Volume 2014 |Article ID 315029 |

Anna Mercy Thoranam Varkey, Sajan Sajeevan, "Efficacy of 2-Phenoxyethanol as an Anaesthetic for Adult Redline Torpedo Fish, Sahyadria denisonii (Day 1865)", International Journal of Zoology, vol. 2014, Article ID 315029, 4 pages, 2014.

Efficacy of 2-Phenoxyethanol as an Anaesthetic for Adult Redline Torpedo Fish, Sahyadria denisonii (Day 1865)

Academic Editor: Thomas Iliffe
Received20 Aug 2014
Revised17 Oct 2014
Accepted12 Nov 2014
Published08 Dec 2014


Efficacy of 2-phenoxyethanol for redline torpedo fish exposed to five concentrations (200, 300, 400, 500, and 600 μlL−1) was evaluated. The time periods necessary for each characteristic stage of induction and recovery were recorded. Results indicated that the induction time of the fish exposed to five anaesthetic concentrations significantly decreased with increasing concentration but recovery time was independent of concentration. Concentration of 500 μlL−1 (induction time: 173 ± 7 and recovery time: 129 ± 41 seconds) was determined as the minimum effective concentration that induces anaesthesia in less than 3 minutes.

1. Introduction

Anaesthesia is an effective method to minimize stress or physical damage caused during handling, transport, grading, weighing, and induction of spawning [1]. L. G. Ross and B. Ross [2] defined anaesthesia as a state caused by an applied external agent resulting in a loss of sensation through depression of the nervous system. The use of anaesthetics is primarily for the purpose of holding fish immobile while the animal is being handled for experiments. When choosing an anaesthetic, a number of considerations should be taken into account such as its efficacy, cost, availability, ease of use, and side effects on fish, humans, and the environment [3, 4]. Presently, there are many anaesthetics available for aquatic animals, including MS-222, benzocaine, quinaldine, chlorobutanol, 2-phenoxyethanol, eugenol, and metomidate [3]. 2-Phenoxyethanol is relatively inexpensive and remains viable in long-term exposure [5] and is also easily available compared to other anaesthetics. Deacon et al. [6] recommended 2-phenoxyethanol as a highly suitable anaesthetic for repeatedly exposed fishes. Over the last three decades, studies have evaluated the anaesthetic efficacy of 2-phenoxyethanol in various fish species [714]. According to Topic Popovic et al. [15], it is necessary to have a better understanding of safety margins, induction, and recovery times for many fish species in order to achieve optimal application.

Sahyadria denisonii (or Puntius denisonii), an ornamental fish popularly known as redline torpedo fish or Miss Kerala, is endemic to the rivers flowing through the Western Ghats hotspots of India. The species is much sought after in the international ornamental fish trade and is listed as “Endangered” in the IUCN Red List of Threatened Species [16]. Captive breeding is considered to be one of the solutions for ensuring sustainability and conserving wild populations of this endangered species [17]. In fisheries, various approaches have been applied to relieve fishes from regular stressful conditions during captive breeding, and anaesthesia has been used as an applicable way to prevent or reduce stress. Efforts to develop captive breeding technology for S. denisonii have shown that the species is very difficult to handle for artificial propagation [17, 18]. Therefore, attempts were made to use anaesthetics to handle the fish during captive breeding. Using clove oil, handling stress was minimized and S. denisonii was bred successfully under hatchery conditions [17, 19]. No literature is available regarding the use of 2-phenoxyethanol to anaesthetise S. denisonii. In the present study, we determined the effective concentration of 2-phenoxyethanol that can be used as an anaesthetic for S. denisonii during artificial propagation.

2. Materials and Methods

The experiments were conducted using adult individuals of S. denisonii with an average weight of 16.5 ± 3.5 gm (mean ± SD; ). Prior to starting the experiment, fishes were acclimatised in cement tanks (2000 litres) for a period of two weeks. Fishes were fed with a formulated diet consisting of crude protein (38%), crude fat (4.0%), crude fibre (3.0%), ash (16%), and moisture (11%) twice a day (at 09:00 and 17:00). Feeding was suspended 24 hours before the start of the experiment. Water quality parameters were maintained within a narrow range of values, temperature (27.0 ± 0.5°C), pH (7.0 ± 0.3), dissolved oxygen (6.50 ± 0.5 mgL−1), alkalinity (65.0 ± 6.0 mgL−1), hardness (70.0 ± 4.0 mgL−1), and ammonia (<0.02 mgL−1) and were checked by using a thermometer for temperature; colour comparator solutions (Nice Chemicals, India) for pH and ammonia; titrimetric method for alkalinity and hardness; and Winkler method for dissolved oxygen using standard procedures [20]. 2-Phenoxyethanol (ethylene glycol monophenyl ether, MERCK-Schuchardt) was used as anaesthetic agent. Dosages of anaesthesia for various teleosts [12] were used as base information and concentrations of 2-phenoxyethanol of 200, 300, 400, 500, and 600 μlL−1 were selected to induce anaesthesia in S. denisonii. Both induction and recovery treatment water were aerated throughout the experimental procedure.

The induction time was considered to be the time period from when an experimental fish is placed in the anaesthetic tank until the time it does not respond to external stimuli. The recovery time is the time period from when an anaesthetized fish is placed in a recovery tank until it recovers from anaesthesia with full equilibrium motion. General fish behavior was assessed for each fish throughout induction and recovery. For practical purposes, four stages of induction and recovery were considered in S. denisonii (Table 1). An induction time of 180 seconds or less with complete recovery within 300 seconds suggested by Marking and Meyer [3] and Trzebiatowski et al. [21] was used to establish anaesthesia induction and recovery stages presented in Table 1. According to L. G. Ross and B. Ross [2], deep anaesthesia (stage IV) is normally used for performing biometric procedures.


Induction stage
 IStart of anaesthetic effect, slow swimming, and physiological position
 IIPartial loss of body balance, body turning to one side, and decreased locomotor activity
 IIIComplete loss of body balance, flank position at the bottom of tank, and reaction only to very strong tactile pressure
 IVNo response to external touch or stimuli and total loss of equilibrium
Recovery stage
 IStart of fin movement and fish still lays on the bottom of the tank
 IIRegular breathing, nonmoving tilting on flank position, and irregular fin movement
 IIIPhysiological position and increased locomotor activity
 IVAbility to swim normally and regular operculum rate

The experimental induction tank (1 × 1 × 1 foot) was filled with water from a similar source to that of the experimental fish holding tanks. Each concentration of 2-phenoxyethanol was initially mixed with a little water in a reagent bottle (50 mL) and then stirred to disperse the chemical to form small droplets before addition to the anaesthetic induction tanks [13]. Each fish was netted carefully with minimum stress and placed in the induction tank. When fish reached stage IV of anaesthesia, it was immediately netted and transferred to recovery tanks, to monitor the recovery stages. The induction and recovery time for each concentration were measured by using an electronic stopwatch (Casio, India). Experiments were repeated eight times to verify the findings with each concentration. The recovered fishes were transferred into the observation tanks (1000 L) for seven days to assess the postrecovery mortality [13]. During the posttreatment period, 50% of the tank water was exchangeddaily and the fishes were fed twice a day to apparent satiation with pelleted feed. Mean induction time and recovery time of anaesthesia were compared using regression analysis at 5% level of significance (), followed by Tukey’s honestly significant difference (HSD) multiple comparison procedure [23]. The results were processed and analysed with SPSS (Windows, Version 15.0).

3. Results and Discussion

In the study, induction and recovery times as well as statistical differences for each anaesthetic concentration in redline torpedo fish are shown in Table 2. The results demonstrate that the induction time of anaesthesia in S. denisonii was decreased with increasing concentrations of 2-phenoxyethanol (). Comparable results were observed in Carassius auratus [9], Tinca tinca [24], Diplodus sargus and Diplodus puntazzo [25], Solea senegalensis [12], Carasobarbus luteus [26], and Hippocampus kuda [13]. The responses to the same anaesthetic can vary considerably among different species, so the characterization of the effective dose of the different anaesthetics in a determined species is an advisable practice [14]. The water quality parameters observed in the present experiment were within the range of our previous studies [17, 18].

2-Phenoxyethanol (μlL-1)

Stages of induction
 I199 ± 11c35 ± 6b26 ± 3a25 ± 4a24 ± 4a
 II92 ± 12d59 ± 16c49 ± 13b39 ± 5a
 III168 ± 65c120 ± 51b85 ± 23a
 IV331 ± 15c173 ± 7b143 ± 24a
Stages of recovery
 I15 ± 4c26 ± 6b33 ± 7a
 II43 ± 7b62 ± 11a64 ± 10a
 III76 ± 9c89 ± 9b100 ± 8a
 IV95 ± 10b141 ± 12a145 ± 14a

Numbers in the same row with the same superscript letter are not significantly different ().

The ideal concentration must be the lowest concentration which enables a transition to anaesthesia in 180 seconds and a full recovery in 300 seconds [3, 10]. In our experiments, minimum effective concentration was observed at 500 μlL−1 of 2-phenoxyethanol (induction and recovery occurred at 173 ± 7 and 129 ± 41 seconds, resp.) and therefore this dose was considered as the minimum effective concentration for handling of adult S. denisonii. These results are consistent with previous studies using 2-phenoxyethanol in teleost fishes [7, 1114, 27]. The effective anaesthetic concentration of 2-phenoxyethanol in a number of fish species has been reported ranging between 200 and 600 μlL−1 [7, 10, 11, 13, 27]. At 500 μlL−1 of 2-phenoxyethanol, the time to reach stage IV of induction (173 ± 7 seconds) was significantly differentfrom all other concentrations at 331 ± 15 seconds in 400 μlL−1 and 143 ± 24 seconds in 600 μlL−1 (Table 2). Tukey’s HSD test revealed that the induction time (stage IV) at 500 μlL−1 was significantly different from that at 400 μlL−1 (331 ± 15 seconds, ) and 600 μlL−1 (143 ± 24 seconds, ). 2-Phenoxyethanol at 200 μlL−1 resulted in sedation (stage I) while at 300 μlL−1 it resulted in sedation (stage II) only and concentration from 400 μlL−1 resulted in progressive anaesthesia. Deep anaesthesia (stage IV) is the stage normally used for performing biometric procedures, induced breeding, and handling procedures [2, 28]; therefore, in S. denisonii, the minimum effective concentration of 2-phenoxyethanol which meets the requirements for handling during hatchery management was 500 μlL−1.

The recovery time was directly proportional to increasing doses of 2-phenoxyethanol (). The longest time for recovery was observed at 600 μlL−1 (145 ± 24 seconds), while the shortest time occurred at 400 μlL−1 (95 ± 3 seconds) (Table 2). Similar results have been reported in Oncorhynchus nerka [29], Rachycentron canadum [30], Hippocampus kuda [13], Dawkinsia filamentosa [31], and Puntius denisonii [17, 18]. However, experiments by Mylonas et al. [4] documented recovery times decreasing with increase in anaesthetic concentration of clove oil and 2-phenoxyethanol in Dicentrarchus labrax and Sparus aurata. Such differences in the respective recovery times might be related to species, size, physiological status, and environmental conditions [2] as well as temperature, pH, salinity, and oxygen and mineral content of the water [9]. The dynamics of recovery times in anaesthetized fish seems to be more complex; for example, in Solea senegalensis, recovery times were weight-dependent for metomidate and dose-dependent for clove oil and MS-222 [12].

According to Pawar et al. [13] and Bambang [32], the recovered fishes should be observed for any abnormal behavior or mortality in posttreatment tanks for seven days. In the present study, recovered S. denisonii monitored in posttreatment tanks for seven days exhibited normal feeding and physiological behaviour, without any mortality or abnormal behaviour. In conclusion, the results indicated that 2-phenoxyethanol can be used as an efficient anaesthetic for the handling of S. denisonii and the lowest effective concentration to induce anaesthesia is 500 μlL−1. This lowest effective concentration induced S. denisonii through all stages of anaesthesia, without causing mortality, and may be helpful in developing techniques for transportation, captive breeding, and other ex situ conservation plans for this endemic and endangered barb.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


The authors wish to thank the College of Fisheries, Kerala University of Fisheries and Ocean Studies, Panangad, Ernakulam, India, for providing the necessary facilities to carry out this work. The second author gratefully acknowledges the Government of Kerala, India, for a scholarship during his doctoral study (2010–2013). The authors also thank Hatchery manager (RGCA), MPEDA, Tamil Nadu, for his help in providing anaesthetics used for this study. Thanks are due to anonymous reviewers for their criticisms which improved the quality of the paper.


  1. G. L. Ross and B. Ross, Anaesthetic and Sedative Techniques for Aquatic Animals, Blackwell Scientific, Oxford, UK, 3rd edition, 2008.
  2. L. G. Ross and B. Ross, Anesthetic and Sedative Techniques for Aquatic Animals, vol. 58, Institute of Aquaculture, University of Stirling, 1999.
  3. L. L. Marking and F. P. Meyer, “A better fish anaesthetics needed in fisheries,” Fisheries, vol. l0, no. 6, pp. 2–5, 1985. View at: Google Scholar
  4. C. C. Mylonas, G. Cardinaletti, I. Sigelaki, and A. Polzonetti-Magni, “Comparative efficacy of clove oil and 2-phenoxyethanol as anesthetics in the aquaculture of European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) at different temperatures,” Aquaculture, vol. 246, no. 1–4, pp. 467–481, 2005. View at: Publisher Site | Google Scholar
  5. H. Kaiser and N. Vine, “The effect of 2-Phenoxyethanol and transport packing density on the post-transport survival rate and metabolic activity in the goldfish (Carassius auratus),” Aquarium Sciences and Conservation, vol. 2, no. 1, pp. 1–7, 1998. View at: Publisher Site | Google Scholar
  6. N. Deacon, H. White, and T. Hecht, “Isolation of the effective concentration of 2-phenoxyethanol for anaesthesia in the spotted grunter, Pomadasys commersonnii, and its effect on growth,” Aquarium Sciences and Conservation, vol. 1, no. 1, pp. 19–27, 1997. View at: Publisher Site | Google Scholar
  7. P. A. Gilderhus and L. L. Marking, “Comparative efficacy of 16 anesthetic chemicals on rainbow trout,” North American Journal of Fisheries Management, vol. 7, pp. 288–292, 1987. View at: Publisher Site | Google Scholar
  8. N. S. Mattson and T. H. Riple, “Metomidate, a better anesthetic for cod (Gadus morhua) in comparison with benzocaine, MS-222, chlorobutanol, and phenoxyethanol,” Aquaculture, vol. 83, no. 1-2, pp. 89–94, 1989. View at: Publisher Site | Google Scholar
  9. O. Weyl, H. Kaiser, and T. Hecht, “On the efficacy and mode of action of 2-phenoxyethanol as an anaesthetic for goldfish, Carassius auratus (L.), at different temperatures and concentrations,” Aquaculture Research, vol. 27, no. 10, pp. 757–764, 1996. View at: Publisher Site | Google Scholar
  10. J. R. Hseu, S. L. Yeh, Y. T. Chu, and Y. Y. Ting, “Comparison of efficacy of five anaesthetic gold lined sea bream, Sparus sarba,” Acta Zoologica et Taiwanica, vol. 9, pp. 35–41, 1998. View at: Google Scholar
  11. R. Kaminski, L. Myszkowski, and J. Wolnicki, “The secrets of 2-phenoxyethanol. IV. Induction and recovery times of early life stages of cyprinids,” Komun. Ryb., vol. 2, pp. 10–11, 2000. View at: Google Scholar
  12. R. A. Weber, J. B. Peleteiro, L. O. G. Martín, and M. Aldegunde, “The efficacy of 2-phenoxyethanol, metomidate, clove oil and MS-222 as anaesthetic agents in the Senegalese sole (Solea senegalensis Kaup 1858),” Aquaculture, vol. 288, no. 1-2, pp. 147–150, 2009. View at: Publisher Site | Google Scholar
  13. H. B. Pawar, S. V. Sanaye, R. A. Sreepada, V. Harish, U. Suryavanshi, and Z. A. Ansari, “Comparative efficacy of four anaesthetic agents in the yellow seahorse, Hippocampus kuda (Bleeker, 1852),” Aquaculture, vol. 311, no. 1–4, pp. 155–161, 2011. View at: Publisher Site | Google Scholar
  14. S. Sajan, V. R. Sreenath, T. V. A. Mercy, and S. Syama, “The efficacy of 2- phenoxyethanol as anaesthetic for juvenile Pearl spot, Etroplus suratensis (Bloch),” Asian Fisheries Science, vol. 26, pp. 178–184, 2013. View at: Google Scholar
  15. N. Topic Popovic, I. Strunjak-Perovic, R. Coz-Rakovac et al., “Tricaine methane-sulfonate (MS-222) application in fish anaesthesia,” Journal of Applied Ichthyology, vol. 28, no. 4, pp. 553–564, 2012. View at: Publisher Site | Google Scholar
  16. A. Ali, N. Dahanukar, and R. Raghavan, “Puntius denisonii. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.1,” View at: Google Scholar
  17. S. Sajan, V. Malika, and T. V. Anna Mercy, “Use of an eco-friendly anaesthetic in the handling of Puntius denisonii (Day, 1865): an endemic ornamental barb of the Western Ghats of India,” Indian Journal of Fisheries, vol. 59, no. 3, pp. 131–135, 2012. View at: Google Scholar
  18. T. V. A. Mercy, V. Malika, and S. Sajan, “Use of tricaine methanesulfonate (MS 222) to induce anaesthesia in Puntius denisonii (Day, 1865) (Teleostei: Cypriniformes: Cyprinidae)—an endangered barb of the Western Ghats Hotspot, India,” Journal of Threatened Taxa, vol. 5, pp. 4414–4419, 2013. View at: Google Scholar
  19. T. V. A. Mercy, V. Malika, and S. Sajan, “Breakthrough in breeding of Puntius denisonii,” Infofish International, vol. 3, no. 4, pp. 14–17, 2010. View at: Google Scholar
  20. APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington, DC, USA, 18th edition, 1992.
  21. R. Trzebiatowski, K. Stepanowska, A. K. Siwicki, and K. Kazun, “The observations of Propiscin efficiency for total anaesthetisation of Wels (Silurus glanis),” Komunikaty Rybackie, vol. 1, pp. 14–18, 1996. View at: Google Scholar
  22. R. C. Summerfelt and L. S. Smith, “Anesthesia, surgery, and related techniques,” in Methods for Fish Biology, C. B. Schreck and P. B. Moyle, Eds., pp. 213–272, American Fisheries Society, Bethesda, Md, USA, 1990. View at: Google Scholar
  23. J. H. Zar, Bio-Statistical Analysis, Prentice Hall, New Jersey, NJ, USA, 4th edition, 1999.
  24. J. Hamackova, A. Lepicova, A. Kozak, Z. Stupka, J. Kouril, and P. Lepic, “The efficacy of various anaesthetics in tench (Tinca tinca L.) related to water temperature,” Veterinarni Medicina, vol. 49, no. 12, pp. 467–472, 2004. View at: Google Scholar
  25. H. Tsantilas, A. D. Galatos, F. Athanassopoulou, N. N. Prassinos, and K. Kousoulaki, “Efficacy of 2-phenoxyethanol as an anaesthetic for two size classes of white sea bream, Diplodus sargus L., and sharp snout sea bream, Diplodus puntazzo C,” Aquaculture, vol. 253, no. 1–4, pp. 64–70, 2006. View at: Publisher Site | Google Scholar
  26. K. Gokcek and F. Ogretmen, “Comparative efficacy of three anesthetic agents in Himri Barbel, Carasobarbus luteus (Heckel, 1843) under controlled conditions,” Journal of Animal and Veterinary Advances, vol. 10, no. 25, pp. 3350–3355, 2011. View at: Publisher Site | Google Scholar
  27. L. A. K. Inoue, C. S. Neto, and M. Moraes, “Standardization of 2-phenoxyethanol as anesthetic for juvenile Brycon cephalus (Gunther, 1869): the use in field procedures,” Clinical Rural, vol. 34, no. 2, pp. 563–565, 2004. View at: Publisher Site | Google Scholar
  28. J. J. J. Bernardes, F. K. Nakagome, F. L. de Mello, S. Garcia, and A. J. Hilton, “Eugenol as an anaesthetic for juvenile common snook,” Pesquisa Agropecuária Brasileira, vol. 48, no. 8, pp. 1140–1144, 2013. View at: Google Scholar
  29. C. A. Woody, J. Nelson, and K. Ramstad, “Clove oil as an anaesthetic for adult sockeye salmon: field trials,” Journal of Fish Biology, vol. 60, no. 2, pp. 340–347, 2002. View at: Publisher Site | Google Scholar
  30. M. Gullian and J. Villanueva, “Efficacy of tricaine methanesulphonate and clove oil as anaesthetics for juvenile cobia Rachycentron canadum,” Aquaculture Research, vol. 40, no. 7, pp. 852–860, 2009. View at: Publisher Site | Google Scholar
  31. P. K. Pramod, A. Ramachandran, T. P. Sajeevan, S. Thampy, and S. S. Pai, “Comparative efficacy of MS-222 and benzocaine as anaesthetics under simulated transport conditions of a tropical ornamental fish Puntius filamentosus (Valenciennes),” Aquaculture Research, vol. 41, no. 2, pp. 309–314, 2010. View at: Publisher Site | Google Scholar
  32. H. Bambang, Application of clove oil as anaesthetic for sae bass (Lates calcarifer, Bloch) [Msc. Thesis], Department of Aquaculture, Kasetsart University, 2003.

Copyright © 2014 Anna Mercy Thoranam Varkey and Sajan Sajeevan. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly as possible. We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. Review articles are excluded from this waiver policy. Sign up here as a reviewer to help fast-track new submissions.