ISSN 1678-2305 online version
Scientific Article
ON DOCTOR FISH, Garra rufa (HECKEL, 1843)
This study aimed to determine the anesthetic efficacy of clove oil and 2-phenoxyethanol on doctor fish
Baki Aydın1
(Garra rufa) at two different water temperatures. Experimental fish (1.2 ± 0.2 g mean weight) were
subjected to 25, 50, 75 and 100 μL L-1 clove oil and 100, 200, 300, 400 and 500 μL L-1 2-phenoxyethanol
Süleyman Akhan1
concentrations at water temperature of 15 and 25 °C, and the induction and recovery times were
Erkan Gümüş1
investigated. Results showed that induction and recovery times in doctor fish were significantly
Mehmet Özbaş1
affected by clove oil and 2-phenoxyethanol concentrations as well as water temperature.
The interaction of anesthetic concentration and water temperature on all induction stage time
was significant in clove oil. Between the anesthetic concentration and temperature interaction was
significant for recovery times in both anesthetic agents. The induction time decreased significantly
with increasing concentration of both anesthetic agents at water temperature of 15 and 25 °C.
1Akdeniz University, Faculty of Fisheries, Department
The lowest effective concentrations that produced induction within 3 min and recovery within
of Aquaculture, 07058, Antalya, Turkey. E-mail:
5 min were 50-75 μL L-1 of clove oil and 300 μL L-1 of 2-phenoxyethanol in both 15 and 25 °C (corresponding author)
respectively. The results also indicated that clove oil was effective at 4-fold lower concentrations
than 2-phenoxyethanol, but the recovery time was longer than 2-phenoxyethanol. These results
suggest that clove oil and 2-phenoxyethanol were effective anesthetics and could be used as
anesthetic agents in doctor fish.
Key words: anesthetic agent; anesthesia; induction time; recovery time; essential oil; Eugenia
Received: February 15, 2019
Approved: July 30, 2019
DOUTOR, Garra rufa (HECKEL, 1843)
Este estudo teve como objetivo determinar a eficácia anestésica do óleo de cravo e do
2-fenoxietanol em peixes medicinais (Garra rufa) em duas diferentes temperaturas da água.
Os peixes (1,2 ± 0,2 g de peso médio) foram expostos a 25, 50, 75 e 100 μL L-1 de óleo de cravo e
100, 200, 300, 400 e 500 μL L-1 de 2-fenoxietanol a 15 e 25 ° C. temperatura da água e os tempos
de indução e recuperação foram investigados. Os resultados mostraram que os tempos de indução
e recuperação nos peixes medicinais foram significativamente afetados pelas concentrações de
óleo de cravo e 2-fenoxietanol, bem como pela temperatura da água. A interação de concentração
e temperatura da água em todos os tempos de estágio de indução foi significativa no óleo de
cravo. Concentração de efeito interativa significativa e temperatura no tempo de recuperação
foram encontradas para cada agente anestésico. O tempo de indução diminuiu significativamente
com o aumento da concentração de ambos os agentes anestésicos a 15 e 25 ° C da temperatura
da água. As menores concentrações efetivas que produziram indução dentro de 3 min e
recuperação dentro de 5 min foram 50-75 μL L-1 de óleo de cravo e 300 μL L-1 de 2-fenoxietanol
em 15 e 25 ° C respectivamente para peixes medicinais. Os resultados também indicaram que o
óleo de cravo-da-índia era eficaz em concentrações 4 vezes menores do que o 2-fenoxietanol, mas
a recuperação foi maior do que o 2-fenoxietanol. Estes resultados sugerem que o óleo de cravo e
o 2-fenoxietanol eram anestésicos eficazes e poderiam ser usados como agentes anestésicos em
peixes medicinais.
Palavras-chave: anestésico; anesthesia; tempo de indução; tempo de recuperação; óleo essencial;
Eugenia caryophyllus.
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
oil, also used as a natural anesthetic drug, does not require any
withdrawal period in contrast to some anesthetics like MS-222,
Garra rufa (Doctor fish) is a subtropical freshwater fish species
and it also has been shown to be safe for humans (Javahery et al.,
belonging to the Cyprinidae and they prefer between 15 and 28 °C
2012). These two anesthetic agents have been evaluated in
water temperature under natural conditions (Baensch and Riehl,
various fish species such as Sparus aurata and Oncorhynchus
1991). In the last decade, G. rufa is getting more popular and
mykiss (Tort et al., 2002), Dicentrarchus labrax and S. aurata
commonly used for fish SPA and fish pedicure in the worldwide.
(Mylonas et al., 2005), Solea senegalensis (Weber et al., 2009),
These species have been used in ichthyotherapy for alternative
Pterophyllum scalare (Mitjana et al., 2014), Argyrosomus regius
treatment of healing of skin diseases such as psoriasis and eczema
(Cárdenas et al., 2016), Acipenser persicus (Adel et al., 2016),
(Ozcelik et al., 2000; Yedier et al., 2016) so these fish are called
Silurus glanis (Gökçek et al., 2016), Amphiprion ocellaris and
“doctor fish”. Also, doctor fish has been used in aquarium fish
Xiphophorus helleri (Hekimoğlu et al., 2017), Poecilia reticulata
sector due to its feeding strategy which cleans the aquarium
(Mitjana et al., 2018).
(Vazirzadeh et al., 2014). The demand for this fish is increasing day
The effective concentrations of anesthetics are depending on
by day in both health tourism and aquaculture sector. The increase
the fish species and anesthetic agent (Zahl et al., 2009; Skår et al.,
in demand for doctor fish subsequently increases the pressure
2017). Temperature, pH, age, size, sex, and interactions among
on natural fish stocks. Doctor fish culture is very important for
these factors also affect the efficacy of anesthetics in fish (Ross and
conservation of natural stocks in terms of sustainable tourism
Ross, 2008; Zahl et al., 2009; Mitjana et al., 2018). It is known that
and aquaculture. Furthermore, culture of this fish has become
the responses of fish to anesthetics can considerably vary between
a significant economic gain worldwide. It is emphasized that
different water temperatures (Akbulut et al., 2012; Santos et al.,
during the aquaculture activities, the use of anesthetic agents
2015; Skår et al., 2017). For this reason, it is very important to
is required to maximize fish welfare during handling process
determine effective anesthetic concentrations for each fish species
(Barata et al., 2016).
at different water temperatures. To the best of our knowledge,
Anesthetic agents, both synthetic and plant originated are used
there is no study on the effects of anesthetic agents on doctor fish.
in aquaculture procedures to minimize fish activity and to avoid
Therefore, the present study aimed to investigate the anesthetic
stress and physical damages caused by handling (Priborsky and
effects of clove oil and 2-phenoxyethanol in doctor fish, and
Velisek, 2018). A good anesthetic agent for fish should induce
determine their effective anesthetic concentrations. In addition, the
anesthesia even at low concentrations in less than 3 min and
effects of interaction between anesthetic concentration and water
allow recovery within 5 min, should also be cheap and easy to
temperature on the efficacy of the anesthetics were investigated.
use (Marking and Meyer, 1985; Kizak et al., 2018). The major
synthetic anesthetics used in aquaculture are 2-phenoxyethanol
(Priborsky and Velisek, 2018), tricaine methanesulphonate (MS-222)
and metomidate (Weber et al., 2009), benzocaine (Gökçek et al.,
2016), etomidate (Rożyński et al., 2018), propofol and quinaldine
Anesthetic agents
sulphate (Priborsky and Velisek, 2018), and ketamine hydrochloride
Clove flower (Eugenia caryophyllus) essential oil (99%
(Adel et al., 2016). Some plant originated essential oils such as basil
purity, Talya Bitkisel Urünler Ind. Co. Ltd., Antalya, Turkey)
and lemongrass (Limma-Netto et al., 2016), camphor (Pedrazzani
and 2-phenoxyethanol (ethylene glycol monophenyl ether,
and Neto, 2016), spearmint and lavender (Metin et al., 2015),
Sigma-Aldrich Inc.) were used as anesthetic agents. According
Myrcia sylvatica and Curcuma longa (Saccol et al., 2017), Aloysia
to producer declaration, clove essential oil consists of 80.56%
triphylla (Batista et al., 2018), Lippia alba (Souza et al., 2018)
eugenol, 9.77% eugenyl acetate, 7.26% β-caryophyllene and
rosewood (Kizak et al., 2018), geranium (Can et al., 2018), and
2.41% other minor constituents.
clove (Javahery et al., 2012; Cunha et al., 2015; Fujimoto et al.,
2018; Mitjana et al., 2018) have recently been studied as potential
anesthetic agents in aquaculture.
Fish and experimental conditions
Clove oil as most popular plant originated essential oil as an
After all experimental protocols’ approval of the Akdeniz University
anesthetic agent is obtained by the distillation of the leaves, stems,
Animal Experiments Local Ethics Committee (Date: 24.01.2018,
and flowers of Eugenia aromatica or Eugenia caryophylata trees and
Decision no: 16), the experiment was carried out in April-June
its active ingredient is eugenol at concentrations of approximately
2018 at the Experimental Fish Unit of Fisheries Faculty, Akdeniz
70-90% by volume (Mylonas et al., 2005; Ross and Ross, 2008;
University (Antalya, Turkey). Prior to the experiments, a total of
Javahery et al., 2012; Mitjana et al., 2014). Another most widely
200 doctor fish (Garra rufa Heckel, 1843) (1.29 ± 0.24 g mean
used anesthetic in aquaculture is 2-phenoxyethanol, which is
body weight) were randomly divided into 2 groups (100 fish each
an aromatic liquid and colorless, and reasonably water-soluble
group) and put into 2 circular fiberglass tanks (200 L) equipped
chemical (Hekimoğlu et al., 2017; Mitjana et al., 2018). Clove
with continuous aeration and external filter. Two different water
oil and 2-phenoxyethanol are increasingly used in aquaculture
temperatures (15 and 25 °C) were applied with the following
operations due to its low cost, availability, efficacy and easy
water quality parameters: pH 7.40 ± 0.11; dissolved oxygen
preparation features in most fish species (Ghanawi et al., 2013;
8.87 ± 0.51 mg L-1; and total ammonia 0.99 ± 0.22 mg L-1. Water
Santos et al., 2015; Adel et al., 2016; Mitjana et al., 2018). Clove
temperature of recirculating tank systems were adjusted to 15 and
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
25 °C with chiller-heater devices (2500 kcal hour-1, Akuakare
Statistical analysis
Products, Mugla, Turkey). Ten percent of water in the tanks was
At first, normality of the data was assessed using a Shapiro-Wilk
renewed daily by dechlorinated tap water. The photoperiod was
test and homogeneity of variance was verified using the Levene
provided under a 12 h light:12 h dark cycle by fluorescent lamps.
test. Significant differences among means were compared
Experimental fish were fed two times a day (9:00 a.m. and 5:00 p.m.)
using ANOVA, followed by the Bonferroni’s post hoc test.
with commercial feed containing 41.0% crude protein, 7.0% crude
Two-way analysis of variance (ANOVA) was used to test for
fat (ArtAkua, İzmir, Turkey) and was allowed for acclimation for
the significance of the effects of anesthetic agent concentrations,
21 days before the anesthetic efficiency experiments.
water temperatures, and its interaction (concentration x water
temperature). The relationship between each of the stages of
anesthesia and anesthetic agent concentration was examined using
Anesthetic efficacy experiment
regression analysis (concentration × time of anesthesia induction;
After acclimation, anesthetic efficacy of clove oil and
concentration × time of recovery from anesthesia). Statistical analyses
were conducted using the SPSS software (v23, IBM Corporation,
2-phenoxyethanol were investigated on doctor fish at two different
New York, USA). The results are presented as means ± SD and
water temperatures. Stock solutions of anesthetic agents were prepared
differences were considered statistically significant when P˂0.05.
before the experiment as follows: clove oil and 2-phenoxyethanol,
each of them was mixed with nine volumes of 95% ethanol to
increase water solubility (Yildiz et al., 2013). Induction process
was conducted in a 5 L glass container (3 L of water) equipped with
aeration. The fish were anesthetized with 25, 50, 75 and 100 μL L-1
At the end of the experimental anesthetic administration,
clove oil and 100, 200, 300, 400 and 500 μL L-1 2-phenoxyethanol
no mortality was detected 48 h after exposure to clove oil
concentrations at two different water temperatures (15 and 25 °C)
and 2-phenoxyethanol concentrations. The induction time for
and induction and recovery time were recorded. Ten fish were
anesthesia stage 1, stage 2, stage 3 and recovery time for doctor
fish at two water temperatures (15 and 25 °C) are given in Table 2.
exposed to each anesthetic concentration for determination of
100 μL L-1 2-phenoxyethanol concentration was not sufficient to
induction time. Each fish was individually caught and placed into
induction (stage 3) to anesthesia in both water temperatures within
the anesthetic container, and used only once. The times required to
15 min. Clove oil was found to be anesthetic at all concentrations
reach the desired stage of anesthesia (induction time) were recorded
(25-100 μL L-1) and 2-phenoxyethanol was found to be anesthetic
based on the fish behavioral responses. The concentration was
at 200 μL L-1 and above concentrations (200-500 μL L-1). However,
considered as insufficient for both anesthetic agents when their
25 μL L-1 of clove oil at 15 °C water temperature was not sufficient
concentrations did not cause any induction within 15 minutes.
to reach to stage 3 within 10 min. The shortest time to induction
The different stages of induction and recovery of anesthesia were
(stage 3) time were 96.2 sec in 15 °C and 68.1 sec in 25 °C at
determined according to a protocol adapted from Keene et al.
100 μL L-1 for the clove oil. For the shortest induction (stage 3)
times for 2-phenoxyethanol at the concentration of 500 μL L-1
(1998) and Cunha et al. (2015) (Table 1). After the induction
was 63.4 sec in 15 °C water temperature and 54.0 sec in 25 °C
period, recovery times were evaluated. The fish were removed from
water temperature. The different anesthetic agents resulted in
anesthesia container and transferred into a 10 L glass container
different induction and recovery times. Induction times decreased
containing 5 L of anesthetic-free water which was supplied from
when two anesthetic agent concentrations increased at 15 °C
15 or 25 °C temperature groups’ tanks with constant aeration.
water temperature (Figure 1). A similar relationship was obtained
It was considered as recovered when the fish regained equilibrium
at 25 °C as well (Figure 1). Whereas, higher anesthetic agent
and started to swim in the container. After recovery, each fish
concentrations caused prolongation on recovery time significantly
was transferred into the aquarium to check mortality for 48 h.
for two anesthetics at both water temperatures (Figure 2). In higher
Table 1. Behavioral observations of anesthesia stages.
Exhibited behavior
Relaxation and no response to stimuli: fish calm and do not respond to tactile touch, but respond to external stimuli
Stage 1
(a blow on the anesthetic chamber); opercular rate increases.
Imbalance swimming: fish loss their equilibrium and show imbalance swimming; normal opercular rate; response to
Stage 2
external stimuli.
Total loss of equilibrium and movement: fish lay on lateral side; no movement; no response to external stimuli; slightly
Stage 3
depressed opercular rate.
Recovery Total behavioural recovery. Fish began to normal swimming behavior in the container.
Behavioral observations of anesthesia stages adapted from Keene et al. (1998) and Cunha et al. (2015).
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
Table 2. Induction and recovery time (second) of different concentrations of clove oil and 2-phenoxyethanol on doctor fish at
different water temperatures.
15 °C water temperature
25 °C water temperature
(μL L-1)
Stage 1
Stage 2
Stage 3
Stage 1
Stage 2
Stage 3
Clove oil
65.8 ± 7.9a
139.4 ± 9.6a
764.4 ± 23.8a
231.3 ± 28.6d
36.5 ± 7.6a
71.8 ± 8.8a
231.4 ± 31.2a 268.7 ± 21.1d
34.2 ± 6.4b
62.1 ± 5.8b
225.6 ± 33.9b
276.8 ± 22.8c
24.6 ± 4.2b
44.2 ± 5.3b
167.7 ± 20.4b 317.5 ± 21.8bc
27.6 ± 4.1c
48.2 ± 3.4bc 162.9 ± 22.8c
322.6 ± 36.9b
19.7 ± 2.4bc 32.3 ± 10.4bc 89.5 ± 13.3bc 345.0 ± 45.7ab
20.6 ± 3.0d
39.8 ± 3.9c
96.2 ± 13.2d
392.5 ± 20.4a
16.4 ± 2.1c
25.5 ± 5.5c
68.1 ± 10.1c 372.5 ± 19.1a
115.6 ± 14.4a 401.4 ± 32.2a
107.1 ± 11.6a382.0 ± 26.2a
71.6 ± 8.8b
109.5 ± 15.6b 236.2 ± 33.6a
181.1 ± 13.7ab 64.1 ± 7.2b
86.2 ± 6.5b
207.8 ± 18.6a 111.5 ± 16.8c
42.1 ± 7.6cd
57.7 ± 7.9c
94.2 ± 14.2b
174.6 ± 12.7bc 36.7 ± 5.2c
51.4 ± 7.7c
80.3 ± 10.0b 119.2 ± 19.9bc
34.6 ± 5.6d
42.9 ± 5.5cd
71.7 ± 6.1c
183.3 ± 12.5ab 25.7 ± 5.8de 35.4 ± 5.3cd
60.5 ± 7.6c
134.1 ± 14.6b
19.5 ± 3.5e
34.0 ± 5.9de
63.4 ± 5.6cd
196.6 ± 15.9a
20.2 ± 3.7e
31.3 ± 3.9de
54.0 ± 7.2cd
168.1 ± 20.8a
*Anesthetic concentration was not sufficient to induction to anesthesia within 15 minutes. Data are expressed as Mean ± SD (N=10). Values with different superscripts
in each column are significantly different (P<0.05).
Figure 1. Relationships between induction time (Stage 3) of doctor
Figure 2. Relationships between recovery time in doctor fish at
fish in different water temperatures exposed to different concentrations
different water temperatures exposed to different concentrations
of clove oil and 2-phenoxyethanol. Mean ± SD (N=10).
of clove oil and 2-phenoxyethanol. Mean ± SD (N=10).
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
water temperature (25 °C) caused shorter recovery times in all
100 μL L-1 2-phenoxyethanol was inadequate for induce anesthesia
2-phenoxyethanol concentrations, apart from fish anesthetized
in doctor fish. It is possible that 100 μL L-1 of 2-phenoxyethanol
with 75-100 μL L-1 concentrations of clove oil. Clove oil and
concentrations (or lower than that) can be used to induce sedation
2-phenoxyethanol concentrations significantly affected all
during transportation or other procedures.
anesthetic induction stages (Stage 1, stage 2 and stage 3) and
Induction times in both water temperature decreased significantly
recovery time at two water temperatures (Table 2). Similarly,
with increasing concentrations of clove oil or 2-phenoxyethanol
water temperature significantly affected the anesthetic induction
(P<0.05). There was a strong negative relationship between
and recovery times for both anesthetic agents. Two-way ANOVA
clove oil concentration and induction time at water temperature
revealed not only anesthetic agent concentrations but also water
of 15 °C (R2 = 0.990) and 25 °C (R2 = 0.867) (Figure 1).
temperatures played a significant role on anesthesia of doctor fish
The relationship was also recorded between induction time and
(P<0.001). Furthermore, the interaction of water temperatures
2-phenoxyethanol concentration at water temperature of 15 °C
and anesthetic concentrations on all induction and recovery times
(R2 = 0.925) and at 25 °C (R2 = 0.954) (Figure 1). Similarly,
for clove oil, and recovery times for 2-phenoxyethanol was also
negative relationship between anesthetic agent concentrations
significant (P<0.001).
and induction time of anesthesia were reported in D. labrax and
S. aurata (Mylonas et al., 2005), S. senegalensis (Weber et al.,
2009), Acipenser gueldenstaedtii (Akbulut et al., 2011), Siganus
rivulatus (Ghanawi et al., 2013), P. scalare (Mitjana et al., 2014),
A. persicus (Adel et al., 2016), S. glanis (Gökçek et al., 2016),
There are several studies which try to determine the effective
Colossoma macropomum (Saccol et al., 2017), Carassius auratus
concentrations of clove oil and 2-phenoxyethanol in S. aurata and
(Kizak et al., 2018).
D. labrax (Mylonas et al., 2005), S. senegalensis (Weber et al.,
In this study, it was determined that the recovery time at
2009), A. persicus (Adel et al., 2016), A. regius (Barata et al.,
both water temperatures was shorter in the 2-phenoxyethanol
2016). However, there is no anesthetic efficacy study in doctor
concentrations than clove oil, which is an important criterion in
fish regarding these anesthetic drugs. According to Marking and
selecting anesthetic agents. Similar result has been demonstrated
Meyer (1985), an ideal anesthetic agent for fish should induce
in D. labrax (Mylonas et al., 2005). While recovery time at 25 °C
anesthesia in less than 3 min and allow recovery in 5 min. Our study
water temperature was less than 5 min in all 2-phenoxyethanol
demonstrated that the effective concentrations in 15 and 25 °C
concentrations in the present study, the time was over 5 min at clove
water temperature that produced induction time (Stage 3) within
oil concentrations except 25 μL L-1. Higher anesthetic concentrations
3 min and recovery time within 5 min were 50-75 μL L-1 for clove
in clove oil and 2-phenoxyethanol resulted in higher recovery
oil and 300 μL L-1 for 2-phenoxyethanol, respectively (Table 2,
times in both water temperatures in the present study (P<0.05)
Figure 1). Similar (Mylonas et al., 2005; Serezli et al., 2012;
(Figure 2). We found a positive relationship between anesthetic
Adel et al., 2016) and different results have been observed by
concentration and recovery time in this study. Similar effects have
other researchers in S. senegalensis (Weber et al., 2009), Huso
been demonstrated in A. gueldenstaedtii (Akbulut et al., 2011),
huso (Shaluei et al., 2012), P. scalare (Mitjana et al., 2014),
A. persicus (Adel et al., 2016), S. glanis (Gökçek et al., 2016)
P. reticulata (Cunha et al., 2015). The effective clove oil and
and C. auratus (Kizak et al., 2018). However, it was reported that
2-phenoxyethanol concentrations to induce anesthesia in fish
similar or shorter recovery time in P. scalare (Mitjana et al., 2014),
species varies between 27-100 μL L-1 and 200-1400 μL L-1,
S. senegalensis (Weber et al., 2009), S. rivulatus (Ghanawi et al.,
respectively (Javahery et al., 2012; Pedrazzani and Neto, 2016;
2013) exposed to clove oil, and C. macropomum (Saccol et al.,
Fujimoto et al., 2018; Mitjana et al., 2018). Effective concentrations
2017) exposed to M. sylvatica and C. longa essential oils. Also,
of anesthetic agents in the present study seem to be among these
Mirghaed et al. (2016) and Bolasina et al. (2017) could not find
values. The ratio of the major components (Eugenol: 80.56%)
positive relationships between anesthetic concentration and
in the content of the clove oil used in the present study seems
recovery time. Weber et al. (2009) and Mitjana et al. (2014)
to be compatible with the eugenol ratios approximately 70-90%
explained that differences among the studies might be clarified
in other studies (Akbulut et al., 2012; Ghanawi et al., 2013).
if the specific properties of each species is taken into account,
Hekimoğlu et al. (2017) stated eugenol ratio in clove oil was
such as the physiological responses of fish to anesthetic agents.
96.1%. As in Hekimoğlu et al. (2017), the ratio of eugenol in
In addition, it is stated that the pharmacokinetics of the anesthetic
clove oil could be different from this range in some studies,
agent may cause differences among studies (Zahl et al., 2009;
and this may cause variations in the results. As reported by
Bolasina et al., 2017).
Mylonas et al. (2005), we found that clove oil was effective at
4-fold lower concentrations than 2-phenoxyethanol on doctor fish.
Anesthetic concentration and various biological or environmental
Barata et al. (2016) explained this situation that clove oil affects
factors significantly affect the anesthesia of fish (Santos et al.,
different type of receptors might justify a higher efficiency and
2015; Li et al., 2018; Mitjana et al., 2018). Changes in water
the use of lower concentrations. It is clear that the clove oil is
temperature have been shown to affect induction and recovery time
advantageous because of its plant origin, safe nature, low price,
in various fish species (Hamackova et al., 2004; Mylonas et al.,
and its effectiveness even at lower concentrations (Mylonas et al.,
2005; Zahl et al., 2009; Santos et al., 2015; Skår et al., 2017).
2005; Javahery et al., 2012; Kizak et al., 2018). In agreement with
In the present experiments, 15 and 25 °C water temperatures were
previous findings for H. huso (Shaluei et al., 2012), concentration of
preferred on doctor fish anesthesia because this cyprinid fish is
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
generally found in the natural habitats at water temperatures of
15-25 °C (Vazirzadeh et al., 2014). Our results showed that a rise
in water temperature from 15 to 25 °C shortened both induction
The authors would like to thank the Talya Bitkisel Ürünler Ind
and recovery times for 2-phenoxyethanol, and induction time for
Co.; Ltd. for the supply of clove oil.
clove oil. For example, the induction time to stage 3 anesthesia
for clove oil concentrations varied from 764 s to 96 s at water
temperature of 15 °C, and from 231 s to 68 s at 25 °C. Water
temperature significantly affects stage 1, stage 2, stage 3, and
Adel, M.; Sadegh, A.B.; Yeganeh, S.; Movafagh, A.N.; Saoud, I.P. 2016.
recovery time in both clove oil and 2-phenoxyethanol (P˂0.05)
Anesthetic efficacy of clove oil, propofol, 2-phenoxyethanol, and
(Table 2). Furthermore, statistically significant interactions were
ketamine hydrochloride on Persian sturgeon, Acipenser persicus,
detected between water temperature and anesthetic concentration
juveniles. Journal of the World Aquaculture Society, 47(6): 812-819.
(P<0.001). A negative interaction between water temperature and .
anesthetic concentration was clearly seen in almost all the results.
Akbulut, B.; Aydın, I.; Çavdar, Y. 2012. Influence of temperature on clove
Similar findings have been reported for clove oil by Hoskonen and
oil anaesthesia in flounder (Platichthys flesus Linnaeus, 1758.
Pirhonen (2004). Shortened induction and recovery times with
Journal of Applied Ichthyology, 28(2): 254-257. http://dx.doi.
increasing water temperature have also been shown for clove oil
org/10.1111/j.1439-0426.2012.01936.x .
or 2-phenoxyethanol in Tinca tinca (Hamackova et al., 2004),
Akbulut, B.; Çavdar, Y.; Çakmak, E.; Aksungur, N. 2011. Use of clove oil to
S. aurata and D. labrax (Mylonas et al., 2005), Epinephelus bruneus
anaesthetize larvae of Russian sturgeon (Acipenser gueldenstaedtii).
(Park et al., 2008), Gadus morhua (Zahl et al., 2009), O. mykiss
Journal of Applied Ichthyology, 27(2): 618-621. http://dx.doi.
(Yildiz et al., 2013) and Siganus rivulatus (Santos et al., 2015).
org/10.1111/j.1439-0426.2010.01653.x .
Reduced induction and recovery times with increased temperatures
Baensch, H.A.; Riehl, R. 1991. Aquarien atlas. Bd. 3. Melle: Mergus, Verlag
from 6 to 12 °C have been reported by Skår et al. (2017) for
für Natur-und Heimtierkunde. 1104 p.
Cyclopterus lumpus anesthetized with metacaine, benzocaine,
Barata, M.; Soares, F.; Aragão, C.; Almeida, A.C.; Pousão-Ferreira, P.;
and isoeugenol. The interactive effects of water temperature and
Ribeiro, L. 2016. Efficiency of 2-phenoxyethanol and clove oil for
anesthetic concentration have been well documented in this study
reducing handling stress in reared meagre, Argyrosomus regius (Pisces:
and literature. This reduction in time of induction and recovery
Sciaenidae). Journal of the World Aquaculture Society, 47(1): 82-92.
of anesthesia as water temperature increases is possibly related .
to the acceleration of the opercular ventilation rate and gill blood
Barbas, L.A.L.; Hamoy, M.; Mello, V.J.; Barbosa, R.P.M.; Lima, H.S.T.; Torres,
flow owing to the increased basal metabolism of fish maintained
M.F.; Nascimento, L.A.S.; Silva, J.K.R.; Andrade, E.H.A.; Gomes,
at higher water temperatures (Zahl et al., 2009; Silva et al., 2012;
M.R.F. 2017. Essential oil of citronella modulates electrophysiological
Santos et al., 2015; Skår et al., 2017). It is thought that changes in
responses in tambaqui Colossoma macropomum: a new anaesthetic
electrocardiographic responses may play a role in obtaining these
for use in fish. Aquaculture (Amsterdam, Netherlands), 479: 60-68.
results due to concentration, temperature, or interaction between .
these factors. Santos et al. (2015) stated that increasing metabolic
Batista, E.S.; Brandão, F.R.; Majolo, C.; Inoue, L.A.K.A.; Maciel, P.O.; Oliveira,
rate accelerates respiration, and increases cardiac output and
M.R.; Chaves, F.C.M.; Chagas, E.C. 2018. Lippia alba essential oil
blood flow through the gills. Furthermore, Barbas et al. (2017)
as anesthetic for tambaqui. Aquaculture (Amsterdam, Netherlands),
reported a depressant effect on cardiac rhythm and decreased
495: 545-549. .
heart rates that occurred during C. macropomum anesthesia with
Bolasina, S.N.; Azevedo, A.; Petry, A.C. 2017. Comparative efficacy of
essential oil of citronella, Cymbopogon nardus. From all these
benzocaine, tricaine methanesulfonate and eugenol as anesthetic
results, it is concluded that the change in fish physiology due to
agents in the guppy Poecilia vivipara. Aquaculture Reports, 6: 56-60.
the change in water temperature significantly affects the duration .
of the anesthesia induction and recovery time.
Can, E.; Kizak, V.; Seyhaneyildiz Can, Ş.; Özçiçek, E. 2018. Anesthetic
potential of geranium (Pelargonium graveolens) oil for two cichlid
species, Sciaenochromis fryeri and Labidochromis caeruleus. Aquaculture
(Amsterdam, Netherlands), 491: 59-64.
In conclusion, the results indicate that clove oil and 2-phenoxyethanol
Cárdenas, C.; Toni, C.; Martos-Sitcha, J.A.; Cárdenas, S.; Heras, V.;
can be used as effective anesthetic agents for doctor fish anesthesia.
Baldisserotto, B.; Heinzmann, B.M.; Vazquez, R.; Mancera, J.M. 2016.
The present study demonstrated that the minimum effective
Effects of clove oil, essential oil of Lippia alba and 2-phe anaesthesia on
concentration of clove oil was determined as 75 μL L-1 at water
juvenile meagre, Argyrosomus regius (Asso, 1801). Journal of Applied
temperature of 15 °C and 50 μL L-1 at 25 °C, and 300 μL L-1 at
Ichthyology, 32(4): 693-700. .
both temperatures for 2-phenoxyethanol. However, further studies
Cunha, L.; Geraldo, A.M.R.; Silva, V.; Cardoso, M.; Tamajusuku, A.S.K.;
are needed to determine the effects of anesthetic agents on doctor
Hoshiba, M.A. 2015. Clove oil as anesthetic for guppy. Boletim do
fish of different sizes and the physiological responses.
Instituto de Pesca, 41: 729-735.
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
Fujimoto, R.Y.; Pereira, D.M.; Silva, J.C.S.; Oliveira, L.C.A.; Inoue, L.A.K.A.;
Mitjana, O.; Bonastre, C.; Insua, D.; Falceto, M.V.; Esteban, J.; Josa, A.;
Hamoy, M.; Mello, V.J.; Torres, M.F.; Barbas, L.A.L. 2018. Clove
Espinosa, E. 2014. The efficacy and effect of repeated exposure
oil induces anaesthesia and blunts muscle contraction power in three
to 2-phenoxyethanol, clove oil and tricaine methanesulphonate as
Amazon fish species. Fish Physiology and Biochemistry, 44(1): 245-
anesthetic agents on juvenile Angelfish (Pterophyllum scalare).
256. PMid:29022202.
Aquaculture (Amsterdam, Netherlands), 433: 491-495. http://dx.doi.
Ghanawi, J.; Monzer, S.; Saoud, I.P. 2013. Anaesthetic efficacy of clove
org/10.1016/j.aquaculture.2014.07.013 .
oil, benzocaine, 2-phenoxyethanol and tricaine methanesulfonate in
Mitjana, O.; Bonastre, C.; Tejedor, M.T.; Garza, L.; Esteban, J.; Falceto,
juvenile marbled spinefoot (Siganus rivulatus). Aquaculture Research,
M.V. 2018. Simultaneous effect of sex and dose on efficacy of clove
44(3): 359-366. .
oil, tricaine methanesulfonate, 2-phenoxyethanol and propofol as
Gökçek, K.; Öğretmen, F.; Kanyilmaz, M. 2016. Efficacy of clove oil,
anaesthetics in guppies, Poecilia reticulata (Peters). Aquaculture
2-phenoxyethanol and benzocaine on European catfish, Silurus glanis
Research, 49(6): 2140-2146. .
linnaeus 1758. Turkish Journal of Fisheries and Aquatic Sciences,
Mylonas, C.C.; Cardinaletti, G.; Sigelaki, I.; Polzonetti-Magni, A. 2005.
16: 129-133.
Comparative efficacy of clove oil and 2-phenoxyethanol as anesthetics
Hamackova, J.; Lepicova, A.; Kozak, P.; Stupka, Z.; Kouril, J.; Lepic, P.
in the aquaculture of European sea bass (Dicentrarchus labrax) and
2004. The efficacy of various anaesthetics in tench (Tinca tinca L.)
gilthead sea bream (Sparus aurata) at different temperatures. Aquaculture
related to water temperature. Veterinarni Medicina, 49(12): 467-472.
(Amsterdam, Netherlands), 246(1-4): 467-481. .
Ozçelik, S.; Polat, H.H.; Akyol, M.; Yalcin, A.N.; Ozcelik, D.; Marufihah,
Hekimoğlu, M.A.; Süzer, C.; Saka, Ş.; Kürşat, F. 2017. Sedative effect of
M. 2000. Kangal hot spring with fish and psoriasis treatment. The
clove oil and 2-phenoxyethanol on marine clownfish (Amphiprion
Journal of Dermatology, 27(6): 386-390. PMid:10920584.
ocellaris) and freshwater swordfish (Xiphophorus helleri). Pakistan
Park, M.O.; Hur, W.J.; Im, S.Y.; Seol, D.W.; Lee, J.; Park, I.S. 2008. Anaesthetic
Journal of Zoology, 49(6): 2209-2216.
efficacy and physiological responses to clove oil-anaesthetized kelp
Hoskonen, P.; Pirhonen, J. 2004. Temperature effects on anaesthesia with
grouper Epinephelus bruneus. Aquaculture Research, 39(8): 877-884.
clove oil in six temperate-zone fishes. Journal of Fish Biology, 64(4): .
1136-1142. .
Pedrazzani, A.S.; Neto, A.O. 2016. The anaesthetic effect of camphor
Javahery, S.; Nekoubin, H.; Moradlu, A.H. 2012. Effect of anaesthesia with
(Cinnamomum camphora), clove (Syzygium aromaticum) and mint
clove oil in fish [review]. Fish Physiology and Biochemistry, 38(6): 1545-
(Mentha arvensis) essential oils on clown anemonefish, Amphiprion
1552. PMid:22752268.
ocellaris (Cuvier 1830). Aquaculture Research, 47(3): 769-776. http://
Keene, J.L.; Noakes, D.L.G.; Moccia, R.D.; Soto, C.G. 1998. The efficacy .
of clove as an anaesthetic for rainbow trout, Oncorhynchus mykiss
Priborsky, J.; Velisek, J. 2018. A review of three commonly used fish
(Walbaum). Aquaculture Research, 29(2): 89-101. http://dx.doi.
anesthetics. Reviews in Fisheries Science & Aquaculture, 8249: 1-26.
org/10.1111/j.1365-2109.1998.tb01113.x . .
Kizak, V.; Can, E.; Danabaş, D.; Can, Ş.S. 2018. Evaluation of anesthetic
Ross, L.; Ross, B. 2008. Anesthetic and sedative techniques for aquatic
potential of rosewood (Aniba rosaeodora) oil as a new anesthetic
animals. London: Wiley-Blackwell. 240 p. http://dx.doi.
agent for goldfish (Carassius auratus). Aquaculture (Amsterdam,
org/10.1002/9781444302264 .
Netherlands), 493: 296-301.
Rożyński, M.; Demska-Zakęś, K.; Sikora, A.; Zakęś, Z. 2018. Impact of
Li, Y.; Liang, S.; She, Q.; Han, Z.; Li, Y.; Li, X. 2018. Influence of temperature
inducing general anesthesia with propiscin (etomidate) on the physiology
and size on menthol anaesthesia in chinese grass shrimp Palaemonetes
and health of European perch (Perca fluviatilis L.). Fish Physiology
sinensis (Sollaud, 1911). Aquaculture Research, 49: 2091-2098.
and Biochemistry, 44(3): 927-937.
Limma-Netto, J.D.; Sena, A.C.; Copatti, C.E. 2016. Essential oils of Ocimum
018-0482-4. PMid:29476378.
basilicum and Cymbopogon flexuosus in the sedation, anesthesia and
recovery of tambacu (Piaractus mesopotamicus male x Colossoma
Saccol, E.M.H.; Toni, C.; Pês, T.S.; Ourique, G.M.; Gressler, L.T.; Silva,
macropomum female). Boletim do Instituto de Pesca, 42(3): 727-733.
L.V.F.; Mourão, R.H.V.; Oliveira, R.B.; Baldisserotto, B.; Pavanato, .
M.A. 2017. Anaesthetic and antioxidant effects of Myrcia sylvatica
(G. Mey.) DC. and Curcuma longa L. essential oils on tambaqui
Marking, L.L.; Meyer, F.P. 1985. Are better anesthetics needed in
(Colossoma macropomum). Aquaculture Research, 48(5): 2012-2031.
fisheries? Fisheries (Bethesda, Md.), 10(6): 2-5. http://dx.doi. .
org/10.1577/1548-8446(1985)010<0002:ABANIF>2.0.CO;2 .
Metin, S.; Didinen, B.I.; Kubilay, A.; Pala, M.; Aker, İ. 2015. Determination
Santos, S.; Ghanawi, J.; Saoud, I.P. 2015. Effects of water temperature and
of anesthetic effects of some medicinal plants on rainbow trout
body weight on anaesthetic efficiency in marbled rabbitfish (Siganus
(Oncorhynchus mykiss Walbaum, 1792). LIMNOFISH Journal of
rivulatus). Aquaculture Research, 46(4): 928-936. http://dx.doi.
Limnology and Freshwater Fisheries Research, 1(1): 37-42.
org/10.1111/are.12249 .
Mirghaed, T.A.; Ghelichpour, M.; Hoseini, S.M. 2016. Myrcene and linalool
Serezli, R.; Basaran, F.; Muhtaroglu, C.G.; Basaran, A.K. 2012. Effects
as new anesthetic and sedative agents in common carp, Cyprinus carpio
of 2-phenoxyethanol anaesthesia on juvenile meagre (Argyrosomus
- Comparison with eugenol. Aquaculture (Amsterdam, Netherlands),
regius). Journal of Applied Ichthyology, 28(1): 87-90. http://dx.doi.
464: 165-170. .
org/10.1111/j.1439-0426.2011.01771.x .
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506
Shaluei, F.; Hedayati, A.; Jahanbakhshi, A.; Baghfalaki, M. 2012. Physiological
Tort, L.; Puigcerver, M.; Crespo, S.; Padrós, F. 2002. Cortisol and haematological
responses of great sturgeon (Huso huso) to different concentrations of
response in sea bream and trout subjected to the anaesthetics clove
2-phenoxyethanol as an anesthetic. Fish Physiology and Biochemistry,
oil and 2-phenoxyethanol. Aquaculture Research, 33(11): 907-910.
38(6): 1627-1634. . .
Vazirzadeh, A.; Zahedinejad, S.; Bahri, A. 2014. Spawning induction in doctor
Silva, L. L.; Parodi, T.V.; Reckziegel, P.; Garcia, V. O.; Bürger, M.E.;
fish, Garra rufa (Heckel, 1843) by ovaprim and captive rearing of
Baldisserotto, B.; Malmann, C.A.; Pereira, A.M.S.; Heinzmann, B.M.
larvae. Iranian Society of Ichthyology, 1(4): 258-265.
2012. Essential oil of Ocimum gratissimum L.: Anesthetic effects,
Weber, R.A.; Peleteiro, J.B.; Martín, L.O.G.; Aldegunde, M. 2009. The
mechanism of action and tolerance in silver catfish, Rhamdia quelen.
efficacy of 2-phenoxyethanol, metomidate, clove oil and MS-222 as
Aquaculture (Amsterdam, Netherlands), 350-353: 91-97. http://dx.doi.
anaesthetic agents in the Senegalese sole (Solea senegalensis Kaup
org/10.1016/j.aquaculture.2012.04.012 .
1858). Aquaculture (Amsterdam, Netherlands), 288(1-2): 147-150.
Skår, M.W.; Haugland, G.T.; Powell, M.D.; Wergeland, H.I.; Samuelsen,
Yedier, S.; Kontaş, S.; Bostanci, D.; Polat, N. 2016. Otolith and scale morphologies
O.B. 2017. Development of anaesthetic protocols for lumpfish
of doctor fish (Garra rufa) inhabiting Kangal Balıklı Çermik thermal
(Cyclopterus lumpus L.): Effect of anaesthetic concentrations, sea
spring. Iranian Journal of Fisheries Science, 15(4): 1593-1608.
water temperature and body weight. PLoS One, 12(7): e0179344. PMid:28678815.
Yildiz, M.; Kayim, M.; Akin, S. 2013. The anesthetic effects of clove oil
and 2-phenoxyethanol on rainbow trout (Oncorhynchus mykiss) at
Souza, C.F.; Baldissera, M.D.; Bianchini, A.E.; Silva, E.G.; Mourão, R.H.V.;
different concentrations and temperatures. Iranian Journal of Fisheries
Silva, L.V.F.; Schmidt, D.; Heinzmann, B.M.; Baldisserotto, B.
Science, 12(4): 947-961.
2018. Citral and linalool chemotypes of Lippia alba essential oil as
anesthetics for fish: a detailed physiological analysis of side effects
Zahl, I.H.; Kiessling, A.; Samuelsen, O.B.; Hansen, M.K. 2009. Anaesthesia
during anesthetic recovery in silver catfish (Rhamdia quelen). Fish
of Atlantic cod (Gadus morhua) - Effect of pre-anaesthetic sedation,
Physiology and Biochemistry, 44(1): 21-34.
and importance of body weight, temperature and stress. Aquaculture
s10695-017-0410-z. PMid:28948452.
(Amsterdam, Netherlands), 295(1-2): 52-59.
Aydın et al. Bol. Inst. Pesca 2019, 45(4): e506. DOI: 10.20950/1678-2305.2019.45.4.506