JOURNAL OF THE Vol. 41, No.

4
WORLD AQUACULTURE SOCIETY August, 2010

The Anesthetic Efficiency of Clove Oil in Caspian Salmon,

Salmo trutta caspius K., Smolts in Dosage–Salinity–pH
Linked Approach

Amir Ghazilou1 , Hasan Saeedi Hasankandi, Farideh Chenary, Afshin

Nateghi, Niloofar Haghi, and Mohammad Reza Sahraeean
Department of Marine Biology, Khorramshahr University of Marine Science and Technology,
PO Box 669, Khorramshahr, Iran

Anesthetics are used in fish culture facili-
ties by biologists and aquaculturists to facilitate
various handling procedures, such as weigh-
ing, sorting, collection of gametes, tagging, as
well as veterinary procedures (Summerfelt and
Smith 1990; Kazu and Siwicki 2001). Regard-
less of the agent, the process of anesthesia in
all fish develops in a similar way and runs
in a progressive pattern (McFarland 1959),
resulting in loss of consciousness, inhibition
of reflex activity, and reduced skeletal mus-
cle tone (McFarland 1960; Isaacs 1983). The
properties addressed for choosing efficient anes-
thetic agent for fish will vary with the objec-
tives and species, but in general, fast induction
(within 3 min), fast recovery (within 15 min),
and no subjected mortality are among the most
prevalent criteria considered in safe anesthesia
(Woody et al. 2002). The most, widely used
fish anesthetics include tricaine methane sul-
fonate, benzocaine, etomidate, metomidate, 2-
phenoxy ethanol, quinaldine, and quinaldine
sulfate. Clove oil has recently been pointed out
as a potential fish anesthetic (Isaacs 1983).
Clove oil is distillated from the stem, leaves,
and buds of the clove tree, Eugenia aromat-
ica (Soto and Burhanuddin 1995), and it has
been demonstrated as an effective anesthetic
agent on several non-salmonid fish (Soto and
Burhanuddin 1995; Munday and Wilson 1997;
Peake 1998; Walsh and Pease 2002; Hajek et al.
2006). Attempts have been made to study prac-
tical clove oil anesthesia on salmonids by vari-
ous researchers. Iversen et al. (2003) evaluated
1 Corresponding author.
 Copyright by the World Aquaculture Society 2010
anesthetic efficiency of different concentrations
of clove oil for salmon and found it as an appro-
priate anesthetic for Atlantic salmon, Salmo
salar, smolts. Velisek et al. (2005) investigated
acute toxicity of clove oil for rainbow trout,
Oncorhynchus mykiss, and estimated LC50s for
different clove oil concentrations. Keene et al.
(1998) recommended 40–60 mg/L clove oil
concentrations as trusted dosage range for juve-
nile rainbow trout.
“Eugenol (4-allyl-2-methoxyphenol),” the
active ingredient of clove oil in fish anesthesia,
is found to be insoluble in water at temperatures
below 15 C (Woody et al. 2002) and its disso-
lution is believed to be completely temperature-
dependent in 16–18 C range (Wen et al. 2005).
Because of weak solubility of eugenol in water,
primary organic solvent (i.e., ethanol) accessed
solutions are much practical in comparison
to direct dissolution method (Anderson et al.
1997; Marking and Meyer 1985). Meanwhile,
application of helping solvents to working anes-
thetic solutions may be less preferred consider-
ing side effects of used organic material (i.e.,
tissue dehydration for ethanol based solutions).
In the other hand, applying helping solvent-free
anesthetic medium with higher primary concen-
trations will bring new concerns on potential
effects of other water physicochemical charac-
teristics such as pH and salinity on consequent
anesthetic efficiency of clove oil. There has
been only one attempt to study the compared
function of water condition on clove oil anes-
thesia in tropical fish performed by Wen et al.
(2005). The aim of this study was to quantify

655
656 GHAZILOU ET AL.
the anesthetic efficiency of clove in Caspian
salmon smolts in a dosage–salinity–pH model.
Materials and Methods
This experimental protocol was approved
by the Local Ethical Committee for Experi-
ments on Animals (Khorramshahr University of
Marine Science, Iran).
Fish
Approximately 450, mixed-sex, potential
1-yr-old Caspian salmon smolts (mean weight =
48.2 g ± 4.3 mean ± SD; mean total length =
16.9 cm ± 2.8 mean ± SD), obtained from local
fish farm, where they were maintained in a
closed freshwater system (Na+ 0.60 mmol/L;
Ca2+ 0.50 mmol/L; Mg2+ 0.25 mmol/L) at 16–
18 C and natural photoperiod and transferred to
the wet laboratories at the Faculty of Science
(Khorramshahr University of Marine Science,
Iran), where they were acclimated for 2 wk to
Khorramshahr city tap water (referred to as
fresh-water) in 400-L tanks at 19 C and the
same photoperiod. The fish were checked for
smolt status following Blackburn and Clarke
(1978) 24-h seawater challenge test and stocked
randomly into three, 400-L flowthrough tanks
filled with either 0.0, 4.0, or 8.0 g/L saltwater
in a closed system and kept for 2 wk for further
acclimation.
Experimental Design
After full acclimation of fish to experimental
salinities, nine 50-L fiberglass treatment aquaria
situated in the same building with the acclima-
tion tanks, were selected and filled with either 0,
4, or 8 g/L saltwater buffered to either 6, 7, or 8
pH levels, respectively. The experimental solu-
tions of clove oil (20, 40, 60, or 80 mg/L) were
achieved by mixing different dosages of clove
oil (89.1% eugenol, Razi Drug Research Insti-
tute, Tabriz, Iran) directly with water in approx-
imately 15-min intervals before each trial. Dur-
ing the experiments, the fish were placed in
test tanks individually and the onset times of
four consecutive phases of induction were eval-
uated for six fish in each trial according to
Thienpoint and Niemegeers (1965) anesthesia
stages scheme:
(1) Phase I: Acceleration and subsequent
deceleration of opercular movements,
a partial loss of reactivity to external
stimuli.
(2) Phase II: Loss of equilibrium, opercular
movements very slow, and fish still
reactive to strong stimuli.
(3) Phase III: Total loss of reactivity, fish
are lying at the tank bottom and do not
respond to handling.
(4) Phase IV: Complete cessation of opercu-
lar movements.
(5) Phase V: Full recovery.
When a fish became anesthetized, it was
immediately taken out, dried with a wet sponge,
and then put into recovery tank containing 50-L
clean aerated water with the same salinity and
pH level and dismissed after full recovery. For
fine monitoring of fish mortality, resident fish in
acclimation tanks were established as blank fish
and observed throughout the test. The experi-
ment was conducted for 4 d during which trials
for each anesthetic concentration were tested
in 1 d. During the experiments, feeding was
stopped and fish were maintained under nat-
ural photoperiod, constant temperature (19 C),
salinity, dissolved O2 (8.3–9.5 mg/L), and pH.
The experimental salinities were achieved by
diluting disinfected seawater (35 g/L) with
dechlorinated fresh tap water.
Data Analysis
Mean induction and recovery times were
compared among treatment groups using
MANOVA, followed by Tukey’s Honestly Sig-
nificant Differences multiple comparison proce-
dure. Residual diagnostics were used to assess
normality and constant variance assumptions
for the final selected models.
Results
All fish exposed to the experimental concen-
trations of clove oil at different salinity and
pH levels were anesthetized to the stage IV
 ANESTHETIC EFFICIENCY OF CLOVE OIL IN CASPIAN SALMON
within 3 min (Table 1). The clove oil admin-
istered at the concentrations ranging from 20
to 80 mg/L resulted in a progressive anesthe-
sia; and increase in the concentration resulted
in the significant shortening of induction time
(Table 1). In the recovery process, all fish
exposed to 20, 40, or 60 mg/L were recovered
from deep anesthesia within 15 min after they
were transferred to recovery tank (Table 1).
Two of the six fish exposed to 80 mg/L clove
oil solution at 4 g/L salinity and basic condition
suffered mortality and one of the individuals
that revived (after 49.1 min), never regained
equilibrium. Anesthesia symptoms faded in a
reverse order and the longest period of regain-
ing full recovery was observed at the highest
concentration (Table 1).
In the salinity trial, a significant varia-
tion (P < 0.05) was recorded among induction
times of applied clove oil dosages adminis-
tered at different salinity levels and increased
induction times along with increased salinity
was found in nearly all applied concentrations
(Table 1). In contrast to induction phase, no
constant dependence could be noted between
recovery time and water salinity (Table 1).
In the pH trial, analysis of quantified data
revealed a significant dependence of induc-
tion time on pH level (Table 1) and resulting
fast inductions were recorded in neutral con-
dition among different clove oil concentrations
(Table 1). In a recovery process, a significant
difference was found among recovery times of
each test concentration at different pH levels
with neutrality leading to fastest recovery times
(Table 1).
Discussion
The experimental trials in this study indicate
that Caspian salmon smolt can be successfully
anesthetized with appropriate concentrations of
clove oil. Our results show that test concentra-
tions which best met the general efficacy criteria
for fish anesthetic (Woody et al. 2002) were
20, 40, and 60 mg/L. The efficacy of clove oil
as an anesthetic was previously reported in a
variety of fish including other salmonids like
rainbow trout juveniles and adults (Anderson
657
et al. 1997) and non-salmonid fishes (Soto and
Burhanuddin 1995; Peake 1998; Hajek et al.
2006). Anderson et al. (1997) indicated clove
oil as a suitable anesthetic for rainbow trout
juveniles and adults. Furthermore, Keene et al.
(1998) presented the results of clove oil appli-
cation in salmonid fishes with anesthesia and
recovery was achieved within 3–4 min and
14 min at of 40–60 mg/L concentration and
10 C temperatures, respectively. The author
indicated that exceeding the time of anesthe-
sia over 5 min had a considerable influence on
the time of recovery. The observed progres-
sion through the various stages of anesthesia
for Caspian salmon smolts in this study was
founded to be consistent with the descriptions
by Keene et al. (1998) modified from Jolly
et al. (1972) and Hikasa et al. (1986) taking
into account the higher water temperature in
our experiments.
According to Trevor and Miller (1987), the
effectiveness of anesthetic on fish primarily
depends on its concentration in the central ner-
vous system tissues. Bonath (1977) demon-
strated that the differences in sensitivity of
various fish species to anesthetic can be related
to the differences in metabolic rate. In this case,
temperature can be considered as one of the
foremost key factors that modifies metabolic
rates and also anesthetic efficiency in fish (Bon-
ath 1977; Wagner et al. 2003). Furthermore,
functional efficiency of an anesthetic in differ-
ent stages from anesthetic agent dissolution to
inclusion into brain cells can be fully affected
by other physicochemical environmental condi-
tion. In this study, when clove oil administered
at different salinity levels, increased induction
times were achieved in higher water salinities.
Zemaitis (1986) revealed that strong precipitat-
ing effect of chlorine ion (Cl− ) on other organic
solutes, known as “salting out” phenomenon,
can put an extraordinary force on appropriate
dissolution of different organic agents in water.
Considering high chlorinity of saltwater, salting
out hypothesis may trigger observed effect of
water salinity on clove oil efficiency. However,
Wen et al. (2005) demonstrated that, in higher
salinity ranges, salinities which share similar
osmotic pressure with marine teleost plasma
 658
Table 1. Timing (min) anesthesia and recovery phases in Caspian salmon smolt exposed to various clove oil concentrations at different salinities and pH levels; mean values ±
SEM.

Dosage (mg/L) Salinity (g/L) pH Phase I Phase II Phase III Phase IV Total induction Total recovery
6 0.19 ± 0.51Aa 0.18 ± 0.16Aa 0.28 ± 0.17Aa 0.50 ± 0.33Aa 1.15 ± 0.43Aa 10.46 ± 1.23Aa
0 7 0.25 ± 0.71Ab 0.24 ± 0.49Ab 0.42 ± 0.22Ab 0.20 ± 0.65Ab 1.11 ± 0.84Aa 8.44 ± 1.60Ab
8 0.30 ± 0.33Ac 0.29 ± 0.65Ac 0.39 ± 0.38Ab 0.38 ± 0.83Ac 1.36 ± 0.67Ab 10.36 ± 2.01Aa
6 0.38 ± 0.50Ba 0.25 ± 0.14Ba 0.43 ± 0.29Ba 0.25 ± 0.21Ba 1.31 ± 0.71Ba 10.48 ± 1.42Aa
20 4 7 0.29 ± 0.24Ab 0.22 ± 0.15Aa 0.33 ± 0.17Bb 0.25 ± 0.88Aa 1.09 ± 0.93Ab 9.71 ± 1.31Bb
8 0.41 ± 0.26Ca 0.37 ± 0.30Bb 0.45 ± 0.12Aa 0.38 ± 0.87Ab 1.61 ± 0.55Bc 10.48 ± 1.11Ba
6 0.75 ± 0.04Ca 0.43 ± 0.15Ca 0.74 ± 0.15Ca 0.60 ± 0.56Ca 2.52 ± 0.69Ca 10.91 ± 2.13Ba
8 7 0.75 ± 0.15Ba 0.43 ± 0.12Ba 0.75 ± 0.81Ca 0.59 ± 0.82Ba 2.52 ± 0.73Ba 9.55 ± 0.98Bb
8 1.05 ± 0.40Cb 0.61 ± 0.46Cb 0.73 ± 0.14Ba 0.40 ± 0.21Ab 2.79 ± 0.49Cb 11.55 ± 0.74Bc
6 0.20 ± 0.81Aa 0.18 ± 0.81Aa 0.26 ± 0.60Aa 0.46 ± 1.23Aa 1.10 ± 0.99Aa 12.37 ± 1.12Aa
0 7 0.26 ± 0.30Ab 0.21 ± 0.35Aab 0.38 ± 0.24Ab 0.17 ± 0.90Ab 1.02 ± 0.83Ab 10.02 ± 1.06Ab
8 0.28 ± 0.41Ab 0.25 ± 0.41Ab 0.44 ± 0.35Ac 0.38 ± 1.12Ac 1.35 ± 0.67Ac 12.34 ± 1.18Aa
6 0.31 ± 0.35Ba 0.29 ± 0.82Ba 0.40 ± 0.29Ba 0.23 ± 0.23Ba 1.23 ± 0.84ABa 12.46 ± 1.21Aa
40 4 7 0.26 ± 0.82Ab 0.22 ± 0.87Ab 0.50 ± 0.69Bb 0.23 ± 0.56Aa 1.21 ± 0.90Ba 11.02 ± 1.30Ab
8 0.26 ± 0.87Ab 0.28 ± 0.20Aa 0.51 ± 0.58Bb 0.30 ± 0.44Ab 1.35 ± 0.79Ab 12.36 ± 1.78Ac

GHAZILOU ET AL.
6 0.70 ± 0.19Ca 0.43 ± 0.15Ca 0.74 ± 0.56Ca 0.55 ± 0.68Ca 2.42 ± 0.71Ba 13.24 ± 1.54Aa
8 7 0.71 ± 0.98Bab 0.40 ± 0.51Ba 0.73 ± 0.85Ca 0.46 ± 0.75Bb 2.30 ± 0.59Cb 11.82 ± 1.24Bb
8 0.74 ± 0.51Bb 0.41 ± 0.66Ba 0.75 ± 0.17Ca 0.61 ± 0.46Bc 2.51 ± 0.82Bc 12.24 ± 1.45Bc
6 0.18 ± 0.14Aa 0.15 ± 0.13Aa 0.38 ± 0.19Aa 0.29 ± 0.55Aa 1.00 ± 0.63Aa 12.80 ± 0.93Aa
0 7 0.21 ± 0.35Aa 0.17 ± 0.11Aab 0.40 ± 0.15Aa 0.14 ± 0.31Ab 0.92 ± 0.50Ab 10.99 ± 1.14Ab
8 0.25 ± 0.10Aa 0.20 ± 0.92Ab 0.45 ± 0.10Ab 0.30 ± 0.87Aa 1.20 ± 0.74Ac 12.65 ± 1.52Aa
6 0.23 ± 0.29Aa 0.22 ± 0.22Ba 0.44 ± 0.46Aa 0.18 ± 0.69Ba 1.07 ± 0.62Aa 12.79 ± 1.81Aa
60 4 7 0.22 ± 0.10Aa 0.22 ± 0.67Ba 0.44 ± 0.29Aa 0.17 ± 0.11Aa 1.05 ± 0.74Ab 9.79 ± 2.20Bb
8 0.34 ± 0.27Ab 0.26 ± 0.12Ba 0.49 ± 0.67Ab 0.25 ± 0.93Ab 1.34 ± 0.88Bc 12.56 ± 2.17Ac
6 0.64 ± 0.72Ba 0.43 ± 0.46Ca 0.68 ± 0.25Ba 0.48 ± 0.51Ba 2.23 ± 0.70Ba 14.91 ± 2.14Ba
8 7 0.63 ± 0.54Ba 0.44 ± 0.18Ca 0.69 ± 0.84Ba 0.48 ± 1.03Ba 2.24 ± 0.46Ba 12.68 ± 2.09Cb
8 0.70 ± 0.16Bb 0.44 ± 0.83Ca 0.69 ± 0.67Ba 0.49 ± 0.41Ba 2.32 ± 0.89Cb 13.24 ± 2.04Bc
6 0.17 ± 0.66Aa 0.13 ± 0.69Aa 0.42 ± 0.23Aa 0.16 ± 0.63Aa 0.88 ± 0.93Aa 19.28 ± 1.32Aa
0 7 0.17 ± 0.10Aa 0.16 ± 0.19Aa 0.33 ± 0.83Ab 0.07 ± 0.31Ab 0.73 ± 0.88Ab 17.77 ± 1.34Ab
8 0.20 ± 0.15Aa 0.17 ± 0.75Aa 0.43 ± 0.82Aa 0.26 ± 0.87Ac 1.06 ± 0.79Ac 20.52 ± 1.03Aa
6 0.18 ± 0.16Aa 0.17 ± 0.33Aa 0.46 ± 0.76Aa 0.09 ± 0.65Ba 0.90 ± 0.96Aa 19.32 ± 1.06Aa
80 4 7 0.20 ± 0.67Aab 0.19 ± 0.51Aa 0.38 ± 0.36Aa 0.13 ± 0.41ABb 1.00 ± 0.94Bb 18.03 ± 2.04ABb
8 0.22 ± 0.75Ab 0.25 ± 0.19Bb 0.53 ± 0.35Bb 0.22 ± 0.43Ac 1.22 ± 0.98Bc 20.66 ± 2.11Ba
6 0.30 ± 0.25Ba 0.42 ± 0.48Ba 0.75 ± 0.58Ba 0.34 ± 0.34Aa 1.81 ± 0.82Ba 19.05 ± 0.98Aa
8 7 0.24 ± 0.43Ab 0.40 ± 0.13Bab 0.56 ± 0.63Bb 0.29 ± 0.43Bb 1.49 ± 0.86Cb 18.22 ± 0.90Bb
8 0.54 ± 0.60Bc 0.37 ± 0.42Cb 0.55 ± 0.21Cb 0.31 ± 0.71Bb 1.77 ± 0.74Cc 21.51 ± 1.39Ac
Mean values having different uppercase superscript letter in the same column are significantly different (P < 0.05) among different pH levels; mean values having different
lowercase superscript letter in the same column are significantly different (P < 0.05) among different salinities.
 ANESTHETIC EFFICIENCY OF CLOVE OIL IN CASPIAN SALMON
will serve as iso-osmotic transport medium in
easy diffusion of the agent to fish blood, as such
shortening the induction time.
Clove oil administered at different pH lev-
els led to fastest inductions in neutral condi-
tion. Hikasa et al. (1986) suggested that run-
ning effect of pH on clove oil anesthesia, can
be discussed in both dissolution and inclu-
sion perspectives. Zemaitis (1986) concluded
that proper dissolution of organic compounds
in water primarily depends on ionization
degree of its molecules. Hence, considering
basic structure of eugenol (guaiacols; phenols),
faster inductions in acidic water can be fully
explained by strong ionization of the agent
in an acidic environment. However, Hikasa
et al. (1986) demonstrated that ionization of
anesthetic agent can probably inhibit sufficient
inclusion of anesthetic molecules into brain
cells. In the case of recovery times, prolonged
recovery times were observed at both sides of
neutrality for all applied concentration. Hargis
(2005) concluded that in rainbow trout, both
acidic and basic pH can result in decreased ven-
tilation rate and subsequent procrastination in
regaining equilibrium. However, adequate evi-
dences do not exist in reasonability of this phe-
nomenon and need further investigation.
Acknowledgment
Authors are grateful for the funding provided
by SRCN organization for the project (Project
No: MB-0812).
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