INDUCTION AND RECOVERY FROM ANESTHESIA IN CYPRINUS
CARPIO IMMERSED TO DIFFERENT CONCENTRATIONS OF 2PHENOXYETHANOL (CLOVE OIL).
Mohammad Shoaib* and Syed Anser Rizvi
Department of Zoology, University of Karachi-75270-Pakistan.
* Corresponding Author: Tel.: +92.300 2589913;
E-mail: Shoaib_11273@yahoo.com
Number of pages
22
Number of Tables
02
Number of Figures
03
1
ABSTRACT
The anesthetic effects of clove oil derived eugenol (2-phenoxyethanol) were studied in
commercially important freshwater fish Cyprinus carpio. %Mortality and effects of
immersion to differential concentrations of eugenol were examined include induction and
recovery from anesthesia. Ten individuals were tested averaging 0.540.25 to 0.970.92 g.
Those fishes immersed to 0.1 and 0.5 ml/l of clove oil exhibit longer duration of induction and
recover within a minute without any mortality. This indicates low administration of eugenol
into body. The induction time proportionally decreased when concentration of clove oil
increase. The recovery from anesthesia observed to be longer due to heavy mixing of eugenol
with water and require more time to flush off from both the gill lamellae and blood. Mortality
rate become high with respect to consective increase of clove oil probably due to deposition of
eugenol into different organs like liver, spleen, brain and kidneys. These findings suggest that
eugenol could be an effective anesthetic for transportation, tagging, induced breeding and
minor surgery. Its benefits include a lower cost; lower required dosage, improved safety for
both fishes and aqua culturist.
Key words: Fish anesthetics, Clove oil, Cyprinus carpio.
2
1. INTRODUCTION
Rapid growth of aquaculture in the world and technological advances
applied in it make exacting demands on newly introduced chemicals and
preparations. Chemicals used in aquaculture are nowadays subject to strict
control, particularly with regard to their safety and efficacy (Taylor and Roberts,
1999). Anesthetics are physical or chemical agents that act on an animal by
initially inducing a calming effect and subsequently inducing loss of
equilibrium, mobility, consciousness, and reflex action (Summerfelt and Smith,
1990). Anesthetics are widely used on hatchery-reared and wild fishes, usually
to reduce stress caused by handling or transporting. A modern fish anesthetics
must meet a number of general requirements, e.g. they must be highly soluble in
water, have short induction time, be non-toxic for both fish and humans, have a
large safety factor, allow an ad lib intensification of anesthesia with a possibility
of spontaneous recovery, and they must leave no residues in fish (BroÏová and
Svobodová, 1986; Ross and Ross, 1999). As such, fish anesthetic research has
been conducted on many compounds, including carbonic acid (Gelwicks et al.
1998), sodium bicarbonate (Peake 1998), quinaldine, benzocaine, and clove oil,
2 phenoxyethanol (Gilderhus and Marking 1987; Iwama et al. 1989.
Clove oil is used as an anaesthetic before handling or treating fish in
breeding, artificial propagation, blood sampling or for some other veterinary
3
interventions. The use of an anaesthetic facilitates the handling of too big or too
agile fish species (Wagner et al. 2002). Clove oil is a dark-brown liquid, a
distillate of flowers, stalks and leaves of the clove tree Eugenia aromatica (Sato
and Burhanuddin, 1995). According to Isaacs (1983), Briozza et al. (1989) and
Keene et al. (1998), clove oil is distilled from stems, leaves and flower buds of
Eugenia caryophyllata, and its active ingredient, i.e. Eugenol (4-allyl-2methoxyphenol), makes up 70 to 90% by weight of clove oil. Clove oil also
contains eugenol acetate (> 17 %) and kariofilen 5 (> 12%).
In fish, anesthetics are absorbed and excreted mainly through gills
(Houston and Woods, 1976; Ferreira et al. 1984). Eugenol and its compounds
and metabolites are quickly removed from the blood and tissues of fish (Fisher
et al. 1990), and the presence of these substances in muscle tissues of fish or
other animals is not considered toxic or mutagenous (Philips 1990; Zheng et al.
1992).
The aim of the present study was to investigate the level at which the
tested fishes respond immediately in terms of induction time (INT) and recovery
time (RET) followed by their mortality along with the effects of clove oil
exposure to person who engaged in these kinds of anesthetic trials.
4
2. MATERIALS AND METHODS
The method used consisted of introducing the active ingredient of clove
oil into fish gills through the water, i.e. “anesthesia by immersion” by following
Brousse, (1974). For the making of working solution, the extract of Eugenol
aromatica was mixed by volume (10:10 ml) with methanol. The working
solution distributed into different concentrations from 0.1, 0.5, 0.75, 1.0, 1.25,
1.5, 1.75, 2.0 and 2.25 ml/l of clove oil. 10 fishes of Cyprinus carpio (Weight
ranged from 0.540.25 to 0.970.92 gms) were immersed into treatment tank
(1’x1’ glass aquarium). A same sized glass aquarium was set as recovery tank
provided with airline access. In the tests, the onsets of individual phases of
anesthesia and recovery rates were studied in term of induction and recovery
time. Evaluations were made in four consective phases.
1) Acceleration and subsequent declaration of opercular movements, a
partial loss of reactivity to external stimuli.
2) Loss of equilibrium, opercular movements very slow, fish still reactive to
strong stimuli.
3) Total loss of reactivity, fish are lying at the tank bottom and do not
respond to handling.
4) Complete cessation of opercular movements, fish die if left in the bath for
too long.
5
Prior to the immersion, they were weighed to investigate the relationship of
body weight and quantity of anesthesia given. % Survival was also observed
throughout the experiment.
Statistical analysis was carried out by using MINITAB 11 to the find the
significant levels between concentration of clove oil, induction and recovery time
and % mortality.
3. RESULTS
3.1. BEHAVIOR OF TESTED FISHES
Cyprinus
carpio
immersed
to
differential
concentrations (0.1-2.25 ml/l) of clove oil exhibited a variety of behaviors.
Following behavioral response of tested fishes were noticed:
Behavioral
response
Normal
Clove oil
concentration (ml/l)
(0.1-0.5 ml/l)
Reactive
sedation
Remarks
to
external
stimuli;
opercular rate and muscle tone
normal.
Light sedation
(0.75 ml/l)
Slight loss of reactivity to external
stimuli;
opercular
rate
slightly
decreased; equilibrium normal.
6
Deep sedation
(1-1.75 ml/l)
Total loss of reactivity to all but
strong erratic; increased opercular
rate; reactivity only to strong tactile
and vibration stimuli.
Total loss of
(2-2.25 ml/l)
equilibrium
Total loss of muscle tone and
equilibrium;
opercular
slow
rate;
loss
but
of
regular
spinal
reflexes.
Loss of reflex
(2.25 ml/l)
reactivity
Total loss of reactivity; opercular
movements slow and irregular; heart
rate very slow; loss of all reflexes.
Medullary
collapse
(2.25 ml/l)
Total loss of reactivity; opercular
movements slow and irregular; heart
rate very slow; loss of all reflexes.
3.2. INDUCTION TIME (INT) OF CLOVE OIL
The time taken for unconscious is presented in Table 1. When the
experimental fishes immersed to low concentration (0.1ml/l) of clove oil, all
respond slowly to become anesthetize and time taken for it was 1120208.0 sec.
This because of the induction of active ingredient of clove oil i.e. 2-
7
phenoxyethanol into blood stream via gills as the mixed water wash the gill
filament. It is clearly shown that there were inversely relationship exit between
INT and concentration of clove oil and statistically it was also indicated that
both parameters were significantly differ (P<0.05). (Table 2 & Fig. 1).
8
Table 1: Anesthetic response of Cyprinus carpio immersed to different
concentrations of clove oil.
Concentration (ml/l)
N
Weight (g)
Induction time (Sec)
Recover time (Sec)
Mortality (%)
0.1
10 0.760.32
1120208.0
22.916.91
00
0.5
10 0.970.92
42.812.72
59.217.07
00
0.75
10 0.540.25
85.247.5
178.895.3
10
1.0
10 0.560.32
54.1018.5
124.7100.9
30
1.25
10 0.580.24
16.86.11
93.759.3
40
1.5
10 0.590.22
13.22.78
599508
40
1.75
10
0.660.3
9.00.667
233.2202.9
50
2.0
10 0.550.41
9.91.72
150.0157.8
60
2.25
10 0.740.30
9.501.35
58.890.2
90
9
Table 2: Regression analysis (Log10) of different clove oil concentrations
with induction time, recovery time and % mortality.
S.No
Regression equation
R-Sq
R-Sq(adj)
F-value
P-value
1
Ylog = 1.47-1.56 xlog
92.7%
91.6%
8.44
0.000
2
Ylog = 2.08+0.628 xlog
42.1%
33.8%
5.08
0.059
%Mortality =-13.8+40.0 Concentration
92.8%
91.7%
89.85
0.000
3*
* Not transformed into Log10 because first two observations were zero.
10
Fig.1: Fitted line polt between clove oil concentration
and induction time.
W = Logten(Induction time), Z = Logten(Clov e oil conc.))
1000
Induction time (Sec)
W = 1.46866 - 1.55768Z
500
R-Sq = 0.927
200
100
50
20
10
0.10
0.15
0.20
0.30
0.40
0.60
0.80 1.00
1.50
2.00
Clove oil concentration (ml/l)
11
Fig.2: Fitted line polt between clove oil concentration and recovery time.
Recovery time (Sec)
600
400
W = Logten(Recovery time), Z = Logten(Clove oil concentration)
300
W = 2.07823 + 0.628420Z
R-Sq = 0.421
200
150
100
80
60
40
30
0.10
0.15
0.20
0.30
0.40
0.60
0.80 1.00
1.50
2.00
Clove oil concentration (ml/l)
12
Fig.3: Fitted line polt between clove oil concentration and % mortalities.
100
Y = -13.7879 + 40.0082X
% Mortalities
R-Sq = 0.928
50
0
0
1
2
Clove oil concentration
13
3.3. RECOVERY TIME (RET) FROM SEDATION
When the tested fish placed into fully aerated, de-chlorinated and clove oil
free water tank the water start to flush off the 2-phenoxyethanol from the gill
filaments. The recover time for lower to higher concentration is found to be
directly proportional to induction time. The reason behind this result may be
concluded that heavy amount of 2-phenoxyethanol enter into blood stream when
the fish immersed to higher concentration and ultimately require more time to
recover from sedation. Some other factors also contribute to recover from
sedation like circulatory collapse and nervous breakdown. Fig. 2 is related to
clarify these results and there were non-significant relationship between
concentration and time taken for recovery (R-Sq 42.1%).
3.4. MORTALITY
During the recovery from an experimental clove oil anesthetic
bath, no mortality occurred in 0.1 & 0.5 ml/l of concentration. Therefore, the
sedation rate was normal. 10, 30, 40 & 50% mortality occurred when the fishes
immersed to 0.75, 1, 1.25, 1.5 & 1.75 ml/l of concentration respectively. All
were under gone in deep sedation. 2 & 2.25 ml/l of concentration caused total
loss of equilibrium while the mortalities occurred 60 and 90%. It was also
observed that 100% of mortality would occur if the fishes will immersed to more
than 2.25 ml/l of clove oil. This will affect reflexes and cause medullary
14
collapse. Moreover, it is concluded from table 1& 2 if 2-phenoxyethanol
introduced into blood stream in more than tolerable doses there will be heavy
mortality observed. The values from regression analysis (R-Sq 91.7% and Pvalue 0.000) also confirm the present assumptions as both were differed
significantly (Fig. 3).
4. DISCUSSION
Clove oil has been used for a number of years to anesthetize fish
(Hikasa et al., 1986). Since in early 1990s, studies on fish have increased
significantly, due largely to the continued expansion of work in the aquaculture
and fishery management arenas and the development of the pet aquaria industry.
Clearly, aquaculture is now the largest growing component of agriculture in the
world whereas in the traditional fisheries are in decline (FAO 2002; USDA
2001).
The active ingredients are phenols derivatives, essentially the C 10H12O2
eugenol compound (Taylor and Roberts, 1999). Anesthetics such as quinaldine
and MS-222 are used widely both by aqua-culturist and fish biologists for the
purpose of minimizing stress and injury during capturing, handling, sorting and
transporting wild and cultured fishes. Stress is a primary factor that affects the
health and welfare of fish. Excessive handling is associated with the activation
15
of hypothalamic-pituitary-inter renal (adreno-corticotropin-cortisol) “stress axis”
(FSBI 2002; Kreiberg 2000; Wedemeyer et al., 1990).
Munday and Wilson 1997 worked on other anesthetics and concluded that
these chemicals not only expensive, and often difficult to obtain in developing
countries. They may also have harmful side effects on humans, especially in
situation where the anesthetic is squister by anglers to capture the fishes.
Therefore, the selection of clove oil as an anesthetic agent was considered for
the present study. In addition to this, Clove oil is both inexpensive and nonharmful to humans, making it attractive as possible alternative anesthetics.
Under stress conditions, physiological and metabolic parameters may alter
and fish will less capable of fighting against various opportunistic pathogens,
including water molds, bacteria and parasites (Powell 2000). Cortisol also
suppresses the immune system suggested by Wendelaor-Bonga, (1997). Kene et
al., 1998 reported that clove oil is highly effective even at low doses compared
with MS-222. Table 1 exhibit the same conclusions i.e. against low
concentrations 0.1 and 0.5 ml/l not even a single fish become dead and only 10
and 30% mortalities occurred against 0.75 and 1.0 ml/l of clove oil. However,
40% of mortality observed against 1.25 and 1.5 ml/l of clove oil. It provides a
much calmer induction to anesthesia than the other chemicals. The present
findings are in line with the findings of Munday and Wilson (1997). Table 1
16
showed that clove oil at the higher concentration of 1.75 and 2.25 ml/l proven
less effective than quinaldine causing 50-90% mortalities. The recovery time
under the influence of clove oil is substantially larger than recovery time from
others. Prince and Powell, 2000 worked on adult rainbow trout and reported that
when rainbow trout immersed to clove oil all specimens were anesthetized
successfully and recovered accordingly.
Clove oil meets seven out of eight criteria for an ideal anesthetic (Marking
and Meyer 1985). Its main advantage is its low price. The use of clove oil,
however, requires that general principles of safe handling of chemicals be
observed. It was observed that a stay in a poorly ventilated room where oil is
used cause headache, nausea and fatigue in vulnerable persons. The same
problems were noticed when the anesthetic trials were carried out. The authors
were felt headache and nausea conditions. Inspite of these drawbacks, if the
trials were conduct in airy laboratories or in the place where cross ventilation
possible the clove oil is proven to be much effective. The ratio between toxic
concentration and therapeutic is termed as therapeutic index (TI) described by
Svobodva and Vykusova, 1999. The TI of clove oil during the whole study
period was relatively low which also show the advantage of clove oil over other
available anesthetic agents.
17
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