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JOURNAL OF ENVIRONMENT AND AQUATIC RESOURCES
2024, Vol. 7, Pages 63-76
https://doi.org/10.48031/msunjear.2024.07.04
Anesthetic Efficacy of Lavender and Lemongrass Essential Oils in
Nile Tilapia (Oreochromis niloticus, i-Excel strain) Fry
Reignier E. Curay, Rimz C. Dadole, Roseller G. Sabilla, Victor R. Navarro,
Kaent Immanuel N. Uba*
Department of Fisheries Science, School of Marine Fisheries and Technology,
Mindanao State University at Naawan, Naawan, Misamis Oriental, 9023 Philippines
*Corresponding author: Email: kaentimmanuel.uba@msunaawan.edu.ph
ABSTRACT
Anesthetics play a crucial role in aquaculture by ensuring the
welfare and health of fish, facilitating various procedures, and
preventing physical injuries during handling. Recently, plant-
based anesthetics for fish are becoming a sustainable alternative
to synthetic anesthetics in aquaculture and fisheries research. The
present study examined the use of lavender (Lavandula
angustifolia) and lemongrass (Cymbopogon citratus) essential
oils as anesthetics for Nile tilapia Oreochromis niloticus (i-Excel strain) fry. Fish (average body
weight = 0.004 g) were immersed in varying concentrations of 40, 50, 60, 70, and 80 μL L-1
and 30, 40, 50, 60, and 70 μL L-1 for lavender and lemongrass essential oils, respectively. The
induction and recovery times were recorded and the best dose was determined based on rapid
induction (<3-4 minutes) and quick recovery (<10 minutes). The results indicated that both
essential oils exhibit dose-dependent effects on sedation and recovery. Lavender essential oil
achieved optimal anesthesia at 60 μL L-1, with an induction time of 229.3±10.7 seconds and a
recovery time of 310.9±16.9 seconds, while lemongrass essential oil induced optimal
anesthesia at 30 μL L-1, with an induction time of 232.5±11.7 seconds and a recovery time of
130.5±8.6 seconds. Both concentrations for both essential oils resulted in 100.0% survival,
while higher doses induced faster anesthesia and recovery but decreased survival rates. These
essential oils offer cost-effective alternatives to synthetic anesthetics for short-term handling
procedures; however, further research on histopathological effects and optimal transport
dosages is needed.
ARTICLE HISTORY
Received: February 7, 2024
Accepted: May 7, 2024
Published May 20, 2024
KEYWORDS
plant-based anesthetics, sedation,
fish handling, stress
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INTRODUCTION
The use of anesthetic is important to secure healthier individuals, provide
welfare, and allow for the prevention of physical injuries and ease of working during
fish farming or for research purposes (Benovit et al. 2012; Zahl et al., 2012).
Anesthetics are used to facilitate handling during biometrics, artificial spawning,
vaccination, sorting of specimens, biopsy, blood collection, surgery, labeling,
transportation, and euthanasia (Javahery et al., 2012). Appropriate anesthetics at
optimal concentrations are expected to minimize the deleterious effects of stress on fish
(Roohi and Imanpoor, 2015). A suitable anesthetic should render fish rapidly
immobilized and result in uneventful recovery. Moreover, it should present high
potency, be widely available, cost-effective, and present low or no toxicity. Anesthetics
should not build up in fish tissues and organs and pose problems for human or animal
consumption. Adding to it, the excretion of the anesthetics from the fish body should
be fast (Javahery et al., 2012; Roohi and Imanpoor, 2015). Prospection of good fish
anesthetics and their best dose-response relationships, along with the behavioral
characterization of the induction and recovery stages, and possible side effects have
been a common point of anesthesia research efforts.
Anesthetic agents, both synthetic and plant-originated, are used in aquaculture
procedures to minimize fish activity and to avoid stress and physical damage caused
by handling (Priborsky and Velisek, 2018). A good anesthetic agent for fish should
induce anesthesia even at low concentrations in less than 3 minutes and allow recovery
within 10 minutes, should also be cheap and easy to use (Park et al., 2003; Kizak et al.,
2018; Spanghero et al., 2019). The major synthetic anesthetics used in aquaculture are
2-phenoxyethanol (Priborsky and Velisek, 2018), tricaine methanesulfonate (MS-222)
and metomidate (Weber et al., 2009), benzocaine (Gökçek et al., 2017), etomidate
(Rożyński et al., 2018), propofol and quinaldine sulfate (Priborsky and Velisek, 2018),
and ketamine hydrochloride (Adel et al., 2016). However, these anesthetics have
undesirable side effects (Palić et al., 2006) such as stressors, hyperactivity, excessive
mucus secretion, corneal damage, and gill irritation, and are also expensive (Aydın and
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J. Environment & Aquatic Resources, Vol 7. (2024)
Barbas, 2020). For this reason, research on the use of effective natural anesthetic
products on fish is intensively carried out. The effective concentrations of anesthetics
depend on the fish species and anesthetic agent (Zahl et al., 2009). Temperature, pH,
age, size, sex, and interactions among these factors also affect the efficacy of
anesthetics in fish (Zahl et al., 2009; Mitjana et al., 2018). It is known that the responses
of fish to anesthetics can considerably vary between different water temperatures
(Santos et al., 2017).
Recently, some studies have been conducted with plant essential oils to find
new anesthetics that are more effective, safer, and less expensive than the currently
available synthetic drugs (Inoue et al., 2003; Aydın and Barbas, 2020; Austin et al.,
2022). Lemongrass Cymbopogon citratus is a plant from the family Gramineae and is
widely cultivated in temperate and tropical regions, especially in Southeast Asia. It is
commonly known as lemongrass and popular in Asian cuisine; frequently used in food
processing as a food flavoring, perfume, and cosmetic industry. The active ingredient
in lemongrass essential oil that is believed to induce anesthesia is called citral. Citral is
a naturally occurring compound found in lemongrass oil and is responsible for its
distinct lemony scent. Recent studies have demonstrated that essential oil from the
genus Cymbopogon has sedative effects on silver catfish (Rhamdia quelen) (Santos et
al., 2017), Tambaqui (Colossoma macropomum) (Barbas et al., 2017), and in
ornamental fish species (Kizak et al., 2018). However, to date, there is still no
information on the anesthetic properties of C. citratus essential oil on Nile tilapia. As
citral oil can produce anxiolytic, sedative, and motor relaxant effects in mice
(Hajizadeh Moghaddam et al., 2023), it was then hypothesized herein that C. citratus
essential oil in appropriate concentrations, would promote an anesthetic effect on Nile
tilapia Oreochromis niloticus as well.
On the other hand, Lavender Lavandula angustifolia is a flowering plant in the
family Lamiaceae. The Lavender essential oil has sedative, antidepressant,
antispasmodic, antibacterial, and local anesthetic effects (Hajhashemi et al., 2003;
Bikmoradi et al., 2015; Can and Sümer, 2019). Linalyl acetate and linalool are
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Curay et al:Anesthetic Efficacy of Lavender and…
considered the primary active constituents and are responsible for the sedative effects
(Denner, 2009). The lavender essential oil has shown an anesthetic effect on blue
dolphin cichlid, Cyrtocara moorii (Can and Sümer, 2019), Convict cichlid Amatitlania
nigrofasciata (Can et al., 2019), Common carp Cyprinus carpio (Krasteva et al.,
2021a), European catfish Silurus glanis (Krasteva et al., 2021b) and Rainbow trout
Oncorhynchus mykiss (Metin et al., 2015). However, lavender (L. officinalis) essential
oil for Convict cichlid Cichlasoma nigrofasciata showed no anesthetic effect (Raisi et
al., 2020).
Nile tilapia O. niloticus (i-Excel strain) adapts easily to different environments
and is one of the most important species of freshwater fish for global aquaculture. The
combination of availability, adaptability, economic importance, research history, and
growth characteristics makes tilapia a suitable species for studying the effects of
anesthetics and developing anesthesia protocols in aquaculture and fish welfare
research. In this sense, the present study investigated the anesthetic effect of the
aforementioned essential oils for Nile tilapia O. niloticus (i-Excel strain) fry.
MATERIALS AND METHODS
Experimental fish
Nile tilapia fry (n=150, average body weight = 0.004 g) of the i-Excel strain
was obtained at the Mindanao State University Naawan Center for Aquaculture
Research and Entrepreneurship Services - Tilapia Nursery. The fish were reared in
tanks with 18 ppt salinity water at a temperature of 28-30°C, fed ad libitum with fry
booster (Tateh Aquafeeds, crude protein – 45.0%) thrice daily, and 100% water change
was done every three days. Fish were acclimatized in an aerated recirculating system
and deprived of food for 24 hours before the experiment.
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Experimental design and procedures
The anesthesia and recovery tests were performed following a completely
randomized experimental design in experimental units (volume: 10L containers) in a
static system with constant aeration. Due to a lack of published data, and to preserve
the well-being of the treated fish and to prevent mortality, the researchers started with
the lowest concentration that showed anesthetic effects within 5 minutes. For lavender
essential oil (EOLA) and lemongrass essential oil (EOCC) it was 40 μL L-1 and 30 μL
L-1, respectively. The final concentrations used were 40, 50, 60, 70, and 80 μL L-1 for
EOLA and 30, 40, 50, 60, and 70 μL L-1 for EOCC. Locally-available commercial
steam-distilled lavender and lemongrass essential oils were used in the experiment. Due
to the hydrophobic nature of the essential oil, a stock solution diluted with 70.0%
ethanol was used at a ratio of 1:4 essential oil:alcohol (v:v) to facilitate dilution in
water.
Each fish (n=15 per concentration at 5 fish per replicate) was exposed
individually to the essential oil concentrations and used only once in each replicate at
a defined concentration. Thereafter, sedated fish were immediately withdrawn from the
container with essential oil and transferred for recovery to containers with only water.
The time to reach stage 4 sedation and stage 5 recovery (Table 1) was recorded in
seconds using a digital timer. Furthermore, fish were considered dead if they did not
exhibit opercular or caudal movements, or if they did not respond to a touch stimulus
10 minutes after transfer to water without the addition of essential oil. The percentage
of survivors was then recorded. Water parameters during the experiment were
maintained at a temperature of 23.8 ± 0.4°C and a pH of 6.8 ± 0.1.
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Table 1. Description of the stages of sedation and recovery used in the present study
(Hikasa et al. 1986).
Stage
Behavior exhibited
Sedation
0
Normal.
1
Sedation. Partial or total loss of reaction in response to external stimuli;
normal equilibrium.
2
Partial loss of equilibrium; erratic swimming.
3
Total loss of equilibrium.
4
Anesthesia; loss of reflex activity.
5
Medullary collapse; respiratory movements cease; fish death.
Recovery
1
The reappearance of opercular movement.
2
Partial recovery of equilibrium with partial recovery of swimming
motion.
3
Total recovery of equilibrium.
4
The reappearance of avoidance swimming motion and reaction in
response to external stimuli, but still the behavioral response is stolid.
5
Total behavioral recovery. Normal swimming.
Data analyses
The data on time to anesthesia induction, recovery, and survival rate were
analyzed using one-way analysis of variance after satisfying assumptions of normality
and homogeneity of variances. Once significant differences were found, Tukey’s test
was used as a post-hoc analysis. All values were expressed as means±standard error of
the means. All statistical tests were conducted using R software version 4.3.2 (R Core
Team, 2023) at a 95% confidence level while all graphs were generated using
SigmaPlot version 15 (Jandel Scientific, Erkrath, Germany).
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RESULTS AND DISCUSSION
Sedation and recovery time of Nile tilapia fry O. niloticus (i-Excel strain)
depended on the dose of the two essential oils (Fig. 1). In using EOLA, the fastest time
to reach stage 4 anesthesia was recorded in 80.0 μL L-1 at 149.8±7.5 s followed by 60.0
μL L-1 at 229.3±10.7 s and 70.0 μL L-1 at 245.8±10.0 s. There were significant
differences among these treatments (p<0.05). Conversely, the slowest time to reach
stage 4 anesthesia was recorded in 40.0 μL L-1 and 50.0 μL L-1 at 332.9±17.5 s and
322.8±11.6 s, respectively, with no significant difference (p>0.05). Furthermore, the
fastest time to reach stage 5 recovery was observed in 40.0 μL L-1 and 50.0 μL L-1 at
160.1±20.0 s and 189.3±13.6 s, respectively, with no significant differences (p>0.05).
Meanwhile, significantly slower recovery was observed in 60.0 μL L-1, 70.0 μL L-1,
and 80.0 μL L-1 at 310.9±16.9 s, 275.9±13.1 s, and 317.8±15.5 s, respectively with no
significant differences (p>0.05).
Figure 1. Induction and recovery time of Nile tilapia Oreochromis niloticus (i-Excel strain) fry
at different concentrations of lavender (left) and lemongrass (right) essential oils. Values are
expressed as means±standard error of the means. Bars with different superscripts indicate
significant differences (One-way ANOVA, post-hoc Tukey’s test, p<0.05).
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Curay et al:Anesthetic Efficacy of Lavender and…
On the other hand, using the EOCC, the fastest time to reach stage 4 anesthesia
was recorded in 50.0 μL L-1 at 128.9±15.4 s followed by 70.0 μL L-1 at 176.5±10.3 s
and 60.0 μL L-1 at 176.5±10.3 s. There were no significant differences among these
treatments (p<0.05). Conversely, the slowest time to reach stage 4 anesthesia was
recorded in 30.0 μL L-1 and 40.0 μL L-1 at 232.5±11.7 s and 331.5±21.9 s, respectively,
with no significant differences (p>0.05). Furthermore, the fastest time to reach stage 5
recovery was observed in 50.0 μL L-1 at 117.1±16.5 s followed by 40 μL L-1, 30 μL L-
1, and 60 μL L-1 at 128.9±18.1 s, 130.5±8.6 s, and 160.2±12.9 s, respectively, with no
significant differences (p>0.05). Meanwhile, significantly slower recovery time was
observed in 70 μL L-1 at 232.1±13.8 s (p<0.05).
In terms of survival (Fig. 2), fish subjected to EOLA had a 100.0±0.0% survival
rate except in 80.0 μL L-1 at 80.0±11.5% which is significantly lower (p<0.05). On the
other hand, in fish subjected to EOCC, 100.0±0.0% survival was recorded in 30 μL L-
1 and 40 μL L-1 while in 50 μL L-1, 60 μL L-1, and 70 μL L-1 significantly lower survival
was recorded at 53.3±29.1%, 66.7±17.6%, and 53.3±6.7%, respectively (p<0.05).
Figure 2. Survival rate of Nile tilapia Oreochromis niloticus (i-Excel strain) fry at
different concentrations of lavender (left) and lemongrass (right) essential oils. Values
are expressed as means±standard error of the means. Bars with different superscripts
indicate significant differences (One-way ANOVA, post-hoc Tukey’s test, p<0.05).
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Furthermore, the increasing concentration of both essential oils resulted in
decreased induction time of anesthesia and increased recovery time. According to the
criteria for selecting an effective anesthetic agent, it is recommended that anesthesia
should be induced rapidly (<3-4 min) and should take a quick recovery (<10 min) (Park
et al., 2003; Kizak et al., 2018; Spanghero et al., 2019). Moreover, it is also crucial to
take into consideration a high rate of survival in a specific dose. Following this, it can
be concluded that the best dose to use to induce anesthesia and quick recovery without
mortality is 60 μL L-1 and 30 μL L-1 for lavender and lemongrass essential oils for Nile
tilapia fry, respectively.
The anesthetic efficacy of essential oils is intricately linked to the composition
of their constituent elements. The pharmacological effects of essential oils, such as
sedation or anesthesia, may arise from the action of a primary compound or the
synergistic interactions among active constituents (Cunha et al., 2017). Specifically,
research suggests that the anesthetic and sedative properties of lavender and lemongrass
essential oils can be attributed to the presence of linalool and citral, respectively (Kizak
et al., 2018; Aydin and Barbas, 2020).
In the present study, we evaluated the anesthetic efficacy of lavender and
lemongrass essential oils on Nile tilapia fry. Our findings revealed that lavender
essential oil elicited optimal anesthesia at a concentration of 60 μL L-1, with an
induction time of 229.3 seconds, a recovery time of 310.9 seconds, and a survival rate
of 100.0%. Conversely, lemongrass essential oil induced optimal anesthesia at 30 μL
L-1, demonstrating an induction time of 232.5 seconds, a recovery time of 130.5
seconds, and a survival rate of 100.0%.
The anesthetic efficacy of lavender essential oil has been extensively
investigated across various fish species (Can and Sümer, 2019; Can et al., 2019; Raisi
et al., 2020; Metin et al., 2022; Yigit et al., 2022; Thabet et al., 2023). Can and Sümer
(2019) documented that a concentration of 300 µl L-1 of L. angustifolia essential oil
induced anesthesia in Cyrtocara moorii, with an induction time of 109.2 s and a
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Curay et al:Anesthetic Efficacy of Lavender and…
recovery time of 420.0 s. Can et al. (2019) similarly demonstrated the anesthetic
efficacy of L. hybrida essential oil at a concentration of 200 μl L-1, eliciting an induction
time of 20.1 s and a recovery time of 162 s in Amatitlania nigrofasciata. Krasteva et
al. (2021b) delineated effective anesthetic concentrations of lavender essential oil for
European channel catfish, ranging from 160-200 mg L-1. Nevertheless, Metin et al.
(2015) and Yigit et al. (2022) noted the sedative effects of lavender oil on
Oncorhynchus mykiss at concentrations of 200 mg L− 1. Moreover, Thabet et al. (2023)
reported the anesthetic effects of lavender essential oil in Tilapia zilli at 200 µl L-1.
Conversely, Raisi et al. (2020) reported the inefficacy of high concentrations (600 and
1200 mg L-1) of L. officinalis oil in inducing anesthesia in Cichlasoma nigrofasciata.
Furthermore, the anesthetic efficacy of lemongrass essential oil has been
investigated in some fish species (Netto et al., 2017; Kizak et al., 2018; Krasteva et al.,
2023). Netto et al. (2017) noted that 600 μL L-1 of lemongrass C. flexuosos induced
anesthesia in Nile tilapia O. niloticus juveniles while Kizak et al. (2018) reported that
at 200 μL L-1 anesthesia was induced by C. citratus essential oil in ornamental fishes,
Sciaenochromis fryeri and Labidochromis caeruleus. Conversely, Krasteva et al.
(2023) reported that a low concentration of C. schoenanthus at 0.20 ml L-1 induced
anesthesia in Common carp Cyprinus carpio.
The observed variations in anesthetic effects and induction time of the essential
oils between the current study and other published studies can be attributed to several
factors. One of the key factors is the different responses of different fish species. Each
fish species may have specific requirements in terms of the concentration of essential
oil needed to achieve the desired sedation or anesthesia. The optimal dosage and
concentration can vary depending on factors such as the species' size, age, and
physiological characteristics. Furthermore, different fish species exhibit distinct
physiological traits that can influence their response to anesthetics. These physiological
characteristics encompass metabolic rates, body size, lipid content, enzyme systems,
and specific physiological processes. These variances in physiological traits can
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J. Environment & Aquatic Resources, Vol 7. (2024)
contribute to differences in how different fish species react to essential oil as an
anesthetic agent. Furthermore, the differences may be due to differences on chemical
variations in the same plant species.
CONCLUSION AND RECOMMENDATIONS
The present study reports the efficacy of two essential oils, lavender and
lemongra ss, as an anesthetic for Nile tila pia fry at 60 μL L-1 and 30 μL L-1 for lavender
essential oil and lemongrass essential oil, respectively. Furthermore, the use of these
essential oils can be a cheap, alternative fish anesthetic agent to the commercial, but
expensive, MS-222. At lower dosages, no mortality was observed and recovery to
normal behavior was evident. Therefore, this established that both essential oils can be
effective anesthetics for Nile tilapia for short-term handling procedures while further
investigations, particularly on possible histopathological effects should be ascertained
to establish its safe use. Furthermore, it is equally interesting to investigate the optimum
dosage for the transport of this fish species.
ACKNOWLEDGEMENTS
This undergraduate thesis work was partly funded by the HABs Watch Project
of MSU at Naawan. Moreover, the technical support of the MSU Naawan Center for
Aquaculture Research and Entrepreneurship Services is greatly appreciated.
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