Veterinary Anaesthesia and Analgesia, 2014, 41, 621–629 doi:10.1111/vaa.

12146

RESEARCH PAPER

S-(+)-Linalool from Lippia alba: sedative and anesthetic for

silver catfish (Rhamdia quelen)

Clarissa G Heldwein*, Lenise de L Silva*, Eduarda Z Gai*, Cassiela Roman*, Thaylise V Parodi†,
Marilise E B€ 
urger†, Bernardo Baldisserotto†, Erico M de M Flores‡ & Berta M Heinzmann*
*Department of Industrial Pharmacy, UFSM, Santa Maria, Brazil
†Department of Physiology and Pharmacology, UFSM, Santa Maria, Brazil
‡Department of Chemistry, UFSM, Santa Maria, Brazil

Correspondence: Berta Maria Heinzmann, Departamento de Farmacia Industrial, Universidade Federal de Santa Maria, Avenida Roraima
1000, Santa Maria, RS, Brazil BR-97105-900. E-mail: berta.heinzmann@gmail.com

Results Linalool had a similar sedation profile to

Abstract
Objective The present study describes the isolation
of linalool from the essential oil of Lippia alba (Mill.)
N. E. Brown, and its anesthetic effect in silver catfish
(Rhamdia quelen) in comparison with essential oil.
The potentiation of depressant effects of linalool with
a benzodiazepine (BDZ) and the involvement of
GABAergic system in its antagonism by flumazenil
were also evaluated.
Study design Prospective experimental study.
Animals Juvenile silver catfish unknown sex
weighing mean 9.24  2.83 g (n = 6 for each
experimental group per experiment).
Methods Column chromatography was used for the
isolation of S-(+)-linalool. Fish (n = 6 for each
concentration) were transferred to aquaria with
linalool (30, 60, and 180 lL L 1) or EO of L. alba
(50, 100, and 300 lL L 1) to determine the induc-
tion time for anesthesia. After induction, the animals
were transferred to anesthetic-free aquaria to assess
their recovery time. To observe the potentiation, fish
were exposed to linalool (30, 60, and 180 lL L 1) in
the presence or absence of BDZ (diazepam 150 lM).
In another experiment, fish exposed to linalool (30
and 180 lL L 1) or BDZ were transferred to an
anesthetic-free aquaria containing flumazenil (5 lM)
or water to assess recovery time.
the essential oil at a proportional concentration in
silver catfish. However, the anesthesia profile was
different. Potentiation of linalool effect occurred
only when tested at low concentration. Fish
exposed to BDZ showed faster anesthesia recovery
in water with flumazenil, but the same did not
occur with linalool.
Conclusions and clinical relevance The use of
linalool as a sedative and anesthetic for silver
catfish was effective at 30 and 180 lL L 1,
respectively. The mechanism of action seems not
to involve the benzodiazepine site of the GABAergic
system.
Keywords anesthesia, benzodiazepine, GABA, seda-
tion, Verbenaceae.
Introduction
The fish culture industry has grown substantially in
recent decades, and is responsible for the production
of 46% of all the consumed fish in 2008. During this
production process, fish are normally submitted to
some procedures, such as handling and transport,
that are able to evoke physiological and biochemical
changes known as stress responses, which directly
influence animal welfare (Bergqvist & Gunnarsson
2013). Sedatives and anesthetic agents are used in
this context to reduce or minimize this stress and to

621
S-(+)-Linalool anesthesia in catfish CG Heldwein et al.
help handling (Cunha et al. 2010a,b; Parodi et al.
2013). Several substances and their combinations,
such as ether, barbiturates, quinaldine, tricaine
methanesulfonate (MS 222), chlorbutanol, and
benzocaine have been used to induce anesthesia in
fish (Heo & Shin 2010; Gressler et al. 2012).
However, these agents have been associated with
undesirable side effects (Gilderhus & Marking 1987;
Zahl et al. 2012). This fact has motivated research-
ers to look for new anesthetic agents that may be
used in aquaculture.
Recent studies have demonstrated the potential
of essential oils (EOs) and their isolated compounds
as anesthetics in aquaculture (Cunha et al. 2010a,
b). Examples of less toxic agents for aquatic
animals are AQUI-S (AQUI-S; New Zealand Ltd.,
New Zealand), which contains isoeugenol (Mein-
ertz et al. 2006), eugenol and the EOs of Aloysia
triphylla, Lippia alba (Cunha et al. 2010a,b; Parodi
et al. 2012, 2013) and Ocimum gratissimum (Silva
et al. 2012).
Many anesthetics have exhibited a regulatory
influence on GABAA receptor activity, including
some natural products of the terpenoid class
(Heldwein et al. 2012). The expression of the
GABAergic system seems to be conserved among
vertebrates, from fishes to mammals (Delgado &
Schmachtenberg 2008). The interaction of the EOs
of L. alba and O. gratissimum with the benzodiaz-
epine (BDZ) site of the GABAA receptor in fish was
reported recently (Heldwein et al. 2012; Silva
et al. 2012).
Linalool (Lin) is a constituent of several essential
oils whose depressor activities on the central nervous
system (CNS) are well described in rodents and in
humans (Dobetsberger & Buchbauer 2011). This
compound can also be found in Brazilian medicinal
species, such as L. alba (Mill.) N. E. Brown (Verben-
aceae), which is commonly used as a tranquilizer in
folk medicine. For the EO of this species, sedative
(Vale et al. 1999) and anticonvulsant (Viana et al.
2000) actions were also demonstrated in mice, in
addition to the anesthetic properties for aquatic
animals (Cunha et al. 2010a; Parodi et al. 2012).
Thus, the present study describes the isolation of
Lin from the EO of L. alba and the comparison of the
time for anesthesia induction and the recovery of Lin
and this EO in silver catfish (Rhamdia quelen).
Furthermore, the possible involvement of the BDZ
site of the GABAergic system on its mechanism of
action was also researched.
622 © 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 41, 621–629
Materials and methods
Plant material
Lippia alba was grown at the CESNORS campus,
Frederico Westphalen-RS, Brazil (geographically
situated a 27º23′26″ South, 53º25′43″ West and
461 m sea level) and collected in January 2009 and
February 2010. The species was identified by Dr. Dr.
Gilberto Dolejal Zanetti, Department of Industrial
Pharmacy, Federal University of Santa Maria
(UFSM). A voucher specimen (SMDB No. 10050)
was deposited in the herbarium of the Department of
Biology, UFSM.
Extraction of the essential oil of Lippia alba
The EO was obtained from the fresh leaves by the
hydrodistillation process with a Clevenger type
apparatus for 2 hours, according to European
Pharmacopoeia (2007). The samples were stored
at 20 °C until the isolation procedure and phar-
macological tests. The EO composition was previ-
ously analyzed by gas chromatography coupled with
mass spectrometry (GC-MS) as described by Heldw-
ein et al. (2012).
Isolation and characterization of S-(+)-linalool (Lin)
The EO of L. alba (5 g) was submitted to a chroma-
tography column (22.5 9 3.2 cm), containing 70 g
of silica-gel 60 (Merck, 70–230 mesh). The com-
pounds were eluted with a mixture of hexane:
acetone (95:5 v/v), at maximum flux. Fractions
(50 mL) were monitored by thin layer chromatogra-
phy (TLC) (silica gel F254, hexane:acetone 95:5 v/v,
detection: anisaldehyde-sulfuric acid), pooled accord-
ing to their chromatographic profiles and then
concentrated under reduced pressure, at 40 °C.
Fraction 4 (150–200 mL, 701 mg) was analyzed
by GC-MS and corresponded to the isolated com-
pound, which was identified as S-(+)-Linalool (Lin)
([a]D20 = +1.9 {concentration of 5.8 g in 100 mL of
chloroform [CHCl3]}). The identification was per-
formed by a comparison of its chromatographic and
mass spectrometric (MS), Hydrogen and 13-Carbon
nuclear magnetic resonance (1H- and 13C-NMR)
spectroscopic data with a mass spectral library
(NIST/EPA/NIH 2005) and literature data (Adams
2001; Barros et al. 2009). Optical rotation ([a]D20)
was obtained on a Perkin Elmer 343 polarimeter.
 S-(+)-Linalool anesthesia in catfish CG Heldwein et al.
Nuclear Magnetic Resonance spectra were recorded
in CDCl3 on Bruker HPX 300 FT-NMR at 400 MHz
for 1H and 100 MHz for 13C with tetramethylsilane
as internal standard.
Animals
Juvenile silver catfish (9.24  2.83 g; 10.25 
1.08 cm) were acquired and transported as
described by Cunha et al. (2010a). Approximately
130 fish were housed in continuously aerated 250 L
tanks with freshwater for 2 weeks before experi-
ments (18.35  0.65 °C, pH 7.45  0.09, total
ammonia levels of 0.086 mg L 1, dissolved oxygen
levels: 8.99  0.41 mg L 1, total hardness level:
26  2 mg L 1 CaCO3, alkalinity level: 42  1 mg
L 1 CaCO3, conductivity: 0.04 lS cm 1). A semi-
static system was used and 50% of the water volume
was changed daily. The fish received a diet of
commercial feed (Vicente Alimentos S.A.; Presidente
Prudente/SP, Brazil) with 3.5% Ca2+, 28.0% crude
protein and 3500 kcal kg 1 digestible energy,
according to the manufacturer. Juveniles were fasted
for 24 hours prior to the experiments. The method-
ology used in this study was approved by the
Institutional Animal Care and Use Committee of
the Federal University of Santa Maria. The number
of animals used in each experiment was the lowest
possible in order to meet the policy of reduction of
experimental animals of the institution.
Comparison between the anesthetic activity of
S-(+)-linalool (Lin) and of the EO of L. alba
The fish were transferred to 1-L aquaria with Lin at
30, 60, and 180 lL L 1 (25.5, 51 and 153 mg L 1,
respectively, considering the density of 0.85 g mL 1)
or EO of L. alba at 50, 100, and 300 lL L 1,
previously solubilized in 95% ethanol (1:10). These
Lin concentrations were chosen in order to maintain
the proportion of Lin in the EO (60%) (Heldwein et al.
2012). Animals were maintained in this bath until
anesthesia, or at most 30 minutes if anesthesia was
not reached. Six juveniles were used for each
concentration tested, and each juvenile was used
only once. Previous investigation determined that the
concentration of ethanol used for dilution had no
effect on the anesthesia induction time (Cunha et al.
2010b). After induction of anesthesia or at the end
of 30 minutes for sedative concentrations, the ani-
mals were transferred to anesthetic-free aquaria to
assess their recovery time.
© 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 41, 621–629
Anesthetic evaluation in fish involves the obser-
vation of behavior parameters that characterize
some stages of induction (Schoettger & Julin
1967). These stages allow identification of: light
and deep sedation (stages 1 and 2, respectively),
total loss of equilibrium (stages 3a and 3b) and deep
anesthesia (stage 4) (Appendix).
Evaluation of the potentiation of the depressant
effects of Lin on the CNS by a BDZ
Different concentrations of Lin (30, 60, and
180 lL L 1) were combined with 150 lM BDZ
(diazepam obtained from DEG, SP, Brazil), previously
solubilized in Tween 80 at 0.033%, in 1-L aquaria.
Controls of BDZ with the vehicle of EO, EO with
vehicle of BDZ and both vehicles (Tween 80 and
ethanol in the same proportions used in the
solutions) were also performed. The number of
animals (n = 6 for each group) and the experimental
parameters evaluated were the same described for
the comparison between the anesthetic activity of
S-(+)-linalool (Lin) and of the EO of L. alba.
Antagonism of the depressor effects of Lin on the
CNS
To evaluate the possible benzodiazepinic-like mech-
anism, the fish were exposed to Lin or BDZ (diazepam)
until they reached stages of sedation (Lin 30 lL L 1
and BDZ 150 lM) or anesthesia (Lin 180 lL L 1)
(Heldwein et al. 2012). After induction, the animals
were separated into two groups (n = 6 per group).
One group was transferred to an anesthetic-free
aquarium with water and the second group was
placed in an aquarium containing 5 lM flumazenil
(Flumazil; Crist
alia, SP, Brazil), a BDZ antagonist. The
fish of both groups were submitted to an external
stimulus to facilitate the observation of their recovery
behavior, by tapping with a glass rod in the bottom of
the aquarium. The results were noted by scores at 1,
5, 10, 15, and 20 minutes. The scores of recovery
from anesthesia in fish according to Heldwein et al.
(2012) were: 0, no sign of recovery; 0.5, reaction
only after a caudal peduncle stimulus; 1, first sign of
recovery but without posture; 1.5, stopped after
erratic swimming; 2, normal swimming but without
reflex after an external stimulus; 2.5, stopped after
normal swimming but without reflex after an exter-
nal stimulus; 3, normal swimming with reflex after
an external stimulus. When the fish showed a stage of
agitation in comparison to animals of the other
623
S-(+)-Linalool anesthesia in catfish CG Heldwein et al.
recovery treatment, 0.5 was added to the score of
each time of observation. Afterwards, the scores of all
times were added for each evaluated fish.
Statistical analyses
To verify the homogeneity of variances, the data
were submitted to a Levene’s test. A t-test was used
to compare the anesthetic activity of the EO of L. alba
with the corresponding activity of Lin. One-way
ANOVA followed by Tukey’s test or Kruskal-Wallis and
Mann-Whitney tests were used in the potentializa-
tion test, when appropriate (version 17.0; SPSS
Statistics, IL, USA). The results of the reversion test
were compared by two-way ANOVA and Tukey’s test
with SigmaPlot (version 11.0; SYSTAT Software
Inc., CA, USA). Significance was set at a level of 95%
(p < 0.05). Data are presented as mean  SEM.
(a)
(b)
(c)
624 © 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 41, 621–629
Results
A relationship between concentration and induction
time was verified for Lin at stage 2 (ln y = 9.0591 –
1.0837 ln[x]) with r2 = 0.999, where y is the time
to reach the stage and x is the concentration of Lin
(lL L 1). Concentrations of 30 and 60 lL L 1 Lin
only achieved stage 2 (about 3.6 minutes) and stage
3a (about 2.6 minutes), respectively, within
30 minutes of observation. Fish reached stage 4
(about 11.8 minutes) only when exposed to
180 lL L 1. The results concerning the recovery
time demonstrated a positive relation between its
duration and the concentration of Lin, but the same
was not observed for the EO (Fig. 1).
There were significant differences between the
lowest concentrations of Lin and EO tested (30 and
50 lL L 1, respectively) in stage 1. Only animals
Figure 1 Time required for
induction and recovery from
anesthesia using the essential oil of
Lippia alba (EO) and linalool (Lin) in
silver catfish juveniles (n = 6 for
each experimental concentration).
(a) EO 50 lL L 1 and Lin
30 lL L 1; (b) EO 100 lL L 1 and
Lin 60 lL L 1; (c) EO 300 lL L 1
and Lin 180 lL L 1. The
maximum observation time was
30 minutes. *Significantly different
from EO in the same stage
(p < 0.05).
 S-(+)-Linalool anesthesia in catfish CG Heldwein et al.

exposed to 50 lL L 1 EO reached stage 3a (Fig. 1a).
At the intermediate concentrations tested
(60 lL L 1 Lin and 100 lL L 1 EO), significant
differences were observed for stages 1 and 2. Lin at
this concentration did not progress beyond stage 3a,
while fish exposed to 100 lL L 1 EO reached stage
3b and 4 (Fig. 1b). However, the highest concen-
trations evaluated (180 lL L 1 Lin and 300 lL L 1
EO) provided similar induction times for stages 2, 3a,
and 3b, whereas fish in Lin took significantly longer
induction time to reach stage 4 (Fig. 1c). Recovery
time from exposure to Lin at 60 lL L 1 was
increased compared to EO (Fig. 1b).
There was a significantly faster onset of sedation
with 30 lL L 1 of Lin + BDZ compared with Lin or
BDZ alone (p < 0.05), but not at higher concentra-
tions of Lin (Fig. 2a). Onset of stage 3a was
significantly faster with Lin + BDZ compared with
Lin or BDZ alone at the lower (30 lL L 1) and
higher (180 lL L 1) concentrations of Lin (Fig. 2b)
(p < 0.05).
Anesthesia (Stage 4) was produced by Lin and Lin
+ BDZ without statistical differences (Fig. 2c). More-
over, the recovery time for these groups did not
follow a concentration-response curve by regression
analysis. Animals treated with Lin + BDZ at concen-
trations of 30 and 60 lL L 1 showed longer recov-
ery times compared to animals treated with Lin and
BDZ alone (Fig. 2d).
The increase in the Lin concentration promoted a
reduction in the induction time to stage 3a. The
same pattern did not occur with Lin + BDZ in stage 2,
since differences were not detected between the
concentrations. The ethanol used for the dilution of
Lin and added proportionally to the controls of
benzodiazepinic drug (BDZ + vehicle of EO group)
had no significant effect on the time to anesthesia
induction of DBZ group. However, the recovery time

(a) (b)

(c) (d)

Figure 2 Time required for induction and recovery from anesthesia using linalool (Lin), diazepam (BDZ) and the
combination linalool + diazepam (Lin + BDZ) in silver catfish juveniles (n = 6 per treatment group). (a) stage 2; (b) stage 3a;
(c) stage 4; (d) recovery. The maximum observation time was 30 minutes. *Significant difference Lin + BDZ from Lin or BDZ
at the same concentration (p < 0.05). a,b,cDifferent lower case letters indicate a significant difference between concentrations
within the same group (Lin, BDZ or Lin + BDZ).

© 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 41, 621–629 625
S-(+)-Linalool anesthesia in catfish CG Heldwein et al.
Figure 3 Sum of the recovery scores for silver catfish
exposed to linalool (Lin) and benzodiazepine control (BDZ)
(n = 6 per treatment group). The maximum score possible
(quickest recovery) is represented by a dotted line at 17.5.
*Significant difference from recovery in water (p < 0.05).
of BDZ with ethanol demonstrated significant differ-
ences among concentrations when ethanol concen-
tration increased (p < 0.05) (Fig. 2d).
Fish exposed to BDZ presented a more rapid
recovery when in contact with flumazenil compared
to water (p < 0.05). Flumazenil had no impact on
recovery from Lin at both concentrations tested
(Fig. 3).
Discussion
The results demonstrated that Lin had sedative
activity in fish at low concentrations and an
anesthetic action at high concentrations. A sedative
effect induced by the inhalation of Lin alone has been
described previously in mice and humans (Sugawara
et al. 1998; Linck et al. 2009).
The results also confirmed the presence of (S)-
(+)-isomer of Lin isolated from EO of L. alba. This has
been previously reported to be present in this plant,
collected in different Brazilian localities (Siani et al.
2002). However, according to Sugawara et al.
(1998), only (R)-(-)-linalool and the racemic mixture
produce a sedative effect on beta waves of the
encephalogram of humans in response to hearing
environmental sound. On the other hand, both
enantiomers (R and S) demonstrated a similar
influence on the salivary cortisol levels of humans
(H€oferl et al. 2006). To the authors’ knowledge,
there are no studies in aquatic animals comparing
the effects of both isomers.
In comparison to other anesthetics, Lin induces
anesthesia at a higher concentration than reported
for eugenol (20–50 mg L 1) in silver catfish (Cunha
626 © 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 41, 621–629
et al. 2010b). In this study, Lin induced anesthesia
at similar times and concentrations to MS-222 in the
same fish species (Gressler et al. 2012), which is the
only substance approved by the Food and Drug
Administration (FDA) as a fish anesthetic agent
(Facßanha & Gomes 2005). In addition, Lin is already
considered as Generally Recognized as Safe (GRAS)
as a food additive by the same organization (Letizia
et al. 2003), which could favor its potential govern-
mental approval as an anesthetic drug for fish.
The time until induction of anesthesia with Lin at
180 lL L 1 does not correspond to the time until
induction of anesthesia observed with the EO of
L. alba at 300 lL L 1 in silver catfish, which
suggests that it is not the only active anesthetic
compound in this sample. Synergic interactions
between constituents were also described for other
plant extracts in relation to their actions on CNS
(Spinella 2002). These findings support the anes-
thetic effect of the EO described in previous studies
(Cunha et al. 2010a; Heldwein et al. 2012), whose
synergistic interaction between constituents may be
responsible for the final effect observed.
The slow recovery from Lin compared to EO at 60
and 180 lL L 1 may be a reflection of drug accu-
mulation in adipose tissue, which would increase the
recovery time after long exposure (Kiessling et al.
2009; Zahl et al. 2012). Similar results were also
observed for other compounds, such as globulol
which was isolated from essential oil of H. mutabilis
(Silva et al. 2013).
In the combination of BDZ with Lin, no significant
effect on the anesthetic activity of Lin was observed.
These results are distinct from those described
previously for the EO of L. alba containing 60% of
Lin (Heldwein et al. 2012) and indicates that the
combined use of Lin + BDZ in fish anesthesia does
not show a considerable advantage in relation to Lin
alone.
The benzodiazepinic receptor located adjacent to
the GABA receptor complex was described as being
the interaction site of the EO of L. alba in mice and fish
(Vale et al. 1999; Heldwein et al. 2012). According
to the animal model used, a higher sum of score in
water with flumazenil corresponds to faster recovery
and indicates a possible benzodiazepinic-like mecha-
nism (Heldwein et al. 2012). However, inclusion of
flumazenil had no impact on recovery scores in this
study indicating that the same interaction was not
observed for Lin. These results strengthen the
hypothesis that the anesthetic effect of the EO of
L. alba is the result of the synergism of different
 S-(+)-Linalool anesthesia in catfish CG Heldwein et al.
components and at least one of their constituents acts
on the BDZ site of the GABAergic system.
Nevertheless, the interaction of Lin with another
site of the GABA receptor or distinct receptor or
channel responsible for its sedative and anesthetic
actions cannot be excluded from these results. Our
results are similar as described by Brum et al. (2001)
that did not detect the direct interaction of Lin with
the GABAA receptors through [3H] muscimol binding
in mouse cortical membranes. The same authors also
did not discard the hypotheses that Lin produces
changes in the GABA-mediated neuronal inhibition
or effects on GABA release and uptake. In this
context, Hossain et al. (2002) verified that Lin
potentiates the GABA action on GABAA receptors
expressed in Xenopus oocytes, but this effect was not
related to the action on BDZ site. Furthermore,
Lin exhibited an inhibitory effect on N-methyl-D-
aspartate (NMDA) glutamate receptors (Brum et al.
2001). The literature describes the occurrence of
NMDA receptor in teleost fish (Linn & Gafka 1999),
and there is a relationship between this receptor and
anesthesia (Wang et al. 2009). Leal-Cardoso et al.
(2010) studied the pharmacologic effects of Lin in
somatic sensory neurons and demonstrated local
anesthetic activity in peripheral nerves and their
sensory ganglia related to the modulation of voltage-
dependent Na+ channels. Thus, the anesthetic mech-
anism of Lin may be related to these other mecha-
nisms, which should be further investigated in fish.
In conclusion, the results show that the anesthetic
effect of Lin isolated from the EO of L. alba in silver
catfish differs from that detected for the EO. While the
EO mediates an anesthetic effect by benzodiazepinic-
like mechanism, the major compound Lin does not
demonstrate this mechanism. These data are proba-
bly due to the complex composition of the plant
extract evaluated, and more than one compound
present may be responsible for the final activity.
Lin was effective in this species as a sedative at
concentrations of 30 lL L 1 and as an anesthetic at
180 lL L 1. More studies are necessary to evaluate
its anesthetic activity in other aquatic animals in
order to determine the ideal concentrations to be
used for sedation and anesthesia.
Acknowledgements
This study was supported by research funds from
~o de Amparo a
the Fundacßa  Pesquisa do Estado do
Rio Grande do Sul (FAPERGS/PRONEX, process
10/0016-8) and Conselho Nacional de Pesquisa e
© 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 41, 621–629
Desenvolvimento Cientıfico (CNPq, process 47
0964/2009-0). B. Baldisserotto, M. E. B€ urger, and
E. M. M. Flores are grateful to CNPq for their research
fellowships; C.G. Heldwein is grateful to Coordenacß~ ao
de Aperfeicßoamento de Pessoal de Nıvel Superior
(CAPES) by M.Sc. fellowship. The authors also thank
Gilberto D. Zanetti for the botanical identification of
L. alba, Br
aulio Otomar Caron and Denise Schmidt for
the cultivation of L. alba, and Ubiratan Flores da Silva
for the GC-MS analysis.
Conflict of interest
The authors have declared that there is no conflict of
interest.
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Appendix
Stages of anesthesia in fish (from Schoettger & Julin 1967)

Stage Description Behavioral response

1 Light sedation Partial loss of reaction to external stimuli

2 Deep sedation Partial loss of equilibrium, no reaction to external stimuli
3a Total loss of equilibrium Fish usually turn over but retain swimming ability
3b Total loss of equilibrium Swimming ability stops but responds to pressure on the
caudal peduncle
4 Anesthesia Loss of reflex activity, no reaction to strong external stimuli

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