Transportation of silver catfish, Rhamdia quelen, in water with eugenol and the essential oil of Lippia alba

Abstract

This study investigated the effectiveness of eugenol and of the essential oil (EO) of Lippia alba when used in the transport of the silver catfish (Rhamdia quelen). These investigations involved measurements of blood (pH, PvO(2), PvCO(2) and HCO(3) (-)) and water parameters, survival and ionoregulatory balance. Fish (301.24 ± 21.40 g, 28.90 ± 1.30 cm) were transported at a loading density of 169.2 g L(-1) for 4 h in fifteen plastic bags (7 L) divided into five treatments: control, 1.5 or 3.0 μL L(-1) of eugenol and 10 or 20 μL L(-1) of EO of L. alba. The water parameters were measured before (0 h) and after (4 h) transportation. The net Na(+), Cl(-) and K(+) losses were higher in fish from the control treatment compared to the other treatments. The PvO(2), PvCO(2) and HCO(3) (-) increased significantly in all of the treatments at the end of the transport period. In conclusion, based on the water (total ammonia nitrogen) and ionoregulatory indicators determined in the present study, our findings indicate that eugenol and the EO of L. alba are recommended for use in the transport of this species because these anesthetics apparently reduce stress.

Full text

Transportation of silver catfish, Rhamdia quelen, in water
with eugenol and the essential oil of Lippia alba
Alexssandro G. Becker •Thaylise V. Parodi •
Clarissa G. Heldwein •Carla C. Zeppenfeld •
Berta M. Heinzmann •Bernardo Baldisserotto
Received: 3 December 2010 / Accepted: 24 September 2011 / Published online: 5 October 2011
ÓSpringer Science+Business Media B.V. 2011
Abstract This study investigated the effectiveness
of eugenol and of the essential oil (EO) of Lippia alba
when used in the transport of the silver catfish
(Rhamdia quelen). These investigations involved
measurements of blood (pH, PvO
2
,PvCO
2
and
HCO
3-
) and water parameters, survival and ionoreg-
ulatory balance. Fish (301.24 ±21.40 g, 28.90 ±
1.30 cm) were transported at a loading density of
169.2 g L
-1
for 4 h in fifteen plastic bags (7 L)
divided into five treatments: control, 1.5 or
3.0 lLL
-1
of eugenol and 10 or 20 lLL
-1
of EO
of L. alba. The water parameters were measured before
(0 h) and after (4 h) transportation. The net Na
?
,Cl
-
and K
?
losses were higher in fish from the control
treatment compared to the other treatments. The PvO
2
,
PvCO
2
and HCO
3-
increased significantly in all of the
treatments at the end of the transport period. In
conclusion, based on the water (total ammonia nitro-
gen) and ionoregulatory indicators determined in the
present study, our findings indicate that eugenol and
the EO of L. alba are recommended for use in the
transport of this species because these anesthetics
apparently reduce stress.
Keywords Anesthesia Sedation Ion fluxes 
Fish transport
Introduction
The transport of fishes is influenced by many factors,
including the duration of transportation, loading
density (Carneiro et al. 2009), temperature (Golombi-
eski et al. 2003), water physicochemical parameters,
size and physical condition of the fish, and duration of
the depuration period before fish transportation (Berka
1986). The transportation of fishes in Brazil involves
the use of plastic bags. The limitations of this system
include the supply of oxygen and the build-up of
ammonia and carbon dioxide produced during trans-
port (Gomes et al. 1999,2006a,b; Golombieski et al.
2003; Carneiro et al. 2009). Previous studies regarding
the transport of silver catfish, R. quelen, in plastic bags
have evaluated different loading densities (Carneiro
et al. 2009), times, temperatures (Golombieski et al.
2003) and salt concentrations (Gomes et al. 1999).
Anesthetics such as MS-222, benzocaine hydrochlo-
ride, 2-phenoxyethanol and lidocaine hydrochloride
have been used to reduce stress responses during live
A. G. Becker T. V. Parodi C. C. Zeppenfeld 
B. Baldisserotto (&)
Departamento de Fisiologia e Farmacologia, Universidade
Federal de Santa Maria, 97105-900 Santa Maria, RS,
Brazil
e-mail: bbaldisserotto@hotmail.com
C. G. Heldwein B. M. Heinzmann
Departamento de Farma
´cia Industrial, Universidade
Federal de Santa Maria, 97105-900 Santa Maria, RS,
Brazil
123
Fish Physiol Biochem (2012) 38:789–796
DOI 10.1007/s10695-011-9562-4

fish transportation (Carmichael et al. 1984; Ferreira et al.
1984; Teo et al. 1989; Singh et al. 2004;Parketal.
2009). Several studies with native Brazilian fishes
reported the use of eugenol [(2-methoxy-4-(2-propenyl)
phenol, the major component of clove oil (70–90% of
weight)] or clove oil, as an anesthetic (Inoue et al. 2005;
Roubach et al. 2005; Vidal et al. 2006; Gonc¸alves et al.
2008; Cunha et al. 2010b). The essential oil (EO) of
L. alba (Mill.) N.E. Brown (Verbenaceae), an aromatic
shrub with important medicinal properties, is a new
anesthetic for fish (Cunha et al. 2010a,2011). Eugenol
and the EO of L. alba can be used to anesthetize silver
catfish. At concentrations of 50 and 300 lLL
-1
(equivalent to 50 and 240 mg L
-1
), respectively, euge-
nol and the EO of L. alba inhibited the increase in
plasma cortisol levels after handling (Cunha et al.
2010a,b). However, no studies on the use of these
anesthetics in fish transportation have been performed.
Therefore, the aim of this study was to investigate the
effectiveness of eugenol and of EO of L. alba for use
during the transport of silver catfish. The study used the
following indicators: blood and water parameters,
survival and ionoregulatory balance.
Materials and methods
Experimental procedure
Silver catfish (301.24 ±21.40 g, 28.90 ±1.30 cm)
were captured from a cage net inside an earth pond at
the fish culture sector at the Universidade Federal de
Santa Maria campus, Santa Maria, Southern Brazil. Fish
did not go through a depuration period because this
procedure, although recommended (Amend et al. 1982),
is not followed by most fish producers in southern Brazil
(Golombieski et al. 2003). Fish were transported at a
loading density of 169.2 g L
-1
for 4 h in fifteen plastic
bags with 7 L of water and 8 L of pure oxygen, and they
were divided into five treatments (three replicates each).
These treatments were as follows: control; 1.5 or
3.0 lLL
-1
of eugenol (Odontofarma
Ò
,PortoAlegre,
Brazil, equivalent to 1.5 or 3.0 mg L
-1
, respectively,
because the density of this anesthetic is about 1.06) and 10
or 20 lLL
-1
of the EO of L. alba (equivalent to 8 or
16 mg L
-1
, respectively, because the density of this EO
is about 0.80) (both first diluted in ethanol; 1:10). The
transport time was chosen to reduce mortality at this
loading density (Golombieski et al. 2003). The
concentrations of the EO of L. alba in water were within
the range that induced only slight sedation in silver catfish
within 6 h of exposure (5–20 lLL
-1
,equivalentto
4–16 mg L
-1
, respectively) (Cunha et al. 2010a). The
eugenol concentrations used in our study were about 10-
to 20-fold lower than those causing deep anesthesia in
silver catfish within 15 min of exposure (20–50 lLL
-1
)
(Cunha et al. 2010b). A pilot study with 10 fish exposed to
1.5 or 3.0 lLL
-1
of eugenol demonstrated that they only
reached slight sedation within 6 h.
Another experiment evaluated the ventilatory fre-
quency (VF) of the fish exposed to all treatments
(n =6 fish by treatment): control, 1.5 or 3.0 lLL
-1
of eugenol, and 10 or 20 lLL
-1
of the EO of L. alba.
The VF was determined following Alvarenga and
Volpato (1995): the VF per minute was quantified by
visually counting 20 successive opercular or buccal
movements, measuring the elapsed time with a
chronometer. The fish (one fish per aquarium) were
maintained in aquaria (19.3 913.7 911 cm) with
1 L of water and the respective anesthetic concentra-
tions. The times chosen to evaluate the VF were 0, 0.5,
1, 2, 3 and 4 h.
The methodology of this experiment was approved
by the Ethical and Animal Welfare Committee of the
Universidade Federal de Santa Maria (Process no
046/2010).
Plant material
L. alba was cultivated in the experimental area of
the Departamento de Fitotecnia, UFSM campus.
The aerial parts of the plant were collected in July
2008. The plant material was identified by botanist
Dr. Gilberto Dolejal Zanetti, Departamento de
Farma
´cia Industrial, UFSM, and a voucher specimen
(SMDB No. 10050) was deposited in the herbarium
of the Departamento de Biologia, UFSM.
Essential oil extraction
Essential oil was obtained from the fresh leaves of the
plant by steam distillation for 2 h using a Clevenger-
type apparatus. In this method, the distillate is
collected in a graduated glass tube, and the aqueous
phase is automatically reused by returning it to the
distillation flask (European Pharmacopoeia 2007).
The EO samples were stored at -20°C in amber glass
bottles.
790 Fish Physiol Biochem (2012) 38:789–796
123

Water sampling and analyses
Water parameters were measured before and after
transportation. Dissolved oxygen (DO) and tempera-
ture were measured with a YSI oxygen meter (Model
Y5512; YSI Inc., Yellow Springs, OH, USA). The pH
was verified with a DMPH-2 pH meter (Digimed, Sa
˜o
Paulo, SP, Brazil). Nesslerization verified total ammo-
nia nitrogen (TAN) levels according to the method of
Eaton et al. (2005). Un-ionized ammonia (NH
3
) levels
were calculated according to Colt (2002).Water hard-
ness was analyzed by the EDTA titrimetric method.
Alkalinity was determined according to Boyd and
Tucker (1992). Carbon dioxide (CO
2
) was calculated
by the method of Wurts and Durborow (1992).
Ion fluxes
Water samples (5 mL) were collected before and
after transportation. Chloride levels were determined
according to Zall et al. (1956), and Na
?
,K
?
and Ca
2?
levels were determined with a B262 flame spectropho-
tometer (Micronal, Sa
˜o Paulo, Brazil). Standard solu-
tions were made with analytical-grade reagents (Vetec
or Merck) dissolved in deionized water, and standard
curves of each ion to be tested were made for five
different concentrations. Net ion fluxes were calcu-
lated according to Gonzalez et al. (1998):
Jnet ¼Vion1
½ion2
½ðÞ
Mt
where [ion
1
] and [ion
2
] are the ion concentrations in
the water of transport at the beginning and end of the
transport period, respectively, Vis the water volume
(in L), Mis the mass of the fish (in kg) and tis the
duration of the transport (in h).
Blood sampling and analyses
The mixed venous-arterial blood samples (1–1.5 mL)
were collected from the caudal vein of each fish using
heparinized 3-mL syringes before and after the
transporting procedure. This caudal vein is commonly
used for the collection of blood samples in many
species of fish, but because of the proximity of the vein
to an artery, samples are often mixtures of venous and
arterial blood (Sladky et al. 2001; Hanley et al. 2010).
The blood samples were kept in ice. The following
variables were measured using a clinical analyzer
(OMNI C 2413, Roche
Ò
, Rio de Janeiro, RJ, Brazil):
pH, PvO
2
,PvCO
2
, hematocrit (Hct) and HCO
3-
.
The temperature of the clinical analyzer is commonly
37°C, but to determine blood gases, it was corrected to
water temperature (20°C) with the assumption that
ambient water temperature and individual fish body
temperatures were equivalent (Hanley et al. 2010). In
addition, Howell et al. (1970) reported that ectotherm
vertebrates, including fish, maintain an acid–base
balance despite changes in body temperature.
Statistical analyses
All data are expressed as mean ±SEM. Homogeneity
of variances among treatments was tested with the
Levene test. Data exhibited homogeneous variances,
so comparisons between different treatments and
times were made using one-way ANOVA and Tukey’s
test. Analysis was performed using the software
Statistica ver. 5.1 (StatSoft, Tulsa, OK), and the
minimum significance level was set at P\0.05.
Results
Water parameters and mortality
No mortality was recorded in any treatment following
transport. After transport, the highest DO levels and
lowest CO
2
levels were found in the control and in the
10 lLL
-1
of EO L. alba treatment, respectively.
Total alkalinity, pH and NH
3
levels in the water did
not exhibit any significant differences between the
treatments at the end of transport. In addition, the TAN
levels were significantly higher in the control com-
pared with the other groups. Water hardness and
temperature did not exhibit any significant differences
between treatments after transport (Table 1).
Ion fluxes through transportation
The net Na
?
,Cl
-
and K
?
effluxes were significantly
higher in fish from the control treatment compared
with fish in the other treatments. Moreover, the lowest
net Cl
-
and K
?
effluxes were found for the treatments
with 1.5 lLL
-1
of eugenol and 10 and 20 lLL
-1
EO
of L. alba, respectively. The net Ca
2?
fluxes did not
show any significant difference between treatments
(Fig. 1).
Fish Physiol Biochem (2012) 38:789–796 791
123

Blood parameters
The highest PvO
2
,PvCO
2
and HCO
3-
values after
transport were found in the treatments with
3.0 lLL
-1
eugenol and 20 lLL
-1
EO of L. alba.
Blood pH was not affected by treatments (Table 2).
Ventilatory frequency
The VF at 0 h was significantly lower in fish from the
control treatment compared with the other treatments.
The highest VF at 0.5 h was found in the treatments
with 3.0 lLL
-1
of eugenol and 20 lLL
-1
EO of
L. alba. After 1 h of exposure, there was no significant
difference between treatments, but at 2, 3 and 4 h, the
VF was significantly lower in all treatments with
anesthetics compared to the control treatment.
In all treatments with anesthetic, there was a
significant increase in the VF after 0.5 h of exposure
when compared with the other times. However, in the
control treatment, there was a significant decrease in
VF in the first half hour. VF remained constant at the
other times (Table 3).
Discussion
The lethal concentrations (96 h) of TAN and NH
3
for
silver catfish in normoxic conditions (total hardness:
20 mg CaCO
3
L
-1
;25°C) are 7.73 and 0.44 mg L
-1
,
respectively, at pH 6.0 (Miron et al. 2008). Total
ammonia and NH
3
levels were much lower at the end of
the transport in the present study than lethal values.
Therefore, silver catfish could be transported for a
longer period without problems due to ammonia
toxicity under the conditions used in these experiments
(weight of 300 g, density of 169.2 g L
-1
, transported
by 4 h). The TAN excretion by silver catfish trans-
ported in our study was 7.92 mg kg
-1
fish h
-1
, about
Table 1 Water parameters before and after transport (4 h) of silver catfish in plastic bags with eugenol and the essential oil of Lippia
alba added to the water
Water parameter Before
transport
After transport (treatments)
Control Eugenol
(1.5 lLL
-1
)
Eugenol
(3.0 lLL
-1
)
L. alba
(10 lLL
-1
)
L. alba
(20 lLL
-1
)
Dissolved oxygen 12.27 ±0.20 7.63 ±0.46*a 6.58 ±0.41*b 5.55 ±0.83*b 7.77 ±0.52*a 6.12 ±0.61*b
Carbon dioxide 12.56 ±0.41 40.51 ±1.09*c 56.72 ±1.16*a 57.55 ±0.53*a 49.63 ±1.06*b 55.26 ±0.61*a
Alkalinity 18.60 ±1.12 27.70 ±0.90*a 25.50 ±1.00*a 27.20 ±0.80*a 25.80 ±0.90*a 26.20 ±0.80*a
Water hardness 29.46 ±1.78 29.60 ±1.90a 30.70 ±1.80a 29.80 ±2.10a 29.30 ±1.70a 32.00 ±1.90a
pH 6.78 ±0.07 5.90 ±0.08*a 5.77 ±0.07*a 5.80 ±0.05*a 5.91 ±0.07*a 5.83 ±0.06*a
Temperature 20.10 ±0.25 20.60 ±0.32a 20.49 ±0.27a 20.53 ±0.34a 20.61 ±0.36a 20.57 ±0.26a
Total ammonia
nitrogen
1.25 ±0.11 5.36 ±0.26*a 4.36 ±0.24*b 4.37 ±0.25*b 4.40 ±0.25*b 4.44 ±0.21*b
Un-ionized
ammonia
0.0030 0.0018*a 0.0010*a 0.0011*a 0.0015*a 0.0012*a
Values are means ±SEM. Asterisks indicate significant differences when compared to values before transport (P\0.05). Different
letters in the rows indicate significant differences between treatments after transport (P\0.05). Dissolved oxygen, carbon dioxide,
total ammonia nitrogen and un-ionized ammonia were expressed as mg N L
-1
. Alkalinity and water hardness were expressed as mg
CaCO
3
L
-1
Net ion fluxes (µmol.kg-1.h-1)
-700
-600
-500
-400
-300
-200
-100
0
100
200
control
eugenol (1.5 µL L-1)
eugenol (3.0 µL L
-1
)
Lippia alba
(10 µL L
-1
)
Lippia alba
(20 µL L
-1
)
a
bb
a
bb
a
b
d
b
c
e
c
d
d
aaaa
a
Na+Cl-K+Ca2+
Fig. 1 Net ion (Na
?
,Cl
-
,K
?
and Ca
2?
) fluxes measured for
the transport of silver catfish in plastic bags with eugenol and the
essential oil of Lippia alba added to the water. Values are
means ±SEM. Different letters indicate significant differences
between treatments for the same ion (P\0.05)
792 Fish Physiol Biochem (2012) 38:789–796
123

2.36-fold lower than reported by Carneiro et al. (2009)
(18.68 mg kg
-1
fish h
-1
) with silver catfish (weight
of 20 g, loading density of 150 g L
-1
, transported by
4 h). This result was expected because ammonia
excretion decreases with increasing fish mass in silver
catfish (Bolner and Baldisserotto 2007).
In our study, the DO levels after 4 h of transport
still remained within a safe range for silver catfish
(control group—7.63 mg L
-1
) (Braun et al. 2006)
because pure oxygen was added to the plastic bags.
Oxygen consumption was lower than observed by
Golombieski et al. (2003) for the transport of silver
catfish for 6 h (weight of 1.0–2.5 g, loading density of
168 g L
-1
). Silver catfish could reach stage 4 of
anesthesia when exposed to concentrations between
20 and 50 lLL
-1
of eugenol and above 100 lLL
-1
(equivalent to 80 mg L
-1
)EOL. alba within 15 min
(Cunha et al. 2010a,b). This stage is characterized by
the loss of reflex activity (i.e., reduction in the
opercular movement) and by a lack of reaction to
strong external stimuli (Schoettger and Julin 1967).
The anesthetic concentrations used in fish transport
must induce, at most, stage 2 of anesthesia (stage of
deep sedation). Partial loss of equilibrium and lack of
reaction to external stimuli are observed in this stage.
Largemouth black bass, Micropterus salmoides,
exposed to MS-222 (tricaine methanesulfonate)
showed enhanced survival of and a reduction in
stress parameters (plasma glucose and corticosteroids
decreased and plasma chloride and osmolality
increased) during transport compared to fish trans-
ported in water without this anesthetic (Carmichael
et al. 1984). Moreover, the use of benzocaine hydro-
chloride (25 mg L
-1
) on Mozambique tilapia,
Oreochromis mossambicus, reduced oxygen con-
sumption at about 1/3 and decreased ammonia and
CO
2
excretion (Ferreira et al. 1984). In the fry of the
Indian carp Catla catla,Labeo rohita and Cirrhinus
Table 2 Blood parameters before and after transport of silver catfish in plastic bags with eugenol and the essential oil of Lippia alba
added to the water
Blood parameter Before
transport
After transport (treatments)
Control Eugenol
(1.5 lLL
-1
)
Eugenol
(3.0 lLL
-1
)
L. alba
(10 lLL
-1
)
L. alba
(20 lLL
-1
)
pH 7.33 ±0.07 7.24 ±0.03a 7.24 ±0.06a 7.25 ±0.05a 7.29 ±0.05a 7.27 ±0.03a
PvO
2
(mm Hg) 8.99 ±0.54 16.47 ±0.68*b 14.25 ±0.61*b 22.59 ±0.89*a 15.24 ±0.82*b 20.53 ±1.13*a
PvCO
2
(mm Hg) 11.54 ±1.33 23.81 ±0.51*b 21.48 ±0.66*b 27.22 ±0.73*a 23.07 ±0.91*b 27.10 ±0.75*a
Hct (%) 32.64 ±0.84 26.16 ±1.25*a 28.09 ±1.00*a 26.47 ±1.07*a 27.72 ±1.00*a 26.56 ±0.62*a
HCO
3-
(mmoL L
-1
)
7.35 ±0.36 12.05 ±0.14*b 12.41 ±0.41*b 14.07 ±0.16*a 12.87 ±0.12*b 14.82 ±0.33*a
Values are means ±SEM. Asterisks indicate significant differences when compared to values before transport (P\0.05). Different
letters in the rows indicate significant differences between treatments after transport (P\0.05)
Table 3 Ventilatory frequency (opercular or buccal movements min
-1
) measured in silver catfish maintained in water with eugenol
and the essential oil of Lippia alba
Time of exposure (h) Treatments
Control Eugenol
(1.5 lLL
-1
)
Eugenol
(3.0 lLL
-1
)
L. alba
(10 lLL
-1
)
L. alba
(20 lLL
-1
)
0 93.02 ±1.02Ba 101.61 ±1.04Ab 100.42 ±1.34Ab 101.44 ±0.82Ab 105.26 ±0.69Ab
0.5 81.24 ±0.44Cb 111.94 ±0.58Ba 126.58 ±0.35Aa 106.10 ±0.51Ba 121.95 ±0.21Aa
1 72.16 ±0.44Ac 76.58 ±0.60Ac 77.77 ±0.70Ac 67.30 ±0.75Ac 71.30 ±0.75Ac
2 65.25 ±0.88Ac 53.52 ±0.78Bd 51.77 ±0.28Bd 51.06 ±1.05Bd 53.31 ±1.05Bd
3 61.60 ±1.20Ac 47.83 ±0.57Be 45.37 ±1.66Be 43.23 ±1.05Be 42.55 ±0.86Be
4 68.03 ±1.17Ac 45.75 ±0.67Be 42.18 ±1.76Be 41.72 ±0.95Be 43.37 ±0.81Be
Values are means ±SEM. Different capital letters in the rows indicate significant differences between treatments in the same time
(P\0.05). Different lowercase letters in the rows indicate significant differences between times in the same treatment (P\0.05)
Fish Physiol Biochem (2012) 38:789–796 793
123

mrigala (0.09 mg L
-1
), this treatment also decreased
NH
3
excretion (Singh et al. 2004). Park et al. (2009)
suggested that lidocaine hydrochloride at concentra-
tions of 5, 10 or 20 mg L
-1
decreased the metabolic
activity of flounder, Pleuronectes americanus,
because this substance reduced ammonia excretion
(about 27.4–30.5%) and oxygen consumption (about
82.7–86%) compared with a control group after 5 h
transport time.
Eugenol and EO of L. alba in the water used in
transport reduced ammonia excretion by silver catfish
during transport. These findings are in agreement with
those reported by Guo et al. (1995) and Park et al.
(2009). These studies found that the overall reduction
in ammonia excretion could be directly related
to a decrease in the metabolic rate produced by
anesthetics.
Stress conditions such as transport and handling
increase gill blood flow and paracellular permeability.
In freshwater fishes, the result of these changes is ionic
loss (Cech et al. 1996; McDonald et al. 1991).
Common salt has been added to the water used in
transport to reduce the osmotic gradient between the
water and fish plasma. This treatment produces
positive results in several species (Barton and Peter
1982; Carneiro and Urbinati 2001) but not in silver
catfish (Gomes et al. 1999) or in pirarucu, Arapaima
gigas (Gomes et al. 2006b). In the present study,
eugenol and the EO of L. alba in the water of transport
reduced ion loss in silver catfish. This effect was
probably the result of lower gill blood flow that
occurred because the fish were less agitated. More-
over, Cunha et al. (2010a,b) reported that the cortisol
levels did not increase in silver catfish subjected to
handling while anesthetized with eugenol or EO of
L. alba. In addition, other studies (Guo et al. 1995;
Singh et al. 2004; Park et al. 2009) also reported that
the anesthetics used for fish transport reduce agitation
and fish stress.
The blood pH values found in the present study,
regardless of treatment, were similar to or slightly
lower than those reported for tambaqui exposed to
different water pHs (Wood et al. 1998): red pacu
(Piaractus brachypomus) exposed to MS-222 and
eugenol at 50, 100 and 200 mg L
-1
(Sladky et al.
2001); and yellow perch (Perca flavescens), walleye
pike and koi (Cyprinus carpio) anesthetized with
MS-222 (150 mg L
-1
) and buffered with NaHCO
3
(75 mg L
-1
) (Hanley et al. 2010). The blood gas
values (PvO
2
,PvCO
2
and HCO
3-
) before transport
were similar to or lower than those reported by other
studies (Sladky et al. 2001; Souza et al. 2001; Hanley
et al. 2010).
Exposure of silver catfish to eugenol or EO of
L. alba apparently decreased metabolic rate because
fish presented significantly lower ammonia excre-
tion, VF (through all transport time) and net ion
loss. However, DO and carbon dioxide levels in the
water of transport of silver catfish transported with
both anesthetics were significantly lower and higher,
respectively, than in the water of transport of control
fish, indicating the opposite: an increase in meta-
bolic rate through transport. A possible explanation
to these conflicting results would be that eugenol
and EO of L. alba would not reduce metabolic rate
but would decrease ammonia excretion. This lower
ammonia excretion would induce an increase in
plasma ammonia levels. High plasma ammonia levels
did not change PaO
2
but increased PaCO
2
and
plasma HCO
3-
in rainbow trout (Zhang and Wood,
2009), and similarly, silver catfish transported in
water with 3 lLL
-1
eugenol or 20 lLL
-1
EO L.
alba exhibited the highest values of PvCO
2
and
HCO
3-
in the blood at the end of transportation.
Nevertheless, plasma ammonia was not measured in
the present experiment, and according to Zhang and
Wood (2009), high plasma ammonia induced hyper-
ventilation in rainbow trout, which was not observed
in silver catfish. Additional experiments are neces-
sary to explain these results.
In the present study, the Hct values (26–33%) were
similar to those found by Carneiro et al. (2009) in the
same species (Hct: 27–30%). The Hct values
decreased after transport but showed no significant
differences between treatments. Transport procedures
are examples of conditions that can produce stress.
Such stress could decrease Hct. These considerations
suggest a hemodilution caused by osmoregulatory
disturbance (Houston et al. 1996; Morgan and Iwama
1997).
In conclusion, on the basis of the findings regarding
water (TAN) and osmoregulatory indicators obtained
by the present study, our results suggest that the use of
eugenol and EO of L. alba is advisable for the
transport of silver catfish. Additional experiments
using higher loading densities would also be of interest
in order to assess the importance of these anesthetics in
more stressful situations.
794 Fish Physiol Biochem (2012) 38:789–796
123

Acknowledgments This study was supported by research
funds from the Fundac¸a
˜o de Amparo a
`Pesquisa do Estado do
Rio Grande do Sul (FAPERGS/PRONEX, process 10/0016-8)
and Conselho Nacional de Pesquisa e Desenvolvimento
Cientı
´fico (CNPq, process 470964/2009-0). B. Baldisserotto
and A. G. Becker received research and PhD fellowships,
respectively, from CNPq. T.V. Parodi and C.G. Heldwein
received PhD and MSc fellowships, respectively, from
Coordenac¸a
˜o de Aperfeic¸oamento de Pessoal de Nı
´vel
Superior (CAPES). The authors are grateful to Dr. Gilberto
Dolejal Zanetti for the identification of L. alba.
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