Anesthesia induces stress in Atlantic salmon (Salmo salar),Atlantic cod (Gadus morhua) and Atlantic halibut

(Hippoglossus hippoglossus)

Inger Hilde Zahl Æ Anders Kiessling Æ

Ole Bent Samuelsen Æ Rolf Erik Olsen

Received: 11 February 2009 / Accepted: 17 June 2009 / Published online: 13 August 2009

� Springer Science+Business Media B.V. 2009

Abstract Stress in response to anesthesia with

benzocaine, MS-222, metomidate and isoeugenol

was studied in Atlantic salmon (Salmo salar), Atlan-

tic halibut (Hippoglossus hippoglossus), and Atlantic

cod (Gadus morhua) with no concomitant stress from

handling or confinement in association with anesthe-

sia or sampling. All of the anesthetics tested induced

a stress response in all species, displayed by a release

of cortisol to the water. MS-222 anesthesia elicited

the highest cortisol release rates, reaching maximum

levels 0.5 h post-exposure and returning to basal

levels after 3–4 h. Benzocaine anesthesia caused a

bimodal response where the initial peak in cortisol

release rate was followed by a second increase lasting

towards the end of the trial (6 h). This bimodality was

more profound in Atlantic salmon than in Atlantic

halibut and Atlantic cod. Metomidate anesthesia

induced the lowest release of cortisol of the agents

tested in both Atlantic halibut and Atlantic cod, but

resulted in a bimodal response in Atlantic salmon

where the initial increase in cortisol release was

followed by a larger increase peaking at 2–2.5 h post

exposure before returning to basal levels after 5 h.

The stress induced in Atlantic salmon by isoeugenol

anesthesia resembled that of MS-222, but did not

reach the same elevated level. Overall, the cortisol

release was most profound in Atlantic salmon

followed by Atlantic halibut and Atlantic cod.

Keywords Anesthesia � Stress � Cortisol �

MS-222 � Benzocaine � Metomidate �

Isoeugenol

Introduction

During commercial fish farming, the fish are sub-

jected to various practices of handling and confine-

ment. All these situations are associated with acute

stress, which in addition to producing some charac-

teristic stress reactions in the fish all give rise to a

non-specific generalized physiological response,

commonly described by three stages (Wendelaar

I. H. Zahl (&) � O. B. Samuelsen � R. E. Olsen

Institute of Marine Research, P.O. Box 1870, Nordnes,

5817 Bergen, Norway

e-mail: inger.hilde.zahl@imr.no

A. Kiessling

Department of Animal and Aquacultural Sciences,

Norwegian University of Life Sciences, P.O. Box 5003,

1432 Aas, Norway

I. H. Zahl � O. B. Samuelsen

Department of Biology, University of Bergen,

P.O. Box 7800, 5020 Bergen, Norway

A. Kiessling

Department of Wildlife, Fish and Environmental Studies,

Swedish University of Agricultural Sciences, 901 83

Umea, Sweden

123

Fish Physiol Biochem (2010) 36:719–730

DOI 10.1007/s10695-009-9346-2

Bonga 1997). The initial reaction is associated with

an activation of neuroendocrine pathways leading to

a massive release of hormones, primarily catechola-

mines and corticosteroids (Donaldson 1981; Mazeaud

and Mazeaud 1981; Mommsen et al. 1999; Reid et al.

1998). This hormonal release results in mobilization

of energy stores to meet the increased energy demand

and stimulates the cardiovascular and respiratory

function, which is important for the fish in overcom-

ing the stressful event and regaining homeostasis.

However, situations of severe stress, and situations of

chronic or recurrent incidents of stress where the fish

are not given time to recover have detrimental effects

which eventually lead to a general impairment of

condition and performance (Barton 2002; Davis

2006; Jentoft et al. 2005; Pickering and Pottinger

1989). These harmful effects from stress are related

to elevated levels of corticosteroids, primarily corti-

sol (Barton and Iwama 1991; Maule et al. 1989;

Mommsen et al. 1999; Pickering and Pottinger 1989).

Cortisol is synthesized and released more slowly than

the catecholamines noradrenaline and adrenaline and

returns more slowly to basal level (Olsen et al. 2002,

2008; Pickering and Pottinger 1989; Sumpter et al.

1986). Cortisol has therefore been widely used as a

parameter in studies of stress in fish and has revealed

that large variations in the magnitude of the response

exist both between and within species (Barton 2000;

Barton and Iwama 1991; Pottinger and Moran 1993;

Pottinger et al. 1994).

In order to facilitate handling, reduce the risk of

injuries, reduce the risk of inflicting pain, and prevent

stress, anesthetic agents are used. While several

studies report that anesthetics are effective in reduc-

ing the stress associated with confinement and

handling (Davis and Griffin 2004; Iversen et al.

2003; Olsen et al. 1995; Sink et al. 2007; Small 2004;

Small and Chatakondi 2005; Thomas and Robertson

1991) some also indicate that the exposure to

anesthetic agents in itself induces a stress response,

measured by increased levels of cortisol (Barton and

Peter 1982; Davidson et al. 2000; Davis and Griffin

2004; Kiessling et al. 2009; Molinero and Gonzalez

1995; Thomas and Robertson 1991). In a preliminary

study carried out at our laboratory, markedly elevated

plasma cortisol levels were found in juvenile Atlantic

salmon (Salmo salar) subjected to stress in the form

of netting although a preventive treatment with

anesthetics had been applied prior to the stressor

(Kiessling, Johansson and Axen, pers. comm.). This,

in combination with work using adult Atlantic salmon

fitted with dorsal aorta cannulae for studying con-

current plasma cortisol and clearance of anesthetic

agents (Kiessling et al. 2009) clearly indicated that

anesthetics could themselves induce a severe stress

reaction, and that the reaction varied consistently

between different agents. However, neither of the

above-mentioned studies was designed to study the

direct causality between exposure to anesthetic

agents and the induction of a stress reaction.

MS-222, benzocaine, metomidate and isoeugenol

are among the most common anesthetic agents used

for fish (Ackerman et al. 2005; Ross and Ross 2008).

MS-222 and benzocaine are local anesthetics that

inhibit neural signal transmission by blocking voltage

sensitive sodium channels (Frazier and Narahashi

1975; Neumcke et al. 1981). Isoeugenol is similar to

eugenol, a widely used analgesic in dentistry which

inhibits sodium, potassium, and calcium channels,

inhibits N-methyl-D-aspartate (NMDA) receptors and

potentiates inhibitory gamma-aminobutyric acid type

A (GABAA) receptors (Aoshima and Hamamoto

1999; Lee et al. 2005; Li et al. 2007; Park et al. 2006;

Wie et al. 1997). Metomidate is a methyl analogue of

etomidate, a non-barbiturate hypnotic that modulates

and activates GABAA receptors (Ashton and Wau-

quier 1985; Grasshoff et al. 2006; Rusch et al. 2004;

Yang and Uchida 1996). Metomidate has been found

to inhibit the cortisol stress response in fish (Olsen

et al. 1995; Thomas and Robertson 1991). The aim of

the current study was to examine whether exposure to

these anesthetic agents would elicit a stress response

in Atlantic salmon, Atlantic cod (Gadus morhua), and

Atlantic halibut (Hippoglossus hippoglossus). The

non-invasive method developed by Ellis et al. (2004)

for assessing the cortisol status of fish by measuring

the level of cortisol released to water was used in

order to obtain relative measurements over time

without disturbing the fish.

Materials and methods

Experimental fish

Atlantic cod and Atlantic halibut were produced at

IMR, Austevoll Research Station, Norway. Upon

reaching an average weight of 400 g (Atlantic cod)

720 Fish Physiol Biochem (2010) 36:719–730

123

and 200 g (Atlantic halibut), 114 Atlantic cod and

180 Atlantic halibut were randomly distributed into

six 600-l tanks to a total biomass of 25 kg m-3 and

20 kg m-3, respectively. The tanks were supplied

with aerated seawater at a flow rate of 20 l min-1

with an average temperature of 9 ± 1�C. The fish

were kept under natural light and were fed daily by

automatic feeders a commercial marine diet (Skrett-

ing, Stavanger, Norway).

Atlantic salmon (Aqua Gen strain) were obtained

from Aqua Gen, Hemne, Norway, and raised to

experimental size at IMR, Matre, Norway. At an

average weight of 420 g the Atlantic salmon were

randomly distributed in three tanks of 1,000 l, 48 fish

per tank, to a total biomass of 20 kg m-3. The tanks

were supplied with a continuous flow of aerated

seawater of 58 ± 6 l min-1 with a temperature of

9 ± 1�C. The fish were kept under natural light and

fed daily with a commercial marine diet (Biomar,

Myre, Norway) by automatic feeders.

The fish were allowed to adapt to the experimental

conditions for 1 month before experiments were

started and were given 1–2 weeks to recover between

each anesthetic exposure.

Experimental procedure

The fish were deprived of diets 24 h prior to the start

of the experiments and triplicates tanks were used

for each treatment. The experiment was initiated by

carefully stopping the water flow. When the valves

were closed the anesthetic was added to each tank

with as little disturbance to the fish as possible. The

water current generated by the newly stopped water

flow caused dispersion of the anesthetic agent

throughout the tanks. Control groups were treated

the same way except that no anesthetic was added.

The water flow was closed for 5 min allowing the

drug to affect the fish and then reopened to the

initial flow washing the anesthetic out. For water

cortisol analysis samples of 1 l were taken from the

inflowing water and from the outlet of each tank

prior to initiation of each experiment and then at

intervals of 0.5 h from 0.5 h to 4 h, and at 5 h and

6 h following administration of the anesthetic. All

samples were collected via the outlet of the tanks

without disturbing the fish and were stored at -20�C

until analyzed.

Anesthetic agents

The following anesthetic agents were tested: benzo-

caine (Benzoak� Vet, A.C.D Pharmaceuticals, Lek-

nes, Norway), tricaine methanesulfonate (MS-222�,

Pharmaq AS, Oslo, Norway), metomidate hydrochlo-

ride (Aquacalm�, Syndel International Inc., Vancou-

ver, Canada) and isoeugenol (Aqui-S�, Scan Aqua

AS, Aarnes, Norway). Manufacturer’s recommenda-

tions of dosages were followed. An overview of the

anesthetics and dosages administrated to each species

is presented in Table 1.

Water sample processing and cortisol analysis

The water samples were processed according to

Greenwood et al. (2001) and Ellis et al. (2004). Using

a peristaltic pump (Watson–Marlow 520S, Watson–

Marlow Bredel Pumps Ltd., Falmouth, UK) set at

25 ml min-1, the tank water samples were first

pumped through a pre-filter (AcroCapTMFilter, pore

size 0.45 lm, Pall Corporation, East Hills, USA) to

remove particles and then through an activated solid-

phase extraction cartridge, SPEC (Sep-pak� PlusC18,

55–105 lm, Waters Corporation, Milford, USA). The

pre-filters were rinsed with 20 ml of de-ionized water

prior to use and the SPECs were conditioned with

10 ml of methanol followed by 10 ml of de-ionized

water. After loading of the samples, the SPECs were

washed with 5 ml of de-ionized water and dried by

aspirating air, and thereafter stored at -20�C until

further processed.

To determine the exact volume of each water

sample, the water bottles were weighed before and

after pumping using a conversion factor of 1. The

Table 1 Anesthetic agents and dosages administrated to

Atlantic cod, Atlantic halibut, and Atlantic salmon

Benzocaine

(mg l-1)

MS-222

(mg l-1)

Metomidate

(mg l-1)

Isoeugenol

(mg l-1)

Atlantic cod 20 55 3 –

Atlantic

halibut

40 80 3 –

Atlantic

salmon

40 55a 3a 5.4a

MS-222, metomidate and isoeugenol were dissolved in water

before being added to the tanks (n = 3)a n = 2

Fish Physiol Biochem (2010) 36:719–730 721

123

SPECs were thawed and processed as described by

Sorensen and Scott (1994) as modified by Ellis et al.

(2004). In brief: the sample was eluted from the

SPECs using 5 ml of ethyl acetate. The eluate was

evaporated to dryness under nitrogen gas at 45�C and

the residue dissolved in 320 ll Calibrator Diluent

RD5-43, a buffered protein base included in the

cortisol immunoassay kit (ParameterTM KGE008,

R&D Systems Europe, Ltd., Abingdon, UK), which

was used in order to determine the cortisol concen-

tration. According to the specification of the immu-

noassay kit, the cross-reactivity with other steroids

was 4.4% with prednisolone, 1.7% with progesterone,

0.2% with cortisone, and\0.1% with corticosterone,

deoxycorticosterone, estradiol, and prednisone. The

intra- and inter-assay precision was 9.2% (n = 20)

and 21.1% (n = 40) (coefficient of variation, CV)

respectively, and the limit of sensitivity (minimum

detectable dose) was 0.071 ng l-1. The analysis was

carried out in compliance with the assay procedure of

the kit.

Calculation of cortisol release rates

The concentration of cortisol in each water sample

was calculated from the standard curve. Background

cortisol concentration of the inflowing water sampled

prior to initiation of each experiment (0.07 ± 0.01

ng l-1) was subtracted. The cortisol release rate

(ng g-1 h-1) of the fish in each tank was then

calculated from the fish biomass of the tank, the tank

water volume and water flow, according to Ellis et al.

(2004) and Folkedal et al. (in prep).

Statistics

The data were analyzed by the main factorial model

(general linear model, Statistical Analysis System

(SAS) for PC (ver.8.2), ANOVA for unbalanced data).

Included in the model as main factors were sample

time (categorical), anesthetics (categorical), tank as

block (categorical). Groups were compared by the ad

hoc variance test (F-test) using the least-squares

means procedure when significant effects were found

in the main model. The level of statistical significance

was set at P\ 0.05. All data were tested for normality

by a normal probability plot (proc univariate).

Results

Atlantic cod

A significant increase in the release of cortisol in

response to anesthesia was seen in all treatment

groups, with the highest measured release rate at 0.5 h

(Fig. 1; Table 2). MS-222 elicited the highest increase

in cortisol with a peak value at 0.5 h of 0.28 ±

0.03 ng g-1 h-1 followed by benzocaine (0.23 ±

0.02 ng g-1 h-1) and metomidate (0.05 ± 0.02 ng

Fig. 1 Cortisol release

rates into water by Atlantic

cod after exposure to

different anesthetic agents,

and control (no anesthetic).

Values are mean ± SE

(n = 3)

722 Fish Physiol Biochem (2010) 36:719–730

123

g-1 h-1). The cortisol release then decreased in a

linear manner returning to the initial basal level 2 h

post-exposure. Although the increase in cortisol

release following benzocaine anesthesia was slightly

lower compared to MS-222 at 0.5 h, the release of

cortisol was maintained at a higher level throughout

the experiment returning to basal levels only after 6 h

post-exposure. The small increase in cortisol secretion

after exposure to metomidate returned quickly to basal

level within 1.5 h of exposure. The temporal halt in

water supply did not seem to elicit any stress response

in the fish as no increase in cortisol release was seen in

the untreated control group during the 4-h period.

Atlantic halibut

The pattern of the response to anesthetics in Atlantic

halibut resembled that of Atlantic cod with a

significant increase in the release rate of cortisol seen

within the first hour in all treatment groups (Fig. 2;

Table 3). Furthermore, the most profound response

was found following exposure to MS-222, where the

level reached 0.68 ± 0.01 ng g-1 h-1 at 0.5 h before

decreasing steadily to 0.01 ± 0.02 ng g-1 h-1 2.5 h

post-exposure. Benzocaine appeared to cause a

bimodal response where cortisol release reached a

maximum of 0.36 ± 0.03 ng g-1 h-1 after 0.5 h,

returned to basal level after 2.5 h and then reached a

second increase of 0.12 ± 0.03 ng g-1 h-1 after 3.5 h

returning to initial levels after 6 h. The rise following

metomidate reached 0.17 ± 0.02 ng g-1 h-1 at 0.5 h

and returned to basal level 2.5 h post-exposure.

Stopping the water flow resulted in a non-significant

(P[ 0.05) elevation of cortisol in the control group

peaking at 0.03 ± 0.02 ng g-1 h-1 after 1 h, return-

ing to initial basal levels after 2 h.

Table 2 Statistical differences between and within anesthetic treatments in Atlantic cod

Time (h) 0 0.5 1 1.5 2 2.5 3 3.5 4 5 6

Benzocaine aA bA bcA cA acA cA cA aA acA acA aA

MS-222 aA bA bA cA aA aB aB aA aB aB aA

Metomidate aA bB bB aB aA aB aB aA aB aB aA

Control aA aB aB aB aA aB aB aA aB

Different uppercase letters indicate significant difference at P\ 0.05 level between different anesthetic treatments, while different

lowercase letters are indicative of the same within each treatment

Fig. 2 Cortisol release

rates into water by Atlantic

halibut after exposure to

different anesthetic agents,

and control (no anesthetic).

Values are mean ± SE

(n = 3 for groups exposed

to anesthetic agents, n = 2

for control group)

Fish Physiol Biochem (2010) 36:719–730 723

123

Atlantic salmon

All treatments resulted in a significant elevation of the

cortisol (Fig. 3; Table 4). MS-222 caused a sharp

increase peaking at 0.47 ± 0.04 ng g-1 h-1 after

0.5 h, which was significantly higher than the other

anesthetics tested. The level then dropped rapidly

until 2.5 h (0.02 ± 0.01 ng g-1 h-1) and remained in

the region of the initial basal level throughout the

experiment. As seen with Atlantic cod and Atlantic

halibut, the increase in cortisol release of the benzo-

caine-treated group was lower than that of MS-222,

and appeared later, peaking at 0.29 ± 0.04 ng

g-1 h-1 after 1.5 h. A reduction in the release seen

after 2 h was immediately followed by a second

increase peaking after 2.5–3 h before slowly declining

Table 3 Statistical differences between and within anesthetic treatments in Atlantic halibut

Time (h) 0 0.5 1 1.5 2 2.5 3 3.5 4 5 6

Benzocaine aA bA bA aA aA aA cA cA aA aA aA

MS-222 aA bB cA dB dB aA eA aeB aeA aA aA

Metomidate aA bC aB aB aB aA aB aB aA aA aA

Control aA aD aB aB aB aA aB aB aA

Different uppercase letters indicate significant difference at P\ 0.05 level between different anesthetic treatments, while different

lowercase letters are indicative of the same within each treatment

Fig. 3 Cortisol release

rates into water by Atlantic

salmon after exposure to

different anesthetic agents,

and control (no anesthetic).

Values are means ± SE

(n = 3 for benzocaine and

control group, n = 2 for

metomidate, MS-222, and

isoeugenol)

Table 4 Statistical differences between and within anesthetic treatments in Atlantic salmon

Time (h) 0 0.5 1 1.5 2 2.5 3 3.5 4 5 6

Benzocaine aA bA bA bA cAB bA bA bcA cA cA cA

MS-222 aA bB cAB dB adA aB aB aB aB aB aB

Metomidate aA bBC cB cB bB bA acB acB acAB aB aB

Isoeugenol aA bC cAB bcB bA abB aB aB aB aB aB

Control aA aD aC aC aC aB aB aB aB

Different uppercase letters indicate significant difference at P\ 0.05 level between different anesthetic treatments, while different

lowercase letters are indicative of the same within each treatment

724 Fish Physiol Biochem (2010) 36:719–730

123

towards the last sampling at 6 h, but without reaching

basal levels. Metomidate appeared to cause a bimodal

response in cortisol secretion, where a small initial

increase was followed by a larger increase reaching

0.31 ± 0.05 ng g-1 h-1 between 1.5 and 3 h after

exposure. The release rate returned to the initial basal

level after 5 h. Isoeugenol was only tested in Atlantic

salmon. The exposure induced the lowest increase of

cortisol of the anesthetics tested. But as for the others,

there was a significant rise within the first 1.5 h, with a

maximum level of 0.23 ± 0.07 ng g-1 h-1 measured

1 h post-exposure returning to initial basal level after

5 h. Only small, and from zero non-significant,

fluctuations in cortisol release were seen in the control

group.

Discussion

Traditionally, the main incentive to use anesthesia for

fish has been to facilitate various forms of handling.

In more recent times, the perspective of fish welfare

has received increasing attention, including the

potential of using anesthetics to reduce or prevent

stress. The anesthetic agents are administered through

the water, either by being added directly to the

holding tanks without subjecting the fish to any

handling or confinement or to separate tanks in which

the fish are subsequently transferred. Subjecting the

fish to handling and confinement prior to anesthesia

would probably elicit a stress response (Olsen et al.

1995; Sumpter et al. 1986). Administration of

anesthetics directly to the tank without any distur-

bance may provide time for detecting the agents

through taste and smell. The agents may also act as

irritants to the skin by causing damage to the mucous

layer. Furthermore, as the anesthetic starts to take its

effect, the initial struggle to avoid losing balance may

possibly also induce stress. The mode of action of the

various agents will probably also affect the stress

response. Anesthetic agents may affect the endocrine

system and themselves induce elevations in plasma

cortisol concentrations (Oyama 1973; Oyama and

Wakayama 1988).

With reference to these variables, it is not

surprising that literature is contradictory with regard

to the amount of stress a given anesthetic may cause.

Stress in response to anesthesia, assessed by

increased levels of cortisol, has been reported for

several anesthetic agents in various fish species.

Exposure to MS-222 has been found to induce a

cortisol response in a range of species, including

Atlantic salmon, rainbow trout (Oncorhynchus

mykiss), channel catfish (Ictalurus punctatus), striped

bass (Morone saxatilis), hybrid striped bass (Morone

chrysops 9 Morone saxatilis), gilthead sea bream

(Sparus aurata), and red drum (Sciaenops ocellatus)

(Barton and Peter 1982; Davis and Griffin 2004;

Davis et al. 1982; Kiessling et al. 2009; Molinero and

Gonzalez 1995; Small 2003; Thomas and Robertson

1991). However, when administered in connection

with handling MS-222 has been found to suppress the

cortisol response in channel catfish, red drum and

black sea bass (Centropristis striata) (King et al.

2005; Small and Chatakondi 2005; Thomas and

Robertson 1991) but not in striped bass and hybrid

striped bass (Davis and Griffin 2004; Davis et al.

1982). Opposing results in rainbow trout indicate that

the stress-reducing potential of MS-222 during han-

dling depends on the nature of the handling (Barton

and Peter 1982; Sink et al. 2007). Metomidate has

been tested during various handling practices and has

been found to reduce the cortisol response in Chinook

salmon (Oncorhynchus tshawytscha), red drum,

channel catfish, black sea bass, hybrid striped bass

and Atlantic salmon (Davis and Griffin 2004; Iversen

et al. 2003; King et al. 2005; Kreiberg and Powell

1991; Olsen et al. 1995; Small 2003, 2004; Thomas

and Robertson 1991). The dosage of metomidate

required for preventing the stress response seems

however to vary both between and within species

(Iversen et al. 2003; Kreiberg and Powell 1991; Olsen

et al. 1995; Sandodden et al. 2001). Isoeugenol has

also been studied with respect to stress-reducing

potential in situations of handling and has been found

to reduce the cortisol response in channel catfish and

Atlantic salmon (Iversen et al. 2003; Small 2004;

Small and Chatakondi 2005). Isoeugenol was how-

ever not found to reduce the stress response to

handling in rainbow trout and hybrid striped bass, and

merely the exposure itself was sufficient for inducing

a cortisol response in these species (Davidson et al.

2000; Davis and Griffin 2004).

In the present study, all anesthetics induced a stress

response in the fish, displayed as a release of cortisol

into the water. Considering that the sampling was not

continuous, it is not possible to determine the exact

time for the cortisol peak. Maximum levels of cortisol

Fish Physiol Biochem (2010) 36:719–730 725

123

were measured at the first sampling point 0.5-h post-

exposure for all agents in Atlantic cod and Atlantic

halibut, whereas in Atlantic salmon the occurrence of

the cortisol peak varied between the anesthetic agents.

Regardless of species, MS-222 appeared to cause the

strongest stress response with cortisol peaking at 0.5 h

before returning to initial basal levels after 3–4 h. A

similar pattern of response has also been reported for

adult Atlantic salmon and Chinook salmon following

MS-222 exposure (Cho and Heath 2000; Kiessling

et al. 2009). The preliminary study with juvenile

Atlantic salmon, mentioned in the introduction,

showed the same trend as found here with MS-222

eliciting the highest increase in plasma cortisol

concentration followed by benzocaine and isoeuge-

nol, respectively, (Kiessling, Johansson and Axen,

pers. comm.). This further underlines that the stress

response induced by these anesthetic agents is

consistent also between species and life stages.

The purpose of administrating the anesthetics

directly to the water of the holding tanks and

collecting water samples from the outlet of the tanks

was to minimize the risk of inducing a stress reaction

due to handling or any other external stimuli that

would mask the effect of the anesthetic agents. The

success of this strategy was supported by the very low

release of cortisol from fish in the control group. The

absence of external stressors may explain the more

rapid clearance of cortisol seen in the present study

compared to earlier studies as these often includes

additional stress from handling in connection to

anesthesia or sample collection in the form of netting

and transfer into and out of anesthesia baths.

As benzocaine and MS-222 have the same mode of

action, it is interesting to note that the stress response

differed when fish were exposed to the two com-

pounds. Firstly, the rapid increase in cortisol release

upon exposure did not reach the same magnitude for

benzocaine as for MS-222. Secondly, the cortisol

release following benzocaine exposure was notably

bimodal with a second peak lasting towards the end of

trial (6 h) a pattern never seen for MS-222. This

bimodality was more profound in Atlantic salmon

than in Atlantic halibut and Atlantic cod. The

bimodality is consistent with findings in Atlantic

salmon anesthetized with benzocaine, given artificial

ventilation during recovery (Kiessling et al. 2009).

Bimodality in the stress response assessed by plasma

cortisol levels has also been observed in rainbow trout

(16 h post-anesthesia) and hybrid striped bass (mea-

sured 6 and 24 h post-anesthesia) anesthetized with

isoeugenol and quinaldine sulphate, respectively

(Davidson et al. 2000; Davis and Griffin 2004). The

dosage of MS-222 used in the current investigation

was higher than of benzocaine, for Atlantic halibut

twice as high. This may possibly contribute to the

difference in response between the two substances

observed immediately following exposure. The effect

of an anesthetic agent is closely related to pharmaco-

kinetic properties, i.e., absorption, distribution, clear-

ance, and elimination. In Atlantic salmon, clearance

rates of 3.1 l kg-1 h-1 and 0.35 l kg-1 h-1 have been

found for MS-222 and benzocaine respectively (Kies-

sling et al. 2009) indicating that the fish may need

longer time to recover from benzocaine than from

MS-222. Benzocaine has higher lipid solubility than

MS-222, and as anesthetics tend to accumulate in

tissues with a high content of fat (Rang et al. 2003)

this may lead to a build-up of benzocaine in fat-rich

tissues, possibly prolonging the effect. A substance

with higher lipid solubility may also more easily pass

through the blood–brain barrier and result in a more

profound central effect, depressing the centers con-

trolling respiration and circulation leading to insuffi-

cient respiration and gill circulation, which may delay

the elimination of the anesthetic, as the major pathway

for drug elimination in fish is over the gills (Hunn and

Allen 1974). The second peak of cortisol could

possibly relate to secondary metabolites formed due

to a delayed elimination or a more pronounced

hypoxia during the initial phase of the anesthesia.

Metomidate induced the lowest increase in cortisol

release of the agents examined here. In Atlantic cod

and Atlantic halibut, the small increase returned to

basal levels within 2 h. In Atlantic salmon, the initial

increase in release rate was followed by a larger

increase peaking at 2–2.5 h post-anesthesia, thus

resulting in a bimodal response not seen in Atlantic

cod or Atlantic halibut. As opposed to the sustained

elevation of cortisol characterizing the bimodal

response following benzocaine anesthesia, the corti-

sol levels returned to basal levels after 5 h. The

increased cortisol release following exposure to the

current dosage of metomidate (3 mg l-1) was rather

unexpected as metomidate has been reported to

prevent the stress response by inhibiting the synthesis

of cortisol (Olsen et al. 1995; Thomas and Robertson

1991). No increase in cortisol level was observed in

726 Fish Physiol Biochem (2010) 36:719–730

123

Atlantic salmon exposed to metomidate following

netting when a dosage of 2 mg l-1 was used (Iversen

et al. 2003), and a dosage of 3 mg l-1 was found to

prevent the cortisol response to handling stress (Olsen

et al. 1995). One can only speculate as to why

exposure to metomidate induced a stress response in

the current study. Since the fish were not subjected to

any stress from handling prior to exposure or in

connection to sampling a slow dispersion, introduc-

ing the fish to low concentrations of the drug might

have elicited a stress response. Low dosages of

metomidate added to the tank (0.5–1 mg l-1) have

previously been found insufficient for inhibiting the

stress response in Atlantic salmon (Eliason et al.

2007; Olsen et al. 1995) and have also been

inadequate for blocking the stress response during

transport (Sandodden et al. 2001). Low dosages of

metomidate (1.5 mg l-1) have on the other hand been

found to inhibit the cortisol response to confinement

stress in both channel catfish and hybrid striped bass

(Davis and Griffin 2004; Small 2004). Metomidate

added to the tanks did not elicit a stress response in

any of these species. There appears thus to be species

differences in the potency of metomidate in reducing

the stress response, supporting the data from the

current investigation. The fact that no further eleva-

tion of cortisol was seen in response to metomidate in

Atlantic cod and Atlantic halibut may indicate that

metomidate was not perceived as a strong stressor by

these species or that the concentration of metomidate

was sufficient for inhibiting any further elevation of

cortisol. The underlying rationale for this apparent

species difference in response may be a result of

behavior. Atlantic salmon is an active swimmer

normally responding to perceived reduction in O2

availability by a strong escape reaction. Metomidate

causes sedation and hypnosis by affecting GABAA

receptors. One of the side effects linked to agents that

act via these receptors is respiratory depression

(Grasshoff et al. 2006; Zeller et al. 2005). If lower

dosages inhibit the respiratory capacity but not the

perception of blood gases indicating a respiratory

insufficiency the fish might respond by stress.

Isoeugenol was only examined in Atlantic salmon.

The peak in cortisol release rate was lower following

exposure to isoeugenol than MS-222 and was mea-

sured 0.5 h later, but paralleled MS-222 in returning

to basal levels 4 h post-exposure. Stress in response to

isoeugenol exposure is consistent with previous

findings in Atlantic salmon (Iversen et al. 2003;

Kiessling et al. 2009, and Kiessling, Johansson and

Axen, pers. comm.). In these studies, however, the fish

were subjected to handling in connection with anes-

thesia. The dosage used in the current study is lower

than used by Kiessling et al. (2009) and Iversen et al.

(2003) but was chosen according to the manufac-

turer’s recommendation in order to obtain sedated and

handleable fish (5.4 mg l-1 isoeugenol corresponds to

10 ml Aqui-S� per 1,000 l).

Atlantic salmon subjected to acute stress in the

form of crowding, netting, and 1.5 min of air exposure

display an immediate release of cortisol, reaching a

maximum level of about 2.8 ng g-1 h-1 approxi-

mately 3 h post-stress (Ellis et al. 2007). In rainbow

trout subjected to a similar stressor, and in European

sea bass (Dicentrarchus labrax) chased for 5 min

prior to 1.5 min of air exposure cortisol release rates

have been found to peak at 1.3 ng g-1 h-1 after 1 h,

returning to basal levels 3 hours post-stress (Ellis et al.

2004; Fanouraki et al. 2008). In these studies the

cortisol release rate and plasma cortisol concentra-

tions were found to correspond. Plasma levels of

cortisol in Atlantic salmon subjected to acute stress

(30 s in air in dip net or chased in the tank for 15 min)

have previously been found to peak 1 hour post-stress,

reaching maximum levels of 110–215 ng ml-1 (Olsen

et al. 2002; Olsen et al. 1995). In Atlantic cod

subjected to the same stressors, increased levels of

plasma cortisol of 80–95 ng ml-1 have been found

0.5–1 h post-stress (King et al. 2006; Olsen et al.

2008). These reports show that identical stressors

elicit a higher cortisol production in Atlantic salmon

than in Atlantic cod, which support the findings in the

current investigation where the magnitude of the

cortisol release in response to the anesthetic agents

was more profound in Atlantic salmon than in Atlantic

cod. Furthermore, the temporal dynamics of the

cortisol response found here, with a maximum peak

in cortisol levels between 0.5 and 1.5 h is in line with

previous studies, both of plasma and water measure-

ments (Barton 2002; Barton and Iwama 1991; Scott

and Ellis 2007; Scott et al. 2008).

Conclusions

This study shows that exposure to anesthetic agents

elicits a stress response in Atlantic salmon, Atlantic

Fish Physiol Biochem (2010) 36:719–730 727

123

halibut, and Atlantic cod. MS-222 induces the most

rapid response in all species while benzocaine

induces a prolonged bimodal response, more pro-

found in Atlantic salmon and Atlantic cod than in

Atlantic halibut. The amount of cortisol released in

response to anesthesia is low compared to what is

reported following netting and air exposure, which

are considered to be strong stressors in fish, but may

represent an extra load during otherwise stressful

circumstances.

Acknowledgments The authors would like to thank the staff

of the Institute of Marine Research, and in particular Ivar

Helge Matre and Rina Helen Skoglund for their valuable

technical assistance, and Grethe Thorsheim for skilful help and

support with water sample processing and analysis. Ole

Folkedal and Thomas Torgersen are highly acknowledged for

proficient support in calculating cortisol release rates. Financial

support was given by the Norwegian Research Council, grant

no. 152898/120.

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