Acute toxicity of carbon dioxide on European seabass (Dicentrarchus labrax): Mortality and effects on plasma ions

Comp. Biochem. Physiol. Vol. 115A, No. 4, pp. 323-727, Copyright 0 1996 Elsevier Science Inc.

1996

ISSN 03OO-9629/96/$15.00 PI1 SO300-9629(96)00100-4

ELSEVIER

Acute Toxicity of Carbon Dioxide on European Seabass (Lkentrarchus labrax): Mortality and Effects on Plasma Ions JonArne

Gr~ttum and Trygve Sigholt

APPLIED CHEMISTRY, CENTER OF AQUACULTURE, N-7034

SINTEF

TRONDHEIM, NURWAY.

ABSTRACT. European Seabass (Dicentrarchus labrax) weighing 40- 120 g were exposed for 120hr to six levels uf CO? (aq) in running seawater at 15 -C l”C, 33-34%, and > 120 mmHg PO! (-75% saturation). Mean water Pco! levels ranged from 0.6mmHg (1.3 mg 1-l) (control tank) to 62.3 mmHg (137.2 mg 1-l). LCzc’s were found to be 51.9,

51.6,

taken

surviving

from

50.4,

and 47.1 fish after

2.9 (SD) mM (control

value)

in [Na+] was observed.

Nat

The

variation

in plasma

No significant

effects

mmHg

for 48, 72, 96 and

120 h, and plasma

to 115 ? 1.8 mM (Pcol increased

from

lactate

with earlier

acid-hase

INTRODUCTION in such

a large and homogeneous

ambient

carbon

spatial

and temporal

estuarine

waters,

ronments

such

dioxide

dimension.

dioxide)

(10-j

to 2 mmHg).

rich in vegetation), have

intoxication

been

tems such as landbased portation, rates

high stocking

oxygen oxygen

density levels

is added in these systems,

ide need

not necessarily as a limiting

The typical vironmental of plasma

and/or

dioxide

plants.

In sys-

live fish trans-

dioxide.

Because

high levels of carbon

be combined

as

low water exchange

of carbon

with

diox-

a low level of

factor. of the acid-base

hypercapnia

is an almost by elevated

plasma pH starts to recover

through

status of fish to enimmediate

plasma

reduction

Pcoz. However,

compensatory

A smaller

Blood

samples

in [Cl-],

from

hut significant

to 200 mM (Pco~ = 50 mmHg

observations

of compensation

were 160 t-

increase exposure).

for respiratory

Copyright 0 1996 Elsevk

observed.

regulation,

ion regulation,

composition. HCO,-/Cl-

acidosis.

Science Inc.

changed

elevation

of [HCO,-1. Marine fish species generally seem to handle hypercapnia acid-base disturbances faster than freshwater species, probably due to differences in environmental water Address repint requests to: J. A. Grottum, SINTEF Applied Chemistry, Center ofAquaculture,N-7034 Trondhelm, Nonvay. Tel. +47-73596385; Fax +47-73596363. E-mall: j,,n.a.grottum~chem.sintef.no

lactate,

in equal amounts

trality

sodium,

chloride

Acid-base regulation is mainly performed by and H+/Na+ ion exchange. The ions are ex-

(9). In marine

in order to maintain

t&h, exchanges

electroneu-

of endogenous

H+,

NH.++, and HCOim against exogenous Na’ and Cl- are ionically inappropriate, and in contrast to the situation in freshwater Cl-

fish, even

add to the passive

into the animal

cordingly,

stagnant as high

carbon

and during

response

pH induced

(e.g.

(16). Acute

in

pressure

of magnitude

Pcoz values

in aquaculture

seafarms,

may lead to toxic

orders

in

in envi-

fluctuations

freshwater

however,

reported

is higher

and Pco~ (partial

In standing

may also occur

as the

in both the

values

diurnal

can vary by three

ponds

60 mmHg

extreme

where

pH can be as great as two units of carbon

variable

This variability

and it may reach as tide pools

environment

is highly

decrease

1996.

KEY WORDS. Sea bass, COI, acute toxicity,

Even

exposure).

value)

were

respectively.

a significant

= 49 mmHg

concentration

COMP BIOCHEMPHYSIOL115A;4:323-327,

ocean,

showed

180 mM (control

ions is in accordance on plasma

120 hr exposures

analysis

vant

along

gradients.

elimination

of acid-base-rele-

the electroneutral

ions and the maintenance

quire mechanisms

of osmotic

for acid-base

loosely be interlocked tion (8). Studies

regulation,

rine teleosts

are scarce.

fish farming

this may be important

sufficient farming

to satisfy is therefore

tion of seabass, is considered. lead

With

the

Seabass

water

increase

partial

for this

combined

with

recirculation

with a small amount of CO?

compared

of gas may to aeration,

of gas lead to an equilibrium

Accumulation tension,

between

of CO! and high temperature

and hypercapnia

may therefore

a problem in rearing this species. The main aim of this study was to identify for seabass centrations

has not been cultiva-

heated

large amounts

information. of seabass

likely to rise (10). In intensive

Oxygenation

air and water.

on ma-

in marine

species.

to an accumulation

where

dioxide

the rise in interest

demand

re-

can only

for osmoregula-

of carbon

of wild stocks

Ac-

equilibrium which

with the mechanisms

of acute toxicity

The exploitation

influx of Na+ and

electrochemical

be

the LCU values

exposed to CO,(aq). Plasma Cl- and Nat conwere measured in order to determine the iono-

osmoregulatory

balance,

and as possible

indicators

of acid-

J. A. Grottum and T. Sigholt

324

base compensation.

Plasma

indicator of anaerobic

lactate

was measured

as an

Mortality

was recorded four times a day. Dead fish were

identified by lack of opercular movement.

metabolism.

the fish were made carefully, MATERIALS European

AND

neously to avoid differences in handling stress between the labrax) (for convenience

‘seabass’ is used in this paper) were brought from a local seafarm as fry and transferred to SINTEF

Center of Aqua-

culture. Until the start of the experiment,

the fish were kept

in 1 m3 tanks (salinity 33-34%, (40-l

T = 15-17°C).

20 g) were transferred to the experimental

groups of fish. After 120 hr, the fish that were still alive were anaesthetized (10 ppm metomidate)

The fish

fin. Blood samples were centrifuged,

and stored at - 80°C until analysis. Blood plasma Cl- concentration was assayed by means of a Radiometer CMT Chloride

Titrator,

Radiometer

had two inlets, both near the wall of the tank, just below the

centration

surface. The main inlet supplied the tank with preheated

heim Cat. No. 139 084).

from a reservoir. This reser-

FLM3 Flame Photometer. was with a L-lactic

The statistical

and plasma was frozen

and Na+ concentration

grey plastic tanks (diam. 60 cm, height 50 cm). Each tank

2 1 min-‘)

and blood from the caudal ves-

sel was collected in heparinized tubes by severing the caudal

tanks 12

days, and starved for 48 hr, before the start of COz exposure. The experiment was carried out in seven flow-through,

seawater (15 2 1°C

of

stress.

Dead fish were removed with dipnets from ail tanks simulta-

METHODS

seabass (Dicentrarchus

Observations

avoiding unnecessary

by means of a

Plasma lactate con-

acid kit (Boehringer

Mann-

analyses of survival were done by Probit

voir also supplied another reservoir, to which carbon diox-

Analysis, which analyses the relationship

ide was added through a diffusor to the water. Carbon diox-

lus (here: dose) and the quanta1 (here: death) (7). The ef-

ide-enriched

fect of COz exposure between

water was added to the tanks through small

between a stimu-

weight and mortality

was

inlets, except for the tank that held the control group. The

tested by linear regression using a t-test for testing against

level of carbon dioxide in each tank was controlled

by the

zero slope. The effects of COz exposure on plasma ions and

flow of CO*-rich

ml l-i,

water, which varied from loo-260

lactate

was tested with one-way ANOVA

depending on COz exposure level. The outlets consisted of

analysis was done using SOLO

a perforated

ANOVA

stand-pipe

surrounded

shaped screen in the center uniform distribution The 120-hr-long

by a perforated

pipe-

(19).

Survival

ver. 4.0, and regression and

by Systat ver. 5.04.

of the tank, which provided

of the water. exposure was conducted

with 14 fish in

RESULTS

each tank. At the start of exposure the volumes of water in

The mean value and range of measured pH and the total

the tanks were decreased by half in order to reduce the time

carbonate

before the expected Pcoz was reached. The Pcoz in the six

tions of COz (aq) and Pcoz are shown in Table 1. The con-

exposure tanks was 21 to 62 mmHg, and the Pcoz in the

trol tank had a Pcoz of 0.6 mmHg, due to excretion bon dioxide by the fish.

control

tank was measured at 0.6 mmHg. Water

(30 ml airtight

water bottles)

samples

were analysed daily with a

Tecator Aquatech 5400 analyzer, to determine the total carbonate concentration (C, = HCOz- + CO:+ COT) (Tecator

Application

note ASN

66-01/83).

The

level of

concentration

(Ct),

and calculated

concentraof car-

After 5 days of exposure, no mortality occurred at Pcoz levels lower than 35 mmHg (77 mg l-l), while 100% mortality was recorded at Pcoz levels above 50 mmHg (111 mg 1-l) (Fig. 1). Th e mean lethal concentrations (LCSJ after

COz (aq) was calculated according to Piedrahita and Seland

48, 72, 96 and 120 hr are shown in Table

( 11 ), and Pcoz according to Colt (5). The pH was measured

no mortality in the control

four times a day (WTW

ship between the wet weight of the fish that died and sur-

air-saturated

pH196).

The alkalinity

used for

2. There

was

group. Analyses of the relation-

seawater was 2.23 mM (3). The pH in the

vival time gave a slope not significantly different from zero.

tanks varied with Pcor but was nearly constant over time at each individual CO, level (pH range SE: 0.021-0.072).

The acute toxicity of COz on seabass did not therefore appear to be weight-dependent, within the range used in this

The Tecator determine

Aquatech

5400 analyzer was also used to

the total ammonia

concentration

in the tank.

Ammonia excretion gave a total ammonia nitrogen (NH3 + NH4+) concentration of less than 8 pg 1-l (
and oxygen concentration

in the water were

experiment. Increased CO, (aq) levels cause a significant reduction in plasma Cl- (P = O.OO), and all exposure groups were significantly different from the control group (P < 0.05). Chloride decreased from a control value of 160 to 115 mM at the highest concentrations that Cl-

concentration

of COz (Fig. I). It seemed

stabilised at 115 mM at the CO*

recorded repeatedly, four times a day (WTW Oxi 196 w/E0 196-1.5 electrode). Water temperature was 15 t

levels in the tanks in which mortality was observed. The increase in plasma Na+ was also significant (P =

1 “C, while the oxygen content was higher than 120 mmHg (- 6 mg ll’, - 75% saturation) during the whole of the experimental period.

0.02), but considerably less than that of the decrease in Cl. Within the COz range to which the fish were exposed, Na+ levels increased from about 180 to about 200 mM, i.e. about

Carbon Dioxide Tolerance

of Seabass

325

TABLE 1. Measured pH and C, (total carbonate) (mg CO1 l-) in the control group and the six exposure groups. The CO2 concentration (mg CO, I-), and Pcor (mmHg) were calculated on the basis of salinity, alkalinity, temperature, pH and total carbonate concentration in the tanks C,

PH Group

Mean

Mean

Range

0 1

7.87 6.37

7.81-7.97 6.36-6.39

2 3 4 5 6

6.15 5.99 5.98 5.98 5.63

6.12-6.16 5.97-6.01 5.95-6.02 5.97-6.00 5.63-5.63

Range

98

95-102

150 181 206 211 212 175

1.3

149-151 176-183 198-211 204-218 205-216 157-193

10% above the control about viving

0.6

45.6 77.4 107.2 111.1 111.1 137.2

value.

30% from the control

cant differences

PC02

CO2(aq)

For comparison, value.

in levels of plasma

fish in the different

20.7 35.2 48.7 50.5 50.5 62.3

groups

There lactate

Cl-

fell by

were no signifibetween

(P = 0.45)

the sur-

(Fig. 1).

DISCUSSION Mortality

In this study mortality that

ranged

decreased 5

EE 180 - rcl~~,-_.......--""i~~j..... .O 160 - a... (itip.....~~~, ii 140 "'I;$......_ ii a 120Ann .__

J

0.6,““““,‘,‘,‘,‘,,

4 a) fV

0.1 0.0

II 0

10

20

30 40 50 Pco,(mmHg)

r’InIs( 0

20

818 40

60

80

I 8 II 100

120

60

II 70

I 80

I I I I 140

160

co,(aq) (mg 1.') FIG. 1. Seabass exposed to 120 hr of various levels of carbon dioxide, given as concentration (COr(aq)) or partial pressure: A The points show cumulative mortality; B Concentration of Na+ and Cl- in plasma (& SD) of surviving fish; C Plasma concentration of lactate (+ SD). Number of samples used in the plasma analysis are shown in brackets. The letters a) and b) are used to distinguishtwo groups with ap proximately the same CO2 level in water.

from

5 1.9 to 47.1 mmHg

as exposure

time

in-

on the acute toxicity of carbon dioxide on marine teleosts is available for comparison. However, the concentration at which mortality was observed is low in comparison with concentrations used for anaesthesia of fish. In carp, for example, all fish completely lost their equilibrium when they were exposed to a Pcor of 125 mmHg but recovered about 15 min after being placed in water with no additional CO2 (18). Studies of the sublethal effects of CO1 are comprehensive (9). Sublethal effects of CO* on the marine teleost Conger conger were observed with Pcoz levels above about 8 mmHg (14), which is about 20% of the LC& 120 hr for seabass. In freshwater fish, Smart et al. (13) reported an increased percentage of nephrocalcinosis in rainbow trout (S&o guirdneri R.) exposed to CO1 (aq) concentrations above 12 mg 1-l ( Pcoz = 4.7 mmHg), which is lower than the reported level for subacute effects on Conger conger. However, in this species, concentrations of 55 mg 1-l (Pcor = 21.5 mmHg) had an effect on growth and conversion ratios, but only after 330 days. Carbon dioxide levels of 47 to 52 mmHg CO? are very unlikely to occur in natural seawater ( 16). However, in conditions related to aquaculture with oxygenation and reuse of water considerably higher levels may be reached, for example during a simulated transport of fish under the following conditions: fish density, 200 kg mm’; oxygen consumption, 3 mg kg-’ min-‘; RQ, 095 mol COz/mol Or (1); AQ, 0.5 mol tot. ammonia/mol Or (12); temperature, 16°C; salinity, 34%; alkalinity 2.3 mM; 80% of produced carbon dioxide removed; no water exchange. The LC50-48 hr (52 creased

B

200 -

varied from 0 to lOO%, with a Pcol from 35 to 62 mmHg after 120 hr. The LC50

from 48 to 120 hr. Limited

information

J. A. Grattum and T. Sigholt

326

TABLE 2. The mean lethal concentration (LC,,) values (*SE) partial pressure (mmHg) and concentration (aq) (mg 1-l )

after 48, 72, 96 and 120 hr of COz (aq) exposure,

48 hr

72 hr

CO2 (as) (mg I-‘)

51.9 2 1.3 115.5 5 2.9

114.9 t 2.9

mmHg) and L(&

120 hr (47 mmHg) might then be reached

Pcoz (mmHg)

51.6 +- 1.3

The CO1 was added to the tank without compensating for the reduced pH. This experimental it is the normal

COr accumulates.

situation

setup was chosen

in fish tanks in which

It is therefore

120 hr

50.4 + 1.4

impossible to distinguish

between the effects of a high level of carbon dioxide and of low pH. In the tank with the highest COz level (62

have resulted in anaerobic metabolism

104.8 -c 5.1

and accumulation

of

are not necessarily

reflected in increased blood levels. So-

called non-release

of lactic acid from muscle to blood has

been observed in several marine teleosts. (15,17) clusions regarding anaerobic

metabolism

CONCLUSIONS

6.4.

The mean lethal partial pressure (L&J was found to be 52-47 dioxide in 48-120

Regulation

Analysis of the plasma Cl- and Na+ levels showed a major

No con-

can therefore

be

drawn.

mmHg), the pH was 5.9 and all fish died within a day. The pH in the other exposure group was in the range of 6.0-

Acid-base

47.1 +- 2.3

112.2 * 3.1

lactate. However, elevated levels of lactic acid in the muscle

within about 30 and 32 hr respectively.

because

96 hr

given as

for carbon dioxide

mmHg for seabass exposed for carbon

hr. This level of Pco2 is unlikely to be

found in natural seawater, but can occur in aquaculture.

A

decrease in plasma Cl-, while Na+ showed a minor increase

raised CO1 (aq) level greatly reduced plasma Cl- concentration and slightly increased in Na’ concentration. No sig-

with increasing concentrations

nificant effects on plasma lactate concentration

in accordance

of CO2 (as). The results are

ing environmental

hypercapnia

in fish (8). Fish appear to

be unable to regulate arterial Pcoz in the face of rises in ambient

Pcoz, by changes

pH is maintained

in ventilation

by ion exchange

(4). The plasma

(8). The respiratory aci-

dosis that results from environmental

hypercapnia

marine teleost Conger conger is almost completely sated for in the extracellular tion of bicarbonate

compartment

mechanism

constant

while

the

sodium

(14). The reduction

HCOI-/ gra-

in plasma chloride

concentration

remains

in plasma Cl- in this experi-

ment may therefore be the result of increased compensation by such a transepithelial

HCO,-/Cl-

with greater

of COr.

exposure

The

exchange smaller

mechanism, increase

in

plasma Na+ shows that any Na+/H+ exchange mechanism to compensate for respiratory acidosis probably plays only a minor role.

Lactate No significant effects on plasma lactate concentrations

observed as a result of high environmental

were

levels of carbon

dioxide. High blood COz level or low pH may reduce the oxygen transport capacity of the blood due to the reduced capacity of the haemoglobin to transport oxygen (Root effect) or haemoglobin fect) (2). Insufficient

We thank Magne Staurnes for valuable comments on the manuscript. This work was catied out as part of a cooperatioe project between SINTEF Center of Aquaculture ana’ Aqua Optima AS financed by The Research Council of Norway (BPEU HB.30137 EU 900). JAG w~ls financed by a grant from SINTEF Strategic Technology Programme of Aquaculture (NTNF-project 26877).

by the accumula-

against the electrochemical

dient, because of a significant reduction concentration

in the

compen-

originating from the ambient water. The

uptake is probably performed by a transepithelial Cl- exchange

were found.

with other studies of acid-base regulation dur-

reduced affinity for oxygen (Bohr efdelivery of oxygen to the tissue may

References

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