Aquaculture Research, 2001, 32, 499±502
SHORT COMMUNICATION
Effects of graded environmental hypercapnia on sea bass (Dicentrarchus labrax L.) feed intake and acid±base balance S Cecchini, M Saroglia, G Caricato, G Terova & L Sileo Department of Animal Production, Aquaculture Section, University of Basilicata, Potenza, Italy
Correspondence: S Cecchini, Department of Sciences of Animal Production, Aquaculture Section, University of Basilicata, Via Nazario Sauro 85, 85100 Potenza, Italy. E-mail:
[email protected]
The effect of environmental hypercapnia on several physiological processes is known. Acute exposure to hypercapnia induces acidosis, which is followed by a compensatory elevation of plasma bicarbonate concentration to restore blood pH to near initial values (Toews, Holeton & Heisler 1983; Cameron 1985). The compensatory phase is accompanied by a reduction in plasma [Cl±] (Toews et al. 1983; Claiborne & Heisler 1986; Dimberg & HoÈglund 1987; Grùttum & Sigholt 1996; Fivelstad, Haavik, Lovik & Olsen 1998), by an increase in plasma [Na+] (Grùttum & Sigholt 1996), possibly to offset H+ excretion and maintain electroneutrality (Cameron 1985) and by an increase in the O2 af®nity of haemoglobin (Peyraud-Waitzenegger & Soulier 1989). Exposure to hypercapnia also induces respiratory and cardiovascular changes, such as increased gill ventilation volume (Janssen & Randall 1975), increased systemic vascular resistance (Perry, Fritsche, Hoagland, Duff & Olson 1999) and an increase of plasma catecholamines in proportion to the severity of blood acidosis (Perry, Kinkead, Gallaugher & Randall 1989). Less information exists about the effect of CO2 on the performance of cultured ®sh, although growth may be affected in ®sh chronically exposed to hypercapnic conditions (Crocker & Cech 1996; Fivelstad et al. 1998). As regards survival during long-term CO2 exposure, Fivelstad, Olsen, Klùften, Ski & Stefansson (1999) ã 2001 Blackwell Science Ltd
observed mortality rates of 1.3%, 4.3% and 7% in Atlantic salmon (Salmo salar) smolts held in fresh water at 7, 19 and 32 mg L±1 respectively. During shorter-term CO2 exposure, Grùttum & Sigholt (1996) indicated the mean lethal concentration (LC50) for sea bass (Dicentrarchus labrax) to be from 115.5 (6 2.9) to 104.8 (6 5.1) for exposure periods of 48±120 h. The aim of the present study was to analyse the effect of graded [CO2] on feed intake and acid±base balance of sea bass held in brackish water. Four groups of ®sh were held in 500-L square tanks connected to a water recirculation system. Three groups of ®ve ®sh each were used to assess feed intake, whilst blood sampling was carried out on a group of 35 ®sh held in the fourth tank. Fish were within the size range of 453±983 g. Initially, temperature was 23° 6 0.2 °C, salinity 12 g L±1, [CO2] 4 mg L±1, dissolved oxygen was maintained at 100% saturation and ®sh were exposed to a photoperiod of 10L:14D. After 2 weeks of acclimatization, [CO2] was subsequently increased at 3-day intervals to 14, 24, 34, 50, 75 and 110 mg L±1 and was then returned to the initial [CO2]. The speci®ed [CO2]s were obtained within 20 min. [CO2] was monitored using an indirect CO2-meter (Model 503 pH/CO2 Analyzer, Royce, New Orleans, USA), which measured pH and temperature, allowing the [CO2] to be calculated when salinity and alkalinity were 499
500 400 415 393 375 350 295 310 280 (6 (6 (6 (6 (6 (6
10.00) 22.05) 4.40) 15.59) 4.76) 9.13)
Water alkalinity (mg L±1) 4 (6 0,65) 14 (6 0.57) 24 (6 0.15) 34 (6 2.23) 50 (6 4.06) 75 (6 1.09) 110 (6 6.68) 2.1 (6 0.22)
Water [CO2] (mg L±1)
*Means are signi®cantly different (P < 0.05) from the starting value. **Means are signi®cantly different (P < 0.01) from the starting value. ***Means are signi®cantly different (P < 0.001) from the starting value. ²Days of exposure correspond with the days from the beginning of the experiment.
0.06) 0.02) 0.06) 0.03) 0.04) 0.01) 0.03) 0.04)
8.03 7.51 7.25 7.08 6.88 6.63 6.48 8.15
1±3 4±6 7±9 10±12 13±15 16±18 19 20±23
(6 (6 (6 (6 (6 (6 (6 (6
Water pH
Days of exposure²
Environmental conditions
0.85 0.89 0.68 0.64 0.57 0.35 0*** ±
(6 (6 (6 (6 (6 (6
0.15) 0.26) 0.24) 0.02) 0.12) 0.06)**
Feed intake (% bw) 7.480 7.594 7.612 7.648 7.658 7.741 7.741 7.492
pH (6 (6 (6 (6 (6 (6 (6 (6
0.05) 0.06) 0.10)* 0.06)** 0.08)** 0.05)*** 0.07)*** 0.05)
Feed intake and acid ±base balance
Table 1 Effects of graded environmental hypercapnia on sea bass feed intake and acid±base balance. Data are expressed as mean (6 SD)
13.94 14.22 16.36 24.14 28.76 31.24 41.04 11.60
(6 (6 (6 (6 (6 (6 (6 (6
PCO2 (mmHg) 1.26) 1.41) 2.77) 3.49)* 6.13)*** 4.27)*** 7.17)*** 1.56)
10.13 (6 0.77) 13.46 (6 1.31) 15.99 (6 1.55)* 25.70 (6 1.16)*** 31.02 (6 0.81)*** 41.21 (6 3.08)*** 54.01 (6 3.09)*** 7.22 (6 1.45)
[HCO3±] (mmol L±1)
Environmental hypercapnia and sea bass S Cecchini et al. Aquaculture Research, 2001, 32, 499±502
ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 499±502
Aquaculture Research, 2001, 32, 499±502
Environmental hypercapnia and sea bass S Cecchini et al.
known. Carbonate alkalinity was determined four times daily using the single acid addition method, described by Parsons, Maita & Lalli (1984) and salinity was determined daily using a salt refractometer with automatic compensation for water temperature. To obtain speci®ed [CO2]s, carbonate alkalinity and pH values were adjusted by addition of calcium bicarbonate and concentrated acid (HCl) or soda solutions injected by a computerized peristaltic pump system. The water conditions during the course of the experiment are shown in Table 1. At each [CO2], blood was sampled from ®ve ®sh to evaluate acid±base balance. With the exception of the ®sh exposed to the highest [CO2], which were sampled after 24 h, blood sampling was performed on the third day of exposure to the given [CO2]. Blood samples (about 150 mL), taken from the branchial artery of anaesthetized ®sh (MS-222, 200 mg L±1) using heparinized glass microtubes (microsampler, AVL, Roswell, GA, USA), were analysed immediately for pH, PCO2 and PO2 using a blood gas analyser (AVLâ, mod. OPTI 2). Plasma [HCO3±] was calculated according to the equation (Operation's Manual-OPTI Critical Care Analyzer):
When [CO2] reached 75 mg L±1, the ®sh became lethargic and there was skin hyperpigmentation; ®sh exposed to the highest [CO2] (110 mg L±1) did not feed, had darkened skin and showed erratic swimming. As regards acid±base balance, there were changes in pH, PCO2 and [HCO3±] that could be linearly related to [CO2] using the following equations:
[HCO3 ± ] (mmol L ± 1) = 0.0307 3 PCO2 3 10(pH ± 6.105) Feed intake was assessed by hand feeding the ®sh to apparent satiation twice a day using commercial extruded feed (Hendrix S.P.A., Verona, Italy) and noting the amount of feed provided. Feed intake at each [CO2] was calculated as the average daily feed consumption in each tank over 3 days. Fish in the tanks used for the assessment of feed consumption were not otherwise used for blood sampling, except for the last sampling. One-way analysis of variance (ANOVA) was used to test for treatment effects followed by Tukey's pairwise comparisons of means. Treatment effects were also examined using linear regression models. No ®sh died during the experiment. Exposure to increasing [CO2] affected feed intake (P < 0.001), pH (P < 0.001), PCO2 (P < 0.001) and plasma [HCO3±] (P < 0.001) (Table 1), but not PO2 (data not presented). The decline in feed intake with the increase [CO2] was described by the following equation: feed intake (% bw) = 0.9302±0.008145 3 [CO2] (r2 = 0.96), ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 499±502
pH = 7.542 + 0.002175 3 [CO2] (r2 = 0.81), PCO2 (mmHg) = 12.41 + 0.2661 3 [CO2] (r2 = 0.95), [HCO3 ± ] (mmol L ± 1) = 8.42 + 0.4263 3 [CO2] (r2 = 0.99). When [CO2] was returned the initial condition at the end of the experiment, the blood parameters became similar to the values recorded at the beginning of the experiment (Table 1). The data indicate that a 3-day exposure to elevated [CO2] produces a compensatory alkalosis in sea bass; increased plasma [HCO3±] leads to increased blood pH. This was seen in the carp (Cyprinus carpio) during long-term hypercapnic exposure (Claiborne & Heisler 1986). Thus, a 3day exposure to hypercapnic conditions might be considered a long-term exposure. During long-term exposure, increased plasma [HCO3±] is attributable to net gain of bicarbonate ions from the environment (Claiborne & Heisler 1986). There was a negative correlation (r2 = 0.96) between feed intake and [CO2] in sea bass, which is in accord with reports that exposure to hypercapnic conditions can result in reduced growth and poorer condition (Crocker & Cech 1996; Fivelstad et al. 1998). However, the effects of hypercapnia on the performance of cultured ®sh seem to be species speci®c. For example, rainbow trout (Oncorhynchus mykiss) does not seem to tolerate [CO2] higher than 12 mg L±1 (Smart, Knox, Harrison, Ralph, Richards & Cowey 1979), whereas Atlantic salmon reared in seawater (Fivelstad et al. 1999), white sturgeon (Acipenser transmontanus) (Crocker & Cech 1996) and sea bass (Blancheton, 2000) seem to be more tolerant. As regards sea bass, more studies are needed to evaluate the effects of chronic exposure to hypercapnic conditions on performance. 501
Environmental hypercapnia and sea bass S Cecchini et al.
References Blancheton J.P. (2000) Developments in recirculation systems for Mediterranean ®sh species. Aquacultural Engineering 22, 17±31. Cameron J.N. (1985) The bone compartment in a teleost ®sh, Ictalurus punctatus: size, composition and acid-base response to hypercapnia. Journal of Experimental Biology 117, 307±318. Claiborne J.B. & Heisler N. (1986) Acid-base regulation and ion transfers in the carp (Cyprinus carpio): pH compensation during graded long- and short-term environmental hypercapnia, and the effect of bicarbonate infusion. Journal of Experimental Biology 126, 41±61. Crocker C.E. & Cech J.J. Jr (1996) The effects of hypercapnia on the growth of juvenile white sturgeon, Acipenser transmontanus. Aquaculture 147, 293±299. Dimberg K. & HoÈglund L.B. (1987) Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard, bicarbonate-rich freshwater. Journal of Comparative Physiolology (B) 157, 405±412. Fivelstad S., Haavik H., Lovik G. & Olsen A.B. (1998) Sublethal effects and safe levels of carbon dioxide in sea water for Atlantic salmon postsmolts (Salmo salar L.): ion regulation and growth. Aquaculture 160, 305±316. Fivelstad S., Olsen A.B., Klùften H., Ski H. & Stefansson S. (1999) Effects of carbon dioxide on Atlantic salmon (Salmo salar L.) smolts, at constant pH in bicarbonate rich freshwater. Aquaculture 178, 171±187. Grùttum J.A. & Sigholt T. (1996) Acute toxicity of carbon dioxide on European sea bass (Dicentrarchus labrax): mortality and effects on plasma ions. Comparative Biochemistry and Physiology 115A, 323±327.
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Aquaculture Research, 2001, 32, 499±502 Janssen R.G. & Randall D.J. (1975) The effects of changes in pH and PCO2 in blood and water on breathing in rainbow trout, Salmo gairdneri. Respiratory Physiology 25, 235±245. Parsons T.R., Maita Y. & Lalli C.M. (1984). A Manual of Chemical and Biological Methods for Seawater Analysis, pp. 141±149, Pergamon Press. Oxford. Peyraud-Waitzenegger M. & Soulier P. (1989) Ventilatory and circulatory adjustments in the European eel (Anguilla anguilla L.) exposed to short term hypoxia. Experimental Biology 48, 107±122. Perry S.F., Kinkead R., Gallaugher P. & Randall D.J. (1989) Evidence that hypoxemia promotes catecholamine release during hypercapnic acidosis in rainbow trout (Salmo gairdneri). Respiratory Physiology 77, 351±363. Perry S.E., Fritsche R., Hoagland T.M., Duff D.W. & Olson K.R. (1999) The control of blood pressure during external hypercapnia in the rainbow trout (Oncorhynchus mykiss). Journal of Experimental Biology 202, 2177±2190. Smart G.R., Knox D., Harrison J.G., Ralph J.A., Richards R.H. & Cowey C.B. (1979) Nephrocalcinosis in rainbow trout Salmo gairdneri Richardson; the effect of exposure to elevated CO2 concentration. Journal of Fish Diseases 2, 279±289. Toews D.P., Holeton G.F. & Heisler N. (1983) Regulation of the acid-base status during environmental hypercapnia in the marine teleost ®sh Conger conger. Journal of Experimental Biology 107, 9±20.
Keywords: acid±base balance, hypercapnia, feed intake, sea bass
ã 2001 Blackwell Science Ltd, Aquaculture Research, 32, 499±502
