THE EFFECTS OF INCREASED CO2 ON POSTSMOLT ATLANTIC SALMON (SALMO SALAR) GROWTH, PHYSIOLOGY AND STRESS RESPONSIVENESS

S. Calabrese1,2*, T.O. Nilsen1,5, S. Fivelstad3, H. Takle4, C. Hosfeld3, L.O.E. Ebbesson5,  S.O. Stefansson1, J. Kolarevic4 , B.F. Terjesen4 and S.O. Handeland5
1Department of Biology, High Technology Centre, University of Bergen, N-5020, Norway, 2Marine Harvest Norway AS, Bergen, Norway; 3Bergen University College, Bergen, Norway, 4Nofima, Ås, Norway; 5Uni Research AS, Bergen, Norway;  
Email: Sara.Calabrese@marineharvest.com

Introduction

Currently, the majority of postsmolt Atlantic salmon production in Norway occurs in sea cages. It is during this phase that the highest losses occur, ~10-15% of the fish that enter seawater do not make it to market size (Gullestad et al., 2011). Extending the time fish spend in controlled environments, such as land-based recirculation systems or semi-closed rearing systems in sea, prior to being stocked in open sea cages, is expected to produce larger and more robust postsmolts, decrease fish losses, shorten production time, and increase overall sustainability.
For semi-closed sea systems to be economically profitable, production intensity needs to be increased from the legislated 25kg/m3 for traditional sea cages. Increased density and reduced water pumping costs per fish will lower investments and overall production costs, but may lead to a buildup of metabolites like CO2 that negatively affect fish health and welfare. Several studies have been done on smolts in land-based systems and the combined effects of high CO2 and low oxygen in open sea cages, however relatively little is known regarding the sole effects of CO2 on postsmolts in semi-closed sea systems (Thorarensen and Farrell, 2011; Terjesen et al., 2013) It is also important to understand how underlying suboptimal conditions like increased CO2 affect fish when challenged with additional stressors like crowding and transfer to open sea cages. Previous studies have shown that chronic mild stress due to poor water quality can reduce normal stress responses and learning when challenged, critical for rapid adaptation to new environments (Grassie et al., 2013). In the present study, we therefore tested effects of increasing CO2 on postsmolt growth, physiology and stress responsiveness. This knowledge will be used for optimizing biological conditions for postsmolts in semi-closed rearing systems.  

Methods

Atlantic salmon smolts weighing 91.5±13.9gr from Lerøy SeaFood were distributed among twelve, square fiberglass tanks (500 L) at the Industrial Laboratory (ILAB), Bergen Norway. Each tank was initially supplied with full-strength seawater at a temperature of 10-11°C under a simulated natural photoperiod. All tanks received a specific water flow of 1.0 L/kg/min and oxygen levels were automatically controlled to maintain saturation above 80% in the outlet water throughout the experiment. Water quality (temperature, salinity, pH, O2, TAN) were monitored. After a 2-week acclimation period pure CO2 (99%) was administered to 6 header tanks to obtain the treatments of 2mg/L (control), 7,15, 20, 25-30 and 30-35mg/L in duplicate tanks. CO2 levels were monitored daily through pH measurements and adjusted in the header tanks. The experiment lasted for a total of 8 weeks and 12 fish per treatment were sampled every second week for blood chemistry (ions and pH, I-STAT analyzer, EC8+ cartridges) and primary stress responses (glucose and cortisol, I-STAT and ELISA). In addition a stress challenge test was done after 4 and 8 weeks of CO2 treatment (2, 15 and 25-30mg/L). The challenge test entailed netting 6 fish/tank and then constraining them in the net (10 L) in a 300 L holding tank (receiving water from the original treatment) for 10 minutes. After the stress challenge and a 30 min recovery period in the 300 L holding tank, the fish were euthanized with a lethal dose of MS222 and blood and organs were quickly sampled. In this report, initial results on growth, blood chemistry and plasma cortisol levels will be presented.

Results and discussion

Preliminary results suggest that SGR is negatively affected when increasing CO2 from 25-30mg/L to 30-35mg/L for an 8-week period. After an 8-week CO2 treatment our results suggest a linear relationship between increased PCO2 in the water and regulatory responses in the blood. Concentrations of blood bicarbonate and pH increased with elevated ambient CO2 and blood chloride ions decreased. Cortisol values before a stress challenge indicated low unstressed values in the control group compared to treatment groups, suggesting that CO2 is a chronic stressor in postsmolt Atlantic salmon. Furthermore, our results indicate a 4-fold increase in plasma cortisol in the control group after a stress challenge; however, this increase was not as pronounced in the 15mg/L and 25-30mg/L group. A similar trend was observed for another primary stress indicator, blood glucose. In the 25-30mg/L group there was no observed elevation of blood glucose after a stress challenge compared to the control group in which values were elevated from 107.6±2.0mg/dL to 116.1±2.7mg/dL. This suggests that increased water CO2 levels reduce the capacity of salmon postsmolts to mobilize energy resources and elicit a stress response when faced with an additional stressor. The ability of postsmolts to cope and adapt in sub-optimal water quality in semi-closed sea systems will be further studied by looking at molecular stress markers in the brain.

In conclusion, our results suggest that growth rate of postsmolts in semi-closed sea systems are reduced by high CO2 levels (30-35mg/L) and that slight increases in CO2 initiate a cascade of physiological regulatory responses that are energy costly. Furthermore, the study shows that CO2 affects the ability of postsmolts to respond to additional stressors.

Acknowledgements

This study was funded by The Research Council of Norway (project 217502/E40   Optimized Postsmolt Production "OPP"), The Norwegian Seafood Research Fund - FHF (project 900816), and Marine Harvest Norway, Lerøy SeaFood, Smøla Klekkeri og Settefisk, Grieg Seafood, Lingalaks, and Erko Settefisk.

References

Grassie, C., Braithwaite, V.A., Nilsson, J., Nilsen, T.O., Teien, H-C., Handeland, S.O.,                      Stefansson, S.O., Tronci, V., Gorissen, M., Flik, G., Ebbesson, L.O.E., 2013. Aluminum exposure impacts brain plasticity and behavior in Atlantic salmon (Salmo salar). Journal of Experimental Biology. 216: 3148-3155

Gullestad, P., Bjørgo, S., Eithun, I., Ervik, A., Gudding, R., Hansen, H., Johansen, R., Osland, A., Rødseth, M., Røsvik, I., Sandersen, H., Skarra, H., Bakke, G., 2011. Effektiv og bærekraftig arealbruk i havbruksnæringen (In Norwegian, "Efficient and sustainable use of areas in Norwegian mariculture"), Oslo, pp. 190.

Terjesen, B.F., Rosten, T.W., Ulgenes, Y., Henriksen, K., Aarhus, I.J., Winther, U., 2013. Betydning av vannmiljøet ved produksjon av laksefisk i lukkede systemer i sjø. Water quality requirements for efficient farming of Atlantic salmon in closed systems. In Norwegian, English abstract . VANN. 48, 14-27.

Thorarensen, H. and Farrell, A.P., 2011. The biological requirements for post-smolt Atlantic salmon in closed-containment systems. Aquaculture. 312, 1-14.