Water Flow Requirements of Post-smolt Atlantic Salmon (Salmo salar L.) Reared in Intensive Seawater Flow-through Systems: A Physiological Perspective^†

Round 1

Reviewer 1 Report

The current study investigated the mass-specific water flow requirement by post-smolts Atlantic salmon. The experiment design is appropriate though 3 replicates for each SWF treatment will be better. The data collection and statistics are reliable. The result analysis, discussion and conclusion is reasonable.

In general, it is a well-prepared scientific paper and would be instructional for post-smolts reared in containment systems.

It is acceptable for publication after tiny revision.

The major flaw of this manuscript is in the experiment setting. There are four mass-specific water flows (0.2, 0.3, 0.4 and 0.5 L kg fish-1 min-1) as experimental treatments with two repetitions, but normally at least three repetitions is statisticly necessary. Please explain why there were only two repetetions for the treatments.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

The current study investigated the mass-specific water flow requirement by post-smolts Atlantic salmon. The experiment design is appropriate though 3 replicates for each SWF treatment will be better. The data collection and statistics are reliable. The result analysis, discussion and conclusion is reasonable.

In general, it is a well-prepared scientific paper and would be instructional for post-smolts reared in containment systems.

It is acceptable for publication after tiny revision.

The major flaw of this manuscript is in the experiment setting. There are four mass-specific water flows (0.2, 0.3, 0.4 and 0.5 L kg fish-1 min-1) as experimental treatments with two repetitions, but normally at least three repetitions is statisticly necessary. Please explain why there were only two repetetions for the treatments.

• It was important to have several flow treatments (dose-response set up) instead of a high and one or two low treatments to find threshold values therefore we choose to have as many treatments as was feasible within the limits of the project and therefor the study had to be limited to two replicates.

Reviewer 2 Report

With the present study “Water flow requirements of post-smolt Atlantic salmon (Salmo salar L.) reared in intensive seawater flow-through systems” the authors analysed the impact of different water flow regimes on the physiological responses of post smolt salmons.

Generally, the study provides a comprehensive broad range approach to the potential resulting effects of the physiological responses, including blood chemistry analysis, gene expression and checks for external welfare. Additionally, for certain experimental factors the study included the acclimatisation phase to higher salinity levels. Even though similar studies are present already, this allows a broader insight into the effects and makes this study interesting for the readers. However, some general questions remain and some small adaptions in the figures and text are necessary. Consequently, I propose the acceptance after a revision.

General questions:

-          Could you provide an explanation for the chosen velocities? Compared to the previous literature, they seem to be in a commonly used range already (compare Fivelstadt et al. 2004). This could be potentially also added in the text.

-          Also: In the time of salinity adaption a higher rate (0.6 L/kg) was used, while the rates were lower during the actual experiment (0.2 – 0.5). This could affect the fishes physiology also. Why was this done? Could there be effects on the following measurements? Please elaborate.

-          L192-194: Given the comments here, only a single household gene was applied. Generally, it is highly recommended to compare to more than one reference gene (Bustin 2009). Even though EFα1a is a commonly used reference gene, using only a single comparison for the relative expression can lead to false readings. Where other reference genes included that could be added? Could otherwise the expression levels of EFα1a be added in the sentence in some way?

-          How transferable is the experimental setting to a final application in a potential industrial scale? Elaborating this, for example in the discussion section, might be interesting for readers from the aquaculture field.

Abstract & Title:

-          The title is a bit misleading. Maybe include that it is focused on the physiological response?

-          L28: TAN? à Explanation first given in the introduction in L65

-          L21: „have sparked“

Materials and Methods:

-          L204-207: How accurate is the measurement of the water flow using the bucket filling time? Was this compared to the measurement using some type of flow-meter for example? Also: Could there be residual areas in the squared tank, where water parameter concentrations differed due to variations in the flow patterns (“squared tank” à L120). If necessary, please address this in the text as well.

-          L251-266: What software was used for the data analysis? Please also add what programs were used to create the graphs.

Results:

-          Fig.1: Significance letters double for some sampling points (C+D). Slight overlay of the x-axis label for A and C.

-          Fig. 2.+3: Significance letters are missing but mentioned in the figure legend.

-          L312: “mRNA” instead of “MRNA” (?)

-          Table2: false line added below “Cataract”

-          Fig.5: x-axis labels need to be aligned with the marked lines

Discussion:

-          L419-422: It is mentioned that parts of the experiments to this study were published previously. It would we good to mention this already in the materials and methods chapter, especially since the same specimen were used.

Sources:

-          L617-618 Sources need to end with a point. Also Line 589

Author Response

Reviewer 2

With the present study “Water flow requirements of post-smolt Atlantic salmon (Salmo salar L.) reared in intensive seawater flow-through systems” the authors analysed the impact of different water flow regimes on the physiological responses of post smolt salmons.

Generally, the study provides a comprehensive broad range approach to the potential resulting effects of the physiological responses, including blood chemistry analysis, gene expression and checks for external welfare. Additionally, for certain experimental factors the study included the acclimatisation phase to higher salinity levels. Even though similar studies are present already, this allows a broader insight into the effects and makes this study interesting for the readers. However, some general questions remain and some small adaptions in the figures and text are necessary. Consequently, I propose the acceptance after a revision.

General questions:

-          Could you provide an explanation for the chosen velocities? Compared to the previous literature, they seem to be in a commonly used range already (compare Fivelstadt et al. 2004). This could be potentially also added in the text.

- Firstly, we would just like to clarify the water velocity (body length/s) was kept stable and equal in all tanks by adjusting the angle of the inlet pipe, this is stated in section 2.6. To make sure water velocity was equal in all tanks, we calibrated the water velocity by marking two points on the tank wall (distance between points ~ 50 cm).  We used a floating object and timed how long it used to float between these two measuring points.  This test was repeated until we had 5 times with equal results.  The reason for 5 measurements was that the object may have a somewhat different entrance to the test at each circulation due to the fact that the tank is square. Hence, the only factor tested in this study was the amount of new sea water added to the tank (L/kg fish/ per minute). As you mention, the ones tested here are in the commonly used range for other lifestages parr/smolt and in freshwater aimed towards -answering questions for land-based production. As mentioned in line 57-60, this has not been tested extensively for post-smolts in sea water. In Fivelstad et al. 2004 both tested salinities are very low (1,6 and 4,5 ppt) versus 34 ppt in the present study. Seawater has a higher buffer capacity for CO2 therefor it may be possible to have fish in lower flows, however post-smolts are in a sensitive stage adapting to a new environment therefore their tolerance to flow is unknown and needs to be tested. A more precise explanation for the chosen SWFs has been added to section 2.2.

-          Also: In the time of salinity adaption a higher rate (0.6 L/kg) was used, while the rates were lower during the actual experiment (0.2 – 0.5). This could affect the fishes physiology also. Why was this done? Could there be effects on the following measurements? Please elaborate.

- A higher rate was used during acclimation to make sure there were no effects of flow while fish were acclimating to sea water and the experimental tanks. Other studies show that 0,5 L/kg fish/min show no effects and low CO2 in the water this was also feedback we received from industry partners, therefore we choose this as our highest treatment. As in all studies we were limited in how many experimental treatments we could choose therefore we thought it was important to have more treatments within the range we hypothesized the threshold would be and one above (0,5 L/kg fish/min). Also initial calculations assured us that we could maintain a good water quality in 0,5 L/kg fish/min treatment. It was important for us to have as many treatments as possible and have equal and fairly small increments between SWF treatments as we wanted to be as precise as possible in SWF rate recommendations. One of the reasons for this, is that for large scale commercial S-CCS where large amounts of water need to be pumped into the system, small adjustments in water flow rate, will have a large economic impact.

-          L192-194: Given the comments here, only a single household gene was applied. Generally, it is highly recommended to compare to more than one reference gene (Bustin 2009). Even though EFα1a is a commonly used reference gene, using only a single comparison for the relative expression can lead to false readings. Where other reference genes included that could be added? Could otherwise the expression levels of EFα1a be added in the sentence in some way?

- Agree with reviewer that a second housekeeping gene is always to prefer, however as the expression levels of EFα1a were very stable in the tissue we used and between treatments and as it is a widely used housekeeping gene for Atlantic salmon, we decided to use only EF1a as mentioned in sentence L192-194. The average EFα1a ct value (all samples) was 19,7±0,6.

          How transferable is the experimental setting to a final application in a potential industrial scale? Elaborating this, for example in the discussion section, might be interesting for readers from the aquaculture field.

• Highly transferable with the limitations discussed in section 4.6 which has been revised to clarify/highlight this.

 Abstract & Title:

-          The title is a bit misleading. Maybe include that it is focused on the physiological response?

-        We agree and have modified the title to “Water flow requirements of post-smolt Atlantic salmon (Salmo salar L.) in seawater flow-through systems, a physiological perspective” 

-          L28: TAN? à Explanation first given in the introduction in L65

- Changed

-          L21: „have sparked“

- Not sure I agree, I haven`t changed

Materials and Methods:

-          L204-207: How accurate is the measurement of the water flow using the bucket filling time? Was this compared to the measurement using some type of flow-meter for example? Also: Could there be residual areas in the squared tank, where water parameter concentrations differed due to variations in the flow patterns (“squared tank” à L120). If necessary, please address this in the text as well.

- This is an accurate method. In fact, we would argue that this is a much better method than using flow meters (which can be relatively inaccurate, depending on calibration, quality and how it is mounted). As mentioned the time to fill a 10L bucket was measured weekly.  Both the bucket volume and timer were accurate. The pressure in the inlet pipe is constant, so we will always get the same amount of water out per min, regardless of when we measure.  At the start we repeated each measurement 5 times and calculated the average value, individual values were so similar that only one measurement per tank was performed once weekly after that. 

• Residual areas were not found in our system. For that, the tank is too small (500l) and the relatively high fish density ensured a good circulation throughout the water column at all times.

-          L251-266: What software was used for the data analysis? Please also add what programs were used to create the graphs.

- Updated

Results:

-          Fig.1: Significance letters double for some sampling points (C+D). Slight overlay of the x-axis label for A and C.

- fixed

-          Fig. 2.+3: Significance letters are missing but mentioned in the figure legend.

- adjusted

-          L312: “mRNA” instead of “MRNA” (?)

- fixed

-          Table2: false line added below “Cataract”

- Could not find this?

-          Fig.5: x-axis labels need to be aligned with the marked lines

- I don`t see this problem in the copy reviewers received? better quality of all graphs has been updated.

Discussion:

-          L419-422: It is mentioned that parts of the experiments to this study were published previously. It would we good to mention this already in the materials and methods chapter, especially since the same specimen were used.

- This has been updated in section 2.2

Sources:

-          L617-618 Sources need to end with a point. Also Line 589

- Fixed.

Author Response File: Author Response.docx

Reviewer 3 Report

The MS investigates the mass-specific water flow requirements for post-smolt Atlantic salmon in semi-closed sea systems, with the aim of informing the design and dimensioning of such systems. The study exposes post-smolts to four levels of specific water flow (0.2, 0.3, 0.4, and 0.5 L kg fish^-1 min^-1) in flow-through seawater, maintaining a stable stocking density. The results indicate that the fish can compensate for reductions in water flow down to 0.2 L kg fish^-1 min^-1 without negative effects on osmoregulation, external welfare, or performance. However, the authors suggest maintaining water flow above 0.3 L kg fish^-1 min^-1 to avoid activating energy-costly physiological regulatory mechanisms.

General comments:

Abstract: The abstract provides a clear and concise overview of the study, including the motivation, experimental design, main findings, and implications for semi-closed sea systems for post-smolt Atlantic salmon. The study's objective, methods, and results are well summarized, and the conclusion offers practical recommendations for large-scale operations.

Clarity: In the sentence describing the experimental conditions, it might be clearer to rephrase the part about flow-through seawater. Consider revising to: "All treatments involved flow-through seawater with full oxygenation, a salinity of 34 ‰, and a mean temperature of 9.3°C."

Terminology: Briefly explain or define the terms "TAN" (total ammonia nitrogen) and "pCO2" (partial pressure of carbon dioxide) in the abstract to ensure that readers understand their significance in the study.

Introduction

The introduction provides a comprehensive background on the importance of developing new production methods for Atlantic salmon post-smolts, focusing on the potential benefits of closed-containment systems (CCS) and semi-closed systems (S-CCS) in terms of environmental impact and fish welfare. The authors highlight the need for establishing safe limits and guidelines for specific water flow (SWF) rates in seawater post-smolt life stages and discuss the potential consequences of reduced water flow on fish physiology, performance, and overall welfare. The introduction sets the context and motivation for the study effectively and clearly states the aim and hypothesis of the study.

Minor suggestions for improvement:

Paragraph structure: Consider dividing the introduction into smaller paragraphs, each focusing on a specific aspect of the background information. For example, one paragraph could discuss the potential benefits of CCS and S-CCS, while another could focus on the challenges associated with reduced water flow. This would improve the overall readability and flow of the introduction. In the last sentence of the introduction, the term "open flow respirometry" is mentioned without any explanation. Briefly explain what open flow respirometry is and why it is relevant to the study.

M&M: Fish stock and rearing conditions (Section 2.1): The paper lacks information on the origin and age of the fish used in the study. Additionally, it would be useful to provide more details about the standard rearing protocols followed, as this could influence the results and their interpretation.

Experimental design (Section 2.2): It is unclear why the experiment was conducted between October and December 2012, and whether this timing could have any impact on the results. Furthermore, it would be helpful to provide justification for the choice of the four SWF rates used in the study.

Blood chemistry sampling & analysis (Section 2.3): Although the ISTAT analyser has been acknowledged to have deviations from conventional laboratory values, it would be useful to provide more information on the limitations and validity of the ISTAT analyser when used for diagnostics in fish. Additionally, it would be helpful to provide references for the mentioned deviations.

Results: 

On line 340, it is stated that there were no significant differences for well-being indicators, with the notation P<0.05. This appears to be incorrect, as for non-significant differences, the correct notation should be P>0.05. Please revise this line accordingly. A similar issue is present on line 338, so please also make the necessary correction there. Additionally, it is unclear why the title of the table indicates that values showing significant differences are not shown. If this is an error, please update the table title to accurately reflect its contents. Alternatively, if this was intentional, kindly provide an explanation in the manuscript for the decision to omit values with significant differences.

Figure 5 is intended to depict an increase in oxygen consumption, but the increase is not easily discernible in the figure as it stands. I would like to suggest that the authors improve the clarity and representation of the data in Figure 5 to more effectively convey the observed differences. By making these adjustments, readers will be better able to understand the results and their significance.
At line 385, please provide a clear explanation and interpretation of the statistical result presented as (P > 0.45, Table 3). 
In Section 3.5, the presentation of the results seems to be somewhat unclear, particularly as Table 3 is located in the preceding section and its corresponding results are mentioned in the initial lines. To enhance the comprehensibility of this information, it would be beneficial to reorganize and clarify the results in Section 3.5, ensuring that the placement of Table 3 and its associated findings are more coherent and easily understood by the readers.

Discussion: In Section 4.1, it would be beneficial to provide a more detailed explanation of the compensatory mechanisms that occur at lower SWFs. Are these mechanisms known to have any long-term negative effects on the fish, or are they simply short-term adaptations? This information would be helpful to better understand the implications of the results.

Author Response

Reviewer 3

The MS investigates the mass-specific water flow requirements for post-smolt Atlantic salmon in semi-closed sea systems, with the aim of informing the design and dimensioning of such systems. The study exposes post-smolts to four levels of specific water flow (0.2, 0.3, 0.4, and 0.5 L kg fish-1 min-1) in flow-through seawater, maintaining a stable stocking density. The results indicate that the fish can compensate for reductions in water flow down to 0.2 L kg fish-1 min-1 without negative effects on osmoregulation, external welfare, or performance. However, the authors suggest maintaining water flow above 0.3 L kg fish-1 min-1 to avoid activating energy-costly physiological regulatory mechanisms.

General comments:

Abstract: The abstract provides a clear and concise overview of the study, including the motivation, experimental design, main findings, and implications for semi-closed sea systems for post-smolt Atlantic salmon. The study's objective, methods, and results are well summarized, and the conclusion offers practical recommendations for large-scale operations.

Clarity: In the sentence describing the experimental conditions, it might be clearer to rephrase the part about flow-through seawater. Consider revising to: "All treatments involved flow-through seawater with full oxygenation, a salinity of 34 ‰, and a mean temperature of 9.3°C."

-Good suggestion à changed

Terminology: Briefly explain or define the terms "TAN" (total ammonia nitrogen) and "pCO2" (partial pressure of carbon dioxide) in the abstract to ensure that readers understand their significance in the study.

-changed

Introduction

The introduction provides a comprehensive background on the importance of developing new production methods for Atlantic salmon post-smolts, focusing on the potential benefits of closed-containment systems (CCS) and semi-closed systems (S-CCS) in terms of environmental impact and fish welfare. The authors highlight the need for establishing safe limits and guidelines for specific water flow (SWF) rates in seawater post-smolt life stages and discuss the potential consequences of reduced water flow on fish physiology, performance, and overall welfare. The introduction sets the context and motivation for the study effectively and clearly states the aim and hypothesis of the study.

Minor suggestions for improvement:

Paragraph structure: Consider dividing the introduction into smaller paragraphs, each focusing on a specific aspect of the background information. For example, one paragraph could discuss the potential benefits of CCS and S-CCS, while another could focus on the challenges associated with reduced water flow. This would improve the overall readability and flow of the introduction. In the last sentence of the introduction, the term "open flow respirometry" is mentioned without any explanation. Briefly explain what open flow respirometry is and why it is relevant to the study.

-We have divided some paragraphs and made a comment in text. Open flow respirometry estimations are explained.

M&M: Fish stock and rearing conditions (Section 2.1): The paper lacks information on the origin and age of the fish used in the study. Additionally, it would be useful to provide more details about the standard rearing protocols followed, as this could influence the results and their interpretation.

• Strain added in section 2.1. I have cited density paper where info on rearing protocol is given. Also attached under if you think we should put it in instead but I think the m&m is already long enough.

• Standard rearing protocol in short: First feeding started in early February 2012 under constant light and in heated water (12–14 °C). Between early May and early August, the fish were maintained indoors in a green 7 m rearing tank (volume: 70 m− 3) at constant light and water temperature (12 °C). All fish were fed ad lib a commercial dry diet (EWOS, Bergen, Norway). A photoperiod regime known to stimulate parr-smolt transition was initiated in the beginning of August (Handeland and Stefansson, 2001). This treatment included a decrease in day-length from LD24:0 to LD12:12 for 5 weeks followed by another 4 weeks on LD24:0.

Experimental design (Section 2.2): It is unclear why the experiment was conducted between October and December 2012, and whether this timing could have any impact on the results. Furthermore, it would be helpful to provide justification for the choice of the four SWF rates used in the study.

• The experiment was conducted between October and December since this was when we had laboratory facilities and fish of the right size/maturity were available to do the experiment. However, the fall is a common period for stocking post-smolts in open cages and S-CCS. Since deep water is commonly pumped in you will get a temperature advantage in the late fall and winter in S-CCS, compared to colder surface layers. When the experiment was performed will mainly affect the water temperature, this is the only parameter that cannot be completely controlled in the lab facility, this is discussed in section 4.6.
• Justification for the SWF rates used is added to section 2.2.

Blood chemistry sampling & analysis (Section 2.3): Although the ISTAT analyser has been acknowledged to have deviations from conventional laboratory values, it would be useful to provide more information on the limitations and validity of the ISTAT analyser when used for diagnostics in fish. Additionally, it would be helpful to provide references for the mentioned deviations.

• The references for both limitations and validity are provided.

Results:

On line 340, it is stated that there were no significant differences for well-being indicators, with the notation P<0.05. This appears to be incorrect, as for non-significant differences, the correct notation should be P>0.05. Please revise this line accordingly.

• Fixed

A similar issue is present on line 338, so please also make the necessary correction there.

• This one was correct.

Additionally, it is unclear why the title of the table indicates that values showing significant differences are not shown. If this is an error, please update the table title to accurately reflect its contents. Alternatively, if this was intentional, kindly provide an explanation in the manuscript for the decision to omit values with significant differences.

• We have updated table 2 so that it is clearer that there were no sign. differences in welfare indicators between treatments.

Figure 5 is intended to depict an increase in oxygen consumption, but the increase is not easily discernible in the figure as it stands. I would like to suggest that the authors improve the clarity and representation of the data in Figure 5 to more effectively convey the observed differences. By making these adjustments, readers will be better able to understand the results and their significance.

• This is done.

At line 385, please provide a clear explanation and interpretation of the statistical result presented as (P > 0.45, Table 3).

• We have tried to clarify.

In Section 3.5, the presentation of the results seems to be somewhat unclear, particularly as Table 3 is located in the preceding section and its corresponding results are mentioned in the initial lines. To enhance the comprehensibility of this information, it would be beneficial to reorganize and clarify the results in Section 3.5, ensuring that the placement of Table 3 and its associated findings are more coherent and easily understood by the readers.

• Editor, please move table 3 to below section 3.5. We have reorganized and clarified section 3.5.

Discussion:

In Section 4.1, it would be beneficial to provide a more detailed explanation of the compensatory mechanisms that occur at lower SWFs. Are these mechanisms known to have any long-term negative effects on the fish, or are they simply short-term adaptations? This information would be helpful to better understand the implications of the results.

• We have made some edits in section 4.1, not sure that this was what the reviewer was after? but it was what I thought was lacking.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The manuscript has been revised in line with the provided recommendations. In my assessment, the ms is now suitable for publication. I'm pleased to see that the authors have taken the feedback into account and made appropriate improvements.