Impact of Temperature Reduction from 14 °C to 12 °C in RASs on Atlantic Salmon: Increased Mineral Accumulation in RASs and Enhanced Growth Post-Transfer to Seawater

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The abstract can be reconstructed for better readability, as a clearer problem statement would enhance the research's focus and impact. In the materials and methods section, the frequency of water quality analysis is not consistently mentioned; this should be corrected to ensure clarity and completeness. Additionally, restructuring certain sections for improved flow and coherence would strengthen the overall presentation of the study.

Comments on the Quality of English Language

English can be improved for better readability and flow can be generated in a storyline manner.

Author Response

Thank you for taking the time to review our manuscript and for your suggestions on how it can be improved.

Comments 1: The abstract can be reconstructed for better readability, as a clearer problem statement would enhance the research's focus and impact.

Response 1: We agree with this and have tried to adjust the opening problem statement in the abstract.

 

Comments 2: In the materials and methods section, the frequency of water quality analysis is not consistently mentioned; this should be corrected to ensure clarity and completeness.

Response 2: Thank you for pointing this out. We have made changes in line 206-208 and adjusted Table 2 to ensure clarity.

 

Comment 3: Additionally, restructuring certain sections for improved flow and coherence would strengthen the overall presentation of the study.

Response 3: Thank you for this suggestion. We have chosen to keep the current structure as we feel it flows good with how the results and discussion are presented. We have also made some improvements on the English language.  

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is straightforwardly written, interesting and within the scope of the journal. It deals with both fish performance and water parameters for salmon RAS rearing in 12 and 14oC and subsequent growth in FTS under common conditions. The experiment is adequately designed although an explanation for needing 9 (!) replicates for the FTS rearing per temperature needs to be added. Analyses and data handling are well performed. Results can be improved. Some issues remain undiscussed (e.g. generally high nitrite and nitrate levels; FCR absence of significance) requiring extra comments in the discussion section. Please find below detailed specific comments.

Abstract: All semicolons (;) are probably commas (,). Please replace.

List of abbreviations: FCR should be corrected to “feed conversion ratio”. Also, TGC should be corrected to “thermal growth coefficient”. Check out the whole manuscript (e.g. ln. 100).

96: Correct to “These parameters are …”

100-101: I suppose, that this is where the decision of the authors to test 12 and 14 oC lies. However, since FCR values obtained were not significant, I think some relative comments regarding FCR in the Discussion section are needed.

132-134: Please add a reasonable explanation for needing 3 replicated FTS tanks for each of the triplicated RAS tank.

191 (subsection 2.4): Water parameters should also include unionized (toxic) ammonia levels.

302-303 (“… tendency … p = 0.08 ... tendency…p = 0.07”): A difference is either significant (P<0.05) or not significant (P> or = 0.05)! In this case, FCR in RAS and FTS rearing is similar and there are no differences between 12 and 14 oC. Please remove the “tendency” sentences.

307 (Table 3): Please, add initial and final weight.

323 (subsection 3.2): Dissolved oxygen concentration (DO in ppm) should also be included, especially when % saturation reported is 84-87%. The authors need to show that DO concentration was not a limiting factor.

326-327: With a P value of 0.61, definitely remove the sentence. However, provide an explanation for the high standard deviation (which probably explains the absence of significance) among triplicated tanks reported for 12 oC.

357-358: Remove this “tendency” sentence.

364-367: Magnesium retention should also be included in Table 6, especially given the difference reported for feces Mg (ln. 371-372).

404 (“…indications of a lower CSI post-transfer for the fish reared at a lower temperature…”): Differences for CSI were not significant. In the present study, there is no indication of lower values in 12oC. Remove the sentence and rephrase to comment on the possible significant differences observed in previous studies.

435: Correct to “These calculations are …”

446-447: Nitrate levels reported here were 244-273 ppm (Table 4). How do the authors say that “…recommended limits for nitrate (100 mg L.1) … were not exceeded”? Rephrase and further comment on levels reported. In addition, provide citations that confirm that 7.8-8.6 ppm of NO2 reported in Table 4 are safe for salmon.

507-520: In this paragraph, comments on Mg (ln. 507-510) should be better linked with following comments on P. Right now, the two are unconnected.

ln. 521-522: For the sentence to be true, please do not forget to add Mg data in Table 6.

Author Response

Thank you for taking the time to review our manuscript and for your suggestions on how we can improve it.

Comments 1: Abstract: All semicolons (;) are probably commas (,). Please replace.

Response 1: All semicolons (;) in the abstract has been updated to commas (,)

 

Comments 2: List of abbreviations: FCR should be corrected to “feed conversion ratio”. Also, TGC should be corrected to “thermal growth coefficient”. Check out the whole manuscript (e.g. ln. 100).

Response 2: Thank you for pointing this out, changes had been made in "List of abbreviations" and in text (line 243 and 332)

 

Comments 3: line 96: Correct to “These parameters are …”

Response 3: This has been corrected (line 109)

 

Comments 4: 100-101: I suppose, that this is where the decision of the authors to test 12 and 14 oC lies. However, since FCR values obtained were not significant, I think some relative comments regarding FCR in the Discussion section are needed.

Response 4: Yes this is correct and we agree with your comment. Its been added a relevant section in discussion on FCR (line 414-419)

 

Comments 5: 132-134: Please add a reasonable explanation for needing 3 replicated FTS tanks for each of the triplicated RAS tank.

Response 5: Thank you for your suggestion, this has been added (line 146-147)

 

Comments 6: 191 (subsection 2.4): Water parameters should also include unionized (toxic) ammonia levels.

Response 6: Thank you for this suggestion. this has now been added (line 212 - 216), table 4 (line 152) and discussed (line 482 - 485)

 

Comments 7: 302-303 (“… tendency … p = 0.08 ... tendency…p = 0.07”): A difference is either significant (P<0.05) or not significant (P> or = 0.05)! In this case, FCR in RAS and FTS rearing is similar and there are no differences between 12 and 14 oC. Please remove the “tendency” sentences

Response 7: Thank you for your comment, we have made adjustment but chosen to keep the term tendency for p values between 0.05 and 0.1.
Among several other topics, the use and judging of statistics in scientific journals have often slightly different approaches. In statistics, the term "tendency" is often used when the p-value is between 0.05 and 0.1 to indicate that there is a suggestive but not definitive evidence of an effect or relationship. By using the term "tendency” when the p-value is between 0.05 and 0.1 suggests that the results are close to being statistically significant but do not meet the conventional threshold of 0.05. This implies that there is some evidence of an effect, but it is not strong enough to be conclusive. By using "tendency," researchers avoid overstating the strength of their findings, but can communicate to the reader that there still will be a probability for differences or relationship given a lower threshold for rejecting your hypothesis.  

Since this is also a term that is already used in articles in the given journal, we hope to keep the use of “tendency” when p-values are between 0.05 and 0.1. 

 

Comments 8: 307 (Table 3): Please, add initial and final weight.

Response 8: Thank you for pointing this out to us. We have added Initial and final weights in table 3 (line 334)

 

Comments 9: 323 (subsection 3.2): Dissolved oxygen concentration (DO in ppm) should also be included, especially when % saturation reported is 84-87%. The authors need to show that DO concentration was not a limiting factor.

Response 9: Thank you for your suggestion. We have updated Table 4 now with DO in ppm (line 352)

Comments 10: 326-327: With a P value of 0.61, definitely remove the sentence. However, provide an explanation for the high standard deviation (which probably explains the absence of significance) among triplicated tanks reported for 12 oC.

Response 10: Thank you for pointing this out. Sentence has been removed.
In terms of the higher standard deviation, this was due to the fact that RAS tank 6 had somewhat higher nitrite compared to the other two tanks (3,2 vs 1,8 and 2,1.) Note that we now have listed nitrite values in Nitrite-Nitrogen (as per comments 15).

 

Comments 11: 357-358: Remove this “tendency” sentence.

Response 11: this has been removed (line 379)

 

Comments 12: 364-367: Magnesium retention should also be included in Table 6, especially given the difference reported for feces Mg (ln. 371-372).

Response 12: Unfortunately due to an oversight, the Mg levels in feed was not analyzed and we therefor cannot estimate the apparent retention. However we still choose to present the concentrations measured in whole body and feces since we still believe they hold some value.  

 

Comments 13: 404 (“…indications of a lower CSI post-transfer for the fish reared at a lower temperature…”): Differences for CSI were not significant. In the present study, there is no indication of lower values in 12oC. Remove the sentence and rephrase to comment on the possible significant differences observed in previous studies.

Response 13: This has been removed (line 432)

 

Comments 14: 435: Correct to “These calculations are …”

Response 14: thank you for pointing this out. This has been corrected (line 467)

 

Comments 15: 446-447: Nitrate levels reported here were 244-273 ppm (Table 4). How do the authors say that “…recommended limits for nitrate (100 mg L.1) … were not exceeded”? Rephrase and further comment on levels reported. In addition, provide citations that confirm that 7.8-8.6 ppm of NO2 reported in Table 4 are safe for salmon

Response 15: Thank you for pointing this out to us. we can see that we have reported in different units. This has now been updated to the same unit of measure to avoid confusion (line 478). nitrite has was also updated and discussed (line 479 - 482)

 

Comments 16: 507-520: In this paragraph, comments on Mg (ln. 507-510) should be better linked with following comments on P. Right now, the two are unconnected.

Response 16: Thank you for pointing this out, we have made changes to improve (line 551)

 

Comments 17: ln. 521-522: For the sentence to be true, please do not forget to add Mg data in Table 6

Response 17: Thank you for pointing this out to us. We have removed this statement since we lack the Mg levels in feed and cannot calculate apparent retention. (line 562).
We have also improved conclusion to fit this (line 588).

Reviewer 3 Report

Comments and Suggestions for Authors

Review of

Impact of Temperature Reduction from 14 °C to 12 °C in RAS on Atlantic Salmon: Enhanced Growth and Physiological Adaptation Post-Transfer to Seawater

*Major concern

Atlantic salmon reared at 12C showed lower growth than those reared at 14C. However, after seawater transfer, salmon previously reared at 12C showed higher growth than those reared at 14C. This is the main finding of the present study, as reflected in the title. This pattern appears to be a typical example of compensatory growth, a well-recognized phenomenon in fish physiology for many decades. However, the manuscript does not mention compensatory growth at all. If the authors believe this phenomenon is unrelated to compensatory growth, they should explicitly clarify their reasoning for skeptical readers. Compensatory growth is not a novel scientific discovery and, therefore, may not be particularly noteworthy on its own. However, from a practical standpoint, the specific method described in this study could have potential value as on-farm technology.

*Specific comments

L12,13,16,17: (for editors) Is it OK to use undefined abbreviations in Highlights?

L45-66: (liner numbers missing ) "The strict regulations...increased productivity."----Meaning unclear.

Figure 1---- should indicate water temperature and other conditions in the figure.

Table 1---- "Water balance -4.1"---- Meaning unclear.

L214-215: Please clearly define the differences among total phosphorus (TP), dissolved phosphorus (DP), and phosphate (PO₄-P). Intuitively, TP and DP should be identical in water samples. Additionally, are there other forms of phosphorus in water besides PO₄-P? This distinction is quite confusing.

L218: "before nitric acid digestion"-----?

L224 and many other places: ----It it necessary to cite so many references? Which one is the most important?

L225: HOUDE

L227: What is "total length, cm3"?

L228: -1

L229: different font

L252: final weight of what?

L253-254: "fine-mesh (12.5 cm ø)"----Is this fine mesh?

L254-255: Please explain what "fecal stability" means. And how it was determined.

L255-256: "detailed in Flo, Cavrois-Rognacki et al. 2024 (Flo, Cavrois-Rogacki et al. 2024)"---Is this a correct way of citation?

L270-273----Meaning unclear.

L278: I could not find the results of proximal composition.

L290: "The fish were not subjected to any treatments beyond their natural experiences."--- This sounds quite odd since all aquaculture conditions are unnatural, not to mention experimental conditions.

Table 3 (and other tables and figures)----- Undefined abbreviations should not be used in tables and figures.

Table 4: Salinity (ppm)----?

L382-401: It appears that similar observations have previously been reported in Atlantic salmon, making this study more of a confirmatory investigation.

L389: "comparable fillet yield"--- Are these sizes of fish processed into fillets? If not, this is irrelevant.

L415-439: Detailed cost calculations are provided; however, the cost depends entirely on the farming site, where water temperature can vary significantly. As a result, this calculation has limited scientific or practical relevance.

L472: "On average, 18 % of the total dietary P is excreted in dissolved form"--- This can also vary depending on diet composition and other factors. Discussing phosphorus excretion in this context is neither informative nor useful.

L528-530--- Meaning unclear.

L542---Increased effluent?

 

 

Comments on the Quality of English Language

as mentioned above.

Author Response

Thank you for your valuable insights and suggestions on how to improve our manuscript.

 

Comments 1: Atlantic salmon reared at 12C showed lower growth than those reared at 14C. However, after seawater transfer, salmon previously reared at 12C showed higher growth than those reared at 14C. This is the main finding of the present study, as reflected in the title. This pattern appears to be a typical example of compensatory growth, a well-recognized phenomenon in fish physiology for many decades. However, the manuscript does not mention compensatory growth at all. If the authors believe this phenomenon is unrelated to compensatory growth, they should explicitly clarify their reasoning for skeptical readers. Compensatory growth is not a novel scientific discovery and, therefore, may not be particularly noteworthy on its own. However, from a practical standpoint, the specific method described in this study could have potential value as on-farm technology.

Response 1: thank you for pointing this out to us. This has been accounted for in the discussion but we did not use the terminology "compensatory growth". We have improved this to make it more clear (line 422) and supplied additional references. 

 

Comments 2: L12,13,16,17: (for editors) Is it OK to use undefined abbreviations in Highlights?

Response 2: undefined abbreviations has been removed from highlights.  

 

Comments 3: L45-66: (liner numbers missing ) "The strict regulations...increased productivity."----Meaning unclear.

Response 3: This seems to be a problem with formatting, please see updated version with improvements in text for clarity (line 74 and line 79)

 

Comments 4: Figure 1---- should indicate water temperature and other conditions in the figure.

Response 4: thank you for this suggestion. Figure 1 has been updated with information (line 156-157)

 

Comments 5: Table 1---- "Water balance -4.1"---- Meaning unclear.

Response 5: There is a balance account/raw material for the sum of water in all the raw materials against the target for water content in the finished product. We must take this into account so that the nutrient content in the recipes is correct and not affected by varying water content in raw materials.

 

Comments 6: L214-215: Please clearly define the differences among total phosphorus (TP), dissolved phosphorus (DP), and phosphate (PO₄-P). Intuitively, TP and DP should be identical in water samples. Additionally, are there other forms of phosphorus in water besides PO₄-P? This distinction is quite confusing

Response 6: Thank you for this suggestion to improve the text. We have updated in line 233.

 

Comments 7: L218: "before nitric acid digestion"-----?

Response 7: This is the referenced procedure for measuring minerals in the sample. For clarity we have improved on the sentence (line 236-237)

 

Comments 8: L224 and many other places: ----It it necessary to cite so many references? Which one is the most important?

Response 8: Thank you for this suggestion. We have removed some excess references (line 82, 110, 243)

 

Comments 9: L225: HOUDE

Response 9: Thank you for pointing this out. Edited reference (line 244)

 

Comments 10: L227: What is "total length, cm3"?

Response 10: thank you for pointing this mistake out. formula has been updated (line 246)

 

Comments 11: L228: -1

Response 11: Thank you for pointing this mistake out to us, formula has been updated (line 247)

 

Comments 12: L229: different font

Response 12: Thank you for pointing this out. Formula has been updated (line 248)

 

Comments 13: L252: final weight of what?

Response 13: thank you for pointing this out. fish has been added to make it clear (line 271)

 

Comments 14: L253-254: "fine-mesh (12.5 cm ø)"----Is this fine mesh?

Response 14: we agree on poor phrasing, "Fine" has been removed (line 273)

 

Comments 15: L254-255: Please explain what "fecal stability" means. And how it was determined.

Response 15: Thank you for your comment. Explanation has been added (line 274 - 277)

 

Comments 16: L255-256: "detailed in Flo, Cavrois-Rognacki et al. 2024 (Flo, Cavrois-Rogacki et al. 2024)"---Is this a correct way of citation?

Response 16: removed (line 277)

 

Comments 17: L270-273----Meaning unclear.

Response 17: Thank you for your comment, rephrasing has been done to improve and make more clear (line 291 - 296)

 

Comments 18: L278: I could not find the results of proximal composition.

Response 18: Thank you for pointing this out. This is a matter of phrasing, we have made adjustments to avoid misinterpretation (line 299-302)

 

Comments 19: L290: "The fish were not subjected to any treatments beyond their natural experiences."--- This sounds quite odd since all aquaculture conditions are unnatural, not to mention experimental conditions.

Response 19: Thank you for your comment. we have removed this sentence (line 313)

 

Comments 20: Table 3 (and other tables and figures)----- Undefined abbreviations should not be used in tables and figures.

Response 20: Thank you for pointing this out. We have updated table 3 (line 333-334) and Figure 3 (line344-345) 

 

Comments 21: Table 4: Salinity (ppm)----?

Response 21: thank you for pointing this out . Salinity is updated to ppt in table 4 (line 353)

 

Comments 22: L382-401: It appears that similar observations have previously been reported in Atlantic salmon, making this study more of a confirmatory investigation.

Response 22: One of the objectives from this study was to investigate the accumulation of minerals in the RAS and from the results it is natural to report growth data even though similar studies has been conducted before. As you also mention, the RAS vs RAS design of the current trial does have a value for on farm technology and also provide new insight in relation to the, not so common, RAS vs RAS trial design on post-transfer performance. 

 

Comments 23: L389: "comparable fillet yield"--- Are these sizes of fish processed into fillets? If not, this is irrelevant.

Response 23: Thank you for your comment, this has been updated (line 414)

 

Comments 24: L415-439: Detailed cost calculations are provided; however, the cost depends entirely on the farming site, where water temperature can vary significantly. As a result, this calculation has limited scientific or practical relevance.

Response 24: We agree that the detailed cost calculation can be dependent on the farming site. Due to heat generation within these systems, cooling is required for most such solutions in production of Atlantic salmon. Since the location and condition of the farming site can significantly influence the total heat generated and thus the different cooling capacities required, our calculations only focuses on the additional cost of cooling water an extra two degrees from 14 to 12°C. We believe this provide some practical relevance to understand how this specific temperature adjustments can affect the energy cost of cooling and hope to keep this in the manuscript. 

 

Comments 25: L472: "On average, 18 % of the total dietary P is excreted in dissolved form"--- This can also vary depending on diet composition and other factors. Discussing phosphorus excretion in this context is neither informative nor useful.

Response 25: Thank you for your comment. We have removed and rephrased this in line 512 - 515. But we wish to keep it as a practical example (541-546) as it is a good way of highlighting how temperature can effect the characteristics of the excrement. We make sure to emphasize that P availability in raw material is the most important factor in determining fractional composition of the P excretion (line 546-548). 

 

Comments 26: L528-530--- Meaning unclear.

Response 26: Thank you for your comment. This has been improved (line 568-571)

 

Comments 27: L542---Increased effluent?

Response 27: thank you for your comment. this has been improved to make it more clear that we address P excretion (line 583)

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Due to the limited time available for review, I can only provide discursive comments. From lines 419–431, it is clear that this study is purely confirmatory, with no new findings reported. The accumulation of minerals in RAS is highly dependent on feed composition, which varies significantly. As a result, this study presents only one of many possible cases of mineral accumulation in RAS, limiting the practical applicability of the reported data and techniques. The Materials and Methods section is highly detailed; however, its practical utility is constrained for the reasons mentioned above. In Table 1, the authors should specify "dry basis" and clarify the meaning of "water balance." Line 233 remains unclear—does "bioavailable P" refer to fish, algae, or bacteria? Additionally, how was bioavailability determined? In Table 2, the row alignment appears inaccurate. Also, the repeated use of "times a week" sounds awkward. Line 236: Is it possible to perform ICP analysis "before" nitric acid digestion? This is what I was referring to previously.

 

Author Response

Again, i would like to thank you for taking the time to review and help improve our manuscript. 

Comments 1: From lines 419–431, it is clear that this study is purely confirmatory, with no new findings reported.

Response 1: We agree that the study confirms previous research on compensatory growth. However, it also highlights the expected compensatory growth at temperature regimes currently utilized and debated in today's commercial RAS production of Atlantic salmon. The finding that fish reared in RAS at 12°C, although initially smaller compared to those reared at 14°C, have the potential to catch up within 10 weeks post-transfer, and that the weight gained is primarily muscle rather than fat, as indicated by a lower VSI, is of high practical significance when farmers are determining a production regimes in RAS. The RAS vs RAS design of the current trial also does have a value for on farm technology and also provide new insight in relation to the, not so common, RAS vs RAS trial design on post-transfer performance.

 

Comments 2: The accumulation of minerals in RAS is highly dependent on feed composition, which varies significantly. As a result, this study presents only one of many possible cases of mineral accumulation in RAS, limiting the practical applicability of the reported data and techniques.

Response 2: We agree that the accumulation of minerals is highly dependent on feed composition, as highlighted in line 523-524. However, our study demonstrates that temperature also plays a significant role in the accumulation tendencies within the system and how this connects to growth both in RAS and post-transfer, which was the purpose of our design. While the composition of diets can have varying effects, understanding that temperature also impacts the accumulation and ultimately the emission levels of minerals (with a focus on phosphorus) is highly relevant for commercial production. This knowledge is of high value since it shows that altering temperature regimes in RAS can directly affect the emission license and potentially be a limiting factor. 

 

Comments 3: In Table 1, the authors should specify "dry basis" and clarify the meaning of "water balance."

Response 3: Thank you for your comment on how to improve the table. Water balance has been specified in line 178-181. We have chosen to present composition and minerals in Table 1 on an "as is" basis and this has been highlighted in table 1. 

 

Comments 4: Line 233 remains unclear—does "bioavailable P" refer to fish, algae, or bacteria? Additionally, how was bioavailability determined?

Response 4: We apologize for using the phrase "bioavailable P" as it leads to more confusion and does not address your previous comment. PO4-P, often referred to as “reactive phosphorus”, is a subset of DP but does not include other forms of P, e.g., condensed P which is more a complex form and need to be hydrolyzed to release PO4-P. This means that all PO₄ are DP but not all DP is PO₄. We chose to measure PO₄-P within the system since it is the simplest form of phosphate and are directly available for biological uptake in aquatic environments. This is therefor an important measure in RAS with a biological filter to understand what is happening with the bacterial development (as described in line 469-471 and 476-479).

 

 

Comments 5: In Table 2, the row alignment appears inaccurate. Also, the repeated use of "times a week" sounds awkward.

Response 5: Thank you for pointing this out to us. We have made changes to the structure in all Tables. The repeated use of "once a week" was a suggestion from another reviewer to avoid misinterpretation, and we chose to keep this in the text. 

 

Comments 6: Line 236: Is it possible to perform ICP analysis "before" nitric acid digestion? This is what I was referring to previously.

Response 6: Thank you for clarifying. Nitric acid digestion occurs before ICP-MS, we have made necessary revisions (line 225)

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

I have no additional comments. 

Author Response

All previous comments addressed.