Performance of Pilot Scale Aquaponic System Supported by Phenotypic Assessment Using Nile Tilapia (Oreochromis niloticus) Wastewater and Blue Mussel Meal as Fish Feed

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review for the paper submitted to “Sustainability”.

Title: Performance of pilot scale aquaponic supported by phenotypic assessment using Nile Tilapia (Oreochromis niloticus) wastewater and blue mussel meal as fish feed

Authors: Maria Berger, Jonathan A. C. Roques, Yenitze Fimbres-Acedo, Vishnukiran Thuraga, Aakash Chawade, Maria Elisabeth Karlsson, Samar Khalil

 

The authors focused on evaluating blue mussel meal as a sustainable feed ingredient within an aquaponic system, specifically examining its effects on Nile tilapia and Tatsoi plant cultivation. Their work centered on comparing this novel diet, which also incorporated pea protein, against a conventional feed containing fishmeal and soy. The authors' study showed that the alternative mussel-based diet successfully supported aquaponic operation, with plant growth in these systems outperforming controlled hydroponics. However, it also indicated a potential trade-off, as fish feed conversion efficiency was lower with the alternative diet compared to the conventional one. Furthermore, the study revealed that the aquaponic environment itself may have suppressive effects on certain microbial pathogens compared to standalone aquaculture systems.The authors' work provides evidence for the feasibility of circular protein sources in integrated food production systems.

 

Suggestions for improving the paper:

 

Abstract. The authors should avoid unnecessary details (L 20-26).

 

Introduction.

 

L 69-71. The authors should explain why Fe and Mn, which are micronutrients, are listed alongside macronutrients (K, P, S) and adjust the text for precision.

 

L 88-89. The authors should clarify if this refers to their ecosystem service in the wild or a potential benefit within an integrated multi-trophic aquaculture (IMTA) context, and how this relates to their use as feed.

 

Materials and Methods.

 

L 118. The authors' statement describing system design "in triplicate" requires clarification. The authors should report whether each of the three system types (Aquaponics, Hydroponic control, RAS control) was replicated three times (n=3 per type) or their repeated each experiment three times.

 

L 124. The authors should explain if this is Photosynthetic Photon Flux Density (PPFD). They should also specify the light source (LED, fluorescent) and report the spectral quality or color temperature if known.

 

L 145. The authors should report the source and composition of the inoculum to ensure reproducibility.

 

L 156-157. The authors should report the concentration achieved in the system water (mg L-1) or the application rate per plant/system volume, as the absolute mass is not interpretable without system water volume.

 

L 161. The authors should report any washout period or system reset procedures between Phase 1 and Phase 2.

 

L 223. The authors should report the duration of the dark adaptation period prior to measuring Quantum Yield.

 

Results.

 

L 278-279. The authors should suggest a reason for this discrepancy in water temperature.

 

Discussion.

 

L 376. The authors should specify which species (Tilapia? Tatsoi? Both?) this refers to and what was previously undiscovered about them in this context.

 

L 387-399. The authors' citation of various FCR values from literature requires synthesis. The authors should position their obtained FCR values (CF: ~0.63, MPF: 0.81-1.07) within these cited ranges and state whether they consider their results typical, high, or low.

 

L 408-409. The authors' suggestion that lower pH in the MPF phase partially explains decreased SGR requires supporting correlation. The authors should discuss if the daily pH data showed a consistent drop coinciding with the feed change and if other factors (like diet digestibility) are considered the primary cause.

 

L 457-461. The authors' explanation for poor hydroponic growth in MPF phase being "linked to issues with the Hoagland solution" is underdeveloped. The authors should move this key methodological explanation from the Discussion to the Results section and provide more details what was the "issue"? Incorrect mixing? Depletion?).

Author Response

Reviewer 1

 The authors focused on evaluating blue mussel meal as a sustainable feed ingredient within an aquaponic system, specifically examining its effects on Nile tilapia and Tatsoi plant cultivation. Their work centered on comparing this novel diet, which also incorporated pea protein, against a conventional feed containing fishmeal and soy. The authors' study showed that the alternative mussel-based diet successfully supported aquaponic operation, with plant growth in these systems outperforming controlled hydroponics. However, it also indicated a potential trade-off, as fish feed conversion efficiency was lower with the alternative diet compared to the conventional one. Furthermore, the study revealed that the aquaponic environment itself may have suppressive effects on certain microbial pathogens compared to standalone aquaculture systems. The authors' work provides evidence for the feasibility of circular protein sources in integrated food production systems.

We thank reviewer 1 for the positive feedback on our manuscript and the suggestions for improvement. Please find below our point-by-point answers to your suggestions in bold. Due to revisions made to the text, the original line numbering has shifted; therefore, we indicate the updated line numbers corresponding to each comment based on the clean revised version of the manuscript. Both a tracked-changes version and a clean version are provided for clarity.

Abstract.

The authors should avoid unnecessary details (L 20-26).

We have deleted the first two sentences and shortened the abstract (L20-42).

Introduction.

 L 69-71. The authors should explain why Fe and Mn, which are micronutrients, are listed alongside macronutrients (K, P, S) and adjust the text for precision.

The review is correct; as requested by both reviewers, we have modified the paragraph L66-72’:

‘Most of the nutrients required by the plants in an aquaponic system are retrieved from fish and feed waste. Nevertheless, plants require certain mineral nutrients that are commonly deficient in fish feed and consequently, in aquaponic systems, as they are often poorly retained in bioavailable form due to precipitation, microbial transformation or uptake inefficiencies under typical aquaponic conditions [11, 12].’ These mineral nutrients include macronutrients such as potassium (K), phosphorus (P) and sulfur (S) as well as micronutrients such as manganese (Mn) and iron (Fe) [12].

 L 88-89. The authors should clarify if this refers to their ecosystem service in the wild or a potential benefit within an integrated multi-trophic aquaculture (IMTA) context, and how this relates to their use as feed.

We have  rephrased this part of the text to enhance clarity L91-95:

‘Blue mussels are filter-feeding mollusks that can mitigate eutrophication through the removal of N in coastal ecosystems [28], thereby providing ecological services. In addition, mussel biomass presents a high-quality marine protein source [29] making blue mussels a potential ingredient in aquaculture.’

Materials and Methods.

L 118. The authors' statement describing system design "in triplicate" requires clarification. The authors should report whether each of the three system types (Aquaponics, Hydroponic control, RAS control) was replicated three times (n=3 per type) or their repeated each experiment three times.

We have rephrased the sentence for more clarity L123-124:

‘Each system was independently replicated three times (n = 3 per system type) and ran simultaneously.’

 L 124. The authors should explain if this is Photosynthetic Photon Flux Density (PPFD). They should also specify the light source (LED, fluorescent) and report the spectral quality or color temperature if known.

The information about PPFD and the type of lamps that have been used have been added L130-135:

‘Nutrient film technique (NFT) was used, where nutrient solutions were circulated. These compartments were also continuously aerated with air stone diffusers. Lighting was provided above the plants using high pressure sodium lamps with a photosynthetic photon flux density (PPFD) emission range of 70 – 215 µmol m^-2 s-¹ in a 14:10 (light: dark) photoperiod. The mean PPFD measured across the plant canopy was 200 µmol m^-2 s-¹.’

L 145. The authors should report the source and composition of the inoculum to ensure reproducibility.

We received the inoculum from the same farm who provided us with the fish. They did not disclose the exact composition of the inoculum, we have adjusted the sentences below, adding these information and providing the name of the farm L159-162.

‘The inoculum consisted of a mature biofilter media with nitrifying bacteria provided by the same commercial fish farm that supplied the fish (Gårdsfisk, https://www.gardsfisk.se/). The exact microbial composition of the inoculum was not further characterized.’

L 156-157. The authors should report the concentration achieved in the system water (mg L-1) or the application rate per plant/system volume, as the absolute mass is not interpretable without system water volume.

We have added the concentration of Fe and MgSO₄ added to our 60 L hydroponic systems L175-179:

‘To address commonly reported micronutrient deficiencies in aquaponic systems, the plants were supplemented with 1.72 g Fe (28.7 mg L⁻¹, YaraTera REXOLIN® D12,Yara International ASA, Oslo, Norway) and 24 g of MgSO4 (400 mg L⁻¹, Epsom salt, CAS number 7487-88-9, Merk, Darmstadt, Germany) applied two and six weeks after transplantation.

L 161. The authors should report any washout period or system reset procedures between Phase 1 and Phase 2.’

 There was no washout period between the two phases. We have added this sentence: ‘No washout period or system reset was applied between the two phases.’ (L164)

L 223. The authors should report the duration of the dark adaptation period prior to measuring Quantum Yield.

The duration of the dark adaptation period was added L243-244:

‘dark-adapted for 15 minutes’

Results.

 L 278-279. The authors should suggest a reason for this discrepancy in water temperature.

We explain in the material and methods that the temperature in the room was set at 24 °C, and that heaters were placed in the fish tanks and biofilters, we have now  made a precision that the heaters were in the systems with fish only, as Nile tilapia thrive at higher temperatures L137-139:

‘Temperature was maintained using aquarium heaters set at of 25 °C placed in the fish tanks and biofilters in the aquaponics and RAS.’

As the experiment was carried out during winter, we believe that despite the temperature set at 24 °C, the greenhouse temperature was closer to 20-21 °C. We have also added a sentence in the results L299-302:

‘Water parameters are presented in Table 2. Water temperature remained stable at the temperature set on the heaters (25 °C) in both aquaponic and RAS systems throughout all phases. In contrast, the hydroponic systems, without heating systems, consistently maintained lower temperatures across the entire experimental period (21.5 °C).’

In addition, we have added heaters in the Figures 1a and 1c.

Discussion.

L 376. The authors should specify which species (Tilapia? Tatsoi? Both?). This refers to what was previously undiscovered about them in this context.

We have deleted this sentence as we believe it was too vague and did not add much to the text (L400).

L 387-399. The authors' citation of various FCR values from literature requires synthesis. The authors should position their obtained FCR values (CF: ~0.63, MPF: 0.81-1.07) within these cited ranges and state whether they consider their results typical, high, or low.

The various FCRs cited were synthetized and regrouped under system type/life stages, and our values were put into context. We have also updated the references. L411-422:

‘Overall, the FCR values obtained in the present study fall within, and in some cases below, the lower range of values reported for Nile tilapia in both RAS and aquaponic systems. Reported FCRs in aquaponic systems typically range from 0.72 in fry [48] to 1.14 – 1.47 in fingerlings [49-51] and up to 1.60 – 1.62 in juvenile fish [50, 52]. RAS studies report FCR as low as 0.65 in fry [48] and 1.27 – 1.78 in fingerlings [53]. The inclusion of pea concentrate in the diet led to FCRs of 0.90 – 1.06 [54]. In this context, the FCR obtained with the CF diet (0.62 – 0.64) can be considered particularly low and comparable to the best values reported for fry under optimized conditions, whereas the MPF treatment (0.81–1.07) falls within the lower-to-mid range reported for fingerling and juvenile Nile tilapia. Thus, the FCRs observed in this study are indicative of efficient feed utilization and are equal to or better than most values reported in comparable RAS and aquaponic production systems.’

L 408-409. The authors' suggestion that lower pH in the MPF phase partially explains decreased SGR requires supporting correlation. The authors should discuss if the daily pH data showed a consistent drop coinciding with the feed change and if other factors (like diet digestibility) are considered the primary cause.

We agree with the reviewer, and it is likely that other factors such as feed change and/or digestibility might have played a role in the decreased SGR, more than the pH changes. We have not done any correlation study and therefore have rephrased our paragraph to include all the possible pists to evaluate in the future, L430-441:

‘While Nile tilapia can tolerate a broad pH spectrum, optimal growth performance and FCR are typically observed within a pH range of 6 – 9 [55, 57]. During the CF phase, pH levels averaged around 6, whereas during the MPF phase, pH declined to approximately 5 – 5.5 in both systems. This is a trend commonly observed in closed systems due to ongoing nitrification and limited buffering capacity [58]. Although suboptimal pH may influence fish physiological performance, including growth, no direct correlation analysis was conducted in the present study, and other factors such as differences in diet composition and digestibility could have played a role and should be investigated in more detail in future studies. Nonetheless, the growth performance and feed efficiency results from this study aligned with values reported in the literature, indicating that the experimental trials were successfully conducted and that both system types provided suitable rearing conditions for Nile tilapia.’

 L 457-461. The authors' explanation for poor hydroponic growth in MPF phase being "linked to issues with the Hoagland solution" is underdeveloped. The authors should move this key methodological explanation from the Discussion to the Results section and provide more details about what was the "issue"? Incorrect mixing? Depletion?).

Although we used the same protocol regarding Hoagland dosing in this phase, it could be that the nutrients were still limited. We have rephrased our sentence L491-493:

‘This decline could be likely linked to an initial underdosing of the Hoagland solution; following its renewal after ten days, plant growth started to improve, indicating previous limitations in nutrient availability.’

Additional changes:

In addition to the modifications following the reviewers’ comments, we have added heaters in the RAS and aquaponic figures (Figures 1a and 1c).

We have deleted the reference to liver and sex determination procedure in the material and methods (not used in this manuscript), previously mentioned at the end of section 2.3.

We have corrected the units for nitrogenous waste products in table 3 (mg L^-1).

‘FORMAS (grant number 2020-00867)’, supporting Jonathan Roques during this project was added in the financial support acknowledgement (L562).

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

 

This paper tries to analyse the effectiveness of replacing conventional fishmeal and soy protein in Nile tilapia feed with a more sustainable alternative composed of blue mussel meal and pea protein concentrate. The study meticulously compares the performance of aquaponic systems (fish and plants, specifically Tatsoi) against control groups like RAS and hydroponic systems. Key findings indicate that the alternative feed supports comparable or superior plant growth in the aquaponic setup, while also leading to a reduction in certain pathogens like Salmonella spp. and Escherichia coli compared to RAS, suggesting its potential to enhance the environmental sustainability and nutrient circularity of integrated food production.

 

The objective of the manuscript, in general, is interesting. However, the manuscript requires some improvements to meet publication standards. The manuscript should become acceptable for publication pending suitable minor revision considering the comments appended below.

 

More specific comments:

                                                                                                                                               

Introduction:

 

Line 58-59: "Among the fish commonly raised in aquaponic systems is Nile tilapia (Oreochromis niloticus) [7].". Try to briefly state why it is commonly raised.

 

Line 59-61: "According to recent FAO data, Nile tilapia is still one of the three most farmed fish species worldwide [1] and has proven suitable for aquaponic farming due to its hardiness and adaptability [7].". This is slightly redundant with the previous point. Please consolidate.

 

Line 69-72: "Nevertheless, plants require certain macronutrients that are commonly deficient in aquaponic systems: potassium (K), phosphorus (P), iron (Fe), manganese (Mn), and sulfur (S) [11]. Especially Fe is essential for vital processes in plants and is commonly added as a supplement [12].". Please briefly explain why these nutrients are deficient, which sets up the rationale for feed optimization.

 

Line 95-97: "While the use of blue mussel meal has been explored in the aquaculture industry, its incorporation into the diet of Nile tilapia within an aquaponic system remains unexplored.". Please clarify the type of aquaculture where mussel meal has been explored (e.g., in recirculating aquaculture systems (RAS) or marine cage farming) to better define the knowledge gap.

 

Line 79-81: "However, these alternatives present their own challenges [18]. They often require large amounts of arable land and freshwater, which raises concerns about their environmental impact and competition with human food crops [18].". Please clarify which alternatives this refers to.

 

Line 98-102: " However, tools of assessment are needed to obtain a deeper knowledge about nutri-98 tional dynamic in aquaponic system in relation to the content in fish feed and plant performance. Phenotypic assessment of the plants … nutritional status and stress conditions [30].". This paragraph introduces the plant assessment methods somewhat abruptly. Try to smooth the transition by stating that assessing plant health is necessary for optimizing the system.

 

Material and methods:

 

Line 121-122: "The hydroponic gutters measured 155 × 13 × 5 cm and accommodated ten plants each.". Please clarify the specific type or material of the hydroponic system used. This should be explicitly stated.

 

Line 144-145: " To promote initial biofiltration, both system types were initiated prior to fish introduction using circulating water with active microbial inoculum.". Please specify the composition of the active microbial inoculum and the duration of the pre-stocking cycling period.

 

Line 147-149: " The hydroponic systems received a standard nutrient solution for hydroponic as described in [31].". Instead of only citing the reference, please briefly describe the composition of the standard nutrient solution.

 

Line 177-180: "Additionally, water samples from the fish tanks, biofilters, and hydroponic units were analyzed by LMI commercial laboratory (Helsingborg, Sweden) for micro- and macronutrients at the beginning, middle and end of each of the experimental phases.". Please specify the analytical method used by the commercial laboratory for analyzing the micro- and macronutrients.

 

Line 192-193: "Eight of the representative fish from each system were selected and placed in the solution.". Define the selection criteria for "representative fish". Were they selected randomly, or were they chosen to match the mean system weight? Explicitly state the method to ensure the subsample for dissection and blood drawing is unbiased.

 

Line 214-217: "At the final plant biometric sampling, eight plants were selected and separated into roots and leaves. Each plant part was wrapped in aluminum foil and weighed to determine wet weight, then dried in a drying oven for five days, after which the dry weight was measured.".  Please specify the temperature used in the drying oven. Drying temperature affects the final dry weight and is a standard reporting requirement.

 

Line 268-274: "Welch’s correction was applied when Levene’s test indicated unequal variances. For comparisons involving more than two groups, one-way ANOVA was performed... Data were log10-transformed when normality assumptions were violated...". Please specify which post-hoc test was used following the one-way ANOVA. Standard practice is to report the specific test (e.g., Tukey's HSD or Bonferroni) for comparing specific group pairs after a significant ANOVA result. The results section later indicates a Bonferroni post-hoc test (Table 2 caption), so this needs to be explicitly mentioned here for completeness.

 

Results:

 

Line 278-280 (Table 2): "In contrast, hydroponic systems consistently maintained lower temperatures across the entire experimental period.". Provide the rationale for this temperature difference. Was the hydroponic reservoir placed in a cooler location, or was it a passive heat loss effect due to not having the fish heaters? Stating the reason here connects the data point to the experimental design.

 

Line 281-283 (Table 2): "Electrical conductivity (EC) and total dissolved solids (TDS) displayed similar patterns, with higher levels in the hydroponic systems across all phases.". Please briefly explain why these values are higher in hydroponics—it's due to the addition of the concentrated commercial nutrient salts. Adding this context clarifies that this is an expected outcome, not a system failure.

 

Line 294-297 (Table 3): "NO₂- levels remained low and similar in both systems during the CF phase but were significantly higher in the 295 MPF phase in the aquaponic system (0.13 ± 0.01 mg L^-1) compared to the RAS (0.06 ± 0.04 296 mg L^-1).". Please offer a potential cause for this surprising difference, as RAS typically has higher waste accumulation. Was this due to a temporary instability in the aquaponic biofilter, or a higher NO₂- leaching from the specific MPF diet? Mentioning a hypothesis here sets up the discussion.

 

Discussion:

 

Line 378-380: "Despite a slightly better FCR in RAS, the other feed utilization and growth performance parameters of were similar during the MPF phase between RAS and aquaponics.". Please clarify the "slightly better FCR in RAS.". Quantify the difference from Table 4 here in the text.

 

Line 416-417: "The dietary shift to an alternative protein source in the MPF phase may have further influenced nutrient digestibility and feed utilization efficiency.". Since the authors did not measure nutrient digestibility, this statement is speculative. Try to rephrase to reflect the lack of direct data.

 

Line 447-451: "There was a clear difference in NO₃- accumulation between the fish rearing system types, with significantly higher levels observed in RAS due to the absence of plants... In aquaponic systems, plant uptake contributes to the regulation of NO₃- concentrations...". Please provide a quantified measure of the plant's effect here, referencing the data from Table 3. State the percentage difference in NO₃- concentration between RAS and Aquaponics during the MPF phase.

 

Line 455-457: "However, during the MPF phase, significant differences emerged, with hydroponic systems showing notably poorer growth performance compared to the aquaponics.". Please use the quantitative data to emphasize the magnitude of the difference.

 

Line 467-469: "Furthermore, the impact of MPF on nutrient digestibility and feed utilization efficiency might have been the causal of positive impact on parameters assessed by phenotypic analyses.". This is a key discussion point but is too vague. Please specify which nutrient from the MPF diet might have caused the improvement.

 

Line 479-480: "This might indicate the suppressive conditions towards these pathogens in the investigated systems.". Please be explicit about which pathogens are being referenced here.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

This paper tries to analyse the effectiveness of replacing conventional fishmeal and soy protein in Nile tilapia feed with a more sustainable alternative composed of blue mussel meal and pea protein concentrate. The study meticulously compares the performance of aquaponic systems (fish and plants, specifically Tatsoi) against control groups like RAS and hydroponic systems. Key findings indicate that the alternative feed supports comparable or superior plant growth in the aquaponic setup, while also leading to a reduction in certain pathogens like Salmonella spp. and Escherichia coli compared to RAS, suggesting its potential to enhance the environmental sustainability and nutrient circularity of integrated food production.

The objective of the manuscript, in general, is interesting. However, the manuscript requires some improvements to meet publication standards. The manuscript should become acceptable for publication pending suitable minor revision considering the comments appended below.

We thank reviewer 2 for the positive feedback on our manuscript and the suggestions for improvement. Please find below our point-by-point answers to your suggestions in bold. Due to revisions made to the text, the original line numbering has shifted; therefore, we indicate the updated line numbers corresponding to each comment based on the clean revised version of the manuscript. Both a tracked-changes version and a clean version are provided for clarity.

Introduction:

 Line 58-59: "Among the fish commonly raised in aquaponic systems is Nile tilapia (Oreochromis niloticus) [7].". Try to briefly state why it is commonly raised, and Line 59-61: "According to recent FAO data, Nile tilapia is still one of the three most farmed fish species worldwide [1] and has proven suitable for aquaponic farming due to its hardiness and adaptability [7].". This is slightly redundant with the previous point. Please consolidate.

We have rephrased this paragraph, removing the redundancy and provided more specific information regarding the suitability of Nile tilapia in aquaponics L54-59:

‘According to recent data from the Food and Agriculture Organization of the United Nations (FAO), Nile tilapia is still one of the three most farmed fish species worldwide [1]. This species has proven suitable for aquaponic farming due to its hardiness and adaptability, including tolerance to suboptimal water quality, robustness under intensive culture conditions, efficient feed conversion and compatibility with plant nutrient requirements [7].’

Line 69-72: "Nevertheless, plants require certain macronutrients that are commonly deficient in aquaponic systems: potassium (K), phosphorus (P), iron (Fe), manganese (Mn), and sulfur (S) [11]. Especially Fe is essential for vital processes in plants and is commonly added as a supplement [12].". Please briefly explain why these nutrients are deficient, which sets up the rationale for feed optimization.

The paragraph has been modified as requested by both reviewers L66-72:

‘Most of the nutrients required by the plants in an aquaponic system are retrieved from fish and feed waste. Nevertheless, plants require certain mineral nutrients that are commonly deficient in fish feed and consequently, in aquaponic systems, as they are often poorly retained in bioavailable form due to precipitation, microbial transformation or uptake inefficiencies under typical aquaponic conditions [11, 12]. These mineral nutrients include macronutrients such as potassium (K), phosphorus (P) and sulfur (S) as well as micronutrients such as manganese (Mn) and iron (Fe) [12].’  

Line 95-97: "While the use of blue mussel meal has been explored in the aquaculture industry, its incorporation into the diet of Nile tilapia within an aquaponic system remains unexplored.". Please clarify the type of aquaculture where mussel meal has been explored (e.g., in recirculating aquaculture systems (RAS) or marine cage farming) to better define the knowledge gap.

We have clarified the type of aquaculture (both cage-based and RAS) and species (marine carnivorous) L99-103:

‘While the use of blue mussel meal has been explored as alternative feed ingredient primarily in in marine aquaculture systems, including cage-based and RAS for carnivorous fish species [31-33], its incorporation into the diet of the omnivorous species Nile tilapia within an aquaponic system has not yet been investigated.’

Line 79-81: "However, these alternatives present their own challenges [18]. They often require large amounts of arable land and freshwater, which raises concerns about their environmental impact and competition with human food crops [18].". Please clarify which alternatives this refers to.

We have clarified which alternatives we referred to (plant-based).  L81-82

‘However, these terrestrial plant-based alternatives present their own challenges [19]’

Line 98-102: " However, tools of assessment are needed to obtain a deeper knowledge about nutritional dynamic in aquaponic system in relation to the content in fish feed and plant performance. Phenotypic assessment of the plants … nutritional status and stress conditions [30].". This paragraph introduces the plant assessment methods somewhat abruptly. Try to smooth the transition by stating that assessing plant health is necessary for optimizing the system.

We have smoothened the transition as suggested by the reviewer L104-108:

‘In order to optimize aquaponic systems, it is essential to monitor plant health and performance in relation to the fish feed composition. Tools for assessing these dynamics, such as phenotypic assessment based on image analyses, measurements of chlorophyll content and leaf area are crucial. These measurements have been implemented as indicators of plant nutritional status and stress conditions [34].’

Material and methods:

Line 121-122: "The hydroponic gutters measured 155 × 13 × 5 cm and accommodated ten plants each.". Please clarify the specific type or material of the hydroponic system used. This should be explicitly stated.

These information have been added L129-132, since the gutter were also present in the aquaponic systems, we have also added it, and we have further provided the total volume of each system L128-131:

‘The aquaponics and hydroponic systems included plastic gutters measuring 155 × 13 × 5 cm (10 L) to accommodate the plants. The total volumes for each system were: aquaponics: 210 L, RAS: 120L, hydroponics: 60 L. Nutrient film technique (NFT) was used, where nutrient solutions were circulated.’

Line 144-145: " To promote initial biofiltration, both system types were initiated prior to fish introduction using circulating water with active microbial inoculum.". Please specify the composition of the active microbial inoculum and the duration of the pre-stocking cycling period.

We received the inoculum from the same farm who provided us with the fish. They did not disclose the exact composition of the inoculum, we have adjusted the sentences below, adding these information and providing the name of the farm L159-162.

‘The inoculum consisted of a mature biofilter media with nitrifying bacteria provided by the same commercial fish farm that supplied the fish (Gårdsfisk, https://www.gardsfisk.se/). The exact microbial composition of the inoculum was not further characterized.’

Line 147-149: " The hydroponic systems received a standard nutrient solution for hydroponic as described in [31].". Instead of only citing the reference, please briefly describe the composition of the standard nutrient solution.

We have used the common hydroponic solution known as the Hoagland solution, we have briefly stated its composition L165-169:

‘The hydroponic systems received a standard mineral nutrient solution for hydroponic as described by Hoagland and Arnon [35], commonly referred to as the Hoagland solution, providing NO₃^-, P, K, S, calcium (Ca) and magnesium (Mg) as macronutrients, along with trace elements including Fe, Mn, zinc (Zn), copper (Cu), boron (B), and molybdenum (Mo).’

Line 177-180: "Additionally, water samples from the fish tanks, biofilters, and hydroponic units were analyzed by LMI commercial laboratory (Helsingborg, Sweden) for micro- and macronutrients at the beginning, middle and end of each of the experimental phases.". Please specify the analytical method used by the commercial laboratory for analyzing the micro- and macronutrients.

We have specified the analytical method used L197-201:

‘Additionally, water samples from the fish tanks, biofilters, and hydroponic units were analyzed by LMI commercial laboratory (Helsingborg, Sweden) using Spurway analyses methods for quantifying the concentration of micro- and macronutrients at the beginning, middle and end of each of the experimental phases.’

Line 192-193: "Eight of the representative fish from each system were selected and placed in the solution.". Define the selection criteria for "representative fish". Were they selected randomly, or were they chosen to match the mean system weight? Explicitly state the method to ensure the subsample for dissection and blood drawing is unbiased.

Yes, we sampled fish which were close to the system’s weight. We also checked for the absence of severe injuries (which we did not detect in any fish), we finally needed fish of sufficient body size to allow reliable blood collection. We have specified L212-215:

‘From each system, eight fish were sampled for blood sampling and subsequent plasma cortisol analysis. Fish were chosen to be representative of the tank population, with body weight close to the system’s mean and no visible sign of severe injury.’ 

Line 214-217: "At the final plant biometric sampling, eight plants were selected and separated into roots and leaves. Each plant part was wrapped in aluminum foil and weighed to determine wet weight, then dried in a drying oven for five days, after which the dry weight was measured.".  Please specify the temperature used in the drying oven. Drying temperature affects the final dry weight and is a standard reporting requirement.

The temperature was specified L235-237:

‘Each plant part was wrapped in aluminum foil and weighed to determine wet weight, then dried in a drying oven at 70 °C for five days, after which the dry weight was measured.’

Line 268-274: "Welch’s correction was applied when Levene’s test indicated unequal variances. For comparisons involving more than two groups, one-way ANOVA was performed... Data were log10-transformed when normality assumptions were violated...". Please specify which post-hoc test was used following the one-way ANOVA. Standard practice is to report the specific test (e.g., Tukey's HSD or Bonferroni) for comparing specific group pairs after a significant ANOVA result. The results section later indicates a Bonferroni post-hoc test (Table 2 caption), so this needs to be explicitly mentioned here for completeness.

The reviewer is correct; we used the Bonferroni correction as post-hoc test. We have specified it in the material and methods L291-292:

‘When ANOVA indicated significant differences, post-hoc pairwise comparisons were conducted using the Bonferroni correction.’

Results:

 Line 278-280 (Table 2): "In contrast, hydroponic systems consistently maintained lower temperatures across the entire experimental period.". Provide the rationale for this temperature difference. Was the hydroponic reservoir placed in a cooler location, or was it a passive heat loss effect due to not having the fish heaters? Stating the reason here connects the data point to the experimental design.

Yes, it was due to the fact that there were no heaters like in the fish tanks. We have modified the paragraph accordingly L299-302:

‘Water parameters are presented in Table 2. Water temperature remained stable at the temperature set on the heaters (25 °C) in both aquaponic and RAS systems throughout all phases. In contrast, the hydroponic systems, without heating systems, consistently maintained lower temperatures across the entire experimental period (21.5 °C).’

Line 281-283 (Table 2): "Electrical conductivity (EC) and total dissolved solids (TDS) displayed similar patterns, with higher levels in the hydroponic systems across all phases.". Please briefly explain why these values are higher in hydroponics—it's due to the addition of the concentrated commercial nutrient salts. Adding this context clarifies that this is an expected outcome, not a system failure.

The reviewer is correct, these higher values throughout the experimental phases were due to the addition of mineral nutrient salts. We have added a sentence to clarify our text L305-308:

 ‘The addition of the Hoagland solution in the hydroponic systems increased the ionic strength of the water and consequently the EC and TDS in these systems. Meanwhile, aquaponic and RAS systems maintained lower, comparable levels that gradually increased over time.’

Line 294-297 (Table 3): "NO₂- levels remained low and similar in both systems during the CF phase but were significantly higher in the MPF phase in the aquaponic system (0.13 ± 0.01 mg L^-1) compared to the RAS (0.06 ± 0.04 296 mg L^-1).". Please offer a potential cause for this surprising difference, as RAS typically has higher waste accumulation. Was this due to a temporary instability in the aquaponic biofilter, or a higher NO₂- leaching from the specific MPF diet? Mentioning a hypothesis here sets up the discussion.

We have provided a potential explanation for this transient accumulation of NO₂^- during the MPF phase, in the discussion L474-479:

‘The relatively high, but still well within tolerable levels for Nile tilapia, NO₂^- levels observed in the aquaponic tanks during the MPF phase may have resulted from a temporary biofilter imbalance following the switch of diet. Since NH₄⁺ levels remained stable during this phase, the transient accumulation likely resulted from a short-term reduction in NO₂^- removal rather than increased N input. However, the observed levels remained low and well within tolerable ranges for Nile tilapia [45].’

Because there was no impact on NH₄⁺, leaching of the MPF is unlikely, but the higher NO₂^- levels are likely due to an unbalance in the filter following the MPF introduction. However, the levels observed are well within the tolerance range for Nile tilapia (~81 mg L⁻¹ for small fish of ~4.4 g and ~8 mg L⁻¹ for large fish ~90.7 g; our experimental fish ~45 g would be expected to have an intermediate tolerance threshold. We have added a reference to backup our statement (Atwood et al, 2001, [45], L321).

Discussion:

Line 378-380: "Despite a slightly better FCR in RAS, the other feed utilization and growth performance parameters of were similar during the MPF phase between RAS and aquaponics.". Please clarify the "slightly better FCR in RAS.". Quantify the difference from Table 4 here in the text.

We have rephrased this sentence, removed ‘slightly’ and quantified the difference between RAS and aquaponics (24.3% improvement in RAS), L401-404:  

‘Despite a better FCR in RAS (0.81± 0.04) compared to aquaponics (1.07 ± 0.14), corresponding to a 24.3% improvement, the other feed utilization and growth performance parameters of were similar during the MPF phase between RAS and aquaponics.

Line 416-417: "The dietary shift to an alternative protein source in the MPF phase may have further influenced nutrient digestibility and feed utilization efficiency.". Since the authors did not measure nutrient digestibility, this statement is speculative. Try to rephrase to reflect the lack of direct data.’

We have rephrased to acknowledge this was speculative and not assessed in our study L442-444:

‘The dietary shift to an alternative protein source in the MPF phase may have further influenced nutrient digestibility and feed utilization efficiency, although these effects were not assessed in this study.’

Line 447-451: "There was a clear difference in NO₃- accumulation between the fish rearing system types, with significantly higher levels observed in RAS due to the absence of plants... In aquaponic systems, plant uptake contributes to the regulation of NO₃- concentrations...". Please provide a quantified measure of the plant's effect here, referencing the data from Table 3. State the percentage difference in NO₃- concentration between RAS and Aquaponics during the MPF phase.

We have added the percentage difference in NO₃⁻  between the two systems, 23.6% higher in RAS compared to aquaponics L481.

Line 455-457: "However, during the MPF phase, significant differences emerged, with hydroponic systems showing notably poorer growth performance compared to the aquaponics.". Please use the quantitative data to emphasize the magnitude of the difference.

We have given the data and relative reduction in biomass accumulation in the hydroponic vs aquaponic system L487-491:

‘However, during the MPF phase, significant differences emerged, with hydroponic systems showing significantly lower growth performance compared to the aquaponics, with a mean biomass accumulation of 1.29 ± 0.33 g per plant, corresponding to a 91.4% reduction in biomass accumulation relative to the aquaponic system.’

Line 467-469: "Furthermore, the impact of MPF on nutrient digestibility and feed utilization efficiency might have been the causal of positive impact on parameters assessed by phenotypic analyses.". This is a key discussion point but is too vague. Please specify which nutrient from the MPF diet might have caused the improvement.

The reviewer is correct, we have rephrased the paragraph, we removed the reference to the nutrient digestibility and feed utilization, which we did not measure, and focused our discussion on  which nutrients from the MPF diet could have had a positive impact on the plant growth in the aquaponic systems L501-510:

‘Furthermore, the MPF diet may have influenced nutrient availability in the aquaponic system, particularly for nutrients linked to photosynthetic performance such as N and Fe [13, 73], thereby contributing to the positive responses on parameters assessed by phenotypic analyses. The improved shoot growth and SPAD indicate an enhanced photosynthetic efficiency, suggesting that the conditions in the aquaponic system with MPF can promote photosynthetic performance towards yield improvement [74]. SPAD measurements also indicated a potential to reduce leaf damage under stress conditions [75] when aquaponic is conducting using MPF as designed in the current study. Previous studies have also reported a similar positive effect of MPF on plant and fish growth and nutrient content in aquaculture [76].’

Line 479-480: "This might indicate the suppressive conditions towards these pathogens in the investigated systems.". Please be explicit about which pathogens are being referenced here.

We have specified which pathogens we were referring to (Oomycetes) L514-516:

‘This might indicate the suppressive conditions towards pathogens related to the fungal group Oomycetes in the investigated systems.’

Additional changes:

In addition to the modifications following the reviewers’ comments, we have added heaters in the RAS and aquaponic figures (Figures 1a and 1c).

We have deleted the reference to liver and sex determination procedure in the material and methods (not used in this manuscript), previously mentioned at the end of section 2.3.

We have corrected the units for nitrogenous waste products in table 3 (mg L^-1).

‘FORMAS (grant number 2020-00867)’, supporting Jonathan Roques during this project was added in the financial support acknowledgement (L562).

Author Response File: Author Response.pdf