Review Reports - Native Fish Inclusion Promotes Nutrient Retention and Productivity in a Biofloc-Based Aquaponic System

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Native fish inclusion improves nutrient retention and productivity in a biofloc-based aquaponic system” is devoted to the study of the presence in biofloc-based aquaponic systems of local small fish - yellowtail lambari (Astyanax bimaculatus) on zootechnical parameters, the growth of lettuce (Lactuca sativa), hematological parameters of Nile tilapia (Oreochromis niloticus), Ah also water quality and nitrogen and phosphorus retention.

Despite the limited knowledge of the functional role of small native fish species, A. bimaculatus are used as potential candidates for integration due to their rapid growth and short production cycles. The authors showed that the co-residence of A. bimaculatus with L. sativa and O. niloticus in a joint biofloc-based aquaponic system led to improved growth and increased biomass of L. sativa lettuce. In addition, the hematological and zootechnical parameters of the main cultivated species, the Nile tilapia O. niloticus, did not deteriorate. There was also an increase in nitrogen and phosphorus retention at the system level and an improvement in water quality due to a decrease in the content of nitrogenous compounds and suspended particles.

The presented work is very relevant and significant in the field of the joint use of two technologies - biofloc and aquaponics, which led to the creation of biofloc-based aquaponic systems.

Despite the well-written manuscript, there are a number of questions and minor comments:

Based on what data did the authors of the manuscript determine the length of stay of small local fish in the experiment?
In the section “3. Results and Discussion”, the authors of the presented work describe in detail the results obtained from the presence of small native fish A. bimaculatus in biofloc-based aquaponic systems and show a decrease in alkalinity, total ammonia nitrogen, toxic ammonia, and total suspended solids in the water. The authors also note that statistically significant differences between the groups were recorded in the last week. Based on all the results obtained, the authors need to provide proposed biochemical explanations for such changes in water quality.
Also in the section “3. Results and Discussion” on page 6, lines 237-239, the authors write “These results suggest that species diversification influenced both the chemical and particular dynamics of the system". The authors need to indicate the variety of which species and which object they are writing about.
In addition, the authors say that the results indicate the influence of species diversity on the chemical composition and dynamics of solid particles in the system. It is necessary to explain these changes.
In the section “3. Results and Discussion”, it is advisable for the authors to divide the results into three subsections.

For example:

3.1. Changes in the main indicators of water quality;

3.2. Changes in the growth indicators of L. sativa lettuce;

3.3. Changes in the indicators of the Nile tilapia O. niloticus.

Authors are recommended to enlarge the section “4. Conclusions”.

Author Response

Dear Editor and Reviewer 1,

We sincerely thank the reviewers for their careful evaluation of our manuscript and for the constructive comments provided. We have revised the manuscript accordingly and believe that these changes have improved its clarity, rigor, and interpretation. All modifications are highlighted in the revised version. Our point-by-point responses are presented below.

Comment 1:
Based on what data did the authors determine the experimental duration?

Response:
The experimental period (35 days) was defined based on previous studies evaluating Nile tilapia performance in biofloc systems, in which this duration is sufficient to detect growth responses and changes in water quality under controlled conditions. This information has been clarified in the revised manuscript.

Comment 2:
Provide biochemical explanations for the observed changes in water quality.

Response:
We agree and have expanded the Discussion to include plausible biochemical explanations. The revised text now considers microbial assimilation processes, in which heterotrophic bacteria incorporate dissolved inorganic nitrogen into microbial biomass, as well as nitrification pathways that contribute to alkalinity consumption through ammonia oxidation. These mechanisms are presented as plausible explanations and not as directly measured processes.

Comment 3:
Clarify which species are being referred to in the discussion.

Response:
The text has been revised to explicitly refer to the integration of Nile tilapia, yellowtail lambari, and lettuce when discussing species diversification and system dynamics.

Comment 4:
Explain the influence of species diversity on chemical composition and particulate dynamics.

Response:
The Discussion has been expanded to clarify that the observed changes in nitrogen compounds and suspended solids may be associated with microbial processes and interactions with particulate organic matter. These explanations are presented cautiously, as they were not directly measured.

Comment 5:
Reorganize the Results and Discussion into subsections.

Response:
We appreciate this suggestion. However, we opted to maintain an integrated structure to preserve the logical connection between water quality, biological performance, and nutrient dynamics within the system. Nevertheless, the manuscript was revised to improve clarity and organization, ensuring that each component is clearly addressed and discussed.

Comment 6:
Expand the Conclusions section.

Response:
The Conclusions section has been expanded to better summarize the findings, reinforce the results related to nutrient retention, and acknowledge the short-term nature of the study.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors

This manuscript evaluates the inclusion of yellowtail lambari (Astyanax bimaculatus) in a biofloc-based aquaponic system integrating Nile tilapia and lettuce. The study addresses a timely and relevant topic: improving nutrient-use efficiency and system productivity through species diversification in integrated aquaculture systems. The experimental design is generally appropriate, and the combination of water-quality monitoring, fish performance, lettuce growth, hematological parameters, and nutrient-retention estimates provides a useful applied dataset.

The results are promising, particularly the lower TAN, NH₃ and TSS values, improved lettuce biomass, and increased system-level N and P retention in the lambari treatment. However, the manuscript would benefit from a more cautious interpretation of the mechanisms behind these effects and clearer framing of the study’s limitations.

1. Distinguish observed effects from inferred mechanisms

The manuscript attributes improvements in water quality and nutrient dynamics to the inclusion of lambari, often invoking microbial activity, biofloc interaction, or altered particulate dynamics. These explanations are plausible and consistent with BFT principles, but they were not directly measured in this experiment.

For example, reduced TAN and NH₃ are interpreted as improved nitrogen utilization, while lower TSS is discussed in relation to possible interaction of lambari with suspended particles or biofloc biomass.

I recommend that the authors clearly distinguish between observed system responses and inferred mechanisms, moderate causal language throughout the Discussion, and explicitly state that microbial community structure, biofloc ingestion, and trophic interactions were not directly assessed.

2. Clarify the interpretation of nutrient retention

The manuscript reports higher system-level nitrogen and phosphorus retention, while no significant differences are observed when Nile tilapia and lettuce are evaluated separately. This is a critical point. The current interpretation suggests improved system efficiency. However, because lambari were present only in one treatment, the higher system-level retention may partly reflect the addition of another biomass compartment rather than improved nutrient-use efficiency alone.

I recommend clarifying the nutrient accounting boundaries, including which nutrient pools were included or excluded from the retention calculation. The authors should also acknowledge that retention in harvested biomass does not represent a complete nutrient mass balance and does not necessarily demonstrate reduced nutrient losses from the system.

3. Experimental duration and system stabilization

The manuscript states that mature biofloc water from a previously stabilized system was used as inoculum. This is an important strength of the experimental design and reduces concerns regarding initial biofloc establishment.

However, the experimental period of 35 days remains relatively short for evaluating whether the newly configured tilapia–lambari–lettuce system reaches a stable long-term plant–microbial–fish equilibrium. Several reported differences, including TAN, NH₃, pH, alkalinity, and TSS, became most evident in the final weeks of the trial. Therefore, the authors should acknowledge that the results demonstrate short-term responses under mature-inoculum conditions, but do not yet confirm the long-term stability or persistence of the observed effects.

4. Define lambari’s functional role more clearly

The manuscript positions lambari as a functional component within the system. However, its exact role remains unclear.

The authors should clarify whether lambari is hypothesized to contribute through direct consumption of suspended particles or biofloc biomass, through additional nutrient storage in lambari biomass, or through indirect effects on microbial and particulate dynamics.

Given that lambari were not externally fed, the assumption that they consumed biofloc or suspended organic matter is central to the manuscript, but it is not directly demonstrated. The authors should therefore avoid implying a confirmed mechanism without direct evidence of biofloc ingestion, changes in floc structure, or microbial modification.

5. Statistical interpretation

The experimental design, with two treatments and four replicates, is acceptable for a controlled aquaculture trial. However, several water-quality variables were compared repeatedly across weeks using t-tests. The authors should briefly justify this approach or acknowledge the possibility of inflated type I error when interpreting weekly differences. This is particularly relevant for variables such as TAN, NH₃, pH, alkalinity, and TSS, where significance is presented across multiple time points.

6. Water-chemistry interpretation

The reductions in TAN, NH₃, and TSS support the conclusion that lambari inclusion affected system dynamics. However, reduced alkalinity should be interpreted with more caution. Lower alkalinity may reflect increased nitrification or microbial activity, but it also indicates greater buffering demand and should not automatically be presented only as an improvement in system performance. A more nuanced interpretation of pH and alkalinity dynamics would strengthen the manuscript.

7. Clarity and conclusion wording

Some sections of the Discussion are repetitive, particularly those dealing with nutrient cycling, system productivity, and sustainability. The linkage between specific results and their interpretation could be made more direct.

The Conclusion also slightly overstates the degree of optimization demonstrated by the experiment. I suggest replacing wording such as “proved effective in optimizing nutrient use and system productivity” with a more cautious formulation, for example:

“The inclusion of yellowtail lambari showed potential to improve nutrient retention and productivity under the tested short-term biofloc-based aquaponic conditions, without negatively affecting Nile tilapia performance.”

Comments on the Quality of English Language

The manuscript is generally clear and understandable, but the English language would benefit from minor revision.

* Some sentences are overly long and could be simplified for clarity.
* Occasional awkward phrasing and grammatical inconsistencies are present.
* Terminology is generally consistent but could be streamlined in sections discussing nutrient dynamics and system processes.

A light language edit is recommended to improve readability and flow.

Author Response

Dear Editor and Reviewer,

Response:
The manuscript has been revised to clearly distinguish between observed results and inferred mechanisms. Statements regarding microbial processes, biofloc interactions, and particulate dynamics were reformulated to indicate that these were not directly measured and should be interpreted as plausible explanations rather than confirmed mechanisms.

Comment 2:
Clarify the interpretation of nutrient retention.

Response:
We clarified that nutrient retention was estimated based on nutrient accumulation in harvested biomass (fish and plants) and does not represent a complete nutrient mass balance. We also acknowledge that the higher system-level retention observed in the lambari treatment may partially reflect the presence of an additional biomass compartment. These points have been incorporated into the Discussion and Conclusions.

Comment 3:
Experimental duration and system stability.

Response:
We now explicitly state that the results represent short-term responses (35 days) under the tested conditions. The use of mature biofloc water as inoculum is highlighted as a strength, but we avoid inferring long-term stability or equilibrium.

Comment 4:
Define the functional role of lambari.

Response:
The manuscript has been revised to describe lambari as a complementary component of the system, potentially contributing through additional biomass and interactions with suspended particles. We explicitly state that these mechanisms were not directly assessed and are inferred from system-level responses.

Comment 5:
Statistical interpretation.

Response:
We acknowledge that analyzing variables at each sampling time using independent t-tests may increase the risk of type I error due to multiple comparisons. This limitation has been explicitly stated in the Statistical Analysis section, and the interpretation of temporal differences has been adjusted to emphasize trends rather than isolated significant results.

Comment 6:
Interpretation of alkalinity.

Response:
The manuscript has been revised to avoid presenting reduced alkalinity as an inherent improvement. We now interpret this parameter in the context of microbial processes, particularly nitrification, which consumes alkalinity and reflects buffering demand within the system.

Comment 7:
Clarity and conclusion wording.

Response:
The Discussion has been revised to reduce repetition and improve clarity. The Conclusions were reformulated to adopt a more cautious tone, emphasizing that the findings represent short-term responses and indicating the potential, rather than confirmed optimization, of lambari inclusion.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

the manuscript addresses a relevant and timely topic in sustainable aquaculture. I really like your work. The introduction provides sufficient background and is well supported by the literature. The research design is appropriate and aligns with the study objectives. The results are clearly presented and easy to follow. Moreover, tables and figures are well prepared and support the findings. The conclusions are consistent with the results. However, the methods section could be described in more detail, especially the statistical approach (eg. the use of the Student’s t-test with a small sample size (n = 4) should be justified, and it should be clarified whether a power analysis was conducted, there is no detailed description of how experimental units were randomly assigned to groups, it is necessary to clarify whether all experimental units were maintained under identical conditions e.g., light intensity, greenhouse microclimate, water flow). Last but not least, some parts of the discussion could be more precise and less repetitive.

All things considered, minor clarifications would improve overall clarity. In my opinion, the manuscript has good potential after moderate revision.

Kind regards :-)

Author Response

Dear Editor and Reviewer,

Comment:
Clarify methodological details, including statistical justification, randomization, and experimental conditions.

Response:
The Methods section has been revised to improve clarity and detail. We clarified that experimental units were randomly assigned to treatments and that all units were maintained under identical environmental and operational conditions, including water circulation, aeration, and temperature control.

Regarding statistical analysis, we confirmed that the data met the assumptions of normality and homoscedasticity and justified the use of the Student’s t-test for comparisons between two treatments. We acknowledge the relatively small sample size (n = 4), which is common in controlled aquaculture experiments, and interpret the results accordingly.

We sincerely appreciate the reviewers’ comments, which have significantly improved the quality and clarity of the manuscript.

Sincerely,
Adolfo Jatobá

(On behalf of all authors)