Combined Effects of Eco-Substrate and Carbon Addition on Water Quality, Fish Performance and Nutrient Budgets in the Pond Polyculture System

Round 1

Reviewer 1 Report

The manuscript describes an experiment where 3 species of carp have been grown together in either standard ponds, or ponds with eco-substrate and carbon addition. The authors have measured water quality, growth, and nutrient levels over the study period of 140 days. The article is well written and concise. I only have minor comments.

Abstract

L14-17 The introduction is a bit generic. What kind of pollution, what kind of traditional aquaculture, what kind of pond polyculture system?

L17-21 These abbreviations need introducing.  

The abstract fails to mention there were 3 species in the pond.

The authors should give some effect sizes, rather than just p values, to help the reader understand the biological significance.   

Introduction

L45 “to be”

Methods

Do the authors have an ethical permit? If so, the details need providing.

L73 Can the authors provide more detail on this substrate, how it was setup, and some background on how it functions?

L79 Were these fish anesthetized when handled?

L81-82 Is this the mean weight of all the fish used in the experiment, irrespective of the pond they went in? Or did the authors know the mean weight of the fish supplied to each pond?

L82 I am assuming that each species was represented in each pond?

L82 How many fish were in each pond, what was the stocking density (start and end), and what was the ratio between the different species?

L81 What was the resolution of the weight measurement, to the nearest gram, ten grams?

L84 Which commercial feed?

L85 From which species was the body weight based on, or was it based on the pond biomass?

L86 When the feed ratio was adjusted, what was it adjusted too? In the line before it says the fish were fed 3% of their body weight daily, but the line after suggests this was adjusted based on feeding levels.

For the statistics, it seems the data has simply been pooled over time. In the figures, it is presented over time. The authors later state that TAN is lower in the experimental than control group, but there is no figure of the pooled data. How can the reader see this? There should be an additional panel with the data pooled over time which matches the stats.

Results

In figure 1A there is no experimental group, or is the data just identical?

L156 FIW should be defined somewhere in the text

Table 3

Several parameters have an SE of 0.00. I think it is more appropriate to use <0.01 as I guess this has just been rounded down to 0 but was not actually 0.

L176 “The nitrogen and phosphorus feed inputs in the treatment group were both lower than those in the control group”. Is this because they were fed less?

Discussion

L218 The authors should provide details on the guidelines for TAN, nitrate and nitrite for the study species.

L230 Was there any observation of the fish eating the eating the biofloc particles? Could it not also be that if the water quality is better, the fish grow better?

Paragraphs 5 and 6 could be combined

Author Response

Response to Reviewer 1 Comments

We greatly appreciate the reviewers' very constructive, detailed and helpful comments and have done necessary changes according to the reviewers' advice (see our responses below on detailed comments by the reviewers).

Responses to Reviewer #1

Comment: The manuscript describes an experiment where 3 species of carp have been grown together in either standard ponds, or ponds with eco-substrate and carbon addition. The authors have measured water quality, growth, and nutrient levels over the study period of 140 days. The article is well written and concise. I only have minor comments.

 Abstract

* L14-17 The introduction is a bit generic. What kind of pollution, what kind of traditional aquaculture, what kind of pond polyculture system?

Answer: As the reviewer said, the introduction is a bit generic. However, due to the strict word limit of the abstract, it is difficult to explain it in detail. The relevant contents are detailed in the introduction.

* L17-21 These abbreviations need introducing.

Answer: These abbreviations have been changed to full names. Line 20-23.

* The abstract fails to mention there were 3 species in the pond.

Answer: There was no significant difference in the growth parameters of C. carpio between the experimental group and the control group, so C. carpio was not mentioned in the abstract. This part has been added to the abstract in Line 24-25. Now, all three species in the pond are mentioned.

* The authors should give some effect sizes, rather than just p values, to help the reader understand the biological significance.

Answer: Thank you for your advice. However, since there are many parameters measured in this experiment, giving specific effect sizes will lead to the number of words in the abstract exceeding the requirements of the journal.

Introduction

* L45 “to be”

Answer: The "tobe" has been changed to the "to be". Line 50.

Methods

* Do the authors have an ethical permit? If so, the details need providing.

Answer: The “ethics statement” has been added in the "Materials and methods". Line 69-74.

* L73 Can the authors provide more detail on this substrate, how it was setup, and some background on how it functions?

Answer: Some details about substrate used in the experiment have been added. Line 84-88.

* L79 Were these fish anesthetized when handled?

Answer: Yes, the fish were treated with tricaine methane sulfonate (MS 222, 200 mg/L) before samping. The missing content has been added. Line 139-140.

* L81-82 Is this the mean weight of all the fish used in the experiment, irrespective of the pond they went in? Or did the authors know the mean weight of the fish supplied to each pond?

Answer: This is the mean weight of all the fish used in the experiment. First, we selected fish with small size difference as experimental fish. Then, the fish were randomly selected and put into each pond, so the size of the fish in each pond were very similar.

* L82 I am assuming that each species was represented in each pond?

Answer: Yes, three kinds of fish with the same size and quantity were represented in each pond on the basis of the general principles of the main culture fish and matching fish in polyculture pond of the north of China.

* L82 How many fish were in each pond, what was the stocking density, and what was the ratio between the different species?

Answer: There were 1000 C. carpio, 70 H. molitrix and 19 A. nobilis, respectively in each pond. The missing content has been added. Line 96-97.

* L81 What was the resolution of the weight measurement, to the nearest gram, ten grams?

Answer: The weight of the fish was accurate to 0.01g. The missing content has been added. Line 106.

* L84 Which commercial feed?

Answer: The commercial feed purchased from Tongwei Feed Company (Shenyang, China). The missing content has been added. Line 98.

* L85 From which species was the body weight based on, or was it based on the pond biomass?

Answer: The feed amount was based on the weight of C. carpio. The content has been corrected. Line 100.

* L86 When the feed ratio was adjusted, what was it adjusted too? In the line before it says the fish were fed 3% of their body weight daily, but the line after suggests this was adjusted based on feeding levels.

Answer: I'm sorry to make this mistake. The amount of food supplied each day was 3% of the body weight of the C. carpio. With the growth of fish, the total amount of feed was adjusted accordingly, and the feed ratio was fixed. The language expression in the manuscript is incorrect and has been revised. Line 101.

* For the statistics, it seems the data has simply been pooled over time. In the figures, it is presented over time. The authors later state that TAN is lower in the experimental than control group, but there is no figure of the pooled data. How can the reader see this? There should be an additional panel with the data pooled over time which matches the stats.

Answer: I'm sorry to make this mistake. Table 1 compares the significance of the water quality parameters in the control and treatment groups. Due to the author's negligence, table 1 was omitted and has been added in the manuscript.

Results

* In figure 1A there is no experimental group, or is the data just identical?

Answer: The water temperature of the experimental group and the control group was identical. Therefore, only one line can be seen in Figure 1.

* L156 FIW should be defined somewhere in the text

Answer: The FIW has been defined in line 105.

Table 3

* Several parameters have an SE of 0.00. I think it is more appropriate to use <0.01 as I guess this has just been rounded down to 0 but was not actually 0.

Answer: Yes, the SE of several parameters just were rounded down to 0 but were not actually 0. The SE of there parameters has been changed to keep 3 significant digits.

* L176 “The nitrogen and phosphorus feed inputs in the treatment group were both lower than those in the control group”. Is this because they were fed less?

Answer: The total amount of feed input in the experimental group and the control group was the same. However, the experimental group had additional starch input, and other inputs were also different between the two groups, so the nitrogen and phosphorus feed inputs ratio in the treatment group were both lower than those in the control group.

Discussion

* L218 The authors should provide details on the guidelines for TAN, nitrate and nitrite for the study species.

Answer: Some content has been added in the manuscript. Line 239-243.

* L230 Was there any observation of the fish eating the eating the biofloc particles? Could it not also be that if the water quality is better, the fish grow better?

Answer: In the experiment, we have not actually carried out research on fish feeding on biofloc particles. However, some research results show that biofloc particles can indeed be eaten by fish, especially filter feeding fish. As the reviewer said, the better the water quality, the faster the fish grow, which is consistent with the results of this study.

* Paragraphs 5 and 6 could be combined

Answer: Thank you for your advice. Paragraphs 5 and 6 have been combined.

Author Response File: Author Response.docx

Reviewer 2 Report

Various relevant fish culture parameters were measured in order to calculate the benefit of adding eco-substrate and carbon to the pond policulture system. Most of the results that are clearly presented were predictable according to very well known theory of  conversion of amino acids from the feed and microbial activities in fish ponds. The results don't bring much new cognition.

The sentence in Discussion: „To date, aquaculture nutrient budgets have been investigated in diverse aquatic animals, including Litopenaeus vannamei [42], Penaeus monodon [21] and Micropterus salmoides [18]. However, there have been no studies on aquaculture nutrient budgets utilizing eco-substrates and carbon addition“, which should explain the main contribution of this article doesn't make much sense because it mixes researched species with researched technology. The contribution of the article must be better explained as well as the benefit of using eco-substrate and carbon. In order to propose the described technology, according to results that do not represent a significant difference between treatment and control, the possible benefit must be better explained.

On the other hand the article is well written with acceptable English.

In Material and methods was not explained how  TN or TP contents in the stocked fish was calculated.

Author Response

Response to Reviewer 2 Comments

We greatly appreciate the reviewers' very constructive, detailed and helpful comments and have done necessary changes according to the reviewers' advice (see our responses below on detailed comments by the reviewers).

Responses to Reviewer #2

* The sentence in Discussion: „To date, aquaculture nutrient budgets have been investigated in diverse aquatic animals, including Litopenaeus vannamei [42], Penaeus monodon [21] and Micropterus salmoides [18]. However, there have been no studies on aquaculture nutrient budgets utilizing eco-substrates and carbon addition“, which should explain the main contribution of this article doesn't make much sense because it mixes researched species with researched technology. The contribution of the article must be better explained as well as the benefit of using eco-substrate and carbon. In order to propose the described technology, according to results that do not represent a significant difference between treatment and control, the possible benefit must be better explained.

Answer: The manuscript was revised as suggested by the reviewer.

* In Material and methods was not explained how TN or TP contents in the stocked fish was calculated.

Answer: The stocked fish were dried at 60°C to constant weight, crushed, and sifted through a 0.15-mm sieve. The TN and TP concentrations in these samples were obtained using the micro-Kjeldahl method. Line 138-142.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have answered my minor concerns. 

L74 - Define CFAS

Author Response

Response to Reviewer 1 Comments

* L74 - Define CAFS

Answer: The "CAFS" is the abbreviation of "Committee for the Welfare and Ethics of Laboratory Animals of Heilongjiang River Fisheries Research Institute". In addition, the "ethics statement" is in a fixed format as required by my organization.

Reviewer 2 Report

In the Introduction, the goal of the experiment was set and the problems caused by the accumulation of nitrogenous compounds in the growing area were theoretically explained. The potential benefit from the use of eco-substrates and carbon addition on water quality was clear, but the results were poorly explained and partially poorly presented. What does Table 1 show? Water quality parameters in different groups during the experimental period with one number? Is that a mean value during the experimental period, or what?

Lines 197-200: Authors presented the difference in final weight at the end of the experiment. What was the difference in weight at the beginning or the mean weight was the same in each treatment? (highly unlikely). Fish were feed 3% of body weight daily in all treatments, according to the Material and Methods, but “the feed consumption was closely monitored so that the feed amount could be adjusted regularly”. How much feed was introduced in each treatment if the feeding was regularly adjusted? If bio flock has to do something with that (more bio flock less feed introduced) than it benefits the water quality and not only the growth of fish.  Maybe this is the reason for: “The main source of nitrogen and phosphorus was the feed. The nitrogen and phosphorus feed inputs in the treatment group were both lower than those in the control group (92.72% and 93.20% of the nitrogen, respectively; 95.97% and 96.34% of the phosphorus, respectively)”.

Lines 275 -285 in the Discussion it was explained “that comprehensive accumulation, including sediment accumulation, adsorption and loss through seepage, was a major nitrogen and phosphorus output, accounting for 59.20–59.65% and 69.30–71.38% of nitrogen and phosphorus outputs, respectively”.

Bacterial activities in the sediment is something completely different that losing pond water through seepage and dilute nitrogen and phosphorus compounds with the necessary addition of new water. Loosing water through seepage depends on the geological structure of the bottom of the pond and is completely different in different geomorphological situations. Similar but not the same is with the structure of the bottom sediment (the amount of active mud) at the beginning of the experiment or the beginning of the growing period. This is why the amount of added feed is generally applicable category and seepage is not.  

The whole beginning of the Discussion is something that belongs to Introduction, while the Discussion should be re-written showing author’s better understanding and ability to explain this problematic.

Author Response

Response to Reviewer 2 Comments

* In the Introduction, the goal of the experiment was set and the problems caused by the accumulation of nitrogenous compounds in the growing area were theoretically explained. The potential benefit from the use of eco-substrates and carbon addition on water quality was clear, but the results were poorly explained and partially poorly presented. What does Table 1 show? Water quality parameters in different groups during the experimental period with one number? Is that a mean value during the experimental period, or what?

Answer: Table 1 displays the water quality parameters in the control and treatment groups at the end of the experiment. More precise statements have been added. Line 159 and Line 164.

 * Lines 197-200: Authors presented the difference in final weight at the end of the experiment. What was the difference in weight at the beginning or the mean weight was the same in each treatment? (highly unlikely). Fish were feed 3% of body weight daily in all treatments, according to the Material and Methods, but “the feed consumption was closely monitored so that the feed amount could be adjusted regularly”. How much feed was introduced in each treatment if the feeding was regularly adjusted? If bio flock has to do something with that (more bio flock less feed introduced) than it benefits the water quality and not only the growth of fish.  Maybe this is the reason for: “The main source of nitrogen and phosphorus was the feed. The nitrogen and phosphorus feed inputs in the treatment group were both lower than those in the control group (92.72% and 93.20% of the nitrogen, respectively; 95.97% and 96.34% of the phosphorus, respectively)”.

Answer: The number and weight of the initial stocking fish in the experimental group and the control group were the same. The missing content has been supplemented in Line 97-99. With the growth of fish, the total amount of feed was adjusted accordingly, and the feed ratio was fixed. The total amount of feed input in the experimental group and the control group was the same. However, the experimental group had additional starch input, and other inputs were also different between the two groups, so the nitrogen and phosphorus feed inputs ratio in the treatment group were both lower than those in the control group.

 * Lines 275 -285 in the Discussion it was explained “that comprehensive accumulation, including sediment accumulation, adsorption and loss through seepage, was a major nitrogen and phosphorus output, accounting for 59.20–59.65% and 69.30–71.38% of nitrogen and phosphorus outputs, respectively”. Bacterial activities in the sediment is something completely different that losing pond water through seepage and dilute nitrogen and phosphorus compounds with the necessary addition of new water. Loosing water through seepage depends on the geological structure of the bottom of the pond and is completely different in different geomorphological situations. Similar but not the same is with the structure of the bottom sediment (the amount of active mud) at the beginning of the experiment or the beginning of the growing period. This is why the amount of added feed is generally applicable category and seepage is not. 

Answer: Thank you for your explanation. As the reviewer said, loosing water through seepage depends on the geological structure of the bottom of the pond and is completely different in different geomorphological situations. In this experiment, in order to ensure the consistent bottom structure of the pond, the experimental pond was re trimmed before the experiment to ensure that the pond conditions were more similar. In addition, comprehensive accumulation wasn't directly measured. was not but was calculated in the study because it was difficult and inaccurate.

* The whole beginning of the Discussion is something that belongs to Introduction, while the Discussion should be re-written showing author’s better understanding and ability to explain this problematic.

Answer: Some part of discussion has been rewritten according to the suggestions of reviewer.

Round 3

Reviewer 2 Report

No suggestions after two revisions.