Fish Welfare in Urban Aquaponics: Effects of Fertilizer for Lettuce (Lactuca sativa L.) on Some Physiological Stress Indicators in Nile Tilapia (Oreochromis niloticus L.)

Round 1

Reviewer 1 Report

Aquaponics has received increased interest in recent years and thus, the paper presents an interested topic. It is well written and is suitable for publication, however, it should be noted that commercial aquaponics relies on the use of decoupled systems where the water from the plant system is not recycled to the fish. Also the article is lacking information about phosphorus. Please find a few comments/suggestions below:

Lines 29-32: Comment to "This mutual exchange depends on the action of two different groups of bacteria such as Nitrosomonas spp. 
and Nitrobacter spp. which oxidize ammonia, excreted by fish, into nitrates, which are more easily absorbed by plant roots": The RAS component of aquaponics depends on this action.

Line 40: Put latin name (Oreochromis niloticus) when tilapia is mentioned first time in the text 

Line 60: Add: and phosphorus

Lin 71: No need to present latin name again

Lines 124 and 172: Check indent

Lines 215-218: Check sentence: This section may be.....

Line 239: Missing space before [14]

Line 254: Change differ to different

Line 271: Check sentence: "a decrease the intensity..."

The article and at least the conclusion should mention decoupled aquaponics systems. Phosphorus  should also be discussed in the paper. 

 

 

Author Response

Ref 1: Thank you for your kind comments and work reading the MS. Below please find our replies/comments in bold. 

Specific comments:

Lines 29-32: Comment to "This mutual exchange depends on the action of two different groups of bacteria such as Nitrosomonas spp. and Nitrobacter spp. which oxidize ammonia, excreted by fish, into nitrates, which are more easily absorbed by plant roots": The RAS component of aquaponics depends on this action.

RAS been added to the sentence

Line 40: Put latin name (Oreochromis niloticus) when tilapia is mentioned first time in the text 

Changed as suggested.

Line 60: Add: and phosphorus

Phosphorous was already included in the list of micronutrients needed for lettuce growth.

Lin 71: No need to present latin name again

Changed as suggested.

Lines 124 and 172: Check indent

Checked.

Lines 215-218: Check sentence: This section may be.....

Eliminated

Line 239: Missing space before [14]

Fixed as suggested.

Line 254: Change differ to different

Changed.

Line 271: Check sentence: "a decrease the intensity..."

Fixed.

The article and at least the conclusion should mention decoupled aquaponics systems. Phosphorus should also be discussed in the paper. 

Both of these have been now been mentioned in the introduction and the discussion. However we have not mentioned it further since, although decoupled aquaponics may be a better commercial option, it was not the aim of this MS to compare coupled and decoupled aquaponics.

Author Response File: Author Response.docx

Reviewer 2 Report

The topic of aquaponics is of great interest economically and ecologically. The authors describe an experiment that compares tanks with and without added fertilizers, used to grow both Tilapia and lettuce. I am intrigued by the concept but have questions about the execution.

First of all, the sample size of the fish is small: only 2 tanks of 8 fish each per treatment. Thus it is not likely to see a difference between treatments for the fish because of low statistical power. In contrast there are 25 lettuce plants per tank.

Also, is the stocking of 8 fish in 200L standard for aquaculture? It seems low.

TABLE 1: There is so much variation between individuals, with such large errors, that it looks like there were no differences between initial and final weights, as well as between the treatment tanks.  By 2x2 ANOVA, were the weights of any of the four groups statistically different from one another? If not, that indicates that the power to test the hypothesis is low.

The stress treatment may not have been sufficient to cause stress, as the fish were tank-mates and may not be stressed by each other even in a crowd for a brief period.  

I am concerned that the fish blood electrolytes were not measured. Particularly the phosphates and calcium balance could be an important biological indicator of effects of the high concentrations of the plant nutrients in the FW tanks.

Table 4 – The nitrates are very high in the FW tank. Why were nitrates added at such high amounts when the fish are supposed to be contributing them? Lines 273, fish waste supplies enough nitrates.

Being in the FW tank exposed them to much higher concentrations of the fertilizer substances and could have affected their health. The number of parameters measured for the lettuce is far greater than the number measured on the fish, basically just weight and the stress indicators.

I am not convinced that this study has the broad implications the authors claim, particularly with the small numbers of fish in the tanks. I would like to see more evidence that the fish had healthy amounts of blood electrolytes before saying that the Fertilizer treatment had no effect.

Tilapia are remarkably adaptable fish but the statement that fertilizing the water has no effect on their growth or health is premature.   

 

Author Response

Thank you for your kind comments and work reading the MS. Below please find our replies/comments. 

Specific comments:

-TABLE 1: There is so much variation between individuals, with such large errors, that it looks like there were no differences between initial and final weights, as well as between the treatment tanks.  By 2x2 ANOVA, were the weights of any of the four groups statistically different from one another? If not, that indicates that the power to test the hypothesis is low.

While we admit that the variation in weight was not ideal, however the main goal of the study was to consider animal welfare not so much growth, which is also partly why the feeding rate was not very high either (around 1%). Regarding growth only, it would have been ideal to include more tanks, and higher densities and number of fish, however, considering the urban aquaculture aspect, we often run into problems of space, both for tanks and for fish. A brief look at the literature on experiments in aquaponics finds a lack of replication of different aquaponics units per treatment, sometimes only with one unit. For all these reasons, although more tanks and fish could have been used, we think that this study design still provides relatively robust results and can help to promote more similar research.

 

-The stress treatment may not have been sufficient to cause stress, as the fish were tank-mates and may not be stressed by each other even in a crowd for a brief period.  

Yes, we agree that the stress treatment was not enough to induce stress, as mentioned in the discussion. However, we chose this time (20 min) based on other studies in out group using rainbow trout, and on the literature, and we did not know, a priori, what the effects would be. In either case, whether a low stress or high stress, both treatments reacted in a similar manner, physiologically speaking, which is a result in itself.  

 

-I am concerned that the fish blood electrolytes were not measured. Particularly the phosphates and calcium balance could be an important biological indicator of effects of the high concentrations of the plant nutrients in the FW tanks.

We agree that it would have been nice to measure fish blood electrolytes. In previous studies by our group, however, we measured in detail several blood electrolytes, including calcium, (Bermejo-Poza R, Fernández-Muela M, De la Fuente J, Pérez C, de Chavarri EG, Díaz MT, Torrent F, Villarroel M. Physio-metabolic response of rainbow trout during prolonged food deprivation before slaughter. Fish physiology and biochemistry. 2019 Feb;45(1):253-65.), and found a lack of response even after 20 days of fasting. Fish, at least trout and other related results from sea-bream, seem to be able to maintain their blood electrolytes within narrow ranges even under high stress, although another option may be to look at ion transporters themselves in the gills, which we did not do, but may be a good idea for the future.  

 

-Table 4 – The nitrates are very high in the FW tank. Why were nitrates added at such high amounts when the fish are supposed to be contributing them? Lines 273, fish waste supplies enough nitrates.

We set out to apply “normal” fertilizer versus nothing, and then measure nitrates in the end, so we did not consider adjusting the nitrate concentrations in the beginning since we would have considered that “tampering” with the commercial protocols. But we have changed line 273 to give more leeway to the idea that fish waste does not always supply enough nitrates.

 

-Being in the FW tank exposed them to much higher concentrations of the fertilizer substances and could have affected their health. The number of parameters measured for the lettuce is far greater than the number measured on the fish, basically just weight and the stress indicators.

Yes, we measured more parameters related to growth in the lettuce and less in the fish, partly since we did not kill the fish as the end of the experiment. The stress indicators are relevant in themselves however, and all point in the same direction, the glucose levels and the triglycerides “follow” the cortisol, which is also important.

 

-I am not convinced that this study has the broad implications the authors claim, particularly with the small numbers of fish in the tanks. I would like to see more evidence that the fish had healthy amounts of blood electrolytes before saying that the Fertilizer treatment had no effect. -Tilapia are remarkably adaptable fish but the statement that fertilizing the water has no effect on their growth or health is premature.   

We agree and have toned-down the implications.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

There is a much clearer rationale for the small number of fish per tank, and a more careful wording of the results, recognizing that stress may increase with more crowded conditions. Making this paper more explicit about the specific conditions is a big improvement.