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Article
Peer-Review Record

Identification of Key Factors Affecting the Trophic State of Four Tropical Small Water Bodies

Water 2020, 12(5), 1454; https://doi.org/10.3390/w12051454
by Homero Cuevas Madrid 1,*, Alfonso Lugo Vázquez 2, Laura Peralta Soriano 2, Josué Morlán Mejía 2, Gloria Vilaclara Fatjó 2, María del Rosario Sánchez Rodríguez 2, Marco Antonio Escobar Oliva 2 and Javier Carmona Jiménez 3
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Water 2020, 12(5), 1454; https://doi.org/10.3390/w12051454
Submission received: 31 March 2020 / Revised: 16 May 2020 / Accepted: 18 May 2020 / Published: 20 May 2020
(This article belongs to the Special Issue Functioning of Small Water Bodies)

Round 1

Reviewer 1 Report

This is a very valuable case study, describing the relationships between various parameters responsible for water quality in small, post-mining water bodies. However, I have some comments to the authors and minor shortcomings that must be considered before publishing.

Regarding the description of the searched water bodies, add information on the vegetation present in them. Were submerged macrophytes there, especially in NL and RL, which had transparency down to the bottom? If the species of these plants were not known, then at least the information on which % of the bottom area is covered by this vegetation would be important.

Authors wrote, “There is no sanitary network that discharges into the lakes”. So, how is wastewater disposal? Are they collected in septic tanks? Alternatively, are they discharged into the ground?

In the chapter Results should be illustrated and discussed the results of the research only. However, their discussion, including comparison with the results of other published data should be presented in the Discussion chapter. That is why literature should be not citied in the Results.

Line 105, 153, 364 – not ‘y’, but ‘and’.

Equation 1 and 4. Do not use spaces in indicators, e.g. not Chlor – a but Chlor-a. The same with TSI – Int.

In equation 4 is TSI Transp, while in the text there is the TSI SDD. Is this the same? If so, then you need to unify them.

Line 171. What is AD? Shouldn't that be DA? The same is in line 310 and 472.

Line 277. What is TSI-I? Shouldn't that be TSI-Int, like in line 116? The same is in line 317.

Line 363 and 365. What is OD? Oxygen demand? Or, it should be DO, i.e. dissolved oxygen?

Line 392. There is 0.013 mg/L. It should be rather 0.13 mg/L.

Line 430. Did you mean here polycyclic aromatic hydrocarbons (PAH) or maybe hydroxyapatite (HAP)?

 

Author Response

Response to Reviewer 1 Comments

This is a very valuable case study, describing the relationships between various parameters responsible for water quality in small, post-mining water bodies. However, I have some comments to the authors and minor shortcomings that must be considered before publishing.

Regarding the description of the searched water bodies, add information on the vegetation present in them. Were submerged macrophytes there, especially in NL and RL, which had transparency down to the bottom? If the species of these plants were not known, then at least the information on which % of the bottom area is covered by this vegetation would be important.

Coverage percentages are not available, but information about the most abundant macrophytes species was added, and also about other groups (phytoplankton, macrophytes, zooplankton, and endemic vertebrate species).

Authors wrote, “There is no sanitary network that discharges into the lakes”. So, how is wastewater disposal? Are they collected in septic tanks? Alternatively, are they discharged into the ground?

A paragraph was added explaining how the wastewater from the EQ is disposed of, not poured into the water bodies.

In the chapter, Results should be illustrated and discussed the results of the research only. However, their discussion, including comparison with the results of other published data should be presented in the Discussion chapter. That is why literature should not be cited in the Results.

Comparisons of the nutrient concentration (N and P) of water that supplies EQ with other areas of the spill were moved to the discussion section..Line 105, 153, 364 – not ‘y’, but ‘and’. Done

Equation 1 and 4. Do not use spaces in indicators, e.g. not Chlor – a but Chlor-a. The same with TSI – Int.

Done

In equation 4 is TSI Transp, while in the text there is the TSI SDD. Is this the same? If so, then you need to unify them.

It was corrected, it remained as TSI SDD

Line 171. What is AD? Shouldn't that be DA? The same is in line 310 and 472.

It is DA, corrected

Line 277. What is TSI-I? Shouldn't that be TSI-Int, like in line 116? The same is in line 317.

It is TSI-Int, corrected

Line 363 and 365. What is OD? Oxygen demand? Or, it should be DO, i.e. dissolved oxygen?

It is DO, corrected

Line 392. There is 0.013 mg/L. It should be rather 0.13 mg/L.

Done

Line 430. Did you mean here polycyclic aromatic hydrocarbons (PAH) or maybe hydroxyapatite (HAP)?

It is HAP, corrected

Author Response File: Author Response.docx

Reviewer 2 Report

The authors analyzed physical and chemical environmental data in four small shallow water bodies for three years. The objective of the study was testing the factors that affected their trophic status by determining which environmental variables explained the variability in chlorophyll during the sample period. The main conclusion was that the water renewal is the main variable controlling the chlorophyll concentration although there were some other variables such as the precipitation of phosphate that would play an important role by regulating the nutrient availability. The authors did a valuable effort of sampling and analysis and presented a worth work to understand the chlorophyll dynamics in the study water bodies. However, I find several flaws that limit the interest of the manuscript as it is presented:

(1)  The Introduction lacks a appropriate scientific focus including the formulation of an hypothesis. As written, it appears that the main objective is testing the utility of PLRS, which would present a limited interest for the reader as this statistic technique is not 'per se' new. In this section, the authors should explain more clearly (i.e. in quantitative terms) which the role of these water bodies would be compared to other systems (lakes and ponds) in the context of the global carbon and nutrient cycles to reinforce the interest of the study. The environmental factors that they are going to test (as they are not taken into account normally in eutrophication studies) and the expected results (i.e. the hypothesis above mentioned) should be better explained and/or justified in the Introduction.

(2) As mentioned the authors, these water bodies depend strongly on the rainfall regimen and possibly on other weather factors. These factors would explain part of the variability observed throughout the study. However, none information about these variables is supplied in the manuscript. Note that this point would be important to do comparisons with other equivalent systems located at different latitude.

(3) Although theoretically the main strength of the manuscript is the statistical analysis, I think that the authors did not take advantage of  their data fully. I would like to see if there was seasonal changes in the water bodies and how they performed in each system. In that sense, I think that at least the raw chlorophyll time series should be shown in the manuscript. Furthermore, it would be illustrative (and useful) showing the bi-plots obtained from PLRS with the correlations for each point as it would inform about the variability in each water body. I would like to see results of PLRS applied to the whole data, just to rate (and discuss) which variable would have higher weight in determining the chlorophyll irrespectively of the particular features of each water body. Additionally, there are some apparently paradoxical results that would be discussed. For instance, according to RDA results, NL is weakly correlated with Factor 1 that mainly determines HRT. However, from the results of PLRS for that water body, it appears that HRT has a relevant role in determining chlorophyll. Irrespectively of these possible additional comparisons to reinforce the Discussion, I have some doubts with the application of PLRS. The main one is about Secchi disc depth. Firstly, I suppose (and the authors mention it, line 121) that in many samplings SDD was not useful to determine the transparency of the water column as it was equal to depth (the authors would specify with which frequency it occurred); I suppose that the authors excluded these data in their statistical analyses. Secondly, I suppose that SDD depends mainly on the chlorophyll concentration, therefore I am not sure that SDD can be used to predict chlorophyll concentration in the PLRS models.  

(4) In the Discussion, I miss a comparative analysis of the obtained results with those presented by other authors in equivalent systems and even in other systems (ponds or lakes) that support more strongly why these small aquatic systems should be studied (the authors only devoted four lines to this comparison). Without this discussion, the interest of the manuscript for a broad range of readers would be limited.    

Other specific comments:

Lines 21-23. The Abstract is little informative and should be reinforced with more information based on the results obtained.

Line 30. I guess that it is not the per se "abundance" the feature of these water bodies that determines their "essential role within global cycles". Additionally, please to specify which this "essential role" is (do they impact the carbon global cycle or any other global process?)

Line 40. Please, to be more explicit in explaining which those other factors are.

Lines 44-50. I am not sure that explaining the theoretical principles of PLRS is necessary.

Lines 51-52. These other variables should be specified.

Line 108. From the reader viewpoint, I would prefer that these methods are described in short here.

Line 121. I think that these modifications were proposed for lakes. I wonder if they are also usable for these smaller water bodies.

Lines 138. I wonder if, at least conceptually, Ca++ and Mg++ concentration are relevant from the eutrophication assessment viewpoint. I do not think so and consequently they should not be included in the statistical analyses. Anyway, I would expect that the authors justify here or in the Introduction their use.

Line 212. This sentence should be rewritten ("all sites shown similar standard deviation").

Line 215. This way to present the comparative results among the different water bodies (RL<NL<CL<SL) results little friendly for the reader.

Lines 250-252. I cannot understand why from "RL and SW4 have similar concentrations" can be concluded that they had "lower phytoplantonic activity". Please, to explain.

Line 292. I think that showing the mean ±1 SD is enough.

Line 299. SD for these means should be also shown.

Line 312. Please, see my previous comment about Ca2+ and Mg+2.

Line 315. I think that HRT was not determined in WS4 (Table 1). Then, I am confused about how this water body was included in this analysis.

Line 330. It is unclear what "direct ratios" means in this context.

Line 344. See my previous comments about the weather variables.

Line 352. Please, to avoid the usage of arrows in the text.

Lines 355-356. This sentence is fairly confused.

Line 383. It is unclear what the authors mean with "phytoplankton biomass" in this sentence.

Line 379. Please, to indicate in the legend the water body at which each panel belong.

Line 392. I suggest to calculate nutrient ratios (N:P) to assess which nutrient (nitrogen or phosphorous) would be limiting the phytoplankton growth.

Line 399. However, the pH is not particularly low in SL, as expected if re-mineralization is intensive. I wonder if it is sure that the water bodies did not receive wastewaters.

Lines 423-424. This sentence is confused. Please, to rewrite

Line 429. That is precisely why information about these "external processes" should be added.

Line 448. It is unclear what "the pH-dependent surface phenomena " means.

Line 452-456. I wonder if the authors have some information about the plankton communities in their systems.

Author Response

Response to Reviewer 2 Comments

The authors analyzed physical and chemical environmental data in four small shallow water bodies for three years. The objective of the study was testing the factors that affected their trophic status by determining which environmental variables explained the variability in chlorophyll during the sample period. The main conclusion was that the water renewal is the main variable controlling the chlorophyll concentration although there were some other variables such as the precipitation of phosphate that would play an important role by regulating the nutrient availability. The authors did a valuable effort of sampling and analysis and presented a worth work to understand the chlorophyll dynamics in the study water bodies. However, I find several flaws that limit the interest of the manuscript as it is presented:

(1)  The Introduction lacks a appropriate scientific focus including the formulation of an hypothesis. As written, it appears that the main objective is testing the utility of PLRS, which would present a limited interest for the reader as this statistic technique is not 'per se' new. In this section, the authors should explain more clearly (i.e. in quantitative terms) which the role of these water bodies would be compared to other systems (lakes and ponds) in the context of the global carbon and nutrient cycles to reinforce the interest of the study. The environmental factors that they are going to test (as they are not taken into account normally in eutrophication studies) and the expected results (i.e. the hypothesis above mentioned) should be better explained and/or justified in the Introduction.

The introduction was rewritten by changing the approach, giving greater weight to the importance of small water bodies. Its role in global processes, ecological importance, and environmental services was briefly described, and a hypothesis was included.

 (2) As mentioned the authors, these water bodies depend strongly on the rainfall regimen and possibly on other weather factors. These factors would explain part of the variability observed throughout the study. However, none information about these variables is supplied in the manuscript. Note that this point would be important to do comparisons with other equivalent systems located at different latitude.

Precipitation data for the studied period were included. We think that the most important influence on lakes is the contribution of water by springs, which does not have such a direct relationship with the amount of rain.

Hypothesis tests were performed to compare differences in the Chlor-a by season (dry and wet), and no significant differences were found (U= 2849.5 p= 0.59), probably due to the hydraulic handling of flow rates that could mask the effect of seasonality, as explained in the next point.

(3) Although theoretically, the main strength of the manuscript is the statistical analysis, I think that the authors did not take advantage of  their data fully. I would like to see if there was seasonal changes in the water bodies and how they performed in each system. In that sense, I think that at least the raw chlorophyll time series should be shown in the manuscript.

The initial approach of research to data processing was time series analysis. However, the results were not satisfactory to explain the differential expression of eutrophication. The following graph shows that there are no apparent relationships of chlorophyll-a with rainfall, and the same is true for some other variables studied, which was corroborated by the analysis of seasonal variation between dry and wet season, in which no significant differences were found, as mentioned above.

 

Furthermore, it would be illustrative (and useful) showing the bi-plots obtained from PLRS with the correlations for each point as it would inform about the variability in each water body.

VIP value is presented instead of correlations because the VIP measure the importance of an explanatory variable for the building of the PLS components, remember that the explanatory variables highly correlated with the response variable are included in the construction of the Beta coefficients.

VIP value is a useful tool for choosing the variables that contribute most to the explanation of the variable response, then reporting correlations could be somewhat redundant, being the reason why the Tannenhaus procedure probably does not considerate reporting them, because if the VIP value is high, the correlation between the explanatory variable and the response variable is also likely to be high and significant.

I would like to see results of PLRS applied to the whole data, just to rate (and discuss) which variable would have higher weight in determining the chlorophyll irrespectively of the particular features of each water body.

The PLSR with whole data (all sites and all variables) models are shown below, where the yellow bars represent the variables retained at the respective iteration, and the left-hand graph is the model with the variables retained at the second iteration and finally, the third graph shows the definitive variables for the model, but it can be observed that variables have different behavior in both the magnitude of the  coefficient and the confidence interval through iterations.

The hydraulic residence time and pH, as in the DA, has important influences in the modeling of chlorophyll-a with PLSR with whole data. Although the time of hydraulic residence shows an important relationship with the concentration of chlorophyll-a, it does not explain the processes that can be developed during the time the water resides in the water bodies. Then, through the identification of the most influential variables in each lake as we did in our study, such processes began to be recognized, is this the reason to apply PLSR on each lake.

Additionally, there are some apparently paradoxical results that would be discussed. For instance, according to RDA results, NL is weakly correlated with Factor 1 that mainly determines HRT. However, from the results of PLRS for that water body, it appears that HRT has a relevant role in determining chlorophyll. Irrespectively of these possible additional comparisons to reinforce the Discussion, I have some doubts with the application of PLRS.

 

The purpose of performing the DA was to highlight the special differences between the water bodies, as you would with ANOVA supplemented with some Post hoc test, but ANOVA with Post hoc does not take into account the interaction between variables, as the DA does.

Due to the consistency between DA and PLSR, the influence of the HRT on the NL is not paradoxical, because although the ratio of HRT in the DA chart to chlorophyll-a is low, however, it is significant, because in PLSR it can be observed that the upper limit of the confidence interval of the HRT coefficient almost acquires zero value, that in such a case if zero was part of the confidence interval of the Beta coefficient the variable would have been discarded. This also indicates some volatility of the variable and in addition, the value of VIP is less than 0.7-0.8 which indicates a low influence, which is consistent with DA.

The main one is about Secchi disc depth. Firstly, I suppose (and the authors mention it, line 121) that in many samplings SDD was not useful to determine the transparency of the water column as it was equal to depth (the authors would specify with which frequency it occurred); I suppose that the authors excluded these data in their statistical analyses. Secondly, I suppose that SDD depends mainly on chlorophyll concentration. Therefore, it is not sure that SDD can be used to predict chlorophyll concentration in the PLRS models.

Some SDD readings were equal to the total depth only at RL and WS4. However, on PLSR models, the SDD contribution was high (VIP high values) and significant because the values of the confidence intervals of the Beta coefficients are far from zero. As if the variable was faulty due to events where SDD=Total depth, the variable would most likely have been discarded during iterations for the retention of significant variables for the final PLSR model, the latter being the only justifiable reason for withdrawing variables. Only one atypical data test (Dixon) was used as the criterion to eliminate observations.

SDD in exploratory correlation analyses showed high correlations with chlorophyll-a, then the PLSR algorithm uses it to construct Beta coefficients, not thus with explanatory variables that are strongly correlated with other explanatory variables that can be omitted, as the TN:TP ratio, and TP and TN concentrations.

(4) In the Discussion, I miss a comparative analysis of the obtained results with those presented by other authors in equivalent systems and even in other systems (ponds or lakes) that support more strongly why these small aquatic systems should be studied (the authors only devoted four lines to this comparison). Without this discussion, the interest of the manuscript for a broad range of readers would be limited.  

The discussion was expanded to compare the findings of this study with results from similar systems in other study sites.

Other specific comments:

Lines 21-23. The Abstract is little informative and should be reinforced with more information based on the results obtained.

The core results of this study and the findings were added in the absctract

Line 30. I guess that it is not the per se "abundance" the feature of these water bodies that determines their "essential role within global cycles". Additionally, please to specify which this "essential role" is (do they impact the carbon global cycle or any other global process?)

The importance of small water bodies was specified for their participation in the carbon cycle, nutrient recycling, and their importance for maintaining regional biodiversity.

Line 40. Please, to be more explicit in explaining which those other factors are.

Such factors were specified.

Lines 44-50. I am not sure that explaining the theoretical principles of PLRS is necessary.

PLSR theorical principles were removed.

Lines 51-52. These other variables should be specified.

Added other variables that may be involved.

Line 108. From the reader viewpoint, I would prefer that these methods are described in short here.

The methods of testing the water parameters were not detailed to make the reading more reader-friendly because they are very common methods that can be consulted in the source. But it briefly explains the method used for TN and TP which is a different reference to the Standard Methods.

Line 121. I think that these modifications were proposed for lakes. I wonder if they are also usable for these smaller water bodies.

The equation can be applied to any body of water that has very high concentrations of phosphorus because the authors do not specify limits for their application. This model, to calculate the TSI weights the chlorophyll-a with greater weight and with less the concentration of phosphorus since the authors of the modification argue that not necessarily the single concentration of nutrients can trigger adverse trophic states, as we have seen in RL and WS 4 which are very transparent waters.

Lines 138. I wonder if, at least, conceptually Ca++ and Mg++ concentration are relevant from the eutrophication assessment viewpoint. I do not think so and, consequently, they should not be included in the statistical analyses. Anyway, I would expect that the authors justify here or in the Introduction their use.

The introduction briefly explains and the methodology reinforces how minerals composition of the most common divalent cations in natural waters (calcium and magnesium) can influence eutrophication.

Line 212. This sentence should be rewritten ("all sites shown similar standard deviation").

The sentence was rewritten.

Line 215. This way to present the comparative results among the different water bodies (RL<NL<CL<SL) the results little friendly for the reader.

We think that this way of presenting the results highlights the first and last position since it is the intermediate positions that usually do not show the most important differences.

Lines 250-252. I cannot understand why from "RL and SW4 have similar concentrations" can be concluded that they had "lower phytoplanktonic activity". Please, to explain.

The phrase was corrected. The correct expression is “lower phytoplanktonic consumption”. Water sprouting in the spring puddle (WS 4) contains high phosphorous concentration and very low chlorophyll a. From the spring, the water flows to the RL in a short time. For this reason, LR also shows high phosphorus levels and chlorophyll-a concentrations just a little bit higher than those of WS4.  

Line 292. I think that showing the mean ±1 SD is enough.

The table was modified to simplify it.

Line 299. SD for these means should be also shown.

SD was calculated based on the three replicates analyzed in the analysis batch

Line 312. Please, see my previous comment about Ca2+ and Mg+2.

The introduction briefly explains, and the methodology reinforces how minerals composed of the most common divalent cations in natural waters (calcium and magnesium) can influence eutrophication.

Line 315. I think that HRT was not determined in WS4 (Table 1). Then, I am confused about how this water body was included in this analysis.

As a lotic system, strictly HRT is not measured, but it is considered that the water resides for a few minutes in the poddle about 3-5 minutes so we consider that the residence time tends to zero days because of 5/1440 min per day =0.00034. Then, when applying the n+1 transformation, there are values of type 1.00034, whose accuracy can be negligible, in any case, the DA is robust enough to process several variables with missing data, constants, or zeros (which do not meet the Normal Distribution) without invalidating the final result. What is supported with both PLSR with total data and DA with total data too, are consistent in their results (HRT as the most significant variable), as explained above,

Line 330. It is unclear what "direct ratios" means in this context.

The term was changed to "direct relationships".

Line 344. See my previous comments about the weather variables.

This point was answered lines above

Line 352. Please, to avoid the usage of arrows in the text.

They were changed to the words Up/Down

Lines 355-356. This sentence is fairly confused.

The sentence was rewritten.

Line 383. It is unclear what the authors mean with "phytoplankton biomass" in this sentence.

The paragraph was rewritten.

Line 379. Please, to indicate in the legend the water body at which each panel belong.

The title of each scatter chart shows the name of the lake next to that of the variable, and in the graphs of the coefficients on the Y-axis also appears the name of the water body next to the title of the axis.

Line 392. I suggest to calculate nutrient ratios (N:P) to assess which nutrient (nitrogen or phosphorous) would be limiting the phytoplankton growth.

TN:TP results were included, but the TN:TP ratios were not significant in the PLSR analysis due probably to the high collinearity they presented with other nutrient species. TN:TP was always discarded from the saturated model as explained above, even when it was removed, the R2 and Q2 values improved.

Line 399. However, the pH is not particularly low in SL, as expected if re-mineralization is intensive. I wonder if it is sure that the water bodies did not receive wastewaters.

Completely safe as it is a protected ecological zone, the origin of nutrients is not from anthropic sources.

Lines 423-424. This sentence is confused. Please, to rewrite.

Done.

Line 429. That is precisely why information about these "external processes" should be added.

The external processes were detailed in the introduction.

Line 448. It is unclear what "the pH-dependent surface phenomena " means.

The paragraph was rewritten to explain the sentence better.

Line 452-456. I wonder if the authors have some information about the plankton communities in their systems.

 A paragraph was added in the description of the area of study of the wealth of species, and not only of phytoplankton, since macrophytes were also included. We also added some data about zooplankton and endemic species of vertebrates.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I thank the authors for considering each my criticism. In my previous review, I pointed out four flaws that limited the quality of the manuscript.

-Two of these comments were relative to the Introduction and Discussion which, from my viewpoint, had to be re-written in order to increase the potential interest of the manuscript. The authors have modified substantially both sections; now, I think that the work and results are adequately put into a wider context and the ms has improved substantially.

-Other main comment was related to check the possible influence of rainfall regimen on the variability in the environmental factors studied by the authors. In the new version, the authors added and analysed data of rainfall and concluded that it had a reduced effect. I am satisfy with this response.

- Finally, I did some considerations related to the statistics tests and their results. I thank the authors for their extensive justification in their reply to this query that helped me to better understand some aspects of their analyses. I only keep some doubts regarding to the appropriateness of using SDD data when it was equal that the total depth. SDD is a proxy of transparency that, obviously, only can be considered a qualitative estimation (transparency is higher than a given value) if it equals the total depth. I just wanted to know the occurrence frequency of SDD data equaling the total depth to assess if it could affect the PLSR results in some of the water masses (irrespectively of the generated PLSR models include or not this variable as I do not know in which extension these tests are sensible to this fact). On other hand, the correlation between SDD and chlorophyll a is expected since chlorophyll concentration influences the water transparency (SDD is not completely independent from chlorophyll). From this viewpoint, the result is little informative. With my previous comment (3) relative to this aspect, I only wanted that the authors re-considered or at least justified using this variable in their models whose objective is understanding the independent environmental factors regulating the chlorophyll concentration.

-The other specific comments have been replied satisfactorily by the authors and the manuscript has been modified accordingly.

 

Consequently, I think that the manuscript is useful for publication in its present form although I encourage the author to add precise information about the occurrence frequency of SDD data equaling the total depth (which permits assessing that it did not affect the PLSR results). Additionally, the usage of SDD in their models for understanding the environmental factors regulating chlorophyll should be justified taking into my previous comment.

Author Response

Response to Reviewer 2 Comments

I thank the authors for considering each my criticism. In my previous review, I pointed out four flaws that limited the quality of the manuscript.

-Two of these comments were relative to the Introduction and Discussion which, from my viewpoint, had to be re-written in order to increase the potential interest of the manuscript. The authors have modified substantially both sections; now, I think that the work and results are adequately put into a wider context and the ms has improved substantially.

-Other main comment was related to check the possible influence of rainfall regimen on the variability in the environmental factors studied by the authors. In the new version, the authors added and analysed data of rainfall and concluded that it had a reduced effect. I am satisfy with this response.

- Finally, I did some considerations related to the statistics tests and their results. I thank the authors for their extensive justification in their reply to this query that helped me to better understand some aspects of their analyses.

-The other specific comments have been replied satisfactorily by the authors and the manuscript has been modified accordingly.

We appreciate your feedback which improved the presentation of our study.

I only keep some doubts regarding to the appropriateness of using SDD data when it was equal that the total depth. SDD is a proxy of transparency that, obviously, only can be considered a qualitative estimation (transparency is higher than a given value) if it equals the total depth. I just wanted to know the occurrence frequency of SDD data equaling the total depth to assess if it could affect the PLSR results in some of the water masses (irrespectively of the generated PLSR models include or not this variable as I do not know in which extension these tests are sensible to this fact). On other hand, the correlation between SDD and chlorophyll a is expected since chlorophyll concentration influences the water transparency (SDD is not completely independent from chlorophyll). From this viewpoint, the result is little informative. With my previous comment (3) relative to this aspect, I only wanted that the authors re-considered or at least justified using this variable in their models whose objective is understanding the independent environmental factors regulating the chlorophyll concentration.

In the paragraph related to the TSI in the results section, there was included the frequency of cases in which it appears SDD=Depth in the respective water bodies.

The SDD=Depth condition only affects the TSI overestimation to some degree the classification of the trophic states of WS 4 (36 cases), which was not analyzed with PLSR, and RL (30 cases), the other water bodies have no such condition.

But as far as the issue is concerned, if the SDD=Depth condition affects the validity of the  PLSR model of RL, we think not, because SDD, as well as the other two variables that are strongly modified by the quantity of primary producers such as pH and DO, may at some point “stagnate” and by increasing the number or activity of primary producers the values of SDD, pH or DO no longer increase, and therefore lose the intensity of the relationship with the modeled variable, but that would not be the case with RL.

For example, if RL were shallower and had a higher degree of eutrophication where the SDD=Depth condition was presented, then the SDD value would become a constant value and would not reflect changes in chlorophyll-a concentration, becoming a useless variable that would be removed from the model through iterations to obtain the final model.

But in the case of RL, where Chlor-a concentrations are normally low, water is clear and SDD=Depth, when higher concentrations of Chlor-a are present changes in transparency are clearly reflected in SDD, as can be seen in the graph, generating a strong association, so much so that PLSR assigns a very high VIP value (the highest among models) to SDD. In short, what makes SDD significant in the RL model is the predominant condition of transparency where changes can be easily noticed. Based on the above, we find it justifiable to use SDD on the RL model.

Besides, SDD is not an explanatory variable of the processes occurring in RL, but a “descriptive” variable used by the PLS algorithm to improve model metrics. This is another reason why we consider it is justifiable to maintain SDD on the RL model.

Also, a review of the writing was made to correct various typographical and language errors, and the reference of precipitation data during the study period was added.

Consequently, I think that the manuscript is useful for publication in its present form although I encourage the author to add precise information about the occurrence frequency of SDD data equaling the total depth (which permits assessing that it did not affect the PLSR results). Additionally, the usage of SDD in their models for understanding the environmental factors regulating chlorophyll should be justified taking into my previous comment.

This point was answered lines above.

The authors thank your time, and valuable commnents.

Author Response File: Author Response.docx

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