Review Reports - Freshwater Diatom Assemblage as Bioindicators of Land-Use Changes in Tropical Andean Streams

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article is devoted to the ecological assessment of the water quality in four Ecuador streams using diatoms as bioindicators. A total of 55 diatom species were identified.

The authors illustrated eight indicator diatom species. Unfortunately, the quality of these images is low. The image of Hannea arcus seems to be compressed. Some diatoms were identified incorrectly. For instance, Navicula notha is actually a species of Gomphonema, Pinnularia subcapitata is not Pinnularia at all. We cannot be sure that other taxa in Table 4 were identified correctly. Since the ecological interpretations depend on the correct identifications I cannot recommend the publication of this manuscript.

Author Response

This article is devoted to the ecological assessment of the water quality in four Ecuador streams using diatoms as bioindicators. A total of 55 diatom species were identified.

The authors illustrated eight indicator diatom species. Unfortunately, the quality of these images is low. The image of Hannea arcus seems to be compressed. Some diatoms were identified incorrectly. For instance, Navicula notha is actually a species of Gomphonema, Pinnularia subcapitata is not Pinnularia at all. We cannot be sure that other taxa in Table 4 were identified correctly. Since the ecological interpretations depend on the correct identifications I cannot recommend the publication of this manuscript.

Response: Thank you for your comments. Species identification was performed following a rigorous taxonomic protocol and subsequently verified by the recognized diatom specialist Dr. Eduardo A. Lobo, from the University of Santa Cruz do Sul (UNISC), Rio Grande do Sul, Brazil, based on the taxonomic framework presented in Freshwater Benthic Diatoms of Central Europe (Cantonati, Kelly, & Lange-Bertalot, Eds., 2017). Following the taxonomic revision, the specimen previously identified as Navicula notha was reidentified as Gomphonema parvulum, while the specimen previously identified as Pinnularia subcapitata was reidentified as Sellaphora lanceolata. Furthermore, in response to the reviewer's suggestions, the table has been updated to reflect the revised taxonomic classification. Following this revision, a total of 54 diatom taxa were identified

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript compiles data on diatom species from specific sites along a river at different points and currents, and various indices have been calculated. The results cited by the authors indicate that water conditions significantly affect the bioindicators; however, this is not supported by the water parameters studied, which diminishes the manuscript's scientific impact. Regarding the methodology, there are aspects that are not well described.

The observations and suggestions are detailed below.

Indicate the province or state and country, as well as the period and year.
In the keywords, modify or remove “Diversity.”
The authors should discuss the importance of richness, abundance, and trophic indices in the introduction. What advantages do these offer? What are their strengths and limitations?
I believe Figure 1 should come first.
Specify the types of livestock activities.
What type of agriculture is practiced, and what are the main crops? The agriculture is intensive, utilizing irrigation systems and agrochemicals.
What are the detection and quantification limits of the equipment? Have the results been previously standardized or validated, in addition to the equipment calibration? Please specify this.
In 2.3, indicate which protocols were followed to identify the species; include bibliographic references.
Write Equation 1 correctly, including its abbreviations.
You must indicate how each value was calculated.
In 3.1, carefully and thoroughly review the results. They are inconsistent. The total exceeds 1,900 individuals. Similarly, in 3.2, 3.3, and 3.4
How were the indices in Table 2 calculated?
Nutrients also have an effect. Additionally, explain why they affected the other parameters, why there were changes, and what caused them
The discussion is very weak; it does not explain the reasons for the variation, how weather conditions have affected it, or how it has changed. How does pH affect the communities, and how does it influence dissolved oxygen (DO)? What conductivity values were obtained, along with those of the other parameters? Are they within the region’s permissible limits for water quality? What are the tolerable limits for the communities? This must be analyzed in detail and compared with other situations.
In the conclusions, highlight the most significant results found (values)

Author Response

The observations and suggestions are detailed below.

Indicate the province or state and country, as well as the period and year.

Response: The information has been incorporated by specifying the monitoring season, the corresponding year, and the months during which monitoring was conducted

In the keywords, modify or remove “Diversity.”

Response: Thank you for your comments. We removed diversity key word in the current manuscript.

The authors should discuss the importance of richness, abundance, and trophic indices in the introduction. What advantages do these offer? What are their strengths and limitations?

Response: Thank you for your comments. We have included a new paragraph in the Introduction that explains the use and applications of richness, abundance, diversity, and trophic indices in diatom research and their relevance as indicators of environmental quality and anthropogenic impacts in freshwater ecosystems.

I believe Figure 1 should come first.

Response: Thank you for your comments. We changed figure 1 and 2 in relation with sugestions.

Specify the types of livestock activities.

Response: Thank you for your valuable comment. The manuscript has been revised to clarify that the predominant livestock activity in the study area is cattle farming, primarily dedicated to dairy production.

What type of agriculture is practiced, and what are the main crops? The agriculture is intensive, utilizing irrigation systems and agrochemicals.

Response: Thank you for your valuable comment. The manuscript has been revised to clarify that agroecological and family farming practices predominate in the study area. The principal crops include corn, legumes, vegetables and fruit trees

What are the detection and quantification limits of the equipment? Have the results been previously standardized or validated, in addition to the equipment calibration? Please specify this.

Response: Thank you for your valuable comment. The Methods section has been revised to clarify that the physicochemical parameters were measured using a Hanna HI98194 multiparameter probe, which was calibrated in the field before each sampling campaign using certified standard solutions. Calibration included NIST-traceable pH buffer solutions (pH 4.01, 7.00, and 10.01), potassium chloride (KCl) conductivity standards, and a sodium sulfite (Na₂SO₃) solution for zero-point dissolved oxygen calibration, followed by air-saturation calibration.

In 2.3, indicate which protocols were followed to identify the species; include bibliographic references.

Response: Thank you for your comment. Section 2.3 has been revised to provide a more detailed description of the protocol used for the taxonomic identification of diatoms. Specifically, it has been clarified that the cleaned samples were prepared for microscopic observation, photographed for taxonomic verification and morphometric analysis, and that species identification was carried out based on the morphological characteristics of the valves using specialized taxonomic keys. In addition, the corresponding bibliographic references have been incorporated.

Write Equation 1 correctly, including its abbreviations.

Response: Equation 1 has been revised and presented in an improved format to enhance its clarity. Furthermore, the abbreviations and definitions of all its components have been provided.

You must indicate how each value was calculated.

Response: Thank you for your comment. The methodology section has been revised to provide a more detailed description of the procedure used to calculate the reported values. Specifically, it has been clarified that the Water Quality Trophic Index (WQTI) was calculated using the trophic values assigned to each diatom species according to Lobo et al. [56]. For species not included in this classification, a default trophic value (tv = 1) was assigned.

In 3.1, carefully and thoroughly review the results. They are inconsistent. The total exceeds 1,900 individuals. Similarly, in 3.2, 3.3, and 3.4

Response: A thorough review of the results presented in Sections 3.1, 3.2, 3.3, and 3.4 was conducted. The values corresponding to the number of individuals and their relative abundances were verified and corrected, ensuring the consistency of the data reported throughout the manuscript.

How were the indices in Table 2 calculated?

Response: Thank you for your comment. We have revised the Methods section to clarify how the Shannon and Simpson indices were calculated.

Nutrients also have an effect. Additionally, explain why they affected the other parameters, why there were changes, and what caused them

Response: Nutrient concentrations (e.g., nitrates and phosphates) were not measured in this study; therefore, it was not possible to assess their influence on the diatom communities or on the other physicochemical parameters. To avoid interpretations that were not supported by the available data, the discussion was restricted exclusively to the variables that were measured, namely temperature, pH, conductivity, total dissolved solids, and dissolved oxygen.

The discussion is very weak; it does not explain the reasons for the variation, how weather conditions have affected it, or how it has changed. How does pH affect the communities, and how does it influence dissolved oxygen (DO)? What conductivity values were obtained, along with those of the other parameters? Are they within the region’s permissible limits for water quality? What are the tolerable limits for the communities? This must be analyzed in detail and compared with other situations.

Response: Thank you for your valuable comment. We have revised and expanded the Discussion to better explain the observed variation in diatom assemblages in relation to environmental and climatic conditions. We now include a more detailed interpretation of how pH influences diatom communities and its indirect relationship with dissolved oxygen (DO). We also added the observed ranges of conductivity and other physicochemical parameters, comparing them with regional water-quality references and previous studies. These additions improve the ecological interpretation and contextualization of our results.

In the conclusions, highlight the most significant results found (values)

Response: Thank you for your valuable comment. We have revised the Conclusions section in accordance with your suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

There is already an extensive literature on the use of diatoms to assess stream condition and this may be a useful contribution to the understanding of this within the region in which the study was performed, but makes few general points that have not been made before. What is lacking, in particular, is a final link between the results obtained and management activities that will drive improvements in the streams. At present, it is a descriptive study rather than an example of a genuine application of diatom-based assessment.

The interesting points for me are the use of a rapid assessment approach (as I interpret the text) in which a relative gentle oxidation is applied, after which temporary (?) slides were prepared in water mounts (please confirm) using Sedgwick Rafter cells rather than permanent mounts using Naphrax or similar? It would be useful to know how much time (and money) is saved by this, and whether comparisions have been made with more traditional and time-consuming approaches in which all rare species are recorded.

I would like to see the Discussion expanded to explain how these results might leverage meaningful change in Ecuadorian rivers. If there are no regulatory instruments available, then this needs to spelt out, as biological assessment needs to be part of a wider process that includes the capacity for results to feed back and drive change.

Detailed comments

Title: it would be better to refer to the diatom assemblage, rather than the diatom community, as diatoms are one part of a broader periphyton community

Lines 132-134: just to be clear: there are no measurements of inorganic nutrients that may support interpretation?

Text on sample analysis seems to be repeated at lines 165-171.

Water Quality Trophic Index – WQTI – is also referred to as TWQI, which is confusing. This is not described in sufficient detail in reference 56, so please add a reference to a more widely-available and comprehensive account. It is difficult to evaluate the paper without having more details of one of your key metrics.

Results: I’m not sure what you mean by an “abundant individual”. I think you mean “individuals that belong to abundant taxa”, making a general point that a few species typically dominate a diatom assemblage.

Fig. 4 adds little to the paper, in my opinion, and could be removed to Supplementary material.

I find the use of Generalized Linear Models to be questionable, in the absence of key water quality and trophic variables.

Table 4 and Fig. 6 could both be moved to Supplementary Material, as there is too much detail in these for most readers to want to digest.

Author Response

Response: Thank you for this valuable comment. We acknowledge that the original version of the manuscript was mainly descriptive. In the revised version, we have strengthened the Discussion and Conclusions by adding a final section that explicitly links the observed diatom responses to potential management and conservation actions. These additions emphasize the applicability of diatom-based assessment as a tool for freshwater ecosystem management in the study region.

Response: The interesting points for me are the use of a rapid assessment approach (as I interpret the text) in which a relative gentle oxidation is applied, after which temporary (?) slides were prepared in water mounts (please confirm) using Sedgwick Rafter cells rather than permanent mounts using Naphrax or similar? It would be useful to know how much time (and money) is saved by this, and whether comparisions have been made with more traditional and time-consuming approaches in which all rare species are recorded.

Response: Thank you for this valuable comment. We rewrite discussion and conclusions in relation with suggestions.

Detailed comments

Title: it would be better to refer to the diatom assemblage, rather than the diatom community, as diatoms are one part of a broader periphyton community

Response: Thank you for this valuable comment. We have replaced the word “community” with “assemblage” in the title.

Lines 132-134: just to be clear: there are no measurements of inorganic nutrients that may support interpretation?

Response: Nutrient concentrations (phosphates and nitrates) were not measured in this study, as its primary objective was to evaluate the structural response of diatom communities and the trophic index across a land-use gradient.

Text on sample analysis seems to be repeated at lines 165-171.

Response: Thank you for your comment. We have carefully checked the manuscript and removed the repeated information in the sample analysis section to avoid redundancy and improve clarity.

Response: The terminology has been standardized to TWQI throughout the manuscript to ensure consistency. In addition, to provide a more comprehensive and accessible description of this metric, the original reference has been retained and a complementary bibliographic source has been added, offering a detailed explanation of the methodology and calculation parameters of the index. This addition facilitates the technical evaluation of the principal tool used in this study.

Response: By this expression, we specifically referred to the abundance of individuals belonging to a given taxon or species within the sample.

Fig. 4 adds little to the paper, in my opinion, and could be removed to Supplementary material.

Response: Thank you for this valuable comment. We have moved Figure 4 to the Appendix.

I find the use of Generalized Linear Models to be questionable, in the absence of key water quality and trophic variables.

Response: Thank you for this important comment. We acknowledge that additional water quality and trophic variables could further improve model performance. However, the GLMs were applied to assess the relationships between richness, diversity indices, abundance, and trophic index with spatial factors (zone and stream) and key physicochemical variables (e.g., pH, conductivity, TDS, and temperature), which are widely recognized as robust proxies of ecological gradients driving diatom assemblages.

Table 4 and Fig. 6 could both be moved to Supplementary Material, as there is too much detail in these for most readers to want to digest.

Response: Thank you for this suggestion. We have moved Table 4 and Figure 6 to the Supplementary Material to improve readability and streamline the main manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors took into account all my comments and significantly revised the manuscript.

Author Response

We sincerely thank the reviewer for the careful evaluation of our manuscript and for acknowledging our revisions. We greatly appreciate your constructive comments and valuable suggestions throughout the review process, which have helped us improve the quality and clarity of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have taken the observations and suggestions into account in a clear and coherent manner. I have no further questions.

Author Response

Reviewer 3 Report

Comments and Suggestions for Authors

Apologies for brevity but I am about to go away, and I understand that deadlines are pressing.

The authors have addressed most of my comments.

There is still almost no information on how this study fits into Ecuador's water management process, which may well mean that there is no obvious means of leveraging biological assessment results into catchment action. It is probably not worth pushing this point. The paper sits along a large literature of diatom-based ecological monitoring that blithely ignores this, so is certainly not an outlier in this respect.

I am still not convinced by the argument supporting their use of GLMs ("... key physicochemical variables (e.g., pH, conductivity, TDS, and temperature), which are widely recognized as robust proxies of ecological gradients driving diatom assemblages"). We really need more information to be sure that these are causal factors rather than correlates. I would suggest removing the GLM section entirely.

Author Response

We appreciate the reviewer's thoughtful comment. We agree that a stronger integration of biological assessment results into water management frameworks would enhance the practical application of ecological monitoring. Although the primary objective of this study was to evaluate diatom-based ecological patterns rather than develop management strategies, we have revised the Discussion to briefly acknowledge the potential role of diatom-based assessments in supporting future freshwater monitoring and management initiatives in Ecuador. We appreciate the reviewer's recognition that this limitation is common within the broader diatom-based ecological monitoring literature.

Response: We appreciate the reviewer's comment. We would like to clarify that the purpose of the GLM analysis was to identify significant associations between environmental variables and diatom community metrics. We believe that the GLM analysis remains an appropriate and valuable approach for evaluating these statistical relationships, as demonstrated in previous studies. For example:

Blanco, S., Olenici, A., Ortega, F., Jiménez-Gómez, F., & Guerrero, F. (2020). Identifying environmental drivers of benthic diatom diversity: the case of Mediterranean mountain ponds. PeerJ, 8, e8825.

Teittinen, A., Kallajoki, L., Meier, S., Stigzelius, T., & Soininen, J. (2016). The roles of elevation and local environmental factors as drivers of diatom diversity in subarctic streams. Freshwater Biology, 61(9), 1509-1521.

Virta, L., & Hedberg, P. (2024). Declining salinity and increasing temperature reduce the diversity and resilience of benthic diatoms. Environmental Microbiology, 26(2), e16569.

Therefore, we decided to retain the GLM analyses because they provide a useful framework for evaluating the relationships between environmental variables and diatom richness, diversity and The Water Quality Trophic Index.