Trophic Structure of Macrozoobenthos in Permanent Streams in the Eastern Balkans

Round 1

Reviewer 1 Report

Overall, this is an interesting paper but the authors fail to highlight its novelty and why it should be of interest to an international audience. – what specifically is it adding to existing knowledge on FFGs in river systems?

The English is difficult to follow in several areas (a few highlighted below). I recommended that the paper be proof read by a native English speaker.

The overall goal or aim of the study needs to be better articulated and linked to the stated specific objectives.  The statistical analysis (Section 2.4) should also be link to the various questions (objectives) asked.  In the same way, it would be helpful to organise the sub-sections in the Results section according to the specific objectives.  Furthermore, it is unclear how and if the trophic structure has enabled the authors to distinguish the role of river types from anthropogenic impacts (as claimed in the abstract).  The discussion could be better structured and shortened to more clearly highlight the key results, the novel findings and their significance to freshwaters outside of the Eastern Balkans.  The same applies to the conclusions.

Lines 54-56: In addition, the Index of Trophic Completeness 54 (ITC) bioassessment approach [13] had good reliability and proved the destructive  processes within the benthic communities caused by different types of anthropogenic impact including alluvial gold mining [14].

What do you mean by ‘destructive processes’?   Should it be? - confirmed degradation in benthic communities caused by………

Lines 77-79: In order to fulfil the set goals in this paper we hypothetise that the FFG's composition is changing within streams and rivers watersheds, or under anthropogenic influence being presented with specific proportion for the mountainous and semi-mountainous rivers

Unclear, needs to be reworded

Do you mean …… changes IN RESPONSE TO
anthropogenic PRESSURES?   What changes in FFGs do you predict?  It would be useful to be more specific.   The change in relation to anthropogenic pressures is not listed as an objective.  Is this an omission?

I am not sure what the following means?   being presented with specific proportion for the
79 mountainous and semi-mountainous rivers

Line 101 - All specimens were preserved in 70% ethanol identified to the possible lowest
taxonomic level.  (lowest possible rather than possible lowest) .   State or tabulate the level that each major group was identified to.

Line 109-110: All the benthic macroinvertebrate taxa (mainly at genus and family level) were assigned to the following FFG's: shredders (SH), scrapers (SC), collectors (CL), filterers (FL), deposit feeders (DF), predators (PR) and parasites (PA).

How did you deal with assignment of trophic groups to genus and family level data where members of that genera or family vary in tropic characteristics?  This needs to be clearly outlined.

Line 114 : The trophic characteristics of the macroinvertebrate species were extracted from the taxa database

What database was used and what do you mean by trophic characteristics?

Use the term macroinvertebrate taxa rather than species unless you have identified the taxa to species level. Apply this throughout the paper.

Line 123-125: Multidimensional scaling (MDS) plot analysis were done to determine the level of similarity in the macro-zoobenthos trophic structure

What statistical method was used to determine the level of similarity?

Cluster analysis is mentioned in the results section but needs to be described in the methods section 2.4.

Line 131-132: the studied  macroinvertebrates’ communities  change to  the studied macroinvertebrate communities

Line 133-134: Furthermore, the dominance of stenobiont species belonging to the groups of shredders and scrapers was registered in the analysis

Reword to read: Furthermore, stenobiont species belonging to the groups of shredders and scrapers were numerically dominant.

Lines 139: 143: We established different proportions of the trophic guilds within the studied watersheds both as ranges of values within each group and in each river basin (Figure 3).
The closest structure with regard of the share of the trophic guilds was observed between
Mesta and Struma Rivers (Figure 3A, B), while in Vardar River basin sites showed more
wider ranges of numbers (Figure 3C)

This paragraph does not adequately or clearly explain the content of Figure 3.

Line 157-160: At R5 sites SH were outnumbered in Struma and Mesta River watershed during the  , while abundance in Vardar River sampling sites did not show significant fluctuations in numbers in both seasons (Figure 4B).

The term ‘significant’ should only be used when statistical analysis has been carried out.  Please clarify.

The quality of Figure 4 needs to be improved.  It is not easy to read.

Line 166-167: Concerning the dynamics of DF abundance, we found this trophic group as more
flexible.

What is meant by the term ‘flexible’?

The quality of Figure 5 needs to be improved. 

Lines 218-220: Jiang et al. [27] present as the main structure-determining factors  in the formation of the bottom communities’ trophic structure are the altitude, bottom substrate, river order and river width.

Reword to read: Jiang et al. [27] noted the main structure-determining factors  in the formation of the trophic structure of benthic communities to be  altitude, bottom substrate, river order and river width.

Lines 222-224: The current study of the trophic structure of macrozoobenthos of semi-mountainous and mountainous rivers in Eastern Balkans referred them to native unaffected river ecosystems.

Please reword – it is not clear what is being reported here.

Lines 231-234: In the process of this restructuring of the FFG,  sensitive taxa which belong to groups of SH and SC decreased or vanished from the invertebrate community, at the expense of increasing
share of more tolerant groups of DF or FL.

Insert comma after FFG . Another edit in bold.

Line 235: Insert comma after ‘obligate groups’

Lines 248-249: CL are relatively tolerant and occur in different habitats and under peculiar environmental conditions [30].  What is meant by peculiar – give examples

Line 265: Their proportion in the surveyed communities was neglected..    What is meant by neglected?  Do you mean negligible?

Line 271: ‘witness’ does not read well in this sentence.

Line 319: The upper river sections have a structure-determining role.  Explain

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript (Ms) addresses relevant issues regarding trophic structure of benthic macroinvertebrates from the Eastern Balkans. Unfortunately, I feel that this valuable information has not been efficiently presented and discussed. In its present form the study is not clear and the data presented is insufficient. To help navigate the Ms and present the results more concisely, the author should create a new table with the following characteristics of the sampling sites: Site code, stream order, stream type, channel width, predominant substrate, percent shading at the sampling site, ecological status according to the national assessment system, main pressures.
I am not a native English speaker, but in my opinion English needs to be heavily revised.
Material and methods
Line 91: The Ecoregion should be covered by reference
Line 97: Authors should clearly indicate when sampling was conducted in spring 2018 and in autumn 2017. Are there differences in the flow regime between the sampling periods?
Statistical analysis
Authors stated (Lines 123-124): Multidimensional scaling (MDS) plot analysis were done to determine the level of similarity in the macrozoobenthos trophic structure between the R3- and R5- type sites.
In my opinion, only sites with good and very good ecological status should be considered in this analysis, as impaired sites generally have uniform macroinvertebrate communities.
Results
Fig. 2 should be omitted.
Discussion
The discussion section is mostly unclear, some statements are not precise enough and need to be more focused and rewritten. I suggest that the authors first discuss the trophic structure of the macroinvertebrate community at unimpacted sampling sites and then at impacted sampling sites.
The authors should discuss the results shown in Fig 3. What is the reason for the difference in the proportion of trophic guilds in the Vardar River?
What is the reason for the seasonal dynamics of Sc and SH in the studied rivers?
Lines 267-268. It would be interesting if the authors could discuss the dominance of passive filtrators (Simuliidae) at site 5_MKD (in autumn).
Line 225. Authors should explain statement: The current study of the trophic structure of macrozoobenthos of semi-mountainous and mountainous rivers in Eastern Balkans referred them to native unaffected river ecosystems.
It is obvious that among investigated sites there are also some impaired sites among those studied. It would be clearer if the table with the characteristics of the sampling sites were included in Ms. I suggest the authors to correct and rewrite the corresponding paragraph (lines 222-234).
Line 240. Please add some species as an example of a stenobiont oligosaprobic benthic species
Line 240-241: Please specify, because the presence of CPOM in the water (at the bottom?) is not always an indicator of unaffected environmental conditions.
Line 264. According to literature reference 39, it can be stated that the filter feeders inhabit exclusively soft and muddy substrates, which is not correct.
Line 248-261 This paragraph should be shortened and rewritten. According to Fig. 2, PR accounted for 6% of the FFG, which is common for the types of river studied.
Lines 279, 283. In discussion section authors shouldn’t refer to figures.
Lines 286-302. It is not clear why the ITC index for small mountain rivers and streams needs further adjustment. The authors should provide more arguments and be more precise in presented arguments.
Please be consistent with names of FFG FL: filterers, filters or filtrators

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

The review of the manuscript entitled: Trophic structure of benthic macroinvertebrates from semi-mountainous and mountainous rivers in the Eastern Balkans: case study in cross-border region between Bulgaria and North Macedonia

Overall, my personal opinion to the manuscript as presented is not acceptable for publication because of weakness in the study's novelty and general weak presentation of sampling design and results. Moreover there is a mess in many parts of the work and it is difficult to understand the main idea of the work. In addition, the authors should improve the quality of the figures as well as its captions.

My comments:

I have a big problem to find something novelty in this paper, e.g. the last sentence in the abstract and in many other parts of the manuscript. Moreover the range of the study is local.

The title is too long, it should be improved.

Line 57 rather aquatic or freshwater macrozoobentos not riverine

Line 72-82 The aims of the study are very obvious and bring nothing new. For example Line 76-79.

Line 88-94 The authors mentioned that the level of disturbance and anthropogenic alternations were presented in the other study but the “Study site” should be describe more detailed in this paper.

Line 96-102 Very general description of macroinvertebrate sampling. It should be improved.

Line 107 “A total of 280 taxa…” - this is a results.

Line 110 I have a question: How do you classify the FFG’s based on the family level? Very often particular macroinvertebrate families are composed with different trophic groups… It should be clarify in the paper.

Line 121 I am not sure that PCA is appropriate analysis. Principal components analysis is a variable-reduction technique that shares many similarities to exploratory factor analysis. Its aim is to reduce a larger set of variables into a smaller set of 'artificial' variables, called 'principal components', which account for most of the variance in the original variables. Maybe better can be use RDA analysis (Redundancy analysis with Monte Carlo permutation test) because it is a method to extract and summarize the variation in a set of response variables that can be explained by a set of explanatory variables.

In the result section there is a lack information about the macroinvertebrate assemblage composition (the table with list of taxa and its abundance and trophic preferences should be added).

Line 189-192 Do you have any statistics which confirm this statement?

Line 195-201 Do you have any statistics which confirm this statement? I can’t find the EQ in the figure 6.

Figures:

The quality of the figures are poor.

Figure 1. The scale should be added, as well as the information about numbers meanings should be added.

Figure 2. I think the pie chart is not appropriate for a research paper, I advise you to change it. Moreover the figure caption should contain an explanation of the abbreviations.

Figure 3. Line 146 …”significant share in communities..” Did you use any statistics confirming this statement? Please add what the whiskers mean.

Figure 4. The quality of the figure is very low. What percentage of the variance is described by the individual PCA axes? The figure caption should contain an explanation of the abbreviations. Why do the particular figures are in different sizes?

Figure 6. The y-axis should be signed. Please add what the whiskers mean. When writing numbers, use a dot instead of a comma (Fig. 6a).

Figure 7. The scale should be added. What is the meaning of the abbreviation? This is a bit annoying and makes it difficult to interpret the figure.

Because of many, serious flaws of the manuscript, which in my opinion cannot be resolved by simple revision, I finish my revision at this place and do not to revised the discussion.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

A main point raised in the first review is that the authors fail to highlight the novelty of the research and why it should be of interest to an international audience. – what specifically is it adding to existing knowledge on FFGs in river systems?  This has not yet been adequately addressed.

The discussion remains a problem as its current structure does not really highlight the main findings and discuss them in the context of previous studies, including highlighting any new insights. Writing a paragraph on each trophic groups is perhaps not the best way to highlight and discuss the main finding.  It is too long and difficult to hold the attention of the reader.

I suggest that the discussion be structured according to the objectives, i.e. what you set out to determine.  Before commencing the discussion it would be good to create a bulleted list (not in the manuscript) of the findings in relation to each objective and then plan how best to order, present and discuss them. Remember to highlight what new insights this study presents that would add to existing knowledge and therefore be of interest outside of the study region.

Secondly, the English is still an issue in several areas and as previously recommended the manuscript should be proof read by a native English speaker.  This will help address areas where the text is still unclear. Here are some examples:

Point 12: Line 166-167: Concerning the dynamics of DF abundance, we found this trophic group as more flexible. What is meant by the term ‘flexible’?   The new term 'plasticity'  is still not clear. 

Lines 222-224: The current study of the trophic structure of macrozoobenthos of semi-mountainous and mountainous rivers in Eastern Balkans referred them to native unaffected river ecosystems.  The slightly reworded sentence is still not clear.

Please also see the comments on the attcahed pdf.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

We transformed the final version of the manuscript according your suggestions and recommendations. Please find the provided answers in the attachment below.

Kind regards,

Biljana

Author Response File: Author Response.docx

Reviewer 2 Report

The revised manuscript (Ms) is much improved, but some important issues are still unclear.

In the Material and Methods section (2.1 Study site) authors stated that investigated sites were apart from evidence of hydromorphological degradation and none of the studied sites had significant sources of pollution. However, according to Rimcheska and Vidinova (2020), 7 sampling sites (out of a total of 38 sampling sites) are under significant antropogenic influence with organic pollution score ≥ 3 and general degradation score > 2,5. This fact shouldn't be neglected, as organic pollution and general degradation (hydromorphology) are usually the dominant factors in shaping benthic macroinvertebrate assemblages.

At the and of the Introduction section, the authors assume that the FFG macroinvertebrate composition is changing within mountainous and semi-mountainous sites from the watersheds studied but it is still not clear whether typological (environmental) characteristics are responsible for the differences in macroinvertebrate FFG composition among river types or whether the differences among river types are mainly anthropogenically influenced, as the authors claime in abstract. For these, I suggest the authors to conduct an additional statistical analysis with exclusion of sampling sites that are significantly antropogenically influenced (with organic pollution score ≥ 3 and a general degradation score > 2,5). The authors neglected this observation in the revised version of the Ms. It is very important to separate the effects of natural conditions and antropogenic influences on aquatic communities because predicting anthropogenic actions resulting in undesirable changes in aquatic systems is crucial to developing effective and sustainable water management strategies.

In addition, the authors ignore my comment about the data on the percentage of shading at sampling sites, which is a very important natural factor affecting the distribution of scrapers at sampling sites.

A next issue that should be more clearly elaborated is the applicability of two FFG metrics for ecological status assessment in mountainous and semi-mountainous streams of the Eastern Balkans. Ecological status based on organic pollution and general degradation is not consistent with ecological status assessment based on two macroinvertebrate FFG metrics. In many cases, investigated sites without antropogenical influence have 3-5 EQ score and this fact is not discussed in the Ms.

The boundaries for ecological quality classes are also not entirely clear, as RETI index values ​​of 0.5 are classified as II EQ-class and as III EQ-class for some sites (see supplementary table).

Lines 247-248 . The lowest RETI index value is characterised by station 5_MKD and not by stations 8_MKD and 4_BG stations as stated.

Author Response

Dear reviewer,

We transformed the final version of the manuscript according to your recommendations and suggestions. Please see the attachment below.

Kind regards,

Biljana

Author Response File: Author Response.docx

Reviewer 3 Report

I think that the authors have done great work to improve the manuscript, which is now ready for publication from my point of view.

Author Response

Dear reviewer,

Thank you for your response. We already made slight changes within the final version of the manuscript according to the recommendations from the other reviewers.

Kind regards,

Bijana

Round 3

Reviewer 1 Report

The manuscript has been well improved but the discussion requires further revision.  It still needs to be better structured to highlight the key results.  It would be useful to structure it in relation to the three scientific questions posed (one paragraph for each question).  As it stands it seems to have a high level of repetition and I think is it still too long to hold the attention of the reader.

Additional edits and comments are marked on the attached annotated pdf.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your notes. We inserted all the suggestions within our manuscript and made the pointed parts more clear adding the necessary explanations. We also incorporated and the suggestions from the other reviewer. We'd like to add that the manuscript text was also proof read by native English speaker and all the necessary changes were done in accordance to his recommendations for the best interpretation of the correct English grammar.

Kind regards,

Biljana

Reviewer 2 Report

The authors provided new data (shading) and additionally improved Ms, but the applicability of FFG indices in ecological status assessment needs further clarification.

In the abstract, the authors state that the comparison between the two trophic indices was done in order to analyze the advantages of using these indices to assess ecological status at the studied sites. But the authors didn't sufficiently clarify this very important issue. The discussion on the explanation of why RETI and ITC values corresponded to the low EQ values at some near-natural sites is still not clear and satisfactory (Lines 364-369). It would be interesting if the authors could discuss the low EQ values of trophic indices in autumn. The authors have suggested further adjustment of the ITC index, but I also expect the authors to make some recommendations on the use of the RETI index in assessing the ecological quality of the river types studied, taking into account some low EQ values at near-natural sites. Are there any limitations to assessing ecological status using the RETI trophic index in river types R-3 and R-5 of ecoregion 7, considering the low EQ values at some undisturbed sites?

Specific comments:

Line 22: I suggest using the word undisturbed, instead of clean

Lines 105-106: The sentence should be omitted. It is related to benthic macroinvertebrate data set.

Line 149: The type of correlation coefficient used should be indicated (Pearson, Spearman ….)

Lines 316-319: I suggest deleting these sentences because the statements are too speculative.

Lines 338-339: The sentence is unclear and should be rearranged.

Line 342: In discussion section authors shouldn’t refer to App.

The English style and language are not good, so I strongly advise the authors to find a native English speaker to proofread the manuscript.

Author Response

Dear reviewer,

Thank you for your notes. We considered all the comments concerning your concerns about the better interpretation of the indices, so we resolved this issue within the manuscript. We believe that now the results are best discussed and all the important findings are highlighted and further expressed in the conclusion part.

The manuscript text was also proof read by native English speaker and all the necessary changes were done in accordance to his recommendations for the best interpretation of the correct English grammar.

Kind regards,

Biljana