Effects of Cumulative Municipal Wastewater Exposure on Benthic Macroinvertebrate Assemblages: An Experimental Stream Approach
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe topic is very interesting, yet this article can be greatly improved.
I like the experiment concept, and the topic is very interesting! Here are my comments:
Line 13 (Abstract): “however responses in these endpoints are not well characterized in systems where MWWE contains diverse chemical mixtures.“ suggestion - change this into: „However, the responses of these metrics in systems with complex chemical mixtures from MWWE are not well understood.“
Line 14: Specifically, just start with “This study…” and then can add “…specifically assessed the effects of cumulative loading of tertiary-treated MWWE on benthic macroinvertebrate communities.”
Line 25-26: Sentence “These differences are consistent as outcomes of nutrient enrichment.” is maybe too general, it doesn’t sound it is conclusion of your study, try to make a more precise statement.
Line 35-36 (Introduction): What do you mean by “is an established urban effluent”? If this should mean “this is a well-known / recognized type of urban effluent”, maybe you can change it for better understanding.
Line 36-37: It is already well-known I suppose, not “increasingly”
Line 39-41: The composition of MWWE is affected by changes in the factors such as human population size and substance use, which can influence (or have an impact on) the effectiveness of wastewater treatment,… I think, this way would be easier to read and understand.
Line 59: variation in flow condition – for me it would be interesting to read in which way, like - in low water regime concentrations of the pollutants and consequently the negative effects can be higher etc., if this papers you are citing have this information you can just expand it a little bit and keep the same references.
Line 62-65: This sounds strange - “utility of utilizing”. Maybe something like this: This complexity indicates the value of using controlled mesocosm systems, especially for refining our understanding of biological endpoints to quantify the impacts of effluent. Artificial streams which are effective at replicating the various environmental conditions exposed to MWWE mixtures have shown that both the composition and total concentrations have a significant impact on benthic macroinvertebrate communities.
Line 95-96: Please provide additional details to elaborate on this important aspect of the methodology. What is the specific treatment process? What is the composition of the treated water? How long did the treatment last (six weeks? when it started when it stopped?)? Please indicate the location of the effluent discharge (represented by the small yellow squares in Figure 1.?) and provide the distances between the sampling sites and the effluent point. Such information is essential for the reader's understanding of the experiment concept.
Line 96-99 (Materials and Methods): First you state that the conditions of the artificial streams are tested before and that they have temperature and dissolved oxygen regimes generally consistent and that PH slightly changes, which is all informative, so it is ok to leave this reference here. However, since this section focuses on the methods of the current study, the subsequent sentence and references related to studies on the Bow River (regarding periphyton growth), would be better placed in the Introduction or Discussion section.
Line 100-101: Same comment as previous, move the sentence (reference 24) to other section of the manuscript were more appropriate. Or along the description of the treatment process if it describes the composition of the treatment effluent composition (Line 95-96).
Line 105 (Figure 1.): Very long caption. You should explain, both in the picture and in the caption, that yellow star represents three baskets and one surber samples and all other descriptive information put in the text. Where the effluence spot is placed – is it at exact spot of the colored squares? – this can be described in the text. Why there is no legend for grey square? “Control streams use Bow River water exposed to upstream WWTP effluents and treatment streams also use Bow River water but 5% of the total stream volume is tertiarily treated MWWE from the nearby Pine Creek WWTP.”–details like “upstream WWTP effluents” and “the nearby Pine Creek WWTP” you should elaborate in text not in the figure caption. If you correctly describe everything in the text than you can shorten the caption like in these examples:
Example 1: “Schematic diagram of the experimental stream facility at Advancing Canadian Water Assets (ACWA). A set of three rock baskets and a Surber samples were collected at each starred sample site. Both the control and treatment streams are fed with water from the Bow River, while the treatment streams received 5% (of total stream volume) of the tertiary treated MWWE.”
Example 2: Schematic diagram of the experimental stream facility at Advancing Canadian Water Assets (ACWA). At each starred sample site, a set of three rock baskets and Surber samples were collected. Both control and treatment streams received water from the Bow River, with treatment streams incorporating 5% tertiary treated municipal wastewater effluent into the total stream volume.
Line 109-114: Since there is comment that the reference was not found, I can assume that you are again referring to earlier data on stream characteristics, but please then merge this information with the information on oxygen and temperature in one place and correct the references. Remember to clearly distinguish the earlier studies from the current study.
Line 120-121: This is too short for an explanation of the merging of data. Please explain this in detail. Since this is your M&M information, more space and more detail is needed here compared to all the information you already gave from previous studies. For instance why 6 and 18? Why you didn’t pool the upstream control data and compared it with downstream control data, and likewise for treatment data? Is the distance between upstream and downstream long enough to maybe record the downstream self-purification improvement?
Line 122: In the heading of the table you say that the data is from 2020, temperature, TOC, TN, TP please also give the reference on previous data source here if needed. Again, it is confusing what is current study and what is old data.
Line 135: Three baskets per sampling site - three upstream and three downstream, at each artificial stream that would be how many? If it is 6 per one stream, and you have 6 streams it would be 36 not 18 as previously stated? And also Surber samples, 6 streams x 2 sampling sites is 12 samples.
Line 138: Why is the material frozen and not fixed in alcohol? How did you do the identification and counting, you should explain this at least in a short sentence.
Line 169-171 (Results): “Total abundance was more than four times higher in the rock basket samples compared to Surber samples, but raw counts were similar for treatments when using the same sampling method and the mean family richness for all samples was almost identical between all treatments and methods (~9 families) (Figure 2).” This is this very long and confusing sentence.
Line 299: This reference should be changed to number.
Line 305: Again, reference to number.
Line 307: Which alternative sampling methods?
Line 319-321: Nice conclusion.
Line 323-325: “Where patterns in established communities (from Surber samples) were not significantly affected by additional MWWE inputs, suggesting that 5% effluent did not stimulate adequate community changes in these conditions, rock baskets communities were affected by cumulative MWWE exposure.” Very confusing sentence, try something like this: “In areas where the established communities ( Surber samples) did not show significant changes due to additional inputs of municipal wastewater (MWWE), this indicates that the 5% wastewater did not sufficiently stimulate community alterations under these conditions. In contrast, the communities in the rock baskets showed a response to the cumulative exposure to MWWE.”
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease see attached document for my comments.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe topic is interesting, but you need better text structure and information flow.
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript has been improved. Here are a few more points to consider.
Abstract: Delete "area limited" and just call them Surber sampler. Rock basket also limit the area sampled. Do this throughout manuscript.
Delete "well" in well established. Not needed
I don't think you study justifies the "multipronged approach" due to the limited number of Surber samples. Revise.
Introduction: Please add questions to the last paragraph of the introduction. Understanding your goals and what questions you asks helps the reader understand and interpret the results.
Figure 1. Remove acronym. Not needed in legend.
Methods: Why did you limit Surber samples to 30 seconds of scrubbing? This would greatly limit the invertebrates you collected and produce lower quality data.
Wow, 850 micron mesh. That is really large and you probably lost a lot of invertebrates. Why did you choose such a large mesh size?
Results: Move to methods " For Surber samples, n = 6 per treatment and for rock baskets, n = 18 per treatment."
Defining you counts of abundance the first time you refer to this int he results would be helpful so the reader completely understand what you mean by this.
Figure 2 appears cut off on the right side. Also change was to were. Remove ) at end.
Strange formatting for table 2 on multiple pages.
Consider making Figure 5 smaller!
Discussion: Add that 3x more rock baskets were collected than Surbers. (line 329)
Author Response
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Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for accepting the corrections. I truly appreciate your effort in enhancing the clarity and flow of the information in the text. The revisions have greatly improved the manuscript, making this interesting experiment and its results more appealing for the scientific community.
Author Response
Thank you very much for your contributions and advice through this process.
