Anthropogenic Impact on Tropical Perennial River in South India: Snapshot of Carbon Dynamics and Bacterial Community Composition

Round 1

Reviewer 1 Report

The Authors undertook an investigation of  anthropogenically induced changes of water quality, the distribution of selected pharmaceuticals, and the effects of pollution on greenhouse gas concentrations and bacterial community composition along Cauvery river, in Southern India. Authors postulate that industrialisation and increased population density influence on changes in water quality, riverine carbon fluxes, and bacterial diversity. Results described in this work could be interesting for readers of Water journal however the quality of the manuscript is not enough high and must be improved before publication. The main drawback of this manuscript are poor description of the results and lack of strong conclusions. It seems that  the work was not thought out and Authors tried to do something with results that they got.

Authors should try to find relation not only between basic parameters but also between abundance of particular members of microbial communities and pharmaceutical concentration. Maybe this kind of analysis will lead to the new findings. Additionally, microbial communities structure and relatedness between samples coming for different stations should be better described.

Other comments:

1. Caption of Figure 2. Please, described what are numbers on the pictures.
2. Lines 139-141. It is not clear why authors did not analyze the concentrations of nitrogen and phosphorus. Both are very important nutrients and its concentrations will provide substantial information about processes in the river.  I did not find in the text information about this nutrients concentrations taken from other sources.
3. Lines 180-183. How many replicates of biomass was taken for 16S rRNA amplicons analysis?
4. Line 194. How Authors removed chimeras and clustered reads? If it was done using iOMICS this sentence should be removed to another paragraph.
5. Line 272. How alpha diversity was calculated and were are values? This sentence should be changed because it is misleading. It is not possible to reveal bacterial phyla by analysis of alpha diversity.
6. First sentence of discussion should be change because Authors do not provide enough information about possible sources of pharmaceutical pollution.
7. Line 390. There is something wrong with this sentence. "... Cyanobacteria abundance was higher..."

Author Response

Response to Reviewer 1 Comments

Point 1: The Authors undertook an investigation of  anthropogenically induced changes of water quality, the distribution of selected pharmaceuticals, and the effects of pollution on greenhouse gas concentrations and bacterial community composition along Cauvery river, in Southern India. Authors postulate that industrialisation and increased population density influence on changes in water quality, riverine carbon fluxes, and bacterial diversity. Results described in this work could be interesting for readers of Water journal however the quality of the manuscript is not enough high and must be improved before publication. The main drawback of this manuscript are poor description of the results and lack of strong conclusions. It seems that  the work was not thought out and Authors tried to do something with results that they got.

Response 1: We appreciate that the reviewer likes the study and find it interesting for readers of Water journal. However, we believe the work was thought out enough! We had a hypothesis and a well-designed sampling plan. For the reviewer, it might look like some data are missing which would help to understand the system better. Additionally, it is important to have in mind that sampling in India is quite different from sampling in developed countries. Starting this field work we had many more parameters on the list. Besides GHG two additional main parameters (and now missing ones) were nutrients and antibiotics. Unfortunately, both failed in analyses. Even though we are aware of this, we were not protected in loosing samples. We hardly tried to get this data anyway, which unfortunately did not work out.

In general, India is lacking data of (polluted) aquatic systems and its consequences. As the title indicates, this study should be rather a descriptive one to get a larger overview including several factors in and around the Cauvery River, combining microbial, biogeochemical and social implications.

We tried to explain and comment our results as good as possible. However, we are limited by our gained data and we did not want to come into a speculative form. That is why we have been rather reserved with interpreting the data more. We now have carefully revised the manuscript and considered all of the reviewer’s comments. We hope that our revised manuscript adequately addresses all the comments and the reviewer now finds the manuscript suitable for publication in Water.

Point 2: Authors should try to find relation not only between basic parameters but also between abundance of particular members of microbial communities and pharmaceutical concentration. Maybe this kind of analysis will lead to the new findings. Additionally, microbial communities structure and relatedness between samples coming for different stations should be better described.

Response 2: We have included microbial communities in the statistical analyses; however, correlations with parameters from Table 2 could only be found for Cyanobacteria, yet we added a table showing these correlations in the supplement of the revised manuscript (Table S1). We refrained from including pharmaceutical concentrations in the statistical analyses owing to the low number of stations analysed for pharmaceutical concentrations due to financial costs.

Point 3: Caption of Figure 2. Please, described what are numbers on the pictures.

Response 3: The following sentences is added to make it clear: ‘The numbers in the pictures present station numbers’

Point 4: Lines 139-141. It is not clear why authors did not analyze the concentrations of nitrogen and phosphorus. Both are very important nutrients and its concentrations will provide substantial information about processes in the river.  I did not find in the text information about this nutrients concentrations taken from other sources.

Response 4: We absolutely agree with the reviewer. This was our initial intention and two important biogeochemical parameters which we wanted to analyse. Unfortunately, the measurements for the N and P samples did not work out due to problems in the laboratory. Nevertheless, we tried to include some nutrient information in the manuscript which we know from the Assessment of Water Quality Index of Cauvery, literature (e.g. Kulandaivel et al. 2009, see line 360 -362) and also by in situ observations (looking at the river side for ecosystems indications (e.g. algae bloom, foam, input of industry and WWT). We now did an additional literature research and included it as Krishan et al. 2016 and Gowda et al. 2016. Since anthropogenic activities, industry and wastewater discharge bring especially in India high pulses of nutrients, we can assume that the river Cauvery has high loads of nutrients. We also now included nutrient concentrations in the text from the literature. Line 362-66.

Point 5: Lines 180-183. How many replicates of biomass was taken for 16S rRNA amplicons analysis?

Response 5: We only took one water replicate for 16S rRNA analyses per station. As most sequencing studies lack replicates due to the high costs for sequencing it holds also true for our study. We could only afford one replicate per stations. We are aware that this is not ideal but it was at least the opportunity to get a view on the microbial data. We think that this is justifiable since the focus of the manuscript is not on microbial studies. We added this information into the text: Water samples for DNA extraction were only taken once per station without replicating. Line 193

Point 6: Line 194. How Authors removed chimeras and clustered reads? If it was done using iOMICS this sentence should be removed to another paragraph.

Response 6: Indeed it was done using iOMICS. We now moved the sentence to lines 214-15.

Point 7: Line 272. How alpha diversity was calculated and were are values? This sentence should be changed because it is misleading. It is not possible to reveal bacterial phyla by analysis of alpha diversity.

Response 7: We agree with the reviewer that alpha diversity cannot be calculated by the relative abundance of bacterial phyla. Therefore, we deleted this part of the manuscript.

Point 8: First sentence of discussion should be change because Authors do not provide enough information about possible sources of pharmaceutical pollution.

Response 8: Thanks. We are aware of this problem. That’s why we wrote that we can only give ‘indications’. However, we see that it could be misleading and changed the first sentence. We removed the pharmaceutical part.

Point 9: Line 390. There is something wrong with this sentence. "... Cyanobacteria abundance was higher..."

Response 9: We agree with the reviewer and removed the sentence.

Author Response File: Author Response.pdf

Reviewer 2 Report

Keywords: You need to add “proteobacteria” and “Cauvery river”.

Global comments on the paper:

The paper is interesting as it focusses on assessment of pharmaceuticals, bacterial community structure, pCO2 and pCH4 in Cauvery river in south of India. Meanwhile the paper/methodology suffers from several key insufficiencies and absolutely needs improvements before any publications. You’ll find here-attached my main comments:

• Comment 1: Lines 39- 40: what do you mean by "global development"?
• Comment 2: Lines 39- 40: You have a lot of other issues such as agriculture, soil losses, water resources overexploitation, biodiversity losses, pollutions...
• Comment 3: Lines 49-50: No wastewaters are firstly from cities, domestic and industrial activities.
• Comment 4: Lines 51-52: It is not totally true. A recent study addressed the issue in south Asian countries. It demonstrated the inaccuracy of pharmaceutical management in these countries.
• Comment 5: Line 72: Replace "give a clear understanding" by "improve the understanding".
• Comment 6: Line 97 Figure 1: Add location of station number 1 - I don't think that you have only so few WWTP on this watershed. Maybe they are the more critical WWTP. In such a case, be so kind to adjust your legend - Add the administrative boundaries of the Karnataka/Tamil Nadu states.
• Comment 7: Lines 108-116: You should also refer to the strong issue of water partition dispute between Tamil Nadu and Karnataka states.
• Comment 8: Line 117: Why didn't you also looked at dry season, as processes and pollution concentrations are supposed to be totally different?
• Comment 9: Line 121: Precise at what dates and hours of the day you performed your sampling at each site?
• Comment 10: Line 122: So, you directly collected water from surface, not under the surface. Could you confirm that?
• Comment 11: Lines 123-124: Why didn't you also look at other key parameters? By only focusing on these specific parameters, you lost the capacity to assess the impacting factors.
• Comment 12: Lines 121-129: What about the flow? Didn't you forget to measure it?
• Comment 13: Lines 139-140: This is clearly an issue.
• Comment 14: Lines 150-153: How did you choose these parameters. Is it because they are the most used, or the most common in Indian rivers? Or because they are classical key pharmaceuticals in waters (as I suspect)? Moreover, I would like to know why you didn’t assess antibiotics, whereas they can strongly impact the microbial composition you tried to assess?
• Comment 15: Lines 215-227: Could you clarify the depth of this river, the number of dams and their impact on the water quality, the mean residence time in the river and dams along the river, in order to understand the potential impact on your results. For instance, what is the volume of Mettur dam, its mean depth, and the mean flow when you performed your sampling?
• Comment 16: Lines 215-227: Do you know if there is vertical evolution of water quality with depth?
• Comment 17: Line 241 Table 2: Most of this table’s results are logics and don’t provide key information. In order to go forward, you should assess the physico-chemical and biological processes.
• Comment 18: Lines 255-257 and line 264: Figure 4: You must add a table with all of the pharmaceuticals and all of the sites. It will be much easier to assess these results than with your partial figures. Add also the detection limits in this table.
• Comment 19: Lines 272-277: Here again be so kind to provide the full table with 17 stations and 12 phyla. Moreover, you should also provide a table with the number/ml. it is a key data that is not provided in your paper.
• Comment 20: Lines 281-285: Could you link that results with these sites specificities?
• Comment 21: Lines 314-315: I understood that you were supposed to perform your sampling in January. Do you still consider that there is a link between an October festival and your results three months later?
• Comment 22: Line 318-319: This is the case in most of the world rivers.
• Comment 23: Lines 343-348: You have forgotten to address the issue of benthic sediments.
• Comment 24: Lines 351-353: Pharmaceutical industries pollute water with pharmaceuticals, whereas WWTP reduce pharmaceutical fluxes to river compared to untreated sources. So, I strongly suggest you modify your sentence.
• Comment 25: Lines 370-372: You are wrong. The studies performed in Europe demonstrate that hospitals represent a limited part of the pharmaceutical fluxes to rivers (except of the pharmaceuticals exclusively used in hospitals).
• Comment 26: Lines 390-392: This is not so simple. Cyanobacteria are phytoplankton cells. They effectively impact pH and DIC, but it is also the case of other phytoplankton cells. You cannot conclude because you have not surveyed phytoplankton.
• Comment 27: Lines 396-398: Why did you speak of lower proportion of taxa of ecological interest abundance? If there is a resilient bacterial community structure in a less polluted station, it should be ecologically interesting. Be so kind to clarify your sentences. Line 408 and 284: Here again you seem to consider that taxa linked with pollution are of ecological interest. It is just amazing!!
• Comment 28: Line 402-403: What kind of nutrient they depend of?
• Comment 29: Lines 408-409: You are right and pinpoint a limitation of your approach. You must provide the total number/ml for each station, and assess these numbers.
• Comment 30: Global comment: You must comment your results and explain the upstream-downstream evolution. It is not sufficient to perform correlation matrix. Assessment includes potential explanation. A part is presented in the discussion step, but you lack of a more in-depth assessment. The reasons are simple. You forgot key data such as flows, residence time, sediments, some key chemical parameters, ecological functioning of the river, and the seasonal patterns. Moreover, you only refer to one single field sampling, whereas the values are totally dependent of meteorology and hydrology. Therefore, you are in a fog and have difficulty to explain your results. These limitations must be presented in the discussion part or in the conclusion with proposal for future complementary studies to perform.

Author Response

Response to Reviewer 2 Comments

Point 1: Keywords: You need to add “proteobacteria” and “Cauvery river”.

Response 1: Thank you for this suggestion. Both suggested words are added to the key words.

Point 2: Global comments on the paper: The paper is interesting as it focusses on assessment of pharmaceuticals, bacterial community structure, pCO2 and pCH4 in Cauvery river in south of India. Meanwhile the paper/methodology suffers from several key insufficiencies and absolutely needs improvements before any publications. You’ll find here-attached my main comments:

Response 2: We appreciate that the reviewer likes the study and generally supports the publication of our results. We are grateful that we get the chance to strengthen the manuscript. We now have carefully revised the manuscript and considered all of your given comments which improved the manuscript. We hope that our revised manuscript sufficiently addresses all the comments and is now suitable for publication in Water.

Point 3: Comment 1: Lines 39- 40: what do you mean by "global development"?

Response 3: We wanted to state with this sentence that the mentioned problems like rapid population increase, urbanization and climate change are a problem which is found in the entire world. To clarify we included the word globalization and removed the words ‘global development.

Point 4: Comment 2: Lines 39- 40: You have a lot of other issues such as agriculture, soil losses, water resources overexploitation, biodiversity losses, pollutions...

Response 4: We agree with the reviewer. However, it would be too much to name all. Furthermore, the mentioned issues by the reviewer are direct consequences of the problems stated by us. Therefore, we included the words ‘…are some of the main threats to..’ to indicate that we only gave a selection.

Point 5: Comment 3: Lines 49-50: No wastewaters are firstly from cities, domestic and industrial activities.

Response 5: Thank you for this comment. We included this in the text and changed the sentences as following: ‘However, inland waters, particularly riverine ecosystems, are constantly polluted with, for example, heavy metals, pharmaceuticals, fertilizers, and wastewater from various sources, such as cities, domestic and industrial activities, and agriculture’ Line 51-54.

Point 6: Comment 4: Lines 51-52: It is not totally true. A recent study addressed the issue in south Asian countries. It demonstrated the inaccuracy of pharmaceutical management in these countries.

Response 6: This is very interesting. It would be nice if we could get the source of the named publication, so that we can include it to our Introduction and change the text accordingly.

Point 7: Comment 5: Line 72: Replace "give a clear understanding" by "improve the understanding".

Response 7: We have rephrased the sentence as suggested.

Point 8: Comment 6: Line 97 Figure 1: Add location of station number 1 - I don't think that you have only so few WWTP on this watershed. Maybe they are the more critical WWTP. In such a case, be so kind to adjust your legend - Add the administrative boundaries of the Karnataka/Tamil Nadu states.

Response 8: We have added the label of station number 1 and the administrative boundaries of Karnataka and Tamil Nadu. We also corrected the legend. Thanks. We included all critical WWTP already and didn’t want to include small ones to not overload the map. The map is not meant as a complete map where all WWTPs has to be included. It should rather give an overview where the main critical points are. We included this information to the figure legend. Line 104.

Point 9: Comment 7: Lines 108-116: You should also refer to the strong issue of water partition dispute between Tamil Nadu and Karnataka states.

Response 9: We are sorry, but this water sharing dispute between the two states is a political issue and has nothing to do with our study. This will influence neither the chemistry nor the biology of the water or the analysis. Thus, we think that it is not important to include this issue in the manuscript.

Point 10: Comment 8: Line 117: Why didn't you also looked at dry season, as processes and pollution concentrations are supposed to be totally different?

Response 10: This is a very good idea. Unfortunately, we only got funding for one sampling campaign. However, as soon as we get funding, we have this for future sampling campaigns in mind. We are open to welcome other interested and well informed researchers to join the project.

Point 11: Comment 9: Line 121: Precise at what dates and hours of the day you performed your sampling at each site?

Response 11: Sampling were done always in the morning from 5 a.m. until latest 12 a.m. We included the dates and the information of time into the text: ‘Sampling was conducted in the morning between 5 and 12 a.m. Sampling was carried out on the following dates: Station 1-5: 19.01.2016; station 6-10: 20.01.2016; station 11-17 21.01.2016; station 18-23 22.01.2016.’  Line: 125-127

Point 12: Comment 10: Line 122: So, you directly collected water from surface, not under the surface. Could you confirm that?

Response 12: No, we did not take surface water directly. We always took the water 20 cm under the surface. Firstly, we washed each sampling vessel three times 20 cm under the water surface. After rinsing we took the sample. We included the following parts into the text: ’… in running water 20 cm beneath the surface..’.  Line 129.

Point 13: Comment 11: Lines 123-124: Why didn't you also look at other key parameters? By only focusing on these specific parameters, you lost the capacity to assess the impacting factors.

Response 13: We totally agree with the reviewer. Initially, we wanted to analyse all important biogeochemical parameters. We did also taken samples for nutrients (N and P). Unfortunately, the measurements did not work out due to problems in the laboratory. This was not the only failure we had to deal with. We run into a lot of other unexpected issues during sampling. Nevertheless, this gave us more experience and we hope that we will succeed with the next campaign. We also wanted to have turbidity and flow velocity data. However, we were very limited in funding/grants and availability of functional equipment for the above mentioned measurements.

We also now included nutrient concentrations in the text from the literature that the reader gets an idea of the Cauverys nutrient concentrations (Krishan et al. 2016, Gowda et al. 2016). Line 361-65.

Point 14: Comment 12: Lines 121-129: What about the flow? Didn't you forget to measure it?

Response 14: This is a good point. No, we didn’t forget. It was a matter of available equipment. We didn’t have one available. Additionally, we also didn’t want to use just the ‘flowing-orange-method’ since flow velocity differs a lot at each side due to the width and unevenness of the river.

Point 15: Comment 13: Lines 139-140: This is clearly an issue.

Response 15: Yes. We are totally aware of that. To overcome this gap, we now included data from literature into the manuscript, thus the reader has an idea of the nutrient concentrations in the river. See also point 13. Line 361-65.

Point 16: Comment 14: Lines 150-153: How did you choose these parameters. Is it because they are the most used, or the most common in Indian rivers? Or because they are classical key pharmaceuticals in waters (as I suspect)? Moreover, I would like to know why you didn’t assess antibiotics, whereas they can strongly impact the microbial composition you tried to assess?

Response 16: As per the literature survey studies undertaken, these were the most prominent pharmaceutical compounds which have been analysed in Indian river systems (Shanmugam, G., Sampath, S., Selvaraj, K. K., Larsson, D. J., & Ramaswamy, B. R. (2014). Non-steroidal anti-inflammatory drugs in Indian rivers. Environmental Science and Pollution Research, 21(2), 921-931). Analgesics and anti-inflammatory drugs are widely used all over India. This is the reason behind choosing these pharmaceuticals compounds. Some of the compounds analysed presently like triclosan and parabens (antimicrobial preservative compounds used in cosmetics, food and pharmaceuticals) have similar effect like antibiotics and they strongly affect microbial community (Selvaraj, K. K., Sivakumar, S., Sampath, S., Shanmugam, G., Sundaresan, U., & Ramaswamy, B. R. (2013). Paraben resistance in bacteria from sewage treatment plant effluents in India. Water science and technology, 68(9), 2067-2073).

We also took samples for antibiotics for each station. This parameter was initially one of our main parameters, next to GHG, nutrients and microbial communities. However, we send the water samples immediately after sampling to SMS labs in Chennai since during this time our lab was not ready for antibiotic analysis. Unluckily it also failed in analyses. We don’t know why and also the company could not give us a solution-oriented explanation. This was very frustrating for us. In a next study, we will try everything to get proper antibiotic results: now, our labs are prepared for it.

Point 17: Comment 15: Lines 215-227: Could you clarify the depth of this river, the number of dams and their impact on the water quality, the mean residence time in the river and dams along the river, in order to understand the potential impact on your results. For instance, what is the volume of Mettur dam, its mean depth, and the mean flow when you performed your sampling?

Response 17: These are interesting parameters, but they exceed the goal of this study. Additionally, this amount of data is only possible to get if you are working in a larger project. It is always nice to have as many data as possible to really understand the system. On the other hand, it also means additional funding, more time and man power with expertise. Thus, as always when you start studying ecosystems you begin with small steps time will bring more data and a better picture.

Replying to the reviewer’s specific questions, the depth of the river really depends where you are as the river is very heterogenous: in its structure, vegetation, width, depth and also anthropogenic use. Therefore, we presented the pictures in Fig. 2 of the manuscript. We have already included all important dams which could influence our results in the map. The volume of the Mettur dam, however, does not play a role for our study since it has no link to the data and is, in our opinion not necessary to include.

Point 18: Comment 16: Lines 215-227: Do you know if there is vertical evolution of water quality with depth?

Response 18: We apologize for not having data to this question, it was beyond our research question. In addition, we are also not aware of papers dealing with this subject in Cauvery.

Point 19: Comment 17: Line 241 Table 2: Most of this table’s results are logics and don’t provide key information. In order to go forward, you should assess the physico-chemical and biological processes.

Response 19: Those are given in table 1 and also in the figures. We think that the manuscript would be too overloaded with data when we bring the data at the same time in table form. We thus decided that it is more useful to visualize them in a figure form. If you really think that it is necessary to bring the data additionally in a table form, we can offer to upload all data on an open source that those are available for everybody (which makes sense anyway).

Point 20: Comment 18: Lines 255-257 and line 264: Figure 4: You must add a table with all of the pharmaceuticals and all of the sites. It will be much easier to assess these results than with your partial figures. Add also the detection limits in this table.

Response 20: See answer 19. Detection limits are added now in material and methods part: ‘The detection limits of the analytes are as follows: Ibuprofen- 10 ng L^-1, paracetamol – 20 ng L^-1, naproxen – 20 ng L^-1, triclosan – 5 ng L^-1, diclofenac - 5 ng L^-1, carbamazepine – 5 ng L^-1, methylparaben – 2 ng L^-1, propylparaben – 2 ng L^-1, nonyl-phenol – 5 ng L^-1, 4-octyl-phenol – 5 ng L^-1 , 2,4’-bisphenol A – 5 ng L^-1.’ Added line: 185-189

Point 21: Comment 19: Lines 272-277: Here again be so kind to provide the full table with 17 stations and 12 phyla. Moreover, you should also provide a table with the number/ml. it is a key data that is not provided in your paper.

Response 21: See answer 19. We will upload the data on an open source.  

Point 22: Comment 20: Lines 281-285: Could you link that results with these sites specificities?

Response 22: In our discussion under chapter 4.4 we link the bacterial composition to site specifics. Line 412-54.

Point 23: Comment 21: Lines 314-315: I understood that you were supposed to perform your sampling in January. Do you still consider that there is a link between an October festival and your results three months later?

Response 23: That would be great to know. At this stage we could only speculate about this idea, since we do not have data from the suggested time.

Point 24: Comment 22: Line 318-319: This is the case in most of the world rivers.

Response 24: Not in all rivers. It depends i.a. on the oxygen level, the nutrient stage, the volume, the air-water-gas exchange and the flow velocity. However, this subject could be discussed in another paper or a workshop by its own.

Point 25: Comment 23: Lines 343-348: You have forgotten to address the issue of benthic sediments.

Response 25: Good point. We added information about this important issue into the text. ‘In addition, elevation, climatic gradient, and benthic sediment mineralization rate may play a role in the different degrees of weathering along the Cauvery river’ Line 357-349.

Point 26: Comment 24: Lines 351-353: Pharmaceutical industries pollute water with pharmaceuticals, whereas WWTP reduce pharmaceutical fluxes to river compared to untreated sources. So, I strongly suggest you modify your sentence.

Response 26: Thank you for this comment. Indeed, compared to industry ect., WWTP reduces the pharmaceuticals. However, we wanted to point out that WWTP release pharmaceuticals despite the fact that they should treat the water. We have modified the sentence as followed: ‘Discharge from pharmaceutical industries into water bodies is considered a major source of pharmaceuticals, followed by agricultural waste and household sewage [50]. However, sewage treatment plants are considered to be an important source despite they reduce pharmaceutical fluxes into the river compared to untreated sources’ Line 370 – 373.

Point 27: Comment 25: Lines 370-372: You are wrong. The studies performed in Europe demonstrate that hospitals represent a limited part of the pharmaceutical fluxes to rivers (except of the pharmaceuticals exclusively used in hospitals).

Response 27: We understand what the reviewer means and have therefore changed the sentences to the following one: ‘The high concentrations of pharmaceuticals such as carbamazepine, ibuprofen, and paracetamol could be attributed to their easy availability’. Line 392-394.

Point 28: Comment 26: Lines 390-392: This is not so simple. Cyanobacteria are phytoplankton cells. They effectively impact pH and DIC, but it is also the case of other phytoplankton cells. You cannot conclude because you have not surveyed phytoplankton.

Response 28: We removed the sentences because it seemed misleading. However, we do not agree that Cyanobacteria belong to phytoplankton. For us Cyanobacteria belong to free-living photosynthetic bacteria. However, Cyanobacteria are part of the phytoplankton group, but phytoplankton is extremely diverse, varying from photosynthesising bacteria (cyanobacteria) to plant-like diatoms. Taxonomically, the diatoms belong to the domain of Eucaryota, whereas cyanobacteria belong to the domain Bacteria (=prokaryota). But this is an old discussion among researchers and it always depends in which field the researchers working. Microbiologists mostly put them to the named phylum whereas researchers working in the subject of Phytoplankton Taxonomy and Ecology sort them to the phytoplankton group.  

Point 29: Comment 27: Lines 396-398: Why did you speak of lower proportion of taxa of ecological interest abundance? If there is a resilient bacterial community structure in a less polluted station, it should be ecologically interesting. Be so kind to clarify your sentences. Line 408 and 284: Here again you seem to consider that taxa linked with pollution are of ecological interest. It is just amazing!!

Response 29: Thank you for this comment. Indeed it is confusing and not well written. We clarified the sentences and included the following one: ‘Hence, this station registered the highest bacterial diversity comprising all bacterial phyla (Figure 5) with less pathogen abundance, indicating a less polluted station with a resilient bacterial-community structure.’ Line 419-21.

Moreover, the reviewer is right that the wording ‘taxa of ecological interest’ doesn’t fit in the context. We thus changed it also in the old lines 408 and 284 through the word ‘pathogens’.  Line 300, 304, 420, 433.

Point 30: Comment 28: Line 402-403: What kind of nutrient they depend of?

Response 30: We are not sure if we understand the question right. If the question refers to the nutrient dependence of Comamonadaceae, we meant N and P.

Point 31: Comment 29: Lines 408-409: You are right and pinpoint a limitation of your approach. You must provide the total number/ml for each station, and assess these numbers.

Response 31: We are sorry, but we do not have the number of sequences per ml available since the analyses were run by InterpretOmics India Pvt. and they could not deliver those data anymore.

Point 32: Comment 30: Global comment: You must comment your results and explain the upstream-downstream evolution. It is not sufficient to perform correlation matrix. Assessment includes potential explanation. A part is presented in the discussion step, but you lack of a more in-depth assessment. The reasons are simple. You forgot key data such as flows, residence time, sediments, some key chemical parameters, ecological functioning of the river, and the seasonal patterns. Moreover, you only refer to one single field sampling, whereas the values are totally dependent of meteorology and hydrology. Therefore, you are in a fog and have difficulty to explain your results. These limitations must be presented in the discussion part or in the conclusion with proposal for future complementary studies to perform.

Response 32: For simple comprehension, we subdivided the River in upstream and downstream of the big Mettur dam. As the reviewer mentioned, our idea was that we can see finally an upstream-downstream evolution. However, no complex statistical analysis could support our hypothesis the change of water quality downstream. As indicated in the manuscript we can only give evidence that we found an increase in EC and pH for this evolution part. However, for microbial part we also run a NMDS, a RDA, a Dendrogram and a PCA. Unfortunately we also could not find a significant impact of the environmental parameters on the OTU composition with the run analysis. There is a slight aggregation of some stations concerning the bacterial composition seen in the NMDS plot and Dendrogram, related to e.g. parameters as elevation (stations in the mountains vs downstream stations), dam associated stations, ect.. Additionally, PCA shows e.g. at very urban stations high methane concentrations and a distinct bacterial community composition. Also the mountain stations and in opposite the valley stations (F1) are accumulated (please see figures below).

We tried to explain and comment our results as good as possible. However, we are limited by our gained data and didn’t want to come into a speculative form. That’s why we have been rather reserved with interpreting the data more. We would be happy about getting an idea of defined parts where the reviewer thinks that they are improvable with the data set we have.

Yes, the reviewer is right that it would be good to have data such as flows, residence time, sediments, some other chemical parameters, ecological functioning of the river, and the seasonal patterns. As we mentioned in our previous comments, this was not a large interdisciplinary project. It was rather a start of investigations to get an idea of the ecosystem and the interactions. Since those data are very new and unique in this region, it is a need to share the data and findings with the community. You will find very seldom paper where an all-in-one solution suitable for every purpose is included. It’s again a matter of money, man power and time.

Referring to only one field sampling is totally fine. Every sampling depends on the in situ conditions during the time of sampling:if you sample Cauvery 10 times a year, you might sample always at the ‘wrong’ time with unusual conditions. You can find several studies where researchers just sample the ecosystem once: this happens not because they do not want to sample more, it is simply due to the fact of financial constraints, lack of time and man power As mentioned before, all investigations start small and have then the potential to expand knowledge and data.

However, we see that we have not addressed the limitations of the study sufficiently. Therefore, we now included the following sentences into the discussion part: ‘In our study, we were restricted in obtaining data about e.g. flow velocity, ecological functioning of the river, and the seasonal patterns. These complementary factors should be considered in future studies in order to understand ecosystem-level interactions, the ecological risk, and implications for human health..’Line 471-74.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has been significantly improved and in this form it could be considered for publication in Water. However, before that authors should focus on the following comments:

1. Sequences of 16S RNA gene should be deposited in a public database (eg. NCBI)
2. Authors wrote in the Abstract that they investigated bacteria diversity. I can not find in the Materials and Methods chapter information how it was done.
3. Figure nr 4 should be improved because there is lack of units in y-xis. Is it pharmaceuticals concentration?

Author Response

Response to Reviewer 1 Comments

Round 2

Point 1: The manuscript has been significantly improved and in this form it could be considered for publication in Water. However, before that authors should focus on the following comments. Sequences of 16S RNA gene should be deposited in a public database (eg. NCBI)

Response 1: We appreciate the reviewer’s comments and his/her support for publication in Water. We have carefully revised the manuscript again and considered all the comments s/he additionally raised.

Additionally, based on reviewers thoughts and suggestions we have made several more changes to improve the manuscript, both in terms of content and language. In order to be able to retrace the revisions, we have made the revision in the track change mode in the manuscript.

We agree that sequences should be deposited in a public database. Unfortunately, we are not able to do so as we have never received the original sequences data from Interpretomics. This company delivered us the final analysed data sets and released the raw data set in a separate file which was only available for a certain time. We haven’t been aware of that and finally the raw data weren’t available anymore. We tried our best, together with the company to rescue the original data but we did not succeed. Thus, we only have the original OTU data from the company and not the sequences. The OTU data however, will be deposited in a supplementary file (Table S2-S3).

Point 2: Authors wrote in the Abstract that they investigated bacteria diversity. I can not find in the Materials and Methods chapter information how it was done.

Response 2: Thank you for this remark. In this study, we did not calculate bacterial diversity, but discussed relative abundances of different phyla, family and classes. In order to avoid confusion, we rephrased the Abstract. Line 33-34.

Point 3: Figure nr 4 should be improved because there is lack of units in y-xis. Is it pharmaceuticals concentration?

Response 3: Thanks a lot for this comment. This mistake is fixed now.

Finally, we would like to thank the reviewer for his/her time, suggestions and thoughts and the improvement of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper has been improved but still needs some improvements. You’ll find here-attached my main comments (I use the same number as in my first assessment for easiness):

• Comment 4: Thanks for your interest. I can refer for instance to this short paper published in 2019 in the IWA journal "Source" https://www.thesourcemagazine.org/study-reveals-antibiotic-levels-in-rivers-globally/.

       I have also in mind a very recent paper, but unfortunately, it is still under reviewing (I am one of the reviewer and I am obliged to confidentiality) and I don't even know if it will be published.

• Comment 7: I don't agree with you. Practically the dispute impacts the level of water that is abstracted (how and when it is abstracted), then the water quality in the river and its ecological functioning.
• Comment 8: I suggest you open to that future work in the discussion part of your paper.
• Comment 11: Be so kind to precise the exact unit. Are they in mg N/l and mg P/l or for instance in mg PO4/l ? Practically your values are extremely high regarding phosphorus.
• Comments 11 and 12: Maybe you should address that issue as a forthcoming adding to the study in the discussion part of the paper.
• Comment 14: Here again, you should add that future complement in the discussion part of the paper.
• Comment 15: You are wrong. Mettur dam and each of the big dams along a river can impact its functioning (both physical, chemical, and biological functioning). I have been working for 35 years on lakes and lakes-rivers interactions in a lot of countries, and I confirm this link can be very impactful. So, I strongly suggest you at least indicate that potential link to look at in your discussion part, to be studied in a future work.
• Comment 16: It is not that no papers deal with that subject that it is not essential. Here again, I suggest you address that issue as a potential subject to look at in future work.
• Comments 17, 18 and 19: Yes, I think it could be valuable to add these data in an accessible internet link.
• Comment 21: The issue is that you provide no data sustaining your hypothesis, as for instance you add no data on residence time or flows. My first feeling is that 3 months is very long, and I am not sure a link can be demonstrated.
• Comment 22: I used the word "most". But ok, just go forward. "The heterotrophic respiration of organic matter" is not the exact wording. "The heterotrophic respiration during the organic matter degradation by microbial biomass" would be better as there is no respiration of organic matter by itself. This process occurs in most of the rivers and lakes. It was the reason of my first comment 22.
• Comment 26: You are true. Meanwhile, Cyanobacteria are both bacteria and phytoplankton. They have photosynthetic pigments and for instance their impact on pH cannot be separated from that of the other phytoplankton cells as you have no data on them. It is the reason for why I said that you cannot conclude on the impact of cyanobacteria on pH and DIC. As you suppress the sentence, it is OK.
• Comment 28: Thanks for your answer. So, you consider they mainly depend on the level of N and P rather than for instance from the level of organic carbon? Could you let me know what are the current levels of N and P that could limit their growth?
• Comment 30: Global comment: Thanks for your detailed explanation. You have understood that I suggested to add all of the limitations of your study in the perspective of future complementary works. It is my request for the above-mentioned comments 8, 11, 12, 14, 15, 16. Some are referred to in your line 471-474 sentences, but other are not yet included. I suggest you adjust your sentence accordingly. Thanks in advance.

Author Response

Response to Reviewer 2 Comments

Round 2

Point 1: The paper has been improved but still needs some improvements. You’ll find here-attached my main comments (I use the same number as in my first assessment for easiness): Comment 4: Thanks for your interest. I can refer for instance to this short paper published in 2019 in the IWA journal "Source" https://www.thesourcemagazine.org/study-reveals-antibiotic-levels-in-rivers-globally/. I have also in mind a very recent paper, but unfortunately, it is still under reviewing (I am one of the reviewer and I am obliged to confidentiality) and I don't even know if it will be published.

Response 1: We appreciate that the reviewer sees the improvement of the manuscript and that s/he supports the publication of a revised manuscript.

Additionally, based on reviewers thoughts and suggestions we have made several more changes to improve the manuscript, both in terms of content and language. In order to be able to retrace the revisions, we have made the revision in the track change mode in the manuscript.

The mentioned report is a really impressive, interesting and alarming study! I hope that publications with detailed information of the study will come out as we are very interested in that subject. We would like to include this study in our paper. However, the link only gave us an article of the study published online in ‘The Source’. We couldn’t find a published paper of it which we can cite. We also looked under the publication list of two of the authors (Prof. Alistair B.A. Boxall, John Wilkinson) who were named in the article, but unfortunately haven’t been successful. Thus, if there is a paper out of this study already a name and a journal would be helpful.

Point 2: Comment 7: I don't agree with you. Practically the dispute impacts the level of water that is abstracted (how and when it is abstracted), then the water quality in the river and its ecological functioning.

Response 2: We fully agree with the reviewer that water level also might impact the water quality and the ecological function. However, since it didn’t impact our sampling campaign as it was not in our focus. . We have now indicated it in the discussion part. Notwithstanding during our analysis we could collect samples in all of the stations. Therefore, we included the following sentence: “The water level of the Cauvery River is further impacted by a strong issue of water partition dispute between the two states Tamil Nadu and Karnataka. This water sharing agreement between the two states is based on riparian rights which are not respected. Most likely due to the weak and seasonal monsoon with increased water demand and the expansion of agricultural land and drinking water schemes. Consequently, water abstraction might not only impact the water level of the Cauvery River, but might also impact the water quality of the river and its ecological functioning.” Line 124-130.

Point 3: Comment 8: I suggest you open to that future work in the discussion part of your paper.

Response 3: We added now the information in the discussion part and included the information of dry season and the benefit for future work. Line 508-512.

Point 4: Comment 11: Be so kind to precise the exact unit. Are they in mg N/l and mg P/l or for instance in mg PO4/l ? Practically your values are extremely high regarding phosphorus.

Response 4: Thanks for the remark. We gave more precise information now. The units are in N and P (for Krishna et al.) and Gowda et al. referred to nitrate and phosphate. Line 386-90.

Point 5: Comments 11 and 12: Maybe you should address that issue as a forthcoming adding to the study in the discussion part of the paper.

Response 5: We addressed now all the issues raised in the discussion part. We agree that it is important to pay attention to the items for future investigations. We have touched on this briefly in the discussion part. Line 508-512.

Point 6: Comment 14: Here again, you should add that future complement in the discussion part of the paper.

Point 6: We also add this future complement about antibiotics in the discussion part. We included the following sentence: “We also took samples for antibiotics for each station. Unfortunately, due to laboratory problems the analyses failed. However, antibiotic analysis should be included in a next approach of a sampling campaign since they can strongly impact the microbial composition and thus ecosystem function.” Line 412-15

Point 7: Comment 15: You are wrong. Mettur dam and each of the big dams along a river can impact its functioning (both physical, chemical, and biological functioning). I have been working for 35 years on lakes and lakes-rivers interactions in a lot of countries, and I confirm this link can be very impactful. So, I strongly suggest you at least indicate that potential link to look at in your discussion part, to be studied in a future work.

Response 7: True, we agree with the reviewer. This cannot be neglected totally. We have included the following sentence: “In future studies, we intend to target the effects of the four large dams on the physical, chemical, and biological functioning of the Cauvery River.” Line 488-490.

Point 8: Comment 16: It is not that no papers deal with that subject that it is not essential. Here again, I suggest you address that issue as a potential subject to look at in future work.

Response 8: Revised as suggested. We addressed the vertical evolution of water quality with depth for a potential subject in the general addition for future work. Line 508-512

Point 9: Comments 17, 18 and 19: Yes, I think it could be valuable to add these data in an accessible internet link.

Response 9: Thanks. For simplicity’s sake we have now included all original data as a supplemental file (Table S2-S5). Since the journal is an open source journal the data are accessible for everyone.

Point 10: Comment 21: The issue is that you provide no data sustaining your hypothesis, as for instance you add no data on residence time or flows. My first feeling is that 3 months is very long, and I am not sure a link can be demonstrated.

Response 10: We misunderstood the previous question. We thought that the October festival might change the water chemistry and biodiversity a lot. However, since our study site is a running water the effect will not hold 3 months along the river but probably locally where the festivals are. Thus, it is unlikely that we saw effects of the festival at our sampling sites. But we think that sampling around festivals along the river would reveal different results due to human impacts. Again, it would be great to sample during festival times and after 3, 7, and 14 days to evaluate the change of water quality. We included the following sentence: “To prove this hypothesis, we suggest future studies to sample shortly after the festival has been held (e.g. after 3, 7 or 14 days).” Line 351-53.

Point 11: Comment 22: I used the word "most". But ok, just go forward. "The heterotrophic respiration of organic matter" is not the exact wording. "The heterotrophic respiration during the organic matter degradation by microbial biomass" would be better as there is no respiration of organic matter by itself. This process occurs in most of the rivers and lakes. It was the reason of my first comment 22.

Response 11: We changed the sentences and included the following suggested sentence instead: “We attribute the negative correlation between pCO₂ concentration with DO saturation (r= –0.68; Table 2) along the Cauvery River to the heterotrophic microbial respiration during the organic matter degradation. “Line 355-357

Point 12: Comment 26: You are true. Meanwhile, Cyanobacteria are both bacteria and phytoplankton. They have photosynthetic pigments and for instance their impact on pH cannot be separated from that of the other phytoplankton cells as you have no data on them. It is the reason for why I said that you cannot conclude on the impact of cyanobacteria on pH and DIC. As you suppress the sentence, it is OK.

Response 12: Thank you for the correction.

Point 13: Comment 28: Thanks for your answer. So, you consider they mainly depend on the level of N and P rather than for instance from the level of organic carbon? Could you let me know what are the current levels of N and P that could limit their growth?

Response 13: Sorry, that was a misunderstanding. C is of course also important for Comamonadaceae. To give an idea of the C,N,P we can refer to a very recent study of a colleague. They did some studies on Comamonadaceae and found out that Comamonadaceae are P limited. Comamonadaceae show higher cell densities, the more P is present in the medium they show a strong homeostasis and the C:P ratio is in the middle range (SRP (stationary): 8.9 µg P/L). The data are not published yet, but we included a table of its stoichiometry to get an idea.

Point 14: Comment 30: Global comment: Thanks for your detailed explanation. You have understood that I suggested to add all of the limitations of your study in the perspective of future complementary works. It is my request for the above-mentioned comments 8, 11, 12, 14, 15, 16. Some are referred to in your line 471-474 sentences, but other are not yet included. I suggest you adjust your sentence accordingly. Thanks in advance.

Response 14: We revised the parts as suggested and added additional missing information for the comments 8, 11, 12, 14, 15, 16. We added the information partly in separate sentences (see also the answers to comments 8, 11, 12, 14, 15, 16) and partly included them in the following sentence which we have adjusted. We hope that we were able to introduce all important points satisfactorily:

“In our study, we were not able in obtain other crucial data such as flow velocity, turbidity, vertical evolution of water quality with depth, ecological functioning of the river, and the seasonal patterns as in dry season processes and pollution concentrations might be different. These complementary factors should be considered in future studies in order to understand ecosystem-level interactions, the ecological risk, and implications for human health.” Line 508-512.

Finally, we are deeply thankful to the reviewer. With her/his in depth comments and broad thinking s/he shaped the manuscript a lot. Additionally s/he provided very interesting views on other related research projects and environmental problems in river systems. Thanks again for her/his time, suggestions and thoughts.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Thanks for your detailed answers and amended paper, and thank you for your data/information on Comamonadaceae.

Most of my comments have been considered. I have only one single remaining request, regarding my comment 11.

I think your sentence is still not clear (lines 386-390). You indicate 3,5 mg PO4/l. As it is indicated, it suggests it is in PO4 not in P, whereas you express that unit in P in your coverletter.

If you effectively have that concentration in P, you should indicate 3,5 mg PO4-P/l.

And similarly for nitrate if it is expressed in mg N/l, you should indicate it in mg NO3-N/l.

Author Response

Response to Reviewer 2 Comments

Round 3

Point 1: Thanks for your detailed answers and amended paper, and thank you for your data/information on Comamonadaceae.

Most of my comments have been considered. I have only one single remaining request, regarding my comment 11.

I think your sentence is still not clear (lines 386-390). You indicate 3,5 mg PO4/l. As it is indicated, it suggests it is in PO4 not in P, whereas you express that unit in P in your coverletter.

If you effectively have that concentration in P, you should indicate 3,5 mg PO4-P/l.

And similarly for nitrate if it is expressed in mg N/l, you should indicate it in mg NO3-N/l.

Response 1: Thank you for this correction. We have now corrected the sentenced as suggested to the following one: “Gowda et al. (2016) found total nitrogen concentrations of 10 mg NO₃ –N L^-1, in average ( 4 to 18 mg L^-1) and total phosphate concentrations of 3.5 mg PO₄ –P L^-1, in average (1 to 7 mg L^-1) in the Mysore plateau of the Cauvery River.” Line 386-88.

Author Response File: Author Response.pdf