Antibiotic Resistance in Wastewater and Its Impact on a Receiving River: A Case Study of WWTP Brno-Modřice, Czech Republic

Round 1

Reviewer 1 Report

The paper "Antibiotic resistance in wastewater and its impact on a receiving river: A case study of WWTP Brno-Modřice, Czech Republic" is interesting, in the scope of Water and could be published only after minor revision.

Specific comments to authors:

General - please arrange reference as follows [xx-xx] when citing more than 3 consecutive new papers. Please arrange the references in increasing order, when they are not consecutive (e.g. 3, 7, 42, 90).

Materials and Methods

line 141: please provide the population equivalent (p.e.) instead of the population served. It is more meaningful. Please include the flow rate (inlet or outlet. Please make clear that the WWTP discharges in the Svratka river.

line 143: Instead of presenting the locations of the sampling points with their coordinates, a simplified scheme could be used instead (check Yotova et al. Molecules, 2019, 24 (12):2274) and remove Table S1.

lines 150-158: Please provide the QC measures for avoiding cross-contamination of the samples - procedures for cleaning of and containers used.

lines 140, 230, 248, etc.: Please use the name of the WWTP consistently throughout.

line 204: Please provide reference to the ISO standard (also in the cited literature), regardless you used the translated Chech version.

Results

lines 240 and 242: It will add value to your paper if you add a table with removal efficiencies (in %) for all the studied parameters, instead of only mentioning "significantly lower compared to..." (line 240) and "removal efficiency of individual ARGs varied..." (line 242).

lines 281-284: In the text, you state that "nonsignificant enrichment of surface water for some ARGs were found. This suggests that despite significant reduction in the ARGs during treatment, river downstream of WWTP was probably slightly enriched by ARGs by the treated effluent"... The same comment as above applies to the "significant reduction". On the other hand, one possible explanation of the nonsignificant enrichment could be found if you calculate the loads. The load from the WWTP effluent (concentration x flow rate) should be compared to the load in the river (concentration x flow rate) upstream and downstream (which are probably the same given the distance apart). A similar approach was used in Venelinov et al. Int. J. BIOautomation, 2021, 25 (2): 169-182.

line 333: Please check the sentence - it is only half...

 

Author Response

The paper "Antibiotic resistance in wastewater and its impact on a receiving river: A case study of WWTP Brno-Modřice, Czech Republic" is interesting, in the scope of Water and could be published only after minor revision.

Specific comments to authors:

General - please arrange reference as follows [xx-xx] when citing more than 3 consecutive new papers. Please arrange the references in increasing order, when they are not consecutive (e.g. 3, 7, 42, 90). 

References corrected.

Materials and Methods

line 141: please provide the population equivalent (p.e.) instead of the population served. It is more meaningful.

Information added.

Please include the flow rate (inlet or outlet)

Information added

Please make clear that the WWTP discharges in the Svratka river.

Corrected

line 143: Instead of presenting the locations of the sampling points with their coordinates, a simplified scheme could be used instead (check Yotova et al. Molecules, 2019, 24 (12):2274) and remove Table S1.

Scheme was added, thaks a lot for the sugestion.

lines 150-158: Please provide the QC measures for avoiding cross-contamination of the samples - procedures for cleaning of and containers used.

The informations were added.

lines 140, 230, 248, etc.: Please use the name of the WWTP consistently throughout.

Corrected.

line 204: Please provide reference to the ISO standard (also in the cited literature), regardless you used the translated Chech version.

Reference added.

Results

lines 240 and 242: It will add value to your paper if you add a table with removal efficiencies (in %) for all the studied parameters, instead of only mentioning "significantly lower compared to..." (line 240) and "removal efficiency of individual ARGs varied..." (line 242).

Table was added, thaks for the sugestion.

lines 281-284: In the text, you state that "nonsignificant enrichment of surface water for some ARGs were found. This suggests that despite significant reduction in the ARGs during treatment, river downstream of WWTP was probably slightly enriched by ARGs by the treated effluent"... The same comment as above applies to the "significant reduction". On the other hand, one possible explanation of the nonsignificant enrichment could be found if you calculate the loads. The load from the WWTP effluent (concentration x flow rate) should be compared to the load in the river (concentration x flow rate) upstream and downstream (which are probably the same given the distance apart). A similar approach was used in Venelinov et al. Int. J. BIOautomation, 2021, 25 (2): 169-182.

Yes, you are right, that the table with removal efficiencies would much improve a value of this MS (see included Table with removal effiencies). We decided not to calculate the loads due to extremely fluctuating discharge of the Svratka River during the 2020 sampling campaign.

line 333: Please check the sentence - it is only half..

The part of the sentence was removed.

 

Reviewer 2 Report

General comments:

• The manuscript contains grammatical errors that should be corrected.

Specific comments:

1. Abstract, line 13: Please add the word “antibiotic”, before resistant bacteria (ARB).
2. Lines 114, 115, 122: What is WTTP? Do the authors mean WWTP?
3. Materials and Methods: What was the frequency of sampling from the river?
4. How many times were the molecular biology analyses repeated for a given sample for statistical analysis purposes?
5. Figure 1: There are no black circles (outliers) in this figure. Therefore, the corresponding text can be removed from the bottom of the figure.
6. Figure 6 is not mentioned or discussed in the text. This should be corrected.
7. The statements on lines 296 to 300 are confusing and need clarification. The statement initially says that the abundance of AR-EC in the River water increased on average about 4.5 times from upstream to downstream of the WWTP discharge point to the river. The statement continues by saying that: statistically significant differences between the number of AR-EC upstream and downstream of the WWTP discharge were not found. Do the authors distinguish between “abundance” and “number” of bacteria? Please clarify.
8. Lines 327-329: Does the statement on these lines refer to the River sediments? Please mention the Rive sediments to avoid confusion with the River water.
9. Lines 311 to 313 mention smaller and non-significant differences in ARG concentration between upstream and downstream of the WWTP discharge. However, lines 327-329 state that “the abundance of AR-EC in the Svratka River increased on average about 7.4 times from upstream to downstream of the WWTP discharge point to the river.” Then, lines 329-330 state: statistically significant differences between the number of AR-EC upstream and downstream of the WWTP discharge were not found. Please clarify the following:
   7. Isn’t the 7.4 times increase in the abundance of AR-EC significant? So, why do you state that statistically significant differences between the number of AR-EC upstream and downstream of the WWTP discharge were not found? As mentioned above, is there a difference between the number and abundance of AR-EC? Please describe.
   8. Isn’t the increase of AR-EC and ARG concentration related? Can AR-EC increase without the increase of ARG, or vice-versa, can ARG increase without the increase of ARB? Please add relevant comments to the text of manuscript.
10. Line 358: Should it be “….sampled WWTP effluent?”
11. Please comment on the potential significance of antibiotics concentration in the influent of WWTP (raw wastewater) on bacterial resistance in general, and on the findings of this study.

Author Response

General comments:

• The manuscript contains grammatical errors that should be corrected.

The manuscript was corrected with using Grammarly software.

Specific comments:

• Abstract, line 13: Please add the word “antibiotic”, before resistant bacteria (ARB).

Added according to the suggestions.

• Lines 114, 115, 122: What is WTTP? Do the authors mean WWTP?

Corrected according to the suggestions.

• Materials and Methods: What was the frequency of sampling from the river?

Added according to the suggestions.

• How many times were the molecular biology analyses repeated for a given sample for statistical analysis purposes?

The information was added.

• Figure 1: There are no black circles (outliers) in this figure. Therefore, the corresponding text can be removed from the bottom of the figure.

Corrected.

• Figure 6 is not mentioned or discussed in the text. This should be corrected

Added according to the suggestions.

• The statements on lines 296 to 300 are confusing and need clarification. The statement initially says that the abundance of AR-EC in the River water increased on average about 4.5 times from upstream to downstream of the WWTP discharge point to the river. The statement continues by saying that: statistically significant differences between the number of AR-EC upstream and downstream of the WWTP discharge were not found. Do the authors distinguish between “abundance” and “number” of bacteria? Please clarify.

Within the manuscript we did not distinguish between abundance and number of bacteria, nevertheless we unified this in the manuscript.

• Lines 327-329: Does the statement on these lines refer to the River sediments? Please mention the Rive sediments to avoid confusion with the River water.

Added according to the suggestions.

• Lines 311 to 313 mention smaller and non-significant differences in ARG concentration between upstream and downstream of the WWTP discharge. However, lines 327-329 state that “the abundance of AR-EC in the Svratka River increased on average about 7.4 times from upstream to downstream of the WWTP discharge point to the river.” Then, lines 329-330 state: statistically significant differences between the number of AR-EC upstream and downstream of the WWTP discharge were not found. Please clarify the following:

Isn’t the 7.4 times increase in the abundance of AR-EC significant? So, why do you state that statistically significant differences between the number of AR-EC upstream and downstream of the WWTP discharge were not found? As mentioned above, is there a difference between the number and abundance of AR-EC? Please describe.

We do not deny the difference between abundance of AR-EC upstream and downstream of the WWTP discharge to be significant, however according to the results of statistical analysis (Wilcoxon test), statistical significant difference was not proved (p>0.05). Within the manuscript we did not distinguish between abundance and number of bacteria, nevertheless we unified this in the manuscript.

Isn’t the increase of AR-EC and ARG concentration related? Can AR-EC increase without the increase of ARG, or vice-versa, can ARG increase without the increase of ARB? Please add relevant comments to the text of manuscript.

Although one man expects close relationships between ARGs and AR-EC concentrations (e.g. Du et al. 2019), it is rather difficult to determinate this relationship in real wastewater samples. A main reason is a fact that some ARGs may occur either as intracellular elements inside the bacterial cells (i.e. as a part of intracellular DNA), while some of the as a free extracellular DNA. Since a method we used for detection of the ARGs in our samples was based on filtration and extraction of the only bacterial cell DNA, we have no idea about how much proportion of free ARGs occurred in the surrounding wastewater. Our previous unpublished experiments suggest that ratio of extracellular DNA to intracellular DNA may vary from 1:4 up to 1:12 depending on type of water (clear natural water vs treated wastewater) or time of sampling, for instance.

The comment was added in to the Discussion.

• Line 358: Should it be “….sampled WWTP effluent?”

Corrected .

• Please comment on the potential significance of antibiotics concentration in the influent of WWTP (raw wastewater) on bacterial resistance in general, and on the findings of this study.

Generally, higher antibiotic residues in WWTPs may significantly affect a fate of ARGs in effluents from WWTP. However, some ARGs showed positive correlations with residual concentration of antibiotics, some negative or no significant correlations (e.g. Xu et al. 2015, Wang et al. 2015). Hence, the high antibiotic residues in treated wastewater may influence the proliferation and fate of ARGs and ARB in the effluents and consequently their fate in receiving river. In this study, however, neither antibiotics concentration were measured in a raw wastewater, nor in WWTP effluent, so we cannot evaluate potential significance of the antibiotics on the abundance of both ARGs and ARB in the effluent of a Brno-Modřice WWTP.

The comment was added in to the Discussion.

 

 

Round 2

Reviewer 2 Report

The manuscript has been improved and I recommend the publication of this manuscript.

Author Response

Dear reviewer,

thank you for your revision. We have approved the english in our manuscript in the WriteFull software (see attachement). 

Sincerely,

authors

Author Response File: Author Response.docx