Long-Term Study of Antibiotic Presence in Ebro River Basin (Spain): Identification of the Emission Sources

Round 1

Reviewer 1 Report

As for my previous comments, the draft would be improved. But, please remove quotation mark in the end of line 304.

Author Response

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Reviewer 2 Report

MATERIAL AND METHODS

• Statistical analysis: What bibliographic support did the authors rely on to use a 10% significance level.

RESULTS

• Results: where "significant at p<0.05 and p<0.01" is: "p-value<0.01", since the p-value is 0.008957.
• also in table 5, it should be "p-value" instead of "p".
• The formatting of the values in tables 4 and 5, as I believe they can be presented with a smaller number of decimal places.
• The legend of table 5 must be completed, since it is not clear what the "M" and "Q" mean.
• Line 213: the authors refer: “Tables 4-5 present the ANOVA and Turkey’s HSD test results in high-pressure live- 213 stock sampling points. Significant differences (p-value<0.1)”. » This is not entirely correct, as for Table 4, the results are presented with a significance level of 1%. On the other hand, it is not clear what justifies the use of the 10% significance level for a particular situation. The significance with which we work must not be adapted to what suits...
• Line 221: the authors refer: “ANOVA and Turkey’s HSD test results in high-pressure WWTPs sampling points 221 showed that there is not a significant correlation between this kind of pressure and the 222 concentration of individual antibiotics in rivers.” » This is not correct, as neither ANOVA nor Tukey's test of multiple comparisons assess the existence of correlations. These tests assess the existence (or not) of statistically significant differences. Authors should clarify and/or review the analysis that was performed.
• Figure 6 has the wrong caption, as the authors indicate that “x” is the “X=arithmetic average marker”, which is really strange, since the last segment of the boxplot is always the maximum value of the sample. Authors must correct the legend/graphic.
• Line 302: the authors refer: “ANOVA test confirms that there is not a significant correlation between the river flow and antibiotic concentration” » » This is not correct, as ANOVA doesn’t evaluates the existence of correlations. This test assesses the existence (or not) of statistically significant differences. Authors should clarify and/or review the analysis that was performed.
• Line 304: there are extra quotes at the end of the sentence.
• The same comments that were made for tables 4 and 5 should be taken into account for tables 6 and 7.
• The location of tables 4, 5, 6 and 7 should be reviewed as they appear before the text where they are cited.
• The same comments that were made for figure 6 should be taken into account for figures 7 and 8.
• Lines 341-344: the authors refer: “Correlating these results with those obtained for surface water samples, where enrofloxacin concentration was relatively high in comparison with other studies [11], [57], the presence of this antibiotic could be definitively associated to intensive farming.”. The authors indicated a correlation and an association. However, statistical analysis is presented. Thus, the authors either redo the text or present the statistical analysis.

Author Response

Find attached the reply, thank you for your comments.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript by Moles and co-authors deals with an important issue related to water pollution, such as the presence of antibiotics in surface waters and wastewaters in relation to urban discharges and intensive farming. I think that the data presented might be of general interest. However, the ms contains some major flaws that should be corrected before being published.

The data presented in the supporting tables show relevant fluctuations in the levels of the drugs analysed between the different sampling carried out in the same sites. This is not made clear in the main text nor even discussed. It is not clear how the authors elaborated the data of the different samplings, which are presented in the charts.

According to pressure identified in the different sites, D19 might be considered as a reference site (even though high levels of enrofloxacin was observed), how do the concentrations of antibiotics vary here compared to other sites?

I am concerned about statistical analysis. ANOVA is not a correlation analysis, so I wonder how authors established correlations and if they made this analysis. The use of p value <0.1 as threshold for significance is unusual, so this choice should be explained providing references.

What is the relevance of comparing the levels of the different drugs? I do not understand the rationale beyond the ANOVA and the Tukey analysis. I would recommend to compare the different sampling points rather than the antibiotics.

I suggest to add also the position of WWTPs sites in the maps showed in fig. 3 and 4.

Author Response

Find attached the reply to your comments, they were very useful, thank you.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Authors improved the manuscript I think that now it can be published after careful check for some editing errors throughout the ms.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors measured four antibiotics in a watershed with active livestock farming. There are some major concerns as described below.

1. Discussions on sources of the antibiotics (e.g., lines 209-274) were not scientific. At least, the authors have to characterize the sampling sites (Table 1) by livestock numbers (Figure 3 & 4) and STP locations (not shown in the draft).
2. Though identification of presence and sources of antibiotics was the main objective of this study (lines 74-75), the authors did not discuss any progress from this viewpoint. At least, they need to compare their outcome with major relevant reports in the world. Though they described “increasing presence of antibiotics” by referring some reports on the same basin (lines 369-371), scheme of this study (i.e., samplings conducted only once or twice a year without obtaining any river flow data) is not strong enough to discuss annual change. In addition, I could not find any “systematic tracing” from this draft, though “systematic tracing” is likely the motivation of this study (line 73).
3. Because there are many reports on the fate of antibiotics in WWTPs, several times spot samplings of four well-known antibiotics in three WWTPs conducted by the authors would be not enough to advance our understanding on the fate of antibiotics in WWTPs. In addition, the authors seem to have already published the same dataset on the three WWTPs (Figure 8 in this draft would be same as Figure 4 in ref. 8).

Author Response

Find attached the pdf with the reponse to your comments. We appreciate your comments and we think we have improved the paper as you have suggested in the first revision.

Author Response File: Author Response.pdf

Reviewer 2 Report

LANGUAGE

I understand that writing in English is not an easy task for non-English native authors. Thus, the manuscript should consequently be corrected by an English proof reader. 

ABSTRACT

• The abstract must be improved/completed, as there is a lack of information on the main results achieved with this study.

MATERIALS AND METHODS

• Table 2 has some header elements cut off; must be corrected.

RESULTS

• Section 3 (3.1 and 3.2) begins with a figure. This should be changed, as a section should never start with a figure and/or table (in a scientific document this should not happen).
• What does the symbol "x" in Figure 6a and 7a represents? This information must be included in the caption.
• There is no interpretation of the results shown in Figure 6 a and b. If so, then what is the point of including Figure 6?
• On lines 206-208 is the following information: “In order to complement the statistic presentation of this research, Tables S1-S4 list quantitative antibiotic concentration results obtained during the 6 sampling campaigns carried out”. However, in the materials and methods section there is no information on statistical analysis. The authors have to clarify this aspect.
• Line 219: the authors refer: “As revealed in Figure 6, the Aragón river sub-basin presented an average concentration of 147 ng/l of enrofloxacin”. Do authors refer to figure 6a or 6b? If it is, the 6a, then the interpretation is not correct, as a boxplot does not give information about the average values. Authors should clarify.
• Line 280: the authors refer: “Concerning average concentration results obtained for antibiotics in wastewater, which are shown in Figure 7, the macrolide azithromycin presented the highest average levels.”. Do authors refer to figure 7a or 7b? If it is, the 7a, then the interpretation is not correct, as a boxplot does not give information about the average values. Authors should clarify.
• Line 290: “Nevertheless, as revealed in Figure 7, azithromycin was also present in smaller urban 290 areas, such as WWTP3. The average concentration of this antibiotic in WWTP3 was higher 291 than 5,000 ng/l.” » idem
• Line 286: the authors refer: “These results are significantly superior to the ones reported in literature for other WWTPs located in Ebro river basin ten years before”. However, the authors cannot indicate that the results are significantly superior, as no inferential statistical analysis is presented.
• Figure 8 should be clarified, as there is no information about what the error bars represent. On the other hand, if the values are in %, as indicated on the vertical axis, then displaying error bars is meaningless. Authors have to clarify/correct the figure.
• Line 308: the authors refer: “Enrofloxacin also presented a significant removal efficiency after the treatment, up to the total suppression.”. Again, authors cannot indicate that the results are significant efficiency, since no inferential statistical analysis is presented. Thus, the authors either redo the text or present the statistical analysis.
• What does the symbol "x" in Figure 9a represent? And the symbol “o”? This information must be included in the caption.
• Line 330: the authors refer: “According to the graph. Do authors refer to figure 9a or 9b?
• Lines 337-339: the authors refer: “Compared to the rest of the studied slaughterhouses, only the duck one, where concentrations exceeding 1,500 ng/l were detected, presented a significant potential emission of azithromycin.”. Again, authors cannot indicate that the results presented a significant potential, since no inferential statistical analysis is presented. Thus, the authors either redo the text or present the statistical analysis.
• Lines 341-344: the authors refer: “Correlating these results with those obtained for surface water samples, where enrofloxacin concentration was relatively high in comparison with other studies [11], [57], the presence of this antibiotic could be definitively associated to intensive farming.”. The authors indicated a correlation and an association. However, statistical analysis is presented. Thus, the authors either redo the text or present the statistical analysis.

SUPPLEMENTARY MATERIAL

• In tables S1, S2, S3 and S4 the legend must be completed, as there is no indication of the meaning of "D" and "n/d".

Author Response

Thank for yor comments,

Find attached the pdf with a concrete reponse to all your comments, thanks for being precise with your comments. We have improved english language and style and also you will se all your comments reponses in the document.

Regards,

SM.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

1. I cannot understand what the authors want to say in discussion on seasonal trend (lines 333-336). Average or range of concentrations of enrofloxacin and sulfadiazine in each season should be described in lines 335-336. According to Figure S1, river flow rate sometimes increases in both the spring and autumn, so I cannot understand which is “rainier season (line 336)”? Or the authors want to say higher concentration in higher flow "day (not season)" for the two antibiotics for veterinary use? As for line 334-335, “the concentration of selected antibiotics is quite similar” between what?

2. The authors should not use the word “WWTPs” in Figure 8. They should describe like this, “Figure 8. (a) Boxplots (X= arithmetic average marker) and (b) average concentration (ng/l) of target antibiotics in a hospital and three slaughterhouse effluents.” In addition, the author should describe how many times they took the effluents samples here or method part.

3. The authors should spell out “ASE” in the footnote of Table 1. In addition, I recommend they to specify the reference of livestock and WWTP pressures not only in line 120, but also in the footnote of Table 1.

4. Please remove or modify “amoxicillin” in line 248.

5. Maybe “groundwaters, and treated and untreated wastewaters” in line 76-77.

Author Response

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Reviewer 2 Report

The authors continue to insist on presenting average values in the boxplot. This is not correct, as a boxplot is a graph composed of maximum value, minimum value, quartiles and median. Additionally, the authors associate descriptive (and fully exploratory) analysis as being statistical analysis of data, which cannot be. 

Author Response

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