A Case Study on Factors Influencing Escherichia coli Concentrations in an Urban River Draining a Fully Sewered Area
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsSuggestions and comments are inserted in the PDF file, which is attached for your reference.
Comments for author File: 
Comments.pdf
Author Response
Reply to reviewer 1
Comment 1) Abbreviate at first appearance on line 37.
Reply 1: The text has been revised into “Escherichia coli” when it appears at the first time in the introduction section.
Comment 2) This map needs further information. First insert a N sign. Also in text is says locations for sampling, however in map only one location is shown. Also add what are these lines indicate. geographical coordinates are also missing.
Reply 2: The north sign and the legends have been added to Figure 1. In addition, geographical coordinates (N 35.50, E139.50 and E139.60) were drawn in the map. “Sampling points” in the text was revised into “Sampling point (Singular form)”.
Comment 3) In case of replicated data it is good to add error bars as to Figure 2.
Reply 3: The monitoring by MLIT is conducted as a monthly routine spot monitoring. Consequently, error bars cannot be added to the figure. Instead, we changed the figure caption and wrote “Monthly spot monitoring” to be more clear.
Comment 4) I would suggest to add correlation between E. coli and resistant ratio to CTX in figure 3.
Reply 4: The significant correlation was not observed in this study. Following sentence has been added to the figure caption to be clearer. “A negative insignificant correlation (r=-0.15, n=29) was found.” These values were also mentioned in the revised main text.
Comment 5) Mention what do dotted and solid lines indicate in figure 4.
Reply 5: All lines were drawn with solid lines in the revised manuscript. In the figure caption, a sentence was added “smoothed curves were drawn to connect the data points”.
6) Conclusion section can be added.
Reply 6: The authors added the conclusion section, as shown below.
- Conclusion
This study presents a case study on the factors influencing E. coli concentrations in an urban river draining a fully sewered area. An approximately 70-fold higher concentration than the average dry-weather level obtained from governmental routine monitoring (1.9 CFU/mL) was observed under wet-weather conditions, probably due to the effects of combined sewer overflows. Very short survival of E. coli (less than one day) was expected in the unfiltered overlying water, likely due to the effects of bacteriophages, protozoan predation, and bacterial competition, whereas longer survival was expected in the sediment. A considerable fraction of E. coli in the overlying water on dry-weather days was probably supplied from the resuspension of the sediment. Highly variable antimicrobial resistance among E. coli populations under dry-weather conditions was possibly caused by the regrowth of a limited number of E. coli individuals in the sediment. Rising temperatures associated with global warming are expected to decrease E. coli concentrations in the target watershed, where E. coli populations are considered to be strongly suppressed by predation and competition. Some of the characteristics of the watershed may be attributed to the fact that treated wastewater accounted for approximately 75% of the dry-weather total flow.
(Other Corrections)
Minor language corrections (“dry-weather” and “wet-weather”) have been made. Several mistypes (like “figure 1” to “figure 4”) has been corrected. The limitations of this study are discussed in the revised manuscript to comply with the suggestions of the other reviewer.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study investigates temporal and environmental factors affecting E. coli concentrations in the Tsurumi River, Japan. Despite full sewer coverage, E. coli levels were observed to increase approximately 70-fold during wet weather, indicating significant influence from combined sewer overflows (CSOs). The findings highlight how rainfall events, sediment resuspension, and temperature significantly govern E. coli behaviour in urban rivers, with potential implications for public health monitoring under climate change.
The research has many limitations listed below, please address carefully:
The author is linking shorter survival of E. coli to global warming without providing any evidence.
Data were collected from one river section near Kamenoko Bridge, limiting the spatial representativeness of results across urban watersheds.
Sampling occurred roughly once per month, which may miss short-term variability during rainfall or CSO events.
While decay rate and temperature were studied, other influential factors such as UV intensity, dissolved organic matter, and nutrient levels were not comprehensively measured.
The study reports decay and concentration changes descriptively but does not employ statistical or hydrological modelling to generalize decay kinetics or predict CSO impacts.
The link between E. coli concentration and CTX resistance was observational and lacked genotypic characterization of resistant strains to confirm sources (human vs. animal).
Wet-weather data may not fully reflect storm variability due to limited rain events captured within sampling periods.
I suggest a conclusion section to summarise the fining and potential future work.
Author Response
Reply to reviewer 2
<General comment>
This study investigates temporal and environmental factors affecting E. coli concentrations in the Tsurumi River, Japan. Despite full sewer coverage, E. coli levels were observed to increase approximately 70-fold during wet weather, indicating significant influence from combined sewer overflows (CSOs). The findings highlight how rainfall events, sediment resuspension, and temperature significantly govern E. coli behaviour in urban rivers, with potential implications for public health monitoring under climate change.
The research has many limitations listed below, please address carefully:
Reply: Thank you for the comments by the reviewer. The authors carefully considered the comments and revised as follows.
<Specific comments>
Comment 1) The author is linking shorter survival of E. coli to global warming without providing any evidence.
Reply 1: The manuscript has been revised to emphasize this study is a case study on the target region. In the revised abstract and in the newly provided conclusion, the authors mentioned this conclusion can be applied only to the target watershed where E. coli populations are strongly suppressed by predation and competition. In addition, the authors mentioned that some of the characteristics of the watershed may be attributed to the fact that treated wastewater accounted for approximately 75% of the dry-weather total flow. Relating this, sections “4.2. Effect of temperature on survival of pathogens” and “4.3. Importance of biological factor to the decay rate” were revised to keep the consistency with the revised conclusion.
Comment 2) Data were collected from one river section near Kamenoko Bridge, limiting the spatial representativeness of results across urban watersheds.
Reply 2: The authors understand the reviewer’s comment on the limitation of the sampling point. In the revised manuscript, the authors emphasized in the abstract and in newly added conclusion that the study is a case study to identify the influencing factors on the dry-weather concentration of E. coli. In addition, the sampling strategy was added in the section of “2.1. Sample Origins” to make the readers smoothly understand the objective of this study.
Comment 3) Sampling occurred roughly once per month, which may miss short-term variability during rainfall or CSO events.
Reply 3: Although the authors understand that time-resolution is an important factor to understand CSO events, the main target of this study is E. coli population on dry-weather conditions. In the revised manuscript, we wrote in the conclusion “Highly variable antimicrobial resistance among E. coli populations under dry-weather conditions was possibly caused by the regrowth of a limited number of E. coli individuals in the sediment” to link the dry-weather E. coli population with wet-weather population.
Comment 4) While decay rate and temperature were studied, other influential factors such as UV intensity, dissolved organic matter, and nutrient levels were not comprehensively measured.
Reply 4: The authors understand that the E. coli decay rates were significantly affected by UV intensity and salt concentrations especially in urban environments with characteristics of relatively long hydraulic retention time and estuarine zone. However, a slightly different situation can be considered in our sampling site. The following sentences were added in the section “4.3. Importance of biological factor to the decay rate”.
Although higher salinity and stronger sunlight (UV) intensity have been shown to accelerate the decay rate [16], they may have only minor effects in the target sampling site which is characterized by complete freshwater with a short hydraulic retention time, because E. coli populations at the sampling site are considered to be strongly suppressed by predation and competition. One possible reason for the short survival of E. coli in overlying water is the situation that treated wastewater accounted for approximately 75% of the dry-weather total flow at the sampling site.
Comment 5) The study reports decay and concentration changes descriptively but does not employ statistical or hydrological modelling to generalize decay kinetics or predict CSO impacts.
Reply 5: The authors understand that to generalize the conclusion in this study, statistical and hydrological modelling is needed. However, the focus of this study is to identify the possible influencing factors on E. coli population on dry-weather conditions. In the section of “4.2. Effect of temperature on survival of pathogens”, the authors added “Note that this discussion is based solely on the decay rates obtained in this study. Hydrological modeling incorporating bacterial source tracking [16,36] is required to distinguish between wildlife and human contributions in a more generalized discussion on temperature-dependent change.” I hope these revisions would be appropriate to satisfy the reviewer’s comment.
Comment 6) The link between E. coli concentration and CTX resistance was observational and lacked genotypic characterization of resistant strains to confirm sources (human vs. animal).
Reply 6: The authors understand the limitation of this work. The addition of genotyping analysis (source tracking of E. coli) may strengthen the conclusion of this research. However, the authors thinks that the resistant ratio to CTX is one of the indicators to distinguish between wildlife and human contributions among E. coli population, as presented in “3.2. Susceptibility of the isolates to antimicrobials”. To show the limitation, the necessity for the source tracking is added at the end section of “4.2. Effect of temperature on survival of pathogens”.
Comment 7) Wet-weather data may not fully reflect storm variability due to limited rain events captured within sampling periods.
Reply 7: To clarify the objective of this study, we added in the section of “2.1. Sample Origins” following sentence. “Both dry-weather and wet-weather days were selected as sampling dates to investigate the residual effects of CSOs on the concentration and antimicrobial resistance profiles of E. coli.” to make the readers smoothly understand the objective of this study.
Comment 8) I suggest a conclusion section to summarize the finding and potential future work.
Reply 8: The authors added the conclusion section, as shown below.
５．Conclusion. This study presents a case study on the factors influencing E. coli concentrations in an urban river draining a fully sewered area. An approximately 70-fold higher concentration than the average dry-weather level obtained from governmental routine monitoring (1.9 CFU/mL) was observed under wet-weather conditions, probably due to the effects of combined sewer overflows. Very short survival of E. coli (less than one day) was expected in the unfiltered overlying water, likely due to the effects of bacteriophages, protozoan predation, and bacterial competition, whereas longer survival was expected in the sediment. A considerable fraction of E. coli in the overlying water on dry-weather days was probably supplied from the resuspension of the sediment. Highly variable antimicrobial resistance among E. coli populations under dry-weather conditions was possibly caused by the regrowth of a limited number of E. coli individuals in the sediment. Rising temperatures associated with global warming are expected to decrease E. coli concentrations in the target watershed, where E. coli populations are considered to be strongly suppressed by predation and competition. Some of the characteristics of the watershed may be attributed to the fact that treated wastewater accounted for approximately 75% of the dry-weather total flow.
(Other Corrections)
Minor language corrections (“dry-weather” and “wet-weather”) have been made. Several mistypes (like “figure 1” to “figure 4”) has been corrected. The captions of the figures and the appearances of the figures were improved in the revised manuscript to comply with the suggestions of the other reviewer.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAccept, the authors have responded sufficiently to reviewers comments.
