Effect of Rainfall and pH on Musty Odor Produced in the Sanbe Reservoir

Round 1

Reviewer 1 Report

The manuscript submitted for review appeared under the same title in Research Square as a preprint (DOI: 10.21203/rs.3.rs-203635/v1) licensed under a CC BY 4.0 License, which has not been completed peer review at a journal („This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints”). The manuscript appears to meet the conditions of “Water” journal to be accepted for publication. (“Water accepts submissions that have previously been made available as preprints provided that they have not undergone peer review”).

The manuscript describes the results of interesting research, the aim of which was to indicate the environmental conditions accompanying the appearance of significant concentrations of geosmin and 2-MIB in the water of the dam reservoir, which are responsible for the unpleasant smell of this water, which matters when this water is used for drinking. This generates increased costs of its treatment.

The authors collected various types of data over a four-year period. They include customary measurements of environmental conditions, but also continuous recording of some parameters, studies of phytoplankton composition and abundance, as well as the cultivation of cultures of selected cyanobacterial species, and, importantly, they presented the iconography of the marked and cultivated species of cyanobacteria. The data set was supplemented by the studies of nucleotide sequences, which finally allowed to identify the taxa responsible for the production of odor substances. Undoubtedly, this collected material must have caused difficulties when trying to arrange an unambiguously clear sequence of the described facts. However, I think that an interesting text of considerable importance eventually emerged, which ends in section 3.5. "Relationship between cyanobacteria and rainfall".

I tried to indicate the places requiring changes in the detailed comments presented below:

Line 16: As these observations were carried out from autumn 2015 to December 2019, and therefore covered only a little more than 4 years, I propose to indicate only the period of these studies (do not write - "five years").

Lines 17-19: I propose, "It was found that geosmin was produced by Dolichospermum crassum ....".

Lines 19-20: Since this influence was indirect, it might be better to write that: "Changes in water temperature and pH caused by rainfall were correlated with changes in the concentration of geosmin and 2-MIB."

Lines 38-39: According to: Izaguirre, G.; Hwang, .J.; Krasner, S,W.; Mcguire, M.J. Geosmin and 2-Methylisoborneol from cyanobacteria in three water supply systems. Applied And Environmental Microbiology, 1982,  43(3): 708-714. - „Geosmin, an earthy-smelling substance, was isolated in 1964 by Gerber and Lechevalier (5). MIB, a musty- or camphorous-smelling compound, was reported in 1969 by Medsker et al. (16) and independently by Rosen et al. in 1970 (21)."

Lines 42-44: I propose: " In 2007, when a high concentration of geosmin occurred at Lake Shinji, producers and wholesalers who harvest and sell of the brackish water bivalve (Corbicula japonica), had difficulty because of the musty odor smell on the bivalve [15,16]."

Line 47-62: I believe this paragraph should be improved. From the current content, it appears that geosmin and 2-MIB is known to be the cause of the undesirable odor of water, but no scientific publication has been provided to state this. Moreover, in the second sentence it was stated that geosmin and 2-MIB are removed with activated carbon, but a publication describing the situation on Kinmen Island in Taiwan (item 18 in the census) and a review publication ("A critical review") were cited to support this. Finally, it is stated that little is known about the relationship between the occurrence of these species and the environmental factors around the reservoir. However, no species in this reservoir have been mentioned so far, so why has it been indicated that this applies to "these species".

Line 72: Why is it indicated that water pH will be discussed in the "around the reservoir" area if only two measuring points (cf. lines 92 and 106) located at the dam itself (Fig. 1) are indicated?

Line 100: Please explain what 'inflow freshwater' means? Is it the sum of rainfall or the volume of water carried into the reservoir by the tributaries?

Lines 100-101: "... and other features from the purification plant ..." - what is behind it?

Lines 114: Why "... brought back ..."?

Lines 125-128: What was the basis of this five point scale? Perhaps, for example, the number or even% of coverage of a microscope slide, or maybe the "relative abundance" was determined only "by eye" (if so, then only one researcher should do it)? The question is justified by the title of the section, announcing the provision of information on "counting of cell numbers for phytoplankton".

Lines 132: Instead of "These phytplankotn" I propose, however, "Isolated specimens of the cyanobacterial trichome ...".

Line 134: A very strange unit of light intensity.

Line 157: The drawings show the change in gesomine content / concentration

Line 158-159 and 160: This exact definition of the depth from which the water samples were taken (reference: Elevation 100 158 m, 1 m above the bottom) should be included in the "Material and methods" section.

Lines 160-161: Are these "a high concentration of musty odor tended to occur in early summer (June to July)", but is it only in these years as for high geosmin concentrations, or each year during the research period? Same question for 2-MIB (lines 164-165).

Lines 173-175: I don't really understand the purpose of Figure 4 and its description. It is based on data from only three depths obtained in five dates (the line for 17 m appears in the diagram in Figure 2 only in April 2017), which may not yet disqualify it, but it certainly does not contribute more information than the graph in Figure 3. Besides, the use of very intense (dark) violet to determine extremely low concentrations of geosmin and 2-MIB (in Figure 5) is contrary to the intuitive perception of such a color (it may mislead the reader). The reference to this figure in the text also does not give any new information with regard to the content contained in lines 166-168. In fact, I have a similar view regarding Figure 5.

In addition. From the diagram in Fig. 3 shows that the course of the line describing changes in the content of 2-MIB at a depth of 17 m presented in Figure 5 indicates a very distant anomaly at this depth in relation to the "normal", i.e. consistent with the expectations, shaping of the geosmin concentration during the 2019 peak year.

Lines 179 and 176: However, I propose to slightly change both pictures, by giving a different format to the numbers on the X axis. In the current version, the description takes up as much space in the drawing as almost 40% of the total variability of the displayed data. Perhaps it is worth replacing the monotonically changing scale "year-month-first day of the month" with a scale covering only the "month". After all, each beginning of a new year is already marked on the chart with vertical dashed lines.

Line 191: What does it mean "although the dominant species did not exist"? After all, even when there is not too much algae in the water, it can usually be indicated that a species dominates / co-dominates. I think it was about the lack of dominance of a species capable of producing geosmin - but it's not the same thing.

Lines 191-192: Were only these two species of cyanobacteria identified at the time? In my opinion, at this point it is more important to indicate that of the species capable of producing geosmin, only Dolichospermum crassum, assessed as rare, has been recorded.

Lines 192-194: Table 1 also shows the presence of Aphanocapsa sp. However, as before, I believe that the most important information is the presence of a species capable of producing 2-MIB.

Lines 194-197: I suggest, similar to above, emphasize the presence of species producing geosmin and 2-MIB more than other species with unknown potential.

Lines 198-200: Did both species mentioned only have these genes in September 2019, or also at other research dates?

Lines 203-213: I suggest you move this paragraph to the beginning of this section (up to line 188). The sentence from lines 207-208 should be moved after the information related to the conducted research (put before the sentence: "The cyanobacteria cf. Geitlerinema sp. and cf. Cuspidothrix sp. ....").

Lines 217-218: The sentence "Dolichospermum and Pseudanabaena were reported as geosmin and 2-MIB producing cyanobacteria, respectively[14]." It is merely a repetition of a sentence from the current lines 207-208, I believe it should be removed.

Lines 201: Table 1. The table does not show the presence of the Pseudanabaena sp. 2 species shown in Figure 6 in the analyzed samples.

If the table shows only species in quantity from "c - abundant" to "rr - very rare",   Why is the "cc - high abundance" mentioned in the "Material and methods" section?

Lines 229-239: Instead of the table, I propose to include a drawing showing the deviations of the average monthly rainfall from the multiannual average (see the file from Figure 6a attached to the review). Please note that in the case of these four years, you can indicate as many as three different types (I assume it is precipitation) of the precipitation abundance cycle in the annual course (I note that I do not study these relationships for this part of the world, I do not know so which of the described types is an anomaly and which is the norm in this region). In the years 2016-2017, two periods are quite clearly visible: wet - when the monthly average of precipitation was greater than the average in the given months from the 4-year period, and dry - when, conversely, the monthly averages were lower. This wet season was from September / October to February and the dry season from March to August / September. The next two years show a completely different type of rainfall. In 2018, from March to September, monthly averages were most often significantly higher than the 4-year averages, while 2019 was exceptionally dry. I think that such a presentation of precipitation anomalies in the analyzed period in this region appeals more to the reader's imagination when it is pointed out that "In 2018, when the concentrations of both geosmin and 2-MIB were low ....", even more so, that the chart shows very clearly this year, higher rainfall was recorded in summer.

Lines 241-245: Graph more accurately than the table and my suggestion for the graph in Fig. 6a, illustrates the situation of the water inflow and the appearance of high concentrations of geosmin in the water of the reservoir. However, it should be clearly stated in the description that the values ​​"14 ng L^{-1 in 2018" and "over 100 ng L-1 in 2016 and 2017" (here, however, the exact values ​​should be specified) are the maximum. I also propose to clearly indicate the days or at least the time period when this "inflow" occurred in May 2018.}

It is also possible to indicate differences in the size of the "inflow" in the period of geosmin occurrence "over 1000 ng L^{-1 in 2016", and even better taking into account also a few days before this period.}

Why do I propose such a description of the situation? Because at the end of June 2016, after a "inflow" of a size comparable to that of 2018, the geosmin of over 1000 ng L^-1 fell to a value below 10 L^-1. However, this fact has not been demonstrated in the text.

Line 247: You must specify what the dashed line in the chart means. The text shows that this is an inflow of 3 m³ s^-1, but the question of what this value results from (please provide the justification in the text in the text).

Lies 249-254: I suggest: “2-MIB concentrations were observed above 10 ng L^-1 from August to December only in 2017 and 2019. They were not detected in 2018, when as many as four tributary peaks above 7 m³ s^-1 were observed from July to October. Based on the results of this analysis on the relationship between precipitation and odorous compounds, it is assumed that the change in the musty smell is influenced by fluctuations in water quality under the influence of the inflow.”

Line 256: In the drawing description, specify what the dashed line in the chart means.

Lines 258-261: I suggest: “The presence of geosmin at a concentration of 10 ng L^-1 or more was observed in a relatively narrow range of water temperature - from 20° to 25°C. 2-MIB in this concentration was recorded in a much wider range of water temperatures - from 10° to 28°C.”

Lines 275: Presentation of the amount of precipitation in the commonly accepted way, i.e. in mm day-1, justifies my earlier request for a more precise explanation of what is actually presented in the graphs Nos. 7 and 8 and in Table 2.

Lines 229-276: I am also wondering about the title of section 3.3. Effect of rainfall on geosmin and 2-MIB, and its content. Two separate issues are clearly discussed here, (1) the amount of precipitation and the concentration of tested substances in the water, as well as (2) the temperature of the water in the reservoir and the amount of rainfall - rather the possibility of cooling the surface water under the influence of rainfall and the ranges of water temperature in which significant concentrations were recorded geosmin and 2-MIB. A very similar range to (2) is found in the next section which describes the effects of pH and temperature on the presence of cyanobacteria. However, please consider whether the content from lines 257-276 should not be moved to section 3.4? I think so, starting with the description and figure 11 in this section, then include the description and figure 10 and finish with figure nr 9 and its description.

Line 297: "for five years (2015–2019)"?

Author Response

Dear Reviewer 1,

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Effect of Rainfall and pH on Musty Odor Produced in the Sanbe Reservoir,” to Water. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted.
Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Authors of the manuscript entitled The "Effect of Rainfall and pH on Musty Odor Produced in the Sanbe Reservoir" presented interesting studies related to the relationship between environmental factors (primarily pH), weather conditions, and cyanobacterial blooms and the associated musty odor from drinking water in the Sanbe reservoir. Authors found that the compounds Geosmin and 2-methylisoborneol (2-MIB) produced by some cyanobacteria caused odor problems. The research methodology adopted in the work is correct. The discussion is interesting, and the final statements in the paper, linking the analyzed problem with low annual rainfall and an increase in pH in the reservoir seem to be justified. Therefore, I believe that the manuscript should be published.

Author Response

Dear Reviewer 2,

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Effect of Rainfall and pH on Musty Odor Produced in the Sanbe Reservoir,” to Water. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Kim et al., presented a study that evaluated the effect of rainfall and pH on musty odor. Samples were collected during five years and two species were found as main contributors of odor in the Sanbe reservoir in Japan

The following comments should be addressed by the authors:

• Line 65. Please add in the caption for figure 1, what represents St. 1. That information was not included.
• Line 52. “…a volume of 2.45x106 m³..). it looks that there is a typo in the volume number (10⁶ instead of 106).
• Line 137. “… by fluorescence microscopy with ethidium bromide staining to confirm axenic strain”. Please provide information about the incubation time, and volume of stain added.
• Figure 2, 3, 7, and 8 did not include visible error bars or standard deviation bars. Please add or make more visible the corresponding bars. In addition specify if the samples were collected and run in duplicates or triplicates.
• Please the make the respective correction for the bicarbonate anion: HCO₃^- instead of HCO₃-
• Can the authors please provide additional information and/or trends about the nutrients (N, P) concentrations found during this study? These data is relevant taking in consideration that the overabundance of these nutrients can promote Harmful cyanobacterial blooms as well.

Author Response

Dear Reviewer 3,

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Effect of Rainfall and pH on Musty Odor Produced in the Sanbe Reservoir,” to Water. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors corrected the text, taking into account all my suggestions. However, re-reading allowed me to spot a few more places that require changes. Moreover, it seems to me that the text requires a thorough revision by an expert linguist before being published.

Detailed comments:

Lines 38-41: The first sentence of lines 38-39 should be combined with the next two. Please note that eg the information "2-MIB was reported in 1969" in two sentences (lines 39 and 40) is identical. Only the publications cited differ. I propose to delete the first sentence and reconcile the quoted items in two new, added sentences.

Line 44: Literature needs to be organized. In the previous paragraph items up to number 17 are cited. Items 20 are cited here. Two items 18 and 19 from the reference list have been omitted.

Lines 49-52: In the current version, I think there is no verb in the subordinate clause regarding geosmin and MIB. Maybe simplify this sentence a bit, e.g. "In Japan, in order to use a reservoir as a water source, when geosmin or 2-MIB is present at a concentration of 10 ng L^-1 or more, ​they must be actively removed in accordance with water quality standards […]".”

Lines 231-235: These two sentences need to be clarified, especially the second sentence. I think, if I figured it out correctly: “In 2018, when both geosmin and 2-MIB concentrations were low (as shown in Figure 3), the annual water inflow to the reservoir was the highest in four years. On the other hand, in 2019, when the average monthly water supply was higher than in September 2016, both geosmin and 2-MIB concentrations were high. "

I admit that I do not know why he refers in this sentence to the average value of September 2016.

Line 236: There is no description of the Y axis in the chart - it seems to be due to the caption, but it can at least specify the unit.

Lines 248: Still no information in drawing caption what the dashed line means.

Lines 240-0242: I propose: "However, the maximum concentration (14 ng L^-1) in 2018 was very low, several times lower than in 2016 and 2017 (over 100 ng L^-1)."

Lines 255-256: I propose: "It is necessary to further investigate whether a inflow of 3 m³ s^-1 day^-1 (and biger) affect the geosmin concentration."

Lines 259: Still no information in drawing caption what the dashed line means.

Lines 255-256: Geosmin? And from the previous sentences it appeared that we are writing about 2-MIB.

Lines 347-354: I believe this paragraph should be in the "Conclusion" section.

Author Response

Dear Reviewer 1

Thank you for your positive comment.

Please see the attachment.

Regards.

Sangyeob Kim 

Author Response File: Author Response.pdf