Microcystin Concentrations and Detection of the mcyA Gene in Water Collected from Agricultural, Urban, and Recreational Areas in a Karst Aquifer in the Yucatan Peninsula of Mexico

Round 1

Reviewer 1 Report

The manuscript was well written. I have two comments that could help improve the paper:

1. Out of all the myc genes, why did the author only focus on mycA? Please add justification in the introduction.

2. Could qPCR provide more information than PCR? That way the relative concentration of mycA could be correlated with MC concentration and other parameters?

Author Response

Thanks for the review on our manuscript. We have addressed the specific comments and below we provide a list of responses to each comment. All revisions and edits were made in the revised main manuscript document and are highlighted in yellow. We hope you find our revised version of the manuscript and our response to queries satisfactory.

1. Out of all the myc genes, why did the author only focus on mycA? Please add justification in the introduction.

RESPONSE: The mcyA gene was selected because in addition to be useful for the detection of toxin producing cyanobacteria, mcyA gene sequences can be used for the selective distinction of various microcystin producing cyanobacteria in field and cultivated samples (Hisbergues et al., 2003 Reference 29). Even though different mcy genes (mcyA, mcyB, mcyE, and mcyT) and intergenic regions (mcyCJ, mcyEG, mcyHA, and mcyTD can be used, it is necessary to validate their usability in detecting MC producing strains in environmental samples (Mbedi et al., 2005). Therefore, we decided to use the mcyA gene for this study, which is one of the few that have been conducted in the Yucatan aquifer to determine the presence of microcystin, Microcystis and mcyA gene.

The following sentence was added in the introduction section as justification: “The latter PCR assay has allowed the identification of various cyanobacteria toxin producing strains and genus, including Microcystis, Dolichospermum (formerly Anabaena), and Nostoc [29], all of which are commonly present in the Yucatan peninsula aquifer [34-36], suggesting that this assay can be used in environmental samples in this aquifer.”

2. Could qPCR provide more information than PCR? That way the relative concentration of mycA could be correlated with MC concentration and other parameters?

RESPONSE: It is correct, qPCR can provide mor information than PCR regarding the concentration or the number of copies of the mcyA gene. However, in this study we focused on the identification of toxin producing cyanobacteria based in PCR amplified mcyA gene fragments, in addition, we used mcyA gene sequences to conduct a phylogenetic reconstruction, all of which was possible through the cloning and sequencing of PCR amplicons.  Moreover, since it has been demonstrated that not always the presence of toxin genes is correlated with the presence of microcystins  in a lake environment [reference 2], we believe that it would be very difficult to correlate the concentration of mcyA gene with the concentration of MC under the current experimental design. A particular study should be conducted to determine if the mcyA gene abundance can be correlated with MC in this karst environment.

Reviewer 2 Report

The aim of this study was to explore the occurrence and concentrations of microcystin and the mcyA gene in water bodies in Yucatan peninsula of Mexico. Water quality was determined by physicochemical, hydrochemical variables and chlorophyll-a (Chl-a). The study has important results because is a baseline about the water quality with the presence of microcystins producing cyanobacteria in different degrees in water urban, agricultural and recreational areas. I have only few comments about the manuscript.

In the abstract Line 31: I suggesting adding that no correlation with the presence of the mcyA gene and microcystins were found. Therefore, a positive correlation was detected between microcystin concentrations with pH and Chl-a.

Line 61-62: “healthy adults”. They were patient in hemodialysis.

Table 3: suggestion: to add the statistics and standard error of each point.

Line 407-422: I suggest a discussing about the permit limit of the legislation for Microcystin in water in Mexico.

Line 411-415:  Is there any study about the risk of the presence of cyanotoxins in this aquifer for human or animal health?

Depends on the literature about risk assessment it is better to write as in conclusion: “ .. indicate a potential health threat to humans and animals.

Author Response

Thanks for the review on our manuscript. We have addressed the specific comments and below we provide a list of responses to each comment. All revisions and edits were made in the revised main manuscript document and are highlighted in yellow. We hope you find our revised version of the manuscript and our response to queries made by the reviewers satisfactory.

In the abstract Line 31: I suggesting adding that no correlation with the presence of the mcyA gene and microcystins were found. Therefore, a positive correlation was detected between microcystin concentrations with pH and Chl-a.

RESPONSE: The suggestion was added in the abstract section as follows: “No correlation with the presence of the mcyA gene and microcystins was found, however, a positive correlation was detected between microcystin concentrations with pH and Chl-a.”

Line 61-62: “healthy adults”. They were patient in hemodialysis.

RESPONSE: The correction was made and “patients in a hemodialysis unit” was used instead of “healthy adults”.

Table 3: suggestion: to add the statistics and standard error of each point.

RESPONSE: In order to follow the reviewer´s suggestion, Table 3 was modified by adding the standard deviation (S.D.), and the standard error (S.E.). An asterisk was added to identify the values below the limit of detection (LoD). The following footnote was added: “*=Below limit of detection (LoD); S.D.= Standard deviation; S.E. = Standard Error.”. In addition, we noticed that a correction needed to be made for P-PO43- mg/L value found in site A6, where the correct value was 2.5, the average was corrected accordingly to 0.78.

Line 407-422: I suggest a discussing about the permit limit of the legislation for Microcystin in water in Mexico.

RESPONSE: In order to discuss about the permit limit of the legislation in Mexico we added the following sentence in line 413 of the discussion section “…. which exceeds the limit established by water quality standards for drinking water in Mexico of 1 μg/L (PROY-NOM-127-SSA1-2017) [20].”

Line 411-415:  Is there any study about the risk of the presence of cyanotoxins in this aquifer for human or animal health? Depends on the literature about risk assessment it is better to write as in conclusion: “ .. indicate a potential health threat to humans and animals.

RESPONSE: Even though Microcystis and other toxin producing cyanobacteria have been reported in the aquifer, very little is known about the risk of the presence of cyanotoxins in human or animal health. We followed the observation and modified accordingly using the sentence “The latter might indicate a potential health threat to humans and animals since microcystin concentrations in the karst aquifer….” instead of “The latter might represent a potential human and animal health risk and since microcystin concentrations in the karst aquifer ...”.

Reviewer 3 Report

Microcystin concentrations and the detection of the mcyA gene…

This a well presented manuscript. My suggestions are with regard to two aspects-

1.     Is the water from the study sites used for human consumption? If not, why should it have <1µg/L of Microcystin?

2.     Although several studies have been conducted on the microcystin concentrations in freshwaters in Mexico (Alillo Sanchez et al., 2014; Zamora Barrios et al., 2015, 2017, 2019), these have not been used for comparisons in the discussion.

Comments for author File: Comments.pdf

There are minor mistakes in English that should be corrected.

Author Response

Thanks for the review on our manuscript. We have addressed the specific comments and below we provide a list of responses to each comment. All revisions and edits were made in the revised main manuscript document and are highlighted in yellow. We hope you find our revised version of the manuscript and our response to queries satisfactory.

1. Is the water from the study sites used for human consumption? If not, why should it have <1µg/L of Microcystin?

RESPONSE: Groundwater (that fills cenotes or sinkholes) in the Yucatan peninsula karst aquifer is the only source of freshwater in the area. Cenotes are considered as potential drinking water sources and are commonly used for recreational activities (Munro et al., 2011) and even though most of the population in the area do not drink the water directly from the cenotes, in rural areas they are the only source for freshwater when piped water is not available or when extreme climatic events (hurricanes) occur.

2. Although several studies have been conducted on the microcystin concentrations in freshwaters in Mexico (Alillo Sanchez et al., 2014; Zamora Barrios et al., 2015, 2017, 2019), these have not been used for comparisons in the discussion.

RESPONSE: We appreciate the suggestion of references to strengthen the comparison with what has been reported in Mexico in the discussion section.  We added the following sentence in lines 429-430 in the discussion section: “…..in concentrations of >1 μg/L in drinking water sources [69], and in concentrations ranging from 0.2 and 2.4 μg/L in lake water [70].”.  We added two from the four references suggested, since we considered them the most comparable with the results presented here. We added references 69 and 79 and made the appropriate corrections in the text and in the reference section.

69. Alillo-Sánchez, J.L.; Gaytán-Herrera, M.L; Martínez-Almeida, V.M.; Ramírez-García, P. Microcystin-LR equivalents and their correlation with Anabaena in the main reservoir of a hydraulic system of Central Mexico. Inland Waters 2014, 4, (3), 327-336 https://doi.org/10.5268/IW-4.3.573
70. Zamora-Barrios, C.A.; Nandini, S.; Sarma, S.S.S. Effect of crude extracts from cyanobacterial blooms in Lake Texcoco (Mexico) on the population growth of Brachionus calyciflorus (Rotifera). Toxicon 2017, 139, 45-53. https://doi.org/10.1016/j.toxicon.2017.09.013.

We also used the pdf from the reviewer to improve the revised manuscript. In addition to the queries above, to improve the quality of the manuscript we detected the following errors and corrected accordingly:

LINE 320: The use of “depth” instead of “depht”

LINE 321: The use of “highest” instead of “hithest”