Are Sterols Useful for the Identification of Sources of Faecal Contamination in Shellfish? A Case Study

Round 1

Reviewer 1 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. 

MATERIALS AND METHODS

- Figure 1 should be mentioned in the section 2.1 (Description of the Sampling Area)

- In the section “2.6. Statistical Analyses”:

- the information about the software used must be information placed at the end of this section;

- the phrase “and statistical significance was determined by either the t-test or analysis of variance (ANOVA) and Tukey test” is not understood, as it is not clear with what purpose each of these statistical tests was used (what was it to study?). On the other hand, it remains to be clarified whether the requirements for the implementation of the methods have been met or not (given that t-test or analysis of variance (ANOVA) are parametric statistical tests and have to validate the necessary requirements for the execution). In addition, it remains to be reported whether the analysis of variance was with one or more factors; additionally, it remains to indicate under which conditions the Tukey test was performed;

- it is necessary to clarify what the authors intend to expose when they say “several correlation tests were carried out”; on the other hand, in this sentence the information about the software used is repeated;

- it remains to be indicated what level of significance was used in the statistical analysis of the data.

RESULTS AND DISCUSSION

- in the legend of figure 2 it remains to indicate what the line segments represented in the bars represent;

the results (whenever applicable) must be presented in the form mean± standard deviation; this information must also appear in the section “2.6. Statistical Analyses”

- in table 2 it is not clear why the data for November and December are underlined;

- having in the discussion phrases that start as follows: “[9] carried 167 out sterol analyses of (…)” or “[26]also measured the concentrations of 170 coprostanol in waters of the Clyde Estuary (…)” is not suitable (unless it is a requirement of the journal);

- on page 10, line 256, the authors begin to present results in the form (for example) “(5.08 ± 5.07%)”, but nowhere is it explained what this means (is the mean± standard deviation?) (the same happens in tables 5 and 7);

- when the authors refer: “As expected, as oysters are filter feeders Pearson correlation analysis (Table 6) confirmed a significant correlation”, it remains to be indicated what the p-value is (neither in the material and methods the level of significance is mentioned for which the results are considered statistically significant);

- table 6 does not indicate what “*” and “**” mean;

- nowhere in the manuscript (specifically, in the results and discussion section) are the results of the statistical tests t-test or analysis of variance or Tukey's test described (very poorly correct) in the material and methods section (“2.6. Statistical Analyzes”). Thus, it is impossible to understand what these statistical tests were used for, or for what purpose; the values of the test statistics, the p-value and whether statistically significant effects are evident must be evident in the text;

- additionally, since the investigation allows having a data with 4 locations, in 12 months, for concentrations of 11 major sterols, then the data analysis should be complemented with a multivariate statistical analysis, namely a principal component analysis (for example).

Author Response

Reviewer 1

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. 

Reply: As suggested by the reviewer, the manuscript has now been proofed and corrected by an English proof-reader.

MATERIALS AND METHODS

- Figure 1 should be mentioned in the section 2.1 (Description of the Sampling Area)

Reply: Figure 1 has now been cited in Section 2.1 as requested. (Page 2, Line 100)

- In the section “2.6. Statistical Analyses”:

- the information about the software used must be information placed at the end of this section;

- the phrase “and statistical significance was determined by either the t-test or analysis of variance (ANOVA) and Tukey test” is not understood, as it is not clear with what purpose each of these statistical tests was used (what was it to study?). On the other hand, it remains to be clarified whether the requirements for the implementation of the methods have been met or not (given that t-test or analysis of variance (ANOVA) are parametric statistical tests and have to validate the necessary requirements for the execution). In addition, it remains to be reported whether the analysis of variance was with one or more factors; additionally, it remains to indicate under which conditions the Tukey test was performed;

- it is necessary to clarify what the authors intend to expose when they say “several correlation tests were carried out”; on the other hand, in this sentence the information about the software used is repeated;

- it remains to be indicated what level of significance was used in the statistical analysis of the data.

Reply: We thank the reviewer for their considered suggestion. We have now taken on board the comments and rewritten the statistics section which now reads

” Data were analysed with IMB SPSS (version 24) Analysis of Variance (ANOVA; p=0.05). The means were separated using Tukey test (p=0.05), where the F-value was significant. To examine whether there was any significant association between the concentrations of faecal sterols (coprostanol and 24-ethylcoprostanol and coprostanol + 24-ethylcoprostanol), several correlation tests were carried out using SPSS version 24. Results are presented as mean ± standard deviation unless otherwise stated. “ Page 4, Lines 140-147”.

RESULTS AND DISCUSSION

- in the legend of figure 2 it remains to indicate what the line segments represented in the bars represent;

Reply: We apologise for the oversight. The requested information has been added to the legend of Figure 2.

the results (whenever applicable) must be presented in the form mean± standard deviation; this information must also appear in the section “2.6. Statistical Analyses”

Reply: As requested, the results (whenever applicable) are presented in the form mean ± standard deviation; this information also appears in the section “2.6. Statistical Analyses”.

- in table 2 it is not clear why the data for November and December are underlined;

We apologise for the oversight; we have removed the underlining.

- having in the discussion phrases that start as follows: “[9] carried 167 out sterol analyses of (…)” or “[26]also measured the concentrations of 170 coprostanol in waters of the Clyde Estuary (…)” is not suitable (unless it is a requirement of the journal);

Reply: We apologise for the oversight; the references in the texts have now been fixed.

- on page 10, line 256, the authors begin to present results in the form (for example) “(5.08 ± 5.07%)”, but nowhere is it explained what this means (is the mean± standard deviation?) (the same happens in tables 5 and 7);

Reply: We thank the reviewer for pointing this out. We have now provided an explanation/interpretation of the results throughout the manuscript.

- when the authors refer: “As expected, as oysters are filter feeders Pearson correlation analysis (Table 6) confirmed a significant correlation”, it remains to be indicated what the p-value is (neither in the material and methods the level of significance is mentioned for which the results are considered statistically significant); and menation the significant

Reply: We apologise for the oversight. The legend and p-value has been added to Table 2 and now it reads” Pearson correlation between sterols in oyster flesh and with potential sources of faecal contamination, including both water and animal faecal samples.  The * and ** showed significantly differences at 0.05 and 0.01 respectively.”

- table 6 does not indicate what “*” and “**” mean;

Reply: The * and ** show significantly differences at 0.05 and 0.01 respectively. This has been added to the legend.

 

- nowhere in the manuscript (specifically, in the results and discussion section) are the results of the statistical tests t-test or analysis of variance or Tukey's test described (very poorly correct) in the material and methods section (“2.6. Statistical Analyzes”). Thus, it is impossible to understand what these statistical tests were used for, or for what purpose; the values of the test statistics, the p-value and whether statistically significant effects are evident must be evident in the text;

Reply: We thank the reviewer for their considered suggestion. We have now taken on board the comments and rewritten the statistics section which now reads:

 

” Data were analysed with IMB SPSS (version 24) Analysis of Variance (ANOVA; p=0.05). The means were separated using Tukey test (p=0.05), where the F-value was significant. To examine whether there was any significant association between the concentrations of faecal sterols (coprostanol and 24-ethylcoprostanol and coprostanol + 24-ethylcoprostanol), several correlation tests were carried out using SPSS version 24. Results are presented as mean ± standard deviation unless otherwise stated. “ Page 4, Lines 140-147”.

 

- additionally, since the investigation allows having a data with 4 locations, in 12 months, for concentrations of 11 major sterols, then the data analysis should be complemented with a multivariate statistical analysis, namely a principal component analysis (for example).

Reply: We thank the reviewer for this question. The aim of this study was is to identify the major source(s) of faecal pollution impacting Salcott Creek oyster-fisheries, UK through the examination of the sterol profiles.

We did investigate the potential of multivariate statistical analysis in addressing this aim but as you can see from the PCA plots below this approach did not shed additional light to assist in addressing the aim of the work. Consequently, correlation analysis was carried out instead leading to significant findings directly relating to the aims of the study.

 

Author Response File: Author Response.docx

Reviewer 2 Report

This work aimed to identify the major source(s) of faecal pollution impacting Salcott Creek oyster-fisheries, UK through the examination of the sterol profiles. The focus is very interesting, although I have struggled a lot to follow it. Specific comments

1-Line 43. The authors mentioned in the Introduction section that oyster have the ability to retain faecal micro-organisms, especially viruses, makeing shellfish potentially susceptible to accumulating faecal pathogens from the overlying water. Please provide a reference for such information

2-Line 124. How many replicate?

 

3-Line 137. Please provide quality control details of GC measurement including detection limit, recovery, etc. 

4-The sampling design must be better explained, for example I see sampling of 8 months for oyster and 12 months for water samples  above oyster. Please specify

 

5- Fig.2 The graph of the total sterol content report that in the months of July and September occur two sampling. Please explain

 

6-In table 4 the authors reported the ratio of sterols for water samples over 12 months. But in Tab. 2 the Concentrations of sterols and total sterols (μg g-1 L water) in water are reported for 10 months. There is a bit of confusion.

 

7- Please check the ratio because many do not match

 

8- Tab 5. Total sterol (μg g-1) or (μg L-1) 


Author Response

Reviewer 2

 

Comments and Suggestions for Authors

This work aimed to identify the major source(s) of faecal pollution impacting Salcott Creek oyster-fisheries, UK through the examination of the sterol profiles. The focus is very interesting, although I have struggled a lot to follow it. Specific comments

1-Line 43. The authors mentioned in the Introduction section that oyster have the ability to retain faecal micro-organisms, especially viruses, makeing shellfish potentially susceptible to accumulating faecal pathogens from the overlying water. Please provide a reference for such information

Reply: We apologise to the reviewer for their difficulty the text. We hope that the significant modifications and correction of the English now assist the reviewer in following the work. We have also included a reference to assist the reader.

2-Line 124. How many replicate?

Reply:  Work was cariied out in triplicate throughout.

3-Line 137. Please provide quality control details of GC measurement including detection limit, recovery, etc. 

Peak identification was based on a relative retention time and total ion mass spectral comparison with an external standard. Sterols were quantified by peak area comparison with the internal standards 5α-cholestane and 19-hydroxycholesterol. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Limit of detection was 0.01 μg L^-1.

Reply: The experimental plan was to sample monthly. Whilst this was possible for water samples, obtaining oyster samples was more difficult; and at 4 sampling times the weather conditions made it impossible to obtain oyster samples. This has now been explained in the Methods section at the end of Tables where appropriate.

5- Fig.2 The graph of the total sterol content report that in the months of July and September occur two sampling. Please explain.

Reply: We apologise for this error. The Figure was corrupted. We have now corrected and updated the figure which now reflects single monthly analysis

6-In table 4 the authors reported the ratio of sterols for water samples over 12 months. But in Tab. 2 the Concentrations of sterols and total sterols (μg g-1 L water) in water are reported for 10 months. There is a bit of confusion.

Reply: The experimental plan was to sample monthly. Whilst this was possible for water samples with exception of August and October, obtaining oyster samples was more difficult; at 4 sampling times the weather conditions made it impossible to obtain oyster samples. This has now been explained in the Methods section at the end of Tables where appropriate.7- Please check the ratio because many do not match. Therefore in Table 4, where water samples were obtained all months are shown but in Table 2 where oysters are used only 8 samples are reported.

 8- Tab 5. Total sterol (μg g-1) or (μg L-1) 


Reply: We apologise for the oversight> In fact for water samples the values are reported as μg L^-1. For faecal material the values are reported as (μg g^-1). We have clarified this in the Table.

 

 

 

Round 2

Reviewer 1 Report

The authors report that: “The means were separated using Tukey test (p=0.05),…” » What does it mean? The Tukey test is used to perform multiple comparisons and, thus, to find out where the statistically significant differences are observed. Therefore, the authors' response remains unclear.

In the statistical analysis section, information about the requirements for ANOVA still missing. Such omission is relevant, since when the requirements are not met, the parametric analysis of variance cannot be performed (being necessary to resort to a non-parametric analysis).

Still in this section the authors answer: “To examine whether there was any significant association between the concentrations of faecal sterols (coprostanol and 24-ethylcoprostanol and coprostanol + 24-ethylcoprostanol), several correlation tests were carried out…” » What does “several correlation tests” mean? This information cannot be written so subjectively! On the other hand, information regarding the level of significance used for the correlation analysis is missing.

Still in this section the authors answer: The information about the software continues to be repeated!

Nowhere in the new manuscript have I seen changes by the authors to the comment made by me, namely: “- nowhere in the manuscript (specifically, in the results and discussion section) are the results of the statistical tests t-test or analysis of variance or Tukey's test described (very poorly correct) in the material and methods section (“2.6. Statistical Analyzes”). Thus, it is impossible to understand what these statistical tests were used for, or for what purpose; the values of the test statistics, the p-value and whether statistically significant effects are evident must be evident in the text;”;

The answer given by the authors to this point (what is it: “Reply: We thank the reviewer for their considered suggestion. We have now taken on board the comments and rewritten the statistics section which now reads: “Data were analysed with IMB SPSS (version 24) Analysis of Variance (ANOVA; p=0.05). The means were separated using Tukey test (p=0.05), where the F-value was significant. To examine whether there was any significant association between the concentrations of faecal sterols (coprostanol and 24-ethylcoprostanol and coprostanol + 24-ethylcoprostanol), several correlation tests were carried out using SPSS version 24. Results are presented as mean ± standard deviation unless otherwise stated. “ Page 4, Lines 140-147”.”) does not correspond to what was asked to change. In other words, it remains unclear in the text where (and for what) statistical tests were used (except for correlation analysis).

Author Response

Reviewer 1

 

The authors report that: “The means were separated using Tukey test (p=0.05),…” » What does it mean? The Tukey test is used to perform multiple comparisons and, thus, to find out where the statistically significant differences are observed. Therefore, the authors' response remains unclear.

In the statistical analysis section, information about the requirements for ANOVA still missing. Such omission is relevant since when the requirements are not met, the parametric analysis of variance cannot be performed (being necessary to resort to a non-parametric analysis).

Still in this section, the authors answer: “To examine whether there was any significant association between the concentrations of faecal sterols (coprostanol and 24-ethylcoprostanol and coprostanol + 24-ethylcoprostanol), several correlation tests were carried out…” » What does “several correlation tests” mean? This information cannot be written so subjectively! On the other hand, information regarding the level of significance used for the correlation analysis is missing.

Still in this section the authors answer: The information about the software continues to be repeated!

Nowhere in the new manuscript have I seen changes by the authors to the comment made by me, namely: “- nowhere in the manuscript (specifically, in the results and discussion section) are the results of the statistical tests t-test or analysis of variance or Tukey's test described (very poorly correct) in the material and methods section (“2.6. Statistical Analyzes”). Thus, it is impossible to understand what these statistical tests were used for, or for what purpose; the values of the test statistics, the p-value and whether statistically significant effects are evident must be evident in the text;”;

 

The answer given by the authors to this point (what is it: “Reply: We thank the reviewer for their considered suggestion. We have now taken on board the comments and rewritten the statistics section which now reads: “Data were analysed with IMB SPSS (version 24) Analysis of Variance (ANOVA; p=0.05). The means were separated using the Tukey test (p=0.05), where the F-value was significant. To examine whether there was any significant association between the concentrations of faecal sterols (coprostanol and 24-ethylcoprostanol and coprostanol + 24-ethylcoprostanol), several correlation tests were carried out using SPSS version 24. Results are presented as mean ± standard deviation unless otherwise stated. “ Page 4, Lines 140-147”.”) does not correspond to what was asked to change. In other words, it remains unclear in the text where (and for what) statistical tests were used (except for correlation analysis).

 

Answer:

We thank the reviewer for their considered suggestion, and we apologise for the oversight. As suggested by reviewer, we clearly explained the statistical analysis and now it reads” Analysis of Variance (ANOVA; p=0.05) was used to determine significant differences among the major sterols and total sterols per month and over the 12-month period. Prior to analysis homogeneity of variance and normal distribution were assessed. In the case of below detection limit, 0 value was used for ANOVA analysis. If normal distribution was not met, data transformation was performed prior to ANOVA (e.g. log transformation) [27]. As ANOVA only identifies significant differences among treatments, to identify treatment effects, the Tukey test (p=0.05) was used. Pearson correlation between sterols in oyster flesh and potential sources of faecal contamination, including both water and animal faecal samples was carried out using SPSS version 24. Both p values 0.01 and 0.05 were used for correlation analysis. Results are presented as mean ± standard error unless otherwise stated.” Page 5, Lines  149-158.

Regarding the p-value, has been added in the results and discussion section (Red font in the results sections.

Reviewer 2 Report

The paper can be published in the present form.

Regards

Author Response

Many thanks for accepting the paper.

Round 3

Reviewer 1 Report

It is not clear whether the variable transformation procedure was performed only when normality was not fulfilled or, if so, it was extensible when there is no homogeneity of variances.

The statement “In the case of below detection limit, 0 value was used for ANOVA analysis.” is not clear. What is the relation of this sentence with the fulfilment (or not) of the requirements of the analysis of variance?

In line 383 (“There was no significant difference the ratio values between the various inputs 383 (p<0.05)”), something is not correct, because if there are no statistically significant differences, then the p-value cannot be less than 0.05.

Author Response

It is not clear whether the variable transformation procedure was performed only when normality was not fulfilled or, if so, it was extensible when there is no homogeneity of variances.

Answer: We thank the reviewer for their considered suggestion. The words “when normality was not fulfilled “has been added to the text

The statement “In the case of below detection limit, 0 value was used for ANOVA analysis.” is not clear. What is the relation of this sentence with the fulfilment (or not) of the requirements of the analysis of variance?

Answer: To clarify, the sentence has been deleted now.

In line 383 (“There was no significant difference the ratio values between the various inputs 383 (p<0.05)”), something is not correct, because if there are no statistically significant differences, then the p-value cannot be less than 0.05.

Answer: We apologise for the oversight. The error has been fixed now.