Occurrence of Selected Known or Suspected Endocrine-Disrupting Pesticides in Portuguese Surface Waters Using SPME-GC-IT/MS
, VirgÃnia Cruz Fernandes 1,*, LuÃsa Correia-Sá 1, Catarina Mansilha 2,3, Cristina Delerue-Matos 1 and Valentina F. Domingues 1Round 1
Reviewer 1 Report
Paper deals with important task. It has a logical structure, all necessary sections. Paper is technically sound. Experimental section is good.
Suggestions:
- It would be good to add point-by-point the main contributions in the end of the Introduction section
- It would be good to add the remainder of this paper
- It would be good to add the Related works section
- Authors should provide a link to open access repository with dataset used for modeling
- Conclusion section should be extended using: 1) numerical results obtained in the paper; 2) limitations of the proposed approach; 3) prospects for the future research.
Author Response
Response to Reviewer 1
We are thankful to all reviewers for the very useful feedback. We made all suggested changes and hope this sufficiently improved the quality of the manuscript and takes away all concerns.
"Please see the attachment word file."
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This study reports the levels of some major pesticides in the environmental water from northern Portugal. Though the proposed analytical method is not a cutting-edge/latest, reported data is valuable in the field of environmental and toxicological science. Because there is a data gap when compared to the other part of EU countries, this paper would provide worthful baseline information on the contamination status of pesticides in Portuguese environment. However, I feel that some critical information should be added. Therefore, revision is necessary before proceeding to further step of publication.
L2 Because most of the target compounds are organochlorines, the title should be more specified. I think you don’t cover full range of known and suspected endocrine disrupting pesticides. To me, this study targeted only some organochlorines with limited other pesticides.
L14 Again, you wrote that known and suspected endocrine disrupting pesticides were measured but I feel the list of analytes is incomplete. Many important ‘known and suspected endocrine disrupting pesticides’ are not listed. First I see only 1 organophosphate pesticide and diazinon is not the only endocrine disrupting pesticide among this group. Second I don’t see important pyrethroids in the list. Third you missed neonicotinoids, which have been widely and intensively used worldwide since 1990s, and they are known as endocrine disrupting pesticides.
L35 “Release” should be “released”.
L55,56 “ECPs” and “ECDs” are typos. You’d better to be careful with typing and grammar.
L74 I don’t see which site is urbanization/agricultural/industrial in the next section.
L107 Did you collect spot samples? If so, you should mention it as the limitation when you discuss the levels of analytes among sampling sites.
L110 Since samples were collected in spring and summer, you can indicate the date and discuss seasonal variation in levels.
L111 Rivers, major cities and other landmark should be shown in the figure. Map on the right side and sampling locations should be shown in larger scale.
L113 Table in Figure 1 can be moved to supplementary information with the fundamental information on the samples such as date/time, water temperature, pH, EC, salinity, etc.
L138 You can claim the originality of the method, otherwise it seems not new, not sensitive, not robust, not wide-targeted, not high-throughput and not reproducible method to me.
L165 Manufacturer and model name of GC and MS should be indicated.
L168 Split ratio should be described.
L176 What was used as the matrix?
L177 Were reported levels corrected by recovery of atrazine? Why did you use only labeled atrazine as an internal standard whereas many labeled pesticides are commercially available and are not such expensive?
L189 Some of results seem to be inconsistent or did not show consistent trends. Please explain the potential reasons clearly.
L200 Figure 2 should be expressed by amount or concentration, not area.
L221 Feel weird to see more than one interception in a graph. Y-axis can be in log-scale.
L275 Internal/external quality control are necessary to validate the method. Reproducibility/repeatability and recovery can be described. In addition, analysis of certified reference materials or the result of inter-lab calibration exercise should be shown.
L308 How did you calculate the total concentration if some of compounds are below LOQ? Is mean appropriate? Did you check the data distribution?
Author Response
Response to Reviewer 2
We are thankful to all reviewers for the very useful feedback. We made all suggested changes and hope this sufficiently improved the quality of the manuscript and takes away all concerns.
"Please see the attachment word file."
Response to Reviewer 2 Comments
Point 1: This study reports the levels of some major pesticides in the environmental water from northern Portugal. Though the proposed analytical method is not a cutting-edge/latest, reported data is valuable in the field of environmental and toxicological science. Because there is a data gap when compared to the other part of EU countries, this paper would provide worthful baseline information on the contamination status of pesticides in Portuguese environment. However, I feel that some critical information should be added. Therefore, revision is necessary before proceeding to further step of publication.
Suggestions:
Response 1: The authors appreciate the comment. We hope that our further effort has improved the manuscript appropriately.
Point 2: L2 Because most of the target compounds are organochlorines, the title should be more specified. I think you don’t cover full range of known and suspected endocrine disrupting pesticides. To me, this study targeted only some organochlorines with limited other pesticides.
Response 2: The authors appreciate the comment. However, the authors are of the opinion that the title uses the term "endocrine disrupting pesticides" precisely to get the reader's attention by showing that more than being pesticides, they are also disruptive. This term should be more evident in the studies to further alert this spread of pesticides in the environment. Some of the studied OCP are known ED and another are suspected.
Point 3: L14 Again, you wrote that known and suspected endocrine disrupting pesticides were measured but I feel the list of analytes is incomplete. Many important ‘known and suspected endocrine disrupting pesticides’ are not listed. First, I see only 1 organophosphate pesticide and diazinon is not the only endocrine disrupting pesticide among this group. Second, I don’t see important pyrethroids in the list. Third you missed neonicotinoids, which have been widely and intensively used worldwide since 1990s, and they are known as endocrine disrupting pesticides.
Response 3: Unfortunately, it is not possible to analyze the entire priority list. Regarding the EU list they are more then 500 (https://ec.europa.eu/environment/chemicals/endocrine/ strategy/substances _en.htm). However, we try to put together 20 pesticides of that list from different categories I, II, and III. Certainly, it could have been another approach. The authors appreciate the comment to take into account in a future selection.
Point 4: L35 “Release” should be “released”.
Response 4: The authors corrected.
Point 5: L55,56 “ECPs” and “ECDs” are typos. You’d better to be careful with typing and grammar.
Response 5: The authors checked the manuscript and corrected.
Point 6: L74 I don’t see which site is urbanization/agricultural/industrial in the next section.
Response 6: The details were described in section 2.3. The authors clarified the information.
Point 7: L107 Did you collect spot samples? If so, you should mention it as the limitation when you discuss the levels of analytes among sampling sites.
Response 7: Yes. They are spot samples. The authors included in the manuscript that information.
Point 8: L110 Since samples were collected in spring and summer, you can indicate the date and discuss seasonal variation in levels.
Response 8: No significant differences were observed between the 2 seasons. That is why there was no division in the discussion. But the authors included that information in the manuscript.
Point 9: L111 Rivers, major cities and other landmark should be shown in the figure. Map on the right side and sampling locations should be shown in larger scale.
Response 9: The figure was improved.
Point 10: L113 Table in Figure 1 can be moved to supplementary information with the fundamental information on the samples such as date/time, water temperature, pH, EC, salinity, etc.
Response 10: The authors included the month of the collection. The other details were not performed.
Point 11: L138 You can claim the originality of the method, otherwise it seems not new, not sensitive, not robust, not wide-targeted, not high-throughput and not reproducible method to me.
Response 11: The authors included in the introduction (L65-66) a sentence/reference highlighting the advantages of the SPME not the originality. The section 3.2 is only focus on the method validation showing the good performance of the present methodology. The goals of this study are described in the end of introduction, line 79-84.
Point 12: L165 Manufacturer and model name of GC and MS should be indicated.
Response 12: The information was included.
Point 13: L168 Split ratio should be described.
Response 13: The information was included.
Point 14: L176 What was used as the matrix?
Response 14: A residue-free surface water sample was used for the preparation of the matrix calibration curve. This information was included in the manuscript.
Point 15: L177 Were reported levels corrected by recovery of atrazine? Why did you use only labeled atrazine as an internal standard whereas many labeled pesticides are commercially available and are not such expensive?
Response 15: At the time of extractions and analyzes it was what we had available. The authors are of the opinion that being a pesticide would be suitable for its use. The results of the atrazine evaluation were used for quality control in terms of GC analysis and extraction procedure. As no deviations were found to justify it, it was not necessary to resort to the correction of values.
Point 16: L189 Some of results seem to be inconsistent or did not show consistent trends. Please explain the potential reasons clearly.
Response 16: The compounds under study belong to different families, hence the differences observed in behavior. Therefore, the authors optimized the extraction conditions and present the results by families of compounds in order to select the best conditions for all.
Point 17: L200 Figure 2 should be expressed by amount or concentration, not area.
Response 17: Thank for the suggestion. It is true that it could be in concentration. We will consider in a future work. The differences in the bars are quite representative in these units as well. The calibration curves were performed after the optimization.
Point 18: L221 Feel weird to see more than one interception in a graph. Y-axis can be in log-scale.
Response 18: The interception in graphs are scale breaks. As we have different quantities in the areas, the visualization is more perceptible.
Point 19: L275 Internal/external quality control are necessary to validate the method. Reproducibility/repeatability and recovery can be described. In addition, analysis of certified reference materials or the result of inter-lab calibration exercise should be shown.
Response 19: The authors carried out an internal laboratory study just like many others carried out in this area (e.g. Ref 25, 35, 36). The experiments of the optimization of the section (3.1) methodology for the study of the efficiency of the method was also carried out in triplicates (see errors bars in graphs). For the validation of the results obtained, all experiments were carried out in triplicate and analyzed in duplicate. Accuracy has been calculated taking all these details into account. Matrix-matched calibration curve, LOD, LOQ and precision were assessed. We don’t have budget for certified reference materials, but we are trying to collaborate with another lab.
Point 20: L308 How did you calculate the total concentration if some of compounds are below LOQ? Is mean appropriate? Did you check the data distribution?
Response 20: Thank you very much for the correction. The authors detected the errors and corrected them. In Table 2, the results were in µg/L and we placed them in ng/L. The section 3.3 was verified.
Round 2
Reviewer 2 Report
I feel that authors revised properly according to the comments and now this manuscript may be accepted for publication after minor modifications. I would strongly recommend that authors will take reviewer’s comments seriously and revise adequately.
Title has a scientific problem. As authors also describe that they cannot cover all the known and suspected endocrine disrupting pesticides in response 3, title would mislead the readers. Of course, I agree that title should be attractive, but it must represent the content of the paper. Therefore, title should be more realistic. For example, “several” or “selected” or number of analytes can be added.
Authors did not answer to the last comment. When total concentrations were calculated, it may often happen that some compounds are below LOQ. Sometimes we add zero or LOQ value or square root of LOQ or impute the data distribution, but it should be described in materials and methods section anyway. Please clearly mention how you calculate the total concentrations with the values below the quantification limits.
In addition, sometimes mean value is not representative when the data distribution is not normal. If you check the data distribution, please mention in the text. If not, please do that and if data shows not normal distribution, you should consider showing log-normal concentration or other non-parametric analyses.
Author Response
Response to Reviewer 2
We are thankful to reviewer 2 for the very useful feedback. We made all suggested changes and hope this sufficiently improved the quality of the manuscript and takes
away all concerns.
Response to Reviewer 2 Comments
Point 1: I feel that authors revised properly according to the comments and now this manuscript may be accepted for publication after minor modifications. I would strongly recommend that authors will take reviewer’s comments seriously and revise adequately.
Response 1: The authors appreciate the comment.
Point 2: Title has a scientific problem. As authors also describe that they cannot cover all the known and suspected endocrine disrupting pesticides in response 3, title would mislead the readers. Of course, I agree that title should be attractive, but it must represent the content of the paper. Therefore, title should be more realistic. For example, “several” or “selected” or number of analytes can be added.
Response 2: The authors agree and changed the title.
Point 3: Authors did not answer to the last comment. When total concentrations were calculated, it may often happen that some compounds are below LOQ. Sometimes we add zero or LOQ value or square root of LOQ or impute the data distribution, but it should be described in materials and methods section anyway. Please clearly mention how you calculate the total concentrations with the values below the quantification limits.
Response 3: The authors added the information in the manuscript taking into account the use of <LOD values.
Point 4: In addition, sometimes mean value is not representative when the data distribution is not normal. If you check the data distribution, please mention in the text. If not, please do that and if data shows not normal distribution, you should consider showing log-normal concentration or other non-parametric analyses.
Response 4: The authors agree and clarified in the manuscript.
