A Comparative Test on the Sensitivity of Freshwater and Marine Microalgae to Benzo-Sulfonamides, -Thiazoles and -Triazoles
, Federica Maraschi 1, Stefano Sangiorgi 2 and Elida Nora Ferri 2,*Round 1
Reviewer 1 Report
A comparative test on the sensitivity of freshwater and marine microalgae to benzo-sulfonamides, -thiazoles and –triazoles
This is interesting manuscript reporting the preliminary data on the differences among three unicellular algae and one luminescent bacteria in their response to heterocyclic pollutants. Tested compounds are widely spread and represent a threat to various aquatic organisms as well as to human health. The main goal of this study was to find the more suitable biotest for environmental toxicity monitoring. Although the selected algae are frequently used organisms in toxicity studies, according to the results these species are not suitable for evaluating the effect of tested pollutants. The results are valuable but i think that some changes must be made before its publishing.
Specific comments are listed below.
Page 1, lines 33-36 – please add some references
Page 2, line 47- please add some reference for “carcinogenic to humans”
Page 3, lines 102-104: The qualitative and quantitative differences among the four microorganisms in sensitivity to the effects of the various compounds were greater than expected, suggesting the need for a preliminary screening before adopting a specific bioassay. – this sentence is not necessary here
Page 3, line 110: 5-methyl-1H-benzotriazole (5TTR) – you mentioned tolyl-triazole (TTR, a mixture of the isomers 4- and 5-methyl-1H-benzotriazole) in the Introduction. Please use the same abbreviation in the Introduction and in Chemicals. Once, when abbreviation was used it is not necessary to repeat full name and abbreviation again. Please correct it.
Page 5, lines 177-180: please insert this description of algae in the last paragraph of the Introduction
Page 5, line 193: whereas the 5 mg L-1 was the lowest concentration of HOBT which reduced in a significant way the population growth – since it is not visible in the figure, please add in the text the % of inhibition when concentration of 5 mg-1 was used
Page 5, lines 196-197: The concentrations in the range 5-100 mg L-1 of BTR reduced the growth rate of the algal population – why do you highlight only these concentrations when the inhibition also can be seen in the lowest concentrations?
Page 7, lines 212-215: please insert this description of algae in the last paragraph of the Introduction and please add some reference for the statements
Page 7, lines 226-228: please insert this description of algae in the last paragraph of the Introduction and please add some reference for the statements
Page 7, lines 233: The BT and HOBT higher concentrations produced inhibition values above 80%. – Is it true for HOBT? According to the figure, it is lower values of inhibition. Please check the results.
Page 7, lines 237-239: ..the 5TTR solutions resulted toxic at all concentrations between the 0.1-100 mg L-1 – please check this statement, according to the figure lower concentration the toxic effect is not highly expressed
Page 8, lines 256- 260: V. fisheri is a bioluminescent marine bacterium which light emission intensity is highly influenced by the conditions of its environment. Based on the ascertained, inversely proportional reduction of the light intensity with the increase of toxicants in solution, the test based on this bacterium represents since a long time the quicker and easier standardized assay for drinking water quality assessment. – please insert this description into the Intoduction
Page 9, Figure 7: please add title on the y axis
Page 9, line 301: …pollution accidents,. – please delete the comma
Page 9, lines 307-310: please confirm this statement with some references
Page 10, line 363: - please check the reference 4. I think that it should be Environ Sci Pollut Res Int. 2015; 22(8): 5711–5741.
Page 11, line 381: please check the reference 10 and correct the name of journal Arch. Environ. Contam. Toxicol. - don’t use a bold
Page 11, line 394: please use a bold for the year of publishing – 2006
Page 13, line 46: please add a point after the … and chlorite (ClO2-)
Page 13, line 465: please correct the journal name – use italic and use a bold for the year of publication
Page 13, line 487: please corecct the journal abbreviation (change into the J. Appl. Phycol.)
Page 14, line 495: please correct the title (correct the phytotoxicity)
Author Response
A comparative test on the sensitivity of freshwater and marine microalgae to benzo-sulfonamides, -thiazoles and –triazoles
This is interesting manuscript reporting the preliminary data on the differences among three unicellular algae and one luminescent bacteria in their response to heterocyclic pollutants. Tested compounds are widely spread and represent a threat to various aquatic organisms as well as to human health. The main goal of this study was to find the more suitable biotest for environmental toxicity monitoring. Although the selected algae are frequently used organisms in toxicity studies, according to the results these species are not suitable for evaluating the effect of tested pollutants. The results are valuable but i think that some changes must be made before its publishing.
The authors are grateful to the Reviewer for the appreciation of our work, for the careful examination of the manuscript and the useful comments and suggestions which allowed us to correct various mistakes and to improve fundamental parts of the paper. The most important changes/additions are in red colour.
The specific answers to the comments are listed below.
Page 1, lines 33-36 – please add some references
A reference has been added.
Page 2, line 47- please add some reference for “carcinogenic to humans”
A reference has been added.
Page 3, lines 102-104: The qualitative and quantitative differences among the four microorganisms in sensitivity to the effects of the various compounds were greater than expected, suggesting the need for a preliminary screening before adopting a specific bioassay. – this sentence is not necessary here
The sentence has been deleted
Page 3, line 110: 5-methyl-1H-benzotriazole (5TTR) – you mentioned tolyl-triazole (TTR, a mixture of the isomers 4- and 5-methyl-1H-benzotriazole) in the Introduction. Please use the same abbreviation in the Introduction and in Chemicals. Once, when abbreviation was used it is not necessary to repeat full name and abbreviation again. Please correct it.
We selected to use the name tolyl-triazole and the abbreviation 5TTR throughout the text, Figure 1 included. Repetitions of full name and abbreviation were deleted.
Page 5, lines 177-180: please insert this description of algae in the last paragraph of the Introduction
We moved this part to the Introduction, as suggested.
Page 5, line 193: whereas the 5 mg L-1 was the lowest concentration of HOBT which reduced in a significant way the population growth – since it is not visible in the figure, please add in the text the % of inhibition when concentration of 5 mg-1 was used
Concerning this point the problem arose during the conversion of the original graphs to those shown in the manuscript: the heading "HOBT" and "MeSBT" were exchanged. Now we employed the original graphs, which are correct and probably clearer.
Page 5, lines 196-197: The concentrations in the range 5-100 mg L-1 of BTR reduced the growth rate of the algal population – why do you highlight only these concentrations when the inhibition also can be seen in the lowest concentrations?
A similar problem occurred during the conversion of these graphs and also in this case we substituted them with the original Excel graphs, on which was prepared the manuscript.
Page 7, lines 212-215: please insert this description of algae in the last paragraph of the Introduction and please add some reference for the statements
We moved also this part to the Introduction and the reference has been added.
Page 7, lines 226-228: please insert this description of algae in the last paragraph of the Introduction and please add some reference for the statements
We moved this part to the Introduction also adding the respective reference.
Page 7, lines 233: The BT and HOBT higher concentrations produced inhibition values above 80%. – Is it true for HOBT? According to the figure, it is lower values of inhibition. Please check the results.
As mentioned above (page 5, line 193...) the mistake was in the headings of the graph, now the correct one is present.
Page 7, lines 237-239: ..the 5TTR solutions resulted toxic at all concentrations between the 0.1-100 mg L-1 – please check this statement, according to the figure lower concentration the toxic effect is not highly expressed
As mentioned above (page 5, lines 196-197...) the graph was substituted by the original one, avoiding the mistakes produced during its conversion to the previous one.
Page 8, lines 256- 260: V. fisheri is a bioluminescent marine bacterium which light emission intensity is highly influenced by the conditions of its environment. Based on the ascertained, inversely proportional reduction of the light intensity with the increase of toxicants in solution, the test based on this bacterium represents since a long time the quicker and easier standardized assay for drinking water quality assessment. – please insert this description into the Intoduction
We moved this part to the Introduction, as suggested.
Page 9, Figure 7: please add title on the y axis
We modified the graph.
Page 9, line 301: …pollution accidents,. – please delete the comma
Comma was deleted.
Page 9, lines 307-310: please confirm this statement with some references
The suitable references have been added
Page 10, line 363: - please check the reference 4. I think that it should be Environ SciPollut Res Int. 2015; 22(8): 5711–5741.
We corrected this citation, actually 2014 was the online publication date.
Page 11, line 381: please check the reference 10 and correct the name of journal Arch. Environ. Contam. Toxicol. - don’t use a bold
We corrected the style of the reference 10 (in the original manuscript).
Page 11, line 394: please use a bold for the year of publishing – 2006
We corrected the style of the reference
Page 13, line 46: please add a point after the … and chlorite (ClO2-)
The point was added
Page 13, line 465: please correct the journal name – use italic and use a bold for the year of publication
We corrected the style of the reference
Page 13, line 487: please correct the journal abbreviation (change into the J. Appl. Phycol.)
We corrected the journal name in the reference
Page 14, line 495: please correct the title (correct the phytotoxicity)
The title was corrected
Reviewer 2 Report
The work presented has scientific value and relevance. The introduction is well written and presents the problematic and the objectives of the work. Nevertheless, material and methods section is rather incomplete and must present more information regarding the assays and procedures performed. Discussion also needs to be improved.
Some notes and questions are described below.
Featured Application
The most suitable instead of “the more suitable”
Introdution
Line 71 contaminants level
Line 77 ecosystems (delete components)
Material and methods
How long did you cultivate the different cultures before the bioassays?
How long did the exposure assays take for each culture? Was optimal density measured only at the beginning and at the end of the assay?
In introduction you refer that concentration of the contaminants can be found within the range of ng/L and ug/L. Can you explain how the concentrations used were chosen? Are they environmentally relevant?
Please include this information in the manuscript.
Were the same concentrations used in the Bioluminescence inhibition assay? Include this information in the manuscript.
Results
Line 167-169 Therefore, the assays were performed not within the usual interval of 72-96 h, but the growth rate of algae was checked at intervals of about 20 ± 2 (interval A), 27 ± 2 (interval B) and 34 ± 2 days (interval C).
This information should be described in material and methods. From the given description, one cannot understand that this was performed.
Figure 3 to 5 should have information regarding the statistical difference to confirm the significantly differences that you mention along the text
EC50 values. You should mention in material and methods that EC50 were calculated – explain how these calculations were made or cite the literature used
Line 261 answers one of the questions that I raised above. Material and methods should include this information and be completed with some detail regarding each followed procedure. For Vibrio fisheri, lower concentrations were used. Why was that?
Line 261-263 this information should be in material and methods and not here
Line 278 more complicated (not most complicated)
Line 326 The most suitable instead of “the more suitable”
Discussion needs some work. Can the inhibition or no inhibition of algae growth or luminescence of vibrio be in the same range of results reported for other contaminants chemically similar?
Were the contaminants used already tested with other algae species? Or with the same algae species or vibrio but at different concentrations and/or assay conditions? If yes, how the reported results can be compared to yours?
Line 323 “no toxicity, no detected risks”
In some assays, you mention 72% inhibition was attained. Is this not relevant? And why HOBT stimulated the light emission intensity? Has this happened for other contaminants?
Author Response
The work presented has scientific value and relevance. The introduction is well written and presents the problematic and the objectives of the work. Nevertheless, material and methods section is rather incomplete and must present more information regarding the assays and procedures performed. Discussion also needs to be improved.
The authors sincerely thank the Reviewer for the overall good evaluation of our work and for the very useful comments underlining the various weak points in our manuscript that we corrected accordingly. The added or deeply modified sentences are in red color.
Some notes and questions are described below.
The answers to the specific comments are listed below.
Featured Application
The most suitable instead of “the more suitable”
The mistake was corrected.
Introduction
Line 71 contaminants level
Line 77 ecosystems (delete components)
Both corrections have been introduced.
Material and methods
How long did you cultivate the different cultures before the bioassays?
We did not use a fixed day; we withdraw a sample from the stock cultures when the number of cells looked adequate for dilution employed in the experiment. Nevertheless, the stock cultures were in the log phase. We introduced this information in the Materials and Method section.
How long did the exposure assays take for each culture? Was optimal density measured only at the beginning and at the end of the assay?
The exposure lasted about one month (34 days ± 2) and we performed three measurements. Now this information is reported in the M&M section.
In introduction you refer that concentration of the contaminants can be found within the range of ng/L and ug/L. Can you explain how the concentrations used were chosen? Are they environmentally relevant?
Please include this information in the manuscript.
The specific information has been added to the 3.1 paragraph, where we explain that we have chosen these concentrations in agreement with literature data since previous work reported that environmental concentrations produced negligible effects and then the studies were carried out employing amounts until hundreds of mg. Suitable references have been cited.
Were the same concentrations used in the Bioluminescence inhibition assay? Include this information in the manuscript.
The concentrations employed in the bioluminescent assay were in the µg L-1 range because of the well-known great sensitivity of this assay. This information has been included in the M&M section.
Results
Line 167-169 Therefore, the assays were performed not within the usual interval of 72-96 h, but the growth rate of algae was checked at intervals of about 20 ± 2 (interval A), 27 ± 2 (interval B) and 34 ± 2 days (interval C).
This information should be described in material and methods. From the given description, one cannot understand that this was performed.
As mentioned above, this information is now reported in the M&M section, paragraph 2.2
Figure 3 to 5 should have information regarding the statistical difference to confirm the significantly differences that you mention along the text
The requested information has been reported in the text mentioning the respective figure and not on the graph itself because these figures already contain a lot of information and adding a further one should create unnecessary confusion.
EC50 values. You should mention in material and methods that EC50 were calculated – explain how these calculations were made or cite the literature used
The information and the respective reference have been mentioned in the M&M section.
Line 261 answers one of the questions that I raised above. Material and methods should include this information and be completed with some detail regarding each followed procedure. For Vibrio fisheri, lower concentrations were used. Why was that?
Actually, various information on the experimental procedures was missed and we included more details on both algae and bacterial assays procedures. The lower concentrations were selected because this assay usually detect contaminants at very low concentrations.
Line 261-263 this information should be in material and methods and not here
The specific information was moved to the M&M.
Line 278 more complicated (not most complicated)
The mistake was corrected.
Line 326 The most suitable instead of “the more suitable”
The mistake was corrected.
Discussion needs some work. Can the inhibition or no inhibition of algae growth or luminescence of vibrio be in the same range of results reported for other contaminants chemically similar?
We never made the attempt to compare the results obtained for similar chemicals from a biotest, since the parameters involved in the biological effects of a compound are some many that it is really complicated to try such a comparison. Surely, data obtained from the tests on a compound rarely will offer information on those produced by a similar one, mainly will be difficult to determine which are the differences that make "similar" or not two molecules. A little change in the structure can influence greatly the interaction of a molecule with the organisms and their metabolism, with unpredictable effects. We mention in this section the reasons for the impossibility to perform a direct comparison with different chemicals and also with data obtained in previous studies carried out in different conditions.
Were the contaminants used already tested with other algae species? Or with the same algae species or vibrio but at different concentrations and/or assay conditions? If yes, how the reported results can be compared to yours?
We reported now information about the previous studies carried out on the same or different algae and the result of the comparison of the trends among them. The conclusion, also reported in the revised manuscript was that, in all studies, the environmental concentrations were not a problem and only higher ones were able to elicit some biological response.
Line 323 “no toxicity, no detected risks”
In some assays, you mention 72% inhibition was attained. Is this not relevant?And why HOBT stimulated the light emission intensity? Has this happened for other contaminants?
Concerning the first question, we agree that the sentence was unclear. Now we specified that we obtained no effect for the short time (acute toxicity) assay, which is the kind of assay applied when using the Vibrio fisheri, especially the MICROTOX® Kit. We detected high values of % of inhibition, produced by various of the tested compounds, but only at the chronic toxicity intervals.
An explanation of the reason because of mainly HOBT stimulated the light emission at 50 and 100 µg L-1 is now reported in the Discussion, together with that explaining the stimulation by all compounds after 24 and 48 hrs from the contact with the 0.1 µg L-1. It is absolutely not rare that organic compounds showing no acute toxicity can be, over time, employed as feed source by bacteria or, at low doses, produce the well-known hormesis effect.
Round 2
Reviewer 2 Report
See the document attached please
Comments for author File: 
Comments.docx
Author Response
The Authors thank the reviewer for the careful pointing out of the various mistakes still present in our text. We introduced the suggested correction and we corrected also various typing mistakes, all highlighted in yellow. In details:
Line 143
Let to grow (not growth) until the cells density required was reached to…
The text has been modified.
Line 162
I have a doubt concerning the formulae presented.
The absorbance of the sample was compared to a blank and not a control sample?
Generally, one uses blank as the medium where the microalgae are grown but without the microalgae. Control is the same medium+microalgae used in the experiment but without the contaminant addiction. Generally, one compares the growth rate of microalgae exposed to contaminants and to microalgae growing in ideal conditions (control) as recommended as OECD procedures. If you mean blank as other definition different from the one given above, you should clarify it in the text.
The same applies to the bioluminescence inhibition.
In fact, in line 199, you say that the comparisons were made between sample and control. Please, check if it was a mistake or clarify the information, please.
We agree with the Reviewer; Because of an incorrect habit, we unfairly used the word blank instead of control. We have replaced the word blank with control throughout the text.
Line 204-206
…since, according to the literature, data reporting negligible toxic effects are generally reported at the environmental concentrations so the employment of higher ones are employed to analyze the in vitro effects [19, 31, 34, 55]
The text has been corrected as suggested.
Line 257
…the most part of
The text has been corrected as suggested.
Line 258
…compounds at the lower concentrations
The text has been corrected as suggested.
Line 325
… directly of our data
The text has been corrected as suggested.
