Assessment of Acute Toxicity of Acid Mine Drainage via Toxicity Identification Evaluation (TIE) Using Daphnia magna and Chlorella vulgaris
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Water
Assessment of acute toxicity of acid mine drainage via Toxicity Identification Evaluation (TIE) using Daphnia magna and Chlorella vulgaris
This investigation deals with Acid mine drainage (AMD) to assess its toxicity, using Daphnia magna and Chlorella vulgaris as indicator organisms. Fractionated AMD was prepared with filtration and aeration at pH 3 and 11. The unfractionated AMD contained low organic matter, which led to a differential toxicity effect on D. Magna, with no observable acute toxicity to C. vulgaris. Cu was determined as the leading cause of acute toxicity to D. magna. The approach is projected as an effective strategy to determine the compound's toxicity.
Specific Comments
- The title needs reframing for a better and more streamlined projection of the theme/content.
- The abstract needs better and more comprehensive yet focused framing.
- Is ‘Toxicity Identification Evaluation (TIE)’ commonly used in toxicity-related investigations? Can it be better used as an ‘Evaluation of toxicity’ or ‘Evaluation of toxicity Identification’
- It would be better to discuss the biological basis of the differential toxicity tolerance of the two indicator organisms more effectively.
- The differential toxicity tolerance of microorganisms is a general phenomenon that sets them as indicator organisms of any polluted environment. What are the specificity and uniqueness of this study?
- Effectively discussing Cu as the leading cause of acute toxicity, specifically to magna, is essential.
- Fig-1 needs redrawing for better representations!
- The conclusion is too long, repetitive, and diffused.
- Overall, although analyzing the toxic components of the environment is essential, the focus should be on discussing the differential toxicity in the indicator organisms rather than the extraction methods and composition of the toxic environment.
Comments on the Quality of English Language
As shown earlier
Author Response
We want to thank the reviewers for carefully reading the manuscript. All the comments were attended and the paper was carefully checked according to the editorial suggestions.
The following comments indicate our response to all of the questions and notes indicated by the Referees and the Editor. Please consider that unless otherwise expressed, the referenced lines correspond to the current numeration after the corrections were made. The revisions according the Reviewer comments have been highlight in yellow color on the revised version of this paper.
REVIEWER # 1
This investigation deals with Acid mine drainage (AMD) to assess its toxicity, using Daphnia magna and Chlorella vulgaris as indicator organisms. Fractionated AMD was prepared with filtration and aeration at pH 3 and 11. The unfractionated AMD contained low organic matter, which led to a differential toxicity effect onD. Magna, with no observable acute toxicity to C. vulgaris. Cu was determined as the leading cause of acute toxicity to D. magna. The approach is projected as an effective strategy to determine the compound's toxicity.
Comment #1: The title needs reframing for a better and more streamlined projection of the theme/content.
Answer: The title was improved.
Comment #2: The abstract needs better and more comprehensive yet focused framing.
Answer: The abstract section was improved.
Comment #3: Is ‘Toxicity Identification Evaluation (TIE)’ commonly used in toxicity-related investigations? Can it be better used as an ‘Evaluation of toxicity’ or ‘Evaluation of toxicity Identification’
Answer: Toxicity Identification Evaluation (TIE) is a specific test with a fully established protocol that is performed in two phases, as presented in this publication. The name "TIE" is the proper name for the test. The different fractionations of Phase I and II were established by Norberg-Kingetal.(1991) and VanSprang and Janssen (1997).
Comment #4 It would be better to discuss the biological basis of the differential toxicity tolerance of the two indicator organisms more effectively.
Answer: To gain a more complete picture, a TIE determination was performed, which identifies the type of compound causing toxicity in bioindicators. This provides a projection of which abatement technology can reduce contamination from a potential AMD spill.
Comment #5: The differential toxicity tolerance of microorganisms is a general phenomenon that sets them as indicator organisms of any polluted environment. What are the specificity and uniqueness of this study?
Answer: This paper studies the effect of AMD on freshwater ecosystems. For this reason, bioindicators well known in the river food chain were used. Daphnia magna, a microcrustacean used as an indicator of river health, is a particular food for invertebrates and predatory fish [1]. In addition, it has advantages such as simplicity of cultivation, a short life cycle, ease of management, and low maintenance cost [2]. Due to the advantages of its reproductive cycle, its assay is widely known and established in toxicology laboratories, and its methodology is fully established by the EPA [3,4].
AMD
- Tkaczyk, A.; Bownik, A.; Dudka, J.; Kowal, K.; Slaska, B. Daphnia magna model in the toxicity assessment of pharmaceuticals: A review. Sci Total Environ 2021, 763, 143038.
- Peng, W.; Liu, Y.; Lin, M.; Liu, Y.; Zhu, C.; Sun, L.; Gui, H. Toxicity of coal fly ash and coal gangue leachate to Daphnia magna: Focusing on typical heavy metals. J Clean Prod 2022, 330, 129946.
- US Environmental Protection Agency (USEPA). Methods for aquatic toxicity identification evaluations: Phase I toxicity characterization procedures, 2nd ed.; EPA-600-6-91-003. Office of Research and Development: Duluth, MN, 1991.
- NCh 2083 Chilean Official Standard. Acute toxicity bioassays by determining inhibition of mobility of Daphnia magna or Daphnia pulex (Crustacea, Cladocera), 1st ed.; National Institute of Standardization: Chile, 1999.
Comment #6: Effectively discussing Cu as the leading cause of acute toxicity, specifically to magna, is essential.
Answer: The explanation is in line 270-272: “The Cu+2 is a leading cause of acute toxicity in Daphnia magna due to its ability to disrupt vital cellular processes, particularly oxidative stress and membrane damage, leading to death”.
Comment #7: Fig-1 needs redrawing for better representations!
Answer: Fig 1 was redrawing.
Comment #8: The conclusion is too long, repetitive, and diffused.
Answer: The conclusion was improved. The conclusion first describes the physiochemical and toxicity characterization of AMD by D. marga and C. vulgaris. The conclusions obtained from TIE fractionation are described in the second section. It is impossible to express the conclusions of this paper in any other way.
Comment #9: Overall, although analyzing the toxic components of the environment is essential, the focus should be on discussing the differential toxicity in the indicator organisms rather than the extraction methods and composition of the toxic environment.
Answer: the objective of this study is to determine the acute toxicity of AMD to D. magna and C. vulgaris via the TIE technique, as well as to assess the effectiveness of this technique at detecting the toxic compounds present in AMD. Although the bioindicators were chosen to understand the potential impact of an AMD flow to a freshwater ecosystem, D. magna and C. vulgaris are indicators at different levels of the food chain of a freshwater system, therefore, the information provided is complementary, but not strictly comparable.
Yours sincerely,
Prof. Gladys Vidal
Engineering and Environmental Biotechnology Group
Environmental Science Faculty & Center EULA-Chile
Universidad de Concepción, Concepción, Chile.
E-mail: glvidal@udec.cl
ww.eula.cl/giba
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Title : Assessment of acute toxicity of acid mine drainage via Toxicity Identification Evaluation (TIE) using Daphnia magna and Chlorella vulgaris
I found the paper very original, well prepared, and interesting, so I support its publication in the journal,after a minor revision. The statitical side in this paper is not clear. Even authors explained they used Kruskall-Wallis ANOVA, I cant find data related to this test (or even post-ANOVA test like Dunn !!). Globaly, I feel the paper is well prepared and a good read and various and appropiate analyses were performed. My specific comments and suggestions are reported below.
Specific recommendations :
-line 1: ‘via’ in italic please.
- lines 1 and 2: is it possible to make a title in an interrogative way? It is more attractive for readers.
- lines 64-68: aythors must give hypotheses at the end of the introduction. Then, in their conclusion section, they must give responses.
-lines 79: How pH can be adjusted and maintained in a constant pH? Is it a little bit variable? If yes give the SD.
-lines 132-133: what kind of transformations authors used?
-line 193: give the exact value of probability please
- line 196: ‘via’ in italic please
- More text must be added to the conclusions to explain the limitations and the global impact of the results obtained.
- I’m not seeing in the text where Kruskall-W was used in the results section!! Also, is there a multiple comparison? If yes, by using which test? Dunn ?
-References: check gain the format of all the references cited to align with MPDI criteria, in case of mistakes.
Author Response
We want to thank the reviewers for carefully reading the manuscript. All the comments were attended and the paper was carefully checked according to the editorial suggestions.
The following comments indicate our response to all of the questions and notes indicated by the Referees and the Editor. Please consider that unless otherwise expressed, the referenced lines correspond to the current numeration after the corrections were made. The revisions according the Reviewer comments have been highlight in yellow color on the revised version of this paper.
REVIEWER # 2
I found the paper very original, well prepared, and interesting, so I support its publication in the journal,after a minor revision. The statitical side in this paper is not clear. Even authors explained they used Kruskall-Wallis ANOVA, I cant find data related to this test (or even post-ANOVA test like Dunn !!). Globaly, I feel the paper is well prepared and a good read and various and appropiate analyses were performed. My specific comments and suggestions are reported below.
Comment #1: line 1: ‘via’ in italic please.
Answer: Now “via” is in italic letter
Comment #2: lines 1 and 2: is it possible to make a title in an interrogative way? It is more attractive for readers.
Answer: The authors were analyzing this very interesting proposal. However, due to the structure of the paper, they decided to keep it in an affirmative sentence structure.
Comment #3: lines 64-68: authors must give hypotheses at the end of the introduction. Then, in their conclusion section, they must give responses.
Answer: Now, at the end of the Introduction section: “The authors hypothesize that the defined indicators D. marga and C. vulgaris are sensitive to the compounds contained in AMD”. Meanwhile in the conclusion section: “The bioindicators D. magna and C. vulgaris were sensitive indicators of AMD toxicity and TIE tests”.
Comment #4. lines 79: How pH can be adjusted and maintained in a constant pH? Is it a little bit variable? If yes give the SD.
Answer: Now, for pH value was given a SD, that is: 4°C ± 0.1 °C.
Comment #5: lines 132-133: what kind of transformations authors used?
Answer: normality was assessed by Shapiro-wilk and homogeneity of variance by Fligner-Killeen. Logarithmic and square root transformations were performed, but still not enough to normalize the data. Therefore, the Kruskal-Wallis nonparametric test was applied. Details of the tests used were added in the methodology section (2.6).
Comment #6: line 193: give the exact value of probability please.
Answer: Thank you very much for the observation. The level of confidence was added.
Comment #7: line 196: ‘via’ in italic please
Answer: Now “vía” is in italic.
Comment #8: More text must be added to the conclusions to explain the limitations and the global impact of the results obtained.
Answer: Thank you very much for your comment. The last paragraph of conclusions was modified to include additional information on the limitations of the study and the overall impact of the results obtained.
Comment #9: I’m not seeing in the text where Kruskall-W was used in the results section!! Also, is there a multiple comparison? If yes, by using which test? Dunn ?
Answer: Thank you very much for your observation. It was indicated in the methodology section (2.6) to which data the Kruskal-Wallis test was applied. On the other hand, since significant differences (p < 0.05) were only found in the EDTA-treated fraction for the CODT and CODs parameters, it was not considered necessary to apply a multiple comparison.
Comment #10: -References: check gain the format of all the references cited to align with MPDI criteria, in case of mistakes.
Answer: The references were checked.
Yours sincerely,
Prof. Gladys Vidal
Engineering and Environmental Biotechnology Group
Environmental Science Faculty & Center EULA-Chile
Universidad de Concepción, Concepción, Chile.
E-mail: glvidal@udec.cl
ww.eula.cl/giba
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
The study titled "Assessment of acute toxicity of acid mine drainage via Toxicity Identification Evaluation (TIE) using Daphnia magna and Chlorella vulgaris" is an interesting study for determining the toxic effect level. However, some revisions are necessary.
1. Key words should be in alphabetical order. Abbreviations should not be used too much in the abstract section.
2. The novelty and differences of the study should be emphasized in more detail in the introduction section. In particular, information should be provided about the living groups used in toxicity.
3. Fish experiments are at the forefront in toxicity studies. Other microorganism-based tests may be more sensitive. However, the resistance of these organisms is low. Why was Vulgaris included while Daphnia was used in classical toxicity? Explain.
4. The purity levels and procurement processes of all chemicals used in the study should be explained. In addition, the brands and models of all measurement devices used should be specified.
5. Comparisons with international standards can also be added to the tables. It is important to know whether the data obtained is above or below the limits.
6. The bibliography section should be checked according to the journal writing rules.
Author Response
We want to thank the reviewers for carefully reading the manuscript. All the comments were attended and the paper was carefully checked according to the editorial suggestions.
The following comments indicate our response to all of the questions and notes indicated by the Referees and the Editor. Please consider that unless otherwise expressed, the referenced lines correspond to the current numeration after the corrections were made. The revisions according the Reviewer comments have been highlight in yellow color on the revised version of this paper.
REVIEWER # 3
The study titled "Assessment of acute toxicity of acid mine drainage via Toxicity Identification Evaluation (TIE) using Daphnia magna and Chlorella vulgaris" is an interesting study for determining the toxic effect level. However, some revisions are necessary.
Comment #1: Key words should be in alphabetical order. Abbreviations should not be used too much in the abstract section.
Answer: Now the key words are in alphabetical order. Abbreviations in the abstract section were reduced.
Comment #2. The novelty and differences of the study should be emphasized in more detail in the introduction section. In particular, information should be provided about the living groups used in toxicity.
Answer: The novelty of this study was incorporated between lines 66-70.
Comment #3: Fish experiments are at the forefront in toxicity studies. Other microorganism-based tests may be more sensitive. However, the resistance of these organisms is low. Why was Vulgaris included while Daphnia was used in classical toxicity? Explain
Answer: This is one of the first studies conducted with AMD and its toxicity detection. Fish populations are not abundant in the high mountains where mining operations are located, so traditional organisms such as Daphnia magna and Chlorella vulgaris were selected due to the biodiversity of these ecosystems.
Comment #4. The purity levels and procurement processes of all chemicals used in the study should be explained. In addition, the brands and models of all measurement devices used should be specified.
Answer: The purity levels of the chemicals and the models of all measurement devices were specified in the Materials and Methods section.
Comment #5. Comparisons with international standards can also be added to the tables. It is important to know whether the data obtained is above or below the limits.
Answer: Due to the type of mining operations around the world and the differences in geology across the planet, AMD toxicity is not comparable. This is why this work is so novel and unique, as it provides scientific evidence of AMD toxicity levels.
Comment #6: The bibliography section should be checked according to the journal writing rules.
Answer: The bibliography was checked according to the journal writing rules.
Yours sincerely,
Prof. Gladys Vidal
Engineering and Environmental Biotechnology Group
Environmental Science Faculty & Center EULA-Chile
Universidad de Concepción, Concepción, Chile.
E-mail: glvidal@udec.cl
ww.eula.cl/giba
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I reviewed the revised manuscript and the point-wise responses. The authors satisfactorily addressed most of the queries, comments, and anomalies. Therefore, I believe the revised version can be accepted for publication.
