Synthesis and Characterization of Humic/Melanin-like Compounds by Oxidative Polymerization of Simple Aromatic Precursors

Round 1

Reviewer 1 Report

Dear Authors,

The manuscript contains interesting and proper information about your investigation into the relationship between molecular and optical properties of humic fractions of DOM through the oxidation and polymerization of simple aromatic structures. The study focused on two types of samples: 1,8-DHN and 5,6-DHI. The study results indicate that 1,8-DHN was successfully oxidized and polymerized after UV irradiation and aging, while 5,6-DHI resulted in the formation of dark insoluble precipitates during the aging process.

Also, this study focuses on the optical and molecular changes in the samples using absorbance and fluorescence spectra, as well as mass spectrometry. Overall, you found that the oxidized 1,8-DHN and 5,6-DHI samples showed similar trends in their absorbance and fluorescence spectra over time compared to the humic fractions of CDOM. Through this study, it would be preferable to shed light on the potential for mimicking the optical properties of humic fractions of CDOM through the oxidation and aging of simple melanin precursors. Your explanations have important implications for understanding the relationship between molecular and optical properties of humic substances and may have applications in various fields such as environmental and materials science.

The work is per ensemble relatively well written, but some improvements, corrections, and changes in the structure of the paper are necessary.

I recommend taking into consideration the following:

1.     Some formulated objectives are necessary for the introduction section about the research study and the novelty in the field of spectral analysis;

2.     To provide additional information regarding work protocols and performance of each analysis equipment and standards, respectively (e.g., performance parameters for measurements on specific values: LOD, LOQ, repeatability, reproducibility, etc.);

3.     A comparative analysis of the research results by correlating the analysis techniques used;

4.     And which are or will be the perspectives based on your research in assessing water quality?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript presents an interesting attempt to produce and to mimic optical properties of humic-like compounds using UV-induced polymerization of simple melanin precursors (1,8-dihydroxy naphthalene (1,8-DHN) and 5,6-dihydroxy indole (5,6-DHI). Topic of manuscript is related to Environmental Chemistry and fits well to “Water Quality and Contamination” Section of the Journal.

Using UV irradiation and aging process authors manage to obtain the mixture of oligomers of starting compounds which mimic the absorption and fluorescence properties of CDOM. ESI-MS and MALDI-MS analysis reveal that dimers/trimers/tetramers are main species in UV-induced polymerization process.

This manuscript could be published, but in current state it is very rough especially from the technical points of view. I have many technical remarks and some questions concerning presentation and discussion of results. Major Revision is recommended according to the remarks below.

Major comments

1)      Authors irradiate their compounds in 1:10 mixture of ACN:water. Is it possible to expect any influence of ACN to composition/properties of final oligomers? Some discussion of this moment will be helpful.

2)      Why 3 hours of UV irradiation were used? How more prolonged (or shortened) irradiation will influence on final composition of oligomers?

3)      All compounds and Suwannee River HA are pH-sensitive, their optical/fluorescence properties can greatly depend on pH. I failed to find in the text information about starting pH of the solution and its changes upon irradiation. Please, indicate pH in the figures’ captions and in the text.

4)      Figure 3 – results for initial 1,8-DHN and 5,6-DHI solutions without irradiation have to be shown. It is common thing in ESI-MS measurements that starting compounds formed dimer/oligomer ions at high initial concentration.

5)      Line 348-350. This conclusion is speculative. Authors don’t investigate the origin of electronic transition in formed oligomers. So alternative point of view “the absorbance and fluorescence spectra of CDOM are formed because of the superposition of multiple individual chromophores” is still in charge. As I understand, there is no preliminary HPLC separation of irradiated mixtures before MS analysis. So different type of oligomers can be produced by oxidation of starting compounds with very different individual absorption spectra. And combination of many absorbing species with different spectra leads to observed structureless spectrum.

6)      Lines 432-433. Here and earlier in the text authors proposed some “charge transfer interactions within the oxidized 1,8-DHN oligomers.” This is pure speculative statement based on analogy with humic substances (HS). Indeed, in HS, which contains different residues of donor and acceptor nature, it is possible to realize CT processes. But I am not sure that it is a case of studied oligomers. Most probably, observed red-shift of absorption and emission spectra is a common effect of elongation of aromatic system upon oligomerization.

7)      Lines 455-359. m/z 331.01 for 1,8-DHN and 255.23 for 5.6-DHI are discussed in the text but such signals are not presented in Figure 3. In opposite, m/z for dimers (319) and trimers (479, 487) of 1,8-DHN and dimers (297) for 5.6-DHI along with unknown signals at 200 and 414 are clearly seen, but is not discussed in the text. How it could be?   

 Technical comments:

 

1.       Line 51, please give abbreviation of “Chromophoric dissolved organic matter” at a first appearance in the text (Line 38).

2.       Probably, formulation of goals (Lines 36-45) of current study is better to move in the end of Introduction (after Line 124).

3.       Table 1. “Concentration in ACN” or in “1:10 ACN:water mixture”? Please, specify.

4.       Line 207 and further in the text. “excitation peaks” it is better to change to “absorption peaks” or “absorption bands with maxima at”

5.       . Line 222-228 All these findings are much clearly described in Lines 371-378 and can be omitted.

6.       The Linear fits (Figure 1, inserts) are poorly seen.  And it is better to add description of these fits to Figure Caption. And something is wrong with this Figure – top pictures for 1,8-DHN are differ from the same pictures in Figure S2. Looks like you mixed them up.

7.       Figure S1, Correct the Figure Caption, results for 5,6-DHI and 1,8-DHN are mixed up. And all figures present results with/without H2O2, why you mentioned them differently?

8.       Figure S2, Quality of figure have to be improved. It is better to indicate spectra not by color, but by number of the day.

9.       Line 223 “samples, demonstrating…” Finish this sentence, please.

10.   Figure S5 – indicate that it is Excitation-emission matrix (EEM) spectra.

11.   Table S1 – structure C16H4N2O2 is impossible, it should be replaced for more adequate.

12.   Information in Tables S1 and S2 correspond to ESI-MS or MALDI-MS results? If former – why there is difference between peaks in Figure 3 and peaks in the Tables?

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

In the manuscript under consideration, the authors attempted to replicate the optical signatures of DOM from simple precursors by oxidizing and initiating the polymerization of the melanin precursors 1,8-dihydroxy naphthalene (DHN) and 5,6-dihydroxy indole (DHI) with the addition of hydrogen peroxide and/or with ultraviolet (UV) irradiation. The manuscript presents information of potential interest to the journal readers. However, there are minor issues to be addressed before the manuscript is considered further:

 

·       It is essential to highlight the material merits and work novelty in the abstract.

·       It is also recommended to either avoid or limit abbreviations in the abstract.

·       The keywords selection should also be reviewed, general keywords and abbreviations are not recommended.

·       The introduction section should be modified to cover other efforts and materials used in this field.

·       The materials and methods section should be supplemented with information detailing how the experiments were arranged, the number of experiments for each case investigated, how was the 5% hydrogen peroxide addition achieved as well as the quality assurance issues should also be highlighted in the manuscript.

·       The reason behind selecting 3 hours as UV exposure time should be highlighted to help the reader.

·       There is insufficient justification for the choice of storage time.

·       All the equipment used in the study should include information about the manufacturer (name, city, country).

·       The quality of the figures should be improved. Uniformity in terms of fonts in the figures should be observed.

The formatting requirements for references should be adhered to by the authors.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

Thank you for your works.

I would like to add the following:

1. Kindly ask the authors to pay attention to the order of section numbering: section 2.5. (L287) is a front of 2.4 (L298)

2. Could the authors specify which type of "concentration in 1:10 ACN: water mixture" refers to in Table 1?

3. The authors should comply with the formatting requirements for references.

 

Author Response

Thank you for your works.

I would like to add the following:

1. Kindly ask the authors to pay attention to the order of section numbering: section 2.5. (L287) is a front of 2.4 (L298)

Thank you for your feedback, this error has been fixed.

1. Could the authors specify which type of "concentration in 1:10 ACN: water mixture" refers to in Table 1

Please see lines 189-190. The concentrations in table 1 refer to the concentrations of the samples prepared for UV irradiation experiments, (diluted by 1:10 from stock solutions).

1. The authors should comply with the formatting requirements for references.

Thank you for your feedback, the references have been modified according to the MDPI handout.

Reviewer 2 Report

Authors greatly improved the mansucript according to the remarks. I am generally satisfied by revised manuscript and now it worth to be published in the Journal. 

Author Response

Thank you very much for your feedback.