Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript evaluates the genotoxicity of size-fractionated urban particulate matter using plant-based assays and compares these findings with mutagenicity data from the Ames test. The topic is relevant to environmental monitoring and public health, and the multi-endpoint experimental design represents a strength of the study. The data appear internally consistent, and the sequential assessment of cytotoxicity and genotoxicity enhances interpretability.

However, the manuscript in its current form has several conceptual and interpretative limitations. In particular, the claim that plant-based assays may serve as an alternative to the Ames test is not sufficiently supported from a mechanistic or comparative perspective. Moreover, the Results and Discussion sections remain largely descriptive and would benefit from clearer quantitative analysis and deeper interpretation. Substantial revisions are required before the manuscript can be considered for publication.

Major comments:

1. The manuscript frames plant-based assays as a potential alternative to the Ames test; however, the scientific basis for this claim is not sufficiently developed. The two systems assess different biological endpoints (bacterial gene mutations in the Ames test vs. eukaryotic DNA strand breaks and chromosomal damage in plant assays) and were applied to chemically distinct extracts (organic vs. aqueous). Therefore, the study does not directly demonstrate equivalence or comparative sensitivity between the two approaches. To substantiate the concept of “alternative,” the Discussion should more clearly address: 1) Whether the endpoints measured by plant assays overlap conceptually with those detected in the Ames test, or instead represent distinct layers of genotoxic information; 2) Whether plant-based assays provide comparable sensitivity to mutagenicity detection, or rather detect additional types of genetic damage not captured by bacterial systems; 3) Whether plant models are intended as substitutes, complements, or screening-level tools in relation to Ames testing. Clarifying this conceptual framework is essential to align the Discussion with the title and central claim of the manuscript.
2. While the Results clearly indicate higher genotoxic activity in finer PM fractions, the presentation remains largely descriptive. Quantitative comparisons among size fractions (e.g., fold change relative to control, relative potency among PM3–10, PM0.5–3, and PM0.5) are not explicitly discussed. Including clearer numerical comparisons would strengthen the interpretation of size-dependent effects.
3. Significant effects are primarily observed at the highest concentration tested (2 m³eq/mL), whereas lower concentrations often do not reach significance. However, dose–response relationships are not explicitly analyzed or interpreted. A brief discussion of potential threshold effects or concentration-dependent trends would improve the robustness of the Results.
4. Both PM0.5–3 and PM0.5 fractions significantly increased primary DNA damage (comet assay), whereas only PM0.5 significantly increased micronucleus frequency. This distinction between transient DNA damage and stable chromosomal alterations is biologically meaningful but is not addressed in the Results section. Highlighting this difference would improve the analytical depth of the presentation.
5. An important methodological aspect concerns the use of different extraction procedures for the two biological systems. The Ames test was performed on organic extracts, whereas plant-based assays were conducted on aqueous extracts. Because these extraction methods yield chemically distinct mixtures, the biological responses observed in the two systems are not directly comparable in terms of sensitivity or potency. While this design is scientifically valid for investigating different chemical fractions, the manuscript should explicitly acknowledge this limitation and clarify that plant assays are not evaluated as direct substitutes for the Ames test using the same exposure matrix. Addressing how this affects the interpretation of the “alternative” concept would substantially strengthen the Discussion.

 

Minor comments:

1. The Abstract is currently too concise and would benefit from additional clarification regarding sampling (information on the type of PM samples used and how they were collected), quantitative results, and the specific added value of plant-based assays relative to Ames testing. Given that the title emphasizes plant-based assays as a potential alternative to the Ames test, the Abstract should more clearly state in what sense plant tests provide an advantage or added value relative to bacterial mutagenicity testing.
2. The Introduction would benefit from additional clarification in several areas. In particular, a brief discussion on how plant-based toxicity and genotoxicity models relate to human health relevance would strengthen the rationale for their use. Moreover, the authors are encouraged to more clearly define the limitations of previous studies and to explicitly state how the present work addresses these gaps. Clarifying the rationale for the selected PM size fractions would also help readers better understand the motivation for the size-resolved approach. Finally, given that the title presents plant-based assays as a potential alternative to the Ames test, the Introduction should more clearly explain which limitations of the Ames test are complemented or addressed by plant-based models.
3. Section 2.1 would benefit from additional clarification. The manuscript does not specify how the three PM size fractions (PM3–10, PM0.5–3, PM0.5) were physically separated during sampling. Please indicate whether the high-volume sampler was equipped with a size-selective inlet or multi-stage impactor, and briefly describe the configuration used to obtain these fractions. In addition, it is unclear whether sampling over the 29-day campaign was performed using a single filter or with daily filter replacement. Clarifying this point, and indicating whether potential pressure drop effects (i.g., change of flow rate during sampling period) were monitored (if applicable), would improve transparency and reproducibility.

 

Author Response

Response to Reviewer 2 Comments

 

We would like to express our gratitude for your review of this manuscript. Please find below the detailed responses, with the corresponding revisions and corrections highlighted in track changes in the resubmitted file.

 

Point-by-point response to Comments and Suggestions for Authors

 

Major comments:

1. The manuscript frames plant-based assays as a potential alternative to the Ames test; however, the scientific basis for this claim is not sufficiently developed. The two systems assess different biological endpoints (bacterial gene mutations in the Ames test vs. eukaryotic DNA strand breaks and chromosomal damage in plant assays) and were applied to chemically distinct extracts (organic vs. aqueous). Therefore, the study does not directly demonstrate equivalence or comparative sensitivity between the two approaches. To substantiate the concept of “alternative,” the Discussion should more clearly address: 1) Whether the endpoints measured by plant assays overlap conceptually with those detected in the Ames test, or instead represent distinct layers of genotoxic information; 2) Whether plant-based assays provide comparable sensitivity to mutagenicity detection, or rather detect additional types of genetic damage not captured by bacterial systems; 3) Whether plant models are intended as substitutes, complements, or screening-level tools in relation to Ames testing. Clarifying this conceptual framework is essential to align the Discussion with the title and central claim of the manuscript.

Response 1: Following the constructive suggestions of the reviewer, we have included the requested clarification in the Discussion section (lines 330-333).

2. While the Results clearly indicate higher genotoxic activity in finer PM fractions, the presentation remains largely descriptive. Quantitative comparisons among size fractions (e.g., fold change relative to control, relative potency among PM3–10, PM0.5–3, and PM0.5) are not explicitly discussed. Including clearer numerical comparisons would strengthen the interpretation of size-dependent effects.

Response 2: We acknowledge the concerns raised by the reviewer. However, in contrast to other tests (e.g., the Ames test) which request or allow this type of data elaboration, the requested elaboration of the genotoxicity data (TI and MN) is not taken into consideration by the major guidelines. The strength of the test data is evaluated by means of a statistical analysis.

3. Significant effects are primarily observed at the highest concentration tested (2 m³eq/mL), whereas lower concentrations often do not reach significance. However, dose–response relationships are not explicitly analyzed or interpreted. A brief discussion of potential threshold effects or concentration-dependent trends would improve the robustness of the Results.

Response 3: Following the reviewer’s suggestion, we analysed the dose–response relationships among the data from genotoxicity tests by a linear correlation test. Almost all the datasets demonstrated a linear correlation among the doses (2, 1, 0 m³_eq/mL):

- Lepidium sativum TI PM3-10: R²=0.9678

- Lepidium sativum TI PM0.5-3: R²=0.9983

- Lepidium sativum TI PM0.5: R²=0.9961

- Allium cepa TI PM3-10: R²=0.8272

- Allium cepa TI PM0.5-3: R²=0.951

- Allium cepa TI PM0.5: R²=0.9892

- Lepidium sativum MN PM3-10: R²=0.9643

- Lepidium sativum MN PM0.5-3: R²=0.5192

- Lepidium sativum MN PM0.5: R²=0.9973

However, despite these concentration-dependent trends, it is not possible (and it is not within the scope of the tests) to determine a potential threshold dose due to the invalidation of the concept in the framework of the genotoxic assessment. It can be concluded from the experimental data that the lowest doses tested do not induce genotoxicity.

Nevertheless, as suggested by the reviewer, we added a specification to improve the Discussion section (lines 325-327).

4. Both PM0.5–3 and PM0.5 fractions significantly increased primary DNA damage (comet assay), whereas only PM0.5 significantly increased micronucleus frequency. This distinction between transient DNA damage and stable chromosomal alterations is biologically meaningful but is not addressed in the Results section. Highlighting this difference would improve the analytical depth of the presentation.

Response 4: Following the valuable input of the reviewer, we have improved the way in which this difference is highlighted in the Discussion section (lines 316-323).

5. An important methodological aspect concerns the use of different extraction procedures for the two biological systems. The Ames test was performed on organic extracts, whereas plant-based assays were conducted on aqueous extracts. Because these extraction methods yield chemically distinct mixtures, the biological responses observed in the two systems are not directly comparable in terms of sensitivity or potency. While this design is scientifically valid for investigating different chemical fractions, the manuscript should explicitly acknowledge this limitation and clarify that plant assays are not evaluated as direct substitutes for the Ames test using the same exposure matrix. Addressing how this affects the interpretation of the “alternative” concept would substantially strengthen the Discussion.

Response 5: Following the reviewer’s suggestions, we have included this clarification in the Discussion section (lines 340-343).

Minor comments:

1. The Abstract is currently too concise and would benefit from additional clarification regarding sampling (information on the type of PM samples used and how they were collected), quantitative results, and the specific added value of plant-based assays relative to Ames testing. Given that the title emphasizes plant-based assays as a potential alternative to the Ames test, the Abstract should more clearly state in what sense plant tests provide an advantage or added value relative to bacterial mutagenicity testing.

Response 1: We have modified the abstract following the reviewer's suggestion, in light of the editorial constraints imposed, which stipulated a maximum length of 200 words.

2. The Introduction would benefit from additional clarification in several areas.

a) In particular, a brief discussion on how plant-based toxicity and genotoxicity models relate to human health relevance would strengthen the rationale for their use.

Response 2a: Following the reviewer’s suggestions, we have included this clarification in the Introduction section (lines 114-118).

b) Moreover, the authors are encouraged to more clearly define the limitations of previous studies and to explicitly state how the present work addresses these gaps.

 Response 2b: We would like to make it clear that we do not consider the previous studies to be limited in any way. It has been observed that there is a lack of studies that have employed this approach. We are in favor of increased use of plant-based models for the evaluation of urban air quality. The present study aims to promote this plant-based approach, rather than fill potential gaps that not necessarily exist.

Following the reviewer’s suggestions, we have clearly stated the aims of this work.

c) Clarifying the rationale for the selected PM size fractions would also help readers better understand the motivation for the size-resolved approach.

 Response 2c: Following the reviewer’s request, details were added in the Introduction section (Lines 50-54).

d) Finally, given that the title presents plant-based assays as a potential alternative to the Ames test, the Introduction should more clearly explain which limitations of the Ames test are complemented or addressed by plant-based models.

Response 2d: We respectfully disagree with the reviewer's suggestion. The proposed in-depth analysis of the disadvantages of the Ames test in the evaluation of the genotoxicological aspect of the air pollutants) should be developed solely in the Discussion section, and not in the Introduction. Indeed, a deeper description had been included in the revision of the manuscript.

3. Section 2.1 would benefit from additional clarification. The manuscript does not specify how the three PM size fractions (PM3–10, PM0.5–3, PM0.5) were physically separated during sampling. Please indicate whether the high-volume sampler was equipped with a size-selective inlet or multi-stage impactor, and briefly describe the configuration used to obtain these fractions. In addition, it is unclear whether sampling over the 29-day campaign was performed using a single filter or with daily filter replacement. Clarifying this point, and indicating whether potential pressure drop effects (i.g., change of flow rate during sampling period) were monitored (if applicable), would improve transparency and reproducibility.

Response 3: Following the reviewer’s request, we included many more details to the Section 2.1.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The study investigated the genotoxic effects of urban fine particulate matter (PM10) using plant models onion bulbs and garden cress seedlings. ​

on my opinion the title could be shorter

Using plants as a potential valuable alternative  to assess the genotoxicity of urban fine particulate matter

 

Please explain the abbreviations from the beginning. Your List is not complete

All formulas must be quoted and explained, including indicating the units

Abstract must be reconsidered. The scope and the novelty must be stressed out and no abbreviations. Also some main ideas -even with figures- as findings - could be revealed.

In the concluding part - Conclusions-  take care to answer all scopes, but based on your own results.

stress the main findings essentially. For example the DNA Damage Detection: and the Micronuclei Formation

Make a clear info/detailed/ for Advantages of Plant-Based Tests, in comparison to standard methods. Also indicate diss-advantages

Both plant models effectively detected DNA damage caused by aqueous extracts of the finest PM fractions (PM0.5-3 and PM0.5) using the comet test. ​ Significant DNA damage was observed in undiluted extracts of PM0.5-3 and PM0.5 fractions.

Please give better comments and figures arguments for your nice ideas that the proposed tests are cost-effective, quicker, and more environmentally friendly, as they avoid the use of organic solvents and lengthy sample preparation. and that​ they also provide a reliable representation of water-leached airborne contaminants. ​

You are indicating that  while the Ames test confirmed mutagenicity of organic PM extracts, the plant-based tests highlighted the genotoxic effects of aqueous PM extracts, emphasizing the complementary nature of these methods. Please explain better this idea.

*The public health implication using plants is an indicator, on my opinion, but can be turned into a useful tool* - Develop this idea in order to attract more readers.

 

Author Response

Response to Reviewer 3 Comments

We would like to express our gratitude for your review of this manuscript. Please find below the detailed responses, with the corresponding revisions and corrections highlighted in track changes in the resubmitted file.

 Point-by-point response to Comments and Suggestions for Authors

Comment 1: The study investigated the genotoxic effects of urban fine particulate matter (PM10) using plant models onion bulbs and garden cress seedlings. ​

on my opinion the title could be shorter

Using plants as a potential valuable alternative  to assess the genotoxicity of urban fine particulate matter

Response 1: We would like to express our gratitude to the reviewer for the insightful feedback. We have partially modified the title according to the suggestion.

Comment 2: Please explain the abbreviations from the beginning. Your List is not complete

Response 2: Following the reviewer's suggestion, the abbreviation list has been amended.

Comment 3: All formulas must be quoted and explained, including indicating the units

 Response 3: The mitotic index alteration percentage (MIA%), which is the sole formula included in the manuscript, has been fully described in the section 2.8, along with the reference of the study in which this formula was described.

Comment 4: Abstract must be reconsidered. The scope and the novelty must be stressed out and no abbreviations.

 Response 4: We have modified the abstract following the reviewer's suggestion, in light of the editorial constraints imposed, which stipulated a maximum length of 200 words. As the only abbreviation used was "PM", we have specified its significance.

Comment 5: Also some main ideas -even with figures- as findings - could be revealed.

Response 5: We regret to inform the reviewer that the preceding statement was not sufficiently clear to enable an appropriate response.

Comment 6: In the concluding part - Conclusions-  take care to answer all scopes, but based on your own results.

stress the main findings essentially. For example the DNA Damage Detection: and the Micronuclei Formation

Response 6: Following the reviewer’s suggestion, we have improved the Conclusions section (lines 431 and 432-436).

Comment 7: Make a clear info/detailed/ for Advantages of Plant-Based Tests, in comparison to standard methods. Also indicate diss-advantages

Response 7: Following the reviewer’s suggestion, we have included a comprehensive description of the advantages and disadvantages of using plants as toxicological models (lines 379-388).

Comment 8: Both plant models effectively detected DNA damage caused by aqueous extracts of the finest PM fractions (PM0.5-3 and PM0.5) using the comet test. ​ Significant DNA damage was observed in undiluted extracts of PM0.5-3 and PM0.5 fractions.

Please give better comments and figures arguments for your nice ideas that the proposed tests are cost-effective, quicker, and more environmentally friendly, as they avoid the use of organic solvents and lengthy sample preparation. and that​ they also provide a reliable representation of water-leached airborne contaminants. ​

Response 8: Following the reviewer’s suggestions, we have the improved Discussion section (lines 359-372).

Comment 9: You are indicating that  while the Ames test confirmed mutagenicity of organic PM extracts, the plant-based tests highlighted the genotoxic effects of aqueous PM extracts, emphasizing the complementary nature of these methods. Please explain better this idea.

Response 9: Following the reviewer’s suggestion, we have better elucidate the concept in the Discussion section (lines 340-343).

Comment 10: *The public health implication using plants is an indicator, on my opinion, but can be turned into a useful tool* - Develop this idea in order to attract more readers.

Response 10: Following the reviewer’s suggestion, we have included some details in the Discussion section (lines 424-428) to emphasize the proposed approach as a valuable instrument within the regulatory public health framework.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors address mutagenicity of atmospheric PM by using plant models. The design of labwork is correct, results are easy to interpret and will provode possible new methodology for a wider audience. 

The weakness of the manuscript is the introduction section. The authors mention possible human health problems induced by air pollution. Instead, information should be given on what chemical components would have mutagenic/carcinogenic effects (PAHs, heavy metals most commonly). I would like to see data regarding the region where the samples were taken. The authors mention that they do not have chemical data (Line 336), still I supppose literature data are available.

Author Response

Response to Reviewer 4 Comments

 

We would like to express our gratitude for your review of this manuscript. Please find below the detailed responses, with the corresponding revisions and corrections highlighted in track changes in the resubmitted file.

 

Point-by-point response to Comments and Suggestions for Authors

Comment 1: The authors address mutagenicity of atmospheric PM by using plant models. The design of labwork is correct, results are easy to interpret and will provode possible new methodology for a wider audience. 

The weakness of the manuscript is the introduction section. The authors mention possible human health problems induced by air pollution. Instead, information should be given on what chemical components would have mutagenic/carcinogenic effects (PAHs, heavy metals most commonly). I would like to see data regarding the region where the samples were taken.

Response 1a: Following the reviewer’s suggestions, the Introduction section has been revised to incorporate additional information regarding air quality monitoring data and the chemical components of air pollution that could have mutagenic or carcinogenic effects related to the study area (the urban area of Brescia) (lines 40-48 and 55-64).

The authors mention that they do not have chemical data (Line 336), still I supppose literature data are available.

Response 1b: As the reviewer correctly observed, the reported references pertain to the chemical composition of Brescia's air over a period of nearly three decades.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed the reviewer’s comments and improved the clarity of the manuscript. In my opinion, the revised version is suitable for publication in its current form.

Author Response

Comments: The authors have adequately addressed the reviewer’s comments and improved the clarity of the manuscript. In my opinion, the revised version is suitable for publication in its current form.

Response 1: We would like to express our gratitude for your review of this manuscript and for the constructive suggestions that you gave us.

Reviewer 2 Report

Comments and Suggestions for Authors

Great corrections and complections. 

Try please yto complete the Conclusions answering to all proposed items raised  from/ in/  the Abstract. What îs important and could be used by readers- Mark it clearly.

Author Response

Comment 1: Great corrections and complections. 

Response 1: We would like to express our gratitude to the reviewer for reviewing this manuscript and for the constructive suggestions provided.

Comment 2: Try please yto complete the Conclusions answering to all proposed items raised  from/ in/  the Abstract. What îs important and could be used by readers- Mark it clearly.

Response 2: The Conclusion section was slightly modified to align more closely with the Abstract.