Comparing the Behavioral Impacts of Heavy Metals and Rare Earth Elements on Black Soldier Fly (Hermetia illucens) Larvae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a really interesting manuscript on the toxicity of two low concentrations of various heavy metals and all the rare earth elements on black soldier fly larvae.  The use of all the lanthanides is particularly interesting.  The increased use industrially of rare earth elements and their corresponding release into the environment is an important area of investigation, making this manuscript significant and timely. The manuscript is easy to read with a  minimum of grammatical, typographical, and related errors. However, the authors have failed to provide essential information on the inorganic chemistry aspects of the experimental section, that require being addressed.  Without this information (as described below), the manuscript is unsuitable for publication so I must recommend major revision.  Other than this, other concerns are relatively minor, although justification of some choices and the limitations thereof must be addressed in greater depth.

 

Major comments: You must indicate that the source of each heavy metals and rare earth are.  Exactly what chemical form (and manufacturer) was used is very important.  For example, whether you used a salt of Cr(III) or chromate (Cr(VI)) is extremely important as these different oxidation states of the element have entirely different chemistry.  As another example, almost all holmium salt are essentially insoluble in water; the form here is important.  The results cannot be properly accessed without this information.

Minor comments:

Abstract -  would be better to state "cobalt as a representative HM" rather than "cobalt (HM)" and "samarium as a representative REE" rather than "samarium (REE)".

Line 113 - "in" should not be bold.

Lines 115-126 - problems with use of semicolons in some places rather than commas.  Might consider breaking into smaller sentences.

Line 159 - "was" should be deleted.

Line 161  - move "all" to before "16" in line 162.

Figure 2 caption -  explicitly state yellow is HM and pink is REE.

Figure 3 - remove "1 ppm" from x-axis ticks for parts A-E and "10 ppm" for parts F-J.  The information is at the top of each box.  This will make the figure cleaner and easier to read.  Similarly, "heavy metals" could be moved to the top of each box rather than being reproduced 10 times.

Figure 4 -reformat as described in Figure 3.

Figures 5 and 6 are too small to read properly.

The limitations on choosing cobalt as a representative HM must be discussed as the mechanisms of toxicity vary greatly across the heavy metals.  

You do not state explicitly why samarium was chosen as the representative REE.

Author Response

Response to Reviewer #1

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments. We have carefully revised the manuscript according to all suggestions. Our detailed responses are provided below.

Major comment

Comment: You must indicate what the source of each heavy metal and rare earth element is. Exactly what chemical form (and manufacturer) was used is very important. For example, whether you used a salt of Cr(III) or chromate (Cr(VI)) is extremely important as these different oxidation states of the element have entirely different chemistry. As another example, almost all holmium salts are essentially insoluble in water; the form here is important. The results cannot be properly assessed without this information.

Response: We appreciate the reviewer for highlighting this important point. We fully agree that the chemical form, oxidation state, and source of each tested compound are essential for the accurate interpretation of toxicity profiles and to ensure experimental reproducibility. Accordingly, we have revised the manuscript to provide complete information for all 23 heavy metal and 16 rare earth element compounds used in this study. Specifically, we have added Supplementary Table S1 (heavy metals) and Supplementary Table S2 (rare earth elements), which provide the metal identity, chloride form, and CAS number for each compound used in this study. These additions enable readers to clearly identify the chemical species tested and facilitate the interpretation, comparison, and reproducibility of the observed neurobehavioral effects. We believe these revisions substantially improve the transparency and methodological rigor of the study.

Minor comments

We sincerely thank the reviewer for these valuable comments and careful evaluation of our manuscript. In response, we have thoroughly revised the manuscript to improve clarity, readability, and scientific rigor. Specifically, in the Abstract, we revised the wording to "cobalt as a representative HM" and "samarium as a representative REE" to better reflect that these compounds were selected as representative case studies rather than being universally representative of their respective metal classes.

We also corrected the formatting issue on Line 113 by removing the unintended bold formatting, rewrote the entire section (Lines 98-113) to improve clarity, eliminate redundant information, enhance logical flow, and present the methodology in a more concise and reader-friendly format, and deleted the unnecessary word "was" on Line 159. The caption of Figure 2 has been revised to explicitly indicate that yellow represents heavy metals (HMs) and pink represents rare earth elements (REEs). Following the reviewer's suggestion, Figures 3 and 4 were reformatted by removing the redundant "1 ppm" and "10 ppm" labels from the x-axis tick labels, as these concentrations are already indicated at the top of each panel, and repetitive group labels were repositioned to improve figure clarity and readability. In addition, the sizes and resolutions of Figures 5 and 6 were substantially increased to ensure that all text, labels, and clustering patterns are clearly legible in both the manuscript and the final published format.

Additionally, in response to this important comment regarding the selection of cobalt as a representative HM, we revised the Materials and Methods (Lines 206-216) as well as the Results (Lines 492-497, and Lines 517-521) to provide a clearer rationale for the transcriptomic case-study selection. Cobalt was selected as the representative heavy metal because it consistently exhibited among the strongest behavioral toxicities across the integrated behavioral analyses. Samarium was chosen as the representative rare earth element due to its pronounced and distinctive behavioral effects. These two compounds were therefore used as representative examples to explore the molecular mechanisms associated with the contrasting behavioral responses observed between HMs and REEs. We also clarified that, because both metal groups encompass chemically diverse compounds, the transcriptomic analyses of cobalt and samarium should be interpreted as representative mechanistic case studies rather than universal molecular signatures of all HMs or REEs (Lines 214–216, Lines 676-683, and Lines 711-714). This clarification defines the scope of the transcriptomic analysis and prevents overgeneralization of the molecular findings to all HMs and REEs.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Comparing the Behavioral Impacts of Heavy Metals and Rare Earth Elements on Black Soldier Fly (Hermetia illucens) Larvae" reports a comparative behavioral and transcriptomic assessment of 23 heavy metals and 16 rare earth elements across two acute exposure concentrations (1 and 10ppm), that are environmentally relevant. The thematic of this work is novel and reflects the recent focus that REEs had in the current global society context. 

The approach performed by the authors was pertinent and the tested endpoints well-chosen. The introduction, methods and discussion were mostly well-detailed and rich. Nonetheless, there some aspects that must be corrected to allow the publication of the manuscript.

 

The main comments/suggestions of the revieweer, are the following ones:

1) The references numbers in the text, must be uniform. In the introduction we can find the numeric representation like this [a;b;c] or this [a],[b],[c]. Please, use the same format throughout the text.

 

2) The last two paragraphs of the introduction do not follow a logic sequence. In the first one (penultimate of the introduction) is mentioned "to achieve the aim...", while the second paragraph (last of the introduction) is mentioned the aim itself. This is not logic. Also, these two paragraphs should be summarized. There is a lot of information that is present in the methods that do not need to be present in the introduction

 

3) Lines 143-145, the information present in this sentence, is already mentioned in the introduction

 

4) Figure 2 caption must depict, with clarity, which color represent each HM or REE. Moreover, there are 17 pink squares in the figure, while there are only mentioned 16 REEs (Line 23). The reviewer interpretates that pink squares are REEs.

 

5) Lines 246-247: please add citation to support the statement

 

6) Figure 5 must have better readability. It is difficult for the reader to visualize the different variables.

 

7) Lines 547-550 report redundant information. These data are already indicated in the introduction.

 

8) Lines 624-625 indicates no mortality was found. However, no indication was given in the methods, that this endpoint was monitored and how it was performed. Please add this information.

 

9) Lines 590-591 mentions dose-response relationship for HM. The reviwer does not agree with this. Looking to Figure 3, for thigmotaxis and fractal dimention, is visible a clear non-monotonic response. This must be discussed and is the opposite pattern described by the authors.

Author Response

Response to Reviewer #2

We sincerely thank the reviewer for the thorough evaluation of our manuscript and for the constructive comments and suggestions. We have carefully revised the manuscript accordingly. Our detailed responses are provided below.

Comment 1: The references numbers in the text, must be uniform. In the introduction we can find the numeric representation like this [a;b;c] or this [a],[b],[c]. Please, use the same format throughout the text.

Response: We sincerely thank the reviewer for identifying this formatting inconsistency. In response, we have revised the citation style throughout the manuscript to ensure a uniform reference format in accordance with the journal guidelines. All multiple citations are now presented consistently using the same notation.

Comment 2: The last two paragraphs of the introduction do not follow a logic sequence. In the first one (penultimate of the introduction) is mentioned "to achieve the aim...", while the second paragraph (last of the introduction) is mentioned the aim itself. This is not logic. Also, these two paragraphs should be summarized. There is a lot of information that is present in the methods that do not need to be present in the introduction.

Response: We appreciate the reviewer's valuable suggestion. The final two paragraphs of the Introduction have been completely reorganized to improve their logical flow. The study rationale and objectives are now presented before the methodological overview. In addition, the Introduction has been substantially condensed by removing methodological details that are more appropriately described in the Materials and Methods section. The revised Introduction now focuses on the study background, existing knowledge gaps, the novelty of the work, and the overall study objectives (Lines 98-113).

Comment 3: Lines 143–145, the information present in this sentence, is already mentioned in the introduction.

Response: Thank you for pointing out this redundancy. The repeated information has been removed from the Methods (Section 2.1) to avoid duplication and improve the overall readability of the manuscript.

Comment 4: Figure 2 caption must depict, with clarity, which color represent each HM or REE. Moreover, there are 17 pink squares in the figure, while there are only mentioned 16 REEs (Line 23). The reviewer interpretates that pink squares are REEs.

Response: We thank the reviewer for this observation. The Figure 2 caption has been revised to explicitly state that yellow represents heavy metals (HMs), whereas pink represents rare earth elements (REEs). To eliminate potential confusion, the figure has been revised to clearly distinguish the control from the REE compounds, and the caption has been updated accordingly (Lines 158-159).

Comment 5: Lines 246–247: please add citation to support the statement.

Response: We sincerely thank the reviewer for this helpful suggestion. In response, we have added the appropriate references to support the statement and improve its scientific basis.

Comment 6: Figure 5 must have better readability. It is difficult for the reader to visualize the different variables.

Response: We thank the reviewer for this helpful suggestion and agree that the original figure could be improved. Accordingly, Figure 5 has been enlarged and reformatted to enhance readability, including increasing the font size and optimizing the panel layout, while maintaining the original data presentation.

Comment 7: Lines 547–550 report redundant information. These data are already indicated in the introduction.

Response: We sincerely appreciate the reviewer's helpful comment. In response, the redundant information in the Discussion has been replaced with a more concise version to eliminate unnecessary repetition. The revised Discussion (Lines 552–555) now places greater emphasis on interpreting the findings rather than reiterating background information already presented in the Introduction.

Comment 8: Lines 624–625 indicates no mortality was found. However, no indication was given in the methods, that this endpoint was monitored and how it was performed. Please add this information.

Response: We sincerely thank the reviewer for identifying this omission. The Materials and Methods section (Lines 149-152) has been revised to describe how mortality was monitored throughout the 24-hour exposure period. Specifically, larval survival was assessed immediately before behavioral recording by counting live and dead larvae in each treatment group. Larvae showing no visible movement or response to gentle mechanical stimulation were considered dead. We also clarified that no mortality was observed in any treatment group at either 1 or 10 ppm (Lines 630-632).

Comment 9: Lines 590–591 mentions dose-response relationship for HM. The reviewer does not agree with this. Looking to Figure 3, for thigmotaxis and fractal dimension, is visible a clear non-monotonic response. This must be discussed and is the opposite pattern described by the authors.

Response: We sincerely thank the reviewer for this important observation. We agree that the original wording could be interpreted as implying a uniform monotonic dose-response across all behavioral endpoints. To address this concern, we have revised throughout the manuscript, including Results (Lines 320-326, Lines 391-395), and Discussion (Lines 608-621, Lines 624-626) to acknowledge the endpoint-specific nature of the observed responses.

For the HM behavior profile, we now clarify that entropy exhibited the clearest concentration-related change, whereas thigmotaxis and fractal dimension displayed metal-specific non-monotonic responses rather than a classical monotonic dose-response pattern. We further emphasize that increasing HM concentration shifted the overall neurobehavioral profile rather than uniformly amplifying every behavioral endpoint. This revised interpretation is now fully consistent with the behavioral patterns presented in Figure 3 and provides a more accurate description of the complexity of HM-induced neurobehavioral toxicity.

Finally, thank you once again for the opportunity to revise and improve our manuscript. The authors sincerely hope that the revised version meets the high standards of Toxics and deeply appreciate the reviewers' insightful comments and your kind consideration of our work for publication. Should any further clarification be needed, the authors would be pleased to provide it.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all my comments; however, while they have included the form of all the metals used, they have put this information in the supplemental section.  This is not appropriate as the readers should not have to go to the supplemental section to understand the basics of what is going on with some of the metals!  The form of each metal must be in the manuscript, not the supplemental material.

Author Response

Response to Reviewer #1

We sincerely thank the reviewer for this valuable suggestion and fully agree that the chemical form of each tested metal is essential information that should be readily accessible to readers. In response, we have revised the manuscript by incorporating the complete list of heavy metal and rare earth element compounds directly into the Materials and Methods (section 2.2) as Table 1 and Table 2, respectively. These tables include the chemical form (including oxidation state where applicable) and CAS number for all compounds used in this study, allowing readers to readily identify the tested substances without referring to the Supplementary Materials.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript was vastly improved. The introduction now follows a logic sequence, the redundant sentences were removed and the results report is more accurate as well as the discussion.

 

The reviewer would like to see more clear figures, but al so understands that is difficult to include all the information and still have the best visibility of the different variables. Nonetheless, there was an improvment comparing to the first version.

 

The reviewer reinforces that the Figure 2. has 17 pink squares (REEs), while the authors mentioned that only 16 REEs were studied. Please correct this. 

Author Response

Response to Reviewer #2

We sincerely thank the reviewer for the positive evaluation of our revised manuscript and for recognizing the improvements made to the Introduction, Results, Discussion, and overall figure presentation. We greatly appreciate these encouraging comments.

In response to the remaining concern, we carefully re-examined Figure 2 and identified the graphical error. The extra pink square was inadvertently included during figure preparation. This has now been removed, and the revised figure correctly displays the 16 rare earth elements (REEs) evaluated in this study, consistent with the manuscript text.

Finally, thank you once again for the opportunity to revise and improve our manuscript. The authors sincerely hope that the revised version meets the high standards of Toxics and deeply appreciate the reviewers' insightful comments and your kind consideration of our work for publication. Should any further clarification be needed, the authors would be pleased to provide it.