Review Reports - Propylene Phenoxetol (1-Phenoxypropan-2-ol): Its Use as an Anesthetic for Echinoderms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

The manuscript's title and core claims should be revised to avoid exceeding the limits of existing evidence; statements suggesting that PP is a generally suitable and safe anesthetic for echinoderms, or that it is superior to MgCl₂, should be presented with greater caution unless directly supported by experimental evidence. To ensure the reproducibility of the literature review, the search strategy, databases, inclusion and exclusion criteria, and the record verification process must be clearly outlined; fluctuations in publication frequency should not be interpreted as direct evidence of actual usage rates or the impact of misinformation. Furthermore, the conceptual and experimental distinctions between the suppression of nerve conduction, immobilization, muscle relaxation, and reversible general anesthesia must be clarified; mechanistic findings derived from isolated tissue and nerve preparations should be treated separately from evidence regarding anesthetic efficacy in the whole organism. The effects of PP and MgCl₂—particularly on mutable collagenous tissue—should be discussed in a balanced manner, and the potential superiority of PP should be presented as a hypothesis requiring testing through comparative experiments rather than as a confirmed conclusion. Additionally, the comparability of existing protocols and the sufficiency of the evidence should be evaluated by preparing a comprehensive table that includes variables such as species, concentration, solvent medium, exposure duration, criteria for anesthesia and recovery, and experimental objectives. Regarding the historical debate over the chemical identity of PP, publications should be categorized based on the strength of the available evidence, and the inability to verify this identity should not be interpreted simply as an error. Inferences regarding the antioxidant effect of PP, its prevention of autolysis in sea cucumbers, and potential commercial applications must also be drawn with due regard for the limitations of the available experimental and toxicological evidence. Alos, "Table S1" in line 194 is unclear and should be checked. The years in line 498, 506, 512, 518, 523, 587, 601, 607, 624, 644, 698, 709, 720, 762, 784, 790, 795, 814 used in the references should be marked in bold. Moreover, More up-to-date references should be included in the text.

The sentence "The absence of comprehensive toxicological data remains an important limitation when evaluating the suitability of chemical agents for aquatic organisms, highlighting the value of biomarker-based approaches and bioaccumulation assessments in aquatic toxicity research (Serdar et al., 2025 - DOI: 10.4194/AQUAST2219)" could be added to the paragraph regarding the lack of toxicological data and/or to the dose-exposure assessment.

The sentence "Aquatic invertebrates such as *Artemia franciscana* are also used in experimental aquaculture research, emphasizing the importance of species-specific methodological considerations when interpreting biological responses across aquatic taxa (Amin et al., 2025 - DOI: 10.4194/AQUAST1995)" could be added to the paragraph in the introduction explaining the importance of aquatic invertebrates in experimental research and/or to the discussion on the mechanism of anesthesia.

Regarding animal welfare, as well as the discussions on physiological safety and recovery, the following sentence could be added: "The evaluation of anesthetic agents should extend beyond the induction of immobility to include physiological stress responses and recovery-related outcomes, as physiological adaptation and oxidative stress are important components of aquatic animal health assessment (Firdaus et al., 2026 - DOI: 10.4194/AQUAST3402)."

The sentence "The importance of evaluating concentration-dependent responses is illustrated by Mercer et al. (2025- DOI: 10.1002/aff2.70134), who assessed relaxation and recovery times alongside behavioral responses in greenlip abalone exposed to different relaxants. Their findings also demonstrate the value of considering post-exposure outcomes, including survival and longer-term growth, rather than relying solely on the immediate induction of relaxation" can be added to the dose, exposure duration and recovery data.

These two references can be considered together to reinforce the PP vs. MgCl₂ debate through both comparative molluscan data and direct echinoderm research: Comparative evidence from greenlip abalone indicates that both propylene phenoxetol and magnesium chloride can be promising relaxants under the tested conditions, although their effects must be evaluated in relation to behavioural responses, recovery, survival, and longer-term outcomes (Mercer et al., 2025). More recently, propylene phenoxetol has also been investigated directly in adult Holothuria (Metriatyla) scabra (Goundar et al., 2026 - DOI: 10.1155/are/5849821). Together, these studies strengthen the rationale for species-specific evaluation of propylene phenoxetol but do not establish its universal superiority over magnesium chloride or its suitability for all echinoderm taxa and experimental application.

Author Response

I thank Reviewer 1 for their helpful comments. The following are my responses to the points raised (numbered 1-13). Line numbers refer to the revised manuscript.

Point 1.

Response 1.

(a) The title has been changed to avoid the word “suitable”.

(b) As stated in the Abstract, the review is intended to endorse PP as a “useful option” for the anesthetization of echinoderms and only suggests it as an alternative to MgCl₂ for experimental work on isolated preparations containing MCT, pending further investigation of the action of MgCl₂ on MCT. This has been clarified by additional wording in the Abstract, in the last paragraph of the Introduction, and in an additional paragraph in Section 4.3.

Point 2.

To ensure the reproducibility of the literature review, the search strategy, databases, inclusion and exclusion criteria, and the record verification process must be clearly outlined; fluctuations in publication frequency should not be interpreted as direct evidence of actual usage rates or the impact of misinformation.

Response 2.

(a) Information on the literature search was provided in the caption of Table S1, which was submitted as a supplementary file together with the original manuscript.

(b) I think that it is reasonable to suggest there might be at least a loose correlation between publication frequency and usage rate, though this could have been better expressed in the original text. Accordingly, the first paragraph of Section 2.2.1 and the last paragraph of Section 4.2 have been reworded to introduce more caution.

Point 3.

Furthermore, the conceptual and experimental distinctions between the suppression of nerve conduction, immobilization, muscle relaxation, and reversible general anesthesia must be clarified; mechanistic findings derived from isolated tissue and nerve preparations should be treated separately from evidence regarding anesthetic efficacy in the whole organism.

Response 3.

The distinction between different anesthetic-induced states has not been made in publications referring to PP use as an echinoderm anesthetic and there have been no related investigations. This deficiency is now highlighted in the third paragraph of Section 2.2.1 and first paragraph of Section 4.3.

Point 4.

The effects of PP and MgCl₂—particularly on mutable collagenous tissue—should be discussed in a balanced manner, and the potential superiority of PP should be presented as a hypothesis requiring testing through comparative experiments rather than as a confirmed conclusion.

Response 4.

Please see Response 1(b).

Point 5.

Additionally, the comparability of existing protocols and the sufficiency of the evidence should be evaluated by preparing a comprehensive table that includes variables such as species, concentration, solvent medium, exposure duration, criteria for anesthesia and recovery, and experimental objectives.

Response 5.

Most of this information was provided in Table S1, which was submitted as a supplementary file together with the original manuscript. Criteria for PP-induced anesthesia and subsequent recovery of whole animals are not addressed explicitly in the literature. The need to establish such criteria has been added in the first paragraph of Section 4.3. The anesthetic action of PP on isolated preparations has been demonstrated by the inhibition of responses to neuro-active agents such as neurotransmitters or elevated K⁺ concentrations, which is now illustrated in an additional Figure (new Figure 2).

Point 6.

Regarding the historical debate over the chemical identity of PP, publications should be categorized based on the strength of the available evidence, and the inability to verify this identity should not be interpreted simply as an error. Inferences regarding the antioxidant effect of PP, its prevention of autolysis in sea cucumbers, and potential commercial applications must also be drawn with due regard for the limitations of the available experimental and toxicological evidence.

Response 6.

(a) I agree that inability to verify that PP was used should not be interpreted as an error. It was O’Neill (1994) who first proposed this as a reason for doubting its identity. Because of that, I err on the side of caution and exclude from the literature analysis (Fig. 1A) the small number of publications in which there is no corroborating evidence that PP was used, while drawing attention to the even smaller number of publications in which there is evidence of confusion (between PP and 2-phenoxyethanol) and therefore genuine cause for doubting the identity of the compound used.

(b) Please note that the claim that PP is an antioxidant is already fully discussed in Section 3.2.1 where it is explained that there is no evidence, experimental or otherwise, for it having antioxidant activity, and that the suggestion that it does arose from a misunderstanding that PP was the same compound as Laborit et al.’s “propylène phénoxétol”.

(c) Evidence for the ability of PP to block autolysis was provided by Hill (1966) and Hill & Reinschmidt (1976) (in the first paragraph of Section 3.2.1). That the evidence includes the results of an experimental investigation has been highlighted by an addition in the first paragraph of Section 3.2.1, and the need for verification has been added to the second paragraph of Section 4.2.

(d) Please note that the discussion in Section 4.2 about potential commercial applications suggests only that “it would be worth exploring its potential” and that “there would seem… to be “scope” for certain uses. The need for further toxicological information is also acknowledged.

Point 7.

"Table S1" in line 194 is unclear and should be checked.

Response 7.

This refers to the supplementary file that was submitted together with the main text.

Point 8.

The years in line 498, 506, 512, 518, 523, 587, 601, 607, 624, 644, 698, 709, 720, 762, 784, 790, 795, 814 used in the references should be marked in bold.

Response 8.

The ‘Biology’ template indicates that book publication years should be in regular font.

Point 9.

More up-to-date references should be included in the text.

Response 10.

The only such reference of which I am aware is Goundar et al. (2026). Please note that I submitted the manuscript on 1.10.26. Goundar et al. (2026) was published on 29.9.26 and I did not receive notification of this until 2.10.26. A mention of it has been added to the third paragraph of Section 2.2.1.

Point 10.

Response 10.

I agree that the first general point here is worth making. A relevant comment has been added to the last paragraph of Section 4.3. Because Serdar et al. (2025) does not refer to anesthetics and introduces new concepts of biomarkers and bioaccumulation assessments, I have cited as an alternative Coleman & Edmands (2024).

Point 11.

Response 11.

In my view, the issue of species-specific methodological considerations and mention of a crustacean arthropod constitute too much of a digression from the text at these locations. A suggestion about potential target species for establishing standardized protocols is already included in Section 4.3.

Point 12.

Response 12.

The suggested comment has been added to the first paragraph of Section 4.3, but citing Carter et al. (2024).

Point 13.

Response 13.

A relevant comment has been added to the first paragraph of Section 4.3, citing Mercer et al. (2025).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive narrative review of the historical and current use of propylene phenoxyethanol (PP, 1-phenoxypropan-2-ol) as an anesthetic for echinoderms. The author addresses a very specific yet often overlooked issue: the sharp decline in PP usage observed since the 1990s, driven by the dissemination of erroneous chemical and physiological information in the scientific literature. The topic aligns with the scope of the journal Biology and is valuable for both invertebrate welfare and marine biology.
Upon reviewing the manuscript, I have the following comments and recommendations:
1. Although this work is a narrative/historical review rather than a rigorous systematic review (and thus does not require a full PRISMA flowchart), the manuscript would benefit from a brief explanation of the literature selection process. In Section 2.2.1, the author mentions a "literature search" covering the period from 1960 to 2026. Adding a short paragraph clearly specifying the databases used (e.g., Google Scholar, Scopus, PubMed) and the search timeframe would enhance the study's transparency and scientific rigor.
2. The author makes a compelling case for returning to the use of PP instead of magnesium chloride (MgCl₂), highlighting the osmotic damage caused by the latter. However, to ensure complete objectivity, the review should also discuss the practical drawbacks of PP in greater detail. For instance, MgCl₂ is available in any laboratory, is non-toxic to humans, and is extremely inexpensive. On the other hand, PP requires careful handling as an industrial chemical, is characterized by certain data gaps regarding human carcinogenicity, and dissolves "slowly" in water. Including a summary table comparing MgCl₂ and PP based on these practical criteria would significantly enhance the article's value for practicing biologists.
3. Line 82: The author notes that all concentration values were converted to percentages. It would be helpful to briefly indicate the standard mass-to-volume ratio in parentheses right at the beginning (e.g., whether 0.1% corresponds to approximately 6.6 mmol/L) so that the information is clear to readers accustomed to molar units.
4. Reference 34: Please verify the year "2026" listed in the bibliography for chronological accuracy, or clarify whether this refers to a publication released online prior to the print version.

Author Response

I thank Reviewer 2 for their helpful comments. The following are my responses to the points raised (numbered 1-4). Line numbers refer to the revised manuscript.

Point 1.

Although this work is a narrative/historical review rather than a rigorous systematic review (and thus does not require a full PRISMA flowchart), the manuscript would benefit from a brief explanation of the literature selection process. In Section 2.2.1, the author mentions a "literature search" covering the period from 1960 to 2026. Adding a short paragraph clearly specifying the databases used (e.g., Google Scholar, Scopus, PubMed) and the search timeframe would enhance the study's transparency and scientific rigor.

Response 1.

Please note that information on the literature search was provided in the caption of Table S1, which was submitted as a supplementary file together with the original manuscript.

Point 2.

The author makes a compelling case for returning to the use of PP instead of magnesium chloride (MgCl₂), highlighting the osmotic damage caused by the latter. However, to ensure complete objectivity, the review should also discuss the practical drawbacks of PP in greater detail. For instance, MgCl₂ is available in any laboratory, is non-toxic to humans, and is extremely inexpensive. On the other hand, PP requires careful handling as an industrial chemical, is characterized by certain data gaps regarding human carcinogenicity, and dissolves "slowly" in water. Including a summary table comparing MgCl₂ and PP based on these practical criteria would significantly enhance the article's value for practicing biologists.

Response 2.

I have provided a new table (Table 2) comparing anesthetic-related aspects of PP, MgCl₂andMS-222.

Point 3.

Line 82: The author notes that all concentration values were converted to percentages. It would be helpful to briefly indicate the standard mass-to-volume ratio in parentheses right at the beginning (e.g., whether 0.1% corresponds to approximately 6.6 mmol/L) so that the information is clear to readers accustomed to molar units.

Response 3.

This information has been added to the last paragraph of the Introduction.

Point 4.

Reference 34: Please verify the year "2026" listed in the bibliography for chronological accuracy, or clarify whether this refers to a publication released online prior to the print version.

Response 4.

“2026” is correct. The journal’s “Cite this article” information (at https://link.springer.com/article/10.1007/s12601-025-00249-3#citeas) is as follows:

Kim, T., Jöst, A.B., Park, HS. et al. Marine Macrobenthos Diversity of Chuuk Atoll in the Federated States of Micronesia, Western Tropical Pacific. Ocean Sci. J. 61, 2 (2026). https://doi.org/10.1007/s12601-025-00249-3

Reviewer 3 Report

Comments and Suggestions for Authors

This is a well-written and valuable review by the leading authority on propylene phenoxetol (PP), making a convincing case that PP has been unjustly neglected as an echinoderm anesthetic due to accumulated misinformation about its identity, solubility, volatility, and mechanism of action. The historical and chemical sections are excellent and represent the paper's strongest contribution.

However, several issues require attention before publication:

1. Bibliometric methodology is weak. The quantitative claims in Figure 1 rest on an unreproducible Google Scholar search with an untested assumption that non-anesthetic citations affect all agents proportionally—likely false for menthol and MS-222. Exact search strings, dates, and absolute counts must be reported.

2. The anti-autolysis claim is overstated.Hill's 1966/1976 findings do not justify the confident recommendations for sea-cucumber production chains, especially given the author's own admission that no antioxidant mechanism is established.

3. The MgCl₂ comparison is one-sided.Mg²⁺ effects on mutable collagenous tissue may be desirable in some protocols. A comparison table across PP, MgCl₂, MS-222, and menthol would help.

4. Safety/regulatory discussion is too brief for applied recommendations. PP lacks carcinogenicity data and food-chain approval; this must be stated prominently, not buried.

5. Practical guidance is missing.A summary box with starting concentration (0.1%), preparation, induction/recovery times, and limitations would greatly increase usefulness.

6. Numerous typos and errors ("echinoiderm," "Amphiuma" for Amphiura, duplicated refs [99]/[143], inconsistent UK/US spelling.

Author Response

I thank Reviewer 3 for their helpful comments. The following are my responses to the points raised (numbered 1-6). Line numbers refer to the revised manuscript.

Point 1.

Bibliometric methodology is weak. The quantitative claims in Figure 1 rest on an unreproducible Google Scholar search with an untested assumption that non-anesthetic citations affect all agents proportionally—likely false for menthol and MS-222. Exact search strings, dates, and absolute counts must be reported.

Response 1.

I have inserted a new Figure 1B which shows the results of an alternative analysis of the literature. The aim of this analysis is to produce relative citation rates for the six most commonly used echinoderm anaesthetics calculated from counts of relevant publications. For practical reasons this employed a more restricted literature search than that used to detect PP publications alone (as illustrated in Figure 1A). The methodology is explained in the caption of Table S1 (supplementary file).

Point 2.

The anti-autolysis claim is overstated. Hill's 1966/1976 findings do not justify the confident recommendations for sea-cucumber production chains, especially given the author's own admission that no antioxidant mechanism is established.

Response 2.

I have tried to avoid the impression I am making confident recommendations for the use of PP in sea-cucumber production chains by rewording the relevant text (second paragraph of Section 4.2):

“Whilst Hill’s evidence that PP blocks autolysis [85,88] (Section 3.2.1) requires verification, it suggests that it may be worth exploring the potential of PP as an anesthetic for employment in sea-cucumber and sea-urchin research methodologies and production chains”.

Please note that I also point out the current limitations on such use that result from inadequate knowledge of PP’s toxicity (second paragraph of Section 4.2), and the need to address this deficiency (last paragraph of Section 4.3).

Point 3.

The MgCl₂ comparison is one-sided. Mg²⁺ effects on mutable collagenous tissue may be desirable in some protocols. A comparison table across PP, MgCl₂, MS-222, and menthol would help.

Response 3.

The possible effects of Mg²⁺ ions on MCT were originally reported in 1982 and 1990 and require to be verified and fully characterised. It is therefore premature to suggest that any such effects might be desirable. A comment on the need for further investigation has been added (third paragraph of Section 4.3). A new table (Table 2) provides a comparison of available information on PP, MgCl₂ and MS-222 (MgCl₂ and MS-222 being the best studied of the alternatives to PP).

Point 4.

Safety/regulatory discussion is too brief for applied recommendations. PP lacks carcinogenicity data and food-chain approval; this must be stated prominently, not buried.

Response 4.

Human toxicity concerns are now highlighted in Table 2.

Point 5.

Practical guidance is missing. A summary box with starting concentration (0.1%), preparation, induction/recovery times, and limitations would greatly increase usefulness.

Response 5.

Please note that information on concentrations and induction and recovery times was provided in Table S1, which was submitted as a supplementary file together with the original manuscript. Limitations of current information are acknowledged in the main text (third paragraph of Section 2.2.1):

“No determination of the dose-response relationship or optimal induction and recovery times for any echinoderm subject – either intact individuals or isolated preparations - has been reported. Neither has there been any investigation of the efficacy of PP use on whole animals for achieving different end-points, such as immobilization (sedation), muscular relaxation and reversible deep anesthesia.”

Point 6.

Numerous typos and errors ("echinoiderm," "Amphiuma" for Amphiura, duplicated refs [99]/[143], inconsistent UK/US spelling.

Response 6.

I have corrected the reference duplication. I am afraid I cannot find "echinoiderm" or "Amphiuma" in the text, and have been unable to detect other typos, errors or inconsistent UK/US spelling. Information on the specific location of these defects would be welcomed.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

Thank you for revising the manuscript and providing detailed responses to the reviewer comments. The revisions address several of the concerns raised during the previous round of review. Nevertheless, some responses primarily explain the authors’ rationale without fully resolving the underlying scientific and methodological issues. Further clarification and revision are therefore necessary before the manuscript can be adequately evaluated for publication.

Although you have removed the word “suitable” from the title, the manuscript continues to advocate propylene phenoxetol (PP) as a useful anesthetic option for echinoderms. However, the evidence presented in the manuscript does not establish standardized dose–response relationships, optimal induction and recovery times, reliable criteria for distinguishing different anesthetic endpoints, or comprehensive physiological and toxicological safety.

Please ensure that the title, Abstract, Simple Summary, and Conclusions consistently reflect these limitations. In particular, you should clearly distinguish between the historical use of PP, evidence of its effects on isolated tissues and nerve preparations, and demonstrated efficacy and safety in intact animals. Although the potential value of PP may be discussed, the wording should not imply a level of validation that has not yet been achieved.

Referring readers to Table S1 does not, in itself, ensure that the literature search is sufficiently transparent and reproducible. Please ensure that the supplementary material and/or Methods section explicitly describes the databases and other sources searched, search terms, search dates, inclusion and exclusion criteria, and procedures used to verify the identity of the compound reported as PP.

Furthermore, the observed decline in publications citing PP cannot independently establish a decline in its actual use, nor can it demonstrate that misinformation caused such a decline. These interpretations should be presented as hypotheses rather than conclusions directly supported by the literature analysis. Please also acknowledge alternative explanations and relevant limitations of the search strategy in the revised Discussion.

You acknowledge that the literature does not adequately distinguish among neural conduction blockade, immobilization, muscle relaxation, and reversible general anesthesia. However, identifying this gap is not sufficient unless its implications for interpreting the available evidence are made explicit.

Please revise the relevant sections to avoid presenting electrophysiological effects observed in isolated preparations as direct evidence of effective or humane anesthesia in intact echinoderms. The criteria required to establish anesthetic depth, recovery, and physiological tolerability should be discussed separately, and the conclusions should be restricted accordingly.

The potential effects of Mg²⁺ on mutable collagenous tissue (MCT) provide a reasonable basis for further investigation. Nevertheless, the available evidence does not establish that PP is generally superior to MgCl₂ in terms of either anesthetic efficacy or overall suitability for echinoderm research.

Please frame this comparison more explicitly as an unresolved research question. Claims concerning the relative advantages of PP should be limited to those supported by direct evidence, and the lack of equivalent comparative experiments should be acknowledged. Potential adverse effects and unresolved physiological actions of both agents should also be discussed in a balanced manner.

You indicate that much of the requested methodological information is already provided in Table S1. Please ensure that this supplementary table enables readers to compare the available studies systematically, including species, PP concentration, exposure medium, exposure duration, experimental purpose, criteria used to determine anesthesia, and recovery observations, wherever these details are available.

Where information was not reported in the original publications, please indicate this explicitly rather than leaving the information ambiguous. The absence of standardized endpoints is itself an important finding of this review and should be clearly distinguished from evidence demonstrating the effectiveness of PP.

Your discussion of the historical confusion surrounding the identity of PP is potentially valuable. However, please clearly distinguish between cases in which compound identification is supported by verifiable evidence and those in which the identity cannot be independently confirmed. An unverified compound identity should not automatically be interpreted as evidence that an earlier study was incorrect.

Similarly, you appropriately acknowledge the lack of empirical evidence supporting antioxidant activity. Please ensure that the reported inhibition of sea-cucumber autolysis is discussed in proportion to the available experimental evidence, with its limitations and the need for independent confirmation clearly stated.

The discussion of potential commercial applications also requires particular caution. Given the acknowledged gaps in toxicological characterization, please avoid language that could be interpreted as endorsing PP for use in food-producing echinoderms before relevant safety, residue, and product-quality assessments have been conducted.

The manuscript would benefit from an increased number of recent and relevant references to ensure reflects current developments in the field. In addition, the entire reference list should be carefully reviewed and revised in accordance with the journal’s author guidelines to ensure consistency and compliance with the required formatting style.

Overall, the manuscript addresses a relevant topic, but its scientific contribution depends on clearly distinguishing established findings from plausible interpretations and hypotheses that require experimental verification. I encourage you to address these issues directly and revise the manuscript accordingly, ensuring that the strength of the conclusions remains proportionate to the available evidence. Such revisions would improve the scientific rigor, transparency, and overall reliability of the manuscript.

Author Response

I thank Reviewer 1 for their comments on my revised manuscript. Below are my responses to their individual points.

Point 1.

Response 1.

(a) I have corrected the Simple Summary, Abstract and last paragraph of the Introduction, in order to remove my endorsement of PP and to emphasise more firmly deficiencies in current knowledge and the need for further research.

(b) The manuscript already explains that, regarding the use of PP as an echinoderm anesthetic, there is a need for research to establish standardized dose–response relationships, optimal induction and recovery times, and criteria for distinguishing different anesthetic endpoints (third paragraph of Section 2.2.1; first paragraph of Section 4.3). The need for more toxicological research is already stated in paragraph 4 of Section 4.3.

Point 2.

Response 2.

(a) The point about the Abstract etc. is addressed in Response 1(a) above.

(b) It has to be emphasised that there has been no research on the efficacy or animal-related safety of PP in the context of its use as an echinoderm anesthetic. The evidence of its effect on isolated preparations is its blockade of responses to neuro-active agents, as illustrated in individual papers. A relevant comment has been added to the second paragraph of Section 2.2.1.

Point 3.

Response 3.

The caption of Table S1 details the sources of information: my own collection of echinoderm literature (which identified 77% of relevant PP-related echinoderm publications), the Scopus database, Google and Google Scholar. It also itemises the search terms and the completion date. Inclusion and exclusion criteria were not applied. Individual papers were read by the author to ensure that PP and other anesthetic agents were used as anesthetics in the reported investigations. The issue of the identity of the compound reported as PP is addressed fully in the fourth paragraph of Section 3.1.1. It would not be appropriate to discuss this in the caption of Table S1. In the caption, relevant publications are defined as those “reporting investigations that refer to the use of propylene phenoxetol to induce anesthesia in echinoderm subjects”.

Point 4.

(a) I agree that evidence from publication rates cannot establish a decline in usage, but I offer the reasonable view that it is suggestive there may have been such a decline, as reflected in my wording “Figure 1B suggests that relative PP usage fell sharply…”. (first paragraph of Section 2.2.1)

“The reason for the marked reduction in the relative citation rate, and therefore possibly usage, of PP since the 1990s is unclear.” (last paragraph of Section 2.2.1).

See also the last sentence of the Summary (Section 2.2.2).

Regarding the possible involvement of misinformation, again I only suggest this as a possible cause (last paragraph of Section 2.2.1).

An alternative explanation for the drop in citation rate (shifts in the focus of echinoderm research) is discussed in the last paragraph of Section 2.2.1.

Point 5.

Response 5.

I am afraid I do not understand this point. I do not know what evidence the interpretation of which is affected by the gap in knowledge is being referred to here.

Point 6.

Response 6.

(a) I do not claim that electrophysiological effects on isolated preparations represent evidence of effective or humane anesthesia.

(b) The need for research to establish criteria required to establish anesthetic depth, recovery, and physiological tolerability is stated in the first paragraph of Section 4.3.

Point 7.

Response 7.

I do not claim that PP is generally superior to MgCl₂. The use of MgCl₂ as an echinoderm anesthetic is discussed fully in the fourth paragraph of Section 2.2.1 and relevant information is included in Table 2. The need for research on the relative merits of MgCl₂ and PP is addressed explicitly in the second paragraph of Section 4.3.

Point 8.

Response 8.

Table S1 shows all such information, with the exception of criteria used to determine anesthesia and recovery observations, which have not been provided in the literature.

Point 9.

Response 9.

The following statement has been added to the third paragraph of Section 2.2.1:

“Criteria to determine anesthesia and recovery have not been described.”

Point 10.

(a) The distinction between cases in which compound identification is supported by verifiable evidence and those in which the identity cannot be independently confirmed is made and discussed fully in the second paragraph of Section 3.1.1

The distinction is also referred to in the second paragraph of the Introduction and in Table S1, which shows separately verified and unverified citations of PP.

(b) The view that an unverified compound identity should not automatically be interpreted as evidence that an earlier study was incorrect is already made in the second paragraph of Section 3.1.1:

“Furthermore, even where authors did not state the source or correct systematic name of the agent, it is unreasonable to question their veracity unless there is other cause for doubt.”

Point 11.

Response 11.

I have addressed this point by changing the wording of the second paragraph of Section 4.2 to the following:

“Hill’s evidence that PP blocks autolysis [85,88] (Section 3.2.1) was derived mainly from a single experiment involving subjective quantification and therefore requires verification.”

Point 12.

Response 12.

This point is already made in the third second paragraph of Section 4.2:

“There would seem, for example, to be scope for its use in post-harvest stages such as grading of stock and transportation to and between culture and holding facilities, though not, at present, to markets: PP has been found to exhibit low acute toxicity but has not been tested for carcinogenicity and, with regard to human health, has been recommended as “a candidate for further work” [161].”

And in the last paragraph of Section 4.3:

“In common with most chemical agents applied to aquatic animals [169], the toxicological properties of PP have still to be fully characterized [161]. Whilst not restraining its use in non-applied research, this limits its range of potential applications in aquaculture and fisheries to procedures chronologically remote from the preparation of processed products or the sale of fresh unprocessed products. First steps might be to determine the dynamics of PP contamination of sea-cucumber body wall and sea-urchin gonads after anesthesia, and relate this information to acceptable daily intake data obtained by standard laboratory animal testing.”

Point 13.

Response 13.

I have tried to include all relevant up-to-date references, including five 2026 references. I am not aware of others. As far as I can determine, the references comply with the required formatting style.