Developmental Toxicity and Thyroid Endocrine Disruption of Polyhexamethylene Guanidine Hydrochloride and Humidifier Disinfectant in Zebrafish Larvae

Round 1

Reviewer 1 Report

This paper focus on the developmental toxicity and thyroid endocrine disruption caused by humidifier disinfectants containing polyhexamethylene guanidine (PHMG) using zebrafish as model organism. Acute lethality, developmental endpoints, whole-body thyroid hormones, and transcription of genes related to the hypothalamus-pituitary-thyroid axis were investigated following a 7-day exposure and the results show the occurrence of developmental effects seen by the increased mortality and also by the disruption of thyroid pathways as seen by the changes in thyroid hormones as well as the associated gene changes. The authors concluded that a chemical modification and/or a feedback mechanism might be responsible for the observed effects.

In general, this is a simple paper which may have some importance for the (eco)toxicology field although the significance and application of this study to the broad community is limited. The introduction should be further worked, and the methods are appropriate but require further edition as some details are missing. The statistical analysis should be reviewed to include the p-values and comparisons made and the discussion needs some clarifications. As such the paper needs to be revised to address the following limitations:

- Is the “Vegetable Home Cleanup HD” the brand of the tested product? The reviewer could not find any information on this product.

- Line 16, which is the “actual-use concentration of HD”?

- Line 22, further clarification should be given as there is no link to the “PHMG-phosphate” in the abstract.

- Line 32, is this product used outside South Korea? In fact, the FDA has approved this agent as a disinfectant for medical devices.

- Line 33-40, what is the relation of the lung-induced deficits/disease with the use of zebrafish to test PHMG? It is clear that zebrafish do not have lungs and that gills are found between 6-14 dpf in zebrafish meaning that exposure below the stage of gill functionality is meaningless. A further clarification of the reported ideas is mandatory. In this respect, the discussion should also be reviewed.

- The reference 13 describes some toxicological effects on zebrafish, namely mortality, in adult zebrafish. This information should be introduced. Also, the reference 13 states that the LC50 was 0.043 mL/L and not 0.043 mg/L as reported in the text.

- The introduction needs to further summarize the zebrafish findings described in the literature. For instance, there are some references describing the effects of PHMG in zebrafish such as doi: 10.1089/zeb.2018.1571 and 10.1016/j.tox.2019.01.001.

- Line 67, the previous study of the group already shows similar effects of PHMG-P as the ones reported in this study. The novelty and differences to the already published work should be highlighted.

- Line 74, in the previous work from the group, the LC50 for PHMG-P was defined as 2.12 mg/L. Why was a higher concentration used in the current study? What is the relation between the selected concentrations and the lung-induced effects?

- Line 87, how were the embryos obtained?

- Line 87, is the sterilization with 30% sodium chloride a common practice? According to my practice, the use of diluted chlorine bleach or chloramine-T are effective strategies to sterilize eggs.

- Line 88, what is the composition of the culture media?

- Line 89, the reported percentages are relative to the amount of PHMG or to the actual HD product? Change the percentages to mg/L.

- Line 90, the OECD guideline 236 is relative to the calculation of the lethal concentration for an exposure of 96h to a selected compound. In this case, the exposure lasted for 7 days and as such, this guideline could not be used to support the experimental design.

- Line 91, how many embryos were put in each well of the plate? Also, how many embryos were used for each technique?

- Line 97, review these endpoints as some could not be measured every 24h, e.g. hatchability and time to hatch.

- Line 100, why the CCAC guidelines were used in this study? Is there no regulation on the use of this model in Republic of Korea?

- Line 102, why were animals used at 7 dpf? What are the ethical implications for the study?

- In the results section, please include the p-values for the comparisons made.

- Figure 1A, at which time were these results collected? Also, it seems strange that the increase in 0.6 resulted in statistical differences but no difference is obtained for the decrease observed in the 0.06 % group. In fact, in this last group, the distance to the control group is higher than that determined in the 0.6 group.

- Figure 1D, review the size of the larvae. It is impossible that a 7 dpf larva has a size of 40 mm (4 cm).

- Please include a figure with the T3/T4 ratio, which has been used as an indicator of toxic contamination.

- Line 208, has this already been described in the literature?

- A further explanation of the gene expression changes should be done to support the results and ideas presented. There are only 9 references in the discussion meaning that the discussion can greatly be improved, and the ideas further explored.

- Line 238, this sentence should be reviewed.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

the manuscript entitled Developmental toxicity and thyroid endocrine disruption of polyhexamethylene guanidine hydrochloride and humidifier
disinfectant in zebrafish larvae, investigated some of the toxicity profiles of mentioned material using zebrafish model. the author demonstrated clear experimental design and appropriate set of assays. however before any conclusion some major concerns need to be addressed. 

• authors in several places mentioned Developmental toxicity. while they have done no developmental at all. no deformity was assessed and no information given regarding the endpoint at different time point of development. the author reported that the observations were made every 24 h but the results are missing. therfore i can not consider it as developmental toxicity.
• Line 79, PHMG-H is a main component of vegetable HD. this is not a precise statement for toxicological study. any component maybe and maybe NOT interfere with the main component. authors need to fully answer the component for the vegetable HD.
• Fig 1D the Asterisk sign is missing for last two concentrations. 
• the main problem with this MS is a high degree of assumption in discussion and strong conclusion through whole discussion. without a proper set of experiments it is very hard to make such statements. here are just some example L 195, L 209, L220, L227, L231-233,L241-242.
• L204 and L216 is not true always. for L 216 you can have a look at https://doi.org/10.1016/j.aquatox.2017.10.002

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed most of the comments raised and have changed the manuscript which is now improved. However, the reviewer still has a major concern related to the product used as no information can be found in the literature. In addition, authors have sent the webpage of the product, but it is in a foreign language without further information on its chemical composition. Furthermore, it seems that the use of these type of products declined from the end of 2011, ten years ago (doi: 10.3390/molecules25143301). Consequently, this seems to not be of particular interest to the wide readers of this journal. In addition, other points need further revision:

- Line 70, include the reference for the previous study of the group.

- Line 83, how can these results be useful for the ecotoxicological risk management? Is the concentration used (0.6%) environmentally relevant? Is this compound detected in aquatic ecosystems? If authors want to explore the environmental associated risks, the introduction should be reviewed to not include epidemiological studies and other mammals studies (line 47-58).

- What is the impact of hydrogen peroxide for bleaching zebrafish eggs and its development? The references cited in the rebuttal letter are relative to rainbow trout and catfish eggs and use higher concentrations which cause hatchability problems. Is this method commonly used for zebrafish bleaching? Please include a reference for this.

- As required in the previous revision, please include the p-values of the comparisons made.

- Again, review the body length units. Again, it is impossible a 7-day larvae to have a size of only 40 um (0.04 mm) when they usually measure around 3-4 mm.

- How was the T3/T4 ratio calculated? Were the results normalized to the control? Please include more information on the methods section.

- Line 209, according to a previous work (doi: 10.3390/molecules25143301), HDs are not used since 2011.

-  Line 212, as referred before, is this compound (PHMG) detected in aquatic ecosystems?

- Line 221-222, what is the relation of HD-associated lung disease to the outcomes of this study? As referred before, if the objective of the work was to explore the environmental-related issues, this should be reviewed.

- Line 264, review the use of italics for gene or proteins.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

the authors provided enough explanation for the comments. 

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

The authors have adressed all my comments and the manuscript can now be accepted for publication.