Microplastic Ingestion from Contaminated Prey in the Bearded Fireworm Hermodice carunculata (Pallas, 1766): Evidence for Rapid Excretion and Low Degradation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for this interesting research!

I believe that addressing my comments in the file I've attached will help improve the manuscript and make it ready for publication.

First evidence of microplastic trophic transfer from a filter feeder to a carnivorous polychaete is clearly emphasised. Therefore, the study is interesting and rather novel, especially in highlighting polychaetes’ role in microplastic cycling. There are a few things that must be improved before acceptance: the methodology needs to be clarified, the discussion needs to be improved with stronger ecological interpretation, and statistical assessment needs to be incorporated.

Comments for author File: Comments.pdf

Author Response

Reviewer #1

Thank you for this interesting research! There are a few things that must be improved before acceptance: the methodology needs to be clarified, the discussion needs to be improved with stronger ecological interpretation, and statistical assessment needs to be incorporated. I believe that addressing my comments in the file I've attached will help improve the manuscript and make it ready for publication.

Comment 1: It is commonly accepted to add the author who has described the species when you mention it for the first time, meaning that Hermodice carnuculata must be Hermodice carnuculata (Pallas, 1766). Isn’t it bearded fireworm?

Response 1: Agreed. We have added the author and the year (Pallas, 1766) next to the species in the title. The common name has been corrected in the manuscript, as suggested. Full scientific name of the species was left at the first mention only (l 67).

Comment 2: From now on you should use only the common English name.

Response 2: Following the reviewer’s suggestion, we have replaced the species name with the common English name throughout the text, after the first mention.

Comment 3: Add the author and year in brackets after the Latin name

Response 3: Author and year of species description have been added (see at lines 58, 231 and 268).

Comment 4: How come there is no paragraph for statistical analyses?

Response 4: We thank the reviewer for pointing this out. Our contribution was submitted as a Brief Report, as results obtained from our pilot study should be regarded as preliminary (l 271), yet providing first evidence of ingestion through contaminated prey in the bearded fireworm. Since only a limited number of organisms was considered, we believe that performing a statistical comparison test would not be appropriate. We reported main descriptive statistics (mean and standard deviation) for MP content in the different samples in the Results section. Following the reviewer’s suggestion, we have added a section on Data analysis (see l 197-203) highlighting this aspect.

Comment 5: What about these mussels? From where did they come from? This is a bit tricky as you might have contaminated your animals already during the process of acclimation. You have to justify this statement. Also, how big were the mussles? Av. lenght of M. galloprovincialis is about 5 cm, but they can be bigger of course. This explanation have to be added for the exposure paragraph too.

Response 5: information about the mussels used for fireworm rearing is now provided in the text (i.e. origin and shell length, see at l 102), while the size of the mussels used in the experiment is reported in the supplementary table S1, as specified at lines 225-226. However, we noted an error in the table number in the text that may have caused some misunderstanding. This has been corrected in the revised version (l 226).

Regarding the reviewer’s concern, we cannot exclude contamination of the animals during acclimation, as microplastics are ubiquitous contaminants. However, for this trophic transfer experiment, we intentionally used manufactured fluorescently labelled polystyrene microspheres, supplied by an external company, as described in section 2.2. These microspheres are of a known size and shape, and they are labelled with a fluorophore, making them easy to identify and trace in biological samples. This allowed us to avoid misidentification during sample observation and microplastic quantification. In addition, fireworms were kept in laboratory close-circulating system of aquaria during rearing, where these polystyrene microspheres were not added. Regarding the experimental setting, upon microscopy observation of control specimens (i.e., mussels and fireworms kept in seawater and not exposed to these microspheres), no fluorescent signal was detected, confirming the lack of contamination from this test material in the animals during incubation.

Comment 6: add the town and country in a similar manner like above - Warrington, USA...

Response 6: Town and country have been included for all instruments and software (see at lines 114-115, 124, 181-182, 184-185, 189, 193).

Comment 7: ethanol, not Ethanol - percentage?

Response 7: The capital letter has been removed, and the percentage specified (l 119).

Comment 8: I am sorry but I didn't understand - how long did the experiment last after the acclimation in total? How long did you give your animals contaminated food?

Response 8: As specified at line 137, mussels were exposed to micro-PS for 3 hours. Bearded fireworms were then fed with these microplastic-contaminated mussels for 3 days (see l 161). As support for the reader, a schematic workflow of the experiment with time-points is reported in figure S1 in the Supplementary materials.

Comment 9: Also, how many fireworms did you investigate in total, did you measure them? usually, the experimental groups have to be from one size/age group...

Response 9: The number of bearded fireworms investigated has been added at l 146 and 161. Measured sizes for each individual, together with the main descriptive statistics, are reported in the supplementary table S2 of the Supplementary material (see l 239).

Comment 10: I think you mention anything about control for the first time here?

Response 10: Control groups are first presented in the Materials and Methods section, see at line 143 for mussels and at line 158 for bearded fireworms.

Comment 11: Author, year in brackets

Response 11: Details have been added (l 268).

Comment 12: Mussels are filter feeders, yes they are constantly exposed not only to waterborne contaminants, but also to contaminants that are stored in sediments as they are relatively sedentary. Yet, I think this hypothesis should be justified with more references. I also think it comes to type of physiology, etc., not a dynamic balance.

Response 12: We thank the reviewer for their observation. This paragraph was actually focused on fireworms, and comparisons with other polychaetes were made. Evidence of dynamic balance between microplastic ingestion and clearance in marine organisms is supported by the cited scientific literature (10.1016/j.envpol.2016.04.012; 10.1016/J.MARPOLBUL.2015.11.053). We have also added references underlining the key role of feeding behaviour and organisms’ physiology in governing microplastic ingestion (see at lines 277-283).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Microplastic ingestion from contaminated prey in the fireworm Hermodice carunculata: evidence for rapid excretion and low degradation” provides a thorough investigation of both the content and the underlying mechanisms. I consider the study to be comprehensive, and once the authors have addressed the reviewers’ comments, I recommend it for publication in Environments.

Specific points to be addressed:

1. In Figure 2, several minor Raman peaks should be clarified with respect to the corresponding elements, such as those near 1600 cm⁻¹ and to the left of 1483 cm⁻¹.

2. In Figure 3, the SEM images lack scale bars, which should be added to indicate dimensions. Moreover, the SEM images presented are not sufficiently clear; higher-quality images should be provided.

3. The authors are encouraged to propose possible solutions or implications in the manuscript to guide readers and enhance the practical relevance of the study.

Author Response

Reviewer #2

The manuscript entitled “Microplastic ingestion from contaminated prey in the fireworm Hermodice carunculata: evidence for rapid excretion and low degradation” provides a thorough investigation of both the content and the underlying mechanisms. I consider the study to be comprehensive, and once the authors have addressed the reviewers’ comments, I recommend it for publication in Environments.

Comment 1: In Figure 2, several minor Raman peaks should be clarified with respect to the corresponding elements, such as those near 1600 cm⁻¹ and to the left of 1483 cm⁻¹.

Response 1: Figure 2 has been implemented with minor Raman peaks as requested. However, in the text only the main peaks of polystyrene were mentioned.

Comment 2: In Figure 3, the SEM images lack scale bars, which should be added to indicate dimensions. Moreover, the SEM images presented are not sufficiently clear; higher-quality images should be provided.

Response 2: We thank the reviewer for bringing this to our attention. The missing scale bar has now been added in the revised manuscript. SEM high-resolution figures are provided as separate files in the submission system, we invite the reviewer to consider them instead of those available in the generated PDF manuscript file.

Comment 3: The authors are encouraged to propose possible solutions or implications in the manuscript to guide readers and enhance the practical relevance of the study.

Response 3: We thank the reviewer for the suggestion. While we had already included some perspectives for future research in the Conclusions section (see l 363-369), we have now expanded the discussion to better highlight the practical implications of our findings. In particular, we emphasized the importance of selecting appropriate sentinel species for biomonitoring and the role of polychaetes in microplastic fate in the seafloor (see l 274-288).

Reviewer 3 Report

Comments and Suggestions for Authors

The article „Microplastic ingestion from contaminated prey….” By Ferrari et al. describes their study in a two level trophic chain and spherical, fluorescent labelled PS particles as model microplastic.

The great value of the publication is that it honestly shares negative results. There is a strongly unbalanced situation in the microplastic research area – and maybe in general in scientific literature – regarding the publication of negative results. Both scientific and general public accepts/believes that microplastics are accumulating in the environment, enter and travel across the trophic chain. Thus, results, that are out of the main-stream discourse often remain hidden and have no chance to add weight to the statistical evaluations.

The weak side of the work is the missing explanation of the presence of these quite large MPs in the body fluids of some of the individuals. More accurate negative controls (adding MPs to the worms shortly before the anesthetizing the specimens) in order to exclude the effect of introducing contamination from the external part of the body during sample preparation, etc. might help to explain these findings. The discussion in lines 236-242 has to be written with care. It is true that similarly to the authors, Ref 51 and 24 has not found MPs in the digestive tract. But it DOES NOT suggest that ingested MP in general might cross cell membranes – as the text states in lines 239-240.  Ref 24 used smaller plastic particle model, where it has higher probability to enter cells but they did not tissue uptake in their animals. Similarly, Ref 51 who used very wide distribution MPs, down to 0.4 µm have no proof for this, they hypotetize: “ It seems that MPs were not retained in worms gut or may be egested quickly, and not accumulated. However, in our study, because of analytical constraints, it was only possible to identify MPs < 20 μm and there is a possibility that smaller particles (small MPs or nanoplastics) have penetrated the tissues.” I suggest to remove the statement about penetration of tissues by MPs in general and valorise the findings of the actual work.

Minor comment: Line 187: in low stable suspensions   Suggested: in low stability suspensions

Author Response

Reviewer #3

The English is fine and does not require any improvement.

The great value of the publication is that it honestly shares negative results. There is a strongly unbalanced situation in the microplastic research area – and maybe in general in scientific literature – regarding the publication of negative results. Both scientific and general public accepts/believes that microplastics are accumulating in the environment, enter and travel across the trophic chain. Thus, results, that are out of the main-stream discourse often remain hidden and have no chance to add weight to the statistical evaluations.

We thank the reviewer for the positive feedback regarding the language quality of our manuscript. We greatly appreciate the reviewer’s recognition of our inclusion of negative results. We agree that, by highlighting them, we contribute to a more transparent and balanced presentation of the data on microplastic research.

Comment 1: More accurate negative controls (adding MPs to the worms shortly before the anesthetizing the specimens) in order to exclude the effect of introducing contamination from the external part of the body during sample preparation, etc. might help to explain these findings.

Response 1: We understand the reviewer’s concern. We believe that the presence of PS microspheres in the body fluids of fireworms could have resulted from the dissection step, as the intestinal tissue wall is very fragile. Accordingly, in Figure 1, data on the number of microplastics in the intestine and in the body fluids are presented together, as we assumed that some microspheres may have leaked in body fluids during dissection, which were collected for this reason. We have revised the text to improve clarity (see l 164-167 and 248-251). Additional negative controls were not included in the experimental design to evaluate potential cross-contamination because, after exposure, fireworms were immediately transferred in clean tanks filled with fresh anesthetizing solution and subsequently dissected.

Comment 2: The discussion in lines 236-242 has to be written with care. It is true that similarly to the authors, Ref 51 and 24 has not found MPs in the digestive tract. But it DOES NOT suggest that ingested MP in general might cross cell membranes – as the text states in lines 239-240.  Ref 24 used smaller plastic particle model, where it has higher probability to enter cells but they did not tissue uptake in their animals. Similarly, Ref 51 who used very wide distribution MPs, down to 0.4 µm have no proof for this, they hypotetize: “ It seems that MPs were not retained in worms gut or may be egested quickly, and not accumulated. However, in our study, because of analytical constraints, it was only possible to identify MPs < 20 μm and there is a possibility that smaller particles (small MPs or nanoplastics) have penetrated the tissues.” I suggest to remove the statement about penetration of tissues by MPs in general and valorise the findings of the actual work.

Response 2: We agree with the reviewer’s remarks. As the statement about microplastics crossing cell membranes could have been misleading, we have removed it from the discussion in the revised manuscript. Instead, now we emphasize that microplastics may not be retained for long periods and can be rapidly egested (see l 269-270).

Comment 3: Line 187: in low stable suspensions   Suggested: in low stability suspensions

Response 3: Thanks, the text was changed accordingly (see l 211).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for addressing all my comments!