Handle with Care—Microplastic Particles in Intestine Samples of Seals from German Waters

Round 1

Reviewer 1 Report

Unfortunately I do not feel that this study is novel or original and it does little to enhance our understanding of microplastics in marine mammals. Suggestions for improving the clarity and flow of the manuscript are listed below.

Introduction:

L37 - 39: This sentence has no context as the authors have not previously introduced the concept of microplastic ingestion by biota.

L44:  It could be argued that sediment samples are also very difficult to deal with - I would suggest editing to 'thus may be more challenging'

L45-46: In ideal world, contamination of samples would be eliminated not just 'kept as low as possible' which a number of studies have achieved. 

 

Materials and Methods:

L70: I am not familiar with the term 'mercy-killed'...perhaps euthanised might be better?

L72: The mention of harbour porpoise is not relevant here as they do not feature in the study. Suggest removing 

L79: I believe it would help with the flow to separate the two types of sample (GIT content and scats) with sub-headings

L80: How was environmental contamination from the sandbank limited/ controlled for?

L96-97: Is this sample collection (ring-shaped piece) relevant to this study? If not I suggest removing.

L138: How many particles were subjected to FTIR? What proportion of the overall number detected?

L144: I would suggest using the term putative/ suspected microplastics unless confirmed by FTIR. Also, there is no need to abbreviate microplastic to MP, it is uneccesary.

L145: Here you say fragment but I think you mean particle as fragment is a specific shape of microplastic and you are talking about fibres in the sentence.

L149: What are the metrics for these variables? How did you measure them?

L152: The concept of the decision tree needs more background context - what is it and why is it a relevant method to use here?

L159: I think you need to specify that this relates to 'recovery rates' and would suggest mentioning it earlier in the methods as I imagine you did this as a pilot prior to using it on your samples?

L162: Why were they sewn into the cloth? The microplastics in your samples would have not been subjected to the same treatment so this control is not comparable and does not represent the conditions to which your samples were exposed.

L167: Typo

168: Were any measures put in place to monitor the effectiveness of this measure?

L189: I don't understand this, further detail/ explanation is needed for clarity. 

Results:

L194: Repetition of the above

L201: Please provide percentage

L202: I think a better title would be 'Better to say 'Efficacy of contamination control measures' or something similar

L204: No need to say at most as it is a range

L207: Better to replace placebo with control

L209: This is some kind or formatting issue here and a number of places within the results section

L214: This title is not a good explanation, I suggest changing to 'Comparison of storage methods for contamination' or something similar

L219: Typo

L222: Formatting error

L243: How were these ground-truthed? Particles can look synthetic but aren't necessary plastic.

L253: Suspected microplastics

L254: zero to 35 microplastics per what?

L257: Formatting error

L257: So 28 of the 31 particles were confirmed as plastic, the rest are putative/ suspected

L262: Formatting error

L280: Improve from what? There are studies which eliminated sources of contamination - this method is not as effective as those so why not just replicate them?

L284: But it doesn't avoid contamination - suggest rewording this

L287: This isn't possible for large samples such as whole digestive tracts/ space constraints/ health and safety. Better to recommend taking samples of the bags and removing any particles that match the characteristics (colour and polymer type) from the results.

L288: Typo

L294-295: I didn't see this mentioned in the methods and should be added

L304: Again not always possible with larger samples

L306: Typo

L367: It is not clear to me how the methods used in this study are interdisciplinary, I suggest removing this

L383: You haven't provided any examples time or costs of your method compared to others so it is not clear whether this is true or not

 

Author Response

Dear Dr. Rodríguez Seijo,

 

many thanks for sending the reviews for our paper entitled “Handle with Care – Microplastic Particles in Intestines Samples of Seals from German Waters”. Please find our rebuttal letter from the next page onwards, replying to all comments of the three reviewers. Please find our responses after every comment in italics. We re-checked scientific language and changed expressions according to reviewer’s comments. Furthermore, a native speaker proofread the manuscript. We send you the manuscript in track change mode. The improved supplement information is additionally attached in a pdf file. All co-authors agreed on the new versions of both.

 

 

With kind regards

 

 

 

 

Prof. Prof. h. c. Dr. Ursula Siebert

 

 

 

 

 

Rebuttal letter

 

Response to Reviewer 1

Unfortunately, I do not feel that this study is novel or original and it does little to enhance our understanding of microplastics in marine mammals. Suggestions for improving the clarity and flow of the manuscript are listed below.

Answer: We are very sorry, that reviewer 1 does not see the value of our study. For clarification, our study does not aim to analyse the burden of marine mammals concerning microplastics. The main outcome is to provide research with opportunities to avoid secondary pollution and to use a standardized protocol for microplastic particle identification. It provides the basis for future research to ensure the real microplastic burden in marine mammals. For enabling research groups without a suitable clean room, new methods are needed to avoid contamination. In our study, we provide new and innovative possibilities to meet the needs of this, even with a low budget. Moreover, the provided decision tree and photo catalogue offer a method to decide which particle is reasonable to isolate and analyse (cost and time effective). In case of a future standardized usage, it can be guaranteed that the results of different research groups are comparable.

Introduction:

L37 - 39: This sentence has no context as the authors have not previously introduced the concept of microplastic ingestion by biota.

Answer: Thank you for this comment. Please find this sentence rewritten: MPs were revealed to be ingested by organisms of lower trophic levels, and even a trophic transfer was detected [14-16].

L44:  It could be argued that sediment samples are also very difficult to deal with - I would suggest editing to 'thus may be more challenging'

Answer: Thank you for mentioning that. We deleted this sentence for clarification.

L45-46: In ideal world, contamination of samples would be eliminated not just 'kept as low as possible' which a number of studies have achieved. 

Answer: We agree with you, but it is almost impossible to guarantee a contamination free sample handling. Therefore, the risk has to be kept as low as possible. It has to be taken into account that microplastic research is only able to inform about an approximate burden since contamination cannot be completely excluded.

 

Materials and Methods:

L70: I am not familiar with the term 'mercy-killed'...perhaps euthanised might be better?

            Answer: Thank you for spotting this. We changed the term to euthanised.

L72: The mention of harbour porpoise is not relevant here as they do not feature in the study. Suggest removing 

Answer: The referring paragraph deals with the information on our data basis and the marine mammals’ occurrence in our waters. Since this is a general information, we would like to keep the sentence as it is.

L79: I believe it would help with the flow to separate the two types of sample (GIT content and scats) with sub-headings

Answer: Thank you for this valuable comment. For clarification, we added the headings “Necropsy Sampling” and “Sandbank Sampling”.

L80: How was environmental contamination from the sandbank limited/ controlled for?

Answer: Thanks for your comment. As mentioned in our material & method part the sandbank is a tide-dependent submerged sandbank that means between the egestion and sandbank collection the time maximum is six hours. Between egestion and sample collection, we cannot control the contamination by air or sand. Nevertheless, to collect samples on a sandbank is rare and thus valuable possibility to investigate in free-ranging marine mammals. For each sample one glass jar and one spoon is used to avoid cross-contamination.

L96-97: Is this sample collection (ring-shaped piece) relevant to this study? If not I suggest removing.

Answer: Thank you for your comment. We totally agree and thus we eliminated the sentence. Since the tissue, we are referring to is not part of this study.

L138: How many particles were subjected to FTIR? What proportion of the overall number detected?

Answer: Please, recheck our material & method part (2.1.5 Polymer Identification) in which we state, that our identification was conducted using µRaman spectroscopy. Furthermore, this information in our opinion belongs to the results section. We state the required information in section 3.3 Isolation of MP in Intestinal and Faecal Samples: A total of 31 suspected plastic particles were analysed via µRaman spectroscopy. 28 particles (90%) of these 31 particles were identified as plastic polymer.

L144: I would suggest using the term putative/ suspected microplastics unless confirmed by FTIR. Also, there is no need to abbreviate microplastic to MP, it is uneccesary.

Answer: Thank you for this recommendation. We amended the term in the whole manuscript if suitable.

L145: Here you say fragment but I think you mean particle as fragment is a specific shape of microplastic and you are talking about fibres in the sentence.

Answer: For clarification as mentioned in the sentence, we defined a particle as either fragment or fibre. We clearly refer to both categories in the whole manuscript when talking about particles.

 

L149: What are the metrics for these variables? How did you measure them?

Answer: The metrics for the mentioned variables were defined by using comparable pictures of already identified particles by µRaman spectroscopy. Thus, for example the higher the fluorescence intensity is, the more likely it is that the particle is a microplastic particle.

L152: The concept of the decision tree needs more background context - what is it and why is it a relevant method to use here?

Answer: The establishing of a decision tree helps to categorize different variables, which are helpful for untrained researchers to gain results that are comparable between research groups. This is essential to compare the burden in different marine environments and biota. Furthermore, the decision tree helps to narrow down suspected microplastic particles out of a bunch of a variety of particles in a sample, which are then used for further analysis. This is cost- and time-effective and a useful tool for encouraging decision makers to support necessary monitoring programme.

L159: I think you need to specify that this relates to 'recovery rates' and would suggest mentioning it earlier in the methods as I imagine you did this as a pilot prior to using it on your samples?

Answer: Thank you for this hint. We agree and moved this section to a new paragraph called “2.1.5 Pre-Trials to Verify the Procedure”.

L162: Why were they sewn into the cloth? The microplastics in your samples would have not been subjected to the same treatment so this control is not comparable and does not represent the conditions to which your samples were exposed.

Answer: Please, keep in mind that the microbeads were used as pre-trial to determine the recovery rate. They were thus treated the same way as the original samples. Have a closer look on the paragraph in the original manuscript (2.1.2 Preparation):

For separating the MP from the biogenic matter, the intestine and faeces samples are washed in self-sewed double layer washing sachets in a commercial washing machine (OK., OWM 15012 A1). Each sample was placed in an inner bag (mesh size 300 µm) which was then placed in the outer nylon bag (mesh size 100 µm). The sachets were made of nylon cloth and were sewn together in the acrylic box with a conventional sewing machine (Singer, Tradition TM 2282) using black cotton yarn. Furthermore, each nylon sachet was used only once to prevent a cross-contamination.

L167: Typo

Answer: Amended.

L 168: Were any measures put in place to monitor the effectiveness of this measure?

Answer: Thank you for this discussion point. The effectiveness of our measure is given by literature research, which shows that a variety of different studies deal with a high share of fibres in their samples. Since in those studies no protecting measures were applied (or not defined) we assume that our measure must be effective to reduce secondary pollution which is indicated by the amount of fibres.

L189: I don't understand this, further detail/ explanation is needed for clarity. 

Answer: We agree that this needed for clarification therefore, we moved the sentence to the following paragraph: 2.1.3 Washing Procedure. Now it should be clear, at what point of the procedure the weighing process takes place and for what reason it is conducted.

 

Results:

L194: Repetition of the above

Answer: Thank you for this hint we erased the sentence.

L201: Please provide percentage

Answer: Thank you for this idea. We would like to keep the used specification since the information we would like to transport to the reader is the range of measured biogenic matter. To inform about the percentage does not make any sense in this context.

L202: I think a better title would be 'Better to say 'Efficacy of contamination control measures' or something similar

Answer: Amended. Changed to “Efficiency of Contamination Control Measures".

L204: No need to say at most as it is a range

Answer: Thank you for spotting this, we have adjusted accordingly.

L207: Better to replace placebo with control

Answer: We agree and amended it.

L209: This is some kind or formatting issue here and a number of places within the results section

Answer: Thank you for spotting this formatting error. We mended this problem.

L214: This title is not a good explanation, I suggest changing to 'Comparison of storage methods for contamination' or something similar

            Answer: Amended.

L219: Typo

Answer: Thank you for spotting this, we corrected it.

L222: Formatting error

Answer: Thank you for spotting this formatting error. We mended this problem.

L243: How were these ground-truthed? Particles can look synthetic but aren't necessary plastic.

Answer: The dataset of particles that were used to build this decision tree have been evaluated independently by two trained researchers. For those particles that were not unanimously classified as either plastic or non-plastic, the classification has been jointly discussed and further expert opinions obtained or investigated by µRaman spectroscopy were undertaken. So that we are confident that true positive rate of 84%, that measures the proportion of MP particles that were identified as possible MP and the true negative rate of 69%, which measures the proportion of non-plastic particles that were identified as being non-plastic, revealed a good performance of the decision tree.

L253: Suspected microplastics

            Answer: Amended.

L254: zero to 35 microplastics per what?

Answer: Thank you for this hint. We clarified the sentence: The share of fibres ranged from zero to 35 per sample (M±SD = 6±7.4) when taking all specimen stored in glass jars into consideration (≥100 µm).

L257: Formatting error

Answer: Thank you for spotting this formatting error. We mended this problem.

L257: So 28 of the 31 particles were confirmed as plastic, the rest are putative/ suspected

Answer: Thank you for this valuable hint. We added the expression “suspected” every time where it was suitable in the manuscript.

L262: Formatting error

Answer: Thank you for spotting this formatting error. We mended this problem.

 

L280: Improve from what? There are studies which eliminated sources of contamination - this method is not as effective as those so why not just replicate them?

L284: But it doesn't avoid contamination - suggest rewording this

Answer: Please, recheck our results of our blanks, which we take into consideration. Please, also check our comment on line 168.

L287: This isn't possible for large samples such as whole digestive tracts/ space constraints/ health and safety. Better to recommend taking samples of the bags and removing any particles that match the characteristics (colour and polymer type) from the results.

Answer: We agree processing larger samples is difficult at least for the step of storing in glass jars. Thus, larger samples have to be processed directly during dissection without storage beforehand. Our results show, that samples stored in plastic bags a more likely to be contaminated but not because of the bag itself but rather because of the contamination of the bag, which is also not checked beforehand in previous studies. We are the first to investigate the risk of secondary contamination by storing samples in plastic sample bags without taking care of prior cleaning measures.

L288: Typo

Answer: Amended.

L294-295: I didn't see this mentioned in the methods and should be added

Answer: Thank you for this hint; we added this in the section 2.1 Sample Collection and 2.2 Evaluation of the Methodical Efficiency.

L304: Again not always possible with larger samples

Answer: Please, check our answer to line 287.

L306: Typo

Answer: Amended.

L367: It is not clear to me how the methods used in this study are interdisciplinary, I suggest removing this

Answer: Please, let us clarify our statement. With interdisciplinary we are referring to both microplastic and diet analysis which are two disciplines profiting from our effective and gentle method.

L383: You haven't provided any examples time or costs of your method compared to others so it is not clear whether this is true or not

Answer: Please, see the following paragraph in section 4. Discussion: “Removing biogenic substances with the help of the washing procedure preserves the original particles, and it is less time- (approx. 90 min) and budget-consuming compared with the application of enzymatic or chemical solutions for the digestion of biogenic material.” In previous studies, the digestion of biogenic material lasts for hours, days or weeks.

Author Response File: Author Response.doc

Reviewer 2 Report

Dear authors,

I firstly apologize if I am making a mistake when understanding the scope of the journal that you are submitting this manuscript. In my opinion, this manuscript does not fit any of the topics that the journal Sustainability have in its scope. Looking at the first paragraph at the discussion section, it made clear to me that statement about the study shows that the manuscript is a purely methodological manuscript for microplastic analysis. I do believe that the study will be really useful for the research community, but I do also think that it needs to be modified for a better understanding and publish in another journal with a clear methodological scope or more pollution specific.

In addition, I found that the number of references used are in a considerable amount. I do believe that for a note that amount should be reduced. I added some suggestions for authors regarding this issue.

Also I included several comments that I think you should be considered for publication.

Despite my comments, I would like to encourage authors to improve the manuscript because I believe it will be worthy for the research community

Regards

Comments for author File: Comments.pdf

Author Response

Dear Dr. Rodríguez Seijo,

 

many thanks for sending the reviews for our paper entitled “Handle with Care – Microplastic Particles in Intestines Samples of Seals from German Waters”. Please find our rebuttal letter from the next page onwards, replying to all comments of the three reviewers. Please find our responses after every comment in italics. We re-checked scientific language and changed expressions according to reviewer’s comments. Furthermore, a native speaker proofread the manuscript. We send you the manuscript in track change mode. The improved supplement information is additionally attached in a pdf file. All co-authors agreed on the new versions of both.

 

 

With kind regards

 

 

 

 

Prof. Prof. h. c. Dr. Ursula Siebert

 

 

 

 

 

Rebuttal letter

 

 

Response to Reviewer 2

 

Dear authors,

I firstly apologize if I am making a mistake when understanding the scope of the journal that you are submitting this manuscript. In my opinion, this manuscript does not fit any of the topics that the journal Sustainability have in its scope. Looking at the first paragraph at the discussion section, it made clear to me that statement about the study shows that the manuscript is a purely methodological manuscript for microplastic analysis. I do believe that the study will be really useful for the research community, but I do also think that it needs to be modified for a better understanding and publish in another journal with a clear methodological scope or more pollution specific.

Answer: Dear Reviewer 2, thank you for bringing this to our attention. We are well aware that the main focus of this special issue is targeting the topic of sustainability. Nevertheless, we believe that our paper, although the focus is on methodology, suits into the scope of the special issue due to the following reasons:

• To reach a sustainability a monitoring is needed, especially to assess the real burden of microplastics in the marine environment, and thus marine mammals.
• To establish a monitoring, it is necessary to keep the procedure applicable for all sorts of research groups and to have it rather time- and cost effective. This is essential to enable research groups around the world to deal with a global problem and to assess this ubiquitous issue.
• Measurements for reducing microplastics are in need of a monitoring to assess the value and effectiveness.

 

In addition, I found that the number of references used are in a considerable amount. I do believe that for a note that amount should be reduced. I added some suggestions for authors regarding this issue.

Answer: Thank you for this comment. We doubled checked the amount of references and erased those who might be dispensable and to make it easier for the reader to keep track on the state of the art.

 

Also I included several comments that I think you should be considered for publication.

Despite my comments, I would like to encourage authors to improve the manuscript because I believe it will be worthy for the research community

Regards

Answer: Thank you for your valuable comments, which we answer in the following paragraph.

 

 

L33: and beads

 

Answer: Thank you for this comment. To summarise all microplastic shapes and characteristics we know use the term particles, to include all categories.

 

L 39: Think ref 70 should be also here

 

Answer: Thank you for double-checking our references. We are not sure if you might have named the wrong reference here, since the reference you are talking about is dealing with polar skuas feeding on penguin carcasses. Thus, it does not fit in the following paragraph: A variety of international studies confirmed the presence of different sized plastic particles in the gastrointestinal tract (GIT) of various marine mammalian species [17-21].

 

L43: The manuscript is about biota and authors should keep their argument with biota. Other environments (sediments and water column) should be mentioned only if it is strictly necessary

           

Answer: Thank you for this comment. We deleted this sentence for sticking with the topic of marine biota as requested.

 

L50: Ref 17 is about macroplastics not microplastics, please remove from here

 

Answer: Please, check the actual aim of the study of Bravo Rebolledo et al. 2013. One of their main results was that they could not identify any microplastics (≥100 µm) even though they were searching for it. The reference is suitable in the sentence we are using it for, since we are referring to digestion protocols rather than microplastic identification.

 

 

L50, 51: Refs 31 and 32 are not for biota and should not be included

           

Answer: Thank you for double-checking the used references. We agreed, and deleted the dispensable one.

 

 

L71: Ref 40 & 41: these are harbour porpoise and common seal refs, authors should included grey seal one, otherwise only include the common seal one as the manuscript is related to seals (with different GIT)

           

Answer: Thank you for this hint we added a new reference, which also investigates grey seals.

 

 

L73: Have been followed any specific protocol since 2014? I do not know any specific one.

In addition, airbone contamination was not estrictly controlled until few years ago and this is not considered anywhere.

 

Answer: We are very sorry that we are not quite sure what the reviewer is referring to. This paper presents the protocol, which is established and used since 2014 for the specific reason to work on microplastic research. For visualising the procedure of removing the rectum, we show pictures in Figure 1 for further information on the procedure after the removal please check the material and method part, and the supplement for clarification.

 

 

L79: How samples have been collected and preserved? By my knowledge, the collection of seal faeces in the area of study are collected regularly at open air beaches. This implies that some airborne contamination might occur and authors seemed not considered that issue.

           

Answer: Thank you for this discussion point. As mentioned in our material & method part the sandbank is a tide-dependent submerged sandbank. That means there is a maximum of six hours between the egestion and sampling on the sandbank (inaccessible for the general public). In this study area, faeces samples are only collected by our institute after the here presented protocol. As described in our manuscript, after sampling we took great care on avoiding contamination.

 

 

L80: Remove, this reference is not needed

 

Answer: We would like to keep this reference to make sure, that interested readers are also informed about the location and procedure on our health monitoring. This helps to inform the reader of the study area and the circumstances.

 

 

L96: Why authors decided 1cm? Where the sample was collected. Why not several ones along the intestine? Authors should explain this method better.

           

Answer: Thank you for this hint. The given information in this sentence, is not important for the presented manuscript, thus it was removed. The collection of a tissue sample with a width of 1 cm was chosen, based on the common procedure used for histopathological analyses. We hope in future projects to be able to investigate tissue samples on lesions and toxic substances related to microplastics.

 

L98: were

           

Answer: Amended.

L98: It is not clear if the whole intestine is put into the bags or only the contents. In the first case, authors should explain how all content can be release from the intestine tissue as microplastics can be retained by intestine folds.

           

Answer: Please check the beginning of the paragraph in which we state that both is washed: For separating the MP from the biogenic matter, the intestine and the faeces samples are washed in self-sewed double layer washing sachets in a commercial washing machine (OK., OWM 15012 A1).

Therefore, we can be sure that all intestine contents are available for analysis. Please also check the sentence under 2.1.4 Isolation: The residue was rinsed with filtered, saturated sodium chloride (NaCl) solution (350 g of table salt dissolved in 1 l of MilliQ water) in glass beakers.

 

L106: Authors should also include a reference or a experiment which shows that microplastics are not affected by the washing powder

           

Answer: Thank you for this discussion point. The used basic protocol of Bravo Rebolledo et al. (2013) used the same enzyme-based washing detergent for macro and microplastic analysis. We agree that a degradation of the particles analysed cannot be excluded. The enzyme-based washing detergent rather effects biogenic material than synthetic structures. We rather believe that the process in the washing machine (shear force) might affect the particle structure and size; therefore, we spare the spin cycle to protect our particles.

 

L107: In my opinion, such detail of this protocol should be included in the appendix section.

           

Answer: We checked the option if it is valuable to move this paragraph to the supplement section. We strongly believe that the washing procedure is one of the major aspects of our microplastic analysis and thus should remain in the main manuscript.

 

 

L138: This reference is not needed and should be remove from here

 

Answer: Thanks for the hint we erased the reference.

 

L153: I apologize, but I got confused with this. If FTIR and Raman says it's a plastic and it's "X" polymer, why authors used this tree decission? This should be more clear why they used different methodologies. I found that in lines 242-245 is slightly explain,, but it should be better explained here.

 

Answer: We would like to clarify for the reviewer our intentions to use a decision tree. We have created criteria to narrow down which characteristics apply to microplastic particles under a fluorescence microscope. These criteria (1) help to categorise particles so that suspected microplastic particles can be finally identified using Raman analysis in the next step and (2) enable reviewers to identify potential plastic particles using the same criteria. If both reviewers come to the same conclusion on specific particles, these ones are going to be further analysed. This helps to eliminate potential non-plastic particles and is thus even more cost-effective.

Please find an additional sentence under 3.2 Protocol Validation to clarify the procedure: Potential microplastic particles identified by using this decision tree are than analysed using µRaman spectroscopy.

 

 

L154: remove marine mammals and replace it by pinnipeds

 

Answer: We agree that this sentence is confusing for the reader, therefore we added the following sentence under 2.1.6 Polymer Identification to make clear that our training subset of samples also include cetaceans: […] with 1185 characterised particles (biogenic or synthetic). This number of particles results of all available samples including pinnipeds and cetaceans, which build the basis for the used parameters in the decision tree.

 

L161: Why authors used beads but not fibers? Beads are difficult to find and may be more difficult to be realsed from sackets, however fibers will give a better idea how many fibers/microplastics can be lost due to the methodology.

 

Answer: The aim of this pre-trial was to guarantee that our procedure of using washing sachets is valuable and not risky to loose possible microplastics. Nevertheless, we wanted to ensure that the particles used for this pre-trial are as realistic as in our samples. Thus, we decided to use particles with a common synthetic polymer structure.

 

L166: Are authors also rinsed material before sampling? This might cause an external contamination.

 

Answer: We are sorry for any confusions. When talking about material in the sentence we are not referring to the sample itself, we are referring to all equipment and instruments used during the procedure. Therefore, we adjusted the sentence accordingly: All material equipment and instruments were rinsed several times with MilliQ water prior to usage.

 

L170: Authors should use biota reference and avoid studies referred to sediment and water. Only if they are strictly necessary should be mentioned.

 

Answer: Thank you for this hint. During double-checking of the named references, we come to the conclusion that both are very valuable to inform the reader on the procedure of dealing with procedural blanks. Therefore, we would like to keep the references as stated.

 

 

L174: These references are not needed. This is the general procedure when doing microplastic analysis.

 

Answer: Thank you we erased the references.

 

L176: Authors should explain better what the mean with "samples of partitioned faecal samples". It is difficult too understand if the mean selected faeces or only part of the faeces and why they did that.

 

Answer: We clarified the sentence by rewriting it: Nine faecal samples were additionally included in this study. These samples were divided: half of each sample was being stored in LDPE-bags, the other half in glass jars for three, six and 12 months.

 

L184: I do think that this part should be removed as it is more a discussion or conclusion statement.

 

Answer: Thank you for this hint. In order to avoid a repetition, we erased this paragraph since our aim of our study is clearly stated in the introduction section.

L188: In my opinion this should be the section 2.1.6 

 

Answer: We agree and moved this paragraph into the section 2.1.3 Washing Procedure since we believe it is more suitable in this section.

 

L194: I do think this is methodology.

 

Answer: We do not agree with the reviewer. The determination of a possible loss of microplastic particles is clearly a result. The procedure itself is described in detail in the suitable section of the material and method part.

 

L197: Authors should explain this in methods.

 

Answer: Please check for detailed information on the use of the hook-and-loop-fastener in the supplement information, as well as the reasons why we discarded this tool.

 

L209: Figure??

Answer: Thank you for spotting this formatting error. We mended this problem.

L210: I found this incidence quite high, and might be a good discussion point

Answer: We agree. Therefore, we took all measurements of the blanks into account to approach the real number of microplastic particles in the samples. We show this in Figure 7 and state that we consider this as contamination in section 3.1.2 Results of procedural Blanks (“Therefore, a mean number of seven 208 particles ≥100 μm (six fragments and one fibre) per sample was considered as contamination.”)

 

L216: crossed Figure 3 and replaced it by Figure 2

 

Answer: Please, recheck this. Figure 3 refers clearly to samples archived in plastic bags as described in this sentence and the figure description. Figure 2 show the potential quantity of microplastics found in blank samples.

 

L219: Authors should be more clear with this methods and results. Samples have been collected since many years (2012?), but then they do the experiment considering months.

In addition, the comparison is only among plastic bags, or also glass jars? why only plastic bags? I my opinion it is a bit confusing because the methodology is not easy to follow.

 

Answer: The usage of plastic bags also in microplastics research is common. It is believed that most research projects also deal with samples stored over month and years before analysis. Therefore, it is important to know what impact a long storage has on the samples.  We wanted to investigate if samples can be contaminated by storage in plastic bags, which would distort the result of the study. The idea on comparing those storage methods developed over the years and was not started straight away in 2014.

 

 

L220: This might be more a discussion point but authors should be more specific why they think is due to high variability of each period group. Is there any differences on collection and/or preservation?

 

Answer: We were not aware that this sentence might cause confusions. The high variability results out of the fact that for each time group (three, six and 12 months) different samples were used. This is the reason why we show the difference between the halved samples stored in glass jars and plastic bags for each period and compare these with the others. We clarified this in the following sentence (4. Discussion):

It has to be taken into account that the used samples already show a basic microplastic contamination. This differs from sample to sample, resulting in the fact that the plastic burden of the samples also varies between the time periods.

 

L222: Figure??

Answer: Thank you for spotting this. We mended this problem.

L233: In my opinion this should be at the method's section.

 

Answer: We carefully checked if this paragraph should be moved to the material and method section. Since the categorisation, which is used for this decision, tree is one of the major results of this paper. We would like to keep it under 3.2 Protocol Validation.

 

L251: Authors should be more clear which procedure they meant.

 

Answer: Thank you for this hint. To clarify which procedure was meant for the reader, we added the following term: In total, 653 potential plastic particles were determined in ten intestinal samples of free-ranging harbour and grey seals, and nine seal faecal samples where the described procedure (sample collection, isolation and identification) was used.

 

L257: Figure

 

Answer: Thank you for this hint we corrected it.

 

 

L262: Figure

Answer: Thank you for spotting this. We mended this problem.

L276: Authors seemed to mention at the beginning that they were comparing FTIR, RAMAN and fluorescence/staining methodologies, but nothing is mentioned about FTIR and RAMAN results

Answer: Thank you for this hint. The sentence the reviewer is referring to, which stated in the introduction section that the use of FT-IR and Raman analysis are two commonly used methods in microplastic research to identify synthetic polymer structures. We are sorry, that we rose the impression that a comparison is happening in our study. We now added commonly to clarify this: Subsequently, MP identification techniques such as the staining and fluorescence microscopic approach [33, 34] and spectroscopic analyses like Fourier transform infrared (FT-IR) [31, 35, 36] or Raman spectroscopy are commonly used [34, 37, 38].

L289: Authors stored frozen and none of these studies said that the physical process of being frozen and defrost afterwards will affect the smaples.

Answer: In our study, all samples are treated the same way, which includes also being stored at -20°C. Our study aims to evaluate the effects of the storing containers either plastic or glass rather than the conditions. We agree with the reviewer that freezing samples containing plastic might lead to the fact that those are even more cracking down into smaller particles. Nevertheless, since all used samples are frozen, this is not affecting our results. To avoid any confusion, we erased the sentence.

 

 

L291: Authors should explain why they think this. Have they checked if plastic bags originally had plastics? they way that they introduce the samples might affect external contamination?

 

Answer: We agree with the reviewer that plastic bags are generally more contaminated when they are used than glass jars, which are at least in our case, washed, disinfected, and only opened for inserting and removing the sample. Furthermore, the reviewer has to keep in mind that we are talking about low-density polyethylene bags (LDPE).

 

 

L295: What it does mean? As they come from seller? I found difficult the justification that plastic bags might come with external contamination if bags have not been checked previously.

 

Answer: The aim of this part of this study was to put the contamination risk by using plastic bags for storage into focus. Please keep in mind that most studies using plastic bags without taking the risk of contamination by these bags into account. Our initial concern was that samples being stored in plastic bags will be contaminated by these plastic bags over time which make them an unsuitable way of storing samples for microplastic analysis.

 

 

L301: Authors should include Lusher et al. 2018 regerence: DOI: 10.1016/j.envpol.2017.09.070

 

Answer: Thanks for this hint we added the named reference.

 

 

L309: Authors should mention this in the method's section.

 

Answer: Please check for detailed information on the use of the hook-and-loop-fastener in the supplement information, as well as the reasons why we discarded this tool.

 

L313: To reduce the number of reference for a note, authors might use other references previously used.

 

Answer: In the mentioned line, we start a completely new topic, which is of course in need of different literature. The literature named until now is thus not suitable.

 

L318: Authors did not included those results in the text.

 

Answer: Please find the requested information both in the material and method section (1) and in the result section (2).

 

1) Single particles were isolated with tweezers and pins, and placed on microscopic slides for further identification by µRaman spectroscopy (ThermoFisher Scientific®, DXR2xi Raman Imaging Microscope).

 

2) For a total of 31 suspected plastic particles, the polymer composition was verified via µRaman spectroscopy. 28 particles (90%) of these 31 particles were identified as plastic polymer. Following polymers were determined: Polyethylene (PE, n = 14), polyethylene terephthalate (PET, n = 5), ethylene-vinyl acetate (EVA, n = 6), polyamide (PA, n = 1) and polypropylene (PP, n = 1).

 

L319: With only two refereces will be enough. I do recommen authors to use only those related to methodology comparison.

 

Answer: We re-checked the mentioned references and would like to keep them, since they support our statement.

 

L327: what about beaks?

 

Answer: We added beaks to the list.

 

L333: This is something expected because faeces collected at beaches are likely to contain sand.

 

Answer: We agree with the reviewer, that the fact of collecting the samples on a sandbank might lead to the risk of integrated sand in the sample. Nevertheless, a mean difference of 4 g cannot be due to the amount of sand collected with samples, which we can assure with our experience of sample taking. The volume of the seal scats on the sandbanks are much higher, compared to the included faeces in the intestine samples collected during necropsies.

 

L339: In my opinion, authors should avoid to talk about cetaceans because their behaviour, diet and GIT is different to seals. In addition, this manuscript is only related to seals.

 

Answer: Thank you for this valuable comment. We erased the sentence.

 

L341: Authors should be careful when doing this statement. In both cases, the contents still being snapshots because both belongs to a meal or consecutive meals. It is not considered snopshot when you have a period of time between collection (it means at least a week in the case of seals).

 

Answer: Thank you for this hint. For clarification, we rewrote the sentence: Thus, the sandbank samples include the faeces of a full egestion of the rectum. In contrast, the intestinal samples represent only a part of the actual excretion, which is here further analysed.

 

L344: This is difficult to argue and authors should be careful with this. See comment above.

 

Answer: Thank you for this comment. We erased the sentence.

 

L349: Authors should include Lusher et al. 2018 regerence: DOI: 0.1016/j.envpol.2017.09.070

 

Answer: Thank you we added the reference.

 

L352: Authors should be careful with this. The use of only one organ (in this case intestine) should be compare with the use of the whole GIT, and obtaining a statistical equation to be used. In this case authors do not know if animals had many microplastics (or even macroplastics) in the rest of the GIT, e.g. stomach) and they should not do such strong statement.

 

Answer: Please, bear in mind that this statement is a general comment on benefits of using necropsy samples. This helps to also get information on the health status of the individuals. We agree that to assess the actual burden, the whole GIT needs to be checked on microplastic occurrence. Nevertheless, our study focuses on giving first evidence of microplastics in marine mammals from German waters and furthermore on sample handling. In addition, it is almost impossible to save the whole GIT for microplastic analysis in all necropsied animals since especially this part of the body also needs to be investigated for other purposes as well, which would increase the risk of contamination. Macroplastic occurrence is noted in all necropsied animals.

 

 

L355: Authors  should be careful here. Cetaceans have several chambers with different surface tissue (folds, graticle, etc) and it might retain more microplastics than seal stomachs which only have one chamber with tissue folds regularly cranial orientated.

 

Answer: We agree with the reviewer that there are differences as also stated in the named reference. Nelms et al. 2019 showed in both cetaceans and seal species a high number of ingested hard parts. Therefore, we would like to keep the sentence.

 

 

L356: Authors should include Lusher et al. 2018 regerence: DOI: 0.1016/j.envpol.2017.09.070

 

Answer: Since we are referring to accumulations of high quantities of hard parts the reference of Lusher et al 2018 is not suitable.

 

 

L356: Again, authors here assumed something that might not occur in seals. By my experience, seals and other marine mammals are "attacked" at the neck or stomach area depending how easy is for the scavenger. The most common area is the neck (close to the face).

 

Answer: Thank you for this discussion point. There are studies dealing with the topics of scavenging on carcasses. Therefore, we rewrote the sentence and used the suitable references for this statement: Nevertheless, the GIT is often specifically opened or removed by scavengers [65, 66].

 

 

L364: Authors should include Lusher et al. 2018 regerence: DOI: 0.1016/j.envpol.2017.09.070

 

Answer: Since we are referring to an amount of positive samples the reference of Lusher et al. 2018 is not suitable.

 

L369: Authors should not do this statement based in only one reference. I suggets to remove this sentence of suggested this possibility as it was suggested in that reference (and even in ref 52).

 

Answer: Thank you for this hint we added two more literature references to       validate our statement.

 

L370: I do recommen authors to be caerful with this statement. Microplastics are difficult to be absorbe and be transferred to other tissues due to the small size to cross membranes. That will be the case of nanoplastics, which is not what the authors are studying.

 

Answer: Please note that the statement the reviewer is referring deals with the accumulation of contaminants being transported or related to microplastics.

 

L371: This has not been confirmed yet. Authors should be careful with statements and should be used more modal verbs.

           

Answer: Please check our comment for line 370.

 

L374: In my opinion this is not needed.

           

Answer: Thanks for this comment. We erased the sentence.

 

L416: Missing editors of the book

 

L418: remove 80 //keine Ahnung wo das Herkunft, formatting error?

 

L470: remove “October 2018”

 

L526: This is a cetacean reference, not a seal

 

L618: I do think this is an old reference in this topic. There are newer ones like: https://doi.org/10.1371/journal.pone.0131085  DOI: 10.1038/srep18573

 

Answer to all comments line 416 - 618: Thank you for this hint. We modified all literature references and added all needed information.

 

L625: Supplementary:

          Figure 9: likely fish spine,

          Figure 10: in my opinion is a clear fish eye lense,

          Figure 11: likely a piece of a fish scale

 

Answer to Figure 9 - 11: Thank you for this assessment. Since we did not do any further analysis, we would like to stick to the used generic term fishbone. We state the possibility of a fish lens in figure 10, but without further analysis, we stick also to the generic term. Furthermore, we cannot narrow the specification of Figure 11 and also want to stick with biogenic fragment.

Author Response File: Author Response.doc

Reviewer 3 Report

Materials and Methods

• Please make clearer at the beginning of this section how many samples did you study
• A Statistical section should be added in which you explain your analysis, why did you use a Wilcoxon test, variables analysed, any checks for normality, etc

Results

• Check a few references “Not found” throughout the text i.e. L 223, 257, 262
• Figure 2 and 4, please be more specific on the Y axis “Quantity” is very broad
• Figure 6. “Microplastic particles from intestinal samples stained with Nile red photographed under a fluorescence microscope” under what specific light parameters? I assume it is green and/or UV fluorescent light? Please specify here

Supplementary information

• Figure 2. Many microbeads are cracked or broken so they have lost their “bead” shape? How did you measure the size of those?

Author Response

Dear Dr. Rodríguez Seijo,

 

many thanks for sending the reviews for our paper entitled “Handle with Care – Microplastic Particles in Intestines Samples of Seals from German Waters”. Please find our rebuttal letter from the next page onwards, replying to all comments of the three reviewers. Please find our responses after every comment in italics. We re-checked scientific language and changed expressions according to reviewer’s comments. Furthermore, a native speaker proofread the manuscript. We send you the manuscript in track change mode. The improved supplement information is additionally attached in a pdf file. All co-authors agreed on the new versions of both.

 

 

With kind regards

 

 

 

 

Prof. Prof. h. c. Dr. Ursula Siebert

 

 

 

 

 

Rebuttal letter

 

 

Response to Reviewer 3

Materials and Methods

Please make clearer at the beginning of this section how many samples did you study

Answer: Thank you for this valuable hint. We added the following sentences: Out of these, ten pinniped samples were used for this study. […] Out of these, nine faecal samples were analysed.

 

A Statistical section should be added in which you explain your analysis, why did you use a Wilcoxon test, variables analysed, any checks for normality, etc

 

Answer: The parametric t-test can only be used if certain conditions such as normal distribution and homogeneity of variance are met. We evaluated this by shapiro.test and var.test in R. This revealed that our variables are neither normally distributed nor present a homogeneous variance, so we had to opt for a non-parametric, distribution-independent test, such as the dependent 2-group Wilcoxon Signed Rank Test.

 

Results

Check a few references “Not found” throughout the text i.e. L 223, 257, 262

Answer: Thank you for this hint we mended the problem.

 

Figure 2 and 4, please be more specific on the Y axis “Quantity” is very broad

Answer: Thank you for this hint. We changed the heading for the Y axis into ”Quantity of suspected microplastics “.

 

Figure 6. “Microplastic particles from intestinal samples stained with Nile red photographed under a fluorescence microscope” under what specific light parameters? I assume it is green and/or UV fluorescent light? Please specify here

Answer: Thank you for that. We added the missing information on the used (TRITC) filter under 2.1.6 Polymer Identification.

 

Supplementary information

Figure 2. Many microbeads are cracked or broken so they have lost their “bead” shape? How did you measure the size of those?

Answer: We agree that the microbeads are cracked or broken. The measurement for these particles was conducted beforehand to check on their size and thus the possibility to get lost during the washing procedure. We just wanted to assure that the cloth with need mesh sizes are suitable for our procedure.

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

Thanks to the authors for modifying their manuscript. However, it feels as though the authors rushed their changes so there are still areas which require further improvement.

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Previous Reviewer comment - L80: How was environmental contamination from the sandbank limited/ controlled for?

Answer: Thanks for your comment. As mentioned in our material & method part the sandbank is a tide-dependent submerged sandbank that means between the egestion and sandbank collection the time maximum is six hours. Between egestion and sample collection, we cannot control the contamination by air or sand. Nevertheless, to collect samples on a sandbank is rare and thus valuable possibility to investigate in free-ranging marine mammals. For each sample one glass jar and one spoon is used to avoid cross-contamination

New Reviewer comment: It is of course possible to account for contamination from this source e.g. take samples of sand and use a damp petri dish to collect air-borne microplastics and compare what you find. This is an oversight.

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Previous reviewer comment - L138: How many particles were subjected to FTIR? What proportion of the overall number detected?

Answer: Please, recheck our material & method part (2.1.5 Polymer Identification) in which we state, that our identification was conducted using µRaman spectroscopy. Furthermore, this information in our opinion belongs to the results section. We state the required information in section 3.3 Isolation of MP in Intestinal and Faecal Samples: A total of 31 suspected plastic particles were analysed via µRaman spectroscopy. 28 particles (90%) of these 31 particles were identified as plastic polymer.

New Reviewer comment: You have still not answered my question. What was the total number of suspected microplastics found in the animal and what proportion of those were subjected to polymer analysis? This information is crucial and should be included in the Methods

------------------------------------------------------------------------------------

Previous Reviewer comment L145: Here you say fragment but I think you mean particle as fragment is a specific shape of microplastic and you are talking about fibres in the sentence.

Answer: For clarification as mentioned in the sentence, we defined a particle as either fragment or fibre. We clearly refer to both categories in the whole manuscript when talking about particles.

New Reviewer comment: This still makes no sense and needs re-wording for clarity

--------------------------------------------------------------------------------

Previous Reviewer comment L149: What are the metrics for these variables? How did you measure them?

Answer: The metrics for the mentioned variables were defined by using comparable pictures of already identified particles by µRaman spectroscopy. Thus, for example the higher the fluorescence intensity is, the more likely it is that the particle is a microplastic particle.

New Reviewer comment: Fluorescence intensity must have a metric/ unit, otherwise it is subjective and not an appropriate method to use. What is the measurement of fluorescence intensity?

------------------------------------------------------------------------------

Previous Reviewer comment L152: The concept of the decision tree needs more background context - what is it and why is it a relevant method to use here?

Answer: The establishing of a decision tree helps to categorize different variables, which are helpful for untrained researchers to gain results that are comparable between research groups. This is essential to compare the burden in different marine environments and biota. Furthermore, the decision tree helps to narrow down suspected microplastic particles out of a bunch of a variety of particles in a sample, which are then used for further analysis. This is cost- and time-effective and a useful tool for encouraging decision makers to support necessary monitoring programme.

New Reviewer comment: This information needs to be included in the manuscript

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Previous Reviewer comment L162: Why were they sewn into the cloth? The microplastics in your samples would have not been subjected to the same treatment so this control is not comparable and does not represent the conditions to which your samples were exposed.

Answer: Please, keep in mind that the microbeads were used as pre-trial to determine the recovery rate. They were thus treated the same way as the original samples. Have a closer look on the paragraph in the original manuscript (2.1.2 Preparation):

For separating the MP from the biogenic matter, the intestine and faeces samples are washed in self-sewed double layer washing sachets in a commercial washing machine (OK., OWM 15012 A1). Each sample was placed in an inner bag (mesh size 300 µm) which was then placed in the outer nylon bag (mesh size 100 µm). The sachets were made of nylon cloth and were sewn together in the acrylic box with a conventional sewing machine (Singer, Tradition TM 2282) using black cotton yarn. Furthermore, each nylon sachet was used only once to prevent a cross-contamination.

New Reviewer comment: Please do not patronise me. If the Methods are unclear it is because you have failed to describe them properly. The onus is on you to make it easy to understand. This text is not clear and I suggest YOU take closer look at what is written. I believe what you mean to say is that the 'nylon cloth enclosing the microbeads was sewn together'.

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Previous Reviewer comment L243: How were these ground-truthed? Particles can look synthetic but aren't necessary plastic.

Answer: The dataset of particles that were used to build this decision tree have been evaluated independently by two trained researchers. For those particles that were not unanimously classified as either plastic or non-plastic, the classification has been jointly discussed and further expert opinions obtained or investigated by µRaman spectroscopy were undertaken. So that we are confident that true positive rate of 84%, that measures the proportion of MP particles that were identified as possible MP and the true negative rate of 69%, which measures the proportion of non-plastic particles that were identified as being non-plastic, revealed a good performance of the decision tree.

New reviewer comment: How were the particles 'evaluated independently by two trained researchers'? This means nothing without transparent methodologies

-----------------------------------------------------------------------------------

Previous Reviewer comment: L280: Improve from what? There are studies which eliminated sources of contamination - this method is not as effective as those so why not just replicate them?

New reviewer comment: No response from authors

Author Response

Dear Dr. Rodríguez Seijo,

 

Many thanks for sending the reviews for our paper entitled “Handle with Care – Microplastic Particles in Intestines Samples of Seals from German Waters”. Please find our rebuttal letter for the second review from the next page onwards, replying to all comments of the remaining two reviewers. Our responses are to be found in italics after every new comment from the reviewers. Please find attached the certificate of the native speaker Ms Sherwood-Brock, who checked our manuscript for grammar and spelling mistake. Furthermore, we are sending you a document in track change mode. All co-authors agreed on the new version of the manuscript. We now hope that we meet the requirements of the reviewers and looking forward to getting a positive answer.

 

 

With kind regards

 

 

 

 

Prof. Prof. h. c. Dr. Ursula Siebert

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rebuttal letter

 

Round 2

Reviewer 1:

Comments and Suggestions for Authors

Thanks to the authors for modifying their manuscript. However, it feels as though the authors rushed their changes so there are still areas which require further improvement.

Previous Reviewer comment - L80: How was environmental contamination from the sandbank limited/ controlled for?

Answer: Thanks for your comment. As mentioned in our material & method part the sandbank is a tide-dependent submerged sandbank that means between the egestion and sandbank collection the time maximum is six hours. Between egestion and sample collection, we cannot control the contamination by air or sand. Nevertheless, to collect samples on a sandbank is rare and thus valuable possibility to investigate in free-ranging marine mammals. For each sample one glass jar and one spoon is used to avoid cross-contamination

New Reviewer comment: It is of course possible to account for contamination from this source e.g. take samples of sand and use a damp petri dish to collect air-borne microplastics and compare what you find. This is an oversight.

Answer: Thank you for this discussion point. For this study, we focussed on establishing a method, which allows for analysing faecal samples without having a clean lab available. Thank you for reminding us of the possibilities of checking airborne contamination. For the burden assessment of seal samples collected on a sandbank we will take into account using only the core of the collected scat to not overestimate the quantity. Since the samples were collected years ago, it is only possible to check for the contamination by additional sand samples and environmental blanks in future, as recommended by the reviewer. 

 

Previous reviewer comment - L138: How many particles were subjected to FTIR? What proportion of the overall number detected?

Answer: Please, recheck our material & method part (2.1.5 Polymer Identification) in which we state, that our identification was conducted using µRaman spectroscopy. Furthermore, this information in our opinion belongs to the results section. We state the required information in section 3.3 Isolation of MP in Intestinal and Faecal Samples: A total of 31 suspected plastic particles were analysed via µRaman spectroscopy. 28 particles (90%) of these 31 particles were identified as plastic polymer.

New Reviewer comment: You have still not answered my question. What was the total number of suspected microplastics found in the animal and what proportion of those were subjected to polymer analysis? This information is crucial and should be included in the Methods

Answer: Thank you for this valuable comment. In the part “3.3 Isolation of Potential MP in Intestinal and Faecal Samples”, the reviewer will find the following information:

Due to the used mesh sizes of the washing sachets (100 µm and 300 µm), all particles smaller than 100 µm were excluded from the study, resulting in 255 identified suspected MP particles (70 fibres and 185 fragments) in 18 out of a total of 19 samples. The share of fibres ranged from zero to 35 per sample (M±SD = 6±7.4) when taking all specimens stored in glass jars into consideration (≥100 µm). The number of fragments in each sample stored in glass jars (≥100 µm) varied from five to 55 parts (M±SD = 13.3±11.3). […] For a total of 31 suspected plastic particles, the polymer composition was verified via µRaman spectroscopy. Twenty-eight particles (90%) of these 31 particles were identified as plastic polymer.

Thus, in our opinion we clearly stated how many particles were further analysed by µRaman spectroscopy. For further clarification, we added the following sentences with the required information:

Those particles were collected randomly from all 19 named samples. Thus 11% of the whole quantity of suspected MPs were analysed. 

We hope that the reviewer can now accept our statement.

 

Previous Reviewer comment L145: Here you say fragment but I think you mean particle as fragment is a specific shape of microplastic and you are talking about fibres in the sentence.

Answer: For clarification as mentioned in the sentence, we defined a particle as either fragment or fibre. We clearly refer to both categories in the whole manuscript when talking about particles.

New Reviewer comment: This still makes no sense and needs re-wording for clarity

Answer: We are sorry that the reviewer still cannot agree with us. We understand that the reviewer has another definition for the word “particles”. We also state in our manuscript the definition we follow throughout the manuscript.

Previous Reviewer comment L149: What are the metrics for these variables? How did you measure them?

Answer: The metrics for the mentioned variables were defined by using comparable pictures of already identified particles by µRaman spectroscopy. Thus, for example the higher the fluorescence intensity is, the more likely it is that the particle is a microplastic particle.

New Reviewer comment: Fluorescence intensity must have a metric/ unit, otherwise it is subjective and not an appropriate method to use. What is the measurement of fluorescence intensity?

Answer: Thank you for this discussion point. The intensity of fluorescence of microplastic particles in all environmental media varies according to polymer type, ageing and additives. For this reason, it is not possible to give a uniform value for the fluorescence intensity, as the differentiation from the background also depends on the respective sample matrix. This is one of the main reasons why automated digital image analysis is only just being developed in this field and must meet these challenges (as can be seen for example in figure 6). In addition to the intensity, the relationship between the RGB values is also decisive within MP analysis. As a threshold in the set-up we used (as described in the Material and Methods section) a value of at least 3000 (max. 16,383) for red and green fluorescence with simultaneous absence of blue wavelengths can be set. This corresponds to the intensity in transferred grey values (and has no physical unit). Nevertheless, this value varies significantly depending on the set-up of the microscope and filter system (and even the light conditions in the respective surrounding), so that this value has to be determined empirically.  For clarification, we have added the following text in the material and method section (2.1.6 Polymer Identification):

Potential MP particles were identified based on their fluorescence and the relation of red and green wavelengths (setting a minimal threshold of intensity of 3000). These particles were photographed (Kern ODC 832 and Canon EOS 80D) under the fluorescence microscope. Subsequently, the identified particles were visually evaluated, measured and counted by using Adobe Photoshop (Version 21.0.3). A subset of identified single particles (n = 31) were isolated with tweezers and pins, and placed on microscopic slides for further identification by µRaman spectroscopy (ThermoFisher Scientific GmbH, DXR2xi Raman Imaging Microscope). […] The following parameters were used for discrimination: fluorescence intensity, RGB spectra relation and appearance of the particle (surface, isolation, completeness, etc.). For additional information, see the supplement.

 

Previous Reviewer comment L152: The concept of the decision tree needs more background context - what is it and why is it a relevant method to use here?

Answer: The establishing of a decision tree helps to categorize different variables, which are helpful for untrained researchers to gain results that are comparable between research groups. This is essential to compare the burden in different marine environments and biota. Furthermore, the decision tree helps to narrow down suspected microplastic particles out of a bunch of a variety of particles in a sample, which are then used for further analysis. This is cost- and time-effective and a useful tool for encouraging decision makers to support necessary monitoring programme.

New Reviewer comment: This information needs to be included in the manuscript

Answer: Thank you for this valuable hint. We added the information discussed in the discussion part.

 

Previous Reviewer comment L162: Why were they sewn into the cloth? The microplastics in your samples would have not been subjected to the same treatment so this control is not comparable and does not represent the conditions to which your samples were exposed.

Answer: Please, keep in mind that the microbeads were used as pre-trial to determine the recovery rate. They were thus treated the same way as the original samples. Have a closer look on the paragraph in the original manuscript (2.1.2 Preparation):

For separating the MP from the biogenic matter, the intestine and faeces samples are washed in self-sewed double layer washing sachets in a commercial washing machine (OK., OWM 15012 A1). Each sample was placed in an inner bag (mesh size 300 µm) which was then placed in the outer nylon bag (mesh size 100 µm). The sachets were made of nylon cloth and were sewn together in the acrylic box with a conventional sewing machine (Singer, Tradition TM 2282) using black cotton yarn. Furthermore, each nylon sachet was used only once to prevent a cross-contamination.

New Reviewer comment: Please do not patronise me. If the Methods are unclear it is because you have failed to describe them properly. The onus is on you to make it easy to understand. This text is not clear and I suggest YOU take closer look at what is written. I believe what you mean to say is that the 'nylon cloth enclosing the microbeads was sewn together'.

Answer: We are sorry that the reviewer feels patronised. That was not our intention. Nevertheless, we do not understand the concerns. We state in the text that our pre-trial samples were treated in the same way as our original samples, especially to have it comparable. Therefore, we wanted to ensure that the reviewer was given the corresponding paragraph in our answer. Both procedures (pre-trial and original samples) were processed in the same way. For further clarification, we have edited the following part (see 2.1.5 Pre-Trials to Verify the Procedure) in the manuscript:

To determine the loss of potential MP, polyethylene microbeads (fragment size: 150 µm - 800 µm) were placed in the nylon sachets and processed in the same manner as the intestinal and scat samples.

 

Previous Reviewer comment L243: How were these ground-truthed? Particles can look synthetic but aren't necessary plastic.

Answer: The dataset of particles that were used to build this decision tree have been evaluated independently by two trained researchers. For those particles that were not unanimously classified as either plastic or non-plastic, the classification has been jointly discussed and further expert opinions obtained or investigated by µRaman spectroscopy were undertaken. So that we are confident that true positive rate of 84%, that measures the proportion of MP particles that were identified as possible MP and the true negative rate of 69%, which measures the proportion of non-plastic particles that were identified as being non-plastic, revealed a good performance of the decision tree.

New reviewer comment: How were the particles 'evaluated independently by two trained researchers'? This means nothing without transparent methodologies

Answer: Thank you for this discussion point. Both researchers identified up to more than 1,500 microplastic particles. This experience was acquired over the last two years and is based on training on visual identification of microplastics given by microplastic experts. Thus, both trained researchers validated the taken photographs of the presented particles independently from each other, based on previous concerted parameters. Afterwards, the results were validated, which resulted in the training set for the presented decision tree. These parameters (fluorescence intensity and appearance of particles) were developed by the authors with the support of the microplastic experts, and were adapted to the used microscope and camera system, and tested by a previous polymer identification by µRaman spectroscopy of test particles. Nevertheless, a given catalogue in the supplement, describing the parameters in text and pictures, allows beginners in microplastic research to have an idea of what could be a microplastic particle or a biogenic one. The use of this catalogue and the idea of a decision tree by using those parameters will narrow down the choice for particles and further analysis like Raman or FT-IR spectroscopy. As a result of this, our methodology is described very transparently and will save unskilled researchers time and money.

 

Previous Reviewer comment: L280: Improve from what? There are studies which eliminated sources of contamination - this method is not as effective as those so why not just replicate them?

New reviewer comment: No response from authors

 

 

L268: check formatted error, I came up again

            Answer: Thank you for spotting this. We corrected it.

 

L350: one dot to be removed

Answer: Amended.

 

In the same line as it was suggested at 2.1, authors seem to justify at the discussion section (L346-L351) that diet and plastics concentrated at the last part of intestines. I have been analyzed GIT (from mouth to annus) for more than 10 years in different marine mammal species, and I have not found any pattern like that. In fact, regularly I found more diet and microplastics in all species in stomachs and first part of the intestines (first 4-5 metres). This is why I cannot understand the methodology approach. I have not found any publication indicating that diet (considering that this is the type of study which have been carried out longer time than MPs) concentrated in the rectum until the full ejection. Authors suggested in that paragraph (L346-L351) that collecting intestines and faeces is a recommendation, however they only used the last part. In my opinion is recommended to use the full digestion tract for a better estimation.

Answer: Please keep in mind, that the approach here is based on evaluating a method for further research. We are part of a large institute collecting a variety of different samples. Therefore, we rarely have the chance to investigate the whole GIT solely for microplastic occurrence since this needs special procedures compared to other topics. Therefore, we decided to use rectum samples WITH content to ensure us having faeces available for further analysis.

 

 

L360-364:

Answer: Thank you for this discussion point. There are studies dealing with the topics of scavenging on carcasses. Therefore, we rewrote the sentence and used the suitable references for this statement: Nevertheless, the GIT is often specifically opened or removed by scavengers [65, 66].

Talking by my experience of more than 10 years attending strandings and reviewing dissection reports of different marine mammals, this is not often everywhere. It is occurring from time to time. The new reference added is the only reference I had in mine and that’s why I argued previously that this circumstance is uncommon. If you still keep the sentence, I do recommend removing again the penguin reference but add the personal communication from your person in charge to do marine mammal dissections in your team. In that case, as a local case it could be justify. But again, there are few references and it does not occur everywhere as it depend on many issues.

Answer: Thank you for this hint. We deleted the paragraph to avoid misunderstandings (Furthermore, it is already known that the stomach of marine mammals accumulates a high quantity of ingested hard parts [20]. Nevertheless, the GIT is often specifically opened or removed by scavengers [65, 66]. Owing to these facts, this study considers only undamaged GITs for establishing this approach in microplastic research although, this presented approach is also applicable for stomach tissue samples and the content.)

 

 

L364: Answer: Since we are referring to an amount of positive samples the reference of Lusher et al. 2018 is not suitable.

I do not understand authors response. The study from Nelms et al and Lusher et al are similar as they follow similar protocols. In fact, in Table 1 (Lusher et al.) indicates which animals contained macrodebris, or microplastics or both.

Answer: Thank you for the hint. Maybe there was a misunderstanding regarding the correct reference. The reviewer is right. We added Lusher et al. 2018 to the sentence as a reference.

 

L371-L378: Answer: Please note that the statement the reviewer is referring to deals with the accumulation of contaminants being transported or related to microplastics.

Apologize me for not being clear, I meant that the statement seems to say that microplastics AND contaminants associated accumulate in predators (see L376: accumulate in top predator species). This is the reason why I said is a strong statement. It is widely known in the community that most of the microplastics are egested (there are many publications about this, in particular in invertebrates). Therefore, the possibility to be accumulated is low. However, the transferred of associated contaminants to the predator itself is something that most of the community suspect that it is occurring. My suggestion was to try to be careful with that sentence.

Answer: We rewrote the sentence to clarify our statement: Since it is proven that microplastics are transported across the food web [14, 16, 68], it is assumed that in particular the contained contaminants accumulate due to absorption and leaching [69] in top predator species as it is already evidenced for example for organochlorides [70, 71].

Author Response File: Author Response.doc

Reviewer 2 Report

The paper needs major corrections again, even after the authors corrections.

Comments for author File: Comments.pdf

Author Response

Dear Dr. Rodríguez Seijo,

 

Many thanks for sending the reviews for our paper entitled “Handle with Care – Microplastic Particles in Intestines Samples of Seals from German Waters”. Please find our rebuttal letter for the second review from the next page onwards, replying to all comments of the remaining two reviewers. Our responses are to be found in italics after every new comment from the reviewers. Please find attached the certificate of the native speaker Ms Sherwood-Brock, who checked our manuscript for grammar and spelling mistake. Furthermore, we are sending you a document in track change mode. All co-authors agreed on the new version of the manuscript. We now hope that we meet the requirements of the reviewers and looking forward to getting a positive answer.

 

 

With kind regards

 

 

 

 

Prof. Prof. h. c. Dr. Ursula Siebert

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rebuttal letter

 

Round 2

 

Reviewer 2:

Second review of the Sustainability Journal Manuscript ID: sustainability-968357 Title: Handle with Care – Microplastic Particles in Intestine Samples of Seals from German Waters

General comments:

I am still thinking that this manuscript has many issues to be amended that authors are not considered. I have strong doubts about the methodology that they suggested to be used for assessment/monitoring using only one part of the intestine. In order to do not repeat myself I justify my concerns below.

I accepted the arguments authors gave related to maintained information and other references. In the last case, I am still thinking that they are too many references and despite it was suggested to avoid references related to studies with no biota authors are still using them. This is not a good practice, because there are many available references related to biota. They should try to use references related to biota.

 

Answer: We carefully checked if this paragraph should be moved to the material and method section. Since the categorisation, which is used for this decision, tree is one of the major results of this paper. We would like to keep it under 3.2 Protocol Validation.

I am still thinking that this will fit better in methods, but the argument by authors is accepted to keep it at results section. Apologize for not understanding the intestine washing procedure. You were right! However, in my opinion it makes the reader going up and down and that’s why I got confused.

Answer: We are glad to hear that the reviewer accepted our decision.

 

Answer: Thank you for this hint. The given information in this sentence, is not important for the presented manuscript, thus it was removed. The collection of a tissue sample with a width of 1 cm was chosen, based on the common procedure used for histopathological analyses. We hope in future projects to be able to investigate tissue samples on lesions and toxic substances related to microplastics.

I do believe that that future research will be very valuable for the community. I am looking forward to seeing that research!

Answer: Thank you for this compliment. We are looking forward to proceeding with our research.

 

In addition, there are some arguments that authors are still not resolving:

2.1: Why authors decided that the last 10 cm of the intestinal region might be representative? I found difficult to justify that using the last 10cm of an intestine can be extrapolate the incidence of microplastics in animals. The only reference up to date combining the different parts of the GIT in marine mammals has been carried out by Lusher et al. 2018 (Doi: 10.1016/j.envpol.2017.09.070) where it is possible to see that microplastics are usually in higher number in the stomach region and not in the last part of it This is the same situation for studies on diet. Most of the diet tend to concentrate in stomachs although some diet remains can be found along intestines. In addition, authors ignored a reference on grey seal intestines related to microplastics (Doi: 10.1016/j.marpolbul.2019.06.014) where authors showed a higher incidence of microplastics at the first part of the intestines (Figure 4 of the publication). (I think also this reference should be included at L354 and see comments about this issue below at results section comments).

Answer: Thank you for this discussion point. We do not plan to extrapolate our data. The main focus of this study is to establish a suitable sample handling and procedure.  We are part of a large institute with a variety of projects concerning health in free-ranging animals. Thus, we rarely have the chance to investigate the whole GIT solely for microplastic occurrence since this needs special procedures compared to other topics such as parasite investigation. Therefore, we decided to use rectum samples WITH content to ensure us having faeces available for further analysis. Certainly, interdisciplinary working groups allows us to have a greater look at the health issues concerning microplastic occurrence.

 

2.1.6: Authors are explaining here that microplastics are identifying using a visual technique; however, they never indicate in the text that those plastics were confirmed with other analytical technique (this is seems to be explained in the Supplementary file). Visual identification is not a good practice for confirming that an anthropogenic particle is plastic. This identification should be confirmed always with another analytical technique. Authors explained in their “Author’s response to Reviewer’s comments” and I think that they should include this brief sentence here saying “After visual identification, uRaman analysis was carried out for particle confirmation”. I’m aware that they mentioned now at the result section (3.2), but this should be included in methodology as it is part of the methodology process.

Answer: Thank you for this idea. We rearranged the sentence: Subsequently, the photos were visually evaluated, measured and counted by using Adobe Photoshop (Version 21.0.3). A subset of identified single particles (n = 31) were isolated with tweezers and pins, and placed on microscopic slides for further identification by µRaman spectroscopy (ThermoFisher Scientific GmbH, DXR2xi Raman Imaging Microscope).

 

2.2: Their answer to previous revision:

Answer: The usage of plastic bags also in microplastics research is common. It is believed that most research projects also deal with samples stored over month and years before analysis. Therefore, it is important to know what impact a long storage has on the samples. We wanted to investigate if samples can be contaminated by storage in plastic bags, which would distort the result of the study. The idea on comparing those storage methods developed over the years and was not started straight away in 2014

Authors indicated that they collected 5 samples since 2012 stored in LDPE bags. They also included another 9 samples, but they didn’t indicate if these new samples were stored in LDPE bags. I do understand that usually this is the procedure, use LDPE bags. In my opinion, this is a weak argument to suggest the release of microplastics from plastics bags over the time. Microplastics can evenly distributed in specific samples with no reason. Firstly, because it is difficult to confirm if microplastic was released from the bag or it was in the sample before it was collected. May be the defrost process might facilitate the release in specific samples, depending the time it was store, how long it was exposed to environmental conditions previous to be stored, how was the scat/faeces (e.g. more or less dry). All these things might affect the potential release of microplastics from the bags. The only reasonable way to know if a bag release microplastics is knowing that our sample is totally free of other particles. This is possible something to justify the results. However, I do agree that using glass jars is better to control plastic contamination, however, the problem arise that glass jars can be broken when transfer to lab facilities (for different reasons) and they should not be frozen (defrost process may cause the glass broken more easily than plastic). In addition, I suggested the possibility of airborne/external contamination when collecting the scats/faeces at beaches. But authors did not mention this possibility.

Answer: Thank you for sharing your ideas and experience with us. We agree that there are many aspects, which have to be taken into account when storing samples. Nevertheless, the common way of storing the samples (even if the focus is on MP occurrence) is in plastic bags. Therefore, it is essential to inform the readers about possible contamination when using plastic bags. We agree that glass jars are fragile but our experience has shown that it is reasonable to take this risk. Apart from that, larger glass particles could easily be removed and Nile Red would not stain the smaller glass particles. Thus, it will not affect the samples.

 

Answer from authors: Thank you for this discussion point. As mentioned in our material & method part the sandbank is a tide-dependent submerged sandbank. That means there is a maximum of six hours between the egestion and sampling on the sandbank (inaccessible for the general public). In this study area, faeces samples are only collected by our institute after the here presented protocol. As described in our manuscript, after sampling we took great care on avoiding contamination.

I would like to ask the same question again: How samples have been collected and preserved? By my knowledege, the collection of seal faeces in the area of study are collected regularly at open air beaches. This implies that some airborne contamination might occur, and authors seemed not considered that issue. Unfortunately for our research, airborne contamination occurs even if there is in a beach with not public access. Winds are able to move those particles from far away areas and even from researchers collecting samples. By my experience, I found particles from my clothes working in the lab with my sample under the microscope without air currents and no people around. This is one of the reasons to work with white clothes (if possible, cotton ones) to remove later from our samples and not considered them. In relation to fragments it will be more complicate the airborne contamination and samples should not be affected, except if there are fragments at the beach (which it is reasonable to assume that we are not collecting them)

Answer: We were not aware that this sentence might cause confusions. The high variability results out of the fact that for each time group (three, six and 12 months) different samples were used. This is the reason why we show the difference between the halved samples stored in glass jars and plastic bags for each period and compare these with the others. We clarified this in the following sentence (4. Discussion): It has to be taken into account that the used samples already show a basic microplastic contamination. This differs from sample to sample, resulting in the fact that the plastic burden of the samples also varies between the time periods.

 

With the small changes now it’s more clear what was the procedure, but I have many doubts about that experimental procedure (see my comments above).

Answer: Thank you for clarifying your question and sharing your experience with us. We are aware of airborne contamination. Thus, in future we will only use the core of the scat for assessing the real burden of microplastics in faeces. This will avoid an overestimation of MPs caused by contamination by sand or the collector. In this concerning case, which is discussed by the reviewer, the scat samples were used to determine differences in sampling storage. Since we deal with GIT samples, the idea was to assess the contamination risk of the used plastic bags by using faeces unprotected by tissue.  

 

 

Results:

Answer: Please, recheck this. Figure 3 refers clearly to samples archived in plastic bags as described in this sentence and the figure description.

Figure 2 show the potential quantity of microplastics found in blank simples I am still confused about Figure 3. Y axe says: differences compared to samples archived in glass. I guess you meant stored. In the X axe are the months that samples were in bags. However, even reading the legend I only see samples stored in bags. I can’t identify which is the difference between storing the sample in glass and bags; I mean compare 3 months bags/glass, 6 months bags/glass, 12 months bags/glass. That was my question previously and that’s why it seems that figures in text are crossed. Apologize me if this is a simple thing but may be the legend could explain a bit more, or the text.

Answer: Thank you for this hint. For clarification, we changed the labelling of the y-axis to “Differential values between samples stored in glass jars and plastic bags”. Furthermore, we checked on the position of mentioning the appropriate figure. We are sorry; we cannot find any misplacing here.

 

L347: Thus, the sandbank samples include the faeces of a full egestion of the rectum. In my opinion, this is still too strong statement. I suggest authors to add: “may included…”.

Answer: Thank you for this statement. We amended the sentence.

 

L268: check formatted error, I came up again

            Answer: Thank you for spotting this. We corrected it.

 

L350: one dot to be removed

Answer: Amended.

 

In the same line as it was suggested at 2.1, authors seem to justify at the discussion section (L346-L351) that diet and plastics concentrated at the last part of intestines. I have been analyzed GIT (from mouth to annus) for more than 10 years in different marine mammal species, and I have not found any pattern like that. In fact, regularly I found more diet and microplastics in all species in stomachs and first part of the intestines (first 4-5 metres). This is why I cannot understand the methodology approach. I have not found any publication indicating that diet (considering that this is the type of study which have been carried out longer time than MPs) concentrated in the rectum until the full ejection. Authors suggested in that paragraph (L346-L351) that collecting intestines and faeces is a recommendation, however they only used the last part. In my opinion is recommended to use the full digestion tract for a better estimation.

Answer: Please keep in mind, that the approach here is based on evaluating a method for further research. We are part of a large institute collecting a variety of different samples. Therefore, we rarely have the chance to investigate the whole GIT solely for microplastic occurrence since this needs special procedures compared to other topics. Therefore, we decided to use rectum samples WITH content to ensure us having faeces available for further analysis.

 

 

L360-364:

Answer: Thank you for this discussion point. There are studies dealing with the topics of scavenging on carcasses. Therefore, we rewrote the sentence and used the suitable references for this statement: Nevertheless, the GIT is often specifically opened or removed by scavengers [65, 66].

Talking by my experience of more than 10 years attending strandings and reviewing dissection reports of different marine mammals, this is not often everywhere. It is occurring from time to time. The new reference added is the only reference I had in mine and that’s why I argued previously that this circumstance is uncommon. If you still keep the sentence, I do recommend removing again the penguin reference but add the personal communication from your person in charge to do marine mammal dissections in your team. In that case, as a local case it could be justify. But again, there are few references and it does not occur everywhere as it depend on many issues.

Answer: Thank you for this hint. We deleted the paragraph to avoid misunderstandings (Furthermore, it is already known that the stomach of marine mammals accumulates a high quantity of ingested hard parts [20]. Nevertheless, the GIT is often specifically opened or removed by scavengers [65, 66]. Owing to these facts, this study considers only undamaged GITs for establishing this approach in microplastic research although, this presented approach is also applicable for stomach tissue samples and the content.)

 

 

L364: Answer: Since we are referring to an amount of positive samples the reference of Lusher et al. 2018 is not suitable.

I do not understand authors response. The study from Nelms et al and Lusher et al are similar as they follow similar protocols. In fact, in Table 1 (Lusher et al.) indicates which animals contained macrodebris, or microplastics or both.

Answer: Thank you for the hint. Maybe there was a misunderstanding regarding the correct reference. The reviewer is right. We added Lusher et al. 2018 to the sentence as a reference.

 

L371-L378: Answer: Please note that the statement the reviewer is referring to deals with the accumulation of contaminants being transported or related to microplastics.

Apologize me for not being clear, I meant that the statement seems to say that microplastics AND contaminants associated accumulate in predators (see L376: accumulate in top predator species). This is the reason why I said is a strong statement. It is widely known in the community that most of the microplastics are egested (there are many publications about this, in particular in invertebrates). Therefore, the possibility to be accumulated is low. However, the transferred of associated contaminants to the predator itself is something that most of the community suspect that it is occurring. My suggestion was to try to be careful with that sentence.

Answer: We rewrote the sentence to clarify our statement: Since it is proven that microplastics are transported across the food web [14, 16, 68], it is assumed that in particular the contained contaminants accumulate due to absorption and leaching [69] in top predator species as it is already evidenced for example for organochlorides [70, 71].

Author Response File: Author Response.doc