Best Practices: Detecting and Quantifying Micro- Nanoplastics (MNP) in Biological Tissues

Fortunately, modern analytical pyrolysis systems have the added capability to thermally remove "smaller" organic molecules that were not removed through digestions and density separations. This "double-shot" capability as mentioned above can provide cleaner, less complex chromatograms enabling higher quality ident/quant of pyrolyzates.

The compatibility of the measurements of MNP's physical and chemical characteristics are still fraught with pitfalls. It will be some time before one may rely on comparisons with confidence.

The list of measurement instruments that have been used to identify MNP is much longer than those listed in the comments. But, each technique has limitations which, with the long list of techniques, is beyond the scope of this manuscript.

Regarding expert 8's assertion that: "Biological components will produce the same marker compounds as PVC in py-GCMS.", recent developments have provided ways to remove such components before ident/quant of the MNPs.

The authors may reflect on their learning journey while building this review and put forth best practices for learning/enhancing ones' one attempts at building best practices consensus.

The experts have provided some ideas that should be melded into the manuscript.

Standards organizations and private vendors have developed file format conversion programs for interoperability between different vendors file formats. In some cases, there is open source software for analyzing analytical data without the need of a specific vendors hardware.

Blanks have been discussed multiple times throughout this review process. Expert 2 brings it together well, "...transparency is vital...  ...accurately interpret..."

The paper suggested by expert 4 is valuable to help one frame experimental parameters. And the standards organizations efforts, while still in infancy, will help guide the MNP practitioner.

With reference to expert 6's concentration based remarks: Yes,, use units meaningful to the analytical objective. But It would also be helpful to also report in units that are prevalent in the science. Readers of studies will make those unit conversions to enhance their understanding of the findings. The study author has a more intimate knowledge of the matrix and analyte results, therefore is better equipped to report in converted units.

In addition to expert 4's debate comment, the authors should be more precise in their use of "methods", "samples", and "small". For example it would be better to use analytes instead of "samples" and "small" is a non quantitative term.

MW analysis or MW determination or MW distribution?  Polymers are by their nature comprised of various molecular weights. 
I concur with expert 8, the information being sought by the question is unclear. Is the question regarding a high confidence determinization of total mass present for each MNP identified?

Expert 2's initial remarks contain some important considerations regarding various digestion schema. These concepts should be included in the manuscript.
And it goes without saying... not point in destroying the analytes while trying to remove the matrix.

Unfortunately, there is no shortcut. In analytical chemistry there is no better way to build confidence than doing the full analytical schema and then sharing and discussing the results with subject matter experts.

Experts have offered some good ideas. 
The readers should also consider their own expertise and experience with various measurement techniques.  Understanding the fundamentals of the measurement technique facilitates being able to identify outliers and errors.

With respect to Py-GC/MS, minimum number/weight depends on the polymers' pyrolyzate fragmentation pattern. PE and PP generate many pyrolyzates and thus any individual pyrolyzate will be more difficult to ident/quant. Whereas ABS produces many fewer pyrolyzates so an individual pyrolyzate can be easier to ident/quant.

I agree with expert 8. There are a multitude on polymer varieties and many variations within a variety. This number becomes unmanageable when additive and modifier formulation differences are factored in.  The authors could address this problem through mentioning annual production output of the 10 - 20 most common polymers as a point of initial focus.

For Py-GC/MS, most lower molecular weight interferences can be removed through thermal desorption. PAH, long chain fatty acids and amines.
Regarding expert 1's comments: the ratio of the triplet peaks can be an indication of whether one is detecting LDPE, HDPE, or a fatty acid. This ratio becomes less reliable as the signal/noise ratios become smaller. Also, as one uses higher molecular weight pyrolyzates for PE ident/quant, the triplet looses chromatographic resolution. There are enough differences in the Retention Indices and mass spectrum between PE pyrolyzates and acid/amide pyrolyzates to allow differentiation upon careful inspection.

The experts have given further explications of relevant generally available knowledge. Authors should caution the reader to identify recent research results for the reader's specific matrix and analyte.

The experts have called out multiple chemical classes that can interfere with optical spectroscopy and Py-GC/MS. The authors would serve the readers well to include these mentions in the manuscript. 

In line with expert 4's comment, it could be added that the false PE signals come long chain organic acids and amides natural products that are prevalent in the air.

The experts have provided some valuable considerations with regards to field blanks that should be incorporated by the authors. As pointed out by experts 2 and 3, there is not just one "field" blank needed. Blanks from multiple sources and steps along the analytical schema will need to be taken to properly account for possible contaminations. This thinking should be incorporated by the authors.

Considering the expenditure of time, money, analytical measurement time, etc., needed to produce an MNP analytical result, that value can be severely diminished by MNP unaware individuals. As mentioned by several experts, training (even rudimentary MNP awareness training) of those who will be doing the sampling, packaging, and transporting to the analytical lab will produce outsized returns in the value of the data produced.

Because to the ubiquity of MNPs, rather than "metal this" and "glass that" the authors should recommend to the reader conduct a thorough evaluation of analyte degradation sources and contamination point sources risks and the measures taken or not taken to lower contamination risk. This also applies to transport/storage, to Extraction (sample processing) section, and to the Particle Quant/Qual section.

Signals from blanks that are above a standard sample LOD should cause the analyst to reexamine the analytical schema for ways to lower those blank signals below the LOD of a standard sample.

I suggest the authors incorporate the detailed and valuable learnings proffered, especially by reviewer 2.

If a blank is subtracted from a measurement result, but the value that is subtracted is not reported, then the reader may come to incorrect conclusions about total MNPs present in the matrix sampled.

The authors should point out as per the reviewers the need for consideration of the components of the matrix (and their breakdown products) when evaluating analytes of interest for possible false signals.

The experts proffer some good considerations. The author can help the readers' analytical thinking by incorporation their comments, highlighting statistical sampling that incorporates consideration for the possible variability of matrix and analytes from various sampling locations and different times.

Until the science evolves beyond the "Hey, I found this (name of polymer) in that (name of matrix)!", the analytical science of recovery and reducibility will take a back seat. Standards organizations' Data Quality Objectives requirements are helping move recovery and reducibility measurement forward. 

If low/high recovery rates are encountered, the analyst should comment on possible reasons (several mentioned by the reviewers) for recoveries that are outside usual published standards. For example "trace" analytes in a complex matrix usually results in higher variability than concentrated analytes in a simple matrix.

It is hard for one to imagine how to value a result without knowing where it lives in relation to the LOQ/LOD.

The authors should caution the reader to take into account that LOD and LOQ are more than just a function of the measurement. The amount of sample processed for a single determination (hence the need for "amount analyte/amount matrix"), the efficiency of the matrix removal process, the effectiveness of the analyte recovery, etc. The analyst must also remember that the measurement systems' native S/N ratio affects LOD and LOQ. Even systems with the same model number can have different S/N ratios because of differences in number samples analyzed, type of samples analyzed, maintenance schedules, as well as aging of the sample introduction, lamps, optics/filters (IR, Raman, MS), detectors, etc.

The reviewers have pointed out some of the possible matrix considerations. The authors should caution the user to carefully consider the various compositional factors of their sample matrix as to chemical class (lipids, protein, carbohydrates, minerals, water, etc.) and their subsequent impact on the analytes of interest through sampling, transportation, storage, matrix removal, analyte enrichment, residual matrix analysis interferences, etc.

Studies have found MPs in ancient soil from lake beds: https://doi.org/10.1126/sciadv.adi8136, and archeological sites boreholes https://doi.org/10.1016/j.scitotenv.2024.169941. Although the authors were not rigorous in explanation of contamination prevention, it highlights the risk in using pre plastic history samples as being truly "Blanks."

If the expense of a "clean room" and "clean scientists" if prohibitive, background MNPs in the laboratory may be reduces with the utilization of residential/commercial "air purifiers." Units equipped with HEPA 14 filters will filter 99.995% of 0.3 - 0.1 micron particles from 186 sq meters (2,000 sq ft). 

Because of the ubiquity of MNP in our lives, one must envision every environment the samples may pass through or reside in. The reviewers have mentioned many sources of contamination, but the section title "...major laboratory sources for background MNP contamination..." constrains thinking to the analysis laboratory. As reviewers point out, the sampling environment, and storage environment are also a source on MNP contamination. One must not forget sample transport, sample "log-in", laboratory storage before processing, etc. One must envision themselves as a MNP and follow the entire life of the particle facilitate envisioning possible sources of contamination that need to be risk evaluated.

In my practice, I do not use LOD to do correction. In general, I just report the number. 

I suggest the author carefully evaluate the comments and incorporate some of them into the revised paper if paired blank correction is important in practice. I have no practical experience about this. 

I suggest that the author carefully evaluate the reviewers' comments and provide reliable information into the revised manuscript to strengthen its overall quality.

Continued research efforts are essential for developing methodologies and addressing current challenges in the field.

I also suggest that the author reevaluate some of the referenced resources and consider incorporating them into the paper to enhance its value for readers.

I agree with the above comments that defining an absolute value for different samples and experiments can be risky. Instead, the paper could suggest an acceptable recovery rate range to provide more flexibility and practical guidance.

Ideally, yes. In practice, this could serve as a useful guideline.

I agree that the paper could benefit from a more detailed discussion of the LOQ. However, as noted earlier, in practice, researchers often need to make compromises based on the conditions and resources available to them.

I agree with the above comments. In many cases, ideal conditions are difficult to achieve, and researchers often need to make compromises and work within the available constraints.

Based on the feedback, it is evident that this field requires further research efforts.

I suggest that the author review the above comments and make slight modifications to incorporate some of the valuable points raised by the evaluators.

Each evaluator has provided suggestions based on their unique background and experience, making them all worthy of consideration. However, it is understandable that not all suggestions can be incorporated into the paper due to potential length constraints. I recommend that the author reflect on the core focus of the paper, selectively integrate some of the suggestions, and develop each key point in greater depth to offer more precise and effective guidance.

 After reviewing all the expert responses, I have nothing to add. Their comments comprehensively addressed the issue. 

 At this time, I have no further comments or additions beyond what I have already stated in my initial response.  

 In my opinion, the presentation of techniques is well done and cover a large area, and could be suitable for being added  to the manuscript. 

Several suggestions were offered, and I particularly agree with expert 8’s recommendation to address the details during the third round of review.  

 There is a broad consensus regarding the methods that are generally considered most suitable. The methods discussed  reflect the current state of the art in analytical techniques, covering the key approaches recognized within the field. 

 I concur with the perspectives expressed by the experts as well as the comments. I possible, I recommend that these valuable insights should be added into the revised manuscript. 

I stand by my  opinion, that a more practical  solution would be to make sure that the raw data could be made available upon request.

 I also fully agree that it is crucial for researchers to explicitly state whether blank correction has been performed, and if so, to provide a detailed explanation of the exact procedures used. This transparency is vital for ensuring the reproducibility and reliability of the results, as well as for allowing readers to accurately interpret the data. 

These resources indicated by the experts are highly valuable, and I encourage the authors to consider including them as references in the revised manuscript. 

 I stand by my initial opinion that as a general principle, analytical methods that compromise the physical or chemical integrity of the plastic particles should be avoided because, preserving the original state of the plastics is essential for ensuring reliable identification, quantification, and comparison across studies.

There is a clear a divergence of opinions among the experts on this point. I think that the numerical values provided should be regarded as indicative. The broader recommendations made by the experts appear to be well-reasoned and balanced, and I believe they offer a sound basis for guiding future work. These insights should be thoughtfully added into the revised manuscript to strengthen its practical relevance.

 A significant number of experts expressed their  disagreement with the proposed 100-particle limit, and I share their concerns. Imposing a fixed numerical threshold is problematic, particularly because analysts cannot predict in advance how many particles will be present in an unknown sample. Such a rigid limit may not reflect the variability encountered in real-world samples and could undermine the accuracy or representativeness of the analysis. A more flexible, context-dependent approach would likely be more appropriate, allowing for adjustments based on sample type, matrix complexity, and analytical objectives. 

 Several options have been presented by the experts. I think that the authors should consider summarizing them and adding the most relevant ones into the manuscript to enhance its clarity and practical usefulness. 

 The importance of appropriate pairing is highlighted by the experts. They  also offer several valuable suggestions in their comments on how this can be effectively implemented. I recommend that these suggestions be clearly summarized and added to the revised manuscript. 

 In my opinion it is essential to include both a field blank and a procedural (or laboratory) blank for each sampling event. Including both types of blanks is a critical component of quality assurance, ensuring the integrity and reliability of the analytical results. 

 The general consensus among the experts appears to be that meaningful progress has been made in advancing understanding on this topic. However, it is equally clear that the work is far from complete. Ongoing research efforts continue to refine methodologies, address unresolved questions, and build a more comprehensive evidence base. While current findings are promising, further investigation is necessary to fully validate conclusions and translate them into practical applications. 

 While the experts agree that a minimum recovery rate should be recommended, there is considerable variation in their opinions regarding what that minimum should be. At this stage, I maintain my position that the  threshold  should be chosen based on the type of samples,  analytical method and the scale of the study.

 There is a clear consensus among the experts on this particular issue, and their collective feedback is both thoughtful and constructive. The authors should carefully consider and add these comments into their revised mansucript. 

Having read the responses of all experts, I have nothing to add to my initial response. The views of the experts cover the issue completely in my opinion.

I have nothing to add to my initial response.

The overview of techniques is complete in my opinion and can be incorporated in the manuscript.

Various suggestions were made and I agree with the comment of expert 8 of discussing the details during round 3.

There is mostly agreement on the methods that are in general most suited. The methods mentioned are complete regarding the current analytical state of the art.

I agree on the views of the experts and the 2nd-round comments. As much as possible, these opinions could be incorporated in the manuscript.

The key issue for me is that in principle we agree on making all data, including the reaw data, available. There might be practical and institutional restrictions but to me these issues can be resolved one way or the other.

I am especially in agreement with the viewpoint of expert 1

The suggestions are helpful and should be considered for inclusion in the manuscript.

The suggestions make sense and should be considered for inclusion in the manuscript on an individual basis.

There is agreement on the options that are available and it might be good to inlcude them in the manuscript

There is consensus but details are oftren lacking.

In general, methods that affect the integrity of the plastics are to be avoided. The 2nd general consensus is on preference for a combination of a strong oxidizer like H2O2, metaloxide like KOH and/or enzymes to be used in the sample preparation steps.

Nothing to add to my initial response after reading the other opinions. 

There is disagreement among the experts. I think that the numbers given are only indicative and the general recommendations made by the expert make sense in my opion and should be follwoed-up in the manuscript.

Looks like there is agreement that such a scheme does not exist. Experts mostly mention issues that need to be part of such a scheme.

Various issues are raised and I agree on them - most of them are of a descriptive nature and are of relevance to the manuscript.

Various examples are provided of impacts on indicator ions for polymers. There is a trend towards using multiple indicator ions to rule out hydrocarbon signals in py-GC/MS as one of the approaches to assess impacts of natural molecules present in the digested sample.

I conclude that there is no general method available to minumize interferecnes. The experts provide issues that need to be made part of a general method to be developed. The further details will depend on the case at hand and will depend on the matrix and the properties (including chemical composition) of the plastic particles of interest.

I agree on the statements made by the experts. They are mostly in line and the key statements could be included in the manuscript.

As already stated, the issue of enzymatic digestion is an important addition.

Again: various suggestions which cover the key issues.

Severl general and practical suggestions are provided. They cover the topic sufficiently well.

I read the suggestions of the experts and the additional comment made in the 2nd round. I have nothing to add an d think the responses are well covering the issues.

Expert 1 points to an important issue. In fact, many biological samples, namely, human samples, are collected and stored resorting to plastic material and it is quite difficult to determine the source of any micro- and nanoplastics found. For the materials used, chemical signatures may be used, but MPs and NPs present due to contamination are harder to trace. This is particular evident for "legacy samples", which have been stored for long time, sometimes, years, and are then explored for the presence of MPs. But, given the these were not collected and handled in accordance with MP/NP analyses guidelines, it is quite difficult to definitively ascertain the origin of any putative MPs. 

Various options are provided. Again: I propose to summarize them and include the most relevant ones in the manuscript.

The need of pairing is recognized by the experts and valuable suggestions are provided on how to do this. I recommend to summarize the suggestions and include in the manuscript.

There is some disagreement on whether or not correction should be made. I think that this is necessary and agree on the opinion of expert 7 of subtracting the blank response from the sample response - i.e. what I call the lowest level of quantification.

Looks like the experts agree on the need of best practises whilst acknowledging that such practises do not yet exist.

I agree on the general opinion that there is a need for field and procedural blanks for every sampling.

The general consensus seems to be that progress is being made on this topic, but research is still ongoing.

Many of the experts pull the discussion back to the challenges of extraction and cleanup.  This is truly a large gap that needs further testing before analytical instruments can be successful. I would also highlight that this question lumps micro and nanoplastics together, but in fact, different methods are needed for different sized particles.

I think a focused discussion/debate over the details shown in this table during round 3 would be informative and help improve the paper.

Various options are suggested by the experts. I have nothing to add at this point.

The experts agree on the need of a minimum recovery rate but disagree on an actual number. My recommendation would be to mention that a recovery rate of 80 % is more or less ideal, with numbers as of 50 or 60 % also being acceptable.

I understand the points of all experts and do not strongly agree or disagree with any of their perspectives.

These are very helpful resources and I encourage the authors to consider adding them as references to the paper to promote and echo reporting guidelines.

This is an area where experts diverge significantly. It's intriguing.  Regarding expert 3's answer, carbonyl index can only be used for certain polymers (e.g. polyethylene) and the method for calculating this weathering indicator is not standardized and within consensus across the field. I would caution the authors to include it as a rule, but suggesting the methods need to improve to quantify weathering indicators so that plastic particles can be categorized by weathering severity would be good.
Expert 1 asks about the different types of plastic polymers - there are 1000s of plastic polymers and within each polymer there are 1000s of different grades and additive packages. Particles should absolutely be identified to the polymer chemical structure and reported by polymer category. Tire wear is an interesting and challenging category, since tires are made of a variety of polymer blends and with other polymer reinforcing fibers.  Oof, they are challenging but I agree that tire and road wear particles get their own category because of the abundance in natural environments.

There is in general, consensus on this issue. I have nothing to add.

There is in general terms, agreement across experts. The details provided are most useful and I agree on them.

Many of the experts disagreed with the 100 particle limit and I agree with them. The analyst cannot predetermine how many particles will be present in an unknown sample. 

I first need to correct my initial response as 'needed' should be replaced by 'available'. I support the general consensus of each type of tissue requiring its own MNP-free control matrix, whilst ackowledging that such matrix may not be available for all tissues. In that case, an alternative is needed that resembles the tissue as much as possible.

I agree strongly with expert 1's statement that "Because studies analyzing MNPs in biological samples are not many, more such interreferences are yet to be discovered."

I too support the suggestions of experts 3 and 4 to state concentrations of KOH and H2O2 as an example, rather than a rule.  

I suspect that making blanks in a medical setting is challenging for so many reasons, but mainly because the microplastic analyst is unaware with the devices and procedures the patient went through before the sample was collected.  If samples are collected over many years, different materials and devices are likely to be used. A tour and full explanation of the medical procedures (from home preparation, surgical preparation and up until tissue sample is taken) would provide great insight to the microplastic analyst.  After this tour and discussion, the analyst could better design proper field blanks.

It is clear from the responses that such a control matrix is hard to find. The suggestions made, make sense to me in general terms.

The comments provide a good addition to what is already written in the manuscript. I agree with the comments and especially like the overall nominator of dust being the major source.

I like the suggestion "buy in" and training for the medical staff before sample collection.  I would suggest detailed discussions between the medical staff and the microplastic analyst.  A visit to the medical facility and collection of unused medical devices (for polymer ID) and understanding of the potential plastic contamination in the air and procedures is important. Sterility is required for tissue sampling from live animals, but this will not prevent plastic background contamination and in fact, it might increase it depending on the methods of sterilization.

sample size should be determined through a statistical power testing method and will depend on the scientific question and the variability across individual samples within a population.

There are many suggestions from the various experts. They touch upon various aspects of the manuscript. I agree with most of them and I can only recommend the author to seriously consider each suggestion. It is to be noted in this respect that especially the numbers given will always be up for discussion, like for instance the number of blancs to be included. Specific for this example, it should be added that numbers are up for discussion.

I found this question a bit confusing, so I think that possibly lead to the 6:3 yes:no answer ratio.  Analyte-like response in procedural blanks is likely less problematic than analyte-like responses in the true samples.  For example, natural polyamides (proteins) will mimic nylons in FTIR analysis.  Assuming the procedural blanks dont have matrix in them, very little to no proteins should be present to amplify the nylon signal.  But in the tissue samples, this signal should be expected.  Ways to avoid those false positives include very close manual examination of the FTIR spectra and comparison to several natural polyamides. The answers seemed to build on and reiterate previous suggestions for how to eliminate background contamination.

The two spike materials suggested by expert 7 are not really made for spiking into tissues, rather into water I believe, so that's not very relevant to this specific question.
I'd also like to ask what the author of the question meant by validated?  Is a method validated if it was tried once in one lab with one technique and resulted in high recovery?  Or would the extraction/cleanup method need to be validated by two or more labs and analytical techniques? 

I've asked colleagues to please write a review article that reviews the use of commercially available or lab-made nano and microplastic particles summarizing which polymers, sizes and shapes are available.  Including a table in this review article would make it very useful and likely highly cited.

I enjoyed reading the different creative ideas to this challenging gap.

I agree with all comments and especially want to echo what expert 1 discusses before and during collection of human samples. This environment is not the laboratory where the samples will be processed and analyzed and will be less controlled for microplastic contamination.  It is key to mention this in the paper and suggest best practices that will control, minimize, cut away, or quantify the background contamination in room for surgery prep and performance.

I dont disagree with any of the experts comments.  All are worthy of consideration.

Potential interferences depend on tissue type and plastic chemistry and the authors need to keep in mind the expert's comments and clarify this in the manuscript by offering additional information on this.

I agree with expert 9. The first thing that should be adressed is the training of the staff that collects the samples. This will help improve considerably the quality of the samples.

The authors should carefully read the suggestions made by the experts and try and incorporate them into the revised manuscript.

The reviewers offer valuable information about potential sources of such particles and appropriately highlight their limitations. The authors are encouraged to address these into the  revised manuscript 

To follow up on the comment made by Expert 1 about "characteristic three peaks for PE", if triplet peaks are not observed or demonstrated by py-GC/MS in a study, it is an indication that the signal could be hydrocarbon signal from matrices or background, not representing PE.  

I would like to provide a visual aid for the authors regarding to the discussion here. (Table 3 and Fig1 from this reference. Qualitative and quantitative analysis of mixtures of microplastics in the presence of calcium carbonate by pyrolysis-GC/MS - ScienceDirect) Commonly used marker for PET, PVC, and PS (BP, Nap, and SSS) are less sensitive than hydrocarbon signal.

In addition to chemical signal identification by mass spectrometry and spectroscopy methods, molecular weight measurement could further help the distinguish between plastics (high MW, often 100k or M Da range) and bio macromolecules (typically in the range of k to 100 k, lower than plastics.) 

This review paper suggests the LOQs are chemistry-dependent: polyethylene (0.5–4 µg), polypropylene (0.3–1.9 µg), polyethylene terephthalate (0.5–5 µg), polyvinyl chloride (0.3–3 µg), polystyrene (0.1–1.2 µg), and polyamide (0.5–9 µg). I also suggest supplying authors references to help cover more views.
Progress in quantitative analysis of microplastics in the environment: A review - ScienceDirect

To building up on previous comments, either spectroscopy or mass spectrometry-based methods are based on chemical identification. Chromatography techniques, such as size exclusion chromatography (SEC) and asymmetrical flow field-flow fractionation (AF4), could contribute to separate NPs and MPs. Thus, advanced hyphenated techniques require further development to comprehensively achieve physical and chemical analysis sequentially or simultaneously. 

I agree with expert 1's comment above that methods to distinguish NPs from MPs is needed.

I agree that it is essential that researchers state clearly whether blank correction has been perfumed and if so, how exactly.

I accept the views expressed that strong acid digestion is not to be recommended.

This clearly remains an area of debate given the varying views expressed.

I also support the comments of experts 3 and 4 regarding enzymatic digestion approaches.

There are some very insightful comments here for the authors to consider. I reiterate my comment that medical staff collecting samples need training in how to minimise or at least take into account MNP contamination during sample collection.

I agree with the views expressed that the minimum of 50 samples collected ethically is problematic. It does depend on the objectives of the study and how difficult such samples are to obtain in an ethical manner.

The reviewers provide some useful information here for consideration by the authors.

I think the reviewers provide useful advice of where such particles may be sourced, as well as cautioning about their flaws. The authors are advised to incorporate these views.

While the reviewers are unanimous that a minimum recovery rate should be recommended; there is quite a range of views on what that minimum should be. I would stand by my view of 60% at the current time, pending the results of a large-scale interlaboratory trial to provide empirically-based data.

The experts are unanimous on this point. They make some useful comments that the authors should take heed of. In particular, I like the comment that it should also be added how this translates into the LOQ in actual samples of a specific weight. This may seem obvious but it should not be assumed that less experienced readers of this article will do this unless specifically instructed.

The reviewers are unanimous on this point. There are some useful detailed comments as well that the authors should take into account.

There appears near universal consensus between the reviewers. I would stress however that in many instances, this will not be practically viable and some extrapolation between tissues will have to be made.

The reviewers concur closely on the difficulty of finding an appropriate MNP-free human tissue for QA/QC use. I see the potential in developing an artificial tissue that could fulfil such a purpose. We work with 3D-human skin equivalent models that are lab-cultured human skin, so I can see this as technically feasible.

The reviewers are pretty consistent in their views and cover the potential sources of contamination well.

The comments from the experts cover similar aspects. I certainly agree that the frequency of blanks should be increased from 1 in 20. For analysis of conventional chemical contaminants, my practice is to conduct 1 blank for each 5 samples as part of ongoing QA/QC. As part of initial method validation, at least 3 and ideally 5 blanks should be conducted. I also very much support a recommendation that governments support the development of suitable reference materials.

I agree with the comments of experts  3 and 4 that a proper attention should be given to the  section regarding the sample's digestion using 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes. The appropriate enzymes for different types of samples or analytical methods should be discussed.  Also a schematic representation of different types of samples and analytical methods and also some validated ratios  of of 10% KOH, 15% H2O2 should be added to the manuscript.

Agree with the comments.  The value of " at least 50 samples" is not scientifically based.  The sample size should be determined based on study goals.  "Representativeness" of the sample should be the thrust here.  

One of the major deficiencies in MNP research on biological samples is the lack of separation of "micro" versus "nano" plastics.  In fact, many current spectroscopy and mass spectrometry based methods do not distinguish nanoplastics from microplastics.  Nanoplastics may be more toxic than microplastics which need to be studied.  A method to clearly identify nanoplastics independently of microplastics is needed

The comments of the evaluators were very precise and well thought. The authors should pay careful attention and try and implement the evaluators suggestions in the revised manuscript. Also, as almost everybody agreed, the issue of "Collect at least 50 samples ethically" should be properly addressed in the revised version.

The evaluators have provided very specific and complementary comments that show where the manuscript could benefit from further clarification, elaboration, or additional information. I strongly recommend that the authors take into account these comments and try and include them during the revision process to improve the quality of the manuscript. 

The comments from experts are complementary and identifies areas that need clarification or addition to the text.  I would suggest that the authors review and consider the comments carefully while revising the manuscript.