Do you recommend any changes to the best practices outlined in Sample Collection sections? If Yes, please explain below and indicate which section you are suggesting changes be made to.

Answer Explanations

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  Expert 3

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	1	0
  Store samples to prevent tissue differentiation loss	1	0
  Avoid cross contamination	1	0

  1) The statement "Total count rather than percentage-based subsampling is recommended when population-level metrics are of interest" should be further explained and supported. Also, in some cases, such as high(er) throughput analysis, total count may not be feasible.  
  2) This is highly dependent of the research question and there is a growing consensus that sample collection (in this case, using meshes), should be fit-for-purpose. And, when talking about aquatic sampling, in particular, there is research showing that grab samples offer a better "picture" of the prevalence of microplastics, as opposed to other sampling techniques. If intended to be suitable for all types of samples, this should be mentioned. 
  3) Given that ethanol can be used to store samples, but that that same ethanol may cause MP fragmentation, then perhaps an appropriate strategy to overcome this may be suggested. Or a "control", to assess this level of potential fragmentation.
  4) Performing all work in biosafety cabinets, whenever possible, is also a usually recommended strategy. 
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  Expert 9

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	0	1
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	0	1
  Store samples to prevent tissue differentiation loss	0	1
  Avoid cross contamination	0	1

  Overall, I find nothing to disagree with here.
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  Expert 2

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	1	0
  Store samples to prevent tissue differentiation loss	1	0
  Avoid cross contamination	0	1

  Collect at least 50 samples ethically. YES
  While 50 samples may be good, the number should be justified according to study goals and statistical power analysis. Additionally, ethical considerations should include permits, minimizing harm, and transparency with communities when relevant. The paper should include a guideline on how to justify alternative sample sizes using statistical power analysis or ethical considerations in the cases where collecting 50 samples is not feasible.
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest. YES
  It should be considered to add  in  this section a guideline on how to select appropriate aperture or mesh size based on particle size distribution data or results from preliminary testing. Also, the importance of minimizing  the particle damage from mesh abrasion during sampling should be  addressed in this section. 
  Store samples to prevent tissue differentiation loss. YES
  A warning about  the use of some fixatives that may interfere with spectroscopic analysis should be added, because some preservatives or fixatives such as formalin or  certain alcohols can modify the polymer surfaces or interfere with spectroscopy analysis. This will greatly  help with the prevention of  analytical complications.
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  Expert 4

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	0	1
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	1	0
  Store samples to prevent tissue differentiation loss	1	0
  Avoid cross contamination	1	0

  1 - Collect at least 50 samples.
  It should be added that this often is not feasible, whilst also requiring a lot of analytical effort and hence time, materials, and funding. Also the issue of pooling of samples could be discussed.
  2 - Use an appropriate sampling device aperture and mesh for the sizes of particles of interest.
  This section is of a detailed-technical nature and rather straightforward and does not really add much. It might be consider to merge this section with other sections on technical issues.
  3 - Store samples to prevent tissue differentiation loss.
  This is a rather open-ended section and it is recommendable to add a closing statement and/or recommendation on the kind of action that is needed to avoid the pitfalls mentioned, like in the case of using ethanol. It could be recommended not to use ethanol or to verify the stability of the MNP after exposure in 20% ethanol.
  4 - Avoid cross contamination.
  This section is not adding anything and should be merged, for instance with the section on creating an environment as free from palstics as possible - even though this section is on a slightly different aspect of contamination of samples.
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  Expert 1

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	1	0
  Store samples to prevent tissue differentiation loss	1	0
  Avoid cross contamination	1	0

  The recommendation to collect at least 50 samples is not scientifically based.  I think this number is subjective.  Instead describe the need for statistically meaningful and representativeness of the sample.  Depending on the study goals and objectives, sample size should be robust to make reliable and reproducible conclusions.  In some studies involving human samples, <10 samples were analyzed and those studies received considerable media attention.  Unfortunately, some such studies lack analytical rigor but media tends to sensitize and often overinterpret the data.  Such sensitive studies involving human samples should have robust sample size and produce reliable and reproducible data.
  
  When discussing about sample collection, it is very important to emphasize that investigators, especially who are experts in analysis of MNPs be involved in sample collection.  Mos studies involving analysis of MNPs in human specimens relied on samples collected by a collaborating partner (mostly physicians) in hospital settings or on achieved biospecimens.  Sample integrity is not well known and therefore data quality is unknown.  For instance, sample collection in hospital settings may involve the need for use of gloves and plastic devices.  Wildlife tissues may be collected following dissection and that procedure may involve use of plastic sheets or tools that may contaminate samples.  Containers used to store samples should be free of plastics.  Therefore, a few sentences describing the need for rigor in sample collection for MNP analysis should be mentioned.  Compromised sample will result in compromised data (irrespective of how good your analytical method is).
  
  Many analytical methods use sieves and often they use mesh size that can remove MNPs from samples or extracts.  For example studies have used 2 mm sieve to remove soil particles above 2 mm, but they still report MNP concentrations, without stating that their data represent only a fraction (<2 mm) of MNPs present in samples.  The studies ignore that  MNPs of size 2-5 mm are not accounted for through this method.  Unless this is documented on their report/publication, data can be misleading.  The authors of this report have used an example of 1 mm i.d. needle for blood collection, and these are all important measures to be taken into account.
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  Expert 5

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	1	0
  Store samples to prevent tissue differentiation loss	1	0
  Avoid cross contamination	1	0

  The amount and type of sampling (including blank samples) should be the result of a statistically designed experiment that is based on the research question and objective. An arbitrary number of samples does not necessarily provide a high confidence sample set. It may over sample or under sample, both of which may not reach ethical goals.
  
  The paragraph on ..."appropriate sampling device aperture and mesh..." is only valid for liquid samples. It would be helpful for the authors to include a best practices paragraph about the 'how' of sampling solids (tissues) and viscous biologicals, after all, the term "tissues" is used int the article title.  There are many factors to consider. For example; scalpel vs punch, surface sample vs tissue layers, homogeneity of the tissue sample, etc.
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  Expert 6

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	0	1
  Store samples to prevent tissue differentiation loss	0	1
  Avoid cross contamination	0	1

  In addition to providing ref 20, can authors add more explanation or calculations to back up why at least 50 samples. Should there be different number for different type of biological tissues.
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  Expert 8

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	0	1
  Store samples to prevent tissue differentiation loss	1	0
  Avoid cross contamination	1	0

  Figure 2 - change the plastic snapcap tube to a glass vial
  Line 251 - "less uncertainty" is not necessarily true. Uncertainty in measurements was discussed above. I suppose you are talking about inherent variability across samples, which is expected to be large. You might mean statistical power here.
  Line 260 - "organisms" - This seems specific to a particular question. That question should be specifically stated here. For example, Savoca et al (2023) suggests which marine organisms are the best for plastic ingestion monitoring in the N Pacific.
  Line 260 - "Total count..." This needs a lot of clarification. It’s vague, uses terminology that may mean something different across disciplines (population-level means something different in basic statistical testing than it does for ecological research).
  Line 262 - "described in the..." Is this a section of the current review paper? If so, it’s not clear which section to me.
  Line 281 - By "fixed," do you mean formaldahyde or ethanol? Many biologists will haphazardly store in anything fixative without thinking about how it will dissolve polymers. To me, this seems dangerous to suggest.
  Line 286 - Ethanol extracts plastic additives away from the plastic particles making them appear different and smaller, especially flexible PVC particles. Our lab does not recommend the use of any fixative chemical. They are to be avoided for polymer incompatibility reasons.  Dry at room temperature is best - I suggest saying that.
  Line 291 - change "is" to "are"
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  Expert 7

  Section	Yes (please explain)	No
  Collect at least 50 samples ethically	1	0
  Use an appropriate sampling device aperture and mesh for the sizes of particles of interest	0	1
  Store samples to prevent tissue differentiation loss	0	1
  Avoid cross contamination	0	1

  I don't believe there should be a fixed number of samples to collect. Each case is different. What's more important is the principle: the more samples you have, the lower the uncertainty will be.