Do you recommend any changes to the best practices outlined in Extraction 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
  Conduct the minimum extraction steps necessary to amplify the plastic	0	1
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	1	0
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	1	0

  1) Enzymes do not seem to be discussed in the paper. 
  2) Broadly, this seems OK, but it should perhaps be noted that the selection of the density separation solution is always a compromise, as even 1.8g/mL leaves out some polymers of higher densities (e.g., PTFE). Again, this goes back to the fit-for-purpose and research question aspects.
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  Expert 9

  Section	Yes (please explain)	No
  Conduct the minimum extraction steps necessary to amplify the plastic	0	1
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	0	1
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	0	1

  Overall, I find nothing of note to take issue with in this section.
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  Expert 4

  Section	Yes (please explain)	No
  Conduct the minimum extraction steps necessary to amplify the plastic	0	1
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	1	0
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	0	1

  With regard to the issue "Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes". It cannot be sufficiently stressed that this is a key issue, depending on the matrix, the polymer composition, and the  analytical method used. In case of for example the combination of any biological tissue, Py-GC/MS, and PE, a virtually 100 % efficviency of the digestion is needed in order not to overestimate the actual amount of PE in the biological tissues. In common practise, this implies that KOH digestion is not suited in this example as the LOD will commonly be much higher that the actual concentrations of PE present. Another issue is the efficiency of the digestion as this needs to be high. In line 324 the term 'high degree' is used. It is to be discussed (possibly on a case by case basis) what degree is actually needed in order not to significanlty affect the count of plastics.
  
  It is also to be noted that enzymatic digestion is also a suited option on top of alkaline digestion.
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  Expert 1

  Section	Yes (please explain)	No
  Conduct the minimum extraction steps necessary to amplify the plastic	1	0
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	1	0
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	1	0

  The section on "conduct minimum extraction......plastic" needs a thorough revision.  It is basically 'preconcentration or enrichment' of extracts to have enough particles to produce signals above the noise.  In small molecule analysis is called preconcentration or analyte enrichment.  
  
  It is also stated that increasing the extraction steps will reduce recovery rates.  This statement is not substantiated. Because this report/manuscript is focused on biological samples, some statements related to minimum extraction steps for biological samples are needed.  For example, digestion of biological tissues with KOH and H2O2, twice or multiple times?
  
  Lines 300-303:  Needs to be rewritten.
  
  Digestion of samples with >5:1 ratio of 10%KOH and 15%H2O2 is suggested.  This ratio and the concentration of KOH and H2O2 can vary depending on the sample type.  Therefore, pinpointing exact concentration of KOH and H2O2 is not needed.  Instead, use those numbers as an example, not as a rule.  Use of trade names, like Alcojet is not recommended or rephrase the sentence to state that some surfactants like Alcojet.  
  
  Lines 342-343: Needs rephrasing and unclear.
    
  Line 338:  A sentence or two describing the principle behind density separation will set stage for the rest of the narrative.  
  Lines 344-346:  Needs rewriting.  Unclear as written.
  Line 348:  Given examples of common density separating fluids.  
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  Expert 5

  Section	Yes (please explain)	No
  Conduct the minimum extraction steps necessary to amplify the plastic	0	1
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	1	0
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	0	1

  careful wording should be used to differentiate between extraction (from the matrix) and enrichment (concentrating the analyte in/on the measurement media)
  One should search prior art to match the appropriate digestion agent with the biological sample to facilitate minimal MNP loss and maximize recoverable MNP.
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  Expert 6

  Section	Yes (please explain)	No
  Conduct the minimum extraction steps necessary to amplify the plastic	0	1
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	1	0
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	1	0

  Please add details if all three approaches should be used together or combination of any. Do we have examples on which method work most effectively on certain sample type.
  
  Is 1-1.8 g/ml referring to the density of particles? If so, please consider polyolefin and enlarge the range, which has density less 1 g/ml. If it is referring to separation medium, please make it clear as well.
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  Expert 8

  Section	Yes (please explain)	No
  Conduct the minimum extraction steps necessary to amplify the plastic	1	0
  Digest samples using > 5:1 ratio of 10% KOH, 15% H2O2, or appropriate enzymes	1	0
  Density separate samples at high enough density (typically 1-1.8 g/mL) for particles of interest	1	0

  Figure 3 - I suggest adding a section and circle to this diagram.  Test all reagents at temperature and duration for compatibility with the polymers of interest before you begin analyzing samples.
  Line 310 - Filtration is an extraction technique, it’s size separation and particle vs liquid separation, so particles from drinking water are extracted.
  Line 316 - It might be worth mentioning that a new apparatus for density separation also reduces the number of transfers of the sample allowing more extraction and cleanup steps  all within one apparatus without loss of particles - Shaw et al 2024 https://doi.org/10.1186/s43591-024-00093-7
  Line 323 - add a list of which polymers were tested by Tsangaris et al - were they diverse? What sizes?
  Line 336 - 337 - You could discuss apparatus selection here, and also the benefits of using vacuum before density separation to remove gases from the particles (Shaw et al)
  Line 348 - Awkward phrasing and I think water alone is not a good example.
  Density separation section in general - Stoke's Law is something that should be considered during density separation and is rarely discussed in microplastic literature.