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

Answer Explanations

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  Expert 9

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	0	1
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	0	1
  Quantify the recovery rate of the method	0	1
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	0	1
  Keep methods consistent across a study as much as possible	0	1
  Use targeted techniques when study goals allow	0	1
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  Surely trip blanks inherently incorporate contamination that would be detected in reagent and equipment blanks? Consequently, some rephrasing of this section is required to clarify this.
  While outsourcing of matrix spikes is the ideal; it comes with resource implications that impact disproportionately on less-resourced laboratories. As the intention here is that the analyst analyses these matrix spikes "blind", an alternative is for a colleague to prepare matrix spikes at levels that the analyst is unaware of.
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  Expert 3

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	0	1
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	0	1
  Quantify the recovery rate of the method	0	1
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	1	0
  Keep methods consistent across a study as much as possible	0	1
  Use targeted techniques when study goals allow	0	1
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  Not clear what "great blanks" is, in this context.
  It is likely best to conduct three, as, in situations in which only 20 samples are analyzed and 1 technique is use,d this would result in 1 blank and 1 spike; so that the findings are statistically relevant, I would increase it to three. 
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  Expert 2

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	1	0
  Create or outsource blank and spike reference materials	0	1
  Quantify the limit of detection of the method	0	1
  Quantify the recovery rate of the method	0	1
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	1	0
  Keep methods consistent across a study as much as possible	0	1
  Use targeted techniques when study goals allow	0	1
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  Create a lab and field environment as free from plastic as possible. Even though the section highlight the important challenges posed by nanoplastic contamination, it does not suggest any  measures to be taken into consideration. 
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique. While this might work well for a big experiment, it may not be a  proper scale for small batch experiments. I would recommend introducing a phrase and a standard that could be accepted for studies that have less than 20 samples.
  
  
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  Expert 4

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	1	0
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	1	0
  Quantify the recovery rate of the method	1	0
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	1	0
  Keep methods consistent across a study as much as possible	1	0
  Use targeted techniques when study goals allow	1	0
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  Overall, the document is clearly written and contains the most relevant aspects of the best practices for detection and quantification of plastics in biological tissues. A general issue is that more strictly the order of (analytical) activities could be maintained within the manuscript, i.e. from sample collection, storage, to sample preparation preparation (including the proper lab environment and the QA/QC of analytical techniques) to final  (FAIR) reporting and storage of data. Samople collection is for instance now in the middle of the document, while it makes sense to start with this section.
  The following observations apply to each of the scetion mentioned above:
  
  1 - Create a lab and field environment as free from plastic as possible
  This is a brief section that in technical terms is fine. For me, however, the key issue is that the lab personnel and lab management needs to have a mindset with regard to creating a plastic-free environment. This mindset translates indeed in technical issues as mentioned in somewhat detail in this section. A detail remark is the suggestion to add "with a foil clear of microplastics" after ".....can covering samples19.". Furthermore, a reference is needed for the statement in line 89.
  2 - Create or outsource blank and spike reference materials
  A minor remark deals with the term 'great blanks'. This might be substituted into for instance 'suited blancs'.
  It might be good to add some more information on the details of the novel immobilization technique discussed in lines 123-125 (reference 39).
  3 - Quantify the limit of detection of the method
  I propose to add some more information on methods/approaches available for determining the Limit of Detection (LoD). As anb alternative, there might be specific referencing to the relevant sections in this document.
  4 - Quantify the recovery rate of the method
  A general key issue is: how to correct for recovery rate? Or, in other words: how are samples corrected for recovery rates?
  Minor issues: line 174 - 'generates' might be more suited than just generate.Furthermore, the option of sorption to equipment and glassware might be mentioned.
  5 - Conduct a minimum of 1 blank and 1 spike per 20 samples or technique
  It might be added that it is also needed to regulaerly check the potency of the reactants like H2O2 as a general QA/QC effort.
  6 - Keep methods consistent across a study as much as possible
  I do not agree on the recommendation in lines 205-209 as this still does not necessarily solve the problem. An alterantive might be to build a database on the combination by analyzing 'many' samples thus allowing for statistical analysis and comparison of the data.
  7 - Use targeted techniques when study goals allow
  I do not think that a separate section is needed on this topic. The information provided can most likely be included in other sections. An example is inclusion in the previous section 'Start with a previously established method....'.
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  Expert 5

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	1	0
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	1	0
  Quantify the recovery rate of the method	0	1
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	0	1
  Keep methods consistent across a study as much as possible	0	1
  Use targeted techniques when study goals allow	0	1
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  laboratory gloves can also produce nylon and latex microplastics. Also consider the shoes, walls, floors, laboratory horizontal and vertical surfaces, laboratory instruments, tables, chairs/stools, waste bins and lining of waste bins, etc. 
  MNP standards are also available from Frontier Laboratories: https://www.frontier-lab.com/products/multi-functional-pyrolysis-system/194709/
  Detection/Quantitation limits: please add formulas and expiations for determining the limit of detection and limit of quantitation for Py-GC/MS
  
  As has been stated by others, but worth reiterating, training and awareness will reduce contamination and false positives.
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  Expert 6

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	0	1
  Create or outsource blank and spike reference materials	0	1
  Quantify the limit of detection of the method	1	0
  Quantify the recovery rate of the method	0	1
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	0	1
  Keep methods consistent across a study as much as possible	0	1
  Use targeted techniques when study goals allow	0	1
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  Current manuscript only talks about number-based calculations. There are also weight-based calculations for concentrations of MPs in samples. Please describe merits associated with weight-based calculation on limit of detection and limit of quantitation. If there are different analytical methods commonly used, it is recommended to brief discuss and compare those.
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  Expert 8

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	0	1
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	1	0
  Quantify the recovery rate of the method	1	0
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	0	1
  Keep methods consistent across a study as much as possible	1	0
  Use targeted techniques when study goals allow	1	0
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	1	0

  Row 96 - There are so many pitfalls for use of museum specimens - namely sample storage
  Row 124 - Reference materials with fixed in place particles cannot be used as a true spike into a matrix. They can only test a particular part of the overall method, not the entire workflow.  Fixed location nanoplastic arrays are being developed at NIST.
  Row 129 - awkward phrasing
  Rows 136-138.  It's bold to suggest criteria like this. There’s a big difference between a sample that’s an entire brain vs a 2 g sample of a brain for blank counts. The recovery range is quite wide but necessary at this stage in microplastic quant. 40% RSD is giant variability.
  Row 138 - When blanks are used for detection limit, yes I can imagine improvement. But detection limits are also set by the instrument used and that will not change by staff experience.
  Row 142 - Background contamination is one piece to detection limits, but not the complete picture. In MP research, I agree background is the biggest player, but the instrument itself also has a detection limit and should not be ignored.
  Eqn 1 &2 - Parentheses are needed because these equations include summing and multiplying
  Row 150 - Is this review only for spectroscopic methods? Py-GCMS measures mass of a polymer so these equations need to be described in both ways to be relevant to both kinds of instruments. Py isnt mentioned for the first time until much later.
  Row 163 - change to "particles’ count or mass recovered" 
  Row 174 - change higher to greater (and do that throughout paper)
  Row 177 - it's important to mention that particles could have also been present in the sample matrix that was used as the basis for the spike.
  Row 203 - It is unclear to me what this means. 40% of samples analyzed? 40% of laboratories doing the analysis?
  Row 211 - The river sampling ASTM method and the ISO py-GCMS calibration method should be cited here. As far as I know, these are the only ones published.
  Row 223 - The graphic suggests not reinventing the wheel. It seems like an oversight to not include that as a goal in the text here, but actually I would warn against discouraging innovation. The microplastic field still needs tinkering and invention. So far the review focuses only on spectroscopy methods. Those methods are likely going to be left behind as mass spectroscopy methods improve. Innovation and tinkering is absolutely still needed today. We are not yet ready for standardized methods and I encourage you to say that in this paper.
  Row 230 - Microfibers are probably the worst example you can provide here. I’d suggest fragments. We are nowhere near being able to accurately count microfibers in samples.
  Row 243 - This is the first mention of pyGCMS. Subsampling introduces HUGE variability. I wouldn’t recommend this unless you have a section discussing the best practices for subsampling, which would be very useful in this review. I found it far below, seems a call out to that section or reorg could be helpful.
  Row 245 - Acknowledging the tradeoff of time is important here. Reanalysis of the same filter doubles the sample analysis time. In some cases, one filter takes us 4-5 days to map. 
  
  
• View answer

  Expert 7

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	1	0
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	0	1
  Quantify the recovery rate of the method	0	1
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	0	1
  Keep methods consistent across a study as much as possible	0	1
  Use targeted techniques when study goals allow	1	0
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  1. Create a lab and field environment as free from plastic as possible
  In line 72, ...ultimately controls the detection limit.. I felt that the detection limit should be limit of quantification. 
  In lines 73 and 74, microplastics from outside the sample (samples)...and equipment (equipments)...
  In line 78, . but should be , but
  In line 81, in the air should be from the air.
  
  2. Create or outsource blank and spike reference materials
  In line 95 and 96, a low plastic biological specimen should be a biological specimen low in plastic.
  In line 99, between found and specialized, there should be a comma.
  
  3. Use targeted techniques when study goals allow.
  For this session, targeted techniques can be developed and established for untargeted questions. Targeted questions and targeted techniques may miss some important information.
  
   
• View answer

  Expert 1

  Section	Yes (please explain)	No
  Create a lab and field environment as free from plastic as possible	0	1
  Create or outsource blank and spike reference materials	1	0
  Quantify the limit of detection of the method	1	0
  Quantify the recovery rate of the method	1	0
  Conduct a minimum of 1 blank and 1 spike per 20 samples or technique	0	1
  Keep methods consistent across a study as much as possible	1	0
  Use targeted techniques when study goals allow	1	0
  Repeat sample analysis for a sample per batch by replicating to assess intra sample variability	0	1

  The quality assurance and quality control section describes practices that are commonly followed in trace chemical analysis lab, although some sections are specific to labs that analyze micro- and nano-plastics (MNPs).  
  
  Establishing a lab environment that is free of MNPs is very important.  A difficult source of avoid MNP contamination is airborne particles.  Controlling/monitoring lab air quality is critical and ISO clean 5 or class 6 clean rooms may help reduce contamination by airborne particles, but such infrastructure can be expensive.  This is critical for labs measuring MNPs at trace levels, especially in human specimens.
  
  In terms of reference materials, I would suggest government organizations/institutions invest in developing certified reference materials for MNPs in biological samples.  Developing such reference materials for a wide range of environmental and biological matrices is needed.  Institutions like AIST (Japan), NIST (USA) and European union may come forward to invest in developing reference materials for MNPs in environmental and biological samples. 
  
  The sections on detection limit and recovery are informative, although the suggested equations should not be treated as the rule.  Detection limits and recoveries vary depending on sample mass and analytical technique used, especially in the analysis of biological samples.  Therefore, these sections may be used as guidance but not the rule.  Authors may want to add some statements that  these are guidelines not rules.  Some discussions made on this section on lines 153-157 are not clear.  
  
  In most of the discussions pertaining to quantification, a denominator is missing.  That is, quantification is often centered around reporting number of particles, not number of particles per unit volume or mass of samples.  This is important for biological sample analysis.  A mere number of particles alone is not suitable for exposure or risk assessment.  It is the number per unit mass of sample makes the results more meaningful for risk assessment and comparison between studies.  In environmental/bio- monitoring studies, measurements are presented in the units of concentration (e.g., ng/g or ng/ml of tissue), which has a denominator.   One study reported MNPs in scat, but presenting number in a scat sample can be meaningless if the number is presented per unit mass.  It is because mass of scat from animals can vary widely.  In the recent techniques of MNP analysis using pyrolysis GCMS, detection limits are reported in the units of ng/g  or ug/g or mg/g tissue or sample.  Majority of the discussion in the QA/QC section is focused on microscopy and spectroscopy based technologies.  The QA/QC section should include more information on pyrolysis GCMS technique as this is gaining more attention due to reliability and reproducibility of the method.