Study Design and Methods: Please rate your confidence (0 = no confidence/fatal flaw; 5=highest confidence) in the study design and methods used. Please explain your rating

Answer Explanations

• 0

  Expert 5
  1. Analytical Techniques: Advanced and sensitive techniques such as pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) were used to quantify microplastics. This method is used for detecting microplastics in the environment and biological tissues. This is a promising technique. 

   

  However, significant number of questions came. There are many challenges of detecting some particular polymers such PET and PVC. This study detected these polymers; however, authors didn’t clarify how they did overcome this detection challenge for the polymers like PVC, PET 

   

  Detecting certain polymers, such as PET (polyethylene terephthalate) and PVC (polyvinyl chloride), poses significant challenges due to their complex chemical structures and similarities to other materials. These challenges often result in difficulties in identification and separation during recycling processes. This study did find these polymers but didn't detail the specific methods or techniques used to overcome these challenges. Without this information, it's unclear how the researchers managed to accurately detect these polymers despite the known difficulties, avoid under- or over-quantification, and reproduce the results. This omission highlights the need for transparency in the methodologies employed, which could provide valuable insights for future research and practical applications in polymer detection and recycling. 

   

  A detailed discussion with references has been given here: 
  
  

  A.   Quantification of Polyvinyl Chloride (PVC) Microplastics: When it comes to quantifying PVC (polyvinyl chloride) using pyrolysis or TGA (thermogravimetric analysis), the current literature often relies on markers like benzene or naphthalene. However, these pyrolysis products are highly nonspecific, as they can also be produced during the pyrolysis of other synthetic polymers, such as PET (polyethylene terephthalate), or even organic substances. This nonspecificity means that severe matrix effects could lead to an overestimation of PVC presence. (Rødland et al., 202; Ribeiro et al., 2020; Goßmann et al., 2022; Kittner et al., 2022; Fischer et al., 2017). Another significant challenge in quantifying microplastics is the effect of different matrix ingredients. These matrix effects further complicate accurate measurement, as they can interfere with the detection of specific polymers. As a result, there is still no alternative analytical method that provides specific and reliable quantification of PVC in environmental samples. (Goßmann et al., 2022; Kittner et al., 2022; Fischer et al., 2017; Lu et al., 2019). A distinctive feature of PVC and related polymers, such as poly(vinylidene chloride) or chlorinated polyethylene (PE), is the presence of chlorine. During pyrolysis, chlorine is released as hydrogen chloride (HCl), making it a promising marker for the quantification of chlorinated polymers, mainly PVC. However, HCl's high volatility, polarity, and acidity make it difficult to analyze via GC-MS (gas chromatography-mass spectrometry). (Miyake et al., 2007; Want et al., 2010) An alternative approach involves trapping HCl in an absorption solvent, followed by analysis using ion chromatography. This method offers a way to quantify chlorinated polymers more reliably, but it still presents challenges that researchers continue to address (Kamp et, 2023). Kamp et al. (2023) became able to quantify PVC MPs via pressurized liquid extraction and combustion ion chromatography under chosen condition. 
  
  

  Furthermore, La Nasa et al. (2020) identified a major issue is that the most common pyrolysis products of PS (polystyrene) and PVC (styrene and benzene, respectively) are not very specific, making it difficult to differentiate between them. On the other hand, the more specific markers, such as styrene-dimer for PS and chlorobenzene for PVC, are produced in much smaller quantities during pyrolysis. This low yield makes them less reliable for detection, as their signals are weaker and harder to measure accurately. As a result, these detection methods suffer from poor detection limits, complicating the identification and analysis of these polymers (La Nasa et al., 2020). 
  
  

   

  Reference: 
  
  

  Rødland, E. S.; Okoffo, E. D.; Rauert, C.; Heier, L. S.; Lind, O. C.; Reid, M.; Thomas, K. V.; Meland, S. Road de-icing salt: Assessment of a potential new source and pathway of microplastics particles from roads. Sci. Total Environ. 2020, 738, 139352  DOI: 10.1016/j.scitotenv.2020.139352 

   

  Ribeiro, F.; Okoffo, E. D.; O’Brien, J. W.; Fraissinet-Tachet, S.; O’Brien, S.; Gallen, M.; Samanipour, S.; Kaserzon, S.; Mueller, J. F.; Galloway, T.; Thomas, K. V. Quantitative Analysis of Selected Plastics in High-Commercial-Value Australian Seafood by Pyrolysis Gas Chromatography Mass Spectrometry. Environ. Sci. Technol. 2020, 54, 9408– 9417,  DOI:10.1021/acs.est.0c02337 

   

  Goßmann, I.; Süßmuth, R.; Scholz-Böttcher, B. M. Plastic in the air?! - Spider webs as spatial and temporal mirror for microplastics including tire wear particles in urban air. Sci. Total Environ. 2022, 832, 155008  DOI: 10.1016/j.scitotenv.2022.155008 

   

  Kittner, M.; Kerndorff, A.; Ricking, M.; Bednarz, M.; Obermaier, N.; Lukas, M.; Asenova, M.; Bordós, G.; Eisentraut, P.; Hohenblum, P.; Hudcova, H.; Humer, F.; István, T. G.; Kirchner, M.; Marushevska, O.; Nemejcová, D.; Oswald, P.; Paunovic, M.; Sengl, M.; Slobodnik, J.; Spanowsky, K.; Tudorache, M.; Wagensonner, H.; Liska, I.; Braun, U.; Bannick, C. G. Microplastics in the Danube River Basin: A First Comprehensive Screening with a Harmonized Analytical Approach. ACS ES&T Water 2022, 2, 1174– 1181,  DOI: 10.1021/acsestwater.1c00439 

   

  Fischer, M.; Scholz-Bottcher, B. M. Simultaneous Trace Identification and Quantification of Common Types of Microplastics in Environmental Samples by Pyrolysis-Gas Chromatography-Mass Spectrometry. Environ. Sci. Technol. 2017, 51, 5052– 5060,  DOI: 10.1021/acs.est.6b06362 

   

  Halbach, M.; Vogel, M.; Tammen, J. K.; Rudel, H.; Koschorreck, J.; Scholz-Bottcher, B. M. 30 years trends of microplastic pollution: Mass-quantitative analysis of archived mussel samples from the North and Baltic Seas. Sci. Total Environ. 2022, 826, 154179  DOI: 10.1016/j.scitotenv.2022.154179 

   

  Lu, P.; Huang, Q.; Bourtsalas, A. C. T.; Themelis, N. J.; Chi, Y.; Yan, J. Review on fate of chlorine during thermal processing of solid wastes. J. Environ. Sci. 2019, 78, 13– 28,  DOI: 10.1016/j.jes.2018.09.003 

   

  Miyake, Y.; Kato, M.; Urano, K. A method for measuring semi- and non-volatile organic halogens by combustion ion chromatography. J. Chromatogr. A 2007, 1139, 63– 69,  DOI: 10.1016/j.chroma.2006.10.078 

   

  Wang, Q.; Makishima, A.; Nakamura, E. Determination of Fluorine and Chlorine by Pyrohydrolysis and Ion Chromatography: Comparison with Alkaline Fusion Digestion and Ion Chromatography. Geostand. Geoanal. Res. 2010, 34, 175– 183,  DOI: 10.1111/j.1751-908X.2010.00043.x 

   

  La Nasa, J., Biale, G., Fabbri, D., Modugno, F., 2020. A review on challenges and developments of analytical pyrolysis and other thermo-analytical techniques for the quali-quantitative determination of microplastics. J. Anal. Appl. Pyrolysis 104841. https://doi.org/10.1016/j.jaap.2020.104841 
  
  

  B.    Quantification of Polyethylene Terephthalate (PET) Microplastics: Lauschke et al., 2023 highlighted the various effects of components in the sample matrix on the pyrolysis of PET (polyethylene terephthalate), and it explores different strategies to address these issues. The presence of impurities alters the distribution of pyrolysis products, reacts with analytes, or reduces the intensity of specific markers used for identifying and quantifying PET. Therefore, separating MPs from sample components is essential. One promising method to remove the inorganic matrix is pressurized liquid extraction. However, during this extraction process, PET undergoes a depolymerization reaction, which affects the recovery and reproducibility of the method. Additionally, using TMAH (tetramethylammonium hydroxide), a common derivatization agent in Py-GC-MS analysis, did not mitigate these matrix effects. According to Lauschke et al., 2023, that fast and reliable thermoanalytical method for precisely quantifying PET in complex environmental matrices could not be recommended at this time. Instead, extensive and time-consuming sample clean-up protocols remain necessary to achieve accurate results. 

  
  

  Reference: 

  Lauschke T, Dierkes G, Ternes TA (2023) Challenges in the quantification of poly(ethylene terephthalate) microplastics via thermoanalytical methods posed by inorganic matrix components. J Anal Appl Pyrol 174:106108. https://doi.org/10.1016/j.jaap.2023.106108 

   

  2. Quality assurance and quality control (QA/QC) results: The study in question has some significant flaws when it comes to quality assurance and quality control (QA/QC), particularly in the absence of detailed information provided in these areas as follows: 

  • No Positive Control and Recovery Test and Results 
  • No Background Contamination and the Digestion Process Control Test and Results 
  • No Negative/Procedural Control Test and Results 
  • No Sample Collection Blank Test and Results 
  • No LOD/LOQ and Results 

  QA/QC measures are crucial in scientific research to ensure the reliability and accuracy of data. Without rigorous QA/QC measures in place, there is a heightened risk of errors, contamination, or inaccuracies in the findings related to MNPs detection. 
  
  

  3. Microplastic Visualization and Characterization: Understanding how MNPs may interact with biological systems is important. For instance, microscopic visualization of the size and shape of MNPs are fundamental parameters for their characterization. For example, particles, smaller than 100 nanometers, can reach nearly all organs once they enter the human body. This raises concerns about the potential long-term health impacts of ongoing microplastic accumulation in humans (FAO, 2023). Determining these features accurately is essential for assessing their distribution, transport mechanisms, translocation, and potential toxicity in biological tissues; and further to provide with a baseline for future mechanistic study. Unfortunately, this study lacks detailed information on the microscopic visualization and size and shape characteristics of the MNPs it investigates. Without this essential data, it is challenging to fully understand their fate, interactions with organisms including human, and potential health risks and toxicity. Enhancing the characterization of MNPs sizes and shapes is essential for advancing our understanding of their health risk and toxicity implications. Application of micro-Raman and/or micro-FTIR coupled with pyro-GC/MS could clarify this issue of MNPs size and shape characterization. 

   

  Reference: Food and Agriculture Organization. Microplastics in food commodities. A food safety review on human exposure through dietary sources. [accessed on 2023 February 20]. Available at: https://www.fao.org/documents/card/en/c/cc2392en. 

   

  4. Microplastic Isolation and Digestion Efficiency: The study mentions that the combined approach of tissue digestion, ultracentrifugation, and Py-GC/MS is still being refined and that a fraction of smaller nanoparticles may have been lost during ultracentrifugation. This could affect the accuracy of microplastic quantification. No details have been provided on whether the particle loss has been investigated during ultracentrifugation. 

   

  5. Polymer Spectra Matching Comparison: This study provided the clarification in the supplementary as follows: 

   

  “Quantification was performed using F-Search MNPs 2.1 software by comparing mass spectra and retention times to a calibration curve constructed with polymer NMPs-CaCO3 calibration standards (Frontier Labs, Koriyama, Japan) at various weights (0.1, 0.2, 0.5, 2, and 4 mg). Once the calibration curve was established, samples were analyzed using F-Search MNPs 2.1 software.  F-search MP 2.1 includes a library of twelve commonly used polymers including polyethylene (PE), polyvinyl chloride (PVC), nylon 66 (N66), nylon 6 (N6), styrene/butadiene rubber (SBR), polyurethane (PU), polypropylene (PP), polymethyl methacrylate (PMMA), acrylonitrile butadiene styrene (ABS) , polyethylene terephthalate (PET) , polycarbonate (PC), and polystyrene (PS)”. 

   

  However, the threshold for the match percentage/proportion between the analyzed polymers and the reference has not been clarified and demonstrated, making it unclear what criteria were used to determine a match. 

   

  Overall Evaluation: Limitations related to analytical techniques, QA/QC, the refinement of microplastic isolation methods, sample size, and potential confounders prevent a higher confidence rating. 
• 1

  Expert 4
  There are several issues related to the study design and methods.  
  
  Methods for the analysis of microplastics in biological tissues are not harmonized and, in many cases, not validated.  This study suffers from the lack of well validated analytical method.  The authors have cited their own publication (Garcia et al. 2024) and provided a very brief description of the method used for the analysis of 12 types of micro-/nano-plastics (MNPs) in testis samples.  Garcia et al. 2024 does not describe a validated method, and is more focused on placental sample analysis.  First, background levels of contamination of microplastics in the analytical procedure is not at all mentioned.  Many chemicals (e.g., KOH), solvents (e.g., ethanol) and supplies (e.g., centrifuge tubes) used in analysis contain plastics, as they are often stored in PVC or other plastic containers (at the production site or at retailers).  There is no mention of what were the concentrations of MNPs found in procedural blanks.  It is imperative to show a chromatogram of procedural blanks and report background levels of MNPs in the analytical procedure.  Any analytical method would require internal and external validation to assure data quality.  The method used by the authors is not validated with spike-recovery tests.  There is no discussion of accuracy and precision.  There is no discussion on method detection limits.  There is no clear and detailed discussion on quantification method, which is very important in quantitative analysis.  There is a short/less than a sentence that mention on calibration with concentrations in "sub/few milligrams" but the reported levels are in "microgram" and that raises the question of how the quantification was performed.  There is no use of surrogate or internal standard.  There are no matrix spikes.  Overall, analytical aspect, which is the 'core' of the study is very weak and raises concerns/questions about the data quality and reliability.  In fact, authors have stated in the limitation section that the method is "still being refined".  Analysis of  critical biological matrix for such sensitive studies without a well refined method can lead to erroneous outcomes.  At least I would expect authors provide extensive, supportive evidences, and clear details of method validation; providing procedural blank chromatograms (documenting what was found in blanks), report on background levels of contamination, calibration standard/quantification method (calibration curves and quantification methods), matrix spike recoveries (for accuracy), surrogate standard recoveries (if any; authors did not seem to have any surrogate or internal standard), limits of detection and it was calculated.  If with such internal validation,  it would only make this study stronger with another layer of confirmatory analysis (using another method) to validate the results/data.  The analytical method overall is very weak.  
  
  There are issues associated with sample collection and storage:
  
  Sample collection:  The testis samples of humans and animals were collected from hospitals (probably by surgeons) and potential contamination that occurred at the time of surgery is not known.  A wide range of plastics are used in medical settings and during surgeries many surgical tools including gowns and gloves worn by physicians and veterinarians can be a source of MNP contamination.  Potential contamination of samples by plastic materials used during collection is not mentioned.   How were the human testis samples collected?? 
  
  Sample storage:  The year of sample collection for canine testis is not provided.  How were human and canine testis samples stored?  frozen? liquid nitrogen?  in what type of containers? Human testis were stored for 7 years and no details about the storage conditions were given.  Long term cold storage can affect/alter the weight of the testis, especially storage for 7 years (due to desiccation).  In that case, weight of testis, used as an outcome parameter, can be prone to errors.
  
  Tissue selection:  It is not clear whether the testis samples were homogenized prior to aliquoting 0.42 g for digestion.  If only a fraction is cut, that may not represent concentration in the entire testis/organ.  No description is made on that regard.
  
  Sperm count:  Sperm count is an important parameter analyzed, but there is no information on how this was done.  A brief phrase on "densities counted on the hemocytometer" but that is not adequate.  If the dogs have some other illnesses, it is possible that sperm count can be affected by the illness.  There is no thorough and detailed information (method of analysis of sperm counts, health condition of dogs) on this important parameter presented on this article.  Furthermore, no data/discussions are presented with regard to normal sperm count in dogs versus what was found by the authors.
  
  Data analysis:  Sample size is small to warrant any cause-effect linkage.  Data are presented in an epidemiological perspective by associating MNPs with sperm count and testis weight, but no confounding factors/covariates are considered.  Data analysis and interpretation are inappropriate, considering the study design.  
• 4

  Expert 1
  The investigators have made considerable efforts to design the study rigorously and analyze the data accurately. However, they acknowledge certain limitations in their methodology. The combined techniques of tissue digestion, ultracentrifugation, and Py-GC/MS require further validation. Specifically, it is crucial to assess the efficiency of the digestion (saponification) process and account for potential losses of nano- or micro-particles during ultracentrifugation. This is particularly important as microplastics, with particle densities ranging from 0.88 to 1.40 g/mL, are more susceptible to loss during centrifugation.
• 0

  Expert 2
  I have extremely limited confidence in the outcome of this study. In my opinion there are several fatal flaws:
  1 - The selection of especially the dog samples tested. A key factor is for instance the age of the dog as age is already associated with reduced sperm counts.
  2 - The sampling procedures: it is essential in any analytical work with nano- and microplastics that care is taken to avoid any contamination of the samples. In this case, the samples were collected by vets and it is highly likely that no attention at all was paid to avoiding cross-contamination of the samples. This is of relevance for the whole chain of dealing with the animals, actual collection of the samples, and storage. No details are given on this aspect.
  3 - Analytical considerations: the method of pyrolysis GC-MS is insufficiently validated for the analysis of plastics in biological samples. Currently, lots of research is ongoing to improve the analytical tool of pyrolysis GC-MS and for instance provide suited reference materials.
  Thereupon, in this specific case the digestion method used was not validated. This implies that any data reported is questionable.
  4 - The main problem is co-variance in various respects. This includes first of all co-variance between different plastic types found in single samples. If only for this reason, it is not allowed to present Figure 4, let alone to draw any conclusions from this figure.
  Secondly, there is co-variance between concentrations of individual plastics and the summed concentration of all plastics in a specific sample.
  Thirdly, there is co-variance between plastics in tissues and additives present in the plastics. It certainly cannot be excluded that actually the additives are causing adverse effects instead of the plastcis, as suggested here. Especially Endocrine Disrupting Chemicals are suspicious in this resepct.
  Fourthly: the authors themselves already detected a source of co-variance as related to the origin of the samples (private versus public veterinary clinics). No doubt, there are more sources of yet non-identified co-variance.
  5 - A relatively minor issue is that many of the plots depicted in figure 4 lack statistical rigor.
• 1

  Expert 3
  Pros
  - They used chemical analysis to assess their samples, not just visual techniques. 
  
  Cons
  - Log (x+1) transformation is a commonly used statistical approach but a better strategy would have been to correct the non detects (zeros) using a standard non detect correction algorithm (https://doi.org/10.1002/etc.4046). 
  - There is nowhere near enough information given about the pygcms setup to assess or reproduce the measurements in the main text. Pyrolyzer, GC, and MS settings and configurations need to be described. The methods description relies heavily on referencing a previous study Garcia 2024 but this study doesn't seem to follow that method exactly because there is no mention of FTIR or Fluorescence in this study.  
  - Limits of detection/quantification for each analyte are not described. 
  - There isn't enough explanation about how percentages were calculated for the polymer types to understand whether they could be skewed by the individuals with the highest abundance. 
  - There is no mention of positive or negative controls for this study, recently editors have suggested not accepting studies without these components (https://doi.org/10.1016/j.scitotenv.2023.168465).