Qualitative analysis
Commodity polymers
Overall, none of the post-treatment polymer spectra, both in FTIR and Raman, showed substantial spectral band changes that would hinder recognition (Fig. 4, Commodity polymers). However, minor alterations were detected in several cases, which are further detailed below.
Using Raman, increased fluorescence or baseline shifting (category 1 changes) were the most frequently seen effects, but did not hinder a successful recognition of the MP’s polymer type. Only for PVC in the H2O2 Fe uncooled treatment clear degradation effects on the polymer basis became visible in multiple measured replicate spectra. Two peaks around 1124 and 1511 cm− 1 (C = C double bonds) strongly increased, which are a direct result of the dehydrochlorination reaction due to thermo-oxidative processes [47]. It is noteworthy that these degradation processes appear to affect individual PVC particles differently, as we see from the varying spectral characteristics between the replicate measurements (see also deposited spectroscopic data at https://doi.org/10.5281/zenodo.4568683 [34]) and also from varying degrees of visual yellowing from white over beige to yellowish brown across particles.
In 10 out of the 125 Raman measurements multiple new peaks appeared which could not be attributed to specific functional group frequencies and were therefore not regarded as a polymer related change (these cases are indicated by * in Fig. 4, compare annotated deposited spectra [34]). These peaks were treatment and polymer unspecific and most frequently appeared in the water control. This lead to the suspicion that the water, used to rinse after the treatment, introduced the impurities. Switching to ultra-purified water for the remaining tests (i.e. H2O2 LT, Pentane, SPT) completely prevented these unknown peaks. They do not carry exploitable information for the question of polymer recognisability and their emphasis in Fig. 4 merely serves the purpose of clarity when comparing the here presented results to the original spectra provided in the deposited spectra [34].
In FTIR, the polymer basis was always clearly identifiable. We detected, however, minor polymer degradation processes in the H2O2 LT treatment. This concerned ABS, PC, PMMA, PP, PVC, TPU where new bands could be assigned to carboxylic acids which may have resulted from oxidising polymer chain ends. In the SPT treatment, similar oxidations were observed for ABS and PVC. The corresponding Raman spectra, however, did not systematically indicate these changes. This is plausible as FTIR is sensitive to polar organic groups, such as C = O and its variations (e.g. esters, anhydrides, amides, imides), while Raman is less sensitive to these.
It appears that for FTIR category 2 changes occur rather treatment-specific, whereas for Raman spectra no treatment-specific impacts were seen. Instead, several cases of category 1 effects appeared in a rather polymer-specific manner. Although category 1 effects were also found in the water control of HDPE and TPU, it is noticeably the actual treatments that got assigned to this category.
As Fig. 5 shows, the HQI of the treated samples lie in the range of the untreated reference for most tested polymer types. For some treatments, the HQI range of the replicates varied more than for others. In the case of LDPE, the HQI distribution reflects the assignment of the categories (compare Fig. 4). That is, the KOH + NaClO treatment introduced category 1 changes, while no significant changes occurred in the other treatments. Only for KOH + NaClO, the HQI drops to around 90–59 for the different replicate spectra. Similarly, the correct material type was found amongst the first ten hits in the library search, except two single replicates of a polymer-treatment-combination. One pentane-treated HDPE was identified as aliphatic fatty acid or alcohol. This is, from our experience, a frequently occurring problem in Raman spectroscopy. This and the fact that it only happened for one in three replicates should not lead to classifying the pentane treatment as harmful for this polymer type. The second not correctly identified spectrum resulted from a KOH + NaClO-treated PET replicate. Here, the occurrence of the inorganic contaminant calcium carbonate led to extra bands that prevented a semi-automated polymer identification. Overall, this confirms the assumption that none of the treatments induced changes that prevented the identification by a manually supervised spectral library search, as it is common in microplastics research. The result of a HQI based polymer attribution depends, however, on the available references, the applied algorithm and search parameters. It is not generalisable between studies and laboratories and therefore not used as a systematic evaluation criterion here.
In summary, our qualitative results indicate that, regarding the spectroscopic recognisability of MP particles of any of the tested commodity polymers, all of the tested treatments are safe to apply, when the chosen analysis technique is either FTIR or Raman spectroscopy. However, effects on the treated polymers spectra, that were less critical to polymer recognition (i.e. category 1, Table 3), occurred frequently. Raman baseline shifts can be caused by heating of the sample during measurement [48]. Fluorescence mainly originates from sample characteristics, but background conditions such as ambient lighting, can play a role as well [49]. Both effects are too unspecific to be assigned to a polymer degradation process. Changes in the ATR-FTIR baseline can occur due to multiple changes in the optical path, e.g. the contact between the sample and the ATR crystal. They reflect physical rather than chemical changes, and again, are not fit to indicate a change in the polymer composition.
Paint resins
Out of the five paints, in all but one (acrylate) the polymer resin was not identifiable, already in the pre-treatment Raman spectra. In the KOH + NaClO as well as in the H2O2 Fe cooled treatment acrylate showed effects of degradation, however, was still recognisable. After the uncooled H2O2 Fe treatment, however, the polymer related bands were not found any longer, making an identification impossible. All other four paint spectra were substantially masked by fluorescence in both pre- and post-treatment spectra. In some cases, pigments (inorganic bands) dominated the spectra. One black (PU) and one grey paint (alkyd) showed an additional band which is indicative for black carbon (1343 cm− 1 and 1581 cm− 1, [47]). But as black carbon can stem from various sources in environmental samples, without any signal of the polymer bases the Raman spectra of these paint resins are unspecific and an identification by an automated detection procedure is not possible. As a result, no conclusions can be drawn concerning effects of the tested treatments on the polymer bases and associated processes of polymer degradation based on Raman spectroscopy.
The polymeric binders were clearly detectable for acrylate and epoxy based paints using FTIR spectroscopy. In these paints none of the treatments lead to a change in spectral quality, except the H2O2 LT treatment, which introduced carboxylic acid salt signatures around 1600, 1350 and 789 cm− 1 in acrylic paint, indicating polymer oxidation processes. While for PU based paint no exploitable spectral properties could be found in neither pre- nor post-treatment spectra, the paints based on rosin and alkyd exhibited some weaker bands that could be attributed to the organic binder matrix by an experienced spectroscopist, however not with sufficient confidence. Hence, we note that only the epoxy and the acrylic paint can be regarded as reliably identifiable by their polymer FTIR bands and are thus useful for evaluating the treatments effects on polymer recognisability. Chemical treatment-caused spectral changes i.e. bands weakening or disappearing were found in acrylic paint for H2O2 (Fe + LT) and SPT treatments. This did, however, not hinder the recognition of the polymeric binder as only inorganic components were affected indicating mineral depletion (CaCO3). Thus, no restricting categories were assigned based on this observation (Fig. 4).
Comparing Raman and FTIR spectra of paints, overall masking was weaker in the FTIR spectra. Where peak changes were present, they were dominatingly caused by pigments or other fillers, for both techniques. Only for acrylic paint the strong thermo-oxidative regime of high temperature Fenton’s catalysed H2O2 treatment made an unambiguous spectral recognition impossible in Raman, however not in FTIR.
In paints based on rosin, polyurethane and alkyd the detection of binder matrix bands was not possible already on the untreated material (at least not to a degree of certainty that would allow the identification if the material was unknown). Thus, degradative effects of the treatments cannot be studied with the here applied techniques. Our conclusions are limited to the observation that in general a reliable detection of paint flakes of these resins using spectroscopic identification is not guaranteed. In contrast to commodity plastics, paint resins are typically composed of high amounts of non-polymeric constituents and only a smaller portion of the polymer itself. Accordingly, it is questionable whether paint resins can be exhaustively and reliably identified in environmental systems using spectroscopic methods [20, 50]. The chemical bonds of many inorganic and organic pigments and other paint constituents are strong Raman scatterers, which renders them well detectable in Raman spectra (also better than with FTIR). On the downside they frequently mask the weaker bands of the polymeric binder due to their strong intensities or additional fluorescence. Large reference libraries containing spectra of large varieties of paint composites preferably from different manufacturers might be able to detect paint flake MP based on their (pigment dominated) fingerprint. This approach has also been taken by other studies [13, 51]. Most pigments are, however, not solely applied in synthetic polymers but in a large diversity of technical applications, which is why an indirect identification of MP via those pigments cannot be done in a systematic way. Furthermore, due to the large variety in different paint compositions, an automatic detection is limited unless the particular paint product is available in the spectral library.
Quantitative analysis
Particle property comparisons
As for the qualitative data, a high level overview on the quantitative data can be gained from the heat maps that summarise the relative changes that we could attribute to the treatments (Fig. 6). Noticeable are elevated values present in various polymers exposed to the H2O2 treatment. Further, for ABS, PC and epoxy based paint there are higher values found across treatments.
To enable a deeper insight into the quantitative dataset, we provide an interactive version of the heat map figures (Fig. 6), augmented with plots of individual particle data showing detailed and aggregated differences between pre- and post-treatment images. It may also be used to understand the necessity and effect of the GLM corrections that were applied to the data. The interactive figure may be downloaded from: https://doi.org/10.5281/zenodo.4568524 [52].
Particle counts
From the GLM corrected data, it can be seen that the majority of polymer treatment combinations did not show any loss significantly different from the negative control. Exceptions are the SPT treated ABS (30%) and the H2O2 treatments of epoxy based paint and PC (46% and 25%, respectively). For these three polymers also the corresponding water treatments show particle losses higher than any of the other polymers. This indicates that for images of these polymers not all of the confounding effects could be removed by the GLM correction, and the actual treatment caused changes must be lower.
For the H2O2 treated epoxy paint particles we conclude that the relative loss in the GLM corrected data is indeed indicative of an actual treatment caused effect. Influences of confounding factors were comparably low for this image pair (npre = 212, BDI = 0). The modal value of particle brightness is reduced in the post-treatment image (\( \mathsf{pre}\_\mathsf{histFGpeak} \) = 183 and \( \mathsf{post}\_\mathsf{histFGpeak} \) = 103 out of maximum of 255, respectively, see Table 5 for explanations), indicating a possible material erosion, which could not be explained by other factors. This is further substantiated by looking at individual particles in a pre-post-comparison, where dark indentations or holes become visible, in places where the particle surface or boundary were priorly intact (Fig. 7).
It is known that certain polyepoxides are susceptible towards peroxides, in fact, this is utilised in certain technical applications. One study used 24% and 50% H2O2 to superficially degrade epoxy resins and expose embedded fibres in a fibre reinforced polymer (FRP) [53]. For the recycling of carbon fibres from FRPs, acetified H2O2 (30%) was deemed effective for an epoxide matrix decomposition in an overnight treatment [54]. When epoxy resin particles are targeted within environmental MP studies and peroxide digestion protocols are applied, we recommend for future studies to note that numbers are likely underrepresenting the actual environmental load for these polymers. In our data the corrected particle loss exceeded that of the epoxy water digestion by 35 percentage points. Hence, it can be assumed, that a reported concentration of epoxy MP particles in a certain environment are only covering approximately 65% of the total load present, at least for particles in the here studied size range.
The effect of H2O2 could also be seen on the epoxy layer that we used to immobilise the particle on the Si wafers. Increased brightness and haziness of the image background was apparent especially around the wafer edges, which made more encroachments by manual image postprocessing necessary in those wafers. Small defects in the layer were promoted to brighter visual appearances after the treatment that could lead to false positive detections, while increased brightness in the near field surrounding of particles, is likely a contributor to the observed particle area growth that was observed in several H2O2 treated polymers. These effects on the layer show that with the epoxy resin used here, we have not yet found the optimal immobilisation adhesive for the purpose of the study. We argue though, that the effects do not constitute a failure of the immobilisation technique, as we know from the positive control wafer (PA6 treated with HCl) the detailed visual characteristics of spots where particles got removed from the layer. Such appearances were not what we could typically observe in the images of H2O2 treated wafers, instead, the image objects at places where particles were expected generally resembled what particles looked like in the other treatments (including the negative control water treatment). This is yet not sufficient evidence to fully exclude that a loosening of particles may have occurred in some places due to the layer being affected, but it shows the this was not predominant process. Hence we conclude: the spin-coated epoxy layer did resist our H2O2 treatment at least to a degree that particles could be held in place and measured after the treatment. It could be further improved by finding an adhesive composition that does not show any visual nor integrity-related impacts from any of the tested treatments. Experiences and advice from our adhesive layer development phase are detailed in the SI (S3).
The wafer of H2O2 treated PC suffered the problem of having a low total particle number (npre = 26), which could not be entirely alleviated by the applied GLM correction. The particle brightness was not reduced on this wafer after the treatment (\( \mathsf{pre}\_ \) and \( \mathsf{post}\_\mathsf{histFGpeak} \) were at 88 and 89, respectively), suggesting that there was no treatment caused effect acting on all particles collectively. Instead the loss was driven by only six particles that could not be matched between the images. The low npre is thus regarded here as the main reason for the resulting significant loss.
The loss of particles from the SPT treated ABS wafer should only with certain reservations be seen as an indicator of particle degradation, as the image quality was on the brink of our exclusion level (BDI = 26). We interpret the observed losses as partly treatment caused and discuss this interpretation below (Comparison of qualitative and quantitative results).
Most prominent, but also expected, is the loss of nearly all particles in the positive control wafer (HCl on PA6), where 69% of pre-treatment particles could no longer be confirmed to be present post treatment (85%, without GLM correction, see also Using a positive control for calibration of particle detection).
Particle areas
In contrast to particle counts, changes of particle area were recorded to differ in negative (area decrease) as well as positive (area increase) direction. The largest decrease of particle area can be seen in the positive control, however, also the SPT treated ABS was recorded with a significant area reduction of 30%. But as noted above, one should take this result with caution, due to the low image quality of this wafer. Several polymers treated with H2O2 revealed significant area increases post treatment (PP, TPU and PC with a 34%, 60% and 220% average increase, respectively). The extreme increase in particle area of PC was mostly caused by two outliers where in the pre-treatment image the particle was only partly detected by the thresholding algorithm. An increase in area or particle volume would not hinder a MP detection, however, it may lead to a distorted representation of particle sizes.
For the H2O2 treated epoxy paint particles, where we described a significant particle loss above, we do not see a change in area for the remaining matched particles. The formation of dark holes in particles (Fig. 7) is not represented in the post-treatment area measurements, because particles are identified as closed shapes by the detection algorithm.
Methodological caveats
The here presented technique for quantitative MP treatment evaluation was limited in two ways. Limitations were caused either by effects of a specific treatment on the adhering epoxy layer, or, by polymer specific characteristics which impeded a contrast rich DIC imaging.
The KOH + NaClO treatment disrupted the adhesion between the spin coated layer and the underlying Si wafer, which lead to a partial or complete detachment of the epoxy film and wrinkling artefacts on the images in several cases. The film itself did not seem to be degraded or otherwise affected as microscopic imaging could confirm that the epoxy matrix and attached MP particles were visually intact. However, the correct placement for a post-treatment imaging was disturbed and the layers were partially outside of the microscope’s focal plane, which resulted in darker areas that could no longer be recognised by the particle detection. This affected especially the KOH + NaClO treated wafers of PP and PET, where such areas needed be mutually excluded from the pre- and post-treatment images in the manual image postprocessing. The H2O2 treatment had a different effect on the epoxy layer, however without affecting the image quality to an extent where the image analysis would be impeded. The wafers treated with H2O2 were showing varying degrees of obliqueness in the otherwise clear epoxy layer, indicating a surface affect or softening of the layer. The resulting implications are discussed in the above section.
For PVC, PMMA, PS and, to a lesser degree, ABS the chosen microscopy mode (reflected DIC) was not able to generate images that have a good particle to background contrast. The image postprocessing did increase the particle brightness for the particle detection algorithm but at the cost of a proportionally gained brightness of the image background. For reasons unbeknown to us, the surface topography of MP particles of these polymers did not create an optical path length difference that would allow for contrast rich DIC images. The size range of the particles of these polymers was not different than for the other polymers and further investigations beyond the scope of the study would be necessary to clarify whether this is indeed due to polymer specific optical characteristics or rather special interactions between the particles and the uncured epoxy layer at the time of application. Unless a solution is found to create particles and immobilisations of these four polymers that yield a sufficient contrast, the more suitable approach would be applying a different microscopic technique. Possible candidates are discussed in the SI (S3).
With the current state of the experimental approach, the polymers of PVC, PS and PMMA, could not be included in the analysis, due to the above-mentioned reasons. PMMA and PVC were excluded due to low image qualities. Of the ABS wafers only the KOH + NaClO treated sample had image qualities that resulted in a BDI exceeding the set cut-off of 27. For PS, however, the BDI was not able to ascertain that the image quality was unsuitable for an analysis, because the image defects in PS were slightly different than in PVC and PMMA. While the latter showed a general and evenly low contrast between particles and background, PS had mostly a stronger contrast but included many optical artefacts (i.e. bright ring-like halos around particles). It was attempted in image postprocessing to remove the artefacts manually, however, the PS images could not be improved sufficiently. Images of PVC and PS can be viewed in the deposited imaging dataset at https://doi.org/10.5281/zenodo.4568488 [55] as an example of the contrast problems.
The above discussed caveats currently pose certain limitations in terms of applicability, explanatory power and required work effort. The true value, however, is present here as the possibility of the immobilisation approach to study effects on individual particles simultaneously on large particle numbers in the smaller MP size ranges. How this compares to other concepts is discussed below in the section Comparison of experimental concepts. Additionally, in the SI we describe more detailed the lessons learned and attempts to further methodological improvements for interested readers (S3).
Comparison of qualitative and quantitative results
While the quantitative particle data provides insights in the particle-based alterations of a treatment, it does not convey whether these can be recorded by the chosen analysis technique (here Raman and FTIR spectroscopy). As an example, particles might still be present after a treatment, but changes on the surface of the particles or the polymer matrix might render them undetectable. The other way round - a polymer type is recognised post treatment but the quantitative measurements reveal particle losses or area changes - is also a possible scenario. In fact, the epoxy based paint resin in our data is an example, where we observed a significant particle loss after a 24 h H2O2 exposure, while FTIR recorded no changes to the polymer spectrum even after a prolonged 2 weeks’ exposure or the uncooled Fenton’s with its 80 + °C temperatures. The two-tiered approach, providing qualitative and quantitative data on the same polymer treatment model systems, therefore allows for a higher certainty on the applicability of the tested treatments. For a comprehensive evaluation both aspects are of importance.
A related study also reported qualitative (spectroscopic) and quantitative (recovery of spiked particles) data [11]. They noted a qualitative change on H2O2 treated PVC (see also below section for further details: Comparison of our results to current literature), however could not confirm that the described polymer degradation would manifest in the quantitative measurements.
In our data, SPT treated ABS that was indicating negative changes in particle numbers in the quantitative result, also showed alterations in the spectra of FTIR (assigned category 2, emergence of new bands or band intensities altered), and Raman (category 1, baseline drift). The treatment with SPT resembles a density separation where HCl is used to acidify the solution to pH 3, in order to prevent sodium poly tungstate precipitation [31]. We know that ABS is attacked by acidic digestions [1], however, this was only shown for highly concentrated and strongly oxidative acids (HNO3 69% and HClO4 70%), neither of which applies to the here deployed solution. As it was demonstrated there, the acidic treatment lead to a bloating and bubbling dissolution of the macroplastic piece that was tested [1]. The present results indicate an area reduction of those particles that were found again after the treatment. A testable conjecture beyond the scope of this study, is that a count and area decrease was detectable – despite working under a much weaker acidic environment – because of the small size range of particles that were used here, and which potentially are more easily damaged by acids.
Comparison of our results to current literature
To our knowledge, polymer chemical resistance tests in qualitative and quantitative manner have not yet been performed on such small MP particles. Partly related studies reported recovery rates of > 90% of spiked particles using fluorescence-labelled microspheres ≤ 30 μm as representatives of small MP for a novel magnetic separation [12] and in a density separation and digestions [10]. Working with aliquoted pipetting of bulk particle suspensions, they noted that direct comparisons of the same particles’ pre- and post-treatment counts was infeasible in this size range [12]. Generally, our overall finding, that, with respect to spectroscopic recognisability, no considerable changes to the polymeric bands were caused by the tested treatments on MP < 70 μm, is in agreement with other studies which used larger MP particles. One study tested the same KOH + NaClO protocol on a similar set of 12 commodity polymers (≥ 5 mm) and performed Raman spectroscopy on pre- and post-treatment particles, finding no relevant changes on the polymer matrix [1]. Dehaut et al. [5] applied a KOH protocol without an additional oxidiser, also reporting no effect on Raman spectra even at 60 °C in all 11 polymers that were comparable between their and our study. Similarly, Hurley et al. [25] found no significant spectral changes applying a Fenton’s protocol (cooled, < 40 °C) to a set of 8 different commodity polymers (~ 3 mm) using FTIR.
In our Raman measurements, the only polymer degradative effect was observed in PVC, where the formation of C = C double bonds (caused by dehydrochlorination) occurred only in the uncooled H2O2 Fe treatment (> 80 °C, 10 min) as a direct consequence of thermo-oxidative processes on the polymer matrix (as we also observed in an early study [47]). Likewise, Raman spectra measured by Karami et al. [11] on PVC particles treated with 50 °C H2O2 were described as being diminished in the C − Cl stretching band, which would be indicative of the same dehydrochlorination process that we observed. But, as mentioned above, PVC exposed to 60 °C in a KOH based treatment (10%, 24 h) exhibited no effect on Raman spectra [5], albeit the presence of a hydrolytic and thermo-oxidative pressure. The difference lies apparently in the stronger oxidising potential of the H2O2, that induces detectable – but non-critical – polymer degradation of PVC. It should be noted though, that the occurrence of PVC thermo-oxidative degradation depends on the specific PVC material and its added stabilisers. It is plausible that for other PVC compositions this effect may be induced alone by temperature rises above 60 °C.
In general, negative impacts of elevated temperatures during treatments have also been demonstrated by various studies. For instance, increased mass loss for PA in H2O2 (among other treatments), was reported for temperatures between 60 and 70 °C [14] and for > 70 °C [25]. In our qualitative analysis PA did not show signs of degradation, however, we cannot infer whether particle losses or area changes may have resulted from these treatments, as no treatments involving elevated temperatures were part of our quantitative analysis. Yet, the non-catalysed H2O2 treatment (room temperature) resulted in no change of PA6 particle numbers or areas.
For the pentane treatment [31] we could now demonstrate polymer resistivity among our test series. The protocol, which involves a weakly acidic digestant (ethanol acidified to pH 3 using acetic acid is added to the sample before pentane), had no impact on the spectra of any of the tested polymers, hence also not on the polyamides, known to be susceptible to acidic degradation. PA6 particles, which we tested quantitatively, suffered no detectable impact by the pentane treatment in particle numbers or areas. Similarly, the acidic SPT was not affecting particle numbers or sizes, except for ABS, where a minor degradative effect could not be excluded, based on our data.
It was reported that PA6.6 melted when applying H2O2 at 70 °C and a significant weight and size loss was measured [25]. In our uncooled catalysed H2O2 treatment (reaching up to 85 °C for the 10 min treatment duration) PA 6 showed a yellowing effect despite not showing significant quantifiable losses in size or area. Potentially, the temperature exposure in our treatment was not long enough to melt the polymer compared to their exposure time (12 h).
Studies exist that found degradative effects of alkaline protocols on PET [5, 14, 25], however also other factors such as elevated temperatures were involved in some of these cases. Our qualitative results of the protocol using KOH + NaClO do not show signs of degradation of PET in FTIR or Raman spectra; neither do we see a significant particle loss or area change in the quantitative data. The quantitative are, however, of lower confidence, as these samples were affected by the KOH + NaClO induced displacement of the epoxy layer, complicating image analyses.
Comparison of experimental concepts
To our knowledge, working with particles immobilised by adhesives to obtain quantitative measurements has not been performed so far. A related approach was developed recently for measuring PS nanoplastic spheres, where etched cavities in Si wafers were serving as a nanofabricated grid [56]. Particles placed in suspension on the cavity array were assembling by capillary forces and thus temporarily arrested for subsequent material and size determination using Raman spectroscopy. Alternative approaches to microscopy have been applied to determine size changes, such as laser sizing [57]. However, when measuring in wet dispersion problems can arise for polymers with a lower density than water as a sufficient particle dispersion can be challenging to achieve. Absolute particle counts cannot be determined using this technology and problems can arise if post-treatment particle numbers fall below the measurement limit of the specific laser sizer [57]. Also Coulter counters or flow cytometry, might be suitable techniques for these kind of measurements. Usually applied for counting plankton or cells, they allow simultaneous count and size measurements. A disadvantage, however, is the necessity for a filtration step to purify the particles for a post-treatment measurement. Finally, direct microscopic imaging of filtered bulk particle suspensions before and after a treatment could be a viable approach as well. However, like all methods using mobile particles entails the problem, that count data is difficult to obtain for small-sized MP. Particles can get lost in transfer steps and effects like breaking apart, aggregation, handling loss or real treatment-caused degradation are difficult to disentangle. This has been observed by others as well. Thiele et al. [13] used MP particles < 600 μm (that could still be manually handled) for spiking and recovery tests in a treatment evaluation and reported issues with particle loss in transfer steps that needed to be mathematically accounted for. With an immobile particle approach these problems do not arise.