1. Processing of MPs from plastic pellets through cryogenic pretreatment and milling

NOTE: This methodology is based on a procedure described elsewhere, employing a PBAT film composed of the same material used for this presented study²⁹.

1. Weigh polymer pellet samples of ~1 g and transfer into a 50 mL glass jar.
2. Place the "rectangular delivery" tube with a 20 mesh (840 µm) sieve in the slot in front of the rotary cutting mill and raise the delivery tube until it hits the stop pin.
3. Position the glass plate over the milling chamber's face and secure it with the adjustable clamp. Next, place a 50 mL glass jar under the mill outlet (Figure 2).
4. Position the sliding side arm support on the mill (located on the right upper side) in the middle of the front glass and tighten with the knurled bolt. Ensure that the front glass of the mill is securely positioned (Figure 2a).
5. Insert the hopper funnel on top of the mill into the opening of the upper milling chamber.
6. Plug a line cord into a power outlet and the press cord switch to start the mill operation.
   NOTE: To prevent jamming, feed only material after the mill is powered on and rotating. Also, wear eye and ear protection during the entire milling procedure.
7. Feed the sample slowly into the hopper (around 10 pellets/min) to prevent slowing down or jamming. After audible noise reduces, add the next batch of pellets (~10 pieces). After processing the pellets (1 g), press the cord switch to stop the mill operation for ~20 min to cool down. Use a wooden plunger to feed the sample and prevent particles' ejection and agglomeration inside the feeding hopper.
   CAUTION: The optimal feed rate varies depending on the type of processing material. Immediately turn off the mill if the processing speed decreases due to particle friction in the cutting chamber, or if formation of molten polymer is observed on the glass plate, to prevent overheating and further melting of the polymer particles.
8. Remove the 20 mesh (840 µm) delivery tube and replace it with the 60 mesh (250 µm) delivery tube upon completion of the first batch (Figure 2b).
9. Reintroduce the collected material into the mill hopper. Follow steps 1.1 and 1.7 for the 250 µm milling fraction.
10. Refeed the collected 250 µm fractions up to three times.
11. Recover the remaining particles in the chamber and add them to the collected main fraction.

2. Processing of plastic films by cryogenic pretreatment and milling

1. Retrieve a film specimen from the roll and cut the specimen into strips of ~120 mm (cross-direction) x 20 mm (machine direction) with a paper cutter.
2. Presoak fragments (~1 g) in 800 mL of deionized (DI) water for 10 min in a 1000 mL glass beaker. This step improves embrittlement for the subsequent cryogenic cooling procedure by presoaking the polymer.
   CAUTION: Handle liquid nitrogen with safety equipment by wearing cryogenic gloves and safety glasses.
3. Slowly add 200 mL of liquid nitrogen (N₂) to a cryogenic container.
4. Transfer the presoaked film particles carefully into the cryogenic container with steel tweezers. Presoak for 3 min in liquid N₂.
5. Transfer the frozen film fragments into a 200 W, 14-speed blender.
6. Process the frozen material at speed level 3 for 10 s to break the frozen glass film structure. To promote further size reduction, add 400 mL of DI water and blend the film-water slurry for 5 min.
7. Transfer the slurry into a Büchner funnel with filter (1 µm mesh) and apply vacuum for at least 1 h.
8. Vacuum-dry solid particles at 30 °C for at least 48 h in an aluminum dish.
9. Feed dry particles into the mill with tweezers. For milling, follow steps 1.1-1.11.

3. Processing of plastic films pretreated through environmentally weathering and milling

1. Lay out plastic film fragments recovered from the field on a smooth surface (lab bench). Carefully remove absorbed soil particles and plant remnants with the soft-bristle brush.
2. Cut the film with scissors into ~4 cm² samples of ~1.0 g.
3. Add film fragments into a 1000 mL beaker filled with 500 mL of DI water. Stir at a rate of 300 min^-1 with 20 mm stirring bar for 1 h.
4. Remove dissolved soil particles by decanting and reintroducing DI water under slight agitation of the beaker into a sink or plastic bucket. Repeat this step three times. Continuous agitation keeps soil particles dispersed in water and can be easier decanted.
5. Transfer samples from the beaker into an aluminum dish. Air-dry the plastic samples for 12 h, and then transfer and dry in a vacuum oven for 24 h at 30 °C. For milling, follow steps 1.1-1.11.

4. Sieving procedure through cascaded sieves

1. Stack the sieves (3 inches in diameter) starting with the pan at the bottom, followed by the finest sieve (#325; 45 µm), and then by increasingly coarser sieves (such as #140; 106 µm, and #60; 250 µm, where the #20; 840 µm sieve is the coarsest), and place the lid on top.
2. Mount all four sieves on the shaker by inserting four pins in the openings of the sieve shaker.
3. Transfer individual fractions collected in either step 1, 2, or 3 on top of the four cascaded sieves. Shake for 10 min at 300 min^-1.
4. Recover the larger (top) fraction separately, which will be subjected to further milling.
   NOTE: Adjust the shaking speed on the shaker as needed. Alternatively, shaking sieves by hand is possible. Use only one sieve at a time, starting with the mesh #20 sieve: hold the bottom and the lid firmly against the sieve by hand, and shake axially and horizontally for 5 min.
5. Reintroduce sieved particles of d_p > 106 µm to the rotary cutting mill as described in steps 1.6-1.10.
6. Recover the bottom fractions from the pan and reintroduce the particles to the next smaller sieve size. Repeat the procedure until 106 µm particles represent the main fraction.
7. Merge the collected 106 µm fractions and store the particles in a dry area (desiccator or air-sealed plastic bag).
   ​NOTE: The 45 µm fraction is part of the 106 µm fraction; however, the former fraction was not isolated and separately analyzed since the yield is generally very low. Yield recoveries and particle size fractions of individual fractions can be determined by gravimetric measurements in wt% for each sieving fraction (mesh #20 – mesh #325) in relation to the initial feeding fraction using a high-precision microbalance.

5. Preparation of an aqueous NP slurry for wet grinding

1. Prepare a slurry of MPs dispersed in DI water by adding 800 mL of distilled water into the 1000 mL glass beaker and inserting a stirring bar (diameter = 8 mm, length = 50.8 mm).
2. Introduce 8 g of the 106 µm plastic fraction from steps 1, 2, 3, or 4 into DI water, producing a 1 wt% slurry.
3. Place the glass beaker on a stirring plate and stir magnetically for 24 h at 400 min^-1 to soak the particles in water to promote particle softening.
4. Transfer the particles into a 1000 mL plastic container.
5. Fill an additional two 1000 mL plastic containers with DI water, which will be used to rinse off adhering particles on the grinder's hopper during the grinding process.

6. Preparation of the wet grinding machine for NP production

1. Place stones with a 46-grain size (grit of a grinding stone = 297-420 µm) in the wet friction grinder and fasten the center nuts hand-tight with a 17 mm wrench.
2. Add the hopper on top and fasten the three nuts and bolts with the 17 mm wrench.
3. Place a 1 L plastic collection jar under the outlet of the collider. Place a second empty 1 L bucket next to the outlet, which will be used for exchanging while processing.
4. Adjust the gauge clearance to + 1.0, corresponding to a positive 0.10 µm shift from the zero position.
5. Switch the power on and carefully turn the adjustment wheel clockwise until hearing the grinding stones touch. Then, adjust the flexible measurement ring to zero and turn the wheel counterclockwise immediately. By default, the speed is adjusted to 1500 min^-1.
   NOTE: Avoid "dry-grinding" of the stones as this creates excessive heat on the grinding stones.
6. Turn the adjustment wheel clockwise until the stones touch and gently fill the water-NP slurry into the hopper. Decrease the gap continually to a clearance gauge of -2.0, corresponding to a negative 0.20 µm shift from the zero position after the slurry was introduced. Plastic particle-water slurries between the two stone disks promote transformation from MPs into NPs and avoid direct friction between the grinding stones.
7. Collect the slurry by exchanging the collection buckets once the filling level in the bucket exceeds 0.5 L.
8. Collect and reintroduce the particles into the grinder between 30-60 times; higher passes (number of reintroductions) result in smaller particle sizes.
9. Wash adhering particles adsorbed to the hopper with the prepared DI water bottle to allow suitable slurry mixing while processing.
   ​NOTE: Collection of intermediate samples during the process is possible by holding 20 mL glass vials into the outlet stream. The individual steps will assess the particle fragmentation mechanisms while process severity (number of passes) increases. Recover the slurry and stir for 4 h at 400 min^-1 at 25 °C for good mixing; let the slurry stand for 48 h to stabilize.

7. Recovery and drying of NPs from the slurry

1. Isolate the bottom fraction (or phase with the highest NP concentration) if multiple layers in the slurry are observed by slowly pouring the slurry into an additional 1000 mL glass beaker.
2. Transfer the fractions into centrifugation vials (50 mL) and centrifuge for 10 min (relative centrifugal force [RCF] = 20 x 10² g). The RCF (also termed g-force) is the generated radial force as a function of the rotor radius and rotor speed, which causes separation of the heavier particles and the water of the slurry.
3. Remove the transparent top layer by decanting it into a separate aluminum pan.
4. Transfer the remaining bottom layer (containing an NP slurry) into an additional aluminum dish and place it in a vacuum oven at 30 °C for 48 h.
5. Recover dried material with a spatula under a fume hood or glove box while wearing a respiratory mask. Transfer the dried content into a 100 mL glass container and seal with a lid.
6. Contain NPs in a vial and store them in an airtight, dry, and cool place (e.g., a desiccator).
   NOTE: MNPs released into the environment during the manufacturing process (here, either during the wet-grinding process or as dried particles) may pose a severe threat to aquatic and terrestrial ecosystems. In particular, regulatory measures are designed to minimize the risk for their production and use for engineered nanomaterials³⁰. Therefore, the formation of MNPs requires specific precautional steps such as material handling in a fume hood or glove box. Furthermore, aqueous waste solutions formed during the isolation of NPs (steps 6.7-6.9) will be subject to an end-of-life disposal procedure performed by the Environmental Health and Safety Department.

8. MP imaging via stereo microscopy

1. Disperse ~20 mg of particles (collected in step 4) on a surface of area ~4 cm². Spread white or translucent MPs on a dark surface and spread black or dark-colored MPs on a white background (paper sheet) to maximize background contrast.
2. Adjust the microscope to the lowest magnification to capture the largest possible area (middle of the particle area). Next, direct the external lamp to the focus center to attain illumination on the regions of interest.
3. Apply a magnification that allows the detection of >50 particles in the middle of the field of view. This amount is recommended to obtain robust statistical evaluation results.
4. Focus on areas with no or minor particle overlap and good color contrast.
5. Capture at least five representative images by focusing on the outer particle shapes. The local computer used for imaging saves high-resolution images as a bitmap in the software.
6. Save the stereomicroscope recorded images in a file format recognized by ImageJ (bitmap, tiff, or jpeg) for the following quantitative data analysis.
   NOTE: Take one reference image at the exact magnification settings for which the main image was taken using a ruler or any other reference object recorded in the image. This procedure will allow easy calibration of the images when preparing and analyzing through ImageJ software.

9. Image analysis through ImageJ

1. Open ImageJ software³¹ and prepare file import by entering (CTRL + L) to open Command finder. Next enter Bio-Formats on the right lower corner. This function activates the menu path File > Import > Bio formats (> refers to navigation steps within the software). Search for the directory of stored image files.
   NOTE: If the Bio-Formats package does not appear in the Command finder, search online under Bio-Formats ImageJ. Follow instructions for downloading and installation of ImageJ. The Bio-Formats importer allows for simple handling of importing/exporting picture files within ImageJ and searching for commands.
2. Open the image (alternatively Bio-Formats import as described in step 9.1) by clicking File > Open > select particle image at the file location collected in step 4.7 and the ruler-reference image described in step 1.6. Creating a duplicate image is recommended by clicking Shift + Command + D to compare with the original image while adjusting the threshold settings of the copy image.
   NOTE: File > Open command opens various formats natively supported by ImageJ as described in step 8.7. Alternatively, select the image location on the computer and drag and drop the file on the main ImageJ window status bar. The image file will open automatically in a separate window.
3. Zoom in and out to the image using CTRL + and CTRL –, respectively.
4. Set the measurements by clicking Analyze > Set Measurements, then select Area and Shape Descriptors as the default values.
5. Define the scale bar by drawing a line straight over the length of the scale bar using the ruler reference image as described in step 8. Press Analyze > Set Scale, and enter the numerical value of the bar length under Known distance and the unit of the corresponding length.
6. Visualize the scale bar on the image by clicking Analyze > Tools > Scale Bar, and adjust settings such as showing crisp contrast on the image. Select a position on the image where the scale bar should be placed for scale bar settings. Select Width to adjust the bar in calibrated units, Height of the bar in pixels, and Font Size of the scale bar label. Select background to adjust the filling color of the label text box.
   NOTE: For micrometers, the entry of µm is sufficient; the program adapts µm automatically in the data output.
7. Transform the image into an 8-bit image by selecting Image > Type > 8-bit.
8. Convert the copied image to 8-bit by selecting Image > Type > 8-bit.
9. Adjust by selecting Image > Adjust > Threshold > Set (compare size to the original image).
10. Determine which measurements to take by selecting Analyze > Set Measurements.
11. Select Analyze particles > 0-infinity, click Display results, and in situ show.
12. Store the ROI (.zip) results under Save Measurements and Select Folder.
13. Save Results (*.csv) under File > Save as > Select Folder.

10. Particle diameter (d_p) and shape factor calculation in spreadsheet software

NOTE: Knowledge of particle diameter and shape factors are essential for particle behavior (fate, transport) in the environment and the determination of surface area. Therefore, geometry is essential when MPs are used for environmental studies. For example, different interaction mechanisms with soil were observed depending on MPs' sizes and shapes, such as MP-MP and MP-soil agglomerations, which influence particle movement in soil^15,32. Therefore, the following steps are suggested to determine the d_p-particle size distribution and geometrical parameter.

1. Import the corresponding *.csv file obtained and saved from ImageJ analysis (step 9.13) into the spreadsheet software.
   NOTE: The numerical values in each column line reflect individual calculations for each particle according to equation 1 and equation 2.
2. Evaluate the average shape parameter values such as circularity (CIR) and aspect ratio (AR) by entering = average (x,y) at the bottom of each column, where x represents the first line and y last line of the column, then press Enter. The CIR values describe the relationship between the projected area and the perfect circle with an individual particle's CIR = 1 (equation 1). The AR represents the particle length/width ratio described by equation 2.
3. Determine if the average AR < 2.5, then calculate d_p values in a new column using equation 3. If AR ≥ 2.5, then calculate d_p values reflecting equation 4. Add a new column to calculate d_p based on the area column received from the ImageJ output.
   NOTE: Selection of the AR threshold values ≥ 2.5 represents more rectangular-shaped particles, whereas AR < 2.5 reflects more round-shaped particles. This selection allows for minimizing the d_p calculation error derived from the area measured by microscopy and determined through ImageJ.
   (1)
   (2)
   (3)
   (4)

11. Statistical analysis for MPs and NPs

1. Open the *.csv data file with the statistical software by File > Open > Select file location of the corresponding file as created in step 9.13.
   NOTE: Alternatively, the table can be directly transferred through the copy-paste feature into the statistical software. Refer to the Table of Materials for the brand and version of the statistical software Edit > Paste with column names.
2. Evaluate the d_p data by selecting Analyze > Distribution.
3. Select d_p, which reflects the column's data, drag and drop into Y columns, and press the OK button. This feature creates a histogram with a statistical output including Summary Statistics, Mean, and Std Dev values in a separate window.
4. Evaluate if the histogram follows a normal distribution (or the best fit for d_p) with the best fit curve by selecting the triangle next to d_p > Continuous Fit, and then select the curve received as the best fit (for example, Fit Normal). This step superimposes the histogram with a normally distributed fit.
5. Determine and report the mean and standard deviation values from the summary statistics output of the average shape parameter values of circularity (Cir), aspect ratio (AR), roundness (Round), and solidity (Sol).
   NOTE: A statistical significance level of α = 0.05 is recommended and was employed for all evaluations. The significance level is the probability of rejecting the null hypothesis when it is true when comparing numerical results.

12. Best fit of d_p size distribution and particle shape factors

1. Load the data set into statistical software and use the same *.csv data set for the distribution of d_p as calculated in step 10.
2. Select Analyze > Reliability and Survival > Life Distribution.
3. Drag the d_p column to the Y, Time to Event field, and select OK. This feature creates an output with a probability plot as a function of d_p.
4. Determine the optimum distribution under Compare Distributions by checking Nonparametric, Lognormal, Weibull, Loglogistic, and Normal.
5. Evaluate the quality of the model fits by the lowest numerical values for the Akaike's and Bayesian information criteria (AIC and BIC, respectively) in the Statistics Model Comparison Table below the graph by the lowest BIC numbers. The best fit model is presented in the first row by default. Parametric or nonparametric estimate output fields for each distribution evaluation are located below the Compare Distributions graph.
6. Save the output script to the data table by selecting the red pull-down triangle on the upper left corner by Save Script > To Data Table. Next, save the original Data Table in the desired file location by selecting File > Save as > *.jmp.

13. Dimensional characterization of NPs through dynamic light scattering

1. Start the dynamic light scattering (DLS) software by double-clicking on the desktop icon. Select File > New > SOP. Add sample name and select material refractive index to 1.33 for distilled water and 1.59 for polymers³³ in the DLS software under Sample Setup. Select Material in the Pull-Down menu then click OK.
   NOTE: Clicking the pull-down menu opens the Materials Manager, which offers to add new samples or modify existing samples by changing the Refractive index and Absorption. Select as dispersant Water.
2. Select the proper cell under Cell > Cell Type and select Reports to determine which output will be presented after each measurement.
3. Start the instrument by closing the instrument lid and switching on the system by closing the lid (if open) and pressing the ON button. Wait after the first beep and wait around 30 min to allow stabilization of the beam.
4. Wait until the initialization routine is completed and wait for a second beep sound, indicating that the pre-set temperature (generally 25 °C) has been reached.
5. Prepare a sample slurry of NPs (as received in step 7) and DI water in a 15 mL vial at ~0.1 wt% concentration by magnetically stirring for ~1 h to allow to mix well.
6. Shake the slurry before transferring ~1.0 mL into the 4.5 mL quartz cuvette and open the lid. Then, carefully insert the sample cell into the sample holder of the DLS instrument.
   NOTE: Prepare three samples from the same slurry batch at the same concentration as described in step 13.5.
7. Perform three measurements (selection in the DLS software) for each sample. Between measurements, remove the sample cell and gently shake the samples for 5 s to allow mixing of the sample.
8. Extract and export data through the DLS software, transfer the dataset into the spreadsheet software, and create histograms for MPs and NPs as described in steps 11.1-11.5 (Figure 1). Copy from the Records View Tab either a Table or Graph by selecting Edit-copy, which can be pasted into another application such as the spreadsheet software.

14. Chemical analysis of MNPs using Fourier transformation infrared (FTIR) spectrometry-attenuated total reflectance (ATR)

NOTE: Chemical analyses of MNPs by Fourier transformation infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies are well-suited tools to assess the impact of wet grinding on chemical bonding properties, as well as the relative amounts of major components and the polymers' monomeric constituents, respectively¹⁰. In addition, thermal properties and the stability of MNPs' polymeric constituents can be assessed through differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), respectively²⁹.

1. Clean the detection system (ATR crystal surface) with ethanol and a lint-free cloth.
2. Start the software and press Background Button in the command bar to perform a Background Scan in the air by clearing the instrument beam path. The background spectrum is displayed shortly after collection.
3. Enter Sample ID and Sample Description in the instrument settings toolbar.
4. Adjust the spectral wavenumber between 4000 cm^-1 and 600 cm^-1 and select a resolution of 2.0 cm^-1 in absorbance mode. Select 32 scans per spectrum and start.
5. Place a plastic sample (~20 mg or ~ 1-3 mm³) of MPs (106 µm) and NPs (~300 nm) inside of a steel washer with an inner diameter of ~10 mm, or equivalent, on the crystal surface.
   NOTE: The washer prevents dispersion on crystal when the sample holder compresses the sample, resulting in material inhomogeneities and data bias due to inconsistent measurements.
6. Place the washer in the center of the ATR crystal and add the polymer sample into the middle of the washer opening with a spatula.
7. Swing the sample lever above into the center of the sample and turn the knob clockwise by monitoring the Force Gauge force between 50-90. The sample shows the preliminary spectra. Press the Scan button a second time to collect the spectrum.
8. Collect between 8-10 spectra by clicking the Scan button and mix the samples carefully after each measurement with a spatula to allow the collection of representative results.
9. Click on the Sample View folder in the Data Explorer to display all collected samples superimposed in the viewing area. First, remove significantly deviating spectra representing outliers. Next, select either Absorbance or Transmittance mode in the toolbar.
10. Save spectra by selecting the Sample View folder containing the spectra and selecting Save As from the file menu. The dialog window enables the file name, destination directory, and the default location change for all spectra.
    NOTE: Alternatively, the spectra can be saved as a *.sp file by selecting a spectrum and right-clicking to display the Binary option. Select Save Binary and browse the final Save location.
11. Perform baseline correction and mean normalization by selecting a single spectrum in the Data Explorer by selecting Process > Normalization in the menu either through the software or in the next step.
    NOTE: Mean normalization compensates for spectral errors due to the thickness or material variation in the sample.
12. Clean the crystal area with ethanol and lint-free cloth upon completion of the data collection.
13. Interpret differences between MPs and NPs according to assigned FTIR vibration bands, assigned and evaluated in a previous publication¹⁰.