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.
2. Processing of plastic films by cryogenic pretreatment and milling
3. Processing of plastic films pretreated through environmentally weathering and milling
4. Sieving procedure through cascaded sieves
5. Preparation of an aqueous NP slurry for wet grinding
6. Preparation of the wet grinding machine for NP production
7. Recovery and drying of NPs from the slurry
8. MP imaging via stereo microscopy
9. Image analysis through ImageJ
10. Particle diameter (dp) 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 soil15,32. Therefore, the following steps are suggested to determine the dp-particle size distribution and geometrical parameter.
11. Statistical analysis for MPs and NPs
12. Best fit of dp size distribution and particle shape factors
13. Dimensional characterization of NPs through dynamic light scattering
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.
To validate the experimental procedure method and analysis, MPs and NPs were formed from pellets and film materials and compared by size and shape using microscopic images. The method described in Figure 1 efficiently formed MPs and NPs from biodegradable plastic pellets and films; this was achieved through cryogenic cooling, milling, and wet-grinding and characterization. The former step was unnecessary for environmentally weathered films because weathering induced embrittlement (Astner et al., unpublished). Pellets were also directly subjected to milling without cryogenic pretreatment. After milling, particles were fractionated through sieving into four size fractions: 840 µm, 250 µm, 106 µm, and 45 µm, as described in protocol step 4. The latter three fractions consisted solely of MPs. Subsequently, particle characterization for each fraction was assessed by determining the distribution of size (dp) and shape factors (i.e., circularity and aspect ratio) of collected stereomicroscopic images using ImageJ software as given in steps 8.1-8.6. Examples of images obtained by a stereomicroscope are shown for the 106 µm sieving fraction for PBAT pellets (Figure 3a,c) and the 250 µm sieve fraction, and for unweathered PBAT film treated with cryogenic exposure (Figure 3b,d).
Statistical analysis of particle dimensions indicated an average dp that was 41 µm smaller than the nominal sieve size (106 µm) for the PBAT pellet, and 137 µm smaller for the PBAT film (250 µm nominal size), suggesting that the smaller sieve fraction represents a more homogenous particle size distribution (Table 1). This observation was also confirmed by a larger value in circularity and lower aspect ratios (suggesting more round-shaped particles) for the processed pellets compared to the film material, which may be attributed to the different properties (density) of the starting materials. A normal distribution was the best model for describing the particle size distribution for both fractions. However, for determining circularity and aspect ratio, the Weibull and Lognormal models were optimal (Figure 4a–d; Table 1). For both feedstocks, a wet grinding process applied to the 106 µm MP sieve fractions formed NPs, and their particle size distribution was measured via DLS. Numerical analysis revealed a bimodal particle size distribution for NPs produced from both feedstocks (Figure 5). The main particle populations for NPs from PBAT pellets were at ~80 nm and 531 nm, and corresponding number density frequency (NDF) values were at 25% and 5%, respectively. On the other hand, NPs derived from PBAT films possessed size maxima at ~50 nm and 106 nm, with corresponding NDF values of 11% and 10%, respectively. The observations suggest that NPs from PBAT pellets yielded more uniform dp values (~50-110 nm) than PBAT films; however, a particle subpopulation between 300 nm and 700 nm, with a maximum at 531 nm, also coexisted (Figure 5).
The chemical bonding properties of the PBAT film were evaluated by FITR spectroscopy. Spectra showed only minor changes due to milling for MPs and wet-grinding for NPs in the regions between 1300 and 700 cm-1. However, a significant decrease in the C-O stretching of starches, reflecting the absorbance of the starch component10, was observed for the mulch film. However, minor changes were observed for the bands representing PBAT, such as C-H and C-O stretching, between 1800 cm-1 and 1230 cm-1, suggesting insignificant changes in structure for the polyester attributed to the wet grinding process (Figure 6).
Figure 1: Flow diagram to form and characterize micro- and nanoplastics. The representation shows the formation process and the subsequent geometrical and chemical particle evaluation. Geometrical properties were determined by combining stereomicroscopy and image analysis (ImageJ), followed by a numerical statistical analysis. Chemical characterization such as molecular bonding was conducted through Fourier transformation infrared spectrometry using attenuated total reflectance (FTIR-ATR). The molecular structure of polymers can be assessed by nuclear magnetic resonance (NMR) spectroscopy as a complementary method (not described in this study). For each step, key points are highlighted in the following procedure. Please click here to view a larger version of this figure.
Figure 2: Rotary cutting mill apparatus. Images of (a) the rotary mill assembly including the feeding hopper, front glass plate, and sieve slot; (b) individual delivery tubes with sieve sizes #20 (840 µm) and #60 (250 µm) are fitted into the mill sieve slot starting with the coarser; and (c) double-layer glass front plate is attached to the front of the grinding chamber. Please click here to view a larger version of this figure.
Figure 3: Stereomicrographs of microplastics (MPs), including software processed images. The images were of MPs derived from (a) PBAT pellets (106 µm sieve fraction) and (b) PBAT film (250 µm sieve fraction) prepared through cryogenic exposure followed by mechanical milling. A black background was selected for imaging white PBAT particles (a), and a white background was selected for a black PBAT film (b). Corresponding images were processed by ImageJ software31 (c) and (d), respectively. A best-fit model of the distribution of dp, depicted in histograms of particles derived from stereographs of (e) PBAT pellets and (f) PBAT film is represented by a normal distribution. Error bars reflect one standard deviation. A stereomicroscope collected stereomicrographs with an integrated camera head. Please click here to view a larger version of this figure.
Figure 4: Particle shape factor distribution histograms with superimposed best curve fitting. The image represents MPs: (a) circularity and (c) aspect ratio for PBAT pellets and (b) circularity and (d) aspect ratio for PBAT film, based on ImageJ analysis31. Stereomicrographs are based on two sieve fractions particles of PBAT pellets (106 µm) and PBAT BDM MPs (250 µm). Numerical analysis was performed in the statistical software, V 15. Stereographs and histograms represent the corresponding images. Please click here to view a larger version of this figure.
Figure 5: Histograms of particle size (dp) for NPs. The figure represents particle distributions derived from PBAT film and PBAT pellets formed from the wet-grinding treatment of the 106 µm MP sieve fraction. Curves represent two-parameter Weibull model fits to size distribution, conducted using the statistical software. Data measurements were performed using dynamic light scattering. Please click here to view a larger version of this figure.
Figure 6. Representative FTIR spectra of MNP comparison among different processing steps. The figure depicts the comparison among the initial conditions of the PBAT film, PBAT-MPs, and PBAT-NPs. The PBAT film was cryogenically treated prior to mechanical milling of MPs consisting of the 106 µm sieve fraction of dry milled plastics; NPs were produced via wet grinding of the 106 µm sieve fraction MPs after dry milling and sieving. Spectral data was collected using a spectrometer fitted with a diamond attenuated total reflectance (ATR) attachment. Spectral data analysis was performed using FTIR spectrum analysis software. Please click here to view a larger version of this figure.
PBAT pellets | PBAT film | |
Sieve fraction, μm | 106 | 250 |
Normal dp, μm | 65 | 113 |
Std Dev, μm | 24 | 58 |
Circularity | 0.68 | 0.47 |
Aspect Ratio | 1.73 | 2.33 |
Best fit, dp | Normal | Normal |
Best fit, Circularity | Weibull | Weibull |
Best fit, Aspect Ratio | Lognormal | Lognormal |
N | 83 | 125 |
Table 1: Representative particle size and shape parameters. Results were derived from statistical analysis for MPs processed from PBAT pellets and PBAT film depicted in Figure 3 and Figure 4.
Aluminum dish, 150 mL | Fisher Scientific, Waltham, MA, USA | 08-732-103 | Drying of collected NPs |
Aluminum dish, 500 mL | VWR International, Radnor, PA, USA | 25433-018 | Collecting NPs after wet-grinding |
Centrifuge | Fisher Scientific, Waltham, MA, USA | Centrific 228 | Container for centrifugation |
Delivery tube, #20, 840 µm | Thomas Scientific, Swedesboro, NJ, USA | 3383M30 | Sieving of the first fraction during milling |
Delivery tube, #60, 250 µm | Thomas Scientific, Swedesboro, NJ, USA | 3383M45 | Sieving of the second fraction (3x) during milling |
Thermomixer, 5350 Mixer | Eppendorf North America, Enfield, CT, USA | 05-400-200 | Analysis of sieving experiments |
FT-IR Spectrum Two, spectrometer with attenuated total reflectance (ATR) | Perkin Elmer, Waltham, MA, USA | L1050228 | Measuring FTIR spectra |
Glass beaker, 1000 mL | DWK Life Sciences, Milville, NJ, USA | 02-555-113 | Stirring of MPs-water slurry before grinding |
Glass front plate | Thomas Scientific, Swedesboro, NJ, USA | 3383N55 | Front cover plaste for Wiley Mini Mill |
Glass jar, 50 mL | Uline, Pleasant Prairie, WI, USA | S-15846P | Collective MPs after milling |
Glove Box, neoprene | Bel-Art-SP Scienceware, Wayne, NJ, USA | BEL-H500290000 | 22-Inch, Size 10 |
Zetasizer Nano ZS 90 size analyzer | Malvern Panalytical, Worcestershire, UK | Zetasizer Nano ZS | Measuring nanoplastics dispersed in DI-water |
Microscope camera | Nikon, Tokyo, 108-6290, Japan | Nikon Digital Sight 10 | Combined with Olympus microscope to receive digital images |
Microscope | Olympus, Shinjuku, Tokyo, Japan | Model SZ 61 | Imaging of MPs |
Nitrogen jar, low form dewar flasks | Cole-Palmer, Vernon Hills, IL, USA | UX-03771-23 | Storage of liquid nitrogen during cryogenic cooling |
Accurate Blend 200, 12-speed blender | Oster, Boca Raton, FL, USA | 6684 | Initiating the size reduction of cryogenically treated plastic film |
PBAT film, – BioAgri™ (Mater-Bi®) | BioBag Americas, Inc, Dunedin, FL, USA | 0.7 mm thick | Feedstock to form MPs and NPs, agricultural mulch film |
PBAT pellets | Mobius, LLC, Lenoir City, TN, USA | Diameter 3 mm | Feedstock to form microplastics (MPs) and nanoplastics (NPs) trough milling and grinding |
Plastic centrifuge tubes, 50 mL | Fisher Scientific, Waltham, MA, USA | 06-443-18 | Centrifugation of slurry after wet-grinding |
Plastic jar, 1000 mL, pre-cleaned, straight sided | Fisher Scientific, Waltham, MA, USA | 05-719-733 | Collection of NPs during and after wet grinding |
Polygon stir bars, diameterø=8 mm, length=50.8 mm | Fisher Scientific, Waltham, MA, USA | 14-512-127 | Stirring of MPs slurry prior to wet-grinding |
Scissors, titanium bonded | Westcott, Shelton, CT, USA | 13901 | Cutting of initial PBAT film feedstocks |
Square glass cell with square aperture and cap, 12 mm O.D. | Malvern Panalytical, Worcestershire, UK | PCS1115 | Measuring of NPs particle size |
Stainless steel bottom, 3 inch, pan | Hogentogler & Co. Inc, Columbia, MD, USA | 8401 | For sieving after Wiley-milling |
Stainless steel sieve, 3 inch, No. 140 (106 µm) | Hogentogler & Co. Inc, Columbia, MD, USA | 1308 | For sieving after Wiley-milling |
Stainless steel sieve, 3 inch, No. 20 (850 µm) | Hogentogler & Co. Inc, Columbia, MD, USA | 1296 | Sieving of MPs after Wiley-milling |
Stainless steel sieve, 3 inch, No. 325 (45 µm) | Hogentogler & Co. Inc, Columbia, MD, USA | 1313 | Sieving of MPs after Wiley-milling |
Stainless steel sieve, 3 inch, No. 60 (250 µm) | Hogentogler & Co. Inc, Columbia, MD, USA | 1303 | Sieving of MPs after Wiley-milling |
Stainless steel top cover, 3 inch | Hogentogler & Co. Inc, Columbia, MD, USA | 8406 | Sieving of MPs after Wiley-milling |
Stainless steel tweezers | Global Industrial, Port Washington, NY, USA | T9FB2264892 | Transferring of frozen film particles from jar into blender |
Vacuum oven, model 281A | Fisher Scientific, Waltham, MA, USA | 13-262-50 | Vacuum oven to dry NPs after wet-grinding |
Friction grinding machine, Supermass Colloider | Masuko Sangyo, Tokyo, Japan | MKCA6-2J | Grinding machine to form NPs from MPs |
Wet-grinding stone, grit size: 297 μm -420 μm | Masuko Sangyo, Tokyo, Japan | MKE6-46DD | Grinding stone to form NPs from MPs |
Wiley Mini Mill, rotary cutting mill | Thomas Scientific, Swedesboro, NJ, USA | NC1346618 | Size reduction of pellets and film into MPs and NPs |
Software | |||
FTIR-Spectroscopy software | Perkin Elmer, Waltham, MA, USA | Spectrum 10 | Collection of spectra from the initial plastic, MPs and NPs |
Image J, image processing program | National Institutes of Health, Bethesda, MD, USA | Version 1.53n | Analysis of digital images received from microscopy |
Microscope software, ds-fi1 software | Malvern Panalytical , Malvern, UK | Firmware DS-U1 Ver3.10 | Recording of digital images |
Microsoft, Windows, Excel 365, spreadsheet software | Microsoft, Redmond, WA, USA | Office 365 | Calculating the average particle size and creating FTIR spectra images |
JMP software, statistical software | SAS Institute Inc., Cary, NC, 1989-2021 | Version 15 | Statistical analysis of particle size and perform best fit of data set |
Unscrambler software | Camo Analytics, Oslo, Norway | Version 9.2 | Normalizing and converting FTIR spectra into .csv fromat |
Microplastics (MPs) and nanoplastics (NPs) dispersed in agricultural ecosystems can pose a severe threat to biota in soil and nearby waterways. In addition, chemicals such as pesticides adsorbed by NPs can harm soil organisms and potentially enter the food chain. In this context, agriculturally utilized plastics such as plastic mulch films contribute significantly to plastic pollution in agricultural ecosystems. However, most fundamental studies of fate and ecotoxicity employ idealized and poorly representative MP materials, such as polystyrene microspheres.
Therefore, as described herein, we developed a lab-scale multi-step procedure to mechanically form representative MPs and NPs for such studies. The plastic material was prepared from commercially available plastic mulch films of polybutyrate adipate-co-terephthalate (PBAT) that were embrittled through either cryogenic treatment (CRYO) or environmental weathering (W), and from untreated PBAT pellets. The plastic materials were then treated by mechanical milling to form MPs with a size of 46-840 µm, mimicking the abrasion of plastic fragments by wind and mechanical machinery. The MPs were then sieved into several size fractions to enable further analysis. Finally, the 106 µm sieve fraction was subjected to wet grinding to generate NPs of 20-900 nm, a process that mimics the slow size reduction process for terrestrial MPs. The dimensions and the shape for MPs were determined through image analysis of stereomicrographs, and dynamic light scattering (DLS) was employed to assess particle size for NPs. MPs and NPs formed through this process possessed irregular shapes, which is in line with the geometric properties of MPs recovered from agricultural fields. Overall, this size reduction method proved efficient for forming MPs and NPs composed of biodegradable plastics such as polybutylene adipate-co-terephthalate (PBAT), representing mulch materials used for agricultural specialty crop production.
Microplastics (MPs) and nanoplastics (NPs) dispersed in agricultural ecosystems can pose a severe threat to biota in soil and nearby waterways. In addition, chemicals such as pesticides adsorbed by NPs can harm soil organisms and potentially enter the food chain. In this context, agriculturally utilized plastics such as plastic mulch films contribute significantly to plastic pollution in agricultural ecosystems. However, most fundamental studies of fate and ecotoxicity employ idealized and poorly representative MP materials, such as polystyrene microspheres.
Therefore, as described herein, we developed a lab-scale multi-step procedure to mechanically form representative MPs and NPs for such studies. The plastic material was prepared from commercially available plastic mulch films of polybutyrate adipate-co-terephthalate (PBAT) that were embrittled through either cryogenic treatment (CRYO) or environmental weathering (W), and from untreated PBAT pellets. The plastic materials were then treated by mechanical milling to form MPs with a size of 46-840 µm, mimicking the abrasion of plastic fragments by wind and mechanical machinery. The MPs were then sieved into several size fractions to enable further analysis. Finally, the 106 µm sieve fraction was subjected to wet grinding to generate NPs of 20-900 nm, a process that mimics the slow size reduction process for terrestrial MPs. The dimensions and the shape for MPs were determined through image analysis of stereomicrographs, and dynamic light scattering (DLS) was employed to assess particle size for NPs. MPs and NPs formed through this process possessed irregular shapes, which is in line with the geometric properties of MPs recovered from agricultural fields. Overall, this size reduction method proved efficient for forming MPs and NPs composed of biodegradable plastics such as polybutylene adipate-co-terephthalate (PBAT), representing mulch materials used for agricultural specialty crop production.
Microplastics (MPs) and nanoplastics (NPs) dispersed in agricultural ecosystems can pose a severe threat to biota in soil and nearby waterways. In addition, chemicals such as pesticides adsorbed by NPs can harm soil organisms and potentially enter the food chain. In this context, agriculturally utilized plastics such as plastic mulch films contribute significantly to plastic pollution in agricultural ecosystems. However, most fundamental studies of fate and ecotoxicity employ idealized and poorly representative MP materials, such as polystyrene microspheres.
Therefore, as described herein, we developed a lab-scale multi-step procedure to mechanically form representative MPs and NPs for such studies. The plastic material was prepared from commercially available plastic mulch films of polybutyrate adipate-co-terephthalate (PBAT) that were embrittled through either cryogenic treatment (CRYO) or environmental weathering (W), and from untreated PBAT pellets. The plastic materials were then treated by mechanical milling to form MPs with a size of 46-840 µm, mimicking the abrasion of plastic fragments by wind and mechanical machinery. The MPs were then sieved into several size fractions to enable further analysis. Finally, the 106 µm sieve fraction was subjected to wet grinding to generate NPs of 20-900 nm, a process that mimics the slow size reduction process for terrestrial MPs. The dimensions and the shape for MPs were determined through image analysis of stereomicrographs, and dynamic light scattering (DLS) was employed to assess particle size for NPs. MPs and NPs formed through this process possessed irregular shapes, which is in line with the geometric properties of MPs recovered from agricultural fields. Overall, this size reduction method proved efficient for forming MPs and NPs composed of biodegradable plastics such as polybutylene adipate-co-terephthalate (PBAT), representing mulch materials used for agricultural specialty crop production.