Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies

Summary

We show the formation and dimensional characterization of micro- and nanoplastics (MPs and NPs, respectively) using a stepwise process of mechanical milling, grinding, and imaging analysis.

Abstract

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.

Introduction

In recent decades, the rapidly increasing global production of plastics and improper disposal and lack of recycling for plastic waste has led to environmental pollution that has impacted marine and terrestrial ecosystems^1,2,3. Plastic materials are essential for contemporary agriculture, particularly to cultivate vegetables, small fruit, and other specialty crops. Their usage as mulch films, high and low tunnel coverings, drip tape, and other applications aim to enhance crop yield and quality, lower production costs, and promote sustainable farming methods^4<....

Protocol

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 ....

Results

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 .......

Discussion

This method describes an effective process initially described in a previous publication²⁹, to prepare MNPs sourced from pellets and mulch films for environmental studies. The size reduction process involved cryogenic cooling (for film only), dry milling, and wet grinding stages, to manufacture model MNPs. We have applied this method to prepare MNPs from a wide range of polymeric feedstocks, including low-density polyethylene (LDPE), polybutyrate adipate-co-terephthalate (PBAT), and polylactic aci.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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