Name: forming micro-and nano-plastics from agricultural plastic films for employment in fundamental research studies
Uploaded: 2022-07-27T14:15:00
Description: Watch this Scientific Journal Video about Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies at JoVE.com

This research was funded by the Herbert College of Agriculture, the Biosystems Engineering and Soil Department, and the Science Alliance at the University of Tennessee, Knoxville. Furthermore, the authors gratefully acknowledge the financial support provided through the USDA Grant 2020-67019-31167 for this research. The initial feedstocks for preparing MNPs of PBAT-based biodegradable mulch film were kindly provided by BioBag Americas, Inc. (Dunevin, FL, USA), and&#160;PBAT pellets by Mobius, LLC (Lenoir City, TN).

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.
<ol>
	<li>Weigh polymer pellet samples of ~1 g and transfer into a 50 mL glass jar.</li>
	<li>Place the &#34;rectangular delivery&#34; tube with a 20 mesh (840 &#181;m) sieve in the slot in front of the rotary cutting...

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...

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 ecosystems1,2,3. 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 methods4,5. However, the expanding employment of &#34;plasticulture&#34; has raised concerns about the formation, distribution, and retention of plastic pieces in agricultural environments. After a continuous fragmentation process caused by embrittlement through environmental degradation during service life, larger plastic fragments form micro- and nanoplastics (MNPs), which persist in soil or migrate to adjacent waterways via water runoff and wind6,7,8. Environmental factors such as ultraviolet (UV) radiation through sunlight, mechanical forces of water, and biological factors trigger plastic embrittlement of environmentally dispersed plastics, resulting in the breakdown of larger plastic fragments into macro- or meso-plastic particles9,10. Further defragmentation forms microplastics (MPs) and nanoplastics (NPs), reflecting particles of average size (nominal diameter; dp) of 1-5000 &#181;m and 1-1000 nm, respectively11. However, the upper dp limit for NPs (i.e., a lower limit for MPs) is not universally agreed upon and&#160;in several papers, this is listed as 100 nm12.
MNPs from plastic waste pose an emerging global threat to soil health and ecosystem services. Adsorption of heavy metals from freshwater by MPs led to an 800-fold higher concentration of heavy metals compared to the surrounding environment13. Furthermore, MPs in aquatic ecosystems pose multiple stressors and contaminants by altering light penetration, causing oxygen depletion, and causing adhesion to various biota, including penetration and accumulation in aquatic organisms14.
Recent studies suggest that MNPs can impact soil geochemistry and biota, including microbial communities and plants15,16,17. Furthermore, NPs threaten the food web17,18,19,20. Since MNPs readily undergo vertical and horizontal transport in soil, they can carry absorbed contaminants such as pesticides, plasticizers, and microorganisms through the soil into groundwater or aquatic ecosystems such as rivers and streams21,22,23,24. Conventional agricultural plastics such as mulch films are made from polyethylene, which must be removed from the field after usage and disposed of in landfills. However, incomplete removal leads to substantial plastic debris accumulation in soils9,25,26. Alternatively, soil-biodegradable plastic mulches (BDMs) are designed to be tilled into the soil after use, where they will degrade over time. However, BDMs persist temporarily in soil and gradually degrade and fragment into MPs and NPs9,27.
Many current environmental ecotoxicological and fate studies employ idealized and non-representative MPs and NPs model materials. The most commonly used surrogate MNPs are monodisperse polystyrene micro- or nanospheres, which do not reflect the actual MNPs residing in the environment12,28. Consequently, the selection of unrepresentative MPs and NPs may result in inaccurate measurements and results. Based on the lack of appropriate model &#924;NPs for terrestrial environmental studies, the authors were motivated to prepare such models from agricultural plastics. We previously reported on the formation of MNPs from BDMs and polyethylene pellets through mechanical milling and grinding of plastic pellets and film materials and the dimensional and molecular characteristics of MNPs29. The current paper provides a more detailed protocol for preparing MNPs that can be applied more broadly to all agricultural plastics, such as mulch films or their pelletized feedstocks (Figure 1). Here, to serve as an example, we chose a mulch film and spherical pellets of the biodegradable polymer polybutylene adipate terephthalate (PBAT) to represent agricultural plastics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum dish, 150 mL&#160;</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>08-732-103</td>
			<td>Drying of collected NPs</td>
		</tr>
		<tr>
			<td>Aluminum dish, 500 mL</td>
			<td>VWR International, Radnor, PA, USA</td>
			<td>25433-018</td>
			<td>Collecting NPs after wet-grinding</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>Centrific 228</td>
			<td>Container for centrifugation</td>
		</tr>
		<tr>
			<td>Delivery tube, #20, 840 &#181;m</td>
			<td>Thomas Scientific, Swedesboro, NJ, USA</td>
			<td>3383M30</td>
			<td>Sieving of the first fraction during milling</td>
		</tr>
		<tr>
			<td>Delivery tube, #60, 250 &#181;m</td>
			<td>Thomas Scientific, Swedesboro, NJ, USA</td>
			<td>3383M45</td>
			<td>Sieving of the second fraction (3x)&#160; during milling</td>
		</tr>
		<tr>
			<td>Thermomixer,&#160; 5350 Mixer</td>
			<td>Eppendorf North America, Enfield, CT, USA</td>
			<td>05-400-200</td>
			<td>Analysis of sieving experiments</td>
		</tr>
		<tr>
			<td>FT-IR Spectrum Two, spectrometer with attenuated total reflectance (ATR)</td>
			<td>Perkin Elmer, Waltham, MA, USA</td>
			<td>L1050228&#160;</td>
			<td>Measuring FTIR spectra</td>
		</tr>
		<tr>
			<td>Glass beaker, 1000 mL</td>
			<td>DWK Life Sciences, Milville, NJ, USA</td>
			<td>02-555-113</td>
			<td>Stirring of MPs-water slurry before grinding</td>
		</tr>
		<tr>
			<td>Glass front plate</td>
			<td>Thomas Scientific, Swedesboro, NJ, USA</td>
			<td>3383N55&#160;</td>
			<td>Front cover plaste for Wiley Mini Mill</td>
		</tr>
		<tr>
			<td>Glass jar, 50 mL</td>
			<td>Uline, Pleasant Prairie, WI, USA</td>
			<td>S-15846P</td>
			<td>Collective MPs after milling</td>
		</tr>
		<tr>
			<td>Glove Box, neoprene</td>
			<td>Bel-Art-SP Scienceware, Wayne, NJ, USA</td>
			<td>BEL-H500290000</td>
			<td>22-Inch, Size 10</td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS 90 size analyzer</td>
			<td>Malvern Panalytical, Worcestershire, UK&#160;</td>
			<td>Zetasizer Nano ZS</td>
			<td>Measuring nanoplastics dispersed in DI-water</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Nikon, Tokyo,&#160;108-6290, Japan</td>
			<td>Nikon Digital Sight 10</td>
			<td>Combined with Olympus microscope to receive digital images</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus, Shinjuku, Tokyo, Japan</td>
			<td>Model SZ 61</td>
			<td>Imaging of MPs</td>
		</tr>
		<tr>
			<td>Nitrogen jar, low form dewar flasks</td>
			<td>Cole-Palmer, Vernon Hills, IL, USA</td>
			<td>UX-03771-23</td>
			<td>Storage of liquid nitrogen during cryogenic cooling</td>
		</tr>
		<tr>
			<td>Accurate Blend 200, 12-speed blender</td>
			<td>Oster, Boca Raton, FL, USA</td>
			<td>6684</td>
			<td>Initiating the size reduction of cryogenically treated plastic film</td>
		</tr>
		<tr>
			<td>PBAT film, - BioAgri&#8482; (Mater-Bi&#174;)</td>
			<td>BioBag Americas, Inc, Dunedin, FL, USA</td>
			<td>0.7 mm thick</td>
			<td>Feedstock to form MPs and NPs, agricultural mulch film</td>
		</tr>
		<tr>
			<td>PBAT pellets</td>
			<td>Mobius, LLC, Lenoir City, TN, USA</td>
			<td>Diameter 3 mm</td>
			<td>Feedstock to form microplastics (MPs) and nanoplastics (NPs) trough milling and grinding</td>
		</tr>
		<tr>
			<td>Plastic centrifuge tubes, 50 mL</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>06-443-18</td>
			<td>Centrifugation of slurry after wet-grinding</td>
		</tr>
		<tr>
			<td>Plastic jar, 1000 mL, pre-cleaned, straight sided</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>05-719-733</td>
			<td>Collection of NPs during and after wet grinding</td>
		</tr>
		<tr>
			<td>Polygon stir bars, diameter&#248;=8 mm, length=50.8 mm&#160;&#160;</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>14-512-127</td>
			<td>Stirring of MPs slurry prior to wet-grinding</td>
		</tr>
		<tr>
			<td>Scissors, titanium bonded</td>
			<td>Westcott, Shelton, CT, USA</td>
			<td>13901</td>
			<td>Cutting of initial PBAT film feedstocks</td>
		</tr>
		<tr>
			<td>Square glass cell with square aperture and cap, 12 mm O.D.</td>
			<td>Malvern Panalytical, Worcestershire, UK&#160;</td>
			<td>PCS1115</td>
			<td>Measuring of NPs particle size</td>
		</tr>
		<tr>
			<td>Stainless steel bottom, 3 inch, pan</td>
			<td>Hogentogler &#38; Co. Inc, Columbia, MD, USA</td>
			<td>8401</td>
			<td>For sieving after Wiley-milling</td>
		</tr>
		<tr>
			<td>Stainless steel sieve, 3 inch, No. 140 (106 &#181;m)</td>
			<td>Hogentogler &#38; Co. Inc, Columbia, MD, USA</td>
			<td>1308</td>
			<td>For sieving after Wiley-milling</td>
		</tr>
		<tr>
			<td>Stainless steel sieve, 3 inch, No. 20 (850 &#181;m)</td>
			<td>Hogentogler &#38; Co. Inc, Columbia, MD, USA</td>
			<td>1296</td>
			<td>Sieving of MPs after Wiley-milling</td>
		</tr>
		<tr>
			<td>Stainless steel sieve, 3 inch, No. 325 (45 &#181;m)</td>
			<td>Hogentogler &#38; Co. Inc, Columbia, MD, USA</td>
			<td>1313</td>
			<td>Sieving of MPs after Wiley-milling</td>
		</tr>
		<tr>
			<td>Stainless steel sieve, 3 inch, No. 60 (250 &#181;m)</td>
			<td>Hogentogler &#38; Co. Inc, Columbia, MD, USA</td>
			<td>1303</td>
			<td>Sieving of MPs after Wiley-milling</td>
		</tr>
		<tr>
			<td>Stainless steel top cover, 3 inch</td>
			<td>Hogentogler &#38; Co. Inc, Columbia, MD, USA</td>
			<td>8406</td>
			<td>Sieving of MPs after Wiley-milling</td>
		</tr>
		<tr>
			<td>Stainless steel tweezers</td>
			<td>Global Industrial, Port Washington, NY, USA</td>
			<td>T9FB2264892</td>
			<td>Transferring of&#160; frozen film particles from jar into blender</td>
		</tr>
		<tr>
			<td>Vacuum oven, model 281A</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>13-262-50</td>
			<td>Vacuum oven to dry NPs after wet-grinding</td>
		</tr>
		<tr>
			<td>Friction grinding machine, Supermass Colloider</td>
			<td>Masuko Sangyo, Tokyo, Japan</td>
			<td>MKCA6-2J</td>
			<td>Grinding machine to form NPs from MPs</td>
		</tr>
		<tr>
			<td>Wet-grinding stone, grit size: 297 &#956;m -420 &#956;m</td>
			<td>Masuko Sangyo, Tokyo, Japan</td>
			<td>MKE6-46DD</td>
			<td>Grinding stone to form NPs from MPs</td>
		</tr>
		<tr>
			<td>Wiley Mini Mill, rotary cutting mill</td>
			<td>Thomas Scientific, Swedesboro, NJ, USA</td>
			<td>NC1346618</td>
			<td>Size reduction of pellets and film into MPs and NPs</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FTIR-Spectroscopy software</td>
			<td>Perkin Elmer, Waltham, MA, USA</td>
			<td>Spectrum 10&#160;</td>
			<td>Collection of spectra from the initial plastic, MPs and NPs</td>
		</tr>
		<tr>
			<td>Image J, image processing program</td>
			<td>National Institutes of Health, Bethesda, MD, USA</td>
			<td>Version 1.53n</td>
			<td>Analysis of digital images received from microscopy&#160;</td>
		</tr>
		<tr>
			<td>Microscope software, ds-fi1 software</td>
			<td>Malvern Panalytical , Malvern, UK</td>
			<td>Firmware DS-U1 Ver3.10</td>
			<td>Recording of digital images</td>
		</tr>
		<tr>
			<td>Microsoft, Windows, &#160;Excel 365, spreadsheet software</td>
			<td>Microsoft, Redmond, WA, USA</td>
			<td>Office 365</td>
			<td>Calculating the average particle size and creating FTIR spectra images</td>
		</tr>
		<tr>
			<td>JMP software, statistical software</td>
			<td>SAS Institute Inc., Cary, NC, 1989-2021</td>
			<td>Version 15</td>
			<td>Statistical analysis of particle size and perform best fit of data set</td>
		</tr>
		<tr>
			<td>Unscrambler software</td>
			<td>Camo Analytics, Oslo, Norway</td>
			<td>Version 9.2</td>
			<td>Normalizing and converting FTIR spectra into .csv fromat</td>
		</tr>
	</tbody>
</table>

forming micro-and nano-plastics from agricultural plastic films for employment in fundamental research studies

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 &#181;m, 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 &#181;m 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.

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 ...

Watch this Scientific Journal Video about Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies at JoVE.com

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

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.

Microplastics (MPs) and nanoplastics (NPs) dispersed in agricultural ecosystems can pose a severe threat to biota in soil and nearby waterways. In ...

Anton Astner:This protocol describes the preparation of engineered micro and nanoplastics sourced from a wide range of polymer feed stocks, such as pellets and films representing authentic model materials. This method efficiently forms micro nanoplastics through a simple mechanical procedure involving cryogenic milling, seething and wet grinding that can serve a surrogate materials for environmental studies. To begin retrieve foam from roll and cut P B A T foam into strips with a paper cutter pre soak fragments in deionized water for 10 minutes and transfer the foam material into a cryogenic container.Slowly add 200 milliliters of liquid nitrogen to a cryogenic container transfer pres soaked foam fragments or pellets carefully into the cryogenic container with steel tweezers. Next, transfer frozen foam fragments or pellets into a blender and process for 10 seconds. Add 400 milliliters of DI water and blend the foam water slurry for five minutes.Transfer slur into a buchner funnel with a filter and apply vacuum for one hour. Then transfer the sample with the funnel into a vacuum oven. Dry at 30 degrees Celsius for at least 48 hours.Weigh polymer film or pellet samples and transfer into a 50 milliliter glass jar. Place the rectangular delivery tube with a 20 mesh seed in the slot in front of the rotary cutting milk and raise the delivery tube until it hits the stock pin. Position the glass plate over the milling chambers face and secure it with the adjustable clamp.Next place a 50 milliliter glass jar under the mill outlet. Position the sliding side arm support on the mill slightly off center on the front glass and tighten with the neural bolt. Insert the hopper funnel on top of the mill into the opening of the upper milling chamber.Plug a line cord into a power outlet and press the cord switch to start the mill operation. Feed the sample slowly and add the next batch of film or pellet fragments after the audible noise reduces. After processing the film or pellet fragments press the cord switch to stop the mill operation for approximately 20 minutes to cool down.Clean the cutting chamber with a spatula and bristle brush And collect particles in the glass jar. Remove the 20 mesh 840 micrometer delivery tube and replace it with the 60 mesh delivery tube. Upon completion of the first batch reintroduce the collected material into the mill hopper.Follow the same procedure for the 60 mash milling fraction. Recover the remaining particles in the chamber and add them to the collected main fraction. Prepare a slurry of microplastics.Start by taking di water into a glass beaker and insert a stirring bar. Introduce eight grams of the collected 250 micrometer plastic fraction. Place the glass beaker on a stirring plate and stir magnetically for 24 hours at 400 rotations per minute.To soak the particles in water transfer the particles into a plastic container. Fill in additional two one liter plastic containers with di water, which will be used to rinse off endearing particles on the grinder's hopper. During the grinding process.Place stones with a 46 green size in the wet friction grinder and fasten the center nut. Hand tight with a 17 millimeter wrench add the hopper on top and fasten the four bolts with the five millimeter Allen wrench. Place a one liter plastic collection jar under the outlet of the collider.Place a second empty one liter bucket next to the outlet which will be used for exchanging while processing. Switch the power on and carefully adjust the gauge clearance by turning the adjustment wheel clockwise, corresponding to a positive 0.10 micrometer shift from the zero position until hearing the grinding stones touch. Next, adjust the flexible measurement ring to zero and turn the wheel counterclockwise immediately.By default, the speed is adjusted to 1500 revolutions per minute. Turn the adjustment wheel clockwise until the stones touch and gently fill the water nanoplastic slurry into the hopper. Decrease the gap continually to a clearance gauge of negative 2.0 corresponding to a negative 0.20 micrometer shift from the zero position after the slurry was introduced.Collect the slurry by exchanging the collection buckets. Once the filling level and the bucket exceeds 0.5 liters collect and reintroduce the particles into the grinder between 30 to to 60 times higher. Passes result in smaller particle size.Wash endearing particles on the hopper with the prepared DI water bottle to allow suitable slurry mixing while processing. Recover the slurry and stir for four hours at 400 rotations per minute at 25 degrees Celsius to allow it to mix well let the slurry stand for 48 hours to stabilize. The numerical analysis revealed the bimodal particle size distribution for nanoplastics produced from both feed stocks.The main particle populations for nanoplastics from P B A T pallets were at approximately 79 and 530 nanometers, and corresponding number density frequency values were at 25 and 5%respectively. On the other hand, nanoplastics derived from P B A T foams possessed size maxima at approximately 50 and 106 nanometers with corresponding number density frequency values of 11 and 10%respectively. FTIR R results for P B A T Nanoplastics indicate a distinct decrease in absorbance values between 980 and 1200 centimeter inverse reflecting the starch component leaching during wet grinding.Consistent with previous study observations Anton Astner:For microplastic formation from plastic film and pellets cryogenic pretreatment induces embrittlement to the plastics. It mimics the impact of environmental weathering but not perfectly, which allows accelerated defragmentation. The cryo milling procedure combined with the wet grinding process allows the processing of numerous differently plastic feed stocks to form authentical micro nanoplastics used in environmental studies studies.

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