Name: sampling, identification and characterization of microplastics release from polypropylene baby feeding bottle during daily use
Uploaded: 2021-07-24T14:45:00
Description: Watch this Scientific Journal Video about Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use at JoVE.com

The authors appreciate the Enterprise Ireland (grant number CF20180870) and Science Foundation Ireland (grants numbers: 20/FIP/PL/8733, 12/RC/2278_P2 and 16/IA/4462) for financial support. We also acknowledge financial support from the School of Engineering Scholarship at Trinity College Dublin and China Scholarship Council (201506210089 and 201608300005). In addition, we appreciate the professional help from Prof. Sarah Mc Cormack and technician teams (David A. McAulay, Mary O&#39;Shea, Patrick L.K. Veale, Robert Fitzpatrick and Mark Gilligan etc.) of Trinity Civil, Structural and Environmental Department and AMBER Research Centre.

1. Hot water preparation
<ol>
	<li>For all hardware that comes into contact with the samples, use clean glass made of borosilicate 3.3 to prevent any potential contamination. Thoroughly clean all the glassware. 
	Caution: Pre-existing scratches or imperfection spots on glassware can release particles during the heating and shaking process. We suggest that users check the glassware and avoid the use of the scratched glassware. Our comparison of glassware made of different glasses (such as soda-lime...

<ol>
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Though the study of MPs in marine and freshwater has been widely reported and the relevant standard protocol has been developed17, the study of daily-use plastic products is an important emerging research area. The different environmental conditions experienced by household plastic products means that extra care and efforts are needed to obtain reliable results. The study protocol must be consistent with the real daily use scenarios. For example, sonication is widely used in lab-tests to clean sam...

Most types of plastics are non-biodegradable but can break down into small pieces due to chemical and physical processes such as oxidation and mechanical friction1,2. Plastic pieces smaller than 5 mm are classified as microplastics (MPs). MPs are ubiquitous and found in almost every corner in the world. They have become a global concern due to the potential risk to humans and wildlife3,4. To date, significant accumulations of MPs have been found in fish, birds, insects5,6 as well as mammals (mouse, in the gut, kidney and liver7,8). Studies found that the exposure and accumulation of MPs can damage the lipid metabolism of mice7,8. A risk assessment focusing on fish found that sub-micron MPs can penetrate the blood-to-brain barrier and cause brain damage9. It should be noted that to date all MP risk results have been obtained from animal studies while the specific risk to human health is still unknown.
In the last 2 years, concerns about the MP threat to human health substantially increased with the confirmation of the levels of human exposure to MPs. The accumulation of MPs has been found in the human colon10, the placenta of pregnant women11 and adult stool12. A precise determination of MP release levels is crucial to identify exposure sources, assess the health risk and evaluate the efficiency of any potential control measures. In the last few years, some case studies reported that daily-use plastics (i.e., the plastic kettle13 and single-use cups14) can release extremely high quantities of MPs. For example, disposable paper cups (with interiors laminated with polyethylene-PE or copolymer films), released approximately 250 micron-sized MPs and 102 million sub-micron-sized particles into each milliliter of liquid following exposure to 85-90 &#176;C&#160;hot water14. A study of polypropylene (PP) food containers reported that up to 7.6 mg of plastic particles is released from the container during a single use15. Even higher levels were recorded from teabags made from polyethylene terephthalate (PET) and nylon, which released approximately 11.6 billion MPs and 3.1 billion nano-sized MPs into a single cup (10 mL) of the beverage16. Given that these daily-use plastic products are designed for food and beverage preparation, the release of high quantities of MPs is likely and their consumption is a potential threat to human health.
Studies on MP release from household plastic products (i.e., the plastic kettle13 and single-use cups14) are at an early stage, but it is expected that this topic will receive increasing attention from researchers and the general public. The methods required in these studies are significantly different from those used in room temperature marine or freshwater studies where well-established protocols already exist17. In contrast, studies involving the daily-use of household plastic products involves much higher temperature (up to 100 &#176;C), with in many cases repeated cycling back to room temperature. Previous studies pointed out that plastics in contact with hot water can release millions of MPs16,18. In addition, the daily-use of plastic products may over time change the properties of the plastic itself. It is therefore crucial to develop a sampling protocol that accurately mimics the most common daily-use scenarios. The detection of micro-sized particles is another major challenge. Previous studies pointed out that MPs release from plastic products are smaller than 20 &#181;m16,19,20. Detection of these types of MPs requires the use of smooth membrane filters with small pore size. In addition, it is necessary to distinguish MPs from possible contaminants captured by the filter. High sensitivity Raman spectroscopy is used for chemical composition analysis, which has the advantage of avoiding the need for high laser power that is known to easily destroy small particles20. Hence, the protocol must combine contamination-free handling procedures with the use of optimal membrane filters and for a characterization method that allows fast and accurate MP identification.
The study reported here focused on the PP-based baby feeding bottle (BFB), one of the most commonly used plastic products in daily life. It was found that a high number of MPs are released from plastic BFB during formula preparation18. For further study of MP release from daily plastics, the sample preparation and detection method for BFB is detailed here. During sample preparation, the standard formula-preparation process (cleaning, sterilizing and mixing) recommended by the WHO21 was carefully followed. By designing the protocols around the WHO guidelines, we ensured that the MP release from BFBs mimicked the baby formula preparation process used by parents. The filter process was designed to accurately collect the MPs released from BFBs. For the chemical identification of MPs, the working conditions for Raman spectroscopy were optimized to obtain clean and easily identified spectra of MPs, while at the same time avoiding the possibility of burning the target particles. Finally, the optimum test procedure and applied force to allow accurate 3-dimensional topography mapping of MPs using atomic force microscopy (AFM) was developed. The protocol (Figure 1) detailed here provides a reliable and cost-effective method for MP sample preparation and detection, which can substantially benefit future studies of plastic products.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AFM cantilever</td>
			<td>NANOSENSORS</td>
			<td>PPP-NCSTAuD-10</td>
			<td>To obtain three-dimensional topography of PP MPs</td>
		</tr>
		<tr>
			<td>Atomic force microscope</td>
			<td>Nova</td>
			<td>NT-MDT</td>
			<td>To obtain three-dimensional topography of PP MPs</td>
		</tr>
		<tr>
			<td>Detergent</td>
			<td>Fairy Original</td>
			<td>1015054</td>
			<td>To clean the brand-new product</td>
		</tr>
		<tr>
			<td>Gold-coated polycarbonate-PC membrane filter-0.8 um</td>
			<td>APC, Germany</td>
			<td>0.8um25mmGold</td>
			<td>To collect microplastics in water and benefit for Raman test</td>
		</tr>
		<tr>
			<td>Gwyddion software</td>
			<td>Gwyddion</td>
			<td>Gwyddion2.54</td>
			<td>To determine MPs topography</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>US National Institutes of Health</td>
			<td>No, free for use</td>
			<td>To determine MPs size</td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>De&#39;longhi, Italy</td>
			<td>815/1195</td>
			<td>Hot water preparation</td>
		</tr>
		<tr>
			<td>Optical microscope, x100</td>
			<td>Mitutoyo, Japan</td>
			<td>46-147</td>
			<td>To find and observe the small MPs</td>
		</tr>
		<tr>
			<td>Raman spectroscopy</td>
			<td>Renishaw</td>
			<td>InVia confocal Raman system</td>
			<td>To checmically determine the PP-MPs</td>
		</tr>
		<tr>
			<td>Shaking bed-SSL2</td>
			<td>Stuart, UK</td>
			<td>51900-64</td>
			<td>To mimic the mixing process during sample preparaton</td>
		</tr>
		<tr>
			<td>Standard polystyrene microplastic spheres</td>
			<td>Polysciences, Europe</td>
			<td>64050-15</td>
			<td>To validate the robusty of current protocol</td>
		</tr>
		<tr>
			<td>Tansfer pipette with glass tip</td>
			<td>Macro, Brand</td>
			<td>26200</td>
			<td>To transfer water sample to glass filter</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Witeg, Germany</td>
			<td>DH.WUC.D06H</td>
			<td>To clean the glassware</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>ILMVAC GmbH</td>
			<td>105697</td>
			<td>To filter the water sample</td>
		</tr>
	</tbody>
</table>

sampling, identification and characterization of microplastics release from polypropylene baby feeding bottle during daily use

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use cups and kettles) indicate that MP release during daily use can be extremely high. Precisely determining the MP release level is a crucial step to identify and quantify the exposure source and assess/control the corresponding risks stemming from this exposure. Though protocols for measuring MP levels in marine or freshwater has been well developed, the conditions experienced by household plastic products can vary widely. Many plastic products are exposed to frequent high temperatures (up to 100 &#176;C) and are cooled back to room temperature during daily use. It is therefore crucial to develop a sampling protocol that mimics the actual daily-use scenario for each particular product. This study focused on widely used polypropylene-based baby feeding bottles to develop a cost-effective protocol for MP release studies of many plastic products. The protocol developed here enables: 1) prevention of the potential contamination during sampling and detection; 2) realistic implementation of daily-use scenarios and accurate collection of the MPs released from baby feeding bottles based on WHO guidelines; and 3) cost-effective chemical determination and physical topography mapping of MPs released from baby feeding bottles. Based on this protocol, the recovery percentage using standard polystyrene MP (diameter of 2 &#181;m) was 92.4-101.2% while the detected size was around 102.2% of the designed size. The protocol detailed here provides a reliable and cost-effective method for MP sample preparation and detection, which can substantially benefit future studies of MP release from plastic products.

To validate this protocol, the water sample was prepared by adding standard polystyrene microplastic spheres (a diameter of 2.0 &#177; 0.1 &#181;m) to DI water. The MP quantity added corresponded to 4,500,000 particles/L, which is similar to the MP release level from BFBs. Following protocol sections 2-3, the MPs were successfully collected (Figure 4A) and the recovery rate was 92.4-101.2%. This recovery rate is comparable to a previous study on MPs23. Using ImageJ, t...

Watch this Scientific Journal Video about Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use at JoVE.com

Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use

This study detailed a reliable and cost-effective protocol for microplastics collection and detection from the daily use of plastic products.

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use ...

Microplastics are becoming a global concern due to the potential risk to human health. This study focused on widely used polypropylene-based baby feeding bottles to develop a cost-effective protocol suitable for an MP study of many plastic products. This protocol provides the details of sample preparation, identification, and characterization.It can substantially benefit future studies of microplastics released from plastic products. Microplastics released during formula preparation. Soak the baby feeding bottle in 95 degrees Celsius deionized water to sterilize the bottle.To avoid the bottle floating, slightly press it using a stainless steel tweezer to ensure the whole bottle body is immersed in the water. After five minutes, take the bottle out and move it to a clean glass disk to await for air-drying. After air-drying, pour 180 milliliters of 70 degrees Celsius deionized water into the bottle.Then cover the bottle immediately using a glass Petri dish and place it onto a shaking bed. To stimulate the formula mixing process, shake the bottle at a speed of 180 rpm for 60 seconds. After shaking, move the bottle to a clean glass plate and allow it to cool down.Sample preparation for microplastics identification and quantification. Membrane filter is critical for microplastics capture, visualization, and detection. We used the gold coated polycarbonate membrane filter to capture microplastics.Place a piece of membrane filter in the middle of a glass base. Then assemble the glass funnel and stainless steel clamp to fix the membrane filter and connect the glass filter with a vacuum pump after gently shaking the cooled water sample in the baby-feeding bottle. Then transfer a certain amount of water sample to the glass funnel using a glass pipette.Switch on the vacuum pump to allow the water sample to filter through the membrane filter slowly. After filtering, wash the interior of the glass funnel using deionized water to ensure there are no particles sticking to the funnel. Disconnect the vacuum pump and disassemble the glass filter.Then, carefully take out the membrane filter using a stainless steel tweezer and move it to a clean cover glass. Store the sample in a clean glass Petri dish immediately. Microplastics identification and quantification.Carefully take out the sample from the glass Petri dish and place the filter sample in the middle of the Raman sample stage. Choose the representative spots in the membrane filter. Observe and photograph the particles on the surface of the membrane filter using an optical microscope.Set up the Raman system and test these particles one by one using 532 nanometer excitation laser at 10%intensity. Compare the Raman spectrum obtained to referenced standard spectra. The AFM and Raman spectroscopy are assembled in one system.Hence switch the system to AFM mode and test the topography of interesting microplastic particles. Microplastics test results. To validate this protocol, the standard polystyrene microplastics spheres were added to DI water and tested using the developed protocol.The PS microplastics were successfully collected and detected. The particle inside the red box was confirmed as a typical polystyrene microplastic. Following the protocol, the microplastics recovery rate was 92 to 101%Hence, the developed protocol is reliable for the baby-feeding bottle test.The protocol was used to test microplastics released from eight popular baby feeding bottle products, and found the microplastics released levels ranged from 1.3 million to 16.2 million particles per liter during formula preparation. The 3D topographic image further showed that the surface texture of the released microplastics is rich with nano sized bumps and valleys, which can substantially increase their absorption capacity.Conclusion. The study showed that plastic products that are used daily are very important sources for human microplastic exposure.The protocol detailed here provides a reliable and cost-effective method for microplastics sample preparation and detection, which can substantially benefit future studies of microplastic release from plastic products.

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Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...