Release Characteristics of Small-Sized Microplastics in Bottled Drinks Using Flow Cytometry Sorting and Nile Red Staining

Abstract

Small-sized microplastics (MPs) pose concerns about potential risks to both the environment and human health. However, research on MP pollution is hampered by limitations in the detection techniques. Also, few studies have provided insight into the release of small-sized MPs from disposable polyethylene terephthalate (PET)-bottled drinks for outdoor usage. Thus, PET bottles’ potential to release small MPs sized 1–100 μm outdoors was studied in relation to physical and chemical parameters (temperature, sunlight irradiation, and drink characteristics) using flow cytometry and Nile Red dye. The results showed that temperatures below 80 °C had little effect on the release of MPs from PET bottles. Sunlight irradiation and alkalinity were prone to promote the generation of MPs, mostly 1–5 μm in size. Moreover, the combined impact test implied that two pairings—acidity with temperature and alkalinity with sunlight—positively affected MP release, with maximum releases of 21,622 ± 2477 particles/L and 31,081 ± 7173 particles/L, respectively. Based on the rapid quantification of small-sized MPs using flow cytometry after Nile Red selection, the results hereby presented will assist researchers in reducing MP release and aid them in the evaluation of MPs’ contamination of aquatic environments.

1. Introduction

Microplastics (MPs; <5 mm in diameter [1]) have been a troubling, emerging contaminant ever since the enormous rise in the consumption of plastic-based goods worldwide and the mismanagement of plastic waste [2]. Particle size, as a key characteristic of MPs, has a significant impact on their ability to adsorb harmful contaminants [3], promote biofilm formation [4], and be ingested by or entangle living organisms [5,6]. Based on previous studies, MP particles less than 100 μm in diameter, especially those sized 1–10 μm, have a higher uptake rate in cells or human tissues [7], including the brain, the lungs, the gastrointestinal tract, the placenta, the blood, and the sperm [8,9,10,11,12]. These small-sized MPs may be eliminated in human feces or accumulate in the body [13,14]. This causes cytotoxicity and systemic toxic effects, including oxidative stress and inflammation, eventually leading to cancer, allergies, and other diseases [15,16]. The size distribution of MPs has been often shown to follow a power law with a negative exponent, implying that numerical concentrations increase dramatically with smaller sizes [17]. Therefore, the environmental and health risks that small-sized MPs (1–100 μm) pose must be addressed.

The research on small-sized MPs is hampered by the limitations of the detection methods, especially for MPs in the size range of 1–5 μm. Some researchers have primarily investigated MPs with sizes of 10 μm and above. For instance, H. Li et al. [18] utilized laser direct infrared spectroscopy (LD-IR) to detect MPs (≥10 μm) in 10 commercially available bottled waters, with 10–50 μm MPs accounting for approximately 68% of all detected particles. While Oßmann et al. [19] successfully detected small-sized MPs measuring 1.5 μm in mineral water bottles using micro-Raman spectroscopy in as early as 2018, the presence of fluorescent background interference and the destructive nature of lasers significantly increase the detection difficulty, leading to longer detection times and higher costs [20]. Flow cytometry, commonly used for microbial cell counting, has gradually been applied for the quantification of small-sized MPs and nano-plastics (NPs) due to its simplicity, rapid counting, and good recognition performance [21,22,23]. Morgana et al. [24] successfully applied flow cytometry for the detection of MPs and NPs in mask leachates, achieving a detection limit as low as 0.1 μm. It is undeniable that flow cytometry holds broad potential for the detection of small-sized MPs and NPs.

Bottled drinks, which utilize approximately 80% polyethylene terephthalate (PET) as packaging [25], are favored by consumers engaging in outdoor sports. The significant issue is that, during outdoor transportation, storage, and use, PET bottles may be exposed to high temperatures and prolonged sunlight irradiation, leading to plastic degradation and the release of MPs into the drinks [26,27]. Previous studies have focused on the release of MPs from the plastic packaging of food, other than PET-bottled drinks, with particular attention given to disposable cups, tea bags, feeding bottles, and takeout containers [28,29]. For example, polypropylene (PP) feeding bottles and plastic baby teats release significant amounts of MPs and NPs due to heat and wear when feeding, leading to early-life human exposure to MPs [30,31]. In addition, regardless of exposure to high temperatures, refrigeration, or intense light, polyethylene (PE) disposable cups have great potential to release MPs into the liquid they carry [26,32]. According to a study conducted by Hee et al. [33], the average human ingests as many as 195,000 MP particles annually from takeaway/eat-in food and drink plastic storage. It remains unclear how different physical and chemical factors affect the release of MPs from the single-use PET bottles used outdoors, particularly when these factors interact in combination. Thus, studying the characteristics of the MPs released from PET bottles is important to assess the risk of MP contamination.

In this study, we considered the effects of temperature, sunlight irradiation, and drink characteristics on the release of small-sized MPs (1–100 μm) from PET bottles outdoors, both individually and in combination. The number, size, and surface morphology of MP particles were identified using flow cytometry and scanning electron microscopy (SEM). The obtained results can be used as a reference for reducing MPs and evaluating MP pollution in aquatic environments.

2. Materials and Methods

2.1. Sample Materials

Bottled drinking water from a top-selling brand was purchased from a well-known Chinese online retail shopping platform (https://www.taobao.com/). The packaging consisted of a transparent disposable plastic bottle with a capacity of 555 mL. Raman spectroscopy (HORIBA Jobin Yvon, LabRAM HR Evolution, Paris, France) was used to determine that the body and cap of the plastic bottles were composed of PET and PE, respectively (hereafter referred to as PET bottles). All the water samples were bought from the same store in December 2022. Before the tests, all the samples were stored in sealed cartons at room temperature and shielded from light.

2.2. MP Release Experiment

2.2.1. Temperature Experiment

To simulate the release of MPs in bottled drinks in a high-temperature outdoor environment, five temperatures of 20, 35, 50, 65, and 80 °C were selected to mimic daily life scenarios (details in Supplementary Note S1). The disposable PET bottles were first placed in a water bath for 24 h. The bottles were then cooled to room temperature (20 °C) to avoid damage to the membrane structure due to high temperatures during subsequent filtration. The plastic bottles were shaken for 1 min using a vortex mixer (Vortex-Genie 2, Mo Bio Laboratories, Inc., San Diego, CA, USA) to prevent sticking to the walls or the agglomeration of MPs during the cooling process.

2.2.2. Sunlight Irradiation

When exposed to sunlight irradiation outdoors, plastics are mainly affected by UV, which accounts for about 5% of solar radiation [23]. Thus, two UV lamps with different powers (15 W and 20 W, respectively; YIXIAN, 300–420 nm) were used in this experiment. The plastic bottles were arranged in a circle with a radius of 10 cm, centered on a lamp, and exposed to radiation for 1, 2, and 4 h, which were equivalent to 1.71–6.84 h of direct sunlight (details are provided in Supplementary Table S1 and Supplementary Note S2).

2.2.3. Drink Characteristics

PET bottles are commonly used for a variety of drinks, including sodas, juices, carbonated soft drinks, and drinking water. A typical drink characteristic, the pH, was chosen as the representative characteristic. Commercially available drinks were replaced with pH-adjusted water to eliminate the interference of additives like sweeteners and pigments. Five experimental sample groups were established, with their pH values adjusted to 2.5, 5.0, 7.0, 8.5, and 11.0 by adding phosphoric acid and sodium hydroxide (AR; Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). These pH values represented acidic drinks such as cola, neutral drinks such as purified water, and alkaline drinks such as herbal teas, respectively. Subsequently, the plastic bottles were stored at room temperature (20 °C) in a light-proof environment for 14 days before conducting the MP analysis.

2.2.4. Combination Experiment

The multifactorial combination experiment was designed to closely simulate the real-life conditions in which plastic bottles are used outdoors, including exposure to high temperatures and sunlight before drink consumption. The pH values of the water samples were adjusted to 2.5, 7.0, and 8.5, as described in Section 2.2.3, before waiting for further processing. Each process was performed in triplicate.

In the experiment investigating the combined effect of pH and temperature, the plastic bottles were subjected to heat treatment following pH adjustment, as described in Section 2.2.1. The temperatures selected for this experiment were 20, 35, 50, and 65 °C. Additionally, in order to compare the results with the single-factor effect of the pH, another set of plastic bottles, with the pH adjustment only, was stored at 20 °C for 24 h before detection.

In the experiment investigating the combined effect of pH and sunlight irradiation, the plastic bottles were subjected to sunlight exposure following pH adjustment, as described in Section 2.2.2. The sunlight exposure time were set to 1.71, 3.42, and 6.84 h. Additionally, a set of pH-adjusted plastic bottles was tested after being stored in a light-proof environment for 1.71, 3.42, and 6.84 h, in order to examine the effect of a single pH factor.

2.3. Extraction of MPs

Each experimental water sample had a volume of 1.11 L (two 555 mL plastic bottles) to ensure an adequate number of particles for detection. Ultrapure Milli-Q water (18.2 MΩ, hereafter “ultrapure water”) was used to replace the original water in all the plastic bottles. As shown in Figure 1, all the experimental water samples were filtered using a mixed-cellulose filter membrane (47 mm diameter, 0.22 μm pore size, Labselect, Hefei, China) with a filtration unit (2 L, Longreen, Shenzhen, China) and a vacuum pump. All the filter membranes were rinsed with ultrapure water three times before use to prevent contamination with impurities and fibers. The filtered membrane was then placed in the mouth of a round-bottom glass centrifuge tube (30 mL, Hangzhou Feiseier, Hangzhou, China), and the front side of the membrane was repeatedly rinsed with 30 mL of ultrapure water, causing the MP particles to come off the membrane and enter the centrifuge tube. To avoid the interference of organic matter and microbial cells in the water, 3 mL of 30% hydrogen peroxide (H₂O₂, Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) solution was added to the centrifuge tube, which was placed in a water bath at 50 °C for 24 h [34]. The centrifuge tube was shaken in a vortex mixer for 1 min, and the flow tube was promptly filled with 1 mL of the water sample. A Nile Red stain was then added vertically to the flow tube for dyeing. Nile Red is capable of specific selection in plastic polymers with hydrophobicity, binding to the MPs’ surface to emit fluorescence, whereas inorganic particles cannot be stained [35,36]. The aforementioned filtration steps were repeated for the remaining water samples, and the membranes were placed on a glass dish with a cover and dried at room temperature for subsequent microscopy testing. All the samples were sealed with aluminum foil before testing.

Procedural blanks were prepared. A total of 1.11 L of ultrapure water underwent the same MP extraction procedure as the experimental water samples (filtration, elution, digestion, and sampling), which was conducted in triplicate. To avoid potential contamination by MPs and impurities, all the glass containers used in the experiments, including the filtration devices, the centrifuge tubes, the glass dishes, and the beakers, were sonicated with an ultrasonic cleaner for 30 min and rinsed three times with ultrapure water before use. During sample preparation and testing, nitrile cleanroom gloves and cotton lab coats were worn, and the sleeves of the laboratory coat were wrapped in the gloves. The procedural blanks showed an average MP concentration of 31,261 ± 1092 particles/L, mainly from plastic products which could not be avoided in the experiment (such as ultrapure water dispenser outlet tubes, plastic pipette tips and tubes, etc.) and ubiquitous air microplastic pollution [37]. The procedural blank values were subtracted from the MP concentrations of all the experimental samples.

2.4. Counting and Partition of Microplastics with Flow Cytometry

All the water samples were analyzed using an analytical flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA) equipped with a blue laser (488 nm) and a red laser (638 nm). Signals from the scattered light signals from the forward-scattering channel (FSC, gain setting = 248), side-scattering channel (SSC, gain setting = 265), and fluorescence channel (PC5.5, gain setting = 544) were analyzed. The sample flow rate was 30 μL/min, and the recording time was 60 s. Each water sample was mixed using a vortex mixer before testing to evenly disperse the particles in the water. The flow cytometer was quickly cleaned three times between each water sample to prevent cross-contamination due to residual particles in the detection channels. The CytExpert 2.3 software (Beckman Coulter, Inc., Brea, CA, USA) was used to analyze the data, and the particle signals were presented as scatter plots with “gates” to distinguish between microplastics of different sizes, shapes, and complexities.

Theoretically, flow cytometry can detect all suspended particles in the size range of 0.2–100 μm. However, the signal of MPs smaller than 1 μm mostly overlapped with the background noise in our test. Thus, four sizes (1, 5, 10, and 20 μm) of polystyrene microplastics (PS, TJDAEKJ Co., Ltd., Tianjin, China) were prepared as reference reagents to set the size-dependent gating strategy on the histograms and density plots. Ultimately, the MP particles in the samples were classified into four size classes: “1–5” (MPs with a theoretical diameter ≥ 1 μm but <5 μm), “5–10” (MPs ≥ 5 μm but <10 μm), “10–20” (MPs ≥ 10 μm but <20 μm), and “>20” (MPs ≥ 20 μm but <100 μm) (Supplementary Figure S1). In flow cytometry, each channel has three parameters that can be recorded: area (A), height (H), and width (W). By utilizing the density plot of the forward-scatter height (FSC-H) versus the forward-scatter area (FSC-A), non-spherical particles and aggregations can be visualized and assessed [24]: for single spherical particles, FSC-H = FSC-A, and, for non-spherical particles or aggregations, FSC-H < FSC-A.

2.5. Analysis of MP Morphology by SEM

To obtain the morphology of the particles released into the water after the heat treatment and the surface morphology of the inner wall of the disposable plastic bottles, high-resolution SEM (QUANTA FEG 650, FEI, Hillsboro, OR, USA) was used to observe the filter membrane and the inner wall of the plastic bottles. SEM images with a wide range of magnifications (from 1000× to 100,000×) were used to observe the different sizes of the particles and aggregates in randomly selected areas of the sample.

2.6. Statistical Analysis

All the statistical analyses were performed using IBM SPSS Statistics 27.0. To investigate the effect of temperature variation on MP release from plastic bottles, an analysis of variance (ANOVA) and Duncan’s multiple tests, including normality distribution and homoscedasticity checks, were used to analyze data variability. An independent samples’ t-test was used to validate the differences in the mean values of MP release under the effect of joint and single factors. A p-value of less than 0.05 was considered a significant difference.

3. Results and Discussion

3.1. Effect of Temperature on MP Release

The release of MPs from PET bottles at different temperatures is shown in Figure 2 and Supplementary Table S2. When the temperature increased from 20 °C to 65 °C, the concentrations of total MPs from the PET bottles did not significantly differ (p > 0.05) from the blank control values, which implied that the release of MP particles in PET bottles was barely affected by the temperature increase (≤65 °C). This phenomenon can be attributed to the thermal resilience of PET, which renders it resistant to damage caused by the energy generated from high temperatures in the summer. Secondary carbon in the carbon atoms of the main chain of polymers, such as PE and PET, is less susceptible to abiotic attacks than the tertiary carbon in PP and PS [38,39]. When no mechanical force (such as squeezing or shaking) was applied, as demonstrated in our experiment, the flaking effect causing by hot water only contributed to a negligible percentage of MP release [40]. However, when the temperature exceeds the glass transition temperature (T_g, 75 °C) of the PET, the mechanical properties of the plastic surface are destroyed, making it easier for small-sized MPs to peel off from the surface and enter the water [41]. We found that the bottles were severely distorted when heated to 80 °C (Supplementary Figure S2), and the concentrations of total MPs at this temperature were significantly higher than those at 20 °C (p = 0.002), with a release of up to 36,239 ± 15,689 particles/L.

The percentage of particles within each size range in the total particle quantities was not constant but exhibited a complex variation process, which was contingent upon the different temperatures (Figure 2). This phenomenon can be attributed to both fragmentation and agglomeration processes, meaning that not only can larger particles break into smaller ones, but smaller particles can also aggregate onto the surfaces of larger particles [24]. Smaller particles have a higher surface energy and a stronger adsorption and, therefore, tend to aggregate more on the surface of larger particles [42]. As seen in the SEM images of the inner walls of the plastic bottles and the surfaces of the filtered membranes before and after heat treatment, in Figure 3, the plastic pieces’ surfaces became rough, and the surfaces of the large particles in the water had numerous tiny particles attached. The same temperature experiments were carried out using glass bottles, and we found that the concentration of total particles initially decreased but eventually stabilized (Supplementary Figure S3), indicating that the aggregation of the particles in the water occurred as a result of temperature effects.

3.2. Effect of Sunlight Irradiation on MP Release

Plastics can degrade under sunlight irradiation, producing plastic chips or even MPs. Therefore, various exposure times were used to assess the effect of sunlight irradiation on the release of MPs from the PET bottles. As shown in Figure 4a–d, the concentration of particles from PET bottles under sunlight irradiation exhibited a fluctuating nonlinear trend. It initially increased and subsequently decreased, reaching a maximum release of 42,793 ± 8273 particles/L at an exposure time of 4.62 h. Particles ranging from 1 to 20 μm showed significant release, with an average release amount ratio of approximately 80:6:1 for particles sized 1–5 μm, 5–10 μm, and 10–20 μm, respectively. However, we did not consistently detect a significant release of particles in the >20 μm size class. There was an inverse relationship between the particle size and the time until peak concentration of the released particles. Specifically, the 10–20 μm size class of particles reached its peak concentration (1171 ± 780 particles/L) at 3.08 h, while the 5–10 μm size class of particles peaked at 3.42 h with a concentration of 3153 ± 624 particles/L, and the 1–5 μm size class of particles took 4.62 h to reach its peak concentration (up to 40,450 ± 6699 particles/L). Subsequently, between 4.62 and 6.84 h, there was a decreasing trend in the concentration of the 1–5 μm and 5–10 μm size classes of particles, while particles larger than 10 μm remained relatively stable, with fluctuations. This behavior may be related to the fragmentation and degradation of the particles. As the irradiation energy on a plastic surface is greater than the dissociation energy of the C–O bonds in the polymer under UV radiation [43], the carbon chain breaks and the molecular weight decreases, changing the mechanical properties of the material, which eventually leads to the appearance of pores and cracks on the plastic surface, breaking into small MPs [44,45]. As irradiation continues, the MP particles formed by fragmentation undergo further photo-oxidative degradation and are fragmented into NPs with smaller sizes (<1 μm) [23] or even other dissolved oligomers [46] which are not detected by flow cytometry. Smaller particles pose greater health risks and can be ingested by smaller organisms or human cells, causing cellular immune damage [10].

Taking into account the results from each particle size class, it was observed that the MP particles in the 1–5 μm size range showed the highest abundance, accounting for 77%–92% of the total count, followed by the 5–10 μm size range, ranging from 6% to 12% (Figure 4e). As previously reported, the concentration of MPs follows an exponential growth pattern as the particle size decreases [17]. With the passage of time, small-sized MPs and NPs are expected to become prevalent in plastic products. The characterization of particle shapes using SEM revealed that the vast majority of the observed particles were found to be spherical and varied in size and degree of abrasion, with a minor number of irregularly shaped particles (Figure 4f). Using flow cytometry, density plots of FSC-H versus FSC-A were performed to support this finding, with the majority of the FSC-H values being equal to the FSC-A values (Supplementary Figure S4).

3.3. Effect of Drink Characteristics on the Release of MPs

The pH, as the chemistry indicator which best separates various drinks, was simulated to explore the effect of the drinks’ characteristics on MP release, and the results are shown in Figure 5. Compared with the neutral group, the total concentration of MPs increased in both the acidic and alkaline groups. The highest released concentration was observed in the pH 8.5 alkali group (8919 ± 1892 particles/L), with particles sized 1–5 μm accounting for approximately 78% of the total count, while particles sized 5–10 μm and 10–20 μm accounted for 11% each. No particle larger than or equal to 20 μm was detected. Consistent with our other findings, the amount of MP contamination in the bottled water was closely related to the pH, with weak alkalinity increasing the number of small-sized MP particles, especially for particles in the 1–5 μm and 5–10 μm size classes [47]. Particle release was also observed within the pH range of 2.5 to 5.0 (3874–6847 particles/L), with the release concentration increasing as the pH decreased. Among these, the release of particles ≥ 20 μm was particularly significant, accounting for 34% of the concentration at a pH of 2.5. The decrease in the water pH may facilitate the aggregation of MP particles [48], whereby particles smaller than 10 μm aggregate between other particles, resulting in the formation of larger-sized particles.

3.4. Combination Effect of pH and Temperature on MP Release

The release of MPs from PET bottles with different pH values under the effect of a heat treatment is shown in Figure 6. We observed that, under both the pH 2.5 acidic and pH 8.5 alkaline groups, the heat treatment promoted the release of MPs from the PET bottles compared to the effect of the pH 7.0 neutral environments. The concentration of released MPs increased with the rising temperature, reaching up to 21,622 ± 2477 particles/L (pH = 2.5, T = 65 °C) and 16,847 ± 9557 particles/L (pH = 8.5, T = 65 °C) in the acidic and alkaline groups, respectively. Specifically, when considering the individual effects of each factor on the results, it was observed that, in the pH 2.5 acidic group, the single acidic factor (T = 20 °C, pH = 2.5) did not induce the release of MPs. In the pH 8.5 alkaline group, the release concentration caused by the single alkaline factor (T = 20 °C, pH = 8.5) showed no significant difference compared to the release concentration at T = 35 °C. This indicates that temperature and an acidic pH may jointly stimulate the release of MP particles from PET bottles, especially for particles of 1–5 μm sizes, and their combined effect is greater than the individual factors alone. It has been previously confirmed that the rate of PET hydrolytic cleavage is enhanced in an acidic environment [49,50], and an increase in temperature promotes the hydrolysis reaction [51], leading to the production of large amounts of MPs. In contrast, in our study, the effects of temperature and an alkaline pH on MP release remained independent between 35 °C and 50 °C, synergistically promoting the release of MP at temperatures exceeding 50 °C.

3.5. Combination Effect of pH and Sunlight Irradiation on MP Release

Figure 7 demonstrates the combined effect of sunlight irradiation and pH on the release of MPs from the PET bottles. In the “pH 2.5 + sunlight” group, the release of MPs of all particle sizes was positively correlated with the irradiation time, while the maximum release concentrations were 23,694 ± 9557 particles/L (1–5 μm), 2793 ± 949 particles/L (5–10 μm), 1622 ± 780 particles/L (10–20 μm), and 1622 ± 780 particles/L (>20 μm). Compared to the “pH 2.5” group, the release concentration of the particles in the “pH2.5 + sunlight” group significantly increased during the 3.42–6.84 h of sunlight exposure. However, when compared to the “sunlight” neutral group, the “pH2.5 + sunlight” group showed a delayed time to reach the peak particle concentration, with no significant difference in the highest total particle concentration (p = 0.479). These findings suggest that, in an acidic environment, the release of MPs induced by sunlight exposure is comparable to that in a neutral environment, with a slower release rate.

In the “pH 8.5 + sunlight” group, the release of MPs peaked at 31,081 ± 7173 particles/L within a shorter irradiation time (1.71 h). In comparison, the “sunlight” neutral group required 3.42 h (29,550 ± 9209 particles/L) while the “pH 8.5” group required 6.84 h (7568 ± 480 particles/L) to reach their respective peak particle concentrations. Put simply, the combined effect of an alkaline pH and sunlight irradiation advanced the peak of MP release compared to the effects of a single pH factor (“pH 8.5” group) and a single sunlight factor (“sunlight” group), indicating that the rate of plastic degradation was accelerated, resulting in a much higher risk of MP ingestion by humans within a short amount of time. Some studies have found that, under photodegradation and alkaline conditions, polyvinyl chloride rainwater pipes show an increase in oxygen-containing functional groups [52], leading to the degradation and loosening of the plastic surface and promoting the release of MPs [53]. We found that the number of MP particles in the 1–5 μm size class gradually decreased, whereas the number of particles in the >5 μm size class consistently increased, indicating that particle aggregation accounts for the main variation.

3.6. Risk Reduction Strategy

After a great deal of plastic bottles are discarded, they remain in the environment for a long time and continue to be broken down by light, heat, microbes, and other environmental forces, generating an abundance of small-sized MPs [50]. The particle abundance of these small-sized MPs increases dramatically with a decreasing size and is relatively stable in an aqueous environment, where they can remain for longer periods of time [54]. Eventually, small-sized MPs create a new ecological niche that can be occupied by microorganisms through adsorption and absorption and act as transporters of various environmental toxins [55]. Furthermore, research has indicated that PET bottles not only release MPs when stimulated by the external environment but may also lead to the leaching of additives, heavy metals, organic chemicals, and other substances [39]. For example, exposure to sunlight has been found to have a profound impact on the levels of phthalates present in bottled water, with the amount increasing significantly [56]. In addition, PET bottles are susceptible to leaching antimony at a higher rate when exposed to higher temperatures and acidic pH levels [57]. With an increased retention time in the environment, PET bottles may be colonized by biota and other plastics [58] and progressively degraded by organisms [59].

Considering the alarming frequency with which PET bottles are utilized worldwide and the grave risks posed to both the ecosystem and human health by the consumption of small-sized MPs, it is imperative that we take action to curb our dependence on disposable PET bottles in our daily routines and, instead, actively seek out and promote sustainable plastic and/or alternatives. Bottled drinks, especially those with high pH levels, should not be exposed to high temperatures or prolonged sunlight when transported, stored, and used, to minimize MP release.

4. Conclusions

Using laboratory simulation experiments and small-sized MP particle-counting techniques, our research demonstrated that outdoor-exposed PET bottles are a major source of MPs (especially those sized 1–5 μm). In this paper, we reported that temperature, sunlight irradiation, and drink characteristics have different degrees of influence on the release of MPs from PET bottles exposed to the outdoors. Our more specific conclusions are as follows: (1) the release of MP particles in PET bottles was barely affected by the temperature increase (≤65 °C); (2) under the influence of a sunlight irradiation alone, the concentration of MP particles in the PET bottles first increased and then decreased, reaching a maximum release at an exposure time of 4.62 h; (3) the alkaline 8.5 pH drink bottles had a higher risk of releasing small-particle-sized MPs than neutral and acidic environments; and (4) there were two pairings with synergistic effects on the MPs released from the PET bottles—an acidic pH with temperature and an alkaline pH with sunlight irradiation—with maximum releases of 21,622 ± 2477 particles/L and 31,081 ± 7173 particles/L, respectively. PET bottles may show some specific release characteristics after extended exposure times that were not taken into account in this study. Future research is required in order to improve our detection techniques and apply them to additional environmental media associated with plastic, like natural water bodies, water supply systems, and drainage networks, to further characterize their microplastic-related fate.

Author Contributions

Data curation, P.Z.; funding acquisition, K.Z.; Investigation, C.C., Y.Z., and Y.S.; methodology, P.Z.; supervision, K.Z. and T.Z.; writing—original draft, P.Z.; and writing—review and editing, K.Z. and T.Z. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures

Figure 1. Schematic illustration showing the process followed by the researchers to analyze the MPs.

Figure 2. Variation in the concentration and percentage of MPs with different sizes under different heat treatments ((a): 1–5 μm, (b): 5–10 μm, (c): 10–20 μm, (d): 20–100 μm). n = three independent samples for each test. The black line represents the percentage of particles with a specific size in the total number of particles of a 1–100 μm size at the same temperature. Different letters denote differences (“a”: p > 0.05, and “b”: p < 0.05).

Figure 3. Typical SEM images of the filter membrane and the plastic bottle’s inner wall before and after heat treatment.

Figure 4. Effect of sunlight irradiation on the release of MPs. (a–d) Impact of sunlight irradiation on the release of MP particles of different sizes; (e) percentage of released MP particles of different sizes; and (f) typical SEM images of the corresponding spherical particles.

Figure 5. Effect of drinks’ characteristics on the release of MPs. (a) Concentration and (b) size distribution of the MPs released from PET bottles with various pH values stored in a dark room for 14 days.

Figure 6. Effect of pH and temperatures on the release of MPs. The concentration of the released MPs of different sizes in the PET bottles with various pH values (2.5, 7.0, and 8.5) under different temperatures (20 °C, 35 °C, 50 °C, and 65 °C): (a) 1–5 μm, (b) 5–10 μm, (c) 10–20 μm, and (d) >20 μm. The black horizontal lines and “x” marks in the box plots represent the median and mean values, respectively, while the boxes represent the standard error, and the whiskers represent the range of data.

Figure 7. Effect of pH and sunlight irradiation on the release of MPs. The concentration of the released MPs of different sizes in the PET bottles with various pH values (2.5, 7.0, 8.5) under sunlight irradiation for 1.71, 3.42, and 6.84 h: (a) 1–5 μm, (b) 5–10 μm, (c) 10–20 μm, and (d) >20 μm. “pH 2.5/8.5” indicates the effect of a single acidic/alkaline factor. The bottles were adjusted to the respective pH levels and then placed in a dark room for 1.71, 3.42, and 6.84 h. “Sunlight” indicates the effect of a single sunlight irradiation factor. The bottles with a pH of 7 were subjected to sunlight irradiation for 1.71, 3.42, and 6.84 h. “pH + sunlight” indicates the combined effect of both pH and sunlight irradiation.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/w16131898/s1: Figure S1: Gating strategy in flow cytometry; Figure S2: Deformation of PET bottles after heat treatment; Figure S3: Count of MP particles in plastic and glass bottles at different temperatures; Figure S4: Log–log density plots of FSC-H and FSC-A; Table S1: Correspondence between the actual sunlight irradiation time and the experimental UV lamp irradiation time; Table S2: The number of MPs released from PET bottles at different temperatures; Table S3: The number of MPs released from PET bottles under sunlight irradiation; Table S4: The release concentration of MP particles in PET bottles with various pH values; Table S5: The number of MPs released from PET bottles with various pH values under different temperatures; Table S6: The number of MPs released from PET bottles with various pH values under sunlight irradiation; Note S1: The rationale for selecting the five specified temperatures; Note S2: The irradiation of UV and sunlight conversion [23].

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