Quantification of microplastics by count, size and morphology in beverage containers using Nile Red and ImageJ

Abundant evidence of microplastics (MP) found in the environment, and its toxicity effect in animals calls for human-related research. However, well-established quantitative controlled studies on the potential route of human exposure to MP are still sparse. MP count, size and morphology in 15 polylactic acid (PLA)-lined plastic cups and 15 PLA-lined paper cups were examined using Nile Red fluorescence tagging, microscopic photography, and morphology assessment and quantification based on ImageJ. In the plastic cups, the count and area of MP fibers were found to be significantly higher compared with blanks (p< 0.05), but not MP particles or total MP. In paper cups, count or area was not significantly different in terms of MP particle, MP fibers or total MP. No interesting trend was observed in the distribution regarding the size of MP particles or fibers. These results indicate that selected paper cups and plastic cups could be considered as safe beverage containers, but further research on the toxicological effects of MPs in different morphologies released from plastic cups on human health is needed.


INTRODUCTION
Microplastics (MPs) are water-insoluble, synthetic polymers of diverse shapes, with sizes ranging from 1 μm to 5 mm (Frias & Nash ). The detection of MPs in human stools also suggests MP consumption results in their transportation through, and contamination of the gastrointestinal system (Schwabl et al. ). These findings raised concern about the impact of MPs on human health and called for research in human toxicity (Smith et al. 

Study design
To meet our objectives, we included one experiment group of 15 plastic cups, one experiment group of 15 paper cups and one control group of 5 procedural blanks in our study design. Previous studies support using Nile Red fluorescence for distinguishing MP from non-polymer materials, and programs like ImageJ have been used to quantify MP using

Sample collection and processing
We examined polylactic acid (PLA)-lined paper cups and PLA-lined plastic cups, which were common beverage containers (manufactured by NatureWorks) that were used and could be purchased in the New England area. Fifteen paper cups and 15 plastic cups were collected from a cafeteria in Harvard T.H. Chan School of Public Health.
To minimize potential contamination from external sources during the experiment, such as airborne fibers and particles, the whole experiment was processed in a fume hood, and the workplace as well as all glassware were sterilized using the standardized biochemistry laboratory protocol. The pre-filtered Milli-Q water was prepared by vacuum filtration of Milli-Q water with the same glass microfiber filter used to filter out MPs in each condition.
Regular personal protection equipment was worn throughout the whole process.
To examine the contamination of MPs from the surface of the containers, 100 mL of pre-filtered Milli-Q water was added to all containers, followed by a slow, 1-min clockwise revolving motion in the fume hood under the laboratory temperature (23-25 C). Afterwards, the filtered water was transferred to a clean beaker, dyed by Nile Red solution (dissolved with acetone) with a working concentration of 10 μg/mL, and incubated for 30 min. This concentration strikes a balance between fluorescent strength of dyed MP and reduction of 'noise' from Whatman filters that were used for filtration (Maes et al. ). Each beaker of incubated solution was then vacuum filtered through a dedicated glass microfiber filter. The processed filters were dried in the Petri dish for at least 24 h in the laminar fume hood.
To account for the potential contamination from ambient air, chemicals, glassware and other testing materials, we introduced five procedural blanks. In blank samples, we used clean beakers that were washed with pre-filtered Milli-Q water and went through the same procedure (rinsing, staining, vacuum filtration and incubation) identical to all the samples in the experiment groups. The level of microplastic contamination found in our procedural blanks suggests accountability of MP contamination from the laboratory environment.

Data collection
Using an Olympus BX-60 with zoom at 10 × , we examined

Data processing
Each recognized cluster of fluorescence was manually classified as either particles or fibers, based on the morphology of the fluorescence, in order to overcome miscounting issues that can arise when using a high threshold for MP classification by ImageJ. With ImageJ, any pixel that did not meet a set level of fluorescence shows up as white, while any pixel meeting the requirement shows up as black (Figure 1). This sometimes leads to fibrous MP being cut into multiple parts, which causes ImageJ to classify one fibrous MP as multiple MPs. We made duplications, separations and 'blacked out' parts of a photo prior to ImageJ analysis to separate fibrous MP from particles in a photograph, which prevented automated overcounting. This process was performed for every sample and was done independently by two researchers whose results of classification were compared and finalized through discussion. Both of them were blinded to the sample types. As a result, we obtained separated folders that contained photos with particles only or with individual fibers for each sample.
All manually separated photos were processed by ImageJ 1.52q. Image processing was based on the ImageJ

Statistical analysis
Data analysis was performed in R v3.5.1. The csv file for every image was read and summarized within samples for particles and fibers, respectively. Files that contained no result indicated that any fluorescence did not meet our absolute threshold, even if identified by the naked eye. Particle, fiber and total MP area of a sample were calculated by summing the area of the particles, fibers or both, respectively. For particle counts, row number in the csv file, which was a detached observation of MP, was summed up. Fiber count was measured by image count because each fiber had a dedicated image modified to remove other fluorescence. Particle area and fiber area were measured by summing up the area for all images within each sample. The feature used for particle inclusion was maximum Feret diameter, the maximum distance possible between any two parallel planes restricting the particle perpendicular to that direction. We included particles with maximum Feret diameter 7 μm, which was the maximum Feret of the smallest recognized MP by human eye, under the microscope across all photos. For fiber inclusion, the threshold was area >0 mm 2 . T-tests were performed to find any statistically significant differences in numbers and areas of MPs between different sample types.

RESULTS
All samples of 15 paper cups, 15 plastic cups and 5 blanks were successfully collected, processed and analyzed.
During the data analysis process, 6,860 images were processed in total. On average, around 30 images per sample were originally mixed with fibers and particles, and around 6-7 images needed to be reprocessed due to misrecognition of background per sample by ImageJ. Figure 1 displays what a typical MP fiber (Figure 1(a)) and an MP particle  indicates that for MP particles, the numbers were quite similar between the three conditions. However, fiber count was significantly higher in plastic cups compared with blanks.
The counts that included MP particles or combined numbers were not significantly different between the three conditions. Figure 3 displays the total MP areas across our three types of samples. We could see that the area of MP fibers from plastic cup samples was significantly higher than that of the blanks (p ¼ 0.032). Total MP area in terms of the sum of particles and fibers was higher in both plastic and paper cups compared with the blanks, but was not significant with p-values of 0.095 and 0.320, respectively. We can, therefore, conclude that there was no significantly elevated level of count and area in plastic or paper cups in terms of MP particle or total MP. Nevertheless, MP fiber count and area were significantly higher when pre-filtered water was exposed to plastic cups, when compared with our blanks.
We also performed an analysis on the distribution of the size of MP in terms of particle maximum Feret diameter, particle area and fiber area. Density plots were made by merging information of all recognized particles and fibers across all samples within each sample type. However, the probability densities of log particle maximum Feret diameter (mm) of three sample types fall on the same curve and fit well. No significant differences were found in the distribution of particle maximum Feret diameter (a), particle area (b) or fiber area (c) in the log scale as shown in Figure 4.
These results indicate that the differences observed previously in counts and areas between sample types were independent to particle or fiber size distributions, and the differences exist in all levels of size of MPs. These results are also supported by comparing the particle median maximum Feret diameter (d), particle median area (e) and fiber median area (f) as shown in Figure 4. No significant difference was found in plastic cups or paper cups compared with blanks according to the result of t-tests.

DISCUSSION
Partially consistent with prior studies, MP fibers are found to be significantly elevated releasing from the surfaces of the PLA-lined plastic cups while being vibrating under the laboratory temperature (around 23 C). A related study demonstrated that steeping a single plastic teabag at brewing temperature (95 C) could release around 11.6 billion MP and 3.1 billion nano-plastics into a single cup of the beverage (Hernandez et al. ). Another study found that opening and closing a series of plastic bottles increased the number of microplastic particles (Winkler et al. ).  Table 1, we could see a tradeoff  speed. This study demonstrated that a smaller limit of detection (7 μm) is compatible with automated counting for each filter, without the need for stitching images.

However, the insignificant result of total MP in this study
The detection limit was set to 50 μm in Prata's study by allowing detection of fluorescence of over 3 px (Prata et al. ). In Mason's study, MPs were visualized as long as 1 px was recognized (Mason et al. ). The reason we did not use a minimum recognized pixel number to define our detection limit is that it would count detached pixels from the main particle that could not be eliminated through 'despeckle' function in ImageJ as independent particles, which would affect the accuracy of quantification greatly.
Additionally  As the regulatory and policy environment shifts toward

CONCLUSIONS
This study provides quantitative estimates to MPs releasing from the surface of PLA-lined beverage containers. The data showed PLA-lined paper cups are not significant sources of MPs. PLA-lined plastic cups were found to be significant sources only for MP fibers, but not MP particles or total MPs. These results indicate that selected paper cups and plastic cups could be considered as safe beverage containers. Further research on the toxicological effects of MPs in different morphologies released from plastic cups on human health is needed.