Determination of microparticles, in particular microplastics in beverages

The usage of plastics makes daily life easier. Being lightweight, resistant, non-conductive and inexpensive, they are used in a wide variety of applications. Consequently, plastic production volumes have increased greatly during the last decades. In keeping with this, the amount of plastics waste has increased dramatically as well. Although some of it is recycled, burned or at least collected and dumped, much plastic finds its way into natural environment. First, larger plastic debris received public attention, as it is unattractive and primarily a threat to various animals. Later, after its first detection in the oceans at the beginning of the 1970s, smaller plastic debris, so-called microplastics, attracted increasing attention. Today, microplastics have been shown to be present in a variety of environmental compartments: in seawater from the Arctic to the Antarctic, in freshwater of rivers and lakes, in corresponding sediments till the deep-sea sediments and also in the air. Moreover, many animals, including food-producing aquatic organisms, ingest these small plastic pieces, potentially causing harm to themselves. Therefore, the questions arose as to whether humans ingest microplastics with their food (aquatic and non-aquatic) and whether doing so may pose risks to human health. Up to now, however, only a few studies reporting the microplastic content in food (e.g., bivalves, honey, German beer and tap water) have been published. Some of these results are viewed sceptically since the researchers did not pay much attention to the prevention of sample contamination and did not clearly identify microplastics as such. Thus, the first aim of the present work was to establish analytical methods for the analysis of microplastics, with special attention to potential unintended sample contamination and with clear identification of the material of particles. Since the first risk assessments of the European Food Safety Authority (EFSA) indicated that the smallest particles (<1.5 µm) in particular may penetrate deeply into organs and cause harm, this size range had to be covered. One reliable analytical technique, which allows identification of the material of particles down to a size of 1 µm, is micro-Raman spectroscopy. Each material, which is Raman-active, has its own characteristic spectrum. From the Raman spectra generated for each particle, the particle’s composition can be identified. For liquid matrices, particle separation can be performed via filtration, and Raman measurements can be done directly on an appropriate filter surface. To simplify analysis, automatic particle detection can be used. However, to obtain high-quality Raman spectra, one must ensure that the filter material does not interfere with the particle spectra. Furthermore, for automatic particle detection, the filter surface must be smooth and flat, and it must show a high optical contrast to the particles. Thus, eight commercially available filter materials and then three newly developed metal-coated polycarbonate membrane filters were tested for applicability. Among all commercially available filters, only gold-coated and black polycarbonate membrane filters had a surface that was sufficiently smooth and that showed particles in high contrast under dark field illumination. However, black filters exhibited an intense Raman spectrum, and gold-coated filters showed weak fluorescence, both of which interfered with spectra of small particles. Furthermore, small particles in particular were easily burnt on both filter materials: they were destroyed by the Raman laser and were no longer identifiable. As the gold coating shielded the spectrum of the underlying polycarbonate, common polycarbonate filters were coated with a thin film of other metals such as nickel, titanium and aluminium using electron beam evaporation. Of these three new filter materials, only aluminium coating showed ideal characteristics: Under dark field illumination, the flat filter surface showed the particles at high contrast, enabling the use of automatic particle detection. The aluminium coating shielded the spectrum of the underlying polycarbonate, and aluminium itself showed no Raman background spectrum and no respectively very weak fluorescence (depending on the laser wavelength used for Raman scattering). Particle burning was observed only with increased laser power. Furthermore, aluminium acted as a mirror; reflected the laser light; and, as a result, significantly enhanced the intensity of the Raman spectra of particles. With this new type of filter material for particle separation and subsequent automatic particle detection and measurement with micro-Raman spectroscopy, identification of particles down to a size of 1 µm became possible. Based on this development, a method was established to analyse bottled mineral water for contamination with particles. After the addition of ethylenediaminetetraacetic acid tetrasodium salt and sodium dodecyl sulphate solutions, an aliquot of each sample was filtered through the above-mentioned newly developed membranes, and a predefined subset of the remaining particles were identified by micro-Raman spectroscopy. Subsequently, 32 samples of bottled mineral water packaged in single use and reusable bottles made of poly(ethylene terephthalate) (PET) or glass were analysed. Water from 21 different brands was tested, and data on contamination with microplastics, additives and pigment particles were collected. On average, over 90% of the detected microplastics and pigment particles were smaller than 5 µm and could be detected for the first time. They have thus not been covered by previous studies. Microplastics were found in water from all bottle types. The levels varied widely between bottles, both within and between brands. On average, water from single use PET bottles was less contaminated (2 649 ± 2 857 microplastics/l) than water from reusable PET bottles (4 889 ± 5 432 microplastics/l) or glass bottles (6 292 ± 10 521 microplastics/l). The predominant polymer type in water from PET bottles was PET, indicating that the bottle itself is a source of contamination. Water from glass bottles was contaminated with microplastics of different polymer types, such as polyethylene (PE), polypropylene (PP) and styrene-butadiene-copolymer. Apart from potential abrasions from the bottle caps, other routes of contamination must also be considered. In water from reusable PET bottles, on average, 708 ± 1 024 particles/l of the additive tris(2,4-di-tert-butylphenyl)phosphite were identified. This additive may have leached from the bottle material. Pigment particles were also detected in water from all bottle types. However, high amounts were found only in reusable paper-labelled bottles (195 047 ± 330 810 pigment particles/l in glass and 23 594 ± 25 518 pigment particles/l in PET bottles). Detected pigment types were identical to the colours used for label printing. During the washing process of reusable bottles, the washing liquor comes into direct contact with the paper labels and the inner surface of the bottles. Thus, bottles may be contaminated with pigment particles detached from the labels via the washing liquor. The bottle cleaning process may also be relevant for the contamination with microplastics and may explain the contamination of mineral water stored in glass bottles. To verify these assumptions, cleaned unfilled glass bottles were investigated for contamination with particles. Since the bottle cleaning process is unaffected by the beverage to be filled into the bottles, empty beer bottles of three breweries were analysed. Three bottles of different ages were chosen from each manufacturer. For one brewery, the analysis was repeated after the exchange of the washing liquor. Cleaned bottles were collected as samples directly at the end of the bottle cleaning process and closed immediately. In the laboratory, they were filled with ultrapure water, which was then analysed similarly to bottled mineral water. This process simulated the bottle filling, and the particle amounts introduced in beverages via contaminated bottles were determined. Whereas microplastics were found in high amounts (up to 26 939 ± 8 456 microplastics/l) in bottles of two brands, bottles of the third brand showed almost no contamination (271 ± 394 microplastics/l). Pigment particles were measured in a range from 117 189 ± 60 379 /l up to more than one million particles/l. The number of pigment particles in the third brand was close to the mean value of all brands. The corresponding washing liquor of the third brand may have been less contaminated with microplastics, potentially because the washing liquor was cleaned by filtration. However, this effect did not influence the pigment content. The microplastic content increased from 9 823 ± 2 906 /l to 26 939 ± 8 456 /l, and the quantity of pigment particles decreased from 236 065 ± 112 631/l to 117 189 ± 60 379 /l in bottles from the other brewery after the exchange of the washing liquor. Thus, microplastics may be already present in fresh washing liquor. The exchange of the washing liquor decreased the pigment particle count, but only for a short period. The bottle age did not affect the concentrations of microplastics and pigments. Apart from phenoxy resin and an unidentified polymer, which were additionally found in cleaned beer bottles, the same polymer types (PE, PP, polystyrene and styrene-butadiene-copolymer) were present in cleaned beer bottles and in mineral water bottled in glass. Accordingly, the pigment types were the same as those detected in mineral water and on the corresponding paper labels. Furthermore, the size distribution for the microplastics and pigments in beer bottles was similar to what was found in glass-bottled mineral water. Overall, it is quite likely that most microplastics and pigment particles were already present in the cleaned glass bottles, subsequently contaminating the mineral water they were filled with. The bottle cleaning process was clearly identified as a contamination pathway for microplastics and pigment particles in mineral water and beer. All beverages stored in cleaned reusable bottles may be contaminated in the same way. Pigment particles most likely originate from the paper labels. Initial sources of microplastics may be, for example, contaminated empties, chemicals or drinking water used in the bottle washer, or also the air. However, further tests must be performed to clearly evaluate initial sources of microplastics. In addition, the effect on product contamination of parameters such as filtration of the washing liquor or the amount of clear water used for the final bottle rinsing steps should be investigated. In addition, humans use tap water to meet their needs for water and to prepare food. If contaminated with microparticles, especially microplastics, drinking water may be an important source for total oral intake of microplastics by humans. Accordingly, the method established for testing bottled mineral water was evaluated for application in testing tap water. Tap water from three households was investigated in a manner similar to that used for bottled water. Two samples were taken in adjacent households with the same water supplier to check for a potential influence of the domestic drinking water system (zinc-coated iron pipes versus PE pipes). With a maximum of 271 microplastics/l, none of the three tap water samples showed higher amounts of microplastics than the corresponding blank samples. Tap water was thus not significantly contaminated with microplastics. However, some polymer types (i.e., polyester, poly(vinyl chloride) and polyoxymethylene) detected in tap water were never identified in blank samples in this study and may thus have originated from the samples. Interestingly, in water distributed via PE pipes, no PE particles were found. Since no significant difference between the microplastic contents of the blanks and samples could be observed, the results have to be confirmed by further experiments. For samples with such low contamination levels, the analytical method must be modified. Either the quantity of microplastics in the blank samples must be reduced, or the concentration of microplastics on the filter surface must be increased. Some non-plastic particles were present in greater numbers in the tap water and could therefore be clearly determined. In all samples, particles with a dominant spectrum of carotene were found. Furthermore, particles of silicon dioxide, iron oxide and titanium dioxide were present in all samples, and potash feldspar was present in two. Particles of three materials could not be identified because there were no matching reference spectra. Most of these particles were likely of natural origin. Some materials such as iron oxide or silicon dioxide may come from the domestic drinking water system respectively the drinking water treatment. To obtain a general overview of particles present in tap water and to generate data about contamination with microplastics, further tests are required. To measure small amounts of microplastics, the analytical method must be modified. Based on the knowledge gained in the present work, further analytical methods can be developed to analyse microparticles in different beverages and in more complex foods. The data on the content of microparticles in beverages can be used to estimate the oral intake of microplastics and other particles by humans via beverages. Subsequently, studies on the toxicological effects and further risk assessments can be performed based on realistic concentrations. Furthermore, the results can be used as a basis for reducing contamination of beverages with particles.