Plastic and Life: Protocols and Tools for Micro-Spectroscopic Analysis of Micro- and Nanoplastics in Maternal and Fetal Matrices

The widespread use of plastics has resulted into environmental plastic pollution. Plastic can break down into smaller particles, called microplastics and nanoplastics, in the micrometer and nanometer size range respectively. These particles have been found inside environmental matrices, food matrices, as well as in the human body. Especially for the unborn child, whose immune system is not fully developed yet, a risk assessment of micro- and nanoplastics (MNPs) is needed. With micro-spectroscopic techniques, we can analyze MNPs’ size, shape, surface groups and numbers, yielding important exposure information. This PhD thesis contributes to the development of micro-spectroscopic techniques to analyze MNPs in maternal and fetal tissues. Chapter 1 describes the necessity of this research and addresses the knowledge gaps. In Chapter 2, a pre-processing protocol is described that was developed to allow for accurate detection of commercially available fluorescently labelled polystyrene (PS) MNPs in placental tissue. These MNPs function as model particles to test the developed method for selectivity and effectiveness. Proteinase K in tris(hydroxymethyl)aminomethane hydrogen chloride (TRIS-HCl) buffer was shown to be the most successful environment to effectively digest placental tissue. Confocal fluorescence microscopy (CFM) images showed PS MNPs particles to be stable throughout all steps used in the digestion protocol and resulted in recovery rates (RRs) for different sizes of PS particles. RRs of 98 % ± 6 for 200 nm PS particles, 148 % ± 8 for 500 nm PS particles, 147 % ± 8 for 1 μm PS particles and 81 % ± 8 for 10 μm PS particles were found. This indicates that tissue residue appears as particulate matter within the 500 nm to 1 µm size range; these residues can then be fluorescently stained by leached dye, probably released due to the conditions used in the digestion protocol. Chapter 3 describes several analytical methods to quantify nanoplastics without losing their morphological information. Five sizes of nanoplastics of different plastic types were imaged with CFM in 12 different concentrations. For each particle size and type, a range was determined in which fluorescence intensity scales linearly with particle concentration. Within these linear ranges, CFM can be used as a practically applicable analytical technique for NP quantification while simultaneously yielding micro-spectroscopic information and particle numbers. In Chapter 4, first steps were made for MNP analysis in real-life samples. Placental perfusion is an excellent method to study uptake and transfer over the placenta. It was shown that in all perfusions, there was minimal to no transfer of PS to the fetal reservoir, but retention in tissue was seen for all perfusions. More retention was seen for smaller particles. As MNP analysis in human matrices has been difficult, and people spend most of their lives indoors, household dust is another relevant matrix to study maternal MNP exposure. In Chapter 5, a method has been developed to analyze microplastics in dust with infrared micro-spectroscopy. Results showed a significantly higher number of particles in samples than in blanks, particles’ size and shape was analyzed, and spectroscopic analysis showed the presence of polyamide.