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Christian Pacher, BSc Microplastics in shore sediments – Development of a suitable extraction and purification technique for citizen science projects Masterarbeit zur Erlangung des akademischen Grades eines Master of Science der Studienrichtung Ökologie und Evolutionsbiologie an der Karl-Franzens-Universität Graz Betreuer: Priv.-Doz. Mag. Dr.rer.nat. Stephan Koblmüller Institut für Biologie Abstract ..................................................................................................................................... 4 Introduction .............................................................................................................................. 5 Plastic – Our omnipresent companion .................................................................................... 5 Dangers of microplastics ........................................................................................................ 6 Plastic distribution in the Mediterranean Sea ......................................................................... 8 Microplastics in shore sediments ............................................................................................ 9 Citizen science ...................................................................................................................... 10 Location of the sampling spot .............................................................................................. 11 Material and methods ............................................................................................................ 12 Field sample collection ......................................................................................................... 12 Contamination prevention .................................................................................................... 13 Density separation ................................................................................................................ 13 Conventional density solutions ......................................................................................... 13 Potassium carbonate (K2CO3) – A novel, cheap and non-toxic solution .......................... 17 Plastic types .......................................................................................................................... 18 Plastic resistance to K2CO3 ................................................................................................... 21 Validation of K2CO3 as density-solution by recovery-experiments ..................................... 21 Digestion of organic pollutants ............................................................................................. 24 In advance excluded digestive agents ............................................................................... 27 Enzymatic treatments ........................................................................................................ 29 Most promising agents ...................................................................................................... 31 Polymer-resistance against shortlisted protocols .............................................................. 33 Comparison between virgin plastics and weathered plastics ............................................ 34 Testing of the six protocols on organic material ............................................................... 35 Testing of the whole protocol on beach samples .................................................................. 36 Results ..................................................................................................................................... 38 Validation of K2CO3 as density-solution by recovery-experiment ...................................... 38 Plastic resistance to K2CO3 ................................................................................................... 39 2 Resistance of different plastic types to different chemicals ................................................. 39 Comparison between virgin plastics and weathered plastics ................................................ 42 Digestion of organics by different chemicals and enzymes ................................................. 43 Testing of the whole protocol on beach samples .................................................................. 44 Discussion ................................................................................................................................ 46 Validation of K2CO3 as density-solution by recovery-experiments ..................................... 46 Plastic resistance to K2CO3 ................................................................................................... 49 Resistance of different plastic types against different chemicals and enzymes ................... 50 Comparison between virgin plastics and weathered plastics ................................................ 50 Digestion of organics by different chemicals and enzymes ................................................. 51 Testing of the whole protocol on beach samples .................................................................. 53 Conclusion and outlook ......................................................................................................... 57 Acknowledgements ................................................................................................................. 58 References ............................................................................................................................... 59 3 Abstract Plastics have been with us for about 70 years. Over time plastics accumulate in the ocean and microplastics get formed and can be found everywhere from fish-intestines, the deep-sea, to sea-ice and beaches. Beaches are good indicators for microplastic distribution. To extract mi- croplastics from beaches a density-separation is the most commonly used method. Usually, a digestive agent is used to get rid of organic pollutants. The aim of this study is to confirm potassium carbonate (K2CO3) as a valid density-medium and to find the most suitable digestive agent. By recovery experiments with PVC, potassium carbonate could be validated as a non- toxic and cheap alternative to conventional density solutions. Potassium hydroxide (KOH) so- lution worked best in digesting organic pollutants, while leaving plastics unbothered. Samples rich in plant material should be treated with Fenton’s reagent. A combination of both protocols proved to be the most efficient approach for beach sediments. Recommendations for future research include the testing of enzymatic digestion protocols and the study of techniques to separate microplastics from tar. Plastik ist bereits seit 70 Jahren unser stetiger Begleiter. In dieser Zeit sammelte sich immer mehr Kunststoff in den Ozeanen an, das sich mit der Zeit in Mikroplastik zersetzte. Mikroplas- tik ist weltweit verbreitet und wurde bereits in Fischinnereien, der Tiefsee, im Polareis und an Stränden nachgewiesen. Strände sind gut geeignet, um die Verteilung von Mikroplastik im Meer zu untersuchen. Um Mikroplastik aus Stränden zu extrahieren, wird meistens eine Dich- teauftrennung durchgeführt. Zur Aufreinigung biologischer Verunreinigungen nutzen viele Stu- dien üblicherweise Chemikalien oder Enzyme. Das Ziel dieser Studie besteht darin, Kalium- karbonat (K2CO3) als neuartiges Dichtemedium zu validieren und das passendste Aufreini- gungsverfahren für ein Citizen Science Projekt zu finden. Durch Rückgewinnungsexperimente mit PVC konnte Kaliumkarbonat als ungiftige und günstige Alternative zu anderen gängigen Dichtemedien etabliert werden. Kaliumhydroxid-Lösung (KOH) erwies sich als erfolgreichste Möglichkeit biologische Verunreinigungen, vor allem tierischen Ursprungs, zu entfernen, ohne Plastik anzugreifen. Proben, die stark mit pflanzlichen Resten verschmutzt sind, sollten mit Fenton‘s Reagenz aufgereinigt werden. Die Kombination beider Protokolle war das effizien- teste Aufreinigungsverfahren in dieser Arbeit. Empfehlungen für zukünftige Arbeiten beinhal- ten die Untersuchung enzymatischer Aufreinigungsprotokolle und Experimente zum Auflösen teerartiger Verunreinigungen. 4 Introduction Plastic – Our omnipresent companion The term plastic applies primarily to synthetic polymers, typically prepared by polymerisation of monomers. These monomers are extracted from oil or gas and various chemical additives are added to gain the desired properties (Cole et al., 2011; Derraik, 2002; Rios et al., 2007; Thompson et al., 2009). The origin of plastic dates back to 1907, when Leo Hendrik Baekeland created the first plastic made from synthetic components, called Bakelite (Baekeland, 1909; Crespy et al., 2008). Since the 1940s and 1950s plastic has become an important part of our daily life (Thompson et al., 2009). Plastic-products are easily mouldable, extremely durable, corrosion-resistant and have good insulation properties, making them versatile for all different kinds of applications, all while being incredibly lightweight (Imhof et al., 2012). In 1950 the yearly plastic production was estimated to be 2 million tonnes per year, which increased to 380 million tonnes per year in 2015, including products like resins and fibres (Geyer et al., 2017). PET-, PA-, PP- and polyacrylic fibres excluded, 322 million tonnes were produced in 2015 (PlasticsEurope, 2016). This amount (excluding fibres) increased to around 359 million tonnes in 2018, with 62 million tonnes thereof produced in Europe (PlasticsEurope, 2019).

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