Microplastics in Shore Sediments – Development of a Suitable Extraction and Purification Technique for Citizen Science Projects

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 microplastics 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) solution 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). The biggest plastic demand is in the industry of packaging production, which uses 39.9% of the plastics produced in Europe (PlasticsEurope, 2019). The cumulative waste generated in the 65 years from 1950 to 2015, amounts to 6.3 billion tonnes, of which only 9% have been recycled and 12% incinerated. 79% of the produced waste were deposited in landfills or ended up somewhere in the environment (Geyer et al., 2017). These huge amounts of plastic in landfills and nature can easily get blown away by the wind, like hurricanes for example, and ultimately reach rivers, streams and eventually the ocean (Imhof et al., 2012; Thompson et al., 2005). In 2010, between 4.8 and 12.7 million tonnes of plastic waste in different forms entered the ocean (Jambeck et al., 2015). These sources, originating in mainland, contribute even more to ocean waste than debris generated by the shipping- and fishing-industry (Imhof et al., 2012; Thompson et al., 2005). Large plastic pieces pose a huge threat for marine animals. They can cause mechanical harm to birds, fish, and marine mammals for example by being ingested. Because of their indigestibility plastics can lead to blockage of the intestines. Large debris can also cause entanglement and block the mobility of animals, interfering with their hunting- or fleeing-ability, lead to serious wounds and infections or even lead to suffocation in marine mammals (Derraik, 2002; Laist, 1987; Thompson et al., 2005; Thompson et al., 2004). Another problem occurs when the plastic

5 pieces, in various sizes, sink to the sea floor, where they can inhibit the gas circulation between sediments and the overlying water. This can lead to dangerous, anoxic conditions in the pore systems, disrupting the normal functioning of benthic ecosystems and preventing oxygen-dependent life from existing (Goldberg, 1994).

Dangers of microplastics Since plastic items typically remain in the marine environment for decades, they are exposed to lots of degrading processes like UV radiation, hydrolysis and biodegradation (Andrady, 2011; Cole et al., 2011; Imhof et al., 2012). UV radiation and mechanical abrasion are the main drivers, which lead to the formation of fragments that get smaller the longer the plastic remains in the ocean. Considering the persistence of plastics these degradation processes are estimated to take hundreds of years, a time that may even be prolonged in the deep-sea, where the absence or at least a shortage of light and oxygen inhibits the processes decomposing the particles (Barnes et al., 2009). Beaches on the other hand are hotspots of microplastic formation, since plastic degradation driven by UV radiation is very effective in such surroundings with enough oxygen available and no inhibiting UV-filtering effects by surrounding water. The absence of cooling water also speeds up the process, resulting in a fast formation of tiny particles (Andrady, 2011).

Because of the continuous and ongoing fragmentation of plastic pieces, the concentration of plastic debris is believed to increase with decreasing size of the particles in the oceans (Kaberi et al., 2013). The particles are typically assigned to one of five different categories, depending on their characteristics: Fragments, beads, fibres, foams and films (Lusher et al., 2017a).

Although there is still an ongoing debate for the size limits of microplastics, this study chose to follow the suggestions by the National Oceanic and Atmospheric Administration (NOAA) (Arthur et al., 2009) and the European Commission (Galgani et al., 2013): Once the plastic fragments reach a size of 5mm or less along the longest dimension, they are considered microplastics. This upper boarder was set to direct the conversation on ecological effects rather than physical effects, like blockage of the digestive system and because this definition is widely accepted by scientists and used in many experimental studies today (Barnes et al., 2009; Besley et al., 2017; Cole et al., 2014; Galgani et al., 2010; Moore, 2008). To enable a citizen science approach of the protocols applied in the present study, in all experiments particles <300µm were discarded. This was done to prevent samples from false positive results, because there is a high risk of contamination with smaller particles deriving from synthetic clothes for example,

6 especially when working with non-professionals. This would lead to an overestimation of microplastic distribution. Furthermore the sampling and analysis of smaller particles gets much more challenging in comparison to larger microplastics (Lenz et al., 2015), making it unpracti- cal for potential citizen science studies.

An important distinction has to be made between primary and secondary microplastics. Primary microplastic-beads are produced in huge volumes as additives in toothpaste and other personal care products and also used in airblast cleaning (Cole et al., 2011; Gregory, 1996; Zitko & Hanlon, 1991). Particles that stem from larger plastic pieces and get fragmented in the ocean are called secondary microplastics (Napper et al., 2015). Microplastics pose many dangers to the environment. Decreasing size leads to a higher risk for marine biota to ingest these particles by accident, for example as bycatch or if the plastic gets confused with sustenance (Browne et al., 2008). Microplastic-particles have already been confirmed to be found in multiple different marine groups of the animal-kingdom including whales (e.g. Besseling et al., 2015; Lusher et al., 2015), squid (e.g. Braid et al., 2012), mussels (e.g. Browne et al., 2008; De Witte et al., 2014; Li et al., 2015; Vandermeersch et al., 2015), turtles (e.g. Tourinho et al., 2010), seabirds (e.g. Tourinho et al., 2010; Van Franeker et al., 2011), crustaceans (e.g. Hämer et al., 2014; Watts et al., 2014, 2015), worms (e.g. Thompson et al., 2004), fish (e.g. Avio et al., 2015; Collard et al., 2015; Foekema et al., 2013; Wagner et al., 2017) or even zooplankton (e.g. Cole et al., 2013, 2014). Ingested particles can lead to a variety of problems, like blockage of the digestive system and reduced sense of hunger, which can lead to physical degeneration or eventually to starvation (Wright et al., 2013).

An underestimated danger of MPs is their potential to serve as vector for chemicals. Because of their particular surface structure different classes of hydrophobic organic contaminants can attach to plastic particles (Teuten et al., 2007). The large surface-to-mass-ratio improves these adhesive properties (Cole et al., 2013) and allows the polymers to adsorb chemicals in up to 105 - 106 times higher concentrations than in surrounding water and to transport them into the intestines of animals (Mato et al., 2001; Teuten et al., 2009). In the digestive system they get released because of the strong changes in pH-milieu, presence of digestive juices and enzymes (Teuten et al., 2007). Polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and dichlorodiphenyldichloroethylene (DDE) (Andrady, 2011; Derraik, 2002; Mato et al., 2001; Rios et al., 2010) are some of the typical chemicals associated with microplastics and are proven to be either carcinogenic, endocrinal active or acutely toxic. These properties can have long-lasting negative effects on organisms, like lowered enzyme production, a retarded growth-rate or disturbed hormonal balance, leading to lowered reproductive success (Azzarello

7 & Van Vleet, 1987; Galgani et al., 2010; Peakall, 1970; Rios et al., 2010; Santodonato, 1997; Yang et al., 2006). Thus, they can negatively impact the whole marine food web and accumulate in bigger predators (Teuten et al., 2009), eventually reaching and influencing humans through the food chain (Farrell & Nelson, 2013; Rios et al., 2010; Setälä et al., 2014). Besides already mentioned chemicals also toxic metals are able to adhere to microplastics and, by being available in a bioaccessible form, to enter the digestive system of marine biota (Ashton et al., 2010). Concentrations on tested plastic particles were measured to be up to 800 times higher than in surrounding water in an experimental study, making these particles possible vectors for heavy metal contamination, enabling an accumulation in the marine food web (Brennecke et al., 2016).

Apart from these accumulating properties, chemicals, incorporated during production, can also leach out of plastics, when exposed to weathering effects (Cole et al., 2013; Talsness et al., 2009; Teuten et al., 2009) or digestive fluids. Nonylphenol is one example for an endocrinological active additive (Mato et al., 2001; Soto et al., 1991). Bisphenol A gained notoriety for being carcinogenic and repressing fertility by being endocrinological active (Keri et al., 2007; Lithner et al., 2009; Talsness et al., 2009; Wagner & Oehlmann, 2009; Yang et al., 2006). Other additives used in plastic production have also been proven to be toxic for reproduction or carcinogenic, but also allergenic, mutagenic, generally toxic or hazardous to the environment (Lithner et al., 2009).

Plastic distribution in the Mediterranean Sea Microplastic seems to be ubiquitous in the ocean (Eriksen et al., 2014) and was found in sea ice (Obbard et al., 2014), marine snow (Zhao et al., 2017) and even deep-sea sediments (Van Cauwenberghe et al., 2013b; Woodall et al., 2014). Floating plastics also accumulate in the big ocean gyres in the Atlantic, the Pacific and the Indian Ocean (e.g. Eriksen et al., 2013; Law et al., 2010; Moore et al., 2001; Sesini, 2011; Van Sebille et al., 2012). The biggest one of them, the so called ‘Great Pacific Garbage Patch’ (GPGP), received lots of media attention, because of the huge loads of plastics caught in it. As early as 1999 Moore et al. (2001) found a mean abundance of around 334.000 plastic fragments/km² in the GPGP with a mean mass of approximately 5kg/km². This corresponds to approximately six times the dry weight of plankton in the same area. Lebreton et al. (2018) estimated the GPGP to contain a total of 1.8 trillion plastic pieces weighing 79.000 tonnes, using a model calculation.

8 The Mediterranean Sea is a centre of plastic accumulation as well (Lots et al., 2017), probably because of the hydrodynamics of the semi-enclosed basin, with outflow mainly occurring at the Strait of Gibraltar through a deep-water layer. These accumulations make the Mediterranean sea comparable to the accumulation zones in the big ocean gyres (Cózar et al., 2015), even leading to the name ‘Mediterranean Plastic Soup’ (Karlberg, 2013; Suaria et al., 2016). For this reason, the Mediterranean Sea has to be the focus of extensive future research. The Mediterra- nean Microplastics Citizen Science Project (MedMicroplastiCS) is a non-profit project, which aims to do that by including the public and citizen scientists. Present study is the first scientific output in the course of this project and as this thesis is written, further studies are planned to be carried out in the near future.

Microplastics in shore sediments Since the trend of the yearly growing output figures of plastic is not always reflected in an increase of the number of floating microplastic particles (Law et al., 2010), Turra et al. (2014) suggested that beaches for example, serve as depositional areas. Surface seawater has an average density of 1.025g/cm³ (Mas-Pla et al., 2013), ranging from 1.020 to 1.029 g/cm³, depending on factors like salinity and temperature of the water. Since many polymers are less dense than seawater (Table 2), they float on the surface and get eventually washed on the beaches, where they accumulate in the sediment. Plastics with a higher density are prone to sink to the bottom of the water basin, where they blend into sediments (Van Cauwenberghe et al., 2015), while also light-weight plastics gain more density and sink after a while, driven by processes like biofouling and mineral adsorption (Corcoran, 2015; Morét-Ferguson et al., 2010). On sandy or rocky shores, a so-called strandline or drift line gets formed. A strandline is a line at the upper extreme of the high tide, which consists of decomposing seaweed, debris, and plastics. Strand- lines are not immovable; they can get shifted by the tides or other factors like wind. Storms and extreme weathers can also have a huge impact on strandlines (EUNIS habitat classification - http://eunis.eea.europa.eu/habitats/5444; accessed on 07. October 2019).

On the strandline plastics and other debris accumulate and can be seen with the naked eye to some extent (Figure 1). Imhof et al. (2018) suggested the drift line as a comparable sampling spot for microplastic studies, because of the accumulation of the microplastics in this zone. Beaches are also easily reachable sampling spots, enabling citizen science studies to take place (Lots et al., 2017) and can reflect the global increase in plastic production (Claessens et al., 2011). Because of the wide variety of methods in microplastic sampling in beach sediments,

9 different studies have not always been comparable in the past, hence this reflects a possible source of errors (Imhof et al., 2018). Costa & Duarte (2017) also highlighted the importance of the strandline, because it reflects the most recent input from the preceding tide. It also shows the importance of storm events and mirrors the floating portion of microplastics in the water column. By examining the strandline, a comparison of microplastics with larger plastic items is enabled, since those items can get sampled on the same beach at the same time. Beach clean- ups are gaining popularity, which can be used in citizen science projects. The strandline also makes it possible to track a seasonal gradient of microplastic distribution, by continuous obser- vation throughout the year and also factors like coastline orientation and wave conditions, which are known to be mainly responsible for debris accumulations (Herrera et al., 2018).

Citizen science Since the strandline is an easily accessible sampling spot, it is possible to include non-professional volunteers in the sampling steps. Volunteers included in scientific projects, who help collect or analyse data, are called citizen scientists (Silvertown, 2009). The integration of such persons has multiple advantages. Raising awareness for the growing plastic problem in the public is an important point and can lead to better environmental consciousness. The importance of recycling and reuse should be underlined, to encourage a more sustainable handling with plastic items. By the inclusion of online tools volunteers can find projects fitting their interests (Kobori et al., 2016). The second big advantage of the assignment of volunteers is the high number of samples obtainable by this strategy, if the citizen scientists are deployed in this step (e.g. Lots et al., 2017). Environmental studies are most often in need of large sample numbers, especially when sampling should happen throughout the year, and very often these can only be gained with citizen science (Silvertown, 2009). Larger sample sizes tend to have a lower error rate, but it has to be acted with caution, to minimize possible sampling errors by differently trained, experienced or biased observers (Dickinson et al., 2010). Since only a limited number of professionals, who need to supervise the sampling process, has to be paid, this can help save money (Cohn, 2008), which can be used for equipment and to bring attention to the cause of the study, to gain a higher interest by the public. Hidalgo-Ruz & Thiel (2013) confirmed that the assignment of citizen scientists can help generate large sample numbers, without suffering from a loss of quality. In their study schoolchildren were able to follow instructions, which helped to gain valid data for spatial and temporal distribution of microplastics. Citizen science projects are a great possibility for volunteers to gain scientific skills and knowledge, like the

10 development of scientific questions, or data collection- and analysis-techniques (Kobori et al., 2016; Rowe et al., 2019).

The aim of this work was to identify the easiest and fastest method to sample beaches and extract the microplastics from the sediment. The following purification process had to be time- efficient and cheap, to keep up with a possible high number of samples, to allow the analysis of samples taken by volunteers. A special focus was the establishment of a non-toxic method to extract the plastics, to allow untrained citizen scientists to conduct this task and to enable an environmentally acceptable approach.

Location of the sampling spot Since plastic is not equally distributed across beaches, the three-dimensional arrangement of the sediment needs to be taken into account (Costa & Duarte, 2017). There are different possi- bilities to take sediment samples from beaches, like core sampling (e.g. Turra et al., 2014) or superficial sampling (e.g. Lots et al., 2017). As mentioned above, the strandline seems to be a comparable sampling spot (Imhof et al., 2018) and was therefore used as point of reference in this study. Regarding the depth of the samples some papers suggested that the upper layer was more promising. Yu et al. (2016) compared superficial surface samples (0 – 2cm depth) with deeper sediment samples (20cm depth). The superficial samples contained more microplastics than deeper samples, but without statistical significance. The results of Carson et al. (2011) coincide with these findings. 50% of plastic fragments were found in the upper 5cm of their sediment cores and nearly 95% were located in the upper 15cm. It has to be mentioned that they only sampled a maximum depth of 25cm. In contrast Cannas et al. (2017) compared the upper 50cm to deeper samples with 100 – 150cm depth. The deeper samples included significantly higher levels of microplastics, but the authors attributed these high levels to a flood event, which took place a few years prior to the study and substantially changed the morphology of the beach. Turra et al. (2014) took samples as deep as 2m and found accumulations in 20cm, 40cm and 60cm depth. Even deeper samples contained significantly less microplastics. They estimated that if other studies took deeper samples than the surface layer in consideration as well, microplastic abundances might be much higher. Fisner et al. (2017) stated that more than 90% of plastic occurred in the top 1m in their study, while the top 2m were sampled. Yu et al. (2016) speculated that the high abundance of microplastic particles in higher layers could result from the direct exchange with sea water that the top layers of sediment undergo. Because of that it is

11 presumable and reasonable that more microplastics end up in the upper sand layers. There is more data needed, to conclude on the best sampling spot.

Material and methods

Field sample collection This study decided to focus on the top sediment layer, because of the easy accessibility, which would also be needed for a citizen-science study. The upper 25cm might be easily accessible for non-professional volunteers with no specialized tools. Since the upper half of this layer seemed to include more plastics (Carson et al., 2011), samples were taken in the superficial 5cm, as recommended by Besley et al. (2017) and Galgani et al. (2013). If the top layer was made up of coarse sediment, pebbles larger than 1cm were removed manually. For sampling a stainless-steel cylinder with 15cm diameter was pushed 5cm into the sediment and a metal spoon was used to transfer the sediment into a jar, which was then closed with a lid. That way it was stored, until it was used.

A B

Figure 1: A: Strandline of Uvala Lakošaše; B: Strandline of Uvala Stoja. Multiple strandlines are visible. The upper one was used for sampling.

12 Contamination prevention To prevent contamination of the samples, they were taken using stainless-steel tools and were stored in a capped glass until analysis. All solutions, including density solutions for the separation of microplastics and sand, and digestive agents, were mixed using distilled water and the respective chemicals in powdery form or used directly from packaging. While not in use, the solutions were also stored in closed glass bottles. During all experiments, a cotton lab coat, safety goggles and nitrile or latex gloves were worn. A clean working area was assured, by cleaning with ethanol before and after each experiment using a cellulose cloth.

When working with non-professional volunteers, contamination with fibres cannot be prevented, because of the use of synthetic clothing and a lack of facemasks for example. Because of the citizen science-friendly approach of this study, in all experiments, particles smaller than 300µm were discarded. Hence this study focused on particles between 300µm and 5mm.

Density separation Conventional density solutions Literature research showed different methods for microplastic isolation from sediment samples (Hidalgo-Ruz et al., 2012; Van Cauwenberghe et al., 2015). Visual sorting is used by many different studies, but tends to be biased and has a high error rate regarding particles with a similar size and shape compared to natural sediments, especially in organic-rich samples (Dekiff et al., 2014; GESAMP, 2015; Lenz et al., 2015; Lorenz, 2014; Tagg et al., 2017). Hidalgo-Ruz et al. (2012) stated that as much as 70% of visually identified microplastic particles cannot be confirmed by FT-IR spectroscopy. Hence density separation seems to be a more reliable method and has been used by multiple studies (Table 1). Density separation is based on the differences in density between plastic particles and sediment. Usually, the aim is to let the plastic float on the surface, while the sediment remains on the bottom of the extraction tank. Recovery rates of plastic are mainly dependent on the density solution used (Hamm et al., 2018). An overview about previously used solutions and their densities is given in table 1.

13 Table 1: An overview about different flotation agents used in different studies for microplastic extraction. The used density range in mentioned literature is given, as well as the German Water Hazard Classification (WGK: Wassergefährdungsklasse) retrieved from Carl Roth, product data sheets and Umweltbundesamt IV 2.6: WGK1 (lowly hazardous to waters), WGK2 (distinctly hazardous to waters) and WGK3 (severely hazardous to waters). GHS-Symbols (Globally Harmonized System of Clas- sification, Labelling and Packaging of Chemicals) provided by the safety data sheets from Sigma Aldrich of regarding chemicals are listed. For a detailed explanation on the hazard symbols visit https://echa.europa.eu/regulations/clp/clp-pictograms. Prices were looked up on https://www.sigmaaldrich.com/catalog in April 2020 for chemicals with 99% purity; exceptions noted. *1: Yu et al. (2016) stated a density of 1.27g/cm³, which is not possible for NaCl-Solution, hence a typing error was assumed and the highest possible density at 0°C was put instead (https://handymath.com/cgi-bin/nacltble.cgi?submit=Entry; accessed on 16. April 2020). *2: Only prices for smaller amounts were given, which were extrapolated to 1kg. *³: Lithium metatungstate was not sold on beforementioned website, but instead it was sold by LMT Liquid, LLC; https://www.lmtliq- uid.com/price-and-order-information.html (accessed on 16. April 2020) as ready-to-use solution. Density- German European Hazard Flotation agent Literature range used Water Hazard Price [€/kg] Symbols [g/cm³] Classification Sodium chloride a-i, ad-ag 1.15-1.207*1 WGK1 - 28.50 (NaCl)

Calcium chloride 99*² (≥ 98.0 j, ai 1.30-1.46 WGK1 (CaCl2) %)

Sodium bromide e 1.37 WGK1 - 67.20*² (NaBr) Sodium tungstate ah 1.5 WGK1 504*² (Na2WO4·2H2O)

Sodium chloride (NaCl) + Sodium c 1.50 WGK3 28.50/330 iodide (NaI)

Potassium for- k 1.5 WGK1 - 77.70 mate (HCO2K)

Potassium car- present 1.54 WGK1 36.20 bonate (K2CO3) study

Lithium metatungstate d 1.62 Not classified 750$/Litre*³ -24 (Li2O13W4 )

Zinc bromide e 1.71 WGK3 185 (≥98%) (ZnBr2)

Sodium polytungstate 2070*² l-n 1.4-1.8 WGK2 (3Na2WO4·9WO3 (≥85%) ·H2O)

Zinc chloride o-w, ad 1.4-1.8 WGK3 232*² (ZnCl2)

14 Sodium iodide b, e, x-z, aa-ad, ai 1.566-1.98 WGK3 330*² (NaI)

a: Carson et al., 2011, b: Dekiff et al., 2014, c: Han et al., 2019, d: Masura et al., 2015, e: Quinn et al., 2017, f: Reddy et al., 2006, g: Thompson et al., 2004, h: Vianello et al., 2013, i: Yu et al., 2016, j: Stolte et al., 2015, k: Zhang et al., 2017, l: Ballent et al., 2016, m: Corcoran et al., 2009, n: Enders et al., 2020, o: Bergmann et al., 2017, p: Imhof et al., 2012, q: Imhof et al., 2013, r: Imhof et al., 2016, s: Imhof et al., 2018, t: Liebezeit & Dubaish, 2012, u: Löder et al., 2017, v: Mintenig et al., 2017, w: Rowe et al., 2019, x: Van Cauwenberghe et al., 2013a, y: Van Cauwenberghe et al., 2013b, z: Claessens et al., 2013, aa: Nuelle et al., 2014, ab: Kedzierski et al., 2017, ac: Fischer & Scholz-Böttcher, 2017, ad: Coppock et al., 2017, ae: Browne et al., 2011, af: Avio et al., 2015, ag: Claessens et al., 2011, ah: Dehaut et al., 2016, ai: Crichton et al., 2017

Sodium chloride (NaCl) solution, which has been used in many experimental studies (as listed in table 1), has a density of maximum 1.207g/cm³. Since many types of plastic have higher densities than NaCl-solution, they do not float on the surface and cannot be extracted using this medium. A NaCl-solution with a density of 1.18g/cm³ for example can only yield recovery rates of 54%, as measured by the European plastic production (Kedzierski et al., 2017). PVC (1.10 – 1.58g/cm³) and PET (1.29 – 1.45g/cm³) (Table 2) for example, are too dense to float in this solution but are ranked among the plastics with the highest yearly production (PlasticsEurope, 2019). To extract 93-98% of plastics a solution of 1.8g/cm³ density is needed (Kedzierski et al., 2017). To achieve higher densities, typically solutions like sodium polytungstate, zinc chloride or sodium iodide are used (Table 1). But also solutions like zinc bromide, sodium tungstate, lithium metatungstate, potassium formate and a mix between sodium chloride and sodium iodide are used to achieve densities of 1.50g/cm³ or higher, which Han et al. (2019) considers necessary to extract the most common types of microplastics. This conforms to table 2, as only PVC, of the most common polymers (PlasticsEurope, 2019) can be denser than 1.50g/cm³.

Multiple studies mentioned the expensiveness of one or more of the listed density media (Claessens et al., 2013; Coppock et al., 2017; Han et al., 2019; Kedzierski et al., 2017; Miller et al., 2017; Nuelle et al., 2014). To ensure comparability, prices for one kilogram of the sub- stances at a purity of ≥99% (exceptions mentioned) were looked up on https://www.sigmaaldrich.com/ in April 2020 (Table 1). It is questionable, if these high purities are necessary, but for standardisation purposes, they were chosen for this table. A trend, for denser solutions to be more expensive, can be seen. This is an important factor for the funding of projects and should be especially considered in citizen science projects. The desirable huge number of samples needs a lot of density separations. Hence, to keep science affordable, a reasonable but rather cheap density solution should be chosen.

15 This study particularly focused on environmental safety as a main property, for the choice of a density medium. For this purpose, the German water hazard classification (Wassergefähr- dungsklasse WGK), as well as hazard symbols provided by safety data sheets of the used chemicals, were compared. The water hazard classification is divided into three classes: WGK1 (lowly hazardous to waters), WGK2 (distinctly hazardous to waters) and WGK3 (severely hazardous to waters). The European hazard symbols are standardised by the European Union and the latest version of the safety data sheet for each chemical was used as reference. Additionally, the GHS (Globally Harmonized System of Classification and Labelling of Chemicals) Hazard Statements are mentioned in the following paragraph, to underline the pictograms (https://pub- chem.ncbi.nlm.nih.gov/ghs/). Again, a trend can be seen, for denser solutions to be more hazardous to the environment.

Zinc chloride, for example, which has been used in many studies, seems to be very dangerous for the environment, especially for waters. It is explicitly mentioned to be very toxic to aquatic life with long lasting effects (GHS hazard statement: H410) and is considered severely hazardous to waters (WGK3). Sodium iodide is marked with WGK3 according to the environmental agency Germany IV 2.6 (=Umweltbundesamt) and the safety date sheet states that it’s very toxic for aquatic life (H400). Nuelle et al. (2014) worked with NaI, because they preferred to work with “environmentally friendly chemicals and to ensure maximum cost-effectiveness”, which cannot be confirmed by the present study. Sodium polytungstate is described as distinctly hazardous to waters (WGK2) and harmful to aquatic life with long lasting effects (H412) and zinc bromide is toxic to aquatic life with long lasting effects (H411) and severely hazardous to waters (WGK3). Furthermore, the aforementioned chemicals are labelled with the phrase “a release to the environment should be avoided” (P273) (Table 1; Sigma Aldrich and Carl Roth, product data sheets).

Potassium formate seems to be the only substance used in past studies, which meets all points of expectations (Table 1), but has only been used in one study (Zhang et al., 2017), which did not test for any advantages or disadvantages like polymer persistency or recovery rates.

A never before used flotation medium, which conforms to all desired properties, is potassium carbonate (K2CO3), as suggested by Gohla et al. (2020). It is even cheaper than potassium formate and is easily obtainable, another advantage regarding a possible usage in a citizen science project.

16 Potassium carbonate (K2CO3) – A novel, cheap and non-toxic solution Potassium carbonate is a cheap, environmentally acceptable alternative (Table 1). It can be easily obtained and has a solubility in water of 112g per 100ml at 20°C (https://www.ilo.org/dyn/icsc/showcard.display?p_version=2&p_card_id=1588; accessed on 29. April 2020) reaching densities up to approximately 1.8g/cm³. For the experiments in this work a solution with a density of 1.54g/cm³ was prepared, assuring a reasonable mixing-time suitable for citizen-science. A density of 1.54g/cm³ is enough for most particles (Han et al.,

2019), including PVC, to float (Table 2; Chapter: ‘Validation of K2CO3 as density-solution by recovery-experiments’). Exceptions include heavy polymers like POM (Polyoxymethylene; 1.41-1.61g/cm³), different polyesters (1.01-2.3g/cm³) or PTFE (Polytetrafluoroethylene; 2.15- 2.20g/cm³) (EPA, 1992; Hamm et al., 2018; Hidalgo-Ruz et al., 2012). These plastics, however, are not very common in usage and are not expected to be found on beaches in high amounts.

Mixing of the solution

To produce one litre of potassium carbonate solution with a density of 1.54g/cm³ 770g of K2CO3 in powdery form have to be dissolved in 500-600ml of distilled water and filled up to 1l. The water was put into a flask first and put in motion using a magnetic stirrer. Under constant stirring the powdery K2CO3 was added in small portions, until everything was in the flask. The stirring process continued for about 20 minutes until the solution was completely clear. Since this is an exotherm reaction, the solution got very hot during mixing. To make sure, no plastics were damaged in the extracting process, the solution was cooled down to room temperature. A concluding density check was performed to confirm the desired density, by weighing a defined volume of the solution (100ml of the solution needed to weigh 154g, to validate the desired density).

Storing of the solution

Before mixing of the solution, the K2CO3 powder was stored in a closed bucket, to protect the powder from air humidity and possible contamination with particles floating in the air. Moist powder clumps together and complicates the handling. To prevent the mixed solution from getting polluted by surrounding microplastics in the air or other pollutants, it was stored in closed, tinted glass bottles at room temperature with a filling quantity of 3l each. By closing the bottles also evaporation of liquid was prevented, which could have had an impact on the density.

17 When potassium carbonate solution is stored, it sometimes tends to crystalize as dihydrate salt. Though not an unusual behaviour, the solution needed to get stirred before each application using a magnetic stirrer. This provided a homogenous distribution of the potassium carbonate in the solution, leading to a uniform density. Before usage, a quick density check was performed, by weighing a defined volume of the solution.

Recycling Multiple studies performed recycling and reuse of their respective flotation media (Enders et al., 2020; Han et al., 2019; Kedzierski et al., 2017; Nuelle et al., 2014), which had no significant impact on background contamination, hence the solution in this study was also recycled to reduce waste and material cost.

The potassium carbonate-solution used in the preceding steps was collected and filtered using a vacuum pump and filter-paper (e.g. 4µm pore size). This assured that no left-over particles from the last sample were transferred into new samples. Because of potassium carbonate stick- ing to filters and the settling process during the extraction the density of the filtered solution was always less than 1.54g/cm³. To regain the original density 1l of the filtered solution was weighed. The difference to 1540g was added in form of new potassium carbonate-powder to the solution. It was dissolved in the solution by stirring with a magnet-stirrer for about 20 minutes, until the solution was completely clear again. On average the filtered solution had a density of 1.50g/cm³ (± 0.02g/cm³) and 36.58g (± 12.59g) of powder was needed to regain the desired density, when 1l of the solution was recycled.

The recycled potassium carbonate-solution was not colourless, like fresh solution, but rather yellow, but still transparent. After a storage-time of more than 24 hours, the recycled solution started to form streaks at ground level and had to be stirred again before usage. The consistent density throughout all experiments was important and tested rigorously, hence a concluding density check was performed, by weighing of a defined volume of the solution.

This recycling-step helped to reduce the produced waste-products, to be consistent with the environmental-friendly approach of this work.

Plastic types Polymers can be roughly separated into thermoplastics, thermosets and elastomers. Thermo- plastics are polymers that become mouldable when heated and harden when cooled again. Ther- mosets on the other hand cannot be re-melted and reformed after initial hardening, because they

18 undergo a chemical change when heated and create a strong close meshed network (PlasticsEurope, 2019). Elastomers are plastics of good dimensional stability, but with high elasticity, like rubber. Since there is a huge number of plastic types, table 2 only lists the plastics with the highest demand in Europe (PlasticsEurope, 2019) and additionally other polymers in- teresting for this study. In table 3 typical applications of the different types of plastic are given.

Table 2: Densities of different polymers according to Andrady (2011), EPA (1992), Hamm et al. (2018), Han et al. (2019), Hidalgo-Ruz et al. (2012), Nuelle et al. (2014), Qiu et al. (2016) and Quinn et al. (2017). European plastic demand according to PlasticsEurope (2019). Main plastics examined in the present study marked in green. Density-range European plastic Recycling Plastic type [g/cm³] demand [%] Code Low-Density Polyethylene 0.89 – 0.94 17.5 04 (LDPE) Polypropylene (PP) 0.83 – 0.946 19.3 05 High-Density Polyethylene 0.94 – 0.97 12.2 02 (HDPE) Polystyrene (PS) 0.96 – 1.10 6.4 06 Expanded Polystyrene (EPS) 0.015 – 0.03 Polyamide (PA) 1.02 – 1.15 ~ 2 07/PA Polycarbonate (PC) 1.2 ~ 2 07 Cellulose acetate (CA) 1.22 – 1.24 Polyurethane (PUR) 1.05 – 1.28 7.9 07 Polyethylene terephthalate 1.29 – 1.45 7.7 01 (PET) Polyvinyl chloride (PVC) 1.10 – 1.58 10 03 Polyoxymethylene (POM) 1.41 – 1.61 07 Polytetrafluoroethylene (PTFE) 2.15 – 2.2 07

Table 3: Typical applications for different plastic types, adapted from Andrady (2011), GESAMP (2015) and Hurley et al. (2018). Plastic type Typical applications (examples) LDPE Plastic bags, six-pack rings, beverage bottles, straws Bottle caps, straps, nettings for different purposes, packaging, food contain- PP ers, rope, textiles, reusable containers HDPE Microbeads in personal care products, milk and juice bottles PS Plastic cutlery, food containers, buoys, foam cups, cool boxes, packaging PA Synthetic fibres (nylon), netting, fishing nets, rope, plastic foil

19 PC Beverage bottles, synthetic glass CA Cigarette filters PUR Mattresses, earplugs, car seats, footwear PET Beverage bottles, plastic containers, synthetic fibres (polyester) PVC Pipes, Plastic foil, beverage bottles, cups, containers POM Eyeglass frames, gear wheels, ski bindings, guns, knife handles PTFE Non-stick coating for pans, containers

This work focused on the plastics with the biggest demand in Europe (PlasticsEurope, 2019), being Low-Density Polyethylene (LDPE), Polypropylene (PP), High-Density Polyethylene (HDPE), Polystyrene (PS), Polyurethane (PUR), Polyethylene terephthalate (PET) and Polyvi- nyl-chloride (PVC). Polyamide (PA) was additionally examined, because of its typical application in fishing nets and the frequent mentioning in other studies, and includes PA6.6, the original Nylon, as well as the often-used PA6, which is also considered to be nylon. It has the same chemical properties, but a different production.

Cellulose acetate (CA) is another material regularly mentioned in studies about digestive agents, because of its usage in filters, especially laboratory and cigarette filters. It gets easily destroyed by digestive solutions (e.g. Dehaut et al., 2016; Prata et al., 2019). Since CA is de- rived from cellulose, it accounts as a natural polymer and for this reason has high potential to get degraded in the environment (Puls et al., 2011). This explains, why CA is not found as microplastic in marine organisms or other environmental studies (Dehaut et al., 2016). Because of this property CA was not investigated in present study.

Secondary microplastic fragments used in our experiments were made by shredding easily obtainable items (Table 4) using various methods. Each item was identified using the resin identification code or other markings on the packaging. Foamed polystyrene (PS) was plucked apart into single beadlets with forceps. The Nylon-string (PA), the packaging material (LDPE), the soft drink bottle (PET), the mattress (PUR) and the shower gel bottle (HDPE) were cut into small pieces with a scissor. Falcon tubes (PP) and the PVC-pipe were shredded with a house- hold-grater. Additionally, weathered plastics from a beach in Pula, Croatia were collected and shredded in the same way as the associated virgin polymer, as well for the comparison between virgin and weathered plastics. The upper size-limit for used particles was 5mm.

20 Table 4: Plastic items used in present study. Weathered plastics have been recovered from a beach in Pula, Croatia and were identified by the resin identification code. Origin Plastic type Virgin plastics Weathered plastics LDPE Packaging material - PP Falcon tubes Yogurt cup HDPE Shower gel bottle Bottle caps PS Foamed polystyrene Foamed polystyrene PA Nylon string Bristles of a brush PUR Mattress Earplug PET Soft drink bottle Soft drink bottle PVC PVC-pipe -

Plastic resistance to K2CO3 For potassium carbonate to be useful for plastic extractions, it needs to be compatible with all important plastics, that can be found on beaches. According to Bürkle GmbH (2020) polycarbonate has limited resistance to K2CO3. Since PC does not belong to the plastics with the highest yearly production in Europe (PlasticsEurope, 2019), it may not play a crucial role in the total abundance of plastic on beaches. Own visual investigation of plastic particles after K2CO3 treatment showed no optical changes in microplastics. To test for changes in weight, a weighed amount of eight different types of microplastic particles (LDPE, PP, HDPE, PS, PA, PUR, PET and PVC) has been mixed into a potassium carbonate-solution, where they remained for 80 minutes, which is four times the approximate duration of the extraction used in this study, including stirring time and waiting period. Particles were rinsed with clear water using a 0.3mm sieve after the experiment, put in a drying chamber at 50°C overnight and weighed again.

Validation of K2CO3 as density-solution by recovery-experiments Mean recovery was tested with beach-sand from Pula, Croatia and with fine aquarium-quartz- sand. The beach-sand was sieved in different size classes, while the quartz-sand was used as a mixture of very fine sediment fractions (Table 5). Before usage, the beach-sand was washed with water and K2CO3-solution. Afterwards it was put in the drying oven at 60°C until the sand was completely dry. This way it was ensured, that no plastics from the environment tampered with the recovery-experiments.

21 Table 5: Exact composition of the quartz-sand. Size fraction Percentage 63-125µm 1.6% 125-250µm 25.8% 250-500µm 71.8% 500-2000µm 0.8%

The microplastic particles used in the recovery-experiments were self-made using a PVC-pipe and a stainless-steel grater. PVC was used because of its high density, since a convenient density solution should be able to extract plastics as dense as PVC. 30 random plastic particles produced this way were measured on the longest axis, using a stereo microscope (Olympus SZ40). The particles (Figure 2) had a size range between 400 and 3900 µm, with a mean of 1646.7 ± 862.9µm, being distinctly under the, in this work used, upper limit for microplastics (5000µm). 170g of each class of sand was polluted with either 0.5g or 0.05g of grated PVC, resulting in six different approaches (Table 6). Each approach was repeated five times.

Figure 2: Nine examples of grated PVC-particles, used for the recovery experiments. Each side of the blue squares equals 5mm.

Table 6: Six different approaches on density separation. Each approach was repeated five times. Size-class

Quartz-sand 250-500µm 500-2000µm 0.5 0.5 0.5 Mass of microplastic [g] 0.05 0.05 0.05

For each polluted 170g portion of sand one overflow cup (Figure 3) was prepared and filled with K2CO3-solution till the 5cm-mark (=115ml). Under continuous stirring the sand was slowly added and, after more solution was added up to 1cm below the escape tube, stirred for

23 another 3 minutes with a glass stirrer. After stirring the solution was left to set for 15 minutes, giving the sand time to settle and the plastics to float. After this waiting period more K2CO3- solution was poured into the overflow-cups carefully, so that the solution and the floating particles descended through the escape tube. The outflow was collected in a folded filter, hold by a funnel. The filtered solution was collected and recycled at a later point, as explained before. The extract was rinsed three times with regular tap water, followed by three washing steps with vinegar to solve any possible remnants of K2CO3. The last three washing steps were conducted using distilled water, to get rid of any remaining vinegar. At last, the filter including the extract was put into the drying chamber at 50°C for at least 12h to evaporate the water. This step left the samples ready to be weighed.

Figure 3: Schematic set-up of the experiments with overflow-cups. Brown: Sediment; Red: Plastic particles in the sediment and floating on the density solution; Grey: Glass stirrer to put sediment in motion; Blue: Potassium carbonate solution; Green: Funnel with filter to collect floating plastic particles. The lower part of the cylinder with a height of 5cm is equivalent to a volume of 115ml.

Digestion of organic pollutants One of the biggest challenges in microplastics-extraction from different media are organic pollutants, reaching from tiny copepods to algae and animal- or plant-fragments. These pollutants are masking microplastics and therefore should be removed. Especially white microplastics are

24 prone to be confused with natural debris, while for example blue fragments can be identified as plastics most of the time (Lavers et al., 2016; Lenz et al., 2015). The type of organic pollutants and the observer experience had distinct impact on the detection probability, which ranged from 60 to 100%, depending on the colour of the polymers (Lavers et al., 2016). Lenz et al. (2015) showed that blue particles were successfully identified with 86% accuracy, while green and red particles were correctly identified in 54% and 52% of cases, respectively. White, grey, black, or transparent particles had a much lower success rate with 41 – 42%. In literature even error rates as high as 70% are mentioned (Hidalgo-Ruz et al., 2012).

Biological pollutants can be removed by concentrated acids and alkalis, which are known to be able to cleave proteins, fats and carbohydrates (Nuelle et al., 2014). Also oxidising agents and enzymes are used on a regular basis for this purpose (Table 7).

After an extensive literature research, a lot of different protocols for the digestion of organic pollutants in microplastic samples were found. Some of these protocols have been used on sediment samples (e.g. Masura et al., 2015; Nuelle et al., 2014), but many studies also worked on different sample matrices like water samples (e.g. Cole et al., 2014), sea food (e.g. Avio et al., 2015; Lusher et al., 2017a; Lusher et al., 2017b) or sewage sludge (e.g. Mintenig et al., 2017).

One goal of this study was to single out the most promising digestion protocols, or rather point out protocols, which should be avoided, because they attack polymers or because of insufficient digestion efficiency.

Table 7: Different chemicals or enzymes used to dissolve organic molecules in microplastic samples. Treatments are often used at different concentrations, temperatures, or reaction times or in combination with each other. Treatment Literature Hydrogen peroxide Avio et al., 2015; Claessens et al., 2013; Hurley et al., 2018; Karami et al., 2017; Klein et al., 2015; Li et al., 2015, 2016; Nuelle et al., 2014; Prata et (H2O2) al., 2019; Zhao et al., 2017 Potassium hydroxide Dehaut et al., 2016; Foekema et al., 2013; Hurley et al., 2018; Karami et al., 2017; Kühn et al., 2017; Lusher et al., 2017a; Prata et al., 2019; (KOH) Wagner et al., 2017; Zhang et al., 2017 Sodium hydroxide Avio et al., 2015; Claessens et al., 2013; Cole et al., 2014; Dehaut et al., 2016; Hurley et al., 2018; Karami et al., 2017; Mintenig et al., 2017; (NaOH) Nuelle et al., 2014 Claessens et al., 2013; Cole et al., 2014; Karami et al., 2017; Nuelle et al., Hydrochloric acid (HCl) 2014 Avio et al., 2015; Claessens et al., 2013; Collard et al., 2015; Davidson & Nitric acid (HNO3) Dudas, 2016; De Witte et al., 2014; Dehaut et al., 2016; Karami et al., 2017; Nuelle et al., 2014; Prata et al., 2019; Vandermeersch et al., 2015

25 Peroxymonosulfuric acid Imhof et al., 2016, 2018; Klein et al., 2015 (H2SO5) Sodium dodecyl sulfate Prata et al., 2019 (SDS; NaC12H25SO4) Sodium hypochlorite Collard et al., 2015; Karami et al., 2017 (NaClO) Potassium persulfate (K2S2O8)/Sodium hy- Dehaut et al., 2016 droxide (NaOH) Hydrofluoric acid (HF) Dubaish & Liebezeit, 2013

Perchloric acid (HClO4) De Witte et al., 2014; Dehaut et al., 2016; Vandermeersch et al., 2015 + Nitric Acid (HNO3) Fenton’s reagent (H2O2 + Bergmann et al., 2017; Dyachenko et al., 2017; Hurley et al., 2018; FeSO4) Masura et al., 2015; Prata et al., 2019; Rowe et al., 2019; Tagg et al., 2017 Trypsin Courtene-Jones et al., 2017 Pepsin Dehaut et al., 2016; Karl et al., 2014 Proteinase-K Cole et al., 2014; Karlsson, 2014; Karlsson et al., 2017 Treatments with several Fischer & Scholz-Böttcher, 2017; Löder et al., 2017; Löder et al., 2015; enzymes and reagents Mintenig et al., 2017

Note that some chemicals listed in table 7 are being applied at different temperatures, because higher temperatures can speed up the digestion process (Karami et al., 2017; Prata et al., 2019). The problem is, that also the aggressiveness against plastic particles rises with temperature (Bürkle GmbH, 2020; Thermo Fisher Scientific, 2016), hence a compromise has to be found. By an extensive literature research, improper chemicals were identified and excluded from further experiments.

As additional source Bürkle GmbH (2020) was used, which lists chemical resistance of different polymers based on specifications by manufacturers. Of the polymers investigated in this study, the following are listed in Bürkle GmbH (2020): LDPE, PP, HDPE, PS, PA, and PVC. PET is only listed in the form of a glycol-modified copolymer, called PETG and PUR is not listed in this table, so Thermo Fisher Scientific (2016) was additionally used for values regarding PUR only. Polymer-tests were listed for 20°C and 50°C. Room temperature was assumed to be 20°C in present study.

Note that Thermo Fisher Scientific (2016) tested for damage after seven or 30 days respectively, which is longer than most of the suggested protocols and Bürkle GmbH (2020) didn’t specify their reaction times. Since those values only refer to laboratory tests of raw material, they can

26 deviate from tests with processed plastics. Thus, those values in the following passages should only serve as a guideline and be regarded with caution.

In advance excluded digestive agents

Hydrochloric acid (HCl) Some kinds of plastic like PS, PA, PUR, PETG and PVC are reported to be non-inert to HCl in concentrations necessary to digest organic particles (Bürkle GmbH, 2020; Thermo Fisher

Scientific, 2016). According to Karami et al. (2017), HCl in low concentrations didn’t reach the required digestion efficiency for organic pollutants, while in higher concentrations it had a destructive impact on plastics. HCl (37%) nearly fully dissolved the polyamides PA6 and PA6.6 and only led to acceptable outcomes for PS and PP, which showed results inside the tolerance. All other tested polymers (LDPE, HDPE, PA6, PA6.6, PET and PVC) got affected by HCl. It was emphasised by this paper, that HCl melted PET fragments and led to some particles clumping together. These findings also conform to results of Nuelle et al. (2014), who worked with HCl of a medium concentration (20%), which also lacked the desired digestion efficiency. For these reasons HCl was excluded from following the experiments, because the chance of plastic fragments getting lost in the purification process was too high.

Nitric acid (HNO3) According to Bürkle GmbH (2020) and Thermo Fisher Scientific (2016) plastics like LDPE,

PP, HDPE, PS, PA, PUR, PETG and PVC are non-resistant to HNO3 in elevated concentrations.

HNO3 in low concentrations didn’t reach the required digestion efficiency for organic pollutants, but had a destructive impact on plastics in higher concentrations (Karami et al., 2017).

The polyamides PA6 and PA6.6 got completely dissolved by HNO3 (69%) and it didn’t produce desired results for any of the tested polymers (LDPE, PP, HDPE, PS, PA6, PA6.6, PET and PVC). They noted that it melted LDPE, HDPE, and PP. Dehaut et al. (2016) also advised against the usage of HNO3, primarily because of its degrading actions against PA. Therefore, HNO3 was not used in present study, because of concerns regarding plastic loss.

Sodium hydroxide (NaOH) NaOH is also known to corrode PUR and PETG and also PVC in elevated temperatures (Bürkle GmbH, 2020; Thermo Fisher Scientific, 2016). NaOH was used by Nuelle et al. (2014) and Karami et al. (2017) in different concentrations and lacked digestion efficiency in comparison to other digestive agents like H2O2. Mintenig et al. (2017) and Cole et al. (2014) also reported

27 an insufficient digestion. Dehaut et al. (2016) noted, that PET was seriously degraded by NaOH. Thus, NaOH was not considered as digestive agent in this work.

Potassium persulfate (K2S2O8) and Sodium hydroxide (NaOH) Based on Maher et al. (2002), Dehaut et al. (2016) developed a protocol consisting of a mix between K2S2O8 (0.27 M) and low concentrated NaOH (0.24 M). K2S2O8 is known to be corrosive against PA and when heated can also attack PUR and PVC (Bürkle GmbH, 2020; Thermo Fisher Scientific, 2016). The protocol required a heating process up to 65°C, which can be too high for some plastics (Munno et al., 2018) and therefore should be avoided if possible. Only CA was attacked by this approach. Even though the tolerance of plastics seems to be quite high, there are other disadvantages of this agent, i.e. the relatively high cost, the difficulty to prevent potassium persulfate-solution from crystalizing and a lack of digestion efficiency (Dehaut et al., 2016). Those reasons led to a rejection of this method as well.

Hydrofluoric acid (HF) Dubaish & Liebezeit (2013) treated their samples with HF (40%), despite their knowledge of the possibility that PC and PS may get lost during the process. According to Bürkle GmbH (2020) and Thermo Fisher Scientific (2016) PC and PS, as well as other plastics like PA, PUR, PETG and PVC are susceptible against HF. Since this agent is highly aggressive against these important plastics, it was excluded from further analysis.

Perchloric acid (HClO4) and Nitric acid (HNO3)

HClO4 (68%) was only used in a 1:4-combination with HNO3 (65%) by De Witte et al. (2014) and Vandermeersch et al. (2015). Their protocol required a 10min boiling step, which can be destructive for various plastics (Munno et al., 2018) and a second boiling step until organic tissues were completely digested. Bürkle GmbH (2020) and Thermo Fisher Scientific (2016) list LDPE, PP, HDPE, PS, PA, PUR, PETG and PVC among others as susceptible against perchloric acid. Therefore, this protocol was also not tested in this study.

Peroxymonosulfuric acid (H2SO5) Imhof et al. (2016) and Imhof et al. (2018) treated their samples with hydrogen peroxide and sulfuric acid, resulting in peroxomonosulfuric acid, also known as piranha solution. A big dis- advantage of their method is the reaction time of one week. Klein et al. (2015) used the same digestive agent, but with a much shorter reaction time; the samples were incubated overnight. Nonetheless this solution was not tested in present study, because it is known to be highly

28 explosive and hazardous (https://web.archive.org/web/20150615054735/ http:/www.ch.cam.ac.uk/sites/ch/files/users/meg27/DSO017C%20Acid%20Piranha%20Solu- tion%200911.pdf; accessed on 23. February 2020) and it is advised against using piranha solution unless absolutely necessary (https://www.drs.illinois.edu/site-documents/Piranha- Waste.pdf; accessed on 23. February 2020), which is not suitable for a citizen science approach.

Sodium dodecyl sulfate (SDS; NaC12H25SO4) SDS as digestive agent was tested by Prata et al. (2019), with the aim to find a shorter, but efficient protocol. It was tested under different temperatures and time spans but was less efficient than the control with distilled water, so this agent was not investigated any further in the present study.

Sodium hypochlorite (NaClO) Karami et al. (2017) tested NaClO (5%) on biological materials like fish muscle and skin. The tests did not result in a satisfying digestion compared to other analysed methods. Because of these results NaClO was not researched any further in this study and no polymer tests were conducted. According to Bürkle GmbH (2020) plastics like LDPE, PP, HDPE, PS, PA, PETG and PVC are sensitive against NaClO, even in diluted form, so usage of a higher concentrated solution might not be reasonable. PUR also seems so be susceptible against NaClO, at least at higher concentrations (12.5%), no values for low concentrations are given (Thermo Fisher Scientific, 2016). Hence this oxidative agent was also not used in present study.

Enzymatic treatments Literature shows many examples of enzymatic treatments of microplastic samples, mainly water samples. Typically, plastic is quite resistant against these protocols, but the problem is the time-consuming working process needed to digest organic material. This makes the application inconvenient for large sample numbers and therefore probably not suitable for citizen science projects. A deeper investigation regarding sediment samples is needed and recommended and is planned to be carried out in form of a different master’s thesis in the course of the MedMi- croplastiCS-Project. In this section a quick overview about typically used protocols is given.

Trypsin Courtene-Jones et al. (2017) tested multiple different enzymes (collagenase, papain, trypsin) under different conditions for their digestion efficiency. Trypsin led to the greatest weight loss at a very low concentration, while not impacting microplastics negatively.

29 Pepsin Pepsin, another enzymatic protocol developed for a different purpose (Karl et al., 2014), was used by Dehaut et al. (2016) in the light of microplastic extraction. Since it didn’t live up to their expectations, no following experiments were conducted.

Proteinase-K (50°C, 2h) Cole et al. (2014) and Karlsson et al. (2017) used an approach with proteinase-K, which had no significant impact on microplastics. The approach seemed promising, especially for water samples, since it yielded digestion efficiencies up to 97% regarding zooplankton (Cole et al., 2014). For digestion of larger organisms the approach is not cost-effective (Avio et al., 2015).

Treatments with several enzymes and reagents Several studies proposed treatments with multiple reacting agents, leading to complicated and long digestion steps. Löder et al. (2017) for example used a basic enzymatic purification protocol, initially developed for water surface samples, and proposed a universal enzymatic purification protocol for different kinds of samples, consisting of multiple different steps and reagents, which should be adapted to the sample matrix. The reagents used include sodium dodecyl sulfate (SDS), protease, lipase, cellulase, amylase, hydrogen peroxide and chitinase. Since each step needs to be carried out under different pH- and temperature-conditions, the duration of the digestion of one sample can take more than two weeks. Mintenig et al. (2017) used a similar protocol, but without amylase. Fischer & Scholz-Böttcher (2017) used a shorter protocol, including SDS, protease, H2O2 and chitinase, leading to a reaction time of a little more than three days.

Löder et al. (2017) specified the digestion rate of their basic enzymatic purification protocol with 98.3 ± 0.1% and the recovery rate, tested with red, fluorescent microplastics, with 84.5 ± 3.3%, which would lead to an exclusion of this protocol for present study.

Since the desired protocol eventually should be able to cope with lots of samples in a short time to be able to help a citizen science project, the time-consuming huge number of working steps makes those protocols not recommendable.

30 Most promising agents Following protocols were shortlisted for further analysis by extensive literature research and tested for polymer resistance, if not already done before, with the aim of receiving the ideal protocol. Details regarding already known plastic compatibility can be seen in table 9.

Protocol 1 – Potassium hydroxide (KOH (10%), 40°C, 96h) Karami et al. (2017) recommended a protocol using a potassium hydroxide solution (KOH 10% w/v) at 40°C for 96h. Bürkle GmbH (2020) reported polymers like PC and PETG to be non- inert to potassium hydroxide (10%). Data for PS and PVC was not given, but Thermo Fisher Scientific (2016) reported incomparability for PUR and PVC at higher concentrations (no values for 10% were given). According to Karami et al. (2017) the recovery rate for PVC was 93.3%, while the other tested polymer-classes (LDPE, PP, HDPE, PS, PA6, PA6.6 and PET) had recovery rates between 95% and 105%.

The big advantage of this approach is the easy methodology. A drawback is the relatively long reaction time of four days.

Protocol 2 – Potassium hydroxide (KOH (10%), 60°C, 24h) A similar protocol was used by Dehaut et al. (2016) and Hurley et al. (2018). They also used a KOH-solution with a concentration of 10% but with a temperature of 60°C. Because of the higher reaction temperature, the digestion was sped up to 24h. Values in Bürkle GmbH (2020) remained the same at 50°C, except for additional missing data for PA. Thermo Fisher Scientific (2016) noted a faster degradation of PVC, specifically that some effects were measured after seven days of exposure at a concentration of 5%. A concentrated solution may even cause immediate damage.

Karami et al. (2017) also used a protocol (additionally to protocol 1) with a reaction temperature of 60°C. In this case the reaction time remained 96h. Plastics that had not been attacked by KOH after 96h were marked as successful for protocol 2 in table 9. Plastics corroded by KOH after 96h were not considered in this protocol, because no information about the condition after 24h is available.

Protocol 2 has an easy methodology, while working at an acceptable time span, making it ad- vantageous over protocol 1. Three previous studies (listed in table 9) employing protocol 2 yielded contradictory results for PS. Hence, we also tested this protocol in the present study.

31 Protocol 3 – Hydrogen peroxide (H2O2 (35%), room temperature, 1 week)

Nuelle et al. (2014) used hydrogen peroxide (H2O2 35%). They stored their H2O2-covered samples for one week at room temperature. Only the size-loss of the particles was analysed in their work. According to Bürkle GmbH (2020) PA is non-inert to H2O2 (different concentrations) at room temperature and PUR also showed some damaging effects after one week, when exposed to H2O2 (30%) (Thermo Fisher Scientific, 2016). The long reaction time of one week seems to be problematic at first glance, but the low number of steps required combined with the easy handling of this protocol still qualifies it as a valid candidate for a citizen science project. Since only size was a tested factor in this study, all plastics were tested again for their resistance in present study.

Protocol 4 – Hydrogen peroxide (H2O2 (30%), 60°C, 24h)

Hurley et al. (2018) also used H2O2 (30%). By increasing the reaction temperature to 60°C, they decreased the reaction time to 24h. PA showed a weight increase of +7.42%, using this protocol and is also marked as not stable by Bürkle GmbH (2020). PUR showed immediate damage at 50°C (Thermo Fisher Scientific, 2016). The recovery rate of the other tested polymers (LDPE, PP, HDPE, PS and PET) was not affected by the H2O2, including PP, which is only limitedly resistant to H2O2 (30%) at 50°C (Bürkle GmbH, 2020).

Protocol 5 – Fenton’s reagent (H2O2 (30%), iron(II) sulfate (FeSO4) and concentrated sulfuric acid (H2SO4)), room temperature, 24h)

Fenton’s reagent, a mix between H2O2, iron(II) sulfate heptahydrate and sulfuric acid, was used by Hurley et al. (2018). Due to the presence of an iron catalyst, the rapid formation of free radicals can increase the digestion efficiency compared to hydrogen peroxide alone, while maintaining mild conditions (Dyachenko et al., 2017; Prata et al., 2019). For their protocol

Hurley et al. (2018) dissolved 20g of FeSO4 in 1l of distilled water and used concentrated H2SO4 to achieve a pH-value of 3.0. The solution was mixed with H2O2 (30%) in a proportion of 1:1. The reaction time of 24h is suitable for a citizen science project with lots of samples.

Of the tested plastic types, only PA was attacked by this approach, which resulted in a weight gain of +5.49%. Missing plastics types were tested in present study.

32 Protocol 6 – Fenton’s reagent (H2O2 (30%)), iron(II) sulfate (FeSO4) and concentrated sulfuric acid (H2SO4)), 50°C, 1h)

Prata et al. (2019) used a different approach with Fenton’s reagent. They mixed 15g of FeSO4 with 1l of distilled water and added 6ml of H2SO4 per litre water according to the protocol of

Masura et al. (2015). This solution was mixed with H2O2 (30%) in proportion 1:1. They used different reaction times of 1h and 6h, and reaction temperatures of room temperature and 50°C. For the present study, the protocol with a reaction time of 1h and temperature of 50°C was chosen, to receive the desired results in an optimized timespan. An increase in time only led to little improvement in digestion of organic pollutants (Prata et al., 2019) and was therefore not conducted in this study.

Table 8: The most promising protocols in short with used chemicals, reaction time and reaction temperature (RT = room 1 temperature). * : H2O2 (30%) was mixed with a FeSO4-solution of 20g/l (pH3; adjusted with H2SO4) in 1:1-ratio. *² H2O2

(30%) was mixed with a FeSO4-solution of 15g/l (including 6ml/l H2SO4) in 1:1-ratio.

Protocol Used Chemical Duration [h] Temperature [°C] 1 Potassium hydroxide solution (KOH 10% w/v) 96 40 2 Potassium hydroxide solution (KOH 10% w/v) 24 60

3 hydrogen peroxide-solution (H2O2 35%) 168 (1 week) RT

4 hydrogen peroxide-solution (H2O2 30%) 24 60 5 Fenton’s reagent*1 24 RT 6 Fenton’s reagent*² 1 50

Polymer-resistance against shortlisted protocols In the next step the most promising protocols (Table 8) were tested for the resilience of the most important plastic particles (PlasticsEurope, 2019) regarding the chemicals used in above mentioned protocols. Many kinds of plastic have already been tested before and concerning studies are cited in table 9. With the aim of completing such an overview-table, the missing polymers were tested.

The particles were dried in a drying chamber for 24h before the start of each experiment. For each approach 0.1 – 0.3g of the regarding polymer (depending on density and form/handling of each polymer) were overlaid with 20ml of the associated chemical. Glass bottles with a lid were used, but not completely closed to allow gases to escape. Experiments were prepared under the fume hood and transported in an incubator afterwards, to ensure constant temperature conditions. The samples were permanently shaken (80rpm), to make sure, that all particles had steady

33 contact with the chemicals. This speed was applied on all protocols, since studies have shown that a reduction of stirring speed did not affect the digestion efficiency (Dehaut et al., 2016). After the appropriate reaction time for each protocol, the experiment was stopped. Polymers were filtered with a stainless-steel sieve (pore size 0.3mm). After rinsing with clear water, the plastics were dried again and weighed after 24h. Each experiment was conducted in three replicates. Additionally, three control-experiments using water instead of KOH, H2O2 or Fenton’s reagent respectively were carried out for every approach. The same temperature and reaction time were maintained.

A recovery rate between 95% and 105% was considered successful, as suggested by Karami et al. (2017). By setting this range, a minor degradation of the tested microplastics or a minor weight-increase by deposition of dried chemicals or water stains, did not lead to an exclusion of the protocol immediately.

Table 9: Overview of the corrosive effects on different polymers by the tested protocols. Plastics were weighed before and after treatment (exceptions mentioned). Recovery rate between 95% and 105% was marked as successful (=green), while others were marked red. Boxes with mixed results by different sources were marked yellow. Boxes with blue frames were examined in present study. *1: Nuelle et al. (2014) only tested for differences in size and not in weight, so these findings needed to get tested again in this study, to ensure comparability with other protocols.

Protocol 1 Protocol 2 Protocol 3 Protocol 4 Protocol 5 Protocol 6 (KOH) (KOH) (H2O2) (H2O2) (Fenton’s reagent) (Fenton’s reagent) PP a a, b, c d *1 c c e

LDPE a a, b, c d *1 c c e

HDPE a a, b, c d *1 c c e

PS a a, c d *1 c c e

PA a a, b, c c (+7.42%) c (+5.49%) e

PUR b d *1

PET a b, c d *1 c c e

PVC a (-6.7%) b d *1 e a: Karami et al., 2017, b: Dehaut et al., 2016, c: Hurley et al., 2018, d: Nuelle et al., 2014, e: Prata et al., 2019

Comparison between virgin plastics and weathered plastics An often mentioned point of criticism is the comparability between laboratory studies and environmental studies with regard to microplastic particles (e.g. Phuong et al., 2016). Some of the experiments conducted in ‘Polymer-resistance against shortlisted protocols’ were also carried out with weathered plastics found on beaches in Pula, Croatia. This was done to check for

34 comparability between vulnerability of environmental and virgin plastics. Since the amount of environmental plastic was limited, not all experiments have been performed on weathered plastics. Table 10 lists the conducted experiments.

Table 10: Protocols tested on environmental plastics for comparison with virgin polymers.

Protocol Weathered plastic 2 PS PP HDPE 3 PS PA PET 4 PUR

Testing of the six protocols on organic material After optical analysis of sediment samples collected in Pula, Croatia, ten organic materials were chosen to represent the organic matter found in the beach sediment. Prata et al. (2019) suggested using algae and wood, to represent plant tissues, fish muscle and feathers, to represent animal tissues, palm oil, as fat and paraffin, as a wax. Nuelle et al. (2014) on the other hand proposed the use of pieces of bones, chitin carapaces and feathers, standing for animal tissue and seed capsules, fruits, leaves and stems, to imitate the plant tissues found in sediment samples. In their paper particles between 0.5 and 1mm and between 2 and 3mm were used.

In the present study ten different types of organic material were chosen, to guarantee a broad- spectrum applicability (Table 11). Since three of the chosen organics are very lightweight (namely salmon skin/scales, feathers, and dead hydrangea leaves), they were used in smaller amounts to prevent them from covering the whole sample. Except for the salmon parts and the tomato-leaves, all particles were dried in a drying closet for 24h at 50°C before the experiments. Aforementioned components were not dried, to remain their original structure, in which they are also likely to be found on beaches. All components of the sample were cut to pieces of 5mm or less, to be in the same size category as microplastics.

35 Table 11: Materials used in the composition of the pooled organic sample. Percentages are calculated with dry weight, except for salmon pieces and fresh tomato-leaves. 1not dried before weighing. Represented organics Material % Sum animal/plant [%] Salmon fillet1 12.5 Salmon skin/scales1 4.17 Animal matter Eggshell 12.5 45.84 Feathers 4.17 Chitin carapace 12.5 Dead hydrangea-leaves 4.17 Fresh tomato-leaves1 12.5 Plant matter 41.67 Toothpick 12.5 Seed capsules 12.5 Wax Paraffin 12.5 Total 100

Each of the six selected protocols was tested in its efficiency of dissolving 1g of the prepared organic sample. Every organic sample was prepared individually to guarantee an even distribution of the ten components. After the treatment, the samples were rinsed with cold water, dried for 24 hours and weighed again. To examine if potential weight loss originates from the used chemicals and can’t be explained by drying effects, each protocol was simultaneously conducted using tap water. The same duration, temperature and handling was applied and only the chemicals were replaced with water. Each approach was conducted with three samples.

The two most promising protocols, for animal and plant tissues respectively, were also conducted in succession on the same samples, to further improve the digestion process. The sample was furthermore rinsed with warm water under 60°C, to help with removal of paraffin.

Testing of the whole protocol on beach samples To test the applicability of the best protocol of the ones evaluated in this study on beach samples, samples were taken in Pula, Croatia. The samples were taken in accordance with the conditions set in chapter ‘Field sample collection’ on two different beaches ‘Uvala Stoja’ (44°51'37.0"N 13°48'54.0"E; three samples) and ‘Uvala Soline’ (44°50'58.8"N 13°50'12.0"E; two samples), a fine sediment and a coarse sediment beach (Table 12). Two of the five samples were taken along the strandline, to improve chances of microplastic presence, as explained before. Sample E was taken 7m below the strandline on Uvala Stoja, to include samples with 36 different starting conditions. Since no strandline was visible in Uvala Soline, the two samples were taken along the waterline. All samples were stored with a closed lid, to prevent any contamination from happening.

Table 12: Overview over sampling spots for tested beach samples. Sample Sample location A Uvala Stoja; Strandline B Uvala Stoja; Strandline C Uvala Soline; Waterline (no strandline visible) D Uvala Soline; Waterline (no strandline visible) E Uvala Stoja; 7m below the strandline

For the extraction process 115ml of sediment was transferred into the overflow cups, which were already filled with 115ml of potassium carbonate. Inside the overflow cups the sediment was overlaid with more potassium carbonate-solution until 1cm below the escape tube. When all overflow cups were filled, all five samples were stirred for a minimum of three minutes. After stirring the samples were given a settling time of 15min. After this waiting period, the overflow cups were filled further with potassium carbonate-solution until the floating particles entered the escape tube and were pushed into the sieves (pore size 0.3mm) by the solution. Solution running through the sieves was filtered once more using a filter with a pore size of 4µm and was recycled afterwards.

The collected extract was thoroughly rinsed with cold, clear water and put in a drying chamber for 12h at 50°C. When dry, the extract was further processed using protocol 2, which was de- termined to be suitable for environmental samples, with no prior knowledge about the pollution, in chapter ‘Digestion of organics by different chemicals and enzymes’. After optically analys- ing the samples after the digestion process, Fenton’s reagent was additionally used to further improve the purification of the samples.

A concluding optical analysis was conducted, to identify particles, suspected to be microplastics.

37 Results

Validation of K2CO3 as density-solution by recovery-experiment

Table 13: Bold numbers show the mean percentage of recovered PVC in five of the six approaches. *cases with more than 100% recovery were reported in this approach. Size-class

Quartz-sand 250-500µm 500-2000µm Mass of micro- 0.5 93.2 ± 2.9 93.2 ± 2.0 93.5 ± 1.9 plastic [g] 0.05 97.5 ± 6.4* - 82.8 ± 8.7

Table 13 shows the mean recovery rates for PVC using K2CO3-Solution with 5 replicates for each approach. Overall, K2CO3-Solution led to a mean recovery rate of 92.0%. Approaches in size-class 250-500µm with 0.05g of PVC couldn’t be carried out successfully, because of the inability to remove organic pollution.

The lowest recovery rate was achieved in the size-class 500-2000µm using 0.05g of PVC, while the approach with quartz-sand and 0.05g of PVC yielded the highest recovery rate. It should be mentioned that in the latter approaches there were two cases in which the recovery rate excelled 100%. The rates were 102.4 and 105.4% respectively (see Figure 4).

120

110

100

Recovery rate Recovery [%] 70

Quartz/0,5g Quartz/0,05g250-500/0,5g 500-2000µm/0,5g 500-2000µm/0,05g

Figure 4: Boxplot of the recovery rates in different approaches using different grain size and different amounts of PVC.

38 Plastic resistance to K2CO3

Table 14: Recovery for each used type of plastic after being exposed to K2CO3 for 80min. Plastic type Recovery [%] LDPE 103.4 PP 100.6 HDPE 101.3 PS 104.7 PA 93.6 PUR 99.2 PET 97.3 PVC 104.6

Of the eight tested polymers only PA showed a weight loss greater than 5% (Table 14). The other polymers showed no noteworthy degradation after being exposed to potassium carbonate.

Resistance of different plastic types to different chemicals In accordance with Karami et al. (2017) the optimum recovery was set between 95 and 105% for these experiments. This range allows a minor degradation of the tested plastic materials or a minor increase in the weight by deposition of dried chemicals or water stains.

Table 15: Results of the plastic resistance tests conducted in this study. Additionally, the results of the control-experiments using H2O are listed. *contains one sample with only 44.3% recovery. Protocol – Control – Weight before [g] Weight after [g] Mean Recov- Mean Recov- ery [%] ery [%] A B C A B C

Protocol 1 PUR 0.2008 0.1998 0.2011 0.2032 0.2022 0.2030 101.1 ± 0.1 99.8 ± 0.8

Protocol 2 PS 0.1004 0.1016 0.1021 0.1026 0.1035 0.1048 102.2 ± 0.3 97.9 ± 0.1

PP 0.2987 0.2996 0.3058 0.2991 0.3006 0.3060 100.2 ± 0.1 100.1 ± 0.2

LDPE 0.3011 0.3000 0.3021 0.2998 0.2998 0.2994 99.5 ± 0.3 101.2 ± 1.1

HDPE 0.2004 0.2012 0.1999 0.2008 0.2016 0.1993 100.0 ± 0.2 100.0 ± 0.1 Protocol 3 PS 0.1028 0.1013 0.0990 0.1029 0.1014 0.0979 100.1 ± 0.1 99.5 ± 1.2

PA 0.2014 0.2000 0.1992 0.2015 0.1995 0.1953 99.3 ± 0.9 99.7 ± 0.3

PUR 0.2020 0.1994 0.2023 0.2010 0.1987 0.1999 99.3 ± 0.4 100.9 ± 0.2

39 PET 0.2007 0.2010 0.2017 0.2009 0.2004 0.2018 100.0 ± 0.2 100.0 ± 0.1

PVC 0.1997 0.2000 0.2006 0.1989 0.1986 0.1995 99.5 ± 0.1 100.4 ± 0.5

PUR 0.2009 0.1999 0.2012 0.1992 0.1931 0.1971 97.9 ± 1.0 75.6 ± 22.2* Protocol 4 PVC 0.2003 0.2002 0.2002 0.1918 0.1918 0.1950 96.3 ± 0.8 98.4 ± 0.7

PUR 0.2022 0.2018 0.2007 0.2017 0.2007 0.1999 99.6 ± 0.1 100.3 ± 0.4 Protocol 5 PVC 0.1998 0.1996 0.2008 0.1993 0.1986 0.2005 99.7 ± 0.2 100.2 ± 0.1

Protocol 6 PUR 0.2007 0.2011 0.2000 0.1998 0.1992 0.1981 99.2 ± 0.2 100.3 ± 0.2

All protocols tested in this study showed results between 95 and 105% recovery and were considered successful and not corrosive against the tested polymers (Table 15). Above listed results were used to complete and recolour table 9, resulting in an updated version (Table 16). This table gives an overview about the six tested protocols.

One of the three control experiments with H2O for protocol 4 conducted with PUR suffered from weight loss of 55.7%. The other two controls showed recovery rates of 92.8% and 89.8%.

Since all other approaches with PUR and H2O showed no weight loss (protocols 1, 3, 5 and 6) at different temperatures and reaction times, a handling error was assumed. A possible source of the mistake seems to be the extractor hood in the clean bench. Protocol 4 was the first to be tested on polymers in this study, hence the extractor hood was turned off during the handling of foamed plastics in following experiments, to prevent this from happening.

Table 16: Overview of the corrosive effects on different polymers by the tested protocols. Plastics were weighed before and after treatment. Recovery rate between 95% and 105% was marked as successful (=green), while others were marked red. Boxes with blue frames were examined in this study. In cases of mixed results in literature, the results conducted in this study were used in the outcome.

Protocol 1 Protocol 2 Protocol 3 Protocol 4 Protocol 5 Protocol 6 (KOH) (KOH) (H2O2) (H2O2) (Fenton’s Reagent) (Fenton’s Reagent) PP a a, b, c c c d

LDPE a a, b, c c c d

HDPE a a, b, c c c d

PS a c c d

PA a a, b, c c (+7.42%) c (+5.49%) d

PUR b

PET a b, c c c d

PVC a (-6.7%) b d a: Karami et al., 2017, b: Dehaut et al., 2016, c: Hurley et al., 2018, d: Prata et al., 2019

40 Protocol 1 Protocol 2 Protocol 3 Protocol 4 Protocol 5 Protocol 6

110

105

100 ______

95 RECOVERY [%] RECOVERY

80 PP LDPE HDPE PS PA PUR PET PVC POLYMERTYPES

Figure 5: Percentages of the remaining weight of the polymers after chemical treatment with different protocols. Pooled data of own experiments and literature (see table 16). For protocol 2 results by Hurley et al. (2018) are applied, except for PUR and PVC, which are extracted from graphs by Dehaut et al. (2016), since exact numbers were not given.

Protocols 2, 3 and 6 didn’t show corrosive effects stronger than 5% against the plastics used in this study, according to above mentioned literature (Table 16) and conducted experiments (Ta- ble 16; Figure 5). When using protocols 4 and 5 PA showed a weight gain of +7.42% and +5.49% respectively (Hurley et al., 2018). PVC showed a weight loss of 6.7% when treated with protocol 1 (Karami et al., 2017).

41 Comparison between virgin plastics and weathered plastics

** virgin weathered 102 ** ns *** ns ns ** 100

94 REMAINING [%]WEIGHTREMAINING

90 2/ePS 3/PP 3/HDPE 3/PS 3/PA 3/PET 4/PUR

PROTOCOL/TYPE OF POLYMER

Figure 6: Percentages of the remaining weight of the polymers after chemical treatment. Significant differences between reaction of virgin and associated weathered polymer are shown (Two-tailed t-test was performed using GraphPad Prism 5.01 for Windows, GraphPad Software, San Diego USA, www.graphpad.com; ns=not significant; ** = p<0.05; *** = p<0.005).

As visible in figure 6 there are significant differences between the effect of tested protocols on virgin and on weathered polymers in four out of seven cases. Only two of the results from weathered plastics are lower than 95%, defined in the present study as lower boundary for plastic resilience: Protocol 3 had a stronger effect on weathered PS and the effect of protocol 4 was stronger on weathered PUR.

42 Digestion of organics by different chemicals and enzymes

100

Chemicalchemical digestion digestion H2O-comparisonH2O-comparison

A B AB AB AB AB AB 80 C D DE EF CD EF

40 REMAINING [%]WEIGHTREMAINING

0 1 2 3 4 5 6 2+6

PROTOCOL NUMBER

Figure 7: Percentages of the remaining weight of the prepared samples treated with the six different protocols and each associated control attempt with water. Protocol 2 was additionally tested in succession with protocol 6. Letters show significant difference between chemical protocols (One-way ANOVA with Tukey’s post-test was performed using GraphPad Prism 5.01 for Windows, GraphPad Software, San Diego USA, www.graphpad.com; p<0.05).

As shown in figure 7, each sample lost weight, no matter if treated with chemicals or water. The weight loss was always higher when chemicals were used. Protocols 1 and 2, both using KOH, achieved the most promising results with a weight loss of 61.6 ± 1.1% and 61.4 ± 0.5%, respectively, with no statistically significant differences between them. Animal matter was dissolved except for eggshell and chitin carapace. The bigger problem turned out to be the plant matter, which was still present in the samples after the digestion process, except for the seed capsules. The mass of the plant matter was visibly lower, as tomato- and hydrangea-leaves for example were notably corroded, although not completely dissolved.

Regarding plant matter protocols 3 – 6 led to better results, but their overall digestion efficiency was significantly lower. Of these four, protocol 6 yielded the best results with a weight loss of 51.7 ± 4.4%. The use of Fenton’s reagent (protocols 5 and 6) led to an orange recolouring of the samples, which is caused by the precipitation of iron oxides.

43 The combination of protocols 2 and 6 led to the highest digestion efficiency, with significantly more successful results than all other protocols alone (p<0.05). Chitin, eggshell and wood-remnants were still present after the extraction.

Testing of the whole protocol on beach samples

A B C D E Figure 8: Extracts of the beach samples before chemical digestion.

The five beach samples showed highly different results. The extract from sample A was rich in organic material, especially plant tissues and snail shells. Extracts from samples B and E included heavy loads of black stone-like clumps, presumably tar. Extracts from samples C and D, representing Uvala Soline, included mainly sediment but also snail shells (Figure 8). Table 17 includes the weight of each extract before and after chemical treatment with protocol 2 and further digestion with protocol 6.

Table 17: Efficiency of the chemical digestion on the beach samples. Weight after Weight after addi- Weight of ex- chemical digestion tional chemical di- Remaining Sample tracted matter [g] with Protocol 2 gestion with Proto- weight [%] [g] col 6 [g] A 0.1261 0.0995 0.0146 11.6 B 0.0269 0.0224 0.0158 58.7 C 0.9588 0.8889 0.5050 52.7 D 0.2825 0.2743 0.2634 93.2 E 0.0174 0.0150 0.0119 68.4

I K

L Figure 9: Close-up of the samples after chemical digestion with KOH. Examples for different typically encountered materials are circled: A, G: snail shell; B: plant material; C, J: suspected polymer; D, F: tar lump; E, L: suspected polymer fibre; H, I, K: either polymer particles or splintered paint.

45 After an additional digestion step using protocol 6, particles suspected to be microplastics were optically sorted, counted, and weighed. Results are given in table 18.

Table 18: Number and weight of suspected microplastic-particles in the samples from beach-sediment. Number of suspected Sample Weight [mg] plastic particles A 9 0.0018 B 24 0.0015 C 18 0.0042 D 13 0.0017 F 6 0.0003

Discussion

Validation of K2CO3 as density-solution by recovery-experiments The biggest problems, that arose during these validation-experiments, were rooted in the usage of beach-sand. The central issue were snail shells, filled with air, leading to them floating on the surface of the density-solution. Even after preceding washing steps there were still shells in the sediment, which complicated the analysis, since no simple scaling was possible. Even an additional vacuum-step didn’t resolve this problem, since the generated vacuum, using a vacuum-pump (VacuMan Vakuumpumpe), was not strong enough to get all the air out of the shells. This made some additional steps necessary.

For the beach-sediment fractions between 500µm and 2000µm optical selection was necessary and satisfactory. Because of the size of the sediment, shells were easily identifiable under the binocular, assuring a time-efficient analysis. After shells were removed, the remaining extracts were weighed.

Approaches with quartz-sand also had difficulties. In detail, this means that very fine sand grains floated on the surface and in the liquid column and didn’t have enough time to sink to the bottom of the overflow cups. To solve this problem the waiting period would have to be significantly extended. To keep this method time-efficient a different approach was used.

Samples with 0.05g of PVC were put in Eppendorf-tubes with K2CO3-Solution and centrifuged for 3min at 14000rpm. Since the PVC has a lower density than the solution it remained floating, while the fine sand-grains settled on the bottom of the tubes. Three of these samples had a 46 recovery rate of higher than 100% after centrifuging, requiring an optical selection step. This led to only two cases, in which the recovery rate was still higher than 100%.

In samples with 0.5g of PVC, bigger tubes (20ml) had to be used, since the amount of floating plastic was too large for Eppendorf-tubes. This required the use of a bigger centrifuge, allowing only 6000rpm. The duration of the centrifugation was elongated in return to 5min, which led to satisfying outcomes.

In the few cases where there was more plastic found after the extraction than the initial plastic weight, one possible explanation might be the extremely fine sand in the approaches conducted with quartz-sand. Because of the spiral form of many of the PVC-fragments (Figure 2), fine grains of sand could get caught inside these fragments, increasing the total weight of the samples.

Since the maximum and minimum values both resulted from experiments conducted with only 0.05g of PVC, there is a possibility that the used analytical balance was not precise enough or had fluctuations influencing the weight of these small amounts of plastics. Dehaut et al. (2016) mentioned the problem of weighing uncertainty and repeated each measurement five times to show an error bar representing the amount of uncertainty. This should be considered in future studies.

Leaving the experiments with 0.05g of PVC aside, a similar recovery rate of 93.3% was achieved, instead of 92.0%.

Overall, the recovery rate is fairly high, especially considering that the experiments were conducted using PVC, which is one of the densest plastics with high demand. Because of this density it can be speculated, that it is most difficult to extract this plastic from sand. With that in mind the extraction rates for other polymers should be even higher, but that’s not entirely true, as can be seen in the following paragraphs.

Compared to NaCl-solution, potassium carbonate has a massive advantage. Recovery rates for different particle types and sediments range between 68.8 and 97.5% using NaCl in repeated extractions (Claessens et al., 2011). Quinn et al. (2017) showed recoveries between 85% and 95% for particles ranging 200-400µm and recoveries below 85% for larger particles (800- 1000µm). These larger particles match the PVC-fragments used in present study better, hence are a better comparison. For heavier polymers like PVC and PET they only showed a recovery around 65-70% using NaCl.

47 Quinn et al. (2017) also tested NaBr which showed recovery rates around 85% in the comparable size category (800-1000µm) for PVC and also overall for different polymers.

CaCl2 was tested using an air-venting technique on different coloured PE-fragments (Stolte et al., 2015), but showed an average recovery rate distinctly lower than K2CO3, with a mean around 55.5%. Crichton et al. (2017) showed an overall recovery rate of 69% for CaCl2. In their paper they also examined PVC and showed a recovery rate of 86.6%, which is still lower than the recovery rate achieved in present study. Nonetheless it is worth to mention, that the recovery rate for PVC in this study was higher, than for fibres made from PE or nylon or for ABS- (Acrylonitrile butadiene styrene), vinyl- or EPS-particles, with distinctly lower densities than PVC.

For NaI recovery rates of 83.3% have been shown including fibres, particles and PVC (Crichton et al., 2017). If only PVC is looked at, the recovery rate reaches 96.7%. It should be noted, that they repeated the extraction process a total of five times, to gain the desired recovery. Nuelle et al. (2014) showed similar results, with rates ranging from 91 (PET) to 99% (PE), except for EPS, where the achieved rate was only 68%. The recovery rate for PVC was 97% in this study. Their results are not accurately comparable, since they used a two-step procedure including air- induced overflow. NaI was also used by Quinn et al. (2017), who achieved recovery rates between 95 and 100% in the size category 200-400µm. For PVC the rate was around 97% in this category. For larger microplastics between 800 and 1000µm the rates were lower with 90-95%, being around 90% for PVC.

Quinn et al. (2017) also worked with ZnBr2, which yielded similar recovery rates. For size category 200-400µm, the recovery was 90-100% for different plastics and around 90-93% for PVC. The bigger category 800-1000µm showed recovery rates of 95-100% for different plastics and around 95% for PVC.

ZnCl2 was used by Imhof et al. (2012) and yielded a recovery rate of 99.7% for microplastics in the size category 1-5mm and a recovery rate of 95.5% for smaller particles. These rates were achieved using the Munich Plastic Sediment Separator (MPSS), an elaborate tool for microplastic analysis, which minimizes the chances of particle loss during handling. Without the MPSS the recovery rate was 99.1% for size category 1-5mm, but only 39.8% for particles smaller than 1mm.

Comparison between different studies proves to be difficult since different approaches have been conducted with different polymer-types and sizes, using different equipment and

48 techniques to improve recovery. Looking at the results, PVC is not always the polymer with the lowest extraction rate, since many factors have influence on the extraction process. Some studies used repeated extractions, others used completely different tools, but overall compared to mentioned solutions potassium carbonate can keep up with the high recovery rates needed to extract microplastics from sediment. If only the rates are considered, K2CO3 yielded better results than NaCl, NaBr or CaCl2. Recovery rates of NaI are partly higher and partly lower than the rates in the present study, depending on which study is looked at. Results of ZnBr2 are similar to the rates in the present study. ZnCl2 produced better rates than K2CO3, while being used in the MPSS, but had problems with recovering particles smaller than 1mm in the classical approach, without using the MPSS. This study used particles in a range from 400 to 3900 µm, with a mean of 1646.7 ± 862.9µm. The usage of potassium carbonate in tools like the MPSS could lead to excellent results and should therefore definitely be considered in future studies.

The biggest advantage of potassium carbonate lies in its environmental sustainability (Table 1), while maintaining high recovery rates. The cheap price and easy availability make it a valuable option for large projects, and it should be taken into consideration.

Potassium carbonate still has its flaws, namely the extraction of polymers denser than 1.54g/cm³. Some polymers like POM (Polyoxymethylene; 1.41-1.61g/cm³), different polyesters (1.01-2.3g/cm³) or PTFE (Polytetrafluoroethylene; 2.15-2.20g/cm³) (EPA, 1992; Hamm et al., 2018; Hidalgo-Ruz et al., 2012) for example reach densities, which can lead to unsatisfactory results in extractions. Only 7% of yearly produced plastics reach densities higher than 1.6g/cm³ and only 2-7% reach densities higher than 1.8 g/cm³ (Kedzierski et al., 2017), hence they only play a small part in environmental pollution. If these polymers need to be examined for various reasons, potassium carbonate can be mixed to reach densities up to approximately 1.8g/cm³.

Plastic resistance to K2CO3 As shown in table 14, only one of eight tested polymers showed degradation after being exposed to potassium carbonate. This weight loss can possibly be explained by the shape of the PA- particles. PA was used in form of a nylon-thread, cut into pieces shorter than 5mm. The cross section of the thread was under 0.3mm, so it was possible for particles to get lost during the washing step, by escaping through the sieve with a pore size of 0.3mm.

Overall plastics don’t seem to get attacked by potassium carbonate within 80min, but more experiments, using a bigger sample size, need to be carried out.

49 The prolonged reaction time, compared to the time needed for the extraction process, gives the possibility to extend the settling time for particles, possibly counteracting the fine-sand-problem, that occurred in approaches with quartz-sand, mentioned in chapter ‘Validation of K2CO3 as density-solution by recovery-experiments’.

Resistance of different plastic types against different chemicals and enzymes Looking at the polymers with the biggest demand in Europe (PlasticsEurope, 2019) only PVC was attacked by one protocol, namely protocol 1 (Table 16; Figure 5). All other protocols seem to be safe to work with, regarding these polymers. If PA is taken into account, protocols 4 and 5 also do a little damage. Since PA only accounts to around 2% of the plastic demand in Europe (PlasticsEurope, 2019), its relative role in shore sediments can be neglected. All three mentioned changes in weight are less than 10% and since they all just affect one polymer type respectively, the impact on the overall outcome may be negligible.

Regarding the results from present study and from literature, protocols 2, 3 and 6 should be chosen to conserve all tested types of polymers.

Comparison between virgin plastics and weathered plastics The stronger effect on weathered polymers can at least partly be explained by dissolving of microbial biofouling. Also the chemical and physical properties of microplastics can change due to biofouling (Morét-Ferguson et al., 2010). Especially plastics on beaches are prone to degradation by environmental factors like UV radiation, since photo-oxidative degradation is initiated. This light-induced oxidation process can continue as long as oxygen is available and is the most efficient degradation processes (Andrady, 2011). The occurring brittle surface layer may be more sensitive to external stressors, like the applied chemicals, therefore a stronger damage to plastic particles cannot be ruled out.

The environmental EPS, which was tested for its resilience against protocol 2, showed corrosive effects under 2%, leading to the assumption that this protocol may be suitable for work on environmental samples. In comparison, protocol 3 corroded weathered EPS stronger than the maximum 5%, the other polymers tested were not attacked. Protocol 6 has already been tested rigorously for its compatibility with weathered polymers (Prata et al., 2019). Weathered PE, PP, HDPE, EPS, PA (Nylon), CA, PVC and additionally polyethylene fibres have been tested for their resistance against Fenton’s reagent at 50°C for 1h. Only CA showed weight loss under

50 this protocol, in weathered and virgin condition. In the same study KOH was tested at 50°C for 1h, which yielded the same results as Fenton’s reagent.

Digestion of organics by different chemicals and enzymes As seen in figure 7, the weight loss in the control experiments using water is not negligible. The reason for the loss of 36.1% (mean of all controls) mass lies at least partly in the drying of the samples. Salmon fillet, salmon skin/scales and tomato-leaves were not dried before the experiments and therefore lost most of their weight during the drying step after the experiment. Prata et al. (2019) found a similar digestion efficiency of water in their experiments and attributed it partly to the inability to retrieve the pooled organic sample from the flask used for the digestion process. Their second reasoning was the loss of soluble fractions, for example in fish material. These factors may also play a role in the present study.

Protocol 1 provided the best results regarding digestion efficiency using KOH and led to a weight loss of 61.6%. Nonetheless, protocol 2, which also used KOH, didn’t cause harm to any of the tested polymers and also showed the second-best digestion efficiency with 61.4% weight loss, regarding the pooled organic sample. The difference between both protocols is within the standard deviation and not statistically significant. The biggest part of the weight loss was due to the degradation of animal tissue, corresponding to the findings of Prata et al. (2019), in whose study feathers and fish tissue were effectively destroyed by KOH. KOH (10%) at 50°C showed a digestion efficiency of 58.3% after one hour on a mixed organic sample in their study, with only little improvement after six hours.

This point should be kept in mind when working with environmental samples. A preliminary optical analysis can help to determine if the organic pollutants are predominantly plant-based or have animal sources. If plant matter outweighs the animal matter, protocol 6 should also be taken into consideration. Coincidently to the findings of Prata et al. (2019) the redox reaction occurring by the use of Fenton’s reagent in protocol 6 shows promising results regarding plant matter. In the present study the efficiency was 51.7% compared to 65.9% achieved by Prata et al. (2019) with the same protocol. The differences can probably be attributed to the differences in the composition of the samples. The samples used in the present study had slightly more animal matter than plant matter, possibly explaining the higher efficiency of KOH in this study compared to Fenton’s reagent.

Overall, protocol 2 seems to be the best choice, if no knowledge about the composition of the organic pollutants is existing prior to the extraction process. Protocol 2 is also a good choice to

51 work with, regarding a possible citizen science approach. The relatively short reaction time of 24h and the easy handling of the solution is essential for the analysis of the possible enormous number of samples, provided by citizen scientists. Dehaut et al. (2016) also described KOH (10%) as the best compromise to extract and identify microplastics, yielding a digestion efficiency of 99.6 – 99.8% for their sample, consisting of mussels, crabs and fish tissue. KOH was also described as the most suitable method for examining biota for their microplastics content, because of its cheapness and effectiveness (Lusher et al., 2017a).

Conversely, the data of Hurley et al. (2018) showed the best digestion efficiency was yielded by Fenton’s reagent with 86.9 ± 9.87% for sludge and 106 ± 13.8% for soil. The protocol used was the same as protocol 5, which showed the poorest efficiency in present study. They used sludge from a wastewater treatment plant and soil from a bank next to an artificial stream. Compared to Fenton’s reagent KOH (as used in protocol 2) showed a poor efficiency with only

56.8 ± 16.6% in sludge and 34.5 ± 22.5% in soil, while H2O2 (as used in protocol 4) showed an efficiency of 80.2 ± 4.2% for sludge and 96.3 ± 14.9% for soil. Hurley et al. (2018) argued that cellulose and chitin, which are more resistant to KOH, as well as alkali-insoluble humins, may be included in their tested sludge and soil. The differences in the composition of sludge and soil are reflected in the different efficiency of the protocols.

Comparability between different studies is very difficult, since often the exact composition of the samples is not given. It’s also a big difference, if samples get treated with chemicals before or after density separation. This study chose to perform the density separation first, since then less chemicals are needed for each sample, securing a more sustainable and economical approach.

If preliminary studies show strong loads of plant material, protocol 6 should be considered instead or additionally to protocol 2, depending on available resources. The application of both protocols on the same samples one after another increased the digestion efficiency significantly (Table 7), but is more time-consuming than only conducting one protocol. Since the application of protocol 6 with a reaction time of only 1h doesn’t need much time, this should still be considered. Additional time is necessary for drying the samples in-between the application of both protocols, but since the digestion steps are conducted with extracts, the drying time needed is quite short. The high digestion efficiency yielded by Prata et al. (2019) with KOH, possibly allows for a time reduction of protocol 2 by shortening the reaction time, without big losses in efficiency.

52 The orange recolouring, happening when Fenton’s reagent is used, could lead to problems regarding the analysis of polymers. But more experiments, using IR-spectroscopy for example, are needed to test this.

The paraffin included in the samples poses a problem, which is easy to solve. Because of the included drying process, the paraffin, which doesn’t get dissolved by any of the tested protocols, melts and then dries, when cooled again, and clumps the sample together. This only happens, when the samples get rinsed with cold water, like conducted in present study. When rinsed with warm water, not hotter than 60°C, to protect plastic particles, the digesting efficiency can be improved by up to 12.5% by washing out the paraffin. This also prevents clumping and facili- tates a possible following optical analysis. Because of this reasoning, the combination of protocols 2 and 6 was rinsed with warm water after the second chemical digestion step, instead of cold water. The improvement of rinsing with warm water is visible when comparing the H2O- comparisons of protocol 2 and protocol 2+6. The weight loss of protocol 2+6 (H2O-comparison) is significantly higher, than protocol 2 (H2O-comparison) alone (Two-tailed t-test was performed using GraphPad Prism 5.01 for Windows, GraphPad Software, San Diego USA, www.graphpad.com; p<0.0005).

Both of the proposed protocols are too dangerous to let young pupils work with them. But the high reactiveness is absolutely crucial for the dissolving of organic matter. Grown-up citizen scientists could at least work under guidance and the use of protective gear since the handling of both protocols is easy. Citizen science studies including students working with Fenton’s reagent (as used in protocol 6) have already been carried out (Rowe et al., 2019).

Both Fenton’s reagent and KOH have the advantage, that they can be easily disposed of down the drain, after an initial adjustment of the pH-value (Rowe et al., 2019; http://www.halbmikrotechnik.de/big/GefStoff_Soest/2_3-17.htm; accessed on 12. November 2020). The pH should be between 5 and 9, which can be easily attained by adding NaOH or HCl, respectively. This makes the work with these protocols easier and even more convenient.

Testing of the whole protocol on beach samples Beaches are a convenient sampling site, because they are easily accessible and reflect the plastic accumulation in the ocean. But plastic can have direct impacts on these habitats as well. Besides the before mentioned possible release of aggregated toxic chemicals, the plastic can also have more physical effects. Carson et al. (2011) discussed the influence of plastic particles on the permeability of sediments: Beaches with higher amounts of microplastic tend to have a higher

53 permeability, which can lead to faster dehydration, causing multiple problems for inhabitants of beaches like crustaceans (Penn & Brockmann, 1994), fish (Quinn, 1999) or molluscs (D’ávila & Bessa, 2005) for example. Besides that, plastic can also have insulating properties. According to Carson et al. (2011) sediments with higher loads of plastic need a longer time to warm up. This can have dramatic effects on the development of animals with temperature-dependent sex- determination like sea turtles for example (Yntema & Mrosovsky, 1982). Even low concentrations of plastic can lead to temperature changes, high enough to influence the sex-ratio of sea turtles (Carson et al., 2011).

Those findings should underline the importance of sediment sampling on beaches, to help conserve habitats essential to vulnerable marine life forms. The usage of potassium carbonate for these sampling procedures helps cut the toxic waste products and additionally enables the work with pupils or other interested participants in form of a citizen-science project. This is necessary to raise awareness and to gather data in higher loads to make different beaches, and by that different sea-sections, comparable.

The results of the extractions in the present study from two sample beaches underline the importance of comparable sampling. Each sample can look completely different, according to where on the beach it has been taken. Present samples A-D have been taken along the strandline or waterline and show strong variability between both beaches. Hence, unnecessary differences should be filtered out, to get comparable results. Preliminary studies should be conducted before beaches get sampled on the grand scale by citizen scientists. Theoretically, the present protocol should work on every beach, but to make things easier some beaches should better be excluded from the start. Beaches with pebbles bigger than 5cm caused the biggest problems in this study, since the fine sediment needed is hidden deep down under the surface. Heavy loads of organic pollutants can be combated with aforementioned chemical protocols, but an additional optical analysis is still needed. By the use of chemical digestion, the workload can be minimized, because especially small organic particles, which are most difficult to separate from plastics, get dissolved. The remaining particles are generally larger, but also snail shells don’t get dissolved. The shells are recognizable by their distinct forms (Figure 9; A and G) and need to be sorted out manually.

Interestingly, the samples from Uvala Stoja featured heavy loads of tar (Figure 9; D and F), a problem already shown for other beaches around the world (e.g. Debrot et al., 2013; Del Sontro et al., 2007; Herrera et al., 2018; Shiber, 1987). The reason for the high abundance on Uvala Stoja is not entirely clear but might be traced back to its usage as boat mooring. The sheltered

54 orientation and the wind and wave conditions on the beach (Herrera et al., 2018) could lead to accumulations similar to microplastic accumulations on the same beach. This study highly rec- ommends examining the high abundance on this beach and to expand the research to other beaches in the Mediterranean Sea. In the light of a citizen science project a possibility to separate tar from microplastics is not only useful, but inevitable to ensure comparability around the world.

The samples also included particles, suspected to be microplastics (Figure 9; C, E, H, I, J, K and L). E and L are examples for plastic fibres, which are expected to be found, since one clothing item can shed more than 1900 fibres per wash (Browne et al., 2011), weighing up to 260mg for a single 660g polyester apparel (Dubaish & Liebezeit, 2013).

Particles H, I and K may look like plastics, but a second possibility is a mix-up with paint- particles. Since the usage of Uvala Stoja as boat mooring, splintered paint can also get accumu- lated in the sediment. Paint particles can also pose a serious health hazard to aquatic animals, since they are prone to consumption and can include toxic or harmful additives (Imhof et al., 2016). Antifouling paints for example often include metals, which can leach to the ocean (Brennecke et al., 2016). To distinguish between paint and microplastics follow-up-analyses are needed.

Particles C, J and L also show characteristics of plastic. Norén (2007) defined microplastics with following criteria: 1. No visibility of cellular or organic structures in the concerning particle. 2. Fibres should have a uniform thickness, while having a three-dimensional bending behaviour. 3. Particles should be homogenously coloured; especially blue, red, black, and yellow particles are suspected to be microplastics. 4. Transparent or whitish particles should be analysed extra carefully with a microscope. Often plastic particles have a shiny texture and a spher- ical shape. They can be flexible or solid, but the uniformity of features is decisive (Lusher et al., 2017a). According to all beforementioned features, all three particles (C, J and L) cannot be ruled out and should be included in possible follow-up-analyses.

More problematic are plastic particles, which have been in the sea for a long time. Embrittle- ment, fragmentation, bleaching and biological crusts can distort the appearance. This makes secondary analysis necessary, to correctly identify these particles (Lusher et al., 2017a).

For the beach samples included in this study, a concluding optical analysis was performed, with regard to the reduced pollution after chemical digestion. Since small particles have been digested, the error rate should be much smaller, compared to studies without a chemical digestion

55 steps. By the use of Fenton’s reagent, the sample was partly orange recoloured. This incident didn’t influence the suspected microplastics, but rather particles that seemed to be of biological descent. Since they didn’t get dissolved it can be speculated, that they are made of chalk or chitin. A further investigation of this topic is necessary.

After the optical analysis, the number of suspected microplastic particles per sample averaged at 13 ± 7.9 for Uvala Stoja and 15.5 ± 2.5 for Uvala Soline. Each sample was taken from 115ml sediment. To compare the results to other studies, this was extrapolated to 250ml, resulting in 28.3 and 33.7 particles per 250ml, respectively. Browne et al. (2011) mentioned samples ranging from 2 (Australia) to 31 particles per 250ml (Portugal, United Kingdom), but no samples from the Mediterranean Sea were included. Their study only focused on particles smaller than 1mm and had no lower boundary, still the results are in a similar size range and suggest a heavy plastic pollution in the northern Adriatic Sea.

For comparison with other studies, present results were converted to particles per kg dry weight sediment using the weight of the 115ml sample-volumes. Uvala Stoja contained 129.8 ± 78.6 particles, while Uvala Soline contained 92.2 ± 14.9 particles per kg dry weight. In the Po River Delta, which is also located in the northern Adriatic Sea, 2.92 ± 4.86 to 23.30 ± 45.43 microplastic particles per kg dry-weight have been found (Piehl et al., 2019), while in the lagoon of Venice abundance of microplastic particles smaller than 1mm ranged from 672 to 2175 per kg dry weight (Vianello et al., 2013). Laglbauer et al. (2014) found a microplastic concentration of 177.8 particles per kg dry weight in beaches in Slovenia, using a particle size range of 0.25mm to 5mm, which is similar to the present study.

Different reviews that also collected values in particles per kg dry weight and converted from other data to this unit, noted ranges from 0.1 to 5000 (Ballent et al., 2016) and from 0 to 5500 (Lots et al., 2017). Overall, the results of the present study are in comparable ranges to values found in the literature. The huge differences between studies can at least partly be attributed to different sampling techniques, purification processes and analysis, and underline the importance of standardisation. To enforce comparability with other studies, it is important that weight and count of microplastics get recorded.

Exact analysis of the particles and a guaranteed verification as microplastics is possible by different analysis techniques. The most commonly used ones are mentioned below.

The simplest technique is the so-called hot needle-test, a fast, inexpensive test in which a hot metal tip gets applied to assumed plastic particles. The melting behaviour gives conclusions

56 about the material of the particle. It is a very subjective method solely based on a visual identification, which doesn’t allow to differentiate between different polymers (Lusher et al., 2017a). The hot needle-test doesn’t help with the recognition of tar particles, since they also melt when heated.

Raman- or Fourier-transform infrared- spectroscopy (FTIR), which both rely on the detection of changes in frequency of electromagnetic radiation, have already been performed by multiple studies (e.g. Browne et al., 2011; Dehaut et al., 2016; Piehl et al., 2019). A big advantage is, that the plastic particles don’t get destroyed by these techniques, making it a favourable method if particles should be weighed after.

Another possibility to validate and assign plastic particles to different groups is pyrolysis combined with gas chromatography and mass spectrometry, which analyses the characteristic ther- mal degradation products of plastics, displaying their chemical composition (e.g. Dehaut et al., 2016; Dekiff et al., 2014; Fischer & Scholz-Böttcher, 2017; Fries et al., 2013; Nuelle et al., 2014).

Scanning electron microscopy plus energy-dispersive X-ray spectroscopy (SEM/EDS), another method used before (Wagner et al., 2017) was successful in characterizing microplastics and enabled a fast differentiation from mineral-based natural particles.

Conclusion and outlook To compare beaches regarding microplastic pollution on a large (temporal and spatial) scale, lots of data is needed. To gain enough data, the help of non-professional volunteers is indispen- sable. With the help of the protocol compiled in this study, a citizen science project can be successful in sampling different beaches over a greater timespan. The use of potassium carbonate as floating medium enables a hazard-free working environment for volunteers, while the work with KOH or Fenton’s reagent helps to distinguish plastics from other pollutants, saving time by shortening the working process, enabling a project to keep up with the much-needed large number of samples. Further improvements could be made by testing enzymatic digestion protocols and their applicability to sediment samples. Also, techniques to remove the tar lumps and possible chalk and chitinous pollutions need to be evaluated to secure comparable sampling.

The MedMicroplastiCS-Project enables young scientists to work in the field of microplastics. Currently two more master’s theses are planned to be conducted with focus on enzymatic digestion, tar pollution and a better purification process for chalk and chitinous particles. Also, a

57 new possibility to identify microplastics should possibly be tested: Nuclear magnetic reso- nance-spectroscopy (NMR-spectroscopy), which could enable samples to be qualitatively and quantitatively measured, if the purification process beforehand can cleanse the samples from pollution.

Acknowledgements First, I would like to thank the core-team of the MedMicroplastiCS-Project Sandra Bracun, Gerwin Gretschl and Jan Gohla for coming up with the idea of examining the beach sediments of the Mediterranean Sea to determine the microplastic pollution. Thank you to Sandra for being a contact person and assistant, whenever I needed one.

Further I want to thank Stephan Koblmüller for supervising this study and for the input given throughout the project, Kristina Sefc for helping with statistics and experimental setup and Markus Deutsch for giving input and being a helping hand during sampling and laboratory work, conducted in Pula.

Thank you to Meeresschule Pula and University of Graz for providing a workplace and material and to the whole team of Meeresschule for helping with sampling or keeping company during laboratory work.

Also thank you to Sarah Piehl and Kevin Schröder for sharing their experience with me and to Klaus Zangger for giving his input on the chemical aspects regarding plastics.

Finally, I want to thank Haus des Meeres Wien for presenting Sandra Bracun, Gerwin Gretschl and Jan Gohla with the Hans und Lotte Hass Prize, enabling the funding of this study.

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