Occurrence of microplastic particles in marine mammals from ...

University of Veterinary Medicine Hannover

Occurrence of microplastic particles

in marine mammals from German waters

and improvement of sample storage

Mikroplastikpartikel in marinen Säugetieren aus deutschen Gewässern und

Etablierung einer geeigneten Probenaufbewahrung

INAUGURAL – DISSERTATION

in fulfilment of the requirements for the degree of

- Doctor rerum naturalium -

(Dr. rer. nat.)

submitted by Carolin Philipp Bremerhaven

Büsum 2021

Scientific supervisor: Prof. Prof. h. c. Dr. Ursula Siebert

Institute for Terrestrial and Aquatic Wildlife Research,

University of Veterinary Medicine Hannover, Foundation.

Prof. Dr. rer. nat. Dieter Steinhagen

Institute of Parasitology, Fish Disease Research Unit,


First supervisors: Prof. Prof. h. c. Dr. Ursula Siebert

Institute for Terrestrial and Aquatic Wildlife Research,


Prof. Dr. rer. nat. Dieter Steinhagen

Institute of Parasitology, Fish Disease Research Unit,

University of Osnabrück.

Second examinor: Assoc. Prof. Dr. Marcus Schulz

Department of Mathematics / Informatics


Date of oral examination: 14.05.2021

Parts of this study were published in a peer reviewed journal:

Philipp, C., Unger, B., Fischer, E. K., Schnitzler, J. G., and Siebert, U. (2020). Handle with

Care—Microplastic Particles in Intestine Samples of Seals from German Waters.

Sustainability, 12, 10424. doi:10.3390/su122410424

Philipp, C., Unger, B., Ehlers, S.M., Koop, J.H.E., Siebert, U. (2021). First Evidence of

Retrospective Findings of Microplastics in Harbour Porpoises (Phocoena phocoena) From

German Waters. Frontiers in Marine Science, 8, Article 682532.

doi: 10.3389/fmars.2021.682532

Results, or parts of the results of this study were published in reports:

Philipp, C., Unger, B., Siebert, U. (2021). Impacts of marine litter on biota – Marine

Mammals. in Assessment and implementation of long-term monitoring of pollution of diverse

marine compartments and biota with marine litter, final report on behalf of the German

Environment Agency, FKZ 3717 25 225 0

Groß, S., Philipp., C., Siebert, U. (2020). Untersuchungen zum Massensterben von

Trottellummen (Uria aalge) und Tordalken (Alca torda) Anfang 2019. Report to the

Ministry of Energy, Agriculture, the Environment, Nature and Digitalization and the Agency

for Coastal Defence, National Park and Marine Conservation Schleswig-Holstein.

Results of this study were presented on the following conferences:

Philipp, C., Unger, B., Fischer, E., Siebert, U. (2020). Proof of the invisible ones –

Microplastic burden in marine mammals from the German North and Baltic Seas. In: talk,

MICRO2020, Lanzarote and Beyond, virtually conference, 23rd to 27th of November, 2020,

abstract book p. 345. https://www.micro.infini.fr/IMG/pdf/micro2020proceedingsbook.pdf

Philipp, C., Unger, B., Fischer, E., Siebert, U. (2019). Microplastics in marine top predators

– A pilot study from German waters. In: poster, World Marine Mammal Conference,

Barcelona, Spain 9th to 12th of December, 2019, abstract book p. 557.

https://www.wmmconference.org/wp-content/uploads/2020/02/WMMC-Book-of-Abstracts-

3.pdf

Philipp, C., Unger, B., Hillmann, M., Siebert, U. (2018). Pilot study on possible micro plastic

occurrence in marine mammals from German waters. In: poster, MICRO 2018, Lanzarote,

Spain, 19th to 23rd of November, 2018, abstract book, p. 348.

https://micro2018.sciencesconf.org/data/pages/micro2018_proceedings_book_1.pdf

For my Parents with Love & Gratitude

“Cherish the natural world,

because you’re a part of it and you depend on it.”

Sir David Attenborough

Table of Contents

List of Abbreviations ................................................................................................................ 9

Introduction ............................................................................................................................ 17

I. History of Plastics ........................................................................................................ 17

II. Production of Plastics ................................................................................................. 18

III. Facing the Mankind Problem .................................................................................... 19

IV. From Macro- to Nanoplastics ................................................................................... 21

V. Marine Litter .............................................................................................................. 22

V.I. Impacts of Marine Litter to the Marine Environment .......................................... 25

V.II. Microplastics and marine mammals .................................................................... 26

VI. Identifying Microplastic Particles ............................................................................ 28

VI.I. Collection techniques .......................................................................................... 29

VI.II. Removing Biogenic Matter ................................................................................ 30

VI.III. Identification of Microplastics .......................................................................... 31

VII. Target species in the German North and Baltic Seas .............................................. 32

VIII. Aims of the Thesis ................................................................................................. 35

Chapter I: Handle with Care – Microplastic Particles in Intestine Samples of Seals from

German Waters ...................................................................................................................... 41

Abstract ........................................................................................................................... 41

Introduction ..................................................................................................................... 42

Materials and Methods .................................................................................................... 43

Results ............................................................................................................................. 50

Discussion ....................................................................................................................... 56

Conclusion ...................................................................................................................... 60

Acknowledgements ......................................................................................................... 61

Supplementary Material .................................................................................................. 62

1. Preparation .................................................................................................................. 62

1.1 Pre-trials ................................................................................................................. 62

1.2 Working in an Acrylic Hood .................................................................................. 64

2. Intestinal and Faecal Samples ..................................................................................... 64

2.1 New design of the washing bags ............................................................................ 64

2.2 Prevention of overcounting .................................................................................... 65

3. Identification Catalogue .............................................................................................. 66

3.1 Categorization of parameters ................................................................................. 73

4. Results ......................................................................................................................... 75

Chapter II: First Evidence of Retrospective Findings of Microplastics in Harbour

Porpoises (Phocoena phocoena) From German Waters ..................................................... 83

Abstract ........................................................................................................................... 83

Introduction ..................................................................................................................... 84

Materials and Methods .................................................................................................... 85

Results ............................................................................................................................. 88

Discussion ....................................................................................................................... 98

Conclusion .................................................................................................................... 104

Acknowledgements ....................................................................................................... 106

Supplementary Material ................................................................................................ 107

Overall Discussion ................................................................................................................ 111

Overall Conclusion ............................................................................................................... 131

Outlook .................................................................................................................................. 135

Summary ............................................................................................................................... 137

Zusammenfassung ................................................................................................................ 139

Bibliography ......................................................................................................................... 141

List of Figures ....................................................................................................................... 183

List of Supplementary Figures ............................................................................................ 185

List of Supplementary Tables ............................................................................................. 186

Acknowledgements ............................................................................................................... 187

Curriculum Vitae ................................................................................................................. 191

List of Abbreviations

List of Abbreviations In addition to the commonly used abbreviations following short forms were used:

ALDFG Abandoned, lost or otherwise discarded fishing gear

ATR Attenuated total reflectance

BS Baltic Sea

CTD Conductivity, temperature and pressure of seawater (depth) measurement device

DDT Dichlorodiphenyltrichloroethane

ECHA European Chemicals Agency

EEA European Environment Agency

EU European Union

FTIR Fourier-transform infrared spectroscopy

GC-MS Gas chromatograph mass spectrometry

GES Good Environmental Status

GIT Gastrointestinal tract

GPGP Great Pacific Garbage Patch

H2O2 Hydrogen peroxide

HDPE High density polyethylene

HELCOM Helsinki Commission

Hg Grey seal (Halichoerus grypus)

HNO3 Nitric acid

HOC Hydrophobic organic contaminants

ICES International Council for the Exploration of the Sea

IMO International Maritime Organization

ITAW Institute for Terrestrial and Aquatic Wildlife Research

KOH Potassium hydroxide

LDC London Dumping Convention

LDPE Low density polyethylene

LLDPE Linear low density polyethylene

MARPOL International Convention for the Prevention of Pollution from Ships (Marine

Pollution)

List of Abbreviations

MSFD Marine Strategy Framework Directive

MP Microplastic

MPA Marine Protected Areas

MPW Mismanaged plastic waste

NaCl Sodium chloride

NS North Sea

OSPAR The convention for the Protection of the Marine Environment of the North-East

Atlantic, which was concluded in Oslo (1972) and updated in Paris (1992) is

managed by the OSPAR Commission.

PA Polyamide

PCB Polychlorinated biphenyls

PE Polyethylene

PEST Polyester

PET Polyethylene terephthalate

PFAS Per- and polyfluoroalkyl substances

POP Persistent organic pollutants

PP Polypropylene

PS Polystyrene

Pv Harbour seal (Phoca vitulina)

PVC Polyvinyl chloride

SAC Special Area of Conservation

SCOS Special Committee on Seals

SH Schleswig-Holstein

TiHo Stiftung Tierärztliche Hochschule Hannover (University of Veterinary

Medicine Hannover, Foundation)

TNR True negative rate

TPR True positive rate

UBA Federal Environment Agency (Umweltbundesamt)

UN United Nations

UNEP United Nations Environmental Programme

WWTP Wastewater treatment plants

Introduction

Introduction

17

Introduction

I. History of Plastics

The history of synthetic polymers reaches back to the early 19th century, when Bakelite was

invented in 1909 (Baekeland, 1909). This discovery could be assumed as the inception of the

‘Plastic Age’ (Yarsley and Couzens, 1945; Thompson et al., 2009), and as a benchmark of the

so-called Anthropocene (Lewis and Maslin, 2015). Firstly invented for the military, the

industrialised fabrication of synthetic polymers conquered the everyday life and businesses of

the western countries in the 1950’s, based on its miscellaneous qualities (Life Magazine, 1955;

Andrady and Neal, 2009; Sperling, 2011; Geyer et al., 2017). Synthetic polymers are

characterised by plasticity, dye ability, lightness, heat-, cold- and weather-resistance. In

addition, they are characterised by low production costs and thus meet the needs in almost all

bygone and recent industries until today (Andrady and Neal, 2009). Since this time, plastic has

become an indispensable part of the everyday life resulting in a global production of almost

400 million tonnes in 2019 (PlasticsEurope, 2020).

As a consequence, the rising plastic production cause a global litter issue (Geyer et al., 2017),

based on high consume and low decomposition properties (Barnes et al., 2009; Andrady, 2015).

Geyer et al. (2017) estimated the weight of all ever produced plastic resin until 2015 of 7,800

tonnes, whereof 60% are not in use anymore and are located in landfills. However, the plastic

production increased steeply since the late 2000’s, thus 12,000 tonnes of plastic waste will be

combusted and additionally 12,000 tonnes are supposed to end in landfills by 2050 (Geyer et

al., 2017).

Back in the early 20th century, synthesised materials were produced and appreciated for their

long durability. Nowadays those qualities are taken for granted, but the consequences of this

durability is poorly understood (Galgani et al., 2000; Andrady, 2015). To overcome the still

increasing utilisation of single-used plastic items, the European Commission enacted a

legislation (Environment and Public Health and Food Safety, 2019). This regulation will

specifically ban single-used items and force the recycling and reusing of all produced packaging

material by 2030. Furthermore, the European Chemicals Agency (ECHA) proposed some

microplastic reduction and restriction approaches to decrease the intentional utilisation (ECHA,

2019a). This proposal was submitted to the European Commission, and numerous EU Member

Introduction

18

States already apply some of the required restrictions (ECHA, 2019b). Nevertheless, especially

in the medical industry, synthesised materials are needed for single-use objects like syringes or

gloves, and are thus indispensable (Lebreton and Andrady, 2019; Patrício Silva et al., 2020).

II. Production of Plastics

Polyethylene (PE; incl. LDPE, LLDPE and HDPE) and polypropylene (PP) have the highest

demand in Europe (PlasticsEurope, 2020). These thermoplastics dominate the plastic use (PE:

29.8%; PP: 19.4%), besides polyethylene terephthalate (PET; 7.9 %), polystyrene (PS; 6.2 %)

and polyvinyl chloride (PVC; 10%) (PlasticsEurope, 2020). In addition, the global production

of PE and PP is steadily growing and is mostly used as packaging material (Gregory and

Andrady, 2004; PlasticsEurope, 2020). Thus, it is not surprisingly that packaging items are the

most frequently found items in landfilling, beach litter or marine litter surveys, due to the fact

that it is used only for a short duration (OSPAR Commission, 2000, 2020; Widmer and

Hennemann, 2010; Schulz et al., 2013; Lebreton and Andrady, 2019; PlasticsEurope, 2020). In

42% of all international produced packaging items, the manufacturing was done by the

utilisation of synthesised microplastic resin (Geyer et al., 2017).

Industrially synthesised small-sized plastic particles are produced for a variety of industries or

for the production of plastic items, melted and moulded on site of application (Galgani et al.,

2015; Karlsson et al., 2018). This so-called primary plastic, plastic resin or further described as

virgin pellets is categorised by its shape as nurdles, pellets or beads, which gives further

information on the application (Karlsson et al., 2018). The usage of beads in facial cleaners and

other cosmetics facilitating the foam or peeling qualities increased since the 1990’s (Zitko and

Hanlon, 1991; Gregory, 1996; Fendall and Sewell, 2009). In addition, these microplastics are

also utilised for grinding purposes (Browne et al., 2011), e.g. in the shipyard industry replacing

sand in sandblasters for removing old paintings or biofouling growth. Nurdles or beads are often

used for manufacturing of new plastic objects, whilst melting down and shaped into a larger

plastic item, whereof PE and PP are also the dominating polymers (PlasticsEurope, 2020).

The transportation via containers and the manufacturing and utilisation on site of virgin pellets

is easier and cheaper based on their small size and low weight (Hopewell et al., 2009; Karlsson

Introduction

19

et al., 2018; Ackerman et al., 2019). Based on a high demand and the global transport ways to

the production sites, occurring spills have to be taken into account as point source for

microplastics (Karlsson et al., 2018).

III. Facing the Mankind Problem

The increased requirements result in an ascending plastic production. As consequence mankind

faces the problem of an increased waste disposal, enhanced by a growing world population

(Jambeck et al., 2015; Lebreton et al., 2017). Especially the removal of synthesised

microplastics in personal care products and lost fibres originating from washed cloths are

difficult to remove in wastewater treatment plants (WWTP) (Bretas Alvim et al., 2020). There

is still no unified, efficient and practicable approach to remove all small-sized particles out of

wastewater from WWTP (Prata, 2018; Sun et al., 2019), although this issue is also known since

the 1990s (Zitko and Hanlon, 1991; Gregory, 1996). Thus, microplastics are still present in the

released water or the sewage sludge (Dubaish and Liebezeit, 2013; Dris et al., 2015; Weithmann

et al., 2018; Corradini et al., 2019). Further, the sludge is often used as field fertilisers,

introducing the microplastics into the environment (Nizzetto et al., 2016b). Several studies

already identified the microplastic burden in crop fields and agricultural soils (Weithmann et

al., 2018; Corradini et al., 2019; Crossman et al., 2020).

The reprocessing of older and unused plastic items is still complicated. Different polymer

networks, additives, toxic substances, and impureness by e.g. household waste, inhibit the

recycling process (Hopewell et al., 2009). Moreover, the only effective and progressive

approach of the removal of already used and manufactured plastics are a disruptive thermal

treatment like incineration (Geyer et al., 2017). Moreover, combustion processes generate

energy and a chemical feedstock, thus saving fossil fuels (Al-Salem et al., 2017; Anuar

Sharuddin et al., 2017). To further successfully decrease the plastic input into the environment

the primary production has to be reduced, which can be decreased by the development of

feasible recycling methods (Geyer et al., 2016). However, recycled plastic polymers for

example PE or PP have the same or similar traits as the virgin ones (Achilias et al., 2007).

Introduction

20

It has to be taken into account that all plastic items ever produced are everlasting thus remain

in the environment, either fractured or in one piece (Thompson et al., 2005). Nevertheless, the

recovering of plastic waste into a recycling loop, ensures a variety of economic benefits like

the conserving of emissions and fossil fuels, next to the protection of the environment

(Hopewell et al., 2009).

Areas with a highly anthropogenic frequency are stressed by intense plastic production,

consume and litter properties (Jambeck et al., 2015). The intentional or the unintentional

littering is supported by the missing of professional dumpsites in mostly developing countries,

which especially in open landfills causes spreading of litter items by wind and rain (Ryan et al.,

2009; Jambeck et al., 2015; Sharma et al., 2019; Nanda and Berruti, 2020). The mishandling

and mismanagement of plastic waste (MPW) is further increasing, unless the prevention of

waste or the recycle processes are improving (Lebreton and Andrady, 2019). Thus, the global

MPW results in approximately 80 million tonnes in 2015, that is roughly half of the produced

amount (47%) (Lebreton and Andrady, 2019). In addition, a recent study of Borelle et al. (2020)

estimated a global influx of 19 to 23 million tonnes of plastic (11% of produced plastic waste)

reaching the marine and limnic environment in 2016. It furthermore cannot be ruled out that

this is an underestimation, since primary microplastics and abandoned fishing gear were not

considered.

The diverse circumstances of landfilling, affect even uninhabited or remote areas, like the Alps,

both Polar regions, distant islands or the deep sea, and even the Marianas Trench is burdened

by marine litter (Slip and Burton, 1991; Barnes, 2005; Eriksen et al., 2014; Tekman et al., 2017;

Chiba et al., 2018; Bergmann et al., 2019; Parolini et al., 2021).Thus, it is not surprising that

litter was shown to impact wildlife. The harmful characteristics were already confirmed in

several studies for different species in the aquatic environment in the last decade (Fossi et al.,

2020; Kühn and van Franeker, 2020), whereas scientific information on terrestrial species is

still lacking (Lu et al., 2019; Kolenda et al., 2021). Although the harmful risk of persistent

plastics was already addressed in the late 1980’s (Bean, 1987).

Introduction

21

IV. From Macro- to Nanoplastics

Plastic items can be categorised by their size. The term macro-litter describes items which are

between 20 mm and 15 cm in size (Gregory and Andrady, 2004), whereas meso-litter is used

for particles in a size range of 5 to 10 mm (Gregory and Andrady, 2003). Gregory and Andrady

(2003) further relate to micro-litter, when a particle measures a size between 67 and 500 µm.

Most studies refer to micro-litter or microplastics when investigating particles smaller than 5

mm (Moore, 2008; Arthur et al., 2009; Fendall and Sewell, 2009). For clarifying, this

dissertation follows the term microplastic describing particles smaller than 5 mm. Until today,

there is no formal term for nano-litter or nanoplastics (Koelmans, 2019). Thus, for classifying

those particles, the attributes and qualities of nanomaterials are used (Klaine et al., 2012).

Further definitions for narrowing down nanoplastic are given by Wagner et al. (2014)

describing particles smaller than 20 µm, and Klaine et al. (2012) categorise nanoplastics if the

particle size is less than 100 nm.

The definitions often refer to already given size ranges in plankton quantification systems.

Particles between 5 mm and 333 µm in size were initially called microplastics, based on the

common mesh sizes of used plankton nets (Arthur et al., 2009). This is similar to nanoplastics,

were the size limit of nanoplankton is set (Sieburth et al., 1978).

Next to primary plastics as described in the former chapter, secondary microplastics is another

form of small-sized particles. Those ones emerges if larger plastic items crack into smaller

pieces due to different physical forces. Especially, weathering processes promote the brittleness

of plastic items. Several laboratory and field studies investigate the degradation of plastic under

different circumstances. In particular UV-radiation, the physical abrasion by wind and waves,

or chemical reactions like oxidation, are confirmed to force the decaying process (Andrady,

1990, 2011). Whereas seawater and biofouling inhibit the ageing process by protecting the

plastic for overheating, UV-radiation and physical abrasion (Andrady, 1990).

Thus, it is not surprising, that microplastics seem to be present and accumulate in the world

oceans since more than 50 years (Carpenter et al., 1972; Thompson, 2004; Geyer et al., 2017).

Based on the concerns of microplastics, the research increased steeply in the last decade

(Cowger et al., 2020).Whereas, knowledge on the presence and health effects of nanoplastic is

still lacking (Paul et al., 2020). Certainly, based on the physical characteristics, nanoplastic

Introduction

22

particles aggregate in water or air to larger clusters, adsorbing pollutants and biogenic material

(Ward and Kach, 2009; Andrady, 2011; Song et al., 2014; Yu et al., 2019). In addition, these

small-sized particles are known to be cell-permeable, thus open up a new concern in

accumulation of pollutants and its health effects in organisms (Jani et al., 1989; LeFevre et al.,

1989; Lehner et al., 2019; Li et al., 2020b; Sarasamma et al., 2020).

V. Marine Litter

When focussing on the marine environment, found garbage is often called marine litter. The

United Nations Environmental Programme (UNEP) is giving the following definition of those

items: “[…] marine litter is defined as any persistent, manufactured or processed solid material

discarded, disposed of or abandoned in the marine and coastal environment (UNEP, 2009).

Pasternak et al. (2017) further proposed: “Marine debris is a consequence of poor or inadequate

solid waste management practices […]”. The entering of debris into the seas is aided by the

transport of rivers, drainage or sewage systems or the wind as proposed by the Marine Strategy

Framework Directive (MSFD) (Galgani et al., 2010). In addition, ecological calamites like

flooding events e.g. tsunamis or river discharges have to be taken into account as pathways of

marine litter (Murray et al., 2018; van Emmerik et al., 2019). It is often cited that 80% of marine

litter in the ocean originates from land-based sources (Jambeck et al., 2015; LI et al., 2016).

Even though the presence, potential entries and pathways of marine litter were already

recognised in the 1970’s (National Research Council, 1975), the global influx of waste into the

oceans is still increasing (Jambeck et al., 2015). This results in a plastic burden in all seven

oceans (Eriksen et al., 2014).

The regulations of the international maritime organisation (IMO) prohibits the discharge of on

board generated waste into the sea in accordance to the MARPOL (Annex V) convention 73/78

(MARPOL, 2017; IMO, 2021). Nevertheless, container ships and fishing vessels, ferries and

cruise liners are considered to be one of the major sources of ocean based litter input (Pruter,

1987), next to generated garbage on oil- and gas platforms or offshore wind turbines (Galgani

et al., 2013).

Introduction

23

The consensus of the issue of marine litter was already addressed in 1972 by several member

states of the United Nations (UN), resulting in the London Dumping Convention (LDC) that

adapt the legislations several times in previous years (IMO, 2002; Butt, 2007). Nevertheless,

the MARPOL (Annex V) convention 73/78 passed legislations to manage the avoidance of

discarded waste and pollutants into the oceans until today (Lentz, 1987; MARPOL, 2017).

Focussing on the study area of this thesis, the Helsinki Convention already took part in

environmental protection for the Baltic Sea in 1974 (HELCOM, 1993), whereas regulations on

dumping waste into the North Sea became effective in 1972 under the Oslo Convention (Lentz,

1987). Furthermore, the North and Baltic Seas are considered as MARPOL Special Areas

(Annex V) since the 1990’s. Within these areas, the disposal of waste (incl. fishing gear, plastic

and garbage) is banned (OSPAR Commission, 2000). In addition, the EU declared several areas

in the North and Baltic Seas as so-called Marine Protected Areas (MPA) since 2008 based on

the MSFD, protecting the marine environment and its biodiversity from several anthropogenic

impacts with special legislations (EEA, 2015). Around 18% of the North Sea are MPAs,

presenting the highest protection status in European seas (EEA, 2015).

Despite these regulations, several publications are still documenting higher abundances of

marine litter along shipping routes and anchor places (Katsanevakis and Katsarou, 2004;

Grøsvik et al., 2018). Furthermore, it is assumed that only 27% of the global ship waste is

disposed as required. The remaining is illegally dumped or burned (Sheavly and Register, 2007;

Øhlenschlaeger et al., 2013).

In addition, a variety of studies confirms a high influx of marine litter from land into the oceans

(Galgani et al., 2015; Jambeck et al., 2015; Lebreton et al., 2017). Estimations demonstrate a

global inflow of plastic waste between 1.15 and 2.41 million tonnes annually into the open sea,

whereof 86% are mainly transported by rivers located in Asia (Lebreton et al., 2017). This is

not surprising since Jambeck et al. (2015) already indicated the missing waste management in

Asia, which is most likely the reason for a high litter input into the marine environment. This

is also enhanced by the import of plastic waste from European countries (Liang et al., 2021).

The remaining 14% of plastic waste is conveyed by rivers of Africa, America, Europe and

Australia (Lebreton et al., 2017). The Yangtze River for example showed the largest amount

with approximately 0.33 million tonnes per year, whereas the estimated annually quantity of

Introduction

24

plastic input by European rivers ranges between 2,310 – 9,320 tonnes (Lebreton et al., 2017).

Regarding the world’s oceans, the study of Eriksen et al. (2014) assumed that an amount of

5.25 trillion drifting plastic particles including all size classes, are located at the surface waters

and sum up to a total weight of 268,940 tons. Those pathways of plastic items are depending

on their density attributes i.e., is the object either buoyant or does it sink to the sediment at once.

Currently, there are several studies investigating the plastic burden in their local study areas,

confirming the seafloor as potential sink for plastics (Matsuguma et al., 2017; Lorenz et al.,

2019). Furthermore, Brandon et al. (2019) confirmed an increased amount of plastic pollution

over several decades in sediment cores from the Santa Barbara Basin (USA, California). To

estimate or define trends for the burden in ocean sediments further research is needed (Eriksen

et al., 2014), since investigating studies in large scales for the sea floor are lacking in recent

years (Galgani et al., 2000).

However, the density of approximately 60% of all produced plastics is lower than seawater,

thus floating on the marine water surface (Andrady, 2011; Lebreton et al., 2018). Furthermore,

biofouling by different invertebrate or botanical species is confirmed to additionally change the

density and stability characteristics of plastics (Andrady, 1990; Muthukumar et al., 2011; Fazey

and Ryan, 2016; Van Melkebeke et al., 2020).

Ocean currents, eddies and winds causing accumulation of marine litter in five subtropical

gyres, the so-called garbage patches (Eriksen et al., 2014; van Sebille, 2015). The Great Pacific

Garbage Patch (GPGP) is the largest and most distinct one. Although still growing, it is already

confirmed as an area with high quantity of plastics (Lebreton et al., 2018). It is important to

note, that the accumulated litter of all size-classes, is distributed in the entire water column,

based on the density characteristics of the items (Andrady, 2011). They are rather floating and

aggregating than building concrete litter islands (Lebreton et al., 2018). Faunal and floral

species in this area are thus affected, no matter if they are living near the surface, in the water

column or near the seabed (Boerger et al., 2010). Sightings of different cetacean species,

including mother-calf pairs were recorded in the GPGP in 2016 (Gibbs et al., 2019), which

highlights the rather loose aggregation of the garbage patch.

Introduction

25

V.I. Impacts of Marine Litter to the Marine Environment

Marine litter is mostly known to influence the marine environment in three different ways: i)

cause harm by entanglement, ii) lead to severe health impacts after ingestion, and iii) increase

the distribution of invasive species. The following chapter gives a more detailed overview on

those impacts.

Floating marine litter are assumed to function as transport devices for alien species and invasive

species, introducing them to new marine habitats (Miralles et al., 2018). Especially adherent

invertebrates like oysters or barnacles are assumed to drift on marine debris over the sea. This

was observed in the Mediterranean Sea (Rech et al., 2018), the Atlantic region (Holmes et al.,

2015) and the Southern Ocean (Barnes and Fraser, 2003). These studies highlight the mobility

and ubiquitous presence of marine litter.

The risk of entanglement in or the ingestion of litter items is of concern for a variety of species,

mainly affecting larger specimens like top predators. One of the first incidents was reported for

a shark trapped in a car tyre in 1931 (Gudger and Hoffmann, 1931). This report describes an

entanglement in anthropogenic manufactured debris of an individual long way before the plastic

production rose commercially. Nowadays, several studies confirm the exposure for different

marine specimens (Laist, 1997; Galgani et al., 2018; Gibbs et al., 2019) and the risk of

abandoned, lost or otherwise discarded fishing gear (ALDFG) (Macfadyen et al., 2009), which

was already noted as risk potential in the beginning of the 1990’s (Andrady, 1990).

Different studies reveal impacts in moving and feeding behaviour for marine mammals,

seabirds, sea turtles and other species around the world (Laist, 1997). When focussing on

marine mammals in the North Atlantic area, especially right whales (Eubalaena glacialis) are

highly affected by ALDFG, where entanglement often ends fatal (Sharp et al., 2019). Other

cetaceans like humpback whales (Megaptera novaeangliae) or harbour porpoises (Phocoena

phocoena) are also reported to be affected (Unger et al., 2017; Basran et al., 2019). Furthermore,

cases of severe and lethal entanglements are also described for the two most occurring seal

species in the North Atlantic region, the harbour seal (Phoca vitulina) and the grey seal

(Halichoerus grypus) (Allen et al., 2012; Unger et al., 2017).

Beside the risk of an entanglement, the ingestion of marine litter is also assumed to have an

impact on marine species of different trophic levels in all seven seas (van Franeker et al., 2011;

Introduction

26

Foekema et al., 2013; Forrest and Hindell, 2018; Kühn and van Franeker, 2020). Top predators

in particular, are exposed to an intentional or unintentional uptake and thus accumulate litter

and ALDFG in the gastrointestinal tract (GIT) (Kühn et al., 2015). Based on the high

accumulation rate of plastic debris in GITs of northern fulmars (Fulmarus glacialis) in the

North Atlantic region (van Franeker et al., 2011), OSPAR appointed this species as indicator

for the environmental quality in 2008 (OSPAR, 2008). In addition, records of litter items in

GITs of different marine mammal species are already recorded for the North and Baltic Seas

(Unger et al., 2016, 2017; van Franeker et al., 2018). In former studies, it is described that

especially ALDFG and sharp-edged indigestible items result in severe lesions, inflammations,

and septicaemia in the whole GIT – from the jaw up to the anus and mostly ends fatal.

V.II. Microplastics and marine mammals

Based on their small size (>5 mm), microplastic particles are particularly proposed to

accumulate within the food web (Browne et al., 2008; Farrell and Nelson, 2013). Whereas the

bioavailability depends on the attributes of the particles like colour, shape and size (Wright et

al., 2013). The size coincides with several phyto- and zooplankton species. Thus, particles are

unintentionally up taken by filtration specimens of all trophic levels from Krill species (Dawson

et al., 2018) as well as top predators like humpback whales (Megaptera novaeangliae)

(Besseling et al., 2015). The microplastic ingestion and resulting health effects are determined

in several laboratory studies with molluscs, crustaceans and fish species (Cole et al., 2013;

Farrell and Nelson, 2013; Lei et al., 2018; Naidoo and Glassom, 2019). Thus, the smaller the

particles are, the higher the bioavailability for several species (Lebreton and Andrady, 2019).

In contrast, knowledge on the impacts of microplastic on larger species, like marine mammals

is widely unknown. Furthermore, the presence of microplastics in wild-ranging invertebrate

specimens is confirmed in all seven seas (Davison and Asch, 2011; Wieczorek et al., 2018;

Arias et al., 2019; Bakir et al., 2020; Daniel et al., 2020; Sfriso et al., 2020). Due to ethical

reasons, laboratory studies investigating microplastic ingestion and resulting effects in marine

mammals is lacking. However, studies confirming the presence of microplastics in wild-ranging

marine mammals increased in the last decade (Zantis et al., 2021). Those studies analysed GITs

of stranded or bycaught individuals. The microplastic presence was already observed in

Introduction

27

different cetaceans in the North Atlantic, e.g. a stranded humpback whale in the North Sea

(Besseling et al., 2015), different toothed whale species like the harbour porpoise or short-

beaked common dolphin (Delphinus delphis) around the British coast (Nelms et al., 2019).

Furthermore, investigations in microplastic presence in GIT samples were conducted in

different areas of the North Atlantic for Phocidae species (Phoca vitulina and Halichoerus

grypus) (Hernandez-Milian et al., 2019; Nelms et al., 2019). The analysis of microplastics

ingested in pinnipeds could also be examined by investigating faecal samples of living

individuals, as it was performed in Otariids from the North Pacific (Callorhinus ursinus)

(Donohue et al., 2019), the South Pacific and the South Atlantic Ocean (Arctocephalus

philippii, Otaria bvronia and Arctocephalus australis) (Perez-Venegas et al., 2020) or in

Phocidae species from the western North Atlantic (Phoca vitulina and Halichoerus grypus)

(Hudak and Sette, 2019).

Certainly, these small-sized particles are assumed to cause irritations and inflammations in the

tissue of the GIT, while settling down in the lumen of the stomach or the intestine, as it is

confirmed in other mammal species like mice (Li et al., 2020a). Those punctual inflammations

or irritations are assumed to cause diseases like gastritis or enteritis, since it is already proven

that polystyrene particles cause pro-inflammatory responses in human gastric epithelial cells

(Forte et al., 2016). Nevertheless, those effects also depend on the size and diameter, and the

digestion rate of an individual. Moreover, Jâms et al. (2020) investigated the correlation

between the body size of various faunal species of different trophic levels and the impact of the

sizes of ingested plastic objects. Thus, the risk of inflammations is depending on the quantity

and size of the ingested plastics, and the dimensions of the GIT from the ingesting individual.

Several health effects triggered by nano- and microplastics are already proven in smaller

species, e.g. zebrafish (Danio rerio) (Lei et al., 2018; Naidoo and Glassom, 2019).In addition,

oxidative stress for several cells, heart complaints, reproduction failure, and behavioural

disorders are confirmed in laboratory studies in mice and rats (Hou et al., 2020; Li et al., 2020c;

An et al., 2021; da Costa Araújo and Malafaia, 2021).

Another risk of microplastics are the sorbing and accumulating abilities for pollutants (e.g.

polychlorinated biphenyls (PCB)) due to their lipophilic attributes and the enlarged surface

(Endo et al., 2005; Song et al., 2014). They serve as vector for chemicals (e.g.

Introduction

28

dichlorodiphenyltrichloroethane (DDT)), bacteria and viruses and end up in ingesting

individuals (Teuten et al., 2009; Viršek et al., 2017; Razanajatovo et al., 2018; Curren and

Leong, 2019). This accumulated bioavailability is especially harmful for marine predators at

the top of the food web. Particularly persistent organic pollutants (POPs), like DDT and PCB

were confirmed to accumulate in marine mammals (Kleivane et al., 1995; Aguilar and Borrell,

2005; Ross, 2006; Dorneles et al., 2013; Jepson et al., 2016), and were also identified on

microplastics (Endo et al., 2005; Bakir et al., 2012). Marine top predators accumulate these

toxic compounds in their fatty tissue and organs, which can cause severe health impacts

(Schmidt et al., 2020b, 2020a; Sonne et al., 2020). Furthermore, Ryan et al. (1988) already

confirmed a linkage between POP-contaminated plastics and the accumulation in the fatty

tissue of seabirds. In addition, the transfer of POPs while gestation and lactation was already

identified in marine mammals and in humans (Wolkers et al., 2004; Desforges et al., 2012;

Aerts et al., 2019; Witczak et al., 2021).

Besides PCB, further hydrophobic organic contaminants (HOC) like per- and polyfluoroalkyl

substances (PFAS) were recently investigated. PFAS are a highly discussed issue in pollutant

research, but only few indices of interactions with microplastics are given (Guo et al., 2012;

Atugoda et al., 2020). There are already reports of PFAS accumulations in marine mammals

(Gebbink et al., 2016; Spaan et al., 2020). Nevertheless, a linkage between microplastics and

PFAS is not confirmed to date (Pramanik et al., 2020).

VI. Identifying Microplastic Particles

In recent years, several techniques were developed to isolate microplastic particles from

biogenic or other organic matter originating from marine or limnic environments. This results

in a high amount of publications and a still increasing interest in this topic (Thompson, 2015).

Thus, the reproducibility between studies is often hampered due to different terminologies, used

size limits or techniques (Löder and Gerdts, 2015; Cowger et al., 2020). Furthermore, with

decreasing particle size, the risk of loss or misidentification is increasing (Shim et al., 2017).

Nevertheless, some protocols are considered as already established in marine litter analysis, and

especially in microplastic research. Some of them are presented in the following paragraph.

Introduction

29

VI.I. Collection techniques

Water samples are commonly collected with plankton nets, e.g. manta trawls or bulk samples

(Eriksen et al., 2014; Fischer et al., 2016; Mani et al., 2016; Tamminga et al., 2019). Those bulk

samples can be collected with simple containers, attached pumps (Desforges et al., 2014) or

CTD-devices (conductivity, temperature and depth measure devices) with affiliated hydrocasts

(Pieper et al., 2020). Samples of the beach or of the tidal flat are often simply collected with a

spoon (Stolte et al., 2015; Hengstmann et al., 2018). Whereas, samples of sediments need

specialised pump systems or other specialised collection systems (Van Cauwenberghe et al.,

2013; Mani et al., 2019; Zhang et al., 2020).

Small specimens of fish or invertebrates like crustaceans are easy to catch, collect or to held in

aquariums (Browne et al., 2008; Farrell and Nelson, 2013; Rummel et al., 2016). In the case of

larger species, such as vertebrates, samples are collected in the course of necropsies of carcasses

resulting from fished, hunted, bycaught, stranded or mercy-killed individuals (Lusher et al.,

2013, 2017b; Fossi et al., 2014; Nelms et al., 2018; Siebert et al., 2020). In addition, faecal

samples are another opportunity (Nelms et al., 2018; Donohue et al., 2019; Hudak and Sette,

2019; Perez-Venegas et al., 2020).

Samples of air however, are collected with positioned containers or petri dishes (Dris et al.,

2016). Furthermore, those procedures are also commonly used for identifying the potential

contamination in the working environment (Torre et al., 2016). In addition, further procedural

control blanks need to be taken into account for avoiding an overestimation of found

microplastics (Norén, 2007; Hidalgo-Ruz et al., 2012; Nuelle et al., 2014; Vandermeersch et

al., 2015).

The storage of those samples depends on the volume and the medium. In several studies, the

samples were stored in plastic bags or other plastic containers (Hidalgo-Ruz and Thiel, 2013;

Lusher et al., 2013; Donohue et al., 2019), whereas other studies recommend the usage of glass

containers (Mani et al., 2016; Hengstmann et al., 2018; Lusher and Hernandez-Milian, 2018;

Tamminga et al., 2018) or a wrapping in aluminium foil (Perez-Venegas et al., 2018). Studies

comparing benefits or disadvantages concerning the contamination risk are still lacking,

although the risk is considered by the ones previously mentioned.

Introduction

30

VI.II. Removing Biogenic Matter

The organic or biogenic material of both faunal and floral origin, has to be taken into account

in all research topics when investigating microplastics in environmental samples (Thompson,

2004; Hidalgo-Ruz et al., 2012; Nuelle et al., 2014). Thus, cleaning procedures for subsequently

isolating microplastic particles from unwanted material have to be conducted to facilitate the

polymer identification (Löder and Gerdts, 2015).

Prior to all cleaning procedures, sieving the sample with different mesh sizes is recommended

in almost all studies to narrow down the number of unwanted material (Hidalgo-Ruz et al.,

2012; Van Cauwenberghe et al., 2013; Mani et al., 2016; Hernandez-Milian et al., 2019). A

density separation is commonly used and highly recommended for sediment or beach samples

(Hidalgo-Ruz et al., 2012; Vermeiren et al., 2020). Especially density separations in saturated

sodium chloride (NaCl, ~ 1.2 g/cm3) solutions are preferred (Hidalgo-Ruz et al., 2012; Nuelle

et al., 2014; Stolte et al., 2015). In addition, an attached air- or liquid circulation system is an

upgrade in conducting density separations (Claessens et al., 2013; Nuelle et al., 2014; Stolte et

al., 2015).

Chemical digestion techniques are used to remove the biogenic matter. For example, hydrogen

peroxide (H2O2), potassium hydroxide (KOH) or nitric acid (HNO3) are common detergents for

eliminating the unwanted material (Claessens et al., 2013; Dehaut et al., 2016; Kühn et al.,

2017; Naidoo et al., 2017; Lusher and Hernandez-Milian, 2018; Tamminga et al., 2018).

Furthermore, faunal samples originating from higher trophic levels have a higher biogenic

content in comparison to low trophic levels. Thus, also enzymatic approaches are proposed for

removing biogenic material (Cole et al., 2014; Mani et al., 2016; Bretas Alvim et al., 2020)

Furthermore, the protocol for investigating vertebrate samples and their microplastic burden

established by Lusher and Hernandez-Milian (2018) describes the basic regulations for

microplastic research. This protocol refers to samples of the GIT and its content. Besides

filtering processes with steel mesh sieves in different sizes (e.g. 1,000 µm, 500 µm and 250

µm), a KOH digestion of the biogenic material is suggested. Based on the digesting volume,

the procedure lasts from days to weeks (Lusher and Hernandez-Milian, 2018).

Introduction

31

The used mesh size in the beginning of collecting and cleaning procedures and the pore size of

the used filters limit the sizes of the investigated parts. This is still one of the major issues in

comparing different studies (Cowger et al., 2020).

VI.III. Identification of Microplastics

Once the samples are cleaned, the identification process is not highly differing. In this

paragraph, the most common identification techniques are presented.

Characterisation takes place by the naked eye, when the samples have to be sorted by e.g. colour

or other attributes, which could be damaged in further processing steps (Lusher et al., 2014).

Especially for larger microplastics (1 – 10 mm) this procedure is applicable (Hidalgo-Ruz and

Thiel, 2013; Hernandez-Milian et al., 2019). Furthermore, this step could be supported by

binoculars or different types of microscopes to enable the identification of smaller particles,

e.g. by stereo microscopy (Norén, 2007; Song et al., 2015; Lusher and Hernandez-Milian,

2018). Certainly, recent studies do not recommend this technique, due to high over- or

underestimation of plastic particles (Löder and Gerdts, 2015). To pre-select particles for further

polymer identification, a staining is advisable. Particularly Nile Red staining is currently one

of the most used procedures to differentiate biogenic materials from synthetic ones (Desforges

et al., 2014; Tamminga et al., 2017; Vermeiren et al., 2020).

As recommended by the MSFD, at least 10% of all found particles needs to be spectroscopically

analysed to reduce miss-identifications (Lusher and Hernandez-Milian, 2018). A case study

examining sediment samples revealed that only 1.4% of all potential microplastic particles

which were identified by visual investigations, could be confirmed as true microplastics by

Fourier-transform infrared (FTIR) spectroscopy (Löder and Gerdts, 2015). Furthermore, plastic

particles found in biogenic material are exposed to several environmental factors leading to

changes in the polymer presence and bonding. Nevertheless, a FTIR spectroscopic analysis

enables to identify those weathered particles despite these changes (Turner and Holmes, 2011).

Next to FTIR analysis, Raman spectroscopy is one of the most common techniques for polymer

identification (Löder and Gerdts, 2015). Raman spectroscopy assesses the attributes of the

particle surface measuring the frequency, intensity and polarisation of back scattered lights

induced by a laser beam (Löder and Gerdts, 2015). In contrast, FTIR spectroscopy energises

Introduction

32

different molecular levels compared to Raman, thus both techniques could support each other

in polymer identification (Löder and Gerdts, 2015). In order to investigate smaller microplastic

particles, µRaman or µFTIR measures are used (Tamminga et al., 2019; Pereira et al., 2020)

Besides those two non-destructive methods, where the particles stay intact for further

processing or archiving, the destructive method of using pyrolysis-gaschromatography linked

with mass spectrometry (GC-MS) are among the three frequently used analysing techniques

(Löder and Gerdts, 2015). This destructive method is a thermal measurement that identifies

occurring incineration products of present polymer bonds (Nuelle et al., 2014; Löder and

Gerdts, 2015).

Thus, a subsequently analysis of the identification and thus confirmation seems to be inevitable

and is already established in microplastic research (Lusher et al., 2013; Lenz et al., 2015; Löder

and Gerdts, 2015). Furthermore, a combination of different identification tools lead to most

reliable results (Löder and Gerdts, 2015; Dehaut et al., 2016; Shim et al., 2017).

VII. Target species in the German North and Baltic Seas

This thesis focusses on three marine mammal species inhabiting the German North and Baltic

Seas. The harbour seal (Phoca vitulina) and the grey seal (Halichoerus grypus) are the two

native pinniped species in German waters (Reijnders and Lankester, 1990). The harbour

porpoise (Phocoena phocoena) is the only regularly occurring and reproducing cetacean in the

German North and Baltic Seas (Hammond et al., 2017; Bjørge and Tolley, 2018).

The harbour seals inhabiting the North and Baltic Seas belonging to the subspecies of the North-

East Atlantic: Phoca vitulina vitulina (Teilmann and Galatius, 2018). When focussing on the

Wadden Sea and the island Helgoland, the trilateral surveys which were conducted in Dutch,

German and Danish waters estimated an abundance of 41,700 individuals in 2020 (Galatius et

al., 2020a). In the Baltic Sea, the harbour seal is mainly inhabiting the southern area and

approximately 2,000 individuals are estimated (Teilmann and Galatius, 2018; ICES, 2019). The

breeding season is followed by the moulting time in summertime (May – September) (Teilmann

and Galatius, 2018). Mating season begins directly after closure of lactation time, followed by

a diapause in females. New-born harbour seals are able to swim from the first day and are

Introduction

33

lactated for three to four weeks (Teilmann and Galatius, 2018). After weaning, the mother

animal is leaving the pup, thus it starts feeding on crustaceans and small fish itself (Hall and

Thompson, 2009; SCOS, 2014). In contrast, the pupping season of grey seals starts in

November and lasts up to January in the Wadden Sea (Brasseur et al., 2020). Whereas the

pupping season in the Baltic Sea lasts from February to March (Galatius et al., 2020b). Grey

seal pups are dependent on lactation for up to three weeks, and are born with lanugo fur which

is pervious for water (Hall and Thompson, 2009; SCOS, 2014). Thus, grey seal pups are unable

to swim and prey on fish until the lanugo is replaced by pelage after the weaning period

(Grajewska et al., 2020).

It has to be noted, that the grey seal is repopulating the North Sea since the 20th century, as

several anthropogenic impacts lead to a decline in the population size (Reijnders et al., 1995).

Since the last decade, the population of grey seals is still increasing in the southern North Sea

7,649 individuals were counted in the Wadden Sea area of the North Sea in the winter season

2019-2020 (Brasseur et al., 2020). A population size of more than 30,000 individuals is

estimated for grey seals in the whole Baltic Sea (ICES, 2019). Furthermore, genetic analyses

of grey seal individuals from the Baltic Sea result in the assumption of different subspecies with

limited gene exchange (Graves et al., 2009). Thus, individuals in the North Atlantic area,

including the North Sea are part of the subspecies Halichoerus grypus atlantica and individuals

of the Baltic Sea form a second subspecies Halichoerus grypus grypus (Olsen et al., 2016;

Galatius et al., 2020b).

Both seal species are typically feeding on demersal or benthic prey species like sandeels, flatfish

and gadoids, with seasonal differences, but also on pelagic species like herring, whiting and

scad (Pierce and Santos, 2003; Wilson and Hammond, 2019). Certainly, both seal species are

assumed to feed in offshore and in coastal areas (Hall and Thompson, 2009; Wilson and

Hammond, 2019).

Harbour porpoises are listed in the EU Habitats Directive in the Annexes II and IV and is subject

to a high conservation status in Europe (EU, 1992). This toothed whale is feeding on benthic

prey species like sandeels, gadoids, gobies, cods and flatfishes, even so on pelagic schooling

fishes like clupeids (Gilles, 2008; Leopold, 2015; Andreasen et al., 2017; Gross et al., 2020).

Feeding grounds of harbour porpoises in the North Sea are identified at “Borkum Reef Ground”

Introduction

34

and the “Dogger Bank tail” (Gilles et al., 2011). Those areas are declared German protection

sites (Special Area of Conservation, SAC) under the EU Natura 2000 Habitats Directive, next

to the important breeding ground at “Sylt Outer Reef” (Gilles et al., 2009, 2011; Federal Agency

for Nature Conservation, 2010; Nachtsheim et al., 2021). The “Fehmarn Belt”, the “Kadet

Trench” and the “Pomeranian Bay with Odra Bank” are identified as important residence

habitats of the harbour porpoise and are additional declared SACs in the Baltic Sea (Federal

Agency for Nature Conservation, 2008, 2020; Viquerat et al., 2014).

Based on different genetic analyses, up to thirteen subpopulations of the harbour porpoise are

expected for the North Atlantic Area (Bjørge and Tolley, 2018). Focussing on the North Sea,

no differences could be identified between the northern and the southern North Sea, whereas

variabilities in gene expression between individuals from the North Sea, compared to the Baltic

Sea are determined (Andersen, 2003; Wiemann et al., 2010; Lah et al., 2016). Furthermore,

genetic analyses of harbour porpoises originating from the Baltic Sea, revealed genetic varieties

and thus confirmed the hypothesis of different sub-populations in the following areas:

Skagerrak, Kattegat, Øresund, Belts and the Baltic Sea Proper (Andersen, 2003; Wiemann et

al., 2010; Lah et al., 2016). An abundance of 345,373 individuals were estimated within the

North Sea, whereas approximately 40,000 - 42,000 individuals are estimated for the western

Baltic Sea (incl. Kattegat, Belt Sea, Western Baltic) (Viquerat et al., 2014; Hammond et al.,

2017). Moreover, a decline of 1.79% of the abundance of harbour porpoises in the German

North Sea was determined by Nachtsheim et al. (2021).

The risk of exposure to macro- and meso-litter for those three marine mammals was already

determined in the North and Baltic Seas (Unger et al., 2017). Some knowledge is gained for the

occurrence of microplastics larger than 1 mm in harbour porpoises and harbour seals from the

southern North Sea (Bravo Rebolledo et al., 2013; van Franeker et al., 2018). However,

information on the occurrence of smaller microplastics are still poorly understood and studies

investigating specimen of the Baltic Sea are scarce.

During regularly conducted necropsies at ITAW of stranded, bycaught or mercy-killed marine

mammals of both seas (Siebert et al., 2001, 2007, 2020), samples of the GIT are collected since

2014. Those samples are analysed in the course of this thesis for the very first time.

Introduction

35

Furthermore, faecal samples of seals were collected at a well-known haul-out site in the North

Sea.

VIII. Aims of the Thesis

This thesis aims at firstly establishing a new protocol for avoiding secondary contamination

while processing samples of the gastrointestinal tract collected from marine mammals.

Secondly, this protocol is implemented for identifying the quantity and quality of microplastic

particles found in marine mammals from German waters (harbour porpoises, harbour seals and

grey seals, see Figure 1).

This thesis consists of the following two chapters:

Chapter 1: Improvement of sample storage and sample handling with the focus on

reducing secondary pollution

The first chapter focuses on a combination and improvement of already established procedures

to decrease the risk of secondary contamination. Therefore, protecting measures already start

during opening the carcasses and end in the stage of staining the filtered samples to avoid an

overestimation of microplastic particles. To ensure a low contamination risk, the samples were

collected in washed and disinfected glass jars since it could not be excluded that the usage of

plastic bags might contaminate the sample. Furthermore, the whole treatment of the intestine

and faecal samples is conducted in a closed acrylic box wearing white cotton gloves above

nitrile ones and a cotton laboratory coat. All used instruments were rinsed several times under

Milli-Q water (Millipore) to ensure that rinsing water can be excluded as a contamination

source. Procedural blanks are used to determine the ambient contamination within the working

environment and narrow down the actual burden of microplastics in marine mammals

inhabiting German waters.

For the isolation of microplastics (>100 µm) in the biogenic material, the samples are washed

in self-sewed washing bags in a conventional washing machine, as already conducted in diet

analysis research. Furthermore, the identification of potential microplastic particles is

conducted using fluorescence microscopy and µRaman spectroscopy.

Introduction

36

Chapter 2: Assessment of the microplastic burden in harbour porpoises from German

waters

This chapter focusses on the assessment of the real burden of microplastics in harbour porpoises

from the German North and Baltic Seas. This is the first time of investigating microplastic

particles smaller than 1 mm in harbour porpoises in a retrospective manner (2014 – 2018).

Intestine samples of 30 juvenile and adult individuals from the German North Sea (n = 14) and

Baltic Sea (n = 16) are analysed after the established protocol in Chapter 1. Certainly, the

polymer identification of found microplastics is conducted via µFTIR spectroscopy. In

addition, information on the health status collected in the conducted necropsies of the

investigated individuals were considered for possible detectable effects.

Figure 1. Overview of examined intestinal and scat samples of harbour seals, grey seals and harbour

porpoises from the North Sea and Baltic Sea.

Chapter I: Handle with Care – Microplastic

Particles in Intestine Samples of

Seals from German Waters

Published:

Philipp, C., Unger, B., Fischer, E.K., Schnitzler, J.G., Siebert, U. (2020).

Handle with Care – Microplastic Particles in Intestine Samples of Seals

from German Waters.

Sustainability.

DOI: 10.3390/su122410424

Chapter I

41

Chapter I: Handle with Care – Microplastic Particles in Intestine Samples of

Seals from German Waters

C. Philipp a, B. Unger a, E.K. Fischer b, J.G. Schnitzler a, U. Siebert a

a Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine

Hannover, Foundation, Werftstraße 6, 25761 Büsum, Germany

b Center for Earth System Research and Sustainability (CEN), University of Hamburg,

Bundesstraße 55, 20146 Hamburg, Germany

Keywords: microplastic; plastic isolation; plastic ingestion; gastrointestinal tract; marine

mammals

Abstract

The Marine Strategy Framework Directive (MSFD) aims to reduce the marine debris burden in

the marine environment by 2020. This requires an assessment of the actual situation, which

includes the occurrence as well as the caused impacts. Information on both is scarce when it

comes to top predators like marine mammals and the burden of microplastic. This is hampered

by the limited access to free ranging marine mammals for collecting samples, as well as sample

handling. The present study investigated gastrointestinal tracts and faecal samples of harbour

seals (Phoca vitulina) and grey seals (Halichoerus grypus) regularly occurring in the German

North Sea and Baltic Sea with the aim of gaining information on the occurrence of

microplastics. In total, 255 particles ≥100 µm (70 fibres, 185 fragments) were found in

exemplary ten intestine and nine faecal samples. The findings ranged from no fibres and six

fragments, up to 35 fibres and 55 fragments per sample. This study established a protocol for

sample handling, microplastic isolation (≥100 µm) and quantification of gastrointestinal tracts

and faecal samples of marine mammals with a low share of contamination. This approach helps

to quantify the presence of microplastics in free-ranging marine mammals and is therefore

applicable to assess the real burden of microplastic presence in the marine environment.

Introduction

42

Introduction

The challenging nature of marine debris pollution is well known (Galgani et al., 2015). Due to

its widespread use, its specific characteristics and discard virtue, synthetic polymers, so-called

“plastics” make up a high share of marine debris in our oceans (Thompson, 2004; Ryan et al.,

2009; Thiel et al., 2013). Microplastic (MP) includes synthetic polymers in the form of particles

smaller than 5 mm (Arthur et al., 2009). These particles originate either from large plastic items

cracking down into smaller fragments due to various forces (secondary MP); or are intentionally

produced in those small sizes (primary MP) (Gregory and Andrady, 2003; Browne et al., 2007;

Cole et al., 2011; Andrady, 2015). The awareness of microplastics started back in the 1970s

(Carpenter et al., 1972; Morris and Hamilton, 1974) but has only recently become more into

the focus of environmental research (Hermsen et al., 2018; Zhang et al., 2019).

MPs were revealed to be ingested by organisms of lower trophic levels, and even a trophic

transfer was detected (Farrell and Nelson, 2013; Setälä et al., 2014; Rummel et al., 2016). A

variety of international studies confirmed the presence of different sized plastic particles in the

gastrointestinal tract (GIT) of various marine mammalian species (Bravo Rebolledo et al., 2013;

Unger et al., 2016, 2017; Donohue et al., 2019; Nelms et al., 2019).

Dealing with faunal samples implies the initial elimination of organic matter enclosing the

particles; their detection otherwise would be obscured (Cole et al., 2014; Löder and Gerdts,

2015). Therefore, the biogenic compounds have to be removed prior to further investigations

in order to obtain convincing and reliable results (Cole et al., 2014; Löder and Gerdts, 2015).

Furthermore, it needs to be ensured that the risk of secondary contamination during sampling,

storing and treating is kept as low as possible. Contamination of samples might occur during

their handling and origin, e.g. from storage containers, laboratory cloths, gloves and the

working environment in general (Roux et al., 2001; Hidalgo-Ruz et al., 2012; Foekema et al.,

2013; Woodall et al., 2014; Vandermeersch et al., 2015).

Previous studies already presented various digestion protocols of biota samples in recent years

(Bravo Rebolledo et al., 2013; Lusher and Hernandez-Milian, 2018; Nelms et al., 2019).

Enzymes were predominantly used to remove the organic matter (Cole et al., 2014; Catarino et

Chapter I

43

al., 2017), in some cases acids or alkaline solutions were applied to digest organic remains

(Claessens et al., 2013; Foekema et al., 2013; Lusher and Hernandez-Milian, 2018).

Subsequently, MP identification techniques such as the staining and fluorescence microscopic

approach (Tamminga et al., 2017; Fischer, 2019) and spectroscopic analyses like Fourier

transform infrared (FT-IR) (Mintenig et al., 2017; Primpke et al., 2017; Jung et al., 2018) or

Raman spectroscopy are commonly used (Lenz et al., 2015; Fischer, 2019; Karbalaei et al.,

2019). A combination of both methods is considered to be a reliable option regarding the

identification of polymer compositions (Shim et al., 2017).

However, information on the presence of MP in and impacts on marine top predator species is

scarce. Therefore, the present study focusses on an establishing a protocol to isolate particles,

evaluate potential contamination and propose a method to discriminate between MP and other

particles in faecal and intestinal samples of seals in order to investigate the presence of MP in

marine mammals in further research. Samples were taken from harbour seals (Phoca vitulina)

and grey seals (Halichoerus grypus), which regularly occur in the North and Baltic Seas. This

study was realised within the framework of the project ‘Assessment and implementation for

long-term monitoring of pollution of diverse marine compartments and biota with marine litter’

(Federal Environment Agency Germany), the aims of which was to optimise sample handling

and to reduce the risk of secondary contamination for detecting microplastic particles in

intestinal samples of marine mammals

Materials and Methods

Sample Collection

The Institute for Terrestrial and Aquatic Wildlife Research (ITAW) at the University of

Veterinary Medicine Hannover (Foundation, Germany) regularly conducts necropsies of

stranded, bycaught or euthanised marine mammals found along the coastline of the Federal

State of Schleswig-Holstein, Germany (Siebert et al., 2001, 2007; Unger et al., 2017). These

are predominantly the three species regularly occurring in German waters; harbour porpoise

(Phocoena phocoena), harbour seal and grey seal.


44

Necropsy Sampling

Concerning microplastic analyses in the GIT of marine mammals, the rectum of harbour seals

and grey seals has been sampled since 2014. Out of these, ten pinniped samples were used for

this study. The caudal part of the rectum was tied off with a drawstring (Figure 2A).

Subsequently, in a cranial direction, an 8 - 10 cm section was tied off. Both ends were cut off

behind the drawstrings to prevent the loss of faeces (Figure 2B). With the help of metallic

tweezers, the samples were transferred to cleaned and disinfected glass jars. Afterwards, the

glass jars were stored at - 20°C until further processing (Figure 2C and D).

Sandbank Sampling

In the course of a regularly conducted health monitoring of live animals at the Lorenzenplate

(Hasselmeier et al., 2008), a tide-dependent submerged sandbank in the North Sea, further

faecal samples of free-ranging harbour seals and grey seals have been collected since 2012. Out

of these, nine faecal samples were analysed. No genetic analyses have been performed to

distinguish between harbour seal and grey seal faeces. Hence, this study consecutively refers to

them as seal faecal samples.

Chapter I

45

Figure 2. Sampling of intestinal samples: (A) The caudal part of the rectum is tied off; (B) An 8-10 cm

section of the rectum is measured in cranial direction and tied off a second time; (C) The intestinal

sample is cut with a scalpel and placed with metal tweezers in a glass jar (D).

The following steps were conducted in a closed acrylic box (see supplementary information)

and are applicable for all further investigations on intestinal and faecal samples. The box was

equipped with two holes, which formed the only openings within the box, these being necessary

for processing the samples. During the whole procedure, the samples were solely processed

with glass or metallic instruments. Furthermore, cotton gloves were worn over nitrile ones

during all processing steps.


46

Preparation

After defrosting the samples in the glass jars, the closed intestinal parts were rinsed with MilliQ

water (Millipore), measured and opened inside the washing sachets on a glass cutting board to

prevent the loss of faeces and potential MP.

For separating the MP from the biogenic matter, the intestine and faeces samples were washed

in self-sewed double layer washing sachets in a commercial washing machine (OK., OWM

15012 A1). Each sample was placed in an inner bag (mesh size 300 µm) which was then placed

in the outer nylon bag (mesh size 100 µm). The sachets were made of nylon cloth and were

sewn together in the acrylic box with a conventional sewing machine (Singer, Tradition TM

2282) using black cotton yarn. Furthermore, each nylon sachet was used only once to prevent

a cross-contamination.

Washing Procedure

The washing procedure of the samples was based on the protocol established by Bravo

Rebolledo et al. (2013). To remove biogenic organic matter, enzyme-based washing powder

(Biotex® stain removing powder, biological detergent, bleach free; 35 g) was added in the pre

wash cycle. Subsequently, conventional detergent (35 g, Gut & Guenstig Classic,Edeka

Zentrale AG & Co. KG) was used in the main wash cycle, assisting the cleaning procedure. The

samples were washed in a delicate wash cycle without spinning at 60°C. A delicate wash cycle

without spinning was chosen in order to prevent particles from escaping from the sachets and

thus not being available for further analysis. For a valid evaluation, all samples were weighed

before and after the washing procedure, for quantify the loss of biogenic compounds (e.g. blood,

faeces, soft tissue).

Prior to each wash cycle, an empty wash cycle was run using disinfectant (Impresan, Brauns-

Heitmann GmbH & Co.KG) at 90°C. This step cleaned the washing machine and additionally

reduced the likelihood of cross-contamination.

Chapter I

47

Isolation

After the washing procedure, the washing sachets containing the sample residues were covered

in aluminium foil and stored in a half-closed box under a fume hood for drying overnight. The

outside of the sachets were cleaned with MilliQ water prior to opening. The residue was rinsed

with a filtered, saturated sodium chloride (NaCl) solution (350 g of table salt dissolved in 1 l of

MilliQ water) in glass beakers. The solution was left overnight for density separation.

Approximately 20 to 30 mL of the surface of the supernatant was pipetted onto a cellulose filter

(Rotilabo®, Typ11A, Ø 55 mm, retention 12 – 15 µm) using a Buechner funnel (Rotilabo®,

porcelain, volume: 70 mL; Ø 55 mm) attached to a vacuum pump. The remaining solution and

the sediment were decanted onto a second filter. Both surface and bottom sample suspensions

were investigated separately in order to avoid an MP underestimation. The filtration steps

resulted in a total of four filters for each sample (inner bag: surface water and water from the

beaker bottom; outer bag: surface water and water from the beaker bottom).

The filters were stored in glass petri dishes (STERIPLAN®, Ø 60 mm) and dried in a heating

cabinet for three hours at 50°C. Until further processing, the closed petri dishes including the

sample filter were stored in a closed and dry environment.

Pre-Trials to Verify the Procedure

The methodical efficiency was evaluated, focussing on potential losses of particles and

secondary contaminations. The use of washing sachets was assessed for potential loss of

particles and loss of nylon fibres and thus the recovery rate. To determine the loss of potential

MP, polyethylene microbeads (fragment size: 150 µm - 800 µm) were placed in the nylon

sachets and processed in the same manner as the intestine and scat samples.

Prior to sample processing, five sachets were solely washed without any detergent and sample

(at 60°C, delicate cycle, no spinning cycle as mentioned above). A Cora ball®, which traps

loose fibres, was added. After the washing procedure using a Cora ball® the inspection showed

no attached fibres.


48

Polymer Identification

For identifying possible MP, the filters were stained with Nile Red diluted in chloroform (1 mg

/ mL in chloroform) in accordance to Tamminga et al. (2017). The stained filters remained

covered for drying for at least 24 hours, before being analysed under a plan fluorescence

microscope (Kern OBN- 148 and Zeiss AX10) using a TRITC HC filter set (F36-503, AHF

Analysetechnik, Tuebingen, Germany) in accordance with Tamminga et al. (2019).

Potential MP particles were photographed (Kern ODC 832 and Canon EOS 80D) under the

fluorescence microscope. Subsequently, the photos were visually evaluated and the suspected

MP were measured and counted by using Adobe Photoshop (Version 21.0.3). After visual

identification, single particles were isolated with tweezers and pins, and placed on microscopic

slides for further identification by µRaman spectroscopy (ThermoFisher Scientific GmbH,

DXR2xi Raman Imaging Microscope).

In general, direct comparisons among different studies are hampered due to varying sample

treatment protocols and mesh sizes being used. To address this problem, the present study

followed the sample processing conducted by Bravo Rebolledo et al. (2013) and focussed on

particle sizes ≥100 µm. In this study, the term MP included synthetic fragments and fibres. The

classification of fibres was used, if a fragment has an elongated cylindrical shape and a

thickness of up to 23 µm.

All particles larger than 100 µm were taken into account for valid results due to the used mesh

size (100 µm) of the outer washing sachet to avoid an overestimation. Two researchers

independently investigated the photos taken of particles to identify the potential MP. The

following parameters were used for discrimination: fluorescence intensity and appearance of

the particle (surface, isolation, completeness, etc.). For additional information, see the

supplement.

Based on the aforementioned discrimination parameters, we used recursive partitioning to

explore decision rules for predicting whether the particle was plastic or not. The decision tree

was created in RStudio (Version 1.1.447) with the package ‘rpart’ R package rpart.plot

(Therneau et al., 2019) with 1185 characterised particles (biogenic or synthetic). This number

of particles results of all available samples including pinnipeds and cetaceans, which build the

basis for the used parameters in the decision tree. The original dataset (n=1185) was randomly

Chapter I

49

split into training (80%) and test (20%) subsets. The training subset was used for model tuning

and training and the performance of the final model was compared using the Test subset.

Evaluation of the Methodical Efficiency

In the present study, the entire working space was located in an acrylic box situated under a

fume hood to decrease the potential secondary contamination of samples and used items. All

equipment and instruments were rinsed several times with MilliQ water prior to usage. The box

was washed with MilliQ water and wiped between each step with cotton cloths, which were

exchanged regularly.

For monitoring secondary contamination, procedural blanks of the applied chemicals and

detergents were used in accordance with Hengstmann et al. (2018) and Nuelle et al. (2014).

Procedural blanks helped to estimate the number of particles being introduced during the

processing; particles on the procedural blank filters were separately quantified, measured and

qualified. Contamination of cloths or instruments was deducted after the quantitative analysis

as performed in former studies.

To prove the possible contamination of plastic bags, five faecal samples frozen which had been

frozen in low density polyethylene (LDPE) bags since 2012. Nine faecal samples were

additionally included in this study. These samples were divided: half of each sample was stored

in LDPE-bags, the other half in glass jars for three, six and 12 months. The plastic bags in which

the samples are stored are not disinfected or cleaned prior usage. The comparison of the found

number of particles in glass and plastic samples was performed using a Wilcoxon signed-rank

test, because of the relation between the samples and the assumed no normal distribution. To

test if the findings of suspected MP increased over time, the plastic samples taken from different

periods were analysed by a non-parametric ANOVA using a Kruskal-Wallis test.

Results

50

Results


Procedural Efficiency of Washing Sachets

The pre-trials showed a loss of 0.358 g (2.99%) was determined for those sachets, which were

closed with a hook-and-loop fastener. Due to this small percentage loss, the usage of nylon

cloth was maintained, but the hook-and-loop fastener was discarded and replaced by a seam.

For further details, refer to the supplementary information.

By comparing the dry weights of the samples (before and after the washing procedure), the

average reduction in biogenic matter in intestinal samples differed between 9.97 g and 0.22 g

(mean = 1.57 g). In the faecal samples, the average reduction varied between 5.07 and 48.55 g

(mean = 13.33 g).

Efficiency of Contamination Control Measures

Procedural blanks analysed for the background contamination of the working space showed a

share ranging from zero to two particles per sample (M±SD = 0.9±0.99). Only one fibre larger

than 100 µm was found on the working environment controls (n = 12). One to two fragments

were found within the NaCl blanks (n = 5), which also included a potential contamination by

the used plastic laboratory bottles (M±SD = 1.4±0.99). Furthermore, five control washing

sachets showed a contamination of two to at most eight particles (M±SD = 4.6±2.7). Therefore,

a mean number of seven particles ≥100 µm (six fragments and one fibre) per sample was

considered as contamination. Figure 3 shows the share of suspected MP particles found in the

procedural blanks.

Chapter I

51

Figure 3. Suspected MP from procedural blank controls (≥100 µm). Working environment: 12 control

filter; Washing sachets: Five blank samples; NaCl Solution: Five control samples.

Comparison of suitable storage methods for avoiding contamination

Each sample archived in a glass jar showed a lower amount of suspected MP compared with

those stored in plastic bags (Figure 4). The Wilcoxon test revealed significant differences,

showing that plastic stored samples included eight particles (≥100 µm) more compared with

those stored in glass jars (particles ≥100 µm: p = 0.012; particles ≤100 µm: p = 0.0104).

The direct comparison of samples stored for different periods in plastic bags (three, six and 12

months) revealed no significant increase in microplastic particles over time (Figure 4). This

was due to a high variability in each period group. However, a comparison with samples, which

had been stored in plastic for eight years, showed no significant differences to the present

samples (Figure 5).

Results

52

Figure 4. Differential values in the quantity of suspected MPs (≥100 µm) in samples stored in plastic

bags, compared to ones stored in glass jars from different storage times.

Chapter I

53

Figure 5. Suspected microplastics in faecal samples stored in glass jars (n = 9) and plastic bags (n = 9)

combined from all storing periods (three, six and 12 months; particles ≥100 µm). In addition, five

samples of seal faeces, which had been stored in LDPE bags for eight years, were presented for

comparison purposes.


The results of the used decision tree (Figure 6) revealed the following parameters to be

decisive: 1) If a particle could be associated with a biogenic shape or structure (e.g. crustacean

limbs or algae). If yes, it was classified as no MP (No-Plastic). If not, the fluorescent colour of

the particle (orange including red and yellow or white) defined the next classification (2). If the

fluorescent colour was white, the particle was classified as suspected MP. If the colour was

yellow or orange, the next category was the appearance: (3) blurred, melted or defined

appearance. If it was defined, the particle was classified as plastic; if not it was identified as

No-Plastic. If, the fluorescent colour the second category was white, the particle was

categorised as plastic. A catalogue of examples and categories is presented in the supplement.

Results

54

Potential microplastic particles identified by using this decision tree are than analysed using

µRaman spectroscopy.

The calculated sensitivity (true positive rate, TPR) of 84%, that measures the proportion of

particles that were identified as possible MP and the specificity (true negative rate, TNR) of

69%, which measures the proportion of particles that were identified as being non-plastic,

revealed a good performance of the decision tree.

Figure 6. Example of a decision tree of the test set-up of 510 particles showing the decisive parameters.

Isolation of Potential MP in Intestinal and Faecal Samples

In total, 653 potential plastic particles were determined in ten intestinal samples of free-ranging

harbour and grey seals, and nine seal faecal samples where the described procedure (sample

collection, isolation and identification) was used. Due to the used mesh sizes of the washing

sachets (100 µm and 300 µm), all particles smaller than 100 µm were excluded from the study,

Chapter I

55

resulting in 255 identified suspected MP particles (70 fibres and 185 fragments) in 18 out of a

total of 19 samples. The share of fibres ranged from zero to 35 per sample (M±SD = 6±7.4)

when taking all specimen stored in glass jars into consideration (≥100 µm). The number of

fragments in each sample stored in glass jars (≥100 µm) varied from five to 55 parts (M±SD =

13.3±11.3).

Some MP findings are shown in Figure 7. For a total of 31 suspected plastic particles, the

polymer composition was verified via µRaman spectroscopy. 28 particles (90%) of these 31

particles were identified as plastic polymer. Following polymers were determined:

Polyethylene (PE, n = 14), polyethylene terephthalate (PET, n = 5), ethylene-vinyl acetate

(EVA, n = 6), polyamide (PA, n = 1) and polypropylene (PP, n = 1). Those particles were

collected randomly from all 19 named samples, thus 11% of the whole quantity of suspected

MPs were analysed.

Figure 7. Identified microplastic particles from intestinal samples stained with Nile Red photographed

under a fluorescence microscope using a TRITC filter: (A) A nylon fibre of the used nylon cloth; (B)

PET-fibre found in a grey seal; (C) EVA particle found in a harbour seal; (D) PP particle found in a

grey seal; (E) PE found in a harbour seal

Discussion

56

Figure 8 shows the higher quantify of particles found in intestinal samples of grey and harbour

seals compared with the collected faecal samples where the species is unknown. It is significant

that the quantity of suspected MP exceeded the maximal assumed secondary contamination of

seven particles per sample (see dotted line in Figure 8).

Figure 8. Suspected microplastics in intestinal samples of harbour (n = 5) and grey seals (n = 5), in

comparison to faecal samples from seals collected on the sandbank (Lorenzenplate, North Sea) (n = 9,

no determined species per sample: faeces originated from either a grey or a harbour seal). All samples

were stored in glass jars. The dotted line show the maximal presumption of secondary contamination of

seven particles per sample.

Discussion

An initial and essential step for studying the effects of MP particles in GITs of marine mammals

is the detection and evaluation of their current presence and distribution. Apart from assessing

the presence of microplastics in marine mammals, the main focus of this study was to improve

the avoidance of secondary contamination during sample handling. This problematic nature of

secondary contamination, which was also shown in previous studies (Bravo Rebolledo et al.,

Chapter I

57

2013; Foekema et al., 2013; Nuelle et al., 2014; Hengstmann et al., 2018; Lusher and

Hernandez-Milian, 2018), needs to be focused on to achieve genuine results. The presented

protocol combines established protocols for isolating MP from biota samples (Bravo Rebolledo

et al., 2013; Lusher and Hernandez-Milian, 2018) new approaches for specifically avoiding

secondary contamination.

The first step for avoiding contamination was to store the samples in glass jars, since they are

prone to contamination when stored in conventional plastic bags. The present study presented

evidences of this aforementioned fact. The results of the direct comparison between archived

samples in plastic bags and in glass jars showed that a contamination by plastic bags is likely.

The amount of plastic particles did not significantly increase over time, but the basis of

contamination from external sources seems to be higher when archiving samples in plastic bags

instead of storing them in glass jars. It has to be taken into account that the used samples already

show a basic microplastic contamination. This differs from sample to sample, resulting in the

fact that the plastic burden of the samples also varies between the time periods. A conceivable

parameter besides the polymer structure of plastic bags is the cleaning aspect. Glass jars were

cleaned and disinfected prior to usage; plastic bags were used in their original state. Based on

the results of this study, it is recommended to use glass jars instead of plastic bags or containers.

A closed working environment was created to improve the measures undertaken in previous

studies to prevent secondary contamination. The procedural blanks analysed for the background

contamination of the working space showed a very low number of particles (0-2 per sample).

Furthermore, the share of fibres was significantly lower in this study compared to similar

approaches in marine mammals (Lusher et al., 2018; Nelms et al., 2018, 2019; Donohue et al.,

2019) or other marine species (Lusher et al., 2013; Steer et al., 2017; Fischer, 2019).

Additionally, it has been previously highlighted, that fibres in particular are an indicator of

secondary contamination, originating from the laboratory clothing and working environment

(Hidalgo-Ruz et al., 2012; Nuelle et al., 2014; Torre et al., 2016; Lusher et al., 2017b;

Hengstmann et al., 2018; Lusher and Hernandez-Milian, 2018). Therefore, the acrylic box

safeguarded the samples from direct contact with the laboratory environment and personnel.

The presented study revealed that closed sealing, like the washing sachet, reduces the loss of

particles. To minimise the risk of losing particles as well as reducing the contamination of the

samples, it is advisable to place the samples into a nylon cloth, which is then sewn together.

Discussion

58

Pre-trials revealed the application of hook-and-loop fasteners to be unsatisfactory (see

supplementary information); similar results are noted for rubber bands (Orr et al., 2003).

The usage of nylon cloths was inevitable due to the favourable mesh sizes and the fact that no

solid material can be used in the washing machine, although polyamide (nylon) is one of the

most abundant polymers (Andrady, 2011; ter Halle et al., 2017). Indeed, fibres of the nylon

washing sachets can be easily identified showing a distinct fibre pattern and were therefore not

included in our study. Thus, there was no need to exclude polyamides from this study, if these

polymers were found. To validate this assumption, a found fibre showing a distinct structure

and fluorescence intensity compared with the fibres of the used nylon cloths was identified as

polyamide (nylon) by µRaman spectroscopy. Therefore, the polymer identification via µRaman

spectroscopy was used to manifest the presence of microplastics, and is an already established

and reliable application in microplastic analysis (Lang et al., 1986; Andrady, 2011; Lenz et al.,

2015; Cabernard et al., 2018). Thus, the benefits of this applied cleaning procedure (1. time and

cost effective, 2. valuable results) outweigh the disadvantages of the used nylon cloths. The

usage of a conventional washing machine and detergent was demonstrated as a successful

application to speed up the purification procedure resulting in clean residues. Removing

biogenic substances with the help of the washing procedure preserves the original particles, and

it is less time- (approx. 90 min) and budget-consuming compared with the application of

enzymatic or chemical solutions for the digestion of biogenic material. Thus, the reduction of

biogenic compounds (e.g. faeces, blood, and soft remains of prey species) was effective to

decrease the sample to the remaining hard parts (e.g. sand grains, fish bones and lenses, beaks,

and microplastics), as also seen by the compared dry weights. These results are also shown by

the comparison of dry weights before and after the washing procedure. In the intestinal samples,

the intestinal tissue and the hard parts remained. A mean difference of 1.75 g was determined.

In comparison to the faecal samples where a mean difference of 13.33 g was ascertained. The

weights were mostly dependent on the quantity of hard parts (e.g. sand grains, fish otoliths, and

lenses, bones or squid beaks) within the respective sample. Furthermore, the weight of pure

faeces was higher in the sandbank faecal samples (mean = 19.52 g) compared to faeces included

in the intestinal tissue (mean = 15.06 g). Firstly, these differences can be explained by the

intestinal physiology of phocid species, with a short length of the large intestine where the

chymus is dehydrated and stored as faeces until excretion (Helm, 1983; Pabst et al., 1999).

Chapter I

59

Secondly, passage times of marine mammals have to be taken into account if an assessment of

the microplastic burden in marine mammals is required (Panti et al., 2019). The passage times

of harbour seals and grey seals were estimated to be longer (6.5 – 30 h) (Prime and Hammond,

1987; Harvey, 1989). Thus, the sandbank samples may include the faeces of a full egestion of

the rectum. In contrast, the intestinal samples represent only a part of the actual excretion, which

is here further analysed. Furthermore, a reduction in size and loss of microplastics is

conceivable like it is confirmed for otoliths (Prime and Hammond, 1990). Combining collected

faecal and intestinal samples is advisable for further investigations as this will display the

burden of microplastics in an extended way.

Besides the method, there are more side parameters which have to be considered while

collecting samples from wild ranging animals. This study only investigated a part of the

intestinal system instead of examining the whole GIT or the stomach as performed in recent

studies (Lusher et al., 2015a, 2018; Nelms et al., 2019). This step was chosen to enable a

potential contamination-free sample although a complete examination of the residual GIT and

the carcass can be conducted. The reason for this is the usage of one organ for only one

assessment is applicable in the fewest of cases during necropsies. Another benefit of this

strategy is the unhindered necropsy of the whole individual, which enables an entire overview

of the health status and possibly provides more background information.

Furthermore, it is already known that the stomach of marine mammals accumulates a high

quantity of ingested hard parts (Nelms et al., 2019).

The amount of positive samples (suspected MP ≥ 100 µm, in glass jars) is higher than in

similar studies of faecal samples of seal species from the northern and southern Pacific Ocean

(Perez-Venegas et al., 2018; Donohue et al., 2019) despite it beings conformable with studies

investigating marine mammals of the north-eastern North Atlantic region (Lusher et al., 2018;

Hernandez-Milian et al., 2019; Nelms et al., 2019). As a consequence of the results of the used

statistical applications (precision of 94% and sensitivity of 84%), in combination with the 90%

chance of a correct classification as MP, this study is assumed to be a valuable approach in MP

monitoring in marine mammals.

The establishment of a decision tree helps to categorize different variables, which are helpful

for untrained researchers to gain results that are comparable between research groups. This is

essential to compare the burden in different marine environments and biota. Furthermore, the

Conclusion

60

decision tree helps to narrow down suspected microplastic particles out of a bunch of a variety

of particles in a sample, which are then used for further analysis. This is cost- and time-effective

and a useful tool for encouraging decision makers to support necessary monitoring

programmes.

In addition, the applied methods comprise a combination of interdisciplinary analysis:

identifying microplastics and their consumed prey (diet analysis of hard parts). This is based

on the conciliatory washing procedure; the retained hard parts can be used for prey species

identification (Orr et al., 2003; Robinson et al., 2018). Since it is proven that microplastics are

transported across the food web (Farrell and Nelson, 2013; Setälä et al., 2014; Ory et al., 2018),

it is assumed that in particular the contained contaminants accumulate due to absorption and

leaching (Rani et al., 2015) in top predator species as it is already evidenced for example for

organochlorides (Ross et al., 2000; Siebert et al., 2012). This allows new insights into the

correlation between microplastic occurrence in marine top predator species and their prey,

which serve as a vector.

Conclusion

The sample protocol applied within this study is applicable for isolating and identifying

microplastics from samples of gastrointestinal tracts of marine mammals and is comparable

with other approaches like diet analysis. Low numbers of detected fibres led to the conclusion

that secondary contamination is low. This seems to be most likely an effect of using a closed

environment, the acrylic box and the usage of glass jars as storage container. Furthermore, the

used isolation procedure is less cost-intensive, more gentle and faster in comparison to an

enzyme or chemical digestion. However, every working environment and samples of different

species and different sizes pose different challenges for microplastic analysis.

It can be assumed that the current approach provides reliable results, reflecting the actual

microplastic burden in the analysed species. Thus, this study presents a possible application for

microplastic monitoring in marine mammal samples, which has to be extended to a higher

amount of samples to achieve convincing findings in different species and water bodies.

Nevertheless, this method is also transferable to other mammalian species.

Chapter I

61

Author Contributions

Conceptualization, B.U. and C.P.; Methodology, B.U. and C.P.; Investigation: C.P.; Formal

analysis and Validation: J.G.S. and C.P.; Supervision: U.S. and E.K.F.; Resources: E.K.F. and

U.S.; Writing—original draft: C.P.; Writing—review and editing: B.U., E.K.F., J.G.S., U.S.

and C.P. All authors have read and agreed to the published version of the manuscript.

Funding

The stranding network and the health monitoring is funded by the Schleswig-Holstein Agency

for Coastal Defence, National Park and Marine Conservation, Germany. The pathological

investigations of the marine mammals are partly funded by the Ministry of Energy, Agriculture,

Environment, Nature and Digitalization of Schleswig-Holstein, Germany. The Federal

Environment Agency (UBA) partly funded the analysis of a number of samples within the

project “Assessment and implementation for long-term monitoring of pollution of diverse

marine compartments and biota with marine litter” (FKZ 3717252250). This publication was

supported by the German Research Funding Organisation (Deutsche Forschungsgemeinschaft)

and the University of Veterinary Medicine Hannover, Foundation (Germany), within the

funding programme Open Access Publishing.

Acknowledgements

We would like to thank all seal rangers in Schleswig-Holstein (SH), Germany for reporting and

collecting carcasses as well as our colleagues in the ITAW necropsy team for collecting and

dissecting carcasses continuously throughout the year. Special thanks go to E. Ludes-

Wehrmeister and S. Gross for advising us regarding tissue sampling and storing. We would like

to thank the “Microplastic Research at CEN – MRC” working group at the University of

Hamburg (Germany), for their assistance and hospitality. Special thanks in particular go to M.

Tamminga for sharing his expertise.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study;

in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the

decision to publish the results.

Supplementary Material

62


1. Preparation

1.1 Pre-trials

Pre-trials for testing the efficiency of the procedure have been carried out beforehand. To

determine the loss of particles in the nylon washing bags, aluminium silicate (200 µm – 500

µm) and polyethylene microbeads (150 µm - 800 µm) were used. Due to the mesh size, the

inner washing bag retains particles ≥300 µm (Supplementary Figure 1A), the outer washing

bag particles ≥100 µm (Supplementary Figure 1B). Therefore, particles smaller than 100 µm

were excluded from the analysis. Using three washing bags five times, each time freshly filled

with aluminium silicate tested the loss of particles. These pre-trial bags were weight before and

after the washing procedure. An average loss of 6.29 g (14.79%) was determined. This high

loss is assumed to result from the sharp form of the aluminium silicate fractions, which damages

the nylon fabrics (Supplementary Figure 1D).

Supplementary Figure 1. The washing bag design used for the pre-trials using pre-sewed bags closed

with stitched hook and loop fastener (A & B). The size fragmentation based on the nylon cloths operates

successful during the washing procedure, what approved the method of different cloths (C). These pre-

trials were conducted with aluminium silicate (200 µm – 500 µm).

A C

B D

Chapter I

63

The loss of 0.358 g (2.99%) was much less while using the less sharp microbeads instead of

aluminium silicate. Here, no damage could be recognised. Since microbeads are more

comparable to actual microplastic in their characteristics, an average loss of 3% have to be

taken into account per sample. Sewing the bags additionally in a cross stitch helps to keep the

loose fibres of the nylon cloth together, and further secures the closeness.

Supplementary Figure 2. The second circle of pre-trials were conducted with microbeads (ca. 150 µm

– 800 µm; see B & C). Oil pellets (green) were removed during washing procedure (see A); the

remaining microbeads were used for determining the loss of particles.


64

1.2 Working in an Acrylic Hood

The sample handling was conducted in an acrylic hood. This closed environment decreases the

risk of contaminating the samples. Holes for the hands, the electric cable and hoses as well as

fixing equipment, were sawn out (Supplementary Figure 3).

Supplementary Figure 3. Work process in an acrylic box. A) Sewing the samples into nylon washing

bags. B) Processing the samples with cotton gloves within an acrylic box situated under a fume hood.

2. Intestinal and Faecal Samples

2.1 New design of the washing bags

For analysing the intestine and faeces samples, a new design of the washing bags was

established (Supplementary Figure 4). The hook and loop fastener as seen in Supplementary

Figure 1 was discarded, to reduce the potential of hold back particles. Furthermore, the samples

are directly sewed into both cloths to decrease the contamination and it turned out to be a faster

method. For distinguishing samples, rinsed metal paperclips where added.

Chapter I

65

Supplementary Figure 4. Sliced intestine sample sewed in the nylon washing bags. Washing bag

consists of two nylon cloths (inner bag: 300 µm and outer bag: 100 µm). Overhanging nylon cloth is

cropped before conducting the washing cycle.

2.2 Prevention of overcounting

Lost fibres of the nylon washing bags can easily be identified, since they are showing a distinct

fibre pattern (Supplementary Figure 5). Black cotton yarn was used for sewing, since the Nile

Red staining does not stain it. Therefore, these nylon fibres and the black cotton were not

included in this study. All particles <100 µm are discarded due to the mesh size of the outer

bag (100 µm) to avoid counting particles entering the sample as contamination.

Supplementary Figure 5. Nylon fibres of the washing bag cloth with an incomparable zigzag shape

and reddish colour.


66

3. Identification Catalogue

Particles are assumed as MP in the presented study after the following criteria:

the particle is self-contained

the structure distinguishes from those associated with a biogenic one (e.g. chitin

structure, plant-based or fish-related ones)

the particle is fluorescent (yellow to whitish colour)

the location of the particle is ca. 3 mm distant from the filter edge (avoidance of

contamination)

The following figures underpin the beforehand described list of issues and can help for

distinguishing MP from biogenic particles.

Supplementary Figure 6. Fish bone.

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67

Supplementary Figure 7. Fish bones and plant-based fragments.

Supplementary Figure 8. Fish vertebrae.


68

Supplementary Figure 9. Fishbone.

Supplementary Figure 10. Biogenic fragment (e.g. plant-based seed or a big fish lens).

Certainly, no microplastic bead.

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Supplementary Figure 11. Biogenic fragment.

Supplementary Figure 12. Plant-based fragment.


70

Supplementary Figure 13. Biogenic fragment.

Supplementary Figure 14. Cellulose fibre.

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Supplementary Figure 15. Microplastic at the edge of a filter.

This fragment is to close located to the edge, thus it was not counted.

Supplementary Figure 16. Countable microplastic fragment.


72

Supplementary Figure 17. Countable microplastic fragment surrounded by fish bones.

Supplementary Figure 18. Countable microplastic fragment surrounded by a cellulose fibre.

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3.1 Categorization of parameters

Supplementary Figure 19. Fluorescence intensity.

Supplementary Figure 20. Surface.

Supplementary Figure 21. Appearence.


74

Supplementary Figure 22. Completeness.

Supplementary Figure 23. Surroundings.

Supplementary Figure 24. Organic matter.

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4. Results

All microplastic particles (≥100 μm) found in intestinal and faecal samples of seals from

German waters are shown in Supplementary Table 1. Labelling is made up of species initials

(Pv = Phoca vitulina; Hg = Halichoerus grypus) and sampling location (e.g. Intestine). All

samples are archived in glass jars.

Faecal samples archived in plastic bags since 2012, are named as “LDPE_Bag_2012”. Faecal

samples of seals (no information on the species) were halved and stored in glass jars or plastic

bags for different time periods, which is also stated in the label (“Glass_3_months” or

“Plastic_3_months”, etc.). The column “identified polymer structures” gives information on the

identified polymers regardless the number of particles analysed. NA (no information available)

refers to samples in which no particles could be further identified by μRaman spectroscopy.

The findings of fragments and fibres, in addition to the identified polymer structure should only

give information on the usefulness of the applied and presented protocol.

Supplementary Table 1. Results of microplastic analysis.


76

Chapter I

77

Chapter II: First Evidence of Retrospective Findings

of Microplastics in Harbour Porpoises

(Phocoena phocoena) From German Waters

Published:

Philipp, C., Unger, B., Ehlers, S.M., Koop, J.H.E., Siebert, U.

First Evidence of Retrospective Findings of Microplastics

in Harbour Porpoises (Phocoena phocoena) From German Waters.

Frontiers in Marine Science.

DOI: 10.3389/fmars.2021.682532

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83

Chapter II: First Evidence of Retrospective Findings of Microplastics in

Harbour Porpoises (Phocoena phocoena) From German Waters

Carolin Philipp a, Bianca Unger a, Sonja M. Ehlersb,c, Jochen H. E. Koopb,c, Ursula Siebert a

a Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine

Hannover, Foundation. Werftstr. 6, 25761 Büsum, Germany

b Department of Animal Ecology, Federal Institute of Hydrology, Am Mainzer Tor 1, 56068

Koblenz, Germany

c Institute for Integrated Natural Sciences, University of Koblenz-Landau, 56070, Koblenz,

Germany

Keywords: microplastic burden; FTIR; marine mammals; cetacean; North Sea; Baltic Sea;

nutritional status; health

Abstract

Microplastic ingestion by lower trophic level organisms is well known, whereas information

on microplastic ingestion, egestion and accumulation by top predators such as cetaceans is still

lacking. This study investigates microplastics in intestinal samples from harbour porpoises

(Phocoena phocoena) found along the coastline of Schleswig- Holstein (Germany) between

2014 and 2018. Out of 30 individuals found along the North Sea (NS) and the Baltic Sea (BS)

coast, 28 specimens contained microplastic. This study found a relationship between the

nutritional status of cetaceans and the amount of found microplastics. Harbour porpoises with

a good or moderate nutritional status contained a higher number of microplastics, when

compared with specimens in a poor nutritional status. In addition, when individuals died

accidently due to suspected bycatch in gillnets, where a feeding event is highly assumed or a

pharyngeal entrapment happened, the microplastic burden was higher. In total, 401

microplastics (≥100 µm), including 202 fibres and 199 fragments were found. Intestines of the

Introduction

84

specimens of the BS contained more microplastics than the ones from the NS. Differences in

the share of fibres could be revealed: for BS fibres constituted 51.44% and for NS, fibres

constituted 47.97%. The polymers polyester, polyethylene, polypropylene, polyamide, acrylic

(with nitrile component) and an acrylic/alkyd paint chip (with styrene and kaolin components)

were identified. This is the first study investigating the occurrence of microplastics in harbour

porpoises from German waters and will, thus, provide valuable information on the actual burden

of microplastics in cetaceans from the North and Baltic Seas.

Introduction

The ubiquitous presence of marine litter, and especially the occurrence of small particles called

microplastics (<5 mm) (Arthur et al., 2009) is already confirmed in different marine habitats

and organisms (Fossi et al., 2014; Lusher et al., 2015b; Pereira et al., 2020). A trophic transfer

of microplastic particles between species of different trophic levels can be assumed as this has

previously been determined by other studies (Farrell and Nelson, 2013; Setälä et al., 2014;

Nelms et al., 2018). In addition, the ingestion and presence of microplastics has been highly

studied throughout the food web in recent years (Miller et al., 2020). Besides the unintentional

uptake of microplastics by prey species, an intentional uptake by organisms caused by a

burdened environment or due to accidental prey resemblance has already been shown (Ory et

al., 2017; Roch et al., 2020).

When focusing on the study area, a microplastic burden in the North-East Atlantic area and its

organisms occupying different trophic levels has also been verified Furthermore, it is assumed

that microplastics and pollutants accumulate in marine top predator species (Fossi et al., 2014;

Jepson et al., 2016; Garcia-Cegarra et al., 2021). The ingestion of marine litter is already

confirmed by previous studies in the North Sea (NS) and Baltic Sea (BS) (Unger et al., 2017;

van Franeker et al., 2018). This is not surprising, since the NS and adjacent waters are assumed

to be highly affected by anthropogenic exploitations (Halpern et al., 2008). Nevertheless, the

knowledge on the burden of microplastics in top predators, particularly in cetaceans of the

eastern North Atlantic region, including the BS, is still lacking. Both seas are different in

topography, salinity, hydrology (Frid et al., 2003; Sjöqvist et al., 2015), and the occurring ship

Chapter II

85

traffic (OSPAR, 2021). Thus, differences in the presence and absence of marine litter and in

particular of microplastics between both seas could be hypothesised.

For investigating this knowledge gap, this study focusses on intestinal samples of harbour

porpoises (Phocoena phocoena) originating from both seas. This species is the only regularly

occurring cetacean in the southern NS, and the only cetacean inhabiting the BS (Hammond et

al., 2017). Therefore, this study aimed to gain knowledge regarding the following three aspects:

(i) assessment of the general risk of microplastic accumulation in harbour porpoises, (ii)

evaluation of potential health impacts, and (iii) the comparison between the individuals

originating from the NS and the BS. The porpoises found along the German NS coast are part

of the North-East Atlantic population, and the Baltic individuals belong to the subspecies of the

western Baltic population (Gaskin, 1984; Andersen, 2003; Lah et al., 2016).

This is the first retrospective investigation of microplastics in relation to the health status of

harbour porpoises from German waters to date, examining particles which are smaller in size

than 1 mm. Based on the collected information on sex, age and health status during necropsies,

this study enables to determine potential relationships between a suspected microplastic burden

and different health aspects in harbour porpoises originating from the NS and the BS for the

first time. In addition, since the polymer types of found particles are determined by micro-

Fourier-transform infrared spectroscopy (µFTIR), possible microplastic sources could be

hypothesised and discussed.


Based on the well-established stranding network in Schleswig- Holstein (Germany), carcasses

of stranded and bycaught harbour porpoises from the NS and BS are collected in the course of

a health monitoring (Siebert et al., 2001, 2020; Lehnert et al., 2005). This monitoring is

established since 1990 at the Institute for Terrestrial and Aquatic Wildlife Research (ITAW),

which regularly conducts necropsies of harbour porpoises using a standardised protocol (Siebert

et al., 2001, 2020). Since 2014, intestinal samples of marine mammals, including harbour

porpoises, were exclusively collected for microplastic analysis. Based on the necropsies, the

age, sex, health status and the location in which each individual was found is assessed and


86

recorded. Thus, this information is available for the investigated intestinal samples from

harbour porpoises found between 2014 and 2018.

The following criteria were applied for choosing the most suitable samples: (i) the

gastrointestinal tract (GIT) had to be intact, (ii) faeces were present, and (iii) the individual was

already weaned. 30 individuals were chosen for analysis: 14 individuals from the NS and 16

individuals from the BS (Figure 9).

Figure 9. Locations where investigated harbour porpoises were found (n = 30).

The intestinal samples were stored in pre-cleaned glass jars at −20°C until further processing.

Then, each defrosted and opened intestinal sample was placed into a double-layered washing

sachet made of nylon cloths. The inner bag of the washing sachet has a mesh size of 300 µm

and the outer bag has a mesh size of 100 µm. Both cloths, including the sample, were sewn

together with the help of a conventional sewing machine, resulting in a so-called washing

sachet. These washing sachets were washed in a conventional washing machine at 60◦C without

Chapter II

87

spinning cycle. For the removal of biogenic matter, an enzyme based detergent and a

conventional detergent were added for facilitating the washing procedure. Subsequently, a

density separation, a vacuum filtration onto cellulose filters (Rotilabo®, Typ 11A, Ø 55 mm,

retention 12–15 µm) and fluorescence microscopy enabled by Nile Red (diluted with

chloroform) staining were conducted for microplastic isolation and identification.

Subsequently, all potential microplastics found on the cellulose filters were photographed,

counted, and measured in size. All steps of sample processing were conducted in a closed

acrylic box to avoid airborne contamination. The whole implementation of sample handling

and processing is described in detail in Philipp et al. (2020).

For polymer identification, selected microplastic particles were manually collected. In addition,

a disinfectant step was conducted to exclude a passing on of bacterial or parasitical zoonosis.

For this purpose, the cellulose filters containing the stained particles were sprayed with ethanol

(70%). After evaporation, the particles showed the same fluorescence qualities as before. Thus,

the potential microplastics were selected and manually collected with tweezers or needle pins

and placed into a droplet of ethanol (70%) onto an aluminium oxide membrane filter (Anodisc,

Ø47 mm, 0.2 µmpore size, Whatman, Freiburg, Germany). The filter was kept still until the

droplet was evaporated and the particles had attached to the filter. Since the transfer of particles

was done manually, a loss of particles needs to be taken into account.

The polymer composition of 77 potential microplastics (incl. fragments and fibres) from

intestinal sample were analysed by using a µFTIR spectroscope (Hyperion 2000, Bruker,

Ettlingen, Germany). All measurements were conducted in transmission mode with 32 co-

added scans (sometimes 100 scans for very thin fibres) and a spectral resolution of4 cm−1 in a

wavenumber range of 4,000–1,250 cm−1 as aluminium oxide membrane filters are infrared

inactive between 3,800 and 1,250 cm−1. For background measurements, the blank aluminium

oxide membrane filter was used. For thick particles, for which transmission mode was not

suitable, the measurements were conducted in attenuated total reflectance (µATR) mode. Those

µATR measurements were conducted between 4,000 and 600 cm−1.

Procedural blanks of the used detergents and materials, e.g., nylon sachets (n = 3), and the

working environment (n = 10) were taken into account for avoiding an overestimation caused

by secondary contamination. The analysed blank filters of the working environment

Results

88

accompanied the samples from time of collection until the staining procedure was finished. On

average, one fibre and seven particles were found in those procedural blanks and were finally

subtracted from the microplastic counts in each parallel sample. Four of those potential

microplastics could be collected, manually placed on the aluminium oxide membrane filter and

were considered for µFTIR analysis. Moreover, the polymer composition of different

equipment materials like the nitrile gloves and shavings of the used acrylic box were

additionally determined by FTIR in ATR mode (Vertex 70; Bruker, Ettlingen, Germany) or by

µFTIR to avoid an overestimation. For the Vertex measurements, ATR measurements were

performed in a wavenumber range of 4,000– 370 cm−1 with 8 co-added scans and a spectral

resolution of 4 cm−1.

The quantity of found microplastics in comparable groups is given in mean ± standard deviation

(M ± SD) to enable a comparison between findings. Moreover, the results were statistically

analysed by determining the Cohen’s d and applying a paired t-test using the package “pwr” in

the R software Version 4.0.2 (Champely et al., 2020; R Core Team, 2020). Thus, results were

described as significant if p <0.05. In addition, the Figures Figure 10, Figure 13Figure

14Figure 15 were visualised using the package ‘ggplot2’ (Wickham, 2016).

Results

Quantity and Size

In total, 30 intestinal samples were available for analyses. An amount of 611 potential

microplastics (incl. fragments and fibres, >100 µm) were found. A secondary contamination of

one fibre and seven fragments were considered and subtracted from each sample. Thus, 401

microplastics were finally determined. This amount of microplastics was found in 28 intestinal

samples, in the remaining an absence of microplastics was noticed. When categorising into

particle type, 202 fibres and 199 fragments were found. Hence, only two intestines were free

from microplastics. Most of the found fibres had a length between 100 and 2,000 µm (Figure

10).

Four additional fibres longer than 5,000 µm, thus defined as mesoplastics (Gregory and

Andrady, 2003), were found. Three of them occurred in a sample of an adult male harbour

Chapter II

89

porpoise found in 2017 (lengths: 8,450, 6,964, and 8,029 µm). The fourth fibre (7,365 µm in

length) was found in a juvenile male stranded in 2014. Both carcasses were found in the BS.

Based on the size, those four fibres were excluded from the results.

Figure 10. Width-length distribution of all suspected microplastic particles in intestine samples of 30

harbour porpoise in the size range of 100 µm up to 5,000 µm (n = 611). Secondary contamination was

not considered.

Results

90

FTIR Results

Out of all 611 potential microplastics found in the 30 intestinal samples from the harbour

porpoises, originally 94 particles (16%) were selected for polymer identification by µFTIR.

Those fibres and fragments were manually collected and placed onto Anodisc membrane filters.

Subsequently, 77 particles (12%, nfibres= 28, nfragments = 49) found in the intestinal samples were

finally analysed by µFTIR. The remaining 17 microplastics (nfibres= 7, nfragments = 10) were either

lost during the sample transport in closed petri dishes to the analysing site or could not be

measured due to their small size. Polyester (PEST) was the most frequently found polymer in

those investigated intestinal samples (nPEST = 30), followed by polyethylene (PE, n = 17) and

polyamide (PA, n = 12) (Figure 11). Furthermore, two polypropylene (PP) particles, one paint

chip (acrylic / alkyd with kaolin and styrene) (see Supplementary Figure 25), one none further

identified polyolefin and one cellulose acetate fibre (which is a semi-synthetic cellulose) were

determined. Three acrylic particles, including two with nitrile component, were additionally

found. A visualisation of the found polymers (n = 67) is given in Figure 12.

Chapter II

91

Figure 11. Microplastic particles stained with Nile Red (A-D) and the corresponding measured µFTIR

spectra of found polymers (in red; Polyamide: PA; Polyethylene: PE; Polypropylene: PP and Polyester:

PEST). Reference spectra from the OPUS software are blue (Bruker, Ettlingen, Germany).

Results

92

Figure 12. Overview of identified polymer compositions in microplastics (n = 67) found in intestinal

samples after analysed by µFTIR spectroscopy: PEST: 30%, polyolefine: 20% (incl. PE and PP), PA:

12%, paint chip: 1%, others: 4% (incl. acrylic with nitrile component, and cellulose acetate). Polymer

composition of microplastics found in procedural blanks was not considered for this overview, albeit

the coincidence of polymers in procedural blanks and samples were considered, and thus not taken into

account.

Moreover, the polymer composition of two fragments (one fragment found in an intestine and

one from a procedural blank) could not be identified. However, both showed strong similarities

and were excluded from the analysis. Only four potential microplastic particles (nfibres = 2,

nfragments = 2) were found on all procedural blank filters, and were additionally analysed by

µFTIR. One fibre from the blanks was lost and one fragment could not be clearly

spectroscopically identified. However, the other fibre was identified as PEST and the second

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93

fragment was determined as varnish with kaolin, styrene and calcium carbonate. Furthermore,

two fragments from the intestinal samples had spectra which were highly similar to the varnish

which was found in a procedural blank. Hence, those particles were excluded from the analysis.

In five cases of potential microplastic particles, biogenic matter was identified and a sixth

particle was clearly different from plastic. In addition, one particle could not be identified due

to its small size, since it was broken during the manual collection and placement on the

aluminium oxide filter.

Differences between Seas

Comparing the samples based on their origin (NS or BS), there was a significantly higher

amount of microplastics in individuals from the BS if compared to the NS (nBS = 278;

M±SD=18.27±14.54; nNS = 123; M±SD = 8.2±7.89; p-value = 0.03). Furthermore, the highest

number of 48 microplastic particles was found in an adult female from the BS. When comparing

the share of fibres in both seas, significant differences could be revealed (BS: 51.44%; NS:

47.97%; p-value = 0.02). The share of fragments, however, was similiar across locations (BS:

48.56%; NS: 52.03%; p-value = 0.1).

Differences per Year

The annual mean values for each sea revealed a higher number of microplastic particles in

harbour porpoises from the BS. Furthermore, the range of microplastics found in individuals

from the NS was mostly between zero and up to 10 particles per individual in 2015, 2016, and

2018. Only in 2014 and 2017, more than 20 particles were found in the intestinal samples from

the NS (2014: 29 and 2017: 21). However, in the BS samples more than 30 particles were found

in 2015, 2017, and 2018. The years 2015 and 2018 were the ones with the highest number of

findings per individual (44 particles in 2015 and 48 particles in 2018). In two cases from the

NS, microplastics were not present (2014 and 2016). In comparison, in the BS microplastic was

found in all samples. All this information is presented in Figure 13. However, no significant

differences could be determined between the two sample sites (NS and BS; p-value = 0.21),

mainly because of the low power of the statistical analysis, which resulted from the low sample

Results

94

size within each year and sea. Following the power analysis, a sample size of at least 12

individuals per year for each sea would be necessary for a reliable trend interpretation (power

80%, p-value = 0.05).

Figure 13. Quantity of suspected microplastics from each year, divided by sea (BS = Baltic Sea; NS =

North Sea; n = 401).

Differences in Age and Sex

This study investigates intestinal samples of 13 female and 17 male harbour porpoises (Figure

14). The microplastic burden in females is slightly higher (M ± SD = 13.38 ± 15.41), when

compared to the amount of microplastics in males (M ± SD = 13.35 ± 10.56). Certainly, no

significant difference in microplastic load could be revealed between sexes (p- value = 0.99).

Moreover, no significant differences between adult harbour porpoises (n = 21; M ± SD = 13.82

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95

± 13.25) and juvenile ones (n = 9; M ± SD = 12.18 ± 12.31) were confirmed (p-value = 0.82),

although the microplastic amount in adult ones seemed higher. The two unburdened samples

from the NS originated from a juvenile male and an adult female. In both age classes the highest

amount of microplastics was found in females (adult: 48 particles; juvenile: 44 particles; see

Figure 14).

Figure 14. Quantity of suspected microplastic particles (n = 401; secondary contamination was

considered and subtracted) separated by sex and age of the investigated harbour porpoises (n = 30) from

German waters (NS and BS).

Results

96

Health Status

The evaluation of the whole GIT revealed an absence of parasite specimens in all investigated

intestines. A mild enteritis was found for seven individuals. In detail, in one of those harbour

porpoises a mild diffuse eosinophilic enteritis was determined. Furthermore, in two cases

(juveniles) a mild diffuse eosinophilic enteritis and a focal mural one were determined. A most

likely parasitic etiology was observed in those two harbour porpoises. A fourth individual was

affected by a diffuse mild lymphocytic-plasmacellular and eosinophilic infiltration of the

lamina propria in combination with a moderate hyperplasia of the Peyer’s plaques. Three further

porpoises showed evidence of gastritis (mild and high grade) and enteritis. Whereof two

individuals suffered from a mild non-suppurative enteritis, and the third one was also affected

by a diffuse moderate lymphocytic- plasmacellular and eosinophilic infiltration ofthe lamina

propria. In total, parasite infestations of e.g. Pholeter gastrophilus and Anisakis simplex in the

multi-chambered stomach was confirmed in 12 harbour porpoises.

Harbour porpoises investigated in this study, which were either accidentally bycaught (Siebert

et al., 2020) or affected by a pharyngeal entrapment (Gross et al., 2020), showed a microplastic

burden of 19.8 particles (SD = 12.77; n = 11) per individual. Compared to the remaining ones

(n = 19), where no accidental death could be diagnosed (incl. the three pregnant females), a

lower mean value of10 (SD = 11.59) microplastics per porpoise was identified. Furthermore, if

the individual was in a good (n = 9) or moderate (n = 14) nutritional status, the mean number

of particles was significantly higher (Meangood = 14.11; Meanmoderate = 16.07) in contrast to a

bad nutritional status (n = 7; Meanbad = 7) (Figure 15). This is also confirmed by the statistical

analysis (p-value = 0.04).

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97

Figure 15. Quantity of suspected microplastics (n = 401) in combination with the nutritional status and

further information about the carcasses (bycaught: the harbour porpoise was (suspected to be) bycaught

by a fishing boat or in a gillnet; flatfish: the harbour porpoise was affected by a pharyngeal entrapment;

na: no extraordinary finding could be revealed; pregnant: pregnancy in female was noticed). The

nutritional status is coded as follows: 1: bad; 2: moderate and 3: good.

Discussion

98

Discussion

This study is the first to evaluate the microplastic burden in marine mammals inhabiting

German waters focussing on particles smaller than 1 mm, in marine mammals inhabiting

German waters. Furthermore, intestinal samples of harbour porpoises originating from the BS

were investigated in microplastic research for the very first time. In total, 93% of all

investigated samples from the NS and the BS show a burden of microplastic particles. Minor

differences in the range of detected fibres, and no differences in the quantity of found fragments

were revealed for both seas. Based on the loss of only 3% of hard parts during sample

processing, and the considered secondary pollution, revealed by the preceding publication, the

results are reliable and not overrated (Philipp et al., 2020).

Evaluation of the Method and Results

The Nile Red staining is a well-established method in microplastic pre-identification to

preselect particles for further investigation (Erni-Cassola et al., 2017). Since some polymer

types are melted or dissolved when stained with Nile Red diluted in chloroform (Tamminga et

al., 2017), a loss of, e.g. polystyrene or cellulose acetate particles is highly likely. Hence, melted

particles were excluded from pre-selection and further polymer identification based on their

deficit in quality and the fragile consistency. Furthermore, based on the manual transferring of

single particles onto the Anodisc filter, a loss of promising polymer particles has to be taken

into account. For future analyses it is advisable to use the Anodisc filter straight away for

filtering the samples, instead of transferring it manually after the filtration process.

Based on the fact that many potential microplastics (91%, 73 out of 80, incl. 77 analysed

particles found in intestines and 3 analysed particles found on procedural blanks) could be

identified as microplastics (incl. nintestinal= 77 and nproceduralsamples= 3) by µFTIR analysis, it is

highly likely that most particles counted in our study were microplastic. Taken this evaluation

and the validation of Philipp et al. (2020) into account (90%), a reliable number of 361

microplastics out of the 401 suspected particles is assumed. Thus, the results show the

applicability of the protocol introduced by Philipp et al. (2020) for intestinal samples of

cetaceans and determine the actual burden in a reliable way.

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Comparison with other Studies

The determined percentage of 93% (28 out of 30 examined intestines are burdened) coincides

with the results of Lusher et al. (2018) investigating carcasses of cetaceans from Irish waters

(98%), and Nelms et al. (2019) analysing marine mammals found along the coastline of Great

Britain (100%). If focussing on harbour porpoises, the study by Lusher et al. (2018) determined

a microplastic presence in only 6.25% of all investigated cetacean carcasses. One explanation

for these differences in microplastic occurrences could be the chosen time period of the review

by Lusher et al. (2018). It was conducted between 1990 and 2015. A second explanation could

be the used mesh size of at least 300 µm, resulting in the loss of smaller particles, which are

included in the study presented herein. Furthermore, no detailed information on the stranding

site is given, which would be useful for comparison purpose, since differences in microplastic

loads around Ireland (Irish Sea, Celtic Sea and the western coastline facing the open North

Atlantic) were determined when the microplastic occurrence was compared at different prawn

fishing grounds in 2016 (Hara et al., 2020). Furthermore, the study by Lusher et al. (2018),

confirmed the microplastic burden in 21 individuals covering six different cetacean species

summing up to 528 investigated GITs. In addition, microplastic (≥ 300 µm) was only found in

Odontoceti species. The study of van Franeker (2018) conducted on harbour porpoises stranded

at the Dutch coast, revealed the presence of marine litter items [incl. macroplastic and

microplastics (≥1 mm)] in 7% of all investigated stomachs. Van Franeker and colleagues are

aware of the fact that due to the mesh size range of used sieves, particles smaller than 1 mm

were lost and not considered during their study (van Franeker et al., 2018). As the here

presented study confirmed that the main part of found microplastics are smaller than 1 mm

(85%), the results of van Franeker et al. (2018) are not comparable with our study. In addition,

the size limits of 100 µm and 5 mm, which are based on the used mesh sizes of the washing

sachets (Philipp et al., 2020) and the definition of microplastics (Arthur et al., 2009), overlap

with the size of zooplankton species, which a variety of invertebrates feed on (Devriese et al.,

2015; Fischer, 2019). Thus, investigations in predatory fish or marine mammal species should

also focus on these small-sized microplastic particles.

Reference studies of microplastics in marine mammals, especially from the BS, are scarce.

Thus, studies investigating fish, sediment and water samples are considered for further

discussion. The microplastic burden in different fish species is higher in the BS (11–22%) (Lenz

Discussion

100

et al., 2016; Beer et al., 2018) when compared to the southern NS (5.4%) (Foekema et al., 2013).

In addition, studies investigating fish species inhabiting waters between Norway and Denmark

determined a low risk of microplastic occurrence in fish (1.2%) (Foekema et al., 2013). In

contrast, the microplastic concentrations in surface waters and sediment samples show higher

concentration in the southern NS (Karlsson et al., 2017; Lorenz et al., 2019), compared to

findings of the BS (Graca et al., 2017; Tamminga et al., 2018). Whereas, a model on the global

fibre distribution in surface waters estimated a higher accumulation in the BS

(∼1,760±4,500m−3), compared to the North Atlantic region (∼1,800 ± 1,720 m−3) (Lima et

al., 2021). Nevertheless, an ubiquitous distribution of synthetic particles along the German BS

coast is assumed (Stolte et al., 2015). Furthermore, the fact that marine mammals of the BS

might be more exposed to marine litter than in the NS was already confirmed (Unger et al.,

2017). Thus, a higher risk of microplastic burden in the BS could be hypothesised. The results

obtained in this study underline the findings of marine litter and support the following

hypothesis: Investigated harbour porpoises of the BS show a higher number of microplastic

particles (incl. fibres and fragments), in contrast to individuals from the NS in each year. In

particular, two females from the BS show a high amount of microplastics (44 and 48 particles).

For avoiding an overassessment in future research, higher sample sizes per sea are highly

recommended.

It will be worth to strive for a reference study investigating the area of the Baltic Proper, since

this area is assumed to accumulate pollutants from the whole BS (Stolte et al., 2015), and the

here occurring harbour porpoise subspecies is critically endangered (Carlén et al., 2018).

Nevertheless, the time span of the available sample collection and the quantity of samples is

still too low for identifying a trend in both seas. After statistical assessment with paired t-test

and Cohen’s d, the samples size has to be increased if reliable comparisons in microplastic

burden of individuals of both seas should be evaluated. Thus, a continuation of the herein

presented approach is advisable and is intended.

Polymer Findings

In this study, the most frequently found polymer was PEST. Based on the fact that six fragments

and 24 fibres were found in the intestinal samples and only one PEST fibre was identified in

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one of the procedural blanks, PEST microplastics were still taken into account and were not

excluded from this study. Additionally, the procedural blanks show a low amount of fibres

(Øfibres = 1 and Øfragments = 7 per procedural blank) and were already subtracted from the results

presented here. To control for microplastic contamination, only cotton gloves and lab coats

were worn while processing the samples (Philipp et al., 2020). Other studies investigating GIT

samples of marine mammals from the North Atlantic found PEST particles, even though high

protective measurements were used (Lusher et al., 2015a; Nelms et al., 2019). In addition, a

high amount of synthetic fibres like PEST fibres were determined in the Northeast Atlantic

region (Thompson, 2004; Lusher et al., 2014), and in inhabiting fish species (Lusher et al.,

2013; McGoran et al., 2017; Pereira et al., 2020). The twelve found polyamide particles were

not excluded from the analysis, since fibres of the used nylon cloth (PA) are obviously

identifiable due to their unique fibre pattern (Philipp et al., 2020), and are clearly different from

the PA particles found in the intestinal samples. Thus, those fibres were immediately excluded

while pre-selecting, counting and collecting particles for microplastic analyses. Furthermore,

not only PA fibres but also PA fragments were found. Previously, high numbers of PA

microplastics were identified in GITs of marine mammals (Nelms et al., 2019) and in fish from

the North-East Atlantic region (Lusher et al., 2013; McGoran et al., 2017).

Both, the NS and the BS are enclosed by highly populated countries (Halpern et al., 2008,

2015). The system of waste management is relatively sophisticated in European countries, but

there is still a high amount of produced waste, thus the likelihood of litter input into the marine

environment is increasing (Andreasi Bassi et al., 2017). Another source of microplastic

particles, and in particular of fibres which originate from washed clothes are waste water

treatment plants (WWTP) (Browne et al., 2011; Bretas Alvim et al., 2020). Moreover, it is well

known that WWTP are not able to hold back microplastic fibres and particles (Dubaish and

Liebezeit, 2013; Mani et al., 2016). Thus, it could be assumed that the found PA and PEST

microplastics originated from waste water, which is transported by rivers and ends up in the

marine environment (Browne et al., 2011; Dubaish and Liebezeit, 2013; Mani et al., 2016).

Besides land-based sources, which are responsible for 80% of marine litter inputs (Jambeck et

al., 2015), sea-based sources like lost fishing gear or waste generated on ships or offshore

installations need to be taken into account (Galgani et al., 2013). Furthermore, both seas are

highly impacted by anthropogenic exploitations, such as fishing activities (Catchpole et al.,

Discussion

102

2005; Halpern et al., 2008, 2015). Thus, the fishing industry is a potential source of found

microplastics such as PA and PEST, as well as PE and PP (Pruter, 1987; Deshpande et al.,

2020).

Globally, the demand for PE is high (PlasticsEurope, 2020). Thus, it is not surprising that a high

amount of PE microplastics was identified in this study. This result is comparable to findings

in surface waters of the BS (Gewert et al., 2017). Studies investigating microplastics in marine

mammals or fish from the North Atlantic found lower amounts of PE and PP (Lusher et al.,

2013; Nelms et al., 2019). Due to the relatively low proportion of microplastics investigated

with µFTIR spectroscopy, no final conclusions can be drawn on differences in polymer

occurrences in the NS and the BS.

One of the microplastics found was identified as an acrylic or alkyd chip with evidence of kaolin

and styrene. Such polymers and additives are commonly used for car (Yang et al., 2012) and

ship paints (Lee et al., 2021). Hence, the microplastic chip that was found in the intestine of

one harbour porpoise originating from the NS could have derived from abraded ship paint. It is

well known that weathered paint from ship and boat surfaces is regularly released into the ocean

(Song et al., 2014; Lacerda et al., 2019). Hence, paint fragments can be ingested by marine

mammals such as cetaceans as also suggested by Lusher et al. (2018). Unfortunately, this pre-

mentioned study did not provide information on paint polymer types. Furthermore, paint chips

have already been found in Australian sea turtles (Caron et al., 2018) or in pelagic fish (Ory et

al., 2017). Thus, an unintentional prey intake of paint chips by, e.g., fish while preying on

zooplankton is assumed (Ory et al., 2017), and the transport through the food web could be

considered.

Trophic Accumulation via the Food Web?

The study of van Franeker et al. (2018) pointed out, that the microplastic presence in harbour

porpoises most likely occurred due to an unintentional intake while feeding on burdened fish

specimens close to the sediment. Beside pelagic species like herring (Clupea harengus),

gadoids (e.g. Merlangius merlangus) and sprat (Sprattus sprattus), different benthic sandeel

species (e.g., Ammodytes marinus) and gobies (Gobiidae) form the main prey of harbour

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porpoises (Leopold, 2015). The linkage between burdened prey species and top predators was

previously confirmed for grey seals preying on gadoids (Hernandez-Milian et al., 2019).

Moreover, three of the here analysed harbour porpoises asphyxiated by pleuronectiforms (e.g.,

Solea solea) and two further individuals were bycaught in gillnets for benthic and demersal fish

species. Those harbour porpoises showed a high mean microplastic amount per individual (M

± SD = 17.7 ± 12.42). Thus, a relationship between preying on benthic fish and the presence of

microplastics in predator species is highly assumed. Furthermore, the bycaught or asphyxiated

porpoises were in a better nutritional status in comparison to the remaining ones. However, a

good nutritional status is not synonymous with a good health of a harbour porpoise (Siebert et

al., 2020). Based on the results presented herein, it can be assumed that a harbour porpoise,

which is not physically hampered in feeding or hunting, will accumulate more microplastics in

the GIT than a diseased one. The results of this study are supported by the assumptions of

Leopold (2015) and Rummel et al. (2016) who claimed that 5,000 individual gobies per day are

needed for maintaining a harbour porpoise’s good physical condition, and the mean burden of

0.03 particles in each benthic fish may result in a daily intake of almost 150 microplastic

particles by one adult harbour porpoise. Kastelein et al. (1998) detected a passage time of 143

- 196 min in captive harbour porpoises. If the assumption is that a full digestion takes ~3 h, a

harbour porpoise will approximately defaecate eight times a day. Thus, the potential

accumulation of 150 microplastics has to be divided by eight, resulting in 18.75 particles per

egestion. Certainly, this is just a rough estimate and many factors influence egestion and

ingestion rates, but this calculation coincides with the findings of microplastics in samples from

the BS (M±SD = 18.27±14.54).

Impact on the Health Status

Across all 30 individuals no intralesional parasites could be identified in the intestines.

However, seven individuals showed an enteritis, whereof two are attributed to parasites and

another one accompanied by a hyperplasia of Peyer’s plaques. These accumulations of follicles

are known to adsorb different kinds of particles (biogenic and synthetic), and to transfer it into

the lymph system when the particle is adequately small in size(Ensign et al., 2012). In total,

only two juvenile harbour porpoises originating from the BS showed changes in the Peyer’s

Conclusion

104

plaques, where a low number of suspected microplastics was found (n = 17 and n = 15), in

comparison to the other 28 specimens. However, the stomachs were affected by parasites in 12

cases, whereof 11 suffered from a gastritis. Two harbour porpoises from the BS showed changes

in the stomach tissue, including gastritis, with no evidence of parasite occurrence. Similar

pathological findings were identified for individuals from the BS (Siebert et al., 2020). Thus, a

relationship between the accumulation of microplastics and intestinal parasites could not be

confirmed in harbour porpoises, as it was previously suggested for grey seals by Hernandez-

Milian et al. (2019). In addition, a parasite infestation in the four-chambered stomach of harbour

porpoises is more likely than an affection of the intestine (Lakemeyer et al., 2020). Furthermore,

only 11 and 27 microplastic particles were found in the intestinal samples in those two cases.

Hence, a relationship between tissue damage and microplastics seems to be unlikely. However,

tissue damage and inflammations are assumed to be caused by micro- and nanoplastic

occurrence (Carr et al., 2012; Stock et al., 2019). In addition, the occurrence of an enteritis in

harbour porpoises seems to be rare, since only 9% of the Baltic individuals found in German

waters seems to be affected (Siebert et al., 2020). Thus, it is still speculative if the presence of

microplastics may cause the tissue damages found in the harbour porpoises as it has already

been observed in beluga whales (Moore et al., 2020).

No positive or negative impacts could be revealed in pregnant females: (I) the observed quantity

differs extremely between those three specimens (4, 7, and 48 microplastics were found per

individual); (II) the number of examined pregnant specimens is, thus, too low. It has to be taken

into account that on the one hand, the occurrence of solid particles (microplastics in this study)

does not necessarily have to be accompanied by tissue damage. On the other hand, the

observation of tissue damage does not absolutely indicate the presence of microplastics.

Moreover, it was shown that synthetic materials adsorb pollutants and toxins, and serve as a

vector (Yu et al., 2019) and, thus, most likely cause contaminant accumulation.

Conclusion

For analysing the microplastic burden in marine mammals most studies investigate the whole

GIT (Lusher et al., 2015a; Nelms et al., 2019). The study presented herein revealed three

benefits of focussing on only one part of the GIT: 1) avoidance of secondary contamination in

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105

smaller samples is easier, 2) the remaining carcass and GIT can be entirely evaluated for a

health monitoring, and 3) the findings in the rectum and faeces confirm the egestion of

microplastic particles. For evaluating the intake and egestion rate, further research is needed.

Nevertheless, microplastic investigations and experiments in mammals, and especially in free-

ranging marine mammals, are complicated based by field conditions. Furthermore, ethical

concerns arise as indicated by Nelms et al. (2018). Thus, samples of carcasses and faecal

samples of alive individuals are the most feasible approach to assess the microplastic burden in

marine mammals.

This is the first study investigating harbour porpoises from different subpopulations for

microplastics, and revealed differences in microplastic presence in the NS and the BS. A higher

risk of exposure to microplastics was revealed for the western Baltic population, if compared

to the North-East Atlantic population. Thus, a higher microplastic burden in the BS is assumed.

Furthermore, evidence for the continuous accumulation of microplastics via the food web was

given, but could not significantly be confirmed in adult individuals, compared to juvenile ones.

Additionally, there is no significant difference in the quantity of synthetic particles in male or

female harbour porpoises. To gain further knowledge on differences in sex or age, the quantity

of samples has to be increased in future research.

An important relationship between a good or moderate nutritional status and the occurrence of

microplastics is demonstrated in this study. Moreover, the egestion and thus, a discharge of

microplastic particles out of the organism could be confirmed. No relationship between

parasites, tissue damage and microplastic presence could be identified. Therefore, a histological

investigation of cell damage or tissue damage localisation with the help of biomarkers would

be advisable in future research. Further investigations are needed for evaluating the rate of

accumulation and burden in harbour porpoises in the different seas. Indeed, this study outlines

first evidence in retrospective microplastic burden. Nevertheless, a higher sample size, as well

as a larger temporal coverage is needed to reliably estimate trends in the microplastic burden in

harbour porpoises. Furthermore, this study supports the need for a comprehensive marine litter

monitoring in predatory species to gain knowledge on accumulation processes and health

assessment in apex species of the marine food web.

Acknowledgements

106

Author Contributions

CP, BU, and US conceptualized the study and acquired funding. CP conducted the laboratory

analysis of processing the samples and isolate microplastics, assisted by BU. CP did the

statistical analysis. JHEK provided the µFTIR and FTIR for polymer type analysis. SME

conducted the polymer identification by µFTIR and FTIR. CP and SME generated the figures.

The manuscript was prepared by CP, and contributed editing with perspectives and arguments

was done by BU, SME, JHEK, and US. All authors approved the submitted version.

Funding

The stranding network and the health monitoring are partly funded by the Schleswig-Holstein

Agency for Coastal Defence, National Park and Marine Conservation, Germany. The

pathological investigations of the marine mammals are partly funded by the Ministry of Energy,

Agriculture, Environment, Nature and Digitalization of Schleswig-Holstein, Germany. The

Federal Environment Agency (UBA) partly funded the analysis of samples within the project

“Assessment and implementation for long-term monitoring of pollution of diverse marine

compartments and biota with marine litter” (FKZ 3717252250).

Acknowledgements

We would like to thank all seal rangers in Schleswig-Holstein, Germany for reporting and

collecting carcasses. Further thanks go to our colleagues in the ITAW necropsy team for

collecting and dissecting carcasses, as well as collecting samples for our purpose continuously

throughout the year. Special thanks go to Dieter Steinhagen for improving this study with his

valuable comments. Besides, we would like to thank the reviewers for their valuable comments

and advice, which helped to improve the manuscript.

Chapter II

107


Supplementary Table 2. Information on all 30 investigated harbour porpoises from German waters

(2014 – 2018). Every individual was examined after Siebert et al. (2001, 2020) and microplastic

investigation was conducted after Philipp et al. (2020).


108

Supplementary Figure 25. Measured spectrum (red) of one paint chip (µFT-IR, Hyperion 2000,

Bruker, Ettlingen, Germany) showing styrene (blue circle) and kaolin (green circle) components. The

reference spectrum of kaolin is blue.

Overall Discussion

Overall Discussion

111

Overall Discussion

The number of studies investigating microplastic particles in the marine environment increased

during recent years (Thompson, 2015). Moreover, in- and egestion rates of microplastics and

associated health effects in invertebrates are well-known, but still a cause of concern. Most

findings are based on laboratory studies with invertebrates or rodents like mice and rats, which

revealed new insights into pathways of microplastic particles within cells and organisms (Carr

et al., 2012; Cole et al., 2013; Setälä et al., 2016; Bhagat et al., 2020; da Costa Araújo and

Malafaia, 2021). However, detailed knowledge of the presence in free-ranging top predators,

like marine mammals, is still lacking. The mentioned knowledge gaps exist due to two aspects:

i) the access to these species is difficult because of their aquatic lifestyle. For cetaceans, the

easiest opportunity is to collect samples from stranded or bycaught specimen (Lusher et al.,

2015a, 2018; Hernandez-Milian et al., 2019; Nelms et al., 2019). The advantage of focussing

on pinnipeds is the possibility to collect scat samples of live individuals on known haul-out

sites, to assess the microplastic burden of a certain population (Donohue et al., 2019; Perez-

Venegas et al., 2020). ii) Laboratory studies, pre-trials or similar investigations are not feasible

due to ethical concerns.

Focussing on the study sites the North and Baltic Seas, investigations in microplastic particles,

in particular in the size range between 300 µm and 100 µm is lacking for all three species

(harbour porpoise, harbour seal and grey seal). Thus, the present study will complement existing

investigations on microplastic occurrence in marine mammals from the North Sea. Since no

data is currently available from the Baltic Sea, so this thesis will provide the first baseline

information on microplastic burden in Baltic marine mammal species.

Besides these listed problems, only limited standardisation is found in the high number of

arising protocols in sample handling, sample processing and microplastic identification in the

literature, which reduces the comparability (Löder and Gerdts, 2015). Different sample

collection, cleaning, and isolation procedures for purification of the synthesised particles for

further polymer identification (loss of biogenic material) are suggested. In addition, to enable a

valuable examination of microplastic presence in different media, an afterwards or so-called

secondary contamination needs to be considered (Norén, 2007; Hidalgo-Ruz et al., 2012; Nuelle

et al., 2014; Vandermeersch et al., 2015; Torre et al., 2016).

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112

Thus, the samples of interest need to be treated with protective care to avoid an overestimation

of particles, which is initiated by airborne contamination or impure detergents and instruments

(Norén, 2007; Hidalgo-Ruz et al., 2012; Nuelle et al., 2014; Vandermeersch et al., 2015; Torre

et al., 2016).

To overcome these problems in sample handling, the first aim of this doctoral thesis was the

establishment of a protocol which is effective in cost and time, and is thus applicable for

different working groups, particularly focussing on gastrointestinal tract (GIT) and scat

samples. To verify the protocol and its outcomes, the six following key questions were

considered in Chapter I:

1) Is it possible to lower the risk of secondary contamination by using selected protective

measures during sample collection and preparation?

2) What are the benefits and disadvantages of using a washing machine for sample

purification in microplastic research?

3) Does the type of sample (intestine or scat) require different sample handling

procedures?

4) How is it possible to avoid an over- or underestimation in microplastic findings?

5) Does the quantity of microplastic particles differ in intestine and scat samples?

1) Is it possible to lower the risk of secondary contamination by using selected protective

measures during sample collection and preparation?

The establishment of an efficient protocol of sample handling and processing, which meets the

standards necessary to prevent secondary contamination and to purify the samples, was

developed by examining intestinal and scat samples of seals from the North and Baltic Seas.

In order to reduce the risk of contamination to the lowest possible level, the sample processing

begins at the stage of sample collection. While some studies give advice to handle samples with

metal instruments and to store them in glass jars (Lusher and Hernandez-Milian, 2018; Nelms

et al., 2019), other studies recommend the usage of plastic containers or bags (Lusher et al.,

Overall Discussion

113

2013; Donohue et al., 2019). Moreover, several researches investigated the change of physical

plastics properties under various environmental conditions such as UV radiation, low and high

temperatures or the exposure to sea water (Andrady, 1990, 2011). To assess the potential effects

of different storage types on the samples, this study is the first one to report the comparison

between the use of glass jars and plastic bags in affecting samples. The results presented here

confirm a lower contamination of samples stored in glass jars compared to samples stored in

plastic bags, for different time periods (3, 6, 12 and 96 months) at -20°C. Further, since no

particles were found in the samples matching the characteristics of the polymer of the used

plastic bags, it can be assumed that the contamination is the result of the bags and further due

to the fact, that the plastic bags were not cleaned prior to usage, as it was carried out for the

glass jars.

Accordingly, secondary contamination can play a vital role in overestimating the real burden

and several studies investigating microplastics give advice to process the samples within a

laminar flow or in a clean laboratory (Nelms et al., 2018; Bergmann et al., 2019). Due to the

fact that this is not always feasible, other studies already introduced low-budget alternatives

like the usage of procedural blanks (Nuelle et al., 2014; Hengstmann et al., 2018; Nelms et al.,

2018), or totally exclude fibres from the analysis (Kühn et al., 2020). Since secondary

contamination is often indicated by a high proportion of fibres, we could show that new

protective measures, such as wearing cotton gloves, next to cotton lab coats and processing the

samples within a closed acrylic box, reduce the proportion of fibres: The low share of fibres in

the analysed procedural blanks (one fibre was found in 12 blanks) and in investigated samples

(M±SD = 6±7.4 fibres) demonstrates the success in largely avoiding secondary contamination

(Nuelle et al., 2014; Donohue et al., 2019; Kühn et al., 2020) by the measures applied.

Moreover, the proportion of detected fibres in this study are lower compared to other studies

investigating GIT samples of marine mammals (Lusher et al., 2018; Donohue et al., 2019;

Nelms et al., 2019), which further supports the established protocol.

Furthermore, all used detergents and materials were analysed, and results were corrected

respectively in the presented method. Those were the first steps to decrease the risk of secondary

contamination. In that respect, we advise to process only parts of the GIT, since experience has

taught, that the use of smaller samples (in size, volume, and weight) further reduces the risk of

Overall Discussion

114

secondary contamination, in as much as smaller samples are simpler to handle, and thus easier

to protect.

2) What are the benefits and disadvantages of using a washing machine for sample

purification in microplastic research?

The choice of a purification method within this doctoral thesis fell on the use of a washing

machine, an already established technique in diet analysis for gaining clean hard parts like

otoliths and fish bones for prey identification (Orr et al., 2003; Leopold et al., 2015).

Investigating GIT samples for diet analysis is thus easily connectable with microplastic

research. Moreover, this benefits microplastic research since the analysis of prey species in

combination with the accumulation of microplastics in the prey and the predator species can

reveal possible relations. Moreover, the utilisation of a washing machine saved time in the

cleaning process (1.5 hours), when compared to other approaches e.g. enzymatic or chemical

digestion, which can last for days to weeks (Foekema et al., 2013; Cole et al., 2014; Lusher and

Hernandez-Milian, 2018). Sewing the washing bags around the sample directly (referred to as

“washing sachet”) will avoid a loss of sample, as it is shown in this study (3%) and is thus

highly recommended for further analyses, since no adequate information on the sealing was

given in former studies dealing with stomach or intestine contents (Orr et al., 2003; Bravo

Rebolledo et al., 2013; Leopold et al., 2015).

Approaching the real burden of microplastics includes the consideration of different size

classes. Since tools for the isolation and identification of nanoplastics (≤20 µm) from marine

mammal tissue is lacking (Wagner et al., 2014; Ferreira et al., 2019; Prüst et al., 2020) and are

not within the scope of this thesis. Even the focus on particles in a size range of 100 µm to 5

mm in this thesis is uncommon in microplastic research, as the majority of microplastic

investigations in marine mammals focussed on particle sizes over 300 µm (Besseling et al.,

2015; Lusher et al., 2018; van Franeker et al., 2018; Donohue et al., 2019; Hudak and Sette,

2019; Perez-Venegas et al., 2020), it complements the existing knowledge in microplastic

presence.

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115

The fact of size limitations to 300 µm is based on the beginning in microplastic analysis, since

first findings of synthetic particles were found in manta trawls (333 µm) (Arthur et al., 2009)

and sediment samples (Thompson, 2004), both focussing on plankton species and in virtue of

the limitations in the applied techniques for isolation and identification (Foekema et al., 2013;

van Franeker et al., 2018; Donohue et al., 2019; Hudak and Sette, 2019). Yet, this study

demonstrated, that when using the presented methods, a detection of particles smaller than 300

µm is well possible without the use of highly advanced techniques.

3) Does the type of sample (intestine or scat) require different sample handling

procedures?

In order to highlight the applicability for different sample types, intestinal tissue (including

faeces) and scat samples were examined. The results show, the used method was suitable for

both sample types. Nevertheless, both types showed different benefits for further research. The

examination of intestinal samples for microplastic burden analysis revealed the following three

advantages: i) the intestinal tissue, which is potentially carrying microplastics within the lumen

is included, ii) the possibility of collecting sub-samples of the intestinal tissue prior to

processing, enabled further investigations, e.g. histological examinations, and iii) samples were

collected during necropsies, which allows to note valuable information on sex, age and health

status of each individual, and thus facilitates further studies in microplastic research related to

health issues. Despite these advantages of intestinal samples, the analysis of scat samples has

also benefit, as the microplastic burden and its suspected effects in live animals can be assessed

and estimated. Since seals are assumed to be philopatric (Pomeroy et al., 2000; Dietz et al.,

2012), information on the used habitat can be incorporated and sample collection repeated over

time. In addition, correlations between the volume of scats and the microplastic burden is

assessable, since the actual load could be determined, which gives new insights in microplastic

egestion. Furthermore, it has to be taken into account that necropsy samples represent only a

small part of the population,, since an unknown share of individuals is dying offshore (Williams

et al., 2011; Peltier et al., 2012), thus bearing the risk of an underestimation, which could be

likely somehow compensated for by examining scat samples.

Overall Discussion

116

4) How is it possible to avoid an over- or underestimation in microplastic findings?

By using the washing machine and self-sewed washing sachets out of fabrics in different mesh

sizes (100 µm and 300 µm), the inclusion of small synthesised particles (≥100 µm) was feasible.

Thus, this study was able to fill the knowledge gaps in occurrence of those smaller particles in

marine mammals. Here it was shown, that the exclusion of these smaller particles results in a

considerable difference: only 37% of suspected microplastics would be considered in 9 out of

10 examined seal intestinal samples (instead 10/10 if no secondary contamination would be

considered; Chapter I), and only 44% of potential microplastics in 24 out of 30 examined

harbour porpoise samples (instead 28/30 if no secondary contamination would be considered;

Chapter II). Particles, which are suspected to originate from secondary contamination were

not subtracted in this part, since in our opinion, the size of a particle plays a subordinate role

compared to the polymer composition or the appearance. Moreover, the size of each particle

(originating from contamination or not) can vary, thus an exclusion of microplastics by size

was not considered in the whole study. Accordingly, applying the suggested protocol has

therefore the potential to considerably reduce the risk of underestimating the true microplastic

burden.

After the inclusion of different size classes, the identification of synthesised particles out of the

biogenic ones plays a major role in avoiding an overestimation. Correspondingly, an approach

to pre-select suspected microplastics out of the total number of particles was additionally

established within this thesis. To pre-select potential microplastics for further polymer

determination, the samples were stained with Nile Red and visually examined under a

fluorescence microscope, which is an already established tool in microplastic research (Erni-

Cassola et al., 2017; Shim et al., 2017; Tamminga et al., 2017). Moreover, it is recognised that

only 1.4% out of particles which were visually inspected by the naked eye or under a traditional

microscope, could be confirmed as synthetic ones after polymer identification spectroscopy

(Löder and Gerdts, 2015).

In order to differentiate whether stained particles are of biogenic or synthetic origin they were

categorised following three different characteristics: i) associations with biogenic material, ii)

fluorescence intensity/colour, and iii) the appearance of the particle. Based on those categories,

1,185 particles (found in GIT samples of different marine mammal species) were examined and

Overall Discussion

117

a decision tree was established. Accordingly, the utilisation of this protocol, catalogue, and the

decision tree results in a reliable determination of microplastics, which was confirmed by

µRaman spectroscopy (match rate of 90%). This decision tree is applicable for untrained

researchers, next to an emerged catalogue of different characteristics, for identifying suspected

microplastics out of biogenic ones. Furthermore, narrowing down the number of potential

microplastic particles prior to polymer identification by e.g. µRaman spectroscopy, decreases

the risk of misidentification, thus saves time and budget. For instance, polyethylene,

polyethylene terephthalate, polypropylene and polyamide are some of the identified polymers

found in the intestinal and scat samples of seals.

5) Does the quantity of microplastic particles differ in intestine and scat samples?

To put the results of this study in relation to other works dealing with intestinal samples of

marine mammals it is advisable to consider investigations from the same study area (North

Atlantic region). Since investigations from the Baltic Sea are lacking, comparisons between our

results (90%, 0 to 83 microplastics per individual) and microplastic findings in different marine

mammals from the North Sea were considered. Accordingly, findings of microplastics in

marine mammals stranded around the British coastline (100%, 1 to 12 microplastics larger than

35 µm per individual) (Nelms et al., 2019) and around Ireland (100%, 1 to 88 microplastics

larger than 300 µm per individual) (Lusher et al., 2018) overlap with the here presented results.

Similar to the results of a study examining scat samples of fur seals (fragments: 68%, fibres:

32%) (Donohue et al., 2019) a higher proportion of fragments (70%) was found, when

compared to the quantity of fibres (30%) within this thesis. Nevertheless, the percentage of

samples containing microplastics in general in this study is higher (100%, when only the scat

samples are considered), compared to the previously mentioned study on fur seals (41%)

(Donohue et al., 2019) or as in studies examined scat samples of seals found in South America

(68%) (Perez-Venegas et al., 2020) or from the south-east coastline of the USA (6%) (Hudak

and Sette, 2019). The main reasons for this could be the investigated sizes of microplastics. The

focussed particles in the aforementioned studies had at least a size of 250 µm or 500 µm, thus

the smaller fractions were lost whereas those microplastics are included in our study (100 µm

– 5 mm). Thus, an underestimation in the other studies is possible (Donohue et al., 2019). In

Overall Discussion

118

addition, differences between seas and species need to be considered, since also our study

confirmed variations in harbour porpoises, harbour seals and grey seals (if Chapter I and II

are considered here).

To further overcome the knowledge gaps in microplastic presence in excretions of seals, a weak

correlation between the weight of scats and the number of detected microplastics could be

determined (Spearmen p = 0.23). In addition, about 1 microplastic particle (100 µm – 5 mm)

could be identified within 2 g of scat (wet weight; Mparticles±SD = 0.5±0.2 per gram). A

comparison between the intestine samples with faeces content and the microplastic load was

not feasible due to differences in size and thickness of the intestinal tissue, thus resulting in

inappropriate quantities. Moreover, intestine samples and scat samples are not comparable with

each other since scat samples are characterised by a higher volume and weight, and scat samples

from a sandbank represent an accumulation of faeces over a longer time period before egestion.

Further a secondary contamination of the scat samples originating from the sandbank during

defecation cannot be fully ruled out and need to be considered. To enable a comparison between

those sample types, the acquisition of dry weights of the faeces should be considered, and a

subsequent correlation with microplastic findings has to be taken into account in future

research.

Moreover, the inclusion of different GIT parts needs to be considered, since studies

investigating stomach samples of marine mammals confirmed the retaining characteristics of

this hollow organ in comparison to intestinal or scat samples (Nelms et al., 2019). This fact

leads to higher numbers of microplastic particles in stomachs, when compared to intestinal or

scat samples (Nelms et al., 2019), what could be also revealed in unpublished data of comparing

stomach contents with the intestinal samples of the same animal. The findings support and

underline the hypothesis as the determined quantity of microplastics is twice as high in the

stomachs compared to the intestines (Philipp et al., unpublished data).

Thus, the outcome of this study complements existing knowledge and reveals new insights in

microplastic occurrence in seals from German waters.

Overall Discussion

119

Chapter II

To enhance the awareness of microplastic presence in marine top predator species, intestinal

samples of harbour porpoises from the North and Baltic Seas were examined and compared in

the course of the second chapter. For processing the samples and isolating potential

microplastics, the established protocol, presented in Chapter I, was used. The aim here was to

gain further knowledge on the microplastic burden in relation to health and life history of the

harbour porpoises from German waters. For accomplish the main objectives of this chapter, the

following key questions will lead through the following discussion parts.

1) Are harbour porpoises of the North and Baltic Seas are exposed to microplastic

particles?

2) Is the microplastic burden linked to life history parameters or the health status?

3) Which polymers could be identified by µFTIR spectroscopy?

1) Are harbour porpoises of the North and Baltic Seas exposed to microplastic particles?

The results of this analysis confirmed the presence of microplastic particles in intestinal samples

from harbour porpoises originating from German waters (28/30). Intestinal samples from 30

individuals from the North Sea (NS) and the Baltic Sea (BS) show differences between the

origin sea and the collection year (2014 – 2018). Only two samples were free of microplastics

and both originated from individuals found in the NS. Furthermore, in each year the mean

quantity of microplastics found were significantly higher in the samples from the BS (M±SD =

18.27±14.54), compared to ones from the NS (M±SD = 8.2±7.89). Based on the low sample

size from each year originating from the NS or BS, no trend analysis could be conducted, since

the power analysis revealed a power of 20%. Thus, analyses to estimate trends would result in

over- or underestimations. Nevertheless, the aim of this study was to gain first evidence of

microplastic burden in relation to the health status of harbour porpoises from German waters.

This aim was accomplished and results are further discussed in the following paragraphs, albeit

trend estimations with an adapted and enlarged sample size should be considered in future

research.

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120

In total, 401 microplastics (incl. 202 fibres and 199 fragments) were determined, besides four

meso-fibres which were longer than 5 mm. Three of those fibres were found in an adult male,

and the fourth fibre was identified in a juvenile male harbour porpoise. Both individuals were

found in the Baltic Sea. Certainly, no significant differences between the share of fibres or

fragments could be determined, when both seas were taken into account. As already shown in

Chapter I, the share of fibres is in this study again lower (50%), if compared to other studies

dealing with marine mammals close to the study area (83 - 85%) (Lusher et al., 2018;

Hernandez-Milian et al., 2019; Nelms et al., 2019).

Comparable studies investigating marine mammals from the BS are missing. Thus, studies

focussing on sea water (Tamminga et al., 2018), sediment (Graca et al., 2017; Näkki et al.,

2019) and fish samples (Lenz et al., 2016; Beer et al., 2018) collected from the BS were

considered for comparison purpose. The microplastic burden in different fish species seems to

be higher in the BS compared to the NS (Lenz et al., 2016; Beer et al., 2018). However,

sediment and water samples of the BS show a lower amount of microplastic particles (Graca et

al., 2017; Tamminga et al., 2018), when compared to samples originating from the NS

(Karlsson et al., 2017; Lorenz et al., 2019). The missing knowledge on microplastic burden in

marine mammals inhabiting the BS underlines the importance of this study. Moreover, this

study provides one of the missing pieces in the puzzle of microplastic distribution, occurrence

and accumulation within the ecosystem of the Baltic Sea.

As already mentioned in Chapter I, investigations in microplastic particles smaller than 300

µm are lacking for the southern NS and for the entire BS, thus comparisons are only possible

to a limited extent. There is only one study focussing on stomach contents of harbour porpoises

and investigated particles larger than 1 mm (van Franeker et al., 2018). Nevertheless, this

research revealed a microplastic burden in 9% of the examined stomachs (van Franeker et al.,

2018). This low percentage is not surprising, since the loss of smaller particles is tremendous if

compared with other studies. Moreover, an underestimation in microplastic occurrence is

assumed by us, and the authors itself (van Franeker et al., 2018). Thus, this study seems to be

negligible in the context of microplastic presence and in further discussion, even though it is

the only one focussing on harbour porpoises inhabiting the southern North Sea.

Overall Discussion

121

Two other studies focussing on marine mammals stranded around Great Britain (≥35 µm) and

Ireland (≥300 µm), revealed both microplastic occurrence in each examined individual (Lusher

et al., 2018; Nelms et al., 2019). Those values are within the range of microplastic burden of all

harbour porpoises examined here. Focussing on smaller particles, the here presented study

revealed that 85% of the detected microplastics range between 100 µm and 1 mm. This

percentage is only comparable with the findings around Great Britain, since the majority of that

study lays within the size classes ranging between 35 µm and 1 mm, giving no accurate

percentage (Nelms et al., 2019). In cetacean carcasses found around Ireland, only 17% of all

detected microplastics are in the size range of 300 µm to 1 mm (Lusher et al., 2018). To

overcome those deficiencies in underestimations and comparability between studies, the

inclusion of particles in size of 100 µm to 1 mm needs to be considered in future studies. In

order to be able to compare microplastic findings, future studies should also pay attention on

the same particle size and agree on uniform size classes.

Moreover, those small-sized particles coincide with the size of various zoo- and phytoplankton

species or their prey species, hence an unintentional uptake by organisms of lower trophic levels

and the potential accumulation in top predator species thereafter can be assumed (Thompson,

2004; Farrell and Nelson, 2013; Cole et al., 2014; Setälä et al., 2014; Dawson et al., 2018). For

instance, one study investigating fish species of the South Pacific Ocean revealed the accidental

uptake of paint chips, instead of feeding on blue-coloured copepods (Ory et al., 2017). This

discovery coincides with the findings of paint chips in intestinal samples of harbour porpoises

originating from the North Sea from this study and additionally emphasises the importance of

focussing on small-sized particles. The accumulation of synthetic particles in different size

classes within the last 50 years in the Northeast Atlantic is evident (Thompson, 2004), yet the

degradation of those particles into smaller ones by different physical forces needs to be

considered (Andrady, 1990, 2011), when investigating microplastics. Thus, further studies need

to focus on small-sized particles (e.g. 100 µm to 1 mm) to give a comprehensive overview in

microplastic burden.

Overall Discussion

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2) Is the microplastic burden linked to life history parameters or the health status?

The study described in this chapter is one of the very first ones, investigating microplastic

burden in relation to life history parameters like age and sex of examined harbour porpoises.

Information on the age and sex of the individuals compared with the amount of microplastics,

could not reveal significant differences. The first hypothesis of accumulating microplastics in

the GIT over the time, resulting in a higher burden in older individuals could not be confirmed.

Since, no correlation between age and number of microplastics was found, the egestion of

particles needs to be considered and is likely. Moreover, the results of the scat samples analysed

in Chapter I support this assumption. Thus, an accumulation of microplastics in the intestinal

lumen, as it is assumed for nanoparticles (Domenech et al., 2020) in particular in the area of

Peyer’s plaques, could likely be excluded for the sizes considered in our study (Jani et al.,

1992a, 1992b; Florence et al., 2000; Stock et al., 2019). In particular, it can be expected that

some smaller particles get stuck in straitened parts of the GIT or that they are held back by

mucosa or microvilli in the lumen of the intestine (Jani et al., 1992b; Florence and Hussain,

2001; Stock et al., 2019). By including the intestinal tissue in the microplastic analysis, all

potential particles attached to the tissue were considered for this investigation. Moreover, a

transport of nanoparticles by the lymph and blood system, and thus the passing on of pollutants

and plastic particles is already confirmed during gestation and lactation for mammals and

humans (Wolkers et al., 2004; Desforges et al., 2012; Aerts et al., 2019; Ragusa et al., 2021;

Witczak et al., 2021). Even though nanoparticles were not considered here, an assessment of

the microplastic presence in females (incl. three pregnant ones) and males was evaluated.

However, no significant differences could be revealed by the statistical analysis when

comparing both sexes. It needs to be highlighted though, that the sample size is too low for a

comprehensive understanding and the confirming of incontrovertible facts. Thus, an increased

sample size and the focus of small-sized particles like nanoparticles is advisable for further

investigations.

Besides the information on the life history, data on the health status and the potential cause of

death could be evaluated and considered in microplastic research. Thus, a linkage between the

health status and microplastic occurrence is expected to enhance the knowledge in microplastic

burden. In particular, the results unveiled a relationship between the nutritional status of a

harbour porpoise and its microplastic burden. Individuals having a good or moderate nutritional

Overall Discussion

123

status showed a higher number of microplastics, when compared with porpoises in a poor

nutritional status. However, a good or moderate nutritional status is not necessarily

accompanied by good health of an individual (Siebert et al., 2020). The fact that harbour

porpoises with a good constitutional status are highly active in feeding and thus the constant

uptake of microplastics need to be considered, since those cetaceans are well-known to feed

and hunt on benthic fish species (Gilles, 2008; Leopold, 2015; Andreasen et al., 2017; van

Franeker et al., 2018; Gross et al., 2020). Hence, due to their feeding strategy the sediment is

stirred up and microplastics are released into the marine environment and can be accidentally

consumed. In general, the burden of microplastic in benthic and pelagic fish species like herring

(Clupea harengus), cod (Gadus morhua) and a variety of pleuronectiforms (e.g. common sole

(Solea solea) and European flounder (Platichthys flesus)) originating from the NS and BS are

already determined and the load does not significantly differ from each other (Foekema et al.,

2013; Lenz et al., 2016; Rummel et al., 2016; Beer et al., 2018; Fischer, 2019).

Several studies already investigated sediments and their load of marine litter, and ascertain that

sediments serve as a sink in particular for microplastics (Woodall et al., 2014; Kanhai et al.,

2019; Näkki et al., 2019). Beside the study of Hernandez-Milian et al. (2019), investigating

GIT samples of grey seals, benthic prey species and its microplastic load, our study gives also

a significant evidence for underlining the hypothesis of an uptake of microplastics in top

predators (such as marine mammals) by feeding on benthic prey species (such as fish).

In order to gain a better understanding of the relationship between nutritional status and

microplastic burden it needs to be highlighted, that in individuals where a pre-mortem feeding

event is assumed (accidental bycaught individuals in gillnets for benthic fish species or

individuals died by a pharyngeal entrapment by benthic flatfish, n = 11) and which were further

considered to be in a good nutritional status, a higher microplastic burden was found (M±SD =

19.18±12.77) in comparison to the remaining ones (n = 19, M±SD = 10±11.59).

Correspondingly, those results reveal a relationship between individuals, which could be likely

considered as healthy, and are thus expected to hunt and feed on prey in a natural and effective

way, and the number of microplastic particles within their GIT in contrast to individuals for

which recent feeding events are considered unlikely. These results show that during a constant

uptake of prey species, the amount of plastic particles is kept high while for individuals not

having fed for a certain period of time due to e.g. sickness, the burden is reduced due to the

Overall Discussion

124

likely constant excretions of particles. This raises the questions whether actual healthy

individuals are exposed to an increased risk of ingesting microplastics and whether pelagic vs.

benthic feeding strategies influence this risk. Moreover, the selection of prey species and their

exposure to microplastics needs also to be further addressed in future studies.

Furthermore, it was already shown for krill species, that they digest microplastics and thus serve

as a source of nanoplastics for other organisms or enhance the uptake of those particles when

ingested by predator species (Dawson et al., 2018). Concerning this matter, the isolation and

detection of nanoplastics by e.g. histological analysis (Ahrendt et al., 2020; Batel et al., 2020)

or the utilisation of biomarkers (Revel et al., 2019; Sarasamma et al., 2020), need to be

considered for future research, since current research is mainly focussing on cell damage

triggered by small-sized particles in organisms of low trophic levels, e.g. specimens of mussels

(Gaspar et al., 2018; Li et al., 2020b; Sendra et al., 2020). In contrast, knowledge in mammals

is based on in vitro experiments, and the available knowledge is thus still scarce (Prata et al.,

2020; Banerjee and Shelver, 2021). Moreover, evidence for lesions or inflammations in the GIT

as the result of microplastic ingestion were found in mice (particle size: 10 – 150 µm) and

zebrafish (particle size: ~70 µm) (Lei et al., 2018; Li et al., 2020a). In addition, the impact of

nanoparticles (44 nm and 100 nm) is assumed to be more serious than microparticles as it is

confirmed by in vitro experiments in human gastric cells (Forte et al., 2016). Furthermore, an

in vivo study using mice detected no evidence of cytotoxicity or lesions in intestinal tissue

(particle size: 1 µm, 4 µm and 10 µm) (Stock et al., 2019). Thus, there has been less evidence

for lesions or inflammations in the GIT triggered by microplastic particles, when compared to

nanoparticles. Moreover, a linkage between information on the health status and the

microplastic occurrence within the organism needs to be correlated with the particle size, and

the body size of the investigated organism.

Around this issue, the comparison between the investigated harbour porpoises diagnosed with

gastritis or/and enteritis and individuals which showed no similar lesions, revealed no

significant evidence of a relation between microplastic presence (≥100 µm) and respective

effects on the health e.g. lesions like ulcers and inflammations. In addition, no correlation in

parasite infestations and microplastic occurrence was found, as it is assumed in grey seals

(Hernandez-Milian et al., 2019).

Overall Discussion

125

3) Which polymers could be identified by µFTIR spectroscopy?

In virtue of the first chapter, where the polymer identification was conducted by µRaman

spectroscopy, a comparison between this technique and the usage of micro-Fourier-transform

infrared (µFTIR) spectroscopy was aimed in Chapter II. Specifically, based on the time saving

aspect, the µFTIR technique seems more practical, although the costs for the specific filter

(aluminium oxide) are high. Nevertheless, they are needed to avoid a scattering by the filter and

thus enable a reliable and fast polymer identification of the particles. In contrast, the used

identification procedure by µRaman was conducted with particles placed on glass object slides,

resulting in a cheap but time intense analysis. Despite the differences in costs and time, both

approaches revealed a similar match rate in determining synthetic particles out of the pre-

selection (µRaman: 90% and µFTIR: 93%). Thus, a combination of both seems to be the most

reliable method to detect and identify different polymer compounds and associated bonds

within one sample due to their different abilities (Turner and Holmes, 2011; Löder and Gerdts,

2015; Shim et al., 2017), as described in detail in the introduction paragraph.

The determination of the specific polymer compounds in microplastics found in intestines of

harbour porpoises by µFTIR spectroscopy enables an allocation to different potential sources.

Besides the paint chip, which presumably originates from ship or car varnish (Yang et al., 2012;

Lee et al., 2021), polyamide, polyester, polyethylene and polypropylene were found. It is highly

likely that those four last-mentioned polymers are originating from abandoned, lost or discarded

fishing gear (ALDFG) (Pruter, 1987; Macfadyen et al., 2009; UNEP, 2016). This further

coincides with some of the bycaught harbour porpoises, found in gillnets (Siebert et al., 2020).

Gillnets, one of the most common types of fishing gear in the Northeast Atlantic, are often made

of polyamide fibres (Brown and Macfadyen, 2007; Macfadyen et al., 2009; Gilam et al., 2016;

Deshpande et al., 2020). Furthermore, gillnets are often reported to end as ALDFG (Deshpande

et al., 2020), thus increasing the risk of entanglement and bycatch of marine mammals. A

potential approach to prevent this threat and to decrease the loss of synthetic fibres, is the

development and usage of biodegradable fibres for gillnets (Grimaldo et al., 2020). The

replacement of biodegradable polymers is also considered but rather discouraged in

aquaculture, since it is another source for microplastics as the time needed for degradation is

still long (Lusher et al., 2017a; Schoof and DeNike, 2017).

Overall Discussion

126

The two thermoplastics polyethylene and polypropylene are the most frequently used synthetic

polymers in particular for packaging items (Gregory and Andrady, 2004; PlasticsEurope, 2020),

next to the usage in fishing gear (Pruter, 1987; Deshpande et al., 2020). Moreover, those kinds

of polymers are the most common synthetics found in beach litter and marine litter surveys

(OSPAR Commission, 2000; Widmer and Hennemann, 2010; Schulz et al., 2013). Hence, it is

not surprising that those polymers were also found in the investigated intestines of harbour

porpoises (Chapter II) and in seals (Chapter I).

Polyester was the dominating polymer in all harbour porpoise samples. One potential

explanation is the loss of fibres during washing procedures in e.g. washing machines (Browne

et al., 2011) or drying procedures in cloth dryers (Kapp and Miller, 2020). Thus, a possible

source of this synthetic kind are wastewaters and the resulting sludge in wastewater treatment

plants (WWTPs) (Dubaish and Liebezeit, 2013; Dris et al., 2015; Weithmann et al., 2018;

Corradini et al., 2019; Masiá et al., 2020). To date, no reliable technique to effectively remove

microplastics from wastewater is available for WWTPs (Prata, 2018; Sun et al., 2019).

Moreover, heavy rain and flooding events are spreading the microplastics after the yielding of

sludge and transfer it to the limnic and ultimately to the marine environment (Galgani et al.,

2010; Jambeck et al., 2015; Nizzetto et al., 2016a).

The next step will be to conduct a toxicological risk assessment to investigate the correlation

between microplastic presence and the burden of toxic pollutants within an individual. This

approach would be reasonable, since microplastics are known to sorb additives, pollutants, toxic

compounds and heavy metals on their large surface (surface area to volume ratio), next to

bacteria and viruses (Teuten et al., 2009; Viršek et al., 2017; Razanajatovo et al., 2018; Curren

and Leong, 2019). Thus, those small particles can act as a vector and provide a toxic

conglomerate after the intake by an individual. Hence, the risk of accumulation of pollutants

and other toxic substances in top predator species is assumed to be higher, if they are exposed

to microplastics (Fossi et al., 2012; Baini et al., 2017; Germanov et al., 2018). Within this

context, the relationship between benthic and pelagic feeding and the exposure to microplastics

and thus toxic compounds needs to be further investigated. Accordingly, the health impacts

seem to be exacerbated if the contamination of the marine environment by microplastics will

Overall Discussion

127

not decrease within the next years. Broadly speaking, this problem can only be addressed if the

utilisation of recycling and reusing techniques is fully developed or improved within the next

years, the use of single-use plastic items is banned and the production of primary microplastics

and resins is reduced in the future.

Therefore, the herein presented results are highly valuable for assessing the microplastic burden

in the three regularly occurring free-ranging marine mammal species in German waters, which

are known to be under frequent anthropogenic pressure (Halpern et al., 2008, 2015). The results

lay the basis for further studies on the impact on marine mammals in relation to microplastic

occurrence. Moreover, investigating marine mammal species as organisms of higher trophic

levels will help to assess the environmental status e.g. of the North and Baltic Seas.

Accordingly, marine mammals should be considered as sentinel species since they represent

exposures of different abiotic and biotic factors, and in particular the pollutant accumulation in

the whole food web within the habitat (Fossi and Panti, 2017; Nelms et al., 2019; Sonne et al.,

2020). Thus, the outcomes of this doctoral thesis are urgently needed to provide a baseline on

habitat condition and marine mammal burden concerning microplastics as requested by

Descriptor 10 in the Marine Strategy Framework Directive (MSFD) of the European Union

(European Union, 2008). Moreover, the conservation and protection of the marine environment

and its sustainable utilisation (Sustainable Development Goal 14) are a major aim of the United

Nations and should be reached by 2030 (United Nations, 2016).

Overall Conclusion

Overall Conclusion

131

Overall Conclusion

The aim of this study was to establish and implement a standardised and scientific sound

protocol focusing on time and budget constraints as well as to reduce the risk of secondary

contamination in order to provide first evidence of the microplastic burden in marine mammals

inhabiting German waters.

The four major outcomes during the establishment of the adapted protocol are i) the use of a

protective hood to reduce contamination, as it is essential and an effective and easy approach

to implement, ii) that the storage in plastic bags does not necessarily lead to a contamination by

the bag itself but rather due to plastic residues still present in the bags following production, iii)

the development of a decision tree based on the characteristics of stained microplastic particles

facilitates the pre-selection prior to polymer identification even for untrained personnel, and iv)

the protocol is applicable for analysing smaller microplastic particles (<1 mm). The results of

this study revealed the importance of those small particles (~80% out of all found ones). Since

these particles coincide with sizes of zoo- and phytoplankton, an accumulation within the food

web can be assumed and should further be considered in future research. Furthermore, an

exclusion of small-sized microplastics (100 – 299 µm) will lead to a drastic underestimation

which will skew the risk assessment (e.g. only 44% out of the identified microplastics identified

here in harbour porpoises would be considered). In addition, the analysis of different parts of

the gastrointestinal tract (stomach, intestine and scat) confirmed the transport of microplastics

through the gastrointestinal passage in marine mammals, resulting in an egestion. Thus,

microplastics are reintroduced into the environment and are once again bioavailable for

organisms.

By applying the established procedure in the analysis of samples originating from marine

mammals from German waters, this study could show that i) the microplastic burden is higher

in marine mammals originating from the Baltic Sea, ii) the nutritional status, thus the fitness of

an individual seems to be linked to the amount of ingested microplastic particles, and iii) the

burden in individuals feeding on flatfish is higher which leads to the assumption that the uptake

rate by feeding on benthic species is increased.

This thesis gives first indices of microplastic exposure and its uptake in marine mammals

inhabiting German waters. Nevertheless, to acquire more information in microplastic burden

Overall Conclusion

132

and its resulting health effects, the sample size of investigated individuals needs to be increased.

Moreover, techniques to analyse lesions in tissues and organs, next to assessments of pollutant

and nanoplastic accumulation in the organism should be considered in future research. Hence,

the gained contributions out of this thesis have wide applicability in the field of microplastic

research, both, in terms of sample handling as well as in risk assessments in top predator

species.

Outlook

Outlook

135

Outlook

This study gives first evidence on the microplastic burden in marine mammals from the North

and Baltic Seas. Furthermore, the results are applicable as a baseline for future monitoring on

the microplastic burden in both seas. Since marine mammals are top predator species in the

aquatic food web, they are sentinels and thus represent the habitat’s microplastic exposure.

Correspondingly, the results of this study are essential for serving as baseline for the definition

of “Good Environmental Status” as required in the course of the implementation of the Marine

Strategy Framework Directive.

For ensuring a reliable assessment in future research, a higher sample size per sea and species

is essential to close existing knowledge gaps in terms of microplastic occurrence and its

potential health impacts on the organisms. In addition, an increased sample size enables the

performance of trend analyses in future research and a retrospective manner. These approaches

help to outline the dimensions of microplastic presence in the selected habitats for the past and

the future.

The here used techniques allow for an analysation of particles of at least 100 µm and larger in

size, and thus cover the sizes of various zoo- and phytoplankton species that present the lowest

trophic levels of the food web. Those size classes were neglected in recent years. However, this

study underlines the importance of these sizes and emphasises the need of focusing on predator-

prey relations and feeding behaviours in future research. Correspondingly, the developed

approaches in microplastic isolation and quantification in this study are applicable as a

methodological roadmap for future studies since they are feasible for different sample types

and lab conditions.

Besides the ingestion, the egestion of microplastic particles in marine mammals is now

confirmed. Nevertheless, information on the excretion rate and the potential harm risk is still

lacking in top predator species. Thus, further research is needed concerning microplastics’

performance within the gastrointestinal tract: their sorbing charcteristics, capabilities of causing

lesions and inflammations, and cell motility especially for even smaller particles, such as

nanoplastics.

Summary

137

Summary

Carolin Philipp - Occurrence of microplastic particles in marine mammals from German

waters and improvement of samples storage

This work aimed to combine interdisciplinary approaches from microplastic research in order

to determine a reliable estimation on the potential exposure of microplastics in marine

mammals. For so far, little is known about the presence and conceivable health effects of

microplastic particles in the organism of top predators.

In order to obtain valuable results, the following two aspects were pursued: i) processing of the

samples need to be affordable and time efficient, ii) the risk of secondary contamination has to

be kept as low as possible. Thus, a protocol of sampled processing and handling was established

(Chapter I) and utilised to assess the microplastic load in gastrointestinal tract and scat samples

of marine mammals inhabiting the German North and Baltic Seas (Chapter I & II).

The developed protocol combines already established procedures in the field of diet analysis of

marine mammals, and approaches to isolate and identify microplastic particles out of different

biogenic media. The cleaning procedure based on a washing cycle and different detergents

result in purified remains within 1.5 hour, which are narrowed down to biogenic hard parts and

synthesised particles. Moreover, a low share of fibres was detected in procedural blank filters,

which underlines a low risk of secondary contamination during sample handling, thus, an

overestimation could be ruled out. To identify particles that are supposed to be of synthetic

origin, a decision tree was established, which can subsequently be applied to a subset to evaluate

the likelihood of a synthetic origin of a particle. Since, polymer identification measurements

like µRaman or µFTIR spectroscopy are cost and time intensive, a pre-selection of suspected

particles is advisable and turns out as effective tool as it is confirmed in Chapter I (chance of

correct classification by 90%). Protective measures like wearing cotton lab coats and gloves,

and processing the samples within a closed acrylic box, seems to be accountable for those

results. Moreover, two additional outcomes were gained within this study: i) there is a low risk

of contamination by using plastic bags for storing samples and ii) the analysis of particles in a

size of at least 100 µm and larger is feasible if dealing with intestinal or scat samples of marine

mammals. Until today, information on these two topics was scarce.

Summary

138

The established protocol was utilised to give first evidence in microplastic burden in harbour

porpoises, harbour seals and grey seals inhabiting the North and Baltic Seas. Microplastic

particles have been detected in the scat samples as well as in the GIT samples of all three

species. In particular, the analysis of the harbour porpoise samples showed a higher microplastic

burden in individuals from the Baltic Sea. Using µRaman and (Chapter I) and µFTIR

spectroscopy (Chapter II), various polymer types such as polyester, polyethylene and

polypropylene and also a paint chip (car or ship paint) could be identified. Accordingly, the

egestion of microplastics out of the mammalian organism is confirmed, but also implies the

reintroduction of microplastics back to environment.

This is the very first time that samples of marine mammals from the Baltic Sea have been

investigated for potential microplastic burden. In addition, the study on harbour porpoises is

characterised by a retrospective analysis (2014 – 2018) including information on age, sex and

the health status of the examined individuals. A correlation between diseases of the GIT

(gastritis and enteritis) or parasite infestation on a macroscopic level and microplastic

occurrence could not be determined. However, the increased intake of microplastic particles

seems far more likely in individuals with good or moderate nutritional and health status, as they

are most likely to be able to feed unhindered and continuously on prey (compare to senile or

diseased individuals) and thus an increased unintentional uptake of microplastics is assumable.

Nevertheless, no correlations could be found between age, sex or the respective medical

conditions.

In conclusion, the results of this thesis emphasise the need of an enhanced monitoring of marine

mammals and its exposure to microplastics. It serves as a proposal for the needed baseline in

microplastic monitoring in marine mammals in the North and Baltic Seas. This is a valuable

approach to take measures for achieving Good Environmental Status (GES) within the

European Marine Strategy Framework Directive (MSFD), if this state has not been reached.

Zusammenfassung

139

Zusammenfassung

Carolin Philipp - Mikroplastikpartikel in marinen Säugetieren aus deutschen Gewässern und

Etablierung einer geeigneten Probenaufbewahrung

Ziel dieser Dissertation war es, interdisziplinäre Ansätze aus der Mikroplastik-Forschung zu

kombinieren, um eine verlässliche Abschätzung über die potenzielle Exposition von

Mikroplastik in marinen Säugetieren zu ermitteln. Denn bisher ist nur wenig über die Präsenz

und die denkbaren gesundheitlichen Auswirkungen von Mikroplastikpartikeln im Organismus

von Topprädatoren bekannt. Um aussagekräftige Ergebnisse zu erhalten, wurden die folgenden

zwei Aspekte verfolgt: i) die Verarbeitung der Proben muss kostengünstig und zeiteffizient sein

und ii) das Risiko einer Sekundärkontamination muss so gering wie möglich gehalten werden.

Daher wurde ein Protokoll zur Probenverarbeitung und -handhabung erstellt (Kapitel I). Dies

wurde genutzt, um die Mikroplastikbelastung in Kotproben, sowie im Gastrointestinal Trakt

(GIT) mariner Säugetiere aus der deutschen Nord- und Ostsee, zu erfassen (Kapitel I & II).

Das entwickelte Protokoll verbindet bereits etablierte Verfahren im Bereich der

Nahrungsanalyse von Meeressäugern und Ansätze zur Isolierung und Identifizierung von

Mikroplastikpartikeln aus verschiedenen biogenen Medien. Das auf einem Waschgang

basierende Reinigungsverfahren führt innerhalb von 1,5 Stunden zu gereinigten biogenen

Hartteilen, sowie synthetischen Partikeln (Mikroplastikfasern und –fragmente). Darüber hinaus

wurde ein geringer Anteil an Fasern in prozessbegleitenden Blindfiltern (engl.: procedural

blanks) nachgewiesen, was das geringe Risiko einer Sekundärkontamination bei der

Probenhandhabung unterstreicht, so dass eine Überschätzung ausgeschlossen werden konnte.

Um Partikel zu identifizieren, die vermutlich synthetischen Ursprungs sind, wurde ein

Entscheidungsbaum (engl.: decision tree) erstellt, dieser ist im Nachgang für eine Teilmenge

(engl.: subset) anwendbar, um die Wahrscheinlichkeit eines synthetischen Ursprungs eines

Partikels zu bewerten. Eine Polymeranalyse mittels µRaman- oder µFTIR-Spektroskopie ist

kosten- und zeitintensiv, daher ist eine Vorselektion verdächtiger Partikel ratsam und erweist

sich als effektives Werkzeug, wie in Kapitel I bestätigt wird (Chance auf korrekte

Klassifizierung bei 90%). Darüber hinaus wurden in dieser Studie zwei weitere Resultate

gewonnen: i) Es besteht ein (geringes) Kontaminationsrisiko durch die Verwendung von

Zusammenfassung

140

Plastikbeuteln für die Lagerung von Proben und ii) die Analyse von Partikeln mit einer

Mindestgröße von 100 µm ist möglich, wenn es sich um Darm- oder Kotproben von

Meeressäugern handelt. Beide Informationen fehlten bis heute.

Das etablierte Protokoll wurde genutzt, um erste Hinweise auf die Mikroplastikbelastung von

Seehunden, Kegelrobben und Schweinswalen aus Nordsee und Ostsee zu geben. Sowohl in den

Kotproben, als auch in den GIT Proben aller drei Arten sind Mikroplastikpartikel nachgewiesen

worden. Besonders die Analyse der Schweinswalproben zeigte eine höhere Belastung in

Individuen aus der Ostsee, im Gegensatz zu untersuchten Individuen aus der Nordsee, auf.

Mittels µFT-Spektroskopie (Kapitel I) und µFT-IR (Kapitel II) konnten diverse Polymere wie

z.B. Polyester, Polyethylen und Polypropylen und auch ein Farbchip (Auto- oder Schiffslack)

identifiziert werden. Dementsprechend ist die Ausscheidung von Mikroplastik aus dem

Säugetierorganismus bestätigt, impliziert jedoch eine Wiedereinführung von Mikroplastik in

die Umwelt.

Dies ist die erste Studie, in der marine Säugertiere aus der Ostsee auf eine potentielle

Mikroplastikbelastung hin untersucht worden sind. Zusätzlich zeichnet sich die Untersuchung

an den Schweinswalen, durch eine retrospektive Analyse (2014 – 2018) unter Einbeziehung

von Informationen zu Alter, Geschlecht und Gesundheitszustand der untersuchten Tiere aus.

Eine Korrelation zwischen Erkrankungen des GIT (Gastritis und Enteritis), sowie des Befalls

von Parasiten konnte auf makroskopischer Ebene nicht festgestellt werden. Allerdings scheint

die erhöhte Aufnahme von Mikroplastikpartikeln in Individuen mit gutem oder moderatem

Ernährungs- und Gesundheitszustand weitaus wahrscheinlicher, da sie höchstwahrscheinlich

ungehindert und kontinuierlich Nahrung und damit Mikroplastik zu sich nehmen können (im

Vergleich zu altersschwachen oder kranken Individuen). Dennoch konnten keine Korrelationen

zwischen dem Alter, dem Geschlecht oder der jeweiligen Krankheitsbilder festgestellt werden.

Zusammenfassend unterstreichen die Ergebnisse dieser Arbeit die Notwendigkeit eines

verstärkten Monitorings von Meeressäugern und ihrer Exposition gegenüber Mikroplastik. Im

Rahmen der Europäischen Meeresstrategie Rahmenrichtlinie kann sie als Basislinie für die

Nord- und Ostsee genutzt werden, um Maßnahmen zur Einhaltung oder Erreichung eines guten

Umweltzustandes (engl.: Good Environmental Status, GES), falls dieser noch nicht vorliegt.

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List of Figures

183

List of Figures

Figure 1. Overview of examined intestinal and scat samples of harbour seals, grey seals and

harbour porpoises from the North Sea and Baltic Sea. ........................................... 36

Figure 2. Sampling of intestinal samples: (A) The caudal part of the rectum is tied off; (B)

An 8-10 cm section of the rectum is measured in cranial direction and tied off a

second time; (C) The intestinal sample is cut with a scalpel and placed with metal

tweezers in a glass jar (D). ...................................................................................... 45

Figure 3. Suspected MP from procedural blank controls (≥100 µm). Working environment:

12 control filter; Washing sachets: Five blank samples; NaCl Solution: Five

control samples. ...................................................................................................... 51

Figure 4. Differential values in the quantity of suspected MPs (≥100 µm) in samples stored

in plastic bags, compared to ones stored in glass jars from different storage times.

................................................................................................................................ 52

Figure 5. Suspected microplastics in faecal samples stored in glass jars (n = 9) and plastic

bags (n = 9) combined from all storing periods (three, six and 12 months; particles

≥100 µm). In addition, five samples of seal faeces, which had been stored in

LDPE bags for eight years, were presented for comparison purposes. .................. 53

Figure 6. Example of a decision tree of the test set-up of 510 particles showing the decisive

parameters. .............................................................................................................. 54

Figure 7. Identified microplastic particles from intestinal samples stained with Nile Red

photographed under a fluorescence microscope using a TRITC filter: (A) A nylon

fibre of the used nylon cloth; (B) PET-fibre found in a grey seal; (C) EVA particle

found in a harbour seal; (D) PP particle found in a grey seal; (E) PE found in a

harbour seal ............................................................................................................. 55

Figure 8. Suspected microplastics in intestinal samples of harbour (n = 5) and grey seals (n

= 5), in comparison to faecal samples from seals collected on the sandbank

(Lorenzenplate, North Sea) (n = 9, no determined species per sample: faeces

originated from either a grey or a harbour seal). All samples were stored in glass

jars. The dotted line show the maximal presumption of secondary contamination

of seven particles per sample. ................................................................................. 56

Figure 9. Locations where investigated harbour porpoises were found (n = 30). ................... 86

List of Figures

184

Figure 10. Width-length distribution of all suspected microplastic particles in intestine

samples of 30 harbour porpoise in the size range of 100 µm up to 5,000 µm (n =

611). Secondary contamination was not considered. .............................................. 89

Figure 11. Microplastic particles stained with Nile Red (A-D) and the corresponding

measured µFTIR spectra of found polymers (in red; Polyamide: PA;

Polyethylene: PE; Polypropylene: PP and Polyester: PEST). Reference spectra

from the OPUS software are blue (Bruker, Ettlingen, Germany). .......................... 91

Figure 12. Overview of identified polymer compositions in microplastics (n = 67) found in

intestinal samples after analysed by µFTIR spectroscopy: PEST: 30%,

polyolefine: 20% (incl. PE and PP), PA: 12%, paint chip: 1%, others: 4% (incl.

acrylic with nitrile component, and cellulose acetate). Polymer composition of

microplastics found in procedural blanks was not considered for this overview,

albeit the coincidence of polymers in procedural blanks and samples were

considered, and thus not taken into account. .......................................................... 92

Figure 13. Quantity of suspected microplastics from each year, divided by sea (BS = Baltic

Sea; NS = North Sea; n = 401). .............................................................................. 94

Figure 14. Quantity of suspected microplastic particles (n = 401; secondary contamination

was considered and subtracted) separated by sex and age of the investigated

harbour porpoises (n = 30) from German waters (NS and BS). ............................. 95

Figure 15. Quantity of suspected microplastics (n = 401) in combination with the nutritional

status and further information about the carcasses (bycaught: the harbour

porpoise was (suspected to be) bycaught by a fishing boat or in a gillnet; flatfish:

the harbour porpoise was affected by a pharyngeal entrapment; na: no

extraordinary finding could be revealed; pregnant: pregnancy in female was

noticed). The nutritional status is coded as follows: 1: bad; 2: moderate and 3:

good. ....................................................................................................................... 97

List of Supplementary Figures

185

List of Supplementary Figures

Supplementary Figure 1. The washing bag design used for the pre-trials using pre-sewed

bags closed with stitched hook and loop fastener (A & B). The size fragmentation

based on the nylon cloths operates successful during the washing procedure, what

approved the method of different cloths (C). These pre-trials were conducted with

aluminium silicate (200 µm – 500 µm). ................................................................. 62

Supplementary Figure 2. The second circle of pre-trials were conducted with microbeads

(ca. 150 µm – 800 µm; see B & C). Oil pellets (green) were removed during

washing procedure (see A); the remaining microbeads were used for determining

the loss of particles. ................................................................................................ 63

Supplementary Figure 3. Work process in an acrylic box. A) Sewing the samples into nylon

washing bags. B) Processing the samples with cotton gloves within an acrylic box

situated under a fume hood. .................................................................................... 64

Supplementary Figure 4. Sliced intestine sample sewed in the nylon washing bags.

Washing bag consists of two nylon cloths (inner bag: 300 µm and outer bag: 100

µm). Overhanging nylon cloth is cropped before conducting the washing cycle. . 65

Supplementary Figure 5. Nylon fibres of the washing bag cloth with an incomparable

zigzag shape and reddish colour. ............................................................................ 65

Supplementary Figure 6. Fish bone. ...................................................................................... 66

Supplementary Figure 7. Fish bones and plant-based fragments. ......................................... 67

Supplementary Figure 8. Fish vertebrae. ............................................................................... 67

Supplementary Figure 9. Fishbone. ....................................................................................... 68

Supplementary Figure 10. Biogenic fragment (e.g. plant-based seed or a big fish lens). ..... 68

Supplementary Figure 11. Biogenic fragment. ..................................................................... 69

Supplementary Figure 12. Plant-based fragment. ................................................................. 69

Supplementary Figure 13. Biogenic fragment. ..................................................................... 70

Supplementary Figure 14. Cellulose fibre. ............................................................................ 70

Supplementary Figure 15. Microplastic at the edge of a filter. ............................................. 71

Supplementary Figure 16. Countable microplastic fragment. ............................................... 71

Supplementary Figure 17. Countable microplastic fragment surrounded by fish bones. ..... 72

List of Supplementary Tables

186

Supplementary Figure 18. Countable microplastic fragment surrounded by a cellulose

fibre. ........................................................................................................................ 72

Supplementary Figure 19. Fluorescence intensity. ............................................................... 73

Supplementary Figure 20. Surface. ....................................................................................... 73

Supplementary Figure 21. Appearence. ................................................................................ 73

Supplementary Figure 22. Completeness. ............................................................................. 74

Supplementary Figure 23. Surroundings. .............................................................................. 74

Supplementary Figure 24. Organic matter. ........................................................................... 74

Supplementary Figure 25. Measured spectrum (red) of one paint chip (µFT-IR, Hyperion

2000, Bruker, Ettlingen, Germany) showing styrene (blue circle) and kaolin

(green circle) components. The reference spectrum of kaolin is blue. ................. 108

List of Supplementary Tables

Supplementary Table 1. Results of microplastic analysis. .................................................... 75

Supplementary Table 2. Information on all 30 investigated harbour porpoises from German

waters (2014 – 2018). Every individual was examined after Siebert et al. (2001,

2020) and microplastic investigation was conducted after Philipp et al. (2020). . 107

Acknowledgements

187

Acknowledgements

Now, I have to acknowledge that dreams do come true. Having started as a bachelor’s student

in biology, the strength and the will to become a “real” marine biologist just commenced when

ITAW times began. Now that I have been allowed to go through various work areas and have

had the opportunity to work on a thesis in such an exciting research field, I think that I take the

final step to see myself as a dedicated marine biologist. Certainly, this success can only be

attributed to the support of my many colleagues and friends. Therefore, I want to thank you all,

from the bottom of my heart, who accompanied me.

First of all, I would like to thank my supervisors, who gave me the opportunity to write my

thesis in this exciting and emergent research field. You both gave me the needed support to

compile and complete this thesis. Many thanks go to Prof. Prof. h. c. Dr. Ursula Siebert for

the trust and the support to work in different research fields, which enabled me to gain

knowledge in various topics and achieve the doctor’s degree. Further thanks go to Prof. Dr.

rer. nat. Dieter Steinhagen, who encouraged and supported me in writing and wording.

I am especially grateful to Abbo van Neer. You brought me here and kept me here at the ITAW.

Since you mentored me in my bachelor thesis, I could always trust in you – in your help, your

knowledge, and your support and to always to have an open ear for me. In addition,

extraordinary thanks are going to Bianca Unger. You encouraged and supported me in every

situation at work and in life since I started here at the ITAW. Thank you for your time, your

nerves and your good mood – which always helped to get through it, no matter the issue. Further

thanks go to Britta Schmidt; there is no better office mate I could wish for. I want to thank you

for always calming me down or agitating with me with your support, and our discussions, in

which we tried to find the right words. Moreover, Dominik Nachtsheim – As I often say, what

would we do without you? No matter if statistical questions had to be analysed or if I needed

advice from you - regarding whatever. Thank you for your trusting nature. Further special

thanks to Johannes Baltzer, who always helped me out if something was missing or not

comprehensible to me. You calmed me down and made me realise that the thesis is realisable

and will be good in time. Thank you all for having my back. There are so many more people

belonging to the often-cited ITAW family – including current and former staff members. I also

want to thank you all for your trust and supportive manner and especially for the great times

Acknowledgements

188

we had – regarding the work, necropsies, conferences, barbecues, pizza evenings, or after-work

beer sessions. You all took part in my doctoral thesis – THANK YOU!

Next to my colleagues, which became friends over time, I would like to thank Franziska

Wilshues de Chavez for always being there if I need you, for being my best friend ever since

we started the Bachelor’s programme together in Hannover. You accompanied me in all life

situations and further encouraged and supported me every step of the way to complete the

doctoral thesis. I am very grateful to have you in my life. Further special thanks to my

hometown crew, the Wulsdorfer, I know that I can always count on you guys, and I do not

want to miss anyone of you. Thank you for still being part of my life and for your support during

the last 15 years.

Last but not least, I would like to thank my parents: Mom & Dad, you always believed in me

and supported me, whatever I wanted to do or dreamed of. Your trust and faith in me let me

grow in life and in my career. Thank you for being on my side.

Curriculum Vitae

Personal details

Name: Carolin Philipp

Birthday: 06.09.1991

Birthplace: Bremerhaven, Germany

Nationality: German

School Education

2008 – 2011 Abitur (A-level, higher education entrance qualification)

School Schulzentrum Geschwister Scholl, Bremerhaven

2002 – 2008 Comprehensive school

School Paula-Modersohn-Gesamtschule, Bremerhaven

1998 – 2002 Primary school

School Altwulsdorfer Grundschule, Bremerhaven

University Education

since 04/ 2019 Doctoral candidate

University Institute for Terrestrial and Aquatic Wildlife Research, University

of Veterinary Medicine Hannover, Foundation

2015 – 2017 Master of Science (Marine Environmental Sciences)

University Carl von Ossietzky University Oldenburg, Germany

Thesis Common minke whales Balaenoptera a. acutorastrata in the

North Sea: Habitat use and distribution patterns

2015 – 2011 Bachelor of Science (Biology)

University Gottfried Wilhelm Leibniz University Hannover, Germany

Thesis Kegelrobben (Halichoerus grypus) und Tourismus:

Verhaltensbeobachtungen auf der Helgoländer Düne

Internships

09/2017 – 10/2017 Volunteer

Institution ORCA Foundation, Plettenberg Bay, South Africa

07/2016 – 09/2016 Intern, Section Biosciences – Functional Ecology

Institution Alfred Wegener Institute, Helmholtz Centre for Polar and Marine

Research, Bremerhaven

Work Experiences

since 01/2018 Research associate in different projects of external funds



04/2017 – 09/2017 Student assistant



06/2016 – 03/2017 Associate of the zoo school in the “Zoo am Meer”, Bremerhaven

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