1. Robyn Elizabeth Jones
MSc Thesis 2014
The Effects of PVC Microplastics on Benthic Organisms
found on the North Wales Coast.
Project Supervisor: Dr Andrew Davies
In collaboration with the GEOMAR Centre for Ocean Research, Kiel.

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DECLARATION
This work has not previously been accepted in substance for any degree and is not being
currently submitted for any degree.
This dissertation is being submitted in partial fulfilment of the requirement of the M.Sc. in
Marine Environmental Protection.
The dissertation is the result of my own independent work / investigation, except where
otherwise stated.
Other sources are acknowledged by footnotes giving explicit references and a bibliography is
appended.
I hereby give consent for my dissertation, if accepted, to be made available for photocopying
and for inter-library loan, and the title and summary to be made available to outside
organisations.
Signed:
Date: 19/09/2014

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The Effects of PVC Microplastics on Benthic Organisms found on the North Wales Coast
Email: rjones7817@gmail.com Tel: 07884265622
Abstract
An increase in plastic production has been observed since the 1970s coinciding with the
rising demand from the human population. This has led to the increase in plastic pollution in
the coastal marine environment. This study aims to investigate the effects of the microplastic
PVC and the pollutant Fluoranthene on the lugworm Arenicola marina and whether a transfer
of PVC through the food web could occur for the mussel Mytilus edulis and the dog whelk
Nucella lapillus as a model food chain. Arenicola marina were exposed to 6 different
Polyvinyl chloride (PVC) concentrations; 3% without pollution, 3% with pollution, 0.3%,
0.03%, 0.003% and 0% for 51 days while monitoring the faecal casts produced, worm
weight, motility and mortality. The mussels were exposed to 3 different PVC concentrations
5g, 1g and 0g for 2 different exposure times 1 hour and 6 hours before being presented to the
dog whelk for a 48 hour period. Results showed that Arenicola marina was significantly
affected by the higher percentages of PVC for its faecal cast weight and also affected by the
presence of pollutant for motility. The weight and mortality of the worm was not however
affected by the amount of PVC. A transfer of PVC was also present between Mytilus edulis
and Nucella lapillus which was significantly influenced by the concentration of PVC initially
exposed to the mussel not the exposure time. These results suggest that the lugworm has
resilience to the presence of PVC as its weight and mortality rate is not affected by the
different PVC concentrations but reduced feeding levels are observed with the decline in
faecal casts. The transfer of particles from the mussel to the dog whelk may signify the
transfer of plastics to higher tropic level organisms including the possible transfer to humans.

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Acknowledgments
I would like to thank the following people for their help during the process of this project. Dr
Andrew Davies for his patience, help and advice throughout my experimental design, data
collection, write up and for introducing me to new and different concepts within Marine
Biology over the last 5 years; Ina Liebetrau for her collaboration from the GAME Institute
(GEOMAR) in Germany who was a huge help for the design of the Arenicola marina
experiment, animal collection and general guidance throughout the project duration; Maike
Nicolai for her help in creating the GoPro footage for the GAME project; Berwyn Roberts for
his help in the aquarium and his professional advice for organism collection sites; the
technical staff Ian Pritchard and Joan Griffiths for the use of equipment; Sandie Hague for
being a constant rock and hub of information and organisation throughout the last 12 months
and Steve Balestrini for his patience and for answering my constant questions about
equipment and the laboratory space.
I would also like to thank the MSc contingent for the coffee breaks throughout the long shifts
in the computer room; my housemates Catherine Sharp and L-J Stokes and parents for
listening to my constant whinging and providing motivation, Ben Dickinson for his proof
reading skills, Illtud Jones for his ‘crack the whip’ approach to make me sit down and work
and finally Murray Gold for creating the perfect writing music.

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Table of Contents
1 Introduction...................................................................................................................11
1.1 Microplastics .........................................................................................................12
1.2 Marine Ingestion....................................................................................................13
1.3 Transfer through the Food Chain...........................................................................14
1.4 Aims and Objectives..............................................................................................15
2 Materials and Methods..................................................................................................17
2.1 Sample Organisms.................................................................................................17
2.2 Mytilus edulis and Nucella lapillus .......................................................................18
2.3 Arenicola marina Ingestion...................................................................................19
2.4 Statistical Analysis ................................................................................................22
2.5 Transfer through the Food Web.............................................................................22
2.6 Statistical Analysis ................................................................................................25
3 Results...........................................................................................................................27
3.1 Arenicola marina Ingestion...................................................................................27
3.2 Transfer through the Food Chain...........................................................................35
4 Discussion.....................................................................................................................42
4.1 Arenicola marina Ingestion...................................................................................42
4.2 The Presence of Pollutants ....................................................................................45
4.3 Transfer through the Food Chain...........................................................................46
4.4 Limitations of Current Project and Recommendations for Future Work ..............48

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4.5 Conclusions ...........................................................................................................49
5 References.....................................................................................................................50
6 Appendix I ....................................................................................................................55
7 Appendix II...................................................................................................................58

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List of Figures
Figure 1: Arenicola marina J-shaped burrow (Marinebio 2014).............................................17
Figure 2: The layout of the experiment. Each tank was labelled 1-60 and colour coded for the
different plastic concentrations. Black-0% (No pollutant), Green- 3% (No Pollution),
Yellow/Black- 3% (Pollutant), Yellow/Red- 0.3% (Pollutant), Red/Green- 0.03% (Pollutant)
and White/Black- 0.003% (Pollutant)......................................................................................20
Figure 3: The presence of PVC in an opened mussel. The red circles indicate the plastic
particles which have accumulated in the soft tissue. ...............................................................24
Figure 4: Average faecal cast weight, digging in time and worm weight for Arenicola marina
for all 6 treatments of microplastic. The * indicates the absence of pollutant. Error bars +/-
1SE...........................................................................................................................................27
Figure 5: The average change in faecal cast weight, digging in times and worm weight for
Arenicola marina for the 6 different microplastic treatments. A positive value shows a
decrease in the cast weight and negative values show an increase in cast weight. * indicates
the absence of pollutant. Error bars +/- 1 SE...........................................................................29
Figure 6: The average slope for each PVC treatment for the faecal cast weight, digging in
times and worm weight for Arenicola marina. Error bars +/- 1 SE. *Indicates the absences of
pollutant. ..................................................................................................................................33
Figure 7: The number of mortalities for each PVC treatment. * Indicates no pollutant..........34
Figure 8: The average number of particles found in the mussel Mytilus edulis for the 3
different concentrations and 2 different exposure times. Error bars +/- 1 SE. ........................35
Figure 9: The average number of particles found in the mantle, gut and gills of the mussel
Mytilus edulis for 3 different plastic concentrations and 2 different exposure times. Error bars
+/- 1 SE. ...................................................................................................................................37

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Figure 10: The percentage of mussel tissue consumed by the dog whelks and the number of
particles transferred to the dog whelk for each of the 3 replicates. An absence of data means
no tissue was consumed and no particles were transferred. 1, 2 and 3 represents the 3
replicate dog whelks for each different treatment for the percentage of the mussel it
consumed. A, B and C represents the same dog whelks but represents the number of particles
transferred from the mussel. ....................................................................................................39
Figure 11: The average number of particles transferred to the dog whelk Nucella lapillus
from Arenicola marina for each PVC concentration and exposure time. Error bars +/- 1 SE.
..................................................................................................................................................40

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List of Tables
Table 1: The amount of sediment and plastic present for each treatment. ..............................20
Table 2: T-tests for the 6 different PVC concentrations exposed to Arenicola marina
comparing the average start and end faecal cast weight..........................................................30
Table 3: T-tests for the 6 different PVC concentrations exposed to Arenicola marina
comparing the average start and end digging in times (*Indicates no pollutant). ...................31
Table 4: T-tests for the 6 different PVC concentrations exposed to Arenicola marina
comparing the average start and end worm weight. (*Indicates no pollutant, **Indicates a
Mann-Whitney U Test)............................................................................................................32
Table 5: A 2-way ANOVA for the concentration of PVC and the exposure time plastic
accumulation in the mussel Mytilus edulis. .............................................................................36
Table 6: Tukey pairwise comparison for the 3 different PVC concentrations ........................36
Table 7: A 3-way ANOVA between PVC concentration, exposure time and type of soft tissue
for PVC accumulation in the mussel Mytilus edulis................................................................38
Table 8: A 2- Way ANOVA for the concentration of PVC and the exposure time plastic
accumulation in the dog whelk Nucella lapillus......................................................................40
Table 9: Tukey pairwise comparison for the particle uptake and accumulation in the dog
whelk Nucella lapillus for the 3 different PVC concentrations...............................................41
Table 10:One-Way ANOVA pairwise comparison test for faecal cast weight using the LSD
test between the 6 different plastic treatments exposed to the worm Arenicola marina. *
indicates the absence of pollutant. ...........................................................................................55
Table 11: One-Way ANOVA pairwise comparison test for digging in times using the LSD
test between the 6 different plastic treatments exposed to the worm Arenicola marina. *
indicates the absence of pollutant ............................................................................................56

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Table 12: Tukey pairwise comparison test for worm weights between the 6 different plastic
treatments exposed to the worm Arenicola marina. * indicates the absence of pollutant .......57

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1 Introduction
Current demand for plastics globally stands at around 245 million tonnes and has been
steadily increasing since the 1940s (Andrady 2011). The term plastic refers to the synthetic
organic polymers which are derived from the polymerisation of monomers extracted from oil
or gas (Cole et al. 2011). Reasons for this increase in demand are due to the material itself as
it is lightweight, strong, inert, low cost, moisture resistant and versatile (Andrady 2011). The
mass production of this material began in the 1940s and has accounted for 8% of oil
production (Cole et al. 2011). These plastics can take various shapes, sizes and colours with
physical characteristics showing a resistance to aging and biological degradation (Moore
2008).
The accidental release of plastics into the marine environment has become a reoccurring issue
through discards and plastic waste (Wright et al. 2013). The main sources of this plastic are
the maritime users of the ocean such as the fishing industry and land based sources such as
beach litter from tourism and urban run-off (Andrady 2011). Coastal areas are hotspots for
the accumulation of these plastics. Further studies into this pollution have shown that
microplastics have increased significantly in abundance over time though examining plankton
samples (Thompson et al. 2004). Plastic resin pellets and plastic fragments are sources and
sinks of xenoestrogens and persistent organic pollutants (POPs) in these coastal areas and
have high environmental risks when ingested by baseline marine organisms (Frias et al.
2010). Microplastics which float on the water surface before sinking to the seabed are
exposed to hydrophobic compounds which can in some cases be concentrated up to 500 times
compared to that of the water present at the bottom of the seabed (Teuten et al. 2007). This
study aims to investigate the effects of microplastic particles and POPs on several common
coastal marine organisms.

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It has been estimated that marine plastic litter in general effects around 267 species globally.
This number only represents studied species, and through more research, this number may
actually be higher (Gorycka 2009).
1.1 Microplastics
Microplastics have not been universally defined and have therefore been defined differently
by various researchers meaning no real continuity is present. For this study, the definition of
microplastics is any plastics which are truly microscopic (Bakir et al. 2012) and can pass
through a 500 µm sieve but retained by a 50 µm sieve (Andrady 2011). These microplastics
are created through the breakdown of larger plastics through mechanical processes such as
internal wave action and biological processes such as animal ingestion as mentioned above
(Cooper and Corcoran 2010). Under marine conditions, microplastics undergo these break
down processes through four changes a) Bio-degradation, by living organisms in the marine
environment b) photodegradation, through the process of light/Ultra-Violet (UV) exposure c)
thermooxidative degradation, through the process of oxidative breakdown d) Thermal
degradation, breakdown through mid to high temperatures from air exposure and e) though
hydrolysis, which involves a chemical reaction with water (Andrady 2011).
As previously mentioned, POPs are present in areas where plastic accumulation is high.
These pollutants are picked up by microplastics through partitioning (Andrady 2011) a
process which involves the sorption of pollutants onto microplastics. Pollutants such as
polychlorinated biphenyls (PCBs) cover a wide range of hydrophobicities and are therefore
an important class of pollutant (Velzeboer et al. 2014). Reasons why microplastics are good
carriers of these pollutants are due to the surface area to volume ratio of the particle and that
pollutants have a higher affinity for the hydrophobic surface of plastic compared to seawater
meaning these particles can become heavily contaminated over time (Wright et al. 2013b).

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1.2 Marine Ingestion
The presence of microplastics in the water column and in the sediment makes marine
organisms vulnerable at all stages of the food web due to the particle size. Biota found at both
ends of the trophic levels are susceptible to ingesting microplastics as in some cases studies
have suggested that they lack the ability to differentiate between microplastics and prey due
to their similar size and colours (Cole et al. 2011, Moore 2008). Current studies have shown
20 species of marine organisms ingesting microplastics with only four being fully studied at
their adult stage; Arenicola marina, Mytilus edulis, Placopecten magellanicus and Talitrus
saltator with results showing significant impacts on feeding rates and weight loss (Kaposi et
al. 2013). Potential impacts of the ingestion of microplastics include the blockage of the
digestive tract if particles pass through the gut which in turn blocks the passage of food
leading to a false sense of satiation and therefore decreasing feeding rate. Further to this, the
release of organic pollutants and additives from the plastics into the body is common,
increasing chemical contamination (Lusher et al. 2013). The toxicity related to these plastics
are attributed to several factors including a) residual monomers from manufacture which
leech out of ingested plastic b) toxicity of intermediates from the partial degradation of
plastics e.g. the burning of polystyrene c) absorbed POPs from seawater and concentrated on
the plastic is an effective transport of pollutants into a marine organism (Andrady 2011).
Microplastics that are suspended in the water column or re-suspended through storm
conditions or turbulence in estuaries are susceptible to uptake by filter feeders (von Moos et
al. 2012). Cole et al. (2011) studied the effects of these plastics on filter feeders such as the
blue mussel Mytilus edulis and reported some cases where the mussels had the ability to filter
out any unwanted particles through the gills which are then transported to the labial palps for
digestion or rejection. Further investigations into this mussel have however have shown a
decrease in lysosomal enzyme stability and increased onset of granulocytoma formation (an

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inflammatory cellular response to environmental pollution) (von Moos et al. 2012, Wenger et
al. 2012).
Microplastics of a higher density sink to the seabed and settle in sediment (Andrady 2011),
therefore these are available for uptake by deposit feeders which mistake them for organic
matter and in turn become primary consumers of microplastics (Murray and Cowie 2011).
The lug worm Arenicola marina has been studied through the use of various experiments
including a mesocosm to create an environment exposing the worm to PVC contaminated and
clean sediments over long term (4 weeks) and short term (48 hours). Results have concluded
that the worm showed supressed feeding activity, longer gut residence times, inflammation
and reduced lipid and energy reserves. These responses resulted in the fasting effect due to
the change in feeding activity and a decline in weight (Wright et al. 2013a).
1.3 Transfer through the Food Chain
The ingestion of microplastics by lower trophic level organisms can potentially be transferred
through the food web if retained for a certain period of time by the primary consumer. This
transfer has been shown with the zooplankton community in the Baltic Sea. Results have
shown that Eurytemora affinis and the mysid shrimp Marenzelleria spp ingested high
amounts of microplastics with 67% and 86% of individuals consuming the plastics
respectively (Setälä et al. 2014). The direct transfer of plastics from the mysid shrimp to other
organisms in the pelagic food web including fish and other zooplankton grazers may then be
present although few studies have been conducted on this transfer (Sherr and Sherr 2002).
Larger organisms such and the blue crab Mytilus edulis and the common shore crab Carcinus
maenas have also shown a transfer of microspheres when the crabs were exposed to
contaminated mussels (Farrell and Nelson 2013). Further studies in the English Channel have
concluded that 36.5% of 10 species of fish contained the microplastic polyester with

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contamination occurring through the ingestion of lower trophic zooplankton (Lusher et al.
2013).
As previously mentioned, at present, few studies have been under taken into the potential
transport of plastic through the food web. The gut retention time of marine organisms has
proved to be an important factor when determining the ability of plastics to transfer between
the gut and tissues of the organism and through the food web (Wright et al. 2013b).
1.4 Aims and Objectives
The presence of microplastics in the marine environment is increasing globally. The aims of
this project are to examine the effects of these plastics on certain benthic marine organisms
through ingestion. Past studies have not been able to conclude what the main effects of
microplastics and pollutants are on benthic species with results varying for different
organisms. Furthermore, little knowledge is present for the transfer of microplastics through
the food chain. In order to research these areas, the two main aims are:
Aim 1: To record the percentage of PVC plastic it takes to significantly reduce motility,
faecal cast production and the lifespan of the lugworm Arenicola marina and whether the
presence of pollutants have any further effect. The two hypotheses which will be used to test
this are
H1
: The larger the percentage of PVC present in the sediment will reduce the motility, faecal
cast weight and weight of the lugworm itself, but increase the mortality rate.
H2
: The presence of the pollutant accumulated with the plastics will have a larger and quicker
effect on the lugworm compared to the plastic on its own.
This will be done by adding the same amount of polyvinyl chloride (PVC) (with and without
the pollutant) and 4 other PVC percentages (6 treatments overall) to the sediment present in

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the worm tanks and assessing their motility, faecal casts, weight and mortality throughout the
exposure period.
Aim 2: To see whether a transport of PVC through the food chain is present between the 2
model organisms the mussel Mytilus edulis and the dog whelk Nucella lapillus. The
hypothesis to test this is:
H3
: The microplastic PVC will transfer from the mussel to the dog whelk with more plastics
being transferred from those mussels exposed for a longer time and to higher PVC
concentrations.
This will be done using PVC and exposing it to the mussels over three different
concentrations and two different time periods. The soft tissue of these mussels will then be
provided to the dog whelk to see whether the plastics pass on to the whelk and also the areas
in which plastic accumulates in the mussel soft tissue.

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2 Materials and Methods
2.1 Sample Organisms
2.1.1 Arenicola marina
The lug worm Arenicola marina is a polycheate worm with a cylindrical body which reach
approximately 12-20cm in length in their adult stages. These worms are found from the high
water neap tidal level to the middle or at the lower sandy shore (Kristensen 2001, Marlin
2014).
These worms are characterised by their J-shaped burrows where it lives head down in the
sediment (Figure 1). It is a deposit feeder, ingesting sub-surface sediment and defecating at
the surface. This leads to the sand above the head of the worm sinking to create a feeding
funnel.
Figure 1: Arenicola marina J-shaped burrow (Marinebio 2014).
These worms are considered ecosystem engineers as they bio-turbate the sediment creating
direct communication between the subsurface layer and deeper sediments through channels
allowing solute exchange (Kristensen 2001). Arenicola marina defecates at the surface of the
sediment in the form of faecal casts. These casts consist of sediment of a low nutritional
value and must handle large amounts in order to satisfy their needs (Kristensen 2001). This

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species was used for this study as they are abundant across the UK coastline and would
therefore be a good indicator of the presence and effects of pollution of North Wales.
The collection of the lugworms took place on the 18/06/14 at Red Wharf Bay, North Wales
(53.2965° N, 4.2071° W). 70 worms were collected using forks and placed into a bucket
containing sand and seawater. The experiment only requires 60 worms but 70 worms were
taken in case any deaths of worms occurred between the collection and the start of the
experiment. Only the worms which had no physical damage and were over a length of 4cm
(full body) were kept and used. Sediment for feeding was also collected at this time in 7
heavy duty plastic bags. Throughout the lugworm experiment, sediment was collected from
Red Wharf Bay once a week in order to supplement the feeding process.
2.2 Mytilus edulis and Nucella lapillus
The blue/common mussel Mytilus edulis is a filter feeding bivalve found around the whole
UK coastline (similar to the lugworm) from the high intertidal to the shallow subtidal on both
rocky shores and open coasts (Marlin 2014). The feeding behaviour of this bivalve is
responsive to both biotic and abiotic factors (Bayne et al. 1993) suggesting that any changes
in environmental parameters may influence feeding behaviour. This species has a high
commercial value and the industry has been well developed throughout Western Europe
(Hernroth et al. 2002). This mussel species was used for this study because it is abundant
across the UK coastline with its predators also being abundant. The lugworm was not used
for this section as its predators tend to be birds which would not have been feasible for this
study. The dog whelk Nucella lapillus is a gastropod found on all wave exposed and sheltered
rocky coastlines around the UK. They can reach up to 6cm in height and are commonly found
amongst mussels and barnacles as these are their main source of prey and are therefore a
main intertidal predator (Marlin 2014).

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These organisms were collected from Aberffraw (53.1880° N, 4.4630° W) where both
mussels and dog whelks are both present and where dog whelks actively feed on mussels.
Past research has shown that dog whelks found in areas where mussels are not present will
not feed on mussels under laboratory conditions as they do not recognise them as a food
source (Largen 1967).
2.3 Arenicola marina Ingestion
The plastic used for both experiments is polyvinyl chloride (PVC) (2ug Number 13280: Cas
Number 9002-86-2). Before the start of this experiment, PVC was incubated for a period of
24 days for particles >50µm. During the incubation period, 250g of PVC was mixed with
1.25 litres of seawater with 0.25ml of Acetone and 0.2ml of Fluoranthene per 1 litre of
seawater. The Acetone pollutant was added to the plastic on the first day of the incubation
period with the Fluoranthene renewed every 4 days and 1 litre of seawater changed every 2
days. Materials were constantly mixed throughout this period with mechanical mixers and
stored in a cold and dark area of the aquarium. Due to the period in which the plastic needed
to be incubated, this section was started and conducted by GAME (GEOMAR Institute)
before commencing this thesis project.
After collection the worms were stored in a bucket with a small amount of seawater and
filtered air for 24 hours. After this period, the each worm was weighed and measured before
being placed in tanks labelled 1-60. The times taken for each worm to start to digging into the
sediment after being added to the tank and the dig in times were then recorded. Timing
stopped once the anterior thoracic region was buried into the sediment. These worms
remained in the tanks for 6 days before any treatments were added.
The following design was used when adding varying concentrations of plastics to the worm
tanks. The 6 different plastic treatments used were; 0% and 3% PVC plastic without any

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pollutant and 3%, 0.3, 0.03 and 0.003% plastic with pollutant present (Figure 2). Plastic was
added to the tanks for feeding once a week by removing 750g of sediment from each tank.
Each tank was labelled 1-60 and colour coded depending on the treatment. 1kg of clean
sediment was always present in each tank with the other 750g being renewed once a week.
The percentage of plastic was taken from the 750g added to the tank and not the total amount.
Figure 2: The layout of the experiment. Each tank was labelled 1-60 and colour coded for the different plastic
concentrations. Black-0% (No pollutant), Green- 3% (No Pollution), Yellow/Black- 3% (Pollutant),
Yellow/Red- 0.3% (Pollutant), Red/Green- 0.03% (Pollutant) and White/Black- 0.003% (Pollutant).
The amount of plastic to be added to each treatment at each feeding time was calculated and
the values are shown in Table 1. The water supply for the tanks was created using plastic
pipes built into 5 squares attached to taps with the ability to provide water for 12 tanks. Small
holes were then drilled in to the pipes to allow water flow over each individual tank.
Table 1: The amount of sediment and plastic present for each treatment.
% PVC Sediment (g) Dry PVC (g)
3 727.5 22.5
0.3 747.88 2.115
0.03 749.77 0.225
0.003 749.97 0.0225
0 750 0

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The polluted water removed from each tank during feeding was filtered through cat litter
before being disposed in order to reduce contamination of the surrounding environment.
Sediment removed was not returned to the Menai Strait but bagged and retained for other
uses. The treatments started on the 25/06/2014 and data collection commenced on the
30/07/2014 allowing 5 days to acclimatise in order for the 1st
data reading to account for the
presence of microplastics.
2.3.1 Performance Metrics
The faecal casts of each worm were removed with a 60ml syringe (Appendix II, video 1).
Preliminary experiments showed that these casts needed to be taken at the same time each
day in order to get an account of the 24 hour period. These casts were oven dried and
weighed every 2 days for a period of 2 months to analyse whether an increase or decrease in
cast weight was present with plastic exposure. On alternate days to the cast weighing, faeces
within the tanks were flattened in order to only collect casts produced over a 24 hour period
for consistency. The casts were dried in the oven for a period of 4-5 hours on 150°C.
However, if casts were left over night, then oven temperature changed to 80°C for
approximately 14-16 hours.
The motility of the worms was observed though digging in times. Worms were placed on the
surface of the sediment times were recorded for the worm to dig itself into the sediment
(Appendix II, video 2). Time started when the worm probed into the sediment. This
observation was undertaken 4 times throughout the experiment including the start and the
end. Worms were dug out by hand in order to reduce any physical damage.
The weight (200g scales) and length (30cm ruler) of each worm was taken 4 times throughout
the experiment including the start and end to see whether decreases in both were seen. In

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order to reduce disturbance to the worms, this data was recorded at the same time as the
digging in times.
The mortality of the worms under each treatment was taken by the number of days each
worm lasted in the experiment. The presence of faecal casts within the tank indicated if the
worm was still alive. If casts did not appear after 4 days, the worm was considered dead and
dug out.
2.4 Statistical Analysis
The data collected during this experiment will be analysed using IMB SPSS Statistics
Software. Univariate analysis using One- Way ANOVA was used to determine whether
significant differences were present between the 6 different PVC treatments overall and for
mortality. Independent t-tests were then used to discover whether significant differences were
present for the 6 different treatments from the start and end observation for the faecal cast
size, motility and weight. Comparisons of the average slope of each treatment were also
undertaken to visualise any trends. The assumptions of equal variance and normal
distribution were met before the ANOVA was run for the different factors.
2.5 Transfer through the Food Web
Several preliminary experiments were conducted prior to the main design. The 1st
experiment
was to check the removal times of plastic from the mussels for 3 different time periods 1, 5
and 12 hours. Mussels were removed from plastic exposure after the 3 different time periods
and results showed that a 12 hour exposure time was too long as the microplastics had been
partially removed from the mussel by this time when compared to the 5 hour exposure.
Furthermore, the PVC had already sunk if no re-suspension process was present. Therefore

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the times 1 hour and 5 hours were used for the final experiment with periodic mixing every
hour occurring in the 5 hour time period.
Preliminary data was also collected for the way in which the mussels were presented to the
dog whelks and how the mussels were to be dissected. The size of the mussels used ranged
from 2-4cm in length as past research has suggested that mussels over a size of 5cm are
usually not eaten by dog whelks (Hughes and Burrows 1993). Mussels were provided to the
dog whelks in 2 separate ways; strands of contaminated soft tissue cut out of the shell and
placed in the tank and by opening the shell and placing in the tank. Observations showed that
the dog whelks consumed more of the mussel which was presented with the shell opened.
2.5.1 Plastic Preparation
Initial experiments used PVC which was <50µm in size. 165g of plastic was mixed with
Fluorescein (free acid) 95% dye content (Sigma-Aldrich) for 24 hours. The dye was added to
400ml of freshwater and heated until boiling.in a glass beaker. The temperature was then
reduced and finally left to cool with the PVC added to the water and dye, then mixed
periodically. The plastic was dried in the oven at a low temperature to remove any excess
water.
Using a fluorescent microscope, observations showed that the dye had not stained the PVC.
Further experiments using Acetone to break down the surface of the plastic also did not allow
the dye to stain the PVC and therefore other options were considered. Preliminary
experiments to observe the mussel plastic uptake did visually show the plastic within the
tissue but with no dye present was difficult to count. Further experiments with >50µm
microplastic PVC showed this size particle to also be consumed by mussels and was easier to
count under a microscope, so was used in the final experiment. Go Pro cameras were used to

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photograph the mussel tissue if needed so detailed counts could be made later on bigger
screens.
The mussel Mytilus edulis was exposed to 3 different microplastic concentrations all of which
were >50µm sized particles for 2 different exposure times. Concentration 1 consisted of 0g
PVC (control), Concentration 2 consisted of 1g PVC and Concentration 3 consisted of 5g
PVC l) mixed with 500ml of seawater. The plastic was given to the mussels supplemented
with seawater. Due to the low exposure times no extra algae was supplemented to the mussels
as algae was naturally present in the seawater. Under each different concentration, the 2
different exposure times 1 hour and 5 hours were used. 3 replicates were used for each
exposure time under each concentration and therefore 18 mussels in total were used for the
initial microplastic ingestion experiment.
Mussels were then dissected and microplastics were counted to estimate the plastic ingestion
for each exposure time and concentration (Figure 3).
Figure 3: The presence of PVC in an opened mussel. The red circles indicate the plastic particles which have
accumulated in the soft tissue.
The microplastic concentration was counted for 3 target soft tissues, gill, stomach/gut and
mantle. Preliminary experiments showed the microplastics to be difficult to count without the

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presence of any fluorescent dye. Where possible microplastics were counted using a
microscope camera in ordered to collect a more accurate account of plastic uptake.
This process was then repeated with the mussel being placed in the tank with the dog whelk
Nucella lapillus with the shell opened. The whelk was left with the mussels for a period of 48
hours in 500ml of fresh seawater (replenished every 24 hours) to observe how much of the
mussel had been consumed by the whelk. The dog whelks were starved for a period of 7 days
prior to the experiment and were frozen after the experiment had finished.
2.5.2 Mussel Dissection
After the 2 exposure times, the mussels were then placed in the freezer for 18 hours to relax.
Mussels were then opened and strands of soft tissue were extracted from the shell using a
scalpel and examined under a Novex ZOOM Stereo Range light microscope to count the
amount of PVC which had accumulated in the tissue. The gut was also finely cut to see
whether plastics had accumulated.
2.5.3 Dog Whelk Ingestion
To investigate is PVC was transferred to the dog whelk, the organisms feeding on the mussels
from the different exposure times and concentrations were pulled from the shell dissected and
examined under a microscope (Novex ZOOM Stereo Range light). Plastic was counted in the
same way as the mussel dissections by looking at the gut and the gills.
2.6 Statistical Analysis
A similar approach was used for the second experiment using a 2 way ANOVA to compare
the concentration of plastic used and the exposure times of the mussels to the plastic. This
allowed analysis into whether differences and interactions were present in the initial uptake
of plastic for the mussels and the transfer to the dog whelks. A 3-way ANOVA was used to

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integrate the type of soft tissue into the other 2 factors (concentration and time) to see the
differences and interactions.

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3 Results
3.1 Arenicola marina Ingestion
Significant differences were present for the 6 different treatments, for the weight of the faecal
casts produced by Arenicola marina (One-Way ANOVA: F(5,119) = 20.221, P = <0.001) over
the 51 day experiment (Figure 4).
Figure 4: Average faecal cast weight, digging in time and worm weight for Arenicola marina for all 6 treatments
of microplastic. The * indicates the absence of pollutant. Error bars +/- 1SE.
Pairwise comparisons (Appendix I, Table 10) show that overall, significant differences were
present between the cast sizes for the treatments 0% and 3%*, 3%, 0.3% (P= <0.001), 3%*
and 0.3% (P= 0.046), 3%* and 0.03% and 0.003% (P= <0.001), 3% and 0.03% and%
0.003% (P= <0.001), 0.3% and 0.003% (P= <0.001), 0.3 and 0.03 (P= 0.02) and finally
0.03% and 0.003% (P= 0.036). The two 3% treatments have therefore been shown to
significantly reduce the cast weight produced by the worms compared to the other smaller
0
20
40
60
80
100
120
0
2
4
6
8
10
12
14
16
18
20
3* 3 0.3 0.03 0.003 0
MeanDiginginTime(s)
MeanWeightofFaecalCastsandWeightofWorm(g)
Faecal
Casts
Weight of
Worm
Digging In

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treatments while the lower concentrations such as 0.003% and 0.03% not showing to be
significantly different from the control and had heavier casts. However, the presence of a
pollutant to the plastic does not significantly affect the cast weight when compared between
the 2 largest PVC (3%) treatments (P= 0.526).
A one-way ANOVA was carried out to see if a significant difference was present for the 6
different treatments regarding motility of Arenicola marina. Initial results failed the
homogeneity test (Levenes test) and so data was transformed using the square root
transformation in SPSS. In total, the data was transformed to 3rd
root with P=0.053 for the
Levenes test. A one-way ANOVA then showed that the motility (dig in times) between the 6
treatments was just significantly different from each other (F(5,23)= 2.640, P= 0.05). When the
data was graphed, only slight differences were visible between the 6 different treatments and
therefore accounting for the P value being so close to 0.05 (Figure 4). Pairwise comparisons
(Appendix I, Table 11) show that overall, significant differences were present between the 2
3% PVC treatments; 3% (with pollutant) and 0.3%; 0.03% and 0.003% suggesting that the
presence of pollutants slightly influences the motility of the worm.
Initial tests to compare the different worm weights did not show the data to be of equal
variances. The data was therefore transformed using the SQRT function in SPSS until 3rd
root
was achieved however; the data was still classed as unequal by the Levenes test so a Kruskal-
Wallis test was therefore used. Results from this test showed that a significant difference was
present between the 6 different treatments (x2
= 15.890, P= 0.013, df= 5) (Figure 4). A post
hoc Tukey pairwise test (Appendix I, Table 12) shows the differences between these
treatments. This pairwise comparison shows that significant differences are present between
the following treatments: 0% and 3%*, 3%, 0.3% and 0.03%; 3%* and 0.3%; 3% and 0.3%;
0.3% and 0.003%. These results suggest that every treatment of plastic differs from the
control with significantly lower weights however as these results were taken at the very start

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of the experiment the 1st
observation is for weights without plastic exposure with the control
average weight being higher than the other treatments. The 5 other treatments had a relatively
similar starting weights and significant differences were seen between the 3% polluted plastic
and the smaller treatment 0.03% and also the non-polluted 3% plastic and 0.3% plastics. The
presence of pollutant to the plastic again has no significant effect on the weight of the worm.
The individual samples (6x10 replicates) of Arenicola marina were selected from every PVC
treatment and were tested to see if significant differences were seen between the start of the
experiment and the end for the 3 performance matrices faecal cast weight, worm weight and
digging in time. The last dry weight reading was subtracted from the start dry weight reading
and the average change in weight for each treatment was recorded in Figure 5.
Figure 5: The average change in faecal cast weight, digging in times and worm weight for Arenicola marina for
the 6 different microplastic treatments. A positive value shows a decrease in the cast weight and negative values
show an increase in cast weight. * indicates the absence of pollutant. Error bars +/- 1 SE.
-70
-50
-30
-10
10
30
50
70
-15
-10
-5
0
5
10
15
3* 3 0.3 0.03 0.003 0
MeanChangeinDigginginTime(s)
MeanChangeinFaecalCastWeightandWormWeight(g)
Faecal
Cast
Weight
of Worm
Digging
In

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Results show that a decrease in faecal cast weight was observed for the two 3% PVC
treatments. The other PVC treatments have however shown an increase in average faecal cast
weight. T-tests for each treatment are shown in Table 2 (Levenes test passed for all
treatments >0.05) which compare the mean weight of the faecal casts at the beginning of the
experiment to that at the end to observe whether a significant change was present between the
1st
and last data collection. Analysis shows that no significant differences are present for the
start and the end of the experiment for all the plastic treatments except the 0.3% PVC
concentration (Table 2). This therefore suggests that over the duration of exposure to
different PVC concentrations, no significant change is seen in the faecal cast weight produced
by Arenicola marina.
Table 2: T-tests for the 6 different PVC concentrations exposed to Arenicola marina comparing the average start
and end faecal cast weight.
Treatment t DF P- Value
0% -1.107 18 0.283
3%* 1.464, 18 0.160
3% 0.351 18 0.729
0.3% -2.988 18 0.008
0.03% -0.81 18 0.936
0.03% -0.368 18 0.717
Results show that with the exception of the 0.3% treatment, the effects of the PVC plastic
concentration on cast weight occurs quickly (during the 5 day acclimatisation period to PVC
before data collection commenced) leading to the significant differences betw

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een the treatments and then only vary slightly throughout the rest of the experiment with the
highest change in cast weight being recorded as an increase of 10.77g for the 3% with
pollutant treatment.
For the motility of the worm, results show that an increase in digging in time is present for
the 3%*, 3% and 0.003% treatments with decreases observed for the control (0%), 0.3% and
0.03% (Figure 5) from the start of the relationship to the end. T-tests comparing the average
digging in time for the start and end of the experiment for each treatment are shown in Table
3 (Levenes test passed for all treatments >0.05). Analysis shows that no significant
differences were present between the start and end of the experiment for all 6 treatments.
Results show that the motility of worms when exposed to the 6 different PVC concentrations
and the pollutant shows that the amount if PVC present in the sediment does not affect the
digging in times of the worms as they also do not differ from start to end.
Table 3: T-tests for the 6 different PVC concentrations exposed to Arenicola marina comparing the average start
and end digging in times (*Indicates no pollutant).
Treatment t DF P- Value
0% 1.272 18 0.221
3%* 0.266 18 0.793
3% 0.63 18 0.554
0.3% 1.429 18 0.170
0.03% 1.768 18 0.094
0.03% 0.852 18 0.405
For the weight of the worm, results show that an increase in weight is seen for all treatments
(Figure 5) from the start to the end of the experiment; however large error is seen for all
treatments. T-tests comparing the average weight of the worm at the start and the end of the

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experiment for each treatment are shown in Table 4. These results show that with the
exception of the 0.03% PVC treatment, no significant differences are present from the start to
the end of the experiment for the different plastic treatments. The differences between the
control and the plastic treatments can be attributed to the 1st
observation weight differences.
Differences between the 3% treatments and the 0.3 and 0.03% suggest that the presence of a
higher quantity restricts weight gain but the presence of pollutant does not.
Table 4: T-tests for the 6 different PVC concentrations exposed to Arenicola marina comparing the average start
and end worm weight. (*Indicates no pollutant, **Indicates a Mann-Whitney U Test)
Treatment t DF P- Value
0% 0.314 18 0.757
3%* 0.360 18 0.665
3% 0.424 18 0.677
0.3% -2,944 18 0.677
0.03%** 18 0.023
0.03% -0.651 18 0.523
Comparisons were made for the average regression slopes of each treatment for the duration
of the experiment for the faecal cast weight, digging in times and worm weight (Figure 6).
Analysis shows that negative slopes were present over all for the two 3% PVC treatments for
the faecal cast size signifying a slight decline in faecal cast weight over time. Positive slopes
were present for the other 4 treatments, however, no significant differences were present
between all 6 different treatments (One-Way ANOVA: F(5,59)=1,450, P= 0.222).

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Figure 6: The average slope for each PVC treatment for the faecal cast weight, digging in times and worm
weight for Arenicola marina. Error bars +/- 1 SE. *Indicates the absences of pollutant.
When comparing the average regression slopes for each treatment for the digging in times, a
large amount of error is observed for all the PVC treatments suggesting that a high amount
variation between the worms within each treatment is present (Figure 6). Results show that
the only treatment to have an increase in digging in time over the experiment period is the 3%
(with pollutant) however, no significant differences were present between the treatments
(One-Way ANOVA: F(5, 59)=0.636, P= 0.673).
When comparing the average regression slopes for each treatment for the weights of the
worm (Figure 6) large error is present once again. Results show that the only 2 treatments to
induce an increase in weight were the 3% (without pollutant) and 0.003%, treatments,
-25
-20
-15
-10
-5
0
5
10
15
20
25
-5
-4
-3
-2
-1
0
1
2
3
4
5
0 3* 3 0.3 0.03 0.003
MeanSlopeforDigginginTime
MeanSlopeforFaecalCastandWormWeight
PVC Concentration (%)
Faecal
Cast
Worm
Weight
Digging
In

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however, no significant differences were found between all 6 different treatments (One-Way
ANOVA: F(5,59)= 0.325, P= 0.896).
The mortality of the worms was decided by the number of days which they remained in the
experiment. Figure 7 shows the average number of Arenicola marina mortalities in the
experiment for each treatment.
Figure 7: The number of mortalities for each PVC treatment. * Indicates no pollutant.
A Kruskall-Wallis test to compare the treatments (unequal variances) shows that there are no
significant differences between the average number of days Arenicola marina remained in the
experiment for the 6 different treatments even though 3% (with pollutant), 0.03% and 0.003%
have lower averages (x2
= 1, P= 0.317, df= 1) suggesting the amount of plastic does not
influence mortality rates.
0
0.5
1
1.5
2
2.5
0 3 * 3 0.3 0.03 0.003
NumberofMortalities
Microplastic Concentration (%)

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3.2 Transfer through the Food Chain
3.2.1 Initial PVC Ingestion
Mussels exposed to 3 different concentrations of PVC for 2 different periods of time are
shown to have differences in the accumulation of particles in their soft tissue depending on
the exposure time and PVC concentration (Figure 8). The control (concentration 1) shows no
plastic accumulation in the mussels for the 2 exposure times.
Figure 8: The average number of particles found in the mussel Mytilus edulis for the 3 different concentrations
and 2 different exposure times. Error bars +/- 1 SE.
Results for a 2-Way ANOVA are shown in Table 5. Significant differences in plastic
accumulation are present between the 3 PVC concentrations (P= 0.002) but not time
(P=0.890). This suggests that 5g (Concentration 3) triggers a higher plastic accumulation in
the mussel soft tissue compared to the lower treatments (Figure 8). The interaction of these 2
factors is however not significant (P= 0.925) suggesting that the 3 different PVC
concentrations do not respond significantly differently to time and the amount of PVC
accumulated in the mussel soft tissue and are not affected by one another.
0
10
20
30
40
50
60
70
80
90
1 Hour 2 Hours 1 Hour 2 Hours 1 Hour 2 Hours
0g 1g 5g
AverageNumberofParcles
Concentration of PVC (g) and Exposure Time (Hours)

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Table 5: A 2-way ANOVA for the concentration of PVC and the exposure time plastic accumulation in the
mussel Mytilus edulis.
Factor D.F MS F-Ratio P- Value
Concentration 2 46652.38 11.66 0.002
Time 1 80.22 0.020 0.890
Interaction 2 314.05 0.078 0.925
Error 12 4000.778
A post hoc Tukey test confirms the differences between the 3 different concentrations (Table
6). Results show that significant differences are present between 0g PVC (concentration 1)
and concentrations 1g (concentration 2) and 5g (concentration 3) (P= 0.028 and <0.001
respectively). However no significant difference was present between the 1g and 5g
concentrations (P= 0.064).
Table 6: Tukey pairwise comparison for the 3 different PVC concentrations
Treatment Contrast Std. Error Sig 95% CL 95% CL
Lower Bound Upper Bound
Con 1 vs Con 2 -95.1667 32.903311 0.028 -180.6315 -9.7018
Con 1 vs Con 3 -176.1667 32.903311 <0.001 -261.6315 90.7018
Con 2 vs Con 3 81.0000 32.903311 0.064 -166.4649 4.4649
Comparisons were also made between the concentrations and time for the different tissues
examined in each mussel (Figure 9). Results show that the amounts of PVC accumulation
differ between the 3 different soft tissues for the different factors.

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Figure 9: The average number of particles found in the mantle, gut and gills of the mussel Mytilus edulis for 3
different plastic concentrations and 2 different exposure times. Error bars +/- 1 SE.
A 3-way ANOVA (Table 7) to test whether differences were present between the PVC
concentration, exposure time and the target soft tissue (mantle, gut and gills) showed that
concentration is again significantly different from each other (P= <0.001) but the exposure
time and soft tissue are not (P= 0.837 and P= 0.974 respectively). Interactions between the
PVC concentrations, time and tissue were found to be non-significant (P= 0.845 and P=
0.852 respectively). However, significant interactions were present between exposure time
and the type of soft tissue and also between all 3 factors (P= <0.001 and P= 0.014
respectively) suggesting that if any one treatment was changed, it would affect the two other
remaining treatments with a 2 way interaction which varies across the 2 levels (concentration
and time) and that of a 3rd
variable (soft tissue).
0
20
40
60
80
100
120
140
1 Hour 2 Hours 1 Hour 2 Hours 1 Hour 2 Hours
0g 1g 5g
AverageNumberofParticlesPresent
Concentration of PVC (g) and Exposure Time (hours)
Mantle
Gut
Gills

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Table 7: A 3-way ANOVA between PVC concentration, exposure time and type of soft tissue for PVC
accumulation in the mussel Mytilus edulis.
Source of Variation D.F MS F- Ratio P-Value
Concentration 2 15550.79 25.06 <0.001
Time 1 26.74 0.043 0.837
Tissue 2 16.51 0.027 0.974
Con x Time 2 104.68 0.169 0.845
Con x Tissue 4 208.96 0.336 0.852
Time x Tissue 2 6454.29 10.40 <0.001
Con x Time x Tissue 4 2255.74 3.63 0.014
Error 36 620.31
Total 54
These results suggest that the exposure time influences the amount of PVC present within
each specific soft tissue and overall, the higher the concentration of PVC present, the larger
the accumulation in the mussel as a whole. The gut of the mussel was shown to have larger
amounts of PVC present when exposed to PVC for longer periods of time whereas the gills
(especially for the highest concentration) and the mantle are shown to have higher amounts of
PVC accumulated when exposed for a shorter time (Figure 9).
3.2.2 Transfer to Nucella lapillus
Dog whelks which fed on mussels which were previously exposed to different PVC
concentrations for different time periods were shown to partially consume the tissue
presented. Figure 10 shows the percentage of mussel tissue consumed by the dog whelk for
the various treatments and the number of particles transferred between the model organisms.

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Figure 10: The percentage of mussel tissue consumed by the dog whelks and the number of particles transferred
to the dog whelk for each of the 3 replicates. An absence of data means no tissue was consumed and no particles
were transferred. 1, 2 and 3 represents the 3 replicate dog whelks for each different treatment for the percentage
of the mussel it consumed. A, B and C represents the same dog whelks but represents the number of particles
transferred from the mussel.
Results showed that the whelks did not consume all the mussel presented to it and in some
cases did not consume any at all over the 48 hour period. This was therefore taken into
account when dissecting the dog whelks for plastic.
The dissected dog whelks showed that a very low transfer of PVC particles was present from
the mussels (Figure 10). Initial results show that the dog whelks that consumed more of the
mussels overall accumulated more plastic compared to those which consumed less or none.
The 3 replicate mussels (1, 2 and 3) from figure 16 coincide with those in figure 10.
The average number of particles of PVC collected in the dog whelks (Figure 11) for the
different treatments was shown to be significantly different (P= 0.005) for the 3 different
0
2
4
6
8
10
12
14
16
18
20
0
10
20
30
40
50
60
70
80
1 Hour 2 Hours 1 Hour 2 Hours 1 Hour 2 Hours
0g 1g 5g
NumberofParticlesPresent
%ofMusselConsumedbytheDogWhelk
PVC Concentration (g) and Exposure Time (Hours)
1
2
3
a
b
c

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concentrations but not significantly different (P= 0.314) for the 2 different exposure times
(Table 8). The interaction between these two treatments was not shown to be significant (P=
0.201) suggesting that the dog whelks which have consumed mussels which have been
exposed to different PVC concentrations for different periods of time do not respond
differently to interactions of exposure time and PVC concentration.
Figure 11: The average number of particles transferred to the dog whelk Nucella lapillus from Arenicola marina
for each PVC concentration and exposure time. Error bars +/- 1 SE.
Table 8: A 2- Way ANOVA for the concentration of PVC and the exposure time plastic accumulation in the dog
whelk Nucella lapillus
Factor D.F MS F-Ratio P- Value
Concentration 2 112.389 8.720 0.005
Time 1 14.222 1.103 0.314
Interaction 2 23.722 1.841 0.201
Error 12 12.889
0
2
4
6
8
10
12
14
16
1 Hour 2 Hours 1 Hour 2 Houra 1 Hour 2 Hours
0g 1g 5g
AverageNumberofParticlesPresent
PVC Concentration (g) and Exposure Time (Hours)

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A Tukey post hoc test to compare the 3 different concentrations is shown in Table 9. Results
here show that significant differences were present between the control and the highest PVC
concentration (5g) (P=0.004) and marginally between concentration 2 (1g) and concentration
3 (5g) (P= 0.045). This therefore suggests that the highest PVC concentration accumulates
significantly more in dog whelks compared to the other lower PVC concentrations.
Table 9: Tukey pairwise comparison for the particle uptake and accumulation in the dog whelk Nucella lapillus
for the 3 different PVC concentrations.
Treatment Contrast Std. Error Sig 95% CL 95% CL
Lower Bound Upper Bound
Con 1 vs Con 2 -2.8333 2.07275 0.388 -8.3632 2.6965
Con 1 vs Con 3 -8.5000 2.07275 0.004 -14.0298 -2.9702
Con 2 vs Con 3 -5.6667 2.07275 0.045 -11.1965 -0.1368
These results therefore suggest that a transfer though the food chain is present but only
slightly. The initial uptake of PVC by the mussels is significant for the higher concentrations
with large clumps of plastic accumulating in the different soft tissue. A very small amount of
these plastics are then transferred to dog whelks when they are consumed. When comparisons
were made between the amount of plastics present over all in the mussels and the dog whelks,
the whelks were shown to have significantly less (One-Way ANOVA: F (1,35)=16.133, P=
<0.001).This suggests that overall compared to the initial ingestion of PVC plastics, the
transfer of plastic to dog whelks is minimal.

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4 Discussion
This study was designed to analyse the effects which PVC microplastics have on benthic
organisms found in North Wales. Results have demonstrated that microplastics do affect
various benthic organisms through ingestion but not to the extent of previous research. The
effects and magnitude of microplastic ingestion can vary between species depending on their
sensitivity to marine pollution (Andrady 2011). This study has focused specifically on 3
species Arenicola marina, Mytilus edulis and Nucella lapillus. These organisms are
commonly found around the coast of the UK and would be susceptible to plastic
contamination if present in the waters around the coast of North Wales.
4.1 Arenicola marina Ingestion
During the course of the Arenicola marina ingestion experiment, the lug worm was subjected
to various concentrations of PVC <50µm for 51 days. During this period the faecal casts,
weight, motility and mortality were monitored. The initial hypothesis H1
stated that ‘The
larger percentage of PVC present in the sediment would reduce the motility, faecal cast
weight and weight of the lugworm itself, but increase the mortality rate’. The results from the
Arenicola marina ingestion experiment have shown that the presence of PVC in high
concentrations in surrounding sediment decreases the faecal casts produced by the worms
compared to lower or concentrations. However, results here suggest that impacts occur
quickly with the exposure to PVC as changes in faecal weight for all treatments did not
change throughout the duration of the experiment but did initially. This suggests that these
worms reduce their consumption of sediment after plastic exposure. The weight and motility
of the lugworms were not however affected by the presence of different PVC treatments.
Other research has demonstrated similar results for Arenicola marina performance matrices.
Investigations into microplastics present in marine sediments have occurred globally and

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affects such as mortality, reduced reproductive success and morbidity are a common
occurrence (Zarfl et al 2011). Research by Wright et al. (2013a) has suggested that lugworms
which are exposed 5% unplasticised polyvinylchloride (UPVC) significantly reduce their
feeding rates compared to exposure to 1% and 0% UPVC. These findings are similar to this
study as results do show a decline in faecal casts with higher PVC concentrations. Wright et
al. (2013a) also concluded that the effects of UPVC ingestion can be recorded in as little as 2
days similar to this study with the effects of PVC being recorded within the 6 day
acclimatisation period (where the worms were subjected to the different PVC concentrations
before data collection started). This therefore suggests that the presence of plastic in high
quantities for this study inhibits food consumption either through less particle uptake or by
lack of adhesion by the PVC to the worms feeding apparatus (Wright et al 2013a). Derraik
(2002) also states that the presence of microplastics in the gut of coastal marine organisms
have previously led to lower steroid hormone levels and diminished feeding stimulus.
Similarly, Tourino et al (2010) also found that blockages of the digestive tract of the green
turtle were observed under the influence of microplastics with 60.5% of the examined turtles
showing these affects. These findings from previous research regarding the blockages of
digestive tracts may provide reason for the results recorded in this study. In the long term, a
reduced feeding rate may in turn decrease the energy reserves and fitness of the exposed
organism Arenicola marina (Wright et al. 2013a).
The results in this study also suggest that the motility of the worm was compromised by the
initial reduction in feeding as a slight significant difference was seen with regards to the
presence of PVC. This slight difference may suggest that it takes longer than 51 days for a
more prominent effect on motility to take place. Other research into the lugworm has also
shown that weight loss is a common occurrence after exposure to PVC microplastic due to

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the reduced feeding activity and also immunity and survival rates (Browne et al. 2013). These
effects were not however observed in the results of this study.
Microplastic contamination is more profound in coastal areas especially harbours and areas
which are susceptible to heavy ship traffic (Claessens et al. 2011). Microplastics have been
found on sandy beaches across the globe including areas such as New Zealand, Canada and
the UK initially in the form of plastic pellets since the 1970s. Analysis into the types of micro
plastics present on beaches have been attributed to sewage outflows and various other
anthropogenic events with PVC and polymeride contributing to approximately 80% of
microplastic samples from 18 beaches around the UK (Ivar do Sul and Costa 2014). The UK
and the North Wales coast is an active area for coastal tourism, both recreational and
commercial fishing and for the passing of large vessels from Liverpool Bay to Holyhead
(Cole et al. 2011). Cefas (1987) has stated that the Irish Sea is prone to high levels of
pollution in the form of metals and plastics due to the amount of different anthropogenic
disturbances present. In the Red Wharf Bay area, pollution sources such as sewage outfalls
from Benllech (approximately 2km north of Red Wharf Bay) (Rees 2004) may influence the
resilience of the lugworms to any form of microplastics transported from the outflow as
previous results suggest that sewage outflows can be a source of microplastics (Claessens et
al. 2011). The combination of close sewage outfalls and the busy shipping lanes from
Liverpool Bay may therefore explain why exposure to PVC did not significantly affect the
weight of the worms and only slightly the motility for the 6 treatments. It may also explain
why no significant changes in performance matrices in the worm Arenicola marina were
recorded for the duration of the experiment.

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4.2 The Presence of Pollutants
The Arenicola marina ingestion experiment was also created to test whether the presence of
pollutants on microplastics affects the faecal cast weight, motility and worm weight. The
initial hypothesis H2
stated that ‘The presence of the pollutant with the plastics will have a
larger and quicker effect on the lugworm compared to the plastic on its own’. Results from
this study suggest that the presence of the pollutant Fluoranthene did not significantly affect
the faecal casts and worm weight as results did not differ between the 3% non-contaminated
treatment and the 3% contaminated PVC; however it did affect the motility as significant
differences were present.
Previous research has shown that the presence of pollutants on microplastics can have
significant impacts if ingested by the lugworm Arenicola marina. Besseling et al. (2012)
focused on the fitness and polychlorinated biphenyls (PCBs) and their effect on Arenicola
marina. Results here show that the presence of pollutants in the sediment reduce the lipid
content of the worms however, it was suggested that the presence of these pollutants unless
they are in high quantities were usually minimal. Microplastics which enter the coastal
environment normally contain small amounts of pollutants on their surface suggesting that in
low magnitudes, the effects of these pollutants on worms are minimal (Besseling et al.
(2012). The results from this study have agreed with this theory as the data was just
significantly different for the two 3% PVC concentrations for motility but not significant for
the weight of the worm and the faecal casts produced suggesting that the worms in Red
Wharf Bay are only affected by the presence of any small scale pollutant in terms of motility.
Welsh Water has confirmed that areas such as Menai Bridge and Beaumaris have around 0.8
ng 1-1of dieldrin hydrocarbons present in the water with no polychlorinated biphenyl
detected in the area (Cefas 1987). Further information from Rees (2004) has suggested that
the water quality in Red Wharf Bay has been increasing over the last 10 years suggesting

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minimal pollutants are present in this area which may account for the slight effect. Other
research has suggested that the leaching of chemicals such as bisphenol A (BA) and
nonylphenol (NP) from microplastics to benthic organisms such as the lugworm do create a
pathway for pollutants. Models have again also shown that in order for these pollutants to be
of any high and recordable risk to the organism an unlikely number of scenarios would need
to occur due to the variability of environmental conditions (Koelmans et al. 2014). Voparil et
al. (2004) states that the bioavailability of polycyclic aromatic hydrocarbons (PAHs) from
particles such as tire rubber or diesel soot ingested by lugworms and found in the gut can
increase the bioavailability of POPs in these species. In order for these POPs to have a
significant impact on the organism, the dose depends on the volume of the microplastic and
its residence time in the organism (Ryan et al. 1988). The presence of Fluoranthene in this
study may not be in high enough concentrations to have a profound effect on the exposed
lugworm as results were only just significant for motility and not for the weight of the worm
and faecal casts produced.
4.3 Transfer through the Food Chain
During the course of the food chain experiment, mussels were exposed to 2 different PVC
concentrations for 2 different exposure times to analyse their PVC uptake before being
present to dog whelks. The initial hypothesis H3
for this experiment stated that ‘The
microplastic PVC will transfer from the mussel to the dog whelk with more plastics being
transferred from those mussels exposed for a longer time and higher concentrations’. The
results from this study have agreed with this hypothesis as the transfer of PVC to mussels is
higher for higher exposure concentrations but is not however affected by the exposure
duration. Further analysis however shows that the exposure time did affect the area of soft
tissue which the plastic accumulated. A shorter exposure time meant that plastic had not

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enough time to accumulate in the gut but only the mantle and gills. A longer exposure time
meant that that accumulation in the gut was higher than the other 2 areas. When exposed to
dog whelks, the transfer of PVC was influenced by the amount of tissue consumed. Analysis
showed that more PVC was transferred to the whelks which consumed the mussels which had
previously been exposed to 5g PVC concentrations but were not again affected by the
exposure time.
The effects of the presence of microplastics on the mussel Mytilus edulis are similar to that of
the lugworm Arenicola marina with the accumulation in the gut and the gills restricting food
intake. Studies have also concluded that the translocation of any microplastics to the
circulatory system of the mussel can have serious effects on predators such as the crab
Carcinus maenas and the dog whelk Nucella lapillus as more plastic is retained in the mussel
for longer (Browne et al. 2008). Past research involving the transfer of microplastics between
other coastal marine organisms have observed similar results to this study. Investigations
using the crab Carcinus maenas and the mussel Mytilus edulis as model organisms have
shown that a transfer of plastic is also present between these 2 species. Results have shown
that microspheres were found in the crabs stomach, hepatopancreas, ovary and gills.
However, unlike the results for the present study, the crabs were left with the mussels for
different time periods which also affected the accumulation of plastic (Farrell and Nelson
2013). Other studies have included the transfer of particles to the Norway lobster (Murray
and Cowie 2011), the transfer between the fur seal Arctocephalus spp and lantern fish
Electona subaspera (Eriksson and Burton 2003) and also primary trophic level species such
as the algae Chorella spp (Bhattacharya et al. 2010).
The impacts of these particles on dog whelks may be similar to those recorded from the
lugworm Arenicola marina with reduced feeding activity. The number of particles transferred
to the dog whelk was dependent on the percentage of the mussel consumed with a higher

48. MSc Thesis 2014
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48
transfer of particles observed with the higher percentage of mussel consumed similar to the
observations recorded by Farrell and Nelson (2013). The potential transfer of contaminates
with these microplastics to dog whelks may induce sexual confusion or ‘imposex’ which
describes the change of male characteristics to parasitized and unparasitized versions of
female gonads (Gibbs et al. 1987, ICES 2012).
The transfer of plastic particles has now been recorded from the mussel Mytilus edulis to both
the dog whelk Nucella lapillus and the shore crab Carcinus maenas. This provides the
potential for the transfer to other coastal marine organisms as the mussel is an ecosystem
engineer which is a favoured prey for a variety of predators including birds, starfish and
humans (Browne et al. 2008). Even though the transfer of particles in this study is relatively
small, it does indicate that a transfer between organisms is present. This may in turn show
potential for the transfer of particles from lower trophic levels to higher level predators and
even humans (Farrell and Nelson 2013).
4.4 Limitations of Current Project and Recommendations for Future Work
This current study was only able to focus on the effects of PVC on Arenicola marina over a
51 day timescale. Research has suggested that a longer exposure period to microplastics has a
greater influence on performance matrices. Furthermore, throughout the duration of the
experiment it became apparent that the PVC did not fully mix into the sediment but settled on
the surface. This in turn may have restricted the influence of PVC on the worms as they spent
the majority of the experiment on the bottom of the tanks. Future experiments may want to
use a heavier type of microplastic or introduce a new mixing method during feeding in order
to fully mix the PVC with sediment in order to get the full exposure.

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4.5 Conclusions
Findings from this study have suggested that ingested microplastics do affect coastal benthic
species around the North Wales coast which agrees with previous research. The coastal
marine environment is a large sink of microplastics as they are areas of urban water discharge
and human activity (Browne et al. 2011). The presence of these plastics in this habitat has in
turn led to the ingestion of particles by benthic invertebrates leading to a range of
toxicological effects with numerous studies being conducted (von Moos et al. 2012).
However, little research has been conducted on the transfer of these plastics from baseline
organisms to those species higher up the food chain.
The aims of this project were to analyse the effects of microplastics on faecal cast weight,
worm weight, mortality and motility for the lugworm Arenicola marina and whether a
transfer of microplastics through the food chain is present for the mussel Mytilus edulis and
the dog whelk Nucella lapillus. Results have showed that restrictions are made for several
performance matrices of the lugworm Arenicola marina but not all and that the presence of
pollutants combined with the microplastics does not always significantly affect the
performance of the worm. Further results have shown that a slight transfer of microplastics
through the food chain is present for the two model benthic species used. A longer
experimental duration may enhance these results by providing temporal data as this study was
restricted by time. This study has also shown that more research is still needed into the effects
of microplastics especially with a growing human population. Future management plans are
also needed to reduce the impacts and amount of plastics within these coastal environments
(Harrison et al. 2011).

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Wright S.L., Rowe D., Thompson R.C. and Galloway T.S. (2013a) Microplastic ingestion
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6 Appendix I
Table 10:One-Way ANOVA pairwise comparison test for faecal cast weight using the LSD test between the 6
different plastic treatments exposed to the worm Arenicola marina. * indicates the absence of pollutant.
Treatment Contrast Std. Error Sig 95% CL 95% CL
Lower Bound Upper Bound
0% vs 3%* 6.323 .920 <0.001 4.500 8.146
0% vs 3% 5.738 .920 <0.001 3.914 7.561
0% vs 0.3 4.469 .920 <0.001 2.646 6.292
0% vs0.03 1.604 .920 .084 -.219 3.427
0% vs 0.003 -3.45 .920 .709 -2.168 1.479
3%* vs 3% -586 .920 .526 -2.409 1.238
3%* vs 0.3 -1.854 .920 .046 -3.677 -.031
3%* vs 0.03 -4.719 .920 <0.001 -6.542 -2.896
3%* vs 0.003 -6.668 .920 <0.001 -8.491 -4.884
3% vs 0.3 -1.268 .920 .171 -3.092 .555
3% vs 0.03 -4.133 .920 <0.001 -5.957 -2310
3% vs 0.003 -6.082 .920 <0.001 -7.905 -4.259
0.3 vs 0.03 -2.865 .920 .002 -4.688 -1.042
0.3 vs 0.003 -4.814 .920 <0.001 -6.637 -2.990
0.03 vs 0.003 -1.949 .920 .036 .125 3.772

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Table 11: One-Way ANOVA pairwise comparison test for digging in times using the LSD test between the 6
different plastic treatments exposed to the worm Arenicola marina. * indicates the absence of pollutant
Treatment Contrast Std. Error Sig 95% CL 95% CL
Lower Bound Upper Bound
0% vs 3%* 0.0417 0.02939 0.173 -0.0201 0.1034
0% vs 3% -0.0496 0.02939 0.109 -0.1113 0.0122
0% vs 0.3 0.0162 0.02939 0.589 -0.0456 0.0779
0% vs0.03 0.0378 0.02939 0.215 -0.0204 0.0995
0% vs 0.003 0.0262 0.02939 0.384 -0.0355 0.0880
3%* vs 3% -0.0913 0.02939 0.006 -0.1530 -0.0295
3%* vs 0.3 -0.0255 0.02939 0.397 -0.0873 0.0362
3%* vs 0.03 -0.0039 0.02939 0.895 -0.0657 0.0578
3%* vs 0.003 -0.0154 0.02939 0.606 -0.0772 0.0463
3% vs 0.3 0.0657 0.02939 0.038 0.0040 0.1275
3% vs 0.03 0.0873 0.02939 0.008 0.0256 0.1491
3% vs 0.003 0.0758 0.02939 0.019 0.0141 0.1376
0.3 vs 0.03 0.0216 0.02939 0.472 -0.0402 0.0833
0.3 vs 0.003 0.0101 0.02939 0.736 -0.0517 0.0718
0.03 vs 0.003 0.0115 0.02939 0.700 -0.0502 0.0733

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Table 12: Tukey pairwise comparison test for worm weights between the 6 different plastic treatments exposed
to the worm Arenicola marina. * indicates the absence of pollutant
Treatment Contrast Std. Error Sig 95% CL 95% CL
Lower Bound Upper Bound
0% vs 3%* 1.070 .30845 0.028 0.0897 2.0503
0% vs 3% 1.170 .30845 0.014 0.1897 2.1503
0% vs 0.3 2.075 .30845 <0.001 1.0947 3.0553
0% vs0.03 1.600 .30845 0.001 0.6197 2.5803
0% vs 0.003 0.6850 .30845 0.276 -0.2953 1.6653
3%* vs 3% 0.100 .30845 0.999 -0.8803 1.0803
3%* vs 0.3 1.005 .30845 0.043 0.0247 1.9853
3%* vs 0.03 0.530 .30845 0.538 -0.4503 1.5103
3%* vs 0.003 -0.3850 .30845 0.808 -1.3653 0.5953
3% vs 0.3 0.9050 .30845 0.080 -0.0753 1.8853
3% vs 0.03 0.430 .30845 0.730 -0.5503 1.4103
3% vs 0.003 -0.4850 .30845 0.625 -1.4653 0.4953
0.3 vs 0.03 -0.4750 .30845 0.645 -1.4553 0.5053
0.3 vs 0.003 -1.390 .30845 0.003 -2.3703 -0.4097
0.03 vs 0.003 -0.915 .30845 0.075 -1.8953 0.0653

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7 Appendix II
Video 1: The sampling of the faecal casts produced by the lug worm Arenicola marina using
a 60ml syringe.
Video 2: The burrowing of the lug worm Arenicola marina as an example of the digging in
performance matrix recorded.
Both videos are provided on the CD attached at the end of this document.