Final project 2015 MP

Final Year Report 2015 Darren Machen

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Microplastic Ingestion by Mytilus edulis Cultivated for Human Consumption

Final Year Report

Darren Machen 12021396


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Contents Page

Title Page Number

Figures, Images and Tables 3 1. Abstract 4 2. Introduction 5 3. Method 10 4. Results 13

4.1. S.E.M Findings 13 4.2. Plastic Abundance 14 4.3. Table of Abundance results 14

5. Statistical Analysis 15 5.1. Homogeneity of Variance 15 5.2. Normal Distributed Residual Values 16

5.3. Is There a Higher Amount of MP in a Sample Based on

the Location? 18 5.4. Plastic Particles Found 19 5.5. Size of Particles 20 5.6. Controlled Blanks 22 5.7. Results Summary 23

6. Discussion 24 6.1. Initial Problems 24 6.2. Identification 26 6.3. Chemical Properties 26 6.4. Size 28 6.5. Depuration 30 6.6. Combined Sewage Overflow 32

6.7. Mussel Health 36 6.8. Human Health 38 6.9. Conclusion 40

7. Acknowledgements 41 8. References 42


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Figures, Images and Tables

Figure Page Number

1. Particles Per Gram Equation 14 2. Test of Homogeneity of Variance 15 3. Histogram of Residual Data 16 4. Histogram of Residual Data 16 5. Normality Plot 16 6. Normality Plot 16 7. ANOVA 18 8. Graph of Particles 19 9. Histogram of Particles 20 10. Statistical Read Out for Size 20 11. Particles in Size Groups 20 12. Size in Groups Report

Image Page Number

1. Spectrum From S.E.M 2015 13 2. Plastic Particle From S.E.M 2015 13 3. Spectrum From S.E.M 2015 27 4. Mass of Particles From S.E.M 2015 29 5. CSO Map of Brixham Area 2015 33 6. CSO Map of Stirling Area 2015 34

Table Page Number

1. Plastic Abundance 14


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1. Abstract

Plastic is now ubiquitously present in the world’s oceans, seas and rivers with pieces

of plastic becoming smaller before finally becoming microplastics (MP). The aim of

this project was to find out if there was MP present in the mussel species Mytilus

edulis and if there was a possibility that this was entering the human food chain. It

would also look to establish if there this would be damaging mussel or human health.

The presence of MP in the marine environment is of particular concern because of

this interaction with and ingestion by marine biota. Mussels that had been grown for

human consumption were purchased from supermarkets and restaurants that had be

cultivated in four different UK locations. The mussel flesh was removed from the

shell, weighed and recorded before being cut into small pieces and digested in nitric

acid. After digestion, the mixture was filtered so the remaining particles could be

examined via scanning electron microscope. All four sample groups showed strong

evidence of plastic particles based on visual and chemical analysis. The smallest

recorded particle was 54.40 µm and the largest was 2140 µm. The mean particle

size was 439.81 µm ± 383.99 µm, falling into the expected limits of what is widely

recognised as MP. The evidence from this report has confirmed the findings of other

studies showing ubiquitously present MP within all of the sample groups. This report

has proven with confidence that the MP is presenting degree of risk on some level,

although further investigation would be beneficial. Increasing the size of the sample

group and using some more in-depth analytical tools would strengthen the work

shown but in no way takes away from what has been achieved as a starting point.


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2. Introduction

Plastics are synthetic organic polymers, and though they have only been produced

for just over a century (Derraik, 2002), their versatility has led to a dramatic increase

in usage throughout the world since the development of the first modern plastic

‘Bakelite’ in 1907 (Cole et al. 2011). The second half of the 20th century has seen

plastic become one of the most universally used, multipurpose materials in the global

economy (Plastics Europe, 2013). Since its introduction, the plastics industry has

experienced continual growth through the last 50 years. Year on year industry

growth of 8.7% (Plastics Europe, 2013) has cemented plastic in the lives of

consumers with plastic in one form or another being present in most products. First

reports in the 1970’s of plastic marine debris drew minimal attention from the

scientific community (Andrady, 2011), however, It has been estimated that 10% of

plastic produced globally now enters the oceans (Cole et al. 2013) while recovery of

material remains low at around 5% (Moore, 2008); this could have drastic

consequences to the variety of marine organisms that inhabit them. Global plastic

production reached 288 million tonnes in 2012, which was a 2.8% increase on 2011

(Plastics Europe, 2013). Based on the 2012 figure from Plastics Europe and the

estimation from Mathew Coles et al. (2013), there could be 28 million 800 thousand

tonnes of plastic debris entering the ocean each year. The five highest production

plastics, which account for approximately 90% of the total demand, are polyethylene

(PE), polypropylene (PP), polyvinylchloride (PVC), polystyrene (PS) and

polyethylene terephthalate (PET) (Zarfl and Matthies, 2010) suggesting that these

varieties will make up the majority of plastic entering the water cycle.


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Plastic is now ubiquitously present in the world’s oceans (Cauwenberghe and

Janssen, 2014), seas and rivers. The impact of large plastic debris, known as

‘macroplastic’, has long been studied due to their aesthetic and economic

repercussions in the tourist industry and the injury, death or ingestion by marine

birds and mammals (Cole et al. 2011). The physical characteristics of plastics show

a high resistance to ageing and minimal biodegradation (Moore, 2008). In fact,

plastic can take decades if not centuries to fully degrade (Cole et al. 2014); meaning

the plastic that is affecting marine biota now could be some of the very first plastic

ever produced. When macroplastics are exposed to UVB radiation in sunlight, the

oxidative properties of the atmosphere and the hydrolytic properties of seawater

(Moore, 2008), plastic polymers become brittle and start to fracture or break apart.

This mechanism of degradation continues until the pieces of plastic become smaller

and smaller (Moore, 2008), finally becoming microplastics (MP). This breakdown

over time defines these particles as secondary microplastics (Cole et al. 2011).

Primary microplastics are made to be of small size and are present in facial-

cleansers and cosmetics (Cole et al. 2011). The impact of MP on marine organisms

will depend on where they are located in the water column (Cauwenberghe and

Janssen, 2014). Typically, high density MP, such as the primary MP in cleansers, will

sink (Cauwenberghe and Janssen, 2014) and lower density particles, such as

secondary MP from degradation, will float (Cauwenberghe and Janssen, 2014) or be

suspended in seawater.


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For the purpose of this paper, the term microplastics (MP) will used to describe

plastic particles that have been subject to degradation by exposure to UVB radiation,

the atmosphere and seawater. The term microplastic (MP) is defined differently by

various researchers (Andrady, 2011). Generally particles that are <5mm are

categorised as MP (Moos, Burkhardt-Holm and Köhler, 2012); as particles of plastics

ranging in dimensions from a few µm to 500 µm (5mm) are commonly present

(Andrady, 2011). MP are barely visible to the naked eye, passing through a 500 µm

sieve but retained by a 67 µm sieve (0.06– 0.5mm) (Andrady, 2011) although plastic

particles smaller than this can be found. Due to their small dimensions, MP have a

similar size to planktonic organisms and other suspended particles (Cauwenberghe

and Janssen, 2014) that can be mistaken for food sources by filter feeding

organisms of a higher trophic level. This makes MP available to an array of marine

invertebrates that would otherwise not be affected from not feeding on larger pieces

of marine debris (Cauwenberghe and Janssen, 2014).

Seawater already contains numerous natural micro- and nano- particles, most of

them <100 nm in size (Andrady, 2011) that have no ill effect. However, MP particles

have the potential to do damage as they differ in nature to other natural particles of

the same or similar dimensions. Flow or run off from land can contain both biogenic

organic matter such as high molecular weight aliphatic hydrocarbons and

anthropogenic pollutants including polychlorinated biphenyls (PCBs). (Kanzari et al.

2014) which are persistent organic pollutants (POP) that can be absorbed by MP.

Ingestion of MP debris has been demonstrated for a range of marine organisms,

including Mytilus edulis (Bakir, Rowland and Thompson, 2012) in laboratory settings.

http://pubs.acs.org/action/doSearch?ContribStored=Burkhardt-Holm%2C+P

http://pubs.acs.org/action/doSearch?ContribStored=K%C3%B6hler%2C+A


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Similarly, other studies have been conducted to understand what the effect of this

ingestion may have on the feeding organism.

Studies have shown the MP adsorb PCBs from surrounding seawater, bound to the

plastic matrix POPs escape rapid degradation and are subject to long range

transportation (Zarlf and Matthies, 2010). There is evidence that some POPs show a

preference to sorption on plastic polymers, showing different affinity according to

polymer type (Bakir, Rowland and Thompson, 2012). When ingested by organisms

there is a possibility that this becomes a biomagnification route for organic chemicals

adsorbed to or contained within the plastics (Zarfl and Matthies, 2010). The

presence of MP in the marine environment is of particular concern because of this

interaction with and ingestion by marine biota (Hidalgo-Ruz et al. 2012). In a

population of Great Shearwaters (Puffinus gravis) the concentration of PCBs was

show to be directly correlated to the amount of plastic that had been consumed (Zarfl

and Matthies, 2010). While MP has been reported in a variety of marine organism,

including M. edulis, the extent of the toxicological hazard to these organisms are not

well known (Hidalgo-Ruz et al. 2012).


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M. edulis is commonly grown in the United Kingdom and Europe for human

consumption meaning that there is a possibility for the transportation of MPs and

POPs to the human food chain. The concentration of POPs may only be a small

amount for a large organism but could be more problematic for M. edulis. The aim of

this study is to discover if mussels cultivated for human consumption contain MP and

how these have come to be ingested. It will look at what environmental,

topographical and biological factors that have attributed to the amount of MP within

the samples and aim to investigate possible damage.


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3. Method

Mussels that had been grown for human consumption were purchased from

supermarkets and restaurants to give a broad range of locations in the United

Kingdom. The four UK locations were the South West coast (Brixham), Ireland,

Stirling and the Shetland isles. Due to the mussels being ready to enter the food

chain, they had likely already been subjected to a depuration period that cleared

their guts of any effluent or possible plastic particles. 5 replicates would be made for

each site; this included 1 blank to ensure what was being found was from inside the

mussel flesh and not outside contamination. The mussel flesh was removed from the

shell, weighed and recorded before being cut into small pieces. A previous batch

test, carried out by myself, had given an ideal mussel weight of between 9 and 12

grams for 20ml of acid. To eliminate the risk of contamination each instrument was

cleaned with deionised water before moving on to the next sample. The resulting

mussel flesh was transferred to warming tubes and capped with loose fitting foil. The

foil would aid the reflux when acid was added by causing condensation of the fumes.

To this, 20ml of 69% nitric acid (HNO3) was added and left for 24 hours to steep in

the fume cupboard. The acid flesh mixture was then slowly heated up to 80˚C and

left for three hours. An adapted version of Classons (2013) method was used to yield

similar digested results. To be sure to achieve the predicted 80% digestion

efficiency, the two hours began after refluxing. Once this was completed the contents

of the warming tube was diluted with 250ml of deionised water heated to 80˚C in a

500ml conical flask. The warming tube was then flushed with deionised water to

remove any residual material (this waste water was added to the beaker). After a


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short cooling period the weakened acid mix was filtered. Filtration was carried out

using a 300ml vaccuum flask, Buchner funnel and 0.65 µm cellulose nitrate

membrane filters (47mm with a capture diameter of 37mm) before flushing the 500ml

beaker. The filters were left to air dry and stored in petriei dishes before be examined

under a Scanning Electron Microscope.

For the S.E.M a Phillips XL30 ESEM was used. The height of the detector was

adjusted to give an acquisition rate of 2 KCPS. The chamber pressure was 0.5 Torr

with an accelerator voltage of 20,000 KV working with the back scatter electron

detector and a spot size of six. Five pencil spots at points relating to 12, 3, 6, and 9,

and in the centre were made on the filter to be used as a rough guide to the centre;

this would show in the S.E.M how close the machine was to centre from the

prediction eliminating any problem of being placed too far off centre in the S.E.M.

From the centre, at 100x magnification each frame was counted out towards the

edge equalling eighteen frames (frame size 133mm x 0.91 mm). On every third

frame, any fibrous material was recorded. This was decided as a fair and unbiased

method against a random frame selection approach. Each suspected plastic fibre

was measured and had a picture taken for reference later (Fig.1). From prior

research there was a good indication of what MP would look like in various forms

allowing assumptions to be made before measurements were taken. This was

carried out for each of the twenty samples, including their blanks, and lab coats were

worn all times to protect the samples from contamination of clothing fibres. Once the

samples had all been finished they were analysed using statistical software.

Differences between location were analysed using a one-way ANOVA, the number


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of particles were log+1 transformed to ensure that assumptions of homogeneity of

variance and normally-distributed residual were met.


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4. Results

4.1. Scanning Electron Microscope (S.E.M) Findings

When looking at the particles under the S.E.M, fibrous strands were discovered that

varied in length. Each of the particles had a very strong peak of carbon (C), with a

smaller peak of Oxygen (O), this suggested it was of organic origin and suggested

that we were looking at a hydrocarbon that was plastic (Image 1). There we also

other peaks within the spectrum, namely Silica (Si) and Chlorine (Cl). The silica was

most likely from the back ground of the filter. In the picture the pieces that glow bright

white are silica and these are organisms that use silica and calcium in their body

construction. The chorine was most likely in the MP particles themselves. This could

suggest that the plastic was polyvinyl chloride (PVC). Image.2 is an example of what

was being found when looking at the S.E.M.

Image 1. Spectrum from S.E.M 2015 Image 2. Picture of Plastic particle from S.E.M 2015


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4.2. Plastic Abundance

To work out the abundance of particles per filter and per gram of mussel weight, the

following equation was applied;

𝐴 = 𝜋𝑟2

𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑓𝑖𝑙𝑡𝑒𝑟 37𝑚𝑚 (3.7𝑐𝑚)

𝜋 × 18.5² = 1075 𝑚𝑚²

1075

0.91 = 1181.31 ( 𝐹𝑟𝑎𝑚𝑒𝑠 𝑝𝑒𝑟 𝐹𝑖𝑙𝑡𝑒𝑟)

1181.32 × 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑁𝑜. 𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 = 𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑝𝑒𝑟 𝐹𝑖𝑙𝑡𝑒𝑟

𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑝𝑒𝑟 𝐹𝑖𝑙𝑡𝑒𝑟

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑀𝑢𝑠𝑠𝑒𝑙= 𝑃𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑝𝑒𝑟 𝐺𝑟𝑎𝑚

4.3. Table of Abundance Results

The table clearly highlights Brixham as having the highest proportion of plastic

particles per gram of mussels at 15.68 ppg. Second highest is Shetland with 8.95

ppg, followed by Ireland with 4.64 ppg and Stirling with 3.62 ppg.

Key

PPF: Particles per filter PPAW: Particles per average weight PPG: Particle per gram

ST: Stirling BR: Brixham SH: Shetland IR: Ireland

Table 1. Particle abundance in differing values.

Figure 1. Particles per gram equation.


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5. Statistical Analysis

A one-way ANOVA can be used to discover is there is a significant difference

between the particles found at each site. Before this can be completed, the data

must be tested for homogeneity of variance and normally distributed residual values.

5.1. Homogeneity of Variance

Both the Bartlett’s and Levene’s test in fig.2. show non-significance (p> 0.05),

meaning there is homogeneity of variance. The assumption of homogeneity of

variance is that the variance within each sample is equal. This indicates that the data

has equal variance despite the indication of excess zero data points.

ST

SH

IR

BR

20151050

SIT

E

95% Bonferroni Confidence Intervals for StDevs

Test Statistic 6.10

P-Value 0.107

Test Statistic 1.09

P-Value 0.380

Bartlett's Test

Levene's Test

Test of Homogeneity (Original Data)

Figure 2. Test of homogeneity of variance.


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5.2. Normally Distributed Residual Values

86420-2-4-6

6

5

4

3

2

1

0

Residual (Original Data)

Fre

qu

en

cy

Mean 2.220446E-17

StDev 2.851

N 20

Histogram(response is PARTICLES)

1.51.00.50.0-0.5-1.0-1.5

6

5

4

3

2

1

0

ResidualFr

eq

ue

ncy

Mean 6.661338E-17

StDev 0.7453

N 20

Histogram(response is Log +1)

1050-5

99

95

90

80

70

60

50

40

30

20

10

5

1

Residual

Pe

rce

nt

Normal Probability Plot(response is PARTICLES)

210-1-2

99

95

90

80

70

60

50

40

30

20

10

5

1

Residual

Pe

rce

nt

Normal Probability Plot(response is Log +1)

For an ANOVA to be a true representation of the data, the residual values need to

show normal distribution. Figure 3 shows a left skew with a distinct tail running off to

the right. The residual values of the original data are not normally distributed. This

can also be seen in the normal probability plot (fig.5); the two tails are clearly defined

at each end of the plot. Figure 4 shows distribution around zero with a good degree

of spread either side. To achieve this, using one-way ANOVA, the numbers of

particles were log+1 transformed to ensure that the need for normally distributed

Figure 5 and Figure 6. Normal probability plot comparison (Original and Log+1 residuals)

Figure 3 and Figure 4. Histogram of Residual Data comparison (Original and Log+1 residuals)


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residual values were met. In the log+1 normality probably plot (fig.5), the tail from the

top right has been significantly reduced and the values have a good correlation.


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5.3. Is there a higher amount of MP for each sample based on their

location?

Proposed hypothesis:

Н1 : There is a significant difference in particles based on location

Null hypothesis:

Н0 : There is not a significant difference in particles based on location

ANOVA

P_LOG_1

Sum of

Squares

df Mean

Square

F Sig.

Between

Groups 5.145 3 1.715 2.600 .088

Within

Groups 10.554 16 .660

Total 15.699 19

There was not a statistically significant difference between groups as determined by

one-way ANOVA (F (3, 16) = 2.600, 𝜌 = .088).

The 𝜌- value .088, so as 𝜌 = > 0.05 there is not enough evidence to support the

proposed hypothesis. There is a not statistically significant difference between the

particles found based on their location.

Figure 7. ANOVA statistical read out.


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5.4. Plastic particles found

Despite the findings of the One-way ANOVA, visually Brixham shows a higher

concentration of plastic particles. The statistics will test each individual value against

the other, showing that there is no significant difference. This is not to say that the

statistics have been incorrectly managed, it is more likely due to the small sample

size and lack of replicates to confidently shows a statistical difference. Fig 8 shows

all of the particles from one mussel group combined and the higher amount for

Brixham.

9

35

20

9

0

5

10

15

20

25

30

35

40

Stirling Brixham Shetland Ireland

Number of

Particles

Cultivation Site

Figure 8. Graph displaying grouped particles.


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5.5. Size of particles

The data from the size of particles has a distinct skew to the left hand side (fig.9)

suggesting that the majority of the articles were of a smaller size. The smallest

recorded particle was 54.40 µm and the largest was 2140 µm. The mean particle

size was 439.81 µm ± 383.99 µm.

Figure 9. Histogram of particle size in µm.

SPSS Statistics 2015

Figure 10. Statistical read out based on size in µm.

SPSS Statistics 2015

Figure 11. Particles in size groups.

Figure 12. Size in groups report.


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By separating the particles into ‘Bins’ it is easier to see the distribution. 30 of the 80

particles recorded were between 51-259 µm, whilst only 15 particles were close to or

larger than 1 mm in size. There was double the amount of smaller range particles

than higher range.


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5.6. Controlled Blanks

To ensure that the plastic that was being discover was not from an outside

contamination source such as clothing, lab coats were worn at all times. However,

on 1 of the blanks a single fiber was found that look very similar to the others; this

sample was part of the Brixham group. There were no other particles viewed on any

of the other blanks during this project


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5.7. Results Summary

The results have proven the existence of plastic particles within the mussels from the

sampled areas, using a combination of statistical and visual analysis. Brixham has

clearly demonstrated a higher amount of plastic in the mussel flesh, but all of the

samples yield large amounts of plastic. The use of SPSS did not show a statistical

difference between all the samples, but this is likely due to the small data set. If this

were to be carried out again more replicates of similar values would probably show a

statistical significance but that would have to be proven. All of the methods of

analysis are viable ways of counting plastic abundance, size and particles per gram.

This indicates that the results can be viewed with a strong degree of confidence to

their accuracy. The fact that 1 particle was viewed on a blank does not disrupt this

confidence in the results. Each filter viewed multiple particles outside the sampling

method that were not counted. This one particle was the only one viewed on the

filter, out of all the blanks, whether it was in the samples frame or not. This 1 particle

may have contaminated the filter during storage although the upmost care was used

throughout to stop this from happening.


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6. Discussion

6.1. Initial problems

It took approximately one hour for the reflux mechanism of the digestion to start in

comparison to the predicted two hour overall time scale. This meant during one of

the trail runs the heat was increased to speed up the digestion. However, this was a

mistake as it caused the samples to boil over and the samples were then not

salvageable and discarded. Due to incomplete digestion, some of the samples were

unable to be used for S.E.M analysis. During the initial set up, another method of

filtration was added to the final step. The use of glass wool was trialled to see if it

was able to filter out any of the lager fatty deposits in the less digested samples.

After these had be looked at under the S.E.M it was found to be unsuccessful as it

caused large amounts of glass fibres to be deposited on the filter paper and was in

fact worse the original. Because on some samples fatty deposits hindered the view

of the electron microscope these were left out. This resulted in 5 usable samples

from each site. This was a frustrating outcome as there was a possibility to make the

project more accurate with a larger data set. However, what has been shown is a

true representation of the samples that were used. More than 20 filters that were

used in the final analysis were made but it was decided to leave these out and keep

five per area. The mussels from Stirling and Brixham were the 2 that had the most

trouble in digestion. This is interesting as it would have been beneficial to see more

samples for these areas as they are the highest and lowest values recorder. To

analyse the approximate size of the fibres, all of the 80 that had been found were


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measured. This would give a better understanding of the size of the particles that

were discovered. There are quite a few zero data points, this made statistical

analysis more of a challenge. This is not to say that the sample contained no plastic,

this is because the method of sampling did not allow the plastic to be recorded. This

was deemed a fair way of sampling the filter and gave the best compromise between

usable results within the time available.


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6.2. Identification

6.3. Chemical properties

The characterisation of MP uses morphological descriptions, size, shape or colour,

with the most reliable technique being infrared spectroscopy which reveals the

chemical composition (Eerkes-Medrano, Thompson and Aldridge, 2015). However,

with the knowledge that plastic is not degraded by NHO3 during the digestion

process, it can be said with a good degree of certainty that what is left behind is

plastic. Using the S.E.M, the plastic can be scanned to reveal the chemical

composition. This is not as sensitive as infrared spectroscopy but it still shows what

elements are within that field of view. The basic structure of plastics is constructed

from monomer units by chemical reaction (Klein, 2011). The monomer units are

organic carbon-based molecules. Besides Carbon (C) and Hydrogen (H) atoms as

main components, plastics can also contain elements like Oxygen (O), Sulphur (S)

or Chlorine (Cl) in the monomer unit (Klein, 2011).


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In image 3, it can be seen that there is a very strong C peak with an O peak of

around 1 quarter of the size. There are also peaks within the Cl and S grouping. This

chemically supports what is known to be contained within plastic polymers and

shows with a good degree of certainty that the particles left after digestion were of

plastic origin. Although it can be confidentially confirmed that what was found is

plastic, it is harder to confirm what type of plastic is contained within the samples.

Image 3. Spectrum from S.E.M 2015


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6.4. Size

The term Microplastic (MP) was first used in 2004 and is a classification based on

the size of the particle (Hidalgo-Ruz et al. 2012). Generally particles that are <5mm

are categorised as MP (Moos, Burkhardt-Holm and Köhler, 2012); as particles of

plastics ranging in dimensions from a few µm to 500 µm (5mm) are commonly

present (Andrady, 2011). The smallest particle found was 54.40 µm and the largest

2140 µm. The largest of the particles was 2.14 mm, which is well within the specified

boundary for a MP. From a size point of view it can be confidently confirmed that

what was found is of MP origin. As M. edulis is a selective filter feeder, laboratory

test have been conducted to find a suggested limit for the size of particle retention

(10-30µm) (Cauwenberghe et al. 2015). The recorded sizes found in this research

have found particles that far exceed the size of this suggested limit. To select

particles of appropriate size, large particles elicit secretion of mucus; this mucus

entangles particles so that they can be excreted via pseudofaeces (Riisgard, Egede

and Saavedra, 2011).




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In image 4, a large mass of tangled fibres is shown from a sample of digested M.

edulis. The higher amount of larges particles recorder may be proof of pseudofaeces

in action and show that these tangled particles are unable to be readily excreted

though this normal process. Microplastic especially in fiber form can cause problems

to the organism that ingests them as they cause blockages in the intestinal tract and

undergo accumulation (Mathalon and Hill, 2014).Trapped inside the intestinal tract of

M. edulis, these particles may be able to untangle during the agitation involved with

digestion. This would explain the high amount of large particles outside of the

suggested limit in laboratory research or the higher amount despite depuration.

Image 4. Mass of Particles S.E.M 2015


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6.5. Depuration

Depuration is the process applied to M. edulis that involves them being placed in

clean sterilised sea water and allowed to continue filtration activities for a set period

of time (FAO, 2010). This ensures the risk of illness when eaten is lowered due to

lower concentrations of faecal contaminants contained within the bivalve (FAO,

2010). This process would also allow MP in M. edulis to be excreted. The decision to

depurate mussels is based on water cleanliness clarification carried out by sanitation

surveys centred on E. coli. If waters are of A grade quality (<230 E.coli/100g),

mussels can be directly consumed without the need for depuration (DEFRA, 2013); a

water rating of B or below indicates mussels have to be depurated. Whether or not a

sample in subjected to the depuration process will have an effect on the recorded

amount of MP. The shellfish waters Directive (2006/113/EEC) ensures that member

states designate water that is in need protection of improvement to support shellfish

growth directly intended from human consumption (HMG, 2012). It recognises that

protecting human health cannot be guaranteed by protecting water quality alone. For

this reason faecal coliforms standards are set for mussel flesh (HMG, 2012),

although as mussels are a bio-indicator, this level could be reflection of water quality.

Based on this idea, the lower recorded values for Ireland and Stirling may suggest

that the water quality is below B grade. The need for depuration has lowered the MP

content that is associated with poorer water quality. Similarly, mussels from Shetland

and Brixham showed the highest recorded levels of MP suggesting that the water

quality in this area may be high enough to warrant consumption without depuration

(A grade). After investigation, it was discovered that mussels that are grown in the


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region of Brixham go through a 42 hour depuration process (Brixham Sea Farms,

2014).The fact that mussels need to have this depuration period in Brixham reveals

that the water quality in that area must be of B grade or lower suggesting that the

high amount found may be due to other underlying factors. In Cauwenberghe’s et al

2014 paper, mussels used for lab based test are depurated. This would give a better

representation of the amount that a person may ingest whilst consuming mussels as

it simulates production practice. Although deputation is conducted for other means

(E.coli risk), the method is proven to result in a safer product for consumption,

however, it inadvertently reduces the concentration of MP. Even with depuration, MP

are present, this is due to ingested MP having the potential to be taken by epithelial

cell in the intestinal tract, even translocating into the circulatory system of the

mussels (Cauwenberghe and Janssen, 2014) and as mussels are eaten whole,

consumption will inevitability be linked to MP ingestion. Furthermore, there may be a

possibility that MPs are forming clumps within the intestines and cannot be readily

excreted as discussed in section 6.4.


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6.6. Combined Sewage Overflow

During periods of heavy rainfall, sewers can become overwhelmed by the volume of

water leading to discharge to the ocean via combined sewer overflow (CSO) (Kay,

2008). This water contains human waste (black water) and water from household

use such as a washing machine (grey water). These overflow events can lead to

bivalves such as M. edulis concentrating and retaining human pathogens (Kay,

2008) meaning they need depuration as previously discussed (section 6.5). At

present, the health effects attributed to the ingestion of and translocation of bacteria

via mussels is well documented; this is why the Shellfish Waters Directive

(2006/113/EC) was created. However, a study conducted by Browne, et al. (2011)

has found that there may be other less noticeable contaminants within CSO. It was

found that an important source of MP was found in sediments near sewage water

outlets (Browne et al. 2011). Further test revealed that a single garment of clothing

can produce >1900 fibres per wash (Browne et al. 2011). This water is then drained

into the combined sewer system and has the possibility to make it in to the marine

environment and suggests that a large proportion of fibrous material is a

consequence of washing clothing (Browne et al. 2011). Furthermore, a quarter of all

sewage sludge was dumped at sea, until a ban in 1998; this would have meant any

MP filtered from waste water would have entered the ocean (DEFRA, 2002). These

findings would explain the visual nature of what was viewed under the S.E.M. All of

the MP particles discovered were fibrous and could very well be clothing fibres,

although the exact origin cannot be confidently confirmed at this stage.


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The image above shows a high concentration of CSOs in the Brixham area, a total of

10 (SAS, 2014) when in fact there are around 19. Mussels are allowed to mature for

18-24 months (Seafish, 2011) which gives them a chance of having multiple

interactions with high rainfall events that lead to CSOs being used. Following the

introduction of the EU Bathing Water Regulations in the early 1990’s, schemes were

recommended to improve bathing waters (Torbay, 2010) that would aim to limit the

frequency of polluting events. The consent was set at 3 spills per bathing season

(Torbay, 2010) signalling that mussels grown in the Torbay area could, at the very

least, be subjected to 6 spills for a 24 months growing period. Furthermore, this does

not include the higher possibility of extreme weather events that happen at other

Image 5. CSO Map of Brixham Area. (SAS, 2015)


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times of the year, leading to the belief that this is a very minimal estimation. This is

reflected in the amount of MP found in the samples from Brixham.

The mussels from Brixam had 15.68 ppg, whilst Stirling had only 3.62 ppg, Brixham

showed around 5 times more MP particles per gram. In image 6, it can be seen that

there is a dramatic difference in the amount of CSOs for the Stirling area. Although it

is known that the Brixham image 5 is an underestimation, if used as a visual tool it

can still represent the clear difference. If you took the 4 CSOs from image 6 and

multiplied it by 5 you would have 20 CSOs, which is close to the real amount.

Although this is just a rough estimation, it is highly likely based on this evidence that

the MP values of these 2 extremes are linked to the concentration of CSOs in the

area of mussels sampled.

Image 6. CSO Map of Stirling Area. (SAS, 2015)


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This problem is only exacerbated by the practice of using sewage sludge that has

been treated as a soil improver. Sewage sludge is increasing added to soil as it is

deemed be economically or environmentally advantageous and is set to increase to

13 million tonnes by 2020 (Jones, 2014). There have been accounts of soils that

have had soil sludge spread being inadvertently contaminated with plastic as a result

of this practice (Thompson, 2009). It is perfectly plausible that like nutrient runoff,

sewage sludge used on land may leech MP that would otherwise have been

captured by the filtration process, allowing it to reach water courses and end up in

the sea.


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6.7. Mussel health

M. edulis have been used extensively as an indicator species for monitoring the

uptake and bioaccumulation of hydrophobic contaminants in the marine

environment, including polycyclic aromatic hydrocarbons (PAHs), chlor-obiphenyls

(CBs) and polybrominated diphenyl ethers (PBDEs) (Webster, et al. 2009). PAHs

and organochlorine pesticides (OCs) are present on the marine environment in the

form of complex mixtures. The ecotoxicological nature of contamination interactions

is poorly understood with most scientific studies formed from single contaminate

exposures (Richardson, et al. 2008), unlike real world interactions where organisms

are exposed to many at one time. Mato et al. (2001) found 100,00 - 1 million time

higher concentration of Polychlorinated byphenyls (PCBs) on polypropylene pieces

compared to the surrounding seawater (Mato, et al. 2001) meaning MP could

provide a route from transport into exposed organisms (Mathalon, et al. 2014). One

study has quantified concentrations from MP particles found from beaches showing

reported values of PAH = 39-1200 ng/g, PCB = 27-980 ng/g and DDT = 22-980 ng/g

(Andrady, 2011). As well as exposure to these potentially harmful chemicals, a

study on the effects of nanopolystyrene (30nm) on the feeding of M. edulis showed

significant increase in pseudofaeces production and decreased filter feeding activity,

reducing energy acquisition leading to possible starvation (Cauwenberghe, et al.

2015). In image 4 there is a mass of fibres which seems to show pseudofaeces and

possibly this natural process causing blockages within the intestines. These

blockages would not allow the natural excretion of larger MP particles and may lead

to excessive leaching of POPs or starvation. Either of these outcomes could have


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detrimental effect to the health of M. edulis or their reproductive activity. However, as

it cannot be confidentially confirmed what plastic has been found during this study, it

is hard to speculate as to which chemicals would be being ingested. A study by Bakir

et al. (2012), showed specific absorption behaviours of polymers PE and PVC, with

PE (Phenanthrene) and DDT (Bakir, et al. 2012) whilst Webster, et al. (2009)

showed interactions with PHs, CBs and PBDEs. Although Webster, et al. (2009) was

not showing this based on MP, it provides evidence that these chemicals are being

ingested purely through normal filtration. This means that MP with higher

concentrations of POPs are putting a further strain on these organisms.


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6.8. Human health

From all the evidence of the previous sections it can be said that there is a high

possibility that MPs are entering the human food chain and that there is a high

chance these will contain POPs in one form or another. The degree to which a

person is exposed to these chemicals will have influence the damage that is caused.

The most persistent PCB congeners (PCB 153/158) have a half-life of around a year

in the human blood, whereas lower chlorinated PCBs can be transformed within

days (Vetter, 2009). Despite any difference in residence time within the body,

contaminants may have effect on fetus health as they are more venerable to

chemical exposure than adults (Vizcaino, et al., 2013). Furthermore, POPs have

been linked to cardiovascular disease (CVD) or cancer and individuals with these

diseases had significantly higher concentrations of PCBs than that of healthy

individuals (Ljunggren et al. 2014). It is hard to fully understand the concentration of

POPs entering the food chain and this would ultimately depend on how much a

person would eat. Areas of the world where seafood is a primary food source would

expect to ingest higher concentrations if the main food source was contaminated

with POPs. To further the problems of POPs, there are also know chemicals within

plastic that are harmful to human health and could have potential risks (Thompson et

al. 2009). Chemicals within plastic and some POPs are classified as endocrine

disruptors meaning they interfere with and mimic hormones within the body and can

cause reproductive issues (Thompson et al. 2009). Whilst these chemicals could be

having a small effect on a large organism like a human there could be even more

detrimental effects to a small organism such as M. edulis. It may not have a large


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impact on the mussels that are cultivated but could pose a threat to natural beds that

will reproduce independently.

6.9. Conclusion


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MP are said to be ubiquitous in the marine environment and the fits very well with the

finding of this study. Every sample contained MP in amounts large and small.

Despite the fact that it cannot be confirmed what plastic is being ingested, there is

overwhelming evidence that suggests POPs are entering the food chain and the

probability that this is aided by MP ingestion is high. However, the use of other

analytical tool would give a better understanding and what type of plastic had been

found and therefore be able to see if it was likely to contain POPs. The size of the

particles that were found were in the right region despite some being larger or

smaller. Also the chemical scan of the particles showed the elements you would

expect with plastic particles. The background research and the finding in this report

show excellent similarities to support finding MP. One area that has not been fully

explored by wider literature but argues a strong case is CSOs. The evidence put

forward that shows there is a likely hood of the pieces of plastic may be coming from

CSO events looks to be the strongest explanation for the high amount for in the

sample in a combination with blockages in the intestinal tract. This would explain

how depuration could still yield high amounts of plastic. The efforts expressed during

this study have made an excellent start on a somewhat under studied field example

area of MP problem. This area would certainly benefit from a lengthy study in the

future that would allow other avenues to be explored in more detail.

7. Acknowledgments


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Dr Mark Steer for his continued support throughout the project. Paul Anstey for the

part he played in development of the method, his efforts in digestion and data

gathering.

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Final project 2015 MP

Documents