Assessment of the Sphyraena viridensis Food Chain Including Surface Water, Boops boops and Trachurus mediterraneus. Case Study of Samos Island in the Eastern Aegean Sea, Greece

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RESEARCH REPORT

Microplastic Presence in the Food Chain of the Yellowmouth Barracuda

(Sphyraena viridensis)

Assessment of the Sphyraena viridensis Food Chain Including Surface Water, Boops boops and Trachurus mediterraneus. Case Study of

Samos Island in the Eastern Aegean Sea, Greece

Sophie Mentzel 25.07.2015

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Research Report

Microplastic Presence in the Food Chain of the Yellowmouth Barracuda

(Sphyraena viridensis)

Assessment of the Sphyraena viridensis Food Chain Including Surface Water, Boops boops and Trachurus mediterraneus.

Case Study of Samos Island in the Eastern Aegean Sea, Greece

S. Mentzel (61516)

HZ University of Applied Sciences M. Michels

Vlissingen, The Netherlands Semester 2, Year 2014/2015

July 2015 Final

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Summary

The main focus of this research study was to investigate the persistence of

microplastic fibres in marine ecosystems in order to do this the food chain of the yellowmouth barracuda (Sphyraena viridensis) was analysed. The location of the research was around the island of Samos in the Eastern Aegean Sea. An analysis was carried out of every trophic level and each was represented by a species within this food chain. The chosen species were zooplankton/ surface water as the first trophic level, Boops boops as the second and Trachurus mediterraneus as the third.

The fish samples were caught by local fisherman who also delivered needed information such as location and catching method. In order to assess the presence of microplastic fibres in a species the stomach contents were analysed and surface water samples were taken.

In all investigated samples fibres were present and some species such as Boops boops, Trachurus mediterraneus and Sphyraena viridensis showed correlation between weight or length and the amount of fibres. Furthermore, microplastic fibres were present in the surface water. The threat resulting from these microplastic is rather high. It can be assumed that there is a threat for humans and environments by possible clogging or transportation of fibres to surrounding tissue.

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Content

1 Introduction ... 1

1.1 Goal and Research Question ... 2

1.2 Objective of Study ... 3

1.3 Structure of the Thesis ... 5

2 Theoretical Framework ... 6

2.1 Description of Plastic and Microplastic Marine Debris, Persistence and Interaction of Pollution ... 7

2.2 Investigated Food Chain ... 11

3 Materials and Methodology ... 17

3.1 Study Area ... 17

3.2 Surface Water Methodology... 18

3.3 Fish Stomach and Gut Methodology ... 20

3.4 Filtration Paper Analysis ... 22

3.5 Method Review ... 23

3.6 Statistical Analysis ... 24

3.7 Knowledge Gap, Limits and Preconditions ... 25

4 Results ... 26

4.1 Attempted Method Development to Dissolve Fish Stomachs ... 26

4.2 Microplastic Fibres in Surface Water ... 26

4.3 Microplastic Fibres in Boops boops ... 27

4.4 Microplastic Fibres in Trachurus mediterraneus ... 28

4.5 Microplastic Fibres in Sphyraena viridensis ... 29

4.6 Overall Food Chain ... 30

5 Discussion ... 31

5.1 Sample Period and Location ... 31

5.2 Correlation Between the Total Fibres and Length/ Weight ... 31

5.3 Quality Control During Research... 32

5.4 Identification Problems of Microplastic Fibres ... 33

5.5 Health Hazard For Human Food Resources ... 34

6 Conclusion ... 35

References ... 38

Appendix 1 Method Used to Dissolve Organic Matter ... 42

Appendix 2 Surface Water Sampling Information ... 46

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Appendix 3 Surface Water Microplastic Fibres Presence ... 47

Appendix 4 B. boops Information Samples... 48

Appendix 5 B. boops Microplastic Fibres Presence ... 49

Appendix 6 T. mediterraneus Information Sample ... 52

Appendix 7 T. mediterraneus Microplastic Fibres Presence ... 53

Appendix 8 S. viridensis Information Sample ... 56

Appendix 9 S. viridensis Microplastic Fibres Presence ... 57

Appendix 10 Control Sample ... 60

Appendix 11 Quality Control ... 63

Appendix 12 Surface Water Sample Standard Deviation ... 69

Appendix 13 Standard Deviation Fish Weight ... 70

Appendix 14 Standard Deviation Fish Fibres per Weight ... 71

Appendix 15 Standard Deviation Fish Length ... 72

Appendix 16 Standard Deviation Fish Fibres Per Length ... 73

Appendix 17 Standard Deviation Fish Fibres ... 74

Appendix 18 Overall Standard Deviation Fish ... 75

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Acronyms

PE Polyethylene

P Polypropylene

PS Polystyrene

PET Thermoplastic Polyester PVC Poly vinyl chloride

M molarity

ANOVA Analysis of variance

PAHs polycyclic aromatic hydrocarbons PCBs polychlorinated biphenyls

r correlation coefficient

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1 Introduction

Every year millions of tons of plastic are produced and countless plastic remnants accumulate in the marine habitats worldwide and already have been present for decades.

The first microplastics were found in samples back in the 1960s (Mitchell, Olsen, &

Thompson, 2004). China has the highest production of plastic in the world whereas Europe is the second highest producer (see Figure 1). Plastics are produced worldwide and lead to a problem with global consequences.

Figure 1 World Plastic Production 2011 (Plastics Europe, 2012)

Plastic pollution occurs in the marine environment, its water, sediments and species.

Attention was already given to macro and mega plastic litter twenty years ago, but was ignored for a long time (Goryska, 2009). Plastic debris undergoes fragmentation on land and at sea which leads to microscopic particles of plastic (Claessens, De Meester, Van Landuyt, De Clerck, & Janssen, 2011). Although, microplastic itself has only recently been defined as a threat to the marine environment, it is a global problem and one of the most significant pollutants. Microplastic is able to accumulate within the food chain, which is not only a threat to wildlife, but also to humans through diet (Goryska, 2009). The increase in human

population coupled with the use of plastic in cosmetics, packaging and households makes it even more urgent to carry out more research on this topic (Goryska, 2009). It is imperative that future studies are undertaken on microplastic pollution in order to spread awareness about the potential risks from overuse of plastics. Policy can then be enforced, education spread and behaviour changed.

In the Mediterranean Sea approximately 16% of species are critically endangered, vulnerable or near threatened, nevertheless, it is a hotspot of biodiversity (Irving, et al., 2014). The fish

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stocks of the Mediterranean Sea are very important food resources as fish is a substantial part of the Mediterranean diet.

Currently the average Greek eats 26 kilogramme of seafood per year and it is expected to increase by another kilo by 2030 (Pierre Failler, 2007).

Samos is located in the Aegean Sea where substantial fishing and tourism takes place. Both of which are very valuable to the Samian economy. Any damage to the ecosystem and fish stocks in this region can greatly influence the wealth of Samos Island and with that the whole Greek economy, presently an issue of tantamount importance. Therefore, it is necessary to reduce pollution in order to achieve an environmentally and economically sustainable future.

Additionally, plastic has possmay contribute to cancer-related issues (Frias, Sobral, &

Ferreira, 2010).

To identify the possible threat to human food resources the food chain of Sphyraena

viridensis, or yellowmouth barracuda, will be investigated for microplastic fibres presence in the Aegean Sea. The different trophic levels are represented by surface water (which contains zooplankton), Boops boops, or Bogue, and Trachurus mediterraneus, or horse mackerel, all of which, apart from surface water, are consumed by humans.

1.1 Goal and Research Question

This study’s motivation was to investigate the presence of microplastic fibres in the local ecosystem, which was limited to a representative food chain. The aim of the research was to obtain information about how much microplastic fibres are present in the investigated food chain, if bioaccumulation occurs and to identify the potential threat to humans and ecosystems. This is particularly important because the fish species and other marine animals are part of the human diet. The outcome of this research can be used to spread awareness of the extent to which microplastic fibres have become imbedded and persist in marine ecosystems. The pollution of these food resources with microplastic and other chemicals which occur within plastic can be transported and can be a hazard to human health (Goryska, 2009). Of course it is also a threat to the local ecosystem; especially due to the fact that an ecosystem can only properly function if a certain ratio exists between the

different trophic levels (Goryska, 2009). Such pollution can interfere with mortality rates and thus affect population size and damage the balance in the community with unforeseeable consequences.

The main question of this research is;

“Are microplastic fibres present in all trophic levels of the Sphyraena viridensis (yellowmouth barracuda) food chain in the eastern Aegean Sea?”

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3 This encompasses the following subquestions:

To what extent are the different trophic levels of the food chain, represented by surface water, Boops boops, Trachurus mediterraneus and Sphyraena viridensis, polluted by microplastic?

Is there a correlation between the size or weight and the amount of microplastic present in the stomachs of the fish in the investigated represented food chain, indicating

bioaccumulation of microplastics?

1.2 Objective of Study

The effects of chemicals absorbed in microplastics on human health and the environment is of great interest today, especially for aquatic food products. Even at low concentrations, toxic elements can be very harmful when ingested over a long time period. In the marine environment, pollutants might accumulate in organisms, sediment and

subsequently be transferred to humans through the food chain. Therefore, it is important to determine presence of microplastic in fish stomachs for human health (Dogan, Tuzen, Mendil, & Saylak, 2007). To prove that there is a significant threat to humans, it is necessary to investigate a food chain where many trophic levels are consumed. There is limited

information on the spread of microplastic in fish in the Aegean Sea. Therefore, the aim of this study was to determine the presence of microplastic found in the stomach of popular fish species in this region, depending on their availability at the time of research. Furthermore, species which have a large impact on the ecosystem have been chosen. Figure 2 shows the relative total impact a species has on an ecosystem. The higher the keystone index, the higher is the total impact.

Bogue is a benthopelagic fish (see figure below number 23) with a keystoneness of about -0,7 and a relative total impact of 0,1. The horse mackerel has the number 31, which is a relatively low keystoneness, so a relatively low impact on the ecosystem. The yellowmouth barracuda is a medium sized pelagic fish which gives number 34. This has a keystoneness of about

-0,5 which means results in a high relative total impact of about 0,35. This species is the top predator of the investigated food chain. Even with the horse mackerel having a relatively low impact on the ecosystem, it is chosen as part of the investigated food chain because it links the B. boops and S. viridensis.

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Figure 2 Keystone Index and Relative Total impact of Each Functional Group of the Model, Circle Size Indicates the % Relative Biomass of Each Group (Tsagarakis, et al., 2010)

The food chain is represented by zooplankton, present in surface water, and the fish species S. Viridensis, T. mediterraneus, B. boops. These species were chosen as they are likely to be caught during spring when this research is being undertaken. This research will help to spread awareness of potential risks of microplastic fibres, especially within the local population which is highly dependent on good quality fish resources and other aquatic

species. It will also indicate the importance of the removal and reduction of plastic pollution in every ecosystem in the world.

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5 Hypothesis

The hypothesis is that the amount of fibres is expected to be higher in larger sized fish in higher trophic levels than in smaller sized fish in lower trophic levels. Therefore the hazard posed to humans from ingesting fish from higher trophic level is bigger compared to that of lower trophic levels when focused on the absolute amount of fibres. On the other hand the threat of lower trophic levels is higher due to the relative higher amount of fibres present per weight or length. Thus it follows, that although consuming an individual which is from a higher trophic level will result in ingestion of more plastic fibres compared to an individual lower level fish, the pattern of the human diet means that several lower trophic level fish will be eaten as opposed to one higher trophic level fish. As such in terms of food resources consuming the same weight lower trophic level fish poses more risk to human food resources.

1.3 Structure of the Thesis

The first chapter of the research report describes the literature background and gives an overall idea about the topic, the species which have been used to analyse the presence of microplastic fibres. It also describes possible threats for ecosystems and human food

resources due to toxins and clogging. After this, the methodology used in the study is

described for the zooplankton sampling and analysis. In addition, it is described how the fish is dissected and its stomach tested on the presence of micro fibres. Afterwards, the results of the study are shown and for each of the species such as B. boops, T. mediterraneus and S. viridensis. Also, the amount of fibres in the surface water is measured to have an idea how many fibres are present in the habitat and near food sources of herbivores species which might ingest them by accident. Furthermore the results of the statistical analysis are described. The discussion chapter draws reasons to the outcomes and gives reasons for occurrence of pollution. In the conclusion chapter assumption are made which were made out of the discussion and results. The recommendation chapter describes what needs to be changed to conduct better research in the future as well as what could limit the occurrence of microplastic fibres.

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2 Theoretical Framework

Annually, about 245 millions of tonnes of plastic are produced every year, nearly a third of which is used for packaging material. Materials for packaging are PE (29 %), PP (19 %), PS (7,5 %), PET (6,5 %) and PVC (11 %) (Andrady, 2011) as figure 3 shows. Nowadays, a countless amount of this plastic is accumulating in the marine habitat worldwide where it persists for centuries. Furthermore, plastic has the potential to absorb, release and transport chemicals which might be harmful to the environment (Mitchell et al., 2004). A disturbing 80% of the beach litters are from land-based sources and 18% of the marine plastic debris is from the fishing industry (Andrady, 2011). Within the last decades the main focus of plastic pollution was on entanglement of marine mammals and other species such as turtles. Other work has been conducted on ingestion of plastics by birds, some of which feed the plasticd to their chicks (Andrady, 2011). Recently there is a shift in attention focusing more on

microplastic rather than large plastic debris in marine ecosystems.

Figure 3 European Plastic Demand by Resin Type 2011 (Plastics Europe, 2012)

Problem Statement

The effects of chemicals absorbed in microplastics on human health and the environment is of great interest today, especially for aquatic food products. Even at low concentrations, toxic elements can be very harmful when ingested over a long time period. In the marine environment, pollutants might accumulate in organisms, sediment and

subsequently transferred to human through the food chain. Therefore, it is important to determine presence of microplastic in fish stomachs for human health (Dogan et al., 2007).

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This research is being carried out as work on the persistence and the bioaccumulation of microplastic in marine ecosystems is limited to date. In order to extend knowledge in this field of research the presence of microplastic in a representative food chain is assessed.

2.1 Description of Plastic and Microplastic Marine Debris, Persistence and Interaction of Pollution

Chemical Characteristics of Plastic

Plastic is created from organic and inorganic raw materials which are usually obtained from oil, coal and natural gas. Plastic consists of long chain polymeric molecules, which make most of the plastics persistent. Depending on the monomers during the polymerization process, different types of plastic are produced. This process can lead to strong or weak hydrocarbon-bond creation. Polystyrene has a crystalline structure and is comprised of strong hydrocarbon-bonds. Whereas other plastics, such as polyethylene, consist of weaker hydrocarbon-bonds which appear like entangled strings as an amorphous structure.

Thermoplastics can repeatedly be thermally softened and hardened by cooling; those plastics can be remoulded and reused practically indefinitely. Those plastics are used for packaging, food containers and other household products. Thermoplastics which are heat resistant and more durable can be used in automobiles, machineries and electrical products.

Another group of plastic, called thermosetting plastics, harden permanently after being heated. Those have higher melting points and are used for high-heat resistant products.

(Goryska, 2009). Plastic is an inexpensive, lightweight, strong, durable and corrosion resistant material (Ivar do Sul & Costa, 2013). It is extremely stable (Shimao, 2001) and resistant to biodegradation (Mitchell et al., 2004). Most of the synthetic polymers are buoyant in water, for example PE and PP, whenever those particles are buoyant enough to float on seawater. Polymers which are denser than seawater, for example PVC, might settle.

Microbial films can develop on those submerged plastics and change their physiochemical properties (Ivar do Sul & Costa, 2013).

Microplastic

Microplastic is defined as small plastic particles conventionally assumed to range between 333 µm and 5 mm. Recent research shows that the size frequency of plastic debris is highly skewed towards smaller particles (Browne, Galloway, & Thompson, 2007). These small plastic particles can be found all around the world in high amounts, not only near highly industrialised or urban areas but also in remote and low industrialised places (Goryska, 2009).

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The first source of microplastic is fragmentation which takes place through chemical

weathering and mechanical erosion. The degradation process is more likely to take place on land, and not in the sea, due to a higher solar radiation and mechanical erosion. In the marine environment, fragmentation could take place through wave action and abrasion from sediment particles. Another source is in cleaning products where it is used as an abrasive scrubber. These make up the smallest plastic particles (Goryska, 2009). Microplastic is easily transported with sewage through the waste water treatment facilities where it enters the aquatic habitat (Goryska, 2009). Microscopic plastic debris, such as fragments or fibres, are widely spread in the marine environment and accumulates in the pelagic, demersal and benthic zones (Mitchell et al., 2004).

The Effects of Microplastic Pollution

There is very little information about the impacts of plastics on the marine environment. It is known that various turtles, seabirds and other marine mammals are affected by plastic litter. Microplastic can be found in mussels as well (Goryska, 2009) and has been detected in fish, probably taken up through their diet which had previously

accumulated plastic. Mussels and fish are food sources for humans, which pose a threat to human health (Goryska, 2009). Microplastics can be ingested by a variety of marine

organisms and could be transferred along the food web.

The effects of microplastic ingestion of marine organisms are divided into three categories:

- Physical clogging and damage of feeding appendages or the digestive tract - Leaching of plastic component chemicals into the organism after digestion - Ingestion and accumulation of adsorbed chemicals by the organism

Ingestion of microplastic that mimics natural food fails to provide nutrition proportionate to its weight or volume. It can lead to starvation due to false feeling of satiation and irritation of stomach lining. With that the individual fails to store fat necessary for migration and

reproduction. Moreover, thin packaging film has the potential to inhibit gas exchange in the gills which might interfere with O2 uptake and CO2 sequestration. (Moore, 2008)

Furthermore, there is a chance of plastic debris changing the composition of the sea floor which would interfere with inhabitants of the sediment. It also fills up and destroys nursery habitat with marine litter, so less new life is able to emerge (Moore, 2008).

Microplastic has the potential of toxicity due to additives and monomers (Setälä, Fleming- Lehtinen, & Lehtiniemi, 2013). It can be highly carcinogenic, reproducing abnormalities in humans, invertebrates and rodents (Goryska, 2009). Plastic can absorb hydrophobic

substances such as PAHs and PCBs which can threat the whole food chain because plastic

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is present in all sizes and depths of the ocean (Frias et al., 2010). PCB congeners for example are not only carcinogenic agents; they are also endocrine disrupters which can affect the immune, reproductive, nervous and endocrine system of animals (Frias et al., 2010).

When exposed to salts in water, polycarbonate might accelerate leaching of the bioactive Bisphenol-A monomer (Moore, 2008). All of these substances could impact the sea-surface micro-layer ecosystems (Frias et al., 2010).

Microplastic especially in fibre form poses threats to organisms that consume them as they can cause clogging in the digestive tract, become translocated to different tissues within the organism, and undergo accumulation (Mathalon & Hill, 2014).

Filter feeders may also take up contaminated particles and with that transport the pollutants upwards onto organisms in higher trophic levels. Chronic negative health effects resulting from ingestion of these organisms can also be experienced by humans. (Frias et al., 2010)

“However, the great majority of people during their lifetime are exposed to complex mixtures of organic compounds at low concentrations. Thus, long-term exposure may be more important in terms of overall public health than short-term exposure.”1

Existing Studies

As Figure 4 shows, research regarding microplastic pollution has already been

conducted worldwide, especially in industrial countries. A lot of studies have been carried out regarding the pollution of sediment and chemical pollution (Ivar do Sul & Costa, 2013).

Microplastics in species have been researched; some of these reports dealt with accumulation of the microplastic within an ecosystem as “Trophic level transfer of

microplastic: Mytilus edulis (L.) to Carcinus meanas (L.)” by Paul Farrell and Kathryn Nelson or “Accumulation and fragmentation of plastic debris in global environments” by David K. A.

Barnes et al. in 2009.

1Frias, J.P.L.; Sobral, P.; Ferreira, A:M.: Organic pollutants from two beaches of the Portuguese coast, 2010 , page 5,

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Figure 4 Microplastic Studies in the World (Ivar do Sul & Costa, 2013)

Environmental and Economic Impact and Health

Presently, the average Greek eats 26 kilograms of seafood per year and this is expected to increase by another kilo by 2030. Other Mediterranean countries show similar behaviour (France 32 kg/capita/year, Italy 26 kg/capita/year, Portugal 59 kg/capita/year and Spain 39 kg/capita/year) (Pierre Failler, 2007). Research showed that 90 percent of the samples from the Mediterranean Sea contain plastics. The movement of the plastic is fairly unknown but microplastic is available to every level of the food web from the primary producer to higher trophic levels. The impact on population levels as a result of ingestion of small plastic particles is undocumented but it is clear that microplastic pollution is threatening the Mediterranean Sea (Ivar do Sul & Costa, 2013). This is a result of being a semi-enclosed sea surrounded by highly industrialised and densely populated countries. Thus chemical pressure is high in the Mediterranean Sea. These chemical contaminants are causing

alteration to marine ecosystems impacting individuals, population, species and food webs by entering at different trophic levels (Cresson, et al., 2014).

Plastic usage increased from 1950 with 1.5 million tons to 245 million tons in 2008 (Rios, Jones, Moore, & Naravan, 2010). The production of plastic depletes a significant proportion of the world’s non-renewable resources. The plastic themselves have a limited durability and longevity and then are thrown away; this increases pressure on the resources. The plastic debris currently poses a significant hazard to wildlife (Krause, von Nordheim, & Brägen,

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2006). Based on the present trend, animals and humans are at risk due to microplastic pollution. Microplastic particles will continue their slow, intricate paths towards the bottom of the ocean and at some point become buried in sand or mud for centuries. Scientists need to find economic and logistical ways to remove this pollutant (Ivar do Sul & Costa, 2013).

2.2 Investigated Food Chain

It has been documented that over 180 species ingest plastic debris such as plastic fragments. The potential for ingestion and accumulation of smaller plastic pieces is far greater than with larger pieces because they are to be taken up by the smaller fish, as such they are able to accumulate up the food chain.

Concentrations of pollutants increase in the aquatic food chain. Phytoplankton, as primary producer plays a key role in transport of organic contaminants through food chains to the higher trophic levels. The primary consumer, as zooplankton for example copepods, plays an important ecological role in aquatic ecosystems by controlling the phytoplankton communities and by acting as a direct or indirect food source for higher trophic animals. The first step in the transfer of chemicals through the food chain is the bioaccumulation at lower trophic levels. High concentration in fish is partly a result of transfer throughout the food chain (Xinhong & Wang, 2005).

Figure 5 Pollutants in a Food web (Ivar do Sul & Costa, 2013)

Some toxic effects are chronic, less evident and act on a long time-scale such as

genotoxicity and reproductive failure. Anyhow they are considered as an important risk to the ecosystem. Bioaccumulation and the change in primary production at the bottom of the food chain have an effect on the concentration of organic matter throughout the whole system. If there is a higher bioavailability of contaminants in the primary producers it has an impact on the whole ecosystem. Environmental perturbation can alter dynamics and coastal structures substantially which can produce the occurrence of trophic cascade and with that the

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extinction of species. Sudden regime shifts and ecosystem collapses are likely to occur in stressed ecosystems, due to top-down (e.g. overfishing) versus bottom-up (e.g. increase of nutrition input that causes eutrophication) (Bacelar, Dueri, Hernandez-Garcia, & Zalddivar, 2008). As shown in figure 6 the representative food chain of the ecosystem in the area and season are S. viridensis, T. mediterraneus, B. boops and zooplankton in surface water.

Figure 6 Investigated Food Chain

Zooplankton

Figure 7 Zooplankton with Ingested Fluorescent Beads (Setälä, Fleming-Lehtinen, & Lehtiniemi, 2013)

The first species, in a traditional marine food web, is phytoplankton as the primary producer. It transfers organic carbon to higher trophic level such as zooplankton (Anjusha, et al., 2013). Zooplankton is a key component in the structure and correct functioning of a marine planktonic food web. It links planktonic primary production with the top pelagic consumer levels. In addition, it plays an important role in nutrient recycling within the water columns and exports particulate matter out of the photic-zone (Saiz, Calbet, Atienza, &

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Alcaraz, 2007). These species can act as an indicator of the heath of an ecosystem. (de Puelles, Alemany, & Jansa, 2007). The zooplankton community is dominated by

crustaceans and copepods (Anjusha, et al., 2013).

There has already been research carried out regarding microplastic presence in zooplankton.

One study showed ingestion of microplastic in various zooplankton communities (Figure 7) (Setälä et al., 2013) however it only identified microplastics which are flouresecent as figure 7 shows.

Boops boops

Figure 8 Boops Boops School (Archipelagos, Institute for Marine Conservation, 2013)

B. boops or Bogue is a fish of the family Sparidae. Characteristically it is 10-25 cm long, fusiform, oval body, big eyes, brownish lateral line and brown blotch on base of pectoral fin. (Archipelagos, Institute for Marine Conservation, 2013). It is a demersal fish, as well as semi-pelagic, species living on all types of bottom, such as sand, mud, rock and sea grass beds. Furthermore, Bogue can be found in a depth until 350 meters, but it is more abundant in the upper 100 meter and coastal waters. Further, it is found in schools (Figure 8). In the Mediterranean Sea it reproduces around April to May (Fisheries and Aquaculture

Department, 2015).

Figure 9 Global Capture Production for Species (Tonnes) (Fisheries and Aquaculture Department, 2015)

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Greece is one of the countries with the largest catch of this species. The global capture has been increasing within the last 60 years as the figure “Global Capture Production for species (tonnes)” shows (Figure 9). B. boops is caught on line gear, with bottom trawls and purse seines but also with beach seins and trammel nets (Fisheries and Aquaculture Department, 2015).

Trachurus mediterraneus

T. mediterraneus or Mediterranean horse mackerel (Figure 10) is a benthopelagic pisivores (Food and Agriculture Organization of the United Nations, 2015) that preys mainly on fish (Cresson, Ruitton, Ourgaud, & Harmelin-Vivien, 2013). It can be found in the Eastern Atlantic and Mediterranean Sea. Also, it mainly occurs in deeper waters of 5 to 250 m, but also in surface waters (Food and Agriculture Organization of the United Nations, 2015).

Figure 10 T. mediterraneus School (Horst, 2012)

Its characteristics are an elongate and fairly compressed body with a large head. Commonly it has a length of 10 to 30 cm. The upper part of the body and top of head are dusky to near black, or grey to bluish green. The lower two thirds of body and head are usually paler, whitish to silvery. The caudal fin is yellowish (Food and Agriculture Organization of the United Nations, 2015).

The Mediterranean horse mackerel migrates in large schools and often shoals with other Trachurus species as T. trachurus or T. picturatus (Food and Agriculture Organization of the United Nations, 2015). T. mediterraneus preys on B. boops, Phycis phycis, Phycis

blennoides, Scopraena porcus, Conger conger and Sepiola sp. (Santic, Jardas, & Pallaora, 2003). As the figure below shows, the peak catch production of this species was in the 80s after European fish stock legislation was introduced in the 1970’s (European Commission, 2015). Nowadays, it is lower, but more or less a steady amount of catches (Figure 11)

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Figure 11 Global Capture Production for Species (Tonnes) (Food and Agriculture Organization of the United Nations, 2015)

Sphyraena viridensis

S. viridensis or yellowmouth barracuda (Figure 12) is one of the most common coastal pelagic predators in the Mediterranean Sea and northeast Atlantic. The geographical range of this species might be wider because it is often confused with S. Sphyraena (Perdro Barreiros, Serrao Santos, & de Borba, 2002).

Figure 12 S. Viridensis School (Koszorek, 2013)

Characteristically the yellowmouth barracuda has a slender fusiform body with conical, hydrodynamical snout. It has a long mouth with low protractile capacity and with a prognatic lower jaw which contains two rows of long canine-like teeth. One characteristic that allows differentiation from S. Sphyraena is the absence of a scale on the preopercullum (Perdro Barreiros et al., 2002). S. viridensis has a darkish grey to bluish dorsally and silvery ventrally body. The upper half has numerous vertical dark bands that extend below the lateral line in the anterior part of the flanks. A juvenile individual of this species is more greenish to dark yellow (Perdro Barreiros et al. , 2002).

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It can be found in schools of larger than 10, small groups of less than 10, pairs or isolated individuals.The most important prey for S. viridensis is Carangidae species which vary in different regions. For example Trachurus picturatus near the Azores (Perdro Barreiros et al., 2002) and Trachurus mediterraneus in the Aegean Sea. Furthermore, it preys on Boops boops, Sardine pilcharduy, Mullus surmuletus, Chromis chromis, Diplodus annularis, Sparisoma cretende, Sepia officinalis, Atherina boyeri and Spica smaris in the Aegean Sea (Kalogirou, Mittermayer, Pihl, & Wennhage, 2012).

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3 Materials and Methodology 3.1 Study Area

Figure 13 Location of the Study Area: Samos Island in the Aegean Sea

The research took place near the institute, in the Eastern Aegean Sea, around the island of Samos, close to the coastline of Turkey. The islands’ population is approximately 34000 inhabitants. It has a Mediterranean climate of a typical Greek island, where summers are hot and winters are mild. Furthermore, it has one of the longest periods of sunshine in Greece. (Archipelagos, Institute for Marine Conservation, 2014). The island hosts a high biodiversity, including the surrounding marine environment. The local fishing industry is considered of great economic importance not only for local trade but also for tourism. (Irving, et al., 2014).

The transect lines 1 to 5 show the area of surface sampling near Mesokampo on Samos Island. Most of the fish were caught in Vathi Bay in the North of Island. The second and third S. viridensis samples were taken near Pythagorio in the south east of the island. The fourth and fifth T. mediterraneus were caught in Kokkari Bay also in the north. The fish samples were caught by local fishermen, the information related to the exact location of the catch, such as the longitudinal and latitudinal coordinates are ill-defined due to the lack of GPS information.

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Figure 14 Sample and Catch Location

3.2 Surface Water Methodology

In the following, the methodology to sample surface water and to identify the presence of micro fibres are described.

Surface Water Sampling Method

Sampling took place on the 6th, 9th and 13th of May when the weather conditions were good with low wind speed and sunshine. The method to determine the distribution of microplastic pollution of the surface ocean is described in the following passage. Zooplankton and phytoplankton are both present in the surface water and are prey of B. Boops. Only the presence of microplastic in the surface water is analysed due to the fact that only ingested fluorecent microplastics are visisble under a microscope.

Used materials to carry out this method are:

- Mesh net (a net for sampling the surface water with 333 µm mesh, radius of the haul is 29 cm and detachable collection container) + boom

- Graduated glass sample jars - 5 mm metal sieve

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The first steps of the sampling were to check the net for holes and tears and to note the weather conditions. Afterwards, the boom was used to deploy the mesh net from the side of the vessel to avoid disturbance from the bow wave. The net had to be hauled horizontally through the upper 20 cm of surface water at an average speed of 2.5 knots if possible, over a distance of about 500 meters. It was then dipped slowly down into the water without

submerging the mouth. This helps rinsing the content into the collecting container. Then, the net was retrieved and large debris/ organisms were removed. To collect all debris and plankton stuck, the mesh was washed with seawater in the collecting container. The collecting container was then removed from the net and the sample was reduced to 0.20 litres in a glass jar. After this, the sample was sieved through a 5 mm mesh, so only particles between 0.333 and 5 mm were considered in a jar containing saltwater solution. Finally, the sample jar was labelled and the data such as dates, time, sampling location, length of haul, mesh size (333 µm) and net mouth dimension, were recorded (Archipelagos, Institute for Marine Conservation, 2014).

Laboratory Analysis of the Surface Water Sample

After the field work was properly carried out, the surface water sample was further treated in the laboratory. The required materials are:

- NaCl (33 g)

- Distilled water (200 ml) - Glass fibre filter paper 1.2 µm - Glass syringe

- Vacuum pump and filtration system

The salt solution was made with a salt concentration of 167 g/l. The solution was stirred until the salt dissolved. The salt solution was then filtered twice. To ensure the accuracy and quality of these experiments a control sample was also produced.

After the sample was sieved into the jar (see surface water sampling method), the sample was left to settle in the glass jar for 24 hours to separate the low-density plastic particles from organic tissue by buoyancy. After settling, a glass syringe held at the sample surface was used to extract the supernatant. This extracted water was then filtered; the retained microplastics were washed with distilled water to remove the salts. Thereupon, the filter paper could dry it was covered while drying to avoid contamination. The microplastic fibres present were expressed in particles per fitered volume (particles/m3). The filtered volume is described with the following formula. 𝐹𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑉𝑜𝑙𝑢𝑚𝑒𝑛 = 𝜋 ∗ 𝑟2∗ 𝑑. Wheras, r is 14,5 cm (Archipelagos, Institute for Marine Conservation, 2014).

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3.3 Fish Stomach and Gut Methodology

The fish stomach and gut methodology not only consists of the dissection of species.

It also includes the analysis of microplastic fibre present in the stomachs of mentioned species. Each individual sample was weighed, measured, dissected and the sex was determined. To ensure reliable results, five stomachs were analysed for each trophic level.

The following dissection description gives an idea on how the stomach was identified and removed. The stomach and gut of each fish was used to determine the microplastic fibres present in the individual.

Dissection Description

This method gives an overview of how the dissection was carried out. The materials used for fish dissections are:

- Dissection microscope with a proper illumination - Dissection set with scissors, scalpel and forceps - Probes (needle with blunt tip)

- Kitchen bakery paper

- Lab coat, gloves (non-plastic) - Security goggles

- Glass Petri dishes.

As shown in figure 15 “Incision with a Sharp Knife”, the first step of a fish dissection, is to make a shallow longitude incision along the ventral left-hand side which extends from the anterior of the anus to below the gill arches. It is important to cut deep enough to gain access to the gut but damaging the organs has to be avoided (Elenbaas, 2014).

Figure 15 Incisions With a Sharp Knife (Elenbaas, 2014)

Afterwards, the hand is put inside the gut cavity and one side of the cage is gently lifted up.

Then, transverse cuts, from the anterior and posterior ends of the longitudinal incision on the

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left side of the specimen are made. It is valid that the transverse cuts extend as far dorsally as required to permit an unobstructed view of the gut cavity. At the end, the stomach and gut is identified (see Figure 16), removed and placed in a Petri glass (Elenbaas, 2014).

Figure 16 Overview Organs in a Fish (Archipelagos, Institute for Marine Conservation, 2014)

Analysis Method for Microplastic in Fish Stomach

As soon as the fish stomachs were removed, the microplastic contamination was evaluated. It was important to clean the materials prior with alcohol and three times with filtered distilled water. The required materials were:

- NaCl

- Distilled water (Cole, et al., 2014) - Graduated glass jars with metal caps - Glass fibres filter paper 1.2 µm - Forceps

- Tape measure scale (Archipelagos, Institute for Marine Conservation, 2014)

The salt solution was made with a salt concentration of 167 g/l. The solution was stirred until the salt was dissolved. The salt solution was then filtered twice. To ensure the accuracy and quality of these experiments a control sample was also produced. The stomach samples were placed into the saltwater solution. The glass jar was shaken for one minute and then left for about 24 hours, so the organic material could sink and the low density plastics float on the surface of the solution. Afterwards, 300 ml of the supernatant was removed with a glass pipette at the surface. This extracted water was filtered and the filter paper was allowed to dry.

The jar was filled with filtered salt solution, again shaken and left for 24 hours, to ensure that

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as much microplastic as possible was collected. Once more about 300 ml of the supernatant was removed and filtered. To dry the filter paper, it was placed in a glass petri dish and covered to avoid contamination (Archipelagos, Institute for Marine Conservation, 2014).

3.4 Filtration Paper Analysis

The filtration paper method was used in both cases, for the analysis of microplastic contamination for fish stomachs and zooplankton.

Figure 17 Method to Review a Filter Paper (Archipelagos, Institute for Marine Conservation, 2014)

After vacuum filtration, the clamp and top piece of the Whatman flask needed to be removed.

The filter paper was transferred into a glass Petri dish by using metal forceps. It was

important not to disturb the sample at any time. For analysis, the filter paper was put onto a glass slide using, again, metal forceps. The slide was then placed under the microscope.

Using a 40x magnification lens the filter paper was systematically viewed, using the method shown in figure “Method to Review a Filter Paper”. During this viewing items of interest were counted and, to ensure accuracy, a second count was carried out (Archipelagos, Institute for Marine Conservation, 2014).

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3.5 Method Review

To prevent contamination during lab work, the distilled water from a plastic container, used to prepare the saltwater solution, was filtered before use. Additionally, the equipment was checked for contamination under a microscope and surface, hands and equipment are cleaned with distilled water before use. During the dissection, nitrile gloves and not plastic ones were worn to prevent manipulation and possible contamination of the stomach with microplastic fibres (Archipelagos, Institute for Marine Conservation, 2014).

The literature research showed different method to dissolve organic matter (described in chapter 4.1). Due to the lack of time, further research on new methods to dissolve a fish stomach and the correct ratio of baking soda to water, stirring and temperature was not able to be tried out. Additionally, the used stomach to test the methods was of a B. boops, it is significantly smaller than one of larger fish, the barracuda and mackerel. The time needed to dissolve the larger stomachs is therefore longer and needs further investigation. Another reason to not continue investigating different methods was that the laboratory equipment did not allow for proper contamination control. The stirring magnet was covered in plastic which might erode and skew the results; the temperature measuring device needed to be placed in the solution whilst heating up; and the baking soda is packed in plastic, all of which may lead to contamination. Other ideas, such as the usage of an ultrasonic bath to dissolve the

organic tissue could not be conducted due to the lack of equipment.

Ultimately the saltwater method, mentioned in various other researches and previously used by Archipelagos, was used to investigate the presence of microplastic fibres in the fish stomachs. Reviewing several reports on microplastic presence in plankton and fish gut showed that the techniques which are used for this research are quite uniform. Similar methods are described in “Microplastics in the Marine Environment: A Review of the

methods Used for Identification and Quantification” ,by Valeria Hidalgo-Ruz et al., “Isolation of microplastic in biota-rich seawater samples and marine organisms” ,by Matthew Cole et al., “The Impact of Plastic debris on Biota of tidal Flats in Ambon Bay”, by Prulley Uneputty and S. M. Evans, and “ Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre”, by Christiana M. Boerger et al.. One disadvantage of this method is that it only enables the detection of low density microplastic fibres, high density fibres cannot be recorded.

Another problem may arise while using a 0.02 µm filter paper, as clogging of the filter may occur due to improperly dissolved tissue and salt crystals. A mesh filter of 50 µm appeared to have the fastest filtration rate. Although the 20 µm mesh-filter theoretically captures smaller microplastic nevertheless those small particles are able to be identified with only a binocular

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microscope. It was therefore not necessary to use such a small mesh size for this study (Cole, et al., 2014).

3.6 Statistical Analysis

The statistical analysis is carried out by determining the standard deviation for weight, length, number of fibres, number of fibres per weight or length. The following formula is used to determine the standard deviation:

𝜎 = √ 1

𝑛 − 1∑(𝑥𝑖− 𝜇)2

𝑛

𝑖=1

Furthermore, the correlation coefficient r is determined by using a power trend line. The correlation coefficient gives an idea about how strongly related one variable to another is. It ranges from - 1,00 to + 1,00. A perfect negative relationship exists between two variables when r is - 1,00. On the other hand, a perfect positive relationship occurs whenever r is + 1,00. The closer r is to - 1,00 and + 1,00 the more correlation exists (Higgins, 2005).

A power trend line is curved and preferably used with a data set that compares

measurements which increase at a specific rate. It can only be used whenever the data contains no zero or negative values (Office, 2015). This is the case with the data set

collected during this study, due to that fact that no fish in existance weights or measures zero or negative weight or size.

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3.7 Knowledge Gap, Limits and Preconditions

It is unknown at what time microplastic started to be present in the sample species.

Another knowledge gap is the extent of microplastic content in the target species of this research. Currently very little research is being carried out with the species being

investigated. Furthermore, it is not possible to clarify if the species ingested microplastic mistakenly, on purpose, or indirectly through their prey. Moreover, pollution might be a result from other prey species and not only the investigated one; microplastic pollution could also be a result of preying on M. surmuletus and not T .mediterraneus. In addition, it is not possible to have exactly the same location for all the samples. It is only possible to have samples from the same region so there might be discrepancies in their contamination with microplastic due to differences in living conditions and surrounding.

The research was limited by weather conditions which influenced the possibility of obtaining fish and taking the surface water samples. The surface water samples are only able to be taken with low wind and wave conditions.

A sodium hydroxide solution with 10 M would be a suitable method to decompose all organic matter, but it potentially leads to damage or discolouring of the plastic particles can occur with such a strong alkaline treatment (Cole, et al., 2014). Further, the lack of a laboratory hood makes it too unsafe to use a sodium hydroxide solution with a concentration of 10 M or at a temperature of 60°C, due to formation of toxic gases (Cole, et al., 2014). As a laboratory hood is not available, neither attempting the sodium hydroxide method nor the potassium hydroxide method are options for this research.

The basic laboratory equipment did not allow any other strong alkaline methods or acidic solutions. Furthermore, the lack of a tap in the laboratory makes it difficult to maintain a clean working environment. Additionally, the microscope can only identify plastic particles which are big enough to be identified with a 40x magnification. This also only allows the

identification of colour and existence of microplastic. The size of the fibres is not able to be determined because they are not present linearly and this cannot be measured. It is also not possible to analyse the type of plastic due to insufficient equipment.

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4 Results

4.1 Attempted Method Development to Dissolve Fish Stomachs

After conducting desk research into the different ways of dissolving fish stomachs, three options became apparent. The first possible method to dissolve the organic tissue is with a sodium hydroxide solution. In February, the first method was tried with a 1 and 2 M sodium hydroxide solution using hydroxide as an reaction agent. The stomach was not dissolved within a week in either of the two solutions. A reason for that might have been the low molarities or temperature. A second trial was carried out at a higher temperature of 25

°C, room temperature in an incubator. The results were the same (see Appendix 1). After contacting some researchers who had already experimented with this method, it was advised to try it at 60 °C or with a potassium hydroxide solution. Some studies used alkaline solution method and showed that optimal alkaline digestion protocol is 1 and 2 M NaOH which digest a sample by 90.0 ± 2.9% and 85.0 ± 5.0%, whereas 10 M NaOH has a digestion efficacy of 91.3 ± 0.4 % (Cole, et al., 2014). As a laboratory hood is not available, neither trying out the sodium hydroxide method at a temperature of 60°C nor changing the concentration nor the potassium hydroxide method are options for this research.

To continue, two more methods were tested: dissolving the fish stomach with coca cola, with phosphoric acid as the active ingredient; and baking soda solutions, also called sodium hydrogen carbonate solution, at a temperature of 25°C in the incubator. The results showed that the baking soda solution dissolved the stomach better than the coca cola, with which the stomach just expanded in size. The same method was tested at a temperature of 41°C and again the baking soda solution dissolved more. To hasten the process, the sodium hydrogen carbonate solution was heated up and stirred for about three hours. The fish stomach

dissolved quite well with this procedure (see Appendix 1).

4.2 Microplastic Fibres in Surface Water

Microplastic fibres have been found in all of the investigated transects and ranged from 5 to 16 fibres see table. This table refers to the five surface water samples taken (Z. 1- 5) and describes the total number of microplastic fibres present. An average of the sample set was taken as well as a standard deviation.

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Table 1 Microplastic Fibres Present in the Surface Water

Sample

Filtered

Volumen (m3) Total Fibres

Total Fibres per 1000 m3

Z. 1 18.30 5.00 273

Z. 2 16.11 13.00 807

Z. 3 14.22 5.00 352

Z. 4 18.61 16.00 860

Z. 5 17.30 9.00 520

Average 16.91 9.60 568

The Appendix 3 “Surface Water Microplastic Fibres Presence” shows that black, blue and transparent fibres were present in all of the samples showing that black fibres are the highest in number.

The average amount of microplastic fibres per 1000 m3 present in the surface water is 568±

264 with an average filtered volume of 16,91±1,79 (see Appendix 12 “Surface Water Sample Standard Deviation” and graph below).

4.3 Microplastic Fibres in Boops boops

For the second trophic level the table below shows the results of microplastic

presence in the representative species Bogue. B. boops stomachs showed microplastic in all of the investigated specimens. The amount of microplastic fibres ranges from 12 to 20 as the table below shows.

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Table 2 Microplastic Fibres Present in B. boops

Sample

Length (cm)

Weight

(g) Total Fibres

Fibres per Length (m)

Fibres per Weight (kg)

B.b. 1 19 73 15 79 205

B.b. 2 20,5 88 17 83 193

B.b. 3 19 69 20 105 290

B.b. 4 18,5 76 13 70 171

B.b. 5 20 72 12 60 167

Average 19,4 76 15 79 204

The colouration of fibres differs between black, blue, red, transparent, green and others. In number, black and transparent are the most abundant with 26 and 24 (see Appendix 5 “B.

boops Microplastic Fibres Presence”). The average weight is 76 ± 7.4, the average length is 19,4 ± 0.8 and average number of fibres is15,4 ± 3,2 in this species (see Appendix 13,15 and 17). The average fibres per weight are 204 ± 49,9 and fibres per length are 79 ± 16,9 (see Appendix 14 and 16).

4.4 Microplastic Fibres in Trachurus mediterraneus

T. mediterraneus represents the third trophic level of the investigation. The analysis of the T. mediterraneus stomachs showed presence of microplastic ranging from 14 to 62 pieces (see table below).

Table 3 Microplastic Fibres in T. mediterraneus

Sample Weight Length Total Fibres

Fibres per Length (m)

Fibres per Weight (kg)

T.m. 1 127 24 62 258 488

T.m. 2 151 25 18 72 119

T.m. 3 157 27 14 52 89

T.m. 4 209 31 22 71 105

T.m. 5 233 30 22 73 94

Average 175,4 27 28 101 157

The fibre colouration within these individuals ranges from blue most numerous, black second highest to yellow with the lowest as shown in the Appendix 7 “T. mediterraneus Microplastic Fibres Presence”. The first individual had the most fibres present. The four other specimens have one third the number of fibres than the first (see table above). The average weight is

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175,4 ± 43,9, the average length is 27 ± 3,1 and average number of fibres is 28 ± 19,5 (see Appendix 13,15 and 17). The average fibres per weight are 157 ± 173,1 and fibres per length 101 ± 86,2 (see Appendix 14 and 16).

4.5 Microplastic Fibres in Sphyraena viridensis

The S. viridensis stomach all contained micro fibres and the amount of fibres range from 23 to 75 (see table below).

Table 4 Microplastic Fibers Present in S. viridensis

Sample Weight Length

Total Microplastic

Fibres per Length (m)

Fibres per Weight (kg)

S.v. 1 229 37 35 95 153

S.v. 2 1750 74 75 101 43

S.v. 3 656 56 48 86 73

S.v. 4 390 53 23 43 59

S.v. 5 819 62 30 48 37

Average 769 56 42 75 55

All kinds of fibre colours are present in the investigated species. The highest amount of microplastic was found in the second individual. It also contains the highest amount of black and transparent fibres see Appendix 9 S. viridensis Microplastic Fibres Presence. The average amount of fibres in this species is 769 ± 594,3 weight, 56 ± 13,5 of length and 42 ± 20,5of total fibres. The average fibres per weight are 55 ± 46,9 and fibres per length 75 ± 26,9.

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4.6 Overall Food Chain

Figure 19 Correlation Length And Number of Fibres

Figure 21 Correlation Weight and Number of Fibres

Comparing weight and length of the average species in the food chain shows that there is a positive correlation between weight or length and the number of fibres due to the correlation coefficient (R) being high (almost 1) in both cases. The fibres per weight and weight show a negative correlation. Contrary to that the fibres per length and specimen length show little correlation due to the correlation coefficient being low (see Figure 20). The graphs above show also that the number of fibres is higher whenever fish are heavier or taller. Conversely the ratio of fibres to length/ weight is greater in smaller fish. This correlation implies that the hypothesis of this investigation, that consumption per gram of lower trophic level fish is more harmful than of higher levels is correct.

y = 1.2292x0.8901 R² = 0.9173 0

10 20 30 40 50 60

0 20 40 60 80

Number of Fibres

Length (cm)

Length

y = 2.765x0.4179 R² = 0.9399 0

10 20 30 40 50 60 70

0 500 1000 1500

Number of Fibres

Weight (g)

Weight

y = 122.92x-0.11 R² = 0.1446

0 50 100 150 200

0 20 40 60 80

Fibres per Length (m)

Length (cm)

Fibres per Length

Figure 18 Correlation Length and Fibres perLength

y = 2765x-0.582 R² = 0.9681

0 50 100 150 200 250 300 350

0 500 1000 1500

Fibres per Weight (kg)

Weight (g)

Fibres per Weight

Figure 20 Correlation Weight and Fibres per Weight

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5 Discussion

As previously shown, the results attested to the presence of microplastic in all trophic levels of the investigated food chain. The results show that partly there is a correlation in size or weight of the species and amount of fibres found in the investigated species. This

supports the statement made in the hypothesis that the consumption of an individual higher trophic level fish is more harmful than an individual lower level species due to greater microplastic presence. Moreover the presence of a greater number of fibres found in higher trophic levels is arguably evidence for the bioaccumulation of microplastic in the food chain of the yellowmouth barracuda, and thus marine ecosystems. The bioaccumulation of microplastic in this food chain in which ultimately humans are the final consumer indicates that microplastic fibres do indeed pose a very serious threat to human health, particularly in maritime areas of heavily pescetarian diet.

5.1 Sample Period and Location

The samples were all taken around Samos Island and the fish are highly likely to travel along the whole coastline even though some samples were taken in the north and some in the south of the Island. The samples were taken over one and a half months, with the exception of zooplankton samples which were taken latest due to the weather conditions;

strong wind, currents or waves. This might have influenced the amount of fibres found because the weather conditions changed significantly from strong winds and 20°C to a moderate climate. Due to this change in conditions the water temperature increased which might have had an influence on the activity of the fish and with that the possibility of ingesting microplastic fibres was higher.

5.2 Correlation Between the Total Fibres and Length/ Weight

The occurring differences in the standard deviations could be caused by different day of catch, location of catch, sex, low difference between size and weight of the individuals.

The B. boops show a small standard deviation which could be a result of them being caught at the same day (see Appendix 4 “B. boops Information Samples”). These individuals were probably part of a fish school with similar eating and traveling behaviour. All those factors result in similar size/ weight and presence of fibres or other contaminants.

T. mediterraneus and S. viridensis standard deviation are much bigger which could be a result of the specimen being caught at different locations and days. This results in different of lifestyle. Though some fish were caught at the same day and showed similar characteristics (see Appendix 6 and 8).

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The findings show that there were more fibres present in the tested yellowmouth barracuda than in horse mackerel, bogue and surface water. This correlates with other research, which states that heavy metals, mercury, and pesticides accumulate within the food chain and are more highly concentrated in high trophic levels than in lower ones (Cresson, et al., 2014). It means that the highest trophic level of this investigated food chain have the most fibres compared lower trophic levels as the B. boops and T. mediterraneus. This correlates with the behaviour of other pollutants which accumulate in a food chain. Considering the average length, weight and amount of fibres also supports this test result. The smallest and lightest fish, B. boops, has the lowest amount of fibres and the tallest or heaviest fish, S. viridensis, has the highest amount.

On the other hand, the results show that the number of fibre per weight or length is higher in smaller or lighter specimens. This correlates with other studies which argue that herbivore accumulate higher concentrations of metals than carnivore species due to their feeding habit (El-Moselhy, Othman, El-Azem, & El-Metwally, 2014).

5.3 Quality Control During Research

The control sample shows little evidence of micro fibres with an average of 1,1 ± 1,2 which indicates almost no contamination from outside (see Appendix Control Sample). This insures the accuracy of properly conducted work. Contamination control measures were:

filtering the distilled water due to its storage in plastic containers; cleaning the needed equipment first with soap or alcohol and afterwards three times with filtered distilled water.

The occurring contamination could be a result of the atmospheric or the tank pollution. These sources of contamination were checked on weekly basis (see Appendix Quality Control).

Further contamination with fibres can result from the salt used for the salt solution which was packaged in plastic, or stirring due plastic coating of the stirring magnet which could erode (see appendix saltwater contamination). To counteract these interferences, every saltwater solution was filtered twice before for the analysis of microplastic fibres present in the fish stomach. Contamination with fibres might have occurred due to filtering the distilled water with contaminated filter paper though the rate of contamination decreased (see Appendix Quality Control). Due to the contamination of the new filter paper with which the distilled water from plastic containers was filtered, some contamination might have occurred. Though, the contamination of filter paper became less over the period of research see Appendix Quality Control, Filter Paper Contamination. Apart from the Surface water filter papers, but the threat of contamination wasn’t as high as from the others (see Graphs in the Appendix Quality Control, Filter Paper Contamination).

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5.4 Identification Problems of Microplastic Fibres

The report “A comparison of microscopic and spectroscopic identification methods for analysis of microplastic in environmental samples” by Young Kyoung Song et al., pointed out the discrepancies in the identification of microplastic fibres and other fibres (see figure below).

Figure 22 Pictures of natural fibres: (a) non-plastic (organic), (b) non-plastic (cotton) and (c) non plastic (rayon) and synthetic fibres: (d) and (e) impact polypropylene (Song, et al., 2015)

Therefore, it can only be assumed that the discovered fibres are from microplastic origin though it cannot be certain but is highly possible.

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