Wp5regionalreportbiota

DERELICT FISHING GEAR MANAGEMENT SYSTEM IN THE ADRIATIC REGION (DEFISHGEAR)

Pilot assessment on microplastic in guts of commercially available fish species and mussels Report for WP5

September, 2016

Derelict Fishing Gear management system in the Adriatic Region Authors: Špela Koren (IWRS), Manca Kovač Viršek (IWRS), Monika Peterlin (IWRS), Andreja Palatinus (IWRS), Petra Horvat (NIC), Andrej Kržan (NIC), Jasna Šiljić (IOF), Dubravka Bojanić Varezić (IOF), Mišo Pavičić (IOF), Pero Tutman (IOF), Cristina Mazziotti (ARPAER), Paola Martini (ARPAER), Catherine Tsangaris (HCMR), Nikoletta Digka (HCMR), Christina Zeri (HCMR), Andrej Gajić (HEIS), Marina Fusco (HEIS), Admir Aladžuz (HEIS), Melina Džalić-Valjevac (HEIS), Gulielm Kroqi (AUT), and Jerina Kolitari (AUT).

Report editors: Špela Koren, Manca Kovač Viršek Contact information: DeFishGear website: http://www.defishgear.net/

Contact persons: Špela Koren, PhD (associate researcher): spela.koren@izvrs.si Manca Kovač Viršek, PhD (WP5 leader and project coordinator on Institute for Waters of the Republic of Slovenia): manca.virsek@izvrs.si Monika Peterlin, PhD (coordinator of Marine Sector at Institute for Waters of the Republic of Slovenia): monika.peterlin@izvrs.si

Institutions: ARPAER: Regional Agency for Environmental Protection in the Emilia-Romagna region, Italy AUT: Agricultural University of Tirana, Laboratory of Fisheries and Aquaculture, Albania CMCC: Euro-Mediterranean Center on Climate Change HCMR: Hellenic Centre for Marine Research, Institute of Oceanography, Greece HEIS: Hydro-Engineering Institute of the Faculty of Civil Engineering, Bosnia and Herzegovina IOF: Institute for Oceanography and Fisheries, Croatia IWRS: Institute for water of the Republic of Slovenia, Slovenia NIC: National Institute of Chemistry, Laboratory for polymer chemistry and technology, Slovenia

Derelict Fishing Gear management system in the Adriatic Region

This document was produced as final report in DeFishGear project in work package 5 (WP5) – Microplastics, by the financial assistance of the IPA Adriatic Cross-Border Cooperation Programme.

DeFishGear: Derelict Fishing Gear Management System in the Adriatic Region Project code: 1°str/00010 Project duration: 1. 11. 2013 – 30. 9. 2016 Programme: IPA Adriatic Cross-border Cooperation Programme 2007 – 2013

Disclaimer This document has been produced with the financial assistance of the IPA Adriatic Cross-Border Cooperation Programme. The contents of this document are the sole responsibility of Institute for Water of the Republic of Slovenia and can under no circumstances be regarded as reflecting the position of the IPA Adriatic Cross-Border Cooperation Programme Authorities.

Institute for water of the Republic of Slovenia (IWRS) has taken due care in the preparation of this report to ensure that all facts and analysis presented are as accurate as possible within the scope of the project. However no guarantee is provided in respect of the information presented, and IWRS is not responsible for decisions or actions taken on the basis of the content of this report.

Derelict Fishing Gear management system in the Adriatic Region

Acronyms FT-IR - Fourier transform infrared spectroscopy GES - Good Environmental Status MP - microplastic MSFD - Marine Strategy Framework Directive (2008/56/EC) NIR - Near infrared spectroscopy PE - Polyethylene PP - Polypropylene

Derelict Fishing Gear management system in the Adriatic Region

Summary This report presents the results of monitoring of microplastics (plastic particles ≤ 5 mm) in marine biota (fish and mussels) of the Adriatic Region collected within the project “Derelict Fishing Gear Management System in the Adriatic Region” or short the “DeFishGear” project (code: 1°STR/ 00010), which was operational between 1/11/2013 and 30/9/2016, and was funded by IPA Adriatic Crossborder Cooperation Programme 2007 – 2013. This final report is the result of work package 5 (WP5), which was led by Institute for Water of the Republic of Slovenia.

The main objectives of the output “Pilot assessment on microplastic in guts of commercially available fish species and mussels” were:  

Development of harmonized sampling and sample analysis methodology for biota, using commercial fish species and mussels.

Estimation of microplastic concentration in the biota of the Adriatic Sea.

Methodology The biota sampling and microplastic separation protocol were done according to the protocol described in the Chapter 4: “Biota sampling and microplastic separation” of the document: Recommendation on regional approach to monitoring and assessment of microplastic in the marine environment, from Manca Kovač Viršek, Andreja Palatinus, Helen Kaberi, Catherine Tsangaris, Cristina Mazziotti (December 2015).

In frame of the DeFishGear project, together 12 different fish species: common pandora (Pagellus erythrinus), gilthead sea bream (Sparus aurata), red mullet (Mullus barbatus), surmullet (Mullus surmuletus), common sole (Solea solea), giant goby (Gobius cobitis), sand sole (Pegusa lascaris), european sprat (Sprattus sprattus), european pilchard (Sardina pilchardus), golden grey mullet (Liza aurata), tub gurnard (Chelidonichthys lucerna), and mediterranean horse mackerel (Trachurus mediterraneus) and 1 species of bivalve mollusc mediterranean mussel (Mytilus galloprovincialis) were examined in the years of 2015 and 2016 for the presence of microplastics (Table 1). Only 10 different fish species (without tub gurnard and mediterranean horse mackerel) and mediterranean mussel are further discussed.

The fish and mussel samples were mainly collected from fishermen or were bought on the fish market, only in Bosnia and Hercegovina and in Greece the fish were sampled by researcher by trawling. The fish and mussel samples were later dissected in the laboratory and the guts were examined for the presence of microplastics. Those were extracted, counted and weighted, categorized into 6 categories (fragments, filaments, foams, films, granules, and pellets) and analysed for their chemical structure with FT-IR. At the end estimation of density of microplastic was calculated per fish or mussel for each species and were compared among all countries in the Adriatic region.

Results The average numbers of microplastic particles in examined fish were in range from 1 to 13 microplastic particles/fish. This minimum and maximum average numbers were observed at the same fish species, Mullus barbatus from Greece and Bosnia and Hercegovina. The average number of particles found in Pagellus erithrinus, that were caught in Croatia and in Greece were 2 and 1 microplastic particles/fish, respectively. The average numbers of particles in Sardina pilchardus that were caught in the two

Derelict Fishing Gear management system in the Adriatic Region different countries by the Adriatic Sea, were 2.5 microplastic particles/fish species in Croatia and Greece, respectively.

12,95

14

Nr of particles/fish

12 10

9,45

9,45

8,4 7,3

8

5,35

6 4

1,9

2

2,68

2,54

1,88

2,4 0,88

1,26

Slovenia

Croatia

BIH

Sardina pilchardus

Pagellus erithrinus

Mullus barbatus

Sprattus sprattus

Pegusa lascaris

Mullus barbatus

Gobius cobitis

Sardina pilchardus

Pagellus erithrinus

Mullus surmuletus

Sparus aurata

Solea solea

Liza aurata

0

Greece

Average numbers of microplastic particles per fish [Nr of particles/fish] on regional level

On regional level the average numbers of microplastic particles in examined mussels were in range from 2 to 4 microplastic particles/mussel, where minimum was observed in mussels from Italy and maximum in mussel from Bosnia and Hercegovina. The average number of microplastic particles in mussels from Greece were closer to average number from Bosnia and Hercegovina, and was 3 microplastic particles/mussels.

Nr particles/mussel

14 12

10 8 6 3,67

3,15

Mytilus galloprovincialis

Mytilus galloprovincialis

Mytilus galloprovincialis

Italy

BIH

Greece

4 2

1,65

0

Average numbers of microplastic particles per mussel [Nr of particles/mussel] on regional level

Derelict Fishing Gear management system in the Adriatic Region

Conclusion Based on this study the following conclusions are presented: 

Commercially available fish and mussels from the Adriatic Sea are contaminated with microplastic. The average content of microplastics extracted were in range from 1 - 13 microparticles/fish and 2 - 4 microparticles/mussels.



The predominant type of microplastics found in fish and mussels from Bosnia and Herzegovina, Croatia and Slovenia were filaments, and the second most common were fragments. In fish from Greece and mussels from Italy, the fragments were predominant type of microplastics and the filaments were the second most common, while in the mussels from Greece the fragments and filaments found were in similar numbers.



Results of microplastics in fish samples from the Adriatic Sea shows high variability among all of 10 compared fish samples. The comparison of different fish species caught on different parts of the Adriatic Sea, that represented different habitat types, shows different type and average number of microplastics per fish among areas. These results lead to the conclusion that the density of microplastic in the marine environment is of more importance than the actual fish habitat (e.g. pelagic, benthic).



Only few microparticles collected from biota samples were successfully measured with FT-IR for the chemical composition. The prevailing chemical composition were polyethylene and polypropylene materials. These results are in correlation with the results of microplastics sampled on sea surface and from rivers in the Adriatic region.



This research, conducted in the frame of DeFishGear project, present the first valuable data on the overall state of the contamination with microplastics in commercially important fish and mussels species in years of 2015 and 2016 in the Adriatic Sea, where first coordinated research was conducted in the countries of Italy, Slovenia, Croatia, Bosnia and Herzegovina, Albania and Greece.

Derelict Fishing Gear management system in the Adriatic Region

Table of contents Table of contents ..................................................................................................................................viii 1

Introduction .................................................................................................................................... 1 1.1

Description of the teams involved in microplastic research................................................... 2

1.1.1

Albania - Agricultural University of Tirana, Tirana .......................................................... 2

1.1.2

Bosnia and Hercegovina - Hydro-Engineering Institute Sarajevo, Sarajevo ................... 2

1.1.3

Croatia - Institute for Oceanography and Fisheries, Split ............................................... 2

1.1.4

Greece - Hellenic Centre for Marine Research, Anavyssos ............................................. 3

1.1.5

Italy - Regional Agency for Environmental Protection in the Emilia-Romagna region, Cesenatico .......................................................................................................................... 3

1.1.6

Slovenia ........................................................................................................................... 3

Institute for Water of the Republic of Slovenia, Ljubljana .............................................................. 3 The National Institute of Chemistry, Ljubljana ............................................................................... 4 1.2

2

1.2.1

History of research on biota in the Adriatic Region ........................................................ 5

1.2.2

Fisheries activities in the Adriatic Sea ............................................................................. 5

Materials and Methods ................................................................................................................... 7 2.1

Biota sampling and sample analysis........................................................................................ 7

2.1.1

Biota sampling................................................................................................................. 8

2.1.2

Separation and analysis of microplastics from biota samples ...................................... 11

2.2 3

Literature review..................................................................................................................... 5

Chemical identification of microparticles ............................................................................. 15

Results ........................................................................................................................................... 16 3.1

Country level ......................................................................................................................... 16

3.1.1

Quantity of microplastics according to the number and weight .................................. 16

3.1.2

Size distribution ............................................................................................................ 24

3.1.3

Identification of plastic type ......................................................................................... 28

3.2

Regional level ........................................................................................................................ 29

4

Discusion ....................................................................................................................................... 31

5

Conclusions ................................................................................................................................... 35

5

References .................................................................................................................................... 36

Derelict Fishing Gear management system in the Adriatic Region

1 Introduction This report presents the results of monitoring of microplastics (plastic particles ≤ 5 mm) in marine biota (fish and mussels) of the Adriatic Region collected within the project “Derelict Fishing Gear Management System in the Adriatic Region” or short the “DeFishGear” project (code: 1°STR/ 00010), which was operational between 1/11/2013 and 30/9/2016, and was funded by IPA Adriatic Crossborder Cooperation Programme 2007 – 2013. This final report is the result of work package 5 (WP5), which was led by Institute for Water of the Republic of Slovenia.

The aim of WP5 was to develop regional approach to monitoring and assessment of microplastic (plastic particles in size ≤ 5 mm) and nanoplastics (≤ 0.3 mm) pollution as well as identification of impacts on marine biota, using commercial fish species. Micro and nanoplastics can damage fish’s digestion tract and enhance bioaccumulation of pollutants (organic and inorganic) adsorbed to the particles, and consequently affect human health when edible species are contaminated. Available recommendations proposed also by MSFD GES Technical Subgroup on Marine Litter was taken into account in selection of common methodology to analyse microplastic and nanoplastics. Several case studies and exchange of knowledge among partners have been performed. Common data base with public access was made and also the proposal for reduction of microplastic pollution was written.

Sampling and analyses of microplastics in marine biota were performed in 6 countries of the Adriatic Region (Albania, Bosnia and Herzegovina, Croatia, Greece, Italy and Slovenia) by 7 project partners from the DeFishGear project.

For the purpose of coordinated sampling and analysis for the presence of microplastics on the sea surface, beach sediments and biota the protocols were developed: 

Recommendation on regional approach to monitoring and assessment of microplastic in the marine environment, from Manca Kovač Viršek, Andreja Palatinus, Helen Kaberi, Catherine Tsangaris, Cristina Mazziotti (December 2015)(Kovač Viršek et al., 2015)

In this document the Chapter 4: “Biota sampling and microplastic separation” describes the methodology for sampling and analysis of microplastics in biota.

Three other reports and a management plan are also the final result of WP5:    

“Pilot assessment on microplastic in sea surface and beach sediment potential accumulation zones” (September 2016) “Pilot assessment on microplastic in fishing areas” (September 2016) “Pilot assessment on microplastic in rivers” (September 2016) “Strategic recommendations for improving marine litter management in the Adriatic Sea in the field of microplastic pollution” (Kovač Viršek, 2016).

Results of biota sampling locations with results of microplastic analysis are presented in the DeFishGear Database http://defishgear.izvrs.si/defishgear/

1

Derelict Fishing Gear management system in the Adriatic Region

1.1 Description of the teams involved in microplastic research These targeted actions related to monitoring and assessment of microplastic pollution were performed in 6 eligible countries in the Adriatic Region (Albania, Bosnia and Herzegovina, Croatia, Greece, Italy and Slovenia) by following 7 project partners from the DeFishGear project:       

Regional Agency for Environmental Protection in the Emilia-Romagna region, Cesenatico, Italy; Institute for Water of the Republic of Slovenia, Ljubljana, Slovenia; National Institute of Chemistry, Ljubljana, Slovenia; Institute for Oceanography and Fisheries, Split, Croatia; Hydro-Engineering Institute Sarajevo, BIH; Agricultural University of Tirana, Albania; Hellenic Centre for Marine Research, Greece.

1.1.1 Albania - Agricultural University of Tirana, Tirana Agricultural University of Tirana (AUT) is a unique center for education in the field of Agriculture in Albania. The Aquaculture and Fisheries Laboratory is the only marine research laboratory in Albania.

Research activities conducted encompass aspects of sea exploration: chemical, biological parameters and fisheries. Nowadays the laboratory employs has about 13 permanent staff members and a number of University academic staff participating in cases of projects and researches, mostly researchers and PhD students. The DeFishGear team from Albania didn’t have any experiences with the microplastics in biota prior to the project.

1.1.2 Bosnia and Hercegovina - Hydro-Engineering Institute Sarajevo, Sarajevo The Hydro-Engineering Institute Sarajevo (HEIS) is independent, market oriented, consulting, designing, scientific, and research institute. The Institute employs about 60 people, of which more than 40 are researchers and designers - experts in different fields. The team working on analysis of microplastics in marine biota in DeFishGear project had no previous experiences on the topic.

Therefore they performed extensive literature review prior to the beginning of the sampling of biota and microplastic analysis to get knowledge about this topic.

1.1.3 Croatia - Institute for Oceanography and Fisheries, Split The Institute of Oceanography and Fisheries (IOF) is a scientific public institute and it is National Reference Centre for the marine research. Institute employs 116 people of whom 71 are experts in different field of which 50 are professionals with PhD. Scientific activities conducted in the IOF are interdisciplinary including biological, chemical, physical, geological and fishery research. The research vessels Bios and Navicula, owned by IOF, are used for oceanographic and fisheries research activities, designed specifically for surveys and data collection for both coastal and open-sea waters.

The IOF team involved in DeFishGear project are employees of two laboratories; Laboratory of ichthyology and coastal fisheries and Laboratory of fisheries science and management of pelagic and demersal resources. The team includes scientists who are experts in the fields of biology and biotechnology, dealing with the biology and ecology of fishes and their developmental stages, trophic 2

Derelict Fishing Gear management system in the Adriatic Region ecology, population dynamics, all aspects of fisheries including stock assessment and management of pelagic, demersal and coastal resources. At the beginning of the DeFishGear project, two people were hired to be fully involved in the activities assigned by the project and they were sent for training in order to specialize in collection and processing samples of microplastics.

1.1.4 Greece - Hellenic Centre for Marine Research, Anavyssos Hellenic Centre for Marine Research (HCMR) has participated in a number of projects related to the topic of marine litter. In the framework of the FP7 project PERSEUS (2011-2015) the seafloor litter was studied in the Eastern Mediterranean and the Black Sea. The methodology for the analysis of microplastics in sediments was developed in the framework of the FP7 project CLEANSEA (2012-2016).

Finally, in the framework of the FP7 SEAS ERA project MERMAID (2012-2015), besides other descriptors, the marine litter descriptor (D10) was studied in the Gulf of Lions (France), Saronikos Gulf (Greece) and the Cilician basin (Turkey), linking the defined targets for marine litter to the corresponding measures.

1.1.5 Italy - Regional Agency for Environmental Protection in the EmiliaRomagna region, Cesenatico Regional Agency for Environmental Protection in the Emili-Romagna region (ArpaER) carries out research about coastal marine systems with emphasis on multidisciplinary ecosystem complexity: the main activities include research, development and application of monitoring programme to comply with regional, national, EU laws. During the 2013 the ArpaER was involved in sea surface microplastics analysis for the Marine Strategy. In the 2015 they started again with the activities about Marine Strategy and therefore they are involved in sea surface microplastics analysis. In Italy the microplastics analysis in the beach sediment were not taken into consideration until now.

The team is composed from marine biologists and technicians that are involved in sampling activities and laboratory analysis with a couple years of experience in this skill.

1.1.6 Slovenia Institute for Water of the Republic of Slovenia, Ljubljana Institute for water of the Republic of Slovenia (IWRS) implements common European water policy and international strategies and conventions, participates in expert working groups of the Common Implementation Strategy process of directives at the DG Environment of the European Commission and activities of the European Environment Agency. IWRS elaborates water management and marine management studies and technical background legal regulations which mainly support the Ministry of Agriculture and the Environment and develops approach to monitoring and management of marine litter pollution, including beach litter and microplastic and has been involved in Clean Coast, Marine Renegades and other NGO projects.

The Institute is involved in marine litter research since 2010 and has the leading role in microplastic analysis in Slovenian sea water since 2011. The team is composed from biologists and technicians from Sector for Marine Waters that are involved in sampling activities and laboratory analysis of microplastics with a couple years of experience in this skill.

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Derelict Fishing Gear management system in the Adriatic Region

The National Institute of Chemistry, Ljubljana The National Institute of Chemistry (NIC) is a public research institution active in all areas of chemistry.

It was the first research institution in Slovenia to obtain ISO 9001 standards in 2003. It offers highlevel research equipment such as NMR spectrometers, ultra-high resolution microscopes, chromatography systems etc. and is routinely involved in national and international research frameworks. The Department for Polymer Chemistry and Technology (D07) with its 17 members is specialized in synthesis and characterization of polymers and plastics. It provides a full array of chemical polymer/plastics characterization expertise: spectroscopy, chromatography, thermal methods, as well as an excellent staff including 9 Ph. D. experts.

The laboratory has a history of activity in the broad area of environmental aspects of plastics and polymers including plastic waste management, plastics recycling, biodegradability of plastics, biobased plastics and bionanocomposites. Through its project involvement D07 has expertise in plastics sustainability issues from its strong involvement in bioplastics as well as the legal, standardization/certification and strategic aspects of plastic waste management, plastic pollution in the environment and environmental solutions such as biodegradable plastics.

Through the waste management and biodegradability issues the team lead by dr. Andrej KrĹžan has had exposure to the issue of marine litter and microplastics. In cooperation with the University of Nova Gorica dr. KrĹžan lead the first studies of microplastics occurrence in the Adriatic sea (Slovenian part), which started in 2011 (two studies finished by 2015). The results of these exploratory studies were included in the national reporting as part of fulfilling the requirements for the Marine Strategy Framework Directive.

4

Derelict Fishing Gear management system in the Adriatic Region

1.2 Literature review 1.2.1 History of research on biota in the Adriatic Region Microplastic pollution is relatively new recognized threat to the environment. It was first identified and documented from in 1970s, when several researches published first findings about microplastics appearance in environment (Carpenter et al., 1972; Colton et al., 1974; Kartar et al., 1973). In 2008 there was an “International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris” which brought microplastics into broader attention of the scientific and political community (Allsopp et al., 2009).

In Albania, Bosnia and Herzegovina, Croatia, Greece and Italy there were no studies done on assessment of microplastic pollution in biota from the Adriatic region prior to the DeFishGear project.

A rare exception in this field are data from Slovenia where one preliminary study was conducted on selected fish species caught in the Fisheries Reserve from Slovenian Sea (Bajt, 2015). Fishes were analysed for their stomach content and several plastic pieces were found in 15% of the stomachs.

Microplastics were mostly found in the stomachs of European seabass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) (40% of stomachs), rarely in common pandora (Pagellus erythrinus), but never in the stomachs of golden grey mullet (Liza aurata), although this species is the most abundant in the area. Microplastic particles found were fibres, pieces of spherical shape, and wires, all determined as polyamide or polyethylene using FTIR and most ranging bellow 500 μm size (Bajt et al., 2015).

Therefore this report presents the first consistent results on presence, quantity and type of microplastic particles in biota (fish and mussels) from the Adriatic Sea for the years of 2015 and 2016.

1.2.2 Fisheries activities in the Adriatic Sea The Adriatic Sea is one of the largest areas of occurrence of demersal and small pelagic shared stocks in the Mediterranean.

The main small pelagic species are sardine (Sardina pilchardus), anchovy (Engraulis encrasicolus), horse mackerel (Trachurus spp.) and mackerel (Scomber spp.). In the northern area, sprat (Sprattus sprattus) is found, although it was more abundant during the 1960s and 1970s than nowadays (United Nations Environment…, 2014).

On the depth from 10 to 50 m (continental shelf) the dominant fish species in terms of biomass are red mullet (Mullus barbatus), poor cod (Trisopterus minutus), various species of triglids, common sole (Solea solea), various species of flatfishes, gobies and pandoras (Pagellus spp.). From 50 to 100 m deep, anglerfish (Lophius spp.), european hake (Merluccius merluccius), greater forkbeard (Phycis blennoides) and red bandfish (Cepola rubescens) are also abundant, as well as blue whiting (Micromesistius poutassou) at 100 to 200 m deep (United Nations Environment…, 2014).

Fishing gears In the Adriatic sea two kind of fishing gears are mainly used to catch small pelagic species (mainly anchovy and sardine): »volante« a mid-water pelagic trawl net towed by two vessels, mostly used in the northern and central areas by Italian fleet. Another type of catching small pelagic species is light attraction purse seine, which is used in the Gulf of Trieste and south of Ancona on the Western side 5

Derelict Fishing Gear management system in the Adriatic Region and it is the main gear used in Slovenia, Croatia and Montenegro and Albania (United Nations Environment…, 2014).

For fishing of demersal species like red mullets, bottom trawls are used, and the »rapido«, another type of bottom gear, is used for the demersal fishery. Bottom trawls and Rapido trawls can induce severe sub-lethal and lethal damages on non-target species. Along the Croatian coast bottom trawl fisheries is mainly regulated by spatial and temporal fisheries over whole year. Also bottom trawl fishery is closed half year in the majority of inner sea (United Nations Environment…, 2014).

National fleets operating in the area Italy: The Italian pelagic fleet is composed primarily of purse seiners (»lampara vessels«) and midwater pelagic pair trawlers (»volante«). Total catches (not discriminated by species) of purse seiners in 2010 were 5,747 tons and of mid-water pair trawlers 44,393 tons (United Nations Environment…, 2014).

Albania: The fishing fleet is composed mainly by bottom trawlers, followed by trammel and gillnetters, hooks and lines, and purse seiners. Catch data is not available, but it is likely to be about 800 tons per year (United Nations Environment…, 2014).

Croatia: The fishing fleet is composed mainly with purse seiner. The main catches are small pelagic fish as sardines and anchovy with recorded landings of 44,614 tons and 14,163 tons in 2011, respectively (United Nations Environment…, 2014).

Slovenia: The fishing fleet is mainly composed of pure seiners that mostly catch sardines and anchovies. The pair trawlers stopped operation in 2012 as a result of the implementation of the measure for permanent cessation of fishing activities. Total catchment was 169 tons of fish and was 25 tons of mollusc, both in 2015 (Statistical office of Republic of Slovenia, http://www.stat.si/statweb/en/home).

Bosnia and Hercegovina: Total fish farms and fish production was 2,853 tons in 2013 (Food and Agriculture…, 2015).

Bivalve mollusc fishery seems to be quite significant in the Adriatic area, especially in the North West basin due to edaphic and trophic conditions. Mussels are usually harvested (collected) by hand and less frequently, where rich mussels’ beds on lagoon bottoms are present, through bottom trawl fishery (United Nations Environment…, 2014).

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Derelict Fishing Gear management system in the Adriatic Region

2 Materials and Methods 2.1 Biota sampling and sample analysis The biota sampling and microplastic separation protocol is described in the Chapter 4: “Biota sampling and microplastic separation” of the document: Recommendation on regional approach to monitoring and assessment of microplastic in the marine environment, from Manca Kovač Viršek, Andreja Palatinus, Helen Kaberi, Catherine Tsangaris, Cristina Mazziotti (December 2015) (Kovač Viršek, 2016).

In frame of the DeFishGear project, together 12 different fish species and 1 species of bivalve mollusc from the Adriatic Sea were examined for the presence of microplastics (Table 1, Figure 1). Only 10 different species are further discussed (without Chelidonichthys lucerna and Trachurus mediterraneus).

Table 1: Sampled fish and bivalve mollusc species in years of 2015 and 2016 by the country.

Country

Sampled fish species

Italy Slovenia

Croatia

Bosnia and Hercegovina

Albania

Greece

Sampled bivalve mollusc species Mytilus galloprovincialis

Liza aurata Solea solea Sparus aurata Mullus surmuletus Pagellus erythrinus Sardina pilchardus Gobius cobitis Mullus barbatus Pegusa lascaris Sprattus sprattus Mullus barbatus Pagellus erythrinus Solea solea Trachurus mediterraneus Triglia lucerna Mullus barbatus Pagellus erythrinus Sardina pilchardus

7

Mytilus galloprovincialis

Mytilus galloprovincialis

Derelict Fishing Gear management system in the Adriatic Region

Figure 1: Locations of all sampling sites of fish and mollusc species in the Adriatic Sea. Source: https://www.google.com/earth/

2.1.1 Biota sampling 2.1.1.1 Albania The fish for biota analyses were bought by local fisherman which also provided the dates and coordinates of the fishing of each specimen. Mullus barbatus and Triglia lucerna were caught on 24th of January 2016. Solea solea and Trachurus mediterraneus were caught on 22th of January 2016 on location near town of Lezha. Pagellus erythrinus and Sardina pilcardus were caught on 2nd of February 2015, near the town of Vlora. Technical data referred to this survey are described in Appendix, Table A1.

2.1.1.2 Bosnia and Herzegovina Fish material used in the studies is presented with three main samples consisting of 20 individuals of following species: Mullus barbatus Linnaeus, 1758, Solea solea (Linnaeus, 1758) and Gobius cobitis Pallas, 1814 caught by 2 DeFishGear teams on a trawl studies conducted in the Neum bay in years 2015 and 2016 (Appendix, Table A2).

Biota sampling was conducted with improvised bottom seine (trawl) in selected polygons within the Neum bay, with variations in total length ranging from 1000 to 3000 m. In order to obtain a more precise and concrete information regarding the presence of both macro- and microplastics within the digestive tube of certain marine taxa, sampling of target taxa was performed at four different sites within the studied area at depth between 18 and 25 m. Total sampling area amounted 105 km2.

Considering the circadian rhythm of the target taxa, trawl samplings was performed in the period 8

Derelict Fishing Gear management system in the Adriatic Region ranging from 20:00 to 03:00. Certainly, considering the specificity of studied habitats, as well as the current conditions during the field sampling, a certain degree of deviation from the planned polygons was allowed.

Upon the trawl sampling biological material were adequately enumerated according to the following principle XX-BA/1Y - where XX present the ordinal number, BA stands for Bosnia and Herzegovina, while Y is the last character of the year in which monitoring was carried out. Thus, samples were enumerated according to the following principle XX-YY/1Z - where XX represent the first (or any other) letters in the scientific name of the species, YY present ordinal number of the sample and Z present last character of the year in which monitoring were performed (Figure 2a and b).

In total 4 different fish species with total of 20 individuals per sample were selected for laboratory analysis: 

Demersal fish: Mullus barbatus (Perciformes: Mullidae) MB-0X/1Y, Pegusa lascaris (Pleuronectiformes: Soleidae) PL-0X/1Y, and Gobius cobitis (Perciformes: Gobiidae) GC0X/1Y



Pelagic fish: Sprattus sprattus (Clupeiformes: Clupeidae) SS-0X/1Y.

The project team bought additional 80 specimens of the Sprattus sprattus (Clupeiformes: Clupeidae) at total five different fish markets in Sarajevo city, Bosnia and Hercegovina.

The basic morphometrical characteristics, life stage, presence of ecto- or endoparasites and any other pathological changes of individuals are presented in Appendix,

9

Derelict Fishing Gear management system in the Adriatic Region Table A4: Data table of Pegusa lascaris specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

Ecto/Endo parasites

Pathological changes

PL-01/15

22.5

95

Adult

/

/

PL-02/15

18.8

35

Adult

/

/

PL-03/15

17.4

35

Adult

/

/

PL-04/15

20.2

65

Adult

/

/

PL-05/15

20.2

75

Adult

/

/

PL-06/15

15.9

30

Juvenile

/

/

PL-07/15

13.2

15

Juvenile

/

/

PL-08/15

8.9

5

Juvenile

/

/

PL-09/15

13.6

15

Juvenile

/

/

PL-10/15

13.4

15

Juvenile

/

/

PL-11/16

10

4.8

Juvenile

/

/

PL-12/16

10

4.7

Juvenile

/

/

PL-13/16

8.5

3.7

Juvenile

/

/

PL-14/16

9.5

4.3

Juvenile

/

/

PL-15/16

8

3.5

Juvenile

/

/

PL-16/16

8

3.7

Juvenile

/

/

PL-17/16

8

3.9

Juvenile

/

/

PL-18/16

9

4.3

Juvenile

/

/

PL-19/16

6

2.4

Juvenile

/

/

PL-20/16

13.8

15

Juvenile

/

/

Sample No.

10

Derelict Fishing Gear management system in the Adriatic Region Table A5: Data table of Gobius cobitis specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

GC-01/16

7.5

15.56

Adult

GC-02/16

10

11.75

Adult

GC-03/16

10.9

12.98

Adult

GC-04/16

10

10.77

Adult

GC-05/16

9.2

6.90

Adult

GC-06/16

9

8.09

Adult

GC-07/16

10.4

11.16

Adult

GC-08/16

9.9

9.82

Adult

GC-09/16

10

9.04

Adult

GC-10/16

9.5

9.68

Adult

GC-11/16

10.5

11.72

Adult

GC-12/16

8.6

10

Adult

GC-13/16

9

7.70

Adult

GC-14/16

8

4.36

Adult

GC-15/16

8.2

5.29

Adult

GC-16/16

9

6.67

Adult

GC-17/16

7

4.80

Adult

GC-18/16

9

8.57

Adult

GC-19/16

8.3

5.05

Adult

GC-20/16

10

9

Adult

Sample No.

11

Ecto/Endo parasites Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine

Pathological changes / / / / / / / / / / / / / / / / / / / /

Derelict Fishing Gear management system in the Adriatic Region Table A6: Data table of Sprattus sprattus specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

Ecto/Endo parasites

Pathological changes

SS-01/16

11

8.34

Adult – F

/

/

SS-02/16

9.33

8.01

Adult – M

/

/

SS-03/16

8.1

5.03

Adult – F

/

/

SS-04/16

9.8

8.23

Adult – M

/

/

SS-05/16

10.1

9.23

Adult – F

/

/

SS-06/16

9.6

9.10

Adult – F

/

/

SS-07/16

10.2

9.90

Adult – M

/

/

SS-08/16

8.9

8.94

Adult – F

/

/

SS-09/16

10.3

9.52

Adult – F

/

/

SS-10/16

10.3

9.44

Adult – M

/

/

SS-11/16

9.9

7.68

Adult – F

/

/

SS-12/16

9.2

7.76

Adult – M

/

/

SS-13/16

9.0

7.2

Adult – M

/

/

SS-14/16

7.2

6.9

Adult – F

/

/

SS-15/16

8.8

6.94

Adult – M

/

/

SS-16/16

8.3

5.05

Adult – F

/

/

SS-17/16

8.9

7.46

Adult – F

/

/

SS-18/16

9.5

9.10

Adult – F

/

/

SS-19/16

8.7

6.82

Adult – M

/

/

SS-20/16

10.7

10.77

Adult – M

/

/

Sample No.

Legend: M: male, F: female

12

Derelict Fishing Gear management system in the Adriatic Region Table A7 - Table A6.

The samples of mussels (total 30 individuals) have been collected from the local aquaculture facilities “Ancora” Inc., Neum. a)

b)

Figure 2: a) The red mullet (Mullus barbatus) and b) the European hake (Merluccius merluccius) – two of the common fish species caught during the trawl studies within the Neum bay. Photo: A. Kahrić.

2.1.1.3 Croatia (Sampling area: Split region) Three commercial fish species were examined for microplastics representing three different habitats, and consequently feeding habits, benthic Mullus surmuletus, benthopelagic Pagellus erythrinus and pelagic Sardina pilchardus. In collaboration with fishermen the samples were collected from April to October 2015 (Appendix, Table A7) along the eastern coast of the Adriatic Sea, representing the most famous fishing areas in Croatia. Thirty individuals of each species (in total 90 specimens) were dissected and investigated for microplastic particles.

The samples of Mullus surmulets were collected by purse seine in two different locations, around Cres Island and Paklenjaci archipelago during June and October 2015, respectively. Individuals of Pagellus erythrinus were captured during September 2015 by trawling in wider Zadar area (around Pašman and in Virsko more) and in Istria area (Rt Kamenjak). Samples of Sardina pilchardus were taken from seine nets in April 2015, around island of Drvenik.

Relatively small individuals of Mullus surmuletus were represented in our sample, and consequently there were found a high number of immature specimens (N = 9) (Appendix, Table A8). The total length of specimens were in range from 10.1 to 25.5 cm, with an average of 19.5 (SD ± 5.33).

The specimens of Pagellus erythrinus were mostly in second maturity stage, with relatively small gonads in both, males and females (Appendix, Table A9). The total length of individuals were in range from 12.5 to 22.3 cm, with an average of 17.37 cm (SD ± 3.06).

The individuals of Sardina pilchardus were mostly in the same size category, with an average value of 13.2 cm (SD ± 0.73). The specimens were in range from 11.5 to 14.6 cm, with an average weigh of 16.87 g (Appendix, Table A10).

13

Derelict Fishing Gear management system in the Adriatic Region

2.1.1.4 Greece (Sampling area: Corfu island) Mussels and fish were sampled in the Northern Ionian Sea (Appendix, Table A11). This area is mainly influenced by agriculture, aquaculture, fisheries, maritime transport and tourism. Fish (Sardina pilchardus, Pagellus erythrinus and Mullus barbatus) were caught by offshore trawling North of Corfu in April 2015. The fish sampling was conducted using the experimental trawling vessel R/V ‘Filia’.

Mussels Mytilus galloprovincialis were collected by hand from the port of Corfu and from a mussel farm in Thesprotia in June and July 2015. Sampling dates and locations are shown in Appendix, Table A11. Mussels and fish were stored at -20°C prior microplastic detection. Fish/mussel length, fish weight, fish gender and fish maturity stage are shown in Appendix, Table A12 - Table A17.

2.1.1.5 Italy (Sampling area: Gulf of Venice and Cesenatico region) The analyses were conducted on two different group of mussels of Mytilus galloprovincialis of 30 individuals each, coming from farms in Chioggia and Cesenatico, respectively (Appendix, Table A18). The mussels were collected directly by farmers in July 2015 and brought to the laboratory. A first comparison of average length and width values revealed that mussels from Chioggia were slightly smaller than those from Cesenatico (Chioggia pool Length 6.12 cm Width 3.01; Cesenatico pool Length 6.78 cm Width 3.25 cm) (Appendix, Table A19

14

Derelict Fishing Gear management system in the Adriatic Region Table A19).

2.1.1.6 Slovenia The fish for biota analyses were bought from local fisherman which also provided the dates and coordinates of the fishing surfvey for each specimen. Golden grey mullet (Liza aurata) was caught on 19th of September 2015 on location near town of Ankaran. Gilthead sea bream (Sparus aurata) and common sole (Solea solea) were caught on 2nd and 4th of November 2015, respectively, and both were caught near the town of Izola. Technical data referred to sampling are described in Appendix, Table A20, and technical data referred to individual fishes from all three fish species are in Appendix, Table A21 - Table A23.

2.1.2 Separation and analysis of microplastics from biota samples Separation of microplastics from biota samples and further analysis was performed according to the protocols (See chapter 2.1 Biota sampling and sample analysis).

National adaptations of the protocols and examples of laboratory work are presented below.

2.1.2.1 Albania The photos of dissection of fish samples (Figure 3).

Figure 3: Dissection of fish samples. Photo: AUT, Albania.

2.1.2.2 Bosnia and Herzegovina Fish samples In order to avoid possible contamination in significant extent, complete team was equipped with surgical caps, masks, long-sleeved laboratory coats and latex surgical gloves (Figure 4).

Autopsy tools used in isolation of target parts of the digestive tubes are: scalpels with size 2, 3 and 4 with the usage of different surgical blades (no. 10, 11, 15 and 24) then surgical and anatomical tweezers, surgical and anatomical scissors, forceps and different probes.

15

Derelict Fishing Gear management system in the Adriatic Region

Figure 4: Treatment of dissected samples of the Sprattus sprattus, Bosnia and Hercegovina. Photo: K. Selman and A. Gajić / Sharklab ADRIA.

Mussel samples The mussel were frozen immediately after sampling and were transported to the laboratory.

Immediately upon defrost the samples were dissected and treated according to the protocol (Monitoring microplastic litter in biota..., 2015).

2.1.2.3 Croatia Due to the lack of appropriate software microplastics image analysis and measures of separated particles on analytical laboratory scale weren’t performed in the laboratory of IOF. Instead the samples were sent to National Institute of Chemistry in Ljubljana, Slovenia.

Special attention was given to avoid contamination from the air, a glass box was used during fish dissection for this propose (Figure 5) and the stereomicroscope had a nylon cover during visual examination.

16

Derelict Fishing Gear management system in the Adriatic Region

Figure 5: Dissection of fish samples in the glass box to avoid contamination from the air. Photo: IOF, Croatia.

2.1.2.4 Greece Flotation separation was omitted after H2O2 digestion of the samples on a hot plate. Digestion of samples with H2O2 on the hot plate was more or less full, so there was no need for separation of microplastics by flotation. Also the samples were heated on the hot plate until H2O2 was evaporated to nearly 1 ml (not fully evaporated) to avoid sample sticking on the bottom of the vial.

The microparticles were not weighed as it was very difficult to handle with forceps due to their small size.

To reduce contamination by air transported fibers, additional precautions were applied (Torre et al., 2016). For initial examination of fish gastrointestinal content, the stereomicroscope observation area was isolated using a plastic cover. This procedure considerably reduced fiber contamination in blank filters placed in the working area. During digestion and when not in use, the samples were always covered. A glove bag was used as working area for sample filtration (Figure 6). These precautions considerably removed fiber contamination in procedural blanks (negative controls). During observation under the stereomicroscope, the filters were covered with glass lids.

17

Derelict Fishing Gear management system in the Adriatic Region

Figure 6: Flotation separation and filtration of samples in the glove box to avoid airborne contamination. Photo: HCMR, Greece.

2.1.2.5 Slovenia Only one adaptation of the protocol (Monitoring microplastic litter in biota..., 2015) was done. In step 5.c in chapter “4.3.2 Laboratory separation of microplastics in fishes” of the protocol the incubation of sample with 20 ml of 30% H2O2 per 1g of wet weight of stomach or intestine was done at room temperature for 24h and not at 55 – 65°C in heated water bath.

Prior to the laboratory work the sticky roller was used to clean all the fibres form laboratory coats in order to reduce contamination of the samples with fibres from clothes.

18

Derelict Fishing Gear management system in the Adriatic Region

2.2 Chemical identification of microparticles Chemical identification of microplastic particles was done by the use of ATR FT-IR microscopy and Near IR spectroscopy on Chemical Institute in Ljubljana, Slovenia. An FTIR microscope combines the function of a microscope and an infrared spectrometer. This allows recording a spectrum on a very small area suitable for analysis of microplastics smaller than 1 mm.

Microplastics particles were placed on a glass filter and the ATR germanium crystal was cleaned using alcohol and lint free cloth. The filter with the microparticles was placed on the automatic scanning table and the joystick was used to locate the sample and to record an optical image. The measured area was 20 x 20 Âľm in size. Spectra of sample area was compared with the spectrum in spectral database to identify the composition of the sample.

Greek samples of microparticles were analysed by the use of FT-IR spectroscopy. This technique was used to confirm the synthetic polymer composition of microparticles that were large enough to handle with forceps and analysed on an Agilent Cary 630 FTIR spectrometer using a self-generated polymer library. However, FT-IR microscopy was required to analyze the majority of microparticles found in mussels and fish.

19

Derelict Fishing Gear management system in the Adriatic Region

3 Results 3.1 Country level 3.1.1 Quantity of microplastics according to the number and weight There were difficulties to weigh-in the particles extracted from biota, since the particles were so low in number and small that they were too light to weight on analytical scale. Apart from this, Greek researchers expressed the number of microparticles also per g of tissue (digestive gland and gills) and Italian researchers expressed the number of microparticles per g of mussel.

3.1.1.1 Albania There were small variability of different microparticles found in all six species of fish, since there were only filaments and smaller fragments.

3.1.1.2 Bosnia and Herzegovina Fish samples During the laboratory studies plastic particles have not been observed within the oral cavity nor the oesophagus. Despite, both macroplastic and microplastic particles were fairly often observed in the pyloric stomach. The average number of collected particles at all fish species were in average in range from 5-13 particles per fish or 12.95 particles/fish at Mullus barbatus, 9.45 particles/fish at Pegusa lascaris, 8.4 particles/fish at Gobius cobitis and 5.35 at Sprattus sprattus (Figure 7).

Significant number of filaments and fragments was observed in all samples, regardless of the size of the individual. In all 4 species, filaments were predominant type of microplastic particles with 83% at Mullus barbatus, 94% at Pegusa lascaris, 89% at Gobius cobitis and 99% at Sprattus sprattus. The second most often in guts of fish were fragments with 14.29% at Mullus barbatus, 5.82% at Pegusa lascaris, 8.93% at Gobius cobitis and 0.93% at Sprattus sprattus (Figure 8).

12

Nr of microparticles/fish

10 8 Fragments

6

Filaments Other

4 2 0 Mullus barbatus Pegusa lascaris

Gobius cobitis Sprattus sprattus

Figure 7: Average number of all microplastic particles per one individual per fish species, Bosnia and Hercegovina (2015-2016).

20

Derelict Fishing Gear management system in the Adriatic Region 100% 80% 60% 40% 20% 0%

Mullus barbatus

Pegusa lascaris

Gobius cobitis

Sprattus sprattus

Other

3,09%

0,00%

2,38%

0,00%

Fragments

14,29%

5,82%

8,93%

0,93%

Filaments

82,63%

94,18%

88,69%

99,07%

Filaments

Fragments

Other

Figure 8: Categories composition of microplastic particles for each fish species, Bosnia and Hercegovina (2015 - 2016).

Mussel samples Within the sample of Mytilus galloprovincialis Lamarck, 1819 (Bivalvia: Mytiloida) composed from 30 individuals, total of 110 filaments or in average 3.67 filaments per individual mussel have been observed, while fragments have not been noticed.

3.1.1.3 Croatia Different number of microplastic particles per fish were in range from 1 to 7. Mostly were found only one, and occasionally a few pieces per gut. In some guts of all examined species occurred more pieces of microplastics. Average number of ingested pieces per gut for Mullus surmuletus and Sardina pilchardus (2.68 ± 1.81 and 2.54 ± 1.12, respectively) were higher than in Pagellus erythrinus (1.88 ± 1.12) (Table 2).

A total of 34 microplastic particles (3 fragments; 26 filaments; 3 foam and 2 other type of microplastics) were visually observed in Pagellus erythrinus. Of 59 reported particles in Mullus surmuletus, 45 were filaments, while fragments, films and foams were found in smaller amounts. In guts of Sardina pilchardus only fragments (N=9) and filaments (N=19) were found (Table 4). In 21 of total 30 (70%) guts of Mullus surmuletus were found plastic pieces and in 15 guts of Pagellus erythrinus (50%). Lower number of microplastics was recorded in Sardina pilchradus, approximately 35% of total examined specimens.

Table 2: Number of microplastics (MP) found in guts in three different fish species in Croatia in 2015.

Mullus surmuletus

Pagellus erythrinus

Sardina pilchardus

No. of guts

30

30

30

No. of guts contained MP

21

15

11

No. of microplastics in all guts

59

34

28

2.68 ± 1.8

1.88 ± 1.4

2.54 ± 1.1

Average No. of MP per gut

21

Derelict Fishing Gear management system in the Adriatic Region Filaments were predominant category found in inspected fish species usually with more than one filament per fish gut. Except the filaments, occasionally were found few fragments in Mullus surmuletus and Pagellus erythrinus. From other categories only one piece per fish gut were found, except the films in Mullus surmuletus (2 pieces per fish) (Figure 9).

4,5

Nr of microparticles/fish

4 3,5

3 2,5 2

1,5 1 0,5 0 Mullus surmuletus Filaments

Pagellus erythrinus

Fragments

Films

Foam

Sardina pilchardus Other

Figure 9: Average number of all microplastic particles per one individual per fish species, Croatia (2015).

In all of three species, filaments were predominant microplastic category with 67.68% (Sardina pilchardus), 76.27% (Mullus surmuletus) and 76.47% (Pagellus erythrinus). A relatively high percentage of fragments were noted in guts of Sardina pilchardus (32.14%), while that type of plastic was less represented in guts of Mullus surmuletus (6.78%) and Pagellus erythrinus (8.82%). Films were also occurred in digestive tract of Mullus surmuletus and Pagellus erythrinus, with a contribution of 6.78% and 8.82%, respectively, while other categories were found in smaller amounts (Figure 10).

100% 80% 60% 40% 20% 0%

Mullus surmuletus

Pagellus erythrinus

Other

Sardina pilchardus

5,88%

Foam

1,69%

Films

6,78%

8,82%

Fragments

6,78%

8,82%

32,14%

Filaments

76,27%

76,47%

67,86%

Filaments

Fragments

Films

Foam

Other

Figure 10: Categories composition of microplastic particles for each fish sample, Croatia (2015).

22

Derelict Fishing Gear management system in the Adriatic Region

3.1.1.4 Greece Items resembling microplastics were found in 14 out of 38 sardine (S. pilchardus), 9 out of 19 common pandora (P. erythrinus), 12 out of 25 red mullet (M. barbatus) and 67 out of 80 mussel (M. galloprovincialis) samples examined. These items were fragments and filaments of various sizes and colours. Items resembling those found in procedural blanks were not taken into account.

The average numbers of filaments and fragments resembling microplastics per fish per species are shown in Figure 11. The average number of all microplastic particles (sum of filaments and fragments) per fish in the three species ranged from 0.88 to 2.53 and was higher in S. pilchardus samples. Among colours, blue, pink, black, grey, transparent, orange, green and brown items were found in the fish samples.

The average numbers of filaments and fragments resembling microplastics per mussel in each sampling area are shown in Figure 12. Blue, pink and transparent items were the most abundant. The average number of all microplastic particles (sum of filaments and fragments) per mussel in the two sampling areas ranged from 3.05 to 3.25 and was slightly higher in the mussels from the mussel farm.

On the other hand, when calculating the average number of microplastics per g of tissue (digestive gland and gills), mussels from Corfu Port showed higher number of microplastics (Figure 13). This is because Corfu Port mussel tissues weighed less (0.17 Âą 0.097) than those of the mussel farm in Thesprotia (0.39 Âą 0.15).

Fragments were the most abundant items in S. Pilchardus and M. barbatus while similar numbers of fragments and filaments were found in P. erythrinus and M. galloprovincialis (Figure 14).

5

Nr of microparticles/ fish

4,5 4 3,5

3 FILAMENTS

2,5

FRAGMENTS

2 1,5 1 0,5 0 Sardina pilchardus

Pagellus erithrinus

Mullus barbatus

Figure 11: Average number of microplastic particles per one individual per fish species in April 2015, Greece.

23

Derelict Fishing Gear management system in the Adriatic Region

4,5

Nr of microparticles/ mussel

4 3,5 FILAMENTS

3

FRAGMENTS

2,5

2 1,5 1 0,5 0 Corfu Port

Mussel farm in Thesprotia

Figure 12: Average number of microplastic particles per one individual of mussel in two areas in June and July 2015, Greece.

20 18

Nr of microparticles

16 14 12 per g tissue

10 8

per mussel

6 4 2 0 Corfu Port

Mussel farm in Thesprotia

Figure 13: Average number of microplastic particles (sum of filaments and fragments) per g of tissue and per one individual of mussel in two areas in June and July 2015, Greece.

24

Derelict Fishing Gear management system in the Adriatic Region 100% 80% 60% FRAGMENTS

40%

FILAMENTS

20% 0% Sardina pilchardus

Pagellus erithrinus

Mullus barbatus

Mytilus galloprovincialis

Figure 14: Categories composition of microplastic particles for each fish and mussel sample collected from April to July 2015, Greece.

3.1.1.5 Italy In Figure 15 we observed that the number of filaments in Cesenatico is lower than in Chioggia, an opposite situation is shown for the fragments. The analysis of number of particles per g in samples in Chioggia showed that the number of fragments and filaments are similar (Figure 16).

The analysis of the composition of microparticles for each mussel pool (Figure 17) show that the percentage of fragments is greater in Cesenatico than in Chioggia.

Average values of fragments (Figure 15) found in Cesenatico (2.6 particles/mussel) are higher than those found in Chioggia (1.4 particles/mussel), while the filaments were greater in Chioggia (1.6 particles/mussel) than in Cesenatico (1.3 particles/mussel). The similar trend is observed also for the number of microplastic per mussel mass (Figure 16). The percentage of fragments are higher in Cesenatico (73.13%) and lower in Chioggia (43.8%) (Figure 17).

25

Derelict Fishing Gear management system in the Adriatic Region

5

Nr of microparticles/mussel

4,5 4 3,5 3

Filaments

2,5

Fragments

2 1,5 1 0,5 0 Cesenatico

Chioggia

Figure 15: Average number of all microplastic particles per one individual of mussel from July 2015, Italy.

Nr of microparticles/g

3 2,5 2 Filaments 1,5

Fragments

1 0,5 0 Cesenatico

Chioggia

Figure 16: Average weight of all microplastic particles per mussel mass [g] in July, Italy.

26

Derelict Fishing Gear management system in the Adriatic Region 100% 80% 60% 40%

20% 0%

Cesenatico

Chioggia

Fragments

73,1%

43,8%

Filaments

26,9%

56,3%

Figure 17: Categories composition of microplastic particles for each mussel pool in 2015, Italy.

3.1.1.6 Slovenia We have managed to acquire the data about the number and type of microplastic particles found in biota. Filaments were predominant microplastic particles found in all three species, with averages in range from 1.9 to 9.45 filaments per fish, with maximum at species Liza aurata and minimum at Solea solea (Figure 18). Predominant type of microplastics were filaments that were in ratio from 94.7% to 98.4% with minimum at Solea solea and maxiumum at Sparus aurata, where only filaments were found (Figure 19). The average number of filaments for Liza aurata, Sparus aurata and Solea solea were 9.3, 7.3 and 1.8, respectively. Also some fragments were extracted from biota samples, but only from Liza aurata (0.15 fragments/fish) and Solea solea species (0.10 fragments/fish), which means 3 and 2 fragments per 20 fishes, respectively (Figure 18 and Figure 19).

18

Nr of microparticles/fish

16 14 12 10

Filaments

8

Fragments

6 4 2 0 Liza aurata

Sparus aurata

Solea solea

Figure 18: Average number of all microplastic particles per one individual per fish species from September and November 2015, Slovenia.

27

Derelict Fishing Gear management system in the Adriatic Region 100% 80% 60% 40% 20%

0%

Liza aurata

Sparus aurata

Solea solea

Fragments

1,59%

0,00%

5,26%

Filaments

98,41%

100,00%

94,74%

Figure 19: Categories composition of microparticles for each sample from September and November 2015, Slovenia.

3.1.2 Size distribution 3.1.2.1 Bosnia and Herzegovina The study points out that there is no direct relationship between the size of the observed individual and the size of the macro- and microplastics found in the digestive system. Size of filaments ranges from approximately 300 μm to more than 15.000 μm (=1.5 cm), regardless of the size of the individual.

The average lengths of collected filaments were in average from 550 to 1500 μm and fragments sizes were in average from 120 to 550 μm (Table 3).

Table 3: Overview of the observed microplastic particles in fish caught in Neum Bay, Bosnia and Hercegovina in season 2015.

Fish sample Mullus barbatus

Pegusa lascaris

Gobius cobitis

Sprattus sprattus

Category of particles Fragments

Total number of Average length particles (μm) 37 550

Min length (μm) cca 100

Max length (μm) cca 1,500

Filaments

214

1,500

350

15,730

Other

8

500

150

1,000

Fragments

11

450

140

1,200

Filaments

178

1,200

250

2,300

Other

N/A

N/A

N/A

N/A

Fragments

15

450

100

1,000

Filaments

149

1,000

300

2,200

Other

4

350

240

700

Fragments

1

120

120

120

Filaments

106

550

250

1,500

Other

N/A

N/A

N/A

N/A

28

Derelict Fishing Gear management system in the Adriatic Region Fragments were mostly angular, sometimes square-like or completely irregular, shape primarily red or transparent. Filaments were of various diameters and primarily red and blue colour (Figure 20 and Figure 21). a)

b)

Figure 20: Filaments as red filament (a) and blue filament (b) are often observed in demersal fish species, primarily on the Mullus barbatus. Photo a) shows an irregular plastic fragment, probably a polyethylene, while photo b) presents an extremely long filament with lateral extensions as a possible results of mechanical degradation and/or abrasion; Bosnia and Hercegovina. Photo: A. Gajić, Sharklab

a)

b)

Figure 21: Both b) blue and b) red filaments have been commonly encountered at all studied samples. Presented figures originates from the digestive tract of the specimens of the pelagic species Sprattus sprattus from the fish market. Similar structures have been identified in other biota samples, as well as the bottom sediment and sea surface studies as well; Bosnia and Hercegovina. Photo: E. KrijeĹĄtorac, Sharklab

29

Derelict Fishing Gear management system in the Adriatic Region

3.1.2.2 Croatia In Table 4 the number of microplastic particles found in different species regarding different categories of microplastics is presented.

Table 4: Number of particles per category of microplastics per fish species, Croatia.

Sample name

Mullus surmuletus

Pagelus erythrinus

Sardina pilchardus

Category

Nr of particles

fragments filaments films foam other fragments filaments films foam other fragments filaments films foam other

4 45 4 1 0 3 26 3 0 2 9 19 0 0 0

3.1.2.3 Greece In fish samples, microplastic particle`s length ranged from 0.089 mm to 1.578 mm for filaments and from 0.24 mm to 1.272 mm for fragments (Table 5). In mussel samples microplastic particle`s length ranged from 0.03 mm to 1.8 mm for filaments and from 0.017 mm to 0.795 mm for fragments (Table 5).

Table 5: Number of particles per category of microplastics per fish or mussel sample, Greece.

Sample name

category

filaments fragments filaments Pagellus erithrinus fragments filaments Mullus barbatus fragments filaments Mytilus galloprovincialis fragments Sardina pilchardus

Nr of particles 29 92 62 12 12 7 15 121 131

average length (mm) 0.661 ± 0,43 68,152 0.127 ± 0.15 0.364 ± 0.22 0.273 ± 0.42 68,152 0.503 ± 0.39 0.190 ± 0.17 1,416666667 0.465 ± 0.42 0.157± 0.17 1,416666667

30

max length (mm) 1.578 0.809 0.877 1.272 1.20 0.63 1.8 0.795

min length (mm) 0.089 0.024 0.133 0.034 0.121 0.034 0.03 0.017

Derelict Fishing Gear management system in the Adriatic Region

3.1.2.4 Italy Within the mussels from Cesenatico, 83% of them contained microplastic particles, while the percent was lower (63%) in mussel sample from Chioggia. In this report average values refer only to mussels in which microplastics were found. In the pool of mussels from Cesenatico we found a total of 68 particles, that is 2.26 particles/individual, in particular 49 fragments, 18 filaments and 1 other.

Average, maximum and minimum lengths of fragments were 0.094, 1.105 and 0.009 mm, respectively (Table 6).

In the pool of mussels from Chioggia 32 particles were found in 30 individuals that is 1.06 No./particles/individual, in particular 14 fragments and 18 filaments with average, maximum and minimum lengths of fragments were of 0.303, 0.952 and 0.063 mm, respectively (Table 6).

Table 6: Number of particles per category of microplastics per mussel sample, Italy.

Sample name M.galloprovincialis (Cesenatico) M.galloprovincialis (Chioggia)

Category fragments filaments other fragments filaments

Nr of particles /30 individuals 49 18 1 14 18

Average Max length length (mm) (mm) 0.0943 1.1055 / / 0.4368 / 0.3028 0.9515 / /

Min length (mm) 0.0089 / / 0.0627 /

3.1.2.5 Slovenia In fish samples, the length of microplastic fragments found in fishes from Slovenia ranged from 0.2 mm to 4.43 mm and was in average 1.77 ± 2.31 for Liza aurata and 0.89 ± 0.98 for Solea solea (Table 7).

Table 7: Number of particles per category of microplastics per fish sample, Slovenia.

Category

No of particles /20 individuals

fragments

3

1.77 ± 2.31

4.43

0.24

filaments

186

/

/

/

Sparus aurata

filaments

146

/

/

/

Solea solea

fragments

2

0.89 ± 0.98

1.59

0.2

filaments

36

/

/

/

Sample name Liza aurata

31

Average Max length length (mm) (mm)

Min length (mm)

Derelict Fishing Gear management system in the Adriatic Region

3.1.3 Identification of plastic type Chemical identification of microplastic particles was successfully done only for the samples from Bosnia and Hercegovina and Greece. The results are presented below.

3.1.3.1 Bosnia and Herzegovina Chemical analyses revealed the chemical composition of mentioned contaminants as primarily polyethylene.

3.1.3.2 Greece Due to size limitations, FT-IR spectroscopy was only applicable in fragments larger than 0.2 mm while in filaments it was not applicable at all. The number and composition of microparticles identified as plastic is shown in Table 8.

Table 8: Chemical composition of microparticles for fish and mussel species from 2015, Greece.

Species

PE

PP

Sardina pilchardus

1

-

Pagellus erithrinus

1

2

Mullus barbatus

-

-

Mytilus galloprovincialis

1

-

Legend: PE - polyethylene, PP – polypropylene

32

Derelict Fishing Gear management system in the Adriatic Region

3.2 Regional level The gathered results on regional level are presented in Figure 22. Among the fish species examined, three were common in different countries: Mullus barbatus, Pagellus erithrinus and Sardina pilchardus. Average numbers of microplastic particles in examined fish were in range from 0.88 to 12.95 microplastic particles/fish. This minimum and maximum average numbers were observed at the same fish species, Mullus barbatus from Greece and Bosnia and Hercegovina. The average number of particles found in Pagellus erithrinus that were caught in Croatia and in Greece, were 1.88 and 1.26 microplastic particles/fish, respectively. Average numbers of microplastics in fish of Sardina pilchardus species were 2.54 and 2.53 microplastic particles/fish in Croatia and Greece, respectively.

12,95

14

Nr of particles/fish

12 10

9,45 7,3

8

9,45

8,4

5,35

6 4

1,9

2

2,68

1,88

2,54

2,4 0,88

1,26

Slovenia

Croatia

BIH

Sardina pilchardus

Pagellus erithrinus

Mullus barbatus

Sprattus sprattus

Pegusa lascaris

Mullus barbatus

Gobius cobitis

Sardina pilchardus

Pagellus erithrinus

Mullus surmuletus

Sparus aurata

Solea solea

Liza aurata

0

Greece

Figure 22: Average numbers of microplastic particles per fish [Nr of particles/fish] on regional level.

On regional level the average numbers of microplastic particles in examined mussels were in range from 1.65 to 3.67 microplastic particles/mussel, where minimum was observed in mussels from Italy and maximum in mussel from Bosnia and Hercegovina (Figure 23). The average number of microplastic particles in mussels from Greece were closer to average number from Bosnia and Hercegovina, and was 3.15 microplastic particles/mussels (Figure 23).

33

Derelict Fishing Gear management system in the Adriatic Region

Nr particles/mussel

14 12 10 8 6 3,67

3,15

Mytilus galloprovincialis

Mytilus galloprovincialis

Mytilus galloprovincialis

Italy

BIH

Greece

4 2

1,65

0

Figure 23: Average numbers of microplastic particles per mussel [Nr of particles/mussel] on regional level.

34

Derelict Fishing Gear management system in the Adriatic Region

4 Discusion Effects of litter ingestion by marine animals are reported from different areas worldwide, for both, larger litter items and microplastics. Microplastics, similar as a large litter items, can cause physical and chemical treats to organism after ingestion (Browne et al., 2008). In the Mediterranean Sea, which is one of the most polluted sea (Costello et al., 2010), the presence of microplastics in shallow and deep waters is already confirmed (Galgani et al., 1996; Waters et al., 2010). Despite the problem of microplastics has been reported decades before (Carpenter et al., 1972; Hoss and Settle, 1990), the microplastic ingestion in the Mediterranean Sea was not systematically investigated so far.

Nevertheless, some authors noticed microplastic presence in stomach contents of different fish species (Deudero, 1998; Massuti et al., 1998; Madurell, 2003). Some of the authors reported negative effects of litter on different organisms in the Mediterranean Sea, related to entanglement, ingestion, suffocation and general debilitation (Deudero and Alomar, 2015). Anastasopoulou et al. (2013) were for the first time in the Mediterranean focused on the presence of plastic debris in digestive tracts of deep-water fishes (both elasmobranchs and teleosts) from Ionian Sea. Other researches in Mediterranean study the litter interaction with other marine species than fishes, except the newest study on the presence of plastic debris in the stomachs of large pelagic fishes (Xiphias gladius, Thunnus thynnus and Thunnus alalunga) (Romeo et al., 2015). On the eastern part of the Adriatic Sea, only few authors reported about ingestion of larger litter items in marine organisms, such as turtles (Buršić et al., 2008; Lazar and Gračan, 2011) and marine mammals (Pribanić et al., 1999). Therefore the study conducted in DefishGear project applying a common protocol for microplastic analysis in biota in years of 2015 – 2016 present the first coordinated study on presence of microplastics in biota species in the level of the entire Adriatic Sea with the focus on commercially interesting fish and bivalve mussel species.

In frame of the DeFishGear project, together 12 different fish species and 1 species of bivalve mollusc from the Adriatic Sea were examined for the presence of microplastics (See: Chapter 1 Introduction) and 10 fish species and 1 bivalve mollusc are further discussed. The first results on the average number of microplastic particles per individual of fish species on national level shows different rates of microplastic ingestion in investigated fish species. The results also suggest that to some extent there is a correlation between the number of ingested microplastic particles per fish and the habitat that fish species inhabit (Jardas, 1996), e.g. pelagic or demersal fish species.

If we compare sampled fish species from Croatia and Greece according to fish habitats (pelagic and demersal - benthic), a difference in amount of plastic intake is apparent. In Croatia a larger amount of ingested microplastics in benthic Mullus surmuletus (in 21 out of 30 specimen or 70%) and Pagellus erythrinus (in 15 of 30 specimen or 50%) were recorded, in comparison to pelagic Sardina pilchardus (in 11 of 30 specimen or 30%), even though the average content of microplastic particles per individual were comparable. The averages for demersal fish species of Mullus surmuletus and Pagellus erythrinus were 2.68 ± 1.8 and 1.88 ± 1.4 particles per fish, respectively, and 2.54 ± 1.1 for pelagic species Sardina pilchardus. Fish sampled in Greece showed higher number of ingested microplastics/fish in the pelagic species Sardina pilchardus (2.53±4.26) in comparison to the demersal species Pagellus erythrinus (1.20±1.44 ) and Mullus barbatus (0.88±1.33.) but the differences were not statistically significant (Man Witney test, P>0.05). Lusher et al. (2013) haven’t found a difference in plastic ingestion among pelagic (38%) and demersal (35%) species, while Naves et al. (2015) reported that benthic species more often take microplastics as food (63.5%), in comparison with pelagic species. In addition, Rummel et al. (2016) recorded higher microplastic ingestion in pelagic than in demersal fish from the 35

Derelict Fishing Gear management system in the Adriatic Region North Sea and Baltic Sea. The fact is that microplastic can be available for both, benthic and pelagic fish.

Also in comparison of demersal and pelagic fish species from Bosnia and Hercegovina the differences among demersal and pelagic species are distinct. Higher number of microplastic particles per fish was observed in demersal fish species Mullus barbatus, Pegusa lascaris, and Gobius cobitis where averages were 12.95, 9.45 and 8.4 microplastic particles per fish, respectively, than at pelagic fish species. The average number of microplastics at pelagic fish species Sprattus sprattus was lower and was 5.35 microplastic particles per individual.

Differences in ingestion of microplastics among some of the examined fish species may be explained by the different feeding habits and given conditions in fishing location. The density of plastic in marine environment can influence the rate of plastic ingestion in pelagic and demersal fish population (Erriksson and Burton, 2003). Higher density of microplastics in fishes feeding habitat therefore increases the possibility of swallowing plastic pieces. The microplastic particles can be located in sediments, throughout the water column and on the sea surface, and could be thus easily and accidentally substitute with natural food sources (Boerger et al., 2010; Lusher et al., 2013). Small microplastics is in similar size category as a zooplankton and it can be potentially consumed by planktivorous feeders (Davison and Asch, 2011), as an example of Clupeiformes (Collard et al., 2015), such as Sardina pilchardus.

On the contrary, when comparing the results of microplastics content in the gut of the same fish species collected in different areas of the Adriatic Sea, namely demersal Mullus barbatus from Bosnia and Hercegovina and from Greece, we can see different trend. In M. barbatus from Bosnia and Hercegovina there were in average 12.95 particles per fish, when in M. barbatus from Greece there were in average only 0.88 particles per fish. Also the ratio between filaments and fragments was inversed, since their ratio in M. barbatus from Bosnia and Hercegovina was 83% to 14%, respectively, and in fish from Greece it was 32% and 68%, respectively.

When comparing the results of plastic microplastics in the gut of Sardina pilchardus from Croatia and Greece, we can also observe few difference in the data. The average numbers of total microplastic particles per fish were lower in fish from Croatia (0.93 particles/fish) than from Greece (2.53 particles/fish). The average number of particles were 0.63 filament/fish and 0.30 fragments/fish in fish from Croatia and 0.805 and 1.72 for filaments and fragments, respectively, in fish from Greece. The ratio of found filaments to fragments in the gut of fish from Croatia was 68% to 32%, respectively, whereas in guts from fishes from Greece this ratio was 32% to 68% in favour to fragments.

Also for the demersal fish species Pagellus erythrinus, sampled in different parts of Adriatic Sea, the results on microplastic content in fish differ among locations. In Pagellus erythrinus from Croatia there were in average more filaments and less fragments per fish than in Greece. There were in average 2.6 filaments/fish, 1.0 fragments/fish, and 1.0 films/fish in fish from Croatia and 0.6 filaments/fish and 0.6 fragments/fish in fish from Greece. The ratio of filaments to fragments in the gut of Pagellus erythrinus was in Croatia 76% to 9%, and in Greece it was 50% to 50%, respectively, and was therefore also different among different parts of the Adriatic Sea as was already observed at Mullus barbatus and Sardina pilchardus.

After the comparison of the results of microplastics in fish samples from the Adriatic Sea, there is noticeable high variability among all of 10 compared fish samples. The average content of microparticles extracted were in range from 0.88 – 12.95 microparticles/fish. After the additional 36

Derelict Fishing Gear management system in the Adriatic Region comparison of three fish species from different parts of the Adriatic Sea (Mullus barbatus, Pagellus erythrinus and Sardina pilchardus), results are suggesting that the density of microplastic in the marine environment is of more importance, than is the actual fish habitat. Namely, microparticles does not simply just sink to the sediment and stays there. Behaviour of microplastic particles in marine environment should be considered in a dynamic and changing perspective, since initially floating particles can sink to sediment, potentially being remobilized to water column by bioturbation, resuspension or hydrodynamic conditions (Avio et al., 2016). Because of remobilization of the microplastic particles, they are “available” again for both, pelagic and demersal fish species.

One commercially interesting bivalve species was examined in this research. This was Mytilus galloprovincialis, which was sampled in Italy, Bosnia and Hercegovina, and Greece. Different contents and different predominant types of microplastics was observed among mussels from different locations. The highest number of microplastic particles per mussel was observed in mussels from Bosnia in Hercegovina where only filaments were found in average of 3.67 particles/mussel. Similar number of particles was found in Greece where in average (both locations) of 3.15 particles/mussel.

The lowest number of particles/mussel was observed in Italy with the average of 1.65 particles/mussel. The average ratio of filaments to fragments differ among in Italy and Bosnia and Hercegovina and Greece, since in Italy the ratio was in favour to fragments (filaments to fragments, 36% to 64%). In Greece the ratio of filaments to fragments was more similar, 52% to 48%, and in Bosnia and Hercegovina only filaments were found in bivalves.

The chemical composition of collected particles from biota was determined only for particles gathered in Greece and Bosnia and Hercegovina. In Greece just a few micro particles were identified as plastic (polyethylene and polypropylene) and in Bosnia and Hercegovina they were primarily identified as polyethylene. This result is in correlation with the results of analysis of chemical composition of microplastic sampled on sea surface (Pilot assessment on microplastic in sea surface…, 2016), sea surface fishing area (Pilot assessment on microplastic in fishing areas, 2016), and from the river outflows in the Adriatic region (Pilot assessment on microplastic in rivers, 2016).

The chemical characterization of microparticles collected from biota is more difficult than e.g. analysis of microparticles gathered on the beach. There are several reasons for this. The particles are usually smaller and filaments are predominant type, which are more difficult to measure. The fragments after chemical treatment with H2O2 for organic matter removal (gut content) usually became more fragile and are hard to handle (extraction of particles from the sample, transfer for FT-IR measurement), since they often break. If we try to retain microparticles on the filter, after digestion with H2O2 and filtration, there are usually problems with measurement, since organic matter can cover the microparticles.

Chemical characterization of microparticles found in biota is a crucial step to confirm if particles from all categories (filaments, fragments, films,…) are from plastic material. They can be from natural material (e.g. cotton fibers, calcium carbonate shells) or any other artificial material (e.g. aluminium foil).

There are several factors that can influence the final results of the study. At purchase of the fish or mussels (if live catch is not an option) it is important that samples are fresh and not already old and in process of advanced deterioration, since the organisms intestine should be as fresh and intact as possible in order to perform quality section of the sample and measurement of correct weigh of the intestine and their content, and also extraction of the microplastics. As already mentioned in the report on assessment of microplastic in sea surface and beach sediment potential accumulation zones (Pilot assessment on microplastic…, 2016), in the process of separation of microplastic particles from 37

Derelict Fishing Gear management system in the Adriatic Region the samples factors, that can influence the quality of microplastic separation are also 1) the quality of stereomicroscope, where polarization light could help to distinguish among plastic and non-plastic particles, 2) the experience and precision of working person, and 3) the appropriate laboratory, which need to be clean and closed, in order to avoid possible contamination of samples with airborne filaments.

During the DeFishGear project, the protocols for sampling and analysis of microplastics from biota were developed. The prevention of contamination of the samples was important. The main concern was the contamination with the fibers, since fibers or microfibers are omnipresent in the marine ecosystems (Phuong et al., 2016), in the atmospheric fallout (Dris et al., 2015) in the everyday human working and living environment and they can be easily transported by the air (Torre et al., 2016). Thus the risk of artefacts by background contamination is a crucial concern, since it can affect objectivity of methodology for microplastic analysis and the out coming results (Torre et al., 2016).

In the laboratories of HCMR, one of the project partners on the DeFishGear project, the study on the topic of prevention of airborne contamination when studying microplastic pollution in marine biota was conducted. They concluded, that the number of filaments decreased by 95.3% when using microscope cover during visual inspection of the sample, and by 86.4% when the glove box was used, as additional precaution during microplastic analysis (Michele, et al., 2016). Applying this methodology, they found out, that the incidence of anthropogenic microfibers in the fish diet in the Ionian Sea is very low (Michele, et al., 2016).

The pioneer research on biota from the Adriatic Sea conducted in the DeFishGear project provide us with valuable first data on the overall state of the contamination with microplastics of commercially important fish and bivalve species throughout the entire Adriatic Region.

38

Derelict Fishing Gear management system in the Adriatic Region

5 Conclusions Based on this study the following conclusions are presented: 

Commercially available fish and mussels from the Adriatic Sea are contaminated with microplastic. The average content of microplastics extracted were in range from 0.88 – 12.95 microparticles/fish and 1.65 – 3.67 microparticles/mussels.



The predominant type of microplastics found in fish and mussels from Bosnia and Herzegovina, Croatia and Slovenia were filaments, and the second most common were fragments. In fish from Greece and mussels from Italy, the fragments were predominant type of microplastics and the filaments were the second most common, while in the mussels from Greece the fragments and filaments found were in similar numbers.



Results of microplastics in fish samples from the Adriatic Sea shows high variability among all of 10 compared fish samples. The comparison of different fish species caught on different parts of the Adriatic Sea, that represented different habitat types, shows different type and average number of microplastics per fish among areas. These results lead to the conclusion that the density of microplastic in the marine environment is of more importance than the actual fish habitat (e.g. pelagic, benthic).



Only few microparticles collected from biota samples were successfully measured with FT-IR for the chemical composition. The prevailing chemical composition were polyethylene and polypropylene materials. These results are in correlation with the results of microplastics sampled on sea surface and from rivers in the Adriatic region.



This research, conducted in the frame of DeFishGear project, present the first valuable data on the overall state of the contamination with microplastics in commercially important fish and mussels species in years of 2015 and 2016 in the Adriatic Sea, where first coordinated research was conducted in the countries of Italy, Slovenia, Croatia, Bosnia and Herzegovina, Albania and Greece.

39

Derelict Fishing Gear management system in the Adriatic Region

5 References Allsopp, M., Walters, A., Santillo, D., Johnston, P. (2006). “Plastic Debris in the World’s Oceans”, Greenpeace Report, Netherlands.

Avio, C.G., Gorbi, S., Regoli, F. (2016). Plastics and microplastics in the oceans: From emerging pollutants to emerged threat. Marine Environmental Research, http://dx.doi.org/10.1016/j.marenvres.2016.05.012.

Bajt, O., Szewc, K., Horvat, P., Pengal, P., Grego, M. (2015). Microplastics in sediments and fish of the Gulf of Trieste. V: Seminar on Microplastics Issues, Piran, May 4-6 2015. Kržan, A., (ur.), Horvat, P., (ur.). Micro 2015: book of abstracts. [Ljubljana]: DeFishGear, 53-54.

Boerger, C., Lattin, G., Moore, S., Moore, C. (2010). Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre. Marine Pollution Bulletin, 60, 2275–2278.

Buršić, M., Gračan, R., Katić, J., Tvrtković, N., Lazar, B. (2008). Ingestion of marine debris by loggerhead sea turtles, Caretta caretta, in the eastern Adriatic Sea. Proceedings of the Twenty-seventh Annual Symposium on Sea Turtle Biology and Conservation (Eds. Rees A.F., Frick M., Panagopolou A., Williams K.) NOAA Technical Memorandum NMFS-SEFSC-569, Miami, pp. 10.

Carpenter, E.J., Anderson, S.J., Harvey, G.R., Miklas, H.P., Peck, B.B. (1972). Polystyrene spherules in coastal waters. Science, 175, 749–750.

Collard, F., Gilbert, B., Eppe, G., Parmentier, E., Das, K. (2015). Detection of anthropogenic particles in fish stomachs: an isolation method adapted to identification by Raman spectroscopy. Archives of Environmental Contamination and Toxicology, 69, 331–339.

Colton J. B., Knapp F., D., Burns B. R. (1974). “Plastic particles in surface waters of the northwestern Atlantic”. Science, 185, pp. 491-497.

Costello, M.J., Coll, M., Danovaro, R., Halpin, P., Ojaveer, H., Miloslavich, P. (2010). A census of marine biodiversity knowledge, resources, and future challenges. PLoS ONE, 5(8), 12110.

Davison, P., Asch, R. (2011). Plastic ingestion by mesopelagic fishes in the North Pacific Subtropical Gyre. Marine Ecology Progress Series, 432,173–180.

Deudero, S. Alomar. C. (2015). Mediterranean marine biodiversity under threat: Reviewing influence of marine litter on species. Marine Pollution Bulletin, 98, 58–68.

Deudero, S. (1998). Relaciones tróficas en las comunidades ícticas asociadas adispositivos agregadores de peces. PhD thesis. Universitat de les Illes Balears. Deudero, S., 2001. Interspecific trophic relationships among pelagic fish species underneath FADs. Journal of Fish Biolgy, 58 (1), 53–67.

Dris, R., Gasperi, J., Rocher, V., Saad, M., Renault, N., Tassin, B. (2015). Microplastic contamination in an urban area: a case study in Greater Paris. Environ. Chem. 12, 592.

Errikson, C., Burton, H. (2003). Origins and biological accumulation of small plastic particles in Furseals from Macquarie Island. Ambio, 6, 380–384.

Food and Agriculture Organization of the United Nations (2015). The Fisheries and Aquaculture Sector in Bosnia and Herzegovina. Preparation of IPARD Forest and Fisheries Sector Reviews in Bosnia and Herzegovina. 2015, pp. 155.

40

Derelict Fishing Gear management system in the Adriatic Region Galgani, F., Souplet, A., Cadiou, Y. (1996). Accumulation of debris on the deep sea floor off the French Mediterranean coast. Marine Ecology Progress Series, 142, 225–234.

Hoss, D.E., Settle, L.R. (1990). Ingestion of plastic by teleost fishes. In: Shomura, R.S., Godrey, M.L. (Eds.), Proceedings of the Second International Conference on Marine Debris 2–7 April 1989, Honolulu, Hawaii. U.S. Department of Commerce. NOAA Technical Memorandum. NMFS, NOAA-TM NMFS-SWFC-154, 693 - 709.

Jardas I. (1996). Jadranska ihtiofauna. Školska knjiga, Zagreb.

Kartar S., Milne R., Sainsbure M. (1973). “Polystyrene waste in the Severn Estuary”, Marine Pollution Bulletin, 4, pp. 144.

Kovač Viršek, M. (2016). Strategic recommendation for improving marine litter management in the Adriatic Sea in the field of microplastic pollution. The document produced within the project DeFishGear (1°str/00010), IPA Adriatic Cross-border Cooperation Programme 2007 – 2013.

Kovač Viršek, M., Palatinus, A., Kaberi, H., Tsangaris, C., Mazziotti C. (2015). Recommendation on regional approach to monitoring and assessment of microplastic in the marine environment. The document produced within the project DeFishGear (1°str/00010), IPA Adriatic Cross-border Cooperation Programme 2007 – 2013.

Lazar, B., Gračan, R. (2011). Ingestion of marine debris by loggerhead seaturtles, Carreta carreta in the Adriatic Sea. Marine Pollution Bulletin, 62, 43–47.

Lusher, A.L., McHugh, M., Thompson, R.C. (2013). Occurrence of microplastics in the gastrointestinal tract of pelagic and demersal fish from the English Channel. Marine Pollution Bulletin, 67, 94–99.

Madurell, T. (2003). Feeding strategies and trophodynamic requirements of deep-sea demersal fish in the Eastern Mediterranean. Ph.D. Thesis, Athens, 251.

Massutí, E., Deudero, S., Sánchez, P., Morales-Nin, B. (1998). Diet and feeding of dolphin (Coryphaena hippurus) in western Mediterranean waters. Bulletin of Marine Science, 63 (2), 329–341.

Neves, D., Sobral, P., Pereira, T. (2015). Marine litter in bottom trawls off the Portuguese coast. Marine Pollution Bulletin, 99, 301–304.

Phuong, N.N., Zalouk-Vergnoux, A., Poirier, L., Kamari, A., Châtel, A., Mouneyrac, C., Lagarde, F. (2016).

Is there any consistency between the microplastics found in the field and those used in laboratory experiments? Environ. Pollut. 211, 111–123.

Pilot assessment on microplastic in fishing areas (2016). The document produced within the project DeFishGear (1°str/00010), IPA Adriatic Cross-border Cooperation Programme 2007 – 2013.

Pilot assessment on microplastic in rivers (2016). The document produced within the project DeFishGear (1°str/00010), IPA Adriatic Cross-border Cooperation Programme 2007 – 2013.

Pilot assessment on microplastic in sea surface and beach sediment potential accumulation zones (2016). The document produced within the project DeFishGear (1°str/00010), IPA Adriatic Crossborder Cooperation Programme 2007 – 2013.

41

Derelict Fishing Gear management system in the Adriatic Region Pribanic, S., Holcer, D., Miokovic, D. (1999). First report on plastic ingestion by striped dolphin (Stenella coerueoalba) in Croatian part of the Adriatic Sea. In: Proceedings of the thirteenth annual conference of the European cetacean society, Valencia, Spain (Eds: Evans P.G.H., Cruz J., Raga, J. A.). pp. 443-446.

Romeo, T., Pietro, B., Pedà, C., Consoli, P., Andaloro, F., Fossi, M.C. (2015). First evidence of presence of plastic debris in stomach of large pelagic fish in the Mediterranean Sea. Marine Pollution Bulletin, 95 (1), 358–361.

Rummel, C.D., Löder, M.G., Fricke, N.F., Lang, T., Griebeler, E.M., Janke, M., Gerdts, G. (2016). Plastic ingestion by pelagic and demersal fish from the North Sea and Baltic Sea. Marine Pollution Bulletin, 102, 134–141.

Statistical office of Republic of Slovenia, http://www.stat.si/statweb/en/home [1/9/2016].

Torre, M., Digka, N., Anastasopoulou, A., Tsangaris, C., Mytilineouet, C. (2016). Anthropogenic microfibres pollution in marine biota. A new and simple methodology to minimize airborne contamination, Marine Pollution Bulletin, http://dx.doi.org/10.1016/j.marpolbul.2016.07.050.

United Nations Environment Programme, Mediterranean action plan (2014). Status and conservation of fisheries in the Adriatic. Draft internal report.

Watters, D. L., Yoklavich, M. M., Love, M. S., Schroeder, D. M. (2010). Assessing marine debris in deep sea floor habitats off California. Marine Pollution Bulletin, 60, 131–138.

42

Derelict Fishing Gear management system in the Adriatic Region

Appendix: Coordinates, technical data for sampling of biota Table A1: Coordinates, technical data for sampling of biota in 2015, Albania.

Mullus barbatus

Sample name GPS coordinates

Lat (X)

Triglia lucerna

Solea solea

Trachurus Pagellus mediterraneus erythrinus

Sardina pilcardus

41.316902 41.201457 41.784148

41.703919

40.660869 40.53415

Lon (Y) 19.150031 19.268802 19.517721

19.280095

19.284421 19.235849

Location name

Durres

Durres

Lezha

Lezha

Vlora

Vlora

Table A2: Coordinates, technical data for sampling of biota in the period of 2015-2016, Bosnia and Herzegovina.

Nr

Beginning

End point

Length

Surface

Depth

Direction

Sediment

L1

42°55'49.03"N 42°54'59.87"N 1.700 mi 17°35'7.02"E 17°37'3.42"E

17.37 mi2

87 feet

S/E

Sludge to Clay

L2

42°55'59.95"N 42°55'11.59"N 1.700 mi 17°34'2.98"E 17°35'58.17"E

17.37 mi2

75 feet

S/E

Sludge to Sand

L3

42°56'0.03"N 42°55'37.56"N 0.930 mi 17°35'13.95"E 17°36'13.46"E

8.68 mi2

80 feet

S/E

Sludge

L4

42°55'23.31"N 42°54'58.89"N 0.930 mi 17°35'50.31"E 17°36'47.22"E

8.68 mi2

85 feet

S/E

Sludge

Derelict Fishing Gear management system in the Adriatic Region Table A3: Data table of Mullus barbatus specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

Ecto/Endo parasites

Pathological changes

MB-01/15

19.7

100

Adult

/

/

MB-02/15

24.1

205

Adult

/

/

MB-03/15

20.4

140

Adult

Several endoparasites present inside the intestine

/

MB-04/15

17

65

Adult

Several endoparasites present inside the intestine

/

MB-05/15

14

35

Adult

/

/

MB-06/15

17

65

Adult

/

/

MB-07/15

16

50

Adult

/

/

MB-08/15

17

40

Adult

Several endoparasites present inside the intestine

/

MB-09/15

15.5

30

Adult

/

/

MB-10/15

14

50

Juvenile

/

/

MB-11/15

16

50

Adult

/

/

MB-12/15

16

95

Adult

/

/

MB-13/15

20

70

Adult

/

/

MB-14/15

18

70

Juvenile

/

/

MB-15/15

18

50

Adult

/

/

MB-16/15

17

45

Adult

/

/

MB-17/15

15

50

Juvenile

/

/

MB-18/15

16

20

Juvenile

/

/

MB-19/15

13.5

25

Juvenile

/

/

MB-20/15

14

75

Juvenile

/

/

Sample No.

Derelict Fishing Gear management system in the Adriatic Region Table A4: Data table of Pegusa lascaris specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

Ecto/Endo parasites

Pathological changes

PL-01/15

22.5

95

Adult

/

/

PL-02/15

18.8

35

Adult

/

/

PL-03/15

17.4

35

Adult

/

/

PL-04/15

20.2

65

Adult

/

/

PL-05/15

20.2

75

Adult

/

/

PL-06/15

15.9

30

Juvenile

/

/

PL-07/15

13.2

15

Juvenile

/

/

PL-08/15

8.9

5

Juvenile

/

/

PL-09/15

13.6

15

Juvenile

/

/

PL-10/15

13.4

15

Juvenile

/

/

PL-11/16

10

4.8

Juvenile

/

/

PL-12/16

10

4.7

Juvenile

/

/

PL-13/16

8.5

3.7

Juvenile

/

/

PL-14/16

9.5

4.3

Juvenile

/

/

PL-15/16

8

3.5

Juvenile

/

/

PL-16/16

8

3.7

Juvenile

/

/

PL-17/16

8

3.9

Juvenile

/

/

PL-18/16

9

4.3

Juvenile

/

/

PL-19/16

6

2.4

Juvenile

/

/

PL-20/16

13.8

15

Juvenile

/

/

Sample No.

Derelict Fishing Gear management system in the Adriatic Region Table A5: Data table of Gobius cobitis specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

GC-01/16

7.5

15.56

Adult

GC-02/16

10

11.75

Adult

GC-03/16

10.9

12.98

Adult

GC-04/16

10

10.77

Adult

GC-05/16

9.2

6.90

Adult

GC-06/16

9

8.09

Adult

GC-07/16

10.4

11.16

Adult

GC-08/16

9.9

9.82

Adult

GC-09/16

10

9.04

Adult

GC-10/16

9.5

9.68

Adult

GC-11/16

10.5

11.72

Adult

GC-12/16

8.6

10

Adult

GC-13/16

9

7.70

Adult

GC-14/16

8

4.36

Adult

GC-15/16

8.2

5.29

Adult

GC-16/16

9

6.67

Adult

GC-17/16

7

4.80

Adult

GC-18/16

9

8.57

Adult

GC-19/16

8.3

5.05

Adult

GC-20/16

10

9

Adult

Sample No.

Ecto/Endo parasites Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine Numerous endoparasites present in the intestine

Pathological changes / / / / / / / / / / / / / / / / / / / /

Derelict Fishing Gear management system in the Adriatic Region Table A6: Data table of Sprattus sprattus specimen caught by trawl sampling in Bosnia and Herzegovina.

Fish length [cm]

Fish weight [g]

Life Stage

Ecto/Endo parasites

Pathological changes

SS-01/16

11

8.34

Adult – F

/

/

SS-02/16

9.33

8.01

Adult – M

/

/

SS-03/16

8.1

5.03

Adult – F

/

/

SS-04/16

9.8

8.23

Adult – M

/

/

SS-05/16

10.1

9.23

Adult – F

/

/

SS-06/16

9.6

9.10

Adult – F

/

/

SS-07/16

10.2

9.90

Adult – M

/

/

SS-08/16

8.9

8.94

Adult – F

/

/

SS-09/16

10.3

9.52

Adult – F

/

/

SS-10/16

10.3

9.44

Adult – M

/

/

SS-11/16

9.9

7.68

Adult – F

/

/

SS-12/16

9.2

7.76

Adult – M

/

/

SS-13/16

9.0

7.2

Adult – M

/

/

SS-14/16

7.2

6.9

Adult – F

/

/

SS-15/16

8.8

6.94

Adult – M

/

/

SS-16/16

8.3

5.05

Adult – F

/

/

SS-17/16

8.9

7.46

Adult – F

/

/

SS-18/16

9.5

9.10

Adult – F

/

/

SS-19/16

8.7

6.82

Adult – M

/

/

SS-20/16

10.7

10.77

Adult – M

/

/

Sample No.

Legend: M: male, F: female

Derelict Fishing Gear management system in the Adriatic Region Table A7: Coordinates, technical data for sampling of three fish from April to October 2015, Croatia.

Fish species Mullus surmuletus Mullus surmuletus Pagellus erythrinus Pagellus erythrinus Pagellus erythrinus Pagellus erythrinus Sardina pilchardus

Type of sampling

Date [dd/mm/yy]

By fishermen

5.7.2015

By fishermen

22.10.2015

By fishermen

5.9.2015

By fishermen

5.9.2015

By fishermen

7.9.2015

By fishermen

8.9.2015

By fishermen

29.4.2015

Location 44,62864 N 14,42122 E 43,14812 N 16,42771 E 43,93438 N 15,31259 E 44,27149 N 14,97391 E 44,73603 N 14,01307 E 44,73329 N 14,36383 E 43,46927 N 16,22295 E

Number of individuals 21 9 25 3 1 1 30

Derelict Fishing Gear management system in the Adriatic Region Table A8: Data table of Mullus surmuletus specimen caught by fishermen in July and October 2015 along the eastern coast of Croatia.

Sample label

Fish length [cm]

Fish weight [g]

Fish gender

Fish maturity stage

Fish deformation

Ms1

21.5

135.29

Female

5

/

Ms2

22.1

134.88

Female

5

/

Ms3

21.2

119.46

Male

5

/

Ms4

20.2

116.60

Female

5

/

Ms5

21.0

136.65

Female

5

/

Ms6

20.0

113.33

Female

5

/

Ms7

21.9

130.00

Female

4

/

Ms8

22.9

146.00

Female

4

/

Ms9

20.3

113.00

Female

4

/

Ms10

23.2

151.00

Male

4

/

Ms11

24.3

194.00

Female

5

/

Ms12

24.2

195.00

Male

4

/

Ms13

25.5

218.00

Female

3

/

Ms14

24.4

176.00

Male

3

/

Ms15

23.1

155.00

Female

5

/

Ms16

23.2

178.00

Female

4

/

Ms17

25.1

195.00

Male

5

/

Ms18

25.2

212.00

Female

5

/

Ms19

22.1

143.00

Female

5

/

Ms20

24.7

202.00

Female

4

/

Ms21

22.2

148.00

Female

4

/

Ms22

13.5

28.94

Female

Immature

/

Ms23

13.6

28.90

Female

Immature

/

Ms24

12.5

24.42

Female

Immature

/

Ms25

12.0

21.33

Male

Immature

/

Ms26

11.8

18.42

Female

Immature

/

Ms27

10.4

11.73

Male

Immature

/

Ms28

10.2

12.10

Female

Immature

/

Ms29

12.7

25.37

Female

Immature

/

Ms30

10.1

11.80

-

Immature

/

Derelict Fishing Gear management system in the Adriatic Region Table A9: Data table of Pagellus erythrinus caught fishermen in September 2015 along the eastern coast of Croatia.

Fish length [cm]

Fish weight [g]

Fish gender

Fish maturity stage

Fish deformation

Pe1

21.1

105.13

Female

2

/

Pe2

18.9

79.45

Male

3

/

Pe3

18.3

75.69

Male

3

/

Pe4

16.7

55.73

Female

3

/

Pe5

15.1

41.29

/

Immature

/

Pe6

22.3

128.38

Male

2

/

Pe7

21.0

102.07

Male

2

/

Pe8

19.6

92.03

Female

4

/

Pe9

20.1

102.14

Male

2

/

Pe10

21.6

111.68

Male

2

/

Pe11

13.1

24.77

Female

1

/

Pe12

15.8

49.84

Female

2

/

Pe13

15.4

48.75

Female

2

/

Pe14

13.9

30.76

Female

2

/

Pe15

12.5

27.53

/

Immature

/

Pe16

15.0

42.73

Female

2

/

Pe17

13.1

26.36

/

Immature

/

Pe18

16.7

67.49

Female

3

/

Pe19

13.2

31.19

/

Immature

/

Pe20

13.6

34.82

Female

1

/

Pe21

16.5

54.95

Female

2

/

Pe22

16.6

58.46

Female

2

/

Pe23

15.8

55.49

Female

2

/

Pe24

14.8

36.28

Female

1

/

Pe25

21.5

117.10

Male

2

/

Pe26

21.4

117.22

Male

3

/

Pe27

20.1

105.86

Male

2

/

Pe28

18.1

76.27

Female

3

/

Pe29

19.0

84.72

Male

1

/

Pe30

20.5

130.10

Male

2

/

Sample label

Derelict Fishing Gear management system in the Adriatic Region Table A10: Data table of Sardina pilchardus caught on by fishermen in April 2015 along the eastern coast of Croatia.

Fish length [cm]

Fish weight [g]

Fish gender

Fish maturity stage

Fish deformation

Sp1

13.3

16.5

Female

2

/

Sp2

12.6

13.71

Male

1

/

Sp3

12.6

15.35

Female

1

/

Sp4

12.5

16.39

Male

1

/

Sp5

13.6

18.85

Female

2

/

Sp6

13.4

16.8

Male

2

/

Sp7

14.1

19.8

Female

2

/

Sp8

14.6

21.15

Male

2

/

Sp9

11.5

15.3

Female

1

/

Sp10

12.7

14.56

Female

1

/

Sp11

13.4

17.09

Male

1

/

Sp12

12.7

15.83

Male

1

/

Sp13

13.9

18.6

Male

1

/

Sp14

12.6

15.26

Male

2

/

Sp15

13.3

15.78

Female

1

/

Sp16

12.3

13.19

Male

1

/

Sp17

14

20.19

Male

6

/

Sp18

13.4

17.87

Female

1

/

Sp19

14.2

20.28

Female

6

/

Sp20

14

19.38

Male

5

/

Sp21

13.1

16.28

Male

6

/

Sp22

11.7

13.99

Female

1

/

Sp23

13.2

15.75

Male

6

/

Sp24

12.7

16.91

Female

1

/

Sp25

12.9

15.73

Female

1

/

Sp26

13.1

16.72

Female

1

/

Sp27

12.6

15.58

Female

1

/

Sp28

12.6

15.51

Male

6

/

Sp29

14

21.73

Male

6

/

Sp30

13

16.1

Male

6

/

Sample label

Derelict Fishing Gear management system in the Adriatic Region Table A11: Species, number of individuals, type, date and location of sampling in the Northern Ionian Sea, Greece.

Species

Type of sampling

Sardina pilchardus

DFG team/ Bottom trawl

Pagellus erithrinus

DFG team/ Bottom trawl

Mullus barbatus

DFG team/ Bottom trawl

Mytilus galloprovincialis

DFG team/ By hand

Date dd/mm/yy

22.04.15

22.04.15 26.04.15 25.04.15

Location Start

End

39,68882 N/ 19,90893 E 39,5819 N/ 20,080E 39,61061 N/ 20,035533 E 39,41603 N/ 19,85897 E 39,5893 N/ 19,9704 E

39,6881 N/ 19,91572 E 39,6045 N/ 20,088 E 39,59295 N/ 20,05615 E 39,42562 N/ 19,83247 E 39,5706 N/ 19,94942 E

Number of individuals 11 25 14 5 25

26.06.15

39.627826 N/ 19.905782 E

40

27.07.15

39.606567 N/ 20.149421 E

40

Derelict Fishing Gear management system in the Adriatic Region Table A12: Fish length, fish weight, fish gender and fish maturity stage of Sardina pilchardus sampled in April 2015 in the Northern Ionian Sea, Greece.

Species

Sardina pilchardus

Sample Nr. 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A 11A 2B 3B 4B 5B 6B 7B 8B 9B 10B 11B 12B 13B 15B 16B 17B 19B 20B 21B 22B 23B 25B 26B 27B 28B 29B

Total length [cm] 11.2 11 11.2 11 11 10.7 10.7 11.2 11.2 11.2 11.1 12 11.2 10 11 10.7 11.8 11.1 11 10 11 11 10.7 11 12.2 11 10.5 11.2 10.4 13 12 11.1 10.9 10.3 10.1 10.7

Legend: M: male, F: female, NI: not identified

Total weight [g] 11.34 9.52 9.22 9.84 9.36 9.4 8.14 9.06 11.21 10.23 9.43 11.5 9.42 8.03 9.37 8.54 11.62 9.38 9.58 6.97 8.69 9.04 9.22 9.51 12.57 8.92 8.66 10.69 8.59 15.04 10.33 9.48 9.42 8.49 8.34 8.58

Gender NI M M F NI F F M NI F M NI NI NI NI NI M NI M M M M M M M F M M M M NI F M NI NI M

Maturity stage / 1 1 1 / 2 2 2 / 2 2 / / / / / 2 / 5 3 2 2 4 4 3 2 2 2 2 2 / 2 2 / / 1

Deformation / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / /

Derelict Fishing Gear management system in the Adriatic Region Table A13: Fish length, weight, gender and maturity stage of Pagellus erithrinus sampled in April 2015 in the Northern Ionian Sea, Greece.

Species

Pagellus erithrinus

Sample Nr.

Total length [cm]

Total weight [g]

Gender

Maturity stage

Deformation

1

20.7

103.48

M

4

/

2

19

82.89

F

4

/

3

16.8

58.73

F

4

/

4

14

37.25

F

5

/

5

17.6

70.37

F

4

/

6

17.5

67.23

F

4

/

8

20.2

127.64

F

4

/

9

14.5

39.8

F

4

/

10

16

46.48

F

3

/

11

20.4

105.49

F

5

/

12

14.6

41.12

F

2

/

13

16.4

52.65

F

4

/

14

11.8

58.06

F

2

/

15

15.6

47.73

F

2

/

16

13.2

35.57

F

2

/

17

12.1

29.53

F

2

/

18

10.4

17.08

F

2

/

19

10.4

17.08

F

2

/

20

11.7

22.51

F

2

/

Legend: M: male, F: female

Derelict Fishing Gear management system in the Adriatic Region Table A14: Fish length, weight, gender and maturity stage of Mullus barbatus sampled in April 2015 in the Northern Ionian Sea, Greece.

Species

Mullus barbatus

Sample Nr.

Total length [cm]

Total weight [g]

Gender

Maturity stage

Deformation

1

11.4

16.27

NI

/

/

2

11.3

16.41

M

5

/

3

10.2

12.24

NI

/

/

4

11.9

18.04

M

5

/

5

10.3

12.71

M

5

/

6

13.7

27.73

M

5

/

7

9.9

10.18

M

5

/

8

13.4

27.12

M

5

/

9

12

19.46

NI

/

/

10

10.3

10.56

M

5

/

11

11

15.82

M

5

/

12

19.5

105.52

F

5

/

13

16

44.86

M

5

/

14

15.7

44.51

M

5

/

15

9.1

8.24

NI

/

/

16

16.4

54.43

M

5

/

17

11

13.98

M

5

/

18

12

15.27

M

5

/

19

13.4

25.12

M

5

/

20

18.5

79.16

F

5

/

21

13.7

27.23

M

5

/

22

16

43.41

M

5

/

23

13

27.68

F

5

/

25

13.5

27.56

M

5

/

26

10

9.87

NI

/

/

Legend: M: male, F: female, NI: not identified

Derelict Fishing Gear management system in the Adriatic Region Table A15: Mussel Mytilus galloprovincialis lengths sampled in June and July in the Northern Ionian Sea, Greece – 1/3.

Species

Mytilus galloprovincialis

Sample Nr.

Total length [cm]

Total weight [g]

Gender

Maturity stage

Deformation

1

4

/

/

/

/

2

3.5

/

/

/

/

3

3.9

/

/

/

/

4

4

/

/

/

/

5

3.45

/

/

/

/

6

4.2

/

/

/

/

7

4.2

/

/

/

/

8

4.5

/

/

/

/

9

3.9

/

/

/

/

10

4.5

/

/

/

/

11

4.2

/

/

/

/

12

3.7

/

/

/

/

13

4

/

/

/

/

14

4.1

/

/

/

/

15

4.6

/

/

/

/

16

4

/

/

/

/

17

4.1

/

/

/

/

18

5.5

/

/

/

/

19

4.6

/

/

/

/

20

4.1

/

/

/

/

21

4.1

/

/

/

/

22

5

/

/

/

/

23

3.9

/

/

/

/

24

4.4

/

/

/

/

25

4

/

/

/

/

26

4.9

/

/

/

/

27

3.6

/

/

/

/

28

4

/

/

/

/

29

3.5

/

/

/

/

30

3.6

/

/

/

/

Derelict Fishing Gear management system in the Adriatic Region Table A16: mussel Mytilus galloprovincialis lengths sampled in June and July in the Northern Ionian Sea, Greece – 2/3.

Species

Mytilus galloprovincialis

Sample Nr.

Total length [cm]

Total weight [g]

Gender

Maturity stage

Deformation

31

3,5

/

/

/

/

32

3,6

/

/

/

/

33

3,8

/

/

/

/

34

3,8

/

/

/

/

35

4,5

/

/

/

/

36

4,2

/

/

/

/

37

3,6

/

/

/

/

38

4,1

/

/

/

/

39

3,5

/

/

/

/

40

3,9

/

/

/

/

41

5,1

/

/

/

/

42

5,3

/

/

/

/

43

5,6

/

/

/

/

44

5,3

/

/

/

/

45

5,4

/

/

/

/

46

5

/

/

/

/

47

5,4

/

/

/

/

48

5,3

/

/

/

/

49

5,1

/

/

/

/

50

4,8

/

/

/

/

51

5,6

/

/

/

/

52

4,9

/

/

/

/

53

5,4

/

/

/

/

54

5

/

/

/

/

55

5,1

/

/

/

/

56

5,2

/

/

/

/

57

5,3

/

/

/

/

58

5,5

/

/

/

/

59

6,2

/

/

/

/

60

4,7

/

/

/

/

61

5,7

/

/

/

/

Derelict Fishing Gear management system in the Adriatic Region Table A17: mussel Mytilus galloprovincialis lengths sampled in June and July in the Northern Ionian Sea, Greece – 3/3.

Species

Mytilus galloprovincialis

Sample Nr.

Total length [cm]

Total weight [g]

Gender

Maturity stage

Deformation

62

4,9

/

/

/

/

63

5,5

/

/

/

/

64

5,1

/

/

/

/

65

5,3

/

/

/

/

66

5,5

/

/

/

/

67

5,4

/

/

/

/

68

4,8

/

/

/

/

69

5,5

/

/

/

/

70

5,8

/

/

/

/

71

5,2

/

/

/

/

72

5,2

/

/

/

/

73

5,4

/

/

/

/

74

5,1

/

/

/

/

75

5,4

/

/

/

/

76

5,2

/

/

/

/

77

5

/

/

/

/

78

5,2

/

/

/

/

79

5,5

/

/

/

/

80

4,9

/

/

/

/

Table A18: Mussel species, number of individuals, type, date and location of sampling Mytilus galloprovincialis in the Gulf of Venice, Italy.

Mussel species Mytilus galloprovincialis Mytilus galloprovincialis

Type of sampling By fishermen By fishermen

Sample Date Location Number of Location No. [dd/mm/yy] (coordinates) individuals 45,12143 N C1-C30 13.07.2015 Chioggia 30 12,44842 E C3044,21725 N 24.07.2015 Cesenatico 30 C60 12,46583 E

Derelict Fishing Gear management system in the Adriatic Region

Derelict Fishing Gear management system in the Adriatic Region Table A19: Length, width and weight of individual mussels of Mytilus galloprovincialis sampled in the Gulf of Venice, Italy.

Mussel width [cm]

Mussel weight [g]

Sample No.

C1

Mussel length [cm] 6.8

Mussel width [cm] 3.7

Mussel weight [g]

C31

Mussel length [cm] 7.7

3.2

1.2

C2

5.6

2.8

1.34

C32

6.1

3.2

1.36

C3

6.8

3.5

1.48

C33

7.2

3.3

2.29

C4

5.6

3

1.15

C34

8.2

3.95

2.49

C5

6.2

3.2

1.4

C35

5.6

2.8

1.12

C6

5.7

3

1.25

C36

6.35

3.3

2

C7

6

3.2

1.68

C37

5.95

2.8

1.68

C8

5.8

2.8

1.08

C38

6.35

3

1.85

C9

6.5

3

1.43

C39

5.8

3.4

2

C10

6

2.8

1.52

C40

6.5

2.8

1.93

C11

5.5

2.8

1.57

C41

6.7

3.3

1.88

C12

5.6

3.3

1.42

C42

7.8

3.9

3.04

C13

5.7

2.5

1.23

C43

6.7

3.3

1.68

C14

6.5

3.2

1.77

C44

8

3.9

2.4

C15

6.2

3.2

1.43

C45

7.3

3.3

2.27

C16

5.8

3

1.12

C46

7.8

3.5

2.42

C17

6.2

2.8

1.5

C47

7.5

3.3

2.35

C18

5.8

3

1.23

C48

7.3

3.3

2.58

C19

7.8

3.8

1.48

C49

6.7

3.4

1.42

C20

6.2

3.8

1.28

C50

7.3

3.2

1.73

C21

7

3.2

1.86

C51

6

3.2

1.33

C22

5.9

2.7

1.06

C52

6.5

3.3

1.99

C23

5.5

2.4

0.84

C53

7.2

3.6

2.68

C24

6.5

2.8

1.2

C54

6.7

3.3

1.53

C25

6.9

3

1.21

C55

6.2

2.7

1.68

C26

5.7

2.8

1.12

C56

5.65

2.8

1.48

C27

5.8

2.8

1.41

C57

7.15

3.45

2.6

C28

5.7

2.9

1.34

C58

6.3

2.8

1.37

C29

5.5

2.8

1.28

C59

6.6

2.8

1.26

C30

6.8

3

1.26

C60

6.4

3.1

1.39

Sample label

2.54

Derelict Fishing Gear management system in the Adriatic Region Table A20: fish species, number of individuals and technical data for sampling of fish in Slovenia in 2015.

Type of sampling

Date [dd/mm/yy]

Location

Liza aurata

By fishermen

19.9.2015

Ankaran

Sparus aurata

By fishermen

2.11.2015

Izola 1

Solea solea

By fishermen

4.11.2015

Izola 2

Fish species

Coordinates

Number of individuals

45,34' 59'' N 13,41' 59,7'' E 45,56 N 13,66 E 45,57 N 13,64 E

20 20 20

Table A21: Data table for individuals for Golden grey mullet (Liza aurata) caught by fishermen in Slovenia in 2015.

Sample label

Fish length [cm]

Fish weight [g]

Fish gender

Fish maturity stage

Fish deformation

C1

38.4

503

F

5

/

C2

38.3

482

F

5

/

C3

34.0

342

F

5

/

C4

35.4

366

F

5

/

C5

35.5

420

F

5

/

C6

34.0

302

M

4

/

C7

32.9

252

F

4

/

C8

33.1

350

F

4

/

C9

37.1

442

F

5

/

C10

34.9

383

F

4

/

C11

31.7

303

F

4

/

C12

31.2

288

M

0

/

C13

37.0

524

F

5

/

C14

31.5

294

F

3

/

C15

33.2

329

M

4

/

C16

30.6

275

F

4

/

C17

35.0

429

F

5

/

C18

34.2

347

F

4

/

C19

32.9

309

F

4

/

C20

34.7

356

F

5

/

Legend: M: male, F: female, /: not identified

Derelict Fishing Gear management system in the Adriatic Region Table A22: Data table for individuals for Gilt-head (sea) bream (Sparus aurata) caught by fishermen in Slovenia in 2015.

Sample label

Fish length [cm]

Fish weight [g]

Fish gender

Fish maturity stage

Fish deformation

O1

29.0

378

F

3

/

O2

29.8

430

F

3

/

O3

30.7

369

F

3

/

O4

27.6

299

F

3

/

O5

26.9

265

F

3

/

O6

28.2

353

F

3

/

O7

27.9

302

F

3

/

O8

27.6

301

F

3

/

O9

27.8

337

F

3

/

O10

28.2

319

F

3

/

O11

30.4

395

F

2

/

O12

29.0

348

F

2

/

O13

26.5

290

F

3

/

O14

29.6

356

F

3

/

O15

27.9

327

F

4

/

O16

29.4

382

F

3

/

O17

27.7

288

F

3

/

O18

27.7

316

F

3

/

O19

29.1

348

F

2

/

O20

29.0

387

F

2

/

Legend: M: male, F: female, /: not identified

Derelict Fishing Gear management system in the Adriatic Region Table A23: Data table for individuals for common sole (Solea solea) caught by fishermen in Slovenia in 2015.

Sample label Fish length [cm] Fish weight [g] Fish gender

Fish maturity stage

Fish deformation

L1

28.7

206

/

1

/

L2

25.9

133

/

1

right finn deformed

L3

27.5

179

/

1

/

L4

26.5

165

/

1

/

L5

28.0

201

F

4

/

L6

27.9

162

/

1

/

L7

25.7

145

/

1

/

L8

26.8

148

M

4

/

L9

27.4

169

/

1

/

L10

25.5

158

/

1

/

L11

27.6

177

/

1

/

L12

27.4

193

/

1

/

L13

27.9

195

/

1

/

L14

26.5

180

/

1

/

L15

28.9

183

/

1

/

L16

27.6

170

M

4

/

L17

25.0

147

M

4

/

L18

27.4

184

/

1

/

L19

28.0

207

/

1

/

L20

26.3

165

/

1

/

Legend: M: male, F: female, /: not identified