Mytilus Edulis) from Skutulsfjörður and Álftafjörður in the Westfjords of Iceland

Master’s Thesis

Understanding the Spatial Distribution of Arsenic, Cadmium, and Mercury Concentrations in Blue Mussels (Mytilus edulis) from Skutulsfjörður and Álftafjörður in the Westfjords of Iceland

Anna N. Hixson

Advisor: Helga Gunnlaugsdóttir Ph.D.

University of Akureyri Faculty of Business and Science University Centre of the Westfjords Master of Resource Management: Coastal and Marine Management Ísafjörður, May 2019

Supervisory Committee

Advisor: Helga Gunnlaugsdóttir, Ph.D.

External Reader: Pernilla Carlsson, Ph.D.

Program Director: Catherine Chambers, Ph.D.

Anna N. Hixson Understanding the Spatial Distribution of Arsenic, Cadmium, and Mercury Concentrations in Blue Mussels (Mytilus edulis) from Skutulsfjörður and Álftafjörður in the Westfjords of Iceland

45 ECTS thesis submitted in partial fulfilment of a Master of Resource Management degree in Coastal and Marine Management at the University Centre of the Westfjords, Suðurgata 12, 400 Ísafjörður, Iceland

Degree accredited by the University of Akureyri, Faculty of Business and Science, Borgir, 600 Akureyri, Iceland

Printing: Háskólaprent, Reykjavík, May 2019

Declaration

I hereby confirm that I am the sole author of this thesis and it is a product of my own academic research.

______

Anna Nicole Hixson

Abstract Trace elements above natural background levels can be a threat to marine ecosystem health and potentially also human health. Long-term global monitoring programs have generated large datasets that allow for a better understanding of how concentrations of trace elements in the marine biosphere change over time. These monitoring programs utilize water, sediment, and/or biota samples to understand how these elements are interacting in the environment. However, these monitoring programs normally do not investigate oddities or unusual conditions further. In the case of the Westfjords Iceland, concentrations of arsenic (As), cadmium (Cd), and mercury (Hg), have been prominent in the local blue mussel (Mytilus edulis) for over a decade (1999-2018) in Skutulsfjörður and Álftafjörður. Although, these concentrations have shown peculiarities when compared to other monitoring sites in Iceland, they have not been extensively studied. This thesis aimed to generate a better understanding of the distribution of As, Cd, and Hg in Skutulsfjörður and Álftafjörður, highlight potential sources of these pollutants, and establish viable strategies for future research and management. This study reveals that high As is not limited to a single site in Skutulsfjörður (24.05-83.87 mg/kg dw); illustrates the relatively constant levels of all elements in Álftafjörður since its last sampling in 2013; the potential role of pollution sources on these concentrations; and the differences in these concentrations between populated fjords and rural fjords. These findings suggest the need for new management strategies including further monitoring and more widespread analysis of trace elements in biota in this area, to better understand and mitigate any potential negative consequences that these elevated concentrations may have on the surrounding coastal environment. Útdráttur Ólífræn snefilefni yfir náttúrulegum bakgrunnsgildum geta verið ógn fyrir vistkerfi sjávar og hugsanlega einnig fyrir heilsu manna. Langtíma alþjóðleg vöktunarverkefni hafa leitt af sér yfirgripsmikil gagnasöfn sem auka skilning á því hvernig styrkur ólífrænna snefilefna í lífríki sjávar breytist með tímanum. Í þessum alþjóðlegu vöktunarverkefnum eru tekin sýni af vatni, seti og/eða dýrum/plöntum úr lífríkinu til að skilja tengsl þessara snefilefna við umhverfið. Hins vegar rannsaka þau yfirleitt ekki nánar óvenjulegar niðurstöður sem stinga í stúf. Á Vestfjörðum hefur styrkur arsens (As), kadmíums (Cd) og kvikasilfurs (Hg) verið hár í kræklingi (Mytilus edulis) í meira en áratug (1999-2018) í Skutulsfirði og Álftafirði. Þrátt fyrir að styrkur þessara efna hafi verði hár í samanburði við aðra vöktunarstaði á Íslandi, þá hafa ekki verið gerðar margar rannsóknir á þessu sviði. Þessi ritgerð miðar að því að skapa betri skilning á dreifingu As, Cd og Hg í Skutulsfirði og Álftafirði, benda á hugsanlega uppsprettur þessara mengunarefna og koma með tillögur að mögulegum stjórnunaraðferðum sem og framtíðar rannsóknum. Niðurstöður þessarar rannsóknar sýna; að hár styrkur As sé ekki einungis bundinn við einn stað í Skutulsfirði (24.05-83.87 mg/kg dw); að styrkur allra þriggja snefilefnanna sé svipaður og þegar síðast voru tekin sýni í Álftafirði árið 2013;

v hugsanleg áhrif mengandi uppspretta á styrk þessara snefilefna og muninn í styrk snefilefnanna í fjörðum sem eru nágrenni íbúabyggðar eða dreifbýli. Þessar niðurstöður benda til þess að þörf sé á nýjum stjórnunaraðferðum þ.m.t. frekari vöktun og eftirlit með styrk ólífrænna snefilefna í lífríki sjávar á þessum slóðum til þess að skilja betur og draga úr hugsanlega neikvæðum afleiðingum sem hár styrkur snefilefna getur haft á strandsvæði og nærliggjandi lífríki.

vi Table of Contents Abstract ...... v List of Tables ...... ix List of Figures...... x Acronyms and Terms ...... xi Acknowledgments ...... xii 1 Introduction ...... 1 1.1 Marine Pollutants from a Global Perspective ...... 1 1.2 Marine Pollutants in the Westfjords of Iceland...... 2 1.3 Research Aims and Questions ...... 5 1.4 Justification of Research ...... 6 2 State of Knowledge ...... 9 2.1 Coastal and Marine Monitoring ...... 9 2.2 Blue Mussels as Biomonitors ...... 9 2.2.1 Biomonitoring Programs ...... 11 2.3 Trace Metals in the Marine Environment ...... 14 2.3.1 Trace Metals in the Arctic ...... 15 2.3.2 Trace Metals in Iceland ...... 16 2.3.3 Arsenic, Cadmium, and Mercury ...... 20 3 Methodology ...... 25 3.1 Sampling ...... 25 3.1.1 Sampling and Study Area ...... 25 3.1.2 Sample Collection ...... 29 3.2 Analyses of trace metals ...... 32 3.2.1 Sample preparation ...... 32 3.2.2 Trace metal analysis ...... 32 3.3 Statistical Analyses ...... 34 4 Results ...... 35 4.1 Temporal Analysis Results ...... 35 4.1.1 Average Concentration of Trace Elements ...... 35 4.1.2 Temporal Trends ...... 35 4.2 Spatial Distribution ...... 39 4.3 Environmental Impacts ...... 41 4.4 Concentrations at three locations in Westfjords ...... 45 5 Discussion ...... 47 5.1 Temporal Trends ...... 47 5.2 Spatial Distribution ...... 48 5.2.1 Environmental Impacts in Skutulsfjörður ...... 50 5.2.2 Three Locations within the Westfjords ...... 51 5.3 Study Limitations and Future Research...... 53 5.3.1 Limitations ...... 53

vii 5.3.2 Future Research ...... 53 5.4 Regional Management Suggestions ...... 56 5.4.1 Public Health ...... 56 5.4.2 Environmentally Protected Areas ...... 56 6 Conclusion ...... 59 References ...... 61 Appendix A ...... 68 Appendix B ...... 69 Appendix C ...... 81 Appendix D ...... 82 Appendix E ...... 103

viii List of Tables

Table 1: Overview of each sampling site and its surroundings ...... 28 Table 2: The values of certified reference materials Dorm-4, Fish Protein and blue mussel tissue collected in 2018 ...... 33 Table 3:LOQ and LOD values for blue mussel tissue analyses in 2018 ...... 34 Table 4: Average concentrations of As, Cd, and Hg in Skutulsfjörður and Álftafjörður over time ...... 35 Table 5: Concentration per element per site for 2018 ...... 39 Table 6: Average concentration of As near sites of impact ...... 43 Table 7: Average concentration of Cd near sites of impact ...... 43 Table 8: Average concentration of Hg near sites of impact ...... 44 Table 9: Concentrations of As, Cd, and Hg in Skutulsfjörður, Álftafjörður, and Skötufjörður in 2018 ...... 46

ix List of Figures

Figure 1: Map of Iceland highlighting the Westfjords in green ...... 3 Figure 2: Skutulsfjörður and the location of the former incinerator ...... 4 Figure 3: Locations of blue mussel sampling sites around Iceland for 2017 ...... 18 Figure 4: The ten sites where blue mussel sampling was conducted in the Westfjords of Iceland in 2018 ...... 24 Figure 5: All sampling site locations with corresponding images of the location depicting the surrounding area within Skutulsfjörður and Álftafjörður ...... 27 Figure 6: Cleaning the mussels of any debris ...... 30 Figure 7: 2.5cm mesh used to suspend the mussels ...... 31 Figure 8: Makeshift depuration bucket ...... 31 Figure 9: Concentrations of As, Cd, and Hg in blue mussels in Skutulsfjörður over time ...... 37 Figure 10: Concentrations of As, Cd, and Hg in blue mussels in Álftafjörður over time ...... 38 Figure 11: Concentrations of As, Cd, and Hg (mg/kg dw) in blue mussels from all sites in Skutulsfjörður for 2018...... 40 Figure 12: Concentrations of As, Cd, and Hg (mg/kg dw) in blue mussels from both sites in Álftafjörður for 2018...... 41 Figure 13: Concentration of As per site located near industry, sewage/river outflows, or no known impacts in Skutulsfjörður...... 42 Figure 14: Concentration of Cd per site located near industry, sewage/river outflows, or no known impacts in Skutulsfjörður...... 43 Figure 15: Concentration of Hg per site located near industry, sewage/river outflows, or no known impacts in Skutulsfjörður...... 44 Figure 16: Locations of blue mussel sampling sites in Skutulsfjörður, Álftafjörður, and Skötufjörður for 2018...... 45 Figure 17: Depiction of the protected area at the base of Pollurinn and the additional suggested sampling sites for future research...... 54

x Acronyms and Terms

AMAP-Arctic Monitoring and Assessment Programme As- Arsenic ATDSR-Agency for Toxic Substances and Disease Registry Cd-Cadmium CEC- Contaminants of Emerging Concern CEMP-Coordinated Environmental Monitoring Programme Cu-Copper Dw-dry weight FAO-Food and Agricultural Organization Hg-Mercury ICES- International Council for Exploration of the Sea ICP-MS-Inductively Coupled Plasma Mass Spectrometer JAMP-Joint Assessment and Monitoring Programme MFRI- Marine and Freshwater Research Institute Iceland NOAA-National Oceanic and Atmospheric Association OSPAR- Convention for the Protection of the Marine Environment of the North-East Atlantic PAH-polycyclic aromatic hydrocarbons Pb- Lead PCB-polychlorinated biphenyls Se-Selenium SMW-State Mussel Watch Program Tn-Tin WFD- EU Water Framework Directive (2000/60/ce)

Ww-wet weight

Zn-Zinc

xi Acknowledgments

I would like to thank my advisor Helga Gunnlaugsdóttir for her guidance throughout this entire process; Erla Sturludottir for sharing her data tables and her overall suggestions and recommendations for the project; Natasa Desnica and Branka Borojevic for guiding me through the analysis preparation methods and assisting in its completion, as well as completing the elemental analysis; The Environmental Agency of Iceland (UST) for giving me access to previous monitoring data; Matis and MFRI for allowing me to use their facilities for preparation and analysis of my samples; the Arctic Fox Centre and Stephen Midgley, Gudbjorg, Anja, and Cristian Gallo for allowing me to borrow equipment for this project; Brack Hale for his suggestions and assistance with maps, and Jennifer Smith for editing assistance; Halldor and Gudbjorg for passing along the sampling guidelines for me to follow; Siggi and Dan for assisting in my statistical analysis processes; Astrid for her guidance with QGIS; and Hlynur for translating some Icelandic documents.

xii 1 Introduction 1.1 Marine Pollutants from a Global Perspective

A nearly endless list of pollutants can be found in the marine environment: plastics, fertilizers, pesticides, antibiotics, hormones, heavy metals, and even items such as rubber ducks or televisions. These pollutants are entering the marine ecosystem through runoff, sewage pipelines, shipping and fishing vessels, and also through atmospheric processes (Beyer et al., 2017; Goldberg, 1975; Kühn & van Franeker, 2012). Some pollutants, such as trace elements, occur naturally in the marine and terrestrial environment (Jörundsdóttir et al., 2014; Richir & Gobert, 2016). Trace elements are naturally present at low levels, also known as background levels, in biota, sediment, and water and in many cases are needed to sustain life (Richir & Gobert, 2016; Wada, 2004). However, when trace elements increase past these background levels, they can become toxic. Commonly monitored elements along European coastlines are lead (Pb), selenium (Se), cadmium (Cd), arsenic (As), mercury (Hg), copper (Cu), and zinc (Zn) (Convention for the Protection of the Marine Environment of the North-East Atlantic [OSPAR], 2018b). All of these elements, although naturally occurring, can be enhanced through anthropogenic processes such as mining, improper battery disposal, smelting, fertilizer and pesticides, and sewage discharge (Furness, 1990; OSPAR Commission, 2009) This can lead to increased stress on marine organisms leading to the death of ecosystems, and long term human exposure to these elements can lead to serious health problems such as cancer (Bach, Sonne, Rigét, Dietz, & Asmund, 2014; International Agency for Research on Cancer & Weltgesundheitsorganisation, 2012; Keil, Berger-Ritchie, & McMillin, 2011).

Marine pollutants cause damage to coral reefs, contribute to ocean acidification, entangle animals such as sea turtles, decreasing species resilience, or, as in the case of plastics, fill the stomachs of fish, birds, and other wildlife. Worldwide, pollutants such as polycyclic aromatic hydrocarbons (PAH’s), polychlorinated biphenyls (PCB’s), trace metals, and other

1 pollutants, such as contaminants of emerging concern (CEC’s) are monitored annually or bi- annually along coastlines (Lanksbury et al. 2014; Jörundsdóttir et al.,2013; Sturludottir et al. 2013; AMAP, 2011, 2017, 2018; ). What happens along coastlines will impact the open ocean, and vice versa. It is important to monitor these pollutants in coastal environments to create baselines to better protect and maintain proper ecosystem function and health.

1.2 Marine Pollutants in the Westfjords of Iceland

Iceland is an island of 357,050 people (Statice.is, 2019) situated on top of the North American and Eurasian tectonic plates. Iceland’s position along this fault line allows for volcanic and geothermal activity throughout the island, which can contribute to trace levels of elements in coastal and freshwater systems (Bárðarson, 2009; Stefansson & Arnórsson, 2005). Although Iceland is quite isolated geographically, various factors can influence the amount and types of pollutants found along the Icelandic coastline, such as the level of industrial and human activity in the country, the degree of volcanic activity, long-range atmospheric transport, and ocean currents (AMAP, 2011; ICES, 2018).

Iceland has been considered a pristine environment due to its small population and location, far removed from heavily industrialized nations (Egilson et al., 1999; Eydal et al., 2013; Jörundsdóttir et al., 2014). Despite this, as a shipping and fishing nation, its many harbours can contribute to both localized and widespread pollution. Marine vessels and harbours can contribute to the dispersal of substances such as oil, PAH’s, and metals such as Tin (Tn) and Cu (Halldórsson, Svavarsson, & Granmo, 2005). Additionally, some of the highest levels of trace elements have been found in blue mussels even in remote regions of Iceland, such as the island of Grimsey (population 71) (Statice.is, 2019; Sturludottir et al., 2013). Annual biomonitoring of the coastlines in Iceland revealed Grimsey had one of the highest concentrations of Hg (0.077 mg/kg, compared to the lowest 0.041 mg/kg at Hvitanes) and the highest concentration of Zn (180 mg/kg, compared to the lowest 120 mg/kg at Botn) in Iceland from the 1990’s until 2010 (Sturludottir et al., 2013).

2 In the Westfjords region of Iceland, located in the far northwest corner of the country, marine pollutants are apparent on a more localized scale. Figure 1 (below) depicts the region of the Westfjords of Iceland in green. The Westfjords of Iceland is home to a population of 7,063 (Statice.is, 2019) and Ísafjörður is the largest town with 2,752 individuals (Statice.is, 2019). Ísafjörður is a part of a larger municipality called Ísafjarðarbær, which includes the towns of Flateyri, Sudereyri, and Thingeyri. Thus, the municipality centralizes many services in Ísafjörður such as the medical facility, major grocery stores and former incinerator.

Figure 1: Map of Iceland, highlighting the Westfjords in green, the red box highlights the large fjord of Isafjordardjup, within this fjord are the two smaller fjords used in this study Skutulsfjörður and Álftafjörður. (Map data: Based on data from National Land Survey of Iceland)

3 In 2010, the municipal incinerator in the town of Ísafjörður in Skutulsfjörður had to close operations due to the release of various dioxins that had contaminated the meat and milk produced by the neighbouring cattle and sheep farms (Halldorsson et al., 2012). Figure 2 pinpoints the location of Ísafjörður and the former incinerator within the Westfjords. This incident was isolated to Skutulsfjörður due to its high mountains and narrow inlets, but it negatively impacted the farms in the area. The livestock were sent to slaughter, but neither the meat nor their products could be sold due to the contamination (Halldorsson et al., 2012). Annual biomonitoring of the region utilized local blue mussels (Mytilus edulis) in one location in both Skutulsfjörður and Álftafjörður as sampling sites. In Skutulsfjörður, near the river Úlfsá (see Figure 2), levels of As have been high (>40 mg/kg) and levels of Hg have hovered around 0.10 mg/kg (dw) for the past twenty years (Sturludottir et al., 2013).

Figure 2:Location of Skutulsfjörður in the Westfjords and the former incinerator within Ísafjörður (red x) and the annual sampling location near Úlfsá (blue circle). (Map data: Based on data from National Land Survey of Iceland) 4 Initially, the high concentration of As was thought to be attributed to the former incinerator, however, further research suggest that the incinerator has not played a role in As concentrations, and that other causes may exist such as old oil tanks in the area (Jörundsdóttir et al., 2013; Sturludottir et al., 2013). In 2010, the highest concentration of Hg in Iceland was found in Skutulsfjörður near the Úlfsá river at 0.081 mg/kg (dw) (Sturludottir et al., 2013). Sewage, runoff, abandoned waste depositories, and/or landfills are potentially impacting the surrounding ecosystem in Skutulsfjörður and could impact these levels (Gharibi, 2011).

This region of Iceland has also experienced high levels of Cd in blue mussels in Arnarfjörður in the Southwest, and in fjords such as Hestfjordur located in Isafjordardjup. These values ranged from 0.95-2.05 mg/kg ww (Gunnlaugsdóttir et al., 2007; Sturludottir et al., 2013). The European Unions limit for consumption for Cd, is 1.0 mg/kg ww, may of these locations have surpassed this limit (Gunnlaugsdóttir et al., 2007). The presence of Cd in Iceland is most likely due to its volcanic activity rather than anthropogenic sources (Egilson et al., 1999). Even though Cd may originate from natural sources, high levels can still be dangerous to ecosystem and human health. Sturludottir et al. (2013) expressed that the concentrations of Cd in Iceland were higher than the average concentrations found in Norway and the Eastern US, but were comparable to those found in China from the 1990s to 2000s. In Arnarfjörður, mussel cultivation was banned due to the concentration of Cd being higher than the European Commissions limit for consumption (1.0 mg/kg wet weight) (Gunnlaugsdóttir et al., 2007). Whether the source is anthropogenic or natural, these concentrations of trace elements in this remote region of Iceland need to be investigated further, especially if the coastal resources are going to be used for human or animal consumption.

1.3 Research Aims and Questions

This thesis aims to generate a better understanding of the spatial distribution of As, Cd, and Hg in Skutulsfjörður and Álftafjörður. This information will generate valuable information for the municipalities of Ísafjörðurbaer and Sudavikhreppur, which could be used to develop

5 appropriate management and mitigation strategies. The main objectives of this thesis are to [1] develop a better understanding of the spatial distribution of As in blue mussel concentrations in Skutulsfjörður [2] assess the levels of As, Cd, and Hg in Álftafjörður since its last sampling (2013), and [3] examine sources of pollution throughout Skutulsfjörður that could potentially contribute to higher concentrations of As, Cd and Hg. Answering the following research questions will fulfil the objectives of this thesis:

 Question 1: What is the distribution of As in blue mussels throughout Skutulsfjörður, are high levels isolated to the singular location (Úlfsá) or widespread?  Question 2: How do As, Cd, and Hg levels in Álftafjörður compare to previous sampling data, last measured in 2013?  Question 3: Do concentration levels of As, Cd, and Hg change due to their proximity to natural or artificial geographical features (i.e. sewage pipes, industrial work areas, river outflows)?  Question 4: How do concentrations of As, Cd, and Hg differ between fjords with low, mid-level and high human presence?

1.4 Justification of Research

As, Cd, and Hg are three toxic trace metals that can enter the marine and coastal system through various ways, such as fluvial discharge, industrial runoff, wastewater, and leaching. All of these metals are listed as priority hazardous substances by the Agency for Toxic Substances and Disease Registry (ATDSR, 2018). Although these metals are monitored in biota around the Icelandic coastline at a national level, their presence is seldom investigated at a more regional level with the exception of Cd measurements in Arnarfjörður (Gunnlaugsdóttir et al., 2007). This lack of further investigation is apparent in Skutulsfjörður and Álftafjörður.

Regular monitoring of the Icelandic coastline has shown that concentrations of As in Skutulsfjörður have been 3.7 times higher than in Álftafjörður, the adjacent fjord (Jörundsdóttir et al., 2014; Sturludottir et al., 2013). The annual sampling for blue mussels

6 always occurs in the same location to maintain a monitoring standard (Sturludottir et al., 2013; Coordinated Environmental Monitoritng Program [CEMP], 2018). It is important to maintain consistent monitoring, however, a historically high concentration at a singular monitoring location warrants further investigation.

Additionally, until 2016 the neighbouring fjord, Álftafjörður was also regularly sampled and exhibited high levels of Cd compared to Skutulsfjörður and the rest of Iceland (Sturludottir et al., 2013). These two neighbouring fjords are located in the geologically oldest area of Iceland, and have historically high levels of two different trace elements. Therefore, it is important to revaluate these areas and update the values of Cd in Álftafjörður and As in Skutulsfjörður.

Furthermore, Sturludottir et al. (2013) observed that the temporal average of Hg was the highest in Skutulsfjörður compared to the rest of Iceland. Although Hg has not surpassed the environmental quality standard of 20 ug/kg (ww) (European Commission, 2006) in Skutulsfjörður or Álftafjörður, it is present and should be investigated further.

An overall inquiry to these elevated values in the remote region of the Westfjords has not been compiled. The current monitoring plan has not investigated the source or the extent of the pollution spatially in either fjord. The concentrations of As, Cd, and Hg are potentially hazardous to individual organisms, ecosystems, and human health. Thus, it is important that the overall extent of this pollution is investigated further.

This thesis illustrates the current state of knowledge regarding the use of blue mussels as biomonitors for coastal pollution, subsequent biomonitoring programs and the state of trace elements in the marine environment and their potential impacts on ecosystem and human health. The methods used are comparable to the aforementioned biomonitoring programs, from blue mussel sampling through to elemental analyses. The data collected highlights the wide spread nature of As concentration within Skutulsfjörður with ranges from 24-85 mg/kg dw. Blue mussels from two other fjords sampled in 2018 ranged from 9.32-11.43 mg/kg dw. This discrepancy suggests the need for further research on this topic leading to future management and mitigation strategies.

2 State of Knowledge

2.1 Coastal and Marine Monitoring

Coastal monitoring and marine monitoring are practices that are used around the world at international, national, and regional levels. The practice promotes sampling of local species ranging from marine fauna to deep-sea fish as well as, water, and sediment to assess known contaminants such as heavy metals, microplastics, chemicals (organic and inorganic), and contaminants of emerging concern (Goldberg, 1975; Maruya et al., 2014; Richir & Gobert, 2016; Scarpato et al., 2010). For nations residing in the EU, the EU Water Framework Directive (WFD) (2000/60/ce) lists marine pollution as one of the largest concerns that is in need of regular monitoring (European Commission, 2000, 2008). The practice of coastal monitoring is important to better understand how industry, agriculture, and development impact the coastal environment and its many species. The regular monitoring of pollutants along coastlines around the world allows for the development of important baselines (Arctic Monitoring and Assessment Program [AMAP], 2011; Jörundsdóttir et al., 2014; Lanksbury et al., 2014; Melwani et al., 2013;). These baselines are likewise important in order to quantify the extent and severity of a hazardous event, such as an oil spill or a natural disaster (Lanksbury et al., 2014; Maruya et al., 2014). Lastly, it is important to have baselines of current contaminant levels to better understand how climate change will interact with pollutants overtime and their overall impact on our marine environment (Coppola et al., 2018; Jörundsdóttir et al., 2014).

2.2 Blue Mussels as Biomonitors

Mytilus edulis, commonly known as the blue mussel, are sessile marine bivalves located in the intertidal zone in Iceland and across the globe (Sturludottir et al., 2013; Chase et al., 2001). The blue mussels sessile nature, resilience, and adaptability to varying salinities and

9 temperatures, along with its overall abundance across the globe, assist this specie to be an appropriate tool for biomonitoring (Chandurvelan et al., 2015). In particular, these characteristics make the blue mussel a reliable species for monitoring trace metals and other contaminants in the marine ecosystem (Chandurvelan et al., 2015; Chase et al., 2001). As bivalves, blue mussels filter water and food through their digestive system with the ebb and flow of tides and currents, picking up particulates and contaminants as they naturally move through the marine system (Gouelletquer, 2004; Chandurvelan et al., 2015; Chase et al., 2001; Lanksbury et al., 2014). Because of the consumption of water and particulates as food, mussels can concentrate 105 more contaminants in their soft tissue than found in the water column or sediment in the same area. This allows for a more accurate image of pollution levels than water samples or sediment samples alone (Andral et al., 2010; Lanksbury et al., 2014). This was apparent in the North Sea when PCB and PAH concentrations were measurable in mussels, but were undetectable in the water column (Lanksbury et al., 2014). The anatomy of blue mussels and other bivalves is also important because of the role of these species in the marine ecosystem. In nutrient-rich ecosystems, mussels can decrease the amount of nutrients in the water column, such as nitrogen (Northern Economics Inc, 2009). In these cases, the nitrogen is fixed in the mussels’ digestive system and removed from the surrounding environment, reducing the potential for eutrophication or “dead zones” and making the area more habitable for other species (Galimany et al., 2017). Consequently, mussels are deemed “cleaners” and add a unique ecological value to the marine ecosystem (Gouelletquer, 2004).

When mussels are used as biomonitors, they can be sampled from coastal zones where they naturally occur or they can be transplanted from mussel farms (Lanksbury et al., 2014; CEMP, 2018). Both methods are adequate; however, using naturally occurring mussels is more cost and time effective. Using transplanted mussels allows for a greater degree of control as well as renders quantifiable the impacts certain pollutants may have on the mussel (Lanksbury et al., 2014).

10 2.2.1 Biomonitoring Programs

International Monitoring Agreements

In Iceland, blue mussels have been collected annually to assess contaminants around the Icelandic coastline (Sturludottir et al., 2013; Jörundsdóttir et al., 2014). This information is compiled annually to oblige the Oslo Paris Agreement (OSPAR). The OSPAR commission was developed in the late 1960s, initially to address pollution in the North Sea, continuing until the late 1990s. After the meetings of the Oslo and Paris Commissions in 1992, OSPAR adopted the Convention for the Protection of the Marine Environment in the North-East Atlantic Ocean, within this convention the new OSPAR Commission was developed (OSPAR, 2018). At present, the commission focuses on biological diversity and ecosystems, hazardous substances and eutrophication, human activities, offshore industry, radioactivity, and cross-cutting issues (OSPAR, 2018b; OSPAR Commission, 2010). Sixteen countries are participants in the OSPAR commission, including Iceland. The commission established the Joint Assessment and Monitoring Program (JAMP) to outline strategies and methods for participating countries to utilize during monitoring and assessment to help them contribute consistent data to the commission. In 2016, JAMP was moved to a broader section of the agreement, called the Coordinated Environmental Monitoring Program (CEMP), and the program was updated under this title in 2018 (OSPAR, 2018a). The CEMP Guidelines for Monitoring Contaminants in Biota (2018) are similar to other monitoring programs, such as those used by Mussel Watch (Goldberg, 1975) in the United States and the previous trans boundary program in the Mediterranean, Evaluation of Coastal Water Contamination Using Biointegrators [MYTILOS] (Andral et al., 2011). These monitoring methods consist of a process of sampling, depuration, dissection, and freezing of blue mussels or other Mytilus spp (CEMP, 2018). The methods used in this thesis are based on OSPAR’s official CEMP Guidelines for Monitoring Contaminants in Biota.

In Iceland, the sampling process is slightly altered due to the need to sample native mussels in remote areas; thus, after depuration the mussels are frozen until analysis. Reference

11 material for these alterations, as well as the official CEMP guidelines, can be found in Appendix B and C. Once compiled, data from all OSPAR participating countries shall be submitted and uploaded to the International Council for Exploration of the Sea (ICES) (ices.dk) database allowing for national and international comparisons.

Additionally, for Iceland and other Arctic nations, the data collected through the OSPAR commission is used in the Arctic Monitoring and Assessment Program (AMAP), which allows for Arctic nations to publish studies related to pollution, climate change, and ecosystem and human health (AMAP, 2016, 2017). Developed in 1991, AMAP is one of six working groups in the Arctic Council. The program’s main goals are to compile regularly monitored data on pollutants and climate through atmospheric, freshwater, seawater, and biota testing to appropriately inform policy decisions in Arctic countries (AMAP, 2017).

In Iceland, blue mussels have been used for over 20 years to monitor trace elements and other contaminants in the marine environment (Jörundsdóttir et al., 2014; OSPAR, 2010; Sturludottir et al., 2013) . Fulfilling OSPAR and participating in AMAP has allowed for the generation of substantial datasets on contaminants in Iceland and across the Arctic, providing valuable comparisons and beneficial temporal data to track changes within the marine and terrestrial environment (AMAP, 2017).

National and Regional Agreements

National and local environmental research programs around the world have and continue to use blue mussels (Mytilus edulis or other Mytilus spp) as biomonitors. Programs using mussels as biomonitors exist in the United States at national, regional, and state levels, as well as in various European countries such as Germany and the Netherlands, and along the Mediterranean coast (Adams et al., 2001; Lanksbury, Niewolny, Carey, & West, 2014; NOAA, 2018; Richir & Gobert, 2016; Stanković, Jović, Milanov, & Joksimović, 2011)

In the United States, the National Oceanic and Atmospheric Association (NOAA) developed the Mussel Watch Program under the department of National Status and Trends (NS&T) in 1986 (Chase et al., 2001; NOAA, 2018) The Mussel Watch Program collects

12 native mussels and oysters and around the coastline of the United States to track trends of various contaminants (Chase et al., 2001; Lanksbury et al., 2014). Mussel Watch has generated valuable temporal data sets tracking contamination at local, regional, and national levels (Chase et al., 2001; Lanksbury et al., 2014; Bricker et al., 2014). The program has also used these temporal trends to better understand the impacts that storm events, such as Hurricane Katrina or Hurricane Sandy, can have on near-shore contamination(Lanksbury et al., 2015; NOAA, 2018) Today, Mussel Watch is the longest standing contamination study within the United States (Lanksbury et al., 2014; Bricker et al., 2014). Due to lessons learned from the Mussel Watch Program, various states and regions have expanded and are now generating more regional or localized data to better assess the needs for intervention, development, and continuous monitoring. For example, Washington State, California, and the Gulf of Maine are areas that have further developed regional approaches to coastal monitoring (see e.g., Lanksbury et al., 2014).

Washington State developed its own pilot program after Mussel Watch called for more action and volunteers in 2012. By including additional sampling locations and changing from native mussels to transplanted mussels, the Mussel Watch Pilot Expansion Program was created (Lanksbury et al., 2013). The move towards transplanted mussels occurred due to the lack of native specimens; it aimed to monitor the effects of contaminants on mussels, studying, for example, rate of survival, growth, and reproduction capability ([American Society for Testing and Materials] ASTM International, 2007; Lanksbury et al., 2014). The program can therefore both measure concentrations of contaminants as well as the overall impact it has on a previously healthy specimen.

Similarly, California developed the State Mussel Watch Program (SMW) in 1977 (Bricker et al., 2014). Although this larger statewide program ended in 2003, a series of sites are still regularly sampled through endowment programs and other partners, such as the regional monitoring program in the San Francisco estuary and the state water resources board (Melwani et al., 2013). Between 2009 and 2010, NOAA wanted to investigate contaminants of emerging concern along the California coastline, such as pharmaceutical products, flame- retardants, and hormones (Bricker et al., 2014). Using similar methods and 25 of the

13 previously used sites from the national Mussel Watch Program, this study investigated contaminants of emerging concern as well as the previously studied “legacy” pollutants (Bricker et al., 2014; Melwani et al., 2013). As in Washington State, transplanted mussels are now used in California to enable more control on the location and exposure time rather than on specific species (Bricker et al., 2014). This in turn has allowed for the development of localized management strategies to assess and mitigate at-risk areas (Melwani et al., 2013; Bricker et al., 2014).

Gulf Watch in the Gulf of Maine is both a regional and an international approach to coastal monitoring because the Gulf of Maine includes not only U.S. states such as Massachusetts, New Hampshire, and Maine, but also encompasses Canadian provinces and Canadian water bodies such as the Bay of Fundy (Chase et al., 2001). Because this area is so large and includes diverse ecosystems, the region created its own additional program, Gulf Watch, which includes information collected from Canada and the United States(Chase et al., 2001). This transboundary monitoring program uses similar methods to the OSPAR protocol, such as collecting naturally occurring specimens of a certain size and in the same season.

2.3 Trace Metals in the Marine Environment

Trace metals are small amounts of natural elements found within terrestrial and marine environments (Richir & Gobert, 2016). Industrialization processes have the ability to increase the quantity of trace elements present in terrestrial and marine ecosystems over time (Halldorsson et al., 2012). Additionally, industrial practices such as mining and refining can alter the chemical structure of trace metals, increasing their toxicity. For example, mining tailings are the organic waste from mines that once exposed to the environment can be hazardous (Ferreira da Silva, Zhang, Serrano Pinto, Patinha, & Reis, 2004). In Portugal, the abandoned Castromil mine has left the surrounding agricultural land ridden with As and Pb. When tailings are left unattended, chemical and environmental weathering has the potential to disperse heavy metals such as As and Cd in to the surrounding environment (Ferreira da Silva et al., 2004).

14 Trace elements in the marine environment are monitored globally because of their overall toxicity when the concentration surpasses background levels (Richir & Gobert, 2016). The toxicity of these metals has the ability to negatively impact not only individual species, but also entire ecosystems. Some trace metals are biologically necessary for survival, such as Zn, which assists in cell division and formation (Furness, 1990). However, all metals can become toxic in high enough concentrations, and this toxicity threshold varies by metal as well as organism (Furness, 1990).

2.3.1 Trace Metals in the Arctic

The Arctic is a unique area with small populations, cold climates, and little industrial activity. Countries in the Arctic that regularly participate in coastal monitoring using blue mussels are Greenland, Iceland, the Faroe Islands, Norway, and Sweden (AMAP, 2011; Rigét et al., 2011; Jörundsdóttir et al., 2014). Comparing trace elements at these sites reveals an important baseline for understanding how pollutants are developing (whether natural or anthropogenic) across the Arctic—a “pristine” area (Jörundsdóttir et al., 2014). Although there are many similarities to these Arctic nations, regional factors, development, long-range atmospheric transport, and currents play major roles in the concentration of trace metals across the Arctic (Rigét et al., 2011 and Jörundsdóttir et al., 2014).

The Arctic in many ways has avoided large-scale development and overuse of fossil fuels (AMAP, 2016; Poppel et al., 2015). However, due to climate change and increased globalization, the Arctic is becoming a hotspot for tourism, oil exploration, and new shipping routes. Additionally, the Arctic is considered a “sink” for global pollutants (AMAP, 2016, 2017). All river and stream effluents in the northern hemisphere enter an ocean that can circulate northwards towards the Arctic, depositing pollutants via naturally occurring oceanic currents. Also, atmospheric transport of particulates from coal power plants, oil refineries, and smelters experience long-range atmospheric transport that are regularly deposited on the Arctic ice sheet (AMAP, 2016). The threats of future development in the Artic in conjunction with natural processes, such as long-range

15 atmospheric transport, are concerning. Much of the human diet in the area comes from the sea; this gives further reason to monitor, assess, and mitigate sources of contamination.

Trace Metals in Bergen, Norway

Blue mussels were used to assess trace element concentrations in Bergen, Norway, in 2004 (Airas, Duinker, & Julshamn, 2004). Prior to 1997, discharge of both municipal waste water and sewage water directly flowed into the Bergen harbour (Airas et al., 2004; Gharibi, 2011). Copper, zinc, and cadmium concentrations were attributed to sewage discharge in Bergen, based on studies prior to 1997 (Airas et al., 2004). Additionally, mercury concentrations were considered to be influenced by industrial activities in the area (Airas et al., 2004). Overall, the study assessed changes of trace metal concentrations since the removal of the raw sewage discharge in to the harbour (Airas et al., 2004). Sampling sites were located all over the Bergen harbour and concentrations of trace metals varied throughout (Airas et al., 2004).

After the removal of the discharge, Airas et al. (2004) found that certain metals such as Zn (15-51 mg/kg ww) and Cu (0.8-4.1 mg/kg ww) although deminished from the previous study, still remained high compared to averages around Norway (Zn: 8.0-23.0 mg/kg ww, and Cu: 0.5-2.3 mg/kg ww), concluding that even after the removal of the discharge these elements remain available for uptake by the mussels, potentially stored within the sediment. The study suggests that the removal of the sewage waste water discharge in the Bergen harbour has reduced the level of several trace elements, including Cd, Cu, and Zn (Airas et al., 2004).

2.3.2 Trace Metals in Iceland

Iceland, a volcanically active country located on the mid Atlantic ridge, can expect environmental readings of trace metals due to the naturally occurring processes that expel gas, lava, and tephra, which can contain these elements (Pope & Brown, 2014). The Westfjords and Eastfjords regions, however, are some of the geologically oldest parts of Iceland and possess minimal geothermal activity (Bárðarson, 2009). This minimal

16 geothermal activity could be a limiting factor in the overall presence of trace metals in the area, but this has not been studied. It is thus noteworthy that Ísafjörður and Súðavík have high concentrations of As and Cd compared to both regional and international data (Sturludottir et al., 2013; Jörundsdóttir et al., 2014). Ísafjörður has maintained an average concentration of 62 mg/kg (dw) in As, whereas other areas in Iceland have an average between 10-17mg/kg (dw) (Sturludottir et al., 2013). Additionally, in 2017 the sampling site at Úlfsá in Skutulsfjörður maintained the highest concentration of As of 56.57 mg/kg (dw) compared to the rest of the sampling locations in Iceland (see Figure 3), with the next highest concentration at 17.29 mg/kg (dw) in Hvalfjordur.

In a 2014 study, Jörundsdóttir et al., (2014) found that when compared to other Arctic nations the concentration of As in blue mussels from Ísafjörður, at 79 mg/kg (dw), was substantially higher than As concentrations found at other locations including Norway(13.0- 32.0 mg/kg dw), Greenland (8.2-11.0 mg/kg dw), and other locations in Iceland (10.0-13.0 mg/kg dw). The next highest value was 35.0 mg/kg (dw) found in Vagur, in the Faroe Islands (Jörundsdóttir et al., 2014).

According to Jörundsdóttir et al. (2014) Cd concentrations were the highest in Mjolifjordur, located in the Eastfjords of Iceland) (4.3 mg/kg dw). Additionally, in Sturludottir et al. (2013), the sampling locations in the Eastfjords (Brekka and Botn) had two of the highest average concentrations of Cd (3.9-4.2 mg/kg dw). Jörundsdóttir et al. (2014) suggest that the levels in Mjolifjordur may be high due to volcanic activity. Álftafjörður had the fourth highest level of Cd in both Sturludottir et al. (2013) and Jörundsdóttir et al. (2014), but this fjord is not located in an area of active volcanism, so there is no clear explanation for these high Cd levels. However, other areas of the Westfjords have also seen high levels of Cd such as Arnarfjörður (Gunnlaugsdóttir et al., 2007). As there are few known anthropogenic sources of Cd in the region, this may have originated from large natural Cd deposits originally from Iceland’s formation, possibly mobilised due to processes such as eroding bedrock (Egilson et al., 1999; Reykdal & Thorlacius, 2001). The significance of Iceland’s geological history’s contribution to the levels of As, Cd, and Hg in the Westfjords has not been studied.

17 Iceland’s geological history poses various questions about the potential release of trace metals in to the environment. According to Tabelin et al. (2018), cases of weathering and excavation can lead to the release of trace elements such as As and Cd into the environment. This can be seen in tunnelling, when creating open space beneath the ground and exposing once buried rocks to new levels of oxygen, different pH levels and other weathering processes (Tabelin et al., 2018). Additionally, the process of erosion on Iceland’s coastline and sediment mobilisation through fluvial systems may expose pockets of trace elements remaining from Iceland’s formation. Similar processes have been noted in New Zealand, which is also a relatively young volcanic country. Nielsen and Nathan (1975) noted two locations approximately 1km apart, where one location had Cd levels twice as high as the other, most likely due to the proximity to a river outflow transporting eroded sediment and thus potential sources of minerals.

Figure 3: Approximate locations of blue mussel sampling sites around Iceland in 2017 (Map data: Based on data from National Land Survey of Iceland)

The Environmental Agency of Iceland (UST), in collaboration with Matís and Hafrannsóknastofnun (Marine and Freshwater Institute (MFRI)) carries out a continuous monitoring program to uphold the OSPAR agreement for contaminants in Icelandic biota (Sturludottir et al., 2013). The blue mussel (Mytilus edulis) is one of the species monitored. This data on the biosphere of Iceland dates back to 1989 (Sturludottir et al., 2013). Figure 3 depicts the sampling areas used in 2017 to fulfil the agreement; some sampling locations have changed over time, however, the site in Skutulsfjörður, Úlfsá has been used since 1999. Some results are considered out of the ordinary; however, they have not been explored further because this is out of scope of the on going monitoring program. The most striking results are as follows:

1. Levels of total As in Úlfsá in Skutulsfjörður have been around 60 mg/kg (dw) since samples were first tested in 1997; while other areas of Iceland have consistent measurements between 10-20mg/kg (dw) (Jörundsdóttir et al., 2013; Sturludottir et al., 2013). 2. Cd, although not overtly present in Skutulsfjörður, has surpassed the ICES 75% baseline of 2.0 mg/kg (dw) for multiple years in Álftafjörður near Súðavík (Jörundsdóttir et al., 2013; AMAP, 2017).

Sturludottir et al. (2013) and AMAP (2017) allude to the high levels of As in Úlfsá being attributed to the former incinerator, Funi. The incinerator did contaminate the surrounding livestock (sheep and cow products); however, Halldorsson et al. (2012) only mention the findings related to dioxins like PCBs. Additionally, the levels of As in Skutulsfjörður remained high after the incinerator closed in 2011 (Halldorsson et al., 2012; Jörundsdóttir et al., 2013).

Cadmium concentrations have been high near Súðavík in the Westfjords, the Eastfjords near the towns of Botn, Brekka, and Dalatangi, as well as the small island of Grimsey off the North coast of Iceland (Sturludottir et al., 2013). All of the previously mentioned locations experience different levels of industrial activity and, according to Sturludottir et al. (2013),

19 Cd concentrations are historically unstable in both Iceland and Norway and have yet to express a general trend. Annual monitoring has created a large dataset ideal for spatial and temporal analysis for all of Iceland, but a more localized approach needs to be developed to assess the high concentrations in Skutulsfjörður and Álftafjörður (Sturludottir et al., 2013).

2.3.3 Arsenic, Cadmium, and Mercury

Arsenic (As, atomic number 33) is a metalloid that occurs in many minerals and originates in the Earth’s crust (IARC, 2012; Murcott, 2012). Arsenic is regularly found in the environment as a sulphuric compound and is found in both an organic and inorganic forms in over 200 mineral species (IARC, 2012). Around 7900 tons of arsenic is released naturally in to the atmosphere every year, primarily due to volcanic activity (IARC, 2012). Anthropogenic sources of arsenic in air can contribute up to 24000 tons per year (IARC, 2012). Arsenic, however, is increasingly complex. Its organic form is commonly found in the natural environment at low levels in water, soil, and air (Davis et al., 2017.). The presence of inorganic As, which has greater toxicity, has increased with the use of pesticides, tobacco production, and mining activity (Ferreira da Silva et al., 2004 and Davis et al., 2017). The major mode of transportation of arsenic through the environment is by water (IARC, 2012).

Marine organisms compared to freshwater organisms may have greater capacity to accumulate As due to abiotic factors such as salinity (Ventura Lima, 2011). Arsenic compounds found within the marine environment are primarily arsenobetaine, followed by arsenosugars and arsenolipids, all of which are considered less toxic (Molin et al., 2012, 2015). The impacts of As on the marine environment are varied; however, the overall impact reduces the organism’s resilience. Ventura Lima (2011) suggest As contamination may increase oxidative stress on marine biota, as seen in the rise of oxidized proteins in the zebra fish. Additionally, As is known to be genotoxic to bivalves, which can cause various genetic mutations, which can also hinder the species resilience (Jörundsdóttir et al., 2014). Coppola et al (2018) examined the impacts of As exposure during periods of high temperatures to better understand the impacts of climate change and pollution on marine organisms, using

20 Mytilus galloprovncialis as a case study. It was confirmed in this study that oxidative stress occurs when exposed to As and warming conditions, individually and collectively. The overall stress on the organism is greater when both factors are acting together, which highlights emerging concern in light of climate change (Coppola et al., 2018).

The movement of As and other trace elements through an organism varies greatly on its age, and in some cases, gender (Ünlü & Fowler, 1979). In the late 1970’s Ünlü and Fowler, (1979) examined the flux of As in the mediterranean mussel Mytilus galloprovincialis. There appeared to be a correlation between arsenic and temperature; higher As concentrations were observed at higher temperatures. Furthermore, after 17 days of exposure to water contaminated with As, the rate of uptake decreased in all samples. This suggests mussels were reaching an As isotopic equilibrium where the As content in the water and the As content in the body are equal. Finally, Ünlü and Fowler (1979) noted that if As is consumed solely through water the rates of concentration are less and are disproportionate to the level of As in the water. If consumed through a food source and water, the rates of accumulation are much higher. This suggests that the mussels food source play a major role in its overall As concentration (Ünlü & Fowler, 1979).

In 2015, As was listed as the top priority hazardous substance in the world (ATDSR, 2018; Coppola et al., 2018) as it is chronically toxic. As does not accumulate due to its short half- life of 10-12 hours in the human body; however, consistent consumption or exposure enables chronic toxicity. The human population is exposed to As through contaminated ground water in areas around Southeast Asia, such as Bangladesh. According to Davis et al., (2017) more than 100 million people are exposed to inorganic As through contaminated groundwater per year. This exposure can lead to various types of cancers and metabolic syndromes.

Cadmium (Cd, atomic number 48) is a metalloid that is similar to mercury and zinc and is considered a heavy metal due to its toxicity and atomic weight. Cadmium is regularly released in to the environment by volcanic eruptions, forest fires, and anthropogenic activity such as agriculture, batter manufacturing, mining and the smelting of metals such as iron

21 and nickel. (Tabelin et al., 2018). Cadmium can be found in basaltic rock generally at levels of 0.6 mg/kg (Tabelin et al., 2018). Cadmium is carcinogenic and can accumulate in the body of any species easily due to its elongated half life, which can be between 4-38 years depending on the point (soil, water, air, food) of accumulation (Kiel et al., 2011). Cadmium accumulates in humans and other organisms in the kidneys and liver where it replaces Zn, which is needed for cell division and growth (Gunnlaugsdóttir et al., 2007), and with continued exposure, can lead to kidney and or liver disease. It inhibits DNA reparations and, similar to As, induces oxidative stress (Maar et al., 2018).

Mercury (Hg, atomic number 80) is a heavy metal, formerly used in thermometers and other medical devices. The presence of mercury has increased since the industrial revolution and is projected to continue (Beyer et al., 2017; Coppola et al., 2017; Lamborg et al., 2014). Mercury tends to bioaccumulate and biomagnifies in marine and terrestrial biota, leading to older and larger specimen having a higher concentration (AMAP, 2011, 2018; Rigét et al., 2011). In the Arctic Hg has been found in marine mammals, birds, humans, water, sediment, etc., Hg is present in the entire ecosystem of the Arctic. AMAP (2018) reports that increased Hg concentrations in marine and terrestrial mammals suppresses immune system function, resulting in overall stress on the organism and making them more susceptible to various infections. Additionally, as the Arctic climate continues to warm, the bioaccumulative properties of Hg are also changing making some species more susceptible to the negative effects of Hg (AMAP, 2018). Hg has been known to lead to neurological disorders and can cause complications during pregnancy in humans and other species.

Hg has many negative effects on humans and other species. These effects have been seen most notably in Minamata, Japan (Ekino et al., 2007). For twenty years citizens of Minamata were consuming low doses of Hg. It was later determined that seafood from Minamata Bay was polluted from industrial runoff from Chisso Co Ltd (Ekino et al., 2007; Harada, 1995). Individuals who ingested too much Hg experienced symptoms such as tremors, ataxia, and impaired vision, and, if pregnant, it could impair foetal development (Harada, 1995). In the 1950s when the disease and source were recognized, measures were taken to reduce the consumption of polluted seafood (Harada, 1995; Ekino et al., 2007).

22 However, year’s later individuals who have suffered from the disease are still experiencing a wide variety of symptoms such as numbness throughout their extremities. The lasting effects of Hg on the human body are still being studied. The UN established the Minamata Convention in 2017 to ensure the world would not experience another incident similar to that of Minamata, Japan. Iceland became the 92nd party to join the convention in 2018, which ensures future research of Hg and its impacts on the environment, phasing out certain Hg products, and bans on future use and exploitation of Hg (United Nations, 2019).

3 Methodology

3.1 Sampling

3.1.1 Sampling and Study Area

Blue mussels (Mytilus edulis) were used in this study to investigate trace metals (As, Cd, Hg) in the marine environment. The selected sampling locations needed to have a sufficient population of mussels of adequate size (4-6cm) and be accessible. The criteria used to determine adequate accessibility were as follows: suitable gradient of the coastline, minimal surf and waves, availability of parking, and shoreline for which permission was given to collect mussels. Table 1 and Figures 4 and 5 illustrate the sampling locations and their surrounding environment.

25 N

Figure 4: The ten sites where sampling was conducted in the Westfjords of Iceland: eight in Skutulsfjörður (sites 1-5,7,9-10) and two in Álftafjörður (sites 8 &6). (Map data: Based on data from National Land Survey of Iceland)

The annual sampling location indicated with a blue square in Figure 4, and the mussels from this site were not collected as a part of this study, however, the results were used as such. All other sampling locations, i.e. red squares in Figure 4, were collected specifically for this study and are not regularly sampled. Past annual biomonitoring of the Icelandic coastline to fulfil the OSPAR agreement has been restricted to single sampling sites in both Skutulsfjörður and Álftafjörður in the Westfjords

26 of Iceland (Sturludottir et al., 2013). Seven of the sites in Skutulsfjörður sampled for this study are new locations (marked with red squares in Figure 4), and one corresponds to the location for annual monitoring of Úlfsá (Site 10). Sites 1,2,4,7 and 10 are located on the water body called Pollurinn, in Icelandic, this means puddle. The area of Pollurinn is somewhat sheltered from the greater fjord due to the Ísafjörður spit (Figure 4 and Figure 5). Mussels from Site 4 were collected along the same 200m of coastline as Site 10, the close proximity of these two sites are to better understand if As concentrations in blue mussels are isolated to the single site at Úlfsá. All samples were collected in the same month as the official sampling in order to use the annual sampling location as a singular site in this study. The addition of new locations within Skutulsfjörður will generate a better understanding of the spatial distribution of As, Cd, and Hg in the area. The eight sites in Skutulsfjörður include areas in the inner and outer sections of the fjord (see Table 1).

27 Table 1: Overview of each blue mussel sampling site and its surroundings in Skutulsfjörður and Álftafjörður

Blue Mussel Sampling Sites and the Surrounding Areas Site # Location Fjord Coordinates Located Comments

Site 1 River Skutulsfjörður 66.063334, -23.174145 River/Sewage River outflow, near sewage pipe, Tunguá protected area Site 2 Kayak Skutulsfjörður 66.067405, -23.128619 Industry Near inner harbour, small boat launch Centre Site 3 Outer Skutulsfjörður 66.069853, -23.117908 Industry Large port for shipping, fishing, and harbour cruise tourism Site 4 Holtahverfi Skutulsfjörður 66.056306, -23.162396 River/Sewage River outflow, near former incinerator, protected area, near fireworks launch Site 5 Road to Skutulsfjörður 66.090931, -23.114176 Remote Western side of Skutulsfjörður, off Hnifsdalur major road way, near abandoned pier Site 6 Valagil Álftafjörður 65.966861, -23.084715 Remote Large river outflow, at the end of spit, near a roadway Site 7 End of Spit Skutulsfjörður 66.063850, -23.127854 Industry Near opening of Pollurinn, former dump, large mussel beds, strong sulphur aroma Site 8 Langeyri Álftafjörður 66.019662, -22.992282 Industry Small harbour next to beach, somewhat industrial area Site 9 Arnardalur Skutulsfjörður 66.094798, -23.052611 Remote Eastern side of Skutulsfjörður, farm land, off major road Site Úlfsá Skutulsfjörður 66.060000, -23.166000 River/Sewage Official sampling location for 10 OSPAR, same 200m of coastline as site 4 Previous studies of trace metals in Álftafjörður were also limited to a single site, Dvergasteinn (Site 6). This site was regularly sampled from 1996 to 2013. In 2016, the number of sampling sites for the annual biomonitoring was reduced and altered to represent defined water bodies along the coast of Iceland in accordance to the legislation on water management instated in 2011. Thus, the sampling location at Dvergasteinn was discontinued and a new sampling site was established at Hvalskurdará in Skötufjörður. Dvergasteinn has not been sampled since, even though Cd levels were elevated compared to other locations in the past (Sturludottir et al., 2013). The locations selected in Álftafjörður were the former annual sampling site in Dvergasteinn near a large river outflow (Site 6), and a location in the

28 harbour of Langeyri (Site 8), which is in closer proximity to the town of Súðavík, a small port town with a population of 163 (Statice.is, 2019) people and historic marine infrastructure.

Figure 5: All sampling site locations with corresponding images of the location depicting the surrounding area within Skutulsfjörður and Álftafjörður. (Map data: Based on data from National Land Survey of Iceland)

3.1.2 Sample Collection

Sampling took place between the 27 August and 9 September 2018 at low tide. During this time, the low tide ranged between 0.1m to 0.8m, exposing most, if not the entire intertidal zone. In order to facilitate comparisons with past annual sampling data and to maintain

29 standardized methods, the sampling procedure was based on the sampling guidelines from the OSPAR commission adapted to the availability of equipment in the Westfjords (CEMP, 2018). These guidelines can be referenced in Appendix B and C.

To ensure a representative sample for the trace metal analysis, 50 mussels between 4-6cm in length were collected per sampling site. The mussels collected were attached firmly to rocks, seaweed, kelp, or other mussels in a sandy bed, this ensured site accuracy. Once collected, the mussels were placed immediately in a bucket of cold seawater collected from the site so they would remain alive. Mussels were cleaned of any debris such as barnacles and beards before depuration to ensure no new material would pass through their intestinal tracts and an effort to make the deshelling process easier during analysis preparation (Figure 6). Once cleaned, the mussels were placed in a small mesh net bag (2.5cm mesh size) (Figure 7) and set for depuration in cold seawater from the collection site for 24 hours. The process of depuration invokes the natural filtration process of the mussels and empties their stomach contents (Lee, Lovatelli, & Ababouch, 2008). A period of 24hr was chosen to ensure that the stomach tissue of the mussel, rather than the stomach content is analysed. Depuration occurred in a twenty-litre bucket with approximately 10 L of ambient seawater from the sampling site and an aquarium air pump attached with an air stone in order to maintain around 5mg/l of dissolved oxygen within the bucket, as recommended by the Food and Agriculture Organization of the United Nations (FAO) (Lee et al., 2008) (Figure 8). Although the air stone increases aeration, it should not interfere with the faecal matter settlement at the bottom of the bucket. To maintain an appropriate temperature, the bucket was kept outside in a cool environment or in a garage with temperatures around 5ºC.

Figure 6: Cleaning the mussels of any debris such as beards or barnacles before setting for depuration. 30

Figure 7: 2.5cm mesh to create bags to suspend the mussels during depuration.

Figure 8: A coat hanger was used as a makeshift holder for the mesh bags to suspend the mussels in the 10 L of ambient seawater.

31 To ensure no freshwater or debris would fall in to the bucket, it was kept lightly covered with painters plastic. After depuration, the mussels were placed on a towel, evenly spaced, and allowed to drain for 5-10 minutes before freezing. The mussels were then placed in a zip lock bag, labelled – with site, day, time and number of mussels - and then frozen and kept frozen at -18 ºC until analysis.

3.2 Analyses of trace metals

3.2.1 Sample preparation

Sample preparation was carried out in trace element analytical laboratory facilities provided by Matis in Reykjavik, Iceland. The analyses preparation procedures were according to OSPAR for the monitoring of trace metals in biota (CEMP, 2018). Before preparation began, each laboratory technician put on nitrile gloves and goggles, following the laboratory safety protocol. The ceramic tools and kitchen blender were cleaned with a 2%

Ethylenediaminetetraacetic acid (EDTA)/ Na3 citrate solution to remove any potential metal contamination and then rinsed with high purity Milli-Q water. One sampling site (50 mussels) was processed at a time. First, the mussels were removed from the freezer to thaw. While thawing each mussel was weighed and the length (4-6cm), width, and height were individually measured to the nearest mm. The flesh and shell were weighed separately to the nearest ±0.01g. The flesh was removed with the ceramic knife. Lastly, the total soft body weight and total shell weight for each sample (50-60 mussels) was recorded to ±0.01g (Appendix D). The soft bodies of 50 individuals were then homogenized in the kitchen mixer, placed in two plastic containers and frozen. Mussel tissue contains around 90% water. Removing it makes the process of homogenization more efficient and easier to analyse. Frozen samples were freezedried (freeze dryer Alph 2-4 LSC/LyoCube 4-8, Martin Christ).

3.2.2 Trace metal analysis

Trace metal analysis took place at Matis in Reykjavik, Iceland. Matis staff members performed the elemental analysis of one sample per site (sites 1-9), resulting in a total of

32 nine analysed samples. The analysis is based on a validated NMKL 186-2007 method as described in Jörundsdóttir (2014).

Briefly the trace metals (Cd, As, Hg) in the samples were determined by inductively coupled mass spectrometry (ICP-MS), using Indium (115In) as an internal standard. To prepare for ICP-MS 150-200 mg of freeze dried samples was weighed in quartz tubes with 3ml of concentrated Nitric Acid (HNO3 ; TraceSelected, 69%) and digested in the Milestone Ultrawave Acid Digestion System, for 10 minutes on 240°C. After digestion, the samples were transferred to 50ml polypropylene tubes and diluted to 30ml with Milli-Q water. Once diluted the samples were placed for analysis in ICP-MS (Agilent 7500ce, Waldbronn, Germany). Calibration was performed using Peak Performance (CPI International) elemental standards. A 10-point calibration curve was made for each element, range 0.1- 200ppb. The following isotopes were analysed: 75As, 111Cd and 202Hg.

To ensure the quality of the method the trace element analytical laboratory at Matís participates in QUASIMEME proficiency scheme annually as well as proficiency schemes organized by EURLs for heavy metals, with satisfactory results. Additionally, certified reference materials (CRM) such as mussel tissue (ERM-CE278, JRC-), DORM (dogfish muscle, IRMM) and blank samples were used to monitor quality of each analytical run and achieve the most accurate results. The CRM’s were analysed in each run with the samples. In cases where Z-score> 2, another CRM is used to confirm the results. When the Z- score>±2 this is considered unsatisfactory. Table 2 expresses the values of the CRM’s run with the samples collected.

Table 2: The values of certified reference materials Dorm-4, Fish Protein and blue mussel tissue collected in 2018 Sample As Cd Hg As Cd Hg Mussel tissue, certified value 6,07 0,348 0,196 ERM-CE278 6,209 0,297 0,212 Z-score 0,19 -0,78 0,40 DORM-4, Fish certified value 6,8 0,306 0,41 protein 6,283 0,241 0,384 Z-score -0,63 -1,11 -0,34

33 Blanks were additionally analysed with each sample. The concentration in the blank samples are recorded and used to determine limit of detection (LOD) and limit of quantitation (LOQ). Both LOD and LOQ are expressed in Table 3 below.

Table 3: LOQ and LOD values for blue mussel tissue analyses in 2018 LOQ mg/kg LOD mg/kg As Cd Hg As Cd Hg 0,04 0,03 0,03 0,013 0,01 0,01

3.3 Statistical Analyses

Statistical analyses were minimal in this study due to the small number of samples. Linear regression models were created using R and R Studio to assess temporal trends of As, Cd, and Hg in Skutulsfjörður (n=17) and Álftafjörður (n=18) using data retrieved from the ICES database (dome.ices.dk). The concentrations of As, Cd, and Hg overtime in these two locations were normally distributed except for As in Skutulsfjörður and Hg in Álftafjörður. The concentration of As in Skutulsfjörður was normalized using the Tukey Transformation function in R. The Tukey transformation finds the value (lambda) that allows the data to become normally distributed; in this case the value was -3.45, thus, lambda < 0. Resulting in the equation (Transformed data=-1*xlambda), where x is the concentration of As. Once both concentrations were transformed they displayed normality and could be analysed using linear regression models. All other concentrations were normally distributed confirmed by using the Anderson-Darling Test.

34 4 Results 4.1 Temporal Analysis Results

4.1.1 Average Concentration of Trace Elements

Sturludottir et al., (2013) established temporal averages for As, Cd, and Hg concentrations for Skutulsfjörður and Álftafjörður from 1999-2010 and 1996-2010 respectively. Table 4 compares the average concentration until 2010 established by Sturludottir et al., (2013) to the updated averages with available data collected by myself and the official blue mussel sampling for these elements from 1996-2018 and 1999-2018 found on the ICES database (dome.ices.dk). Due to the change of sampling location in Dvergasteinn (site 6), the data collection ended in 2013. Additionally, the data from Skutulsfjörður lacks sampling data from 2014 and 2015. Thus the temporal data for Skutulsfjörður consists of a total of 17 years (n=17; 1999-2018) and Álftafjörður a total of 18 years (n=18; 1996-2018).

Table 4: Average concentrations of As, Cd, Hg in blue mussels in Skutulsfjörður over a 17- year period and for Álftafjörður an 18-year period, 2010 averages determined by Sturludottir et al., (2013)

Average Concentration for Each Element in Blue Mussels from the 1990’s to 2010 and updated for the 1990’s to 2018 Skutulsfjörður (n=17) Álftafjörður (n=18) mg/kg dry weight Year As Cd Hg As Cd Hg 2010 69.74 1.39 0.099 16.09 3.87 0.055 2018 64.71 1.27 0.095 15.83 3.85 0.059

Average concentrations of As, Cd, and Hg seem to have decreased since 2010.

4.1.2 Temporal Trends

Using past elemental concentration data from the ICES database (ices.dk) and additional data collected in 2018 from Sites 6 and 10, linear regressions were used to assess any trends

35 in the concentrations of As, Cd, and Hg over time, Figure 4 depicts the two sampling locations used for this temporal analysis (Site 6 and Site 10). In the statistical analyses all p- values less than 0.05 were considered significant. Additionally standard bar charts express the concentration recorder per year of sampling at each location.

In Skutulsfjörður, only Cd displayed a significant decrease over time (Figure 9; p- value=0.031). Trends for As and Hg in Skutulsfjörður were insignificant. Figures 10 and 11 expresses the linear models and concentration per year for each element in Skutulsfjörður and Álftafjörður respectively. In Álftafjörður, only As displayed a significant decrease over time (Figure 10; p-value=0.027). Trends for Cd and Hg in Álftafjörður were insignificant. In both Skutulsfjörður and Álftafjörður, As and Cd spiked in 2006. Hg did not show the same increase in either location.

Figure 9: Linear models of the concentrationsof As, Cd, and Hg found in blue mussels in Skutulsfjörður over time and the concentrations of As, Cd, and Hg measured in blue mussels in Skutulsfjörður each year until 2018. Data from (dome.ices.dk) and UST. 37

Figure 10: Linear models of the concentrations of As, Cd, and Hg found in blue mussels in Álftafjörður over time and the concentrations of As, Cd, and Hg measured in blue mussels in Álftafjörður each year until 2018. Data from (dome.ices.dk) and UST.

38 4.2 Spatial Distribution

All trace elements were present in the mussel tissue from each sampling site. Table 5 displays all sampling locations and their concentrations in dry weight. As concentrations were consistently higher throughout Pollurinn in Skutulsfjörður (54.45-83.87 mg/kg (dw)), compared to the northern part of the fjord had around half of the concentration seen in Pollurinn (24.05 and 30.65 mg/kg (dw)). Cd concentrations were higher at the two locations in Álftafjörður (3.41 and 5.31 mg/kg (dw)) and at Site 3 in Skutulsfjörður (8.68 mg/kg (dw)) compared to sites within Pollurinn (1.04-1.62 mg/kg (dw)). The concentrations in Álftafjörður (sites 6 and 8) are comparable to the historic data near Dvergasteinn (Site 6).

Table 5:Concentrations per element per site from blue mussel sampling in 2018

Concentration of each element in blue mussels per site and average per fjord Mg/ kg Dry weight

Skutulsfjörður As Cd Hg

Site 1 61.45 1.59 0.088 Site 2 54.44 1.62 0.066 Site 3 55.84 8.68 0.089 Site 4 66.18 1.04 0.076 Site 5 24.05 3.92 0.063 Site 7 83.87 1.29 0.067 Site 9 30.65 3.47 0.063 Site 10 59.06 1.28 0.069 Skutulsfjörður Average 54.44 2.86 0.073 Standard Deviation 19.15 2.59 0.011 Álftafjörður As Cd Hg

Site 6 11.43 3.41 0.051 Site 8 9.41 5.31 0.032 Álftafjörður Average 10.42 4.36 0.041 Standard Deviation 1.43 1.34 0.014 Figures 11 and 12 express the concentrations of each element found in blue mussels at each sampling site. No singular site maintained the highest concentration of all three elements i.e. Site 7 had the highest concentration of As (83.88mg/kg), Site 3 had the highest concentration of Cd (8.68mg/kg) and Hg (0.088mg/kg). The average concentrations of As

39 and Hg were higher in Skutulsfjörður. Álftafjörður, even with only two sampling sites maintained a higher average concentration of Cd.

Figure 11: Concentrations of As, Cd, and Hg (mg/kg dw) measured in blue mussels from all sites in Skutulsfjörður in 2018.

Figure 12: Concentrations of As, Cd, and Hg (mg/kg dw) measured in blue mussels from both sites in Álftafjörður in 2018.

4.3 Environmental Impacts

Industrial activity, fresh water inputs, and sewage discharge all have the potential to impact trace element concentration in coastal and marine environments (Airas et al., 2004; Cabral- Oliveira et al., 2016; Cabral-Oliveira, Pratas, Mendes, & Pardal, 2015). The collection sites in Skutulsfjörður were categorized by their proximity to a potential impact i.e. industry, sewage/rivers, or no known impact. In this case, in Pollurinn sites located near rivers were also dually impacted by sewage outflows (Sites 1,4,and 10). Tables 6-8 represent the average concentration of each element for sites within immediate proximity to industry,

41 sewage/rivers, or no known impact. To enable this comparison the results from Sites 5 and 9 were used as references of no known impacts. Similarly, Figures 13-15 show the total concentrations of As, Cd, and Hg within immediate proximity to industry, sewage/rivers, or no known impacts.

The two sites (Sites 5 and 9) with no known impacts were lower in As, but maintained similar averages to the other sites in Cd and Hg concentrations. The highest average of As and Hg were found at the sites near sewage and river outflows. The greatest average Cd concentration was found near industrial activity.

Figure 13: Concentration of As in blue mussels per site located near industry, sewage/river outflows, or no known impacts in Skutulsfjörður.

42 Table 6: Average concentration of As in blue mussels near sites of impact

Avg. Concentration mg/kg (dw) of As near potential impact sites Type Industry Sewage/Rivers No Known Impact Location(s) Sites 2,3,7 Sites 1,4,10 Sites 5 and 9 Average (mg/kg) 64.72 62.23 27.35

Figure 14: Concentration of Cd in blue mussels per site located near industry, sewage/river outflows, or no known impacts in Skutulsfjörður.

Table 7: Average concentration of Cd in blue mussels near sites of impact

Avg. Concentration of Cd mg/kg (dw) near potential impact sites Type Industry Sewage/Rivers No Known Impact Location(s) Sites 2,3,7 Sites 1,4,10 Sites 5 and 9 Average mg/kg 3.86 1.30 3.70

Figure 15: Concentration of Hg in blue mussels per site located near industry, sewage/river outflows, or no known impacts in Skutulsfjörður.

Table 8: Average concentration of Hg in blue mussels near sites of impact

Avg. Concentration of Hg mg/kg (dw) near potential impact sites Type Industry Sewage/Rivers No Influence Location(s) Sites 2,3,7 Sites 1,4,10 Sites 5 and 9 Average (mg/kg) 0.07 0.08 0.06

44 4.4 Concentrations at three locations in Westfjords

For 2018, blue mussel samples were collected in Skötufjörður, Álftafjörður, and Skutulsfjörður. The collection in Álftafjörður (Site 6) was performed as a part of this study, while the samples collected in the other two fjords are the annual samples collected for the area. Using the data collected for this study in Álftafjörður allows for comparisons across three different fjords. Comparing the concentrations in Skötufjörður, Álftafjörður, and Skutulsfjörður (Figure 16) may lend itself to understand how human activity can impact trace element concentration in biota and the environment in the Westfjords. The new annual monitoring location in Skötufjörður (established in 2016) has very few inhabitants compared to both Álftafjörður and Skutulsfjörður and can act as a valuable comparison to both fjords

Figure 16: Locations of blue mussel sampling sites in Skutulsfjörður, Álftafjörður, and Skötufjörður for 2018. (Map data: Based on data from National Land Survey of Iceland)

45 in light of future development. The 2018 values for As, Cd, and Hg for the three fjords are expressed in Table 9.

Table 9: Concentrations of As, Cd, and Hg found in blue mussels in Skutulsfjörður, Álftafjörður, and Skötufjörður in 2018.

Concentration of each element in 2018 (mg/kg, dw) Location As Cd Hg

Skutulsfjörður 59.06 1.28 0.069 Álftafjörður 11.43 3.41 0.051 Skötufjörður 9.32 11.92 0.045

46 5 Discussion 5.1 Temporal Trends

The annual biomonitoring of Iceland has allowed for the generation of large temporal datasets, providing scientists and policy makers benchmark data for many areas of the country. It is important to note that there is always a certain variability and uncertainty in the data obtained. This variability lies for example in: sampling time, weather events, age and size of the mussels collected, and the analytical method; this in turn can all effect the average concentrations of As, Cd and Hg. The CEMP guidelines are designed to minimise this variability as much as possible.

As a result, temporal trends need to be subjected to further statistical analysis. This will elucidate the significance of an apparent trend such as the decrease of the elements investigated here in the timeframe from the late 1990’s to 2018 in both Skutulsfjörður and Álftafjörður (see Figures 10 and 11).

Twenty years ago, the range for Cd was 0.38-8.80 mg/kg dw and for Hg 0.0001-0.104 mg/kg dw throughout Iceland (Egilson et al., 1999). The averages and the overall values collected between both fjords in 2018 are within these ranges. Unfortunately, in Egilson et al. (1999) there was no assessment of As concentrations throughout Iceland. There are very few apparent temporal trends in the concentrations of As, Cd, and Hg in Skutulsfjörður and Álftafjörður. The concentration of Cd is the only significant decreasing trend (Figure 9; p- value=0.031) in Skutulsfjörður. Additionally, the decrease of As is the only significant trend in Álftafjörður (Figure 10; p-value=0.027). Interestingly, these trends and overall concentrations contrast with each other. Skutulsfjörður, although significantly decreasing in Cd, is high in As. While Álftafjörður is high in Cd, and significantly decreasing in As concentration. This information and lack of trends, aligns with the analyses performed by Sturludottir et al., (2013), which pointed out that Cd in Álftafjörður had increased until 2003, then began to decrease, as well as, Skutulsfjörður maintaining the highest As and Hg

47 concentrations in Iceland at the time. Additionally, there are other outliers in the temporal data (see data tables in Appendix E). For example, in 2006 in both Skutulsfjörður and Álftafjörður, concentrations of As and Cd spiked, whereas Hg did not experience the same increase. The values in 2006 were the highest values seen in both fjords i.e. As in Skutulsfjörður 153.26 mg/kg dw and Cd in Álftafjörður 8.03mg/kg dw (Appendix E). Unfortunately, because these samples are only taken once a year it is unclear why this occurred.

In Álftafjörður the concentrations found in 2018 are of little concern and are comparable to the previous years that the site was sampled (Figures 11 and 13, Appendix E). It may be of Sudavikhreppur’s interest to further test the area for heavy metals and other pollutants if the development of the maerl-farming factory is to commence. This maerl factory will be the largest in Iceland and increase development and industrial processes in the area of Súðavík (Haraldsson, 2015). This initial result from the site at Langeyri (Site 8) could be baseline data to better understand the impacts of industrial activity from construction, processing, and seafloor disturbances that may ensue through the development of this factory.

5.2 Spatial Distribution

The average concentrations of As and Hg were higher in Skutulsfjörður than in Álftafjörður (Table 5). However, higher average concentrations of Cd were obtained at the two collection sites in Álftafjörður (Table 5). The highest concentration of Cd in Álftafjörður was found at Site 8 (5.31 mg/kg dw; 0.69 mg/kg ww) near the historical port (Table 5, Figures 4 & 5). This concentration is nearing the 1.0 mg/kg ww environmental standard set by the EU (European Commission, 2006), and further studies should examine its consistency for the area. This area, named Langeyri by locals, is currently undergoing construction and development for a potential factory, the actions of digging or building materials could contribute to the already seemingly high natural levels in the area.

In Skutulsfjörður it is apparent that the high concentrations of As (50-83.87 mg/kg dw) are not spatially bound to the historically studied site of Úlfsá (Site 4). Higher than average (10- 17 mg/kg dw (Sturludottir et al., 2013)) concentrations of As were found to be present at

48 multiple locations inside Pollurinn and outside Pollurinn at the large harbour, with the highest concentration found at Site 7 at the end of the spit (Table 5, Figure 4).

There are many potential reasons for these elevated concentrations such as: untreated municipal waste from the households in the town Ísafjörður, the various industrial activities that occur (shipping, fishing, cruise ships, metal working, ship building) the presence of the landfill at the end of the spit, and additionally the area could be lacking dilution from a regular influx and out flux of seawater from the greater fjord (Eydal et al., 2013). Recent studies on the area (Pétursson, 2018) have addressed the surface flow rate of this fjord utilizing modelling technology simulating dissolved waste from a salmon farm in the fjord. Using modelling technology based on tidal fluctuations, Pétursson (2018) highlights the limited mixing that occurs between Pollurinn and the outer fjord in no wind to 10m/s wind scenarios. This new study supports the previous report by Eydal et al., (2013) suggesting limited mixing between Pollurinn and the outer fjord of Skutulsfjörður. With the planned expansion of the outer harbour, this mixing rate might change so it is important to have the data from this study as a baseline. There were no other apparent trends for the trace elements studied across sites. Hg concentrations across space were relatively stable, the highest concentrations were found in the outer harbour of Ísafjörður (Site 3), and near the river Tunguá (Site 1)(Table 5, Figure 4). These higher levels could be influenced by the industrial activities of the harbour, the dredging that has occurred near the mouth of Tunguá, tunnel excavation and building, or runoff from nearby roads.

No single site in Skutulsfjörður had considerably elevated concentrations of all three trace elements. However, for the majority of sites the concentrations within Pollurinn were higher than those outside of Pollurinn. This suggests that Pollurinn is more regularly exposed to these elements. In 2014, the Environmental Agency of Iceland labelled sites that are “at risk”, where appropriate management measures need to be proposed or “potentially at risk”, where monitoring, risk assessment and data quality evaluation need to occur, within river basins, lakes, and coastal water bodies (Jonsson, Jensson, Weisshappel, Þórðarsson, & Karlsdottir, 2014). This process was completed in Iceland as a compliance indicator for Article 5 in the WFD to record the potential impacts inhibiting the water bodies from

49 maintaining a “good” status (Jonsson et al., 2014). In this report, the inner part of Skutulsfjörður, Pollurinn, was highlighted as “potentially at risk” for point source pollution including bacteria load and industrial waste (Jonsson et al., 2014). The higher than average concentrations of As (50-83.87 mg/kg dw) detected in mussels from this water body concur with this assessment. Additionally, Eydal et al., (2013) labelled this area of Skutulsfjörður “protected” and “closed” suggesting little water exchange with the greater fjord. This suggests that any heavy metal contamination is less likely to be dispersed and/or diluted into the outer sea.

5.2.1 Environmental Impacts in Skutulsfjörður

In Skutulsfjörður, higher than average As concentrations were observed at sites near river/sewage outflows, Sites 1, 4, and 10, into Pollurinn i.e. near the river Tunguá and Úlfsá and two sewage pipelines for the two suburban areas (Holtahverfi and Seljalandshverfi) (Table 5; Figure 14). However, the highest As concentration was found near an area of industrial activity, a former dumpsite at the end of the spit (Site 7) in Ísafjörður (Tables 5 and 6, Figure 14). This dump ground at Sudurtangi was formerly used as an incinerator, after this, general waste was put there in the 1970’s and 1980’s and created a landfill that now supports various forms of infrastructure and industry, with future plans of development (Teiknistofan Eik ehf., 2014) Similarly, the highest concentration of Cd was found in an area of industrial activity, near the larger harbour (Site 3) in Ísafjörður (Tables 3 and 5; Figure 15). This harbour has a lot of boat traffic as well as a refuelling station, various mechanic shops, and fish factories. This area is a highly urbanized section of the town, with the groundcover consisting of primarily impervious surfaces such as concrete or asphalt. As a result, any pollutants from industry and runoff water from streets are discharged directly into the harbour. Finally, concentrations of Hg were some of the highest near sewage/river systems from 0.088 mg/kg (dw) to 0.076 mg/kg (dw) (Tables 5 and 8; Figure 16).

Generally, the average concentrations of all elements except Hg were higher near industry, however, the average Cd concentration in the area of no known impacts (Sites 5 and 9, see Table 7), is close to the amount seen near industry, which indicates a high background value

50 for Cd in this area. Site 5 is located on the North western side of Skutulsfjörður the sample in this location consisted of fewer than 50 mussels, below the recommended size range 4- 6cm (Appendix D), but the third highest concentration of Cd was found at this site. This area needs to be investigated further with an adequate sample of 50+ mussels between 4-6 cm for comparison. The concentration of Hg was consistent within Pollurinn to the outer harbour in Ísafjörður, suggesting concentrations of Hg may be impacted by human activity in harbours and/or sewage and river outflows in the area (Figure 15).

5.2.2 Three Locations within the Westfjords

Skötufjörður, Álftafjörður, and Skutulsfjörður are all within the same water region of Iceland (101), which includes the entirety of the Westfjords (Jonsson et al., 2013). However, it is difficult to determine if they are classified as the same type of water body. In the municipal plan for Ísafjarðarbær, Pollurinn is considered an estuary with prominent bird life (Teiknistofan Eik ehf., 2009) and other literature suggests that Skutulsfjörður and Álftafjörður are the same type of water body as depicted in the maps in Jónsson et al., (2013). However, the official documentation on Álftafjörður, classifies it as a transitional water body due to its large salinity gradient (0->30ppm) (Eydal & Jóhannesdóttir, 2014). The mapping technology available from the Icelandic Meteorological Organization, suggest that both Álftafjörður and Pollurinn are “sheltered” coastal water bodies(Veðurstofa Íslands, 2018). It is difficult to determine through the literature and legal framework that is accessible the appropriate classification according to the WFD in Iceland. Although the classifications of these three water bodies are indistinguishable, what is known are the variable levels of human activity in each of these fjords; Skutulsfjörður having the highest amount of human activity and Skötufjörður the least. While comparing data from these three fjords for 2018, it can be inferred that the Cd concentrations are most likely derived from natural sources (Egilson et al., 1999; Gunnlaugsdóttir et al., 2007). Previous research has shown that Cd concentrations have been apparent in less populous areas of the Westfjords of Iceland such as Arnafjordur. Gunnlaugsdóttir et al. (2007) discovered the average concentration of Cd was higher than the European Commission limit of 1.0mg/kg ww (EC No 1881/2006) for molluscs in Arnafjordur in NW-Iceland(European Commission, 2006).

51 Gunnlaugsdóttir et al. (2007) also found that concentrations of Cd within four other fjord systems in NW-Iceland were close to this limit. These high concentrations are apparent in Skötufjörður and Álftafjörður, both with small populations but higher levels of Cd compared to Skutulsfjörður and Straumur an area near Reykjavik that in 2017 had a Cd concentration of 2.06 mg/kg dw (see Table 9). Secondly, fjords with intensified levels of development seem to have higher concentrations of As in the Westfjords, as seen in Skutulsfjörður (Tables 5 and 9). In Skutulsfjörður the highest concentrations in both As and Hg were observed followed by Álftafjörður then Skötufjörður (Table 9). In comparison, the Cd concentration does the opposite; with the highest concentration found in Skötufjörður and the lowest in Skutulsfjörður.

The concentration of As at the end of the Ísafjörður spit (Site 7) was almost ten times higher than the concentration found in Skötufjörður (85.6mg/kg and 9.3 mg/kg dw, respectively). This is a notable difference and should not be ignored. This high concentration of As in Skutulsfjörður is in agreement with the long term analysis performed by Sturludottir et al., (2013), that suggests As concentrations varied by location, with the location at Úlfsá in Skutulsfjörður being the greatest concentration (62 mg/kg dw) found in Iceland at the time, compared to other values ranging from 10-17 mg/kg dw, including areas such as Reykjavik, with the greatest population in Iceland. Highlighting the uniqueness of Ísafjörður and Skutulsfjörður that needs further investigation.

Unfortunately, there is a substantial lack of knowledge on how the geology of the area of the Westfjords affects heavy metal concentrations. What is known is that the majority of the bedrock in the area is basalt and ranges from .8million to 3.3million years old (Jonsson et al., 2013), but what this releases during erosion processes is unknown for the area. Therefore, one cannot ignore the potential influence the geological history may have on As, Cd, and Hg concentrations in Skutulsfjörður, Álftafjörður, and Skötufjörður.

52 5.3 Study Limitations and Future Research

5.3.1 Limitations

The limitations to this study included overall cost, timing, mussel availability, and available information on the geology of the Westfjords. Unfortunately it was not possible to get external funding for this study, hence the out of pocket cost for trace elemental analyses was quite expensive and this limited the number of tests and sites that could be included in the study. Additionally, to follow the CEMP guidelines and maintain consistency with the annual sampling required sampling within a Spring tide. Spring tides occur during full and new moons increasing gravitational pull on the Earth. Thus, these tides expose more of the intertidal zone during the low tides. This time constraint left little room for extreme weather events. Additionally, conducting the sampling alone over multiple locations became increasingly difficult. Locations with appropriately sized mussels were also difficult to find, thus limiting the number of sampling locations and also led to exclusion of more sampling sites outside of Pollurinn. Also, some areas of beach are private land and one needs permission to be able to access the area. Lastly, having little knowledge of Icelandic may have limited the number of papers found on trace elements, geology of the Westfjords, municipality plans, environmental protected areas, etc. This lack of knowledge of the local language was also hindering when papers were found, as translating the work took time and occasionally lacked clarity.

5.3.2 Future Research

Future research in this region should also specifically examine inorganic As. The annually monitored samples and the samples used in this study assessed total As concentrations in blue mussels. Examining inorganic As specifically would allow for a better understanding of the potential source of the concentrations, with high quantities of inorganic As pointing towards more anthropogenic sources (such as hospital waste, paints, batteries, etc.) (Han et al., 2003). Additionally, monitoring should resume in Álftafjörður, especially if the development of the maerl factory is approved. Since it would then be of even greater value

53 to have more data on the status of trace elements in marine biota in this area prior to this development, in order to obtain a robust baseline of heavy metals in the area. As well as throughout the construction and processing phases of the business; this would aid in understanding how this industry affects these levels within the fjord.

Studying multiple collection sites of blue mussels around Skutulsfjörður should be repeated to confirm the findings in this study. This may for example be of interest to better understand the effects of sewage outflow on As, Cd, and Hg concentrations (Airas et al., 2004;Cabral-Oliveira et al., 2015). Likewise, if the sewage outflow is redirected, data collected during and after could measure the contribution sewage has had on the heavy metal concentration in blue mussels, and any lasting impacts it may have had. Any study in the area would also benefit from analysing the sediment and water column for these trace metals. As mentioned in Ünlü & Fowler (1979), the food source of the mussels play an important role in their accumulation of various elements, thus it would be of best interest to sample the water column, sediment, and biota such as mussels to generate a more complete understanding of where the trace elements are located. Also, to generate a more thorough spatial understanding, the number of sampling locations along the coastline should be increased. Some of the areas that should be considered for this in-depth study are represented in Figure 17.

54 Pollurinn

Figure 17: Depiction of the protected area at the base of Pollurinn and the additional suggested sampling sites for future research. (Map data: Based on data from National Land Survey of Iceland)

Utilizing transplanted mussels throughout Skutulsfjörður would also be worthwhile to generate a spatial distribution, as well as the ability to place them not only along the coast but also at depth. Also, placing and retrieving the mussels at different times of the year would allow for a better understanding of how seasonal variation can impact the overall concentration of trace elements. Additionally, using transplanted mussels would create a better understanding of the impacts on the mussels when exposed to these high concentrations and would create a better picture of ecosystem health as reported by Lanksbury et al., (2014). Lanksbury et al., (2014) utilised Mytilus spp to understand biological endpoints of the mussels (mortality, growth, and condition index), as well as the overall concentration of contaminants such as PCB’s and trace elements. It was found that mortality increased along a contaminant gradient from rural to urban areas, suggesting contaminants decrease the overall fitness and resilience of the mussel to withstand contaminated waters.

55 5.4 Regional Management Suggestions

Assessing the end of the Ísafjörður spit and the sewage outlets should be the priority to the municipality before any new development within Ísafjörður continues. The previous assessments of the area of Pollurinn from Eydal et al. (2013) and Jonsson et al. (2014), highlight the delicate and lagoon nature of the area. It is imperative that more action is taken to understand the issues within Pollurinn, including the heavy metals studied here and potential other consequences of sewage discharge. The concentrations of metals (especially As) in the blue mussels from Skutulsförður are far beyond background levels for a “pristine” environment and the existing long-term monitoring data (from 1999-2018) shows that this is not an isolated incident. This study has highlighted the distribution of these concentrations in Skutulsfjörður and suggests further investigation by the municipality.

5.4.1 Public Health

In light of Iceland partaking in the WFD (2000/60/ce), additional data on the water column would complement the current status on the biota. First and foremost, because there is little knowledge on the state of the water column, limiting human exposure to the waters in Pollurinn is advisable, due to the direct sewage outflows consisting of industrial and municipal waste. This includes the annual swimming race that takes place in the summer and advising individuals to not eat fish or mussels from the area. Also, assessing the overall structural integrity of the landfill at the end of the spit is advisable. This area should be assessed for overall leaching and impermissible dumping. A clean up of Pollurinn due to the potential risks of human and ecosystem health is imperative for Ísafjarðarbær. The quickest solutions would include: limiting chemical use within drainage systems, collecting industrial waste rather than dumping, and minimizing dredging and or dumping of material in to Pollurinn.

5.4.2 Environmentally Protected Areas

In Ísafjarðarbær’s town plan, part of Pollurinn is considered an area of environmental importance, in Icelandic “náttúruminjaskrá” (see Figure 17). This area includes the southern

56 beach of Pollurinn East towards the airport and additionally the bay of Engidalur (known as H6). Within the municipal plan it states these are areas of importance due to the vast bird life that utilise these beaches and mud flats, including the valuable Eider duck. If these birds are eating mussels high in As and/or Hg, these metals can negatively impact their ability to reproduce, overall immunity, and may be cause for shorter lifespans (Coppola et al., 2017, 2018; Keil et al., 2011; Ventura-Lima, Bogo, & Monserrat, 2011). Additionally the plan states that no new garbage disposal sites or mines should be located in the areas of environmental protection. Although this is positive for the future it does not address the current state of sewage and drainage pipes emptying into the area. This area at the south end of Pollurinn is experiencing pollution from sewage and drainage pipes in conjunction with other activities that occur in town that may negatively impact this area, despite its listing as an area to be preserved.

6 Conclusion

The presence of As in blue mussels in Skutulsfjörður is not isolated to the original sampling location at Úlfsá. Concentrations of As ranging from 30-85 mg/kg (dw) were found throughout Skutulsfjörður (Table 5), with the highest concentrations of As found within Pollurinn. The temporal data in conjunction with new spatial data give enough information for the locality to investigate the sources of pollution (natural or anthropogenic) in greater detail.

Concentrations of As, Cd, and Hg in Álftafjörður at the original sampling site are consistent with previous temporal data. There is a significant decrease in As over time at the original sampling location (Figure 10). The second site in Álftafjörður had somewhat higher concentrations of Cd compared to the original sampling site (Table 5). Monitoring should continue at both locations, to understand the consequences of construction, development, processing and benthic disturbances due to the potential maerl factory to be put in place.

Concentrations of As, Cd, and Hg varied in their proximity to known sources of pollution in Skutulsfjörður. The highest concentration of As was found near a former dumping ground that now acts as a landfill; the highest concentration of Cd was located near the large harbour; and the greatest concentrations of Hg were found near river and sewage outflows. Industry and sewage potentially impact the concentrations of As, Cd, and Hg in the fjord. These impact sources need further investigation and testing to ensure their presence and overall impact on the environment is negligible.

The concentrations of As, Cd, and Hg differed between the three fjords sampled in the region. The concentrations of As and Hg were highest in Skutulsfjörður. The concentration of Cd on the other hand was highest in Skötufjörður. Due to the differences in population, geography, geology of these areas, these findings highlight the need for further research and understanding of the human and natural geological impacts on trace element concentration within the marine environment of the Westfjords of Iceland.

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Appendix A

Research ethics training and clearance

University Centre of the Westfjords Suðurgata 12 400 Ísafjörður, Iceland +354 450 3040 [email protected]

This letter certifies that Anna Hixson has completed the following modules of: (x) Basic ethics in research (x) Human subjects research (x) Animal subjects research Furthermore, the Masters Study Committee has determined that the proposed masters research entitled Understanding the Spatial Distribution of Arsenic, Cadmium, and Mercury Concentrations in Blue Mussels (Mytilus edulis) from Skutulsfjörður and Álftafjörður in the Westfjords of Iceland meets the ethics and research integrity standards of the University Centre of the Westfjords. Throughout the course of his or her research, the student has the continued responsibility to adhere to basic ethical principles for the responsible conduct of research and discipline specific professional standards.

University Centre of the Westfjords ethics training certification and research ethics clearance is valid for one year past the date of issue.

Effective Date: 15 June 2018 Expiration Date: 15 June 2019

68 Prior to making substantive changes to the scope of research, research tools, or methods, the student is required to contact the Masters Study Committee to determine whether or not additional review is required

Appendix B

The following is the technical annex from the CEMP guidelines for sampling, transport, and analyses of trace elements in Mytilus edulis downloaded from the OSPAR website. https://www.ospar.org/work-areas/cross-cutting-issues/cemp

Technical Annex 2: Metals

This annex is intended as a supplement to the general guidelines. It is not a complete description or a substitute for detailed analytical instructions. Advice and recommendations given in documents prepared through the QUASIMEME project (Quality Assurance of Information for Marine Environmental Monitoring in Europe) are frequently cited.

1. Species

1.1 Fish and shellfish

1.1.1 Criteria for the selection of species for temporal trend monitoring

Species for temporal trend monitoring can only be selected in the light of information on fish stock composition and history. It is essential that long time series with one species are obtained. Care should be taken that the sample is representative of the population and can be repeated annually. Fish and shellfish species currently used for trend monitoring are listed in Tables 1 and 2 of the main guidelines.

1.1.2 Criteria for the selection of species for spatial distribution monitoring

To standardise results the first choice species Limanda limanda, Gadus morhua and Mytilus edulis or M. galloprovincialis should be used if possible. The second choice species Merlangius merlangus, Merluccius merluccius, Platichthys flesus and Crassostrea gigas should only be used when none of the first choice species are available.

First choice species

Limanda limanda (dab)

69 Dab is a ground dwelling species confined to the shelf seas. It has replaced the previously recommended plaice and flounder for the following reasons:

a. its migration is less pronounced, thus it is more likely to represent the area in which it is caught;

b. it has been used successfully in disease studies, thus complementary information from such studies would be available (in fish disease studies a length range for individual fish of 20-25 cm is used).

The southern distribution limit of dab is the north coast of Spain.

Gadus morhua (cod)

Cod normally live near the seabed but may also be pelagic. Cod occur in coastal areas and to 600 m depth. Cod may also be found in the open ocean and so may also be used for monitoring oceanic regions of the Maritime Area. The southern distribution limit of cod is at 45°N. A sampling size range of 30-45 cm is specified because cod of that size and age tend to feed on a fairly uniform diet.

Mytilus sp. (mussel)

Mytilus edulis occurs in shallow waters along almost all coasts of the Contracting Parties. It is therefore suitable for monitoring in nearshore waters. No distinction is made between M. edulis and M. galloprovincialis because the latter, which may occur along Spanish and Portuguese coasts, cannot easily be discerned from M. edulis. A sampling size range of 3-6 cm is specified to ensure availability throughout the whole maritime area. For monitoring in polluted areas, mussels may be transplanted from an unpolluted area and then left in the polluted area (Benedicto et al., 2011; Søndergaard et al., 2011) for e.g. one year before sampling and analyses. The results will reflect the last years contamination in contrast to resident mussels that will reflect several years of contamination.

Second choice species

Platichthys flesus (flounder)

The distribution of flounder extends further south than that of dab and might therefore represent the flatfish of choice for certain Portuguese coastal areas and Spain’s northwestern coastal areas. Flounder is not suitable for monitoring in open sea areas due to its migration pattern. A sampling size range of 15-35 cm ensures individuals of the 2-year age class.

Merlangius merlangus (whiting)

Whiting can be caught in coastal waters and up to 200 m depth. Its distribution is from Portugal to Iceland and Norway, thus covering all the maritime area subject to monitoring

70 by Contracting Parties. It is a suitable substitute for cod. The sampling size range, 20-35 cm, may need adjustment in the light of future experience.

Merluccius merluccius (hake)

Hake live at 100-300 m along the shelf margins. The sampling size range is 20-35 cm. The sampling size interval suggested is arbitrary and may need adjustment in the light of future experience.

Crassostrea gigas (Pacific oyster)

The Pacific oyster should be sampled in areas where Mytilus sp. is not available. The sampling size should be within the length range 9-14 cm to ensure individuals of the 2 year age class.

2. Sampling

Two alternative sampling strategies are described: sampling to minimise natural variability and length-stratified sampling.

2.1 Sampling to minimise natural variability

Gain in precision of the contaminant data can be obtained by minimising variance from the biological covariables. For fish, this can be achieved by sampling and analysing individually at least 12 young fish of the same sex, e.g. 2-3 year old female fish. To assist the selection of the relevant length range in order to find individuals of the recommended age, it is advised to produce specific species and region related correlation graphs by use of existing data from the respective monitoring data base. An example is given in Appendix 1.

For shellfish, a sample should be collected with the number of individuals large enough to be divided into at least 3 equal pools with each pool consisting of at least 20 animals and enough soft tissue for all analyses. The length of the individuals collected should be constant from year to year at each station, or should at least fall within a very narrow range, e.g. within 5 mm. To reflect recent levels of contamination, young individuals should be chosen. In selecting the sample, care should be taken to ensure that it is representative of the population and that it can be obtained annually.

2.2 Length-stratified sampling

Where successfully ongoing, length-stratified time series should be continued.

2.2.1 Shellfish

For shellfish, the upper limit should be chosen in such a way that at least 20 mussels in the largest length interval can easily be found. The length stratification should be determined in such a way that it can be maintained over many years. The length interval should be at least 5 mm in size. The length range should be split into at least 3 length intervals (small, medium

71 and large) which are of equal size after log transformation. For example, if the length range is 40-70 mm, then the interval boundaries could be (rounded to 1 mm) as follows: a. 5 intervals: 40 - 45 46 - 50 51 - 56 57 - 63 64 - 70 b. 3 intervals: 40 - 48 49 - 58 59 – 70.

3. Transportation

3.1 Fish and shellfish

Samples should be kept cool and frozen at below -20°C as soon as possible after collection. Length and weight should be determined before freezing. Live mussels should be transported in closed containers at temperatures between 5-15°C, preferably below 10°C. Frozen samples should be transported in closed containers at temperatures below -20°C. More rigorous conditions will be necessary for samples for biological effects monitoring, e.g. storage in liquid nitrogen.

4. Pre-treatment and storage

4.1 Contamination

Sample contamination may occur during sampling, sample handling, pre-treatment and analysis (Oehlenschläger, 1994a), due to the environment, the containers or packing material used, the instruments used during sample preparation or from the chemical reagents used during the analytical procedures. Controlled conditions are therefore required for all procedures, including the dissection of fish organs on board ship. Relevant references concerning clean laboratories include Moody (1982), Mitchell (1982a), Boutron (1990) and Schmidt and Gerwinski (1994).

4.3 Shellfish

4.3.1 Depuration

Mussels should be placed on a polyethylene tray elevated above the bottom of a glass aquarium. The aquarium should be filled with sub-surface sea water collected from the same site as the samples and which has not been subject to contamination from point sources if possible. The aquarium should be aerated and the mussels left for 20-24 hours at water temperatures and salinity close to those from which the samples were removed.

4.3.2 Opening of the shells

Mussels should be shucked live and opened with minimum tissue damage by detaching the adductor muscles from the interior of one valve. The mussels should be inverted and allowed to drain on a clean towel or funnel for at least 5 minutes in order to minimise influence on dry weight determinations.

72 4.3.3 Dissection and storage

The soft tissues should be removed and deep frozen (-20C) as soon as possible in containers appropriate to the intended analysis. The dissection must always be done by trained personnel on a clean bench wearing clean gloves and using clean stainless steel knives which may be equipped with blades made of ceramics or titanium to reduce the risk of Cr and Ni contamination. Colourless polyethylene tweezers are recommended for holding tissues during dissection. After each sample has been prepared, the tools should be cleaned regularly. The following procedure is recommended: wash in acetone or alcohol and high purity water; wash non-stainless steel tools in HNO3 p.a./high purity water 1+1 (for tweezers 1+6); rinse with high purity water.

5. Analysis

5.1 Preparation of equipment and reagents

Glassware and Teflon equipment should be washed extensively with dilute nitric acid, distilled water and acidified metal-free deionised water, and should be rinsed immediately before use with the acids or solvents used according to the following procedure. The blank from all plastic and glassware after the purification procedure should be controlled. Acids, solvents, chemicals and adsorption materials should be free of trace metals or organometallic compounds. If not they should be purified by appropriate methods. Acids should be checked by measuring blanks using the analytical procedure applied to the samples. If necessary, the acids should be purified by distillation, preferably under sub- boiling point conditions in a quartz distillation apparatus. If appropriate, chemicals and adsorption materials should be purified by exhaustive extraction with the solvents used for extraction of the metal compounds. Care should be taken to avoid contamination from laboratory air dust particles. Relevant references concerning reagents and materials Moody et al. (1982; 1989); Tschöpel et al. (1980), Kosta (1982), Mitchell (1982b), Paulsen et al. (1989) and Luque de Castro and Luque García (2002).

5.2 Dry weight determination

Dry weight determinations should be carried out by air-drying homogenised sub-samples of the material to be analysed to constant weight at 105°C. Freeze-drying could also be used for the dry weight determination.

5.3 Determination of metals

5.3.1 Homogenisation and drying

73 When the analysis is undertaken, all fluids that may initially separate on thawing should be included with the materials homogenised. Wet or freeze-dried tissues should be homogenised. Homogenisation of wet tissues should be performed immediately prior to any subdividing of the sample. Fresh tissue should be thoroughly homogenised to include any moisture and lipids that may have separated from the solid parts of the sample. Aliquots should be taken as soon as possible, either for direct analysis or for drying. When grinding samples after drying, classical techniques using a ball mill made of different materials should be used. Relevant references concerning homogenisation include Iyengar, 1976; Iyengar et al., 1977 and Klussmann et al., 1985. References concerning sample pretreatment include Klussmann et al. (1985), Luque de Castro and Luque García (2002) and Larsen et al. (2011).

5.3.2 Digestion

The minimum requirements for the digestion procedure are the following: complete destruction of all organic material and mineralisation of the sample; avoidance of loss of the elements to be determined; avoidance of contamination; a sampling size of minimum 200 mg dry material

The following aspects should be considered as well:

• Digestion methods will be favoured with use only small amounts of ultra-pure reagents and chemicals;

• The method should be safe to handle (e.g. avoiding hydroperchloric acid);

• Some methods analyse directly and dissolution is not necessary e.g. AMA-

254 for mercury analyses.

• A microwave digestion closed system is preferred for biota samples.

• The use of automated procedures is preferred.

Trace element analysis in biological tissues normally involves digestion of the sample with acids. Very pure acids are essential to ensure acceptable blanks. If “matrix-effects” prevail after sample digestion, three strategies may be followed: standard addition for calibration; chemical separation procedures;

74 matrix modifiers.

5.3.3 Instrumental determination

The appropriate instrumental equipment has to be chosen with regard to (i) the elements to be analysed (ii) the concentration levels to be detected (iii) the matrix and the sampling processing prior to the measurement (e.g. digestion, pre-cleaning), but for economic reasons also taken into account (iv) the typical throughput number of samples and (v) investigation and operational costs.

For marine biota samples, all relevant monitoring programmes include mercury, cadmium and lead as mandatory parameters. For analysing Cd and Pb from open sea samples, e.g. flatfish liver of dab and plaice, Graphite Furnace Atomic Absorption Spectometry (GFAAS) and ICP-MS are appropriate. For higher concentrated metals such as Cu and Zn, Flame- AAS, ICP-AES or ICP-OES (weak for Pb) and Total Refection X-Ray Spectrometry (TXRF, weak for Cd) may also be used, but are not suitable to cover all obligating measurements in the required concentration range at very low concentrations without additional preconcentrating procedures.

For mercury cold vapour AAS-systems are commonly used, as stand-alone device or addition to AAS-systems. In recent years, direct measuring systems for analysing mercury from liquid and solid samples without any preceding digestion have become available (e.g. AMA, PE SMS 100 and MLS DMA-80), which have been proven to produce accurate and reliable results. Also a GFAAS-system equipped with a solid sample (autosampler) device for direct measuring and a high-resolution continuum source has become available, which reduces the pretreatment of the samples and has only one source for all elements. Direct methods for analysing mercury using pyrolysis combined with a gold trap and fluorescence or atomic absorption detection are sensitive enough to measure biota sample directly (Carbonell et al., 2009; Maggi et al., 2009; Torres et al., 2012). For the detection of hydride forming elements, such as arsenic, selenium or antimony, nearly all manufacturers of AAS offer additional hydride add-on devices.

6. Analytical quality assurance

The programme planners must decide on the accuracy, precision, repeatability, limit of detection and limit of determination required for each specific programme. Achievable limits of determination are as follows:

Cd 5 g/kg wet weight;

Hg 10 g/kg wet weight;

Pb 20 g/kg wet weight;

Cu 200 g/kg wet weight.

75 Relevant references concerning QA include HELCOM (1988), QUASIMEME (1992), Harms (1994) and ICES (1995).

6.1 Calibration and preparation of calibrands

For calibration purposes, single element standard stock solutions at a concentration of 1000 mg/l are commercially available or can be prepared from the highest quality elements available (generally 99,999% purity) dissolved in high purity acid (usually 1 molar nitric acid). Single or mixed working element standard solutions for calibration purposes are prepared by taking aliquots of the standard stock solutions which are diluted using diluted acid as required. Both standard stock and working solutions are stored in polyethylene, borosilicate or silica volumetric flasks. Borosilicate flasks must not be cleaned with alkaline solutions or heated above 70°C.

Working standard solutions at concentrations less than 100 µg/l should be prepared immediately before use. The actual concentration of the element should be stated on the label together with the date of the preparation of all standard solutions. The calibration procedure must meet some basic criteria or assumptions in order to give a best estimate of the true (but unknown) element content of the sample analysed. These are as follows: the masses or concentrations of standards for the establishment of the calibration function must be prepared without bias; the chemical and physical properties of the calibration standards must closely resemble those of the sample under investigation; sample and calibration standard must be subject to the same operational steps of the analytical procedure; signals of repeatedly analysed calibration standards must be randomly distributed on either side of the calibration line.

Application of chemical separation procedures. Although relatively simple standards with a minimum of matrix matching are required, separation procedures that consist of several stages are prone to systematic errors due to both uncontrollable contamination and analyte losses, respectively.

6.2 Blanks

A procedural blank should be measured for each sample series and should be prepared simultaneously using the same chemicals and solvents as for the samples. Its purpose is to indicate sample contamination by interfering compounds, which will lead to errors in quantification. Detailed information how to reduce and control contamination is given by ICES (1995).

6.3 Accuracy and precision

76 A laboratory reference material (LRM), preferably a Certified Reference Material (CRM), should be included in the analyses, at least one LRM/CRM sample for each series of identically prepared samples.

The LRM must be homogeneous, well characterised for the determinands in question and stability tests must have shown that it produces consistent results over time. The LRM should be of the same type of matrix (e.g. liver, muscle tissue, fat or lean fish) as the samples, and the determinand concentrations should occur in a comparable range to those of the samples. If the range of determinand concentrations in the sample is large (> factor of 5) two reference materials should be included in each batch of analyses to cover the lower and upper concentrations. It is good practice to run duplicate analyses of a reference material to check within-batch analytical variability. The use of a freeze-dried LRM is a practicable alternative to a homogenised and frozen LRM. However, the efficiency of the preceding steps such as homogenisation and drying cannot be checked. A quality control chart should be recorded for each metal. When introducing a new LRM or when it is suspected from the control chart, that there is a systematic error possibly due to an alteration of the LRM, another LRM (preferably a CRM) with a matrix as close as possible to the material analysed, should be used to check the reference material. Table 1 contains information on CRMs commercially available for use in marine monitoring.

Table 1: Certified Reference Materials for metals in marine organisms.

Code Organization Matrix

1 ERM-CE278k IRMM Mussel tissue

ERM-BB422 IRMM Fish muscle

BCR-463 IRMM Tuna fish

2 DOLT-4 NRC Dogfish liver

DORM-4 NRC Fish

LUTS-1 NRC Non defatted lobster hepatopancreas

TORT-3 NRC Lobster hepatopancreas

3 SRM 2976 NIST Mussel tissue

SRM 1946 NIST Lake fish tissue

1) IRMM: Institute for Reference Materials and Measurements (Europe)

2) NRC: National Research Council (Canada)

77 3) NIST: The National Institute of Standards and Technology (USA)

Additionally a duplicate of at least one sample should be run with every batch of samples. Each laboratory should participate in inter laboratory comparison studies and proficiency testing schemes on a regular basis, preferably at an international level.

6.4 Data collection and transfer

Data collection, handling and transfer must take place using quality controlled procedures.

7. Data recording and reporting parameters

Data reporting should be in accordance with the requirements for national comments and with the latest ICES reporting formats. Results should be reported according to the precision required for the programme. In practice, the number of significant figures is defined by the performance of the procedure.

The following parameters should be recorded although they may serve different purposes, e.g. internal sampling protocols, and QA or requirements of the database of the assessing body:

78 7.1 Sampling and biological parameters

Shellfish location of sampling site (name, latitude and longitude); date and time of sampling (GMT); sampling depth with respect to low tide (for sub-tidal sites only); irregularities and unusual conditions; name and institution of sampling personnel; number of pooled samples; number of individuals in pool; mean, minimum and maximum length and standard deviation; mean dry shell weight; mean soft tissue weight (wet weight); condition index.

7.2 Analytical and quality assurance parameters

LRM and CRM results for the metals listed in section 7.3 reported on a wet weight basis;

Uncertainty as U2 in the units of the result for use in the OSPAR assessment tool mean soft tissue dry weight and method of determining water content if this differs from air drying to constant weight at 105C; descriptions of the digestion and instrumental determination methods used; the determination limit for each element. The limits should not exceed the values in Section 6; the relevant QA information according to the requirements specified in the programme; the mean tissue lipid weight and method of extraction could also provide valuable

79 information.

7.3 Parameters

Elements of interest for monitoring programmes for which these guidelines apply:

- cadmium (total);

- mercury (total);

- lead (total);

- zinc;

- copper.

80 Appendix C

The following are the adjusted guidelines derived from the CEMP sampling guidelines for the annual monitoring of Mytilus edulis in Iceland. Jörundsdóttir et al., (2013) used these guidelines for the annual sampling report produced by Matis and were obtained by personal communication with Halldor Halldorsson.

September 2016 - Halldór Pálmar Halldórsson Upplýsingar frá Svanhildi Egilsdóttur á Hafró

Leiðbeiningar varðandi kræklingasöfnun – mengunarvöktun UST / AMSUM

Áhöld: - Plastfata/poki til að safna í - Plasttunna 50-60 L. - Netpokar eða plastfötur með mörgum götum (ca 1 cm í þvermál) - Skíðmál eða reglustika - Plastpokar og merkimiðar - Fiskabúrsloftdæla með slöngu og "loftsteini"

Söfnun Safna á 50 kræklingum sem eru 40 til 60 mm að skellengd, eða sem næst 50 mm að meðallengd ef hægt er. Ágætt þó að safna ríflegum fjölda (ca 60 stk) og ganga svo úr skugga um að amk 50 séu lifandi að lokinni hreinsun í sjó sem hér er lýst. Kræklinginn á að setja í netpoka (eða götótta fötu) sem er hengdur ofarlega í plasttunnuna sem þarf að vera full af hreinum sjó af söfnunarstað. Lofti þarf að dæla í tunnuna megnið af tímanum. Þarna á kræklingurinn að vera í sólarhring svo hann tæmi meltingarveginn og passa þarf að sjórinn hitni ekki að ráði, þ.e. hafa tunnuna úti við í skugga eða í kæli. Að því loknu er kræklingurinn tekinn úr sjónum, honum pakkað í plastpoka og sýnið merkt vel: „AMSUM mengunarvöktun“, staður og dagsetning. Sýnið á síðan að frysta. Það þarf að skrifa þessar upplýsingar á miða sem fer inn í pokann og tússa sömu upplýsingar utan á pokann eða skrifa á annan miða sem settur er utan á pokann, og er annað hvort límdur á hann eða festur við hann með bandi.

Þegar búið er að frysta sýnin þarf að senda þau sem fyrst til Matís (Vínlandsleið 12, 113 Reykjavík) en ganga þarf úr skugga um að þau berist þangað fyrir kl. 16 á virkum degi. Best er að hringja í Halldór Pálmar í síma 848 8811 áður en sýnin verða send af stað. Pakka þarf sýnunum vel inn í dagblöð svo þau þiðni ekki, setja þau í kassa (með kælikubbum ef lengur en 3-4 klst á leiðinni) og merkja kassann vel.

81 Appendix D

The following are the raw data tables of all of the mussels collected from this study (Sites 1- 9), except for the official samples collected from Site 10. Each table contains the number of mussels, length width, height, total weight, weight of soft body, and weight of shell for each mussel collected at each sampling site.

Blue mussel (Mytilus Date of Species: edulis) sampling: 25.08.2018 Sampled Length: 4-6 cm by: A.Hixson Date of Site 1 River preparation Location: Tunga : 11.09.2018 Coordinate s: IFL#: 5 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 49.8 25.3 22.1 12.47 5.81 6.42 2 50.3 25.9 21.4 12.07 5.57 6.43 3 47.0 23.1 19.8 10.85 5.10 5.72 4 47.0 24.2 20.4 11.12 4.58 6.54 5 47.0 24.8 20.3 9.50 4.02 5.46 6 51.4 25.3 22.6 12.61 5.11 7.36 7 44.8 24.3 20.4 10.69 5.17 5.48 8 47.6 23.7 20.3 10.19 3.95 6.16 9 46.4 24.2 21.4 10.52 4.69 5.40 10 50.9 24.8 19.2 10.11 4.33 5.67 11 52.0 24.3 22.0 11.25 3.94 7.28 12 50.9 25.3 20.4 12.66 4.51 8.08 13 45.4 24.2 22.1 11.18 4.42 6.65 14 47.6 23.1 19.8 11.75 5.81 5.82 15 44.2 23.1 17.6 7.28 2.72 4.44 16 49.2 26.4 24.2 15.82 5.05 10.70 17 52.6 26.4 21.50. 15.81 7.35 8.25 18 55.3 27.6 20.9 16.88 8.23 8.44 19 44.8 23.2 17.6 8.38 2.77 5.34 20 43.6 22.1 18.2 7.55 2.74 4.56

82 21 56.5 24.2 22.6 15.87 6.60 8.67 22 49.7 25.4 21.4 12.85 5.13 7.58 23 47.0 22.6 22.6 12.67 5.85 6.61 24 49.8 25.3 21.5 9.73 3.71 5.71 25 46.5 25.3 19.8 11.73 5.36 6.34 26 51.4 26.9 21.9 14.41 6.10 8.12 27 49.7 24.8 21.4 13.81 5.70 7.81 28 45.3 22.0 16.4 7.04 2.24 4.67 29 43.1 21.4 16.4 6.05 2.64 3.26 30 56.4 23.1 24.2 18.53 8.90 9.25 31 47.6 26.4 21.5 10.75 3.75 6.87 32 46.4 27.1 23.1 19.82 9.66 9.89 33 44.8 24.8 19.2 8.60 2.52 5.91 34 44.9 22.2 21.0 9.15 3.94 5.04 35 43.5 22.1 18.7 7.37 2.24 4.90 36 47.1 25.5 22.5 9.58 3.55 5.95 37 42.9 22.0 17.7 8.16 2.49 5.51 38 44.9 22.2 21.1 10.02 3.14 6.64 39 46.7 23.4 22.6 11.92 4.27 7.47 40 47.9 22.1 19.0 10.83 5.19 5.35 41 43.2 27.2 23.5 18.05 8.37 9.55 42 52.2 25.3 22.8 10.56 3.00 7.49 43 53.1 26.0 22.5 13.74 5.84 7.68 44 44.0 21.9 17.5 9.39 4.19 4.91 45 53.3 27.9 20.2 15.47 6.69 8.48 46 55.2 26.3 24.9 21.31 9.77 11.27 47 54.1 24.5 25.1 15.14 4.73 10.06 48 56.5 25.3 24.2 18.53 8.04 10.32 49 59.2 28.2 24.9 21.75 10.26 11.19 50 59.9 24.3 22.9 18.64 6.92 11.54 Total Weight soft Weight Length Width Height weight body shell Average 49.01 24.54 21.09 12.40 5.13 7.08 Stdev 4.43 1.76 2.22 3.84 2.05 1.99 Min 42.93 21.44 16.37 6.05 2.24 3.26 Max 59.93 28.20 25.13 21.75 10.26 11.54 Total Weight 255.88 Softbody: Total 351.17 Weight

83 Shell:

84 Blue mussel (Mytilus Date of Species: edulis) sampling: 27.08.2018 Length: 4-6 cm Sampled by: A.Hixson site 2:kayak-dive Date of Location: centre preparation: 10.9.2018 Coordina tes: IFL#: 4 Weight soft Weight Length Width Height Total weight body shell (mm) (mm) (mm) (g) (g) (g) 1 53.6 26.1 20.3 17.14 9.94 7.04 2 41.7 21.9 14.9 7.64 4.44 3.11 3 42.1 22.6 15.1 7.67 3.50 3.74 4 43.8 21.4 15.4 8.39 4.70 3.28 5 52.3 27.1 17.1 13.62 7.58 5.77 6 43.8 21.9 15.4 8.27 4.86 3.38 7 45.2 22.0 16.3 9.18 5.62 4.07 8 39.9 21.2 15.6 8.28 4.71 3.46 9 55.9 27.7 20.1 17.87 9.80 7.47 10 52.6 23.9 20.4 14.47 8.11 6.18 11 47.6 22.7 16.8 10.76 6.54 4.10 12 47.7 23.4 16.6 10.73 6.14 4.54 13 50.1 21.8 17.4 11.38 6.04 5.26 14 44.2 22.2 15.5 8.74 5.46 3.26 15 55.2 21.9 18.8 13.21 7.71 5.40 16 55.9 26.7 18.8 15.52 8.77 6.61 17 52.7 22.8 19.9 13.13 7.63 5.40 18 46.0 21.5 18.2 11.15 5.97 5.10 19 47.1 22.7 18.2 11.63 7.24 4.32 20 45.7 22.2 16.8 9.86 5.56 4.22 21 40.5 20.4 15.3 6.25 3.80 2.34 22 44.9 22.2 17.6 10.07 5.51 4.38 23 39.4 18.7 12.7 5.88 3.25 2.57 24 38.0 18.0 15.7 6.18 3.60 2.53 25 41.5 19.8 15.4 6.69 3.99 2.54 26 45.3 23.2 17.1 10.03 5.42 4.33 27 41.5 20.3 14.2 6.73 3.61 2.79 28 49.7 25.8 19.1 11.93 7.09 4.50 29 49.6 24.1 16.9 10.70 5.97 4.32 30 51.9 24.7 17.1 13.08 7.09 5.42 31 54.7 25.9 19.7 14.00 8.60 5.05 32 59.8 29.7 21.9 18.22 11.49 6.34 33 44.8 21.5 17.0 9.84 5.07 4.43 34 47.1 23.6 16.9 10.37 5.82 4.21 35 50.9 23.1 17.0 11.21 6.48 4.37 36 44.8 20.8 16.5 8.33 4.53 3.40 37 40.9 20.8 16.0 5.89 3.33 2.27 38 40.9 20.8 14.8 6.75 3.52 2.81

85 39 41.9 21.9 13.7 6.60 3.92 2.33 40 37.0 19.1 13.7 5.55 2.87 2.26 41 46.9 23.1 17.1 10.22 5.83 4.05 42 42.0 22.0 15.3 7.88 4.34 3.13 43 40.3 20.4 15.8 6.60 3.85 2.23 44 47.1 22.6 16.5 10.53 5.91 4.20 45 48.7 24.7 17.1 11.11 6.50 4.29 46 49.7 25.4 20.2 13.78 8.04 5.66 47 51.5 25.4 17.0 12.63 8.10 4.37 48 47.0 22.0 15.9 9.57 5.33 4.09 49 45.9 22.6 17.0 10.03 5.73 4.05 50 59.7 29.7 20.9 18.13 10.63 7.21 Weight soft Weight Length Width Height Total weight body shell Average 46.94 22.92 16.96 10.47 5.99 4.24 Stdev 5.55 2.53 2.01 3.34 2.03 1.36 Min 37.04 17.97 12.67 5.55 2.87 2.23 Max 59.75 29.74 21.85 18.22 11.49 7.47 Total Weight Soft 298.4 body: Total Weight 208.78 Shell:

86 Blue mussel (Mytilus Date of Species: edulis) sampling: 29.08.2018 1304 Sampled Length: 4-6 cm by: A.Hixson Date of site 3 outer preparation Location: harbour : 10.09.2018 Coordinates : IFL#: 8 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 58.66 29.12 18.09 16.64 10.52 5.76 2 57.44 29.91 19.01 15.44 9.60 5.45 3 56.82 27.01 19.96 17.86 9.95 7.56 4 40.68 21.05 14.44 7.01 3.92 2.94 5 54.31 26.80 19.98 12.66 8.19 4.28 6 51.19 24.69 15.95 10.63 6.18 4.30 7 47.13 25.30 17.11 10.03 5.80 4.09 8 54.85 25.34 21.45 13.97 7.98 5.79 9 54.75 26.55 21.05 12.80 7.19 5.43 10 52.52 25.35 19.66 15.19 7.59 7.33 11 60.36 30.33 18.65 16.25 8.28 7.70 12 55.95 29.69 20.32 18.97 11.29 7.51 13 60.34 32.55 20.85 22.93 12.86 9.69 14 52.54 27.62 20.35 13.36 5.29 7.93 15 56.95 28.64 19.71 16.39 9.25 6.94 16 57.56 29.66 15.90 18.20 10.04 7.90 17 43.64 22.52 13.12 7.39 4.45 2.91 18 61.44 29.74 21.41 21.37 13.17 8.06 19 58.74 32.05 16.45 21.59 12.08 9.14 20 55.9 29.66 18.65 15.51 8.78 6.61 21 51.42 27.69 20.23 11.50 5.03 5.72 22 46.45 23.66 15.33 9.82 5.26 4.38 23 41.94 24.76 11.55 5.85 3.19 2.45 24 51.96 27.51 15.90 12.18 6.62 5.20 25 52.54 27.60 14.75 11.96 5.97 5.58 26 57.60 29.75 23.19 18.84 10.76 7.99 27 53.64 29.72 19.75 15.86 9.07 6.49 28 61.45 31.98 17.10 21.16 13.18 7.75 29 53.17 29.15 15.33 14.37 8.58 5.80 30 59.67 30.30 19.15 18.04 10.53 7.33 31 53.65 27.59 19.74 15.44 8.57 6.75 32 53.13 26.50 17.44 12.31 7.61 4.59

87 33 45.31 25.80 16.50 11.22 6.55 4.49 34 45.84 24.75 12.95 10.15 4.87 4.91 35 52.55 26.50 16.97 11.55 6.45 5.02 36 52.56 26.50 16.60 13.60 8.24 5.07 37 49.75 25.86 17.55 12.29 7.2 5.07 38 50.75 27.50 18.12 12.13 6.62 5.39 39 42.55 23.70 13.62 6.81 4.14 2.47 40 59.66 31.42 22.50 20.75 12.18 8.31 41 60.34 30.33 21.42 20.93 11.84 8.92 42 60.85 32.55 20.85 18.28 9.01 8.95 43 63.65 30.30 19.80 14.89 6.97 7.75 44 62.60 30.86 20.85 18.38 9.50 8.52 45 63.20 32.10 19.20 20.06 11.38 8.50 46 62.52 29.78 21.50 19.39 11.12 8.10 47 64.75 32.07 25.90 29.40 17.25 11.48 48 39.65 21.50 14.73 6.13 3.32 2.49 49 36.42 18.12 10.35 3.95 1.85 1.95 50 37.5 21.44 11.95 5.54 2.8 2.44 Total Weight soft Weight Length Width Height weight body shell Average 53.58 27.62 18.06 14.54 8.16 6.14 Stdev 7.19 3.33 3.23 5.22 3.17 2.19 Min 36.42 18.12 10.35 3.95 1.85 1.95 Max 64.75 32.55 25.90 29.40 17.25 11.48 Total Weight Soft 406.4

body: Total Weight 304.36

Shell:

88 Blue mussel (Mytilus Date of Species: edulis) sampling: 31.08. 2017 Length: 4-6 cm Sampled by: A.Hixson Date of Location: Site 4: Úlfsá preparation: 11.09.2018 Coordina tes: IFL#: R17-2779-7 Weight soft Weight Length Width Height Total weight body shell (mm) (mm) (mm) (g) (g) (g) 1 46.31 25.95 20.10 11.95 7.10 4.64 2 41.51 21.87 16.29 7.56 3.22 3.70 3 46.84 23.53 19.62 8.79 4.21 4.33 4 42.90 24.43 18.03 9.53 4.94 4.57 5 47.36 25.24 20.60 10.55 6.10 4.39 6 42.34 22.72 16.84 7.90 4.19 3.62 7 43.73 23.01 16.13 7.23 3.36 3.90 8 42.71 23.43 17.02 8.18 4.57 3.60 9 45.26 24.03 17.80 9.22 5.69 3.47 10 45.73 23.75 18.34 7.32 3.78 3.50 11 45.91 22.85 19.16 7.85 4.23 3.58 12 48.58 22.17 19.17 6.44 2.05 4.26 13 47.28 26.30 17.32 10.18 5.42 4.19 14 54.31 25.36 21.20 15.75 8.63 7.09 15 45.54 22.64 19.63 7.86 3.73 4.15 16 40.32 21.75 17.19 7.28 3.45 3.67 17 56.53 25.96 23.58 17.72 10.46 7.19 18 43.29 21.79 16.59 6.90 3.53 3.27 19 41.56 21.43 17.42 7.78 4.41 3.26 20 40.60 19.91 16.90 6.65 3.37 3.18 21 42.02 20.95 16.72 7.62 4.07 3.54 22 44.79 22.91 17.17 8.23 4.53 3.13 23 43.99 22.10 15.78 7.91 4.45 3.39 24 40.00 22.49 18.13 7.02 4.17 2.73 25 51.63 24.60 21.29 14.61 7.04 7.49 26 42.75 22.25 17.10 7.81 4.68 3.00 27 40.32 20.84 17.29 5.82 2.56 2.98 28 51.59 26.66 18.99 11.92 7.12 4.81 29 44.28 21.52 16.32 7.59 4.47 3.11 30 40.18 18.99 14.70 4.50 2.47 1.98 31 43.88 22.13 17.60 8.13 4.62 3.48 32 42.46 22.61 17.36 8.22 4.67 3.45 33 46.97 23.83 18.44 10.03 5.92 4.07 34 42.70 20.37 18.39 8.21 4.38 3.73 35 59.28 27.87 23.23 18.32 10.98 7.23 36 42.41 22.42 17.58 7.53 4.54 2.88

89 37 39.73 19.11 14.83 6.01 3.54 2.49 38 41.64 22.29 16.67 5.83 2.88 2.88 39 40.32 20.49 15.39 5.10 2.58 2.44 40 42.64 20.99 16.01 5.47 2.60 2.71 41 40.12 22.80 17.26 8.27 3.73 4.45 42 42.31 22.69 15.98 6.50 2.70 3.32 43 41.22 22.42 15.71 5.54 2.19 3.10 44 40.66 23.63 15.87 6.22 2.92 2.94 45 39.02 21.32 14.03 4.61 2.23 2.28 46 39.27 20.29 14.01 4.65 2.25 2.30 47 39.50 18.71 13.98 4.35 2.01 2.27 48 38.84 20.17 16.67 6.36 3.24 3.00 49 38.06 20.93 18.08 5.82 2.17 3.49 50 38.42 19.54 15.00 4.33 1.89 2.37 Weight soft Weight Length Width Height Total weight body shell Average 43.79 22.52 17.49 8.10 4.28 3.69 Stdev 4.57 2.05 2.12 3.11 1.99 1.25 Min 38.06 18.71 13.98 4.33 1.89 1.98 Max 59.28 27.87 23.58 18.32 10.98 7.49 Total Weight 213.33 Soft body: Total Weight 183.08 Shells:

90 Blue mussel (Mytilus Date of Species: edulis) sampling: 02.09.2018 Sampled Length: 2-5cm by: A.hixson Date of site 5: preparation Location: hnifsdalur : 13.09.2018 Coordinate s: IFL#: 1 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 35.9 18.5 14.9 5.24 2.94 2.20 2 31.8 16.7 12.9 3.51 1.77 1.64 3 28.4 14.7 12.8 2.37 1.15 1.16 4 24.3 13.0 11.1 1.74 0.91 0.79 5 23.8 11.6 10.6 1.82 0.79 0.97 6 24.7 12.1 12.5 2.23 0.96 1.20 7 27.5 13.8 10.7 2.06 1.09 0.94 8 29.6 14.0 12.9 2.80 1.52 1.26 9 30.8 15.4 13.5 3.45 1.75 1.61 10 28.7 15.6 11.1 2.31 1.19 0.97 11 27.6 14.5 11.9 2.54 1.26 1.24 12 30.6 17.7 16.0 4.21 1.95 2.18 13 30.9 16.5 13.3 3.26 1.94 1.26 14 31.8 17.4 13.3 3.41 1.86 1.52 15 30.1 17.1 13.2 3.08 1.79 1.22 16 29.3 15.9 13.4 3.17 1.73 1.39 17 34.4 19.4 17.0 5.08 2.66 2.33 18 34.8 15.4 14.9 4.25 1.97 2.12 19 30.4 15.9 14.5 3.73 2.09 1.60 20 32.3 16.7 13.8 2.79 1.31 1.42 21 33.2 17.6 13.3 3.64 2.12 1.47 22 33.1 18.0 14.0 4.43 2.13 2.18 23 34.9 16.5 15.5 3.90 1.95 1.76 24 37.2 18.1 14.3 4.71 2.74 1.84 25 38.6 20.2 14.3 5.55 2.49 2.98 26 34.7 18.4 15.2 4.43 2.42 1.94 27 37.6 20.3 15.3 5.93 3.08 2.70 28 34.9 19.4 19.3 6.28 2.94 3.19 29 34.3 18.6 15.0 4.96 2.45 2.37 30 38.6 21.4 15.4 5.96 3.21 2.67

91 31 39.48 20.3 14.8 5.64 2.93 2.59 32 38.8 20.0 20.6 8.99 3.81 4.89 33 39.6 21.0 18.1 7.58 4.27 3.26 34 40.0 20.3 15.8 5.59 2.64 2.75 35 42.9 21.4 17.8 7.81 4.73 3.01 36 45.9 23.1 18.3 9.92 5.45 4.42 37 42.8 25.1 18.5 8.85 4.33 4.34 38 45.4 23.0 19.2 9.32 4.6 4.57 Total Weight soft Weight Length Width Height weight body shell Average 33.94 17.75 14.71 4.65 2.39 2.16 Stdev 5.61 3.10 2.49 2.20 1.15 1.07 Min 23.84 11.60 10.62 1.74 0.79 0.79 Max 45.94 25.05 20.58 9.92 5.45 4.89 Total Weight 90.41

Soft body:

Total

Weight 81.45

Shells:

92 Blue mussel (Mytilus Date of Species: edulis) sampling: 05.09.2018 Sampled Length: 4-6 cm by: A.Hixson Date of Site 6 preparation Location: Valagil : 12.09.2018 Coordinate s: IFL#: 3 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 48.4 26.9 18.3 10.11 6.40 3.62 2 50.7 25.8 18.8 11.15 7.00 4.03 3 46.6 22.6 18.4 6.73 4.08 2.65 4 46.1 24.2 18.7 9.06 5.58 3.39 5 50.4 25.5 22.1 13.29 8.90 4.34 6 45.7 22.1 17.8 8.48 5.26 3.14 7 46.1 22.2 18.0 6.98 4.06 2.88 8 46.9 24.4 20.1 10.61 6.64 3.95 9 44.8 23.0 18.0 8.78 5.80 3.57 10 44.0 22.2 16.2 7.44 4.49 2.89 11 44.7 21.7 15.4 6.74 3.80 2.87 12 44.5 22.0 17.6 6.83 4.12 2.50 13 48.7 23.3 20.3 10.85 6.60 4.13 14 41.6 22.2 16.2 6.91 3.97 2.81 15 42.5 22.1 17.1 7.28 4.39 2.84 16 53.6 26.9 21.1 11.05 6.07 4.88 17 47.0 22.7 17.0 8.14 5.09 2.94 18 44.8 22.0 18.9 8.39 5.28 3.05 19 41.4 21.5 16.0 6.03 3.67 2.24 20 41.3 21.4 15.5 6.28 3.87 2.35 21 40.5 28.7 14.9 5.79 3.11 2.49 22 41.6 20.1 16.8 6.49 4.33 2.40 23 42.1 21.5 16.4 5.96 3.47 2.40 24 44.5 24.3 18.7 8.51 5.19 3.30 25 47.2 23.3 19.7 9.22 5.43 3.69 26 44.7 21.6 17.4 7.76 4.74 2.96 27 42.3 21.5 17.3 7.37 4.22 3.12 28 42.0 20.8 16.3 6.45 3.45 2.64 29 43.7 21.3 16.8 6.71 4.06 2.59 30 42.1 20.8 17.2 6.50 4.35 2.09 31 43.5 22.3 16.0 6.68 3.78 2.66

93 32 51.1 25.2 20.1 12.66 7.36 5.15 33 45.9 26.0 17.4 8.99 5.35 3.17 34 42.2 21.5 18.5 7.11 4.55 2.50 35 44.3 23.4 17.5 8.22 5.68 2.48 36 46.0 21.8 17.1 8.31 4.96 3.16 37 42.2 21.1 14.7 6.22 3.67 2.34 38 47.3 23.8 17.6 9.53 5.57 3.94 39 46.1 24.4 17.1 8.86 5.19 3.48 40 43.6 22.0 17.3 8.22 5.06 3.09 41 40.3 20.3 14.4 4.16 2.31 1.69 42 41.3 19.8 17.4 6.70 4.32 2.27 43 54.5 22.6 17.2 8.29 5.16 3.07 44 40.1 21.2 16.1 5.16 2.74 2.33 45 40.4 21.4 16.5 6.52 3.85 2.50 46 40.0 19.5 17.0 5.89 3.79 1.93 47 38.5 20.4 14.8 4.39 2.13 1.96 48 39.1 20.9 14.8 5.66 3.38 2.04 49 41.2 19.9 17.0 5.39 2.92 2.43 50 38.8 20.7 14.2 4.99 2.99 1.90 Total Weight soft Weight Length Width Height weight body shell Average 44.33 22.53 17.31 7.68 4.64 2.94 Stdev 3.67 2.02 1.69 2.00 1.33 0.75 Min 38.46 19.52 14.21 4.16 2.13 1.69 Max 54.52 28.67 22.06 13.29 8.90 5.15 Total Weight 231.25

Soft body: Total Weight 145.93

Shells:

Blue mussel (Mytilus Date of Species: edulis) sampling: 04.09.2018 Sampled Length: 4-6 cm by: A.Hixson Date of Site 7 End preparation Location: of Spit : 12.09.2017 Coordinate s: IFL#: 6 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 43.5 21.7 16.1 7.72 4.23 3.31 2 42.1 20.2 16.0 6.38 3.88 2.36 3 43.9 22.3 17.8 9.06 5.52 3.49 4 44.0 23.3 17.0 8.50 4.12 4.35 5 50.6 26.7 20.1 13.51 7.26 6.14 6 44.8 25.3 19.9 10.30 6.19 4.09 7 41.5 19.8 14.9 5.94 3.39 2.53 8 42.4 20.2 14.1 5.66 3.41 2.26 9 46.2 21.9 18.9 9.92 5.11 4.76 10 43.9 20.7 17.2 8.08 4.49 3.61 11 40.9 21.2 16.2 7.05 4.24 2.79 12 45.1 21.7 19.2 9.25 5.43 3.75 13 41.6 20.6 15.9 6.91 3.80 3.10 14 41.5 19.1 16.3 6.45 3.98 2.45 15 42.7 22.0 18.5 8.24 4.78 3.42 16 51.6 26.6 21.0 14.24 8.23 6.25 17 45.5 24.7 18.0 9.78 5.25 4.40 18 46.2 22.8 21.1 11.54 6.85 4.90 19 43.4 22.8 15.8 7.55 4.07 3.66 20 41.2 21.1 17.9 7.88 4.32 3.49 21 46.1 23.2 19.3 10.75 5.61 5.11 22 42.5 21.1 17.5 7.79 4.37 3.27 23 43.2 21.4 18.3 8.49 4.25 4.17 24 43.5 21.4 18.9 9.08 4.26 4.66 25 41.3 18.7 16.1 6.91 3.32 3.42 26 42.5 19.9 17.1 6.98 4.07 2.82 27 43.0 21.4 16.6 7.66 4.25 3.31 28 41.0 21.6 16.5 7.32 4.00 3.18 29 40.7 20.7 14.2 5.88 3.24 2.54 30 40.4 20.9 15.8 7.22 4.14 2.82 31 39.23 18.9 15.4 5.56 3.32 2.14 32 39.8 20.8 15.3 4.41 2.15 2.21 33 39.2 20.7 14.9 6.00 3.01 2.87

95 34 38.9 20.1 15.1 5.47 3.17 2.20 35 40.0 19.6 16.2 6.21 3.44 2.72 36 39.0 20.9 16.8 5.87 2.37 3.39 37 39.3 18.8 15.7 5.60 2.58 2.89 38 38.9 18.6 15.6 4.71 1.97 2.52 39 38.3 19.5 15.0 5.22 2.33 2.73 40 39.6 20.1 16.7 6.69 3.78 2.90 41 37.1 19.6 14.7 5.28 2.73 2.22 42 38.1 17.8 14.3 4.82 2.33 2.35 43 38.4 19.1 16.1 5.89 3.06 2.76 44 39.1 20.7 15.7 5.98 3.30 2.64 45 37.7 18.6 14.0 4.80 2.58 2.02 46 38.2 19.1 12.9 4.43 2.59 1.66 47 37.3 18.3 13.7 5.14 2.60 2.45 48 39.3 19.3 16.2 6.51 3.57 2.88 49 37.2 18.3 13.9 4.85 2.47 2.21 50 37.44 17.91 15.1 5.09 2.69 2.31 Total Weight soft Weight Length Width Height weight body shell Average 41.57 20.82 16.50 7.21 3.92 3.21 Stdev 3.24 2.03 1.91 2.22 1.34 1.02 Min 37.07 17.83 12.88 4.41 1.97 1.66 Max 51.59 26.65 21.06 14.24 8.23 6.25 Total Weight 195.62

Soft body: Total Weight 159.17

shells:

96 Blue mussel (Mytilus Date of Species: edulis) sampling: 05.09.2018 Sampled Length: 4-6 cm by: A.Hixson site8: Langeyri Date of Location: sudavik preparation: 10.09.2018 Coordinate s: IFL#: 2 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 34.80 18.15 14.08 5.06 2.45 2.49 2 43.83 21.35 17.73 9.41 5.79 3.56 3 38.93 17.45 15.39 6.17 2.53 3.30 4 37.34 16.93 13.46 4.46 2.61 1.63 5 35.43 17.61 13.38 4.82 1.96 4.13 6 38.07 17.73 13.10 5.26 2.73 4.83 7 51.74 24.33 19.80 15.04 8.76 6.01 8 42.04 20.77 14.10 7.51 3.93 3.22 9 36.70 17.93 12.60 5.19 2.62 2.35 10 40.05 20.45 17.23 8.28 4.79 3.33 11 42.59 20.99 17.32 10.16 5.93 4.02 12 43.84 23.37 18.26 10.21 6.54 3.42 13 39.80 19.81 14.13 5.89 3.42 2.17 14 40.11 18.79 15.18 7.13 3.96 2.83 15 37.43 16.54 12.57 5.15 2.58 2.05 16 45.38 20.60 15.65 7.90 4.26 3.14 17 38.03 18.06 15.33 6.23 3.12 2.73 18 36.24 16.66 14.16 5.05 2.66 1.87 19 35.00 16.58 13.62 3.34 1.54 1.68 20 36.19 17.50 12.35 4.72 2.47 1.79 21 35.76 17.89 14.84 4.95 2.08 2.45 22 34.83 18.03 12.49 4.50 2.3 1.98 23 32.86 15.86 13.65 3.64 1.50 1.86 24 35.19 18.84 12.29 3.58 1.87 1.48 25 36.24 17.80 11.61 3.11 1.43 1.53 26 36.77 16.33 13.12 3.89 1.29 2.06 27 36.27 17.95 12.09 4.13 1.89 2.00 28 32.58 16.58 11.17 3.08 1.34 1.41

97 29 31.87 15.86 10.90 2.76 1.00 1.27 30 31.64 15.30 11.64 2.85 1.23 1.29 31 33.30 17.65 13.36 4.40 2.31 1.75 32 35.96 17.96 12.06 4.51 2.16 1.95 33 33.89 16.12 11.51 2.93 1.17 1.59 34 33.97 15.37 13.13 4.19 2.19 1.72 35 34.26 16.28 11.46 3.85 2.09 1.44 36 35.53 18.42 12.63 5.15 2.76 2.09 37 32.68 15.32 10.58 3.27 1.51 1.52 38 34.58 17.15 12.95 3.92 1.61 2.00 39 35.63 16.73 12.42 3.94 2.09 1.55 40 33.91 17.82 12.40 4.54 2.05 2.01 41 33.54 15.92 13.14 3.29 1.27 1.51 42 36.36 18.24 12.65 4.90 2.64 1.86 43 34.63 16.79 10.56 3.56 1.61 1.67 44 35.22 18.44 10.72 3.99 2.02 1.59 45 35.22 16.55 12.03 3.37 1.31 1.76 46 34.57 15.45 11.82 3.19 1.27 1.68 47 33.61 16.65 10.95 2.44 0.95 1.31 48 32.01 17.14 12.90 3.48 1.48 1.70 49 32.42 16.84 10.98 2.97 1.30 1.35 50 32.24 15.83 10.31 2.77 1.37 1.22 Total Weight soft Weight Length Width Height weight body shell Average 36.42 17.77 13.24 4.96 2.51 2.22 Stdev 3.93 1.96 2.11 2.37 1.56 0.99 Min 31.64 15.30 10.31 2.44 0.95 1.22 Max 51.74 24.33 19.80 15.04 8.76 6.01 Total Weight 124.16

Soft body: Total Weight 104.74

Shells:

Blue mussel (Mytilus Date of Species: edulis) sampling: 06.09.2018 Sampled Length: 4-6 cm by: A.hixson site 9: Date of ulufurs preparation Location: beach : 12.09.2018 Coordinates : IFL#: 1 Total Weight soft Weight Length Width Height weight body shell (mm) (mm) (mm) (g) (g) (g) 1 47.5 23.7 17.6 10.45 5.25 5.01 2 49.8 27.6 21.2 12.99 7.23 5.66 3 58.3 27.8 24.0 21.56 11.89 9.36 4 53.1 27.8 20.4 16.91 9.14 7.58 5 60.1 32.8 24.3 24.99 12.83 11.84 6 50.9 24.7 16.8 12.26 6.75 5.34 7 53.6 26.3 22.3 17.58 9.26 8.10 8 58.4 29.4 19.9 18.27 10.01 8.19 9 48.9 25.6 17.0 10.05 4.66 5.01 10 51.8 25.8 17.7 13.56 7.52 5.86 11 48.1 24.6 16.9 10.18 5.38 4.34 12 46.2 24.0 16.2 9.94 4.72 4.90 13 46.4 24.9 16.3 6.96 2.71 3.87 14 47.5 25.7 22.5 9.41 3.92 5.32 15 53.9 27.0 17.6 12.29 5.75 6.21 16 47.3 24.9 19.0 11.50 6.04 5.19 17 56.0 29.4 19.6 18.66 11.27 7.15 18 47.2 26.2 18.4 9.33 4.85 4.40 19 45.7 23.5 16.5 6.62 1.59 4.89 20 49.5 24.8 18.5 10.78 5.10 5.56 21 51.9 26.1 19.7 14.25 7.04 7.14 22 40.7 21.1 13.8 5.99 3.27 2.59 23 40.5 20.5 15.5 6.56 3.40 2.84 24 50.6 25.3 17.3 10.94 5.47 5.37 25 52.2 25.0 17.8 12.00 6.68 5.26 26 43.9 22.5 15.2 8.03 4.58 3.37

99 27 44.2 24.2 19.3 10.71 5.80 4.87 28 41.3 21.3 15.2 5.55 2.68 2.73 29 40.1 20.8 16.3 6.94 3.73 2.81 30 40.5 22.6 18.5 4.43 1.38 2.88 31 43.68 21.0 15.0 7.03 3.44 3.47 32 46.0 23.3 15.2 7.30 2.98 4.05 33 44.8 19.7 15.6 7.74 3.70 3.82 34 45.9 24.8 18.5 11.42 6.04 5.03 35 41.6 22.5 14.8 8.02 4.34 3.53 36 42.6 22.3 13.2 8.51 4.27 3.91 37 43.1 21.3 17.1 6.64 3.37 3.14 38 44.7 21.3 15.1 8.14 4.07 3.85 39 41.1 20.7 14.1 6.67 3.48 2.73 40 40.9 19.9 17.9 6.37 3.53 2.78 41 40.1 22.1 14.2 6.12 3.15 2.74 42 40.5 21.3 14.8 6.67 3.43 2.91 43 40.4 20.7 15.4 6.96 3.33 3.33 44 40.1 20.3 14.1 6.15 3.19 2.72 45 38.8 20.2 17.1 4.59 1.87 2.58 46 38.2 19.6 17.3 6.38 2.91 3.09 47 38.0 19.4 16.6 6.12 3.26 2.69 48 39.0 21.6 16.0 7.18 4.00 2.96 49 38.3 18.5 16.0 5.23 2.15 2.88 50 38.50 19.96 13.6 5.10 2.29 2.50 Total Weight soft Weight Length Width Height weight body shell Average 45.85 23.53 17.25 9.76 4.97 4.57 Stdev 5.89 3.08 2.59 4.53 2.64 2.00 Min 38.01 18.51 13.23 4.43 1.38 2.50 Max 60.12 32.75 24.28 24.99 12.83 11.84 Total Weight Soft 246.49

body: Total Weight 226.42

Shells:

100 Appendix E

The following are the temporal values for As, Cd, and Hg in both Skutulsfjörður and Álftafjörður as found in the ICES database (dome.ices.dk) and used in Sturludottir et al. (2013). The data concentrations for 2018 in Álftafjörður were collected for this study and the 2018 concentrations for Skutulsfjörður were collected as a part of the annual monitoring program for OSPAR.

Trace Element Concentration per Annual Sampling of Blue Mussels: Skutulsfjörður mg/kg (dw) As Cd Hg 1999 53.5 1.63 0.071 2000 54.8 1.57 0.139 2001 57.7 1.59 0.078 2003 70.7 1.59 0.101 2004 73.8 1.41 0.098 2005 63.3 1.47 0.126 2006 153.26 2.19 0.126 2007 60.92 0.893 0.101

2008 73 1.1 0.081 2009 65.1 0.99 0.068 2010 59.74 0.84 0.070 2011 48.18 0.78 0.080 2012 53.9 0.69 0.070 2013 55 1.15 0.111 2016 55.2 1.1 0.090 2017 56.57 1.26 0.087 2018 59.061 1.283 0.069

101 Trace Element Concentration per Annual Sampling of Blue Mussels: Álftafjörður mg/kg (dw) As Cd Hg 1996 12.04 2.34 0.047 1997 17.50 3.77 0.058 1999 18.80 0.95 0.063 2000 19.20 4.38 0.093

2001 25.00 3.03 0.055 2002 25.60 5.13 0.055 2003 20.10 5.54 0.046 2004 28.40 4.15 0.058 2005 14.00 3.90 0.054 2006 25.17 8.03 0.052 2007 8.27 3.12 0.052 2008 9.00 2.90 0.042 2009 8.00 2.76 0.030 2010 13.39 3.98 0.070 2011 10.11 3.55 0.070 2012 10.00 3.03 0.100 2013 9.00 5.24 0.060

2018 11.43 3.41 0.051

102