and for Food and Feed

Transkript

1 of March The Potential of Ulva for Bioremediation st and for Food and Teis Boderskov Supervisors: Jens J. Sloth, Annette Bruhn, DTU, National Food Institute AU, Department of Bioscience Esben Rimi Christiansen Esben Rimi Christiansen au au AARHUS UNIVERSITET AARHUS UNIVERSITET Denmark Master s Thesis M.Sc.Eng. Esben Rimi Christiansen

2 Abstract Bioremediation strategies can be implemented in clean-up processes of environment and a potential organism for this process has been proven to be the opportunistic and fast growing green algae Ulva spp. Addressing Ulva spp. in bioremediation strategies has shown high potential in improving the quality in marine waters but without action Ulva spp. can become a nuisance during the formation of Green Tides. The biomass yields that come by exploiting the bioremediation potential of Ulva spp. have many applications, e.g. in pharmaceuticals, as a fertiliser or for food and feed products. The purpose of this study was to evaluate the potential of Ulva spp. in Danish waters 1) for bioremediation of nutrients and 2) for following to utilise the biomass for food and feed applications. Therefore, spores were extracted from an Ulva sp., seeded on substrates and deployed at sea. Finally, the bioremediation potential of coastally cultivated Ulva sp. and naturally harvested Ulva spp. was assessed. The food and feed safety was evaluated regarding the tissue contents of As, Cd, Pb and Hg found in collected Ulva spp. by this and other European studies. N- and P-sequestering was higher in naturally occurring Ulva spp. at non-exposed sites than in Ulva sp. cultivated at an exposed site. Biomass yields through coastal cultivations were modest and therefore, the Ulva spp. was concluded to have the highest bioremediation potential through harvest during Green Tides allowing for large biomass yields and large nutrient removal. Furthermore, it was found, that the bioremediation potential of these metals was very low and as long Ulva spp. are not collected in areas influenced by harbours, industry and other sources of contamination, there is a high potential that maximum levels for toxic metals do not prevent Ulva spp. in food and feed products. 1/86

3 Resumé Strategier inden for bioremediering kan anvendes til at foretage oprensningsprocesser af miljøet, og en potentiel organisme til disse processer har vist sig at være den opportunistiske og hurtigt voksende grønalge Ulva spp. Anvendelsen af Ulva spp. i bioremediering har vist højt potentiale i at forbedre kvaliteten i marine miljøer, men uden handling kan Ulva spp. blive en gene under dannelsen af så kaldte Green Tides. Biomasseudbytterne, der kan komme af at udnytte bioremedieringspotentialet for Ulva spp., har mange anvendelsesmuligheder, f.eks. i medicinalindustrien, som gødning eller i foder- og fødevareindustrien. Formålet med dette studie er at evaluere potentialet af Ulva spp. i danske marine miljøer 1) for bioremediering af næringsstof og 2) for efterfølgende anvendelse af biomassen som foder eller fødevare. Derfor blev spore ekstraheret fra en Ulva sp., sået på nogle materialer og udsat til havs. Til sidst blev bioremedieringspotentialet vurderet for Ulva sp. kultiveret til havs og Ulva spp. samlet naturligt. Foder- og fødevaresikkerheden blev evalueret mht. giftige metalindhold (As, Cd, Pb og Hg) i vævet for dyrket og naturligt forekommende Ulva spp. fundet igennem dette studie og rapporteret fra lignende europæiske studier. N- og P-isolering var højere i naturligt forekommende Ulva spp. ved ikke-eksponerede steder i forhold til Ulva sp. dyrket et eksponeret sted. Biomasseudbytter igennem dyrkning til havs var beskedne og derfor kunne det konkluderes, at det største bioremedieringspotentiale for Ulva spp. hviler i muligheden for en omfattende høst i forbindelse med Green Tides, hvor fjernelsen af store mængder biomasse ligeledes fører til fjernelsen af meget næringsstof. Endvidere kunne det konkluderes, at Ulva spp. har et meget lavt bioremedieringspotentiale for de nævnte metaller, og så længe Ulva spp. ikke fremtræder i forurenede områder, så er der et højt potentiale for, at grænseværdierne for disse metaller ikke forhindrer anvendelsen i foder og fødevarer. 2/86

4 Table of Contents Abstract... 1 Resumé... 2 Preface... 7 Abbreviations... 8 Definitions... 9 Background Introduction Green Tides The Ulva blooms Impact and management of Green Tides Bioremediation potential of Ulva Controlled growth of Ulva Land-based and coastal cultivation of Ulva Chemical composition of Ulva Nutritional value of Ulva Ulva as a bioindicator Sources of metals in Ulva Seasonal and species dependent variation in Ulva metal content Priority pollutants Arsenic in Ulva Iodine in Ulva The potential for using Ulva in food or feed applications Project aim Materials and Methods Terms and classifications Collection of Ulva Thalli for induction of sporulation Thalli for biochemical characterisation of Danish Ulva Cultivation of Ulva Discs for sporulation Blended material for sporulation Seeding of spores Ribbon /86

5 2.4.2 Rope Growth of Ulva Laboratory Nursing and coastal cultivation Tissue analyses Dry matter content (DMC) Ash fraction and total P content C and N content Metal analyses Sample digestion and preparation Apparatus for metal analyses Data analysis Bioremediation and food and feed potential Assessment of Danish Green Tides Bioremediation capacity of macro nutrients and metals Food and feed safety regarding As, Cd, Pb and Hg Food and feed safety regarding ias Literature review on toxic metals in European Ulva Statistics Results Cultivation of Ulva Sporulation of Ulva Settling of spores Growth of seedlings Coastal cultivation Ulva tissue biochemistry Moisture content, dry matter and ash fraction Macro nutrients in Ulva: C, N and P Priority pollutants in Danish Ulva spp Priority pollutants in European Ulva spp Inorganic Arsenic Species dependent variation Bioremediation Coastal cultivation /86

6 3.3.2 Danish Green Tides Site dependent bioremediation capacity of Ulva Food and feed potential of Danish and European Ulva Arsenic Cadmium Lead Mercury Discussion Bioremediation potential of Ulva Preparation of lines for coastal cultivation of Ulva Growing Ulva on lines at sea Ulva tissue contents of macro nutrients Bioremediation potential of coastal cultivations of Ulva Bioremediation potential of Ulva related to Danish Green Tides Bioremediation capacity of Ulva for nutrients Food and feed potential of Ulva Arsenic Cadmium Lead Mercury Seasonal and species dependent variation in Ulva metal contents Iodine Future perspectives of Ulva for bioremediation food and feed Conclusion Ulva can be harvested from nature and cultivated on structures The species accumulate nutrients and metals Acknowledgements References Appendices Collection of Ulva Coastal cultivation at Grenaa (O) Kortlægning af søsalat (Ulva lactuca) i Mariager Fjord og Seden Strand /86

7 6.4 References for literature review Evaluation of methods (Results and Discussion) C, N measurements accuracy P analyses ICP-MS accuracy ICP-MS precision Comparison of Danish and European Ulva Personal activities and involvements connected to the field of topic Participation at the Seventh Nordic Seaweed Conference Participation in the annual Nature Science Festival at Kattegatcenteret Part time job at the project Hovedet i Havet Media coverage /86

8 Preface This study has been carried out as the final project (30 ECTS) for completing the education of a Master of Science in Engineering in Biotechnology at DTU. The work of the project has been conducted partly at the Technical University of Denmark (DTU) and partly at Aarhus University (AU). The motivation of this project comes from an interest in discovering the bioremediation potential of seaweeds and the aim of this study is to provide new knowledge on how Sea Lettuce can be used for an improvement of Danish marine waters while contributing to a database for future European regulation on toxic metals in seaweed for human and animal consumption. 7/86

9 Abbreviations Chemical elements and compounds (total) Arsenic Inorganic arsenic Arsenate Arsenite Organic arsenic Monomethylarsenic Dimethylarsenic Cadmium (total) Carbon Hydrogen chloride Lead Mercury (total) Nitrogen As ias As(V) As(III) oas MMAs DMAs Cd C HCl Pb Hg N Nitric acid HNO 3 (total) Phosphorous P Rhodium Rh Materials and methods Average AV counts per second cps Dry weight DW Dry matter content DMC Demineralised water DemW Inductively coupled plasma mass spectrum ICP-MS Internal standard solution ISTD Milli-Q water MQ-water Population N Sea water SW Standard error SE Sterilised sea water SSW Ulva spp./sp. Ulva Wet weight WW 8/86

10 Definitions A collection of definitions has been gathered for the terms used within the study (Table 1). Table 1. The terms used throughout the study and how they are interpreted based on definitions cited from other papers. Whenever definitions are pulled out of contents or pieces are missing for the relevance of this study, this information is added in italic in parenthesis as of: (e.g. [comment]). Term Definition Bioindicators: The core principle of using bioindicators is that they give a direct measure of the effect of pollution on the organisms. (Wan et al. 2017) (Bioindicators are [comment]) sessile or sedentary; tolerant to high levels of contaminants andwide ranges of salinity, permitting laboratory studies of the kinetics of contaminants; abundant within the study area, easy to identify and collect and should provide sufficient amounts of tissue for analysis; and there should be a simple correlation between the concentration of the contaminant in the tissues of the organism and the average concentration bioavailable in the environment. (Villares et al. 2001) Bioremediation: uses biological organisms under controlled conditions to degrade, neutralize, and/or remove harmful contaminants from a polluted site. (Neveux et al. 2017) Bioremediation capacity: The nutrient (and metal [comment]) remediation capacity of seaweeds is determined by the amount of nutrients (and metals [comment]) removed from the sea by harvesting the seaweed biomass. (Neveux et al. 2017) Gamete: Gametophyte: A haploid reproductive cell; gametes fuse in pairs to form zygotes, which are diploid. (Chaffey 2014) In plants that have an alternation of generations, the haploid (1N) gameteproducing generation, or phase. (Chaffey 2014) Green Tides: are caused by very large accumulations of green macro-algae that occur under suitable conditions, in particular eutrophication. (Gao et al. 2010) 9/86

11 Memory effect: Prebiotics: Priority pollutants: The memory effect due to the retention of analyte in the sample injection system or the torch will induce additional signal intensity of the subsequent sample or increase the detection limit. (Jin et al. 2003) Prebiotics are non-digestible food ingredients that selectively stimulate the growth and/or activity of one or a limited number of beneficial bacteria (probiotics) in the colon (Hayes & Tiwari 2015) priority pollutants (e.g. arsenic, cadmium, chromium, and lead) can be a risk to both ecosystems and human health. (Wan et al. 2017) In this study the term will specifically refer to those toxic metals covered by EU regulation on maximum levels, i.e. As, Cd, Pb and Hg (European Commission 2015, European Commission 2017). Spore: Sporophyte: A reproductive cell, usually unicellular, capable of developing into an adult without fusion with another cell. (Chaffey 2014) The spore-producing, diploid (2N) phase in a life cycle characterised by alternation of generations. (Chaffey 2014) Ulva (sp./spp.): Common name: (Sea Lettuce), Synonym: (Enteromorpha), Lineage: (Eukaryota; Viridiplantae; Chlorophyta; Ulvophyceae; Ulvales; Ulvaceae) (NCBI taxonomy 2017). Zoospores: A motile spore, found among algae, oomycetes, and chytrids. (Chaffey 2014) Zygote: The diploid (2N) cell resulting from the fusion of male and female gametes (Chaffey 2014) 10/86

12 Background A consequence of anthropogenic activity (agriculture, industry etc.) is a discharge of wastewater into the environment, which can be reflected by the water quality at coastal areas (Ohlendorf et al. 1988). The water quality can be improved by the implementation of bioremediation, which involves the use of biological organisms to clean up contaminated sites (Neveux et al. 2017). This study will target the use of the seaweed, Ulva spp., as a potential organism for bioremediation. The bioremediation potential will be evaluated on its ability to accumulate pollutants associated with anthropogenic and the potential for subsequent use of the biomass will be evaluated (Figure 1). Examining the bioremediation potential of Ulva spp. is of high interest since the species is considered opportunistic and fast growing and at some places even a nuisance (Ye et al. 2011, Bolton et al. 2016). Figure 1. Schematic representation of how seaweed bioremediation can act as a key link in circular management of water (blue) and resources (red) (Neveux et al. 2017). There are already many applications of seaweed and since at least BC 6000 seaweeds have been consumed by humans (Nisizawa et al. 1987). Today, Korea has the highest annual seaweed consumption globally of 8.5 kg per person (Hwang et al. 2017) and the demand for seaweed is extending globally (Besada et al. 2009, Tabarsa et al. 2012). Seaweeds already are used in European products, e.g. is the seaweed carbohydrate, carrageenan, commonly used as a thickening and stabilising agent in especially foods (Necas & Bartosikova 2013), e.g. Danish chocolate milk (Matilde Original Kakaomælk (Arla 2018)). Ulva spp. have been reported commonly used in salads and in soups (Nisizawa et al. 1987, Tabarsa et al. 2012). Thus, edible seaweed already is easily available for human consumption, but the European legislation does not yet cover maximum levels for toxic metals in seaweed. Hopefully, this study can contribute with more data on some toxic metals in Ulva spp. to support the development of the European regulation on maximum levels for these elements in seaweed for food and feed. 11/86

13 1 Introduction 1.1 Green Tides Worldwide, a concern is growing to the blooms of green opportunistic macroalgae forming the so called Green Tides occurring at coastal areas (Ye et al. 2011). One of the most intensive events of Green Tides reports approximately 20 million tons algal biomass in wet weight (WW) covering more than 3500 km 2 off the coast in the Yellow Sea in China, 2008 (Gao et al. 2010) and in 2013 the green tide was estimated to spread over twice the area as for in 2008 (Jacobs 2013). While the impact of Green Tides appears to be highest along the coast of the Yellow Sea (Smetacek & Zingone 2013), countries all over the world have reported invasions of the green macroalgae blooms (Figure 2) (Wkhlu et al. 2016, Wan et al. 2017, Charlier et al. 2008), and in most of the cases the phenomenon comes down to one dominant genus of macroalgae; Ulva (Smetacek & Zingone 2013, Ye et al. 2011, Wan et al. 2017). Figure 2. Coastal sites around the world with frequent Green Tides reported (Ye et al. 2011) The Ulva blooms Ulva has been described as a weed in sheltered, eutrophic coastal areas where they tend to form Green Tides (Bolton et al. 2016). Species of Ulva are commonly known as Sea Lettuce, which on higher order belongs to the family Ulvaceae of the phylum Chlorophyta, green algae (NCBI taxonomy 2017). The genus Ulva is characterised as green seaweeds, which are either tubular or have foliose blades. The species with tubular morphology have previously been known under the genus name Enteromorpha but as for the species of the genus Chloropelta (Tanner 1980), the genus was reduced to synonymy with Ulva (Hayden et al. 2003). 12/86

14 The morphology of Ulva spp. is simple but taxonomically they can be very hard to distinguish (Heesch et al. 2009, Wan et al. 2017). Furthermore, morphological features of Ulva have been found to depend on the salinity and the nutrient concentrations (Messyasz & Rybak 2011). Thus, genetic analyses are needed in many cases for correct classification of Ulva spp. (Heesch et al. 2009, Wan et al. 2017). Hence, the names of Ulva species used in the literature are often dubious (Bolton et al. 2016), and all species of Ulva will therefore be reported generically as Ulva throughout this report, unless very specific cases are considered, e.g. crucial factors governing growth (section 1.2.2). Increased concentrations of nitrates in freshwater can ameliorate the impacts of low salinity on Ulva, thus allowing them to thrive in freshwater, estuaries, sewage sources and brackish water as well as in coastal seas (Ichihara et al. 2013, Kamer & Fong 2001). The persistent Ulva blooms will occur at places, at which the species are under favourable environmental conditions, i.e. eutrophic water with elevated temperature and irradiance (Bo et al. 2012, Gao et al. 2017a, Kamer & Fong 2001). Marine ecosystems in Europe are under pressure from eutrophication (Hering et al. 2010) and with increasing water temperatures as a result from global warming an increase in future Green Tides is expected (Wan et al. 2017, Smetacek & Zingone 2013, Gao et al. 2017a) Impact and management of Green Tides The Green Tides carry large economical and ecological consequences. Economical losses can be a consequence of removing and disposing Ulva blooms smothering shorelines and of negative effects on tourism (Charlier et al. 2012, Smetacek & Zingone 2013). Ecological consequences are first of all reflected through the high competition of dense Ulva mats towards other organisms living in the littoral zone (Messyasz & Rybak 2011, Quillien et al. 2015, Li et al. 2017), e.g. slow growing algae like Fucus vesiculosus are hampered due to shading from the fast growing Ulva (Pedersen & Borum 1997). Eventually, the natural decomposition of Ulva biomass can cause a release of noxious sulphuric gasses (Nedergaard et al. 2002, Wang et al. 2011) posing a threat to the health of humans, animals and the local marine ecosystem (Malone Rubright et al. 2017, Reiffenstein et al. 1992). The death of two people and several animals has already been linked to toxic fumes from Ulva morasses at the Brittany coast in France (Bevan & Walker 2017, BBC 2009). Thus, for the sake of the ecosystems, recreational value and human health at areas under impact of Green Tides, it must be understood how to reduce the occurrences of Green Tides and how to utilise removed biomass. At the Brittany coast in France 100,000 m 3 of Ulva biomass is harvested annually, but so far the high expenses of the removal are only justified by the environmental improvement (Charlier et al. 2008). The removed biomass can be applied for biofuel, pharmaceuticals, human consumption and as a fertilizer 13/86

15 or an animal feed (Holdt & Kraan 2011, Ye et al. 2011), which will be clarified later, as it is the aim of this report to further investigate the benefits of Ulva biomass removal and its potential for food and feed. 1.2 Bioremediation potential of Ulva The bioremediation potential of seaweed is dependent on two factors: (1) the bioremediation capacity, i.e. the amount of contaminants (e.g. nutrients and metals) removed when harvesting the seaweed biomass and (2) the areal biomass production, i.e. how much biomass can be harvested per area per day (Neveux et al. 2017). Thus, an organism with high potential for bioremediation can accumulate large amounts of contaminants and can be harvested in a big scale. Recent reviews have covered the bioremediation potential of Ulva (Bolton et al. 2016, Neveux et al. 2017). Overall, seaweed farming has potential for climate change mitigation (e.g. by nutrient sequestration and methane emission reduction) and adaption (e.g. increasing ph at sites impacted by ocean acidification and production of oxygen, thus, reducing oxygen depletion) (Duarte et al. 2017). The particular potential of Ulva lies e.g. within its efficiency in nutrient uptake (particularly ammonium) and its cultivation efficiency in closed systems (Neveux et al. 2017, Lamprianidou et al. 2015). Ulva harvesting in closed systems can be done simply with nets and thus, is much simpler in comparison to microalgae harvests, and the potential production levels of Ulva have even been reported comparable to those of microalgae (Bolton et al. 2016). A challenge for Ulva seems to be the cultivation at sea (Castelar et al. 2014), but for Ulva and as well for other seaweeds, the life history must be controlled for a commercial viable production (Bolton et al. 2009) Controlled growth of Ulva The reproductive events of Ulva depend on two life stages, which includes gametophytes (1N) and sporophytes (2N) characterised by a similar morphology (Nordby & Hoxmark 1972). One of the differences between the two life stages is that sporophytes release zoospores (1N) that grow into isomorphic gametophytes, which release gametes that may unite with gametes of the opposite sex to produce a zygote (2N) that will grow into a sporophyte. If gametes are not united, then there will be no sexual reproduction and they will simply grow into a gametophyte (Figure 3) (Nordby & Hoxmark 1972, Chaffey 2014). 14/86

16 Figure 3. Ulva exits in two isomers, i.e. sporophytes (2N) and gametophytes (1N), which are distinguished between their reproductive events. Sporophytes produce zoospores (1N) and gametophytes produce gametes (1N), which potentially fuse into zygotes (2N) (Rocktopus 2017). Sporulation of Ulva repeats weekly during the summer, forth weekly during the winter and spore release is specially high during spring (Lüning et al. 2008). It has been suggested that sporulation is controlled by two sporulation inhibitors, SI-I and SI-II, at which the first inhibits spore production (gametogenesis) and the latter promotes vegetative growth (Stratmann et al. 1996, Nordby & Hoxmark 1972). In the Thermaikos Gulf, Ulva population densities reached minimum values during summer and early fall (July September /October) and in early fall new individuals appeared, which would then reach maximum population densities in spring (April) (Malea & Haritonidis 1999). This has been explained by Ulva sporulating away during the summer and autumn leaving only the basal zone of the thalli for vegetative growth during spring (Stratmann et al. 1996, Lüning et al. 2008). It has been argued, that Ulva thalli are buried during winter and in spring these thalli are liberated initiating a bloom that cause a rapid increase in Ulva biomass (Lentz 1998, Schories 1995). The understanding of the reproductive events and the seasonality of Ulva is crucial to the management of Green Tides (Gao et al. 2017b, Vesty et al. 2015) and controlled growth of Ulva for land-based and coastal 15/86

17 systems (Carl et al. 2016a, Praeger & de Nys 2017). The reproductive events of Ulva can cause biomass to be lost as spores but it can also allow for seeding of Ulva onto substrates (Carl et al. 2014, Castelar et al. 2014). Ulva spores settle on substrates by adhesion, at which the flagella are discharged (Callow & Callow 2006). The settling success has been found to increase with temperatures up to a maximum of 23⁰C (Christie & Shaw 1968, Gao et al. 2017a). Seeding experiments on bioballs showed that relatively high seeding densities over time did not affect the biomass yield nor the specific growth rates (Praeger & de Nys 2017) Land-based and coastal cultivation of Ulva Crucial factors governing growth of Ulva is nutrient availability, light and temperature. The minimum requirement of light for U. lactuca is 2.5 μmol m ² s ¹ (Sand-Jensen 1988) and optimal growth for U. flexusa Wulfen has been observed to be approximately 80 μmol m ² s ¹ (Imchen 2012). Optimal growth rates of Ulva range between 10⁰C and 20⁰C (Kalita & Tytlianov 2003, Mohsen et al. 1973) but too high temperatures as well as too high light intensities can decrease the nutritional value of the biomass (Mohsen et al. 1974, Mohsen et al. 1973). Temperatures of 5⁰C have demonstrated to be the most favourable for maintaining U. fenestra at vegetative growth (Kalita & Tytlianov 2003) and an increase in temperature has been reported to lead to an increase in germination and spore release, consequently resulting in a loss of Ulva biomass (Gao et al. 2017a). High salinities (20-35 ) have been reported to promote germination and growth of Ulva (Sousa et al. 2007, Martins et al. 1999, Jie et al. 2016) and finally, the nutrient availability has been described to be positively correlated with growth (Imchen 2012). Growth of Ulva has been found to be higher when grown in tanks rather than at sea, which has been explained by lack of competition in batch cultivations together with higher nutrient availability (Castelar et al. 2014). There are many advantages of land-based cultivations of Ulva. It can be integrated in land-based aquaculture to re-use nutrients in effluent water (Lawton et al. 2013, Bolton et al. 2009), it can be grown with manure as a nutrient source (Nielsen et al. 2012) and controlled conditions have demonstrated high growth rates, e.g. 600% per week (Israel et al. 1995) or g DW m -2 d -1 (Msuya & Neori 2008, Cohen & Neori 1991) and high total inorganic nitrogen (TIN) and total ammonium nitrogen (TAN) removal rates, e.g. 1.4 g TAN m -2 d -1 and 2.16 g m -2 d -1, respectively (Ben-Ari et al. 2014). Ulva has been found to have a superior ability for ammonium removal compared to biofilm (Cahill et al. 2010) but has been reported less efficient in phosphor (P) removal (Cohen & Neori 1991, Nielsen et al. 2012). Nevertheless, growth and nutrient removal in aquaculture systems can be optimised depending on flow-, aeration- and water exchange rates (Neveux et al. 2017). A study reports that only from abalone effluents, 1 to 1.5 tons biomass WW can be produced monthly in 30 m long paddle-raceways (Bolton et al. 2016). Even though the current biomass 16/86

18 yields at sea are not yet compatible to land-based systems, one way of optimising coastal cultivation of Ulva would be by using nets, thus, upscaling the cultivation surface (Castelar et al. 2014). 1.3 Chemical composition of Ulva Nutritional value of Ulva A high part of Ulva consist of water, namely 78-80% (Lamare & Wing 2001, Marsham et al. 2007), and the ash fraction has been found to vary between 11-52% of dry weight (DW) (Ortiz et al. 2006, Foster & Hodgson 1998) (Table 2). Most studies of naturally collected Ulva agree that Ulva has a very low total lipid content, % of DW (Wong & Cheung 2000, Marsham et al. 2007, Ortiz et al. 2006, Foster & Hodgson 1998), but Ulva has also been found with total lipid contents up to 8% of DW, which has been suggested to be attributable to genetic and environmental variation (Yaich et al. 2011, Maehre et al. 2014). It has been estimated that Ulva has carbohydrate contents of 15-62% of DW (Wong & Cheung 2000, Ortiz et al. 2006), which mainly consist of dietary fibre (Carl et al. 2016b), i.e % of DW (Rasyid 2017, Ortiz et al. 2006). Protein concentrations and total phosphorous (P) contents are in the range of 6-29% of DW (Foster & Hodgson 1998, Marsham et al. 2007, Smith et al. 2017) and % of DW (Rasyid 2017, Maehre et al. 2014), respectively. Finally, nitrogen (N) contents have been found in the range % of DW (Kamer et al. 2004, Van Alstyne 2016). Protein and N contents have been reported to be even higher in Ulva from tank cultivations, e.g. reaching contents of 44.3% N of DW and 5.9% protein of DW, respectively (Nielsen et al. 2012, Msuya & Neori 2008). Table 2. Overall nutritional value of naturally collected Ulva. Component Value Moisture 78 a -80 b % of WW Ash 11 c -52 d % of DW Carbohydrate 15 e -62 c % of DW Dietary fibre 14 f -61 c % of DW Total lipid 0.3 c,d -8 g,h % of DW Protein 6 d -29 b % of DW P 0.05 f -5 g % of DW N 1.2 i -3.2 j % of DW a (Lamare & Wing 2001), b (Marsham et al. 2007), c (Ortiz et al. 2006), d (Foster & Hodgson 1998), e (Wong & Cheung 2000), f (Rasyid 2017), g (Maehre et al. 2014), h (Yaich et al. 2011), i (Kamer et al. 2004), j (Van Alstyne 2016). Abbreviations: dry weight (DW), wet weight (WW). 17/86

19 High contents of carbohydrates in Ulva give high potential for biofuel production (Ghadiryanfar et al. 2016, Milledge & Nielsen 2016) and high levels of dietary fibre together with other bioactive components make Ulva a good source of prebiotics (Filomena et al. 2016, Lawlor et al. 2010). Prebiotics endorse the growth of favourable bacteria in the gut system (Holdt & Kraan 2011), which can help preventing human and animal diseases (Filomena et al. 2016). Ulvans are among the most studied polysaccharides in Ulva (Hayes & Tiwari 2015) and do not only feature prebiotic effects but also represent very potent bioactive compounds reported to have anti-influenza properties, potential for treatment of gastric ulcers, hepatoprotective effects and antitumor capacities, etc. (Holdt & Kraan 2011, De Jesus Raposo et al. 2015, Hardouin et al. 2016). Bacterial strains with probiotic and anti-inflammatory effects have also been isolated from Ulva tissue (Ramasany S. y Kumar 2009). Furthermore, Ulva has been found to be a good source of antioxidants and vitamins, of which they are specially rich on vitamin A, C, E and B vitamins, except for vitamin B 3 and B 6 (Taboada et al. 2010, Ortiz et al. 2006, Nunes et al. 2017). The same studies also endorse Ulva as a good source of minerals, but toxic metals have also been reported to accumulate in biomass tissue (Boubonari et al. 2008, Ho 1990, El-din & Mohamedein 2014) (section 1.3.5) Ulva as a bioindicator For Ulva to have a potential in food and animal feed applications, the nutritional value must be assessed together with the content of harmful metals, e.g. As, Cd, Pb and Hg. High contents of harmful metals can compromise the use of Ulva for food or feed. Unfortunately for this matter, the use of Ulva as a bioindicator has been reported. Bioindicators are various types of biota, which are used to reflect the effect of environmental pollution and are defined by exhibiting a significant correlation between contaminants, e.g. toxic metals, in water and sediments and those in an ubiquity organism (Wan et al. 2017, Sawidis et al. 2001, Conti & Cecchetti 2003, Villares et al. 2001). Several studies report the use of Ulva as a bioindicator, e.g. for contamination with Pb in marine systems (Favero & Frigo 2002, Leal et al. 1997, Malea & Haritonidis 2000) or Ni in freshwater and marine systems (Rybak et al. 2012b). Although Ulva might not be an ideal bioindicator for all metals, it has repeatedly been reported to contain elevated contents of trace elements compared to other macroalgae as well as to similar species at distinct locations. The frequent reported elevated contents of elements in Ulva are Cd, Co, Cr, Cu, Fe, Hg, Mn and Zn (Ferreira 1991, Leal et al. 1997, Malea & Haritonidis 2000, Ho 1990, Laib & Leghouchi 2012, Ustunada et al. 2011, Abdallah & Abdallah 2008). However, the highly toxic priority pollutants i.e. As (and ias), Hg, Cd and even Pb have commonly been found in Ulva at concentrations, which could cause a potential health risk to humans if consumed (Phaneuf et al. 1999, Malea & Haritonidis 2000, Pell et al. 2013). 18/86

20 1.3.3 Sources of metals in Ulva There are several indications of when to expect metal contents to be elevated in Ulva. Studies along the Bulgarian Black Sea coast reported that serious contamination of Ulva tissue could be traced to areas with direct human impact (Strezov & Nonova 2005). Contamination sources such as industry waste sewers, shipyards, harbours and high ship traffic intensity were reported to influences the contents of Cd, Cu, Pb, Fe and Zn in Ulva (Malea & Haritonidis 1999, Ustunada et al. 2011, Rodr & Ignacio 2006, Gaudry et al. 2007, Charlier et al. 2012). Other cases included harbour docks with chromated cobber arsenate wood, where accumulations of Cr, Cu and As was found (Weis & Weis 1992) and industrial or urban discharges resulting in high tissue Hg concentrations (Coelho et al. 2009, Diop & Amara 2016, Zhang & Wong 2007). Furthermore, agricultural nutrient and metal effluents (Ohlendorf et al. 1988) have been suggested to influence the Ulva biochemistry (Wan et al. 2017, Diop & Amara 2016, Maehre et al. 2014). Hence, the already listed metals As, Cd, Cr, Cu, Fe, Hg, Pb, and Zn and also Mn are commonly associated with anthropogenic activity (Ohlendorf et al. 1988) but in many cases these pollutants can also occur in Ulva from geogenic sources (Rodr & Ignacio 2006, Wan et al. 2017), e.g. Ulva collected at a rocky shore relatively away from mining areas, industrial sites and harbours has been reported with elevated Cd contents (Schintu et al. 2010). In the rare case of Ulva occurring in inland freshwater systems with very low salinity, thalli have been found to accumulate Ca, Mg, Ni and Cd to a much higher extend than in marine systems (Rybak et al. 2012a). Similar studies have also confirmed, that there is a correlation between high concentrations of Fe, Zn and Cd and low salinity, which has been argued to be a cause of the inverse relationship between ion activity and salinity (Favero et al. 1996) Seasonal and species dependent variation in Ulva metal content Not only is the elemental composition of Ulva site dependent, it is also claimed to follow seasonal patterns (Schintu et al. 2010, Malea et al. 2015, Haritonidis & Malea 1999, Favero et al. 1996) Trace elements in Ulva are generally at a minimum during summer and at a maximum at autumn/winter, which has been explained by a dilution effect associated with growth dynamics. The leading argument is that an increase in Ulva biomass will lead to a decrease in its metal concentration (Villares et al. 2002, Haritonidis & Malea 1999). Finally, the metal content seems to depend on the specific species of Ulva, which is generally distinguished between filamentous or tubular species (previously known under the genus Enteromorpha) and foliose species, where the foliose species are reported to have a lower metal uptake (Trifan & Chimie 2015, Malea & Haritonidis 2000). 19/86

21 1.3.5 Priority pollutants Despite the many elevated elemental concentrations reported in Ulva, the EU-regulation for harmful metals in seaweed covers only few of the most toxic metals, i.e. As, Cd, Pb and Hg, which will be referred to as priority pollutants (European Commission 2015, European Commission 2017). No regulation has yet been made specifically for maximum levels for As, Pb and Hg in seaweed for human consumption, but Cd contents in food supplements may not exceed 3 mg kg -1 DW -1 (European Commission 2017). For feed materials derived from seaweed a maximum level for total arsenic (As) and inorganic arsenic (ias) has been set at 40 and 2 mg kg -1, respectively (European Commission 2015). Thus, the only legislation for priority pollutants in seaweeds is Cd in food supplements and As and ias in feed materials. Nevertheless, an overview has been made of the legislation for maximum levels for priority pollutants in somehow similar food or feed materials, which will be used to evaluate the food and feed safety of Ulva (Table 3). Table 3. European maximum levels for priority pollutants in food and feed (European Commission 2015, European Commission 2017). Metals food (mg kg -1 WW -1 ) feed (mg kg -1 relative to a moisture content of 12%) ias a <2 b As No level defined 40 b Cd 3 c 0.5 d Pb e 5-10 d Hg f g a Different rice products, b feed materials derived from seaweed, c food supplements from dried seaweed (mg kg -1 DW -1 ), d complementary or complete feed, e leaf vegetables or food supplements, f fishery products, g any feed material or fishery products, abbreviations: wet weight (WW). Bolt represents regulation for pollutants in seaweed Arsenic in Ulva When considering As it is distinguished between organic arsenic (oas) and ias, i.e. the sum of arsenate (As(V)) and arsenite (As(III)), of which As(V) dominates under aerobic conditions and As(III) under anaerobic conditions (Jiang et al. 2009). Both mentioned species of ias are extremely potent carcinogens to humans (Lin et al. 2002, Lièvremont et al. 2009, Hirata & Toshimitsu 2007) and the maximum levels for ias in food and feed products are therefore much lower than for As (European Commission 2015, European Commission 2017). Organic As compounds are generally considered non-toxic except for trivalent mono- 20/86

22 and dimethylated arsenic compounds, i.e. monomethylarsenite (MMAs(III)) and dimethylarsenite (DMAs(III)) (Petrick et al. 2000, Petrick et al. 2001). Nevertheless, these compounds are considered unstable over longer time (Segura et al. 2002). Exposure of As in edible seaweed has been analysed in context to cooking in boiling water for 5 min (Sartal et al. 2012). Out of four studied seaweeds, Kombu, Wakame, Nori and Ulva it was found that short cooking released up to 71% of the As to the cooking water (Figure 4). Other studies report of edible seaweeds with contents of priority pollutants that should preclude their consumption for humans and animals and therefore legislation is needed for maximum levels to evaluate the safety of these products being on the market (Besada et al. 2009). Studies have also reported on sheep exposure of arsenic through a complete seaweed diet. It was estimated that the sheep consumed 2-4 kg seaweed each day, which equalled to a daily As intake of approximately mg. It was concluded that As did not accumulate in concerning amounts in the sheep (Feldmann et al. 2000). Figure 4. As contents in four seaweeds including Ulva (Sea Lettuce) before and after cooking and the As contents in the cooking water and in dialysates after in-vitro gastrointestinal digestion of cooked seaweed (Sartal et al. 2012). 21/86

23 1.3.7 Iodine in Ulva Iodine concentrations in Ulva have been reported in a range of mg kg -1 DW -1 (Hou et al. 1997, Hortas et al. 2011, Desideri et al. 2016, Mairh et al. 1989). Brown seaweeds are known to have even higher iodine contents, examples are Laminaria digitata with 7316 mg kg -1 DW -1 (Desideri et al. 2016), L. jamonica with 3040 mg kg -1 DW -1 (Hou et al. 1997) and Saccharina latissima with 6138 mg kg -1 DW -1 (Hortas et al. 2011). Iodine is essential to humans and iodine deficiency is known to cause severe goitre and has been reported a public health problem in developing countries as well as in Denmark (Bimenya et al. 2002, Thilly et al. 1992, Ministry of Environment and Food of Denmark 2017a). Therefore, it has been common to add iodine to salt and in Denmark the recommended daily intake of iodine for adults is 150 µg with a recommended upper level for the daily intake of 600 µg (Ministry of Environment and Food of Denmark 2017a). Assuming Ulva contains around 25 mg Iodine kg -1 DW -1, this will allow an adult to consume approximately 6 g of dried Ulva to meet the recommended daily intake of iodine and 24 g of dried Ulva to reach the maximum daily intake. 1.4 The potential for using Ulva in food or feed applications In the eastern Asian kitchen, it is already very common to use Ulva and other seaweeds for cooking (Nisizawa et al. 1987, Hwang et al. 2017) and the trend is slowly expanding to the rest of the world (Besada et al. 2009, Tabarsa et al. 2012). Many benefits of using Ulva for human consumption have already been mentioned (section 1.3.1) and effects of using Ulva as an animal feed have also been studied (Michalak & Chojnacka 2009, Naidoo et al. 2006, Maia et al. 2016, Machado et al. 2014). A study on sheep reported that Ulva supplementation had no positive effect on several factors, e.g. feed conversion, gas production, estimated energy etc. The study concluded that higher Ulva contents of the diet could potentially give more positive results (El-Waziry et al. 2015). Nevertheless, this has not been proven the case for the use of Ulva as a complete feed for goats, where Ulva was found to resemble medium quality hay (Ventura & Castañón 1998). Nor with geese has a complete diet of Ulva shown any detectable effects (Rowcliffe et al. 2001). Feed with different contents of raw Ulva was given to poultry, which showed that higher Ulva contents resulted in a reduction in poultry growth rates (Ventura et al. 1994). Thus, using Ulva as a complete feed does not appear to be an optimal solution, but positive results have been obtained by using Ulva as micro-elemental feed supplement in the diet for livestock, e.g. laying hens and their eggs increased in weight through the inclusion of Ulva in their diet (Michalak & Chojnacka 2009). 22/86

24 Fresh Ulva in a mixed diet has also been found to produce increased growth in abalone (Naidoo et al. 2006) and Ulva can as well be used as supplementary feed for e.g. shrimps, European seabass and rainbow trout (Pallaoro et al. 2016, Wassef et al. 2013, Cruz-SuÁrez et al. 2009, Güroy et al. 2013). Finally, studies suggest that Ulva potentially has a more significant influence on microbial communities compared to whey (Jung et al. 2016) and that inclusion of Ulva in feed can reduce methane production in rumen fluids (Machado et al. 2014, Maia et al. 2016). 1.5 Project aim The aims of this study were: 1) to examine the potential of U. lactuca for bioremediation of nutrients in coastal ecosystems, by either harvesting natural populations of Ulva or by cultivating Ulva in coastal waters; and 2) to evaluate the quality of the biomass for subsequent use as food or feed. For Ulva to be implemented in bioremediation strategies two hypothesis must be fulfilled: (H1) the species can be harvested from nature and/or cultivated on structures (H2) the species biomass accumulates nutrients and metals Thus, the bioremediation potential of Ulva will be evaluated on its potential for successive growth and its nutrient and metal uptake, which will be drawn in perspective to food or feed safety. 23/86

25 2 Materials and Methods 2.1 Terms and classifications The target organism of this study is Ulva sp. All values will be represented as AV±SE unless anything else is annotated. Study sites will be categorised in three: Harbour/Industry, Open sea and Protected sites. The first category includes all sites placed in or close to a harbour or an industrial source. Sites with no such influence that are connected to open water are referred to as Open sea and the term Protected sites covers fiords, lagoons, broads etc., i.e. non-exposed sites (Table 4). Table 4. The extend of the terms used to categorise the study sites of this study. Each categorised site will be abbreviated by the initial letter, i.e. (H), (P) and (O), when put in context to sample locations. Harbour/industry Protected sites Open sea Definition Any site that could be affected by a harbour or an industrial source. Non-exposed sites that do not have a rapid water exchange with the sea. Exposed sites that are fully connected to the sea. Examples In or close to a harbour or near by a sewage outlet. Fiords, lagoons, broads and bays with low water exchange. Bays with rapid water exchange and cultivations at open sea. 2.2 Collection of Ulva Thalli of Ulva was throughout this study collected for two purposess: Induction of sporulation (section 2.2.1) and for analysis of C-, N-, P- and metal content (section 2.2.2) Thalli for induction of sporulation Whole and healthy-looking thalli, i.e. not fragmented or grassed, from a single population of U. lactuca was collected by hand on the 9 th of August 2017 in depths of app. 0.5 m in the lagoon at Kattegatcenteret, Grenaa, Denmark ( N, E). Thalli were during midday collected in a sealed bucket containing seawater (SW) from the site and transported within 2 hours to 10⁰C where they were kept in the bucket overnight prior to induction of sporulation (section 2.3) Thalli for biochemical characterisation of Danish Ulva Natural Danish populations of Ulva were collected by hand in depths of m at ten stations, and at two stations, Grenaa (GreO) and Hjarnø (HjaO), Ulva was cultivated on rope (Table 5, Figure 5). Samples from Fredericia (FreH), Aarhus Harbour (AarH), Horsens Fiord (HorH) and Vejle Harbour (VejH) were sampled close to or in a harbour or an industrial area. Three samples represented Open water, i.e. the site at 24/86

26 Aarhus Beach (O) and harvested biomass from the coastal cultivation in Kattegat close to Grenaa (Grenaa (O)). Thalli collection from these sites were all sampled in triplicates. Ulva cultivated at Hjarnø (O) was not included in tissue biochemistry analyses but only used for examination of the growth potential. The sites: Mariager Fiord (MarP), Halkær Broad (HalP), Seden Beach (SedP) and Norsminde Fiord (NorP) were all categorised as protected sites and represented in replicates of 9-10 except for the latter (NorP), at which only one sample was collected. Sampling at all sites was done during September 2017 except for samples from Grenaa (O), where sampling was done during July The sampling procedure was to collect biomass of g in a net, which was swung in circles (r= 0.5 m) ten times over ten seconds. Subsequently, the biomass was transferred to a sealed plastic bag, which later would be transferred to storage at -18⁰C. No extensive washing was done even though sand or dirt in some cases was present on thalli. Sand and dirt was removed in later steps (section 2.6.1) For all samples from protected sites, surface and bottom temperatures and oxygen levels were measured together with salinity and macroalgae cover. At these sampling points biomass densities per m 2 were assessed by measuring the weight of all Ulva within an area of 0.25 m 2 (appendix 6.1). Samples from Fredericia (H), Aarhus (H), Aarhus Beach (O) and Grenaa (O) were of young thalli and samples from Vejle (H), Horsens (H), and all the protected sites were of old thalli and thalli from Norsminde Fiord (P) were decomposing. Table 5. The geographical locations of the study sampling stations. Station Sample site Sample time Site description Coordinates ( N; E) a N b FreH Fredericia 22-Sep-17 Harbour/Industry 55 33'11, 9 43'48 3 VejH Vejle harbour 22-Sep-17 Harbour/Industry 55 42'24, 9 32'36 3 HorH Horsens Fiord 22-Sep-17 Harbour/Industry 55 51'12, 9 52'09 3 AarH Aarhus harbour 22-Sep-17 Harbour/Industry 56 10'02, 10 13'44 3 AarO Aarhus beach 22-Sep-17 Open sea 56 07'02, 10 13'43 3 GreO Grenaa, Kattegat 5-Jul-17 Open sea 56 32'33, 10 59'53 3 MarP Mariager Fiord 04-Sep-2017 Protected sites 56 41'54, 10 11'52 10 HalP Halkær Broad 22-Sep-2017 Protected sites 56 56'57, 9 33'32 10 SedP Seden Beach 19-Sep-2017 Protected sites 55 26'53, 10 26'01 9 NorP Norsminde Fiord 07-Sep-2017 Protected sites 56 00'43, 10 13'57 1 a DATUM: WGS84, b Number of collected samples 25/86

27 Figure 5. The Ulva sampling sites of this study and sites for coastal cultivation (Grenaa (GreO), Hjarnø (HjaO) and Kerteminde (KerO)), map from Simplemaps (2017). Pictures: Esben Rimi Christiansen. 2.3 Cultivation of Ulva On the day after collection, Ulva was prepared for induction of sporulation, first by a rough washing in SW using hands to clean thalli followed by an extensive washing in demineralised water (DemW). Washed Ulva was then incubated either as discs from thalli (section 2.3.1) or blended thalli (section 2.3.2). Incubation was done in 1 L beakers containing 1 L of f/2 medium (Guillard & Ryther 1962, Guillard 1975) prepared from sterile seawater (SSW) (sterilised by tyndallisation (Aminot & Kérouel 1997)) and the growth 26/86

28 conditions were 10⁰C, 50 µmol photons m -2 s -1, 12:12 hour light:dark cycles, salinity 23 and aeration. Aeration was added equally to all beakers to ensure water movement of discs and blended material. From day five and on every morning the number of released spores were counted by microscopy (40x, LEICA DM LB). On day seven, the medium was changed into fresh f/2 medium and on day eight spores were collected for seeding (section 2.4) Discs for sporulation Two incubations were made with 9 mm diameter discs cut from the marginal apical zone of 20 individuals of Ulva (Lüning et al. 2008). The WW of the 20 discs was 0.6 g (Figure 6) Blended material for sporulation The same 20 individuals of Ulva from section were cut in half for a duplicate of the incubation. Thalli were blended in 500 ml SSW using a hand blender (Braun, Control plus). Fragments <1 mm were discarded by pouring the blended material through a 1 mm filter. Fragments were finally in the size-range of 1-3 mm. After a final washing step using 500 ml DemW incubation of the blended material, 18 g WW per replicate, was initiated (Figure 6). Figure 6. Overview of preparation for induction of sporulation with discs and blended thalli. Pictures of Ulva: Esben Rimi Christiansen. 27/86

29 2.4 Seeding of spores Seeding was done with (1.1±0.1, N=6) 10 5 spores ml -1 (AV±SE, N) from discs (section 2.3.1) and with (1.3±0.1, N=6) 10 5 spores ml -1 from the blended thalli (section 2.3.2). Discs and blended material was filtered and removed from the media using a 1 ml filter followed by a 63 µm filter. One litre of spore containing media from each incubation was used for seeding of spores onto two materials, ribbon (section 2.4.1) and rope (section 2.4.2). While spores had not been seeded, medium was kept on ice to prevent settling of spores (Gao et al. 2017c). The density of settled spores was assessed after two and three weeks of incubation. High densities were simply annotated as 20 seedlings per mm 2 making an underestimate of the real density Ribbon Two times 3 m ribbons (width=5 cm, Algaetex, SION Industries) was drawn through 0.5 L of each of the two spore extracts, section and section The procedure was repeated until complete soaking of media and the ribbons were coiled and left in sealed plastic bags for two hours in darkness at 10⁰C allowing for settling of spores (Callow et al. 1997) Rope Another 0.5 L from each spore extract, section and section 2.3.2, was poured over 30 m coiled nylon rope (d=4 mm, Algaetex, SION Industries) in a 2 L container, which was filled with artificial seawater (ASW) (salinity 20 ), thus giving a concentration of approximately spores ml -1 for both spore extracts. Like for ribbons, the ropes were left in darkness for 2 h at 10⁰C (Callow et al. 1997). 2.5 Growth of Ulva Ulva seeded on ribbons and ropes was grown under controlled conditions in the laboratory and deployed at coastal sites. Before coastal deployment, the seeded material was nursed under controlled conditions Laboratory Thirty centimetres of rope and 3 cm ribbon from section 2.3 were transferred to 0.3 L beakers containing 0.2 L of f/2 medium prepared from artificial seawater (ASW), at which triplicates would be cultivated under three conditions: (1) 18⁰C without movement and 10⁰C (2) with and (3) without movement (120 rpm by a shaking table (Laboshake, Gernhard)). Light and salinity were constant factors for all three conditions, i.e. 50 µmol photons m -2 s -1, 12:12 hour light:dark cycles and a salinity of 2 (Table 6). 28/86

30 Table 6. The experimental setup for growth of seedlings. 18⁰C 10⁰C 10⁰C Spores from Seeded on (Warm+Still) (Cold + Still) (Cold + Movement) Ribbon - CSA(D) (1-3) CMA(D) (1-3) Discs Rope - - CMB(D) (1-3) Ribbon WSA (1-3) CSA (1-3) CMA (1-3) Blended thalli Rope - - CMB (1-3) C/W: Cold/Warm, M/S: Movement/Still, A/B: Ribbon/Rope, (D): Spores from disc incubation. Seedling densities were measured after 2 and 3 weeks by microscopy (10x, LEICA MZ 125). A random area of 5mm 2 on the substrate was identified and the number of seedlings was counted. Seedling lengths were measured weekly from week 3 to 6 by microscopy (10x, LEICA MZ 125) connected to a camera (NIKON, DS- Fi1) using the program (NIS-Elements D 3.2) by choosing only the longest ten individuals within a randomly selected area of 5mm 2 (Figure 7). Figure 7. Length measurements of Ulva seedlings within a square of 5 mm Nursing and coastal cultivation Cultivation at Grenaa (O) was conducted by Teis Boderskov (appendix 6.2) and coastal deployment at Hjarnø (O) and Kerteminde (O) has been done throughout this study as follows. By the end of the laboratory experiment (section 2.5.1) all substrates were deployed at Kerteminde (O). Before deployment at Hjarnø (O), ribbons were covered by f/2 medium ASW in small tanks and rolls of 30 m rope were kept vertically in transparent tanks filled with f/2 medium ASW. Media was not changed over a period of 2 months as substrate was consecutively deployed, but DemW was added repeatedly to ensure constant salinity. Seedlings were counted and measured as described (section 2.5.1) before deployment at sea (Table 7). 29/86

31 Table 7. Coastal cultivation sites and substrate deployments. St a Coordinates b Site description Deployment Substrates 56 32'33, Open water, exposed, 28-May-2017 GreO 10 59'53 strong currents '44, Open water, partly 20-Sep-2017 HjaO 10 14'35 protected , Open water, partly 14-Oct-2017 KerO protected. a Grenaa, Hjarnø, Kerteminde: Open site, b ( N; E), DATUM: WGS84 3 2m rope 2 2m cm rope and 3 30cm ribbon 6 30cm rope and 6 30cm ribbon 2.6 Tissue analyses Dry matter content (DMC) Known amounts of collected Ulva (section 2.2.2) was freeze-dried at -40⁰C and moisture and DMC was calculated as of Equation 1 and Equation 2, respectively. After freeze-drying foreign matters such as sand released from thalli was discarded. The dry Ulva was homogenised by dry milling (ISO 9001, Retsch). Equation 1. Equation 2. Moisture content (%) = (WW DW) WW 100% Drymatter content (%) = DW WW 100% Ash fraction and total P content A few grams of DW (section 2.6.1) were incinerated at 520⁰C for 2 h. The ash fraction of DW was calculated as of Equation 3 and the ash was used for a spectrophotometric determination of total P content (Hansen & Koroleff 2007). Prior to spectrophotometric analysis, the ash was dissolved in 1 M HCl acid (100 mg for 25 ml acid), brought to boil for 15 min and then filtered through 25 mm glass microfiber filters (Whatman TM ). Equation 3. Ash Fraction (%) = ash weight DW 100% Not reference material was included in the analysis, but P contents were also analysed by ICP-MS to evaluate the reliability of the results (section 2.7, appendix 6.5.2). 30/86

32 2.6.3 C and N content Freeze dried Ulva (section 2.6.1) was used for analysis of C and N contents following Dumas combustion procedure (Etheridge et al. 1998). Samples were weighed into a tin capsule (Sercon, 8x5 mm), where after they were combusted and analysed (Vario El Cube Elementar Analyzer, Elementar Analysensysteme GMBH, Germany). Five nitrogenous compounds with known C and N contents were included in the analysis to evaluate method accuracy (appendix 6.5.1). Data was treated in Excel 2016 where a linear regression was made from a standard curve based on a standard in five increasing concentrations and their response during the run. Based on these standards C and N contents were assessed in the study samples and contents were transformed to % of DW. Five selected controls were used to evaluate the accuracy (%) of the method (Equation 4). 2.7 Metal analyses Total As will be referred to as: As Sample digestion and preparation. Sample digestion (Hansen et al. 2009) was conducted using disposable glass vials (MG5, Anton Paar, Graz, Austria) and approximately 20 mg of each sample (section 2.6.1) collected with disposable spatulas (VWR) and weighted by a high-accuracy balance (Sartorius, GENIUS F474). A certified reference material, Bladderwrack, (ERM-CD200) Fucus vesiculosus (Institute of Reference Materials and Measurements, Geel, Belgium), was included in triplicates for analytical quality assurance and reagent blanks were included to enable correction of results. Each vial was added 500 µl 66-69% HNO 3 (SCP Science, France) and sealed with PTFE lids (Lab support, Hillerød, Denmark) and screw caps (MG5, PEEK screw cap, Lab support). All vials were placed in a 64-position carousel (64MG5, Anton Paar) and the micro-waved (Anton Paar, Multiwave 3000) assisted digestion was achieved at 500 W by holding a temperature of 140⁰C at 20 bar for 80 min with a two-step ramping period of totally 25 min at 200 W. After at least one day of cooling of the samples, they were diluted in falcon tubes (Sarstedt) with MQ-water (Millipore) to reach an acid concentration of approximately 2% HNO 3. Samples for Hg measurements (5 ml) were furthermore added 35 µl of 37% HCl (Plasma PURE) to reach a final concentration of approximately 1% HCl. To every sample an internal standard solution (ISTD) of Rhodium (Rh, SCP Science, France) at 100 µg L -1 in 2% HNO 3 was added to reach final concentrations of 1 µg L /86

33 Samples were stored at room temperature until inductively coupled plasma mass spectrum (ICP-MS) analysis Apparatus for metal analyses Standards curves for Hg was made from a 1000 mg L -1 stock solution (SCP Science, France) and added 37% HCl for a final concentration of 1%. All other elements (P, As, Rh, Cd and Pb) were made from 1000 mg L -1 stock solutions (SCP Science) and no HCl was added. Standards were diluted with 2% HNO 3. The analyses were run by ICP-MS (Thermo icapq) in two modes, KED (with Helium added as collision gas (CCT2 gas)) and STD (no gas added). The dwell time was 0.01 sec for all elements except for P and Rh, of which it was 0.1 sec. The carrier gas flow was 0.91 L min -1 for both KED and STD mode, and distinctively, the KED mode was run with a collision gas flow of 5.75 L min -1. The temperature in the spray chamber was 2.7 ⁰C and the plasma power was 1550 W (Table 8). Table 8. ICP-MS settings for the determination of metals (As, Cd, Hg and Pb), phosphor (P) and the internal standard, Rhodium (Rh). Parameters P As Rh Cd Hg Pb m/z (u) & Dwell time (s) Mode KED KED & STD STD Collision gas (L min -1 ) & 0 0 Nebulizer gas (L min -1 ) 0.91 Plasma power (W) 1550 Spray chamber temp. (⁰C) Data analysis All data were analysed with Excel Signal intensity for each element (in cps counts per second) were corrected for according to the ISTD and known concentrations of standards were plotted against cps using LINEST to form the calibration curves. Using the known dilution factors and the regression line from the standards the concentrations (mg kg -1 ) for all analyte elements in the samples were calculated. All data was 32/86

34 evaluated by assessing the accuracy (%) of Hg, Cd, Pb and As by Equation 4 determined from the reference material and the precision calculated using duplicate analysis of samples by Equation 5 (appendix ). The reference material was analysed at the end of each analytical sequence. Finally, blanks of 2% HNO 3 were run frequently among samples with the As-analysis but by mistake not with the other metal analyses. Equation 4. Accuracy (%) = [metal] Ref,found [metal] Ref,reported 100% Equation 5. Precision (%) = SE([metal] SampleA;B) 100% A; B represents dublicates. AV([metal] SampleA;B ) 2.8 Bioremediation and food and feed potential Assessment of Danish Green Tides The abundance of Ulva was assessed at Mariager Fiord (P) and Seden Beach (P) using drone and ortho photos (Figure 8). Macroalgae cover was used as ground truth points in the photo analyses and a maximum likelihood classification. The method was conducted by Michael Bo Rasmussen (appendix 6.3). Biomass yields in retro perspective were concluded on this year s biomass density measurements as these methods had not been conducted the previous years. Figure 8. (Left) Orthophoto from Mariager Fiord and (Right) drone records from Seden Beach. Light green areas indicate Ulva coverage of 0-50% and dark green indicates coverage of %. 33/86

35 2.8.2 Bioremediation capacity of macro nutrients and metals. Macro nutrient concentrations were transformed to WW from percentage concentrations of DW in a twostep process (Equation 6, Equation 7), whereas metals only needed a conversion from DW to WW (Equation 7). Finally, the amount of remediated macro nutrients and metals were calculated (Equation 8). X{unit} = C, N, P and Y{unit} = C, N, P, As, Cd, Pb and Hg. Equation 6. [X]{mg kg 1 DW 1 } = [X]{% of DW} 10,000 Equation 7. [Y]{mg kg 1 WW 1 } = [Y]{mg kg 1 DW 1 } DM Y {%} Equation 8. Y{mg} = [Y]{mg kg 1 WW 1 } Biomass{kg WW 1 } Biomass densities and biochemical analyses of Ulva tissue were conducted and assessed only in 2017 but this year s estimates were assumed to be representative for previous years. Likewise, thalli from Hjarnø (O) and Kerteminde (O) had not been analysed but estimates were made based on this year s means from the respective classification, i.e. Open sea Food and feed safety regarding As, Cd, Pb and Hg. When food and feed safety was assessed contents represented in DW were transformed to WW (Equation 7) and a moisture content of 12% (Equation 9). For WW transformation of all European Ulva a moisture content of 82.6% based on this study mean was applied (Equation 10). Equation 9. [metal]{mg kg 1 WW 1 } = [metal]{mg kg 1 DW 1 } Equation 10. [metal]{mg kg 1 WW 1 } = [metal]{mg kg 1 DW 1 } Food and feed safety regarding ias To assess the food or feed safety regarding ias, estimates were calculated assuming that 2.6% of As is ias. 2.6% was the highest fraction of ias found in European Ulva. This is based on data with low extraction efficiencies of As ranging between 20-40% with one exception of 65%. 34/86

36 2.9 Literature review on toxic metals in European Ulva The toxic metal contents of Danish Ulva were compared to toxic metal contents in European Ulva. The data on European Ulva was found through the Web of Science (September 2017) using the following keywords: (Ulva OR sea lettuce OR Enteromorpha) AND (metal* OR trace element* OR Hg OR mercury OR Iod* OR arsen* OR Pb OR Cd OR cadmium) Only studies concerning the As-, Cd-, Pb- or Hg contents in Ulva spp. sampled from natural European populations were included in the study. In studies with several data points from samples from same location, only mean values were used. In studies where only ranges were represented, the middle value was used (appendix 6.5.5) Statistics Statistical analyses were performed with JMP (SAS Inc.). The variables for sporulation and growth of Ulva: Sporulation method, material, temperature, movement and time were tested in a model as fixed factors. All variables were considered categorical except for time, which was continuous. Measures of density (n=6) and lengths (n=10) were log10 transformed and analysed in a 5-degree factorial design. Log10 transformation of response variables allowed residuals to follow assumptions regarding normality and homoscedasticity. The assumption of normality regarding metal contents in Ulva was did not apply for all stations of this study nor for the geographic groupings in neither this or other studies all together. Differences in metal contents were therefore assessed based a nonparametric overall comparison and for each pair using the Wilcoxon method. The null hypothesis was that two means in a pair are the same and all possible individual comparisons were tested, excluding Norsminde Fiord since this station was only represented by one sample. All statistical differences were based on a significance level of α= /86

37 3 Results All values will be represented as AV±SE unless anything else is annotated. 3.1 Cultivation of Ulva Sporulation was successfully induced in Ulva and spores were seeded on two materials that were grown under controlled conditions in the laboratory and deployed for coastal cultivation Sporulation of Ulva Spores from blended thalli and 9 mm discs of thalli were released in high numbers on day 8 with the highest yield of (1.1±0.1, N=6) 10 5 spores ml -1 for one of the duplicates of the disc incubation and (1.3±0.1, N=6) 10 5 spores ml -1 for one of the duplicates of the blended thalli incubation. Hence, the spore release was relatively equal for both treatments Settling of spores The number of spores mm -2 increased during the weeks (slope=2.45 µm) and density was highest for treatments at 18⁰C. Spores from disc incubation had a higher settling success than spores from blended thalli, and spores settled more densely on ropes rather than on ribbons. Movement only had a significant effect for the settling density (Table 9).The treatment resulting in highest density was spores from discs settled on ropes incubated at 10⁰C and movement (Figure 10) and free floating thalli were observed after 4 weeks (Figure 9). Table 9. Mixed model results for density (log transformed). Time: continuous, others: fixed. Source DF Error F ratio p a Estimate b Time (weeks) < Spores [Blend; Disc] <.0001 [-0.26; 0] Temp [10⁰C; 18⁰C] <.0001 [-0.39; 0] Movement [Yes; No] [0.05; 0] Material [Ribbon; Rope] <.0001 [-0.14; 0] a The probability value, b the correlation estimates are presented in a one- or two-dimensional vector (A) or [A; B] that refers to the source, e.g. [Blend; Disc] = [-0.26; 0], of which the second-dimension, presented as zero, serves as reference to the first. Thus, a negative correlation indicates that A is less favourable than B Growth of seedlings The length of seedlings increased by weeks (slope=1.41 µm after 2.5 weeks) and length growth was statistically unaffected by the substrate and the sporulation method. Water movement did influence growth positively and the optimal temperature was 18⁰C rather than 10⁰C (Table 10, Figure 10). 36/86

38 Figure 9. Highest density sample, CMB(D), after 2, 3 and 4 weeks (left to right). Pictures: Esben Rimi Christiansen. Table 10. Mixed model results for length (log transformed). Time: continuous, others: fixed. Source DF Error F ratio p a Estimate b Time (weeks) < Spores [Blend; Disc] [0.01; 0] Temp [10⁰C; 18⁰C] <.0001 [-0.05; 0] Movement [Yes; No] <.0001 [0.17; 0] Material [Ribbon; Rope] [0.004; 0] a The probability value, b the correlation estimates are presented in a one- or two-dimensional vector (A) or [A; B] that refers to the source, e.g. [Blend; Disc] = [0.01; 0], of which the second-dimension, presented as zero, serves as reference to the first. Thus, a negative correlation indicates that A is less favourable than B. After 4.5 weeks the growth rate of CSA was considered insignificant and for WSA and CMB(D) growth rates were declining. For the rest of the samples the growth rates were increasing (Table 11, Figure 10). Table 11. Growth rates of each sample after 4.5 weeks (log transformed). Sample a DF Error t ratio p b Estimate CMA < CMA(D) CMB < CMB(D) < CSA WSA < a C/W: Cold/Warm, M/S: Movement/Still, A/B: Ribbon/Rope, (D): Spores from disc incubation, b the probability value. 37/86

39 Figure 10. Density (left) and lengths (right) measurements (AV±SE) after 2-3 weeks and 3-6 weeks, respectively. C/W: Cold/Warm, M/S: Movement/Still, A/B: Ribbon/Rope, (D): Spores from disc incubation Coastal cultivation Lines of rope and ribbon with seeded Ulva was deployed at sea at three stations, Grenaa (O), Hjarnø (O) and Kerteminde (O), at separate times (Table 12). Growth was observed at all stations but on rope only. The highest biomass yield was 225.9±14.5 g m -1 at Grenaa (O), of which replicates for the latter due to practical mistakes were taken as one. Thalli at Hjarnø (O) after 84 days during autumn deployment reached 14.4±1 cm in length and 1.5±0.2 cm in width but growth was very dispersed, whereas spring deployment at Grenaa (O) resulted in thalli dimensions of 14±0.7 cm in length, 0.97±0.03 cm in width and dense growth. At Kerteminde (O) ropes and ribbons from the laboratory experiment (section 3.1.3) were deployed, of which growth was observed on rope (i.e. CMB and CMB(D)). The lines with relatively densely seeded Ulva yielded 22.8±4.8 g m -1 and lines with less densely seeded Ulva yielded the double 52.8±1.5 g m -1 and reached the highest width of this study (4.3±0.3 cm). Table 12. Final weight and length of Ulva grown on rope at different sites on various dates (AV±SE). St Deployment Sampling Days Seedlings (mm) Length (cm) Width (cm) Weight per m. seeded line (g m -1 ) GreO a >1 14± ± ±14.5 HjaO ± ±1 1.5± KerO ± ± ± ±1.5 KerO a ± ± ± ±4.8 a Relatively densely seeded Ulva. 38/86

40 Growth on ribbons at all stations was considered unsuccessful. After 8 weeks of deployment at Hjarnø (O) seedlings on ribbons had hardly grown any bigger (Figure 11) considering the result of seedling growth on ropes over the same amount of time (Figure 12). Hence, no successive growth of Ulva on ribbons deployed at sea has been reported throughout this study. Measurements for Ulva growth at Hjarnø (O) were only conceded after 8 weeks (15 December) but based on personal observations it appeared that there had been no significant further growth on ropes after 5 weeks of deployment (24 November) (Figure 12). Figure 11. Ulva seedlings on ribbons by the day of deployment (21-Sep-17) at Hjarnø and after 5 (24-Nov- 17) and 8 weeks (15-Dec-17) (left to right). Pictures: Esben Rimi Christiansen and Teis Boderskov. Figure 12. Ulva seedlings on ropes by the day of deployment (21-Sep-17) at Hjarnø and after 5 (24-Nov-17) and 8 weeks (15-Dec-17) (left to right). Pictures: Esben Rimi Christiansen and Teis Boderskov. 39/86

41 3.2 Ulva tissue biochemistry Moisture content, dry matter and ash fraction Danish Ulva was throughout this study found to have a mean moisture content of 82.6±0.4% of WW (range of means: % of WW), consequently a dry matter content of 17.3±0.4% of WW (range of means: % of WW) (Table 13). Statistically, there was an overall significant difference between moisture and location. The moisture content was highest for Ulva collected in Vejle (H), 87.5±0.5% of WW and the lowest for Ulva from Aarhus (H), Halkær Broad (P) and Seden Beach (P) (approximately 81% of WW). Considering the ash fractions there was an overall statistical significant difference between the locations with a mean of 28.3±1.2% of DW. Ulva with the highest ash fraction was found in Horsens (H) (45.2±3.3% of DW) and the lowest in Fredericia (H), Aarhus (H) and Seden Beach (P) (approximately 21-22% of DW). Table 13. Moisture content, dry matter and ash fraction for Ulva at study sites. Station N a Moisture (% of WW) Dry matter (% of WW) Ash fraction (% of DW) FreH ± ± ± 0.8 VejH ± ± ± 2.4 HorH ± ± ± 3.3 AarH ± ± ± 0.1 AarO ± ± ± 1.3 GreO ± ± ± 0.3 MarP ± ± ± 2.3 HalP ± ± ± 2.8 SedP ± ± ± 0.7 NorP Total ± ± ± 1.2 a Number of samples, (-) no SE due to lack of replicates, (N) Number of replicates. 40/86

42 3.2.2 Macro nutrients in Ulva: C, N and P Mean C, N and P contents found in Danish Ulva (Table 14, Figure 13) were 27.9±1.0% C of DW, 3.5±0.3% N of DW, 0.29±0.03% P of DW, respectively, with means in the range of % C of DW, % N of DW and % P of DW, respectively. The C and N contents in Ulva from Seden Beach (P) were significantly higher than in Ulva from all other stations except from Fredericia (H), which also had high nutrient content. There was a statistical significant difference among C, N and P contents in Ulva from the various locations. The lowest N content was found at the coastal cultivation site at Grenaa (O) (0.6±0.1% N of DW) followed by Mariager Fiord (P) (1.6±0.2% N of DW). The highest P contents in Ulva were found at Halkær Broad (P) 0.61±0.04% P of DW, followed by Horsens (H) and Vejle (H). Again, Mariager Fiord (P) and Grenaa (O) had the lowest means, 0.15±0.01% P of DW and 0.05±<0.01% P of DW, respectively. A correlation between salinity and % P of DW was observed with a goodness of fit of R 2 = 0.73, which applied only for samples from protected sites as salinity had not been measured elsewhere. The tendency suggested that the P contents in Ulva are inversely proportional with the salinity (Figure 13). No correlation between salinity and C or N contents was observed. Table 14. C, N and P analyses on Ulva from the study stations (AV±SE, % of DW). Station N a C N P FreH ± ± ± <0.01 VejH ± ± ± 0.01 HorH ± ± ± 0.01 AarH ± ± ± <0.01 AarO ± ± ± 0.01 GreO ± ± ± <0.01 MarP ± ± ± 0.01 HalP ± ± ± 0.04 SedP ± ± ± 0.01 NorP ± ± ± - Total ± ± ± 0.03 a Number of samples, (-) no SE due to lack of replicates. 41/86

43 Figure 13. (a) C, (b) N and (c) P contents in Ulva (% of DW) in respect to the study stations (interquartile ranges and outliers) and (d) the correlation between P contents plotted against salinity ( ). Statistical significant differences are represented by different letters, and the location(s) at which Ulva has the statistical significant highest contents is(are) annotated with A. 42/86

44 3.2.3 Priority pollutants in Danish Ulva spp. In Horsens (H), the Ulva contents of all metals were significantly highest. At this location the average contents of As, Cd, Pb and Hg were 15.0±1.2, 0.49±0.04, 29.7±4.1 and 0.199±0.01 mg kg -1 DW -1, respectively (Table 15, Figure 14). Thalli from VejH, which were also collected within an industrial zone, showed relatively high contents of As (10.1±1.9 mg kg -1 DW -1 ) and Pb (0.8±0.1 mg kg -1 DW -1 ) but contents of Cd (0.12 ±0.02 mg kg -1 DW -1 ) and Hg (0.039±0.010 mg kg -1 DW -1 ) did not seem particularly affected by the location. Relatively high contents of Hg were found in Ulva from Mariager Fiord (P) (0.136±0.027 mg Hg kg -1 DW -1 ). The lowest contents of As were in Ulva from Mariager Fiord (P) (3.0±0.3 mg As kg -1 DW -1 ) and at the coastal cultivation in Grenaa (O) (1.7±0.2 mg As kg -1 DW -1 ), and regarding Cd and Pb the least contaminated Ulva was from Mariager Fiord (P) (0.06±<0.01 mg Cd kg -1 DW -1, 0.7±0.1 mg Pb kg -1 DW -1 ), Fredericia (H) (0.03 ±0.01 mg Cd kg -1 DW -1, 0.7±0.1 mg Pb kg -1 DW -1 ), Aarhus (H) (0.04±<0.01 mg Cd kg -1 DW -1, 0.8±0.1 mg Pb kg -1 DW -1 ) and Aarhus Beach (O) (0.03±0.01 mg Cd kg -1 DW -1, 0.5±<0.1 mg Pb kg -1 DW -1 ). Pb contents in Ulva from Grenaa (O) had high variation (10±9.1 mg Pb kg -1 DW -1 ) due to a single outlier at 37 mg kg -1 DW -1. Samples from Norsminde Fiord (P) were not included in the statistics. Table 15. Contents of priority pollutants (AV±SE, mg kg -1 DW -1 ) in Ulva tissue at the study stations. St N a [As] [Cd] [Pb] [Hg] FreH ± ± ± ± VejH ± ± ± ± HorH ± ± ± ± AarH ± ± < ± 0.1 BDL AarO ± ± ± < ± GreO ± ± ± ± MarP ± ± < ± ± HalP ± ± ± ± SedP ± ± ± ± NorP ± ± ± ± <0.001 Total ± ± ± ± a Number of analyses, (BDL) Below detection limits. 43/86

45 Figure 14. Contents of (a) As, (b) Cd, (c) Pb, and (d) Hg in Danish Ulva in from different sampling locations (interquartile ranges and outliers). Statistical significant differences are represented by different letters, and the location(s) at which Ulva has the statistical significant highest contents is(are) annotated with A Priority pollutants in European Ulva spp. The data of this study made on Danish Ulva spp. including 56 data points was compared to European Ulva, of which a total of 136 data points were extracted from 35 studies, of which 4 data points representing freshwater Ulva were excluded (Figure 15, appendix 6.4). Except for Hg data, most of the data points were reported from either protected sites or open sea sites and only few studies report samples collected near or in harbours or sites under industrial influence. For this study, the majority of Ulva were collected at 44/86

46 protected sites and in harbours or industrial areas and all elements were analyses for in all samples. Regarding European Ulva, the most frequently studied priority pollutants were Cd and Pb. Figure 15. Distribution of data points regarding As, Cd, Pb and Hg, respectively, throughout other European studies (blue border) and this study (red border) grouped in sampling site categories. Data points of European Ulva represent depending on the study both single and mean values of sample numbers and data points of Danish Ulva represent the actual sample number belonging to each of the categorised sites. As summary of the literature review, maximum concentrations of As, Cd, Pb and Hg were 21.9 mg As kg -1 DW -1 (Michalak & Chojnacka 2010), 23.6 mg Cd kg -1 DW -1 (Schintu et al. 2010), 386 mg Pb kg -1 DW -1 (Schintu et al. 2010) and mg Hg kg -1 DW -1 (Ferreira 1991, Coelho et al. 2009), respectively. These maximum contents were mainly reported from sources close to harbours or areas reported with industrial influence. Mean contents of As in European Ulva did not vary significantly between the three categories but for Danish Ulva As contents were highest close to harbours or industry and lowest at open sea (Figure 16). Cd contents in Danish Ulva were only slightly elevated in the fiords in contrast to European Ulva, at which protected sites resulted in the highest mean contents despite outliers. It was also noteworthy that all three categories Danish Ulva had lower Cd contents than European Ulva. Pb contents showed a large variation among all the categories in European Ulva, i.e. highest by harbours and industrial areas, lower at protected sites and lowest at open sea. No difference was observed in Danish Ulva Pb contents among the three categories, except at open sea Pb where contents were significantly lower in Danish Ulva. As for Hg contents in Danish Ulva, no difference was found for the classifications, but European Ulva showed relatively high contents by harbours and industrial areas and at open sea. 45/86

47 Figure 16. European (blue) and Danish (red) Ulva contents of (a) As, (b) Cd, (c) Pb, and (d) Hg in context to the source, i.e. samples from protected sites, harbour- or industrial influenced areas or at open sea (interquartile ranges and outliers). Statistical significant differences between Danish and European Ulva at each categorised site is annotated by signs (>, = and <). Statistical significant differences between the categorised sites among European and Danish Ulva, respectively, are represented by different letters and colours, and the site(s) at which Ulva has the statistical significant highest contents is(are) annotated with A. (N): Data points of European Ulva (N EU ) represent depending on the study both single and mean values of sample numbers and data points of Danish Ulva (N Dk ) represent the actual sample number belonging to each of the categorised sites. 46/86

48 3.2.5 Inorganic Arsenic European studies of Ulva report that extractable ias contents accounts for maximum 2.6% and in average 1.1% of extractable As contents (Figure 17). oas contents ranged between 2.3 and 23% with a mean of 12% of As, though it was not counted for the exact same oas compounds among the studies and not all oas compounds were considered. Furthermore, the results are not fully representative since only a fraction of 20-40% of As could be extracted (Garcia-Sartal et al. 2012, Ka & Elteren 2006) and in one case did the extraction efficiency reach 65% of As (Pell et al. 2013). Figure 17. ias and oas contents (mg kg -1 DW -1 and %) in European Ulva (interquartile ranges and outliers). N=10 (The number of data points represent single values and not means). The results accounts for only 20-40% of As and in one case 65% of As Species dependent variation European Ulva was furthermore used to examine species dependent variation, which is commonly addressed to occur between foliose and tubular species. All species reported as of the genus Enteromorpha were assumed to be tubular species and species reported as Ulva sp. were assumed to be foliose species. A Wilcoxon analysis showed no statistically significant difference between As, Cd, Pb and Hg contents in tubular and folios species (Figure 18). 47/86

49 Figure 18. Contents of As, Cd, Pb and Hg in European foliose or tubular Ulva species (AV±SE). (N): Data points of European Ulva represent a mixture of single and mean values depending on the study. 3.3 Bioremediation The bioremediation potential was examined for coastal cultivated Ulva and for the Danish Green Tides Coastal cultivation Only biomass from Grenaa (O) was studied for nutrients and priority pollutants but estimates were made for Ulva grown at Hjarnø (O) and Kerteminde (O) (Table 16). Based on these estimates it was found, that through coastal cultivation of Ulva on rope there was a minimum potential of remediating 1 g C m -1, 0.07 g N m -1 and g P m -1. The maximum bioremediation potential for macro nutrients was 7.4 g C m -1, 0.19 g N m -1, 0.02 g P m -1 for Ulva grown in Kattegat near Grenaa (Grenaa (O)). The minimal remediation potential of priority pollutants was estimated to 21 µg As m -1, 1.8 µg Cd m -1, 30 µg Pb m -1 and 0.5 Hg µg m -1. The remediation potential of Cd at Grenaa (O) was lower (1.8 µg m -1 ) than what it expected at Hjarnø (O) and Kerteminde (O) (2.3 µg m -1 ) even though the biomass yield was highest at Grenaa (O). Bioremediation of Pb at Grenaa (O) (434 µg m -1 ) showed the highest potential of priority pollutant remediation. 48/86

50 Table 16. Bioremediation capacity (µg m -1 ) of nutrients and priority pollutants (µg m -1 ) by coastal cultivation of Ulva on rope. St Year Biomass (g m -1 ) C N P As Cd Pb Hg GreO ,361, ,487 17, HjaO a ,040,060 74,077 4, KerO a ,257, ,818 8, KerO a ,013,392 72,178 3, a calculated on this study means of biochemistry of Ulva thalli collected at open sea Danish Green Tides From a total study area of 6.70 km 2 and 0.32 km 2 for Mariager Fiord (P) and Seden Beach (P), respectively, it was found that the potential biomass yield for 2017 was around 2400 and 515 tons WW (Table 17). This potential yield was estimated to be the same in 2012 at Mariager Fiord (P). For Seden Beach (P) there was a tendency of a potential increasing biomass yield since Table 17. Biomass densities at Mariager Fiord (MarP) and Seden Beach (SedP), 2012, 2014, 2016 and Ulva coverage 0-50% Ulva coverage % Total St Year Biomass (kg m -2 ) Area (km 2 ) Biomass a (tons WW) Biomass (kg m -2 ) Area (km 2 ) Biomass a (tons WW) Biomass (tons WW) MarP b SedP a calculated on 2017 biomass measurements, b calculated directly on drone records, (-) no data. 49/86

51 Ulva collected at Seden Beach (P) had generally higher contents of nutrients and priority pollutants than Ulva collected at Mariager Fiord (P) but Mariager Fiord (P) considered a 21-fold larger study area. Thus, the highest potential nutrient removal by collection of Ulva could have been achieved at the study area at Mariager Fiord (P) in 2012 and 2017 where removal of around 2400 tons WW would have allowed for removal of 94 tons C, 5.9 tons N and 0.6 tons P (Table 18). Furthermore, 1.1 kg As, 23 g Cd, 272 g Pb and 50 g Hg would have been removed. If 515 tons WW had been removed from Seden Beach (P) in 2017, then 35 tons C, 5.8 tons N and 0.25 tons P would have been removed simultaneously together with 0.6 kg As, 14 g Cd, 251 g Pb and 8 g Hg. Table 18. Potential bioremediation potential for nutrients (tons) and priority pollutants (g) by complete harvest of Ulva biomass (tons WW) at Mariager Fiord (MarP) and Seden Beach (SedP). Ulva thalli biochemical analyses were only conducted in Station Year Biomass C N P As Cd Pb Hg MarP SedP Site dependent bioremediation capacity of Ulva The potential bioremediation capacity of Ulva for macro nutrients and priority pollutants was assessed for each of the categorised sites in considering a hypothetical harvest of 1 tons of biomass WW. Considering bioremediation of nutrients, then the highest bioremediation capacity for Danish Ulva is estimated to be at protected sites or at sites in connection to harbours or industry (Table 19). The C-sequestering for Danish Ulva was somehow the same at all sites (44-50 kg C tons -1 WW -1 ) with no significant difference (p=0.806), but the mean N contents in Danish Ulva were two-fold lower at Open sea (3.2 kg N 50/86

52 tons -1 WW -1 ) compared to both other categorised sites ( kg N tons -1 WW -1 ). Nevertheless, N-contents in Danish Ulva were only significantly lower from Open sea compared to Ulva from Harbour/Industry (p=0.013). The mean P contents in Danish Ulva at protected sites (0.17 kg P tons -1 WW -1 ) were approximately two- and four- times lower (with statistical support) than in Danish Ulva from the other categorised sites, Harbour/Industry (0.42 kg P tons -1 WW -1, p=0.001) and Protected sites (0.64 kg P tons -1 WW -1, p=0.004), respectively. No data was collected on macro nutrients in European Ulva. The bioremediation capacity of metals in both Danish and European Ulva was found to have a low potential at all three categorised sites. The highest potential occurred to be remediation of Pb in harbours or industrial areas, at which 1 tons of fresh European Ulva on average potentially could remove approximately 19 g Pb. In all other cases and for both European and Danish Ulva less than 1.5 g of each of the priority pollutants could potentially be removed with the harvest of 1 tons Ulva biomass WW. Table 19. The bioremediation capacity of Danish and European Ulva collected at the three categorised sites considering a potential harvest of 1 tons of biomass WW and the consequent removal of macro nutrients (kg tons -1 WW -1 ) and priority pollutants (g tons -1 WW -1 ). Study Categorised sites C N P As Cd Pb Hg Protected sites Danish Harbours/Industry Open sea Protected sites European Harbours/Industry n.d Open sea Food and feed potential of Danish and European Ulva Food and feed safety was evaluated by comparing European maximum levels defined for seaweed or most similar class with mean and maximum values of European and Danish Ulva collected at the three categorised sites of this study (Table 20-Table 24) Arsenic There are currently no defined maximum levels for total arsenic in food for human consumption but the contents for feed is set to 40 mg kg -1. Rice products for food have a defined maximum level for ias, i.e mg kg -1 WW -1, and feed materials derived from seaweed may not exceed 2 mg kg /86

53 In this perspective, total As contents in both European and Danish Ulva did not seem to conflict with food or feed safety, since the highest recorded value was 21.9 mg kg -1 DW -1 (Table 20). Likewise, contents of ias did not exceed any of the defined maximum levels in rice products (Table 21). Table 20. As contents in Danish and European Ulva at the three categorised sites (AV±SE, max). Site Study Dried (mg kg -1 DW -1 ) Food (mg kg -1 WW -1 ) Feed (mg kg -1 relative to 12% moisture) a N b Protected sites EU 4.9 ± ± ± DK 6.1 ± ± ± Harbour/Industry EU 4.2 ± ± < ± DK 8.9 ± ± ± Open sea EU 5.7 ± ± ± DK 3.3 ± ± ± a Different rice products may not exceed 40 mg kg -1 WW -1, b Number of samples. NB: No contents exceed the European maximum levels for priority pollutants in feed and no limits have been defined for food. (EU) European Ulva, (DK) Danish Ulva collected in this study. Table 21. Estimated ias contents of mean- and maximum As-values in Danish and European Ulva at the three categorised sites assuming that 2.6% of As exists as ias (AV, max). Site Study Food (mg kg -1 WW -1 ) a Feed (mg kg -1 relative to 12% moisture) b N c Protected sites EU DK Harbour/Industry EU DK Open sea EU DK a Different rice products may not exceed mg kg -1 WW -1, b feed materials derived from seaweed may not exceed 2 mg kg -1 relative to a moisture content of 12%, c Number of samples. NB: No contents exceed than the European maximum levels for priority pollutants in food or feed. (EU) European Ulva, (DK) Danish Ulva collected in this study. 52/86

54 3.4.2 Cadmium Maximum levels for Cd have been set at 3 mg kg -1 DW -1 for food supplements from dried seaweed and complementary or complete feed have been set at 0.5 mg kg -1. These limits were not exceeded by Danish Ulva, though Cd contents in Ulva collected from Horsens (H) reached the limits for feed (Table 22). The only mean Cd contents higher than what is allowed for feed was found in European Ulva collected near harbours or an industrial source. The maximum contents of Cd found in European Ulva collected at all three categorised sites exceed the limit for food and feed, except for European Ulva collected at protected sites, which did not exceed the maximum levels for food supplements derived from seaweed. Table 22. Cd contents in Danish and European Ulva at the three categorised sites (AV±SE, max). Site Study Dried (mg kg -1 DW -1 ) a Food (mg kg -1 WW -1 ) Feed (mg kg -1 relative to 12% moisture) b Protected sites EU 0.6 ± ± < ± DK 0.1 ± < <0.1 ± < ± < Harbour/Industry EU 4.0 ± ± ± DK 0.1 ± < <0.1 ± ± < Open sea EU 0.5 ± ± < ± DK 0.1 ± <0.1 ± < ± < a Food supplements from dried seaweed may not exceed 3 mg kg -1 DW -1, b complementary or complete feed may not exceed 0.5 mg kg -1 relative to a moisture content of 12%, c Number of samples. Bold represents contents exceeding the European maximum levels for priority pollutants in food or feed. (EU) European Ulva, (DK) Danish Ulva collected in this study. N c Lead Seaweed has no defined maximum levels for Pb and the nearest defined category for food is leaf vegetables and food supplements at mg kg -1 WW -1 and complete feed or complementary feed may not exceed 5-10 mg kg -1. These limits for food and feed were not exceeded in Danish Ulva collected at protected sites, though Danish Ulva collected from Horsens (H) and cultivated at Grenaa (O) was found with too high contents (Table 23). The average values of Cd in European Ulva collected at harbours or industrial areas are too high to be used as complete or complementary feed, but it could be accepted as a food supplement. 53/86

55 Considering the highest found contents of Cd in European Ulva, the maximum levels were exceeded by far. European Ulva from a harbour was found with Cd contents of 66 mg kg -1 WW -1, which exceeded the maximum level for food supplements 22-fold and the maximum level for leaf vegetables 220-fold. Table 23. Pb contents in Danish and European Ulva at the three categorised sites (AV±SE, max). Site Study Dried (mg kg -1 DW -1 ) Food (mg kg -1 WW -1 ) a Feed (mg kg -1 relative to 12% moisture) b N c Protected sites EU 6.9 ± ± ± DK 1.6 ± ± < ± Harbour/Industry EU 35.7 ± ± ± DK 7.5 ± ± ± Open sea EU 6.3 ± ± ± DK 6.0 ± ± ± a Leaf vegetables or food supplements may not exceed mg kg -1 WW -1, b complete or complementary feed may not exceed 5-10 mg kg -1 relative to a moisture content of 12%, c Number of samples. Bold represents contents exceeding the European maximum levels for priority pollutants in food or feed. (EU) European Ulva, (DK) Danish Ulva collected in this study Mercury No maximum levels for Hg in seaweed for food or feed have been defined, but fishery products for food cannot exceed mg kg -1 WW -1 and fishery products for feed are limited to mg kg -1. Hg contents in neither European or Danish Ulva from any of the three categorised sites compromised the food safety in context to fishery products, but European Ulva collected in context to a harbour or an industrial contaminated site exceed the maximum levels defined for fishery products for feed (Table 24). 54/86

56 Table 24. Hg contents in Danish and European Ulva at the three categorised sites (AV±SE, max). Site Stu dy Dried (mg kg -1 DW -1 ) Food (mg kg -1 WW -1 ) a Feed (mg kg -1 relative to 12% moisture) b N c Protected sites EU 0.06 ± <.01 ± < ± DK 0.08 ± ± < ± Harbour/Industry EU 0.41 ± ± ± DK 0.07 ± ± < ± Open sea EU 0.14 ± ± ± DK 0.07 ± ± < ± a Fishery products may not exceed mg kg -1 WW -1, b any feed material or fishery products may not exceed mg kg -1 relative to a moisture content of 12%, c Number of samples. Bold represents contents exceeding the European maximum levels for priority pollutants in food or feed. (EU) European Ulva, (DK) Danish Ulva collected in this study. 55/86

57 4 Discussion 4.1 Bioremediation potential of Ulva Preparation of lines for coastal cultivation of Ulva The optimal seeding was statistically dependent on the sporulation method, the temperature and the substrate. Other studies support that settling success is increased by higher temperature (Gao et al. 2017a) until a maximum of 23⁰C (Christie & Shaw 1968) and other substrates have also been taken in use, e.g. freefloating bioballs (Praeger & de Nys 2017). Studies have also reported that fragmentation increases the spore release of Ulva but the reason for the higher seeding density when using spore extracts from discs cannot be fully explained (Gao et al. 2010). Nevertheless, it can be argued that (1) spores from discs have been healthier in contrast to spores produced from highly fragmented thalli and that (2) there is a higher risk of contamination when using blended material possessing doubt on if all counted cells where actually spores from Ulva. The growth of Ulva seedlings was statistically only affected by the temperature and water movement. Best growth was theoretically achieved at 18⁰C and with water movement while the substrate and the seeding method was statistically insignificant to influence growth. Other studies have confirmed the importance of temperature (Gao et al. 2017a) and water movement (Neushul et al. 1992). More factors to consider would be light (Sand-Jensen 1988, Mohsen et al. 1974), salinity (Sousa et al. 2007, Martins et al. 1999, Jie et al. 2016) and dissolved CO 2 (Gao et al. 2017a). Growth rates ceased after 4.5 weeks for seedlings growing without water movement and for seedlings densely seeded on the substrate. Thus, densely seeded Ulva or seedlings growing without water movement need a maximum nursery period of 4.5 weeks before coastal deployment Growing Ulva on lines at sea Ulva is not annual (Lüning et al. 2008, Malea & Haritonidis 1999) and has been recorded to have increased growth rates at high temperatures and water movement or nutrient exchange, and other studies have reported light as an important factor for growth (Sand-Jensen 1988, Mohsen et al. 1974). Thus, there are many indications that Ulva is a good summer crop. Nevertheless, deployment of lines seeded with Ulva and deployed at summer was in all cases unsuccessful, but deployment during autumn and spring did yield Ulva biomass. In contrast to the controlled experiment in the laboratory, growth of Ulva deployed at sea did depend on the substrate. No growth was at all observed on deployed ribbons at either Kerteminde (O) or 56/86

58 Hjarnø (O), e.g. 2 mm seedlings on ribbons at Hjarnø (O) had not grown any bigger after 8 weeks. An explanation could be that ribbons are too prone to catch foreign organisms and other algae outcompete Ulva. It has previously been reported that deployment of artificial substrata can result in the growth of many diverse organisms (Suutari et al. 2017), but even non-fouled ribbons at Kerteminde (O) and Hjarnø (O) did not yield any Ulva biomass. Thus, growth of Ulva deployed at sea was only achieved on rope. The low biomass yield at Hjarnø (O) was assumed to be a result of low density of deployed seedlings. The biomass yield at Kerteminde (O) was equally low for densely seeded Ulva and a little higher for less densely seeded Ulva. Densely seeded Ulva at Kerteminde (O) grew longer but less densely seeded Ulva grew thicker. A study of Ulva seeded on bioballs reported that high seeding densities did not affect the specific growth rates (Praeger & de Nys 2017). The seedling length by the time of deployment did not appear to have a high influence on the final lengths and biomass yields of Ulva after days. Nevertheless, a more controlled experiment with simultaneous deployment and sampling times of lines with Ulva of different seedling lengths are needed to clarify this. Personal observations of this study also reported that free floating thalli appeared 4 weeks after seeding, which could be a relevant note for further investigations. It is not of interest that settled Ulva seedlings are released, which in extreme cases can lead to outbreaks of Green Tides (Smetacek & Zingone 2013). Thus, understanding of how, when and to what degree Ulva seedlings or adult thalli are released from the substrates is of high importance Ulva tissue contents of macro nutrients Ulva collected at Seden Beach (P) and Fredericia (H) was found to have the highest contents of C and N. The highest P contents were found at Halkær Broad (P). The negative correlation between high salinities and high phosphorous contents might be explained by runout of P. It has been hypothesised that freshwater runouts cause a decrease in salinity at protected sites while nutrient rich runouts simultaneously result in an increased P availability, which is then reflected by the P contents in Ulva. No correlation was found 57/86

59 between salinity and any of the other macro nutrients, but other studies have emphasised the influence of nutrient rich estuaries on Ulva (McAvoy & Klug 2005, Cohen & Fong 2018). The lowest reported carbohydrate contents in Ulva was 15% of DW, which is in agreement with the lowest C contents of this study (14.4% C of DW) found in Ulva samples from Halkær Broad (P) (Wong & Cheung 2000). The study s lowest N contents (0.5% of DW) in Ulva was from the cultivation at Grenaa (O) and from the same site P contents also met the lowest P contents reported by other studies (0.05% P of DW), (Rasyid 2017). The macro nutrient contents in Ulva at Grenaa (O) were expectedly low because of low access of nutrients because of the growth period during the beginning of the summer Bioremediation potential of coastal cultivations of Ulva The bioremediation potential of seeded Ulva in this study was lower than for non-seeded artificial substrata deployed at sea for one year (Suutari et al. 2017) and in comparison did one year of cultivation of Saccharina latissima at Hjarnø (O) lead to the potential removal of approximately 5.5 g N m -1 and 1 g N m -1, i.e 79- and 250-fold higher amounts than expected for Ulva grown over 85 days on the same location (Marinho et al. 2015). The bioremediation capacity of Ulva for priority pollutants at open sea was compared to the other categories (i.e. protected sites and sites in context to harbours and industry) not very high regarding As, Cd and partly Hg but one outlier for Pb contents measurements resulted in a relatively high bioremediating capacity of Pb at open sea. High Pb contents were only found in one of the replicates of Ulva from Grenaa (O) and might therefore simply be a result of a contamination and not be representative of the true bioremediation capacity of Ulva at that location. Thus, the bioremediation potential of priority pollutant for coastal cultivated Ulva is estimated to be relatively low but this might increase the value as a food or feed product Bioremediation potential of Ulva related to Danish Green Tides Two fiords have been examined in context to Danish Green Tides. If the total study area of Mariager Fiord (P) in 2017 was cleaned from Ulva this would result in a removal of 2400 tons biomass WW, of which 93 tons would be C, 5.9 tons would be N and 0.58 tons would be P. By a potential full removal of the 515 tons biomass WW from Seden Beach (P) in 2017 just as much N would be removed and around half as much P (0.25 tons) and C (35 tons). 58/86

60 Thus, Ulva at Seden Beach (P) has the highest bioremediation capacity and since Seden Beach (P) only represents a small area of Odense Fiord, the total area of the fiord accounts for much more, emphasising the high bioremediation potential of Ulva at this location. Regarding priority pollutants, only minor amounts of Cd and Hg could potentially be removed, i.e g by complete biomass harvest of each of the study areas. Potential amounts for removal of Pb and As were relatively higher, but a potential harvest of 2400 tons biomass WW from Mariager Fiord (P) would lead to the removal of 1.1 kg As and 269 g Pb. Thus, it is not believed that extensive Ulva biomass removal will improve the water quality concerning priority pollutants at Mariager Fiord (P) and Seden Beach (P) Bioremediation capacity of Ulva for nutrients Metal contents were too low for proper amounts to be removed simultaneously with Ulva biomass harvest, but Ulva showed a high bioremediation capacity for macro nutrients. According to the three categorised sites of this study, C-sequestering of Ulva was almost the same at all sites but reflected by the N and P contents, Ulva harvested at protected sites and in areas connected to harbours or industry showed the highest bioremediation capacity. This could be connected to the fact, that a higher nutrient availability causes higher nutrient contents in Ulva (Nielsen et al. 2012, Msuya & Neori 2008). Thus, it is expected, that the bioremediation capacity of Ulva at open sea is lowest due to a reduced nutrient availability in the water compared to protected sites. 4.2 Food and feed potential of Ulva The food and feed safety of the harvested Ulva was evaluated upon the found contents of priority pollutants in Danish and European Ulva, and since iodine frequently appear in high concentrations in seaweed (mainly brown algae), this concern will be evaluated (Hou et al. 1997, Hortas et al. 2011, Desideri et al. 59/86

61 2016). The contents of priority pollutants found throughout this study were compared to European maximum levels for food and feed (European Commission 2015, European Commission 2017) Arsenic Ulva with the highest As contents of in the study was collected from Horsens (H) and Vejle (H) and the lowest As contents was in Ulva from the cultivation at Grenaa (O). It was significant that arsenic contents were highest in Ulva collected at sites in context to harbours and industrial areas and lowest at open sea. European Ulva collected from the three categorised study sites did not show any significant difference in As contents but Danish Ulva collected at sites in context to harbours and industrial areas had a significantly higher content of As compared to European Ulva. Currently no rules apply for maximum levels for As in food but none of the found As contents exceed the limit for feed materials derived from seaweed. An overestimate of the ias ratio was used to assess the food and feed safety according to European maximum levels for ias. The literature reports low extraction efficiencies of As, but despite that it is estimated that ias contents in Danish and European Ulva will not exceed the maximum levels. Thus, As and ias are not expected to compromise the food and feed safety of Ulva, which is also confirmed by experiments with in-vitro gastrointestinal digestions of cooked Ulva (Sartal et al. 2012) Cadmium Cd contents in Ulva was significantly highest at Horsens (H) and lowest in Aarhus Beach (O), Aarhus (H) and Fredericia (H). It was not expected that Ulva from Aarhus (H) and Fredericia (H) near harbours and industrial areas had the lowest Cd contents of Ulva collected throughout this study. An explanation might be that the collection sites for at Aarhus (H) and Fredericia (H) have large water exchange, thus, balancing between the two categories Harbour/Industry and Open sea. Overall, Cd contents in Ulva was only significantly higher from protected sites compared to the other two categories. Studies of European Ulva supported the statement that Cd contents from areas influenced by harbours and industry are relatively high (Zhang & Wong 2007, Charlier et al. 2012). Cd contents in European Ulva was in all three categories higher than in Danish Ulva. If Ulva was to be used as a food supplement, then it is not recommended that Ulva is collected near harbours or industrial areas, since Cd contents in European Ulva at these sites are too high according to EU- 60/86

62 legislation for maximum levels in dried seaweed as a food supplement. Too high contents have also been observed in European Ulva collected at exposed sites ( Open sea ) but Danish Ulva has not been recorded with too high Cd contents. The same story applies for use of Ulva as a feed and regarding these legislations Cd contents have also been recorded to be too high in European Ulva at protected sites. Thus, Cd in Danish Ulva would not preclude its consumption by either humans or animals, contrary some European Ulva, of which some samples have too high contents of Cd Lead One sample from Grenaa (O) might potentially be an outlier that potentially has been contaminated during the transportation or sampling preparation. The absolute lowest Pb contents in Danish Ulva was in samples collected at Aarhus Beach (O). Overall there was no significant variation among Pb contents in Danish Ulva collected at the three categorised sites, though Pb contents had been expected to be significantly higher in and near harbours (Zhang & Wong 2007, Charlier et al. 2012). European Ulva seemed to have significantly higher contents of Pb at sites belonging to the category Harbour/Industry, and Pb contents in Ulva at open sea were the significant lowest. European Ulva contained more Pb than Danish Ulva at all categorised sites except for at Open sea. Neither Danish nor European Ulva from any of the categorised sites had potential as a food product if it was considered a leaf vegetable. Although, if it was to be used as a food supplement, then average Pb contents would for all sites be accepted but both in Danish and European Ulva outliers have been found, which would compromise the safety. Almost the same story applies for Ulva as a feed product. Only Danish Ulva collected from protected sites can be used as a complete feed but in the other two categories and as well for European Ulva did mean contents exceed the maximum value for a complete feed product. Ulva could potentially be used as a complementary feed, but in both Danish and European Ulva contents have exceeded the maximum levels. Thus, the Pb contents in Ulva seem to only allow for its use as a food supplement or as a complementary feed, but there is an elevated risk that Pb contents will be too high. 61/86

63 4.2.4 Mercury Mean Hg contents of Danish Ulva only varied from European Ulva at sites influenced by industrial contaminations and harbours, at which European Ulva had significantly higher contents. If European Ulva was compared among the categorised sites, then it was concluded that there was no significant difference between Ulva from Open sea and Harbour/Industry, but Hg contents in Ulva from protected sites were significantly lower, but the maximum contents in Ulva from industrial areas and harbours were relatively high. In a food and feed perspective when comparing Ulva to a fishery product then there is only one conflict with the safety of it, i.e. the use of Ulva from industrial contaminated sites. Thus, the recommendation would be to avoid using Ulva from industry contaminated areas Seasonal and species dependent variation in Ulva metal contents Seasonal dependent variation has not specifically been studied throughout this study but reported contents of priority pollutants in European Ulva collected in all seasons have been analysed together. It has been suggested that metal contents in Ulva are only indirectly seasonal dependent, i.e. the direct factor relies upon the growth dynamics of Ulva where increasing biomass is negative proportional with the metal contents as a result of dilution (Schintu et al. 2010, Haritonidis & Malea 1999, Favero et al. 1996). Further investigations could be relevant at areas with elevated contents of priority pollutants in Ulva to examine if the season of harvest could increase the potential as a food or feed product. It has been suggested that tubular species have higher metal uptakes than foliose species (Trifan & Chimie 2015, Malea & Haritonidis 2000), which could lead to the hypothesis that tubular species generally have higher contents of priority pollutants compared to foliose species. Nevertheless, based on the data of European Ulva it was found that it cannot be statistically proven that any of the priority pollutants generally occur in higher contents in tubular species compared to foliose species. This suggests that both tubular and foliose species can be grouped together when comparing their food and feed safety and be considered under the same terms. 62/86

64 4.2.6 Iodine Iodine concentrations are approximately 50 to 1000-fold lower in Ulva (7-70 mg kg -1 DW -1 ) compared to brown algae such as Laminaria spp. ( mg kg -1 DW -1 ) (Hou et al. 1997, Desideri et al. 2016). Nevertheless, even with these concentrations the daily recommended intake for an adult can potentially be exceeded through consumption of relatively few grams of Ulva. Iodine contents in Danish Ulva were not analysed in this study, but by completing this investigation an assessment can be made on a recommended daily intake of Ulva to avoid consumers getting overexposed to iodine. 63/86

65 4.3 Future perspectives of Ulva for bioremediation Ulva could at two of the three cultivations of this study not be grown successfully seeded on lines during the summer period but seemed to thrive perfectly in autumn and spring while pausing its growth during winter. One of the biggest challenges of this study when growing Ulva during the summer period was fouling. Deployed lines were quickly covered by unwanted organisms exploiting the space that the cultivated Ulva was supposed to fill. It is therefore doubtful if Ulva will become a good summer crop for coastal cultivations in Denmark and since other studies suggest that Ulva hardly grows during winter, it is recommended that Ulva is cultivated and harvested over two seasons, namely autumn and spring (Rowcliffe et al. 2001, Lüning et al. 2008). The highest biomass yield of cultivated Ulva throughout this study was achieved by harvest of Ulva in July. If Ulva according to other studies reaches optimal biomass densities during spring, then this is the most important growth season for deployment of Ulva (Lentz 1998, Malea & Haritonidis 1999). A strategy could potentially be deployment of Ulva in autumn and harvest come the end of the following spring. However, biomass yields are restricted to the short growth period and big biomass yields might never compare to cultivation of other seaweeds, e.g. Saccharina latissima (Neveux et al. 2017, Marinho et al. 2015). It must be emphasized that bioremediation with Ulva through coastal cultivation and biomass removal can only be considered to have local and short-timed effects, since the potential nutrient discharge reduction is very low compared to the total nutrient load on the Danish marine waters (Jouvenel & Pollard 2001, Cederwall & Elmgren 1990), but the nutrient load can be further reduced through land-based systems of Ulva cultivations, e.g. on aquaculture waste waters (Lawton et al. 2013, Bolton et al. 2009) and manure from agriculture (Nielsen et al. 2012). The bioremediation potential for Ulva at protected sites is much higher than at open sea and Green Tides allow for potential harvest of huge biomasses of Ulva. Thus, the author suggests that the full bioremediation potential of Ulva is carried out through three events: (1) harvest of naturally occurring Ulva in the outbreaks of Green Tides, (2) land-based systems of Ulva cultivation in a set-up that allow for a reduction in anthropogenic nutrient discharge to the environment and (3) coastal cultivations of Ulva to decrease local nutrient loads at places to affected by Green Tides. 64/86

66 4.3.2 food and feed What appears to be the biggest problem with Ulva is potential high Pb contents. By now, it all comes to turn with what Ulva is classed together with among similar food and feed products when determining the potential, but the food and feed safety of Ulva can only finally be assessed when maximum levels for all the priority pollutants have been settled for regarding seaweed products. Other metals frequently reported elevated such as Co, Cr and Cu will also be relevant for further examination to fully understand if these metals are prone occur in high contents in Ulva tissue in perspectives of possessing a potential health risk. However, if metal contents are too high, then the preparation method can reduce the tissue contents, e.g. have As contents in raw seaweed been reduced markedly through cooking (Sartal et al. 2012). Another aspect is microbial contamination of Ulva, e.g. by Salmonella or Escherichia coli but a study conducted by the Ministry of Environment and Food of Denmark concludes that by avoiding sources of contamination such as harbours and sewage outlets then Danish Ulva should be safe for human consumption (Ministry of Environment and Food of Denmark 2017b). Finally, studies report that Ulva does not have any distinctive effects as a feed for sheep or goats (El-Waziry et al. 2015, Ventura & Castañón 1998) but promising health results have been achieved with Ulva as a feed supplement for livestock and abalone (Naidoo et al. 2006, Michalak & Chojnacka 2009) and even better, the inclusion of Ulva in feed for ruminants has been suggested to reduce their methane emission (Maia et al. 2016, Machado et al. 2014). 65/86

67 5 Conclusion Natural and coastally cultivated populations of Ulva have throughout this study been successfully harvested and chemically analysed for macro nutrients (C, N and P) and priority pollutants (As, Cd, Pb and Hg). Both study hypotheses have been accepted, i.e. (H1) the species can be harvested from nature and/or cultivated on structures (H2) the species biomass accumulates nutrients and metals. The bioremediation capacity and the food and feed potential of Ulva has been assessed based on the state of art. 5.1 Ulva can be harvested from nature and cultivated on structures In the laboratory there was no significant difference in growth rates of seedlings on ropes or ribbons but at coastal cultivations biomass was only yielded on ropes. Thus, ribbon does not appear to be a suitable structure for coastal cultivation of Ulva. The highest biomass yield was achieved by cultivating Ulva during spring at a very exposed site, though nutrient contents in Ulva from the coastal cultivation was lower than from protected sites, e.g. fiords. Thus, due to a higher nutrient content and a higher potential biomass harvest the bioremediation potential of Ulva was much higher at protected sites for natural occurring populations rather than for populations cultivated at sea. 5.2 The species accumulate nutrients and metals Ulva showed a poor bioremediation capacity of the environmental harmful metals, As, Cd, Pb and Hg but potential health risks associated with Hg, As and ias contents in Ulva were negligible. Relatively high contents of Cd were only a problem in areas associated with harbours or industry, and in both Danish and European Ulva independently on the sampling locations Pb contents were detected too high for human or animal consumption. Problematically, even cultivated Ulva at sea was found with relatively high Pb contents and if this is not the result of a single outlier then the potential of Ulva for food and feed could potentially be compromised. Considering Ulva in feed products, several studies suggest that Ulva should not consist of a complete diet but rather as a fraction of a feed product, which potentially carries high environmental benefits with it, e.g. through methane emission reduction for ruminants. Thus, when deciding for European maximum levels for priority pollutants concerning Ulva (and other seaweeds), it is recommended that Ulva is not considered a complete diet but rather a food or feed supplement. 66/86

68 Acknowledgements While travelling around in Australia I had the fortune of meeting Annette Bruhn when visiting at James Cook s University in Townsville. I was instantly inspired by her enthusiasm and her perspectives on how seaweed can make a difference for the environment. Now, one year later I have handed in my Master s Thesis in a co-work with Annette and through our team work I have truly learned a lot. The project has also been supervised by Jens J. Sloth, which I know from previous work and re-establishing the collaboration has been an immense pleasure. I would like to thank both my supervisors for their extensive help and the interest they have dedicated to the project. Sampling of Ulva at Mariager Fiord, Seden Beach, Norsminde Fiord and Halkær Broad has been completed thanks to Leo Mosgaard Nielsen and Michael Bo Rasmussen. Another thanks to Michalael for conducting ortho- and drone photo analyses assessing biomass coverage and potential biomass harvest at Mariager Fiord and Seden Beach. Teis Boderskov has contributed to the work of this study by cultivating and harvesting Ulva in Kattegat at Grenaa and he has as well been a huge support in the project development and has always shown an interest in discussing my ideas and in staying up to date with my progress. Most of the laboratory work has been conducted at the Institute of Bioscience (AU) in Silkeborg, at which Kitte Linding Gerlich has been much involved and very helpful. From the same institute I must also thank Thorsten Johannes Skovbjerg Balsby who has taught me a lot about statistical performances e.g. factorial designs. Metal analyses have been conducted at DTU Lyngby at the National Food Institute thanks to the laboratory support of Annette Landin and Birgitte Koch Herbst who have also contributed to good mood on work days. Thanks, both to the Research Group of Marine Ecology in Silkeborg and the Research Group for Nano-Bioscience for use of their lab and other facilities. Finally, I would like to thank Johan Cassias for teaching me arts in graphic design, which has assigned a new value to my data presentation in the report, and Bærnt Kjær Sørensen for his help in deploying my lines seeded with Ulva at Kerteminde but also for our many talks about Ulva, which have provided me with new perspectives on the extend, to which Ulva from a commercial perspective can be used to improve the environmental conditions locally and even worldwide. 67/86

69 References ABDALLAH, M.A.M. & ABDALLAH, A.M.A Biomonitoring study of heavy metals in biota and sediments in the South Eastern coast of Mediterranean sea, Egypt. Environ Monit Assess, , /s AMINOT, A. & KÉROUEL, R Assessment of heat treatment for nutrient preservation in seawater samples. Analytical Chimica Acta, ARLA Matilde Kakao Mælk Available at: [Accessed February 20, 2018]. BBC Seaweed suspected in French death. BBC NEWS Available at: BEN-ARI, T., NEORI, A., BEN-EZRA, D., SHAULI, L., ODINTSOV, V. & SHPIGEL, M Management of Ulva lactuca as a biofilter of mariculture effluents in IMTA system. Aquaculture, 434, , /j.aquaculture BESADA, V., ANDRADE, J.M., SCHULTZE, F. & GONZÁLEZ, J.J Heavy metals in edible seaweeds commercialised for human consumption. Journal of Marine Systems, 75, , /j.jmarsys BEVAN, T. & WALKER, M Jogger dies after being overcome by fumes from toxic seaweed as deadly as cyanide. Mirror Available at: BIMENYA, G.S., KAVIRI, D., MBONA, N. & BYARUGABA, W Monitoring the severity of iodine deficiency disorders in Uganda. African Health Science, 2, BO, M., LIU, F. & LI, Z Growth and nutrient uptake capacity of two co-occurring species, Ulva prolifera and Ulva linza. Aquatic Botany, 100, 18 24, /j.aquabot BOLTON, J.J., CYRUS, M.D., BRAND, M.J., JOUBERT, M. & MACEY, B.M Why grow Ulva? Its potential role in the future of aquaculture. Perspectives in Phycology, 3, , /pip/2016/0058. BOLTON, J.J., ROBERTSON-ANDERSSON, D. V., SHUULUKA, D. & KANDJENGO, L Growing ulva (chlorophyta) in integrated systems as a commercial crop for abalone feed in South africa: A swot analysis. Journal of Applied Phycology, 21, , /s BOUBONARI, T., MALEA, P. & KEVREKIDIS, T The green seaweed Ulva rigida as a bioindicator of metals (Zn, Cu, Pb and Cd) in a low-salinity coastal environment. Botanica Marina, 51, , /BOT CAHILL, P.L., HURD, C.L. & LOKMAN, M Keeping the water clean - Seaweed biofiltration outperforms traditional bacterial biofilms in recirculating aquaculture. Aquaculture, 306, , /j.aquaculture CALICETI, M., ARGESE, E., SFRISO, A. & PAVONI, B Heavy metal contamination in the seaweeds of the Venice lagoon. Chemosphere, 47, CALLOW, J.A. & CALLOW, M.E Spore Adhesive System. In Biological Adhesives CALLOW, M.E., CALLOW, J.A., PICKETT-HEAPS, J.D. & WETHERBEE, R PRIMARY ADHESION OF ENTEROMORPHA (CHLOROPHYTA, ULVALES) PROPAGULES: QUANTITATIVE SETTLEMENT STUDIES AND VIDEO MICROSCOPY. Journal of Phycology, 947, /86

70 CARL, C., DE NYS, R. & PAUL, N.A The seeding and cultivation of a tropical species of filamentous Ulva for algal biomass production. PLoS ONE, 9, /journal.pone CARL, C., LAWTON, R.J., PAUL, N.A. & DE NYS, R. 2016a. Reproductive output and productivity of filamentous tropical Ulva over time. Journal of Applied Phycology, 28, , /s CARL, C., MAGNUSSON, M., PAUL, N.A. & DE NYS, R. 2016b. The yield and quality of multiple harvests of filamentous Ulva tepida. Journal of Applied Phycology, 28, , /s CASTELAR, B., REIS, R.P. & DOS SANTOS CALHEIROS, A.C Ulva lactuca and U. flexuosa (Chlorophyta, Ulvophyceae) cultivation in Brazilian tropical waters: Recruitment, growth, and ulvan yield. Journal of Applied Phycology, 26, , /s z. CEDERWALL, H. & ELMGREN, R Biological Effects of Eutrophication in the Baltic Sea, Particularly the Coastal Zone. Ambio, 19, CHAFFEY, N Raven biology of plants. 8th ed. Annals of Botany, /aob/mcu090. CHARLIER, R.H., FINKL, C.W. & KRYSTOSYK-GROMADZINSKA, A Throw It Overboard: A Commentary on Coastal Pollution and Bioremediation. Journal of Coastal Research, 28, , CHARLIER, R.H., MORAND, P. & FINKL, C.W How Brittany and Florida coasts cope with green tides. International Journal of Environmental Studies, 65, , / CHRISTIE, A.O. & SHAW, M Settlement experiments with zoospores of Enteromorpha intestinalis (L.) link. British Phycological Bulletin, 3, , / COELHO, J.P., PEREIRA, M.E., DUARTE, A.C. & PARDAL, M.A Contribution of primary producers to mercury trophic transfer in estuarine ecosystems: Possible effects of eutrophication. Marine Pollution Bulletin, 58, , /j.marpolbul COHEN, I. & NEORI, A Ulva lactuca Biofilters for Marine Fishpond Effluents (I. Ammonia Uptake Kinetics and Nitrogen Content). Botanica Marina, 34, COHEN, R.A. & FONG, P Using Opportunistic Green Macroalgae as Indicators of Nitrogen Supply and Sources to Estuaries. Ecological Applications, 16, CONTI, M.E. & CECCHETTI, G A biomonitoring study: trace metals in algae and molluscs from Tyrrhenian coastal areas. Environmental Research, 93, , /S (03) CRUZ-SUÁREZ, L.E., TAPIA-SALAZAR, M., NIETO-LÓPEZ, M.G., GUAJARDO-BARBOSA, C. & RICQUE-MARIE, D Comparison of Ulva clathrata and the kelps Macrocystis pyrifera and Ascophyllum nodosum as ingredients in shrimp feeds. Aquaculture Nutrition, 15, , /j x. DE JESUS RAPOSO, M.F., DE MORAIS, A.M.B. & DE MORAIS, R.M.S.C Marine polysaccharides from algae with potential biomedical applications. Marine Drugs, 13, , /md DESIDERI, D., CANTALUPPI, C., CECCOTTO, F., MELI, M.A., ROSELLI, C. & FEDUZI, L Essential and toxic elements in seaweeds for human consumption. Journal of Toxicology and Environmental Health, 79, , / DIOP, M. & AMARA, R Mercury concentrations in the coastal marine food web along the Senegalese coast. Environmental Science and Pollution Research, , /s x. DUARTE, C.M., WU, J., XIAO, X., BRUHN, A. & KRAUSE-JENSEN, D Can Seaweed Farming Play a Role in 69/86

71 Climate Change Mitigation and Adaptation? Frontiers in Marine Science, 4, /fmars EL-DIN, N.G.S. & MOHAMEDEIN, L.I Seaweeds as bioindicators of heavy metals off a hot spot area on the Egyptian Mediterranean Coast during Environ Monit Assess, , /s EL-WAZIRY, A., AL-HAIDARY, A., OKAB, A., SAMARA, E. & ABDOUN, K Effect of dietary seaweed (Ulva lactuca) supplementation on growth performance of sheep and on in vitro gas production kinetics. Turkish Journal of Veterinary and Animal Sciences, 39, 81 86, /vet ETHERIDGE, R.., PESTI, G.. & FOSTER, E A comparison of nitrogen values obtained utilizing the Kjeldahl nitrogen and Dumas combustion methodologies (Leco CNS 2000) on samples typical of an animal nutrition analytical laboratory. Animal Feed Science and Technology, 73, 21 28, /S (98) EUROPEAN COMMISSION Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Communitites, L364, 5 24, /dose-response Hanekamp. EUROPEAN COMMISSION Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed - Council statement. Official Journal of the European Communitites, FALANDYSZ, J Mercury concentrations in benthic animals and plants inhabiting the Gulf of Gdafisk, Baltic Sea. The Science of Total Environment, 141, FAVERO, N., CATTALINI, F., BERTAGGIA, D. & ALBERGONI, V Metal Accumulation in a Biological Indicator (Ulva rigida) from the Lagoon of Venice (Italy). Arch. Environ. Contain. Toxicol, 18, FAVERO, N. & FRIGO, M.G BIOMONITORING OF METAL AVAILABILITY IN THE SOUTHERN BASIN OF THE LAGOON OF VENICE (ITALY) BY MEANS OF MACROALGAE. Water, Air, and Soil Pollution, FELDMANN, J., JOHN, K. & PENGPRECHA, P Arsenic metabolism in seaweed-eating sheep from Northern Scotland. Fresenius Journal of Analytical Chemistry, 368, , /s FERREIRA, J.G Factors governing mercury accumulation in three species of marine macroalgae. Aquatic Botany, 39, FILOMENA, M., RAPOSO, D.J., MARIA, A., BERNARDO, M., MANUEL, R. & COSTA, S Emergent Sources of Prebiotics: Seaweeds and Microalgae. Marine Drugs, 1 27, /md FOSTER, G.G. & HODGSON, A.N Consumption and apparent dry matter digestibility of six intertidal macroalgae by Turbo sarmaticus (Mollusca: Vetigastropoda: Turbinidae). Aquaculture, 167, , /S (98) FYTIANOS, K., EVGENIDOU, E. & ZACHARIADIS, G Use of Macroalgae as Biological Indicators of Heavy Metal Pollution in Thermaikos Gulf, Greece. Bulletin of Environmental Contamination and Toxicology, GAO, G., CLARE, A.S., ROSE, C. & CALDWELL, G.S. 2017a. Eutrophication and warming-driven green tides (Ulva rigida) are predicted to increase under future climate change scenarios. Marine Pollution Bulletin, 114, , /j.marpolbul GAO, G., CLARE, A.S., ROSE, C. & CALDWELL, G.S. 2017b. Intrinsic and extrinsic control of reproduction in the green tide-forming alga, Ulva rigida. Environmental and Experimental Botany, 139, 14 22, 70/86

72 /j.envexpbot GAO, G., CLARE, A.S., ROSE, C. & CALDWELL, G.S. 2017c. Non-cryogenic preservation of thalli, germlings, and gametes of the green seaweed Ulva rigida. Aquaculture, 473, , /j.aquaculture GAO, S., CHEN, X., YI, Q., WANG, G., PAN, G., LIN, A. & PENG, G A strategy for the proliferation of Ulva prolifera, main causative species of green tides, with formation of sporangia by fragmentation. PLoS ONE, 5, 1 7, /journal.pone GARAI, T., LOCATELLI, C. & TORSI, G Determination of heavy metals in environmental bio-indicators by voltammetric and spectroscopic techniques. Fresenius journal of analytical chemistry, GARCIA-SARTAL, C., TAEBUNPAKUL, S., STOKES, E., BARCIELA-ALONSO, M. DEL C., BERMEJO-BARRERA, P. & GOENAGA- INFANTE, H Two-dimensional HPLC coupled to ICP-MS and electrospray ionisation (ESI)-MS/MS for investigating the bioavailability in vitro of arsenic species from edible seaweed. Analytical and Bioanalytical Chemistry, , /s GAUDRY, A., ZEROUAL, S., GAIE-LEVREL, F., MOSKURA, M., BOUJRHAL, F.Z., EL MOURSLI, R.C., GUESSOUS, A., MOURADI, A., GIVERNAUD, T. & DELMAS, R Heavy metals pollution of the atlantic marine environment by the Moroccan phosphate industry, as observed through their bioaccumulation in Ulva lactuca. Water, Air, and Soil Pollution, 178, , /s GHADIRYANFAR, M., ROSENTRATER, K.A., KEYHANI, A. & OMID, M A review of macroalgae production, with potential applications in biofuels and bioenergy. Renewable and Sustainable Energy Reviews, 54, , /j.rser GUILLARD, R.R.L Vulture of phyto plankton for feeding marine invertebrates. Smith, W.L. & Matoira, H.C., eds. Culture of Marine Invertebrate Animals, pp. GUILLARD, R.R.L. & RYTHER, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology, 8(2), GÜROY, B., ERGÜN, S., MERRIFIELD, D.L. & GÜROY, D Effect of autoclaved Ulva meal on growth performance, nutrient utilization and fatty acid profile of rainbow trout, Oncorhynchus mykiss. Aquaculture International, 21, , /s HANSEN, H.P. & KOROLEFF, F Determination of nutrients. In Methods of Seawater Analysis: Third, Completely Revised and Extended Edition , / ch10. HANSEN, T.H., LAURSEN, K.H., PERSSON, D.P., PEDAS, P., HUSTED, S. & SCHJOERRING, J.K Micro-scaled high throughput digestion of plant tissue samples for multi-elemental analysis. Plant Methods, 12, 1 11, / HARDOUIN, K., BEDOUX, G., BURLOT, A.S., DONNAY-MORENO, C., BERGÉ, J.P., NYVALL-COLLÉN, P. & BOURGOUGNON, N Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae). Algal Research, 16, , /j.algal HARITONIDIS, S. & MALEA, P Bioaccumulation of metals by the green alga Ulva rigida from Thermaikos Gulf, Greece. Environmental Pollution, 104, HAYDEN, H.S., BLOMSTER, J., MAGGS, C.A., SILVA, P.C., STANHOPE, M.J. & WAALAND, J.R Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera. European Journal of Phycology, 38, , / /86

73 HAYES, M. & TIWARI, B.K Bioactive carbohydrates and peptides in foods: An overview of sources, downstream processing steps and associated bioactivities. International Journal of Molecular Sciences, 16, , /ijms HEESCH, S., BROOM, J.E.S., NEILL, K.F., FARR, T.J., DALEN, J.L. & NELSON, W.A Ulva, Umbraulva and Gemina: Genetic survey of New Zealand taxa reveals diversity and introduced species. European Journal of Phycology, 44, , / HERING, D., BORJA, A., CARSTENSEN, J., CARVALHO, L., ELLIOTT, M., FELD, C.K., HEISKANEN, A.S., et al The European Water Framework Directive at the age of 10: A critical review of the achievements with recommendations for the future. Science of the Total Environment, 408, , /j.scitotenv HIRATA, S. & TOSHIMITSU, H Determination of arsenic species and arsenosugars in marine samples by HPLC-ICP-MS. Applied Organometallic Chemistry, 21, , /aoc HO, Y.B Ulva lactuca as bioindicator of metal contamination in intertidal waters in Hong Kong. Hydrobiologia, 203, HOLDT, S.L. & KRAAN, S Bioactive compounds in seaweed: functional food applications and legislation. Journal of Applied Phycology, , /s HORTAS, V.R., GARCÍA-SARTAL, C., BARCIELA-ALONSO, M.C., DOMÍNGUEZ-GONZÁLEZ, R., MOREDA-PIÑEIRO, A. & BERMEJO-BARRERA, P Bioavailability study using an in-vitro method of iodine and bromine in edible seaweed. Food Chemistry, 124, , /j.foodchem HOU, X., CHAI, C., QIAN, Q., YAN, X. & FAN, X Determination of chemical species of iodine in some seaweeds (I). The Science of the Total Environment, 204, HWANG, Y.O., PARK, S.G., PARK, G.Y., CHOI, S.M., KIM, M.Y., PARK, S.G., PARK, G.Y., CHOI, S.M. & TOTAL, M.Y.K Total arsenic, mercury, lead, and cadmium contents in edible dried seaweed in Korea. Food Additives & Contaminants, 3210, 7 13, / ICHIHARA, K., MIYAJI, K. & SHIMADA, S Comparing the low-salinity tolerance of Ulva species distributed in different environments , /j x. IMCHEN, T Recruitment Potential of a Green Alga Ulva flexuosa Wulfen Dark Preserved Zoospore and Its Development. PLoS ONE, 7, 1 5, /journal.pone ISRAEL, A.A., FRIEDLANDER, M. & NEORI, A Biomass Yield, Photosynthesis and Morphological Expression of Ulva lactuca. Botanica Marina, 38, JACOBS, A With Surf Like Turf, Huge Algae Bloom Befouls China Coast. The New York Times. JIANG, J., BAUER, I., PAUL, A. & KAPPLER, A Arsenic redox changes by microbially and chemically formed semiquinone radicals and hydroquinones in a humic substance model quinone. Environmental Science and Technology, 43, , /es803112a. JIE, X., XIAOHONG, Z., CHUNLEI, G.A.O., MEIJIE, J., RUIXIANG, L.I., ZONGLING, W., YAN, L.I., SHILIANG, F.A.N. & XUELEI, Z Effect of temperature, salinity and irradiance on growth and photosynthesis of Ulva prolifera. Acta Oecologica, 35, , /s JOUVENEL, J.Y. & POLLARD, D.A Survey of macroalgal mats in the Gulf of Finland, Baltic Sea. Aquatic Conservation: Marine and Freshwater Ecosystems, 11, 11 18, /aqc.428. JUNG, H., KIM, J. & LEE, C Continuous anaerobic co-digestion of Ulva biomass and cheese whey at 72/86

74 varying substrate mixing ratios: Different responses in two reactors with different operating regimes. Bioresource Technology, 221, , /j.biortech KA, E. & ELTEREN, J.T. VAN Arsenosugars and other arsenic compounds in littoral zone algae from the Adriatic Sea. Chemosphere, 63, , /j.chemosphere KALITA, T.L. & TYTLIANOV, E.A Effect of temperature and illumination on growth and reproduction of the green alga Ulva fenestrata. Russian Journal of Marine Biology, 29, KAMER, K. & FONG, P Nitrogen enrichment ameliorates the negative effects of reduced salinity on the green macroalga Enteromorpha intestinalis. Marine Ecology Progress Series, 218, 87 93, /meps KAMER, K., FONG, P., KENNISON, R. & SCHIFF, K Nutrient limitation of the macroalga Enteromorpha intestinalis collected along a resource gradient in a highly eutrophic estuary. Estuaries and Coasts, 27, , /BF LAIB, E. & LEGHOUCHI, E Cd, Cr, Cu, Pb, and Zn concentrations in Ulva lactuca, Codium fragile, Jania rubens, and Dictyota dichotoma from Rabta Bay, Jijel (Algeria). Environ Monit Assess, 184, , /s LAMARE, M.D. & WING, S.R Calorific content of New Zealand marine macrophytes. New Zealand Journal of Marine and Freshwater Research, 8330, / LAMPRIANIDOU, F., TELFER, T. & ROSS, L.G A model for optimization of the productivity and bioremediation efficiency of marine integrated multitrophic aquaculture. Estuarine, Coastal and Shelf Science, 164, , /j.ecss LAWLOR, P.G., HUGHES, H. & GARDINER, G.E Prebiotics from Marine Macroalgae for Human and Animal Health Applications , /md LAWTON, R.J., MATA, L., DE NYS, R. & PAUL, N.A Algal Bioremediation of Waste Waters from Land-Based Aquaculture Using Ulva: Selecting Target Species and Strains. PLoS ONE, 8, /journal.pone LEAL, M.C.F., VASCONCELOS, M.T., SOUSA-PINTOT, I. & CABRALT, J.P.S Biomonitoring with Benthic Macroalgae and Direct Assay of Heavy Metals in Seawater of the Oporto Coast (Northwest Portugal). Marine Pollution Bulletin, 34. LENTZ, V.L.F Role of cold resistance and burial for winter survival and spring initiation of an Ulva spp. (Chlorophyta) bloom in a eutrophic lagoon (Veerse Meer lagoon, The Netherlands). Marine Biology, LI, H., ZHANG, Y., TANG, H., SHI, X., RIVKIN, R.B. & LEGENDRE, L Spatiotemporal variations of inorganic nutrients along the Jiangsu coast, China, and the occurrence of macroalgal blooms (green tides) in the southern Yellow Sea. Harmful Algae, 63, , /j.hal LIÈVREMONT, D., BERTIN, P.N. & LETT, M.C Arsenic in contaminated waters: Biogeochemical cycle, microbial metabolism and biotreatment processes. Biochimie, 91, , /j.biochi LIN, S., SHI, Q., BRENT NIX, F., STYBLO, M., BECK, M. A., HERBIN-DAVIS, K.M., HALL, L.L., SIMEONSSON, J.B. & THOMAS, D.J A novel S-adenosyl-L-methionine:arsenic(III) methyltransferase from rat liver cytosol. Journal of Biological Chemistry, 277, , /jbc.M LLORENTE-MIRANDES, T., RUIZ-CHANCHO, M.J., BARBERO, M., RUBIO, R. & LÓPEZ-SÁNCHEZ, J.F Measurement 73/86

75 of arsenic compounds in littoral zone algae from the Western Mediterranean Sea. Occurrence of arsenobetaine. Chemosphere, 81, , /j.chemosphere LÜNING, K., KADEL, P. & PANG, S Control of reproduction rhythmicity by environmental and endogenous signals in Ulva pseudocurvata (Chlorophyta). Journal of Phycology, 44, , /j x. MACHADO, L., MAGNUSSON, M., PAUL, N.A., DE NYS, R. & TOMKINS, N Effects of marine and freshwater macroalgae on in vitro total gas and methane production. PLoS ONE, 9, /journal.pone MAEHRE, H.K., MALDE, M.K., EILERTSEN, K.-E. & ELVEVOLL, E.O Characterization of protein, lipid and mineral contents in common Norwegian seaweeds and evaluation of their potential as food and feed. Journal of the Science of Food and Agriculture, 94, , /jsfa MAIA, M.R.G., FONSECA, A.J.M., OLIVEIRA, H.M., MENDONÇA, C. & CABRITA, A.R.J The potential role of seaweeds in the natural manipulation of rumen fermentation and methane production. Scientific Reports, 6, 1 10, /srep MAIRH, O.P., RAMAVAT, B.K., TEWARI, A., OZA, R.M. & JOSHI, H. V Seasonal variation, bioaccumulation and prevention of loss of iodine in seaweeds. Phytochemistry, 28, , / (89)80336-X. MALEA, P., CHATZIAPOSTOLOU, A. & KEVREKIDIS, T Trace element seasonality in marine macroalgae of different functional-form groups. Marine Environmental Research, 103, 18 26, /j.marenvres MALEA, P. & HARITONIDIS, S Metal content in Enteromorpha linza (Linnaeus) in Thermaikos Gulf (Greece). Hydrobiologia, MALEA, P. & HARITONIDIS, S Use of the green alga Ulva rigida C. Agardh as an indicator species to reassess metal pollution in the Thermaikos Gulf, Greece, after 13 years. Journal of Applied Phycology, 120, MALONE RUBRIGHT, S.L., PEARCE, L.L. & PETERSON, J Environmental toxicology of hydrogen sulfide. Nitric Oxide, 71, 1 13, /j.niox MARINA, B. & HÄGERHÄLL, B Marine Botanical-hydrographical Trace Element Studies in the Öresund Area. Botanica Marina, XVI. MARINHO, G.S., HOLDT, S.L., BIRKELAND, M.J. & ANGELIDAKI, I Commercial cultivation and bioremediation potential of sugar kelp, Saccharina latissima, in Danish waters. Journal of Applied Phycology, 27, , /s MARSHAM, S., SCOTT, G.W. & TOBIN, M.L Comparison of nutritive chemistry of a range of temperate seaweeds. Food Chemistry, 100, , /j.foodchem MARTINS, I., MIGUEL, J., FLINDT, M.R. & CARLOS, J The effect of salinity on the growth rate of the macroalgae Enteromorpha intestinalis (Chlorophyta) in the Mondego estuary (west Portugal). Acta Oecologica, 20, MCAVOY, K.M. & KLUG, J.L Positive and negative effects of riverine input on the estuarine green alga Ulva intestinalis (syn. Enteromorpha intestinalis) (Linneaus). Hydrobiologia, 545, 1 9, /s MESSYASZ, B. & RYBAK, A Abiotic factors affecting the development of Ulva sp. (Ulvophyceae; 74/86

76 Chlorophyta) in freshwater ecosystems. Aquatic Ecology, 75 87, /s MICHALAK, I. & CHOJNACKA, K Edible macroalga Ulva prolifera as microelemental feed supplement for livestock: The fundamental assumptions of the production method. World Journal of Microbiology and Biotechnology, 25, , /s MICHALAK, I. & CHOJNACKA, K Multielemental analysis of macroalgae from the Baltic Sea by ICP-OES to monitor environmental pollution and assess their potential uses. International Journal of Environmental Analytical Chemistry, 89, , / MILLEDGE, J.J. & NIELSEN, B. V High-value products from macroalgae : the potential uses of the invasive brown seaweed, Sargassum muticum. Reviews in Environmental Science and Bio/Technology, 15, 67 88, /s MINISTRY OF ENVIRONMENT AND FOOD OF DENMARK. 2017a. Jod i fødevarer Available at: [Accessed February 20, 2018]. MINISTRY OF ENVIRONMENT AND FOOD OF DENMARK. 2017b. Prøveresultater - hygiejnisk kvalitet af spiselig tang Available at: er_fisk_og_fiskeprodukter_spiselig_tang.aspx [Accessed February 9, 2018]. MOHSEN, A.F., KHALEATA, A.F., HASHEM, M.A. & METWALLI, A Effect of Different Nitrogen Sources on Growth, Reproduction, Amino Acid, Fat and Sugar Contents in Ulva fasciata Delile (Part III). Botanica Marina, 17, 1 2, /botm MOHSEN, A.F., NASR, A.H. & METWALLI, A.M Effect of temperature variations on growth, reproduction, amino acid synthesis, fat and sugar content in ulva fasciata delile plants. Hydrobiologia, 42, , /BF MSUYA, F.E. & NEORI, A Effect of water aeration and nutrient load level on biomass yield, N uptake and protein content of the seaweed Ulva lactuca cultured in seawater tanks. Journal of Applied Phycology, 20, , /s NAIDOO, K., MANEVELDT, G., RUCK, K. & BOLTON, J.J A comparison of various seaweed-based diets and formulated feed on growth rate of abalone in a land-based aquaculture system. Journal of Applied Phycology, 18, , /s NCBI TAXONOMY Ulva Available at: ep=1&srchmode=1&unlock [Accessed November 13, 2017]. NECAS, J. & BARTOSIKOVA, L Carrageenan: a review. Veterinarni Medicina, 2013, NEDERGAARD, R.I., RISGAARD-PETERSEN, N. & FINSTER, K The importance of sulfate reduction associated with Ulva lactuca thalli during decomposition: A mesocosm experiment. Journal of Experimental Marine Biology and Ecology, 275, 15 29, /S (02) NEUSHUL, M., BENSON, J., HARGER, B.W.W. & CHARTERS, A.C Macroalgal farming in the sea: water motion and nitrate uptake. Journal of Applied Phycology, NEVEUX, N., BOLTON, J.J., BRUHN, A., ROBERTS, D.A. & RAS, M The bioremediation potential of seaweeds: Recycling nitrogen, phosphorus and other waste products. Blue technology: Production and uses of marine molecules, /86

77 NIELSEN, M.M., BRUHN, A., RASMUSSEN, M.B., OLESEN, B., LARSEN, M.M. & MØLLER, H.B Cultivation of Ulva lactuca with manure for simultaneous bioremediation and biomass production. Journal of Applied Phycology, , /s z. NISIZAWA, K., NODA, H., KIKUCHI, R. & WATANABE, T The main seaweed foods in Japan. Hydrobiologia, 29, NONOVA, T. & TOSHEVA, Z Sr, 210 Pb, 210 Po and Ra isotopes in marine macroalgae and mussel Mytilus galloprovincialis from the Bulgarian Black Sea zone. Journal of Radioanalytical and Nuclear Chemistry, 307, , /s x. NORDBY, Ø. & HOXMARK, R.C Changes in cellular parameters during synchronous meiosis in Ulva mutabilis Føyn. Experimental Cell Research, 75, NUNES, N., FERRAZ, S., VALENTE, S., BARRETO, M.C. & PINHEIRO DE CARVALHO, M.A.A Biochemical composition, nutritional value, and antioxidant properties of seven seaweed species from the Madeira Archipelago. Journal of Applied Phycology, 29, , /s x. OHLENDORF, D.H., LIPSCOMB, J.D. & WEBER, P.C Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature, 336, , /332141a0. ORTIZ, J., ROMERO, N., ROBERT, P., ARAYA, J., LOPEZ-HERNÁNDEZ, J., BOZZO, C., NAVARRETE, E., OSORIO, A. & RIOS, A Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chemistry, 99, , /j.foodchem PALLAORO, M.F., DO NASCIMENTO VIEIRA, F. & HAYASHI, L Ulva lactuca (Chlorophyta Ulvales) as co-feed for Pacific white shrimp. Journal of Applied Phycology, 28, , /s PEDERSEN, M.F. & BORUM, J Nutrient control of algal growth in estuarine waters. Nutrient limitation and the importance of nitrogen requirements... Oceanographic Literature Review, 44, 315, /meps PELL, A., KOKKINIS, G., MALEA, P., PERGANTIS, S.A., RUBIO, R. & LÓPEZ-SÁNCHEZ, J.F LC ICP MS analysis of arsenic compounds in dominant seaweeds from the Thermaikos Gulf (Northern Aegean Sea, Greece). Chemosphere, 93, , /j.chemosphere PETRICK, J.S., JAGADISH, B., MASH, E. A & APOSHIAN, H. V Monomethylarsonous acid (MMA(III)) and arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate dehydrogenase. Chemical research in toxicology, 14, , /tx000264z. PETRICK, J.S., AYALA-FIERRO, F., CULLEN, W.R., CARTER, D.E. & VASKEN APOSHIAN, H Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes. Toxicology and applied pharmacology, 163, , /taap PHANEUF, D., CO, I., DUMAS, P., FERRON, L.A. & LEBLANC, A Evaluation of the Contamination of Marine Algae (Seaweed) from the St. Lawrence River and Likely to Be Consumed by Humans. Environmental Research Section A, 80, PRAEGER, C. & DE NYS, R Seeding filamentous Ulva tepida on free-floating surfaces: A novel cultivation method. Algal Research, 24, 81 88, /j.algal QUILLIEN, N., NORDSTRÖM, M.C., GUYONNET, B., MAGUER, M., LE GARREC, V., BONSDORFF, E. & GRALL, J Large-scale effects of green tides on macrotidal sandy beaches: Habitat-specific responses of zoobenthos. Estuarine, Coastal and Shelf Science, 164, , /j.ecss RAMASANY S. Y KUMAR Anti Inflamatory, Antinociceptive and Central Nervous System Depressant 76/86

78 Activites of Marine Bacterial Extracts. Journnal of Pharmacology and Toxicology, 4, RASYID, A Evaluation of nutritional composition of the dried seaweed Ulva lactuca from Pameungpeuk waters, Indonesia. Tropical Life Sciences Research, 28, , /tlsr REIFFENSTEIN, R.J., HULBERT, W.C. & ROTH, S.H Toxicology of Hydrogen Sulfide. Annual Review of Pharmacology and Toxicology, 32, , /annurev.pa ROCKTOPUS, M Ulva Life Cycle Available at: [Accessed October 19, 2017]. RODR, A.P. & IGNACIO, S Element concentrations in some species of seaweeds from La Paz Bay and La Paz Lagoon, south-western Baja California, Mexico , /s z. ROWCLIFFE, J.M., WATKINSON, A.R., SUTHERLAND, W.J. & VICKERY, J.A The depletion of algal beds by geese: A predictive model and test. Oecologia, 127, , /s RYAN, S., MCLOUGHLIN, P. & DONOVAN, O.O A comprehensive study of metal distribution in three main classes of seaweed. Environmental Pollution, 167, , /j.envpol RYBAK, A., MESSYASZ, B. & ŁESKA, B. 2012a. Bioaccumulation of alkaline soil metals (Ca, Mg) and heavy metals (Cd, Ni, Pb) patterns expressed by freshwater species of Ulva (Wielkopolska, Poland). Internat. Rev. Hydrobiol., , /iroh RYBAK, A., MESSYASZ, B. & ŁESKA, B. 2012b. Freshwater Ulva (Chlorophyta) as a bioaccumulator of selected heavy metals (Cd, Ni and Pb) and alkaline earth metals (Ca and Mg). Chemosphere, 89, , /j.chemosphere SAND-JENSEN, K Minimum light requirements for growth in Ulva lactuca. Marine Ecology Progress Series, 50, SARTAL, C.G., BARCIELA-ALONSO, M.C. & BERMEJO-BARRERA, P Effect of the cooking procedure on the arsenic speciation in the bioavailable (dialyzable) fraction from seaweed. Microchemical Journal, 105, 65 71, /j.microc SAWIDIS, T., BROWN, M.T., ZACHARIADIS, G. & SRATIS, I Trace metal concentrations in marine macroalgae from different biotopes in the Aegean Sea. Environment International, 27, SCHINTU, M., MARRAS, B., DURANTE, L., MELONI, P. & CONTU, A Macroalgae and DGT as indicators of available trace metals in marine coastal waters near a lead zinc smelter. Environ Monit Assess, , /s SCHORIES, D Sporulation of Enteromorpha spp. (chlorophyta) and overwintering of spores in sediments of the Wadden Sea. Netherlands Journal of Aquatic Ecology, 29, SEGURA, M., MUÑOZ, J., MADRID, Y. & CÁMARA, C Stability study of As(III), As(V), MMA and DMA by anion exchange chromatography and HG-AFS in wastewater samples. Analytical and Bioanalytical Chemistry, 374, , /s SFRISO, A., MARCOMINI, A. & ZANETTE, M Heavy Metals in Sediments, SPM and Phytozoobenthos of the Lagoon of Venice. Marine Pollution Bulletin, 30. SIMPLEMAPS Free Denmark SVG Map Available at: [Accessed November 17, 2017]. 77/86

79 SMETACEK, V. & ZINGONE, A Green and golden seaweed tides on the rise. Nature, 504, 84 88, /nature SMITH, J.L., SUMMERS, G. & WONG, R Nutrient and heavy metal content of edible seaweeds in New Zealand. New Zealand Journal of Crop and Horticultural Science, 38, 19 28, / SOUSA, A.I., MARTINS, I., LILLEBØ, A.I., FLINDT, M.R. & PARDAL, M.A Influence of salinity, nutrients and light on the germination and growth of Enteromorpha sp. spores. Journal of Experimental Marine Biology and Ecology, 341, , /j.jembe STORELLI, M.M., STORELLI, A. & MARCOTRIGIANO, G.O Heavy metals in the aquatic environment of the Southern Adriatic Sea, Italy Macroalgae, sediments and benthic species. Environment International, 26, STRATMANN, J., PAPUTSOGLU, G. & OERTEL, W Differentiation of Ulva Mutabilis (Chlorophyta) Gametangia and Gamete Release Are Controlled By Extracellular Inhibitors1. Journal of Phycology, 32, , /j x. STREZOV, A. & NONOVA, T Influence of macroalgal diversity on accumulation of radionuclides and heavy metals in Bulgarian Black Sea ecosystems. Journal of Environmental Radioactivity, 100, , /j.jenvrad STREZOV, A.S. & NONOVA, T.P Comparative analysis of heavy metal and radionuclide contaminants in Black Sea green and red macroalgae. Water Science & Technology, 51, 1 8. SUUTARI, M., LESKINEN, E., SPILLING, K., KOSTAMO, K. & SEPPÄLÄ, J Nutrient removal by biomass accumulation on artificial substrata in the northern Baltic Sea. Journal of Applied Phycology, 29, , /s SZEFER, P Concentration of elements in some seaweeds from Coastal region of the Southern Baltic and in the Żarnowiec Lake. Oceanologia, 25. TABARSA, M., REZAEI, M., RAMEZANPOUR, Z. & WAALAND, J.R Chemical compositions of the marine algae Gracilaria salicornia (Rhodophyta) and Ulva lactuca (Chlorophyta) as a potential food source. Journal of the Science of Food and Agriculture, 92, , /jsfa TABOADA, C., MILLÁN, R. & MÍGUEZ, I Composition, nutritional aspects and effect on serum parameters of marine algae Ulva rigida. Journal of the Science of Food and Agriculture, 90, , /jsfa TANNER, C.E Chloropelta Gen. Nov., an Ulvaceous Green Alga With a Different Type.of Development. Journal of Phycology, 16, , /j tb03007.x. THILLY, C.H., VANDERPAS, J.B., BEBE, N., NTAMBUE, K., CONTEMPRE, B., SWENNEN, B., MORENO-REYES, R., BOURDOUX, P. & DELANGE, F Iodine Deficiency, Other Trace Elements, and Goitrogenic Factors in the Etiopathogeny of lodine Deficiency Disorders (IDD). The Humana Press, TRIFAN, A. & CHIMIE, R.R. DE Heavy metal content in macroalgae from Roumanian Black Sea. Revue Romaine de Chimie, 60, USTUNADA, M., ERDUGAN, H., YILMAZ, S., AKGUL, R. & AYSEL, V Seasonal concentrations of some heavy metals (Cd, Pb, Zn, and Cu) in Ulva rigida J. Agardh (Chlorophyta) from Dardanelles (Canakkale, Turkey). Environ Monit Assess, , /s VAN ALSTYNE, K.L Seasonal changes in nutrient limitation and nitrate sources in the green macroalga 78/86

80 Ulva lactuca at sites with and without green tides in a northeastern Pacific embayment. Marine Pollution Bulletin, 103, , /j.marpolbul VENTURA, M.. & CASTAÑÓN, J.I The nutritive value of seaweed (Ulva lactuca) for goats. Small Ruminant Research, 29, , /S (97)00134-X. VENTURA, M.R., CASTAÑON, J.I.R. & MCNAB, J.M Nutritional value of seaweed (Ulva rigida) for poultry. Animal Feed Science and Technology, 49, 87 92, / (94) VESTY, E.F., KESSLER, R.W., WICHARD, T. & COATES, J.C Regulation of gametogenesis and zoosporogenesis in Ulva linza (Chlorophyta): comparison with Ulva mutabilis and potential for laboratory culture. Frontiers in plant science, 6, 15, /fpls VILLARES, R., PUENTE, X. & CARBALLEIRA, A Seasonal variation and background levels of heavy metals in two green seaweeds. 119, VILLARES, R., PUENTE, X. & CARBALLEIRA, A Ulva and Enteromorpha as indicators of heavy metal pollution WAN, A.H.L., WILKES, R.J., HEESCH, S., BERMEJO, R., JOHNSON, M.P. & MORRISON, L Assessment and characterisation of Ireland s green tides (Ulva species). PLoS ONE, 12, /journal.pone WANG, C., YU, R. & ZHOU, M Acute toxicity of live and decomposing green alga Ulva (Enteromorpha) prolifera to abalone Haliotis discus hannai. Chinese Journal of Oceanology and Limnology, 29, , /s WASSEF, E.A., EL-SAYED, A.F.M. & SAKR, E.M Pterocladia (Rhodophyta) and Ulva (Chlorophyta) as feed supplements for European seabass, Dicentrarchus labrax L., fry. Journal of Applied Phycology, 25, , /s WEIS, J.S. & WEIS, P Transfer of contaminants from CCA-treated lumber to aquatic biota. J. Exp. Mar. Biol. EcoL, 161, WKHLU, D.Q.G., RU, G., OHDVW, D.W., GXH, D.U.H., WKDQDWRFRHQRVHV, W.R. & GLQRIODJHOODWHV, R.I Green tides on the Brittany Coasts. In IEEE US/EU Baltic International Symposium, BALTIC 2006, /BALTIC WONG, K.H. & CHEUNG, P.C.K Nutritional evaluation of some subtropical red and green seaweeds. Food Chemistry, 71, , /S (00) YAICH, H., GARNA, H., BESBES, S., PAQUOT, M., BLECKER, C. & ATTIA, H Chemical composition and functional properties of Ulva lactuca seaweed collected in Tunisia. Food Chemistry, 128, , /j.foodchem YE, N. HAO, ZHANG, X. WEN, MAO, Y. ZE, LIANG, C. WEI, XU, D., ZOU, J., ZHUANG, Z. MENG & WANG, Q. YIN Green tides are overwhelming the coastline of our blue planet: Taking the world s largest example. Ecological Research, 26, , /s ZBIKOWSKI, R., SZEFER, P. & LATA, A Distribution and relationships between selected chemical elements in green alga Enteromorpha sp. from the southern Baltic. Science Direct, 143, , /j.envpol ZHANG, L. & WONG, M.H Environmental mercury contamination in China: Sources and impacts. Environment International, 33, , /j.envint /86

81 6 Appendices 6.1 Collection of Ulva Figure 19. Biomass density measuring within an area of 0.25 m 2, picture Michael Bo Rasmussen. 6.2 Coastal cultivation at Grenaa (O) Fertilization: 9 mm Disks were stamped from the apical part of randomly chosen individuals and poured into 3x2L glass bottles (21 disks per bottles) with 2L of PES enriched sterilized seawater. They were hereafter cultured using 80 PAR and 12 hours light pr day at 10 degrees for 7 days. After 7 days the color of the disks was evaluated, and yellow disks were classified as fertile and green disks as non-fertile. During the experiment 2 disks disappeared from one of the culture flasks. Seeding: On the 27 th of March 2017, a mixture of zygotospores and gametospores from all three replicates in the fertilization experiment were used to seed three spools, with 30 meters of 3 mm Danline twine each, and a 3 x 2 meter Algaetex net with 50 cm mesh size. The spools were placed in a PE basin with app. 30 Liters of Filtered and UV treated seawater, and the net within a 10 L bucket with Filtered and UV treated seawater. 2L of the mixture was poured into the basin with spoles and 1L of the mixture into the bucket, and the basins was darkened for 20 hours. After 20 hours the light was set on again (12 hours light pr day) and the net was transferred to a larger basin (55x75 cm) with appr. 50 L of nutrient enriched seawater at 10 degree celcius. The net and spools were kept under these conditions until deployment with regular nutrient addition and water change (app. every 14 days) Written by Teis Boderskov. 80/86

82 6.3 Kortlægning af søsalat (Ulva lactuca) i Mariager Fjord og Seden Strand Metode Kortlægningen af forekomster af søsalat i Mariager Fjord og Seden strand er foretaget ved hjælp af droneoptagelser, analyse af ortofoto fra 2012, g 2016 samt på baggrund 10 ground truth stationer på hver lokalitet, hvor der er foretaget indsamlinger og bestemmelse af søsalatbiomassens vådvægt og dækningsgrad. Biomasseprøverne er i laboratoriet efterfølgende blevet analyseret for N, P, C, tørstof- og tungmetalindhold. Desuden blev salinitet, temperatur, og iltforhold målt i overfladen og over bunden. Drone flyvninger er udført den 4. september 2017 i Mariager Fjord og den 19. september 2017 i Seden Strand. Optagelserne er udført med Inspire 2 drone langs et udlagt grid med en flyhøjde på m, hvilket giver en pixelopløsning på billederne på mellem 2 og 3 cm. De enkelte billeder er blevet stykket sammen ved hjælp af softwaren Agisoft PhotoScan Professional. Analyse af ortofotos udført på fotos fra 2012, 2014 og 2016 (kilde: Produktblad (DDO2016), COWI, 2016) med en opløsning på cm pr. pixel. Udbredelsen af søsalat på baggrund af droneoptagelserne er blevet analyseret i ArcMap på baggrund af billedernes RGB værdi ved hjælp af en Maximum Likelihood klassificering. Til klassificeringen er anvendt RGB signalet fra de før omtalte ground truth stationer. Klassificeringen er endvidere blevet overført på ortofoto fra 2012, 2014 og Da der ikke forekommer ground truth informationer sammen med disse optagelser, vil analysen af ortofoto være behæftet med større unøjagtigheder end droneoptagelserne. Written by Michael Bo Rasmussen. 6.4 References for literature review (Malea et al. 2015, Pell et al. 2013, Garcia-Sartal et al. 2012, Llorente-mirandes et al. 2010, Strezov & Nonova 2009, Schintu et al. 2010, Boubonari et al. 2008, Favero & Frigo 2002, Caliceti et al. 2002, Sawidis et al. 2001, Storelli et al. 2001, Malea & Haritonidis 2000, Malea & Haritonidis 1999, Garai et al. 1999, Fytianos et al. 1999, Haritonidis & Malea 1999, Leal et al. 1997, Sfriso et al. 1995, Ferreira 1991, Strezov & Nonova 2005, Rybak et al. 2012b, Rybak et al. 2012a, Trifan & Chimie 2015, Marina & Hägerhäll 1973, Falandysz 1994, Coelho et al. 2009, Ryan et al. 2012, Ka & Elteren 2006, Zbikowski et al. 2006, Besada et al. 2009, Szefer 1988, Michalak & Chojnacka 2010, Conti & Cecchetti 2003, Wan et al. 2017, Nonova & Tosheva 2016) 81/86

83 6.5 Evaluation of methods (Results and Discussion) C, N measurements accuracy Five references were used for assessment of C and N contents, Cysteine, Atropine, Sulphanilamide, Acetanilide and Tomato. The found values C and N were in the range of 2 to 7% too low and 5 to 6% too high, respectively (Table 25). It is not assumed that uncertainty in accuracy is high enough to compromise the results for C and N contents in unknown samples. Table 25. Results of this study C and N analysis on references including reported values and the accuracy. Found (%) Reported (%) Accuracy (%) Reference C N C N C N Cysteine Atropine Sulphanilamide Acetanilide Tomato P analyses No reference material was used for assessment of P and therefore two different analytical methods were used, one spectrophotometrically (Hansen & Koroleff 2007) and another by ICP-MS (Table 26). For most of the sample locations there was an agreement between the two methods and overall there was no statistical difference, but P contents found in Ulva at FerH by the method of (Hansen & Koroleff 2007) resulted in a statistical significant higher P contents than found by ICP-MS, i.e vs. 0.24% of DW, respectively. For Ulva samples from Aarhus Beach (O), Grenaa (O) and Seden Beach (P) spectrophotometrical analysis resulted in a statistical significant lower content of P compared to the analysis by ICP-MS, i.e vs. 0.24% of DW, 0.05 vs. 0.06% of DW and 0.24 vs. 0.33% of DW, respectively. Despite the significant difference between the two methods according to P-contents in Ulva from Fredericia (H), Aarhus Beach (O) and Grenaa (O) these differences are very small and therefore not assumed to be of importance. Nevertheless, the difference in the two methods for the analysis of Ulva from Seden Beach (P) was high. P contents found by ICP-MS on average were almost 50% higher than the results of the spectrophotometric analysis. High contents of P in Ulva analysed by ICP-MS exceeded the limits of the standard curve, thus, the results of the spectrophotometric analysis were used for the investigations of this study. 82/86

84 Table 26. The location means for two P-analysis throughout this study and the statistical correleations. Location N a % P of DW b % P of DW c Wilcoxon Prob>ChiSq FreH ±< ±<0.01 P b > P c VejH ± ±0.02 n.s. HorH ± ±0.03 n.s. AarH ±< ±0.01 n.s. AarO ± ±<0.01 P b < P c GreO ±< ±<0.01 P b < P c MarP ± ±0.01 n.s. HalP ± ±0.06 n.s. SedP ± ±0.01 P b < P c NorP ± ± - n.s. Total ± ±0.03 n.s. a Number of samples, b (Hansen & Koroleff 2007), c ICP-MS, (-) no SE due to lack of replicates, (n.s.) not significantly different ICP-MS accuracy The digestion method was tested using the reference: Bladderwrack, (ERM) Fucus vesiculosus [CD200]. A good match was found between reported values of Cd and Pb (Table 27). The detected values of As were a little too high (61.8±1.5 mg kg -1 DW -1 ) according to the reported value (55±4 mg kg -1 DW -1 ), which resulted in an accuracy 113%. The found Hg levels in the reference material could not be accepted as they were under the limit of detection for the analytical method. Table 27. Found and reported elemental contents (mg kg -1 DW -1 ) in reference material, ERM-CD200. Element Found Precision (%) Reported Accuracy (%) 202Hg BDL BDL BDL ± ± Cd ± ± Pb ± ± As ± ± (BDL) Below detection limit. 83/86

85 The best accuracy was achieved for Pb (100%) but this was dependent on a perfect mean of 0.51 mg kg -1 DW -1 though the triplicates ranged from mg kg -1 DW -1. This study followed a rule of thumb that the found values may vary with around 10% from the mean, for the method to be accepted. It is noteworthy that the good accuracy of the method is based on the inclusion of triplicates. The Hg contents in the reference material could not be validated as the values were below the detection limit, so only precision validation could support the results of Hg contents in Danish Ulva ICP-MS precision The precision of metal analyses was tested through duplicates of selected samples (Table 28). The first sample run for Cd, Pb and Hg analysis was removed from the data due to lack of blank between standard and sample resulting in the first sample being target of the memory effect. The average precision for As (range 1-8%) and Hg (range 0-7%) was high and varied only with 3%. Cd had a generally high precision (10%) despite two outliers at 21% (Aarhus (H)) and 28% (Grenaa (O)) and precision of Pb was on average 9%, with four outliers ranging between 12-15%, i.e. Aarhus (H), Grenaa (O), Mariager Fiord (P) and Halkær Broad (P). The outlier variations have in common that they are comparing duplicates with very low contents and it can therefore also be expected that the sensitivity will be decreased. Thus, even some precisions seem to be bad this is assumed to be a low sensitivity of the very low contents and does therefore not seem to compromise the reliability of the results of this study. Table 28. Precision (%) for duplicates between samples. ID Location N As Cd Pb Hg 1 FreH VejH AarH GreO MarP HalP SedP NorP Total (Red, >10%): Poor precision, (-) removed due to memory effect 84/86

86 6.5.5 Comparison of Danish and European Ulva For the study of priority pollutants of European Ulva to be fully reliable results of all analysed samples should be included. However, this was not possible, as many studies reported only mean values. Therefore, whenever a mean value representing only one of the categorised sites was given in a study, this value was included, so mean values of other studies would not be outweighed. When comparing European Ulva to Danish Ulva all the data of this study is included and not only means. Consequently, Danish Ulva is somehow weighed equally against European Ulva, which does not fully represent the truth, but since this comparison serves only in giving an indication of what can be expected, this should not be a problem compromising the conclusions drawn. 6.6 Personal activities and involvements connected to the field of topic Participation at the Seventh Nordic Seaweed Conference 2017 Presented a poster attached in the end of the report. (Title: Toxic metals in European Ulva spp. evaluation of potential use in food and feed applications ). Designed a welcome drink containing spirulina attached in the end of the report. (Title for Danish version: Den Spirulina-inspirerede gin & tonic, Title for English version: The Spirulina inspired gin & tonic ) Participation in the annual Nature Science Festival at Kattegatcenteret 2017 Taught students from primary school how to cultivate sea lettuce attached in the end of the report. (Title: Dyrk din egen søsalat ) Figure 20. The setup for visiting students from primary school. Photo by Lone Thybo Mouritsen. 85/86

87 6.6.3 Part time job at the project Hovedet i Havet Figure 21. Teaching students at a primary school how to grow sea lettuce and how to cook bladder wrack Media coverage Covered by Politiken on the 22 nd of October 2017 in the article: Forskere: Vi står ved foden af et tangeventyr and by Aarhus Stiftstidende on the 8 th of December in the article: Skoleelever indtog skibsdækket (Figure 22). Figure 22. Young students learning to grow their own sea lettuce. Photo from article, linked below. accessed the 1 st of March /86

88 Toxic metals in European Ulva spp. evaluation of potential use in food and feed applications Esben Rimi Christiansen1, Annette Bruhn2 & Jens J. Sloth3 1 Technical University of Denmark, Denmark 2Aarhus University, Department of Bioscience, Denmark 3DTU Food, Technical University of Denmark, Denmark Introduction Results There is an increased interest in Europe to understand and evaluate the commercial potential of high-yielding European seaweed species such as Ulva spp. (European Commission, 2017). This study presents a literature analysis of the content of selected toxic trace metals in European Ulva spp. to assess its potential for application in the food or feed sector. In total 137 data points were extracted from 35 studies. The maximum concentrations of As, Cd, Pb and Hg were 22, 23.6, 748 and 2.2 mg/kg dry weight (DW), respectively. Average concentrations of the complete dataset of the study (Fig 1) and a dataset excluding contaminated sites (Figs 2-5) were evaluated with respect to EU maximum levels (European Parliament, 2015 and 2017). Only a few studies report of concentrations of inorganic arsenic (ias), but a mean value of ± mg/kg DW was found for samples from the Mediterranean Sea. Data collection and analysis Table 1. Mean contents of toxic metals in wild European Ulva spp. collected at non-contaminated sites and European maximum levels for food and feed. The data of this study has been found through the Web of Science (September 2017) using the following keywords: (Ulva OR sea lettuce OR Enteromorpha) AND (metal* OR trace element* OR Hg OR mercury OR Iod* OR arsen* OR Pb OR Cd OR cadmium). Only studies concerning the As-, Cd-, Pb- or Hg contents in Ulva spp. sampled from natural European populations were included in the study. In studies with several data points from samples from same location, only mean values were used. Statistical analyses were performed with JMP (SAS Inc.) (a) No level defined 3 (c) (e) (g) 0.1 (N=7) 5.55 (N=27) 0.81 (N=96) 7.32 (N=96) 0.15 (N=35) <2 (b) 40 (b) 0.5 (d) 5 10 (f ) (h) (a) different rice products, (b) feed materials derived from seaweed, (c) food supplements from dried seaweed, (d) complementary or complete feed, (e) leaf vegetables or food supplements, (f ) complete feed or complementary feed, (g) fishery products, (h) any feed material or fishery products. Abbreviations: dry weight (DW), wet weight (WW) Atlantic Ocean (N=11) Baltic Sea (N=5) Black Sea (N=0) Freshwater (N=0) Atlantic Ocean (N=13) Mediterranean Sea (N=11) Figure 2. Arsenic concentration in wild European Ulva spp. from different non-contaminated marine areas and freshwater (interquartile ranges and outliers). Baltic Sea (N=17) Black Sea (N=7) Freshwater (N=3) Mediterranean Sea (N=56) Figure 4. Lead concentrations in wild European Ulva spp. from different non-contaminated marine areas and freshwater (Interquartile ranges and outliers) Cd (mg/kg DW) Baltic Sea Black Sea Freshwater Mediterranean Sea Figure 1. Concentrations of As. Cd. Pb and Hg in samples of wild European Ulva spp. from different marine areas and freshwater (AV±SE) Atlantic Ocean Hg (mg/kg DW) Cd (mg/kg DW) Pb (mg/kg DW) ias As Cd Pb Hg Pb (mg/kg DW) Hg (mg/kg DW) Mean contents in European Ulva spp. (mg/kgdw) 12 As (mg/kg DW) As (mg/kg DW) 15 Metals Maximum levels (European Parliament, 2015 & 2017) Food Feed (mg/kgww) (mg/kg relative to a moisture content of 12 %) 0 Atlantic Ocean (N=13) Baltic Sea (N=17) Black Sea (N=7) Freshwater (N=3) Mediterranean Sea (N=56) Figure 3. Cadmium concentrations in wild European Ulva spp. from different non-contaminated marine areas and freshwater (interquartile ranges and outliers). Atlantic Ocean (N=19) Baltic Sea (N=11) Black Sea (N=1) Freshwater (N=0) Mediterranean Sea (N=4) Figure 5. Mercury concentrations in wild European Ulva spp. from different non-contaminated marine areas and freshwater (interquartile ranges and outliers). Discussion and concluding remarks Concentrations of total As, ias and Cd in European Ulva spp. sampled at non-contaminated areas do not exceed the EU maximum levels. In contrast, concentrations of Pb and Hg exceeding the maximum levels have been reported, which could potentially limit the potential use of European Ulva spp. in food and/or feed applications. Further investigations are needed to further understand the factors that influence the concentration of toxic metals in Ulva spp. Acknowledgements This study has been conducted as a part of Tang.nu supported by The Velux Foundations and Macrofuels. References European Commission. (2017) GENIALG, Project ID: , web page: last visited 6 Oct 2017 European Parliament. (2015). DIRECTIVE 2002/32/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 7 May 2002 on undesirable substances in animal feed European Parliament. (2017). COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs

89 Nordic Seaweed Konference 17 præsenterer: Den Spirulina-inspirerede gin & tonic Spirulina-essens: ½ tsk Spirulina 1 cl soda water z Spirulina reagerer voldsomt med kulsyre. Derfor er det vigtigt at få den afbruset ved først at lave en essens med danskvand. Dette hjælper også til at få Spirulina til at forekomme homogent i drinken, da den ellers kan give problemer med at koagulere. Det obligatoriske: Gin og agurk 2 cl og en skive. Spirulina har umami, men smager alene ikke godt. Gin lægger sig som et naturligt element med spirulinaen og agurken afrunder smagen så den bliver frisk. Tonic 3 cl Til gin hører tonic, og associationen til den velkendte drink vellidt af mange vil give Spirulinaudviklingen et forspring. Cava/Brut/(Asti) 6 cl Hvis der er noget der kan ødelægge en cocktail er det asti og ekstrem sødme. Min anbefaling er cava eller brut det tørre og sprudlende. Designed and developed by Esben Rimi Christiansen Mulige tilvalg: Blue caracao Få dråber Blandes i spirulinaessensen. Henvender sig til den stilede. Giver et flot farvespil i lagdelingen. Æblecider 3 cl Et lille strejf af sødme fra en æblecider (evt. somersby) kan afrunde den ellers tørre kombination af tonic og cava. Hint: Spirulina-essensen og det obligatoriske kan forberedes individuelt på forhånd og så blandes sammen ved servering. Når først spirulinaen er tilført vil drinken bruse op og senere lægge sig kedeligt. Hvis den går hen og ser kedelig ud kan den blot toppes med mousserende. 1) Den fuldendte Bygget op på spirulinaessensen. 2) Den stilede Først blandes det obligatoriske og så tilføjes spirulinaessensen som et shot.

90 Nordic Seaweed Konference 17 presents: The Spirulina inspired gin & tonic Spirulina-essence: ½ tsp. Spirulina 1 cl soda water z Spirulina reacts strongly with carbonic acid. A bit of the pressure of this reaction can be punched by premixing Spirulina in soda water before adding the other sparkling ingredients. Another advantage will be an anti-coagulation of Spirulina in your desired drink. The mandatory: Gin and cucumber - 2 cl and a slice Spirulina has umami, which is considered the last basic taste to complete an culinary experience. Gin, with a flavor extracted from botanicals, becomes a natural element to Spirulina and a slice of cucumber brings freshness to the yet dry and savory taste. Tonic - 3 cl Say gin and people will think tonic. We are now presenting an irresistable upgrade of the regular G&T. Cava/Brut/(Asti) - 6 cl One thing to ruin a cocktail is extreem sweetness. Bartender s recommendation is cava or brut. The dry and sparkling choice. - Don t ruin it with asti Designed and developed by Esben Rimi Christiansen The secondary: Blue caracao - Few Drops Is mixed in the Spirulina-essence to apply an authentic and natural look of an Spirulina-environment. Apple cider - 3 cl A fruity sweet finish by an apple cider rounds of the dry notes of G&T and cava. Hint: The Spirulina-essence and the mandatory can be prepared seperately in advance and mixed at the time of serving. As soon as Spirulina has been introduced in an environment of disolved carbon dioxide a chemical reaction resulting in a beatiful foam which does not stay for long. If the drink should loose its shine in the wait then top it with more sparkles. 1) The complete Build on a shot of the Spirulina-essence. 2) The stylished Finished with a shot of the Spirulina-essence.

91 Dyrk din egen søsalat A) Find søsalat Gå evt. ind på og søg på søsalat under råvarer for at finde informationer om forekomster og kendetegn. (?) Hvordan kendetegnes søsalat? Det ligner lidt salatblade Det er grønt og gror i søer B) Fremstil kunstigt havvand 20 g. HAVsalt blandes om i 1 L vand og blandes grundigt. Gødning tilføjes saltvandet ud fra instrukserne på pakken. Opbevar på køl. Hint: Giv vandet dobbelt så meget gødning som anvist. (?) Hvorfor bruge kunstigt havvand? Fordi det vokser bedst i søvand Fordi det er renere end rigtig havvand C) Vask og forbered søsalat Skyl søsalaten under hanen til du har et rent salatblad. Klip små stykker (0.5*0.5cm) af siderne af og brug dem i D. Hint: klip små stykker fra flere forskellige søsalatsblade. Klip i siden (?) Hvorfor skylle søsalaten? Så det kun er søsalat, der gror For at fjerne alt saltet D) Start dyrkning Inden fjerde skift skulle du kunne se babysøsalat begynde at gro Kom 30 cm snor ned i et syltetøjsglas. Fyld glasset med kunstigt havvand fra B og smid de vaskede stykker søsalat ned i glasset. Henstil i et vindue overtrukket med film. Hint: Er der ikke nok lys i dit vindue? Stil en lampe over den og hav den tændt, når du er hjemme. (?) Hvor gror søsalat bedst? Hvor der er meget lys og høj næring Hvor der er middel lys og lidt næring E) Skift kunstigt havvand ca. hver 2. uge Første gang vandet skiftes kan søsalaten fra D smides ud. Fra nu af vil søsalaten gro op fra snoren og siderne på glasset. Hint: Ryst glasset blidt en gang om dagen. (?) Hvorfor skal det kunstige havvand skiftes jævnligt? For at sørge for, at der hele tiden er nok salt og næring For at forhindre små fisk i at spise de nye spire af søsalat Intet grønt ved 4. skift? Dette kan være årsagen: For lidt/meget lys Ikke anvendt havsalt Succesraten er størst i foråret. Det vides endnu ikke helt! Dyrk søsalat og vær med i forskningen! Er det lykkedes? Send mig et billede! Esben Rimi Christiansen, s123138@student.dtu.dk