Challenges for shellfish aquaculture in Mediterranean coastal areas

PhD program in Marine Sciences Challenges for shellfish aquaculture in Mediterranean coastal areas Article based thesis Doctoral thesis by: Margarita Fernández Tejedor Thesis advisor: Manuel Espino Infantes, Jorge Diogène Fadini Thesis submitted to obtain the title of Doctor by the Universitat Politècnica de Catalunya Department of Civil and Environmental Engineering, Barcelona School of Civil Engineering (ETSECCPB) Barcelona, October 2023 2 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Errata page _______________________________________________________________________________ 3 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Errata page Abstract _______________________________________________________________________________ 5 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Abstract The increase in the production of marine bivalves targets the sustainable development of aquaculture to improve global food and nutrition security. Bivalve aquaculture is currently facing challenges and threats that are limiting its growth and even its maintenance. Summer marine heat waves produce important mortalities endangering the harvest of the year and the seed of the following harvest. There is a need for expansion to deeper areas less affected by these events. We have developed a methodology to select these areas based on a combination of in situ and remote sensing data to build carrying capacity model for a coastal area in the Western Mediterranean. Harmful algal blooms (HABs) have an impact on bivalve aquaculture through the production of toxins that can accumulate in the bivalves affecting human health, to avoid that, shellfish closures are enforced in areas where toxins are detected over regulatory levels. Direct effects of HABs also threaten bivalve aquaculture producing mortality of farmed bivalves. We have evaluated the risk and trends of HABs in the Mediterranean Sea using bibliographic references and records of HABs events. The results show a low risk of toxic blooms but a higher risk of high biomass blooms without clear trends. Unexplained mortalities of farmed bivalves occur in the Mediterranean Sea, the presence of pathogens alone does not always explain these events. We have detected the presence of Perkinsus olseni for the first time in Mytilus galloprovincialis from the Mediterranean Sea. Warming may stimulate its proliferation, but we cannot conclude that its presence alone was the trigger of the mortality event. Translocation of bivalves from other geographical areas may be a risk for the introduction of pathogens and invasive species. In addition, the production of local seed may have other benefits due to bivalve adaptation to local environmental conditions. The replacement of live microalgae diets with artificial diets to simplify hatchery-nursery procedures has been attempted by different teams during the last decades. Artificial diets have been successful for fish and crustaceans but not yet for bivalves. We have tested the efficacy of an artificial diet to feed larvae of Ostrea stentina. Our results show significant differences in the growth and survival of the same larvae feed with live microalgae ponting out that it is not adequate for the hatchery and nursery production of Ostrea stentina larvae. We have addressed various challenges of the bivalve aquaculture in the Mediterranean Sea and have provided tools and evaluations to overcome the imitations and facilitate its development. Key words: Bivalve aquaculture, Mediterranean Sea, Ebro delta, Carrying capacity, Remote sensing, Harmful Algal Blooms, Shellfish pathogens, Perkinsus, Mytilus galloprovincialis, Chlorophyll, _______________________________________________________________________________ 6 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas _______________________________________________________________________________ 7 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Preface Exploring and observing the nature, we can find new ways of progress improving our lives and minimising our negative impacts. I am grateful to all of those who shared their knowledge with me. Table of contents _______________________________________________________________________________ 9 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Table of contents List of illustrations and tables. ......................................................................................................... 14 List of abbreviations and symbols .................................................................................................... 23 Glossary ........................................................................................................................................... 29 Chapter 1. General introduction. ...................................................................................................... 32 1. Aquaculture production. ........................................................................................................... 33 1.1. Global aquaculture. ........................................................................................................... 33 1.2. Aquaculture production of bivalves in the Mediterranean Sea. ........................................ 34 1.3. Marine Aquaculture in Spain. ........................................................................................... 36 2. The Mediterranean Sea ............................................................................................................ 39 3. The Ebro delta as a model. ....................................................................................................... 40 4. Thesis objectives. ..................................................................................................................... 44 4.1. Hypothesis. ........................................................................................................................ 46 4.3. Specific objectives. ........................................................................................................... 46 4.4. Long-term objective. ......................................................................................................... 47 Chapter 2. New shellfish growing areas in open waters. ................................................................. 59 1. Introduction .............................................................................................................................. 61 2. Materials and Methods ............................................................................................................. 64 2.1. Study Area ......................................................................................................................... 64 2.2. In Situ Measurements ........................................................................................................ 65 2.3. Maps of Chlorophyll-a from Sentinel-2 ............................................................................ 66 2.4. Carrying Capacity Model .................................................................................................. 68 3. Results ...................................................................................................................................... 71 3.1. In Situ Measurements ........................................................................................................ 71 3.2. Maps of Chlorophyll-a from Sentinel-2 ............................................................................ 73 3.3. Carrying Capacity Model .................................................................................................. 76 4. Discussion ................................................................................................................................ 80 5. Conclusions .............................................................................................................................. 83 References .................................................................................................................................... 83 Chapter 3. Trends in toxic phytoplankton in the Mediterranean. ..................................................... 95 1. Introduction .............................................................................................................................. 98 2. HABs in the Mediterranean Sea: toxic species and harmful event distribution ..................... 100 2.1. Toxic species ................................................................................................................... 100 2.2. Toxic events..................................................................................................................... 107 _______________________________________________________________________________ 16 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas renewal time of each embayment. The dots on the lines at the bottom of the figure correspond to the CC calculated for the average renewal time of each external area (F1–F6, A1–A6). ...................... 79 Figures in chapter 3. Figure 31. Examples of toxic species from the Mediterranean Sea. A) Alexandrium minutum stained with calcofluor. B) Azadinium dexteroporum. C) Dinophysis sacculus. D) Fibrocapsa japonica. E) Ostreopsis fattorussoi stained with calcofluor (courtesy of S. Accoroni). F) Prorocentrum lima. G) Pseudo-nitzschia multistriata. Scale bars in A and B: 5 µm; in C, D, E, F and G: 20 µm. ............ 103 Figure 32. Cumulative numbers of known toxic species in the Mediterranean Sea in different years. ........................................................................................................................................................ 106 Figure 33. Geographic range of potentially toxic species in the Mediterranean Sea. Distribution of species known to produce toxins related to: A) Diarrhoetic shellfish poisoning (DSP), Dinophysis spp. and the benthic species Prorocentrum lima and P. rhathymum. B) Paralytic shellfish poisoning (PSP), Alexandrium spp. and Gymnodinium catenatum. C) Amnesic shellfish poisoning (ASP), Pseudo-nitzschia spp. and Nitzschia bizertensis. For the genera Dinophysis, Pseudo-nitzschia and Alexandrium, which include both toxic and non-toxic species, the maps represent only toxic species and, in case of cryptic or problematic species, only the records validated by electron microscopy, molecular methods and/or toxin production. ................................................................................. 108 Figure 34. Fig. 4. Geographic range of potentially toxic species in the Mediterranean Sea. A) Ostreopsis spp. (mostly O. cf. ovata) and other benthic dinoflagellate species related to the ciguatera fish poisoning (CFP). B) Species producing ichthyotoxins (Alexandrium pseudogonyaulax, Karenia spp., Karlodinium spp., Chattonella spp., Vicicitus globosus, Prymnesium spp., etc.) and other toxins. The latter include mainly a few widespread dinoflagellate species that produce yessotoxins (Lingulodinium polyedra, Gonyaulax spinifera and Protoceratium reticulatum), but also other dinoflagellates producing azaspiracids (Azadinum spp.), pinnatoxins (Vulcanodinium rugosum) and other toxins with poorly known effects (e.g., Prorocentrum spp.). See Table 1 for a complete list. ......................................................................................................................................................... 112 Figure 35. A) Mat of Oscillatoria acutissima in the Eastern Harbour of Alexandria (Egypt). B) Bloom of Noctiluca scintillans in Thermaikos Gulf (Thessaloniki, Greece). C) Discoloration caused by Euglena viridis in the Golden Horn Estuary (Sea of Marmara, Turkey). D) Shellfish mortality in Ras El-Bar (Egypt) in 2011 due to the proliferation of N. scintillans and consequent oxygen depletion. E) Pelagic mucilages in the Gulf of Naples (Italy). ........................................................................ 116 _______________________________________________________________________________ 17 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 36. Distribution of potentially toxic species, mucilages and discolorations in the Mediterranean Sea. A) Distribution of species known to be toxic and harmful events until 1995 as reported in Jacques and Sournia (1978-1979) and Honsell et al. (1995). B) Distribution of potentially toxic species (excluding Ostreopsis and CFP species) and harmful events updated to the present status of knowledge. The position of the circles in several cases has been slightly modified to reduce overlapping...................................................................................................................................... 119 Figure 37. Harmful events related to microalgae in the Mediterranean Sea (n = 501) based on records in the Harmful Algae Event Database HAEDAT (http://haedat.iode.org/). High density phytoplankton blooms with no impacts were not considered. A) Relative abundance of different types of nuisance with details of seafood toxicity. B) Interannual variations of ASP, DSP and PSP toxicity events. ............................................................................................................................................ 120 Figures in chapter 4. Figure 41. Locations of sampling areas in Italy and Spain: Bay of Naples and Bay of Alfacs. .. 152 Figure 42. Histopathology of mussels affected by Perkinsosis in mussels (M. galloprovincialis) in Italy and Spain. (A, B) typical feature of the inflammatory lesion (big arrows) with haemocytes (*) related to Perkinsus (small arrows): haemocytes nodulation in gonadal follicle (A) and the interstitial space of digestive tubules (B) big arrows. (C) detail of the reactive connective tissue, underlined by Mallory Trichrome in light blue with inflammatory capsules (arrows); (D) detail of a capsule in the connective tissue space with haemocyte (h) phagocyting trophozoite of Perkinsus (arrow); (E) inflammatory capsule displaying apoptotic haemocytes (arrow) with visible trophozoite. (F) Perkinsus (arrows) in the gill (G) haemal vessel in samples from Spain; E, epithelium. .............. 156 Figure 43. Ray’s fluid thioglycolate medium (RFTM) assay. (A) Perkinsus hypnospores in very heavy infection in M. galloprovincialis in Campania in digestive tissue (DG), muscle (M) and mantle (A) and connective tissue close to the gills (G) (B). ...................................................................... 157 Figure 44. Evolutionary analysis by Maximum Likelihood method of Perkinsus spp. ITS sequences. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 41 nucleotide sequences. There was a total of 794 positions in the final dataset. ........................................................................................................................................................ 159 Figure 45. TCS network of 163 ITS1 haplotypes of Perkinsus olseni estimated by PopART. Each line between haplotypes indicates a single nucleotide substitution. The size of each circle is _______________________________________________________________________________ 18 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas proportional to the absolute haplotype frequency, small black circles represent missing haplotypes, and color shows localities where each haplotype was observed. ................................................... 159 Figures in chapter 5. Figure 51. Close up of a P. nobilis shell showing two attached adult individuals of O. stentina used for maturation. ................................................................................................................................ 173 Figure 52. Neighbor joining (NJ) tree based on Maximum Composite Likelihood (MCL) distances showing the phylogenetic affinity of the Ostrea sp. specimens used for this study (Ostrea sp.-IRTA - Ebro Delta) to Ostrea stentina. Numbers near nodes give bootstrap support (%). The inclusion in the different groups of dwarf oysters proposed by Hu et al. (2019) is indicated. Groups 1 and 2 are O. equestris and O. aupouria from close but divergent populations from the Americas and Asian Pacific, respectively. Group 3 is a new species Ostrea neostentina sp. nov. found in southeastern Spain (Mar Menor lagoon), eastern Tunisia, Hong Kong, and Japan, and group 4 is O. stentina occurring in northern (Avilés in the Atlantic Ocean) and northeastern Spain (Alfacs Bay in the Mediterranean Sea), Portugal, Morocco, and northern Tunisia. ............................................................................. 177 Figure 53. (A) Larval size (first 15 days of life) of individuals fed with live microalgae vs. those fed the Shellfish Diet 1800®. For the live diet, the size of a group of larvae that remained in the tank while the majority was taken to fixation is also indicated. (B) Temporal changes in the number of larvae in of O. stentina feed with each type of diet. Error bars are mean standard error. .............. 178 Figure 54. Complete larval development of O. stentina raised with the live diet. (A) Veliger larvae with 4 days of life; (B) Larvae with 6 days of life starting to show the umbo (um); (C and D) umbonate larvae of 8 to 11 days of life; (E) early pediveliger (13 days of life); and (F) pediveliger stage, with some of the larvae showing an eyespot (ey) and the umbo leaning asymmetrically towards one side of the shell. ..................................................................................................................................... 180 Figure 55. (A) Seed size of O. stentina raised with the live diet in the three settlement cylinders. The settlement period is indicated in grey and corresponds to the time frame indicated in panel 5B. Larvae in cylinder 3 were set to settle 1 week later than those in cylinders 1–2. (B) Rates of settlement during the first 23 days of benthic growth. The 70 % success is indicated with a dashed line. Error bars are mean standard error. ......................................................................................................... 181 Figure 56. (A) Seed of ca. 5 mm size in early November and (B) seed of ca. 14 mm size in early January already showing some juvenile traits such as a banded dark and clear pattern on the right flat valve. .............................................................................................................................................. 181 _______________________________________________________________________________ 19 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 57. Histology of different tissue of O. stentina: (A and B) visible hermaphroditic gonad with ripe female phase and developing male phase; vitellogenic ovocytes (Ov) and spermatogonia (Spg) are indicated; (C) digestive gland (DG) show a thick adsorbing epithelium sign of an active feeding; (D) Details of mantle epithelium (E) showing well developed mucous cells stained with acid alcian Blue Pas (black arrow) and eosinophilic granular cells (white arrow) part of the oyster first line of defense. CT: connective tissue. ...................................................................................................... 182 Figures in chapter 6. Figure 61. Time series of monthly mean (blue line) and 24-month filtered (red line) sea surface temperature anomalies in the Mediterranean Sea during the period 1993-2021. Anomalies are relative to the climatological period 1993-2014 and built from the CMEMS SST_MED_SST_L4_REP_OBSERVATIONS_010_021 satellite product. https://doi.org/10.48670/moi-00268............................................................................................... 198 Figure 62. Mediterranean Sea Chlorophyll-a time series and trend from Observations Reprocessing OMI_HEALTH_CHL_MEDSEA_OCEANCOLOUR_area_averaged_mean. https://doi.org/10.48670/moi-00259............................................................................................... 199 Figure 63. Mediterranean Sea Mean Sea Level time series and trend from Observations Reprocessing. MEDSEA_OMI_SL_area_averaged_anomalies. https://doi.org/10.48670/moi-00264. ........................................................................................................................................................ 200 Tables in chapter 2. Table 21. Spectral bands and spectral combinations tested for chlorophyll-a estimation. ............ 68 Table 22. Values used for the DEB model. .................................................................................... 69 Table 23. Best performing models per C2-Net and spectral band combination. ........................... 73 Table 24. Carrying capacity (CC) calculated for each area together with the corresponding surface and renewal times (Tr) and Clearance rate times (Cr). Note that the surface in AI and FI corresponds to the masked areas not to the whole surface of the embayments. ................................................... 79 Tables in chapter 3. _______________________________________________________________________________ 20 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Table 31. Potentially toxic species in the Mediterranean Sea and associated types of syndromes or impacts (see Moestrup et al. (2009) and Lassus et al. (2016) for details). ASP, amnesic shellfish poisoning; AZP, azaspiracid shellfish poisoning; DSP, diarrhoetic shellfish poisoning; PSP, paralytic shellfish poisoning; CFP, ciguatera fish poisoning. ‘Other toxins’ include unknown toxins or toxins with poorly known effects. ............................................................................................................. 100 Table 32. Potentially toxic species described from the Mediterranean Sea. ................................ 103 Tables in chapter 4. Table 41. List of the primers used to detect Perkinsus spp. with PCR and qPCR in this study. .. 153 Table 42. Survey results of Perkinsus infection in the blue mussel, M. galloprovincialis collected from Campania region and Ebro Delta. n.p.: analysis not performed. ........................................... 154 Tables in chapter 5. Table 51. Results of two-tailed t-test for independent samples testing for differences between larval growth with the live microalgae diet and the Shellfish Diet 1800® at each experimental date. Significant differences are indicated in bold. ................................................................................. 178 Supplementary materials. Supplementary materials from chapter 1. Figure 1S1. Aquaculture production of marine finfish in Spain: Atlantic and Mediterranean coasts (JACUMAR, 2023). ....................................................................................................................... 217 Figure 1S2. Value of the aquaculture production of marine finfish in Spain: Atlantic and Mediterranean coasts (JACUMAR, 2023). .................................................................................... 217 Figure 1S3. Aquaculture production of bivalves in Spain: Atlantic and Mediterranean coasts (JACUMAR, 2023). ....................................................................................................................... 218 Supplementary materials from chapter 2. _______________________________________________________________________________ 21 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 2S1. Secchi depth (m) at the different sampling stations. Note that the axis of sampling stations is reversed in the graphs showing chlorophyll-a concentration from Figure 3. ................ 220 Figure 2S2. Chlorophyll-a concentration, from Sentinel-2 images, averaged for each polygon and date outside the embayments. ........................................................................................................ 221 Figure 2S3. Matrix showing the results of the statistical analysis used to detect differences on the chlorophyll-a concentration, from Sentinel-2 images, averaged for each polygon and date outside the embayments. The squares colored in green correspond to polygons where statistical significant differences have been found, red color is for polygons where no differences have been detected, orange is for those polygons where no significant differences were found although they may exist. ........................................................................................................................................................ 222 Figure 2S4. Chlorophyll-a concentration, from Sentinel-2 images, averaged for each of the 3 parts of each polygon outside the embayments, A-C are areas at different distances from the shore inside each polygon. ................................................................................................................................. 223 Supplementary materials from chapter 3. Figure 3S1. Distribution of Pseudo-nitzschia spp., Nitzschia spp. and Halamphora spp. in the Mediterranean Sea. The 3 genera include both species that produce the toxin Domoic Acid and nontoxic species, and are often identified only at the genus level. Data from OBIS (https://obis.org/). ........................................................................................................................................................ 240 Table 3S1. Water discoloration events along the Mediterranean coasts. Species names have been updated following the currently accepted taxonomical nomenclature. .......................................... 225 Supplementary materials from chapter 4. Table 4S1. The pairwise genetic distance between isolates ......................................................... 241 List of abbreviations and symbols _______________________________________________________________________________ 23 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas List of abbreviations and symbols 𝐶𝑅: Clearance rates of mussels. 𝑃󰇗𝑥 : is the ingestion rate of mussels. AFLP: Amplified Fragment Length Polymorphism. APD: average percentage difference apig: pigment absorption product. AS: Adriatic Sea. ASC. Aquaculture Stewardship Council. ASP: Amnesic shellfish poisoning. Aura OMI NASA: The Ozone Monitoring Instrument (OMI) aboard NASA's Aura satellite measures ozone from Earth's surface to top-of-atmosphere (https://www.earthdata.nasa.gov/learn/finddata/near-real-time/omi). AZP: Azaspiracid poisoning. B01: Coastal aerosol, 442.7 nm (S2A), 442.3 nm (S2B), 60m (https://docs.sentinelhub.com/api/latest/data/sentinel-2-l1c/). B02: Blue band, 492.4 nm (S2A), 492.1 nm (S2B), 10m resolution. B03: Green band, 559.8 nm (S2A), 559.0 nm (S2B), 10m resolution. B04: Red band, 664.6 nm (S2A), 665.0 nm (S2B),10m resolution. B05: Vegetation red edge band, 704.1 nm (S2A), 703.8 nm (S2B), 20m resolution. B06: Vegetation red edge band, 740.5 nm (S2A), 739.1 nm (S2B), 20m resolution. B07: Vegetation red edge band, 782.8 nm (S2A), 779.7 nm (S2B), 20m resolution. B08: NIR band, 832.8 nm (S2A), 833.0 nm (S2B), 10m resolution. B09: Water vapour band, 945.1 nm (S2A), 943.2 nm (S2B), 60m resolution. B10: SWIR – Cirrus band, 1373.5 nm (S2A), 1376.9 nm (S2B), 60m resolution. B11: SWIR band, 1613.7 nm (S2A), 1610.4 nm (S2B), 20m resolution. B12: SWIR band, 2202.4 nm (S2A), 2185.7 nm (S2B), 20m resolution. List of abbreviations and symbols _______________________________________________________________________________ 24 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas B8A: Narrow NIR band, 864.7 nm (S2A), 864.0 nm (S2B), 20m resolution. BG: Blue to Green ratio. BG2: Blue to Green ratio 2. BHR: Broad host range. BIAS. is the tendency of a statistic to overestimate or underestimate the parameter or measurement. BLAST: Basic Local Alignment Search Tool. BLAST: Basic Local Alignment Search Tool. C2-Nets: Atmospheric correction processors derived from the original Case 2 Regional Coast Colour. C2RCC: Case 2 Regional Coast Colour. C2XC: C2X-COMPLEX processors cal: calibration. CC: carrying capacity. CDOM: Colored dissolved organic matter. CFP: Ciguatera. CLM: Cloud masks (more) data, 160m resolution. CLP: Cloud probability data, based on s2cloudless (more), 160m resolution. CLUSTAL: software for multiple sequence alignments. COI sequence: Mitochondrial cytochrome c oxidase subunit I. CT: clearance time. DA: Domoic acid. DEB: Dynamic Energy Budget. DNA: Deoxyribonucleic acid. dNTP: Deoxynucleotide triphosphate. DSP: Diarrhetic Shellfish Poisoning. DTX: dinophysistoxin. DU: Dobson units eDNA: Environmental DNA. EOVs: Essential Ocean Variables. List of abbreviations and symbols _______________________________________________________________________________ 25 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas ESA: European Space Agency. EU: European Union. f/2 medium: this microalgae culture medium contains the concentration of the original formulation, termed "f Medium" (Guillard and Ryther 1962), reduced by half. FSW: Filtered seawater. GenBank: an annotated collection of all publicly available DNA sequences (https://www.ncbi.nlm.nih.gov/genbank/). GIT: 3 band model from Gitelson et al 2011. GTP: Graphic Processing Tool. HAB: harmful algal bloom. HABMAP: Harmful Algal Bloom Map. HABs: harmful algal blooms. HAEDAT: Harmful Algal Event Database. HB-HABs: High biomass harmful algal blooms. iCOR: is a software to atmospherically correct Earth observation data. IOC: Intergovernmental Oceanographic Commission. ITS: Internal Transcriber Spacer. L1C: Level 1C products are a compilation of elementary granules of fixed size, within a single orbit. A granule is the minimum indivisible partition of a product (containing all possible spectral bands). The granules, also called tiles, are 100x100km2 ortho-images in UTM/WGS84 projection. The UTM (Universal Transverse Mercator) system divides the Earth’s surface into 60 zones. Each UTM zone has a vertical width of 6° of longitude and horizontal width of 8° of latitude. Tiles are approximately 700 MB in size. Tiles can be fully or partially covered by image data. Partially covered tiles correspond to those at the edge or top and bottom of the Datastrip (Copernicus Sentinel-2 Collection 1 MSI Level-1C (L1C) , https://sentinels.copernicus.eu/web/sentinel/sentinel-data-access/sentinelproducts/sentinel-2-data-products/collection-1-level-1c) Copernicus Sentinel-2 (processed by ESA), 2021, MSI Level-1C TOA Reflectance Product. Collection 1. European Space Agency, https://doi.org/10.5270/S2_-742ikth. MAE: mean average error. MEGA: Molecular Evolutionary Genetics Analysis. MHW: Marine heat wave. Chapter 1. General introduction. _______________________________________________________________________________ 32 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Chapter 1. General introduction. Aquaculture production has been increasing in the last decades to meet the increasing global demand for seafood. The forecast of global aquaculture fish production for the year 2023 is estimated in 96 million tonnes of live weight while the total capture of fish is estimated in 89.5 million of tonnes (FAO, 2023). The global meat production forecast for the year 2023 is 364 million tonnes of carcass weight equivalent, it includes bovine, poultry, pig and ovine meat. Globally, aquatic foods provide about 17 percent of animal protein, reaching over 50 percent in several countries in Asia and Africa (FAO, 2022a). Fisheries production systems are under considerable stress from overfishing, habitat loss, pollution, invasive species, water abstraction, damming, and disruption of fisheries by climate change (Allison et al., 2009). Marine aquaculture is seen as a key sector to improve global food and nutrition security due to its great potential for reducing food system environmental stressors (Gephart et al., 2021). Among the different marine aquaculture products, bivalves could contribute 43 percent of aquatic food by 2050 (Costello et al., 2020). The General Fisheries Commission for the Mediterranean (GFCM) of the Food and Agriculture Organization of the United Nations (FAO) designed a strategy to achieve the sustainable management of marine resources in the Mediterranean and the Black Sea (FAO, 2021a). One of the targets of this strategy, target 3, aims at the sustainable development of aquaculture and its contribution to sustainable food systems, working towards the resilience of the sector against global challenges such as climate change and pollution. One of the expected outcomes of the FAO Blue Transformation roadmap is to reach at least 35 percent growth in global sustainable aquaculture production by 2030 (FAO, 2022b). To achieve these objectives, it is necessary to increase the production of marine bivalves. Marine bivalves are low trophic species, and their production system can be classified as non-fed aquaculture. The production of bivalves is frequently referred as shellfish aquaculture which includes other molluscs, crustaceans, and echinoderms. Bivalve aquaculture is currently facing challenges related to: 1) the need for expansion to new coastal zones resilient to climate change (Filgueira et al., 2016); 2) the increase in mortalities associated to new pathogens (Elston et al., 2008) and marine heat waves (Lacoue-Labarthe et al., 2016); 3) chemical (Saldaña-Serrano et al., 2022; Squadrone et al., 2016) and microbial pollution (Hunt et al., 2023; Rowan, 2023) of the coastal waters; 4) harmful algal blooms (Brown et al., 2020; Gianella et al., 2021; Rolton et al., 2022; Stoner et al., 2023; Tan et al., 2023); 5) the difficulties in obtaining local seed (Avdelas et al., 2021); 6) the translocation of invasive species (Cahill et al., 2022; Lacoste and Gaertner-Mazouni, 2015). Scientific research needs to focus on these challenges to help the aquaculture industry to achieve the objectives of increasing bivalve production. Chapter 1. General introduction. _______________________________________________________________________________ 33 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas 1. Aquaculture production. 1.1. Global aquaculture. World aquaculture production in 2019 consisted of 56.3 million tonnes of finfish, 17.6 million tonnes of molluscs, 10.5 million tonnes of crustaceans, 977 thousand tonnes of other aquatic animal species and 34 million tonnes of seaweeds (Figure 11). Finfish production from inland aquaculture represented 56.7 percent of world total aquaculture production of aquatic animals in 2019. The annual growth rate for the global fish and shellfish aquaculture production was on average 3.7 percent during 2016–2019. The contribution of aquaculture to the total production of aquatic animals from capture and aquaculture combined has risen steadily from 39.9 percent in 2010 to 48.0 percent in 2019. Preliminary estimates from FAO for 2019 indicate a growth in per capita consumption of fish to about 20.5 kg, with the share of aquaculture production in total available food fish supply overtaking that of capture fisheries (11.1 kg vs 9.5 kg). In 2019 1 , world aquatic production from aquaculture was 120 million tonnes while capture production was 93 million tonnes (Figure 12). This comparison may be biased due to the differences in the proportions between capture and aquaculture for the different groups of species. The edible content of molluscs in low in comparison to finfish and the amount of capture production of fish (freshwater, diadromous and marine fishes) was 79 million tonnes and 56 million tonnes were produced by the aquaculture industry. For the rest of species (crustaceans, molluscs, aquatic plants and others) the total amount produced by aquaculture was much higher than the captured production, 64 and 14 million tonnes respectively (FAO, 2021b). Figure 11. World aquaculture production of fish, crustaceans, molluscs and seaweeds (FAO, 2021b). 1 The FAO Fishery and Aquaculture Statistics – Yearbook 2020 was published at the end of September 2023, after the completion of this manuscript. In the year 2020, world aquatic production from aquaculture was 123 million tonnes while capture production was 91 million tonnes. FAO. 2023.Fishery and Aquaculture Statistics – Yearbook 2020. FAO Yearbook of Fishery and Aquaculture Statistics. Rome. Chapter 1. General introduction. _______________________________________________________________________________ 34 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 12. World aquatic production by capture (FAO, 2021b). 1.2. Aquaculture production of bivalves in the Mediterranean Sea. In the Mediterranean Sea, the countries that have reported aquaculture production of bivalves during 2010-2019 are Albania, Algeria, Bosnia, Croatia, France, Greece, Italy, Montenegro, Morocco, Slovenia, Spain, Tunisia, and Turkey (FAO, 2021b). The species produced are Mytilus galloprovincialis Lamarck, Crassostrea gigas Thunberg, Ostrea edulis Linnaeus, Ruditapes decussatus Linnaeus, and Ruditapes philippinarum Adams & Reeve (FAO, 2021b). The total production in the Mediterranean Sea during the year 2019, in thousands of tonnes was 105 for M. galloprovincialis, 31 for R. philippinarum and 5.7 for C. gigas (Figure 13). The production of O. edulis and R. decussatus was 93 and 73 tonnes respectively (FAO, 2021b). For Spain and France, we include only the production from the regions bordering the Mediterranean Sea excluding the production from the Atlantic regions (Agreste, 2021; JACUMAR, 2023). Among the different Mediterranean countries, it is Italy the major producer of farmed bivalves. During the year 2020, Italy produced 50 thousand tonnes of M. galloprovincialis, 24 thousand tonnes of R. philippinarum and 182 tonnes of C. gigas. (Ispra, 2022). Bivalve production in Italy occurs mainly in Emilia Romagna (16 thousand tonnes) and Veneto (32 thousand tonnes), smaller productions are localized in Friuli Venezia Giulia, Liguria, Marche. Lazio, Abruzzo, Molise, Campania, Puglia, Calabria, Sicilia and Sardegna (Ispra, 2022). Bivalve production in Italy which is higher than finfish production, has decreased in Italy during the last 15 years (Ispra, 2022). Chapter 1. General introduction. _______________________________________________________________________________ 35 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 13. Aquaculture production of bivalves in the Mediterranean Sea (FAO, 2021b). Italy produces 59 percent of the total Mediterranean aquaculture production of M. galloprovincialis (Figure 14) followed by Greece (22 percent), France (6 percent), Spain (5 percent) and Turkey (4 percent). Figure 14. Aquaculture production of M. galloprovincialis in the Mediterranean Sea (FAO, 2021b). Chapter 1. General introduction. _______________________________________________________________________________ 36 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas There is a high seasonal demand of mussels in the Mediterranean countries due to tourism. Local aquaculture production is not sufficient. In spite of the high demand, mussel aquaculture in the Mediterranean has not increased in the last years in contrast to the increase of the global production. Research for the identification of suitable farming areas is needed in some countries (Avdelas et al., 2021). 1.3. Marine Aquaculture in Spain. In the year 2022, Spain produced 59 thousand tonnes of finfish for a value of 541 million Euros, 378 tonnes of crustaceans (2,6 million Euros), 227 thousand tonnes of bivalve molluscs (176 million Euros), 2 tonnes of microalgae (1,7 million Euros) and 19 tonnes of seaweed (152 thousand Euros) (Figure 15, Figure 16, Figure 1S1, Figure 1S2, Figure 1S3). The species produced for each of these groups are the following (JACUMAR, 2023); Finfish: Sparus aurata Linnaeus; Solea senegalensis Kaup; Dicentrarchus labrax Linnaeus; Mugil Linnaeus; Chelon labrosus Risso; Seriola dumerili Risso; Scophthalmus maximus Linnaeus; Diplodus sargus Linnaeus; Argyrosomus regius Asso; Dicentrarchus punctatus Bloch; Thunnus thynnus Linnaeus; Anguilla anguilla Linnaeus; Crustaceans: Penaeus japonicus Spence Bate; Penaeus vannamei Boone; Penaeus kerathurus Forskål; Palaemon varians Leach; Bivalve molluscs: Aequipecten opercularis Linnaeus; Ostrea edulis Linnaeus; Crassostrea gigas Thunberg; Ensis Schumacher; Mytilus galloprovincialis Lamarck; Venus verrucosa Linnaeus; Cerastoderma edule Linnaeus; Ruditapes philippinarum A. Adams & Reeve; Ruditapes decussatus Linnaeus; Venerupis corrugata Gmelin; Microalgae: Dunaliella salina (Dunal) Teodoresco; Tetraselmis F.Stein; Tetraselmis chui Butcher; Microchloropsis gaditana (L.M.Lubián) M.W.Fawley, I.Jameson & K.P.Fawley; Seaweed: Ulva lactuca Linnaeus; Ulva rigida C. Agardh; Gracilaria Greville; Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders; Codium tomentosum Stackhouse; Codium vermilara (Olivi) Delle Chiaje; Gracilaria dura (C. Agardh) J. Agardh; Gracilariopsis longissima (S.G. Gmelin) Steentoft, L.M. Irvine & Farnham; Some species are produced in both, the Mediterranean and the Atlantic coasts of Spain but from the species listed above, the following species are not produced in the regions bordering the Mediterranean coast of Spain or at least not in a relevant amount: Penaeus vannamei, Dunaliella salina, Tetraselmis chui, Saccharina latissima, Ulva rigida, Codium tomentosum, Codium vermilara, Gracilaria dura, Gracilariopsis longissima, Scophthalmus maximus, Aequipecten opercularis, Ostrea edulis, Cerastoderma edule, Venerupis corrugata (Figure 15, Figure 17, Figure 18). Chapter 1. General introduction. _______________________________________________________________________________ 37 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Catalonia reports the production of razor clams as produced in aquaculture, since we are aware that this production has been captured by shellfish harvesters, we have excluded this production from the total Spanish production on bivalve aquaculture (2010-2022). Most of the aquaculture production of crustaceans in Spain is located in Andalusia (370 tonnes, 2.6 million euros), as well as the production of microalgae (1.57 tonnes, 1.6 million Euros), Andalusia produces 7 tonnes of seaweed (Ulva lactuca and Gracilaria) for a value of 118 thousand Euros. The production of marine finfish in Spain is mainly located in the Mediterranean coast (43 thousand tonnes, 405 million Euros). Among the different species, the production of Sparus aurata and Dicentrarchus labrax is mainly located along the Mediterranean coast, in the regions of Andalusia, Murcia, Valencia and Catalonia. There is a small production of Solea senegalensis (363 tonnes), Mugil and Chelon labrosus (45 tonnes), Diplodus sargus (1 tonnes) and Dicentrarchus punctatus (0.38 tonnes) in Andalusia and a small production (147 tonnes) of Seriola dumerili in Murcia and Valencia. Argyrosomus regius (4.7 tonnes) is produced in Andalusia, Murcia and Valencia. The whole Spanish production of Thunnus thynnus is located in Murcia (8 thousand tonnes) and Catalonia (2. 6 thousand tonnes). There is a small production of Anguilla anguilla (335 tonnes) in Valencia. The production of bivalves in Spain is mainly located in the Atlantic coast (222 thousand tonnes), only 6 thousand tonnes are produced in the Mediterranean coast for a value of 10 million Euros. Along the Mediterranean coast, Crassotrea gigas is produced in Andalusia (28 tonnes), Valencia (42 tonnes) and Catalonia (397 tonnes); Ruditapes philippinarum is produced in Andalusia (4.67 tonnes) and Catalonia (6 tonnes) (Figure 18); Mytilus galloprovincialis is produced in the Balearic Islands (104 tonnes), Andalusia (640 tonnes), Valencia (1.8 thousand tonnes), Catalonia (2.9 thousand tonnes) (Figure 19). Figure 15. Aquaculture production of marine finfish in the Mediterranean coast of Spain (JACUMAR, 2023). Chapter 1. General introduction. _______________________________________________________________________________ 38 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 16. Value of the aquaculture production of marine finfish in the Mediterranean coast of Spain (JACUMAR, 2023). Figure 17. Aquaculture production of bivalve molluscs in the Mediterranean coast of Spain (JACUMAR, 2023). Chapter 1. General introduction. _______________________________________________________________________________ 39 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 18. Aquaculture production of bivalves, excluding mussels, in the Mediterranean coast of Spain (JACUMAR, 2023). Figure 19. Mussel production in the different regions of the Mediterranean coast of Spain (JACUMAR, 2023). 2. The Mediterranean Sea The Mediterranean Sea is connected with the North Atlantic Ocean through the Strait of Gibraltar, with the Black Sea through the Turkish Strait System (Dardanelles, Marmara Sea and Bosphorus Strait). It is a concentration basin where evaporation exceeds precipitation. The surface Atlantic water entering through the Strait of Gibraltar circulates along the North African coast, the main part flows through the Sardinia Channel into the Tyrrhenian Sea and a smaller part flows through the Sicily Channel into the eastern Mediterranean Sea. In the northern Tyrrhenian Sea one branch flows through the Corsica channel. The principal flow in the eastern Mediterranean is in a cyclonic circulation but Chapter 1. General introduction. _______________________________________________________________________________ 40 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas in the northern Ionian Sea, the North Ionian Gyre shifts between cyclonic and anti-cyclonic on decadal time scales. The surface Atlantic Water is transformed in Levantine Intermediate Water in the area east of Rhodes and in the Cretan Sea and from there it spreads to the Levantine basin and to the Ionian Sea, it passes the Sicily channel, tourns around the Tyrrenian Sea and enters the western Mediterranean through the Sardinia Channel, a fraction flows out the Tyrrhenian Sea northwards entering the Ligurian Sea and the Provençal Basin (Tanhua et al., 2013). Mesoscale processes play a key role in determining the large-scale circulation, the distribution of water masses and their mixing, meanders, eddies, and filaments mainly originate as instabilities of large-scale currents and fronts (Mason et al., 2023). Salinity ranges from 36.1 in the Atlantic water at the entrance in the Strait of Gibraltar, to 39.1 at the surface in the eastern Mediterranean Sea (Schroeder et al., 2023). The influence of the major rivers (Po, Rhone and Nile) can be observed in the Sea Surface Salinity. The Mediterranean Sea is oligotrophic, even ultra-oligotrophic and the ratio N:P is higher than in the world ocean. The deep Mediterranean water exported to the Atlantic Ocean has a higher concentration of nutrients than the Atlantic Surface water entering through the Strait of Gibraltar (Tanhua et al., 2013). The Mediterranean exports inorganic N and P to the Atlantic, and imports organic N and P from the Atlantic, less bioavailable for phytoplankton, mineralization processes are predominant (Álvarez et al., 2023). The Mediterranean Sea is becoming warmer and saltier, future projections predict that SST will continue increasing throughout the basin as well as the surface salinity in the Levantine basin and the Aegean Sea. 3. The Ebro delta as a model. The Ebro delta concentrates most of the bivalve aquaculture production from the Mediterranean coast of Spain. Mussels (M. galloprovincialis) and oysters (C. gigas) are grown in ropes hanging from rafts in the two coastal embayments of Alfacs Bay and Fangar Bay (Figure 110). Mussel production for the year 2022 was 2,272 tonnes in Alfacs Bay and 552 tonnes in Fangar Bay. The production of oyster for the same year was 25 tonnes in Alfacs Bay and 372 tonnes in Fangar Bay. In some of the rafts, shellfish farmers hang mussel seed collectors but most of the mussel seed is imported from Italy and Greece. Oyster seed is imported from shellfish hatcheries and natural collection, both from France. In these two coastal embayments there are some areas dedicated to grow clams (R. philippinarum) but its production has been highly impacted by the invasion of the blue crab (Callinectes sapidus Rathbun). There is also mussel aquaculture outside these two coastal bays. A long-line is installed in the south of the Ebro delta (80 tonnes/year 2022) and there is high interest in installing new farms in the northern area outside Fangar Bay. Chapter 1. General introduction. _______________________________________________________________________________ 41 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 110. Maps showing the location of the Ebro delta in the Western Mediterranean and the bivalve farms in Alfacs Bay and Fangar Bay, the red rectangles show the rafts where shellfish farmers grow oysters and mussels. Massive mussel mortalities occur in summer when seawater temperature rises above 28 °C (Ramon et al., 2007). The first known mussel mortality event that occurred in the Ebro delta associated to high seawater temperature happened in 1994 in Alfacs Bay, the second occurred in the year 2003 and affected both bays. These events of high seawater temperature in summer, are increasing in frequency and duration (Figure 111). Shellfish farmers have adapted the calendar of seeding and harvesting with the objective of harvesting the whole production before seawater temperature in the two coastal embayments reaches 28 °C. The mussel seed collected within the embayments at the beginning of each year will reach commercial size 12-18 months later, the mussel seed collected and grown in the two embayments does not survive in years when seawater temperature rises above 28 °C during more than one week. Some farmers prefer to dedicate the whole farm to grow imported seed since they face the risk of not only a losing the production of the year but also the production of the year after. Marine heat wave events are also increasing in their geographical extent and therefore other Mediterranean coastal areas could be affected at the same time hampering the obtention of mussel seed for starting new cycles of production. Some shellfish farmers requested to open a channel at the interior of Alfacs Bay to connect the inner part of the Bay with the open sea. The results of the simulations showed that the integrated values of seawater temperature over the bay did not show significant variations between the tests and the control case, with differences smaller than 0.07 °C (Cerralbo et al., 2019). Finding new appropriate locations to maintain the seed and to grow mussels up to commercial size during summer is a requirement not only to increase mussel production but also to maintain the current production in the future years. Chapter 1. General introduction. _______________________________________________________________________________ 48 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Oral bioaccessibility of toxic and essential elements in raw and cooked commercial seafood species available in European markets. Food Chemistry 267, 15-27. Avdelas, L., Avdic-Mravlje, E., Borges Marques, A.C., Cano, S., Capelle, J.J., Carvalho, N., Cozzolino, M., Dennis, J., Ellis, T., Fernández Polanco, J.M., Guillen, J., Lasner, T., Le Bihan, V., Llorente, I., Mol, A., Nicheva, S., Nielsen, R., van Oostenbrugge, H., Villasante, S., Visnic, S., Zhelev, K., Asche, F., 2021. The decline of mussel aquaculture in the European Union: causes, economic impacts and opportunities. Reviews in Aquaculture 13(1), 91-118. Aznar-Alemany, O., Trabalon, L., Jacobs, S., Barbosa, V.L., Tejedor, M.F., Granby, K., Kwadijk, C., Cunha, S.C., Ferrari, F., Vandermeersch, G., Sioen, I., Verbeke, W., Vilavert, L., Domingo, J.L., Eljarrat, E., Barcelo, D., 2017. Occurrence of halogenated flame retardants in commercial seafood species available in European markets. Food Chem. Toxicol. 104, 35-47. Barbosa, V., Maulvault, A.L., Alves, R.N., Kwadijk, C., Kotterman, M., Tediosi, A., FernandezTejedor, M., Sloth, J.J., Granby, K., Rasmussen, R.R., Robbens, J., De Witte, B., Trabalon, L., Fernandes, J.O., Cunha, S.C., Marque, A., 2018. Effects of steaming on contaminants of emerging concern levels in seafood. Food Chem. Toxicol. 118, 490-504. Bergholtz, T., Daugbjerg, N., Moestrup, O., Fernandez-Tejedor, M., 2006. On the identity of Karlodinium veneficum and description of Karlodinium armiger sp nov (Dinophyceae), based on light and electron microscopy, nuclear-encoded LSU rDNA, and pigment composition. Journal of Phycology 42(1), 170-193. Brenner, M., Fraser, D., Van Nieuwenhove, K., O'Beirn, F., Buck, B.H., Mazurie, J., Thorarinsdottir, G., Dolmer, P., Sanchez-Mata, A., Strand, O., Flimlin, G., Miossec, L., Kamermans, P., 2014. Bivalve aquaculture transfers in Atlantic Europe. Part B: Environmental impacts of transfer activities. Ocean & Coastal Management 89, 139-146. Brigolin, D., Lourguioui, H., Taji, M.A., Venier, C., Mangin, A., Pastres, R., 2015. Space allocation for coastal aquaculture in North Africa: Data constraints, industry requirements and conservation issues. Ocean & Coastal Management 116, 89-97. Brown, A.R., Lilley, M., Shutler, J., Lowe, C., Artioli, Y., Torres, R., Berdalet, E., Tyler, C.R., 2020. Assessing risks and mitigating impacts of harmful algal blooms on mariculture and marine fisheries. Reviews in Aquaculture 12(3), 1663-1688. Cahill, P.L., Davidson, I.C., Atalah, J.A., Cornelisen, C., Hopkins, G.A., 2022. Toward integrated pest management in bivalve aquaculture. Pest Management Science 78(11), 4427-4437. Carrasco, N., Arzul, I., Berthe, F.C.J., Fernandez-Tejedor, M., Durfort, M., Furones, M.D., 2008. Delta de l'Ebre is a natural bay model for Marteilia spp. (Paramyxea) dynamics and life-cycle studies. Dis. Aquat. Org. 79(1), 65-73. Chapter 1. General introduction. _______________________________________________________________________________ 49 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Carrasco, N., Gairin, I., Perez, J., Andree, K.B., Roque, A., Fernandez-Tejedor, M., Rodgers, C.J., Aguilera, C., Furones, M.D., 2017. A Production Calendar Based on Water Temperature, Spat Size, and Husbandry Practices Reduce OsHV-1 mu var Impact on Cultured Pacific Oyster Crassostrea gigas in the Ebro Delta (Catalonia), Mediterranean Coast of Spain. Frontiers in Physiology 8, 10. Casso, M., Navarro, M., Ordonez, V., Fernandez-Tejedor, M., Pascual, M., Turon, X., 2018. Seasonal patterns of settlement and growth of introduced and native ascidians in bivalve cultures in the Ebro Delta (NE Iberian Peninsula). Regional Studies in Marine Science 23, 12-22. Cerralbo, P., Balsells, M.F.P., Mestres, M., Fernandez, M., Espino, M., Grifoll, M., SanchezArcilla, A., 2019. Use of a hydrodynamic model for the management of water renovation in a coastal system. Ocean Science 15(2), 215-226. Costello, C., Cao, L., Gelcich, S., Cisneros-Mata, M.Á., Free, C.M., Froehlich, H.E., Golden, C.D., Ishimura, G., Maier, J., Macadam-Somer, I., Mangin, T., Melnychuk, M.C., Miyahara, M., de Moor, C.L., Naylor, R., Nøstbakken, L., Ojea, E., O’Reilly, E., Parma, A.M., Plantinga, A.J., Thilsted, S.H., Lubchenco, J., 2020. The future of food from the sea. Nature 588(7836), 95-100. Cunha, S.C., Fernandes, J.O., Vallecillos, L., Cano-Sancho, G., Domingo, J.L., Pocurull, E., Borrull, F., Maulvault, A.L., Ferrari, F., Fernandez-Tejedor, M., Van den Heuvel, F., Kotterman, M., 2015. Co-occurrence of musk fragrances and UV-filters in seafood and macroalgae collected in European hotspots. Environmental Research 143, 65-71. Cunha, S.C., Trabalon, L., Jacobs, S., Castro, M., Fernandez-Tejedor, M., Granby, K., Verbeke, W., Kwadijk, C., Ferrari, F., Robbens, J., Sioen, I., Pocurull, E., Marques, A., Fernandes, J.O., Domingo, J.L., 2018. UV-filters and musk fragrances in seafood commercialized in Europe Union: Occurrence, risk and exposure assessment. Environmental Research 161, 399-408. Dayan, H., McAdam, R., Juza, M., Masina, S., Speich, S., 2023. Marine heat waves in the Mediterranean Sea: An assessment from the surface to the subsurface to meet national needs. Frontiers in Marine Science 10. Delgado, M., Alcaraz, M., 1999. Interactions between red tide microalgae and herbivorous zooplankton: the noxious effects of Gyrodinium corsicum (Dinophyceae) on Acartia grani (Copepoda : Calanoida). Journal of Plankton Research 21(12), 2361-2371. Delgado, M., Estrada, M., Camp, J., Fernández, J.V., Santmartí, M., Lletí, C., 1990. Development of a toxic Alexandrium minutum Halim (Dinophyceae) bloom in the harbour of Sant Carles de la Ràpita (Ebro Delta, northwestern Mediterranean). Scientia Marina 54(1), 1-7. Delisle, L., Pauletto, M., Vidal-Dupiol, J., Petton, B., Bargelloni, L., Montagnani, C., Pernet, F., Corporeau, C., Fleury, E., 2020. High temperature induces transcriptomic changes in Crassostrea gigas that hinder progress of ostreid herpesvirus (OsHV-1) and promote survival. Journal of Experimental Biology 223(20). Chapter 1. General introduction. _______________________________________________________________________________ 50 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Delisle, L., Petton, B., Burguin, J.F., Morga, B., Corporeau, C., Pernet, F., 2018. Temperature modulate disease susceptibility of the Pacific oyster Crassostrea gigas and virulence of the Ostreid herpesvirus type 1. Fish & Shellfish Immunology 80, 71-79. Elston, R.A., Hasegawa, H., Humphrey, K.L., Polyak, I.K., Häse, C.C., 2008. Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management. Dis. Aquat. Org. 82(2), 119-134. FAO, 2021a. GFCM 2030 Strategy for sustainable fisheries and aquaculture in the Mediterranean and the Black Sea. FAO, Rome, Italy. FAO, 2021b. FAO Yearbook. Fishery and Aquaculture Statistics 2019/FAO annuaire. Statistiques des pêches et de l'aquaculture 2019/FAO anuario. Estadísticas de pesca y acuicultura 2019, Rome. FAO, 2022a. The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. FAO, Rome, Italy. FAO, 2022b. Blue Transformation - Roadmap 2022–2030: A vision for FAO’s work on aquatic food systems. Rome. FAO, 2023. Food Outlook – Biannual report on global food markets, Rome, Italy. Fernández-Tejedor, M., Delgado, M., Garcés, E., Camp, J., Diogène, J., 2010. Toxic phytoplankton response to warming in two Mediterranean bays of the Ebro Delta. Commission Internationale pour l'Exploration Scientifique de la mer Méditerranée. Fernandez-Tejedor, M., Soubrier-Pedreno, M.A., Furones, M.D., 2004. Acute LD50 of a Gyrodinium corsicum natural population for Sparus aurata and Dicentrarchus labrax. Harmful Algae 3(1), 1-9. Fernandez-Tejedor, M., Soubrier-Pedreno, M.A., Furones, M.D., 2007. Mitigation of lethal effects of Karlodinium veneficum and K. armiger on Sparus aurata: changes in haematocrit and plasma osmolality. Dis. Aquat. Org. 77(1), 53-59. Filgueira, R., Guyondet, T., Comeau, L.A., Tremblay, R., 2016. Bivalve aquacultureenvironment interactions in the context of climate change. Global Change Biology 22(12), 39013913. Gagnaire, B., Thomas-Guyon, H., Burgeot, T., Renault, T., 2006. Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: Screening of 23 molecules using flow cytometry. Cell Biology and Toxicology 22(1), 1-14. Chapter 1. General introduction. _______________________________________________________________________________ 51 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Garces, E., Delgado, M., Maso, M., Camp, J., 1999. In situ growth rate and distribution of the ichthyotoxic dinoflagellate Gyrodinium corsicum Paulmier in an estuarine embayment (Alfacs Bay, NW Mediterranean Sea). Journal of Plankton Research 21(10), 1977-1991. Garces, E., Fernandez, M., Penna, A., Van Lenning, K., Gutierrez, A., Camp, J., Zapata, M., 2006. Characterization of NW Mediterranean Karlodinium spp. (Dinophyceae) strains using morphological, molecular, chemical, and physiological methodologies. Journal of Phycology 42(5), 1096-1112. García-Altares, M., Casanova, A., Fernández-Tejedor, M., Diogène, J., de la Iglesia, P., 2016. Bloom of Dinophysis spp. dominated by D. sacculus and its related diarrhetic shellfish poisoning (DSP) outbreak in Alfacs Bay (Catalonia, NW Mediterranean Sea): Identification of DSP toxins in phytoplankton, shellfish and passive samplers. Regional Studies in Marine Science 6, 19-28. Garcia-Pimentel, M., Campillo, J.A., Castano-Ortiz, J.M., Llorca, M., Leon, V.M., 2023. Occurrence and distribution of contaminants of legacy and emerging concern in surface waters of two Western Mediterranean coastal areas: Mar Menor Lagoon and Ebro Delta. Marine pollution bulletin 187, 114542-114542. Garcia, C., Thébault, A., Dégremont, L., Arzul, I., Miossec, L., Robert, M., Chollet, B., François, C., Joly, J.-P., Ferrand, S., Kerdudou, N., Renault, T., 2011. Ostreid herpesvirus 1 detection and relationship with Crassostrea gigas spat mortality in France between 1998 and 2006. Veterinary Research 42(1), 73. Garrabou, J., Gómez-Gras, D., Medrano, A., Cerrano, C., Ponti, M., Schlegel, R., Bensoussan, N., Turicchia, E., Sini, M., Gerovasileiou, V., Teixido, N., Mirasole, A., Tamburello, L., Cebrian, E., Rilov, G., Ledoux, J.-B., Souissi, J.B., Khamassi, F., Ghanem, R., Benabdi, M., Grimes, S., Ocaña, O., Bazairi, H., Hereu, B., Linares, C., Kersting, D.K., la Rovira, G., Ortega, J., Casals, D., Pagès-Escolà, M., Margarit, N., Capdevila, P., Verdura, J., Ramos, A., Izquierdo, A., Barbera, C., Rubio-Portillo, E., Anton, I., López-Sendino, P., Díaz, D., Vázquez-Luis, M., Duarte, C., Marbà, N., Aspillaga, E., Espinosa, F., Grech, D., Guala, I., Azzurro, E., Farina, S., Cristina Gambi, M., Chimienti, G., Montefalcone, M., Azzola, A., Mantas, T.P., Fraschetti, S., Ceccherelli, G., Kipson, S., Bakran-Petricioli, T., Petricioli, D., Jimenez, C., Katsanevakis, S., Kizilkaya, I.T., Kizilkaya, Z., Sartoretto, S., Elodie, R., Ruitton, S., Comeau, S., Gattuso, J.-P., Harmelin, J.-G., 2022. Marine heatwaves drive recurrent mass mortalities in the Mediterranean Sea. Global Change Biology 28(19), 5708-5725. Gephart, J.A., Henriksson, P.J.G., Parker, R.W.R., Shepon, A., Gorospe, K.D., Bergman, K., Eshel, G., Golden, C.D., Halpern, B.S., Hornborg, S., Jonell, M., Metian, M., Mifflin, K., Newton, R., Tyedmers, P., Zhang, W., Ziegler, F., Troell, M., 2021. Environmental performance of blue foods. Nature 597(7876), 360-365. Gianella, F., Burrows, M.T., Swan, S.C., Turner, A.D., Davidson, K., 2021. Temporal and Spatial Patterns of Harmful Algae Affecting Scottish Shellfish Aquaculture. Frontiers in Marine Science 8. Gimenez Papiol, G., Casanova, A., Fernandez-Tejedor, M., de la Iglesia, P., Diogene, J., 2013. Management of domoic acid monitoring in shellfish from the Catalan coast. Environmental Monitoring and Assessment 185(8), 6653-6666. Chapter 1. General introduction. _______________________________________________________________________________ 52 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Gobler, C.J., Doherty, O.M., Hattenrath-Lehmann, T.K., Griffith, A.W., Kang, Y., Litaker, R.W., 2017. Ocean warming since 1982 has expanded the niche of toxic algal blooms in the North Atlantic and North Pacific oceans. Proceedings of the National Academy of Sciences 114(19), 4975-4980. Guallar, C., Delgado, M., Diogene, J., Fernandez-Tejedor, M., 2016. Artificial neural network approach to population dynamics of harmful algal blooms in Alfacs Bay (NW Mediterranean): Case studies of Karlodinium and Pseudo-nitzschia. Ecological Modelling 338, 37-50. Hallegraeff, G.M., Anderson, D.M., Belin, C., Bottein, M.-Y.D., Bresnan, E., Chinain, M., Enevoldsen, H., Iwataki, M., Karlson, B., McKenzie, C.H., Sunesen, I., Pitcher, G.C., Provoost, P., Richardson, A., Schweibold, L., Tester, P.A., Trainer, V.L., Yñiguez, A.T., Zingone, A., 2021. Perceived global increase in algal blooms is attributable to intensified monitoring and emerging bloom impacts. Communications Earth & Environment 2(1), 117. Hassan, M.M., Parks, V., Laramore, S., 2021. Assessment of microalgae concentrate as diet for hard clam, Mercenaria mercenaria, larvae. Aquaculture Nutrition 27(6), 1871-1879. Hunt, K., Doré, B., Keaveney, S., Rupnik, A., Butler, F., 2023. A quantitative exposure assessment model for norovirus in oysters harvested from a classified production area. Microbial Risk Analysis 23, 100247. Ispra, 2022. Aziende in acquacoltura e produzioni 2019-2020. JACUMAR, 2023. Producción de acuicultura. Ministerio de Agricultura, Pesca y Alimentación. Krause, G., Le Vay, L., Buck, B.H., Costa-Pierce, B.A., Dewhurst, T., Heasman, K.G., Nevejan, N., Nielsen, P., Nielsen, K.N., Park, K., Schupp, M.F., Thomas, J.B., Troell, M., Webb, J., Wrange, A.L., Ziegler, F., Strand, A., 2022. Prospects of Low Trophic Marine Aquaculture Contributing to Food Security in a Net Zero-Carbon World. Frontiers in Sustainable Food Systems 6. Krock, B., Busch, J.A., Tillmann, U., Garcia-Camacho, F., Sanchez-Miron, A., GallardoRodriguez, J.J., Lopez-Rosales, L., Andree, K.B., Fernandez-Tejedor, M., Witt, M., Cembella, A.D., Place, A.R., 2017. LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean). Marine Drugs 15(12). Lacoste, E., Gaertner-Mazouni, N., 2015. Biofouling impact on production and ecosystem functioning: a review for bivalve aquaculture. Reviews in Aquaculture 7(3), 187-196. Lacoue-Labarthe, T., Nunes, P.A.L.D., Ziveri, P., Cinar, M., Gazeau, F., Hall-Spencer, J.M., Hilmi, N., Moschella, P., Safa, A., Sauzade, D., Turley, C., 2016. Impacts of ocean acidification in a warming Mediterranean Sea: An overview. Regional Studies in Marine Science 5, 1-11. Chapter 1. General introduction. _______________________________________________________________________________ 53 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Liu, Y., Saitoh, S.-I., Igarashi, H., Hirawake, T., 2014. The regional impacts of climate change on coastal environments and the aquaculture of Japanese scallops in northeast Asia: case studies from Dalian, China, and Funka Bay, Japan. International Journal of Remote Sensing 35(11-12), 44224440. Lupo, C., Bougeard, S., Le Bihan, V., Blin, J.L., Allain, G., Azéma, P., Benoit, F., Béchemin, C., Bernard, I., Blachier, P., Brieau, L., Danion, M., Garcia, A., Gervasoni, E., Glize, P., Lainé, A., Lapègue, S., Mablouké, C., Poirier, L., Raymond, J.C., Treilles, M., Chauvin, C., Le Bouquin, S., 2021. Mortality of marine mussels Mytilus edulis and M. galloprovincialis: systematic literature review of risk factors and recommendations for future research. Reviews in Aquaculture 13(1), 504-536. Masanja, F., Yang, K., Xu, Y., He, G., Liu, X., Xu, X., Xiaoyan, J., Xin, L., Mkuye, R., Deng, Y., Zhao, L., 2023. Impacts of marine heat extremes on bivalves. Frontiers in Marine Science 10. Mason, E., Barceló-Llull, B., Sánchez-Román, A., Rodríguez-Tarry, D., Cutolo, E., Delepoulle, A., Ruiz, S., Pascual, A., 2023. Chapter 8 - Fronts, eddies and mesoscale circulation in the Mediterranean Sea, In: Schroeder, K., Chiggiato, J. (Eds.), Oceanography of the Mediterranean Sea. Elsevier, pp. 263-287. Maulvault, A.L., Anacleto, P., Barbosa, V., Sloth, J.J., Rasmussen, R.R., Tediosi, A., FernandezTejedor, M., van den Heuvel, F.H.M., Kotterman, M., Marques, A., 2015. Toxic elements and speciation in seafood samples from different contaminated sites in Europe. Environmental Research 143, 72-81. Maurin, C.E., Byron, C.J., Wilson, K.A., St Gelais, A.T., 2019. Food webs and species biodiversity of the fouling community associated with bivalve aquaculture farms compared to analogous non-farm structures. Marine Environmental Research 147, 49-61. Moreau, P., Faury, N., Burgeot, T., Renault, T., 2015. Pesticides and Ostreid Herpesvirus 1 Infection in the Pacific Oyster, Crassostrea gigas. PLOS ONE 10(6), e0130628. Moreira, R., Balseiro, P., Forn-Cuní, G., Milan, M., Bargelloni, L., Novoa, B., Figueras, A., 2018. Bivalve transcriptomics reveal pathogen sequences and a powerful immune response of the Mediterranean mussel (Mytilus galloprovincialis). Marine Biology 165(4), 61. Naylor, R.L., Hardy, R.W., Buschmann, A.H., Bush, S.R., Cao, L., Klinger, D.H., Little, D.C., Lubchenco, J., Shumway, S.E., Troell, M., 2021. A 20-year retrospective review of global aquaculture. Nature 591(7851), 551-+. Newell, C.R., Hawkins, A.J.S., Morris, K., Boss, E., Thomas, A.C., Kiffney, T.J., Brady, D.C., 2021. Using High-Resolution Remote Sensing to Characterize Suspended Particulate Organic Matter as Bivalve Food for Aquaculture Site Selection. Journal of Shellfish Research 40(1), 113-118, 116. Ordonez, V., Pascual, M., Fernandez-Tejedor, M., Pineda, M.C., Tagliapietra, D., Turon, X., 2015. Ongoing expansion of the worldwide invader Didemnum vexillum (Ascidiacea) in the Chapter 1. General introduction. _______________________________________________________________________________ 54 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Mediterranean Sea: high plasticity of its biological cycle promotes establishment in warm waters. Biological Invasions 17(7), 2075-2085. Ordonez, V., Pascual, M., Fernandez-Tejedor, M., Turon, X., 2016. When invasion biology meets taxonomy: Clavelina oblonga (Ascidiacea) is an old invader in the Mediterranean Sea. Biological Invasions 18(4), 1203-1215. Palumbi, S.R., Evans, T.G., Pespeni, M.H., Somero, G.N., 2019. Present and future adaptation of marine species assemblages: DNA-based insights into climate change from studies of physiology, genomics, and evolution. Oceanography 32(3), 82–93. Peris, A., Barbieri, M.V., Postigo, C., Rambla-Alegre, M., Lopez de Alda, M., Eljarrat, E., 2022. Pesticides in sediments of the Ebro River Delta cultivated area (NE Spain): Occurrence and risk assessment for aquatic organisms. Environmental Pollution 305. Ramon, M., Fernandez, M., Galimany, E., 2007. Development of mussel (Mytilus galloprovincialis) seed from two different origins in a semi-enclosed Mediterranean Bay (NE Spain). Aquaculture 264(1-4), 148-159. Renault, T., 2011. Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know?, In: Margarita, S. (Ed.), Pesticides in the Modern World. IntechOpen, Rijeka, p. Ch. 12. Rey-Campos, M., Moreira, R., Gerdol, M., Pallavicini, A., Novoa, B., Figueras, A., 2019. Immune Tolerance in Mytilus galloprovincialis Hemocytes After Repeated Contact With Vibrio splendidus. Front. Immunol. 10. Rolton, A., Rhodes, L., Hutson, K.S., Biessy, L., Bui, T., MacKenzie, L., Symonds, J.E., Smith, K.F., 2022. Effects of Harmful Algal Blooms on Fish and Shellfish Species: A Case Study of New Zealand in a Changing Environment. Toxins 14(5), 341. Romero, A., Costa Md, M., Forn-Cuni, G., Balseiro, P., Chamorro, R., Dios, S., Figueras, A., Novoa, B., 2014. Occurrence, seasonality and infectivity of Vibrio strains in natural populations of mussels Mytilus galloprovincialis. Dis. Aquat. Org. 108(2), 149-163. Roque, A., Carrasco, N., Andree, K.B., Lacuesta, B., Elandaloussi, L., Gairin, I., Rodgers, C.J., Furones, M.D., 2012. First report of OsHV-1 microvar in Pacific oyster (Crassostrea gigas) cultured in Spain. Aquaculture 324, 303-306. Rowan, N.J., 2023. Current decontamination challenges and potentially complementary solutions to safeguard the vulnerable seafood industry from recalcitrant human norovirus in live shellfish: Quo Vadis? Science of The Total Environment 874, 162380. Saldaña-Serrano, M., Bastolla, C.L.V., Mattos, J.J., Lima, D., Freire, T.B., Nogueira, D.J., De-laTorre, G.E., Righetti, B.P.H., Zacchi, F.L., Gomes, C.H.A.M., Taniguchi, S., Bícego, M.C., Bainy, A.C.D., Chapter 1. General introduction. _______________________________________________________________________________ 55 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas 2022. Microplastics and linear alkylbenzene levels in oysters Crassostrea gigas driven by sewage contamination at an important aquaculture area of Brazil. Chemosphere 307, 136039. Schroeder, K., Tanhua, T., Chiggiato, J., Velaoras, D., Josey, S.A., García Lafuente, J., VargasYáñez, M., 2023. Chapter 4 - The forcings of the Mediterranean Sea and the physical properties of its water masses, In: Schroeder, K., Chiggiato, J. (Eds.), Oceanography of the Mediterranean Sea. Elsevier, pp. 93-123. Slater, M., James, P., 2023. Low trophic species in aquaculture—growth and research challenges. Journal of the World Aquaculture Society 54(1), 4-6. Snyder, J., Boss, E., Weatherbee, R., Thomas, A.C., Brady, D., Newell, C., 2017. Oyster Aquaculture Site Selection Using Landsat 8-Derived Sea Surface Temperature, Turbidity, and Chlorophyll a. Frontiers in Marine Science 4. Squadrone, S., Brizio, P., Stella, C., Prearo, M., Pastorino, P., Serracca, L., Ercolini, C., Abete, M.C., 2016. Presence of trace metals in aquaculture marine ecosystems of the northwestern Mediterranean Sea (Italy). Environmental Pollution 215, 77-83. Stoner, O., Economou, T., Torres, R., Ashton, I., Brown, A.R., 2023. Quantifying Spatiotemporal risk of Harmful Algal Blooms and their impacts on bivalve shellfish mariculture using a data-driven modelling approach. Harmful Algae 121, 102363. Supono, S., Mugica, M., Spreitzenbarth, S., Jeffs, A., 2023. Potential for Concentrated Microalgae as Replacement Diets for Juvenile Green-Lipped Mussels, Perna canaliculus. Aquaculture Research 2023, 9841172. Tan, K., Sun, Y., Zhang, H., Zheng, H., 2023. Effects of harmful algal blooms on the physiological, immunity and resistance to environmental stress of bivalves: Special focus on paralytic shellfish poisoning and diarrhetic shellfish poisoning. Aquaculture 563, 739000. Tanhua, T., Hainbucher, D., Schroeder, K., Cardin, V., Álvarez, M., Civitarese, G., 2013. The Mediterranean Sea system: a review and an introduction to the special issue. Ocean Sci. 9(5), 789803. Thomsen, J., Stapp, L.S., Haynert, K., Schade, H., Danelli, M., Lannig, G., Wegner, K.M., Melzner, F., 2017. Naturally acidified habitat selects for ocean acidification-tolerant mussels. Sci Adv 3(4), e1602411. Tissot, A.G., Granek, E.F., Thompson, A.W., Hladik, M.L., Moran, P.W., Scully-Engelmeyer, K., 2022. The silence of the clams: Forestry registered pesticides as multiple stressors on soft-shell clams. Science of The Total Environment 819, 152053. Chapter 1. General introduction. _______________________________________________________________________________ 56 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas van der Schatte Olivier, A., Jones, L., Vay, L.L., Christie, M., Wilson, J., Malham, S.K., 2020. A global review of the ecosystem services provided by bivalve aquaculture. Reviews in Aquaculture 12(1), 3-25. Venier, P., Varotto, L., Rosani, U., Millino, C., Celegato, B., Bernante, F., Lanfranchi, G., Novoa, B., Roch, P., Figueras, A., Pallavicini, A., 2011. Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis. BMC Genomics 12(1), 69. Vittoria Barbieri, M., Peris, A., Postigo, C., Moya-Garces, A., Simon Monllor-Alcaraz, L., Rambla-Alegre, M., Eljarrat, E., Lopez de Alda, M., 2021. Evaluation of the occurrence and fate of pesticides in a typical Mediterranean delta ecosystem (Ebro River Delta) and risk assessment for aquatic organisms. Environmental Pollution 274. Willer, D., Aldridge, D.C., 2017. Microencapsulated diets to improve bivalve shellfish aquaculture. Royal Society Open Science 4(11), 171142. Willer, D.F., Aldridge, D.C., 2019. Microencapsulated diets to improve bivalve shellfish aquaculture for global food security. Global Food Security 23, 64-73. _______________________________________________________________________________ 57 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 64 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas the suitability of the nearby Mediterranean waters for shellfish aquaculture. These are crucial topics for the future of shellfish aquaculture in the region of the Ebro delta and other Mediterranean areas. This study focuses on the development of a methodology to create, calibrate, and validate an algorithm that provides an accurate estimation of chlorophyll-a concentration in the coastal areas of the Ebro delta (NW Mediterranean) using atmospherically corrected Sentinel 2 (S2) remote sensing reflectances (Rrs) and a tool to calculate the carrying capacity of the potential new aquaculture areas and to evaluate its suitability. For this purpose, the DEB model has been implemented using twoyear estimates of chlorophyll-a concentration derived from Sentinel-2 imagery and additional sitespecific auxiliary data. The proposed approach is based on open-source data and open-source software tools. The use of remote sensing data will help the low trophic aquaculture industry to expand their activities to new suitable areas. 2. Materials and Methods 2.1. Study Area The Ebro delta extends 25 km from the mainland and forms two embayments, Alfacs and Fangar (Figure 21). Fangar Bay, located in the north, has an extension of 12 km2 and a mean depth of 2 m, it contains 16 × 106 m3 of seawater, and the mouth of the Bay is 1 km wide. Alfacs Bay is in the south, its extension is 50 km2, the mean depth is 4 m, it contains 200 × 106 m3 of seawater, and its mouth is 3 km wide. Seawater temperature inside the embayments presents wide fluctuations in comparison to open waters. Both bays receive freshwater inputs from the agriculture drainage channels, favoring stratification during calm conditions (Camp and Delgado, 1987). The average salinity is similar in both bays: 34.6 in Fangar Bay and 34.8 in Alfacs. Freshwater inputs are 228 × 106 m3/year and 365 × 106 m3/year in Fangar and Alfacs, respectively. Water renewal time is approximately 1–2 days in Fangar and 10 days in Alfacs Bay (Camp and Delgado, 1987). Chlorophyll-a concentration increases during wind events due to horizontal mixing and bottom resuspension (Balsells et al., 2021). Both embayments currently hold mussel and oyster production; the location of mussels and oyster rafts is shown in Figure 22. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 65 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 21. Map showing the location of the sampling stations that were visited for in situ measurements and collection of water samples. Sampling stations in the southern area are numbered from 1 to 10, sampling stations in the northern area are numbered from 11 to 20. Figure 22. Map showing the extent of the area of study divided in the area inside (AI, FI) and outside the coastal embayments. The external area is divided into different polygons (A1–A6, F1–F6). 2.2. In Situ Measurements Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 66 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Sampling cruises were conducted, coinciding with a Sentinel-2 (S2) pass when the weather forecast was favorable. A total of 17 sampling cruises were carried out during the period 2 September 2020 to 27 October 2021, 9 of them covering the northern area and 8 for the southern area of the Ebro delta (western Mediterranean). Ten sampling stations were visited during each cruise, including waters inside and outside the bays. The location of the 20 sampling stations is shown in Figure 1. At each station, bottom depth, and the Secchi disk depth (ZSD) were measured; the profiles of temperature and salinity were obtained using a SeaBird19plus CTD and water samples were taken for chlorophyll-a analysis. Upon arrival at the laboratory, water samples were filtered through Whatman® glass microfiber filters, Grade GF/F (0.7 μm) 47 mm, using low vacuum. The filters were maintained at -80 °C until analysis. Filter contents were extracted in acetone 90%, the absorbance of the extract was measured in a Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer, and the chlorophyll-a concentration was calculated using the formula Chlorophyll-a = 11.85 E664 − 1.54 E647 − 0.08 E630 (Jeffrey and Humphrey, 1975). 2.3. Maps of Chlorophyll-a from Sentinel-2 The Sentinel-2 (S2) constellation consists of two satellites (S2A and S2B) operated by The European Space Agency (ESA). Each satellite has on-board the MultiSpectral Instrument (S2-MSI). The S2MSI Level-1 (L1C, Top of atmosphere) imagery includes 13 spectral bands centered at different wavelengths from 443 nm to 2200 nm with different spatial resolutions (10, 20, and 60 m). All available S2-L1C images between 1 October 2019 and 30 September 2021 were downloaded from the Copernicus Services Data Hub (https://cophub.copernicus.eu/dhus/#/home, accessed on 14 July 2022). The only orbit completely covering the study area was orbit #51. Every day the data from this orbit were available, the four tiles needed to cover the area were downloaded. This amounted to a total of 560 images (140 images per tile), covering a surface of 100 × 100 km2 for each tile. Each image was visually checked for clouds and shadows over the region of study, leading to the rejection of 350 images (62.5 %), implying the rejection of 46 % of dates with chlorophyll-a in situ data. Due to project administrative deadlines, only images until September 2021 were used to accomplish the study. The pre-processing of S2 imagery was performed through the Graphic Processing Tool (GTP) of SNAP v8.0 (SNAP-ESA) and Rcore 3.6 (RCoreTeam). For atmospheric correction of S2-MSI L1C imagery, the Case 2 Regional Coast Colour (C2RCC) and the C2X-COMPLEX (C2XC) processors (C2-Nets) (Brockmann et al., 2016) included in SNAP were applied on all valid S2 images. These processors are based on a multi-sensor per-pixel artificial neural network (NN) method, and differ in their training ranges of inherent optical properties (Warren et al., 2019). The parametrization for the atmospheric correction of each image included: (a) pressure (hPa) from NCEP/DOE Reanalysis II data provided by the NOAA PSL, Boulder, Colorado, USA, from their website at https://psl.noaa.gov Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 67 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas (accessed on 16 July 2022) (Kanamitsu et al., 2002); (b) atmospheric ozone in Dobson units (DU) from the Aura OMI NASA dataset (NASA Goddard Space Flight Center), downloaded for each location and date from https://oceancolor.gsfc.nasa.gov/ (accessed on 17 July 2022) (NASA Ocean Color Data). Surface seawater temperature and salinity were obtained from the sampling cruises. For land/water segmentation, the valid pixel expression was set as a threshold on the short-wave infrared (SWIR) band of S2-MSI L1C images centered at 1600 nm (B11). The threshold was defined independently for each image with the triangle threshold method. From each C2-Net, remote sensing reflectance (Rrs) of visible and near infrared bands and the pigment absorption product (apig) were generated. C2-Nets flags, which include codes for quality control of pixels, were also exported. For C2RCC and C2XC, independently, images from the same date were merged when more than one tile were available. A procedure to select match-ups between chlorophyll-a concentration measured from water samples and S2 Rrs was performed for the two C2-Nets. This procedure involves several steps. Firstly a 3 × 3 pixel window, centered at the coordinates of in situ measurements, was extracted for each date and sampling location, and C2-nets were quality-checked in all extracted pixels by applying the recommended flags (Pereira-Sandoval et al., 2019). Then, flagged pixels as well as pixels with negative Rrs at bands B1, B2, B3, and B4 were removed from the analysis, as recommended by Cui et al. (Cui et al., 2010). The number of remaining pixels within each pixel window was checked; windows with less than 5 remaining pixels were removed from the analysis. Outliers were defined through Boxplot analysis applied to each available pixel window and spectral band (B1-B7 and B8A). Finally, the remaining pixel windows with less than 5 valid pixels were removed from the analysis. A set of band combinations in the form of spectral indices were computed for all valid Rrs matchups (Table 21). These spectral indices include visible and red edge S2 spectral bands (B1 to B6; ~443 nm to ~740 nm), exploiting specific chlorophyll-a absorption peaks in the blue and red spectral regions. The red edge bands are used for mitigating the effect of absorption by non-algal particles, yellow substances, and backscattering. For each C2-Net, chlorophyll-a was modelled with all the computed spectral indices and the apig band. Seventy percent of the data were used for model calibration (cal) and 30% for model validation (val). The cal/val datasets were generated randomly. Models were developed for raw and log-transformed Rrs/apig and chlorophyll-a data. Linear, linear piecewise (1 breakpoint), polynomial (2nd, 3rd, and 4th order), logarithmic, power, and exponential models were tested. The entire process was iterated 100 times with varying cal/val datasets. For each model (C2-Net × Band Combination × Type of fitting), performance was evaluated by means of the following statistics: mean average error (MAE), root mean squared error (RMSE), average percentage difference (APD), BIAS, and Pearson’s r. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 68 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Table 21. Spectral bands and spectral combinations tested for chlorophyll-a estimation. Bands combination Reference Equations Red to Green ratio (RG) (Cairo et al., 2020) Rrs(B4)/Rrs(B3) Blue to Green ratio (BG) Based on OC2 (O'Reilly and Werdell, 2019) Rrs (B2)/Rrs(B3) Blue to Green ratio 2 (BG2) Based on OC3 (O'Reilly and Werdell, 2019) max (Rrs (B1), Rrs(B2))/Rrs(B3) Red Edge 1 to Blue (REB1) (O'Reilly and Werdell, 2019) Rrs (B5)/Rrs(B2) Red Edge 1 to Green (REG1) (Cairo et al., 2020) Rrs (B5)/Rrs(B3) Red Edge 1 to Red (RER) (Cairo et al., 2020) (Gitelson et al., 2011) Rrs (B5)/Rrs(B4) Red Edge 2 to Green (REG2) (Cairo et al., 2020) Rrs (B6)/Rrs(B3) GIT (3 band model) (Gitelson et al., 2011) (1/Rrs(B4) - 1/Rrs(B5))*Rrs(B6) Normalized Difference Chlorophyll Index (NDCI) (Mishra and Mishra, 2012) (Rrs(B6) - Rrs(B5))/(Rrs(B6) + Rrs(B5)) apig (apig) (Niroumand-Jadidi et al., 2021) apig To generate the maps of chlorophyll-a, all flagged pixels (atmospheric correction flagging) were removed, as were pixels with negative Rrs in any of the spectral bands of each valid S2 image processed with the C2-Nets. The best performant model was selected according to lowest MAE, APD, RMSE, and BIAS, in this order, and it was applied to all valid images and pixels. Pixels with negative chlorophyll-a values were set to 0 mg/m3 and pixels with unreliably high chlorophyll-a concentration (>30 mg/m3) were removed. Pixels corresponding to the mussel farm structures or rafts were masked out using an available shapefile to mitigate/avoid pixel mixing problems. 2.4. Carrying Capacity Model For the analysis and extraction of data from the generated images, QGIS 3.24 was used (QGIS.org, 2022). The area of interest corresponding to the masked images was divided into 2 different regions inside and outside the two coastal embayments (Figure 22). The studied area comprises 37.5 km2 inside and 37.8 km2 outside Alfacs Bay (southern area) and 2.7 km2 inside and 19.7 km2 outside Fangar Bay (northern area). The external area was divided into 12 polygons, 6 outside each embayment (Figure 22). The area inside each polygon was 5.5, 7.5, 7, 6.8, 4.8, 6.1 km2 for A1–A6 and 1.8, 2.5, 2.6, 2.9, 3.8, 6.1 km2 for F1–F6. Monthly chlorophyll-a concentration was averaged for each area of study, combining the available images for the period. The external areas were divided into different parts (polygons) to evaluate the gradient in chlorophyll-a concentration at different distances from the mouth of the bay. Each polygon was divided into three parts to evaluate the impact of the distance to the shore on the chlorophyll-a concentration. The chlorophyll-a concentration, retrieved from the S2-L1C images treated with the algorithm calibrated and validated in this study, was used to determine the suitability of the different areas and their carrying capacity. To determine the suitability of each polygon for mussel aquaculture, the DEB model was applied. The model allows us to calculate the amount of energy used for growth and Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 69 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas reproduction through the ingestion of the available food using the version from Rosland et al. (2009) and the notation from Kooijman (1986). Mussel ingestion rate is proportional to the surface of the mussel, expressed as structural volume. 𝑃󰇗𝑥={𝑝󰇗𝑋𝑚} 𝑓 𝑇𝑑 𝑉23 ⁄ (1) 𝑓= 𝜒 𝜒+𝜒𝑘 (2) 𝜒𝑘= {𝑝󰇗𝑋𝑚} 𝑉23 ⁄ 𝐶𝑓 𝐶𝑅 (3) 𝐿=𝑉13 ⁄ 𝛿𝑚 (4) 𝑇𝑑=exp(𝑇𝐴 𝑇𝐼−𝑇𝐴 𝑇) 1+𝑒𝑥𝑝(𝑇𝐴𝐿 𝑇−𝑇𝐴𝐿 𝑇𝐿)+𝑒𝑥𝑝(𝑇𝐴𝐻 𝑇𝐻−𝑇𝐴𝐻 𝑇) (5) where 𝑃󰇗𝑥 is the ingestion rate; the values used for each parameter were obtained from van der Veer et al. (2006) and are shown in Table 22. Clearance rates (𝐶𝑅) were obtained from Galimany et al. (2011). The measured in situ seawater temperature (°C) at 1 m depth was converted to Kelvin degrees (K). In Equation (2), f is the Michaelis–Menten Equation to scale the ingestion rate to the food concentrations (X); this term scales the amount of food ingested as a function of the food (chlorophyll-a) available. In Equation (4), L is mussel length (cm), where δM is the dimensionless shape coefficient. Td in Equation (5) is the Arrhenius temperature function. The model (Equations (1)–(5)) was computed in Python. Table 22. Values used for the DEB model. Parameter Unit Definition Value {𝒑󰇗𝑿𝒎} J cm-2 d-1 Maximum Surface area-specific assimilation rate 273 𝝌𝒌 mg C m-3 Half saturation coefficient calculated 1 𝑻𝑨 K Arrhenius temperature 5800 𝑻𝑰 K Reference temperature 289 𝑻𝑳 K Lower boundary of tolerance range 275 𝑻𝑯 K Upper boundary of tolerance range 296 𝑻𝑨𝑳 K Arrhenius temperature for rate of decrease at lower boundary 45430 𝑻𝑨𝑯 K Arrhenius temperature for rate of decrease at upper boundary 31376 𝜹𝒎 Shape coefficient 0.29 V cm3 Bivalve volume 0.06 Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 70 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas 𝐶𝑓 Jµg-1 chla 0.419 1 Calculated using Formula (3) In a previous study (Ibáñez-Solé, 2014), the DEB model was found to better reflect field observations of mussel ingestion in Alfacs Bay, in comparison with the Scope for Growth (SFG). Carrying capacity was calculated by applying the recommendations of the Aquaculture Stewardship Council (2019). This standard provides indicators for a first approach to characterize the potential of an area. In the present study, the carrying capacity was defined as the number of rafts/ropes/mussels that can be grown in each area and was calculated as a function of the food availability measured through the chlorophyll-a concentration and the renewal time of the water masses. Chlorophyll-a concentration was obtained from the S2 images treated with the algorithm in this study, and the renewal time was calculated using the current speed at the different external areas. 𝐶𝐶(𝑚𝑢𝑠𝑠𝑒𝑙𝑠)=[𝐶ℎ𝑙𝑎(𝑚𝑔)∗0.75 𝐶𝑟(𝑙ℎ,𝑑𝑎𝑡𝑒)𝑚𝑢𝑠𝑠𝑒𝑙𝑠∗𝐶ℎ𝑙𝑎(𝑚𝑔 𝑙,𝑑𝑎𝑡𝑒). ] 𝑇𝑟(ℎ) (6) 𝐶𝐶(𝑟𝑜𝑝𝑒𝑠)=𝐶𝐶(𝑚𝑢𝑠𝑠𝑒𝑙𝑠) 𝑟𝑜𝑝𝑒 (7) In formula (6), 𝑇𝑟(ℎ) is the renewal time of the water in each area, 𝐶ℎ𝑙𝑎(𝑚𝑔) is the total amount of chlorophyll-a in the whole volume of each area, and 𝐶ℎ𝑙𝑎(𝑚𝑔 𝑙) is the chlorophyll-a concentration of each area obtained from the Satellite images. 𝐶ℎ𝑙𝑎(𝑚𝑔)=𝑉𝑜𝑙𝑢𝑚𝑒 (𝑙) 𝑥 𝐶ℎ𝑙𝑎(𝑚𝑔 𝑙)(8) Different values of clearance rate, rate 𝐶𝑟(𝑙ℎ,𝑑𝑎𝑡𝑒)𝑚𝑢𝑠𝑠𝑒𝑙𝑠 were applied by selecting the corresponding values for each time of the year. The percentage of chlorophyll-a available for the cultivated mussels was assumed to be 0.75, and 0.25 was the remnant percentage. A standardized length of 3 m per rope that holds 1250 mussels was also used. These are the usual characteristics of the mussel ropes used in the Ebro delta. For the external areas, the average renewal times were calculated from the Copernicus Marine Service (Clementi et al., 2021) using data from the years 2019 to 2021. For all areas, the volume was calculated for 3 m of depth as standard length of mussel Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 71 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas ropes. The renewal times used for the areas inside the embayments were obtained from the bibliography (Delgado, 1987). We have not included primary production time because renewal times are short in the external areas. The following diagram Figure 23 shows the relation between in situ data, remote sensing data, and the different models used in this study. Figure 23. Diagram showing the relation between in situ data, remote sensing data, and the different models. 3. Results 3.1. In Situ Measurements The results from the in situ measurements in October 2021 were not used for the calibration and validation of the satellite images but are included here to provide a wider view of the in situ environmental conditions of the area. The average depth of the 16 sampling stations was 7.7 ± 0.2 m (Mean ± Std. Error) and the range was 3.7–14.8 m. The average Secchi depth was 3.5 ± 0.2 m (Mean ± Std. Error) and the range was 1.3–10.4 m (Figure 2S1). The average chlorophyll-a concentration in the water samples was 2.4 ± 0.1 mg/m3 (Mean ± Std. Error) and the range was 0.1-8.9 mg/m3 (Figure 24). There was a statistically significant difference in chlorophyll-a concentration and Secchi disk depth (p ≤ 0.001) between the interior and the exterior sampling stations from the whole studied area (Kruskal–Wallis One Way Analysis of Variance on Ranks). In the southern area, there was no statistically significant difference in the mean values of chlorophyll-a concentration among the different sampling stations (p = 0.118) and dates (p = 0.251) (Two Way Analysis of Variance). In the northern area, there was a statistically significant difference (p ≤ 0.001) between sampling stations and dates. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 72 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 24. Concentration of chlorophyll-a (mg/m3) at the different sampling stations. The range in seawater temperature was 10.9–28.2 °C in the northern area outside Fangar Bay and 11.7–29.2 °C in the southern area outside Alfacs Bay. Minimum values were registered in January 2020 and maximum values in August 2020 (Figure 25). Higher temperature is measured in the southern area in comparison to the northern area. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 73 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 25. Seawater temperature (°C) in the external areas of the Ebro delta during the period of study. 3.2. Maps of Chlorophyll-a from Sentinel-2 Models performed better with C2XC than with C2RCC, particularly coupled with band combinations including spectral bands in the blue or green, and red or red edge regions. The best performance models for each C2-Net and band combinations are shown in Table 3. The best model for chlorophylla estimation was found with the C2XC processor, applying the REB1 band ratio (see Table 23) with a 2nd polynomial fitting ([chlorophyll-a] = −0.615 + 10.88 × REB1 + 8.704 × REB12). Table 23. Best performing models per C2-Net and spectral band combination. Bands combination C2-Nets FIT 1 MAE (mg/m3) APD (%) RMSE (mg/m3) BIAS (mg/m3) r RG C2RCC Pm4 0.736 39.843 1.107 -0.075 0.820 RG C2XC Pm4 0.703 59.572 0.949 0.083 0.870 BG C2RCC Lm 0.726 39.815 1.107 -0.070 0.817 BG C2XC LLPm3 0.734 39.634 1.120 -0.102 0.816 BG2 C2RCC Lm 0.751 59.752 1.013 -0.061 0.850 BG2 C2XC LLPm3 0.734 39.634 1.120 -0.102 0.816 REB1 C2RCC Lm 0.741 39.530 1.101 0.018 0.820 REB1 C2XC Pm2 0.598 36.216 0.887 0.054 0.887 REG1 C2RCC Lm 0.738 38.898 1.097 -0.027 0.820 REG1 C2XC Pm4 0.616 42.844 0.878 -0.022 0.889 RER C2RCC Lm 0.745 51.303 1.090 -0.056 0.825 RER C2XC LLPm4 1.005 73.194 1.428 -0.263 0.682 REG2 C2RCC Lm 0.742 51.294 1.047 0.039 0.839 REG2 C2XC LLPm2 0.669 50.039 0.947 -0.048 0.870 GIT C2RCC Lm 0.748 54.933 1.038 0.019 0.842 GIT C2XC LLPm4 1.052 79.078 1.486 -0.223 0.644 NDCI C2RCC Lm 0.749 42.445 1.104 0.095 0.820 NDCI C2XC Pm4 1.358 113.510 1.880 -0.297 0.251 apig C2RCC Lm 0.753 59.581 1.010 -0.017 0.850 apig C2XC LLPm4 1.064 78.601 1.483 -0.193 0.661 1 Lm: Linear model; PmX: X degree polynomial; LLPmX: Log-Log X degree polynomial. The calibration and validation plots, as well as the fit between observed and estimated chlorophyll-a concentration values, are presented in Figure 26. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 80 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas A3 7 x 106 11.1 478 15 A4 6.8 x 106 10.3 494 14 A5 4.8 x 106 7.3 460 10 A6 6.1 x 106 9.2 437 14 FI 2.7 x 106 24.5 85 64 F1 1.8 x 106 13 107 17 F2 2.5 x 106 15.5 120 21 F3 2.6 x 106 13.8 107 15 F4 2.9 x 106 12.5 174 17 F5 3.8 x 106 11.4 223 17 F6 6.1 x 106 12.9 325 19 4. Discussion The values of in situ Secchi disk depth and chlorophyll-a concentration measured in this study (2019– 2021) differed slightly from those found in a previous study (Delgado, 1987) conducted during the period 1982–1983. The Secchi disk depth (ZSD), at that time, was measured at some of the same sampling stations used for our study. The values obtained in (Delgado, 1987) were 0.7–1.9 m deeper than our measurements when comparing the average values for each station. The same authors measured chlorophyll-a concentration and found yearly averages for the inner areas of 3.2 mg/m3 for AI and 3.44 mg/m3 for FI; these values are slightly lower than those found in our study: 5.2 mg/m3 for AI and 4 mg/m3 for FI. Therefore, the differences in Secchi disk depth can be partially explained by differences in chlorophyll-a concentration. Secchi disk depth (ZSD) is influenced by the phytoplankton biomass and other optical variables such as the colored dissolved organic matter (CDOM) and suspended particulate matter (SPM). Resuspension of sediments and the brownification of coastal waters influence ZSD, and therefore its relationship with chlorophyll-a concentration is not a straight inverse relation. The same authors detected maximum chlorophyll-a values in June– July (3.5–11 mg/m3) and September–October (25 mg/m3), and values in the range of 1–3.7 mg/m3 for the rest of the year. The maximum chlorophyll-a concentration in water samples measured in our study was 8.9 mg/m3 in December 2020 (AI). Higher values were observed from the satellite images. It is important to highlight that the values of chlorophyll-a retrieved from the Sentinel-2 images for the different polygons located at different distances from the mouth of the bays reflect the gradients observed in the in situ measurements of chlorophyll-a concentration. Different methods exist for atmospheric correction in coastal waters, and some of them (C2-Nets, iCOR) are available in open-source tools such as SNAP. This feature opens the door to nonspecialized users to generate atmospherically corrected S2 images by using this tool and following the proposed methodology. The model proposed here for mapping chlorophyll-a concentration, and the pixels quality control procedure, can also be implemented using the band-math’s functions provided by this tool. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 81 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas The proposed methodology allows one to estimate chlorophyll-a concentration from Sentinel-2 with an accuracy higher than 70 % in most cases (Table 23 and Figure 26). The best performant model was achieved with C2XC, which showed more consistent and accurate Rrs estimates than C2RCC in prior research (Soriano-Gonzalez et al., 2022). However, empirical algorithms, such as the one used, can be expected to perform well only inside their range and for the area they were derived for. They are also limited in their ability to discriminate between non-unique signals from parameters that may be covariant, for example TSM and chlorophyll-a concentration (Matthews, 2011). The developed model is less reliable in conditions of high concentration of TSM and/or CDOM, which may occur after strong winds or storms (increased water turbulence, sediment resuspension, land runoff), as observed on 5 February 2020 (Figure 27). In these cases, the selected model tends to overestimate chlorophyll-a concentration because the red edge reflectance increases at high TSM values overlapping the absorption of chlorophyll-a in the blue region of the spectrum, which is also affected by greater CDOM concentration (CDOM strongly absorbs light up to 500 nm) (Ligi et al., 2017; Niroumand-Jadidi et al., 2021). In these cases, other empirical or semi-analytical algorithms may be more accurate. Further research should focus on the development of a multi-algorithm blending approach, which has proven to be more suitable across different types of water optical properties (Moore et al., 2014), albeit it will involve a more complex procedure. The development of an algorithm switching-based method requires the accurate definition of the optical water type pixel by pixel, accounting for the spectral shape, magnitude, and distinctive Rrs spectral features (Spyrakos et al., 2018; Uudeberg et al., 2019) and considering the uncertainty of the Rrs retrieval from C2-Nets across different optical water types (Soriano-Gonzalez et al., 2022). In addition, in situ data covering all different scenarios are desirable, which may be difficult to reach due to complex logistics and meteorological limitations (i.e., cloud coverage), as occurred in our study. The gradient observed in chlorophyll-a concentration from the inner areas towards the open sea is also observed in the values of ingestion rate (𝑃𝑥󰇗). The values of 𝑃𝑥󰇗. integrate the effect of chlorophylla concentration and seawater temperature on mussel physiology. Lower seawater temperature in summer in open waters in comparison to the inner areas could compensate the effect of having less phytoplankton available in these waters, obtaining similar or even better growth when high seawater temperature decreases mussel filtration rates. When we observe the mean values for the period of study in each area, in the north, we can see that the gradient decreases from 9.1 Jd−1 to 6.7 Jd−1 from area F1 to area F5, and it increases slightly to 6.9 Jd−1 in area F6. The gradient is less stepped in the southern area, from 9.3 Jd−1 in A1 to 8.3 Jd−1 in A6. We can employ the value of 𝑃𝑥󰇗. to rank the suitability of the different polygons for mussel aquaculture; the most suitable would be those with the highest 𝑃𝑥󰇗, which are A1 and F1 followed by A2–5. The less suitable would be F3–6. We have based our model on the chlorophyll-a concentration measured in each area. The DEB model reflects well the decrease in food ingestion during summer months due to limiting seawater temperatures Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 82 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas (Anestis et al., 2007). The use of chlorophyll-a as a proxy does not consider changes in food quality due, for example, to the presence of certain phytoplankton species with a different nutritional value. Some authors have detected gaps in DEB model parametrization and proposed to improve seston characterization (Filgueira et al., 2020; Filgueira et al., 2019), which should be tackled in further research. A shellfish farm may exceed the ecological carrying capacity when the removal of phytoplankton biomass exceeds the renewal, resulting in a phytoplankton depleted water mass. In the proposed CC model, the renewal time of each area is shorter than the clearance rate time, thus complying with the ASC bivalve standard (Aquaculture Stewardship Council, 2019) that recommends comparing how long it takes to clear the body (CT) of water with the renewal time (RT), the ratio CT/RT should be >1. The same standard recommends that in locations where CT < RT, then the ratio CT/PPT should be >3. PPT is the number of days required for the replacement of phytoplankton biomass (PB) in the water body considering phytoplankton growth (PB/PPP; PPP = phytoplankton primary production). When the clearance rate is higher than the renewal rate plus the primary production, the body of water is depleted of phytoplankton and the bivalve stock will have less food available. Other authors suggest employing the regulation ratio defined as a fraction of the phytoplankton turnover rate RR = (1/CT)/(1/PT), where PT is the time it takes to renew the phytoplankton stock in an area (Filgueira et al., 2019; Smaal and van Duren, 2019). We have applied the same formula for CC in the internal and external areas without considering primary production time. Renewal time in the internal areas is longer and it would be appropriate to include primary production time in the calculations. There are already mussel and oyster farms inside the embayments, therefore the chlorophyll-a concentration measured is affected already by the consumption of the bivalves from these farms. More complex models are needed to assess, with more detail, the carrying capacity inside the embayments. Renewal times are not uniform inside the embayments where hydrodynamic models such as the Regional Ocean Model System (ROMS) coupled to a biogeochemical nutrient-phytoplankton-zooplanktondetritus model (NPZD) such as in Dabrowski et al. (2013) will provide a more accurate evaluation. Hydrodynamical modeling coupled to ecological modeling has been applied in other geographical areas to decide the most appropriate locations of the bivalve farms inside an embayment to avoid negative consequences for the ecosystem (Filgueira et al., 2021). We have shown how small changes in renewal time affect the carrying capacity of the different water bodies in our study. The standard from the Aquaculture Stewardship Council (Aquaculture Stewardship Council, 2019) recommends to use retention time calculated as the number of days for tides to flush a volume of water equal to the volume of the area. In the Mediterranean Sea, tides have low amplitude, and so flushing time has to be calculated by other means. The results on the modeled number of rafts in each area show that these external areas can hold a significant production of mussels in waters that are less impacted by warm events in summer. Mussel Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 83 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas growth in these areas may be slower in comparison to the embayments due to lower chlorophyll-a concentration. The differences in seawater temperature measured during this study in summer were 1–2 °C for the same day and depth between internal and external areas; this small difference may be crucial to reduce mussel mortality in summer. 5. Conclusions The proposed methodology can be reproduced using open-source tools such as SNAP software, enabling end-users to obtain their own maps of chlorophyll-a concentration from Sentinel-2 images. This methodology allows one to estimate chlorophyll-a concentration from Sentinel-2 with an accuracy higher than 70 % in most cases. The best performant model was achieved with C2XC for atmospheric correction of S2-MSI L1C imagery. We have shown that the application of the DEB theory using multispectral remote sensing imagery allows one to rank the suitability of the different areas for shellfish aquaculture. It also allows one to determine the periods of the year with favorable and unfavorable conditions for shellfish growth. The results presented in this study show that the carrying capacity of the different areas is highly variable depending on the renewal times of the water, which at the same time are highly variable during the year. Nevertheless, the external areas located close to the Ebro delta embayments can hold a significant mussel production that would allow the reduction of mussel mortality during summer months due to the lower seawater temperature in these areas. This study shows the potential of the combined use of satellite remote sensing, in situ sampling, and carrying capacity models as a tool for helping the low trophic aquaculture industry to expand their activities to new suitable areas. Funding: This research was funded by the European Commission though the project New Technologies, Tools and Strategies for a Sustainable, Resilient and Innovative European Aquaculture (NewTechAqua), European Commission, Grant agreement ID: 862658 from Horizon 2020 Societal Challenges. Acknowledgments: We acknowledge the support given by the technical staff of IRTA. References Anestis, A., Lazou, A., Portner, H.O., Michaelidis, B., 2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. American Journal of Physiology-Regulatory Integrative and Comparative Physiology 293(2), R911-R921. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 84 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Angelats, E., Soriano-Gonzalez, J., Fernandez-Tejedor, M., Alcaraz, C., 2022. Combined flooding and water quality monitoring during short extreme events using Sentinel-2: the case study of Gloria storm in Ebro delta., 24th ISPRS Congress on Imaging Today, Foreseeing Tomorrow, Nice, FRANCE, pp. 361-368. Aquaculture Stewardship Council, 2019. ASC Bivalve Standard version 1.1. Babin, M., Roesler, C.S., Cullen, J.J., 2008. Real-time Coastal Observing Systems for Marine Ecosystem Dynamics and Harmful Algal Blooms: theory, instrumentation and modelling. UNESCO, Paris, France. Balsells, M.F.P., Grifoll, M., Fernandez-Tejedor, M., Espino, M., 2021. Short-Term Response of Chlorophyll a Concentration Due to Intense Wind and Freshwater Peak Episodes in Estuaries: The Case of Fangar Bay (Ebro Delta). Water 13(5). Barille, L., Le Bris, A., Goulletquer, P., Thomas, Y., Glize, P., Kane, F., Falconer, L., Guillotreau, P., Trouillet, B., Palmer, S., Gernez, P., 2020. Biological, socio-economic, and administrative opportunities and challenges to moving aquaculture offshore for small French oyster-farming companies. Aquaculture 521. Brigolin, D., Porporato, E.M.D., Prioli, G., Pastres, R., 2017. Making space for shellfish farming along the Adriatic coast. Ices Journal of Marine Science 74(6), 1540-1551. Brockmann, C., Doerffer, R., Peters, M., Kerstin, S., Embacher, S., Ruescas, A., 2016. Evolution of the C2RCC Neural Network for Sentinel 2 and 3 for the Retrieval of Ocean Colour Products in Normal and Extreme Optically Complex Waters, Living Planet Symposium, p. 54. Cairo, C., Barbosa, C., Lobo, F., Novo, E., Carlos, F., Maciel, D., Flores, R., Silva, E., Curtarelli, V., 2020. Hybrid Chlorophyll-a Algorithm for Assessing Trophic States of a Tropical Brazilian Reservoir Based on MSI/Sentinel-2 Data. Remote Sensing 12(1). Camp, J., Delgado, M., 1987. Hidrografía de las bahías del delta del Ebro. Investigación Pesquera 51(3), 351-369. Cardoso, J., van der Veer, H.W., Kooijman, S., 2006a. Body-size scaling relationships in bivalve species: A comparison of field data with predictions by the Dynamic Energy Budget (DEB) theory. Journal of Sea Research 56(2), 125-139. Cardoso, J., Witte, J.I., van der Veer, H.W., 2006b. Intraand interspecies comparison of energy flow in bivalve species in Dutch coastal waters by means of the Dynamic Energy Budget (DEB) theory. Journal of Sea Research 56(2), 182-197. Cerralbo, P., Espino, M., Grifoll, M., 2016. Modeling circulation patterns induced by spatial cross-shore wind variability in a small-size coastal embayment. Ocean Modelling 104, 84-98. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 85 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Chen, J., Zhang, M.W., Cui, T.W., Wen, Z.H., 2013. A Review of Some Important Technical Problems in Respect of Satellite Remote Sensing of Chlorophyll-a Concentration in Coastal Waters. Ieee Journal of Selected Topics in Applied Earth Observations and Remote Sensing 6(5), 2275-2289. Clementi, E., Aydogdu, A., Goglio, A.C., Pistoia, J., Escudier, R., Drudi, M., Grandi, A., Mariani, A., Lyubartsev, V., Lecci, R., Cretí, S., Coppini, G., Masina, S., Pinardi, N., 2021. Mediterranean Sea Physical Analysis and Forecast (CMEMS MED-Currents, EAS6 system) (Version 1) set. Copernicus Monitoring Environment Marine Service (CMEMS). Cui, T.W., Zhang, J., Groom, S., Sun, L., Smyth, T., Sathyendranath, S., 2010. Validation of MERIS ocean-color products in the Bohai Sea: A case study for turbid coastal waters. Remote Sensing of Environment 114(10), 2326-2336. Dabrowski, T., Lyons, K., Cure, M., Berry, A., Nolan, G., 2013. Numerical modelling of spatiotemporal variability of growth of Mytilus edulis (L.) and influence of its cultivation on ecosystem functioning. Journal of Sea Research 76, 5-21. Dame, R.F., Prins, T.C., 1997. Bivalve carrying capacity in coastal ecosystems. Aquatic Ecology 31(4), 409-421. Delgado, M., 1987. Fitoplancton de las bahías del delta del Ebro. Investigación Pesquera 51(4), 517-548. DeLuca, N.M., Zaitchik, B.F., Guikema, S.D., Jacobs, J.M., Davis, B.J.K., Curriero, F.C., 2020. Evaluation of remotely sensed prediction and forecast models for Vibrio parahaemolyticus in the Chesapeake Bay. Remote Sensing of Environment 250. Dong, S.P., Wang, F., Zhang, D.X., Yu, L.Y., Pu, W.J., Shang, Y.K., 2022. Growth performance and ecological services evaluation of razor clams based on dynamic energy budget model. Journal of Environmental Management 306. Duarte, P., Fernandez-Reiriz, M.J., Labarta, U., 2012. Modelling mussel growth in ecosystems with low suspended matter loads using a Dynamic Energy Budget approach. Journal of Sea Research 67(1), 44-57. FAO, 2022a. The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. FAO, Rome, Italy. Filgueira, R., Chica, M., Palacios, J.J., Strohmeier, T., Lavaud, R., Aguera, A., Damas, S., Strand, O., 2020. Embracing multimodal optimization to enhance Dynamic Energy Budget parameterization. Ecological Modelling 431. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 86 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Filgueira, R., Guyondet, T., Comeau, L.A., Grant, J., 2014. A fully-spatial ecosystem-DEB model of oyster (Crassostrea virginica) carrying capacity in the Richibucto Estuary, Eastern Canada. Journal of Marine Systems 136, 42-54. Filgueira, R., Guyondet, T., Thupaki, P., Sakamaki, T., Grant, J., 2021. The effect of embayment complexity on ecological carrying capacity estimations in bivalve aquaculture sites. Journal of Cleaner Production 288. Filgueira, R., Strople, L.C., Strohmeier, T., Rastrick, S., Strand, O., 2019. Mussels or tunicates: That is the question. Evaluating efficient and sustainable resource use by low-trophic species in aquaculture settings. Journal of Cleaner Production 231, 132-143. Fuentes-Santos, I., Labarta, U., Alvarez-Salgado, X.A., 2019. Modelling mussel shell and flesh growth using a dynamic net production approach. Aquaculture 506, 84-93. Galimany, E., Ramon, M., Ibarrola, I., 2011. Feeding behavior of the mussel Mytilus galloprovincialis (L.) in a Mediterranean estuary: A field study. Aquaculture 314(1-4), 236-243. Gencat, 2022. Evolució de la producció aqüícola marina. Departament d'Acció Climàtica, Alimentació i Agenda Rural. Gernez, P., Barille, L., Lerouxel, A., Mazeran, C., Lucas, A., Doxaran, D., 2014. Remote sensing of suspended particulate matter in turbid oyster-farming ecosystems. Journal of Geophysical Research-Oceans 119(10), 7277-7294. Giordano, L., Portacci, G., Caroppo, C., 2019. Multidisciplinary tools for sustainable management of an ecosystem service: The case study of mussel farming in the Mar Piccolo of Taranto (Mediterranean, Ionian Sea). Ocean & Coastal Management 176, 11-23. Gitelson, A.A., Gao, B.C., Li, R.R., Berdnikov, S., Saprygin, V., 2011. Estimation of chlorophylla concentration in productive turbid waters using a Hyperspectral Imager for the Coastal Ocean-the Azov Sea case study. Environmental Research Letters 6(2). Gordon, H.R., Morel, A.Y., 1983. Remote Assessment of Ocean Color for Interpretation of Satellite Visible Imagery: A Review. Springer US, New York, NY. Graham, P., Falconer, L., Telfer, T., Mossone, P., Viale, I., Carboni, S., 2020. A modelling approach to classify the suitability of shallow Mediterranean lagoons for pacific oyster, Crassostrea gigas (Thunberg, 1793) farming. Ocean & Coastal Management 192. Guyondet, T., Roy, S., Koutitonsky, V.G., Grant, J., Tita, G., 2010. Integrating multiple spatial scales in the carrying capacity assessment of a coastal ecosystem for bivalve aquaculture. Journal of Sea Research 64(3), 341-359. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 87 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Habbane, M., ElSabh, M.I., Dubois, J.M., 1997. Determination of potential for aqua-culture activities via passive teledetection and a grid-based geographical information system - Application to coastal waters to the Baie-des-Chaleurs (Eastern Canada). International Journal of Remote Sensing 18(16), 3439-3457. Harley, C.D.G., 2008. Tidal dynamics, topographic orientation, and temperature-mediated mass mortalities on rocky shores. Marine Ecology Progress Series 371, 37-46. Hou, Y.X., Zhao, G., Chen, X.H., Yu, X., 2022. Improving Satellite Retrieval of Coastal Aquaculture Pond by Adding Water Quality Parameters. Remote Sensing 14(14). Huot, Y., Babin, M., Bruyant, F., Grob, C., Twardowski, M.S., Claustre, H., 2007. Relationship between photosynthetic parameters and different proxies of phytoplankton biomass in the subtropical ocean. Biogeosciences 4(5), 853-868. Ibáñez-Solé, J., 2014. Modelo dinámico para el cálculo de la capacidad de carga de una bahía costera para el cultivo de bivalvos, In: Fernández-Tejedor, M., García-Ybarra, P.L. (Eds.). Universidad Nacional de Educación a Distancia (España). Facultad de Ciencias. Jeffrey, S.W., Humphrey, G.F., 1975. New spectrometric equations for determining chlorophylls a, b, c1 and c2 in higher-plants, algae and natural phytoplankton. Biochemie Und Physiologie Der Pflanzen 167(2), 191-194. Juza, M., Fernandez-Mora, A., Tintore, J., 2022. Sub-Regional Marine Heat Waves in the Mediterranean Sea From Observations: Long-Term Surface Changes, Sub-Surface and Coastal Responses. Frontiers in Marine Science 9. Juza, M., Tintore, J., 2021. Multivariate Sub-Regional Ocean Indicators in the Mediterranean Sea: From Event Detection to Climate Change Estimations. Frontiers in Marine Science 8. Kanamitsu, M., Ebisuzaki, W., Woollen, J., Yang, S.K., Hnilo, J.J., Fiorino, M., Potter, G.L., 2002. NCEP-DOE AMIP-II reanalysis (R-2). Bulletin of the American Meteorological Society 83(11), 1631-1643. Kooijman, S., 1986. Energy budgets can explain body size relations. Journal of Theoretical Biology 121(3), 269-282. Krause, G., Le Vay, L., Buck, B.H., Costa-Pierce, B.A., Dewhurst, T., Heasman, K.G., Nevejan, N., Nielsen, P., Nielsen, K.N., Park, K., Schupp, M.F., Thomas, J.B., Troell, M., Webb, J., Wrange, A.L., Ziegler, F., Strand, A., 2022. Prospects of Low Trophic Marine Aquaculture Contributing to Food Security in a Net Zero-Carbon World. Frontiers in Sustainable Food Systems 6. Ligi, M., Kutser, T., Kallio, K., Attila, J., Koponen, S., Paavel, B., Soomets, T., Reinart, A., 2017. Testing the performance of empirical remote sensing algorithms in the Baltic Sea waters with modelled and in situ reflectance data. Oceanologia 59(1), 57-68. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 88 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Lucas, L.V., Cloern, J.E., Thompson, J.K., Stacey, M.T., Koseff, J.R., 2016. Bivalve Grazing Can Shape Phytoplankton Communities. Frontiers in Marine Science 3. Matthews, M.W., 2011. A current review of empirical procedures of remote sensing in inland and near-coastal transitional waters. International Journal of Remote Sensing 32(21), 6855-6899. Miloslavich, P., Bax, N.J., Simmons, S.E., Klein, E., Appeltans, W., Aburto-Oropeza, O., Garcia, M.A., Batten, S.D., Benedetti-Cecchi, L., Checkley, D.M., Chiba, S., Duffy, J.E., Dunn, D.C., Fischer, A., Gunn, J., Kudela, R., Marsac, F., Muller-Karger, F.E., Obura, D., Shin, Y.J., 2018. Essential ocean variables for global sustained observations of biodiversity and ecosystem changes. Global Change Biology 24(6), 2416-2433. Mishra, S., Mishra, D.R., 2012. Normalized difference chlorophyll index: A novel model for remote estimation of chlorophyll-a concentration in turbid productive waters. Remote Sensing of Environment 117, 394-406. Moore, T.S., Dowell, M.D., Bradt, S., Verdu, A.R., 2014. An optical water type framework for selecting and blending retrievals from bio-optical algorithms in lakes and coastal waters. Remote Sensing of Environment 143, 97-111. Muller-Karger, F.E., Miloslavich, P., Bax, N.J., Simmons, S., Costello, M.J., Pinto, I.S., Canonico, G., Turner, W., Gill, M., Montes, E., Best, B.D., Pearlman, J., Halpin, P., Dunn, D., Benson, A., Martin, C.S., Weatherdon, L.V., Appeltans, W., Provoost, P., Klein, E., Kelble, C.R., Miller, R.J., Chavez, F.P., Iken, K., Chiba, S., Obura, D., Navarro, L.M., Pereira, H.M., Allain, V., Batten, S., Benedetti-Checchi, L., Duffy, J.E., Kudela, R.M., Rebelo, L.M., Shin, Y., Geller, G., 2018. Advancing Marine Biological Observations and Data Requirements of the Complementary Essential Ocean Variables (EOVs) and Essential Biodiversity Variables (EBVs) Frameworks. Frontiers in Marine Science 5. NASA Goddard Space Flight Center, O.E.L., Ocean Biology Processing Group., Ancillary Meteorological Ocean Color Data, NASA OB.DAAC. 2018. Available online: https://disc.gsfc.nasa.gov/datasets/OMTO3_003/summary. NASA Ocean Color Data, Available online: https://oceandata.sci.gsfc.nasa.gov (accessed on 27 September 2021). Naylor, R.L., Hardy, R.W., Buschmann, A.H., Bush, S.R., Cao, L., Klinger, D.H., Little, D.C., Lubchenco, J., Shumway, S.E., Troell, M., 2021. A 20-year retrospective review of global aquaculture. Nature 591(7851), 551-+. Niroumand-Jadidi, M., Bovolo, F., Bruzzone, L., Gege, P., 2021. Inter-Comparison of Methods for Chlorophyll-a Retrieval: Sentinel-2 Time-Series Analysis in Italian Lakes. Remote Sensing 13(12). O'Reilly, J.E., Werdell, P.J., 2019. Chlorophyll algorithms for ocean color sensors-OC4, OC5 & OC6. Remote Sensing of Environment 229, 32-47. Chapter 2. New shellfish growing areas in open waters. _______________________________________________________________________________ 89 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Pahlevan, N., Mangin, A., Balasubramanian, S.V., Smith, B., Alikas, K., Arai, K., Barbosa, C., Belanger, S., Binding, C., Bresciani, M., Giardino, C., Gurlin, D., Fan, Y.Z., Harmel, T., Hunter, P., Ishikaza, J., Kratzer, S., Lehmann, M.K., Ligi, M., Ma, R.H., Martin-Lauzer, F.R., Olmanson, L., Oppelt, N., Pan, Y.Q., Peters, S., Reynaud, N., de Carvalho, L.A.S., Simis, S., Spyrakos, E., Steinmetz, F., Stelzer, K., Sterckx, S., Tormos, T., Tyler, A., Vanhellemont, Q., Warren, M., 2021. ACIX-Aqua: A global assessment of atmospheric correction methods for Landsat-8 and Sentinel-2 over lakes, rivers, and coastal waters. Remote Sensing of Environment 258. Palmer, S.C.J., Gornez, P.M., Thomas, Y., Simis, S., Miller, P.I., Glize, P., Barille, L., 2020. Remote Sensing-Driven Pacific Oyster (Crassostrea gigas) Growth Modeling to Inform Offshore Aquaculture Site Selection. Frontiers in Marine Science 6. Pereira-Sandoval, M., Ruescas, A., Urrego, P., Ruiz-Verdu, A., Delegido, J., Tenjo, C., SoriaPerpinya, X., Vicente, E., Soria, J., Moreno, J., 2019. Evaluation of Atmospheric Correction Algorithms over Spanish Inland Waters for Sentinel-2 Multi Spectral Imagery Data. Remote Sensing 11(12). Pete, R., Guyondet, T., Bec, B., Derolez, V., Cesmat, L., Lagarde, F., Pouvreau, S., Fiandrino, A., Richard, M., 2020. A box-model of carrying capacity of the Thau lagoon in the context of ecological status regulations and sustainable shellfish cultures. Ecological Modelling 426. QGIS.org, 2022. QGIS Geographic Information System. QGIS Association. http://www.qgis.org. Radiarta, I., Saitoh, S.I., 2009. Biophysical models for Japanese scallop, Mizuhopecten yessoensis, aquaculture site selection in Funka Bay, Hokkaido, Japan, using remotely sensed data and geographic information system. Aquaculture International 17(5), 403-419. Ramon, M., Fernandez, M., Galimany, E., 2007. Development of mussel (Mytilus galloprovincialis) seed from two different origins in a semi-enclosed Mediterranean Bay (NE Spain). Aquaculture 264(1-4), 148-159. Raymond, W.W., Barber, J.S., Dethier, M.N., Hayford, H.A., Harley, C.D.G., King, T.L., Paul, B., Speck, C.A., Tobin, E.D., Raymond, A.E.T., McDonald, P.S., 2022. Assessment of the impacts of an unprecedented heatwave on intertidal shellfish of the Salish Sea. Ecology 103(10). RCoreTeam, A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org. Rosland, R., Strand, O., Alunno-Bruscia, M., Bacher, C., Strohmeier, T., 2009. Applying Dynamic Energy Budget (DEB) theory to simulate growth and bio-energetics of blue mussels under low seston conditions. Journal of Sea Research 62(2-3), 49-61. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 97 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Highlights • The Mediterranean Sea harbors more than 80 toxin-producing species, with a few cases of DSP and PSP and about 300 toxic events in 31 years, with an impact mainly on aquaculture. • The number of toxic species detected in the area has remarkably increased since the 1980s, but with no clear trend in toxin-related harmful events. • Large amounts of palytoxin-like toxins produced by Ostreopsis accumulate along rocky shores in summer since the 1990s, with sporadic problems caused by direct contact or aerosol. • Non-toxic events such as seawater discolorations and mucilages show wide fluctuations over the years with no clear trends, but represent the main risk in the MS for their possible impact on tourism and recreational activities. Abstract We review the spatial distribution of toxic marine microalgal species and the impacts of all types of harmful algal events (Harmful Algal Blooms, HABs) in the Mediterranean Sea (MS), including the Black Sea, the Sea of Marmara, coastal lagoons and transitional waters, based on two databases compiled in the Ocean Biogeographic Information System (OBIS). Eighty-four potentially toxic species have been detected in the MS (2,350 records), of which 16 described from these waters between 1860 and 2014 and a few suspected to have been introduced. More than half of these species (46) produce toxins that may affect human health, the remainders ichthyotoxic substances (29) or other types of toxins (9). Nevertheless, toxicity-related events are not frequent in the MS (308 records in 31 years), and mainly consist of impacts on aquaculture, caused by the dinoflagellates Dinophysis and Alexandrium, along with a few actual shellfish poisoning cases. Pseudo-nitzschia blooms are widespread, but domoic acid in shellfish rarely exceeds regulatory levels. Fish kills are probably less sporadic than reported, representing a problem at a few places along the southern MS coasts and in the Ebro River Delta. Since the last decade of the 20th century, blooms of the benthic dinoflagellates Ostreopsis cf. ovata have regularly occurred all along rocky shores of the MS, at times with human health problems caused by toxic aerosol. New records of Gambierdiscus and Fukuyoa, until now reported for the westernmost and easternmost MS coasts, raise concerns about the risk of ciguatera, a syndrome so far known only for subtropical and tropical areas. Recent discoveries are the dinoflagellates Vulcanodinium rugosum, responsible for the presence of pinnatoxins in French lagoons’ shellfish, and the azaspiracid-producers Azadinium spp. Mucilages and discolorations have a major impact on tourism in summer. Reports of toxic species and HABs have apparently increased in the MS over the last half century, which is likely related to the increased awareness and monitoring operations rather than to an actual increase of these phenomena. Indeed, while the case of Ostreopsis Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 98 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas appears as a sudden upsurge rather than a trend, no actual increase of toxic or noxious events has so far emerged in intensively studied areas, such as the French and Spanish coasts or the Adriatic Sea. Moreover, some cases of decrease are reported, e.g., for Alexandrium minutum blooms disappearing from the Harbour of Alexandria. Overall, main HAB risks derive from cases of massive development of microalgal biomass and consequent impacts of reduced coastal water quality on tourism, which represents the largest part of the marine economy along the MS coasts. Keywords HABs, Mediterranean Sea, Microalgae, Toxicity, OBIS 1. Introduction The Mediterranean Sea (MS, from the Latin mare Mediterraneum = the sea surrounded by land) is an enclosed basin surrounded on the north by southern Europe and Anatolia, on the south by North Africa and on the east by the Levant. It occupies an area of approximately 2510,000 km2 lying between latitudes 30° and 46° N. The narrow and shallow Strait of Gibraltar to the west connects it with the Atlantic Ocean, the Dardanelles to the east with the Black Sea through the Sea of Marmara and the Bosporus, while to the south-east the Suez Canal, opened in 1869 and recently expanded, allows the exchange with the Red Sea. In spite of its geographic position within the northern temperate latitudes, the quite shallow sill (170 m) at the Atlantic boundary blocks the entrance of deep, cold oceanic waters and determines temperate-subtropical conditions in the whole area, with minimum temperatures rarely and only at certain locations going below 12 °C. The size, location, and morphology of the MS are at the base of its complex physical dynamics with a distinctive thermohaline circulation and permanent or semi-permanent sub-basin gyres. A marked oligotrophy, increasing along both the west-east and the north-south directions, characterizes the MS (Siokou-Frangou et al., 2010). However, along the Mediterranean coasts there are densely populated areas while a number of large rivers with extended catchment basins flow in the MS (e.g., the Po in the northern Adriatic, the Nile in Egypt, the Ebro in Spain, and the Rhone in France). This implies that mesoand eutrophic conditions, and at times pollution, can affect various coastal areas (UNEPMAP, 2012). The MS has been the crossroad of various cultures since the very beginning of the human colonization and the development of ancient civilizations. Trading routes, migrations, invasions and the struggle for power have shaped the dynamic history of populations around the basin for millennia. The population grew from 281 million in 1970 to 419 million in 2000 and 472 million in 2010, and is predicted to reach 572 million by 2030. Coastal administrative entities make less than 12 % of the surface area of the Mediterranean countries, but host more than a third of the population of the whole Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 99 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas region. Coastal population grew from about 100 million in 1980 to 150 million in 2005 and could reach 200 million by 2030 (UNEP-MAP, 2017). The MS also represents a unique geographic landscape that generates wealth but requires cooperation among the different countries to preserve the environment and the biological resources. The conservative value of the economic assets of the MS has been estimated to be in the order of US$ 5.6 trillion, generating an annual economic value of US$ 450 billion (Randone et al., 2017). A large fraction of the economic value is represented by tourism and related activities; fisheries come as second but >80% of the fish stock is presently threatened. Aquaculture in the MS has considerably expanded over the last decades reaching about 1.3 million tons in 2009 with an estimated value of US$ 3700 million (Rosa et al., 2012). Most of the marine aquaculture production comes from the north Mediterranean countries, which are also the most intensively monitored, but it is rapidly expanding also in Turkey and Egypt. In spite of the dramatic alteration of habitats, depletion of natural resources and increased number of alien species, the MS is still characterized by high biodiversity in most animal and algal groups and a considerable number of endemic species (Coll et al., 2010). The rate at which climatic conditions (e.g., surface temperature, heat waves and sea level) have changed in the MS over the last decades is higher than the global average (Cramer et al., 2018). These changes, coupled with increased population size, urbanization and changes in land use at many coastal places, may pose at serious risk the quality of the environment, the quality and quantity of food and consequently the health and safety of the local populations (Cramer et al., 2018). Especially in view of the growing need to exploit marine resources, HABs and toxic species may represent an increasing risk for human health and economic activities. Few are the papers reviewing the occurrence of harmful species and/or events at the scale of the whole Mediterranean basin. Fifty years ago, Jacques and Sournia (1978-1979) published a first account of the cases of water discoloration (‘eaux rouges’) and the species involved. The overview included mainly dinoflagellate blooms, along with a few cases of anoxia but with no evidence of toxic effects in humans or marine fauna in those years when microalgal toxins were still almost unknown. In an overview of nearly twenty years later, cases of PSP and DSP – mainly attributable to Alexandrium minutum and Dinophysis spp., respectively – were reported from the northern coasts of the basin, along with the records of various potentially toxic or ichthyotoxic dinoflagellates at different sites (Honsell et al., 1995). A subsequent overview of toxic and harmful microalgae covering up to 2009 pointed at the sudden spreading of Ostreopsis cf. ovata blooms along the rocky Mediterranean shores (Zingone, 2010). The present overview covers the MS distribution of marine, toxin-producing microalgae, as included in the IOC-UNESCO Taxonomic Reference List of Harmful Micro Algae (Moestrup et al., 2009) and Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 100 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas the cases of toxin-related harmful events (Sections 2.1 and 2.2), including direct impact on human health or natural resources or indirect impact to aquaculture industry. In addition, we review nontoxic events that include high biomass harmful algal blooms (HB-HABs) causing seawater discolorations, anoxia or any other damages to the environment or human activities (Section 2.3). Finally, we discuss the trends of HABs in the MS in general and particularly in the Adriatic Sea, which is considered a HAB hotspot (Section 3). The overview is based on information from more than 600 scientific publications and technical reports collected in two curated databases in the Ocean Biogeographic Information System OBIS (Zingone et al., 2022): the MS-HABMAP-OBIS (https://obis.org/), gathering records of toxic species occurrence, and the Harmful Events Database (HAEDAT, http://haedat.iode.org/), collecting information of either toxic or non-toxic events, i.e., cases of intoxications, closures of aquaculture plants, seawater discolorations and mucilages. The present review is a contribution to a first appraisal of the current knowledge of HAB occurrences across the world seas, namely, the Global HAB Status Report, (Hallegraeff et al., 2017; Zingone et al., 2017). The requirement for such an assessment has emerged from the apparent worldwide increase and spreading of HABs and their negative impacts contrasted by the lack of an overview founded on a robust basis of data. 2. HABs in the Mediterranean Sea: toxic species and harmful event distribution 2.1. Toxic species Of the more than 140 potentially toxic species listed in the IOC-UNESCO taxonomic reference list (Moestrup et al., 2009), 84 have been found in the MS so far: 17 diatoms, 54 dinoflagellates, 3 dictyochophytes, 6 haptophytes, and 4 raphidophytes (Table 31), and some examples in Figure 31. These records cover both species actually found to produce toxins in the MS and species known to be toxic from other areas. Given the known variability in toxin production among strains of the same species, non-tested local populations are only ‘potentially toxic’ in most cases, but for brevity they will be referred to as ‘toxic’ in the context of this paper. Sixteen of the toxic species have actually been discovered and described from the MS (Table 32), the first ones (Prorocentrum lima, Dinophysis caudata, D. sacculus and D. tripos) in the second half of the 19th century and the most recent ones (Vulcanodinium rugosum, Azadinium dexteroporum, Nitzschia bizertensis and Ostreopsis fattorussoi) in the current decade. Some of the HAB species of the MS, such as D. caudata and Chattonella subsalsa, are widely distributed worldwide while others, including the recently described N. bizertensis and O. fattorussoi, so far seem to be restricted to specific areas of the MS. Table 31. Potentially toxic species in the Mediterranean Sea and associated types of syndromes or impacts (see Moestrup et al. (2009) and Lassus et al. (2016) for details). ASP, amnesic shellfish Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 101 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas poisoning; AZP, azaspiracid shellfish poisoning; DSP, diarrhoetic shellfish poisoning; PSP, paralytic shellfish poisoning; CFP, ciguatera fish poisoning. ‘Other toxins’ include unknown toxins or toxins with poorly known effects. Bacillariophyceae Halamphora coffeaeformis ASP Nitzschia bizertensis ASP Pseudo-nitzschia australis ASP Pseudo-nitzschia brasiliana ASP Pseudo-nitzschia caciantha ASP Pseudo-nitzschia calliantha ASP Pseudo-nitzschia cuspidata ASP Pseudo-nitzschia delicatissima ASP Pseudo-nitzschia fraudulenta ASP Pseudo-nitzschia galaxiae ASP Pseudo-nitzschia hasleana ASP Pseudo-nitzschia multiseries ASP Pseudo-nitzschia multistriata ASP Pseudo-nitzschia pseudodelicatissima ASP Pseudo-nitzschia pungens1 ASP Pseudo-nitzschia subfraudulenta ASP Pseudo-nitzschia subpacifica ASP Dictyochophyceae Pseudochattonella farcimen Ichthyotoxicity Pseudochattonella verruculosa Ichthyotoxicity Vicicitus globosus Ichthyotoxicity Dinophyceae Alexandrium andersonii PSP Alexandrium balechii Ichthyotoxicity Alexandrium minutum PSP Alexandrium ostenfeldii PSP Alexandrium pacificum2 PSP Alexandrium pseudogonyaulax Ichthyotoxicity Alexandrium tamarense2 PSP Alexandrium taylorii PSP Amphidinium carterae Ichthyotoxicity Amphidinium klebsii Ichthyotoxicity Azadinium dexteroporum AZP Azadinium poporum AZP Dinophysis acuminata DSP Dinophysis acuta DSP Dinophysis caudata DSP Dinophysis fortii DSP Dinophysis infundibulum DSP Dinophysis ovum DSP Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 102 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Dinophysis sacculus DSP Dinophysis tripos DSP Fukuyoa paulensis CFP Gambierdiscus australes CFP Gambierdiscus cf. belizeanus CFP Gambierdiscus carolinianus CFP Gambierdiscus silvae CFP Gonyaulax spinifera Other toxins Gymnodinium catenatum PSP Karenia bicuneiformis Ichthyotoxicity Karenia brevis Ichthyotoxicity Karenia cristata Ichthyotoxicity Karenia longicanalis Ichthyotoxicity Karenia mikimotoi Ichthyotoxicity Karenia papilionacea Ichthyotoxicity Karenia selliformis Ichthyotoxicity Karlodinium armiger Ichthyotoxicity Karlodinium corsicum Ichthyotoxicity Karlodinium veneficum Ichthyotoxicity Lingulodinium polyedra Other toxins Margalefidinium polykrikoides Ichthyotoxicity Ostreopsis fattorussoi Airborne disease Ostreopsis cf. ovata Airborne disease Ostreopsis cf. siamensis Airborne disease Pfiesteria piscicida Ichthyotoxicity Phalacroma mitra DSP Phalacroma rotundatum DSP Polykrikos hartmannii Ichthyotoxicity Prorocentrum borbonicum Other toxins Prorocentrum cordatum Other toxins Prorocentrum emarginatum Other toxins Prorocentrum lima DSP Prorocentrum mexicanum Other toxins? Prorocentrum rhathymum DSP Protoceratium reticulatum Other toxins Vulcanodinium rugosum Other toxins Haptophyceae Chrysochromulina leadbeateri Ichthyotoxicity Phaeocystis cf. globosa Other toxins Prymnesium calathiferum Ichthyotoxicity Prymnesium faveolatum Ichthyotoxicity Prymnesium parvum Ichthyotoxicity Prymnesium polylepis Ichthyotoxicity Raphidophyceae Chattonella marina3 Ichthyotoxicity Chattonella subsalsa Ichthyotoxicity Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 103 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Heterosigma akashiwo Ichthyotoxicity Fibrocapsa japonica Ichthyotoxicity 1 Including P. pungens var. aveirensis. 2 A. pacificum (group IV) and A. tamarense (group III), following the ribotype group designation in John et al. (2014) and Litaker et al. (2018). 3 Including Chattonella marina var. antiqua. Figure 31. Examples of toxic species from the Mediterranean Sea. A) Alexandrium minutum stained with calcofluor. B) Azadinium dexteroporum. C) Dinophysis sacculus. D) Fibrocapsa japonica. E) Ostreopsis fattorussoi stained with calcofluor (courtesy of S. Accoroni). F) Prorocentrum lima. G) Pseudo-nitzschia multistriata. Scale bars in A and B: 5 µm; in C, D, E, F and G: 20 µm. Table 32. Potentially toxic species described from the Mediterranean Sea. Species name Described in Described as Type locality Alexandrium minutum Halim Halim (1960a) Harbour of Alexandria, Egypt Alexandrium pseudogonyaulax (Biecheler) Horiguchi ex K.Yuki & Y.Fukuyo Biecheler (1952) Goniodoma pseudogonyaula x Thau Lagoon, Gulf of Lion, France Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 104 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Azadinium dexteroporum Percopo & Zingone Percopo et al. (2013) Gulf of Naples, Italy Chattonella subsalsa Biecheler* Biecheler (1936) Saltern of Villeroy, Sète, France Dinophysis caudata Kent Kent (1881) Nearby Fano, Marche Region, Italy Dinophysis fortii Pavill. Pavillard (1923) Thau Lagoon and/or Sète harbour, France Dinophysis infundibulum J.Schiller Schiller (1928) Southern Adriatic Sea Dinophysis sacculus F.Stein Stein (1883) Kvarner Gulf, Croatia Dinophysis tripos Gourret Gourret (1883) South of Ratonneau, Gulf of Marseille, France Karlodinium armiger Bergholtz, Daugbjerg & Moestrup (Bergholtz et al., 2006) Alfacs Bay, Catalonia, Spain Karlodinium corsicum (Paulmier, Berland, Billard & Nézan) Siano & Zingone Paulmier et al. (1995) Gyrodinium corsicum Diana Lagoon, Corse, France Nitzschia bizertensis Bouchouicha-Smida, Lundholm, Hlaili & Mabrouk Bouchouicha-Smida et al. (2014) Bizerte Lagoon, Tunisia Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 105 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Ostreopsis fattorussoi Accoroni, Romagnoli & Totti Accoroni et al. (2016) Batroun, Lebanon Prorocentrum lima (Ehrenb.) F.Stein Ehrenberg (1860) Cryptomonas lima Sorrento, Gulf of Naples, Italy Prymnesium faveolatum Fresnel Fresnel et al. (2001) Beach of Roquebrun, Cap Martin, France Vulcanodinium rugosum Nézan & Chomérat** Nezan and Chomerat (2011) Ingril Lagoon, France ⁎ A second, distinct genotype also discovered in Mediterranean waters (Klopper et al., 2013). ⁎⁎ First report in Rhodes et al. (2010) from New Zealand. The discovery of potentially toxic species in the MS has undergone an evident escalation over the years (Figure 32), from the first descriptions of more than a century before the discovery of their toxicity to the rapid increase after the 1960s and the most recent findings. Information on their distribution has also markedly increased along with the intensification of monitoring operations and studies on planktonic and benthic microalgae (e.g., Aligizaki et al. (2009); Balkis and Tas (2016); Fernández et al. (2019); Pistocchi et al. (2012); Zingone et al. (2006)) and of their resting stages in the sediments (Bravo et al., 2006; Satta et al., 2013) or sediment traps (Montresor et al., 1998). Yet the actual range of most toxic species in the MS is far from being known. Indeed, the identification of some of the most represented genera in the MS, such as Alexandrium, Karenia, Karlodinium and Pseudo-nitzschia, as well as of many other flagellates, is quite problematic. In many cases the observation of live material or methods more complex than light microscopy are needed. Cryptic diversity discovered in many microalgal taxa over the last decades also concerns several harmful genera and species, which have undergone careful taxonomic investigations more than other nontoxic taxa. This trend has led to the discovery of non-toxic taxa morphologically similar to toxic ones, such as several species in the P. delicatissima and P. pseudodelicatissima species-complexes (Bates et al., 2018), the non-toxic A. tamutum hardly distinguishable from A. minutum (Figure. 1A, Montresor et al. (2004)) and the non-toxic, chain-forming Gymnodinium impudicum (as Gyrodinium impudicum, Fraga et al. (1995)) which was misidentified as Gymnodinium catenatum in studies predating its discovery (e.g., Carrada et al. (1991)). Recent studies coupling detailed morphological Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 112 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 34. Fig. 4. Geographic range of potentially toxic species in the Mediterranean Sea. A) Ostreopsis spp. (mostly O. cf. ovata) and other benthic dinoflagellate species related to the ciguatera fish poisoning (CFP). B) Species producing ichthyotoxins (Alexandrium pseudogonyaulax, Karenia spp., Karlodinium spp., Chattonella spp., Vicicitus globosus, Prymnesium spp., etc.) and other toxins. The latter include mainly a few widespread dinoflagellate species that produce yessotoxins (Lingulodinium polyedra, Gonyaulax spinifera and Protoceratium reticulatum), but also other dinoflagellates producing azaspiracids (Azadinum spp.), pinnatoxins (Vulcanodinium rugosum) and other toxins with poorly known effects (e.g., Prorocentrum spp.). See Table 1 for a complete list. First problems caused by Ostreopsis in the MS were fish and invertebrate kills in 1998 along the coasts of Tuscany (northern Tyrrhenian Sea) (Sansoni et al., 2003; Simoni et al., 2003). Some years later (2002) more than 200 people coming from the beach of the city of Genoa (Ligurian Sea) were hospitalized with fever, red eyes and wheeze (Ciminiello et al., 2006). The only known problems caused by benthic microalgae at that time were those related to ciguatera fish poisoning (CFP) in subtropical areas, whereas cases of toxic aerosol were only known for planktonic Karenia brevis blooms in the Gulf of Mexico. In those years, similar human health problems and dermatitis cases Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 113 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas were reported from the Catalonia and Balearic Islands (Vila et al., 2008), French (Cohu et al., 2013) and Algerian coasts (Illoul et al., 2012), and are still reported nowadays at several MS places (e.g., Croatian coast, (Ninčević Gladan et al., 2019). Both the presence of toxins in the aerosol (Ciminiello et al., 2014) and toxicological data on the effects of inhalation exposure in mice (Poli et al., 2018) support a link between Ostreopsis toxins and the respiratory symptoms reported during blooms. However, those health problems do not occur during all phases of a bloom (Vila et al., 2016) and are quite sporadic compared to the widespread and often massive presence of the suspected causative species. The presence of Ostreopsis toxins in marine animals used as food and their impacts on the animal health are relevant for their sanitary implications, which are still controversial (Tubaro et al., 2011). Apparently healthy organisms (e.g., mussels and sea urchins) during Ostreopsis blooms can accumulate fairly large amount of toxins (Aligizaki et al. (2008); E. Fattorusso & V. Soprano, pers. comm.), but macroscopic damages have been reported for various benthic organisms in the MS (Accoroni et al., 2016; Sansoni et al., 2003; Simoni et al., 2003) and elsewhere (Shears and Ross, 2009). In mussels, Ostreopsis can induce important and not completely reversible ultrastructural damages (Carella et al., 2015) and immunological, histological and oxidative responses (Gorbi et al., 2013) while in sea urchins Ostreopsis blooms affect reproduction and offspring health (Migliaccio et al., 2016). Four species of the dinoflagellate genus Gambierdiscus, which can produce CFP toxins, have recently been found in the MS. Gambierdiscus australes, G. cf. belizeanus, G. carolinianus, G. silvae and some unidentified Gambierdiscus spp., have been reported from the Balearic Islands (Tudó et al., 2018), Greece and Cyprus (Aligizaki et al., 2018; Aligizaki and Nikolaidis, 2008; Holland et al., 2013; Tudó et al., 2018), with the highest diversity in Crete. Fukuyoa paulensis also has been found in the Balearic Islands (Laza-Martínez et al., 2016) and Cyprus (Tudó et al., 2018). Yet CFP cases are not known in the MS countries with the exception of a suspected case of ciguatoxins in rabbitfish (Siganus rivolutus) reported from Israeli coasts (Bentur and Spanier, 2007). 2.2.5. Azaspiracid shellfish poisoning (AZP) The toxins azaspiracids (AZAs), produced by a number of dinoflagellate species of the genera Azadinium and Amphidoma, and the human syndrome they can cause, AZP, have been discovered at the beginning of this century (James et al., 2002). Subsequently AZAs have been reported in shellfish from numerous sites, including the MS (Bacchiocchi et al., 2015). A new species described from the MS, A. dexteroporum ((Percopo et al. (2013); Figure 31), produces a whole suite of AZAs that can cause direct harm to molluscs (Giuliani et al., 2019; Rossi et al., 2017). Another toxic Azadinium, A. poporum, has been found in Greek waters (Luo et al., 2018) but no impacts related to AZAs have been reported so far. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 114 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas 2.2.6. Ichthyotoxicity About half of the potentially toxic MS species produce a variety of toxins that differ from those related to the syndromes mentioned in the previous sections. Of these, the majority (29 species, Table 31) produce substances that have been associated with fish and/or shellfish kills. With a few exceptions, species in this list are unarmoured dinoflagellates, e.g., Karenia and Karlodinium, and other flagellates belonging to the prymnesiophytes, raphidophytes and dictyochophytes, which are all hardly identifiable in fixed material under the light microscope, and hence are overlooked in most monitoring and ecological investigations. The large majority of the information on the presence of these ichthyotoxic species (Figure 34) comes from fish mortality events, mainly located near fishfarming plants, in which the identification of the culprit became necessary. The few fish mortality events in the MS known before 1975 were related to HB-HABs of nonichthyotoxic species causing anoxia in bottom waters (see Section 2.3.1) rather than to ichthyotoxic species (Jacques and Sournia, 1978-1979). In the subsequent years, fish kills by ichthyotoxic species were reported sporadically from Catalan coasts, Spain (Garcés et al., 1999a), caused by Karlodinium spp., and Sardinia (Italy), caused by Chattonella subsalsa (Stacca et al., 2016). Occasional fish mortality events were related to Prymnesium spp., in the Ebro Delta (Spain, Comín and Ferrer (1978)) and in a Tuscany lagoon (Italy, Mattioli and Simoni (1999)), Karenia selliformis in the Gulf of Gabes (Tunisia, (Feki et al., 2013; Romdhane et al., 1998)) and Karenia brevis and Pseudochattonella cf. verruculosa in Greece (Ignatiades and Gotsis-Skretas, 2010). In other cases, fish kills occurred during blooms of species toxic to humans, like in Egypt in 1987 (Labib and Halim, 1995; Zaghloul and Halim, 1992) where Alexandrium minutum was the culprit. No fish or shellfish kill accidents in the MS have ever been associated with blooms of two potentially ichthyotoxic Alexandrium species, A. balechii and A. pseudogonyaulax. Benthic cyanobacteria are poorly investigated in Mediterranean waters, but blooms of filamentous cyanobacteria have been the cause of massive fish mortalities in Alexandria waters (Egypt) during spring 2005 (Ismael, 2012). 2.2.7. Other toxins The dinoflagellates Gonyaulax spinifera, Lingulodinium polyedra and Protoceratium reticulatum, which are quite widespread in the MS (Figure 34), produce yessotoxins (YTX). These substances were initially associated to DSP because their presence gives similar positive results in mouse bioassay, but they are not considered toxic to humans (Tubaro et al., 2010). However, YTXs caused economic impacts in 2002, 2004 and 2007, when mussel harvesting was halted for a long time (average closure 153 days) in the north-western Adriatic Sea (Poletti et al., 2008). Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 115 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Vulcanodinium rugosum produces pinnatoxins (Nezan and Chomerat, 2011; Rhodes et al., 2010) a neurotoxin that has lethal effects on sea urchin larvae, oysters and Artemia. Currently there are no problems related to this species, while toxic effects on humans are not known. 2.3. Non-toxic events Independent from toxin production, all microalgae may exert a negative impact when they reach a high biomass producing seawater discolorations, mucilages or anoxia in bottom waters (Zingone and Enevoldsen, 2000). Although several microalgal species are frequently associated with these HBHABs, as detailed in the next sections, the number of species that may cause harm with no specific toxin production is in theory unlimited, and can vary from place to place. For this reason, it is not possible to define a global or regional list of non-toxic harmful microalgae. In addition to HB-HABformers, some non-toxic species, mainly diatoms, may cause mechanical harm to invertebrates’ gills (Bell, 1961), but no information on such events is available for the MS. In case of fish or invertebrate kills, at time it is hard to discern whether the cause has been anoxia, toxic substances or mechanical damages. In many cases, species known to produce toxins may produce non-toxic HB-HABs, which have no impact on human or marine fauna health but important consequences for tourism. For all these reasons, the boundaries between events described in the previous and next sections cannot always be well defined. 2.3.1. Discolorations In the MS, discoloration or anoxia have frequently been caused by unarmoured dinoflagellates either toxic (e.g., Margalefidinium polykrikoides) or non-toxic (e.g., Noctiluca scintillans), but also by numerous armored dinoflagellates, diatoms, prasinophytes, prymnesiophytes and raphidophytes (Table 3S1). Changes of seawater colour caused by HB-HABs (Figure 35) have been noticed since the first half of the XX century in both lagoons and coastal sites, where they were given several names (purga de mar, punti verdi) before the one of red tides gained popularity. The oldest records include discolorations caused by Chattonella subsalsa in 1956 in the Algiers harbour (Hollande and Enjumet, 1957), Alexandrium minutum in 1957 in the Alexandria harbour (Halim, 1960a) and Prorocentrum cordatum in the Gulf of Naples in September 1962 (Yamazi, 1964). Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 116 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 35. A) Mat of Oscillatoria acutissima in the Eastern Harbour of Alexandria (Egypt). B) Bloom of Noctiluca scintillans in Thermaikos Gulf (Thessaloniki, Greece). C) Discoloration caused by Euglena viridis in the Golden Horn Estuary (Sea of Marmara, Turkey). D) Shellfish mortality in Ras El-Bar (Egypt) in 2011 due to the proliferation of N. scintillans and consequent oxygen depletion. E) Pelagic mucilages in the Gulf of Naples (Italy). Different dinoflagellates (e.g., Alexandrium spp., Noctiluca scintillans, Karlodinium spp.), raphidophytes (Chattonella subsalsa and Fibrocapsa japonica, Figure 31) and chlorophytes (Tetraselmis wettsteinii and Pyramimonas spp.) occasionally produced discoloration (Table 3S1, Figure 35), which in some cases were also associated with fish kills and/or massive death of marine invertebrates caused by anoxic conditions (e.g., (Arzul et al., 1994; Garcés et al., 1999a; Halim and Labib, 1996)). A couple of such cases of fish mortality events attributed to anoxia were already reported in the review by Jacques and Sournia (1978-1979): in Izmir Bay (Nümann, 1955, in Jacques and Sournia (1978-1979)) and in the Adriatic Sea (Froglia, 1970; Piccinetti and Manfrin, 1969), during blooms of Gymnodinium sp. and Protoperidinium depressum, respectively. Discolorations were particularly frequent in the northern Adriatic Sea in summer in the 1970−‘80s, when dinoflagellate blooms (e.g., Lingulodinium polyedra, Alexandrium mediterraneum and Lepidodinium chlorophorum) turned the sea into various colours (Boni (1983), Table 3S1), at times extending offshore as in the case of N. scintillans in 1980 (Fonda Umani et al., 2004) and L. chlorophorum in 1984 (Artegiani et al., 1985). Some summer blooms were caused by diatoms (e.g., Skeletonema marinoi and Chaetoceros spp.), particularly after intense freshwater inputs (Boni, 1983; Regione Emilia-Romagna, 1982-2018). Over the last decades blooms of F. japonica (Figure 31) became common in late summer (Cucchiari et al., 2008) in shallow coastal waters where they lasted Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 117 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas up to 20–40 days. Along the eastern Adriatic coast, ‘red tides’ were limited to eutrophicated semienclosed bays (Marasović et al., 1991) or to unusual phenomena such as bloom of the silicoflagellate Octactis (formerly Distephanus) speculum in summer 1983 in bottom waters in the Gulf of Trieste, causing anoxia (Fanuko, 1989). An increasing number of discolorations have been observed over two decades in the Golden Horn Estuary of the Sea of Marmara (Taş et al., 2016). An unusual bloom of the coccolithophore Holococcolithophora sphaeroidea (as Calyptrosphaera sphaeroidea) caused a white-green-turquoise discoloration in a vast area off the Tarragona harbour (Spain, Cros et al. (2002)). The most recent event has been a long-lasting bloom of Margalefidinium cf. polykrikoides that produced a yellow brownish discoloration in a touristic area of the Ionian Sea (Italy) in JulyAugust 2018, recurring in the same place in summer 2019 (Roselli et al., 2020). In summer, discolorations can be a serious problem along Mediterranean beaches where they have an impact on tourism and recreational use of the sea. This is the case of the recurrent Alexandrium taylorii blooms along the Sicilian and Sardinian coasts (Italy) and in the Balearic Islands (Spain) (e.g., (Basterretxea et al., 2005; Giacobbe et al., 2007; Sampedro i Roig, 2018; Satta et al., 2010)). 2.3.2. Mucilages In the MS, a number of cases of mucilaginous aggregate formation related to microalgal growth have been described, the most conspicuous of which occurred in the northern Adriatic Sea in the 1990s. Mucilaginous macroaggregates represent the last stage of aggregation of organic matter, mainly refractory polysaccharides derived from phytoplankton exudates (Myklestad, 1995) and/or from bacterial capsular material (Stoderegger and Herndl, 1998) whose hydrolysis cannot be sustained by phosphorus-limited bacteria (Danovaro et al., 2005). Whereas marine snow (aggregates of 0.5–1 cm diameter) is common in all the oceans (Simon et al., 2002), the mucilage event in the northern Adriatic Sea was unique in that those aggregates covered hundred square kilometres of both coastal and offshore areas. The formation of larger aggregates was favored by the strong stratification of the water column and reduced circulation that retained freshwater in the northern Adriatic basin (Russo et al., 2005). The direct responsible of the phenomenon were often thought to be the most abundant phytoplankton species in the aggregates, such as Cylindrotheca closterium (Revelante and Gilmartin, 1991) and Gonyaulax fragilis (Pompei et al., 2003), both capable to produce large amounts of refractory polysaccharides (Pistocchi et al., 2005; Urbani et al., 2005). In fact, phytoplankton communities associated with mucilage aggregates largely vary, depending on sampling area and period (Totti et al. (2005), and references therein), while the aggregates represent a self-sustained microcosm hosting a rich microorganism community (Simon et al., 2002). Pelagic mucilages have been reported at several other Mediterranean sites, such as the Greek (GotsisSkretas, 1995; Nikolaidis et al., 2006) and Catalan coasts (Sampedro et al., 2007) where Gonyaulax fragilis was thought to be involved in their production, and the Sea of Marmara (Turkey) where Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 118 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Cylindrotheca closterium, Skeletonema costatum and Gonyaulax fragilis were indicated as the most abundant species (Tüfekçi et al., 2010). In the Tyrrhenian Sea, extensive pelagic aggregates were observed in 1991, 2000 and 2012 (Figure 35, (Calvo et al., 1991; Escalera et al., 2018; Innamorati et al., 1993)). Foam accumulated massively along the Catalan coast in March 2006 during a Phaeocystis sp. bloom, an event that was related to anomalous hydrographic winter conditions (Arin et al., 2014). Massive mucilage events have also concerned the benthic environment. Ostreopsis cf. ovata during intense blooms forms a network-shaped mucilaginous biofilm that can harm benthic invertebrates (Schiaparelli et al., 2007). In the Tyrrhenian and Ligurian Seas (western MS), benthic mucilages have occurred since 1991 (Sartoni and Sonni, 1991), and have been attributed to the massive growth of several macroand microalgae such as the filamentous brown alga Acinetospora crinita and the colonial pelagophytes Nematochrysopsis marina and Chrysonephos lewisii (Giuliani et al., 2005; Schiaparelli et al., 2007). The allochthonous pelagophyte Chrysophaeum taylorii, recorded in the western MS since 2005, in recent years was involved in the formation of dense layers of mucous covering macroalgae, gorgonians and the surrounding rocks (Caronni et al., 2015; Luglié et al., 2008). 3. Trends in the Mediterranean HABs 3.1. General trends The MS has undergone profound changes over the last centuries. Human action has mainly been visible along the coasts of the basin, which have become increasingly populated and deeply modified by coastal and riverine engineering and deforestation which, along with cultural eutrophication, are all potential drivers of deep changes in phytoplankton communities (Garcés and Camp, 2012). Natural and/or man-induced meteorological and climatic variations superimpose to these changes often with an amplifying effect. The most striking characteristic of the MS HABs over the last 50 yrs, which approximately correspond to the time since when they have been studied more intensively, is the remarkable increase of the toxic species list, from a few taxa to the more than 80 of the present review (Figure 32). Over the same period, the records of these species across the MS have also remarkably increased (Figure 36). This trend is parallel to that of the increased list of toxic species and of their records worldwide, which is an obvious result of the intensification of the taxonomic and toxin studies on marine microalgae (Zingone et al., 2017). The increase of the records of actual HAB events from the less than 30 cases listed by Jacques and Sournia (1978-1979) and Honsell et al. (1995) to the several hundred cases of halted aquaculture operations, seawater discoloration and minor human health accidents presently recorded in HAEDAT is also impressive (Figure 37). Damages to aquaculture caused by ASP and PSP toxins in mussels have been limited over the last 30 years while DSP cases have represented about 75% of the harmful events, with an increase between Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 119 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas the decade 1987–1997 and the two following ones (Figure 37). This trend should however be interpreted with caution because it has been paralleled by a remarkable growth of the coastal MS population (Section 1), much more intensive use of marine resources, and consequent raise of the level of attention to the integrity and safety of marine resources. Figure 36. Distribution of potentially toxic species, mucilages and discolorations in the Mediterranean Sea. A) Distribution of species known to be toxic and harmful events until 1995 as reported in Jacques and Sournia (1978-1979) and Honsell et al. (1995). B) Distribution of potentially toxic species (excluding Ostreopsis and CFP species) and harmful events updated to the present status of knowledge. The position of the circles in several cases has been slightly modified to reduce overlapping. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 120 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 37. Harmful events related to microalgae in the Mediterranean Sea (n = 501) based on records in the Harmful Algae Event Database HAEDAT (http://haedat.iode.org/). High density phytoplankton blooms with no impacts were not considered. A) Relative abundance of different types of nuisance with details of seafood toxicity. B) Interannual variations of ASP, DSP and PSP toxicity events. In fact, toxic blooms as well as mucilage events and discolorations in the MS have generally shown an unpredictable interannual periodicity, like in the case of the conspicuous blooms of Noctiluca scintillans in the Adriatic Sea (Fonda Umani et al., 2004), Moroccan (Tahri-Joutei et al., 2003), Catalan (Lopez and Arte, 1971) and French coasts (M.-O. Soyer in Jacques and Sournia (19781979)). There are cases of decreases, e.g., the blooms of Alexandrium pacificum occurring on the Catalan coast from 1996 to 1998 (Vila et al., 2000) but rarely recorded afterwards (Sampedro i Roig, 2018). Blooms of A. minutum were recurrent in Egyptian waters but not recorded any longer after 1994 (Ismael and Halim, 2000), while their frequency doubled from 2000 to 2012 along the Catalan coast (Sampedro i Roig, 2018). Blooms of the ciliate Mesodinium rubrum hosting cryptophyte chloroplasts were not recorded in the MS (Jacques and Sournia, 1978-1979) until their occurrence in both the Adriatic (Sorokin et al., 1999) and Tyrrhenian Seas (Siano et al., 2006), and afterwards have only been observed in 2017 in the North Aegean Sea (Genitsaris et al., 2019). In the case of Ostreopsis cf. ovata, rather than an increase the phenomenon in the MS has shown a sudden upsurge around the 2000, followed by an expansion of the known range for the species in the next years and a relative stability in the following decade. Indeed Ostreopsis cf. ovata provides the most evident case of range expansion and increased impact over time in the MS. Although benthic microalgae have received scarce attention until the late 20th century, it is unlikely that the species Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 121 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas might have been abundant but undetected before. The apparent sudden range expansion and impact of Ostreopsis cf. ovata is in line with an increasing trend of species of the same genus in New Zealand and some other temperate areas around the world (Parsons et al., 2012). On the other hand, no clear increase of the impact or of species abundance has been reported since the 2000 outburst, while the above-mentioned range expansion has coincided with a dramatic increase in monitoring programs and research projects focused on benthic microalgae. Initially, the sudden relevance of the phenomenon was associated with an increase of temperature in the MS, based on the belief that all Ostreopsis species were of tropical origin. In fact, Ostreopsis cf. ovata and its close relatives are widely distributed in temperate areas, also matching the apparent preference of the species for moderately high rather than very high temperature (Mangialajo et al., 2011; Scalco et al., 2012). Overall, the trend observed for this species in the MS, with an outburst followed by a stabilizing trend, recalls that of an invasive species rather than that of a species favored by a temperature increase. 3.2. HAB trends in the Adriatic Sea, a case study The Adriatic Sea (AS) represents a unique system for its semi-enclosed morphology, shallow depth and oligotrophic nature in most parts but with eutrophic characteristics along the north-western coasts driven by inputs from the Po River and other rivers (Cozzi and Giani, 2011; Mozetič et al., 2010). The AS is considered one of the hotspots of MS HABs (Garcés and Camp, 2012), in terms of both occurrence and impacts. However, compared to the great variety of potentially toxic species (Mozetič et al., 2019), toxicity cases are limited, and the most common toxins found above the regulatory limits in the Adriatic shellfish to date are DSP toxins (okadaic acid group) and other lipophilic toxins (yessotoxins and pectenotoxins). Because of the early development of sea-related activities, there is a wealth of information from the area dating back to the last century, which allows some insights on possible HAB trends. Phytoplankton in certain areas of the AS (e.g., Gulf of Trieste, Gulf of Venice, Senigallia-Susak transect, Kaštela Bay) have been extensively studied for decades (Bernardi Aubry et al., 2012; Cerino et al., 2019; Marić et al., 2012; Mozetič et al., 2012; Ninčević Gladan et al., 2010; Totti et al., 2019b), highlighting a number of changes, such as trends or regime shifts in main phytoplankton groups (Mozetič et al., 2010; Totti et al., 2019b) and in bloom forming species (Cabrini et al., 2012). However, no trends specifically related to toxic species is evident from these long-term studies, neither in terms of increased frequency nor of abundance. In fact, most studies on HAB species are snapshots of isolated toxic episodes (Pistocchi et al. (2012), and references therein). Similar conclusions can be drawn also from toxicity events: aquaculture operations have been halted frequently over the last 20 years (Section 2.2.1), but without any significant trend for DSP events. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 128 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Carrada, G.C., Casotti, R., Modigh, M., Saggiomo, V., 1991. Presence of Gymnodinium catenatum (Dinophyceae) in a coastal mediterranean lagoon. Journal of Plankton Research 13(1), 229-238. Cerino, F., Fornasaro, D., Kralj, M., Giani, M., Cabrini, M., 2019. Phytoplankton temporal dynamics in the coastal waters of the north-eastern Adriatic Sea (Mediterranean Sea) from 2010 to 2017. Nature Conservation 34, 343-372. Cerino, F., Orsini, L., Sarno, D., Dell’Aversano, C., Tartaglione, L., Zingone, A., 2005. The alternation of different morphotypes in the seasonal cycle of the toxic diatom Pseudo-nitzschia galaxiae. Harmful Algae 4(1), 33-48. Ciminiello, P., Dell'Aversano, C., Fattorusso, E., Forino, M., Magno, G.S., Tartaglione, L., Grillo, C., Melchiorre, N., 2006. The Genoa 2005 Outbreak. Determination of Putative Palytoxin in Mediterranean Ostreopsis ovata by a New Liquid Chromatography Tandem Mass Spectrometry Method. Analytical Chemistry 78(17), 6153-6159. Ciminiello, P., Dell'Aversano, C., Fattorusso, E., Forino, M., Magno, G.S., Tartaglione, L., Quilliam, M.A., Tubaro, A., Poletti, R., 2005. Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish. Rapid Communications in Mass Spectrometry 19(14), 2030-2038. Ciminiello, P., Dell’Aversano, C., Iacovo, E.D., Fattorusso, E., Forino, M., Tartaglione, L., Benedettini, G., Onorari, M., Serena, F., Battocchi, C., Casabianca, S., Penna, A., 2014. First Finding of Ostreopsis cf. ovata Toxins in Marine Aerosols. Environmental Science & Technology 48(6), 35323540. Cohu, S., Mangialajo, L., Thibaut, T., Blanfuné, A., Marro, S., Lemée, R., 2013. Proliferation of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth, biotic substrate and environmental factors in the North West Mediterranean Sea. Harmful Algae 24, 32-44. Coll, M., Piroddi, C., Steenbeek, J., Kaschner, K., Ben Rais Lasram, F., Aguzzi, J., Ballesteros, E., Bianchi, C.N., Corbera, J., Dailianis, T., Danovaro, R., Estrada, M., Froglia, C., Galil, B.S., Gasol, J.M., Gertwagen, R., Gil, J., Guilhaumon, F., Kesner-Reyes, K., Kitsos, M.-S., Koukouras, A., Lampadariou, N., Laxamana, E., López-Fé de la Cuadra, C.M., Lotze, H.K., Martin, D., Mouillot, D., Oro, D., Raicevich, S., Rius-Barile, J., Saiz-Salinas, J.I., San Vicente, C., Somot, S., Templado, J., Turon, X., Vafidis, D., Villanueva, R., Voultsiadou, E., 2010. The Biodiversity of the Mediterranean Sea: Estimates, Patterns, and Threats. PLOS ONE 5(8), e11842. Comín, F.A., Ferrer, X., 1978. Desarrollo masivo del fitoflagelado Prymnesium parvum Carter (Haptophyceae) en una laguna costera del delta del Ebro. Conversi, A., Fonda Umani, S., Peluso, T., Molinero, J.C., Santojanni, A., Edwards, M., 2010. The Mediterranean Sea Regime Shift at the End of the 1980s, and Intriguing Parallelisms with Other European Basins. PLOS ONE 5(5), e10633. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 129 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Cozzi, S., Giani, M., 2011. River water and nutrient discharges in the Northern Adriatic Sea: Current importance and long term changes. Continental Shelf Research 31(18), 1881-1893. Cozzi, S., Ibáñez, C., Lazar, L., Raimbault, P., Giani, M., 2019. Flow Regime and NutrientLoading Trends from the Largest South European Watersheds: Implications for the Productivity of Mediterranean and Black Sea’s Coastal Areas. Water 11(1), 1. Cramer, W., Guiot, J., Fader, M., Garrabou, J., Gattuso, J.-P., Iglesias, A., Lange, M.A., Lionello, P., Llasat, M.C., Paz, S., Peñuelas, J., Snoussi, M., Toreti, A., Tsimplis, M.N., Xoplaki, E., 2018. Climate change and interconnected risks to sustainable development in the Mediterranean. Nature Climate Change 8(11), 972-980. Cros, L., Arbós, D., Arin, L., Camp, J., Estrada, M., Alós, J.M.F., Morales-Blake, A., Sampedro, N., Riba, J., Vila, M., 2002. A Calyptrosphaera sphaeroidea bloom in waters of the Tarragona harbour (NW Mediterranean). Cucchiari, E., Guerrini, F., Penna, A., Totti, C., Pistocchi, R., 2008. Effect of salinity, temperature, organic and inorganic nutrients on growth of cultured Fibrocapsa japonica (Raphidophyceae) from the northern Adriatic Sea. Harmful Algae 7(4), 405-414. D'Alelio, D., d'Alcala, M.R., Dubroca, L., Sarno, D., Zingone, A., Montresor, M., 2010. The time for sex: A biennial life cycle in a marine planktonic diatom. Limnology and Oceanography 55(1), 106-114. Danovaro, R., Armeni, M., Luna, G.M., Corinaldesi, C., Dell'Anno, A., Ferrari, C.R., Fiordelmondo, C., Gambi, C., Gismondi, M., Manini, E., Mecozzi, M., Perrone, F.M., Pusceddu, A., Giani, M., 2005. Exo-enzymatic activities and dissolved organic pools in relation with mucilage development in the Northern Adriatic Sea. Science of The Total Environment 353(1), 189-203. Danovaro, R., Fonda Umani, S., Pusceddu, A., 2009. Climate Change and the Potential Spreading of Marine Mucilage and Microbial Pathogens in the Mediterranean Sea. PLOS ONE 4(9), e7006. Degobbis, D., Precali, R., Ferrari, C.R., Djakovac, T., Rinaldi, A., Ivančić, I., Gismondi, M., Smodlaka, N., 2005. Changes in nutrient concentrations and ratios during mucilage events in the period 1999–2002. Science of The Total Environment 353(1), 103-114. Delgado, M., Estrada, M., Camp, J., Fernández, J.V., Santmartí, M., Lletí, C., 1990. Development of a toxic Alexandrium minutum Halim (Dinophyceae) bloom in the harbour of Sant Carles de la Ràpita (Ebro Delta, northwestern Mediterranean). Scientia Marina 54(1), 1-7. Dell’Aversano, C., Tartaglione, L., Polito, G., Dean, K., Giacobbe, M., Casabianca, S., Capellacci, S., Penna, A., Turner, A.D., 2019. First detection of tetrodotoxin and high levels of Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 130 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas paralytic shellfish poisoning toxins in shellfish from Sicily (Italy) by three different analytical methods. Chemosphere 215, 881-892. Della Loggia, R., Cabrini, M., Del Negro, P., Honsell, G., Tubaro, A., 1993. Relationship between Dinophysis spp. in seawater and DSP toxins in mussels in the Northern Adriatic Sea, Toxic Phytoplankton Blooms in the Sea, pp. 483-488. Dzhembekova, N., Urusizaki, S., Moncheva, S., Ivanova, P., Nagai, S., 2017. Applicability of massively parallel sequencing on monitoring harmful algae at Varna Bay in the Black Sea. Harmful Algae 68, 40-51. Ehrenberg, C., 1860. Nachtrag zu Hrn. Ehrenbergs Mittheilung vom 8. Decbr. 1859. Verzeichniss der Leuchtthierchen. . Monatsberichte der Koniglichen Preitssischen Akademie der Wissenschaften zu Berlin, 791-793. Escalera, L., Italiano, A., Pistocchi, R., Montresor, M., Zingone, A., 2018. Gonyaulax hyalina and Gonyaulax fragilis (Dinoflagellata), two names associated with ‘mare sporco’, indicate the same species. Phycologia 57(4), 453-464. Fanuko, N., 1989. Possible relation between a bloom of Distephanus speculum (Silicoflagellata) and anoxia in bottom waters in the Northern Adriatic, 1983. Journal of Plankton Research 11(1), 75-84. Feki, W., Hamza, A., Frossard, V., Abdennadher, M., Hannachi, I., Jacquot, M., Belhassen, M., Aleya, L., 2013. What are the potential drivers of blooms of the toxic dinoflagellate Karenia selliformis? A 10-year study in the Gulf of Gabes, Tunisia, southwestern Mediterranean Sea. Harmful Algae 23, 8-18. Fernández, R., Maman, L., Jaen, D., Fuentes, L.F., Ocana, M.A., Gordillo, M.M., 2019. Dinophysis Species and Diarrhetic Shellfish Toxins: 20 Years of Monitoring Program in Andalusia, South of Spain. Toxins 11(4). Fertouna-Bellakhal, M., Dhib, A., Fathalli, A., Bellakhal, M., Chomérat, N., Masseret, E., Laabir, M., Turki, S., Aleya, L., 2015. Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia, Southern Mediterranean Sea). Harmful Algae 48, 69-82. Fonda Umani, S., Beran, A., Parlato, S., Virgilio, D., Zollet, T., De Olazabal, A., Lazzarini, B., Cabrini, M., 2004. Noctiluca scintillans Macartney in the Northern Adriatic Sea: long-term dynamics, relationships with temperature and eutrophication, and role in the food web. Journal of Plankton Research 26(5), 545-561. Forteza, V., Quetglas, G., Delgado, M., Reyero, M.I., Fraga, S., Franco, M.J., Cacho, E., 1998. Toxic Alexandrium minutum bloom in Palma de Mallorca harbour, (Balearic Islands), westerm Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 131 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Mediterranean, 8th International Conference on Harmful Algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Vigo, Spain, pp. 58-59. Fraga, S., Bravo, I., Delgado, M., Franco, J.M., Zapata, M., 1995. Gyrodinium impudicum sp nov (Dinophyceae), a non toxic, chain-forming, red tide dinoflagellate. Phycologia 34(6), 514-521. France, J., Mozetič, P., 2006. Ecological characterization of toxic phytoplankton species (Dinophysis spp., Dinophyceae) in Slovenian mariculture areas (Gulf of Trieste, Adriatic Sea) and the implications for monitoring. Marine Pollution Bulletin 52(11), 1504-1516. Francé, J., Petelin, B., Mozetič, P., 2018. Can we track climate related changes in the HAB species assemblage in a highly variable coastal sea (Gulf of Trieste, Adriatic Sea), Book of Abstracts of the 4 th International Symposium. The Effects of Climate Change on the World’s Oceans, Washington, DC, p. 179. Frehi, H., Couté, A., Mascarell, G., Perrette-Gallet, C., Ayada, M., Kara, M.H., 2007. Dinoflagellés toxiques et/ou responsables de blooms dans la baie d'Annaba (Algérie). Comptes Rendus Biologies 330(8), 615-628. Fresnel, J., Probert, I., Billard, C., 2001. Prymnesium faveolatum sp nov (Prymnesiophyceae), a new toxic species from the Mediterranean Sea. Vie Et Milieu-Life and Environment 51(1-2), 89-97. Froglia, C., 1970. Fitoplanctonti Raccolti in Occasione Della Moria di Pesci Verificatasi Lungo la Costa Marchigiano-Romagnola Nel Maggio 1969. Giornale Botanico Italiano 104(5), 389-396. Garcés, E., Camp, J., 2012. Habitat changes in the Mediterranean Sea and the consequences for harmful algal blooms formation. Life in the Mediterranean Sea: a look at habitat changes, 519541. Garcés, E., Delgado, M., Masó, M., J., C., 1999a. In situ growth rate and distribution of the ichthyotoxic dinoflagellate Gyrodinium corsicum Paulmier in an estuarine embayment (Alfacs Bay, NW Mediterranean Sea). Journal of Plankton Research 21(10), 1977-1991. García-Altares, M., Casanova, A., Fernández-Tejedor, M., Diogène, J., de la Iglesia, P., 2016. Bloom of Dinophysis spp. dominated by D. sacculus and its related diarrhetic shellfish poisoning (DSP) outbreak in Alfacs Bay (Catalonia, NW Mediterranean Sea): Identification of DSP toxins in phytoplankton, shellfish and passive samplers. Regional Studies in Marine Science 6, 19-28. Genitsaris, S., Stefanidou, N., Sommer, U., Moustaka-Gouni, M., 2019. Phytoplankton Blooms, Red Tides and Mucilaginous Aggregates in the Urban Thessaloniki Bay, Eastern Mediterranean. Diversity 11(8), 136. Giacobbe, M.G., Penna, A., Gangemi, E., Masò, M., Garcés, E., Fraga, S., Bravo, I., Azzaro, F., Penna, N., 2007. Recurrent high-biomass blooms of Alexandrium taylorii (Dinophyceae), a HAB species expanding in the Mediterranean. Hydrobiologia 580(1), 125-133. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 132 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Giani, M., Rinaldi, A., Degobbis, D., 2005. Mucilages in the Adriatic and Tyrrhenian Sea: An introduction. Science of The Total Environment 353(1), 3-9. Giuliani, M.E., Accoroni, S., Mezzelani, M., Lugarini, F., Bacchiocchi, S., Siracusa, M., Tavoloni, T., Piersanti, A., Totti, C., Regoli, F., Rossi, R., Zingone, A., Gorbi, S., 2019. Biological Effects of the Azaspiracid-Producing Dinoflagellate Azadinium dexteroporum in Mytilus galloprovincialis from the Mediterranean Sea. Marine Drugs 17(10), 595. Giuliani, S., Lamberti, C.V., Sonni, C., Pellegrini, D., 2005. Mucilage impact on gorgonians in the Tyrrhenian sea. Science of the Total Environment 353(1-3), 340-349. Gómez, F., 2003. The toxic dinoflagellate Gymnodinium catenatum: an invader in the Mediterranean Sea. Acta Botanica Croatica 62(2), 65-72. Gomis, C., Alcober, J., Bernabeu, A., 1996. Seguimiento de las poblaciones fitoplanctónicas en las bateas mejilloneras del puerto de Valencia, 1991–1994. IV Reunion Iberica de fitoplancton toxico y biotoxinas St. Carles de la Rapita (Tarragona): Generalitat de Catalunya. Department d’Agricultura, Ramaderia i Pesca, 29-38. Gorbi, S., Avio, G.C., Benedetti, M., Totti, C., Accoroni, S., Pichierri, S., Bacchiocchi, S., Orletti, R., Graziosi, T., Regoli, F., 2013. Effects of harmful dinoflagellate Ostreopsis cf. ovata exposure on immunological, histological and oxidative responses of mussels Mytilus galloprovincialis. Fish & Shellfish Immunology 35(3), 941-950. Gotsis-Skretas, O., 1995. Mucilage appearances in Greek waters during 1982–1994. Science of The Total Environment 165(1), 229-230. Gourret, P., 1883. Sur les Péridiniens du Golfe de Marseille. Annales du Musée d'Histoire Naturelle de Marseille, Zoologie 1(8), 1-114. Grzebyk, D., Audic, S., Lasserre, B., Abadie, E., de Vargas, C., Bec, B., 2017. Insights into the harmful algal flora in northwestern Mediterranean coastal lagoons revealed by pyrosequencing metabarcodes of the 28S rRNA gene. Harmful Algae 68, 1-16. Halim, Y., 1960a. Alexandrium minutum nov. g. nov. sp. dinoflagellé provocant des ‘eaux rouges’. Vie et Milieu 11(1), 102-105. Halim, Y., Labib, W., 1996. First recorded toxic Alexandrium minutum Halim. Harmful Algae News(16). Hallegraeff, G., Bresnan, E., Enevoldsen, H., Schweibold, L., Zingone, A., 2017. Call to contribute to Global HAB Status Report. Harmful Algae News 58, 1-3. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 133 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Holland, W.C., Litaker, R.W., Tomas, C.R., Kibler, S.R., Place, A.R., Davenport, E.D., Tester, P.A., 2013. Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay. Toxicon 65, 15-33. Hollande, A., Enjumet, M., 1957. Sur une invasion des eaux du port d’Alger par Chattonella subsalsa (Hornellia marina Sub.) Biecheler. Remarques sur la toxicité de cette Chloromonadine. Bull. Trav. Stat. Aquicult. Pêche Castiglione 8, 271-280. Honsell, G., Boni, L., Cabrini, M., Pompei, M., 1992. Toxic or potentially toxic dinoflagellates from the Northern Adriatic Sea, In: Vollenweider, R.A., Marchetti, R., Viviani, R. (Eds.), Marine Coastal Eutrophication. Elsevier, Amsterdam, pp. 107-114. Honsell, G., Dell’Aversano, C., Vuerich, F., Sosa, S., Tartaglione, L., Tubaro, A., 2008. Pseudonitzschia and ASP in the northern Adriatic Sea, Proceedings of the 12th International Conference on harmful algae, pp. 4-6. Honsell, G., Nichetto, P., Sidari, L., Tubaro, A., 1995. Toxic dinoflagellates in the Mediterranean sea. Giornale botanico italiano 129(1), 391-403. Honsell, G., Poletti, R., Pompei, M., Sidari, L., Milandri, A., Casadei, C., Viviani, R., 1996. Alexandrium minutum Halim and PSP contamination in the northern Adriatic Sea (Mediterranean Sea), In: Yasumoto, T., Oshima, Y., Fukuyo, Y. (Eds.), 7th International Conference on Toxic Phytoplankton. Intergovernmental Oceanographic Commission of UNESCO, Sendai, Japan. Ignatiades, L., Gotsis-Skretas, O., 2010. A Review on Toxic and Harmful Algae in Greek Coastal Waters (E. Mediterranean Sea). Toxins 2(5), 1019-1037. Illoul, H., Hernandez, F.R., Vila, M., Adjas, N., Younes, A.A., Bournissa, M., Koroghli, A., Marouf, N., Rabia, S., Ameur, F.L.K., 2012. The genus Ostreopsis along the Algerian coastal waters (SW Mediterranean Sea) associated with a human respiratory intoxication episode. Cryptogamie Algologie 33(2), 209-216. Innamorati, M., Raddi, E., Buzzichelli, S., Melley, A., Balzi, M., 1993. Le mucillagini nel Mar Tirreno. Biol. Mar. Suppl. Notiziario SIBM 1, 23-26. Ismael, A., Halim, Y., 2000. Occurrence and succession of potentially harmful phytoplankton species in the eastern harbour of Alexandria, Egypt, In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), 9th International Conference on Harmful Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Hobart, Tasmania, Australia. Ismael, A.A., 2012. Benthic bloom of cyanobacteria associated with fish mortality in Alexandria waters. The Egyptian Journal of Aquatic Research 38(4), 241-247. Italiano, A., Di Cioccio, D., Borra, M., Biffali, E., Procaccini, G., Zingone, A., 2012. AFLP reveals intraspecific variations in geographically diverse Ostreopsis cf. ovata populations, In: Kim, H.G. (Ed.), Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 134 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas 15th International Conference on Harmful Algae. International Society for the Study of Harmful Algae, , Changwon, Korea, pp. 63-66. Jacques, G., Sournia, A., 1978-1979. Les «eaux rouges» dues au phytoplancton en Méditerranée. Vie et Milieu, 175-187. James, K.J., Furey, A., Lehane, M., Ramstad, H., Aune, T., Hovgaard, P., Morris, S., Higman, W., Satake, M., Yasumoto, T., 2002. First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish. Toxicon 40(7), 909-915. John, U., Litaker, R.W., Montresor, M., Murray, S., Brosnahan, M.L., Anderson, D.M., 2014. Formal Revision of the Alexandrium tamarense Species Complex (Dinophyceae) Taxonomy: The Introduction of Five Species with Emphasis on Molecular-based (rDNA) Classification. Protist 165(6), 779-804. Kaniou-Grigoriadou, I., Mouratidou, T., Katikou, P., 2005. Investigation on the presence of domoic acid in Greek shellfish. Harmful Algae 4(4), 717-723. Kent, W.S., 1881. A manual of the Infusoria: including a description of all known Flagellate, Ciliate, and Tentaculiferous Protozoa, British and foreign, and an account of the organization and the affinities of the sponges. David Bogue. Klopper, S., John, U., Zingone, A., Mangoni, O., Kooistra, W., Cembella, A.D., 2013. Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa? European Journal of Phycology 48(1), 79-92. Koukaras, K., Nikolaidis, G., 2004. Dinophysis blooms in Greek coastal waters (Thermaikos Gulf, NW Aegean Sea). Journal of Plankton Research 26(4), 445-457. Labib, W., Halim, Y., 1995. Diel vertical migration and toxicity of Alexandrium minutum Halim red tide, in Alexandria, Egypt. Marine life (Marseille) 5(1), 11-17. Lassus, P., Chomérat, N., Hess, P., Nézan, E., 2016. Toxic and Harmful Microalgae of the World Ocean / Micro-algues toxiques et nuisibles de l’océan mondial. International Society for the Study of Harmful Algae/ Intergovernmental Oceanographic Commission of UNESCO, Denmark. Laza-Martínez, A., David, H., Riobó, P., Miguel, I., Orive, E., 2016. Characterization of a Strain of Fukuyoa paulensis (Dinophyceae) from the Western Mediterranean Sea. J. Eukaryot. Microbiol. 63(4), 481-497. Lilly, E.L., Kulis, D.M., Gentien, P., Anderson, D.M., 2002. Paralytic shellfish poisoning toxins in France linked to a human-introduced strain of Alexandrium catenella from the western Pacific: evidence from DNAand toxin analysis. Journal of Plankton Research 24(5), 443-452. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 135 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Litaker, R., Fraga, S., Montresor, M., Brosnahan, M., Hoppenrath, M., Murray, S., Calado, A., 2018. A practical guide to new nomenclature for species within the “Alexandrium tamarense species complex”. Harmful Algae News 61, 13-15. Ljubešić, Z., Bosak, S., Viličić, D., Borojević, K.K., Marić, D., Godrijan, J., Ujević, I., Peharec, P., Đakovac, T., 2011. Ecology and taxonomy of potentially toxic Pseudo-nitzschia species in Lim Bay (north-eastern Adriatic Sea). Harmful Algae 10(6), 713-722. Lopez, J., Arte, P., 1971. Red tide on the NE coast of Spain. Investigacion Pesquera 35(2), 699-&. Lugliè, A., Giacobbe, M., Sannio, A., Fiocca, F., Sechi, N., 2003. First record of the dinoflagellate Alexandrium catenella (Whedon & Kofoid) Balech (Dinophyta), a potential producer of paralytic shellfish poisoning. Italian waters (Sardinia, Tyrrhenian Sea). Bocconea 16(2), 10451051. Luglie, A., Giacobbe, M.G., Riccardi, E., Bruno, M., Pigozzi, S., Mariani, M.A., Satta, C.T., Stacca, D., Bazzoni, A.M., Caddeo, T., Farina, P., Padedda, B.M., Pulina, S., Sechi, N., Milandri, A., 2017. Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean: New Data from Sardinia and Sicily (Italy). Microorganisms 5(4). Luglié, A., Satta, C., Padedda, B., Pulina, S., Sechi, N., 2008. What is Chrysophaeum taylorii Lewis & Bryan doing in Sardinia (Tyrrhenian Sea, Mediterranean)? Harmful Algae News 36, 4-6. Lugliè, A., Satta, C.T., Pulina, S., Bazzoni, A.M., Padedda, B.M., Sechi, N., 2011. Le problematiche degli Harmful Algal Blooms (HABs) in Sardegna/Harmful Algal Blooms in Sardinia. Biologia marina mediterranea 18(1), 2. Luo, Z.H., Krock, B., Giannakourou, A., Venetsanopoulou, A., Pagou, K., Tillmann, U., Gu, H.F., 2018. Sympatric occurrence of two Azadinium poporum ribotypes in the Eastern Mediterranean Sea. Harmful Algae 78, 75-85. Mangialajo, L., Ganzin, N., Accoroni, S., Asnaghi, V., Blanfuné, A., Cabrini, M., CattaneoVietti, R., Chavanon, F., Chiantore, M., Cohu, S., Costa, E., Fornasaro, D., Grossel, H., Marco-Miralles, F., Masó, M., Reñé, A., Rossi, A.M., Sala, M.M., Thibaut, T., Totti, C., Vila, M., Lemée, R., 2011. Trends in Ostreopsis proliferation along the Northern Mediterranean coasts. Toxicon 57(3), 408-420. Marasović, I., Gačić, M., Kovačević, V., Krstulović, N., Kušpilić, G., Pucher-Petković, T., Odẑak, N., Šolić, M., 1991. Development of the red tide in the Kaštela Bay (Adriatic Sea). Marine Chemistry 32(2), 375-387. Marasovic, I., Nincevic-Gladan, Z., Skejic, S., Grbec, B., Buzancic, M., Ujevic, I., 2007. Temporal distribution of Dinophysis spp. in relation to diarrhetic shellfish poisoning shellfish toxicity. International Journal of Environment and Health 1(3), 493-506. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 136 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Margalef, R., Estrada, M., 1987. Synoptic distribution of summer microplankton (Algae and Protozoa) across the principal front in the Western Mediterranean. Investigación Pesquera 51(1), 121-140. Marić, D., Kraus, R., Godrijan, J., Supić, N., Djakovac, T., Precali, R., 2012. Phytoplankton response to climatic and anthropogenic influences in the north-eastern Adriatic during the last four decades. Estuarine, Coastal and Shelf Science 115, 98-112. Marić, D., Ljubešić, Z., Godrijan, J., Viličić, D., Ujević, I., Precali, R., 2011. Blooms of the potentially toxic diatom Pseudo-nitzschia calliantha Lundholm, Moestrup & Hasle in coastal waters of the northern Adriatic Sea (Croatia). Estuarine, Coastal and Shelf Science 92(3), 323-331. Mattioli, M., Simoni, F., 1999. Toxicity of Prymnesium in the shallow lake of Massaciuccoli (Migliarino-San Rossore-Massaciuccoli Regional Park-Pisa-Tuscany). Harmful Algae News 19(7). Migliaccio, O., Castellano, I., Di Cioccio, D., Tedeschi, G., Negri, A., Cirino, P., Romano, G., Zingone, A., Palumbo, A., 2016. Subtle reproductive impairment through nitric oxide-mediated mechanisms in sea urchins from an area affected by harmful algal blooms. Scientific Reports 6(1), 26086. Moestrup, Ø., Akselman, R., Cronberg, G., Elbraechter, M., Fraga, S., Halim, Y., Hansen, G., Hoppenrath, M., Larsen, J., Lundholm, N., 2009. onwards. IOC-UNESCO Taxonomic Reference List of Harmful Micro Algae Accessed at http://www.marinespecies.org/hab on 2019-12-18. Montresor, M., John, U., Beran, A., Medlin, L.K., 2004. Alexandrium tamutum sp nov (Dinophyceae): A new nontoxic species in the genus Alexandrium. Journal of Phycology 40(2), 398411. Montresor, M., Zingone, A., Sarno, D., 1998. Dinoflagellate cyst production at a coastal Mediterranean site. Journal of Plankton Research 20(12), 2291-2312. Moschandreou, K.K., Nikolaidis, G., 2010. The genus Pseudo-nitzschia (Bacillariophyceae) in Greek coastal waters. Botanica Marina 53(2), 159-172. Mozetič, P., Cangini, M., Francé, J., Bastianini, M., Bernardi Aubry, F., Bužančić, M., Cabrini, M., Cerino, F., Čalić, M., D'Adamo, R., Drakulović, D., Finotto, S., Fornasaro, D., Grilli, F., Kraus, R., Kužat, N., Marić Pfannkuchen, D., Ninčević Gladan, Ž., Pompei, M., Rotter, A., Servadei, I., Skejić, S., 2019. Phytoplankton diversity in Adriatic ports: Lessons from the port baseline survey for the management of harmful algal species. Marine Pollution Bulletin 147, 117-132. Mozetič, P., Francé, J., Kogovšek, T., Talaber, I., Malej, A., 2012. Plankton trends and community changes in a coastal sea (northern Adriatic): Bottom-up vs. top-down control in relation to environmental drivers. Estuarine, Coastal and Shelf Science 115, 138-148. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 137 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Mozetič, P., Solidoro, C., Cossarini, G., Socal, G., Precali, R., Francé, J., Bianchi, F., De Vittor, C., Smodlaka, N., Fonda Umani, S., 2010. Recent Trends Towards Oligotrophication of the Northern Adriatic: Evidence from Chlorophyll a Time Series. Estuaries and Coasts 33(2), 362-375. Myklestad, S.M., 1995. Release of extracellular products by phytoplankton with special emphasis on polysaccharides. Science of the Total Environment 165(1-3), 155-164. Nezan, E., Chomerat, N., 2011. Vulcanodinium rugosum gen. nov., sp. nov. (Dinophyceae): a new marine dinoflagellate from the French Mediterranean coast. Cryptogamie Algologie 32(1), 318. Nikolaidis, G., Aligizaki, K., Koukaras, K., Moschandreu, K., 2006. Mucilage phenomena in the North Aegean Sea, Greece: another harmful effect of dinoflagellates, In: Moestrup, Ø., Doucette, G., Enevoldsen, H., Godhe, A., Hallegraeff, G., Luckas, B. (Eds.), 12th International Conference on Harmful Algae. International Society for the Study of Harmful Algae and Intergovernamental Oceanographic Commission of UNESCO, Copenhagen, Denmark, pp. 219-222. Nincevic-Gladan, Z., Skejic, S., Buzancic, M., Marasovic, I., Arapov, J., Ujevic, I., Bojanic, N., Grbec, B., Kuspilic, G., Vidjak, O., 2008. Seasonal variability in Dinophysis spp. abundances and diarrhetic shellfish poisoning outbreaks along the eastern Adriatic coast. Botanica Marina 51(6), 449-463. Ninčević Gladan, Ž., Arapov, J., Casabianca, S., Penna, A., Honsell, G., Brovedani, V., Pelin, M., Tartaglione, L., Sosa, S., Dell’Aversano, C., Tubaro, A., Žuljević, A., Grbec, B., Čavar, M., Bužančić, M., Bakrač, A., Skejić, S., 2019. Massive Occurrence of the Harmful Benthic Dinoflagellate Ostreopsis cf. ovata in the Eastern Adriatic Sea. Toxins 11(5), 300. Ninčević Gladan, Ž., Marasović, I., Grbec, B., Skejić, S., Bužančić, M., Kušpilić, G., Matijević, S., Matić, F., 2010. Inter-decadal Variability in Phytoplankton Community in the Middle Adriatic (Kaštela Bay) in Relation to the North Atlantic Oscillation. Estuaries and Coasts 33(2), 376-383. Orhanovic, S., Nincevic, Z., Marasovic, I., PavelaVrancic, M., 1996. Phytoplankton toxins in the Central Adriatic Sea. Croatica Chemica Acta 69(1), 291-303. Parsons, M.L., Aligizaki, K., Bottein, M.Y.D., Fraga, S., Morton, S.L., Penna, A., Rhodes, L., 2012. Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology. Harmful Algae 14, 107-129. Paulmier, G., Berland, B., Billard, C., Nezan, E., 1995. Gyrodinium corsicum nov. sp. (Gymnodiniales, Dinophycées), organisme responsable d'une "eau verte" dans l'étang marin de Diana (Corse), en avril 1994. Cryptogamie, Algologie 16, 77-94. Pavillard, J., 1923. A propos de la systématique des Péridiniens. Bulletin de la Société Botanique de France 70(5), 876-882. Chapter 3. Trends in toxic phytoplankton in the Mediterranean. _______________________________________________________________________________ 144 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Zingone, A., 2010. Harmful Algal Blooms in the Mediterranean: an historical overview, Phytoplankton responses to Mediterranean environmental changes. CIESM Workshop Monographs, pp. 19-24. Zingone, A., Enevoldsen, H., Hallegraeff, G.M., 2017. Are HABs and their societal impacts expanding and intensifying? A call for answers from the HAB scientific community, Marine and Fresh-Water Harmful Algae-Proceedings of the 17th International Conference on Harmful Algae. International Society for the Study of Harmful Algae and Intergovernmental Oceanographic Commission of UNESCO, Florianópolis, Brazil, pp. 14-17. Zingone, A., Enevoldsen, H.O., 2000. The diversity of harmful algal blooms: a challenge for science and management. Ocean & Coastal Management 43(8-9), 725-748. Zingone, A., Escalera, L., Bresnan, E., Enevoldsen, H., Provoost, P., Richardson, A.J., Hallegraeff, G., 2022. Datasets for the study of harmful algae, their global distribution and trends. Zingone, A., Siano, R., D’Alelio, D., Sarno, D., 2006. Potentially toxic and harmful microalgae from coastal waters of the Campania region (Tyrrhenian Sea, Mediterranean Sea). Harmful Algae 5(3), 321-337. Zingone, A., Wyatt, T., 2005. Harmful algal blooms: keys to the understanding of phytoplankton ecology. The Sea. Harvard University Press, Harvard, 867-926. _______________________________________________________________________________ 145 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Chapter 4. New pathologies _______________________________________________________________________________ 147 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Chapter 4. New pathologies. The contents of this chapter were published in the journal Frontiers in Marine Science. Title: The endoparasite Perkinsus olseni affecting the Mediterranean mussels (Mytilus galloprovincialis) in the Italian and Spanish waters: A new possible threat for mussel aquaculture and wild animal population. Authors: Francesca Carella, Margarita Fernandez Tejedor, Grazia Villari, Karl Blyth Andree and Gionata De Vico. Reference: Carella F, Fernandez Tejedor M, Villari G, Andree KB and De Vico G (2023) The endoparasite Perkinsus olseni affecting the Mediterranean mussels (Mytilus galloprovincialis) in the Italian and Spanish waters: A new possible threat for mussel aquaculture and wild animal population. Front. Mar. Sci. 10:1116837. doi: 10.3389/fmars.2023.1116837 Copyright © 2023 Carella, Fernandez Tejedor, Villari, Andree and De Vico. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Chapter 4. New pathologies _______________________________________________________________________________ 149 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Abstract Dinoflagellates belonging to the Perkinsus genus are OIE (World Organization for animal Health)- listed pathogens extremely virulent for clams and oysters in many marine ecosystems throughout the world. During the monitoring activities of the Mediterranean mussel (Mytilus galloprovincialis) in Campania region (Italy), the presence of typical trophozoites of Perkinsus sp. was observed in mussels from farms and natural banks. Simultaneously, following mussel mortality in the Spanish waters of Catalonia, histopathological studies revealed the presence of the same parasite. Although perkinsosis is an endemic disease in clams in Italy (with prevalence from 40 to 80 %), there are no reports to date of its presence in Mediterranean mussels and of the effect on this species. For this study, histopathology, Ray’s Fluid Thioglycollate Medium (RFTM), and molecular diagnostics with conventional Polimerase Chain Reaction (PCR) and qPCR were performed. In samples from Italy, histopathology in the mussel from one farm revealed a prevalence of 26 % in February 2019, 40 % in February 2020, 16 % in November 2020, and 23 % in April 2021. In a natural bank, Perkinsus was also detected in May 2020 but in lower prevalence. In Spain, in July 2020, the presence of the parasite was 20 % in one site and 10 % in a second site and related to animal mortality. In both areas, Perkinsus sp. elicited multiple inflammatory capsules of different size or infiltrates at the level of the digestive gland and gonad. Molecular diagnostics of the Internal Transcriber Spacer (ITS) region of the rDNA (ITS1, 5.8S, and ITS2) showed a 97 % similarity of P. olseni from Italy with samples from New Zealand, Australia, and Uruguay and in bivalves such as Pitar rostrata, Astrovenus sp., and Haliotis sp., whereas in Spain the identity was 99 % samples from South Korean venerids such as Anadara granosa. Phylogenetic analysis group together P. olseni from Italian and Spanish mussels but place them distant from other P. olseni described in the clams from Europe (Italy, France, and Spain). Direct impact of transboundary animal diseases in aquaculture constitutes a serious consequence for export living animals and their products, as well for international trade. This compromises food security, also causing a high socioeconomic impact on aquaculture exporting nations. P. olseni is a generalist pathogen able to infect different bivalve species, possibly passing from clams to oysters and mussels. Recognized international organizations should take this into account in the view of possible cross-infection. Other studies are needed to define pathogen virulence in this species. 1. Introduction In the past years, infectious diseases are emerging significantly in marine and freshwater environments. The elements involved in this emergence are different. Cultivated animals are involved in the global trading, facilitating the introduction of serious infectious diseases, called transboundary diseases (TD). Several transboundary aquatic animal diseases (TAADS) have swept Chapter 4. New pathologies _______________________________________________________________________________ 150 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas regions over the past 30 years causing massive economic and social losses, responsible for the introduction, establishment, and spread of pathogens into new geographic areas. Nowadays, there are several international codes of practice and guidelines to reduce the risk of introducing pathogens. WOHA (World Organization of Animal Health) has developed recommendations and protocols in the International Aquatic Animal Health Code, which deals with the health surveillance of aquatic animals (OIE, 2021). Emerging disease in mussels has been reported repeatedly in the past years, as in other bivalve species. Recently, the potentially zoonotic bacteria Nocardia crassotreae were reported in the Mediterranean mussel M. galloprovincialis (Carella et al., 2013a; De Vico and Carella, 2019), the OIE listed parasite Marteilia refringens have been observed in the area (Carella et al., 2010), and many other emerging disease conditions have been also reported in other bivalve species in the same area (Carella et al., 2013a; Carella et al., 2013b). Perkinsosis is an important disease that has been reported worldwide in bivalves and gastropods. Perkinsus pathogens can infect a wide range of hosts and possibly are responsible for mortality events for their extensive invasive ability and virulence. Nowadays, seven species within the genus Perkinsus have been reported including P. marinus, P. olseni, P. qugwadi, P. chesapeaki, P. mediterraneus, P. honshuensis, and P. beihaiensis (Ramilo et al., 2015) with only P. olseni and P. marinus listed notifiable parasites listed by OIE (OIE, 2021). Pathogens can display a highly flexible ranges of hosts, called multi-host or generalist pathogens, or can infect only one or a few related species and called specialist pathogens. P. olseni has been reported in 30 mollusc species, bivalves, and gastropods over a wide range of geographical locations (Itoiz et al., 2022). It is generally associated with mass mortality of clams such as Manila clams Ruditapes philippinarum in Europe, the venerid clam R. philippinarum in Asia, the cockle Austrovenus stutchburyi in New Zealand (Dungan et al., 2007), and in the abalone Haliotis spp in Australia. Recently, reports of perkinsosis in mussels have been increasing; Itoh et al. (2019) reported P. beihaiensis infection in the invasive M. galloprovincialis in Japan, whereas Vazquez et al. (2022) reported P. olseni in M. chilensis in Argentina. In this study, we report, for the first time, the presence of the parasite Perkinsus sp. in the Mediterranean mussel M. galloprovincialis in Europe. First detection was in mussels from Italy, in Campania region, in mussel farms from 2019 to 2021 and later in natural beds in 2020. During the study, we also observed the presence of Perkinsus sp. like cells in mussel samples from Catalonia (Spain) following a mussel episode of mortality. Within the past few years, more data on the genetic variation within some of the Perkinsus species have become available, and many ITS (internal transcribed spaces) regions now described have that allowed to assess intraspecific variation and to compare the dissimilarity of sequence with the differences observed among the Perkinsus species. Chapter 4. New pathologies _______________________________________________________________________________ 151 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas During the surveys, we conducted phylogenetic analyses to estimate the relationships with the group of Perkinsus spp. in mussels from Italy and Spain and haplotype characterization along with animal histopathology to define host response and possible Perkinsus pathogenicity. 2. Materials and methods 2.1. Sampling of Mediterranean mussels in Italy and Spain During 2018–2021, a field survey targeting infectious agents of the Mediterranean mussel Mytilus galloprovincialis was conducted in which mussels were manually collected in one mussel farm and one natural bank on the north coast of the Campania region (Italy). In Italy, collections were made in a mussel farm in Campania Region (Naples Bay) in February 2019 (N = 20), February 2020 (N = 20), November 2020 (N = 30), and April 2021 (N = 30). A sampling was performed in a close natural bank in May 2020 (N = 30). Alfacs Bay, located in the south of the Ebro delta (Western Mediterranean), is a shellfish growing area where mussels and oysters are grown in ropes hanging from rafts. Sampling in Alfacs Bay was conducted on 21 July 2020, 2 weeks after the beginning of the mortality event on 6 July 2020 as reported by the mussel farmers. Sampling in Alfacs Bay was conducted in two sites, A (40°37’15.28’’N; 0°39’14.58’’E) and B (40°37’1.98”N; 0°37’51.66”E) (Figure 41). The mussels were transported to the laboratory alive in isothermal boxes. Prior to processing, the animals were measured for animal shell length, total weight, and meat weight (MW) according to Galtsoff (1964). Chapter 4. New pathologies _______________________________________________________________________________ 152 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas Figure 41. Locations of sampling areas in Italy and Spain: Bay of Naples and Bay of Alfacs. 2.2. Light microscopy For animal histopathology, from each animal, a transverse section including digestive tissue, gill, foot, and gonad was obtained and fixed in Davidson’s solution for 72–96h. Fixed tissues were embedded in paraffin blocks and sectioned at 3 µm with a rotary microtome (Bioptica, Italy). Tissue sections were deparaffinised, stained with Carazzi haematoxylin and eosin and a special stain such as Mallory’s trichrome (Mazzi, 1977). Digital images and measurements were obtained using an integrated Axioscope A1 (Zeiss, Germany) and camera Axiocam 208. 2.3. PCR and qPCR for Perkinsus species identification and presence evaluation From each specimen, 25–30 mg of gonad, digestive tissue and gills, preserved in TE buffer at −20°C, was taken for total genomic DNA extraction using QIAamp DNA Mini Kit (QIAGEN, Germany), according to the manufacturer’s instructions (tissue protocol). The DNA quality and quantity were measured with the use of a Nanodrop spectrophotometer (Thermo Fisher Scientific) and stored at Chapter 4. New pathologies _______________________________________________________________________________ 153 Margarita Fernández Tejedor. Challenges for shellfish aquaculture in Mediterranean coastal areas −80 °C for long-term preservation. Primers used in the study are listed in Table 41. More in detail, PCR assays using the generic primers PerkITS750/PerkITS85 (Casas et al., 2002) were carried out first to detect Perkinsus spp. in all sampled mussels. PCR reactions were carried out in 50 μl of final volume using the Mastermix GoTaq polymerase (Promega) following the instructions of the manufacturer. A positive control provided by IRTA institute constituted by a clam infected P. olseni was included in each reaction along with a negative control (master mix with no DNA). Amplification parameters were performed as follows: An initial denaturation of 4 min at 94°C followed by 35 cycle amplifications (1 min at 94 °C, 1 min at 53 °C, and 3 min at 68 °C) and a final extension of 5 min at 68 °C. The resulting PCR products were purified and sent to an external sequencing facility (Eurofins Genomics, Germany). Table 41. List of the primers used to detect Perkinsus spp. with PCR and qPCR in this study. To better define pathogen presence in the Italian samples, a more sensitive procedure of real-time quantitative PCR (qPCR) was also performed using primers Perk-ITS-qF1/Perk-ITS-qR2 (Rios et al., 2020) that amplifies the internal transcribed spacer region (ITS-1 and ITS-2) of the gene complex that codes for ribosomal RNAs in P. olseni. Wells were filled to a final volume of 10 µl, using 1 µl of DNA, 5 µl of Taq Universal SYBR green mix (Applied Biosystem), 0.5 µl of each primer (10 µM), and 3 µl of distilled water. Amplification was performed under the following conditions: denaturation for 10 min at 95 °C, amplification by 40 cycles of 15 s at 95 °C and 60 s at 60 °C, melting curve evaluation 1 min at 95 °C, and increase of 0.5 °C each 30 s starting in 60 °C, end at 95 °C for 15 s. All reactions were performed using two technical replicates. 2.4. Phylogeny and haplotype analysis of Perkinsus based on the ribosomal ITS region The resulting ITS chromatograms (648 bp) were analyzed using BioEdit software (v. 7.2). All generated sequences were searched for identity using BLAST (Basic Local Alignment Search Tool) through web servers of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The sequences were also aligned with the available sequences for Perkinsus spp. found in the GenBank database using the MUSCLE algorithm. Maximum likelihood (ML) analysis was conducted using MEGA version X software (Kumar et al., 2004) with 1,000 replicates for calculating bootstrap values.