toxins

toxins Article

Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Toxins Evanthia Mantzouki 1, *, Miquel Lürling 2,3 ID , Jutta Fastner 4 , Lisette de Senerpont Domis 2,3 , El˙zbieta Wilk-Wo´zniak 5 , Judita Koreivien˙e 6 , Laura Seelen 2,3 ID , Sven Teurlincx 3 ID , ¯ e Karosien˙e 6 , Yvon Verstijnen 2 , Wojciech Krzton´ 5 , Edward Walusiak 5 , Jurat˙ 6 6 ¯ e Kasperoviˇcien˙e , Ksenija Savadova , Irma Vitonyt˙e 6 , Carmen Cillero-Castro 7 ID , Jurat˙ 8 ID , Agnieszka Budzynska 8 , Ryszard Goldyn 8 ID , Anna Kozak 8 ID , Joanna Rosinska ´ 8 8 8 ID El˙zbieta Szelag-Wasielewska ˛ , Piotr Domek , Natalia Jakubowska-Krepska , Kinga Kwasizur 9 ID , Beata Messyasz 9 , Aleksandra Pełechata 9 , Mariusz Pełechaty 9 , Mikolaj Kokocinski 9 , Ana García-Murcia 10 , Monserrat Real 10 , Elvira Romans 10 , Jordi Noguero-Ribes 10 , David Parreño Duque 10 , Elísabeth Fernández-Morán 10 ID , Nusret Karakaya 11 , Kerstin Häggqvist 12 , Nilsun Demir 13 ID , Meryem Beklioglu ˘ 14 , Nur Filiz 14 , 14 14 14 14 ID Eti E. Levi , Ugur ˘ Iskin , Gizem Bezirci , Ülkü Nihan Tav¸sanoglu ˘ , Koray Özhan 15 ID , 16 16 17 Spyros Gkelis ID , Manthos Panou , Özden Fakioglu , Christos Avagianos 18 , Triantafyllos Kaloudis 18 ID , Kemal Çelik 19 , Mete Yilmaz 20 , Rafael Marcé 21 , Nuria Catalán 21,22 ID , Andrea G. Bravo 22 ID , Moritz Buck 22 , William Colom-Montero 23 , Kristiina Mustonen 23 , Don Pierson 23 , Yang Yang 23 , Pedro M. Raposeiro 24 , Vítor Gonçalves 24 ID , Maria G. Antoniou 25 , Nikoletta Tsiarta 25 , Valerie McCarthy 26 , Victor C. Perello 26 , Tõnu Feldmann 27 , Alo Laas 27 ID , Kristel Panksep 27 , Lea Tuvikene 27 , Ilona Gagala 28 , Joana Mankiewicz-Boczek 28 ID , Meral Apaydın Yagcı ˘ 29 ID , Sakir ¸ Çınar 29 , Kadir Çapkın 29 , 29 29 29 Abdulkadir Yagcı ˘ , Mehmet Cesur , Fuat Bilgin , Cafer Bulut 29 , Rahmi Uysal 29 , 30 Ulrike Obertegger ID , Adriano Boscaini 30 , Giovanna Flaim 30 ID , Nico Salmaso 30 ID , Leonardo Cerasino 30 , Jessica Richardson 31 , Petra M. Visser 32 , Jolanda M. H. Verspagen 32 , Tünay Karan 33 , Elif Neyran Soylu 34 ID , Faruk Mara¸slıoglu ˘ 35 ID , Agnieszka Napiórkowska-Krzebietke 36 ID , Agnieszka Ochocka 37 , Agnieszka Pasztaleniec 37 , Ana M. Antão-Geraldes 38 , Vitor Vasconcelos 39 ID , João Morais 39 ID , Micaela Vale 39 ID , Latife Köker 40 , Reyhan Akçaalan 40 ID , Meriç Albay 40 , Dubravka Špoljari´c Maroni´c 41 , Filip Stevi´c 41 , Tanja Žuna Pfeiffer 41 , Jeremy Fonvielle 42 , Dietmar Straile 43 ID , Karl-Otto Rothhaupt 43 , Lars-Anders Hansson 44 , Pablo Urrutia-Cordero 22,44 ID , Ludˇek Bláha 45 , Rodan Geriš 46 , Markéta Fránková 47 , Mehmet Ali Turan Koçer 48 , Mehmet Tahir Alp 49 , Spela Remec-Rekar 50 , Tina Elersek 51 , Theodoros Triantis 52 ID , Sevasti-Kiriaki Zervou 52 ID , Anastasia Hiskia 52 , Sigrid Haande 53 , Birger Skjelbred 53 , Beata Madrecka 54 , Hana Nemova 55 , Iveta Drastichova 55 , Lucia Chomova 55 , Christine Edwards 56 , Tugba ˘ Ongun Sevindik 57 , 57 57 58 Hatice Tunca , Burçin Önem , Boris Aleksovski , Svetislav Krsti´c 58 , Itana Bokan Vuceli´c 59 , Lidia Nawrocka 60 , Pauliina Salmi 61 , Danielle Machado-Vieira 62 , Alinne Gurjão de Oliveira 62 , Jordi Delgado-Martín 63 , David García 63 , Jose Luís Cereijo 63 , Joan Gomà 64 ID , Mari Carmen Trapote 64 , Teresa Vegas-Vilarrúbia 64 ID , Biel Obrador 64 , Magdalena Grabowska 65 , Maciej Karpowicz 65 ID , Damian Chmura 66 , Bárbara Úbeda 67 , José Ángel Gálvez 67 ID , Arda Özen 68 ID , Kirsten Seestern Christoffersen 69 ID , Trine Perlt Warming 69 , Justyna Kobos 70 , Hanna Mazur-Marzec 70 , Carmen Pérez-Martínez 71 ID , Eloísa Ramos-Rodríguez 71 ID , Lauri Arvola 72 , Pablo Alcaraz-Párraga 73 , Magdalena Toporowska 74 , Barbara Pawlik-Skowronska 74 , Michał Nied´zwiecki 74 , Wojciech P˛eczuła 74 , Manel Leira 75 ID , Armand Hernández 76 ID , Enrique Moreno-Ostos 77 , José María Blanco 77 , Valeriano Rodríguez 77 , Jorge Juan Montes-Pérez 77 , Roberto L. Palomino 77 , Estela Rodríguez-Pérez 77 , Rafael Carballeira 78 , Antonio Camacho 79 ID , Antonio Picazo 79 , Carlos Rochera 79 ID , Anna C. Santamans 79 , Carmen Ferriol 79 , Susana Romo 80 , Juan Miguel Soria 80 ID

Toxins 2018, 10, 156; doi:10.3390/toxins10040156

www.mdpi.com/journal/toxins

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81 , Daniel Szymanski 81 , Marek Kruk 82 ID , Julita Dunalska 81 , Justyna Sienska ´ ´ Iwona Kostrzewska-Szlakowska 83 , Iwona Jasser 84 , Petar Žutini´c 85 , Marija Gligora Udoviˇc 85 , 87 , Anđelka Plenkovi´c-Moraj 85 , Magdalena Frak ˛ 86 , Agnieszka Bankowska-Sobczak ´ 87 88 2,89,90 ID Michał Wasilewicz , Korhan Özkan , Valentini Maliaka , Kersti Kangro 27,91 , Hans-Peter Grossart 42,92 ID , Hans W. Paerl 93 ID , Cayelan C. Carey 94 ID and Bas W. Ibelings 1 1 2

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Department F.-A. Forel for Environmental and Aquatic Sciences, University of Geneva, 1205 Geneva, Switzerland; [email protected] Department of Environmental Sciences, Wageningen University & Research, 6700 Wageningen, The Netherlands; [email protected] (M.L.); [email protected] (L.d.S.D.); [email protected] (L.S.); [email protected] (Y.V.); [email protected] (V.M.) Department of Aquatic Ecology, Netherlands Institute of Ecology (NIOO-KNAW), 6700 Wageningen, The Netherlands; [email protected] German Environment Agency, Unit Drinking Water Resources and Water Treatment, Corrensplatz 1, 14195 Berlin, Germany; [email protected] Institute of Nature Conservation, Polish Academy of Sciences, 31-120 Krakow, Poland; [email protected] (E.W.-W.); [email protected] (W.K.); [email protected] (E.W.) Institute of Botany, Nature Research Centre, Vilnius 08412, Lithuania; [email protected] (J.K.); [email protected] (J.K.); [email protected] (J.K.); [email protected] (K.S.); [email protected] (I.V.) R&D Department Environmental Engineering, 3edata, 27004 Lugo, Spain; [email protected] Department of Water Protection, Adam Mickiewicz University, 61614 Poznan, Poland; [email protected] (A.B.); [email protected] (R.G.); [email protected] (A.K.); [email protected] (J.R.); [email protected] (E.S.-W.); [email protected] (P.D.); [email protected] (N.J.-K.) Department of Hydrobiology, Adam Mickiewicz University, 61614 Poznan, Poland; [email protected] (K.K.); [email protected] (B.M.); [email protected] (A.P.); [email protected] (M.P.); [email protected] (M.K.) Department of Limnology and Water Quality, AECOM U.R.S, 08036 Barcelona, Spain; [email protected] (A.G.-M.); [email protected] (M.R.); [email protected] (E.R.); [email protected] (J.N.-R.); [email protected] (D.P.D.); [email protected] (E.F.-M.) Department of Environmental Engineering, Abant Izzet Baysal University, 14280 Bolu, Turkey; [email protected] Department of Science and Engineering, Åbo Akademi University, 20520 Åbo, Finland; [email protected] Department of Fisheries and Aquaculture, Ankara University, 6100 Ankara, Turkey; [email protected] Department of biology, Middle East Technical University, 6800 Ankara, Turkey; [email protected] (M.B.); [email protected] (N.F.); [email protected] (E.E.L.); [email protected] (U.I.); [email protected] (G.B.); [email protected] (Ü.-N.T) Institute of Marine Sciences, Department of Oceanography, Middle East Technical University, 06800 Ankara, Turkey; [email protected] Department of Botany, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; [email protected] (S.G.); [email protected] (M.P.) Department of Basic Science, Ataturk University, 25240 Erzurum, Turkey; [email protected] Water Quality Department, Athens Water Supply and Sewerage Company, 11146 Athens, Greece; [email protected] (C.A.); [email protected] (T.K.) Department of Biology, Balikesir University, 10145 Balikesir, Turkey; [email protected] Department of Bioengineering, Bursa Technical University, 16310 Bursa, Turkey; [email protected] Catalan Institute for Water Research (ICRA), 17003 Girona, Spain; [email protected] (R.M.); [email protected] (N.C.) Department of Ecology and Genetics, Limnology, Uppsala University, 75236 Uppsala, Sweden; [email protected] (A.G.B.); [email protected] (M.B.); [email protected] (P.U.-C.) Department of Ecology and Genetics, Erken Laboratory, Uppsala University, 76173 Norrtalje, Sweden; [email protected] (W.C.-M.); [email protected] (K.M.); [email protected] (D.P.); [email protected] (Y.Y.)

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Research Center in Biodiversity and Genetic Resources (CIBIO-Azores), InBIO Associated Laboratory, Faculty of Sciences and Technology, University of the Azores, 9501-801 Ponta Delgada, Portugal; [email protected] (P.M.R.); [email protected] (V.G.) Department of Environmental Science and Technology, Cyprus University of Technology, 3036 Lemesos, Cyprus; [email protected] (M.G.A.); [email protected] (N.T.) Centre for Freshwater and Environmental Studies, Dundalk Institute of Technology, A91 K584 Dundalk, Ireland; [email protected] (V.M.); [email protected] (V.C.P.) Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, 51014 Tartu, Estonia; [email protected] (T.F.); [email protected] (A.L.); [email protected] (K.P.); [email protected] (L.T.); [email protected] (K.K.) European Regional Centre for Ecohydrology of the Polish Academy of Sciences, 90364 Lodz, Poland; [email protected] (I.G.); [email protected] (J.M.-B.) Republic of Turkey Ministry of Food Agriculture, Fisheries Research Institute, 32500 Egirdir, ˘ Isparta, Turkey; [email protected] (M.A.Y.); [email protected] (¸S.Ç.); [email protected] (K.Ç.); [email protected] (A.Y.); [email protected] (M.C.); [email protected] (F.B.); [email protected] (C.B.); [email protected] (R.U.) Department of Sustainable Ecosystems and Bioresources, Fondazione Edmund Mach, 38010 San Michele all’Adige, Italy; [email protected] (U.O.); [email protected] (A.B.); [email protected] (G.F.); [email protected] (N.S.); [email protected] (L.C.) Department of Biological and Environmental Sciences, University of Stirling, Stirling FK9 4LA, UK; [email protected] Department of Freshwater and Marine Ecology, University of Amsterdam, 1090 GE Amsterdam, The Netherlands; [email protected] (P.M.V.); [email protected] (J.M.H.V.) Department of Molecular Biology and Genetics, Gaziosmanpasa University, 60250 Merkez, Turkey; [email protected] Department of Biology, Giresun University, 28100 Giresun, Turkey; [email protected] Department of Biology, Hitit University, 19040 Corum, Turkey; [email protected] Department of Icthyology, Hydrobiology and Aquatic Ecology, Inland Fisheries Institute, 10719 Olsztyn, Poland; [email protected] Department of Freshwater Protection, Institute of Environmental Protection- National Research Institute, 01-692 Warsaw, Poland; [email protected] (A.O.); [email protected] (A.P.) Centro de Investigação da Montanha, Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal; [email protected] Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) and University of Porto, 4450-208 Matosinhos, Portugal; [email protected] (V.V.); [email protected] (J.M.); [email protected] (M.V.) Department of Freshwater Resource and Management, Faculty of Aquatic Sciences, Istanbul University, 34134 Istanbul, Turkey; [email protected] (L.K.); [email protected] (R.A.); [email protected] (M.A.) Department of Biology, Josip Juraj Strossmayer University of Osijek, 31000 Osijek, Croatia; [email protected] (D.Š.M.); [email protected] (F.S.); [email protected] (T.Ž.P.) Department of Experimental Limnology, Leibniz Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany; [email protected] (J.F.); [email protected] (H.-P.G.) Department of Biology, Limnological Institute, University of Konstanz, 78464 Konstanz, Germany; [email protected] (D.S.); [email protected] (K.-O.R.) Department of Biology, Lund University, 22362 Lund, Sweden; [email protected] RECETOX, Faculty of Science, Masaryk University, 62500 Brno, Czech Republic; [email protected] Department of Hydrobiology, Morava Board Authority, 60200 Brno, Czech Republic; [email protected] Laboratory of Paleoecology, Institute of Botany, The Czech Academy of Sciences, 60200 Brno, Czech Republic; [email protected] Department of Environment and Resource Management, Mediterranean Fisheries Research Production and Training Institute, 7090 Antalya, Turkey; [email protected] Faculty of Aquaculture, Mersin University, 33160 Mersin, Turkey; [email protected] Department of Water Quality, Slovenian Environmental Agency, 1000 Ljubljana, Slovenia; [email protected]

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Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, 1000 Ljubljana, Slovenia; [email protected] Institute of Nanoscience and Nanotechnology, National Center for Scientific Research «DEMOKRITOS», 15341 Attiki, Greece; [email protected] (T.T.); [email protected] (S.-K.Z.); [email protected] (A.H.) Department of Freshwater Ecology, Norwegian Institute for Water Research, 0349 Oslo, Norway; [email protected] (S.H.); [email protected] (B.S.) Institute of Environmental Engineering, Poznan University of Technology, 60965 Poznan, Poland; [email protected] National Reference Center for Hydrobiology, Public Health Authority of the Slovak Republic, 82645 Bratislava, Slovakia; [email protected] (H.N.); [email protected] (I.D.); [email protected] (L.C.) School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen AB10 7GJ, UK; [email protected] Department of Biology, Sakarya University, 54187 Sakarya, Turkey; [email protected] (T.O.S.); [email protected] (H.T.); [email protected] (B.Ö.) Faculty of Natural Sciences and Mathematics, SS Cyril and Methodius University, 1000 Skopje, Macedonia; [email protected] (B.A.); [email protected] (S.K.) Department for Ecotoxicology, Teaching Institute of Public Health of Primorje-Gorski Kotar County, 51000 Rijeka, Croatia; [email protected] Institute of Technology, The State University of Applied Sciences, 82300 Elblag, Poland; [email protected] Department of Biological and Environmental Science, University of Jyväskylä, 40014 Jyväskylä, Finland; [email protected] Departamento de Sistemática e Ecologia, Universidade Federal da Paraíba, 58059-970 Paraíba, Brasil; [email protected] (D.M.-V.); [email protected] (A.G.d.O.) Department of Civil Engineering, University of A Coruña, 15192 A Coruña, Spain; [email protected] (J.D.-M.); [email protected] (D.G.); [email protected] (J.L.C.) Department of Evolutionary Biology, Ecology, and Environmental Sciences, University of Barcelona, 08028 Barcelona, Spain; [email protected] (J.G.); [email protected] (M.C.T.); [email protected] (T.V.-V.); [email protected] (B.O.) Department of Hydrobiology, University of Bialystok, 15245 Bialystok, Poland; [email protected] (M.G.); [email protected] (M.K.) Institute of Environmental Protection and Engineering, University of Bielsko-Biala, 43309 Bielsko-Biala, Poland; [email protected] Department of Biology, University of Cádiz, 11510 Puerto Real, Cádiz, Spain; [email protected] (B.Ú.); [email protected] (J.Á.G.) Department of Forest Engineering, University of Cankiri Karatekin, 18200 Cankiri, Turkey; [email protected] Freshwater Biological Laboratory, Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark; [email protected] (K.S.C.); [email protected] (T.P.W.) Department of Marine Biotechnology, University of Gdansk, 81378 Gdynia, Poland; [email protected] (J.K.); [email protected] (H.M.-M.) Department of Ecology, University of Granada, 18071 Granada, Spain; [email protected] (C.P.-M.); [email protected] (E.R.-R.) Lammi Biological Station, University of Helsinki, 16900 Lammi, Finland; [email protected] Department of Animal Biology, Plant Biology and Ecology, University of Jaen, 23701 Jaen, Spain; [email protected] Department of Hydrobiology and Protection of Ecosystems, University of Life Sciences in Lublin, 20262 Lublin, Poland; [email protected] (M.T.); [email protected] (B.P.-S.); [email protected] (M.N.); [email protected] (W.P.) Instituto Dom Luiz, University of Lisbon, 1749016 Lisbon, Portugal; [email protected] Institute of Earth Sciences Jaume Almera, ICTJA, CSIC, 08028 Barcelona, Spain; [email protected] Department of Ecology, University of Malaga, 29071 Malaga, Spain; [email protected] (E.M.-O.); [email protected] (J.M.B.); [email protected] (V.R.); [email protected] (J.J.M.-P.); [email protected] (R.L.P.); [email protected] (E.R-P.)

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Centro de Investigacións Cientificas Avanzadas (CICA), Facultade de Ciencias, Universidade da Coruña, 15071 A Coruña, Spain; [email protected] Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, 46980 Paterna Valencia, Spain; [email protected] (A.C.); [email protected] (A.P.); [email protected] (C.R.); [email protected] (A.C.S.); [email protected] (C.F.) Department of Microbiology and Ecology, University of Valencia, 46100 Burjassot, Spain; [email protected] (S.R.); [email protected] (J.M.S.) Department of Water Protection Engineering, University of Warmia and Mazury, 10-720 Olsztyn, Poland; [email protected] (J.D.); [email protected] (J.S.); [email protected] (D.S.) Department of Tourism, Recreation and Ecology, University of Warmia and Mazury, 10-720 Olsztyn, Poland; [email protected] Faculty of Biology, University of Warsaw, 02-096 Warsaw, Poland; [email protected] Department of Plant Ecology and Environmental Conservation, Faculty of Biology, University of Warsaw, 02-089 Warsaw, Poland; [email protected] Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia; [email protected] (P.Ž.); [email protected] (M.G.U.); [email protected] (A.P.-M.) Department of Environmental Improvement, Faculty of Civil and Environmental Engineering, Warsaw University of Life Sciences—SGGW, 02-787 Warsaw, Poland; [email protected] Department of Hydraulic Engineering, Faculty of Civil and Environmental Engineering, Warsaw University of Life Sciences—SGGW, 02-787 Warsaw, Poland; [email protected] (A.B.-S.); [email protected] (M.W.) Institute of Marine Sciences, Marine Biology and Fisheries, Middle East Technical University, 06800 Ankara, Turkey; [email protected] Society for the Protection of Prespa, 53077 Agios Germanos, Greece Institute for Water and Wetland Research, Department of Aquatic Ecology and Environmental Biology, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands Tartu Observatory, Faculty of Science and Technology, University of Tartu, 61602 Tartu, Estonia Institute of Biochemistry and Biology, Potsdam University, 14469 Potsdam, Germany Institute of Marine Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 28557, USA; [email protected] Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA; [email protected] Correspondence: [email protected]

Received: 15 February 2018; Accepted: 29 March 2018; Published: 13 April 2018







Abstract: Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains. Keywords: microcystin; anatoxin; cylindrospermopsin; temperature; direct effects; indirect effects; spatial distribution; European Multi Lake Survey

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Key Contribution: This manuscript describes the outcomes of the European Multi Lake Survey (EMLS) in which the effects of temperature and nutrients on the variability of production of multiple classes of cyanobacterial toxins were assessed. At this continental scale, temperature through direct and indirect effects, was found to be the main driver of cyanobacterial toxin distribution and toxin quota.

1. Introduction As a consequence of human population growth, along with associated agricultural, urban and industrial activities, harmful algal blooms worldwide are on the increase [1]. Eutrophication, one major outcome of anthropogenic activities in the catchments of aquatic ecosystems, is consistently recognized as the main driver of cyanobacterial blooms [2,3]. In addition, damage to ecosystems and loss of natural resources (e.g., in Lake Taihu, China [4] and references within [5]) are also attributed to on-going climatic change [6]. A synergistic interaction between increased nutrients and climate-related changes is predicted [7] based on experimental [8] and field studies [9], potentially further exacerbating the occurrence of cyanobacterial blooms. The long history of cyanobacterial adaptation to a wide range of environmental conditions including extremes [10] supports their successful occurrence in a variety of lake ecosystems. These adaptations come in the form of functional traits such as phosphorus storage, buoyancy regulation, nitrogen fixation and the formation of akinetes (resting spores). Extensive research has linked the prevalence of species with specific functional traits to certain sets of environmental conditions [11,12]. For example, Microcystis aeruginosa can rapidly float up in the illuminated near surface layers under conditions of enhanced water column stability [13], and through buoyancy regulation gain access to both nutrients at deeper layers and light at the surface [14]. Toxin production, by the production of hepatotoxins (e.g., microcystins (MCs) and nodularin (NOD)), cytotoxins (cylindrospermopsins (CYN)) and neurotoxins (e.g., anatoxins (ATX)), is another common trait of a large number of cyanobacterial species. Although numerous studies have elucidated the chemical properties, biosynthesis and genetics of the most well-known toxins [15–17], still little is known why toxins are produced and what determines their presence in field populations. There is evidence that production of these secondary metabolites might provide a competitive advantage for example through providing resistance against grazing [18–20] or a physiological benefit e.g., in enhancing nutrient uptake or offering protection against oxidative stress (references within [21]). The abundance of toxins during blooms depends on the presence of toxic strains within the cyanobacterial population [22]. Different species have been shown to produce specific toxins or even variants [23–25]. However, in toxic strains, the environmental factors controlling expression of the toxin synthetase genes are still a contentious issue [26]. Lack of consistency in experimental findings, along with a lack of standardization in surveying and sampling design in field studies, so far has hindered a coherent understanding of how environmental stressors are linked to cyanobacterial toxin production and toxin quota (toxin concentration per cell or unit algal biomass). Various studies have shown contradictory responses of toxin producing taxa to similar environmental parameters. For example, experiments with MC-producing Planktothrix agardhii showed that high nitrogen concentrations (one factor in the study to vary among others like phosphorus, temperature, pH, light) were correlated to high MC production in batch cultures [27]. However, in experiments with Microcystis aeruginosa, N-limited chemostat experiments triggered an increase in MC content, with smaller and faster growing cells being mostly promoted as a response to favourable growing conditions [28]. Similarly, an experiment with ATX producers showed that temperature dependent optimal growth conditions did not necessarily result in higher toxin concentration [29]. In this study, while Aphanizomenon cultures proliferated at 30 ◦ C, Dolichospermum (formerly Anabaena) cultures suffered at this high temperature [29]. Nevertheless, ATX production was reduced by both

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tested species [29]. A subsequent study on ATX or MC producing strains of Dolichospermum indicated strain-specific responses to temperature and light limited conditions [30]. Consequently, there is no easy way to deduce from environmental conditions which toxins will come to dominate in a bloom, nor whether toxin concentrations will be high. Microcystins (MCs), as the largest, best described and most diverse group of cyanobacterial toxins [26], have been the focus of management and mitigation guidelines. For drinking water, the World Health Organisation (WHO) has set a provisional guideline value-maintained at the same level in the EU Drinking Water Directive-of 1 µg/L for MC-LR, a value that is accepted in most countries. In recreational waters, however, there is less consistency, even for MC-LR, and national authorities use a variety of risk assessment schemes and criteria to inform management decisions/practice [31]. MC-LR is the best studied MC variant [32,33], yet other variants such as MC-YR [34], -LW and -LF [35] can also be highly toxic and may contribute significantly to the total MCs in a lake. MC-RR, although reported to be ten times less toxic than MC-LR after intraperitoneal injection in mice (LD50 ), is one of the most frequently reported toxin in the lakes along with MC-LR and MC-YR [36] and may be more harmful to aquatic biota than MC-LR [37]. Good toxicity data are lacking for the vast majority of MC variants (presently more than 250 [23]). Good toxicological data on other toxins such as CYN and ATX are necessary to include a full spectrum of cyanotoxins in human and ecosystem risk assessment (reviewed in [36,38]). Although a fair number of toxin surveys at the national level have been carried out [39], studies have rarely investigated the spatial distribution of different classes of cyanotoxins at larger geographical scales, encompassing lakes of widely different characteristics and environmental diversity. According to studies [40,41], the climate in Europe is already shifting north (e.g., central European countries will experience longer hot summers, similar to presently in Mediterranean countries) and, as such, a study on cyanotoxins over a large latitudinal gradient may offer insights into their future distribution. In the European Multi Lake Survey (EMLS), lakes across the continent were sampled once in a snapshot approach, for physical, chemical and biological parameters, during the summer of 2015. Standardized and commonly practiced field protocols along with centralized laboratory analyses for all parameters other than microscopy were undertaken for all samples, avoiding inconsistencies between data in this large dataset. In addition, we addressed our research questions minimizing confounding effects of seasonality, by sampling all lakes during the locally two warmest weeks of summer based on at least 10-year air temperature records. In this study, we investigated how the distribution of toxin concentration and toxin quota were defined by environmental parameters. We hypothesize that, in an unusually warm summer—like 2015—was in parts of Europe [42]—temperature, either through direct (surface temperature, epilimnetic temperature) or indirect (water stability expressed as maximum buoyancy frequency) effects, strongly influences the distribution of toxin concentrations and toxin quota. Additionally, we hypothesize that, under high temperature stress, the stringent selection of specific well-adapted strains of cyanobacteria reduces toxin diversity, potentially promoting dominance by a few highly toxic variants. 2. Results 2.1. Toxin Distribution on a Continental Scale In the subset of the 137 EMLS lakes used in our analysis, all seven toxins analysed were detected in samples from only three lakes, which shows that it is possible but rare to have such a diverse number of toxins present in one lake at one moment in time. The presence of four, five or six toxins was found in 34, 26 and 25 lakes, respectively. Finally, 18 lakes had two toxins and 13 lakes only one toxin. MC variants were found in 93% of the 137 EMLS lakes (Table 1). MC-YR was the most common of the five MCs (in 82% of the subset). Although the variant MC-dmRR was the rarest variant encountered, it had the highest concentrations compared to any other toxin variant (14.89 µg/L in Polish Lake Syczynskie). ´ The MC variants MC-LW, MC-LF and MC-LY, and the toxin nodularin (NOD) were

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also analysed, but they were not included in the analysis as they were too scarce (see materials and methods). The MC-LF was present in two Spanish reservoirs (Abegondo and As Forcadas), while MC-LW and MC-LY were not found in any of the EMLS lakes. Nodularin was found only in two Spanish reservoirs (As Forcadas and Valdecanas) in concentrations close to the limit of quantification (0.007 µg/L). Table 1. Summary of toxin variants (Total microcystin: MC-Tot, microcystin YR: MC-YR, microcystin dmLR: MC-dmLR, microcystin LR: MC-LR, microcystin RR: MC-RR, anatoxin: ATX, cylindrospermopsin: CYN, microcystin dmRR: MC-dmRR) ordered by decreasing number of presence in the investigated 137 EMLS lakes.

Toxin Variant

Present (n Lakes)

Concentration Range (µg/L)

Limit of Quantification 1 (µg/L)

MC-Tot MC-YR MC-dmLR MC-LR MC-RR ATX CYN MC-dmRR

127 113 108 93 67 54 53 52

0–17.18 0–4.92 0–3.16 0–3.97 0–3.31 0–1.33 0–2.01 0–14.89

0.0050 0.0054 0.0086 0.0358 0.0004 0.0004 0.0489

1

Mean

Stdv

1.20 0.14 0.15 0.20 0.20 0.03 0.05 0.52

2.70 0.56 0.50 0.55 0.50 0.12 0.20 1.83

limit of quantification (LOQ) of the LC-MS/MS method measured for an average filtered volume = 250 mL.

CYN was detected in 39% of the 137 EMLS lakes (Table 1). One German Lake (Grosser Dabelowsee), three Polish Lakes (Bninskie, ´ Lusowskie and Probarskie), and two Turkish lakes (Caycoren and Mollakoy) (Figure 1a) had solely CYN, in relatively low concentrations ( 0.05). MC-LR was the only toxin variant that showed a negative trend (red arrow) to increases in toxin diversity, although lacking statistical significance. Similarly, all toxin quota were tested against Toxin Richness. In this case, all toxin quota increased significantly with toxin richness apart from MC-dmLR and MC-LR that, although showing a positive trend, were not significant (Table 4).

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Table 4. Statistical results of the negative binomial generalized linear model, showing the response of the toxin quota (MC-YR, MC-dmLR, MC-LR, MC-RR, MC-dmRR, ATX and CYN over chlorophyll-a) to increases in Toxin Diversity Index (TDI) and Toxin Richness. Black upward arrows correspond to increases of the toxin variant to increases in the TDI and Toxin Richness, red downward arrows correspond to decreases of the toxin variant when TDI increases. Statistically significant effects are shown in bold (p < 0.05).

Toxin Quota

Response When TDI ↑

X2

p

Response When Richness ↑

X2

p

MC-YR MC-dmLR MC-LR MC-RR ATX CYN MC-dmRR

↑ ↑ ↓ ↑ ↑ *** ↑ ** ↑

0.10 0.10 0.09 0.44 0.15 0.38 0.2

0.02 0.02 0.54 0.06 0.0002 0.007 0.85

↑ ↑ ↑ ↑ ** ↑ *** ↑ *** ↑

0.10 0.06 0.3 0.90 0.19 0.56 0.41

0.01 0.06 0.2 0.003