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Investigations on the Impact of Toxic Cyanobacteria on Fish : As INVESTIGATIONS ON THE IMPACT OF TOXIC CYANOBACTERIA ON FISH - AS EXEMPLIFIED BY THE COREGONIDS IN LAKE AMMERSEE - DISSERTATION Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften an der Universität Konstanz Fachbereich Biologie Vorgelegt von BERNHARD ERNST Tag der mündlichen Prüfung: 05. Nov. 2008 Referent: Prof. Dr. Daniel Dietrich Referent: Prof. Dr. Karl-Otto Rothhaupt Referent: Prof. Dr. Alexander Bürkle 2 »Erst seit gestern und nur für einen Tag auf diesem Planeten weilend, können wir nur hoffen, einen Blick auf das Wissen zu erhaschen, das wir vermutlich nie erlangen werden« Horace-Bénédict de Saussure (1740-1799) Pionier der modernen Alpenforschung & Wegbereiter des Alpinismus 3 ZUSAMMENFASSUNG Giftige Cyanobakterien beeinträchtigen Organismen verschiedenster Entwicklungsstufen und trophischer Ebenen. Besonders bedroht sind aquatische Organismen, weil sie von Cyanobakterien sehr vielfältig beeinflussbar sind und ihnen zudem oft nur sehr begrenzt ausweichen können. Zu den toxinreichsten Cyanobakterien gehören Arten der Gattung Planktothrix. Hierzu zählt auch die Burgunderblutalge Planktothrix rubescens, eine Cyanobakterienart die über die letzten Jahrzehnte im Besonderen in den Seen der Voralpenregionen zunehmend an Bedeutung gewonnen hat. An einigen dieser Voralpenseen treten seit dem Erstarken von P. rubescens existenzielle, fischereiwirtschaftliche Probleme auf, die wesentlich auf markante Wachstumseinbrüche bei den Coregonenbeständen (Coregonus sp.; i.e. Renken, Felchen, etc.) zurückzuführen sind. So auch am Ammersee, wo die beschriebenen Wachstumseinbrüche vermeintlich sogar regelmäßig zum vorzeitigen Verenden bestimmter Coregonenjahrgänge führen. Interessanterweise hatten die Coregonen im Ammersee wiederholt einen außergewöhnlich blau gefärbten Darminhalt. Diese auffällige Färbung wird vermutlich von cyanobakteriellen Farbpigmenten verursacht und deutet darauf hin, dass die Coregonen im Ammersee mit Cyanobakterien in Kontakt kommen. Es scheint daher grundsätzlich nicht abwegig, dass die Schwierigkeiten der Ammersee-Coregonen in kausalem Zusammenhang zum Auftreten giftiger P. rubescens Filamente stehen könnten. Ziel des Dissertationsprojektes war es daher, • das Aufkommen, die Verteilung und die Toxizität von P. rubescens im Ammersee über einen aussagekräftigen Zeitraum detailliert zu erfassen, • in an die natürlichen Verhältnisse angelehnten Laborexperimente zu untersuchen, ob die vorgefundenen P. rubescens-Dichten Coregonenpopulationen beeinträchtigen und gesundheitlich schädigen können und schließlich, • zu prüfen, ob es Hinweise auf P. rubescens Expositionen und dadurch verursachte Schädigungen wildlebender Coregonen im Ammersee gibt. Zur Bestimmung der Planktothrix-Dichte im Ammersee war es zunächst notwendig, ein Verfahren zu etablieren, bei dem die in Wasserproben enthaltenen Planktothrix-Filamente auf Filtern mittels Fluoreszenzmikroskopie und digitaler Bildverarbeitung einfach, schnell und akkurat quantifiziert werden können. Dieses Verfahren ermöglichte eine aufwendige Beprobung und damit eine aussagekräftige Beschreibung des zeitlichen und räumlichen Verteilungsmusters von P. rubescens im See. Bei den von April 1999 bis September 2004 durchgeführten Beprobungen zeigte sich dann, dass P. rubescens-Filamente im Ammersee – wenn auch in unterschiedlicher Dichte – durchgehend vorhanden waren. Der Bereich maximaler Zelldichten korrelierte jeweils von Mai bis Oktober 4 ZUSAMMENFASSUNG _________________________________________________________________________________________________________________ signifikant mit der unteren Grenze der euphotischen Tiefe und dem Beginn des Metalimnion. P. rubescens erreichte regelmäßig im Sommer maximale Dichten (zeitweilig bis zu 75.000 Zellen/ml). Darüber hinaus konnte P. rubescens auch während der winterlichen Vollzirkulation über den Wasserkörper verteilt in Zelldichten von bis zu 15.000 Zellen/ml nachgewiesen werden. In welchem Ausmaß sich P. rubescens entwickeln kann, scheint wesentlich von der Illumination des Metalimnion und damit von der Transparenz des Wassers abzuhängen. Zudem scheint P. rubescens auch von regelmäßigen Phosphat-Auszehrung und den hohen Stickstoffkonzentrationen in dem re-oligotrophierten See zu profitieren. In 27 bzw. 38 von 54 Planktonproben aus verschiedenen Monaten konnten mittels HPLC und ELISA Toxinanalyse diverse fischgiftige Microcystine (in erster Linie Microcystin-RR Varianten) nachgewiesen werden. Nahezu konstante Microcystin/Phycoerythrin Verhältnisse verdeutlichten, dass die von P. rubescens produzierten Microcystinmengen weitgehend unveränderlich sind. Dies bedeutet, dass bei einem Aufkommen von P. rubescens auch von einem Auftreten messbarer Microcystin-Belastungen auszugehen ist. Die Auswirkungen von P. rubescens auf Coregonen wurden in Laborexperimenten untersucht, wobei die im Ammersee erfassten P. rubescens Dichten und verschiedene Expositionsformen berücksichtigt wurden. Vorversuche verdeutlichten, dass aufgrund einer kovalenten Bindung des Microcystins im Gewebe eine aussagekräftige Quantifizierung geringer Microcystinmengen in Fischgeweben generell schwierig und via ELISA-, HPLC- und PPAssay-Toxinanalytik unmöglich ist. Microcystine konnten im Fischgewebe aber qualitativ, mittels immunhistochemischer Anfärbung mit anti-Microcystin Antikörpern lokalisiert werden. Die exponierten Renken reagierten mit auffälligem Verhalten, einer gesteigerten Atemfrequenz und wiesen erhöhte Serumglukose-Konzentrationen auf. Merkmale, die in ihrer Gesamtheit als eindeutige Stressindikatoren zu bewerten sind. Pathologische Auffälligkeiten in der Leber, Niere und im Gastrointestinaltrakt verdeutlichen zudem beträchtliche Organschäden die auf nachhaltige Auswirkungen auf Organfunktionen schließen lassen. Die Tatsache, dass die geschädigten Gewebebereiche gemäß immunhistologischer Anfärbung vielfach auch Microcystin enthielten, veranschaulicht einen kausalen Zusammenhang von Gewebeschäden und der offensichtlichen Aufnahme von Microcystin. Eine erhöhte Empfindlichkeit gegenüber Ektoparasiten und eine erhöhte Mortalitätsrate deuten weiter daraufhin, dass durch die Wirkung von P. rubescens letztendlich auch die Kondition der experimentell exponierten Coregonen beeinträchtigt wurde. Insgesamt betrachtet war die Symptomatik der Auswirkungen in den verschiedenen Expositionsansätzen vergleichbar – die Intensität der Wirkung war hingegen dosisabhängig. Dies verdeutlicht, dass sich mit zunehmender P. rubescens-Zelldichte entsprechende Effekte früher und deutlicher ausprägen. Nichtsdestoweniger zeigte sich auch bei vergleichsweise geringer P. rubescens Dichten (≈1500 Zellen/ml) eine fischgiftige Wirkung. 5 ZUSAMMENFASSUNG _________________________________________________________________________________________________________________ P. rubescens Zelldichten von mindestens 1500 Zellen/ml waren im Ammersee in etwa zur Hälfte der 261 Wochen andauernden Beobachtungsperiode festzustellen, was verdeutlicht, dass die Coregonen im Ammersee tatsächlich regelmäßig mit schädlichen P. rubescens Zelldichten konfrontiert sind. Dies scheint vor allem dann problematisch, wenn die Coregonenpopulation einer P. rubescens-Exposition nicht aktiv ausweichen kann (z.B. bei P. rubescens Entwicklungen die den gesamten Wasserkörper umfassen). In der Tat, auch im See selbst ergaben sich Anhaltspunkte für eine P. rubescens Exposition der Coregonen. So konnte gezeigt werden, dass die Fische regelmäßig P. rubescens Filamente inkorporieren und verdauen. Dadurch werden im Darm der Fische die in den Filamenten enthaltenen Metabolite freigesetzt. Die Freisetzung von Phycocyanin bewirkt die auffällige Blaufärbung des Darminhalts und die Freisetzung der Microcystine verursacht eine Microcystin- Exposition der Coregonen. Da Microcystin stichprobenartig zudem auch in Leberhomogenaten von Ammersee Coregonen nachzuweisen war, ist wahrscheinlich, dass das im Darm freigesetzte Microcystin über das Darmepithel in den Organismus gelangen und sich entsprechend nachhaltig auf den Gesundheitszustand und die physiologische Kondition der Coregonen auswirken kann. Man kann daher davon auszugehen, dass die in den Laborexperimenten aufgezeigten Microcystin-Schäden auch in den Fischen im See auftreten und sich dauerhafte P. rubescens Vorkommen entsprechend substanziell auf die Coregonen auswirken. Hinzu kommt, dass neben den experimentell aufgezeigten unmittelbaren Schädigungen weiter auch indirekte Einflüsse (z.B. P. rubescens bedingte Veränderungen in der Umwelt der Coregonen) eine entscheidende Rolle spielen können. Insgesamt betrachtet unterstützen die bisherigen Erkenntnisse damit die Vermutung, dass das anhaltende Aufkommen von P. rubescens eine wesentliche Ursache für den Wachstumseinbruch und die schlechte physiologische Kondition der Coregonen in Voralpenseen wie dem Ammersee ist. 6 SUMMARY Toxic cyanobacteria affect organisms of almost all stages of development and trophic levels. Especially threatened are aquatic organisms such as fish, as they can be affected by toxic cyanobacteria via multiple routes, and their options for exposure avoidance in waters containing toxic cyanobacteria are limited. Among the most toxic cyanobacteria are species of the genus Planktothrix, including Planktothrix rubescens. During the last decades, P. rubescens has become one of the predominant species of the phytoplankton community in several lakes in the pre-alpine regions. In some of those lakes (e.g. Lake Ammersee) the rise of P. rubescens has been observed to coincide with pronounced slumps
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