T h e You are kindly invited n to attend the public e defence of my PhD thesis u r o t o The neurotoxin BMAA in x The neurotoxin BMAA i n in aquatic systems B aquatic systems M analysis, occurrence A and effects A i analysis, occurrence and effects n a July 8th, 2016 q 8:30 a.m. u a t i c In the Aula of s y Wageningen University s t Generaal Foulkesweg 1a e Wageningen m s Elisabeth J. Faassen [email protected] E l i s a b Paranymphs e t h J Sarian Kosten . [email protected] F Elisabeth J. Faassen a a Tineke Meijers-Faassen s s [email protected] e n THE NEUROTOXIN BMAA IN AQUatIC SYSTEMS ANALYSIS, OCCURRENCE AND EFFECTS Elisabeth J. Faassen THESIS COMMITTEE Promotor Prof. Dr M. Scheffer Professor of Aquatic Ecology and Water Quality Management Wageningen University Co-promotor Dr M.F.L.L.W. Lürling Assistant professor, Aquatic Ecology and Water Quality Management Group Wageningen University Other members Prof. Dr C. Kroeze, Wageningen University Prof. Dr E. von Elert, University of Köln, Germany Prof. Dr L. Lawton, Robert Gordon University, United Kingdom Prof. Dr O. Ploux, Paris Diderot University, France This research was conducted under the auspices of the Graduate School for Socio-Economic and Natural Sciences of the Environment (SENSE) THE NEUROTOXIN BMAA IN AQUatIC SYSTEMS ANALYSIS, OCCURRENCE AND EFFECTS Elisabeth J. Faassen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday July 8th, 2016 at 8.30 a.m. in the Aula. Elisabeth J. Faassen The neurotoxin BMAA in aquatic systems. Analysis, occurrence and effects 196 pages. PhD thesis Wageningen University, Wageningen, NL (2016) With references, with summary in English ISBN 978-94-6257-785-5 DOI 10.18174/378695 “Forget injuries, never forget kindnesses” Confucius TABLE OF CONTENTS 1 General introduction 9 2 Presence of BMAA in Dutch urban waters 25 3 Comparing three analytical methods for BMAA quantification 37 4 Evaluation of a commercially available ELISA for BMAA determination 55 5 Presence of BMAA in aquatic systems: what do we really know? 71 6 Trans generational effects of BMAA on Daphnia magna 103 7 A collaborative evaluation of LC-MS/MS based methods for BMAA analysis 125 8 Standard operating procedure: Extraction and LC-MS/MS analysis of 149 underivatised BMAA 9 Synthesis 159 Summary 169 Cited literature 171 Acknowledgements 187 About the author 189 Sense certificate 193 “Nobody said it was easy No one ever said it would be so hard I’m going back to the start” Coldplay, The Scientist CHAPTER 1 GENERAL INTRODUCTION 1.1 EUTROPHICATION AND PHYTOPLANKTON BLOOMS A B Aquatic systems are the habitat of many species and provide important ecosystem services, 30 m such as provision of drinking water and food, and coast protection [1, 2]. However, in Planktothrix rubescens many regions in the world, aquatic systems suffer heavily from increasing human pressure [3] and as a consequence, ecosystem functioning and biodiversity have decreased [4, Karlodinium 5]. Eutrophication, the pollution with nutrients, is one of the drivers of aquatic system veneficum degradation and is regarded as one of the most important water quality issues in both Raphidophyte freshwater and marine systems [6, 7]. Woronichinia naegeliana Eutrophication changes food webs, as primary producers, and mostly phytoplankton species, benefit from the high level of available nutrients. Zooplankton can control excessive phytoplankton growth by grazing and by doing so, they transfer nutrients and energy to higher trophic levels [8]. However, when zooplankton grazing is reduced, for instance because zooplankton is intensely preyed upon by fish [8], or because of the phytoplankton’s toxicity, hard to handle morphology or poor nutritional value [9], phytoplankton can proliferate. In this situation, the trophic coupling between phyto- and zooplankton is distorted [10], and phytoplankton may reach very high densities. In freshwater systems, such phytoplankton blooms mainly consist of cyanobacteria, which can accumulate at water surfaces and lee- side shores in thick scums [11], while in marine systems, diatoms and dinoflagellates are the main blooming species (Figure 1.1). Excessive phytoplankton growth can reduce water transparency, reduce macrophyte growth and may cause anoxia and fish kills at night or upon decay [6, 7, 12]. Moreover, as will be explained in the next paragraph, phytoplankton blooms can be dangerous to humans, pets and wildlife because some phytoplankton species can produce potent toxins. Blooms of toxic species and blooms that have other detrimental effects on aquatic systems or ecosystem services are therefore often referred to as harmful (algal or cyanobacterial) blooms [6, 13, 14]. 1.2 PHYCOTOXINS One of our main concerns with phytoplankton blooms, as outlined above, is that they can be toxic. A variety of marine and freshwater phytoplankton species are capable of producing so called phycotoxins. In marine systems, phycotoxins are mainly produced by dinoflagellates and diatoms whereas in freshwater ecosystems, most toxins are produced by cyanobacteria [13]. Phycotoxins differ greatly in their structure, mode of action and level of toxicity. For instance, some toxins are protein phosphatase inhibitors (microcystins [15], okadaic acid [16]), while others block sodium channels (saxitoxins [17]), inhibit acetyl-choline esterase (anatoxin-a(s) [18]) or bind to glutamate receptors (domoic acid [19]). Main targets of phycotoxins are the nervous system and organs like liver, kidney and skin [11, 19, 20] and some toxins are tumour promotors [20, 21]. The effects of algal toxin exposure can range 10 GENERAL INTRODUCTION A B 30 m Planktothrix rubescens 1 Karlodinium veneficum Raphidophyte Woronichinia naegeliana Figure 1.1. Two freshwater cyanobacteria (A, Planktothrix rubescens and Woronichinia naegeliana) and two marine blooming phytoplankton species, a raphidophyte and the dinoflagellate Karlodinium veneficum (B, preserved in Lugol’s solution). Credit: Miquel Lürling (A) and Nathan S. Hall (B). from mild - like nausea and local numbness - to severe, such as respiratory difficulties, paralysis and death [19, 21-23]. Many phycotoxins can be transferred from phytoplankton to higher trophic levels such as shellfish, zooplankton and fish. Humans are therefore not only exposed to phycotoxins through direct contact with toxic algal blooms, but ingestion of contaminated food can be a major exposure route as well [13, 24]. Yearly, phycotoxins are responsible for over 60.000 human intoxications worldwide [25]. Moreover, phycotoxins can kill wildlife (e.g. [26, 27]) and pets (e.g. [28, 29]). Phytoplankton blooms also have a negative economic impact: in Europe, marine blooms alone are estimated to yearly cause a 813 M€ economic loss, mainly to recreation and tourism (637 M€) and commercial fisheries (147 M€) [30]. Although hundreds of algal toxins have been identified already, new compounds are still being discovered. One of the compounds that have recently been added to the list of known phycotoxins is the non-proteinogenic, neurotoxic amino acid β-N-methylamino-L-alanine (BMAA, Figure 1.2). BMAA was discovered in 1967 in a terrestrial ecosystem, in the seeds of the cycad Cycas micronesica on the island of Guam [31]. In 2003, BMAA was found in the cyanobacterium Nostoc living in symbiosis with this cycad [32]. This first report of BMAA in a cyanobacterium was followed by positive reports for BMAA in free living and symbiotic cyanobacteria, as well as in cyanobacteria dominated field samples [33-36]. Given the putative role of BMAA in neurodegenerative illnesses [37], these findings of widespread occurrence of BMAA in virtually all tested cyanobacteria at sometimes alarming high concentrations led to the assumption that BMAA may pose a worldwide significant risk to human health [34, 38, 39]. GENERAL INTRODUCTION 11 Figure 1.2. Chemical structure of BMAA. 1.3 THE ROLE OF BMAA IN THE AETIOLOGY OF NEURODEGENERATIVE DISEASES BMAA research started on Guam [31], in search for a cause of the high incidence of amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC), a combination of amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD), among the indigenous Chamorro people. ALS-PDC incidence among the Chamorro people was about 100 times higher than in the continental United States [40], and was related to the Chamorro traditional diet, of which cycad seeds were a main constituent [41]. BMAA was found in dietary items such as cycad flour that was prepared from washed seeds, and flying foxes, a bat species that foraged on cycad seeds and that was regarded as a delicacy by the Chamorro people [42, 43]. In subsequent studies, BMAA was shown to be neurotoxic (e.g. [31, 44]), and BMAA exposure is at present regarded as one of the possible causes of ALS/PDC on Guam [45]. BMAA research expanded beyond Guam when its presence was reported in free living cyanobacteria originating from all over the world [33]. This finding implied that human exposure to BMAA could occur globally, and not only in the few ALS/PDC hotspots in the western Pacific, where the use of cycads was integrated in the traditional way of living [46-48]. BMAA exposure was now suggested to (also) play a role in the globally occurring neurodegenerative diseases AD, PD and ALS [37]. AD, PD and ALS are fatal, age-related, progressive neurodegenerative diseases. AD and PD have a high incidence: in the United States, 4.5 million people were suffering from AD in 2005, and AD incidence is expected to increase in the US to 11-16 million cases in 2050 [49]. Approximately one million Americans were affected by PD in 2005, and this number is expected to increase to 4 million in 2040 [49].