Eutrophication is a major water quality issue and in many aquatic systems, it leads to the proliferation of toxic phytoplankton species. The neurotoxin β-N-methylamino-L-alanine (BMAA) is one of the compounds that can be present in phytoplankton. BMAA has been suggested to play a role in the neurodegenerative diseases Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis, although this hypothesis still needs to be confirmed. It is expected that the main human exposure pathways to BMAA are through direct contact with BMAA containing phytoplankton and through ingestion of BMAA contaminated food, such as fish and shellfish. However, reports on the occurrence of BMAA in aquatic systems have been conflicting and the cause of these reported differences was heavily debated. The use of different analytical methods seems to play a crucial role in the observed discrepancies, but initially, there was little consensus on which method produced most reliable results. The objectives of the work presented in this thesis therefore were to find out what has caused the differences in published results on BMAA concentrations, and to identify and produce reliable data on the presence of BMAA in aquatic systems. In addition, I aimed to determine the effect of BMAA exposure on a key species in many freshwater ecosystems, the grazer Daphnia magna.
The performances of different analytical techniques were compared, and LC-MS/MS analysis, either preceded by derivatisation or not, was found to produce most reliable results. LC-FLD and ELISA should not be used for BMAA analysis, as both methods risk misidentifying BMAA or overestimating its concentrations due to their low selectivity. When reviewing literature on the presence of BMAA in aquatic systems, it was found that the observed discrepancies in results could be explained by the use of unselective analytical methods in some studies, and by severe reporting deficiencies in others. When only studies that used appropriate analytical techniques and that correctly reported their work were taken into account, it was shown that BMAA could be present in phytoplankton and higher aquatic organisms, in concentrations of µg/g dry weight or lower. These results are in agreement with our findings of BMAA in cyanobacterial scums from Dutch urban waters. In a 2008 screening, BMAA was found to be present in 9 out of 21 analysed cyanobacterial scums, at concentrations ranging from 4 to 42 µg/g dry weight. When this screening was repeated 8 years later with 52 similar samples, BMAA was detected below the quantification limit in one sample and quantified in another sample at 0.6 µg/g dry weight.
In order to perform the work presented in this thesis, sensitive and selective analytical methods, mostly based on LC-MS/MS analysis without derivatisation, were developed. This resulted in a standard operating procedure for the underivatised LC-MS/MS analysis of BMAA in cyanobacteria. Also, a CYANOCOST initiated workshop was given, in which a group of scientists from 17 independent laboratories evaluated LC-MS/MS based methods in different matrices. A bound BMAA from found in the supernatant was the most abundant fraction in the positive samples that were tested: cycad seed, seafood and exposed D. magna. In addition, it was found that the deuterated internal standard used for quantification was not a good indicator for the release of BMAA from bound forms, resulting in unprecise quantification of total BMAA.
BMAA was found to reduce survival, somatic growth, reproduction and population growth in D. magna. Animals did not adapt to BMAA exposure: exposed animals born from exposed mothers had a lower brood viability and neonate weight than animals exposed to BMAA, but born from unexposed mothers. In addition, D. magna was shown to take up BMAA from the growth medium and to transfer it to its offspring. D. magna therefore might be an important vector for BMAA transfer along the pelagic food chain, but whether BMAA plays a role in preventing zooplankton from controlling cyanobacterial blooms needs further investigation.
Although BMAA research has much progressed between the start of this thesis’ work and its completion, some important questions still require an answer. Most urgently, it should be determined whether BMAA is indeed involved in the neurological diseases mentioned above, and if so, which doses trigger the onset of these diseases. Human exposure pathways should then be more systematically quantified, and it might be prudent to investigate if the occurrence of BMAA is restricted to aquatic systems, or whether sources from terrestrial systems contribute to BMAA exposure as well.