Elisabeth J. Faassen

Recovery rates reflect distance to a tipping point in a living system

Tipping points, at which complex systems can shift abruptly from one state to another, are notoriously difficult to predict. Theory proposes that early warning signals may be based on the phenomenon that recovery rates from small perturbations should tend to zero when approaching a tipping point; however, evidence that this happens in living systems is lacking. Here we test such ‘critical slowing down’ using a microcosm in which photo-inhibition drives a cyanobacterial population to a classical tipping point when a critical light level is exceeded. We show that over a large range of conditions, recovery from small perturbations becomes slower as the system comes closer to the critical point. In addition, autocorrelation in the subtle fluctuations of the system’s state rose towards the tipping point, supporting the idea that this metric can be used as an indirect indicator of slowing down. Although stochasticity prohibits prediction of the timing of critical transitions, our results suggest that indicators of slowing down may be used to rank complex systems on a broad scale from resilient to fragile.

Determination of the neurotoxins BMAA (ß-N-methylamino-L-alanine) and DAB (a-,¿-diaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms

Abstract We aimed to determine concentrations of the neurotoxic amino acids β-N-methylamino-L-alanine (BMAA) and α-,γ-diaminobutyric acid (DAB) in mixed species scum material from Dutch urban waters that suffer from cyanobacterial blooms. BMAA and DAB were analysed in scum material without derivatization by LC-MSMS (liquid chromatography coupled to tandem mass spectrometry) using hydrophilic interaction chromatography (HILIC). Our method showed high selectivity, good recovery of added compounds after sample extraction (86% for BMAA and 85% for DAB), acceptable recovery after sample hydrolysation (70% for BMAA and 56% for DAB) and acceptable precision. BMAA and DAB could be detected at an injected amount of 0.34 pmol. Free BMAA was detected in nine of the 21 sampled locations with a maximum concentration of 42 μg/g DW. Free DAB was detected in two locations with a maximum concentration of 4 μg/g DW. No protein-associated forms were detected. This study is the first to detect underivatized BMAA in cyanobacterial scum material using LC-MSMS. Ubiquity of BMAA in cyanobacteria scums of Dutch urban waters could not be confirmed, where BMAA and DAB concentrations were relatively low; however, co-occurrence with other cyanobacterial neurotoxins might pose a serious health risk including chronic effects from low-level doses.

A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria.

The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) has been considered a serious health threat because of its putative role in multiple neurodegenerative diseases. First reports on BMAA concentrations in cyanobacteria were alarming: nearly all cyanobacteria were assumed to contain high BMAA concentrations, implying ubiquitous exposure. Recent studies however question this presence of high BMAA concentrations in cyanobacteria. To assess the real risk of BMAA to human health, this discrepancy must be resolved. We therefore tested whether the differences found could be caused by the analytical methods used in different studies. Eight cyanobacterial samples and two control samples were analyzed by three commonly used methods: HPLC-FLD analysis and LC-MS/MS analysis of both derivatized and underivatized samples. In line with published results, HPLC-FLD detected relatively high BMAA concentrations in some cyanobacterial samples, while both LC-MS/MS methods only detected BMAA in the positive control (cycad seed sarcotesta). Because we could eliminate the use of different samples and treatments as causal factors, we demonstrate that the observed differences were caused by the analytical methods. We conclude that HPLC-FLD overestimated BMAA concentrations in some cyanobacterial samples due to its low selectivity and propose that BMAA might be present in (some) cyanobacteria, but in the low µg/g or ng/g range instead of the high µg/g range as sometimes reported before. We therefore recommend to use only selective and sensitive analytical methods like LC-MS/MS for BMAA analysis. Although possibly present in low concentrations in cyanobacteria, BMAA can still form a health risk. Recent evidence on BMAA accumulation in aquatic food chains suggests human exposure through consumption of fish and shellfish which expectedly exceeds exposure through cyanobacteria.

Presence of the Neurotoxin BMAA in Aquatic Ecosystems: What Do We Really Know?

The neurotoxin β-N-methylamino-l-alanine (BMAA) is suspected to play a role in the neurological diseases amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. BMAA production by cyanobacteria has been reported and contact with cyanobacteria infested waters or consumption of aquatic organisms are possible pathways to human exposure. However, there is little consensus regarding whether BMAA is present in cyanobacteria or not, and if so, at what concentrations. The aim of this review is to indicate the current state of knowledge on the presence of BMAA in aquatic ecosystems. Some studies have convincingly shown that BMAA can be present in aquatic samples at the µg/g dry weight level, which is around the detection limit of some equally credible studies in which no BMAA was detected. However, for the majority of the reviewed articles, it was unclear whether BMAA was correctly identified, either because inadequate analytical methods were used, or because poor reporting of analyses made it impossible to verify the results. Poor analysis, reporting and prolific errors have shaken the foundations of BMAA research. First steps towards estimation of human BMAA exposure are to develop and use selective, inter-laboratory validated methods and to correctly report the analytical work.

Controlling toxic cyanobacteria: Effects of dredging and phosphorus-binding clay on cyanobacteria and microcystins

Sediment dredging and Phoslock(®) addition were applied individually and in combination in an enclosure experiment in a Dutch hypertrophic urban pond. These measures were applied to control eutrophication and reduce the risk of exposure to cyanobacterial toxins. Over the 58 days course of the experiment, cyanobacteria (predominantly Microcystis aeruginosa) gradually decreased until they dropped below the level of detection in the combined treated enclosures, they were reduced in dredged enclosures, but remained flourishing in controls and Phoslock(®) treated enclosures. Cyanobacteria were, however, less abundant in the enclosures (medians chlorophyll-a 30-87 μg l(-1)) than in the pond (median chlorophyll-a 162 μg l(-1)), where also a thick surface scum covered one-third of the pond for many weeks. Soluble reactive phosphorus (SRP), total phosphorus and total nitrogen concentrations were significantly lower in the combined dredged and Phoslock(®) treated enclosures than in controls. Median SRP concentrations were 24 μg P l(-1) in the combined treatment, 58 μg P l(-1) in dredged enclosures, and 90 μg P l(-1) in controls and 95 μg P l(-1) in Phoslock(®) treated enclosures. Hence, the combined treatment was most effective in decreasing SRP and TP, and in lowering cyanobacterial biomass. Microcystin (MC) concentrations were analyzed by LC-MS/MS. MC concentrations and cyanobacterial biomass were positively correlated in all treatments. Mean MC concentrations in controls (71 μg l(-1)), Phoslock(®) treated enclosures (37 μg l(-1)) and dredged enclosures (25 μg l(-1)) exceeded the provisional guideline of 20 μg l(-1), whereas mean MC concentrations were 13 μg l(-1) in the combined treated enclosures. All samples contained the MC variants dmMC-RR, MC-RR, MC-YR, dmMC-LR and MC-LR; traces of MC-LY and nodularin were detected in few samples. The different treatments did not change the relative contribution of the variants to the MC pool; MC profiles in all treatments and the pond showed dominance of MC-RR followed by MC-LR. In the surface scum of the pond, total MC concentration was extremely high (64000 μg l(-1) or 1300 μg g(-1) DW), which poses a serious health hazard to children playing on the banks of the pond. Based on our results and pond characteristics, we propose combined sediment dredging and Phoslock(®) addition, fish removal and strong reduction of duck feeding by the neighborhood as most promising measures controlling cyanobacterial hazards in this pond.

Dog Poisonings Associated with a Microcystis aeruginosa Bloom in the Netherlands

In early autumn 2011, three dogs died after they had been exposed to a Microcystis aeruginosa bloom on Lake Amstelmeer, The Netherlands. The cyanobacterial scum from the lake contained up to 5.27 × 103μg g−1 dry-weight microcystin, the vomit of one of the dogs contained on average 94 µg microcystin g−1 dry-weight. In both cases, microcystin-LR was the most abundant variant. This is the first report of dog deaths associated with a Microcystis bloom and microcystin poisoning in The Netherlands.

Occurrence of the Microcystins MC-LW and MC-LF in Dutch Surface Waters and Their Contribution to Total Microcystin Toxicity

Microcystins (MCs) are the most frequently found cyanobacterial toxins in freshwater systems. Many MC variants have been identified and variants differ in their toxicity. Recent studies showed that the variants MC-LW and MC-LF might be more toxic than MC-LR, the variant that is most abundant and mostly used for risk assessments. As little is known about the presence of these two variants in The Netherlands, we determined their occurrence by analyzing 88 water samples and 10 scum samples for eight MC variants ((dm-7-)MC-RR, MC-YR, (dm-7-)MC-LR, MC-LY, MC-LW and MC-LF) by liquid chromatography with tandem mass spectrometry detection. All analyzed MC variants were detected, and MC-LW and/or MC-LF were present in 32% of the MC containing water samples. When MC-LW and MC-LF were present, they contributed to nearly 10% of the total MC concentrations, but due to their suspected high toxicity, their average contribution to the total MC toxicity was estimated to be at least 45%. Given the frequent occurrence and possible high toxicity of MC-LW and MC-LF, it seems better to base health risk assessments on the toxicity contributions of different MC variants than on MC-LR concentrations alone.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Effects of hydrogen peroxide and ultrasound on biomass reduction and toxin release in the cyanobacterium, Microcystis aeruginosa.

Cyanobacterial blooms are expected to increase, and the toxins they produce threaten human health and impair ecosystem services. The reduction of the nutrient load of surface waters is the preferred way to prevent these blooms; however, this is not always feasible. Quick curative measures are therefore preferred in some cases. Two of these proposed measures, peroxide and ultrasound, were tested for their efficiency in reducing cyanobacterial biomass and potential release of cyanotoxins. Hereto, laboratory assays with a microcystin (MC)-producing cyanobacterium (Microcystis aeruginosa) were conducted. Peroxide effectively reduced M. aeruginosa biomass when dosed at 4 or 8 mg L−1, but not at 1 and 2 mg L−1. Peroxide dosed at 4 or 8 mg L−1 lowered total MC concentrations by 23%, yet led to a significant release of MCs into the water. Dissolved MC concentrations were nine-times (4 mg L−1) and 12-times (8 mg L−1 H2O2) higher than in the control. Cell lysis moreover increased the proportion of the dissolved hydrophobic variants, MC-LW and MC-LF (where L = Leucine, W = tryptophan, F = phenylalanine). Ultrasound treatment with commercial transducers sold for clearing ponds and lakes only caused minimal growth inhibition and some release of MCs into the water. Commercial ultrasound transducers are therefore ineffective at controlling cyanobacteria.

Evaluation of a commercial enzyme linked immunosorbent assay (ELISA) for the determination of the neurotoxin BMAA in surface waters.

The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA.