Methods for simultaneous detection of the cyanotoxins BMAA, DABA, and anatoxin-a in environmental samples

Highlights

• Methods for simultaneous detection of cyanotoxins BMAA, DABA, and anatoxin-a.

• Detection limits for open water samples ranged from 5 to 7 μg/L using HPLC/FD.

• Detection limits for biological (fish and plant) samples ranged from 0.8 to 3.2 μg/L.

• This method can also be used to distinguish free versus bound forms.

Abstract

Blue-green algae, also known as cyanobacteria, can produce several different groups of toxins in the environment including hepatotoxins (microcystins), neurotoxic non-protein amino acids β-methylamino-l-alanine (BMAA), and 2,4-diaminobutyric (DABA), as well as the bicyclic amine alkaloid anatoxin-a. Few studies have addressed the methods necessary for an accurate determination of cyanotoxins in environmental samples, and none have been published that can detect these cyanotoxins together in a single sample. Cyanotoxins occur in a wide range of environmental samples including water, fish, and aquatic plant samples. Using polymeric cation exchange solid phase extraction (SPE) coupled with liquid chromatography and fluorescence detection (HPLC/FD), and liquid chromatography ion trap tandem mass spectrometry (LC/MS/MS), these compounds can for the first time be simultaneously quantified in a variety of environmental sample types. The extraction method for biological samples can distinguish bound and free cyanotoxins. Detection limits for water ranged from 5 to 7 μg/L using HPLC/FD, while detection limits for and LC/MS were in the range of 0.8–3.2 μg/L.

Introduction

Cyanobacteria are a diverse and ubiquitous group of prokaryotic organisms capable of photosynthesis as well as heterotrophic pathways of respiration. Many aquatic cyanobacteria have been shown to produce chemicals that are potentially toxic to humans and other organisms in marine and freshwater environments (Huisman et al., 2005). Commonly identified freshwater cyanotoxins that impact the nervous system thus far include: anatoxin-a and saxitoxins. Saxitoxins have been found in a limited number of freshwater cyanobacteria (5 genera) (Chorus and Bartram, 1999), and will not be discussed further here. Each of these neurotoxins has a different mode of action. Anatoxin-a is a low molecular weight alkaloid with acute neurotoxicity, that is produced by at least four genera of cyanobacteria worldwide (Wood et al., 2007). Anatoxin-a's mode of action is binding with the nicotinic acetylcholine receptor, where it acts as an analogue of acetylcholine. The molecule is not degraded by cholinesterase, thus causing permanent stimulation of muscle cells leading to paralysis and respiratory arrest.

BMAA (β-methylamino-l-alanine) and DABA (2,4-diaminobutyric acid) are non-protein amino acids that are found in both marine and freshwaters (Banack et al., 2010). BMAA is produced by almost all cyanobacteria; including the common genera Anabaena and Nostoc, which also live symbiotically in plant roots (Fogg et al., 1973, Cox et al., 2005). DABA is also ubiquitous in nature, which is not surprising considering that it is a metabolite of BMAA.

BMAA is considered an “excitotoxin” and its suggested mode of action is killing neurons, thus long-term exposure is thought to lead to human neurodegenerative diseases like Parkinson's, Alzheimer's, and ALS (Lou Gehrig's Disease) (Ince and Codd, 2005, Rao et al., 2006, Lobner et al., 2007, Liu et al., 2009).

Many algae have formed symbiotic relationships with other organisms; in which algae provide organic material such as nitrogenous compounds and energy via photosynthesis to the host, while the host provides physical protection and carbon. Cyanobacterial symbionts are found in higher plants like Gunnera (Fogg et al., 1973), as well as lichens, coral reefs, and sea sponges (Brodo et al., 2001, Taylor, 1983). Cyanobacteria seem to have developed symbiotic relationships more often than other algal groups and can also be found with protozoa such as Cyanophora paradoxa (Fogg et al., 1973, Keeling, 2004). Bergman et al. (1996) showed that nitrogen-fixing Nostoc symbionts are known to occur with fungi, bryophytes, and aquatic ferns such as Azolla, gymnosperms such as cycads, and in angiosperms like Gunnera. Potential effects of exposure, Lindholm et al. (1999) reported extensive fish mortality in a brackish-water lake in Finland potentially due to exposure from multiple algal toxins. Shumway (1990) also reported fish and shellfish death related to algal toxins. Williams et al. (2007) reported on the occurrence of algal toxins in freshwater systems in Florida. Their data showed the presence of microcystin (0.1–3.6 μg/L) in all 12 months of the years, and anatoxin-a was identified in 2002–03 (30%, 0.05–7.0 μg/L), but not in 2003–04. Vega et al. (1968) and Polsky et al. (1972) were the first to show BMAA neurotoxicity. BMAA was extracted from cycad seeds, which were traditionally used by the Chamorro people in Guam as food and rarely as a medicinal plant. Also Murch et al. (2004a) suggested that BMAA can be incorporated into plant and animal proteins (Karamyan and Speth, 2008).

Cox et al. (2005) showed that the cyanotoxin BMAA may be produced by all known groups of cyanobacteria in both symbiotic and free-living forms. Their research showed that BMAA was present in cycads with Nostoc as the symbiont, and in some flowering aquatic plants such as Gunnera (Cox et al., 2003). BMAA was also shown to occur in other free-living cyanobacteria such as Synechocystis and Anabaena, often found in freshwaters in the U.S. Cox et al. (2003) reported that free-living cyanobacteria can produce 0.3 μg/g of free BMAA and 2–37 μg/g as symbionts in the coralloid roots of cycad trees. The BMAA tended to be concentrated in developing reproductive tissue of cycads (Banack and Cox, 2003).

Human exposure to BMAA, documented through consumption of seeds or flour, has been thought to be associated with ALS/Parkinsonism–Dementia Complex (PDC), though widespread linkages have yet to be demonstrated. BMAA was detected in the brain tissue of nine Canadian Alzheimer's (AD) patients, suggesting that BMAA may occur in different sources, and that there are perhaps alternative ecological pathways for bioaccumulation of BMAA in aquatic or terrestrial ecosystems (Cox et al., 2005, Murch et al., 2004a, Murch et al., 2004b). Murch et al. (2004b) explained that the mechanism of slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam, by suggesting that the BMAA neurotoxin produced by Nostoc can be concentrated within the protein of cycads seeds. Grinding the seeds for flour used for their daily diet, BMAA was concentrated over time in brain tissue, resulting in the death to Chamorro people due to ALS and/or PDC. Pablo et al. (2009) looked for a relationship between BMAA occurrences in patients with different neurological diseases in North America including Alzheimer's disease (AD), ALS, and Huntington's disease (HD). The data showed that BMAA occurred in ALS and AD patients, which suggested a possible gene/environmental interaction, in which BMAA triggered neurodegeneration in vulnerable individuals. The Chamorro traditional food also included flying fox bats, which feed on cycad seeds. They suggested that, “the plant and animal proteins provide unrecognized reservoirs for the slow release of this toxin”. Many studies have suggested the presence of cyanotoxins such as BMAA in patients in Guam and North America, and indicated that there is likely more than one route of human exposure to these toxins other than cycad seed ingestion (Pablo et al., 2009).

Previous analytical methods, Banack et al. (2007) described five different analytical methods to detect BMAA in marine cyanobacteria including: liquid chromatography with fluorescence, ultraviolet, and mass spectrometric detection (HPLC/FD, UPLC–UV, LC/MS and triple quadrupole LC/MS/MS) and an amino acid analyzer. HPLC/FD and UPLC–UV pre-column derivatization of BMAA was performed using AQC (6-aminoquinolyli-N-hydroxysuccinimidyl) derivatization. Metcalf et al. (2008) measured BMAA in environmental samples collected between 1990 and 2004 in the UK using HPLC/FD and LC–MS/MS. Ten of the samples were shown to include other cyanotoxins including anatoxin-a and saxitoxin. Table 1 provides a summary of methods used for detecting cyanotoxins in different environmental matrices.

In light of these various approaches, demonstrating the utility of a single simplified method designed to simultaneously detect multiple cyanotoxins in water and aquatic tissue could be very useful in evaluating the production, occurrence and causative relationships between cyanotoxins and cyanobacteria, as well as their impacts on other organisms in freshwater ecosystems (Chorus and Bartram, 1999, Huisman et al., 2005). For example, no LD₅₀ determinations are available for fish in these ecosystems, and no LC₅₀ data are currently available for human risk assessments.