these separate pathways include production of alkyl iminium salts, acyl iminium salts and tosyl iminium salts. enyne metathesis enyne metathesis of anatoxin-a involves the use of a ring closing mechanism and is one of the more recent advances in anatoxin-a synthesis. in all methods involving this pathway, pyroglutamic acid is used as a starting material in conjunction with a grubb's catalyst. similar to iminium cyclization, the first attempted synthesis of anatoxin-a using this pathway used a 2,5-cis-pyrrolidine as an intermediate. biosynthesis anatoxin-a is synthesized in vivo in the species anabaena flos aquae, as well as several other genera of cyanobacteria. anatoxin-a and related chemical structures are produced using acetate and glutamate. further enzymatic reduction of these precursors results in the formation of anatoxin-a. homoanatoxin, a similar chemical, is produced by oscillatoria formosa and utilizes the same precursor. however, homoanatoxin undergoes a methyl addition by s-adenosyl-l_methionine instead of an addition of electrons, resulting in a similar analogue. the biosynthetic gene cluster (bgc) for anatoxin-a was described from oscillatoria pcc 6506 in 2009. stability and degradation anatoxin-a is unstable in water and other natural conditions, and in the presence of uv light undergoes photodegradation, being converted to the less toxic products dihydroanatoxin-a and epoxyanatoxin-a. the photodegradation of anatoxin-a is dependent on ph and sunlight intensity but independent of oxygen, indicating that the degradation by light is not achieved through the process of photo-oxidation. studies have shown that some microorganisms are capable of degrading anatoxin-a. a study done by kiviranta and colleagues in 1991 showed that the bacterial genus pseudomonas was capable of degrading anatoxin-a at a rate of 2–10 μg/ml per day. later experiments done by rapala and colleagues (1994) supported these results. they compared the effects of sterilized and non-sterilized sediments on anatoxin-a degradation over the course of 22 days, and found that after that time vials with the sterilized sediments showed similar levels of anatoxin-a as at the commencement of the experiment, while vials with non-sterilized sediment showed a 25-48% decrease. detection there are two categories of anatoxin-a detection methods. biological methods have involved administration of samples to mice and other organisms more commonly used in ecotoxicological testing, such as brine shrimp (artemia salina), larvae of the freshwater crustacean thamnocephalus platyurus, and various insect larvae. problems with this methodology include an inability to determine whether it is anatoxin-a or another neurotoxin that causes the resulting deaths. large amounts of sample material are also needed for such testing. in addition to the biological methods, scientists have used chromatography to detect anatoxin-a. this is complicated by the rapid degradation of the toxin and the lack of commercially available standards for anatoxin-a. public health despite the relatively low frequency of anatoxin-a relative to other cyanotoxins, its high toxicity (the lethal dose is not known for humans, but is estimated to be less than 5 mg for an adult male) means that it is still considered a serious threat to terrestrial and aquatic organisms, most significantly to livestock and to humans. anatoxin-a is suspected to have been involved in the death of at least one person. the threat posed by anatoxin-a and other cyanotoxins is increasing as both fertilizer runoff, leading to eutrophication in lakes and rivers, and higher global temperatures contribute to a greater frequency and prevalence of cyanobacterial blooms. water regulations the world health organization in 1999 and epa in 2006 both came to the conclusion that there was not enough toxicity data for anatoxin-a to establish a formal tolerable daily intake (tdi) level, though some places have implemented levels of their own. drinking water advisory levels anatoxin-a is not regulated under the safe drinking water act, but states are allowed to create their own standards for contaminants that are unregulated. currently there are four states that have set drinking water advisory levels for anatoxin-a as seen in the table below. on october 8, 2009 the epa published the third drinking water contaminant candidate list (ccl) which included anatoxin-a (among other cyanotoxins), indicating that anatoxin-a may be present in public water systems but is not regulated by the epa. anatoxin-a's presence on the ccl means that it may need to be regulated by the epa in the future, pending further information on its health effects in humans. drinking water advisory levels state concentration (µg/l) minnesota 0.1 ohio 20 oregon 0.7 vermont 0.5 recreational water advisory levels in 2008 the state of washington implemented a recreational advisory level for anatoxin-a of 1 µg/l in order to better manage algal blooms in lakes and protect users from exposure to the blooms. canada the canadian province of québec has a drinking water maximum accepted value for anatoxin-a of 3.7 µg/l. new zealand new zealand has a drinking water maximum accepted value for anatoxin-a of 6 µg/l. water treatment as of now, there is no official guideline level for anatoxin-a, although scientists estimate that a level of 1 μg l−1 would be sufficiently low. likewise, there are no official guidelines regarding testing for anatoxin-a. among methods of reducing the risk for cyanotoxins, including anatoxin-a, scientists look favorably on biological treatment methods because they do not require complicated technology, are low maintenance, and have low running costs. few biological treatment options have been tested for anatoxin-a specifically, although a species of pseudomonas, capable of biodegrading anatoxin-a at a rate of 2–10 μg ml−1 d−1, has been identified. biological (granular) activated carbon (bac) has also been tested as a method of biodegradation, but it is inconclusive whether biodegradation occurred or if anatoxin-a was simply adsorbing the activated carbon. others have called for additional studies to determine more about how to use activated carbon effectively. chemical treatment methods are more common in drinking water treatment compared to biological treatment, and numerous processes have been suggested for anatoxin-a. oxidants such as potassium permanganate, ozone, and advanced oxidation processes (aops) have worked in lowering levels of anatoxin-a, but others, including photocatalysis, uv photolysis, and chlorination, have not shown great efficacy. directly removing the cyanobacteria in the water treatment process through physical treatment (e.g., membrane filtration) is another option because most of the anatoxin-a is contained within the cells when the bloom is growing. however, anatoxin-a is released from cyanobacteria into water when they senesce and lyse, so physical treatment may not remove all of the anatoxin-a present. additional research needs to be done to find more reliable and efficient methods of both detection and treatment. laboratory uses anatoxin-a is a very powerful nicotinic acetylcholine receptor agonist and as such has been extensively studied for medicinal purposes. it is mainly used as a pharmacological probe in order to investigate diseases characterized by low acetylcholine levels, such as muscular dystrophy, myasthenia gravis, alzheimer disease, and parkinson disease. further research on anatoxin-a and other less potent analogues are being tested as possible replacements for acetylcholine. genera of cyanobacteria that produce anatoxin-a anabaena (dolichospermum) aphanizomenon cylindrospermopsis cylindrospermum lyngbya microcystis nostoc oscillatoria microcoleus (phormidium) planktothrix raphidiopsis tychonema woronichinia