WHO/SDE/WSH/xxxx English only Cyanobacterial toxins: Anatoxin-a Background document for development of WHO Guidelines for Drinking-water Quality and Guidelines for Safe Recreational Water Environments Version for Public Review Nov 2019 © World Health Organization 20XX Preface Information on cyanobacterial toxins, including anatoxins, is comprehensively reviewed in a recent volume to be published by the World Health Organization, “Toxic Cyanobacteria in Water” (TCiW; Chorus & Welker, in press). This covers chemical properties of the toxins and information on the cyanobacteria producing them as well as guidance on assessing the risks of their occurrence, monitoring and management. In contrast, this background document focuses on reviewing the toxicological information available for guideline derivation and the considerations for deriving the guideline values for microcystins in water. Sections 1-3 and 8 are largely summaries of respective chapters in TCiW and references to original studies can be found therein. To be written by WHO Secretariat Acknowledgements To be written by WHO Secretariat Abbreviations used in text AF drinking-water allocation factor ALP alkaline phosphatase ALT alanine aminotransferase ASP aspartate aminotransferase ATX anatoxin-a bw body weight C daily drinking-water consumption CYN cylindrospermopsin dw dry weight DWI daily water intake ELISA enzyme-linked immunosorbent assay FSH follicle stimulating hormone GD gestational day GGT gamma-glutamyl transferase GSH glutathione GST-P glutathione S-transferase placental form-positive GTX gonyautoxin GV guideline value HBV hepatitis B virus HPLC high performance liquid chromatography HTX homoanatoxin i.p. intraperitoneal i.v. intravenous LC-MS liquid chromatography – mass spectrometry LDH lactate dehydrogenase LH luteinizing hormone LOAEL lowest-observed-adverse-affect level MC microcystin(s) NOAEL no-observed-adverse-affect level P fraction of the TDI allocated to drinking-water PoD point of departure PP1 protein phosphatase-1 PP2A protein phosphatase-2A PSP Paralytic Shellfish Poisoning PST paralytic shellfish toxin STX saxitoxin STXOL saxitoxinol STXs saxitoxins, comprising all analogues TDI tolerable daily intake UF uncertainty factor Table of Contents 1.0 EXECUTIVE SUMMARY ...................................................................................................... 7 2.0 GENERAL DESCRIPTION ..................................................................................................... 7 2.1 Identity .......................................................................................................................... 7 2.2 Physical and Chemical Properties ................................................................................. 7 2.3 Organoleptic Properties ................................................................................................ 7 2.4 Major Uses and Sources................................................................................................ 8 2 ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE ............................................................. 8 2.1 Air .................................................................................................................................. 8 2.2 Food .............................................................................................................................. 8 2.3 Water ............................................................................................................................ 9 2.4 Estimated total exposure and relative contribution of drinking-water ........................ 9 3 KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS ............................. 9 Absorption ................................................................................................................................ 9 Distribution ............................................................................................................................. 10 Metabolism ............................................................................................................................. 10 Elimination .............................................................................................................................. 10 4 EFFECTS ON HUMANS ......................................................................................................... 10 5 EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO SYSTEMS ........................................ 10 Acute exposure ....................................................................................................................... 10 Short-term exposure ............................................................................................................... 11 Genotoxicity and carcinogenicity ............................................................................................ 12 In vitro systems ....................................................................................................................... 12 Mode of action ........................................................................................................................ 12 6 OVERALL DATABASE AND QUALITY OF EVIDENCE .............................................................. 12 6.1 Summary of Health Effects ......................................................................................... 12 6.2 Quality of Evidence ..................................................................................................... 12 7 PRACTICAL CONSIDERATIONS ............................................................................................. 13 7.1 Source control ............................................................................................................. 13 7.2 Monitoring .................................................................................................................. 14 7.3 Analytical methods and achievability ......................................................................... 14 7.4 Treatment methods and performance ....................................................................... 15 8 CONCLUSIONS ..................................................................................................................... 15 8.1 Derivation of the guideline-value ............................................................................... 15 8.2 Considerations in applying the health-based reference values ................................. 16 REFERENCES (still need some tidying up) .............................................................................. 17 1.0 EXECUTIVE SUMMARY [to be completed] 2.0 GENERAL DESCRIPTION 2.1 Identity Anatoxin-a (ATX; CAS Reference Number 64285-06-9), or 2-acetyl-9-azabicyclo[4:2:1]non- 2-ene, is a tropane-related bicyclic, secondary amine alkaloid. Figure 2.1 shows the presence of an additional methyl group (CH) on carbon atom 11 (C11) which differentiates homoanatoxin- a (HTX; CAS Reference Number 142926-86-1; Fig. 2.1) from its analog ATX. Both molecules share almost identical toxicological properties. Other derivatives of ATX have been identified in cyanobacterial cultures or in field samples, including 2,3-epoxy-anatoxin-a, 4-hydroxy- and 4-oxo-derivatives, dihydroanatoxin-a and dihydrohomoanatoxin-a, that possibly represent degradation products (TCiW; (Testai, in press). H H a N b N O O Figure 2.1: Structures of anatoxin-a (a) and homoanatoxin-a (b) 2.2 Physical and Chemical Properties ATX has a molecular formula of C10H15NO and an average molecular weight of 165.232 Da (monoisotopic MW 165.115 Da). ATX is highly soluble in water with a computed Kow of 0.8 and has a high boiling point of 291°C. It has a density of 1.04 and a low vapor pressure of 0.002. Other physico-chemical properties such as the soil adsorption coefficient (Koc), how it volatizes from water, and its distribution in the atmosphere (Henry’s Law constant) are unknown. Limited information on the chemical breakdown, biodegradation and distribution in the environment is available (TCiW; (Testai, in press). Table 2.1: Physical and chemical properties of anatoxins. N/A: not applicable. Property anatoxin-a homoanatoxin CASRN1 64285-06-9 142926-86-1 Chemical Formula C10H15NO C11H17NO Average MW2 (g/mole) 165.237 179.264 Monoisotopic MW (g/mole) 165.115 179.131 Color/Physical State Boiling Point 291°C N/A 3 Kow 0.8 1.3 Solubility in Water High High Solubility in Other Solvents 1 Chemical Abstracts Service Registry Number 2 Molecular Weight 3logP computation with XLogP3 (Cheng et al., 2007) 2.3 Organoleptic Properties While none of the known cyanobacterial toxins have been shown to affect the taste or odour of water, some cyanobacterial species produce other compounds such as geosmin and methyl- isoborneol that do cause taste and odour of water, thus indicating the presence of cyanobacteria in raw water. However, as this applies only to some species, the absence of these typical tastes 7 and odours are not a reliable indicator for the absence of cyanotoxins. Taste or odour thresholds in water are 0.004 ppb for geosmin and 0.006 ppb for methyl-isoborneol (TCiW, Kaloudis, in press). 2.4 Major Uses and Sources ATX occurs naturally (although high concentrations are typical for waterbodies influenced by human activity, i.e. effluents from wastewater or run-off from agricultural land) and there are no known commercial applications. ATX is produced by a variety of cyanobacteria species belonging to Nostocales (Anabaena flos-aquae, A. lemmermannii,