Chapter 2 Cyanobacterial toxins CONTENTS Introduction and general considerations 15 References 19 2.1 Hepatotoxic cyclic peptides – microcystins and nodularins 21 2.1.1 Chemical structures 21 2.1.2 Toxicity: mode of action 24 2.1.3 Derivation of provisional guideline values 25 2.1.4 Production 28 2.1.4.1 Producing cyanobacteria 28 2.1.4.2 Microcystin/nodularin profiles 31 2.1.4.3 Biosynthesis 32 2.1.4.4 Regulation of biosynthesis 32 2.1.5 Occurrence in water environments 35 2.1.5.1 Bioaccumulation 36 2.1.6 Environmental fate 36 2.1.6.1 Partitioning between cells and water 36 2.1.6.2 Chemical breakdown 37 2.1.6.3 Biodegradation 38 References 39 2.2 Cylindrospermopsins 53 2.2.1 Chemical structures 53 2.2.2 Toxicity: mode of action 53 2.2.3 Derivation of provisional guideline values 54 2.2.4 Production 57 2.2.4.1 Producing cyanobacteria 57 2.2.4.2 Cylindrospermopsin profiles 58 2.2.4.3 Biosynthesis 58 2.2.4.4 Regulation of biosynthesis 59 2.2.5 Occurrence in water environments 61 2.2.5.1 Bioaccumulation 62 2.2.6 Environmental fate 62 2.2.6.1 Partitioning between cells and water 62 13 14 Toxic Cyanobacteria in Water 2.2.6.2 Chemical breakdown 62 2.2.6.3 Biodegradation 63 References 64 2.3 Anatoxin-a and analogues 72 2.3.1 Chemical structures 72 2.3.2 Toxicity: mode of action 73 2.3.3 Derivation of health- based reference values 73 2.3.4 Production 76 2.3.4.1 Producing cyanobacteria 76 2.3.4.2 Toxin profiles 76 2.3.4.3 Biosynthesis and regulation 81 2.3.5 Occurrence in water environments 82 2.3.5.1 Bioaccumulation 84 2.3.6 Environmental fate 84 2.3.6.1 Partitioning between cells and water 84 2.3.6.2 Chemical breakdown 85 2.3.6.3 Biodegradation 85 References 86 2.4 Saxitoxins or Paralytic Shellfish Poisons 94 2.4.1 Chemical structures 94 2.4.2 Toxicity: mode of action 95 2.4.3 Derivation of guideline values 97 2.4.4 Production 100 2.4.4.1 Producing cyanobacteria 100 2.4.4.2 Toxin profiles 100 2.4.4.3 Biosynthesis and regulation 101 2.4.5 Occurrence in water environments 102 2.4.5.1 Bioaccumulation 103 2.4.6 Environmental fate 104 References 104 2.5 Anatoxin-a(S) 109 2.5.1 Chemical structure 109 2.5.2 Toxicity: mode of action 109 2.5.3 Derivation of guideline values for anatoxin-a(S) in water 110 2.5.4 Production, occurrence and environmental fate 110 References 111 2.6 Marine dermatotoxins 113 2.6.1 Chemical structures 114 2.6.2 Toxicity 115 2.6.3 Incidents of human injury through marine cyanobacterial dermatotoxins 116 2.6.4 Biosynthesis and occurrence in the environment 118 References 119 2 The cyanotoxins 15 2.7 β-Methylamino-L-alanine (BMAA) 123 2.7.1 Discrepancies introduced by incorrect BMAA analysis 123 2.7.2 The BMAA-human neurodegenerative disease hypothesis 125 2.7.2.1 ALS/PDC attributed to BMAA versus other manifestations of neurodegenerative disease 127 2.7.3 Postulated human exposure and BMAA mechanism of action 128 2.7.4 Conclusions 130 References 132 2.8 Cyanobacterial lipopolysaccharides (LPS) 137 2.8.1 General characteristics of bacterial LPS 137 2.8.2 What is known about bioactivity of cyanobacterial LPS? 139 2.8.3 Methodological problems of studies on cyanobacterial LPS 141 2.8.4 Possible exposure routes to cyanobacterial LPS 143 2.8.5 Conclusions 144 References 145 2.9 Cyanobacterial taste and odour compounds in water 149 2.9.1 Chemistry and toxicity 149 2.9.2 Analysis 150 2.9.3 Producing organisms 151 2.9.4 Biosynthesis 152 2.9.5 Geosmin and MIB concentrations in aquatic environments 152 2.9.6 Removal of geosmin and MIB by water treatment processes 152 2.9.7 Co-occurrence of T&O compounds and cyanotoxins 153 References 154 2.10 Unspecified toxicity and other cyanobacterial metabolites 156 2.10.1 Bioactive metabolites produced by cyanobacteria 156 2.10.2 Toxicity of cyanobacteria beyond known cyanotoxins 158 References 159 INTRODUCTION AND GENERAL CONSIDERATIONS The following sections provide an overview of the individual types of cyanobacterial toxins, focusing on toxins that have been confirmed to, or suggested to have implications for human health, namely, microcystins, cylindrospermopsins, anatoxins, saxitoxins, anatoxin-a(S) and dermatotoxins, the latter primarily produced by marine cyanobacteria. Two further cyanobacterial metabolites, lipopolysaccharides (LPS) and β- methylamino-alanine (BMAA), are discussed in respective sections with 16 Toxic Cyanobacteria in Water the conclusion that the available evidence does not show their proposed toxic effects to occur in dose ranges relevant to the concentrations found in cyanobacterial blooms. A further section includes information on taste and odour compounds produced by cyanobacteria because, while actually not toxic, they sometimes indicate the presence of cyanobacteria. Finally, recognising that there are many cyanobacterial metabolites and further toxic effects of cyanobacterial cells that have been observed which cannot be attributed to any of the known cyanotoxins, a section covers “additional toxicity” and bioactive cyanobacterial metabolites. The sections on the major toxin types review the chemistry, toxicology and mode of action, producing cyanobacteria and biosynthesis, occurrence and environmental fate. Given the document’s scope, the individual sec- tions discuss ecotoxicological data only briefly. This, however, does not imply that cyanotoxins do not play an important role in aquatic ecosystems. Further, possible benefits of toxin biosynthesis for the producing cyanobac- teria are currently discussed but not yet understood, and this remains an important field of research but is discussed in this volume only briefly. For microcystins, cylindrospermopsins and saxitoxins guideline values (GVs) have been derived based on the toxicological data available and con- sidering there is credible evidence of their occurrence in water to which people may be exposed. For anatoxin-a, although GVs cannot be derived due to inadequate data, a “bounding value”, or health-based reference value, has been derived. For anatoxin-a(S) and the dermatotoxins, the tox- icological data for deriving such values are not sufficient, and hence, no such values are proposed. BOX 2.1: HOW ARE GUIDELINE VALUES DERIVED? For most chemicals that may occur in water, including for the known cya- notoxins, it is assumed that no adverse effect will occur below a threshold dose. For these chemicals, a tolerable daily intake (TDI) can be derived. TDIs represent an estimate of an amount of a substance, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk. TDIs are usually based on animal studies because, for most chem- icals, the available epidemiological data are not sufficiently robust, mainly because the dose to which people were exposed is poorly quantified and because it is scarcely possible to exclude all confounding factors (including simultaneous exposure to other substances) that may have influenced differ- ences between those exposed and the control group. TDIs based on animal studies are based on long-term exposure, preferably spanning a whole life cycle or at least a major part of it, exposing groups of animals (frequently mice or rats) to a series of defined doses applied orally via drinking-water or 2 The cyanotoxins 17 gavage. The highest dose for which no adverse effects in the exposed animals were detected is the no observed adverse effect level (NOAEL), generally expressed in dose per body weight and per day (e.g., 40 μg/kg bw per day). Sometimes no NOAEL is available, while the lowest observed adverse effect level (LOAEL) can be considered in establishing the TDI. The LOAEL is defined as the lowest dose in a series of doses causing adverse effects. An alternative approach for the derivation of a TDI is the determination of a benchmark dose (BMD), in particular, the lower confidence limit of the benchmark dose (BMDL; WHO, 2009a). A BMDL can be higher or lower than NOAEL for individual studies (Davis et al., 2011). A NOAEL (or LOAEL or BMDL) obtained from animal studies cannot be directly applied to determine “safe” levels in humans for several rea- sons such as differences in susceptibility between species (i.e., humans vs. mice or rats), variability between individuals, limited exposure times in the experiments or specific uncertainties in the toxicological data. For example, for the cyanotoxins for which WHO has established GVs, exposure times did not span a whole life cycle because the amount of pure toxin needed for such a long study – a few hundred grams – was simply not available or would be extremely costly to purchase. To account for these uncertainties, a NOAEL is divided by uncertainty factors (UFs). The total UF generally comprises two 10-fold factors, one for interspecies differences and one for interindividual variability in humans. Further uncertainty factors may be incorporated to allow for database deficiencies (e.g., less than lifetime exposure of the animals in the assay, use of a LOAEL rather than a NOAEL, or for incomplete assessment of particular endpoints such as lack of data on reproduction) and for the severity or irreversibility of effects (e.g., for uncertainty regarding carcinogenicity or tumour promotion). Where ade- quate data is available, chemical specific adjustment factors (CSAFs) can be used for interspecies and intraspecies extrapolations, rather than the use of the default UFs. The TDI is calculated using the following formula: NOAEL or LOAEL or BMDL TDI= UF12N ×× UF UF or CSAFs The unit of TDI generally is the amount of toxin per bodyweight (bw) per day, for example, 0.1 μg/kg bw per day.