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Critical Reviews in Toxicology, 38:97 125, 2008 Copyright c 2008 Informa Healthcare USA, Inc. ISSN: 1040-8444 print / 1547-6898 online DOI: 10.1080/10408440701749454 Human Health Risk Assessment Related to Cyanotoxins Exposure Enzo Funari and Emanuela Testai Environment and Primary Prevention Department, Istituto Superiore di Sanità, Rome, Italy This review focuses on the risk assessment associated with human exposure to cyanotoxins, secondary metabolites of an ubiquitous group of photosynthetic procariota. Cyanobacteria occurr especially in eutrophic inland and coastal surface waters, where under favorable conditions they attain high densities and may form blooms and scums. Cyanotoxins can be grouped according to their biological effects into hepatotoxins, neurotoxins, cytotoxins, and toxins with irritating potential, also acting on the gastrointestinal system. The chemical and toxicological properties of the main cyanotoxins, relevant for the evaluation of possible risks for human health, are presented. Humans may be exposed to cyanotoxins via several routes, with the oral one being by far the most important, occurring by ingesting contaminated drinking water, food, some dietary supplements, or water during recreational activities. Acute and short-term toxic effects have been associated in humans with exposure to high levels of cyanotoxins in drinking and bathing waters. However, the chronic exposure to low cyanotoxin levels remains a critical issue. This article identifies the actual risky exposure scenarios, provides toxicologically derived reference values, and discusses open issues and research needs. Keywords Cyanobacteria, Cyanotoxins, Human Health, Toxicological Risk Assessment 1. INTRODUCTION Cyanobacteria are a group of ubiquitous photosynthetic procariota. They occur especially in surface waters, where they can tolerate remarkable changes of salinity and temperature and photosynthesize under conditions of low light intensity, that is, high turbidity (Rai, 1990). In favorable conditions for their growth (i.e., nutrient availability, temperature, light), cyanobacteria form blooms, giving rise to biomass accumulation and scum (Ressom et al., 1994). Planktonic cyanobacteria produce, as secondary metabolites, a high variety of toxins known as cyanotoxins that give rise to some concern for human health. Indeed, cyanobacteria have been included among emerging pathogenic microorganisms, even though they do not colonize and invade the host (OECD, 2005). Cyanobacteria are characterized by a wide morphological variability (Chorus and Bartram, 1999; Sivonen and Jones, 1999). In most cases their cells are surrounded by a gelatinous stratum, which increases their chance to survive even in harsh environmental conditions (Whitton, 1992). In the last years many papers have been published, reporting the occurrence of cyanobacteria in surface waters and envi- Address correspondence to E. Funari/E. Testai, Istituto Superiore di Sanità, Environment and Primary Prevention Department, Viale Regina Elena, 299, 00161 Roma Italy. E-mail: enzo.funari@iss.it; emanuela.testai@iss.it ronmental factors influencing their production, reviewing toxicological and ecotoxicological properties of selected toxins, and reporting methods of detection (van Apeldoorn et al., 2007; Codd et al., 2005; Dittmann and Wiegand, 2006; Zurawell et al., 2005; Duy et al., 2000). The readers are invited to consult the cited papers for a detailed description of those issues, which are only mentioned or not treated here. Indeed, in this article attention is focused on the evaluation of the risk for human health associated with the different sources and routes of exposures to those cyanotoxins known so far. Besides this introductory section, the review consists of four parts: Section 2 very briefly introduces the different cyanobacteria producing toxins and their occurrence in the environment; section 3 describes the toxicological profile of the main known cyanotoxins; from this, in section 4 the risks for human health deriving from different exposure scenarios are presented; and finally, in section 5, based on the analysis of the available data, some research needs and open issues are highlighted, although the list is far from exhaustive. 2. TOXIC CYANOBACTERIA AND KNOWN CYANOTOXINS 2.1. Cyanotoxins Classification Cyanotoxins can be classified into categories that reflect their biological effects on the systems and organs that they affect most strongly (Codd et al., 2005). 97
98 E. FUNARI AND E. TESTAI TABLE 1 Toxigenic cyanobacteria from marine, brackish and freshwaters Cyanotoxin Main producing cyanobacteria Bibliographic source Microcystins Most of Microcystis spp and Planktothrix spp, some Anabaena, Nostoc and Synechocystis and Cyanobium bacillare, Arthrospira fusiformis, Limnothrix redekei, Phormidium formosum, Hapalosiphon hibernicus Sivonen and Jones, 1999; Cronberg et al., 2003; Odebrecht et al., 2002; Ballot et al, 2004; Gkelis et al., 2001; Steffensen et al., 2001; Prinsep et al., 1992 Nodularins Nodularia spumigena (in transitional waters) Rinheart et al., 1988 Cylindrospermopsin Cylindrospermopsis raciborskii, Umezakia natans, Aphanizomenon ovalisporum, Aphanizomenon flos-aquae, Rhaphidiopsis curvata, Anabaena lapponica, Anabaena bergii Anatoxin-a Most of Anabaena spp., some Aphanizomenon (A. flos-aquae, A. issatschenkoi), Cylindrospermum, Microcystis and Planktothrix spp. and Raphidiopsis mediterranea Ohtani et al., 1992; Harada et al., 1994; Banker et al., 1997; Schembri et al., 2001; Li et al., 2001; Fastner et al., 2007; Spoof et al., 2006 Edwards et al., 1992; Sivonen et al., 1989; Park et al., 1993; Namikoshi et al., 2003; Wood et al., 2007 Homoanatoxin-a Oscillatoria formosa, Raphidiopsis mediterranea Skulberg et al., 1992; Steffensen et al., 2001; Namikoshi et al., 2003 Anatoxin a-(s) Anabaena flos-aquae and A. lemmermannii Carmichael and Gorham, 1978; Henriksen et al., 1997 Saxitoxins (PSP) Aphanizomenon, Anabaena, Lyngbya and Humpage et al., 1994 Cylindrospermopsis spp. LPS endotoxins All cyanobacteria McElhiney and Lawton, 2005 Aplysiatoxin, Lyngbyatoxin Debromoaplysiatoxin Lyngbya majuscula (marine waters), Oscillatoria nigro-vridis Serdula et al., 1982; Mynderse et al., 1997 Microviridin J Microcystis spp Rohrlack et al., 2003 β-n-methylamino-lalanine Microcystis, Anabaena, Nostoc and Planktothrix spp Cox et al., 2005 and most of cyanobacteria symbionts tested In this sense, they include: Hepatotoxins (more than 70 microcystin variants, 6 known nodularine variants). Neurotoxins (anatoxin-a, homoanatoxin-a, anatoxin a-(s), 21 known saxitoxin variants, known also as paralytic shellfish poisoning toxins). Cytotoxins: cilyndrospermopsin. Irritants and gastrointestinal toxins: aplysiatoxin, debromoaplysiatoxin, lyngbyatoxin (produced by marine cyanobacteria); lipopolysaccharidic (LPS) endotoxins. Other cyanotoxins whose toxicological or ecotoxicological profile is still only partially known, such as microviridin J and β-n-methylamino-l-alanine (BMAA). At present it is not clear which is the proportion of known versus unknown cyanotoxins. 2.2. Cyanotoxin Production Each cyanotoxin can be produced by more than one cyanobacterial species; likewise, the same species is able to produce more than one toxin (Table 1). Moreover, within a single species, different genotypes occur, some of which possess the gene for a given toxin and others that do not. This was first demonstrated for microcystins (MCs) (Kurmayer et al., 2002). In 50 75% of cyanobacterial blooms, the toxicity is associated with a simultaneous production of diverse cyanotoxins (An and Carmichael, 1994), whose relative importance and spatial distribution are subjected to a wide variability. The toxicity of a given bloom is determined by its strain composition, i.e., the relative share of toxic versus nontoxic genotypes. The dynamics of toxigenic cyanobacteria in surface water bodies can be studied by means of DNA-based tests; available data on a German lake indicate that remarkable variations of the genotype ratio can be found, even on a weekly or biweekly scale (Kurmayer and Kutzenberger, 2003). Therefore, the variations of the MC concentrations detected in the lakes might be a result of population dynamics, altering the proportion of toxic genotypes within the population of cyanobacteria (Dittmann and Börner, 2005). The amount of MC production by a cyanobacterial population in culture appears to be directly proportional to its growth
HUMAN HEALTH RISK OF CYANOTOXINS EXPOSURE 99 TABLE 2 Maximum levels of different cyanotoxins detected in blooms material found in the literature Cyanotoxins Maximum levels Location References Microcystins 7,300 µg/g 15,600 25,000 µg/l China and Portugal Japan, Germany Nagata et al., 1997; Fastner et al., 1999; Chorus et al., 1998 Nodularins 18,000 µg/g Baltic Sea Kononen et al., 1993 Cilyndrospermopsin 5,500 µg/g Australia Sivonen and Jones, 1999 Anatoxin-a 2,600 4,400 µg/g Finland, Japan Sivonen et al., 1989; Harada et al., 1993 Saxitoxins 2,040 3,400 µg/g Australia Negri et al., 1997; Humpage et al., 1994 Anatoxin-a (s) 3,300 µg/g Denmark Henriksen et al., 1997 rate, with the highest amount being produced during the late logarithmic phase; the amount of MCs contained in a single cell of Microcystis aeruginosa is constant within a narrow range (twoto threefold) (van Apeldoorn et al., 2007). Beside the population dynamics, the remarkable variability in MC concentrations in water bodies has been attributed to environmental condition variation, which can influence cyanotoxin production rate (Sivonen and Jones, 1999). Yet the role of environmental factors in cyanotoxin production is not sufficiently understood. Some studies showed that the variations of parameters such as light, culture age, temperature, ph, and nutrients give rise to differences in the cellular MC content not exceeding a factor of five (Sivonen and Jones, 1999). The production of cyanotoxins by cyanobacteria has been confirmed by laboratory tests using mono-cyanobacteria isolated cultures; however, the possibility that associated eterothropic bacteria may have a role in cyanotoxin production or in its modulation cannot be definitely ruled out (Codd et al., 2005). 2.3. Cyanotoxins in Surface Waters A high number of studies have been published on the occurrence of cyanotoxins, particularly MCs, in surface waters, which have been extensively reviewed in dedicated publications (Chorus and Bartram, 1999; van Apeldoorn et al., 2007). This subsection only represents a brief synthesis of the available data, relevant to the assessment of the risks to human health associated with exposure to cyanotoxins. Specific data on different sources of human exposure to cyanotoxins are reported into each specific paragraph. Cyanotoxins may be localized both within the cyanobacterial cells and dissolved in the water, depending on both the nature of the toxin and the growth stage (Chorus and Bartram, 1999; van Apeldoorn et al., 2007). The highest total (intracellular plus dissolved) cyanotoxin levels have been found in blooms and scums (Table 2). Total MC concentrations in surface waters vary in a very wide range of values (from trace to several milligrams per liter), being strongly influenced by the occurrence of these forms of biomass. Total MC concentrations of 10 50 µg/l, up to 350 µg/l, have been reported in surface waters in Germany (Fastner et al., 1999), but much higher levels (up to 25,000 µg/l) elsewhere (Sivonen and Jones, 1999). Total cylindrospermopsin (CYN) and anatoxina(s) concentrations up to 12.1 and 3300 µg/l have been determined in surface waters (Rücker et al., 2007; Sivonen and Jones, 1999). The sum of intracellular plus dissolved cyanotoxin level is generally the most relevant index of exposure to be considered in risk evaluation; nevertheless, in some cases it may be necessary to differentiate between particulate and dissolved form. As an example, when for drinkin-water purposes simple treatments are in place to remove cells, the levels of dissolved cyanotoxins are of interest in order to evaluate human exposure. Intracellular MC content is generally higher than that dissolved in the surrounding water (van Apeldoorn et al., 2007; Ibelings and Chorus, 2007), where they are partially released, probably due to active transport (Rapala et al., 1997). On the contrary, CYN may often be found at higher levels in dissolved form than within cells (Rücker et al., 2007); limited or no information is available about the proportion of dissolved form with respect to the total level for the other cyanotoxins. After a collapse of aging, declining blooms or their treatment with algaecides, high concentrations of dissolved cyanotoxins can be found in the surrounding water (van Apeldoorn et al., 2007; Jones and Orr, 1994). However, these high levels are usually not long-lasting, due to strong dilution in the water body, wind mixing, adsorption to the sediment, and (bio)degradation. Indeed, once released in the water, cyanotoxins persist in the environment, depending on the efficiency of degradation (i.e., photolysis, hydrolysis and bacterial degradation). MCs and nodularins (NODs) can persist in water for relatively long times, ranging between 21 days and 2 3 months (Ressom et al., 1994; Jones and Orr, 1994), and up to 6 months in dry scum (Jones et al., 1995). A half-life of 11 15 days has been reported for CYN in surface waters (Chiswell et al., 1999). Similarly anatoxin-a showed a half-life of about 14 days in normal light conditions with basic ph and low initial concentrations (Smith and Sutton, 1993), whereas a much shorter half-life (1 2 hours) has been shown in the presence of high light intensity (Stevens
100 E. FUNARI AND E. TESTAI and Krieger, 1991), indicating a high efficiency of the photolysis reaction. Anatoxin-a(s) is relatively stable under neutral and acidic conditions (Matsunaga et al., 1989). A persistence of 1 2 months has been reported for saxitoxin (STX) in surface water (Batoreu et al., 2005). 3. CHEMICAL AND TOXICOLOGICAL PROFILES OF CYANOTOXINS A basic understanding of cyanotoxin chemical properties and toxicological potential is crucial for the human health risk assessment associated with their exposure. Therefore, a brief description of the most relevant features of the different cyanotoxins is in the following, summarizing general chemical structure information, mechanism of action, toxicokinetics, acute and repeated toxicity data, and, when available, data on genotoxicity, reproductive toxicity and carcinogenicity potential. A synoptic view of the major features of main cyanotoxins is presented in Table 3. 3.1. Mycrocistins and Nodularins Microcystins and NODs are cyclic peptides consisting of seven (MC) or five (NOD) amino acids. A common characteristic of both hepatotoxins is the amino acid Adda, which is unique for cyanobacteria. Structural variations occur by changing of two (MC) or one (NOD) amino acid(s), and several other changes in small side groups: these differences give rise to more than 70 MC variants and about 6 NOD variants (Sivonen and Jones, 1999). The chemical structure of MC-LR is shown in Figure 1; this is the most common MC congener, characterized by the presence of leucin (L) and arginin (R) as L-amino acids in positions 2 and 4 (Carmichael, 1988). On the basis of acute toxicity, MC-LR is considered among the most potent hepatotoxins within the different variants and is by far the most studied. Microcystin mechanism of action is associated with specific inhibition of protein serine/threonine phosphatases (PP1 and PP2A), altering phosphorilation of cellular proteins involved in signal trasduction (Gehringer, 2004). At high levels of exposure (representative of acute intoxication), MC-LR produces a cascade of events (cytoskeleton alterations, lipid peroxidation, oxidative stress, apoptosis) leading to centrolobular toxicity with intrahepatic hemorrhagic areas due to damage of sinusoidal capillaries. At low doses (typical of long-term exposure), phosphatases inhibition induces cellular proliferation and hepatic hypertrophy (Gehringer, 2004). MC-LR is able to induce oxidative stress and apoptosis in human cell lines (Botha et al., 2004). The binding of the hepatotoxin to the human hepatic mitochondrial aldehyde dehydrogenases (ALDH2) has recently been shown: Since the alteration of this enzymatic activity is involved in oxidative stress induction, it has been hypothesized that the enzyme could be one of the main targets of MC-LR mechanism of toxicity in humans (Chen et al., 2006). The degree of severity of MC-induced toxicity depends on the levels and duration of exposure, determined by the balance between MC absorption, detoxification, and excretion. MC-LR is highly hydrophilic and can not enter cell membranes by passive transport. It is actively absorbed by the intestinal mucosa, thanks to the organic anion transport system (OATP) and then enters hepatocytes through to the activity of bile acid transporters and OATP (Fisher et al., 2005). These active transporters are expressed also in the kidney and in the blood brain barrier, partially explaining some neurological disorders observed in humans during a fatal incident in Brazil (Azevedo et al., 2002). MC are conjugated with reduced glutathione in the liver of both aquatic organisms (Pflugmacher et al., 1998) and mammals (Kondo et al., 1992, 1996). The reaction, catalyzed by glutathione S-transferases, involves the methyl group of N-methyldihydroalanine (opposed to Adda): Conjugates retain only a minimal residual inhibitory activity with respect to the parent compound and are mainly excreted in the urine (Dittmann and Wiegand, 2006). The MC-LR acute toxicity after intraperitoneal (ip) administration to mice results in a LD 50 = 50 µg/kg bw; when given by the oral route, MC-LR is less toxic (LD 50 = 5000 µg/kg bw and even higher in the rat) (Fawell et al., 1994, 1999a). The lower acute toxicity (30- to 100-fold) shown by the oral route is likely due to toxicokinetic differences: The active transport system used for the absorption through the gastrointestinal (GI) mucosa is bypassed by ip injection and MC-LR is directly available for internalization into hepatocytes. This observation is partially supported by studies on organ distribution after oral and ip administration in mice, indicating a 80-fold difference in hepatic content of radiolabeled 3 H-dihydro-MC-LR (Nishiwaki et al., 1994; Robinson et al., 1989; Robinson et al., 1991). Therefore, ip administration is not fully representative of the actual conditions of human exposure, mainly associated with consumption of possibly contaminated drinking water and food, and has only a limited value for risk assessment. Acute toxicity is highly variable among MC variants: Some of them, such as MC-LA, -YR, and -YM, show ip LD 50 similar to MC-LR; for the other congeners, LD 50 s are spread in a wide range of values (from 50 up to 1200 µg/kg) (Table 4), due to the presence of different substituents. As an example, for MC-RR, containing polar amino acids in positions 2 and 4, the LD 50 is 10- fold higher than for MC-LR (Kotak et al., 1993; Wolf and Frank, 2002), whereas the presence of hydrophobic amino acids, such as alanine or phenylalanine, does not affect the acute toxicity (Zurawell et al., 2005). Adda group stereochemistry and its double bonds configuration are crucial for MC-induced PP1 and PP2A inhibition, due to a covalent binding between a protein cysteine and the Adda-glu group (Harada et al., 1990). However, Adda per se is not able to inhibit PP1 and PP2A, and it is not toxic when ip injected in mice even at very high doses (10 mg/kg bw) (Harada et al., 2004). Freeze-dried algal aqueous suspensions from both Microcystis and Anabaena blooms showed very low potential for skin irritation, and gave contrasting results for eye irritation, while clear positive results were obtained in the skin sensitization test.
TABLE 3 Main toxicological data of some cyanotoxins i.p. LD50 Oral LD50 Target organ and mechanism of LOEL ADI/TDI Cyanotoxin (µg/kg b.w.) (µg/kg b.w.) action NOEL (µg/kg/d) (µg/kg/d) (µg/kg/d) Microcystin (MC) 50 1200 5000 Liver (PP1 and PP2A phosphatases inhibition-tumor promoting activity) Nodularin (NOD) 50 ND Liver (PP1 and PP2A phosphatases inhibition-tumor promoting activity) Cylindrospermopsin (CYN) 2100 (24 h) 200 (6 days) 4400 6900 (2 6 days) Kidney, liver (Parent compound: protein synthesis inhibition; Metabolites: different but unknown mechanism; possible genotoxicity) Anatoxin-a 375 5000 Neuromuscular system (Depolarizing effect due to binding to nicotinic Ach receptor) 40 (MC-LR; mice; 13 weeks; gavage) 330 (MC-LR in BGAS extracts; mice; 13 weeks; dietary) 200 0.04 (UF = 1000) ND (refer to MC-LR) ND 30 (Mice; 11 weeks; gavage) C. raciborskii extracts more toxic than pure CYN >510 (mice; 54 days; drinking water) Limited chronic risk Homoanatoxin-a 330 ND Similar to anatoxin-a ND Limited chronic risk Anatoxin a-(s) 20 40 Peripheral nervous system (AChE inhibition; nerve hyper-excitability) Saxitoxin (STX) 10 20 263 Neuromuscular system (Membrane ion LPS Endotoxins 40 190 mg/kg bw channel block) Human: 0.144 0.304 mg/person: mild symptoms 0.456 12 mg/person: from moderate symptoms up to paralysis and death ND Skin and mucosa (irritation, topic effects) ND Limited chronic risk ND Acute risk > chronic 60 0.03 (UF = 1000) ND 0.51 (UF = 1000) ND i.p. = intraperitoneal; ND = Not determined; UF = uncertainty factor. Note: bibliographic references are available within the text. 101
102 E. FUNARI AND E. TESTAI FIG. 1. Chemical structure of microcystin-lr, characterized by the presence of leucin (L) and arginin (R) as L-amino acids in positions 2 and 4. However, components other than cyanotoxins are likely to be present in the algal extract, with possible irritation and sensitizing potential. Indeed, no correlation was found between the toxin content and the allergenic character; pure MC-LR showed only slight skin sensitizing potential, even when tested at high concentrations (1.5 mg/ml) (Torokne et al., 2001). In addition, the axenic strains were not allergenic at all (Torokne et al., 2001). Among the available studies on repeated toxicity, the most relevant one has been carried out with mice, shown to be more susceptible to MC-induced acute effects than other rodent species. Microcystin-LR was administered orally (by gavage) for 90 days at 3 different doses (Fawell et al., 1994). The study allowed the identification of a no-observed-effect level (NOEL) of 40 µg/kg bw/day (Fawell et al., 1994). Slight hepatic damages were observed at the lowest-observed-effect level (LOEL) of 200 µg/kg bw/day in a limited number of treated animals, whereas at the highest dose tested (1 mg/kg bw/day) all the animals show hepatic lesions, consistent with the known action of MC-LR. When mice were subchronically administered with MC-LR-containing extracts through the diet, a regimen more similar to human exposure, the NOAEL value was higher (333 µg/kg bw/day) (Schaeffer et al., 1999), due to toxicokinetic differences. Indeed, gavage corresponds to a bolus dose, resulting in tissue concentrations higher than those attained after the TABLE 4 Differences in acute toxicity of MC variants Toxin i.p. LD 50 (µg/kg) M.W. Structure MC-LR 50 994 cyclo-(d-ala-l-leu-d-measp-l-arg-adda-d-glu-mdha-) [D-Asp 3 ]MC-LR 50 970 cyclo-(d-ala-l-leu-d-asp-l-arg-adda-d-glu-mdha-) [L-MeLan 7 ]MC-LR 1000 1115 cyclo-(d-ala-l-leu-d-measp-l-arg-adda-d-glu-l-melan-) [6(Z)-Adda 5 ]MC-LR >1200 994 cyclo-(d-ala-l-leu-d-measp-l-arg-6(z)adda-d-glu-mdha) MC-LA 50 909 cyclo-(d-ala-l-leu-d-measp-l-ala-adda-d-glu-mdha-) MC-RR 500 1037 cyclo-(d-ala-l-arg-d-measp-l-arg-adda-d-glu-mdha-) [Dha 7 ]MC-RR 180 980 cyclo-(d-ala-l-arg-d-measp-l-arg-adda-d-glu-dha-) [6(Z)-Adda 5 ]MC-RR >1200 1037 cyclo-(d-ala-l-arg-d-measp-l-arg-6(z)adda-d-glu-mdha) MC-YR 150 200 1044 cyclo-(d-ala-l-tyr-d-measp-l-arg-adda-d-glu-mdha-) MC-YA 60 70 959 cyclo-(d-ala-l-tyr-d-measp-l-ala-adda-d-glu-mdha-) MC-AR 250 952 cyclo-(d-ala-l-ala-d-measp-l-arg-adda-d-glu-mdha-) MC-M(O)R 700 800 1028 cyclo-(d-ala-l-met(o)-d-measp-l-arg-adda-d-glu-mdha-) Aminoacidic differences with respect to MC-LR are indicated in bold. i.p. = intraperitoneal; M.W. = Molecular Weight. Data from Zurawell et al. (2005).
HUMAN HEALTH RISK OF CYANOTOXINS EXPOSURE 103 FIG. 2. Chemical structure of the most common congener of nodularin. more gradual introduction of a dietary treatment, giving time to the detoxification/excretion systems to be efficient. As for the other toxicological properties relevant to the risk assessment, pure MC-LR was not teratogenic in mice, in cultured mouse embryos and in frog embryos (Chernoff et al., 2002). Other studies found MC-producing cyanobacteria extracts to be embryotoxic and teratogenic in frog (Dvorakova et al., 2002; Buryskova et al., 2006). The evidence was strong, but unrelated to MC content, suggesting that the extracts may contain bioactive compounds other than MCs. Furthermore, a direct interaction with DNA, responsible for genotoxic activity, can be reasonably excluded (Runnegar and Falconer, 1982; Repavich et al., 1990). Indeed, contrasting results have been reported and positive results have been obtained both in vivo and in vitro only at highly cytotoxic doses, suggesting the involvement of DNA endonucleases (Ding et al., 1999; Rao et al., 1998; Zhan et al., 2004). The possibility exists that MC-LR induces oxidative stress, resulting in indirect oxidative DNA damage (Lankoff et al., 2004). The tumor-promoting activity of MC-LR was described already early in cyanotoxin research (Falconer, 1991; Nishiwaki- Matsushima et al., 1992) and more recently confirmed by cyanotoxin administration with known carcinogenic compounds, such as aflatoxin B1 and diethyl-nitrosamine (Wanght and Zhuth, 1996; Sekijima et al., 1999). On the contrary, MC-LR did not show any tumor induction when the cyanotoxin was given to mice by gavage for 28 weeks (80 µg/kg bw/day) (Ito et al., 1997). The International Agency for Research on Cancer (IARC) recently reviewed available data on MC-LR carcinogenity, concluding that there is inadequate evidence in both humans and experimental animals for the carcinogenicity of MC-LR; however, on the basis of data on tumor promoting mechanisms, IARC has classified MC-LR as possibly carcinogenic to humans (Group 2B) (IARC, 2006). Nodularins share with MCs not only a similar chemical structure, which is depicted in Figure 2, but also the mechanism of action, that is, phosphates inhibition (Yoshizawa et al., 1990). However, they have not been studied as extensively as MCs. Nodularins display cumulative toxicity and are tumor promoters without any initiation capability (Ohta et al., 1994; Song et al., 1999; Sueoka et al., 1997). However, according to the IARC evaluation, NODs are not classifiable as to their carcinogenicity to humans (Group 3), due to the scant amount of data available (IARC, 2006). The ip LD 50 in mice is similar to the one calculated for MC- LR (50 70 µg/kg bw), but no data on repeated toxicity relevant for risk assessment are available. However, it is reasonable to consider that, at least as a worst case, the situation can be compared with MC-LR and therefore to adopt the NOEL value identified for MC-LR, expressing NOD concentrations as MC-LR equivalents. 3.2. Cylindrospermopsin The cylindrospermopsin (CYN) molecule consists of a tricyclic guanidine group combined with a hydroximethyl uracil (Figure 3). It is considered a cytotoxin, since it produces both hepatotoxic and nephrotoxic effects, although other organs may also be FIG. 3. Chemical structure of cylindrospermopsin.
104 E. FUNARI AND E. TESTAI damaged following exposure to the toxin (Falconer et al., 1999; Ohtani et al., 1992; Seawright et al., 1999; Terao et al., 1994). Cylindrospermopsin is highly hydrophilic; its intestinal absorption needs active transport systems as well as the entrance into hepatocytes, making use of the bile acid transport system (Chong et al., 2002). However, due to the small size of the molecule, a limited passive diffusion through biological membranes occurs, as indicated by in vitro studies, showing cytotoxic effects in a cell line, not expressing bile acid transport system (Chong et al., 2002). When [ 14 C]-CYN (0.2 mg/kg bw) was ip injected to mice, radioactivity was recovered mainly in the liver and, to a lesser extent, in the kidney (20.6% and 4.3% of the administered dose 6 hours after the treatment, respectively). In the liver about 2% of the injected dose was still detectable a week after dosing. Urinary excretion is the major route of elimination: 50 70% of administered radioactivity was present in the urine within 6 12 hours. Most of the urinary radiolabel (about 72%, corresponding to around 50% of the administered dose) was associated with the parent compound (Norris et al., 2001), indirectly indicating that about one-half of the parent compound undergoes biotrasformation. Experimental evidences, produced both in vivo on rodent species (Norris et al., 2001, 2002) and in vitro in primary hepatocytes (Runnegar et al., 1994, 1995), indicated that CYN is bioactivated by cytochrome P-450 (P450). The substantial GSH depletion, observed after CYN oral administration to rats, leads to the hypothesis that CYN can be further metabolized by GSH conjugation (Runnegar et al., 1994). Nevertheless, the reduction in GSH content may be also attributed to the inhibition of GSH synthesis (Runnegar et al., 1995). However, metabolic information about CYN is very limited and based mainly on indirect observations. Cylindrospermopsin has a late and progressive acute toxicity: After treatment with a lethal dose, death usually occurs 24 120 hours after treatment. Indeed, LD 50 in mice after ip injection of pure CYN is 2.1 mg/kg bw after 24 h, but it is 10-fold lower (LD 50 = 0.2 mg/kg bw) when the observation period is prolonged to 120 144 hours (Ohtani et al., 1992). A similar trend was seen when Cylindrospermopsis raciborskii extracts containing CYN were injected to mice: The LD 50 value at 7 days corresponded approximately to 0.18 mg CYN equivalent/kg bw. When mice were dosed with freeze-dried extracts from C. raciborskii via the oral route, the acute toxicity was lower (oral LD 50 = 4.4-6.9 mg CYN equivalent/kg bw after 2 6 days), likely due to toxicokinetics differences (Seawright et al., 1999; Chorus and Bartram, 1999). Acute hepatic damage is localized in the centrilobular areas, being characterized by hepatocyte vacuolization and increased pigmentation of nuclei and cytoplasm. Necrosis and increased lumen of proximal tubules and alterations in the glomerulus are the main features of renal toxicity (Falconer et al., 1999). The mode of action of CYN as such has been associated with inhibition of protein synthesis (Terao et al., 1994), whereas metabolites very likely act with a different mechanism (Froscio et al., 2001). Indeed, in mice hepatocytes, CYN concentrations 0.5 µm were able to inhibit protein synthesis in 4 hours; necrosis occurred only at 10-fold higher concentrations (in 18 hours). The presence of P450 inhibitors prevented cell death (observed in 18 hours) but not protein synthesis inhibition. This suggest that at low CYN concentrations toxicity is mainly due to the parent compound through protein synthesis inhibition, whereas at higher concentrations the toxicologically relevant compounds are very likely represented by CYN metabolites (Froscio et al., 2003). CYN irritation potential was tested in rabbits in an intradermal test, by injecting 0.2 ml lyophilized extract from Aphanizomenon ovalisporum. Results showed a moderate skin irritation response (Torokne et al., 2001). When lyophilized C. raciborskii extract containing 0.015 mg CYN/g and a nontoxic Aphanizomenon strain extract were tested in a maximization test for skin sensitization, a clear positive result was obtained in both cases (Torokne et al., 2001). Therefore, the high sensitizing action displayed by the cyanobacterial extracts cannot be associated with the presence of CYN, and may be attributed to other cellular constituents, such as LPS endotoxins. Among the available repeated toxicity studies, two have been considered relevant for the risk assessment: (1) mice treated for 10 weeks with 3 doses of CYN-containing C. raciborskii extracts dissolved in drinking water (corresponding to 0, 216, 432, and 657 µg toxin equivalents/kg/day bw), and (2) mice treated for 11 weeks by gavage with purified CYN (0, 30, 60, 120, and 240 µg/kg/day bw) (Humpage and Falconer, 2003). Both treatments resulted in a dose-dependent increase in liver and kidney weight, alteration in plasma enzymes (used as markers for hepatic and renal toxicity), and consistent hystopathological changes at the high doses. No NOEL could be derived from the study with extract, since effects were evidenced in all treated animals at each dose. In the study with pure CYN, renal effects occurred at lower doses: The no-observed-adverse-effect level (NOAEL) of 30 µg/kg bw per day has been identified on the basis of increased kidney weight observed at the immediately higher dose tested (i.e., 60 µg/kg bw per day). When comparing the two studies, at similar levels of toxin equivalents (i.e., administration of 216 and 240 µg toxin equivalents/kg/day bw), the degree of severity of the effects was higher following administration of the extract rather than the pure toxin. The result suggests that cyanobacterial constituents other than CYN may contribute to toxicity. An additional subchronic study is available, describing oral toxicity in mice exposed to CYN in drinking water for 42 weeks (Sukenik et al., 2006): Results qualitatively support the already mentioned findings, confirming the liver and the kidney as the major targets for CYN-induced toxicity. In addition, increase in cholesterol levels in the plasma and liver and variations in blood parameters (e.g., elevated hematocrit and deformation of red blood cells) were also reported. In this study, the animals in the experimental group (one for gender) received CYN at gradually increasing daily doses, ranging from 10 to 55 µg/kg bw, with
changes occurring every 8 weeks (Sukenik et al., 2006). Due to the nonstandard experimental design for a toxicity test, it is not possible to derive and adequate NOAEL or lowest-observedadverse-effect level (LOAEL). Regarding the other toxicological endpoints relevant for the risk assessment, a possible interaction with DNA, leading eventually to genotoxic activity, may be suggested by the chemical structure of the toxin (presence of guanidine-like groups and potentially reactive sulphates). In a limphoblastoid cell line, lacking metabolic competence, CYN induced cytogenetic damage (micronuclei formation and chromosomal loss) but only at concentrations (1 6 µm) at which protein synthesis inhibition and overt cytotoxicity were already evident (Humpage et al., 2000). Similar results were obtained also in CHO K1 cells, lacking any P450-mediated metabolic activity (Fessard and Bernard, 2003). On the contrary, a possible genotoxic activity, not secondary to cytotoxic effects, was suggested by positive comet assay results obtained with murine hepatocytes treated with nontoxic low CYN concentrations ( 0.05 µm) (Humpage et al., 2005). Hepatocytes differ from the previously used cell lines in that they are metabolically competent; indeed, positivity in comet assay was prevented by treating hepatocytes with P450 inhibitors (Humpage et al., 2005), suggesting that the genotoxic potential is very likely dependent on CYN bioactivation. In vivo DNAadducts formation (Shaw et al., 2000) and DNA fragmentation in the liver (Shen et al., 2002) have also been reported, but since the doses tested were similar to or slightly less than the LD 50 value, they can be very likely a consequence of cytotoxicity and do not allow any final conclusion on CYN genotoxicity. A study has been conducted in order to evidence a possible carcinogenic activity induced by CYN (Falconer and Humpage, 2001): C. raciborskii extracts containing CYN (500 and 1500 µg/kg bw) have been orally administered to mice (1 dose every 2 weeks for 3 times) followed by administration of 10 µg O-tetradecanoyl-forbole 13-acetate (TPA), a known tumor promoter, twice a week for 30 weeks (Falconer and Humpage, 2001). The results suggest some tumorigenic activity, although the unusual study design (very high doses and frequency of administration of an extract instead of the toxin), the limited number of animal tested, and the lack of both dose dependence and statistical significance of results do not allow us, at present, to draw any conclusions. Although the study has been considered a preliminary one, no other report on CYN carcinogenicity has been published in the mean time. No reproductive effects induced by CYN have been described up to now. HUMAN HEALTH RISK OF CYANOTOXINS EXPOSURE 105 FIG. 4. Chemical structure of (A) anatoxin-a and (B) homoanatoxin. 3.3.1. Anatoxins-a and Homoanatoxin-a Anatoxin-a is a bicyclic alkaloid; the presence of an additional methyl group is the only difference with homoanatoxin-a (Figure 4); the toxicological properties of the two structurally related molecules are almost identical. Anatoxin-a is a potent pre- and postsynaptic depolarizing agent, acting by binding to nicotinic receptors for acetylcholine in the central and peripheral nervous system, and in neuromuscular junctions (Carmichael, 1998). The toxin has a high acute toxicity: The ip LD 50 in mice is 375 µg/kg bw; death is due to muscular paralysis and respiratory failure in a very short time (Fawell and James, 1994), whereas after oral administration the LD 50 is >5000 µg/kg bw and death occurrs after a latency period (Fitzgeorge et al., 1994). The direct intravenous (iv) injection results in a higher toxicity (iv LD 50 < 100 µg/kg bw) and more rapid death (Astrachan et al., 1980). The toxin is rapidly absorbed after ingestion and it is readily degraded, and therefore a low bioaccumulating potential can be anticipated. The acute toxicity of homoanatoxin-a is similar to its analogous toxin (ip LD 50 = 330 µg/kg bw in mice), with overlapping symptoms and death within 5 10 minutes (Namikoshi et al., 2003). Some repeated toxicity studies (treating periods <2 months) are available on anatoxin-a (Fawell et al., 1999b; Astrachan et al., 1980); in all cases no effect was observed even at the highest doses tested (120 and 510 µg/kg bw per day in the 2 studies, respectively). Therefore a NOEL value could not be derived. It can be concluded that acute effects seem to represent the major concern for human health. When anatoxin-a was administered to pregnant rodents in the appropriate gestational days (i.e., the susceptibility windows for neurological development), no fetal abnormalities neither neurobehavioral late effects have been evidenced after in utero exposure, neither any effects of maternal toxicity (Fawell et al., 1999b; Rogers et al., 2005; MacPhail et al., 2005). 3.3. Neurotoxins Although with different mechanisms, all the known neurotoxins act on the neuromuscolar system, by blocking skeletal and respiratory muscles, leading to death for respiratory failure. The major groups are anatoxins and saxitoxins, whose major toxicological features are described in the following. 3.3.2. Anatoxins-a(s) Anatoxin-a(s) is the phosphoric ester of N-hydroxyguanidine (Figure 5); similarly to organophosphorous insecticides, to which is structurally related, it irreversibly inhibits acethylcholinesterase (AChE) activity in the neuromuscular junctions (Carmichael and Falconer, 1993), blocking hydrolysis of the
106 E. FUNARI AND E. TESTAI FIG. 5. Chemical structure of anatoxin-a(s). neurotransmitter. As a consequence, acethylcholine accumulates, leading to nerve hyperexcitability. Anticholinesterase activity of anatoxin-a(s) is limited to peripheral nervous system; indeed, the brain AChE activity is unaffected also at neurotoxin lethal doses (Cook et al., 1988). The affinity for the human AChE in red blood cells is relatively high, and therefore the risk of an acute intoxication for the human population is not negligible, higher than the one shown by some aquatic species (Carmichael, 1990). The LD 50 in mice after ip injection is 20 40 µg/kg bw (surviving time 5 30 min) (Mahmood and Carmichael, 1987; Matsunaga et al., 1989; Cook et al., 1988). Data on oral administration as well as on subchronic and/or chronic toxicity are not available. 3.3.3. Saxitoxins Paralytic shelfish poisoning (PSP) toxins are a family of more than 20 congeners of the same molecule, consisting of a tetrahydropurine group and 2 guanidine subunits; saxitoxin (STX) and neosaxitoxin structure is similar to that of carbamates (Figure 6). The great majority of reported toxicological data have been obtained with STX produced by marine organisms and only limited information is available for STX produced by freshwater cyanobacteria. However, the chemical structure and the toxicological profile of the toxins are the same, independently on their source. The various PSP toxins significantly differ in toxicity, with STX being the most toxic; they prevent electrical transmission (within the peripheral nerves and skeletal or cardiac muscles), followed by muscle and respiratory paralysis (Kao, 1993; Su et al., 2004; Wang et al., 2003). The mechanism of action is FIG. 6. General structure of PSP toxin. R4-1: carbamate toxins, including STX and neo-saxitoxin; R4-2: N-sulfocarbamoyl (or sulfamate) toxins, including GTX5 and GTX6; R4-2 and R4-3 decarbamoyl toxins, including dcstx. based on the blocking of Na channels in neuronal cells (Kao, 1993) and on Ca 2+ and K + channels blocking in cardiac cells, thus preventing the propagation of the action potential (Su et al., 2004; Wang et al., 2003). The cause of death is asphyxiation due to progressive respiratory muscle paralysis. The 7,8,9-guanidine function has been identified as the one involved in the channel blockade, whereas the removal of the carbamoyl group side-chain gives rise to a molecule with about 60% of the original toxic activity. The biological mechanism of action has been clarified for 50% of the natural analogues, suggesting that it could be basically the same for all the toxins within the PSP family. Saxitoxins are readily absorbed by GI tract; they diffuse through the blood brain barrier and are excreted mainly in the urine. Studies on their metabolism are very scant. However, clinical observations in patients surviving PSP intoxication for 24 hours suggest that PSPs undergo either rapid excretion, metabolism, or both. They can bioaccumulate in crustaceans and mollusks, which seem to be resistant to their toxic effects (Llewellyn et al., 1997); this feature determines the possibility of high levels of exposure for predators, including humans (Negri and Jones, 1995). In mice the ip LD 50 for STX is 10 µg/kg bw and the oral LD 50 is 263 µg/kg bw (Mons et al., 1998). The level at which PSP intoxications occur in humans varies considerably; indeed, an oral consumption of about 300 µg PSP toxin per person in one case was reported as nontoxic, whereas in another one it was fatal (FAO, 2004). According to the FAO report (2004), a total acute ingestion in the range 0.144 1.66 mg STX-eq (STX equivalants) per person mainly induces mild symptoms, whereas for consumption in a similar overlapping range of doses (0.456 12.4 mg STX-eq/person) a broad spectrum of effects has been described, ranging from moderate symptoms up to paralysis and death (Shumway, 1995; FAO, 2004). This high variability can be attributed to the different approaches and methods applied to quantify the actual level of exposure as well as to differences in individual susceptibility. The determinants of the severity of the effects are the specific toxicity of the PSP toxin(s) in the ingested food, the amount of food ingested, and the rate of elimination of the PSP toxin(s) from the body. When symptoms are mild to moderate, recovery from an STX intoxication is usually complete (Orr et al., 2004). In fatal cases, respiratory paralysis occurs within 2 to 12 hours after consumption of the PSP-contaminated food (FAO, 2004). The N-sulfocarbamoyl compounds are appreciably less toxic than the corresponding carbamoyl toxins. However, under acidic conditions, such as those in the gastric environment, the SO 3 group is lost, converting the toxins in the carbamoyl analogue (Aune, 2001), with increases in toxicity of up to 40-fold. This conversion may therefore have relevant health consequences, since fish containing weakly toxic N-sulfocarbamoyl toxins could result in severe poisoning episodes after ingestion and acidic hydrolysis in the stomach. No repeated toxicity data are available at present, nor are data on the genotoxic potential.
HUMAN HEALTH RISK OF CYANOTOXINS EXPOSURE 107 FIG. 7. General structure of LPS endotoxins. Some evidences of teratogenic activity have been provided in fish and amphibian larvae, in which STX concentrations 10 µg/l caused growth retardation; malformation and mortality occur at 500 µg/l (IPCS, 1984; Oberemm et al., 1999). However, no data are available on mammals. 3.4. LPS Endotoxins LPS endoxins are external components of cell membranes of most cyanobacteria as well as gram-negative bacteria. The molecule consists of three regions: an internal acylated glycolipid (termed lipid A), and a central area of liposaccharides, linking the internal subunit with the external specific carbohydrate polymer (O-specific chain) (Jann and Jann, 1984) (Figure 7). Among bacteria this external subunit shows the most diversity and is the basis for serological specificity, but also the lipid A moiety is variable. Cyanobacterial LPS endotoxins slightly differ from those typical of other bacteria in the three components, including the presence of small quantities of phosphates (Mayer and Weckesser, 1984; Kaya, 1996). LPS endotoxins exposure has been associated with local effects due to direct contact, such as skin or eye irritation, gastrointestinal problems or allergic reactions. However, these types of effects have never been experimentally reproduced and the LPS endotoxin mechanism of action is unknown. Indeed, the potential induction of gastroenteritis and inflammation problems has been often assumed in analogy with known effects of LPS from gram-negative bacteria, which have been extensively studied. It has been shown that they bind to transmembrane receptors within the Toll-like receptor family, initiating a cascade of host-mediated responses, among which the release of cytokines and other inflammatory mediators, stimulation of monocytes and macrophages, and congregation of neutrophils and platelets microcapillaries, followed by vascular injury (Heumann et al., 2002). Therefore, LPS endotoxins are not directly toxic, but their toxicity is associated with the interaction with host-mediated factors. Among different gram-negative bacteria, the lipid A moiety is considered the LPS component responsible for toxic effects, which can be extremely variable, up to totally inactive (Stewart et al., 2006a). Since the structure of the lipid A subunit in the cyanobacterial LPS molecule has not been clearly identified so far, no definite conclusion should be drawn on the degree of their
108 E. FUNARI AND E. TESTAI toxic potential, although some indirect data seem to suggest a role for LPS in cyanobacterial intoxication. As already mentioned, results from maximization tests clearly indicated that different cyanobacterial extracts induce high degree of skin sensitization, independent from the production of intracellular cyanotoxins (Torokne et al., 2001), suggesting a possible role for other cyanobacterial constituents, among which LPS endotoxins. However, during cyanobacterial blooms many other organic compounds (such as aldehydes, terpenoids and ketons), some of which are endowed with irritating and sensitizing properties, are dissolved in water. Therefore, it is possible that irritating and sensitizing effects observed so far were due to the concurrent presence of different etiologic agents. Very little is known on LPS endotoxins systemic effects; some data on lethality in mice after injection of LPS extracts from different cyanobacteria indicate that LD 50 values range between 40 and 190 mg/kg bw, although some exctracts caused no death at 250 mg/kg bw; in addition, qualitative dermonecrotic lesions in rabbit skin were described, following sequential subcutaneous and iv injections of the same LPS extracts (Stewart et al., 2006a). Due to the fact that cell wall fragments are readily aerosolized, inhalation of LPS might contribute in explaining cyanobacterialrelated adverse effects known as flue-like symptoms, characterized by cough, chills, sore throat, and fever. However, no clear association have been found and therefore it can be concluded that the health implications of cyanobacterial LPS are poorly understood and this topic requires more research. 3.5. Other Toxins Beside the already mentioned toxins, which have been detected in freshwater and brackish waters worldwide, other cyanotoxins have been identified, mainly in marine coastal areas in Hawaii and the Indo-Pacific, among which are aplysiatoxin, debromoaplysiatoxin, and lyngbyatoxin. These toxins have been indicated as the causative agents of contact dermatitis (swimmer s itch) in Hawaii (Serdula et al., 1982) and of intoxications due to the ingestion of contaminated meat from Chelonia midas, a marine turtle (Yasumoto, 1998). In the years 1993 1998 in Madagascar, consumption of meat from marine turtles led to some poisoning episodes; 414 people were intoxicated, 29 of whom died. Described symptoms included acute gastritis, mouth ulcers, burning of buccal mucosa and tongue with appearance of papule, salivation, headache, weakness, and fever (Champetier et al., 1998). Aplysiatoxin is a phenolic bislactone (Figure 8). When ip injected to mice, it showed an LD 50 value around 100 120 µg/kg bw; the cause of death was bleeding from the small intestine preceded by dilation of the lymphatic vessel and congestion of capillaries in the lamina propria (Ito and Nagai, 1998, 2000). At sublethal doses the major effect was diarrhea, induced by hypersecretion from edema in the caecum (Ito and Nagai, 1998). After oral administration, toxicity was reduced and sublethal effects on the small intestine were observed at much higher doses (3 mg/kg bw). When the toxin was iv injected (100 mg/kg), FIG. 8. Chemical structure of aplysiatoxin. the target vessels were in the lung: Fibrin deposition in the dilated pulmonary artery caused the appearance of a gap in the artery wall and, consequently, bleeding. In addition, bleeding effects on the small intestine were also present, due to fibrin deposition in the lumen via distension of the capillary wall (Ito and Nagai, 2000). Lyngbyatoxin has many similarities with aplysiatoxin in its mechanism of toxicity; indeed, ip injection of lethal doses in mice (250 µg/kg bw) induced severe damages in the villi capillaries, leading to bleeding in the small intestine; sublethal doses caused erosion in the stomach, small and large intestine and inflammation in the lung (Ito et al., 2002). After oral administration, the pathological outcome at sublethal doses was almost the same, but effects occured at higher doses (600 1000 µg/kg bw). No data on aplysiatoxin- or lyngbyatoxin-induced repeated toxicity are available at the moment, but it is known that they are potent tumor promoters, as well as debromoaplysiatoxin, acting through potentiation of protein kinase C as 12-Otetradecanoylphorbol-13- acetate (TPA) does (Fujiki et al., 1981, 1982). Cyanobacteria may also produce a number of other bioactive peptides, including microviridins, microginins, and cyanopeptolides. Their function, actual presence in the environment, and impact on human and environmental health are poorly known (Welker and von Doeren, 2006). Among the already mentioned peptides, microviridin J and BMAA have been the subject of some recent publications. Microviridin J, another metabolite of Microcystis spp., has been indicated as the cause for a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. The toxin consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Proximal hydrophobic interactions between Arg and other regions of the molecule result in the formation and stabilization of an additional ring system. The presence of Arg and its distinctive conformational interactions has been associated with microviridin J inhibition of trypsin, chymotrypsin, and trypsin-like proteases in the daphnid, presumably linked to the molting disruption. No data are available on the toxicological profile of this toxin in mammals (Rohrlack et al., 2003, 2004) It has been recently demonstrated that a wide variety of both free-living and symbiont cyanobacteria are able to produce β-nmethylamino-l-alanine (BMAA), a nonessential amino acid, at significant levels (Cox et al., 2005). Although with contrasting opinions, neurotoxic effects have been attributed to BMAA