Cyanotoxins Potentially Present in Cyanobacterial Food Supplements

Cyanobacteria used as dietary supplements can be a source of cyanotoxins even when the main ingredient is considered nontoxic, such as Artbrospira maxima. Nonetheless, some studies suggest a potential for “Spirulina ” products to contain cyanotoxins, possibly via contamination of cultures with other, toxigenic cyanobacteria: the anatoxin-a analogs epoxyanatoxin-a and dihydrohomoanatoxin-a have been identified at concentrations ranging from nondetectable to 19 pg/g dry weight in “Spirulina”-based dietary supplements (Salazar et al., 1996; Salazar et ah, 1998; Draisci et ah, 2001). A market analysis demonstrated concentrations of anatoxin-a ranging between 2.50 and 33 pg/g, whereby these included products intended for human and animal consumption (Rellan et ah, 2009). In alkaline crater lakes in Kenya, Arthrospira fusiformis was found to produce small amounts of both microcystins and anatoxin-a (Ballot et ah, 2004; Ballot et ah, 2005), and ELISA results were positive for microcystins in “Spirulina” food supplements, suggesting a contamination with a microcystin producer (Gilroy et ah, 2000). There are no proven cases of human injury as a result of ingesting “Spirulina"-based food supplements, although these were proposed as the cause of liver injury of a 52-year-old Japanese (Iwasa et ah, 2002). However, consumption of “Spirulina” as well as other cyanobacteria-based food supplements are frequently accompanied by massive diarrhoea, nausea, abdominal pain and skin rash (Rzymski & Jaskiewicz, 2017).

Nostoc commune produced by indigenous people of Peru were found to contain fi-methyl-amino-alanine (BMAA; Johnson et ah, 2008). However, the analytical method used is now known to substantially overestimate BMAA concentrations, and the toxic potential of BMAA is debated highly controversially. The conclusion of section 2.7 of the present volume is that, at present, the weight of evidence suggests that BMAA is present in insufficiently high concentrations to cause neurogenerative diseases.

Aph. flosaquae can contain cylindrospermopsins, anatoxin-a and saxi- toxins as well as toxicity not attributable to any of the known cyanotoxins (see Heussner et ah, 2012, and Chapter 2). Although microcystin production has not been observed for Aphanizomenon sp., in natural blooms, Aphanizomenon sp. is often found associated with other cyanobacteria which are known to be toxigenic.

Common cyanobacteria associated with blooms of Aphanizomenon sp. are Microcystis sp. and Dolichospermum sp., that is, species that potentially produce microcystins (Ekman-Ekebom et ah, 1992; Teubner et ah, 1999; Wood et ah, 2011; Shams et ah, 2015; Chapter 4). Analysis of Aph. flosaquae samples taken from Lake Klamath for dietary supplement production demonstrated that approximately 80% of the samples taken between 1994 and 1998 contained >1 pg MC-LR equivalents per gram dry weight, which is the maximum acceptable content established by the state of Oregon in the USA (Gilroy et ah, 2000). Further studies showed higher as well as lower microcystin contents (Table 5.7), which is partly attributed to shifts in taxonomic composition within the blooms in Lake Klamath dominated by Aph. flosaquae, in particular, the variable share of toxigenic Microcystis sp. in bulk phytoplankton biomass. The studies summarised in Table 5.7 show a trend to lower maximum microcystin contents over time.

Table 5.7 Microcystin concentration in Aphanizomenon sp. dietary supplements from the market

Number of Samples

% samples exceeding 10 iglg DW

Microcystin content Mg/g DW

Detection

method

Reference

87

72

2.2-10.9

ELISA

Gilroy et al. (2000)

52

50

0-35.7

ELISA

Lawrence et al. (2001)

0-49.0

cPPA

0-35.7

LC-MS/MS

6

100

1 1-24.7

ELISA, cPPA HPLC

Schaeffer et al. (1999)

18

80

0.3-8.3

Adda-ELISA

Hoeger & Dietrich

0.5-5.9

cPPA

(2004)

12

33

0.1 -4.7

ELISA

Saker et al. (2005) Saker et al. (2007)

26

35

LC-MS/MS

Vichi et al. (2012)

10

60

Adda-ELISA

Heussner et al. (2012)

50

cPPA

40

1 1.0

LC-MS/MS

60

6

0-3.0

LC-MS/MS

Marsan et al. (2018)

7

<0.25-2.8

PPA

DW: dry weight; LoD: limit of detection; ELISA: enzyme-linked immunosorbent assay; cPPA: colorimetric protein phosphatase inhibition assay, HPLC: high-pressure liquid chromatography; LC-MS/MS: liquid chromatography-mass spectrometry; Adda-ELISA: enzyme-linked immunosorbent assay with a recognition antibody specifically directed against the Adda-moiety of microcystins.

Assessing and Managing the Risk of Cyanotoxin Exposure through Food Supplements

In the studies summarised in Table 5.7, maximum contents of microcystin per gram dry weight range between 3.0 and 49 pg/g, and therefore, a risk of exposure to cyanotoxins cannot be ignored. A detailed assessment, however, is difficult, firstly, because the manufacturer’s recommendations for daily consumption vary widely from 0.5 to 15 g/day with some products indicating no maximum limit (iMarsan et al., 2018) and, secondly, because individual consumption also varies and may largely exceed recommendations. However, based on reported possible toxin contents and a consumption of a few grams per day, exposure may well be at levels exceeding the provisional tolerable daily intake (TDI) of 0.04 pg/kg (see section 2.1) for adults and especially for children. Further, in deriving its drinking-water guideline values (GVs) for lifetime exposure, 20% of intake are allocated to sources other than drinking-water, which may not be appropriate for persons consuming cyanobacterial products on a regular basis (see sections 2.1 and 2.2). Dietrich and Hoeger (2005) discuss these aspects for varying levels of microcystin contamination of food supplements and propose corresponding maximum amounts that can be safely consumed by infants, children and adults.

As with other health risks, animal poisoning indicate potential adverse health effects in humans (Hilborn & Beasley, 2015). The case of an 11-year-old female spayed pug dog, weighing 8.95kg and presenting with abnormally high alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) activities and serious liver dysfunction, indicates uptake of a hepatotoxin. This dog was fed single to multiple daily rations of 1 gram of 100% certified organic Aph. flosaquae for approximately three and a half weeks. The analysis of the powder via LC-MS/MS revealed 0.166 pg/g of MC-LR and 0.962 pg/g of MC-LA, while no other MCs were reported (Bautista et al., 2015). Thus, the MC content would approximate the Oregon provisional guidance value of 1 pg/g dw (Gilroy et al., 2000). However, with an analytical method including more microcystin variants, as suggested in section 14.3, a higher actual total MC content may have been found. Further, neither the number of daily rations nor any further potential source of the dog’s exposure - such as cyanobac- terial blooms in a waterbody - are known, making it difficult to estimate retrospectively whether the undoubted exposure to microcystins through dietary supplements was enough to explain the observed symptoms in this single study on one animal.

A further issue in this context is the as of yet very incomplete understanding of the bioactivity of cyanobacterial metabolites beyond the known toxins. Underdal et al. (1999) found protracted toxic response in test animals exposed to extracts of Aph. flosaquae but could not identify any toxins. Similarly, Heussner et al. (2012) found cytotoxicity in Aph. flosaquae product extracts that were not associated with any of the known cyanobacterial toxins. Indeed, particularly Apbanizomenon species are known for inducing effects not yet explained by any identified cyanobacterial metabolite, for example, malformation of fish embryos (Oberemm et al., 1997; Berry et al., 2009). While such effects cannot be quantitatively used for a human health risk assessment, they do indicate potential presence of further hazards to clarify.

Further, field collections of cyanobacteria and, possibly to a lesser extent, cyanobacteria harvested from open tanks contain a high diversity of hetero- trophic bacteria, including human pathogens (Berg et al., 2009) that may cause further health hazards.

Approaches to Assessing and Controlling the Potential Cyanotoxin Hazards

The regulation of dietary supplements is generally less strict compared to regulations for food, pharmaceutical or drinking-water, and only few regulatory schemes are in place. For example, since 1994, dietary supplements have been regulated in the USA under the Dietary Supplement Health and Education Act (DSHEA; FDA, 2017). Because cyanobacteria are capable of producing toxins and their presence has been confirmed in some dietary supplements, it is appropriate to regulate and monitor these toxins in dietary supplements, including the provision of adequate information to consumers. Considerations include the following:

Testing for cyanotoxin content: Biomass collected from natural blooms or open tank incubators should be tested, lot by lot as recommended by the regulatory authority, for possible contamination with potentially toxigenic cyanobacteria, for example, Microcystis sp. in blooms dominated by Apb. flosaquae. Production lots should be managed by unique identifying numbers and production dates. For potential subsequent reanalysis by regulatory authorities, producers should be mandated to retain representative samples of each charge produced and to make these available upon official request.

Testing for other contaminants: Dietary supplement products should be tested for other potential contaminants, including indicators for pathogenic bacteria and protozoa, where and when contamination is expected. This is best based on an assessment of contamination risks from the catchment or the culture conditions. Examples of contamination sources include excreta of migrating birds or surface runoff following rainfall.

Claims on possible effects: The proposed beneficial effects of the consumption of cyanobacterial food supplements have not been demonstrated in scientifically sound studies; only subjective and anecdotal evidence is proposed by the vendors. Therefore, product information should not suggest that consumption of larger amounts would produce more positive effects.

Consumer information: Producers should clearly inform the consumers which quality control procedures are in place and give access to the test results. Further they should give a clear maximum daily doses, specified for infants, children and adults. None of these measures, however, can serve to protect from negative effects of known and yet unknown bioactive substances in cyanobacteria, as discussed in section 2.10.

 
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