Toxicity: Mode of Action
The toxic effects of microcystin, summarised in the following, are described in detail in the WHO Background Document on Microcystins (WHO, 2020; see there for further information and references). In summary, microcystins need a membrane transporter to enter cells - that is, the organic acid transporter polypeptides (OATP) which are expressed particularly in the liver but also in the intestinal tract and in some other tissues. Experiments have shown that when OATP is inhibited or lacking, liver damage is reduced. The essential role of OATP explains why most of the MCs ingested are taken up by the liver. While detoxification occurs in the liver, clearance of MCs seems to take a long time, up to weeks. Once in cells, MCs cause protein phosphatase (PP1, PP2A and PP5) inhibition, resulting in destabilisation of the cyto- skeleton followed by cellular apoptosis and necrosis. High acute doses thus cause haemorrhage in the liver due to the damage of sinusoidal capillaries. At low doses (below 20 pg/kg bw) and with repeated long-term exposure, phosphatase inhibition induces cellular proliferation, hepatic hypertrophy and tumour promoting activity.
There is a growing body of evidence indicating harmful microcystin-related neurological and reproductive effects, but the data are not yet robust enough to use as a basis for guideline development.
While some cyanobacterial extracts show genotoxicity, pure microcys- tins do not, and cellular DNA damage observed after in vitro treatments with pure MC may be due to the induction of apoptosis and cytotoxicity rather than direct effects on the DNA. On this basis, IARC has classified microcystins as Group 2B, possibly carcinogenic to humans (IARC, 2010), based on their tumour promoting activity mediated via protein phosphatase inhibition (a threshold effect) rather than genotoxicity.
Derivation of Provisional Guideline Values
The following section is taken directly from the WHO chemicals background document on microcystins which discusses the considerations for the derivation of provisional guideline values for exposure to microcystins in more detail (WHO, 2020). Insufficient data are available to derive a GV for MC variants except MC-LR. The two key oral toxicity studies of the effects of MC-LR on liver toxicity on which human health-based guideline values can be calculated are the following:
- • Fawell et al. (1999): Mice of both sexes given MC-LR by gavage at 40 pg/kg bw per day for 13 weeks did not show treatment-related effects in the parameters measured. Only slight hepatic damage was observed at the lowest observed effect level (LOAEL) of 200 pg/kg bw per day in a limited number of treated animals, whereas at the highest dose tested (1 mg/kg bw per day), all the animals showed hepatic lesions, consistent with the known action of MC-LR.
- • Heinze (1999): Exposure of male rats (females were not included) to MC-LR in drinking-water for 28 days at doses as low as 50 pg/kg bw per day (identified as the LOAEL) resulted in increased liver weight, liver lesions (with haemorrhages) and increased ALP (alkaline phosphatase) and LDH (lactate dehydrogenase), but no changes were measured in the mean levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) which are early markers for hepatotoxic- ity. Some of the histological effects, including Kupffer cell activation and PAS staining, showed no dose response since all 10 animals at the low and high doses displayed a similar degree of damage.
Although the duration of the Heinze (1999) study was shorter and more applicable to the exposure duration envisaged for application of the shortterm guideline value, the advantage of the Fawell et al. (1999) study is that an additional uncertainty factor is not needed for extrapolation from a LOAEL to a NOAEL, which would increase the total uncertainty and reduce the confidence in the derivation of the short-term guideline value. For this reason, the NOAEL derived by Fawell et al. (1999) was selected as the basis for the short-term and recreational guideline values as well as the lifetime guideline value.
The GVs for MC-LR are considered provisional due to inadequacies in the database as reflected in section 6.2 of the background document (WHO, 2020) and the database uncertainty factor (UF) of 1000 for the lifetime GV.
Calculation of provisional tolerable daily intake for microcystin-LR
where
TDIMC chronic = tolerable daily intake for chronic exposure
NOAEL = no-observed-adverse-effect level (40 pg/kg bw per day, based on Fawell et al., 1999)
UF = uncertainty factor (1000 = 10 for interspecies variation x 10 for intraspecies variation x 10 for database deficiencies, including use of a subchronic study)
For comparison, if the LOAEL from Heinze (1999) is used as the point of departure and incorporating uncertainty factors of 10 for inter- and intraspecies variability and 10 for database uncertainties including the use of a LOAEL (as per WHO policy), then the TDI would be 0.05 pg/kg per day.
Calculation of provisional lifetime drinking-water guideline value for microcystin-LR
where
GVchronk = guideline value for chronic (lifetime) exposure
NOAEL = no-observed-adverse-effect level (40 pg/kg bw per day, based on Fawell et al., 1999)
bw = body weight (default = 60 kg for an adult)
P = fraction of exposure allocated to drinking-water (80%, as other sources of exposure such as air, food and soil are considered minor for lifetime exposure)
UF = uncertainty factor (1000 = 10 for interspecies variation x 10 for intraspecies variation x 10 for database deficiencies, including use of a subchronic study)
C = daily drinking-water consumption (default = 2 L for an adult).
Calculation of provisional short-term drinking-water guideline value for microcystin-LR
where
GVshort.term = guideline value for short-term exposure
NOAEL = no-observed-adverse-effect level (40 pg/kg bw per day, based on Fawell et al., 1999)
bw = body weight (default = 60 kg for an adult)
P = fraction of exposure allocated to drinking-water (default for shortterm exposure = 100%, as drinking-water is expected to be the most significant source of exposure)
UF = uncertainty factor (100 = 10 for interspecies variation x 10 for intraspecies variation)
C = daily drinking-water consumption (default = 2 L for an adult).
Calculation of provisional recreational water guideline value for microcystin-LR
where
GVrecreation = guideline value for recreational exposure NOAEL = no-observed-adverse-effect level (40 pg/kg bw per day, based on Fawell et al., 1999)
bw = body weight (default = 15 kg for a child)
UF = uncertainty factor (100 = 10 for interspecies variation x 10 for intraspecies variation)
C = daily incidental water consumption (default = 250 mL for a child).
The provisional recreational guideline value, aimed to protect from systemic effects, is based on exposure of a child because the lower body weight and higher likely water intake (as a function of body weight) were considered worst case, and on a conservative scenario of a 15 kg child swallowing 250 mL of water (WHO, 2003b).
Considerations in applying the provisional guideline values
The provisional guideline values are based on toxicological data for MC-LR. However, MCs usually occur as mixtures. In the absence of oral toxicity data for other congeners, it is recommended that these values be applied to total MCs as gravimetric or molar equivalents based on the assumption that all MCs have similar toxicity to MC-LR. The kinetic differences among variants mean that further investigation of the oral toxicity of MC variants other than MC-LR is warranted reduce this relevant source of uncertainty.
In some regions, other sources of exposure besides drinking-water can be significant (see section 2.4). This includes food from locations where blooms have a long duration and there is high consumption of locally affected food items (e.g. fish eaten with viscera, or shellfish). In such situations, it may be appropriate to consider reducing the allocation factor for the lifetime and short-term drinking-water GVs based on relative exposure data for the population.
The short-term drinking-water GV is intended to provide guidance on how much the lifetime GV can be exceeded for short periods of about 2 weeks until enhanced water treatment or other measures can be implemented. It is not intended to allow for repeated seasonal exceedances of the lifetime GV.
The short-term drinking-water guideline value is based on exposure of adults. Since infants and children can ingest a significantly larger volume of water per body weight (e.g., up to 5 times more drinking-water/kg bw for bottle-fed infants compared to adults), it is recommended that alternative water sources such as bottled water are provided for bottle-fed infants and small children when MC concentrations are greater than 3 pg/L for short periods, as a precautionary measure.
