Cyanobacterial Toxins -- Microcystin-LR

Toxin Production and Persistence

The factors inducing the production of toxins by cyanobacteria are not well known. Laboratory studies demonstrate that some of the same environmental factors as above, such as temperature, light, nitrogen concentrations, carbon availability (in the form of bicarbonate, carbonate and carbon dioxide), phosphate concentrations and pH, could be important. As toxin production varies greatly among different strains of the same species, genetic differences and metabolic processes may also be important in the production of these secondary metabo-lites. Studies have shown that the ability to produce toxins can vary temporally and spatially at a particular site.⁶

Cyanobacterial toxins tend to be associated with cyanobacterial cells and may be membrane bound or occur free within the cells. In laboratory studies, most of the toxin release occurs as cells age and die and passively leak their cellular contents; some active release of toxins can also occur from young, growing cells.^5,11

Toxin levels do not necessarily coincide with maximum algal biomass; there can be significant variation in the amount of toxin per unit biomass of cyanobacteria over time, which is independent of changes in the blue-green algal population. Kotak et al.¹³ found higher concentrations of microcystins in blooms taken during the day than at night, whereas a study in Australia found no significant difference in toxin concentrations from cyanobacteria incubated for 24 hours at different depths in a reservoir.¹⁴

Microcystins are relatively persistent in the aquatic environment. Studies in Australia have shown that microcystin-LR was present up to 21 days following treatment of a Microcystin aeruginosa bloom with an algicide.¹⁵ Studies conducted in natural waters in the United Kingdom indicated that five days were required for the destruction of 50% of the toxin.¹⁶ Biodegradation and photolysis are means by which released micro-cystin-LR can naturally decrease in concentration.^17,18 Cousins et al.¹⁹ demonstrated that the primary biodegradation of microcystin-LR in reservoir water had a first half-life of approximately four days. It was noted, however, that the half-life of this toxin in natural waters would likely vary considerably with changes in water temperature and the size of the microbial population.

Levels of microcystin-LR in Alberta lakes and dug-out ponds, measured using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (according to the method of Harada et al.,²⁰ as modified slightly by Kenefick et al.²¹), ranged from 4 to 605 µg/g dry weight (dw) of biomass²² or up to 1500 µg/g.¹⁰ Similarly, microcystin-LR levels in natural blooms of Microcystis in Japan between 1989 and 1991 ranged from 27 to 622 µg/g dw of biomass.¹² In the same blooms, the levels of microcystin-RR and microcystin-YR ranged from 11 to 979 and from 9 to 356 µg/g dw of biomass, respectively, with a total maximum micro-cystin level of 1732 µg/g dw of biomass.¹²

Exposure to Microcystins

The major route of human exposure to cyano-bacterial toxins is the consumption of drinking water. Minor exposure routes are the recreational use of lakes and rivers (oral and dermal routes) and, for some people, the consumption of certain algal food supplements (oral route); products containing non-Spirulina blue-green algae harvested from natural lakes have been found to contain microcystin toxins.²³ Another minor route of exposure would be from the use of showers (inhalation). The extent to which cyanobacterial toxins move up the food chain (freshwater mussels and fish) has not been widely investigated, although covalently bound microcystins have been detected in the tissues of both freshwater and saltwater mussels^24,25 and in the liver of salmon²⁶ in recent studies. There is also some preliminary experimental evidence that the toxins, or their transformation products, can move up the aquatic food chain. Fish consuming Daphnia magna exposed to radiolabelled microcystin-LR accumulated the radio-label in their tissues.²⁷ The duration of exposure to microcystins by these various routes would generally be shorter in countries such as Canada than in those with milder climates, such as Australia.

Absorption of microcystin-LR through skin contact is unlikely, as microcystin-LR does not readily cross cell membranes.²⁸ Microcystin-LR is water soluble and non-volatile; therefore, inhalation and absorption through the lungs are unlikely, unless microcystin-LR is present as an aqueous aerosol in air.²⁹ Although for some people intake may occur through the ingestion of blue-green algal products and possibly fish or seafood, for most people the major route of exposure to microcystin-LR is drinking water consumption.

The levels of microcystin in the raw intake water for two Alberta drinking water supplies (communities not specified), as measured by the sensitive phosphatase inhibition bioassay, ranged from 0.15 to 4.3 µg/L, with a large coefficient of variation (59%) for hourly fluctuations over an 11.5-hour period; in treated water, levels ranged from 0.09 to 0.64 µg/L, with a small coefficient of variation (10%). Over a five-week period, similar coefficients of variation were obtained in the two types of samples.¹⁰

In the summer of 1993, microcystin-LR was detected (detection limit 0.05 µg/L) in raw water samples (maximum 0.45 µg/L) collected from Shoal Lake, Ontario, and from within the treated tap water distribution system (maximum 0.55 µg/L) following the identification of Microcystis aeruginosa blooms in Shoal Lake.^30,31 No toxin-producing algae were detected in Deacon Reservoir, Winnipeg's main storage facility for water from Shoal Lake. In 1995, 160 surface water supplies, located mainly in southwestern Manitoba, were chosen for algal study. Microcystin-LR was detected (detection limit 0.1 µg/L) in 70% of the raw water supplies, 93% of the municipal water supplies, 57% of the dugouts used for domestic and shared domestic plus livestock water consumption, 84% of the dugouts used exclusively for livestock and 44% of the recreational use sites. Treated water samples were analysed only if the raw water supplies were found to have detectable levels of toxins. Toxin was present in 68% of the treated water samples collected from both the municipal sites and the dugout sites. Thus, it appears that conventional water treatment methods may be only partially successful in removing the toxins. Toxin concentrations ranged from <0.1 to 1.0 µg/L in raw water samples and from <0.1 to 0.6 µg/L in treated water samples. Dugouts used extensively for livestock consumption had the highest levels of toxin. Two samples taken directly from an algal bloom on a livestock dugout had toxin levels of 1.5 and 8.0 µg/L. A much higher microcystin-LR level has also been reported. On September 9, 1996, a sample collected at Victoria Beach on Lake Winnipeg (used for domestic and recreational purposes) had a microcystin-LR concentration of 300 µg/L, whereas microcystin-LR levels had declined to 0.2 µg/L in a follow-up sample taken later in the month.³² At that time, a large algal mass had been concentrated by the wind into the shore area; Aphanizomenon flos-aqua, Microcystis flos-aqua and Microcystis aeruginosa were the dominant algal species.

In a follow-up study, Jones et al.³¹ investigated microcystin-LR in a smaller number of water supplies (n = 7) over a longer period of time (June-December). The data indicated that microcystin-LR was present throughout the entire sampling period and that it could persist for long periods (more than two months) following the decline of the algal population. No relationship was found between levels of microcystin-LR and blue-green algal densities or environmental variables, such as physico-chemical properties or nutrient loads. Microcystin-LR concentrations were -1 µg/L in all treated samples.

In a biweekly survey of dugouts in the Peace River area of northern Alberta in the summer of 1997, 11 of 12 dugouts (all used for domestic purposes, usually without treatment) had concentrations of microcystin-LR above 0.5 µg/L at least once during the sampling period (July 23 - August 20), with six having levels above 1.5 µg/L on at least one occasion.³³ In a follow-up study during the summer of 1998, 18 dugouts and nine municipal reservoirs were sampled on a biweekly basis between July 7 and September 24.³⁴ The study was designed to examine the effects of dugout aeration and nutrient loading on toxin levels. In contrast to the previous year's study, 97% of the samples had microcystin-LR levels below the detection limit (0.3 µg/L); the maximum reported concentration was 0.5 µg/L. The authors were unable to assess the effects of aeration or nutrient loading on toxin formation.

It is apparent that levels of microcystin-LR exceeding 0.5 µg/L occur from time to time in those provinces where measurements have been made so far. It is likely that microcystins also occur in the surface water supplies in other Canadian provinces, but no monitoring data are available. With appropriate treatment, the maximum concentration of total microcystins in drinking water in Canada is probably less than 1 µg/L, based on the above data. Average concentrations would probably be well below this.

A two-year American Water Works Association Research Foundation (AWWARF) survey on 45 utility waters in the United States and Canada was carried out between 1996 and January 1998.³⁵ Eighty percent of the 677 samples received contained microcystins (using enzyme-linked immunosorbent assay [ELISA]), with 4.3% of these having microcystin concentrations above 1 µg/L. Only two finished water samples had microcystin levels of >1 µg/L, which indicated that most water treatment plants involved with the survey had adequate treatment regimes at the time to reduce toxin levels.

Analytical Methods

The analysis of microcystins in drinking water is an emerging area of research, and there are few "standard methods" available (for a brief review, see Ressom et al.⁶). For several methods, there is a need for inter-laboratory collaborative trials.

In comparing the various analytical methods being used for microcystin-LR and other microcystins and their detection limits, it may be useful to distinguish screening methods -- such as the mouse bioassay, ELISA and the phosphatase bioassay, which are conducted prior to cleanup and which indicate the presence of toxins in samples -- from methods that are conducted for the identification and quantification of the various individual microcystins.³⁶ Often more than one toxin may be present in a sample. The consensus among those using analytical methods is that a single method will not suffice. The best approach for monitoring is to use a combination of screening and more sophisticated and costly specific chemical methods.

Although the guideline is specific to microcystin-LR, it is important that one measures total microcystins. "Total microcystins" includes all microcystin variants, not just microcystin-LR, that are occurring free in the water and are bound to or inside cyanobacterial cells. Thus, sample preparation may need to include sonification (to break up cells) and a variety of extraction procedures in order to isolate the different (i.e., more lipophilic or polar) microcystins. So far, published studies on the levels of microcystins in water supplies have generally not clearly indicated whether total microcystins or free microcystins were measured. It is also important that samples be taken at a post-treatment site.

Purified standards are commercially available for microcystin-LR, -RR, -YR, -LW and -LF toxins.

Screening

The mouse bioassay still plays an important role as a screening method, as it gives the total toxic potential of the sample within a few hours, and it is possible to distinguish hepatotoxins from neurotoxins.¹ For screening, many scientists prefer the protein phosphatase bioassay. This method is sensitive to sub-nanogram levels of microcystins^29,37,38 and can be conducted on 30 µL of sample. With this method, 100 samples can be analysed in one day.³⁹ The method, however, is not specific to microcystins and will indicate the presence of other substances inhibiting protein phosphatases. This should not be a problem, however, when monitoring a particular area where the potentially occurring

species and their possible toxins are known. In conjunction with HPLC, the method is useful in identifying toxic fractions. A colorimetric adaptation is also in use.⁴⁰ A published ELISA method, using polyclonal antibodies, is available,⁴¹ with a detection limit of 0.2 ng/mL; this method is commercially available. It is likely that methods using monoclonal or polyclonal antibodies raised against a single toxin (e.g., microcystin-LR) will have problems of cross-reactivity with other microcystins. Using the method of Chu et al.,⁴¹ Carmichael⁴² found that most of the important micro-cystins reacted with the antibody, but about 10% did not. A method using polyclonal antibodies is also under development in Canada (Prairie Biological Research Laboratories, Edmonton) and is awaiting validation.⁴³ A method using monoclonal antibodies is under development in Japan.⁴⁴

Identification

For identification, Harada³⁹ has used improved frit micro liquid chromatography/mass spectrometry (LC/MS) with fast atom bombardment (FAB), with a detection limit of 1 ng.⁴⁵ After isolation and purification by HPLC and thin-layer chromatography (TLC), Sivonen⁴⁶ assigned structures to many microcystins and their variants by FABMS, FABMS/MS and ¹H nuclear magnetic resonance (NMR).

Separation and Quantification

There are several HPLC methods, many of which are variations on methods developed by Siegelman et al.⁴⁷ and Harada et al.²⁰ In the United Kingdom, an official HPLC method with UV detection and a detection limit of 0.5 µg/L has been developed by the Water Research Centre (WRc) in Medmenham. This method has been tested in a limited collaborative trial with five laboratories for drinking water and reservoir water.³It is designed to measure only free microcystin-LR; it does not measure total microcystin-LR (i.e., free plus cell bound) or micro-cystins other than microcystin-LR. It could be modified, however, to measure total microcystins.

For separation and quantification, HPLC with fluorescence or chemoluminescence detection (sub-nanogram detection limit) and micro LC/MS with FAB (single ion monitoring, or SIM) (detection limit ~1 ng) are the preferred methods, partly because of their lower detection limits compared with the UK HPLC method.³⁹ An HPLC method with photodiode array detection is being used by G. Codd (University of Dundee) but has not been published (see Edwards et al.⁴⁸).