A problem blooms…

Microcystins are cyclic peptides produced by cyanobacteria in surface water that experience seasonal algal bloom. They are toxic to humans and animals, with effects ranging from liver damage to increased occurrences of liver cancer – but how best to detect and monitor them? K. H. Cha and Kefei Wang have some answers… Microcystins (MCs) are a natural substance produced by cyanobacteria, a microorganism that forms a part of many freshwater and marine ecosystems. Cyanobacteria produce a whole range of toxins known collectively as cyanotoxins, which are contained within the cell during its life and released during cell breakdown or lysis. The toxins tend to enter water in large quantities following the end of a cyanobacteria bloom, when the bacteria have multiplied to high densities in water rich in nutrients and exposed to sunlight. Of these cyanotoxins, microcystins are the most commonly found in surface water. The contamination of drinking water with microcystins is a truly global problem to public health. Microcystins have been found on every continent and in almost every part of the world. Their presence in surface water depends on the timing and duration of the blooms that release them, which may range from two to four months in temperate climates to almost all year round in dry subtropical regions¹. The physical qualities of microcystins make their presence in drinking water even more problematic. Microcystins are cyclic peptides, formed of seven amino acids with the two terminal amino acids of the linear peptide joined to form a cyclic compound. They are a relatively large natural product, with a molecular weight of approximately 800-1,100¹, water soluble and extremely stable. MCs remain potent even after water is boiled and in dark natural waters may persist for months or even years. Although microcystins do not affect aquatic life, they are known to be toxic to mammals. Acute MC poisoning has been observed in animals ranging in size from ducks and other waterfowl, all the way up to rhinoceroses². The impact of the toxin on humans has also been noted for many hundreds of years. During the Han dynasty around 1,000 years ago, General Zhu Ge-Ling led his troops to a river crossing during a military campaign in Southern China. Many of his troops were then said to have died from poisoning after drinking the river water, which had turned green¹. Microcystins’ first appearance in scientific literature came in 1878, when George Francis recorded the acute cyanotoxic poisoning of his domestic animals on Lake Alexandrina, Australia³. [caption id="attachment_37747" align="alignleft" width="200"] Figure 1. Full scan spectrum of MCs[/caption] Subsequently, extensive research has been conducted into the toxic impact of MCs on humans. Microcystins primarily impact the liver; lower concentrations result in liver damage, while acute exposure to high concentrations can cause death from liver haemorrhage or liver failure. Gastrointestinal illness has been reliably attributed to microcystin toxins in the water supply. In Zimbabwe, children living in one area of the city of Harare became sick each year with gastroenteritis; this was found to be caused by the decay of the natural microcystin bloom in the reservoir supplying that district⁴. The kidneys, lungs and intestines may also be affected. Long-term exposure to even low levels of MCs can be damaging, and has been linked, for instance, to liver cancer. In Southern China, liver cancer mortality rates vary greatly from village to village, depending on whether the water supply is sourced from deep wells or surface water, such as ponds and ditches. Since cyanobacteria are abundant in the surface water of this region, it has been proposed that microcystins in drinking water are responsible for this variation⁵. Studies have suggested a connection between MCs and tumour promotion, although further research is required to understand the genotoxic and tumour promoting potential of these toxins⁶. [caption id="attachment_37749" align="alignright" width="200"] Figure 2. Chromatograph of 0.5 ppb mixed standard of MCs.[/caption] Humans most frequently come into contact with microcystins through the consumption of contaminated drinking water. People may also be exposed during recreational water use and there is a limited body of research into inhalation through aerosols, such as while showering, although more work needs to be done in this area⁷. Some groups of people are at particular risk to the toxin, including children, because they drink a higher volume of water than adults in proportion to their body weight. MCs present greater danger to people with existing injury to susceptible organs, through hepatitis, liver cirrhosis or kidney damage for example. Kidney dialysis patients are especially vulnerable if exposed to microcystins in the water used for their dialysis, as they are exposed intravenously to large volumes⁸. [caption id="attachment_37751" align="alignleft" width="200"] Figure 3. Calibration of MCs.[/caption] Due to the toxic effects of microcystins, WHO has established a guideline of 1.0 µg/l to regulate the common variant MC-LR¹. This threshold value has been adopted by many countries, including China, France and Brazil. Countries such as Italy and South Africa have chosen to set an even lower acceptable limit⁹. The detection of microcystins is frequently conducted through bioassay. However, for the monitoring of drinking water, WHO recommends the use of physiochemical analysis because of the need to rapidly screen a large number of samples on a regular basis and to detect very low concentrations of the toxin. Moreover, liquid chromatography coupled with mass spectrometry (LC-MS) is highlighted as the technique offering the most effective combination of sensitivity and selectivity¹. The following case study demonstrates how an LC-MS may be used to detect MC levels as low as 0.05 ppb, far exceeding the WHO guidelines to ensure the safety of drinking water. Case study The Bruker EVOQ was used to quantify three common cyanobacterial toxins, MC-LR, MC-YR and MC-RR, in drinking water, by direct injection. To prepare the samples, Microcystin RR, LR and YR mixture (5 ug/mL in Methanol) was purchased from Sigma-Aldrich (St. Louis, MO) and diluted with ultrapure water for preparation of calibration standards from 0.050 to 50 ppb. Instrument calibration:

Mass Spectrometer (EVOQ Elite)
ESI	+4500 V
Probe temperature	350°C
Probe gas	50 units
Nebuliser gas	50 units
Cone gas temperature	200°C
Cone gas	15 units
Active exhaust	On
Collision gas	Argon at 1.5 mTorr
Optimised MRM transitions:
MC-RR (MW: 1038)MC-LR (MW: 995) MC-YR (MW: 1045)	m/z 520 > 135 (CE = 24V)m/z 498 > 135 (CE = 11V) m/z 523 > 135 (CE = 9V)

 

Chromatograph (Advance UHPLC)
Column	ACE® Excel 2 C18, 100mm x 2.1 mm i.d.
Injection volume	50 µL
Flow rate	0.4 mL/min
Column temperature	40°C
Mobile phase A	Water with 0.1% Formic acid
Mobile phase B	Acetonitrile
Gradient conditions:
0.00 min1.00 min 7.00 min 7.10 min 10.00 min	30% B30% B 95% B 30% B 30% B

The full scan spectra of three MCs are characterised by their predominant doubly charged ions as shown in Figure 1. These were used as precursor ions for matrix reaction monitoring (MRM) method development due to their high sensitivity. The MCs share the common product ion of m/z 135. [PhCH₂CH (OCH₃)]⁺ as a result of cleavage of the methoxy group of the ADDA residue. The LC-MS/MS method has proved sensitive to detect 0.05 ppb of MCs in water with good repeatability (RSD ? 10%, n=7). Figure 2 shows the representative chromatogram of a 0.05 ppb standards. Figure 3 shows the MC calibration curves from 0.05 ppb to 50 ppb, demonstrating excellent linearity with R2>0.997. The presence of microcystins in drinking water is a truly global issue. Exposure to the toxin has serious consequences for human and public health; hence it is vital that levels are monitored to preserve the safety of drinking water. The EVOQ Elite can quickly and accurately analyse large quantities of water samples using the LC-MS/MS method, and has the exceptional sensitivity to easily exceed the guidelines set by WHO. References

1. WHO, Toxic Cyanobacteria in Water: A guide to their public health consequences, monitoring and management, World Health Organisation, Geneva, 1999.
2. Carmichael, W.W. 1992 A Status Report on Planktonic Cyanobacteria (Blue Green Algae) and their Toxins. EPA/600/R-92/079, Environmental Monitoring Systems Laboratory, Office of Research and Development, US Environmental Protection Agency, Cincinnati, Ohio.
3. Francis, G. 1878 Poisonous Australian lake. Nature 18,11-12.
4. Zilberg, B. 1966 Gastroenteritis in Salisbury European children - a five-year study. Cent. Afr. J. Med., 12(9), 164-168.
5. Yu, S. -Z. 1989 Drinking water and primary liver cancer. In: Z.Y. Tang, M.C. Wu and S.S. Xia [Eds] Primary Liver Cancer. China Academic Publishers, New York, 30-37; Yu, S.-Z. 1995 Primary prevention of hepatocellular carcinoma. J. Gastroenterol Hepatol., 10(6), 674-82.
6. Falconer, I.R. 1991 Tumor promotion and liver injury caused by oral consumption of cyanobacteria. Environ. Toxicol. Water Qual., 6(2), 177-184.
7. Fitzgeorge, R.B., Clark, S.A., and Keevil, C.W. 1994 Routes of intoxication. In: G.A. Codd, T. M. Jeffries, C.W. Keevil and E. Potter [Eds] 1st International Symposium on Detection Methods for Cyanobacterial (Blue-Green Algal) Toxins, Royal Society of Chemistry, Cambridge, UK, 69-74.
8. Jochimsen, E.M., Carmichael, W.W., An, J., Cardo, D.M., Cookson, S.T., Holmes, C.E.M., Antunes, M.B. de C., Filho, D.A. de M., Lyra, T.M., Barreto, V.S.T., Azevedo, S.M.F.O. and Jarvis, W. R. 1998 Liver failure and death after exposure to microcystins at a haemodialysis center in Brazil. New Engl. J. Med., 338(13), 873-878.
9. http://www.epa.gov/cyano_habs_symposium/monograph/Ch36.pdf accessed at 11:30 24 January 2013.

  Authors K. H. Chafrom Chemical and Applied Market, Bruker Korea, and Kefei Wang, Chemical and Applied Markets, Bruker Fremont, CA USA.