New section for Microcystin-LR background document:

Treatment and control measures and technical achievability

Microcystins are largely cell-bound, with usually more than 95% of the toxin
contained within healthy cells. Dying and decaying cyanobacteria may release
microcystins into the water, but the data available indicate that usually
biodegradation will be sufficiently effective to preclude the build-up of high
concentrations of extracellular microcystin dissolved in water, unless cell lysis is
induced artificially (for such a case, see Jones & Orr 1994). A very effective way to
deal with high microcystin concentrations therefore is to remove the cells, intact and
without damage (Drikas et al. 2001; Hart et al. 1998). Any damage, such as that
caused by preoxidation, may lead to cell leakage, and consequently in an increase of
the dissolved toxin concentration entering the treatment plant. This may be critical,
as dissolved toxin is not removed by conventional treatment technologies.

Conventional treatment using coagulation will remove cyanobacteria cells; however,

sludge containing toxic cyanobacteria should be isolated from the treatment process
as cells contained in sludge can break down rapidly and release dissolved toxin
(Chow et al. 1999). Experimental and full-scale studies for the removal of
cyanobacteria using membranes are scarce. In general, micro- and ultra-filtration
membranes could be expected to remove cyanobacterial cells effectively. Membrane
filtration of toxic cyanobacteria should be carried out with frequent backwashing, and
isolation of the backwash water from the plant due to the risk of the cells releasing
dissolved toxin (Chow et al. 1997; Gijsbertsen-Abrahamse et al. 2006). The
treatments mentioned above will not remove extracellular, or dissolved toxin to a
significant extent.

Most of the common microcystin variants are well removed by activated carbon (Hart
et al 1998; UKWIR 1996; Cook and Newcombe 2002). The exception is microcystin
LA which is not readily removed and other processes are recommended (Cook and
Newcombe 2002). For other microcystins wood-based, chemically activated carbon
is the most effective, or a carbon with similar physical properties. Doses of powdered
activated carbon required for removal to below the guideline value will depend on
water quality, and site specific tests are recommended. Granular activated carbon
filtration displays a limited lifetime for all toxins. This can vary between 2 months to
more than one year depending on the type of toxin and the water quality (Newcombe
2002; UKWIR, 1996)

Dissolved microcystins have been shown to be removed by some reverse osmosis

and nanofiltration membranes. As removal will depend of membrane pore size
distribution and water quality, site specific tests are recommended (Smith et al 2002;
Gijsbertsen-Abrahamse et al. 2006; Muntisov and Trimboli 1996; Neumann and
Weckesser 1998).

Chlorination and ozonation are effective for the removal of microcystins. A residual of
at least 0.3 mg L-1 of ozone for 5 minutes will be sufficient for all of the most common
microcystins. For chlorine a dose of 3 mg L-1 applied to obtain a residual of 0.5 mg
L-1 for at least 30 minutes will be effective (Nicholson et al. 1994; Newcombe 2002;
Rositano et al. 1998: Rositano et al. 2001; Ho et al. 2006a; Acero et al. 2005).
Microcystin LA may require a higher residual, as it is slightly less susceptible to
oxidation by chlorine (Ho et al. 2006). Potassium permanganate is effective for
microcystins, and chlorine dioxide and chloramine are ineffective (Rositano et al.
1998).

Riverbank filtration and slow sand filtration have proven very effective in removing
microcystins, as cyanobacterial cells are retained and dissolved toxin is degraded in
the uppermost substrate layers. Grtzmacher et al. (2006) show that a travel time of
several days is likely to suffice, particularly if the underground consists of fine- to
middle-grained sand and conditions are aerobic, not below 10C, and some clogging
layer (i.e. biofilm) is present.

Biological filtration can be very effective for the removal of most toxins. However,
factors affecting the removal such as biofilm mass and composition, acclimation
periods, temperature and water quality cannot be easily controlled (Ho et al., 2006b).

References
Acero J., Rodriguez E. and Meriluoto J (2005) Kinetics of reactions between chlorine
and the cyanobacterial toxins microcystins. Water Research, 39, 1628-1638.
Chow, C. W. K., Drikas, M., House, J., Burch, M. D. and Velzeboer, R. M. A. (1999)
The impact of conventional water treatment processes on cells of the
cyanobacterium Microcystis aeruginosa. Water Research 33(15), 3253-3262.
Chow, C. W. K., Panglisch, S., House, J., Drikas, M., Burch, M. D. and Gimbel, R.
(1997) A study of membrane filtration for the removal of cyanobacterial cells.
Journal of Water SRT Aqua 46(6), 324-334.
Cook, D. and Newcombe, G. (2002) Removal of microcystin variants with powdered
activated carbon. Water Science & Technology: Water Supply 2(5/6), 201-
207.
Drikas, M., Chow, C. W. K., House, J. and Burch, M. D. (2001) Using coagulation,
flocculation and settling to remove toxic cyanobacteria. Journal of the
American Water Works Association 93(2), 100-111.
Hart, J., Fawell, J. K. and Croll, B. (1998) The fate of both intra- and extracellular
toxins during drinking water treatment. Water Supply 16(1/2), 611-616.
Ho L, Onstad G, von Gunten U, Rinck-Pfeiffer S, Craig K and Newcombe G (2006a)
Differences in the chlorine reactivity of four microcystin analogues. Water
Research 40 (6): 1200-1209.
Ho L, Meyn T, Keegan A, Hoefel D, Brookes J, Saint CP and Newcombe G (2006b)
Bacterial degradation of microcystin toxins within a biologically active sand
filter. Water Research 40 (4): 768-774.
Newcombe, G. (2002) Removal of algal toxins from drinking water using ozone and
GAC. AWWA Research Foundation Report, American Water Works
Association, Denver, CO.
Gijsbertsen-Abrahamse AJ, Schmidt W, Chorus I, Heijman SGJ. (2006) Removal of
cyanotoxins by ultrafiltration and nanofiltration Journal of Membrane Science
276 (1-2), 252-259
Grtzmacher, G., Wessel, G., Bartel, H. & Chorus, I. (2006): Final report NASRI:
Retention and elimination of cyanobacterial toxins (microcystins) through
artificial recharge and bank filtration. KompetenzZentrum Wasser Berlin.
Muntisov, M. and Trimboli, P. (1996) Removal of algal toxins using membrane
technology. Water Journal of the Australian Water Association 23(3), 34.
Neumann, U. and Weckesser, J. (1998) Elimination of microcystin peptide toxins
from water by reverse osmosis. Environmental Toxicology and Water Quality
13, 143-148.
Nicholson, B. C., Rositano, J. and Burch, M. D. (1994) Destruction of cyanobacterial
peptide hepatotoxins by chlorine and chloramine. Water Research 28(6),
1297-1303.
Rositano, J., Nicholson, B. C. and Pieronne, P. (1998) Destruction of cyanobacterial
toxins by ozone. Ozone: Science & Engineering 20, 223-238.
Rositano, J., Newcombe, G., Nicholson, B. and Sztajnbok, P. (2001) Ozonation of
NOM and algal toxins in four treated waters. Water Research 35(1), 23-32.
Smith, D. P., Falls, V., Levine, A. D., MacLeod, B., Simpson, M. and Champlin, T. L.
(2002) Nanofiltration to augment conventional treatment for removal of algal
toxins, taste and odor compounds, and natural organic matter. In Proceedings
of the Water Quality Technology Conference, November 10-14, 2002, Seattle,
Washington, USA. CD ROM.
UKWIR (1996) Pilot scale GAC tests to evaluate toxin removal. UK Water Industry
Research Ltd. Report No. 96/DW/07/1, London, UK.
Analytical methods for Microcystins:
Drafted by Ingrid Chorus, Yasumoto Magara, Jutta Fastner

Although the provisional WHO GV is specifically for microcystin-LR, most samples contain
several of microcystin variants, and for hazard analysis it is important to know the
concentrations of all of them. Methods for determining microcystins include
1. Physico-chemical analysis by chromatographic separation (HPLC, GC, LC) and
detection either by UV absorbance (photodiode array detector) or mass spectrometry.
2. Immunoassay (ELISA) for which several kits are commercially available
3. Enzyme assay using protein phosphatase inhibition.
An ISO-method for microcystin analysis by HPLC is available (ISO 20179 2005).
While chemical analysis differentiates between the structural variants of microcystin, immuno-
and enzyme assays detect the sum of all microcystins in a sample. The errors associated with
these assays are due to differences in reactivity between variants, but they are usually quicker to
perform, require less elaborate equipment and may be cheaper when analyzing large numbers
of samples.
Sampling:
For sampling raw water, it is important to take into account that some cyanobacterial taxa such
as Microcystis and Anabaena may accumulate as scums, therefore, sampling needs to consider
the purpose of determination. To determine the maximum concentration of microcystin in
water bodies, it may be adequate to directly sample the scum or the surface water, taking a grab
sample. To determine the mean concentration of microcystins in water bodies, a composite
sample mixed from samples taken at different depths from the surface to the bottom of the
water-body may be most representative. To determine concentrations in raw water, the choice
may be the site and depth of the drinking-water offtake.
Sample Pretreatment:
The major share of microcystins in a raw water sample is usually cell-bound, i.e. occurs in the
cyanobacterial biomass, however, particularly for assessing removal and/or breakthrough in
drinking-water treatment, analysis of dissolved microcystins is important. Furthermore, the
provisional WHO Guideline value is for the sum of cell-bound and dissolved microcystin-LR,
sometimes termed total microcystin (caution is required here because total microcystin is
also used by some authors for the sum of all structural variants). While analytical methods are
the same for both fractions, they do require different sample pre-treatment.
For separating cells from dissolved microcystins, the sample is filtered (pore size 0.45 m, at
most 1 m; the recommended volume to filter will depend on the cell density, frequently 50 ml
to 100 ml will suffice).
Microcystins bound in biomass need to be extracted. This may be achieved through sequential
extraction of the cells on the filter with aqueous methanol (to improve extraction efficiency
freeze-thawing cycles prior to extraction can be added or the use of sonication).
Dissolved microcystins can be detected directly in the filtrate with immuno- and enzyme assays
as well as with highly sensitive mass spectrometry usually down to 0.1 g/L. For detection of
lower concentrations as well as for UV-detection following chromatography, a concentration
step is usually needed. This can be achieved through solid phase extraction (e.g. with ODS-C18
(see ISO 20179:2005).
Chemical analysis:
Determination of microcystins by UV
HPLC coupled with UV-detection, standardized by the above-mentioned ISO-method, is the
most frequently used approach. It involves separation of the microcystins with a reversed phase
column using a gradient elution. Absorption spectra are acquired between 200 nm and 300 nm
(photodiode array detector), and microcystins are identified both by their characteristic
absorption spectrum (see ISO TC 147/SC 2 N 0689) and retention time (when the standards are
available). In absence of access to standards, verification of a peak as microcystin may be
achieved through analyzing individual peaks from characteristic samples with the protein
phosphatase assay or mass spectrometry, with the latter also providing information on the
structural variant. Quantification is achieved by measuring the chromatographic signals of each
microcystin against calibration curves produced from standards. Concentrations of further
microcystins for which no quantitative standards are available may be inferred by relating their
signal against that of MCYST-LR and then be reported as MCYST-LR concentration
equivalents (As response factors of microcystins in UV detection appear to vary by a factor of
maximally two, the uncertainty of this approach is negligible for hazard characterization).
The detection limit of this method is somewhat variable, depending on the matrix, but 0.1 g/l
may be achieved in most cases.
Determination of microcystins by mass spectrometry:
Unambiguous identification of microcystins may be achieved with a mass spectrometry (MS)
detector, ideally with tandem mass spectrometry (MS-MS) in order to obtain information both
on the molecular signal and fragments. Detection limits of 0.1 g/l and less can be achieved.
The drawback in relation to UV-detection is the potentially much higher variability of response
factors, so that estimates of quantification are not possible without standards for the specific
variant.
Determination of total microcystins by LC/MS or GC/MS:
Determination of microcystins is also possible through GC/MS and LC/MS after oxidation of
microcystin to 2-methyl-3-methoxy-4-phenyl butyric acid (MMPB), ionization with ESI
(atmospheric pressure ionization) for LC or chemical ionization for GC, and subsequent mass
spectrometric detection. This method is highly sensitive as it detects MMPB as surrogate,
achieving a detection limit of 0.1 g/L.
Determination of total microcystins with immunoassay (ELISA):
Several ELISA kits are commercially available which have plates or vials prepared with
microcystin antibodies attached to their walls. These determine total microcystins and do not
differentiate between structural variants. Most of the ELISA kits can produce results within 2
hrs in a routine laboratory. Their detection limit is in the range of 0.1 - 0.2 g/l.
Determination of total microcystins with enzyme assay (Protein Phosphatase Assay; PPA):
Colorimetric assays using the ability of microcystins to specifically inhibit type-1 protein
phosphatase are available. They allow visual confirmation of the presence or absence of
microcystin in a water sample by comparing the colour reaction against a control sample as well
as to roughly estimate the amount of toxin present in the sample (positive control is 1.5 g/L
microcystin-LR). False positives may be possible due to other PPA inhibitors in the sample, but
in practice have rarely proved problematic, particularly if information about potentially
occurring cyanobacterial species is available and indicates a likelihood of microcystins to
occur.

References:
ISO (2005): Water Quality: Determination of microcystins - method using solid phase
extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV)
detection. ISO, Geneva, Switzerland (ISO 20179:2005).