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Determination of Cyanotoxins in Drinking and Ambient Freshwaters

Initial detection of freshwater harmful algal bloom (HAB) events relies on qualitative, visual observations of blooms formation, including:
  • surface water discoloration (e.g., a red, green, or brown tint)
  • thick, mat-like accumulations on the shoreline and surface
  • fish kills

EPA developed the Cyanobacteria Assessment Network (CyAN) Mobile Application for the early detection of algal blooms in U.S water bodies and to help local and state water quality managers make faster and better-informed management decisions related to cyanobacterial blooms. It provides an easy-to-use, customizable interface for accessing algal bloom satellite data for over 2,000 of the largest lakes and reservoirs in the United States. The CyAN app is free and available for download in the Google Play store for Android™ devices.

Sample Collection

When collecting samples of water with cyanobacteria and /or cyanotoxins, samples should reflect the water source conditions and handled properly to ensure reliable results. If cyanobacteria and/or their cyanotoxins are suspected in surface water supplying a public water system, among the most important sample handling considerations are the following:

  • Collection – Bottle type, volume, and preservative used depend on the laboratory doing the analysis. Generally, samples should be collected and stored in amber glass containers to avoid potential cyanotoxin adsorption associated with plastic containers and to minimize exposure to sunlight.
  • Quenching – samples (particularly “finished” drinking water samples) that include a residual disinfectant, e.g., chlorine, should be quenched immediately upon sampling. Sodium thiosulfate or ascorbic acid are commonly used as quenching agents and their appropriateness can be specific to the analytical method selected to meet the monitoring data quality objectives. The different approaches are deliberate and designed to meet method performance goals that include established criteria for sample hold times.
  • Chilling – samples should be cooled immediately after collection, during shipping, and pending analysis at the laboratory. Depending on the analytical method being used, sample freezing may be appropriate to extend holding times, taking precautions to avoid breakage.

Analytical Methods

There is a diverse range of rapid screen tests and laboratory methods available to detect and identify cyanobacteria cells and cyanotoxins in water. These methods can vary greatly in their degree of sophistication and the information they provide. These methods include:

  • Enzyme–linked immunosorbent assays (ELISA)
  • Protein phosphatase inhibition assay (PPIA)
  • Reversed-phase high performance liquid chromatographic methods (HPLC) combined with mass spectrometric (MS, MS/MS) or ultraviolet/photodiode array detectors (UV/PDA).
  • Liquid chromatography/mass spectrometry (LC/MS)
  • Conventional polymerase chain reaction (PCR), quantitative real–time PCR (qPCR) and microarrays/DNA chips

Many of these methods have been developed to analyze for microcystins and its congeners, however, relatively little work has been done on methods for detection of other toxins, including anatoxins and cylindrospermopsins. Saxitoxins are the exception, as they also occur widely in the marine environment and many methods have been developed for their detection in shellfish.

EPA developed the following procedures for the detection of cyanotoxins in drinking water and ambient freshwater:

The table below describes the techiques available for cyanotoxin measurement in freshwater. Commercially available Enzyme-Linked Immunosorbent Assay (ELISA) test kits are one of the more commonly utilized cyanotoxin testing methods, since they do not require expensive equipment or extensive training to run. Semi-quantitative field screening ELISA kits are available for the presence or absence of cyanotoxins. If cyanotoxins are detected by a field screening kit, repeat analysis is recommended using either a quantitative ELISA test or one of the other analytical methods identified in the Table.

More precise, more quantitative ELISA test kits are available for microcystins/nodularins (including ADDA-ELISA), saxitoxin, anatoxin-a, and cylindrospermopsin. Although they provide rapid results, ELISA kits generally have limitations in selectivity and are not congener specific. In addition, the ability of ELISA to recognize different variants or congeners of cyanotoxins can vary quantitatively due to different cross-reactivities. The microcystins/nodularins (ADDA) kit is based on the ADDA structure within the microcystin molecule and is designed to detect over 100 microcystin congeners identified to date (but cannot distinguish between congeners).

Methods that utilize liquid chromatography combined with mass spectrometry (LC/MS) can precisely and accurately identify specific microcystin congeners for which standards are available. LC/MS methods have also been designed to minimize matrix interference. At this time there are only standards for a limited number of the known microcystin congeners. If congener-specific information is needed, an LC/MS (ion-trap, TOF, tandem mass spectrometry) method should be considered. HPLC-PDA methods are less selective than LC/MS methods and the quantitation is more problematic due to lower selectivity and to sample matrix interference. However, when analytical toxin standards are available for confirmation, they could provide a measure of resolution of the congeners present.

The following table describes the techniques available for cyanotoxin measurement in freshwater.

Freshwater Cyanotoxins
Techniques Anatoxins Cylindrospermopsins Microcystins Saxitoxins
Biological Assays
Mouse      Yes                   Yes          Yes  
Protein Phosphatase Inhibition Assays (PPIA)      No                    No          Yes  
Neurochemical      Yes                    No           No  
Enzyme-Linked Immunosorbent Assays
      Yes                    Yes           Yes       Yes
Chromatographic Methods
Gas Chromatography
Gas Chromatography with Flame
Ionization Detection (GC/FID)
      Yes                   No            No         No
Gas Chromatography with Mass
Spectrometry (GC/MS)
      Yes                  No            No         No
Liquid Chromatography  
Liquid Chromatography / Ultraviolet-
Visible Detection (LC/UV or LC/PDA)
       Yes                  Yes            Yes         Yes
Liquid Chromatography/Fluorescence (LC/FL)        Yes                  No            No         Yes
Liquid Chromatography Combined with Mass Spectrometry
Liquid Chromatography Ion Trap Mass        Yes                  Yes            Yes         Yes
Liquid Chromatography Time-of-Flight
Mass Spectrometry (LC/TOF MS)
       Yes                  Yes            Yes         Yes
Liquid Chromatography Single
Quadrupole Mass Spectrometry (LC/MS)
       Yes                  Yes            Yes        Yes
Liquid Chromatography Triple
Quadrupole Mass Spectrometry (LC/MS/MS)
        Yes                  Yes            Yes        Yes

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