HAB Methods
This page provides a description of current methods used to sample, monitor, detect and identify cyanobacteria cells, cyanotoxin producing genes and cyanotoxins in drinking and ambient freshwaters. It highlights important considerations during sample collection and provides an overview of analytical methods useful in freshwater cyanotoxin identification.
Detecting Cyanobacteria and Cyanotoxins in Drinking and Ambient Freshwaters
Initial detection of freshwater harmful algal bloom (HAB) events often 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
More quantitative detection methods employ satellites and surface water monitoring, both response oriented and routine.
The EPA developed the Cyanobacteria Assessment Network (CyAN) 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 addition, the National Aquatic Resource Surveys (NARS) collaborative programs between the EPA, state, and Tribal monitoring programs provide comprehensive, probabilistic estimates of condition in the nation’s waters. These programs include monitoring of algae and algal toxins. Information on these monitoring program results can be found at:
Many federal, state and Tribal water quality programs collect routine and targeted algal and algal toxins monitoring data. These entities submit this data through the Water Quality Exchange (WQX) where it is then made available for retrieval through the Water Quality Portal (WQP). These resources can be accessed through:
Monitoring
Monitoring for harmful algae and algal toxins will depend in some part on what waterbodies, what application, and what question any entity wants to address. Separate and distinct approaches and sampling methods exist for streams, lakes, wetland, and coastal systems. Similarly, different temporal and spatial sampling strategies will be required for monitoring drinking water facilities and their source waters than for recreational waters. Lastly, different approaches may be needed for surveillance as opposed to trends or specific research questions.
The EPA has a wide range of resources related to monitoring surface and drinking water quality for a range of audiences.
The Interstate Technology Regulatory Council (ITRC) also has a thorough discussion of cyanobacterial monitoring considerations and approaches for a variety of applications and waterbodies.
- Strategies for Preventing and Managing Harmful Cyanobacterial Blooms (HCB-1) (lakes and reservoirs)
- Strategies for Preventing and Managing Benthic Harmful Cyanobacterial Blooms (HCB-2) (streams)
Sample Collection
When collecting samples of water with cyanobacteria and /or cyanotoxins, samples should reflect the water source conditions and be 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 and the toxin being analyzed. 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.
More details on sampling collection requirements are provided in the method descriptions referenced on this page.
Analytical Methods for Toxin Analysis
There is a diverse range of rapid screen tests and laboratory methods available to detect and identify cyanobacteria cells, cyanotoxin producing genes 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 microcystin 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.
The EPA developed the following procedures for the detection of cyanotoxins in drinking water and ambient freshwater:
- Method 544: Determination of Microcystins and Nodularin in Drinking Water
- Method 545: Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water
- Method 546: Determination of Total Microcystins and Nodularins in Drinking and Ambient Waters
- Method for Determination of Cylindrospermopsin and Anatoxin-a in Ambient Freshwaters (pdf)
- Method for Determination of Microcystins and Nodularin in Ambient Freshwaters (pdf)
The table describes the techniques 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 cyanotoxin 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 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 (ELISA) |
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 |
Molecular tools, such as qPCR and variations of PCR, also exist for identifying and quantifying cyanobacteria – both targeted taxa as well as assemblage analysis based methods. Such methods are not always easy to translate into biomass or abundance, but they yield relative quantitative measures of abundance based on the copies of genes. These methods are also very sensitive and can identify taxa not always seen with traditional microscopic techniques.
Similar molecular tools can also be used to identify genes for toxin-production. While not the same as measuring toxin concentrations directly, they offer resource efficient ways of assessing the potential for the cyanobacteria present in a waterbody to produce toxins. Moreover, recent research suggests that the number of copies of toxin producing genes may also be a good predictor of future toxin concentration (e.g. a week ahead), giving water resources managers much needed time to prepare for in situ toxin sampling and measurement to protect human health.
To learn more about detection methods for cyanobacterial blooms and their toxins visit: